sirolimus and Breast-Neoplasms

Hormone receptor-positive (HR+) disease is the most frequently diagnosed subtype of breast cancer. Among tumor subtypes, natural course of HR+ breast cancer is indolent with favorable prognosis compared to other subtypes such as human epidermal growth factor protein 2-positive disease and triple-negative disease. HR+ tumors are dependent on steroid hormone signaling and endocrine therapy is the main treatment option. Recently, the discovery of cyclin-dependent kinase 4/6 inhibitors and their synergistic effects with endocrine therapy has dramatically improved treatment outcome of advanced HR+ breast cancer. The demonstrated efficacy of additional nonhormonal agents, such as targeted therapy against mammalian target of rapamycin and phosphatidylinositol 3-kinase signaling, poly(ADP-ribose) polymerase inhibitors, antibody-drug conjugates, and immunotherapeutic agents have further expanded the available therapeutic options. This article reviews the latest advancements in the treatment of HR+ breast cancer, and in doing so discusses not only the development of currently available treatment regimens but also emerging therapies that invite future research opportunities in the field.

Topics: Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Female; Humans; Prognosis; Protein Kinase Inhibitors; Sirolimus; Treatment Outcome

Breast cancer (BC) and prostate cancer (PC) are at the top of the list when it comes to the most common types of cancers worldwide. The phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway is important, in that it strongly influences the development and progression of these tumors. Previous studies have emphasized the key role of inhibitors of the PIK3/AKT/mTOR signaling pathway in the treatment of BC and PC, and it remains to be a crucial method of treatment. In this review, the inhibitors of these signaling pathways are compared, as well as their effectiveness in therapy and potential as therapeutic agents. The use of these inhibitors as polytherapy is evaluated, especially with the use of hormonal therapy, which has shown promising results.

Topics: Breast Neoplasms; Hormones; Humans; MTOR Inhibitors; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Prostatic Neoplasms; Proto-Oncogene Proteins c-akt; Sirolimus; TOR Serine-Threonine Kinases

The primary source of failure of cancer therapies is multidrug resistance (MDR), which can be caused by different mechanisms, including the overexpression of ABC transporters in cancer cells. Among the 48 human ABC proteins, the breast cancer resistance protein (BCRP/ABCG2) has been described as a pivotal player in cancer resistance. The use of functional inhibitors and expression modulators is a promising strategy to overcome the MDR caused by ABCG2. Despite the lack of clinical trials using ABCG2 inhibitors, many compounds have already been discovered. This review presents an overview about various ABCG2 inhibitors that have been identified, discussing some chemical aspects and the main experimental methods used to identify and characterize the mechanisms of new inhibitors. In addition, some biological requirements to pursue preclinical tests are described. Finally, we discuss the potential use of ABCG2 inhibitors in cancer stem cells (CSC) for improving the objective response rate and the mechanism of ABCG2 modulators at transcriptional and protein expression levels.

Topics: Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily G, Member 2; Breast Neoplasms; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Female; Humans; Neoplasm Proteins; Neoplastic Stem Cells

The coronavirus disease 2019 (COVID-19) has affected approximately 2 million individuals worldwide; however, data regarding fatal cases have been limited.. To report the clinical features of 162 fatal cases of COVID-19 from 5 hospitals in Wuhan between December 30, 2019 and March 12, 2020.. The demographic data, signs and symptoms, clinical course, comorbidities, laboratory findings, computed tomographic (CT) scans, treatments, and complications of the patients with fatal cases were retrieved from electronic medical records.. Young patients with moderate COVID-19 without comorbidity at admission could also develop fatal outcomes. The in-hospital survival time of the fatal cases was similar among the hospitals of different levels in Wuhan.

Topics: Adolescent; Adult; Animals; Asthma; Atrial Fibrillation; Autoantibodies; Biomarkers; Breast Neoplasms; Child; Conjunctivitis, Allergic; Cornea; COVID-19; Cyclosporine; Cytokines; Death, Sudden, Cardiac; Defibrillators, Implantable; Diet; Disease Models, Animal; Docetaxel; Double-Blind Method; Dry Eye Syndromes; Educational Status; Emulsions; Female; Fluorescein Angiography; Fluoresceins; Focus Groups; Heart Failure; Hemothorax; Humans; Inflammation; Keratoconus; Male; Meibomian Glands; Mice; Middle Aged; Multiple Sclerosis; Myocardial Infarction; Myocardium; Nerve Fibers; Nigeria; Obesity; Overweight; Pandemics; Primary Prevention; Prospective Studies; Qualitative Research; Registries; Retinal Ganglion Cells; Retinal Vessels; Schools; Sirolimus; Tertiary Care Centers; Th1 Cells; Th2 Cells; Tomography, Optical Coherence; Troponin I; Tumor Necrosis Factor-alpha; United States; Ventricular Remodeling

Patients with metastatic breast cancer may develop oral morbidities that result from therapeutic interventions. Mammalian target of rapamycin (mTOR) inhibitor-associated stomatitis (mIAS) is a common adverse event (AE), secondary to mTOR inhibitor therapy, that can have a negative impact on treatment adherence, quality of life, and health care costs. A multidisciplinary team approach is important to minimize mIAS and to maximize treatment benefits to patients with breast cancer. In this review, we discuss the pathophysiology, diagnosis, and natural history of mIAS. Current and new management strategies for the prevention and treatment of mIAS are described in the context of fostering a coordinated team care approach to optimizing patient care.. The authors conducted a PubMed search from 2007 through 2017 using the terms "stomatitis," "mIAS," "everolimus," "mTOR," "metastatic breast cancer," and "oral care." They selected articles published in peer-reviewed journals that reported controlled trials and evidence-based guidelines.. mIAS can be distinguished from mucositis caused by cytotoxic chemotherapy or radiotherapy on the basis of cause, clinical presentation, and treatment paradigms. Specific preventive and therapeutic management strategies can be implemented across the continuum of patient oral health care.. Oral health care providers are on the frontline of oral health care for patients with metastatic breast cancer and are uniquely positioned to provide patient education, advocate accurate reporting of mIAS, and support early identification, monitoring, and prompt intervention to mitigate the severity and duration of this manageable, potentially dose-limiting AE.

Topics: Antineoplastic Agents; Breast Neoplasms; Humans; Quality of Life; Sirolimus; Stomatitis; TOR Serine-Threonine Kinases

Metastatic breast cancer (MBC) is the leading cause of cancer-related morbidity and mortality among women worldwide. Endocrine therapy is the standard of care for the most common subtype of MBC, hormone-receptor positive (HR+), human epidermal growth factor receptor 2-negative (HER2-) disease. Advances in treating this type of MBC have focused on improving the efficacy of endocrine therapy by adding agents that target specific molecular pathways of breast cancer cell growth and survival. The combination of the aromatase inhibitor exemestane and the mammalian target of rapamycin inhibitor, everolimus, more than doubled median progression-free survival compared with exemestane alone (7.8 vs 3.2 months, respectively; hazard ratio 0.45 [95% confidence interval 0.38-0.54]; log rank P < 0.0001) in the BOLERO-2 study in postmenopausal women with HR+, HER2- locally advanced or metastatic breast cancer that had recurred or progressed on prior non-steroidal aromatase inhibitor therapy. In addition, everolimus plus exemestane was associated with a manageable safety profile. The results of BOLERO-2 led to regulatory approval of everolimus plus exemestane. Additional everolimus-based combinations have been or are under investigation in the HR+, HER2- MBC setting, including combinations with letrozole, fulvestrant, ribociclib, tamoxifen, and chemotherapy. This review summarizes key data on everolimus-based combinations focusing on efficacy, safety, biomarkers, quality of life, and health economic outcomes. These data are discussed in the context of the changing MBC treatment algorithm to provide insights into the clinical relevance of everolimus-based combinations.

Topics: Androstadienes; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Everolimus; Female; Humans; Prognosis; Receptor, ErbB-2; Receptors, Estrogen; Receptors, Progesterone; Sirolimus; Tamoxifen

Mechanistic target of rapamycin controls cell growth, metabolism, and aging in response to nutrients, cellular energy stage, and growth factors. In cancers including breast cancer, mechanistic target of rapamycin is frequently upregulated. Blocking mechanistic target of rapamycin with rapamycin, first-generation and second-generation mechanistic target of rapamycin inhibitors, called rapalogs, have shown potent reduction of breast cancer tumor growth in preclinical models and clinical trials. In this review, we summarize the fundamental role of the mechanistic target of rapamycin pathway in driving breast tumors. Moreover, we also review key molecules involved with aberrant mechanistic target of rapamycin pathway activation in breast cancer and current efforts to target these components for therapeutic gain. Further development of predictive biomarkers will be useful in the selection of patients who will benefit from inhibition of the mechanistic target of rapamycin pathway.

Topics: Breast Neoplasms; Cell Proliferation; Female; Humans; Molecular Targeted Therapy; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Middle East Respiratory Syndrome (MERS) is a novel respiratory illness firstly reported in Saudi Arabia in 2012. It is caused by a new corona virus, called MERS corona virus (MERS-CoV). Most people who have MERS-CoV infection developed severe acute respiratory illness.. This work is done to determine the clinical characteristics and the outcome of intensive care unit (ICU) admitted patients with confirmed MERS-CoV infection.. This study included 32 laboratory confirmed MERS corona virus infected patients who were admitted into ICU. It included 20 (62.50%) males and 12 (37.50%) females. The mean age was 43.99 ± 13.03 years. Diagnosis was done by real-time reverse transcription polymerase chain reaction (rRT-PCR) test for corona virus on throat swab, sputum, tracheal aspirate, or bronchoalveolar lavage specimens. Clinical characteristics, co-morbidities and outcome were reported for all subjects.. Most MERS corona patients present with fever, cough, dyspnea, sore throat, runny nose and sputum. The presence of abdominal symptoms may indicate bad prognosis. Prolonged duration of symptoms before patients' hospitalization, prolonged duration of mechanical ventilation and hospital stay, bilateral radiological pulmonary infiltrates, and hypoxemic respiratory failure were found to be strong predictors of mortality in such patients. Also, old age, current smoking, smoking severity, presence of associated co-morbidities like obesity, diabetes mellitus, chronic heart diseases, COPD, malignancy, renal failure, renal transplantation and liver cirrhosis are associated with a poor outcome of ICU admitted MERS corona virus infected patients.. Plasma HO-1, ferritin, p21, and NQO1 were all elevated at baseline in CKD participants. Plasma HO-1 and urine NQO1 levels each inversely correlated with eGFR (. SnPP can be safely administered and, after its injection, the resulting changes in plasma HO-1, NQO1, ferritin, and p21 concentrations can provide information as to antioxidant gene responsiveness/reserves in subjects with and without kidney disease.. A Study with RBT-1, in Healthy Volunteers and Subjects with Stage 3-4 Chronic Kidney Disease, NCT0363002 and NCT03893799.. HFNC did not significantly modify work of breathing in healthy subjects. However, a significant reduction in the minute volume was achieved, capillary [Formula: see text] remaining constant, which suggests a reduction in dead-space ventilation with flows > 20 L/min. (ClinicalTrials.gov registration NCT02495675).. 3 组患者手术时间、术中显性失血量及术后 1 周血红蛋白下降量比较差异均无统计学意义（. 对于肥胖和超重的膝关节单间室骨关节炎患者，采用 UKA 术后可获满意短中期疗效，远期疗效尚需进一步随访观察。.. Decreased muscle strength was identified at both time points in patients with hEDS/HSD. The evolution of most muscle strength parameters over time did not significantly differ between groups. Future studies should focus on the effectiveness of different types of muscle training strategies in hEDS/HSD patients.. These findings support previous adverse findings of e-cigarette exposure on neurodevelopment in a mouse model and provide substantial evidence of persistent adverse behavioral and neuroimmunological consequences to adult offspring following maternal e-cigarette exposure during pregnancy. https://doi.org/10.1289/EHP6067.. This RCT directly compares a neoadjuvant chemotherapy regimen with a standard CROSS regimen in terms of overall survival for patients with locally advanced ESCC. The results of this RCT will provide an answer for the controversy regarding the survival benefits between the two treatment strategies.. NCT04138212, date of registration: October 24, 2019.. Results of current investigation indicated that milk type and post fermentation cooling patterns had a pronounced effect on antioxidant characteristics, fatty acid profile, lipid oxidation and textural characteristics of yoghurt. Buffalo milk based yoghurt had more fat, protein, higher antioxidant capacity and vitamin content. Antioxidant and sensory characteristics of T. If milk is exposed to excessive amounts of light, Vitamins B. The two concentration of ZnO nanoparticles in the ambient air produced two different outcomes. The lower concentration resulted in significant increases in Zn content of the liver while the higher concentration significantly increased Zn in the lungs (p < 0.05). Additionally, at the lower concentration, Zn content was found to be lower in brain tissue (p < 0.05). Using TEM/EDX we detected ZnO nanoparticles inside the cells in the lungs, kidney and liver. Inhaling ZnO NP at the higher concentration increased the levels of mRNA of the following genes in the lungs: Mt2 (2.56 fold), Slc30a1 (1.52 fold) and Slc30a5 (2.34 fold). At the lower ZnO nanoparticle concentration, only Slc30a7 mRNA levels in the lungs were up (1.74 fold). Thus the two air concentrations of ZnO nanoparticles produced distinct effects on the expression of the Zn-homeostasis related genes.. Until adverse health effects of ZnO nanoparticles deposited in organs such as lungs are further investigated and/or ruled out, the exposure to ZnO nanoparticles in aerosols should be avoided or minimised.

Topics: A549 Cells; Acetylmuramyl-Alanyl-Isoglutamine; Acinetobacter baumannii; Acute Lung Injury; Adaptor Proteins, Signal Transducing; Adenine; Adenocarcinoma; Adipogenesis; Administration, Cutaneous; Administration, Ophthalmic; Adolescent; Adsorption; Adult; Aeromonas hydrophila; Aerosols; Aged; Aged, 80 and over; Aging; Agriculture; Air Pollutants; Air Pollution; Airway Remodeling; Alanine Transaminase; Albuminuria; Aldehyde Dehydrogenase 1 Family; Algorithms; AlkB Homolog 2, Alpha-Ketoglutarate-Dependent Dioxygenase; Alzheimer Disease; Amino Acid Sequence; Ammonia; Ammonium Compounds; Anaerobiosis; Anesthetics, Dissociative; Anesthetics, Inhalation; Animals; Anti-Bacterial Agents; Anti-HIV Agents; Anti-Infective Agents; Anti-Inflammatory Agents; Antibiotics, Antineoplastic; Antibodies, Antineutrophil Cytoplasmic; Antibodies, Monoclonal, Humanized; Antifungal Agents; Antigens, Bacterial; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Antimetabolites, Antineoplastic; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Antioxidants; Antitubercular Agents; Antiviral Agents; Apolipoproteins E; Apoptosis; Arabidopsis; Arabidopsis Proteins; Arsenic; Arthritis, Rheumatoid; Asthma; Atherosclerosis; ATP-Dependent Proteases; Attitude of Health Personnel; Australia; Austria; Autophagy; Axitinib; Bacteria; Bacterial Outer Membrane Proteins; Bacterial Proteins; Bacterial Toxins; Bacterial Typing Techniques; Bariatric Surgery; Base Composition; Bayes Theorem; Benzoxazoles; Benzylamines; beta Catenin; Betacoronavirus; Betula; Binding Sites; Biological Availability; Biological Oxygen Demand Analysis; Biomarkers; Biomarkers, Tumor; Biopsy; Bioreactors; Biosensing Techniques; Birth Weight; Blindness; Blood Chemical Analysis; Blood Gas Analysis; Blood Glucose; Blood Pressure; Blood Pressure Monitoring, Ambulatory; Blood-Brain Barrier; Blotting, Western; Body Mass Index; Body Weight; Bone and Bones; Bone Density; Bone Resorption; Borates; Brain; Brain Infarction; Brain Injuries, Traumatic; Brain Neoplasms; Breakfast; Breast Milk Expression; Breast Neoplasms; Bronchi; Bronchoalveolar Lavage Fluid; Buffaloes; Cadherins; Calcification, Physiologic; Calcium Compounds; Calcium, Dietary; Cannula; Caprolactam; Carbon; Carbon Dioxide; Carboplatin; Carcinogenesis; Carcinoma, Ductal; Carcinoma, Ehrlich Tumor; Carcinoma, Hepatocellular; Carcinoma, Non-Small-Cell Lung; Carcinoma, Pancreatic Ductal; Carcinoma, Renal Cell; Cardiovascular Diseases; Carps; Carrageenan; Case-Control Studies; Catalysis; Catalytic Domain; Cattle; CD8-Positive T-Lymphocytes; Cell Adhesion; Cell Cycle Proteins; Cell Death; Cell Differentiation; Cell Line; Cell Line, Tumor; Cell Movement; Cell Nucleus; Cell Phone Use; Cell Proliferation; Cell Survival; Cell Transformation, Neoplastic; Cell Transformation, Viral; Cells, Cultured; Cellulose; Chemical Phenomena; Chemoradiotherapy; Child; Child Development; Child, Preschool; China; Chitosan; Chlorocebus aethiops; Cholecalciferol; Chromatography, Liquid; Circadian Clocks; Circadian Rhythm; Circular Dichroism; Cisplatin; Citric Acid; Clinical Competence; Clinical Laboratory Techniques; Clinical Trials, Phase I as Topic; Clinical Trials, Phase II as Topic; Clostridioides difficile; Clostridium Infections; Coculture Techniques; Cohort Studies; Cold Temperature; Colitis; Collagen Type I; Collagen Type I, alpha 1 Chain; Collagen Type XI; Color; Connective Tissue Diseases; Copper; Coronary Angiography; Coronavirus 3C Proteases; Coronavirus Infections; Cost of Illness; Counselors; COVID-19; COVID-19 Testing; Creatine Kinase; Creatinine; Cross-Over Studies; Cross-Sectional Studies; Cryoelectron Microscopy; Cryosurgery; Crystallography, X-Ray; Cues; Cultural Competency; Cultural Diversity; Curriculum; Cyclic AMP Response Element-Binding Protein; Cyclin-Dependent Kinase Inhibitor p21; Cycloparaffins; Cysteine Endopeptidases; Cytokines; Cytoplasm; Cytoprotection; Databases, Factual; Denitrification; Deoxycytidine; Diabetes Complications; Diabetes Mellitus; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type 2; Diagnosis, Differential; Diatoms; Diet; Diet, High-Fat; Dietary Exposure; Diffusion Magnetic Resonance Imaging; Diketopiperazines; Dipeptidyl Peptidase 4; Dipeptidyl-Peptidase IV Inhibitors; Disease Models, Animal; Disease Progression; Disease-Free Survival; DNA; DNA Damage; DNA Glycosylases; DNA Repair; DNA-Binding Proteins; DNA, Bacterial; DNA, Viral; Docetaxel; Dose Fractionation, Radiation; Dose-Response Relationship, Drug; Down-Regulation; Doxorubicin; Drosophila; Drosophila melanogaster; Drug Carriers; Drug Delivery Systems; Drug Liberation; Drug Repositioning; Drug Resistance, Bacterial; Drug Resistance, Multiple, Bacterial; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Drug Synergism; Drug Therapy, Combination; Edema; Edible Grain; Education, Graduate; Education, Medical, Graduate; Education, Pharmacy; Ehlers-Danlos Syndrome; Electron Transport Complex III; Electron Transport Complex IV; Electronic Nicotine Delivery Systems; Emergency Service, Hospital; Empathy; Emulsions; Endothelial Cells; Endurance Training; Energy Intake; Enterovirus A, Human; Environment; Environmental Monitoring; Enzyme Assays; Enzyme Inhibitors; Epithelial Cells; Epithelial-Mesenchymal Transition; Epoxide Hydrolases; Epoxy Compounds; Erythrocyte Count; Erythrocytes; Escherichia coli; Escherichia coli Infections; Escherichia coli Proteins; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Esophagectomy; Estrogens; Etanercept; Ethiopia; Ethnicity; Ethylenes; Exanthema; Exercise; Exercise Test; Exercise Tolerance; Extracellular Matrix; Extracorporeal Membrane Oxygenation; Eye Infections, Fungal; False Negative Reactions; Fatty Acids; Fecal Microbiota Transplantation; Feces; Female; Femur Neck; Fermentation; Ferritins; Fetal Development; Fibroblast Growth Factor-23; Fibroblast Growth Factors; Fibroblasts; Fibroins; Fish Proteins; Flavanones; Flavonoids; Focus Groups; Follow-Up Studies; Food Handling; Food Supply; Food, Formulated; Forced Expiratory Volume; Forests; Fractures, Bone; Fruit and Vegetable Juices; Fusobacteria; G1 Phase Cell Cycle Checkpoints; G2 Phase Cell Cycle Checkpoints; Gamma Rays; Gastrectomy; Gastrointestinal Microbiome; Gastrointestinal Stromal Tumors; Gefitinib; Gels; Gemcitabine; Gene Amplification; Gene Expression; Gene Expression Regulation; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Neoplastic; Gene Expression Regulation, Plant; Gene Knockdown Techniques; Gene-Environment Interaction; Genotype; Germany; Glioma; Glomerular Filtration Rate; Glucagon; Glucocorticoids; Glycemic Control; Glycerol; Glycogen Synthase Kinase 3 beta; Glycolipids; Glycolysis; Goblet Cells; Gram-Negative Bacterial Infections; Granulocyte Colony-Stimulating Factor; Graphite; Greenhouse Effect; Guanidines; Haemophilus influenzae; HCT116 Cells; Health Knowledge, Attitudes, Practice; Health Personnel; Health Services Accessibility; Health Services Needs and Demand; Health Status Disparities; Healthy Volunteers; Heart Failure; Heart Rate; Heart Transplantation; Heart-Assist Devices; HEK293 Cells; Heme; Heme Oxygenase-1; Hemolysis; Hemorrhage; Hepatitis B; Hepatitis B e Antigens; Hepatitis B Surface Antigens; Hepatitis B virus; Hepatitis B, Chronic; Hepatocytes; Hexoses; High-Throughput Nucleotide Sequencing; Hippo Signaling Pathway; Histamine; Histamine Agonists; Histidine; Histone Deacetylase 2; HIV Infections; HIV Reverse Transcriptase; HIV-1; Homebound Persons; Homeodomain Proteins; Homosexuality, Male; Hospice and Palliative Care Nursing; HSP70 Heat-Shock Proteins; Humans; Hyaluronan Receptors; Hydrogen; Hydrogen Peroxide; Hydrogen-Ion Concentration; Hydrolysis; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Hypoglycemia; Hypoglycemic Agents; Hypoxia; Idiopathic Interstitial Pneumonias; Imaging, Three-Dimensional; Imatinib Mesylate; Immunotherapy; Implementation Science; Incidence; INDEL Mutation; Induced Pluripotent Stem Cells; Industrial Waste; Infant; Infant, Newborn; Inflammation; Inflammation Mediators; Infliximab; Infusions, Intravenous; Inhibitory Concentration 50; Injections; Insecticides; Insulin-Like Growth Factor Binding Protein 5; Insulin-Secreting Cells; Interleukin-1; Interleukin-17; Interleukin-8; Internship and Residency; Intestines; Intracellular Signaling Peptides and Proteins; Ion Transport; Iridaceae; Iridoid Glucosides; Islets of Langerhans Transplantation; Isodon; Isoflurane; Isotopes; Italy; Joint Instability; Ketamine; Kidney; Kidney Failure, Chronic; Kidney Function Tests; Kidney Neoplasms; Kinetics; Klebsiella pneumoniae; Knee Joint; Kruppel-Like Factor 4; Kruppel-Like Transcription Factors; Lactate Dehydrogenase 5; Laparoscopy; Laser Therapy; Lasers, Semiconductor; Lasers, Solid-State; Laurates; Lead; Leukocyte L1 Antigen Complex; Leukocytes, Mononuclear; Light; Lipid Peroxidation; Lipopolysaccharides; Liposomes; Liver; Liver Cirrhosis; Liver Neoplasms; Liver Transplantation; Locomotion; Longitudinal Studies; Lopinavir; Lower Urinary Tract Symptoms; Lubricants; Lung; Lung Diseases, Interstitial; Lung Neoplasms; Lymphocyte Activation; Lymphocytes, Tumor-Infiltrating; Lymphoma, Mantle-Cell; Lysosomes; Macrophages; Male; Manganese Compounds; MAP Kinase Kinase 4; Mass Screening; Maternal Health; Medicine, Chinese Traditional; Melanoma, Experimental; Memantine; Membrane Glycoproteins; Membrane Proteins; Mesenchymal Stem Cell Transplantation; Metal Nanoparticles; Metalloendopeptidases; Metalloporphyrins; Methadone; Methane; Methicillin-Resistant Staphylococcus aureus; Mexico; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Inbred ICR; Mice, Knockout; Mice, Nude; Mice, SCID; Mice, Transgenic; Microarray Analysis; Microbial Sensitivity Tests; Microbiota; Micronutrients; MicroRNAs; Microscopy, Confocal; Microsomes, Liver; Middle Aged; Milk; Milk, Human; Minority Groups; Mitochondria; Mitochondrial Membranes; Mitochondrial Proteins; Models, Animal; Models, Molecular; Molecular Conformation; Molecular Docking Simulation; Molecular Dynamics Simulation; Molecular Epidemiology; Molecular Structure; Molecular Weight; Multilocus Sequence Typing; Multimodal Imaging; Muscle Strength; Muscle, Skeletal; Muscular Diseases; Mutation; Mycobacterium tuberculosis; Myocardial Stunning; Myristates; NAD(P)H Dehydrogenase (Quinone); Nanocomposites; Nanogels; Nanoparticles; Nanotechnology; Naphthalenes; Nasal Cavity; National Health Programs; Necrosis; Needs Assessment; Neoadjuvant Therapy; Neonicotinoids; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Proteins; Neoplasm Recurrence, Local; Neoplasm Staging; Neoplasm Transplantation; Neoplasms; Neoplastic Stem Cells; Netherlands; Neuroblastoma; Neuroprotective Agents; Neutrophils; NF-kappa B; NFATC Transcription Factors; Nicotiana; Nicotine; Nitrates; Nitrification; Nitrites; Nitro Compounds; Nitrogen; Nitrogen Dioxide; North Carolina; Nuclear Magnetic Resonance, Biomolecular; Nuclear Proteins; Nucleic Acid Hybridization; Nucleosomes; Nutrients; Obesity; Obesity, Morbid; Oceans and Seas; Oncogene Protein v-akt; Oncogenes; Oocytes; Open Reading Frames; Osteoclasts; Osteogenesis; Osteoporosis; Osteoporosis, Postmenopausal; Outpatients; Ovarian Neoplasms; Ovariectomy; Overweight; Oxazines; Oxidants; Oxidation-Reduction; Oxidative Stress; Oxides; Oxidoreductases; Oxygen; Oxygen Inhalation Therapy; Oxygenators, Membrane; Ozone; Paclitaxel; Paenibacillus; Pain Measurement; Palliative Care; Pancreatic Neoplasms; Pandemics; Parasympathetic Nervous System; Particulate Matter; Pasteurization; Patient Preference; Patient Satisfaction; Pediatric Obesity; Permeability; Peroxiredoxins; Peroxynitrous Acid; Pharmaceutical Services; Pharmacists; Pharmacy; Phaseolus; Phenotype; Phoeniceae; Phosphates; Phosphatidylinositol 3-Kinases; Phospholipid Transfer Proteins; Phospholipids; Phosphorus; Phosphorylation; Photoperiod; Photosynthesis; Phylogeny; Physical Endurance; Physicians; Pilot Projects; Piperidines; Pituitary Adenylate Cyclase-Activating Polypeptide; Plant Extracts; Plant Leaves; Plant Proteins; Plant Roots; Plaque, Atherosclerotic; Pneumonia; Pneumonia, Viral; Point-of-Care Testing; Polyethylene Glycols; Polymers; Polysorbates; Pore Forming Cytotoxic Proteins; Positron Emission Tomography Computed Tomography; Positron-Emission Tomography; Postprandial Period; Poverty; Pre-Exposure Prophylaxis; Prediabetic State; Predictive Value of Tests; Pregnancy; Pregnancy Trimester, First; Pregnancy, High-Risk; Prenatal Exposure Delayed Effects; Pressure; Prevalence; Primary Graft Dysfunction; Primary Health Care; Professional Role; Professionalism; Prognosis; Progression-Free Survival; Prolactin; Promoter Regions, Genetic; Proof of Concept Study; Proportional Hazards Models; Propylene Glycol; Prospective Studies; Prostate; Protein Binding; Protein Biosynthesis; Protein Isoforms; Protein Kinase Inhibitors; Protein Phosphatase 2; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Protein Structure, Tertiary; Protein Transport; Proteoglycans; Proteome; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-myc; Proto-Oncogene Proteins c-ret; Proto-Oncogene Proteins p21(ras); Proton Pumps; Protons; Protoporphyrins; Pseudomonas aeruginosa; Pseudomonas fluorescens; Pulmonary Artery; Pulmonary Disease, Chronic Obstructive; Pulmonary Gas Exchange; Pulmonary Veins; Pyrazoles; Pyridines; Pyrimidines; Qualitative Research; Quinoxalines; Rabbits; Random Allocation; Rats; Rats, Sprague-Dawley; Rats, Wistar; Receptors, Histamine H3; Receptors, Immunologic; Receptors, Transferrin; Recombinant Proteins; Recurrence; Reference Values; Referral and Consultation; Regional Blood Flow; Registries; Regulon; Renal Insufficiency, Chronic; Reperfusion Injury; Repressor Proteins; Reproducibility of Results; Republic of Korea; Research Design; Resistance Training; Respiration, Artificial; Respiratory Distress Syndrome; Respiratory Insufficiency; Resuscitation; Retinal Dehydrogenase; Retreatment; Retrospective Studies; Reverse Transcriptase Inhibitors; Rhinitis, Allergic; Ribosomal Proteins; Ribosomes; Risk Assessment; Risk Factors; Ritonavir; Rivers; RNA Interference; RNA-Seq; RNA, Messenger; RNA, Ribosomal, 16S; RNA, Small Interfering; Rosuvastatin Calcium; Rural Population; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Salivary Ducts; Salivary Gland Neoplasms; San Francisco; SARS-CoV-2; Satiation; Satiety Response; Schools; Schools, Pharmacy; Seasons; Seawater; Selection, Genetic; Sequence Analysis, DNA; Serine-Threonine Kinase 3; Sewage; Sheep; Sheep, Domestic; Shock, Hemorrhagic; Signal Transduction; Silver; Silymarin; Single Photon Emission Computed Tomography Computed Tomography; Sirolimus; Sirtuin 1; Skin; Skin Neoplasms; Skin Physiological Phenomena; Sleep Initiation and Maintenance Disorders; Social Class; Social Participation; Social Support; Soil; Soil Microbiology; Solutions; Somatomedins; Soot; Specimen Handling; Spectrophotometry, Ultraviolet; Spectroscopy, Fourier Transform Infrared; Spectrum Analysis; Spinal Fractures; Spirometry; Staphylococcus aureus; STAT1 Transcription Factor; STAT3 Transcription Factor; Streptomyces coelicolor; Stress, Psychological; Stroke; Stroke Volume; Structure-Activity Relationship; Students, Medical; Students, Pharmacy; Substance Abuse Treatment Centers; Sulfur Dioxide; Surface Properties; Surface-Active Agents; Surveys and Questionnaires; Survival Analysis; Survival Rate; Survivin; Sweden; Swine; Swine, Miniature; Sympathetic Nervous System; T-Lymphocytes, Regulatory; Talaromyces; Tandem Mass Spectrometry; tau Proteins; Telemedicine; Telomerase; Telomere; Telomere Homeostasis; Temperature; Terminally Ill; Th1 Cells; Thiamethoxam; Thiazoles; Thiophenes; Thioredoxin Reductase 1; Thrombosis; Thulium; Thyroid Cancer, Papillary; Thyroid Carcinoma, Anaplastic; Thyroid Neoplasms; Time Factors; Titanium; Tomography, Emission-Computed, Single-Photon; Tomography, X-Ray Computed; TOR Serine-Threonine Kinases; Transcription Factor AP-1; Transcription Factors; Transcription, Genetic; Transcriptional Activation; Transcriptome; Transforming Growth Factor beta1; Transistors, Electronic; Translational Research, Biomedical; Transplantation Tolerance; Transplantation, Homologous; Transportation; Treatment Outcome; Tretinoin; Tuberculosis, Multidrug-Resistant; Tuberculosis, Pulmonary; Tubulin Modulators; Tumor Microenvironment; Tumor Necrosis Factor Inhibitors; Tumor Necrosis Factor-alpha; Twins; Ultrasonic Therapy; Ultrasonography; Ultraviolet Rays; United States; Up-Regulation; Uranium; Urethra; Urinary Bladder; Urodynamics; Uromodulin; Uveitis; Vasoconstrictor Agents; Ventricular Function, Left; Vero Cells; Vesicular Transport Proteins; Viral Nonstructural Proteins; Visual Acuity; Vital Capacity; Vitamin D; Vitamin D Deficiency; Vitamin K 2; Vitamins; Volatilization; Voriconazole; Waiting Lists; Waste Disposal, Fluid; Wastewater; Water Pollutants, Chemical; Whole Genome Sequencing; Wine; Wnt Signaling Pathway; Wound Healing; Wounds and Injuries; WW Domains; X-linked Nuclear Protein; X-Ray Diffraction; Xanthines; Xenograft Model Antitumor Assays; YAP-Signaling Proteins; Yogurt; Young Adult; Zebrafish; Zebrafish Proteins; Ziziphus

We evaluated if standard hormonal therapy (HT) could be improved by the addition of mammalian target of rapamycin inhibitors (mTOR-I) in metastatic luminal breast cancer. A meta-analysis on 4 phase II-III randomized clinical trials was performed. Pooled hazard ratio (HR) for progression free survival (PFS)/ time to progression (TTP) was 0.62 in favor of mTOR-I+HT arm (95% confidence interval [CI] 0.55-0.70; p<0.0001). There was significant heterogeneity for PFS/TTP (Cochran's Q 32, p<0.0001, I2 index 90.6%). Pooled HR for overall survival (OS) was 0.84 in favor of the combination arm (95% CI 0.71-0.99; p=0.04). Heterogeneity was not significant (Cochran's Q 4.47, p=0.1, I2 index 55.3%). Pooled risk ratio (RR) for objective response rate (ORR) was 0.88 in favor of experimental arm (95% CI 0.85-0.91; p<0.0001). Heterogeneity was not significant (Cochran's Q 2.11, p=0.3, I2 index 5.2%). Adverse events (AEs), in particular those of grade 3-4, mostly occurred in mTOR-I+HT arm. Combination therapy of HT plus mTOR-I improves the outcome of metastatic luminal breast cancer patients. Our results provide evidence of a class-effect of these targeting molecules.

Topics: Antineoplastic Agents, Hormonal; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Clinical Trials, Phase II as Topic; Clinical Trials, Phase III as Topic; Disease-Free Survival; Humans; Neoplasm Metastasis; Outcome Assessment, Health Care; Proportional Hazards Models; Randomized Controlled Trials as Topic; Receptor, ErbB-2; Sirolimus; Tamoxifen; TOR Serine-Threonine Kinases

Disease progression despite existing endocrine therapies remains a major challenge to the effective management of hormone-receptor-positive (HR(+)), human epidermal growth factor receptor-2-negative (HER2(-)), advanced breast cancer. Recent advances in elucidating the molecular mechanisms of disease progression have identified the existence of adaptive "cross-talk" between the estrogen receptor (ER) and various growth factor receptor and intracellular signaling pathways, allowing breast cancer cells to escape the inhibitory effects of endocrine therapy. These findings provide the clinical rationale for enhancing or extending endocrine sensitivity by combining endocrine therapy with a targeted agent against a compensatory pathway. In BOLERO-2, adding the mTOR inhibitor everolimus to endocrine therapy significantly improved progression-free survival (PFS) in patients with HR(+) advanced breast cancer previously treated with nonsteroidal aromatase inhibitor therapy. Notably, PFS benefits were comparable in subgroup analyses of first- and later-line settings. These results contrast with those of the large first-line HORIZON study, wherein adding the mTOR inhibitor temsirolimus to endocrine therapy did not improve PFS. Therefore, it is unclear whether a targeted agent should only be combined with endocrine therapy to restore endocrine sensitivity or whether it may also prevent or delay resistance in hormone-sensitive advanced breast cancer. Numerous additional targeted agents are currently being evaluated in combination with endocrine therapies, including PI3K, cyclin-dependent kinase 4/6, SRC, and histone deacetylase inhibitors. Appropriate patient selection based on prior treatment history will become increasingly important in maximizing the incremental benefit derived from these new agents combined with existing endocrine therapies in HR(+) advanced breast cancer.

Topics: Anastrozole; Androstadienes; Antibodies, Monoclonal, Humanized; Antineoplastic Agents, Hormonal; Antineoplastic Combined Chemotherapy Protocols; Aromatase Inhibitors; Bevacizumab; Biomarkers, Tumor; Breast Neoplasms; Clinical Trials as Topic; Disease-Free Survival; Estradiol; Everolimus; Female; Fulvestrant; Humans; Molecular Targeted Therapy; Nitriles; Patient Selection; Postmenopause; Receptor, ErbB-2; Receptors, Estrogen; Receptors, Progesterone; Sirolimus; Treatment Failure; Treatment Outcome; Triazoles

Endocrine therapy is the recommended systemic therapy for hormone receptor (HR) positive metastatic breast cancer (MBC). However so far the limited number of endocrine agents and the onset of endocrine resistance have severely limited the therapeutic options for this patients. In the last years many targeted agents have been investigated to prevent or overcome endocrine resistance; only a few of them have been found effective in HR positive MBC, such as everolimus, CK4/6 inhibitors and HDAC inhibitors. Furthermore, translational medicine studies using next generation sequencing technologies have evaluated genetic variations of a broad panel of cancer-related genes and explored their correlations with targeted agents benefit. In some studies predictive biomarkers have been identified and many ongoing studies are evaluating the efficacy of targeted drugs in HR positive MBC patients selected for biomarkers or stratified by pathways amplification.

Topics: Antineoplastic Agents, Hormonal; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Clinical Trials, Phase II as Topic; Clinical Trials, Phase III as Topic; Everolimus; Female; Humans; Neoplasm Metastasis; Receptor, ErbB-2; Receptors, Estrogen; Receptors, Progesterone; Sirolimus; Translational Research, Biomedical; Treatment Outcome

Hormone-dependent breast cancer is the first example of cancer treated by targeted therapy for more than 30 years. Blocking estrogen pathway was the first therapeutical strategy for this subtype of breast cancer, and remains the principle of current standard treatment. Despite the efficacy of drugs used in endocrine therapy, hormone resistance is a major problem for the management of patients with hormone-dependent breast cancer. In this review, we will discuss the development of strategies targeting the PI3K/Akt/mTOR pathway, CDK4/6 (Cyclin Dependent Kinase 4/6) and FGFR (Fibroblast Growth Factor Receptor) in hormone-dependent metastatic breast cancer (ER+). Recent results of clinical trials showed that combination of endocrine therapy with such pharmacological inhibitors is a promising strategy to overcome endocrine resistance. Mutated forms and isoforms of ERα have been recently discovered and its targeting could represent an therapeutic alternative. Future progress will focus on the identification of new compounds and combinations with other targeted therapies to improve the efficacy of such inhibitors in clinical practice.

Topics: Antineoplastic Agents, Hormonal; Aromatase Inhibitors; Breast Neoplasms; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Drug Resistance, Neoplasm; Elafin; Estrogen Receptor Antagonists; Female; Humans; Molecular Targeted Therapy; Neoplasms, Hormone-Dependent; Proto-Oncogene Proteins c-akt; Receptors, Fibroblast Growth Factor; Selective Estrogen Receptor Modulators; Sirolimus; TOR Serine-Threonine Kinases

Despite improvements in early detection, surgery and systemic therapy, metastatic breast cancer remains a major cause of death. Luminal type breast cancers expressing hormone estrogen receptor (ER) or progesterone (PR) and without HER2 overexpression are generally sensitive to endocrine therapy, but raise the issue of the occurrence of resistance to treatment, particularly at metastatic stage. A better understanding of hormone resistance may guide the development of new therapeutics. New strategies aim at enhancing and prolonging of endocrine sensitivity, by optimizing existing schemes, or by combining an endocrine therapy with a targeted therapies specific to hormone resistance pathways: ER signaling, PI3K/AKT/mTOR and Cyclin Dependent Kinase (CDK). Key corners of 2014 include confirmation of benefit of high dose fulvestrant, and commercialization of everolimus as the first mTOR inhibitor in this indication. Other strategies are being tested dealing with new endocrine therapies or new molecular targets such as PI3K inhibitors, insulin-like growth factor receptor (IGF-R) and histone deacetylase (HDAC) inhibitors. Coming years may be fruitful and might radically change our way to treat these patients.

Topics: Antineoplastic Agents, Hormonal; Breast Neoplasms; Estradiol; Estrogen Receptor Antagonists; Everolimus; Female; Fulvestrant; Histone Demethylases; Humans; Molecular Targeted Therapy; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase Inhibitors; Randomized Controlled Trials as Topic; Receptor, ErbB-2; Receptor, IGF Type 1; Receptors, Estrogen; Receptors, Progesterone; Sirolimus; TOR Serine-Threonine Kinases

Owing to development of next generation sequencer (NGS), deep biological insights of breast cancer have been provided. Information of genomic mutations and rearrangements in patients' tumors is required to understand the mechanism in resistance of molecular targeted medicine. To date, NGS analyses illustrated not only base substitution patterns and lists of driver mutations and key rearrangements, but also a manner of tumor evolution. Breast cancer genome is dynamically changing and evolving during cancer development course and treatment procedures. Liquid biopsy is a non-invasive method to detect the genomic evolution of breast cancer, which is now under development for future application to clinical practice of cancer treatment. In this article, latest knowledge regarding breast cancer genome, especially in terms of 'evolution', is summarized.

Topics: Antibodies, Monoclonal, Humanized; Bevacizumab; Breast Neoplasms; Denosumab; Everolimus; Evolution, Molecular; Female; Gene Rearrangement; Genome, Human; Genomics; Humans; Molecular Targeted Therapy; Mutation; RANK Ligand; Receptor, ErbB-2; Sirolimus; TOR Serine-Threonine Kinases; Trastuzumab; Vascular Endothelial Growth Factor A

The mammalian target of rapamycin (mTOR) inhibitor class of drugs represents the newest addition to the armamentarium of therapies for hormonally driven breast cancer. It has recently been shown that the addition of mTOR inhibitor everolimus to aromatase inhibitors in hormone receptor-positive breast cancers improves progression-free survival. However, a clinically significant toxicity associated with this class of drugs is the development of noninfectious pneumonitis (NIP). Although generally mild and manageable, everolimus-induced NIP requires prompt diagnosis and management. This article will provide a brief overview of data relating to dysregulation of the phosphatidylinositol-3-kinase/protein kinase B/mTOR pathway in breast cancer; review the literature relating to the efficacy and safety of mTOR inhibitors in breast cancer; and evaluate the incidence, severity, and optimal management of mTOR inhibitor-related NIP in breast cancer.

Topics: Antineoplastic Agents; Breast Neoplasms; Cough; Dyspnea; Elafin; Everolimus; Female; Humans; Incidence; Pneumonia; Proto-Oncogene Proteins c-akt; Severity of Illness Index; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Breast cancer cells can develop resistance to standard hormonal treatment and chemotherapy with the activation of the mTOR pathway; this is supported by results of preclinical and clinical studies. In clinical trials, the addition of everolimus to hormonal treatment or anti-HER2 treatment improved the outcomes of breast cancer patients. The aim of this review is to discuss the efficacy and safety data of everolimus in all categories of breast cancer in recent published studies.. Everolimus showed positive results in clinical studies. A literature search was made from PubMed, ASCO and San Antonio Breast Cancer Symposium Meeting abstracts by using the following search key words: 'everolimus', 'RAD001', 'mTOR inhibitor', 'breast cancer' 'endocrine therapy resistance' and 'HER-2 targeted therapies'. The last search was on June 10, 2013. The most important limitation of our review is that most of the data on everolimus rely on phase I and II trials.. Preclinical studies showed that mTOR activation can be the responsible mechanism in all subgroups of breast cancer. Results of both the TAMRAD and BOLERO-2 studies have showed that mTOR inhibition in combination with endocrine therapy can be a new treatment strategy for MBC patients who are resistant to aromatase inhibitors. In the BOLERO-2 study, time to deterioration in health-related quality of life was also significantly higher in the everolimus and exemestane arm compared to the exemestane plus placebo arm. The recently completed BOLERO-3 study showed that mTOR inhibition in combination with trastuzumab plus vinorelbine treatment significantly improved PFS compared to trastuzumab plus vinorelbine alone in trastuzumab-resistant MBC patients.. Recent trials have shown that everolimus has produced promising anti-tumor activity in combination with trastuzumab in HER2-positive metastatic breast cancer and in combination with exemestane in patients with hormone-receptor-positive metastatic breast cancer who had recurrence or progression while receiving a nonsteroidal aromatase inhibitor. Results of ongoing studies with everolimus show evidence that using everolimus in earlier stages of the disease, namely in the adjuvant and neoadjuvant settings, could be benefical.

Topics: Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Carboplatin; Disease-Free Survival; Drug Resistance, Neoplasm; Everolimus; Female; Humans; Immunosuppressive Agents; Neoplasm Recurrence, Local; Paclitaxel; Receptor, ErbB-2; Sirolimus; TOR Serine-Threonine Kinases; Trastuzumab

Human epidermal growth factor receptor 2 (HER2) is overexpresed in 15-20% of all breast cancers. Treatment with trastuzumab has led to an improved outcome and prolonged survival of HER2-positive breast cancer patients and today the drug is established as standard of care in both the adjuvant and metastatic settings. However, trastuzumab resistance is common and a major focus in the treatment of HER2-positive breast cancer has been developing therapeutic agents to either potentiate the effect of trastuzumab or to target cells which have become resistant to trastuzumab. The present review addresses efficacy and toxicity of dual targeting in HER2-positive breast cancer.. A computer-based literature search was carried out using PubMed; data reported at international meetings and clinicaltrials.gov was included.. This paper describes efficacy and safety of lapatinib, pertuzumab or trastuzumab-DM1 in combination with trastuzumab in the (neo)adjuvant and metastatic settings. Furthermore, combinations of trastuzumab and drugs targeting the downstream pathway are described.. Dual blockade is likely to represent a substantial advance for patients with HER2-positive breast cancer. However, the relevant subpopulation remains to be defined and side effects including cardiotoxicity might be a limiting factor to the use. There is an urgent need for prospective biomarker-driven trials to identify patients for whom dual targeting is cost-effective.

Topics: Ado-Trastuzumab Emtansine; Afatinib; Antibodies, Monoclonal, Humanized; Antineoplastic Combined Chemotherapy Protocols; Biomarkers, Tumor; Breast Neoplasms; Clinical Trials as Topic; Disease-Free Survival; Everolimus; Female; Gene Expression Regulation, Neoplastic; Humans; Lapatinib; Maytansine; Molecular Targeted Therapy; Neoadjuvant Therapy; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Quinazolines; Quinolines; Receptor, ErbB-2; Sirolimus; TOR Serine-Threonine Kinases; Trastuzumab; Treatment Outcome; Up-Regulation

The estrogen steroid hormone receptor (ER) and human epithelial growth factor receptor 2 membrane tyrosine kinase growth factor receptor (HER2) are the mediators of two key pathways involved in breast carcinogenesis, invasive behavior and cell growth. Co-expression of these receptors results in specific biological features that are not fully understood, but include relative resistance to hormonal therapy and chemotherapy as well as better long-term outcome imparted by ER and worse outcome by HER2 expression. The ER and HER2 signaling pathways interact with each other as do many biological networks, and this creates opportunities for therapeutic co-targeting with agents that modulate these respective pathways. However, relatively few studies have been conducted to test concurrent manipulation of ER and HER2. The avoidance of chemotherapy side effects is an attractive feature that has further spurred explorations in this strategy. Still, the only dually targeted strategy approved by some regulatory agencies is the combination of hormonal therapy using aromatase inhibition and the HER2 kinase inhibitor lapatinib. Other dual combinations have also demonstrated a benefit, although most of the testing has compared hormonal therapy with or without HER2-directed agents and not the other way around, limiting the applicability of this concept in routine clinical practice, especially when chemotherapy is also used. Newer generation signal transduction inhibitors can augment the efficacy of hormonal therapy, with one such example of mTOR blockade using everolimus now in the clinic. The logical extension of ER and HER2 co-targeting is the discovery and clinical testing of "synthetic lethal" combinations attacking diverse pathways that produce quantum improvements over either therapy alone. Molecular annotation of human cancers can further inform personalized combinatorial regimens based on the unique circuitry of an individual patient's tumor, with the potential to yield much more than incremental gains in survival.

Topics: Anastrozole; Androstadienes; Antibodies, Monoclonal, Humanized; Antineoplastic Combined Chemotherapy Protocols; Aromatase Inhibitors; Breast Neoplasms; Estradiol; Everolimus; Female; Fulvestrant; Gefitinib; Humans; Lapatinib; Letrozole; Molecular Targeted Therapy; Nitriles; Quinazolines; Receptor, ErbB-2; Receptors, Estrogen; Signal Transduction; Sirolimus; Tamoxifen; Trastuzumab; Triazoles

Postmenopausal women with advanced breast cancer recurring/progressing on or after initial (adjuvant or first-line) endocrine therapy may be treated multiple times with one of several endocrine or combinatorial targeted treatment options before initiating chemotherapy. In the absence of direct head-to-head comparisons of these treatment options, an indirect comparison can inform treatment choice. This network meta-analysis compared the efficacy of everolimus plus exemestane with that of fulvestrant 250 and 500 mg in the advanced breast cancer setting following adjuvant or first-line endocrine therapy. The reported hazard ratios (HRs) for progression-free survival (PFS) or time to progression from six studies that formed a network to compare everolimus plus exemestane (BOLERO-2 trial) with fulvestrant were analyzed by means of a Bayesian network meta-analysis. In the primary comparison (PFS analysis based on the local review of disease progression from BOLERO-2 with the data from the other studies), everolimus plus exemestane appeared to be more efficacious than both fulvestrant 250 mg (HR = 0.47; 95 % credible interval [CrI] 0.38-0.58) and 500 mg (HR = 0.59; 95 % CrI 0.45-0.77). Similar results were obtained in an alternate comparison based on central review of disease progression from BOLERO-2 with the data from the other studies (HR = 0.40; 95 % CrI 0.31-0.51 and HR = 0.50; 95 % CrI 0.37-0.67, respectively), and in a subgroup analysis of patients who had received prior aromatase inhibitor therapy (HR = 0.47; 95 % CrI 0.38-0.58 and HR = 0.55; 95 % CrI 0.40-0.76, respectively). These results suggest that everolimus plus exemestane may be more efficacious than fulvestrant in patients with advanced breast cancer who progress on or after adjuvant or first-line therapy with a nonsteroidal aromatase inhibitor.

Topics: Androstadienes; Antineoplastic Combined Chemotherapy Protocols; Aromatase Inhibitors; Breast Neoplasms; Clinical Trials as Topic; Disease Progression; Estradiol; Everolimus; Female; Fulvestrant; Humans; Neoplasm Recurrence, Local; Neoplasm Staging; Proportional Hazards Models; Receptor, ErbB-2; Receptors, Estrogen; Receptors, Progesterone; Sirolimus; Treatment Outcome

Most breast cancer (BC) patients have tumors that express hormone receptors (HRs). Although endocrine therapy, such as aromatase inhibitors, is very effective, most patients with metastatic HR-positive (HR(+)) BC become resistant to endocrine therapy at some point in their treatment and subsequently require chemotherapy. The PI3K/mTOR pathway is often upregulated in endocrine-resistant BC patients and, therefore, has been one of the targets for development of new agents. Recently, a Phase III trial (BOLERO-2) in aromatase inhibitor-resistant BC patients showed a significant improvement in time to progression with the combination of everolimus and exemestane compared with exemestane alone, confirming the importance of the PI3K/mTOR pathway in endocrine-resistant BC. Side effects from mTOR inhibitors are manageable, but early detection and proactive management are required to ensure patients' safety, compliance and continuity of treatment. Thus, mTOR inhibitors offer a new hope and promise for patients with HR(+) BC.

Topics: Antineoplastic Agents, Hormonal; Breast Neoplasms; Drug Resistance, Neoplasm; Drug Synergism; Everolimus; Female; Humans; Indoles; Purines; Sirolimus; TOR Serine-Threonine Kinases

Everolimus, an orally administered rapamycin analogue, inhibits the mammalian target of rapamycin (mTOR), a highly conserved intracellular serine-threonine kinase that is a central node in a network of signaling pathways controlling cellular metabolism, growth, survival, proliferation, angiogenesis, and immune function. Everolimus has demonstrated substantial clinical benefit in randomized, controlled, phase III studies leading to approval for the treatment of advanced renal cell carcinoma, advanced neuroendocrine tumors of pancreatic origin, renal angiomyolipoma and subependymal giant-cell astrocytoma associated with tuberous sclerosis complex, as well as advanced hormone-receptor-positive (HR(+)) and human epidermal growth factor receptor-2-negative advanced breast cancer.. We discuss clinically relevant everolimus-related adverse events from the phase III studies, including stomatitis, noninfectious pneumonitis, rash, selected metabolic abnormalities, and infections, with focus on appropriate clinical management of these events and specific considerations in patients with breast cancer.. The majority of adverse events experienced during everolimus therapy are of mild to moderate severity. The safety profile and protocols for toxicity management are well established. The class-effect adverse event profile observed with everolimus plus endocrine therapy in breast cancer is (as expected) distinct from that of endocrine therapy alone, but is similar to that observed with everolimus in other solid tumors. Information gained from the experience in other carcinomas on prompt diagnosis and treatments to optimize drug exposure, treatment outcomes, and patients' quality of life also applies to the patient population with advanced breast cancer.. As with all orally administered agents, education of both physicians and patients in the management of adverse events for patients receiving everolimus is critical to achieving optimal exposure and clinical benefit. Active monitoring for early identification of everolimus-related adverse events combined with aggressive and appropriate intervention should lead to a reduction in the severity and duration of the event.

Topics: Administration, Oral; Breast Neoplasms; Drug-Related Side Effects and Adverse Reactions; Everolimus; Female; Humans; Neoplasm Staging; Sirolimus

Phosphatidylinositol-3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway is commonly deregulated in breast cancer and has been involved in resistance to endocrine therapy. In the BOLERO-2 study, the addition of everolimus, a selective inhibitor of mTOR protein, to exemestane was associated with a significant improvement in progression-free survival, compared to exemestane plus placebo, in patients with hormone receptor-positive, HER2-negative metastatic breast cancer, and resistant to non-steroidal aromatase inhibitor therapy. However, adverse events and treatment stops were more often observed with the combination therapy, suggesting the need for a careful benefit/risk evaluation before initiating this new combination. This review aims at synthesizing the biological basis of the everolimus-exemestane association, presenting the main validated and ongoing therapeutic trials, interests and limits, as well as the multiple potential therapeutic perspectives.

Topics: Androstadienes; Antineoplastic Combined Chemotherapy Protocols; Aromatase Inhibitors; Breast Neoplasms; Drug Resistance, Neoplasm; Everolimus; Female; Humans; Neoplasm Proteins; Phosphatidylinositol 3-Kinases; Randomized Controlled Trials as Topic; Sirolimus; TOR Serine-Threonine Kinases

Endocrine therapy remains a mainstay in the treatment of hormone-sensitive metastatic breast cancer. Nevertheless, acquired resistance to endocrine therapy is an important clinical problem. Understanding the mechanisms of resistance is fundamental in order to develop new therapeutic strategies such as mTOR inhibition through everolimus. Its efficacy in association with endocrine therapy has been shown in two randomized trials. However, the addition of everolimus to endocrine therapy is accompanied by a significant increase in potentially severe side effects. Identifying and adequately addressing these side effects is crucial to decrease toxicity of these new therapies.

Topics: Antineoplastic Agents, Hormonal; Breast Neoplasms; Clinical Trials as Topic; Drug Resistance, Neoplasm; Everolimus; Female; Humans; Neoplasm Metastasis; Sirolimus; TOR Serine-Threonine Kinases

Breast cancer (BC) is diagnosed in nearly 1 in 3 women with cancer in the United States; one third of these patients have regional lymph node metastases at the time of diagnosis. The 5-year survival rate of patients with metastatic BC is very low, and approximately 40,000 women were expected to die of the disease in 2013. About 75% of patients with BC have hormone receptor-positive (HR(+)) disease, which is often managed with endocrine therapy; however, most patients eventually have resistance to these therapies. Recently, the mammalian target of rapamycin (mTOR) inhibitor everolimus, in combination with exemestane, improved progression-free survival (PFS) of patients with advanced BC, leading to its approval by the US Food and Drug Administration. Because adverse events (AEs) associated with everolimus might differ from AEs that oncologists who treat patients with BC are more familiar with, everolimus AEs and their effective management are reviewed in this article. Possible dose adjustments of everolimus for patients with renal or hepatic impairment and strategies for minimizing potential interactions of everolimus with other drugs and food are also discussed.

Topics: Antineoplastic Agents; Breast Neoplasms; Everolimus; Female; Humans; Medical Oncology; Sirolimus; TOR Serine-Threonine Kinases

The combination of exemestane and everolimus is a new treatment option for metastatic hormone-sensitive breast cancer. This treatment is used after progression on non-steroidal aromatase inhibitors. The treatment is generally well tolerated, but sometimes leads to minor or even serious side effects. It is important to be aware of these side effects and to treat them. We describe two patients who had to cope with various forms of toxicity: a 73-year-old woman with aphthous mouth lesions and a 49-year-old woman with pneumonitis. We then discuss the efficacy of the combination exemestane and everolimus and its positioning in the treatment of metastatic hormone-sensitive breast cancer. Finally, some common and some potentially serious side effects will be discussed, along with recommendations for their management and indications for distinguishing side effects from disease progression.

Topics: Aged; Androstadienes; Antineoplastic Agents; Breast Neoplasms; Drug Therapy, Combination; Everolimus; Fatigue; Female; Humans; Sirolimus

Skeletal metastases are an incurable complication afflicting the majority of patients who die from advanced breast cancer. They are most often osteolytic, characterized by net bone destruction and suppressed new bone formation. Life expectancy from first diagnosis of breast cancer bone metastases is several years, during which time skeletal-related events - including pain, fracture, hypercalcemia, and spinal cord compression - significantly degrade quality of life. The bone marrow niche can also confer hormonal and chemo-resistance. Most treatments for skeletal metastases target bone-destroying osteoclasts and are palliative. Recent results from the Breast cancer trials of Oral Everolimus-2 trial suggest that agents such as the mammalian target of rapamycin inhibitor everolimus may have efficacy against breast cancer bone metastases in part via stimulating osteoblasts as well as by inhibiting tumor growth. Selective estrogen receptor modulators similarly inhibit growth of estrogen receptor-positive breast cancers while having positive effects on the skeleton. This review discusses the future role of bone-anabolic agents for the specific treatment of osteolytic breast cancer metastases. Agents with both anti-tumor and bone-anabolic actions have been tested in the setting of multiple myeloma, a hematological malignancy that causes severe osteolytic bone loss and suppression of osteoblastic new bone formation. Stimulation of osteoblast activity inhibits multiple myeloma growth - a strategy that might decrease breast cancer burden in osteolytic bone metastases. Proteasome inhibitors (bortezomib and carfilzomib) inhibit the growth of myeloma directly and are anabolic for bone. Drugs with limited anti-tumor activity but which are anabolic for bone include intermittent parathyroid hormone and antibodies that neutralize the WNT inhibitors DKK1 and sclerostin, as well as the activin A blocker sotatercept and the osteoporosis drug strontium ranelate. Transforming growth factor-beta inhibitors have little tumor antiproliferative activity but block breast cancer production of osteolytic factors and are also anabolic for bone. Some of these treatments are already in clinical trials. This review provides an overview of agents with bone-anabolic properties, which may have utility in the treatment of breast cancer metastatic to the skeleton.

Topics: Antineoplastic Agents; Bone Density Conservation Agents; Bone Neoplasms; Boronic Acids; Bortezomib; Breast Neoplasms; Everolimus; Female; Humans; Oligopeptides; Osteoblasts; Parathyroid Hormone; Proteasome Inhibitors; Pyrazines; Recombinant Fusion Proteins; Sirolimus; Thiophenes; Transforming Growth Factor beta

Sequential endocrine treatments are recommended for estrogen receptor (ER) positive human epidermal growth factor receptor 2 (HER 2) negative metastatic breast cancers except in the case of symptomatic visceral disease. However, patients who suffer from disease progression while receiving a non-steroidal aromatase inhibitor (NSAI) have a very poor prognosis with standard endocrine therapy alone. Recently, based onthe results of the BOLERO 2 trial, the mammalian target of rapamycin (mTOR) inhibitor everolimus, combined with exemestane, a steroidal aromatase inhibitor, has been approved in Europe and the US for patients suffering from ER positive HER2 negative advanced breast cancer previously treated by a NSAI. The median progression-free survival (PFS) increased from 3.2 to 7.8 months in patients receiving everolimus and exemestane compared to placebo and exemestane. The magnitude of benefit was consistent in all pre-specified subgroups. Side effects were manageable and the quality of life was at least maintained. Everolimus has also beenrecently studied in HER2 positive locally advanced or metastatic disease in heavily pretreated patients (BOLERO 3 trial). This trial met its primary endpoint. The median PFS was increased in patients receiving trastuzumab, vinorelbine and everolimus compared to patients receiving trastuzumab, vinorelbine and placebo. We review pharmacological data and side effects of the drug. We also review the most important clinical trials leading to reimbursement of everolimus in metastatic breast cancer.

Topics: Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Drug-Related Side Effects and Adverse Reactions; Everolimus; Female; Humans; Neoplasm Metastasis; Receptor, ErbB-2; Sirolimus

Although patients with hormone receptor (HR)-positive breast cancer are successfully treated with endocrine therapy, many tumors go on to develop resistance to these agents. Studies have determined that mechanisms of resistance to endocrine therapy are quite complex and can involve a multitude of signal transduction pathways, either through direct association with the estrogen receptor or through cross-talk with other pathways. Preclinical studies have suggested the therapeutic importance of the mammalian target of rapamycin (mTOR) pathway and that inhibiting this pathway may restore sensitivity to endocrine therapy. The oral mTOR inhibitor everolimus has been extensively studied for breast cancer. Clinical studies suggest that everolimus in combination with endocrine therapy improves progression-free survival and is well tolerated. A combined approach, targeting both mTOR signal transduction and the HR pathways, promises to take clinical research in a new direction for the treatment of HR-positive advanced breast cancer.

Topics: Antineoplastic Agents; Antineoplastic Agents, Hormonal; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Carcinoma; Drug Resistance, Neoplasm; Everolimus; Female; Humans; Receptor, ErbB-2; Receptors, Estrogen; Receptors, Progesterone; Signal Transduction; Sirolimus; Tamoxifen; TOR Serine-Threonine Kinases; Treatment Outcome

Oral everolimus (Afinitor(®)) in combination with exemestane is indicated for the treatment of hormone receptor-positive, human epidermal growth factor receptor (HER) 2-negative advanced breast cancer in postmenopausal women after failure of treatment with letrozole or anastrozole (in the USA) or after recurrence of progression following a nonsteroidal aromatase inhibitor (AI) in women without symptomatic visceral disease (in the EU). Everolimus, a selective inhibitor of mammalian target of rapamycin (mTOR), inhibits the downstream signalling events of the mTOR pathway. This review summarizes the pharmacology of everolimus and reviews its efficacy and tolerability when administered in combination with exemestane in postmenopausal women with oestrogen receptor-positive, HER2-negative advanced breast cancer refractory to nonsteroidal AIs. In the well-designed BOLERO-2 study, the addition of everolimus to exemestane was shown to significantly prolong progression-free survival in this patient population. However, treatment-emergent adverse events and treatment discontinuations occurred more frequently with combination therapy than with exemestane alone, suggesting a need for careful benefit/risk assessment prior to initiating therapy. Mature survival data from this study are awaited and additional studies would help to further demonstrate the benefit of combination therapy. Nevertheless, current evidence suggests that everolimus plus exemestane combination therapy may be a useful treatment option in patients with postmenopausal hormone receptor-positive, HER2-negative, advanced breast cancer refractory to nonsteroidal AIs.

Topics: Androstadienes; Antineoplastic Combined Chemotherapy Protocols; Aromatase Inhibitors; Breast Neoplasms; Everolimus; Female; Humans; Postmenopause; Randomized Controlled Trials as Topic; Receptor, ErbB-2; Secondary Prevention; Sirolimus; Treatment Outcome

Everolimus is a mammalian target of rapamycin (mTOR) inhibitor approved for the treatment of advanced renal cell carcinoma, pancreatic neuroendocrine tumors, subependymal giant cell astrocytoma associated with tuberous sclerosis complex, renal angiomyolipoma and tuberous sclerosis complex, and, in combination with exemestane, for hormone receptor-positive HER2-negative advanced breast cancer after failure of treatment with letrozole or anastrozole. Results from the phase III BOLERO-2 trial demonstrated that everolimus in combination with exemestane provided significant clinical benefit to patients with advanced hormone receptor-positive breast cancer. Although everolimus is generally well tolerated, as with most therapies administered in an advanced cancer setting, drug-related adverse events (AEs) inevitably occur. Most common AEs observed in the everolimus studies include stomatitis, rash, infection, noninfectious pneumonitis, and hyperglycemia. Clinical awareness and early identification of such AEs by oncology nurses are essential to dosing (interruptions, reduction, and treatment discontinuation); quality of life; and, ultimately, patient outcomes. Because everolimus has already been shown to significantly improve clinical efficacy in patients with advanced breast cancer, a proactive approach to the practical management of AEs associated with this mTOR inhibitor as well as other most common AEs observed in this patient population has been reviewed and outlined here.

Topics: Adult; Androstadienes; Antineoplastic Agents; Breast Neoplasms; Clinical Trials, Phase III as Topic; Everolimus; Exanthema; Female; Humans; Hyperglycemia; Pneumonia; Sirolimus; Stomatitis; Treatment Outcome

Patients with breast cancer face substantial challenges to bone health from bone metastases, as well as from chemotherapy and endocrine therapies that generally elicit disease control at the cost of increased bone turnover. Consequently, maintaining bone health is of critical importance for these patients. Recently reported results from BOLERO-2 showed significant clinical benefits with adding everolimus to exemestane therapy in postmenopausal women with estrogen-receptor-positive breast cancer recurring or progressing despite nonsteroidal aromatase inhibitor therapy. Moreover, exploratory analyses from BOLERO-2 showed that adding everolimus may have beneficial effects on bone turnover and progressive disease in bone in this patient population. These results are supported by preclinical studies in which mTOR inhibition was associated with decreased osteoclast survival and activity. Thus, everolimus therapy may be able to ameliorate the negative effects of estrogen suppression on bone health. This review discusses the effects of mTOR inhibition on bone health during endocrine therapy.

Topics: Animals; Antineoplastic Agents; Bone and Bones; Bone Neoplasms; Breast Neoplasms; Everolimus; Female; Humans; Sirolimus; TOR Serine-Threonine Kinases

Approximately 75% of patients with breast cancer present hormone receptor-positive tumors. This subtype of breast cancer initially shows a high overall response rate to hormonal treatments. However, resistance eventually develops, resulting in tumor progression. The PI3K/Akt/mTOR pathway regulates several cellular functions in cancer such as cell growth, survival, and proliferation. In addition, a high activation level of the PI3K/Akt/mTOR pathway is related to resistance to conventional chemotherapy and hormone therapy. The mTOR inhibitor everolimus, in combination with hormonal treatments, has led to excellent results in progression-free survival in patients with metastatic breast cancer resistant to hormone therapies. Therefore, everolimus has entered the National Comprehensive Cancer Network (NCCN) guidelines 2012 and its combination with exemestane was approved recently by the US Food and Drug Administration and the European Medicines Agency. This is the first time that a drug will have been approved for the restoration of hormone sensitivity in breast cancer.

Topics: Androstadienes; Animals; Antineoplastic Combined Chemotherapy Protocols; Aromatase Inhibitors; Biomarkers, Tumor; Breast Neoplasms; Disease-Free Survival; Everolimus; Female; Humans; Molecular Targeted Therapy; Protein Kinase Inhibitors; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Treatment Outcome

Everolimus is an orally available inhibitor of the mammalian target of rapamycin (mTOR), which has been approved in combination with exemestane for hormone receptor-positive (HR) breast cancer after failure of treatment with non-steroidal aromatase inhibitors. Everolimus is generally very well tolerated with most common side effects including stomatitis, rash, fatigue, hyperglycemia, hyperlipidemia, and myelosuppression. Most of these side effects are mild and resolve with dose interruptions or dose reductions. Symptomatic non-infectious pneumonitis is a relatively uncommon class effect of mTOR inhibitors, which can be life threatening. Given the efficacy of everolimus in HR-positive metastatic breast cancer, it is crucial for physicians to recognize toxicities related to everolimus and start timely interventions. This review will focus on the adverse events reported with everolimus in breast cancer trials and will provide practical guidelines for the management of these adverse events.

Topics: Antineoplastic Agents; Breast Neoplasms; Everolimus; Fatigue; Female; Humans; Hyperglycemia; Hyperlipidemias; Pneumonia; Sirolimus; Stomatitis; TOR Serine-Threonine Kinases

Hepatitis B reactivation can occur with cytotoxic chemotherapy in patients with hepatitis B and cancer. Reactivation can occur in a patient with chronic hepatitis, an inactive carrier, or one with resolved hepatitis. Clinical presentation may range from subclinical elevation of liver enzymes to fatal fulminant hepatic failure. Mammalian target of rapamycin inhibitors, which include everolimus, are a new generation of targeted agents that are currently approved for many cancers (since March 2009) including advanced hormone receptor positive, human epidermal growth factor receptor 2-negative breast cancer, in conjunction with exemestane (as of July 2012). We are therefore still learning the various adverse events that occur with this new class of agents. Here, we present an unfortunate case of fatal hepatitis B reactivation in a woman with metastatic breast cancer treated with everolimus and exemestane. We have detailed the controversies around hepatitis B screening prior to immunosuppressive therapy. Clinicians and patients should be aware of this rare but fatal complication prior to everolimus use, and a detailed history, screening for hepatitis B and prophylactic antiviral treatment should be considered.

Topics: Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Disease Progression; Everolimus; Fatal Outcome; Female; Hepatitis B; Hepatitis B virus; Humans; Middle Aged; Neoplasm Metastasis; Sirolimus; Treatment Outcome; Virus Activation

To review the studies addressing mammalian target of rapamycin (mTOR) inhibitors in breast cancer and resistance to rapalogs. Preclinical and clinical studies have suggested mTOR inhibitors may help overcome the resistance to endocrine therapy and trastuzumab. Despite much interest, knowledge of the mechanism and molecular response to mTOR inhibitors is incomplete.. Resistance to mTOR inhibitors has been explored in preclinical studies and can be defined as primary, associated with amplifications or mutations of different kinases, or secondary, in which rapalog activates the feedback loops involving the insulin-like growth factor I receptor (IGF-IR), platelet-derived growth factor receptor and mitogen-activated protein kinase (MAPK) pathway. Current clinical trials are testing the combinations of rapamycin with other kinase inhibitors including IGF-IR, phosphoinositide 3-kinase and MAPK-extracellular signal-regulated kinase inhibitors.. Recent findings on the resistance to rapalogs have stimulated the assessment of combinations of inhibitors in clinical trials. This review summarizes the current knowledge of primary and secondary rapalog resistance, and the current efforts to overcome this resistance.

Topics: Androstadienes; Antibodies, Monoclonal, Humanized; Antineoplastic Combined Chemotherapy Protocols; Biomarkers, Tumor; Breast Neoplasms; Clinical Trials as Topic; Drug Therapy, Combination; Everolimus; Female; Humans; Mitogen-Activated Protein Kinases; Molecular Targeted Therapy; Protein Kinase Inhibitors; Receptor, IGF Type 1; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Trastuzumab; Treatment Outcome

In the last decade, the targeted therapy of breast cancer became part of routine clinical protocols all over the globe. Options in today's targeted therapy include hormonal therapy and the modulation of the EGFR/HER-pathway. Of the four HER receptors, HER2 is the target of currently used treatment strategies. HER2 activates multiple intracellular pathways via RAS, RAF and PI3K. We give a comprehensive summary of approved monoclonal antibodies and tyrosine kinase inhibitors acting over HER2, including trastuzumab, lapatinib and pertuzumab. We elaborate on their mechanism of action and on clinical trials behind their approval. Agents in third phase clinical studies (neratinib, afatinib) are also described. We give a brief overview of agents currently in phase I and phase II studies; these are acting over the PI3K pathway, over IGFR1 and over HSP90. Furthermore, currently validated negative biomarkers (markers predicting lack of response) in clinical use are also summarized. Finally, the major bottlenecks of clinical application including tumor heterogeneity and the high diversity of clinical studies are discussed. For a breakthrough we will need to identify new positive biomarkers of therapy response.. Az utóbbi évtizedben az emlõrák célzott terápiája világszerte a klinikai rutin részévé vált. A jelenleg elérhetõ célzott terápiás kezelések célpontjai a hormonreceptorok és az EGFR/HER útvonal. A négy HER receptor közül a HER2 alkalmazott terápiás célpont, mely a RAS, RAF és PI3K molekulákon keresztül aktiválja a sejten belüli jelátviteli útvonalakat. Összefoglaló írásunkban röviden ismertetjük a HER2-n ható, az emlõrák terápiájában már elfogadott monoklonális antitesteket (trastuzumab, pertuzumab) és tirozinkináz-gátlót (lapatinib), valamint az elfogadásukhoz vezetõ klinikai vizsgálatok eredményeit. A harmadik fázisban lévõ klinikai vizsgálatokba bevont szerek (neratinib, afatinib) is bemutatásra kerülnek, illetve röviden bemutatjuk az elsõ és második fázisban lévõ, az mTOR, a PI3K, az IGFR1 és a HSP90 molekulákon ható szereket is. Kitérünk a klinikai gyakorlatban alkalmazható negatív biomarkerekre (amelyek a terápiás válasz hiányát tudják elõre jelezni). További klinikai áttöréshez új terápiás választ elõre jelzõ pozitív biomarkerekre lesz szükség.

Topics: Ado-Trastuzumab Emtansine; Afatinib; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Bevacizumab; Biomarkers, Tumor; Breast Neoplasms; Clinical Trials as Topic; Everolimus; Female; Gene Amplification; Gene Expression Regulation, Neoplastic; Humans; Lapatinib; Maytansine; Molecular Targeted Therapy; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Quinazolines; Quinolines; Receptor, ErbB-2; Sirolimus; TOR Serine-Threonine Kinases; Trastuzumab

In recent years, a number of new molecules - commonly known as biological therapies - have been approved or are in late stages of regulatory evaluation for the treatment of advanced breast cancer. These innovative compounds have improved treatment efficacy and have probably contributed to the increase in survival length observed in some breast cancer subtypes. However, these agents are not deprived of toxicity, which can impair quality of life and may occasionally be life-threatening. In this article, we reviewed the most common toxicities associated with these drugs and provided a number of practical recommendations on their optimal clinical management.

Topics: Ado-Trastuzumab Emtansine; Angiogenesis Inhibitors; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Bevacizumab; Biological Therapy; Breast Neoplasms; Drug-Related Side Effects and Adverse Reactions; Everolimus; Humans; Immunosuppressive Agents; Lapatinib; Maytansine; Quinazolines; Sirolimus; Trastuzumab

Mammalian target of rapamycin (mTOR) is a crucial mediator of tumor progression and may be a promising target in a significant proportion of patients with breast cancer. More specifically, the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT)/mTOR pathway plays a critical role in multiple cellular functions including metabolism, proliferation, growth and survival. This pathway is higly active in many types of cancer and is linked to resistance to many types of therapy. Direct blockade of the mTOR pathway is a new area in breast cancer therapy, with the potential to modulate growth factor- and estrogen-dependent and estrogen-independent pathways, which contribute to the pathogenesis and progression of tumors. Thus, inhibitors of mTOR are of interest as potential therapeutic agents for patients with breast cancer, everolimus and temsirolimus being the main representatives of this category. This review of the literature analyzes the available data emerging from trials and evaluates the efficacy and safety of mTOR inhibitors in all subtypes of breast cancer.

Topics: Breast Neoplasms; Clinical Trials as Topic; Drug Resistance, Neoplasm; Estrogens; Everolimus; Female; Humans; Molecular Targeted Therapy; Neoplasms, Hormone-Dependent; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Receptor, ErbB-2; Sirolimus; TOR Serine-Threonine Kinases

The PI3K-AKT-mTOR network has been the major focus of attention for cancer researchers (both in the clinic and the laboratory) in the last decade. An incomplete knowledge of the molecular biology of this complex network has seen an expansion of first generation allosteric mTOR inhibitors, rapalogues, but also biomarker studies designed to identify the best responders of these agents. Currently, research in this pathway has focused on the dual nature of mTOR that is integrated by mTOR-RAPTOR complex (mTORC1) and mTOR-RICTOR complex (mTORC2). These two complexes are regulated by different upstream proteins and also regulated by multiple different compensatory feedback loops. The related advantage of feedback regulation of signaling systems is that it allows diversification in signal response. This deeper understanding has facilitated the development of a novel second generation of inhibitors that are able to affect both mTORC1 and mTORC2, and their downstream effectors, through inhibition of their catalytic activity (ATP competitive inhibitors, attacking the kinase domain of this protein) than binding to the FKBP12 regulatory proteins as for rapalogues. This article reviews the newest insight in the signaling network of the mTOR pathway, preclinical/clinical status of mTOR inhibitors (including second generation of kinase inhibitors) and then focuses on the development of a new wave of research related to combination therapies in subset specific breast tumors.

Topics: Antibiotics, Antineoplastic; Breast Neoplasms; Humans; Phosphatidylinositol 3-Kinases; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

The mammalian target of rapamycin (mTOR) is a signaling kinase of the phosphatidylinositol 3-kinase/protein kinase B (also known as Akt) signaling pathway that mediates cell growth and metabolism. Dysregulation of the mTOR pathway creates a favorable environment for the development and progression of many cancers, including breast cancer, and is associated with the development of resistance to endocrine therapy and to the anti-human epidermal growth factor receptor-2 (HER2) monoclonal antibody trastuzumab. Therefore, the addition of mTOR inhibitors to conventional breast cancer therapy has the potential to enhance therapeutic efficacy and/or overcome innate or acquired resistance. Everolimus, an mTOR inhibitor with demonstrated preclinical activity against breast cancer cell lines, has been shown to reverse Akt-induced resistance to hormonal therapy and trastuzumab. Phase I-II clinical trials have demonstrated that everolimus has promising clinical activity in women with HER2-positive, HER2-negative, and estrogen receptor-positive breast cancer when combined with HER2-targeted therapy, cytotoxic chemotherapy, and hormonal therapy, respectively. Everolimus is generally well tolerated; hematologic abnormalities and stomatitis are most common adverse events when this drug is combined with cytotoxic chemotherapy. Based on these promising results, everolimus is currently under evaluation in a series of phase III Breast Cancer Trials of Oral Everolimus (BOLERO) trials of women with HER2-positive and estrogen receptor-positive breast cancer. Results of these trials will help to establish the role of everolimus in the treatment of clinically important breast cancer subtypes.

Topics: Breast Neoplasms; Clinical Trials as Topic; Clinical Trials, Phase III as Topic; Drug Interactions; Everolimus; Female; Humans; Immunosuppressive Agents; Receptor, ErbB-2; Receptors, Estrogen; Sirolimus; TOR Serine-Threonine Kinases

Topics: Ado-Trastuzumab Emtansine; Antibodies, Monoclonal, Humanized; Breast Neoplasms; Denosumab; Drug Design; Drug Therapy, Combination; Everolimus; Humans; Lapatinib; Letrozole; Maytansine; Molecular Targeted Therapy; Nitriles; Quinazolines; Receptor, ErbB-2; Sirolimus; Tamoxifen; Trastuzumab; Triazoles

Approximately 15%-23% of breast cancers overexpress human epidermal growth factor receptor 2 (HER2), which leads to the activation of signaling pathways that stimulate cell proliferation and survival. HER2-targeted therapy has substantially improved outcomes in patients with HER2-positive breast cancer. However, both de novo and acquired resistance are observed.. A literature search was performed to identify proposed mechanisms of resistance to HER2-targeted therapy and identified novel targets in clinical development for treating HER2-resistant disease.. Proposed HER2-resistance mechanisms include impediments to HER2-inhibitor binding, signaling through alternative pathways, upregulation of signaling pathways downstream of HER2, and failure to elicit an appropriate immune response. Although continuing HER2 inhibition beyond progression may provide an additional clinical benefit, the availability of novel therapies targeting different mechanisms of action could improve outcomes. The developmental strategy with the most available data is targeting the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (mTOR) pathway. The oral mTOR inhibitor everolimus has shown promising activity in combination with chemotherapy and trastuzumab in trastuzumab-refractory, advanced breast cancer.. Non-HER2-targeted therapy is a promising means of overcoming resistance to HER2-targeted treatment. Ongoing clinical studies will provide additional information on the efficacy and safety of novel targeted therapies in HER2-resistant advanced breast cancer.

Topics: Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Breast Neoplasms; Cell Proliferation; Drug Resistance, Neoplasm; Everolimus; Female; Humans; Immunosuppressive Agents; Phosphatidylinositol 3-Kinase; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Receptor, ErbB-2; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Trastuzumab

mTOR inhibitors are currently used in the treatment of solid malignancies. Since their approval, several cases of pulmonary toxicity (PT) have been described. This analysis aims to report the incidence and the risk of PT in published randomized controlled trials.. PubMed and Scopus were reviewed for phase II-III randomized controlled trials with temsirolimus and everolimus. The characteristic of each study and incidence of all- and high-grades PT were collected.. A total of 2233 patients were available for meta-analysis: 989 had breast cancer, 833 had neuroendocrine tumor and 411 had metastatic renal cell carcinoma. In patients taking mTOR inhibitors, the incidence of all- and high-grades PT was 10.4% and 2.4%, respectively. Compared to controls, the relative risk for all- and high-grades PT was 31- and 8.8-folds, respectively. No significant heterogeneity was observed between the studies. Not any relationship was found between the incidence of lung metastases, treatment exposure and the incidence of PT.. The high grade PT is a rare event and 10% of patients may experience mild grade toxicity with a worsening of quality of life and interruption of therapy in some cases. We recommend monitoring of PT in patients treated with mTOR inhibitors.

Topics: Antineoplastic Agents; Breast Neoplasms; Carcinoma, Renal Cell; Clinical Trials, Phase II as Topic; Clinical Trials, Phase III as Topic; Everolimus; Humans; Incidence; Kidney Neoplasms; Lung; Neoplasms; Neuroendocrine Tumors; Pneumonia; Randomized Controlled Trials as Topic; Respiratory Function Tests; Risk; Sirolimus; TOR Serine-Threonine Kinases

Recent data from clinical trials evaluating mammalian target of rapamycin (mTOR) inhibitors in the setting of endocrine resistance in luminal (estrogen receptor-positive, human epidermal growth factor receptor 2-negative) breast cancers have validated this pathway as a bona-fide therapeutic target in this setting. There are currently many agents under clinical investigation that inhibit the phosphatidylinositol 3-kinase (PI3K) pathway. We review these findings in the context of the preclinical data and the current status of biomarker development in this field.. Clinical trials in the neoadjuvant (RAD2222) and metastatic setting (TAMRAD, BOLERO-2) have reported improved clinical outcome of patients with unselected luminal breast cancer through the addition of mTOR inhibitors to standard endocrine treatment. PI3K molecular aberrations are frequently found in luminal breast cancer, yet the role of these in defining patients' prognosis and response to PI3K/AKT/mTOR inhibitors remains to be determined.. Therapeutic targeting of the PI3K pathway promises improved clinical outcome for patients with luminal breast cancer. Correspondingly, agents that target this pathway are entering the clinic at an unprecedented rate. Future clinical trials that incorporate correlative translational research will help us decipher important information critical for successful development of these agents in breast cancer: which part of the pathway should be targeted and in which clinical scenario; and which patients are more likely to benefit from these drugs, particularly in the adjuvant setting.

Topics: Antineoplastic Agents; Breast Neoplasms; Clinical Trials as Topic; Drug Resistance, Neoplasm; Female; Humans; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase Inhibitors; Receptor, ErbB-2; Receptors, Estrogen; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

PI3K/AKT/mTOR pathway is a crucial mediator of tumor progression. As the PI3K/Akt pathway is heavily deregulated in breast cancer, the application of mTOR inhibitors in breast cancer patients seems warranted. This is the first systematic review according to PRISMA guidelines to synthesize all available data of mTOR inhibitors in all subcategories of breast cancer. The search strategy retrieved 16 studies evaluating everolimus (1492 patients), seven studies examining temsirolimus (1245 patients), one study evaluating sirolimus (400 patients) and two studies evaluating MKC-1 (60 patients). The Breast Cancer Trials of Oral Everolimus-2 (BOLERO-2) study has marked a turning point in the evaluation of everolimus in the treatment of estrogen receptor positive breast cancer. Given the positive results, everolimus has entered NCCN 2012 guidelines, and its approval of its combination with exemestane by FDA and EMA is imminent. In addition, the promising antitumor activity and long-term disease control further suggest that mTOR inhibition with everolimus may provide an avenue for achieving long-lasting benefit from trastuzumab-based therapy in HER2-positive patients. Regarding temsirolimus, it seems that the agent may play, in the future, a role in the treatment of metastatic breast cancer; importantly, however, there is an unmet need to find its optimal target subpopulation.

Topics: Breast Neoplasms; Everolimus; Humans; Receptor, ErbB-2; Receptors, Estrogen; Sirolimus; TOR Serine-Threonine Kinases

The phosphoinositide triphosphate kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) is a central regulatory pathway involved in cell proliferation, growth, differentiation, metabolism and survival. Deregulation of this pathway is well described in breast cancer and is associated to the development of endocrine resistance among hormone receptor (HR)-positive tumors. Everolimus , an mTOR-inhibitor has clinical activity against breast cancer and has shown to restore sensitivity to endocrine therapy.. We review the clinical data and the results of the recently published clinical trials evaluating the use of everolimus in HR-positive breast cancer patients in combination with endocrine therapy. We discuss the data regarding efficacy but also describe in detail the side effect profile of this drug.. Everolimus represents a new therapeutic alternative for the treatment of HR-positive metastatic breast cancer. Everolimus is in general a well-tolerated drug, however, stomatitis, fatigue and hematological abnormalities are common. It is still unclear if there are specific subgroups of patients that receive greater benefit from everolimus and whether there is a relationship between the presence of PIK3CA mutations and efficacy. The results of biomarker studies will hopefully provide information that will help us determine which patients are most likely to benefit from this treatment.

Topics: Antineoplastic Agents; Breast Neoplasms; Everolimus; Humans; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Receptors, Steroid; Sirolimus; TOR Serine-Threonine Kinases

Approaches to treatment for many patients with advanced breast cancer are based on the expression of specific receptors. Treatments targeting the hormone receptor (typically the estrogen receptor) are used to reduce signaling through these receptors and thereby inhibit proliferation of breast cancer cells expressing these receptors. Although these treatments are effective for many patients, resistance to treatment is common. Recent clinical trials suggest that using multiple agents targeting the same pathway is not sufficient to overcome resistance. New treatment approaches are needed for these patients. Inhibition of the mTOR signaling pathway, a key point of confluence for multiple signaling cascades, offers a promising approach to restoring sensitivity to endocrine therapy in breast cancer. This article reviews the current data from studies of mTOR inhibitors everolimus and temsirolimus in combination with endocrine therapies to overcome treatment resistance in patients with advanced breast cancer.

Topics: Antineoplastic Agents, Hormonal; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Drug Resistance, Neoplasm; Everolimus; Female; Humans; Sirolimus; TOR Serine-Threonine Kinases

Topics: Adenocarcinoma; Angiomyolipoma; Antibiotics, Antineoplastic; Astrocytoma; Brain Neoplasms; Breast Neoplasms; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Humans; Kidney Neoplasms; Lung Neoplasms; Mutation; Sirolimus; TOR Serine-Threonine Kinases; Tuberous Sclerosis; Tuberous Sclerosis Complex 1 Protein; Tuberous Sclerosis Complex 2 Protein; Tumor Suppressor Proteins

The human epidermal growth factor receptor (HER-2) oncogene encodes a transmembrane tyrosine kinase receptor that has evolved as a major classifier of invasive breast cancer and target of therapy for the disease. The validation of the general prognostic significance of HER-2 gene amplification and protein overexpression in the absence of anti-HER-2 targeted therapy is discussed in a study of 107 published studies involving 39,730 patients, which produced an overall HER-2-positive rate of 22.2% and a mean relative risk for overall survival (OS) of 2.74. The issue of HER-2 status in primary versus metastatic breast cancer is considered along with a section on the features of metastatic HER-2-positive disease. The major marketed slide-based HER-2 testing approaches, immunohistochemistry, fluorescence in situ hybridization, and chromogenic in situ hybridization, are presented and contrasted in detail against the background of the published American Society of Clinical Oncology-College of American Pathologists guidelines for HER-2 testing. Testing issues, such as the impact of chromosome 17 polysomy and local versus central HER-2 testing, are also discussed. Emerging novel HER-2 testing techniques, including mRNA-based testing by real-time polymerase chain reaction and DNA microarray methods, HER-2 receptor dimerization, phosphorylated HER-2 receptors, and HER-2 status in circulating tumor cells, are also considered. A series of biomarkers potentially associated with resistance to trastuzumab is discussed with emphasis on the phosphatase and tensin homologue deleted on chromosome ten/Akt and insulin-like growth factor receptor pathways. The efficacy results for the more recently approved small molecule HER-1/HER-2 kinase inhibitor lapatinib are also presented along with a more limited review of markers of resistance for this agent. Additional topics in this section include combinations of both anti-HER-2 targeted therapies together as well as with novel agents including bevacizumab, everolimus, and tenespimycin. A series of novel HER-2-targeting agents is also presented, including pertuzumab, ertumaxomab, HER-2 vaccines, and recently discovered tyrosine kinase inhibitors. Biomarkers predictive of HER-2 targeted therapy toxicity are included, and the review concludes with a consideration of HER-2 status in the prediction of response to non-HER-2 targeted treatments including hormonal therapy, anthracyclines, and taxanes.

Topics: Anthracyclines; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Combined Chemotherapy Protocols; Bevacizumab; Biomarkers, Tumor; Breast Neoplasms; Everolimus; Evidence-Based Medicine; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; In Situ Hybridization; Lapatinib; Neoplasm Staging; Polymerase Chain Reaction; Practice Guidelines as Topic; Prognosis; Pyrazoles; Pyrimidines; Quinazolines; Receptor, ErbB-2; RNA, Messenger; Sirolimus; Survival Analysis; Taxoids; Trastuzumab; Treatment Outcome; Up-Regulation

To review data supporting the effectiveness of emerging treatment options for metastatic breast cancer.. Recent research has focused on several signal-transduction pathways important in the pathogenesis of breast cancer. Mammalian target of rapamycin (mTOR) is a serine-threonine protein kinase that is involved in cell growth and survival. Everolimus, an orally active inhibitor of mTOR, has demonstrated promising efficacy results and a favorable safety profile in initial studies. Epidermal growth factor receptor (EGFR), a cell-surface molecule that has been implicated in the pathogenesis of breast cancer, may also be important in the emergence of resistance to endocrine therapy. Initial clinical studies have suggested that EGFR inhibitors such as gefitinib may delay the development of resistance to endocrine therapy in patients with breast cancer when given concurrently with tamoxifen or an aromatase inhibitor. Finally, considerable recent research has examined the role of epigenetic gene silencing, in which acetylation or deacetylation of DNA modifies the expression of tumor-suppressing genes. The enzyme histone deacetylase (HDAC) suppresses gene transcription by modifying chromatin into a more compact form. HDAC inhibitors have emerged as a potential new treatment option for several cancer types, including breast cancer. The HDAC inhibitor vorinostat has recently been examined in combination with other treatments, including cytotoxic agents and bevacizumab, for the treatment of breast cancer. In one small Phase I and II study, first-line treatment with the combination of vorinostat, paclitaxel, and bevacizumab produced objective responses (partial or complete) in more than 50% of patients with recurrent or metastatic breast cancer.. Although the results of the described studies are promising, randomized controlled clinical trials are needed to better understand the efficacy and safety of emerging treatment options for patients with metastatic breast cancer.

Topics: Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Drug Delivery Systems; Drugs, Investigational; ErbB Receptors; Everolimus; Female; Gefitinib; Histone Deacetylase Inhibitors; Humans; Intracellular Signaling Peptides and Proteins; Models, Biological; Protein Serine-Threonine Kinases; Quinazolines; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

The introduction of bisphosphonates represents an important advance in the care of patients with metastatic bone disease. Nonetheless, we remain unable to prevent metastatic bone destruction. This review will discuss several novel therapies, including inhibitors of receptor activator of nuclear factor-kappabeta, c-Src, mammalian target of rapamycin, cathepsin K, and alpha(5)beta(3) integrins, which could improve our control over this devastating complication.

Topics: Antineoplastic Agents; Bone Density Conservation Agents; Bone Neoplasms; Bone Remodeling; Breast Neoplasms; Cathepsin K; Cathepsins; Diphosphonates; Drugs, Investigational; Female; Humans; Integrin alpha Chains; Integrin beta Chains; Sirolimus

Therapeutics that interfere with estrogen receptor function (antiestrogens, eg, tamoxifen; aromatase inhibitors, eg, letrozole) have contributed to a dramatic reduction in breast cancer mortality; however, not all estrogen-receptor-positive breast cancers respond. The mammalian target-of-rapamycin (mTOR) is emerging as an important target molecule in the treatment of breast cancer. Furthermore, activation of growth-factor signaling pathways that involve mTOR may contribute to both the failure of endocrine therapy as well as the development of resistance. RAD001 (everolimus) is a potent, orally bioavailable inhibitor of the mTOR pathway. Preclinical data show that RAD001 effectively inhibits the proliferation and growth of a number of cancer cell lines in vitro and a range of tumor types in experimental animal models of cancer. Moreover, RAD001 exhibits an antiangiogenic activity, which may also contribute to its anticancer activity. The aromatase inhibitor letrozole is a potent endocrine therapy for breast cancer that acts to inhibit the aromatization of androgens, thereby reducing plasma and tumor estrogen levels. Combining RAD001 with letrozole is a rational approach to the treatment of advanced breast cancer, offering the potential for inhibition of tumor cell growth/proliferation and angiogenesis while at the same time potentially preventing the development of letrozole resistance. Preclinical data, derived from aromatase-expressing, estrogen-receptor-positive breast tumor models, suggest a synergistic interaction between RAD001 and letrozole that results in more profound effects on proliferation and the induction of tumor cell death. Importantly, early clinical data show no pharmacokinetic interaction or increase in toxicity with combined treatment, as compared with treatment with RAD001 alone, and there is evidence of antitumor activity. Enrollment into phase II studies is presently underway.

Topics: Aromatase Inhibitors; Breast Neoplasms; Clinical Trials as Topic; Everolimus; Female; Forecasting; Humans; Immunosuppressive Agents; Letrozole; Neoplasms, Hormone-Dependent; Nitriles; Receptors, Estrogen; Sirolimus; Triazoles

Systemic therapy for breast cancer is undergoing many interesting advances. In recent years, a better understanding of the underlying biology of cancer has led to the identification of several opportunities to target cellular pathways for therapeutic advantage. Hormonal therapy is often chosen as the first line of therapy because it is generally very well tolerated. However, most patients eventually become unresponsive to endocrine manipulation. Improved understanding of the mechanisms of hormonal resistance, and of the interplay between hormone receptors and cellular growth pathways, provides the rationale for combining signal transduction inhibitors and hormonal agents to overcome resistance. Emerging data from preclinical studies suggest that this goal might be achievable in the clinic. Antiangiogenic therapy has shown efficacy in colorectal and lung cancer and appears promising in breast cancer, although improved overall survival has not yet been shown. There have also been advances in cytotoxic chemotherapy. Reformulation of paclitaxel in polyoxy-castor-oil-free albumin nanoparticles (ABI-007) has allowed more convenient, safer, and enhanced drug delivery with associated improvement in outcomes. New agents with novel mechanisms of action are also in development and appear to have activity in cancer cells that are resistant to currently available agents.

Topics: Angiogenesis Inhibitors; Aromatase Inhibitors; Breast Neoplasms; ErbB Receptors; Estradiol; Female; Fulvestrant; Humans; MAP Kinase Signaling System; Paclitaxel; Sirolimus; Vinblastine

There is currently a wealth of information regarding the mutations that contribute to cancer development. Most of these mutations alter the expression and activity of signal transduction proteins. The current goal in cancer therapy is to use our knowledge of the molecular alterations in a cancer cell to choose the most appropriate signal transduction inhibitor for an individual patient. The topic of this review is the mammalian target of rapamycin (mTOR) kinase signaling pathway, which is aberrantly activated in many types of human cancer. We will discuss the mTOR pathway and the potential mechanisms that contribute to its activation in cancer, together with data relating to the potential for inhibitors targeting the mTOR-signaling pathway to impact on breast cancer therapy.

Topics: Antibiotics, Antineoplastic; Breast Neoplasms; Female; Humans; Protein Kinases; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Temsirolimus (CCI779), an intravenous analog of rapamycin, presents immunosuppressive properties and also antiproliferative activity. Its principal target is the mTOR serine/threonin kinase which controls the initiation of the transcription of many ARNm implicated in carcinogenesis. Breast cancers, glioblastoma and renal cell carcinoma were particularly studied with response rates from 10 to 20 %. In haematology, mantle-cell lymphoma is of particular interest because of constitutional activation of cyclin D1 (response rate of 40 %). As a whole these data define temsirolimus as a promising new drug. Current and further developments are based on its association with chemotherapy in a concomitant or sequential way.

Topics: Antineoplastic Agents; Breast Neoplasms; Cyclin D1; Glioblastoma; Humans; Kidney Neoplasms; Lymphoma, Mantle-Cell; Protein Kinase Inhibitors; Protein Kinases; Sirolimus; TOR Serine-Threonine Kinases

Topics: Antibiotics, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Drug Interactions; Female; Humans; Oncogene Protein v-akt; Prognosis; Protein Kinases; Receptor, ErbB-2; Sirolimus; TOR Serine-Threonine Kinases; Treatment Outcome

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Female; Genetic Markers; Humans; Intracellular Signaling Peptides and Proteins; Patient Selection; Protein Kinases; Sirolimus; TOR Serine-Threonine Kinases

Endocrine therapy is the most effective systemic treatment for patients with hormone-receptor-positive (HR(+)) breast cancer. Unfortunately, efficacy is often limited by the onset of resistance, which is almost inevitable for patients with advanced disease. Several patterns of endocrine resistance are recognizable clinically, including: A) tumors that are inherently insensitive to all attempts at estrogen receptor (ER) targeting despite expression of ER (pan-endocrine therapy resistance); B) tumors that are estrogen dependent but resistant to one or more specific endocrine therapies (agent-selective resistance); and C) tumors that initially respond but subsequently progress (acquired resistance). Current insights into the molecular basis for these resistance patterns are rudimentary, but are most clearly illuminated by investigations that focus on the crosstalk between the ErbB or HER peptide growth factor family and the ER. The data are sufficiently compelling to be addressed by ongoing clinical trials that examine combinations of endocrine agents and either trastuzumab (Herceptin; Genentech, Inc.; South San Francisco, CA) or ErbB-specific tyrosine kinase (TK) inhibitors. Preliminary data from a small "proof of concept" phase II study of letrozole (Femara; Novartis Pharmaceuticals Corporation; East Hanover, NJ) and trastuzumab demonstrated durable responses despite tamoxifen (Nolvadex; AstraZeneca Pharmaceuticals; Wilmington, DE) resistance. Efficacy was variable, however, despite the selection of patients on the basis of ER and ErbB-2 coexpression. Complicating matters further, resistance often occurs in the absence of any evidence for ErbB TK family member expression. In the absence of a clear target, common downstream signal transduction proteins that are known to intersect with the ER pathway can be inhibited to address resistance, including G proteins with farnesyltransferase inhibitors and molecular target of rapamycin (mTOR) with rapamycin analogues. With a number of phase III clinical trials now under way, major advances in the endocrine treatment of advanced disease are possible.

Topics: Alkyl and Aryl Transferases; Antineoplastic Agents; Aromatase Inhibitors; Breast Neoplasms; Drug Resistance, Neoplasm; Farnesyltranstransferase; Humans; Immunosuppressive Agents; Letrozole; Nitriles; Receptor, ErbB-2; Receptors, Estrogen; Sirolimus; Tamoxifen; Triazoles

Mammalian target of rapamycin (mTOR) is a serine-threonine kinase member of the cellular phosphatidylinositol 3-kinase (PI3K) pathway, which is involved in multiple biologic functions such as transcriptional and translational control. mTOR is a downstream mediator in the PI3K/Akt signaling pathway and plays a critical role in cell survival. In breast cancer this pathway can be activated by membrane receptors, including the HER (or ErbB) family of growth factor receptors, the insulin-like growth factor receptor, and the estrogen receptor. There is evidence suggesting that Akt promotes breast cancer cell survival and resistance to chemotherapy, trastuzumab, and tamoxifen. Rapamycin is a specific mTOR antagonist that targets this pathway and blocks the downstream signaling elements, resulting in cell cycle arrest in the G1 phase. Targeting the Akt/PI3K pathway with mTOR antagonists may increase the therapeutic efficacy of breast cancer therapy.

Topics: Animals; Antibiotics, Antineoplastic; Breast Neoplasms; Cell Cycle; Humans; Phosphatidylinositol 3-Kinases; Protein Kinase Inhibitors; Protein Kinases; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Wyeth (formerly American Home Products) is developing temsirolimus [Cell cycle inhibitor-779, CCI 779], an ester analogue of sirolimus, for the treatment of cancer, multiple sclerosis and rheumatoid arthritis. Temsirolimus binds to the cytosolic protein, FKBP, which subsequently inhibits mTOR (mammalian target of rapamycin). Inhibition of mTOR blocks a number of signal transduction pathways that suppress translation of several key proteins regulating the cell cycle. These effects lead to a cell cycle block at the G1 phase. In animal models of human cancers, temsirolimus inhibited the growth of a diverse range of cancer types even when an intermittent dosing schedule was used. The compound also appears to have potential for the blockade of inflammatory responses associated with autoimmune and rheumatic diseases by inhibiting T-cell proliferation. On 11 March 2002, American Home Products changed its name and the name of its subsidiary Wyeth-Ayerst to Wyeth. During the first half of 2004, Wyeth initiated ongoing recruitment into a US phase III trial comparing orally administered temsirolimus plus letrozole versus letrozole alone as first-line treatment among approximately 1200 postmenopausal women with advanced breast cancer. The multicentre, randomised, double-blind, placebo-controlled trial is estimated to last 34 months. All subjects will have the option of participating in the long-term follow-up phase of the trial that involves follow-up every 3 months until disease progression; the primary endpoint is overall progression-free survival. In August 2004, the US FDA granted temsirolimus fast-track status for the first-line treatment of poor-prognosis patients with advanced renal cell carcinoma. Previously in March 2002, temsirolimus received fast-track status from the FDA for the treatment of renal cell carcinoma in patients who failed to respond to interleukin-2 treatment. Wyeth intends to file a NDA for temsirolimus for this indication by 2006. Researchers from Wyeth presented the findings from a preclinical study of temsirolimus at the 67th Annual Scientific Meeting of the American College of Rheumatology and the 38th Annual Meeting Association of Rheumatology Health Professionals (ACR/ARHP-2003) [Orlando, FL, USA; October 2003]. The aim of this study was to determine the effect of temsirolimus on lymphocyte proliferation and cytokine production. Since lymphocytes and cytokines are significantly involved in the pathogenesis of rheumatoid arthritis, tems

Topics: Animals; Antibiotics, Antineoplastic; Breast Neoplasms; Clinical Trials, Phase I as Topic; Clinical Trials, Phase II as Topic; Female; Humans; Immunosuppressive Agents; Neoplasms; Rheumatic Diseases; Sirolimus

The mammalian target of rapamycin (mTOR), a downstream effector of the phosphatidylinositol 3-kinase (PI3K)/Akt (protein kinase B) signaling pathway that mediates cell survival and proliferation, is a prime strategic target for anticancer therapeutic development. By targeting mTOR, the immunosuppressant and antiproliferative agent rapamycin inhibits signals required for cell cycle progression, cell growth, and proliferation. Both rapamycin and novel rapamycin analogues with more favorable pharmaceutical properties, such as CCI-779, RAD 001, and AP23573, are highly specific inhibitors of mTOR. In essence, these agents gain function by binding to the immunophilin FK506 binding protein 12 and the resultant complex inhibits the activity of mTOR. Because mTOR activates both the 40S ribosomal protein S6 kinase (p70s6k) and the eukaryotic initiation factor 4E-binding protein-1, rapamycin-like compounds block the actions of these downstream signaling elements, which results in cell cycle arrest in the G1 phase. Rapamycin and its analogues also prevent cyclin-dependent kinase (CDK) activation, inhibit retinoblastoma protein phosphorylation, and accelerate the turnover of cyclin D1, leading to a deficiency of active CDK4/cyclin D1 complexes, all of which potentially contribute to the prominent inhibitory effects of rapamycin at the G1/S boundary of the cell cycle. Rapamycin and rapamycin analogues have demonstrated impressive growth-inhibitory effects against a broad range of human cancers, including breast cancer, in preclinical and early clinical evaluations. In breast cancer cells, PI3K/Akt and mTOR pathways seem to be critical for the proliferative responses mediated by the epidermal growth factor receptor, the insulin growth factor receptor, and the estrogen receptor. Furthermore, these pathways may be constitutively activated in cancers with many types of aberrations, including those with loss of PTEN suppressor gene function. Therefore, the development of inhibitors of mTOR and related pathways is a rational therapeutic strategy for breast and other malignancies that possess a wide range of aberrant molecular constituents. This review will summarize the principal mechanisms of action of rapamycin and rapamycin derivatives, as well as the potential utility of these agents as anticancer therapeutic agents with an emphasis on breast cancer. The preliminary results of early clinical evaluations with rapamycin analogues and the unique developmental challenges tha

Topics: Antibiotics, Antineoplastic; Breast Neoplasms; Clinical Trials, Phase II as Topic; Clinical Trials, Phase III as Topic; Female; Humans; Protein Kinase Inhibitors; Protein Kinases; Sirolimus; TOR Serine-Threonine Kinases

Stomatitis is one of the main reasons to discontinue everolimus in patients with hormone receptor-positive (HR+)/human epidermal growth factor receptor 2-negative (HER2-) metastatic breast cancer (mBC). To decrease stomatitis and subsequently early treatment discontinuations or dose reductions, the DESIREE trial investigated the use of a stepwise dose-escalation schedule of everolimus (EVE esc).. DESIREE is a phase II, multicentre, randomised, double-blind, placebo-controlled trial in patients with HR+/HER2- mBC and progression/relapse after nonsteroidal aromatase inhibitor treatment. Patients were randomised to EVE esc (2.5 mg/day, week 1; 5 mg/day, week 2; 7.5 mg/day, week 3; 10 mg/day, weeks 4-24) or everolimus 10 mg/day (EVE 10mg) for 24 weeks plus exemestane. The primary endpoint was the incidence of stomatitis episodes grade ≥2 within 12 weeks of treatment. The secondary endpoints included toxicity, relative total dose intensity (RTDI) and quality of life (QoL).. A total of 160 patients were randomised and 156 started treatment (EVE esc: 80; EVE 10mg: 76). The median age of patients was 64 years (range 33-85), 56.3% patients in the EVE esc arm versus 42.1% in the EVE 10mg arm had liver metastasis (P = 0.081) and 62.5% versus 51.3% received over one metastatic therapy line (P = 0.196). Within 12 weeks, the incidence of stomatitis episodes grade ≥2 was significantly lower in the EVE esc arm compared with the EVE 10mg arm (28.8% versus 46.1%; odds ratio 0.47, 95% confidence interval 0.24-0.92; P = 0.026). Toxicity was in line with the known safety profile without new safety concerns. The median RTDI was 91.1% in the EVE esc arm versus 80.0% in the EVE 10mg arm (P = 0.329). Discontinuation rate in the first 3 weeks was 6.3% versus 15.8%, respectively (P = 0.073). QoL was comparable between the two treatment arms.. A dose-escalation schema of everolimus over 3 weeks can be successfully used to reduce the incidence of high-grade stomatitis in the first 12 weeks of treatment in patients with HR+/HER2- mBC.. ClinicalTrials.govNCT02387099; https://clinicaltrials.gov/ct2/show/NCT02387099.

Topics: Adult; Aged; Aged, 80 and over; Breast Neoplasms; Everolimus; Female; Humans; Middle Aged; Neoplasm Recurrence, Local; Quality of Life; Receptor, ErbB-2; Sirolimus; Stomatitis

The coronavirus disease 2019 (COVID-19) has affected approximately 2 million individuals worldwide; however, data regarding fatal cases have been limited.. To report the clinical features of 162 fatal cases of COVID-19 from 5 hospitals in Wuhan between December 30, 2019 and March 12, 2020.. The demographic data, signs and symptoms, clinical course, comorbidities, laboratory findings, computed tomographic (CT) scans, treatments, and complications of the patients with fatal cases were retrieved from electronic medical records.. Young patients with moderate COVID-19 without comorbidity at admission could also develop fatal outcomes. The in-hospital survival time of the fatal cases was similar among the hospitals of different levels in Wuhan.

Topics: Adolescent; Adult; Animals; Asthma; Atrial Fibrillation; Autoantibodies; Biomarkers; Breast Neoplasms; Child; Conjunctivitis, Allergic; Cornea; COVID-19; Cyclosporine; Cytokines; Death, Sudden, Cardiac; Defibrillators, Implantable; Diet; Disease Models, Animal; Docetaxel; Double-Blind Method; Dry Eye Syndromes; Educational Status; Emulsions; Female; Fluorescein Angiography; Fluoresceins; Focus Groups; Heart Failure; Hemothorax; Humans; Inflammation; Keratoconus; Male; Meibomian Glands; Mice; Middle Aged; Multiple Sclerosis; Myocardial Infarction; Myocardium; Nerve Fibers; Nigeria; Obesity; Overweight; Pandemics; Primary Prevention; Prospective Studies; Qualitative Research; Registries; Retinal Ganglion Cells; Retinal Vessels; Schools; Sirolimus; Tertiary Care Centers; Th1 Cells; Th2 Cells; Tomography, Optical Coherence; Troponin I; Tumor Necrosis Factor-alpha; United States; Ventricular Remodeling

Combining the mTOR inhibitor ridaforolimus and the anti-IGFR antibody dalotuzumab demonstrated antitumor activity, including partial responses, in estrogen receptor (ER)-positive advanced breast cancer, especially in high proliferation tumors (Ki67 > 15%).. This randomized, multicenter, international, phase II study enrolled postmenopausal women with advanced ER-positive breast cancer previously treated with a nonsteroidal aromatase inhibitor (NCT01234857). Patients were randomized to either oral ridaforolimus 30 mg daily for 5 of 7 days (once daily [qd] × 5 days/week) plus intravenous dalotuzumab 10 mg/kg/week or oral exemestane 25 mg/day, and stratified by Ki67 status. Due to a high incidence of stomatitis in the ridaforolimus-dalotuzumab group, two sequential, nonrandomized, reduced-dose cohorts were explored with ridaforolimus 20 and 10 mg qd × 5 days/week. The primary endpoint was progression-free survival (PFS).. Median PFS was 21.4 weeks for ridaforolimus 30 mg qd × 5 days/week plus dalotuzumab 10 mg/kg (n = 29) and 24.3 weeks for exemestane (n = 33; hazard ratio = 1.00; P = 0.5). Overall survival and objective response rates were similar between treatment arms. The incidence of drug-related, nonserious, and serious adverse events was higher with ridaforolimus/dalotuzumab (any ridaforolimus dose) than with exemestane. Lowering the ridaforolimus dose reduced the incidence of grade 3 stomatitis, but overall toxicity remained higher than acceptable at all doses without improved efficacy.. The combination of ridaforolimus plus dalotuzumab was no more effective than exemestane in patients with advanced ER-positive breast cancer, and the incidence of adverse events was higher. Therefore, the combination is not being further pursued.

Topics: Adult; Aged; Androstadienes; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Combined Chemotherapy Protocols; Aromatase Inhibitors; Breast Neoplasms; Disease-Free Survival; Female; Humans; Middle Aged; Protein Kinase Inhibitors; Receptors, Estrogen; Sirolimus; Stomatitis

To evaluate whether adding humanized monoclonal insulin growth factor-1 receptor (IGF-1R) antibody (dalotuzumab) to mammalian target of rapamycin (mTOR) inhibitor (ridaforolimus) plus aromatase inhibitor (exemestane) improves outcomes in patients with estrogen receptor (ER)-positive advanced/metastatic breast cancer.. This randomized, open-label, phase II trial enrolled 80 postmenopausal women with high-proliferation (Ki67 index staining ≥15%), ER-positive breast cancer that progressed after a non-steroidal aromatase inhibitor (NCT01605396). Randomly assigned patients were given oral ridaforolimus 10 mg QD 5 ×/week, intravenous dalotuzumab 10 mg/kg/week, and oral exemestane 25 mg/day (R/D/E, n = 40), or ridaforolimus 30 mg QD 5 ×/week and exemestane 25 mg/day (R/E; n = 40). Primary end point was progression-free survival (PFS).. Median PFS was 23.3 weeks for R/D/E versus 31.9 weeks for R/E (hazard ratio 1.18; 80% CI 0.81-1.72; P = 0.565). Grade 3-5 adverse events were reported in 67.5% of patients in the R/E arm and 59.0% in the R/D/E arm. Stomatitis (95.0 vs. 76.9%; P = 0.021) and pneumonitis (22.5 vs. 5.1%; P = 0.027) occurred more frequently in the R/E than the R/D/E arm; hyperglycemia (27.5 vs. 28.2%) occurred at a similar rate.. R/D/E did not improve PFS compared with R/E. Because the PFS reported for R/E was similar to that reported for everolimus plus exemestane in patients with advanced breast cancer, it is possible that lower-dose ridaforolimus in the R/D/E arm (from overlapping toxicities with IGF1R inhibitor) contributed to lack of improved PFS.

Topics: Adult; Aged; Aged, 80 and over; Androstadienes; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Combined Chemotherapy Protocols; Biomarkers, Tumor; Breast Neoplasms; Female; Humans; Middle Aged; Neoplasm Staging; Retreatment; Sirolimus; Treatment Outcome

Middle East Respiratory Syndrome (MERS) is a novel respiratory illness firstly reported in Saudi Arabia in 2012. It is caused by a new corona virus, called MERS corona virus (MERS-CoV). Most people who have MERS-CoV infection developed severe acute respiratory illness.. This work is done to determine the clinical characteristics and the outcome of intensive care unit (ICU) admitted patients with confirmed MERS-CoV infection.. This study included 32 laboratory confirmed MERS corona virus infected patients who were admitted into ICU. It included 20 (62.50%) males and 12 (37.50%) females. The mean age was 43.99 ± 13.03 years. Diagnosis was done by real-time reverse transcription polymerase chain reaction (rRT-PCR) test for corona virus on throat swab, sputum, tracheal aspirate, or bronchoalveolar lavage specimens. Clinical characteristics, co-morbidities and outcome were reported for all subjects.. Most MERS corona patients present with fever, cough, dyspnea, sore throat, runny nose and sputum. The presence of abdominal symptoms may indicate bad prognosis. Prolonged duration of symptoms before patients' hospitalization, prolonged duration of mechanical ventilation and hospital stay, bilateral radiological pulmonary infiltrates, and hypoxemic respiratory failure were found to be strong predictors of mortality in such patients. Also, old age, current smoking, smoking severity, presence of associated co-morbidities like obesity, diabetes mellitus, chronic heart diseases, COPD, malignancy, renal failure, renal transplantation and liver cirrhosis are associated with a poor outcome of ICU admitted MERS corona virus infected patients.. Plasma HO-1, ferritin, p21, and NQO1 were all elevated at baseline in CKD participants. Plasma HO-1 and urine NQO1 levels each inversely correlated with eGFR (. SnPP can be safely administered and, after its injection, the resulting changes in plasma HO-1, NQO1, ferritin, and p21 concentrations can provide information as to antioxidant gene responsiveness/reserves in subjects with and without kidney disease.. A Study with RBT-1, in Healthy Volunteers and Subjects with Stage 3-4 Chronic Kidney Disease, NCT0363002 and NCT03893799.. HFNC did not significantly modify work of breathing in healthy subjects. However, a significant reduction in the minute volume was achieved, capillary [Formula: see text] remaining constant, which suggests a reduction in dead-space ventilation with flows > 20 L/min. (ClinicalTrials.gov registration NCT02495675).. 3 组患者手术时间、术中显性失血量及术后 1 周血红蛋白下降量比较差异均无统计学意义（. 对于肥胖和超重的膝关节单间室骨关节炎患者，采用 UKA 术后可获满意短中期疗效，远期疗效尚需进一步随访观察。.. Decreased muscle strength was identified at both time points in patients with hEDS/HSD. The evolution of most muscle strength parameters over time did not significantly differ between groups. Future studies should focus on the effectiveness of different types of muscle training strategies in hEDS/HSD patients.. These findings support previous adverse findings of e-cigarette exposure on neurodevelopment in a mouse model and provide substantial evidence of persistent adverse behavioral and neuroimmunological consequences to adult offspring following maternal e-cigarette exposure during pregnancy. https://doi.org/10.1289/EHP6067.. This RCT directly compares a neoadjuvant chemotherapy regimen with a standard CROSS regimen in terms of overall survival for patients with locally advanced ESCC. The results of this RCT will provide an answer for the controversy regarding the survival benefits between the two treatment strategies.. NCT04138212, date of registration: October 24, 2019.. Results of current investigation indicated that milk type and post fermentation cooling patterns had a pronounced effect on antioxidant characteristics, fatty acid profile, lipid oxidation and textural characteristics of yoghurt. Buffalo milk based yoghurt had more fat, protein, higher antioxidant capacity and vitamin content. Antioxidant and sensory characteristics of T. If milk is exposed to excessive amounts of light, Vitamins B. The two concentration of ZnO nanoparticles in the ambient air produced two different outcomes. The lower concentration resulted in significant increases in Zn content of the liver while the higher concentration significantly increased Zn in the lungs (p < 0.05). Additionally, at the lower concentration, Zn content was found to be lower in brain tissue (p < 0.05). Using TEM/EDX we detected ZnO nanoparticles inside the cells in the lungs, kidney and liver. Inhaling ZnO NP at the higher concentration increased the levels of mRNA of the following genes in the lungs: Mt2 (2.56 fold), Slc30a1 (1.52 fold) and Slc30a5 (2.34 fold). At the lower ZnO nanoparticle concentration, only Slc30a7 mRNA levels in the lungs were up (1.74 fold). Thus the two air concentrations of ZnO nanoparticles produced distinct effects on the expression of the Zn-homeostasis related genes.. Until adverse health effects of ZnO nanoparticles deposited in organs such as lungs are further investigated and/or ruled out, the exposure to ZnO nanoparticles in aerosols should be avoided or minimised.

Topics: A549 Cells; Acetylmuramyl-Alanyl-Isoglutamine; Acinetobacter baumannii; Acute Lung Injury; Adaptor Proteins, Signal Transducing; Adenine; Adenocarcinoma; Adipogenesis; Administration, Cutaneous; Administration, Ophthalmic; Adolescent; Adsorption; Adult; Aeromonas hydrophila; Aerosols; Aged; Aged, 80 and over; Aging; Agriculture; Air Pollutants; Air Pollution; Airway Remodeling; Alanine Transaminase; Albuminuria; Aldehyde Dehydrogenase 1 Family; Algorithms; AlkB Homolog 2, Alpha-Ketoglutarate-Dependent Dioxygenase; Alzheimer Disease; Amino Acid Sequence; Ammonia; Ammonium Compounds; Anaerobiosis; Anesthetics, Dissociative; Anesthetics, Inhalation; Animals; Anti-Bacterial Agents; Anti-HIV Agents; Anti-Infective Agents; Anti-Inflammatory Agents; Antibiotics, Antineoplastic; Antibodies, Antineutrophil Cytoplasmic; Antibodies, Monoclonal, Humanized; Antifungal Agents; Antigens, Bacterial; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Antimetabolites, Antineoplastic; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Antioxidants; Antitubercular Agents; Antiviral Agents; Apolipoproteins E; Apoptosis; Arabidopsis; Arabidopsis Proteins; Arsenic; Arthritis, Rheumatoid; Asthma; Atherosclerosis; ATP-Dependent Proteases; Attitude of Health Personnel; Australia; Austria; Autophagy; Axitinib; Bacteria; Bacterial Outer Membrane Proteins; Bacterial Proteins; Bacterial Toxins; Bacterial Typing Techniques; Bariatric Surgery; Base Composition; Bayes Theorem; Benzoxazoles; Benzylamines; beta Catenin; Betacoronavirus; Betula; Binding Sites; Biological Availability; Biological Oxygen Demand Analysis; Biomarkers; Biomarkers, Tumor; Biopsy; Bioreactors; Biosensing Techniques; Birth Weight; Blindness; Blood Chemical Analysis; Blood Gas Analysis; Blood Glucose; Blood Pressure; Blood Pressure Monitoring, Ambulatory; Blood-Brain Barrier; Blotting, Western; Body Mass Index; Body Weight; Bone and Bones; Bone Density; Bone Resorption; Borates; Brain; Brain Infarction; Brain Injuries, Traumatic; Brain Neoplasms; Breakfast; Breast Milk Expression; Breast Neoplasms; Bronchi; Bronchoalveolar Lavage Fluid; Buffaloes; Cadherins; Calcification, Physiologic; Calcium Compounds; Calcium, Dietary; Cannula; Caprolactam; Carbon; Carbon Dioxide; Carboplatin; Carcinogenesis; Carcinoma, Ductal; Carcinoma, Ehrlich Tumor; Carcinoma, Hepatocellular; Carcinoma, Non-Small-Cell Lung; Carcinoma, Pancreatic Ductal; Carcinoma, Renal Cell; Cardiovascular Diseases; Carps; Carrageenan; Case-Control Studies; Catalysis; Catalytic Domain; Cattle; CD8-Positive T-Lymphocytes; Cell Adhesion; Cell Cycle Proteins; Cell Death; Cell Differentiation; Cell Line; Cell Line, Tumor; Cell Movement; Cell Nucleus; Cell Phone Use; Cell Proliferation; Cell Survival; Cell Transformation, Neoplastic; Cell Transformation, Viral; Cells, Cultured; Cellulose; Chemical Phenomena; Chemoradiotherapy; Child; Child Development; Child, Preschool; China; Chitosan; Chlorocebus aethiops; Cholecalciferol; Chromatography, Liquid; Circadian Clocks; Circadian Rhythm; Circular Dichroism; Cisplatin; Citric Acid; Clinical Competence; Clinical Laboratory Techniques; Clinical Trials, Phase I as Topic; Clinical Trials, Phase II as Topic; Clostridioides difficile; Clostridium Infections; Coculture Techniques; Cohort Studies; Cold Temperature; Colitis; Collagen Type I; Collagen Type I, alpha 1 Chain; Collagen Type XI; Color; Connective Tissue Diseases; Copper; Coronary Angiography; Coronavirus 3C Proteases; Coronavirus Infections; Cost of Illness; Counselors; COVID-19; COVID-19 Testing; Creatine Kinase; Creatinine; Cross-Over Studies; Cross-Sectional Studies; Cryoelectron Microscopy; Cryosurgery; Crystallography, X-Ray; Cues; Cultural Competency; Cultural Diversity; Curriculum; Cyclic AMP Response Element-Binding Protein; Cyclin-Dependent Kinase Inhibitor p21; Cycloparaffins; Cysteine Endopeptidases; Cytokines; Cytoplasm; Cytoprotection; Databases, Factual; Denitrification; Deoxycytidine; Diabetes Complications; Diabetes Mellitus; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type 2; Diagnosis, Differential; Diatoms; Diet; Diet, High-Fat; Dietary Exposure; Diffusion Magnetic Resonance Imaging; Diketopiperazines; Dipeptidyl Peptidase 4; Dipeptidyl-Peptidase IV Inhibitors; Disease Models, Animal; Disease Progression; Disease-Free Survival; DNA; DNA Damage; DNA Glycosylases; DNA Repair; DNA-Binding Proteins; DNA, Bacterial; DNA, Viral; Docetaxel; Dose Fractionation, Radiation; Dose-Response Relationship, Drug; Down-Regulation; Doxorubicin; Drosophila; Drosophila melanogaster; Drug Carriers; Drug Delivery Systems; Drug Liberation; Drug Repositioning; Drug Resistance, Bacterial; Drug Resistance, Multiple, Bacterial; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Drug Synergism; Drug Therapy, Combination; Edema; Edible Grain; Education, Graduate; Education, Medical, Graduate; Education, Pharmacy; Ehlers-Danlos Syndrome; Electron Transport Complex III; Electron Transport Complex IV; Electronic Nicotine Delivery Systems; Emergency Service, Hospital; Empathy; Emulsions; Endothelial Cells; Endurance Training; Energy Intake; Enterovirus A, Human; Environment; Environmental Monitoring; Enzyme Assays; Enzyme Inhibitors; Epithelial Cells; Epithelial-Mesenchymal Transition; Epoxide Hydrolases; Epoxy Compounds; Erythrocyte Count; Erythrocytes; Escherichia coli; Escherichia coli Infections; Escherichia coli Proteins; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Esophagectomy; Estrogens; Etanercept; Ethiopia; Ethnicity; Ethylenes; Exanthema; Exercise; Exercise Test; Exercise Tolerance; Extracellular Matrix; Extracorporeal Membrane Oxygenation; Eye Infections, Fungal; False Negative Reactions; Fatty Acids; Fecal Microbiota Transplantation; Feces; Female; Femur Neck; Fermentation; Ferritins; Fetal Development; Fibroblast Growth Factor-23; Fibroblast Growth Factors; Fibroblasts; Fibroins; Fish Proteins; Flavanones; Flavonoids; Focus Groups; Follow-Up Studies; Food Handling; Food Supply; Food, Formulated; Forced Expiratory Volume; Forests; Fractures, Bone; Fruit and Vegetable Juices; Fusobacteria; G1 Phase Cell Cycle Checkpoints; G2 Phase Cell Cycle Checkpoints; Gamma Rays; Gastrectomy; Gastrointestinal Microbiome; Gastrointestinal Stromal Tumors; Gefitinib; Gels; Gemcitabine; Gene Amplification; Gene Expression; Gene Expression Regulation; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Neoplastic; Gene Expression Regulation, Plant; Gene Knockdown Techniques; Gene-Environment Interaction; Genotype; Germany; Glioma; Glomerular Filtration Rate; Glucagon; Glucocorticoids; Glycemic Control; Glycerol; Glycogen Synthase Kinase 3 beta; Glycolipids; Glycolysis; Goblet Cells; Gram-Negative Bacterial Infections; Granulocyte Colony-Stimulating Factor; Graphite; Greenhouse Effect; Guanidines; Haemophilus influenzae; HCT116 Cells; Health Knowledge, Attitudes, Practice; Health Personnel; Health Services Accessibility; Health Services Needs and Demand; Health Status Disparities; Healthy Volunteers; Heart Failure; Heart Rate; Heart Transplantation; Heart-Assist Devices; HEK293 Cells; Heme; Heme Oxygenase-1; Hemolysis; Hemorrhage; Hepatitis B; Hepatitis B e Antigens; Hepatitis B Surface Antigens; Hepatitis B virus; Hepatitis B, Chronic; Hepatocytes; Hexoses; High-Throughput Nucleotide Sequencing; Hippo Signaling Pathway; Histamine; Histamine Agonists; Histidine; Histone Deacetylase 2; HIV Infections; HIV Reverse Transcriptase; HIV-1; Homebound Persons; Homeodomain Proteins; Homosexuality, Male; Hospice and Palliative Care Nursing; HSP70 Heat-Shock Proteins; Humans; Hyaluronan Receptors; Hydrogen; Hydrogen Peroxide; Hydrogen-Ion Concentration; Hydrolysis; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Hypoglycemia; Hypoglycemic Agents; Hypoxia; Idiopathic Interstitial Pneumonias; Imaging, Three-Dimensional; Imatinib Mesylate; Immunotherapy; Implementation Science; Incidence; INDEL Mutation; Induced Pluripotent Stem Cells; Industrial Waste; Infant; Infant, Newborn; Inflammation; Inflammation Mediators; Infliximab; Infusions, Intravenous; Inhibitory Concentration 50; Injections; Insecticides; Insulin-Like Growth Factor Binding Protein 5; Insulin-Secreting Cells; Interleukin-1; Interleukin-17; Interleukin-8; Internship and Residency; Intestines; Intracellular Signaling Peptides and Proteins; Ion Transport; Iridaceae; Iridoid Glucosides; Islets of Langerhans Transplantation; Isodon; Isoflurane; Isotopes; Italy; Joint Instability; Ketamine; Kidney; Kidney Failure, Chronic; Kidney Function Tests; Kidney Neoplasms; Kinetics; Klebsiella pneumoniae; Knee Joint; Kruppel-Like Factor 4; Kruppel-Like Transcription Factors; Lactate Dehydrogenase 5; Laparoscopy; Laser Therapy; Lasers, Semiconductor; Lasers, Solid-State; Laurates; Lead; Leukocyte L1 Antigen Complex; Leukocytes, Mononuclear; Light; Lipid Peroxidation; Lipopolysaccharides; Liposomes; Liver; Liver Cirrhosis; Liver Neoplasms; Liver Transplantation; Locomotion; Longitudinal Studies; Lopinavir; Lower Urinary Tract Symptoms; Lubricants; Lung; Lung Diseases, Interstitial; Lung Neoplasms; Lymphocyte Activation; Lymphocytes, Tumor-Infiltrating; Lymphoma, Mantle-Cell; Lysosomes; Macrophages; Male; Manganese Compounds; MAP Kinase Kinase 4; Mass Screening; Maternal Health; Medicine, Chinese Traditional; Melanoma, Experimental; Memantine; Membrane Glycoproteins; Membrane Proteins; Mesenchymal Stem Cell Transplantation; Metal Nanoparticles; Metalloendopeptidases; Metalloporphyrins; Methadone; Methane; Methicillin-Resistant Staphylococcus aureus; Mexico; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Inbred ICR; Mice, Knockout; Mice, Nude; Mice, SCID; Mice, Transgenic; Microarray Analysis; Microbial Sensitivity Tests; Microbiota; Micronutrients; MicroRNAs; Microscopy, Confocal; Microsomes, Liver; Middle Aged; Milk; Milk, Human; Minority Groups; Mitochondria; Mitochondrial Membranes; Mitochondrial Proteins; Models, Animal; Models, Molecular; Molecular Conformation; Molecular Docking Simulation; Molecular Dynamics Simulation; Molecular Epidemiology; Molecular Structure; Molecular Weight; Multilocus Sequence Typing; Multimodal Imaging; Muscle Strength; Muscle, Skeletal; Muscular Diseases; Mutation; Mycobacterium tuberculosis; Myocardial Stunning; Myristates; NAD(P)H Dehydrogenase (Quinone); Nanocomposites; Nanogels; Nanoparticles; Nanotechnology; Naphthalenes; Nasal Cavity; National Health Programs; Necrosis; Needs Assessment; Neoadjuvant Therapy; Neonicotinoids; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Proteins; Neoplasm Recurrence, Local; Neoplasm Staging; Neoplasm Transplantation; Neoplasms; Neoplastic Stem Cells; Netherlands; Neuroblastoma; Neuroprotective Agents; Neutrophils; NF-kappa B; NFATC Transcription Factors; Nicotiana; Nicotine; Nitrates; Nitrification; Nitrites; Nitro Compounds; Nitrogen; Nitrogen Dioxide; North Carolina; Nuclear Magnetic Resonance, Biomolecular; Nuclear Proteins; Nucleic Acid Hybridization; Nucleosomes; Nutrients; Obesity; Obesity, Morbid; Oceans and Seas; Oncogene Protein v-akt; Oncogenes; Oocytes; Open Reading Frames; Osteoclasts; Osteogenesis; Osteoporosis; Osteoporosis, Postmenopausal; Outpatients; Ovarian Neoplasms; Ovariectomy; Overweight; Oxazines; Oxidants; Oxidation-Reduction; Oxidative Stress; Oxides; Oxidoreductases; Oxygen; Oxygen Inhalation Therapy; Oxygenators, Membrane; Ozone; Paclitaxel; Paenibacillus; Pain Measurement; Palliative Care; Pancreatic Neoplasms; Pandemics; Parasympathetic Nervous System; Particulate Matter; Pasteurization; Patient Preference; Patient Satisfaction; Pediatric Obesity; Permeability; Peroxiredoxins; Peroxynitrous Acid; Pharmaceutical Services; Pharmacists; Pharmacy; Phaseolus; Phenotype; Phoeniceae; Phosphates; Phosphatidylinositol 3-Kinases; Phospholipid Transfer Proteins; Phospholipids; Phosphorus; Phosphorylation; Photoperiod; Photosynthesis; Phylogeny; Physical Endurance; Physicians; Pilot Projects; Piperidines; Pituitary Adenylate Cyclase-Activating Polypeptide; Plant Extracts; Plant Leaves; Plant Proteins; Plant Roots; Plaque, Atherosclerotic; Pneumonia; Pneumonia, Viral; Point-of-Care Testing; Polyethylene Glycols; Polymers; Polysorbates; Pore Forming Cytotoxic Proteins; Positron Emission Tomography Computed Tomography; Positron-Emission Tomography; Postprandial Period; Poverty; Pre-Exposure Prophylaxis; Prediabetic State; Predictive Value of Tests; Pregnancy; Pregnancy Trimester, First; Pregnancy, High-Risk; Prenatal Exposure Delayed Effects; Pressure; Prevalence; Primary Graft Dysfunction; Primary Health Care; Professional Role; Professionalism; Prognosis; Progression-Free Survival; Prolactin; Promoter Regions, Genetic; Proof of Concept Study; Proportional Hazards Models; Propylene Glycol; Prospective Studies; Prostate; Protein Binding; Protein Biosynthesis; Protein Isoforms; Protein Kinase Inhibitors; Protein Phosphatase 2; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Protein Structure, Tertiary; Protein Transport; Proteoglycans; Proteome; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-myc; Proto-Oncogene Proteins c-ret; Proto-Oncogene Proteins p21(ras); Proton Pumps; Protons; Protoporphyrins; Pseudomonas aeruginosa; Pseudomonas fluorescens; Pulmonary Artery; Pulmonary Disease, Chronic Obstructive; Pulmonary Gas Exchange; Pulmonary Veins; Pyrazoles; Pyridines; Pyrimidines; Qualitative Research; Quinoxalines; Rabbits; Random Allocation; Rats; Rats, Sprague-Dawley; Rats, Wistar; Receptors, Histamine H3; Receptors, Immunologic; Receptors, Transferrin; Recombinant Proteins; Recurrence; Reference Values; Referral and Consultation; Regional Blood Flow; Registries; Regulon; Renal Insufficiency, Chronic; Reperfusion Injury; Repressor Proteins; Reproducibility of Results; Republic of Korea; Research Design; Resistance Training; Respiration, Artificial; Respiratory Distress Syndrome; Respiratory Insufficiency; Resuscitation; Retinal Dehydrogenase; Retreatment; Retrospective Studies; Reverse Transcriptase Inhibitors; Rhinitis, Allergic; Ribosomal Proteins; Ribosomes; Risk Assessment; Risk Factors; Ritonavir; Rivers; RNA Interference; RNA-Seq; RNA, Messenger; RNA, Ribosomal, 16S; RNA, Small Interfering; Rosuvastatin Calcium; Rural Population; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Salivary Ducts; Salivary Gland Neoplasms; San Francisco; SARS-CoV-2; Satiation; Satiety Response; Schools; Schools, Pharmacy; Seasons; Seawater; Selection, Genetic; Sequence Analysis, DNA; Serine-Threonine Kinase 3; Sewage; Sheep; Sheep, Domestic; Shock, Hemorrhagic; Signal Transduction; Silver; Silymarin; Single Photon Emission Computed Tomography Computed Tomography; Sirolimus; Sirtuin 1; Skin; Skin Neoplasms; Skin Physiological Phenomena; Sleep Initiation and Maintenance Disorders; Social Class; Social Participation; Social Support; Soil; Soil Microbiology; Solutions; Somatomedins; Soot; Specimen Handling; Spectrophotometry, Ultraviolet; Spectroscopy, Fourier Transform Infrared; Spectrum Analysis; Spinal Fractures; Spirometry; Staphylococcus aureus; STAT1 Transcription Factor; STAT3 Transcription Factor; Streptomyces coelicolor; Stress, Psychological; Stroke; Stroke Volume; Structure-Activity Relationship; Students, Medical; Students, Pharmacy; Substance Abuse Treatment Centers; Sulfur Dioxide; Surface Properties; Surface-Active Agents; Surveys and Questionnaires; Survival Analysis; Survival Rate; Survivin; Sweden; Swine; Swine, Miniature; Sympathetic Nervous System; T-Lymphocytes, Regulatory; Talaromyces; Tandem Mass Spectrometry; tau Proteins; Telemedicine; Telomerase; Telomere; Telomere Homeostasis; Temperature; Terminally Ill; Th1 Cells; Thiamethoxam; Thiazoles; Thiophenes; Thioredoxin Reductase 1; Thrombosis; Thulium; Thyroid Cancer, Papillary; Thyroid Carcinoma, Anaplastic; Thyroid Neoplasms; Time Factors; Titanium; Tomography, Emission-Computed, Single-Photon; Tomography, X-Ray Computed; TOR Serine-Threonine Kinases; Transcription Factor AP-1; Transcription Factors; Transcription, Genetic; Transcriptional Activation; Transcriptome; Transforming Growth Factor beta1; Transistors, Electronic; Translational Research, Biomedical; Transplantation Tolerance; Transplantation, Homologous; Transportation; Treatment Outcome; Tretinoin; Tuberculosis, Multidrug-Resistant; Tuberculosis, Pulmonary; Tubulin Modulators; Tumor Microenvironment; Tumor Necrosis Factor Inhibitors; Tumor Necrosis Factor-alpha; Twins; Ultrasonic Therapy; Ultrasonography; Ultraviolet Rays; United States; Up-Regulation; Uranium; Urethra; Urinary Bladder; Urodynamics; Uromodulin; Uveitis; Vasoconstrictor Agents; Ventricular Function, Left; Vero Cells; Vesicular Transport Proteins; Viral Nonstructural Proteins; Visual Acuity; Vital Capacity; Vitamin D; Vitamin D Deficiency; Vitamin K 2; Vitamins; Volatilization; Voriconazole; Waiting Lists; Waste Disposal, Fluid; Wastewater; Water Pollutants, Chemical; Whole Genome Sequencing; Wine; Wnt Signaling Pathway; Wound Healing; Wounds and Injuries; WW Domains; X-linked Nuclear Protein; X-Ray Diffraction; Xanthines; Xenograft Model Antitumor Assays; YAP-Signaling Proteins; Yogurt; Young Adult; Zebrafish; Zebrafish Proteins; Ziziphus

This European phase IIIb, expanded-access multicenter trial evaluated the safety of EVE plus EXE in a patient population similar to BOLERO-2.. Post-menopausal women aged ≥18 years with hormone receptor-positive, human epidermal growth factor-receptor-2-negative advanced breast cancer (ABC) recurring/progressing during/after prior non-steroidal aromatase inhibitors were enrolled. The primary objective was safety of EVE plus EXE based on frequency of adverse events (AEs), and serious AEs (SAEs). The secondary objective was to evaluate AEs of grade 3/4 severity.. The median treatment duration was 5.1 months [95% confidence interval (CI) 4.8-5.6] for EVE and 5.3 months (95% CI 4.8-5.6) for EXE. Overall, 2131 patients were included in the analysis; 81.8% of patients experienced EVE- or EXE-related or EVE/EXE-related AEs (investigator assessed); 27.2% were of grade 3/4 severity. The most frequently reported non-hematologic AEs were (overall %, % EVE-related) stomatitis (52.8%; 50.8%) and asthenia (22.8%; 14.6%). The most frequently reported hematologic AEs were (overall %, % EVE-related) anemia (14.4%; 8.1%) and thrombocytopenia (5.9%; 4.6%). AE-related treatment discontinuations were higher in elderly (≥70 years) versus non-elderly patients (23.8% versus 13.0%). The incidence of EVE-related AEs in both elderly and non-elderly patients appeared to be lower in first-line ABC versus later lines. The incidence of AEs (including stomatitis/pneumonitis) was independent of BMI status (post hoc analysis). Overall, 8.5% of patients experienced at least one EVE-related SAE. Of the 121 on-treatment deaths (5.7%), 66 (3.1%) deaths were due to disease progression and 46 (2.2%) due to AEs; 4 deaths were suspected to be EVE-related.. This is the largest ever reported safety dataset on a general patient population presenting ABC treated with EVE plus EXE and included a sizeable elderly subset. Although the patients were more heavily pretreated, the safety profile of EVE plus EXE in BALLET was consistent with BOLERO-2.. EudraCT Number: 2012-000073-23.

Topics: Aged; Aged, 80 and over; Androstadienes; Antineoplastic Combined Chemotherapy Protocols; Aromatase Inhibitors; Breast Neoplasms; Disease-Free Survival; Drug-Related Side Effects and Adverse Reactions; ErbB Receptors; Everolimus; Female; Humans; Male; Neoplasm Metastasis; Neoplasm Recurrence, Local; Postmenopause; Receptor, ErbB-2; Receptors, Estrogen; Sirolimus

Everolimus, which inhibits the mammalian target of rapamycin (mTOR), is increasingly used in breast cancer and familiarity with its full range of toxicity is critical for practicing oncologists.. We studied hematologic changes in 31 patients with metastatic breast cancer treated in a phase II clinical trial using everolimus. Complete blood counts were collected at baseline, 2 weeks, 4 weeks, every 4 weeks during treatment, and 1 month after discontinuation. Adverse events were defined using Common Toxicity Criteria version 3. Linear mixed models with fixed effects of time and random intercepts and slopes were used to study trends and comparisons were conducted using paired t tests.. Anemia was reported in 22 patients (71%), thrombocytopenia in 17 (55%), and leukopenia in 14 (45%). These were predominantly grade 1 or 2 and did not require dose modification. Red blood cell mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) both decreased significantly over time (P < .0001) starting at 2 weeks with no significant change in mean corpuscular hemoglobin concentration (MCHC) (P = .104). Both MCV and MCH increased 1 month after treatment discontinuation (P values < .0001 and .0003, respectively) indicating reversibility of this effect. Although total leukocyte counts remained largely stable, lymphocyte percentage progressively decreased over time with a trend for increased neutrophils.. In addition to anemia, leukopenia, and thrombocytopenia, everolimus consistently induces red cell microcytosis and reduced hemoglobin content. Lymphopenia may contribute to immune suppression and increased risk of infection. Familiarity with these hematologic changes is prudent as more patients are treated with this class of drugs.

Topics: Anemia; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Erythrocyte Count; Erythrocyte Indices; Estradiol; Everolimus; Female; Fulvestrant; Hematologic Diseases; Humans; Leukocyte Count; Leukopenia; Neoplasm Metastasis; Sirolimus; Thrombocytopenia

Although trastuzumab-containing therapies prolong survival in patients with metastatic breast cancer (MBC), most tumors develop trastuzumab resistance, potentially mediated by aberrant phosphatidylinositide 3-kinase (PI3K)/AKT signaling. Ridaforolimus (a mammalian target of rapamycin [mTOR] inhibitor) may overcome trastuzumab resistance by inhibiting PI3K signaling.. A single-arm, phase IIb trial was conducted to evaluate the efficacy and safety of ridaforolimus-trastuzumab in human epidermal growth factor receptor 2-positive (HER2(+)) trastuzumab-refractory MBC (NCT00736970). Ridaforolimus was administered orally (40 mg daily) for 5 d/wk plus weekly trastuzumab. The primary end point was objective response (OR).. Thirty-four patients were enrolled (91% had received 1 or 2 previous trastuzumab-based therapies, whereas 9% had received 3 previous therapies). The most common reasons for discontinuation were disease progression (62%) and adverse events (AEs; 24%). Three patients died; 1 because of bowel perforation, which was possibly ridaforolimus related. Partial response was observed in 5 patients (15%). Median duration of response was 19.1 weeks (range, 15.9-80.1 weeks). Fourteen patients (41%) achieved stable disease (SD); 7 patients (21%) maintained SD for ≥ 24 weeks. The clinical benefit response (CBR) rate was 34.3%. Median progression-free survival (PFS) and overall survival (OS) were 5.4 months (range, 0-20.3 months; 95% confidence interval [CI], 2.0-7.4) and 17.7 months (range, 0-25.9 months; 95% CI, 8.8-20.8), respectively. PFS rate at 6 months was 37%. The most common treatment-related AEs were stomatitis (59%), diarrhea (27%), and rash (27%).. Ridaforolimus-trastuzumab was well tolerated and demonstrated antitumor activity in trastuzumab-resistant HER2(+) MBC.

Topics: Administration, Oral; Adult; Aged; Aged, 80 and over; Antibodies, Monoclonal, Humanized; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Carcinoma, Ductal, Breast; Drug Resistance, Neoplasm; Female; Humans; Middle Aged; Neoplasm Metastasis; Receptor, ErbB-2; Sirolimus; Survival Analysis; Trastuzumab

Mammalian target of rapamycin (mTOR) inhibition activates compensatory insulin-like growth factor receptor (IGFR) signaling. We evaluated the ridaforolimus (mTOR inhibitor) and dalotuzumab (anti-IGF1R antibody) combination.. In vitro and in vivo models, and a phase I study in which patients with advanced cancer received ridaforolimus (10-40 mg/day every day × 5/week) and dalotuzumab (10 mg/kg/week or 7.5 mg/kg/every other week) were explored.. Preclinical studies demonstrated enhanced pathway inhibition with ridaforolimus and dalotuzumab. With 87 patients treated in the phase I study, main dose-limiting toxicities (DLT) of the combination were primarily mTOR-related stomatitis and asthenia at doses of ridaforolimus lower than expected, suggesting blockade of compensatory pathways in normal tissues. Six confirmed partial responses were reported (3 patients with breast cancer); 10 of 23 patients with breast cancer and 6 of 11 patients with ER(+)/high-proliferative breast cancer showed antitumor activity.. Our study provides proof-of-concept that inhibiting the IGF1R compensatory response to mTOR inhibition is feasible with promising clinical activity in heavily pretreated advanced cancer, particularly in ER(+)/high-proliferative breast cancer (ClinicalTrials.gov identifier: NCT00730379).

Topics: Adult; Aged; Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Humans; Middle Aged; Receptor, IGF Type 1; Receptors, Somatomedin; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays

Everolimus is an agent frequently associated with specific toxicities. Predictive markers of efficacy are needed to help define which patients could benefit from it. The goal of this exploratory study was to identify potential predictive biomarkers in the mammalian target of rapamycin (mTOR) complex 1 (mTORC1) activation pathway using primary tumor samples collected during the phase II tamoxifen plus everolimus (TAMRAD) trial.. Tumor tissues were collected retrospectively from the TAMRAD trial. Immunohistochemistry was carried out using specific antibodies directed toward proteins that result in mTORC1 activation [canonical phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mTOR or alternative pathways]. DNA was extracted from the tumor tissue; mutation screening in the PIK3CA gene (exons 9 and 20) and the KRAS gene (exons 2 and 3) was first carried out using Sanger direct sequencing, and then completed by next-generation sequencing for PIK3CA. An exploratory analysis of everolimus efficacy in terms of a time-to-progression (TTP) increase was carried out in each biomarker subgroup (high versus low expression referring to the median percentage of marked cells).. A total of 55 primary tumor samples from the TAMRAD trial—25 from the tamoxifen-alone group and 30 from the tamoxifen/everolimus group—were evaluated for biomarkers. The subgroups most likely to have an improvement in TTP with tamoxifen/everolimus therapy, compared with tamoxifen alone, were patients with high p4EBP1, low 4EBP1, low liver kinase B1, low pAkt, and low PI3K. Among the 45 samples screened for mutation status, nine samples (20%; 95% CI 9.6-34.6) had a PIK3CA mutation. KRAS mutation was observed in one patient.. A positive correlation between late effectors of mTORC1 activation, a positive correlation between Akt-independent mTORC1 activation, and an inverse correlation between canonical PI3K/Akt/mTOR pathway and everolimus efficacy were observed in this exploratory analysis. However, these correlations need to be validated in larger studies before applying the findings to routine clinical practice.

Topics: Adaptor Proteins, Signal Transducing; Adult; Aged; Aged, 80 and over; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Base Sequence; Biomarkers, Tumor; Breast Neoplasms; Cell Cycle Proteins; Class I Phosphatidylinositol 3-Kinases; Everolimus; Female; Humans; Mechanistic Target of Rapamycin Complex 1; Middle Aged; Multiprotein Complexes; Phosphatidylinositol 3-Kinases; Phosphoproteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins p21(ras); ras Proteins; Retrospective Studies; Sequence Analysis, DNA; Sirolimus; Tamoxifen; TOR Serine-Threonine Kinases

In a phase 3, double-blind, randomized, international study (the BOLERO-2), the addition of mTOR inhibitor everolimus to exemestane was evaluated in postmenopausal women with estrogen-receptor-positive (ER⁺) advanced/recurrent breast cancer that was refractory to any nonsteroidal aromatase inhibitor (NSAI). This report presents the safety and updated (18- month) efficacy results from the Japanese subset (n=106) of BOLERO-2. After a median follow-up of 18 months, the median progression-free survival time was 8.5 months with everolimus plus exemestane compared to 4.2 months with placebo plus exemestane. The most common adverse events (AEs) with everolimus plus exemestane were stomatitis, rash, dysgeusia, and non-infectious lung disease. The AEs reported with the combination therapy were mostly of grade 1 or 2 and manageable with appropriate intervention. In conclusion, this combination could be a useful addition to the armamentarium of treatments for Japanese postmenopausal women with ER⁺ advanced/recurrent breast cancer progressing on NSAIs.

Topics: Adult; Aged; Androstadienes; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Double-Blind Method; Everolimus; Female; Humans; Middle Aged; Postmenopause; Receptors, Estrogen; Sirolimus

Constitutive activation of the PI3K/Akt/mTOR pathway has been suggested as a mechanism of resistance to trastuzumab therapy. This phase II trial was designed to evaluate the safety and clinical activity of daily oral sirolimus, a mammalian target of rapamycin (mTOR) inhibitor, in combination with trastuzumab in HER2-positive metastatic breast cancer following disease progression on prior trastuzumab therapy. Sirolimus 6 mg oral daily dose was administered with a standard dose and schedule of trastuzumab weekly or every 3 weeks. Pharmacodynamic studies included Western blot analysis of S6K1, phosphoS6K1, and mTOR in peripheral mononuclear cells, circulating tumor cells (CTC), and endothelial cells (CEC). Eleven patients were evaluable for safety; and nine were evaluable for response assessment. Subsequent enrollment was stopped due to slow accrual. Study treatment-related grade 3 toxicity included pneumonitis, myelosuppression (leukopenia/anemia), and dermatologic reactions (mucositis, nail changes and rash), with no grade 4 events. One patient received eight cycles (58 weeks) and achieved a partial response. Five patients treated for a total of 101 weeks (median 12 weeks, range 8-47 weeks) achieved stable disease as best response. Overall response rate was 1/9 (11 %) and clinical benefit rate was 4/9 (44 %). There was no statistically significant correlation between response and post-treatment change in levels of the mTOR pathway biomarkers, CTCs, HER2 CTCs, or CECs. Sirolimus 6 mg administered daily with trastuzumab appears to be well tolerated in patients with metastatic HER2-positive breast cancer following disease progression on prior trastuzumab therapy, with evidence of disease activity. mTOR inhibition may overcome resistance to trastuzumab in some HER2-positive tumors.

Topics: Adult; Antineoplastic Combined Chemotherapy Protocols; Biomarkers; Breast Neoplasms; Disease Progression; Female; Humans; Immunohistochemistry; Leukocytes, Mononuclear; Middle Aged; Neoplasm Metastasis; Neoplastic Cells, Circulating; Receptor, ErbB-2; Retreatment; Sirolimus; TOR Serine-Threonine Kinases; Trastuzumab; Treatment Outcome

mTOR inhibition reverses trastuzumab resistance via the hyperactivated PIK/AKT/mTOR pathway due to PTEN loss, by sensitising PTEN-deficient tumours to trastuzumab. The BOLERO-1 study assessed the efficacy and safety of adding everolimus to trastuzumab and paclitaxel as first-line treatment for patients with HER2-positive advanced breast cancer.. In this phase 3, randomised, double-blind trial, patients were enrolled across 141 sites in 28 countries. Eligible patients were aged 18 years or older, with locally assessed HER2-positive advanced breast cancer, with Eastern Cooperative Oncology Group (ECOG) performance status of 0-1, who had not received previous trastuzumab or chemotherapy for advanced breast cancer within 12 months of randomisation, had measurable disease as per Response Evaluation Criteria in Solid Tumors (RECIST) or bone lesions in the absence of measurable disease, without previous systemic treatment for advanced disease except endocrine therapy. Patients were randomly assigned (2:1) with an interactive voice and web response system to receive either 10 mg everolimus once a day orally or placebo plus weekly trastuzumab intravenously at 4 mg/kg loading dose on day 1 with subsequent weekly doses of 2 mg/kg of each 4 week cycle plus paclitaxel intravenously at a dose of 80 mg/m(2) on days 1, 8, and 15 of each 4 week cycle. Randomisation was stratified according to previous use of trastuzumab and visceral metastasis. Patients and investigators were masked to the assigned treatments. Identity of experimental treatments was concealed by use of everolimus and placebo that were identical in packaging, labelling, appearance, and administration schedule. The two primary objectives were investigator-assessed progression-free survival in the full study population and in the subset of patients with hormone receptor-negative breast cancer at baseline; the latter was added during the course of the study, before unmasking based on new clinical and biological findings from other studies. All efficacy analyses were based on the intention-to-treat population. Enrolment for this trial is closed and results of the final progression-free survival analyses are presented here. This trial is registered with ClinicalTrials.gov, number NCT00876395.. Between Sept 10, 2009, and Dec 16, 2012, 719 patients were randomly assigned to receive everolimus (n=480) or placebo (n=239). Median follow-up was 41·3 months (IQR 35·4-46·6). In the full population, median progression-free survival was 14·95 months (95% CI 14·55-17·91) with everolimus versus 14·49 months (12·29-17·08) with placebo (hazard ratio 0·89, 95% CI 0·73-1·08; p=0·1166). In the HR-negative subpopulation (n=311), median progression-free survival with everolimus was 20·27 months (95% CI 14·95-24·08) versus 13·08 months (10·05-16·56) with placebo (hazard ratio 0·66, 95% CI 0·48-0·91; p=0·0049); however, the protocol-specified significance threshold (p=0·0044) was not crossed. The most common adverse events with everolimus were stomatitis (314 [67%] of 472 patients in the everolimus group vs 77 [32%] of 238 patients in the placebo group), diarrhoea (267 [57%] vs 111 [47%] patients), and alopecia (221 [47%] vs 125 [53%]). The most frequently reported grade 3 or 4 adverse events in the everolimus group versus the placebo group were neutropenia (117 [25%] vs 35 [15%]), stomatitis (59 [13%] vs three [1%]), anaemia (46 [10%] vs six [3%]) and diarrhoea (43 [9%] vs 10 [4%]) On-treatment adverse event-related deaths were reported in 17 (4%) patients in the everolimus group and none in the placebo group.. Although progression-free survival was not significantly different between groups in the full analysis population, the 7·2 months prolongation we noted with the addition of everolimus in the HR-negative, HER2-positive population warrants further investigation, even if it did not meet prespecified criteria for significance. The safety profile was generally consistent with what was previously reported in BOLERO-3. Proactive monitoring and early management of adverse events in patients given everolimus and chemotherapy is crucial.. Novartis Pharmaceuticals.

Topics: Adult; Aged; Aged, 80 and over; Antibodies, Monoclonal, Humanized; Antineoplastic Combined Chemotherapy Protocols; Biomarkers, Tumor; Breast Neoplasms; Disease-Free Survival; Dose-Response Relationship, Drug; Double-Blind Method; Drug Administration Schedule; Everolimus; Female; Humans; Kaplan-Meier Estimate; Maximum Tolerated Dose; Middle Aged; Neoplasm Invasiveness; Neoplasm Staging; Paclitaxel; Proportional Hazards Models; Receptor, ErbB-2; Sirolimus; Survival Analysis; Trastuzumab; Treatment Outcome; Young Adult

In the randomized phase 2 study there was evaluated the efficacy of neoadjuvant endocrine treatment (anastrozole, exemestane) in comparison with chemotherapy (doxorubicin plus paclitaxel). Preoperative endocrine therapy was well tolerated. There was a trend towards higher overall rates of objective response and breast conserving surgery (BCS) among patients with tumors expressing high levels of ER (luminal A) in endocrine therapy group compared with chemotherapy group (43% vs 24%; p = 0,054). In HER2-positive breast cancer patients the addition of trastuzumab to neoadjuvant chemotherapy improved the overall and pathological complete response. Trastuzumab made possible an increasing number of breast conserving surgery (23% vs 13%; p = 0,022). No patient treated with trastuzumab and with chemotherapy had a local recurrence after BCS.

Topics: Adult; Aged; Anastrozole; Androstadienes; Antibodies, Monoclonal, Humanized; Antineoplastic Agents, Hormonal; Antineoplastic Combined Chemotherapy Protocols; Biomarkers, Tumor; Breast Neoplasms; Doxorubicin; Everolimus; Female; Humans; Mastectomy, Segmental; Middle Aged; Neoadjuvant Therapy; Nitriles; Paclitaxel; Postmenopause; Receptor, ErbB-2; Receptors, Estrogen; Sirolimus; TOR Serine-Threonine Kinases; Trastuzumab; Treatment Outcome; Triazoles

The addition of mTOR inhibitor everolimus (EVE) to exemestane (EXE) was evaluated in an international, phase 3 study (BOLERO-2) in patients with hormone-receptor-positive (HR(+)) breast cancer refractory to letrozole or anastrozole. The safety and efficacy of anticancer treatments may be influenced by ethnicity (Sekine et al. in Br J Cancer 99:1757-62, 2008). Safety and efficacy results from Asian versus non-Asian patients in BOLERO-2 are reported.. Patients were randomized (2:1) to 10 mg/day EVE + EXE or placebo (PBO) + EXE. Primary endpoint was progression-free survival (PFS). Secondary endpoints included overall survival, response rate, clinical benefit rate, and safety.. Of 143 Asian patients, 98 received EVE + EXE and 45 received PBO + EXE. Treatment with EVE + EXE significantly improved median PFS versus PBO + EXE among Asian patients by 38 % (HR = 0.62; 95 % CI, 0.41-0.94). Median PFS was also improved among non-Asian patients by 59 % (HR = 0.41; 95 % CI, 0.33-0.50). Median PFS duration among EVE-treated Asian patients was 8.48 versus 4.14 months for PBO + EXE, and 7.33 versus 2.83 months, respectively, in non-Asian patients. The most common grade 3/4 adverse events (stomatitis, anemia, elevated liver enzymes, hyperglycemia, and dyspnea) occurred at similar frequencies in Asian and non-Asian patients. Grade 1/2 interstitial lung disease occurred more frequently in Asian patients. Quality of life was similar between treatment arms in Asian patients.. Adding EVE to EXE provided substantial clinical benefit in both Asian and non-Asian patients with similar safety profiles. This combination represents an improvement in the management of postmenopausal women with HR(+)/HER2(-) advanced breast cancer progressing on nonsteroidal aromatase inhibitors, regardless of ethnicity.

Topics: Adult; Aged; Androstadienes; Antineoplastic Combined Chemotherapy Protocols; Asian People; Breast Neoplasms; Everolimus; Female; Humans; Middle Aged; Quality of Life; Receptor, ErbB-2; Sirolimus; TOR Serine-Threonine Kinases; Treatment Outcome

Human epidermal growth factor (HER) -mediated signaling is critical in many cancers, including subsets of breast and lung cancer. HER family members signal via the phosphatidylinositide 3-kinase (PI3K) -AKT/protein kinase B-mammalian target of rapamycin (mTOR) cascade; mTOR activation is critical for the expression of multiple contributors to tumor growth and invasion. On the basis of preclinical data suggesting synergy of HER2 inhibition and mTOR inhibition in breast and lung cancer models, we conducted a phase I combination study of neratinib, a small-molecule irreversible pan-HER tyrosine kinase inhibitor, and temsirolimus, an mTOR inhibitor, in patients with advanced solid tumors.. This study enrolled patients to dosing combinations of neratinib and temsirolimus. The primary objective was to estimate the toxicity contour of the combination and establish recommended phase II doses.. Sixty patients were treated on 12 of 16 possible dosing combinations. Diarrhea was the most common drug-related (93%) and dose-limiting toxicity (DLT), constituting four of 10 DLTs. Dose-limiting grade 3 metabolic abnormalities were also observed. Other frequent drug-related toxicities included nausea, stomatitis (both 53%), and anemia (48%). Two maximum-tolerated dose combinations were identified: 200 mg of neratinib/25 mg of temsirolimus and 160 mg of neratinib/50 mg of temsirolimus. Responses were noted in patients with HER2-amplified breast cancer resistant to trastuzumab, HER2-mutant non-small-cell lung cancer, and tumor types without identified mutations in the HER-PI3K-mTOR pathway.. The combination of neratinib and temsirolimus was tolerable and demonstrated antitumor activity in multiple tumor types, warranting further evaluation.

Topics: Aged; Anemia; Antineoplastic Combined Chemotherapy Protocols; Area Under Curve; Breast Neoplasms; Diarrhea; Dose-Response Relationship, Drug; Female; Gene Amplification; Humans; Lung Neoplasms; Male; Middle Aged; Mutation; Nausea; Neoplasms; Phosphatidylinositol 3-Kinases; Quinolines; Receptor, ErbB-2; Signal Transduction; Sirolimus; Stomatitis; TOR Serine-Threonine Kinases; Treatment Outcome

Fulvestrant, which degrades ER, is used after AI failure in metastatic breast cancer but resistance develops quickly. We hypothesized that using everolimus to inhibit mTOR, a key signaling pathway in endocrine resistance, may delay fulvestrant resistance in patients and thus improve its efficacy. We conducted a phase II trial of combined fulvestrant and everolimus in postmenopausal women with disease progression or relapse after an AI. Primary endpoint was time to progression (TTP) and secondary endpoints included objective response rate, clinical benefit rate (CBR), safety, and biomarker correlates. Tumor blocks were collected and biopsy of accessible tumor was done for future biomarker analysis. Of 33 patients enrolled two were ruled ineligible after enrollment and were excluded from study analysis, for a total of 31 evaluable patients. Median age was 54 years (range 45-85). Prior therapy included tamoxifen (81 %), chemotherapy (71 %), with 26 % of patients having received 3 or more endocrine agents. Median TTP was 7.4 months (95 % CI 1.9-12.1) with an objective response rate of 13 % and CBR of 49 %. Of particular note, 32 % of patients exhibited de novo resistance to study treatment with disease progression as their best response. Most common adverse events (AEs) were elevated AST (87 %) and ALT (77 %), anemia (74 %), hyperglycemia (71 %), and hypercholesterolemia (68 %). Prominent clinical toxicities were mucositis (58 %), weight loss (48 %), and rash (42 %). Most AEs were grade 1 or 2 and largely reversible with infrequent need for everolimus dose reduction. To conclude, everolimus plus fulvestrant is effective after AI failure in heavily pretreated metastatic ER-positive breast cancer and has manageable toxicity. Further study of this combination is warranted in randomized studies. Since not all patients experience benefit, and in view of potential toxicities, biomarker examination is critical to help select patients most likely to benefit from this strategy in future studies.

Topics: Aged; Aged, 80 and over; Antineoplastic Agents, Hormonal; Antineoplastic Combined Chemotherapy Protocols; Aromatase Inhibitors; Breast Neoplasms; Estradiol; Everolimus; Female; Fulvestrant; Humans; Immunosuppressive Agents; Middle Aged; Neoplasm Recurrence, Local; Receptors, Estrogen; Sirolimus; TOR Serine-Threonine Kinases; Treatment Failure

The present exploratory analysis examined the efficacy, safety, and quality-of-life effects of everolimus (EVE) + exemestane (EXE) in the subgroup of patients in BOLERO-2 whose last treatment before study entry was in the (neo)adjuvant setting. In BOLERO-2, patients with hormone-receptor-positive (HR(+)), human epidermal growth factor receptor-2-negative (HER2(-)) advanced breast cancer recurring/progressing after a nonsteroidal aromatase inhibitor (NSAI) were randomly assigned (2:1) to receive EVE (10 mg/day) + EXE (25 mg/day) or placebo (PBO) + EXE. The primary endpoint was progression-free survival (PFS) by local assessment. Overall, 137 patients received first-line EVE + EXE (n = 100) or PBO + EXE (n = 37). Median PFS by local investigator assessment nearly tripled to 11.5 months with EVE + EXE from 4.1 months with PBO + EXE (hazard ratio = 0.39; 95 % CI 0.25-0.62), while maintaining quality of life. This was confirmed by central assessment (15.2 vs 4.2 months; hazard ratio = 0.32; 95 % CI 0.18-0.57). The marked PFS improvement in patients receiving EVE + EXE as first-line therapy for disease recurrence during or after (neo)adjuvant NSAI therapy supports the efficacy of this combination in the first-line setting. Furthermore, the results highlight the potential benefit of early introduction of EVE + EXE in the management of HR(+), HER2(-) advanced breast cancer in postmenopausal patients.

Topics: Aged; Aged, 80 and over; Androstadienes; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Clinical Trials as Topic; Disease-Free Survival; Everolimus; Female; Humans; Kaplan-Meier Estimate; Middle Aged; Neoplasm Recurrence, Local; Neoplasms, Hormone-Dependent; Receptors, Estrogen; Receptors, Progesterone; Sirolimus

Pegylated liposomal doxorubicin (PLD) is active in breast, endometrial, and ovarian cancer. Preclinical data suggest that the combination of PLD with a mammalian target of rapamycin (mTOR) inhibitor has an additive effect. The safety and recommended phase two dose (RPTD) of temsirolimus in combination with PLD were assessed. (18) F-fluorodeoxyglucose (FDG) positron emission tomography (PET)/CT was performed for early response monitoring. Nineteen patients with advanced breast, endometrial, and ovarian cancer were treated with increasing doses of temsirolimus (10, 15, or 20 mg once weekly) and PLD (30 or 40 mg/m(2) once every 4 weeks). PLD was initiated 2 weeks after start of temsirolimus. FDG-PET/CT was performed at baseline, after 2 and 6 weeks. Standardized uptake values (SUV), metabolic volume, and total lesion glycolysis (TLG, SUV × metabolic volume) were calculated. The RPTD was 15 mg temsirolimus and 40 mg/m(2) PLD. Dose-limiting toxicities (DLT) were thrombocytopenia grade 3 with nose bleeding and skin toxicity grade 3. Most frequent treatment-related toxicities were nausea, fatigue, mucositis, and skin toxicity. Changes in TLG after 2 weeks predicted partial response (PR) after 10 weeks (p = 0.037). A rise in SUV between the second and sixth week predicted progression (PD) (p = 0.034) and was associated with worse progression free survival (PFS) (HR 1.068; p = 0.013). The RPTD was established at 15 mg temsirolimus weekly and PLD 40 mg/m(2) once every 4 weeks and the combination was safe. Early response evaluation with FDG-PET/CT may predict subsequent radiological PR and PD. This trial is registered under number NCT0098263.

Topics: Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Disease-Free Survival; Dose-Response Relationship, Drug; Doxorubicin; Endometrial Neoplasms; Female; Fluorodeoxyglucose F18; Glycolysis; Humans; Middle Aged; Multimodal Imaging; Ovarian Neoplasms; Polyethylene Glycols; Positron-Emission Tomography; Protein Kinase Inhibitors; Sirolimus; Tomography, X-Ray Computed; TOR Serine-Threonine Kinases; Treatment Outcome; Young Adult

In the BOLERO-2 trial, everolimus (EVE), an inhibitor of mammalian target of rapamycin, demonstrated significant clinical benefit with an acceptable safety profile when administered with exemestane (EXE) in postmenopausal women with hormone receptor-positive (HR(+)) advanced breast cancer. We report on the incidence, time course, severity, and resolution of treatment-emergent adverse events (AEs) as well as incidence of dose modifications during the extended follow-up of this study.. Patients were randomized (2:1) to receive EVE 10 mg/day or placebo (PBO), with open-label EXE 25 mg/day (n = 724). The primary end point was progression-free survival. Secondary end points included overall survival, objective response rate, and safety. Safety evaluations included recording of AEs, laboratory values, dose interruptions/adjustments, and study drug discontinuations.. The safety population comprised 720 patients (EVE + EXE, 482; PBO + EXE, 238). The median follow-up was 18 months. Class-effect toxicities, including stomatitis, pneumonitis, and hyperglycemia, were generally of mild or moderate severity and occurred relatively early after treatment initiation (except pneumonitis); incidence tapered off thereafter. EVE dose reduction and interruption (360 and 705 events, respectively) required for AE management were independent of patient age. The median duration of dose interruption was 7 days. Discontinuation of both study drugs because of AEs was higher with EVE + EXE (9%) versus PBO + EXE (3%).. Most EVE-associated AEs occur soon after initiation of therapy, are typically of mild or moderate severity, and are generally manageable with dose reduction and interruption. Discontinuation due to toxicity was uncommon. Understanding the time course of class-effect AEs will help inform preventive and monitoring strategies as well as patient education.. NCT00863655.

Topics: Adult; Aged; Aged, 80 and over; Breast Neoplasms; Disease-Free Survival; Drug-Related Side Effects and Adverse Reactions; Everolimus; Female; Humans; Middle Aged; Neoplasm Staging; Postmenopause; Sirolimus; TOR Serine-Threonine Kinases

Disease progression in patients with HER2-positive breast cancer receiving trastuzumab might be associated with activation of the PI3K/Akt/mTOR intracellular signalling pathway. We aimed to assess whether the addition of the mTOR inhibitor everolimus to trastuzumab might restore sensitivity to trastuzumab.. In this randomised, double-blind, placebo-controlled, phase 3 trial, we recruited women with HER2-positive, trastuzumab-resistant, advanced breast carcinoma who had previously received taxane therapy. Eligible patients were randomly assigned (1:1) using a central patient screening and randomisation system to daily everolimus (5 mg/day) plus weekly trastuzumab (2 mg/kg) and vinorelbine (25 mg/m(2)) or to placebo plus trastuzumab plus vinorelbine, in 3-week cycles, stratified by previous lapatinib use. The primary endpoint was progression-free survival (PFS) by local assessment in the intention-to-treat population. We report the final analysis for PFS; overall survival follow-up is still in progress. This trial is registered with ClinicalTrials.gov, number NCT01007942.. Between Oct 26, 2009, and May 23, 2012, 569 patients were randomly assigned to everolimus (n=284) or placebo (n=285). Median follow-up at the time of analysis was 20.2 months (IQR 15.0-27.1). Median PFS was 7.00 months (95% CI 6.74-8.18) with everolimus and 5.78 months (5.49-6.90) with placebo (hazard ratio 0.78 [95% CI 0.65-0.95]; p=0.0067). The most common grade 3-4 adverse events were neutropenia (204 [73%] of 280 patients in the everolimus group vs 175 [62%] of 282 patients in the placebo group), leucopenia (106 [38%] vs 82 [29%]), anaemia (53 [19%] vs 17 [6%]), febrile neutropenia (44 [16%] vs ten [4%]), stomatitis (37 [13%] vs four [1%]), and fatigue (34 [12%] vs 11 [4%]). Serious adverse events were reported in 117 (42%) patients in the everolimus group and 55 (20%) in the placebo group; two on-treatment deaths due to adverse events occurred in each group.. The addition of everolimus to trastuzumab plus vinorelbine significantly prolongs PFS in patients with trastuzumab-resistant and taxane-pretreated, HER2-positive, advanced breast cancer. The clinical benefit should be considered in the context of the adverse event profile in this population.

Topics: Adult; Aged; Aged, 80 and over; Antibodies, Monoclonal, Humanized; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Disease-Free Survival; Double-Blind Method; Drug Resistance, Neoplasm; Everolimus; Female; Genes, erbB-2; Humans; Kaplan-Meier Estimate; Middle Aged; Proportional Hazards Models; Sirolimus; Trastuzumab; Treatment Outcome

Since PI3K/AKT/mTOR pathway activation diminishes the effects of hormone therapy, combining aromatase inhibitors (anatrozole) with mTOR inhibitors (everolimus) was investigated.. We evaluated anastrozole and everolimus in 55 patients with metastatic estrogen (ER) and/or progesterone receptor (PR)-positive breast and gynecologic tumors. Endpoints were safety, antitumor activity and molecular correlates.. Full doses of anastrozole (1 mg PO daily) and everolimus (10 mg PO daily) were well tolerated. Twelve of 50 evaluable patients (24%) (median = 3 prior therapies) achieved stable disease (SD) ≥ 6 months/partial response (PR)/complete response (CR) (n = 5 (10%) with PR/CR): 9 of 32 (28%) with breast cancer (n=5 (16%) with PR/CR); 2 of 10 (20%), ovarian cancer; and 1 of 6 (17%), endometrial cancer. Six of 22 patients (27%) with molecular alterations in the PI3K/AKT/mTOR pathway achieved SD ≥ 6 months/PR/CR. Six of 8 patients (75%) with SD ≥ 6 months/PR/CR with molecular testing demonstrated at least one alteration in the PI3K/AKT/mTOR pathway: mutations in PIK3CA (n=3) and AKT1 (n=1) or PTEN loss (n=3). All three responders (CR (n = 1); PR (n=2)) who had next generation sequencing demonstrated additional alterations: amplifications in CCNE1, IRS2, MCL1, CCND1, FGFR1 and MYC and a rearrangement in PRKDC.. Combination anastrozole and everolimus is well tolerated at full approved doses, and is active in heavily-pretreated patients with ER and/or PR-positive breast, ovarian and endometrial cancers. Responses were observed in patients with multiple molecular aberrations. CLINICAL TRAILS INCLUDED: NCT01197170.

Topics: Adult; Aged; Aged, 80 and over; Anastrozole; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Everolimus; Female; Genital Neoplasms, Female; Humans; Kaplan-Meier Estimate; Middle Aged; Nitriles; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Treatment Outcome; Triazoles; Young Adult

Pegylated liposomal doxorubicin (PLD) is used to treat patients with breast and gynecological cancers. In order to optimize treatment with PLD, we assessed the prognostic and predictive factors for efficacy of PLD.. Seventeen patients treated with PLD 30 or 40 mg/m(2) underwent pharmacokinetic sampling during the first cycle of treatment. PLD exposure was calculated. An univariate analysis was performed with the variables: hand-foot syndrome, mucositis, rash, neutropenia, age, tumor type, number of previous therapies, ECOG performance status and progression-free survival (PFS). Candidate variables with p ≤ 0.1 were selected for the multivariate analysis.. Based on the results of the multivariate analysis, the PLD exposure (log AUC) was higher in patients who experienced rash (p = 0.002) and mucositis (p = 0.001) compared to those who did not have these adverse events. The development of hand-foot syndrome was significantly related to a lower risk of disease progression (HR 0.1; 95 % CI 0.02-0.64). Patients with an ECOG status of 0 had a longer PFS than the patients with an ECOG status of 1 (HR 5.4; 95 % CI 1.3-22.8). Moreover, PLD exposure (ln AUC) was also positively related to PFS (HR 0.001; 95 % CI 0.00-0.42).. The extent of the exposure to PLD was correlated with more adverse events and longer PFS. This has important clinical implications, since dose reductions or interruptions might thus negatively affect treatment outcomes. More attention should be paid to preventive and supportive measures of adverse events of PLD to keep the exposure to PLD as high as possible.

Topics: Adult; Aged; Antibiotics, Antineoplastic; Area Under Curve; Breast Neoplasms; Disease-Free Survival; Doxorubicin; Drug Administration Schedule; Endometrial Neoplasms; Female; Hand-Foot Syndrome; Humans; Middle Aged; Mucositis; Multivariate Analysis; Ovarian Neoplasms; Polyethylene Glycols; Sirolimus; Treatment Outcome; Young Adult

The aim of this study is to estimate the quality-adjusted progression-free survival (QAPFS) as an effectiveness measure for the treatment arms of the BOLERO-2 trial. For each treatment arm of the trial, QAPFS was estimated by multiplying the overall health utility weights associated with progression-free survival (PFS) (accounting for utility decrements associated with the adverse events of treatments) by the corresponding mean PFS time. Health utility data were obtained from the literature, while mean PFS times were estimated through a survival analysis of the reconstructed individual patient data of the BOLERO-2 trial. PFS (robust mean, (95 % robust confidence interval)) was 44.73 weeks (41.03; 48.43) for Everolimus + Exemestane and 22.98 weeks (19.88; 26.08) for Placebo + Exemestane. The QAPFS (robust mean, (95 % robust confidence interval)) for the treatment arms of the trial was 30.09 (27.60; 32.58) for Everolimus + Exemestane and 16.27 (14.07; 18.46) for Placebo + Exemestane, respectively. Using QAPFS as an outcome measure provides a complete picture of the benefit induced by the treatment arms of the BOLERO-2 trial. The benefit of Everolimus + Exemestane over Placebo + Exemestane observed in the trial is maintained in this analysis. The approach and estimates obtained as part of our analysis can serve as a basis for cost effectiveness analyses of the treatment arms of the BOLERO-2 trial.

Topics: Androstadienes; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Disease-Free Survival; Everolimus; Female; Humans; Receptor, ErbB-2; Receptors, Estrogen; Receptors, Progesterone; Sirolimus; Thiocarbamates; Treatment Outcome

The GeparQuinto study showed that adding bevacizumab to 24 weeks of anthracycline-taxane-based neoadjuvant chemotherapy increases pathological complete response (pCR) rates overall and specifically in patients with triple-negative breast cancer (TNBC). No difference in pCR rate was observed for adding everolimus to paclitaxel in nonearly responding patients. Here, we present disease-free (DFS) and overall survival (OS) analyses.. Patients (n = 1948) with HER2-negative tumors of a median tumor size of 4 cm were randomly assigned to neoadjuvant treatment with epirubicin/cyclophosphamide followed by docetaxel (EC-T) with or without eight infusions of bevacizumab every 3 weeks before surgery. Patients without clinical response to EC ± Bevacizumab were randomized to 12 weekly cycles paclitaxel with or without everolimus 5 mg/day. To detect a hazard ratio (HR) of 0.75 (α = 0.05, β = 0.8) 379 events had to be observed in the bevacizumab arms.. With a median follow-up of 3.8 years, 3-year DFS was 80.8% and 3-year OS was 89.7%. Outcome was not different for patients receiving bevacizumab (HR 1.03; P = 0.784 for DFS and HR 0.974; P = 0.842 for OS) compared with patients receiving chemotherapy alone. Patients with TNBC similarly showed no improvement in DFS (HR = 0.99; P = 0.941) and OS (HR = 1.02; P = 0.891) when treated with bevacizumab. No other predefined subgroup (HR+/HER2-; locally advanced (cT4 or cN3) or not; cT1-3 or cT4; pCR or not) showed a significant benefit. No difference in DFS (HR 0.997; P = 0.987) and OS (HR 1.11; P = 0.658) was observed for nonearly responding patients receiving paclitaxel with or without everolimus overall as well as in subgroups.. Long-term results, in opposite to the results of pCR, do not support the neoadjuvant use of bevacizumab in addition to an anthracycline-taxane-based chemotherapy or everolimus in addition to paclitaxel for nonearly responding patients.. NCT 00567554, www.clinicaltrials.gov.

Topics: Adult; Angiogenesis Inhibitors; Antibodies, Monoclonal, Humanized; Bevacizumab; Breast Neoplasms; Chemotherapy, Adjuvant; Drug Therapy, Combination; Everolimus; Female; Humans; Middle Aged; Receptor, ErbB-2; Sirolimus; Survival Analysis

The BOLERO-2 study previously demonstrated that adding everolimus (EVE) to exemestane (EXE) significantly improved progression-free survival (PFS) by more than twofold in patients with hormone-receptor-positive (HR(+)), HER2-negative advanced breast cancer that recurred or progressed during/after treatment with nonsteroidal aromatase inhibitors (NSAIs). The overall survival (OS) analysis is presented here.. BOLERO-2 is a phase III, double-blind, randomized international trial comparing EVE 10 mg/day plus EXE 25 mg/day versus placebo (PBO) + EXE 25 mg/day in postmenopausal women with HR(+) advanced breast cancer with prior exposure to NSAIs. The primary end point was PFS by local investigator assessment; OS was a key secondary end point.. At the time of data cutoff (3 October 2013), 410 deaths had occurred and 13 patients remained on treatment. Median OS in patients receiving EVE + EXE was 31.0 months [95% confidence interval (CI) 28.0-34.6 months] compared with 26.6 months (95% CI 22.6-33.1 months) in patients receiving PBO + EXE (hazard ratio = 0.89; 95% CI 0.73-1.10; log-rank P = 0.14). Poststudy treatments were received by 84% of patients in the EVE + EXE arm versus 90% of patients in the PBO + EXE arm. Types of poststudy therapies were balanced across arms, except for chemotherapy (53% EVE + EXE versus 63% PBO + EXE). No new safety concerns were identified.. In BOLERO-2, adding EVE to EXE did not confer a statistically significant improvement in the secondary end point OS despite producing a clinically meaningful and statistically significant improvement in the primary end point, PFS (4.6-months prolongation in median PFS; P < 0.0001). Ongoing translational research should further refine the benefit of mTOR inhibition and related pathways in this treatment setting.. NCT00863655.

Topics: Androstadienes; Breast Neoplasms; Double-Blind Method; ErbB Receptors; Everolimus; Female; Humans; Placebos; Sirolimus; Survival Analysis

Breast Cancer Trials of Oral Everolimus 2 (BOLERO-2), a phase III study in postmenopausal women with estrogen receptor-positive breast cancer progressing despite nonsteroidal aromatase inhibitor therapy, showed statistically significant benefits with adding everolimus to exemestane. Moreover, in preclinical studies, mammalian target of rapamycin inhibition was associated with decreased osteoclast survival and activity. Exploratory analyses in BOLERO-2 evaluated the effect of everolimus on bone marker levels and progressive disease in bone.. Patients were treated with exemestane (25mg/day) and randomized (2:1) to everolimus (10mg/day; combination) or placebo (exemestane only). Exploratory endpoints included changes in bone turnover marker levels vs baseline and progressive disease in bone, defined as unequivocal progression of a preexisting bone lesion or the appearance of a new bone lesion.. Baseline disease characteristics were well balanced between arms (N = 724); baseline bisphosphonate use was not (43.9% combination vs 54.0% exemestane only). At a median of 18 months of follow-up, median progression-free survival (primary endpoint) was statistically significantly longer with the combination vs exemestane only (Cox proportional hazard ratio = 0.45, 95% confidence interval = 0.38 to 0.54; log-rank, 1-sided P < .0001). Bone marker levels at 6 and 12 weeks increased with exemestane only, as expected, but decreased with the combination. The cumulative incidence rate of progressive disease in bone was lower in the combination arm. Bone-related adverse events occurred with similar frequency in both arms (3.3% combination vs 4.2% exemestane only).. These exploratory analyses suggest that everolimus has beneficial effects on bone turnover and progressive disease in bone in patients receiving exemestane for hormone receptor-positive breast cancer progressing during/after nonsteroidal aromatase inhibitor therapy.

Topics: Aged; Alkaline Phosphatase; Androstadienes; Antineoplastic Agents; Aromatase Inhibitors; Bone Density; Bone Density Conservation Agents; Bone Neoplasms; Bone Remodeling; Bone Resorption; Breast Neoplasms; Collagen Type I; Confounding Factors, Epidemiologic; Disease Progression; Disease-Free Survival; Drug Administration Schedule; Everolimus; Female; Follow-Up Studies; Fractures, Spontaneous; Humans; Middle Aged; Odds Ratio; Osteogenesis; Postmenopause; Procollagen; Proportional Hazards Models; Receptors, Estrogen; Sirolimus; TOR Serine-Threonine Kinases; Treatment Failure; Treatment Outcome

The randomized, controlled BOLERO-2 (Breast Cancer Trials of Oral Everolimus) trial demonstrated significantly improved progression-free survival with the use of everolimus plus exemestane (EVE + EXE) versus placebo plus exemestane (PBO + EXE) in patients with advanced breast cancer who developed disease progression after treatment with nonsteroidal aromatase inhibitors. This analysis investigated the treatment effects on health-related quality of life (HRQOL).. Using the European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire-Core 30 (EORTC QLQ-C30) questionnaire, HRQOL was assessed at baseline and every 6 weeks thereafter until disease progression and/or treatment discontinuation. The 30 items in 15 subscales of the QLQ-C30 include global health status wherein higher scores (range, 0-100) indicate better HRQOL. This analysis included a protocol-specified time to definitive deterioration (TDD) analysis at a 5% decrease in HRQOL versus baseline, with no subsequent increase above this threshold. The authors report additional sensitivity analyses using 10-point minimal important difference decreases in the global health status score versus baseline. Treatment arms were compared using the stratified log-rank test and Cox proportional hazards model adjusted for trial stratum (visceral metastases, previous hormone sensitivity), age, sex, race, baseline global health status score and Eastern Cooperative Oncology Group performance status, prognostic risk factors, and treatment history.. Baseline global health status scores were found to be similar between treatment groups (64.7 vs 65.3). The median TDD in HRQOL was 8.3 months with EVE + EXE versus 5.8 months with PBO + EXE (hazard ratio, 0.74; P = .0084). At the 10-point minimal important difference, the median TDD with EVE + EXE was 11.7 months versus 8.4 months with PBO + EXE (hazard ratio, 0.80; P = .1017).. In patients with advanced breast cancer who develop disease progression after treatment with nonsteroidal aromatase inhibitors, EVE + EXE was associated with a longer TDD in global HRQOL versus PBO + EXE.

Topics: Adult; Aged; Androstadienes; Antineoplastic Combined Chemotherapy Protocols; Aromatase Inhibitors; Breast Neoplasms; Disease Progression; Double-Blind Method; Drug Administration Schedule; Everolimus; Female; Health Status; Humans; Middle Aged; Odds Ratio; Postmenopause; Proportional Hazards Models; Quality of Life; Sirolimus; Surveys and Questionnaires; Treatment Outcome

We tested the oral mammalian target of rapamycin (mTOR) inhibitor everolimus in addition to paclitaxel in patients with HER2-negative tumours not responding to initial neoadjuvant cytotoxic and anti-angiogenic treatment.. Patients with primary HER2-negative tumours received four neoadjuvant cycles of epirubicin/cyclophosphamide (EC) with or without bevacizumab. Patients without clinical response were randomised to receive weekly paclitaxel (80 mg/m(2)) with or without everolimus (5mg p.o. daily, after a step-wise dose-escalation starting from 2.5mg bid) for 12 weeks before surgery. To detect an increase in pathological complete response (pCR; ypT0 ypN0) from 5% to 12.1% (odds ratio 2.62) 566 patients had to be recruited. The trial was stopped prematurely due to completion of accrual in the main study.. Of 1948 patients initially starting neoadjuvant treatment 403 were randomised. A total of 18 (4.6%) patients, 7 (3.6%) treated with paclitaxel and everolimus and 11 (5.6%) treated with paclitaxel alone had a pCR (odds ratio 0.36 (OR) (95% confidence interval (CI), 0.24-1.6) p=0.34). Overall response rate in breast and lymph nodes at surgery was 52.2% after paclitaxel plus everolimus and 61.7% after paclitaxel alone (p=0.063). Breast conserving treatment was performed in 54.4% of patients with the combination treatment and 61.9% with paclitaxel alone (p=0.20). Mucosal inflammation, thrombocytopenia, neutropenia, infection, and skin rash were more frequent when everolimus was added to paclitaxel.. Neoadjuvant therapy with everolimus and paclitaxel for patients with HER2-negative disease unresponsive to EC with or without bevacizumab did not improve the pCR rate. Long-term outcome is awaited.. Novartis, Roche, and Sanofi-Aventis.

Topics: Adult; Aged; Antibodies, Monoclonal, Humanized; Antineoplastic Combined Chemotherapy Protocols; Bevacizumab; Breast Neoplasms; Cyclophosphamide; Dose-Response Relationship, Drug; Drug Administration Schedule; Drug Resistance, Neoplasm; Epirubicin; Everolimus; Exanthema; Female; Humans; Mastectomy, Segmental; Middle Aged; Mucositis; Neoadjuvant Therapy; Neutropenia; Paclitaxel; Sirolimus; Thrombocytopenia; Treatment Outcome

The mammalian target of rapamycin (mTOR) plays a critical role in promoting tumor cell growth and is frequently activated in breast cancer. In preclinical studies, the antitumor activity of mTOR inhibitors is attenuated by feedback up-regulation of AKT mediated in part by Insulin-like growth factor type 1 receptor (IGF-1R). We designed a phase I trial to determine the maximum-tolerated dose (MTD) and pharmacodynamic effects of the IGF-1R antibody Cixutumumab in combination with temsirolimus in patients with metastatic breast cancer refractory to standard therapies. A 3 + 3 Phase I design was chosen. Temsirolimus and Cixutumumab were administered intravenously on days 1, 8, 15, and 22 of a 4-week cycle. Of the 26 patients enrolled, four did not complete cycle 1 because of disease progression (n = 3) or comorbid condition (n = 1) and were replaced. The MTD was determined from the remaining 22 patients, aged 34-72 (median 48) years. Most patients (86 %) had estrogen receptor positive cancer. The median number of prior chemotherapy regimens for metastatic disease was 3. The MTD was determined to be Cixutumumab 4 mg/kg and temsirolimus 15 mg weekly. Dose-limiting toxicities (DLTs) included mucositis, neutropenia, and thrombocytopenia. Other adverse events included grade 1/2 fatigue, anemia, and hyperglycemia. No objective responses were observed, but four patients experienced stable disease that lasted for at least 4 months. Compared with baseline, there was a significant increase in the serum levels of IGF-1 (p < 0.001) and IGFBP-3 (p = 0.019) on day 2. Compared with day 2, there were significant increases in the serum levels of IGF-1 (p < 0.001), IGF-2 (p = 0.001), and IGFBP-3 (p = 0.019) on day 8. A phase II study in women with metastatic breast cancer is ongoing.

Topics: Adult; Aged; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Female; Humans; Insulin-Like Growth Factor Binding Protein 3; Insulin-Like Growth Factor I; Insulin-Like Growth Factor II; Maximum Tolerated Dose; Middle Aged; Neoplasm Metastasis; Receptor, IGF Type 1; Sirolimus

Everolimus (EVE)+exemestane (EXE; n = 485) more than doubled median progression-free survival versus placebo (PBO) + EXE (n = 239), with a manageable safety profile and no deterioration in health-related quality-of-life (HRQOL) in patients with hormone-receptor-positive (HR(+)) advanced breast cancer (ABC) who recurred or progressed on/after nonsteroidal aromatase inhibitor (NSAI) therapy. To further evaluate EVE + EXE impact on disease burden, we conducted additional post-hoc analyses of patient-reported HRQOL.. HRQOL was assessed using EORTC QLQ-C30 and QLQ-BR23 questionnaires at baseline and every 6 weeks thereafter until treatment discontinuation because of disease progression, toxicity, or consent withdrawal. Endpoints included the QLQ-C30 Global Health Status (QL2) scale, the QLQ-BR23 breast symptom (BRBS), and arm symptom (BRAS) scales. Between-group differences in change from baseline were assessed using linear mixed models with selected covariates. Sensitivity analysis using pattern-mixture models determined the effect of study discontinuation on/before week 24. Treatment arms were compared using differences of least squares mean (LSM) changes from baseline and 95% confidence intervals (CIs) at each timepoint and overall.. Clinicaltrials.gov: NCT00863655.. Progression-free survival, survival, response rate, safety, and HRQOL.. Linear mixed models (primary model) demonstrated no statistically significant overall difference between EVE + EXE and PBO + EXE for QL2 (LSM difference = -1.91; 95% CI = -4.61, 0.78), BRBS (LSM difference = -0.18; 95% CI = -1.98, 1.62), or BRAS (LSM difference = -0.42; 95% CI = -2.94, 2.10). Based on pattern-mixture models, patients who dropped out early had worse QL2 decline on both treatments. In the expanded pattern-mixture model, EVE + EXE-treated patients who did not drop out early had stable BRBS and BRAS relative to PBO + EXE.. HRQOL data were not collected after disease progression.. These analyses confirm that EVE + EXE provides clinical benefit without adversely impacting HRQOL in patients with HR(+) ABC who recurred/progressed on prior NSAIs versus endocrine therapy alone.

Topics: Adult; Aged; Androstadienes; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Disease Progression; Disease-Free Survival; Everolimus; Female; Health Status; Humans; Immunosuppressive Agents; Middle Aged; Placebos; Quality of Life; Receptor, ErbB-2; Receptors, Estrogen; Receptors, Progesterone; Sirolimus; Surveys and Questionnaires; Treatment Outcome

Everolimus has shown to stop formation and activity of osteoclasts. Breast cancer patients with bone metastases only are candidates for effective but low toxic treatment.. We evaluated everolimus in a double-blind, placebo-controlled, phase II, randomized discontinuation study in breast cancer patients with HER2 negative breast cancer patients with bone metastases only. After being stable on 8 weeks of everolimus 10 mg/day, patients were randomized to everolimus-continuation or placebo. Primary outcome was time (from randomization) to progression (TTP). Seventy-six patients would have had to be randomized to show a hazard ration (HR) of 0.5 for everolimus-continuation.. Eighty-nine patients were enrolled in 4 years. Thirty-nine patients with SD after 8 weeks on everolimus were randomized to everolimus-continuation or placebo. TTP in patients with everolimus-continuation was 37.0 (95 % CI 16.7-40.3) versus 12.6 weeks (95 % CI 7.1-17.9) with placebo [HR 0.554 (95 % CI 0.282-1.09) p = 0.0818], adjusted for endocrine therapy [HR 0.464 (95 % CI 0.226-0.954) p = 0.037]. TTP in everolimus responders (n = 6) was 86 weeks.. The RADAR study is mainly hypothesis generating. It suggests that everolimus has single-agent activity, and patients with bone metastases only may retrieve long-term benefit from everolimus if they do not progress within 8 weeks of treatment.

Topics: Adult; Aged; Antineoplastic Agents; Bone Neoplasms; Breast Neoplasms; Disease Progression; Double-Blind Method; Drug-Related Side Effects and Adverse Reactions; Everolimus; Female; Humans; Middle Aged; Placebos; Sirolimus

Increased activation of the PI3K/Akt/mTOR pathway is a common factor in putative mechanisms of trastuzumab resistance, resulting in dysregulation of cell migration, growth, proliferation, and survival. Data from preclinical and phase 1/2 clinical studies suggest that adding everolimus (an oral mTOR inhibitor) to trastuzumab plus chemotherapy may enhance the efficacy of, and restore sensitivity to, trastuzumab-based therapy. In this phase 2 multicenter study, adult patients with HER2-positive advanced breast cancer resistant to trastuzumab and pretreated with a taxane received everolimus 10 mg/day in combination with paclitaxel (80 mg/m(2) days 1, 8, and 15 every 4 weeks) and trastuzumab (4 mg/kg loading dose followed by 2 mg/kg weekly), administered in 28-day cycles. Endpoints included overall response rate (ORR), progression-free survival (PFS), overall survival (OS), and safety. Fifty-five patients were enrolled; one remained on study treatment at the time of data cutoff. The median number of prior chemotherapy lines for advanced disease was 3.5 (range 1-11). The ORR was 21.8 %, the clinical benefit rate was 36.4 %, the median PFS estimate was 5.5 months (95 % confidence interval [CI]: 4.99-7.69 months), and the median OS estimate was 18.1 months (95 % CI: 12.85-24.11 months). Hematologic grade 3/4 adverse events (AEs) included neutropenia (25.5 % grade 3, 3.6 % grade 4), anemia (7.3 % grade 3), and thrombocytopenia (5.5 % grade 3, 1.8 % grade 4). Nonhematologic grade 3/4 AEs included stomatitis (20.0 %), diarrhea (5.5 %), vomiting (5.5 %), fatigue (5.5 %), and pneumonia (5.5 %), all grade 3. These findings suggest that the combination of everolimus plus trastuzumab and paclitaxel is feasible, with promising activity in patients with highly resistant HER2-positive advanced breast cancer. This combination is currently under investigation in the BOLERO-1 phase 3 trial.

Topics: Adult; Aged; Aged, 80 and over; Antibodies, Monoclonal, Humanized; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Disease Progression; Disease-Free Survival; Everolimus; Female; Humans; Kaplan-Meier Estimate; Middle Aged; Neoplasm Staging; Paclitaxel; Receptor, ErbB-2; Sirolimus; Taxoids; Trastuzumab; Treatment Failure

Effective treatments for hormone-receptor-positive (HR(+)) breast cancer (BC) following relapse/progression on nonsteroidal aromatase inhibitor (NSAI) therapy are needed. Initial Breast Cancer Trials of OraL EveROlimus-2 (BOLERO-2) trial data demonstrated that everolimus and exemestane significantly prolonged progression-free survival (PFS) versus placebo plus exemestane alone in this patient population.. BOLERO-2 is a phase 3, double-blind, randomized, international trial comparing everolimus (10 mg/day) plus exemestane (25 mg/day) versus placebo plus exemestane in postmenopausal women with HR(+) advanced BC with recurrence/progression during or after NSAIs. The primary endpoint was PFS by local investigator review, and was confirmed by independent central radiology review. Overall survival, response rate, and clinical benefit rate were secondary endpoints.. Final study results with median 18-month follow-up show that median PFS remained significantly longer with everolimus plus exemestane versus placebo plus exemestane [investigator review: 7.8 versus 3.2 months, respectively; hazard ratio = 0.45 (95% confidence interval 0.38-0.54); log-rank P < 0.0001; central review: 11.0 versus 4.1 months, respectively; hazard ratio = 0.38 (95% confidence interval 0.31-0.48); log-rank P < 0.0001] in the overall population and in all prospectively defined subgroups, including patients with visceral metastases, [corrected] and irrespective of age. The incidence and severity of adverse events were consistent with those reported at the interim analysis and in other everolimus trials.. The addition of everolimus to exemestane markedly prolonged PFS in patients with HR(+) advanced BC with disease recurrence/progression following prior NSAIs. These results further support the use of everolimus plus exemestane in this patient population. ClinicalTrials.gov #NCT00863655.

Topics: Adult; Aged; Aged, 80 and over; Androstadienes; Antineoplastic Combined Chemotherapy Protocols; Bone Neoplasms; Breast Neoplasms; Disease-Free Survival; Double-Blind Method; Everolimus; Female; Humans; Liver Neoplasms; Lung Neoplasms; Middle Aged; Postmenopause; Receptors, Estrogen; Receptors, Progesterone; Sirolimus; Treatment Outcome

Postmenopausal women with hormone receptor-positive (HR(+)) breast cancer in whom disease progresses or there is recurrence while taking a nonsteroidal aromatase inhibitor (NSAI) are usually treated with exemestane (EXE), but no single standard of care exists in this setting. The BOLERO-2 trial demonstrated that adding everolimus (EVE) to EXE improved progression-free survival (PFS) while maintaining quality of life when compared with EXE alone. Because many women with HR(+) advanced breast cancer are elderly, the tolerability profile of EVE plus EXE in this population is of interest.. BOLERO-2, a phase III randomized trial, compared EVE (10 mg/d) and placebo (PBO), both plus EXE (25 mg/d), in 724 postmenopausal women with HR(+) advanced breast cancer recurring/progressing after treatment with NSAIs. Safety and efficacy data in elderly patients are reported at 18-month median follow-up.. Baseline disease characteristics and treatment histories among the elderly subsets (≥ 65 years, n = 275; ≥ 70 years, n = 164) were generally comparable with younger patients. The addition of EVE to EXE improved PFS regardless of age (hazard ratio, 0.59 [≥ 65 years] and 0.45 [≥ 70 years]). Adverse events (AEs) of special interest (all grades) that occurred more frequently with EVE than with PBO included stomatitis, infections, rash, pneumonitis, and hyperglycemia. Elderly EVE-treated patients had similar incidences of these AEs as did younger patients but had more on-treatment deaths.. Adding EVE to EXE offers substantially improved PFS over EXE and was generally well tolerated in elderly patients with HR(+) advanced breast cancer. Careful monitoring and appropriate dose reductions or interruptions for AE management are recommended during treatment with EVE in this patient population.

Topics: Aged; Aged, 80 and over; Androstadienes; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Everolimus; Female; Follow-Up Studies; Humans; International Agencies; Neoplasm Metastasis; Neoplasm Recurrence, Local; Neoplasm Staging; Prognosis; Receptor, ErbB-2; Receptors, Estrogen; Receptors, Progesterone; Safety; Sirolimus; Survival Rate

Recent data showed improvement in progression-free survival (PFS) when adding everolimus to exemestane in patients with advanced breast cancer experiencing recurrence/progression after nonsteroidal aromatase inhibitor (AI) therapy. Here, we report clinical outcomes of combining the mammalian target of rapamycin (mTOR) inhibitor temsirolimus with letrozole in AI-naive patients.. This phase III randomized placebo-controlled study tested efficacy/safety of first-line oral letrozole 2.5 mg daily/temsirolimus 30 mg daily (5 days every 2 weeks) versus letrozole/placebo in 1,112 patients with AI-naive, hormone receptor-positive advanced disease. An independent data monitoring committee recommended study termination for futility at the second preplanned interim analysis (382 PFS events).. Patients were balanced (median age, 63 years; 10% stage III, 40% had received adjuvant endocrine therapy). Those on letrozole/temsirolimus experienced more grade 3 to 4 events (37% v 24%). There was no overall improvement in primary end point PFS (median, 9 months; hazard ratio [HR], 0.90; 95% CI, 0.76 to 1.07; P = .25) nor in the 40% patient subset with prior adjuvant endocrine therapy. An exploratory analysis showed improved PFS favoring letrozole/temsirolimus in patients ≤ age 65 years (9.0 v 5.6 months; HR, 0.75; 95% CI, 0.60 to 0.93; P = .009), which was separately examined by an exploratory analysis of 5-month PFS using subpopulation treatment effect pattern plot methodology (P = .003).. Adding temsirolimus to letrozole did not improve PFS as first-line therapy in patients with AI-naive advanced breast cancer. Exploratory analyses of benefit in younger postmenopausal patients require external confirmation.

Topics: Administration, Oral; Adult; Aged; Aged, 80 and over; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Disease-Free Survival; Double-Blind Method; Female; Humans; Letrozole; Middle Aged; Neoplasm Metastasis; Nitriles; Placebos; Postmenopause; Sirolimus; Treatment Outcome; Triazoles

The phosphatidylinositol 3' kinase (PI3K) pathway is commonly activated in breast cancer and aberrations such as PI3K mutations are common. Recent exciting clinical trial results in advanced estrogen receptor-positive (ER) breast cancer support mTOR activation is a major means of estrogen-independent tumor growth. Hence the means to identify a responsive breast cancer population that would most benefit from these compounds in the adjuvant or earlier stage setting is of high interest. Here we study PIK3CA genotype as well as a previously reported PI3K/mTOR-pathway gene signature (PIK3CA-GS) and their ability to estimate the level of PI3K pathway activation in two clinical trials of newly diagnosed ER-positive breast cancer patients- a total of 81 patients- one of which was randomized between letrozole and placebo vs letrozole and everolimus. The main objectives were to correlate the baseline PIK3CA genotype and GS with the relative change from baseline to day 15 in Ki67 (which has been shown to be prognostic in breast cancer) and phosphorylated S6 (S240) immunohistochemistry (a substrate of mTOR). In the randomized dataset, the PIK3CA-GS could identify those patients with the largest relative decreases in Ki67 to letrozole/everolimus (R = -0.43, p = 0.008) compared with letrozole/placebo (R = 0.07, p = 0.58; interaction test p = 0.02). In a second dataset of pre-surgical everolimus alone, the PIK3CA-GS was not significantly correlated with relative change in Ki67 (R = -0.11, p = 0.37) but with relative change in phosphorlyated S6 (S240) (R = -0.46, p = 0.028). PIK3CA genotype was not significantly associated with any endpoint in either datasets. Our results suggest that the PIK3CA-GS has potential to identify those ER-positive BCs who may benefit from the addition of everolimus to letrozole. Further evaluation of the PIK3CA-GS as a predictive biomarker is warranted as it may facilitate better selection of responsive patient populations for mTOR inhibition in combination with letrozole.

Topics: Aged; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Cell Proliferation; Class I Phosphatidylinositol 3-Kinases; Everolimus; Female; Gene Expression Regulation, Neoplastic; Genotype; Humans; Letrozole; Middle Aged; Mutation; Nitriles; Phosphatidylinositol 3-Kinases; Receptors, Estrogen; Sirolimus; TOR Serine-Threonine Kinases; Triazoles

Inhibition of mammalian target of rapamycin with everolimus may improve the efficacy of taxanes. Everolimus and docetaxel are both metabolized by CYP3A4, which could result in a pharmacokinetic (PK) interaction.. Fifteen patients with metastatic breast cancer were treated with docetaxel (doses of 40-75 mg/m(2) intravenously on day 1 of a 21-day cycle) in combination with everolimus (doses ranging from 20 to 50 mg orally on days 1 and 8 of a 21-day cycle) in a phase 1 trial using the continuous reassessment method to determine maximum tolerated dose. The first 2 patients developed a dose-limiting toxicity (neutropenic infection), prompting a mandatory dose reduction and PK evaluation of both everolimus and docetaxel for patients enrolled in subsequent dosing cohorts.. Fifteen patients were treated. Dose-limiting toxicity included grade 3 mucositis (n = 1), prolonged grade 4 neutropenia (n = 1), and grade 3 infection/febrile neutropenia (n = 3). Day 8 of everolimus was commonly held for neutropenia despite a dose reduction in docetaxel to 40 mg/m(2). Eleven patients underwent complete PK evaluation for everolimus, and 9 patients underwent complete PK evaluation for both everolimus and docetaxel. Widely variable changes in clearance were seen for both drugs, and the study was terminated because of lack of efficacy and concerns regarding toxicity seen with the combination.. Weekly everolimus in combination with docetaxel every 3 weeks was associated with excessive neutropenia and variable clearance of both drugs, making combination therapy unpredictable, even at low doses of both drugs.

Topics: Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Docetaxel; Drug Administration Schedule; Early Termination of Clinical Trials; Everolimus; Female; Humans; Maximum Tolerated Dose; Middle Aged; Neutropenia; Sirolimus; Taxoids

Resistance to endocrine therapy in breast cancer is associated with activation of the mammalian target of rapamycin (mTOR) intracellular signaling pathway. In early studies, the mTOR inhibitor everolimus added to endocrine therapy showed antitumor activity.. In this phase 3, randomized trial, we compared everolimus and exemestane versus exemestane and placebo (randomly assigned in a 2:1 ratio) in 724 patients with hormone-receptor-positive advanced breast cancer who had recurrence or progression while receiving previous therapy with a nonsteroidal aromatase inhibitor in the adjuvant setting or to treat advanced disease (or both). The primary end point was progression-free survival. Secondary end points included survival, response rate, and safety. A preplanned interim analysis was performed by an independent data and safety monitoring committee after 359 progression-free survival events were observed.. Baseline characteristics were well balanced between the two study groups. The median age was 62 years, 56% had visceral involvement, and 84% had hormone-sensitive disease. Previous therapy included letrozole or anastrozole (100%), tamoxifen (48%), fulvestrant (16%), and chemotherapy (68%). The most common grade 3 or 4 adverse events were stomatitis (8% in the everolimus-plus-exemestane group vs. 1% in the placebo-plus-exemestane group), anemia (6% vs. <1%), dyspnea (4% vs. 1%), hyperglycemia (4% vs. <1%), fatigue (4% vs. 1%), and pneumonitis (3% vs. 0%). At the interim analysis, median progression-free survival was 6.9 months with everolimus plus exemestane and 2.8 months with placebo plus exemestane, according to assessments by local investigators (hazard ratio for progression or death, 0.43; 95% confidence interval [CI], 0.35 to 0.54; P<0.001). Median progression-free survival was 10.6 months and 4.1 months, respectively, according to central assessment (hazard ratio, 0.36; 95% CI, 0.27 to 0.47; P<0.001).. Everolimus combined with an aromatase inhibitor improved progression-free survival in patients with hormone-receptor-positive advanced breast cancer previously treated with nonsteroidal aromatase inhibitors. (Funded by Novartis; BOLERO-2 ClinicalTrials.gov number, NCT00863655.).

Topics: Adult; Aged; Aged, 80 and over; Androstadienes; Antineoplastic Combined Chemotherapy Protocols; Aromatase Inhibitors; Bone Neoplasms; Breast Neoplasms; Disease-Free Survival; ErbB Receptors; Everolimus; Female; Humans; Kaplan-Meier Estimate; Middle Aged; Postmenopause; Recurrence; Sirolimus; Stomatitis; TOR Serine-Threonine Kinases

Preclinical models suggested that activating mutations of the PIK3CA gene are associated with sensitivity to inhibitors of the mammalian target of rapamycin (mTOR). In breast cancers, PIK3CA mutations are associated with estrogen receptor (ER) positivity. We therefore performed an open-label single arm phase II study of the rapamycin analog, temsirolimus, at a dose of 25 mg weekly, in women with pretreated breast cancers that were positive for ER, PR, or HER2. Archived formalin-fixed paraffin embedded tumor was collected for immunohistochemical evaluation of components of the PI3K/Akt/mTOR pathway and PIK3CA mutation analysis. Thirty-one patients were enrolled. There were no major objective responses; however, three patients had stable disease for over 24 weeks. Twenty-three tumor samples were available for mutational analysis. There were five tumors with PIK3CA mutations; no association was found between prolonged stable disease and PIK3CA mutation or any immunohistochemical marker. There was a trend toward improved progression free survival (PFS) for patients with positive nuclear staining for phospho-Akt308. One patient remains on study four and a half years after starting therapy; her tumor did not have a PIK3CA mutation. We conclude that single agent temsirolimus has minimal activity in a population of women with heavily pretreated breast cancer. We found no evidence that either absence of immunohistochemical staining for PTEN or mutations in the hotspot domains of PIK3CA in the primary tumor were associated with clinical benefit.

Topics: Antineoplastic Agents; Breast Neoplasms; Disease Progression; Female; Humans; Mutation; Neoplasm Metastasis; Protein Kinase Inhibitors; Sirolimus; Treatment Outcome

Cross-talk between signal transduction pathways likely contributes to hormone resistance in metastatic breast cancer (mBC). Everolimus, an oral inhibitor of the mammalian target of rapamycin, has restored sensitivity in endocrine-resistance models and shown anticancer activity in early-phase mBC clinical trials. This analysis evaluated efficacy and safety of everolimus in combination with tamoxifen in patients with mBC resistant to aromatase inhibitors (AIs).. This open-label, phase II study randomly assigned postmenopausal women with hormone receptor-positive, human epidermal growth factor receptor 2-negative, AI-resistant mBC to tamoxifen 20 mg/d plus everolimus 10 mg/d (n = 54) or tamoxifen 20 mg/d alone (n = 57). Randomization was stratified by primary and secondary hormone resistance. Primary end point was clinical benefit rate (CBR), defined as the percentage of all patients with a complete or partial response or stable disease at 6 months. No formal statistical comparison between groups was planned.. The 6-month CBR was 61% (95% CI, 47 to 74) with tamoxifen plus everolimus and 42% (95% CI, 29 to 56) with tamoxifen alone. Time to progression (TTP) increased from 4.5 months with tamoxifen alone to 8.6 months with tamoxifen plus everolimus, corresponding to a 46% reduction in risk of progression with the combination (hazard ratio [HR], 0.54; 95% CI, 0.36 to 0.81). Risk of death was reduced by 55% with tamoxifen plus everolimus versus tamoxifen alone (HR, 0.45; 95% CI, 0.24 to 0.81). The main toxicities associated with tamoxifen plus everolimus were fatigue (72% v 53% with tamoxifen alone), stomatitis (56% v 7%), rash (44% v 7%), anorexia (43% v 18%), and diarrhea (39% v 11%).. This study suggests that tamoxifen plus everolimus increased CBR, TTP, and overall survival compared with tamoxifen alone in postmenopausal women with AI-resistant mBC.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Combined Chemotherapy Protocols; Aromatase Inhibitors; Breast Neoplasms; Everolimus; Female; Humans; Middle Aged; Neoplasm Metastasis; Receptor, ErbB-2; Receptors, Estrogen; Receptors, Progesterone; Sirolimus; Tamoxifen

The combination of the mTOR inhibitor everolimus with the aromatase inhibitor exemestane was evaluated in the randomized Phase III BOLERO-2 trial. Research has indicated that aberrant signaling through the mTOR pathway is associated with resistance to endocrine therapies. The BOLERO-2 trial examined the effects on progression-free survival of the addition of everolimus to exemestane in a patient population of postmenopausal, hormone receptor-positive, advanced breast cancer. At the interim analysis, the median progression-free survival assessed by local investigators was 6.9 months for everolimus plus exemestane versus 2.8 months for placebo plus exemestane (hazard ratio: 0.43; p < 0.001), and by central assessment was 10.6 versus 4.1 months, respectively (hazard ratio: 0.36; p < 0.001). The everolimus plus exemestane arm showed greater number of grade 3 and 4 adverse events. This study suggests that the addition of everolimus to exemestane is a potential viable treatment option for this patient population.

Topics: Adult; Aged; Androstadienes; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Everolimus; Female; Humans; Middle Aged; Neoplasm Staging; Postmenopause; Receptor, ErbB-2; Receptors, Estrogen; Sirolimus; Treatment Outcome

Despite advances in the the first- and second-line treatment of metastatic breast cancer, there remains a large unmet need for additional treatment options. As preclinical studies have suggested that combining everolimus with carboplatin may produce higher activity than each drug by itself, we initiated a phase I study of this combination.. Patients with pre-treated metastatic breast cancer received weekly carboplatin at AUC2 and daily oral everolimus at different dose-levels (level I: 2.5 mg; II: 5 mg; III: 7.5 mg; IV: 10 mg). Three patients were assigned to dose-levels I to III, and six to dose-level IV. The primary end-point was to determine the maximum tolerated dose (MTD).. Fifteen patients were recruited to the study. The median number of previous chemotherapies was four (range: 1-11). No dose-limiting toxicity occurred at levels I-III during the first cycle. Based on the pre-determined definition, the maximum planned dose-level IV was selected as the MTD. Patients received a median of four cycles of treatment (range 1-13). Most frequent grade 3 and 4 toxicities included leukopenia, thrombocytopenia and infection. Response rates were as follows: 21% partial response, 43% stable disease, and 36% progressive disease.. Carboplatin and everolimus is a well-tolerated combination for heavily pre-treated metastatic breast cancer. Everolimus (10 mg/d) and carboplatin (AUC2 weekly) were defined as the MTD. This dose is currently being employed in an ongoing phase II trial.

Topics: Aged; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Carboplatin; Everolimus; Female; Humans; Middle Aged; Neoplasm Metastasis; Sirolimus; TOR Serine-Threonine Kinases

Preclinical data suggest that combining the mTOR/hypoxia-inducible factor (HIF) inhibitor temsirolimus and the antiangiogenesis antibody bevacizumab may augment antitumor activity as well as resensitize cells to anthracyclines.. We initiated a phase I study of bevacizumab and temsirolimus plus liposomal doxorubicin in patients with advanced malignancies. Patients (N = 136) were enrolled according to a modified 3 + 3 design plus dose expansion in responsive tumor types.. The most common cancers were breast (n = 29), epithelial ovarian (n = 23), and colorectal cancer (n = 17). The median number of prior chemotherapy regimens was four (range: 0-16). Grade 3 or higher adverse events (> 5%) included pancytopenia, mucositis, hand-foot syndrome, hypertension, and fistula. This regimen led to a 21% (n = 28) stable disease (SD) ≥ 6 months and 21% (n = 29) rate of partial or complete remission [PR/CR; (total SD ≥ 6 months/PR/CR = 42% (n = 57)]. PR/CR was most common in parotid gland adenocarcinoma (4/6, 67%), metaplastic breast cancer (5/12, 42%), endometrial endometrioid carcinoma (6/15, 40%), and in patients with a PIK3CA mutation and/or a PTEN mutation/loss (11/28, 39%). The maximum tolerated dose was liposomal doxorubicin 30 mg/m(2) and bevacizumab 15 mg/kg every three weeks with temsirolimus 25 mg weekly.. Patients tolerated bevacizumab and temsirolimus together with liposomal doxorubicin. Further evaluation, especially in patients with parotid, metaplastic breast, and endometrial endometrioid cancer, and in patients with PIK3CA and/or PTEN aberrations is warranted.

Topics: Adolescent; Adult; Aged; Angiogenesis Inhibitors; Antibiotics, Antineoplastic; Antibodies, Monoclonal, Humanized; Antineoplastic Combined Chemotherapy Protocols; Bevacizumab; Breast Neoplasms; Carcinoma, Ovarian Epithelial; Colorectal Neoplasms; Disease-Free Survival; Doxorubicin; Drug-Related Side Effects and Adverse Reactions; Female; Humans; Maximum Tolerated Dose; Middle Aged; Neoplasms, Glandular and Epithelial; Ovarian Neoplasms; Protein Kinase Inhibitors; Sirolimus

mTOR plays a key role in tumor cell cycle control, proliferation, and survival. RAD001 (everolimus) is a novel macrolide that inhibits mTOR and thus downstream signaling pathways. 31 post-menopausal women with early breast cancer were given 5 mg RAD001 once daily for 14 days prior to surgery. Biopsies were taken at diagnosis and at surgery (post 14 days of treatment) and assessed for immunohistochemical changes in proliferation (Ki67), apoptosis (active caspase-3), p-AKT (s473), p-S6 (s235/236 and s240/244), p-mTOR (s2448), ER, and PR. Five patients did not complete the 2-week treatment period due to adverse events. All adverse events were grade 1 or 2 (NCIC-CTC scale). RAD001 treatment significantly decreased proliferation (geometric mean reduction 74% from baseline (p = 0.019)), particularly in HER-2 positive tumors. High Ki67 pre-treatment correlated with reduction in Ki67, an increase in apoptosis, a reduction in p-AKT (cytoplasmic) and reduction in p-mTOR following treatment. Nuclear expression of p-AKT was significantly reduced with treatment. Tumors that had a reduction in Ki67 with treatment exhibited a significant reduction in cytoplasmic p-AKT. p-S6 staining was significantly reduced independently of Ki67 (p < 0.001 for two sites of phosphorylation). RAD001 5 mg/daily is safe and tolerable in postmenopausal early breast cancer patients and inhibits the mTOR pathway and its downstream effectors, significantly reducing tumor cell proliferation. Tumors with high Ki67, high p-AKT, and HER-2 positivity may be more responsive to mTOR inhibition with RAD001. This is the first study to report results of RAD001 5 mg as a single agent in early breast cancer.

Topics: Aged; Aged, 80 and over; Antineoplastic Agents; Breast Neoplasms; Everolimus; Female; Humans; Ki-67 Antigen; Middle Aged; Neoplasm Staging; Proto-Oncogene Proteins c-akt; Receptor, ErbB-2; Ribosomal Protein S6 Kinases; Sirolimus; TOR Serine-Threonine Kinases

To determine the feasible dose and schedule for everolimus, an oral mTOR inhibitor, combined with vinorelbine and trastuzumab for patients with HER2-overexpressing metastatic breast cancer pretreated with trastuzumab. In this phase Ib multicenter, Bayesian dose-escalation study, 50 patients received everolimus 5 mg/day, 20 mg/week, or 30 mg/week plus vinorelbine (25 mg/m² on day 1 and 8 every 3 weeks) and trastuzumab (2 mg/kg weekly). Endpoints included end-of-cycle-1 dose-limiting toxicity (DLT) rate (primary endpoint), safety, relative dose intensity, overall response rate (ORR), and pharmacokinetics. Grade 3/4 neutropenia was the most common end-of-cycle-1 DLT and occurred in 10 of 30 and 4 of 14 patients in the 5 mg/day and 30 mg/week cohorts, respectively. Other end-of-cycle-1 DLTs included single cases of febrile neutropenia, grade 3 stomatitis with concomitant fatigue, grade 2 stomatitis, grade 3 anorexia, and grade 2 acneiform dermatitis, all in the 5-mg/day cohort. Based on the recorded DLTs and global safety, everolimus 5 mg/day and 30 mg/week were chosen as the optimal dose levels for the daily and weekly arms. Forty-seven patients were evaluable for efficacy. ORR was 19.1%, with a disease control rate of 83.0% and median progression-free survival of 30.7 weeks. No drug interaction was observed between everolimus and vinorelbine. Everolimus combined with weekly vinorelbine and trastuzumab generally was well tolerated and had encouraging antitumor activity in heavily pretreated patients with HER2-overexpressing metastatic breast cancer that progressed on trastuzumab (NCT00426530).

Topics: Adult; Aged; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Disease-Free Survival; Drug Administration Schedule; Everolimus; Female; Genes, erbB-2; Humans; Middle Aged; Neoplasm Metastasis; Prognosis; Sirolimus; Survival Analysis; TOR Serine-Threonine Kinases; Trastuzumab; Treatment Outcome; Vinblastine; Vinorelbine

Topics: Aged; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Combined Chemotherapy Protocols; Bevacizumab; Breast Neoplasms; Carcinoma; Doxorubicin; Female; Humans; Metaplasia; Middle Aged; Neoplasm Metastasis; Prognosis; Receptor, ErbB-2; Receptors, Estrogen; Receptors, Progesterone; Sirolimus; Stem Cell Transplantation; Treatment Outcome

Trastuzumab resistance has been linked to activation of the phosphoinositol 3-kinase (PI3K) pathway. Phosphatase and tensin homolog (PTEN) is a dual phosphatase that counteracts the PI3K function; PTEN loss leads to activation of the Akt cascade and the downstream mammalian target of rapamycin (mTOR). Preclinical studies demonstrated that mTOR inhibition sensitized the response to trastuzumab in mice with HER2 overexpressing and PTEN-deficient breast xenografts. Our trial evaluated the safety and efficacy of the combination of everolimus and trastuzumab in women with HER2-overexpressing metastatic breast cancer (MBC) that progressed on trastuzumab-based therapy.. This represents a pooled analysis (n = 47), stemming from two trials that occurred concurrently in The University of Texas MD Anderson Cancer Center, Beth Israel Deaconess Medical Center, and Dana-Farber Cancer Institute. Patients with HER2-overexpressing MBC who had progressed on trastuzumab-based therapy received trastuzumab every 3 weeks in combination with daily everolimus.. Among 47 patients, the combination of everolimus and trastuzumab provided partial responses in seven patients (15%) and persistent stable disease (lasting 6 months or longer) in nine patients (19%), resulting in a clinical benefit rate of 34%. The median progression-free survival (PFS) was 4.1 month. Fatigue, infection, and mucositis were the predominant nonhematologic toxicities. Trastuzumab did not have significant influence on the pharmacokinetic profile of everolimus. Patients with PTEN loss demonstrated decreased overall survival (P = .048). However, PFS was not affected by PTEN loss.. Inhibition of mTOR results in clinical benefit and disease response in patients with trastuzumab-resistant HER2-overexpressing MBC.

Topics: Adult; Aged; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Combined Chemotherapy Protocols; Biomarkers, Tumor; Breast Neoplasms; Disease-Free Survival; Everolimus; Female; Humans; Immunohistochemistry; Kaplan-Meier Estimate; Middle Aged; Neoplasm Metastasis; PTEN Phosphohydrolase; Receptor, ErbB-2; Salvage Therapy; Sirolimus; TOR Serine-Threonine Kinases; Trastuzumab

Liposomal doxorubicin (D) and bevacizumab (A) are active single agents in gynecologic and breast malignancies which share a resistance mechanism: upregulation of hypoxia inducible factor (HIF-1α). We, therefore, added temsirolimus (T), which inhibits HIF-1α, to D and A (DAT). Trial objectives were assessment of safety, preliminary efficacy, and identification of biological response correlates.. Cycle length was 21 days, with IV D, A, and T on day 1; T on days 8 and 15 (3+3 dose-escalation design with expansion cohorts). Mutational assays for PIK3CA, BRAF, KRAS, and immunhistochemistry for PTEN loss were conducted.. This article details 74 patients with gynecologic and breast malignancies who received at least one dose of drug on study. Median patient age: 52 (27-79); prior regimens: 4 (1-11). Responses: 1 (1.4%) complete response (CR), 14 (18.9%) partial responses (PR), and 13 (17.6%) with stable disease (SD) ≥ 6 months (total = 37.9%). The most common grade 1 toxicities were fatigue (27%) and anemia (20.2%). Notable grade 3/4 toxicities: thrombocytopenia (9.5%), mucositis (6.7%), and bowel perforation (2.7%). PIK3CA mutations or PTEN loss were identified in 25 of 59 (42.3%) of tested patients. Among these, nine (36%) achieved CR/PR and four (16%) had SD ≥ 6 months (CR+PR+SD ≥ 6 months = 52%).. DAT is well tolerated with manageable side effects. Responses observed warrant further evaluation. Mutational analyses were notable for a high percentage of responders with phosphoinositide-3-kinase pathway aberrations.

Topics: Adult; Aged; Antibodies, Monoclonal, Humanized; Antineoplastic Combined Chemotherapy Protocols; Bevacizumab; Breast Neoplasms; Class I Phosphatidylinositol 3-Kinases; Dose-Response Relationship, Drug; Doxorubicin; Drug Administration Schedule; Female; Genital Neoplasms, Female; Humans; Middle Aged; Mutation; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins p21(ras); PTEN Phosphohydrolase; ras Proteins; Sirolimus; Survival Rate

Diffusion-weighted magnetic resonance imaging data obtained early in the course of therapy can be used to estimate tumor proliferation rates, and the estimated rates can be used to predict tumor cellularity at the conclusion of therapy. Six patients underwent diffusion-weighted magnetic resonance imaging immediately before, after one cycle, and after all cycles of neoadjuvant chemotherapy. Apparent diffusion coefficient values were calculated for each voxel and for a whole tumor region of interest. Proliferation rates were estimated using the apparent diffusion coefficient data from the first two time points and then used with the logistic model of tumor growth to predict cellularity after therapy. The predicted number of tumor cells was then correlated to the corresponding experimental data. Pearson's correlation coefficient for the region of interest analysis yielded 0.95 (P = 0.004), and, after applying a 3 × 3 mean filter to the apparent diffusion coefficient data, the voxel-by-voxel analysis yielded a Pearson correlation coefficient of 0.70 ± 0.10 (P < 0.05).

Topics: Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Cell Survival; Chemotherapy, Adjuvant; Cisplatin; Computer Simulation; Diffusion Magnetic Resonance Imaging; Drug Therapy, Computer-Assisted; Everolimus; Female; Humans; Image Enhancement; Image Interpretation, Computer-Assisted; Middle Aged; Models, Biological; Paclitaxel; Prognosis; Reproducibility of Results; Sensitivity and Specificity; Sirolimus; Systems Integration; Treatment Outcome

There is growing evidence that uncontrolled activation of the PI3K/Akt/mTOR pathway contributes to the development and progression of breast cancer. Inhibition of this pathway has antitumour effects in preclinical studies and efficacy in combination with other agents in breast cancer patients. The aim of this study is to characterise the effects of pre-operative everolimus treatment in primary breast cancer patients and to identify potential molecular predictors of response. Twenty-seven patients with oestrogen receptor (ER)-positive breast cancer completed 11-14 days of neoadjuvant treatment with 5-mg everolimus. Core biopsies were taken before and after treatment and analysed using Illumina HumanRef-8 v2 Expression BeadChips. Changes in proliferation (Ki67) and phospho-AKT were measured on diagnostic core biopsies/resection samples embedded in paraffin by immunohistochemistry to determine response to treatment. Patients that responded to everolimus treatment with significant reductions in proliferation (fall in % Ki67 positive cells) also had significant decreases in the expression of genes involved in cell cycle (P = 8.70E-09) and p53 signalling (P = 0.01) pathways. Highly proliferating tumours that have a poor prognosis exhibited dramatic reductions in the expression of cell cycle genes following everolimus treatment. The genes that most clearly separated responding from non-responding pre-treatment tumours were those involved with protein modification and dephosphorylation, including DYNLRB2, ERBB4, PTPN13, ULK2 and DUSP16. The majority of ER-positive breast tumours treated with everolimus showed a significant reduction in genes involved with proliferation, these may serve as markers of response and predict which patients will derive most benefit from mTOR inhibition.

Topics: Antibiotics, Antineoplastic; Biopsy; Breast Neoplasms; Cell Cycle; Cell Proliferation; Chemotherapy, Adjuvant; Everolimus; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Regulatory Networks; Humans; Immunohistochemistry; Intracellular Signaling Peptides and Proteins; Ki-67 Antigen; Neoadjuvant Therapy; Oligonucleotide Array Sequence Analysis; Phosphorylation; Postmenopause; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Receptors, Estrogen; Sirolimus; Time Factors; TOR Serine-Threonine Kinases; Treatment Outcome

To determine the recommended dose of everolimus, a mammalian target of rapamycin inhibitor, combined with paclitaxel and trastuzumab in patients with human epidermal growth factor receptor 2 (HER2)-overexpressing metastatic breast cancer pretreated with trastuzumab.. In this phase Ib, multicenter, dose-escalation study, patients were treated with everolimus 5 mg/d, 10 mg/d, or 30 mg/wk in combination with paclitaxel (80 mg/m(2) days 1, 8, and 15 every 4 weeks) and trastuzumab (2 mg/kg weekly). End points included end-of-cycle 1 dose-limiting toxicity (DLT) rate (primary end point), safety, relative dose intensity of study drugs, overall response rate (ORR), and pharmacokinetics.. Of 33 patients enrolled, 31 were pretreated with taxanes, and 32 were resistant to trastuzumab. Patients received a median of two lines of chemotherapy in the metastatic setting (range, 0 to 17 lines). Three patients experienced cycle 1 DLTs: febrile neutropenia (5 mg/d), stomatitis (10 mg/d), and confusion (30 mg/wk). Grade 3 to 4 neutropenia was the most common toxicity observed (n = 17 patients [52%]). On the basis of observed DLTs and overall safety, 10 mg/d was recommended for additional development. Twenty-seven patients had measurable disease and were evaluable for efficacy. Among these patients, ORR was 44%. Overall disease was controlled for 6 months or more in 74%. Median progression-free survival was 34 weeks (95% CI, 29.1 to 40.7 weeks). Among 11 patients who were resistant to both trastuzumab and taxane, a similar level of antitumor activity was observed (ORR, 55%).. Everolimus combined with weekly paclitaxel and trastuzumab was generally well tolerated and had encouraging antitumor activity in patients with trastuzumab-pretreated and -resistant metastatic HER2-overexpressing breast cancer.

Topics: Adult; Aged; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Combined Chemotherapy Protocols; Area Under Curve; Breast Neoplasms; Disease-Free Survival; Everolimus; Female; Humans; Kaplan-Meier Estimate; Maximum Tolerated Dose; Middle Aged; Paclitaxel; Sirolimus; Trastuzumab; Treatment Outcome

Cross-talk between the estrogen receptor (ER) and the phosphoinositide-3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathways is a mechanism of resistance to endocrine therapy, and blockade of both pathways enhances antitumor activity in preclinical models. This study explored whether sensitivity to letrozole was enhanced with the oral mTOR inhibitor, everolimus (RAD001).. Two hundred seventy postmenopausal women with operable ER-positive breast cancer were randomly assigned to receive 4 months of neoadjuvant treatment with letrozole (2.5 mg/day) and either everolimus (10 mg/day) or placebo. The primary end point was clinical response by palpation. Mandatory biopsies were obtained at baseline and after 2 weeks of treatment (ie, day 15). Samples were assessed for PI3K mutation status (PIK3CA) and for pharmacodynamic changes of Ki67, phospho-S6, cyclin D1, and progesterone receptor (PgR) by immunohistochemistry.. Response rate by clinical palpation in the everolimus arm was higher than that with letrozole alone (ie, placebo; 68.1% v 59.1%), which was statistically significant at the preplanned, one-sided, alpha = 0.1 level (P = .062). Marked reductions in progesterone receptor and cyclin D1 expression occurred in both treatment arms, and dramatic downregulation of phospho-S6 occurred only in the everolimus arm. An antiproliferative response, as defined by a reduction in Ki67 expression to natural logarithm of percentage positive Ki67 of less than 1 at day 15, occurred in 52 (57%) of 91 patients in the everolimus arm and in 25 (30%) of 82 patients in the placebo arm (P < .01). The safety profile was consistent with historical results of everolimus monotherapy; grades 3 to 4 adverse events occurred in 22.6% of patients who received everolimus and in 3.8% of patients who received placebo.. Everolimus significantly increased letrozole efficacy in neoadjuvant therapy of patients with ER-positive breast cancer.

Topics: Adult; Aged; Aged, 80 and over; Antibiotics, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Aromatase Inhibitors; Biomarkers, Tumor; Biopsy; Breast Neoplasms; Cell Proliferation; Chemotherapy, Adjuvant; Class I Phosphatidylinositol 3-Kinases; Cyclin D1; Double-Blind Method; Europe; Everolimus; Female; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Ki-67 Antigen; Letrozole; Middle Aged; Mutation; Neoadjuvant Therapy; Nitriles; Palpation; Phosphatidylinositol 3-Kinases; Phosphorylation; Postmenopause; Receptors, Estrogen; Receptors, Progesterone; Ribosomal Protein S6 Kinases; Sirolimus; Time Factors; Treatment Outcome; Triazoles; United States

To evaluate the safety and efficacy of oral everolimus, a mammalian target of rapamycin (mTOR) inhibitor, in two different schedules in minimally pretreated patients with metastatic breast cancer and to explore for possible biologic correlates of response.. Patients who received no or one prior chemotherapy regimen for metastatic breast cancer were entered onto this multicenter, noncomparative, randomized phase II study of everolimus 10 mg daily versus 70 mg weekly; the multinomial end points of response and progression were evaluated at 8 weeks. A two-stage accrual design was used, with 15 evaluable patients in each schedule in stage 1. Only daily therapy met criteria for continuing, and another 15 patients were added. pAKT, PTEN, carbonic anhydrase 9, estrogen receptor (ER), progesterone receptor, and human epidermal growth factor receptor 2 (HER2) were evaluated for possible correlation with response.. The most common drug-related toxicities were fatigue, rash, anorexia, diarrhea, stomatitis, cough, and pneumonitis. Pneumonitis occurred at higher than expected rates and seemed to be schedule dependent, with the highest incidence on the daily schedule. Response rate with daily therapy was 12% (95% CI, 3.4% to 28.2%) compared with 0% (95% CI, 0.0% to 20.6%) for weekly therapy. Twenty-seven percent of patients on daily therapy discontinued treatment compared with 13% on weekly therapy (16% v 6% with pneumonitis, respectively). No biologic correlates of response could be identified, although there were trends favoring benefit in ER-positive and HER2-negative metastatic breast cancer.. Oral everolimus has activity in metastatic breast cancer that is schedule dependent. Daily therapy with 10 mg is worthy of further study in this patient population.

Topics: Adult; Aged; Antineoplastic Agents; Breast Neoplasms; Dose-Response Relationship, Drug; Everolimus; Female; Humans; Immunosuppressive Agents; Middle Aged; Sirolimus

To investigate the safety and pharmacokinetics (PK) of combined treatment with letrozole and the oral mTOR inhibitor RAD001 in patients with metastatic breast cancer stable or progressing after > or = 4 months on letrozole alone.. Eighteen patients received letrozole (2.5 mg/day) and RAD001 at 5 mg/day (cohort 1) or 10 mg/day (cohort 2). In the absence of DLT in cohort 1, cohort 2 was expanded to 12 patients to obtain additional safety and PK data.. Most common adverse events were stomatitis (50.0% of patients), fatigue (44.4%), anorexia and/or decreased appetite (44.4%), diarrhoea (38.9%), headache (33.3%) and rash (33.3%). There was 1 DLT, a grade 3 thrombocytopaenia in cohort 2. No clinically relevant PK interaction was detected. Seven patients received the combination therapy for >6 months. One patient had a complete response, and one had a 28% reduction in liver metastases, both in cohort 2.. Daily therapy with RAD001 plus letrozole is promising: the results suggest anti-tumour activity with no PK interactions. The overall safety profile of the combination is consistent with that expected for RAD001 monotherapy. A daily dose of RAD001 10mg is recommended for further trials.

Topics: Administration, Oral; Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Cohort Studies; Drug Interactions; Everolimus; Feasibility Studies; Female; Humans; Immunosuppressive Agents; Letrozole; Male; Middle Aged; Nitriles; Sirolimus; Treatment Outcome; Triazoles

In this study, two doses of temsirolimus (CCI-779), a novel inhibitor of the mammalian target of rapamycin, were evaluated for efficacy, safety, and pharmacokinetics in patients with locally advanced or metastatic breast cancer who had been heavily pretreated.. Patients (n = 109) were randomly assigned to receive 75 or 250 mg of temsirolimus weekly as a 30-minute intravenous infusion. Patients were evaluated for tumor response, time to tumor progression, adverse events, and pharmacokinetics of temsirolimus.. Temsirolimus produced an objective response rate of 9.2% (10 partial responses) in the intent-to-treat population. Median time to tumor progression was 12.0 weeks. Efficacy was similar for both dose levels but toxicity was more common with the higher dose level, especially grade 3 or 4 depression (10% of patients at the 250-mg dose level, 0% at the 75-mg dose level). The most common temsirolimus-related adverse events of all grades were mucositis (70%), maculopapular rash (51%), and nausea (43%). The most common, clinically important grade 3 or 4 adverse events were mucositis (9%), leukopenia (7%), hyperglycemia (7%), somnolence (6%), thrombocytopenia (5%), and depression (5%).. In heavily pretreated patients with locally advanced or metastatic breast cancer, 75 and 250 mg temsirolimus showed antitumor activity and 75 mg temsirolimus showed a generally tolerable safety profile.

Topics: Adult; Aged; Antineoplastic Agents; Area Under Curve; Bone Neoplasms; Breast Neoplasms; Female; Humans; Male; Middle Aged; Protein Kinase Inhibitors; Protein Kinases; Safety; Salvage Therapy; Sirolimus; Soft Tissue Neoplasms; TOR Serine-Threonine Kinases; Treatment Outcome

Breast cancer is the most common malignancy among women. It is a complex condition with many subtypes based on the hormone receptor. The mammalian target of the rapamycin (mTOR) pathway regulates cell survival, metabolism, growth, and protein synthesis in response to upstream signals in both normal physiological and pathological situations, primarily in cancer. The objective of this study was to screen for a potential target to inhibit the mTOR using a variety of inhibitors derived from Cichorium intybus and to identify the one with the highest binding affinity for the receptor protein. Initially, AutoDock Vina was used to perform structure-based virtual screening, as protein-like interactions are critical in drug development. For the comparative analysis, 110 components of Cichorium intybus were employed and ten FDA-approved anticancer medicines, including everolimus, an mTOR inhibitor. Further, the drug-likeness and ADMET properties were investigated to evaluate the anti-breast cancer activity by applying Lipinski's rule of five to the selected molecules. The promising candidates were then subjected to three replica molecular dynamics simulations run for 100 ns, followed by binding free energy estimation using MM-GBSA. The data were analyzed using root-mean-square deviation (RMSD), root-mean-square fluctuation (RMSF), and protein-ligand interactions to determine the stability of the protein-ligand complex. Based on the results, taraxerone (98) revealed optimum binding affinities with mTOR, followed by stigmasterol (110) and rutin (104), which compared favorably to the control compounds. Subsequently, bioactive compounds derived from Cichorium intybus may serve as lead molecules for developing potent and effective mTOR inhibitors to treat breast cancer.

Topics: Breast Neoplasms; Cichorium intybus; Female; Humans; Ligands; Molecular Docking Simulation; Molecular Dynamics Simulation; MTOR Inhibitors; Sirolimus; TOR Serine-Threonine Kinases

Autophagy disorders are linked to human cancer, and the details of their mechanisms remain unclear.. To investigate the regulatory role of PRMT5 in the autophagy of breast cancer cells.. Human breast adenocarcinoma cell lines (MDA-MB-231, MCF7) were cultured. Plasmids of overexpression and down-regulation of PRMT5 were transfected into MDA-MB-231 and MCF7 cells. The MTT assay was used to determine the proliferation of MDA-MB-231 and MCF7 cells. A western blotting assay was used to verify the expression of autophagy-associated molecules. Immunofluorescence was applied to observe the expression of GFP-LC3.. The expression of PRMT5 decreased the sensitivity to rapamycin and nutrient deprivation. PRMT5 acts as an oncogene to promote cell proliferation and influences migration and stamness. PRMT5 expression elevated the autophagic activity initiated by EBSS and Rapamycin. PRMT5 was necessary and sufficient to enhance stress-induced autophagy. PRMT5 could improve several autophagy- related gene expressions. Atg5 expression could be regulated by activating the PRMT5 and PDCD4 molecules. The PRMT5 molecule could mediate the regulation of ULK1 expression.. PRMT5 influenced multiple stages of autophagy in controlling autophagy and tumorigenesis. Autophagy-related PRMT5 might be a respected target for therapeutic interventions in cancers. This study would provide new ideas for treating and selecting breast cancer targets.

Topics: Apoptosis Regulatory Proteins; Autophagy; Breast Neoplasms; Carcinogenesis; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Female; Humans; Protein-Arginine N-Methyltransferases; RNA-Binding Proteins; Sirolimus

One of the most common malignancies diagnosed and the leading cause of death for cancer-stricken women globally is breast cancer. The molecular subtype affects therapy options because it is a complex disorder with multiple subtypes. By concentrating on receptor activation, mTOR (mammalian target of rapamycin) can be employed as a therapeutic target. The goal of this work was to screen a number of inhibitors produced from Hibiscus rosa-sinensis for possible target to inhibit the mTOR and to determine which has the greatest affinity for the receptor. Primarily, the ionization states of the chosen compounds were predicted using the ChemAxon web platform, and their pKa values were estimated. Given the significance of interactions between proteins in the development of drugs, structure-based virtual screening was done using AutoDock Vina. Approximately 120 Hibiscus components and ten approved anti-cancer drugs, including the mTOR inhibitor everolimus, were used in the comparative analysis. By using Lipinski's rule of five to the chosen compounds, the ADMET profile and drug-likeness characteristics were further examined to assess the anti-breast cancer activity. The compounds with the highest ranked binding poses were loaded using the SeeSAR tool and the HYDE scoring to give interactive, desolvation, and visual ΔG estimation for ligand binding affinity assessment. Following, the prospective candidates underwent three replicas of 100 ns long molecular dynamics simulations, preceded with MM-GBSA binding free energy calculation. The stability of the protein-ligand complex was determined using root mean square deviation (RMSD), root mean square fluctuation (RMSF), and protein-ligand interactions. The results demonstrated that the best mTOR binding affinities were found for stigmastadienol (107), lupeol (66), and taraxasterol acetate (111), which all performed well in comparison to the control compounds. Thus, bioactive compounds isolated from Hibiscus rosa-sinensis could serve as lead molecules for the creation of potent and effective mTOR inhibitors for the breast cancer therapy.

Topics: Breast Neoplasms; Female; Flowers; Hibiscus; Humans; Ligands; Molecular Docking Simulation; Molecular Dynamics Simulation; Rosa; Sirolimus; TOR Serine-Threonine Kinases

We previously identified the mechanistic target of rapamycin complex 2 (mTORC2) as an effector of Ras for the control of directed cell migration in

Topics: Animals; Breast Neoplasms; Cell Movement; Dictyostelium; Epithelial Cells; Female; Humans; Mammals; Mechanistic Target of Rapamycin Complex 2; ras Proteins; Sirolimus

Mammary carcinogenesis, being ranked second in cancer-related mortality and the inadequacy of existing chemotherapy advocates the development of a novel treatment approach targeting its molecular signalling. Hyperactivation of mammalian target of rapamycin (mTOR) has a critical role in developing invasive mammary cancer and it can be a potential target.. This experiment was to explore the efficacy of mTOR-specific siRNA on therapeutic targeting of the mTOR gene, assess its proficiency in suppressing in vitro breast cancer and determine underlying molecular mechanisms.. Specific siRNA targeting mTOR was transfected into MDA-MB-231 cells and mTOR downregulation was validated through qRT-PCR and western blot analysis. Cell proliferation was analysed by MTT assay and confocal microscopy. Apoptosis was studied through flow cytometry and S6K, GSK-3β and caspase 3 expression were estimated. Further, the effect of mTOR blockade on cell cycle progression was determined.. Following transfection of mTOR-siRNA into the MDA-MB-231 cells, cell viability and apoptosis were examined which indicates that clinically relevant concentration of mTOR-siRNA inhibited cell growth and proliferation and promote apoptosis, resulting from the suppression of mTOR. This leads to the downregulation of mTOR downstream S6K and upregulation of GSK-3β. An increased level of caspase 3 symbolises that the apoptotic activity is mediated through caspasedependent pathway. Further, mTOR downregulation causes cell cycle arrest in G0/G1 phase as observed in the flow cytometry study.. With these results, we can conclude that mTOR-siRNA exerts direct 'anti-breast cancer' activity propagated by the S6K-GSK-3β- caspase 3 mediated apoptosis and by inducing cell cycle arrest.

Topics: Apoptosis; Breast Neoplasms; Caspase 3; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Female; Glycogen Synthase Kinase 3 beta; Humans; RNA, Small Interfering; Sirolimus; TOR Serine-Threonine Kinases

Topics: Antioxidants; Breast Neoplasms; Cell Line, Tumor; Female; Humans; Liposomes; Resveratrol; Sirolimus

Activation of the PI3K-mTOR pathway is central to breast cancer pathogenesis including resistance to many targeted therapies. The mTOR kinase forms two distinct complexes, mTORC1 and mTORC2, and understanding which is required for the survival of malignant cells has been limited by tools to selectively and completely impair either subcomplex. To address this, we used RMC-6272, a bi-steric molecule with a rapamycin-like moiety linked to an mTOR active-site inhibitor that displays >25-fold selectivity for mTORC1 over mTORC2 substrates. Complete suppression of mTORC1 by RMC-6272 causes apoptosis in ER+/HER2- breast cancer cell lines, particularly in those that harbor mutations in PIK3CA or PTEN, due to inhibition of the rapamycin resistant, mTORC1 substrate 4EBP1 and reduction of the pro-survival protein MCL1. RMC-6272 reduced translation of ribosomal mRNAs, MYC target genes, and components of the CDK4/6 pathway, suggesting enhanced impairment of oncogenic pathways compared to the partial mTORC1 inhibitor everolimus. RMC-6272 maintained efficacy in hormone therapy-resistant acquired cell lines and patient-derived xenografts (PDX), showed increased efficacy in CDK4/6 inhibitor treated acquired resistant cell lines versus their parental counterparts, and was efficacious in a PDX from a patient experiencing resistance to CDK4/6 inhibition. Bi-steric mTORC1-selective inhibition may be effective in overcoming multiple forms of therapy-resistance in ER+ breast cancers.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Drug Resistance; Female; Humans; Mechanistic Target of Rapamycin Complex 1; Mechanistic Target of Rapamycin Complex 2; Multiprotein Complexes; Sirolimus; TOR Serine-Threonine Kinases

Human epidermal growth factor receptor 2 (HER2) overexpression is an independent prognostic factor of poor prognosis and a predictor of efficacy of anti-HER2 therapy. A limited number of patients can receive standard second-line therapy (DS-8201 or T-DM1) for metastatic HER2-positive in some parts of the world, including China, due to many factors, such as cost-benefit ratios.. A 51-year-old premenopausal woman was diagnosed with HER2-positive breast cancer. The pathological stage was ypT3N2M0 and stage IIIA. Trastuzumab targeted therapy combined with goserelin depot was started along with letrozole endocrine therapy. After eight courses of treatment, magnetic resonance imaging (MRI) examination revealed new multiple metastases in the liver, and progression disease (PD) was evaluated. Due to abnormal activation of the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway in the patient, treatment was changed to the mammalian target of rapamycin (mTOR) inhibitor combined with the anti-HER-2 agents inetetamab and paclitaxel, while partial response (PR) was evaluated after 6 cycles of treatment. As the patient was hormone receptor (HR) positive, treatment was changed to the inetetamab + rapamycin + exemestane regimen. The lesion continued to shrink and PR was evaluated for 8 cycles. The original regimen was continued, PR was evaluated after 12 courses of treatment. The abdominal MRI performed showed an increase in the volume of intrahepatic multiple metastatic tumor lesion. Efficacy was used to assess for PD and the progression-free survival (PFS) was 317 days.. A phosphatidylinositol-4, 5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) mutation in trastuzumab-treated metastatic HER2-positive breast cancer female had a long PFS by treating with the mammalian target of rapamycin inhibitor in combination with the anti-HER-2 agent inetetamab.

Topics: Breast Neoplasms; Female; Humans; Middle Aged; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Sirolimus; TOR Serine-Threonine Kinases; Trastuzumab

Mammary physiology is distinguished in containing adult stem/progenitor cells that are actively amending the breast tissue throughout the reproductive lifespan of women. Despite their importance in both mammary gland development, physiological maintenance, and reproduction, the exact role of mammary stem/progenitor cells in mammary tumorigenesis has not been fully elucidated in humans or animal models. The implications of modulating adult stem/progenitor cells in women could lead to a better understanding of not only their function, but also toward possible breast cancer prevention led us to evaluate the efficacy of rapamycin in reducing mammary stem/progenitor cell activity and malignant progression markers.. We analyzed a large number of human breast tissues for their basal and luminal cell composition with flow cytometry and their stem and progenitor cell function with sphere formation assay with respect to age and menopausal status in connection with a clinical study (NCT02642094) involving a low-dose (2 mg/day) and short-term (5-7 days) treatment of the mTOR inhibitor sirolimus. The expression of biomarkers in biopsies and surgical breast samples were measured with quantitative analysis of immunohistochemistry.. Sirolimus treatment significantly abrogated mammary stem cell activity, particularly in postmenopausal patients. It did not affect the frequency of luminal progenitors but decreased their self-renewal capacity. While sirolimus had no effect on basal cell population, it decreased luminal cell population, particularly in postmenopausal patients. It also significantly diminished prognostic biomarkers associated with breast cancer progression from ductal carcinoma in situ to invasive breast cancer including p16INK4A, COX-2, and Ki67, as well as markers of the senescence-associated secretary phenotype, thereby possibly functioning in preventing early breast cancer progression.. Overall, these findings indicate a link from mTOR signaling to mammary stem and progenitor cell activity and cancer progression. Trial registration This study involves a clinical trial registered under the ClinicalTrials.gov identifier NCT02642094 registered December 30, 2015.

Topics: Animals; Biomarkers; Breast Neoplasms; Epithelial Cells; Female; Humans; Mammary Glands, Animal; Sirolimus; Stem Cells; TOR Serine-Threonine Kinases

Autophagy is an important regulator of cellular homeostasis and its dysregulation often results in cancer. Aberrant glycosylation induced by oncogenic transformation contributes to tumor invasion and metastasis. In a previous study, we have demonstrated that EpCAM, a glycosylation protein, is associated with cell growth and metastasis in breast cancer. But the effect of EpCAM glycosylation on autophagy is not clear. the precise mechanism of regulation remains largely unknown. In this study, breast cancer cells were transfected with N-glycosylation mutation EpCAM plasmid to express deglycosylated EpCAM. The result showed that deglycosylated EpCAM promoted autophagy in breast cancer cells. We further confirmed this conclusion with the activator (Rapamycin, RAP) and inhibitor (Wortmannin) of autophagy. We also found that deglycosylated EpCAM promoted apoptosis and inhibited proliferation through activating autophagy by suppressing Akt/mTOR signaling pathway in breast cancer cells. These findings represent a novel mechanism by which deglycosylated EpCAM inhibits proliferation by enhancing autophagy of breast cancer cells via PI3K/Akt/mTOR pathway. In conclusion, the combination of autophagy modulation and EpCAM targeted therapy is a promising therapeutic strategy in the treatment of breast cancer.

Topics: Antifungal Agents; Autophagy; Biomarkers; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Epithelial Cell Adhesion Molecule; Female; Gene Expression Regulation, Neoplastic; Gene Silencing; Heterocyclic Compounds, 3-Ring; Humans; Phosphatidylinositol 3-Kinases; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Sirolimus; TOR Serine-Threonine Kinases; Wortmannin

Autophagy drives drug resistance and drug-induced cancer cell cytotoxicity. Targeting the autophagy process could greatly improve chemotherapy outcomes. The discovery of specific inhibitors or activators has been hindered by challenges with reliably measuring autophagy levels in a clinical setting. We investigated drug-induced autophagy in breast cancer cell lines with differing ER/PR/Her2 receptor status by exposing them to known but divergent autophagy inducers each with a unique molecular target, tamoxifen, trastuzumab, bortezomib or rapamycin. Differential gene expression analysis from total RNA extracted during the earliest sign of autophagy flux showed both cell- and drug-specific changes. We analyzed the list of differentially expressed genes to find a common, cell- and drug-agnostic autophagy signature. Twelve mRNAs were significantly modulated by all the drugs and 11 were orthogonally verified with Q-RT-PCR (Klhl24, Hbp1, Crebrf, Ypel2, Fbxo32, Gdf15, Cdc25a, Ddit4, Psat1, Cd22, Ypel3). The drug agnostic mRNA signature was similarly induced by a mitochondrially targeted agent, MitoQ. In-silico analysis on the KM-plotter cancer database showed that the levels of these mRNAs are detectable in human samples and associated with breast cancer prognosis outcomes of Relapse-Free Survival in all patients (RSF), Overall Survival in all patients (OS), and Relapse-Free Survival in ER+ Patients (RSF ER+). High levels of Klhl24, Hbp1, Crebrf, Ypel2, CD22 and Ypel3 were correlated with better outcomes, whereas lower levels of Gdf15, Cdc25a, Ddit4 and Psat1 were associated with better prognosis in breast cancer patients. This gene signature uncovers candidate autophagy biomarkers that could be tested during preclinical and clinical studies to monitor the autophagy process.

Topics: Antineoplastic Agents; Autophagy; Biomarkers, Tumor; Bortezomib; Breast Neoplasms; Cell Line, Tumor; Drug Resistance, Neoplasm; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Regulatory Networks; Humans; MCF-7 Cells; Organophosphorus Compounds; Receptor, ErbB-2; Receptors, Estrogen; Receptors, Progesterone; Sequence Analysis, RNA; Sirolimus; Tamoxifen; Trastuzumab; Ubiquinone

Rapalogues are powerful therapeutic modalities for breast cancer; however, they suffer from low solubility and dose-limiting side effects. To overcome these challenges, we developed a long-circulating multiheaded drug carrier called 5FA, which contains rapamycin-binding domains linked with elastin-like polypeptides (ELPs). To target these "Hydra-ELPs" toward breast cancer, we here linked 5FA with four distinct peptides which are reported to engage the cell surface form of the 78 kDa glucose-regulated protein (csGRP78). To determine if these peptides affected the carrier solubility, this library was characterized by light scattering and mass spectrometry. To guide in vitro selection of the most potent functional carrier for rapamycin, its uptake and inhibition of mTORC1 were monitored in a ductal breast cancer model (BT474). Using flow cytometry to track cellular association, it was found that only the targeted carriers enhanced cellular uptake and were susceptible to proteolysis by SubA, which specifically targets csGRP78. The functional inhibition of mTOR was monitored by Western blot for pS6K, whereby the best carrier L-5FA reduced mTOR activity by 3-fold compared to 5FA or free rapamycin. L-5FA was further visualized using super-resolution confocal laser scanning microscopy, which revealed that targeting increased exposure to the carrier by ∼8-fold. This study demonstrates how peptide ligands for GRP78, such as the L peptide (RLLDTNRPLLPY), may be incorporated into protein-based drug carriers to enhance targeting.

Topics: Animals; Breast Neoplasms; Drug Carriers; Elastin; Endoplasmic Reticulum Chaperone BiP; Female; Humans; Hydra; Peptides; Sirolimus; TOR Serine-Threonine Kinases

To investigate the effects of everolimus, a mechanistic/mammalian target of rapamycin (mTOR) inhibitor, on tumor growth and immune response in a mouse model of breast cancer. Human hormone receptor-positive (HR+)/human epidermal growth receptor 2-negative (HER2-) MC4-L2 cell line was used to establish a mouse model of breast cancer. The inhibitory effects of high (10 mg/kg) and low (5 mg/kg) doses of everolimus were investigated on tumor growth. Additionally, the frequency of CD4+Foxp3+ regulatory T cells (Tregs), CD8+Foxp3+ Tregs, and CD4+ and CD8+ T cells expressing cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) was explored by flow cytometry in bone marrow, lymph nodes, and spleen. Our results showed that both 10 mg/kg and 5 mg/kg doses of everolimus efficiently inhibited tumor growth, resulting in reduced breast tumor volume. In addition, it was revealed that everolimus-treated mice induced a higher frequency of CD4+Foxp3+ Tregs, CD8+Foxp3+ Tregs, and CD4+Foxp3+CTLA-4+ Tregs as well as CD4+ and CD8+ T cells expressing CTLA-4 in their bone marrow, lymph nodes, and spleen compared with standard control (vehicle-treated) in a dose-dependent manner. Furthermore, we found that everolimus treatment with 10 mg/kg and 5 mg/kg increased the frequency of Helios+Foxp3+ Tregs in the bone marrow of treated mice compared with the control group. Our results indicate that treatment with everolimus not only inhibits tumor growth but also exerts an immunomodulatory effect by inducing Tregs in the lymphoid organs of breast cancer-bearing mice. The combination of therapy with other anti-cancer agents may negate immune suppression and improve the efficacy of mTOR-targeted breast cancer therapy.

Topics: Animals; Breast Neoplasms; CTLA-4 Antigen; Disease Models, Animal; Everolimus; Female; Forkhead Transcription Factors; Humans; Mammals; Mice; Sirolimus; TOR Serine-Threonine Kinases

Phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway (PAM pathway) plays an important role in the development of breast cancer and are closely associated with the resistance to endocrine therapy in advanced breast cancer. Therefore, anti-cancer treatment targeting key molecules in this signaling pathway has become research hot-spot in recent years. Randomized clinical trials have demonstrated that PI3K/AKT/mTOR inhibitors bring significant clinical benefit to patients with advanced breast cancer, especially to those with hormone receptor (HR)-positive, human epidermal growth factor receptor (HER) 2-negative advanced breast cancer. Alpelisib, a PI3K inhibitor, and everolimus, an mTOR inhibitor, have been approved by Food and Drug Administration. Based on their high efficacy and relatively good safety profile, expanded indication of everolimus in breast cancer have been approved by National Medical Products Administration. Alpelisib is expected to be approved in China in the near future. The members of the consensus expert panel reached this consensus to comprehensively define the role of PI3K/AKT/mTOR signaling pathway in breast cancer, efficacy and clinical applications of PI3K/AKT/mTOR inhibitors, management of adverse reactions, and PIK3CA mutation detection, in order to promote the understanding of PI3K/AKT/mTOR inhibitors for Chinese oncologists, improve clinical decision-making, and prolong the survival of target patient population.. 磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(AKT)/哺乳动物雷帕霉素靶蛋白(mTOR)信号通路(PAM信号通路)在乳腺癌发生发展中发挥重要作用，与晚期乳腺癌内分泌治疗耐药密切相关，以PAM信号通路中的关键分子为靶点的抗癌治疗成为了近几年的研究热点。靶向PAM信号通路抑制剂为晚期乳腺癌患者尤其是激素受体阳性人表皮生长因子受体2阴性患者带来明显临床获益。目前，美国食品和药物管理局批准上市的PAM信号通路抑制剂包括PI3K抑制剂Alpelisib和mTOR抑制剂依维莫司，同时，依维莫司也在国内获得批准用于乳腺癌新适应证，Alpelisib有望不久在中国上市。鉴于此，专家制定了PAM信号通路抑制剂治疗晚期乳腺癌的临床应用专家共识，系统地介绍了PAM信号通路的机制、PAM信号通路抑制剂的疗效、临床应用、不良事件管理及PIK3CA基因检测建议，旨在提高中国临床肿瘤医师对PAM信号通路抑制剂的认知，推进临床决策的精准性，达到延长患者生存时间的目标。.

Topics: Breast Neoplasms; Consensus; Everolimus; Female; Humans; MTOR Inhibitors; Phosphatidylinositol 3-Kinase; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Sirolimus; TOR Serine-Threonine Kinases

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Female; Humans; Mechanistic Target of Rapamycin Complex 1; Mechanistic Target of Rapamycin Complex 2; Proton Magnetic Resonance Spectroscopy; Protons; Sirolimus; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays

We investigated the activity and inhibition of mTOR and other metabolic pathways with their clinical significance in human breast tumors (using ten cell lines and nearly a hundred biopsy samples).Based on our results, the metabolic and mTOR inhibitor treatments showed a moderate tumor growth inhibitory effect in the cell lines subtype independently, which indicates tumor cell and tissue adaptation. Providing human tissue samples, we found a subtype independent correlation between high mTOR activity and protein expression characterizing alternative metabolic pathways with increased expression and the poor prognosis of breast tumors. Breast tumors are characterized by metabolic heterogeneity and significant metabolic plasticity, which can be targeted by combining anti-metabolic treatments and new therapies. Concerning these, an immunohistochemical evaluation (IHC panel) can be recommended, which is suitable for both metabolic plasticity evaluation and recognition of cases that may require stricter follow-up or metabolic targeted therapy due to the expected poor prognosis.

Topics: Breast Neoplasms; Cell Line, Tumor; Female; Humans; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Our study aimed to develop and find out the best drug candidate against the mechanistic target of rapamycin (mTOR/FRB) domain having a critical role in the aetiology of breast cancer. The FKBP12-rapamycin-binding (FRB) domain in the essential phosphoinositide 3 kinase/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway has been a vital player in the disease progression in breast cancer. By using structure-based drug designing , the best possible targets have been identified and developed. The three-dimensional structure of the target protein was generated using I-TASSER. The ligands were generated against the most suitable target active site using standard tools for active site identification. Furthermore, the seed molecule was drawn using Chemsketch, which was then grown into the pocket using Ligbuilder. The obtained ligands were further validated using online programs for bioavailability and toxicity, followed by molecular dynamic simulations. The study concludes that the equilibrated NVT-NPT complexes indicate LIG2 stability over LIG3. RMSD and RMSF have shown that the complex of LIG2 is more stable than LIG3. LIG2 has the potential antagonistic properties to target the mTOR/FRB domain and has therapeutic implications for breast cancer.

Topics: Breast Neoplasms; Female; Humans; Ligands; Molecular Dynamics Simulation; Phosphatidylinositol 3-Kinases; Sirolimus; TOR Serine-Threonine Kinases

Topics: Anti-Bacterial Agents; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Female; Humans; MCF-7 Cells; Quinolines; Receptor, Bradykinin B2

Breast cancer is the most common cancer in women and the leading cause of cancer mortality in women over 40 it's the year. The existence of the PI3K/AKT/mTOR pathway aberrations in more than 70% of breast cancer has caused to become a therapeutic target. AZD3463 is an anti-cancer agent used as a potential inhibitor of ALK/IGF1R. It also induces apoptosis and autophagy of the PI3K/AKT/mTOR pathway in cancer cells. Although the mTOR signaling might be inhibited by rapamycin treatment, signals transmitted from the upstream pathway supports cell survival and proliferation. The WST-1 assay test was performed to evaluate the anti-proliferative effects of rapamycin and AZD3463. Besides, the effects of them on apoptosis, autophagy, cytostatic, and metabolism in MCF7 breast cancer cells were investigated. Also, changes in the expression of apoptotic regulatory genes, cell cycle, and metabolism in the PI3K/AKT/mTOR Pathway were determined by Quantitative RT-PCR. The results showed that rapamycin and AZD3463 treatments significantly reduced survival in MCF7 cells. Also, apoptosis, autophagy, and cell population in the G0/G1 stage in the MCF7 cell category in the treatment group showed an increase compared to the control group. The combination of rapamycin and AZD3463 (AZD-RAPA) was determined as an additive according to isobologram analysis. In the combination of rapamycin with AZD3463, the expression of CDKN1B, PTEN, FOXO3, and APC genes increases, and the expression of PRKCB and PIK3CG genes decreases. Our results showed that the use of AZD-RAPA reduced the resistance of cancer cells to treatment and it leads cancer cells to apoptosis.

Topics: Apoptosis; Autophagosomes; Autophagy; Breast Neoplasms; Cell Cycle; Cell Survival; Drug Resistance, Neoplasm; Female; Gene Expression Regulation, Neoplastic; Glycolysis; Humans; Inhibitory Concentration 50; MCF-7 Cells; Oxygen Consumption; Phenotype; Sirolimus

The combination of everolimus (EVE) and exemestane (EXE) is approved for the treatment of patients with metastatic hormone receptor-positive breast cancer (mHRBC) who progress on nonsteroidal aromatase inhibitor (NSAI) therapy. However, none of the patients enrolled in the trial that led to this approval (BOLERO-2) had previously received CDK4/6 inhibitors (CDK4/6is), which have since become a frontline standard of care for mHRBC. As such, the clinical benefit of EVE plus EXE in patients who have previously received CDK4/6is remains unknown.. Adult patients with mHRBC at our institution who progressed on an NSAI plus CDK4/6i or NSAI therapy alone and were treated with at least one cycle of EVE plus EXE between 2012 and 2018 were analyzed. Collected data included patient demographics, treatment history, adverse events, and clinical outcomes. Primary objectives were to compare progression-free survival (PFS) and overall survival (OS) between patients who received prior NSAI plus CDK4/6i therapy versus an NSAI alone.. Among 43 patients, 17 had prior CDK4/6i exposure. With the exception of de novo metastatic disease, patient and disease characteristics were comparable across treatment cohorts. There was no significant difference in PFS (median, 3.6 vs. 4.2 months) or OS (median, 15.6 vs. 11.3 months) between patients who had received prior CDK4/6is and those who had not, respectively.. Prior exposure to CDK4/6i therapy did not impact survival outcomes for patients with mHRBC taking EVE plus EXE. However, there was a trend toward improved OS in the CDK4/6i cohort that should be evaluated in larger cohorts.. The use of CDK4/6 inhibitors in combination with a nonsteroidal aromatase inhibitor has become a standard frontline therapy in metastatic hormone receptor-positive breast cancer. An approved subsequent line of therapy is everolimus plus exemestane; however, the original data supporting this therapy predated approval of CDK4/6 inhibitors. As such, the clinical benefit of everolimus and exemestane in patients previously treated with a CDK4/6 inhibitor was unknown. This retrospective cohort study offers real-world data demonstrating prior CDK4/6 inhibitor exposure does not impact survival outcomes for everolimus plus exemestane.

Topics: Adult; Androstadienes; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Cyclin-Dependent Kinase 4; Everolimus; Female; Hormones; Humans; Receptor, ErbB-2; Retrospective Studies; Sirolimus

In tumors, nutrient availability and metabolism are known to be important modulators of growth signaling. However, it remains elusive whether cancer cells that are growing out in the metastatic niche rely on the same nutrients and metabolic pathways to activate growth signaling as cancer cells within the primary tumor. We discovered that breast-cancer-derived lung metastases, but not the corresponding primary breast tumors, use the serine biosynthesis pathway to support mTORC1 growth signaling. Mechanistically, pyruvate uptake through Mct2 supported mTORC1 signaling by fueling serine biosynthesis-derived α-ketoglutarate production in breast-cancer-derived lung metastases. Consequently, expression of the serine biosynthesis enzyme PHGDH was required for sensitivity to the mTORC1 inhibitor rapamycin in breast-cancer-derived lung tumors, but not in primary breast tumors. In summary, we provide in vivo evidence that the metabolic and nutrient requirements to activate growth signaling differ between the lung metastatic niche and the primary breast cancer site.

Topics: Animals; Antibiotics, Antineoplastic; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Drug Resistance, Neoplasm; Female; Gene Expression Regulation, Neoplastic; Humans; Ketoglutaric Acids; Lung Neoplasms; Mammary Neoplasms, Experimental; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Inbred BALB C; Mice, Nude; Monocarboxylic Acid Transporters; Phosphoglycerate Dehydrogenase; Pyruvic Acid; RNA, Small Interfering; Serine; Signal Transduction; Sirolimus

The new therapeutic solutions for breast cancer treatment are needed, for example, combined therapy consisted of several drugs that characterize different mechanisms of action and modern drug delivery systems. Therefore, we used combination of epothilone B (EpoB) and rapamycin (Rap) to analyze the cytotoxic effect against breast cancer cells (MCF-7; MDA-MB-231). Also, the effect of drugs co-delivered in bioresorbable micelles functionalized with biotin (PLA-PEG-BIO; poly(lactide)-co-poly(ethylene glycol)-biotin) was studied. The comparison of effects of the mixture of free drugs and the micelles co-loaded with EpoB and Rap revealed a significant decrease in the cell metabolic activity and survival. Moreover, the dual drug-loaded PLA-PEG-BIO micelles enhanced the cytotoxicity of EpoB and Rap against the tested cells as compared with the free drugs. The blank PLA-PEG-BIO micelles did not affect the tested cells. We expect that mixture of EpoB and Rap may be promising in breast cancer treatment and PLA-PEG-BIO micelles as carrier of these two drugs can be applicable for successful targeted delivery.

Topics: Biocompatible Materials; Breast Neoplasms; Cell Death; Cell Line, Tumor; Cell Survival; Drug Carriers; Drug Delivery Systems; Epothilones; Female; Humans; Micelles; Nanoparticles; Proton Magnetic Resonance Spectroscopy; Sirolimus

This study was conducted to collect clinical safety, tolerability, and efficacy data with the use of everolimus (EVE) combined with exemestane (EXE) in patients with advanced breast cancer (ABC).. The EVEREXES trial initiated in 2012, provided early access to the first dual blockade treatment with EVE + EXE in patients with HR+, HER2 - ABC in Asia and other emerging growth countries. Postmenopausal women with HR+, HER2 - ABC who had documented recurrence or progression, following a nonsteroidal aromatase inhibitor therapy, were treated with EVE (10 mg/day) + EXE (25 mg/day) orally.. A total of 235 patients received ≥ 1 dose of study medication. At the end of the study, all patients ceased the treatment. Disease progression (66.0%) was the primary reason of discontinuation. The most common AEs (≥ 20%) were stomatitis, decreased appetite, hyperglycemia, rash, aspartate aminotransferase increased, anemia, alanine aminotransferase increased, cough, and fatigue. No new safety concerns were identified in the current study. Median progression-free survival (PFS) in the Asian subset was similar to that of the overall population (9.3 months in both groups). Confirmed overall response rate (ORR) was achieved for 19.6% of the patients. Efficacy of EVE + EXE across subgroups (prior CT, line of treatment, and presence of visceral metastases) was maintained.. The safety and efficacy results from EVEREXES trial are consistent to data previously reported in BOLERO-2. These results support that EVE + EXE could be a viable treatment option for the postmenopausal women with HR+, HER2 - ABC in Asian region.

Topics: Androstadienes; Antineoplastic Combined Chemotherapy Protocols; Asia; Breast Neoplasms; Everolimus; Female; Humans; Neoplasm Recurrence, Local; Postmenopause; Receptor, ErbB-2; Sirolimus

Design and examine the effect of sirolimus-PEGylated (Stealth) liposomes for breast cancer treatment. In this study, we developed conventional and Stealth liposome nanoparticles comprising of distearoylphosphatidylcholine (DSPC) or dipalmitoyl-phosphatidylcholine (DPPC) and DSPE-MPEG-2000 lipids loaded with sirolimus as an anticancer agent. The effect of lipid grade, drug loading and incubation times were evaluated.. Particle size distribution, encapsulation efficiency of conventional and Stealth liposomes were studied followed by cytotoxicity evaluation. The cellular uptake and internal localisation of liposome formulations were investigated using confocal microscopy.. The designed Stealth liposome formulations loaded with sirolimus demonstrated an effective in vitro anticancer therapy compared with conventional liposomes while the length of the acyl chain affected the cell viability. Anticancer activity was found to be related on the drug loading amounts and incubation times. Cell internalization was observed after 5 h while significant cellular uptake of liposome was detected after 24 h with liposome particles been located in the cytoplasm round the cell nucleus. Sirolimus Stealth liposomes induced cell apoptosis.. The design and evaluation of sirolimus-loaded PEGylated liposome nanoparticles demonstrated their capacity as drug delivery carrier for the treatment of breast cancer tumours.

Topics: 1,2-Dipalmitoylphosphatidylcholine; 3T3 Cells; Animals; Antibiotics, Antineoplastic; Breast Neoplasms; Cell Line, Tumor; Cell Survival; Drug Delivery Systems; Female; Humans; Liposomes; Mice; Nanoparticles; Phosphatidylcholines; Phosphatidylethanolamines; Polyethylene Glycols; Sirolimus; Time Factors

Topics: Administration, Cutaneous; Aged; Antibiotics, Antineoplastic; Breast Neoplasms; Endometrial Neoplasms; Female; Humans; Lymphangioma; Middle Aged; Radiotherapy; Sirolimus; Skin Neoplasms; Thoracic Wall; Vulvar Neoplasms

Cooperative photothermal therapy (PTT) and photodynamic therapy (PDT) represents a promising strategy to conquer tumor with synergistic effect, while their long-term efficacy has been strictly limited by the multiple resistances of tumor. Here, we reported a core-shell nanoplatform for enhanced PTT/PDT combination against metastatic breast cancer. The nanosystem had photosensitizer chlorin e6 (Ce6) and rapamycin (RAP) pure drugs core and the polydopamine (PDA) shell, with surface PEGylation. Notably, we found that RAP was a highly robust sensitizer to boost the efficacy of both PTT and PDT by inhibiting the expression of heat shock protein 70 (HSP 70) and hypoxia inducible factor-1α (HIF-1α), respectively, resulting in cooperatively enhanced antitumor efficiency. Moreover, metastasis, the fatal risk of breast cancer, was also inhibited by virtue of RAP-mediated matrix metalloproteinases-2 (MMP-2) suppression. Upon intravenous injection, the nanosystem could passively accumulate into the tumor and impose potent phototherapies upon dual laser irradiations for complete tumor elimination and metastasis inhibition, giving rise to 100% mice survival over a long observation period. Collectively, this work offers a general solution to address the key limitations of tumor-resistant phototherapies and provides a highly promising nanoplatform for the management of metastatic cancer.

Topics: Animals; Antibiotics, Antineoplastic; Apoptosis; Breast Neoplasms; Cell Movement; Cell Proliferation; Female; Humans; Lung Neoplasms; Mice; Nanoparticles; Photosensitizing Agents; Phototherapy; Sirolimus; Tumor Cells, Cultured; Wound Healing; Xenograft Model Antitumor Assays

Bioluminescence resonance energy transfer (BRET) is a commonly used assay system for studying protein-protein interactions. The present protocol introduces a conceptually unique ligand-activatable BRET system (termed BRET9), where a full-length artificial luciferase variant 23 (ALuc23), acting as the energy donor, is sandwiched in between a protein pair of interest, FRB and FKBP, and further linked to a fluorescent protein as the energy acceptor for studying protein-protein interaction. A specific ligand, rapamycin, which initiates intramolecular interactions of FRB and FKBP inside the probe, which develops molecular strain in the sandwiched ALuc23 to complete its folding, thus, the probe system greatly enhances both the overall bioluminescence (BL) spectrum and the BRET signal in the far-red (FR) region. This new BRET system provides a robust ligand-activatable platform that efficiently reports FR-BL signals in mammalian cells.

Topics: Apoptosis; Breast Neoplasms; Cell Proliferation; Female; Fluorescence Resonance Energy Transfer; Humans; Immunosuppressive Agents; Luciferases; Luminescent Agents; Luminescent Measurements; Optical Imaging; Protein Interaction Mapping; Sirolimus; Tacrolimus Binding Proteins; TOR Serine-Threonine Kinases; Tumor Cells, Cultured

Topics: Antineoplastic Combined Chemotherapy Protocols; Autophagy; Breast Neoplasms; Cell Proliferation; Doxycycline; Female; HT29 Cells; Humans; MCF-7 Cells; Mitochondria; Neoplasm Proteins; Sirolimus

The clinical utility of rapamycin (Rapa) is limited by solubility, bioavailability, and side effects. To overcome this, our team recently reported an elastin-like polypeptide (ELP) nanoparticle with high affinity, noncovalent drug binding, and integrin-mediated cellular uptake. Given the scarcity of pharmacology/toxicology studies of ELP-based drug carriers, this article explores safety and efficacy of ELP-Rapa. ELP-Rapa nanoparticles tested negative for hemolysis, did not interfere in plasma coagulation nor in platelet function, and did not activate the complement. Upon incubation with HepG2 cells, ELP-Rapa revealed significant cellular uptake and trafficking to acidic organelles, consistent with lysosomes. Internalized ELP-Rapa nanoparticles increased oxidative stress 4-fold compared to free drug or free ELP controls. However, mice bearing orthotopic hormone receptor positive BT-474 breast tumors, given a high dose (∼10-fold above therapeutic dose) of 1 month administration of ELP-Rapa, did not induce hepatotoxicity. On the other hand, tumor growth and mTOR signaling were suppressed without affecting body weight. Nanoparticles assembled using ELP technology appear to be a safe and efficient strategy for delivering Rapa.

Topics: Animals; Breast Neoplasms; Drug Carriers; Elastin; Female; Humans; Mice; Peptides; Sirolimus

To investigate if ICI 182,780 (fulvestrant), a selective estrogen receptor alpha/beta (ERα/ERβ) antagonist, and G-1, a selective G-protein-coupled receptor (GPER) agonist, can potentially induce autophagy in breast cancer cell lines MCF-7 and SKBr3, and how G-1 affects cell viability.. Cell viability in MCF-7 and SKBr3 cells was assessed by the MTT assay. To investigate the autophagy flux, MCF-7 cells were transfected with GFP-LC3, a marker of autophagosomes, and analyzed by real-time fluorescence microscopy. MCF-7 and SKBr3 cells were incubated with acridine orange for staining of acidic vesicular organelles and analyzed by flow cytometry as an indicator of autophagy.. Regarding cell viability in MCF-7 cells, ICI 182,780 and rapamycin, after 48 hours, led to decreased cell proliferation whereas G-1 did not change viability over the same period. The data showed that neither ICI 182,780 nor G-1 led to increased GFP-LC3 puncta in MCF-7 cells over the 4-hour observation period. The cytometry assay showed that ICI 182,780 led to a higher number of acidic vesicular organelles in MCF-7 cells. G-1, in turn, did not have this effect in any of the cell lines. In contrast, ICI 182,780 and G-1 did not decrease cell viability of SKBr3 cells or induce formation of acidic vesicular organelles, which corresponds to the final step of the autophagy process in this cell line.. The effect of ICI 182,780 on increasing acidic vesicular organelles in estrogen receptor-positive breast cancer cells appears to be associated with its inhibitory effect on estrogen receptors, and GPER does notseem to be involved. Understanding these mechanisms may guide further investigations of these receptors' involvement in cellular processes of breast cancer resistance.

Topics: Analysis of Variance; Autophagy; Blotting, Western; Breast Neoplasms; Cell Proliferation; Cell Survival; Estrogen Receptor alpha; Estrogen Receptor Antagonists; Estrogen Receptor beta; Female; Flow Cytometry; Fulvestrant; Humans; MCF-7 Cells; Receptors, G-Protein-Coupled; Reproducibility of Results; Sirolimus; Time Factors; Transfection

We performed a retrospective study on the efficacy and safety of sirolimus (an mTOR inhibitor) in hormone receptor (HR)-positive advanced breast cancer and searched for biomarkers to predict its efficacy.. All patients with HR-positive metastatic breast cancer treated with sirolimus plus endocrine therapy between December 2017 and July 2018 at the Cancer Hospital, Chinese Academy of Medical Sciences were consecutively and retrospectively reviewed. Mutations in circulating tumour DNA (ctDNA) were assayed for 1021 tumour-related genes via gene panel target capture-based next-generation sequencing.. Thirty-six patients with metastatic breast cancer treated with sirolimus plus endocrine therapy were included. The progression-free survival (PFS) rates between the sirolimus group and everolimus group were similar, and the median PFS was 4.9 months and 5.5 months, respectively (hazard ratio 1.56, 95% CI 0.86-2.81, P = 0.142). The objective response rate in the 36 patients was 19.4%, and the clinical benefit rate was 41.7%. Lipid metabolism disorder was the most common adverse event (69.4%), and 13.9% of patients had stomatitis. Most (94.4%) adverse events were grade 1-2. Twenty patients (55.6%) underwent ctDNA analysis before receiving sirolimus therapy. For patients who received less than 3 lines of chemotherapy, those with PI3K/Akt/mTOR pathway alterations had a better response to sirolimus than those without alterations, with a median PFS of 7.0 months vs 4.3 months (hazard ratio = 0.01, 95% CI 0.00-0.34, P = 0.010).. Sirolimus is a potentially effective treatment option for patients with HR-positive advanced breast cancer.

Topics: Adult; Antineoplastic Combined Chemotherapy Protocols; Aromatase Inhibitors; Biomarkers, Tumor; Breast Neoplasms; China; Circulating Tumor DNA; Everolimus; Female; Fulvestrant; Humans; Middle Aged; Progression-Free Survival; Retrospective Studies; Signal Transduction; Sirolimus; Tamoxifen; TOR Serine-Threonine Kinases; Toremifene

linc-ROR is reported to be a potential biomarker of breast cancer, but the detailed mechanism of linc-ROR-mediated breast cancer regulation has not been fully studied. We aimed to explore how linc-ROR affects proliferation, metastasis, and drug sensitivity in breast cancer. Cell lines in which linc-ROR was overexpressed or knocked down were constructed, and the cell proliferation, colony formation, cell migration, and invasion abilities of these lines were explored. A CCK-8 assay was performed to determine the sensitivity of the breast cancer cells to rapamycin. Next-generation sequencing was conducted to explore the detailed regulatory mechanism of linc-ROR; differentially expressed RNAs in the linc-ROR-overexpressing cell line compared with the negative control were screened out, and their target genes were chosen to perform Gene Ontology analysis, Kyoto Encyclopedia of Genes and Genomes analysis, protein-protein interaction network analysis, and competing endogenous RNA (ceRNA) network analysis. The ceRNA mechanism of linc-ROR for miR-194-3p, which targets MECP2, was determined through dual-luciferase reporter assay, RT-qPCR, western blot, and rescue experiments. Finally, we found that linc-ROR was upregulated in breast tumor tissues. linc-ROR promoted the cell proliferation, colony formation, cell migration, and invasion of breast cancer and decreased the sensitivity of breast cancer cells to rapamycin. The overexpression of linc-ROR triggered changes in the whole transcriptome of breast cancer cells, and a total of 85 lncRNAs, 414 microRNAs, 490 mRNAs, and 92 circRNAs were differentially expressed in the linc-ROR-overexpressing cell line compared with the negative control. Through a series of bioinformatic analyses, the 'linc-ROR/miR-194-3p/MECP2' ceRNA regulatory axis was confirmed to be involved in the linc-ROR-mediated progression and drug sensitivity of breast cancer. In conclusion, linc-ROR serves as an onco-lncRNA in breast cancer and promotes the survival of breast cancer cells during rapamycin treatment by functioning as a ceRNA sponge for miR-194-3p, which targets MECP2.

Topics: Adult; Aged; Base Sequence; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Disease Progression; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Gene Ontology; Gene Regulatory Networks; Humans; Methyl-CpG-Binding Protein 2; MicroRNAs; Middle Aged; Neoplasm Invasiveness; Reproducibility of Results; RNA, Long Noncoding; Sirolimus; TOR Serine-Threonine Kinases; Up-Regulation

The aim of this study was to analyze the effects of rapamycin treatment on apoptosis via mTOR pathway in metastatic and non-metastatic human breast cancer cell lines by immunohistochemical and TUNEL analysis.. MCF-7 and MDA-MB 231 cell lines were incubated under standard conditions forming Rapamycin and control groups. In immunohistochemical evaluation; mTOR pathway was evaluated with anti-IGF1, anti-PI3K, anti-pAKT1/2/3, anti-mTORC1, anti-mTORC2 and anti-ERK1 antibodies. The effect of apoptosis was also confirmed by TUNEL method.. In this study, activation of PI3K/AKT/mTOR and related molecular pathways in the MDA-MB 231 and MCF-7 breast cancer cell line was evaluated and it was observed that these pathways could play a key role in cancer development. Increased apoptotic cells were observed in mTORC1 inhibition by Rapamycin administration.. Targeting the mTOR pathway in breast cancer treatment may be a treatment option. In addition, the demonstration and confirmation of increased apoptosis in Rapamycin treated groups suggested that Rapamycin, an inhibitor of mTOR, is promising in the treatment of breast cancer (Tab. 2, Fig. 3, Ref. 66).

Topics: Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Humans; MCF-7 Cells; Neoplasm Metastasis; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Sirolimus; TOR Serine-Threonine Kinases

Mammary and extramammary Paget's Diseases (PD) are a malignant skin cancer characterized by the appearance of Paget cells. Although easily diagnosed, its pathogenesis remains unknown. Here, single-cell RNA-sequencing identified distinct cellular states, novel biomarkers, and signaling pathways - including mTOR, associated with extramammary PD. Interestingly, we identified MSI1 ectopic overexpression in basal epithelial cells of human PD skin, and show that Msi1 overexpression in the epidermal basal layer of mice phenocopies human PD at histopathological, single-cell and molecular levels. Using this mouse model, we identified novel biomarkers of Paget-like cells that translated to human Paget cells. Furthermore, single-cell trajectory, RNA velocity and lineage-tracing analyses revealed a putative keratinocyte-to-Paget-like cell conversion, supporting the in situ transformation theory of disease pathogenesis. Mechanistically, the Msi1-mTOR pathway drives keratinocyte-Paget-like cell conversion, and suppression of mTOR signaling with Rapamycin significantly rescued the Paget-like phenotype in Msi1-overexpressing transgenic mice. Topical Rapamycin treatment improved extramammary PD-associated symptoms in humans, suggesting mTOR inhibition as a novel therapeutic treatment in PD.

Topics: Adult; Aged; Animals; Antibiotics, Antineoplastic; Biomarkers; Breast Neoplasms; Female; Humans; Male; Mice; Middle Aged; Nerve Tissue Proteins; Paget Disease, Extramammary; RNA-Binding Proteins; Sirolimus; TOR Serine-Threonine Kinases

There is limited literature on patterns of everolimus use and subsequent hospitalizations and emergency room (ER) visits in real-world clinical practice. In this study, we describe patterns of everolimus use and hospitalizations and ER visits in a large cohort of patients with breast cancer (BC).. Patients with BC treated with everolimus were identified in the MarketScan database from 2009 to 2016. The pattern of everolimus use and frequency of associated ER visits and hospitalizations during treatment (between the first claim and 30 days after the last claim for everolimus) were identified. Descriptive statistics and regression models were used.. A total of 3,556 everolimus users were identified (median age of 60 years; median days of use, 112). The initial prescribed dose was 10 mg in 74.8% of the patients. Compared with the initial dose, 23.5% of patients had a dose change. Forty-six percent of patients were hospitalized or had an ER visit during the treatment with everolimus. Age greater than 71, higher comorbidity score, treatment year prior to 2012, and lower initial dose were found to be significantly associated with ER visit/hospitalization in the regression models.. A significant proportion of patients receiving everolimus had an ER visit or hospitalization during the use of everolimus. These results provide data regarding risks and benefits of treatment with everolimus. These results will be helpful in identifying patients at higher risk of hospitalizations or ER visits and facilitate evidence-based decision making to avoid serious complications.. Everolimus, a mammalian target of rapamycin inhibitor, is approved in combination with exemestane in patients with hormone receptor-positive tumors previously treated with anastrozole or letrozole. As new drugs become available, it is crucial to understand the adverse events and potential complications associated with the use of such drugs in the general population, outside of the controlled clinical trial setting. This study describes the patterns of everolimus use and adverse events, including hospitalization and emergency room visits, in a large cohort of patients with metastatic breast cancer in routine practice.

Topics: Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Everolimus; Female; Humans; Letrozole; Middle Aged; Sirolimus

Rapalogues are a unique class of drugs with both cytostatic and immunosuppressive properties. Two founding members, rapamycin (Rapa) and its chemical derivative everolimus (Eve), are extremely potent, but their clinical use presents multiple challenges. Being water-insoluble, administration is restricted to the oral route, which results in a low bioavailability of <10%. Human studies of rapalogues are reported to yield a high blood to plasma ratio and poor correlation between blood concentration and dose. Moreover, treatment results in dose-limiting toxicities such as stomatitis and pneumonitis, which often leads to discontinuation of therapy. We previously reported an elastin-like polypeptide decorated with two-headed FKBP rapalogue-binding domains. Called "FAF", this biomacromolecular drug-carrier solubilizes, retargets, and releases rapalogues within disease sites. FAF-rapalogue formulations are free of cosolvents or surfactants, which promotes their parenteral administration. In this study, subcutaneously given FAF-Rapa significantly suppressed tumor growth in a mouse model of hormone receptor positive (HR+) breast cancer, compared to an oral formulation of Eve (Affinitor). Additionally, mTOR, the pharmacological target of rapalogues, was inhibited to a greater extent in tumors of FAF-Rapa and FAF-Eve groups compared to mice that received oral Eve. No signaling suppression was detected in the liver and spleen, which were evaluated to represent off-target organs exposed to the circulating formulation.

Topics: Animals; Breast Neoplasms; Everolimus; Female; Humans; Mechanistic Target of Rapamycin Complex 1; Mice; Peptides; Sirolimus

AKT-mTORC1 (mammalian target of rapamycin complex 1) signaling pathway plays a critical role in tumorigenesis and can be targeted by rapamycin. However, the underlying mechanism of how long noncoding RNA (lncRNAs) regulate the AKT-mTORC1 pathway remains unclear. EPIC1 (epigenetically-induced lncRNA 1) is a Myc-binding lncRNA, which has been previously demonstrated to be overexpressed in multiple cancer types. In a pathway analysis including 4962 cancer patients, we observed that lncRNA EPIC1 expression was positively correlated with the AKT-mTORC1 signaling pathway in more than 10 cancer types, including breast and ovarian cancers. RNA-seq analysis of breast and ovarian cancer cells demonstrated that EPIC1-knockdown led to the downregulation of genes in the AKT-mTORC1 signaling pathway. In MCF-7, OVCAR4, and A2780cis cell lines, EPIC1 knockdown and overexpression, respectively, inhibited and activated phosphorylated AKT and the downstream phosphorylation levels of 4EBP1 and S6K. Further knockdown of Myc abolished the EPIC1's regulation of AKT-mTORC1 signaling; suggested that the regulation of phosphorylation level of AKT, 4EBP1, and S6K by EPIC1 depended on the expression of Myc. Moreover, EPIC1 overexpressed MCF-7, A2780cis, and OVCAR4 cells treated with rapamycin showed a significant decreasing in rapamycin mediated inhibition of p-S6K and p-S6 comparing with the control group. In addition, Colony Formation assay and MTT assay indicated that EPIC1 overexpression led to rapamycin resistance in breast and ovarian cancer cell lines. Our results demonstrated the lncRNA EPIC1 expression activated the AKT-mTORC1 signaling pathway through Myc and led to rapamycin resistance in breast and ovarian cancer.

Topics: Antibiotics, Antineoplastic; Apoptosis; Biomarkers, Tumor; Breast Neoplasms; Cell Proliferation; Drug Resistance, Neoplasm; Female; Gene Expression Regulation, Neoplastic; Humans; Mechanistic Target of Rapamycin Complex 1; Ovarian Neoplasms; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-myc; RNA, Long Noncoding; Sirolimus; Tumor Cells, Cultured

mTOR inhibitors are considered today to be one of the most promising anticancer drugs. Here to study the mechanism of the acquired resistance of MCF-7 breast cancer cells to mTOR inhibitors two different models of the cell resistance were used: rapamycin-resistant MCF-7/Rap subline developed under long-term rapamycin treatment, and metformin-resistant MCF-7/M subline obtained by long-term metformin treatment. We have found that both resistant sublines were characterized by common features: increased expression of mTOR-interacting Raptor protein, increased phosphorylation of Akt, and activation of growth-related transcriptional factor AP-1. Cell response to mTOR inhibitors was partially restored under treatment with PI3K inhibitor wortmannin supporting the direct connection between Akt activation and poor cell response to therapeutic drugs. Transfection of mir-181c, one of the positive regulators of Akt and mTOR, led to an increase in the cell resistance to both mTOR inhibitors, rapamycin and metformin, which correlated with Raptor overexpression and activation of Akt/AP-1 signaling. In general, the effect of Raptor overexpression in the resistant cells, as well as the ability of mir-181c to modulate the Raptor expression, can open novel perspectives in the treatment of rapalogues-resistant cancers, based on the drugs design targeting mir-181c/Raptor axis.

Topics: Antibiotics, Antineoplastic; Breast Neoplasms; Cell Line, Tumor; Drug Resistance, Neoplasm; Humans; MCF-7 Cells; MicroRNAs; Proto-Oncogene Proteins c-akt; Regulatory-Associated Protein of mTOR; Signal Transduction; Sirolimus; Up-Regulation

The effects of diet in cancer, in general, and breast cancer in particular, are not well understood. Insulin inhibition in ketogenic, high fat diets, modulate downstream signaling molecules and are postulated to have therapeutic benefits. Obesity and diabetes have been associated with higher incidence of breast cancer. Addition of anti-cancer drugs together with diet is also not well studied.. Two diets, one ketogenic, the other standard mouse chow, were tested in a spontaneous breast cancer model in 34 mice. Subgroups of 3-9 mice were assigned, in which the diet were implemented either with or without added rapamycin, an mTOR inhibitor and potential anti-cancer drug.. Blood glucose and insulin concentrations in mice ingesting the ketogenic diet (KD) were significantly lower, whereas beta hydroxybutyrate (BHB) levels were significantly higher, respectively, than in mice on the standard diet (SD). Growth of primary breast tumors and lung metastases were inhibited, and lifespans were longer in the KD mice compared to mice on the SD (p<0.005). Rapamycin improved survival in both mouse diet groups, but when combined with the KD was more effective than when combined with the SD.. The study provides proof of principle that a ketogenic diet a) results in serum insulin reduction and ketosis in a spontaneous breast cancer mouse model; b) can serve as a therapeutic anti-cancer agent; and c) can enhance the effects of rapamycin, an anti-cancer drug, permitting dose reduction for comparable effect. Further, the ketogenic diet in this model produces superior cancer control than standard mouse chow whether with or without added rapamycin.

Topics: 3-Hydroxybutyric Acid; Animals; Antineoplastic Agents; Blood Glucose; Breast Neoplasms; Diet, Ketogenic; Disease Models, Animal; Female; Insulin; Ketosis; Mice; Sirolimus

Marine ecosystems are the most prevalent ecosystems on the planet, providing a diversity of living organisms and resources. The development of nanotechnology may provide solutions for utilizing these thousands of potential compounds as marine pharmaceuticals. Here, we designed a liposomal glycol chitosan formulation to load both doxorubicin (DOX) and rapamycin (RAPA), and then evaluated its therapeutic potential in a prepared drug-resistant cell model. We explored the stability of the drug delivery system by changing the physiological conditions and characterized its physicochemical properties. The electrostatic complexation between DOX-glycol chitosan and docosahexaenoic acid RAPA-liposomes (GC-DOX/RAPA ω-liposomes) was precisely regulated, resulting in particle size of 131.3 nm and zeta potential of -14.5 mV. The well-characterized structure of GC-DOX/RAPA ω-liposomes led to high loading efficiencies of 4.1% for DOX and 6.2% for RAPA. Also, GC-DOX/RAPA ω-liposomes exhibited high colloidal stability under physiological conditions and synergistic anti-cancer effects on DOX-resistant MDA-MB-231 cells, while showing pH-sensitive drug release behavior. Our results provided a viable example of marine pharmaceuticals with therapeutic potential for treating drug-resistant tumors using an efficient and safe drug delivery system.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Chitosan; Docosahexaenoic Acids; Doxorubicin; Drug Delivery Systems; Drug Liberation; Drug Resistance, Neoplasm; Ecosystem; Female; Humans; Liposomes; Nanoparticles; Particle Size; Sirolimus

A total of 70% of breast cancers express the estrogen receptor (ER)α; therefore, targeting the ER may be an effective endocrine therapy with which to inhibit breast cancer growth. Tamoxifen is the most common‑used clinically used drug for the treatment of advanced or metastatic ER‑positive (ER+) breast cancer. However, a substantial proportion of patients become resistant to endocrine therapies. To overcome this limitation, in this stud, we sought to maximize the benefits associated with tamoxifen therapy via drug combination strategies. We demonstrated that rapamycin, an FDA‑approved mammalian target of rapamycin (mTOR) inhibitor, enhanced the effects of endocrine therapy with tamoxifen, and the concentration of tamoxifen required for ER+ breast cancer cell growth inhibition was substantially reduced. Moreover, treatment with rapamycin plus tamoxifen significantly inhibited tumor growth in vivo. In addition, this synergistic effect may be mediated by the induction of p73. We revealed a novel mechanism in which p73 increases ERα expression by directly binding to the promoter region of the ERα gene. Taken together, the findings of this study indicate that combination therapy with rapamycin and tamoxifen underlying p73‑mediated ERα expression may provide new insight into the drug combination for the treatment of ER+ breast cancer.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Cell Line, Tumor; Drug Resistance, Neoplasm; Drug Synergism; Estrogen Receptor alpha; Female; Gene Expression Regulation, Neoplastic; Humans; MCF-7 Cells; Mice; Mice, Nude; RNA, Small Interfering; Sirolimus; Tamoxifen; Tumor Protein p73; Up-Regulation; Xenograft Model Antitumor Assays

Topics: Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Everolimus; Humans; Immunohistochemistry; Immunoprecipitation; MCF-7 Cells; Mechanistic Target of Rapamycin Complex 1; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Sirolimus

In an attempt to develop effective and safe anticancer agents, we designed, synthesized and examined 23 novel quinacrine (QC) derivatives by combining the 9-aminoacridine scaffold and the [1,3]thiazinan-4-ones group. Most of these hybrids showed strong anticancer activities, among which 3-(3-(6-chloro-2-methoxyacridin-9-ylamino)propyl)-2-(thiophen-2-yl)-1,3-thiazinan-4-one (25; VR151) effectively killed many different cancer cell types, including eight breast cancer cell lines with different genetic background, two prostate cancer and two lung cancer cell lines. In contrast, compound 25 is less effective against non-cancer cells, suggesting it may be less toxic to humans. Our data showed that cancer cells are arrested in S phase for a prolonged period due to the down-regulation of DNA replication, leading to eventual cell death. We have also shown that the S phase arrest may be resulted by the down-regulation of cyclin A coupled with the continued up-regulation of cyclin E, which coincide with the down-regulation of mTor-S6K and mTor-4EBP1 pathways.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Drug Design; Drug Screening Assays, Antitumor; Female; Humans; Molecular Structure; Quinacrine; Structure-Activity Relationship; Thiazines

Breast cancer development and progression are influenced by insulin-like growth factor receptor 1 (IGF1R) and insulin receptor (InsR) signaling, which drive cancer phenotypes such as cell growth, proliferation, and migration. IGF1R and InsR form IGF1R/InsR hybrid receptors (HybRs) consisting of one molecule of IGF1R and one molecule of InsR. The specific signaling and functions of HybR are largely unknown, as HybR is activated by both IGF1 and insulin, and no cellular system expresses HybR in the absence of holo-IGF1R or holo-InsR. Here we studied the role of HybR by constructing inducible chimeric receptors and compared HybR signaling with that of holo-IGF1R and holo-InsR. We cloned chemically inducible chimeric IGF1R and InsR constructs consisting of the extracellular domains of the p75 nerve growth factor receptor fused to the intracellular β subunit of IGF1R or InsR and a dimerization domain. Dimerization with the drugs AP20187 or AP21967 allowed specific and independent activation of holo-IGF1R, holo-InsR, or HybR, resulting in activation of the PI3K pathway. Holo-IGF1R and HybR both promoted cell proliferation and glucose uptake, whereas holo-InsR only promoted glucose uptake, and only holo-IGF1R showed anti-apoptotic effects. We also found that the three receptors differentially regulated gene expression: holo-IGF1R and HybR up-regulated EGR3; holo-InsR specifically down-regulated JUN and BCL2L1; holo-InsR down-regulated but HybR up-regulated HK2; and HybR specifically up-regulated FHL2, ITGA6, and PCK2. Our findings suggest that, when expressed and activated in mammary epithelial cells, HybR acts in a manner similar to IGF1R and support further investigation of the role of HybR in breast cancer.

Topics: Animals; Breast Neoplasms; Cell Line, Transformed; Cell Proliferation; Female; Humans; Indicators and Reagents; Insulin; Insulin-Like Growth Factor I; Mammary Glands, Human; MCF-7 Cells; Mice; Models, Molecular; Neoplasm Proteins; Peptide Fragments; Protein Interaction Domains and Motifs; Protein Multimerization; Receptor, Insulin; Receptors, Somatomedin; Recombinant Fusion Proteins; Signal Transduction; Sirolimus; Tacrolimus

Protein-based micelles have shown significant potential for tumor-targeted delivery of anti-cancer drugs. In this light, self-assembled nanocarriers based on GRAS (Generally recognized as safe) amphiphilic protein co-polymers were synthesized via carbodiimide coupling reaction. The new nano-platform is composed of the following key components: (i) hydrophobic zein core to encapsulate the hydrophobic drugs rapamycin (RAP) and wogonin (WOG) with high encapsulation efficiency, (ii) hydrophilic lactoferrin (Lf) corona to enhance the tumor targeting, and prolong systemic circulation of the nanocarriers, and (iii) glutaraldehyde (GLA)-crosslinking to reduce the particle size and improve micellar stability. Zein-Lf micelles showed relatively rapid release of WOG followed by slower diffusion of RAP from zein core. This sequential release may aid in efflux pump inhibition by WOG thus sensitizing tumor cells to RAP action. Interestingly, these micelles showed good hemocompatibility as well as enhanced serum stability owing to the brush-like architecture of Lf shell. Moreover, this combined nano-delivery system maximized synergistic cytotoxicity of RAP and WOG in terms of tumor inhibition in MCF-7 breast cancer cells and Ehrlich ascites tumor animal model as a result of enhanced active targeting. Collectively, GLA-crosslinked zein-Lf micelles hold great promise for combined RAP/WOG delivery to breast cancer with reduced drug dose, minimized side effects and maximized anti-tumor efficacy.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Carcinoma, Ehrlich Tumor; Cross-Linking Reagents; Drug Carriers; Female; Flavanones; Glutaral; Humans; Hydrophobic and Hydrophilic Interactions; Lactoferrin; MCF-7 Cells; Micelles; Nanoparticles; Scutellaria; Sirolimus; Zein

Metastatic estrogen receptor alpha positive (ERα+) cancers account for most breast cancer mortality. Cancer stem cells (CSCs) and dense/stiff extracellular matrices are implicated in aggression and therapy resistance. We examined this interplay and response to mTOR inhibition using ERα+ adenocarcinomas from NRL-PRL females in combination with Col1a1

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Collagen; Collagen Type I; Collagen Type I, alpha 1 Chain; Estrogen Receptor alpha; Female; Fulvestrant; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Mice; Neoplasm Transplantation; Neoplastic Stem Cells; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Tumor Burden; Tumor Microenvironment

Rapamycin (Rp), the main mammalian target of rapamycin complex inhibitor, is a promising therapeutic agent for breast cancer. However, metabolic disorders and drug resistance reduce its efficacy. Epidemiological, clinical, and experimental studies have demonstrated that omega-3 polyunsaturated fatty acids (ω-3 PUFAs) significantly reduce the incidence and mortality of breast cancer and improve metabolic disorders.. Three breast cancer cell lines and immunocompetent and immunodeficient mice were used to evaluate the therapeutic effects of Rp plus ω-3 PUFA treatment. The production of reactive oxygen species (ROS) and glucose uptake were examined by flow cytometry. Metabolic shift was examined by metabonomics, seahorse experiments, and western blot analysis.. We found that ω-3 PUFAs and Rp synergistically induced cell cycle arrest and apoptosis in vitro and in vivo, accompanied by autophagy blockage. In addition, Rp-induced hypertriglyceridemia and hypercholesterolemia were completely abolished by ω-3 PUFA supplementation. Moreover, the combined treatment of ω-3 PUFA and Rp significantly inhibited glycolysis and glutamine metabolism. The anti-tumor effects of this combination treatment were dependent on ROS production, which was increased by β-oxidation and oxidative phosphorylation.. Our study revealed that ω-3 PUFA enhanced the anti-tumor activity of Rp while minimizing its side effects in vitro and in vivo. These results provide novel insights into the mechanisms underlying the potential beneficial effects of Rp combined with ω-3 PUFAs on the prevention of breast cancer.

Topics: Adenosine Triphosphate; Animals; Apoptosis; Breast Neoplasms; Cell Cycle Checkpoints; Cell Line, Tumor; Disease Models, Animal; Fatty Acids, Omega-3; Female; Humans; Lactic Acid; Malondialdehyde; MCF-7 Cells; Metabolomics; Mice; Microtubule-Associated Proteins; Oxidative Phosphorylation; Reactive Oxygen Species; Sirolimus

Everolimus has been shown to overcome endocrine resistance in hormone receptor positive advanced breast cancer patients. Predictive biomarkers of everolimus efficacy have been investigated in primary breast cancer tissue without finding univocal results. The goal of this study was to investigate the mutational burden in the metastatic site of endocrine-resistant tumors treated with everolimus plus exemestane.. Mass Array Sequenom platform was used to analyse genetic status of 18 cancer-related genes in 25 archival tumor specimens from metastatic lesions and available primary matched breast cancer tissue of patients treated with everolimus and exemestane for advanced disease. An exploratory analysis of everolimus efficacy in terms of progression free survival benefit and single gene mutation was carried out.. The mutational status of breast cancer recurrence allows the identification of some genes potentially correlating tumor response/resistance to everolimus. The most frequently mutated genes were involved in the PI3K/AKT/mTOR pathway highlighting that the deregulation of this pathway in the relapse plays a crucial role in the mechanisms of everolimus resistance/sensitivity. Owing to the small sample size and the retrospective nature of the study, these correlations need to be validated in a large prospective study.

Topics: Aged; Androstadienes; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; DNA Mutational Analysis; Everolimus; Female; Genes, Neoplasm; Humans; Middle Aged; Neoplasm Recurrence, Local; Phosphatidylinositol 3-Kinases; Prospective Studies; Retrospective Studies; Sirolimus

Breast cancer is the most common cancer in women worldwide. Hormone receptor breast cancers are the most common ones and, about 2 out of every 3 cases of breast cancer are estrogen receptor (ER) positive. Selective ER modulators, such as tamoxifen, are the first line of endocrine treatment of breast cancer. Despite the expression of hormone receptors some patients develop tamoxifen resistance and 50% present de novo tamoxifen resistance. Recently, we have demonstrated that activated mammalian target of rapamycin (mTOR) is positively associated with overall survival and recurrence free survival in ER positive breast cancer patients who were later treated with tamoxifen. Since altered expression of protein kinase B (PKB)/Akt in breast cancer cells affect N-myristoyltransferase 1 (NMT1) expression and activity, we investigated whether mTOR, a downstream target of PKB/Akt, regulates NMT1 in ER positive breast cancer cells (MCF7 cells). We inhibited mTOR by treating MCF7 cells with rapamycin and observed that the expression of NMT1 increased with rapamycin treatment over the period of time with a concomitant decrease in mTOR phosphorylation. We further employed mathematical modelling to investigate hitherto not known relationship of mTOR with NMT1. We report here for the first time a collection of models and data validating regulation of NMT1 by mTOR.

Topics: Acyltransferases; Adenocarcinoma; Breast Neoplasms; Enzyme Induction; Estrogens; Female; Gene Expression Regulation, Neoplastic; Humans; MCF-7 Cells; Models, Biological; Neoplasm Proteins; Neoplasms, Hormone-Dependent; Phosphorylation; Protein Processing, Post-Translational; Proto-Oncogene Proteins c-akt; Receptors, Estrogen; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Direct reversion of cancers into normal-like tissues is an ideal strategy for cancer treatment. Recent reports have showed that defined transcription factors can induce reprogramming of cancer cells into pluripotent stem cells, supporting this notion. Here, we have developed a reprogramming method that uses a conceptually unique strategy for breast cancer cell treatment. We have screened a kinase inhibitor library and found that Rho-associated protein kinase (ROCK) and mammalian target of rapamycin (mTOR) kinase inhibitors can substitute for all transcription factors to be sufficient to reprogram breast cancer cells into progenitor cells. Furthermore, ROCK-mTOR inhibitors could reprogram breast cancer cells to another terminal lineage-adipogenic cells. Genome-wide transcriptional analysis shows that the induced fat-like cells have a profile different from breast cancer cells and similar to that of normal adipocytes. In vitro and in vivo tumorigenesis assays have shown that induced fat-like cells lose proliferation and tumorigenicity. Moreover, reprogramming treatment with ROCK-mTOR inhibitors prevents breast cancer local recurrence in mice. Currently, ROCK-mTOR inhibitors are already used as antitumor drugs in patients, thus, this reprogramming strategy has significant potential to move rapidly toward clinical trials for breast cancer treatment.

Topics: Adipocytes; Amides; Animals; Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cellular Reprogramming; Cellular Reprogramming Techniques; Female; Humans; Mice; Mice, Inbred NOD; Mice, SCID; Pluripotent Stem Cells; Pyridines; rho-Associated Kinases; Sirolimus; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays

The mTOR pathway and the enzyme telomerase are two key players commonly upregulated in cancers. They render survival and proliferative advantage to cancer cells, and are regarded as attractive anticancer targets. Rapamycin, a macrolide antibiotic and mTOR inhibitor, has recently also been implicated in telomerase inhibition and telomere attrition, although the mechanisms remain poorly understood. Using breast cancer cells (MCF-7 and MDA-MB-231) wherein telomerase activity and mTOR pathway are concurrently overexpressed, this study sought to unravel novel mechanisms by which rapamycin may affect these pathways. Short term treatment with an acute dose of rapamycin inhibited the mTOR pathway and telomerase activity and induced G1 arrest. This arrest was independent of cyclin D1 and p21 levels and was not mediated by DNA damage in both cell types. While long term treatment with a clinically relevant dose of rapamycin resulted in compromised population doubling capacity and mTOR pathway inhibition, there was no effect on telomere functionality and telomerase activity as evidenced by our assessments of hTERT protein levels, in vitro telomerase activity, telomere length and telomere FISH analyses. We also found that sustained rapamycin treatment leading to Akt activation may play a role in resistance in the more invasive MDA-MB-231 cells. In summary, rapamycin specifically inhibits the activation of mTOR pathway. Moreover, we show for the first time that while acute short-term treatment with rapamycin induces telomerase inhibition, it does not affect telomerase activity nor does it inflict telomere dysfunction in breast cancer cells upon chronic long-term treatment with a clinically relevant dose. These findings may be useful while designing combinatorial treatment strategies with rapamycin inhibition in the clinic.

Topics: Antibiotics, Antineoplastic; Breast Neoplasms; Cell Cycle; DNA Damage; Female; Gene Expression Regulation, Neoplastic; Humans; Sirolimus; Telomerase; Telomere Homeostasis; TOR Serine-Threonine Kinases; Tumor Cells, Cultured

The crosstalk between inflammation and autophagy is an emerging phenomenon observed during tumorigenesis. Activation of NF-κB and IRF3 plays a key role in the regulation of cytokines that are involved in tumor growth and progression. The genes of innate immunity are known to regulate the master transcription factors like NF-κB and IRF3. Innate immunity pathways at the same time regulate the genes of the autophagy pathway which are essential for tumor cell metabolism. In the current study, we studied the role of MITA (Mediator of IRF3 Activation), a regulator of innate immunity, in the regulation of autophagy and its implication in cell death of breast cancer cells. Here, we report that MITA inhibits the fusion of autophagosome with lysosome as evident from different autophagy flux assays. The expression of MITA induces the translocation of p62 and NDP52 to mitochondria which further recruits LC3 for autophagosome formation. The expression of MITA decreased mitochondrial number and enhances mitochondrial ROS by increasing complex-I activity. The enhancement of autophagy flux with rapamycin or TFEB expression normalized MITA induced cell death. The evidences clearly show that MITA regulates autophagy flux and modulates mitochondrial turnover through mitophagy.

Topics: Autophagosomes; Autophagy; Breast Neoplasms; Carcinogenesis; Cell Line, Tumor; Female; Gene Expression Regulation, Neoplastic; Humans; Immunity, Innate; Inflammation; Interferon Regulatory Factor-3; Lysosomes; Membrane Proteins; Mitochondria; Mitophagy; NF-kappa B; Signal Transduction; Sirolimus

Trastuzumab is a therapeutic monoclonal antibody that selectively recognizes HER2/neu receptor for targeting breast cancers. In this study, we aimed to present a strategy to combine chemo and phototherapy and targeted delivery via monoclonal antibody for enhanced anticancer effects. We co-loaded a chemotherapeutic agent, rapamycin, and a photosensitizer, polypyrrole, in trastuzumab-conjugated liposomes (LRPmAb) for combined chemo-photothermal therapy. LRPmAb had small size (172.2±9.6nm), narrow distribution, and negative ζ-potential (-12.0±0.3mV). In addition, LRPmAb showed pH- and temperature-dependent release profiles. LRPmAb showed significantly enhanced uptake in BT-474 cells, a natural HER2/neu expressing cell line. We found that these LRPmAb were effective in delivering rapamycin and showed higher therapeutic efficacy in breast cancer cells overexpressing HER2/neu receptors compared with cells that did not overexpress these receptors. Furthermore, LRPmAb showed synergistic activity against rapamycin-sensitive and resistant cell lines in vitro. These findings indicated that LRPmAb-mediated drug delivery could improve the therapeutic efficacy against breast cancer and overcome drug resistance.

Topics: Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Drug Carriers; Humans; Liposomes; Photochemotherapy; Polymers; Pyrroles; Receptor, ErbB-2; Sirolimus; Trastuzumab

The mammalian target of rapamycin inhibitor everolimus is approved as an antitumor agent in advanced estrogen receptor-positive breast cancer. Surrogate bone marker data from clinical trials suggest effects on bone metabolism, but the mode of action of everolimus in bone biology remains unclear. In this study, we assessed potential bone-protective effects of everolimus in the context of osteotropic tumors.. The effects of everolimus on cancer cell viability in vitro and on tumor growth in vivo were assessed. Everolimus-regulated osteoclastogenesis and osteoblastogenesis were also assessed in vitro before we assessed the bone-protective effect of everolimus in a model where bone loss was induced in ovariectomized (OVX) mice. Finally, the role of everolimus in the progression of osteolytic bone disease was assessed in an intracardiac model of breast cancer bone metastases.. At low concentrations (1 nM) in vitro, everolimus reduced the viability of human and murine cancer cell lines and impaired the osteoclastogenesis of osteoclast progenitors as assessed by quantitative real-time polymerase chain reaction and counting tartrate-resistant acid phosphatase-positive, multinucleated osteoclasts (p < 0.001). Everolimus had little or no deleterious effect on osteoblastogenesis in vitro, with concentrations of 1 and 10 nM increasing the messenger RNA expression of osteoblast marker genes (p ≤ 0.05) and leaving mineralization in differentiated human mesenchymal stem cells unchanged. Everolimus treatment (1 mg/kg body weight/day) prevented the bone loss observed in OVX mice and concurrently inhibited the metastatic growth of MDA-MB-231 cells by 70% (p < 0.002) while preserving bone mass in an intracardiac model of bone metastasis.. These results underline the antitumor effects of everolimus and highlight its bone-protective efficacy, warranting further research on the potential implications on bone health in populations prone to osteoporosis and bone metastases, such as postmenopausal women with breast cancer.

Topics: Animals; Bone Neoplasms; Breast Neoplasms; Cell Differentiation; Cell Line, Tumor; Cell Survival; Everolimus; Female; Humans; Mice; Osteoblasts; Osteoporosis; RAW 264.7 Cells; Sirolimus

Recombinant protein-polymer scaffolds such as elastin-like polypeptides (ELPs) offer drug-delivery opportunities including biocompatibility, monodispersity, and multifunctionality. We recently reported that the fusion of FK-506 binding protein 12 (FKBP) to an ELP nanoparticle (FSI) increases rapamycin (Rapa) solubility, suppresses tumor growth in breast cancer xenografts, and reduces side effects observed with free-drug controls. This new report significantly advances this carrier strategy by demonstrating the coassembly of two different ELP diblock copolymers containing drug-loading and tumor-targeting domains. A new ELP nanoparticle (ISR) was synthesized that includes the canonical integrin-targeting ligand (Arg-Gly-Asp, RGD). FSI and ISR mixed in a 1:1 molar ratio coassemble into bifunctional nanoparticles containing both the FKBP domain for Rapa loading and the RGD ligand for integrin binding. Coassembled nanoparticles were evaluated for bifunctionality by performing in vitro cell-binding and drug-retention assays and in vivo MDA-MB-468 breast tumor regression and tumor-accumulation studies. The bifunctional nanoparticle demonstrated superior cell target binding and similar drug retention to FSI; however, it enhanced the formulation potency, such that tumor growth was suppressed at a 3-fold lower dose compared to an untargeted FSI-Rapa control. This data suggests that ELP-mediated scaffolds are useful tools for generating multifunctional nanomedicines with potential activity in cancer.

Topics: Animals; Antibiotics, Antineoplastic; Breast; Breast Neoplasms; Cell Line, Tumor; Drug Carriers; Drug Delivery Systems; Elastin; Female; Humans; Integrins; Mice; Mice, Nude; Nanoparticles; Peptides; Sirolimus

Recombinant Elastin-Like Polypeptides (ELPs) serve as attractive scaffolds for nanoformulations because they can be charge-neutral, water soluble, high molecular weight, monodisperse, biodegradable, and decorated with functional proteins. We recently reported that fusion of the FK-506 binding protein 12 (FKBP) to an ELP nanoparticle (FSI) reduces rapamycin (Rapa) toxicity and enables intravenous (IV) therapy in both a xenograft breast cancer model and a murine autoimmune disease model. Rapa has poor solubility, which leads to variable oral bioavailability or drug precipitation following parenteral administration. While IV administration is routine during chemotherapy, cytostatic molecules like Rapa would require repeat administrations in clinical settings. To optimize FKBP/Rapa for subcutaneous (SC) administration, this manuscript expands upon first-generation FSI nanoparticles (

Topics: Animals; Breast Neoplasms; Female; Mice; Nanoparticles; Optical Imaging; Peptides; Sirolimus; Tacrolimus Binding Proteins; Triple Negative Breast Neoplasms

Liposomes have been employed to improve pharmacokinetics and reduce side effects of drugs. They can be functionalized with antibodies for targeted delivery. While the monoclonal antibody trastuzumab has been employed in the therapy of HER2-positive breast cancer, the resistance developed during treatment has been reported. Rapamycin could be used in combination with trastuzumab for improved therapeutic response.. In this study, we aimed to develop rapamycin-loaded liposomes and immunoliposomes with trastuzumab, characterize them and evaluate their in vitro cytotoxicity.. Formulations were prepared by the thin film hydration method and immunoliposome was conjugated to antibody by covalent bond. Characterization involved particle size, polydispersity, zeta potential, encapsulation efficiency, functionalization efficiency, DSC and FTIR assays. Cell studies were conducted through the MTT assay.. SPC:Chol:DSPE-PEG formulation prepared at 1:10 drug to lipid ratio presented high encapsulation efficiency, appropriate particle size, low polydispersity, negative zeta potential and colloidal stability. Rapamycin exhibited intermolecular interactions with lipids and underwent crystallinity reduction. Rapamycin-loaded immunoliposomes were prepared with high trastuzumab functionalization efficiency and antibody stability. Cytotoxicity studies showed that the HER2-positive SK-BR-3 cell line was sensitive to trastuzumab, either as free drug or in the context of immunoliposomes, and is more sensitive to rapamycin than the triple negative MDA-MB-231 cells. For MDA-MB-231, the liposomal rapamycin was more cytotoxic than the free drug. Furthermore, the immunoliposomes showed potent cytotoxicity against SK-BR-3 cells. Finally, rapamycin and trastuzumab exhibited in vitro synergistic effect, particularly through immunoliposomes.. The formulation developed herein has potential for in vivo evaluation.

Topics: Antibiotics, Antineoplastic; Breast; Breast Neoplasms; Cell Line, Tumor; Female; Humans; Immunoconjugates; Liposomes; Phosphatidylethanolamines; Polyethylene Glycols; Receptor, ErbB-2; Sirolimus; Trastuzumab

Breast cancer is a complex disease with at least five different molecular subtypes identified. The breast tumor molecular subtypes guide stratification of patients for specific targeted therapy regimens and each subtype is associated with significantly different patient outcomes. For example, patients with the HER2 positive molecular subtype benefit from the HER2 targeted therapy trastuzumab. Unfortunately, women with the HER2 positive molecular subtype have the worst overall prognosis and nearly 70% of women with HER2 positive breast cancer exhibit de novo or acquired resistance to trastuzumab. Identification of tumor markers predicting trastuzumab response can be used to further stratify patients for life-saving personalized therapeutic options. The aim of this study was to identify clinically useful tumor markers predicting de novo tumor cell resistance to trastuzumab treatment. To identify oncogenic signaling pathways activated in response to trastuzumab treatment, we performed a Human Phospho-Kinase Proteome Profiler Array analysis comparing trastuzumab sensitive MCF-7/HER2.2 and trastuzumab resistant MCF-7/HER2Δ16H cells following acute treatment with 20 μg/ml of trastuzumab for 2 h. We found that of the 43 phosphorylation activated human kinases represented on the array, S6K1 was the only kinase altered greater than 1.5-fold in response to trastuzumab treatment of the trastuzumab resistant MCF-7/HER2Δ16H cells. Trastuzumab activation of S6K1 was confirmed in the two trastuzumab resistant SUM190 and SUM225 cell lines. Significantly, trastuzumab failed to stimulate S6K1 activation in the trastuzumab sensitive MCF-7/HER2.2, BT474, and SKBR3 cell lines suggesting that trastuzumab activation of S6K1 is a tumor cell marker for trastuzumab resistance. Consistent with a role for mTORC1/S6K1 signaling promoting trastuzumab resistance, all cell lines were sensitive to S6K1 inactivation with significant growth inhibition following treatment with the mTORC1 inhibitor rapamycin. In conclusion, characterizing rapid trastuzumab induced molecular alterations resulted in the identification of activated S6K1 as an early breast tumor cell marker for trastuzumab resistance. Our results further suggest that trastuzumab resistant breast tumor cells are addicted to mTORC1/S6K1 oncogenic signaling and targeting mTORC1 with rapamycin reverses trastuzumab resistance.

Topics: Antineoplastic Agents; Biomarkers, Tumor; Breast Neoplasms; Drug Resistance, Neoplasm; Humans; MCF-7 Cells; Mechanistic Target of Rapamycin Complex 1; Multiprotein Complexes; Ribosomal Protein S6 Kinases, 70-kDa; Sirolimus; TOR Serine-Threonine Kinases; Trastuzumab

(+)-Usnic acid (1) is a common bioactive lichen-derived secondary metabolite with a characteristic dibenzofuran scaffold. It displayed low micromolar antiproliferative activity levels and, notably, induced autophagy in a panel of diverse breast cancer cell lines, suggesting the mechanistic (formerly "mammalian") target of rapamycin (mTOR) as a potential macromolecular target. The cellular autophagic markers were significantly upregulated due to the inhibition of mTOR downstream effectors. Additionally, 1 showed an optimal binding pose at the mTOR kinase pocket aided by multiple interactions to critical amino acids. Rationally designed benzylidene analogues of 1 displayed excellent fitting into a targeted deep hydrophobic pocket at the core of the kinase cleft, through stacking with the phenolic side chain of the Tyr2225 residue. Several potent analogues were generated, including 52, that exhibited potent (nM concentrations) antiproliferative, antimigratory, and anti-invasive activities against cells from multiple breast cancer clonal lines, without affecting the nontumorigenic MCF-10A mammary epithelial cells. Analogue 52 also exhibited potent mTOR inhibition and autophagy induction. Furthermore, 52 showed potent in vivo antitumor activity in two athymic nude mice breast cancer xenograft models. Collectively, usnic acid and analogues are potential lead mTOR inhibitors appropriate for future use to control breast malignancies.

Topics: Animals; Apoptosis; Autophagy; Benzofurans; Benzylidene Compounds; Breast Neoplasms; Cell Proliferation; Crystallography, X-Ray; Disease Models, Animal; Female; Humans; Lichens; Mice; Mice, Nude; Models, Molecular; Molecular Conformation; Molecular Structure; Protein Kinase Inhibitors; Sirolimus; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays

Breast cancer is the second leading cause of cancer deaths among women. Paclitaxel (PTX) is used for its treatment, however non-selectivity, rapid systemic clearance and hypersensitivity to the commercially available formulation are major drawbacks. Rapamycin (RAP), an mTOR inhibitor, acts synergistically with PTX, and thus could be used in combination with it. Drug loading into nanocarriers, particularly liposomes, has proven to enhance efficacy and reduce side-effects of chemotherapeutic drugs. Within this context, the functionalization of liposomes with antibodies for overexpressed receptors on tumor surface is a potential strategy to increase specificity and reduce side-effects. Specifically, active targeting of HER2(+) breast cancer cells can be achieved by immunoliposomes consisting of liposomes coated with an anti-HER2 monoclonal antibody, Trastuzumab. Herein, we have synthesized PTX/RAP co-loaded immunoliposomes coated with Trastuzumab, performed physicochemical characterization, and evaluated the formulations for cytotoxicity and uptake in 4T1 (triple negative) and SKBR3 (HER2 positive) cell lines. Furthermore, we aimed to compare the immunoliposomes with liposomes and solution of PTX/RAP in vivo, employing human xenograft HER2-overexpressing tumors in mouse model. The co-loaded immunoliposomes had a mean particle size of 140.3nm, a zeta potential of -9.85mV and drug encapsulation efficiency of 55.87 and 69.51, respectively for PTX and RAP. The functionalization efficiency of Trastuzumab was higher than 70% and the antibody retained HER2 binding activity. Cell studies showed increased cytotoxicity of PTX/RAP for the immunoliposome, compared to the control liposomes in SKBR3 cells, which could be attributed to enhanced uptake mediated through HER2 binding. Furthermore, immunoliposomes were better able to control tumor growth in vivo, with tumor volume averages corresponding to 25.27, 44.38 and 47.78% of tumor volumes of untreated control, PTX/RAP solution and control liposomes, respectively. Taken together, our results support the clinical development of immunoliposomes for targeted delivery of PTX and RAP to HER2-positive breast cancer.

Topics: Animals; Antibodies, Monoclonal; Breast Neoplasms; Cell Line, Tumor; Female; Humans; Liposomes; Mice; Paclitaxel; Receptor, ErbB-2; Sirolimus; Trastuzumab

Neovascularisation supports the metastatic switch in many aggressive solid cancers. Tumour neovessels are mostly lined by endothelial cells sprouting from nearby capillaries, but they could also be contributed by circulating endothelial progenitor cells (EPCs). However, scant information is available about tumour-derived EPCs.. We carried out the first thorough, unbiased comparison of phenotype, function and genotype of normal versus tumour-derived endothelial colony forming cells (ECFCs), a truly endothelial EPC subtype. We used healthy donors-derived ECFCs (N-ECFCs) as control for breast cancer (BC)- and renal cell carcinoma (RCC)-derived ECFCs.. We found that both BC- and RCC-ECFCs belong to the endothelial lineage. Normal and tumour-derived ECFCs did not differ in terms of proliferative and tubulogenic rates. However, RCC-ECFCs were more resistant to rapamycin-induced apoptosis, whereas BC-ECFCs were more sensitive as compared with N-ECFCs. Gene expression profiling revealed 382 differentially expressed genes (DEGs; 192 upregulated and 150 downregulated) and 71 DEGs (33 upregulated, 38 downregulated) when comparing, respectively, BC- and RCC-ECFCs with N-ECFCs. Nonetheless, BC- and RCC-derived ECFCs shared 35 DEGs, 10 of which were validated by qRT-PCR; such 35 DEGs are organised in a gene network centred on FOS.. These results provide the first clear-cut evidence that BC- and RCC-derived ECFCs exhibit an altered gene expression profile as compared with N-ECFCs; yet, they share a common gene signature that could highlight novel and more specific targets to suppress tumour vascularisation.

Topics: Adult; Aged; Aged, 80 and over; Antibiotics, Antineoplastic; Breast Carcinoma In Situ; Breast Neoplasms; Carcinoma, Ductal, Breast; Carcinoma, Renal Cell; Down-Regulation; Endothelial Progenitor Cells; Female; Gene Expression Regulation, Neoplastic; Genotype; Humans; Kidney Neoplasms; Male; Middle Aged; Neovascularization, Pathologic; Phenotype; Sirolimus; Transcriptome; Tumor Cells, Cultured; Up-Regulation

P-glycoprotein (P-gp) efflux is the major cause of multidrug resistance (MDR) in tumors when using anticancer drugs, moreover, poor bioavailability of few drugs is also due to P-gp efflux in the gut. Rapamycin (RPM) is in the clinical trials for breast cancer treatment, but its P-gp substrate property leads to poor oral bioavailability and efficacy. The objective of this study is to formulate and evaluate nanoparticles of RPM, along with a chemosensitizer (piperine, PIP) for improved oral bioavailability and efficacy. Poly(d,l-lactide-co-glycolide) (PLGA) was selected as polymer as it has moderate MDR reversal activity, which may provide additional benefits. The nanoprecipitation method was used to prepare PLGA nanoparticles with particle size below 150 nm, loaded with both drugs (RPM and PIP). Prepared nanoparticles showed sustained in vitro drug release for weeks, with initial release kinetics of zero order with non-Fickian transport, subsequently followed by Higuchi kinetics with Fickian diffusion. An everted gut sac method was used to study the effect of P-gp efflux on drug transport. This reveals that the uptake of the RPM (P-gp substrate) has been increased in the presence of chemosensitizer. Pharmacokinetic studies showed better absorption profile of RPM from polymeric nanoparticles compared to its suspension counterpart and improved bioavailability of 4.8-folds in combination with a chemosensitizer. An in vitro cell line study indicates higher efficacy of nanoparticles compared to free drug solution. Results suggest that the use of a combination of PIP with RPM nanoparticles would be a promising approach in the treatment of breast cancer.

Topics: Alkaloids; Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily B, Member 1; Benzodioxoles; Biological Availability; Breast Neoplasms; Cell Line, Tumor; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Female; Humans; Lactic Acid; Nanoparticles; Piperidines; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Polyunsaturated Alkamides; Sirolimus

The importance of mTOR signaling in tumor biology is widely accepted and a number of agents that selectively target mTOR are being developed in cancer therapy. On the other hand, it has been demonstrated that mTOR can act as an angiogenic agent. Thus, we hypothesized that the mTOR inhibitor-induced anticancer effect is affected by expression of a key angiogenic factor, vascular endothelial growth factor (VEGF) and investigated the anticancer effect underlying mTOR using an in vitro assay. The mTOR inhibitor rapamycin dose-dependently reduced the cell viability of the breast cancer cell line, MCF-7, but did not reduce the cell viability of the colon cancer cell line, HT-29. Rapamycin reduced the VEGF expression in the culture medium of MCF-7, while rapamycin did not contribute VEGF expression in the culture medium of HT-29. VEGF stimulated cell viability and VEGF inhibition reduced cell viability of MCF-7, and rapamycin dose-dependently restored the cell viability of MCF-7 reduced by rapamycin. These findings suggest that mTOR acts as a direct anticancer agent and that the mTOR-inhibitor-induced anticancer effect involved the reduced expression of VEGF in MCF-7. Our results imply that mTOR regulates the expression of VEGF and is involved in breast cancer progression.

Topics: Breast Neoplasms; Female; Gene Expression Regulation, Neoplastic; Humans; MCF-7 Cells; Protein Kinase Inhibitors; Sirolimus; TOR Serine-Threonine Kinases; Vascular Endothelial Growth Factor A

The sensitization of breast cancer stem cells (BrCSCs) to the inhibitive effects of radiotherapy through adjuvant therapy which targets oncogenic pathways represents a prospective strategy for improving the effect of radiation in patients with triple-negative breast cancer (TNBC). Mammalian target of rapamycin (mTOR) activation is one of the most frequent events in human malignancies, and is critical for sustaining the self‑renewing ability of cancer stem cells (CSCs); inhibition by rapamycin is an effective and promising strategy in anticancer treatments. In the present study, we found that mTOR activity was closely related to the self-renewal ability of BrCSCs, and in triple negative MDA-MB-453 and MDA-MB‑468 cells, rapamycin repression of mTOR phosphorylation decreased the number of mammospheres and helped to sensitize the resistant CSCs to low-dose radiation therapy. By inhibiting mTOR and mitochondrial manganese superoxide dismutase (MnSOD), we confirmed that rapamycin functioned through the mTOR/MnSOD/reactive oxygen species (ROS) signaling pathway, and the existence of Akt governed the rapamycin‑induced asymmetric division (AD) of stem cells in cases of radiation‑treated breast cancer. The synergic effects of rapamycin and low-dose radiation induced the AD of stem cells, which then resulted in a decrease in the number of mammospheres, and both were mediated by MnSOD. Governed by Akt, the consequent inhibition of ROS formation and oxidative stress preserved the AD mode of stem cells, which is critical for an improved radiotherapy response in clinical treatment, as the tumor group is thus easier to eliminate with radiation therapy. We posit that an in-depth understanding of the interaction of radiation with CSCs has enormous potential and will make radiation even better and more effective.

Topics: Apoptosis; Asymmetric Cell Division; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Female; Gene Expression Regulation, Neoplastic; Humans; Neoplastic Stem Cells; Phosphorylation; Proto-Oncogene Proteins c-akt; Radiation; Radiation Tolerance; Reactive Oxygen Species; Sirolimus; Superoxide Dismutase; TOR Serine-Threonine Kinases

RB1CC1/FIP200 was essential for autophagosome formation. Therefore, RB1CC1/FIP200 cellular levels are critical for the activation of the autophagy pathways. Following the screen of miRNAs affecting RB1CC1/FIP200 level and rapamycin-induced autophagy, we discovered miR-20a and miR-20b could regulate autophagy by targeting RB1CC1/FIP200.. Inhibitory effect of miR-20a and 20b on basal and rapamycin-stimulated autophagy was demonstrated using various autophagic tests including GFP-LC3 puncta analysis, LC3II/LC3I gel shift and TEM observation.. We discovered RB1CC1/FIP200 as cellular targets of miR-20a and miR-20b. Upon miR-20a and miR-20b overexpression, both mRNA and protein levels of RB1CC1/FIP200 decreased. miR-20a and miR-20b target sequences present in the 3' UTR of RB1CC1/FIP200 mRNAs and introduction of mutations abolished the miR-20a and miR-20b responsiveness. In MCF7 and MDA-MB-231 breast cancer cells, miR-20a and miR-20b over-expression attenuated basal and rapamycin-induced autophagy; while suppression of miR-20a or miR-20b by specific antagomir showed normal rapamycin-induced autophagic activity.. To our knowledge, this is the first study showing the significance of miR-20a and miR-20b regulating autophagy by targeting RB1CC1/FIP200.

Topics: Autophagy; Autophagy-Related Proteins; Breast Neoplasms; Cell Line; Cell Line, Tumor; Female; Gene Expression Regulation, Neoplastic; Humans; MCF-7 Cells; MicroRNAs; Protein-Tyrosine Kinases; RNA, Messenger; Sirolimus

The mechanistic target of rapamycin complex 1 (mTORC1) plays a crucial role in controlling cell growth and homeostasis. Deregulation of mTOR signaling is frequently observed in some cancers, making it an attractive drug target for cancer therapy. Although mTORC1 inhibitor rapalog-based therapy has shown positive results in various pre-clinical animal cancer studies, tumors rebound upon treatment discontinuation. Moreover, several recent clinical trials showed that the mTORC1 inhibitors rapamycin and rapalog only reduce the capacity for cell proliferation without promoting cell death, consistent with the concept that rapamycin is cytostatic and reduces disease progression but is not cytotoxic. It is imperative that rapamycin-regulated events and additional targets for more effective drug combinations be identified. Here, we report that rapamycin treatment promotes a compensatory increase in transglutaminase 2 (TGM2) levels in mTORC1-driven tumors. TGM2 inhibition potently sensitizes mTORC1-hyperactive cancer cells to rapamycin treatment, and a rapamycin-induced autophagy blockade inhibits the compensatory TGM2 upregulation. More importantly, tumor regression was observed in MCF-7-xenograft tumor-bearing mice treated with both mTORC1 and TGM2 inhibitors compared with those treated with either a single inhibitor or the vehicle control. These results demonstrate a critical role for the compensatory increase in transglutaminase 2 levels in promoting mTORC1 inhibitor resistance and suggest that rational combination therapy may potentially suppress cancer therapy resistance.

Topics: Animals; Antibiotics, Antineoplastic; Breast; Breast Neoplasms; Cell Line, Tumor; Female; Gene Expression Regulation, Neoplastic; GTP-Binding Proteins; Humans; Mechanistic Target of Rapamycin Complex 1; Mice, SCID; Multiprotein Complexes; Protein Glutamine gamma Glutamyltransferase 2; Protein Kinase Inhibitors; Sirolimus; TOR Serine-Threonine Kinases; Transglutaminases

Protein arginine methylation is a common posttranslational modification resulting in the generation of asymmetric dimethylarginine (aDMA) and symmetric dimethylarginine (sDMA). Currently, the regulation of aDMA or sDMA by hypoxia, nutrient stavation or cytokines in the tumor microenvironment remains largely unknown. Here we show that p90aDMA, p70aDMA and p90sDMA, endogenous proteins containing aDMA or sDMA with mass 70 or 90 kDa, were widely and dominantly expressed in breast cancer cell lines. Notably, it was p90aDMA rather than p90sDMA that accumulated in the nucleus upon stimulation of cancer cells with interleukin (IL)-2, IL-4, IL-6 but not IL-8. In addition, the p90aDMA accumulation could be inhibited after treatment with a global methyltrasferase inhibitor, adenosine-2',3'-dialdehyde (AdOx). It seemed that some endogenous proteins in cancer cells were asymmetrically arginine-methylated upon exposure to some cytokines.. Furthermore, endogenous proteins of aDMA, such as p90aDMA and p70aDMA, were degraded in response to hypoxia, nutrient starvation and rapamycin treatment in breast and cervical cancer cells. IL-2/4/6 slightly increased basal autophagy but slightly decreased the rapamycin‑induced autophagy in cancer cells, suggesting that IL-2/4/6 and autophagy inducers play distinct roles in the regulation of aDMA of proteins. Conversely, rapamycin accumulated p90sDMA in MDA-MB‑231 and MCF-7 cells. Taken together, our results add a new dimension to the complexity of arginine methylated regulation in response to various stimuli and provide the first evidence that aDMA serves as one specific degradation signal of selective autophagy.

Topics: Arginine; Autophagy; Breast Neoplasms; Cell Line, Tumor; Female; Humans; Interleukin-2; Interleukin-4; Interleukin-6; MCF-7 Cells; Molecular Weight; Nuclear Proteins; Protein Processing, Post-Translational; Sirolimus; Tumor Microenvironment; Uterine Cervical Neoplasms

Targeting mTORC1 is a highly promising strategy in cancer therapy. Suppression of mTORC1 activity leads to rapid dephosphorylation of eIF4E-binding proteins (4E-BP1-3) and subsequent inhibition of mRNA translation. However, how the different 4E-BPs affect translation during prolonged use of mTOR inhibitors is not known. Here we show that the expression of 4E-BP3, but not that of 4E-BP1 or 4E-BP2, is transcriptionally induced during prolonged mTORC1 inhibition in vitro and in vivo. Mechanistically, our data reveal that 4E-BP3 expression is controlled by the transcription factor TFE3 through a cis-regulatory element in the EIF4EBP3 gene promoter. CRISPR/Cas9-mediated EIF4EBP3 gene disruption in human cancer cells mitigated the inhibition of translation and proliferation caused by prolonged treatment with mTOR inhibitors. Our findings show that 4E-BP3 is an important effector of mTORC1 and a robust predictive biomarker of therapeutic response to prolonged treatment with mTOR-targeting drugs in cancer.

Topics: Adaptor Proteins, Signal Transducing; Animals; Antibiotics, Antineoplastic; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Breast Neoplasms; Carrier Proteins; Cell Cycle Proteins; Cell Proliferation; CRISPR-Cas Systems; Databases, Genetic; Eukaryotic Initiation Factors; Female; Gene Editing; Gene Expression Regulation, Neoplastic; HeLa Cells; Hep G2 Cells; Humans; Indoles; Male; MCF-7 Cells; Mice; Mice, Inbred C57BL; Phosphoproteins; Protein Biosynthesis; Purines; Signal Transduction; Sirolimus; Survival Analysis; TOR Serine-Threonine Kinases

The PI3K-Akt-mTOR signaling pathway has been identified as a key driver of carcinogenesis in several cancer types. As such, a major area of focus in cancer biology is the development of genomic biomarkers that can measure the activity level of the PI3K-Akt-mTOR pathway. In this study, we systematically estimate PI3K-Akt-mTOR pathway activity in breast primary tumor samples using transcriptomic profiles derived from drug treatment in MCF7 cell lines. We demonstrate that gene expression profiles derived from chemically-induced protein inhibition allows us to measure PI3K-Akt-mTOR pathway activity in patient tumor samples. With this approach, we predict prognosis and response to chemotherapy in cancer patients, and screen for potential pharmacological modulators of PI3K-Akt-mTOR pathway inhibitors.

Topics: Algorithms; Androstadienes; Antibiotics, Antineoplastic; Breast Neoplasms; Cell Line, Tumor; Chromones; Female; Gene Expression Regulation, Neoplastic; HL-60 Cells; Humans; Kaplan-Meier Estimate; MCF-7 Cells; Models, Genetic; Morpholines; Phosphatidylinositol 3-Kinases; Prognosis; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Transcriptome; Wortmannin

Mammalian target of rapamycin (mTOR) represents a key downstream intermediate for a myriad of oncogenic receptor tyrosine kinases. In the case of the insulin-like growth factor (IGF) pathway, the mTOR complex (mTORC1) mediates IGF-1 receptor (IGF-1R)-induced estrogen receptor alpha (ERα) phosphorylation/activation and leads to increased proliferation and growth in breast cancer cells. As a result, the prevalence of mTOR inhibitors combined with hormonal therapy has increased in recent years. Conversely, activated mTORC1 provides negative feedback regulation of IGF signaling via insulin receptor substrate (IRS)-1/2 serine phosphorylation and subsequent proteasomal degradation. Thus, the IGF pathway may provide escape (e.g. de novo or acquired resistance) from mTORC1 inhibitors. It is therefore plausible that combined inhibition of mTORC1 and IGF-1R for select subsets of ER-positive breast cancer patients presents as a viable therapeutic option.. Using hormone-sensitive breast cancer cells stably transfected with the aromatase gene (MCF-7/AC-1), works presented herein describe the in vitro and in vivo antitumor efficacy of the following compounds: dalotuzumab (DALO; "MK-0646"; anti-IGF-1R antibody), ridaforolimus (RIDA; "MK-8669"; mTORC1 small molecule inhibitor) and letrozole ("LET", aromatase inhibitor).. With the exception of MK-0646, all single agent and combination treatment arms effectively inhibited xenograft tumor growth, albeit to varying degrees. Correlative tissue analyses revealed MK-0646 alone and in combination with LET induced insulin receptor alpha A (InsR-A) isoform upregulation (both mRNA and protein expression), thereby further supporting a triple therapy approach.. These data provide preclinical rationalization towards the combined triple therapy of LET plus MK-0646 plus MK-8669 as an efficacious anti-tumor strategy for ER-positive breast tumors.

Topics: Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antigens, CD; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Blotting, Western; Breast Neoplasms; Cell Proliferation; Drug Synergism; Female; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasms, Hormone-Dependent; Phosphorylation; Proto-Oncogene Proteins c-akt; Real-Time Polymerase Chain Reaction; Receptor, IGF Type 1; Receptor, Insulin; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Hyperactivation of the mechanistic target of rapamycin complex 1 (mTORC1) is a frequent event in breast cancer and current efforts are aimed at targeting the mTORC1 signaling pathway in combination with other targeted therapies. However, patients often develop drug resistance in part due to activation of the oncogenic Akt signaling and upregulation of autophagy, which protects cancer cells from apoptosis. In the present study we investigated the effects of combination therapy of rapamycin (an allosteric mTORC1 inhibitor) together with resveratrol (a phytoestrogen that inhibits autophagy). Our results show that combination of these drugs maintains inhibition of mTORC1 signaling, while preventing upregulation of Akt activation and autophagy, causing apoptosis. Additionally, this combination was effective in estrogen receptor positive and negative breast cancer cells, underscoring its versatility.

Topics: Apoptosis; Autophagy; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Drug Synergism; Estrogen Receptor alpha; Female; Humans; Mechanistic Target of Rapamycin Complex 1; Models, Biological; Multiprotein Complexes; Proto-Oncogene Proteins c-akt; Resveratrol; Ribosomal Protein S6 Kinases; Signal Transduction; Sirolimus; Stilbenes; TOR Serine-Threonine Kinases; Up-Regulation

Topics: Breast Neoplasms; Drug-Related Side Effects and Adverse Reactions; Female; Humans; Sirolimus

Mammalian targets of rapamycin inhibitors (mTOR inhibitors, mTORI) are indicated for the management of several cancer types, including hormone receptor--positive or HER2-negative breast cancer, advanced renal cell carcinoma, advanced neuroendocrine tumors of pancreatic origin, and tuberous sclerosis complex-related tumors. Among the most common adverse events of mTORI medication are discrete, large, solitary or multiple, superficial ulcers, almost exclusively situated on nonkeratinized oral mucosa, described as mTORI-associated stomatitis (mIAS). We describe the clinical presentation, course, and management of mIAS in three patients receiving the mTORI everolimus (Afinitor, Novartis, East Hanover, NJ). In two patients, mIAS manifested 9 and 30 days after first using everolimus, respectively, whereas in the third patient, it recurred 3 months after re-introduction of everolimus. Oral rinses with a "magic mouthwash" solution (dexamethasone oral drops solution 2 mg/mL × 10 mL, lidocaine gel 2% × 30 g, doxycycline suspension 50 mg/5 mL × 60 mL, and sucralfate oral suspension 1000 mg/5 mL × 150 mL, dissolved in sodium chloride 0.9% × 2000 mL) four times daily proved helpful in alleviating the symptoms, and the ulcers healed in 4 to 15 days. No side effects were recorded, and dose reduction or discontinuation of everolimus was not necessitated in two cases.

Topics: Aged; Antineoplastic Agents; Breast Neoplasms; Carcinoma, Renal Cell; Everolimus; Female; Humans; Kidney Neoplasms; Male; Middle Aged; Neoplasm Metastasis; Sirolimus; Stomatitis

The mammalian target of rapamycin complex 1 (mTORC1) is a critical regulator of G1 cell cycle progression. Two key substrates of mTORC1 are ribosomal subunit S6 kinase (S6K) and eukaryotic initiation factor 4E (eIF4E) binding protein-1 (4E-BP1). We reported previously that simultaneous knockdown of S6K and eIF4E causes a transforming growth factor-β (TGF-β)-dependent G1 cell cycle arrest in MDA-MB-231 human breast cancer cells. Rapamycin inhibits the phosphorylation of S6K at nano-molar concentrations in MDA-MB-231 cells; however, micro-molar concentrations of rapamycin are required to inhibit phosphorylation of 4E-BP1 - the phosphorylation of which liberates eIF4E to initiate translation. Micro-molar doses of rapamycin are required for complete G1 cell cycle arrest - indicating that 4E-BP1 is a critical target of mTOR for promoting cell cycle progression. Data are provided demonstrating that G1 cell cycle arrest induced by rapamycin is due to up-regulation of TGF-β signaling and down-regulation of Rb phosphorylation via phosphorylation of the mTORC1 substrates S6K and 4E-BP1 respectively. These findings enhance the current understanding of the cytostatic effects of mTORC1 suppression with therapeutic implications.

Topics: Antibiotics, Antineoplastic; Breast Neoplasms; Cell Line, Tumor; Cyclin D1; Dose-Response Relationship, Drug; Female; G1 Phase Cell Cycle Checkpoints; Humans; MCF-7 Cells; Phosphorylation; Retinoblastoma Protein; Sirolimus; Transforming Growth Factor beta

Everolimus (RAD001, Afinitor(®)) is an oral, selective mTOR inhibitor recently approved by the US-FDA in combination with exemestane for treatment of hormone receptor positive advanced breast cancer. To date, no molecular predictors of response to everolimus in breast cancer have been identified. We hypothesized predictive markers could be identified using preclinical models. Using a molecularly characterized panel of human breast cancer and immortalized breast epithelial cell lines, we determined sensitivity to everolimus alone or in combination with ER- or HER2- targeted therapy. Gene expression microarrays and comparative genomic hybridization were performed on the cell lines to identify predictors of response to everolimus. Among 13 everolimus-sensitive cell lines, 10/13(77 %) were luminal, while in 26 resistant cell lines, 16/26(62 %) were non-luminal, and 10/26(38 %) were luminal. Only 3/24 non-luminal lines were sensitive, two of which were HER2+. Everolimus enhanced the anti-proliferative effect of both tamoxifen (TAM) and fulvestrant (FUL) in ER+ breast cancer cell lines, as well as trastuzumab in HER2+ cell lines. Everolimus + FUL but not everolimus + TAM reversed acquired resistance to TAM. Everolimus inhibited mTOR in tested cell lines by decreasing S6 phosphorylation, mediating its anti-proliferative effect by G0/G1 cell cycle arrest and induction of apoptosis. Chromosomal amplifications of AURKA (p value = 0.04) and HER2 (p value = 0.03) were each associated with increased sensitivity to everolimus. Transcript expression microarrays identified GSK3A, PIK3R3, KLF8, and MAPK10 among the genes overexpressed in sensitive luminal lines, while PGP, RPL38, GPT, and GFAP were among the genes overexpressed in resistant luminal cell lines. These preclinical in vitro data provide further support for continued clinical development of everolimus in luminal (ER+ or HER2+) breast cancer in combination with targeted therapies. We identified several potential molecular markers associated with response to everolimus that will require validation in clinical material.

Topics: Animals; Antibodies, Monoclonal, Humanized; Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Drug Resistance, Neoplasm; Estradiol; Everolimus; Female; Fulvestrant; Gene Expression Regulation, Neoplastic; Humans; Mice; Molecular Targeted Therapy; Receptor, ErbB-2; Receptors, Estrogen; Sirolimus; Tamoxifen; TOR Serine-Threonine Kinases; Trastuzumab; Xenograft Model Antitumor Assays

Evidence has been accumulating for a role for metformin in reducing breast cancer risk in post-menopausal women. It inhibits growth of breast cancer cells via several mechanisms, primarily the AMPK/mTOR signalling pathway. Another possible protective mechanism may be the ability of metformin to inhibit aromatase activity. In the present study, we investigated the effects of metformin on the basal growth of MCF-7 cells, after oestradiol (E2) stimulation and after the inhibition of mTOR by rapamycin. Secondly, we investigated the effects of metformin on the activity of a number of steroidogenic enzymes and the mRNA expression of aromatase and steroid sulphatase (STS). High doses of metformin significantly inhibited both basal and oestrogen-stimulated cell division. Low-dose rapamycin (10-10 M) did not inhibit growth, but the addition of metformin induced a significant reduction in growth. High-dose rapamycin (10-8 M) inhibited growth, and this was further attenuated by the addition of metformin. Exposure to low (10-7 M) and high (10-4 M) doses of metformin for 7-10 days significantly reduced the conversion of androstenedione (ANDRO) and testosterone (TESTO) (both requiring aromatase), but not the conversion of oestrone or oestrone sulphate (ES) via 17β-hydroxysteroid dehydrogenase/sulphatase to E2. This attenuation was via a downregulation in the expression of total aromatase mRNA and promoter II, whilst the expression of sulphatase was unaffected by metformin. In conclusion, plasma levels of metformin have a dual therapeutic action, first by directly inhibiting cell proliferation which can be augmented by rapamycin analogues, and secondly, by inhibiting aromatase activity and reducing the local conversion of androgens to E2.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Estradiol; Female; Gene Expression Regulation, Neoplastic; Humans; MCF-7 Cells; Metformin; Sirolimus

Blockade of mammalian target of rapamycin (mTOR) is a promising area in breast cancer therapy. However, in clinical trials, objective response rate with mTOR inhibitor monotherapy in breast cancer was modest. Biomarker studies designed to identify the responders of rapalogs are of increasing interest. We validated p27KIP1 expression levels as a candidate predictive biomarker of response to rapalogs. We also analyzed the correlation between rapamycin activity and p27KIP1 expression in the primary breast cancer cells and the patient-derived breast tumor xenograft models. The cells isolated from the breast tumor tissues expressing high levels of p27KIP1 were sensitive to rapamycin, whereas the cells from the tissues expressing low levels of p27KIP1 exhibited resistance to rapamycin. The correlation between p27KIP1 expression and rapamycin antitumor activity was also observed in the patient-derived breast tumor xenograft models. Moreover, we also found rapamycin significantly decreased phosphorylated p70S6K1 and phosphorylated 4EBP1 in both samples. It seemed that the different sensitivity of tumor cells to rapamycin did not owe to its different potency against mTOR activity. We further propose p27KIP1 expression level may be also a candidate predictive biomarker of rapalogs for breast cancer therapy, which requires additional clinical validation.

Topics: Adult; Aged; Animals; Antineoplastic Agents; Biomarkers, Tumor; Breast Neoplasms; Cyclin-Dependent Kinase Inhibitor p27; Female; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Middle Aged; Phosphorylation; Sirolimus; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays

Histone deacetylase 1 (HDAC1) is an important epigenetic controller involved in transcriptional regulation through modification of chromatin structure. Genetic and epigenetic changes and deregulation of signal transduction pathways have been implicated in the development of breast cancer. Downregulation of estrogen receptor α (ERα) expression is one of the mechanisms behind the acquisition of endocrine resistance. Sustained and increased hormone and growth factor receptor signaling in breast cancer cells contribute to resistance to endocrine therapy. Both HDACs and the PI3K/mTOR signaling pathway are becoming promising targets in breast cancer, reversing also acquired hormone resistance. Here we show how mitogens, activating the PI3K/mTOR pathway, trigger the phosphorylation of HDAC1 in breast cancer cells, which is completely dependent on the activity of the p70 S6 kinase (S6K1). Our findings show that S6K1, overexpressed in many breast cancers, controls HDAC1-dependent transcriptional regulation of ERα levels upon mitogenic stimuli, controlling HDAC1 recruitment to the ERα promoter. Furthermore, cell treatment with both mTOR and HDACs inhibitors shows an additive effect in inhibiting breast cancer proliferation. This confirms the novel cross-talk between the HDAC1 and PI3K pathways with clinical implications towards the treatment of this malignant disease.

Topics: Breast Neoplasms; Cell Survival; Estrogen Receptor alpha; Female; Gene Expression; Gene Expression Regulation, Neoplastic; Histone Deacetylase 1; Humans; MCF-7 Cells; Mitogens; Phosphatidylinositol 3-Kinases; Phosphorylation; Protein Processing, Post-Translational; Ribosomal Protein S6 Kinases, 70-kDa; Sirolimus; TOR Serine-Threonine Kinases

Lipogenesis inhibition was reported to induce apoptosis and repress proliferation of cancer cells while barely affecting normal cells. Lipins exhibit dual function as enzymes catalyzing the dephosphorylation of phosphatidic acid to diacylglycerol and as co-transcriptional regulators. Thus, they are able to regulate lipid homeostasis at several nodal points. Here, we show that lipin-1 is up-regulated in several cancer cell lines and overexpressed in 50 % of high grade prostate cancers. The proliferation of prostate and breast cancer cells, but not of non-tumorigenic cells, was repressed upon lipin-1 knock-down. Lipin-1 depletion also decreased cancer cell migration through RhoA activation. Lipin-1 silencing did not significantly affect global lipid synthesis but enhanced the cellular concentration of phosphatidic acid. In parallel, autophagy was induced while AKT and ribosomal protein S6 phosphorylation were repressed. We also observed a compensatory regulation between lipin-1 and lipin-2 and demonstrated that their co-silencing aggravates the phenotype induced by lipin-1 silencing alone. Most interestingly, lipin-1 depletion or lipins inhibition with propranolol sensitized cancer cells to rapamycin. These data indicate that lipin-1 controls main cellular processes involved in cancer progression and that its targeting, alone or in combination with other treatments, could open new avenues in anticancer therapy.

Topics: Antibiotics, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Autophagy; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Dose-Response Relationship, Drug; Female; Humans; Lipogenesis; Male; Molecular Targeted Therapy; Nuclear Proteins; Phosphatidate Phosphatase; Phosphorylation; Propranolol; Prostatic Neoplasms; Proto-Oncogene Proteins c-akt; rhoA GTP-Binding Protein; Ribosomal Protein S6; RNA Interference; Signal Transduction; Sirolimus; Time Factors; TOR Serine-Threonine Kinases; Transfection

Mesenchymal/metaplastic breast cancers (MpBCs) are often triple-negative (TNBC), and chemo-refractory, and can harbor phosphoinositide 3-kinase (PI3kinase) alterations; thus, therapy with mTor inhibitors may demonstrate activity.. Patients with mesenchymal/MpBC treated with temsirolimus-based regimens were evaluated. Mutational analyses [polymerase chain reaction (PCR)-based DNA sequencing method, mass spectrometric detection (Sequenom MassARRAY), or next-generation sequencing] as well as loss of phosphatase and tensin homolog (PTEN) (immunohistochemistry) were performed (archived tissue when available).. Twenty-three patients (one of whom was on two separate trials) were treated using temsirolimus-containing regimens: temsirolimus alone (n = 1 patient) or combined with the following: liposomal doxorubicin and bevacizumab (DAT, n = 18); liposomal doxorubicin (DT, n = 1); paclitaxel and bevacizumab (TAT, n = 2); paclitaxel (TT, n = 1); carboplatin and bevacizumab (CAT, n = 1). Response rate [complete response (CR) + partial response (PR)] was 25% across all regimens; 32% in the anthracycline-based regimens [DAT and DT (CR = 2, PR = 4; N = 19)]. An additional two patients achieved stable disease (SD) ≥6 months [total SD ≥6 months/CR/PR = 8 (33%)]. Molecular aberrations in the PI3K pathway were common: PIK3CA mutation = 6/15 (40%), PTEN mutation = 3/11 (27%), and PTEN loss = 2/11 (18%). A point mutation in the NF2 gene (K159fs*16; NF2 alterations can activate mTor) was found in one patient who attained CR (3+ years). Of the eight patients who achieved SD ≥6 months/CR/PR, all 4 patients with available tissue had a molecular aberration that activate the PIK3CA/Akt/mTOR axis: PIK3CA mutation = 2; PTEN loss = 1; NF2 aberration = 1.. DAT has activity in MpBCs including complete CRs. Molecular aberrations that can activate the PI3 K/Akt/mTOR axis are common in MpBC.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Combined Chemotherapy Protocols; Bevacizumab; Breast Neoplasms; Carboplatin; Class I Phosphatidylinositol 3-Kinases; Doxorubicin; Female; Follow-Up Studies; Humans; Mesoderm; Metaplasia; Middle Aged; Neoplasm Metastasis; Neoplasm Staging; Paclitaxel; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Polyethylene Glycols; Polymerase Chain Reaction; Prognosis; PTEN Phosphohydrolase; Sirolimus; Survival Rate; Young Adult

Focal adhesion kinase (FAK) controls cell growth and survival downstream of integrin-matrix receptors. Upon adhesion loss or FAK inhibition, FAK can translocate to the nucleus. The nucleolus is a non-membrane nuclear structure that regulates ribosome biogenesis and cell proliferation. Nucleostemin (NS), a nucleolar-localized protein, modulates cell cycle progression, stemness, and three-dimensional tumor spheroid formation. The signaling pathways that regulate NS levels in tumors remain undefined.. Human breast carcinoma cells were evaluated for growth in culture (adherent and anchorage-independent spheroid) and as orthotopic tumors. FAK signaling was evaluated by pharmacological FAK inhibitor addition (PF-271, IC50~0.1 μM) and by small hairpin RNA (shRNA) knockdown followed by re-expression of FAK wildtype (WT) or a kinase-dead (KD, K454R) FAK point mutant. Immunoblotting was used to evaluate FAK, NS, nucleolar phosphoprotein B23, and nucleolin levels. Total and phosphospecific antibody imunoblotting were used to detect changes in FAK, Akt kinase (Akt also known as protein kinase B), and 4E-binding protein 1 (4E-BP1) phosphorylation, a translation repressor protein and target of the mammalian target of rapamycin (mTOR) complex. Immunohistochemical, co-immunoprecipitation, and cellular fractionation analyses were used to evaluate FAK association with nucleoli.. Pharmacological (0.1 μM PF-271) or genetic inhibition of FAK activity prevents MDA-MB-231 and 4T1L breast carcinoma growth as spheroids and as orthotopic tumors. FAK inhibition triggers proteasome-mediated decreased NS levels but no changes in other nucleolar proteins such as B23 (nucleophosmin) or nucleolin. Active FAK was associated with purified nucleoli of anchorage-independent cells and present within nucleoli of human invasive ductal carcinoma tumor samples. FAK co-immunoprecipitated with B23 that binds NS and a complex between FAK, NS, Akt, and mTOR was detected. Constitutively-active Akt kinase promoted tumor spheroid growth, stabilized NS levels, and promoted pS65 4E-BP1 phosphorylation in the presence of inhibited FAK. Rapamycin lowered NS levels and inhibited pS65 4E-BP1 phosphorylation in cells with activated Akt-mTOR signaling.. FAK signaling occurs in the nucleolus, active FAK protects NS, and Akt-mTOR pathway regulates NS protein stability needed for breast carcinoma spheroid and tumor growth.

Topics: Animals; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Disease Models, Animal; Enzyme Activation; Female; Focal Adhesion Protein-Tyrosine Kinases; GTP-Binding Proteins; Humans; Mice; Nuclear Proteins; Nucleophosmin; Protein Kinase Inhibitors; Protein Transport; Proto-Oncogene Proteins c-akt; RNA Interference; RNA, Messenger; RNA, Small Interfering; Sirolimus; Spheroids, Cellular; Tumor Burden; Tumor Cells, Cultured; Tumor Stem Cell Assay

With tremendous advances in sequencing and analysis in recent years, a wealth of genetic information has become available to identify and classify breast cancer into five main subtypes - luminal A, luminal B, claudin-low, human epidermal growth factor receptor 2-enriched, and basal-like. Current treatment decisions are often based on these classifications, and while more beneficial than any single treatment for all breast cancers, targeted therapeutics have exhibited limited success with most of the subtypes. Luminal B breast cancers are associated with early relapse following endocrine therapy and often exhibit a poor prognosis that is similar to that of the aggressive basal-like breast cancers. Identifying genetic components that contribute to the luminal B endocrine resistant phenotype has become imperative. To this end, numerous groups have identified activation of the phosphatidylinositol 3-kinase (PI3K) pathway as a common recurring event in luminal B cancers with poor outcome. Examining the pathways downstream of PI3K, Fu and colleagues have recreated a human model of the luminal B subtype of breast cancer. The authors were able to reduce expression of phosphatase and tensin homolog (PTEN), the negative regulator of PI3K, using inducible short hairpin RNAs. By varying the expression of PTEN, the authors effectively conferred endocrine resistance and recapitulated the luminal B gene expression signature. Using this system in vitro and in vivo, they then tested the ability of selective kinase inhibitors downstream of PI3K to enhance current endocrine therapies. A combination of fulvestrant, which blocks ligand-dependent and -independent estrogen receptor signaling, with protein kinase B inhibition was found to overcome endocrine resistance. These findings squarely place PTEN expression levels at the nexus of luminal B breast cancers and indicates that patients with PTEN-low estrogen receptor-positive tumors might benefit from combined endocrine and PI3K pathway therapies.

Topics: Animals; Antineoplastic Agents, Hormonal; Breast Neoplasms; Female; Humans; PTEN Phosphohydrolase; Sirolimus

Nowadays, emerging aspects of cancer therapy involve both diagnostic and therapeutic modules in a single setting. Targeted theranostic nanoplatforms have emerged globally as frontier research for the improvement of cancer therapy. Trastuzumab (Tmab), a humanized monoclonal antibody is now being used to target human epidermal growth factor receptor-2 (HER 2) positive cancer cells. In the present study, we have analysed the imaging and theragnosis potentiality of Tmab functionalized lipid based nanoparticles (NPs) loaded with anticancer drug rapamycin and imaging agent (quantum dots) for targeted cancer therapy and imaging. The therapeutic evaluation of drug loaded NPs were evaluated through various in vitro cellular studies. The results showed enhanced therapeutic efficacy of targeted drug loaded NPs over native drug and unconjugated NPs in HER 2 positive SKBR 3 breast cancer cell line. Moreover, exploration of the therapeutic benefits of rapamycin loaded Tmab conjugated NPs (Tmab-rapa-NPs) at molecular level, revealed augmented down regulation of mTOR signalling pathway thereby, inducing more cell death. Above all, our targeted multifunctional NPs have shown an excellent bio-imaging modality both in 2D monolayer and 3D tumor spheroid model. Thus, we can anticipate that such a multimodal nanotheranostic approach may be a useful tool for better cancer management in future.

Topics: Antibiotics, Antineoplastic; Breast; Breast Neoplasms; Cell Line, Tumor; Cell Survival; Drug Delivery Systems; Female; Humans; Lipids; Nanoparticles; Quantum Dots; Receptor, ErbB-2; Sirolimus; TOR Serine-Threonine Kinases; Trastuzumab

Rapamycin analogues have antitumor efficacy in several tumor types, however few patients demonstrate tumor regression. Thus, there is a pressing need for markers of intrinsic response/resistance and rational combination therapies. We hypothesized that epithelial-to-mesenchymal transition (EMT) confers rapamycin resistance. We found that the epithelial marker E-cadherin protein is higher in rapamycin sensitive (RS) cells and mesenchymal breast cancer cell lines selected by transcriptional EMT signatures are less sensitive to rapamycin. MCF7 cells, transfected with constitutively active mutant Snail, had increased rapamycin resistance (RR) compared to cells transfected with wild-type Snail. Conversely, we transfected two RR mesenchymal cell lines-ACHN and MDA-MB-231-with miR-200b/c or ZEB1 siRNA to promote mesenchymal-to-epithelial transition. This induced E-cadherin expression in both cell lines, and ACHN demonstrated a significant increase in RS. Treatment of ACHN and MDA-MB-231 with trametinib modulated EMT in ACHN cells in vitro. Treatment of MDA-MB-231 and ACHN xenografts with trametinib in combination with rapamycin resulted in significant growth inhibition in both but without an apparent effect on EMT. Future studies are needed to determine whether EMT status is predictive of sensitivity to rapalogs and to determine whether combination therapy with EMT modulating agents can enhance antitumor effects of PI3K/mTOR inhibitors.

Topics: Animals; Antigens, CD; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Cadherins; Cell Proliferation; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; Extracellular Signal-Regulated MAP Kinases; Female; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Homeodomain Proteins; Humans; MCF-7 Cells; Mice, Nude; MicroRNAs; Mitogen-Activated Protein Kinase Kinases; Mutation; Phosphorylation; Protein Kinase Inhibitors; Pyridones; Pyrimidinones; RNA Interference; Sirolimus; Snail Family Transcription Factors; Time Factors; TOR Serine-Threonine Kinases; Transcription Factors; Transfection; Tumor Burden; Xenograft Model Antitumor Assays; Zinc Finger E-box-Binding Homeobox 1

The mTORC1 inhibitors, rapamycin and its analogs, are known to show only modest antitumor activity in clinic, but the underlying mechanisms remain largely elusive. Here, we found that activated AKT signaling is associated with rapamycin resistance in breast and colon cancers by sustained phosphorylation of the translational repressor 4E-BP1. Treatment of tumor cells with rapamycin or the AKT inhibitor MK2206 showed a limited activity in inhibiting 4E-BP1 phosphorylation, cap-dependent translation, cell growth and motility. However, treatment with both drugs resulted in profound effects in vitro and in vivo. Mechanistic investigation demonstrated that the combination treatment was required to effectively inhibit PRAS40 phosphorylation on both Ser183 and Thr246 mediated by mTORC1 and AKT respectively, and with the combined treatment, dephosphorylated PRAS40 binding to the raptor/mTOR complex was enhanced, leading to dramatic repression of mTORC1-regulated 4E-BP1 phosphorylation and translation. Knockdown of PRAS40 or 4E-BP1 expression markedly reduced the dependence of tumor cells on AKT/mTORC1 signaling for translation and survival. Together, these findings reveal a critical role of PRAS40 as an integrator of mTORC1 and AKT signaling for 4E-BP1-mediated translational regulation of tumor cell growth and motility, and highlight PRAS40 phosphorylation as a potential biomarker to evaluate the therapeutic response to mTOR/AKT inhibitors.

Topics: Adaptor Proteins, Signal Transducing; Animals; Breast Neoplasms; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Drug Synergism; Female; Heterocyclic Compounds, 3-Ring; Humans; MCF-7 Cells; Mechanistic Target of Rapamycin Complex 1; Mice, Nude; Multiprotein Complexes; Phosphoproteins; Proto-Oncogene Proteins c-akt; Random Allocation; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Transcription Factors; Xenograft Model Antitumor Assays

The normal process of epithelial mesenchymal transition (EMT) is subverted by carcinoma cells to facilitate metastatic spread. Cancer cells rarely undergo a full conversion to the mesenchymal phenotype, and instead adopt positions along the epithelial-mesenchymal axis, a propensity we refer to as epithelial mesenchymal plasticity (EMP). EMP is associated with increased risk of metastasis in breast cancer and consequent poor prognosis. Drivers towards the mesenchymal state in malignant cells include growth factor stimulation or exposure to hypoxic conditions.. We have examined EMP in two cell line models of breast cancer: the PMC42 system (PMC42-ET and PMC42-LA sublines) and MDA-MB-468 cells. Transition to a mesenchymal phenotype was induced across all three cell lines using epidermal growth factor (EGF) stimulation, and in MDA-MB-468 cells by hypoxia. We used RNA sequencing to identify gene expression changes that occur as cells transition to a more-mesenchymal phenotype, and identified the cell signalling pathways regulated across these experimental systems. We then used inhibitors to modulate signalling through these pathways, verifying the conclusions of our transcriptomic analysis.. We found that EGF and hypoxia both drive MDA-MB-468 cells to phenotypically similar mesenchymal states. Comparing the transcriptional response to EGF and hypoxia, we have identified differences in the cellular signalling pathways that mediate, and are influenced by, EMT. Significant differences were observed for a number of important cellular signalling components previously implicated in EMT, such as HBEGF and VEGFA. We have shown that EGF- and hypoxia-induced transitions respond differently to treatment with chemical inhibitors (presented individually and in combinations) in these breast cancer cells. Unexpectedly, MDA-MB-468 cells grown under hypoxic growth conditions became even more mesenchymal following exposure to certain kinase inhibitors that prevent growth-factor induced EMT, including the mTOR inhibitor everolimus and the AKT1/2/3 inhibitor AZD5363.. While resulting in a common phenotype, EGF and hypoxia induced subtly different signalling systems in breast cancer cells. Our findings have important implications for the use of kinase inhibitor-based therapeutic interventions in breast cancers, where these heterogeneous signalling landscapes will influence the therapeutic response.

Topics: Breast Neoplasms; Cell Hypoxia; Cell Line, Tumor; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; Everolimus; Female; Humans; Immunosuppressive Agents; Neoplasm Metastasis; Proto-Oncogene Proteins c-akt; Pyrimidines; Pyrroles; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

With this study we aimed to research the effects of immunosuppressive drugs, their cumulative doses, and viral infections on development of malign tumors in patients who have undergone treatment for 5 years.. We examined 100 patients who underwent renal transplantation from 2004 to 2009. Patients had mycophenolate mofetil and steroid in addition to cyclosporine, sirolimus, or tacrolimus as immunosuppressive treatment. For malignancy screening, physical examination, radiologic and endoscopic screening were done, and immunosuppressive drugs and their cumulative doses, age, sex, body mass index (BMI), dialysis history, and viral infection history were investigated.. The mean age of patients was 42.03 ± 11.30 years. There were 1 colon cancer patient, 1 retroperitoneal liposarcoma, 1 renal oncocytoma, 3 Kaposi sarcoma patients treated with cyclosporine; in those treated with Tac there were 1 basal cell carcinoma, 1 Kaposi sarcoma, 2 thyroid carcinoma, 1 breast carcinoma, 1 bladder carcinoma, 1 renal cell carcinoma, and 1 colon carcinoma patients. The mean age of patients having carcinoma was statistically significant compared with those without cancer (P < .01). The prednisolone cumulative dose was significantly higher in carcinoma patients than in patients without carcinoma (P < .01).. The use of long-term chronic immunosuppressive therapy may increase the development of cancer. The risk of carcinoma increases with increasing drug dose and time period of the immunosuppressive drug. There was not a negative effect on cancer prevalence in patients with cyclosporine or tacrolimus. But the cumulative dose of steroids significantly increased malignancy occurence.

Topics: Adult; Breast Neoplasms; Carcinoma; Colonic Neoplasms; Cyclosporine; Early Detection of Cancer; Female; Humans; Immunosuppressive Agents; Kidney Transplantation; Male; Middle Aged; Mycophenolic Acid; Neoplasms; Retroperitoneal Neoplasms; Sarcoma, Kaposi; Sirolimus; Steroids; Tacrolimus; Thyroid Neoplasms; Time Factors; Urologic Neoplasms

Blocking the enzyme Fatty Acid Synthase (FASN) leads to apoptosis of HER2-positive breast carcinoma cells. The hypothesis is that blocking FASN, in combination with anti-HER2 signaling agents, would be an effective antitumor strategy in preclinical HER2+ breast cancer models of trastuzumab and lapatinib resistance. We developed and molecularly characterized in vitro HER2+ models of resistance to trastuzumab (SKTR), lapatinib (SKLR) and both (SKLTR). The cellular interactions of combining anti-FASN polyphenolic compounds (EGCG and the synthetic G28UCM) with anti-HER2 signaling drugs (trastuzumab plus pertuzumab and temsirolimus) were analyzed. Tumor growth inhibition after treatment with EGCG, pertuzumab, temsirolimus or the combination was evaluated in two in vivo orthoxenopatients: one derived from a HER2+ patient and another from a patient who relapsed on trastuzumab and lapatinib-based therapy. SKTR, SKLR and SKLTR showed hyperactivation of EGFR and p-ERK1/2 and PI3KCA mutations. Dual-resistant cells (SKLTR) also showed hyperactivation of HER4 and recovered levels of p-AKT compared with mono-resistant cells. mTOR, p-mTOR and FASN expression remained stable in SKTR, SKLR and SKLTR. In vitro, anti-FASN compounds plus pertuzumab showed synergistic interactions in lapatinib- and dual- resistant cells and improved the results of pertuzumab plus trastuzumab co-treatment. FASN inhibitors combined with temsirolimus displayed the strongest synergistic interactions in resistant cells. In vivo, both orthoxenopatients showed strong response to the antitumor activity of the combination of EGCG with pertuzumab or temsirolimus, without signs of toxicity. We showed that the simultaneous blockade of FASN and HER2 pathways is effective in cells and in breast cancer models refractory to anti-HER2 therapies.

Topics: Animals; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Breast Neoplasms; Catechin; Cell Adhesion; Cell Line, Tumor; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Fatty Acid Synthase, Type I; Female; Gallic Acid; Humans; Lapatinib; Mice; Mice, Inbred NOD; Mice, SCID; Mutation; Naphthalenes; Neoplasm Invasiveness; Neoplasm Transplantation; Quinazolines; Receptor, ErbB-2; Signal Transduction; Sirolimus; Trastuzumab

Breast cancer cells with stem cell characteristics (CSC) are a distinct cell population with phenotypic similarities to mammary stem cells. CSCs are important drivers of tumorigenesis and the metastatic process. Tamoxifen is the most widely used hormonal therapy for estrogen receptor (ER) positive cancers. In our study, tamoxifen was effective in reducing proliferation of ER + adherent cancer cells, but not their CSC population. We isolated, expanded and incubated CSC from seven breast cancers with or without tamoxifen. By genome-wide transcriptional analysis we identified tamoxifen-induced transcriptional pathways associated with ribosomal biogenesis and mRNA translation, both regulated by the mTOR-pathway. We observed induction of the key mTOR downstream targets S6K1, S6RP and 4E-BP1 in-patient derived CSCs by tamoxifen on protein level. Using the mTOR inhibitors rapamycin, everolimus and PF-04691502 (a dual PI3K/mTOR inhibitor) and in combination with tamoxifen, significant reduction in mammosphere formation was observed. Hence, we suggest that the CSC population play a significant role during endocrine resistance through activity of the mTOR pathway. In addition, tamoxifen further stimulates the mTOR-pathway but can be antagonized using mTOR-inhibitors.

Topics: Antineoplastic Agents, Hormonal; Breast Neoplasms; Cell Separation; Cell Survival; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Enzyme Activation; Estrogen Antagonists; Everolimus; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; MCF-7 Cells; Neoplastic Stem Cells; Protein Kinase Inhibitors; Pyridones; Pyrimidines; RNA, Messenger; Signal Transduction; Sirolimus; Spheroids, Cellular; Tamoxifen; TOR Serine-Threonine Kinases; Transcription, Genetic; Transcriptional Activation; Tumor Cells, Cultured

The established anticancer and neuroprotective properties of oleocanthal combined with the reported role of mammalian target of rapamycin (mTOR) in cancer and Alzheimer's disease development encouraged us to examine the possibility that oleocanthal inhibits mTOR. To validate this hypothesis, we docked oleocanthal into the adenosine triphosphate binding pocket of a close mTOR protein homologue, namely, PI3K-γ. Apparently, oleocanthal shared nine out of ten critical binding interactions with a potent dual PIK3-γ/mTOR natural inhibitor. Subsequent experimental validation indicated that oleocanthal indeed inhibited the enzymatic activity of mTOR with an IC50 value of 708 nM. Oleocanthal inhibits the growth of several breast cancer cell lines at low micromolar concentration in a dose-dependent manner. Oleocanthal treatment caused a marked downregulation of phosphorylated mTOR in metastatic breast cancer cell line (MDA-MB-231). These results strongly indicate that mTOR inhibition is at least one of the factors of the reported anticancer and neuroprotective properties of oleocanthal.

Topics: Aldehydes; Animals; Breast Neoplasms; Cyclopentane Monoterpenes; Female; Humans; Models, Molecular; Olive Oil; Phenols; Phosphatidylinositol 3-Kinases; Phosphorylation; Sirolimus; TOR Serine-Threonine Kinases

The clinical impact of HER2 inhibitors in the treatment of HER2-amplified breast cancers has been largely confined to chemotherapy combination regimens, since HER2 inhibitors appear to have very modest efficacies by themselves. This is due to the resilient nature of the functionally relevant HER2-HER3 tumor driver, bidirectionally linked with downstream PI3K/Akt pathway signaling, which can break through the inhibitory effects of most current HER2 or HER3 targeting therapies. A vertical combination approach targeting HER2 and a downstream pathway is a highly rational strategy for much more effective targeted therapy of this disease. However the importance of these downstream pathways in many human tissues and cells significant limits their usefulness as secondary targets by narrowing the therapeutic index of such combination therapies. The secondary target that can afford the highest potential for clinical translation is the one with the highest synergy against tumor cells in combination with HER2-inhibition, allowing the widest therapeutic index for clinical translation. We conducted a comparative analysis of such secondary targets in combination with the HER2 inhibitor lapatinib and find that the inhibition of mTor affords the highest degree of synergy. In further dissecting the individual roles of TORC1 and TORC2 complexes using pharmacologic and genetic tools, we find that it is specifically the inactivation of TORC2 that most synergistically enhances the efficacy of lapatinib. Although inhibitors that selectively target TORC2 are not currently available, these data make a compelling case for their development.

Topics: Antineoplastic Agents; Apoptosis; Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Cell Survival; Drug Synergism; Humans; Imidazoles; Indoles; Lapatinib; Mechanistic Target of Rapamycin Complex 1; Mechanistic Target of Rapamycin Complex 2; Multiprotein Complexes; Naphthyridines; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Purines; Quinazolines; Quinolines; Receptor, ErbB-2; Receptor, ErbB-3; RNA Interference; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Topics: Administration, Intravenous; Adult; Antineoplastic Agents; Breast Neoplasms; Carcinoma; Compassionate Use Trials; Endometrial Neoplasms; Female; Follow-Up Studies; Humans; Liver Neoplasms; Lung Neoplasms; Male; Middle Aged; Retrospective Studies; Sirolimus; Survival Analysis; Treatment Outcome

This study evaluated the effects of an mTOR inhibitor everolimus alone or in combination with letrozole on MCF-7/Aro (MCF-7 cells stably transfected with CYP19) in vitro and in vivo. In vitro studies, full-length CYP19 (aromatase) was cloned in a plasmid transfer vector pH ß-Aro and then transfected into MCF-7 stem cells which were ESA(+)CD44(+)CD24(-/low) sorted by flow cytometry. MTT assays were used to quantify the inhibitory effect of the drugs on MCF-7/Aro stem cells (SCs) and non-stem cells (NSCs). Apoptosis and the cell cycle distributions of stem cells were examined by flow cytometry. The tumorigenicity of stem cells after treatment was investigated by soft agar colony formation assays. In vivo studies, the BALB/c mice were injected with MCF-7/Aro SCs, and the different treatments were administered. After necropsy, the expression of KI67, CD31, AKT1, phospho-AKT (Thr308), and mTOR was analyzed by immunohistochemistry. In vitro, compared with MCF-7/Aro NSCs, there were greater resistance to the standard treatment doses of letrozole and everolimus in MCF-7/Aro SCs (17- and 15-fold, respectively). Treatment with everolimus or letrozole resulted in growth inhibition of SCs in a dose-dependent manner. Compared with single-agent therapy, the combination of everolimus with letrozole was more effective in the inhibition of cell growth (P < 0.001) and tumorigenicity (P < 0.01). In addition, an increase in G1 cell cycle arrest and increases in early apoptosis were observed in the combination treatment group compared with either single-agent group. In vivo, the xenograft tumor sizes were significantly decreased in everolimus alone group compared to control group, and everolimus plus letrozole therapy was much more effective compared with either single agent alone (P < 0.01). Compared with everolimus alone, the combination of everolimus and letrozole reduced the expression of KI67, mTOR, and phospho-AKT (Thr308; P < 0.01). Everolimus has effective inhibition on aromatase-overexpressing stem cell in vitro and in vivo. The combination everolimus and letrozole could be more effective than either drug alone.

Topics: Animals; Apoptosis; Aromatase; Breast Neoplasms; Cell Proliferation; Drug Synergism; Elafin; Everolimus; Female; Gene Expression Regulation, Neoplastic; Humans; Letrozole; MCF-7 Cells; Mice; Nitriles; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Triazoles; Xenograft Model Antitumor Assays

Topics: Antineoplastic Agents; Breast Neoplasms; Drug Monitoring; Everolimus; Female; Humans; Lung Diseases, Interstitial; Mucin-1; Sirolimus; Treatment Outcome

Thiamine-dependent enzymes (TDEs) control metabolic pathways that are frequently altered in cancer and therefore present cancer-relevant targets. We have previously shown that the recombinant enzyme thiaminase cleaves and depletes intracellular thiamine, has growth inhibitory activity against leukemia and breast cancer cell lines, and that its growth inhibitory effects were reversed in leukemia cell lines by rapamycin. Now, we first show further evidence of thiaminase therapeutic potential by demonstrating its activity against breast and leukemia xenografts, and against a primary leukemia xenograft. We therefore further explored the metabolic effects of thiaminase in combination with rapamycin in leukemia and breast cell lines. Thiaminase decreased oxygen consumption rate and increased extracellular acidification rate, consistent with the inhibitory effect of acute thiamine depletion on the activity of the TDEs pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes; these effects were reversed by rapamycin. Metabolomic studies demonstrated intracellular thiamine depletion and the presence of the thiazole cleavage product in thiaminase-treated cells, providing validation of the experimental procedures. Accumulation of ribose and ribulose in both cell lines support the thiaminase-mediated suppression of the TDE transketolase. Interestingly, thiaminase suppression of another TDE, branched chain amino ketoacid dehydrogenase (BCKDH), showed very different patterns in the two cell lines: in RS4 leukemia cells it led to an increase in BCKDH substrates, and in MCF-7 breast cancer cells it led to a decrease in BCKDH products. Immunoblot analyses showed corresponding differences in expression of BCKDH pathway enzymes, and partial protection of thiaminase growth inhibition by gabapentin indicated that BCKDH inhibition may be a mechanism of thiaminase-mediated toxicity. Surprisingly, most of thiaminase-mediated metabolomic effects were also reversed by rapamycin. Thus, these studies demonstrate that acute intracellular thiamine depletion by recombinant thiaminase results in metabolic changes in thiamine-dependent metabolism, and demonstrate a previously unrecognized role of mTOR signaling in the regulation of thiamine-dependent metabolism.

Topics: Amino Acids, Aromatic; Amino Acids, Branched-Chain; Animals; Antibiotics, Antineoplastic; Breast Neoplasms; Cell Survival; Female; Humans; Hydrolases; Leukemia; MCF-7 Cells; Mice; Mice, Nude; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Sirolimus; Thiamine; Xenograft Model Antitumor Assays

Upregulation of PI3K/Akt/mTOR signalling in endocrine-resistant breast cancer (BC) has identified mTOR as an attractive target alongside anti-hormones to control resistance. RAD001 (everolimus/Afinitor®), an allosteric mTOR inhibitor, is proving valuable in this setting; however, some patients are inherently refractory or relapse during treatment requiring alternative strategies. Here we evaluate the potential for novel dual mTORC1/2 mTOR kinase inhibitors, exemplified by AZD8055, by comparison with RAD001 in ER + endocrine resistant BC cells.. In vitro models of tamoxifen (TamR) or oestrogen deprivation resistance (MCF7-X) were treated with RAD001 or AZD8055 alone or combined with anti-hormone fulvestrant. Endpoints included growth, cell proliferation (Ki67), viability and migration, with PI3K/AKT/mTOR signalling impact monitored by Western blotting. Potential ER cross-talk was investigated by immunocytochemistry and RT-PCR.. RAD001 was a poor growth inhibitor of MCF7-derived TamR and MCF7-X cells (IC50 ≥1 μM), rapidly inhibiting mTORC1 but not mTORC2/AKT signalling. In contrast AZD8055, which rapidly inhibited both mTORC1 and mTORC2/AKT activity, was a highly effective (P <0.001) growth inhibitor of TamR (IC50 18 nM) and MCF7-X (IC50 24 nM), and of a further T47D-derived tamoxifen resistant model T47D-tamR (IC50 19 nM). AZD8055 significantly (P <0.05) inhibited resistant cell proliferation, increased cell death and reduced migration. Furthermore, dual treatment of TamR or MCF7-X cells with AZD8055 plus fulvestrant provided superior control of resistant growth versus either agent alone (P <0.05). Co-treating with AZD8055 alongside tamoxifen (P <0.01) or oestrogen deprivation (P <0.05) also effectively inhibited endocrine responsive MCF-7 cells. Although AZD8055 inhibited oestrogen receptor (ER) ser167 phosphorylation in TamR and MCF7-X, it had no effect on ER ser118 activity or expression of several ER-regulated genes, suggesting the mTOR kinase inhibitor impact was largely ER-independent. The capacity of AZD8055 for ER-independent activity was further evidenced by growth inhibition (IC5018 and 20 nM) of two acquired fulvestrant resistant models lacking ER.. This is the first report demonstrating dual mTORC1/2 mTOR kinase inhibitors have potential to control acquired endocrine resistant BC, even under conditions where everolimus fails. Such inhibitors may prove of particular benefit when used alongside anti-hormonal treatment as second-line therapy in endocrine resistant disease, and also potentially alongside anti-hormones during the earlier endocrine responsive phase to hinder development of resistance.

Topics: Antineoplastic Agents, Hormonal; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Drug Resistance, Neoplasm; Drug Synergism; Estradiol; Estrogen Antagonists; Estrogen Receptor Antagonists; Everolimus; Female; Fulvestrant; Humans; Immunosuppressive Agents; MCF-7 Cells; Mechanistic Target of Rapamycin Complex 1; Mechanistic Target of Rapamycin Complex 2; Morpholines; Multiprotein Complexes; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Receptors, Estrogen; Sirolimus; Tamoxifen; TOR Serine-Threonine Kinases

Topics: Antineoplastic Agents; Breast Neoplasms; Budgets; Drug Costs; ErbB Receptors; Female; Humans; Receptor, ErbB-2; Sirolimus

Catalase is an antioxidant enzyme that catalyzes mainly the transformation of hydrogen peroxide into water and oxygen. Although catalase is frequently down-regulated in tumors the underlying mechanism remains unclear. Few transcription factors have been reported to directly bind the human catalase promoter. Among them FoxO3a has been proposed as a positive regulator of catalase expression. Therefore, we decided to study the role of the transcription factor FoxO3a and the phosphatidylinositol-3 kinase (PI3K) signaling pathway, which regulates FoxO3a, in the expression of catalase. To this end, we developed an experimental model of mammary breast MCF-7 cancer cells that acquire resistance to oxidative stress, the so-called Resox cells, in which catalase is overexpressed as compared with MCF-7 parental cell line. In Resox cells, Akt expression is decreased but its phosphorylation is enhanced when compared with MCF-7 cells. A similar profile is observed for FoxO3a, with less total protein but more phosphorylated FoxO3a in Resox cells, correlating with its higher Akt activity. The modulation of FoxO3a expression by knockdown and overexpression strategies did not affect catalase expression, neither in MCF-7 nor in Resox cells. Inhibition of PI3K and mTOR by LY295002 and rapamycin, respectively, decreases the phosphorylation of downstream targets (i.e. GSK3β and p70S6K) and leads to an increase of catalase expression only in MCF-7 but not in Resox cells. In conclusion, FoxO3a does not appear to play a critical role in the regulation of catalase expression in both cancer cells. Only MCF-7 cells are sensitive and dependent on PI3K/Akt/mTOR signaling.

Topics: Breast Neoplasms; Catalase; Female; Gene Expression Regulation, Enzymologic; Humans; MCF-7 Cells; Oxidative Stress; Phosphatidylinositol 3-Kinase; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

In contrast with the conventional targeting of nanoparticles to cancer cells with antibody or peptide conjugates, a hyaluronic acid (HA) matrix nanoparticle with intrinsic-CD44-tropism was developed to deliver rapamycin for localized CD44-positive breast cancer treatment. Rapamycin was chemically conjugated to the particle surface via a novel sustained-release linker, 3-amino-4-methoxy-benzoic acid. The release of the drug from the HA nanoparticle was improved by 42-fold compared to HA-temsirolimus in buffered saline. In CD44-positive MDA-MB-468 cells, using HA as drug delivery carrier, the cell viability was significantly decreased compared to free rapamycin and CD44-blocked controls. Rat pharmacokinetics showed that the area under the curve of HA nanoparticle formulation was 2.96-fold greater than that of the free drug, and the concomitant total body clearance was 8.82-fold slower. Moreover, in immunocompetent BALB/c mice bearing CD44-positive 4T1.2neu breast cancer, the rapamycin-loaded HA particles significantly improved animal survival, suppressed tumor growth and reduced the prevalence of lung metastasis.. This study demonstrates increased efficiency of rapamycin delivery and consequential treatment effects in a breast cancer model by hyaluronic acid - L-rapamycin conjugates with intrinsic tropism for CD44-positive cells.

Topics: Animals; Antibiotics, Antineoplastic; Breast; Breast Neoplasms; Cell Line, Tumor; Drug Carriers; Drug Delivery Systems; Female; Hyaluronan Receptors; Hyaluronic Acid; Mice, Inbred BALB C; Nanoparticles; Rats; Rats, Sprague-Dawley; Sirolimus

Topics: Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Drug Resistance, Neoplasm; Everolimus; Female; Humans; Sirolimus

Topics: Antineoplastic Agents; Breast Neoplasms; Budgets; Drug Costs; ErbB Receptors; Female; Humans; Receptor, ErbB-2; Sirolimus

Inhibition of poly(ADP-ribose) polymerase (PARP) is a promising therapeutic strategy for BRCA1 deficient cancers, however, the development of drug resistance limits clinical efficacy. Previously we found that the BRCA1-AKT1 pathway contributes to tumorigenesis and that the AKT1/mTOR is a novel therapeutic target for BRCA1-deficient cancers. Here, we report that phosphorylation of ribosomal protein S6, a mTOR downstream effector, is greatly increased in BRCA1 deficient cells resistant to PARP inhibition. Phosphorylation of S6 is associated with DNA damage and repair signaling during PARP inhibitor treatment. In BRCA1 deficient cells, expression of S6 lacking all five phosphorylatable sites renders the cells sensitive to PARP inhibitor and increases DNA damage signals. In addition, the S6 mutations reduce tumor formation induced by Brca1-deficiency in mice. Inhibition of S6 phosphorylation by rapamycin restores PARP sensitivity to resistant cells. Combined treatment with rapamycin and PARP inhibitor effectively suppresses BRCA1-deficient tumor growth in mice. These results provide evidence for a novel mechanism by which BRCA1 deficient cancers acquire drug resistance and suggest a new therapeutic strategy to circumvent resistance.

Topics: Animals; Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Drug Resistance, Neoplasm; Female; Fluorescent Antibody Technique; Gene Knock-In Techniques; Humans; Immunohistochemistry; Mice; Phosphorylation; Phthalazines; Piperazines; Poly(ADP-ribose) Polymerase Inhibitors; Ribosomal Protein S6; Sirolimus; Ubiquitin-Protein Ligases; Xenograft Model Antitumor Assays

Patients with ER/HER2-positive breast cancer have a poor prognosis and are less responsive to selective estrogen receptor modulators; this is presumably due to the crosstalk between ER and HER2. Fatty acid synthase (FASN) is essential for the survival and maintenance of the malignant phenotype of breast cancer cells. An intimate relationship exists between FASN, ER and HER2. We hypothesized that FASN may be the downstream effector underlying ER/HER2 crosstalk through the PI3K/AKT/mTOR pathway in ER/HER2-positive breast cancer. The present study implicated the PI3K/AKT/mTOR pathway in the regulation of FASN expression in ER/HER2-positive breast cancer cells and demonstrated that rapamycin, an mTOR inhibitor, inhibited FASN expression. Cerulenin, a FASN inhibitor, synergized with rapamycin to induce apoptosis and inhibit cell migration and tumorigenesis in ER/HER2-positive breast cancer cells. Our findings suggest that inhibiting the mTOR-FASN axis is a promising new strategy for treating ER/HER2-positive breast cancer.

Topics: Animals; Apoptosis; Blotting, Western; Breast Neoplasms; Cell Movement; Cell Proliferation; Cerulenin; Drug Synergism; Estrogen Receptor alpha; Fatty Acid Synthase, Type I; Female; Fluorescent Antibody Technique, Indirect; Gene Expression Regulation, Neoplastic; Humans; Mice; Mice, Nude; Protein Kinase Inhibitors; Real-Time Polymerase Chain Reaction; Receptor, ErbB-2; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sirolimus; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Topics: Breast Neoplasms; Drug-Related Side Effects and Adverse Reactions; Female; Humans; Sirolimus

The aim of this study is to outline a general process for assessing the feasibility of performing a valid network meta-analysis (NMA) of randomized controlled trials (RCTs) to synthesize direct and indirect evidence for alternative treatments for a specific disease population.. Several steps to assess the feasibility of an NMA are proposed based on existing recommendations. Next, a case study is used to illustrate this NMA feasibility assessment process in order to compare everolimus in combination with hormonal therapy to alternative chemotherapies in terms of progression-free survival for women with advanced breast cancer.. A general process for assessing the feasibility of an NMA is outlined that incorporates explicit steps to visualize the heterogeneity in terms of treatment and outcome characteristics (Part A) as well as the study and patient characteristics (Part B). Additionally, steps are performed to illustrate differences within and across different types of direct comparisons in terms of baseline risk (Part C) and observed treatment effects (Part D) since there is a risk that the treatment effect modifiers identified may not explain the observed heterogeneity or inconsistency in the results due to unexpected, unreported or unmeasured differences. Depending on the data available, alternative approaches are suggested: list assumptions, perform a meta-regression analysis, subgroup analysis, sensitivity analyses, or summarize why an NMA is not feasible.. The process outlined to assess the feasibility of an NMA provides a stepwise framework that will help to ensure that the underlying assumptions are systematically explored and that the risks (and benefits) of pooling and indirectly comparing treatment effects from RCTs for a particular research question are transparent.

Topics: Antineoplastic Agents; Antineoplastic Agents, Hormonal; Antineoplastic Combined Chemotherapy Protocols; Bayes Theorem; Breast Neoplasms; Disease-Free Survival; Everolimus; Feasibility Studies; Female; Humans; Meta-Analysis as Topic; Middle Aged; Randomized Controlled Trials as Topic; Sirolimus; Treatment Outcome

Obesity increases the risk of cancer death among postmenopausal women with estrogen receptor-positive (ER+) breast cancer, but the direct evidence for the mechanisms is lacking. The purpose of this study is to demonstrate direct evidence for the mechanisms mediating this epidemiologic phenomenon.. We analyzed transcriptomic profiles of pretreatment biopsies from a prospective cohort of 137 ER+ breast cancer patients. We generated transgenic (MMTV-TGFα;A (y) /a) and orthotopic/syngeneic (A (y) /a) obese mouse models to investigate the effect of obesity on tumorigenesis and tumor progression and to determine biological mechanisms using whole-genome transcriptome microarrays and protein analyses. We used a coculture system to examine the impact of adipocytes/adipokines on breast cancer cell proliferation. All statistical tests were two-sided.. Functional transcriptomic analysis of patients revealed the association of obesity with 59 biological functional changes (P < .05) linked to cancer hallmarks. Gene enrichment analysis revealed enrichment of AKT-target genes (P = .04) and epithelial-mesenchymal transition genes (P = .03) in patients. Our obese mouse models demonstrated activation of the AKT/mTOR pathway in obesity-accelerated mammary tumor growth (3.7- to 7.0-fold; P < .001; n = 6-7 mice per group). Metformin or everolimus can suppress obesity-induced secretion of adipokines and breast tumor formation and growth (0.5-fold, P = .04; 0.3-fold, P < .001, respectively; n = 6-8 mice per group). The coculture model revealed that adipocyte-secreted adipokines (eg, TIMP-1) regulate adipocyte-induced breast cancer cell proliferation and invasion. Metformin suppress adipocyte-induced cell proliferation and adipocyte-secreted adipokines in vitro.. Adipokine secretion and AKT/mTOR activation play important roles in obesity-accelerated breast cancer aggressiveness in addition to hyperinsulinemia, estrogen signaling, and inflammation. Metformin and everolimus have potential for therapeutic interventions of ER+ breast cancer patients with obesity.

Topics: Adipocytes; Adipokines; Aged; Animals; Antineoplastic Agents; Biomarkers, Tumor; Breast Neoplasms; Cell Proliferation; Disease Models, Animal; Everolimus; Female; Humans; Kaplan-Meier Estimate; Metformin; Mice; Mice, Transgenic; Middle Aged; Obesity; Postmenopause; Prospective Studies; Proto-Oncogene Proteins c-akt; Receptors, Estrogen; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Transcriptome

Topics: Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Drug Resistance, Neoplasm; Female; Humans; Sirolimus

Topics: Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Drug Resistance, Neoplasm; Female; Humans; Sirolimus

The expansion of molecular genetic knowledge leads to targeted therapy which is a common part of cancer treatment. Targeted agents also exist for breast cancer. However, new efficient molecules are urgently needed. On one hand, there are cancer subgroups where we do not have efficient targeted drugs, on the other hand, the results of established cancer therapies can be improved by interfering with another signal transduction pathway. Everolimus is a targeted agent which was effective in several clinical trials and became registered for treatment of hormone receptor positive breast cancer. This article summarizes the results of published breast cancer everolimus trials. The results of two phase 3 trials are available. In the BOLERO-2 trial exemestane was compared with the combination of exemestane and everolimus, while in BOLERO-3 trial trastuzumab and vinorelbine were investigated with or without everolimus. In both trials the progression-free survival was significantly longer in the experimental arm. The overall survival data of BOLERO-2 trial, a secondary end point, are also available. The 4.4 month benefit in the experimental arm is clinically important but it has not reached the level of statistical significance. We have not had biomarkers so far that could help us to identify a subgroup of cancers sensitive to everolimus.. A molekuláris genetikai ismeretek bõvülésével a daganatok célzott kezelése a mindennapi gyógyító tevékenység részévé vált. Az emlõdaganatok kezelésében is rendelkezünk célzottan ható molekulákkal, de új hatékony szerekre mégis égetõ szükség van, mert egyrészrõl vannak olyan betegcsoportok, akiknél nem rendelkezünk igazoltan hatékony célzott kezeléssel, másrészrõl a már meglévõ célzott kezelések eredményeit is javíthatja, ha több támadásponton, több jelátviteli úton avatkozunk be a daganat mûködésébe. Az everolimus egy olyan célzottan ható molekula, mely több vizsgálatban is hatékonynak és elõnyösnek mutatkozott, és hormonreceptor-pozitív emlõdaganatok kezelésére törzskönyvezésre is került. A jelen közlemény az emlõtumorokkal kapcsolatban publikált everolimusvizsgálatok adatait foglalja össze. Jelenleg két fázis 3 vizsgálat eredményei állnak rendelkezésre. A BOLERO-2 vizsgálatban önmagában adott exemestant hasonlították össze az everolimusszal kombinált kezeléssel, míg a BOLERO-3 vizsgálatban a trastuzumab és vinorelbin mellett alkalmazott everolimus hatását elemezték. Mindkét vizsgálatban a progressziómentes túlélés szignifikánsan hosszabb volt a kombinációs karokon. A BOLERO-2 vizsgálatban már teljes túlélési eredményekkel is rendelkezünk, amely másodlagos végpont volt. A vizsgálati karon elért 4,4 hónapos elõny klinikailag releváns, de a statisztikai szignifikanciaszintet nem érte el. Egyelõre nem rendelkezünk olyan biomarkerrel, amely segítségével az everolimusra érzékeny daganatok csoportja meghatározható lenne, az ezt célzó transzlációs vizsgálatok folyamatban vannak.

Topics: Adult; Aged; Androstadienes; Antibodies, Monoclonal, Humanized; Antineoplastic Combined Chemotherapy Protocols; Aromatase Inhibitors; Breast Neoplasms; Clinical Trials, Phase III as Topic; Disease Progression; Disease-Free Survival; Drug Administration Schedule; Drug Resistance, Neoplasm; Everolimus; Female; Humans; Kaplan-Meier Estimate; Middle Aged; Molecular Targeted Therapy; Postmenopause; Randomized Controlled Trials as Topic; Receptors, Estrogen; Receptors, Progesterone; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Trastuzumab; Treatment Outcome; Vinblastine; Vinorelbine

Everolimus in combination with exemestane significantly improved progression-free survival compared to exemestane alone in patients previously treated with non-steroidal aromatase inhibitors in the BOLERO-2 trial. As a result, this combination has been approved by the food and drug administration to treat postmenopausal women with hormone receptor positive and HER2 negative metastatic breast cancer. A cost-effectiveness analysis was conducted to determine whether everolimus represents good value for money, utilizing data from BOLERO-2. A decision-analytic model was used to estimate the incremental cost-effectiveness ratio between treatment arms of the BOLERO-2 trial. Costs were obtained from the Center for Medicare Services drug payment table and physician fee schedule. Benefits were expressed as quality-adjusted progression-free survival weeks (QAPFW) and quality-adjusted progression-free years (QAPFY), with utilities/disutilities derived from the literature. Deterministic and probabilistic sensitivity analyses were performed. A willingness to pay threshold of 1-3 times the per capita gross domestic product was adopted, as per the definition of the World Health Organization. The U.S. per capita gross domestic product in 2013 was $49,965; thus, a threshold varying between $49,965 and $149,895 was considered. Everolimus/exemestane had an incremental benefit of 11.88 QAPFW (0.22 QAPFY) compared to exemestane and an incremental cost of $60,574. This translated into an ICER of $265,498.5/QAPFY. Univariate sensitivity analyses showed important variations of the ICER, ranging between $189,836.4 and $530,947/QAPFY. A tornado analysis suggested that the key drivers of our model, by order of importance, included health utility value for stable disease, everolimus acquisition costs, and transition probabilities from the stable to the progression states. The Monte-Carlo simulation showed results that were similar to the base-case analysis. This cost-effectiveness analysis showed that everolimus plus exemestane is not cost-effective compared to exemestane alone. Further research is needed to investigate the cost-effectiveness of the drug combination within sub-groups of the population studied in BOLERO-2.

Topics: Androstadienes; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Cost-Benefit Analysis; Disease Progression; Disease-Free Survival; Everolimus; Female; Humans; Markov Chains; Neoplasm Metastasis; Sirolimus

Resistance to receptor tyrosine kinase (RTK) blockade in breast cancer is often mediated by activation of bypass pathways that sustain growth. Src and mammalian target of rapamycin (mTOR) are two intrinsic targets that are downstream of most RTKs. To date, limited clinical efficacy has been observed with either Src or mTOR inhibitors when used as single agents. Resistance to mTOR inhibitors is associated with loss of negative feedback regulation, resulting in phosphorylation and activation of AKT. Herein, we describe a novel role for Src in contributing to rapalog-induced AKT activation. We found that dual activation of Src and the mTOR pathway occurs in nearly half of all breast cancers, suggesting potential cross-talk. As expected, rapamycin inhibition of mTOR results in feedback activation of AKT in breast cancer cell lines. Addition of the Src/c-Abl inhibitor, dasatinib, completely blocks this feedback activation, confirming convergence between Src and the mTOR pathway. Analysis in vivo revealed that dual Src and mTOR inhibition is highly effective in two mouse models of breast cancer. In a luminal disease model, combined dasatinib and rapamycin is more effective at inducing regression than either single agent. Furthermore, the combination of dasatinib and rapamycin delays tumor recurrence following the cessation of treatment. In a model of human EGFR-2-positive (HER2(+)) disease, dasatinib alone is ineffective, but potentiates the efficacy of rapamycin. These data suggest that combining mTOR and Src inhibitors may provide a new approach for treating multiple breast cancer subtypes that may circumvent resistance to targeted RTK therapies.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Dasatinib; Drug Resistance, Neoplasm; Female; Genes, src; Humans; MCF-7 Cells; Mice; Phosphorylation; Proto-Oncogene Proteins c-abl; Proto-Oncogene Proteins c-akt; Pyrimidines; Rats; Signal Transduction; Sirolimus; Thiazoles; TOR Serine-Threonine Kinases

Topics: Breast Neoplasms; Drug-Related Side Effects and Adverse Reactions; Female; Humans; Sirolimus

Topics: Breast Neoplasms; Drug-Related Side Effects and Adverse Reactions; Female; Humans; Sirolimus

Everolimus is an orally administered anti-cancer drug that inhibits the mammalian target of rapamycin (mTOR) signal transduction route. Use of everolimus may be associated with insulin resistance, manifesting in impaired glucose tolerance or hyperglycaemia.. A 74-year-old female patient with a locally recurrent breast cancer developed hyperglycaemia, which started 2 weeks after the initiation of treatment with everolimus 10 mg once daily. Metformin and insulin were administered to restore normoglycaemia.. At the initiation of treatment with an mTOR inhibitor such as everolimus the treating physician should be aware of the occurrence of hyperglycaemia. Metformin is then the medicine of first choice.

Topics: Aged; Antineoplastic Agents; Breast Neoplasms; Everolimus; Female; Humans; Hyperglycemia; Hypoglycemic Agents; Metformin; Neoplasm Recurrence, Local; Sirolimus; TOR Serine-Threonine Kinases

Treatment options for recurrent or progressive hormone receptor-positive (HR+) advanced breast cancer include chemotherapy and everolimus plus exemestane (EVE + EXE). This study estimates the costs of managing adverse events (AEs) during EVE + EXE therapy and single-agent chemotherapy in Western Europe.. An economic model was developed to estimate the per patient cost of managing grade 3/4 AEs for patients who were treated with EVE + EXE or chemotherapies. AE rates for patients receiving EVE + EXE were collected from the phase III BOLERO-2 trial. AE rates for single-agent chemotherapy, capecitabine, docetaxel, or doxorubicin were collected from published clinical trial data. AEs with at least 2% prevalence for any of the treatments were included in the model. A literature search was conducted to obtain costs of managing each AE, which were then averaged across Western European countries (when available). Per patient costs for managing AEs among patients receiving different therapies were reported in 2012 euros (€).. The EVE + EXE combination had the lowest average per patient cost of managing AEs (€730) compared to all chemotherapies during the first year of treatment (doxorubicin: €1230; capecitabine: €1721; docetaxel: €2390). The most costly adverse event among all patients treated with EVE + EXE was anemia (on average €152 per patient). The most costly adverse event among all patients treated with capecitabine, docetaxel, or doxorubicin was lymphocytopenia (€861 per patient), neutropenia (€821 per patient), and leukopenia (€382 per patient), respectively.. The current model estimates that AE management during the treatment of HR+ advanced breast cancer will cost one-half to one-third less for EVE + EXE patients than for chemotherapy patients. The consideration of AE costs could have important implications in the context of healthcare spending for advanced breast cancer treatment.

Topics: Androstadienes; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Databases, Factual; Europe; Everolimus; Female; Humans; Models, Economic; Sirolimus

Cellular signaling pathways involving mTOR, PI3K and ERK have dominated recent studies of breast cancer biology, and inhibitors of these pathways have formed a focus of numerous clinical trials. We have chosen trametinib, a drug targeting MEK in the ERK pathway, to address two questions. Firstly, does inhibition of a signaling pathway, as measured by protein phosphorylation, predict the antiproliferative activity of trametinib? Secondly, do inhibitors of the mTOR and PI3K pathways synergize with trametinib in their effects on cell proliferation? A panel of 30 human breast cancer cell lines was chosen to include lines that could be classified according to whether they were ER and PR positive, HER2 over-expressing, and "triple negative". Everolimus (targeting mTOR), NVP-BEZ235 and GSK2126458 (both targeting PI3K/mTOR) were chosen for combination experiments. Inhibition of cell proliferation was measured by IC50 values and pathway utilization was measured by phosphorylation of signaling kinases. Overall, no correlation was found between trametinib IC50 values and inhibition of ERK signaling. Inhibition of ERK phosphorylation was observed at trametinib concentrations not affecting proliferation, and sensitivity of cell proliferation to trametinib was found in cell lines with low ERK phosphorylation. Evidence was found for synergy between trametinib and either everolimus, NVP-BEZ235 or GSK2126458, but this was cell line specific. The results have implications for the clinical application of PI3K/mTOR and MEK inhibitors.

Topics: Antineoplastic Agents; Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Drug Synergism; Everolimus; Extracellular Signal-Regulated MAP Kinases; Female; Humans; Imidazoles; Inhibitory Concentration 50; MAP Kinase Signaling System; MCF-7 Cells; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Proto-Oncogene Proteins c-akt; Pyridazines; Pyridones; Pyrimidinones; Quinolines; Signal Transduction; Sirolimus; Sulfonamides; TOR Serine-Threonine Kinases

The use of molecularly targeted drugs as single agents has shown limited utility in many tumor types, largely due to the complex and redundant nature of oncogenic signaling networks. Targeting of the PI3K/AKT/mTOR pathway through inhibition of mTOR in combination with aromatase inhibitors has seen success in particular sub-types of breast cancer and there is a need to identify additional synergistic combinations to maximize the clinical potential of mTOR inhibitors. We have used loss-of-function RNAi screens of the mTOR inhibitor rapamycin to identify sensitizers of mTOR inhibition. RNAi screens conducted in combination with rapamycin in multiple breast cancer cell lines identified six genes, AURKB, PLK1, PIK3R1, MAPK12, PRKD2, and PTK6 that when silenced, each enhanced the sensitivity of multiple breast cancer lines to rapamycin. Using selective pharmacological agents we confirmed that inhibition of AURKB or PLK1 synergizes with rapamycin. Compound-associated gene expression data suggested histone deacetylation (HDAC) inhibition as a strategy for reducing the expression of several of the rapamycin-sensitizing genes, and we tested and validated this using the HDAC inhibitor entinostat in vitro and in vivo. Our findings indicate new approaches for enhancing the efficacy of rapamycin including the use of combining its application with HDAC inhibition.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Aurora Kinase B; Benzamides; Breast Neoplasms; Cell Cycle Proteins; Cell Line, Tumor; Class Ia Phosphatidylinositol 3-Kinase; Drug Screening Assays, Antitumor; Drug Synergism; Female; Humans; MCF-7 Cells; Mice; Mice, SCID; Mitogen-Activated Protein Kinase 12; Neoplasm Proteins; Phosphoinositide-3 Kinase Inhibitors; Polo-Like Kinase 1; Protein Kinase D2; Protein Kinase Inhibitors; Protein Kinases; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Pyridines; Random Allocation; RNA Interference; Sirolimus; Xenograft Model Antitumor Assays

Activation of the phosphatidylinositol 3-kinase (PI3K) pathway in estrogen receptor α (ER)-positive breast cancer is associated with reduced ER expression and activity, luminal B subtype, and poor outcome. Phosphatase and tensin homolog (PTEN), a negative regulator of this pathway, is typically lost in ER-negative breast cancer. We set out to clarify the role of reduced PTEN levels in endocrine resistance, and to explore the combination of newly developed PI3K downstream kinase inhibitors to overcome this resistance.. Altered cellular signaling, gene expression, and endocrine sensitivity were determined in inducible PTEN-knockdown ER-positive/human epidermal growth factor receptor 2 (HER2)-negative breast cancer cell and/or xenograft models. Single or two-agent combinations of kinase inhibitors were examined to improve endocrine therapy.. Moderate PTEN reduction was sufficient to enhance PI3K signaling, generate a gene signature associated with the luminal B subtype of breast cancer, and cause endocrine resistance in vitro and in vivo. The mammalian target of rapamycin (mTOR), protein kinase B (AKT), or mitogen-activated protein kinase kinase (MEK) inhibitors, alone or in combination, improved endocrine therapy, but the efficacy varied by PTEN levels, type of endocrine therapy, and the specific inhibitor(s). A single-agent AKT inhibitor combined with fulvestrant conferred superior efficacy in overcoming resistance, inducing apoptosis and tumor regression.. Moderate reduction in PTEN, without complete loss, can activate the PI3K pathway to cause endocrine resistance in ER-positive breast cancer, which can be overcome by combining endocrine therapy with inhibitors of the PI3K pathway. Our data suggests that the ER degrader fulvestrant, to block both ligand-dependent and -independent ER signaling, combined with an AKT inhibitor is an effective strategy to test in patients.

Topics: Animals; Antineoplastic Agents, Hormonal; Breast Neoplasms; Doxycycline; Drug Resistance, Neoplasm; Estradiol; Female; Fulvestrant; Gene Expression; Gene Knockdown Techniques; Humans; MCF-7 Cells; Mice, Nude; Mitogen-Activated Protein Kinases; Molecular Targeted Therapy; Neoplasms, Hormone-Dependent; Phosphatidylinositol 3-Kinases; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Receptors, Estrogen; Signal Transduction; Sirolimus; Tamoxifen; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays

Topics: Androstadienes; Antineoplastic Agents; Bone Neoplasms; Breast Neoplasms; Drug Therapy, Combination; Everolimus; Female; Humans; Liver Neoplasms; Middle Aged; Neoplasms, Second Primary; Radiodermatitis; Radiotherapy, Adjuvant; Sirolimus

The AKT/mammalian target of rapamycin (mTOR) signaling pathway is regulated by 17α-estradiol (E2) signaling and mediates E2-induced proliferation and progesterone receptor (PgR) expression in breast cancer.. Here we use deep sequencing analysis of previously published data from The Cancer Genome Atlas to demonstrate that expression of a key component of mTOR signaling, rapamycin-insensitive companion of mTOR (Rictor), positively correlated with an estrogen receptor-α positive (ERα+) breast tumor signature. Through increased microRNA-155 (miR-155) expression in the ERα+ breast cancer cells we demonstrate repression of Rictor enhanced activation of mTOR complex 1 (mTORC1) signaling with both qPCR and western blot. miR-155-mediated mTOR signaling resulted in deregulated ERα signaling both in cultured cells in vitro and in xenografts in vivo in addition to repressed PgR expression and activity. Furthermore we observed that miR-155 enhanced mTORC1 signaling (observed through western blot for increased phosphorylation on mTOR S2448) and induced inhibition of mTORC2 signaling (evident through repressed Rictor and tuberous sclerosis 1 (TSC1) gene expression). mTORC1 induced deregulation of E2 signaling was confirmed using qPCR and the mTORC1-specific inhibitor RAD001. Co-treatment of MCF7 breast cancer cells stably overexpressing miR-155 with RAD001 and E2 restored E2-induced PgR gene expression. RAD001 treatment of SCID/CB17 mice inhibited E2-induced tumorigenesis of the MCF7 miR-155 overexpressing cell line. Finally we demonstrated a strong positive correlation between Rictor and PgR expression and a negative correlation with Raptor expression in Luminal B breast cancer samples, a breast cancer histological subtype known for having an altered ERα-signaling pathway.. miRNA mediated alterations in mTOR and ERα signaling establishes a new mechanism for altered estrogen responses independent of growth factor stimulation.

Topics: Animals; Breast Neoplasms; Estrogen Receptor alpha; Estrogens; Everolimus; Female; Gene Expression Regulation, Neoplastic; Humans; MCF-7 Cells; Mechanistic Target of Rapamycin Complex 1; Mechanistic Target of Rapamycin Complex 2; Mice; MicroRNAs; Multiprotein Complexes; Phenotype; Phosphorylation; Proto-Oncogene Proteins c-akt; Receptors, Progesterone; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Everolimus, a mammalian target of Rapamycin inhibitor, has recently been approved for the treatment of metastatic estrogen receptor-positive breast cancer, in combination with exemestane at a daily dose of 10mg. In the literature, few cases of acute kidney injury have been reported related to everolimus use, but none of them in a patient with breast cancer as we report here. Our case report of acute kidney injury demonstrates the potential nephrotoxic effects of everolimus therapy, necessitating close monitoring of renal function prior to, during and after discontinuation of the drug.. We report the first published case of acute kidney injury shortly after initiation of exemestane and everolimus for metastatic breast cancer resistant to letrozole in a 69-year-old Caucasian woman, initially treated for a stage IIB estrogen receptor-positive breast cancer in 1997. Within 2 weeks of therapy, she developed grade 1 to 2 diarrhea, lower extremity edema, lethargy, and anorexia. After 4 weeks of therapy, her blood pressure was 85/59 mmHg and she lost 4 kg bodyweight. Her serum creatinine was 3.34 mg/dL. Everolimus was stopped, and she was hospitalized for rehydration. Her serum creatinine levels peaked at 8.85 mg/dL 8 days after treatment discontinuation, with a calculated creatinine clearance of 7 mL/minute. Dialysis was not required. A month later, her serum creatinine levels slowly dropped to 2.26 mg/dL but did not return to baseline. No re-challenge of everolimus was attempted.. Extreme vigilance should be used when prescribing everolimus for metastatic breast cancer. Although the exact cause of acute kidney injury in our case is unknown, dehydration must be avoided and renal function closely monitored after initiating therapy. Spontaneous recovery after drug discontinuation is possible.

Topics: Acute Kidney Injury; Aged; Androstadienes; Antineoplastic Agents; Breast Neoplasms; Creatinine; Drug Therapy, Combination; Everolimus; Female; Humans; Sirolimus

Phorbol ester (PMA or TPA), a tumor promoter, can cause either proliferation or cell cycle arrest, depending on cellular context. For example, in SKBr3 breast cancer cells, PMA hyper-activates the MEK/MAPK pathway, thus inducing p21 and cell cycle arrest. Here we showed that PMA-induced arrest was followed by conversion to cellular senescence (geroconversion). Geroconversion was associated with active mTOR and S6 kinase (S6K). Rapamycin suppressed geroconversion, maintaining quiescence instead. In this model, PMA induced arrest (step one of a senescence program), whereas constitutively active mTOR drove geroconversion (step two). Without affecting Akt phosphorylation, PMA increased phosphorylation of S6K (T389) and S6 (S240/244), and that was completely prevented by rapamycin. Yet, T421/S424 and S235/236 (p-S6K and p-S6, respectively) phosphorylation became rapamycin-insensitive in the presence of PMA. Either MEK or mTOR was sufficient to phosphorylate these PMA-induced rapamycin-resistant sites because co-treatment with U0126 and rapamycin was required to abrogate them. We next tested whether activation of rapamycin-insensitive pathways would shift quiescence towards senescence. In HT-p21 cells, cell cycle arrest was caused by IPTG-inducible p21 and was spontaneously followed by mTOR-dependent geroconversion. Rapamycin suppressed geroconversion, whereas PMA partially counteracted the effect of rapamycin, revealing the involvement of rapamycin-insensitive gerogenic pathways. In normal RPE cells arrested by serum withdrawal, the mTOR/pS6 pathway was inhibited and cells remained quiescent. PMA transiently activated mTOR, enabling partial geroconversion. We conclude that PMA can initiate a senescent program by either inducing arrest or fostering geroconversion or both. Rapamycin can decrease gero-conversion by PMA, without preventing PMA-induced arrest. The tumor promoter PMA is a gero-promoter, which may be useful to study aging in mammals.

Topics: Breast Neoplasms; Carcinogens; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cellular Senescence; Drug Interactions; Female; Fibrosarcoma; Humans; MAP Kinase Signaling System; Phosphorylation; Sirolimus; Tetradecanoylphorbol Acetate

Rapamycin is an effective anti-angiogenic drug. However, the mode of its action remains unclear. Therefore, in this study, we aimed to elucidate the antitumor mechanism of rapamycin, hypothetically via apoptotic promotion, using MCF-7 breast cancer cells.. MCF-7 cells were plated at a density of 15105 cells/well in 6-well plates. After 24h, cells were treated with a series of concentrations of rapamycin while only adding DMEM medium with PEG for the control regiment and grown at 37oC, 5% CO2 and 95% air for 72h. Trypan blue was used to determine the cell viability and proliferation. Untreated and rapamycin-treated MCF-7 cells were also examined for morphological changes with an inverted-phase contrast microscope. Alteration in cell morphology was ascertained, along with a stage in the cell cycle and proliferation. In addition, cytotoxicity testing was performed using normal mouse breast mammary pads.. Our results clearly showed that rapamycin exhibited inhibitory activity on MCF-7 cell lines. The IC50 value of rapamycin on the MCF-7 cells was determined as 0.4μg/ml (p<0.05). Direct observation by inverted microscopy demonstrated that the MCF-7 cells treated with rapamycin showed characteristic features of apoptosis including cell shrinkage, vascularization and autophagy. Cells underwent early apoptosis up to 24% after 72h. Analysis of the cell cycle showed an increase in the G0G1 phase cell population and a corresponding decrease in the S and G2M phase populations, from 81.5% to 91.3% and 17.3% to 7.9%, respectively.. This study demonstrated that rapamycin may potentially act as an anti-cancer agent via the inhibition of growth with some morphological changes of the MCF-7 cancer cells, arrest cell cycle progression at G0/G1 phase and induction of apoptosis in late stage of apoptosis. Further studies are needed to further characterize the mode of action of rapamycin as an anti-cancer agent.

Topics: Animals; Antibiotics, Antineoplastic; Apoptosis; Breast Neoplasms; Cell Cycle; Cell Proliferation; Female; Flow Cytometry; Humans; Mammary Glands, Animal; MCF-7 Cells; Mice; Sirolimus

Acquisition of resistance to aromatase inhibitors (AIs) remains a major drawback in the treatment of estrogen receptor alpha (ERα)-positive breast cancers. The Res-Ana cells, a new model of acquired resistance to anastrozole, were established by long-term exposure of aromatase-overexpressing MCF-7 cells to this drug. These resistant cells developed ER-independent mechanisms of resistance and decreased sensitivity to the AI letrozole or to ERα antagonists. They also displayed a constitutive activation of the PI3K/Akt/mTOR pathway and a deregulated expression of several ErbB receptors. An observed increase in the phospho-Akt/Akt ratio between primary and matched recurrent breast tumors of patients who relapsed under anastrozole adjuvant therapy also argued for a pivotal role of the Akt pathway in acquired resistance to anastrozole. Ectopic overexpression of constitutively active Akt1 in control cells was sufficient to induce de novo resistance to anastrozole. Strikingly, combining anastrozole with the highly selective and allosteric Akt inhibitor MK-2206 or with the mTOR inhibitor rapamycin increased sensitivity to this AI in the control cells and was sufficient to overcome resistance and restore sensitivity to endocrine therapy in the resistant cells. Our findings lead to us proposing a model of anastrozole-acquired resistance based on the selection of cancer-initiating-like cells possessing self-renewing properties, intrinsic resistance to anastrozole and sensitivity to MK-2206. Altogether, our work demonstrated that the Akt/mTOR pathway plays a key role in resistance to anastrozole and that combining anastrozole with Akt/mTOR pathway inhibitors represents a promising strategy in the clinical management of hormone-dependent breast cancer patients.

Topics: Anastrozole; Antineoplastic Agents, Hormonal; Aromatase Inhibitors; Breast Neoplasms; Cell Line, Tumor; Drug Resistance, Neoplasm; ErbB Receptors; Estrogen Receptor alpha; Female; Heterocyclic Compounds, 3-Ring; Humans; MCF-7 Cells; Neoplasm Recurrence, Local; Nitriles; Proto-Oncogene Proteins c-akt; Receptor, ErbB-2; Receptor, ErbB-3; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Triazoles

Rapamycin is a selective inhibitor of the mammalian target of rapamycin (mTOR), a regulator kinase that integrates growth factors signaling via the phosphoinositide-3-kinase pathway and that has emerged as a novel therapeutic modality in breast cancer (BC). We propose a pre-clinical "ex-vivo" personalized organotypic culture of BC that preserves the microenvironment to evaluate rapamycin-mediated gene expression changes. Freshly excised ductal invasive BC slices, 400 μm thick (n=30), were cultured in the presence or absence (control) of rapamycin (20 nM) for 24 h. Some slices were formalin-fixed for immunohistochemical determinations and some were processed for microarray analysis. Control slices in culture retained their tissue morphology and tissue viability (detected by BrdU uptake). The percentage of proliferating cells (assessed by Ki67) did not change up to 24 h of treatment. Immunohistochemical evaluation of p-AKT, p-mTOR, p-4EBP1 and p-S6K1 indicated that AKT/mTOR pathway activation was maintained during cultivation. For microarray analysis, slices were divided into two groups, according to the presence/absence of epidermal growth factor receptor-type 2 and analyzed separately. Limited overlap was seen among differentially expressed genes after treatment (P<0.01) in both groups suggesting different responses to rapamycin between these BC subtypes. Ontology analysis indicated that genes involved in biosynthetic processes were commonly reduced by rapamycin. Our network analysis suggested that concerted expression of these genes might distinguish controls from treated slices. Thus, breast carcinoma slices constitute a suitable physiological tool to evaluate the short-term effects of rapamycin on the gene profile of individual BC samples.

Topics: Adult; Aged; Aged, 80 and over; Breast Neoplasms; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Regulatory Networks; Humans; Immunohistochemistry; Ki-67 Antigen; Middle Aged; Models, Biological; Neoplasm Proteins; Phosphoproteins; Receptor, ErbB-2; Sirolimus; Tissue Culture Techniques

We report the clinical features and management outcomes in 7 patients with everolimus-related stomatitis.. Fifteen women with hormone-receptor-positive advanced breast cancer receiving everolimus combined with exemestane were prospectively evaluated to assess the development of stomatitis. Oral ulcers were diagnosed based on established criteria.. Seven patients developed stomatitis (46.6%). All patients were treated with topical dexamethasone solution, while everolimus was temporarily discontinued in 4 patients. Stomatitis resolved within 1-2 weeks. Two of the 4 patients, who had interrupted everolimus, developed recurrent stomatitis following drug resume and everolimus was again discontinued and restarted after 2 weeks. To date, 5 patients receive everolimus in full dose. The 2 patients, who developed recurrent stomatitis, received a reduced dose.. Everolimus-related oral ulcers were frequent and led to dose modifications. Controlled trials, endorsing a consensus in terminology, are needed to evaluate measures on prevention and management of this unique toxicity.

Topics: Aged; Androstadienes; Anti-Inflammatory Agents; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Carcinoma; Dexamethasone; Everolimus; Female; Follow-Up Studies; Glucocorticoids; Humans; Middle Aged; Oral Ulcer; Prospective Studies; Recurrence; Sirolimus; Stomatitis; Stomatitis, Aphthous; TOR Serine-Threonine Kinases; Treatment Outcome

Basal-like breast cancer is an aggressive disease for which targeted therapies are lacking. Recent studies showed that basal-like breast cancer is frequently associated with an increased activity of the phosphatidylinositol 3-kinase (PI3K) pathway, which is critical for cell growth, survival, and angiogenesis. To investigate the therapeutic potential of PI3K pathway inhibition in the treatment of basal-like breast cancer, we evaluated the antitumor effect of the mTOR inhibitor MK-8669 and AKT inhibitor MK-2206 in WU-BC4 and WU-BC5, two patient-derived xenograft models of basal-like breast cancer. Both models showed high levels of AKT phosphorylation and loss of PTEN expression. We observed a synergistic effect of MK-8669 and MK-2206 on tumor growth and cell proliferation in vivo. In addition, MK-8669 and MK-2206 inhibited angiogenesis as determined by CD31 immunohistochemistry. Biomarker studies indicated that treatment with MK-2206 inhibited AKT activation induced by MK-8669. To evaluate the effect of loss of PTEN on tumor cell sensitivity to PI3K pathway inhibition, we knocked down PTEN in WU-BC3, a basal-like breast cancer cell line with intact PTEN. Compared with control (GFP) knockdown, PTEN knockdown led to a more dramatic reduction in cell proliferation and tumor growth inhibition in response to MK-8669 and MK-2206 both in vitro and in vivo. Furthermore, a synergistic effect of these two agents on tumor volume was observed in WU-BC3 with PTEN knockdown. Our results provide a preclinical rationale for future clinical investigation of this combination in basal-like breast cancer with loss of PTEN.

Topics: Animals; Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Disease Models, Animal; Drug Resistance, Neoplasm; Drug Synergism; Enzyme Activation; Female; Gene Knockdown Techniques; Heterocyclic Compounds, 3-Ring; Humans; Inhibitory Concentration 50; Neoplasms, Basal Cell; Neovascularization, Pathologic; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Tumor Burden; Xenograft Model Antitumor Assays

Topics: Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Everolimus; Female; Humans; Randomized Controlled Trials as Topic; Sirolimus; TOR Serine-Threonine Kinases

Numerous nanocarriers of small molecules depend on either non-specific physical encapsulation or direct covalent linkage. In contrast, this manuscript explores an alternative encapsulation strategy based on high-specificity avidity between a small molecule drug and its cognate protein target fused to the corona of protein polymer nanoparticles. With the new strategy, the drug associates tightly to the carrier and releases slowly, which may decrease toxicity and promote tumor accumulation via the enhanced permeability and retention effect. To test this hypothesis, the drug Rapamycin (Rapa) was selected for its potent anti-proliferative properties, which give it immunosuppressant and anti-tumor activity. Despite its potency, Rapa has low solubility, low oral bioavailability, and rapid systemic clearance, which make it an excellent candidate for nanoparticulate drug delivery. To explore this approach, genetically engineered diblock copolymers were constructed from elastin-like polypeptides (ELPs) that assemble small (<100nm) nanoparticles. ELPs are protein polymers of the sequence (Val-Pro-Gly-Xaa-Gly)n, where the identity of Xaa and n determine their assembly properties. Initially, a screening assay for model drug encapsulation in ELP nanoparticles was developed, which showed that Rose Bengal and Rapa have high non-specific encapsulation in the core of ELP nanoparticles with a sequence where Xaa=Ile or Phe. While excellent at entrapping these drugs, their release was relatively fast (2.2h half-life) compared to their intended mean residence time in the human body. Having determined that Rapa can be non-specifically entrapped in the core of ELP nanoparticles, FK506 binding protein 12 (FKBP), which is the cognate protein target of Rapa, was genetically fused to the surface of these nanoparticles (FSI) to enhance their avidity towards Rapa. The fusion of FKBP to these nanoparticles slowed the terminal half-life of drug release to 57.8h. To determine if this class of drug carriers has potential applications in vivo, FSI/Rapa was administered to mice carrying a human breast cancer model (MDA-MB-468). Compared to free drug, FSI encapsulation significantly decreased gross toxicity and enhanced the anti-cancer activity. In conclusion, protein polymer nanoparticles decorated with the cognate receptor of a high potency, low solubility drug (Rapa) efficiently improved drug loading capacity and its release. This approach has applications to the delivery of Rapa and its

Topics: Amino Acid Sequence; Animals; Antibiotics, Antineoplastic; Breast; Breast Neoplasms; Cell Line, Tumor; Drug Carriers; Elastin; Female; Humans; Mice; Mice, Nude; Molecular Sequence Data; Nanoparticles; Peptides; Sirolimus; TOR Serine-Threonine Kinases

Everolimus (EVE; an inhibitor of mammalian target of rapamycin [mTOR]) enhances treatment options for postmenopausal women with hormone-receptor-positive (HR(+)), human epidermal growth factor receptor-2-negative (HER2(-)) advanced breast cancer (ABC) who progress on a non-steroidal aromatase inhibitor (NSAI). This is especially true for patients with visceral disease, which is associated with poor prognosis. The BOLERO-2 (Breast cancer trial of OraLEveROlimus-2) trial showed that combination treatment with EVE and exemestane (EXE) versus placebo (PBO)+EXE prolonged progression-free survival (PFS) by both investigator (7.8 versus 3.2 months, respectively) and independent (11.0 versus 4.1 months, respectively) central assessment in postmenopausal women with HR(+), HER2(-) ABC recurring/progressing during/after NSAI therapy. The BOLERO-2 trial included a substantial proportion of patients with visceral metastases (56%).. Prespecified exploratory subgroup analysis conducted to evaluate the efficacy and safety of EVE+EXE versus PBO+EXE in a prospectively defined subgroup of patients with visceral metastases.. At a median follow-up of 18 months, EVE+EXE significantly prolonged median PFS compared with PBO+EXE both in patients with visceral metastases (N=406; 6.8 versus 2.8 months) and in those without visceral metastases (N=318; 9.9 versus 4.2 months). Improvements in PFS with EVE+EXE versus PBO+EXE were also observed in patients with visceral metastases regardless of Eastern Cooperative Oncology Group performance status (ECOG PS). Patients with visceral metastases and ECOG PS 0 had a median PFS of 6.8 months with EVE+EXE versus 2.8 months with PBO+EXE. Among patients with visceral metastases and ECOG PS ≥1, EVE+EXE treatment more than tripled median PFS compared with PBO+EXE (6.8 versus 1.5 months).. Adding EVE to EXE markedly extended PFS by ≥4 months among patients with HR(+) HER2(-) ABC regardless of the presence of visceral metastases.

Topics: Aged; Androstadienes; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Clinical Trials, Phase III as Topic; Everolimus; Exanthema; Fatigue; Female; Follow-Up Studies; Humans; Kaplan-Meier Estimate; Middle Aged; Multicenter Studies as Topic; Neoplasm Metastasis; Placebos; Postmenopause; Prognosis; Randomized Controlled Trials as Topic; Receptor, ErbB-2; Receptors, Steroid; Sirolimus; Stomatitis; Treatment Outcome; Viscera

Everolimus, an mTOR inhibitor, showed great clinical efficacy in combination with tamoxifen, letrozole, or exemestane for the treatment of estrogen receptor-positive (ER+) breast cancer. However, its antitumor activity was shown to be compromised by a compensatory process involving AKT activation. Here, it was determined whether combining an additional PI3K inhibitor can reverse this phenomenon and improve treatment efficacy. In breast cancer cells (MCF-7 and BT474), everolimus inhibited the mTOR downstream activity by limiting phosphorylation of p70S6K and 4EBP1, which resulted in p-Ser473-AKT activation. However, addition of a LY294002, a PI3K inhibitor, to tamoxifen and everolimus treatment improved the antitumor effect compared with tamoxifen alone or the other two agents in combination. Moreover, LY294002 suppressed the activity of the PI3K/AKT/mTOR axis and mitigated the p-Ser473-AKT activation feedback loop in both cell lines. Critically, this combination scheme also significantly inhibited the expression of HIF-1a, an angiogenesis marker, under hypoxic conditions and reduced blood vessel sprout formation in vitro. Finally, it was shown that the three-agent cocktail had the greatest efficacy in inhibiting MCF-7 xenograft tumor growth and angiogenesis. Taken together, these results suggest that inhibition of PI3K and mTOR may further improve therapy in ER(+) breast cancer cells.. Combinatorial inhibition of the PI3K/AKT/mTOR signaling axis may enhance endocrine-based therapy in breast cancer.

Topics: Aminopyridines; Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Chromones; Everolimus; Female; Humans; Imidazoles; Mammary Neoplasms, Experimental; Mice; Mice, Nude; Morpholines; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Quinolines; Signal Transduction; Sirolimus; Tamoxifen; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays

Activating mutations of the PIK3CA gene occur frequently in breast cancer, and inhibitors that are specific for phosphatidylinositol 3-kinase (PI3K) p110α, such as BYL719, are being investigated in clinical trials. In a search for correlates of sensitivity to p110α inhibition among PIK3CA-mutant breast cancer cell lines, we observed that sensitivity to BYL719 (as assessed by cell proliferation) was associated with full inhibition of signaling through the TORC1 pathway. Conversely, cancer cells that were resistant to BYL719 had persistently active mTORC1 signaling, although Akt phosphorylation was inhibited. Similarly, in patients, pS6 (residues 240/4) expression (a marker of mTORC1 signaling) was associated with tumor response to BYL719, and mTORC1 was found to be reactivated in tumors from patients whose disease progressed after treatment. In PIK3CA-mutant cancer cell lines with persistent mTORC1 signaling despite PI3K p110α blockade (that is, resistance), the addition of the allosteric mTORC1 inhibitor RAD001 to the cells along with BYL719 resulted in reversal of resistance in vitro and in vivo. Finally, we found that growth factors such as insulin-like growth factor 1 and neuregulin 1 can activate mammalian target of rapamycin (mTOR) and mediate resistance to BYL719. Our findings suggest that simultaneous administration of mTORC1 inhibitors may enhance the clinical activity of p110α-targeted drugs and delay the appearance of resistance.

Topics: Adult; Animals; Breast Neoplasms; Cell Line, Tumor; Class I Phosphatidylinositol 3-Kinases; Drug Resistance, Neoplasm; Everolimus; Female; Humans; Inhibitory Concentration 50; Insulin-Like Growth Factor I; Mechanistic Target of Rapamycin Complex 1; Mice; Middle Aged; Multiprotein Complexes; Mutation; Neuregulin-1; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase Inhibitors; Ribosomal Protein S6; Sirolimus; TOR Serine-Threonine Kinases; Treatment Outcome

Novel methods to control and treat metastatic breast cancer are needed. Interleukin (IL)-15 is a promising cytokine for cancer immunotherapy, and everolimus is an orally administered mammalian target of rapamycin (mTOR) inhibitor, which is already approved for cancer treatment. In the present study, we investigated the efficacy of IL-15 gene therapy and explored the possibility of combining IL-15 therapy with everolimus to treat metastatic breast cancer.. A plasmid encoding IL-15 and everolimus were given to mice inoculated with 4 T1 mouse breast cancer cells. Tumor size and metastasis were monitored to assess the effect of different treatment regimens. Immunohistochemistry was used to detect CD4⁺, CD8⁺ and NKG2D⁺ cells and also the expression of Ki-67 in tumor tissue; these analyses helped establish the immunization status and tumor proliferation rate of different treatment groups. Terminal deoxynucleotidyl transferase dUTP nick end labeling assays were performed to assess cellular apoptosis in tumor tissues.. Both IL-15 and everolimus significantly decreased tumor size. IL-15 gene therapy increased the proportion of CD4⁺ T and natural killer (NK) cells but had no effect on CD8⁺ T cells. By contrast, everolimus decreased the number of CD8⁺ T cells but had no effect on CD4⁺ T and NK cells compared to the control group. Both IL-15 and everolimus decreased expression of Ki-67 and increased rates of apoptosis. Although effective on their own, no synergistic effect was observed with a combined treatment of everolimus and IL-15 gene therapy.. IL-15 gene therapy was potentially useful for the treatment of metastatic breast cancer. The possibility of combining immunotherapy with everolimus requires further study.

Topics: Animals; Antineoplastic Agents; Apoptosis; Breast Neoplasms; Cell Line; Cell Proliferation; Combined Modality Therapy; Disease Models, Animal; Dose-Response Relationship, Drug; Everolimus; Female; Gene Order; Genetic Therapy; Genetic Vectors; Immunotherapy; Interleukin-15; Mice; Neoplasm Metastasis; Protein Kinase Inhibitors; Sirolimus; TOR Serine-Threonine Kinases; Tumor Burden

Hypoxia can activate autophagy, a self-digest adaptive process that maintains cell turnover. Mammalian target of rapamycin (mTOR) inhibitors are used to treat cancer but also stimulate autophagy.. Human mammary cancer cells and derived xenografts were used to examine whether hypoxia could exacerbate autophagy-mediated resistance to the mTOR inhibitor rapamycin.. Rapamycin exerted potent antitumour effects in MCF-7 and MDA-MB-231 mammary tumours through a marked inhibition of angiogenesis, but the autophagy inhibitor chloroquine (CQ) failed to further sensitise tumours to mTOR inhibition. Rapamycin treatment actually led to tumour reoxygenation, thereby preventing the development of autophagy. Chloroquine alone, however, blocked the growth of MCF-7 tumours and in vitro blunted the hypoxia-induced component of autophagy in these cells. Finally, when initiating CQ treatment in large, hypoxic tumours, a robust antitumour effect could be observed, which also further increased the antiproliferative effects of rapamycin.. The mTOR inhibitor rapamycin significantly contributes to tumour growth inhibition and normalisation of the tumour vasculature through potent antiangiogenic effects. The resulting reduction in hypoxia accounts for a lack of sensitisation by the autophagy inhibitor CQ, except if the tumours are already at an advanced stage, and thus largely hypoxic at the initiation of the combination of rapamycin and CQ treatment.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Autophagy; Breast Neoplasms; Cell Hypoxia; Chloroquine; Female; Humans; Mammary Neoplasms, Experimental; Mice, Nude; Sirolimus; TOR Serine-Threonine Kinases; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Topics: Aged; Antineoplastic Agents; Breast Neoplasms; Everolimus; Female; Hemorrhage; Humans; Lung Diseases; Lung Injury; Neoplasm Recurrence, Local; Sirolimus; Tomography, X-Ray Computed

Inhibitors of the mammalian target of rapamycin (mTORi) have clinical activity; however, the benefits of mTOR inhibition by rapamycin and rapamycin-derivatives (rapalogs) may be limited by a feedback mechanism that results in AKT activation. Increased AKT activity resulting from mTOR inhibition can be a result of increased signaling via the mTOR complex, TORC2. Previously, we published that arsenic trioxide (ATO) inhibits AKT activity and in some cases, decreases AKT protein expression. Therefore, we propose that combining ATO and rapamycin may circumvent the AKT feedback loop and increase the anti-tumor effects. Using a panel of breast cancer cell lines, we find that ATO, at clinically-achievable doses, can enhance the inhibitory activity of the mTORi temsirolimus. In all cell lines, temsirolimus treatment resulted in AKT activation, which was decreased by concomitant ATO treatment only in those cell lines where ATO enhanced growth inhibition. Treatment with rapalog also results in activated ERK signaling, which is decreased with ATO co-treatment in all cell lines tested. We next tested the toxicity and efficacy of rapamycin plus ATO combination therapy in a MDA-MB-468 breast cancer xenograft model. The drug combination was well-tolerated, and rapamycin did not increase ATO-induced liver enzyme levels. In addition, combination of these drugs was significantly more effective at inhibiting tumor growth compared to individual drug treatments, which corresponded with diminished phospho-Akt and phospho-ERK levels when compared with rapamycin-treated tumors. Therefore, we propose that combining ATO and mTORi may overcome the feedback loop by decreasing activation of the MAPK and AKT signaling pathways.

Topics: Animals; Antineoplastic Agents; Arsenic Trioxide; Arsenicals; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Feedback, Physiological; Humans; Mice; Oxides; Phosphorylation; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirolimus; Xenograft Model Antitumor Assays

In one trial, combination therapy with everolimus and exemestane prolonged progression-free survival a few months (on the basis of radiological studies) compared with exemestane alone, which is not the standard treatment. However, the addition of everolimus greatly increased the incidence of serious adverse effects. As of mid-2013, the effect on overall survival is uncertain.

Topics: Antineoplastic Agents; Breast Neoplasms; Everolimus; Female; Humans; Neoplasm Metastasis; Sirolimus

Topics: Antibodies, Monoclonal; Antineoplastic Agents; Breast; Breast Neoplasms; Drug Discovery; Everolimus; Female; HSP90 Heat-Shock Proteins; Humans; Immunoconjugates; Neoplasm Metastasis; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Quinolines; Sirolimus

The mammalian target of rapamycin (mTOR) signaling pathway is upregulated in the pathogenesis of many cancers. Arachidonic acid (AA) and its metabolites play critical role in the development of breast cancer, but the mechanisms through which AA promotes mammary tumorigenesis and progression are poorly understood. We found that the levels of AA and cytosolic phospholipase A2 (cPLA2) strongly correlated with the signaling activity of mTORC1 and mTORC2 as well as the expression levels of vascular epithelial growth factor (VEGF) in human breast tumor tissues. In cultured breast cancer cells, AA effectively activated both mTOR complex 1 (mTORC1) and mTORC2. Interestingly, AA-stimulated mTORC1 activation was independent of amino acids, phosphatidylinositol 3-kinase (PI3-K) and tuberous sclerosis complex 2 (TSC2), which suggests a novel mechanism for mTORC1 activation. Further studies revealed that AA stimulated mTORC1 activity through destabilization of mTOR-raptor association in ras homolog enriched in brain (Rheb)-dependent mechanism. Moreover, we showed that AA-stimulated cell proliferation and angiogenesis required mTOR activity and that the effect of AA was mediated by lipoxygenase (LOX) but not cyclooxygenase-2 (COX-2). In animal models, AA-enhanced incidences of rat mammary tumorigenesis, tumor weights and angiogenesis were inhibited by rapamycin. Our findings suggest that AA is an effective intracellular stimulus of mTOR and that AA-activated mTOR plays critical roles in angiogenesis and tumorigenesis of breast cancer.

Topics: Animals; Arachidonic Acid; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Chick Embryo; Cyclooxygenase 2; Female; Humans; Lipoxygenase; MCF-7 Cells; Mechanistic Target of Rapamycin Complex 1; Mechanistic Target of Rapamycin Complex 2; Multiprotein Complexes; Neovascularization, Pathologic; Phosphatidylinositol 3-Kinase; Phospholipases A2; Proteins; Rats; Rats, Sprague-Dawley; RNA Interference; RNA, Small Interfering; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Tuberous Sclerosis Complex 2 Protein; Tumor Suppressor Proteins; Vascular Endothelial Growth Factor A

The purpose of this study was to clarify the mechanism of acquired resistance to the insulin-like growth factor-1 receptor (IGF-1R) tyrosine kinase inhibitor NVP-AEW541. We developed an acquired resistant model by continuously exposing MCF-7 breast cancer cells to NVP-AEW541 (MCF-7-NR). MCF-7 and MCF-7-NR were comparatively analyzed for cell signaling and cell growth. While phosphorylation of Akt was completely inhibited by 3 μM NVP-AEW541 in both MCF-7 and MCF-7-NR, phosphorylation of S6K remained high only in MCF-7-NR, suggesting a disconnection between Akt and S6K in MCF-7-NR. Consistently, the mTOR inhibitor everolimus inhibited phosphorylation of S6K and cell growth equally in both lines. Screening of both lines for phosphorylation of 42 receptor tyrosine kinases with and without NVP-AEW541 showed that Tyro3 phosphorylation remained high only in MCF-7-NR. Protein expression of Tyro3 was found to be higher in MCF-7-NR than in MCF-7. Gene silencing of Tyro3 using siRNA resulted in reduced cell growth and cyclin D1 expression in both lines. While Tyro3 expression was inhibited by NVP-AEW541 and everolimus in MCF-7, it was reduced only by everolimus in MCF-7-NR. These findings suggested that cyclin D1 expression was regulated in a S6K/Tyro3-dependent manner in both MCF-7 and MCF-7-NR, and that the disconnection between IGF-1R/Akt and S6K may enable MCF-7-NR to keep cyclin D1 high in the presence of NVP-AEW541. In summary, acquired resistance to NVP-AEW541 appears to result from IGF-1R/Akt-independent activation of S6K and expression of Tyro3 and cyclin D1.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Blotting, Western; Breast Neoplasms; Cell Proliferation; Drug Resistance, Neoplasm; Drug Synergism; Everolimus; Female; Humans; Immunosuppressive Agents; Phosphorylation; Pyrimidines; Pyrroles; Receptor, IGF Type 1; Ribosomal Protein S6 Kinases; Sirolimus; Tumor Cells, Cultured

To estimate the budget impact of everolimus as the first and second treatment option after letrozole or anastrozole (L/A) failure for post-menopausal women with hormone receptor positive (HR+), human epidermal growth factor receptor-2 negative (HER2-) advanced breast cancer (ABC).. Pharmacy and medical budget impacts (2011 USD) were estimated over the first year of everolimus use in HR+, HER2- ABC from a US payer perspective. Epidemiology data were used to estimate target population size. Pre-everolimus entry treatment options included exemestane, fulvestrant, and tamoxifen. Pre- and post-everolimus entry market shares were estimated based on market research and assumptions. Drug costs were based on wholesale acquisition cost. Patients were assumed to be on treatment until progression or death. Annual medical costs were calculated as the average of pre- and post-progression medical costs weighted by the time in each period, adjusted for survival. One-way and two-way sensitivity analyses were conducted to assess the model robustness.. In a hypothetical 1,000,000 member plan, 72 and 159 patients were expected to be candidates for everolimus treatment as first and second treatment option, respectively, after L/A failure. The total budget impact for the first year post-everolimus entry was $0.044 per member per month [PMPM] (pharmacy budget: $0.058 PMPM; medical budget: -$0.014 PMPM), assuming 10% of the target population would receive everolimus. The total budget impacts for the first and second treatment options after L/A failure were $0.014 PMPM (pharmacy budget: $0.018; medical budget: -$0.004) and $0.030 PMPM (pharmacy budget: $0.040; medical budget: -$0.010), respectively. Results remained robust in sensitivity analyses.. Assumptions about some model input parameters were necessary and may impact results.. Increased pharmacy costs for HR+, HER2- ABC following everolimus entry are expected to be partially offset by reduced medical service costs. Pharmacy and total budget increases were modest.

Topics: Anastrozole; Antineoplastic Agents; Breast Neoplasms; Budgets; Cost-Benefit Analysis; Drug Costs; ErbB Receptors; Everolimus; Female; Health Care Costs; Humans; Letrozole; Middle Aged; Nitriles; Receptor, ErbB-2; Severity of Illness Index; Sirolimus; Treatment Failure; Triazoles; United States

The mammalian target of rapamycin (mTOR) is a key regulator of cell growth and its uncontrolled activation is a hallmark of cancer. Moreover, mTOR activation has been implicated in the resistance of cancer cells to many anticancer drugs, rendering this pathway a promising pharmacotherapeutic target. Here we explored the capability of a semisynthetic compound to intercept mTOR signaling. We synthesized and chemically characterized a novel, semisynthetic triterpenoid derivative, 3-cinnamoyl-11-keto-β-boswellic acid (C-KβBA). Its pharmacodynamic effects on mTOR and several other signaling pathways were assessed in a number of prostate and breast cancer cell lines as well as in normal prostate epithelial cells. C-KβBA exhibits specific antiproliferative and proapoptotic effects in cancer cell lines in vitro as well as in PC-3 prostate cancer xenografts in vivo. Mechanistically, the compound significantly inhibits the cap-dependent transition machinery, decreases expression of eukaryotic translation initiation factor 4E and cyclin D1, and induces G(1) cell-cycle arrest. In contrast to conventional mTOR inhibitors, C-KβBA downregulates the phosphorylation of p70 ribosomal S6 kinase, the major downstream target of mTOR complex 1, without concomitant activation of mTOR complex 2/Akt and extracellular signal-regulated kinase pathways, and independently of protein phosphatase 2A, liver kinase B1/AMP-activated protein kinase/tuberous sclerosis complex, and F12-protein binding. At the molecular level, the compound binds to the FKBP12-rapamycin-binding domain of mTOR with high affinity, thereby competing with the endogenous mTOR activator phosphatidic acid. C-KβBA represents a new type of proapoptotic mTOR inhibitor that, due to its special mechanistic profile, might overcome the therapeutic drawbacks of conventional mTOR inhibitors.

Topics: Apoptosis; Breast Neoplasms; Cell Cycle Checkpoints; Cell Proliferation; Down-Regulation; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Epithelial Cells; Female; G1 Phase; Humans; Male; Phosphorylation; Prostatic Neoplasms; Protein Interaction Domains and Motifs; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Triterpenes

Cotylenin A, a plant growth regulator, and rapa-mycin, an inhibitor of the mammalian target of rapamycin, are potent inducers of differentiation in myeloid leukemia cells and also synergistically inhibit the proliferation of several human breast cancer cell lines including MCF-7 in vitro and in vivo. However, the mechanisms of the combined effects of cotylenin A and rapamycin are still unknown. Activated Akt induced by rapamycin has been suggested to attenuate the growth-inhibitory effects of rapamycin, serving as a negative feedback mechanism. In this study, we found that cotylenin A could suppress rapamycin-induced phosphorylation of Akt (Ser473) in MCF-7 cells and lung carcinoma A549 cells and that cotylenin A also enhanced the rapamycin-induced growth inhibition of MCF-7 and A549 cells. ISIR-005 (a synthetic cotylenin A-derivative) was able to enhance rapamycin‑induced growth inhibition and could also markedly inhibit rapamycin-induced phosphorylation of Akt. We also found that the HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) or arsenic trioxide (ATO) in combination with rapamycin markedly inhibited the growth of MCF-7 cells and 17-AAG or ATO suppressed rapamycin-induced phosphorylation of Akt. The PI3K inhibitor LY294002 also suppressed rapamycin-induced phosphorylation of Akt and combined treatment showed synergistic growth inhibition of MCF-7 cells. Rapamycin inhibited growth more significantly in Akt siRNA-transfected MCF-7 cells than in control siRNA-transfected MCF-7 cells. These results suggest that the inhibition of rapamycin-induced Akt phosphorylation by cotylenin A correlates with their effective growth inhibition of cancer cells.

Topics: Benzoquinones; Breast Neoplasms; Cell Proliferation; Chromones; Diterpenes; Drug Synergism; Female; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Lung Neoplasms; MCF-7 Cells; Morpholines; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Proto-Oncogene Proteins c-akt; Sirolimus

Autophagy is an evolutionarily conserved lysosomal degradation pathway and plays a critical role in the homeostatic process of recycling proteins and organelles. Functional relationships have been described between apoptosis and autophagy. Perturbations in the apoptotic machinery have been reported to induce autophagic cell deaths. Inhibition of autophagy in cancer cells has resulted in cell deaths that manifested hallmarks of apoptosis. However, the molecular relationships and the circumstances of which molecular pathways dictate the choice between apoptosis and autophagy are currently unknown. This study aims to identify specific gene expression of rapamycin-induced autophagy and the effects of rapamycin when the autophagy process is inhibited. In this study, we have demonstrated that rapamycin is capable of inducing autophagy in T-47D breast carcinoma cells. However, when the autophagy process was inhibited by 3-MA, the effects of rapamycin became apoptotic. The Phlda1 gene was found to be up-regulated in both autophagy and apoptosis and silencing this gene was found to reduce both activities, strongly suggests that Phlda1 mediates and positively regulates both autophagy and apoptosis pathways.

Topics: Adenine; Apoptosis; Autophagy; Breast Neoplasms; Cell Line, Tumor; Humans; Oligonucleotide Array Sequence Analysis; Sirolimus; Transcription Factors; Up-Regulation

mTOR inhibitor rapamycin and its analogs are lipophilic, demonstrate blood-brain barrier penetration, and have shown promising antitumor effects in several types of refractory tumors. We thus try to explore the therapeutic effects of mTOR inhibitors on brain metastasis models. We examined the effects of different dose of mTOR inhibitors (rapamycin, Temsirolimus-CCI-779) on cell invasion in two brain metastatic breast cancer cell lines (MDA-MB231-BR and CN34-BrM2). Antibody microarray and immunoblotting were applied to detect signaling pathways underlying the dose differential drug effects. The in vivo effects of single drug (CCI-779), and drug combination of CCI-779 with SL327 (a brain penetrant MEK inhibitor) to eliminate the unfavorable activation of MAPK pathway were evaluated in MDA-MB231-BR brain metastases xenograft mice. The two mTOR inhibitors, rapamycin and CCI-779, inhibited the invasion of brain metastatic cells only at a moderate concentration level, which was lost at higher concentrations secondary to activation of the MAPK signaling pathway. Pharmacological inhibition of ERK1/2 by PD98059 and SL327 restored the anti-invasion effects of mTOR inhibition in vitro. In vivo, a significant decrease was noted in the average number of micro and large metastatic lesions as well as the whole brain GFP expression in the CCI-779 1 mg/kg/day treated group compared with that in the vehicle group (P < 0.05). However, 10 mg/kg CCI-779 treatment did not show significant anti-metastasis effect on the animal model. High-dose CCI-779 eliciting the ERK MAPK activation in the brain metastatic lesion was corroborated. Combined with the brain penetrant MEK inhibitor SL327, high-dose CCI-779 significantly reduces the brain metastasis, and the combination treatment prohibited perivascular invasion of tumor cells and inhibits tumor angiogenesis in vivo. This study provides evidence on the potential value of CCI-779 as well as CCI-779 + SL327 in prohibiting breast cancer brain metastasis.

Topics: Aminoacetonitrile; Animals; Antineoplastic Agents; Brain Neoplasms; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Female; Humans; MAP Kinase Signaling System; Mice; Mice, Inbred BALB C; Mice, Nude; Mitogen-Activated Protein Kinase Kinases; Neoplasm Invasiveness; Neovascularization, Pathologic; Protein Kinase Inhibitors; Sirolimus; TOR Serine-Threonine Kinases

HER-2-positive breast cancers frequently sustain elevated AKT/mTOR signaling, which has been associated with resistance to doxorubicin treatment. Here, we investigated whether rapamycin, an mTOR inhibitor, increased the sensitivity to doxorubicin therapy in two HER-2-overexpressing cell lines: C5.2, which was derived from the parental HB4a by transfection with HER-2 and SKBR3, which exhibits HER-2 amplification. The epithelial mammary cell line HB4a was also analyzed. The combined treatment using 20 nmol/L of rapamycin and 30 nmol/L of doxorubicin arrested HB4a and C5.2 cells in S to G(2)-M, whereas SKBR3 cells showed an increase in the G(0)-G(1) phase. Rapamycin increased the sensitivity to doxorubicin in HER-2-overexpressing cells by approximately 2-fold, suggesting that the combination displayed a more effective antiproliferative action. Gene expression profiling showed that these results might reflect alterations in genes involved in canonical pathways related to purine metabolism, oxidative phosphorylation, protein ubiquitination, and mitochondrial dysfunction. A set of 122 genes modulated by the combined treatment and specifically related to HER-2 overexpression was determined by finding genes commonly regulated in both C5.2 and SKBR3 that were not affected in HB4a cells. Network analysis of this particular set showed a smaller subgroup of genes in which coexpression pattern in HB4a cells was disrupted in C5.2 and SKBR3. Altogether, our data showed a subset of genes that might be more robust than individual markers in predicting the response of HER-2-overexpressing breast cancers to doxorubicin and rapamycin combination.

Topics: Antibiotics, Antineoplastic; Breast; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Doxorubicin; Drug Synergism; Epithelial Cells; Female; Gene Expression Profiling; Gene Regulatory Networks; Humans; Models, Genetic; Oligonucleotide Array Sequence Analysis; Receptor, ErbB-2; Reverse Transcriptase Polymerase Chain Reaction; Sirolimus

PI3K/Akt/mTOR and p53 signaling pathways are frequently deregulated in tumors. The anticancer drug RAD001 (everolimus) is a known mTOR-inhibitor, but mTOR-inhibition leads to phosphorylation of Akt inducing resistance against RAD001 treatment. There is growing evidence that conflicting signals transduced by the oncogene Akt and the tumorsuppressor p53 are integrated via negative feedback between the two pathways. We previously showed that the anti-malarial Chloroquine, a 4-alkylamino substituted quinoline, is a p53 activator and reduced the incidence of breast tumors in animal models. Additionally, Chloroquine is an effective chemosensitizer when used in combination with PI3K/Akt inhibitors but the mechanism is unknown. Therefore, our aim was to test, if Chloroquine could inhibit tumor growth and prevent RAD001-induced Akt activation. Chloroquine and RAD001 caused G1 cell cycle arrest in luminal MCF7 but not in mesenchymal MDA-MB-231 breast cancer cells, they significantly reduced MCF7 cell proliferation on a collagen matrix and mammospheroid formation. In a murine MCF7 xenograft model, combined treatment of Chloroquine and RAD001 significantly reduced mammary tumor growth by 4.6-fold (p = 0.0002) compared to controls. Chloroquine and RAD001 inhibited phosphorylation of mTOR and its downstream target, S6K1. Furthermore, Chloroquine was able to block the RAD001-induced phosphorylation of Akt serine 473. The Chloroquine effect of overcoming the RAD001-induced activation of the oncogene Akt, as well as the promising antitumor activity in our mammary tumor animal model present Chloroquine as an interesting combination partner for the mTOR-inhibitor RAD001.

Topics: Animals; Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Chloroquine; Everolimus; Female; G1 Phase Cell Cycle Checkpoints; Humans; Immunosuppressive Agents; Mice; Mice, Nude; Neoplasms, Experimental; Oncogene Protein v-akt; Sirolimus; TOR Serine-Threonine Kinases; Tumor Suppressor Protein p53

Chloroquine (CQ) is a 4-aminoquinoline drug used for the treatment of diverse diseases. It inhibits lysosomal acidification and therefore prevents autophagy by blocking autophagosome fusion and degradation. In cancer treatment, CQ is often used in combination with chemotherapeutic drugs and radiation because it has been shown to enhance the efficacy of tumor cell killing. Since CQ and its derivatives are the only inhibitors of autophagy that are available for use in the clinic, multiple ongoing clinical trials are currently using CQ or hydroxychloroquine (HCQ) for this purpose, either alone, or in combination with other anticancer drugs. Here we show that in the mouse breast cancer cell lines, 67NR and 4T1, autophagy is induced by the DNA damaging agent cisplatin or by drugs that selectively target autophagy regulation, the PtdIns3K inhibitor LY294002, and the mTOR inhibitor rapamycin. In combination with these drugs, CQ sensitized to these treatments, though this effect was more evident with LY294002 and rapamycin treatment. Surprisingly, however, in these experiments CQ sensitization occurred independent of autophagy inhibition, since sensitization was not mimicked by Atg12, Beclin 1 knockdown or bafilomycin treatment, and occurred even in the absence of Atg12. We therefore propose that although CQ might be helpful in combination with cancer therapeutic drugs, its sensitizing effects can occur independently of autophagy inhibition. Consequently, this possibility should be considered in the ongoing clinical trials where CQ or HCQ are used in the treatment of cancer, and caution is warranted when CQ treatment is used in cytotoxic assays in autophagy research.

Topics: Animals; Antineoplastic Agents; Autophagy; Autophagy-Related Protein 12; Breast Neoplasms; Cell Line, Tumor; Cell Survival; Chloroquine; Chromones; Cisplatin; Drug Screening Assays, Antitumor; Female; Gene Knockdown Techniques; Humans; Macrolides; Mice; Morpholines; Proteins; Sirolimus; Starvation

Taxanes remain first line chemotherapy in management of metastatic breast cancer and have a key role in epithelial ovarian cancer, with increasingly common use of weekly paclitaxel dosing regimens. However, their clinical utility is limited by the development of chemoresistance. To address this, we modelled in vitro paclitaxel resistance in MCF-7 cells. We show that at clinically relevant drug doses, emerging paclitaxel resistance is associated with profound changes in cell death responses and a switch from apoptosis to autophagy as the principal mechanism of drug-induced cytotoxicity. This was characterised by a complete absence of caspase-mediated apoptotic cell death (using the pan-caspase-inhibitor Z-VAD) in paclitaxel-resistant MCF-7TaxR cells, compared with parent MCF-7 or MDA-MB-231 cell lines on paclitaxel challenge, downregulation of caspase-7, caspase-9 and BCl2-interacting mediator of cell death (BIM) expression. Silencing with small interfering RNA to BIM in MCF-7 parental cells was sufficient to confer paclitaxel resistance, inferring the significance in downregulation of this protein in contributing to the resistant phenotype of the MCF-7TaxR cell line. Conversely, there was an increased autophagic response in the MCF-7TaxR cell line with reduced phospho-mTOR and relative resistance to the mTOR inhibitors rapamycin and RAD001. In conclusion, we show for the first time that paclitaxel resistance is associated with profound changes in cell death response with deletion of multiple apoptotic factors balanced by upregulation of the autophagic pathway and collateral sensitivity to platinum.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Apoptosis Regulatory Proteins; Autophagy; Bcl-2-Like Protein 11; Breast Neoplasms; Caspase 7; Caspase 9; Cell Line, Tumor; Cell Survival; Drug Resistance, Neoplasm; Everolimus; Female; Gene Expression Regulation, Neoplastic; Humans; Immunosuppressive Agents; Membrane Proteins; Paclitaxel; Proto-Oncogene Proteins; RNA, Small Interfering; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Molecularly targeted therapies have emerged as the leading theme in cancer therapeutics. Multi-cytotoxic drug regimens have been highly successful, yet many studies in targeted therapeutics have centered on a single agent. We investigated whether the Src/Abl kinase inhibitor dasatinib displays synergy with other agents in molecularly heterogeneous breast cancer cell lines. MCF-7, SKBR-3, and MDA-MB-231 display different signaling and gene signatures profiles due to expression of the estrogen receptor, ErbB2, or neither. Cell proliferation was measured following treatment with dasatinib±cytotoxic (paclitaxel, ixabepilone) or molecularly targeted agents (tamoxifen, rapamycin, sorafenib, pan PI3K inhibitor LY294002, and MEK/ERK inhibitor U0126). Dose-responses for single or combination drugs were calculated and analyzed by the Chou-Talalay method. The drugs with the greatest level of synergy with dasatinib were rapamycin, ixabepilone, and sorafenib, for the MDA-MB-231, MCF-7, and SK-BR-3 cell lines respectively. However, dasatinib synergized with both cytotoxic and molecularly targeted agents in all three molecularly heterogeneous breast cancer cell lines. These results suggest that effectiveness of rationally designed therapies may not entirely rest on precise identification of gene signatures or molecular profiling. Since a systems analysis that reveals emergent properties cannot be easily performed for each cancer case, multi-drug regimens in the near future will still involve empirical design.

Topics: Antineoplastic Combined Chemotherapy Protocols; Benzenesulfonates; Breast Neoplasms; Butadienes; Cell Line, Tumor; Chromones; Dasatinib; Dose-Response Relationship, Drug; Drug Synergism; Enzyme Inhibitors; Epothilones; Female; Humans; Morpholines; Niacinamide; Nitriles; Paclitaxel; Phenylurea Compounds; Pyridines; Pyrimidines; Signal Transduction; Sirolimus; Sorafenib; Tamoxifen; Thiazoles

Autophagy is one of the major intracellular catabolic pathways, but little is known about the composition of autophagosomes. To study the associated proteins, we isolated autophagosomes from human breast cancer cells using two different biochemical methods and three stimulus types: amino acid deprivation or rapamycin or concanamycin A treatment. The autophagosome-associated proteins were dependent on stimulus, but a core set of proteins was stimulus-independent. Remarkably, proteasomal proteins were abundant among the stimulus-independent common autophagosome-associated proteins, and the activation of autophagy significantly decreased the cellular proteasome level and activity supporting interplay between the two degradation pathways. A screen of yeast strains defective in the orthologs of the human genes encoding for a common set of autophagosome-associated proteins revealed several regulators of autophagy, including subunits of the retromer complex. The combined spatiotemporal proteomic and genetic data sets presented here provide a basis for further characterization of autophagosome biogenesis and cargo selection.

Topics: Amino Acids; Antibodies, Monoclonal; Antiviral Agents; Autophagy; Breast Neoplasms; Electrophoresis, Polyacrylamide Gel; Female; Genetic Testing; Green Fluorescent Proteins; Humans; Immunoprecipitation; Immunosuppressive Agents; Isotope Labeling; Lysosomes; Macrolides; Phagosomes; Proteins; Proteomics; Saccharomyces cerevisiae; Sirolimus; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Starvation; Tumor Cells, Cultured

Topics: Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Breast Neoplasms; Everolimus; Female; Humans; Immunosuppressive Agents; Receptor, ErbB-2; Receptors, Estrogen; Sirolimus; Women's Health

Recent evidence has suggested that breast cancer contains a rare population of cells called cancer stem cells (CSCs), which have an extensive self-renewal ability and contribute to metastasis and therapeutic resistance. This study evaluated the in vitro and in vivo effects of RAD001 (Everolimus) alone or in combination with docetaxel on stem cells from primary breast cancer cells and two breast cancer cell lines (MCF-7 and MDA-MB-231). In In vitro studies, we sorted ESA(+)CD44(+)CD24(-/low) cells as stem cells using flow cytometry from primary breast cancer cells, MCF-7 and MDA-MB-231 cell lines. MTT assays were used to quantify the inhibitory effect of the drugs on total cells and stem cells. Apoptosis and the cell cycle distributions of stem cells were examined by flow cytometry. The tumourigenicity of stem cells after treatment was investigated by soft agar colony formation assays. In In vivo studies, the BALB/c mice were injected with MDA-MB-231 stem cells and the different treatments were administered. After necropsy, the expression of Ki67, CD31, AKT1, and phospho-AKT (Thr308) was analysed by immunohistochemistry. In In vitro studies, all three populations of stem cells were resistant to the standard treatment doses of docetaxel compared with total cells treated with the same drug. Treatment with RAD001 resulted in growth inhibition of all stem cells in a dose-dependent manner. An additive growth inhibitory effect of the combination treatment on the three stem cells was observed in in vitro compared with treatment with RAD001 alone (P<0.001). In addition, an increase in G2/M cell cycle arrest and an increased population of cells in early apoptosis were seen in the combination treatment group compared with either single-agent group (P<0.01). In vivo, the volumes of the xenograft tumours significantly decreased in RAD001 alone group compared to control group (P=0.008), and RAD001 plus docetaxel therapy was much more effective at reducing tumour volume in mice compared with either single-agent alone (P<0.05). Compared with RAD001 alone, the combination of RAD001 and docetaxel reduced the expression of Ki67, CD31, AKT1 and phospho-AKT (Thr308) (P<0.05). We conclude that the combination treatment of RAD001 and docetaxel can inhibit the growth of stem cells in vitro and in vivo by inhibiting cell proliferation, inducing apoptosis, cell cycle arrest and reducing the expression of Ki67, CD31, AKT1 and phospho-AKT (Thr308). These data indicate that combinati

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Docetaxel; Dose-Response Relationship, Drug; Everolimus; Female; Humans; Immunohistochemistry; Immunosuppressive Agents; Mice; Mice, Inbred BALB C; Mice, Nude; Sirolimus; Stem Cells; Taxoids; Tetrazolium Salts; Thiazoles

We sought to determine whether phosphoinositide 3-kinase (PI3K) pathway mutation or activation state and rapamycin-induced feedback loop activation of Akt is associated with rapamycin sensitivity or resistance.. Cancer cell lines were tested for rapamycin sensitivity, Akt phosphorylation, and mTOR target inhibition. Mice injected with breast or neuroendocrine cancer cells and patients with neuroendocrine tumor (NET) were treated with rapalogs and Akt phosphorylation was assessed.. Thirty-one cell lines were rapamycin sensitive (RS) and 12 were relatively rapamycin resistant (RR; IC(50) > 100 nmol/L). Cells with PIK3CA and/or PTEN mutations were more likely to be RS (P = 0.0123). Akt phosphorylation (S473 and T308) was significantly higher in RS cells (P < 0.0001). Rapamycin led to a significantly greater pathway inhibition and greater increase in p-Akt T308 (P < 0.0001) and p-Akt S473 (P = 0.0009) in RS cells. Rapamycin and everolimus significantly increased Akt phosphorylation but inhibited growth in an in vivo NET model (BON). In patients with NETs treated with everolimus and octreotide, progression-free survival correlated with p-Akt T308 in pretreatment (R = 0.4762, P = 0.0533) and on-treatment tumor biopsies (R = 0.6041, P = 0.0102). Patients who had a documented partial response were more likely to have an increase in p-Akt T308 with treatment compared with nonresponders (P = 0.0146).. PIK3CA/PTEN genomic aberrations and high p-Akt levels are associated with rapamycin sensitivity in vitro. Rapamycin-mediated Akt activation is greater in RS cells, with a similar observation in patients with clinical responses on exploratory biomarker analysis; thus feedback loop activation of Akt is not a marker of resistance but rather may function as an indicator of rapamycin activity.

Topics: Animals; Antibiotics, Antineoplastic; Biomarkers, Tumor; Breast Neoplasms; Carcinoma, Neuroendocrine; Cell Line, Tumor; Class I Phosphatidylinositol 3-Kinases; Drug Resistance, Neoplasm; Everolimus; Female; Humans; Mice; Mice, Nude; Mutation; Phosphatidylinositol 3-Kinases; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Sirolimus; TOR Serine-Threonine Kinases; Transplantation, Heterologous

Elastase-mediated cleavage of cyclin E generates low molecular weight cyclin E (LMW-E) isoforms exhibiting enhanced CDK2-associated kinase activity and resistance to inhibition by CDK inhibitors p21 and p27. Approximately 27% of breast cancers express high LMW-E protein levels, which significantly correlates with poor survival. The objective of this study was to identify the signaling pathway(s) deregulated by LMW-E expression in breast cancer patients and to identify pharmaceutical agents to effectively target this pathway. Ectopic LMW-E expression in nontumorigenic human mammary epithelial cells (hMECs) was sufficient to generate xenografts with greater tumorigenic potential than full-length cyclin E, and the tumorigenicity was augmented by in vivo passaging. However, cyclin E mutants unable to interact with CDK2 protected hMECs from tumor development. When hMECs were cultured on Matrigel, LMW-E mediated aberrant acinar morphogenesis, including enlargement of acinar structures and formation of multi-acinar complexes, as denoted by reduced BIM and elevated Ki67 expression. Similarly, inducible expression of LMW-E in transgenic mice generated hyper-proliferative terminal end buds resulting in enhanced mammary tumor development. Reverse-phase protein array assay of 276 breast tumor patient samples and cells cultured on monolayer and in three-dimensional Matrigel demonstrated that, in terms of protein expression profile, hMECs cultured in Matrigel more closely resembled patient tissues than did cells cultured on monolayer. Additionally, the b-Raf-ERK1/2-mTOR pathway was activated in LMW-E-expressing patient samples, and activation of this pathway was associated with poor disease-specific survival. Combination treatment using roscovitine (CDK inhibitor) plus either rapamycin (mTOR inhibitor) or sorafenib (a pan kinase inhibitor targeting b-Raf) effectively prevented aberrant acinar formation in LMW-E-expressing cells by inducing G1/S cell cycle arrest. LMW-E requires CDK2-associated kinase activity to induce mammary tumor formation by disrupting acinar development. The b-Raf-ERK1/2-mTOR signaling pathway is aberrantly activated in breast cancer and can be suppressed by combination treatment with roscovitine plus either rapamycin or sorafenib.

Topics: Acinar Cells; Animals; Benzenesulfonates; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Cyclin E; Cyclin-Dependent Kinase 2; Female; Gene Expression Regulation, Neoplastic; Humans; Kaplan-Meier Estimate; Mammary Glands, Animal; MAP Kinase Signaling System; Mice; Mice, Nude; Neoplasm Invasiveness; Niacinamide; Phenylurea Compounds; Prognosis; Protein Isoforms; Proto-Oncogene Proteins B-raf; Purines; Pyridines; Retrospective Studies; Roscovitine; Sirolimus; Sorafenib; TOR Serine-Threonine Kinases

Development of resistance to endocrine therapy is a clinical issue in estrogen receptor (ER)-positive breast cancer. Here we show that persistent activation of AKT/mTOR signaling is crucial to the acquisition of letrozole resistance in cell clones generated from MCF-7/AROM-1 aromatase-expressing breast cancer cells after prolonged letrozole exposure. ERα plays a marginal role in this context. As a proof of concept, the association between PI3K/AKT/mTOR signaling and insensitivity to endocrine therapies was confirmed in breast cancer patients who developed early letrozole resistance in neoadjuvant setting. In addition our results suggest that, regardless of the mechanism mediating the activation of AKT/mTOR pathway, either RAD001 or NVP-BEZ235 treatment may represent a promising strategy to overcome acquired resistance to letrozole in breast cancers dependent on AKT/mTOR signaling.

Topics: Aged; Antineoplastic Agents; Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Drug Resistance, Neoplasm; Everolimus; Female; Humans; Imidazoles; Immunosuppressive Agents; Letrozole; Neoadjuvant Therapy; Nitriles; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Quinolines; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Triazoles

This study evaluated the effects of a mammalian target of mTOR inhibitor everolimus alone or in combination with trastuzumab on stem cells from HER2-overexpressing primary breast cancer cells and the BT474 breast cancer cell line in vitro and in vivo. For the in vitro studies, we sorted ESA(+)CD44(+)CD24(-/low) cells as stem cells from primary breast cancer cells and BT474 cells using flow cytometry. The MTT assay was used to quantify the inhibitory effect of the drugs on total cells and stem cells specifically. Stem cell apoptosis, cell cycle distributions, and their tumorigenicity after treatment were investigated by flow cytometry or soft agar colony formation assays. For the in vivo studies, BALB/c mice were injected with BT474 stem cells, and the different treatments were administered. After necropsy, the expression of Ki67, CD31, AKT1, and phospho-AKT (Thr308) was analyzed by immunohistochemistry. For the in vitro studies, Treatment with everolimus resulted in stem cell growth inhibition in a dose-dependent manner. The combination of everolimus with trastuzumab was more effective at inhibiting cell growth (P < 0.001) and tumorigenicity (P < 0.001) compared with single-agent therapy. In addition, an increase in G1 cell cycle arrest and an increased population of cells in early apoptosis were seen in the combination treatment group compared with either of the single-agent groups (P < 0.01). For the in vivo studies, everolimus plus trastuzumab therapy was much more effective at reducing tumor volume in mice compared with either single agent alone (P < 0.05). Compared with everolimus alone, the combination of everolimus and trastuzumab reduced the expression of Ki67, AKT1, and phospho-AKT (Thr308) (P < 0.05). We conclude that everolimus has effective inhibitory effects on HER2-overexpressing stem cells in vitro and vivo. Everolimus plus trastuzumab is a rational combination treatment that may be promising in human clinical trials.

Topics: Animals; Antibodies, Monoclonal, Humanized; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Everolimus; Female; Humans; Inhibitory Concentration 50; Ki-67 Antigen; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplastic Stem Cells; Phosphorylation; Platelet Endothelial Cell Adhesion Molecule-1; Proto-Oncogene Proteins c-akt; Receptor, ErbB-2; Sirolimus; TOR Serine-Threonine Kinases; Trastuzumab; Xenograft Model Antitumor Assays

Topics: Androstadienes; Antineoplastic Combined Chemotherapy Protocols; Aromatase Inhibitors; Breast Neoplasms; Female; Humans; Sirolimus; TOR Serine-Threonine Kinases

Topics: Androstadienes; Antineoplastic Combined Chemotherapy Protocols; Aromatase Inhibitors; Breast Neoplasms; Female; Humans; Sirolimus; TOR Serine-Threonine Kinases

Metformin appears to interfere directly with cell proliferation and apoptosis in cancer cells in a non-insulin-mediated manner. One of the key mechanisms of metformin's action is the activation of adenosine monophosphate activated protein kinase (AMPK). AMPK is linked with the phosphatidylinositol 3-kinase (PI3K)/ phosphatase and tensin homolog (PTEN)/protein kinase B (AKT) pathway and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinases (ERK) cascades--all known for being frequently dysregulated in breast cancer. Therefore, simultaneously targeting AMPK through metformin and the PI3K/AKT/mTOR pathway by an mTOR inhibitor could become a therapeutic approach. The aim of this study was to evaluate the anticancer effect of metformin alone and in combination with chemotherapeutic drugs and the mTOR inhibitor RAD001.. The proliferation of breast cancer cells was measured with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay; and the cell apoptosis with enzyme-linked immunosorbent assay (ELISA). Gene expression at the protein level was determined by western blot.. We tested metformin alone and in combination with RAD001 and/or chemotherapeutic agents (carboplatin, paclitaxel and doxorubicin, respectively) on several human breast cancer cell lines with respect to cell proliferation, apoptosis and autophagy. Metformin alone inhibited cell proliferation and induced apoptosis in different breast cancer cell lines (ERα-positive, HER2-positive, and triple-negative). The cytotoxic effect of metformin was more remarkable in triple-negative breast cancer cell lines than in other cell lines. The cell apoptosis induced by metformin is, at least partly, caspase-dependent and apoptosis inducing factor (AIF)-dependent. Interestingly, we demonstrated that metformin induced cell autophagy. Inhibiting autophagy with chloroquine, enhanced the treatment efficacy of metformin, indicating that autophagy induced by metformin may protect breast cancer cells from apoptosis. We further demonstrated that co-administration of metformin with chemotherapeutic agents and RAD001 intensified the inhibition of cell proliferation. The analysis of cell cycle-regulating proteins cyclin D, cyclin E and p27 by western blot indicated that the synergistic inhibition of G1 phase of the cell cycle by the combination treatment of metformin, chemotherapeutic drugs and/or RAD001 contributed to the synergistic inhibition of cell proliferation.. Our investigation provides a rationale for the clinical application of metformin within treatment regimens for breast cancer.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Autophagy; Breast Neoplasms; Carboplatin; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Everolimus; Female; Humans; Metformin; Sirolimus; TOR Serine-Threonine Kinases

Patients with triple-negative breast cancers (TNBCs) typically have a poor prognosis because such cancers have no effective therapeutic targets, such as estrogen receptors for endocrine therapy or human epidermal growth factor receptor 2 (HER2) receptors for anti-HER2 therapy. As the phosphatidylinositol 3' kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) cascade is activated in TNBCs, mTOR is a potential molecular target for anticancer therapy. In this study, we investigated the antitumor activities of everolimus, an oral mTOR inhibitor, in nine TNBC cell lines. Everolimus effectively inhibited cell growth at concentrations under 100 nM (IC(50)) in five cell lines and even in the 1-nM range in three of the five cell lines. To identify specific characteristics that could be used as predictive markers of efficacy, we evaluated the expressions of proteins in the mTOR cascade, basal markers, and cancer stem cell markers using western blotting, fluorescent in situ hybridization (FISH), or immunohistochemistry. All five of the sensitive cell lines were categorized as a basal-like subtype positive for either epidermal growth factor receptor (EGFR) or CK5/6, although resistant cell lines were not of this subtype and tended to exhibit the characteristics of cancer stem cells, with decreased E-cadherin and the increased expression of Snail or Twist. In vivo assays demonstrated antitumor activity in a mouse xenograft model of basal-like breast cancer, rather than non-basal breast cancer. These results suggest that everolimus has favorable activity against basal-like subtypes of TNBCs. Epidermal growth factor receptor and CK5/6 are positive predictive markers of the TNBC response to everolimus, while cancer stem cell markers are negative predictive markers.

Topics: Animals; Antineoplastic Agents; Biomarkers, Tumor; Breast Neoplasms; Cell Line, Tumor; Drug Resistance, Neoplasm; ErbB Receptors; Everolimus; Female; Gene Expression; Gene Silencing; Humans; Inhibitory Concentration 50; Mice; Mice, Nude; Neoplasms, Basal Cell; Neoplastic Stem Cells; Protein Kinase Inhibitors; PTEN Phosphohydrolase; Receptor, ErbB-2; Receptors, Estrogen; Receptors, Progesterone; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays

The benefit of endocrine therapy has always been limited by the eventual development of acquired resistance. For the first time, clinical research has identified a therapeutic agent, everolimus, that targets the mammalian target of rapamycin (mTOR), which in combination with the aromatase inhibitor exemestane can substantially reduce the risk of disease progression and seemingly circumvent endocrine resistance. The magnitude of the benefit represents a quantum shift in how we should use endocrine therapy in future, and potentially defines a new standard of care in this setting.

Topics: Androstadienes; Antineoplastic Combined Chemotherapy Protocols; Aromatase Inhibitors; Breast Neoplasms; Everolimus; Female; Humans; Immunosuppressive Agents; Sirolimus; TOR Serine-Threonine Kinases

Mammalian target of rapamycin (mTOR) is an attractive target for cancer treatment. While rapamycin and its derivatives (e.g., everolimus) have been shown to inhibit mTOR signaling and cell proliferation in preclinical models of breast cancer, mTOR inhibition has demonstrated variable clinical efficacy with a trend toward better responses in estrogen receptor alpha positive (ERα+) compared to ERα negative (ERα-) tumors. Recently, serum- and glucocorticoid-regulated kinase 1 (SGK1) was identified as a substrate of mTOR kinase activity. Previous studies have alternatively suggested that either mTORC1 or mTORC2 is exclusively required for SGK1's Ser422 phosphorylation and activation in breast cancer cells. We investigated the effect of rapamycin on the growth of several ERα+ and ERα- breast cancer cell lines and examined differences in the phosphorylation of mTOR substrates (SGK1, p70S6K, and Akt) that might account for the differing sensitivity of these cell lines to rapamycin. We also examined which mTOR complex contributes to SGK1-Ser422 phosphorylation in ERα+ versus ERα- breast cell lines. We then assessed whether inhibiting SGK1 activity added to rapamycin-mediated cell growth inhibition by either using the SGK1 inhibitor GSK650394A or expressing an SGK1 shRNA. We observed sensitivity to rapamycin-mediated growth inhibition and inactivation of insulin-mediated SGK1-Ser422 phosphorylation in ERα+ MCF-7 and T47D cells, but not in ERα- MDA-MB-231 or MCF10A-Myc cells. In addition, either depleting SGK1 with shRNA or inhibiting SGK1 with GSK650394A preferentially sensitized MDA-MB-231 cells to rapamycin. Finally, we found that rapamycin-sensitive SGK1-Ser422 phosphorylation required ERα expression in MCF-7 derived cell lines. Therefore, targeting SGK1 activity may improve the efficacy of rapamycin and its analogs in the treatment of ERα- breast cancer.

Topics: Antineoplastic Agents, Hormonal; Breast Neoplasms; Dexamethasone; Drug Resistance, Neoplasm; Enzyme Activation; Enzyme Activators; Estrogen Receptor alpha; Female; Furans; Humans; Immediate-Early Proteins; Insulin; MCF-7 Cells; Mechanistic Target of Rapamycin Complex 1; Mechanistic Target of Rapamycin Complex 2; Multiprotein Complexes; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Proteins; Pyridines; Pyrimidines; Sirolimus; Thapsigargin; TOR Serine-Threonine Kinases

Angiogenesis has become an important target in the treatment of several solid tumors, including breast cancer. As monotherapy, antiangiogenic agents have demonstrated limited activity in metastatic breast cancer (MBC); therefore, they have generally been developed for use in combination with chemotherapies. Thus far, the experience with antiangiogenic agents for MBC has been mixed. The results from one study assessing addition of the monoclonal antibody bevacizumab to paclitaxel led to approval of bevacizumab for MBC. However, the modest improvement of progression-free survival rates in subsequent MBC studies has led to reappraisal of bevacizumab. Phase III studies have not produced evidence supporting use of the multikinase inhibitor sunitinib alone or in combination with MBC chemotherapy. Experience with sorafenib in a phase IIb program indicates potential when used in select combinations, particularly with capecitabine; however, phase III confirmatory data are needed. Although antiangiogenic therapies combined with chemotherapy have increased progression-free survival rates for patients with MBC, increases in overall survival times have not been observed. Some studies have tried to combine antiangiogenic agents such as bevacizumab and sunitinib or sorafenib, but that approach has been limited because of toxicity concerns. Sequential use of antiangiogenic agents with differing mechanisms of action may be an effective approach. Despite setbacks, angiogenesis will likely remain an important target of treatment for selected patients with MBC.

Topics: Angiogenesis Inhibitors; Antibodies, Monoclonal, Humanized; Antineoplastic Combined Chemotherapy Protocols; Bevacizumab; Breast Neoplasms; Capecitabine; Clinical Trials, Phase III as Topic; Deoxycytidine; Disease-Free Survival; Everolimus; Female; Fluorouracil; Humans; Indoles; Neoplasm Metastasis; Neovascularization, Pathologic; Niacinamide; Paclitaxel; Phenylurea Compounds; Pyrroles; Sirolimus; Sorafenib; Sunitinib; TOR Serine-Threonine Kinases; Vascular Endothelial Growth Factor A

Metastasis is a major cause of death from malignant diseases, and the underlying mechanisms are still largely not known. A detailed probe into the factors which may regulate tumour invasion and metastasis contributes to novel anti-metastatic therapies. We previously identified a novel metastasis-associated gene 1 (mag-1) by means of metastatic phenotype cloning. Then we characterized the gene expression profile of mag-1 and showed that it promoted cell migration, adhesion and invasion in vitro. Importantly, the disruption of mag-1 via RNA interference not only inhibited cellular metastatic behaviours but also significantly reduced tumour weight and restrained mouse breast cancer cells to metastasize to lungs in spontaneous metastatic assay in vivo. Furthermore, we proved that mag-1 integrates dual regulating mechanisms through the stabilization of HIF-1α and the activation of mTOR signalling pathway. We also found that mag-1-induced metastatic promotion could be abrogated by mTOR specific inhibitor, rapamycin. Taken together, the findings identified a direct role that mag-1 played in metastasis and implicated its function in cellular adaptation to tumour microenvironment.

Topics: 1-Acylglycerol-3-Phosphate O-Acyltransferase; Animals; Breast Neoplasms; Cell Adhesion; Cell Line, Tumor; Cell Movement; Chlorocebus aethiops; COS Cells; Female; Gene Expression Regulation, Neoplastic; HEK293 Cells; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Lung Neoplasms; Mice; Mice, Inbred BALB C; Neoplasm Invasiveness; Neoplasm Metastasis; RNA Interference; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Transcriptome; Tumor Microenvironment

The phosphatidylinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway is a key potential target in breast cancer therapy. Because some cancer cell lines are resistant to mTOR inhibition, we combined the mTOR inhibitor with the PI3K inhibitor and assayed the inhibitory effect of this combination versus that of a single inhibitor.. The proliferation of MCF7, SK-BR-3, T-47D, and MDA-MB-231 cells was measured by MTT assay in the presence of GDC-0941 and/or rapamycin. Afterwards, we determined the visible changes in signaling in the PI3K/AKT/mTOR pathway by Western blotting.. GDC-0941 exhibited excellent inhibition on MCF7, T-47D and SK-BR-3 cells with different characteristics. In addition, GDC-0941 blocked the feedback of PI3K/Akt through S6K1, resulting in decreased Akt activity by rapamycin activation. The combination of GDC-0941 and rapamycin downregulated the key components of the cell cycle machinery, such as cyclin D1 and upregulated the apoptotic markers.. Our findings suggest that GDC-0941, either alone or in combination with rapamycin, may serve as a new, promising approach for breast cancer treatment.

Topics: Antibiotics, Antineoplastic; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Down-Regulation; Female; Humans; Indazoles; MCF-7 Cells; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Sirolimus; Sulfonamides; TOR Serine-Threonine Kinases

We recently reported that a combination of dietary grape polyphenols resveratrol, quercetin, and catechin (RQC), at low concentrations, was effective at inhibiting metastatic cancer progression. Herein, we investigate the molecular mechanisms of RQC in breast cancer and explore the potential of RQC as a potentiation agent for the epidermal growth factor receptor (EGFR) therapeutic gefitinib. Our in vitro experiments showed RQC induced apoptosis in gefitinib-resistant breast cancer cells via regulation of a myriad of proapoptotic proteins. Because the Akt/mammalian target of rapamycin (mTOR) signaling pathway is often elevated during development of anti-EGFR therapy resistance, the effect of RQC on the mTOR upstream effector Akt and the negative regulator AMP kinase (AMPK) was investigated. RQC was found to reduce Akt activity, induce the activation of AMPK, and inhibit mTOR signaling in breast cancer cells. Combined RQC and gefitinib decreased gefitinib resistant breast cancer cell viability to a greater extent than RQC or gefitinib alone. Moreover, RQC inhibited Akt and mTOR and activated AMPK even in the presence of gefitinib. Our in vivo experiments showed combined RQC and gefitinib was more effective than the individual treatments at inhibiting mammary tumor growth and metastasis in nude mice. Therefore, RQC treatment inhibits breast cancer progression and may potentiate anti-EGFR therapy by inhibition of Akt/mTOR signaling.

Topics: Adenylate Kinase; Animals; Antibiotics, Antineoplastic; Antineoplastic Agents; Apoptosis; Breast Neoplasms; Caspase 3; Catechin; Cell Line, Tumor; Cell Proliferation; ErbB Receptors; Female; Gefitinib; Gene Expression Regulation; Humans; Mice; Mice, SCID; Polyphenols; Proto-Oncogene Proteins c-akt; Quercetin; Quinazolines; Resveratrol; Signal Transduction; Sirolimus; Stilbenes; TOR Serine-Threonine Kinases; Vitis; Xenograft Model Antitumor Assays

Strategies to improve the efficacy of endocrine agents in breast cancer (BC) therapy and to delay the onset of resistance include concomitant targeting of the estrogen receptor alpha (ER) and the mammalian target of rapamycin complex 1 (mTORC1), which regulate cell-cycle progression and are supported by recent clinical results.. BC cell lines expressing aromatase (AROM) and modeling endocrine-sensitive (MCF7-AROM1) and human epidermal growth factor receptor 2 (HER2)-dependent de novo resistant disease (BT474-AROM3) and long-term estrogen-deprived (LTED) MCF7 cells that had acquired resistance associated with HER2 overexpression were treated in vitro and as subcutaneous xenografts with everolimus (RAD001-mTORC1 inhibitor), in combination with tamoxifen or letrozole. End points included proliferation, cell-cycle arrest, cell signaling, and effects on ER-mediated transactivation.. Everolimus caused a concentration-dependent decrease in proliferation in all cell lines, which was associated with reductions in S6 phosphorylation. Everolimus plus letrozole or tamoxifen enhanced the antiproliferative effect and G1-accumulation compared with monotherapy, as well as increased phosphorylation (Ser10) and nuclear accumulation of p27 and pronounced dephosphorylation of Rb. Sensitivity was greatest to everolimus in the LTED cells but was reduced by added estrogen. Increased pAKT occurred in all circumstances with everolimus and, in the BT474 and LTED cells, was associated with increased pHER3. Decreased ER transactivation suggested that the effectiveness of everolimus might be partly related to interrupting cross-talk between growth-factor signaling and ER. In MCF7-AROM1 xenografts, letrozole plus everolimus showed a trend toward enhanced tumor regression, versus the single agents. In BT474-AROM3 xenografts, everolimus alone was equally effective at reducing tumor volume as were the combination therapies.. The results provide mechanistic support for recent positive clinical data on the combination of everolimus and endocrine therapy, as well as data on potential routes of escape via enhanced HER2/3 signaling. This merits investigation for further improvements in treatment efficacy.

Topics: Animals; Antineoplastic Agents, Hormonal; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Nucleolus; Cell Proliferation; Disease Models, Animal; Drug Resistance, Neoplasm; Everolimus; Female; Humans; Letrozole; Mechanistic Target of Rapamycin Complex 1; Mice; Multiprotein Complexes; Nitriles; Phosphorylation; Protein Kinase Inhibitors; Protein Transport; Proto-Oncogene Proteins c-akt; Receptor, ErbB-3; Receptors, Estrogen; Signal Transduction; Sirolimus; Tamoxifen; TOR Serine-Threonine Kinases; Triazoles; Xenograft Model Antitumor Assays

Advances in targeted therapies have improved progression-free and overall survival in women with metastatic breast cancer; however, regardless of efficacy, resistance almost always occurs eventually. Upregulation of the PI3K/AKT/mTOR pathway, which promotes cell growth and proliferation, is a means of escaping responsiveness to hormone therapy in hormone receptor-positive disease, or trastuzumab in HER2-positive disease. Everolimus, an inhibitor of mTOR, has shown promise in early clinical trials in metastatic breast cancer and is currently being studied in larger Phase II and III clinical trials, combined with hormone therapy or trastuzumab with or without cytotoxic chemotherapy. In this article, we discuss the mechanistic and preclinical data for everolimus, efficacy and safety results of clinical trials, and the landscape looking forward.

Topics: Animals; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Breast Neoplasms; Clinical Trials as Topic; Drug Evaluation, Preclinical; Drug Resistance, Neoplasm; Everolimus; Female; Humans; Neoplasm Metastasis; Phosphatidylinositol 3-Kinases; Receptor, ErbB-2; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Trastuzumab; Treatment Outcome

Neutrophil gelatinase-associated lipocalin (NGAL, a.k.a Lnc2) is a member of the lipocalin family and has diverse roles. NGAL can stabilize matrix metalloproteinase-9 from autodegradation. NGAL is considered as a siderocalin that is important in the transport of iron. NGAL expression has also been associated with certain neoplasias and is implicated in the metastasis of breast cancer. In a previous study, we examined whether ectopic NGAL expression would alter the sensitivity of breast epithelial, breast and colorectal cancer cells to the effects of the chemotherapeutic drug doxorubicin. While abundant NGAL expression was detected in all the cells infected with a retrovirus encoding NGAL, this expression did not alter the sensitivity of these cells to doxorubicin as compared with empty vector-transduced cells. We were also interested in determining the effects of ectopic NGAL expression on the sensitivity to small-molecule inhibitors targeting key signaling molecules. Ectopic NGAL expression increased the sensitivity of MCF-7 breast cancer cells to EGFR, Bcl-2 and calmodulin kinase inhibitors as well as the natural plant product berberine. Furthermore, when suboptimal concentrations of certain inhibitors were combined with doxorubicin, a reduction in the doxorubicin IC 50 was frequently observed. An exception was observed when doxorubicin was combined with rapamycin, as doxorubicin suppressed the sensitivity of the NGAL-transduced MCF-7 cells to rapamycin when compared with the empty vector controls. In contrast, changes in the sensitivities of the NGAL-transduced HT-29 colorectal cancer cell line and the breast epithelial MCF-10A cell line were not detected compared with empty vector-transduced cells. Doxorubicin-resistant MCF-7/Dox (R) cells were examined in these experiments as a control drug-resistant line; it displayed increased sensitivity to EGFR and Bcl-2 inhibitors compared with empty vector transduced MCF-7 cells. These results indicate that NGAL expression can alter the sensitivity of certain cancer cells to small-molecule inhibitors, suggesting that patients whose tumors exhibit elevated NGAL expression or have become drug-resistant may display altered responses to certain small-molecule inhibitors.

Topics: Acute-Phase Proteins; Antibiotics, Antineoplastic; Benzylamines; Berberine; Biphenyl Compounds; Breast Neoplasms; Calcium-Calmodulin-Dependent Protein Kinases; Cell Line, Tumor; Doxorubicin; Drug Resistance, Neoplasm; ErbB Receptors; Female; Gene Expression; HT29 Cells; Humans; Lipocalin-2; Lipocalins; MCF-7 Cells; Nitrophenols; Piperazines; Protein Kinase Inhibitors; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Quinazolines; Sirolimus; Sulfonamides; Tyrphostins

Topics: Aged; Antineoplastic Agents, Hormonal; Aromatase Inhibitors; Breast Neoplasms; Drug Resistance, Neoplasm; Everolimus; Female; Humans; Neoplasms, Hormone-Dependent; Sirolimus

Safety data for combining bevacizumab, everolimus, or lapatinib with anthracycline- and taxane-based neoadjuvant chemotherapy for breast cancer are limited.. The neoadjuvant GeparQuinto trial investigates the addition of (i) bevacizumab to four cycles epirubicin/cyclophosphamide (EC) followed by four cycles docetaxel (Taxotere) in patients with human epithelial growth factor receptor (HER)2-negative tumors, (ii) everolimus to weekly paclitaxel in patients with HER2-negative tumors not responding to EC ± bevacizumab, and (iii) lapatinib instead of trastuzumab to EC-docetaxel in patients with HER2-positive tumors to improve the rate of pathological complete response. Tolerable dose, need for supportive treatments, and early signals for toxic effect were evaluated in a planned safety analysis of 270 patients.. Treatment with chemotherapy plus bevacizumab, everolimus, or lapatinib was discontinued in 23.0%, 25.8%, and 34.5% compared with chemotherapy alone or plus trastuzumab in 19.4%, 24.1%, 3.2%, respectively. More leukopenia, infections, mucositis, and hypertension but less edema was observed by adding bevacizumab; a trend toward more thrombocytopenia, leukopenia, skin changes, and hyperlipidemia by adding everolimus; and more diarrhea, skin changes, and hot flushes but no cardiac events by substituting trastuzumab by lapatinib.. Adding bevacizumab and everolimus to chemotherapy appeared feasible. Lapatinib at 1250 mg resulted in an increased rate of treatment discontinuations and was subsequently dose reduced to 1000 mg.

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Combined Chemotherapy Protocols; Bevacizumab; Breast Neoplasms; Chemotherapy, Adjuvant; Everolimus; Female; Humans; Lapatinib; Quinazolines; Sirolimus

Recent gene expression profiling studies have identified five breast cancer subtypes, of which the basal-like subtype is the most aggressive. Basal-like breast cancer poses serious clinical challenges as there are currently no targeted therapies available to treat it. Although there is increasing evidence that these tumors possess specific sensitivity to cisplatin, its success is often compromised due to its dose-limiting nephrotoxicity and the development of drug resistance. To overcome this limitation, our goal was to maximize the benefits associated with cisplatin therapy through drug combination strategies. Using a validated kinase inhibitor library, we showed that inhibition of the mTOR, TGFβRI, NFκB, PI3K/AKT, and MAPK pathways sensitized basal-like MDA-MB-468 cells to cisplatin treatment. Further analysis demonstrated that the combination of the mTOR inhibitor rapamycin and cisplatin generated significant drug synergism in basal-like MDA-MB-468, MDA-MB-231, and HCC1937 cells but not in luminal-like T47D or MCF-7 cells. We further showed that the synergistic effect of rapamycin plus cisplatin on basal-like breast cancer cells was mediated through the induction of p73. Depletion of endogenous p73 in basal-like cells abolished these synergistic effects. In conclusion, combination therapy with mTOR inhibitors and cisplatin may be a useful therapeutic strategy in the treatment of basal-like breast cancers.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Blotting, Western; Breast Neoplasms; Carcinoma, Basal Cell; Cell Line, Tumor; Cell Proliferation; Cisplatin; DNA-Binding Proteins; Drug Synergism; Humans; NF-kappa B; Nuclear Proteins; p38 Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinases; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Sirolimus; TOR Serine-Threonine Kinases; Tumor Protein p73; Tumor Suppressor Proteins

Topics: Animals; Antineoplastic Agents; Breast Neoplasms; Drug Resistance, Neoplasm; Everolimus; Female; Humans; Ribosomal Protein S6 Kinases, 70-kDa; Sirolimus; TOR Serine-Threonine Kinases

α-Tocopherol ether-linked acetic acid (α-TEA) is a promising agent for cancer prevention/therapy based on its antitumour actions in a variety of cancers.. Human breast cancer cells, MCF-7 and HCC-1954, were used to study the effect of α-TEA using Annexin V/PI staining, western blot analyses, and siRNA knockdown techniques.. α-Tocopherol ether-linked acetic acid suppressed constitutively active basal levels of pAKT, pERK, pmTOR, and their downstream targets, as well as induced both cell types to undergo apoptosis. Phosphoinositide 3-kinase (PI3K) inhibitor wortmannin suppressed pAKT, pERK, pmTOR, and their downstream targets, indicating PI3K to be a common upstream mediator. In addition, α-TEA induced increased levels of pIRS-1 (Ser-307), a phosphorylation site correlated with insulin receptor substrate-1 (IRS-1) inactivation, and decreased levels of total IRS-1. Small interfering RNA (siRNA) knockdown of JNK blocked the impact of α-TEA on pIRS-1 and total IRS-1 and impeded its ability to downregulate the phosphorylated status of AKT, ERK, and mTOR. Combinations of α-TEA+MEK or mTOR inhibitor acted cooperatively to induce apoptosis and reduce basal levels of pERK and pmTOR. Importantly, inhibition of MEK and mTOR resulted in increased levels of pAKT and IRS-1, and α-TEA blocked them.. Downregulation of IRS-1/PI3K pathways via JNK are critical for α-TEA and α-TEA+MEK or mTOR inhibitor-induced apoptosis in human MCF-7 and HCC-1954 breast cancer cells.

Topics: 1-Phosphatidylinositol 4-Kinase; alpha-Tocopherol; Androstadienes; Anthracenes; Antioxidants; Apoptosis; Blotting, Western; Breast Neoplasms; Butadienes; Drug Synergism; Drug Therapy, Combination; Enzyme Inhibitors; Female; Humans; Immunosuppressive Agents; Insulin Receptor Substrate Proteins; MAP Kinase Kinase 4; Mitogen-Activated Protein Kinase Kinases; Nitriles; Phosphorylation; RNA, Small Interfering; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Tumor Cells, Cultured; Wortmannin

The anti-tumor activity of rapamycin is compromised by the feedback-loop-relevant hyperactive PI3K and ERK-MAPK pathway signaling. In breast cancer cells treated with rapamycin, we observed a moderate increase of AKT phosphorylation (P-AKT) in a rapamycin resistant cell line, MDA-MB-231, as well as a slight increase of P-AKT in a rapamycin sensitive cell line, MCF-7. We found that resveratrol, a natural phytoalexin, suppressed the phosphorylation and activation of the PI3K/AKT pathway in all the three breast cancer cell lines that we tested. It also had a weak inhibitory effect on the activation of the mTOR/p70S6K pathway in two cell lines expressing wildtype PTEN, MCF-7 and MDA-MB-231. The combined use of resveratrol and rapamycin resulted in modest additive inhibitory effects on the growth of breast cancer cells, mainly through suppressing rapamycin-induced AKT activation. We, therefore, reveal a novel combination whereby resveratrol potentiates the growth inhibitory effect of rapamycin, with the added benefit of preventing eventual resistance to rapamycin, likely by suppressing AKT signaling. We also present data herein that PTEN is an important contributor to resveratrol's growth suppressive effects and its potentiation of rapamycin in this therapeutic scenario, as resveratrol's suppression of rapamycin-mediated induction of P-AKT is both PTEN-dependent and -independent. Thus, the resveratrol-rapamycin combination may have therapeutic value in treating breast cancer and perhaps other processes where mTOR is activated.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; Dose-Response Relationship, Drug; Drug Synergism; Extracellular Signal-Regulated MAP Kinases; Female; Humans; Immunoblotting; Inhibitory Concentration 50; Membrane Proteins; Phosphatidylinositol 3-Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Resveratrol; Signal Transduction; Sirolimus; Stilbenes; TOR Serine-Threonine Kinases

mTOR (mammalian target of rapamycin) signaling plays a key role in the development of many tumor types. Therefore, mTOR is an attractive target for cancer therapeutics. Although mTOR inhibitors are thought to have radiosensitization activity, the molecular bases remain largely unknown. Here we show that treating MCF7 breast cancer cells with rapamycin (an mTOR inhibitor) results in significant suppression of homologous recombination (HR) and nonhomologous end joining (NHEJ), two major mechanisms required for repairing ionizing radiation-induced DNA DSBs. We observed that rapamycin impaired recruitment of BRCA1 and Rad51 to DNA repair foci, both essential for HR. Moreover, consistent with the suppressive role of rapamycin on both HR and NHEJ, persistent radiation-induced DSBs were detected in cells pretreated with rapamycin. Furthermore, the frequency of chromosome and chromatid breaks was increased in cells treated with rapamycin before and after irradiation. Thus our results show that radiosensitization by mTOR inhibitors occurs via disruption of the major two DNA DSB repair pathways.

Topics: BRCA1 Protein; Breast Neoplasms; Cell Line, Tumor; Chromosome Aberrations; DNA Breaks, Double-Stranded; DNA Repair; Female; Humans; Rad51 Recombinase; Recombination, Genetic; Sirolimus; TOR Serine-Threonine Kinases

Inhibitors of the kinase mTOR, such as rapamycin and everolimus, have been used as cancer therapeutics with limited success since some tumours are resistant. Efforts to establish predictive markers to allow selection of patients with tumours likely to respond have centred on determining phosphorylation states of mTOR or its targets 4E-BP1 and S6K in cancer cells. In an alternative approach we estimated eIF4E activity, a key effector of mTOR function, and tested the hypothesis that eIF4E activity predicts sensitivity to mTOR inhibition in cell lines and in breast tumours.. We found a greater than three fold difference in sensitivity of representative colon, lung and breast cell lines to rapamycin. Using an assay to quantify influences of eIF4E on the translational efficiency specified by structured 5'UTRs, we showed that this estimate of eIF4E activity was a significant predictor of rapamycin sensitivity, with higher eIF4E activities indicative of enhanced sensitivity. Surprisingly, non-transformed cell lines were not less sensitive to rapamycin and did not have lower eIF4E activities than cancer lines, suggesting the mTOR/4E-BP1/eIF4E axis is deregulated in these non-transformed cells. In the context of clinical breast cancers, we estimated eIF4E activity by analysing expression of eIF4E and its functional regulators within tumour cells and combining these scores to reflect inhibitory and activating influences on eIF4E. Estimates of eIF4E activity in cancer biopsies taken at diagnosis did not predict sensitivity to 11-14 days of pre-operative everolimus treatment, as assessed by change in tumour cell proliferation from diagnosis to surgical excision. However, higher pre-treatment eIF4E activity was significantly associated with dramatic post-treatment changes in expression of eIF4E and 4E-binding proteins, suggesting that eIF4E is further deregulated in these tumours in response to mTOR inhibition.. Estimates of eIF4E activity predict sensitivity to mTOR inhibition in cell lines but breast tumours with high estimated eIF4E activity gain changes in eIF4E regulation in order to enhance resistance.

Topics: 5' Untranslated Regions; Adaptor Proteins, Signal Transducing; Breast Neoplasms; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Drug Screening Assays, Antitumor; Eukaryotic Initiation Factor-4E; Everolimus; Female; Gene Expression Regulation, Neoplastic; Genes, Reporter; Humans; Phosphoproteins; Phosphorylation; Preoperative Care; Protein Biosynthesis; Sirolimus; Tissue Culture Techniques; TOR Serine-Threonine Kinases

The phosphatidylinositol 3-kinase (PI3K) pathway is commonly activated in breast cancers due to frequent mutations in PIK3CA, loss of expression of PTEN or over-expression of receptor tyrosine kinases. PI3K pathway activation leads to stimulation of the key growth and proliferation regulatory kinase mammalian target of rapamycin (mTOR), which can be inhibited by rapamycin analogues and by kinase inhibitors; the effectiveness of these drugs in breast cancer treatment is currently being tested in clinical trials. To identify the molecular determinants of response to inhibitors that target mTOR via different mechanisms in breast cancer cells, we investigated the effects of pharmacological inhibition of mTOR using the allosteric mTORC1 inhibitor everolimus and the active-site mTORC1/mTORC2 kinase inhibitor PP242 on a panel of 31 breast cancer cell lines. We demonstrate here that breast cancer cells harbouring PIK3CA mutations are selectively sensitive to mTOR allosteric and kinase inhibitors. However, cells with PTEN loss of function are not sensitive to these drugs, suggesting that the functional consequences of these two mechanisms of activation of the mTOR pathway are quite distinct. In addition, a subset of HER2-amplified cell lines showed increased sensitivity to PP242, but not to everolimus, irrespective of the PIK3CA/PTEN status. These selective sensitivities were confirmed in more physiologically relevant three-dimensional cell culture models. Our findings provide a rationale to guide selection of breast cancer patients who may benefit from mTOR inhibitor therapy and highlight the importance of accurately assessing the expression of PTEN protein and not just its mutational status.

Topics: Animals; Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Class I Phosphatidylinositol 3-Kinases; Drug Screening Assays, Antitumor; Everolimus; G1 Phase; Humans; Mechanistic Target of Rapamycin Complex 1; Multiprotein Complexes; Mutation; Phosphatidylinositol 3-Kinases; Proteins; PTEN Phosphohydrolase; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Transcription Factors

Inhibition of phosphatidylinositol-3-kinase (PI3K) induces apoptosis when combined with estrogen deprivation in estrogen receptor (ER)-positive breast cancer. The aims of the present study were to identify effective PI3K pathway inhibitor and endocrine therapy combinations, to evaluate the effect of PI3K pathway mutations and estrogen dependency on tumor response, and to determine the relevance of PIK3CA mutation in recurrent disease.. The PI3K catalytic subunit inhibitor BKM120, the mammalian target of rapamycin (mTOR) inhibitor RAD001 and the dual PI3K/mTOR inhibitor BGT226 were tested against ER-positive breast cancer cell lines before and after long-term estrogen deprivation (LTED). The impact of estradiol deprivation and the ER downregulator fulvestrant on PI3K pathway inhibitor-induced apoptosis was assessed. PIK3CA hotspot mutation analysis was performed in 51 recurrent or metastatic breast cancers and correlated with ER status and survival.. Drug-induced apoptosis was most marked in short-term estrogen-deprived cells with PIK3CA mutation and phosphatase and tensin homolog loss. Apoptosis was most highly induced by BGT226, followed by BKM120, and then RAD001. Estradiol antagonized PI3K inhibitor-induced apoptosis following short-term estrogen deprivation, emphasizing a role for estrogen-deprivation therapy in promoting PI3K inhibitor activity in the first-line setting. ER-positive MCF7 LTED cells exhibited relative resistance to PI3K pathway inhibition that was reversed by fulvestrant. In contrast, T47D LTED cells exhibited ER loss and ER-independent PI3K agent sensitivity. PIK3CA mutation was prevalent in relapsed ER-positive disease (48%) and was associated with persistent ER positivity and a late relapse pattern.. Estrogen deprivation increased the apoptotic effects of PI3K and dual PI3K/mTOR inhibitors in ER-positive disease, providing a rationale for PI3K/aromatase inhibitor combinations as first-line therapy. In LTED cells, differential effects on ER expression may be a relevant consideration. When ER was persistently expressed, fulvestrant strongly promoted PI3K drug activity. When ER was lost, PI3K inhibitor monotherapy was sufficient to induce high-level apoptosis. Although tumors with PIK3CA mutation had a late recurrence pattern, these mutations were common in metastatic disease and were most often associated with persistent ER expression. Targeting PIK3CA mutant tumors with a PI3K pathway inhibitor and fulvestrant is therefore a feasible strategy for aromatase-inhibitor-resistant ER-positive relapsed breast cancer.

Topics: Aminopyridines; Antineoplastic Agents, Hormonal; Apoptosis; Aromatase Inhibitors; Breast Neoplasms; Cell Line, Tumor; Class I Phosphatidylinositol 3-Kinases; Estradiol; Estrogens; Everolimus; Female; Fulvestrant; Humans; Imidazoles; Morpholines; Mutation; Neoplasm Recurrence, Local; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; PTEN Phosphohydrolase; Quinolines; Receptors, Estrogen; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Topics: Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents, Hormonal; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Everolimus; Female; Humans; Lapatinib; Molecular Targeted Therapy; Quinazolines; Sirolimus; Trastuzumab

Stromal cells, such as mesenchymal stem cells (MSCs) and carcinoma-associated fibroblasts (CAFs), play a role in cancer progression. To analyze their ability to modulate drug response, we generated spheroids of MCF-7 or MDA-MB-231 breast cancer cells in the absence or presence of human (h)MSCs or hCAFs and tested the susceptibility of the breast cancer cells to three different kinase inhibitors (TKI258, RAD001 and RAF265) used in cancer therapy. While stromal cells did not affect the response of either breast cancer cell line to the PDGFR/FGFR/VEGFR inhibitor TKI258, they sensitized breast cancer cells to the mTOR inhibitor RAD001. In MCF-7 cells, this was accompanied by increased apoptosis. hMSCs and to a lesser extent hCAFs also enhanced the cytotoxic effect of RAF inhibitor RAF265 on MDA-MB-231 cells. Searching for the mechanism that underlies the effect of stromal cells on RAF265 response we found that stromal cells inhibited RAF265-induced increase in ERK1/2 phosphorylation, supported RAF265-dependent downregulation of PKCα (protein kinase Cα) and prevented RAF265-induced conversion of LC3B, a marker of autophagy. To mimic the changes in ERK1/2 phosphorylation and PKCα expression in response to the stromal cells, we treated cells with MEK1 inhibitor U0126 or PKCα inhibitor Gö6976, respectively. U0126, but not Gö6976, was as effective as hMSCs in sensitizing MDA-MB-231 cells to RAF265. This suggests that hMSCs and hCAFs increased the cytotoxic effect of RAF265 on MDA-MB-231 cells by downregulating ERK1/2 phosphorylation. In summary, this study shows that hMSCs are able to render breast cancer cells more susceptible to kinase inhibitors and that, to the most part, hCAFs to which hMSCs can differentiate are able to mimic the drug-sensitizing effects of hMSCs.

Topics: Breast Neoplasms; Cell Communication; Cell Line, Tumor; Cells, Cultured; Coculture Techniques; Everolimus; Female; Fibroblasts; Humans; Imidazoles; Mesenchymal Stem Cells; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Phosphorylation; Protein Kinase Inhibitors; Pyridines; raf Kinases; Sirolimus; Stromal Cells; TOR Serine-Threonine Kinases

Triple-negative breast cancers, which lack estrogen receptor, progesterone receptor, and HER2/neu overexpression, account for approximately 15% of breast cancers, but occur more commonly in African Americans. The poor survival outcomes seen with triple-negative breast cancers patients are, in part, due to a lack of therapeutic targets. Epidermal growth factor receptor (EGFR) is overexpressed in 50% of triple-negative breast cancers, but EGFR inhibitors have not been effective in patients with metastatic breast cancers. However, mTOR inhibition has been shown to reverse resistance to EGFR inhibitors. We examined the combination effects of mTOR inhibition with EGFR inhibition in triple-negative breast cancer in vitro and in vivo. The combination of EGFR inhibition by using lapatinib and mTOR inhibition with rapamycin resulted in significantly greater cytotoxicity than the single agents alone and these effects were synergistic in vitro. The combination of rapamycin and lapatinib significantly decreased growth of triple-negative breast cancers in vivo compared with either agent alone. EGFR inhibition abrogated the expression of rapamycin-induced activated Akt in triple-negative breast cancer cells in vitro. The combination of EGFR and mTOR inhibition resulted in increased apoptosis in some, but not all, triple-negative cell lines, and these apoptotic effects correlated with a decrease in activated eukaryotic translation initiation factor (eIF4E). These results suggest that mTOR inhibitors could sensitize a subset of triple-negative breast cancers to EGFR inhibitors. Given the paucity of effective targeted agents in triple-negative breast cancers, these results warrant further evaluation.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Drug Synergism; ErbB Receptors; Female; Humans; Lapatinib; Mice; Mice, Nude; Quinazolines; Receptor, ErbB-2; Receptors, Estrogen; Receptors, Progesterone; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Tumor Burden; Xenograft Model Antitumor Assays

Elucidating the response of breast cancer cells to chemotherapeutic and hormonal based drugs is clearly important as these are frequently used therapeutic approaches. A signaling pathway often involved in chemo- and hormonal-resistance is the Ras/PI3K/PTEN/Akt/mTOR cascades. In the studies presented in this report, we have examined the effects of constitutive activation of Akt on the sensitivity of MCF-7 breast cancer cells to chemotherapeutic- and hormonal-based drugs as well as mTOR inhibitors. MCF-7 cells which expressed a constitutively-activated Akt-1 gene [∆Akt-1(CA)] were more resistant to doxorubicin, etoposide and 4-OH-tamoxifen (4HT) than cells lacking ∆Akt-1(CA). Cells which expressed ∆Akt-1(CA) were hypersensitive to the mTOR inhibitor rapamycin. Furthermore, rapamycin lowered the IC50s for doxorubicin, etoposide and 4HT in the cells which expressed ∆Akt-1(CA), demonstrating a potential improved method for treating certain breast cancers which have deregulated PI3K/PTEN/Akt/mTOR signaling. Understanding how breast cancers respond to chemo- and hormonal-based therapies and the mechanisms by which they can become drug resistant may enhance our ability to treat breast cancer. These results also document the potential importance of knowledge of the mutations present in certain cancers which may permit more effective therapies.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Doxorubicin; Drug Resistance, Neoplasm; Etoposide; Female; Humans; Molecular Targeted Therapy; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirolimus; Tamoxifen; TOR Serine-Threonine Kinases

The present study was performed to investigate the possible role of mTOR inhibitors in restoring chemosensitivity to adriamycin/cisplatin and elucidate the underlying mechanism. Combining adriamycin/cisplatin with torisel synergistically inhibited the cell proliferation in human oropharyngeal carcinoma cell line KB and its multidrug-resistant subclone KB/7D. Combining adriamycin and torisel inhibited the phosphorylation of 4EBP-1 and p70S6K, the proteins involved in mTOR pathway, increased expression of γH2AX indicative of DNA damage, triggered cell cycle arrest at G2/M and apoptosis. We conclude that chromatin decondensation by DNA damage provided an easy access for torisel to block the translation of proteins essential for DNA repair thereby restoring the chemosensitivity.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cisplatin; DNA Damage; DNA Repair; DNA Topoisomerases, Type II; Doxorubicin; Drug Resistance, Neoplasm; Drug Synergism; Eukaryotic Initiation Factors; Everolimus; Female; Head and Neck Neoplasms; Humans; Phosphorylation; Ribosomal Protein S6 Kinases, 70-kDa; Sirolimus; Topoisomerase II Inhibitors; TOR Serine-Threonine Kinases

The phosphatidylinositol 3-kinase (PI3K)/protein kinase B(AKT)/mammalian target of rapamycin (mTOR) signaling pathway is aberrantly activated in many types of cancer, including breast cancer. It is recognized that breast cancer cells develop resistance to a variety of standard therapies through the activation of this pathway. We hypothesized that targeting this signaling by the mTOR inhibitor RAD001 may potentiate the cytotoxicity of a conventional chemotherapeutic drug, carboplatin, and enhance the treatment efficacy for breast cancer.. Cell proliferation was measured with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay; cell apoptosis with enzyme-linked immunosorbent assay (ELISA). Flow cytometry was used for the analysis of cell cycle distribution and mitochondrial membrane function. Gene expression at the protein level was determined by Western blot.. MTOR inhibitor RAD001 enhanced the sensitivity of breast cancer cells to carboplatin. RAD001 in combination with carboplatin resulted in synergistic inhibition of cell proliferation and caspase-independent apoptosis in these cells. Moreover, in MCF-7 and BT-474 cells, synergistic effects of this combination on G₂/M cell cycle arrest and regulation of different molecules responsible for cell cycle transition and apoptosis were observed. The p53 pathway was involved in the synergism of RAD001 and carboplatin on breast cancer cell proliferation and apoptosis, since the synergistic effect was demonstrated in all tested breast cancer cell lines with wild-type p53 and the use of p53 inhibitor partially antagonized the effect of RAD001 and carboplatin on p53 and p21 expression, as well as their inhibitory effect on cell proliferation. However, a synergistic effect of the combination of the two drugs on cell proliferation was observed in two p53-mutated cell lines with high AKT expression, suggesting that an alternative mechanism underlying the observed synergism exists.. Our results suggest that the combination of RAD001 and carboplatin is a promising treatment approach for breast cancer. On the basis of these results, we have initiated a phase I/II clinical trial with the combination of carboplatin and RAD001 in patients with metastatic breast cancer.

Topics: Antineoplastic Agents; Apoptosis; Blotting, Western; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Drug Synergism; Enzyme-Linked Immunosorbent Assay; Everolimus; Female; Flow Cytometry; Genes, p53; Humans; Membrane Potentials; Sirolimus; TOR Serine-Threonine Kinases

Elucidating the response of breast cancer cells to chemotherapeutic and hormonal based drugs and radiation is clearly important as these are common treatment approaches. Signaling cascades often involved in chemo-, hormonal- and radiation resistance are the Ras/PI3K/PTEN/Akt/mTOR, Ras/Raf/MEK/ERK and p53 pathways. In the following studies we have examined the effects of activation of the Ras/PI3K/PTEN/Akt/mTOR cascade in the response of MCF-7 breast cancer cells to chemotherapeutic- and hormonal-based drugs and radiation. Activation of Akt by introduction of conditionally-activated Akt-1 gene could result in resistance to chemotherapeutic and hormonal based drugs as well as radiation. We have determined that chemotherapeutic drugs such as doxorubicin or the hormone based drug tamoxifen, both used to treat breast cancer, resulted in the activation of the Raf/MEK/ERK pathway which is often associated with a pro-proliferative, anti-apoptotic response. In drug sensitive MCF-7 cells which have wild-type p53; ERK, p53 and downstream p21 (Cip-1 ) were induced upon exposure to doxorubicin. In contrast, in the drug resistant cells which expressed activated Akt-1, much lower levels of p53 and p21 (Cip1) were induced upon exposure to doxorubicin. These results indicate the involvement of the Ras/PI3K/PTEN/Akt/mTOR, Ras/Raf/MEK/ERK and p53 pathways in the response to chemotherapeutic and hormonal based drugs. Understanding how breast cancers respond to chemo- and hormonal-based therapies and radiation may enhance the ability to treat breast cancer more effectively.

Topics: Breast Neoplasms; Cell Culture Techniques; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p21; Doxorubicin; Drug Resistance, Neoplasm; Female; Gene Expression Regulation, Neoplastic; Humans; Phosphatidylinositol 3-Kinase; Phosphorylation; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Radiation Tolerance; Retroviridae; Signal Transduction; Sirolimus; Tamoxifen; TOR Serine-Threonine Kinases; Transfection; Tumor Suppressor Protein p53

Anti-apoptotic signals induced downstream of HER2 are known to contribute to the resistance to current treatments of breast cancer cells that overexpress this member of the EGFR family. Whether or not some of these signals are also involved in tumor maintenance by counteracting constitutive death signals is much less understood. To address this, we investigated what role anti- and pro-apoptotic Bcl-2 family members, key regulators of cancer cell survival, might play in the viability of HER2 overexpressing breast cancer cells.. We used cell lines as an in vitro model of HER2-overexpressing cells in order to evaluate how anti-apoptotic Bcl-2, Bcl-xL and Mcl-1, and pro-apoptotic Puma and Bim impact on their survival, and to investigate how the constitutive expression of these proteins is regulated. Expression of the proteins of interest was confirmed using lysates from HER2-overexpressing tumors and through analysis of publicly available RNA expression data.. We show that the depletion of Mcl-1 is sufficient to induce apoptosis in HER2-overexpressing breast cancer cells. This Mcl-1 dependence is due to Bim expression and it directly results from oncogenic signaling, as depletion of the oncoprotein c-Myc, which occupies regions of the Bim promoter as evaluated in ChIP assays, decreases Bim levels and mitigates Mcl-1 dependence. Consistently, a reduction of c-Myc expression by inhibition of mTORC1 activity abrogates occupancy of the Bim promoter by c-Myc, decreases Bim expression and promotes tolerance to Mcl-1 depletion. Western blot analysis confirms that naïve HER2-overexpressing tumors constitutively express detectable levels of Mcl-1 and Bim, while expression data hint on enrichment for Mcl-1 transcripts in these tumors.. This work establishes that, in HER2-overexpressing tumors, it is necessary, and maybe sufficient, to therapeutically impact on the Mcl-1/Bim balance for efficient induction of cancer cell death.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; bcl-X Protein; Breast Neoplasms; Cell Aggregation; Cell Line, Tumor; Cell Survival; Everolimus; Female; Gene Expression; Gene Knockdown Techniques; Humans; Mechanistic Target of Rapamycin Complex 1; Membrane Proteins; Multiprotein Complexes; Myeloid Cell Leukemia Sequence 1 Protein; Promoter Regions, Genetic; Proteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-myc; Receptor, ErbB-2; RNA Interference; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Autophagy is an evolutionarily conserved mechanism of cellular self-digestion in which proteins and organelles are degraded through delivery to lysosomes. Defects in this process are implicated in numerous human diseases including cancer. To further elucidate regulatory mechanisms of autophagy, we performed a functional screen in search of microRNAs (miRNAs), which regulate the autophagic flux in breast cancer cells. In this study, we identified the tumour suppressive miRNA, miR-101, as a potent inhibitor of basal, etoposide- and rapamycin-induced autophagy. Through transcriptome profiling, we identified three novel miR-101 targets, STMN1, RAB5A and ATG4D. siRNA-mediated depletion of these genes phenocopied the effect of miR-101 overexpression, demonstrating their importance in autophagy regulation. Importantly, overexpression of STMN1 could partially rescue cells from miR-101-mediated inhibition of autophagy, indicating a functional importance for this target. Finally, we show that miR-101-mediated inhibition of autophagy can sensitize breast cancer cells to 4-hydroxytamoxifen (4-OHT)-mediated cell death. Collectively, these data establish a novel link between two highly important and rapidly growing research fields and present a new role for miR-101 as a key regulator of autophagy.

Topics: Autophagy; Autophagy-Related Proteins; Breast Neoplasms; Cell Line, Tumor; Cysteine Endopeptidases; Etoposide; Female; Gene Expression Profiling; Gene Expression Regulation; Humans; MicroRNAs; Oligonucleotide Array Sequence Analysis; rab5 GTP-Binding Proteins; RNA Interference; RNA, Small Interfering; Sirolimus; Stathmin; Tamoxifen

HER2-positive breast cancers exhibit high rates of innate and acquired resistance to trastuzumab (TZ), a HER2-directed antibody used as a first line treatment for this disease. TZ resistance may in part be mediated by frequent co-expression of EGFR and by sustained activation of the mammalian target of rapamycin (mTOR) pathway. Here, we assessed feasibility of combining the EGFR inhibitor gefitinib and the mTOR inhibitor everolimus (RAD001) for treating HER2 overexpressing breast cancers with different sensitivity to TZ.. The gefitinib and RAD001 combination was broadly evaluated in TZ sensitive (SKBR3 and MCF7-HER2) and TZ resistant (JIMT-1) breast cancer models. The effects on cell growth were measured in cell based assays using the fixed molar ratio design and the median effect principle. In vivo studies were performed in Rag2M mice bearing established tumors. Analysis of cell cycle, changes in targeted signaling pathways and tumor characteristics were conducted to assess gefitinib and RAD001 interactions.. The gefitinib and RAD001 combination inhibited cell growth in vitro in a synergistic fashion as defined by the Chou and Talalay median effect principle and increased tumor xenograft growth delay. The improvement in therapeutic efficacy by the combination was associated in vitro with cell line dependent increases in cytotoxicity and cytostasis while treatment in vivo promoted cytostasis. The most striking and consistent therapeutic effect of the combination was increased inhibition of the mTOR pathway (in vitro and in vivo) and EGFR signaling in vivo relative to the single drugs.. The gefitinib and RAD001 combination provides effective control over growth of HER2 overexpressing cells and tumors irrespective of the TZ sensitivity status.

Topics: Animals; Antibodies, Monoclonal, Humanized; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Cell Line, Tumor; Cell Survival; Drug Resistance, Neoplasm; Drug Synergism; ErbB Receptors; Everolimus; Female; Gefitinib; Gene Expression Regulation, Neoplastic; Humans; Mice; Phosphorylation; Quinazolines; Receptor, ErbB-2; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Trastuzumab; Xenograft Model Antitumor Assays

Neoadjuvant therapy in breast cancer has emerged as an important setting for the development of targeted drugs. Because tumor material is available before treatment, at the moment of surgery, and possibly during treatment, precise correlations can be made between target identification, target blockade, and tumor response. Significant improvements have already been achieved by introducing targeted agents to neoadjuvant modalities. In the HER2 patient population, anti-HER2 targeted therapies have consistently demonstrated increased rates of pathological complete response. In the hormone receptor-positive setting, identifying early surrogate markers able to predict response to treatment has the potential to accelerate the development of targeted therapies. Ongoing neoadjuvant research programs such as NeoBIG and I-SPY 2 (Investigation of Serial Studies to Predict Your Therapeutic Response with Imaging And moLecular Analysis 2) are scientifically strong and will most likely demonstrate that the "neoadjuvant step" can lead directly to large, phase III adjuvant registration trials. This implies that the time between drug discovery and regulatory approval can be significantly shortened, which ultimately benefits patients.

Topics: Antibodies, Monoclonal, Humanized; Antineoplastic Combined Chemotherapy Protocols; Biomarkers, Tumor; Breast Neoplasms; Chemotherapy, Adjuvant; Disease-Free Survival; DNA Repair; Drug Discovery; Everolimus; Female; Genetic Predisposition to Disease; Humans; Lapatinib; Letrozole; Molecular Targeted Therapy; Neoadjuvant Therapy; Nitriles; Quinazolines; Randomized Controlled Trials as Topic; Receptor, ErbB-2; Receptors, Estrogen; Sirolimus; Trastuzumab; Triazoles

Hormonal dependence of breast cancer has been known for a long time, yet about half of breast cancers with estrogen receptor will not respond to antihormonal therapy. Now, we know that this resistance may be related to a dysfunction of the estrogen pathway, or that of growth factors and particularly the pathway of cell activation PI3K/Akt/mTOR. Prevention of these different mechanisms of resistance could involve combination therapies such as anti-estrogens (SERMs, aromatase inhibitors) with inhibitors of the activity of growth factors that are particularly temsirolimus and everolimus for the activation pathway cell PI3K/Akt/mTOR.

Topics: Antineoplastic Agents; Breast Neoplasms; Drug Resistance, Neoplasm; Everolimus; Female; Humans; Neoplasm Proteins; Neoplasms, Hormone-Dependent; Phosphatidylinositol 3-Kinases; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Sirolimus; TOR Serine-Threonine Kinases

Antisense oligonucleotides (oligos) have demonstrated their efficacy in inhibiting the growth of prostate and breast tumor cells. Previous studies employed first generation, phosphorothioated, cDNA oligos synthesized complimentary to mRNA encoding transforming growth factor-alpha (TGF-alpha), epidermal growth factor receptor (EGFR), the anti-apoptosis protein bcl-2, and the androgen receptor (AR). In an effort to construct oligos with greater than one mRNA binding site, bi-specifics have been developed which target combinations of the above proteins, and these have been shown at least as effective as the mono-specific oligos from which their sequences were derived. While all bi-specifics have inhibitory effects, which can be enhanced by the combined administration of an additional chemotherapeutic agent, those bi-specifics which target bcl-2 and EGFR were reported to be the most effective. The experiments presented here are an effort to evaluate a new group of bi-specifics whose targets include the chaperone protein clusterin, whose expression is up regulated in many tumors and activity is known to inhibit apoptosis. Of particular interest were those bi-specifics constructed to target both clusterin and bcl-2 (also an apoptosis inhibitory protein). Cell lines targeted included both prostate LNCaP and PC-3, as well as the breast derived MCF-7. In order to identify agents which enhance oligo activity, but contribute less toxicity, oligos were tested both alone and in combination with either the immune inhibitor Rapamycin, or the chemotherapeutic (and more toxic) Taxol. Results indicate that bi-specifics targeting clusterin are statistically effective, and are similarly enhanced by Rapamycin, or Taxol. When bi-specifics including clusterin as a target, were tested against LNCaP and MCF-7 cells, the level of activity was intermediate between that of the mono-specific compounds tested separately. In experiments which compared both, bi-specifics which included a target for clusterin had inhibitory activity similar to the previously described bi-specifics directed towards bcl-2 and EGFR.

Topics: Adenocarcinoma; Antineoplastic Agents; Apoptosis; Breast Neoplasms; Cell Line, Tumor; Clusterin; Drug Delivery Systems; Drug Screening Assays, Antitumor; Female; Humans; Male; Neoplasm Proteins; Oligonucleotides, Antisense; Paclitaxel; Phosphorothioate Oligonucleotides; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; RNA, Messenger; RNA, Neoplasm; Sirolimus; Substrate Specificity

Rapamycin and its analogues inhibit mTOR, which leads to decreased protein synthesis and decreased cancer cell proliferation in many experimental systems. Adenosine 5'- monophosphate-activated protein kinase (AMPK) activators such as metformin have similar actions, in keeping with the TSC2/1 pathway linking activation of AMPK to inhibition of mTOR. As mTOR inhibition by rapamycin is associated with attenuation of negative feedback to IRS-1, rapamycin is known to increase activation of AKT, which may reduce its anti-neoplastic activity. We observed that metformin exposure decreases AKT activation, an action opposite to that of rapamycin. We show that metformin (but not rapamycin) exposure leads to increased phosphorylation of IRS-1 at Ser(789), a site previously reported to inhibit downstream signaling and to be an AMPK substrate phosphorylated under conditions of cellular energy depletion. siRNA methods confirmed that reduction of AMPK levels attenuates both the IRS-1 Ser(789) phosphorylation and the inhibition of AKT activation associated with metformin exposure. Although both rapamycin and metformin inhibit mTOR (the former directly and the latter through AMPK signaling), our results demonstrate previously unrecognized differences between these agents. The data are consistent with the observation that maximal induction of apoptosis and inhibition of proliferation are greater for metformin than rapamycin.

Topics: Antineoplastic Agents; Apoptosis; Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Separation; Enzyme Activation; Female; Flow Cytometry; Humans; Intracellular Signaling Peptides and Proteins; Metformin; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Transfection

Patients with estrogen receptor-positive (ER+) breast cancers are often treated with aromatase inhibitors or by antiestrogens such as tamoxifen to prevent disease recurrence. Resistant tumors nevertheless develop and it is commonly assumed that they arise by the induction of mutations. However, it is also possible that resistant tumors grow from preexisting variant populations within the original tumor. We have investigated this possibility in the case of the MCF-7 breast cancer cell line. The line was cultured for a prolonged period either in the presence of tamoxifen to block the action of oestrogen or in the absence of estrogen to mimic the action of oophorectomy or treatment with aromatase inhibitors. Both treatments led to growth inhibition followed by eventual outgrowth of sub-lines. Five of these sub-lines were developed and characterized for sensitivity to tamoxifen and to the antibiotic rapamycin, expression of HE R2 and PAX2, and phosphorylation of Akt, p70S6K, 4E-BP1, rpS6, EGFR1, Erk and HE R2. All six lines were ER+ and could be divided into four phenotypes distinguished by cell volume, DNA content (ploidy) and cell cycle time. In two cases, selection with tamoxifen and selection in the absence of estrogen produced similar phenotypes. Rapamycin resistance was a feature of the sub-lines developed under estrogen deprivation and was associated with loss of active phospho-HE R2 and acquisition of PAX2 expression. The results support the conclusion that the MCF-7 cell line is heterogeneous and that the selection conditions allow the growth of pre-existing phenotypes.

Topics: Antineoplastic Agents, Hormonal; Apoptosis; Blotting, Western; Breast Neoplasms; Cell Proliferation; DNA, Neoplasm; Drug Resistance, Neoplasm; Female; Flow Cytometry; Humans; Immunosuppressive Agents; PAX2 Transcription Factor; Phenotype; Phosphorylation; Receptor, ErbB-2; Sirolimus; Tamoxifen; Tumor Cells, Cultured

Akt and mTOR are therapeutic targets for the treatment of cancer. The effects of inhibiting mTOR, with rapamycin, and Akt, with A-443654, concurrently, on cell morphology, cell proliferation, the cell cycle, and apoptosis were examined using the benign MCF10A and malignant MCF10CA1a human breast epithelial cells. Rapamycin and A-443654 in combination produced the greatest morphological changes and inhibited cell proliferation by G2/M arrest. Rapamycin and A-443654 in combination induced apoptosis at earlier times and at lower A-443654 concentrations in MCF10CA1a tumor cells than in the benign MCF10A cells. Rapamycin and A-443654 increased p53 and p15(INK4B) protein levels, decreased anti-apoptotic Bcl-2 levels, and increased Bad levels in the MCF10CA1a tumor cells by approximately 5-fold. These results suggest that the combined inhibition of Akt and mTOR may have beneficial therapeutic and safety margin effects.

Topics: Breast Neoplasms; Cell Cycle; Cell Division; Cell Line, Tumor; Enzyme Activation; Epithelial Cells; Female; Fibrocystic Breast Disease; Flow Cytometry; Humans; Immunoblotting; Indazoles; Indoles; Neoplasm Metastasis; Proto-Oncogene Proteins c-akt; Sirolimus; Survivors

PIK3CA mutations are reported to be present in approximately 25% of breast cancer (BC), particularly the estrogen receptor-positive (ER+) and HER2-overexpressing (HER2+) subtypes, making them one of the most common genetic aberrations in BC. In experimental models, these mutations have been shown to activate AKT and induce oncogenic transformation, and hence these lesions have been hypothesized to render tumors highly sensitive to therapeutic PI3K/mTOR inhibition. By analyzing gene expression and protein data from nearly 1,800 human BCs, we report that a PIK3CA mutation-associated gene signature (PIK3CA-GS) derived from exon 20 (kinase domain) mutations was able to predict PIK3CA mutation status in two independent datasets, strongly suggesting a characteristic set of gene expression-induced changes. However, in ER+/HER2- BC despite pathway activation, PIK3CA mutations were associated with a phenotype of relatively low mTORC1 signaling and a good prognosis with tamoxifen monotherapy. The relationship between clinical outcome and the PIK3CA-GS was also assessed. Although the PIK3CA-GS was not associated with prognosis in ER- and HER2+ BC, it could identify better clinical outcomes in ER+/HER2- disease. In ER+ BC cell lines, PIK3CA mutations were also associated with sensitivity to tamoxifen. These findings could have important implications for the treatment of PIK3CA-mutant BCs and the development of PI3K/mTOR inhibitors.

Topics: Antibiotics, Antineoplastic; Antineoplastic Agents, Hormonal; Base Sequence; Breast Neoplasms; Cell Line, Tumor; Class I Phosphatidylinositol 3-Kinases; DNA Primers; Female; Gene Expression Profiling; Humans; Mechanistic Target of Rapamycin Complex 1; Multiprotein Complexes; Mutation; Neoplasms, Hormone-Dependent; Oligonucleotide Array Sequence Analysis; Phosphatidylinositol 3-Kinases; Prognosis; Proteins; Proto-Oncogene Proteins c-akt; Receptor, ErbB-2; Receptors, Estrogen; Signal Transduction; Sirolimus; Tamoxifen; TOR Serine-Threonine Kinases; Transcription Factors

Rapamycin analogs temsirolimus and everolimus have been approved for the treatment of advanced renal cancer and are being tested in a wide spectrum of human tumors. However, objective response rates with rapalogs in clinical trials were modest and variable. Identification of biomarkers capable of predicting response to rapalogs is of increasing interest. We analyzed pairwise Pearson correlation coefficients (r) between rapalogs activity and gene expression profile for each NCI-60 cell line. p27 showed the highest positive correlation among 9,706 gene probes tested. At cellular levels, breast cancer MCF-7, T47D, and BT-474 cells, expressing high levels of p27, were sensitive to rapalogs, whereas the cells expressed low levels of p27, such as MDA-MB-231, MDA-MB-468, and MDA-MB-435 cells, exhibited resistance to rapalogs. Mechanistic study indicated that this correlation is likely determined by the basal level of p27 regardless of the phosphorylation or redistribution of p27 upon rapalogs treatment, which may provide a putative threshold to block G1/S transition. Consistently, down-regulation of p27 by siRNA conferred MCF-7 and BT-474 cells insensitive to rapalogs. Moreover, a significant positive correlation between p27 gene expression and rapamycin anti-tumor activity was also observed in mice bearing different human cancer cell xenografts. In conclusion, p27 expression level is positively correlated with the anticancer activity of rapalogs in vitro and in vivo. We propose p27 expression level may be also a candidate predictive biomarker for patient selection for rapalogs-based therapy, which requires clinical validation in a series of patients treated with rapalogs.

Topics: Animals; Biomarkers; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p27; Female; Humans; Immunoprecipitation; Mice; Protein Kinase Inhibitors; Sirolimus; TOR Serine-Threonine Kinases; Transplantation, Heterologous

The malignant phenotype in breast cancer is driven by aberrant signal transduction pathways. Mixed-lineage kinase-3 (MLK3) is a mammalian mitogen-activated protein kinase kinase kinase (MAP3K) that activates multiple MAPK pathways. Depending on the cellular context, MLK3 has been implicated in apoptosis, proliferation, migration and differentiation. Here we investigated the effect of MLK3 and its signaling to MAPKs in the acquisition of malignancy in breast cancer. We show that MLK3 is highly expressed in breast cancer cells. We provide evidence that MLK3's catalytic activity and signaling to c-jun N-terminal kinase (JNK) is required for migration of highly invasive breast cancer cells and for MLK3-induced migration of mammary epithelial cells. Expression of active MLK3 is sufficient to induce the invasion of mammary epithelial cells, which requires AP-1 activity and is accompanied by the expression of several proteins corresponding to AP-1-regulated invasion genes. To assess MLK3's contribution to the breast cancer malignant phenotype in a more physiological setting, we implemented a strategy to inducibly express active MLK3 in the preformed acini of MCF10A cells grown in 3D Matrigel. Induction of MLK3 expression dramatically increases acinar size and modestly perturbs apicobasal polarity. Remarkably, MLK3 expression induces luminal repopulation and suppresses the expression of the pro-apoptotic protein BimEL, as has been observed in Her2/Neu-expressing acini. Taken together, our data show that MLK3-JNK-AP-1 signaling is critical for breast cancer cell migration and invasion. Our current study uncovers both a proliferative and novel antiapoptotic role for MLK3 in the acquisition of a malignant phenotype in mammary epithelial cells. Thus, MLK3 may be an important therapeutic target for the treatment of invasive breast cancer.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Culture Techniques; Cell Movement; Cell Transformation, Neoplastic; Cells, Cultured; Drug Evaluation, Preclinical; Female; Gene Expression Regulation, Neoplastic; Humans; Mammary Glands, Human; MAP Kinase Kinase Kinases; Mitogen-Activated Protein Kinase Kinase Kinase 11; Neoplasm Invasiveness; Phenotype; RNA, Small Interfering; Sirolimus; Transfection

Many breast cancers exhibit a degree of dependence on estrogen for tumor growth. Although several therapies have been developed to treat individuals with estrogen-dependent breast cancers, some tumors show de novo or acquired resistance, rendering them particularly elusive to current therapeutic strategies. Understanding the mechanisms by which these cancers develop resistance would enable the development of new and effective therapeutics. In order to determine mechanisms of escape from hormone dependence in estrogen receptor-positive (ER-positive) breast cancer, we established 4 human breast cancer cell lines after long-term estrogen deprivation (LTED). LTED cells showed variable changes in ER levels and sensitivity to 17beta-estradiol. Proteomic profiling of LTED cells revealed increased phosphorylation of the mammalian target of rapamycin (mTOR) substrates p70S6 kinase and p85S6 kinase as well as the PI3K substrate AKT. Inhibition of PI3K and mTOR induced LTED cell apoptosis and prevented the emergence of hormone-independent cells. Using reverse-phase protein microarrays, we identified a breast tumor protein signature of PI3K pathway activation that predicted poor outcome after adjuvant endocrine therapy in patients. Our data suggest that upon adaptation to hormone deprivation, breast cancer cells rely heavily on PI3K signaling. Our findings also imply that acquired resistance to endocrine therapy in breast cancer may be abrogated by combination therapies targeting both ER and PI3K pathways.

Topics: Apoptosis; Breast Neoplasms; Estradiol; Estrogen Receptor alpha; Estrogens; Female; Hormones; Humans; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Proto-Oncogene Proteins c-akt; Receptors, Estrogen; Signal Transduction; Sirolimus

To determine a novel chemotherapeutic concept for hormone receptor-negative and HER2-positive breast cancer, a high progression form of the disease for which treatment has been difficult. A combination therapy of 5-fluorouracil (5-FU) with rapamycin (Rap) was examined.. The growth inhibitory effect of treatment was evaluated by an MTT assay and cellular signal/apoptotic pathways were investigated by Western blotting and Hoechst 33342 staining.. Rap was shown to induce an inhibitory effect on the phosphorylation of mTOR and p70S6K. The expression of thymidine synthase (TS) was decreased by Rap. The addition of 5-FU to Rap was found to increase cell death. The Hoechst 33342 assay showed that apoptosis was increased by the combination of 5-FU and Rap in comparison to 5FU alone.. 5-FU is more effective in combination with the TS-reducing action of Rap, even for highly HER2-expressing breast cancer cells.

Topics: Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Cell Line, Tumor; Drug Synergism; Extracellular Signal-Regulated MAP Kinases; Female; Fluorouracil; Humans; Intracellular Signaling Peptides and Proteins; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Receptor, ErbB-2; Receptors, Estrogen; Receptors, Progesterone; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

To utilize a powerful new technology for target discovery, Random Homozygous Gene Perturbation (RHGP), and to identify novel targets that cause tumor cells to become chemoresistant.. RHGP was used to identify and validate genetic changes that cause chemoresistance of tumor cells to Rapamycin.. A series of targets was identified that allowed tumor cells to survive treatment with Rapamycin. We validated these targets and focused on Annexin A13, a target where decreased expression caused tumor cell insensitivity to Rapamycin. Ectopic overexpression of Annexin A13 was likewise sufficient to sensitize malignant breast cancer cells to treatment with Rapamycin.. These findings expand our knowledge of mechanisms that allow tumor cell drug resistance and demonstrate the power of RHGP to identify novel targets and mechanisms.

Topics: Annexins; Antineoplastic Agents; Breast Neoplasms; Cell Survival; Chromosome Mapping; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Female; Gene Library; Gene Targeting; Genetic Vectors; Humans; Sirolimus

Type 2 diabetes increases breast cancer risk and mortality, and hyperinsulinemia is a major mediator of this effect. The mammalian target of rapamycin (mTOR) is activated by insulin and is a key regulator of mammary tumor progression. Pharmacological mTOR inhibition suppresses tumor growth in numerous mammary tumor models in the non-diabetic setting. However, the role of the mTOR pathway in type 2 diabetes-induced tumor growth remains elusive. Herein, we investigated whether the mTOR pathway is implicated in insulin-induced mammary tumor progression in a transgenic mouse model of type 2 diabetes (MKR mice) and evaluated the impact of mTOR inhibition on the diabetic state. Mammary tumor progression was studied in the double transgenic MMTV-Polyoma Virus middle T antigen (PyVmT)/MKR mice and by orthotopic inoculation of PyVmT- and Neu/ErbB2-driven mammary tumor cells (Met-1 and MCNeuA cells respectively). mTOR inhibition by rapamycin markedly suppressed tumor growth in both wild-type and MKR mice. In diabetic animals, however, the promoting action of insulin on tumor growth was completely blunted by rapamycin, despite a worsening of the carbohydrate and lipid metabolism. Taken together, pharmacological mTOR blockade is sufficient to abrogate mammary tumor progression in the setting of hyperinsulinemia, and thus mTOR inhibitors may be an attractive therapeutic modality for breast cancer patients with type 2 diabetes. Careful monitoring of the metabolic state, however, is important as dose adaptations of glucose- and/or lipid-lowering therapy might be necessary.

Topics: Animals; Antibiotics, Antineoplastic; Apoptosis; Blood Glucose; Body Weight; Breast Neoplasms; Diabetes Mellitus, Type 2; Disease Models, Animal; Eating; Female; Insulin; Mice; Mice, Transgenic; Sirolimus; TOR Serine-Threonine Kinases; Triglycerides

Mammalian target of rapamycin (mTOR) signaling is a central regulator of protein translation, cell growth, and metabolism. Alterations of the mTOR signaling pathway are common in cancer, making mTOR a promising therapeutic target. In clinical trials, rapamycin analogs have shown modest response rates for most cancer types, including breast cancer. Therefore, there is an urgent need to better understand the mechanism of action of rapamycin to improve patient selection and to monitor pathway inhibition. To identify novel pharmacodynamic markers of rapamycin activity, we carried out transcriptional profiling of total and polysome-associated RNA in three breast cancer cell lines representing different subtypes. In all three cell lines, we found that rapamycin significantly decreased polysome-associated mRNA for stearoyl-CoA desaturase 1 (SCD1), the rate-limiting enzyme in monounsaturated fatty acid synthesis. Activators of mTOR increased SCD1 protein expression, whereas rapamycin, LY294002, and BEZ235 decreased SCD1 protein expression. Rapamycin decreased total SCD1 RNA expression without inducing a significant decline in its relative polysomal recruitment (polysome/total ratio). Rapamycin did not alter SCD1 mRNA stability. Instead, rapamycin inhibited SCD1 promoter activity and decreased expression of mature transcription factor sterol regulatory element binding protein 1 (SREBP1). Eukaryotic initiation factor 4E (eIF4E) small interfering RNA (siRNA) decreased both SCD1 and SREBP1 expression, suggesting that SCD1 may be regulated through the mTOR/eIF4E-binding protein 1 axis. Furthermore, SCD1 siRNA knockdown inhibited breast cancer cell growth, whereas overexpression increased growth. Taken together these findings show that rapamycin decreases SCD1 expression, establishing an important link between cell signaling and cancer cell fatty acid synthesis and growth.

Topics: Base Sequence; Blotting, Northern; Blotting, Western; Breast Neoplasms; Cell Line, Tumor; DNA Primers; Female; Humans; Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Sirolimus; Stearoyl-CoA Desaturase

Repeated administration of chemotherapeutics is typically required for the effective treatment of highly aggressive tumors and often results in systemic toxicity. We have created a copper-doxorubicin complex within the core of liposomes and applied the resulting particle in multidose therapy. Copper and doxorubicin concentrations in the blood pool were similar at 24 h (∼40% of the injected dose), indicating stable circulation of the complex. Highly quenched doxorubicin fluorescence remained in the blood pool over tens of hours, with fluorescence increasing only with the combination of liposome disruption and copper trans-chelation. At 48 h after injection, doxorubicin fluorescence within the heart and skin was one-fifth and one-half, respectively, of fluorescence observed with ammonium sulfate-loaded doxorubicin liposomes. After 28 days of twice per week doxorubicin administration of 6 mg/kg, systemic toxicity (cardiac hypertrophy and weight and hair loss) was not detected with the copper-doxorubicin liposomes but was substantial with ammonium sulfate-loaded doxorubicin liposomes. We then incorporated two strategies designed to enhance efficacy, mTOR inhibition (rapamycin) to slow proliferation and therapeutic ultrasound to enhance accumulation and local diffusion. Tumor accumulation was ∼10% ID/g and was enhanced approximately 2-fold with the addition of therapeutic ultrasound. After the 28-day course of therapy, syngeneic tumors regressed to a premalignant phenotype of ∼(1 mm)(3) or could not be detected.

Topics: Animals; Antineoplastic Agents; Breast Neoplasms; Copper; Disease Models, Animal; Doxorubicin; Female; Liposomes; Mice; Nanoparticles; Sirolimus; Ultrasonic Therapy; Xenograft Model Antitumor Assays

Previous studies have demonstrated that monospecific antisense oligonucleotides (oligos) directed against mRNA encoding proteins associated with tumor growth, death, and survival are efficacious against breast and prostate tumors. Targeted proteins, associated with different signal transduction pathways, have included transforming growth factor-alpha [TGF-alpha (MR(1))], its binding site the epidermal growth factor receptor [EGFR (MR(2))] sharing sequence homology to the breast cancer prognostic marker Her-2/neu, an apoptosis inhibiting protein [bcl-2 (MR(4))], and the androgen receptor [AR (MR(5))]. In attempts to enhance antisense therapy, recent reports describe how two of the binding sites mentioned above can be sequentially placed within a single complementary (bispecific) strand and administered either in the presence or absence of additional therapeutic agents. When tested against breast and prostate tumor cell lines specific differences were noted: MCF-7 breast cancer cells were more receptive to the inhibitory effects of monospecific oligos, whereas PC-3 and LNCaP prostate cells were particularly responsive to bispecifics. In an effort to identify agents which enhance the activity of oligos and which possess less toxicity than traditionally employed chemotherapeutics, Rapamycin, an immunosuppressive agent known to regulate tumor growth and signal transduction mediated by the mTOR receptor, is compared to paclitaxel in combination therapy employing monospecific or bispecific oligos. Bispecifics were constructed recognizing the binding sites for TGF-alpha and EGFR mRNA [TGF-alpha/EGFR (MR(12)) and EGFR/TGF-alpha (MR(21))]; another pair recognized binding sites for EGFR and bcl-2 [EGFR/bcl-2 (MR(24)) and bcl-2/EGFR (MR(42))]; while a third pair employed only against the LNCaP prostate cell line recognized bcl-2 and the androgen receptor [bcl-2/AR (MR4(45)) and AR/bcl-2 (MR(54))]. Oligo pairs differ in their 5'-3' linear binding site orientations, and were tested in vitro against MCF-7 breast and PC-3 and LNCaP prostate tumor cell lines. Following cell attachment, incubations were done for 2 days with the agents followed by 2 days in their absence. Five experiments evaluated the effect of monospecific or bispecific antisense oligos in combination with an LD(50) dosage of either Rapamycin or paclitaxel and led to the conclusion that although these agents act via different mechanisms, they are comparable in effectiveness.

Topics: Androgen Receptor Antagonists; Antibiotics, Antineoplastic; Antineoplastic Agents, Phytogenic; Apoptosis Regulatory Proteins; Breast Neoplasms; Cell Line, Tumor; Combined Modality Therapy; ErbB Receptors; Female; Gene Targeting; Humans; Male; Oligonucleotides, Antisense; Paclitaxel; Prostatic Neoplasms; Receptors, Androgen; Signal Transduction; Sirolimus; Transforming Growth Factor alpha

The 40 S ribosomal S6 kinase 1 (S6K1) acts downstream of mTOR (mammalian target of rapamycin) and is sensitive to inhibition by rapamycin. The chromosomal region 17q23 containing the RPS6KB1 gene is frequently amplified in breast cancer cells, leading to S6K1 overexpression. The role of S6K1 in disease development and progression is supported by the observation that S6K1 overexpression is associated with poor prognosis in breast cancer patients. However, the identity of mammary cell-specific S6K1 targets is not well understood. In this study, we report that overexpression of S6K1 endows breast cancer cells with a proliferative advantage in low serum conditions and enhanced sensitivity to rapamycin. We investigate the molecular mechanism behind this observation to show that S6K1 regulates estrogen receptor alpha (ERalpha) by phosphorylating it on serine 167, leading to transcriptional activation of ERalpha. By contributing to the activation of ERalpha, S6K1 promotes ERalpha-mediated cell proliferation and may be a target of therapeutic intervention in breast cancer.

Topics: Antibiotics, Antineoplastic; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Chromosomes, Human, Pair 17; Estrogen Receptor alpha; Estrogens; Female; Gene Expression Regulation, Neoplastic; Humans; Phosphorylation; Ribosomal Protein S6 Kinases, 70-kDa; Sirolimus; Transcription, Genetic

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Breast Neoplasms; Diphosphonates; Everolimus; Female; Humans; Imidazoles; Lapatinib; Letrozole; Nitriles; Quinazolines; Sirolimus; Tamoxifen; Trastuzumab; Triazoles; Zoledronic Acid

Because the mammalian target of rapamycin (mTOR) pathway is commonly deregulated in human cancer, mTOR inhibitors, rapamycin and its derivatives, are being actively tested in cancer clinical trials. Clinical updates indicate that the anticancer effect of these drugs is limited, perhaps due to rapamycin-dependent induction of oncogenic cascades by an as yet unclear mechanism. As such, we investigated rapamycin-dependent phosphoproteomics and discovered that 250 phosphosites in 161 cellular proteins were sensitive to rapamycin. Among these, rapamycin regulated four kinases and four phosphatases. A siRNA-dependent screen of these proteins showed that AKT induction by rapamycin was attenuated by depleting cellular CDC25B phosphatase. Rapamycin induces the phosphorylation of CDC25B at Serine375, and mutating this site to Alanine substantially reduced CDC25B phosphatase activity. Additionally, expression of CDC25B (S375A) inhibited the AKT activation by rapamycin, indicating that phosphorylation of CDC25B is critical for CDC25B activity and its ability to transduce rapamycin-induced oncogenic AKT activity. Importantly, we also found that CDC25B depletion in various cancer cell lines enhanced the anticancer effect of rapamycin. Together, using rapamycin phosphoproteomics, we not only advance the global mechanistic understanding of the action of rapamycin but also show that CDC25B may serve as a drug target for improving mTOR-targeted cancer therapies.

Topics: Antibiotics, Antineoplastic; Breast Neoplasms; cdc25 Phosphatases; Cell Line, Tumor; Enzyme Activation; HeLa Cells; Humans; Male; Phosphorylation; Prostatic Neoplasms; Protein Kinases; Proteomics; Proto-Oncogene Proteins c-akt; Sirolimus; TOR Serine-Threonine Kinases

Rapamycin, an inhibitor of mTOR, is in clinical trials for treatment of cancer. Rapamycin resistance has been reported in human breast epithelial tumor cells. Rapamycin effects on mTOR signaling and resistance were examined using benign, premalignant and tumor human breast epithelial cells. Rapamycin inhibition of cell proliferation, the cell cycle and mTOR signaling, including p70S6 and S6RP phosphorylation, was most effective in benign (MCF10A) and premalignant (MCF10AT; MCF10ATG3B) human breast epithelial cells, relative to MCF10CA1a tumor cells. Rapamycin resistance was reflected by reduced inhibition of p70S6K and S6RP phosphorylation in MCF10CA1a tumor cells, with RS6P showing the least response to rapamycin in the tumor cells. Rapamycin differentially inhibited STAT3 phosphorylation in this cell lineage. These data suggest that inhibition of mTOR signaling and STAT3 phosphorylation in benign and premalignant cells may be effective in the treatment of proliferative breast disease (PBD) and in the prevention of tumorigenesis and tumor recurrence.

Topics: Antibiotics, Antineoplastic; Breast; Breast Neoplasms; Cell Cycle; Cell Proliferation; Cells, Cultured; Flow Cytometry; Humans; Immunoblotting; Phosphorylation; Precancerous Conditions; Protein Kinases; Proto-Oncogene Proteins c-akt; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Sirolimus; STAT1 Transcription Factor; TOR Serine-Threonine Kinases

Selective drug delivery is an important approach with great potential for overcoming problems associated with the systemic toxicity and poor bioavailability of antineoplastic drugs. Nanomedicine plays a pivotal role by delivering drugs in a targeted manner to the malignant tumor cells thereby reducing the systemic toxicity of the anticancer drugs. The objective of this study was to prepare and characterize rapamycin loaded polymeric poly(lactide-co-glycolide) (PLGA) nanoparticles (NP) that were surface conjugated with antibodies to epidermal growth factor receptor (EGFR), highly expressed on breast cancer cells, using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) mediated cross linking agents. To potentiate the anticancer efficiency of the formulations, in vitro cytotoxicity of native rapamycin, rapamycin loaded nanoparticles and EGFR antibody conjugated rapamycin loaded nanoparticles (EGFR-Rapa-NPs) were evaluated on malignant MCF 7 breast cancer cell lines. IC(50) doses as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) assay showed the superior antiproliferative activity of EGFR-Rapa-NPs over unconjugated nanoparticles and native rapamycin due to higher cellular uptake on malignant breast cancer cells. Cell cycle arrest and cellular apoptosis induced by the above formulations were confirmed by flow cytometry. Molecular basis of apoptosis studied by western blotting revealed the involvement of a cytoplasmic protein in activating the programmed cell death pathway. Thus it was concluded that EGFR-Rapa-NPs provide an efficient and targeted delivery of anticancer drugs, presenting a promising active targeting carrier for tumor selective therapeutic treatment in near future.

Topics: Antibiotics, Antineoplastic; Breast Neoplasms; Cell Line, Tumor; Cell Survival; Drug Carriers; ErbB Receptors; Female; Humans; Lactic Acid; Materials Testing; Nanostructures; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Sirolimus

Optimal scheduling of cycle-active chemotherapy with (initially cytostatic) molecular-targeted agents is important to maximize clinical benefit. Concurrent scheduling might allow up-regulation of cell death pathways at the time of chemotherapy, whereas sequential treatments might maximize inhibition of repopulation and avoid putting tumor cells out of cycle when administering cycle-active chemotherapy. We compared the effects of concurrent and sequential administration of chemotherapy and the mammalian target of rapamycin (mTOR) inhibitor temsirolimus (CCI-779) on tumor cells and xenografts.. Human prostate cancer PC-3 and LnCaP, and human breast cancer MDA-468 cells and xenografts were treated with chemotherapy (docetaxel and 5-fluorouracil, respectively) and temsirolimus, using concurrent and sequential treatment schedules. Cell killing and repopulation were evaluated by clonogenic assays. Cell cycle analysis was done using flow cytometry. Effects on xenografts were assessed by tumor growth delay.. The proliferation of all cell lines was inhibited by temsirolimus in a dose-dependent manner; PTEN negative PC-3 and mutant LnCaP cells were more sensitive than PTEN-negative MDA-468 cells. Temsirolimus inhibited cell cycle progression from G(1) to S phase in all cell lines. Combined treatment had greater effects than temsirolimus or chemotherapy alone: for PC-3 and LnCaP xenografts, concurrent treatment seemed superior to sequential scheduling, whereas MDA-468 cells and xenograft tumors did not show schedule dependence.. Combined treatment with temsirolimus and chemotherapy had a greater therapeutic effect than monotherapy; concurrent scheduling was more effective for PC-3 and LnCaP cells and xenografts that were sensitive to temsirolimus.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Docetaxel; Drug Synergism; Female; Fluorouracil; Humans; Male; Mice; Mice, Nude; Prostatic Neoplasms; Protein Kinase Inhibitors; Protein Kinases; Sirolimus; Taxoids; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays

Mammalian target of rapamycin (mTOR) is a serine/threonine kinase involved in multiple intracellular signaling pathways promoting tumor growth. mTOR is aberrantly activated in a significant portion of breast cancers and is a promising target for treatment. Rapamycin and its analogues are in clinical trials for breast cancer treatment. Patterns of gene expression (metagenes) may also be used to simulate a biologic process or effects of a drug treatment. In this study, we tested the hypothesis that the gene-expression signature regulated by rapamycin could predict disease outcome for patients with breast cancer.. Colony formation and sulforhodamine B (IC50 < 1 nM) assays, and xenograft animals showed that MDA-MB-468 cells were sensitive to treatment with rapamycin. The comparison of in vitro and in vivo gene expression data identified a signature, termed rapamycin metagene index (RMI), of 31 genes upregulated by rapamycin treatment in vitro as well as in vivo (false discovery rate of 10%). In the Miller dataset, RMI did not correlate with tumor size or lymph node status. High (>75th percentile) RMI was significantly associated with longer survival (P = 0.015). On multivariate analysis, RMI (P = 0.029), tumor size (P = 0.015) and lymph node status (P = 0.001) were prognostic. In van 't Veer study, RMI was not associated with the time to develop distant metastasis (P = 0.41). In the Wang dataset, RMI predicted time to disease relapse (P = 0.009).. Rapamycin-regulated gene expression signature predicts clinical outcome in breast cancer. This supports the central role of mTOR signaling in breast cancer biology and provides further impetus to pursue mTOR-targeted therapies for breast cancer treatment.

Topics: Animals; Antibiotics, Antineoplastic; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Mammary Neoplasms, Experimental; Mice; Mice, Nude; Prognosis; Proportional Hazards Models; Sirolimus; Survival Analysis; Time Factors; Tumor Stem Cell Assay; Xenograft Model Antitumor Assays

Topics: Antibiotics, Antineoplastic; Breast Neoplasms; Cell Line, Tumor; Clinical Trials as Topic; Drug Delivery Systems; Female; Humans; Metformin; Protein Kinases; Receptor, IGF Type 1; Risk Assessment; Sensitivity and Specificity; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Rapamycin (Sirolimus) is a macrolide lactone with antifungal, immunosuppressant, and antiproliferative actions. The mechanism of rapamycin action involves the inhibition of mTOR and subsequent cytostasis. Rapamycin also prevents angiogenesis in tumors and can prevent cancer cells' resistance to other chemotherapeutic agents. However, very poor water solubility, bioavailability, only slight solubility in acceptable parenteral excipients, chemical instability, and major sequestration (95%) of free rapamycin into the erythrocytes have prevented its development as an anticancer drug. To address these problems, it was attempted to develop liposomal rapamycin delivery systems in this study. Conventional and pegylated liposomes were prepared with various lipid and cholesterol ratios. They were then characterized; these liposomes contained 0.68-0.90 mg of rapamycin per milliliter of liposome suspension. Having suitable particle size, these liposomes successfully retained the entrapped drug. Both types of liposomes were found to be effective; however, conventional liposomes showed better antiproliferative activity against MCF-7 cells than pegylated liposomes. But, pegylated liposome showed better stability than conventional liposomes. In conclusion, the enhanced permeability and retention effects of tumors should provide the opportunity for pegylated liposomal rapamycin to be applied as an intravenous drug-delivery system for targeted delivery to cancer cells, avoiding the major sequestration of free rapamycin into the erythrocytes.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Chromatography, High Pressure Liquid; Drug Carriers; Humans; Immunosuppressive Agents; Liposomes; Sirolimus

Topics: Antineoplastic Agents; Breast Neoplasms; Female; Humans; Imidazoles; Intracellular Signaling Peptides and Proteins; Phosphatidylinositol 3-Kinases; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Quinolines; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Honokiol, an active component isolated and purified from Chinese traditional herb magnolia, was demonstrated to inhibit growth and induce apoptosis of different cancer cell lines such as human leukaemia, colon, and lung cancer cell lines; to attenuate the angiogenic activities of human endothelial cells in vitro; and to efficiently suppress the growth of angiosarcoma in nude mice. In this study, we have demonstrated that treatment of different human breast cancer cell lines with honokiol resulted in a time- and concentration-dependent growth inhibition in both estrogen receptor-positive and -negative breast cancer cell lines, as well as in drug-resistant breast cancer cell lines such as adriamycin-resistant and tamoxifen-resistant cell lines. The inhibition of growth was associated with a G1-phase cell cycle arrest and induction of caspase-dependent apoptosis. The effects of honokiol might be reversely related to the expression level of human epidermal growth receptor 2, (HER-2, also known as erbB2, c-erbB2) since knockdown of her-2 expression by siRNA significantly enhanced the sensitivity of the her-2 over-expressed BT-474 cells to the honokiol-induced apoptosis. Furthermore, inhibition of HER-2 signalling by specific human epidermal growth receptor 1/HER-2 (EGFR/HER-2) kinase inhibitor lapatinib synergistically enhanced the anti-cancer effects of honokiol in her-2 over-expressed breast cancer cells. Finally, we showed that honokiol was able to attenuate the PI3K/Akt/mTOR (Phosphoinositide 3-kinases/Akt/mammalian target of rapamycin) signalling by down-regulation of Akt phosphorylation and upregulation of PTEN (Phosphatase and Tensin homolog deleted on chromosome Ten) expression. Combination of honokiol with the mTOR inhibitor rapamycin presented synergistic effects on induction of apoptosis of breast cancer cells. In conclusion, honokiol, either alone or in combination with other therapeutics, could serve as a new, promising approach for breast cancer treatment.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Biphenyl Compounds; Breast Neoplasms; Cell Line, Tumor; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Drug Synergism; Female; Gene Expression Regulation, Neoplastic; Humans; Lapatinib; Lignans; Magnolia; Medicine, Chinese Traditional; Quinazolines; Signal Transduction; Sirolimus; Time Factors

Increased Akt phosphorylation was reported in cancer cell lines and tumor tissues of patients exposed to rapamycin, a response likely contributing to the attenuated antitumor activity of rapamycin. It is, therefore, necessary to develop and validate combination strategies to reverse rapamycin-induced Akt signaling. We now report that Akt activation in response to rapamycin is abrogated by 17-allylamino-17-demethoxygeldanamycin (17-AAG), a heat shock protein 90 (HSP90) inhibitor. Rapamycin/17-AAG combination results in an enhanced antiproliferative activity in both MCF-7 and MDA-MB-231 breast cancer cells. In combination 17-AAG confers potent suppression of Raf-MEK-extracellular signal-regulated kinase signaling, a pathway that is otherwise not inhibited by rapamycin individually. Importantly, 17-AAG cooperates with rapamycin to block the phosphorylation of the mammalian target of rapamycin at Ser2448, as well as its downstream effectors ribosomal p70 S6 kinase and eukaryotic initiation factor 4E binding protein 1, which is accompanied by a substantial reduction in cyclins D1 and E. The potency of rapamycin/17-AAG combination is not affected by the activation of insulin-like growth factor 1 receptor signaling, which has been previously shown to diminish the antiproliferative activity of rapamycin. Rapamycin/17-AAG combination alleviates the induction of HSP90 protein, a heat shock response frequently associated with 17-AAG monotherapy. Our findings establish a mechanistic rationale for a combination approach using rapamycin and 17-AAG in the treatment of breast cancer.

Topics: Benzoquinones; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin E; Extracellular Signal-Regulated MAP Kinases; Female; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Protein Kinases; Proto-Oncogene Proteins c-akt; raf Kinases; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Tissue factor (TF) is a transmembrane glycoprotein that initiates blood coagulation when complexed with activated factor VII (FVIIa). TF is constitutively expressed in a variety of tumor cells and has been implicated in cellular signaling, angiogenesis, and tumor progression. Formation of TF-FVIIa complex and generation of downstream coagulation proteases, including activated factor X (FXa) and thrombin, initiate signaling by activation of protease-activated receptors (PARs). We have previously shown that TF-FVIIa-Xa complex formation promotes phosphorylation of p44/42 mitogen-activated protein kinase and Akt/protein kinase B in human breast cancer cells. In the present study, we show that formation of TF-FVIIa-FXa complex induces phosphorylation of mammalian target of rapamycin (mTOR) and p70 S6 kinase in a human breast cancer cell line, Adr-MCF-7. Activation of the mTOR pathway, which is probably mediated by PAR1 and/or PAR2, was associated with enhanced cell migration, a key step in the metastatic cascade. Inhibition of this pathway with the specific mTOR inhibitor, rapamycin, markedly decreased cell migration induced by formation of TF-FVIIa-FXa complex. These studies suggest that TF-FVIIa-mediated signaling modulates mTOR pathway activation, which regulates in part breast cancer cell migration. Targeting the TF-mediated cell signaling pathway might represent a novel strategy for the treatment of breast cancer.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Movement; Chromones; Factor VIIa; Factor Xa; Female; Humans; Morpholines; Neoplasm Invasiveness; Peptides; Phosphorylation; Protein Kinase Inhibitors; Protein Kinases; Proto-Oncogene Proteins c-akt; Receptor, PAR-1; Receptor, PAR-2; Recombinant Proteins; Ribosomal Protein S6 Kinases, 70-kDa; Sirolimus; Thromboplastin; TOR Serine-Threonine Kinases

The 70 kDa ribosomal protein S6 kinase (RPS6KB1), located at 17q23, is amplified and overexpressed in 10-30% of primary breast cancers and breast cancer cell lines. p70S6K is a serine/threonine kinase regulated by PI3K/mTOR pathway, which plays a crucial role in control of cell cycle, growth and survival. Our aim was to determine p70S6K and PI3K/mTOR/p70S6K pathway dependent gene expression profiles by microarrays using five breast cancer cell lines with predefined gene copy number and gene expression alterations. The p70S6K dependent profiles were determined by siRNA silencing of RPS6KB1 in two breast cancer cell lines overexpressing p70S6K. These profiles were further correlated with gene expression alterations caused by inhibition of PI3K/mTOR pathway with PI3K inhibitor Ly294002 or mTOR inhibitor rapamycin.. Altogether, the silencing of p70S6K altered the expression of 109 and 173 genes in two breast cancer cell lines and 67 genes were altered in both cell lines in addition to RPS6KB1. Furthermore, 17 genes including VTCN1 and CDKN2B showed overlap with genes differentially expressed after PI3K or mTOR inhibition. The gene expression signatures responsive to both PI3K/mTOR pathway and p70S6K inhibitions revealed previously unidentified genes suggesting novel downstream targets for PI3K/mTOR/p70S6K pathway.. Since p70S6K overexpression is associated with aggressive disease and poor prognosis of breast cancer patients, the potential downstream targets of p70S6K and the whole PI3K/mTOR/p70S6K pathway identified in our study may have diagnostic value.

Topics: Antibiotics, Antineoplastic; Apoptosis; Breast Neoplasms; Cell Line, Tumor; Chromones; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Morpholines; Oligonucleotide Array Sequence Analysis; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Protein Kinases; Ribosomal Protein S6 Kinases, 70-kDa; RNA, Small Interfering; Sirolimus; TOR Serine-Threonine Kinases

The enzyme mTOR (mammalian target of rapamycin) is a major target for therapeutic intervention to treat many human diseases, including cancer, but very little is known about the processes that control levels of mTOR protein. Here, we show that mTOR is targeted for ubiquitination and consequent degradation by binding to the tumor suppressor protein FBXW7. Human breast cancer cell lines and primary tumors showed a reciprocal relation between loss of FBXW7 and deletion or mutation of PTEN (phosphatase and tensin homolog), which also activates mTOR. Tumor cell lines harboring deletions or mutations in FBXW7 are particularly sensitive to rapamycin treatment, which suggests that loss of FBXW7 may be a biomarker for human cancers susceptible to treatment with inhibitors of the mTOR pathway.

Topics: Animals; Breast Neoplasms; Cell Cycle Proteins; Cell Line; Cell Line, Tumor; F-Box Proteins; F-Box-WD Repeat-Containing Protein 7; Gene Deletion; Gene Dosage; Gene Silencing; Genes, Tumor Suppressor; Humans; Mice; Mice, Nude; Mutation; Neoplasm Transplantation; Phosphorylation; Protein Binding; Protein Kinases; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Transfection; Tumor Suppressor Proteins; Ubiquitin-Protein Ligases; Ubiquitination

Activation of the Akt/mammalian target of rapamycin (mTOR) pathway has been shown to be associated with resistance to endocrine therapy in estrogen receptor alpha (ERalpha)-positive breast cancer patients. Utmost importance is attached to strategies aimed at overcoming treatment resistance. In this context, this work aimed to investigate whether, in breast cancer cells, the use of an mTOR inhibitor would be sufficient to reverse the resistance acquired after exposure to endocrine therapy. The ERalpha-positive human breast adenocarcinoma derived-MCF-7 cells used in this study have acquired both cross-resistance to hydroxy-tamoxifen (OH-Tam) and to fulvestrant and strong activation of the Akt/mTOR pathway. Cell proliferation tests in control cells demonstrated that the mTOR inhibitor rapamycin enhanced cell sensitivity to endocrine therapy when combined to OH-Tam or to fulvestrant. In resistant cells, rapamycin used alone greatly inhibited cell proliferation and reversed resistance to endocrine therapy by blocking the agonist-like activity of OH-Tam on cell proliferation and bypassing fulvestrant resistance. Reversion of resistance by rapamycin was associated with increased ERalpha protein expression levels and modification of the balance of phospho-ser167 ERalpha/total ERalpha ratio. Pangenomic DNA array experiments demonstrated that the cotreatment of resistant cells with fulvestrant and rapamycin allowed the restoration of 40% of the fulvestrant gene-expression signature. Taken together, data presented herein strongly support the idea that mTOR inhibitor might be one of the promising therapeutic approaches for patients with ERalpha-positive endocrine therapy-resistant breast cancers.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Drug Resistance, Neoplasm; Female; Gene Expression Regulation, Neoplastic; Humans; Models, Genetic; Receptors, Estrogen; Sirolimus

Overexpression of HER-2/Neu occurs in about 25-30% of breast cancer patients and is indicative of poor prognosis. While Her2/Neu overexpression is primarily a result of erbB2 amplification, it has recently been recognized that erbB2 levels are also regulated on the protein level. However, factors that regulate Her2/Neu protein stability are less well understood. The prolyl isomerase Pin1 catalyzes the isomerization of specific pSer/Thr-Pro motifs that have been phosphorylated in response to mitogenic signaling. We have previously reported that Pin1-catalyzed post-phosphorylational modification of signal transduction modulates the oncogenic pathways downstream from c-neu. The goal of this study was to examine the expression of prolyl isomerase Pin1 in human Her2+ breast cancer, and to study if Pin1 affects the expression of Her2/Neu itself.. Immunohistochemistry for Her2 and Pin1 were performed on two hundred twenty-three human breast cancers, with 59% of the specimen from primary cancers and 41% from metastatic sites. Pin1 inhibition was achieved using siRNA in Her2+ breast cancer cell lines, and its effects were studied using cell viability assays, immunoblotting and immunofluorescence.. Sixty-four samples (28.7%) stained positive for Her2 (IHC 3+), and 54% (122/223) of all breast cancers stained positive for Pin1. Of the Her2-positive cancers 40 (62.5%) were also Pin1-positive, based on strong nuclear or nuclear and cytoplasmic staining. Inhibition of Pin1 via RNAi resulted in significant suppression of Her2-positive tumor cell growth in BT474, SKBR3 and AU565 cells. Pin1 inhibition greatly increased the sensitivity of Her2-positive breast cancer cells to the mTOR inhibitor Rapamycin, while it did not increase their sensitivity to Trastuzumab, suggesting that Pin1 might act on Her2 signaling. We found that Pin1 interacted with the protein complex that contains ubiquitinated erbB2 and that Pin1 inhibition accelerated erbB2 degradation, which could be prevented by treatments with the proteasome inhibitor ALLnL.. Pin1 is a novel regulator of erbB2 that modulates the ubiquitin-mediated degradation of erbB2. The overexpression of Pin1 in a majority of Her2-overexpressing breast cancer may contribute to maintain erbB2 levels. Pin1 inhibition alone and in conjunction with mTOR inhibition suppresses the growth of Her2+ breast cancer cells.

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Down-Regulation; Humans; Immunohistochemistry; NIMA-Interacting Peptidylprolyl Isomerase; Peptidylprolyl Isomerase; Proteasome Inhibitors; Protein Processing, Post-Translational; Protein Stability; Receptor, ErbB-2; Sirolimus; Transcription, Genetic; Trastuzumab

mTOR, the mammalian target of rapamycin, is a critical target of survival signals in many human cancers. In the absence of serum, rapamycin induces apoptosis in MDA-MB-231 human breast cancer cells. However, in the presence of serum, rapamycin induces G(1) cell cycle arrest-indicating that a factor(s) in serum suppresses rapamycin-induced apoptosis. We report here that transforming growth factor-beta (TGF-beta) suppresses rapamycin-induced apoptosis in serum-deprived MDA-MB-231 cells in a protein kinase Cdelta (PKCdelta)-dependent manner. Importantly, if TGF-beta signaling or PKCdelta was suppressed, rapamycin induced apoptosis rather than G(1) arrest in the presence of serum. And, if cells were allowed to progress into S phase, rapamycin induced apoptosis in the presence of serum. BT-549 and MDA-MB-468 breast, and SW-480 colon cancer cells have defects in TGF-beta signaling and rapamycin induced apoptosis in these cells in the presence of either serum or TGF-beta. Thus, in the absence of TGF-beta signaling, rapamycin becomes cytotoxic rather than cytostatic. Importantly, this study provides evidence indicating that tumors with defective TGF-beta signaling--common in colon and pancreatic cancers--will be selectively sensitive to rapamycin or other strategies that target mTOR.

Topics: Antibiotics, Antineoplastic; Apoptosis; Breast Neoplasms; Cell Line, Tumor; Dose-Response Relationship, Drug; Humans; Signal Transduction; Sirolimus; Transforming Growth Factor beta

There is currently substantial interest in the regulation of cell function by mammalian target of rapamycin (mTOR), especially effects linked to the rapamycin-sensitive mTOR complex 1 (mTORC1). Rapamycin induces G(1) arrest and blocks proliferation of many tumor cells, suggesting that the inhibition of mTORC1 signaling may be useful in cancer therapy. In MCF7 breast adenocarcinoma cells, rapamycin decreases levels of cyclin D1, without affecting cytoplasmic levels of its mRNA. In some cell-types, rapamycin does not affect cyclin D1 levels, whereas the starvation for leucine (which impairs mTORC1 signaling more profoundly than rapamycin) does. This pattern correlates with the behavior of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1, an mTORC1 target that regulates translation initiation). siRNA-mediated knock-down of 4E-BP1 abrogates the effect of rapamycin on cyclin D1 expression and increases the polysomal association of the cyclin D1 mRNA. Our data identify 4E-BP1 as a key regulator of cyclin D1 expression, indicate that this effect is not mediated through the changes in cytoplasmic levels of cyclin D1 mRNA and suggest that, in some cell types, interfering with the amino acid input to mTORC1, rather than using rapamycin, may inhibit proliferation.

Topics: Adaptor Proteins, Signal Transducing; Antibiotics, Antineoplastic; Breast Neoplasms; Cell Cycle Proteins; Cell Line; Cell Line, Tumor; Cyclin D1; Eukaryotic Initiation Factor-4E; Gene Expression Regulation, Neoplastic; Humans; Mechanistic Target of Rapamycin Complex 1; Multiprotein Complexes; Phosphoproteins; Proteins; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Transcription Factors

Rapamycin and its analogues are currently being tested in clinical trials as novel-targeted anticancer agents. Pre-clinical studies that used breast cancer cell lines have suggested that p-Akt or p-S6K1 expressing tumors, as well as PTEN negative tumors, were sensitive to rapamycin. The aims of this study were to determine the proportion of breast cancer that could be candidates for rapamycin treatment and to elucidate the clinicopathologic characteristics and prognosis of potentially rapamycin-sensitive tumors.. We evaluated the expressions of PTEN, p-Akt and p-S6K1 by performing immunohistochemistry in 122 breast cancer tissues. We analyzed the association of the expression of these proteins with the cliniopathologic variables and the disease-free survival.. PTEN negative tumors, p-Akt expressing tumors and p-S6K1 expressing tumors constituted 4.1% (5/122), 41.0% (50/122), and 36.1% (44/122) of the total tumors, respectively. The proportion of tumors that met the criteria of rapamycin sensitivity was 54.9% (67/122). We could not find any significant correlation between the expression of these proteins and the other prognostic factors. However, the prognosis of tumors with a p-S6K1 expression was significantly worse than that of the p-S6K1 negative tumors.. Based on the status of the PTEN, p-Akt and p-S6K1 expressions as predictors of rapamycin sensitivity, this study suggested that over 50% of breast cancer patients could be potential candidates for rapamycin treatment. In addition, the p-S6K1 expression may constitute an independent prognostic factor for disease-free survival.

Topics: Adult; Aged; Antibiotics, Antineoplastic; Biomarkers, Tumor; Breast Neoplasms; Disease-Free Survival; Drug Resistance, Neoplasm; Female; Humans; Immunohistochemistry; Middle Aged; Prognosis; Protein Kinases; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Ribosomal Protein S6 Kinases; Signal Transduction; Sirolimus; Tissue Array Analysis; TOR Serine-Threonine Kinases

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Capecitabine; Deoxycytidine; Docetaxel; Epothilones; Everolimus; Female; Fluorouracil; Humans; Letrozole; Nitriles; Sirolimus; Tamoxifen; Taxoids; Trastuzumab; Triazoles

Ectopic expression of mutant forms of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) lacking lipid (G129E) or lipid and protein (C124S) phosphatase activity decreased sensitivity of MCF-7 breast cancer cells, which have wild-type PTEN, to doxorubicin and increased sensitivity to the mammalian target of rapamycin (mTOR) inhibitor rapamycin. Cells transfected with a mutant PTEN gene lacking both lipid and protein phosphatase activities were more resistant to doxorubicin than cells transfected with the PTEN mutant lacking lipid phosphatase activity indicating that the protein phosphatase activity of PTEN was also important in controlling the sensitivity to doxorubicin, while no difference was observed between the lipid (G129E) and lipid and protein (C124S) phosphatase PTEN mutants in terms of sensitivity to rapamycin. A synergistic inhibitory interaction was observed when doxorubicin was combined with rapamycin in the phosphatase-deficient PTEN-transfected cells. Interference with the lipid phosphatase activity of PTEN was sufficient to activate Akt/mTOR/p70S6K signaling. These studies indicate that disruption of the normal activity of the PTEN phosphatase can have dramatic effects on the therapeutic sensitivity of breast cancer cells. Mutations in the key residues which control PTEN lipid and protein phosphatase may act as dominant-negative mutants to suppress endogenous PTEN and alter the sensitivity of breast cancer patients to chemo- and targeted therapies.

Topics: Amino Acid Substitution; Antibiotics, Antineoplastic; Breast Neoplasms; Cell Line, Tumor; Doxorubicin; Drug Resistance, Neoplasm; Drug Synergism; Female; Gene Expression; Humans; Mutation, Missense; Protein Kinases; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Transfection

The Akt/mammalian target of the rapamycin (mTOR) signaling pathway represents a promising target for cancer therapy. The phosphorylation status of Akt and of mTOR's phosphorylation target eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) is often used to assess the activity of Akt and mTOR signaling. The purpose of this study was to determine whether primary tumors differ from their metastasis in their expression of pAkt and p4E-BP1.. Primary breast tumors and their distant metastases surgically resected from the same patients were evaluated with immunohistochemical analysis (IHC) for pAkt (Ser473) and p4E-BP1 (Ser65). The agreement between the IHC results for the primary tumor and metastases was evaluated with Cohen kappa (kappa).. Most primary breast tumors and metastatic tumors expressed pAkt (76% of each). Of the 23 matched evaluable pairs, however, 11 (47.8%) had discordant IHC results (kappa -0.31; 95% confidence interval [CI], -0.49 to -0.13). Similarly, although most of the primary and metastatic tumors were positive for p4E-BP1 (75% and 74%), of the 23 matched evaluable pairs, 8 (47.8%) were discordant (kappa 0.10; 95% CI, -0.33-0.52).. In this series, most primary breast tumors and metastases expressed pAkt and p4E-BP1 by IHC. Concordance between IHC findings in primary tumors and metastases was poor, however. Further work is needed to determine whether this reflects true biological heterogeneity or poor reproducibility of IHC with phosphospecific antibodies, and to identify which biomarkers can be assessed most reproducibly in primary tumors to predict activity of Akt/mTOR signaling and sensitivity to pathway inhibitors.

Topics: Adaptor Proteins, Signal Transducing; Adult; Aged; Breast Neoplasms; Cell Cycle Proteins; Cytoplasm; Female; Humans; Immunoenzyme Techniques; Liver Neoplasms; Lung Neoplasms; Middle Aged; Peritoneal Neoplasms; Phosphoproteins; Phosphorylation; Protein Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

The mammalian target of rapamycin (mTOR) inhibitor CCI-779 (temsirolimus) is a recently Food and Drug Administration-approved anticancer drug with efficacy in certain solid tumors and hematologic malignancies. In cell culture studies, CCI-779 at the commonly used nanomolar concentrations generally confers a modest and selective antiproliferative activity. Here, we report that, at clinically relevant low micromolar concentrations, CCI-779 completely suppressed proliferation of a broad panel of tumor cells. This "high-dose" drug effect did not require FKBP12 and correlated with an FKBP12-independent suppression of mTOR signaling. An FKBP12-rapamycin binding domain (FRB) binding-deficient rapamycin analogue failed to elicit both the nanomolar and micromolar inhibitions of growth and mTOR signaling, implicating FRB binding in both actions. Biochemical assays indicated that CCI-779 and rapamycin directly inhibited mTOR kinase activity with IC(50) values of 1.76 +/- 0.15 and 1.74 +/- 0.34 micromol/L, respectively. Interestingly, a CCI-779-resistant mTOR mutant (mTOR-SI) displayed an 11-fold resistance to the micromolar CCI-779 in vitro (IC(50), 20 +/- 3.4 micromol/L) and conferred a partial protection in cells exposed to micromolar CCI-779. Treatment of cancer cells with micromolar but not nanomolar concentrations of CCI-779 caused a marked decline in global protein synthesis and disassembly of polyribosomes. The profound inhibition of protein synthesis was accompanied by rapid increase in the phosphorylation of translation elongation factor eEF2 and the translation initiation factor eIF2 alpha. These findings suggest that high-dose CCI-779 inhibits mTOR signaling through an FKBP12-independent mechanism that leads to profound translational repression. This distinctive high-dose drug effect could be directly related to the antitumor activities of CCI-779 and other rapalogues in human cancer patients.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Division; Cell Line; Cell Line, Tumor; Colonic Neoplasms; Female; Humans; Kidney; Lung Neoplasms; Male; Prostatic Neoplasms; Protein Biosynthesis; Protein Kinases; Sirolimus; Tacrolimus Binding Protein 1A; TOR Serine-Threonine Kinases

Estrogen-receptor (ER)-positive breast cancers comprise the majority of sporadic breast cancers. Although 50% respond to antihormonal treatment, both primary and acquired resistance limit the utility of this therapy, and other agents are needed. Rapamycin, an inhibitor of the mammalian Target of Rapamycin (mTOR), possesses antitumor activity against many tumors including breast tumors, and particularly against ER-positive breast cancer cell lines. The sensitivity of these cells to rapamycin has been attributed to activation of the PI3K/Akt/mTOR pathway by nongenomic ER signaling. The purpose of this study was to evaluate the efficacy of rapamycin against ER-positive breast cancer, particularly under 17beta-estradiol (E2)-dependent conditions, and to investigate mechanisms of rapamycin-sensitivity in ER-positive cells.. Breast cancer cell lines were tested for sensitivity to rapamycin. Antiproliferative effects of rapamycin, alone and in combination with tamoxifen, were assessed under E2-dependent conditions. Western blot analysis was used to detect activation of mTOR by nongenomic ER signaling.. Rapamycin effectively inhibits proliferation of the ER-positive MCF-7 cell line. In our system, this sensitivity is probably not due to nongenomic ER activation of the PI3K/Akt/mTOR pathway; rapid stimulation of mTOR occurred nonspecifically after medium replacement, and addition of E2 stimulated mTOR only after 1 h. Combining rapamycin and tamoxifen under E2-dependent conditions yielded additive/synergistic effects at effective concentrations.. These results suggest that rapamycin may be an effective treatment for ER-positive breast cancer, either alone or in combination with tamoxifen, and also may be a potential therapy for tamoxifen-resistant cancers.

Topics: Antibiotics, Antineoplastic; Antineoplastic Agents, Hormonal; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Cell Division; Cell Line, Tumor; Cell Survival; Drug Resistance, Neoplasm; Estradiol; Humans; Phosphorylation; Protein Kinases; Receptors, Estrogen; Ribosomal Protein S6 Kinases; Signal Transduction; Sirolimus; Tamoxifen; TOR Serine-Threonine Kinases

The objective of this study was to assess the anti-tumor efficacy of rapamycin alone or in combination with herceptin in breast cancer. A total of 20 human breast cancer lines were examined for expression of various receptor tyrosine kinases and activation of their down stream signaling molecules, as well as for their invasion and colony forming ability. The ErbB2 and PI3 kinase pathway inhibitors were tested for the inhibition on breast cancer cell growth and tumor development. Seven of the 20 lines displayed an elevated level of ErbB2, others had varying level of EGF, IGF-1 or insulin receptor. Over 30% of the lines also had constitutive activation of Akt and MAP kinase. The lines displayed a wide range of colony forming and invasion ability. The PI3 kinase pathway inhibitors LY294002 and rapamycin inhibited the colony forming ability of all of the lines with the ErbB2 overexpressing lines having a higher sensitivity. A similar trend was observed for inhibition of invasion by LY294002. Rapamycin alone and additively together with herceptin inhibited the breast cancer cell growth especially in ErbB2 overexpressing cells. Rapamycin and herceptin synergistically inhibited tumor growth and endpoint tumor load in a xenograft model using a MCF-7 subline and in a MMTV-ErbB2 transgenic model. Rapamycin and herceptin significantly reduced the level of cyclin D1 and D3 and increased the cleavage of caspase 3 suggesting an increased apoptosis. Our results suggest that rapamycin together with herceptin has an enhanced anti-cancer effect and could be developed as an improved therapeutic regimen for breast cancer.

Topics: Animals; Antineoplastic Agents; Breast Neoplasms; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin D3; Cyclins; Female; Humans; Intercellular Signaling Peptides and Proteins; Mice; Mice, Inbred BALB C; Mitogen-Activated Protein Kinases; Neoplasm Invasiveness; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Receptor Protein-Tyrosine Kinases; Receptor, ErbB-2; Signal Transduction; Sirolimus; Xenograft Model Antitumor Assays

Developing novel synergistic and more effective combination treatments is necessary for better management of breast cancer in the clinic. It is established that HER-2 overexpressing breast cancers are sensitive to the HER-1 (epidermal growth factor receptor (EGFR)) inhibitor gefitinib, but that this targeted agent produces only moderate therapeutic effects in vivo. Here, we use a model of ER(+) HER-2 overexpressing MCF-7 breast cancer (MCF-7(HER-2)) to identify, as broadly as possible, the in vivo microenvironmental and molecular therapeutic responses to gefitinib to predict a therapeutically viable target for gefitinib-based combination treatment. Our data show a link between in vivo reductions in tumor hypoxia (3-fold decrease, P = 0.002) and elevated activity of the mTOR pathway (3.8-fold increase in phospho-p70-S6K protein, P = 0.006) in gefitinib treated MCF-7(HER-2) tumors. Despite decreased levels of phosphorylated EGFR, HER-2 and Erk1/2 (P = 0.081, 0.005 and 0.034, respectively) the expression of phospho-AKT was not reduced in MCF-7(HER-2) tumors after gefitinib treatment. Levels of ERalpha receptor were, however, 1.8-fold higher in gefitinib treated compared to control tumors (P = 0.008). Based on these results we predict that gefitinib activity against ER(+) HER-2 overexpressing EGFR co-expressing breast cancers should be enhanced if used with agents that target the mTOR pathway. In vitro studies using MCF-7(HER-2) and BT474 breast cancer cells exposed to gefitinib and rapamycin in combination show that this combination produced significantly greater growth inhibitory effects than either of the drugs alone. Chou and Talalay analysis of the data suggested that combination of gefitinib and rapamycin was synergistic (CI < 1) at a number of selected drug ratios and over a broad range of effective doses.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Cell Hypoxia; Cell Line, Tumor; ErbB Receptors; Female; Gefitinib; Humans; Mice; Protein Kinases; Proto-Oncogene Proteins c-akt; Quinazolines; Receptor, ErbB-2; Receptors, Estrogen; Sirolimus; TOR Serine-Threonine Kinases

Resistance to endocrine therapy is a major impediment in breast cancer therapeutics. The Phosphatidylinositol-3-OH kinase (PI3K)/Protein kinase B (Akt/PKB) kinase signaling pathway has been implicated in altering breast cancer response to multiple therapies. How Akt modulates response is an area of significant clinical relevance.. We have used an in vitro model to discern the effects of robust Akt activity on breast cancer cellular response to endocrine therapies.. High levels of Akt activity confer resistance to the aromatase inhibitor Letrozole (Let) and the selective estrogen receptor (ER) down-regulator Fulvestrant (ICI). Akt-induced resistance is not due to failure of these endocrine agents to inhibit estrogen receptor alpha activity. Instead, resistance is characterized by altered cell cycle and apoptotic response. Cotreatment with low concentrations of the mTOR inhibitor RAD-001 and either Let or ICI restores response of the resistant cells to levels observed in the responsive cells treated with either Let or ICI as a single agent.. Our preliminary findings in experiments with RAD-001 indicate that cotreatment with mTOR inhibitors and either Let or ICI reverses the Akt-mediated resistance and restores responsiveness to antiestrogens. Concurrent ER and mTOR inhibition is therefore an effective strategy to overcome growth factor-induced resistance and bears significant implications for optimal clinical development of these agents in breast cancer treatment.

Topics: Antineoplastic Agents, Hormonal; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Aromatase Inhibitors; Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Drug Resistance, Neoplasm; Estradiol; Everolimus; Female; Flow Cytometry; Fulvestrant; Humans; Letrozole; Nitriles; Protein Kinases; Proto-Oncogene Proteins c-akt; Sirolimus; TOR Serine-Threonine Kinases; Triazoles

Several studies have demonstrated significant interactions between immunosuppressants (e.g., cyclosporin A) and chemotherapeutic drugs that are BCRP substrates (e.g., irinotecan), resulting in increased bioavailability and reduced clearance of these agents. One possible mechanism underlying this observation is that the immunosuppressants modulate the pharmacokinetics of these drugs by inhibiting BCRP. Therefore, the aim of this study was to determine whether the immunosuppressants cyclosporin A, tacrolimus and sirolimus are inhibitors and/or substrates of BCRP.. First, the effect of the immunosuppressants on BCRP efflux activity in BCRP-expressing HEK cells was measured by flow cytometry.. Cyclosporin A, tacrolimus and sirolimus significantly inhibited BCRP-mediated efflux of pheophorbide A, mitoxantrone and BODIPY-prazosin. The EC(50) values of cyclosporin A, tacrolimus and sirolimus for inhibition of BCRP-mediated pheophorbide A efflux were 4.3 +/- 1.9 microM, 3.6 +/- 1.8 microM and 1.9 +/- 0.4 microM, respectively. Cyclosporin A, tacrolimus and sirolimus also effectively reversed resistance of HEK cells to topotecan and mitoxantrone conferred by BCRP. When direct efflux of cyclosporin A, tacrolimus and sirolimus was measured, these compounds were found not to be transported by BCRP. Consistent with this finding, BCRP did not confer resistance to the immunosuppressants in HEK cells.. These results indicate that cyclosporin A, tacrolimus and sirolimus are effective inhibitors but not substrates of BCRP. These findings could explain the altered pharmacokinetics of BCRP substrate drugs when co-administered with the immunosuppressants and suggest that pharmacokinetic modulation by the immunosuppressants may improve the therapeutic outcome of these drugs.

Topics: Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Breast Neoplasms; Cell Line, Tumor; Cell Survival; Cyclosporine; Drug Resistance, Neoplasm; Female; Flow Cytometry; Humans; Immunosuppressive Agents; Mitoxantrone; Neoplasm Proteins; Sirolimus; Substrate Specificity; Tacrolimus; Topotecan

Interactions between estrogen receptor signaling and the PI3K/Akt pathway are present in estrogen-dependent cancer cells. Therapeutical inhibition of each of these pathways has been proven to exert antitumoral effects. Inhibition of mammalian target of rapamycin (mTOR), a downstream target of Akt, is able to restore tamoxifen response in tamoxifen-resistant breast cancer cells. Given that Akt and mTOR phosphorylation also is frequently detected in ovarian and endometrial cancer, we intended to find out to what extent mTOR inhibitor RAD001 (everolimus) and tamoxifen add to each other's effects on growth and apoptosis of cancer cell lines derived from these tissues when given concomitantly.. OVCAR-3 and SK-OV-3 ovarian cancer cells, HEC-1A endometrial adenocarcinoma cells and MCF-7 breast cancer cells were treated with different concentrations of mTOR inhibitor RAD001 alone or in combination with 4-OH tamoxifen. Relative numbers of viable cells were assessed by means of the resazurin-based Cell Titer Blue assay, cellular apoptosis was examined by measurement of activated caspases 3 and 7 by means of the luminometric Caspase-Glo assay.. Treatment with RAD001 resulted in growth inhibition of all employed cancer cell lines in a dose-dependent manner, and SK-OV-3 ovarian cancer cells proved to be most sensitive to this drug. Moreover, we report the observation of additive, but not synergistical growth inhibitory effects of a combination treatment with RAD001 and 4-OH TAM on SK-OV-3 and OVCAR-3 ovarian cancer cells and MCF-7 breast cancer cells in vitro, whereas no such effect was observed in HEC-1A endometrial adenocarcinoma cells. Combination treatment with both drugs was demonstrated to be superior to single treatment with lower concentrations (0.1 and 1 nM) of RAD001 or standard concentrations of 4-OH TAM. Furthermore, RAD001 increased the apoptotic effect triggered by high 4-OH TAM concentrations in SK-OV-3 ovarian cancer cells.. Combination treatment with RAD001 and 4-OH TAM in vitro exerts an additive antitumoral effect on ovarian cancer cells and MCF-7 breast cancer cells. The significance of these data in the clinical situation has to be evaluated in further studies.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Breast Neoplasms; Cell Growth Processes; Cell Line, Tumor; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Drug Synergism; Endometrial Neoplasms; Everolimus; Female; Humans; Ovarian Neoplasms; Protein Kinases; RNA, Messenger; Sirolimus; Tamoxifen; TOR Serine-Threonine Kinases

Notch signaling is believed to promote cell survival in general. However, the mechanism is not clearly understood. Here, we show that cells expressing intracellular domain of human Notch1 (NIC-1) are chemoresistant in a wild-type p53-dependent manner. NIC-1 inhibited p53 by inhibiting its activating phosphorylations at Ser(15), Ser(20), and Ser(392) as well as nuclear localization. In addition, we found that inhibition of p53 by NIC-1 mainly occurs through mammalian target of rapamycin (mTOR) using phosphatidylinositol 3-kinase (PI3K)-Akt/protein kinase B (PKB) pathway as the mTOR inhibitor, rapamycin treatment abrogated NIC-1 inhibition of p53 and reversed the chemoresistance. Consistent with this, rapamycin failed to reverse NIC-1-induced chemoresistance in cells expressing rapamycin-resistant mTOR. Further, ectopic expression of eukaryotic initiation factor 4E (eIF4E), a translational regulator that acts downstream of mTOR, inhibited p53-induced apoptosis and conferred protection against p53-mediated cytotoxicity to similar extent as that of NIC-1 overexpression but was not reversed by rapamycin, which indicates that eIF4E is the major target of mTOR in Notch1-mediated survival signaling. Finally, we show that MCF7 (breast cancer) and MOLT4 (T-cell acute lymphoblastic leukemia) cells having aberrant Notch1 signaling are chemoresistant, which can be reversed by both PI3K and mTOR inhibitors. These results establish that Notch1 signaling confers chemoresistance by inhibiting p53 pathway through mTOR-dependent PI3K-Akt/PKB pathway and imply that p53 status perhaps is an important determinant in combination therapeutic strategies, which use mTOR inhibitors and chemotherapy.

Topics: Breast Neoplasms; Cell Nucleus; Cell Survival; Drug Resistance, Neoplasm; Eukaryotic Initiation Factor-4E; Humans; Leukemia-Lymphoma, Adult T-Cell; Phosphatidylinositol 3-Kinases; Phosphorylation; Protein Kinases; Protein Processing, Post-Translational; Proto-Oncogene Proteins c-akt; Receptor, Notch1; RNA, Small Interfering; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Transfection; Tumor Suppressor Protein p53

The phosphatidylinositol 3-kinase (PI3K)/Akt pathway is known to be activated by radiation. The mammalian target of rapamycin (mTOR) is downstream of Akt, and we investigated the effects of radiation on Akt/mTOR signaling in breast cancer cell models. RAD001 (everolimus), a potent derivative of the mTOR inhibitor rapamycin, was used to study the effects of mTOR inhibition, as the role of mTOR inhibition in enhancing radiation remains unexplored. RAD001 decreased clonogenic cell survival in both breast cancer cell lines MDA-MB-231 and MCF-7, although the effect is greater in MDA-MB-231 cells. Irradiation induced Akt and mTOR signaling, and this signaling is attenuated by RAD001. The radiation-induced signaling activation is mediated by PI3K because inhibition of PI3K with LY294002 inhibited the increase in downstream mTOR signaling. Additionally, caspase-dependent apoptosis is an important mechanism of cell death when RAD001 is combined with 3 Gy radiation, as shown by induction of caspase-3 cleavage. An increase in G(2)-M cell cycle arrest was seen in the combination treatment group when compared with controls, suggesting that cell cycle arrest may have been a contributing factor in the increased radiosensitization seen in this study. We conclude that RAD001 attenuates radiation-induced prosurvival Akt/mTOR signaling and enhances the cytotoxic effects of radiation in breast cancer cell models, showing promise as a method of radiosensitization of breast cancer.

Topics: Breast Neoplasms; Caspase 3; Caspases; Cell Death; Cell Survival; Everolimus; Female; Humans; Protein Kinases; Proto-Oncogene Proteins c-akt; Radiation-Sensitizing Agents; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Tumor Cells, Cultured

Mammalian target of rapamycin (mTOR) is increasingly recognized as a master regulator of fundamental cellular functions, whose deregulation may underlie neoplastic transformation and progression. Hence, mTOR has recently emerged as a promising target for therapeutic anticancer interventions in several human tumors, including breast cancer. Here, we investigated the antiangiogenic potential of temsirolimus (also known as CCI-779), a novel mTOR inhibitor currently in clinical development for the treatment of breast cancer and other solid tumors. Consistent with previous reports, sensitivity to temsirolimus-mediated growth inhibition varied widely among different breast cancer cell lines and was primarily due to inhibition of proliferation with little, if any, effect on apoptosis induction. In the HER-2 gene-amplified breast cancer cell line BT474, temsirolimus inhibited vascular endothelial growth factor (VEGF) production in vitro under both normoxic and hypoxic conditions through inhibition of hypoxia-stimulated hypoxia-inducible factor (HIF)-1alpha expression and transcriptional activation. Interestingly, these effects were also observed in the MDA-MB-231 cell line, independent of its inherent sensitivity to the growth-inhibitory effects of temsirolimus. A central role for mTOR (and the critical regulator of cap-dependent protein translation, eIF4E) in the regulation of VEGF production by BT474 cells was further confirmed using a small interfering RNA approach to silence mTOR and eIF4E protein expression. In addition to its effect on HIF-1alpha-mediated VEGF production, temsirolimus also directly inhibited serum- and/or VEGF-driven endothelial cell proliferation and morphogenesis in vitro and vessel formation in a Matrigel assay in vivo. Overall, these results suggest that antiangiogenic effects may substantially contribute to the antitumor activity observed with temsirolimus in breast cancer.

Topics: Angiogenesis Inhibitors; Breast Neoplasms; Cell Growth Processes; Cell Line, Tumor; Enzyme-Linked Immunosorbent Assay; Humans; Neovascularization, Pathologic; Protein Kinase Inhibitors; Protein Kinases; Sirolimus; TOR Serine-Threonine Kinases; Vascular Endothelial Growth Factor A

Loss of the cyclin-dependent kinase inhibitor p27 is associated with poor prognosis in breast cancer. The decrease in p27 levels is mainly the result of enhanced proteasome-dependent degradation mediated by its specific ubiquitin ligase subunit S phase kinase protein 2 (Skp2). The mammalian target of rapamycin (mTOR) is a downstream mediator in the phosphoinositol 3' kinase (PI3K)/Akt pathway that down-regulates p27 levels in breast cancer. Rapamycin was found to stabilize p27 levels in breast cancer, but whether this effect is mediated through changes in Skp2 expression is unknown.. The expression of Skp2 mRNA and protein levels were examined in rapamycin-treated breast cancer cell lines. The effect of rapamycin on the degradation rate of Skp2 expression was examined in cycloheximide-treated cells and in relationship to the anaphase promoting complex/Cdh1 (APC\\C) inhibitor Emi1.. Rapamycin significantly decreased Skp2 mRNA and protein levels in a dose and time-dependent fashion, depending on the sensitivity of the cell line to rapamycin. The decrease in Skp2 levels in the different cell lines was followed by cell growth arrest at G1. In addition, rapamycin enhanced the degradation rate of Skp2 and down-regulated the expression of the APC\\C inhibitor Emi1.. These results suggest that Skp2, an important oncogene in the development and progression of breast cancer, may be a novel target for rapamycin treatment.

Topics: Breast Neoplasms; Cell Line, Tumor; Down-Regulation; Enzyme Inhibitors; Female; Humans; Ligases; Protein Kinases; S-Phase Kinase-Associated Proteins; Sirolimus; TOR Serine-Threonine Kinases; Ubiquitin

The effect of combinations of a mammalian target of rapamycin (mTOR) inhibitor, temsirolimus, and an estrogen receptor-alpha (ERalpha) antagonist, ERA-923, on breast carcinoma in culture and in a xenograft model has been studied. Phase III trials are underway using temsirolimus for several cancers. ERA-923 was studied in a phase I trial for tamoxifen refractory metastatic breast cancer and was shown to have good safety profiles. Combination of noninhibitory doses of temsirolimus with suboptimal doses of ERA-923 synergistically inhibited the growth of MCF-7 cells. Synergy was found across a wide range of doses and could also be achieved by combining temsirolimus with other antiestrogens such as raloxifene and 4-hydroxytamoxifen. In vivo combination of temsirolimus and ERA-923 at certain doses and schedules completely inhibited tumor growth, while individual agents were only partially effective. Although the mechanism underlying the synergism remains to be understood, the results were associated with the ability of temsirolimus to block the transcriptional activity mediated by ERalpha as well as an increase in G1 arrest when it was combined with ERA-923. Results demonstrated for the first time that the combination of temsirolimus and a pure antiestrogen has excellent anticancer activity in preclinical models and, therefore, may have clinical use in treating hormone-dependent tumors.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Cell Cycle; Cell Division; Cell Line, Tumor; Estrogen Receptor Modulators; Female; Genetic Markers; Humans; Indoles; Mice; Mice, Nude; Ovariectomy; Piperidines; Polymerase Chain Reaction; Restriction Mapping; Sirolimus; Thymectomy; Transfection

Rapamycin and its analogues are being tested as new antitumor agents. Rapamycin binds to FKBP-12 and this complex inhibits the activity of FRAP/mammalian target of rapamycin, which leads to dephosphorylation of 4EBP1 and p70 S6 kinase, resulting in blockade of translation initiation. We have found that RAP inhibits the growth of HER-2-overexpressing breast cancer cells. The phosphorylation of mammalian target of rapamycin, p70 S6 kinase, and 4EBP1 is inhibited by rapamycin and cells are arrested in the G1 phase, as determined by growth assays, fluorescence-activated cell sorting analysis, and bromodeoxyuridine incorporation studies. Rapamycin causes down-regulation of cyclin D3 protein, retinoblastoma hypophosphorylation, loss of cyclin-dependent kinase (cdk) 4, cdk6, and cdk2 activity. The half-life of cyclin D3 protein decreases after rapamycin treatment, but not its synthesis, whereas the synthesis or half-life of cyclin D1 protein is not affected by the drug. Additionally, rapamycin caused accumulation of ubiquitinated forms of cyclin D3 protein, proteasome inhibitors blocked the effect of rapamycin on cyclin D3, and rapamycin stimulated the activity of the proteasome, showing that the effect of rapamycin on cyclin D3 is proteasome proteolysis dependent. This effect depends on the activity of HER-2 because Herceptin, a neutralizing antibody against HER-2, is able to block both the induction of proteasome activity and the cyclin D3 down-regulation due to rapamycin. Furthermore, inhibition of HER-2 gene expression by using small interfering RNA blocked the rapamycin effects on cyclin D3. These data indicate that rapamycin causes a G1 arrest in HER-2-overexpressing breast cancer cells that is associated with a differential destabilization and subsequent down-regulation of cyclin D3 protein.

Topics: Antibiotics, Antineoplastic; Breast Neoplasms; Cell Cycle; Cell Growth Processes; Cell Line, Tumor; Cyclin D3; Cyclin-Dependent Kinase 2; Cyclins; Down-Regulation; Humans; Oncogene Protein v-akt; Phosphatidylinositol 3-Kinases; Proteasome Inhibitors; Protein Kinases; Receptor, ErbB-2; Retinoblastoma; RNA, Messenger; Sirolimus; TOR Serine-Threonine Kinases; Ubiquitins

Interactions between chemokines and chemokine receptors have been proposed recently to be of importance in the development and progression of cancer. Human breast cancer cells express the chemokine CXCL10 (IP-10) and also its receptor CXCR3. In this study, we have investigated the role of Ras activation in the regulation of CXCL10 and its receptor splice variant CXCR3-B in two human breast cancer cell lines MDA-MB-435 and MCF-7. In cotransfection assays, using a full-length CXCL10 promoter-luciferase construct, we found that the activated form of Ras, Ha-Ras(12V), promoted CXCL10 transcriptional activation. Ras significantly increased CXCL10 mRNA and protein expression as observed by real-time PCR, fluorescence-activated cell sorting analysis, and ELISA. Selective inhibition of Ha-Ras by small interfering RNA (siRNA) decreased CXCL10 mRNA expression in a dose-dependent manner. Further, using effector domain mutants of Ras, we found that Ras-induced overexpression of CXCL10 is mediated primarily through the Raf and phosphatidylinositol 3-kinase signaling pathways. We also observed that the expression of the splice variant CXCR3-B, known to inhibit cell proliferation, was significantly down-regulated by Ras. Selective inhibition of CXCR3-B using siRNA resulted in an increase in CXCL10-mediated breast cancer cell proliferation through G(i) proteins and likely involving CXCR3-A. Finally, we observed intense expression of CXCL10 and CXCR3 in association with human breast cancer in situ, indicating that these observations may be of pathophysiologic significance. Together, these results suggest that activation of Ras plays a critical role in modulating the expression of both CXCL10 and CXCR3-B, which may have important consequences in the development of breast tumors through cancer cell proliferation.

Topics: Adenocarcinoma; Alternative Splicing; Breast Neoplasms; Carcinoma, Ductal, Breast; Carcinoma, Intraductal, Noninfiltrating; Cell Division; Cell Line, Tumor; Chemokine CXCL10; Chemokines, CXC; Female; Gene Expression Regulation, Neoplastic; Genes, ras; Humans; Neoplasm Proteins; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Isoforms; Proto-Oncogene Proteins c-raf; Proto-Oncogene Proteins p21(ras); Receptors, Chemokine; Receptors, CXCR3; Recombinant Fusion Proteins; RNA, Small Interfering; Signal Transduction; Sirolimus; Transfection

Breast cancer is the most common malignancy and the second most common cause of cancer-related death in women. Endocrine therapy has been used for more than a century to treat advanced-stage breast cancer. The results obtained with the third-generation aromatase inhibitor letrozole demonstrated an actual improvement in patient outcome compared with tamoxifen. This benefit translates into disease-free survival improvement for adjuvant treatment and overall survival in patients with metastatic disease. The present clinical situation of hormonal therapy is stable; however, recently, new anticancer agents (temsirolimus and everolimus) that inhibit mammalian target of rapamycin protein kinase have been developed and seem to be very promising because of their synergistic activity with letrozole. The phase II study of a combination of temsirolimus or everolimus with letrozole demonstrated a better progression-free survival in the combination arm than in the letrozole alone arm. Consequently, the results of ongoing phase III studies are eagerly awaited.

Topics: Antineoplastic Agents; Antineoplastic Agents, Hormonal; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Carcinoma; Clinical Trials, Phase I as Topic; Clinical Trials, Phase II as Topic; Drug Resistance, Neoplasm; Everolimus; Female; Humans; Letrozole; Nitriles; Oncogene Protein v-akt; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase Inhibitors; Protein Kinases; Sirolimus; TOR Serine-Threonine Kinases; Treatment Outcome; Triazoles

The receptor for epidermal growth factor (EGFR) is overexpressed in many cancers. One important signaling pathway regulated by EGFR is the phosphatidylinositol 3'-kinase (PI3K)-phosphoinositide-dependent kinase 1-Akt pathway. Activation of Akt leads to the stimulation of antiapoptotic pathways, promoting cell survival. Akt also regulates the mammalian target of rapamycin (mTOR)-S6K-S6 pathway to control cell growth in response to growth factors and nutrients. Recent reports have shown that the sensitivity of non-small-cell lung cancer cell lines to EGFR inhibitors such as erlotinib (Tarceva, OSI Pharmaceuticals) is dependent on inhibition of the phosphatidylinositol 3'-kinase-phosphoinositide-dependent kinase 1-Akt-mTOR pathway. There can be multiple inputs to this pathway as activity can be regulated by other receptors or upstream mutations. Therefore, inhibiting EGFR alone may not be sufficient for substantial inhibition of all tumor cells, highlighting the need for multipoint intervention. Herein, we sought to determine if rapamycin, an inhibitor of mTOR, could enhance erlotinib sensitivity for cell lines derived from a variety of tissue types (non-small-cell lung, pancreatic, colon, and breast). Erlotinib could inhibit extracellular signal-regulated kinase, Akt, and S6 only in cell lines that were the most sensitive. Rapamycin could fully inhibit S6 in all cell lines, but this was accompanied by activation of Akt phosphorylation. However, combination with erlotinib could down-modulate rapamycin-stimulated Akt activity. Therefore, in select cell lines, inhibition of both S6 and Akt was achieved only with the combination of erlotinib and rapamycin. This produced a synergistic effect on cell growth inhibition, observations that extended in vivo using xenograft models. These results suggest that combining rapamycin with erlotinib might be clinically useful to enhance response to erlotinib.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Dose-Response Relationship, Drug; Drug Synergism; ErbB Receptors; Erlotinib Hydrochloride; Female; HCT116 Cells; HT29 Cells; Humans; Lung Neoplasms; Mice; Mice, Transgenic; Pancreatic Neoplasms; Protein Kinase Inhibitors; Quinazolines; Sirolimus

This study is the first to investigate the anticancer effect of plumbagin in human breast cancer cells. Plumbagin exhibited cell proliferation inhibition by inducing cells to undergo G2-M arrest and autophagic cell death. Blockade of the cell cycle was associated with increased p21/WAF1 expression and Chk2 activation, and reduced amounts of cyclin B1, cyclin A, Cdc2, and Cdc25C. Plumbagin also reduced Cdc2 function by increasing the association of p21/WAF1/Cdc2 complex and the levels of inactivated phospho-Cdc2 and phospho-Cdc25C by Chk2 activation. Plumbagin triggered autophagic cell death but not predominantly apoptosis. Pretreatment of cells with autophagy inhibitor bafilomycin suppressed plumbagin-mediated cell death. We also found that plumbagin inhibited survival signaling through the phosphatidylinositol 3-kinase/AKT signaling pathway by blocking the activation of AKT and downstream targets, including the mammalian target of rapamycin, forkhead transcription factors, and glycogen synthase kinase 3beta. Phosphorylation of both of mammalian target of rapamycin downstream targets, p70 ribosomal protein S6 kinase and 4E-BP1, was also diminished. Overexpression of AKT by AKT cDNA transfection decreased plumbagin-mediated autophagic cell death, whereas reduction of AKT expression by small interfering RNA potentiated the effect of plumbagin, supporting the inhibition of AKT being beneficial to autophagy. Furthermore, suppression of AKT by plumbagin enhanced the activation of Chk2, resulting in increased inactive phosphorylation of Cdc25C and Cdc2. Further investigation revealed that plumbagin inhibition of cell growth was also evident in a nude mouse model. Taken together, these results imply a critical role for AKT inhibition in plumbagin-induced G2-M arrest and autophagy of human breast cancer cells.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Autophagy; Breast Neoplasms; Cell Division; Cell Growth Processes; Cell Line, Tumor; Female; G2 Phase; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Naphthoquinones; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Kinases; Proto-Oncogene Proteins c-akt; RNA, Small Interfering; Sirolimus; TOR Serine-Threonine Kinases

Cancer cells generate survival signals to suppress default apoptotic programs that protect from cancer. Phosphatidylinositol-3-kinase (PI3K) generates a survival signal that is frequently dysregulated in human cancers. Phospholipase D (PLD) has also been implicated in signals that promote survival. One of the targets of PLD signaling is mTOR (mammalian target of rapamycin), a critical regulator of cell cycle progression and cell growth. We report here that elevated PLD activity in the MDA-MB-231 human breast cancer cell line generates an mTOR-dependent survival signal that is independent of PI3K. In contrast, MDA-MB-435S breast cancer cells, which have very low levels of PLD activity, are dependent on PI3K for survival signals. The data presented here identify an alternative survival signal that is dependent on PLD and mTOR and is active in a breast cancer cell line where the PI3K survival pathway is not active.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Survival; Culture Media, Serum-Free; Gene Expression Regulation, Neoplastic; Humans; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phospholipase D; Protein Kinase Inhibitors; Protein Kinases; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

The mammalian target of rapamycin is a serine-threonine kinase that regulates cell cycle progression. Rapamycin and its analogues inhibit the mammalian target of rapamycin and are being actively investigated in clinical trials as novel targeted anticancer agents. Although cyclin D1 is down-regulated by rapamycin, the role of this down-regulation in rapamycin-mediated growth inhibition and the mechanism of cyclin D1 down-regulation are not well understood. Here, we show that overexpression of cyclin D1 partially overcomes rapamycin-induced cell cycle arrest and inhibition of anchorage-dependent growth in breast cancer cells. Rapamycin not only decreases endogenous cyclin D1 levels but also decreases the expression of transfected cyclin D1, suggesting that this is at least in part caused by accelerated proteolysis. Indeed, rapamycin decreases the half-life of cyclin D1 protein, and the rapamycin-induced decrease in cyclin D1 levels is partially abrogated by proteasome inhibitor N-acetyl-leucyl-leucyl-norleucinal. Rapamycin treatment leads to an increase in the kinase activity of glycogen synthase kinase 3beta (GSK3beta), a known regulator of cyclin D1 proteolysis. Rapamycin-induced down-regulation of cyclin D1 is inhibited by the GSK3beta inhibitors lithium chloride, SB216763, and SB415286. Rapamycin-induced G1 arrest is abrogated by nonspecific GSK3beta inhibitor lithium chloride but not by selective inhibitor SB216763, suggesting that GSK3beta is not essential for rapamycin-mediated G1 arrest. However, rapamycin inhibits cell growth significantly more in GSK3beta wild-type cells than in GSK3beta-null cells, suggesting that GSK3beta enhances rapamycin-mediated growth inhibition. In addition, rapamycin enhances paclitaxel-induced apoptosis through the mitochondrial death pathway; this is inhibited by selective GSK3beta inhibitors SB216763 and SB415286. Furthermore, rapamycin significantly enhances paclitaxel-induced cytotoxicity in GSK3beta wild-type but not in GSK3beta-null cells, suggesting a critical role for GSK3beta in rapamycin-mediated paclitaxel-sensitization. Taken together, these results show that GSK3beta plays an important role in rapamycin-mediated cell cycle regulation and chemosensitivity and thus significantly potentiates the antitumor effects of rapamycin.

Topics: Aminophenols; Antibiotics, Antineoplastic; Antimanic Agents; Apoptosis; Breast Neoplasms; Cell Cycle; Cyclin D1; Cysteine Proteinase Inhibitors; Down-Regulation; Drug Resistance, Neoplasm; Female; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Half-Life; Humans; Indoles; Leupeptins; Lithium Chloride; Maleimides; Mitochondria; NF-kappa B; Paclitaxel; Proteasome Inhibitors; Sirolimus

The mammalian target of rapamycin (mTOR) coordinates cell growth with the growth factor and nutrient/energy status of the cell. The phosphatidylinositol 3-kinase-AKT pathway is centrally involved in the transmission of mitogenic signals to mTOR. Previous studies have shown that mTOR is a direct substrate for the AKT kinase and identified Ser-2448 as the AKT target site in mTOR. In this study, we demonstrate that rapamycin, a specific inhibitor of mTOR function, blocks serum-stimulated Ser-2448 phosphorylation and that this drug effect is not explained by the inhibition of AKT. Furthermore, the phosphorylation of Ser-2448 was dependent on mTOR kinase activity, suggesting that mTOR itself or a protein kinase downstream from mTOR was responsible for the modification of Ser-2448. Here we show that p70S6 kinase phosphorylates mTOR at Ser-2448 in vitro and that ectopic expression of rapamycin-resistant p70S6 kinase restores Ser-2448 phosphorylation in rapamycin-treated cells. In addition, we show that cellular amino acid status, which modulates p70S6 kinase (S6K1) activity via the TSC/Rheb pathway, regulates Ser-2448 phosphorylation. Finally, small interfering RNA-mediated depletion of p70S6 kinase reduces Ser-2448 phosphorylation in cells. Taken together, these results suggest that p70S6 kinase is a major effector of mTOR phosphorylation at Ser-2448 in response to both mitogen- and nutrient-derived stimuli.

Topics: Anti-Bacterial Agents; Blood Proteins; Breast Neoplasms; Drug Resistance; HeLa Cells; Humans; In Vitro Techniques; Kidney; Phosphorylation; Protein Kinases; Ribosomal Protein S6 Kinases, 70-kDa; RNA, Small Interfering; Serine; Sirolimus; TOR Serine-Threonine Kinases

The ErbB2 (Neu) receptor tyrosine kinase is frequently overexpressed in human breast cancers, and this phenotype correlates with a poor clinical prognosis. We examined the effects of the mammalian target of rapamycin inhibitor, rapamycin, on mammary tumorigenesis in transgenic mice bearing an activated ErbB2 (NeuYD) transgene in the absence or presence of a second transgene encoding vascular endothelial growth factor (VEGF). Treatment of NeuYD or NeuYD x VEGF mice with rapamycin dramatically inhibited tumor growth accompanied by a marked decrease in tumor vascularization. Two key events that may underlie the antitumor activity of rapamycin were decreased expression of ErbB3 and inhibition of hypoxia-inducible factor-1-dependent responses to hypoxic stress. Rapamycin exposure caused only a modest inhibition of the proliferation of tumor-derived cell lines in standard monolayer cultures, but dramatically inhibited the growth of the same cells in three-dimensional cultures, due in part to the induction of apoptotic cell death. These studies underscore the therapeutic potential of mammalian target of rapamycin inhibitors in ErbB2-positive breast cancers and indicate that, relative to monolayer cultures, three-dimensional cell cultures are more predictive in vitro models for studies of the antitumor mechanisms of rapamycin and related compounds.

Topics: Animals; Apoptosis; Breast Neoplasms; Cell Proliferation; DNA-Binding Proteins; Female; Humans; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Male; Mammary Neoplasms, Experimental; Mice; Mice, Transgenic; Neovascularization, Pathologic; Nuclear Proteins; Phosphorylation; Protein Kinases; Receptor, ErbB-2; Receptor, ErbB-3; Sirolimus; Spheroids, Cellular; TOR Serine-Threonine Kinases; Transcription Factors; Vascular Endothelial Growth Factor A

RAD001 (everolimus), a mammalian target of rapamycin (mTOR) pathway inhibitor in phase II clinical trials in oncology, exerts potent antiproliferative/antitumor activities. Many breast cancers are dependent for proliferation on estrogens synthesized from androgens (i.e., androstenedione) by aromatase. Letrozole (Femara) is an aromatase inhibitor used for treatment of postmenopausal women with hormone-dependent breast cancers. The role of the mTOR pathway in estrogen-driven proliferation and effects of combining RAD001 and letrozole were examined in vitro in two breast cancer models.. The role of the mTOR pathway in estrogen response was evaluated in aromatase-expressing MCF7/Aro breast cancer cells by immunoblotting. Effects of RAD001 and letrozole (alone and in combination) on the proliferation and survival of MCF7/Aro and T47D/Aro cells were evaluated using proliferation assays, flow cytometry, immunoblotting, and apoptosis analyses.. Treatment of MCF7/Aro cells with estradiol or androstenedione caused modulation of the mTOR pathway, a phenomenon reversed by letrozole or RAD001. In MCF7/Aro and T47D/Aro cells, both agents inhibited androstenedione-induced proliferation; however, in combination, this was significantly augmented (P < 0.001, two-way ANOVA, synergy by isobologram analysis). Increased activity of the combination correlated with more profound effects on G1 progression and a significant decrease in cell viability (P < 0.01, two-way ANOVA) defined as apoptosis (P < 0.05, Friedman test). Increased cell death was particularly evident with optimal drug concentrations.. mTOR signaling is required for estrogen-induced breast tumor cell proliferation. Moreover, RAD001-letrozole combinations can act in a synergistic manner to inhibit proliferation and trigger apoptotic cell death. This combination holds promise for the treatment of hormone-dependent breast cancers.

Topics: Antineoplastic Agents; Apoptosis; Breast Neoplasms; Carcinoma; Cell Proliferation; Cell Survival; Drug Interactions; Estrogens; Everolimus; Female; Humans; Immunosuppressive Agents; Letrozole; Nitriles; Protein Kinases; Receptors, Estrogen; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Triazoles; Tumor Cells, Cultured

Recent studies indicate that constitutive signaling through the phosphatidylinositol 3'-kinase (PI3K) pathway is a cause of treatment resistance in breast cancer patients. This implies that patients with tumors that exhibit aberrant PI3K signaling may benefit from targeted pathway inhibitors. The first agents to make it to the clinic are the rapamycin analogs. These compounds inhibit the downstream PI3K effector mTOR (mammalian target of rapamycin). A study presented in this issue of Breast Cancer Research suggests that recently developed inhibitors of phosphoinositide-dependent protein kinase 1, a more proximal target of the PI3K pathway, may provide an alternative route to effective PI3K pathway inhibition for breast cancer treatment.

Topics: Antibiotics, Antineoplastic; Breast Neoplasms; Enzyme Inhibitors; Female; Gene Amplification; Gene Expression Regulation, Neoplastic; Humans; Phosphoinositide-3 Kinase Inhibitors; Signal Transduction; Sirolimus

To study the expression of proline-rich Akt-substrate PRAS40 in the cell survival pathway and tumor progression.. The effects of three key kinase inhibitors on PRAS40 activity in the cell survival pathway, serum withdrawal, H(2)O(2) and overexpression of Akt were tested. The expression of PRAS40, Akt, Raf and 14-3-3 in normal cells and cancer cell lines was determined by Western blot.. The PI3K inhibitors wortmannin and Ly294002, but not rapamycin, completely inhibited the phosphorylation of Akt and PRAS40. The phosphorylation level of Akt decreased after serum withdrawal and treatment with the MEK inhibitor Uo126, but increased after treatment with H(2)O(2) at low concentration, whereas none of these treatments changed PRAS40 activity. 14-3-3 is a PRAS40 binding protein, and the expression of 14-3-3, like that of PRAS40, was higher in HeLa cells than in HEK293 cells; PRAS40 had a stronger phosphorylation activity in A549 and HeLa cancer cells than in HEK293 normal cells. In the breast cancer model (MCF10A/MCF7) and lung cancer model (BEAS/H1198/H1170) we also found the same result: PRAS40 was constitutively active in H1198/H1170 and MCF7 pre-malignant and malignant cancer cells, but weakly expressed in MCF10A and BEAS normal cell. We also discussed PRAS40 activity in other NSCLC cell lines.. The PI3K-Akt survival pathway is the main pathway that PRAS40 is involved in; PRAS40 is a substrate for Akt, but can also be activated by an Akt-independent mechanisms. PRAS40 activation is an early event during breast and lung carcinogenesis.

Topics: 14-3-3 Proteins; Adaptor Proteins, Signal Transducing; Breast Neoplasms; Butadienes; Carcinoma, Non-Small-Cell Lung; Cell Line; Cell Line, Tumor; Chromones; Enzyme Inhibitors; Gene Expression Regulation; Humans; Hydrogen Peroxide; Lung Neoplasms; Morpholines; Nitriles; Phosphoinositide-3 Kinase Inhibitors; Phosphoproteins; Phosphorylation; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirolimus; Transfection

Radiation-induced inhibition of rapamycin-sensitive pathway and its effect on the cellular response to radiation were studied in the human breast cancer cell line MCF-7. Both radiation and rapamycin shared molecular targets and induced similar physiologic responses. Each of these treatments increased immunostaining of mammalian target of rapamycin (mTOR) in the nucleus, and radiation led to decreased phosphorylation of its autophosphorylation site Ser2481. In addition to dephosphorylation of established mTOR downstream effectors 4E-binding protein 1 and p70 ribosomal S6 kinase, both treatments decreased the level of eukaryotic initiation factor 4G. Experiments with the potentiometric dye, JC-1, revealed an oligomycin-dependent increase in mitochondrial membrane potential following radiation or rapamycin treatment, suggesting that both lead to reversal of F0F1ATPase activity. Both radiation and rapamycin induced sequestration of cytoplasmic material in autophagic vacuoles. In both cases, appearance of autophagic vacuoles involved the participation of microtubule-associated protein 1 light chain 3 (LC3). Transient cotransfection of green fluorescent protein-LC3 with either wild-type or dominant-negative mTOR further showed that inactivation of mTOR pathway is sufficient to induce autophagy in these cells. Finally, administration of rapamycin in combination with radiation led to enhanced mitochondria hyperpolarization, p53 phosphorylation, and increased cell death. Taken together, these experiments show that radiation-induced inhibition of rapamycin-sensitive pathway in MCF-7 cells causes changes in mitochondria metabolism, development of autophagy, and an overall decrease in cell survival.

Topics: Adenocarcinoma; Antibiotics, Antineoplastic; Autophagy; Breast Neoplasms; Cell Line, Tumor; Cell Survival; Cytoplasm; Humans; Intracellular Membranes; Intracellular Signaling Peptides and Proteins; Membrane Potentials; Mitochondria; Phosphorylation; Protein Kinases; Sirolimus; TOR Serine-Threonine Kinases; Tumor Suppressor Protein p53; Vacuoles

Rapamycin, an inhibitor of the serine/threonine kinase target of rapamycin, induces G1 arrest and/or apoptosis. Although rapamycin and its analogues are attractive candidates for cancer therapy, their sensitivities with respect to growth inhibition differ markedly among various cancer cells. Using human breast carcinoma cell line MCF-7 as an experimental model system, we examined the growth-inhibitory effects of combinations of various agents and rapamycin to find the agent that most potently enhances the growth-inhibitory effect of rapamycin.. We evaluated the growth-inhibitory effect of rapamycin plus various agents, including cotylenin A (a novel inducer of differentiation of myeloid leukaemia cells) to MCF-7 cells, using either MTT assay or trypan blue dye exclusion test. The cell cycle was analyzed using propidium iodide-stained nuclei. Expressions of several genes in MCF-7 cells with rapamycin plus cotylenin A were studied using cDNA microarray analysis and RT-PCR. The in vitro results of MCF-7 cells treated with rapamycin plus cotylenin A were further confirmed in vivo in a mouse xenograft model.. We found that the sensitivity of rapamycin to MCF-7 cells was markedly affected by cotylenin A. This treatment induced growth arrest of the cells at the G1 phase, rather than apoptosis, and induced senescence-associated beta-galactosidase activity. We examined the gene expression profiles associated with exposure to rapamycin and cotylenin A using cDNA microarrays. We found that expressions of cyclin G2, transforming growth factor-beta-induced 68 kDa protein, BCL2-interacting killer, and growth factor receptor-bound 7 were markedly induced in MCF-7 cells treated with rapamycin plus cotylenin A. Furthermore, combined treatment with rapamycin and cotylenin A significantly inhibited the growth of MCF-7 cells as xenografts, without apparent adverse effects.. Rapamycin and cotylenin A cooperatively induced growth arrest in breast carcinoma MCF-7 cells in vitro, and treatment with rapamycin and cotylenin A combined more strongly inhibited the growth of MCF-7 cells as xenografts in vivo than treatment with rapamycin or cotylenin A alone, suggesting that this combination may have therapeutic value in treating breast cancer. We also identified several genes that were markedly modulated in MCF-7 cells treated with rapamycin plus cotylenin A.

Topics: Animals; Antibiotics, Antineoplastic; Breast Neoplasms; Carcinoma; Cell Line, Tumor; Cell Proliferation; Diterpenes; Drug Interactions; Female; Gene Expression Regulation, Neoplastic; Genes, Neoplasm; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Sirolimus; Transplantation, Heterologous

Rapamycin inhibits the serine-threonine kinase mammalian target of rapamycin (mTOR), blocking phosphorylation of p70 S6 kinase (S6K1) and 4E-binding protein 1 (4E-BP1) and inhibiting protein translation and cell cycle progression. Rapamycin and its analogues are currently being tested in clinical trials as novel-targeted anticancer agents. Although rapamycin analogues show activity in clinical trials, only some of the treated patients respond. The purpose of this study is to identify determinants of rapamycin sensitivity that may assist the selection of appropriate patients for therapy.. Breast cancer cell lines representing a spectrum of aberrations in the mTOR signaling pathway were tested for rapamycin sensitivity. The expression and phosphorylation state of multiple components of the pathway were tested by Western blot analysis, in the presence and absence of rapamycin.. Cell proliferation was significantly inhibited in response to rapamycin in 12 of 15 breast cancer cell lines. The ratio of total protein levels of 4E-BP1 to its binding partner eukaryotic initiation factor 4E did not predict rapamycin sensitivity. In contrast, overexpression of S6K1, and phosphorylated Akt independent of phosphatase and tensin homologue deleted from chromosome 10 status, were associated with rapamycin sensitivity. Targeting S6K1 and Akt with small interfering RNA and dominant-negative constructs, respectively, decreased rapamycin sensitivity. Rapamycin inhibited the phosphorylation of S6K1, ribosomal S6 protein, and 4E-BP1 in rapamycin-resistant as well as -sensitive cells, indicating that its ability to inhibit the mTOR pathway is not sufficient to confer sensitivity to rapamycin. In contrast, rapamycin treatment was associated with decreased cyclin D1 levels in the rapamycin-sensitive cells but not in rapamycin-resistant cells.. Overexpression of S6K1 and expression of phosphorylated Akt should be evaluated as predictors of rapamycin sensitivity in breast cancer patients. Furthermore, changes in cyclin D1 levels provide a potential pharmacodynamic marker of response to rapamycin.

Topics: Adaptor Proteins, Signal Transducing; Antibiotics, Antineoplastic; Blotting, Western; Breast Neoplasms; Carrier Proteins; Cell Cycle; Cell Cycle Proteins; Cell Division; Cell Line, Tumor; Cyclin D1; Dose-Response Relationship, Drug; Genes, Dominant; Humans; Phosphoproteins; Phosphorylation; Protein Kinases; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Ribosomal Protein S6 Kinases, 70-kDa; RNA, Small Interfering; Signal Transduction; Sirolimus; Time Factors; TOR Serine-Threonine Kinases

Topics: Antineoplastic Agents; Breast Neoplasms; Clinical Trials as Topic; Humans; Research Design; Sirolimus

The PTEN protein is a lipid phosphatase with putative tumor suppressing abilities, including inhibition of the PI3K/Akt signaling pathway. Inactivating mutations or deletions of the PTEN gene, which result in hyper-activation of the PI3K/Akt signaling pathway, are increasingly being reported in human malignancies, including breast cancer, and have been related to features of poor prognosis and resistance to chemotherapy and hormone therapy. Prior studies in different tumor models have shown that, under conditions of PTEN deficiency, the PI3K/Akt signaling pathway becomes a fundamental proliferative and survival pathway, and that pharmacological inhibition of this pathway results in tumor growth inhibition. This study aimed to explore further this hypothesis in breast cancer cells. To this end, we have determined the growth response to inhibition of the PI3K/Akt signaling pathway in a series of breast cancer cell lines with different PTEN levels. The PTEN-negative cell line displayed greater sensitivity to the growth inhibitory effects of the PI3K inhibitor, LY294002 and rapamycin, an inhibitor of the PI3K/Akt downstream mediator mTOR, compared with the PTEN-positive cell lines. To determine whether or not these differences in response are specifically due to effects of PTEN, we developed a series of cell lines with reduced PTEN protein expression compared with the parental cell line. These reduced PTEN cells demonstrated an increased sensitivity to the anti-proliferative effects induced by LY294002 and rapamycin compared with the parental cells, which corresponded to alterations in cell cycle response. These findings indicate that inhibitors of mTOR, some of which are already in clinical development (CCI-779, an ester of rapamycin), have the potential to be effective in the treatment of breast cancer patients with PTEN-negative tumors and should be evaluated in this setting.

Topics: Antibiotics, Antineoplastic; Breast Neoplasms; Cell Cycle; Cell Proliferation; Chromones; Enzyme Inhibitors; Female; Genes, Tumor Suppressor; Humans; Morpholines; Phosphatidylinositol 3-Kinases; Phosphoric Monoester Hydrolases; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Signal Transduction; Sirolimus; Tumor Cells, Cultured; Tumor Suppressor Proteins

To identify markers sensitive to inhibitors of the farnesylation pathway, we used 3 breast cancer cell lines (SKBR-3, MDA-175, and MDA-231) to evaluate the in vitro effects of pamidronate, an inhibitor of farnesyl diphosphate synthase. In response to pamidronate, there was significant inhibition of cell proliferation in MDA-231 and SKBR-3 cells, compared to MDA-175 cells. This correlated with their respective basal levels of N-ras and H-ras. N-ras and H-ras protein levels were both reduced in MDA-231 cells, and to lesser extent in SKBR-3 cells, following exposure to pamidronate, whereas these markers were not altered in MDA-175 cells. Combinatorial therapy with pamidronate and Gleevec, an inhibitor of several tyrosine kinases; Velcade, a proteasome inhibitor; or rapamycin, an inhibitor of the mammalian target of rapamycin (m-TOR) all showed additive effects in causing proliferative inhibition in MDA-175 cells. In summary, resistance to pamidronate may result from low levels of GTPase-activating proteins, such as N-ras and H-ras, in tumor cells. Combinatorial therapies directed against other signaling pathways, not dependent upon ras, may be required to overcome such resistance.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Benzamides; Boronic Acids; Bortezomib; Breast Neoplasms; Carcinoma; Cell Line, Tumor; Cell Proliferation; Diphosphonates; Drug Combinations; Drug Resistance, Neoplasm; Female; Humans; Imatinib Mesylate; Pamidronate; Piperazines; Pyrazines; Pyrimidines; ras Proteins; Sirolimus

The Akt/mammalian target of rapamycin (mTOR)/4E-BP1 pathway is considered to be a central regulator of protein synthesis, involving the regulation of cell proliferation, differentiation, and survival. The inhibitors of mTOR as anticancer reagents are undergoing active evaluation in various malignancies including breast cancer. However, the activation status of the Akt/mTOR/4E-BP1 pathway and its potential roles in breast cancers remain unknown. Thus, we examined 165 invasive breast cancers with specific antibodies for the phosphorylation of Akt, mTOR, and 4E-BP1 by immunohistochemistry and compared them with normal breast epithelium, fibroadenoma, intraductal hyperplasia, and ductal carcinoma in situ. We discovered that the phosphorylation of Akt, mTOR, and 4E-BP1 increased progressively from normal breast epithelium to hyperplasia and abnormal hyperplasia to tumor invasion. Phosphorylated Akt, mTOR, and 4E-BP1 were positively associated with ErbB2 overexpression. Survival analysis showed that phosphorylation of each of these three markers was associated with poor disease-free survival independently. In vitro, we further confirmed the causal relationship between ErbB2 overexpression and mTOR activation, which was associated with enhanced invasive ability and sensitivity to a mTOR inhibitor, rapamycin. Our results, for the first time, demonstrate the following: (a) high levels of phosphorylation of Akt, mTOR, and 4E-BP1 in breast cancers, indicating activation of the Akt/mTOR/4E-BP1 pathway in breast cancer development and progression; (b) a link between ErbB2 and the Akt/mTOR/4E-BP1 pathway in breast cancers in vitro and in vivo, indicating the possible role of Akt/mTOR activation in ErbB2-mediated breast cancer progression; and (c) a potential role for this pathway in predicting the prognosis of patients with breast cancer, especially those treated with mTOR inhibitors.

Topics: Adaptor Proteins, Signal Transducing; Adult; Aged; Antibiotics, Antineoplastic; Biomarkers, Tumor; Breast Neoplasms; Carcinoma, Intraductal, Noninfiltrating; Carrier Proteins; Cell Cycle Proteins; Cell Proliferation; Disease Progression; Female; Gene Expression Profiling; Genes, erbB-2; Humans; Hyperplasia; Immunohistochemistry; Middle Aged; Neoplasm Invasiveness; Phosphoproteins; Phosphorylation; Protein Kinases; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Sirolimus; Survival Analysis; TOR Serine-Threonine Kinases

The serine-threonine kinase mammalian target of rapamycin has emerged as a potential target for cancer therapy. Rapamycin and rapamycin analogs are undergoing clinical trials and have induced clinical responses in a subgroup of patients. Rapamycin has also been reported to enhance the efficacy of several cytotoxic agents. The aim of this study was to determine the nature of the interactions between rapamycin and chemotherapeutic agents used as first- and second-line agents against breast cancer.. We performed a multiple drug effect/combination index isobologram analysis in cells sensitive and resistant to rapamycin alone in vitro, and we evaluated the in vivo efficacy of combination therapy in a rapamycin-sensitive model.. In vitro, synergistic interactions were observed in combinations with paclitaxel, carboplatin, and vinorelbine. Additive effects were observed in combinations with doxorubicin and gemcitabine. Rapamycin dramatically enhanced paclitaxel- and carboplatin-induced apoptosis. This effect was sequence dependent and mediated at least partly through caspase activation. Furthermore, rapamycin enhanced chemosensitivity to paclitaxel and carboplatin in HER2/neu-overexpressing cells, suggesting a potential approach to these poorly behaving tumors. Cell lines that are resistant to the growth-inhibitory effect of rapamycin were also resistant to rapamycin-mediated chemosensitization. In vivo, rapamycin combined with paclitaxel resulted in a significant reduction in tumor volume compared with either agent alone in rapamycin-sensitive tumors.. Rapamycin potentiates the cytotoxicity of selected chemotherapeutic agents in cell lines sensitive to the effects of rapamycin due to aberrations in the phosphatidylinositol 3'-kinase/Akt pathway, suggesting that combination therapy may be effective in patients selected for aberrations in this pathway.

Topics: Antibiotics, Antineoplastic; Antineoplastic Agents, Phytogenic; Breast Neoplasms; Drug Interactions; Drug Resistance, Neoplasm; Female; Humans; Protein Kinases; Sirolimus; TOR Serine-Threonine Kinases; Tumor Cells, Cultured

The Akt kinase is a serine/threonine protein kinase that has been implicated in mediating a variety of biological responses. Studies show that high Akt activity in breast carcinoma is associated with a poor pathophenotype, as well as hormone and chemotherapy resistance. Additionally, high Akt activity is associated with other features of poor prognosis. Thus, a chemotherapeutic agent directed specifically toward tumors with high Akt activity could prove extremely potent in treating those breast tumors with the most aggressive phenotypes. Several studies have demonstrated that rapamycin, which inhibits mammalian target of rapamycin (mTOR), a downstream target of Akt, sensitizes certain resistant cancer cells to chemotherapeutic agents. This study evaluated the efficacy of mTOR inhibition in the treatment of tamoxifen-resistant breast carcinoma characterized by high Akt activity. We found that MCF-7 breast cancer cell lines expressing a constitutively active Akt are able to proliferate under reduced estrogen conditions and are resistant to the growth inhibitory effects of tamoxifen, both in vitro as well as in vivo in xenograft models. Cotreatment with the mTOR inhibitor rapamycin in vitro, or the ester of rapamycin, CCI-779 (Wyeth) in vivo, inhibited mTOR activity and restored sensitivity to tamoxifen, suggesting that Akt-induced tamoxifen resistance is mediated in part by signaling through the mTOR pathway. Although the mechanism underlying the synergism remains to be understood, the results were associated with rapamycin's ability to block transcriptional activity mediated by estrogen receptor alpha, as assessed by reporter gene assays with estrogen-responsive element luciferase. These data corroborate prior findings indicating that Akt activation induces resistance to tamoxifen in breast cancer cells. Importantly, these data indicate a novel mechanism for tamoxifen resistance and suggest that blockage of the phosphatidylinositol 3'-kinase/Akt signaling pathway by mTOR inhibition effectively restores the susceptibility of these cells to tamoxifen. These data may have implication for future clinical studies of mTOR inhibition in breast carcinoma.

Topics: Animals; Apoptosis; Breast Neoplasms; Cell Cycle; Cell Proliferation; Drug Resistance, Neoplasm; Enzyme Activation; Female; Gene Expression Regulation, Neoplastic; Humans; Mice; Mice, Nude; Phosphatidylinositol 3-Kinases; Promoter Regions, Genetic; Protein Kinases; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Receptors, Estrogen; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Sirolimus; Tamoxifen; TOR Serine-Threonine Kinases; Transcription, Genetic; Transplantation, Heterologous; Tumor Cells, Cultured

Although interactions between estrogen and growth factor signaling pathways have been studied extensively, how growth factors and progesterone regulate each other is less clear. In this study, we found that IGF-I sharply lowers progesterone receptor (PR) mRNA and protein levels in breast cancer cells. Other growth factors, such as epidermal growth factor, also showed the same effect. The decrease of PR levels was associated with reduced PR activity. Unlike progestins, IGF-I does not utilize the proteasome for down-regulating PR. Instead, the IGF-I-mediated decrease in PR levels is via an inhibition of PR gene transcription. In addition, the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway was found to be specifically involved in this IGF-I effect. Our data also suggest that the IGF-I down-regulation of PR is not mediated via a reduction of estrogen receptor (ER) levels or activity. First, IGF-I induced ligand-independent ER activity while reducing ER-dependent PR levels. Second, whereas PR and cyclin D1 are both ER up-regulated, IGF-I increased cyclin D1 levels while decreasing PR levels. Third, constitutively active PI3K or Akt induced ER activity but reduced PR levels and activity. Taken together, our data indicate that IGF-I inhibits PR expression in breast cancer cells via the PI3K/Akt/mTOR pathway. Because low or absent PR in primary breast cancer is associated with poor prognosis and response to hormone therapy, our results suggest that low PR status may serve as an indicator of activated growth factor signaling in breast tumor cells, and therefore of an aggressive tumor phenotype and resistance against hormonal therapy.

Topics: Breast Neoplasms; Cyclin D1; Enzyme Inhibitors; Female; Growth Substances; Humans; Insulin-Like Growth Factor I; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Predictive Value of Tests; Protein Kinases; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Receptors, Estrogen; Receptors, Progesterone; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Transcription, Genetic; Tumor Cells, Cultured

Previously, we have demonstrated that the two mitogenic growth factors epidermal growth factor and IGF-I can activate Akt and estrogen receptor-alpha (ERalpha) in the hormone-dependent breast cancer cell line, MCF-7. In this report we now show that estradiol can also rapidly activate phosphatidylinositol 3-kinase (PI 3-K)/Akt and that this effect is mediated by the ErbB2 signaling pathway. Treatment of cells with estradiol resulted in phosphorylation of Akt and a 9-fold increase in Akt activity in 10 min. Akt activation was blocked by wortmannin and LY 294,002, two inhibitors of PI 3-K; by genistein, a protein tyrosine kinase inhibitor and an ER agonist; by AG825, a selective ErbB2 inhibitor; and by the antiestrogens ICI 182,780 and 4-hydroxy-tamoxifen; but not by rapamycin, an inhibitor of the ribosomal protein kinase p70S6K; nor by AG30, a selective epidermal growth factor receptor inhibitor. Akt activation by estradiol was abrogated by an arginine-to-cysteine mutation in the pleckstrin homology domain of Akt (R25C). Growth factors also activated Akt in the ER-negative variant of MCF-7, MCF-7/ADR, but estradiol did not induce Akt activity in these cells. Transient transfection of ERalpha into these cells restored Akt activation by estradiol, suggesting that estradiol activation of Akt requires the ERalpha. Estradiol did not activate Akt in MCF-7 cells stably transfected with an anti-ErbB2-targeted ribozyme, further confirming a role for ErbB2. In vitro kinase assays using immunoprecipitation and anti-Akt1, -Akt2, and -Akt3-specific antibodies demonstrated that Akt1 is activated by estradiol in MCF-7 cells whereas Akt3 is the activated isoform in ER-negative MDA-MB231 cells, implying that selective activation of Akt subtypes plays a role in the actions of estradiol. Taken together, our data suggest that estradiol, bound to membrane ERalpha, interacts with and activates an ErbB dimer containing ErbB2, inducing activation of PI 3-K/Akt.

Topics: Androstadienes; Breast Neoplasms; Chromones; Enzyme Inhibitors; Estradiol; Estrogen Receptor alpha; Estrogen Receptor Modulators; Female; Fulvestrant; Genistein; Humans; Morpholines; Mutation; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Protein Isoforms; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Receptor, ErbB-2; Receptors, Estrogen; Serine; Signal Transduction; Sirolimus; Tumor Cells, Cultured; Tyrphostins; Wortmannin

mTOR (mammalian target of rapamycin) is a protein kinase that regulates cell cycle progression and cell growth. Rapamycin is a highly specific inhibitor of mTOR in clinical trials for the treatment of breast and other cancers. mTOR signaling was reported to require phosphatidic acid (PA), the metabolic product of phospholipase D (PLD). PLD, like mTOR, has been implicated in survival signaling and the regulation of cell cycle progression. PLD activity is frequently elevated in breast cancer. We have investigated the effect of rapamycin on breast cancer cell lines with different levels of PLD activity. MCF-7 cells, with relatively low levels of PLD activity, were highly sensitive to the growth-arresting effects of rapamycin, whereas MDA-MB-231 cells, with a 10-fold higher PLD activity than MCF-7 cells, were highly resistant to rapamycin. Elevating PLD activity in MCF-7 cells led to rapamycin resistance; and inhibition of PLD activity in MDA-MB-231 cells increased rapamycin sensitivity. Elevated PLD activity in MCF-7 cells also caused rapamycin resistance for S6 kinase phosphorylation and serum-induced Myc expression. These data implicate mTOR as a critical target for survival signals generated by PLD and suggest that PLD levels in breast cancer could be a valuable indicator of the likely efficacy of rapamycin treatment.

Topics: Adenocarcinoma; Animals; Breast Neoplasms; Cell Division; Culture Media, Serum-Free; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Female; Humans; Mice; Neoplasm Proteins; Phospholipase D; Phosphorylation; Protein Kinases; Protein Processing, Post-Translational; Recombinant Fusion Proteins; Ribosomal Protein S6 Kinases; Sirolimus; TOR Serine-Threonine Kinases

Expression of constitutively active Akt3 was found to increase the size of MCF-7 cells approximately twofold both in vitro and in vivo. A regulatable version of Akt1 (MER-Akt) was also found capable of inducing a twofold increase in the size of H4IIE rat hepatoma cells. Rapamycin, a specific inhibitor of mTOR function, was found to inhibit the Akt-induced increase in cell size by 70%, presumably via inhibition of the Akt-induced increase in protein synthesis. To determine whether Akt could be inhibiting protein degradation, thereby contributing to its ability to induce an increase in cell size, we conducted protein degradation experiments in the H4IIE cell line. Activation of MER-Akt was found to inhibit protein degradation to a degree comparable to insulin treatment. The effects of these two agents on protein degradation were not additive, thereby suggesting that they were acting on a similar pathway. An inhibitor of the phosphatidylinositol 3-kinase pathway, LY-294002, blocked both insulin- and Akt-induced inhibition of protein degradation, again consistent with the hypothesis that both agents were acting on the same pathway. In contrast, rapamycin did not block the ability of either agent to inhibit protein degradation. These results indicate that Akt increases cell size through both mTOR-dependent and -independent pathways and that the latter involves inhibition of protein degradation. These studies are also consistent with the hypothesis that insulin's ability to regulate protein degradation is to a large extent mediated via Akt.

Topics: Animals; Breast Neoplasms; Cell Count; Cell Cycle; Cell Size; Chromones; Enzyme Activation; Insulin; Liver Neoplasms, Experimental; Morpholines; Oncogene Proteins; Phosphoinositide-3 Kinase Inhibitors; Protein Biosynthesis; Protein Kinase Inhibitors; Protein Kinases; Protein Serine-Threonine Kinases; Proteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Rats; Sirolimus; TOR Serine-Threonine Kinases; Transfection; Tumor Cells, Cultured

We describe a severe preoperative cardiogenic shock in a patient scheduled for a breast surgery. The cardiogenic shock was in relation with thrombosis of two sirolimus-eluting stents received 3 months ago. A percutaneous transluminal coronary angioplasty was successfully performed. The patient recovered well after a 1-day treatment including intraaortic balloon counter pulsation and dobutamine infusion. We discuss about the ideal timing to plan surgery and how to manage the shift of antiplatelet agents.

Topics: Aged; Angioplasty, Balloon, Coronary; Breast; Breast Neoplasms; Cardiotonic Agents; Dobutamine; Female; Humans; Immunosuppressive Agents; Shock, Cardiogenic; Sirolimus; Stents; Thrombosis

Anchorage-independent growth is a hallmark of oncogenic transformation. We reported that the mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) inhibitor U0126 inhibited anchorage-independent growth of Ki-ras-transformed rat fibroblasts by simultaneously blocking both extracellular signal-regulated kinase (ERK) and mammalian target of rapamycin (mTOR)-p70(S6K) pathways. Here, we examined the effects of U0126 on the growth of eight human breast cancer cell lines. U0126 selectively repressed anchorage-independent growth of MDA-MB231 and HBC4 cells, two lines with constitutively activated ERK. Loss of contact with substratum triggers apoptosis in many normal cell types, a phenomenon termed anoikis. U0126 sensitized MDA-MB231 and HBC4 to anoikis, i.e., upon treatment with U0126, cells deprived of anchorage entered apoptosis, whereas adherent cells remained viable. Another MEK inhibitor PD98059 also induced anoikis sensitivity in MDA-MB231 cells but not in HBC4 cells. However, HBC4 cells were sensitized to anoikis when PD98059 was combined with the mTOR inhibitor rapamycin. To study the biochemical basis for induction of anoikis sensitivity, we examined the effects of the MEK inhibitors on ERK and p70(S6K) pathways in anchored versus nonanchored cells. As in Ki-ras-transformed rat fibroblasts, U0126 reduced activation of both ERK and p70(S6K) in MDA-MB231 and HBC4 cells, irrespective of anchorage. PD98059, in anchored cells, was more selective for the ERK pathway and did not significantly block the p70(S6K) pathway. Removal of anchorage substantially sensitized p70(S6K) to PD98059 in MDA-MB231 cells, whereas p70(S6K) in suspended HBC4 cells remained fairly refractory. U0126 was either without effect or less inhibitory on p70(S6K) in MDA-MB453 and SKBR3, two cell lines in which anoikis sensitivity was not induced. Thus, susceptibility of the p70(S6K) pathway to MEK inhibitors appeared to be an important determinant of anoikis sensitivity. The results indicate that concurrent inhibition of MEK-ERK and mTOR-p70(S6K) pathways induces apoptosis in MDA-MB231 and HBC4 cells when cells are deprived of anchorage but not when anchored. Inhibitors of MEK-ERK and mTOR-p70(S6K) pathways may provide a therapeutic strategy to selectively target neoplasms proliferating at ectopic locations, with acceptable effects on normal cells in their proper tissue context.

Topics: Animals; Anoikis; Antineoplastic Agents; Breast Neoplasms; Butadienes; Cell Division; Cell Line, Transformed; DNA Fragmentation; Dose-Response Relationship, Drug; Doxorubicin; Enzyme Inhibitors; Fibroblasts; Flavonoids; Immunoblotting; MAP Kinase Kinase Kinases; Mitogen-Activated Protein Kinases; Nitriles; Nucleosomes; Phosphorylation; Protein Kinases; Rats; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Sirolimus; Time Factors; TOR Serine-Threonine Kinases; Tumor Cells, Cultured

PTEN is a tumor suppressor that antagonizes phosphatidylinositol-3 kinase (PI3K) by dephosphorylating the D3 position of phosphatidylinositol (3,4,5)-triphosphate (PtdIns-3,4,5-P3). Given the importance of PTEN in regulating PtdIns-3,4,5-P3 levels, we used Affymetrix GeneChip arrays to identify genes regulated by PTEN. PTEN expression rapidly reduced the activity of Akt, which was followed by a G(1) arrest and eventually apoptosis. The gene encoding insulin receptor substrate 2 (IRS-2), a mediator of insulin signaling, was found to be the most induced gene at all time points. A PI3K-specific inhibitor, LY294002, also upregulated IRS-2, providing evidence that it was the suppression of the PI3K pathway that was responsible for the message upregulation. In addition, PTEN, LY294002, and rapamycin, an inhibitor of mammalian target of rapamycin, caused a reduction in the molecular weight of IRS-2 and an increase in the association of IRS-2 with PI3K. Apparently, PTEN inhibits a negative regulator of IRS-2 to upregulate the IRS-2-PI3K interaction. These studies suggest that PtdIns-3,4,5-P3 levels regulate the specific activity and amount of IRS-2 available for insulin signaling.

Topics: Apoptosis; Breast Neoplasms; Cell Cycle; Cell Line; Chromones; Enzyme Inhibitors; Feedback; Female; Genes, Tumor Suppressor; Humans; Insulin Receptor Substrate Proteins; Intracellular Signaling Peptides and Proteins; Models, Biological; Morpholines; Oligonucleotide Array Sequence Analysis; Phosphatidylinositol 3-Kinases; Phosphatidylinositol Phosphates; Phosphoinositide-3 Kinase Inhibitors; Phosphoproteins; Phosphoric Monoester Hydrolases; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Signal Transduction; Sirolimus; Tumor Cells, Cultured; Tumor Suppressor Proteins; Up-Regulation

The mammalian target of rapamycin (mTOR) is a central regulator of G1 cell cycle protein synthesis that precedes commitment to normal cellular replication. We have studied the effect of cell cycle inhibitor-779 (CCI-779), a rapamycin ester that inhibits mTOR function, on the proliferation of a panel of breast cancer cell lines. Six of eight lines studied were sensitive (IC(50)< or = 50 nM) and two lines were resistant (IC(50)>1.0 microM) to CCI-779. Sensitive lines were estrogen dependent (MCF-7, BT-474, T-47D), or lacked expression of the tumor suppressor PTEN (MDA-MB-468, BT-549), and/or overexpressed the Her-2/neu oncogene (SKBR-3, BT-474). Resistant lines (MDA-MB-435, MDA-MB-231) shared none of these properties. CCI-779 (50 nM) inhibited mTOR function in both a sensitive and a resistant line. In nu/nu mouse xenografts, CCI-779 inhibited growth of MDA-MB-468 (sensitive) but not MDA-MB-435 resistant tumors. Treatment of sensitive lines with CCI-779 resulted in a decrease in D-type cyclin and c-myc levels and an increase in p27(kip-1) levels. There was good correlation between activation of the Akt pathway and sensitivity to CCI-779. Amplification of mTOR-regulated p70S6 kinase, which is downstream of Akt, may also have conferred CCI-779 sensitivity to MCF-7 cells. Taken together, the data suggest that mTOR may be a good target for breast cancer therapy, especially in tumors with Akt activation resulting from either growth factor dependency or loss of PTEN function.

Topics: Animals; Antineoplastic Agents; Breast Neoplasms; Drug Evaluation, Preclinical; Drug Resistance, Neoplasm; Female; Mice; Mice, Nude; Protein Kinase Inhibitors; Protein Kinases; Sirolimus; TOR Serine-Threonine Kinases; Tumor Cells, Cultured

The proto-oncogene ErbB2 is known to be amplified and to play an important role in the development of about one-third of human breast cancers. Phosphatidylinositol 3-kinase (PI3K), which is often activated in ErbB2-overexpressing breast cancer cells, is known to regulate cell proliferation and cell survival. Selective inhibitors of the PI3K pathway were used to assess the relevance of PI3K signaling in the anchorage-independent growth of a series of human mammary carcinoma cell lines. Wortmannin, LY294002, and rapamycin at concentrations that did not affect MAPK phosphorylation but substantially inhibited PI3K, Akt, and p70(S6K) significantly suppressed the soft agar growth of tumor cell lines that overexpress ErbB2 but not the growth of tumor lines with low ErbB2 expression. A similar growth inhibition of ErbB2-overexpressing carcinoma lines was observed when a dominant negative p85(PI3K) mutant was introduced into these cells. Forced expression of ErbB2 in breast cancer lines originally expressing low ErbB2 levels augmented receptor expression and sensitized those lines to LY294002- and rapamycin-mediated inhibition of colony formation. Furthermore, treatment with LY294002 resulted in the selective increase of cyclin-dependent kinase inhibitors p21(Cip1) or p27(Kip1) and suppression of cyclin E-associated Cdk2 kinase activity in ErbB2-overexpressing lines, which may account for their hypersensitivity toward inhibitors of the PI3K pathway in anchorage-independent growth. Our results indicate that the PI3K/Akt/p70(S6K) pathway plays an enhanced role in the anchorage-independent growth of ErbB2-overexpressing breast cancer cells, therefore providing a molecular basis for the selective targeting of this signaling pathway in the treatment of ErbB2-related human breast malignancies.

Topics: Androstadienes; Antibiotics, Antineoplastic; Breast Neoplasms; Cell Adhesion; Cell Division; Cell Survival; Chromones; Cyclin E; Enzyme Inhibitors; Genes, Dominant; Humans; Immunoblotting; MAP Kinase Signaling System; Morpholines; Phosphatidylinositol 3-Kinases; Phosphorylation; Protein Binding; Proto-Oncogene Mas; Receptor, ErbB-2; Signal Transduction; Sirolimus; Transfection; Tumor Cells, Cultured; Wortmannin

The contribution of estrogen (and progesterone) in driving cell cycle progression of hormone dependent breast cancer cells is well documented, however, the roles of the various relevant signal transduction pathways remain unclear. The immunosuppressant rapamycin is a potent inhibitor of cell cycle progression and has been used to define signal transduction pathways. In this study we have determined rapamycin's effects on cell cycle progression in estrogen dependent breast cancer cells using a novel method of inducing S-phase. In this method estradiol-17-beta alone induced S-phase without mitogen support. In our studies the T47D cells were quite sensitive to estradiol-17-beta, with half-maximal induction in the picomolar range. indicating that the estrogen can induce S-phase in the absence of mitogens such as insulin. The estrogen response does not seem to be particularly specific because estriol estrone and estradiol-17-beta-BSA were about as effective as estradiol-17-beta. R5020, a progestin also induced S-Phase, while rapamycin blocked steroid driven transition of cells from G1 to S-phase.

Topics: Antibiotics, Antineoplastic; Breast Neoplasms; Cell Division; Estradiol; Female; Humans; In Vitro Techniques; Promegestone; Receptors, Estrogen; S Phase; Sirolimus; Tumor Cells, Cultured

Insulin-like growth factor I (IGF-I) is a well-established mitogen in human breast cancer cells. We show here that human breast cancer MCF-7 cells, which were prevented from attaching to the substratum and were floating in medium, responded to IGF-I and initiated DNA synthesis. The addition of IGF-I to floating cells induced activation of protein kinase B (PKB)/Akt, as to cells attached to the substratum. In addition, mitogen-activated protein kinase (MAPK)/extracellular response kinase (ERK) and its upstream kinases, ERK kinase (MEK) and Raf-1, were activated by IGF-I in floating cells. While the IGF-I-induced activation of PKB/Akt was inhibited by PI3-K inhibitor LY294002 but not by MEK inhibitor PD98059, the activation of both MEK and ERK by IGF-I was inhibited by both. These findings suggest that the IGF-I signal that leads to stimulation of DNA synthesis of MCF-7 cells is transduced to ERK through PI3-K, only when they are anchorage-deficient.

Topics: Breast Neoplasms; Cell Adhesion; Chromones; DNA, Neoplasm; Enzyme Activation; Enzyme Inhibitors; Female; Flavonoids; Humans; Insulin-Like Growth Factor I; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Morpholines; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-raf; Ribosomal Protein S6 Kinases; Signal Transduction; Sirolimus; Tumor Cells, Cultured

The effects of immunosuppressants and inhibitors of specific calcium/calmodulin kinase (CaMK) of types II and IV on progestin/glucocorticosteroid-induced transcription were studied in two human stably transfected breast cancer T47D cell lines. The lines contain the chloramphenicol acetyl transferase (CAT) gene under control either of the mouse mammary tumor virus promoter (T47D-MMTV-CAT), or the minimal promoter containing five glucocorticosteroid/progestin hormone response elements [T47D-(GRE)5-CAT]. Progestin- and triamcinolone acetonide (TA)-induced CAT gene expression was inhibited in a dose-dependent manner in both lines by preincubation with rapamycin (Rap) and, to a lesser extent, with FK506, but not with cyclosporin A. CaMK II and/or IV inhibitors KN62 and KN93 also inhibited progestin- and TA-stimulated transcription in both lines. None of these drugs had any effect on basal transcription. The antagonist RU486 inhibited all the effects of both progestin and TA, suggesting that progesterone receptor (PR)-, as well as glucocorticosteroid receptor (GR)- mediated transactivation are targets of immunosuppressants and CaMKs in T47D cells. Indeed, Northern analysis showed that Rap, KN62, and, to a lesser degree, FK506 inhibited progestin stimulation of Cyclin D1 mRNA levels, but not those of the non-steroid-regulated glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene. Addition of Rap or KN62 after exposure of cells to progesterone agonist Org 2058 had no effect on induction of CAT activity. Taken together, these data indicate that Rap and FK506, as well as CaMK inhibitors, inhibit steroid-induced activities of exogenous, as well as of some endogenous, steroid receptor-regulated genes by a mechanism preceding hormone-induced receptor activation. Rap appeared to stabilize a 9S form of [3H]Org 2058-PR complexes isolated from T47D (GRE)5CAT cell nuclei. By contrast, the progesterone receptor (PR) was isolated from cells treated with KN62 as a 5S entity, undistinguishable from the 5S PR species extracted from cells treated with progestin only. The nuclear 9S-[3H]Org2058-PR resulting from cells exposed to Rap, contained, in addition to the heat shock proteins of 90 kDa and 70 kDa (hsp90 and hsp70), the FK506-binding immunophilin FKBP52 but not FKBP51, although the latter was part of unliganded PR heterocomplex associated with hsp90. These results suggest that Rap and KN62 act upon the PR by distinct mechanisms, with only Rap impeding progestin-induced PR t

Topics: Animals; Breast Neoplasms; Calcium-Calmodulin-Dependent Protein Kinases; Chloramphenicol O-Acetyltransferase; Enzyme Inhibitors; HSP70 Heat-Shock Proteins; HSP90 Heat-Shock Proteins; Humans; Immunosuppressive Agents; Macromolecular Substances; Mice; Polyenes; Promegestone; Receptors, Glucocorticoid; Receptors, Progesterone; RNA, Messenger; Sirolimus; Tacrolimus; Transcription, Genetic; Triamcinolone Acetonide; Tumor Cells, Cultured