sirolimus and Carcinoma

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

To evaluate activity and toxicity of mTOR inhibitor temsirolimus in patients with platinum-refractory/resistant ovarian cancer (OC) or advanced/recurrent endometrial carcinoma (EC).. Women with epithelial ovarian, fallopian tube or primary peritoneal cancer were eligible, when they had progression during treatment with a platinum based regimen or within 6 months after receiving a platinum based regimen and a previous taxane treatment. Women with advanced/recurrent EC, no longer amenable to curative surgery and/or radiotherapy were eligible when they had no previous or only adjuvant chemotherapy. Preceding endocrine therapy for metastatic/recurrent disease was allowed. Patients received weekly IV infusions of 25mg temsirolimus. Primary endpoint was progression free survival rate after 4 months (OC) or 6 months (EC). A two stage design was applied.. Forty-four patients (OC: n=22; EC: n=22) were enrolled and received temsirolimus treatment. Median age was 56 years (OC) or 63 years (EC). After eight weeks of treatment, 10 of 21 evaluable patients in the OC cohort and 8 of 20 evaluable patients in the EC cohort had progressive disease. Thus efficacy did not meet the predefined levels during the first stage of recruitment and the trial was stopped. Some patients in both cohorts had long lasting PFS (>7 months). Toxicity of temsirolimus was mild.. Temsirolimus treatment was well tolerated in our patients, but did not meet the predefined efficacy criteria. In our study as in other trials on rapalogs in OC or EC, a few patients had long lasting disease stabilisations.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Agents; Carcinoma; Disease-Free Survival; Drug Resistance, Neoplasm; Early Termination of Clinical Trials; Endometrial Neoplasms; Female; Humans; Middle Aged; Neoplasm Recurrence, Local; Ovarian Neoplasms; Platinum Compounds; Response Evaluation Criteria in Solid Tumors; Retreatment; Sirolimus; Young Adult

Bevacizumab and temsirolimus are active agents in gynecologic tumors. Temsirolimus attenuates upregulation of HIF-1α levels, a resistance mechanism for antiangiogenics, and targets the PI3-kinase/AKT/mTOR axis, commonly aberrant in these tumors.. We analyzed safety and responses in 41 patients with gynecologic cancers treated as part of a Phase I study of bevacizumab and temsirolimus.. Median age of the 41 women was 60 years (range, 33-80 years); median number of prior systemic therapies was 4 (1-11). Grade 3 or 4 treatment-related toxicities included: thrombocytopenia (10%), mucositis (2%), hypertension (2%), hypercholesterolemia (2%), fatigue (7%), elevated aspartate aminotransferase (2%), and neutropenia (2%). Twenty-nine patients (71%) experienced no treatment-related toxicity greater than grade 2. Full FDA-approved doses of both drugs (bevacizumab 15mg/kg IV Q3weeks and temsirolimus 25mg IV weekly) were administered without dose-limiting toxicity. Eight patients (20%) achieved stable disease (SD) > 6 months and 7 patients (17%), a partial response (PR) [total = 15/41 patients (37%)]. Eight of 13 patients (62%) with high-grade serous histology (ovarian or primary peritoneal) achieved SD > 6 months/PR.. Bevacizumab and temsirolimus was well tolerated. Thirty-seven percent of heavily-pretreated patients achieved SD > 6 months/PR, suggesting that this combination warrants further study.

Topics: Adult; Aged; Aged, 80 and over; Antibodies, Monoclonal, Humanized; Antineoplastic Combined Chemotherapy Protocols; Aspartate Aminotransferases; Bevacizumab; Carcinoma; Fatigue; Female; Humans; Hypercholesterolemia; Hypertension; Male; Middle Aged; Mucositis; Neutropenia; Ovarian Neoplasms; Sirolimus; Thrombocytopenia; Uterine Cervical Neoplasms

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

Topics: Antineoplastic Combined Chemotherapy Protocols; Carcinoma; Everolimus; Humans; Paclitaxel; Sirolimus; Treatment Outcome; Urinary Bladder Neoplasms

Topics: Antineoplastic Combined Chemotherapy Protocols; Carcinoma; Everolimus; Humans; Paclitaxel; Sirolimus; Treatment Outcome; Urinary Bladder Neoplasms

Topics: Angiogenesis Inhibitors; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Bevacizumab; Carcinoma; Costs and Cost Analysis; Double-Blind Method; Humans; Indoles; Interferon alpha-2; Interferon-alpha; Kidney Neoplasms; Pyrroles; Recombinant Proteins; Sirolimus; Sunitinib; Survival Analysis; Vascular Endothelial Growth Factor A

Ovarian cancer is the second most common gynaecological malignancy and was diagnosed in over 7,000 women in 2011 in the UK. There are currently no reliable biomarkers available for use in a regular screening assay for ovarian cancer and due to characteristic late presentation (78% in stages III and IV) ovarian cancer has a low survival rate (35% after 10 years). The mTOR pathway is a central regulator of growth, proliferation, apoptosis and angiogenesis; providing balance between available resources such as amino acids and growth factors, and stresses such as hypoxia, to control cellular behaviour accordingly. Emerging data links mTOR with the aetiopathogenesis of ovarian cancer. We hypothesised that mTOR inhibitors could play a therapeutic role in ovarian cancer treatment. In this study we began by validating the expression of four main mTOR pathway components, mTOR, DEPTOR, rictor and raptor, at gene and protein level in in vitro models of endometrioid (MDAH‑2774) and clear cell (SKOV3) ovarian cancer using qPCR and ImageStream technology. Using a wound healing assay we show that inhibition of the mTOR pathway using rapamycin, rapalogues, resveratrol and NVP BEZ-235 induces a cytostatic and not cytotoxic response up to 18 h in these cell lines. We extended these findings up to 72 h with a proliferation assay and show that the effects of inhibition of the mTOR pathway are primarily mediated by the dephosphorylation of p70S6 kinase. We show that mTOR inhibition does not involve alteration of mTOR pathway components or induce caspase 9 cleavage. Preclinical studies including ovarian tissue of ovarian cancer patients, unaffected controls and patients with unrelated gynaecological conditions show that DEPTOR is reliably upregulated in ovarian cancer.

Topics: Adaptor Proteins, Signal Transducing; Apoptosis; Carcinoma; Carrier Proteins; Cell Line, Tumor; Cell Proliferation; Female; Gene Expression Regulation, Neoplastic; Humans; Imidazoles; Intracellular Signaling Peptides and Proteins; Ovarian Neoplasms; Protein Kinase Inhibitors; Quinolines; Rapamycin-Insensitive Companion of mTOR Protein; Regulatory-Associated Protein of mTOR; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Focal adhesion kinase (FAK) mediates survival of normal pancreatic islets through activation of AKT. Upon malignant transformation of islet cells into pancreatic neuroendocrine tumors (PanNETs), AKT is frequently overexpressed and mutations in the AKT/mTOR pathway are detected. Because mTOR inhibitors rarely induce PanNET tumor regression, partly because of feedback activation of AKT, novel combination strategies are needed to target FAK/AKT/mTOR signaling.. We characterized the activation of FAK in PanNETs using immunohistochemistry and Western blot analysis and tested the FAK inhibitor PF-04554878 in human PanNET cells in vitro and in vivo (at least three mice per group). In addition, we evaluated the effect of combined FAK and mTOR inhibition on PanNET viability and apoptosis. All statistical tests were two-sided.. We found that FAK is overexpressed and hyperphosphorylated in human PanNETs and that PF-04554878 strongly inhibited FAK (Tyr397) autophosphorylation in a dose-dependent manner. We found that PF-04554878 inhibited cell proliferation and clonogenicity and induced apoptosis in PanNET cells. Moreover, oral administration of PF-04554878 statistically significantly reduced tumor growth in a patient-derived xenograft model of PanNET (P = .02) and in a human PanNET xenograft model of peritoneal carcinomatosis (P = .03). Importantly, PF-04554878 synergized with the mTOR inhibitor everolimus by preventing feedback AKT activation.. We demonstrate for the first time that FAK is overexpressed in PanNETs and that inhibition of FAK activity induces apoptosis and inhibits PanNET proliferation. We found that the novel FAK inhibitor PF-04554878 synergizes with everolimus, a US Food and Drug Administration-approved agent for PanNETs. Our findings warrant the clinical investigation of combined FAK and mTOR inhibition in PanNETs.

Topics: Animals; Antineoplastic Agents; Apoptosis; Benzamides; Carcinoma; Cell Line, Tumor; Cell Proliferation; Cell Survival; Drug Resistance, Neoplasm; Drug Synergism; Everolimus; Female; Focal Adhesion Kinase 1; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Male; Mice; Mice, Inbred NOD; Mice, SCID; Molecular Targeted Therapy; Neuroendocrine Tumors; Organic Chemicals; Pancreatic Neoplasms; Peritoneal Neoplasms; Protein Kinase Inhibitors; Pyrazines; Signal Transduction; Sirolimus; Sulfonamides; TOR Serine-Threonine Kinases; Up-Regulation; Xenograft Model Antitumor Assays

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

To study the mTOR expression of cancer stem cells(CSCs) in nasopharyngeal carcinoma and preliminarily explore the mechanism of inhibiting its proliferation with rapamycin.. Nasopharyngeal carcinoma spherical cells were gathered by using serum-free suspension culture method, CCK8 assay was used to detect cell proliferation, Western blot assay was used to detect the expression of CD44, OCT4, SOX2 and mTOR signaling. The spherical cells and CNE2 were treated with rapamycin in concentrations of 0, 0.1, 1.0, 10.0, 100.0, 1000.0 nmol/L, CCK8 assay was used to detect cell inhibition ratio, Western blot assay was used to detect the expression of mTOR signaling of nasopharyngeal carcinoma spherical cells.. Compared with CNE2, the spherical cells exhibited a high proliferation rate in RPMI 1640 medium supplemented with fetal bovine serum, and overexpressed in OCT4, SOX2 (P < 0.05), but not that of CD44 (P > 0.05). Although the expression levels of mTOR, P70S6, 4EBP1 were not significantly different between the two kinds of cells (P > 0.05) the proteins of phosphorylation activation form of them (P-mTOR, P-P70S6, P-4EBP1) were highly expressed in spherical cells (P < 0.05). The spherical cells and CNE2 were treated with rapamycin in different concentrations, the concentrations for 50% of maximal effect of spherical cells and CNE2 were 2.59 nmol/L and 78.12 nmol/L respectively, rapamycin inhibited the spherical cells more strongly compared with CNEZ. The expression levels of P-mTOR, P-70S6, P-4EBP1 in spherical cells were gradually decreased with increasing of the concentrations of rapamycin, but the difference of the expression levels of mTOR, P70S6, 4EBP1 were not significant.. The proteins of mTOR signaling pathway of CSCs in nasopharyngeal carcinoma are overexpressed, and rapamycin can effectively inhibit cell proliferation of CSCs in nasopharyngeal carcinoma by blocking mTOR signaling pathway.

Topics: Adaptor Proteins, Signal Transducing; Carcinoma; Cell Cycle Proteins; Cell Proliferation; Humans; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; Neoplastic Stem Cells; Phosphoproteins; Phosphorylation; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Tumor Cells, Cultured

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

The underlying causes of endometrial cancer (EMC) are poorly understood, and treatment options for patients with advanced stages of the disease are limited. Mutations in the phosphatase and tensin homologue gene are frequently detected in EMC. Cyclooxygenase 2 (Cox2) and mammalian target of rapamycin complex 1 (mTORC1) are known downstream targets of the phosphatase and tensin homologue protein, and their activities are up-regulated in EMC. However, it is not clear whether Cox2 and mTORC1 are crucial players in cancer progression or whether they work in parallel or cooperatively. In this study, we used a Cox2 inhibitor, celecoxib, and an mTORC1 inhibitor, rapamycin, in mouse models of EMC and in human EMC cell lines to explore the interactive roles of Cox2 and mTORC1 signaling. We found that a combined treatment with celecoxib and rapamycin markedly reduces EMC progression. We also observed that rapamycin reduces Cox2 expression, whereas celecoxib reduces mTORC1 activity. These results suggest that Cox2 and mTORC1 signaling is cross-regulated and cooperatively exacerbate EMC.

Topics: Animals; Antibiotics, Antineoplastic; Apoptosis; Blotting, Western; Carcinoma; Celecoxib; Cell Line, Tumor; Cell Survival; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Disease Models, Animal; Endometrial Neoplasms; Female; Humans; Immunohistochemistry; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Knockout; Multiprotein Complexes; Pyrazoles; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Sirolimus; Sulfonamides; TOR Serine-Threonine Kinases

Mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase and a key element in the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway. Moreover, it is a negative regulator of autophagy and acts as a central regulator in cell growth. For the treatment of cancer, mTOR is a novel and validated therapeutic target. Previous studies have shown that Akt is frequently activated in nasopharyngeal carcinoma (NPC) tissues; thus, the inhibition of mTOR may be a treatment strategy for this tumor type. To evaluate the effect of the mTOR inhibitor RAD001 on NPC cell lines, we performed 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) assays, lactate dehydrogenase (LDH) assays, western blotting and flow cytometry to evaluate the mechanisms of cell death. The growth of both CNE-1 and HONE-1 cells was inhibited in a time- and dose-dependent manner. CNE-1 was more sensitive, with a 50% growth inhibition (GI50) of 30.0±1.0 µM compared to HONE-1, cells which had a GI50 of 56.9±13.1 µM. RAD001 induced apoptosis and autophagy in both cell lines. RAD001 induced a significant increase in growth inhibition in the two cell lines when used in combination with the autophagy inhibitor, 3-methyladenine; however, the percentages of apoptotic cells decreased when RAD001 was combined with the caspase inhibitor, z-VAD-fmk. In conclusion, the main mechanism of the mTOR inhibitor RAD001 in these two NPC cells was apoptotic, not autophagic cell death. The combination of RAD001 with autophagy inhibitors may be a useful therapeutic strategy for nasopharyngeal carcinoma.

Topics: Amino Acid Chloromethyl Ketones; Analysis of Variance; Antineoplastic Agents; Apoptosis; Autophagy; Carcinoma; Cell Line, Tumor; Cell Proliferation; Cell Shape; Everolimus; Flow Cytometry; Humans; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; Sirolimus; TOR Serine-Threonine Kinases

The aim of the present study was to investigate the role of autophagy in response to ionizing radiation (IR) in CNE-2 human nasopharyngeal carcinoma cells and to demonstrate the function of poly(ADP-ribose) polymerase-1 (PARP-1) in the regulation of IR-induced autophagy. Microtubule-associated protein 1 light chain 3 (LC3) and poly(ADP-ribose) (PAR) were assessed using western blotting. Ultrastructural analysis was performed using transmission electron microscopy (TEM). The percentage of apoptotic cells was assessed by flow cytometry. The MTT method was used to detect cell viability of CNE-2 cells at different time points after IR. Clonogenic survival assays were used to evaluate the radiosensitivity of nasopharyngeal carcinoma cells treated with IR and IR combined with autophagy inhibitor (chloroquine phosphate), with autophagy inducer (rapamycin) or with PARP-1 inhibitor 3-amino benzamide (3AB). IR induced a massive accumulation of autophagosomes detected by TEM and intensified the conversion of cytosolic LC3-I to LC3-II. PARP-1 activation was accompanied by strong upregulation of PAR and LC3-II expression in CNE-2 cells. Compared with radiation alone, chloroquine phosphate (CDP) or 3AB combined with IR significantly decreased cell viability, as well as the autophagic ratio and LC3-II protein levels. Inhibition of autophagy increased radiation-induced apoptosis; rapamycin (RAPA) significantly decreased cell viability as well, but RAPA increased the autophagic ratio and LC3-II protein levels; induction of autophagy increased radiation-induced apoptosis. To conclude, PARP-1 regulates IR-induced autophagy, and PARP-1 inhibitor contributes to the radiation sensitization of CNE-2 cells. Blockade of autophagy with CDP enhanced the cytotoxicity of radiotherapy in CNE-2 cells. This suggests that inhibition of autophagy or PARP-1 may be used as an adjuvant therapy to treat nasopharyngeal carcinoma.

Topics: Antineoplastic Agents; Apoptosis; Autophagy; Benzamides; Carcinoma; Cell Line, Tumor; Cell Survival; Chloroquine; Humans; Microtubule-Associated Proteins; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; Phagosomes; Poly (ADP-Ribose) Polymerase-1; Poly Adenosine Diphosphate Ribose; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Radiation Tolerance; Radiation-Sensitizing Agents; Sirolimus

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

The mammalian target of rapamycin (mTOR) signaling is a key pathway in the progression of different cancers and in the homeostasis of stem cells. Here, we investigated the link between mTOR signaling and cancer stem cells (CSCs) in nasopharyngeal carcinoma (NPC). We found that human primary NPC expressed embryonic stem cell (ESC) markers: CD133, SOX2 and OCT4 as well as pmTOR and pS6. Primary ESC-positive NPC cells could form secondary NPC in BALB/c nude mice. Rapamycin, an mTOR inhibitor, significantly suppressed ESC-positive NPC cell growth in vitro and tumor formation in vivo. Our findings suggest that mTOR signaling is activated in CSC-like cells and plays an important role in NPC growth.

Topics: AC133 Antigen; Animals; Antibiotics, Antineoplastic; Antigens, CD; Blotting, Western; Carcinoma; Cell Survival; Dose-Response Relationship, Drug; Enzyme Activation; Glycoproteins; Humans; Immunohistochemistry; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Middle Aged; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; Neoplastic Stem Cells; Octamer Transcription Factor-3; Peptides; Signal Transduction; Sirolimus; SOXB1 Transcription Factors; Time Factors; TOR Serine-Threonine Kinases; Tumor Burden; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Radioresistance of EBV-associated nasopharyngeal carcinoma (NPC) is associated with poor prognosis for patients with this form of cancer. Here, we found that NPC patients had increased serum levels of leukemia inhibitory factor (LIF) and that higher LIF levels correlated with local tumor recurrence. Furthermore, in vitro studies with NPC cells and in vivo xenograft mouse studies demonstrated that LIF critically contributes to NPC tumor growth and radioresistance. Using these model systems, we found that LIF treatment activated the mTORC1/p70S6K signaling pathway, enhanced tumor growth, inhibited DNA damage responses, and enhanced radioresistance. Treatment with either soluble LIF receptor (sLIFR), a LIF antagonist, or the mTOR inhibitor rapamycin reversed LIF-mediated effects, resulting in growth arrest and increased sensitivity to γ irradiation. Immunohistochemical (IHC) analyses of human NPC biopsies revealed that LIF and LIFR were overexpressed in tumor cells and that LIF expression correlated with the presence of the activated p-p70S6K. Finally, we found that the EBV-encoded protein latent membrane protein 1 (LMP1) enhances LIF production. Together, our findings indicate that LIF promotes NPC tumorigenesis and suggest that serum LIF levels may predict local recurrence and radiosensitivity in NPC patients.

Topics: Animals; Carcinoma; Disease Progression; DNA Damage; Gamma Rays; Gene Expression Regulation, Viral; Heterografts; Humans; Leukemia Inhibitory Factor; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Inbred NOD; Mice, SCID; Multiprotein Complexes; Nasopharyngeal Neoplasms; Neoplasm Proteins; Neoplasm Recurrence, Local; Prognosis; Radiation Tolerance; Receptors, OSM-LIF; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Tumor Cells, Cultured; Tumor Microenvironment; Viral Matrix Proteins

Clinical trials using kinase inhibitors have demonstrated transient partial responses and disease control in patients with progressive medullary thyroid cancer (MTC). The goal of this study was to identify potential combinatorial strategies to improve on these results using sorafenib, a multikinase inhibitor with activity in MTC, as a base compound to explore signaling that might predict synergystic interactions. Two human MTC cell lines, TT and MZ-CRC-1, which harbor endogenous C634W or M918T RET mutations, respectively, were exposed to sorafenib, everolimus, and AZD6244 alone and in combination. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide (MTT) and poly (ADP-ribose) polymerase (PARP) cleavage assays were performed to measure cell survival and apoptosis. Western blots were performed to confirm activity of the compounds and to determine possible mechanisms of resistance and predictors of synergy. As a solitary agent, sorafenib was the most active compound on MTT assay. Western blots confirmed that sorafenib, everolimus, and AZD6244 inhibited their anticipated targets. At concentrations below its IC(50), sorafenib-treated TT and MZ-CRC-1 cells demonstrated transient inhibition and then re-activation of Erk over 6 h. In concordance, synergistic effects were only identified using sorafenib in combination with the Mek inhibitor AZD6244 (P<0.001 for each cell line). Cells treated with everolimus demonstrated activation of Akt and Ret via TORC2 complex-dependent and TORC2 complex-independent mechanisms respectively. Everolimus was neither additive nor syngergistic in combination with sorafenib or AZD6244. In conclusion, sorafenib combined with a Mek inhibitor demonstrated synergy in MTC cells in vitro. Mechanisms of resistance to everolimus in MTC cells likely involved TORC2-dependent and TORC2-independent pathways.

Topics: Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Benzenesulfonates; Benzimidazoles; Carcinoma; Carcinoma, Neuroendocrine; Cell Line, Tumor; Cell Survival; Drug Synergism; Everolimus; Extracellular Signal-Regulated MAP Kinases; Humans; MAP Kinase Signaling System; Niacinamide; Phenylurea Compounds; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-ret; Pyridines; Sirolimus; Sorafenib; Thyroid Neoplasms; TOR Serine-Threonine Kinases

Treatment options are insufficient in patients with adrenocortical carcinoma (ACC). Based on the efficacy of sorafenib, a tyrosine kinase inhibitor, and everolimus, an inhibitor of the mammalian target of rapamycin in tumors of different histotype, we aimed at testing these drugs in adrenocortical cancer models. The expression of vascular endothelial growth factor and its receptors (VEGFR1-2) was studied in 18 ACCs, 33 aldosterone-producing adenomas, 12 cortisol-producing adenomas, and six normal adrenal cortex by real-time PCR and immunohistochemistry and by immunoblotting in SW13 and H295R cancer cell lines. The effects of sorafenib and everolimus, alone or in combination, were tested on primary adrenocortical cultures and SW13 and H295R cells by evaluating cell viability and apoptosis in vitro and tumor growth inhibition of tumor cell line xenografts in immunodeficient mice in vivo. VEGF and VEGFR1-2 were detected in all samples and appeared over-expressed in two-thirds of ACC specimens. Dose-dependent inhibition of cell viability was observed particularly in SW13 cells after 24 h treatment with either drug; drug combination produced markedly synergistic growth inhibition. Increasing apoptosis was observed in tumor cells treated with the drugs, particularly with sorafenib. Finally, a significant mass reduction and increased survival were observed in SW13 xenograft model undergoing treatment with the drugs in combination. Our data suggest that an autocrine VEGF loop may exist within ACC. Furthermore, a combination of molecularly targeted agents may have both antiangiogenic and direct antitumor effects and thus could represent a new therapeutic tool for the treatment of ACC.

Topics: Adenoma; Adolescent; Adrenal Cortex Neoplasms; Adult; Aged; Animals; Antineoplastic Combined Chemotherapy Protocols; Benzenesulfonates; Carcinoma; Cell Line, Tumor; Child; Child, Preschool; Everolimus; Female; Humans; Infant; Male; Mice; Mice, Knockout; Mice, SCID; Middle Aged; Niacinamide; Phenylurea Compounds; Pyridines; Sirolimus; Sorafenib; Treatment Outcome; Xenograft Model Antitumor Assays; Young Adult

Understanding the molecular pathogenesis of medullary thyroid carcinoma (MTC) is prerequisite to the design of targeted therapies for patients with advanced disease.. We studied by immunohistochemistry the phosphorylation status of proteins of the RAS/MEK/ERK and PI3K/AKT/mTOR pathways in 53 MTC tissues (18 hereditary, 35 sporadic), including 51 primary MTCs and 2 cases with only lymph node metastases (LNM). We also studied 21 autologous LNMs, matched to 21 primary MTCs. Staining was graded on a 0 to 4 scale (S score) based on the percentage of positive cells. We also studied the functional relevance of the mTOR pathway by measuring cell viability, motility, and tumorigenicity upon mTOR chemical blockade.. Phosphorylation of ribosomal protein S6 (pS6), a downstream target of mTOR, was evident (S ≥ 1) in 49 (96%) of 51 primary MTC samples. This was associated with activation of AKT (phospho-Ser473, S > 1) in 79% of cases studied. Activation of pS6 was also observed (S ≥ 1) in 7 (70%) of 10 hereditary C-cell hyperplasia specimens, possibly representing an early stage of C-cell transformation. It is noteworthy that 22 (96%) of 23 LNMs had a high pS6 positivity (S ≥ 3), which was increased compared with autologous matched primary MTCs (P = 0.024). Chemical mTOR blockade blunted viability (P < 0.01), motility (P < 0.01), and tumorigenicity (P < 0.01) of human MTC cells.. The AKT/mTOR pathway is activated in MTC, particularly, in LNMs. This pathway sustains malignant features of MTC cell models. These findings suggest that targeting mTOR might be efficacious in patients with advanced MTC.

Topics: Animals; Carcinoma; Carcinoma, Neuroendocrine; Cell Line, Tumor; Cell Movement; Cell Survival; DNA Mutational Analysis; Female; Humans; Lymphatic Metastasis; Mice; Mice, Inbred NOD; Mice, SCID; Mitogen-Activated Protein Kinases; Phosphorylation; Protein Processing, Post-Translational; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-ret; Ribosomal Protein S6 Kinases; Signal Transduction; Sirolimus; Statistics, Nonparametric; Thymus Hyperplasia; Thyroid Neoplasms; Tissue Array Analysis; TOR Serine-Threonine Kinases; Tumor Burden; Xenograft Model Antitumor Assays

To investigate the effect of mechanistic target of rapamycin (mTOR)-specific small interfering RNA (siRNA) and rapamycin on tumour size and levels of hypoxia inducible factor 1α(HIF-1α), vascular endothelial growth factor (VEGF) and mTOR proteins, and mTOR mRNA, in a mouse xenograft model of human oesophageal carcinoma.. Tumours were induced in BALB/c nude mice using the human oesophageal squamous cell carcinoma cell line, EC1, injected subcutaneously. Animals were divided into four treatment groups (n = 5 per group) after 7 days: control (phosphate buffered saline, daily intraperitoneal [i.p.] injection); 50 μg/kg rapamycin, daily i.p. injection; 3 μg/kg mTOR siRNA, daily i.p. injection; combined mTOR siRNA and rapamycin, daily i.p. injections. Tumour volume was measured 21 days after xenograft. Levels of mTOR, VEGF and HIF-1α were assessed via immunohistochemistry and in situ hybridization.. mTOR siRNA and/or rapamycin significantly decreased tumour volume and levels of HIF-1α, VEGF and mTOR protein, and mTOR mRNA. Combination treatment was significantly more effective than either treatment alone.. mTOR siRNA and/or rapamycin inhibited the growth of oesophageal carcinoma in vivo. This may represent a novel and effective treatment strategy for oesophageal carcinoma.

Topics: Animals; Antibiotics, Antineoplastic; Carcinoma; Cell Line, Tumor; Esophageal Neoplasms; Female; Gene Expression; Gene Knockdown Techniques; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Mice; Mice, Inbred BALB C; Mice, Nude; RNA, Small Interfering; Sirolimus; TOR Serine-Threonine Kinases; Tumor Burden; Vascular Endothelial Growth Factor A; Xenograft Model Antitumor Assays

mTOR (mammalian target of rapamycin) inhibitors were recently found to be effective in the treatment of various human non-Hodgkin's lymphomas (NHLs). We recently reported that RAD001, an mTOR inhibitor, suppressed the growth of lymphoma cells at concentrations much lower than those required for carcinomas. However, the basis for the enhanced sensitivity to RAD001 is unknown. Seven aggressive NHL cell lines and seven carcinoma cell lines were used in this study. Cell cycle was analysed by flow cytometry. pAKT (phosphorylated AKT) (Ser(473) and Thr(308)), p-p70S6K, p-4E-BP1, p-mTOR, p-eIF4E (phosphorylated eIF4E), cyclin A, cyclin E, cyclin D3, c-Myc and insulin receptor substrate-1 (IRS-1) protein expression were assessed by immunoblotting. The PI3K/AKT/mTOR (phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin) signalling pathway was constitutively expressed in all seven lymphoma cell lines. RAD001 down-regulated p-mTOR, p-p70S6K, p-4E-BP1, cyclin A, cyclin E, cyclin D3, and c-Myc, but did not affect IRS-1. In parallel with RAD001-induced inhibition of cell viability, a dose- and schedule- dependent down-regulation of pAKT and p-eIF4E expressions was demonstrated. In contrast, a compensatory activation of pAKT and p-eIF4E, was observed in seven carcinoma cells. These findings indicate that the basis for enhanced activity of mTOR inhibitors in NHL may be the lack of compensatory activation of AKT and eIF4E phosphorylation in lymphoma cells.

Topics: Apoptosis; Carcinoma; Cell Cycle; Cell Line, Tumor; Eukaryotic Initiation Factor-4E; Everolimus; Gene Expression Regulation, Neoplastic; Humans; Immunosuppressive Agents; Jurkat Cells; Lymphoma; Models, Biological; Phosphorylation; Proto-Oncogene Proteins c-akt; Sirolimus; Tetrazolium Salts; Thiazoles; TOR Serine-Threonine Kinases

Topics: Antineoplastic Agents; Carcinoma; Carcinoma, Papillary; Carcinoma, Renal Cell; Dermatologic Agents; Fatal Outcome; Female; Humans; Hydrocortisone; Keratin-7; Kidney Neoplasms; Lymphatic Metastasis; Middle Aged; Paired Box Transcription Factors; PAX8 Transcription Factor; Sirolimus; Skin Neoplasms; Thyroid Cancer, Papillary; Thyroid Neoplasms

Because mammalian target of rapamycin (mTOR) may be involved in thyroid carcinogenesis, we investigated the expression and activation patterns of mTOR signaling proteins in thyroid carcinoma cells and tumors and their association with tumor histology and aggressiveness.. Tissue specimens from 50 patients with thyroid cancer were analyzed for eIF4E, a critical downstream target of the mTOR pathway, using immunohistochemistry. In addition, fresh-frozen samples from patients, and primary tumor cell cultures were analyzed for expression and activation of mTOR signaling proteins by Western blot. Moreover, pharmacologic studies with rapamycin were performed.. High expression of eIF4E was observed in medullary thyroid carcinomas (MTC) and in aggressive variants of papillary thyroid carcinomas (PTC) as compared with conventional PTC and follicular thyroid carcinomas (P < .0001). The level of eIF4E expression also correlated with tumor stage (P = .002). Using Western blot analysis, p-rpS6, p-4EBP1, 4EBP1, and eIF4E were detected at higher levels in aggressive PTC and MTC cells. Treatment of MTC cells with increasing concentrations of rapamycin resulted in significant cell death and in decreased cell growth associated with deactivation of the mTOR pathway.. mTOR signaling, which controls protein synthesis through regulation of translation initiation, is activated in aggressive PTC and MTC and represents a promising target for investigational therapies in these patients.

Topics: Adult; Blotting, Western; Carcinoma; Carcinoma, Neuroendocrine; Carcinoma, Papillary; Cell Death; Cell Line, Tumor; Eukaryotic Initiation Factor-4E; Female; Humans; Immunohistochemistry; Male; Middle Aged; Neoplasm Staging; Signal Transduction; Sirolimus; Thyroid Cancer, Papillary; Thyroid Neoplasms; TOR Serine-Threonine Kinases; Tumor Cells, Cultured

Retinoic acid presently is the most advanced agent able to improve the efficacy of radioiodine therapy in differentiated thyroid carcinoma. In order to identify compounds with higher efficacy a panel of pharmacologically well-characterized compounds with antitumour action in solid cancer cell lines was screened.. The effects of the compounds on iodide uptake, cell number, proliferation and apoptosis were evaluated.. In general, compounds were more effective in cell lines derived from more aggressive tumours. The effectiveness in terms of number of responsive cell lines and maximal increase in iodide uptake achieved decreased in the order: APHA > valproic acid approximately sirolimus approximately arsenic trioxide > retinoic acid approximately lovastatin > apicidine approximately azacytidine approximately retinol approximately rosiglitazone approximately bortezomib.. We hypothesize that testing of cells from primary tumours or metastases in patients may be a way to identify compounds with optimum therapeutic efficacy for individualized treatment.

Topics: Antineoplastic Agents; Apoptosis; Arsenic Trioxide; Arsenicals; Boronic Acids; Bortezomib; Carcinoma; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Drug Screening Assays, Antitumor; Humans; Iodides; Lovastatin; Neoplasm Metastasis; Oxides; Pyrazines; Sirolimus; Thymus Gland; Thyroid Neoplasms; Tretinoin

The PI3K/Akt/mTOR prosurvival pathway is frequently up-regulated in soft tissue sarcoma. Mammalian target of rapamycin (mTOR) inhibitors, such as rapamycin, have recently shown clinical benefit in soft tissue sarcoma, and mTOR inhibition has also been associated with radiosensitization of carcinoma and endothelial cells. This study tested the hypothesis that rapamycin radiosensitizes soft tissue sarcoma and endothelial cells in vitro and in vivo through the inhibition of mTOR.. Colony formation assays were done to determine the radiosensitizing properties of rapamycin on three human soft tissue sarcoma cell lines (SK-LMS-1, SW-872, and HT-1080) and human dermal microvascular endothelial cells (HDMEC). The functional effects of rapamycin and radiation on the endothelial compartment were evaluated with microvascular sprouting assays. The in vivo radiosensitizing activity of rapamycin was assessed with s.c. SK-LMS-1 nude mice xenografts treated with concurrent daily rapamycin, radiation, or both for three weeks.. In vitro radiosensitization was shown in all three soft tissue sarcoma cell lines with minimally cytotoxic doses of rapamycin. SK-LMS-1 xenografts displayed significant tumor growth delay with rapamycin and radiation compared with either treatment alone. Radiation resulted in transient increased mTOR function, whereas rapamycin abolished this signaling in irradiated and unirradiated samples. In HDMEC, rapamycin and radiation reduced microvessel sprouting, but did not alter colony formation.. Minimally cytotoxic concentrations of rapamycin inhibited the mTOR cascade in culture and in vivo while radiosensitizing soft tissue sarcoma, and produced synergistic effects with radiation on HDMEC microvessel formation. By targeting both tumor and endothelial compartments, rapamycin produced potent radiosensitization of soft tissue sarcoma xenografts. Clinical trials combining rapamycin and radiotherapy in soft tissue sarcoma are warranted.

Topics: Animals; Antineoplastic Agents; Carcinoma; Cell Line, Tumor; Dose-Response Relationship, Radiation; Endothelial Cells; Endothelium; Humans; Mice; Mice, Nude; Microcirculation; Models, Biological; Neoplasm Transplantation; Protein Kinases; Radiation-Sensitizing Agents; Sarcoma; Sirolimus; TOR Serine-Threonine Kinases

Activated mTOR was implicated to play a role in the carcinogenesis of nasopharyngeal carcinoma (NPC). However, the mechanism of activated mTOR/Complex1(mTORC1) signaling pathway in NPC development has not been well established. In this study, we correlated the expression of mTORC1 signal molecules and Cyclin D1 in NPC. We also investigated the effect of blocking mTORC1 signal with rapamycin and mTOR siRNA on Cyclin D1 expression in CNE-2 cells, as well as cell apoptosis and viability. We found a positive association of mTORC1 signal molecules and Cyclin D1 in NPC. Also, we found blockage mTORC1 inhibited Cyclin D1 expression in CNE-2 cells and enhanced cell apoptosis. Our results suggested that mTORC1 signal pathway might be a potential target for NPC therapy.

Topics: Adaptor Proteins, Signal Transducing; Adult; Apoptosis; Carcinoma; Case-Control Studies; Cell Cycle Proteins; Cell Line, Tumor; Cell Survival; Cyclin D1; Dose-Response Relationship, Drug; Female; Humans; Male; Middle Aged; Multiprotein Complexes; Nasopharyngeal Neoplasms; Phosphoproteins; Phosphorylation; Protein Kinases; RNA Interference; RNA, Small Interfering; Signal Transduction; Sirolimus; Time Factors; TOR Serine-Threonine Kinases; Transfection; Up-Regulation

For almost the last two decades, interleukin-2 and interferon-alpha have been the only systemic treatment options available for metastatic renal cell carcinoma. However, in recent years, five new targeted therapies namely sunitinib, sorafenib, temsirolimus, everolimus and bevacizumab have demonstrated clinical activity in these patients. With the availability of new targeted agents that are active in this disease, there is a need to continuously update the treatment algorithm of the disease. Due to the important advances obtained, the Spanish Oncology Genitourinary Group (SOGUG) has considered it would be useful to review the current status of the disease, including the genetic and molecular biology factors involved, the current predicting models for development of metastases as well as the role of surgery, radiotherapy and systemic therapies in the early- or late management of the disease. Based on this previous work, a treatment algorithm was developed.

Topics: Algorithms; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Benzenesulfonates; Bevacizumab; Carcinoma; Clinical Trials, Phase II as Topic; Clinical Trials, Phase III as Topic; Everolimus; Humans; Indoles; Kidney Neoplasms; Linear Models; Neoplasm Metastasis; Neoplasm Staging; Niacinamide; Phenylurea Compounds; Protein Kinase Inhibitors; Protein Kinases; Pyridines; Pyrroles; Receptor Protein-Tyrosine Kinases; Sirolimus; Sorafenib; Sunitinib; TOR Serine-Threonine Kinases; Vascular Endothelial Growth Factors

Activation of the phosphatidylinositol-3-kinase (PI3K) signaling cascade is increasingly recognized as a common feature of thyroid follicular neoplasms. Among the PI3K downstream effectors, the main kinase, directly responsible for the increased cell growth and proliferation, is called mammalian target of rapamycin (mTOR). This central kinase might be directly inhibited via rapamycin and its derivatives. The aim of the present study was to examine whether RAD001 (everolimus) can selectively suppress the proliferation of different anaplastic thyroid cancer (ATC) cells. Five different human ATC cell lines were exposed to different concentrations of RAD001. Importantly, we found a dose-dependent growth inhibition in two ATC cell lines at concentrations of 43.5 and 94.5 nM although not as intensive as within the RAD001 responding K562cell line. The other cell lines revealed a GI (50) between 168 to 234 nM. In parallel, quantitative PCR of PCNA displayed a reduced expression of PCNA within the responding cell lines, respectively. In summary, we found a good responding effect in a part of ATC cell lines, which may have a clinical impact.

Topics: Carcinoma; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Everolimus; Humans; Immunosuppressive Agents; Proliferating Cell Nuclear Antigen; Protein Kinases; Reverse Transcriptase Polymerase Chain Reaction; RNA; Sirolimus; Thyroid Neoplasms; TOR Serine-Threonine Kinases

Differentiated thyroid cancer and anaplastic thyroid cancer tumors frequently have activation of the ras/raf /MAPK kinase (MEK)/ERK and phosphatidylinositol 3-kinase (PI-3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathways.. The objective of the study was to investigate the efficacy of MEK and mTOR inhibitors in preclinical thyroid cancer treatment models with defined mutation status.. The MEK inhibitor AZD6244 (ARRY-142886) and mTOR inhibitor rapamycin were tested separately and in combination in 10 differentiated thyroid cancer and anaplastic thyroid cancer cell lines and in a xenograft model for evidence of pathway inhibition, growth inhibition, apoptosis, and long-range adaptation and resistance.. Seven of 10 tested lines had evidence of significant basal activity of the PI-3K/AKT/mTOR pathway, with elevated phosphorylated AKT and phosphorylated p70 S6 kinase. Activation of ras/RAF/MEK/ERK was equally common in this panel. All 10 lines exhibited better than 60% growth inhibition with combined MEK and mTOR inhibition, including lines with BRAF, Ret-PTC, ras, and PTEN mutations. Rapamycin or AZD6244 alone achieved this threshold in six and two lines, respectively. Dual-pathway inhibition in the Ret-PTC mutant cell line TPC1 caused an intense G(1) arrest in cell culture and reversible cytostatic inhibition in a xenograft model. We did not observe significant feedback up-regulation of AKT activation in either acute or prolonged exposures.. These preclinical results support the inclusion of thyroid cancer patients in early-phase clinical trials combining RAS/RAF/MEK/ERK and PI-3K/AKT/mTOR pathway inhibition.

Topics: Animals; Antibiotics, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Benzimidazoles; Blotting, Western; Carcinoma; Cell Line, Tumor; Flow Cytometry; Humans; Kaplan-Meier Estimate; Mice; Mice, Nude; Mitogen-Activated Protein Kinase Kinases; Signal Transduction; Sirolimus; Thyroid Neoplasms; Transplantation, Heterologous

To evaluate the efficacy and the safety of combined 5-Fluorouracil, irinotecan, bevacizumab and sirolimus in refractory advanced colorectal carcinoma.. We initiated a regimen with at day 1 an injection (iv) of bevacizumab at 5 mg/kg, followed by 180 mg/m(2) irinotecan, followed by Leucovorin 400 mg/m(2), followed by a 5-Fluorouracil bolus 400 mg/m(2); and a 46-h infusion 2400 mg/m(2). Sirolimus was given orally as continuous administration of 2 mg twice a day every days. This treatment was repeated every 14 d.. A total of 12 patients were enrolled. All patients presented with metastatic disease that had failed at least three lines of chemotherapy that contained oxaliplatin, irinotecan and bevacizumab. Cetuximab failure was also observed in all K-Ras wild-type patients. The median number of cycles was 8.5 (range 2-20) and clinical benefit was observed in eight patients. The median time to progression was 5 mo and the median survival was 8 mo. Grade 3 neutropenia developed in four patients, and grade 3 diarrhea and stomatitis in two.. The combination regimen of 5-Fluorouracil, irinotecan, bevacizumab and sirolimus in advanced colorectal carcinoma after failure of classical treatment is feasible and promising. Further evaluation of this combination is required.

Topics: Aged; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Combined Chemotherapy Protocols; Bevacizumab; Camptothecin; Carcinoma; Colorectal Neoplasms; Female; Fluorouracil; Humans; Irinotecan; Male; Middle Aged; Pilot Projects; Sirolimus; Treatment Outcome

Topics: Acute Kidney Injury; Antineoplastic Agents; Carcinoma; Humans; Kidney Neoplasms; Male; Middle Aged; Sirolimus

We examined whether mTOR inhibition by RAD001 (Everolimus) could be therapeutically efficacious in the treatment of bladder cancer. RAD001 markedly inhibited proliferation of nine human urothelial carcinoma cell lines in dose- and sensitivity-dependent manners in vitro. FACS analysis showed that treatment with RAD001 for 48 h induced a cell cycle arrest in the G(0)/G(1) phase in all cell lines, without eliciting apoptosis. Additionally, RAD001 significantly inhibited the phosphorylation of S6 downstream of mTOR and VEGF production in all cell lines. We also found tumor weights from nude mice bearing human KU-7 subcutaneous xenografts treated with RAD001 were significantly reduced as compared to placebo-treated mice. This tumor growth inhibition was associated with significant decrease in cell proliferation rate and angiogenesis without changes in cell death. In conclusion inhibition of mTOR signaling in bladder cancer models demonstrated remarkable antitumor activity both in vitro and in vivo. This is the first study showing that RAD001 could be exploited as a potential therapeutic strategy in bladder cancer.

Topics: Animals; Apoptosis; Carcinoma; Cell Cycle; Cell Separation; Dose-Response Relationship, Drug; Everolimus; Female; Flow Cytometry; Humans; Intracellular Signaling Peptides and Proteins; Mice; Neoplasm Transplantation; Neovascularization, Pathologic; Protein Serine-Threonine Kinases; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Urinary Bladder Neoplasms; Urothelium

Topics: Adult; Carcinoma; Carcinoma, Squamous Cell; Fatal Outcome; Graft Rejection; Humans; Immunosuppressive Agents; Kidney Transplantation; Lymphangitis; Male; Neoplasm Metastasis; Reoperation; Sirolimus; Skin Neoplasms

Sirolimus(SRL, Rapamune(â), rapamycin) is a highly specific inhibitor of mammalian target of rapamycin (mTOR). Sirolimus exerts its biological activity by inhibiting the serine-threonine kinase mammalian target of rapamycin (mTOR), which regulates important cellular processes such as control of cell cycle, cell size, translation initiation and transcription. The ability of sirolimus to inhibit cancer cell proliferation has led to efforts to develop rapamycin and related mTOR inhibitors as anticancer agents. Here, we made mTOR as a controlling target, using a new immunosuppressant named sirolimus, to investigate the inhibitory effects of sirolimus on cell proliferation and the expression of mTOR, Hypoxia-inducible factor-1 alpha(HIF-1alpha) and vascular endothelial growth factor (VEGF) in human pancreatic carcinoma SW1990 cell line. Sirolimus is effective in vivo against pancreatic carcinoma and demonstrates that the effect of sirolimus on the inhibition of tumor cell proliferation is associated with the suppression of the mTOR/HIF-1alpha/vascular endothelial growth factor (VEGF) pathway. Thus, the targeting of mTOR signaling has been exploited as a novel therapy for human cancers.

Topics: Antibiotics, Antineoplastic; Carcinoma; Cell Line, Tumor; Cell Proliferation; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Pancreatic Neoplasms; Protein Kinases; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Vascular Endothelial Growth Factor A

Green tea extract and its major component (-)-epigallocatechin-3-gallate (EGCG) exhibit antiangiogenic activities in various experimental tumor models. A growing body of evidence has established that hypoxia-inducible factor-1alpha (HIF-1alpha) and its downstream target, vascular endothelial growth factor (VEGF), play a critical role in tumor angiogenesis. In this study, we investigated the effect of green tea extract and EGCG on HIF-1alpha and VEGF expression in human cervical carcinoma (HeLa) and hepatoma (HepG2) cells. Our results showed that green tea extract and EGCG significantly inhibited hypoxia- and serum-induced HIF-1alpha protein accumulation in these cancer cells but had no effects on HIF-1alpha mRNA expression. Suppression of HIF-1alpha protein by green tea extract and EGCG also resulted in a drastic decrease in VEGF expression at both mRNA and protein levels. The mechanisms of green tea extract and EGCG inhibition of hypoxia-induced HIF-1alpha protein accumulation seem to involve the blocking of both phosphatidylinositol 3-kinase/Akt and extracellular signal-regulated kinase 1/2 signaling pathways and the enhancing of HIF-1alpha protein degradation through the proteasome system. In addition, green tea extract and EGCG inhibited serum-induced HIF-1alpha protein and VEGF expression by interfering with the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin signaling pathways, which play a crucial role in the protein translational machinery cascade. Functionally, green tea extract and EGCG abolished both chemoattractant- and hypoxia-stimulated HeLa cell migration. Our data suggested that HIF-1alpha/VEGF function as therapeutic target for green tea extract and EGCG in the context of cancer chemoprevention and anticancer therapy.

Topics: Angiogenesis Inhibitors; Anticarcinogenic Agents; Carcinoma; Carcinoma, Hepatocellular; Catechin; Cell Hypoxia; Culture Media; Extracellular Signal-Regulated MAP Kinases; Female; HeLa Cells; Humans; Hypoxia-Inducible Factor 1; Liver Neoplasms; Phosphatidylinositol 3-Kinases; Plant Extracts; RNA, Messenger; Sirolimus; Uterine Cervical Neoplasms; Vascular Endothelial Growth Factor A

Effective development of targeted anticancer agents includes the definition of the optimal biological dose and biomarkers of drug activity. Currently available preclinical models are not optimal to this end. We aimed at generating a model for translational drug development using pancreatic cancer as a prototype. Resected pancreatic cancers from 14 patients were xenografted and expanded in successive groups of nude mice to develop cohorts of tumor-bearing mice suitable for drug therapy in simulated early clinical trials. The xenografted tumors maintain their fundamental genotypic features despite serial passages and recapitulate the genetic heterogeneity of pancreatic cancer. The in vivo platform is useful for integrating drug screening with biomarker discovery. Passages of tumors in successive cohorts of mice do not change their susceptibility to anticancer agents and represent a perpetual live bank, facilitating the application of new technologies that will result in the creation of an integrated stable database of tumor-drug response data and biomarkers.

Topics: Animals; Antineoplastic Agents; Benzamides; Carcinoma; Deoxycytidine; Disease Models, Animal; Female; Gemcitabine; Humans; Injections, Intraperitoneal; Injections, Subcutaneous; Kinetics; Mice; Mice, Nude; Pancreatic Neoplasms; Predictive Value of Tests; Sirolimus; Transplantation, Heterologous; Xenograft Model Antitumor Assays

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

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

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

We used an experimental in vitro model of human ovarian surface epithelium (OSE), the tissue of origin of >90% of ovarian cancers, to more precisely define the contribution of hepatocyte growth factor (HGF) to various OSE phenotypes at different stages of neoplastic progression. Neoplastic transformation of OSE in cultures was achieved by multiple genetic manipulations, resulting in the nontumorigenic line IOSE-29, the tumorigenic IOSE-Ov29, and the tumor-derived, more highly malignant IOSE-Ov29/T4. We demonstrate here that, compared to IOSE-29, IOSE-Ov29 and IOSE-Ov29/T4 exhibited higher levels of the HGF receptor Met and an increasing duration of ERK1/2 activation with malignant progression, in conjunction with other neoplastic properties. HGF activated Met signaling in all lines but elicited different responses: HGF induced cell dispersion (scattering) and collagen gel invasion in IOSE-Ov29 and IOSE-Ov29/T4 but did not alter the growth pattern of IOSE-29. Inhibition with PD98059 and LY294002 independently prevented HGF-induced invasive growth. Furthermore, our results show that HGF-induced invasion can be mediated through a rapamycin-sensitive p70 S6K cascade, which demonstrates that p70S6K can regulate cell motility in addition to its well-established role in protein synthesis. Taken together, our data correlate specific responses to HGF-mediated signaling with specific signaling pathways and with progressive neoplastic changes.

Topics: Carcinoma; Cell Line, Transformed; Cell Line, Tumor; Cell Movement; Cell Transformation, Neoplastic; Enzyme Inhibitors; Epithelial Cells; Female; Hepatocyte Growth Factor; Humans; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Neoplasm Invasiveness; Neoplasm Metastasis; Ovarian Neoplasms; Ovary; Proteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-met; Receptors, Growth Factor; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Sirolimus

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

Molecular markers enabling the prediction of sensitivity/resistance to rapamycin may facilitate further clinical development of rapamycin and its derivatives as anticancer agents. In this study, several human ovarian cancer cell lines (IGROV1, OVCAR-3, A2780, SK-OV-3) were evaluated for susceptibility to rapamycin-mediated growth inhibition. The differential expression profiles of genes coding for proteins known to be involved in the mTOR signaling pathway, cell cycle control and apoptosis were studied before and after drug exposure by RT-PCR. In cells exposed to rapamycin, we observed a dose-dependent downregulation of CCND1 (cyclin D1) and CDK4 gene expression and late G1 cell cycle arrest. Among these cell lines, SK-OV-3 cells resistant to both rapamycin and RAD001 were the sole to show the expression of the anti-apoptotic gene Bcl-2. Bcl-2/bclxL-specific antisense oligonucleotides restored the sensitivity of SK-OV-3 cells to apoptosis induction by rapamycin and RAD001. These results indicate that baseline Bcl-2 expression and therapy-induced downexpression of CCND1 and CDK4 may be regarded as molecular markers enabling the prediction and follow-up of the cellular effects on cell cycle and apoptosis induction of rapamycin in ovarian cancer. Furthermore, strategies to down regulate Bcl-2 in ovarian cancer may prove useful in combination with rapamycin or RAD001 for ovarian cancer.

Topics: Antibiotics, Antineoplastic; Blotting, Western; Carcinoma; Cell Death; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinases; Everolimus; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Inhibitory Concentration 50; Oligonucleotides, Antisense; Ovarian Neoplasms; Polymerase Chain Reaction; Protein Kinase Inhibitors; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; RNA, Messenger; Sirolimus; Transfection

Transforming growth factor beta (TGF-beta) induces cell cycle arrest of most nontransformed epithelial cell lines. In contrast, many human carcinomas are refractory to the growth-inhibitory effect of TGF-beta. TGF-beta overexpression inhibits tumorigenesis, and abolition of TGF-beta signaling accelerates tumorigenesis, suggesting that TGF-beta acts as a tumor suppressor in mouse models of cancer. A screen to identify agents that potentiate TGF-beta-induced growth arrest demonstrated that the potential anticancer agent rapamycin cooperated with TGF-beta to induce growth arrest in multiple cell lines. Rapamycin also augmented the ability of TGF-beta to inhibit the proliferation of E2F1-, c-Myc-, and (V12)H-Ras-transformed cells, even though these cells were insensitive to TGF-beta-mediated growth arrest in the absence of rapamycin. Rapamycin potentiation of TGF-beta-induced growth arrest could not be explained by increases in TGF-beta receptor levels or rapamycin-induced dissociation of FKBP12 from the TGF-beta type I receptor. Significantly, TGF-beta and rapamycin cooperated to induce growth inhibition of human carcinoma cells that are resistant to TGF-beta-induced growth arrest, and arrest correlated with a suppression of Cdk2 kinase activity. Inhibition of Cdk2 activity was associated with increased binding of p21 and p27 to Cdk2 and decreased phosphorylation of Cdk2 on Thr(160). Increased p21 and p27 binding to Cdk2 was accompanied by decreased p130, p107, and E2F4 binding to Cdk2. Together, these results indicate that rapamycin and TGF-beta cooperate to inhibit the proliferation of nontransformed cells and cancer cells by acting in concert to inhibit Cdk2 activity.

Topics: Animals; Antibiotics, Antineoplastic; Carcinoma; CDC2-CDC28 Kinases; Cell Cycle Proteins; Cell Division; Cell Line; Cell Transformation, Neoplastic; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Cyclins; DNA-Binding Proteins; E2F4 Transcription Factor; Enzyme Inhibitors; Epithelial Cells; Genes, Reporter; Growth Inhibitors; Humans; Nuclear Proteins; Phosphoproteins; Protein Binding; Protein Serine-Threonine Kinases; Proteins; Retinoblastoma Protein; Retinoblastoma-Like Protein p107; Retinoblastoma-Like Protein p130; Signal Transduction; Sirolimus; Tacrolimus Binding Proteins; Transcription Factors; Transforming Growth Factor beta; Tumor Suppressor Proteins

The FRAP-p70s6K signaling pathway was found to be constitutively phosphorylated/active in MiaPaCa-2 and Panc-1 human pancreatic cancer cells and a pancreatic cancer tissue sample as judged by the retarded electrophoretic mobility of the two major FRAP downstream targets, p70s6K and 4E-BP1. Treatment of cells with rapamycin, a selective FRAP Inhibitor, inhibited basal p70s6K kinase activity and induced dephosphorylation of p70s6K and 4E-BP1. Moreover, rapamycin inhibited DNA synthesis as well as anchorage-dependent and -independent proliferation in MiaPaCa-2 and Panc-1 cells. Finally, rapamycin strikingly inhibited cyclin D1 expression in pancreatic cancer cells. Thus, inhibitors of the constitutively active FRAP-p70s6K pathway may provide a novel therapeutic approach for pancreatic cancer.

Topics: Calcium-Calmodulin-Dependent Protein Kinases; Carcinoma; Cell Cycle; Cell Cycle Proteins; Culture Media, Serum-Free; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase Inhibitor p27; Humans; Microtubule-Associated Proteins; Mitogen-Activated Protein Kinase 1; Neoplasm Proteins; Pancreatic Neoplasms; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Protein Kinases; Protein Processing, Post-Translational; Ribosomal Protein S6 Kinases; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Tumor Cells, Cultured; Tumor Suppressor Proteins