epidermal-growth-factor and Lung-Neoplasms

Most anticancer drugs often fail in clinical trials due to poor solubility, poor bioavailability, lack of targeted delivery and several off-target effects. Polymeric nanoparticles such as poly(lactide), poly(lactic-. Globally, 2 million people are dying annually due to lung cancer and it is the leading cause of death among men in 93 countries. Currently, lung cancer medicine does not reach tumor sites and induces several side effects. Therefore, lung cancer medicines are not effectively reducing lung cancer. One of the efficient ways of delivering anticancer drugs to improve targeted delivery is the conjugation of drugs with cancer cell surface-targeting moieties and encapsulation of unique nanocarriers/nanoparticles. Specific nanoencapsulated drugs selectively target EGF receptors, folic acid receptors, transferrin receptors, sigma receptors and urokinase plasminogen activator receptors on the lung cancer cell surface and deliver the anticancer drugs, leading to cancer regression.

Topics: Antineoplastic Agents; Cell Line, Tumor; Drug Carriers; Drug Delivery Systems; Epidermal Growth Factor; Humans; Lung Neoplasms; Nanoparticles; Paclitaxel; Pharmaceutical Preparations

Lung carcinoma (LC) is the third most common cancer diagnosis and accounted for the most cancer-related mortality worldwide in 2018. Based on the type of cells from which it originates, LC is commonly classified into non-small cell lung cancers (NSCLC) and small cell lung cancers (SCLC). NSCLC account for the majority of LC and can be further categories into adenocarcinoma, large cell carcinoma, and squamous cell carcinoma. Accurate classification of LC is critical for its adequate treatment and therapeutic outcome. Since NSCLC express more epidermal growth factor receptor (EGFR) with activation mutations, targeted therapy EGFR-tyrosine kinase inhibitors (TKIs) have been considered as primary option of NSCLC patients with activation EGFR mutation. In this review, we present the genetic alterations, reported mutations in EGFR, and TKIs treatment in NSCLC patients with an emphasis on the downstream signaling pathways in NSCLC progression. Among the signaling pathways identified, mitogen activation protein kinase (MAPK), known also as extracellular signal-regulated protein kinase (Erk) pathway, is the most investigated among the related pathways. EGFR activation leads to the autophosphorylation of its kinase domain and subsequent activation of Ras, phosphorylation of Raf and MEK1/2, and the activation of ERK1/2. Phosphatidylinositol 3-kinase (PI3K)/Akt is another signal pathway that regulates cell cycle and has been linked to NSCLC progression. Currently, three generations of EGFR TKIs have been developed as a first-line treatment of NSCLC patients with EGFR activation and mutation in which these treatment options will be further discussed in this review. The Supplementary Appendix for this article is available at http://links.lww.com/JCMA/A138.

Topics: Carcinoma, Non-Small-Cell Lung; Drug Resistance, Neoplasm; Epidermal Growth Factor; Humans; Lung Neoplasms; Mutation; Phosphatidylinositol 3-Kinases; Protein Kinase Inhibitors; Signal Transduction

Numerous peptides including bombesin (BB), endothelin (ET), neurotensin (NTS) and pituitary adenylate cyclase-activating polypeptide (PACAP) are growth factors for lung cancer cells. The peptides bind to G protein-coupled receptors (GPCRs) resulting in elevated cAMP and/or phosphatidylinositol (PI) turnover. In contrast, growth factors such as epidermal growth factor (EGF) or neuregulin (NRG)-1 bind to receptor tyrosine kinases (RTKs) such as the EGFR or HER3, increasing tyrosine kinase activity, resulting in the phosphorylation of protein substrates such as PI3K or phospholipase (PL)C. Peptide GPCRs can transactivate numerous RTKs, especially members of the EGFR/HER family resulting in increased phosphorylation of ERK, leading to cellular proliferation or increased phosphorylation of AKT, leading to cellular survival. GRCR antagonists and tyrosine kinase inhibitors are useful agents to prevent RTK transactivation and inhibit proliferation of cancer cells.

Topics: Bombesin; Endothelins; Epidermal Growth Factor; ErbB Receptors; Humans; Lung Neoplasms; Neuregulin-1; Neurotensin; Phosphatidylinositol 3-Kinases; Pituitary Adenylate Cyclase-Activating Polypeptide; Receptor, ErbB-3; Receptors, G-Protein-Coupled; Type C Phospholipases

To explore the association between epidermal growth factor (EGF) 61A/G polymorphism and lung cancer.All eligible case-control studies published up to August, 2019 were identified by searching PubMed, The excerpta medica database, China Academic Journals Full-text Database, China Biology Medicine, China National Knowledge Infrastructure, China Science and Technology Journal Database, and Wanfang databases. Two researchers independently identified the literature, extracted data, and evaluated quality according to inclusion and exclusion criteria. Meta-analysis was performed by Stata 15.0.A total of 6 studies is included, including 1487 cases and 2044 control subjects. Compared with allele A, allele G was considered to have no association with the risk of lung cancer, odds ratio = 1.07 (95% confidence interval: 0.98-1.15). GG recessive genotype, GG + GA dominant genotype, GG homozygote genotype and GA heterozygote genotype were found out that all of them are not associated with the risk of lung cancer. No association between EGF 61A/G polymorphism and lung cancer was found out by ethnical subgroup analysis. However, in view of the limitations of this study, such as the results of quantitative and sensitivity analysis may be lack of accuracy, so the conclusions of allele model and recessive gene model should be made carefully.It suggested that there was no association between polymorphism of EGF 61A/G and susceptibility of lung cancer.

Topics: Alleles; Epidermal Growth Factor; Genetic Predisposition to Disease; Genotype; Humans; Lung Neoplasms; Observational Studies as Topic; Polymorphism, Single Nucleotide; Risk Factors

The discovery of oncogenic driver gene mutations, including epidermal growth factor receptor (EGFR) mutation, anaplastic lymphoma kinase (ALK) fusion, ROS proto-oncogene 1 (ROS1) fusion, and ret proto-oncogene (RET) fusion, has led to the development of molecularly targeted therapy for non-small-cell lung cancer (NSCLC). This therapy has changed the standard of care for NSCLC. Despite the dramatic response to molecularly targeted therapy, almost all patients ultimately develop resistance to the drugs. To understand the mechanisms of resistance to molecularly targeted agents, it is essential to understand the molecular pathways of NSCLC. Here, we review the mechanisms of resistance to molecularly targeted therapy and discuss strategies to overcome drug resistance.

Topics: Anaplastic Lymphoma Kinase; Carcinoma, Non-Small-Cell Lung; Epidermal Growth Factor; Gene Fusion; Humans; Lung Neoplasms; Molecular Targeted Therapy; Mutation; Protein-Tyrosine Kinases; Proto-Oncogene Mas; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-ret

Bronchoscopy is one of the main techniques used for sampling lung tumor biopsies. In recent years, a large number of tumor specimens have been required to determine the best chemotherapy regimen for each patient; this personalized approach is known as precision medicine. In this review, radial endobronchial ultrasound; bronchoscopic navigation systems, including virtual bronchoscopic navigation and electromagnetic navigation; ultrathin bronchoscope,; and endobronchial ultrasound-guided transbronchial needle aspiration are highlighted as techniques used to increase the diagnostic yield. Personalized therapy includes tests for analysis of epidermal growth factor mutations, anaplastic lymphoma kinase or ROS proto-oncogene 1 fusion gene, and programmed death ligand 1 expression. In cryobiopsy, a relatively large amount of tissue is collected from endobronchial lung cancer and peripheral pulmonary lesions, and it is a promising technique for analyzing these tissues using molecular tests.

Topics: Anaplastic Lymphoma Kinase; Biomarkers, Tumor; Biopsy, Fine-Needle; Bronchoscopy; Epidermal Growth Factor; Gene Expression; Humans; Image-Guided Biopsy; Lung Neoplasms; Mutation; Precision Medicine; Proto-Oncogene Mas; Ultrasonography

Epidermal growth factor (EGFR) tyrosine kinase inhibitors (TKIs), such as gefitinib and erlotinib, show excellent clinical efficacy for patients with non-small cell lung cancer (NSCLC) with EGFR mutations, including Exon 19 deletion and single-point substitution, and L858R of exon 21. The reason for the reduction in effectiveness of these EGFR TKIs is the T790M gatekeeper mutation in the ATP-binding pocket of Exon 20, which increases the affinity of EGFR for ATP. Newer EGFR TKIs, such as afatinib, osimertinib, rociletinib, EGF816 and ASP8273, selectively target T790M mutants, sparing wild-type EGFR. EGFR TKIs have fewer adverse effects than chemotherapy and also improve progression-free survival. Combination therapy of EGFR TKIs with anti-EGFR antibodies is recommended for overcoming the problem of resistance to some extent. This review could help medicinal chemists to design novel EGFR TKIs against NSCLC.

Topics: Animals; Carcinoma, Non-Small-Cell Lung; Disease-Free Survival; Drug Resistance, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Humans; Lung Neoplasms; Protein Kinase Inhibitors

The past few years have witnessed rapid advances in the immunotherapies for non-small-cell lung cancer (NSCLC). CIMAvax-EGF is a therapeutic vaccine against lung cancer independently developed by Cuba. It can exert its anti-tumor effect by forming epidermal growth factor (EGF) antibodies to block the binding of EGF to EGF receptor. So far stage both phases Ⅱ and Ⅲ trials have proved its effectiveness and long-term safety,and phases Ⅲ and Ⅳ trials are underway. A deeper understanding of the role of CIMAvax-EGF in NSCLC will accelerate the application of immunotherapy. This article summarizes the recent advances of CIMAvax-EGF R&D and its application in treating NSCLC.

Topics: Antibodies; Cancer Vaccines; Carcinoma, Non-Small-Cell Lung; Epidermal Growth Factor; ErbB Receptors; Humans; Lung Neoplasms

Lung cancer remains one of the leading causes of cancer-related deaths. Non-small cell lung cancer (NSCLC) is the most common histologic type of lung cancer. Medical and scientific progress has led to longer survival in an increasing number of patients suffering from cancer. Concerning patients with advanced NSCLC, there is a subgroup with long-term survival. The human epidermal growth factor receptor (EGFR) family plays a key role in tumor development. This cluster of genes is associated with augmented angiogenesis and enhanced proliferation, survival, and migration of tumor cells. The CIMAvax-EGF vaccine consists of a chemical conjugate of the EGF with the P64 protein derived from the Meningitis B bacteria and the Montanide ISA 51, as adjuvant. The vaccine induces antibodies against EGF that results in EGF withdrawal. CIMAvax-EGF has been demonstrated to be safe and immunogenic in advanced NSCLC patients. Here we summarize the current knowledge of the mechanism of action of CIMAvax-EGF, highlighting the impact of this anti-EGF-based vaccine on the long-term survival of advanced NSCLC patients.

Topics: Cancer Vaccines; Carcinoma, Non-Small-Cell Lung; Epidermal Growth Factor; ErbB Receptors; Humans; Immunotherapy, Active; Lung Neoplasms; Models, Immunological; Survival Analysis

The EGFR is one of the most popular targets for anticancer therapies and many drugs, such as erlotinib and gefitinib, have got enormous success in clinical treatments of cancer in past decade. However, the efficacy of these agents is often limited because of the quick emergence of drug resistance. Fundamental structure researches of EGFR in recent years have generally elucidated the mechanism of drug resistance. In this review, based on systematic resolution of full structures of EGFR and their variants via single crystal x-ray crystallography, the working and drug resistance mechanism of EGFR-targeted drugs are fully illustrated. Moreover, new strategies for avoiding EGFR drug resistance in cancer treatments are also discussed.

Topics: Antineoplastic Agents; Crystallography, X-Ray; Drug Resistance, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Humans; Lung Neoplasms; Mutation; Protein Domains; Protein Kinase Inhibitors; Signal Transduction

Epidermal growth factor receptor (EGFR) is overexpressed in many epithelial tumors and its role in the development of non-small-cell lung cancer (NSCLC) is widely documented. CIMAvax-EGF is a therapeutic cancer vaccine composed by recombinant EGF conjugated to a carrier protein and emulsified in Montanide ISA51. Vaccination induces antibodies against self-EGF that block EGF-EGFR interaction and inhibit EGFR phosphorylation. Five clinical trials were conducted to optimize vaccine formulation and schedule. Then, two randomized studies were completed in advanced NSCLC, where CIMAvax-EGF was administered after chemotherapy, as 'switch maintenance'. The vaccine was very well tolerated and the most frequent adverse events consisted of grade 1/2 injection site reactions, fever, headache, vomiting and chills. CIMAvax was immunogenic and EGF concentration was reduced after vaccination. Subjects receiving a minimum of 4 vaccine doses had a significant survival advantage. NSCLC patients with high EGF concentration at baseline had the largest benefit, comparable with best maintenance therapies.

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Cancer Vaccines; Carcinoma, Non-Small-Cell Lung; Cetuximab; Epidermal Growth Factor; ErbB Receptors; Humans; Immunotherapy, Active; Lung Neoplasms; Phosphorylation; Vaccination

Modulation of a patient's immune system so that it acts against lung cancer cells has not been successful in the past decades. Advances in our understanding of the immune response to tumors resulted in the development of different kinds of novel immunotherapeutic agents. This has resulted in the development of two major approaches. First, antigen-specific immunotherapy or cancer vaccination, with the MAGE-A3 vaccine in resected early-stage non-small-cell lung cancer (NSCLC), the L-BLP25 vaccine in locally advanced NSCLC after chemoradiotherapy and belagenpumatucel-L and the TG4010 vaccine in advanced-stage NSCLC. Second, non-antigen-specific immunotherapy or cancer immunomodulation is reviewed, including how monoclonal antibodies modulate the interaction between antigen-presenting cells, T-lymphocytes and tumor cells (e.g., antibodies against CTLA-4, or against PD-1 receptor or its ligands). Recent Phase II trials with these treatments have shown promising results of efficacy and tolerability, which has led to testing in several large Phase III trials. Some of these are fully recruited, while others are still ongoing, and important results are be expected in the near future.

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Murine-Derived; Antigens, Neoplasm; Cancer Vaccines; Clinical Trials as Topic; CTLA-4 Antigen; Epidermal Growth Factor; Humans; Immunotherapy; Ipilimumab; Lung Neoplasms; Membrane Glycoproteins; Neoplasm Proteins; Nivolumab

Traditional anti-cancer therapies (surgery, radiotherapy and chemotherapy) have limited effectiveness in curbing progression of advanced tumors. However, with advances in immunology and molecular biology in the last two decades, the prognosis of cancer immunotherapy has improved. An emerging therapy is the cancer vaccine as adjunctive therapy. The purpose of this paper is to review this therapeutic modality for non-small cell lung cancer.

Topics: Antigens, Neoplasm; Bacterial Vaccines; Cancer Vaccines; Carcinoma, Non-Small-Cell Lung; Clinical Trials as Topic; CTLA-4 Antigen; Dendritic Cells; Epidermal Growth Factor; Humans; Immunotherapy; Lung Neoplasms; Mucin-1; Toll-Like Receptors

The epidermal growth factor receptor (EGFR) was among the first receptor tyrosine kinases (RTKs) for which ligand binding was studied and for which the importance of ligand-induced dimerization was established. As a result, EGFR and its relatives have frequently been termed "prototypical" RTKs. Many years of mechanistic studies, however, have revealed that--far from being prototypical--the EGFR family is quite unique. As we discuss in this review, the EGFR family uses a distinctive "receptor-mediated" dimerization mechanism, with ligand binding inducing a dramatic conformational change that exposes a dimerization arm. Intracellular kinase domain regulation in this family is also unique, being driven by allosteric changes induced by asymmetric dimer formation rather than the more typical activation-loop phosphorylation. EGFR family members also distinguish themselves from other RTKs in having an intracellular juxtamembrane (JM) domain that activates (rather than autoinhibits) the receptor and a very large carboxy-terminal tail that contains autophosphorylation sites and serves an autoregulatory function. We discuss recent advances in mechanistic aspects of all of these components of EGFR family members, attempting to integrate them into a view of how RTKs in this important class are regulated at the cell surface.

Topics: Dimerization; Enzyme Activation; Epidermal Growth Factor; Humans; Ligands; Lung Neoplasms; Models, Biological; Models, Molecular; Phosphorylation; Protein Structure, Tertiary

Non-small-cell lung cancer usually carries a dismal prognosis. Novel treatment approaches are clearly warranted. Immunotherapy has emerged as a promising area of research developing agents that manipulate the immune system to induce antitumor responses while avoiding major toxicity. New vaccines and checkpoint inhibitors are currently undergoing investigation in phase II and phase III clinical trials. In advanced non-small-cell lung cancer (NSCLC), belagenpumatucel-L, an allogeneic cell vaccine directed against transforming growth factor β in the tumor microenvironment, knocks down the immune suppression caused by the tumor and has demonstrated a dose- and time-dependent efficacy in some subgroups of patients. L-BLP25 and TG4010 are both antigenic vaccines that target mucin 1, whose encoding proto-oncogene is commonly mutated in solid tumors. The L-BLP25 vaccine achieved a significant improvement in overall survival in the subgroup of patients with stage IIIB NSCLC treated with chemoradiotherapy. TG4010 vaccination resulted in better progression-free survival when added to cisplatin-gemcitabine chemotherapy. These results are being addressed in the currently ongoing phase III TIME trial. In the adjuvant setting, MAGE-A3, an antigen-based vaccine, showed promising results in melanoma-associated antigen A3 positive lung cancer patients who underwent resection in the phase II study; however, no improvement in progression-free survival was observed in the phase III MAGRIT study. CIMAVax is a recombinant human epidermal growth factor (EGF) vaccine that induces anti-EGF antibody production and prevents EGF from binding to its receptor. It has improved overall survival in patients with advanced NSCLC who achieve seroconversion. Ipilimumab, an immune checkpoint inhibitor that targets cytotoxic T-lymphocyte antigen 4, demonstrated improved progression-free survival in advanced NSCLC patients who received the drug after chemotherapy in a phased regimen. Finally, anti-programmed death receptor 1 agents have achieved durable response rates in phase I studies. This review gives an overview of the current data and the most promissory immunotherapeutic agents for NSCLC.

Topics: Antibodies, Monoclonal; Antigens, Neoplasm; Cancer Vaccines; Carcinoma, Non-Small-Cell Lung; Clinical Trials as Topic; Epidermal Growth Factor; Humans; Immunologic Factors; Immunotherapy, Active; Lung Neoplasms; Proto-Oncogene Mas

Non-small-cell lung cancer (NSCLC) subtypes are driven by specific genetic aberrations. For reasons such as this, there is a call for treatment personalization. The ability to instigate NSCLC fragmentation poses new methodological problems, and new 'driver' molecular aberrations are being discovered at an unprecedented pace.. This article describes the clinical development of epidermal growth factor-tyrosine kinase inhibitors (EGFR-TKIs) and crizotinib for EGFR-mutant and anaplastic lymphoma kinase (ALK)-rearranged NSCLC. Further, the authors briefly describe the emerging molecular targets in NSCLC, in terms of both rationale for therapeutic targeting and strategies, for clinical development.. Target identification and validation in NSCLC still requires considerable effort, as not all of the molecular alterations are clear 'drivers' nor can they be efficiently targeted with available drugs. However, 50% of the NSCLC cases are without clear-defined molecular aberrations. Clinical trial methodology will need to develop novel paradigms for targeted drug development, aiming at the validation of an ideal 'biology-to-trial' approach. Despite significant challenges, a truly 'personalized' approach to NSCLC therapy appears to be within our reach.

Topics: Anaplastic Lymphoma Kinase; Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Drug Design; Drug Discovery; Epidermal Growth Factor; Humans; Lung Neoplasms; Molecular Targeted Therapy; Precision Medicine; Receptor Protein-Tyrosine Kinases; Signal Transduction

Lung cancer is the most common cause of cancer-related death in the United States, yet traditional chemotherapy fails to provide long-term benefit for many patients. New approaches are needed to improve overall survival beyond the current standard of care.. This review discusses recent clinical trials using immunotherapy techniques to treat both non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) and highlights ongoing immunotherapy research efforts at our center.. For NSCLC, phase II clinical trials have examined allogeneic vaccines that target either mucin 1 (MUC1), epidermal growth factor or melanoma-associated antigen 3. These vaccines are now undergoing larger phase III trials. An autologous cellular therapy directed against transforming growth factor beta-2 and a recombinant protein with antitumor properties have also shown promise in prolonging survival in NSCLC in phase II trials. The monoclonal antibodies ipilimumab, BMS-936558 (anti-PD-1), and BMS936559 (anti-PD-L1) lead to enhanced T-cell-mediated antitumor effects and have produced objective responses in early-phase clinical trials. Studies for SCLC also exist, such as a novel vaccine therapy targeting p53.. Recent clinical trials in lung cancer demonstrate the potential of immunotherapeutics to increase overall survival in patients with lung cancer compared with the current standard of care.

Topics: Antibodies, Monoclonal; Antigens, Neoplasm; Cancer Vaccines; Carcinoma, Non-Small-Cell Lung; Clinical Trials as Topic; Epidermal Growth Factor; Humans; Immunotherapy; Ipilimumab; Lung Neoplasms; Mucin-1; Neoplasm Proteins; Nivolumab; Small Cell Lung Carcinoma; Transforming Growth Factor beta2

Tumor inflammatory microenvironment is considered to play the role in the sensitivity of tumor cells to therapies and prognosis of lung cancer patients. Interleukin-8 (IL-8) is one of critical chemo-attractants responsible for leukocyte recruitment, cancer proliferation, and angiogenesis. The present study aimed at investigating potential mechanism of IL-8 production from human non-small cell lung cancer (NSCLC) SPC-A1 cells. We initially found that EGF could directly stimulate IL-8 production, proliferation, and bio-behaviors of lung cancer cells through the activation of EGFR, PI3K, Akt, and Erk signal pathway. EGF-stimulated IL-8 production, phosphorylation of Akt and Erk, and cell proliferation and movement could be inhibited by EGFR inhibitor (Erlotinib), PI3K inhibitor (GDC-0941 BEZ-235 and SHBM1009), and ERK1/2 inhibitor (PD98059). Our data indicate that IL-8 production from lung cancer cells could be initiated by their own produced factors, leading to the recruitment of inflammatory cells in the cancer tissue, and the formation of inflammatory microenvironment. Thus, it seems that the signal pathway of EGFR-PI3K-Akt-Erk can be the potential target of therapies for inflammatory microenvironment in lung cancer.

Topics: Animals; Carcinoma, Non-Small-Cell Lung; Epidermal Growth Factor; ErbB Receptors; Humans; Interleukin-8; Lung Neoplasms; MAP Kinase Signaling System; Phosphatidylinositol 3-Kinases; Signal Transduction; Tumor Microenvironment

Brain metastases (BM) are a common occurrence in patients with non-small cell lung cancer (NSCLC). Standard therapy options include whole brain radiotherapy and, in selected patients, surgery or stereotactic radiosurgery. The role of systemic treatment is controversial. There is a strong clinical rationale for the use of targeted therapies, because patients often have a poor performance status, and are not candidates for cytotoxic chemotherapy or radiotherapy, yet treatment is required to improve the extra-cranial disease. The efficacy of epidermal growth factor receptor (EGFR) inhibitors in the treatment of patients with BM from NSCLC has been reported mainly in case reports or small retrospective case series, with only a few prospective trials. Current evidence suggests that the use of EGFR tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib should be considered in patients with asymptomatic CNS involvement, when clinical characteristics suggest a high likelihood of response; these characteristics are adenocarcinoma histology, never-smoker status, female gender and East Asian ethnicity. Upfront therapy with EGFR TKIs should be strongly considered in asymptomatic patients harboring activating EGFR mutations. In symptomatic BM, radiotherapy (RT) remains the standard treatment. Based on currently available data, treatment with concurrent RT and EGFR TKIs should be investigated in experimental trials only.

Topics: Animals; Carcinoma, Non-Small-Cell Lung; Central Nervous System Neoplasms; Clinical Trials as Topic; Epidermal Growth Factor; ErbB Receptors; Humans; Lung Neoplasms; Protein Kinase Inhibitors; Treatment Outcome

Cetuximab is a chimeric human-mouse anti-EGF receptor monoclonal antibody. In Phase I studies, no dose-limiting toxicities were observed with cetuximab as a single agent or combined with chemotherapy; pharmacokinetic and pharmacodynamic analyses supported 250 mg/m(2) weekly administration. Skin toxicity, diarrhea and fatigue were the most common toxicities. The positive results obtained in Phase II trials in patients with advanced non-small-cell lung cancer prompted two randomized Phase III trials evaluating cetuximab in addition to first-line chemotherapy. Both trials showed a small benefit in overall survival for the experimental treatment, which was considered insufficient by the EMA for marketing approval. However, a subgroup analysis of the FLEX Phase III trial recently demonstrated a larger survival benefit from the experimental treatment in patients with high immunohistochemical EGF receptor expression. This finding, if confirmed prospectively, could represent a new opportunity for positioning cetuximab into the standard treatment of advanced non-small-cell lung carcinoma.

Topics: Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Cetuximab; Clinical Trials as Topic; Epidermal Growth Factor; ErbB Receptors; Humans; Lung Neoplasms

After many years of uncertainty regarding the role of immunotherapy in cancer, we finally have vaccines approved for the treatment of some malignancies (e.g., prostate cancer and melanoma). In non-small-cell lung cancer, several vaccines are being studied in randomized Phase III clinical trials due to their promising results seen in the clinic, such as BLP-25 and melanoma-associated antigen A3. Traditionally, non-small-cell lung cancer has not been considered a good target for immunotherapy due to lack of immunogenicity and the strong presence of regulatory T cells, which do not allow an adequate immune response in the host. EGF vaccination is a novel area of immunotherapy for this disease. Thus far, there has been success in generating immune and clinical responses with this vaccine in several clinical trials, and we will review in depth the efficacy and toxicity of this novel agent.

Topics: Cancer Vaccines; Carcinoma, Non-Small-Cell Lung; Clinical Trials as Topic; Epidermal Growth Factor; Humans; Lung Neoplasms

Therapeutic vaccines continue to be one of the most active fields in cancer research. However, despite clear evidence of antitumor effect in laboratory animals, and despite the ability of current vaccine candidates to elicit tumor specific antibodies and T-cells in humans, objective responses in the clinical trials are rare. The role of therapeutic vaccines in advanced cancer patients, if any, would be to decrease the rate of disease progression and to increase survival and quality of life. Due to the redundant regulatory loops contracting the immune response to antigens that cannot be eliminated, such a role would require chronic vaccination, which is at first sight at odds with the classic experience of vaccinology. During the last decade our team has been developing a therapeutic vaccine for advanced lung cancer, which consists in human recombinant Epidermal Growth Factor (EGF) chemically conjugated to a carrier protein from Neisseria meningitides. Several clinical trials have been carried out, showing increase in anti-EGF antibody titters, decrease in plasma EGF concentration and survival advantage in vaccinated patients. In the present paper we review data from 58 patients who were vaccinated monthly for more than one or two years. Long term vaccination was feasible and safe, and there was no evidence of cumulative toxicity. Patients kept high anti-EGF antibody titters during all the time of vaccination, without evidence of immune response exhaustion. Continued vaccination increased the probability to get a high antibody response, which has been previously shown to be, in turn, associated with a better survival. Observations done in this series of patients suggest that long term therapeutic vaccination is a feasible strategy, worth to be further explored in the aim of transforming advanced cancer into a chronic disease.

Topics: Animals; Antibodies; Cancer Vaccines; Clinical Trials as Topic; Epidermal Growth Factor; Humans; Lung Neoplasms; Vaccination

Non-small cell lung cancer (NSCLC) is a poor-prognosis malignancy for which more effective treatments are needed, with accumulating clinical experiences supporting benefits of receptor tyrosine kinase inhibitors for patients with tumors harboring an epidermal growth factor receptor (EGFR) mutation or anaplastic lymphoma kinase ( ALK ) rearrangement.. To review completed and ongoing clinical trials of EGFR tyrosine kinase inhibitors for EGFR mutation-positive NSCLC and an ALK inhibitor for those with ALK rearrangement, while also exploring practical issues surrounding the implementation of molecular testing as a routine component of the diagnostic workup of NSCLC in the United States.. Published biomedical literature, abstracts presented at recent major oncology meetings, and ClinicalTrials.gov.. Continually evolving evidence indicates the possible efficacy of molecularly targeted agents for the treatment of advanced NSCLC, especially adenocarcinoma. To identify patients who will most likely benefit from the targeted therapy, routine determination of the corresponding genetic alterations after histologic diagnosis of NSCLC (reflex molecular testing for EGFR mutations and ALK rearrangement) should be considered.

Topics: Anaplastic Lymphoma Kinase; Carcinoma, Non-Small-Cell Lung; Clinical Trials as Topic; Clinical Trials, Phase III as Topic; Epidermal Growth Factor; ErbB Receptors; Humans; In Situ Hybridization, Fluorescence; Lung Neoplasms; Molecular Targeted Therapy; Mutation; Prognosis; Protein Kinase Inhibitors; Receptor Protein-Tyrosine Kinases

The trend of increased survival in advanced tumors suggests the possibility of the transformation of cancer into a chronic disease. That goal will require therapeutic weapons with low toxicity that can be used chronically. Here we summarize the development of a therapeutic vaccine consisting in recombinant EGF chemically linked to a protein from Neisseria meningitides. In mice, the vaccine elicited antibodies to self-EGF and had anti-tumor activity. Clinical trials have shown that the vaccine is also immunogenic and well tolerated in humans. The vaccination produced a decrease in plasma EGF concentration. Advanced lung cancer patients eliciting high antibody titers of EGF had better survival. The vaccine can be used long term and integrated with other treatment modalities.

Topics: Animals; Autoantibodies; Bacterial Outer Membrane Proteins; Cancer Vaccines; Chronic Disease; Epidermal Growth Factor; Humans; Lung Neoplasms; Neisseria meningitidis; Treatment Outcome; Vaccines, Synthetic

Lung cancer is the most common cause of cancer-related death in the United States; however, recent clinical advances may change this outcome. New data on low-dose computed tomography for lung cancer screening, and technologic advances in surgery and radiation, have improved outcomes for those with early-stage disease. Identification of driver mutations in lung cancer has led to the development of molecular targeted therapy to improve survival of subsets of patients with metastatic disease. These advances now allow for treatment of many patients with lung cancer with comorbidities or poor performance status who would have had limited options in the past.

Topics: Biopsy, Needle; Bronchoscopy; Carcinoma, Non-Small-Cell Lung; Comorbidity; Epidermal Growth Factor; Global Health; Humans; Lung Neoplasms; Mutation; Neoplasm Staging; Occupational Exposure; Tomography, Spiral Computed; United States

CIMAvax EGF is a therapeutic anticancer vaccine developed entirely in Cuba and licensed in Cuba for use in adult patients with stage IIIB/IV non-small-cell lung cancer (NSCLC). The vaccine is based on active immunotherapy by which an individual's immune response is manipulated to release its own effector antibodies (Abs) against the epidermal growth factor (EGF).. Review pre-clinical and clinical research conducted during development of CIMAvax EGF, primarily studies published by Cuban investigators in international peer-reviewed scientific journals. Methods An automated search for "vaccine" and "EGF" was conducted in PubMed, resulting in 17 articles published by Cuban authors between January 1, 1994 and September 30, 2009. Main findings were described and discussed, along with unpublished preliminary findings of an initial ongoing phase III clinical trial.. Articles reviewed describe five phase I/II and one phase II clinical trials conducted in Cuba in 1995-2005. A non-controlled 1995-1996 study resulted in the earliest published scientific evidence of the feasibility of inducing an immune response against autologous EGF in patients with different advanced stage tumors. Subsequent controlled, randomized trials included patients with advanced stage (IIIB/IV) NSCLC. The 2 and 3rd phase I/II trials differentiated immunized patients as poor antibody responders (PAR) and good antibody responders (GAR), according to their anti-EGF antibody response, and confirmed greater immunogenicity with Montanide ISA 51 adjuvant in the vaccine formulation, as well as the benefits of low-dose cyclophosphamide treatment 72 hours before the first immunization. The 4th phase I/II trial found increased immunogenicity with an increased dose divided in 2 anatomical sites and also established correlation between Ab titers, serum EGF concentration and length of survival. In the first 4 phase I/II trials and the phase II trial, vaccine was administered after chemotherapy (ChTVV schedule). In the 5th phase I/III trial, longer survival and increased immunogenicity were achieved using a VChTV schedule and dividing the vaccine dose in 4 anatomical sites. The phase II clinical trial confirmed results of earlier studies as well as the mild-to-moderate adverse event profile associated with CIMAvax EGF Longer survival was observed in all vaccinated patients compared to controls, and the difference was significant (p < 0.05) in the group aged <60 years.. CIMAvax EGF's benefits in earlier NSCLC stages and in other tumor locations, as well as in patients unfit for chemotherapy, need to be evaluated. Evidence of the vaccine's safety for chronic use also needs to be systemized.. Introducción CIMAvax EGF es una vacuna terapéutica contra el cáncer enteramente desarrollada en Cuba y licenciada en el país para su uso en pacientes adultos con cáncer de pulmón de células no pequeñas (CPCNP) en etapas IIIB/IV. La vacuna se basa en la inmunoterapia activa; o sea, manipula la respuesta inmune de un individuo para que genere sus propios anticuerpos efectores (Acs) contra el factor de crecimiento epidérmico (EGF). Objetivo Revisar los estudios clínicos y preclínicos realizados durante el desarrollo de CIMAvax EGF, principalmente los publicados por investigadores cubanos en revistas científicas internacionales arbitradas. Métodos Se efectuó una búsqueda automatizada en PubMed con el uso de las palabras claves “vacuna” y “EGF”, que dio como resultado 17 artículos publicados por autores cubanos entre el 1 de enero de 1994 y el 30 de septiembre del 2009. Se describieron y discutieron los principales resultados, junto con los hallazgos preliminares no publicados aún de un ensayo clínico inicial fase III en ejecución. Resultados Los artículos revisados describen cinco ensayos clínicos fase I/II y uno fase II realizados en Cuba de 1995–2005. Un estudio no controlado de 1995–1996 fue la primera evidencia científicas de la factibilidad de inducir una respuesta inmune contra el EGF autólogo en pacientes con diferentes tumores en etapas avanzadas. Ensayos posteriores controlados y aleatorizados incluyeron pacientes con CPCNP en etapas avanzadas (IIIB/IV). En los ensayos segundo y tercero de fase I/ II, los pacientes inmunizados se diferenciaron en buenos respondedores (BR) y malos respondedores (MR) según las respuestas de anticuerpos contra el EGF y se conformó mayor inmunogenicidad al utilizar el adyuvante Montanide ISA 51 en la formulación vacunal, así como los beneficios del tratamiento de ciclofosfamida a baja dosis 72 horas antes de la primera inmunización. En el cuarto ensayo fase I/II se encontró un aumento de la inmunogenicidad con el aumento de la dosis, dividida en dos sitios anatómicos, y además se estableció la correlación entre los títulos de Acs, la concentración de EGF sérico y la supervivencia. En los primeros cuatro ensayos fase I/II, la vacuna se administró después de la quimioterapia (esquema QVV). En el quinto ensayo fase I/II, se lograron mayor supervivencia e inmunogenicidad utilizando un esquema VQV y dividiendo la dosis vacunal en cuatro sitios anatómicos. El ensayo clínico de fase II conforrmó los resultados de los estudios a

Topics: Adult; Cancer Vaccines; Carcinoma, Non-Small-Cell Lung; Clinical Trials, Phase I as Topic; Clinical Trials, Phase II as Topic; Epidermal Growth Factor; Humans; Immunotherapy, Active; Lung Neoplasms; Randomized Controlled Trials as Topic; Survival Analysis

Lung cancer is the leading cause of cancer death worldwide. Survival remains poor as approximately 80% of cases present with advanced stage disease. However, new treatments are emerging which offer hope to patients with advanced disease. Insights into cell biology have identified numerous intracellular and extracellular peptides that are pivotal in cancer cell signalling. Disrupting the function of these peptides inhibits intracellular signal transduction and diminishes uncontrolled proliferation, resistance to apoptosis and tumour angiogenesis. The most widely studied signalling pathway is the Epidermal Growth Factor (EGF) pathway. EGF signalling can be disrupted at numerous points. Blockade of the cell surface receptor is achieved by the monoclonal antibody cetuximab; intracellular tyrosine kinase activity is inhibited by erlotinib. Vascular Endothelial Growth Factor (VEGF) regulates another pathway important for tumour growth. Inhibition of VEGF impairs angiogenesis and disrupts metastatic spread. Bevacizumab is a monoclonal antibody that binds to VEGF and blocks interaction with its cell surface receptor. Clinical trials have demonstrated that disruption of these signalling pathways can improve survival in advanced lung cancer. New compounds including folate antimetabolites such as pemetrexed, proteasome inhibitors such as bortezomib, modified glutathione analogues such as TLK286, and other agents such as epothilones and other small molecules are currently being evaluated in patients with lung cancer. As more and more signalling peptides are targeted for manipulation, it is hoped that a new era is dawning in the treatment of advanced stage lung cancer. This review will focus on emerging new therapies in the management of lung cancer.

Topics: Antineoplastic Agents; Epidermal Growth Factor; Humans; Lung Neoplasms; Molecular Structure; Signal Transduction; Vascular Endothelial Growth Factor A

To summarize the available knowledge about nonsmall cell lung cancer (NSCLC) in never smokers in terms of biological and clinical-pathological findings.. Overall in newly diagnosed NSCLC, 10% of men and 20% of women, with a much higher proportion among Asiatic women, are never smokers and among them an overwhelming proportion have adenocarcinoma. Several environmental, genetic, hormonal and viral factors have been associated with an increased risk of NSCLC in never smokers, but for none of them there is definitive evidence. The incidence of epidermal growth factor receptor mutations is higher in never smokers, whereas K-ras mutations are rarely detected in this group of never smoking patients. The role of never smoking status in NSCLC as a positive prognostic factor or predictive of a better chemosensitivity to systemic treatments is still undefined.. Epidemiological, molecular and clinical-pathological features indicate NSCLC in never smokers as a distinct entity. Future preclinical studies should address more deeply the biological differences between NSCLC in smokers and never smokers and, to avoid biased results due to differences in survival outcomes, smoking status should be considered among stratification factors in future clinical studies.

Topics: Adenocarcinoma; Asian People; Carcinoma, Non-Small-Cell Lung; Epidermal Growth Factor; Female; Humans; Lung Neoplasms; Male; Risk Factors; Smoking

Molecularly targeted therapies have recently expanded the options available for patients with advanced non-small-cell lung cancer (NSCLC). Two cancer cell pathways in particular have been exploited, the epidermal growth factor receptor (EGFR) and the vascular endothelial growth factor (VEGF) pathway. The former has emerged as a key regulator of cancer cell proliferation and invasion, and several EGFR inhibitors have been developed. Erlotinib, a small-molecule inhibitor of the EGFR intracellular tyrosine kinase, has been found to improve survival compared with placebo in previously treated patients with advanced NSCLC and is Food and Drug Administration (FDA)-approved in this setting. Clinical and molecular predictors of response to erlotinib, such as a history of never smoking and EGFR gene mutation or amplification, are presently being evaluated to select patients for earlier therapy with erlotinib. Additional EGFR inhibitors are also being examined in randomized trials. The VEGF pathway, a key mediator of angiogenesis, has become an attractive target in multiple malignancies, including lung cancer. Bevacizumab, a monoclonal antibody to VEGF, received FDA approval for use in advanced non-squamous-cell NSCLC in 2006 after a phase III trial reported a significant survival advantage when bevacizumab was added to standard first-line chemotherapy. Small-molecule inhibitors of the VEGF receptor tyrosine kinase, such as sunitinib and sorafenib, have also shown promise in phase II trials and are being further investigated in phase III studies. Because preclinical data suggest a synergistic effect when VEGF and EGFR inhibitors are combined, the concurrent use of erlotinib and bevacizumab has additionally been evaluated in a phase II trial, with encouraging early results suggesting at least equivalent activity to standard salvage chemotherapy, with less toxicity. Several other novel agents are being examined, including inhibitors of histone deacteylases and the 26S proteosome. Research efforts are currently focusing on tailoring such therapies according to predictive clinical and molecular markers.

Topics: Angiogenesis Inhibitors; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Benzenesulfonates; Bevacizumab; Carcinoma, Non-Small-Cell Lung; Drug Delivery Systems; Epidermal Growth Factor; Erlotinib Hydrochloride; Humans; Indoles; Lung Neoplasms; Niacinamide; Phenylurea Compounds; Piperidines; Protein Kinase Inhibitors; Pyridines; Pyrroles; Quinazolines; Signal Transduction; Sorafenib; Sunitinib; Treatment Outcome; Vascular Endothelial Growth Factor A

Despite improvements in conventional therapy for non-small cell lung cancer (NSCLC) with surgery, radiotherapy, and cytotoxic chemotherapy, survival remains poor and further improvements are needed. Targeted therapy with biologic agents offers a novel treatment strategy. In the first-line treatment of advanced disease, the most promising results to date have been reported with bevacizumab [an antivascular endothelial growth factor (anti-VEGF) antibody] plus chemotherapy, with significant improvement in survival, compared with chemotherapy alone, in a selected population of patients with nonsquamous histology. Trials incorporating epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors with chemotherapy failed to produce a survival benefit. Nevertheless, cetuximab (an anti-EGFR IgG1 monoclonal antibody) plus chemotherapy has shown promising results in initial studies and continues to be evaluated in larger trials. The benefits of EGFR and VEGF inhibitors in advanced disease have propelled them into evaluation in early stages of NSCLC. Integration of these agents into bi- and tri-modality treatments is currently under investigation in these settings. Finally, emerging data combining EGFR and VEGF inhibitors suggest that multiple pathway inhibition may be more effective than targeting a single pathway.

Topics: Angiogenesis Inhibitors; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Bevacizumab; Carcinoma, Non-Small-Cell Lung; Cetuximab; Epidermal Growth Factor; Humans; Lung Neoplasms; Treatment Outcome; Vascular Endothelial Growth Factor A

Worldwide, approximately 1.3 billion individuals are current smokers, and smoking is the second major cause of death. Currently, lung cancer is the most common type of cancer in Europe, and the third in the U.S. Until now, cytotoxic chemotherapy has had a limited impact on survival in metastatic non-small cell lung cancer (NSCLC). The central role of epidermal growth factor (EGF) and its receptor (EGFR) in lung carcinogenesis is well recognized. Genetic polymorphisms are variants in individual genomes that may be responsible for different functional molecular roles and contribute to variability in drug pharmacokinetic and pharmacodynamic processes. Herein, we review the literature on EGF and EGFR functions and activities, particularly the current role of their functional polymorphisms as related to NSCLC.

Topics: Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Epidermal Growth Factor; ErbB Receptors; Humans; Lung Neoplasms; Polymorphism, Genetic; Protein Kinase Inhibitors; Receptor Protein-Tyrosine Kinases

Malignancies arising from the aerodigestive epithelium, including lung, head and neck, and esophageal carcinomas, are the leading causes of cancer-related mortality worldwide. Given the biological importance of epidermal growth factor receptor (EGFR) in cancer development and progression, EGFR inhibitors have emerged as promising novel therapies.. To summarize the current status of EGFR inhibitors in aerodigestive carcinomas (ADCs), highlight ongoing research designed to optimize their therapeutic effectiveness, and consider the future role of these agents.. Systematic MEDLINE search of English-language literature (1966-April 2007) performed using the terms EGFR, EGFR inhibitors, monoclonal antibodies, tyrosine kinase inhibitors, lung cancer, head and neck cancer, esophageal cancer, and EGFR predictive factors. Quality assessment of selected studies included clinical pertinence, with an emphasis on controlled study design, publication in peer-reviewed journals, adequate number of enrolled patients, objectivity of measurements, and techniques used to minimize bias.. The role of EGFR in ADC pathogenesis has been extensively studied, and multiple EGFR inhibition strategies are under evaluation. Erlotinib, an EGFR tyrosine kinase inhibitor used as a single agent, and cetuximab, an anti-EGFR monoclonal antibody used in combination with radiation, have conferred survival benefit in 1 trial of patients with advanced non-small cell lung cancer (median survival, 6.7 vs 4.7 months; hazard ratio, 0.70; 95% confidence interval, 0.58-0.87; P < .001) and in 1 trial of patients with locally advanced head and neck squamous cell carcinoma (median survival, 49 vs 29.3 months; hazard ratio, 0.74; 95% confidence interval, 0.57-0.97; P = .03), respectively. However, other trials have not shown these degrees of improvement. EGFR inhibitors toxicities include rash, diarrhea, and hypomagnesemia. Somatic mutations and other molecular tumoral characteristics offer opportunities for treatment individualization and optimal patient selection for anti-EGFR therapy.. EGFR is a promising therapeutic target in ADC. Further translational research is needed to optimize ways of inhibiting EGFR using single-agent or combination regimens and to identify patients who benefit the most from these therapies.

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Cetuximab; Epidermal Growth Factor; ErbB Receptors; Erlotinib Hydrochloride; Esophageal Neoplasms; Head and Neck Neoplasms; Humans; Lung Neoplasms; Protein Kinase Inhibitors; Quinazolines

Non-small cell lung cancer (NSCLC) remains a major problem in the western civilization and developing countries. Since most patients with NSCLC have advanced disease at diagnosis, to date, chemotherapy, with third-generation platinum-based doublets, represents the standard of care. Advances in the knowledge of tumour biology and mechanisms of oncogenesis has granted the singling out of several molecular targets for NSCLC treatment. Epidermal growth factor receptor (EGFR), a member of ErbB family, is one of the most studied target. Cetuximab is a chimeric (human-murine) monoclonal antibody directed against the extracellular domain of the EGFR that blocks ligand (TGF-alpha, EGF) access to the receptor. In the present paper we discuss about the activity, tolerability and efficacy of cetuximab, the EGFR monoclonal blocking antibody with the largest amount of clinical data being available on the treatment of advanced NSCLC.

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Carcinoma, Non-Small-Cell Lung; Cetuximab; Clinical Trials as Topic; Epidermal Growth Factor; ErbB Receptors; Humans; Lung Neoplasms; Neoplasm Staging; Transforming Growth Factor alpha

Bevacizumab is a recombinant, humanized monoclonal antibody against vascular endothelial growth factor. Erlotinib HCl is a reversible, highly selective epidermal growth factor receptor tyrosine kinase inhibitor. Additionally, both agents have shown benefit in patients with previously treated non-small cell lung cancer (NSCLC). Preclinical data in xenograft models produced greater growth inhibition with the combination than with either agent alone. A phase I/II study in two centers examined combined erlotinib and bevacizumab treatment in patients with nonsquamous stage IIIB/IV NSCLC with one or more prior chemotherapy. In phase I, 150 mg/d erlotinib orally plus 15 mg/kg bevacizumab i.v. every 21 days was established as the phase II dose. A total of 40 patients were enrolled and treated in this study (34 patients at phase II dose): 21 were female, 30 had adenocarcinoma histology, 9 were never smokers, and 22 had two or more prior regimens. The most common adverse events were mild to moderate rash, diarrhea, and proteinuria. Preliminary data showed no pharmacokinetic interaction between erlotinib and bevacizumab. Eight patients (20.0%) had partial responses and 26 (65.0%) had stable disease as their best response. The median overall survival for the 34 patients treated at the phase II dose was 12.6 months, with progression-free survival of 6.2 months. Encouraging antitumor activity and safety of erlotinib plus bevacizumab support further development of this combination for patients with advanced NSCLC. A randomized phase II trial has been completed, and a phase III trial is ongoing.

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Combined Chemotherapy Protocols; Bevacizumab; Carcinoma, Non-Small-Cell Lung; Clinical Trials, Phase I as Topic; Clinical Trials, Phase II as Topic; Drug Synergism; Epidermal Growth Factor; Erlotinib Hydrochloride; Female; Humans; Lung Neoplasms; Male; Quinazolines; Vascular Endothelial Growth Factor A

Epidermal growth factor receptor (EGFR) Inhibitors have shown promising results in patients with advanced non-small cell lung cancers (NSCLC) who previously have failed on chemotherapy. Objective response is achieved in 10 to 28% of the patients, and about 30% will achieve stable disease. A major problem is how to select the patients, who will benefit from treatment, and who will not.. The predictive role of EGFR protein expression assessed by IHC is still debated. Specific EGFR gene mutations have been identified associated with response to gefitinib (Iressa(R)), but seem not to be associated with stable disease. No studies have yet demonstrated any association between EGFR gene mutations and survival. In this review we describe other marker studies, which are associated with sensitivity to EGFR inhibitors. Increased EGFR gene copy number based on FISH analysis is demonstrated to be a good predictive marker for response, stable disease, time to progression, and survival.. EGFR/FISH seems today to be the best predictive marker for clinical benefit from EGFR inhibitors in NSCLC. Prospective large scale clinical studies must identify the most optimal paradigm for selection of patients.

Topics: Antineoplastic Agents; Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Drug Resistance, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Humans; Lung Neoplasms; Predictive Value of Tests; Protein Kinase Inhibitors; Sensitivity and Specificity

Targeted therapies are emerging as important drugs in the treatment of advanced non-small cell lung cancer (NSCLC). Within the past months, there have been considerable contributions to this topic. The results of several important clinical trials have been published. Furthermore, laboratory results have significantly contributed to clear out some molecular mechanisms regulating sensitivity or resistance to these drugs and to provide rational basis for further clinical studies.. A great part of recently published research on targeted agents in NSCLC regards EGFR inhibitors. Following the demonstration of activity of gefitinib in patients pretreated with chemotherapy, four large randomized trials testing the addition of gefitinib or erlotinib to first-line chemotherapy have been conducted, but failed to show any advantage. Interestingly, erlotinib has shown efficacy compared with placebo in pretreated patients. Mutations in the EGFR gene have shown a strong predictive role for sensitivity to EGFR inhibitors. A number of other targeted agents are currently under investigation: most of the phase II trials maintain a traditional methodology, with response rate as primary measure of activity.. Recent advances will lead to a rapid expansion of further studies aimed to define the best way to use targeted agents in NSCLC. Several methodological issues are still open. The proper selection of patients, the choice of the best study design and the most appropriate end-point for early clinical trials, and the correct modality to integrate these drugs with traditional chemotherapy represent the most challenging points that research is called to answer in the near future.

Topics: Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Clinical Trials as Topic; Epidermal Growth Factor; ErbB Receptors; Humans; Lung Neoplasms; Protein Kinase Inhibitors

Non-small cell lung cancer (NSCLC) may be considered typical of advanced age. Most cases of NSCLC are diagnosed in the advanced or locally advanced stage. It has been shown that combined chemo-radiotherapy is more efficient than either chemotherapy alone or radiation alone, for the therapeutic management of localized unresectable NSCLC. However, chemo-radiotherapy, even if given with sequential approach, in clinical practice can be contraindicated in elderly patients. In fact, this patient population often present at diagnosis with cardiovascular and/or pulmonary comorbidities that increase the risk of severe side effects from chemo-radiotherapy. The present review aims at focusing the currently available evidences on the treatment of elderly patients affected by locally advanced NSCLC and at giving future perspectives on this topic.. Very few specific prospective data are available on the treatment of locally advanced NSCLC in the elderly. Some phase II studies suggest that low-dose chemotherapy given concurrently with radiotherapy could be safely administered to this patient population. Retrospective analyses on full-dose sequential and concurrent chemo-radiation are to be considered globally ambiguous and at risk of selection bias.. Only specifically designed prospective studies will elucidate the real role and feasibility of combined chemo-radiotherapy in the treatment of locally advanced NSCLC in the elderly. Future perspectives on this topic include the evaluation of alternative schedules of chemo-radiotherapy, innovative radiation techniques more suitable to elderly patients, and the introduction of new, well-tolerated, molecularly targeted agents combined with standard treatments.

Topics: Aged; Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Chemotherapy, Adjuvant; Epidermal Growth Factor; ErbB Receptors; Humans; Lung Neoplasms; Protein Kinase Inhibitors; Radiotherapy, Adjuvant

A beneficial role for palliative chemotherapy in patients with advanced non-small cell lung cancer and a good performance status (ECOG 0 or 1) is now well established. In this article, we focus on the available literature for patients with a PS of 2, in whom a role for chemotherapy has yet to be defined.. In the past, the results of randomized trials of comparative standard platinum-based combination chemotherapy regimens have demonstrated inferior survival rates in PS 2 patients compared with those with PS 0 or 1. Consequently, a general view has emerged that the side effects of treatment outweigh the benefits, and chemotherapy has not been recommended as a standard of care. Although few studies have been designed specifically for PS 2 patients, gemcitabine, vinorelbine or taxane monotherapy, dose-attenuated platinum combination regimens, and epidermal growth factor receptor inhibitors may provide a clinical benefit with less toxicity. For example, although the median survival of PS 2 patients treated with best supportive care is 2-3 months, chemotherapy regimens are associated with median survivals ranging from 4 to 6 months. These data provide encouragement to revisit the role of chemotherapy in this group of patients.. There is potential with cytotoxic treatment to improve the palliative options for PS 2 patients with advanced non-small cell lung cancer. Further trials designed specifically for PS 2 patients that include measurement of symptoms, quality of life, and survival and toxicity are required to define the most active but least toxic regimens.

Topics: Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Chemotherapy, Adjuvant; Epidermal Growth Factor; ErbB Receptors; Humans; Lung Neoplasms; Protein Kinase Inhibitors

Lung cancer is the leading cause of cancer deaths in the United States and throughout the world; globally, there are more than 1.1 million deaths each year. Treatment modalities currently employed are significantly limited; 50% of patients experience disease recurrence after surgery, and less than a quarter of patients respond to systemic chemotherapy. These statistics have fueled the search for a safer, more effective treatment modality. Despite significant advances in our understanding of the molecular basis of cancer immunology, many obstacles remain. However, encouraging clinical results in patients immunized with autologous tumor cell vaccines expressing granulocyte macrophage colony-stimulating factor strongly advocate further investigation of immunotherapy in non-small-cell lung cancer (NSCLC). Further studies are needed to demonstrate whether these novel therapies can potentially complement or even replace current therapeutic approaches. We present a review of the various vaccine-based strategies employed to target and treat NSCLC.

Topics: Animals; Antigens; Antigens, Neoplasm; BCG Vaccine; Cancer Vaccines; Carcinoma, Non-Small-Cell Lung; Dendritic Cells; Epidermal Growth Factor; Genes, erbB-2; Glycoproteins; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Immunotherapy; Lung Neoplasms; Mucin-1; Mucins

Current standard therapy for advanced or metastatic non-small cell lung cancer (NSCLC) includes chemotherapy, which is often associated with limited efficacy and toxicity. Thus, there is an unmet need for novel anticancer agents that are both effective and well tolerated in patients with NSCLC. Gefitinib (Iressa) is an orally active epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor in advanced clinical development, and the first agent of its kind to receive approval. Extensive evidence indicates that gefitinib provides significant antitumor activity in previously treated advanced NSCLC, along with fast symptom improvement. This orally administered agent is generally well tolerated. Gefitinib has also shown promising efficacy in other solid tumors that rely on EGFR-related mechanisms for growth and survival. This article reviews the profile of gefitinib and the evidence supporting its use in the treatment of NSCLC.

Topics: Animals; Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Clinical Trials as Topic; Epidermal Growth Factor; Gefitinib; Humans; Lung Neoplasms; Protein-Tyrosine Kinases; Quinazolines

The role of dysregulated epidermal growth factor receptor-tyrosine kinase (EGFR-TK) activity in promoting tumor resistance to radiation therapy is discussed, and evidence supporting the rationale for the use of gefitinib (IRESSA, ZD1839) to enhance tumor radiosensitivity is reviewed.. A review of the literature regarding the role of EGFR-TK signaling in tumor response to radiation therapy was conducted, and results were summarized from preclinical and clinical studies of gefitinib in the treatment of solid tumors alone and in combination with radiation therapy.. Preclinical results indicate that EGFR-TK activity in tumors can block the cytotoxic effects of radiation therapy and enhance tumor repopulation, resulting in failure of local tumor control. In xenograft tumor models, gefitinib in combination with ionizing radiation resulted in additive to synergistic growth inhibition. In randomized clinical trials, gefitinib has demonstrated efficacy with favorable tolerability as monotherapy for patients with advanced non-small-cell lung cancer or head-and-neck carcinomas who had previously received standard therapies.. These results indicate that there is potential for improved responses by combining gefitinib with radiation therapy in non-small-cell lung cancer, head-and-neck cancers, and other solid tumors.

Topics: Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Combined Modality Therapy; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Gefitinib; Glioblastoma; Head and Neck Neoplasms; Humans; Lung Neoplasms; Neoplasm Proteins; Neoplasms; Quinazolines; Radiation Tolerance; Radiation-Sensitizing Agents

Several novel biologic agents have been designed to specifically inhibit different aspects of tumor growth and progression. The epidermal growth factor receptor has a pivotal role in tumor biology and thus is an important anticancer target. Gefitinib is an orally active epidermal growth factor receptor tyrosine kinase inhibitor that blocks signal-transduction pathways implicated in the proliferation and survival of cancer cells. Clinically meaningful antitumor activity, symptom relief, and a good tolerability profile have been shown in phase II trials of gefitinib monotherapy in patients with recurrent non-small cell lung cancer. Clinical trials are ongoing to investigate further applications of this novel agent for the treatment of non-small cell lung cancer.

Topics: Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Carcinoma, Non-Small-Cell Lung; Chemotherapy, Adjuvant; Epidermal Growth Factor; Gefitinib; Humans; Lung Neoplasms; Protein-Tyrosine Kinases; Quinazolines

Traditional cytotoxic anticancer therapies do not differentiate between tumour and host cells, and research efforts have been focused on finding new agents that target tumour tissue. Gefitinib ('Iressa', ZD1839) is an orally active epidermal growth factor receptor tyrosine kinase inhibitor that blocks signal pathways implicated in solid tumour growth and metastasis. In phase II trials, gefitinib 250 mg/day demonstrated efficacy in the control of advanced non-small-cell lung cancer (NSCLC) in patients who had undergone prior chemotherapy. Response rates were 18.4 and 11.8%, and disease control rates were 54.4 and 42.2%, at 250 mg/day in two multicentre trials - IDEAL 1 and 2. Gefitinib also caused rapid relief from the symptoms of NSCLC in approximately 40% of patients, while displaying a generally good tolerability profile that most commonly included mild, reversible gastrointestinal and skin adverse events. Gefitinib 250 mg/day has been approved for use in advanced, previously treated NSCLC in several countries including the USA, Japan and Australia. As a monotherapy and combination therapy, it is being investigated for the treatment of several common tumour types in addition to NSCLC. The pharmacokinetics of gefitinib have shown it to be suitable for once daily dosing, with a terminal half-life of approximately 48 h in patients with cancer. Steady-state exposure is achieved after 10 days dosing, and exposure is dose proportional up to 250 mg/day. Gefitinib is cleared principally by the biliary route and in part by metabolism. This review summarizes relevant data from studies of gefitinib that inform its clinical administration.

Topics: Administration, Oral; Antineoplastic Agents; Area Under Curve; Carcinoma, Non-Small-Cell Lung; Clinical Trials, Phase I as Topic; Clinical Trials, Phase II as Topic; Epidermal Growth Factor; Gefitinib; Humans; Lung Neoplasms; Quinazolines; Survival Analysis; Treatment Outcome

Lung cancer is the leading cause of death from neoplasia in men and women in the United States. Some studies suggest that women are more susceptible than men to tobacco-induced carcinogenesis and may show higher risk than men for lung cancer development from smoking. More recently, increasing biochemical and genetic data have supported this male-female difference in response to tobacco. Estrogens may be involved in lung carcinogenesis, and estrogen receptors (ERs), mainly ERb, are present and functional in normal lung and tumor cell lines and tissues. Estrogen can directly stimulate the transcription of estrogen-responsive genes in the nucleus of lung cells, and it can also transactivate growth factor signaling pathways, in particular the epidermal growth factor pathway. Lung cancer patients currently have few effective therapeutic options. An understanding of these new developments in estrogen signaling and cross-talk pathways may pave the way for innovative combinatorial approaches for treatment of lung cancer and possibly chemoprevention.

Topics: Age Factors; Animals; Antineoplastic Agents; Epidermal Growth Factor; Estrogen Receptor alpha; Estrogen Receptor beta; Estrogens; Female; Gefitinib; Humans; Lung Neoplasms; Male; Quinazolines; Receptors, Estrogen; Risk Factors; Sex Distribution; Sex Factors; Signal Transduction; Smoking; Survival Rate; United States

Gefitinib (Iressa) is a novel targeted therapy that inhibits the tyrosine kinase activity of the epidermal growth factor receptor by competitively blocking the ATP binding site. In preclinical studies gefitinib has shown potent activity in a number of tumor models, including several lung cancer cell lines and xenografts. Two large randomized Phase II studies (IDEAL 1 and IDEAL 2) in pretreated non-small cell lung cancer reported a response rate approaching 20% in second-line patients and approximately 10% in those pretreated with two or more chemotherapy regimens. The median survival in these two studies approached 6-8 months. As a first-line therapy, gefitinib has been assessed in combination with two different chemotherapy regimens in two large randomized studies (INTACT 1 and INTACT 2). Both studies failed to show an improvement in survival on a total patient accrual of >1000 patients in each study. Other end points (e.g., time to progression and response rate) were also not improved by the addition of gefitinib. Additional studies are indicated to assess the possible role of gefitinib in the maintenance of patients who received chemotherapy or chemoradiotherapy. Studies investigating gefitinib as first-line monotherapy are also required.

Topics: Animals; Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Disease Models, Animal; Disease Progression; Epidermal Growth Factor; Gefitinib; Humans; Lung Neoplasms; Protein-Tyrosine Kinases; Quinazolines; Randomized Controlled Trials as Topic; Survival Analysis

Every year more than 370,000 new cases of lung cancer occur in Europe. About 70% of the patients are not curable because of local or distant spread of tumor cells. Despite the use of chemotherapy, median survival of these patients is less than one year In the last two decades, important advances in cancer research have been achieved. This led to the development of a new class of potential anti-cancer agents, selectively targeting molecules which are important for the growth and the spread of tumor cells. Focussing on lung cancer, this review presents compounds that are furthest in clinical development, covering epidermal growth factor receptor inhibitors, receptor tyrosin kinase inhibitors, anti-angiogenic agents, matrix metalloproteinase inhibitors, gene therapy and antisense therapy.

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Division; Collagenases; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Genetic Therapy; Humans; Lung Neoplasms; Matrix Metalloproteinase Inhibitors; Neovascularization, Pathologic; Receptor Protein-Tyrosine Kinases

Treatment of solid tumors with chemotherapy regimens commonly is associated with debilitating or life-threatening side effects. Careful patient management, appropriate and prompt management of side effects, and interruption of therapy frequently are required for patients receiving chemotherapy. Furthermore, the systemic toxicity associated with chemotherapy may result in irreversible and incapacitating side effects, such as peripheral neuropathy, that lead to poor quality of life in patients. Gefitinib (Iressa, ZD1839, AstraZeneca Pharmaceuticals LP, Wilmington, DE) is a biologically based, molecular targeted therapy with a novel mechanism of action: selective inhibition of the epidermal growth factor receptortyrosine kinase (EGFR-TK) activity. Once-daily oral treatment with gefitinib is well tolerated. In clinical trials, treatment with gefitinib resulted in durable tumor responses and improvement in lung cancer-related symptoms in patients with advanced non-small cell lung cancer who had received prior chemotherapy. Trials are under way to explore the full potential of gefitinib and additional EGFR-TK inhibitors for other solid tumors and in other treatment settings, including prevention. Biologically based, molecular-targeted therapies such as gefitinib are providing new treatment options for patients and adding a new dimension to clinical practice for oncology nurses.

Topics: Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Dose-Response Relationship, Drug; Epidermal Growth Factor; Gefitinib; Humans; Lung Neoplasms; Patient Education as Topic; Protein-Tyrosine Kinases; Quinazolines

Twenty years after the epidermal growth factor receptor (EGFR) was identified as a potential anticancer target, the EGFR inhibitor gefitinib (Iressa; AstraZeneca) has been approved for the treatment of patients with advanced non-small-cell lung cancer in many countries. Studies have indicated its potential for treating patients with other types of solid tumours. Investigation of gefitinib has not only increased our knowledge about the biology of EGFR signalling, but is contributing to our evolving understanding of which tumours are EGFR dependent.

Topics: Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Epidermal Growth Factor; ErbB Receptors; Gefitinib; Humans; Lung Neoplasms; Protein-Tyrosine Kinases; Quinazolines

Topics: Adenocarcinoma; Antineoplastic Agents; Epidermal Growth Factor; ErbB Receptors; Gefitinib; Genes, erbB-1; Humans; Lung Neoplasms; Mutation; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-raf; Quinazolines

Although treatment with cytotoxic agents has produced modest survival improvement in patients with stage III and IV non-small cell lung cancer (NSCLC), it appears that a plateau has been reached with currently available chemotherapeutic regimens. Increasing knowledge regarding the properties of malignant neoplasms has identified a number of potential therapeutic targets. The epidermal growth factor receptor (EGFR) is one of these targets. Preclinical models have revealed that tumour growth can be inhibited by monoclonal antibodies directed against EGFR and EGFR-specific tyrosine kinase inhibitors. Erlotinib (Tarceva trade mark; OSI Pharmaceuticals, Genentech and Roche), a quinazoline derivative with good oral absorption, is one of several EGFR tyrosine kinases that has been studied in clinical trials. In a Phase I study, mild diarrhoea and mild rash were the most common toxicities. At a dose of 200 mg/day, diarrhoea was the dose-limiting toxicity. The observation that EGFR overexpression is relatively common in NSCLC led to a Phase II trial of erlotinib at the maximum-tolerated dose (150 mg/day) in previously treated NSCLC patients. Erlotinib produced a 12% response rate and there was no apparent relationship between response and tumour EGFR levels. More recent reports suggest that patients who develop a rash have higher responses. Based on its single agent activity, erlotinib has been evaluated in two Phase III trials which compared erlotinib plus chemotherapy to chemotherapy alone in previously untreated NSCLC patients. Erlotinib has also been compared to placebo in a Phase III trial which was limited to advanced stage NSCLC patients whose disease had progressed after two previous chemotherapy regimens. The optimum use of erlotinib in NSCLC will be determined by the results of the completed and future Phase III trials.

Topics: Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Carcinoma, Non-Small-Cell Lung; Cell Survival; Drug Screening Assays, Antitumor; Epidermal Growth Factor; Erlotinib Hydrochloride; Humans; Lung Neoplasms; Quinazolines; Randomized Controlled Trials as Topic; Tumor Cells, Cultured

Gefitinib is an epidermal growth factor receptor (EGFR) inhibitor that is reported to be well tolerated and active in patients with chemotherapy-resistant non small cell lung cancer. On the other hand, gefitinib was also reported to produce a severe adverse event, interstitial lung disease, in less than 2% of treated patients. Given these circumstances, it is important to evaluate this drug and to establish its use clinically. We do not have sufficient data to evaluate gefitinib at this time. Phase III study of second/third line or maintenance therapy using gefitinib is required for such an evaluation. The development of individualized therapy with gefitinib might be also be required.

Topics: Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Epidermal Growth Factor; Gefitinib; Humans; Lung Neoplasms; Quinazolines

The epidermal growth factor receptor (EGFR) has emerged in recent years as a key target of molecular therapy for solid tumors. The postembryonic role of EGFR is normally limited. In cancer, however, abnormal EGFR-tyrosine kinase (TK) activity plays a central role in many of the processes involved in tumor progression, such as proliferation, angiogenesis, invasiveness, decreased apoptosis, and loss of differentiation. Several different approaches have been taken to inhibit EGFR-mediated activity in tumor cells, including monoclonal antibodies directed at the ligand-binding portion of the EGFR and small-molecule agents that directly inhibit the intracellular TK domain of EGFR. Two of these TK inhibitors, gefitinib and erlotinib (OSI-774, Tarceva ), have shown antitumor activity and good tolerability across several tumor types in early dose-finding clinical trials, particularly for non-small-cell lung cancer (NSCLC). In heavily pretreated patients with advanced NSCLC, gefitinib showed clinically significant tumor responses and symptom relief with good tolerability. Based on these results, gefitinib has now been approved for the third-line treatment of advanced NSCLC. The use of gefitinib in standard treatment programs or combined with other molecular targeted agents may substantially improve the outlook for patients with NSCLC or other types of solid tumors

Topics: Antibodies, Monoclonal; Antineoplastic Agents; Apoptosis; Carcinoma, Non-Small-Cell Lung; Clinical Trials as Topic; Disease Progression; Epidermal Growth Factor; ErbB Receptors; Female; Gefitinib; Humans; Ligands; Lung Neoplasms; Male; Middle Aged; Neoplasm Invasiveness; Neovascularization, Pathologic; Protein-Tyrosine Kinases; Quinazolines

Although advances in chemotherapy have provided some improvement in overall survival for patients with advanced non-small-cell lung cancer (NSCLC), outcomes remain poor. Several targeted therapies for lung cancer are in development, and it is hoped that these new approaches will continue to improve survival for patients with advanced NSCLC. These new therapies are targeted specifically to the molecular pathways and processes that characterize tumor growth and progression, including uncontrolled cell growth, invasion, metastasis, angiogenesis, and resistance to apoptosis. Some molecules, such as protein kinase C and the epidermal growth factor receptor-tyrosine kinase, play central roles in cellular activity and are involved in many of the different signaling pathways underlying malignant transformation. Other molecules are dedicated to a specific process, such as the role of vascular endothelial growth factor in angiogenesis. New approaches use a variety of technologies to target these molecules, including small-molecule inhibitors, antibodies, and antisense oligonucleotides, among others. Many of these therapies are currently being tested alone or in combination with each other and with standard treatments for a variety of tumors. This article summarizes data on treatment of NSCLC with some of the agents that are the furthest along in clinical development. It is possible to envision a future in which a combination of therapies treat lung cancer on multiple fronts, significantly enhancing tumor responses and improving survival beyond current expectations.

Topics: Angiogenesis Inhibitors; Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Gefitinib; Humans; Lung Neoplasms; Protein Kinase C; Protein-Tyrosine Kinases; Quinazolines; Signal Transduction

Even when diagnosed at the earliest stages, non-small-cell lung cancer (NSCLC) has often begun to metastasize, leading to frequent systemic relapses and a poor prognosis. There is an urgent need for effective adjuvant systemic therapy in conjunction with surgery to reverse or control further growth of micrometastases in early-stage NSCLC. Several approaches have been investigated in the search for such a therapy, including various chemotherapies, applied preoperatively or postoperatively, chemopreventive agents, and molecular-targeted agents. This article presents an overview of clinical trials for these adjunctive therapies, including completed trials and trials whose results are anticipated. Although standard postoperative chemotherapy has been found to offer little benefit, likely because of the acquisition of resistance in advanced tumors, some clinical trials with neoadjuvant or alternative chemotherapies have produced encouraging results. Targeted agents such as the epidermal growth factor receptor/tyrosine kinase inhibitor gefitinib have shown early promise for effective disease control. A combination of these new approaches and standard therapy for NSCLC may improve long-term survival in patients with lung cancer

Topics: Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Carcinoma, Non-Small-Cell Lung; Chemoprevention; Chemotherapy, Adjuvant; Drug Resistance; Epidermal Growth Factor; Gefitinib; Humans; Lung Neoplasms; Neoadjuvant Therapy; Neoplasm Metastasis; Prognosis; Protein-Tyrosine Kinases; Quinazolines; Radiotherapy, Adjuvant; Selenium; Survival Analysis

The aims of chemoprevention in lung cancer are to prevent the appearance of disease (primary prevention) and to stop or reverse the progression of premalignant lesions (secondary prevention). Until recently, there was little hope that these goals could be attained. However, the results achieved with tamoxifen in the prevention of breast cancer, and the emergence of new therapies specifically targeted to molecules involved in the pathogenesis of lung cancer have set the stage for investigation of these agents for chemoprevention of lung cancer. Two of these new molecular targeted agents are gefitinib, an inhibitor of epidermal growth factor receptor-tyrosine kinase activity, and tipifarnib (R115777, Zarnestra ), an inhibitor of the farnesyltransferase enzyme, which is required for the proper localization and function of the ras oncogene. Tumor responses and disease stabilization have been achieved with both agents in clinical trials. In the Iressa Dose Evaluation in Advanced Lung Cancer (IDEAL)-1 and IDEAL-2 phase II trials, gefitinib was demonstrated to be effective for disease control in patients with advanced non-small-cell lung cancer. The SPORE (Specialized Program of Research Excellence) Trials of Lung Cancer Prevention (STOP) are 2 parallel studies that will investigate the potential effectiveness of gefitinib and tipifarnib in preventing the appearance and progression of premalignant lesions in former or current smokers with a history of smoking-related cancer. These trials should provide information not only about the potential role of gefitinib and tipifarnib in lung cancer chemoprevention, but also about the molecular changes that underlie tumorigenesis and that may serve as markers of disease progression. The STOP trial objectives are to evaluate the effect of gefitinib and tipifarnib on histologic and biologic parameters in patients with evidence of sputum atypia, to evaluate various parameters as potential predictors of the effectiveness of these agents, and to evaluate the tolerability of these agents over a 6-month course of treatment. Histologic response, defined as prevention of appearance or progression of premalignant lesions, is the primary endpoint of these trials. New targeted molecular therapies such as gefitinib and tipifarnib may offer the opportunity to make chemoprevention a viable treatment modality in lung cancer as well as in other human solid tumors.

Topics: Antineoplastic Agents; Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Chemoprevention; Clinical Trials as Topic; Disease Progression; Epidermal Growth Factor; Gefitinib; Humans; Lung Neoplasms; Precancerous Conditions; Protein-Tyrosine Kinases; Quinazolines; Quinolones

Topics: Alkyl and Aryl Transferases; Angiogenesis Inhibitors; Antineoplastic Agents; Benzamides; Benzodiazepines; Clinical Trials as Topic; Clinical Trials, Phase I as Topic; Clinical Trials, Phase II as Topic; Clinical Trials, Phase III as Topic; Endothelial Growth Factors; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Erlotinib Hydrochloride; Farnesyltranstransferase; Gefitinib; Humans; Imatinib Mesylate; Imidazoles; Intercellular Signaling Peptides and Proteins; Lung Neoplasms; Lymphokines; Metalloendopeptidases; Multicenter Studies as Topic; Piperazines; Polyisoprenyl Phosphates; Protein-Tyrosine Kinases; Pyrimidines; Quinazolines; Quinolones; Randomized Controlled Trials as Topic; Receptor Protein-Tyrosine Kinases; Sesquiterpenes; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Topics: Angiogenesis Inhibitors; Antineoplastic Agents; Antineoplastic Agents, Phytogenic; Antineoplastic Combined Chemotherapy Protocols; Carboplatin; Carcinoma, Non-Small-Cell Lung; Clinical Trials, Phase II as Topic; Clinical Trials, Phase III as Topic; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Lung Neoplasms; Metalloendopeptidases; Neovascularization, Pathologic; Organic Chemicals; Paclitaxel; Prognosis; Randomized Controlled Trials as Topic; Research; Time Factors

Investigators and clinicians have recently called attention not only to the clinical and morphological parameters, but to the parameters characterizing the biological activity of nonsmall-cell carcinoma of the lung (NSCCL) from biochemical and molecular biological points of view. These include production of epidermal growth factor (EGF) receptors (EGFR) and their ligands which are important auto/paracrine regulation of lung tissue formation in health and tumor growth. Active studies of EGFR and EGF-like peptides (mainly, EGF and alpha-TGF) have failed to gain an insight into their role in the pathogenesis of NSCCL. Most authors suppose that tumor EGFR production increases as cell atypical features enhance and tumors show EGFR hyperexpression as compared with intact lung tissue. The expression of EGF and alpha-TGF is associated with poor prognosis in NSCCL. Attempts at designing and clinically testing the agents that block the transmission of EGFR ligands within the tumor cell are well-known, which open up new possibilities for antitumor therapy of patients with NSCCL.

Topics: Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Cell Division; Epidermal Growth Factor; ErbB Receptors; Humans; Lung Neoplasms; Prognosis

Topics: Antigens, Neoplasm; Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Cell Division; Epidermal Growth Factor; Gene Amplification; Genes, p53; Genes, ras; Humans; Lung Neoplasms; Lymphatic Metastasis; Prognosis; Proto-Oncogenes; Survival Rate

Topics: Animals; Brain Neoplasms; Breast Neoplasms; Cell Division; Cell Transformation, Neoplastic; Digestive System Neoplasms; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Growth Substances; Head and Neck Neoplasms; Humans; Lung Neoplasms; Melanoma; Neoplasm Proteins; Neoplasms; Neoplasms, Experimental; Oncogenes; Rats; Receptor Protein-Tyrosine Kinases; Receptors, Growth Factor; Signal Transduction; Urogenital Neoplasms

Topics: Amino Acid Sequence; Carcinoma, Non-Small-Cell Lung; Carcinoma, Small Cell; Epidermal Growth Factor; Gastrin-Releasing Peptide; Growth Substances; Humans; Insulin-Like Growth Factor I; Lung Neoplasms; Molecular Sequence Data; Peptides

A wide range of genetic and phenotypic abnormalities have been identified in lung cancer. However, only a few are known to have an impact on patient outcome and thus may influence choice of therapy. Biologic and molecular factors known in this regard include the epidermal growth factor family and its receptors, markers of neuroendocrine differentiation in non-small cell lung cancer, and mutations of the ras gene family. None of these factors, however, can be considered a standard for selection of patients for therapy until additional information is gleaned from ongoing prospective studies.

Topics: Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Epidermal Growth Factor; ErbB Receptors; Genes, ras; Growth Substances; Humans; Lung Neoplasms; Mutation; Prognosis; Survival Rate; Transforming Growth Factor alpha

Epithelial cell layers exhibit an ordered polarized architecture. However, such structures are disrupted during malignant transformation, which generally coincides with a loss of regulate cell growth. We are investigating how the cadherin cell adhesion system controls these processes. Cadherins form a molecular complex with alpha-catenin, and beta-catenin or plakoglobin at the cytoplasmic side in normal cells. Lung carcinoma PC9 cells express E-cadherin. Although they express other catenins, they lack alpha-catenin and cannot firmly aggregate, suggesting that their E-cadherin is inactive. Transfection of the PC9 cells with alpha-catenin cDNA leads to activation of the E-cadherin, inducing their compact aggregation. In these aggregates, an almost complete epithelial-specific architecture is organized, including the formation of microvilli and a junctional complex. We also studied the effect of hepatocyte growth factor/scatter factor (HGF/SF) on cell-cell contacts in keratinocyte cell lines, and found that this growth factor can disrupt desmosomal cell-cell contacts. HGF/SF, and also epidermal growth factor, enhance tyrosine phosphorylation of beta-catenin or plakoglobin in human carcinoma lines as they induce scattering of these cells. These findings suggest that the cadherin adhesion system is central in organizing epithelial structures and that tyrosine phosphorylation of catenins may modulate this organization process.

Topics: alpha Catenin; Cadherins; Cell Adhesion; Cell Polarity; Cytoskeletal Proteins; Desmoplakins; Desmosomes; Epidermal Growth Factor; Epithelial Cells; Epithelium; gamma Catenin; Hepatocyte Growth Factor; Humans; Lung Neoplasms; Macromolecular Substances; Phosphorylation; Protein Processing, Post-Translational; Transfection

Growth factors play an important role in the pathogenesis and progression of all histologic types of lung cancer. Ideas for the exploitation of growth factors in lung cancer management are growing. The inhibition of the interaction between growth factors and their receptors, utilization of negative growth factors, interruption of the signal transduction pathways, or effecting decreased growth factor and/or receptor expression, could result in cell death, and all seem logical possibilities for new and specific treatment approaches. There can be no question that observations of the abnormal expression of growth factors have made a startling impact in every aspect of cancer research. The elucidation of their role in cell proliferation, coupled with our growing knowledge of the functions of oncogenes, has given birth to a unifying concept for the etiology of malignant transformation, which hopefully will translate into new, less toxic, more effective, and desperately needed lung cancer treatment.

Topics: Amino Acid Sequence; Carcinoma, Non-Small-Cell Lung; Carcinoma, Small Cell; Epidermal Growth Factor; ErbB Receptors; Gene Expression; Growth Substances; Humans; Insulin-Like Growth Factor I; Lung Neoplasms; Molecular Sequence Data; Neuropeptides; Signal Transduction; Smoking

Topics: Asbestos; Bombesin; Epidermal Growth Factor; Gene Expression; Humans; Insulin-Like Growth Factor I; Lung Neoplasms; Platelet-Derived Growth Factor; Transforming Growth Factors

Topics: Animals; Antibodies, Monoclonal; Bombesin; Clinical Trials as Topic; Epidermal Growth Factor; Gastrin-Releasing Peptide; Growth Inhibitors; Growth Substances; Humans; Insulin-Like Growth Factor I; Lung Neoplasms; Peptides; Transferrin

Topics: Aminoacridines; Amsacrine; Animals; Anthracenes; Antineoplastic Agents; Blood Physiological Phenomena; Bone Marrow; Breast Neoplasms; Colony-Forming Units Assay; Drug Evaluation; Drug Evaluation, Preclinical; Drug Stability; Epidermal Growth Factor; Female; Head and Neck Neoplasms; Humans; Interferons; Karyotyping; Leukemia; Lung Neoplasms; Melanoma; Mice; Mice, Nude; Multiple Myeloma; Neoplasm Transplantation; Neoplasms; Ovarian Neoplasms; Platelet-Derived Growth Factor; Suspensions; Transplantation, Heterologous; Tumor Stem Cell Assay

Chemotherapeutic assays, using nitrosoureas, performed on tumor bearing rats have shown a regression of local tumor, accompanied with an amplification of pulmonary metastases, demonstrating that the treatment of metastasis differs from the treatment of a local tumor. Cells organizing a tumor are heterogeneous for their drug resistance, and for a series of properties including their ability to form metastasis. Metastatic cells have to leave the tumoral tissue, to traverse biological barriers, to resist to immune system, to implant and growth in the target tissue. An experimental model has been used to characterize metastatic cells. Metastatic potential has been defined as the ability to invade lungs. Highly metastatic cloned cell lines, such as subline 6, were strongly stimulated to proliferate by EGF, expressed fibronectin, actively degraded the extracellular matrix, rapidly attached to endothelial vascular cells, and resisted to natural killer lymphocytes. Inversely, weakly metastatic lines, such as subline 8, were preferentially stimulated by FGF and EDGF, poorly expressed fibronectin, did not degrade extracellular matrix, slowly attached to vascular cells, and were killed by NK lymphocytes. Studies on a large series of clones showed a diversity between them, and that no one property was determinant. Modulation of these characters by growth factors, hormones and immune state of the host is discussed, and leads to conclude that the expression of metastatic potential of a tumor depends on genetically defined characters and also on influences excerted by the host.

Topics: Animals; Cell Adhesion; Cell Division; Cell Line; Cytotoxicity, Immunologic; Epidermal Growth Factor; Extracellular Matrix; Fibroblast Growth Factors; Fibronectins; Growth Substances; Killer Cells, Natural; Laminin; Lung Neoplasms; Neoplasm Metastasis; Neoplasm Staging; Rats; Rats, Inbred Strains; Rhabdomyosarcoma

Hu-rhEGF-rP64k/Mont is a biotechnology product for the treatment of advanced non-small cell lung cancer (NSCLC). The vaccine induces a neutralizing antibody-mediated immune response, against the normal circulating self-protein antigen epidermal growth factor (EGF), which prevents its binding to and activation of the EGF receptor, inhibiting the transduction of the signals that drive cancer cell proliferation, survival and spread. This phase I study aimed to evaluate the safety and the immunological response of Hu-rhEGF-rP64k vaccine in NSCLC patients.. The Hu-rhEGF-rP64k/Mont vaccine showed to be safe and well tolerated, with dizziness, injection-site reactions and tremors being the most commonly reported adverse event. No severe adverse events or death were related to the vaccination. Immune monitoring demonstrated the generation of anti-EGF antibody titers and as a consequence the patients exhibited a decrease in the EGF concentration. In 80% of the vaccinated patients stable disease was achieved.. Hu-rhEGF-rP64k/Mont elicited a valuable immune response, with good safety profile assuring further clinical development of the vaccine in this population to further confirm the potential benefits on survival.. Chinese Clinical Trial Registry, ChiCTR-OID-17014048 , date 2017/12/20 (retrospectively registered); Chinese Food and Drug Administration, CFDA 2009 L02105, date 2009/04/03; China Drug Trial, CTR20131039 .

Topics: Antibodies, Neutralizing; Cancer Vaccines; Carcinoma, Non-Small-Cell Lung; China; Epidermal Growth Factor; Female; Humans; Immune Tolerance; Immunotherapy, Active; Lung Neoplasms; Male; Middle Aged; Retrospective Studies; Treatment Outcome

The efficacy of erlotinib, the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, has been demonstrated in patients with non-small cell lung cancer (NSCLC) and pancreatic cancer (PC). In the present study, we evaluated the effect of epidermal growth factor (EGF) ointment on erlotinib-related skin effects (ERSEs).. This was an open-label, non-comparative, multicenter, phase II trial. The patients included those diagnosed with NSCLC or PC who were treated with erlotinib. The effectiveness of the ointment was defined as follows: (1) grade 2, 3, or 4 ERSEs downgraded to ≤ grade 1 or (2) grade 3 or 4 ERSEs downgraded to grade 2 and persisted for at least 2 weeks.. Fifty-two patients from seven institutes in Korea were enrolled with informed consent. The final assessment included 46 patients (30 males, 16 females). According to the definition of effectiveness, the EGF ointment was effective in 36 (69.2%) intention to treat patients. There were no statistically significant differences in the effectiveness of the EGF ointment by gender (p = 0.465), age (p = 0.547), tumor type (p = 0.085), erlotinib dosage (p = 0.117), and number of prior chemotherapy sessions (p = 0.547). The grading for the average National Cancer Institute's Common Terminology Criteria for Adverse Events (NCI-CTCAE) rating of rash/acne and itching improved from 2.02 ± 0.83 to 1.13 ± 0.89 and 1.52 ± 0.84 to 0.67 ± 0.90, respectively (p < 0.001). The most common reason for discontinuing the study was progression of cancer (37%).. Based on the results, the EGF ointment is effective for ERSEs, regardless of gender, age, type of tumor, and dosage of erlotinib. The EGF ointment evenly improved all kinds of symptoms of ERSEs.. ClinicalTrials.gov identifier: NCT01593995.

Topics: Aged; Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Dermatologic Agents; Disease Progression; Disease-Free Survival; Drug Eruptions; Epidermal Growth Factor; Erlotinib Hydrochloride; Female; Humans; Lung Neoplasms; Male; Middle Aged; Ointments; Pancreatic Neoplasms; Protein Kinase Inhibitors; Republic of Korea; Treatment Outcome

EGFR is a well-validated target for patients with non-small cell lung cancer (NSCLC). CIMAvax-EGF is a therapeutic cancer vaccine composed of human recombinant EGF conjugated to a carrier protein and Montanide ISA51 as adjuvant. The vaccine is intended to induce antibodies against self EGFs that block EGF-EGFR interaction.. To evaluate overall survival, safety, immunogenicity, and EGF concentration in serum after CIMAvax-EGF, a randomized phase III trial was done in patients with advanced NSCLC. Four to 6 weeks after first-line chemotherapy, 405 patients with stage IIIB/IV NSCLC were randomly assigned to a vaccine group, which received CIMAvax-EGF or a control group, treated with best supportive care.. Long-term vaccination was very safe. Most frequent adverse reactions were grade 1 or 2 injection-site pain, fever, vomiting, and headache. Vaccination induced anti-EGF antibodies and decreased serum EGF concentration. In the safety population, median survival time (MST) was 10.83 months in the vaccine arm versus 8.86 months in the control arm. These differences were not significant according the standard log rank (HR, 0.82; P = 0.100), but according a weighted log rank (P = 0.04) that was applied once the nonproportionality of the HR was verified. Survival benefit was significant (HR, 0.77; P = 0.036) in the per-protocol setting (patients receiving at least four vaccine doses): MST was 12.43 months for the vaccine arm versus 9.43 months for the control arm. MST was higher (14.66 months) for vaccinated patients with high EGF concentration at baseline.. Switch maintenance with CIMAvax-EGF was well tolerated and significantly increased MST of patients that completed induction vaccination. Baseline EGF concentration predicted survival benefit. Clin Cancer Res; 22(15); 3782-90. ©2016 AACR.

Topics: Adjuvants, Immunologic; Cancer Vaccines; Carcinoma, Non-Small-Cell Lung; Epidermal Growth Factor; Female; Humans; Immunotherapy, Active; Kaplan-Meier Estimate; Lung Neoplasms; Male; Neoplasm Staging; Prognosis; Retreatment; Treatment Outcome

An epidermal growth factor (EGF) vaccine was given before and after standard first line chemotherapy to patients with advanced nonsmall cell lung cancer (NSCLC), to investigate the immunologic and clinical results in a phase 1 study. Twenty patients diagnosed with advanced NSCLC were recruited. Two vaccinations were given before the first line of chemotherapy treatment, with subsequent monthly vaccination after concluding chemotherapy. The EGF vaccination dose was increased compared with previous trials; the primary end points were immunogenicity and safety. Anti-EGF antibody titers were more than 20 times higher than those previously obtained, without any increase in adverse events, serum EGF concentration decreased to undetectable levels in all patients. Ninety-two percent of the evaluated patients (n=13) showed an immunodominant antibody response against the central region on the EGF molecule. High percentages of EGF/EGF receptor binding inhibition were observed, which significantly positively correlated with the increased antibody response against the EGF immunodominant region. Survival of the patients in this study correlates positively with antibody titers. This study has shown that combination of EGF vaccination at high dose, with chemotherapy is feasible and well tolerated higher anti-EGF antibody titers and reduction of serum EGF concentration seen; do not entail an increase in severe adverse events. The correlation of survival with antibody titers observed is being confirmed confirmation in a wider and randomized trial currently ongoing.

Topics: Adult; Aged; Antibodies; Antineoplastic Agents, Alkylating; Cancer Vaccines; Carcinoma, Non-Small-Cell Lung; Cyclophosphamide; Drug Therapy, Combination; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Lung Neoplasms; Male; Middle Aged; Pilot Projects; Recombinant Proteins

Epidermal growth factor (EGF) might be a suitable immunotherapeutic target in non-small-cell lung cancer (NSCLC). Our approach consists of active immunotherapy with EGF. The aim of the study is to characterize the humoral response and its effects on signal transduction in relation with the clinical outcome.. Eighty NSCLC patients treated with first-line chemotherapy were randomized to receive the EGF vaccine or supportive care. EGF concentration in sera, anti-EGF antibodies and their capacity to inhibit the binding between EGF/EGF receptor (EGFR), and the EGFR phosphorylation were measured.. Seventy-three percent of vaccinated patients developed a good antibody response, whereas none of the controls did. In good antibody-responder patients, self EGF in sera was significantly reduced. In 58% of vaccinated patients, the post-immune sera inhibited EGF/EGFR binding; in the control group, no inhibition occurred. Post-immune sera inhibited the EGFR phosphorylation whereas sera from control patients did not have this capacity. Good antibody-responder patients younger than 60 years had a significantly better survival. A high correlation between anti-EGF antibody titers, EGFR phosphorylation inhibition, and EGF/EGFR binding inhibition was found. There was a significantly better survival for vaccinated patients that showed the higher capacity to inhibit EGF/EGFR binding and for those who showed an immunodominance by the central region of EGF molecule.. Immunization with the EGF vaccine induced neutralizing anti-EGF antibodies capable of inhibiting EGFR phosphorylation. There was a significant positive correlation between antibody titers, EGF/EGFR binding inhibition, immunodominance of anti-EGF antibodies, and survival in advanced NSCLC patients.

Topics: Antibodies; Cancer Vaccines; Carcinoma, Non-Small-Cell Lung; Epidermal Growth Factor; ErbB Receptors; Humans; Lung Neoplasms; Patient Selection; Phosphorylation; Recombinant Proteins; Survival Analysis

We show the result of a randomized phase II clinical trial with an epidermal growth factor (EGF)-based cancer vaccine in advanced non-small-cell lung cancer (NSCLC) patients, evaluating immunogenicity, safety, and effect on survival.. Eighty patients with stage IIIB/IV NSCLC after finishing first-line chemotherapy were randomly assigned to receive best supportive care or EGF vaccinations.. Vaccination was safe. Adverse events were observed in less than 25% of cases and were grade 1 or 2 according to National Cancer Institute Common Toxicity Criteria. Good anti-EGF antibody response (GAR) was obtained in 51.3% of vaccinated patients and in none of the control group. Serum EGF concentration showed a major decrease in 64.3% of vaccinated patients. GAR patients survived significantly more than those with poor antibody response (PAR). Also, patients whose serum EGF dropped below 168 pg/mL survived significantly more than the rest. There was a trend to an increased survival for vaccinated patients compared with controls. The survival advantage for vaccinated patients compared with controls was statistically significant in the subgroup of patients with age younger than 60 years.. Vaccination with EGF was safe and provoked an increase in anti-EGF antibody titers and a decrease in serum EGF. There was a direct correlation between antibody response and survival. There was a direct correlation between decrease in serum EGF and survival. In patients younger than 60 years, vaccination was associated with increased survival.

Topics: Adenocarcinoma; Adult; Aged; Cancer Vaccines; Carcinoma, Large Cell; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Immunotherapy, Active; Lung Neoplasms; Male; Middle Aged; Neoplasm Staging; Survival Analysis; Treatment Outcome

Epidermal Growth Factor (EGF) promotes tumor cell proliferation and survival upon binding to its receptor. We have developed a new active specific immunotherapy based on EGF deprivation. In the present paper, we show the results of a Phase I trial in 43 patients with advanced non-small cell lung cancer (NSCLC) who received the EGF vaccine. Patients who had already received first line therapy were randomized to receive a single or double dose of the EGF vaccine, weekly for four weeks and monthly thereafter. No significant toxicity was seen after vaccination. Adverse events consisted primarily of fever, chills, nausea, vomiting and flushing. Fifteen patients (39%) developed a good antibody response (GAR) against EGF. The geometric mean of the antibody titer was higher in the double dose group. EGF concentration was quantified in serum. An inverse correlation between anti-EGF antibody titers and EGF concentration was seen after immunization. Vaccinated patients achieved median survival times of 8.23 months from randomization. Patients who received the double dose of treatment showed a trend toward increased survival in comparison with patients who received the single dose. GAR and patients in whom the serum EGF decreased below the 168 pg/ml cut-off point had a significantly better survival when compared to poor responders or patients in which the EGF levels were not considerably reduced. Our results confirm the immunogenicity of the EGF vaccine in the treatment of patients with advanced stage NSCLC. Antibody titers and serum EGF levels appear to correlate with patient survival.

Topics: Aged; Antibodies; Cancer Vaccines; Carcinoma, Non-Small-Cell Lung; Epidermal Growth Factor; Female; Humans; Immunotherapy, Active; Lung Neoplasms; Male; Middle Aged

Gefitinib has shown meaningful antitumor activity with tolerable toxicity in chemotherapy-refractory NSCLC in previous studies. Moreover, EGFR expression failed to show a correlation with response. In an attempt to identify predictive markers of response, we have investigated the tumoral expression of key signaling molecules of EGFR (EGFR, p-EGFR, p-Akt, p-Erk, p-STAT3) by immunohistochemistry and analyzed their correlations with response. Of 65 patients who received gefitinib (250 mg/day) for chemotherapy-refractory NSCLC, there were 14 partial responses (21.5%), 21 stable diseases (32.3%) and 21 progressive diseases (32.3%). Median durations of overall survival and time to progression were 6.7 months and 2.8 months, respectively. Immunohistochemistry was performed in 34 patients with evaluable tissue specimens. EGFR was overexpressed (2+ or 3+) in 32.4% and p-EGFR was positive in 26.5%. The expressions of p-Akt, p-Erk and p-STAT3 were positive (1+ or 2+) in 50%, 38.2% and 79.4%, respectively. The EGFR expression was not correlated with p-EGFR or the downstream molecules. EGFR or p-EGFR status did not correlate with response. Positive expression of p-Erk was significantly associated with poor response (38.1% in -, 14.3% in 1+, 0% in 2+; p = 0.046). Furthermore, tumors with positive p-Akt and negative p-Erk nuclear expression exhibited the best response (60%), whereas there was no response in the opposite [p-Akt (-), p-Erk (+)] cases. Intense nuclear staining of p-Akt (2+) was associated with prolonged TTP (HR 0.25, 95% confidence interval [CI] 0.08-0.79, p = 0.018) and OS (HR 0.16, 95% CI 0.04-0.62, p = 0.008). These results support the assumption that gefitinib responsiveness might be predicted by activated EGFR downstream molecules such as p-Akt and p-Erk.

Topics: Adult; Aged; Antineoplastic Agents; Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Disease Progression; Drug Resistance, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Female; Gefitinib; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Lung Neoplasms; Male; Middle Aged; Predictive Value of Tests; Quinazolines; Survival Analysis

Prostaglandins (PGs) and thromboxane (TX) produced by cyclooxygenase (COX) have a great influence on vascular systems and platelet functions. The serum levels of epidermal growth factor (EGF) and PGs were measured in patients with lung cancer treated with gefitinib, and the influence of EGF on platelet aggregation was investigated. Twenty patients were investigated. The serum level of TXB(2) increased significantly in all patients who received gefitinib for 2 weeks (before vs. after = 94.1 +/- 47.3 vs. 190.9 +/- 54.3, p<0.01). TXB(2) also increased significantly in responders without concurrent chemotherapy (before vs. after = 79.3 +/- 35.5 vs. 194.5 +/- 58.1, p<0.05), but not in non-responders (before vs. after = 106. 5 +/- 65.8 vs. 162.2 +/- 52.8, N.S.). PG 6-keto F1alpha and PGE(2) did not exhibit significant changes. Furthermore, EGF showed no significant change (after vs. before = 234 +/- 35 vs. 276 +/- 72, N.S.). Although there was no correlation between the levels of EGF and TXB(2) (N.S.), the PG 6-keto F2alpha/TXB(2) ratio decreased significantly (before vs. after = 0.054 +/- 0.018 vs 0.033 +/- 0.015, p<0.05). The secondary platelet aggregation observed after high-dose adenosine diphosphate stimulation was inhibited after a 1-minute preincubation with EGF. Platelet aggregation in patients after gefitinib administration tended to accelerate and secondary aggregation was observed after low-dose adenosine diphosphate stimulation. We conclude that careful observation is needed for patients with chronic obstructive pulmonary disease, pulmonary fibrosis, and thromboembolic diseases receiving gefitinib. Furthermore, measurement of prostanoids may be a good predictor of the beneficial and adverse effects. Moreover, the combination of gefitinib with a COX inhibitor that regulates TXA(2)/PGI(2) balance should be evaluated.

Topics: Adult; Aged; Aged, 80 and over; Blood Platelets; Blood Vessels; Epidermal Growth Factor; Female; Gefitinib; Humans; Lung Neoplasms; Male; Middle Aged; Platelet Aggregation; Prostaglandins; Quinazolines

To investigate the anti-tumor activity and toxicity of the epidermal growth factor receptor (EGFR) inhibitor gefitinib (ZD1839 or Iressa; AstraZeneca Pharmaceuticals, Wilmington, DE), in patients with advanced non-small cell lung cancer (NSCLC).. This was an open label, expanded access program (EAP) of oral gefitinib administered at 250 mg per day continuously until evidence of undue toxicity or disease progression.. Two hundred consecutive patients with advanced NSCLC were enrolled in this study. The median number of prior chemotherapy regimens was 2 (range 0-6). One hundred seventy-two patients were treated with gefitinib; 23 expired from disease progression prior to treatment and 5 withdrew their consent. One hundred fifty-four patients are evaluable for toxicity; 8 (5.2%) experienced grade 3/4 toxicity; 2 patients discontinued therapy for grade 3 rash and one for grade 3 nausea. Of 172 patients evaluable for efficacy, 7 (4.1%; 95% CI; 1.7-8.2%) experienced a partial response (PR); 60 patients (34.9%) had stable disease (SD) as their best response. Median survival for all patients was 4.5 months (95% CI; 4.1-4.9 months). One-year survival was 29%. Significant independent prognostic factors associated with improved survival were female gender, good performance status (0/1), and adenocarcinoma histology.. Gefitinib has anti-tumor activity, in a heterogeneous population of NSCLC patients treated on the EAP study. Treatment with gefitinib in this population is associated with a longer survival in women, those with good performance status and in patients with adenocarcinomas. These findings need to be further validated in additional clinical studies.

Topics: Adenocarcinoma; Administration, Oral; Adult; Aged; Aged, 80 and over; Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Disease Progression; Epidermal Growth Factor; Female; Gefitinib; Health Status; Humans; Lung Neoplasms; Male; Middle Aged; Prognosis; Protein-Tyrosine Kinases; Quinazolines; Sex Factors; Survival Analysis

The role that growth factors and their receptors play in human cancer growth and progression makes them interesting targets for novel treatment modalities. Our approach consisted of active immunotherapy with the epidermal growth factor (EGF). Two pilot clinical trials were conducted to examine the safety and immunogenicity of a five-dose immunization protocol and to compare different adjuvants and treatment designs.. Forty patients with advanced non-small-cell lung cancer were enrolled in both trials. They were randomized to be treated with aluminum hydroxide or montanide ISA 51 as adjuvants in the EGF vaccine preparation. The use of cyclophosphamide prevaccination treatment was evaluated in the second trial.. Pooled data from both trials showed that the use of montanide as adjuvant increased the percentage of good antibody responders (GAR). Cyclophosphamide prevaccination treatment did not provoke improvements in antibody response. GAR had a significant increase in survival as compared with poor antibody responders. Response duration was also related to a significant improvement in survival rates.. Vaccination with five doses of EGF vaccine is safe and immunogenic. Montanide ISA 51 increased the percentage of GAR. There is a direct relationship between anti-EGF antibody titers and immune response duration with survival time.

Topics: Adjuvants, Immunologic; Adult; Aged; Aged, 80 and over; Aluminum Hydroxide; Antibody Formation; Cancer Vaccines; Carcinoma, Non-Small-Cell Lung; Epidermal Growth Factor; Female; Humans; Lung Neoplasms; Male; Mannitol; Middle Aged; Oleic Acids; Survival Analysis; Vaccination

To evaluate the efficacy and tolerability of two doses of gefitinib (Iressa [ZD1839]; AstraZeneca, Wilmington, DE), a novel epidermal growth factor receptor tyrosine kinase inhibitor, in patients with pretreated advanced non-small-cell lung cancer (NSCLC).. This was a randomized, double-blind, parallel-group, multicenter phase II trial. Two hundred ten patients with advanced NSCLC who were previously treated with one or two chemotherapy regimens (at least one containing platinum) were randomly assigned to receive either 250-mg or 500-mg oral doses of gefitinib once daily.. Efficacy was similar for the 250- and 500-mg/d groups. Objective tumor response rates were 18.4% (95% confidence interval [CI], 11.5 to 27.3) and 19.0% (95% CI, 12.1 to 27.9); among evaluable patients, symptom improvement rates were 40.3% (95% CI, 28.5 to 53.0) and 37.0% (95% CI, 26.0 to 49.1); median progression-free survival times were 2.7 and 2.8 months; and median overall survival times were 7.6 and 8.0 months, respectively. Symptom improvements were recorded for 69.2% (250 mg/d) and 85.7% (500 mg/d) of patients with a tumor response. Adverse events (AEs) at both dose levels were generally mild (grade 1 or 2) and consisted mainly of skin reactions and diarrhea. Drug-related toxicities were more frequent in the higher-dose group. Withdrawal due to drug-related AEs was 1.9% and 9.4% for patients receiving gefitinib 250 and 500 mg/d, respectively.. Gefitinib showed clinically meaningful antitumor activity and provided symptom relief as second- and third-line treatment in these patients. At 250 mg/d, gefitinib had a favorable AE profile. Gefitinib 250 mg/d is an important, novel treatment option for patients with pretreated advanced NSCLC [corrected]

Topics: Administration, Oral; Adult; Aged; Aged, 80 and over; Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Double-Blind Method; Epidermal Growth Factor; Female; Gefitinib; Humans; Logistic Models; Lung Neoplasms; Male; Middle Aged; Proportional Hazards Models; Protein-Tyrosine Kinases; Quality of Life; Quinazolines; Survival Rate; Treatment Outcome

Gefitinib is an oral agent that inhibits the tyrosine kinase of the epidermal growth factor receptor. In phase I trials gefitinib was well tolerated and antitumor activity was seen in pretreated non-small-cell lung cancer (NSCLC) patients. Preclinical studies indicated enhanced effects when gefitnib was added to carboplatin or paclitaxel. This pilot trial combined gefitinib with carboplatin and paclitaxel to define the toxicities of the combination and assess drug-drug interactions in untreated advanced NSCLC patients.. Initially (part 1) patients were randomly assigned to receive intermittent gefitinib with cycle 1 or 2 of chemotherapy. Thereafter (part 2), the highest dose of gefitinib that was given without dose-limiting toxicity (DLT) from part 1 was administered continuously beginning with the first cycle of chemotherapy. Three sequentially enrolled cohorts received gefitinib 250 and 500 mg (intermittently) and 500 mg (continuously).. We treated 24 patients; nine patients with 250 mg and 15 patients with 500 mg (nine patients continuous). Two occurrences of DLT were observed. One patient (500 mg, part 1) developed grade 3 rash and another patient (part 2) developed prolonged neutropenia. Steady-state gefitinib levels did not affect exposure to chemotherapy. In a limited sample, chemotherapy modestly increased the gefitinib area under concentration-time curve at steady-state and minimum steady-state trough concentration. Partial responses were observed in five of 24 patients. The median survival was 8 months.. The gefitinib with carboplatin and paclitaxel regimen was generally well tolerated and no unanticipated toxicities or clinically relevant pharmacokinetic interactions were observed. Both doses of gefitinib were believed to be safe for further study with chemotherapy. This regimen was thus tested in a completed randomized phase III trial.

Topics: Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Area Under Curve; Carboplatin; Carcinoma, Non-Small-Cell Lung; Dose-Response Relationship, Drug; Epidermal Growth Factor; Female; Gefitinib; Humans; Lung Neoplasms; Male; Middle Aged; Paclitaxel; Pilot Projects; Protein-Tyrosine Kinases; Quinazolines; Survival Rate; United States

We evaluated the efficacy and tolerability of the orally active, selective epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) ZD1839 in patients with pretreated advanced non-small cell lung cancer (NSCLC) participating in a compassionate use program.. Thirty-one patients with advanced, unresectable and progressive NSCLC, previously treated with one or two chemotherapy regimens, received ZD1839 250 mg orally once daily. Patients who had received only one prior chemotherapy regimen had to be considered unsuitable for second-line chemotherapy.. The disease control rate was 32% (95% CI: 15.8-48.7) (1/31 patients had a partial response and 9/31 patients had stable disease) and the median overall survival 23 weeks (range 4-40). Symptom improvement was reported by 39% of patients overall and by 83% of patients who achieved disease control. The median time to symptom improvement was 3 weeks (range 2-4). Adverse events were generally mild (grade I or II) and reversible and consisted mostly of skin rash, diarrhea and fatigue.. ZD1839 demonstrated clinically meaningful antitumor activity with significant improvement in symptoms in this heavily pretreated group of patients with advanced NSCLC. Furthermore, ZD1839 showed a favorable toxicity profile, with the majority of adverse events being mild and reversible.

Topics: Administration, Oral; Adult; Aged; Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Epidermal Growth Factor; Female; Gefitinib; Humans; Lung Neoplasms; Male; Middle Aged; Protein-Tyrosine Kinases; Quinazolines; Salvage Therapy; Treatment Outcome

We report four cases of Brain Metastases (BM) from non-small cell lung cancer (NSCLC) responding to ZD 1839 therapy after standard therapy failure.. Four patients with BM from NSCLC, pretreated with two or more lines of chemotherapy, received ZD 1839 (Iressa), on a compassionate use basis, at the daily dose of 250 mg until disease progression. Three patients received Iressa after whole brain radiotherapy (WBRT) failure.. After 3 months of ZD 1839 therapy, one patient had complete response on the brain with stabilization of extracranial disease, while the other three patients had partial response both on the brain and on extracranial sites. At the time of this analysis, two patients discontinued the treatment after 5 and 7 months for disease progression, while two patients are still on treatment with no evidence of treatment failure after 3+ and 12+ months. ZD 1839 was generally well tolerated, with skin toxicity recorded in two patients.. ZD 1839 may be effective in NSCLC patients with pretreated BM. Large and prospective trials need to clarify the role of ZD 1839 in the treatment of BM from NSCLC.

Topics: Aged; Antineoplastic Agents; Brain Neoplasms; Carcinoma, Non-Small-Cell Lung; Enzyme Inhibitors; Epidermal Growth Factor; Female; Gefitinib; Humans; Lung Neoplasms; Male; Middle Aged; Palliative Care; Quinazolines; Tomography, X-Ray Computed; Treatment Outcome

Even when diagnosed at the earliest stages, non-small-cell lung cancer (NSCLC) has often begun to metastasize, leading to frequent systemic relapses and a poor prognosis. There is an urgent need for effective adjuvant systemic therapy in conjunction with surgery to reverse or control further growth of micrometastases in early-stage NSCLC. Several approaches have been investigated in the search for such a therapy, including various chemotherapies, applied preoperatively or postoperatively, chemopreventive agents, and molecular-targeted agents. This article presents an overview of clinical trials for these adjunctive therapies, including completed trials and trials whose results are anticipated. Although standard postoperative chemotherapy has been found to offer little benefit, likely because of the acquisition of resistance in advanced tumors, some clinical trials with neoadjuvant or alternative chemotherapies have produced encouraging results. Targeted agents such as the epidermal growth factor receptor/tyrosine kinase inhibitor gefitinib have shown early promise for effective disease control. A combination of these new approaches and standard therapy for NSCLC may improve long-term survival in patients with lung cancer

Topics: Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Carcinoma, Non-Small-Cell Lung; Chemoprevention; Chemotherapy, Adjuvant; Drug Resistance; Epidermal Growth Factor; Gefitinib; Humans; Lung Neoplasms; Neoadjuvant Therapy; Neoplasm Metastasis; Prognosis; Protein-Tyrosine Kinases; Quinazolines; Radiotherapy, Adjuvant; Selenium; Survival Analysis

There is evidence of a relationship between epidermal growth factor (EGF) and tumor cell proliferation, such as the overexpression of EGF receptor (EGF-R) in different human tumors, which makes this system an interesting target for cancer treatment. Up to now, passive immunotherapy with monoclonal antibodies against the EGF-R has been assayed in clinics. Our approach consists of active immunotherapy with human EGF (hu-EGF). We conducted a pilot clinical trial to define the safety, toxicity and immunogenicity of vaccination with hu-EGF coupled to a carrier protein.. Ten patients with histologically-proven malignant carcinomas (colon, lung, stomach and prostate) in advanced clinical stages were enrolled. Patients were immunized twice (on days 0 and 15) with hu-EGF linked to either tetanic toxoid (TT, five patients) or P64K Neisseria Meningitidis recombinant protein (P64k, five patients), intradermically, using aluminium hydroxyde as adjuvant.. In both groups 60% of patients developed anti-EGF antibody titers without evidence of toxicity. Secondary reactions were very mild, limited to erythema and itching at the site of injection, which disappeared without medication.. We conclude that the proposed vaccination with hu-EGF was well tolerated and that antibody titers against self EGF were developed. The results of this trial may be useful in the design of new clinical trials with higher dose immunization protocols and using more effective adjuvants.

Topics: Aged; Cancer Vaccines; Carcinoma; Carrier Proteins; Colonic Neoplasms; Epidermal Growth Factor; Female; Humans; Immunotherapy; Lung Neoplasms; Male; Middle Aged; Pilot Projects; Prostatic Neoplasms; Stomach Neoplasms; Treatment Outcome

The value of tumor angiogenesis, EGFR and c-erbB-2 oncoprotein, a long with p 53 protein expression for predicting relapse-free survival was investigated in 110 node-negative breast cancer patients. The grade of neovascularization was assessed by the microvessel density which was obtained by an immunocytochemical staining by factor VIII-related antigen. EGFR, c-erbB-2 oncoprotein and p 53 oncoprotein were also determined by immunocytochemical assay. Univariate analysis showed no statistical significance of EGFR, c-erbB-2 and p53 status as a prognostic indicator. However, the microvessel density was a significant predictor of relapse-free survival. Patients with over 100 counts of factor VIII-RA positive cells per mm2 field in the most active areas of neovascularization showed significantly poorer prognosis compared to those with less than 100 counts (p < 0.005). Multivariate analysis demonstrated that microvessel density was an independent prognostic indicator in node-negative breast cancer patients (p < 0.0005). It was suggested that microvessel density might be of use in selecting the high-risk group in node-negative breast cancer patients needing adjuvant therapies.

Topics: Adenocarcinoma; Adenocarcinoma, Scirrhous; Axilla; Bone Neoplasms; Breast Neoplasms; Carcinoma, Papillary; Epidermal Growth Factor; Female; Humans; Immunohistochemistry; Lung Neoplasms; Lymph Nodes; Neovascularization, Pathologic; Prognosis; Receptor, ErbB-2; Regression Analysis; Tumor Suppressor Protein p53

Topics: Animals; Antibodies, Monoclonal; Bombesin; Clinical Trials as Topic; Epidermal Growth Factor; Gastrin-Releasing Peptide; Growth Inhibitors; Growth Substances; Humans; Insulin-Like Growth Factor I; Lung Neoplasms; Peptides; Transferrin

To investigate the mechanism of the effect of Astragalus membranaceus (A. membranaceus) on lung adenocarcinoma at the molecular level to elucidate the specific targets according to the network pharmacology approach.. The active components of A. membranaceus and their potential targets were collected from the Traditional Chinese Medicine Systems Pharmacology Database. Lung adenocarcinoma-associated genes were acquired based on GeneCards, Online Mendelian Inheritance in Man (OMIM), PharmGKB, and Therapeutic Targets databases. The PI3K/AKT signaling pathway-related genes were obtained using Reactome portal. Networks of "ingredient-target" and "ingredient-target-pathway-disease" were constructed using the Cytoscape3.6.0 software. The relationships among targets were analyzed according protein-protein interaction (PPI) network. Finally, molecular docking was applied to construct the binding conformation between active ingredients and core targets. Cell counting kit 8 (CCK8) and Western blot assays were performed to determine the mechanism of the key ingredient of A. membranaceus.. A total of 20 active components and their 329 targets, and 7,501 lung adenocarcinoma-related genes and 130 PI3K/AKT signaling pathway-related genes were obtained. According to Venn diagram and PPI network analysis, 2 mainly active ingredients, including kaempferol and quercetin, and 6 core targets, including TP53, MAPK1, EGF, AKT1, ERBB2, and EGFR, were identified. The two important active ingredients of A. membranaceus, kaempferol and quercetin, exert the therapeutic effect in lung adenocarcinoma partly by acting on the 6 core targets (TP53, MAPK1, EGF, AKT1, ERBB2, and EGFR) of PI3K/AKT signaling pathway. Expressions of potential targets in lung adenocarcinoma and normal samples were analyzed by using UALCAN portal and found that ERBB2 was overexpressed in lung adenocarcinoma tissues and upregulation of it correlated with clinicopathological characteristics. Finally, quercetin repressed viabilities of lung adenocarcinoma cells by targeting ERBB2 on PI3K/AKT signaling confirmed by CCK8 and Western blot.. Our finding unraveled that an active ingredient of A. membranaceus, quercetin, significantly inhibited the lung adenocarcinoma cells proliferation by repressing ERBB2 level and inactivating the PI3K/AKT signaling pathway.

Topics: Adenocarcinoma of Lung; Astragalus propinquus; Drugs, Chinese Herbal; Epidermal Growth Factor; ErbB Receptors; Humans; Kaempferols; Lung Neoplasms; Molecular Docking Simulation; Network Pharmacology; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Quercetin; Signal Transduction

Signal-transducing adaptor family member-2 (STAP-2) is an adaptor protein that regulates various intracellular signals. We previously demonstrated that STAP-2 binds to epidermal growth factor receptor (EGFR) and facilitates its stability and activation of EGFR signaling in prostate cancer cells. Inhibition of this interaction may be a promising direction for cancer treatment. Here, we found that 2D5 peptide, a STAP-2-derived peptide, blocked STAP-2-EGFR interactions and suppressed EGFR-mediated proliferation in several cancer cell lines. 2D5 peptide inhibited tumor growth of human prostate cancer cell line DU145 and human lung cancer cell line A549 in murine xenograft models. Additionally, we determined that EGFR signaling and its stability were decreased by 2D5 peptide treatment during EGF stimulation. In conclusion, our study shows that 2D5 peptide is a novel anticancer peptide that inhibits STAP-2-mediated activation of EGFR signaling and suppresses prostate and lung cancer progression.

Topics: A549 Cells; Adaptor Proteins, Signal Transducing; Animals; Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Humans; Lung Neoplasms; Male; Mice; Peptides; Prostatic Neoplasms; Signal Transduction

Epidermal growth factor receptor (EGFR) mutations (EGFRm) are common oncogene drivers in non-small cell lung cancer (NSCLC). This real-world study explored treatment patterns and time to receive EGFRm test results in patients with advanced EGFRm NSCLC.. A cross-sectional medical chart review was completed May-August 2020 in Australia, Canada, Germany, Italy, South Korea, Taiwan, UK, and USA. Eligible patients had advanced NSCLC and a positive EGFRm test result January-December 2017. Data were abstracted from NSCLC diagnosis to end of follow-up (31 March 2020) or patient's death whichever occurred earlier. The index date was the date of EGFRm confirmation.. 223 physicians provided data for 1,793 patients. Patients' mean age was 64.7 years, 54 % were male, 30.7 % had no history of smoking. Overall, 78 % of EGFRm test results were received ≤ 2 weeks after request (range of median 7-14 days across countries). Median time from advanced NSCLC diagnosis to EGFRm test result was 18 days (median range 10-22 days across countries). Over a third (37 %) of patients received a systemic treatment prior to EGFRm result; chemotherapy (25 %) and EGFR-TKI (15 %) were most commonly prescribed; post-EGFR test-result was EGFR-TKI (68 %); 80 % of patients initiated EGFR-TKI at any time point post-NSCLC diagnosis. Of those receiving a first-line EGFR-TKI post-EGFRm testing, 84 % received a TKI alone, 12 % in combination with chemotherapy, and 3 % with other treatments. Median time from first-line EGFR-TKI initiation post-EGFRm testing to first subsequent treatment was 19.8 months.. Over one-fifth of patients wait >14 days for their EGFRm test results, affecting their likelihood of receiving first-line EGFR-TKI with 20 % of patients never receiving EGFR TKI treatment. There was significant inter-country variability in the proportion of patients receiving EGFR TKIs. Our study highlights the need to improve EGFRm testing turnaround times and treatment initiation across countries.

Topics: Carcinoma, Non-Small-Cell Lung; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Lung Neoplasms; Male; Middle Aged; Mutation; Protein Kinase Inhibitors

Drug resistance severely limits the clinical therapeutic value of molecularly targeted drugs. Growth factors gain a tremendous amount of focus due to the ability to promote drug resistance in non-small-cell lung cancer (NSCLC). However, whether tumor cells themselves can mediate drug resistance by secreting growth factors needs further clarification. Here, we first screened growth factors to identify autocrine epidermal growth factor (EGF) and transforming growth factor alpha (TGF-α) that caused primary resistance to the ALK inhibitor TAE684 in H3122 cells and the c-MET-specific inhibitor SGX-523 in EBC-1 cells. Next, we discovered increased autocrine production of EGF and TGF-α in established acquired resistant H3122/TR and EBC-1/SR cells. Importantly, overexpression of EGF and TGF-α in two NSCLC cell lines produced resistance to TAE684 and SGX-523. Clinically, NSCLC patients with high expression of EGF and TGF-α developed primary resistance to crizotinib. Mechanistically, autocrine EGF and TGF-α activated EGFR signaling pathways to survive targeted c-Met and ALK inhibition. Furthermore, combined treatment with gefitinib circumvented EGF- and TGF-α-mediated primary and acquired resistance to TAE684/SGX-523. Taken together, these results suggested increased autocrine EGF and TGF-α conferred primary and acquired resistance to ALK/c-Met kinase inhibitors in NSCLC.

Topics: Anaplastic Lymphoma Kinase; Carcinoma, Non-Small-Cell Lung; Epidermal Growth Factor; ErbB Receptors; Humans; Lung Neoplasms; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-met; Transforming Growth Factor alpha

Current evidence indicates that immune checkpoint inhibitors (ICIs) have a limited efficacy in patients with lung cancer harboring epidermal growth factor receptor (EGFR) mutations. However, there is a lack of data on the efficacy of ICIs after osimertinib treatment, and the predictors of ICI efficacy are unclear.. We retrospectively assessed consecutive patients with EGFR-mutant NSCLC who received ICI-based therapy after osimertinib treatment at 10 institutions in Japan, between March 2016 and March 2021. Immunohistochemical staining was used to evaluate the expression of p53 and AXL. The deletions of exon 19 and the exon 21 L858R point mutation in EGFR were defined as common mutations; other mutations were defined as uncommon mutations.. A total of 36 patients with advanced or recurrent EGFR-mutant NSCLC were analyzed. In multivariate analysis, p53 expression in tumors was an independent predictor of PFS after ICI-based therapy (p = 0.002). In patients with common EGFR mutations, high AXL expression was a predictor of shorter PFS and overall survival after ICI-based therapy (log-rank test; p = 0.04 and p = 0.02, respectively).. The levels of p53 in pretreatment tumors may be a predictor of ICI-based therapy outcomes in patients with EGFR-mutant NSCLC after osimertinib treatment. High levels of AXL in tumors may also be a predictor of ICI-based therapy outcomes, specifically for patients with common EGFR mutations. Further prospective large-scale investigations on the predictors of ICI efficacy following osimertinib treatment are warranted.

Topics: Carcinoma, Non-Small-Cell Lung; Epidermal Growth Factor; ErbB Receptors; Humans; Immune Checkpoint Inhibitors; Lung Neoplasms; Mutation; Neoplasm Recurrence, Local; Protein Kinase Inhibitors; Retrospective Studies; Tumor Suppressor Protein p53

WW domain containing E3 ubiquitin protein ligase 2 (WWP2) is a member of the NEDD4 E3 ubiquitin ligase family. WWP2 ligase activity is regulated by the 2, 3-linker auto-inhibition. Tyrosine phosphorylation of the 2, 3-linker was identified as an activating means for releasing the auto-inhibition of WWP2. However, the tyrosine kinase (TK) for the phosphorylation and activation remains unknown. In this report, we have found that non-receptor TK ACK1 binds to the WW3 domain of WWP2 and phosphorylates WWP2. ACK1 phosphorylates WWP2 at the 2, 3-linker and partially activates the ubiquitination ligase activity. Unexpectedly, tyrosine phosphorylation of the 2, 3-linker seems not a major mode for activation of WWP2, as ACK1 causes much higher activation of the 2, 3-linker tyrosine phosphorylation defective mutants of WWP2 than that of wild-type WWP2. Furthermore, epidermal growth factor (EGF) stimulates tyrosine phosphorylation of WWP2 and this EGF-stimulated phosphorylation of WWP2 is mediated by ACK1. Finally, knockdown of WWP2 by shWWP2 inhibits the EGF-dependent cell proliferation of lung cancer A549 cells, suggesting that WWP2 may function in the EGFR signaling in lung cancer progression. Taken together, our findings have revealed a novel mechanism underlying activation of WWP2.

Topics: Epidermal Growth Factor; Humans; Lung Neoplasms; Protein-Tyrosine Kinases; Tyrosine; Ubiquitin-Protein Ligases

Abnormal expression of bicellular tight junction claudins, including claudin-2 are observed during carcinogenesis in human lung adenocarcinoma. However, little is known about the role of tricellular tight junction molecule angulin-1/lipolysis-stimulated lipoprotein receptor (LSR). In the lung adenocarcinoma tissues examined in the present study, expression of claudin-2 was higher than in normal lung tissues, while angulin-1/LSR was poorly or faintly expressed. We investigated how loss of angulin-1/LSR affects the malignancy of lung adenocarcinoma cell line A549 and normal human lung epithelial (HLE) cells. The EGF receptor tyrosine kinase inhibitor AG1478 prevented the increase of claudin-2 expression induced by EGF in A549 cells. Knockdown of LSR induced expression of claudin-2 at the protein and mRNA levels and AG1478 prevented the upregulation of claudin-2 in A549 cells. Knockdown of LSR induced cell proliferation, cell migration and cell metabolism in A549 cells. Knockdown of claudin-2 inhibited the cell proliferation but did not affect the cell migration or cell metabolism of A549 cells. The TGF-β type I receptor inhibitor EW-7197 prevented the decrease of LSR and claudin-2 induced by TGF-β1 in A549 cells and 2D culture of normal HLE cells. EW-7197 prevented the increase of cell migration and cell metabolism induced by TGF-β1 in A549 cells. EW-7197 prevented the increase of epithelial permeability of FITC-4kD dextran induced by TGF-β1 in 2.5D culture of normal HLE cells. In conclusion, downregulation of angulin-1/LSR induces malignancy via EGF-dependent claudin-2 and TGF-β-dependent cell metabolism in human lung adenocarcinoma.

Topics: A549 Cells; Adenocarcinoma of Lung; Claudin-2; Down-Regulation; Epidermal Growth Factor; Humans; Lung Neoplasms; Tight Junctions; Transforming Growth Factor beta; Transforming Growth Factor beta1; Up-Regulation

Metabolic remodeling is crucial in carcinogenesis and cancer progression. Oncogenic mutations may promote metabolic reprogramming in cancer cells to support their energy and biomass requirements. EGFR mutations are commonly found in non-small cell lung cancer (NSCLC) and may induce NSCLC metabolic rewiring. Whether EGFR-driven metabolic reprogramming triggers cell vulnerabilities with therapeutic potential remains unknown.. The role of EGFR signaling activation by EGF was investigated using NSCLC cell lines with different EGFR and KRAS status: A549 (EGFR WT and KRAS c.34G > A), H292 (EGFR WT and KRAS WT) and PC-9 (EGFR exon 19 E746-A750 deletion and KRAS WT). The effect of EGF on NSCLC cell death and cell cycle was evaluated using flow cytometry, and cell migration was assessed through wound healing. EGFR, HER2, MCT1, and MCT4 expression was analyzed through immunofluorescence or western blotting. We explored the impact of glucose and lactate bioavailability on NSCLC cells' metabolic profile using nuclear magnetic resonance (NMR) spectroscopy. Moreover, the expression of several relevant metabolic genes in NSCLC cells or patient samples was determined by RT-qPCR.. We showed that cell lines presented different metabolic profiles, and PC-9 cells were the most responsive to EGF stimulus, as they showed higher rates of cell proliferation and migration, together with altered metabolic behavior. By inhibiting EGFR with gefitinib, a decrease in glucose consumption was observed, which may be related to the fact that despite PC-9 harbor EGFR mutation, they still express the EGFR WT allele. The analysis of NSCLC patients' RNA showed a correlation between MCT1/MCT4 and GLUT1 expression in most cases, indicating that the metabolic information can serve as a reference in patients' follow-up.. Together, this study shows that NSCLC cell lines have heterogeneous metabolic profiles, which may be underlaid by different genetic profiles, revealing an opportunity to identify and stratify patients who can benefit from metabolism-targeted therapies.

Topics: Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Humans; Lung Neoplasms; Mutation; Proto-Oncogene Proteins p21(ras)

Epidermal growth factor receptor (EGFR) abnormalities relevant to tumor progression. A newly developed strategy for cancer therapy is induction of EGFR degradation. GMI, an immunomodulatory protein from the medicinal mushroom Ganoderma microsporum, exhibits anticancer activity. However, its role in the intracellular trafficking and degradation of EGFR remains unclear. In this study, we discovered that GMI inhibits the phosphorylation of multiple tyrosine kinases. Specifically, GMI was discovered to suppress lung cancer cells harboring both wild-type and mutant EGFR by inhibiting EGFR dimerization and eliminating EGFR-mediated signaling. Functional studies revealed that GMI binds to the extracellular segment of EGFR. GMI interacts with EGFR to induce phosphorylation of EGFR at tyrosine1045, which triggers clathrin-dependent endocytosis and degradation of EGFR. Furthermore, in the mouse models, GMI was discovered to suppress tumor growth. Knockdown of EGFR in lung cancer cells abolishes GMI's anticancer activity in vivo and in vitro. Our results reveal the interaction mechanisms through which GMI induces EGFR degradation and abolishes EGFR-mediated intracellular pathway. Our study indicates that GMI is an EGFR degrader for inhibiting EGFR-expressing tumor growth.

Topics: Animals; Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Ganoderma; Lung Neoplasms; Mice; Phosphorylation

Signaling bias is the ability of a receptor to differentially activate downstream signaling pathways in response to different ligands. Bias investigations have been hindered by inconsistent results in different cellular contexts. Here we introduce a methodology to identify and quantify bias in signal transduction across the plasma membrane without contributions from feedback loops and system bias. We apply the methodology to quantify phosphorylation efficiencies and determine absolute bias coefficients. We show that the signaling of epidermal growth factor receptor (EGFR) to EGF and TGFα is biased towards Y1068 and against Y1173 phosphorylation, but has no bias for epiregulin. We further show that the L834R mutation found in non-small-cell lung cancer induces signaling bias as it switches the preferences to Y1173 phosphorylation. The knowledge gained here challenges the current understanding of EGFR signaling in health and disease and opens avenues for the exploration of biased inhibitors as anti-cancer therapies.

Topics: Carcinoma, Non-Small-Cell Lung; Epidermal Growth Factor; ErbB Receptors; Humans; Ligands; Lung Neoplasms; Mutation; Phosphorylation

Circular RNA (circRNA) is a covalently closed endogenous RNA that participates in disease progression. However, its role in lung cancer is largely undetermined. In the present study, we found an onctogenic circRNA in lung cancer, FAT atypical cadherin 3 (FAT3) circRNA (circ-FAT3) was remarkably upregulated in lung cancer in comparison to paired normal tissues. High circ-FAT3 was closely linked to larger tumour size, lymph node metastasis, later clinical stage, as well as dismal outcome. Stable knockdown of circ-FAT3 inhibited cell proliferation and metastasis both in vitro and in vivo. RNA binding protein ELAV like RNA binding protein 1 (HuR) was found to bind to introns flanking circ-FAT3, promoting the cyclization and generation of circ-FAT3. Further, circ-FAT3 was able to sponge miR-136-5p by acting as a competing endogenous RNA (ceRNA), alleviating the repressive effect of miR-136-5p on HuR mRNA at the transcriptional and post-transcriptional levels. Moreover, circ-FAT3 expression in lung cancer tissues was strongly positively and negatively correlated with HuR and miR-136-5p expression, respectively. Overall, our data reveal the previously uncharacterized regulatory loop of circ-FAT3/miR-136-5p/HuR in lung cancer and provide novel evidence for the importance of circRNA as a ceRNA in tumorigenesis.

Topics: Cadherins; Carcinogenesis; Cell Line, Tumor; Cell Proliferation; ELAV-Like Protein 1; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; MicroRNAs; RNA-Binding Proteins; RNA, Circular

Lung cancer is the leading cause of cancer-related deaths with non-small cell lung cancer (NSCLC) being the most common of them. About a third of NSCLC cases have an epidermal growth factor (EGFR) mutation, which is usually susceptible to tyrosine kinase inhibitors (TKIs). In rare cases where patients progress through TKI therapy, the use of immune checkpoint inhibitors (ICIs) remains controversial.. We describe a case of a patient with significant history of smoking and EGFR mutated programmed death ligand-1 (PD-L1) positive NSCLC who was initially treated with TKI therapy.. While patient progressed on TKI therapy, he was able to achieve a durable response with a single PD-L1 agent, pembrolizumab. Contrary to the available evidence, the presented EGFR mutant NSCLC responded to PD-L1 pathway inhibition.. From our observation Pembrolizumab could be promising in patients with rare EGFR mutations who do not respond to EGFR directed therapy. Our report provides supporting data for the use of immunotherapies in patients with EGFR mutated NSCLC.

Topics: Antibodies, Monoclonal, Humanized; B7-H1 Antigen; Carcinoma, Non-Small-Cell Lung; Epidermal Growth Factor; ErbB Receptors; Humans; Lung Neoplasms; Male; Mutagenesis, Insertional; Mutation; Protein Kinase Inhibitors

Topics: Afatinib; Epidermal Growth Factor; Humans; Lung Neoplasms; Mutation; Small Cell Lung Carcinoma

YAP1, a key mediator of the Hippo pathway, plays an important role in tumorigenesis. Alternative splicing of human YAP1 mRNA results in two major isoforms: YAP1-1, which contains a single WW domain, and YAP1-2, which contains two WW domains, respectively. We here investigated the functions and the underlying regulatory mechanisms of the two YAP1 isoforms in the context of EGF-induced epithelial-mesenchymal transition (EMT) in non-small cell lung cancer (NSCLC). Human NSCLC cell lines express both YAP1-1 and YAP1-2 isoforms-although when compared to YAP1-1, YAP1-2 mRNA levels are higher while its protein expression levels are lower. EGF treatment significantly promoted YAP1 expression as well as EMT process in NSCLCs, whereas EGF-induced EMT phenotype was significantly alleviated upon YAP1 knockdown. Under normal culture condition, YAP1-1 stable expression cells exhibited a stronger migration ability than YAP1-2 expressing cells. However, upon EGF treatment, YAP1-2 stable cells showed more robust migration than YAP1-1 expressing cells. The protein stability and nuclear localization of YAP1-2 were preferentially enhanced with EGF treatment. Moreover, EGF-induced EMT and YAP1-2 activity were suppressed by inhibitor of AKT. Our results suggest that YAP1-2 is the main isoform that is functionally relevant in promoting EGF-induced EMT and ultimately NSCLC progression.

Topics: Adaptor Proteins, Signal Transducing; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; Humans; Lung Neoplasms; Transcription Factors; YAP-Signaling Proteins

Osimertinib as first-line treatment for patients with non-small cell lung cancer (NSCLC) harboring epidermal growth factor (EGFR) mutations remains controversial. Sequential EGFR-tyrosine kinase inhibitor (TKI) might be superior to the first line osimertinib in patients at risk of developing acquired T790M mutations.. We enrolled consecutive patients with EGFR-mutated (deletion 19 or L858R) advanced NSCLC treated with first-line drugs and evaluated predictive markers using classification and regression tree (CART) for the detection of T790M mutations based on patient backgrounds prior to initial treatment.. Patients without acquired T790M mutations had worse outcomes than those with T790M mutations (median OS: 798 days vs. not reached; HR: 2.70; P < 0.001). CART identified three distinct groups based on variables associated with acquired T790M mutations (age, CYF, WBC, liver metastasis, and LDH; AUROC: 0.77). Based on certain variables, CART identified three distinct groups in deletion 19 (albumin, LDH, bone metastasis, pleural effusion, and WBC; AUROC: 0.81) and two distinct groups in L858R (age, CEA, and ALP; AUROC: 0.80). The T790M detection frequencies after TKI resistance of afatinib and first-generation EGFR-TKIs were similar (35.3% vs. 37.4%, P = 0.933). Afatinib demonstrated longer PFS (398 vs. 279 days; HR: 0.67; P = 0.004) and OS (1053 vs. 956 days; HR: 0.68; P = 0.051) than first-generation EGFR-TKIs.. Identification of patients at risk of acquiring T790M mutations after EGFR-TKI failure may aid in choice of first-line EGFR-TKI. Furthermore, afatinib may be the more effective 1st-line EGFR-TKI treatment for patients at risk of developing T790M as initial EGFR-TKI resistance.

Topics: Afatinib; Carcinoma, Non-Small-Cell Lung; Epidermal Growth Factor; ErbB Receptors; Humans; Lung Neoplasms; Mutation; Protein Kinase Inhibitors

Chemotherapy is the primary treatment for advanced non-small-cell lung cancer (NSCLC). However, related dose-dependent toxicity limits its clinical use. Therefore, it is necessary to explore new strategies for improving the clinical outcomes while reducing the side effects of chemotherapy in the treatment of NSCLC. In this study, we designed and synthesized epidermal growth factor (EGF)-modified doxorubicin nanoparticles (EGF@DOX-NPs) that selectively targets the epidermal growth factor receptor (EGFR) overexpressed in lung tumor cells. When administered in combination with low-dose X-ray radiotherapy (RT), the NPs preferentially accumulated at the tumor site due to radiation-induced outburst of the local intra-tumoral blood vessels. Compared with DOX alone, EGF@DOX-NPs significantly decreased the viability and migration and enhanced the apoptosis rates of tumor cells

Topics: Animals; Antibiotics, Antineoplastic; Apoptosis; Biomimetics; Carcinoma, Non-Small-Cell Lung; Cell Proliferation; Chemistry, Pharmaceutical; Chemoradiotherapy; Doxorubicin; Drug Carriers; Drug Liberation; Epidermal Growth Factor; ErbB Receptors; Lung Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Nanoparticles; Particle Size; Surface Properties; Xenograft Model Antitumor Assays

There has been great interest in identifying the biological substrate for light-cell interaction and their relations to cancer treatment. In this study, a near-infrared (NIR) laser is focused into the nucleus (nNIR) or cytoplasm (cNIR) of a single living cell by a high numerical aperture condenser to dissect the novel role of cell nucleus in mediating NIR effects on mitochondrial dynamics of A549 non-small cell lung cancer cells. Our analysis showed that nNIR, but not cNIR, triggered mitochondrial fission in 10 min. In contrast, the fission/fusion balance of mitochondria directly exposed to cNIR does not change. While the same phenomenon is also triggered by single molecular interactions between epidermal growth factor (EGF) and its receptor EGFR, pharmacological studies with cetuximab, PD153035, and caffeine suggest EGF signaling crosstalk to DNA damaging response to mediate rapid mitochondrial fission as a result of nNIR irradiation. These results suggest that nuclear DNA integrity is a novel biological target for cellular response to NIR.

Topics: A549 Cells; Cell Nucleus; DNA Damage; Epidermal Growth Factor; ErbB Receptors; Humans; Lung Neoplasms; Mitochondrial Dynamics; Radiation

Non-small cell lung cancer (NSCLC) accounts for the majority (80-85%) of all lung cancers. All current available treatments have limited efficacy. The epidermal growth factor receptor (EGFR) plays a critical role in the development and progression of NSCLC, with high EGFR expression associated with increased cell proliferation and poor prognosis. Thus, interfering with EGFR signaling has been shown to effectively reduce cell proliferation and help in the treatment of NSCLC. We previously demonstrated that the progesterone receptor (PR) contains a polyproline domain (PPD) that directly interacts with Src homology 3 (SH3) domain-containing molecules and expression of PR-PPD peptides inhibits NSCLC cell proliferation. In this study, we investigated whether the introduction of PR-PPD by cell-penetrating peptides (CPPs) could inhibit EGF-induced cell proliferation in NSCLC cells. PR-PPD was attached to a cancer-specific CPP, Buforin2 (BR2), to help deliver the PR-PPD into NSCLC cells. Interestingly, addition of BR2-2xPPD peptides containing two PR-PPD repeats was more effective in inhibiting NSCLC proliferation and significantly reduced EGF-induced phosphorylation of Erk1/2. BR2-2xPPD treatment induced cell cycle arrest by inhibiting the expression of cyclin D1 and CDK2 genes in EGFR-wild type A549 cells. Furthermore, the combination treatment of EGFR-tyrosine kinase inhibitors (TKIs), including Gefitinib or Erlotinib, with BR2-2xPPD peptides further suppressed the growth of NSCLC PC9 cells harboring EGFR mutations as compared to EGFR-TKIs treatment alone. Importantly, BR2-2xPPD peptides mediated growth inhibition in acquired Gefitinib- and Erlotinib- resistant lung adenocarcinoma cells. Our data suggests that PR-PPD is the minimal protein domain sufficient to inhibit NSCLC cell growth and has the potential to be developed as a novel NSCLC therapeutic agent.

Topics: Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Cell-Penetrating Peptides; Drug Resistance, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Erlotinib Hydrochloride; Gefitinib; Humans; Lung Neoplasms; Peptides; Protein Kinase Inhibitors; Receptors, Progesterone

Epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) resistance may be acquired via genotypic and/or phenotypic transformations. Herein, we report an extremely uncommon case with sequential small cell transformation and EGFR T790M mutation, in an elderly female with EGFR exon 21 L858R-mutant lung adenocarcinoma, following treatment with a first-generation EGFR-TKI.. A 67-year-old female never-smoker presented with a cough and dyspnoea of 2 months' duration. Computerised tomography revealed a 39 mm lesion in the upper lobe of the right lung with pleural effusion. Pleural fluid cytology revealed metastatic lung adenocarcinoma, and EGFR testing revealed exon 21 L858R mutation. She was started on gefitinib. After a progression-free survival of 31 months, she presented with disease progression and multiple extra-thoracic metastases. Fine needle aspiration cytology of a chest wall lesion revealed metastatic small cell carcinoma. EGFR testing on this aspirate revealed persistent L858R mutation only. In view of small cell transformation, chemotherapy (etoposide and carboplatin) was administered. After 4 months, ascitic fluid cytology revealed metastatic adenocarcinoma with persistent L858R mutation and an acquired T790M mutation (both detected on liquid biopsy as well) indicating amplification of the adenocarcinoma clone and regression of the small cell carcinoma clone. She was then initiated on osimertinib.. The index case highlights the significance of serial EGFR genotyping along with repeated tissue and/or blood sampling in the prompt detection of genetic and phenotypic resistance mechanisms to EGFR-TKIs. Furthermore, it lends evidence in support of the upfront treatment approaches targeting the heterogeneity of acquired EGFR-TKI resistance mechanisms.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Aged; Carboplatin; Carcinoma, Small Cell; Drug Resistance, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Etoposide; Female; Gefitinib; Humans; Lung Neoplasms; Mutation; Protein Kinase Inhibitors

Topics: Avena; Cell Line, Tumor; Edible Grain; Epidermal Growth Factor; ErbB Receptors; Humans; Lung Neoplasms; ortho-Aminobenzoates; Protein Kinase Inhibitors

Topics: Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Drug Resistance, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Humans; Lung Neoplasms; Mutation; Protein Kinase Inhibitors; Signal Transduction

The epidermal growth factor receptor (EGFR) represents a top therapeutic target in the treatment of non-small cell lung cancer. EGFR expression is intricately modulated by receptor endocytosis, during which EGFR ubiquitylation and deubiquitylation play fundamental roles to govern receptor fate. This study aims to uncover novel aspects of the endocytic regulation of EGFR trafficking by deubiquitylases.. The expression and ubiquitylation of EGFR in non-small cell lung cancer cells treated with deubiquitylase inhibitors were assessed by immunoblotting, immunoprecipitation and mass spectrometry analyses. The intracellular EGFR distribution was investigated using immunofluorescence and confocal microscopy assays, and colocalizations with endocytic compartments were examined using GFP-tagged Rab proteins as markers. The influence of the proteasomal deubiquitylase inhibitor b-AP15 on EGF- and HSP90 inhibitor-induced EGFR downregulation was evaluated by immunoblotting. The anticancer effects of b-AP15 were assessed by cell proliferation, colony formation and flow cytometry assays, as well as xenograft animal models.. We found that b-AP15 caused a dramatically enhanced ubiquitylation of EGFR in lung cancer cells. Treatment with b-AP15 decreased cell surface EGFR levels and accumulated EGFR on recycling endosomes marked with Rab4A and Rab11A. b-AP15 effectively repressed EGF- and HSP90 inhibitor-induced EGFR degradation. Lung cancer cells exposed to b-AP15 showed markedly reduced cell propagation and significantly increased cell apoptosis. Furthermore, b-AP15 effectively inhibited tumor xenograft growth in nude mice.. Proteasomal USP14 and UCHL5 act collectively to promote cell surface recovery of EGFR. Inhibition of proteasomal deubiquitylase activity induces increased EGFR ubiquitylation and retention on recycling endosomes. The USP14 and UCHL5 dual inhibitor b-AP15 elicits potent tumor-suppressive effects to deter cell proliferation and induce apoptotic cell death in lung cancer.

Topics: Animals; Antineoplastic Agents; Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Humans; Lung Neoplasms; Mice; Mice, Nude; Proteasome Inhibitors; Ubiquitin Thiolesterase

Topics: B7 Antigens; B7-H1 Antigen; Epidermal Growth Factor; Humans; Lung Neoplasms; Neoplastic Cells, Circulating; Neoplastic Stem Cells; Nivolumab; Pilot Projects; Programmed Cell Death 1 Receptor; Small Cell Lung Carcinoma

Cr(VI) is broadly applied in industry. Cr(VI) exposure places a big burden on public health, thereby increasing the risk of lung squamous cell carcinoma (LUSC). The mechanisms underlying Cr(VI)-induced LUSC remain largely elusive. Here, we report that the cancer stem cell (CSC)/tumour-initiating cell (TIC)-like subgroup within Cr(VI)-transformed bronchial epithelial cells (CrT) promotes lung cancer tumourigenesis. Mechanistically, Cr(VI) exposure specifically increases the expression levels of aldehyde dehydrogenase 1A1 (ALDH1A1), a CSC marker, through KLF4-mediated transcription. ALDH1A1 maintains self-renewal of CrT/TICs and facilitates the expression and secretion of EGF from CrT/TICs, which subsequently promotes the activation of EGFR signalling in differentiated cancer cells and tumour growth of LUSC. In addition, the ALDH1A1 inhibitor A37 and gemcitabine synergistically suppress LUSC progression. Importantly, high ALDH1A1 expression levels are positively correlated with advanced clinical stages and predict poor survival in LUSC patients. These findings elucidate how ALDH1A1 modulates EGF secretion from TICs to facilitate LUSC tumourigenesis, highlighting new therapeutic strategies for malignant lung cancers.

Topics: Aldehyde Dehydrogenase; Aldehyde Dehydrogenase 1 Family; Carcinogenesis; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Cell Transformation, Neoplastic; Epidermal Growth Factor; Humans; Lung; Lung Neoplasms; Neoplastic Processes; Retinal Dehydrogenase; Tics

Extracellular vesicles (EVs) are cell-derived structures surrounded by a lipid bilayer that carry RNA and DNA as potential templates for molecular diagnostics, e.g., in cancer genotyping. While it has been established that DNA templates appear on the outside of EVs, no consensus exists on which nucleic acid species inside small EVs (<200 nm, sEVs) are sufficiently abundant and accessible for developing genotyping protocols. We investigated this by extracting total intravesicular nucleic acid content from sEVs isolated from the conditioned cell medium of the human NCI-H1975 cell line containing the epidermal growth factor (EGFR) gene mutation T790M as a model system for non-small cell lung cancer. We observed that mainly short genomic DNA (<35−100 bp) present in the sEVs served as a template. Using qEV size exclusion chromatography (SEC), significantly lower yield and higher purity of isolated sEV fractions were obtained as compared to exoEasy membrane affinity purification and ultracentrifugation. Nevertheless, we detected the EGFR T790M mutation in the sEVs’ lumen with similar sensitivity using digital PCR. When applying SEC-based sEV separation prior to cell-free DNA extraction on spiked human plasma samples, we found significantly higher mutant allele frequencies as compared to standard cell-free DNA extraction, which in part was due to co-purification of circulating tumor DNA. We conclude that intravesicular genomic DNA can be exploited next to ctDNA to enhance EGFR T790M mutation detection sensitivity by adding a fast and easy-to-use sEV separation method, such as SEC, upstream of standard clinical cell-free DNA workflows.

Topics: Carcinoma, Non-Small-Cell Lung; Cell-Free Nucleic Acids; Chromatography, Gel; Circulating Tumor DNA; Epidermal Growth Factor; ErbB Receptors; Genomics; Humans; Lung Neoplasms; Mutation; Oncogenes; Protein Kinase Inhibitors

Topics: Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Endothelial Cells; Epidermal Growth Factor; ErbB Receptors; Humans; Ligands; Lung Neoplasms; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-2; Xenograft Model Antitumor Assays

Topics: Antineoplastic Combined Chemotherapy Protocols; Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Clinical Trials, Phase IV as Topic; Cytokines; Epidermal Growth Factor; Humans; Lung Neoplasms; Lymphocyte Subsets; Prognosis

The most frequent mechanism of resistance after 1st/2nd-generation (G) epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) is secondary point mutation Thr790Met (T790M) in EGFR. Afatinib followed by osimertinib (Afa group) may provide better outcomes for T790M-positive non-small cell lung cancer (NSCLC) than 1st-G EGFR-TKI followed by osimertinib (1st-G group). We studied 111 consecutive NSCLC patients with T790M mutation treated with osimertinib after progression following 1st/2nd-G EGFR-TKI between March 28, 2016 and March 31, 2018. We analyzed the ratio of T790M to EGFR-activating mutation (T790M ratio) in post EGFR-TKI resistance re-biopsy tissue using droplet digital polymerase chain reaction. And investigated whether afatinib purified the T790M mutation more than 1st-G EGFR-TKI. Among 60 patients with preserved re-biopsy tissue, we analyzed 38 having adequate DNA content. The response rate in Afa group was 81.8% (n = 11) and 1st-G group was 85.2% (n = 27). The mean T790M ratio in total population was 0.3643. The ratio in those with response to osimertinib was significantly higher than in the non-responders (0.395, 0.202; p = 0.0231) and was similar in Afa and 1st-G group (0.371, 0.362; p = 0.9693). T790M ratio significantly correlated with osimertinib response and was similar between the 1st/2nd-G EGFR-TKIs in 1st/2nd-G EGFR-TKI-refractory tumors.

Topics: Acrylamides; Adult; Aged; Aged, 80 and over; Aniline Compounds; Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Epidermal Growth Factor; Female; Humans; Lung Neoplasms; Male; Middle Aged; Mutation; Protein Kinase Inhibitors; Treatment Outcome

Epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) can effectively inhibit the growth of EGFR-dependent mutant non-small cell lung cancer (NSCLC). Unfortunately, NSCLC patients often develop severe drug resistance after long-term EGFR-TKI treatment. Studies have shown that the disorder of energy metabolism in tumor cells can induce EGFR-TKI resistance. Due to the drug action, gene mutation and other factors, tumor cells undergo metabolic reprogramming, which increases the metabolic rate and intensity of tumor cells, promotes the intake and synthesis of nutrients (such as sugar, fat and glutamine), forms a microenvironment conducive to tumor growth, enhances the bypass activation, phenotype transformation and abnormal proliferation of tumor cells, and inhibits the activity of immune cells and apoptosis of tumor cells, ultimately leading to drug resistance of tumor cells to EGFR-TKI. Therefore, targeting energy metabolism of NSCLC may be a potential way to alleviate TKI resistance.. 表皮生长因子受体酪氨酸激酶抑制剂(epidermal growth factor receptor tyrosine kinase inhibitor，EGFR-TKI)能够有效地抑制EGFR基因突变驱动型非小细胞肺癌(non-small cell lung cancer，NSCLC)的生长，但NSCLC患者在经过长时间持续的EGFR-TKI治疗后往往会发生严重的耐药。研究表明肿瘤细胞能量代谢紊乱对EGFR-TKI耐药的产生具有重要的诱导作用。在药物、基因突变等多种因素的作用下肿瘤细胞会发生代谢重编程，上调肿瘤细胞的代谢速率和强度，促进肿瘤对糖、脂肪、谷氨酰胺等营养物质的摄入和利用，并形成有利于肿瘤生长的微环境，促进肿瘤细胞的旁路激活、表型转化、异常增殖等，从而抑制免疫细胞的活性和肿瘤细胞的凋亡，导致肿瘤细胞对EGFR-TKI产生耐药性。.

Topics: Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Drug Resistance, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Humans; Lung Neoplasms; Mutation; Protein Kinase Inhibitors; Tumor Microenvironment

The serine protease prostasin is a negative regulator of lipopolysaccharide-induced inflammation and has a role in the regulation of cellular immunity. Prostasin expression in cancer cells inhibits migration and metastasis, and reduces epithelial-mesenchymal transition. Programmed death-ligand 1 (PD-L1) is a negative regulator of the immune response and its expression in cancer cells interferes with immune surveillance. The aim of the present study was to investigate if prostasin regulates PD-L1 expression. We established sublines overexpressing various forms of prostasin as well as a subline deficient for the prostasin gene from the Calu-3 human lung cancer cells. We report here that PD-L1 expression induced by interferon-γ (IFNγ) is further enhanced in cells overexpressing the wildtype membrane-anchored prostasin. The PD-L1 protein was localized on the cell surface and released into the culture medium in extracellular vesicles (EVs) with the protease-active prostasin. The epidermal growth factor-epidermal growth factor receptor (EGF-EGFR), protein kinase C (PKC), and mitogen-activated protein kinase (MAPK) participated in the prostasin-mediated up-regulation of PD-L1 expression. A Gene Set Enrichment Analysis (GSEA) of patient lung tumors in The Cancer Genome Atlas (TCGA) database revealed that prostasin and PD-L1 regulate common signaling pathways during tumorigenesis and tumor progression.

Topics: Adenocarcinoma of Lung; B7-H1 Antigen; Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Extracellular Vesicles; Gene Expression Regulation, Neoplastic; Humans; Interferon-gamma; Lung Neoplasms; Mitogen-Activated Protein Kinases; Protein Kinase C; Serine Endopeptidases; Signal Transduction; Up-Regulation

Topics: Carcinoma, Non-Small-Cell Lung; Epidermal Growth Factor; ErbB Receptors; Humans; Lung Neoplasms; Mutation

Thrombopoietin (TPO) is a haematopoietic cytokine mainly produced by the liver and kidneys, which stimulates the production and maturation of megakaryocytes. In the past decade, numerous studies have investigated the effects of TPO outside the haematopoietic system; however, the role of TPO in the progression of solid cancer, particularly lung cancer, has not been well studied. Exogenous TPO does not affect non-small-cell lung cancer (NSCLC) cells as these cells show no or extremely low TPO receptor expression; therefore, in this study, we focused on endogenous TPO produced by NSCLC cells. Immunohistochemical analysis of 150 paired NSCLC and adjacent normal tissues indicated that TPO was highly expressed in NSCLC tissues and correlated with clinicopathological parameters including differentiation, P-TNM stage, lymph node metastasis and tumour size. Suppressing endogenous TPO by small interfering RNA inhibited the proliferation and migration of NSCLC cells. Moreover, TPO interacted with the EGFR protein and delayed ligand-induced EGFR degradation, thus enhancing EGFR signalling. Notably, overexpressing TPO in EGF-stimulated NSCLC cells facilitated cell proliferation and migration, whereas no obvious changes were observed without EGF stimulation. Our results suggest that endogenous TPO promotes tumorigenicity of NSCLC via regulating EGFR signalling and thus could be a therapeutic target for treating NSCLC.

Topics: Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Ligands; Lung Neoplasms; Male; Middle Aged; Phosphatidylinositol 3-Kinases; Protein Binding; Proteolysis; Proto-Oncogene Proteins c-akt; Signal Transduction; Subcellular Fractions; Thrombopoietin

Ascofuranone, an isoprenoid antibiotic initially purified from a culture broth of Ascochyta viciae, has multiple anticancer effects. However, the impacts of ascofuranone on the epithelial-mesenchymal transition (EMT) and epidermal growth factor (EGF)-induced effects on human lung cancer cell lines have not been previously reported. Here, we show that ascofuranone exerts its anticancer effects by inhibiting the EGF-induced EMT and cell migration in human lung cancer cell lines. Ascofuranone significantly inhibited EGF-induced migration and invasion by lung cancer cells, and suppressed EGF-induced morphologic changes by regulating the expression of EMT-associated proteins. In addition, ascofuranone upregulated E-cadherin, and downregulated fibronectin, vimentin, Slug, Snail, and Twist. Inhibition of ERK/AKT/mTOR promoted EGF-induced E-cadherin downregulation and inhibited EGF-induced vimentin upregulation in response to ascofuranone, implying that inhibition of the EGF-induced EMT by ascofuranone was mediated by the ERK and AKT/mTOR pathways. Inhibition of c-Myc suppressed EGF-induced vimentin upregulation, suggesting the involvement of c-Myc. Collectively, these findings suggest that ascofuranone inhibits tumor growth by blocking the EGF-induced EMT through a regulatory mechanism involving ERK, AKT/mTOR, and c-Myc in lung cancer cells.

Topics: Antineoplastic Agents; Cell Line, Tumor; Cell Movement; Cell Survival; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; Extracellular Signal-Regulated MAP Kinases; Humans; Lung Neoplasms; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-myc; Sesquiterpenes; TOR Serine-Threonine Kinases; Wound Healing

Although new generations of EGFR-tyrosine kinase inhibitors (EGFR-TKI) have been developed for the treatment of patients with non-small cell lung cancer (NSCLC) with EGFR-mutant tumors, TKI resistance often returns as a result of additional EGFR mutations. In addition to seeking for next-generation EGFR-TKI, developing novel EGFR-targeting strategies may hold the key to overcome the vicious cycle of TKI resistance. Endocan is known as a receptor tyrosine kinase ligand enhancer in tumorigenesis, but the impact of endocan on EGFR-driven NSCLC progression remains unknown. In this study, higher endocan levels were found in lung tumors compared with cancer-free tissues and correlated with poor prognosis in patients with NSCLC harboring mutant EGFR; circulating endocan levels were also significantly higher in patients with mutant EGFR. Endocan facilitated EGFR signaling via direct binding and enhancing of the EGF-EGFR interaction and supported the growth of tumors driven by mutated EGFR. Activated EGFR in turn upregulated expression of endocan via JAK/STAT3 and ERK/ELK cascades, thus forming a positive regulatory loop of endocan-EGFR signaling. On the basis of the binding region between endocan and EGFR, we designed therapeutic peptides and demonstrated promising therapeutic effects in xenografts harboring EGFR mutations including TKI-resistant T790M. Together, our findings highlight the novel interaction between endocan and EGFR and new opportunities to effectively target endocan-EGFR regulatory axis in patients with TKI-resistant NSCLC. SIGNIFICANCE: Endocan is a novel and critical regulator of EGF/EGFR signaling and serves as an alternative target of EGFR-TKI resistance in NSCLC.

Topics: Animals; Carcinoma, Non-Small-Cell Lung; cdc25 Phosphatases; Cell Line, Tumor; Cell Proliferation; Cell Survival; Disease Progression; Drug Resistance, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Gene Expression; Genes, ras; Heterografts; Humans; Janus Kinases; Lung; Lung Neoplasms; MAP Kinase Signaling System; Mice; Mice, Inbred NOD; Mice, SCID; Mutation; Neoplasm Proteins; Prognosis; Protein Kinase Inhibitors; Proteoglycans; Receptor Cross-Talk; RNA, Messenger; STAT3 Transcription Factor; Up-Regulation

Immunosenescence biomarkers and peripheral blood parameters are evaluated separately as possible predictive markers of immunotherapy. Here, we illustrate the use of a causal inference model to identify predictive biomarkers of CIMAvaxEGF success in the treatment of Non-Small Cell Lung Cancer Patients.. Data from a controlled clinical trial evaluating the effect of CIMAvax-EGF were analyzed retrospectively, following a causal inference approach. Pre-treatment potential predictive biomarkers included basal serum EGF concentration, peripheral blood parameters and immunosenescence biomarkers. The proportion of CD8 + CD28- T cells, CD4+ and CD8+ T cells, CD4/CD8 ratio and CD19+ B cells. The 33 patients with complete information were included. The predictive causal information (PCI) was calculated for all possible models. The model with a minimum number of predictors, but with high prediction accuracy (PCI > 0.7) was selected. Good, rare and poor responder patients were identified using the predictive probability of treatment success.. The mean of PCI increased from 0.486, when only one predictor is considered, to 0.98 using the multivariate approach with all predictors. The model considering the proportion of CD4+ T cell, basal Epidermal Growth Factor (EGF) concentration, neutrophil to lymphocyte ratio, Monocytes, and Neutrophils as predictors were selected (PCI > 0.74). Patients predicted as good responders according to the pre-treatment biomarkers values treated with CIMAvax-EGF had a significant higher observed survival compared with the control group (p = 0.03). No difference was observed for bad responders.. Peripheral blood parameters and immunosenescence biomarkers together with basal EGF concentration in serum resulted in good predictors of the CIMAvax-EGF success in advanced NSCLC. Future research should explore molecular and genetic profile as biomarkers for CIMAvax-EGF and it combination with immune-checkpoint inhibitors. The study illustrates the application of a new methodology, based on causal inference, to evaluate multivariate pre-treatment predictors. The multivariate approach allows realistic predictions of the clinical benefit of patients and should be introduced in daily clinical practice.

Topics: Aged; Antineoplastic Combined Chemotherapy Protocols; Biomarkers, Tumor; Cancer Vaccines; Carcinoma, Non-Small-Cell Lung; CD4 Lymphocyte Count; CD4-Positive T-Lymphocytes; Clinical Trials, Phase III as Topic; Combined Modality Therapy; Epidermal Growth Factor; Female; Humans; Immunosenescence; Lung Neoplasms; Male; Middle Aged; Models, Statistical; Predictive Value of Tests; Prognosis; Randomized Controlled Trials as Topic; Retrospective Studies

Non-small cell lung cancer (NSCLC), one of the leading causes of cancer-related death, has a low 5-year survival rate owing to the inevitable acquired resistance toward antitumor drugs, platinum-based chemotherapy, and targeted therapy. Epidermal growth factor (EGF)-EGF receptor (EGFR) signaling activates downstream events leading to phospholipase C/inositol trisphosphate (IP3)/Ca2+ release from IP3-sensitive Ca2+ stores to modulate cell proliferation, motility, and invasion. However, the role of EGFR-mediated Ca2+ signaling in acquired drug resistance is not fully understood. Here, we analyzed alterations of intracellular Ca2+ ([Ca2+]i) responses between gefitinib-sensitive NSCLC PC-9 cells and gefitinib-resistant NSCLC PC-9/GR cells, and we found that acute EGF treatment elicited intracellular Ca2+ ([Ca2+]i) oscillations in PC-9 cells but not in PC-9/GR cells. PC-9/GR cells presented a more sustained basal [Ca2+]i level, lower endoplasmic reticulum Ca2+ level, and higher spontaneous extracellular Ca2+ ([Ca2+]e) influx than PC-9 cells. Notably, restricting [Ca2+]e in both cell types induced identical [Ca2+]i oscillations, dependent on phospholipase C and EGFR activation. Consequently, restricting [Ca2+]e in PC-9/GR cells upregulated gefitinib-mediated poly (ADP-ribose) polymerase cleavage, an increase in Bax/Bcl-2 ratio, cytotoxicity, and apoptosis. In addition, nuclear factor of activated T cell (NFAT1) induction in response to EGF was inhibited by gefitinib in PC-9 cells, whereas EGF-mediated NFAT1 induction in PC-9/GR cells was sustained regardless of gefitinib treatment. Restricting [Ca2+]e in PC-9/GR cells significantly reduced EGF-mediated NFAT1 induction. These findings indicate that spontaneous [Ca2+]e influx in NSCLC cells plays a pivotal role in developing acquired drug resistance and suggest that restricting [Ca2+]e may be a potential strategy for modulating drug-sensitivity.

Topics: Antineoplastic Agents; Calcium Signaling; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Drug Resistance, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Estrenes; Gefitinib; Humans; Lung Neoplasms; NFATC Transcription Factors; Pyrrolidinones; Type C Phospholipases

Epidermal growth factor (EGF) and its receptor (EGFR) play a paramount role in lung carcinogenesis. The polymorphism in the EGF promoter region EGF+61A>G (rs4444903) has been associated with cancer susceptibility, but its role in lung cancer patients treated with tyrosine kinase inhibitors (TKIs) remains unknown. Here, we aimed to evaluate the predictive and prognostic role of EGF+61A>G SNP in lung cancer from Brazilian EGFR-mutated TKI-treated patients. Herein, patients carrying EGFR-sensitizing mutations submitted to TKI treatment (gefitinib/erlotinib) were analyzed (n = 111) for EGF+61A>G genotype by TaqMan genotyping assay. TKI treatment was classified as partial response (PR), stable disease (SD), and disease progression (DP), according to RECIST1.1. Association analysis was assessed by chi-square and Fisher's test (univariate) and multinomial model (multivariate) and survival analysis by Kaplan-Meier method and log-rank test. The EGF+61A>G genotype frequencies observed were: AA = 31.5% (n = 35), AG = 49.6% (n = 55) and GG = 18.9% (n = 21). The allelic frequencies were 56.3% for A, and 43.7% for G and the population was in Hardy-Weinberg equilibrium (P = 0.94). EGF+61A>G codominant model (AA vs. AG vs. GG) was associated with a response to TKIs (P = 0.046), as well as a recessive model (AA vs. AG + GG; P = 0.023). The multinomial regression showed an association between the codominant model (AG) and recessive model (AG + GG) with SD compared with DP (P = 0.01;OR = 0.08; 95% CI = 0.01-0.60 and P = 0.02;OR = 0.12; 95% CI = 0.20-0.72, respectively). No association between genotypes and progression-free or overall survival was observed. In conclusion, the EGF+61 polymorphism (AG and AG + GG) was independently associated with stable disease in lung cancer patients although it was not associated with the overall response rate to first-generation TKIs or patient outcome.

Topics: Epidermal Growth Factor; Genetic Predisposition to Disease; Humans; Lung Neoplasms; Middle Aged; Protein Kinase Inhibitors; Retrospective Studies

ZNF322A is an oncogenic zinc-finger transcription factor. Our published results show that ZNF322A positively regulates transcription of alpha-adducin (ADD1) and cyclin D1 (CCND1) to promote tumorgenicity of lung cancer. However, the upstream regulatory mechanisms of ZNF322A protein function remain elusive. Here, we demonstrate that AKT could phosphorylate ZNF322A by in vitro kinase assay and cell-based mass spectrometry analysis. Overexpression of AKT promoted ZNF322A protein stability and transcriptional activity, whereas these effects were inhibited by knockdown of AKT or treating with AKT inhibitor. We studied AKT-mediated phosphorylation sites, viz. Thr-150, Ser-224, Thr-234, and Thr-262. ZNF322A phosphorylation at Thr-262 by AKT promoted ZNF322A protein stability thus increased ADD1 promoter activity. Interestingly, phosphorylation at Thr-150, Ser-224, and Thr-234 enhanced transcription activity without affecting protein stability of ZNF322A. Chromatin immunoprecipitation and DNA affinity precipitation assays showed that ZNF322A phosphorylation defective mutants Thr-150A, Ser-224A, and Thr-234A attenuated chromatin binding and DNA binding affinity to ADD1 and CCND1 promoters compared with wild-type ZNF322A. Furthermore, AKT-mediated Thr-150, Ser-224, Thr-234, and Thr-262 phosphorylation promoted lung cancer cell growth and metastasis in vitro and in vivo. Clinically, expression of phosphorylated ZNF322A (p-ZNF) correlated with actively phosphorylated AKT (p-AKT) in tumor specimens from 150 lung cancer patients. Multivariate Cox regression analysis indicated that combined p-AKT and p-ZNF expression profile was an independent factor to predict the clinical outcome in lung cancer patients. Our results reveal a new mechanism of AKT signaling in promoting ZNF322A protein stability and transcriptional activity in lung cancer cell, xenograft, and clinical models.

Topics: Cell Line, Tumor; Cell Proliferation; Epidermal Growth Factor; Humans; Lung Neoplasms; Neoplasm Metastasis; Oncogene Proteins; Phosphorylation; Prognosis; Promoter Regions, Genetic; Protein Stability; Proto-Oncogene Proteins c-akt; Signal Transduction; Transcription Factors; Transcription, Genetic

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Topics: A549 Cells; Bee Venoms; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; Humans; Lung Neoplasms; Neoplasm Invasiveness; Snail Family Transcription Factors; Zinc Finger E-box Binding Homeobox 2

Aberrant activation of EGFR represents a common event in non-small cell lung carcinoma (NSCLC) and activates various downstream signaling pathways. While EGFR activation of β-catenin signaling was previously reported, the mediating mechanism remains unclear. Our current study found that EGFR activation in NSCLC cells releases SHC-binging protein 1 (SHCBP1) from SHC adaptor protein 1 (SHC1), which subsequently translocates into the nucleus and directly promotes the transactivating activity of β-catenin, consequently resulting in development of NSCLC cell stemness and malignant progression. Furthermore, SHCBP1 promotes β-catenin activity through enhancing the CBP/β-catenin interaction, and most interestingly, a candidate drug that blocks the CBP/β-catenin binding effectively abrogates the aforementioned biological effects of SHCBP1. Clinically, SHCBP1 level in NSCLC tumors was found to inversely correlate with patient survival. Together, our study establishes a novel convergence between EGFR and β-catenin pathways and highlights a potential significance of SHCBP1 as a prognostic biomarker and a therapeutic target.

Topics: Active Transport, Cell Nucleus; beta Catenin; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Nucleus; Epidermal Growth Factor; Humans; Lung Neoplasms; Neoplasm Proteins; Shc Signaling Adaptor Proteins

In this issue, Cramer et al. introduce 3D culture models of metastatic mesothelioma to investigate basic cancer biology and new combination therapies for combating this complex and lethal disease. The results suggest that erlotinib-enhanced photodynamic therapy could further improve the efficacy of intraoperative light-activation to mop up residual tumor deposits in the clinic following surgical removal of macroscopic mesothelioma metastases.

Topics: Combined Modality Therapy; Epidermal Growth Factor; Humans; Lung Neoplasms; Mesothelioma; Photochemotherapy; Pleural Neoplasms

Erlotinib is a widely used, reversible tyrosine kinase inhibitor (TKI), targeting pro-proliferative signaling of epidermal growth factor receptor (EGFR). The drug is approved for the first-line treatment of patients with metastatic non-small cell lung cancer with EGFR mutations. Extracellular glycans can affect EGFR expression, dimerization, phosphorylation, and EGF binding. In this study we investigated the effects of EGF and erlotinib on the cell cycle of naive and sialidase (alpha-neuraminidase)-pretreated human A549 alveolar epithelial cells. A549 cells were labeled with propidium iodide, and fractions of cells in different phases of cycle were quantified by flow cytometry. We found that neither did desialilation nor EGF, as well as erlotinib treatment, increase the number of damaged cells (subG0/G1 cell fraction), while erlotinib did significantly increase the number of G0/G1 cells and decrease S + G2/M cell fractions. In naive cells, EGF increased proliferating cell numbers by more than 40%, and this effect was blocked by erlotinib. In desialylated cells, however, proliferation was significantly decreased by about 29%, and EGF and erlotinib did not exert significant effects. We conclude that changes in alveolar epithelial cell membrane glycosylation may affect function of growth-promoting receptors and erlotinib effectiveness.

Topics: Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Epidermal Growth Factor; Epithelial Cells; Erlotinib Hydrochloride; Humans; Lung Neoplasms; Neuraminidase; Protein Kinase Inhibitors; Quinazolines

Epidermal growth factor (EGF) and its receptor (EGFR) play an important role in lung carcinogenesis. A functional single nucleotide polymorphism (SNP) in EGF promoter region (EGF+61 A>G-rs4444903) has been associated with cancer susceptibility. Yet, in lung cancer, the EGF+61 A>G role is unclear. The aim of this study was to evaluate the risk of lung cancer associated with EGF+61 A>G SNP in the Brazilian population. For that, 669 lung cancer patients and 1104 controls were analyzed. EGF+61 A>G genotype was assessed by PCR-RFLP and TaqMan genotyping assay. Both patients and controls were in Hardy-Weinberg equilibrium. As expected, uni- and multivariate analyses showed that tobacco consumption and age were significant risk factors for lung cancer. The genotype frequencies in lung cancer patients were 27.3% of AA, 47.4% of AG and 25.3% of GG, and for controls were 25.3% of AA, 51.6% of AG and 23.1% of GG. The allele frequencies were 51.1% of A and 48.9% of G for both cases and controls. No significant differences for the three genotypes (AA, AG and GG-codominant model) were observed between cases and controls. We then grouped AG and GG (recessive model) genotypes, as well as AA and AG (dominant model), and again, no significant differences were also found. This is the largest study to explore EGF+61 A>G polymorphism association with lung cancer risk and suggests that this SNP is not a risk factor for lung cancer in the Brazilian population.

Topics: Adult; Aged; Aged, 80 and over; Alleles; Brazil; Case-Control Studies; Epidermal Growth Factor; ErbB Receptors; Female; Gene Frequency; Genetic Predisposition to Disease; Genetics, Population; Genotype; Humans; Lung Neoplasms; Male; Middle Aged; Polymorphism, Single Nucleotide; Promoter Regions, Genetic; Risk Factors

Mammalian target of rapamycin (mTOR) has a pivotal role in carcinogenesis and cancer cell proliferation in diverse human cancers. In this study, we observed that epimagnolin, a natural compound abundantly found in Shin-Yi, suppressed cell proliferation by inhibition of epidermal growth factor (EGF)-induced G1/S cell-cycle phase transition in JB6 Cl41 cells. Interestingly, epimagnolin suppressed EGF-induced Akt phosphorylation strongly at Ser473 and weakly at Thr308 without alteration of phosphorylation of MAPK/ERK kinases (MEKs), extracellular signal-regulated kinase (ERKs), and RSK1, resulting in abrogation of the phosphorylation of GSK3β at Ser9 and p70S6K at Thr389. Moreover, we found that epimagnolin suppressed c-Jun phosphorylation at Ser63/73, resulting in the inhibition of activator protein 1 (AP-1) transactivation activity. Computational docking indicated that epimagnolin targeted an active pocket of the mTOR kinase domain by forming three hydrogen bonds and three hydrophobic interactions. The prediction was confirmed by using in vitro kinase and adenosine triphosphate-bead competition assays. The inhibition of mTOR kinase activity resulted in the suppression of anchorage-independent cell transformation. Importantly, epimagnolin efficiently suppressed cell proliferation and anchorage-independent colony growth of H1650 rather than H460 lung cancer cells with dependency of total and phosphorylated protein levels of mTOR and Akt. Inhibitory signaling of epimagnolin on cell proliferation of lung cancer cells was observed mainly in mTOR-Akt-p70S6K and mTOR-Akt-GSK3β-AP-1, which was similar to that shown in JB6 Cl41 cells. Taken together, our results indicate that epimagnolin potentiates as chemopreventive or therapeutic agents by direct active pocket targeting of mTOR kinase, resulting in sensitizing cancer cells harboring enhanced phosphorylation of the mTORC2-Akt-p70S6k signaling pathway.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Chemoprevention; Drugs, Chinese Herbal; Epidermal Growth Factor; Extracellular Signal-Regulated MAP Kinases; G1 Phase Cell Cycle Checkpoints; Glycogen Synthase Kinase 3 beta; HEK293 Cells; Humans; JNK Mitogen-Activated Protein Kinases; Lignans; Lung Neoplasms; Mice; Molecular Docking Simulation; Phosphorylation; Protein Conformation; Proto-Oncogene Proteins c-akt; Ribosomal Protein S6 Kinases, 70-kDa; Ribosomal Protein S6 Kinases, 90-kDa; RNA Interference; RNA, Small Interfering; TOR Serine-Threonine Kinases

FBXW2 inhibits proliferation of lung cancer cells by targeting SKP2 for degradation. Whether and how FBXW2 regulates tumor invasion and metastasis is previously unknown. Here, we report that FBXW2 is an E3 ligase for β-catenin. FBXW2 binds to β-catenin upon EGF-AKT1-mediated phosphorylation on Ser

Topics: A549 Cells; beta Catenin; Cell Line, Tumor; Cell Movement; Epidermal Growth Factor; F-Box Proteins; Gene Knockdown Techniques; HEK293 Cells; HeLa Cells; Humans; In Vitro Techniques; Lung Neoplasms; Lymphatic Metastasis; Matrix Metalloproteinases; Neoplasm Invasiveness; Neoplasm Metastasis; Phosphorylation; Proto-Oncogene Proteins c-akt; Survival Rate; Ubiquitination

Immune checkpoint inhibitors targeting programmed cell death protein 1 (PD-1) and programmed death-ligand 1 (PD-L1) have improved the survival of patients with non-small cell lung cancer (NSCLC). Still, many patients do not respond to these inhibitors. PD-L1 (CD274) expression, one of the factors that influences the efficacy of immune checkpoint inhibitors, is dynamic. Here, we studied the regulation of PD-L1 expression in NSCLC without targetable genetic alterations in EGFR, ALK, BRAF, ROS1, MET, ERBB2 and RET. Analysis of RNA sequencing data from these NSCLCs revealed that inferred IFNγ, EGFR and MAPK signaling correlated with CD274 gene expression in lung adenocarcinoma. In a representative lung adenocarcinoma cell line panel, stimulation with EGF or IFNγ increased CD274 mRNA and PD-L1 protein and membrane levels, which were further enhanced by combining EGF and IFNγ. Similarly, tumor cell PD-L1 membrane levels increased after coculture with activated peripheral blood mononuclear cells. Inhibition of the MAPK pathway, using EGFR inhibitors cetuximab and erlotinib or the MEK 1 and 2 inhibitor selumetinib, prevented EGF- and IFNγ-induced CD274 mRNA and PD-L1 protein and membrane upregulation, but had no effect on IFNγ-induced MHC-I upregulation. Interestingly, although IFNγ increases transcriptional activity of CD274, MAPK signaling also increased stabilization of CD274 mRNA. In conclusion, MAPK pathway activity plays a key role in EGF- and IFNγ-induced PD-L1 expression in lung adenocarcinoma without targetable genetic alterations and may present a target to improve the efficacy of immunotherapy. © 2019 The Authors. Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Topics: A549 Cells; Adenocarcinoma of Lung; Antineoplastic Agents; B7-H1 Antigen; Coculture Techniques; Epidermal Growth Factor; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Hepatocyte Growth Factor; Humans; Interferon-gamma; Lung Neoplasms; Mitogen-Activated Protein Kinases; Protein Kinase Inhibitors; RNA Stability; RNA, Messenger; Signal Transduction

Reseptor tyrosine kinases (cMET and EGFR) are important in lung cancer targeted therapy. We believe if we can use them as markers for clinicians to help decide the diagnosis of lung cancer. This parameter will be important in serum samples of patients with lung cancer diagnosis and treatment. The aim of this study is aimed to evaluate the clinical utility of serum protein and circulating mRNA of cMET and HGF in lung cancer patients. We also analyzed the correlation of mRNA expression with clinicopathologic parameters.. We performed enzyme-linked immunosorbent assay (ELISA) to measure and compare serum protein and circulating mRNA of cMET and HGF levels in peripheral blood from 60 lung cancer patients and 40 healthy control group.. We found that both protein and gene expression levels of serum c-MET, HGF and EGFR were significantly higher in patients with lung cancer than control group. There was no association between HGF, cMET, EGF, EGFR (both protein and gene) expression levels with age, gender, smoking habit, COPD, pathological types or tumor size, stage, metastatic-non metastatic adenocarcinoma-squamous carcinoma, SCLC-NSCLC. As a result of ROC analysis, serum cMET (AUC: 0.892) and HGF protein (AUC: 0.784) were diagnosed in lung cancer patients (Fig. 1). The AUC values of serum EGF and EGFR proteins were calculated to be 0.631 and 0.692, respectively.. To our knowledge this is the first study comparing the levels of protein and mRNA in the serum material of HGF, c-MET, EGF and EGFR parameters in lung cancer patients' blood samples. Further prospective studies with more participants for better understanding of mechanism and effect for HGF and c-MET inhibitors in lung cancer will help us to identify of these biomarkers role for guiding us to sellect individualized itargeted therapies.

Topics: Adult; Aged; Biomarkers, Tumor; Case-Control Studies; Circulating Tumor DNA; Epidermal Growth Factor; ErbB Receptors; Female; Hepatocyte Growth Factor; Humans; Lung Neoplasms; Male; Middle Aged; Neoplasm Grading; Neoplasm Metastasis; Neoplasm Staging; Precision Medicine; Proto-Oncogene Proteins c-met; ROC Curve

Mutation of human receptor tyrosine kinase epidermal growth factor receptor-2 (HER2) is a rare event, found in approximately 1% non-small cell lung cancers (NSCLC). The objective was to investigate the clinical characteristics and management of HER2-mutated NSCLCs in a real-life setting.. This multicenter study described NSCLCs harboring HER2 mutations diagnosed between January 2012 and December 2014, according their clinical characteristics, management, and outcomes: response rate (RR), progression-free survival (PFS), and overall survival (OS).. Thirty patients were included: 66.7% women; median age 65.2 ± 12 years; never or former smokers 73.3%. Of the stage IV patients (n = 23), 86% received first-line platin doublet chemotherapy: RR 61.5% and PFS 6.7 (95% CI 5.9-9.5) months; 52.1% received a second-line therapy: RR 18.2% and PFS 4.9 (95% CI 2.5-11.9) months. Median OS of stage IV was 10.7 months and 2-year OS was 27.2% (95% CI 11.7-63.2). All patients with stage I-III NSCLCs were alive at 2 years.. The rarity of HER2-mutated NSCLCs requires specific studies.

Topics: Adult; Aged; Carcinoma, Non-Small-Cell Lung; Cohort Studies; Epidermal Growth Factor; Female; Humans; Lung Neoplasms; Male; Middle Aged; Mutation; Protein Kinase Inhibitors; Receptor Protein-Tyrosine Kinases; Receptor, ErbB-2

Anoikis acts as a critical barrier to metastasis by inducing cell death upon cancer cell detachment from the extracellular matrix (ECM), thereby preventing tumor cell dissemination to secondary sites. The induction of anoikis requires the lysosomal-mediated downregulation of epidermal growth factor receptors (EGFRs) leading to termination of pro-survival signaling. In this study, we demonstrate that depletion of pre-mRNA splicing factor 4 kinase (PRP4K; also known as PRPF4B) causes dysregulation of EGFR trafficking and anoikis resistance. We also report a novel cytoplasmic localization of PRP4K at the late endosome, and demonstrate both nuclear and cytoplasmic localization in breast, lung and ovarian cancer tissue. Mechanistically, depletion of PRP4K leads to reduced EGFR degradation following cell detachment from the ECM and correlates with increased TrkB, vimentin and Zeb1 expression. As a result, PRP4K loss promotes sustained growth factor signaling and increased cellular resistance to anoikis in vitro and in a novel zebrafish xenotransplantation model of anoikis sensitivity, as well as increased metastasis in a mouse model of ovarian cancer. Thus, PRP4K may serve as a potential biomarker of anoikis sensitivity in ovarian and other epithelial cancers.

Topics: Animals; Anoikis; Biomarkers, Tumor; Breast Neoplasms; Cell Line, Tumor; Cell Nucleus; Down-Regulation; Endosomes; Epidermal Growth Factor; Epithelial Cells; ErbB Receptors; Female; Humans; Lung Neoplasms; Mice; Mice, Inbred C57BL; Neoplasm Invasiveness; Ovarian Neoplasms; Protein Serine-Threonine Kinases; Ribonucleoprotein, U4-U6 Small Nuclear; RNA, Small Interfering; Signal Transduction; Xenograft Model Antitumor Assays; Zebrafish

The coxsackievirus and adenovirus receptor (CAR) is a transmembrane receptor that plays a key role in cell-cell adhesion. CAR is found in normal epithelial cells and is increased in abundance in various human tumors, including lung carcinomas. We investigated the potential mechanisms by which CAR contributes to cancer cell growth and found that depletion of CAR in human lung cancer cells reduced anchorage-independent growth, epidermal growth factor (EGF)-dependent proliferation, and tumor growth in vivo. EGF induced the phosphorylation of CAR and its subsequent relocalization to cell junctions through the activation of the kinase PKCδ. EGF promoted the binding of CAR to the chromokinesin KIF22. KIF22-dependent regulation of microtubule dynamics led to delayed EGFR internalization, enhanced EGFR signaling, and coordination of CAR dynamics at cell-cell junctions. These data suggest a role for KIF22 in the coordination of membrane receptors and provide potential new therapeutic strategies to combat lung tumor growth.

Topics: Adenocarcinoma; Animals; Apoptosis; Cell Proliferation; Coxsackie and Adenovirus Receptor-Like Membrane Protein; DNA-Binding Proteins; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Kinesins; Lung Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

EGFR-dependent cell migration plays an important role in lung cancer progression. Our previous study observed that the HECT E3 ubiquitin ligase NEDD4 is significantly correlated with tumor metastasis and required for migration and invasion signaling of EGFR in gastric cancer cells. However, how NEDD4 promotes the EGFR-dependent lung cancer cell migration is unknown. This study is to elucidate the mechanism by which NEDD4 mediates the EGFR lung cancer migration signaling.. Lentiviral vector-loaded NEDD4 shRNA was used to deplete endogenous NEDD4 in lung cancer cell lines. Effects of the NEDD4 knockdown on the EGFR-dependent or independent lung cancer cell migration were determined using the wound-healing and transwell assays. Association of NEDD4 with activated EGFR was assayed by co-immunoprecipitation. Co-expression of NEDD4 with EGFR or PTEN was determined by immunohistochemical (IHC) staining in 63 lung adenocarcinoma tissue samples. Effects of NEDD4 ectopic expression or knockdown on PTEN ubiquitination and down-regulation, AKT activation and lysosomal secretion were examined using the GST-Uba pulldown assay, immunoblotting, immunofluorescent staining and a human cathepsin B ELISA assay respectively. The specific cathepsin B inhibitor CA-074Me was used for assessing the role of cathepsin B in lung cancer cell migration.. Knockdown of NEDD4 significantly reduced EGF-stimulated cell migration in non-small cell lung carcinoma (NSCLC) cells. Co-immunoprecipitation assay found that NEDD4 is associated with EGFR complex upon EGF stimulation, and IHC staining indicates that NEDD4 is co-expressed with EGFR in lung adenocarcinoma tumor tissues, suggesting that NEDD4 might mediate lung cancer cell migration by interaction with the EGFR signaling complex. Interestingly, NEDD4 promotes the EGF-induced cathepsin B secretion, possibly through lysosomal exocytosis, as overexpression of the ligase-dead mutant of NEDD4 impedes lysosomal secretion, and knockdown of NEDD4 significantly reduced extracellular amount of cathepsin B induced by EGF. Consistent with the role of NEDD4, cathepsin B is pivotal for both basal and the EGF-stimulated lung cancer cell migration. Our studies propose a novel mechanism underlying the EGFR-promoted lung cancer cell migration that is mediated by NEDD4 through regulation of cathepsin B secretion.. NEDD4 mediates the EGFR lung cancer cell migration signaling through promoting lysosomal secretion of cathepsin B.

Topics: Cathepsin B; Cell Line, Tumor; Cell Movement; Epidermal Growth Factor; ErbB Receptors; Humans; Immunohistochemistry; Lung Neoplasms; Lysosomes; Models, Biological; Nedd4 Ubiquitin Protein Ligases; Phosphatidylinositol 3-Kinases; Protein Binding; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Signal Transduction

The management of skin toxicity is crucial for efficient afatinib treatment, but the role of tetracycline class antibiotics (TCs) in managing these rashes is relatively unknown.. We reviewed the clinical records of patients who were administered afatinib for the treatment of non-small cell lung cancer harboring epidermal growth factor receptor mutations between October 2014 and November 2016. Twenty-five patients, who received TCs for the management of afatinib-related skin disorders, were enrolled.. Minocycline was administered orally to participants. Afatinib-related toxic effects, such as rash, diarrhea, and paronychia, were observed in 92%, 92%, and 40% of cases, respectively. Although 24% of diarrhea and 4% of paronychia cases were rated grade 3 or higher, no severe cases of rash were observed during afatinib treatment. Of the 18 afatinib dose reductions, 14 (78%), three (17%), and one (6%) resulted from diarrhea, paronychia, and stomatitis, respectively; no patients required a dose reduction because of rash. When minocycline treatment started, 21 patients (84%) had a rash of grade 1 or less, and three patients had a grade 2 rash. A response to afatinib was observed in 18 patients (72%) and the median duration of afatinib administration was 501 days. An adverse event related to minocycline (grade 1 nausea) was observed in one patient.. A large proportion of the study patients started minocycline before grade 2 rash development and the severity of afatinib-related rash was lower than that previously reported. Oral TCs may be beneficial, especially if started early.

Topics: Administration, Oral; Adult; Aged; Aged, 80 and over; Anti-Bacterial Agents; Carcinoma, Non-Small-Cell Lung; Diarrhea; Epidermal Growth Factor; Exanthema; Female; Humans; Lung Neoplasms; Male; Middle Aged; Minocycline; Mutation; Paronychia; Pyridines; Severity of Illness Index; Thiazoles

Topics: Adenocarcinoma of Lung; Adult; Bronchoscopy; Epidermal Growth Factor; Humans; Lung; Lung Neoplasms; Male; Tomography, X-Ray Computed; Ultrasonography

Malignant pleural mesothelioma (MPM), an aggressive malignancy affecting pleural surfaces, occurs in three main histological subtypes. The epithelioid and sarcomatoid subtypes are characterized by cuboid and fibroblastoid cells, respectively. The biphasic subtype contains a mixture of both. The sarcomatoid subtype expresses markers of epithelial-mesenchymal transition (EMT) and confers the worst prognosis, but the signals and pathways controlling EMT in MPM are not well understood. We demonstrate that treatment with FGF2 or EGF induced a fibroblastoid morphology in several cell lines from biphasic MPM, accompanied by scattering, decreased cell adhesion and increased invasiveness. This depended on the MAP-kinase pathway but was independent of TGFβ or PI3-kinase signaling. In addition to changes in known EMT markers, microarray analysis demonstrated differential expression of MMP1, ESM1, ETV4, PDL1 and BDKR2B in response to both growth factors and in epithelioid versus sarcomatoid MPM. Inhibition of MMP1 prevented FGF2-induced scattering and invasiveness. Moreover, in MPM cells with sarcomatoid morphology, inhibition of FGF/MAP-kinase signaling induced a more epithelioid morphology and gene expression pattern. Our findings suggest a critical role of the MAP-kinase axis in the morphological and behavioral plasticity of mesothelioma.

Topics: Cell Line, Tumor; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; Extracellular Signal-Regulated MAP Kinases; Fibroblast Growth Factor 2; Humans; Lung Neoplasms; Matrix Metalloproteinase 1; Mesothelioma; Mesothelioma, Malignant; Pleural Neoplasms; Signal Transduction

Epidermal Growth Factor Receptor (EGFR) signaling regulates multiple cellular processes including proliferation, survival and apoptosis, and is attenuated by lysosomal receptor degradation. EGFR is a potent oncogene and activating mutations of EGFR are critical determinants of oncogenic transformation as well as therapeutic targets in non-small cell lung cancer. We previously demonstrated that wild type and mutant EGFRs repress the expression of the ARF tumor suppressor to promote the survival of lung tumor cells. In this study, using transient transfection systems in CHO EGFR-null cells as well as in various lung tumor cell lines carrying wild type or activated mutant EGFR, we show that ARF downregulates the expression of EGFR protein by reducing its half life. In wild type EGFR cells, ARF promotes canonical lysosomal degradation of the receptor through enhanced phosphorylation of EGFR-Y1045 and Cbl-Y731. In contrast, in mutant EGFR cells, ARF induces EGFR degradation by activating a non-canonical AKT-dependent lysosomal pathway. Taken together, these results uncover a feedback loop by which ARF may control EGFR turnover to restrain oncogenic signaling. They also highlight distinct degradation promoting pathways between wild type and mutant EGFRs in response to ARF.

Topics: ADP-Ribosylation Factors; Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Down-Regulation; Epidermal Growth Factor; ErbB Receptors; Humans; Lung Neoplasms; Lysosomes; Mutation; Phosphorylation; Reading Frames; Signal Transduction

Lung cancer is one of the most prevalent malignancies in the world. The 5-year survival rate for non-small cell lung cancer (NSCLC) patients is only approximately 15%, with metastasis as the primary cause of death. This study was aimed to investigate cytotoxic effect of external qi of Yan Xin Qigong (YXQ-EQ) toward human lung adenocarcinoma A549 cells as well as its effect on signaling pathways promoting migration, invasion and epithelial-to-mesenchymal transition (EMT) in A549 cells.. Cytotoxic effect of YXQ-EQ was evaluated using MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] and cologenic assays. Apoptosis of treated cells was determined by Annexin V/propidium iodide staining and flow cytometry analysis, while cell migration and invasion were determined using transwell assays and EMT was assessed by morphological changes in cells. Protein expression and phosphorylation were examined by immunoblot analyses.. YXQ-EQ induced apoptosis in A549 cells, resulting in a pronounced reduction in viability and clonogenic formation. This was associated with inhibition of phosphorylation of AKT and ERK1/2 and reduced expression of anti-apoptotic proteins BCL-xL, XIAP and survivin. Furthermore, YXQ-EQ inhibited EGF/EGFR signaling and EGF mediated migration and invasion of A549 cells. While TGF-β1 induced phosphorylation of SMAD2/3 and EMT in A549 cells, YXQ-EQ suppressed TGF-β/SMAD signaling and induced cell death in these cells in the presence of TGF-β1.. Our findings suggest that YXQ-EQ could exert anti-lung cancer effects via inhibiting signaling pathways that are important for NSCLC cell survival and NSCLC metastasis.

Topics: A549 Cells; Apoptosis; bcl-X Protein; Carcinoma, Non-Small-Cell Lung; Cell Movement; Drugs, Chinese Herbal; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; ErbB Receptors; Humans; Lung Neoplasms; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Proto-Oncogene Proteins c-akt; Signal Transduction; Smad2 Protein; Transforming Growth Factor beta1

RAS family GTPases contribute directly to the regulation of type I phosphoinositide 3-kinases (PI3Ks) via RAS-binding domains in the PI3K catalytic p110 subunits. Disruption of this domain of p110α impairs RAS-mutant-oncogene-driven tumor formation and maintenance. Here, we test the effect of blocking the interaction of RAS with p110α on epidermal growth factor receptor (EGFR)-mutant-driven lung tumorigenesis. Disrupting the RAS-PI3K interaction inhibits activation of both AKT and RAC1 in EGFR-mutant lung cancer cells, leading to reduced growth and survival, and inhibits EGFR-mutant-induced tumor onset and promotes major regression of established tumors in an autochthonous mouse model of EGFR-mutant-induced lung adenocarcinoma. The RAS-PI3K interaction is thus an important signaling node and potential therapeutic target in EGFR-mutant lung cancer, even though RAS oncogenes are not themselves mutated in this setting, suggesting different strategies for tackling tyrosine kinase inhibitor resistance in lung cancer.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Disease Models, Animal; Epidermal Growth Factor; ErbB Receptors; Humans; Lung Neoplasms; Mice, Inbred C57BL; Mutation; Phosphatidylinositol 3-Kinases; Protein Binding; Protein Domains; ras Proteins

Malignant pleural mesothelioma is an aggressive cancer, characterized by rapid progression and high mortality. Persistence of tumor-initiating cells (TICs, or cancer stem cells) after cytotoxic drug treatment is responsible for tumor relapse, and represents one of the main reasons for the poor prognosis of mesothelioma. In fact, identification of the molecules affecting TIC viability is still a significant challenge.. TIC-enriched cultures were obtained from 10 human malignant pleural mesotheliomas and cultured in vitro. Three fully characterized tumorigenic cultures, named MM1, MM3, and MM4, were selected and used to assess antiproliferative effects of the multi-kinase inhibitor sorafenib. Cell viability was investigated by MTT assay, and cell cycle analysis as well as induction of apoptosis were determined by flow cytometry. Western blotting was performed to reveal the modulation of protein expression and the phosphorylation status of pathways associated with sorafenib treatment.. We analyzed the molecular mechanisms of the antiproliferative effects of sorafenib in mesothelioma TIC cultures. Sorafenib inhibited cell cycle progression in all cultures, but only in MM3 and MM4 cells was this effect associated with Mcl-1-dependent apoptosis. To investigate the mechanisms of sorafenib-mediated antiproliferative activity, TICs were treated with epidermal growth factor (EGF) or basic fibroblast growth factor (bFGF) causing, in MM3 and MM4 cells, MEK, ERK1/2, Akt, and STAT3 phosphorylation. These effects were abolished by sorafenib only in bFGF-treated cells, while a modest inhibition occurred after EGF stimulation, suggesting that sorafenib effects are mainly due to FGF receptor (FGFR) inhibition. Indeed, FGFR1 phosphorylation was inhibited by sorafenib. Moreover, in MM1 cells, which release high levels of bFGF and showed autocrine activation of FGFR1 and constitutive phosphorylation/activation of MEK-ERK1/2, sorafenib induced a more effective antiproliferative response, confirming that the main target of the drug is the inhibition of FGFR1 activity.. These results suggest that, in malignant pleural mesothelioma TICs, bFGF signaling is the main target of the antiproliferative response of sorafenib, acting directly on the FGFR1 activation. Patients with constitutive FGFR1 activation via an autocrine loop may be more sensitive to sorafenib treatment and the analysis of this possibility warrants further clinical investigation.

Topics: Animals; Apoptosis; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cell Separation; Cell Survival; Down-Regulation; Epidermal Growth Factor; ErbB Receptors; Fibroblast Growth Factor 2; Humans; Lung Neoplasms; Mesothelioma; Mesothelioma, Malignant; Mice, Inbred NOD; Mice, SCID; Models, Biological; Myeloid Cell Leukemia Sequence 1 Protein; Neoplastic Stem Cells; Niacinamide; Phenylurea Compounds; Pleural Neoplasms; Receptor, Fibroblast Growth Factor, Type 1; Signal Transduction; Sorafenib; Time Factors

Long non-coding RNAs are being increasingly recognised as important molecules involved in regulating a diverse array of biological functions. For example, many long non-coding RNAs have been associated with tumourigenesis and in this context their molecular functions often involves impacting on chromatin and transcriptional control processes. One important cellular control system that is often deregulated in cancer cells is the ERK MAP kinase pathway. Here we have investigated whether ERK pathway signaling in response to EGF stimulation, leads to changes in the production of long non-coding RNAs. We identify several different classes of EGF pathway-regulated lncRNAs. We focus on one of the inducible lincRNAs, EGF inducible long intergenic non-coding RNA 1 (EINCR1). EINCR1 is predominantly nuclear and shows delayed activation kinetics compared to other immediate-early EGF-inducible genes. In humans it is expressed in a tissue-specific manner and is mainly confined to the heart but it exhibits little evolutionary conservation. Importantly, in several cancers EINCR1 shows elevated expression levels which correlate with poor survival in lung adenocarcinoma patients. In the context of lung adenocarcinomas, EINCR1 expression is anti-correlated with the expression of several protein coding EGF-regulated genes. A potential functional connection is demonstrated as EINCR1 overexpression is shown to reduce the expression of EGF-regulated protein coding genes including FOS and FOSB.

Topics: A549 Cells; Adenocarcinoma; Adenocarcinoma of Lung; Cell Line; Cell Line, Tumor; Chromatin; Epidermal Growth Factor; Gene Expression Regulation; HEK293 Cells; Humans; Lung Neoplasms; MAP Kinase Signaling System; RNA, Long Noncoding

Tyrosine kinase receptors such as the epidermal growth factor receptor (EGFR) transduce information from the microenvironment into the cell and activate homeostatic signaling pathways. Internalization and degradation of EGFR after ligand binding limits the intensity of proliferative signaling, thereby helping to maintain cell integrity. In cancer cells, deregulation of EGFR trafficking has a variety of effects on tumor progression. Here we report that sortilin is a key regulator of EGFR internalization. Loss of sortilin in tumor cells promoted cell proliferation by sustaining EGFR signaling at the cell surface, ultimately accelerating tumor growth. In lung cancer patients, sortilin expression decreased with increased pathologic grade, and expression of sortilin was strongly correlated with survival, especially in patients with high EGFR expression. Sortilin is therefore a regulator of EGFR intracellular trafficking that promotes receptor internalization and limits signaling, which in turn impacts tumor growth.

Topics: A549 Cells; Adaptor Proteins, Vesicular Transport; Animals; Carcinoma, Non-Small-Cell Lung; Cell Membrane; Epidermal Growth Factor; ErbB Receptors; Female; HEK293 Cells; Humans; Lung Neoplasms; Mice, Nude; Prognosis

In a patient with previous radically treated lung adenocarcinoma, the detection of a new lung cancer raises the question whether recurrence or a second primary lung cancer is involved. Current criteria for differentiating multiple lung tumors lack a biologic and molecular basis and may lead to misclassification with impact on survival.. We report the case of a female patient with a recent diagnosis of lung adenocarcinoma and a previous lung adenocarcinoma submitted to curative surgical therapy 4 years before. As both lesions were resected, were of the same histologic subtype and presented the same immunohistochemistry profile; we decided to perform mutational analysis of the epidermal growth factor (EGFR) gene to differentiate between recurrence and second primary lung cancer.. The EGFR gene was screened for mutations in exons 18, 19, 20 and 21 using direct sequencing of polymerase chain reaction products in DNA obtained from paraffin preserved cells from both tumors.. Mutational analysis of the EGFR gene, revealed different mutations in each tumor (both on exon 19) allowing the confirmation of the diagnosis of two metachronous primary lung cancers.. In this patient, mutational analysis of the EGFR gene was superior to histologic and immunohistochemistry characterization in differentiating between recurrent lung cancer and second primary lung cancer; allowing confirmation of the diagnosis of two metachronous primary lung cancers.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; DNA Mutational Analysis; Epidermal Growth Factor; Female; Humans; Immunohistochemistry; Lung Neoplasms; Middle Aged; Mutation; Neoplasm Recurrence, Local; Neoplasms, Second Primary; Tomography, X-Ray Computed

Rearrangement of the proto-oncogene rearranged during transfection (RET) has been newly identified potential driver mutation in lung adenocarcinoma. Clinically available tyrosine kinase inhibitors (TKIs) target RET kinase activity, which suggests that patients with RET fusion genes may be treatable with a kinase inhibitor. Nevertheless, the mechanisms of resistance to these agents remain largely unknown. Thus, the present study aimed to determine whether epidermal growth factor (EGF) and hepatocyte growth factor (HGF) trigger RET inhibitor resistance in LC-2/ad cells with CCDC6-RET fusion genes.. The effects of EGF and HGF on the susceptibility of a CCDC6-RET lung cancer cell line to RET inhibitors (sunitinib, E7080, vandetanib, and sorafenib) were examined.. CCDC6-RET lung cancer cells were highly sensitive to RET inhibitors. EGF activated epidermal growth factor receptor (EGFR) and triggered resistance to sunitinib, E7080, vandetanib, and sorafenib by transducing bypass survival signaling through ERK and AKT. Reversible EGFR-TKI (gefitinib) resensitized cancer cells to RET inhibitors, even in the presence of EGF. Endothelial cells, which are known to produce EGF, decreased the sensitivity of CCDC6-RET lung cancer cells to RET inhibitors, an effect that was inhibited by EGFR small interfering RNA (siRNA), anti-EGFR antibody (cetuximab), and EGFR-TKI (Iressa). HGF had relatively little effect on the sensitivity to RET inhibitors.. EGF could trigger resistance to RET inhibition in CCDC6-RET lung cancer cells, and endothelial cells may confer resistance to RET inhibitors by EGF. E7080 and other RET inhibitors may provide therapeutic benefits in the treatment of RET-positive lung cancer patients.

Topics: Adenocarcinoma; Cell Line, Tumor; Cetuximab; Drug Resistance, Neoplasm; Epidermal Growth Factor; ErbB Receptors; fms-Like Tyrosine Kinase 3; Gefitinib; Gene Rearrangement; Hepatocyte Growth Factor; Humans; Indoles; Lung Neoplasms; MAP Kinase Signaling System; Mutation; Niacinamide; Phenylurea Compounds; Piperidines; Protein Kinase Inhibitors; Proto-Oncogene Mas; Proto-Oncogene Proteins c-ret; Pyrroles; Quinazolines; RNA, Small Interfering; Signal Transduction; Sorafenib; Sunitinib

In cancer cells, the reversible nature of the stemness status in terms of chemoresistance has been poorly characterized. In this study, we have simulated one cycle of environmental conditions to study such reversibility by first generating floating tumorspheres (FTs) from lung and breast cancer cells by culturing them in serum-free media without the addition of any external mitogenic stimulation, and subsequently (after 2 weeks) re-incubating them back in serum-containing media to simulate routine culture conditions (RCCs). We found that cancer cells are extremely plastic: cells grown under RCCs become multidrug-resistant when grown as FTs, but upon re-incubation under RCCs quickly re-attach and lose the acquired resistance. These phenotypic changes are accompanied by concomitant changes in the expression of key proteins associated with multiple pathways important for chemoresistance, survival, and stemness maintenance. Therefore, our strategy provides an excellent experimental model to study environmental factors that modulate the plasticity of cancer cells. J. Cell. Physiol. 232: 2280-2286, 2017. © 2016 Wiley Periodicals, Inc.

Topics: Antineoplastic Agents; Biomarkers, Tumor; Breast Neoplasms; Cell Adhesion; Cell Plasticity; Cell Proliferation; Culture Media, Serum-Free; Dose-Response Relationship, Drug; Down-Regulation; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Lung Neoplasms; MCF-7 Cells; Neoplastic Stem Cells; Phenotype; Protein Kinase Inhibitors; Signal Transduction; Spheroids, Cellular; Time Factors

Epidermal growth factor (EGF) signaling promotes cell proliferation and survival in several types of cancer. Here, however, we showed that EGF inhibits proliferation and promotes programmed cell death in non-small cell lung cancer (NSCLC) cells. In A549 cells, EGF increased redox factor-1 (Ref-1) expression and the association of Ref-1 with zinc finger-containing transcriptional regulator (EGR1) via activation of p22phox, RAC1, and an NADPH oxidase subunit. EGF increased p22phox and RAC1 expression through activation of purinergic receptors (P2Y). Elevated Ref-1/EGR1 levels increased phosphatase and tensin homolog (PTEN) levels, leading to inhibition of the Akt pathway. EGF-induced PTEN upregulation increased apoptosis and autophagy-induced damage in A549 cells, whereas Ref-1 knockdown blocked EGF-induced PTEN upregulation in an NADPH oxidase p22phox subunit-independent manner. In addition, p22phox knockdown restored EGF-induced effects, implying that changes in P2Y activity caused by EGF, which activates NADPH oxidase via RAC1, influenced Ref-1-mediated redox regulation. Finally, EGF similarly attenuated cell proliferation and promoted autophagy and apoptosis in vivo in a xenograft model using A549 cells. These findings reveal that EGF-induced redox signaling is linked to Ref-1-induced death in NSCLC cells.

Topics: A549 Cells; Animals; Apoptosis; Cell Line, Tumor; Cell Proliferation; Cell Survival; DNA-(Apurinic or Apyrimidinic Site) Lyase; Early Growth Response Protein 1; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Mice; NADPH Oxidases; PTEN Phosphohydrolase; Signal Transduction; Up-Regulation; Xenograft Model Antitumor Assays

Nanoparticles (NPs) have been widely used as carriers to deliver siRNA and chemotherapeutic agents. Bcl-2 siRNA has been widely used to induce cancer cell apoptosis, and doxorubicin (Dox) can destroy cancer cells by binding with cancer cell DNA.. To investigate the therapeutic effect on lung cancer of simultaneously delivering Dox and Bcl-2-siRNA using epidermal growth factor (EGF) modified monomethoxy (polyethylene glycol)-poly (D, L-lactide-co-glycolide)-poly(L-lysine) (mPEG-PLGA-PLL, PEAL) NPs (EGF-PEAL).. EGF-PEAL NPs were characterized with respect to size, zeta potential and morphology. Cytotoxicity and drug (or siRNA) loading capacity of EGF-PEAL NPs were analyzed. Cellular uptake, drug release profile, cell killing effects of Dox and Bcl-2-siRNA-loaded EGF-PEAL NPs were assessed. Biodistribution and therapeutic effects of Dox and Bcl-2-siRNA EGF-PEAL NPs were evaluated in H1299 tumor-bearing mice.. EGF-PEAL NPs or PEAL NPs had nearly negligible cytotoxicity toward H1299 cells. Dox and Bcl-2-siRNA gradually released from EGF-PEAL NPs and exhibited sustained release patterns. Dox and Bcl-2-siRNA-loaded NPs were taken up by cells and induced the apoptosis of H1299 cells more effectively than using Dox or Bcl-2 siRNA alone. With the intravenous injection of PEAL NPs into H1299 xenografted mice, we found that combination treatment suppressed lung cancer growth and reduced Bcl-2 expression in tumor tissue, and EGF-PEAL NPs concentrated in lung tumor much more than non-targeted PEAL NPs.. We conclude that co-delivery of Dox and Bcl-2-siRNA by tumor-targeted EGF-PEAL NPs could significantly inhibit lung cancer growth.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Doxorubicin; Drug Carriers; Epidermal Growth Factor; Lung Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Nanoparticles; Particle Size; Polyesters; Polyethylene Glycols; Polylysine; Proto-Oncogene Proteins c-bcl-2; RNA, Small Interfering; Tissue Distribution

Recent evidence has suggested a possible role for progesterone receptor (PR) in the progression of non-small cell lung cancer (NSCLC). However, little is known concerning roles of PR in NSCLC. PR contains a polyproline domain (PPD), which directly binds to the SH3 domain of signaling molecules. Because PPD-SH3 interactions are essential for EGFR signaling, we hypothesized that the presence of PR-PPD interfered with EGFR-mediated signaling and cell proliferation. We examined the role of PR-PPD in cell proliferation and signaling by stably expressing PR-B, or PR-B with disrupting mutations in the PPD (PR-BΔSH3), from a tetracycline-regulated promoter in A549 NSCLC cells. PR-B dose-dependently inhibited cell growth in the absence of ligand, and progestin (R5020) treatment further suppressed the growth. Treatment with RU486 abolished PR-B- and R5020-mediated inhibition of cell proliferation. Expression of PR-BΔSH3 and treatment with R5020 or RU486 had no effect on cell proliferation. Furthermore, PR-B expression but not PR-BΔSH3 expression reduced EGF-induced A549 proliferation and activation of ERK1/2, in the absence of ligand. Taken together, our data demonstrated the significance of PR extranuclear signaling through PPD interactions in EGFR-mediated proliferation and signaling in NSCLC.

Topics: Breast Neoplasms; Carcinoma, Ductal, Breast; Carcinoma, Non-Small-Cell Lung; Cell Growth Processes; Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Female; HEK293 Cells; Humans; Lung Neoplasms; Proline-Rich Protein Domains; Receptors, Progesterone; Signal Transduction

As targeted cancer therapies and molecular profiling become widespread, the era of "precision oncology" is at hand. However, cancer genomes are complex, making mutation-specific outcomes difficult to track. We created a proof-of-principle, CUSTOM-SEQ: Continuously Updating System for Tracking Outcome by Mutation, to Support Evidence-based Querying, to automatically calculate and display mutation-specific survival statistics from electronic health record data.. Patients with cancer genotyping were included, and clinical data was extracted through a variety of algorithms. Results were refreshed regularly and injected into a standard reporting platform. Significant results were highlighted for visual cueing. A subset was additionally stratified by stage, smoking status, and treatment exposure.. By August 2015, 4310 patients with a median follow-up of 17 months had sufficient data for survival calculation. As expected, epidermal growth factor receptor (EGFR) mutations in lung cancer were associated with superior overall survival, hazard ratio (HR) = 0.53 (P < .001), validating the approach. Guanine nucleotide binding protein (G protein), q polypeptide (GNAQ) mutations in melanoma were associated with inferior overall survival, a novel finding (HR = 3.42, P < .001). Smoking status was not prognostic for epidermal growth factor receptor-mutated lung cancer patients, who also lived significantly longer than their counterparts, even with advanced disease (HR = 0.54, P = .001).. CUSTOM-SEQ represents a novel rapid learning system for a precision oncology environment. Retrospective studies are often limited by study of specific time periods and can lead to incomplete conclusions. Because data is continuously updated in CUSTOM-SEQ, the evidence base is constantly growing. Future work will allow users to interactively explore populations by demographics and treatment exposure, in order to further investigate significant mutation-specific signals.

Topics: Algorithms; Cohort Studies; Computational Biology; DNA, Neoplasm; Electronic Health Records; Epidermal Growth Factor; Follow-Up Studies; Genotype; Humans; Information Storage and Retrieval; Kaplan-Meier Estimate; Lung Neoplasms; Mutation; Neoplasms; Precision Medicine; Proportional Hazards Models; Tobacco Smoking

Epidermal growth factor receptor (EGFR) is an effective molecular target for cancer treatment. Boehmenan, a lignan from the dried stems of Clematis armandii, exhibited the potent cytotoxic effects against many cancer cell lines in previous studies. However, the effects and underlying mechanism of boehmenan on non-small cell lung cancer (NSCLC) remains unclear.. The present study was designed to determine the in vitro anti-cancer properties and underlying molecular mechanisms of boehmenan on A549 NSCLC cells.. Cellular viability and chemoattractive properties of macrophages were investigated by using MTT and transwell migration assay, respectively. Mitochondrial membrane potential (ΔΨm), apoptotic ratio, and cell cycle were measured by flow cytometry. Protein expression was visualized by Western blot using specific antibodies.. Boehmenan concentration-dependently suppressed proliferation and induced G1 phase arrest in A549 NSCLC cells, which were accompanied by reduction of migration, colony formation and increase of apoptosis in A549 cells. In addition, boehmenan treatment markedly modulated apoptosis-related protein (p53, p21, cleaved caspase 3, and cleaved PARP) and cyclin D1 expression and induced ΔΨm collapse in a concentration dependent manner. Furthermore, boehmenan concentration-dependently inhibited EGF-induced activation of EGFR and its downstream signaling molecules, including MEK, Akt, ERK1/2, and STAT3.. Taken together, our results suggested that boehmenan-mediated anti-tumor property was mediated by modulation of mitochondria and EGFR signaling pathway in A549 NSCLC cells.

Topics: Apoptosis; Carcinoma, Non-Small-Cell Lung; Caspase 3; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Clematis; Epidermal Growth Factor; ErbB Receptors; Humans; Lignans; Lung Neoplasms; Membrane Potential, Mitochondrial; Mitochondria; Molecular Structure; Plants, Medicinal; Signal Transduction

The efficacy of EGFR-tyrosine kinase inhibitors (TKIs) is significantly limited by various resistance mechanisms to those drugs. The resistance to EGFR-TKI is largely divided by two classes; acquired resistance after EGFR-TKI treatment, and primary resistance marked by cancer cell's dependence on other oncogene, such as KRAS. YAP has emerged as critical oncogene in conferring drug resistance against targeted therapy. In this study, we evaluated the role of YAP in primary and acquired EGFR-TKI resistance using gefitinib-resistant A549 and PC9 cells and their parental cell lines. Our study revealed that EGFR-TKI resistance is associated with enhanced YAP activity. Notably, YAP activation was independent of the Hippo pathway. We confirmed that AXL is a downstream target of YAP that confers EGFR-TKI resistance. And our results showed that YAP can induce ERK activation in lung adenocarcinoma. The combination of YAP inhibition with EGFR-TKI overcomes primary and acquired EGFR-TKI resistance. We also found increased YAP expression in human lung cancer after acquiring EGFR-TKI resistance. Collectively, we suggest a novel EGFR-TKI resistance mechanism involving YAP activation and suggest targeting YAP and EGFR simultaneously may be a breakthrough treatment of primary and acquired EGFR-TKI resistant lung cancer.

Topics: A549 Cells; Adaptor Proteins, Signal Transducing; Adenocarcinoma; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Epidermal Growth Factor; Hippo Signaling Pathway; Humans; Lung Neoplasms; Phosphoproteins; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Signal Transduction; Transcription Factors; Treatment Outcome; YAP-Signaling Proteins

Non-small cell lung cancer (NSCLC) remains one of the most metastasizing tumors, and directional cell migration is critical for targeting tumor metastasis. GIT2 has been known to bind to Paxillin to control cell polarization and directional migration. However, the molecular mechanisms underlying roles of GIT2 in controlling cell polarization and directional migration remain elusive. Here we demonstrated GIT2 control cell polarization and direction dependent on the regulation of Golgi through RUSC2. RUSC2 interacts with SHD of GIT2 in various lung cancer cells, and stabilizes GIT2 (Mazaki et al., 2006; Yu et al., 2009) by decreasing degradation and increasing its phosphorylation. Silencing of RUSC2 showed reduced stability of GIT2, defective Golgi reorientation toward the wound edge and decreased directional migration. Moreover, short-term EGF stimulation can increase the interaction between RUSC2 and GIT2, prolonged stimulation leads to a decrease of their interaction through activating Rab35. Silencing of Rab35 also reduced stability and phosphorylation of GIT2 and decreased cell migration. Taken together, our study indicated that RUSC2 participates in EGFR signaling and regulates lung cancer progression, and may be a new therapeutic target against lung cancer metastasis.

Topics: A549 Cells; Carcinoma, Non-Small-Cell Lung; Carrier Proteins; Cell Movement; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Golgi Apparatus; GTPase-Activating Proteins; HEK293 Cells; Humans; Lung Neoplasms; Phosphorylation; Protein Binding; Protein Interaction Domains and Motifs; Protein Stability; Protein Transport; rab GTP-Binding Proteins; RNA Interference; Signal Transduction; src Homology Domains; Time Factors; Transfection

Green synthesized silver nanoparticles have significant potential in the pharmaceutical field because of their biological functions such as antioxidant and anticancer activities. Novel silver nanoparticles synthesized from Dendropanax morbifera Léveille leaves (D-AgNPs) exhibit antimicrobial activity and reduce the viability of cancer cells without affecting the viability of RAW 264.7 macrophage-like cells. In this study, we evaluated the anticancer effect of D-AgNPs by measuring the levels of reactive oxygen species (ROS) production and toxicity against A549 and HepG2 cell lines. The effect of D-AgNPs on cell migration, induction of apoptosis, and modification of gene and/or protein expression of cancer-related markers was determined using A549 cells. D-AgNPs exhibited cytotoxicity in A549 and HepG2 cell at different concentrations and enhanced the production of ROS in both cell lines. An increase in cell apoptosis and a reduction in cell migration in A549 cells were also observed after D-AgNP treatment. Furthermore, the effect of D-AgNPs in A549 cells was shown to be related to modification of the EGFR/p38 MAPK pathway. Our data provide the first evidence supporting the potential of D-AgNPs as a possible anticancer agent, particularly for the treatment of non-small cell lung carcinoma.

Topics: A549 Cells; Apoptosis; Araliaceae; Cell Movement; Cell Survival; Down-Regulation; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Lung Neoplasms; Metal Nanoparticles; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Reactive Oxygen Species; Silver

It has been shown that epidermal growth factor receptor (EGFR) mutation status is associated with 5-fluorouracil (5-FU) sensitivity in non-small-cell lung cancer (NSCLC). However, the relationship between EGFR mutation status and dihydropyrimidine dehydrogenase (DPD), a 5-FU degrading enzyme, is unknown.. We elucidated the crosstalk among the EGFR signal cascade, the DPD gene (DPYD), and DPD protein expression via the transcription factor Sp1 and the effect of EGFR mutation status on the crosstalk.. In the PC9 (exon19 E746-A750) study, EGF treatment induced up-regulation of both Sp1 and DPD; gefitinib, an EGFR-tyrosine kinase inhibitor (EGFR-TKI), and mithramycin A, a specific Sp-1 inhibitor, suppressed them. Among EGFR-mutated (PC9, HCC827; exon19 E746-A750 and H1975; exon21 L858R, T790M, gefitinib resistant) and -non-mutated (H1437, H1299) cell lines, EGF administration increased DPYD mRNA expression only in mutated cells (p < 0.05). Accordingly, gefitinib inhibited DPD protein expression only in PC9 and HCC827 cells, and mithramycin A inhibited it in EGFR-mutated cell lines, but not in wild-type. FU treatment decreased the level of cell viability more in gefitinib-treated EGFR-TKI sensitive cell lines. Further, combination treatment of FU and mithramycin A suppressed cell viability even in a gefitinib resistant cell line.. The EGFR signal cascade regulates DPD expression via Sp1 in EGFR mutant cells. These results might be a step towards new therapies targeting Sp1 and DPD in NSCLC with different EGFR mutant status.

Topics: Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Survival; Dihydrouracil Dehydrogenase (NADP); Drug Resistance, Neoplasm; Drug Synergism; Epidermal Growth Factor; ErbB Receptors; Fluorouracil; Gefitinib; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Mutation; Plicamycin; Quinazolines; Signal Transduction; Sp1 Transcription Factor

Abnormalities in the epidermal growth factor (EGF) and EGFR pathway promote progression of NSCLC. Immunization with EGF vaccine induces specific, neutralizing anti-EGF antibodies that prevent binding of the ligand to its receptor. This concept of pathway targeted immunotherapy (PTI) was validated in vitro by dose-related suppression of EGFR, Akt, and Erk1/2 phosphorylation in cell lines with different mutations. A randomized phase II trial showed improved overall survival (OS) in subgroups with advanced NSCLC showing a clear immunologic response. By per-protocol analysis of the ensuing phase IIb trial, patients receiving EGF PTI survived 3 months longer than controls (12.43 versus 9.43 months; hazard ratio = 0.77 [95% confidence interval, 0.61-0.98]). These data were confirmed in a larger trial showing an OS benefit over control of >3 months. The variable most strongly correlated with efficacy was circulating EGF at enrolment. Patients with serum EGF levels >250 pg/mL benefited most from treatment with EGF PTI. Of 188 patients tested, 94 were above this biomarker threshold. The OS benefit from active versus control treatment was 6.7 months. More than 15% of patients had responses for >5 years. Long-term survivors are seen in all EGF PTI trials. Treatment is well-tolerated, induces high anti-EGF antibody titers, reduces levels of circulating serum EGF, achieves durable responses, and significantly prolongs OS. A threshold of 250 pg/mL has been set to enrich the study population in the ongoing pivotal trial. This biomarker-guided study in an enriched population of patients with both squamous and nonsquamous stage IV NSCLC aims to replicate the favorable efficacy/tolerability balance of earlier studies.

Topics: Carcinoma, Non-Small-Cell Lung; Epidermal Growth Factor; Female; Humans; Immunotherapy; Lung Neoplasms; Male; Middle Aged

In advanced stages of cancers, TGF-β promotes tumor progression in conjunction with inputs from receptor tyrosine kinase pathways. However, mechanisms that underpin the signaling cooperation and convert TGF-β from a potent growth inhibitor to a tumor promoter are not fully understood. We report here that TGF-β directly regulates alternative splicing of cancer stem cell marker CD44 through a phosphorylated T179 of SMAD3-mediated interaction with RNA-binding protein PCBP1. We show that TGF-β and EGF respectively induce SMAD3 and PCBP1 to colocalize in SC35-positive nuclear speckles, and the two proteins interact in the variable exon region of CD44 pre-mRNA to inhibit spliceosome assembly in favor of expressing the mesenchymal isoform CD44s over the epithelial isoform CD44E. We further show that the SMAD3-mediated alternative splicing is essential to the tumor-promoting role of TGF-β and has a global influence on protein products of genes instrumental to epithelial-to-mesenchymal transition and metastasis.

Topics: Alternative Splicing; Animals; Cell Line, Tumor; DNA-Binding Proteins; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; Exons; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Heterogeneous-Nuclear Ribonucleoproteins; Humans; Hyaluronan Receptors; Lung Neoplasms; Mice; Mice, Nude; Neoplasm Transplantation; Neoplastic Stem Cells; Phosphorylation; Promoter Regions, Genetic; Protein Isoforms; RNA Precursors; RNA-Binding Proteins; Signal Transduction; Smad3 Protein; Threonine; Transforming Growth Factor beta

Carthamus tinctorius L. is a traditional Chinese medicine that activates blood circulation and dissipates blood stasis, and has been extensively used as antitumor treatment in a clinical setting in single or in compound preparation form. However, empirical evidence and a better understanding of the possible mechanisms involved are still required. Here, we investigated the role of safflower yellow (SY), the active ingredient of C. tinctorius, in the pulmonary metastasis of breast cancer, and the underlying mechanism of action. EGF-meditated time- and dose-dependent cell response profiles were applied to screen for the activity of SY in vitro, while orthotopic lung metastasis and intravenous injection were used to evaluate the antimetastatic role of SY in vivo. SY could dose-dependently inhibit EGF-mediated time- and dose-dependent cell response profiles by inhibiting cytoskeletal rearrangement. We also found that SY significantly inhibited the migration of breast cancer cells in vitro and pulmonary metastasis of breast cancer cells in vivo. Consistent with these phenotypes, formation of invadopodia and the expression of MMP-9 and p-Src proteins were decreased after EGF stimulation in MBA-MD-231 cells treat with SY, as well as in lung metastatic foci. Additionally, circulating tumor cells retained in lung capillaries were also reduced. These results suggest that the antimetastatic effect of SY is due to its inhibition of invadopodia formation, which occurs mainly through Src-dependent cytoskeleton rearrangement. We suggest that SY should be considered as a potential novel therapeutic agent for the treatment of breast cancer.

Topics: Animals; Breast Neoplasms; Carthamus tinctorius; Chalcone; Chlorocebus aethiops; COS Cells; Disease Models, Animal; Dose-Response Relationship, Drug; Epidermal Growth Factor; Female; Humans; Lung Neoplasms; Matrix Metalloproteinase 9; MCF-7 Cells; Mice; Mice, Inbred BALB C; Phytotherapy; Podosomes; Proto-Oncogene Proteins pp60(c-src)

Hepatoblastoma (HBL), the most common childhood liver cancer is cured with surgical resection after chemotherapy or with liver transplantation if local invasion and multifocality preclude resection. However, variable survival rates of 60-80% and debilitating chemotherapy sequelae argue for more informed treatment selection, which is not possible by grading the Wnt-β-catenin over activity present in most HBL tumors. A hypothesis-generating whole transcriptome analysis shows that HBL tumors removed at transplantation are enriched most for cancer signaling pathways which depend predominantly on epidermal growth factor (EGF) signaling, and to a lesser extent, on aberrant Wnt-β-catenin signaling. We therefore evaluated whether EGFR, ASAP1, ERBB2 and ERBB4, which signal downstream after ligation of EGF, and which show aberrant expression in several other invasive cancers, would also predict HBL tumor invasiveness. Immunohistochemistry of HBL tumors (n = 60), which are histologically heterogeneous, shows that compared with well-differentiated fetal cells, less differentiated embryonal and undifferentiated small cells (SCU) progressively lose EGFR and ASAP1 expression. This trend is exaggerated in unresectable, locally invasive or metastatic tumors, in which embryonal tumor cells are EGFR-negative, while SCU cells are EGFR-negative and ASAP1-negative. Loss of EGFR-ASAP1 signaling characterizes undifferentiated and invasive HBL. EGFR-expressing HBL tumors present novel therapeutic targeting opportunities.

Topics: Adaptor Proteins, Signal Transducing; Adolescent; beta Catenin; Child; Child, Preschool; Epidermal Growth Factor; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Hepatoblastoma; Humans; Infant; Liver Neoplasms; Liver Transplantation; Lung Neoplasms; Male; Receptor, ErbB-2; Receptor, ErbB-4; Survival Analysis; Transcriptome; Wnt Signaling Pathway

Surfactant protein D (SP-D) is a member of the collectin family that has an important role in maintaining pulmonary homeostasis. In this study, we demonstrated that SP-D inhibited the proliferation, migration and invasion of A549 human lung adenocarcinoma cells. We found that SP-D suppressed epidermal growth factor (EGF) signaling in A549 cells, H441 human lung adenocarcinoma cells and human EGF receptor (EGFR) stable expression CHO-K1 cells. A binding study using (125)I-EGF demonstrated that SP-D downregulated the binding of EGF to EGFR. A ligand blot indicated that SP-D bound to EGFR, and a lectin blot suggested that EGFR in A549 cells had both high-mannose type and complex type N-glycans. We purified the recombinant extracellular domain of EGFR (soluble EGFR=soluble EGFR (sEGFR)), and demonstrated that SP-D directly bound to sEGFR in a Ca(2+)-dependent manner. The binding of SP-D to sEGFR was suppressed by EDTA, mannose or N-glycopeptidase F treatment. Mass spectrometric analysis indicated that N-glycans in domain III of EGFR were of a high-mannose type. These data suggest that SP-D reduces EGF binding to EGFR through the interaction between the carbohydrate recognition domain of SP-D and N-glycans of EGFR, and downregulates EGF signaling. Our finding suggests the novel type of regulation system of EGF signaling involving lectin-to-carbohydrate interaction and downregulation of ligand binding.

Topics: Animals; Calcium; Cell Line, Tumor; CHO Cells; Cricetinae; Cricetulus; Down-Regulation; Epidermal Growth Factor; ErbB Receptors; Humans; Lung Neoplasms; Neoplasm Proteins; Pulmonary Surfactant-Associated Protein D; Signal Transduction

C-terminal tensin-like (Cten) protein, a component of focal adhesions, contributes to cell motility and invasion in multiple human cancers. Epidermal growth factor can activate signal transducer and activator of transcription 3, and both contribute to invasion through focal adhesion interactions. We hypothesize that Cten may mediate invasion of lung cancer cells provided by epidermal growth factor via signal transducer and activator of transcription 3.. Four human non-small cell lung cancer cell lines were treated with epidermal growth factor to evaluate activation of the signal transducer and activator of transcription 3 pathway and induction of Cten expression. Chemical inhibition of signal transducer and activator of transcription 3 was used to evaluate the effect on epidermal growth factor-induced Cten expression. Protein expression was quantified by Western blot. H125 and A549 cells were transduced with short-hairpin RNA via lentiviral vector to knockdown expression of Cten. An in vitro transwell invasion assay was used to assess the effects of Cten knockdown on cell invasion (n = 3 for all experiments).. Stimulation of lung cancer cells with epidermal growth factor activated the signal transducer and activator of transcription 3 pathway and induced expression of Cten in all cell lines. Signal transducer and activator of transcription 3 inhibition significantly reduced epidermal growth factor-induced expression of Cten in H125 (P < .0001), H358 (P = .006), and H441 (P = .014) cells in a dose-dependent manner. Knockdown of Cten expression resulted in significant decreases in cellular invasion in both H125 (P = .0036) and A549 (P = .0006) cells.. These are the first findings in lung cancer to demonstrate that Cten expression mediates invasion of human lung cancer cells and is upregulated by epidermal growth factor via signal transducer and activator of transcription 3 pathway. Cten should be considered a potential therapeutic target for lung cancer.

Topics: Aminosalicylic Acids; Benzenesulfonates; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Movement; Dose-Response Relationship, Drug; Epidermal Growth Factor; Humans; Lung Neoplasms; Microfilament Proteins; Neoplasm Invasiveness; RNA Interference; Signal Transduction; STAT3 Transcription Factor; Tensins; Time Factors; Transfection

The receptor tyrosine kinase epidermal growth factor receptor (EGFR) and its ligand epidermal growth factor (EGF) are known to play important roles in malignant tumor cells, and the EGFR signaling pathway is one of the most important targets in various tumors, including non-small cell lung cancer (NSCLC). We reported recently that an aberration in certain steps of EGF-stimulated phosphorylated epidermal growth factor receptor (pEGFR) endocytic trafficking from the early endosomes to the late endosomes occurs in the gefitinib-resistant NSCLC cells, in which large amounts of sorting nexin 1 (SNX1) are colocalized with EGFR in the aggregated early endosomes where the internalized pEGFR is also accumulated of these cells. To further investigate the role of SNX1 in EGF‑stimulated pEGFR endocytosis, followed by downstream signaling leading to the activation of phosphatidylinositol 3-kinase (PI3K)--the serine/threonine kinase AKT pathway, we examined the effect of depletion of SNX1 knock-down expression by siRNA and an inhibition of targeting membrane recycling using monensin. Using immunofluorescence, we observed an efficient endocytic transport of pEGFR from early endosomes to late endosomes/lysosomes after EGF-stimulation in the cells transfected with siRNA‑SNX1, whereas the delayed endocytic delivery of pEGFR was evident in the siRNA-control-transfected cells. Furthermore, a large amount of endocytosed pEGFR was accumulated in the presence of monensin in the early endosomes of the SNX1 knock-down cells. In western blot analysis, EGF stimulation of both control and cells transfected with siRNA-SNX1 resulted in rapid phosphorylation of EGFR and enhanced AKT phosphorylation. Monensin-dependent inhibition of AKT phosphorylation was stronger in SNX1 knock-down cells than in controls. In contrast, however, monensin had no effect on AKT phosphorylation triggered by activation of the MET receptor tyrosine kinase. Collectively, we suggest that EGF-stimulated recycling of EGFR to the plasma membrane induces downstream signaling leading to AKT phosphorylation. Suppression of EGFR membrane recycling by SNX1 appears to be critical for the activation of EGFR/PI3K/AKT signaling pathway in human lung cancer cells.

Topics: Cell Line, Tumor; Cell Membrane; Drug Resistance, Neoplasm; Endocytosis; Epidermal Growth Factor; ErbB Receptors; Gefitinib; Gene Knockdown Techniques; Humans; Lung Neoplasms; Monensin; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Quinazolines; Signal Transduction; Sorting Nexins

The anti-tumor role and mechanisms of Cannabidiol (CBD), a non-psychotropic cannabinoid compound, are not well studied especially in triple-negative breast cancer (TNBC). In the present study, we analyzed CBD's anti-tumorigenic activity against highly aggressive breast cancer cell lines including TNBC subtype. We show here -for the first time-that CBD significantly inhibits epidermal growth factor (EGF)-induced proliferation and chemotaxis of breast cancer cells. Further studies revealed that CBD inhibits EGF-induced activation of EGFR, ERK, AKT and NF-kB signaling pathways as well as MMP2 and MMP9 secretion. In addition, we demonstrated that CBD inhibits tumor growth and metastasis in different mouse model systems. Analysis of molecular mechanisms revealed that CBD significantly inhibits the recruitment of tumor-associated macrophages in primary tumor stroma and secondary lung metastases. Similarly, our in vitro studies showed a significant reduction in the number of migrated RAW 264.7 cells towards the conditioned medium of CBD-treated cancer cells. The conditioned medium of CBD-treated cancer cells also showed lower levels of GM-CSF and CCL3 cytokines which are important for macrophage recruitment and activation. In summary, our study shows -for the first time-that CBD inhibits breast cancer growth and metastasis through novel mechanisms by inhibiting EGF/EGFR signaling and modulating the tumor microenvironment. These results also indicate that CBD can be used as a novel therapeutic option to inhibit growth and metastasis of highly aggressive breast cancer subtypes including TNBC, which currently have limited therapeutic options and are associated with poor prognosis and low survival rates.

Topics: Animals; Antineoplastic Agents; Breast Neoplasms; Cannabidiol; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cytokines; Disease Models, Animal; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Lung Neoplasms; Macrophages; Mice; Models, Biological; Neoplasm Invasiveness; Signal Transduction; Triple Negative Breast Neoplasms; Tumor Microenvironment

Growth signals are directly or indirectly involved in telomerase regulation. In this study, we investigated molecular mechanisms of the effect of EGF (epidermal growth factor) on regulating hTERT (human telomerase reverse transcriptase) expression. To elucidate specific transcription factors involved in EGF-stimulated hTERT transcription in A549 and H1299 lung cancer cells, transcription factors drives hTERT promoter activity, such as Myc, Mad, and Ets-2, was evaluated on luciferase reporter assay. The upregulation of hTERT promoter by Ets-2 and Myc were abolished by Mad. Using DAPA (DNA affinity precipitation assay), Ets-2 binding to SNP (T) was stronger than Ets-2 binding to SNP (C) at -245 bp upstream of the transcription start site within the core promoter of hTERT. Ets-2 silence by siRNA decreased hTERT expression at mRNA and protein levels. The regulation of hTERT promoter by EGF/Ets-2 was diminished via the EGFR kinase signal pathway-specific inhibitors AG1478 and Iressa. Inhibitors of Erk and Akt inhibited Ets-2-activated hTERT promoter activity. These data suggested that Ets-2, a genuine cancer-specific transcription factor, is actively involved in EGFR kinase-induced hTERT overexpression pathway in lung cancer cells. Blockage of this pathway may contribute to targeted gene therapy in lung cancer.

Topics: Cell Line, Tumor; DNA-Binding Proteins; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Enzymologic; Humans; Lung Neoplasms; Polymorphism, Single Nucleotide; Promoter Regions, Genetic; Proto-Oncogene Protein c-ets-2; Proto-Oncogene Proteins c-myc; Signal Transduction; Telomerase; Transcription, Genetic

Topics: Adenocarcinoma; Aged; Anaplastic Lymphoma Kinase; Brain Neoplasms; Endometrial Neoplasms; Epidermal Growth Factor; Female; Genotype; Humans; Lung Neoplasms; Precision Medicine; Radiography; Receptor Protein-Tyrosine Kinases

We investigated effects of genetic alterations in epidermal growth factor receptor (EGFR), anaplastic lymphoma kinase (ALK), and Kirsten rat sarcoma viral oncogene homolog (KRAS) on overall survival (OS) and local control after stereotactic radiosurgery for brain metastases in non-small cell lung cancer (NSCLC).. A cohort of 89 out of 262 NSCLC patients (2003-2013) treated with gamma knife radiosurgery for brain metastases had genotyping available and were selected as our study population.. Median follow-up was 12 months. Median OS rates for the EGFR, KRAS, echinoderm microtubule-associated protein-like 4 (EML4)-ALK mutated, and wild-type cohorts were 17, 7, 27, and 12 months, respectively (P = .019), and for targeted versus nontargeted therapy 21 and 11 months, respectively (P = .071). Targeted therapy was a strong predictor of increased OS on univariate (P = .037) and multivariate (P = .022) analysis. Gender, primary tumor controlled status, recursive partitioning analysis class, and graded prognostic assessment score were associated with OS (P < .05). On multivariate analysis, positive EGFR mutational status was a highly significant predictor for decreased survival (hazard ratio: 8.2; 95% CI: 2.0-33.7; P = .003). However, when we recategorized EGFR-mutant cases based on whether they received tyrosine kinase inhibitor, OS was no longer significantly shorter (hazard ratio: 1.5; P = .471). Median OS for patients with and without local failure was 17 and 12 months, respectively (P = .577). Local failure rates for EGFR, KRAS, EML4-ALK mutated, and wild-type cohorts by lesion were 8.7%, 5.4%, 4.3%, and 5.1%, respectively.. This study suggests that EGFR tyrosine kinase mutation and ALK translocation results in improved survival to targeted therapies and that mutation status itself does not predict survival and local control in patients with brain metastases from NSCLC.

Topics: Adult; Aged; Aged, 80 and over; Anaplastic Lymphoma Kinase; Brain Neoplasms; Carcinoma, Non-Small-Cell Lung; Cell Cycle Proteins; Epidermal Growth Factor; Female; Humans; Lung Neoplasms; Male; Microtubule-Associated Proteins; Middle Aged; Mutation; Proportional Hazards Models; Proto-Oncogene Proteins p21(ras); Radiosurgery; Receptor Protein-Tyrosine Kinases; Serine Endopeptidases

Lung cancer is the most lethal neoplasia, and an early diagnosis is the best way for improving survival. Symptomatic patients attending Pulmonary Services could be diagnosed with lung cancer earlier if high-risk individuals are promptly separated from healthy individuals and patients with benign respiratory pathologies. We searched for a convenient non-invasive serum test to define which patients should have more immediate clinical tests. Six cancer-associated molecules (HB-EGF, EGF, EGFR, sCD26, VEGF, and Calprotectin) were investigated in this study. Markers were measured in serum by specific ELISAs, in an unselected population that included 72 lung cancer patients of different histological types and 56 control subjects (healthy individuals and patients with benign pulmonary pathologies). Boosted regression and random forests analysis were conducted for the selection of the best candidate biomarkers. A remarkable discriminatory capacity was observed for EGF, sCD26, and especially for Calprotectin, these three molecules constituting a marker panel boasting a sensitivity of 83% and specificity of 87%, resulting in an associated misclassification rate of 15%. Finally, an algorithm derived by logistic regression and a nomogram allowed generating classification scores in terms of the risk of a patient of suffering lung cancer. In conclusion, we propose a non-invasive test to identify patients at high-risk for lung cancer from a non-selected population attending a Pulmonary Service. The efficacy of this three-marker panel must be tested in a larger population for lung cancer.

Topics: Aged; Aged, 80 and over; Algorithms; Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Dipeptidyl Peptidase 4; Early Detection of Cancer; Epidermal Growth Factor; Female; Humans; Leukocyte L1 Antigen Complex; Logistic Models; Lung Neoplasms; Male; Middle Aged; Prospective Studies; Sensitivity and Specificity

Epithelial-mesenchymal transition (EMT), a process closely related to tumor development, is regulated by a variety of signaling pathways and growth factors, such as transforming growth factor-β1 (TGF-β1) and epidermal growth factor (EGF). Hyaluronan (HA) has been shown to induce EMT through either TGF-β1 or EGF signaling and to be a regulator of the crosstalk between these two pathways in fibroblasts. In this study, in order to clarify whether HA has the same effect in tumor cells, we utilized the lung cancer cell line, A549, and the breast cancer cell line, MCF-7, and found that the effects of stimulation with TGF-β1 were more potent than those of EGF in regulating the expression of EMT-associated proteins and in enhancing cell migration and invasion. In addition, we observed that TGF-β1 activated EGF receptor (EGFR) and its downstream AKT and extracellular signal-regulated kinase (ERK) pathways. Furthermore, we found that TGF-β1 upregulated the expression of hyaluronan synthases (HAS1, HAS2 and HAS3) and promoted the expression of CD44, a cell surface receptor for HA, which interacts with EGFR, resulting in the activation of the downstream AKT and ERK pathways. Conversely, treatment with 4-methylumbelliferone (4-MU; an inhibitor of HAS) prior to stimulation with TGF-β1, inhibited the expression of CD44 and EGFR, abolished the interaction between CD44 and EGFR. Furthermore, the use of shRNA targeting CD44 impaired the expression of EGFR, deactivated the AKT and ERK pathways, reversed EMT and decreased the migration and invasion ability of cells. In conclusion, our data demonstrate that TGF-β1 induces EMT by the transactivation of EGF signaling through HA/CD44 in lung and breast cancer cells.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Movement; Enzyme Activation; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Female; Glucuronosyltransferase; Humans; Hyaluronan Receptors; Hyaluronan Synthases; Hyaluronic Acid; Hymecromone; Lung Neoplasms; MCF-7 Cells; Neoplasm Invasiveness; Proto-Oncogene Proteins c-akt; RNA Interference; RNA, Small Interfering; Signal Transduction; Transforming Growth Factor beta1

Sur8 (also known as Shoc2) is a Ras-Raf scaffold protein that modulates signaling through extracellular signal-regulated kinase (ERK) pathway. Although Sur8 has been shown to be a scaffold protein of the Ras-ERK pathway, its interaction with other signaling pathways and its involvement in tumor malignancy has not been reported. We identified that Sur8 interacts with the p110α subunit of phosphatidylinositol 3-kinase (PI3K), as well as with Ras and Raf, and these interactions are increased in an epidermal growth factor (EGF)- and oncogenic Ras-dependent manner. Sur8 regulates cell migration and invasion via activation of Rac and matrix metalloproteinases (MMPs). Interestingly, using inhibitors of MEK and PI3K we found Sur8 mediates these cellular behaviors predominantly through PI3K pathway. We further found that human metastatic melanoma tissues had higher Sur8 content followed by activations of Akt, ERK, and Rac. Lentivirus-mediated Sur8-knockdown attenuated metastatic potential of highly invasive B16-F10 melanoma cells indicating the role of Sur8 in melanoma metastasis. This is the first report to identify the role of scaffold protein Sur8 in regulating cell motility, invasion, and metastasis through activation of both ERK and PI3K pathways.

Topics: Animals; Cell Line, Tumor; Cell Movement; Epidermal Growth Factor; HEK293 Cells; Humans; Intracellular Signaling Peptides and Proteins; Lung Neoplasms; Melanoma, Experimental; Mice; Neoplasm Metastasis; NIH 3T3 Cells; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; raf Kinases; ras Proteins; Signal Transduction

Dysfunction of the mitochondria is well-known for being associated with cancer progression. In the present study, we analyzed the mitochondria proteomics of lung cancer cell lines with different invasion abilities and found that EGFR is highly expressed in the mitochondria of highly invasive non-small-cell lung cancer (NSCLC) cells. EGF induces the mitochondrial translocation of EGFR; further, it leads to mitochondrial fission and redistribution in the lamellipodia, upregulates cellular ATP production, and enhances motility in vitro and in vivo. Moreover, EGFR can regulate mitochondrial dynamics by interacting with Mfn1 and disturbing Mfn1 polymerization. Overexpression of Mfn1 reverses the phenotypes resulting from EGFR mitochondrial translocation. We show that the mitochondrial EGFR expressions are higher in paired samples of the metastatic lymph node as compared with primary lung tumor and are inversely correlated with the overall survival in NSCLC patients. Therefore, our results demonstrate that besides the canonical role of EGFR as a receptor tyrosine, the mitochondrial translocation of EGFR may enhance cancer invasion and metastasis through regulating mitochondria dynamics.

Topics: Animals; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Movement; Dose-Response Relationship, Drug; Endocytosis; Energy Metabolism; Epidermal Growth Factor; ErbB Receptors; Genotype; GTP Phosphohydrolases; Heterografts; Humans; Lung Neoplasms; Lymphatic Metastasis; Male; Mice, Inbred NOD; Mice, SCID; Mitochondria; Mitochondrial Dynamics; Mitochondrial Membrane Transport Proteins; Phenotype; Protein Transport; Proteomics; Signal Transduction; Time Factors; Transfection

The epidermal growth factor (EGF) plays important roles in non-small cell lung cancer (NSCLC) susceptibility and functional polymorphism in the EGF (+61A/G) gene has been linked to increased risk of NSCLC. This study aimed to evaluate the role of the EGF +61A/G polymorphism in risk of NSCLC adenocarcinoma (ADC) occurrence and survival in an Indian population.. This case- control study included 100 histopathologically confirmed NSCLC (ADC) patients and 100 healthy controls. EGF (A61G) was genotyped by AS-PCR to elucidate putative associations with clinical outcomes. The association of the polymorphism with the survival of NSCLC patients was estimated by Kaplan-Meier curves.. It was found that EGF 61AG heterozygous and GG homozygous genotype is significantly associated with increased risk of NSCLC (ADC) occurrence compared to AA genotype, [OR 2.61 (1.31-5.18) and 3.25 (1.31-8.06), RR 1.51(1.15-2.0) and 1.72 (1.08-2.73) and RD 23.2 (6.90-39.5) and 28.53(7.0-50.1) for heterozygous AG (p=0.005) and homozygous GG (p=0.009)]. Patients homozygous for the G allele exhibited a significantly poor overall survival. The median survival time for patients with EGF 61 AA, AG, and GG genotypes was 10.5, 7.4, and 7.1 months (p=0.02), respectively. NSCLC (ADC) patients with GG + AG exhibited 7.3 months median survival compared to the AA genotype (p=0.009).. The present study revealed that the EGF A61G genotype may be a novel independent prognostic marker to identify patients at higher risk of occurrence and an unfavourable clinical outcome.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Case-Control Studies; Epidermal Growth Factor; Female; Genetic Predisposition to Disease; Genotype; Humans; India; Lung Neoplasms; Male; Middle Aged; Polymorphism, Single Nucleotide; Prognosis; Risk

Inhibition of EGFR-EGF interactions forms an important therapeutic rationale in treatment of non-small cell lung carcinoma. Established inhibitors have been successful in reducing proliferative processes observed in NSCLC, however patients suffer serious side effects. Considering the narrow therapeutic window of present EGFR inhibitors, the present study centred on identifying high efficacy EGFR inhibitors through structure based virtual screening strategies. Established inhibitors - Afatinib, Dacomitinib, Erlotinib, Lapatinib, Rociletinib formed parent compounds to retrieve similar compounds by linear fingerprint based tanimoto search with a threshold of 90%. The compounds (parents and respective similars) were docked at the EGF binding cleft of EGFR. Patch dock supervised protein-protein interactions were established between EGF and ligand (query and similar) bound and free states of EGFR. Compounds ADS103317, AKOS024836912, AGN-PC-0MXVWT, GNF-Pf-3539, SCHEMBL15205939 were retrieved respectively similar to Afatinib, Dacomitinib, Erlotinib, Lapatinib, Rociletinib. Compound- AGN-PC-0MXVWT akin to Erlotinib showed highest affinity against EGFR amongst all the compounds (parent and similar) assessed in the study. Further, AGN-PC-0MXVWT brought about significant blocking of EGFR-EGF interactions in addition showed appreciable ADMET properties and pharmacophoric features. In the study, we report AGN-PC-0MXVWT to be an efficient and high efficacy inhibitor of EGFR-EGF interactions identified through computational approaches.

Topics: Carcinoma, Non-Small-Cell Lung; Computer Simulation; Drug Evaluation, Preclinical; Epidermal Growth Factor; ErbB Receptors; Humans; Lung Neoplasms; Models, Molecular; Molecular Docking Simulation; Protein Conformation; Protein Kinase Inhibitors; Small Molecule Libraries

Sprouty1 protein belongs to a family of receptor tyrosine kinase-mediated signaling inhibitors, whose members are usually regulated by growth factors to form a negative feedback loop. Correspondingly fluctuations of Sprouty1 mRNA in response to single growth factors have been observed. In this report, we investigate Sprouty1 protein levels and show that in non-small cell lung carcinoma-derived cells, the expression levels are unaffected by the serum content in the cellular environment. Although cells harboring K-Ras mutations express insignificant higher Sprouty1 levels, ectopic expression of constitutive active Ras in normal human lung fibroblasts fails to augment Sprouty1 protein content. Furthermore, serum starvation for three days has no influence on Sprouty1 expression and addition of serum or of singular growth factors leaves Sprouty protein levels unchanged. Cell cycle analysis reveals that Sprouty1 levels remain constant throughout the whole cell cycle. These data demonstrate that Sprouty1 expression is not connected with mitogenic signaling and cell proliferation.

Topics: Carcinoma, Non-Small-Cell Lung; Cell Cycle; Cell Line, Tumor; Culture Media, Serum-Free; Epidermal Growth Factor; Fibroblast Growth Factor 2; Fibroblast Growth Factor 9; Genes, ras; Humans; Lung Neoplasms; Membrane Proteins; Mitosis; Mutation; Phosphoproteins; Receptor Protein-Tyrosine Kinases; Signal Transduction

Three-dimensional (3D) cell culture is gaining acceptance in response to the need for cellular models that better mimic physiologic tissues. Spheroids are one such 3D model where clusters of cells will undergo self-assembly to form viable, 3D tumor-like structures. However, to date little is known about how spheroid biology compares to that of the more traditional and widely utilized 2D monolayer cultures. Therefore, the goal of this study was to characterize the phenotypic and functional differences between lung tumor cells grown as 2D monolayer cultures, versus cells grown as 3D spheroids. Eight lung tumor cell lines, displaying varying levels of epidermal growth factor receptor (EGFR) and cMET protein expression, were used to develop a 3D spheroid cell culture model using low attachment U-bottom plates. The 3D spheroids were compared with cells grown in monolayer for 1) EGFR and cMET receptor expression, as determined by flow cytometry, 2) EGFR and cMET phosphorylation by MSD assay, and 3) cell proliferation in response to epidermal growth factor (EGF) and hepatocyte growth factor (HGF). In addition, drug responsiveness to EGFR and cMET inhibitors (Erlotinib, Crizotinib, Cetuximab [Erbitux] and Onartuzumab [MetMab]) was evaluated by measuring the extent of cell proliferation and migration. Data showed that EGFR and cMET expression is reduced at day four of untreated spheroid culture compared to monolayer. Basal phosphorylation of EGFR and cMET was higher in spheroids compared to monolayer cultures. Spheroids showed reduced EGFR and cMET phosphorylation when stimulated with ligand compared to 2D cultures. Spheroids showed an altered cell proliferation response to HGF, as well as to EGFR and cMET inhibitors, compared to monolayer cultures. Finally, spheroid cultures showed exceptional utility in a cell migration assay. Overall, the 3D spheroid culture changed the cellular response to drugs and growth factors and may more accurately mimic the natural tumor microenvironment.

Topics: Antineoplastic Agents; Cell Culture Techniques; Cell Movement; Cell Proliferation; Cell Survival; Drug Discovery; Epidermal Growth Factor; ErbB Receptors; Hepatocyte Growth Factor; Humans; Ligands; Lung Neoplasms; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-met; Reproducibility of Results; Spheroids, Cellular; Treatment Outcome; Tumor Cells, Cultured; Tumor Microenvironment

Oncogenic alterations of epidermal growth factor receptor (EGFR) signaling are frequently observed in lung cancer patients with worse differentiation and poor prognosis. However, the therapeutic efficacy of EGFR-tyrosine kinase inhibitors (TKIs) is currently limited in selected patients with EGFR mutations. Therefore, in this study, we investigated the potential molecular mechanism that contributes to cell viability and the response of gefitinib, one of the EGFR-TKIs, in lung cancer models with wide-type EGFR (wtEGFR). Interestingly, we found that EGF-induced EGFR endocytosis is existed differently between gefitinib-sensitive and -insensitive lung cancer cell lines. Suppressing EGFR endocytos decreased cell viability and increased apoptotic cell death in gefitinib-insensitive lung cancer with wtEGFR in vitro and in vivo. In addition, we found that Rab25 was differentially expressed in between gefitinib-sensitive and -insensitive lung cancer cells. Rab25 knockdown caused the changed EGFR endocytosis and reverted the gefitinib response in gefitinib-sensitive lung cancer with wtEGFR in vitro and in vivo. Taken together, our findings suggest a novel insight that EGFR endocytosis is a rational therapeutic target in lung cancer with wtEGFR, in which the combined efficacy with gefitinib is expected. Furthermore, we demonstrated that Rab25 plays an important role in EGFR endocytosis and gefitinib therapy.

Topics: Animals; Apoptosis; Cell Line, Tumor; Cell Survival; Drug Resistance, Neoplasm; Dynamins; Endocytosis; Epidermal Growth Factor; ErbB Receptors; Gefitinib; Gene Expression Profiling; Gene Knockdown Techniques; Humans; Hydrazones; Lung Neoplasms; Male; Mice; Molecular Targeted Therapy; Protein Kinase Inhibitors; Quinazolines; rab GTP-Binding Proteins; Signal Transduction

Recent epidemiologic studies implying differences in cancer recurrence based on anesthetic regimens raise the possibility that the mu opioid receptor (MOR) can influence cancer progression. Based on our previous observations that overexpression of MOR in human non-small cell lung cancer (NSCLC) cells increased tumor growth and metastasis, this study examined whether MOR regulates growth factor receptor signaling and epithelial mesenchymal transition (EMT) in human NSCLC cells. We utilized specific siRNA, shRNA, chemical inhibitors and overexpression vectors in human H358 NSCLC cells that were either untreated or treated with various concentrations of DAMGO, morphine, fentanyl, EGF or IGF. Cell function assays, immunoblot and immunoprecipitation assays were then performed. Our results indicate MOR regulates opioid and growth factor-induced EGF receptor signaling (Src, Gab-1, PI3K, Akt and STAT3 activation) which is crucial for consequent human NSCLC cell proliferation and migration. In addition, human NSCLC cells treated with opioids, growth factors or MOR overexpression exhibited an increase in snail, slug and vimentin and decrease ZO-1 and claudin-1 protein levels, results consistent with an EMT phenotype. Further, these effects were reversed with silencing (shRNA) or chemical inhibition of MOR, Src, Gab-1, PI3K, Akt and STAT3 (p<0.05). Our data suggest a possible direct effect of MOR on opioid and growth factor-signaling and consequent proliferation, migration and EMT transition during lung cancer progression. Such an effect provides a plausible explanation for the epidemiologic findings.

Topics: Analgesics, Opioid; Anesthetics; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Movement; Cell Proliferation; Disease Progression; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; ErbB Receptors; Gene Silencing; Humans; Lung Neoplasms; Receptors, Opioid, mu; Signal Transduction

Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKI) are effective for non-small cell lung cancers (NSCLC) with EGFR-activating mutations. However, most responders develop resistance. Efatutazone, a novel peroxisome proliferator-activated receptor gamma (PPARγ) agonist, is currently under clinical evaluation; it has antiproliferative effects and induces cellular morphological changes and differentiation. The present study investigated the effects of efatutazone in EGFR-TKI-resistant NSCLC cells, while focusing on cell motility. The PC-9-derived NSCLC cell lines PC-9ER and PC-9ZD, resistant to EGFR-TKI due to v-crk avian sarcoma virus CT10 oncogene homolog-like (CRKL) amplification-induced phosphatidylinositol 3-kinase (PI3K)/v-akt murine thymoma viral oncogene homolog (AKT) activation and an EGFR T790M mutation, respectively, were used. These cells exhibit enhanced cell motility due to transforming growth factor β (TGF-β)/Smad2 family member 2 (Smad2) pathway activation. Efatutazone had no growth-inhibitory effect on the tested cells but inhibited the motility of EGFR-TKI-resistant cells in wound closure and transwell assays. Efatutazone plus erlotinib treatment provided greater inhibition of PC-9ER cell migration than efatutazone or erlotinib alone. Efatutazone suppressed increased TGF-β2 secretion from both cell lines (shown by ELISA) and downregulation of TGF-β2 transcription (observed by quantitative RT-PCR). Immunoblot analysis and luciferase assays revealed that efatutazone suppressed Smad2 phosphorylation and its transcriptional activity. These results suggest that efatutazone inhibits cell motility by antagonizing the TGF-β/Smad2 pathway and effectively prevents metastasis in NSCLC patients with acquired resistance to EGFR-TKI regardless of the resistance mechanism.

Topics: Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Cell Differentiation; Cell Line, Tumor; Cell Movement; Cell Proliferation; Down-Regulation; Drug Resistance, Neoplasm; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Erlotinib Hydrochloride; Humans; Lung Neoplasms; Phosphatidylinositol 3-Kinases; PPAR gamma; Protein Kinase Inhibitors; Quinazolines; Smad2 Protein; Thiazolidinediones; Transforming Growth Factor beta2; Wound Healing

The endocannabinoid anandamide (AEA), a neurotransmitter was shown to have anti-cancer effects. Fatty acid amide hydrolase (FAAH) metabolizes AEA and decreases its anti-tumorigenic activity. In this study, we have analyzed the role of FAAH inhibition in non-small cell lung cancer (NSCLC). We have shown that FAAH and CB1 receptor which is activated by AEA are expressed in lung adenocarcinoma patient samples and NSCLC cell lines A549 and H460. Since the synthetic analogue of anandamide (Met-F-AEA) did not possess significant anti-tumorigenic effects, we used Met-F-AEA in combination with FAAH inhibitor URB597 which significantly reduced EGF (epidermal growth factor)-induced proliferative and chemotactic activities in vitro when compared to anti-tumorigenic activity of Met-F-AEA alone. Further analysis of signaling mechanisms revealed that Met-F-AEA in combination with URB597 inhibits activation of EGFR and its downstream signaling ERK, AKT and NF-kB. In addition, it inhibited MMP2 secretion and stress fiber formation. We have also shown that the Met-F-AEA in combination with URB597 induces G0/G1 cell cycle arrest by downregulating cyclin D1 and CDK4 expressions, ultimately leading to apoptosis via activation of caspase-9 and PARP. Furthermore, the combination treatment inhibited tumor growth in a xenograft nude mouse model system. Tumors derived from Met-F-AEA and URB597 combination treated mice showed reduced EGFR, AKT and ERK activation and MMP2/MMP9 expressions when compared to Met-F-AEA or URB597 alone. Taken together, these data suggest in EGFR overexpressing NSCLC that the combination of Met-F-AEA with FAAH inhibitor resulted in superior therapeutic response compared to individual compound activity alone.

Topics: Adenocarcinoma; Amidohydrolases; Animals; Apoptosis; Arachidonic Acids; Blotting, Western; Calcium Channel Blockers; Carcinoma, Non-Small-Cell Lung; Cell Adhesion; Cell Cycle; Cell Movement; Cell Proliferation; Chemotaxis; Drug Resistance, Neoplasm; Endocannabinoids; Epidermal Growth Factor; ErbB Receptors; Fluorescent Antibody Technique; Humans; Immunoenzyme Techniques; Lung Neoplasms; Male; Mice; Mice, Nude; NF-kappa B; Polyunsaturated Alkamides; Real-Time Polymerase Chain Reaction; Receptor, Cannabinoid, CB1; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Tissue Array Analysis; Tumor Cells, Cultured; Wound Healing; Xenograft Model Antitumor Assays

Genetic mutations in tumor cells cause several unique metabolic phenotypes that are critical for cancer cell proliferation. Mutations in the tyrosine kinase epidermal growth factor receptor (EGFR) induce oncogenic addiction in lung adenocarcinoma (LAD). However, the linkage between oncogenic mutated EGFR and cancer cell metabolism has not yet been clearly elucidated. Here we show that EGFR signaling plays an important role in aerobic glycolysis in EGFR-mutated LAD cells. EGFR-tyrosine kinase inhibitors (TKIs) decreased lactate production, glucose consumption, and the glucose-induced extracellular acidification rate (ECAR), indicating that EGFR signaling maintained aerobic glycolysis in LAD cells. Metabolomic analysis revealed that metabolites in the glycolysis, pentose phosphate pathway (PPP), pyrimidine biosynthesis, and redox metabolism were significantly decreased after treatment of LAD cells with EGFRTKI. On a molecular basis, the glucose transport carried out by glucose transporter 3 (GLUT3) was downregulated in TKI-sensitive LAD cells. Moreover, EGFR signaling activated carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD), which catalyzes the first step in de novo pyrimidine synthesis. We conclude that EGFR signaling regulates the global metabolic pathway in EGFR-mutated LAD cells. Our data provide evidence that may link therapeutic response to the regulation of metabolism, which is an attractive target for the development of more effective targeted therapies to treat patients with EGFR-mutated LAD.

Topics: Adenocarcinoma; Aspartate Carbamoyltransferase; Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing); Cell Line, Tumor; Dihydroorotase; Epidermal Growth Factor; ErbB Receptors; Glucose; Glucose Transporter Type 3; Glycolysis; Humans; Lactic Acid; Lung Neoplasms; Neoplasm Proteins; Pentose Phosphate Pathway; Signal Transduction

Alectinib is a new generation ALK inhibitor with activity against the gatekeeper L1196M mutation that showed remarkable activity in a phase I/II study with echinoderm microtubule associated protein-like 4 (EML4)--anaplastic lymphoma kinase (ALK) non-small cell lung cancer (NSCLC) patients. However, alectinib resistance may eventually develop. Here, we found that EGFR ligands and HGF, a ligand of the MET receptor, activate EGFR and MET, respectively, as alternative pathways, and thereby induce resistance to alectinib. Additionally, the heat shock protein 90 (Hsp90) inhibitor suppressed protein expression of ALK, MET, EGFR, and AKT, and thereby induced apoptosis in EML4-ALK NSCLC cells, even in the presence of EGFR ligands or HGF. These results suggest that Hsp90 inhibitors may overcome ligand-triggered resistance to new generation ALK inhibitors and may result in more successful treatment of NSCLC patients with EML4-ALK.

Topics: Benzoquinones; Blotting, Western; Carbazoles; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Hepatocyte Growth Factor; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Ligands; Lung Neoplasms; Mutation; Oncogene Proteins, Fusion; Phosphorylation; Piperidines; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-met; Transforming Growth Factor alpha; Triazoles

TrkB mediates the effects of brain-derived neurotrophic factor (BDNF) in neuronal and nonnneuronal cells. Based on recent reports that TrkB can also be transactivated through epidermal growth-factor receptor (EGFR) signaling and thus regulates migration of early neurons, we investigated the role of TrkB in migration of lung tumor cells. Early metastasis remains a major challenge in the clinical management of non-small cell lung cancer (NSCLC). TrkB receptor signaling is associated with metastasis and poor patient prognosis in NSCLC. Expression of this receptor in A549 cells and in another adenocarcinoma cell line, NCI-H441, promoted enhanced migratory capacity in wound healing assays in the presence of the TrkB ligand BDNF. Furthermore, TrkB expression in A549 cells potentiated the stimulatory effect of EGF in wound healing and in Boyden chamber migration experiments. Consistent with a potential loss of cell polarity upon TrkB expression, cell dispersal and de-clustering was induced in A549 cells independently of exogeneous BDNF. Morphological transformation involved extensive cytoskeletal changes, reduced E-cadherin expression and suppression of E-cadherin expression on the cell surface in TrkB expressing tumor cells. This function depended on MEK and Akt kinase activity but was independent of Src. These data indicate that TrkB expression in lung adenoma cells is an early step in tumor cell dissemination, and thus could represent a target for therapy development.

Topics: Cadherins; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Movement; Cell Polarity; Epidermal Growth Factor; Humans; Lung Neoplasms; Membrane Glycoproteins; Neoplasm Metastasis; Protein-Tyrosine Kinases; Receptor, trkB; Signal Transduction

Inhibitor of DNA binding/Differentiation 1 (ID1) is a helix loop helix transcription factor that lacks the basic DNA binding domain. Over-expression of ID1 has been correlated with a variety of human cancers; our earlier studies had shown that reported ID1 is induced by nicotine or EGF stimulation of non-small cell lung cancer (NSCLC) cells and its down regulation abrogates cell proliferation, invasion and migration. Here we made attempts to identify downstream targets of ID1 that mediate these effects.. A microarray analysis was done on two different NSCLC cell lines (A549 and H1650) that were transfected with a siRNA to ID1 or a control, non-targeting siRNA. Cells were stimulated with nicotine and genes that were differentially expressed upon nicotine stimulation and ID1 depletion were analyzed to identify potential downstream targets of ID1. The prospective role of the identified genes was validated by RT-PCR. Additional functional assays were conducted to assess the role of these genes in nicotine induced proliferation, invasion and migration. Experiments were also conducted to elucidate the role of ID1, which does not bind to DNA directly, affects the expression of these genes at transcriptional level.. A microarray analysis showed multiple genes are affected by the depletion of ID1; we focused on two of them: Stathmin-like3 (STMN3), a microtubule destabilizing protein, and GSPT1, a protein involved in translation termination; these proteins were induced by both nicotine and EGF in an ID1 dependent fashion. Overexpression of ID1 in two different cell lines induced STMN3 and GSPT1 at the transcriptional level, while depletion of ID1 reduced their expression. STMN3 and GSPT1 were found to facilitate the proliferation, invasion and migration of NSCLC cells in response to nAChR activation. Attempts made to assess how ID1, which is a transcriptional repressor, induces these genes showed that ID1 down regulates the expression of two transcriptional co-repressors, NRSF and ZBP89, involved in the repression of these genes.. Collectively, our data suggests that nicotine and EGF induce genes such as STMN3 and GSPT1 to promote the proliferation, invasion and migration of NSCLC, thus enhancing their tumorigenic properties. These studies thus reveal a central role for ID1 and its downstream targets in facilitating lung cancer progression.

Topics: Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Movement; Cell Proliferation; DNA-Binding Proteins; Down-Regulation; Epidermal Growth Factor; Fluorescent Antibody Technique; Gene Expression Regulation, Neoplastic; Glutathione S-Transferase pi; Humans; Inhibitor of Differentiation Protein 1; Lung Neoplasms; Neoplasm Invasiveness; Nicotine; Promoter Regions, Genetic; Repressor Proteins; RNA, Small Interfering; Stathmin; Transcription Factors; Up-Regulation; Wound Healing

Aberrant epidermal growth factor (EGF)-dependent signaling plays a key role in the progression of human carcinomas. We found that TIP30, a tumor suppressor protein, translocated into the nucleus of human lung adenocarcinoma cells following EGF treatment, and the selective inhibitors of EGFR signaling pathways blocked this effect. Chromatin immunoprecipitation assays revealed that TIP30 negatively regulated EGF-dependent transcriptional activation of CCND1 through a HDAC1-dependent mechanism. In lung adenocarcinoma patients, the level of nuclear TIP30 was inversely correlated with that of EGFR and cyclin D1. These findings suggest that nuclear TIP30-induced downregulation of cyclin D1 transcription antagonizes EGFR signaling and suppresses tumorigenesis.

Topics: Acetyltransferases; Active Transport, Cell Nucleus; Adenocarcinoma; Animals; Cell Line, Tumor; Cell Nucleus; Cyclin D1; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Neoplastic; HEK293 Cells; Humans; Lung Neoplasms; Mice; Protein Binding; Signal Transduction; Transcription Factors; Transcription, Genetic

Inhalation of chemotherapeutic drugs directly into the lungs augments the drug exposure to lung cancers. The inhalation of free drugs however results in over exposure and causes severe adverse effect to normal cells. In the present study, epidermal growth factor (EGF)-modified gelatin nanoparticles (EGNP) was developed to administer doxorubicin (DOX) to lung cancers.. The EGNP released DOX in a sustained manner and effectively internalized in EGFR overexpressing A549 and H226 lung cancer cells via a receptor-mediated endocytosis. In vitro cytotoxicity assay showed that EGNP effectively inhibited the growth of A549 and H226 cells in a dose-dependent manner. In vivo biocompatibility study showed that both GNP and EGNP did not activate the inflammatory response and had a low propensity to cause immune response. Additionally, EGNP maintained a high therapeutic concentration in lungs throughout up to 24 h comparing to that of free drug and GNP, implying the effect of ligand-targeted tumor delivery. Mice treated with EGNP remarkably suppressed the tumor growth (~90% tumor inhibition) with 100% mice survival rate. Furthermore, inhalation of EGNP resulted in elevated levels of cleaved caspase-3 (apoptotic marker), while MMP-9 level significantly reduced comparing to that of control group.. Overall, results suggest that EGF surface-modified nanocarriers could be delivered to lungs via inhalation and controlled delivery of drugs in the lungs will greatly improve the therapeutic options in lung cancer therapy. This ligand-targeted nanoparticulate system could be promising for the lung cancer treatment.

Topics: Animals; Antineoplastic Agents; Caspase 3; Cell Line, Tumor; Doxorubicin; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Lung Neoplasms; Matrix Metalloproteinase 9; Mice; Mice, Inbred BALB C; Mice, Nude; Nanoparticles; Neoplasm Metastasis; Xenograft Model Antitumor Assays

Non-small cell lung cancer (NSCLC) affects millions of patients each year worldwide. Existing therapies include epidermal growth factor receptor (EGFR) inhibition using small molecules or antibodies with good efficacy. Unfortunately, intrinsic and acquired resistance to EGFR therapy remains a persistent complication for disease treatment. A greater understanding of the role of EGFR in NSCLC etiology is crucial to improving patient outcomes. In this study, the role of EGFR in tumor angiogenesis was examined in H292 NSCLC cells under the pretense that confluent cells would exhibit a more angiogenic and growth-centered phenotype. Indeed, confluent H292 cells potentiated endothelial cell angiogenesis in co-culture models in an EGFR-dependent manner. While confluent H292 cells did not exhibit any change in EGFR protein expression, EGFR localization to the extracellular membrane was increased. EGFR membrane localization coincided with a comparable potentiation of maximal EGFR phosphorylation and was followed by a 3-fold increase in vascular endothelial growth factor A (VEGF-A) production as compared to subconfluent cells. EGFR-mediated VEGF-A production was determined to be dependent on signal transducer and activator of transcription 3 (STAT3) activation and not phosphoinositide 3-kinase (PI3K) signaling. These results identify unique cell density dependent phenotypes within a monoclonal NSCLC cell line and provide a potential mechanism of resistance to anti-EGFR therapy in metastatic NSCLC.

Topics: Antibodies, Monoclonal, Humanized; Carcinoma, Non-Small-Cell Lung; Cell Line; Cell Proliferation; Cetuximab; Coculture Techniques; Epidermal Growth Factor; ErbB Receptors; Humans; Lung Neoplasms; Neovascularization, Physiologic; Phosphorylation; Proto-Oncogene Proteins c-met; RNA, Messenger; Signal Transduction; STAT3 Transcription Factor; Up-Regulation; Vascular Endothelial Growth Factor A

Promyelocytic leukemia protein (PML) is emerging as an important tumor suppressor. Its expression is lost during the progression of several types of cancer, including lung cancer. The EGF receptor (EGFR), a membrane-bound receptor tyrosine kinase, transduces intracellular signals responsible for cell proliferation, differentiation and migration. EGFR activity is frequently abnormally upregulated in lung adenocarcinoma (LAC) and thus is considered to be a driving oncogene for LAC. EGFR translocates into the nucleus and transcriptionally activates genes, such as CCND1, that promote cell growth. Recently, we demonstrated that PML interacted with nuclear EGFR (nEGFR) and suppressed the nEGFR-mediated transcriptional activation of CCND1 in lung cancer cells, thereby restraining cell growth. When we further investigated the interplay between PML and EGFR in lung cancer metastasis, we found that the matrix metalloprotease-2 gene (MMP2) was a novel nEGFR target gene and was repressed by PML. We provide evidence that nEGFR bound to the AT-rich sequence (ATRS) in the MMP2 promoter and enhanced its transcriptional activity. In addition, we demonstrated that PML repressed nEGFR-induced MMP2 transcription and reduced cell invasion. PML was recruited by nEGFR to the MMP2 promoter where it reduced histone acetylation, leading to the transcriptional repression of MMP2. Finally, we demonstrated that PML upregulation by interferon-β (IFNβ) in lung cancer cells decreased MMP2 expression and cell invasion. Together, our results suggested that IFNβ induced PML to inhibit lung cancer metastasis by repressing the nEGFR-mediated transcriptional activation of MMP2.

Topics: Adenocarcinoma; Animals; Base Sequence; Binding Sites; Cell Line, Tumor; Cell Nucleus; Cyclin D1; Epidermal Growth Factor; ErbB Receptors; HEK293 Cells; Histones; Humans; Interferon-beta; Lung Neoplasms; Male; Matrix Metalloproteinase 2; Mice; Mice, Inbred NOD; Mice, SCID; Neoplasm Metastasis; Nuclear Proteins; Promoter Regions, Genetic; Promyelocytic Leukemia Protein; RNA, Small Interfering; STAT3 Transcription Factor; Transcription Factors; Transcriptional Activation; Tumor Suppressor Proteins; Up-Regulation

Growth factor signaling is deregulated in cancer and often leads to invasion, yet receptor tyrosine kinase signaling pathways driving invasion under different growth factor conditions are not well understood. To identify specific signaling molecules regulating invasion of A549 non-small cell lung carcinoma (NSCLC) cells downstream of the epidermal growth factor receptor (EGFR) and Met, quantitative site-specific mass spectrometric analysis of tyrosine phosphorylation was performed following epidermal growth factor (EGF) or hepatocyte growth factor (HGF) stimulation, at three different growth factor concentrations and at two time points. Through this analysis, the temporal and concentration dependent phosphorylation profiles were obtained for 131 and 139 sites downstream of EGF and HGF stimulation, respectively. To characterize the effect of these signaling network alterations, we quantified 3D cell migration/invasion through Matrigel. Partial least-squares regression (PLSR) analysis was performed to identify the tyrosine phosphorylation sites most strongly correlated with EGF and/or HGF mediated invasion. Potential common and specific signaling events required for driving invasion downstream of EGFR and Met were identified using either a combined or two independent PLSR models, based on the quantitative EGF or HGF data. Our data highlight the integration and compartmentalization of signaling required for invasion in cancer.

Topics: Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Movement; Epidermal Growth Factor; ErbB Receptors; Hepatocyte Growth Factor; Humans; Lung Neoplasms; Phosphorylation; Phosphotyrosine; Proto-Oncogene Proteins c-met; Signal Transduction

Invadopodia or invasive feet, which are actin-rich membrane protrusions with matrix degradation activity formed by invasive cancer cells, are a key determinant in the malignant invasive progression of tumors and represent an important target for cancer therapies. In this work, we presented a microfluidic 3D culture device with continuous supplement of fresh media via a syringe pump. The device mimicked tumor microenvironment in vivo and could be used to assay invadopodia formation and to study the mechanism of human lung cancer invasion. With this device, we investigated the effects of epidermal growth factor (EGF) and matrix metalloproteinase (MMP) inhibitor, GM6001 on invadopodia formation by human non-small cell lung cancer cell line A549 in 3D matrix model. This device was composed of three units that were capable of achieving the assays on one control group and two experimental groups' cells, which were simultaneously pretreated with EGF or GM6001 in parallel. Immunofluorescence analysis of invadopodia formation and extracellular matrix degradation was conducted using confocal imaging system. We observed that EGF promoted invadopodia formation by A549 cells in 3D matrix and that GM6001 inhibited the process. These results demonstrated that epidermal growth factor receptor (EGFR) signaling played a significant role in invadopodia formation and related ECM degradation activity. Meanwhile, it was suggested that MMP inhibitor (GM6001) might be a powerful therapeutic agent targeting invadopodia formation in tumor invasion. This work clearly demonstrated that the microfluidic-based 3D culture device provided an applicable platform for elucidating the mechanism of cancer invasion and could be used in testing other anti-invasion agents.

Topics: Apoptosis; Carcinoma; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Survival; Dipeptides; Epidermal Growth Factor; Extracellular Matrix; Humans; Lung Neoplasms; Microfluidic Analytical Techniques; Tumor Cells, Cultured

Low-grade inflammation and emphysema have been shown to be associated with an increased risk of lung cancer. However, the systemic inflammatory response in patients with emphysema is still unknown.. TO COMPARE THE PLASMA CYTOKINE PROFILES IN TWO GROUPS OF CURRENT OR FORMER SMOKERS WITHOUT AIRWAY OBSTRUCTION: a control group of individuals without computed tomography (CT) detected emphysema vs. a study group of individuals with CT detected emphysema.. Subjects underwent a chest CT, spirometry, and determination of EGF, IL-15, IL-1ra, IL-8, MCP-1, MIP-1β, TGFα, TNFα, and VEGF levels in plasma. Cytokine levels in each group were compared adjusting for confounding factors.. 160 current smokers and former smokers without airway obstruction participated in the study: 80 without emphysema and 80 subjects with emphysema. Adjusted group comparisons revealed significant reductions in EGF (-0.317, p = 0.01), IL-15 (-0.21, p = 0.01), IL-8 (-0.180, p = 0.02) and IL-1ra (-0.220, p = 0.03) in subjects with emphysema and normal spirometry.. Current or former smokers expressing a well-defined disease characteristic such as emphysema, has a specific plasma cytokine profile. This includes a decrease of cytokines mainly implicated in activation of apoptosis or decrease of immunosurveillance. This information should be taken into account when evaluated patients with tobacco respiratory diseases.

Topics: Cytokines; Epidermal Growth Factor; Female; Humans; Interleukin 1 Receptor Antagonist Protein; Interleukin-15; Interleukin-8; Lung Neoplasms; Male; Mass Screening; Middle Aged; Multivariate Analysis; Pulmonary Emphysema; Smoking; Tomography, X-Ray Computed

It has been proved that cyclooxygenase-2 (COX-2) is a key factor in lung cancer oncogenesis. COX-2 can be induced by a number of cytokines and growth factors and can be regulated by the JAK/STAT signaling pathway. Inhibiting the expression of COX-2 can prevent the development of lung cancer. The aim fo this study is to investigate whether the epidermal growth factor (EGF) can stimulate the signal transducers and activators of transcription 5 (STAT5) as well as to discover the effects of the STAT5 signaling pathway on the COX-2 in human lung adenocarcinoma A549 cells.. The phenomenon of STAT5 activation stimulated by the EGF was assayed through immunofluorescence and Western blot. The adenovirus containing the wild-type (WT)-STAT5 (AdWT-STAT5) plasmid, dominant-negative (DN)-STAT5 (Ad-CMV5Stat5aΔ740) plasmid, and STAT5 siRNA were transfected into A549 cells. The latter two groups were stimulated using EGF. Reverse transcriptase polymerase chain reaction was used to detect the mRNA expression of COX-2.. STAT5 was not activated in A549 cells in vitro. EGF stimulation significantly increased the level of the p-STAT5 protein and induces the shuttling of p-STAT5 from the cytoplasm into the nucleus. STAT5 activation was crucial for the COX-2 expression induced by the EGF. STAT5 was required for COX-2 expression, but can mediated the effects of the COX-2 expression through pathways that were independent of transcriptional activation.. COX-2 expression is dependent on STAT5 phosphorylation. A second pathway does not require STAT5 phosphorylation.. 背景与目的 已有的研究表明COX-2在肺癌发生发展过程中起关键作用，它被一些细胞因子和生长因子所诱导产生，并受到JAK/STAT等信号通路的调控，抑制COX-2的表达能阻止肺癌的发展。本研究旨在探讨表皮生长因子（epidermal growth factor, EGF）在人肺腺癌A549细胞中对STAT5激活效应，以及STAT5信号通路对COX-2调控机制。方法 应用免疫荧光法及Western印迹法检测人肺腺癌A549细胞中EGF对STAT5的激活现象。分别用野生型STAT5（AdWT STAT5），STAT5显性负突变体（AdCMV5 Stat5a△740）以及STAT5 siRNA转染A549细胞，并用EGF对后两组转染细胞加以刺激，使STAT5及p-STAT5的表达发生变化，再用RT-PCR检测A549细胞中的COX-2 mRNA表达。结果 在体外A549细胞中STAT5无激活；EGF可以诱导STAT5的激活，促使磷酸化的STAT5穿梭入核；STAT5的激活是EGF诱导COX-2上调表达的必要条件；非磷酸化的STAT5可能通过非转录激活的途径参与了COX-2表达的调控。结论 在A549细胞中STAT5可以通过磷酸化和非磷酸化两种途径来实现对COX-2的调控。

Topics: Adenocarcinoma; Blotting, Western; Cell Line, Tumor; Cyclooxygenase 2; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Microscopy, Fluorescence; Models, Genetic; Phosphorylation; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Signal Transduction; STAT5 Transcription Factor

Tid1 (DNAJA3), a DnaJ cochaperone, may promote degradation of oncogenic kinases. Tid1 has 2 isoforms, Tid1-L and Tid1-S, that may function differently. In this study, we investigated the role of the Tid1 isoforms in regulating EGF receptor (EGFR) signaling and lung cancer progression. We found that both Tid1-L and Tid1-S expressions were reduced in patients with non-small cell lung cancer compared with normal counterparts. Tid1-L expression correlated inversely with EGFR expression. Low Tid1-L/high EGFR expression predicted poor overall survival in patients with lung adenocarcinoma. Tid1-L overexpression in lung cancer cells attenuated EGFR signaling and inhibited cell proliferation, colony formation, and tumor growth in subcutaneous and orthotropic xenograft models. Conversely, depletion of Tid1 restored EGFR signaling and increased cell proliferation and colony formation. Tid1-L, but not Tid1-S, interacted with EGFR/HSP70/HSP90 through the DnaJ domain, counteracting the EGFR regulatory function of HSP90 by causing EGFR ubiquitinylation and proteasomal degradation. Tid1-L inhibited EGFR signaling even more than the HSP90 inhibitor 17-allylamino-demethoxy geldanamycin. We concluded that Tid1-L acted as a tumor suppressor by inhibiting EGFR signaling through interaction with EGFR/HSP70/HSP90 and enhancing EGFR ubiquitinylation and degradation.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Animals; Apoptosis; Cell Line, Tumor; Cell Proliferation; Epidermal Growth Factor; ErbB Receptors; Female; Gene Expression; HSP40 Heat-Shock Proteins; HSP70 Heat-Shock Proteins; HSP90 Heat-Shock Proteins; Humans; Kaplan-Meier Estimate; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Middle Aged; Multivariate Analysis; Neoplasm Transplantation; Polyubiquitin; Protein Isoforms; Protein Structure, Tertiary; Proteolysis; Signal Transduction; Ubiquitination

Activation of EGF receptor (EGFR) triggers signaling pathways regulating various cellular events that contribute to tissue development and function. Aberrant activation of EGFR contributes to tumor progression as well as therapeutic resistance in patients with cancer. C-terminal tensin-like (CTEN; TNS4) is a focal adhesion molecule that is a member of the tensin family. Its expression is upregulated by EGF and elevated CTEN mediates EGF-induced cell migration. In the presence of CTEN, we found that EGF treatment elevated the level of EGFR protein but not mRNA. The extended half-life of activated EGFR sustained its signaling cascades. CTEN reduced ligand-induced EGFR degradation by binding to the E3 ubiquitin ligase c-Cbl and decreasing the ubiquitination of EGFR. The Src homology 2 domain of CTEN is not only required for binding to the phosphorylated tyrosine residue at codon 774 of c-Cbl, but is also essential for the tumorigenicity observed in the presence of CTEN. Public database analyses indicated that CTEN mRNA levels are elevated in breast, colon, lung, and pancreas cancers, but not correlated with EGFR mRNA levels in these cancers. In contrast, immunohistochemistry analyses of lung cancer specimens showed that CTEN and EGFR protein levels were positively associated, in support of our finding that CTEN regulates EGFR protein levels through a posttranslational mechanism. Overall, this work defines a function for CTEN in prolonging signaling from EGFR by reducing its ligand-induced degradation.

Topics: Cell Line; Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; HCT116 Cells; HEK293 Cells; Humans; Ligands; Lung Neoplasms; Microfilament Proteins; Phosphorylation; Protein Binding; Protein Interaction Domains and Motifs; Protein Processing, Post-Translational; Proteolysis; Proto-Oncogene Proteins c-cbl; RNA, Messenger; Signal Transduction; src Homology Domains; Tensins; Tyrosine; Ubiquitination

MET amplification as a mechanism of acquired resistance to EGF receptor (EGFR)-targeted therapies in non-small cell lung carcinoma (NSCLC) led to investigation of novel combinations of EGFR and MET kinase inhibitors. However, promiscuous interactions between MET and ERBB family members have made it difficult to evaluate the effects of MET on EGFR signaling, both independent of drug treatment and in the context of drug resistance. We addressed this issue by establishing a 32D model cell system wherein ERBBs or MET are expressed alone and in combination. Using this model, we determined that EGFR signaling is sufficient to induce MET phosphorylation, although MET activation is enhanced by coexpression of ERBB3. EGFR-MET cross-talk was not direct, but occurred by a combined regulation of MET levels and intermediary signaling through mitogen-activated protein kinases (MAPK). In NSCLCs harboring either wild-type or mutant EGFR, inhibiting EGFR or MAPK reduced MET activation and protein levels. Furthermore, MET signaling promoted EGFR-driven migration and invasion. Finally, EGFR-MET signaling was enhanced in a highly metastatic EGFR-mutant cell subpopulation, compared with the indolent parental line, and MET attenuation decreased the incidence of brain metastasis. Overall, our results establish that EGFR-MET signaling is critical for aggressive behavior of NSCLCs and rationalize its continued investigation as a therapeutic target for tumors harboring both wild-type and mutant EGFR at early stages of progression.

Topics: Animals; Brain Neoplasms; Carcinoma, Non-Small-Cell Lung; Cell Movement; Epidermal Growth Factor; ErbB Receptors; Humans; Lung Neoplasms; Mice; Mitogen-Activated Protein Kinases; Neoplasm Invasiveness; Neoplasm Metastasis; Oncogene Proteins v-erbB; Phosphorylation; Proto-Oncogene Proteins c-met; Receptor, ErbB-3

To investigate the relationship between EGFR activation and down-regulation of miRNA-145 in lung cancer.. Normal human lung epithelia cell line (BEAS-2B), human lung adenocarcinoma cell lines with wild-type EGFR (A549 and H292) and human lung adenocarcinoma cell lines with EGFR mutation (H1975 and H1650) were chosen in this study. The levels of miRNA-145 and p-EGFR were determined by quantitative real-time PCR (qRT-PCR) and Western blotting, respectively, and the relationship between p-EGFR and miRNA-145 levels was analyzed. The miRNA-145 levels were determined by qRT-PCR after activating EGFR with EGF or blocking EGFR signal pathway with AG1478. In addition, ERK1/2 inhibitor U0126 was used to inhibit ERK1/2 activation and then the expression of miRNA-145 was detected.. The miRNA-145 levels were closely negatively related with p-EGFR in lung cancer cells (r = -0.926, P = 0.024). EGF down-regulated miRNA-145 expression, particularly in BEAS-2B cells (53.0%; t = 30.993, P = 0.001) and A549 cells (42.6%; t = 14.326, P = 0.005).The miRNA-145 was up-regulated after inhibiting p-EGFR with AG1478, and significantly enhanced by 67.5% in H1975 cells when treated with AG1478 (t = 8.269, P = 0.014). The ERK1/2 signal pathway was activated by p-EGFR. U0126 restored the miRNA-145 down-regulation induced by EGFR-activation in lung cancer cells.. The activation of EGFR down-regulates miRNA-145 expression through ERK1/2 in lung cancer cells.

Topics: Butadienes; Carcinoma, Non-Small-Cell Lung; Cell Line; Cell Line, Tumor; Down-Regulation; Enzyme Activation; Enzyme Inhibitors; Epidermal Growth Factor; Epithelial Cells; ErbB Receptors; Humans; Lung; Lung Neoplasms; MAP Kinase Signaling System; MicroRNAs; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nitriles; Phosphorylation; Quinazolines; Tyrphostins

Physiological electric field (EF) plays a pivotal role in tissue development and regeneration. In vitro, cells under direct-current electric field (dcEF) stimulation may demonstrate directional migration (electrotaxis) and long axis reorientation (electro-alignment). Although the biophysical models and biochemical signaling pathways behind cell electrotaxis have been investigated in numerous normal cells and cancer cells, the molecular signaling mechanisms in CL1 lung adenocarcinoma cells have not been identified. Two subclones of CL1 cells, the low invasive CL1-0 cells and the highly invasive CL 1-5 cells, were investigated in the present study. CL1-0 cells are non-electrotactic while the CL 1-5 cells are anodally electrotactic and have high expression level of epidermal growth factor receptor (EGFR), in this study, we investigated the generally accepted hypothesis of receptor tyrosine kinase (RTK) activation in the two cell lines under dcEF stimulation. Erbitux, a therapeutic drug containing an anti-EGFR monoclonal antibody, cetuximab, was used to investigate the EGFR signaling in the electrotaxis of CL 1-5 cells. To investigate RTK phosphorylation and intracellular signaling in the CL1 cells, large amount of cellular proteins were collected in an airtight dcEF stimulation device, which has advantages of large culture area, uniform EF distribution, easy operation, easy cell collection, no contamination, and no medium evaporation. Commercial antibody arrays and Western blotting were used to study the phosphorylation profiles of major proteins in CL1 cells under dcEF stimulation. We found that electrotaxis of CL 1-5 cells is serum independent and EGFR independent. Moreover, the phosphorylation of Akt and S6 ribosomal protein (rpS6) in dcEF-stimulated CL1 cells are different from that in EGF-stimulated cells. This result suggests that CL1 cells' response to dcEF stimulation is not through EGFR-triggered pathways. The new large-scale dcEF stimulation device developed in the present work will aid the sample preparation for protein-based experiments.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Cell Line, Tumor; Cell Movement; Cetuximab; Electric Stimulation; Electromagnetic Fields; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation; Humans; Lung Neoplasms; Phosphorylation; Proto-Oncogene Proteins c-akt; Ribosomal Protein S6; Signal Transduction

Various ion channels are expressed in human cancers where they are intimately involved in proliferation, angiogenesis, invasion and metastasis. Expression of functional voltage-gated Na(+) channels (Nav) is implicated in the metastatic potential of breast, prostate, lung and colon cancer cells. However, the cellular mechanisms that regulate Nav expression in cancer remain largely unknown. Growth factors are attractive candidates; they not only play crucial roles in cancer progression but are also key regulators of ion channel expression and activity in non-cancerous cells. Here, we examine the role of epidermal growth factor receptor (EGFR) signalling and Nav in non-small cell lung carcinoma (NSCLC) cell lines. We show unequivocally, that functional expression of the α subunit Nav1.7 promotes invasion in H460 NSCLC cells. Inhibition of Nav1.7 activity (using tetrodotoxin) or expression (by using small interfering RNA), reduces H460 cell invasion by up to 50%. Crucially, non-invasive wild type A549 cells lack functional Nav, whereas exogenous overexpression of the Nav1.7 α subunit is sufficient to promote TTX-sensitive invasion of these cells. EGF/EGFR signalling enhances proliferation, migration and invasion of H460 cells but we find that, specifically, EGFR-mediated upregulation of Nav1.7 is necessary for invasive behaviour in these cells. Examination of Nav1.7 expression at mRNA, protein and functional levels further reveals that EGF/EGFR signalling via the ERK1/2 pathway controls transcriptional regulation of channel expression to promote cellular invasion. Immunohistochemistry of patient biopsies confirms the clinical relevance of Nav1.7 expression in NSCLC. Thus, Nav1.7 has significant potential as a new target for therapeutic intervention and/or as a diagnostic or prognostic marker in NSCLC.

Topics: Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Movement; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; NAV1.7 Voltage-Gated Sodium Channel; Neoplasm Invasiveness; Signal Transduction

Epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor 2 (VEGFR2) have emerged as two effective clinical targets for non-small-cell lung cancer (NSCLC). In the present study, we found that delphinidin, an anthocyanidin, present in pigmented fruits and vegetables, is a potent inhibitor of both EGFR and VEGFR2 in NSCLC cells that overexpress EGFR/VEGFR2. Using these cells, we next determined the effects of delphinidin on cell growth and apoptosis in vitro and on tumor growth and angiogenesis in vivo. Delphinidin (5-60 µM) treatment of NSCLC cells inhibited the activation of PI3K, and phosphorylation of AKT and MAPKs. Additionally, treatment of NSCLC cells with delphinidin resulted in inhibition of cell growth without having significant toxic effects on normal human bronchial epithelial cells. Specifically, treatment of NCI-H441 and SK-MES-1 cells with delphindin (5-60 µM) resulted in (i) cleavage of PARP protein, (ii) activation of caspase-3 and -9, (iii) downregulation of anti-apoptotic proteins (Bcl2, Bcl-xL and Mcl-1), (iv) upregulation of pro-apoptotic proteins (Bax and Bak), and (v) decreased expression of PCNA and cyclin D1. Furthermore, in athymic nude mice subcutaneously implanted with human NSCLC cells, delphinidin treatment caused a (i) significant inhibition of tumor growth, (ii) decrease in the expression of markers for cell proliferation (Ki67 and PCNA) and angiogenesis (CD31 and VEGF), and (iii) induction of apoptosis, when compared with control mice. Based on these observations, we suggest that delphinidin, alone or as an adjuvant to current therapies, could be used for the management of NSCLC, especially those that overexpress EGFR and VEGFR2.

Topics: Animals; Anthocyanins; Antineoplastic Agents, Phytogenic; Apoptosis; Carcinoma, Non-Small-Cell Lung; Caspases; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Disease Models, Animal; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Lung Neoplasms; Mice; Mitogen-Activated Protein Kinases; Neovascularization, Pathologic; Phosphatidylinositol 3-Kinases; Phosphorylation; Poly(ADP-ribose) Polymerases; Proliferating Cell Nuclear Antigen; Proteolysis; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Tumor Burden; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-2; Xenograft Model Antitumor Assays

The epidermal growth factor receptor (EGFR) signaling system is frequently unbalanced in human malignancies due to increased ligand production, receptor overexpression, receptor mutations, and/or cross-talk with other receptor systems. For this reason, the EGFR is an attractive target for anticancer therapy. The epidermal growth factor also plays an important role in regulating multiple facets of cutaneous wound healing, including inflammation, wound contraction, proliferation, migration, and angiogenesis. In the Center of Molecular Immunology, a cancer vaccine is produced (CIMAvax® EGF) that blocks the binding of EGF to its receptor. This blockade causes a significant inverse association between the anti-EGF antibody titers and EGF concentration. Around 1,500 patients with non-small cell lung cancer have been treated, showing that this vaccine is safe, immunogenic, increases survival and improves quality of life. Taking into account the therapeutic benefits of CIMAvax® EGF vaccination and the role of EGF-EGFR system in the wound healing process, we decided to conduct a retrospective research with the aim of determining the effect to the CIMAvax® EGF vaccine on the wound healing process in patients undergoing surgical treatment.. Medical records of 452 vaccinated patients were reviewed and only six patients receiving surgical treatment were identified. Further information about these six patients was obtained from source documents, including medical records and operative reports using an observational list that included different variables. Post-surgical wound healing complications were identified using the National Cancer Institute Common Toxicity Criteria for Adverse Events (NCI-CTC) version 3.0.. None of the six patients operated on presented adverse events related to the wound healing, that is to say, no wound dehiscence, wound infection, delayed wound healing, fistula formation, abscess formation or hemorrhage/bleeding associated with surgery during treatment with CIMAvax® EGF occurred.. These results suggest that the use of CIMAvax® EGF does not produce a deleterious effect in the wound healing process.

Topics: Aged; Cancer Vaccines; Carcinoma, Non-Small-Cell Lung; Epidermal Growth Factor; ErbB Receptors; Female; Follow-Up Studies; Humans; Lung Neoplasms; Male; Middle Aged; Prognosis; Retrospective Studies; Signal Transduction; Wound Healing

Somatic mutations in the EGFR proto-oncogene occur in ~15% of human lung adenocarcinomas and the importance of EGFR mutations for the initiation and maintenance of lung cancer is well established from mouse models and cancer therapy trials in human lung cancer patients. Recently, we identified DOK2 as a lung adenocarcinoma tumor suppressor gene. Here we show that genomic loss of DOK2 is associated with EGFR mutations in human lung adenocarcinoma, and we hypothesized that loss of DOK2 might therefore cooperate with EGFR mutations to promote lung tumorigenesis. We tested this hypothesis using genetically engineered mouse models and find that loss of Dok2 in the mouse accelerates lung tumorigenesis initiated by oncogenic EGFR, but not that initiated by mutated Kras. Moreover, we find that DOK2 participates in a negative feedback loop that opposes mutated EGFR; EGFR mutation leads to recruitment of DOK2 to EGFR and DOK2-mediated inhibition of downstream activation of RAS. These data identify DOK2 as a tumor suppressor in EGFR-mutant lung adenocarcinoma.

Topics: Adaptor Proteins, Signal Transducing; Adenocarcinoma; Adenocarcinoma of Lung; Animals; Carcinogenesis; Cell Line; Epidermal Growth Factor; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Gene Knockout Techniques; Genomics; Humans; Lung Neoplasms; Male; Mice; Mice, Transgenic; Mutation; Phosphoproteins; Proto-Oncogene Mas; ras Proteins; Tumor Suppressor Proteins

The targeting of oncogenic 'driver' kinases with small molecule inhibitors has proven to be a highly effective therapeutic strategy in selected non-small cell lung cancer (NSCLC) patients. However, acquired resistance to targeted therapies invariably arises and is a major limitation to patient care. ROS1 fusion proteins are a recently described class of oncogenic driver, and NSCLC patients that express these fusions generally respond well to ROS1-targeted therapy. In this study, we sought to determine mechanisms of acquired resistance to ROS1 inhibition. To accomplish this, we analyzed tumor samples from a patient who initially responded to the ROS1 inhibitor crizotinib but eventually developed acquired resistance. In addition, we generated a ROS1 inhibition-resistant derivative of the initially sensitive NSCLC cell line HCC78. Previously described mechanisms of acquired resistance to tyrosine kinase inhibitors including target kinase-domain mutation, target copy number gain, epithelial-mesenchymal transition, and conversion to small cell lung cancer histology were found to not underlie resistance in the patient sample or resistant cell line. However, we did observe a switch in the control of growth and survival signaling pathways from ROS1 to EGFR in the resistant cell line. As a result of this switch, ROS1 inhibition-resistant HCC78 cells became sensitive to EGFR inhibition, an effect that was enhanced by co-treatment with a ROS1 inhibitor. Our results suggest that co-inhibition of ROS1 and EGFR may be an effective strategy to combat resistance to targeted therapy in some ROS1 fusion-positive NSCLC patients.

Topics: Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Cell Survival; Crizotinib; Drug Resistance, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Gene Amplification; Humans; Lung Neoplasms; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Pyrazoles; Pyridines; Signal Transduction

Our aims were to investigate whether the association between smoking and survival is significant when adjusted for prognostic factors including use of epidermal growth factor tyrosine kinase inhibitors and the Glasgow Prognostic Score, an established score for inflammation, and to explore prognostic factors.. We analyzed 244 patients with stage IIIB or IV non-small-cell lung cancer in a registry, including only chemotherapy-receiving outpatients with performance status zero.. Of 244 patients, 170 had died and the median follow-up time for the 74 surviving patients was 12.0 months. In multivariate Cox regression, smoker (hazard ratio compared to never-smoker: 1.67, P < 0.01), stage IV (hazard ratio compared to IIIB: 1.72, P < 0.01), and elevated C-reactive protein level (hazard ratio per 1 mg/dL increase: 1.08, P < 0.01) were significantly associated with shorter survival. The association between survival and smoking was significant, even after adjustment for the Glasgow Prognostic Score and regimens of chemotherapy (hazard ratio: 1.72, P = 0.02). In never-smokers, increased neutrophils were a major determinant of shorter survival and the interaction test between smoking and neutrophils was significant (hazard ratio per 1,000/mm(3) increase for smokers: 1.01; hazard ratio per 1,000/mm(3) increase for never-smokers: 1.44, P for interaction <0.01).. Known factors including treatment response or inflammatory process are not responsible for the fact that advanced non-small-cell lung cancer patients without any history of smoking have better survival than those who have smoked.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Non-Small-Cell Lung; Epidermal Growth Factor; Female; Humans; Lung Neoplasms; Male; Middle Aged; Neoplasm Staging; Neutrophils; Prognosis; Protein Kinase Inhibitors; Smoking; Treatment Outcome

KRAS mutations are one of the most common driver mutations in non-small-cell lung cancer (NSCLC) and finding druggable target molecules to inhibit oncogenic KRAS signaling is a significant challenge in NSCLC therapy. We recently identified epiregulin (EREG) as one of several putative transcriptional targets of oncogenic KRAS signaling in both KRAS-mutant NSCLC cells and immortalized bronchial epithelial cells expressing ectopic mutant KRAS. In the current study, we found that EREG is overexpressed in NSCLCs harboring KRAS, BRAF or EGFR mutations compared with NSCLCs with wild-type KRAS/BRAF/EGFR. Small interfering RNAs (siRNAs) targeting mutant KRAS, but not an siRNA targeting wild-type KRAS, significantly reduced EREG expression in KRAS-mutant and EREG-overexpressing NSCLC cell lines. In these cell lines, EREG expression was downregulated by MEK and ERK inhibitors. Importantly, EREG expression significantly correlated with KRAS expression or KRAS copy number in KRAS-mutant NSCLC cell lines. Further expression analysis using 89 NSCLC specimens showed that EREG was predominantly expressed in NSCLCs with pleural involvement, lymphatic permeation or vascular invasion and in KRAS-mutant adenocarcinomas. In addition, multivariate analysis revealed that EREG expression is an independent prognostic marker and EREG overexpression in combination with KRAS mutations was associated with an unfavorable prognosis for lung adenocarcinoma patients. In KRAS-mutant and EREG overexpressing NSCLC cells, siRNA-mediated EREG silencing inhibited anchorage-dependent and -independent growth and induced apoptosis. Our findings suggest that oncogenic KRAS-induced EREG overexpression contributes to an aggressive phenotype and could be a promising therapeutic target in oncogenic KRAS-driven NSCLC.

Topics: Aged; Apoptosis; Butadienes; Carcinoma, Non-Small-Cell Lung; Cell Line; Cell Line, Tumor; Epidermal Growth Factor; Epiregulin; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Kaplan-Meier Estimate; Lung Neoplasms; Male; Mitogen-Activated Protein Kinases; Mutation; Nitriles; Phenotype; Proto-Oncogene Proteins; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins p21(ras); Pyrazoles; Pyridazines; ras Proteins; RNA Interference

Epidermal growth factor (EGF) receptor plays a crucial role in the biology of human cancer, and is a highly appropriate target for anticancer agents. We have previously designed oligopeptides containing the amino acid sequences around autophosphorylation sites of EGF receptor to identify a specific inhibitor of this receptor. We found that Ac-ENAEYLR-NH(2) and Ac-NYQQN-NH(2) suppressed phosphorylation of purified EGF receptor in a non-ATP-competitive manner whereas Ac-QNAQYLR-NH(2) and Ac-DYQQD-NH(2) caused inhibition in an ATP-competitive manner. The aim of this study was to observe the effects of these peptides on the proliferation, cell death, and apoptosis of human lung carcinoma A549 cells. To facilitate transfer of these inhibitory peptides into A549 cells, the cell-penetrating peptide, human immunodeficiency virus type 1-transactivator of transcription (Tat), was linked to the peptides. When A549 cells were treated with each Tat-conjugated peptide, the peptides penetrated the cells and EGF-stimulated tyrosine phosphorylation of EGF receptor was significantly suppressed. These Tat-conjugated peptides played a suppressive role in EGF-stimulated A549 cell responses. In particular, Tat-epsilon-aminocaproic acid (acp)-ENAEYLR-NH(2) significantly inhibited proliferation and showed cytotoxicity, while Tat-acp-NYQQN-NH(2) and Tat-acp-DYQQD-NH(2) suppressed the anti-apoptotic effect of EGF. In addition, we found that Tat-acp-ENAEYLR-NH(2) also inhibited the phosphorylation of epidermal growth factor receptor 2 (ErbB2) as well as EGF receptor in A549 cells. In conclusion, membrane-permeable synthetic peptides derived from EGF receptor autophosphorylation sites have the potential to suppress EGF receptor function in A549 cells and to be developed into novel and useful agents for cancer therapy.

Topics: Amino Acid Sequence; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Proliferation; Epidermal Growth Factor; ErbB Receptors; Gene Products, tat; Humans; Lung Neoplasms; Oligopeptides; Permeability; Phosphorylation

Norcantharidin is the demethylated analog of cantharidin isolated from blister beetles (Mylabris phalerata Pall.). In this study, we evaluated whether norcantharidin exhibits anticancer effects against the human non-small cell lung cancer cell lines A549 (epidermal growth factor receptor (EGFR) mutation-negative) and PC9 (EGFR mutation-positive). Our results revealed that norcantharidin dose-dependently retards cell growth, arrests cell cycle at G2/M phase, reduces cell migration, and even induces apoptosis at the concentration of 100 µM. Moreover, we found that norcantharidin enhances the anticancer effects of gefitinib and cisplatin. Norcantharidin exhibited similar potency of anticancer effects against the two cell lines with different EGFR mutation status and did not affect EGF-induced EGFR phosphorylation, suggesting that the EGFR signaling may not be the target of norcantharidin. In conclusion, our results suggest that norcantharidin exhibits anticancer effects against non-small cell lung cancer cells in vitro and support its potential as a chemotherapeutic agent for treating non-small cell lung cancer.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Blotting, Western; Bridged Bicyclo Compounds, Heterocyclic; Carcinoma, Non-Small-Cell Lung; Cell Adhesion; Cell Cycle; Cell Movement; Cell Proliferation; Cisplatin; Epidermal Growth Factor; ErbB Receptors; Flow Cytometry; Fluorescent Antibody Technique; Gefitinib; Humans; Lung Neoplasms; Mutation; Phosphorylation; Quinazolines; Signal Transduction; Tumor Cells, Cultured

Tissue Inhibitor of Metalloproteinase 2 (TIMP-2) plays an essential role in regulating matrix remodeling, cell growth, differentiation, angiogenesis and apoptosis in vitro and in vivo. We have recently shown that TIMP-2-mediated inhibition of tumor growth is independent of matrix metalloproteinase-mediated mechanisms, and is a consequence of modulating both the tumor cells and the tumor microenvironment. In the current study we aim to identify the molecular pathways associated with these effects. We analyzed the transcriptional profile of the human lung cancer cell line A549 upon overexpression of TIMP-2 and Ala+TIMP-2 (mutant that does not inhibit MMP activity), and we found changes in gene expression predominantly related to decreased tumor development and metastasis. Increased E-cadherin expression in response to both TIMP-2 and Ala+TIMP-2 expression was confirmed by real time quantitative RT-PCR and immunoblotting. A549 cells treated with epidermal growth factor (EGF) displayed loss of cobblestone morphology and cell-cell contact, while cells overexpressing TIMP-2 or Ala+TIMP-2 were resistant to EGF-induced morphological changes. Moreover, exogenous treatment with recombinant Ala+TIMP-2 blocked EGF induced down-regulation of E-cadherin. In vivo, immunohistochemistry of A549 xenografts expressing either TIMP-2 or Ala+TIMP-2 demonstrated increased E-cadherin protein levels. More importantly, transcriptional profile analysis of tumor tissue revealed critical pathways associated with effects on tumor-host interaction and inhibition of tumor growth. In conclusion, we show that TIMP-2 promotes an anti-tumoral transcriptional profile in vitro and in vivo, including upregulation of E-cadherin, in A549 lung cancer cells.

Topics: Animals; beta Catenin; Cadherins; Cell Adhesion; Cell Line, Tumor; Drug Resistance, Neoplasm; Epidermal Growth Factor; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Immunoblotting; Lung Neoplasms; Mice; Mice, Inbred NOD; Mice, SCID; Microscopy, Confocal; Mutation; Oligonucleotide Array Sequence Analysis; Protease Inhibitors; Reverse Transcriptase Polymerase Chain Reaction; Tissue Inhibitor of Metalloproteinase-2; Transplantation, Heterologous

PHA-E is a natural product extracted from red kidney beans, and it has been reported to induce cell apoptosis by blocking EGFR in lung cancer cells. Because EGF is the major in vivo competitor to PHA-E in clinical application, PHA-E must be proved that has better affinity to EGFR than EGF. This study would focus on how PHA-E tightly bind to EGFR and the results would compare with EGF. The adhesion force, measured by AFM, between EGFR and PHA-E was 207.14±74.42 pN that was higher than EGF (183.65±86.93 pN). The equilibrium dissociation constant of PHA-E and EGF to EGFR was 2.4 10(-9)±1.4 10(-9) and 7.3 10(-8)±2.7 10(-8), respectively, that could evaluate binding affinity. The result showed that binding affinity of PHA-E to EGFR was one order higher than EGF to EGFR. In the results of flow cytometer and confocal microscope, we found binding efficiency of EGF to EGFR was decrease as the concentration of PHA-E increased. In the analysis of Western blot, treatment of A-549 cells with PHA-E resulted in a dose-dependent decrease in EGFR phosphorylation. In conclusion, we found that PHA-E had better adhesion force and binding affinity to EGFR than that of the EGF. The interaction between PHA-E and EGFR could block EGF binding and then inhibit EGFR phosphorylation. PHA-E could be developed into a new target molecule for lung cancer treatment that could be immobilized on the drug carrier to guide therapeutic particles to the tumor site.

Topics: Antineoplastic Agents, Phytogenic; Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Humans; Lung Neoplasms; Phosphorylation; Phytohemagglutinins; Protein Binding

We have recently shown that the adaptor protein p140Cap regulates tumor properties in terms of cell motility and growth. Here, by using the highly metastatic rat adenocarcinoma cell line MTLn3-epidermal growth factor receptor (EGFR), we assess the role of p140Cap in metastasis formation. Orthotopic transplantation of MTLn3-EGFR cells over-expressing p140Cap in Rag2(-/-)γ(c)(-/-) mice resulted in normal primary tumor growth compared with the controls. Strikingly, p140Cap over-expression causes an 80% inhibition in the number of lung metastases. p140Cap over-expressing cells display a 50% reduction in directional cell migration, an increased number and size of focal adhesions, and a strong impairment in the ability to invade in a 3D matrix. p140Cap over-expression affects EGFR signaling and tyrosine phosphorylation of cortactin in response to EGF stimulation. Intriguingly, p140Cap associates with cortactin via interaction with its second proline-rich domain to the cortactin SH3 domain. The phosphomimetic cortactin tyrosine 421 mutant rescues migration and invasive properties in p140Cap over-expressing cells. Taken together, these data demonstrate that p140Cap suppresses the invasive properties of highly metastatic breast carcinoma cells by inhibiting cortactin-dependent cell motility.

Topics: Adaptor Proteins, Vesicular Transport; Adenocarcinoma; Animals; Cell Line, Tumor; Cell Movement; Cortactin; Epidermal Growth Factor; ErbB Receptors; Green Fluorescent Proteins; HEK293 Cells; Humans; Immunohistochemistry; Lung Neoplasms; Mammary Neoplasms, Experimental; Mice; Mice, Knockout; Microscopy, Fluorescence, Multiphoton; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Transplantation; Phosphorylation; Protein Binding; Rats; RNA Interference; Transplantation, Heterologous

A study of patients with advanced non-squamous non-small cell lung cancer (NSCLC) evaluated epidermal growth factor receptor (EGFR) mutation status and serum hepatocyte growth factor (HGF) for their associations with response to gefitinib therapy and prognostic impact. An enzyme-linked immunosorbent assay was used to determine levels of HGF in serum from 96 Japanese patients with advanced non-squamous NSCLC. The peptic nucleic acid-locked nucleic acid clamp method was used to determine their EGFR somatic mutation status. We evaluated the relationship between each independent clinicopathological variable and the response to gefitinib therapy and risk factors associated with prognosis. HGF-positive serum status (hazard ratio, 1.536; 95% confidence interval, 1.042-2.400; P = 0.0295) had a significant and independent negative effect on progression-free survival among patients with wild-type EGFR. We demonstrate that having HGF-positive serum is predictive of a negative response to gefitinib therapy in patients with advanced NSCLC who harbor wild-type EGFR.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Agents; Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Disease-Free Survival; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; Erlotinib Hydrochloride; Female; Gefitinib; Hepatocyte Growth Factor; Humans; Kaplan-Meier Estimate; Lung Neoplasms; Male; Middle Aged; Mutation; Prognosis; Proportional Hazards Models; Quinazolines

Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) show dramatic antitumor activity in a subset of patients with non-small cell lung cancer who have an active mutation in the epidermal growth factor receptor (EGFR) gene. On the other hand, some lung cancer patients with wild type EGFR also respond to EGFR-TKIs, suggesting that EGFR-TKIs have an effect on host cells as well as tumor cells. However, the effect of EGFR-TKIs on host microenvironments is largely unknown. A multiple organ metastasis model was previously established in natural killer cell-depleted severe combined immunodeficient mice using human lung cancer cells. This model was used to investigate the therapeutic efficacy of erlotinib, an EGFR-TKI, on multiple organ metastases induced by human small cell lung cancer cells (SBC-5 cells) that did not express EGFR. Although erlotinib did not have any effect on the proliferation of SBC-5 cells in vitro, it significantly suppressed bone and lung metastases in vivo, but not liver metastases. An immunohistochemical analysis revealed that, erlotinib significantly suppressed the number of osteoclasts in bone metastases, whereas no difference was seen in microvessel density. Moreover, erlotinib inhibited EGF-induced receptor activator of nuclear factor kappa-B expression in an osteoblastic cell line (MC3T3-E1 cells). These results strongly suggested that erlotinib prevented bone metastases by affecting host microenvironments irrespective of its direct effect on tumor cells.

Topics: Animals; Bone Neoplasms; Carcinoma, Small Cell; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epidermal Growth Factor; ErbB Receptors; Erlotinib Hydrochloride; Humans; Lung Neoplasms; Male; Mice; Neoplasm Metastasis; Neovascularization, Pathologic; Osteoblasts; Osteoclasts; Protein Kinase Inhibitors; Quinazolines; RANK Ligand

A high-throughput 32D(L858R/T790M) cell-based assay to identify inhibitors of the L858R/T790M mutant epidermal growth factor receptor (EGFR) pathway was established. After screening, ten hits from among 60,000 compounds in our in-house compound library were initially identified. In the secondary assays, one hit, 1-[2-(decyloxy)-2-oxoethyl]-3-methyl-2-[(4-methylphenoxy) methyl]-1H-benzimidazol-3-ium, was confirmed to directly inhibit the kinase activity of recombinant L858R/T790M EGFR and the phosphorylation of EGFR-L858R/T790M in gefitinib-resistant H1975 cells. Thus, this high-throughput assay system may be useful for identifying novel inhibitors which suppress mutant EGFR-T790M signalling and for overcoming T790M-mediated acquired resistance for future anticancer drug discovery.

Topics: Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Drug Resistance, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Gefitinib; High-Throughput Screening Assays; Humans; Lung Neoplasms; Mutation; Phosphorylation; Protein Kinase Inhibitors; Quinazolines; Signal Transduction

Overexpression of the oxygen-responsive transcription factor hypoxia-inducible factor 1α (HIF-1α) correlates with poor prognosis in breast cancer patients. The mouse mammary tumor virus polyoma virus middle T (MMTV-PyMT) mouse is a widely utilized preclinical mouse model that resembles human luminal breast cancer and is highly metastatic. Prior studies in which the PyMT model was used demonstrated that HIF-1α is essential to promoting carcinoma onset and lung metastasis, although no differences in primary tumor end point size were observed. Using a refined model system, we investigated whether HIF-1α is directly implicated in the regulation of tumor-initiating cells (TICs) in breast cancer.. Mammary tumor epithelial cells were created from MMTV-PyMT mice harboring conditional alleles of Hif1a, followed by transduction ex vivo with either adenovirus β-galactosidase or adenovirus Cre to generate wild-type (WT) and HIF-1α-null (KO) cells, respectively. The impact of HIF-1α deletion on tumor-initiating potential was investigated using tumorsphere assays, limiting dilution transplantation and gene expression analysis.. Efficient deletion of HIF-1α reduced primary tumor growth and suppressed lung metastases, prolonging survival. Loss of HIF-1α led to reduced expression of markers of the basal lineage (K5/K14) in cells and tumors and of multiple genes involved in the epithelial-to-mesenchymal transition. HIF-1α also enhanced tumorsphere formation at normoxia and hypoxia. Decreased expression of several genes in the Notch pathway as well as Vegf and Prominin-1 (CD133)was observed in response to Hif1a deletion. Immunohistochemistry confirmed that CD133 expression was reduced in KO cells and in tumorspheres. Tumorsphere formation was enhanced in CD133hi versus CD133neg cells sorted from PyMT tumors. Limiting dilution transplantation of WT and KO tumor cells into immunocompetent recipients revealed > 30-fold enrichment of TICs in WT cells.. These results demonstrate that HIF-1α plays a key role in promoting primary mammary tumor growth and metastasis, in part through regulation of TICs. HIF-1α regulates expression of several members of the Notch pathway, CD133 and markers of the basal lineage in mammary tumors. Our results suggest that CD133, which has not been profiled extensively in breast cancer, may be a useful marker of TICs in the PyMT mouse model. These data reveal for the first time that HIF-1α directly regulates breast TIC activity in vivo.

Topics: Animals; Caspase 3; Cell Proliferation; Cell Separation; Epidermal Growth Factor; Epithelial Cells; Estrogen Receptor alpha; Female; Flow Cytometry; Gene Expression; Hypoxia-Inducible Factor 1, alpha Subunit; Ki-67 Antigen; Lung Neoplasms; Mammary Neoplasms, Experimental; Mice; Mice, 129 Strain; Mice, Inbred C57BL; Mice, Knockout; Neoplasm Invasiveness; Neoplasm Transplantation; Neoplastic Stem Cells; Phosphoproteins; Spheroids, Cellular; Trans-Activators; Tumor Burden; Tumor Cells, Cultured

Metastasis is a multistep process and the main cause of mortality in lung cancer patients. We previously showed that EGFR mutations were associated with a copy number gain at a locus encompassing the TWIST1 gene on chromosome 7. TWIST1 is a highly conserved developmental gene involved in embryogenesis that may be reactivated in cancers promoting both malignant conversion and cancer progression through an epithelial to mesenchymal transition (EMT). The aim of this study was to investigate the possible implication of TWIST1 reactivation on the acquisition of a mesenchymal phenotype in EGFR mutated lung cancer. We studied a series of consecutive lung adenocarcinoma from Caucasian non-smokers for which surgical frozen samples were available (n = 33) and showed that TWIST1 expression was linked to EGFR mutations (P<0.001), to low CDH1 expression (P<0.05) and low disease free survival (P = 0.044). To validate that TWIST1 is a driver of EMT in EGFR mutated lung cancer, we used five human lung cancer cell lines and demonstrated that EMT and the associated cell mobility were dependent upon TWIST1 expression in cells with EGFR mutation. Moreover a decrease of EGFR pathway stimulation through EGF retrieval or an inhibition of TWIST1 expression by small RNA technology reversed the phenomenon. Collectively, our in vivo and in vitro findings support that TWIST1 collaborates with the EGF pathway in promoting EMT in EGFR mutated lung adenocarcinoma and that large series of EGFR mutated lung cancer patients are needed to further define the prognostic role of TWIST1 reactivation in this subgroup.

Topics: Adenocarcinoma; Aged; Aged, 80 and over; Antigens, CD; Blotting, Western; Cadherins; Cell Line, Tumor; Cell Movement; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Kaplan-Meier Estimate; Lung Neoplasms; Male; Middle Aged; Mutation; Nuclear Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Twist-Related Protein 1

Sphingosine-1-phosphate (S1P) regulates a wide array of biological functions. However, the role of S1P signaling in tumorigenesis remains to be elucidated. In this study, we show that S1P receptor subtype 3 (S1P₃) is markedly up-regulated in a subset of lung adenocarcinoma cells compared to normal lung epithelial cells. Specific knockdown of S1P₃ receptors inhibits proliferation and anchorage-independent growth of lung adenocarcinoma cells. Mechanistically, we demonstrate that S1P₃ signaling increases epidermal growth factor receptor (EGFR) expression via the Rho kinase (ROCK) pathway in lung adenocarcinoma cells. Nuclear run-off analysis indicates that S1P/S1P₃ signaling transcriptionally increases EGFR expression. Knockdown of S1P₃ receptors diminishes the S1P-stimulated EGFR expression in lung adenocarcinoma cells. Moreover, S1P treatment greatly enhances EGF-stimulated colony formation, proliferation and invasion of lung adenocarcinoma cells. Together, these results suggest that the enhanced S1P₃-EGFR signaling axis may contribute to the tumorigenesis or progression of lung adenocarcinomas.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Animals; Carcinoma, Lewis Lung; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Lysophospholipids; Mice; Neoplasm Invasiveness; Receptors, Lysosphingolipid; rho-Associated Kinases; RNA Interference; RNA, Messenger; Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors; Time Factors; Transcriptional Activation; Transfection; Up-Regulation

To determine whether factors secreted by irradiated liver nonparenchymal cells (NPCs) may influence invasiveness and/or metastatic potential of hepatocellular carcinoma (HCC) cells and to elucidate a possible mechanism for such effect.. Primary rat NPCs were cultured and divided into irradiated (10-Gy X-ray) and nonirradiated groups. Forty-eight hours after irradiation, conditioned medium from irradiated (SR) or nonirradiated (SnonR) cultures were collected and added to sublethally irradiated cultures of the hepatoma McA-RH7777 cell line. Then, hepatoma cells were continuously passaged for eight generations (RH10Gy-SR and RH10Gy-SnonR). The invasiveness and metastatic potential of McA-RH7777, RH10Gy-SnonR, and RH10Gy-SR cells were evaluated using an in vitro gelatinous protein (Matrigel) invasion and an in vivo metastasis assay. In addition, SR and SnonR were tested using rat cytokine antibody arrays and enzyme-linked immunosorbent assay (ELISA).. In vitro gelatinous protein invasion assay indicated that the numbers of invading cells was significantly higher in RH10Gy-SR (40 ± 4.74) than in RH10Gy-SnonR (30.6 ± 3.85) cells, and lowest in McA-RH7777 (11.4 ± 3.56) cells. The same pattern was observed in vivo in a lung metastasis assay, as evaluated by number of metastatic lung nodules seen with RH10Gy-SR (28.83 ± 5.38), RH10Gy-SnonR (22.17 ± 4.26), and McA-RH7777 (8.3 ± 3.8) cells. Rat cytokine antibody arrays and ELISA demonstrated that metastasis-promoting cytokines (tumor necrosis factor-α and interleukin-6), circulating growth factors (vascular endothelial growth factor and epidermal growth factor), and metalloproteinases (MMP-2 and MMP-9) were upregulated in SR compared with SnonR.. Radiation can increase invasiveness and metastatic potential of sublethally irradiated hepatoma cells, and soluble mediators released from irradiated NPCs promote this potential. Increased secretion of metastasis-related cytokines and factors from NPCs after irradiation may be a possible mechanism for the radiation-induced invasiveness and metastatic potential of HCC.

Topics: Animals; Carcinoma, Hepatocellular; Cell Culture Techniques; Collagen; Cytokines; Drug Combinations; Epidermal Growth Factor; Hepatocytes; Interleukin-6; Laminin; Liver Neoplasms; Lung Neoplasms; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Proteoglycans; Rats; Rats, Sprague-Dawley; Tumor Necrosis Factor-alpha; Vascular Endothelial Growth Factor A

Epidermal growth factor (EGF) and its receptor play critical roles in non-small cell lung cancer (NSCLC) carcinogenesis. A functional polymorphism in the EGF gene has been linked to increased cancer susceptibility. This study aimed to evaluate the role of the EGF +61A/G polymorphism as risk factors in NSCLC patients. For the present case-control study, we analyzed 112 NSCLC and 126 cancer-free controls from Portugal. Following DNA isolation from peripheral blood, EGF +61A/G polymorphism was assessed by polymerase chain reaction-restriction fragment length polymorphism. Univariate and multivariate logistic regression analyses were used to calculate odds ratio (OR) and 95 % confidence intervals (95 % CI). False-positive report probability was also assessed. The EGF +61 genotypes frequencies in NSCLC were AA (23.2 %), AG (51.8 %), and GG (25 %) and in controls, AA (40.5 %), AG (41.3 %), and GG (18.3 %). When compared to the reference genotype (EGF +61A/A), we found a statistically significant association between EGF +61 A/G (OR = 2.142, 95 % CI 1.170-3.924) and EGF +61G/G (OR = 2.398, 95 % CI 1.157-4.968) genotypes and susceptibility to development of NSCLC. Furthermore, stratification by sex revealed a trend to increased risk of males carrying +61A/G genotype for developing NSCLC (OR = 2.044, 95 % CI 0.998-4.188) when compared to A/A genotype. Our data suggest an increased risk to develop NSCLC in Portuguese population carrying the EGF +61A/G and +61G/G genotypes.

Topics: Adult; Aged; Aged, 80 and over; Alleles; Carcinoma, Non-Small-Cell Lung; Case-Control Studies; Epidermal Growth Factor; Female; Genetic Predisposition to Disease; Genotype; Humans; Lung Neoplasms; Male; Middle Aged; Neoplasm Staging; Polymorphism, Single Nucleotide; Portugal; Reproducibility of Results; Risk

In human adenocarcinoma, claudin-2 expression is higher than that in normal lung tissue, but the regulatory mechanism of its expression has not been clarified. In human adenocarcinoma A549 cells, claudin-2 level time-dependently increased under the control conditions. In contrast, claudin-1 expression remained constant for 24h. The concentration of epidermal growth factor (EGF) in medium time-dependently increased, which was inhibited by matrix metalloproteinase (MMP) inhibitor II, an inhibitor of MMP-1, 3, 7, and 9. MMP inhibitor II decreased claudin-2 and phosphorylated ERK1/2 (p-ERK1/2) levels, which were recovered by EGF. Both claudin-2 and p-ERK1/2 levels were decreased by EGF neutralizing antibody, EGF receptor (EGFR) siRNA, AG1478, an inhibitor of EGFR, U0126, an inhibitor of MEK, and the exogenous expression of dominant negative-MEK. These results suggest that EGF is secreted from A549 cells by MMP and increases claudin-2 expression mediated via the activation of an EGFR/MEK/ERK pathway. The inhibition of the signaling pathway decreased phosphorylated c-Fos and nuclear c-Fos levels. The introduction of c-Fos siRNA decreased claudin-2 level without affecting claudin-1. The promoter activity of human claudin-2 was decreased by AG1478 and U0126. Furthermore, the activity was decreased by the deletion or mutation of the AP-1 binding site of claudin-2 promoter. Chromatin immunoprecipitation and avidin-biotin conjugated DNA assays showed that c-Fos binds to the AP-1 binding site. We suggest that a secreted EGF up-regulates the transcriptional activity of claudin-2 mediated by the activation of an EGFR/MEK/ERK/c-Fos pathway in A549 cells.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Binding Sites; Butadienes; Cell Line, Tumor; Claudins; Epidermal Growth Factor; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Male; MAP Kinase Signaling System; Nitriles; Promoter Regions, Genetic; Protease Inhibitors; Protein Binding; Proto-Oncogene Proteins c-fos; Quinazolines; RNA, Messenger; RNA, Small Interfering; Transcription Factor AP-1; Tyrphostins

Phosphorylation of protein plays a key role in the regulation of cellular signal transduction and gene expression. In recent years, targeted mass spectrometry facilitates functional phosphoproteomics by allowing specific protein modifications of target proteins in complex samples to be characterized. In this study, we employed multiple reaction monitoring (MRM) to examine the influence of gefitinib (also known as Iressa) on the phosphorylation sites of EGFR protein before and after EGF treatment. By coupling MRM to MS/MS, 5 phosphotyrosine (Y1110, Y1172, Y1197, Y1069, and Y1092) and 1 S/T (T693) sites were identified on EGFR. Y1197 and T693 were constitutively phosphorylated. All phosphorylation sites were sensitive to gefitinib treatment except T693. Interestingly, gefitinib treatment induced phosphorylation of S1166 only in the presence of EGF. We further showed that lung cancer cells overexpressing phosphomimic S1166D EGFR mutant possessed significantly lower growth and proliferation property compared to wildtype EGFR-expressing cells. While the function and mode of regulation of S1166 remain unclear, our data supports the notion that S1166 represents a regulatory site that exerts a negative regulation on growth and proliferation of cancer cells. The data presented has implication in our understanding of dynamic drug (gefitinib)-target (EGFR) interaction and in improving the efficacy of target-directed therapeutics.

Topics: Amino Acid Motifs; Amino Acid Sequence; Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Epidermal Growth Factor; ErbB Receptors; Gefitinib; Humans; Lung Neoplasms; Molecular Sequence Data; Peptide Fragments; Phosphorylation; Protein Processing, Post-Translational; Quinazolines; Serine; Tandem Mass Spectrometry; Tyrosine

Ceftriaxone, an FDA-approved third-generation cephalosporin antibiotic, has antimicrobial activity against both gram-positive and gram-negative organisms. Generally, ceftriaxone is used for a variety of infections such as community-acquired pneumonia, meningitis and gonorrhea. Its primary molecular targets are the penicillin-binding proteins. However, other activities of ceftriaxone remain unknown. Herein, we report for the first time that ceftriaxone has antitumor activity in vitro and in vivo. Kinase profiling results predicted that Aurora B might be a potential 'off' target of ceftriaxone. Pull-down assay data confirmed that ceftriaxone could bind with Aurora B in vitro and in A549 cells. Furthermore, ceftriaxone (500 µM) suppressed anchorage-independent cell growth by targeting Aurora B in A549, H520 and H1650 lung cancer cells. Importantly, in vivo xenograft animal model results showed that ceftriaxone effectively suppressed A549 and H520 lung tumor growth by inhibiting Aurora B. These data suggest the anticancer efficacy of ceftriaxone for the treatment of lung cancers through its inhibition of Aurora B.

Topics: Animals; Anti-Bacterial Agents; Aurora Kinase B; Aurora Kinases; Ceftriaxone; Cell Line, Tumor; Epidermal Growth Factor; Humans; Lung Neoplasms; Mice; Protein Serine-Threonine Kinases

Epidermal growth factor receptor (EGFR) inhibitors have been used as anticancer agents for the treatment of a variety of solid tumors. Related skin toxicities are the most common adverse effects and occur with all EGFR inhibitors. Several treatment approaches, such as antiseptic soaps, topical and oral antibiotics, and topical and oral corticosteroids, have been reported; however, the responses have been varied. Acneiform eruption induced by EGFR inhibitor treatment results from disturbed normal keratinocyte and hair follicle biology and may therefore benefit from local restoration of EGF pathway.. We treated HaCaT cells with EGFR inhibitor and evaluated the expression of EGFR. After treatment of cells with EGFR inhibitor, EGFR expression was increased in a dose-dependent manner. We hypothesized that newly synthesized EGFR, not inhibited by EGFR inhibitors, may perform their biological action in keratinocytes in the presence of additional EGF. In this study, we therefore treated acneiform eruption patients with topical recombinant human EGF (rhEGF) with institutional review board approval. Here, we report three cases of such eruptions who responded to topical rhEGF.. Topical rhEGF may be an effective treatment option for EGFR inhibitor-induced acneiform eruption.

Topics: Acneiform Eruptions; Antineoplastic Agents; Blotting, Western; Carcinoma, Non-Small-Cell Lung; Drug Eruptions; Epidermal Growth Factor; ErbB Receptors; Gefitinib; Humans; Lung Neoplasms; Male; Middle Aged; Quinazolines; Treatment Outcome; Tumor Cells, Cultured

To identify stage I lung adenocarcinoma patients with a poor prognosis who will benefit from adjuvant therapy.. Whole gene expression profiles were obtained at 19 time points over a 48-hour time course from human primary lung epithelial cells that were stimulated with epidermal growth factor (EGF) in the presence or absence of a clinically used EGF receptor tyrosine kinase (RTK)-specific inhibitor, gefitinib. The data were subjected to a mathematical simulation using the State Space Model (SSM). "Gefitinib-sensitive" genes, the expressional dynamics of which were altered by addition of gefitinib, were identified. A risk scoring model was constructed to classify high- or low-risk patients based on expression signatures of 139 gefitinib-sensitive genes in lung cancer using a training data set of 253 lung adenocarcinomas of North American cohort. The predictive ability of the risk scoring model was examined in independent cohorts of surgical specimens of lung cancer.. The risk scoring model enabled the identification of high-risk stage IA and IB cases in another North American cohort for overall survival (OS) with a hazard ratio (HR) of 7.16 (P = 0.029) and 3.26 (P = 0.0072), respectively. It also enabled the identification of high-risk stage I cases without bronchioalveolar carcinoma (BAC) histology in a Japanese cohort for OS and recurrence-free survival (RFS) with HRs of 8.79 (P = 0.001) and 3.72 (P = 0.0049), respectively.. The set of 139 gefitinib-sensitive genes includes many genes known to be involved in biological aspects of cancer phenotypes, but not known to be involved in EGF signaling. The present result strongly re-emphasizes that EGF signaling status in cancer cells underlies an aggressive phenotype of cancer cells, which is useful for the selection of early-stage lung adenocarcinoma patients with a poor prognosis.. The Gene Expression Omnibus (GEO) GSE31210.

Topics: Adenocarcinoma; Cell Line, Tumor; Computational Biology; Drug Resistance, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Gefitinib; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Neoplasm Staging; Prognosis; Quinazolines; Reproducibility of Results

In a substantial population of non-small cell lung cancer (NSCLC), expression and activation of EGF receptor (EGFR) have been reported and is regarded as a novel molecular target. A growing body of evidence has shown the signaling crosstalk between EGFR and integrins in cellular migration and invasion. NEDD9 is an integrin signaling adaptor protein composed of multiple domains serving as substrate for a variety of tyrosine kinases. In the present study, we aimed at elucidating a role of NEDD9 in the signaling crosstalk between EGFR and integrins.. Using NSCLC cell lines, we conducted immunoblotting and cellular migration/invasion assay in vitro. Next, we analyzed metastasis assays in vivo by the use of xenograft transplantation model. Finally, we retrospectively evaluated clinical samples and records of patients with NSCLCs.. We showed that tyrosine phosphorylation of NEDD9 was reduced by the inhibition of EGFR in NSCLC cell lines. Overexpression of constitutively active EGFR caused tyrosine phosphorylation of NEDD9 in the absence of integrin stimulation. By gene transfer and gene knockdown, we showed that NEDD9 plays a pivotal role in cell migration and invasion of those cells in vitro. Furthermore, overexpression of NEDD9 promoted lung metastasis of an NSCLC cell line in NOD/Shi-scid, IL-2Rγ(null) mice (NOG) mice. Finally, univariate and multivariate Cox model analysis of NSCLC clinical specimens revealed a strong correlation between NEDD9 expression and recurrence-free survival as well as overall survival.. Our data thus suggest that NEDD9 is a promising biomarker for the prognosis of NSCLCs and its expression can promote NSCLC metastasis.

Topics: Adaptor Proteins, Signal Transducing; Animals; Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Movement; Crk-Associated Substrate Protein; Disease-Free Survival; Epidermal Growth Factor; ErbB Receptors; Female; Gefitinib; Gene Knockdown Techniques; Humans; Integrins; Kaplan-Meier Estimate; Lung Neoplasms; Mice; Mice, Inbred NOD; Mice, SCID; Multivariate Analysis; Neoplasm Transplantation; Phosphoproteins; Phosphorylation; Proportional Hazards Models; Protein Processing, Post-Translational; Quinazolines; Receptor Cross-Talk; Retrospective Studies; RNA, Small Interfering; Signal Transduction

Topics: Epidermal Growth Factor; Forecasting; Humans; Lung Neoplasms; Polymorphism, Genetic; Risk Factors

Chemotherapy drug resistance is a major obstacle in the treatment of cancer. It can result from an increase in levels of cellular drug efflux pumps, such as P-glycoprotein (P-gp). Lapatinib, a growth factor receptor tyrosine kinase inhibitor, is currently in clinical trials for treatment of breast cancer. We examined the impact of co-incubation of chemotherapy drugs in combination with lapatinib in P-gp over-expressing drug resistant cells. Unexpectedly, lapatinib treatment, at clinically relevant concentrations, increased levels of the P-gp drug transporter in a dose- and time-responsive manner. Conversely, exposure to the epidermal growth factor (EGF), an endogenous growth factor receptor ligand, resulted in a decrease in P-gp expression. Despite the lapatinib-induced alteration in P-gp expression, use of accumulation, efflux and toxicity assays demonstrated that the induced alteration in P-gp expression by lapatinib had little direct impact on drug resistance.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Antineoplastic Combined Chemotherapy Protocols; ATP Binding Cassette Transporter, Subfamily B, Member 1; Carcinoma, Squamous Cell; Cell Line, Tumor; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Humans; Lapatinib; Lung Neoplasms; Male; Middle Aged; Protein Kinase Inhibitors; Quinazolines; Time Factors

The c-Met receptor is a potential therapeutic target for non-small cell lung cancer (NSCLC). Signaling interactions between c-Met and the mutant epidermal growth factor receptor (EGFR) have been studied extensively, but signaling intermediates and biological consequences of lateral signaling to c-Met in EGFR wild-type tumors are minimally understood. Our observations indicate that delayed c-Met activation in NSCLC cell lines is initiated by wild-type EGFR, the receptor most often found in NSCLC tumors. EGFR ligands induce accumulation of activated c-Met, which begins at 8 h and continues for 48 h. This effect is accompanied by an increase in c-Met expression and phosphorylation of critical c-Met tyrosine residues without activation of mitogen-activated protein kinase (MAPK) or Akt. Gene transcription is required for delayed c-Met activation; however, phosphorylation of c-Met by EGFR occurs without production of hepatocyte growth factor (HGF) or another secreted factor, supporting a ligand-independent mechanism. Lateral signaling is blocked by two selective c-Met tyrosine kinase inhibitors (TKIs), PF2341066 and SU11274, or with gefitinib, an EGFR TKI, suggesting kinase activity of both receptors is required for this effect. Prolonged c-Src phosphorylation is observed, and c-Src pathway is essential for EGFR to c-Met communication. Pretreatment with pan-Src family kinase inhibitors, PP2 and dasatinib, abolishes delayed c-Met phosphorylation. A c-Src dominant-negative construct reduces EGF-induced c-Met phosphorylation compared with control, further confirming a c-Src requirement. Inhibition of c-Met with PF2341066 and siRNA decreases EGF-induced phenotypes of invasion by ~86% and motility by ~81%, suggesting that a novel form of c-Met activation is utilized by EGFR to maximize these biological effects. Combined targeting of c-Met and EGFR leads to increased xenograft antitumor activity, demonstrating that inhibition of downstream and lateral signaling from the EGFR-c-Src-c-Met axis might be effective in treatment of NSCLC.

Topics: Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; CSK Tyrosine-Protein Kinase; Epidermal Growth Factor; ErbB Receptors; Hepatocyte Growth Factor; Humans; Lung Neoplasms; Phosphorylation; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-myc; Signal Transduction; src-Family Kinases

Lung cancer is the leading cause of cancer death worldwide. The epidermal growth factor receptor (EGFR) represents the main target for non-small cell lung cancer (NSCLC) therapy, as its overexpression or constitutive activation contributes to malignancy and correlates with poor prognosis. Our previous work demonstrated that in epithelial cells β1 integrin is required for propagating EGFR signaling from the plasma membrane to the nucleus. In this study, we silenced β1 integrin in human NSCLC A549 cells. The β1 integrin-silenced cells show a defective activation of the EGFR signaling cascade, leading to decreased in vitro proliferation, enhanced sensitivity to cisplatin and Gefitinib, impaired migration and invasive behavior. Inhibitory effects on tumor growth and on the EGFR pathway were also observed in in vivo experiments. Moreover, β1 integrin silencing increases the amount of EGFR on the cell surface, suggesting that β1 integrin is required for efficient constitutive EGFR turnover at the cell membrane. Although the rate of EGF internalization and recycling is not affected in silenced cells, EGFR signaling is recovered only by expression of the Rab-coupling protein RCP, indicating that β1 integrin sustains the endocytic machinery required for EGFR signaling. Overall, these results show that β1 integrin is an essential regulator of EGFR signaling and tumorigenic properties of lung cancer cells, and that its silencing might represent an adjuvant approach to anti-EGFR therapy.

Topics: Adaptor Proteins, Signal Transducing; Animals; Antibodies, Monoclonal; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cisplatin; Epidermal Growth Factor; ErbB Receptors; Gefitinib; Humans; Integrin beta1; Lung Neoplasms; Membrane Proteins; Mice; Mice, SCID; Neoplasm Invasiveness; Neoplasm Transplantation; Quinazolines; RNA Interference; RNA, Small Interfering; Signal Transduction; Transplantation, Heterologous

Estrogens contribute to the pathogenesis of female lung cancer and function mainly through estrogen receptor-β (ERβ). However, the way in which ERβ expression is regulated in lung cancer cells remains to be explored. We have found that signal transducer and activator of transcription 3 (Stat3) activation up-regulates ERβ expression in PC14PE6/AS2 lung cancer cells in a preliminary Affymetrix oligonucleotide array study, and we sought to confirm the findings. In this study, we show that IL-6 induced ERβ mRNA and protein expression in lung cancer cells. The induction of ERβ in response to IL-6 was abolished by Janus kinase 2 inhibitor-AG490, dominant-negative mutant of Stat3, and Stat3-targeting short interfering RNA. The luciferase reporter assay and chromatin immunoprecipitation assay confirmed that IL-6-activated Stat3 binds to the ERβ promoter. Besides the Janus kinase 2/Stat3 pathway, the MEK/Erk pathway contributes to ERβ up-regulation induced by IL-6; however, the phosphoinositide 3'-kinase/Akt pathway does not. We also found that epidermal growth factor (EGF) stimulation or L858R mutation in EGF receptor (EGFR) induced Stat3 activation as well as ERβ expression in lung cancer cells. Inhibiting Stat3 activity by pharmacological or genetic approaches reduced EGF- and L858R mutant EGFR-induced ERβ expression, indicating that Stat3 activation is required for EGFR signaling-mediated ERβ up-regulation. Silencing ERβ decreased cell proliferation in lung cancer cells that overexpress L858R mutant EGFR. In conclusion, we have identified that Stat3 activation is essential for ERβ induction by IL-6, EGF, and the presence of EGFR mutation. The findings shed light on new therapeutic targets for female lung cancer, especially for those with EGFR mutations.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Cell Line, Tumor; Cell Proliferation; Epidermal Growth Factor; ErbB Receptors; Estrogen Receptor beta; Female; Gene Expression Regulation, Neoplastic; Humans; Interleukin-6; Lung Neoplasms; MAP Kinase Signaling System; Mutation, Missense; Neoplasms, Hormone-Dependent; Phosphorylation; Protein Binding; RNA Stability; STAT3 Transcription Factor; Transcription, Genetic; Up-Regulation

Expression of ID1 (inhibitor of differentiation) has been correlated with the progression of a variety of cancers, but little information is available on its role in non-small cell lung cancer (NSCLC). Here we show that ID1 is induced by nicotinic acetylcholine receptor (nAChR) and epidermal growth factor receptor (EGFR) signaling in a panel of NSCLC cell lines and primary cells from the lung. ID1 induction was Src dependent and mediated through the α7 subunit of nAChR; transfection of K-Ras or EGFR to primary cells induced ID1. ID1 depletion prevented nicotine- and EGF-induced proliferation, migration, and invasion of NSCLC cells and angiogenic tubule formation of human microvascular endothelial cells from lungs (HMEC-Ls). ID1 could induce the expression of mesenchymal markers such as vimentin and fibronectin by downregulating ZBP-89, a zinc finger repressor protein. ID1 levels were elevated in tumors from mice that were exposed to nicotine. Further, human lung tissue microarrays (TMAs) showed elevated levels of ID1 in NSCLC samples, with maximal levels in metastatic lung cancers. Quantitative reverse transcription-PCR (RT-PCR) performed on patient lung tumors showed that ID1 levels were elevated in advanced stages of NSCLC and correlated with elevated expression of vimentin and fibronectin, irrespective of smoking history.

Topics: Animals; Carcinoma, Non-Small-Cell Lung; Cell Movement; Cell Proliferation; Cells, Cultured; Disease Progression; DNA-Binding Proteins; Epidermal Growth Factor; ErbB Receptors; Fibronectins; Gene Expression Regulation, Neoplastic; Humans; Inhibitor of Differentiation Protein 1; Lung; Lung Neoplasms; Neoplasm Metastasis; Neovascularization, Pathologic; Nicotine; Nicotinic Agonists; Protein Subunits; Receptors, Nicotinic; RNA, Small Interfering; Signal Transduction; src-Family Kinases; Transcription Factors; Vimentin

The epidermal growth factor receptor (EGFR) can be activated by several growth factors within the tumor microenvironment, and it can activate several signaling pathways. For tumor development, these EGFR-related signaling pathways may converge on several common nuclear transcription factors, one such transcription factor being STAT5. STAT5 plays an important role in the oncogenic signal transduction pathway in non-small cell lung cancer. In this study, we examined whether the epidermal growth factor (EGF) can stimulate cyclooxygenase-2 (COX-2) expression in human lung adeno-carcinoma A549 cells transfected with or without STAT5 siRNA or dominant-negative (DN)-STAT5, and identified the pathways involved in this response. We found that STAT5 siRNA significantly reduced EGF-induced COX-2 expression, and STAT5 phosphorylation. STAT5 phosphorylation predominantly mediates EGF-induced COX-2 promoter activity. STAT5 siRNA was found to inhibit COX-2 expression in resting A549 cells despite the absence of detectable activated phosphorylated STAT5. Using an adenoviral system, we expressed DN-STAT5 in human lung adenocarcinoma A549 cells in order to broaden the investigation and to determine the role of STAT5 in EGF-mediated COX-2 gene expression. The overexpression of DN-STAT5 significantly inhibited EGF-induced COX-2 expression, and we found that EGF induced the tyrosine phosphorylation of STAT5 and up- regulated COX-2 expression. DN-STAT5 also blocked COX-2 promoter activity. Our results demonstrate that EGF stimulates COX-2 expression in human lung adenocarcinoma A549 cells via the activation of the STAT5 pathway and that COX-2 expression may be independent of phosphorylated STAT5 in A549 cells in vitro.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Cell Line, Tumor; Cyclooxygenase 2; DNA-Binding Proteins; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Phosphorylation; Promoter Regions, Genetic; RNA, Small Interfering; Signal Transduction; STAT5 Transcription Factor

Lung cancer is one of the leading causes of death in the world. Some tumor events are attributed to an important group of molecules (cadherins and integrins). We evaluated the interactions of cell adhesion molecules in cell lines from lung cancer. Two lung cancer cell lines were nonmetastatic (H358 and H441) and two were metastatic (H1299 and H292). All cell lines were treated with epidermal growth factor (EGF), and Western blot analysis was performed to assess the interactions between these proteins. The bronchoalveolar cells H358 showed the three analyzed proteins: E-cadherin, β-catenin, and p120 catenin. The adenocarcinoma cells H441 did not present p120 catenin, and carcinoma cells did not show E-cadherin (H1299) or p120 catenin (H292). FAK (pTyr925) was dephosphorylated in adenocarcinoma cells H441, absent in carcinoma cells H1299, and upregulated in the other carcinoma cells H292. p130Cas showed no difference when the cell lines were treated with EGF for 30 min; it was absent in the metastatic carcinoma cells H1299. Paxillin was dephosphorylated in adenocarcinoma cells H441 and also absent in other metastatic carcinoma cells H292. Vinculin showed the same results, and talin was downregulated in adenocarcinoma cells H441 when the cells were treated with EGF. Rap1 was downregulated and PYK2 was upregulated in the same cell line. Our data help to comprehend the mechanism involved in cell migration to the blood and metastasis generation. In conclusion, the expression patterns of cell-cell adhesion were not affected by EGF treatment but it affected cell-extracellular matrix adhesion.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; beta Catenin; Cadherins; Carcinoma, Non-Small-Cell Lung; Catenins; Cell Adhesion Molecules; Cell Line, Tumor; Crk-Associated Substrate Protein; Delta Catenin; Epidermal Growth Factor; Focal Adhesion Kinase 1; Humans; Lung Neoplasms; Paxillin; rap1 GTP-Binding Proteins; Talin; Vinculin

Topics: Adenocarcinoma; Aged; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Camptothecin; Cetuximab; Colorectal Neoplasms; Epidermal Growth Factor; Female; Humans; Irinotecan; Keratitis; Lung Neoplasms; Ophthalmic Solutions

CK2 interacts and phosphorylates >300 proteins, including Stat3, and is linked to a number of human cancers. Constitutively activated Stat3 has been reported in 50% of human lung cancers. Inhibition of CK2 activity can induce apoptosis and suppression of Stat3 activation in cancer cells. This study examined the effects of CK2 inhibitors on growth inhibition of lung cancer cells and the therapeutic potential on lung cancer. The CK2 inhibitor and radiation both suppressed cancer cell growth in a dose-dependent manner. Besides, the cytotoxic effect of irradiation could be augmented by CK2 inhibitors (p<0.05, two-way analysis of variance and Tukey's Honestly Significant Difference). Moreover, the growth inhibition of CK2 inhibitor and irradiation was both associated with suppression of Stat3 activation. Taken together, inhibition of CK2 activity appears to be a promising treatment strategy for non-small cell lung cancer and CK2 inhibition results in reduced Stat3 activation. Our data warrant further effort to develop CK2-targeted radiosensitizer for lung cancer treatment.

Topics: Carcinoma, Non-Small-Cell Lung; Casein Kinase II; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; Dose-Response Relationship, Radiation; Enzyme Inhibitors; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Radiation Tolerance; Radiation-Sensitizing Agents; STAT3 Transcription Factor

Single disseminated tumor cells (DTC) can be detected in the bone marrow (BM) from 20% to 60% of patients with various tumors including non-small cell lung cancer (NSCLC). Detection of DTC in the BM of NSCLC patients is associated with poor prognosis and may be responsible for metastatic relapse. However, the functional properties of DTC are widely unknown. Here, we performed the first functional analysis of DTC focusing on the activation of the PI3K/Akt signalling pathway and the functional roles of Akt isoforms. In vitro kinase assays revealed a high activity of Akt3 in NSCLC-derived DTC. Proliferation and survival of DTC was reduced by depletion of Akt3 and to a lesser extend by Akt1, but not after depletion of Akt2. The major effect of Akt3 on the proliferation of DTC was associated with an Akt3-mediated regulation of both, cyclin D1 and cyclin D3, whereas Akt1 regulated the expression of cyclin D1 only. In contrast all three Akt isoforms, especially Akt2, were involved in the regulation of migration. Analysis of signalling events downstream of distinct Akt isoforms revealed that expression levels of urokinase-type plasminogen activator and its receptor were decreased after knockdown of Akt1 and Akt3. In addition, EGF-stimulated proliferative and anti-apoptotic signals are mediated by Akt1 and Akt3 in DTC. Finally, by immunofluorescence staining of primary DTC from BM samples of lung cancer patients, pAkt(S473) and Akt3 positive DTC were detected in vivo. Our data demonstrate that Akt1 and notably Akt3 regulate proliferation, survival, migration and EGF-mediated signal transduction in NSCLC-derived DTC.

Topics: Bone Marrow Neoplasms; Breast Neoplasms; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Enzyme Assays; Epidermal Growth Factor; ErbB Receptors; Female; Gene Expression; Gene Knockdown Techniques; Glycogen Synthase Kinase 3; Humans; Isoenzymes; Lung Neoplasms; Phosphorylation; Proto-Oncogene Proteins c-akt; Receptor, ErbB-2; Receptor, ErbB-3; Receptors, Urokinase Plasminogen Activator; RNA Interference; Signal Transduction; Urokinase-Type Plasminogen Activator

Ion channels involved in the migration of tumor cells that is required for their invasion and metastasis. In this paper, we describe the interaction of TRPM7 channel and epidermal growth factor (EGF), an important player in cancer development in the migration of lung cancer cells. The TRPM7 currents in A549 cells were first characterized by means of electrophysiology, pharmacology and RNA interference. Removing Ca(2+) from the extracellular solution not only potentiated a large inward current, but also abolished the outward rectification. 200μM 2-APB inhibited the outward and the inward TRPM7 currents and at the same time restored the property of outward rectification. EGF greatly enhanced the migration of A549 cells, and also markedly up-regulated the membrane protein expression of TRPM7 and the amplitude of TRPM7 currents. Depressing the function of TRPM7 with RNA interference or pharmacological agents not only reversed the EGF-enhanced migration of A549 cells but also inhibited the basal migration of A549 cells in the absence of EGF. Thus it seems that TRPM7 plays a pivotal role in the migration of A549 cells induced by EGF and thus could be a potential therapeutic target in lung cancers.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Blotting, Western; Calcium; Cell Line, Tumor; Cell Movement; Epidermal Growth Factor; Humans; Lung Neoplasms; Membrane Potentials; Patch-Clamp Techniques; Protein Serine-Threonine Kinases; RNA, Small Interfering; TRPM Cation Channels; Up-Regulation

Epidermal growth factor receptor (EGFR) is coactivated by the μ-opioid receptor (MOR), expressed on non-small cell lung cancer (NSCLC) cells and human lung cancer. We hypothesized that clinically used opioid analgesics that are MOR agonists coactivate EGFR, resulting in growth- and survival-promoting signaling.. We used H2009, a human adenocarcinoma NSCLC cell line, with constitutive EGFR phosphorylation, which showed increased expression of MOR and the δ-opioid receptor by reverse transcriptase polymerase chain reaction. We used Western immunoblotting, magnetic bead-based Bio-Plex cytokine assay, immunofluorescent staining, BrdU incorporation enzyme-linked immunosorbent assay, and BioCoat™ Matrigel™ invasion assay to examine cell signaling, cytokine expression, colocalization of MOR and EGFR in human lung cancer, and cell proliferation and invasion, respectively.. Similar to epidermal growth factor (EGF), morphine stimulated phosphorylation of EGFR, Akt/protein kinase B (Akt), and mitogen-activated protein kinase/extracellular signal regulated kinase (MAPK/ERK) signaling in H2009 cells. Opioid receptor (OR) antagonist, naloxone, EGFR tyrosine kinase inhibitor, erlotinib, and silencing of MOR and δ-opioid receptor abrogated morphine- and EGF-induced phosphorylation of signaling, suggestive of OR-mediated coactivation of EGFR. H2009 cells secreted significantly higher levels of cytokines compared with control Beas2B epithelial cells. H2009-conditioned medium stimulated MOR expression in Beas2B cells, suggesting that cytokines secreted by H2009 may be associated with increased OR expression in H2009. We observed colocalization of EGFR and MOR, in human NSCLC tissue. Functionally, morphine- and EGF-induced proliferation and invasion of H2009 cells was ameliorated by naloxone as well as erlotinib.. Morphine-induced phosphorylation of EGFR occurs via ORs, leading to downstream MAPK/ERK, Akt phosphorylation, cell proliferation, and increased invasion. Notably, ORs are also associated with EGF-induced phosphorylation of EGFR. Increased coexpression of MOR and EGFR in human lung cancer suggests that morphine may have a growth-promoting effect in lung cancer.

Topics: Carcinoma, Non-Small-Cell Lung; Cell Line, Transformed; Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Erlotinib Hydrochloride; Humans; Lung Neoplasms; MAP Kinase Signaling System; Morphine; Quinazolines; Receptors, Opioid, mu

Epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that is commonly activated by mutation in non-small cell lung cancer. The mechanism of this oncogenic activation is not completely understood, but in contrast to that of the wild-type EGFR, it is proposed to be independent of kinase domain dimerization. Mechanistic studies on EGFR have mainly relied on cell-based assays or isolated kinase domain measurements. Here we show, using purified, near full-length human EGFR proteins (tEGFRs), that two oncogenic mutants are fully active independently of EGF and highly resistant to the therapeutic and endogenous inhibitors cetuximab, lapatinib and MIG6. Based on the pattern of inhibition and the effects of additional asymmetric kinase dimer interface mutations, we propose that these oncogenic EGFR mutants drive and strongly depend on the formation of the asymmetric kinase dimer for activation, which has implications for drug design and cancer treatment strategies.

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Cetuximab; Epidermal Growth Factor; ErbB Receptors; HEK293 Cells; Humans; Kinetics; Lapatinib; Lung Neoplasms; Models, Molecular; Mutation; Quinazolines; Tumor Suppressor Proteins

In cancer research, it is important to evaluate the performance of a biomarker (e.g., molecular, genetic, or imaging) that correlates patients' prognosis or predicts patients' response to treatment in a large prospective study. Due to overall budget constraint and high cost associated with bioassays, investigators often have to select a subset from all registered patients for biomarker assessment. To detect a potentially moderate association between the biomarker and the outcome, investigators need to decide how to select the subset of a fixed size such that the study efficiency can be enhanced. We show that, instead of drawing a simple random sample from the study cohort, greater efficiency can be achieved by allowing the selection probability to depend on the outcome and an auxiliary variable; we refer to such a sampling scheme as outcome and auxiliary-dependent subsampling (OADS). This article is motivated by the need to analyze data from a lung cancer biomarker study that adopts the OADS design to assess epidermal growth factor receptor (EGFR) mutations as a predictive biomarker for whether a subject responds to a greater extent to EGFR inhibitor drugs. We propose an estimated maximum-likelihood method that accommodates the OADS design and utilizes all observed information, especially those contained in the likelihood score of EGFR mutations (an auxiliary variable of EGFR mutations) that is available to all patients. We derive the asymptotic properties of the proposed estimator and evaluate its finite sample properties via simulation. We illustrate the proposed method with a data example.

Topics: Biomarkers, Tumor; Computer Simulation; Epidermal Growth Factor; Humans; Lung Neoplasms; Methods; Mutation; Neoplasms; Patient Selection; Precision Medicine; Predictive Value of Tests; Prognosis

Like many signaling pathways in development, the Notch receptor pathway plays an important role in cancer pathobiology when it is dysregulated. Potential ligand-binding sites within the epidermal growth factor (EGF)-like repeats of Notch1 have been identified, but the ligand-binding domains in Notch3, which is implicated in lung cancer, are not known. In screening a library of 155 peptides representing all 34 EGF-like repeats in Notch3, we discovered two distinct ligand-binding regions involving the 7-10 and 21-22 repeats that are distinct from the putative ligand-binding domain of Notch1. In cell-based assays, peptides from these regions induced apoptosis and reduced expression of the Notch3-dependent gene Hey1. They also bound directly to the Notch ligand Jagged1, suggesting that their mechanism of action involves disrupting interactions between Notch3 and Jagged1. Recombinant Fc fusion peptides engineered for in vivo testing showed that the Notch3 peptides defined could trigger apoptosis and suppress tumor growth in tumor xenograft assays. These findings rationalize a mechanistic approach to lung cancer treatment based on Notch3 receptor-targeted therapeutic development.

Topics: Apoptosis; Basic Helix-Loop-Helix Transcription Factors; Binding Sites; Calcium-Binding Proteins; Cell Cycle Proteins; Cell Growth Processes; Drug Delivery Systems; Epidermal Growth Factor; HeLa Cells; Humans; Immunoglobulin Fc Fragments; Intercellular Signaling Peptides and Proteins; Jagged-1 Protein; Ligands; Lung Neoplasms; Membrane Proteins; Peptide Fragments; Peptide Library; Protein Structure, Tertiary; Receptor, Notch3; Receptors, Notch; Recombinant Fusion Proteins; Serrate-Jagged Proteins

The interaction between tumor cells and surrounding normal cells is very important in the prognosis of tumors. The object of this study was to determine the effect of recipient bed of tumor xenograft on tumor metastasis using a novel parotid gland tumor model. HeLa cells were xenografted into the parotid gland or subcutaneous tissues of athymic mice. Eight weeks after the transplantation, all mice were euthanized and specimens were immunostained with antibodies for angiogenic factors. FGF-2 was given to HeLa cells and the effect on the cellular migration was determined. EGF was also given to HeLa cells for the evaluation of FGF-2 induction. Immunohistochemical staining was done for the vascular metastasis-related factors on 26 human salivary gland tumors. HeLa cells showed significantly higher lung metastatic potential in the parotid gland than in the subcutis. Immunohistochemical staining revealed overexpression of FGF-2 in the parotid tumors compared to the subcutaneous tumors. The application of FGF-2 to implanting HeLa cells increased the cellular invasion in a dose-dependent manner. The application of EGF increases FGF-2 expression in HeLa cells. In clinical specimens, EGF and VEGF signaling proteins were more expressed than in normal salivary glandular tissues. In conclusion, a parotid tumor model of HeLa cells was successfully developed and it closely mimics high metastasis. These results indicate that recipient bed with intense EGF expression increases tumor metastasis through upregulation of FGF-2 expression.

Topics: Animals; Carcinoma, Adenoid Cystic; Epidermal Growth Factor; ErbB Receptors; Fibroblast Growth Factor 2; HeLa Cells; Humans; Lung Neoplasms; Male; Mice; Mice, Nude; Neoplasm Transplantation; Parotid Neoplasms; Transplantation, Heterologous; Xenograft Model Antitumor Assays

An adaptor protein FRS2beta inhibits epidermal growth factor-receptor (EGFR) tyrosine kinase without being phosphorylated at tyrosine residues after EGF stimulation. Although binding to ERK appears to be important for this inhibition, the precise molecular mechanisms and the role of FRS2beta in signal transduction mediated by other EGFR family members, as well as its role in human cancer, remain unclear. In this study, we demonstrate that FRS2beta inhibits anchorage-independent cell growth induced by oncogenic ErbB2, another member of EGFR family, and that it inhibits heterodimer formation between EGFR and ErbB2. We mapped the residues important for the FRS2beta and ERK interaction to two docking (D) domain-like sequences on FRS2beta and two aspartic acid residues in the common docking (CD) domain of ERK. Moreover, in response to EGF, ERK translocated to the plasma membrane in cells expressing FRS2beta but not an FRS2beta mutant in which four arginine residues in the D domains were replaced with alanines, suggesting that FRS2beta serves as a plasma membrane anchor for activated ERK. Finally, a low mRNA expression level of FRS2beta was significantly correlated with poor prognosis in a cohort of 60 non-small cell lung cancer patients. Therefore, we have identified the molecular mechanisms by which FRS2beta acts as a feedback inhibitor of EGFR family members and suggest a role for FRS2beta as a tumor suppressor.

Topics: Adaptor Proteins, Signal Transducing; Carcinoma, Non-Small-Cell Lung; Cell Culture Techniques; Cell Division; Cell Membrane; Colony-Forming Units Assay; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation, Neoplastic; Humans; Kinetics; Lung Neoplasms; Mitogen-Activated Protein Kinase 3; Myristic Acid; Prognosis; Protein Binding; RNA, Messenger

The diagnosis of multiple primary lung cancer is sometimes difficult when multiple lung tumors with the same histologic type are identified. We now present a case of synchronous double primary lung adenocarcinomas (one in the right upper lobe and another in the right middle lobe) diagnosed based on mutational analysis of the epidermal growth factor receptor (EGFR) gene, although clinico-pathological findings suggested the diagnosis of intrapulmonary metastasis. After complete resection, pathological sections revealed the similar pathological features of two adenocarcinomas and unexpected subcarinal nodal metastasis. As the L858R mutation within exon 21 of the EGFR gene was identified in the middle-lobe tumor and the subcarinal node but not in the upper-lobe tumor, we diagnosed as double primary cancers. Local mediastinal recurrence after operation has been well-controlled with administration of gefitinib, a EGFR-tyrosine kinase inhibitor, and mutational analysis of the EGFR gene provided important information not only in the diagnosis of double primary cancers but also in decision-making of selection of chemotherapeutic agent.

Topics: Adenocarcinoma; Aged; Diagnosis, Differential; DNA Mutational Analysis; Epidermal Growth Factor; Female; Gefitinib; Humans; Lung Neoplasms; Neoplasm Metastasis; Neoplasm Recurrence, Local; Neoplasm Staging; Neoplasms, Multiple Primary; Pneumonectomy; Quinazolines; Radiography, Thoracic

Epidermal growth factor receptor (EGFR) overexpression and activation are hallmarks of non-small cell lung carcinoma (NSCLC). Although EGFR-targeted therapies are used, the prognosis of NSCLC remains poor. ADAM17 induces activation of the EGFR through ligand cleavage. However, we show that inhibition or knockdown of ADAM17 markedly reduces tumorigenesis and survival to a large part independently from EGFR ligand shedding in NSCLC cells. These findings strongly indicate additional oncogenic mechanisms regulated by ADAM17. We identified Notch1 signaling as an ADAM17-controlled pathway and a critical regulator of anchorage-independent growth by using both Notch1 shRNA and ectopic expression of the active intracellular Notch1 fragment. Strikingly, Notch1 knockdown led to a strong reduction of EGFR expression in all analyzed cell lines. Proliferation, survival, and colony formation of Notch1-deficient cells were insensitive to EGF stimulation. Moreover, targeting Notch1 or ADAM17 resulted in substantial cell death, whereas EGFR inhibition predominantly induced cell cycle arrest. Immunohistochemical analysis of primary human tissue revealed a significant correlation between ADAM17, Notch1 signaling, and high EGFR expression levels. In conclusion, this article describes a novel molecular circuitry in NSCLC, incorporating ADAM17 as a regulator of EGFR expression through the activation of Notch1. Due to their central role in tumorigenesis and survival of NSCLC cells, both ADAM17 and Notch1 constitute promising targets for the treatment of NSCLC.

Topics: ADAM Proteins; ADAM17 Protein; Animals; Basic Helix-Loop-Helix Transcription Factors; Carcinoma, Non-Small-Cell Lung; Cell Growth Processes; Cell Line, Tumor; Cell Survival; Cell Transformation, Neoplastic; Epidermal Growth Factor; ErbB Receptors; Homeodomain Proteins; Humans; Lung Neoplasms; Mice; Receptor, Notch1; Signal Transduction; Transcription Factor HES-1; Transplantation, Heterologous

We analyzed the gene expression profiles of clinical lung carcinomas using a cDNA microarray containing 27,648 genes or expressed sequence tags, and identified CDCA5 (cell division cycle associated 5) to be upregulated in the majority of lung cancers. Tumor tissue microarray analysis of 262 non-small cell lung cancer patients revealed that CDCA5 positivity was an independent prognostic factor for lung cancer patients. Suppression of CDCA5 expression with siRNAs inhibited the growth of lung cancer cells; concordantly, induction of exogenous expression of CDCA5 conferred growth-promoting activity in mammalian cells. We also found that extracellular signal-regulated kinase (ERK) kinase phosphorylated CDCA5 at Ser79 and Ser209 in vivo. Exogenous expression of phospho-mimicking CDCA5 protein whose Ser209 residue was replaced with glutamine acid further enhanced the growth of cancer cells. In addition, functional inhibition of the interaction between CDCA5 and ERK kinase by a cell-permeable peptide corresponding to a 20-amino-acid sequence part of CDCA5, which included the Ser209 phosphorylation site by ERK, significantly reduced phosphorylation of CDCA5 and resulted in growth suppression of lung cancer cells. Our data suggest that transactivation of CDCA5 and its phosphorylation at Ser209 by ERK play an important role in lung cancer proliferation, and that the selective suppression of the ERK-CDCA5 pathway could be a promising strategy for cancer therapy.

Topics: Adaptor Proteins, Signal Transducing; Amino Acid Sequence; Animals; Carcinoma, Non-Small-Cell Lung; Carcinoma, Small Cell; Cell Cycle Proteins; Cell Growth Processes; Cell Line, Tumor; Cell Transformation, Neoplastic; Chlorocebus aethiops; COS Cells; Epidermal Growth Factor; Extracellular Signal-Regulated MAP Kinases; Gene Expression Profiling; Gene Knockdown Techniques; Humans; Lung Neoplasms; MAP Kinase Signaling System; Molecular Sequence Data; Peptides; Phosphorylation; RNA, Small Interfering; Transcriptional Activation

In mammalian cells repair of radiation-induced DNA damage appears to be also controlled by the epidermal growth factor receptor (EGFR) with a special impact on DNA double-strand break (DSB) repair. Aim of this study was to demonstrate this interaction between EGFR signalling and DNA DSB repair and to identify the underlying downstream pathways. We especially wanted to know in how far non-homologous end-joining (NHEJ) as the most important DSB repair pathway is involved in this interaction. Overall DSB repair was determined by counting gammaH2AX foci remaining 24 after irradiation, while NHEJ activity was monitored by using a specially designed repair construct stably integrated into the genome. The overall DSB repair capacity was clearly enhanced when EGFR was activated by its natural ligand EGF and, vice versa, was reduced when EGFR was blocked either by the specific antibody Cetuximab or the tyrosine kinase inhibitor erlotinib, whereby reduction was clearly stronger for erlotinib. There was also a difference in the pathways affected. While erlotinib lead to a block of both, MAPK as well as AKT signalling, Cetuximab only affected MAPK. As demonstrated by specific inhibitors (PD98059, AKTIII) EGFR interacts with DSB repair mostly via MAPK pathway. Also for NHEJ activity, there was a substantial increase, when EGFR was activated by EGF as determined for two different reporter cell lines (A549.EJ and H1299.EJ) and, vice versa, a reduction was seen when EGFR signalling was blocked by Cetuximab or erlotinib. There was, however, no difference for the two inhibitors used. This regulation of NHEJ by EGFR was only blocked when ERK was affected by siRNA but not when AKT was knocked down. These data indicate that EGFR modulates DSB repair by regulating NHEJ via MAPK signalling.

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Bronchi; Cell Line, Tumor; Cetuximab; DNA Breaks, Double-Stranded; DNA Damage; DNA Repair; Epidermal Growth Factor; ErbB Receptors; Erlotinib Hydrochloride; Humans; Lung Neoplasms; Mitogen-Activated Protein Kinase Kinases; Quinazolines; RNA, Small Interfering; Signal Transduction

Invasion and metastasis are the primary causes of death in patients with pulmonary carcinoma. The epidermal growth factor (EGF) stimulates A549 cells invasion greatly through activating ERK and PI3K-Akt signaling pathway. The aim of this study is to elucidate the inhibitory effect of Resveratrol on EGF-induced invasive ability of A549 cells in vitro and explore the molecular mechanism.. The cytotoxicity of Resveratrol was evaluated by methyl thiazolyltetrazolium (MTT) assay. Then, the A549 cells were treated with EGF and non-cytotoxic concentration of Resveratrol. The cells' invasion were detected by Boyden chamber assay; MMP-2 activity was determined by gelatine zymography assay; the changes of the related proteins were detected by Western blot.. Resveratrol was not toxic to A549 cells at the concentration between 0 to 30 microM. The invasion ability of EGF-induced A549 cells was decreased after treatment with 20 microM resveratrol for 24 h, accompanied by the inhibition of MMP-2 secretion. And the levels of p-ERK1/2, PI3K (within 6 h) were suppressed too.. 20 microM Resveratrol inhibits A549 cells' invasion possibly through the suppression of the activation of ERK and PI3K-Akt signaling pathways, subsequently exerting inhibitory effect on MMP-2.

Topics: Adenocarcinoma; Anticarcinogenic Agents; Blotting, Western; Cell Line, Tumor; Epidermal Growth Factor; Humans; Lung Neoplasms; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Phosphatidylinositol 3-Kinases; Resveratrol; Signal Transduction; Stilbenes

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is an important mediator in the differentiation, maturation, and survival of inflammatory cells. It might play a significant role in the pathogenesis of asthma. We have shown previously that cytosolic phospholipase A2 group IV A (cPLA2alpha) is able to activate gene expression, through peroxisome proliferator-activated receptor (PPAR)-gamma response elements (PPRE). In the promoter regions of GM-CSF gene (CSF2), we have found potential PPRE. The goal of the current study was to investigate the influence of cPLA2 overexpression and activation on CSF2 gene expression in human lung cells.. Subconfluent A549 cells were transfected with GM-CSF reporter gene and cPLA2alpha overexpression vector. Transfected cells were treated with or without calcium ionophore, A23187, or epidermal growth factor (EGF). Cell lysates were collected and assayed for dual luciferase activity. Some cultures were preincubated with a potent cPLA2alpha inhibitor, methyl arachidonyl fluorophosphonate (MAFP); a secretory phospholipase A2 inhibitor, thioetheramide-PC; a calcium-independent phospholipase A2 inhibitor, bromoenol lactone (BEL); or vehicle. After preincubation, cells were treated with A23187. Expression of GM-CSF messenger RNA (mRNA) was measured using real-time polymerase chain reaction mRNA quantification.. Overexpression of cPLA2alpha or activation of endogenous cPLA2alpha by calcium ionophore or EGF caused a significant increase in GM-CSF relative luciferase activity. Calcium ionophore significantly increased GM-CSF mRNA in A549 human lung cells. These effects were at least in part inhibited by MAFP treatment, but not by BEL or thioetheramide-PC.. These preliminary data might suggest an influence of cPLA2alpha on GM-CSF gene expression in human lung cells, which could play a role in the pathogenesis of asthma and other inflammatory diseases.

Topics: Animals; Cell Line, Tumor; Enzyme Inhibitors; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Genes, Reporter; Granulocyte-Macrophage Colony-Stimulating Factor; Group IV Phospholipases A2; Humans; Lung; Lung Neoplasms; Pilot Projects; Rats; Time Factors

Epidermal growth factor (EGF) mediates breast cancer cell chemotaxis and metastasis through mechanisms that involve the growth-regulatory mammalian target of rapamycin (mTOR) complex mTORC2, but the mechanisms involved remain obscure. Here, we report that the rapamycin-insensitive mTORC2 component protein Rictor is a critical mediator of metastasis in breast cancer cells. In patients with ductal carcinoma, Rictor expression was associated with increased lymph node metastasis. EGF induced translocation and colocalization of Rictor with protein kinase Cζ (PKCζ), a pivotal molecule in chemotaxis signaling. Further, Rictor coimmunoprecipitated with PKCζ in the absence of the mTORC2 complex. Small interfering RNA-mediated knockdown of Rictor inhibited EGF-induced PKCζ phosphorylation and translocation along with phosphorylation of the key F-actin binding protein cofilin. In parallel, Rictor knockdown reduced cellular chemotactic capacity and ablated pulmonary metastasis in a xenograft mouse model of breast cancer. Our findings identify Rictor as an important mediator of chemotaxis and metastasis in breast cancer cells.

Topics: Animals; Breast Neoplasms; Carcinoma, Ductal, Breast; Carrier Proteins; Cell Line, Tumor; Cell Movement; Chemotaxis; Epidermal Growth Factor; Female; Gene Expression Regulation, Neoplastic; HEK293 Cells; Humans; Immunohistochemistry; Lung Neoplasms; Lymphatic Metastasis; Mammary Neoplasms, Experimental; Mice; Mice, SCID; Microscopy, Confocal; Phosphorylation; Protein Binding; Protein Kinase C; Rapamycin-Insensitive Companion of mTOR Protein; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Transplantation, Heterologous

The actin binding protein Mammalian enabled (Mena), has been implicated in the metastatic progression of solid tumors in humans. Mena expression level in primary tumors is correlated with metastasis in breast, cervical, colorectal and pancreatic cancers. Cells expressing high Mena levels are part of the tumor microenvironment for metastasis (TMEM), an anatomical structure that is predictive for risk of breast cancer metastasis. Previously we have shown that forced expression of Mena adenocarcinoma cells enhances invasion and metastasis in xenograft mice. Whether Mena is required for tumor progression is still unknown. Here we report the effects of Mena deficiency on tumor progression, metastasis and on normal mammary gland development.. To investigate the role of Mena in tumor progression and metastasis, Mena deficient mice were intercrossed with mice carrying a transgene expressing the polyoma middle T oncoprotein, driven by the mouse mammary tumor virus. The progeny were investigated for the effects of Mena deficiency on tumor progression via staging of primary mammary tumors and by evaluation of morbidity. Stages of metastatic progression were investigated using an in vivo invasion assay, intravital multiphoton microscopy, circulating tumor cell burden, and lung metastases. Mammary gland development was studied in whole mount mammary glands of wild type and Mena deficient mice.. Mena deficiency decreased morbidity and metastatic dissemination. Loss of Mena increased mammary tumor latency but had no affect on mammary tumor burden or histologic progression to carcinoma. Elimination of Mena also significantly decreased epidermal growth factor (EGF) induced in vivo invasion, in vivo motility, intravasation and metastasis. Non-tumor bearing mice deficient for Mena also showed defects in mammary gland terminal end bud formation and branching.. Deficiency of Mena decreases metastasis by slowing tumor progression and reducing tumor cell invasion and intravasation. Mena deficiency during development causes defects in invasive processes involved in mammary gland development. These findings suggest that functional intervention targeting Mena in breast cancer patients may provide a valuable treatment option to delay tumor progression and decrease invasion and metastatic spread leading to an improved prognostic outcome.

Topics: Animals; Antigens, Polyomavirus Transforming; Cytoskeletal Proteins; Disease Progression; Epidermal Growth Factor; Female; Gene Expression; Lung Neoplasms; Macrophages; Mammary Glands, Animal; Mammary Neoplasms, Experimental; Mammary Tumor Virus, Mouse; Mice; Mice, SCID; Mice, Transgenic; Microfilament Proteins; Neoplasm Invasiveness; Neoplasm Metastasis; Polymerase Chain Reaction; Tumor Microenvironment

In a previously published in vitro study based on top-down proteomics we found that the calcium-binding proteins S100A6 and S100A4 were affected by exposure to ionizing radiation in a p53-dependent fashion. Both proteins showed post-translational modification changes, and S100A6 also showed increased expression and translocation in response to irradiation. Aim of the present study was to evaluate the expression of S100A6 and S100A4 in non-small-cell lung cancer (NSCLC).. S100A6 expression on archival tumor cell lysates from 39 patients with radically resected NSCLC was assessed with SELDI-TOF-MS. S100A6 identity was confirmed using a SELDI-based antibody-capture method on lysates from the A549 lung cancer cell line, cell lysates from two freshly prepared NSCLC samples, four plasma samples and one pleural effusion sample. Immunostainings for S100A6, S100A4 and p53 were performed on tissue microarrays containing 103 stage I surgically resected NSCLC cases and 14 normal lung parenchyma specimens.. The presence of post-translationally modified S100A6 forms was confirmed with SELDI-MS on enriched tumor cell lysates, as well as in plasma and pleural effusion samples. In addition, high S100A6 peak intensity was associated with longer median survival (35 months vs. 18 months for high and low peak intensity, respectively; p=n.s.). The immunohistochemical analysis showed that 25% of tumors were S100A6 positive. S100A6 expression correlated directly with non-squamous histology (p<0.0001) and S100A4 expression (p=0.005), and inversely with p53 expression (p=0.01). S100A6-positive cases showed a trend of longer survival compared with S100A6-negative cases (p=0.07). This difference became significant when the analysis was restricted to p53-negative cases (n=72). In this subgroup of patients, whose tumors likely exhibit a functional p53, S100A6 was an independent prognostic factor of improved survival at multivariate analysis (HR 0.49, 95% CI 0.27-0.81, p=0.017).. In this study we have validated on clinical material our previous findings on cell lines in terms of S100A6 expression and post-translational modifications pattern in NSCLC. Moreover, the survival results obtained in p53-negative stage I NSCLC cases support the proposed pro-apoptotic function of S100A6 and suggest the hypothesis of a cross regulation between these two proteins.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Non-Small-Cell Lung; Cell Cycle Proteins; DNA, Neoplasm; Epidermal Growth Factor; Female; Follow-Up Studies; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Lung Neoplasms; Male; Middle Aged; Neoplasm Staging; Oligonucleotide Array Sequence Analysis; Prognosis; S100 Calcium Binding Protein A6; S100 Proteins; Survival Rate; Sweden

Hematogenous metastasis is one of the most important factors determining the outcome of the patients with salivary adenoid cystic carcinoma (SACC). In the present study, we examined expression profile of genes in SACC cell lines to look for molecules responsible for its unique metastatic trait. A transcriptomic microarray analysis between the lower lung-metastatic rate cell line SACC-83 and the higher lung-metastatic rate cell line SACC-LM were performed, and eight genes, showed by microarray to be highly expressed in SACC-LM, were picked for validation by quantitative real-time PCR. Among the genes, the expression of epiregulin, a novel member of epidermal growth factor family, was 350-folds higher in SACC-LM than in SACC-83. Accordingly, we examined the effects of epiregulin on migration and invasion in SACC-83 as well as its targeted downstream molecules, and found that epiregulin could promote in vitro migration and invasion in SACC-83. Furthermore, epiregulin not only induced activation of both ERK1/2 and Akt, but also expression of COX-2. In addition, all these effects could be partially blocked by U0126, a specific inhibitor of mitogen-activated protein kinase kinase (MEK or MAPKK), or LY294002, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K). Conclusively, the results suggest that epiregulin may play an important role in lung metastasis of SACC.

Topics: Carcinoma, Adenoid Cystic; Cell Line, Tumor; Cell Movement; Cyclooxygenase 2 Inhibitors; Epidermal Growth Factor; Epiregulin; Humans; Lung Neoplasms; Microarray Analysis; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neoplasm Invasiveness; Proto-Oncogene Proteins c-akt; Reverse Transcriptase Polymerase Chain Reaction; Salivary Gland Neoplasms

Metastasis is the major cause of mortality in lung cancer. Chemotaxis plays a vital role in cancer cell metastasis. In the current study, we reported that epidermal growth factor (EGF) induced a robust chemotaxis of A549 and H1299 cells, two representative human non-small cell lung cancer (NSCLC) cells. Chelerythrine chloride, an inhibitor of all protein kinase C (PKC) isozymes, significantly reduced the chemotactic capacity of NSCLC cells while inhibitors of classical or novel PKC isozymes, such as Gö6976, calphostin C, or Gö6850, showed no effect, which suggested that atypical PKC might be involved in the chemotactic process of NSCLC cells. EGF-elicited translocation and phosphorylation of atypical PKCzeta, indicating that EGF could activate PKCzeta. Treatment with a PKCzeta specific inhibitor, a myristoylated pseudosubstrate, blocked the chemotaxis in a dose-dependent manner, further confirming that atypical PKCzeta was required for NSCLC chemotaxis. Mechanistic studies suggested that PKCzeta was regulated by phosphatidylinositol 3 kinase (PI3K)/Akt. Furthermore, PKCzeta-mediated chemotaxis by regulating actin polymerization and cell adhesion. Taken together, our study suggested that PKCzeta was required in NSCLC cell chemotaxis, thus could be used as a target to develop anti-lung cancer metastasis therapies.

Topics: Cell Line, Tumor; Chemokine CXCL12; Chemotaxis; Down-Regulation; Epidermal Growth Factor; ErbB Receptors; Humans; Lung Neoplasms; Phosphatidylinositol 3-Kinases; Protein Kinase C; Proto-Oncogene Proteins c-akt; Receptors, CXCR4; Signal Transduction

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Cetuximab; Epidermal Growth Factor; Humans; In Situ Hybridization, Fluorescence; Lung Neoplasms

Arsenic is an established lung carcinogen, however, the carcinogenic mechanisms are currently under investigation. Phosphorylation of the epidermal growth factor receptor (EGFR) has been reported with arsenic exposure in bladder cells. EGFR is a tyrosine kinase transmembrane receptor that regulates important processes in carcinogenesis, including cell survival, cell cycle progression, tumor invasion, and angiogenesis. We investigated the mechanisms of EGFR pathway activation by levels of arsenic relevant to human exposure scenarios both in vitro using cultured lung epithelial cells, and in lung tumors samples from New England Lung Cancer Study participants. Toenail arsenic levels were used as an internal biomarker of arsenic exposure. Our in vitro data suggest that arsenic increases levels of the EGFR ligand, heparin binding-EGF, and activate EGFR phosphorylation in the lung. Downstream of EGFR, arsenic exposure increased pERK and cyclin D1 levels. These effects were inhibited by treatment of cultured cells with the EGFR tyrosine kinase inhibitor, Tarceva (erlotinib). In a consecutive series of human lung tumor specimens, pEGFR protein levels were higher in subjects with elevated toenail arsenic levels compared to those with low exposure (odds ratio adjusted for other factors, OR 4.1 (95% confidence interval 1.1-15.6) (p = 0.04). These data suggest that arsenic exposure may stimulate EGFR pathway activation in the lung. Moreover, the tumors that arise in arsenic-exposed individuals also exhibit signs of EGFR pathway dysregulation. Further work is needed to assess the clinical utility of targeting the EGFR pathway in subgroups of lung cancer patients who have been exposed to elevated levels of arsenic.

Topics: Adult; Aged; Amphiregulin; Analysis of Variance; Arsenic; Arsenites; Biomarkers; Bronchi; Cells, Cultured; Cyclin D1; Cycloheximide; EGF Family of Proteins; Epidermal Growth Factor; Epithelial Cells; ErbB Receptors; Erlotinib Hydrochloride; Gene Expression; Glycoproteins; Heparin-binding EGF-like Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Lung; Lung Neoplasms; Middle Aged; Nails; Phosphorylation; Protein Kinase Inhibitors; Quinazolines; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Sodium Compounds

Tyrosine kinase receptors and integrins play essential roles in tumor cell invasion and metastasis. Previously, we showed that epidermal growth factor (EGF) stimulation of pancreatic carcinoma cells led to invasion and metastasis that was blocked by antagonists of integrin alpha(v)beta(5). Here, we show that EGF stimulates metastasis of carcinoma cells via a Src-dependent phosphorylation of p130 CAS leading to activation of Rap1, a small GTPase involved in integrin activation. Specifically, EGF receptor (EGFR)-induced Src activity leads to phosphorylation of a region within the CAS substrate domain, which is essential for Rap1 and alpha(v)beta(5) activation. This pathway induces alpha(v)beta(5)-mediated invasion and metastasis in vivo yet does not influence primary tumor growth or activation of other integrins on these cells. These findings show cross-talk between a tyrosine kinase receptor and an integrin involved in carcinoma cell invasion and metastasis and may explain in part how inhibitors of EGFR affect malignant disease.

Topics: Animals; Carcinoma; Cell Movement; Chick Embryo; DNA Primers; Epidermal Growth Factor; ErbB Receptors; Gene Knockdown Techniques; Humans; Inverted Repeat Sequences; Lung; Lung Neoplasms; Mutation; Neoplasm Invasiveness; Neoplasm Metastasis; Pancreatic Neoplasms; Polymerase Chain Reaction; Receptor Cross-Talk; Receptors, Vitronectin; RNA, Neoplasm; Tumor Cells, Cultured

Lung cancer has become increasingly common in women, and gender differences in the physiology and pathogenesis of the disease have suggested a role for estrogens. In the lung recent data have shown local production of estrogens from androgens via the action of aromatase enzyme and higher levels of estrogen in tumor tissue as compared with surrounding normal lung tissue. High levels of aromatase expression are also maintained in metastases as compared with primary tumors. Consistent with these findings, clinical studies suggest that aromatase expression may be a useful predictive biomarker for prognosis in the management of non-small cell lung cancer (NSCLC), the most common form of lung malignancy. Low levels of aromatase associate with a higher probability of long-term survival in older women with early stage NSCLC. Treatment of lung NSCLC xenografts in vivo with an aromatase inhibitor (exemestane) alone or combined with standard cisplatin chemotherapy elicits a significant reduction in tumor progression as compared to paired controls. Further, lung cancer progression is also governed by complex interactions between estrogen and growth factor signaling pathways to stimulate the growth of NSCLC as well as tumor-associated angiogenesis. We find that combination therapy with the multitargeted growth factor receptor inhibitor vandetanib and the estrogen receptor antagonist fulvestrant inhibit tumor growth more effectively than either treatment administered alone. Thus, incorporation of antiestrogen treatment strategies in standard antitumor therapies for NSCLC may contribute to improved patient outcome, an approach that deserves to be tested in clinical trials.

Topics: Animals; Antineoplastic Agents; Aromatase; Carcinoma, Non-Small-Cell Lung; Cell Division; Cell Line, Tumor; Disease Progression; Epidermal Growth Factor; Estrogen Receptor Modulators; Estrogens; Humans; Immunohistochemistry; Lung Neoplasms; Mice; Mice, Nude; Neoplasm Metastasis; Signal Transduction; Transplantation, Heterologous

Cetuximab, which blocks ligand binding to epidermal growth factor receptor (EGFR), is currently being studied as a novel treatment for non-small cell lung cancer (NSCLC). However, its mechanisms of action toward metastasis, and markers of drug sensitivity, have not been fully elucidated. This study was conducted to (a) determine the effect of Cetuximab on invasion and NSCLC-metastasis; (b) investigate urokinase-type plasminogen activator receptor (u-PAR), a major molecule promoting invasion and metastasis, as a target molecule; (c) delineate molecular mediators of Cetuximab-induced metastasis inhibition; and (d) identify biomarkers of drug sensitivity in NSCLC. Cetuximab treatment resulted in reduced growth and Matrigel invasion of H1395 and A549 NSCLC cell lines, in parallel with reduced u-PAR mRNA and protein. u-PAR down-regulation was brought about by suppressing the binding of JunD and c-Jun to u-PAR promoter motif -190/-171 in vivo, and an inhibition of MAP/ERK kinase signaling. Furthermore, Cetuximab inhibited NSCLC proliferation and metastasis to distant organs in vivo as indicated by the chicken embryo metastasis assay. Low E-cadherin and high u-PAR, but not EGFR, was associated with resistance to Cetuximab in seven NSCLC cell lines. Furthermore, siRNA knockdown of u-PAR led to a resensitization to Cetuximab. Moreover, low E-cadherin and high u-PAR was found in 63% of resected tumor tissues of NSCLC patients progressing under Cetuximab therapy. This is the first study to show u-PAR as a target and marker of sensitivity to Cetuximab, and to delineate novel mechanisms leading to metastasis suppression of NSCLC by Cetuximab.

Topics: Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Biomarkers, Tumor; Cadherins; Carcinoma, Non-Small-Cell Lung; Cetuximab; Chick Embryo; Epidermal Growth Factor; ErbB Receptors; Gene Expression; Humans; JNK Mitogen-Activated Protein Kinases; Lung Neoplasms; MAP Kinase Signaling System; Neoplasm Metastasis; Promoter Regions, Genetic; Proto-Oncogene Proteins c-jun; Receptors, Urokinase Plasminogen Activator

We use novel super-resolution bright-field optical microscopy to observe the filopodium activities of human lung cancer cells in a multi-gradient cell culture chip. Temporal variations of the filopodium numbers are measured without fluorescent labelling. By carefully designing the fluidic field inside the culture chip, we establish stable concentration gradients of the injected reagents. The reagents are injected via a separated central inlet, and the concentration gradients are different at different positions in the chip. The same chip can be used for both control and treated experiments. Using epidermal growth factor as the treatment, we verify that the protrusions of filopodia indicate the direction of concentration gradients experienced by a living cancer cell; while the treatment of bovine serum albumin shows no specific effect on the growth of filopodia. The combination of label-free, high-resolution optical microscopy and a micro cell culture chip establishes a convenient and versatile platform for dynamical cancer-cell analyses.

Topics: Animals; Cattle; Cell Line, Tumor; Epidermal Growth Factor; Growth Substances; Humans; Lung Neoplasms; Microfluidic Analytical Techniques; Pseudopodia; Serum Albumin, Bovine; Small Cell Lung Carcinoma; Staining and Labeling

Coexistence of pulmonary tuberculosis (TB) and lung cancer in clinic poses significant challenges for the diagnostic and treatment of both diseases. Although association of chronic inflammation and cancer is well-documented, causal relationship between TB infection and lung cancer are not understood. We present experimental evidence that chronic TB infection induces cell dysplasia and squamous cell carcinoma (SCC) in a lung-specific manner. First, squamous cell aggregates consistently appeared within the lung tissue associated with chronic TB lesions, and in some cases resembled SCCs. A transplantable tumor was established after the transfer of cells isolated from TB lung lesions into syngeneic recipients. Second, the (Mycobacterium tuberculosis) MTB-infected macrophages play a pivotal role in TB-induced carcinogenesis by inducing DNA damage in their vicinity and by the production of a potent epidermal growth factor epiregulin, which may serve as a paracrine survival and growth factor responsible for squamous metaplasia and tumorigenesis. Third, lung carcinogenesis during the course of chronic TB infection was more pronounced in animals with severe lung tissue damage mediated by TB-susceptibility locus sst1. Together, our experimental findings showed a causal link between pulmonary TB and lung tumorigenesis and established a genetic model for further analysis of carcinogenic mechanisms activated by TB infection.

Topics: Animals; Antitubercular Agents; Carcinoma, Squamous Cell; Cell Transformation, Neoplastic; Chronic Disease; Disease Models, Animal; Epidermal Growth Factor; Epiregulin; Female; Gene Expression; Genetic Predisposition to Disease; Host-Pathogen Interactions; Isoniazid; Lung; Lung Neoplasms; Macrophages; Male; Mice; Mice, Congenic; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Inbred Strains; Mycobacterium tuberculosis; Reverse Transcriptase Polymerase Chain Reaction; Time Factors; Tuberculosis, Pulmonary

To develop a polymer-anticancer drug conjugate, we employed gelatin nanoparticles (GPs) as carriers of cisplatin (CDDP) with anticipated improved therapeutic effect and reduced side effects. The anticancer activities of CDDP-incorporated in GPs (GP-Pt) with biotinylated-EGF (bEGF) modification (GP-Pt-bEGF) were studied. GP-Pt-bEGF with EGFR affinity produced much higher Pt concentrations in A549 cells (high EGFR expression) than in HFL1 cells (low EGFR expression). An in vitro anticancer study showed that GP-Pt-bEGF was more potent than free CDDP or GP-Pt because of its rapid effect on the cell cycle as well as a lower IC(50) (1.2microg/ml) that inhibits A549 cell growth. PI staining showed that cells treated with GP-Pt-bEGF for only 4h had the highest sub-G1 population. The CDDP formulations - free CDDP, GP-Pt, and GP-Pt-bEGF - were given by intratumorous injections to SCID mice in a subcutaneous model. This treatment showed that GP-Pt-bEGF had stronger anti-tumor activity and was less toxic than free CDDP in vivo. Mice treated with GP-Pt-bEGF showed slight body weight loss, whereas free CDDP treatment at the same dose caused a body weight loss of 20-30%. Furthermore, these formulations were given to mice with lung cancer via aerosol delivery. This treatment showed that inhaled GP-Pt-bEGF could target EGFR-overexpressing cells to achieve high cisplatin dosage in cancerous lungs. To summarize, gelatin nanoparticles loaded with CDDP and decorated with EGF tumor-specific ligand were successfully developed. Their in vitro and in vivo targeting ability and anticancer effect were confirmed. The aerosol delivery of the nanodrug carrier was demonstrated. Simple aerosol delivery of targeted drug carriers may prove useful for the clinical treatment of lung cancer patients.

Topics: Administration, Inhalation; Animals; Antineoplastic Agents; Biocompatible Materials; Biotinylation; Cell Cycle; Cell Line, Tumor; Cisplatin; Drug Carriers; Drug Delivery Systems; Epidermal Growth Factor; Gelatin; Humans; Lung Neoplasms; Male; Materials Testing; Mice; Mice, SCID; Molecular Structure; Nanoparticles; Xenograft Model Antitumor Assays

High expression of 3-phosphoinositide-dependent protein kinase-1 (PDK1) has been detected in various invasive cancers. In the current study, we investigated its role in cancer cell migration and experimental metastasis. Down-regulation of PDK1 expression by small interference RNA markedly inhibited spontaneous migration and epidermal growth factor (EGF)-induced chemotaxis of human breast cancer cells. The defects were rescued by expressing wild-type PDK1. PDK1-depleted cells showed impaired EGF-induced actin polymerization and adhesion, probably due to a decrease in phosphorylation of LIM kinase/cofilin and integrin beta1. Confocal microscopy revealed that EGF induced cotranslocation of PDK1 with Akt and protein kinase Czeta (PKCzeta), regulators of LIM kinase, and integrin beta1. Furthermore, PDK1 depletion dampened EGF-induced phosphorylation and translocation of Akt and PKCzeta, suggesting that Akt and PKCzeta functioned downstream of PDK1 in the chemotactic signaling pathway. In severe combined immunodeficiency mice, PDK1-depleted human breast cancer cells formed more slowly growing tumors and were defective in extravasation to mouse lungs after i.v. injection. Our results indicate that PDK1 plays an important role in regulating the malignant behavior of breast cancer cells, including their motility, through activation of Akt and PKCzeta. Thus, PDK1, which increases its expression in cancer cells, can be used as a target for the development of novel therapies.

Topics: 3-Phosphoinositide-Dependent Protein Kinases; Actins; Animals; Breast Neoplasms; Cell Adhesion; Cell Line, Tumor; Cell Movement; Chemotaxis; Down-Regulation; Epidermal Growth Factor; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Lung Neoplasms; Mice; Mice, SCID; Microscopy, Fluorescence; Neoplasm Invasiveness; Neoplasm Metastasis; Protein Kinase C-delta; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; RNA, Small Interfering

We present a multiscale agent-based non-small cell lung cancer model that consists of a 3D environment with which cancer cells interact while processing phenotypic changes. At the molecular level, transforming growth factor beta (TGFbeta) has been integrated into our previously developed in silico model as a second extrinsic input in addition to epidermal growth factor (EGF). The main aim of this study is to investigate how the effects of individual and combinatorial change in EGF and TGFbeta concentrations at the molecular level alter tumor growth dynamics on the multi-cellular level, specifically tumor volume and expansion rate. Our simulation results show that separate EGF and TGFbeta fluctuations trigger competing multi-cellular phenotypes, yet synchronous EGF and TGFbeta signaling yields a spatially more aggressive tumor that overall exhibits an EGF-driven phenotype. By altering EGF and TGFbeta concentration levels simultaneously and asynchronously, we discovered a particular region of EGF-TGFbeta profiles that ensures phenotypic stability of the tumor system. Within this region, concentration changes in EGF and TGFbeta do not impact the resulting multi-cellular response substantially, while outside these concentration ranges, a change at the molecular level will substantially alter either tumor volume or tumor expansion rate, or both. By evaluating tumor growth dynamics across different scales, we show that, under certain conditions, therapeutic targeting of only one signaling pathway may be insufficient. Potential implications of these in silico results for future clinico-pharmacological applications are discussed.

Topics: Computational Biology; Epidermal Growth Factor; Lung Neoplasms; Signal Transduction; Transforming Growth Factor beta

During breast cancer development, increased presence of leukocytes in neoplastic stroma parallels disease progression; however, the functional significance of leukocytes in regulating protumor versus antitumor immunity in the breast remains poorly understood. Utilizing the MMTV-PyMT model of mammary carcinogenesis, we demonstrate that IL-4-expressing CD4(+) T lymphocytes indirectly promote invasion and subsequent metastasis of mammary adenocarcinomas by directly regulating the phenotype and effector function of tumor-associated CD11b(+)Gr1(-)F4/80(+) macrophages that in turn enhance metastasis through activation of epidermal growth factor receptor signaling in malignant mammary epithelial cells. Together, these data indicate that antitumor acquired immune programs can be usurped in protumor microenvironments and instead promote malignancy by engaging cellular components of the innate immune system functionally involved in regulating epithelial cell behavior.

Topics: Adenocarcinoma; Animals; CD4-Positive T-Lymphocytes; Epidermal Growth Factor; Female; Interleukin-4; Lung Neoplasms; Macrophages; Mammary Neoplasms, Experimental; Mice; Mice, Transgenic; Myeloid Cells; Phenotype; Signal Transduction; Th2 Cells

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Antineoplastic Agents, Phytogenic; Antineoplastic Combined Chemotherapy Protocols; Carcinoma, Non-Small-Cell Lung; Cetuximab; Cisplatin; Clinical Trials, Phase III as Topic; Dose-Response Relationship, Drug; Epidermal Growth Factor; ErbB Receptors; Follow-Up Studies; Humans; Lung Neoplasms; Proportional Hazards Models; Survival Analysis; Time Factors; Treatment Outcome; Vinblastine; Vinorelbine

EGFR is a major anticancer drug target in human epithelial tumors. One effective class of agents is the tyrosine kinase inhibitors (TKIs), such as gefitinib and erlotinib. These drugs induce dramatic responses in individuals with lung adenocarcinomas characterized by mutations in exons encoding the EGFR tyrosine kinase domain, but disease progression invariably occurs. A major reason for such acquired resistance is the outgrowth of tumor cells with additional TKI-resistant EGFR mutations. Here we used relevant transgenic mouse lung tumor models to evaluate strategies to overcome the most common EGFR TKI resistance mutation, T790M. We treated mice bearing tumors harboring EGFR mutations with a variety of anticancer agents, including a new irreversible EGFR TKI that is under development (BIBW-2992) and the EGFR-specific antibody cetuximab. Surprisingly, we found that only the combination of both agents together induced dramatic shrinkage of erlotinib-resistant tumors harboring the T790M mutation, because together they efficiently depleted both phosphorylated and total EGFR. We suggest that these studies have immediate therapeutic implications for lung cancer patients, as dual targeting with cetuximab and a second-generation EGFR TKI may be an effective strategy to overcome T790M-mediated drug resistance. Moreover, this approach could serve as an important model for targeting other receptor tyrosine kinases activated in human cancers.

Topics: Afatinib; Amphiregulin; Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Cetuximab; Disease Models, Animal; Drug Resistance, Neoplasm; EGF Family of Proteins; Epidermal Growth Factor; Epiregulin; ErbB Receptors; Erlotinib Hydrochloride; Gene Expression Profiling; Glycoproteins; Humans; Intercellular Signaling Peptides and Proteins; Lung Neoplasms; Male; Mice; Mice, Nude; Mice, Transgenic; Mutation; Neoplasm Transplantation; Paclitaxel; Protein Kinase Inhibitors; Quinazolines; Transplantation, Heterologous; Tumor Cells, Cultured

To report persisting corneal erosion and trichomegaly as ocular side effects of cetuximab (Erbitux) treatment, a monoclonal antibody against the epidermal growth factor (EGF) receptor.. A 62-year-old patient was treated with combined chemotherapy including cetuximab for colorectal carcinoma and showed accelerated growth of eyelashes with persistent bilateral corneal erosions resistant to conservative treatment. Topical EGF was successfully applied as an experimental treatment to antagonize the inhibitory effect of the EGF receptor antibody.. Topical application of EGF led to rapid improvement, with complete healing of the epithelial defect after 7 (left eye) and 19 days (right eye) while application of cetuximab was continued.. Cetuximab (Erbitux) can cause impairment of corneal wound healing as an ocular side effect. Patients concerned may benefit from application of EGF eyedrops.

Topics: Administration, Topical; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Cetuximab; Colorectal Neoplasms; Corneal Diseases; Epidermal Growth Factor; ErbB Receptors; Eyelashes; Humans; Hypertrichosis; Liver Neoplasms; Lung Neoplasms; Male; Middle Aged; Recombinant Proteins; Splenic Neoplasms; Wound Healing

Common genetic variation may play an important role in altering lung cancer risk. We conducted a pathway-based candidate gene evaluation to identify genetic variations that may be associated with lung cancer in a population-based case-control study in Xuan Wei, China (122 cases and 111 controls). A total of 1260 single-nucleotide polymorphisms (SNPs) in 380 candidate genes for lung cancer were successfully genotyped and assigned to one of 10 pathways based on gene ontology. Logistic regression was used to assess the marginal effect of each SNP on lung cancer susceptibility. The minP test was used to identify statistically significant associations at the gene level. Important pathways were identified using a test of proportions and the rank truncated product methods. The cell cycle pathway was found as the most important pathway (P = 0.044) with four genes significantly associated with lung cancer (PLA2G6 minP = 0.001, CCNA2 minP = 0.006, GSK3 beta minP = 0.007 and EGF minP = 0.013), after adjusting for multiple comparisons. Interestingly, most cell cycle genes that were associated with lung cancer in this analysis were concentrated in the AKT signaling pathway, which is essential for regulation of cell cycle progression and cellular survival, and may be important in lung cancer etiology in Xuan Wei. These results should be viewed as exploratory until they are replicated in a larger study.

Topics: Adult; Aged; Case-Control Studies; Cell Cycle; Epidermal Growth Factor; Female; Genetic Predisposition to Disease; Genotype; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Logistic Models; Lung Neoplasms; Male; Middle Aged; Polymorphism, Single Nucleotide; Proto-Oncogene Proteins c-akt; Signal Transduction

Little is known about lung carcinoma epidermal growth factor (EGF) kinase pathway signaling within the context of the tissue microenvironment. We quantitatively profiled the phosphorylation and abundance of signal pathway proteins relevant to the EGF receptor within laser capture microdissected untreated, human non-small cell lung cancer (NSCLC) (n = 25) of known epidermal growth factor receptor (EGFR) tyrosine kinase domain mutation status. We measured six phosphorylation sites on EGFR to evaluate whether EGFR mutation status in vivo was associated with the coordinated phosphorylation of specific multiple phosphorylation sites on the EGFR and downstream proteins. Reverse phase protein array quantitation of NSCLC revealed simultaneous increased phosphorylation of EGFR residues Tyr-1148 (p < 0.044) and Tyr-1068 (p < 0.026) and decreased phosphorylation of EGFR Tyr-1045 (p < 0.002), HER2 Tyr-1248 (p < 0.015), IRS-1 Ser-612 (p < 0.001), and SMAD Ser-465/467 (p < 0.011) across all classes of mutated EGFR patient samples compared with wild type. To explore which subset of correlations was influenced by ligand induction versus an intrinsic phenotype of the EGFR mutants, we profiled the time course of 115 cellular signal proteins for EGF ligand-stimulated (three dosages) NSCLC mutant and wild type cultured cell lines. EGFR mutant cell lines (H1975 L858R) displayed a pattern of EGFR Tyr-1045 and HER2 Tyr-1248 phosphorylation similar to that found in tissue. Persistence of phosphorylation for AKT Ser-473 following ligand stimulation was found for the mutant. These data suggest that a higher proportion of the EGFR mutant carcinoma cells may exhibit activation of the phosphatidylinositol 3-kinase/protein kinase B (AKT)/mammalian target of rapamycin (MTOR) pathway through Tyr-1148 and Tyr-1068 and suppression of IRS-1 Ser-612, altered heterodimerization with ERBB2, reduced response to transforming growth factor beta suppression, and reduced ubiquitination/degradation of the EGFR through EGFR Tyr-1045, thus providing a survival advantage. This is the first comparison of multiple, site-specific phosphoproteins with the EGFR tyrosine kinase domain mutation status in vivo.

Topics: Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cluster Analysis; Dose-Response Relationship, Drug; Epidermal Growth Factor; ErbB Receptors; Genome, Human; Humans; Lasers; Ligands; Lung Neoplasms; Microdissection; Mutant Proteins; Mutation; Phosphorylation; Polymerase Chain Reaction; Protein Array Analysis; Reproducibility of Results; Sequence Analysis, DNA; Signal Transduction; Time Factors

Nowadays, biological cancer treatments represent the major advance in non-small cell lung cancer therapeutic strategies. During the last decade, more than 15 randomized trials associating chemo with biological treatments, in first line setting, have included more than 12,073 NSCLC patients, and as much in phase 2-3 trials in second and third line setting. Very few were positive, but currently anti-angiogenic strategy using the humanized monoclonal antibody bevacizumab has been approved in association with chemotherapy, in first line treatment of carefully selected NSCLC patients (with non proximal tumors, without cerebral metastasis, and of non-squamous histology). On the same way, monotherapy by the EGFR tyrosine kinase inhibitor erlotinib has been approved in second and third line setting, with comparable results as chemotherapy. 2008 was the year of new targeted therapies with cetuximab, the chimeric monoclonal antibody directed against EFGR, in association with chemotherapy in first line setting, whereas EGFR TKI are also tested in first line, in patients selected on the ground of the molecular properties of their tumors (with EGFR mutation or positive EGFR FISH). New generation EGFR TKI (more potent if not more selective) are developed in new settings (neo-adjuvant or adjuvant treatment), with promising results in phase 2 trials, whereas active immunotherapy directed toward MUC1 or MAGE-A3 are tested in large phase 3 randomized trials in adjuvant setting (post-surgery or post-radiotherapy), since phase 2 results were appealing. Therefore, during the last few years, targeted therapies quit science-fiction to enter in our current practice, leading clinicians to learn how to treat new kinds of toxicities and to select patients on molecular grounds.

Topics: Antibodies, Monoclonal; Biological Therapy; Carcinoma, Non-Small-Cell Lung; Epidermal Growth Factor; Forecasting; Humans; Lung Neoplasms; Neoplasm Staging; Vascular Endothelial Growth Factor A

The introduction of targeted biological agents represents the most promising approach to improve the disease control and outcome for patients with non-small cell lung cancer. The epidermal growth factor and the vascular endothelial growth factor signaling pathways have been successfully targeted using both orally administered small molecule tyrosine kinase inhibitors and monoclonal antibodies, with associated improvement in overall survival. Although the trials that established the efficacy of these agents allowed the enrollment of patients older than 70 years, the elderly patients constituted the minority. Given the stringent enrolment criteria in terms of organ function and performance status for most clinical trials, the elderly patients on clinical trials are not entirely representative of the overall elderly patient population. Therefore, the applicability of these data to the overall patient population deserves critical appraisal in the absence of trials dedicated specifically to the elderly. Preplanned and unplanned subset analysis of registration trial data is becoming increasingly common as a substitute measure to provide valuable information to guide the use of targeted agents in the elderly. Using this approach, it has been demonstrated that elderly patients are able to tolerate targeted biological therapies but suffer increased rate of toxicities. However, they derive benefit from such agents when they are carefully selected and have their drug doses adjusted appropriately to minimize potential toxicities. This article reviews the use of targeted agents for the treatment of NSCLC in elderly patients.

Topics: Age Factors; Aged; Angiogenesis Inhibitors; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Bevacizumab; Carcinoma, Non-Small-Cell Lung; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Erlotinib Hydrochloride; Gefitinib; Humans; Lung Neoplasms; Protein-Tyrosine Kinases; Quinazolines; Signal Transduction; Vascular Endothelial Growth Factor A

The spread of cancer during metastatic disease requires that tumor cells subvert normal regulatory networks governing cell motility to invade surrounding tissues and migrate toward blood and lymphatic vessels. Enabled (Ena)/vasodilator-stimulated phosphoprotein (VASP) proteins regulate cell motility by controlling the geometry of assembling actin networks. Mena, an Ena/VASP protein, is upregulated in the invasive subpopulation of breast cancer cells. In addition, Mena is alternately spliced to produce an invasion isoform, Mena(INV). Here we show that Mena and Mena(INV) promote carcinoma cell motility and invasiveness in vivo and in vitro, and increase lung metastasis. Mena and Mena(INV) potentiate epidermal growth factor (EGF)-induced membrane protrusion and increase the matrix degradation activity of tumor cells. Interestingly, Mena(INV) is significantly more effective than Mena in driving metastases and sensitizing cells to EGF-dependent invasion and protrusion. Upregulation of Mena(INV) could therefore enable tumor cells to invade in response to otherwise benign EGF stimulus levels.

Topics: Alternative Splicing; Animals; Carcinoma; Cell Movement; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Macrophages; Mice; Microfilament Proteins; Models, Biological; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Transplantation; Protein Isoforms

Non-small cell lung cancer (NSCLC) cells with activating epidermal growth factor receptor (EGFR) somatic mutations have unique biological properties, including high expression of the ErbB ligand epiregulin; however, the biological role of epiregulin in these cells has not been elucidated. To examine its role, we used an immunohistochemical approach to detect epiregulin expression in NSCLC biopsy samples and pharmacologic and genetic approaches to inhibit epiregulin in cultured NSCLC cells. In NSCLC biopsy samples, epiregulin was detected in 237 of 366 (64.7%) tumors, which correlated with nodal metastasis and a shorter duration of survival. In EGFR-mutant NSCLC cell lines, treatment with a small-molecule EGFR tyrosine kinase inhibitor diminished mRNA levels of the gene encoding epiregulin (EREG). The ability of EGFR-mutant NSCLC cells to invade through Matrigel in vitro was inhibited by treatment with an anti-epiregulin neutralizing antibody or by transfection with an EREG short hairpin RNA. Collectively, these findings show that epiregulin expression correlated with advanced disease, was EGFR dependent, and conferred invasive properties on NSCLC cells. Additional studies are warranted in NSCLC patients to evaluate whether epiregulin expression predicts the metastatic potential of primary tumors and whether anti-epiregulin treatment strategies are efficacious in the prevention of metastasis.

Topics: Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Survival; Disease Progression; Disease-Free Survival; Epidermal Growth Factor; Epiregulin; Gene Expression Regulation, Neoplastic; Genes, erbB-1; Humans; Lung Neoplasms; Lymphatic Metastasis; Mutation; Tissue Array Analysis

Delta(9)-Tetrahydrocannabinol (THC) is the primary cannabinoid of marijuana and has been shown to either potentiate or inhibit tumor growth, depending on the type of cancer and its pathogenesis. Little is known about the activity of cannabinoids like THC on epidermal growth factor receptor-overexpressing lung cancers, which are often highly aggressive and resistant to chemotherapy. In this study, we characterized the effects of THC on the EGF-induced growth and metastasis of human non-small cell lung cancer using the cell lines A549 and SW-1573 as in vitro models. We found that these cells express the cannabinoid receptors CB(1) and CB(2), known targets for THC action, and that THC inhibited EGF-induced growth, chemotaxis and chemoinvasion. Moreover, signaling studies indicated that THC may act by inhibiting the EGF-induced phosphorylation of ERK1/2, JNK1/2 and AKT. THC also induced the phosphorylation of focal adhesion kinase at tyrosine 397. Additionally, in in vivo studies in severe combined immunodeficient mice, there was significant inhibition of the subcutaneous tumor growth and lung metastasis of A549 cells in THC-treated animals as compared to vehicle-treated controls. Tumor samples from THC-treated animals revealed antiproliferative and antiangiogenic effects of THC. Our study suggests that cannabinoids like THC should be explored as novel therapeutic molecules in controlling the growth and metastasis of certain lung cancers.

Topics: Animals; Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Chemotaxis; Dronabinol; Epidermal Growth Factor; Lung Neoplasms; Mice; Mice, SCID; Mitogen-Activated Protein Kinases; Phosphorylation; Receptors, Cannabinoid

Epithelial-to-mesenchymal transition (EMT) is a fundamental biological process whereby epithelial cells lose their polarity and undergo a transition to a mesenchymal phenotype. When cancer cells invade adjacent tissues, they use a mechanism akin to EMT, and understanding the molecular mechanisms that drive this transition will facilitate studies into new targets for prevention of metastasis. Extracellular stimuli, such as growth factors, and their cytosolic effectors cooperate to promote EMT. In highly fibrotic cancers like lung cancer, it is thought that extracellular matrix molecules, including collagen, can initiate signals that promote EMT. Here, we present data showing that collagen I induces EMT in non-small cell lung cancer cell lines, which is prevented by blocking transforming growth factor (TGF)-beta3 signaling. In addition, we show that collagen I-induced EMT is prevented by inhibitors of phosphoinositide 3-kinase and extracellular signal-related kinase signaling, which promotes transcription of TGF-beta3 mRNA in these cells. Thus, our data are consistent with the hypothesis that collagen I induces EMT in lung cancer cells by activating autocrine TGF-beta3 signaling. Epidermal growth factor also seems to initiate EMT via a TGF-dependent mechanism.

Topics: Animals; Autocrine Communication; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Polarity; Collagen Type I; Epidermal Growth Factor; Epithelial Cells; Humans; Lung Neoplasms; MAP Kinase Signaling System; Mice; Neoplasm Metastasis; Neoplasm Proteins; Phosphatidylinositol 3-Kinases; RNA, Messenger; RNA, Neoplasm; Transforming Growth Factor beta3

The epidermal growth factor receptor (EGFR) serves as a molecular target for novel cancer therapeutics such as tyrosine kinase inhibitors (TKI) and EGFR Abs. Recently, specific mutations in the EGFR kinase domain of lung cancers were identified, which altered the signaling capacity of the receptor and which correlated with clinical response or resistance to TKI therapy. In the present study, we investigated the impact of such EGFR mutations on antitumor cell activity of EGFR Abs. Thus, an EGFR-responsive cell line model was established, in which cells with tumor-derived EGFR mutations (L858R, G719S, delE746-A750) were significantly more sensitive to TKI than wild-type EGFR-expressing cells. A clinically relevant secondary mutation (T790M) abolished TKI sensitivity. Significantly, antitumor effects of EGFR Abs, including signaling and growth inhibition and Ab-dependent cellular cytotoxicity, were not affected by any of these mutations. Somatic tumor-associated EGFR kinase mutations, which modulate growth inhibition by TKI, therefore do not impact the activity of therapeutic Abs in vitro.

Topics: Animals; Antibodies, Neoplasm; Antibody-Dependent Cell Cytotoxicity; Cell Death; Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Growth Inhibitors; Humans; Lung Neoplasms; Mice; Mutation; Protein Kinase Inhibitors; Signal Transduction

Lung cancer is the most malignant cancer today; in order to develop an effective drug delivery system for lung cancer therapy, gelatin nanoparticles (GPs) were modified with NeutrAvidin(FITC)-biotinylated epidermal growth factor (EGF) to form EGF receptor (EGFR)-seeking nanoparticles (GP-Av-bEGF). Aerosol droplets of the GP-Av-bEGF were generated by using a nebulizer and were delivered to mice model of lung cancer via aerosol delivery. Analysis of the aerosol size revealed that 99% of the nanoparticles after nebulization had a mass median aerodynamic diameter (MMAD) within the suitable range (0.5-5 microm) for lower airway deposition. The safety of inhaled nanoparticles was examined by lung edema and myeloperoxidase (MPO) activity assay. There's no finding suggestive of acute lung inflammation following inhalation. The fluorescence images obtained from live mice showed that the GP-Av-bEGF could target the cancerous lungs in a more specific manner. Fluorescence analysis of the organs revealed that the GP-Av-bEGF was mainly distributed in cancerous lungs. In contrast, nanoparticle accumulation was lower in normal lungs. The histological results indicated that the fluorescent GP-Av-bEGF was colocalized with the anti-EGFR-immunostain due to EGFR binding. The results of this study revealed that GP-Av-bEGF could target to the EGFR-overexpression cancer cells in vivo and may prove to be beneficial drug carriers when administered by simple aerosol delivery for the treatment of lung cancer.

Topics: Aerosols; Animals; Biotin; Epidermal Growth Factor; Gelatin; Lung Neoplasms; Male; Mice; Mice, Nude; Microscopy, Electron, Scanning; Nanoparticles; Tissue Distribution

Gefitinib (Iressa)-a specific inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase-has been shown to suppress the activation of EGFR signaling required for cell survival and proliferation in non-small cell lung cancer (NSCLC) cell lines. We recently provided novel evidence that gefitinib-sensitive PC9 cells show normal endocytosis of EGFR: internalized EGF-EGFR complexes were transported to late endosomes/lysosomes 15 min after EGF stimulation, and then degraded within the lysosomes. However, gefitinib-resistant QG56 cells showed internalized EGFR accumulation in early endosomes after 60 min of internalization, instead of its trafficking to lysosomes, indicating an aberration in some steps of EGF-EGFR trafficking from the early endosomes to late endosomes/lysosomes. Therefore, we postulate that impairment in some steps of EGF-EGFR trafficking from early endosomes to late endosomes/lysosomes might confer gefitinib-resistance in NSCLC cell lines. To further substantiate the detailed internalization mechanism of gefitinib-sensitive and gefitinib-resistant cells, using confocal immunofluorescence microscopy, we examined the endocytic trafficking of phosphorylated EGFR (pEGFR) in the absence or presence of gefitinib. In PC9 and QG56 cells without EGF stimulation, a large number of pEGFR-positive small vesicular structures not colocalized with late endosomes/lysosomes were spread throughout the cytoplasm, and some pEGFR staining was distributed in the nucleus. This implies a novel intracellular trafficking pathway for pEGFR from cytoplasmic vesicles to the nucleus. Furthermore, an aggregated vesicular structure of early endosomes was observed in the perinuclear region of QG56 cells; it was revealed to be associated with SNX1, originally identified as a protein that interacts with EGFR. Therefore, we confirmed our previous data that an aberration in some steps of EGF-EGFR trafficking from the early endosomes to late endosomes/lysosomes occurs in QG56 cells. Furthermore, in PC9 cells, efficient phosphorylation of EGFR and rapid internalization of pEGFR was observed at 3 min after EGF stimulation; these internalized pEGFR-positive vesicles were trafficked to late endosomes at 15 min, indicating rapid trafficking of EGF-pEGFR complexes from early to late endosomes in PC9 cells. Gefitinib treatment strongly reduced the phosphorylation level of EGFR, and subsequent endocytosis of EGFR was significantly suppressed in PC9 cells. In contrast, in QG56 cel

Topics: Biological Transport; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Endocytosis; Endosomes; Epidermal Growth Factor; ErbB Receptors; Gefitinib; Humans; Lung Neoplasms; Lysosomes; Microscopy, Fluorescence; Phosphorylation; Quinazolines; Signal Transduction; Sorting Nexins; Vesicular Transport Proteins

Several somatic mutations within the tyrosine kinase domain of epidermal growth factor receptor (EGFR) have been identified that predict clinical response of non-small-cell lung carcinoma (NSCLC) patients to gefitinib. To test the hypothesis that these mutations cause constitutive EGF receptor signaling, and to investigate its mechanistic basis, we expressed representative examples in a null background and analysed their biochemical properties. Each mutation caused significant EGF-independent tyrosine phosphorylation of EGFR, and allowed the receptor to promote Ba/F3 cell mitogenesis in the absence of EGF, arguing that these are oncogenic mutations. Active mutated receptors are present at the cell surface and are fully competent to bind EGF. Recent structural studies show that the inactive EGFR tyrosine kinase domain is autoinhibited by intramolecular interactions between its activation loop and alphaC helix. We find that mutations predicted to disrupt this autoinhibitory interaction (including several that have not been described in NSCLC) elevate EGF-independent tyrosine kinase activity, thus providing new insight into how somatic mutations activate EGFR and other ErbB family members.

Topics: Antineoplastic Agents; Cell Line, Tumor; Cell Membrane; Cell Proliferation; Epidermal Growth Factor; ErbB Receptors; Gefitinib; Humans; Interleukin-3; Lung Neoplasms; Models, Molecular; Mutation; Phosphorylation; Protein Binding; Quinazolines

Activation of the epidermal growth factor receptor (EGFR) contributes to the pathogenesis of non-small-cell lung carcinomas (NSCLC) and gefitinib, a selective reversible EGFR inhibitor, is effective in treating patients with NSCLC. However, clinical resistance to gefitinib is a frequent occurrence highlighting the need for improved therapeutic strategies. Melanoma differentiation associated gene-7 (mda-7)/Interleukin-24 (IL-24) (mda-7/IL-24) displays cancer-selective apoptosis induction when delivered via a replication-incompetent adenovirus (Ad.mda-7). In this study, the effect of Ad.mda-7 infection, either alone or in combination with gefitinib, was analyzed in a panel of NSCLC cell lines carrying wild-type EGFR (H-460 and H-2030) or mutant EGFR (H-1650 and H-1975). While H-2030 and H-1650 cells were sensitive, H-460 and H-1975 cells were resistance to growth inhibition by Ad.mda-7, which was reversed by the combination of Ad.mda-7 and gefitinib. This combination increased MDA-7/IL-24 and downstream effector double-stranded RNA-activated protein kinase (PKR) protein expression, promoting apoptosis induction of NSCLC cells. Inhibition of PKR significantly inhibited apoptosis induction by Ad.mda-7 when administered alone but not when used in combination with gefitinib. The combination treatment also augmented inhibition of EGFR signaling. Our findings indicate that a combinatorial treatment with Ad.mda-7 and gefitinib may provide benefit in the treatment of NSCLC, especially in patients displaying resistance to clinically used EGFR inhibitors.

Topics: Adenoviridae; Adenoviridae Infections; Antineoplastic Agents; Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Drug Resistance, Neoplasm; eIF-2 Kinase; Enzyme Inhibitors; Epidermal Growth Factor; Gefitinib; Gene Expression Regulation, Neoplastic; Genetic Therapy; Genetic Vectors; Humans; Interleukins; Lung Neoplasms; Mutation; Quinazolines

Ceramide-1-phosphate (C1P) is emerging as a new addition to the family of bioactive sphingolipid metabolites. At low concentrations, C1P enhanced survival of NIH 3T3 fibroblasts and A549 lung cancer cells, while at high concentrations, it reduced survival and induced apoptosis. Apoptosis correlated with degradation of C1P to pro-apoptotic ceramide. To examine the role of endogenous C1P, expression of ceramide kinase, the enzyme that produces C1P, was downregulated, which reduced cellular proliferation, progression into S phase and enhanced apoptosis induced by serum starvation. Our results suggest that ceramide kinase determines the balance between pro-apoptotic ceramide and anti-apoptotic C1P to regulate cell fate, reminiscent of its function in plants.

Topics: Adenocarcinoma; Animals; Apoptosis; Cattle; Cell Cycle; Cell Proliferation; Cell Survival; Ceramides; Down-Regulation; Epidermal Growth Factor; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Mice; NIH 3T3 Cells; Phosphotransferases (Alcohol Group Acceptor); RNA, Messenger; RNA, Small Interfering

Topics: Animals; Breast Neoplasms; Cyclooxygenase 2; Epidermal Growth Factor; Epiregulin; Humans; Lung Neoplasms; Matrix Metalloproteinase 1; Matrix Metalloproteinase 2; Mice; Neovascularization, Pathologic

Metastasis entails numerous biological functions that collectively enable cancerous cells from a primary site to disseminate and overtake distant organs. Using genetic and pharmacological approaches, we show that the epidermal growth factor receptor ligand epiregulin, the cyclooxygenase COX2, and the matrix metalloproteinases 1 and 2, when expressed in human breast cancer cells, collectively facilitate the assembly of new tumour blood vessels, the release of tumour cells into the circulation, and the breaching of lung capillaries by circulating tumour cells to seed pulmonary metastasis. These findings reveal how aggressive primary tumorigenic functions can be mechanistically coupled to greater lung metastatic potential, and how such biological activities may be therapeutically targeted with specific drug combinations.

Topics: Animals; Breast Neoplasms; Capillaries; Cell Line, Tumor; Cyclooxygenase 2; Epidermal Growth Factor; Epiregulin; Female; Humans; Lung; Lung Neoplasms; Matrix Metalloproteinase 1; Matrix Metalloproteinase 2; Mice; Neovascularization, Pathologic

Serum levels of the soluble epidermal growth factor receptor (sEGFR) and its ligands epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha) and amphiregulin (AR) were measured in healthy donors and patients with non-small cell lung cancer (NSCLC) and head and neck carcinoma (HNC). In NSCLC, we found sEGFR and EGF levels significantly lowered in patients with respect to healthy donors. In HNC patients, significantly diminished levels were found in the case of sEGFR, EGF and also AR. In both malignancies, no significant association was found between the serum levels of the molecules and the patients' gender, age or smoking habit. Only a significant association was found between the decrease of sEGFR and the absence of distant metastasis in NSCLC and the tumour stage in HNC. The most interesting result was that combining sEGFR and EGF, sensitivities of 88% in NSCLC and 100% in HNC were reached without losing specificity (97.8% in both cases). The use of discriminant analysis and logistic regression improved the sensitivity for NSCLC and the specificity for HNC. These data demonstrate a potentially interesting value of the serum levels of sEGFR and EGF, especially when combined, as markers for NSCLC and HNC.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Carcinoma; Carcinoma, Non-Small-Cell Lung; Epidermal Growth Factor; ErbB Receptors; Female; Head and Neck Neoplasms; Humans; Ligands; Lung Neoplasms; Male; Middle Aged; Receptors, Androgen; Transforming Growth Factor alpha

Since lung cancer is the most malignant cancer today, a specific drug-delivery system has been developed for superior outcome. In this study, gelatin nanoparticles (GPs) employed as native carriers were grafted with NeutrAvidin(FITC) on the particle's surface (GP-Av). Next, the biotinylated epithelial growth factor (EGF) molecules were conjugated with NeutrAvidin(FITC), forming a core-shell-like structure (GP-Av-bEGF) to achieve the enhancement of targeting efficiency. These nanoparticles were applied as an EGF receptor (EGFR)-seeking agent to detect lung adenocarcinoma. The results showed that the modification process had no significant influence on particle size (220 nm) and zeta potential (-9.3 mV). By the in vitro cell culture test, GP-Av-bEGF resulted in higher entrance efficiency on adenocarcinoma cells (A549) than that on normal lung cells (HFL1) because A549 possessed greater amounts of EGFR. We also found that uptake of GP-Av-bEGF by A549 cells was time and dose dependent. Confocal microscopy confirmed the cellular internalization of GP-Av-bEGF, and more fluorescent spots of GP-Av-bEGF nanoparticles were obviously observed as well as lysosomal entrapment in A549. Finally, the delivery was demonstrated by in vivo aerosol administration to cancerous lung of the SCID mice model, and specific accumulation in cancerous lung was confirmed by image quantification. The targeting ability of GP-Av-bEGF was proved in vitro and in vivo, which holds promise for further anti-cancer drug applications.

Topics: Biotinylation; Cell Line, Tumor; Drug Carriers; Drug Delivery Systems; Epidermal Growth Factor; Gelatin; Humans; Lung Neoplasms; Materials Testing; Nanoparticles; Particle Size

A humanized anti-HER2 monoclonal antibody pertuzumab (Omnitarg, 2C4), binding to a different HER2 epitope than trastuzumab, is known as an inhibitor of heterodimerization of the HER receptors. Potent antitumor activity against HER2-expressing breast and prostate cancer cell lines has been clarified, but this potential is not clear against lung cancers. The authors investigated the in vitro anti-tumor activity of pertuzumab against eight non-small cell lung cancer cells expressing various members of the HER receptors. A lung cancer 11_18 cell line expressed a large amount of HER2 and HER3, and its cell growth was stimulated by an HER3 ligand, heregulin (HRG)-alpha. Pertuzumab significantly inhibited the HRG-alpha-stimulated cellular growth of the 11_18 cells. Pertuzumab blocked HRG-alpha-stimulated phosphorylation of HER3, mitogen-activated protein kinase (MAPK), and Akt. In contrast, pertuzumab failed to block epidermal growth factor (EGF)-stimulated phosphorylation of EGF receptor (EGFR) and MAPK. Immunoprecipitation showed that pertuzumab inhibited HRG-alpha-stimulated HER2/HER3 heterodimer formation. HRG-alpha-stimulated HER3 phosphorylation was also observed in the PC-9 cells co-overexpressing EGFR, HER2, and HER3, but the cell growth was neither stimulated by HRG-alpha nor inhibited by pertuzumab. The present results suggest that pertuzumab is effective against HRG-alpha-dependent cell growth in lung cancer cells through inhibition of HRG-alpha-stimulated HER2/HER3 signaling.

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Carcinoma, Non-Small-Cell Lung; Cell Division; Cell Line, Tumor; Cell Proliferation; Dimerization; Epidermal Growth Factor; ErbB Receptors; Growth Inhibitors; Humans; Ligands; Lung Neoplasms; Neuregulin-1; Phosphorylation; Receptor, ErbB-3; Signal Transduction

Some non-small cell lung cancers (NSCLC) with epidermal growth factor receptor (EGFR) tyrosine kinase domain mutations require altered signaling through the EGFR for cell survival and are exquisitely sensitive to tyrosine kinase inhibitors. EGFR down-regulation was impaired in two NSCLCs with EGFR tyrosine kinase domain mutations. The mutant receptors were poorly ubiquitylated and exhibited decreased association with the ubiquitin ligase Cbl. Overexpression of Cbl increased the degradation of EGFR. Treatment with geldanamycin, an inhibitor of the chaperone heat shock protein 90, also increased both wild-type and mutant EGFR degradation without affecting internalization. The down-regulation of the mutant EGFRs was still impaired when they were stably expressed in normal human bronchial epithelial cells. Thus, the mutations that altered signaling also decreased the interaction of EGFRs with the mechanisms responsible for endosomal sorting.

Topics: Benzoquinones; Carcinoma, Non-Small-Cell Lung; Down-Regulation; Epidermal Growth Factor; ErbB Receptors; HeLa Cells; Humans; Lactams, Macrocyclic; Lung Neoplasms; Mutation; Protein Structure, Tertiary; Proto-Oncogene Proteins c-cbl; Signal Transduction; Ubiquitin

Chemotaxis plays an important role in metastasis of cancer cells. In the current study, we investigated the role of PTEN, a tumor suppressor, in chemotaxis of human breast cancer cells. Over-expression of PTEN inhibited EGF-induced chemotaxis, probably due to an overall reduction of PIP(3) levels. Disruption of PTEN by siRNA caused a marked decrease in chemokinesis, cell adhesion, and membrane spreading, resulting in a severe defect in chemotaxis. In PTEN disrupted cells, PDK1, AKT, and PKCzeta exhibited elevated basal activities, which prevented EGF-induced further activation of these molecules. In the absence of EGF, active PDK1 was detected on multiple directions of the plasma membranes of PTEN disrupted cells, which competed against EGF-induced gradient sensing. To confirm the biological relevance of in vitro studies, both PTEN disrupted cells and its parental human breast cancer cells were injected into tail veins of SCID mice. Mice injected with PTEN disrupted cancer cells showed a marked decrease in lung metastasis. Taken together, our data show that PTEN plays a non-redundant role in EGF-induced chemotaxis of human breast cancer cells, and an optimal level of PTEN is required in these responses.

Topics: Animals; Breast Neoplasms; Cell Adhesion; Cell Line, Tumor; Cell Membrane; Chemotaxis; Epidermal Growth Factor; Gene Expression; Humans; Lung Neoplasms; Mice; Mice, SCID; Neoplasm Metastasis; PTEN Phosphohydrolase; RNA, Small Interfering; Signal Transduction

Conventional therapies for patients with lung cancer have reached a therapeutic plateau. We therefore evaluated the feasibility of combined vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) and epidermal growth factor (EGF) receptor (EGFR) targeting with radiation therapy in an orthotopic model that closely recapitulates the clinical presentation of human lung cancer.. Effects of irradiation and/or ZD6474, a small-molecule inhibitor of VEGFR2 and EGFR tyrosine kinases, were studied in vitro for human lung adenocarcinoma cells by using proliferation and clonogenic assays. The feasibility of combining ZD6474 with radiation therapy was then evaluated in an orthotopic model of human lung adenocarcinoma. Lung tumor burden and spread within the thorax were assessed, and tumor and adjacent tissues were analyzed by means of immunohistochemical staining for multiple parameters, including CD31, VEGF, VEGFR2, EGF, EGFR, matrix metalloproteinase-2 and -9, and basic fibroblast growth factor.. ZD6474 enhanced the radioresponse of NCI-H441 human lung adenocarcinoma cells by a factor of 1.37 and markedly inhibited sublethal damage repair. In vivo, the combined blockade of VEGFR2 and EGFR by ZD6474 blocked pleural effusion formation and angiogenesis and enhanced the antivascular and antitumor effects of radiation therapy in the orthotopic human lung cancer model and was superior to chemoradiotherapy.. When radiation therapy is combined with VEGFR2 and EGFR blockade, significant enhancement of antiangiogenic, antivascular, and antitumor effects are seen in an orthotopic model of lung cancer. These data provide support for clinical trials of biologically targeted and conventional therapies for human lung cancer.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Combined Modality Therapy; DNA Repair; Epidermal Growth Factor; ErbB Receptors; Feasibility Studies; Humans; Lung Neoplasms; Male; Mice; Mice, Nude; Neovascularization, Pathologic; Piperidines; Pleural Effusion; Quinazolines; Radiation Tolerance; Radiation-Sensitizing Agents; Receptors, Vascular Endothelial Growth Factor; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-2; Xenograft Model Antitumor Assays

Epidermal growth factor receptor (EGFR) has been extensively targeted in the treatment of non-small cell lung cancer, producing responses in a small number of patients. To study the role of ligand expression in mediating response to EGFR antagonism, we injected NCI-H441 [EGFR and EGF/transforming growth factor-alpha (TGF-alpha) positive] or PC14-PE6 (EGFR positive and EGF/TGF-alpha negative) human lung adenocarcinoma cells into the lungs of nude mice. We randomized the mice to receive treatment with the EGFR tyrosine kinase inhibitors gefitinib or AEE788 or vehicle. Treatment of mice bearing NCI-H441 but not PC14-PE6 lung tumors resulted in a significant reduction in primary tumor growth, pleural effusion, and lymph node metastasis. Immunohistochemical analyses revealed that NCI-H441 and PC14-PE6 cells expressed EGFR but that the expression of EGF/TGF-alpha was high in NCI-H441 cells and very low in PC14-PE6 cells. Consequently, EGFR was activated in both tumor and tumor-associated endothelial cells in the NCI-H441 tumors but not in the PC14-PE6 tumors. Antagonism of EGFR signaling by treatment of mice with AEE788 decreased proliferation and increased apoptosis of both tumor cells and tumor-associated endothelial cells in NCI-H441 tumors but not in PC14-PE6 tumors. However, after transfection of PC14-PE6 cells with TGF-alpha, lung tumors derived from the transfected cells expressed and activated EGFR in both tumor and tumor-associated endothelial cells and tumors responded to treatment with AEE788. Collectively, these results strongly suggest that the response of human lung cancers growing orthotopically in mice to the inhibition of EGFR signaling is determined by ligand (EGF/TGF-alpha) expression by tumor cells. Our findings provide an additional explanation for the susceptibility of lung cancers to treatment with EGFR tyrosine kinase inhibitors.

Topics: Adenocarcinoma; Animals; Antineoplastic Agents; Blotting, Western; Cell Proliferation; Epidermal Growth Factor; ErbB Receptors; Gefitinib; Gene Dosage; Humans; Lung Neoplasms; Male; Mice; Mice, Nude; Phosphorylation; Purines; Quinazolines; Reverse Transcriptase Polymerase Chain Reaction; Transforming Growth Factor alpha; Xenograft Model Antitumor Assays

Topics: Adenocarcinoma; Back; Carcinoma, Non-Small-Cell Lung; Chemotherapy, Adjuvant; Drug Eruptions; Epidermal Growth Factor; Female; Humans; Lung Neoplasms; Lymphatic Metastasis; Middle Aged; Protein Kinase Inhibitors; Quinazolines; Radiotherapy, Adjuvant; Skin

Epidermal growth factor (EGF) plays an important role in tumourigenesis by binding with its receptor, EGFR. Variations in the DNA sequence in the EGF gene can lead to an alteration in EGF production and/or activity, which can affect an individual's susceptibility to lung cancer. To test this hypothesis, this study examined the association between the +61 A>G polymorphism in the 5'-untranslated region of the EGF gene and the risk of lung cancer in a Korean population.. The EGF+61 A>G genotype was determined in 432 lung cancer patients and 432 healthy age- and gender-matched control subjects.. The +61 AA and +61 AG genotypes were not significantly associated with the risk of lung cancer compared with the +61 GG genotype (adjusted OR = 1.02, 95% CI: 0.77-1.37; and adjusted OR = 0.81, 95% CI: 0.51-1.29, respectively). In addition to the reference model, the EGF+61 A>G polymorphism had no significant association with the risk of lung cancer under both dominant and recessive models for the +61A allele (adjusted OR = 0.98, 95% CI: 0.74-1.29; and adjusted OR = 0.80, 95% CI: 0.51-1.24, respectively).. These results suggest that the EGF+61 A>G polymorphism may not significantly affect the susceptibility to lung cancer in the Korean population.

Topics: Adenocarcinoma; Carcinoma, Small Cell; Carcinoma, Squamous Cell; Epidermal Growth Factor; Female; Genetic Predisposition to Disease; Genotype; Humans; Lung Neoplasms; Male; Middle Aged; Neoplasm Staging; Polymorphism, Single Nucleotide; Risk Factors

The induction of senescence-like growth arrest has emerged as a putative contributor to the anticancer effects of chemotherapeutic agents. Clinical trials are underway to evaluate the efficacy of inhibitors for class I and II histone deacetylases to treat malignancies. However, a potential antiproliferative effect of inhibitor for Sirt1, which is an NAD(+)-dependent deacetylase and belongs to class III histone deacetylases, has not yet been explored. Here, we show that Sirt1 inhibitor, Sirtinol, induced senescence-like growth arrest characterized by induction of senescence-associated beta-galactosidase activity and increased expression of plasminogen activator inhibitor 1 in human breast cancer MCF-7 cells and lung cancer H1299 cells. Sirtinol-induced senescence-like growth arrest was accompanied by impaired activation of mitogen-activated protein kinase (MAPK) pathways, namely, extracellular-regulated protein kinase, c-jun N-terminal kinase and p38 MAPK, in response to epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I). Active Ras was reduced in Sirtinol-treated senescent cells compared with untreated cells. However, tyrosine phosphorylation of the receptors for EGF and IGF-I and Akt/PKB activation were unaltered by Sirtinol treatment. These results suggest that inhibitors for Sirt1 may have anticancer potential, and that impaired activation of Ras-MAPK pathway might take part in a senescence-like growth arrest program induced by Sirtinol.

Topics: Benzamides; beta-Galactosidase; Breast Neoplasms; Cellular Senescence; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Genes, ras; Histone Deacetylase Inhibitors; Humans; Insulin-Like Growth Factor I; Lung Neoplasms; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Naphthols; p38 Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinases; Phosphorylation; Plasminogen Activator Inhibitor 1; Proto-Oncogene Proteins c-akt; Receptor, IGF Type 1; Signal Transduction; Sirtuin 1; Sirtuins; Tumor Cells, Cultured; Tyrosine

Mutations in the kinase domain of epidermal growth factor receptor (EGFR) are associated with clinical responsiveness to gefitinib in patients with non-small-cell lung cancers (NSCLC). Recently, we have identified many novel EGFR mutations in NSCLC tissues. In this study, we found that gefitinib could suppress the tyrosine phosphorylation of most EGFR mutants better than the wild-type receptor. However, gefitinib had quite variable growth-suppressive effects on different EGFR mutant-expressing cells. All tested EGFR mutants have high basal phosphorylation at multiple tyrosine residues. Upon EGF stimulation, the mutated EGFRs did not have apparently stronger phosphorylation at tyrosines 845, 992, 1,068, and 1,173 than the wild-type receptor. However, stronger phosphorylation at tyrosine 1,045 was observed in the S768I, L861Q, E709G, and G719S mutants. The E746-A750 deletion mutant was less responsive to EGF than the wild-type and other mutant receptors. The S768I, L861Q, E709G, and G719S mutants were refractory to EGF-induced ubiquitination and had more sustained tyrosine phosphorylation. E709G and G719S also lacked EGF-induced receptor downregulation. Our results indicate that, in addition to sensitivity to gefitinib, EGFR mutations also caused various changes in EGFR's regulatory mechanisms, which may contribute to the constitutive activation of EGFR mutants and oncogenesis in NSCLC.

Topics: Animals; Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Chlorocebus aethiops; COS Cells; Epidermal Growth Factor; ErbB Receptors; Gefitinib; Immunoprecipitation; Lung Neoplasms; Mutation; Phosphorylation; Quinazolines; Tumor Cells, Cultured; Tyrosine; Ubiquitin

The exact role of a disintegrin and metalloproteinase with thrombospondin motifs-1 (ADAMTS-1) and the underlying mechanism of its involvement in tumor metastasis have not been established. We have now demonstrated that overexpression of ADAMTS-1 promotes pulmonary metastasis of TA3 mammary carcinoma and Lewis lung carcinoma cells and that a proteinase-dead mutant of ADAMTS-1 (ADAMTS-1E/Q) inhibits their metastasis, indicating that the prometastatic activity of ADAMTS-1 requires its metalloproteinase activity. Overexpression of ADAMTS-1 in these cells promoted tumor angiogenesis and invasion, shedding of the transmembrane precursors of heparin-binding epidermal growth factor (EGF) and amphiregulin (AR), and activation of the EGF receptor and ErbB-2, while overexpression of ADAMTS-1E/Q inhibited these events. Furthermore, we found that ADAMTS-1 undergoes auto-proteolytic cleavage to generate the NH(2)- and COOH-terminal cleavage fragments containing at least one thrombospondin-type-I-like motif and that overexpression of the NH(2)-terminal ADAMTS-1 fragment and the COOH-terminal ADAMTS-1 fragment can inhibit pulmonary tumor metastasis. These fragments also inhibited Erk1/2 kinase activation induced by soluble heparin-binding EGF and AR. Taken together, our results suggest that the proteolytic status of ADAMTS-1 determines its effect on tumor metastasis, and that the ADAMTS-1E/Q and the ADAMTS-1 fragments likely inhibit tumor metastasis by negatively regulating the availability and activity of soluble heparin-binding EGF and AR.

Topics: ADAM Proteins; ADAMTS1 Protein; Amino Acid Sequence; Amphiregulin; Animals; Apoptosis; Carcinoma, Lewis Lung; Cell Proliferation; Collagen; Drug Combinations; EGF Family of Proteins; Epidermal Growth Factor; ErbB Receptors; Female; Glycoproteins; Heparin-binding EGF-like Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Laminin; Lung Neoplasms; Mammary Neoplasms, Experimental; Mice; Mice, Inbred C57BL; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Molecular Sequence Data; Neoplasm Invasiveness; Neovascularization, Pathologic; Proteoglycans; Receptor, ErbB-2; Sequence Deletion; Sequence Homology, Amino Acid; Signal Transduction

We evaluated the contribution of three genetic alterations (p53 knockdown, K-RAS(V12), and mutant EGFR) to lung tumorigenesis using human bronchial epithelial cells (HBEC) immortalized with telomerase and Cdk4-mediated p16 bypass. RNA interference p53 knockdown or oncogenic K-RAS(V12) resulted in enhanced anchorage-independent growth and increased saturation density of HBECs. The combination of p53 knockdown and K-RAS(V12) further enhanced the tumorigenic phenotype with increased growth in soft agar and an invasive phenotype in three-dimensional organotypic cultures but failed to cause HBECs to form tumors in nude mice. Growth of HBECs was highly dependent on epidermal growth factor (EGF) and completely inhibited by EGF receptor (EGFR) tyrosine kinase inhibitors, which induced G1 arrest. Introduction of EGFR mutations E746-A750 del and L858R progressed HBECs toward malignancy as measured by soft agar growth, including EGF-independent growth, but failed to induce tumor formation. Mutant EGFRs were associated with higher levels of phospho-Akt, phospho-signal transducers and activators of transcription 3 [but not phospho-extracellular signal-regulated kinase (ERK) 1/2], and increased expression of DUSP6/MKP-3 phosphatase (an inhibitor of phospho-ERK1/2). These results indicate that (a) the HBEC model system is a powerful new approach to assess the contribution of individual and combinations of genetic alterations to lung cancer pathogenesis; (b) a combination of four genetic alterations, including human telomerase reverse transcriptase overexpression, bypass of p16/RB and p53 pathways, and mutant K-RAS(V12) or mutant EGFR, is still not sufficient for HBECs to completely transform to cancer; and (c) EGFR tyrosine kinase inhibitors inhibit the growth of preneoplastic HBEC cells, suggesting their potential for chemoprevention.

Topics: Aged; Bronchi; Cell Adhesion; Cell Growth Processes; Cell Transformation, Neoplastic; Dual Specificity Phosphatase 6; Epidermal Growth Factor; Epithelial Cells; ErbB Receptors; Erlotinib Hydrochloride; Female; Gefitinib; Genes, erbB-1; Genes, p16; Genes, p53; Genes, ras; Humans; Lung Neoplasms; Mitogen-Activated Protein Kinase Kinases; Oncogenes; Phosphorylation; Protein Tyrosine Phosphatases; Proto-Oncogene Proteins c-akt; Quinazolines; RNA Interference; STAT3 Transcription Factor; Telomerase; Transfection; Up-Regulation

Capillaries expressing the laminin alpha2 chain in basement membranes may be considered early developing vessels in normal and neoplastic human tissues. Therefore, we investigated whether up-regulation of this extracellular matrix protein favors transendothelial migration of neoplastic cells and then metastasis. In lung small and large cell neuroendocrine carcinomas, which exhibit a stronger metastatic tendency among carcinomas, laminin alpha2 chain-positive vessels were more numerous than in carcinoid tumors and supraglottis, breast, and lung non-small cell carcinomas, suggesting a direct relationship between these vessels and metastasis. In vitro studies showed that epidermal growth factor (EGF) induced a more efficient migration of the AE-2 lung neuroendocrine carcinoma cell line through the purified laminin alpha2 chain rather than through the laminin beta1 chain and fibronectin. AE-2 cells constitutively expressed all EGF receptors and the alpha6beta1 integrin, which is one of the laminin alpha2 chain receptors. EGF up-regulated alpha6beta1 expression in several tumors. In this regard, we show that EGF increased the chemo-kinetic migration of AE-2 cells through EAHY endothelial monolayers, which was inhibited by the anti-alpha6 integrin chain monoclonal antibody. These data indicate that laminin alpha2 chain and alpha6beta1 may be mutually involved in EGF-dependent migration of AE-2 cells and that laminin alpha2 chain-positive vessels may favor metastasis of EGF-dependent tumors.

Topics: Antibodies, Monoclonal; Capillaries; Carcinoid Tumor; Carcinoma, Neuroendocrine; Cell Line, Tumor; Cell Movement; Epidermal Growth Factor; Gene Expression; Humans; Laminin; Lung Neoplasms; Models, Biological; Neoplasm Invasiveness; Neoplasm Metastasis; Up-Regulation

This study was conducted to gain insight into the relationship between expression profiles and underlying genetic changes, which are known to be important for the pathogenesis of lung cancers.. Expression profiles of 18,175 unique genes and three major targets for genetic changes, p53, epidermal growth factor receptor (EGFR), and K-ras, were investigated in 149 patients with non-small-cell lung cancer, including 90 patients with adenocarcinoma to determine their relationships with various clinicopathologic features and Gene Ontology (GO) terms.. This study successfully established a basis for expression profile-defined classification, which can classify adenocarcinomas into two major types, terminal respiratory unit (TRU) type and non-TRU type. Our GO term-based identifier of particular biologic processes, molecular functions, and cellular compartments clearly showed characteristic retention of normal peripheral lung features in TRU type, in sharp contrast to the significant association of non-TRU type with cell cycling and proliferation-related features. While significantly higher frequency of EGFR mutation was observed in TRU type, we found that the presence of EGFR mutations was a significant predictor of shorter postoperative survival for TRU type, independent of disease stage. We were also able to identify a set of genes in vivo with significant upregulation in the presence of EGFR mutations.. This study has shed light on heterogeneity in lung cancers, especially in adenocarcinomas, by establishing a molecularly, genetically, and clinically relevant, expression profile-defined classification. Future studies using independent patient cohorts are warranted to confirm the prognostic significance of EGFR mutations in TRU-type adenocarcinoma.

Topics: Adenocarcinoma; Epidermal Growth Factor; Genes, ras; Humans; Lung Neoplasms; Molecular Biology; Mutation; Oligonucleotide Array Sequence Analysis

Topics: Carcinoma, Non-Small-Cell Lung; Clinical Trials, Phase I as Topic; Epidermal Growth Factor; Female; Humans; Immunotherapy, Active; Lung Neoplasms; Male

Understanding the mechanisms controlling cancer cell invasion and metastasis constitutes a fundamental step in setting new strategies for diagnosis, prognosis, and therapy of metastatic cancers. LIM kinase1 (LIMK1) is a member of a novel class of serine-threonine protein kinases. Cofilin, a LIMK1 substrate, is essential for the regulation of actin polymerization and depolymerization during cell migration. Previous studies have made opposite conclusions as to the role of LIMK1 in tumor cell motility and metastasis, claiming either an increase or decrease in cell motility and metastasis as a result of LIMK1 over expression (Zebda, N., O. Bernard, M. Bailly, S. Welti, D.S. Lawrence, and J.S. Condeelis. 2000. J. Cell Biol. 151:1119-1128; Davila, M., A.R. Frost, W.E. Grizzle, and R. Chakrabarti. 2003. J. Biol. Chem. 278:36868-36875; Yoshioka, K., V. Foletta, O. Bernard, and K. Itoh. 2003. Proc. Natl. Acad. Sci. USA. 100:7247-7252; Nishita, M., C. Tomizawa, M. Yamamoto, Y. Horita, K. Ohashi, and K. Mizuno. 2005. J. Cell Biol. 171:349-359). We resolve this paradox by showing that the effects of LIMK1 expression on migration, intravasation, and metastasis of cancer cells can be most simply explained by its regulation of the output of the cofilin pathway. LIMK1-mediated decreases or increases in the activity of the cofilin pathway are shown to cause proportional decreases or increases in motility, intravasation, and metastasis of tumor cells.

Topics: Animals; Cell Line, Tumor; Cell Movement; Cell Surface Extensions; Chemotaxis; Cofilin 1; Epidermal Growth Factor; Female; Green Fluorescent Proteins; Lim Kinases; Lung Neoplasms; Mammary Neoplasms, Experimental; Mutation; Neoplasm Invasiveness; Neoplasm Metastasis; Phosphorylation; Protein Kinases; Rats; Rats, Inbred F344; RNA, Small Interfering; Survival Analysis; Transfection

Connective tissue growth factor (CTGF) is a secreted protein that belongs to CCN family. The proteins in this family are implicated in various biological processes, such as angiogenesis, adhesion, migration, and apoptosis. In this study, we explored the roles of CTGF in lung tumorigenesis. The expression levels of CTGF in 58 lung cancer samples were reduced by >2 fold in 57% of the samples compared with matched normal samples using real-time reverse transcription-PCR. These results were confirmed by immunohistochemical staining for CTGF in normal lung epithelia and lung cancer. Cellular proliferation was inhibited in non-small cell lung cancer (NSCLC) cell lines NCI-H460, NCI-H520, NCI-H1299, and SK-MES-1 by CTGF overexpression. Partially purified CTGF suppressed lung cancer cell growth. The growth inhibition caused by CTGF overexpression was associated with growth arrest at G(0)-G(1) and prominent induction of p53 and ADP ribosylation factor. Most interestingly, overexpression of CTGF suppressed insulin-like growth factor-I-dependent Akt phosphorylation and epidermal growth factor-dependent extracellular signal-regulated kinase 1/2 phosphorylation. In summary, NSCLC cells expressed decreased levels of CTGF compared with normal lung cells; this lower expression has an effect on lung cancer cell proliferation and its cellular response to growth factors. Our data suggest that CTGF may behave as a secreted tumor suppressor protein in the normal lung, and its expression is suppressed in many NSCLCs.

Topics: Carcinoma, Non-Small-Cell Lung; Cell Cycle; Cell Proliferation; Connective Tissue Growth Factor; Culture Media, Conditioned; Epidermal Growth Factor; Gene Expression; Growth Inhibitors; Humans; Immediate-Early Proteins; Insulin-Like Growth Factor I; Intercellular Signaling Peptides and Proteins; Lung Neoplasms; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Oncogene Protein v-akt; Phosphorylation; Signal Transduction; Transfection; Tumor Cells, Cultured

Human lung cancer cell lines are widely used to test anticancer drugs. These in-vitro tests, however, preclude the detection of responses to paracrine factors from surrounding stroma. We have cocultured pulmonary fibroblasts CCD-19Lu, from a healthy donor, or HLF-A, from a patient with epidermoid carcinoma of the lung, with two human pulmonary adenocarcinoma cell lines to test the hypothesis that the fibroblasts stimulate the growth of the tumor cells. Both fibroblast cell lines significantly increased the proliferation of the pulmonary adenocarcinoma cell lines in 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide assays, with HLF-A fibroblasts yielding the most pronounced responses. The proliferation of the pulmonary adenocarcinoma cell lines in coculture with fibroblasts was blocked by antibodies against the transforming growth factor-alpha and amphiregulin. In addition, reverse transcription-polymerase chain reaction showed expression of mRNA for amphiregulin and transforming growth factor-alpha in all cell lines, whereas mRNA for the epidermal growth factor was detected only in pulmonary adenocarcinoma cell lines. Western blot analysis revealed that medium containing growth factors released by each fibroblast cell line activated extracellular signal-regulated kinase 1/2 in the both tested pulmonary adenocarcinoma cell lines, but activated Akt kinase only in A549 cells. Assessment of protein levels for cyclin D1 and cyclin E by Western blots demonstrated pronounced increases of both proteins in each pulmonary adenocarcinoma cell line, whereas protein levels for cyclin-dependent kinase inhibitor p21 remained unchanged. Immunocytochemical analysis showed positive immunoreactivity for P-extracellular signal-regulated kinase 1/2, cyclin D1 and cyclin E in pulmonary adenocarcinoma cells cocultured with fibroblasts or exposed to fibroblast-conditioned media. Our data suggest that the growth of pulmonary adenocarcinoma is stimulated by amphiregulin and transforming growth factor-alpha released from pulmonary fibroblasts. This may contribute to the disappointing clinical responses to anticancer drugs, which have shown promise in tests with lung cancer cell lines.

Topics: Adenocarcinoma; Amphiregulin; Animals; Antibodies, Blocking; Cell Line, Tumor; Cell Proliferation; Coculture Techniques; Culture Media, Conditioned; Cyclin D1; Cyclin E; EGF Family of Proteins; Enzyme Activation; Epidermal Growth Factor; Extracellular Signal-Regulated MAP Kinases; Fibroblasts; Glycoproteins; Immunohistochemistry; Intercellular Signaling Peptides and Proteins; Lung; Lung Neoplasms; Mice; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tetrazolium Salts; Thiazoles; Transforming Growth Factor alpha

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Cetuximab; Epidermal Growth Factor; ErbB Receptors; Humans; Lung Neoplasms

Low incidence of cancer in schizophrenia is one of the interesting puzzles in psychiatric field over decades. Analysis of genetic difference between schizophrenia and lung cancer might provide us with possible clues to understand molecular mechanisms of pathophysiology of schizophrenia. Epidermal growth factor (EGF), one of the potent growth promoting factors, has been studied for its roles in cancer development. EGF is also known to be involved in cognitive function. In order to analyze the genetic difference between schizophrenia and lung cancer, polymorphism of EGF gene was studied from 174 schizophrenia patients, 122 lung cancer patients and 132 controls in Korean population. Genotype frequency analysis of EGF gene (AluI restriction site, 5'-UTR, rs4444903) in the EGF gene was studied. The genotype and allele frequencies of the AluI polymorphism showed significant differences between schizophrenia and lung cancer patients [p<0.0001; p<0.0001, odds ratio (95% CI), 0.3690 (0.2600-0.5236)]. When compared with controls, schizophrenia patients showed no significant differences from controls in genotype and allele frequencies [p=0.5151; p=0.3516, odds ratio (95% CI), 0.8589 (0.6235-1.1830)]. However, lung cancer patients showed significant differences from controls in genotype and allele frequencies [p<0.0001; p<0.0001, odds ratio (95% CI), 2.3275 (1.6082-3.3687)]. These results indicate that schizophrenia is not associated with AluI polymorphism of EGF gene and EGF gene polymorphism is different between schizophrenia and lung cancer patients.

Topics: 5' Untranslated Regions; Adult; Case-Control Studies; Epidermal Growth Factor; Female; Gene Frequency; Genetic Predisposition to Disease; Genotype; Humans; Korea; Lung Neoplasms; Male; Middle Aged; Odds Ratio; Polymorphism, Restriction Fragment Length; Schizophrenia

The epidermal growth factor receptor (EGFR) overexpressed in approximately 80% of non-small cell lung cancers (NSCLC) is a target for novel therapeutics. Concurrent chemoradiation is the current standard of care for treatment of patients with locally advanced NSCLC. However, < 20% of patients remain disease-free at 5 years despite this aggressive treatment. Cetuximab is a humanized monoclonal antibody that recognizes the human EGFR, and in previous studies, inhibited the growth of EGFR-expressing human cancer cell lines. In this report, we investigated the cellular and molecular effects of cetuximab alone and in combination with radiation and/or chemotherapy in human NSCLC cell lines with varying levels of EGFR overexpression in vitro and in vivo.. We evaluated the EGFR status of a panel of human NSCLC cancer cell lines by immunohistochemistry and flow cytometry. We then evaluated cetuximab effects on growth, cell cycle distribution, and downstream intracellular signaling molecules in this panel of NSCLC cancer cell lines. NSCLC cell lines were treated with cetuximab alone or in combination with radiation, chemotherapy, or chemoradiation to determine the cooperative effects of cetuximab both in vitro and in vivo in athymic nude mice bearing NSCLC xenografts.. Cetuximab alone inhibited the in vitro growth of some but not all EGFR-expressing NSCLC cell lines in a dose-dependent manner. Flow cytometric analysis of cell cycle distribution after 24 hours of cetuximab treatment revealed a shift into the G(0)/G(1) phase of the cell cycle in cetuximab-sensitive EGFR-expressing cell lines and at concentrations that were growth-inhibitory. There were no cell cycle changes in the EGFR-negative cell lines. After 4 hours of exposure, cetuximab reduced epidermal growth factor (EGF)-induced phosphorylation of EGFR (pEGFR) and HER-2 (pHER2) in cetuximab-sensitive cell lines but not in cetuximab-resistant cell lines. Cetuximab reduced EGF-induced phosphorylation of extracellular signal-regulated kinase-1/2 (pERK) in all EGFR-expressing cell lines. In the absence of EGF, cetuximab alone increased the level of pEGFR and pHER2 above that seen in untreated control cells in both sensitive and resistant cell lines that were EGFR- and HER2-positive, but not in EGFR- or HER2-negative lines. Despite the cetuximab-induced increase in phosphorylation of EGFR and HER2, peak EGF-induced levels of pEGFR and pHER2 were reduced by cetuximab in the cetuximab-sensitive lines but not in the resistant lines. Cooperative (combination index values < 1.0) growth inhibitory effects were observed in vitro combination assays with cetuximab and radiation only in cetuximab-sensitive NSCLC cell lines. A lack of cooperation was seen in cetuximab-insensitive NSCLC cell lines. Similar findings were observed with in vitro combination studies of cetuximab plus cisplatin or paclitaxel. In nude mice bearing EGFR-expressing, cetuximab-sensitive, NSCLC cell line xenografts, cetuximab plus radiation induced a marked improvement in tumor growth inhibition over either agent alone. The growth inhibitory effects of cetuximab-radiation were similar to the growth inhibitory effects of concurrent chemoradiation. Triple combination therapy of cetuximab and chemoradiation yielded a nonsignificant advantage in tumor growth control over doublet combinations (cetuximab and radiation or chemoradiation) in vivo.. Similar results in tumor growth inhibition observed in mice treated with cetuximab-radiation and cisplatin-radiation provide a rationale for the clinical investigation of cetuximab with concurrent radiation in selected patients with locally advanced NSCLC. Local tumor control and treatment toxicity should be evaluated between cetuximab-radiation and chemoradiation regimens. Proper patient selection will be critical to the success of such trials and further studies are needed to identify optimal patient selection criteria.

Topics: Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Cell Proliferation; Cetuximab; Combined Modality Therapy; Drug Resistance, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Female; Flow Cytometry; G1 Phase; Humans; Immunoenzyme Techniques; Lung Neoplasms; Mice; Mice, Nude; Phosphorylation; Radiation, Ionizing; Receptor, ErbB-2; Resting Phase, Cell Cycle; Transplantation, Heterologous

Identifying new effective therapeutic treatments for lung cancer is critical to improving overall patient survival. We have targeted both the estrogen receptor (ER) and the epidermal growth factor receptor (EGFR) pathways using an ER antagonist, fulvestrant ("Faslodex"), and the selective EGFR tyrosine kinase inhibitor, gefitinib ("Iressa"), in non-small cell lung cancer (NSCLC) cells. Rapid activation of phospho-EGFR and phospho-p44/p42 mitogen-activated protein kinase by estrogen was observed, indicating nonnuclear ER transactivation of EGFR. Additionally, EGFR protein expression was down-regulated in response to estrogen and up-regulated in response to fulvestrant in vitro, suggesting that the EGFR pathway is activated when estrogen is depleted in NSCLC cells. Cell growth and apoptosis were examined in several NSCLC lines that express varying amounts of ERbeta, EGFR, and Neu but no full-length ERalpha. One cell line contained an EGFR mutation. Cells were exposed to 10 nmol/L estrogen and 10 ng/mL EGF and either 1 mumol/L fulvestrant or 1 mumol/L gefitinib alone or in combination. In all cell lines, the drug combination decreased cell proliferation up to 90% and increased apoptosis 2-fold. The relative responses to gefitinib and fulvestrant were similar regardless of ER and EGFR expression and mutation status. In an in vivo lung tumor xenograft model, the drug combination decreased tumor volume in severe combined immunodeficient mice by approximately 60% compared with 49% and 32% for gefitinib and fulvestrant treatment alone, respectively. Antitumor effects of the combination therapy were accompanied by biochemical and histologic evidence of increased apoptosis, decreased phospho-p44/p42 mitogen-activated protein kinase expression, and increased Ki-67 expression compared with individual treatment. These studies provide evidence of a functional interaction between the ER and the EGFR pathways in NSCLC.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Growth Processes; Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Estradiol; Female; Fulvestrant; Gefitinib; Humans; Lung Neoplasms; Male; Mice; Mice, SCID; Mitogen-Activated Protein Kinase 3; Quinazolines; Receptors, Estrogen; Xenograft Model Antitumor Assays

Gefitinib ("Iressa", ZD1839) is an orally active, selective epidermal growth factor receptor tyrosine kinase inhibitor, and the single agent is clinically effective in non-small cell lung cancer. Although gefitinib combined with various cytotoxic agents has been reported to enhance cytotoxicity in vitro and in mouse models, the mechanism remains undetermined. Here, to explore the mechanism with topoisomerase I inhibitors, we focused on the efflux pump of the breast cancer resistance protein (BCRP/ABCG2), and then examined whether gefitinib restored drug sensitivity in multidrug-resistant cancer cells overexpressing BCRP. We used PC-6 human small cell lung cancer cells and multidrug-resistant PC-6/SN2-5H cells selected with SN-38 of the active metabolite of irinotecan, and BCRP-overexpressing MCF-7/MX cells selected with mitoxantrone and BCRP cDNA transfectant MCF-7/clone 8 cells. Drug sensitivity against anticancer drugs was determined by tetrazolium dye assay, and intracellular topotecan accumulation by FACScan. The topotecan transport study was done using the plasma membrane vesicles of PC-6/SN2-5H cells. The resistant PC-6/SN2-5H cells overexpressed BCRP but not epidermal growth factor receptor mRNA. Ten micromoles of gefitinib reversed topotecan, SN-38, and mitoxantrone resistance, and increased the intracellular topotecan accumulation in the resistant cells but not in the parental cells. Furthermore, gefitinib inhibited the topotecan transport into the vesicles, and the K(i) value was 1.01 +/- 0.09 micromol/L in the Dixon plot analysis, indicating direct inhibition of BCRP by gefitinib. However, gefitinib was not transported into the vesicles with the high-performance liquid chromatography method. These results indicate that gefitinib reverses BCRP-mediated drug resistance by direct inhibition other than competitive inhibition as a BCRP substrate. Combination of gefitinib and topoisomerase I inhibitors could be clinically effective in cancers expressing BCRP.

Topics: Adenosine Triphosphate; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Biological Transport, Active; Breast Neoplasms; Camptothecin; Carcinoma, Small Cell; Cell Line, Tumor; Cell Membrane; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Epidermal Growth Factor; ErbB Receptors; Gefitinib; Humans; Inhibitory Concentration 50; Irinotecan; Lung Neoplasms; Mitoxantrone; Neoplasm Proteins; Protein Kinase Inhibitors; Quinazolines; Topotecan

Estrogen receptor (ER) agonists and antagonists elicit distinct responses in non-small cell lung cancer (NSCLC) cells. To determine how such responses are generated, the expression of ERalpha, ERbeta, and ER coregulators in human lung fibroblasts and human NSCLC cell lines was evaluated by immunoblot. Ligand-dependent estrogenic responses in NSCLC cells are probably generated via ERbeta and the p160 coactivator GRIP1/TIF2, because expression of these proteins was detected, but not full-length ERalpha or the p160 coactivator SRC-1. ERbeta and GRIP1/TIF2 are shown to interact in vitro in a ligand-dependent manner and thus may form functional transcription complexes in NSCLC cells. Furthermore, the capacity of ER ligands to regulate gene expression in NSCLC cells was explored using gene miniarrays. Expression profiles were examined after treatment with ER agonist 17-beta-estradiol (E2), the pure ER antagonist ICI 182,780 (fulvestrant, Faslodex), or epidermal growth factor, which served as a positive control for an alternative growth stimulus. E-cadherin and inhibitor of differentiation 2 were differentially regulated by E2 versus ICI 182,780 in 201T and 273T NSCLC cell lines. Epidermal growth factor also stimulated proliferation of these cells but had no effect on expression of E-cadherin and inhibitor of differentiation 2, suggesting they are specific targets of ER signaling. These data show that NSCLC cells respond to estrogens/antiestrogens by altering endogenous gene expression and support a model in which ICI 182,780 reduces proliferation of NSCLC cells via its ability to disrupt ER signaling. ICI 182,780 may therefore have therapeutic benefit in NSCLC.

Topics: Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Epidermal Growth Factor; Estradiol; Estrogen Receptor alpha; Estrogen Receptor beta; Estrogen Receptor Modulators; Fulvestrant; Gene Expression Regulation, Neoplastic; Humans; Ligands; Lung Neoplasms; Nuclear Receptor Coactivator 2; Receptors, Estrogen; Transcription Factors

The epidermal growth factor receptor (EGFR) is a key regulator of growth, differentiation, and survival of epithelial cancers. In a small subset of tumors, the presence of activating mutations within the ATP binding site confers increased susceptibility to gefitinib, a potent tyrosine kinase inhibitor of EGFR. Agents that can inhibit EGFR function through different mechanisms may enhance gefitinib activity in patients lacking these mutations. Mevalonate metabolites play significant roles in the function of the EGFR; therefore, mevalonate pathway inhibitors may potentiate EGFR-targeted therapies.. In this study, we evaluated the effect of lovastatin on EGFR function and on gefitinib activity. Effects on EGFR function were analyzed by Western blot analysis using phosphospecific antibodies to EGFR, AKT, and extracellular signal-regulated kinase. Cytotoxic effects of lovastatin and/or gefitinib were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and flow cytometry.. Lovastatin treatment inhibited EGF-induced EGFR autophosphorylation by 24 hours that was reversed by the coadministration of mevalonate. Combining lovastatin and gefitinib treatments showed enhanced inhibition of AKT activation by EGF in SCC9 cells. The combination of 10 mumol/L lovastatin and 10 mumol/L gefitinib treatments showed cooperative cytotoxicity in all 8 squamous cell carcinomas, 4 of 4 non-small cell lung carcinoma and 4 of 4 colon carcinoma cell lines tested. Isobologram and flow cytometric analyses of three representative cell lines with wild-type EGFR ATP binding sites confirmed that this combination was synergistic inducing a potent apoptotic response.. Taken together, these results show that targeting the mevalonate pathway can inhibit EGFR function. They also suggest the potential utility of combining these clinically relevant therapeutic approaches.

Topics: Adenosine Triphosphate; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Binding Sites; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Colonic Neoplasms; Drug Synergism; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Gefitinib; Humans; Lovastatin; Lung Neoplasms; Mevalonic Acid; Mitogen-Activated Protein Kinases; Mutation; Phosphorylation; Quinazolines; Signal Transduction; Tumor Cells, Cultured

Non-small cell lung cancer (NSCLC) expresses a particularly aggressive metastatic phenotype, and patients with this disease have a poor prognosis. CXC chemokine receptor 4 (CXCR4) is a cell surface receptor that has been shown to mediate the metastasis of many solid tumors including lung, breast, kidney, and prostate. In addition, overexpression of the epidermal growth factor receptor (EGFR) is associated with the majority of NSCLC and has been implicated in the process of malignant transformation by promoting cell proliferation, cell survival, and motility. Here we show for the first time that activation of the EGFR by EGF increases CXCR4 expression and the migratory capacity of NSCLC cells. Furthermore, many solid tumors are associated with low oxygen tension, and when NSCLC cells were cultured with EGF under hypoxic conditions, CXCR4 expression was dramatically enhanced. A molecular analysis of these events indicated that augmented CXCR4 expression was regulated by the phosphatidylinositol 3-kinase/PTEN/AKT/mammalian target of rapamycin signal transduction pathway, activation of hypoxia inducible factor (HIF) 1alpha, and ultimately HIF-1-dependent transcription of the CXCR4 gene. Thus, a combination of low oxygen tension and overexpression of EGFR within the primary tumor of NSCLC may provide the microenvironmental signals necessary to upregulate CXCR4 expression and promote metastasis.

Topics: Blotting, Western; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Cell Separation; Cell Survival; Chemokine CXCL12; Chemokines, CXC; Chemotaxis; Dose-Response Relationship, Drug; Epidermal Growth Factor; Flow Cytometry; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Lung Neoplasms; Neoplasm Metastasis; Oxygen; Phosphatidylinositol 3-Kinases; Phosphoric Monoester Hydrolases; Promoter Regions, Genetic; Protein Kinases; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Receptors, CXCR4; RNA, Messenger; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Transcription Factors; Transcription, Genetic; Transcriptional Activation; Transfection; Tumor Suppressor Proteins; Up-Regulation

Topics: Adenocarcinoma; Antineoplastic Agents; Epidermal Growth Factor; ErbB Receptors; Gefitinib; Humans; Ligands; Lung Neoplasms; Mutation, Missense; Quinazolines; Sequence Deletion; Stromal Cells; Transforming Growth Factor alpha

Sialyl-Lewis x epitopes and MUC5AC protein are known to be overexpressed in mucins secreted by patients suffering from various respiratory diseases. To investigate the mechanisms by which airway inflammatory agents mediate the expression of sialyl-Lewis x epitopes and MUC5AC mucin, we examined the effects of tumor necrosis factor (TNF)-alpha and epidermal growth factor (EGF) in the human lung carcinoma cell line, NCI-H292. Basal expression levels of hST3GalIV, FUT3 and C2/4GnT mRNA, involved in the biosynthesis of sialyl-Lewis x, were higher than those of other glycosyltransferases in NCI-H292 cells. TNF-alpha induced expression of hST3GalIV, FUT3, C2/4GnT and MUC5AC mRNAs in NCI-H292 cells. When cells were pretreated with U73122, a phosphatidylinositol-phospholipase C (PI-PLC) inhibitor, the expression of these glycosyltransferase mRNAs was suppressed. Treating cells with EGF induced the down-regulation of these glycosyltransferase mRNAs and sialyl-Lewis x epitopes, while inducing an increase in expression of MUC5AC mRNA. These EGF-mediated effects on the glycosyltransferase and MUC5AC mRNAs were blocked when cells were first exposed to AG1478, an EGF receptor tyrosine kinase inhibitor. These findings suggest that the expression of sialyl-Lewis x epitopes, which is regulated separately from the expression of MUC5AC protein, may be controlled through pathways such as the EGF receptor tyrosine kinase and PI-PLC signaling cascades in NCI-H292 cells.

Topics: Cell Line, Tumor; Epidermal Growth Factor; Epitopes; ErbB Receptors; Gene Expression Regulation; Humans; Lung Neoplasms; Mucin 5AC; Mucins; Oligosaccharides; Phosphatidylinositol Diacylglycerol-Lyase; Phosphoinositide Phospholipase C; RNA, Messenger; Sialyl Lewis X Antigen; Tumor Necrosis Factor-alpha

Inflammation is commonly associated with lung tumors. Since inflammatory mediators, including members of the interleukin-6 (IL-6) cytokine family, suppress proliferation of normal epithelial cells, we hypothesized that epithelial cells must develop mechanisms to evade this inhibition during the tumorigenesis. This study compared the cytokine responses of normal epithelial cells to that of premalignant cells.. Short-term primary cultures of epithelial cells were established from bronchial brushings. Paired sets of brushings were obtained from areas of normal bronchial epithelium and from areas of metaplastic or dysplastic epithelium, or areas of frank endobronchial carcinoma. In 43 paired cultures, the signalling through the signal transducer and activator of transcription (STAT) and extracellular regulated kinase (ERK) pathways and growth regulation by IL-6, leukemia inhibitory factor (LIF), oncostatin M (OSM), interferon-gamma (IFNgamma) or epidermal growth factor (EGF) were determined. Inducible expression and function of the leukemia inhibitory factor receptor was assessed by treatment with the histone deacetylase inhibitor depsipeptide.. Normal epithelial cells respond strongly to OSM, IFNgamma and EGF, and respond moderately to IL-6, and do not exhibit a detectable response to LIF. In preneoplastic cells, the aberrant signaling that was detected most frequently was an elevated activation of ERK, a reduced or increased IL-6 and EGF response, and an increased LIF response. Some of these changes in preneoplastic cell signaling approach those observed in established lung cancer cell lines. Epigenetic control of LIF receptor expression by histone acetylation can account for the gain of LIF responsiveness. OSM and macrophage-derived cytokines suppressed proliferation of normal epithelial cells, but reduced inhibition or even stimulated proliferation was noted for preneoplastic cells. These alterations likely contribute to the supporting effects that inflammation has on lung tumor progression.. This study indicates that during the earliest stage of premalignant transformation, a modified response to cytokines and EGF is evident. Some of the altered cytokine responses in primary premalignant cells are comparable to those seen in established lung cancer cell lines.

Topics: Cell Line, Transformed; Cell Line, Tumor; Cell Transformation, Neoplastic; Cells, Cultured; Cytokines; Densitometry; Epidermal Growth Factor; Epithelial Cells; Extracellular Signal-Regulated MAP Kinases; Fibroblasts; Histones; Humans; Inflammation; Interferon-gamma; Interleukin-6; Lung; Lung Neoplasms; Macrophages; Oncostatin M; Signal Transduction; Time Factors; Treatment Outcome

The development of several targeted agents that play a critical role in the growth and survival of carcinomas has paved the way for a new era in the treatment of patients with thoracic malignancies. The novel antimetabolite pemetrexed has emerged as a key agent in the treatment of advanced malignant pleural mesothelioma (MPM). Inhibitors of the epidermal growth factor receptor (EGFR) are one of many promising targeted therapies for non-small-cell lung cancer (NSCLC). By utilizing agents that specifically target the biochemical and molecular changes underlying cancer, it is possible to envision a future in which combinations of therapies treat cancer on multiple fronts, significantly enhancing tumor responses and improving survival beyond current expectations.

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Combined Chemotherapy Protocols; Bevacizumab; Carcinoma, Non-Small-Cell Lung; Clinical Trials as Topic; Epidermal Growth Factor; Humans; Lung Neoplasms; Thoracic Neoplasms

Gefitinib (Iressa(TM), ZD1839) is an orally active, selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor. Phase I studies showed that it is well tolerated, with evidence of tumor regression in patients with advanced non-small-cell lung cancer (NSCLC). Therefore, we aimed to assess the antitumor activity and tolerability of gefitinib in a series of patients with previously treated, advanced NSCLC, as a part of a compassionate use program.. To be eligible, all patients were required to have histologically or cytologically proven advanced or metastatic NSCLC, prior chemotherapy with at least one cisplatin-containing chemotherapy regimen or contraindication to cytotoxic drugs, Eastern Cooperative Oncology Group performance status < or =2, and adequate hematological, renal and hepatic parameters. All patients provided signed informed consent. Patient re-evaluation was performed every 4-6 weeks.. Seventy-three consecutive patients were enrolled. Response rate, including complete and partial response, was 9.6%; an additional 43.8% of patients achieved stable disease, for an overall disease control of 53.4%. EGFR1 status was evaluated by immunocytochemistry in 25 patients. According to EGFR1 immunoreactivity all responses were observed with medium/strong imunoreactivity while three out of four responses were observed in high expressive patients. Median survival for all patients was 4 months while it reached 6 months for patients with disease control. The 1-year survival rate was 13.1% for the entire series and 23.2% for patients with disease control. Non-hematological toxicity was generally mild.. Gefitinib has promising activity with a good toxicity profile in patients with progressive NSCLC who have received one or two prior chemotherapy regimens. A possible relationship within response and EGFR1 expression is suggested.

Topics: Adenocarcinoma; Adult; Aged; Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Disease Progression; Epidermal Growth Factor; ErbB Receptors; Female; Gefitinib; Humans; Immunohistochemistry; Lung Neoplasms; Male; Middle Aged; Neoplasm Recurrence, Local; Protein-Tyrosine Kinases; Quinazolines; Survival Analysis

Topics: Antibodies, Monoclonal; Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Clinical Trials as Topic; Epidermal Growth Factor; ErbB Receptors; Erlotinib Hydrochloride; Gefitinib; Humans; Lung Neoplasms; Protein-Tyrosine Kinases; Quinazolines

Gefitinib, an inhibitor of the epidermal growth factor receptor tyrosine kinase, induces radiographic regressions and symptomatic improvement in patients with non-small-cell lung cancer (NSCLC). Phase II trials suggested female sex and adenocarcinoma were associated with response. We undertook this analysis to identify additional clinical and pathologic features associated with sensitivity to gefitinib.. We reviewed medical records, pathologic material, and imaging studies of all 139 NSCLC patients treated on one of three consecutive studies of gefitinib monotherapy performed at our institution. We identified patients experiencing a major objective response and compared their clinical and pathologic features with the others. Univariate and multivariable analyses were performed on potential predictive features associated with sensitivity to gefitinib.. Of 139 patients, 21 (15%; 95% CI, 9% to 21%), experienced a partial radiographic response. Variables identified as significant in univariate analysis included adenocarcinoma versus other NSCLC (19% v 0%; P=.004), adenocarcinoma with bronchioloalveolar features versus other adenocarcinomas (38% v 14%; P<.001), never smoker status versus former/current (36% v 8%; P<.001), and Karnofsky performance status > or =80% versus < or =70% (22% v 8%; P=.03). Multivariable analysis revealed the presence of adenocarcinoma with any bronchioloalveolar features (P=.004) and being a never smoker (P=.006) were independent predictors of response.. Our data suggest that individuals in whom gefitinib is efficacious are more likely to have adenocarcinomas of the bronchioloalveolar subtype and to be never smokers. These observations may provide clues to mechanisms determining sensitivity to this agent and suggest that NSCLC has a different biology in patients who never smoked and those with bronchioloalveolar carcinoma.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Agents; Bronchoalveolar Lavage Fluid; Carcinoma, Bronchogenic; Carcinoma, Non-Small-Cell Lung; Epidermal Growth Factor; Female; Gefitinib; Humans; Logistic Models; Lung Neoplasms; Male; Middle Aged; Multivariate Analysis; Protein-Tyrosine Kinases; Quinazolines; Retrospective Studies; Smoking

Cannabinoids, the active components of marijuana and their endogenous counterparts were reported as useful analgetic agents to accompany primary cancer treatment by preventing nausea, vomiting, and pain and by stimulating appetite. Moreover, they have been shown to inhibit cell growth and to induce apoptosis in tumor cells. Here, we demonstrate that anandamide, Delta(9)-tetrahydrocannabinol (THC), HU-210, and Win55,212-2 promote mitogenic kinase signaling in cancer cells. Treatment of the glioblastoma cell line U373-MG and the lung carcinoma cell line NCI-H292 with nanomolar concentrations of THC led to accelerated cell proliferation that was completely dependent on metalloprotease and epidermal growth factor receptor (EGFR) activity. EGFR signal transactivation was identified as the mechanistic link between cannabinoid receptors and the activation of the mitogen-activated protein kinases extracellular signal-regulated kinase 1/2 as well as prosurvival protein kinase B (Akt/PKB) signaling. Depending on the cellular context, signal cross-communication was mediated by shedding of proAmphiregulin (proAR) and/or proHeparin-binding epidermal growth factor-like growth factor (proHB-EGF) by tumor necrosis factor alpha converting enzyme (TACE/ADAM17). Taken together, our data show that concentrations of THC comparable with those detected in the serum of patients after THC administration accelerate proliferation of cancer cells instead of apoptosis and thereby contribute to cancer progression in patients.

Topics: ADAM Proteins; ADAM17 Protein; Amphiregulin; Apoptosis; Benzoxazines; Cannabinoids; Cell Division; Dronabinol; EGF Family of Proteins; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Glioblastoma; Glycoproteins; Heparin-binding EGF-like Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Lung Neoplasms; Metalloendopeptidases; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Morpholines; Naphthalenes; Phosphorylation; Proto-Oncogene Proteins; RNA Interference; Signal Transduction; Transcriptional Activation; Tumor Cells, Cultured

We report the cutaneous side effects of Iressa (ZD1839), a new anti-cancer agent that acts by inhibiting epidermal growth factor receptor signal transduction. The most common cutaneous adverse effect was the development of an acneiform eruption on the face, anterior trunk and back (39%). The second most common side effect was xerosis or desquamation of the face, body or distal parts of the fingers or toes (36%). Additional cutaneous side effects included multiple ingrown paronychial inflammation of the toes and fingers (6%), small ulcers of the oral mucosa or nasal mucosa, and urticaria. The cutaneous adverse effects of Iressa are similar to those of other epidermal growth factor receptor-targeted agents and result from direct interference with the functions of epidermal growth factor receptor signalling in the skin. Iressa-induced acne may be related to excessive follicular hyperkeratosis, follicular plugging, obstructions of the follicular ostium and alteration of hair cycle progression, which lead to an inflammatory response. Xerosis or desquamation reflects a disturbance of the equilibrium between proliferation and differentiation of epidermis. The mechanism by which Iressa leads to the development of paronychia and ingrown nail remains unclear.

Topics: Acneiform Eruptions; Adult; Aged; Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Enzyme Inhibitors; Epidermal Growth Factor; Female; Gefitinib; Humans; Lung Neoplasms; Male; Middle Aged; Paronychia; Quinazolines; Retrospective Studies; Signal Transduction; Skin; Urticaria

Gefitinib is approved as monotherapy for the treatment of locally advanced or metastatic non-small cell lung cancer (NSCLC) in patients who have failed prior platinum-based treatment and docetaxel chemotherapy. Its efficacy when compared to standard treatment and best supportive care is unproven. Common drug-related adverse effects include gastrointestinal and skin disorders. Rare but serious drug-related adverse events, such as shock, thrombophlebitis, thrombocytopenia and interstitial lung disease, have been reported.

Topics: Antineoplastic Agents; Australia; Canada; Carcinoma, Non-Small-Cell Lung; Drug Approval; Drug Costs; Epidermal Growth Factor; Humans; Japan; Lung Neoplasms; Neoplasm Staging; Protein-Tyrosine Kinases; Quinazolines; Randomized Controlled Trials as Topic; Treatment Outcome; United States

Most patients with non-small-cell lung cancer have no response to the tyrosine kinase inhibitor gefitinib, which targets the epidermal growth factor receptor (EGFR). However, about 10 percent of patients have a rapid and often dramatic clinical response. The molecular mechanisms underlying sensitivity to gefitinib are unknown.. We searched for mutations in the EGFR gene in primary tumors from patients with non-small-cell lung cancer who had a response to gefitinib, those who did not have a response, and those who had not been exposed to gefitinib. The functional consequences of identified mutations were evaluated after the mutant proteins were expressed in cultured cells.. Somatic mutations were identified in the tyrosine kinase domain of the EGFR gene in eight of nine patients with gefitinib-responsive lung cancer, as compared with none of the seven patients with no response (P<0.001). Mutations were either small, in-frame deletions or amino acid substitutions clustered around the ATP-binding pocket of the tyrosine kinase domain. Similar mutations were detected in tumors from 2 of 25 patients with primary non-small-cell lung cancer who had not been exposed to gefitinib (8 percent). All mutations were heterozygous, and identical mutations were observed in multiple patients, suggesting an additive specific gain of function. In vitro, EGFR mutants demonstrated enhanced tyrosine kinase activity in response to epidermal growth factor and increased sensitivity to inhibition by gefitinib.. A subgroup of patients with non-small-cell lung cancer have specific mutations in the EGFR gene, which correlate with clinical responsiveness to the tyrosine kinase inhibitor gefitinib. These mutations lead to increased growth factor signaling and confer susceptibility to the inhibitor. Screening for such mutations in lung cancers may identify patients who will have a response to gefitinib.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Amino Acid Sequence; Antineoplastic Agents; Base Sequence; Carcinoma, Non-Small-Cell Lung; DNA Mutational Analysis; Drug Resistance, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Female; Gefitinib; Genes, erbB-1; Heterozygote; Humans; Lung Neoplasms; Male; Middle Aged; Molecular Sequence Data; Mutation; Protein-Tyrosine Kinases; Quinazolines; Sequence Deletion

Topics: Adenocarcinoma; Amino Acid Substitution; Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Controlled Clinical Trials as Topic; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Gefitinib; Genes, erbB-1; Humans; Japan; Ligands; Lung Neoplasms; Mutation; Phosphorylation; Protein Structure, Tertiary; Quinazolines; Sequence Deletion; Smoking; Treatment Outcome; United States

Gefitinib ('Iressa', ZD1839) is an orally active epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor that has demonstrated antitumour activity and favourable tolerability in Phase II studies. We investigated whether EGFR expression levels could predict for response to gefitinib in patients with advanced non-small-cell lung cancer (NSCLC), who received gefitinib (250 mg day(-1)) as part of a worldwide compassionate-use programme. Tissue samples were analysed by immunohistochemistry to assess membrane EGFR immunoreactivity. Of 147 patients enrolled in our institution, 50 patients were evaluable for assessment of both clinical response and EGFR expression. The objective tumour response rate was 10% and disease control was achieved in 50% of patients. Although high EGFR expression was more common in squamous-cell carcinomas than adenocarcinomas, all objective responses were observed in patients with adenocarcinoma. Response and disease control with gefitinib were not associated with high EGFR expression. Overall, median survival was 4 months, and the 1-year survival rate was 18%. Strong EGFR staining correlated with shorter survival time for all patients. Gefitinib demonstrated promising clinical activity in this group of patients with NSCLC. These results have also shown that EGFR expression is not a significant predictive factor for response to gefitinib.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Cell Membrane; Epidermal Growth Factor; ErbB Receptors; Female; Gefitinib; Humans; Immunoenzyme Techniques; Lung Neoplasms; Male; Middle Aged; Predictive Value of Tests; Prognosis; Protein-Tyrosine Kinases; Quinazolines; Survival Rate

Topics: Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Female; Gefitinib; Humans; Lung Neoplasms; Mutation; Pharmacogenetics; Quinazolines; Randomized Controlled Trials as Topic

This work was motivated by the need to find surrogate endpoints for survival of patients in oncology studies. The goal of this article is to determine associations between five time-to-event outcomes coming from three clinical trials for non-small cell lung cancer. To this end, we propose to use the multivariate Dale model for time-to-event data introduced by Tibaldi et al. (Stat. Med. 2003). We fit the model to these data, using a pseudo-likelihood approach to estimate the model parameters. We evaluate and compare the performance of different dimensional models and we relate the Dale model association parameter, i.e. the odds ratio, to well-known quantities such as Kendall's tau and Spearman's rho. Finally, the results are discussed with a perspective on surrogate marker validation. Some suggestions are made regarding further studies in this field.

Topics: Adjuvants, Immunologic; Biomarkers; Cancer Vaccines; Carcinoma, Non-Small-Cell Lung; Epidermal Growth Factor; Humans; Likelihood Functions; Lung Neoplasms; Middle Aged; Models, Immunological; Models, Statistical; Multivariate Analysis; Pilot Projects; Randomized Controlled Trials as Topic; Survival Analysis

Targeting the tumor vasculature may offer an alternative or complementary therapeutic approach to targeting growth factor signaling in lung cancer. The aim of these studies was to evaluate the antitumor effects in vivo of the combination of ZD6126, a tumor-selective vascular-targeting agent; ZD1839 (gefitinib, Iressa), an epidermal growth factor receptor tyrosine kinase inhibitor; and ionizing radiation in the treatment of non-small cell lung cancer xenograft model.. Athymic nude mice with established flank A549 human non-small cell lung cancer xenograft model xenografts were treated with fractionated radiation therapy, ZD6126, ZD1839, or combinations of each treatment. ZD6126 (150 mg/kg) was given i.p. the day after each course of radiation. Animals treated with ZD1839 received 100 mg/kg per dose per animal, 5 or 7 days/wk for 2 weeks. Immunohistochemistry was done to evaluate the effects on tumor growth using an anti-Ki67 monoclonal antibody. Effects on tumor-induced vascularization were quantified using an anti-factor VIII-related antigen monoclonal antibody.. ZD6126 attenuated the growth of human A549 flank xenografts compared with untreated animals. Marked antitumor effects were observed when animals were treated with a combination of ZD6126 and fractionated radiation therapy with protracted tumor regression. ZD6126 + ZD1839 resulted in a greater tumor growth delay than either agent alone. Similar additive effects were seen with ZD1839 + fractionated radiation. Finally, the addition of ZD6126 to ZD1839 and radiation therapy seemed to further improve tumor growth control, with a significant tumor growth delay compared with animals treated with single agent or with double combinations. Immunohistochemistry showed that ZD1839 induced a marked reduction in A549 tumor cell proliferation. Both ZD1839 and ZD6126 treatment substantially reduced tumor-induced angiogenesis. ZD6126 caused marked vessel destruction through loss of endothelial cells and thrombosis, substantially increasing the level of necrosis seen when combined with radiation therapy. The combination of radiation therapy, ZD6126, and ZD1839 induced the greatest effects on tumor growth and angiogenesis.. This first report shows that a selective vascular-targeting agent (ZD6126) + an anti-epidermal growth factor receptor agent (ZD1839) and radiation have additive in vivo effects in a human cancer model. Targeting the tumor vasculature offers an excellent strategy to enhance radiation cytotoxicity. Polytargeted therapy with agents that interfere with both growth factor and angiogenic signaling warrants further investigation.

Topics: Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Endothelium, Vascular; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Female; Gefitinib; Immunohistochemistry; Ki-67 Antigen; Lung Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Necrosis; Neoplasm Transplantation; Neovascularization, Pathologic; Organophosphorus Compounds; Protein Kinase Inhibitors; Quinazolines; Signal Transduction; Time Factors

Topics: Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Combined Modality Therapy; Early Diagnosis; Epidermal Growth Factor; Humans; Lung Neoplasms; Neoplasm Staging; Radiotherapy Dosage

Pulmonary metastases frequently develop in patients with aggressive bladder cancer, yet investigation of this process at the molecular level suffers from the poor availability of human metastatic tumor tissue and the absence of suitable animal models. To address this, we developed progressively more metastatic human bladder cancer cell lines and an in vivo bladder-cancer lung-metastasis model, and we successfully used these to identify genes of which the expression levels change according to the degree of pulmonary metastatic potential. By initially intravenously injecting the poorly metastatic T24T human urothelial cancer cells into nude mice, and then serially reintroducing and reisolating the human tumor cells from the resultant mouse lung tumors, three derivative human lines with increasingly metastatic phenotypes, designated FL1, FL2, and FL3, were sequentially isolated. To identify the genes associated with the most lung-metastatic phenotype, the RNA complement from the parental and derivative cells was evaluated with oligonucleotide microarrays. In doing so, we found 121 genes to be progressively up-regulated during the transition from T24T to FL3, whereas 43 genes were progressively down-regulated. As expected, many of the genes identified in these groups could, according to the ascribed functions of their protein product, theoretically participate in tissue invasion and metastasis. In addition, the magnitude of gene expression changes observed during the metastatic transition correlated with the in vivo propensity for earlier lung colonization and decreased host survival. To additionally define which genes found in the experimental system were of relevance to human bladder cancer lung metastasis, we evaluated gene expression profiles of 23 primary human bladder tumors of various stages and grades, and then we compared these gene expression profiles to the altered profiles in our model cell lines. Here we found that the expression of epiregulin, urokinase-type plasminogen activator (uPA), matrix metalloproteinase (MMP)14, and tissue inhibitor of metalloproteinase (TIMP-2) were consistently and progressively up-regulated when viewed as a function of tumor stage in tissues of patients versus the metastatic potential seen in the mouse lung model. The strong correlation of these four markers between the experimental and clinical situations helps validate this system as a useful tool for the study of lung metastasis and defines targets of therapy that

Topics: Animals; Cell Line, Tumor; Epidermal Growth Factor; Epiregulin; Female; Gene Expression Profiling; Humans; Lung Neoplasms; Mice; Phenotype; Tissue Inhibitor of Metalloproteinase-2; Urinary Bladder Neoplasms

Topics: Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; DNA Mutational Analysis; Epidermal Growth Factor; ErbB Receptors; Gefitinib; Humans; Lung Neoplasms; Protein-Tyrosine Kinases; Quinazolines; Receptor, ErbB-2

Topics: Adult; Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Epidermal Growth Factor; Female; Gefitinib; Humans; Hyperpigmentation; Lung Neoplasms; Middle Aged; Paronychia; Protein-Tyrosine Kinases; Quinazolines

Topics: Antineoplastic Agents; Apoptosis; Blotting, Western; Carcinoma, Non-Small-Cell Lung; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Humans; Lung Neoplasms; Phosphorylation; Precipitin Tests; Protein Tyrosine Phosphatases; Quinazolines; Signal Transduction; Tumor Cells, Cultured; Tyrphostins

Topics: Carcinoma, Non-Small-Cell Lung; Epidermal Growth Factor; Erythema; Female; Gefitinib; Humans; Lung Neoplasms; Middle Aged; Protein-Tyrosine Kinases; Quinazolines

We describe the case of a 52-year-old Japanese woman with advanced adenocarcinoma of the lung, in whom once-daily treatment with 250 mg ZD1839 ('Iressa') demonstrated a marked antitumour effect. She had initially achieved a partial response with cisplatin-based combination chemotherapy, but had subsequently progressed and had failed to respond to salvage chemotherapy. She had also received whole-brain irradiation for brain metastases. On admission, the patient was confined to bed due to dyspnoea and had rapidly progressing hypoxia secondary to lymphangitis carcinomatosa and a massive right pleural effusion. She was treated with oxygen supplementation and oral ZD1839, which, within a week, led to marked tumour regression and gradually improving dyspnoea. The main adverse event observed was a grade 2 rash. A month after starting ZD1839 treatment, the patient was discharged without the need for oxygen supplementation and had since returned to full-time work. This is a demonstration of ZD1839 producing a dramatic clinical response when administered to a patient with poor performance status who had received extensive prior treatment with cytotoxic agents.'Iressa' is a trademark of the AstraZeneca group of companies.

Topics: Adenocarcinoma; Antineoplastic Combined Chemotherapy Protocols; Camptothecin; Carcinoma, Non-Small-Cell Lung; Cisplatin; Docetaxel; Enzyme Inhibitors; Epidermal Growth Factor; Female; Gefitinib; Humans; Irinotecan; Lung Neoplasms; Middle Aged; Neoplasm Staging; Paclitaxel; Protein-Tyrosine Kinases; Quinazolines; Salvage Therapy; Taxoids; Treatment Outcome

Topics: Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Epidermal Growth Factor; Gefitinib; Germany; Lung Neoplasms; Protein-Tyrosine Kinases; Quinazolines

As previously described, SPC/myc transgenic mice developed bronchioloalveolar adenocarcinomas derived from alveolar type II (AT II) cells within 10-14 months, whereas SPC/IgEGF transgenic mice developed hyperplasias. Our purpose was to determine the potential interplay of environmental and genetic factors in lung tumorigenesis.. Six-week-old SPC/myc and SPC/IgEGF transgenic mice, overexpressing c-myc and a secretable form of the epidermal growth factor (IgEGF) under the control of the surfactant protein C (SPC) promoter, were treated with a single dose of the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). As control groups, SPC/myc and SPC/IgEGF transgenic mice were treated with NaCl and non-transgenic littermates were treated with NNK or NaCl, respectively.. After 6 months, none of the NaCl-treated transgenic littermates showed bronchioloalveolar hyperplasia and adenocarcinoma formation, whereas 100% of the NNK-treated SPC/myc transgenic mice did. The effect of NNK on SPC/IgEGF transgenic mice was less pronounced, inducing hyperplasia in the lung in only 16.7% of them. In 90% of the NNK-treated non-transgenic littermates no neoplastic changes were detected in the lung.. These results demonstrate that the progression of pulmonary bronchioloalveolar adenocarcinomas, induced by expression of c-myc as a transgene, was accelerated by NNK, suggesting that c-myc cooperates with NNK-induced mutations.

Topics: Adenocarcinoma, Bronchiolo-Alveolar; Animals; Carcinogens; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Hyperplasia; Intercellular Signaling Peptides and Proteins; Lung; Lung Neoplasms; Mice; Mice, Transgenic; Nitrosamines; Peptides; Proto-Oncogene Proteins c-myc; Pulmonary Surfactant-Associated Protein C; Pulmonary Surfactants

Topics: Animals; Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Clinical Trials, Phase II as Topic; Epidermal Growth Factor; Gefitinib; Humans; Lung Neoplasms; Quinazolines; Randomized Controlled Trials as Topic; Survival Rate

ZD1839 is an orally active inhibitor selective for the epidermal growth factor receptor tyrosine kinase and has shown promise in the treatment of non-small cell lung cancer (NSCLC). We now present a case of diffuse alveolar damage (DAD) that developed in a 67-year-old man treated with ZD1839. On day 8 of ZD1839 administration, the patient complained of dyspnea and a new-ground glass opacity was apparent on a chest X-ray and computed tomography scan. Despite high-dose steroid therapy, the patient died 13 days after the first administration of ZD1839. Postmortem analysis of lung tissue revealed a pattern of DAD. No evidence of infection or of other specific etiologies was apparent. This case is the first reported of respiratory failure after ZD1839 treatment in a patient with NSCLC. Physicians should therefore be aware of the potential pulmonary toxicity of ZD1839.

Topics: Administration, Oral; Aged; Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Epidermal Growth Factor; Fatal Outcome; Gefitinib; Humans; Lung Neoplasms; Male; Protein-Tyrosine Kinases; Pulmonary Alveoli; Quinazolines

Bronchioloalveolar carcinoma (BAC), a form of pulmonary adenocarcinoma, presents unique clinical features, such as endobronchial spread and bronchorrhea in advanced stages. The prognosis for BAC patients in advanced stages is poor, as is the case for patients with other non-small-cell lung cancer (NSCLC) types, because of low susceptibility to conventional chemotherapy. Recently, an orally active, selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (EGFR-TKI), ZD1839 ("Iressa"), has been investigated in phase II clinical studies (IDEAL 1 and IDEAL 2) as monotherapy against chemotherapy-refractory NSCLC, and provided clinically significant antitumor activity. In this study, we examined the therapeutic efficiency of ZD1839 in chemotherapy-refractory BAC patients with bronchorrhea. Two female BAC patients with bronchorrhea were treated once daily with ZD1839 (250 mg/day). In both cases, serous sputum production was dramatically reduced within 3 days of starting the treatment, and hypoxia and radiographic signs of bilateral lung consolidation were visibly improved within 7 days. Following more than 8 months of treatment, no evidence of recurrence or severe adverse events has been observed. These results suggest that this selective EGFR-TKI, ZD1839, may be a powerful agent for treatment of chemotherapy-refractory BAC patients with bronchorrhea.

Topics: Adenocarcinoma, Bronchiolo-Alveolar; Aged; Antineoplastic Agents; Drug Resistance, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Female; Gefitinib; Humans; Hypoxia; Lung Neoplasms; Middle Aged; Quinazolines; Radiography; Sputum

Overexpression of receptor tyrosine kinases including the epidermal growth factor receptor (EGF-R) as well as nonreceptor tyrosine kinases, such as Src, have been implicated in the formation of human lung cancers. In addition, cytokines like interleukin-6 (IL-6) have been demonstrated to modulate lung cancer cell growth and elevated levels of IL-6 have been shown to be an adverse prognostic factor for patients with lung cancer. Despite a large body of evidence pointing to their potential importance, few direct studies into the role of signal transducers and activators of transcription (STAT) pathways in human lung cancer have been undertaken. Here we demonstrate that multiple nonsmall cell lung cancer cell lines demonstrate constitutive Stat3 DNA-binding activity. Stat3 DNA-binding activity is specifically upregulated by the addition of epidermal growth factor (EGF), IL-6, and hepatocyte-derived growth factor (HGF). Furthermore, the stimulation of Stat3 DNA-binding activity by EGF requires the activity of EGF-R tyrosine kinase as well as Src-kinase, while the upregulation of Stat3 activity by IL-6 or HGF requires only Src-kinase activity. Treatment of A549 lung cancer cells with PD180970 or SU6656, both pharmacological inhibitors of Src-kinase, resulted in reduced Src and Stat3 activity, cell cycle arrest in G2, and reduced viability of cells accompanied by induction of apoptosis. Treatment of Stat3-positive A549 and H358 cells with antisense Stat3 oligonucleotides results in complete loss of Stat3 DNA-binding activity and apoptosis, while Stat3-positive H1299 cells remained healthy. Finally, an adenoviral vector expressing a dominant-negative Stat3 isoform results in loss of Stat3 DNA-binding activity, apoptosis, and reduced cellular viability. These results demonstrate a role of Stat3 in transducing survival signals downstream of tyrosine kinases such as Src, EGF-R, and c-Met, as well as cytokines such as IL-6, in human nonsmall cell lung cancers.

Topics: Adenoviridae; Apoptosis; Blotting, Western; Carcinoma, Non-Small-Cell Lung; Cell Cycle; Cell Nucleus; Cell Survival; Cytokines; DNA; DNA-Binding Proteins; Dose-Response Relationship, Drug; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; G2 Phase; Genes, Dominant; Hepatocyte Growth Factor; Humans; Interleukin-6; Lung Neoplasms; Models, Biological; Oligonucleotides, Antisense; Protein Binding; Protein-Tyrosine Kinases; Receptor Protein-Tyrosine Kinases; STAT3 Transcription Factor; Time Factors; Trans-Activators; Transfection; Tumor Cells, Cultured

Topics: Administration, Oral; Aged; Carcinoma, Non-Small-Cell Lung; Chemotherapy, Adjuvant; Epidermal Growth Factor; Female; Follow-Up Studies; Gefitinib; Humans; Lung Neoplasms; Male; Pneumonectomy; Postoperative Care; Quinazolines; Risk Assessment; Sampling Studies; Wound Healing

ZD1839 ('Iressa') is an epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) that inhibits EGFR signaling. Emerging evidence indicates that ZD1839 has clinical potential in lung cancer, but very little is known about the molecular characteristics of lung cancers that may determine sensitivity to ZD1839. We examined a panel of 19 lung cancer cell lines to investigate possible association between ZD1839 sensitivity and histological type, expression level and constitutive phosphorylation of EGFR and K-ras gene status. Our results indicate that neither expression level nor constitutive activation status of EGFR seems to predict sensitivity to ZD1839. In addition, ZD1839 sensitivity was not associated with expression of human epidermal growth factor receptor-2 (HER-2), another member of this tyrosine kinase receptor family nor with co-expression of EGFR and HER-2. Finally, no correlation was found between the presence of activating mutations of the K-ras gene, an important downstream mediator of the EGFR-transduced signals and the relative resistance to ZD1839. These findings warrant future study to clarify how ZD1839 inhibits lung cancer cell growth and to find a useful marker for prediction of sensitivity to this novel and promising agent for the treatment of lung cancers.

Topics: Antineoplastic Agents; Drug Resistance, Neoplasm; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Gefitinib; Genes, ras; Humans; Lung Neoplasms; Phosphorylation; Protein-Tyrosine Kinases; Quinazolines; Receptor, ErbB-2; Tumor Cells, Cultured

TR3, also known as NGFI-B or nur77, is an immediate-early response gene and an orphan member of the steroid/thyroid/retinoid receptor superfamily. We previously reported that TR3 expression was induced by apoptotic stimuli and was required for their apoptotic effect in lung cancer cells. Here, we present evidence that TR3 was also induced by epidermal growth factor (EGF) and serum and was required for their mitogenic effect in lung cancer cells. Ectopic expression of TR3 in both H460 and Calu-6 lung cancer cell lines promoted their cell cycle progression and BrdU incorporation, while inhibition of TR3 expression by the small interfering RNA approach suppressed the mitogenic effect of EGF and serum. Analysis of TR3 mutants showed that both TR3 DNA binding and transactivation were required for its mitogenic effect. In contrast, they were dispensable for its apoptotic activity. Furthermore, confocal microscopy analysis demonstrated that TR3 functioned in the nucleus to induce cell proliferation, whereas it acted on mitochondria to induce apoptosis. In examining the signaling that regulates the mitogenic function of TR3, we observed that coexpression of constitutive-active MEKK1 inhibited TR3 transcriptional activity and TR3-induced proliferation. The inhibitory effect of MEKK1 was mediated through activation of Jun N-terminal kinase, which efficiently phosphorylated TR3, resulting in loss of its DNA binding. Together, our results demonstrate that TR3 is capable of inducing both proliferation and apoptosis in the same cells depending on the stimuli and its cellular localization.

Topics: Apoptosis; Base Sequence; Cell Line, Tumor; Culture Media; DNA, Neoplasm; Epidermal Growth Factor; Gene Expression; Humans; JNK Mitogen-Activated Protein Kinases; Lung Neoplasms; MAP Kinase Kinase Kinase 1; Mitogen-Activated Protein Kinases; Mitosis; Nuclear Receptor Subfamily 4, Group A, Member 1; Phosphorylation; Protein Serine-Threonine Kinases; Receptors, Steroid; Receptors, Thyroid Hormone; Subcellular Fractions; Transcriptional Activation

We described a 70 years old patient with pericardial effusion due to adenocarcinoma of the lung, in whom gefitinib, which is an oral selective inhibitor of the epidermal growth factor receptor of tyrosine kinase, demonstrated a marked antitumor effect. We recommend possible consideration of a treatment with gefitinib for female patients with pericarditis carcinomatosa due to lung adenocarcinoma, even if they have a poor performance status and are not indicated for other intensive therapy.

Topics: Adenocarcinoma; Aged; Antineoplastic Agents; Epidermal Growth Factor; Female; Gefitinib; Humans; Lung Neoplasms; Pericardial Effusion; Protein-Tyrosine Kinases; Quinazolines

Although the mechanism is unknown, infiltration anesthetics are believed to have membrane-stabilizing action. We report here that such a most commonly used anesthetic, lidocaine, effectively inhibited the invasive ability of human cancer (HT1080, HOS, and RPMI-7951) cells at concentrations used in surgical operations (5-20 mM). Ectodomain shedding of heparin-binding epidermal growth factor-like growth factor (HB-EGF) from the cell surface plays an important role in invasion by HT1080 cells. Lidocaine reduced the invasion ability of these cells by partly inhibiting the shedding of HB-EGF from the cell surface and modulation of intracellular Ca2+ concentration contributed to this action. The anesthetic action of lidocaine (sodium channel blocking ability) did not contribute to this anti-invasive action. In addition, lidocaine (5-30 mM), infiltrated around the inoculation site, inhibited pulmonary metastases of murine osteosarcoma (LM 8) cells in vivo. These data point to previously unrecognized beneficial actions of lidocaine and suggest that lidocaine might be an ideal infiltration anesthetic for surgical cancer operations.

Topics: Anesthetics, Local; Animals; Calcium; Cell Membrane; Cell Movement; Epidermal Growth Factor; ErbB Receptors; Heparin; Heparin-binding EGF-like Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Lidocaine; Lung Neoplasms; Mice; Mice, Inbred C3H; Neoplasm Invasiveness; Transcriptional Activation; Tumor Cells, Cultured

Malignant pleural mesothelioma (MPM) is a rare malignancy with no known curative modality. Approximately 70% of MPMs have high levels of expression of the epidermal growth factor receptor (EGFR), and a subset of cell lines derived from MPM patients express both EGFR and transforming growth factor alpha, suggesting an autocrine role for EGFR in MPM. We have determined the effects of EGFR inhibition in MPM cell lines in vitro, using four MPM cell lines derived from previously untreated patients with epithelial (H2461 and H2591), sarcomatoid (H2373), and biphasic (MSTO-211H) MPM. All four cell lines expressed EGFR at levels comparable with the non-small cell lung carcinoma (NSCLC) cell line A549, as shown by Western blot analysis. ZD1839 significantly inhibited epidermal growth factor-dependent cell signaling including phosphorylation of AKT and extracellular signal-regulated kinases 1 and 2 in all MPM cell lines. Furthermore, treatment with ZD1839 led to a significant dose-dependent reduction of colony formation (41-89% at 10 microM) when MPM cells were grown in soft agarose. MSTO-211H, H2461, and H2373 were more sensitive to the growth-inhibitory effects of ZD1839 than was the NSCLC cell line A549, whereas H2591 had similar sensitivity to A549. This variability in growth-inhibitory effects is not related to the amount of EGFR present on MPM cells or to the degree of inhibition of EGFR phosphorylation by ZD1839. We show that H2373 MPM cells, which show 89% growth inhibition at 10 microM ZD1839, undergo a dose-dependent arrest at the G(1)-S phase of the cell cycle and a corresponding increase in p27 levels. However, H2591 cell lines, which show 41% growth inhibition at 10 microM ZD1839, undergo no significant cell cycle changes or changes in p27 levels. Our findings demonstrate that in vitro, ZD1839 is as effective or more effective against MPM cell lines as it is against the NSCLC cell line A549 and suggest that ZD1839 may be an effective therapeutic option for patients with MPM.

Topics: Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Cell Cycle; Cell Division; Epidermal Growth Factor; ErbB Receptors; Gefitinib; Humans; Lung Neoplasms; Mesothelioma; Phosphorylation; Pleural Neoplasms; Quinazolines; Signal Transduction; Tumor Cells, Cultured

Topics: Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Cisplatin; Clinical Trials, Phase III as Topic; Epidermal Growth Factor; Gefitinib; Humans; Lung Neoplasms; Protein-Tyrosine Kinases; Quality of Life; Quinazolines; Survival Analysis; Treatment Outcome

Topics: Advisory Committees; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Benzamides; Carcinoma, Non-Small-Cell Lung; Clinical Trials as Topic; Drug Approval; Drug Industry; Epidermal Growth Factor; Erlotinib Hydrochloride; Gefitinib; Humans; Imatinib Mesylate; Lung Neoplasms; Neoplasms; Patient Selection; Piperazines; Pyrimidines; Quinazolines; United States; United States Food and Drug Administration

Topics: Administration, Oral; Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Epidermal Growth Factor; ErbB Receptors; Gefitinib; Humans; Lung Neoplasms; Neoplasms; Protein-Tyrosine Kinases; Quinazolines

The 11p15 mucin genes (MUC2, MUC5AC, MUC5B and MUC6) possess a cell-specific pattern of expression in normal lung that is altered during carcinogenesis. Growth factors of the epidermal growth factor family are known to target key genes that in turn may affect the homeostasis of lung mucosae. Our aim was to study the regulation of the 11p15 mucin genes both at the promoter and protein levels to assess whether their altered expression may represent a key event during lung carcinogenesis. Studies were performed in the mucoepidermoid NCI-H292 lung cancer cell line. Cell treatment with epidermal growth factor (EGF), transforming growth factor alpha (TGF-alpha), or tumor necrosis factor alpha (TNF-alpha) resulted in a dramatic increase of MUC2 and MUC5AC mRNAs levels, promoter activity, and apomucin expression, whereas those of MUC5B and MUC6 were unchanged. pGL3 deletion mutants of MUC2, MUC5AC, and MUC5B promoters were constructed and used in transient transfection assays to characterize EGF- and TGF-alpha-responsive regulatory regions within the promoters. They were located in the -2627/-2097 and -202/-1 regions of MUC2 and MUC5AC promoters, respectively. Finally, we demonstrate that transcription factor Sp1 not only binds and activates MUC2 and MUC5AC promoters but also participates to their EGF- and TGF-alpha-mediated up-regulation. We also show that Sp3 is a strong inhibitor of 11p15 mucin gene transcription. In conclusion, MUC2 and MUC5AC are two target genes of EGFR ligands in lung cancer cells, and up-regulation of these two genes goes through concomitant activation of the EGFR/Ras/Raf/Extracellular Signal-regulated Kinase-signaling pathway and Sp1 binding to their promoters.

Topics: Base Sequence; Carcinoma, Squamous Cell; DNA Primers; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Flavonoids; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Mucin 5AC; Mucin-2; Mucins; Polymerase Chain Reaction; ras Proteins; Sp1 Transcription Factor; Transforming Growth Factor alpha; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

The extension of lamellipodia has been triggered by the application of epidermal growth factor (EGF). We have used an atomic force microscope (AFM) to investigate this lamellipodial extension. During extension we could detect an increase in height from about 500 nm for the stable lamellipodium to typical values of 600-800 nm for the extending lamellipodium. The AFM was also used to determine the mechanical properties of the lamellipodium where we found a decrease of the elastic modulus by a factor of 1.4 at the same location within the same cell. Both findings are consistent with the cortical expansion hypothesis, suggesting that severing of actin filaments, leading to a swelling of the cytoskeleton, generates the protrusive force during lamellipodial extension.

Topics: Adenocarcinoma; Animals; Chemotaxis; Elasticity; Epidermal Growth Factor; Lung Neoplasms; Microscopy, Atomic Force; Pseudopodia; Rats; Tumor Cells, Cultured

Transgenic mouse models were established to study tumorigenesis of bronchiolo-alveolar adenocarcinomas derived from alveolar type II pneumocytes (AT-II cells). Transgenic lines expressing the murine oncogene c- myc under the control of the lung-specific surfactant protein C promoter developed multifocal bronchiolo-alveolar hyperplasias, adenomas and carcinomas respectively, whereas transgenic lines expressing a secretable form of the epidermal growth factor (IgEGF), a structural and functional homologue of transforming growth factor alpha (TGF alpha), developed hyperplasias of the alveolar epithelium. Since the oncogenes c- myc and TGF alpha are frequently overexpressed in human lung bronchiolo-alveolar adenocarcinomas, these mouse lines are useful as models for human lung bronchiolo-alveolar adenocarcinomas. The average life expectancies of hemizygous and homozygous c- myc transgenics were 14.25 months and 9.2 months, respectively, suggesting that a dosage effect of c- myc caused an accelerated bronchiolo-alveolar adenocarcinoma formation. First analyses of double transgenics, hemizygous for both c- myc and IgEGF, show that these mice develop bronchiolo-alveolar adenocarcinomas at the average age of 9 months, indicating that these oncogenes cooperate during the lung cancer formation. Our results demonstrate that c- myc and EGF are directly involved and cooperate with one another during formation of bronchiolo-alveolar adenocarcinomas in the lung.

Topics: Adenocarcinoma; Animals; Bronchial Neoplasms; Cloning, Molecular; Epidermal Growth Factor; Gene Expression; Genes, myc; Lung Neoplasms; Mice; Mice, Transgenic; Phenotype; Pulmonary Alveoli

To evaluate the role of the NF-kappaB signaling pathway in oncogenic transformation, we expressed IkappaBbeta, a specific inhibitor of NF-kappaB, in two human lung adenocarcinoma cell lines, A549 and H441. Expression of IkappaBbeta significantly reduced NF-kappaB activation induced by cotransfection with p65/RelA or TNF-alpha and abrogated the basal NF-kappaB activity in A549 cells. Transfection of IkappaBbeta into A549, H441 and K-ras-transformed NIH3T3 cells suppressed anchorage-independent growth as measured by colony formation in soft agar. Anchorage-independent growth of vector-transfected A549 cells in reduced serum could be enhanced by both EGF and IGF-I. In contrast, only EGF but not IGF-I could induce anchorage-independent growth of IkappaBbeta-expressing A549 cells, suggesting that the IGF-I signaling pathway regulating growth and survival may be blocked by IkappaBbeta. Interestingly, expression of IkappaBbeta suppressed growth of A549 cells in low serum in vitro without affecting in vivo growth subcutaneously in nude mice. However, metastatic growth of IkappaBbeta-expressing A549 cells in the lungs of nude mice was significantly inhibited. These results provide evidence that NFkappaB plays an important role in anchorage-independent growth and metastatic growth of lung carcinoma cells.

Topics: 3T3 Cells; Adenocarcinoma; Animals; Cell Adhesion; Cell Division; Cell Line, Transformed; Cell Transformation, Neoplastic; DNA-Binding Proteins; Epidermal Growth Factor; Humans; I-kappa B Proteins; Lung Neoplasms; Mice; Mice, Nude; NF-kappa B; Signal Transduction; Transcription Factor RelA; Transfection; Transplantation, Heterologous; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Several inhibitors of EGF receptor (EGFR) tyrosine kinase activity have been developed that compete with ATP at its binding site such as the quinazolines PD 153035 and ZD 1839 or the 4,5-dianilino-phthalimides DAPH1 and DAPH2. When tested on human A431 cells, the quinazolines completely blocked EGF-induced receptor phosphorylation at 100 nM, whereas it was inhibited by DAPH1 and DAPH2 by only 20% at 3 microM. Quinazoline-treated A431 as well as tumor cells expressing less EGFR (A549, MDA MB 231, and T47D) bound 3- to 6-fold more (125)I-labeled EGF than untreated intact control cells. Scatchard analysis revealed the disappearance of low- and high-affinity EGFR on A431 cells upon PD 153035 treatment. A single receptor class of intermediate ligand binding affinity emerged and its number corresponded to the sum of the two classes. DAPH1 and DAPH2 did not change ligand binding properties of EGFR. PD 153035 exerted the most potent effects on EGF binding to A431 or on inhibiting EGF-stimulated growth of rat MTLn3 cells at low ligand concentrations. Cross-linking of EGFR on PD 153035-treated A431 cells indicated the formation of inactive dimers that further increased upon addition of EGF. Chemical cross-linking of (125)I-labeled EGF to PD 153035-treated A431 cells revealed increased binding to monomeric and dimeric EGFR. Thus, the quinazolines sequestered EGFR plus the ligand into inactive receptor/ligand complexes. This novel mode of action of quinazoline tyrosine kinase inhibitors may be the basis for their extraordinary potency especially in conditions when the ligand is present in limiting amounts.

Topics: Animals; Antineoplastic Agents; Breast Neoplasms; Carcinoma, Squamous Cell; Cell Division; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Gefitinib; Humans; Kinetics; Ligands; Lung Neoplasms; Mammary Neoplasms, Experimental; Phthalimides; Quinazolines; Rats; Signal Transduction; Tumor Cells, Cultured

AstraZeneca is developing ZD-1839, an inhibitor of epidermal growth factor receptor 1 (EGFR1) tyrosine kinase, for potential treatment of cancers which overexpress EGF receptors, including non-small cell lung cancer (NSCLC) and pancreatic cancer [196279], [270898]. Phase III studies had started by August 2000 [349551], [350295], [353050], [377656], with first results being expected at the 2001 meetings of the American Association for Cancer Research (AACR) and the American Society for Clinical Oncology (ASCO). The US FDA has issued ZD-1839 with Fast Track status [350295], [353050]. In September 2000, the company expected global NDA filing to take place at the end of 2001, with launch in the next four to five years [383469]. In January 1999, ABN Amro predicted sales of US $25 million in 2004 rising to $82 million in 2005 [316250]. In March 1999, Lehman Brothers predicted a 30% probability that the drug would reach worldwide markets and be launched onto the market in 2004 [336599]. In June 2000, Deutsche Bank predicted sales of US $8 million in 2002, rising to $100 million in 2003 [374500]. In September 2000, analysts Merrill Lynch predicted a launch in 2002 with sales estimated at UK 50 million pounds, rising to 360 million pounds in 2004, while in December 2000, the analysts predicted a filing date in the fourth quarter of 2001 [383742], [396280]. Also in December 2000, Lehman Brothers predicted a filing date late in 2001, and a possible Fast Track review. They also estimated peak sales of US $1 billion [394606].

Topics: Anemia; Animals; Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Clinical Trials, Phase I as Topic; Diarrhea; Disease Models, Animal; Drug Evaluation, Preclinical; Drug Tolerance; Drugs, Investigational; Economics, Pharmaceutical; Epidermal Growth Factor; ErbB Receptors; Gefitinib; Humans; Lung Neoplasms; Nausea; Pancreatic Neoplasms; Quinazolines; Tumor Cells, Cultured

Relatively few genes have been shown to directly affect the metastatic phenotype of breast cancer epithelial cells in vivo. The Rho family of proteins, incluing the Rho, Rac and Cdc42 subfamilies, are related to the small GTP binding protein Ras and regulated diverse biological processes including gene transcription, cytoskeletal organization, cell proliferation and transformation. The effects of Cdc42, Rac and Rho on the actin cytoskeleton suggested a possible role for Rho proteins in cellular motility and metastasis; however, a formal analysis of the role of Rho proteins in breast cancer cellular growth and metastasis in vivo had not previously been performed.. We generated a panel of MTLn3 rat mammary adenocarcinoma cells that expressed similar levels of dominant inhibitory mutants of Cdc42-, Rac- and Rho-dependent signaling, to examine the contribution of these GTPases to cell spreading, guided chemotaxis, and metastasis in vivo. The ability of Rho proteins to regulate intravasation into the peripheral blood was determined by implanting MTLn3 cell stable dominant negative lines in nude mice and measuring the formation of breast cancer cell colonies grown from the peripheral blood. Serial sectioning of the lungs was performed to determine the presence of metastasis in mice in which mammary tumors expressing the dominant negative Rho family proteins had grown to a similar size.. Cell spreading of MTLn3 cells was selectively abrogated by N17Rac1. N19RhoA and N17Cdc42 reduced the number of focal contacts (FCs) and disrupted the co-localization of vinculin with phosphotyrosine at FCs. While N17Rac1 and N17Cdc42 preferentially inhibited colony formation in soft agar, all three GTPases affected cell growth in vivo. To distinguish effects on tumorigenicity from intravasation into the bloodstream, implanted tumors were grown to the same size in nude mice. Each dominant inhibitory Rho protein reduced intravasation into the peripheral blood. Lung metastasis of MTLn3 cells was also abrogated by the dominant inhibitory Rho proteins, despite the presence of residual CFU.. These studies demonstrate for the first time a critical role for the Rho GTPases involving independent signaling pathways to limit mammary tumor cellular growth and metastasis in vivo.

Topics: Adenocarcinoma; Animals; cdc42 GTP-Binding Protein; Cell Division; Chemotaxis; Epidermal Growth Factor; Epithelium; Lung Neoplasms; Mammary Neoplasms, Experimental; Multigene Family; Rats; rho GTP-Binding Proteins; Signal Transduction; Stress Fibers; Transfection; Tumor Cells, Cultured; Tyrosine

Although human small cell lung carcinoma (SCLC) cell lines are typically anchorage-independent and do not attach on most extracellular matrix proteins, OH-1, and several other SCLC cell lines attached on substrates coated with thrombospondin-1 (TSP1). SCLC cells grew long-term as adherent cells on a TSP1-coated substrate. Adhesion of SCLC cells on TSP1 was inhibited by heparin, function-blocking antibodies recognizing alpha3 or beta1 integrin subunits, and by soluble alpha3beta1 integrin ligands. SCLC cells extended neurite-like processes on a TSP1 substrate, which was also mediated by alpha3beta1 integrin. Process formation on a TSP1 substrate was specifically stimulated by epidermal growth factor and somatostatin. Adhesion on TSP1 weakly inhibited SCLC cell proliferation, but this inhibition was strongly enhanced in the presence of epidermal growth factor. TSP1 and an alpha3beta1 integrin-binding peptide from TSP1 also inhibited proliferation when added in solution. High-affinity binding of 125I-labeled TSP1 to OH-1 cells was heparin-dependent and may be mediated by sulfated glycolipids, which are the major sulfated glycoconjugates synthesized by these cells. Synthesis or secretion of TSP1 by SCLC cells could not be detected. On the basis of these results, the alpha3beta1 integrin and sulfated glycolipids cooperate to mediate adhesion of SCLC cells on TSP1. Interaction with TSP1 through this integrin inhibits growth and induces neurotypic differentiation, which suggests that this response to TSP1 may be exploited to inhibit the progression of SCLC.

Topics: Carcinoma, Small Cell; Cell Adhesion; Cell Division; Epidermal Growth Factor; Humans; Integrin alpha3beta1; Integrins; Kinetics; Lung Neoplasms; Neurites; Somatostatin; Thrombospondin 1; Tumor Cells, Cultured

Poor transfection efficiency is the major drawback of lipofection. We showed previously that addition of transferrin (TF) to Lipofectin enhanced the expression of a reporter gene in HeLa cells by 120-fold and achieved close to 100% transfection efficiency. The purpose of this study was to determine whether TF and other ligands could improve the efficiency of lipofection in lung carcinoma cells. Confluent A549, Calu3, and H292 cells were transfected for 18 hours with a plasmid DNA (pCMVlacZ) using Lipofectin plus TF, insulin, or epidermal growth factor as the vector. The transfected cells were assessed for transfection efficiency by beta-galactosidase activity (light units/microg protein) and the percentage of blue cells following 5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside staining. Lipofectin supplemented with epidermal growth factor yielded the largest enhancement of lipofection efficiency (< or =23-fold over that by Lipofectin alone) in all three cell lines. Insulin significantly enhanced the lipofection efficiency in A549 and Calu3 cells but not in H292 cells, whereas TF showed significant lipofection efficiency-enhancing effect in Calu3 and H292 cells but not in A549 cells. The transfection efficiency correlated well with the amounts of DNA delivered to the nucleus as well as the amounts of the receptor. These results indicate that the gene delivery strategy employing ligand-facilitated lipofection can achieve high transfection efficiency in human lung carcinoma cells. In addition, enhancement of the expression of the receptor may be a possible strategy for increasing the efficiency of gene targeting.

Topics: Cell Nucleus; DNA; Epidermal Growth Factor; Gene Transfer Techniques; Genetic Therapy; Humans; Insulin; Ligands; Liposomes; Lung Neoplasms; Transfection; Transferrin; Tumor Cells, Cultured

Matrix metalloproteinase (MMP)-9 is an endopeptidase that digests basement membrane type IV collagen. Enhanced expression has been related to tumor progression both in vitro and in vivo. The control of MMP transcription is complex, but recently, epidermal growth factor receptor (EGFR) expression has been implicated in up-regulation of MMP-9 in tumor cells in vitro. Our objective was to evaluate the relationship between MMP-9 and EGFR expression in non-small cell lung cancer (NSCLC) and to assess the impact of expression on clinicopathological parameters and survival. This is a retrospective study of 169 patients who underwent resection for stage I-IIIa NSCLC with a postoperative survival >60 days. Minimum follow-up was 2 years. Standard avidin-biotin complex immunohistochemistry was performed on 4-microm paraffin-embedded sections from the tumor periphery using monoclonal antibodies to EGFR and MMP-9. MMP-9 was expressed in the tumor cells of 88 of 169 (52%) cases. EGFR expression was found in 94 of 169 (56%) cases [membranous, 55 of 169 (33%); cytoplasmic, 39 of 169 (23%)]. MMP-9 expression was associated with poor outcome in univariate (P = 0.0023) and multivariate (P = 0.027) analysis. Membranous, cytoplasmic, and overall EGFR expression were not associated with outcome (P = 0.13, 0.99, and 0.17, respectively). MMP-9 expression showed a strong correlation with EGFR expression (P < 0.0001) and EGFR membranous expression (P = 0.002) but not with cytoplasmic EGFR expression (P = 0.18). Co-expression of MMP-9 and EGFR (37%) conferred a worse prognosis (P = 0.0001). Subset analysis revealed only MMP-9 and membranous EGFR co-expression (22%) was associated with poor outcome (P = 0.0019). Our results show that a significant proportion of NSCLC tumors co-express MMP-9 and EGFR. The co-expression of these markers confers a poor prognosis. This finding suggests that EGFR signaling pathway may play an important role in the invasive behavior of NSCLC via specific up-regulation of MMP-9.

Topics: Adenocarcinoma; Adult; Age Factors; Aged; Carcinoma, Large Cell; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Cell Membrane; Cytoplasm; Disease Progression; Epidermal Growth Factor; Female; Humans; Immunohistochemistry; Lung Neoplasms; Male; Matrix Metalloproteinase 9; Middle Aged; Multivariate Analysis; Prognosis; Retrospective Studies; Sex Factors; Signal Transduction; Time Factors; Up-Regulation

Topics: Antibodies; Antibody Formation; Canada; Cancer Vaccines; Carcinoma, Non-Small-Cell Lung; Cuba; Epidermal Growth Factor; Humans; Lung Neoplasms; Randomized Controlled Trials as Topic

The erbB-2/HER2 oncogene is overexpressed in a significant fraction of human carcinomas of the breast, ovary, and lung in a manner that correlates with poor prognosis. Although the encoded protein resembles several receptors for growth factors, no high affinity ligand of ErbB-2 has so far been fully characterized. However, several lines of evidence have raised the possibility that ErbB-2 can augment signal transduction initiated by binding of certain growth factors to their direct receptors. Here, we contrasted these two models of ErbB-2 function: First, examination of a large series of epidermal growth factor (EGF)-like ligands and neuregulins, including virus-encoded ligands as well as related motifs derived from the precursor of EGF, failed to detect interactions with ErbB-2 when this protein was singly expressed. Second, by using antibodies that block inter-ErbB interactions and cells devoid of surface ErbB-2, we learned that signaling by all ligands examined, except those derived from the precursor of EGF, was enhanced by the oncoprotein. These results imply that ErbB-2 evolved as a shared receptor subunit of all ErbB-specific growth factors. Thus, oncogenicity of ErbB-2 in human epithelia may not rely on the existence of a specific ligand but rather on its ability to act as a coreceptor for multiple stroma-derived growth factors.

Topics: Breast Neoplasms; Carcinoma; Epidermal Growth Factor; Female; Genes, erbB-2; Glycoproteins; Humans; Ligands; Lung Neoplasms; Nerve Growth Factors; Neuregulins; Ovarian Neoplasms; Receptor, ErbB-2; Receptors, Cytoplasmic and Nuclear; Signal Transduction; Stromal Cells; Tumor Cells, Cultured

Topics: Antibodies, Monoclonal; Cell Movement; Cytological Techniques; Epidermal Growth Factor; ErbB Receptors; Humans; Image Processing, Computer-Assisted; Ink; Internet; Lung Neoplasms; Microscopy, Phase-Contrast; Software; Tumor Cells, Cultured

We recently demonstrated that human lung epithelial cells, overexpressing ErbB-2, formed tumors in nude mice only when high levels of transforming growth factor alpha (TGFalpha) were produced. Cells transfected with a TGFalpha antisense vector failed to form tumors in nude mice. In order to further evaluate the importance, for tumorigenicity, of TGFalpha and its stimulation of ErbB family signalling, the production of other EGF family growth factors by these human lung epithelial cells was studied. We demonstrate for the first time that both tumorigenic and non-tumorigenic human lung epithelial cells produced, in addition to TGFalpha, amphiregulin, betacellulin, heparin-binding EGF and heregulin. These data suggest that human lung epithelial cells have the potential for multifactorial modulation of ErbB receptor family signalling through control of ligand as well as receptor production. In this system, the probable importance of TGFalpha-stimulated signaling for tumorigenicity is supported by its 13-fold higher production in tumorigenic as compared with non-tumorigenic cells and the 2-fold or lower differences observed in production of the other epidermal growth factor (EGF) family ligands.

Topics: Animals; Cell Transformation, Neoplastic; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Genes, erbB-2; Humans; Ligands; Lung; Lung Neoplasms; Mice; Neoplasms, Experimental; Receptor, ErbB-2; Transforming Growth Factor alpha

Vascular endothelial growth factor (VEGF) plays a crucial role in physiological and neoplastic angiogenesis. Moreover, VEGF has been found to be upregulated by conditions associated with the generation of free radicals and reactive oxygen intermediates. In patients with cancer, studies to evaluate VEGF as a measure of tumour activity were carried out. We tested the hypothesis that VEGF is additionally affected by oxidative stress due to anticancer therapy. Moreover, the suitability of epidermal growth factor (EGF) to estimate tumour activity was studied.. 60 patients with non-small cell lung cancer (NSCLC) covering different therapy progress and modalities underwent bronchoalveolar lavage. VEGF-, EGF-, albumin- and total protein-concentrations in bronchoalveolar lavage fluid (BALF) and VEGF-levels in blood plasma were studied.. BALF VEGF-levels were increased in patients with advanced NSCLC before and in anticancer therapy. In patients who had received radiotherapy to the lung prior to chemotherapy, VEGF concentrations were noticeably higher than under sole chemotherapy. Pulmonary endothelial hyperpermeability was found in patients with recently diagnosed tumours and patients undergoing anti-cancer therapy. Evaluation of EGF-levels in BALF revealed no significant influence of tumour activity or cancer therapy on this parameter.. BALF-levels of VEGF are affected by tumour activity and oxidative stress due to anticancer therapy.

Topics: Adult; Aged; Aged, 80 and over; Albumins; Alkaloids; Antineoplastic Combined Chemotherapy Protocols; Bronchial Neoplasms; Bronchoalveolar Lavage Fluid; Carcinoma, Non-Small-Cell Lung; Combined Modality Therapy; Deoxycytidine; Endothelial Growth Factors; Epidermal Growth Factor; Etoposide; Female; Gemcitabine; Humans; Lung Neoplasms; Lymphokines; Male; Middle Aged; Neoplasm Proteins; Neovascularization, Pathologic; Oxidative Stress; Proteins; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors; Vindesine

The levels of EGFR and its ligands have been assayed in tumor and adjacent lung tissues in NSCLC patients. Both EGFR and EGF-like peptides were found in tumor more frequently than in unaltered tissue. It was shown (Kaplan-Meyer) that simultaneous expression of EGFR and its ligands in tumor and adjacent lung tissues was associated with lower overall and relapse-free survival in NSCLC patients.

Topics: Adult; Aged; Carcinoma, Non-Small-Cell Lung; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Lung Neoplasms; Male; Middle Aged; Predictive Value of Tests; Prognosis; Survival Analysis

PTH-related peptide (PTHrP) mediates the syndrome of humoral hypercalcemia of malignancy, a frequent complication of squamous cell carcinomas of the lung. This study was undertaken to determine whether 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] and two nonhypercalcemic analogs, EB1089 and 22-oxa-1,25-(OH)2D3 (22-oxacalcitriol), suppress serum- and epidermal growth factor (EGF)-induced PTHrP gene expression in a human lung squamous cancer cell line, NCI H520. PTHrP expression was up-regulated by serum and EGF in a concentration- and time-dependent manner. Nuclear run-on analysis showed that this induction was mediated via a transcriptional mechanism, and that sequences within promoter 1 were responsible. All three vitamin D3 compounds decreased both basal and serum- and EGF-induced steady state PTHrP messenger RNA and secreted peptide levels. These effects were again mediated via a transcriptional mechanism through sequences within promoter 1. All three vitamin D3 compounds also decreased the proliferation of NCI H520 cells in a concentration- and time-dependent manner. 1,25-(OH)2D3 is hypercalcemic in vivo. However, the noncalcemic analogs EB1089 and 22-oxa-1,25-(OH)2D3 have therapeutic potential, as they suppress not only the basal but also the growth factor-stimulated levels of PTHrP in a cancer cell line associated with hypercalcemia.

Topics: Blood Physiological Phenomena; Calcitriol; Cell Division; Epidermal Growth Factor; Gene Expression Regulation; Humans; Lung Neoplasms; Parathyroid Hormone-Related Protein; Promoter Regions, Genetic; Proteins; Tumor Cells, Cultured

EGF receptor (EGFR) is a transmembrane glycoprotein with trosine kinase activity that is overexpressed in many human cancers, including lung. In the present study, we evaluated the effect of EGF and genistein, a tyrosine kinase inhibitor, on cell proliferation, EGFR phosphorylation and its downstream signal MAP kinase activation and investigated the involvement of these processes in programmed cell death in a human pulmonary adenosquamous carcinoma cell line, NCI-H596. Treatment with EGF resulted in phosphorylation of EGFR, activation of MAP kinase, phosphorylation of ERK 2 (an isoform of MAP kinase), increased cell proliferation and induction of cross-linked envelope (CLE) competence. Genistein abolished the ability of EGF to induce EGFR phosphorylation, to activate MAP kinase and to increase cell proliferation. Genistein alone stimulated CLE competence, but apparently by a different mechanism than EGF since genistein prevented EGF-stimulated CLE competence. The genistein-stimulated CLE competence was accompanied by a decrease in cell proliferation and increased DNA fragmentation. These results demonstrate that genistein antagonizes growth stimulatory EGF signaling upstream of MAP kinase and may simultaneously stimulate an apoptotic pathway. Furthermore, EGF appears to stimulate an alternate, growth related programmed cell death pathway, not involving DNA fragmentation, but characterized by rapid proliferation and genistein-sensitive CLE competence.

Topics: Antineoplastic Agents; Apoptosis; Calcium-Calmodulin-Dependent Protein Kinases; Carcinoma, Non-Small-Cell Lung; Cell Division; DNA Fragmentation; Enzyme Activation; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Genistein; Humans; Lung Neoplasms; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Phosphorylation; Signal Transduction; Tumor Cells, Cultured; Tyrosine

Smad6 and Smad7 function as intracellular antagonists in transforming growth factor-beta (TGF-beta) signaling. Here we report the isolation of human Smad6, which is closely related to Smad7. Smad6 and Smad7 mRNAs were differentially expressed in lung cancer cell lines and were rapidly and directly induced by TGF-beta1, activin and bone morphogenetic protein-7. Cross-talk between TGF-beta and other signaling pathways was demonstrated by the finding that epidermal growth factor (EGF) induced the expression of inhibitory SMAD mRNA. Moreover, whereas the phorbol ester PMA alone had no effect, it potentiated the TGF-beta1-induced expression of Smad7 mRNA. Ectopic expression of anti-sense Smad7 RNA was found to increase the effect of TGF-beta1, supporting its role as a negative regulator in TGF-beta signaling. Thus, expression of inhibitory Smads is induced by multiple stimuli, including the various TGF-beta family members, whose action they antagonize.

Topics: Activins; Animals; Bone Morphogenetic Proteins; Cell Line; DNA-Binding Proteins; Drug Synergism; Epidermal Growth Factor; Epithelial Cells; Gene Expression; Humans; Inhibins; Keratinocytes; Lung; Lung Neoplasms; Mink; RNA, Messenger; Signal Transduction; Smad6 Protein; Smad7 Protein; Tetradecanoylphorbol Acetate; Trans-Activators; Transforming Growth Factor beta; Tumor Cells, Cultured

Growth of the EGF receptor-expressing non-small-cell lung carcinoma cell line H125 seems to be at least partially driven by autocrine activation of the resident EGF receptors. Thus, the possibility of an EGF receptor-directed antiproliferative treatment was investigated in vitro using a monoclonal antibody (alpha EGFR ior egf/r3) against the human EGF receptor and gangliosides which are known to possess antiproliferative and anti-tyrosine kinase activity. The moderate growth-inhibitory effect of alpha EGFR ior egf/r3 was strongly potentiated by the addition of monosialoganglioside GM3. Likewise, the combination of alpha EGFR ior egf/r3 and GM3 inhibited EGF receptor autophosphorylation activity in H125 cells more strongly than either agent alone. A synergistic inhibition of EGF receptor autophosphorylation by alpha EGFR ior egf/r3 and GM3 was also observed in the human epidermoid carcinoma cell line A431. In both cell lines, the inhibition of EGF receptor autophosphorylation by GM3 was prevented by pretreatment of the cells with pervanadate, a potent inhibitor of protein tyrosine phosphatases (PTPases). Also, GM3 accelerated EGF receptor dephosphorylation in isolated A431 cell membranes. These findings indicate that GM3 has the capacity to activate EGF receptor-directed PTPase activity and suggest a novel possible mechanism for the regulation of cellular PTPases.

Topics: Adenocarcinoma; Benzylidene Compounds; Carcinoma, Non-Small-Cell Lung; Cell Division; Cell Membrane; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; G(M3) Ganglioside; Humans; Lung Neoplasms; Nitriles; Phosphorylation; Protein Tyrosine Phosphatases; Quinazolines; Radioligand Assay; Signal Transduction; Tumor Cells, Cultured; Tyrphostins

Antagonists of bombesin/gastrin-releasing peptide (BN/GRP) have been developed to inhibit the stimulatory effects of BN/GRP on the mitogenesis of tumor cells such as human small-cell lung carcinoma (SCLC). The mode of action of these antagonists is not completely understood. In this study, we evaluated the effect of BN/GRP antagonist RC-3095 on receptors for BN/GRP and epidermal growth factor (EGF) in H-128 human SCLC line xenografted into nude mice. Treatment with RC-3095, administered s.c. at a dose of 20 microg/day per animal for 4 weeks caused a 70% reduction in tumor volume and weight. Membrane receptors for BN/GRP and EGF were characterized in untreated and treated animals. In the control group, [125I-Tyr4]BN was bound to a single class of specific, high affinity binding sites with a dissociation constant (Kd) = 6.55 +/- 0.93 nM and maximal binding capacity (Bmax) = 512.8 +/- 34.8 fmol/mg membrane protein. Therapy with RC-3095 decreased the concentration of BN/GRP receptors on H-128 SCLC tumor membranes. Specific, high affinity binding sites for EGF with Kd = 1.78 +/- 0.26 nM and Bmax = 216.8 +/- 19.6 fmol/mg membrane protein were also found on the untreated H-128 SCLC tumors. Treatment with RC-3095 significantly decreased Bmax of receptors for EGF. Our results indicate that the suppression of growth of H-128 SCLC by BN antagonist RC-3095 is accompanied by a decrease in the number of receptors for both BN/GRP and EGF. These observations are in agreement with the results obtained in other experimental cancers. The findings on antagonist RC-3095 reinforce the view that both BN/GRP and EGF receptors participate in a cascade of events involved in the growth of SCLC and other cancers. Although the complete mechanisms of action of antagonist RC-3095 remain to be elucidated, the antitumor effect could be the result of the fall in the EGF receptor number, which might lead to a decrease in EGF receptor autophosphorylation.

Topics: Animals; Antineoplastic Agents; Body Weight; Bombesin; Carcinoma, Small Cell; Down-Regulation; Epidermal Growth Factor; ErbB Receptors; Humans; Lung Neoplasms; Male; Mice; Mice, Nude; Neoplasm Transplantation; Peptide Fragments; Protein Binding; Receptors, Bombesin; Transplantation, Heterologous; Tumor Cells, Cultured

Epidermal growth factor (EGF) enhances fetal lung development in vivo and in vitro. Ligand binding to the EGF receptor stimulates an intrinsic receptor tyrosine kinase initiating a signal transduction cascade. We hypothesized that blocking EGF receptor function with tyrosine kinase inhibitors would decrease the expression of surfactant protein A in human pulmonary epithelial cells. Human pulmonary adenocarcinoma cells (NCI-H441) were exposed to genistein (a broad range inhibitor of tyrosine kinases) and tyrphostin AG1478 (a specific inhibitor of EGF receptor tyrosine kinase). Genistein significantly decreased surfactant protein A (SP-A) and SP-A mRNA levels in H441 cells without affecting cell viability. The inhibitory effect of genistein on SP-A content was reversible. In contrast, tyrphostin AG1478 had no effect on SP-A levels despite a greater inhibitory effect than genistein on EGF receptor tyrosine autophosphorylation. Furthermore, treatment of H441 cells with exogenous EGF did not increase SP-A content or mRNA levels beyond baseline. We conclude that inhibition of tyrosine kinase activity other than the EGF receptor decreases the expression of surfactant protein A at a pretranslational level in human pulmonary adenocarcinoma cells. These results suggest the importance of tyrosine kinases in modulating human SP-A synthesis.

Topics: Benzylidene Compounds; Binding, Competitive; Blotting, Northern; Blotting, Western; Cell Survival; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Genistein; Humans; Isoflavones; Lung; Lung Neoplasms; Nitriles; Phosphorylation; Protein-Tyrosine Kinases; Proteolipids; Pulmonary Surfactant-Associated Protein A; Pulmonary Surfactant-Associated Proteins; Pulmonary Surfactants; Quinazolines; RNA, Messenger; Tumor Cells, Cultured; Tyrphostins

Apoptosis has become a basic tool in developing cancer research and establishing new cancer strategies. However, the molecular mechanism of apoptosis is not well understood. Recently, the authors found that epidermal growth factor (EGF) induces apoptosis in various cancer cells and that there is a novel signal pathway mediated through p53 in signal transduction of EGF. The effect of antisense gene therapy to p53 tumor suppressor on EGF-dependent apoptosis was investigated in cultured NCI-H 596 human non-small cell lung cancer cells with a wild-type p53 gene. Results showed that EGF plus p53 sense oligonucleotides induced EGF-dependent and p53-dependent apoptosis in NCI-H 596 cells within 8 hours. On the other hand, antisense gene therapy using antisense oligonucleotides to p53 tumor suppressor suppressed the induction of EGF-dependent and p53-dependent apoptosis. Mutated p53 antisense-containing mutated CG dinucleotides had the same effect as that of p53 antisense on suppression of apoptosis in NCI-H 596 cells. We found that a new nucleic acid drug, another mutated p53 antisense-containing mutation at three bases immediately 5' and 3' from the CG dinucleotides, potentiated the induction of apoptosis and failed to suppress the induction of EGF-dependent apoptosis. These results suggest that gene therapy using antisense oligonucleotides to the p53 tumor suppressor is effective on EGF-dependent apoptosis of NCI-H 596 human non-small cell lung cancer.

Topics: Antineoplastic Agents; Apoptosis; Blotting, Western; Carcinoma, Non-Small-Cell Lung; Epidermal Growth Factor; Genes, p53; Genetic Therapy; Humans; Lung Neoplasms; Oligonucleotides, Antisense; Tumor Cells, Cultured

To clarify the role of tyrosine phosphorylation of cellular proteins in human lung cancer cells, phosphotyrosine (PTYR)-containing proteins in lung cancer cell lines and in paired tissues resected from cancerous and normal lungs were studied by immunoblotting with an anti-PTYR antibody. We found that the profiles of protein phosphorylation were very similar among those cell lines which had different histological features. The major PTYR-containing proteins (180-190 KDa, 120-130 KD, and 95-100 KDa) were detected in lung cancer cell lines. The expression of EGF receptor (EGF-r) (p185) and o-erb B2 protein, and tyrosine phosphorylation of p125FAK were examined in cancerous lung tissues and normal lung tissues. In surgical specimens, approximately half of the samples of lung cancer tissues showed clear elevation of tyrosine phosphorylation. In these cancerous tissues, no clear amplification of EGF-r and c-erb B2 protein expression was observed. However, elevation of tyrosine phosphorylation of p125FAK was observed in cancerous lung tissues but not in normal lung tissues, and its phosphorylation was closely correlated with the nodal involvement of cancer and disease-free survival time. These results suggested that the intracellular signaling pathway via tyrosine phosphorylation plays a role in the generation and immortalization of lung cancer, and assessment of tyrosine phosphorylation of cellular proteins. especially p125FAK, may be available clinically as a prognostic factor.

Topics: Adult; Aged; Cell Adhesion Molecules; Epidermal Growth Factor; ErbB Receptors; Female; Fluorescent Antibody Technique, Indirect; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Humans; Immunohistochemistry; Lung; Lung Neoplasms; Male; Middle Aged; Phosphorylation; Phosphotyrosine; Protein-Tyrosine Kinases; Proteins; Receptor, ErbB-2; Survival Rate; Tumor Cells, Cultured

Transforming growth factor-alpha (TGF-alpha), a member of the epidermal growth factor (EGF) family that binds to the EGF receptor (EGFR), is thought to function in an autocrine manner in non-small cell lung cancers (NSCLC). Heparin-binding EGF-like growth factor (HB-EGF), a novel member of the EGF family, also binds to EGFR. To compare the expression of HB-EGF, TGF-alpha and EGFR genes in NSCLC and normal lung tissue, we measured the levels of messenger RNA (mRNA) for these genes in human NSCLC and normal lung tissues by Northern hybridization, reverse transcription-polymerase chain reaction (RT-PCR), and in situ hybridization. A total of eight specimens (paired tumor tissue and normal lung tissue) were harvested from four patients who underwent resection of primary resectable NSCLC. HB-EGF was not expressed in either tumor tissue or normal lung tissue, while EGFR and TGF-alpha were expressed in all samples. TGF-alpha was overexpressed in all tumor tissue samples by several hundred-fold, while the expression of EGFR was not significantly different in tumor tissue and normal lung tissue. There was no correlation between the expression of TGF-alpha and EGFR. In situ hybridization showed that TGF-alpha mRNA was localized mainly in the cancer cells of tumor tissues and in the macrophages of alveoli in normal lung tissue. Our results showed that HB-EGF plays no role in the growth of NSCLC, and that there was no significant overexpression of EGFR in tumor tissue. TGF-alpha may play a major role in the growth of NSCLC. This supports a new direction in rational NSCLC treatment.

Topics: Carcinoma, Non-Small-Cell Lung; Epidermal Growth Factor; ErbB Receptors; Female; Heparin; Heparin-binding EGF-like Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Lung; Lung Neoplasms; Male; Middle Aged; RNA, Messenger; Transforming Growth Factor alpha

Chromosome microdissection-fluorescence in situ hybridization and comparative genomic hybridization (CGH) were performed in parallel to identify the native location of amplified DNA in a human non-small cell lung cancer (NSCLC) cell line exhibiting a homogeneously staining region (hsr) and double minutes (dmin). The native locations of microdissected DNA from the hsr and dmin were 7p12-13 and 8q24, respectively. Southern analysis revealed coamplification of EGFR (7p12) and MYC (8q24). CGH detected amplification of DNA not only from 7p12-13 and 8q24, but also from 9p24 and 10q22.

Topics: Blotting, Southern; Carcinoma, Non-Small-Cell Lung; Chromosome Banding; Chromosome Mapping; Chromosomes, Human, Pair 10; Chromosomes, Human, Pair 7; Chromosomes, Human, Pair 8; Chromosomes, Human, Pair 9; DNA, Neoplasm; Epidermal Growth Factor; Gene Amplification; Genes, myc; Humans; In Situ Hybridization, Fluorescence; Lung Neoplasms; Polymerase Chain Reaction; Tumor Cells, Cultured

Glutathione plays an essential role in protecting the pulmonary system from toxic insults. gamma-Glutamyl transpeptidase-related enzyme (GGT-rel) is a novel protein capable of cleaving the gamma-glutamyl peptide bond of glutathione and of converting leukotriene C4 to leukotriene D4. A rat homologue of GGT-rel was identified and was found to be highly expressed in cultures of differentiating rat tracheal epithelial (RTE) cells. The 2.6-kb cDNA predicts a 572-amino acid protein with 79% identity to human GGT-rel. GGT-rel was weakly expressed in normal trachea but was strongly induced by epidermal growth factor in cultures of RTE cells. GGT-rel was also highly expressed in lung tumors induced by inhalation of isobutyl nitrite. These results demonstrate that GGT-rel 1) is expressed in normal tracheal cells, 2) can be induced by epidermal growth factor, and 3) is elevated after chemical exposure. The induction of high levels of GGT-rel may play an important role in protecting the lung from oxidative stress or other toxic insults.

Topics: Amino Acid Sequence; Animals; Base Sequence; Carcinogens; Cell Differentiation; Cells, Cultured; DNA Primers; Epidermal Growth Factor; Epithelial Cells; gamma-Glutamyltransferase; Gene Expression Regulation, Enzymologic; Humans; Lung Neoplasms; Molecular Sequence Data; Nitrites; Organ Specificity; Polymerase Chain Reaction; Rats; Sequence Alignment; Sequence Homology, Amino Acid; Trachea

Epidermal growth factor (EGF) plays a major role in non-small cell lung cancer cell autocrine growth and has been reported to activate the JUN kinase/stress-activated protein kinase (JNK/SAPK) pathway in model cells. Activation of JNK/SAPK leads to the phosphorylation of c-JUN protooncogene on serines 63 and 73. This mechanism is required for and cooperates in the transformation of rat embryo fibroblasts by Ha-RAS. However, the function of JNK/SAPK in human tumor growth is unknown. We have tested several lung carcinoma cell lines. All exhibited UV-C-inducible JNK/SAPK activity; two exhibited constitutive activity in low serum, and two (M103 and A549) exhibited EGF-inducible JNK/SAPK activity. In A549 cells, EGF induced a rapid and prolonged (up to 24 h) activation of the JNK/SAPK pathway that correlated with a 150-190% growth stimulation. Stably transfected clones of A549 cells expressing c-JUN(S63A,S73A), a transdominant inhibitor of c-JUN, completely blocked the EGF-stimulated proliferation effect but did not alter the basal proliferation rate. Consistent with these results JNK antisense oligonucleotides targeted to JNK1 and JNK2 entirely eliminated the EGF-stimulated JNK/SAPK activity and blocked EGF-stimulated growth but not basal growth. In contrast, specific inhibition of the RAF/ERK pathway by PD98059 (MEK1 inhibitor) completely blocked ERK activation by EGF and basal cell growth but not EGF-stimulated growth, thereby dissociating the growth-promoting roles of each pathway. Our observations indicate, for the first time, that JNK/SAPK may be a preferential effector pathway for the growth properties of EGF in A549 cells.

Topics: Animals; Calcium-Calmodulin-Dependent Protein Kinases; Cell Division; Enzyme Activation; Enzyme Inhibitors; Epidermal Growth Factor; Flavonoids; Humans; JNK Mitogen-Activated Protein Kinases; Lung Neoplasms; Mitogen-Activated Protein Kinase 9; Mitogen-Activated Protein Kinases; Oligonucleotides, Antisense; Protein Kinase Inhibitors; Protein Kinases; Rats; Tumor Cells, Cultured

After acute lung injury, altered bronchioloalveolar epithelia must be repaired quickly in order to restore lung function. During reepithelialization, type II cells initially appear to migrate and spread over a remodeled matrix; then a secondary proliferative phase occurs. It was hypothesized that 1) type II cells can develop locomotion in vitro that is modulated by growth factors, proinflammatory cytokines, and substrate adhesion molecules and 2) migration and proliferation of type II cells can occur as distinctive processes. Chemotaxis assays were elaborated using short term cultures of rat type II pneumocytes. Epidermal growth factor (EGF), transforming growth factor-alpha, laminin, fibronectin were found to be the main attractants for type II cells with respective increases of approximately 8.5-, 10.5-, 8-, and 7-fold in cell migration (P<0.05 vs. control). Laminin induced gradient-dependent and random cell migration. Addition of laminin with EGF had a synergistic effect in promoting cell migration (approximately 30-fold increase over control, P<0.05). Interferon-gamma and interleukin-6 inhibited EGF-induced type II cell migration, whereas tumor necrosis factor-alpha and interleukin-1beta acted as primers for type II cell migration (approximately 1.5-fold increase over control, P<0.05. Type II cells did not need to be in a proliferative phase in order to exhibit motility. New insights regarding the regulatory processes for type II cell migration are especially relevant in our understanding of early events occurring during epithelial repair after acute lung injury.

Topics: Adenocarcinoma; Analysis of Variance; Animals; Cells, Cultured; Chemotaxis; Collagen; Cytokines; Epidermal Growth Factor; Epithelial Cells; Epithelium; Extracellular Matrix Proteins; Fibronectins; Growth Substances; Humans; Interferon-gamma; Interleukin-6; Kinetics; Laminin; Lung Neoplasms; Macrophages, Alveolar; Platelet-Derived Growth Factor; Pulmonary Alveoli; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Tumor Cells, Cultured

Mammalian pancreatic ribonuclease (RNase) was conjugated chemically via a disulfide bond to human or murine epidermal growth factor (EGF). The conjugation between EGF and RNase was ascertained by SDS-PAGE using reduced and nonreduced conjugates. The EGF-RNase conjugate retained potent RNase activity and competed with 125I-EGF for binding to EGFR to the same extent as unconjugated EGF. Both the human and murine EGF-RNase conjugates showed dose-dependent cytotoxicity against EGFR-overexpressing A431 human squamous carcinoma cells with IC50 values of 3 x 10(-7) M and 6 x 10(-7) M, respectively, whereas free RNase had an IC50 of 10(-4) M. Against the EGFR-deficient small-cell lung cancer cell line H69, the EGF-RNase conjugate had no cytotoxic effect. The Human EGF-RNase conjugate showed dose-dependent cytotoxicity against other squamous carcinoma cell lines (TE-5, TE-1) and breast cancer cell lines (BT-20, SK-BR-3, MCF-7) and the cytotoxicity of the conjugate correlated positively with the level of expression of EGFR by each cell line. An unconjugated mixture of EGF and RNase had no greater effect than RNase alone on any cell line. Excess free EGF blocked EGF-RNase conjugate cytotoxicity against A431 cells. These results suggest that the EGF-RNase conjugate may be a more effective anticancer agent with less immunogenicity than coventional chimeric toxins.

Topics: Antineoplastic Agents; Breast Neoplasms; Carcinoma, Small Cell; Carcinoma, Squamous Cell; Drug Screening Assays, Antitumor; Epidermal Growth Factor; ErbB Receptors; Esophageal Neoplasms; Humans; Immunotoxins; Lung Neoplasms; Ribonucleases; Tumor Cells, Cultured

Overexpression of epidermal growth factor receptor (EGFr) in squamous carcinomas has been demonstrated extensively. Preliminary clinical studies have shown that radiolabeled anti-EGFr monoclonal antibodies can localize to these tumors. The aims of this study were to determine the tolerance, pharmacokinetics, and radiolocalization properties of 131I-labeled EGF in patients (n = 9) with advanced squamous lung cancer. Patients' vital signs and symptoms were monitored regularly for 3 days. Daily scintigrams and biological samples for pharmacokinetic analysis were obtained for 3-4 days. 99mTc-labeled human serum albumin was administered to patients with positive tumor scans. Six patients had positive tumor scans, and five of them had received >/=1.0 mg EGF. In all of these cases, tumors were visualized the same day of the infusion, although best tumor-background contrast was obtained at 50-74 h. There were no false-positive images. Whole-body radioactivity retention rose significantly with increasing EGF doses; most labeled EGF was eliminated by urinary excretion. Tumor:normal tissue uptake ratios increased during the course of the study. All patients presented self-limited, dose-related gastrointestinal adverse effects. In conclusion, recombinant 131I-labeled EGF administered i.v. can localize to squamous lung cancer efficiently, can be administered safely to patients, and has more advantageous pharmacokinetic properties than monoclonal antibodies. Further studies are warranted to determine more accurately the potential of EGF and EGF-related peptides in the imaging and/or therapy of EGFr-overexpressing human cancers.

Topics: Aged; Aged, 80 and over; Carcinoma, Squamous Cell; Epidermal Growth Factor; Humans; Iodine Radioisotopes; Lung Neoplasms; Middle Aged; Radionuclide Imaging

ErbB-2 and EGFR (epidermal growth factor receptor) are expressed in lung adenocarcinomas and associated with a poor prognosis. Immunocytochemical analysis revealed erbB-2 and EGFR coexperession as a characteristic feature of most lung adenocarcinomas, and at levels of receptor expression present in bronchial epithelial cells. In primary lung tumours and cell lines, erbB-2 detected using Western blot analysis demonstrated low-level phosphotyrosine staining of the 185 kDa band, as compared with breast cancer cell lines. A549 and A427 lung adenocarcinoma cells treated with neu differentiation factor (NDF) showed increased erbB-2 phosphotyrosine staining, but to a much lesser extent than breast cancer cells. The lung cells were examined for expression of the potential autocrine growth factors NDF and transforming growth factor alpha (TGF-alpha) by Northern blot analysis. Both NDF and TFG-alpha mRNA were abundantly expressed in the A549 cells. NDF mRNA was highest during active cell proliferation and decreased in confluent cells or after treatment with the growth-inhibitory steroid dexamethasone. Primary tumours and cell lines expressed EGFR, showing higher basal level phosphotyrosine staining than erbB-2. Treatment with NDF and EGF (epidermal growth factor) stimulated cell growth, and in A549 cells the presence of both factors provided an additive increase in cell growth. The growth stimulus that ligand-activated erbB-2 and EGFR provides to lung adenocarcinoma cells may establish a background of continued cell proliferation over which other critical transforming events may occur.

Topics: Adenocarcinoma; Epidermal Growth Factor; ErbB Receptors; Glycoproteins; Humans; Lung Neoplasms; Neuregulins; Receptor, ErbB-2; Tumor Cells, Cultured

We examined immunohistochemically 111 cases of primary adenocarcinoma of the lung, for transforming growth factor alpha (TGF alpha) or epidermal growth factor (EGF), and argyrophilic nucleolar organizer regions (AgNORs). The presence of more than 75% positive cells for both growth factors was designated as a high-GF, while all others were considered to be a low-GF. If AgNORs counts were more than 5.00, it was considered to be a high-AgNORs group, while less than 5.00 was designated as a low-AgNORs group. In our 111 examined specimens, there were 51 (46%) cases of high-GF, and 64 (58%) with high AgNORs. The 5-year survival rates of the patients with a high-GF and low-GF were 34% and 57% (P < 0.05) respectively, while those with high-AgNORs and low-AgNORs were 21% and 81% (P < 0.001), respectively. In the cases of high-AgNORs, the 5-year survival rates of the patients with high-GF and low-GF were 0% and 36% (P < 0.05), respectively. However, in the cases of low-AgNORs, the 5-year survival rates of the patients with high-GF and low-GF were 83% and 79%, respectively. These data suggest that growth factors might be related to the biological malignancy of tumours with a high cell proliferation.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Cell Division; Epidermal Growth Factor; Female; Humans; Lung Neoplasms; Male; Middle Aged; Nucleolus Organizer Region; Silver Staining; Survival Analysis; Transforming Growth Factor alpha

Increased expression of EGF receptor (EGFR) in metastases of human mammary carcinoma as compared to cells of the primary cancer suggests a contribution of EGFR to mammary carcinoma metastasis. To test for a positive causative link, we investigated 13762NF rat mammary adenocarcinoma cloned tumor cell lines of high (MTLn3) or low (MTC) metastatic potential. While MTC cells expressed barely detectable amounts of EGFR, MTLn3 cells expressed readily detectable levels of functional receptors. A full length cDNA of the human EGFR (HER) was introduced by infection with a retroviral vector into MTC cells. Expression of HER was stable and receptors were functional with respect to surface expression, ligand binding and EGF-stimulated phosphorylation. Independent clones of the transfectants were isolated and characterized. Ligand stimulation of MTC HER cells and derived clones led to enhanced adhesion of cells to extracellular matrix proteins. Implantation of cells intravenously into female nu/nu mice revealed ligand-dependent enhancement of lung colonizing potential of EGFR-expressing cells.

Topics: Adenocarcinoma; Animals; Cell Adhesion; Epidermal Growth Factor; ErbB Receptors; Extracellular Matrix; Female; Ligands; Lung Neoplasms; Mammary Neoplasms, Experimental; Mice; Mice, Nude; Neoplasm Metastasis; Neoplasm Transplantation; Phosphorylation; Rats; Transfection

The metastatic variants of human lung adenocarcinoma cell line DMS4C were established by selection in vivo. Lung, brain, spleen and liver metastatic tumors derived from intracarotid inoculation of athymic BALB/c mice were collected, and their corresponding variant cell lines established. The epidermal growth factor (EGF) receptor expression of the parental cell line as well as the metastatic variant cell lines were investigated. 125I-labeled EGF binding assays showed that there were two types of EGF receptors in both parental and metastatic variants. Compared to DMS4C, the EGF binding capacities were found to be down-regulated by 70, 79, 85 and 89% for lung, spleen, liver and brain variant, respectively. The dissociation constants of spleen, liver and brain EGF receptors were distinct from that of the parental cell line. The EGF receptor autophosphorylation activity of lung variant was shown to be down-regulated as shown by immune complex kinase assay that corresponded to EGF receptor numbers whereas kinase activities of the liver, spleen and brain variants EGF receptors were completely abolished. However, the 170 kilodalton EGF receptor was shown to be unaltered during metastasis. The results indicated that, during metastasis progression, the proliferation of adenocarcinoma cells may have adopted a different growth regulation that is independent of EGF receptor kinase-modulated autocrine pathway. The result also implies that other oncogene may emerge as the major growth regulator for distant metastasis of adenocarcinoma cancer cells. This work provides a model for understanding tumor metastasis progression of human lung cancer.

Topics: Adenocarcinoma; Animals; Brain; Down-Regulation; Epidermal Growth Factor; ErbB Receptors; Humans; Iodine Radioisotopes; Liver; Lung; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Metastasis; Neoplasm Transplantation; Spleen; Tumor Cells, Cultured

Gene expression of growth factors including epidermal growth factor (EGF), transforming growth factor alpha (TGF alpha), epidermal growth factor receptor (EGFR), oncogenes such as c-myc, N-ras, c-erbB2 and tumor suppressor gene P53 were studied in 4 human lung cancer cell lines using Northern blot technique. Among these cell lines were 2 adenocarcinoma cell lines, one large cell carcinoma cell line and one small cell carcinoma cell line. Expression of EGF and TGF alpha mRNAs were found in all 4 cell lines and EFGR mRNA was seen in 3 out of 4 cell lines. Among these cell lines, 2 cell lines with weaker expression of EGF and TGF alpha, expressed c-myc mRNA. Another 2 cell lines had no c-myc but expressed large amounts of EGF and TGF alpha mRNA. No expression of N-ras, c-erbB2 and p53 were found in these cell lines. The results indicate the presence of autocrine loop of growth factors in these cancer cells. The autostimulation of growth factors may be the main cause for the uncontrolled growth of cancer cells. After treating the cancer cells with EGF, anti-EGF and anti-EGFR antibodies, EGF was found to exert a mild stimulating effect on the growth of one cell line, but no effect on the other cell lines. Anti-EGF and anti-EGFR antibodies inhibited the cell growth on all cell lines. These results provided further evidence for the presence of autocrine loop of growth factors in these lung cancer cell lines.

Topics: Adenocarcinoma; Antibodies, Neoplasm; Carcinoma, Non-Small-Cell Lung; Carcinoma, Small Cell; Epidermal Growth Factor; ErbB Receptors; Gene Expression; Genes, Tumor Suppressor; Humans; Lung Neoplasms; RNA, Messenger; Transforming Growth Factor alpha; Tumor Cells, Cultured

We examined the effects of epidermal growth factor (EGF) on metastatic and in vitro invasive capacity of weakly malignant ER-1 cells derived from a rat mammary carcinoma cell line, c-SST-2. EGF enhanced the metastatic capacity and in vitro invasiveness to reconstituted basement membrane, Matrigel, of ER-1 cells in a dose-dependent fashion. EGF-stimulated invasiveness was inhibited by anti-EGF antibody, which is able to neutralize the binding of EGF to EGF receptor, in the invasion assay system. EGF stimulated chemotactic migration toward fibronectin, laminin or newborn rat fibroblast-conditioned medium which was used as a chemoattractant in the in vitro invasion assay, but showed neither adhesion to Matrigel nor production of gelatinase and plasminogen activators. These results suggested that the increased metastatic and invasive capacity of ER-1 cells by EGF might be due to the increase in cell motility.

Topics: Animals; Basement Membrane; Cell Adhesion; Cell Movement; Collagen; Drug Combinations; Epidermal Growth Factor; ErbB Receptors; Female; Laminin; Lung Neoplasms; Mammary Neoplasms, Experimental; Neoplasm Invasiveness; Proteoglycans; Rats; Rats, Inbred SHR; Tumor Cells, Cultured

Interleukin-4 (IL-4) plays an important role in activating the immune system against malignant cells. The human interleukin-4 receptor (hIL-4R) is not only expressed by hematopoietic cells but also on a large number of tissue specimens which include colon, breast and lung carcinomas. In this study we report that rhIL-4 has an antiproliferative effect on 2 out of 3 non-small cell lung carcinoma (NSCLC) cell lines in vitro as measured by human tumor cloning assays (HTCA). In comparison, rhIL-4 had no effect on the growth of small cell lung carcinoma cell lines (SCLC) in vitro. The response towards the cytokine is correlated with expression of at least 1500 high affinity receptors/cell for hIL-4 on the responsive cell lines. Xenotransplanting the human lung tumor cell lines into nude mice followed by 12 days of systemic treatment of the mice with rhIL-4 revealed a significant growth retardation of the IL-4R positive NSCLC cell lines when compared with the controls, whereas the growth of the IL-4R negative SCLC cell lines was unaffected also in vivo. Studies of possible mechanisms involved in the antiproliferative effect of rhIL-4 showed that rhIL-4 does not induce apoptosis or modulation of the transcription factor c myc in the responsive NSCLC cell lines. Additionally, the expression of the epidermal growth factor receptor (EGFR), which is discussed as mediating autocrine/paracrine growth stimulation of NSCLC, is unaffected by rhIL-4. However, we have observed that rhIL-4 inhibited G1-S-phase cell cycle progression. We conclude that rhIL-4 has an antiproliferative effect on the growth of some NSCLC in vitro and in vivo. The mechanisms involved remain to be further elucidated.

Topics: Animals; Apoptosis; Carcinoma, Non-Small-Cell Lung; Carcinoma, Small Cell; Cell Cycle; DNA, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Neoplastic; Growth Inhibitors; HL-60 Cells; Humans; Interleukin-4; Lung Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Transplantation; Proto-Oncogene Proteins c-myc; Recombinant Proteins; RNA, Messenger; RNA, Neoplasm; Transplantation, Heterologous; Tumor Cells, Cultured

a rare case of primary choriocarcinoma of the lung in a male is described with immunohistochemistry for human chorionic gonadotropin (hCG), epidermal growth factor (EGF) and EGF-receptor. The extragonadal trophoblastic origin of this pulmonary carcinoma was definitely confirmed by an autopsy examination, and hCG-production and hCG-positive staining of the tumor cells. Furthermore, the tumor cells clearly expressed EGF and its receptor which play an important role in the proliferation and differentiation of normal and neoplastic trophoblasts of the uterus. Our present case suggests that EGF may act in an autocrine manner in the tumor cells of primary pulmonary choriocarcinoma.

Topics: Aged; Choriocarcinoma; Chorionic Gonadotropin; Epidermal Growth Factor; ErbB Receptors; Fatal Outcome; Humans; Immunohistochemistry; Lung Neoplasms; Male

Histochemical activity and immunoreactivity of nitric oxide synthase (NOS, EC 1.14.13.39) have been recently demonstrated in human lung epithelium. However, the molecular nature of NOS and the regulation and function of the enzyme(s) in the airway is not known. A549 cells (human alveolar type II epithelium-like), BEAS 2B cells (transformed human bronchial epithelial cells), and primary cultures of human bronchial epithelial cells all exhibited constitutive NOS activity that was calcium dependent and inhibitable by the NOS inhibitor NG-monomethyl-L-arginine. Nitric oxide production by epithelial cells was enhanced by culture in the presence of interferon gamma, interleukin 1 beta, tumor necrosis factor alpha, and lipopolysaccharide; the NOS activity expressed under these conditions showed less dependence on calcium, reminiscent of other inducible forms of NOS. Two distinct NOS mRNA species, homologous to previously identified constitutive brain (type I) and inducible hepatic (type II) NOS, were demonstrated by reverse transcription-polymerase chain reaction in all cell lines. Northern analysis confirmed the expression of inducible NOS mRNA. Cell culture with epidermal growth factor, a principal regulator of epithelial cell function, decreased inducible NOS activity by posttranscriptional action but did not affect constitutive NOS activity. The coexistence of constitutive and inducible NOS in human alveolar and bronchial epithelial cells is consistent with a complex mechanism evolved by epithelial cells to protect the host from microbial assault at the air/surface interface while shielding the host from the induction of airway hyperreactivity.

Topics: Adenocarcinoma; Amino Acid Oxidoreductases; Base Sequence; Cell Line; Cytokines; DNA Primers; Enzyme Induction; Epidermal Growth Factor; Epithelium; Gene Expression Regulation, Enzymologic; Humans; Interferon-gamma; Interleukin-1; Lung; Lung Neoplasms; Molecular Sequence Data; Nitric Oxide Synthase; Polymerase Chain Reaction; Pulmonary Alveoli; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Malignant cells frequently acquire a certain independency of exogenous growth factors via the coexpression of epidermal growth factor receptor (EGFR) and epidermal growth factor (EGF)-related molecules. In the present study we investigate a possible involvement of EGF-related molecules in the growth of human lung mesothelioma. Four well-characterised cell lines are analysed for their responsiveness to exogenous EGF and transforming growth factor alpha (TGF-alpha) as well as for coexpression of EGFR and EGF/TGF-alpha. Both growth factors are able to stimulate DNA synthesis in three cell lines, although the degree of responsiveness is very variable, but neither EGF nor TGF-alpha has an effect on the cell line ZL34. In contrast, no heterogeneity is observed in the expression of EGFR, which is similarly high in all cell lines. Analysis of cell supernatants reveals that, whereas no EGF is detected, TGF-alpha is released by two cell lines. Furthermore, these two cell lines, ZL5 and ZL34, are shown to express the membrane anchored precursor pro-TGF-alpha. Thus, coexpression of EGFR and TGF-alpha is observed on two mesothelioma cell lines. The potential autocrine mitogenic role of TGF-alpha in these two cell lines was tested using neutralising antibodies against TGF-alpha and EGFR. In ZL5 cells DNA synthesis was not affected by the presence of neutralising antibodies, indicating that an external autocrine mitogenic pathway is not active in these cells. In ZL34 cells, however, the potential autocrine loop could be disrupted, as DNA synthesis was significantly reduced in the presence of neutralising antibodies. This result gives strong evidence for an autocrine role of TGF-alpha in the growth of the mesothelioma cell line ZL34.

Topics: Antibodies, Neoplasm; Cell Division; Cell Membrane; Culture Media; DNA, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Flow Cytometry; Humans; Immunoblotting; Lung Neoplasms; Mesothelioma; Protein Precursors; Transforming Growth Factor alpha; Tumor Cells, Cultured

Topics: Animals; Cell Division; Cytokines; Epidermal Growth Factor; Lung Neoplasms; Lymph Nodes; Macrophages; Mammary Neoplasms, Experimental; Mutation; Neoplasm Invasiveness; Rats; Tumor Cells, Cultured

Gastrin-releasing peptide (GRP), the mammalian form of the amphibian peptide bombesin, may act as a growth-regulatory peptide in the lung. Cultured human bronchial epithelial (HBE) cells have previously been found to proliferate in response to GRP or bombesin. Three receptors have been identified for bombesin and its analogs, namely gastrin-releasing peptide receptor (GRPR), neuromedin B receptor (NMBR), and bombesin receptor subtype 3 (BRS-3). The human genes for these receptors have been cloned and sequenced. We used the polymerase chain reaction to detect transcripts for these three receptor subtypes in HBE cells cultured from bronchial mucosal biopsies. Primers specific for each receptor subtype were used to amplify DNA fragments from reverse-transcribed mRNA isolated from these cultures. An amplified fragment was detected for GRPR in seven of nine cases, for NMBR in six of 10 cases, and the BRS-3 in three of nine cases. We have also amplified message for NMBR in five of five fresh bronchial biopsies, but message for GRPR could not be detected. In preliminary evaluation of two biopsies, there was also no evidence of BRS-3 expression. Additionally, we have detected transcripts for GRPR and NMBR in four of seven and seven of seven cases, respectively, of cultured non-small cell lung carcinomas (NSCLC). BRS-3 expression was seen in one of seven carcinomas in culture. Identity of the amplified fragments was confirmed by restriction enzyme digestion and Southern blotting. Furthermore, GRP induced expression of the early-response gene, c-jun, in an HBE cell culture and an NSCLC cell line. GRP also promoted colony formation in the NSCLC cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Base Sequence; Bronchi; Carcinoma, Small Cell; Epidermal Growth Factor; Epithelium; Gastrin-Releasing Peptide; Gene Expression; Genes, jun; Humans; Lung Neoplasms; Molecular Sequence Data; Peptides; Polymerase Chain Reaction; Receptors, Bombesin; RNA, Messenger; Tumor Cells, Cultured

The prognosis of patients with follicular (FTC) and papillary (PTC) thyroid cancer depends on age and the size and extent of the tumor. Differentiated thyroid cancers bind more epidermal growth factor (EGF) than normal thyroid tissue, but the role of EGF in the proliferation and invasion of thyroid cancer is unknown. We investigated the effects of EGF on growth, migration, and invasion in a follicular thyroid cancer that metastasized to cervical lymph nodes and the lung (FTC 133, primary; FTC 236, lymph node; and FTC 238, lung metastasis) and in a papillary thyroid cancer (PTC-UC3). As measured by the formazan method (dimethylthiazol-diphenyltetrazolium bromide), EGF caused a dose- and time-dependent increase in the growth of FTC 133 and PTC-UC3 by 25%, but its stimulatory effect on growth of the metastatic FTC subclones was smaller (FTC 236, 14%; FTC 238, 8%; P < 0.001). EGF also enhanced the ability of all cell lines to migrate (through 8-microns pore membranes without Matrigel) or invade (membranes with Matrigel). Migration of FTC 133 was enhanced from 86% migrated tumor cells to 95% after 72 h (P < 0.02). Again, stimulation by EGF was lower in FTC 236 and FTC 238. EGF increased migration in PTC-UC3 from 49% to 58%. EGF stimulated invasion of FTC 133 from 17.5% to 24.9%. In the absence of EGF, FTC 238 was the most invasive tumor, but, again, the EGF stimulatory effect was less pronounced than in the primary tumor. EGF stimulated the invasion of PTC-UC3 from 10.9% to 14.3% (P < 0.03). EGF also stimulated the growth of thyroid cancer xenografts in nude mice. Although all FTC cell lines were 100% tumorigenic in nude mice, PTC-UC3 was less tumorigenic. However, after sc inoculation of EGF-pretreated tumor cells, 7 of 10 animals developed tumors (mean size, 2.3 cm3) compared to 2 of 10 animals (mean size, 1.4 cm3) in the control group (P < 0.02). In summary, EGF stimulates the growth and invasion of differentiated thyroid cancer cells in culture and in nude mice. Escape from growth factor control, such as in FTC 236 and FTC 238, may be an important step in the development of metastatic thyroid cancer.

Topics: Adenocarcinoma, Follicular; Animals; Carcinoma, Papillary; Cell Division; Cell Movement; Epidermal Growth Factor; Humans; Lung Neoplasms; Lymphatic Metastasis; Mice; Mice, Nude; Neoplasm Invasiveness; Neoplasm Transplantation; Thyroid Neoplasms; Tumor Cells, Cultured

The distribution of epidermal growth factor (EGF) and EGF receptor (EGFR) in adult human lung was examined by immunohistochemistry and immunoelectron microscopy. EGF immunoreactivity was observed in secretory granules and endoplasmic reticulum of serous acinar cells of bronchial glands. EGFR immunoreactivity was detected at the cell membrane of the basal cells and the bronchial surface nonciliated cells (indifferent cells) in the bronchus, the nonciliated bronchiolar cells (Clara cells) and the type II pneumocytes in peripheral lung. These results suggest that EGF is secreted into bronchial surface fluid from the bronchial glands and stimulates proliferation and/or differentiation of basal cells and indifferent cells of the bronchus, although the source of EGF responsive to Clara cells and the type II pneumocytes of peripheral lung was not determined.

Topics: Aged; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Immunohistochemistry; Lung; Lung Neoplasms; Male; Microscopy, Immunoelectron; Middle Aged

The regulation of parathyroid hormone-related protein (PTH-rP) mRNA levels and immunoreactive (ir)PTH-rP formation by peptide growth factors, particularly transforming growth factor-beta (TGF-beta) and epidermal growth factor (EGF), in squamous cell carcinomas of gynecologic origin is largely unknown.. PTH-rP mRNA levels were evaluated by Northern analysis in A431 cells (derived from a human vulvar epidermoid carcinoma) and ME-180 cells (derived from a human papillomavirus-infected squamous cell carcinoma of the cervix). PTH-rP protein levels in cell culture media were evaluated using both radioimmunoassay and immunoradiometric assay techniques. These results were compared with those from a lung carcinoid cell line known to produce PTH-rP, namely, NCI-H727 cells.. TGF-beta 1 or EGF treatment caused an increase in the levels of PTH-rP mRNA in A431 cells; these increases in PTH-rP mRNA were detectable after 60 minutes of treatment, were maximal at approximately 4-8 hours, and were approximately additive. Immunoreactive PTH-rP was not detectable (using two different PTH-rP immunoassays) in the culture medium or cell sonicates of A431 cells before or after treatment with TGF-beta 1, EGF, or TGF-beta 1 plus EGF. ME-180 cells responded to EGF (but not to TGF-beta 1) with an increase in the level of PTH-rP mRNA as early as 2 hours; irPTH-rP was present (by use of either immunoassay) in the medium of these cells at 8 and 24 hours. In NCI-H727 (human lung carcinoid) cells, TGF-beta 1 and EGF acted alone and synergistically to effect increases in PTH-rP mRNA and the accumulation of irPTH-rP.. TGF-beta 1 and EGF regulation of PTH-rP gene expression in squamous cell carcinomas of gynecologic origin is unique for each cell line studied and different from that in human lung carcinoid cells.

Topics: Base Sequence; Carcinoid Tumor; Carcinoma, Squamous Cell; Epidermal Growth Factor; Female; Gene Expression Regulation, Neoplastic; Genital Neoplasms, Female; Humans; Lung Neoplasms; Molecular Sequence Data; Neoplasm Proteins; Parathyroid Hormone; Parathyroid Hormone-Related Protein; Proteins; RNA, Messenger; Transforming Growth Factor beta; Tumor Cells, Cultured; Uterine Cervical Neoplasms; Vulvar Neoplasms

Recently, several investigators reported the effects of suramin, a drug used in the treatment of trypanosomiasis, onchocerciasis, and an inhibition of the binding of some growth factors to cell surface receptors. In present study, therefore, we examined the effects of epidermal growth factor(EGF) and suramin on cell proliferation and cell cycle kinetics of an established cell line from human lung cancer(PC-13). EGF showed a stimulatory effect on cell proliferation of PC-13, suggesting EGF behaves as a growth factor of PC-13. On the other hand, suramin inhibited the stimulatory effect of EGF to PC-13 in a dose dependent manner. In addition, it was also observed that suramin inhibits the cell cycle progression from G0/G1 phase to S phase. These results indicate that suramin may withhold the cell proliferation and cell growth via suppression of the EGF cell stimulatory effects.

Topics: Cell Cycle; Cell Division; Epidermal Growth Factor; Humans; Lung Neoplasms; Suramin; Tumor Cells, Cultured

A series of rat monoclonal antibodies (MAbs) has been generated against the extracellular domain of the receptor for EGF which block the binding of EGF and TGF alpha to the receptor and inhibit the growth in vitro of a range of carcinoma cell lines that over-express the receptor for EGF. Some of these antibodies were able also to induce the complete regression of xenografts of EGFR-over-expressing tumours when treatment was started, either at the time of tumour inoculation or later when the tumours were established. The most effective of these antibodies was ICR62, which was also able to activate host immune effector functions. We conclude that antibodies which block growth-factor-ligand interaction can have a profound influence on the proliferative capacity of tumour cells in vivo and may have useful clinical application.

Topics: Animals; Antibodies, Monoclonal; Binding Sites; Breast Neoplasms; Carcinoma; Cell Division; Cell Line; Epidermal Growth Factor; ErbB Receptors; Female; Fibroblasts; Head and Neck Neoplasms; Humans; Immunotherapy; Lung Neoplasms; Mice; Mice, Nude; Ovarian Neoplasms; Rats; Rats, Inbred Strains; Recombinant Proteins; Transforming Growth Factor alpha; Transplantation, Heterologous; Tumor Cells, Cultured; Urinary Bladder Neoplasms; Vulvar Neoplasms

Cluster 13 was defined by 2 independently derived murine monoclonal antibodies (MAbs), RS7 (IgG1) and MR54 (IgG2a), which were raised against human squamous-cell carcinoma of the lung and a human ovarian-carcinoma cell line, respectively. Immunologic and biochemical evidence demonstrated that RS7 and MR54, as well as 2 additional MAbs, MR6 (IgG2a) and MR23 (IgG1), generated in the same fusion as MR54, recognize the same antigen, a 46- to 48-kDa glycoprotein. Evaluation of the expression of antigen on the surface of tumor cell lines, Western blotting analyses, competitive binding studies, and double-determinant ELISA assays, support this conclusion. Two distinct epitopes are defined by these MAbs. In order to further characterize this antigen, amino-acid-sequence analyses were performed on peptides derived from antigen purified by affinity chromatography with MAb RS7. The sequence data obtained from 2 peptides, which were independently generated by CNBr cleavage and trypsin digestion respectively indicated identity to GA733-1. The GA733-1 genomic DNA sequence predicted a type-1 membrane protein of 35 kDa, with 4 potential N-linked glycosylation sites. The GA733-1 protein product has not been identified previously, and MAbs to this tumor-associated antigen were not previously known.

Topics: Amino Acid Sequence; Animals; Antibodies, Monoclonal; Antigens, Neoplasm; Blotting, Western; Carcinoma, Squamous Cell; Cell Adhesion Molecules; Cell Line; Epidermal Growth Factor; Epithelial Cell Adhesion Molecule; Female; Humans; Immunoglobulin G; Lung Neoplasms; Mice; Molecular Sequence Data; Multigene Family; Neoplasms; Ovarian Neoplasms; Sequence Homology, Amino Acid; Tumor Cells, Cultured

The sensitivity of human breast and lung cancer cell lines to TGF-alpha-PE40, a novel chimeric recombinant cytotoxin composed of two independent domains, (i) TGF-alpha and (ii) a 40 kDa segment of the Pseudomonas exotoxin protein, PE-40, was investigated. Toxicity varied widely, correlated with epidermal growth factor receptor (EGFR) levels (P = 0.01) and was greatly reduced by EGF, indicating that binding of TGF-alpha-PE40 to EGFR is important in mediating toxicity. Cell lines expressing low EGFR levels were most highly protected by EGF, indicating that normal (low EGFR-expressing) tissue may be selectively protected by EGF in vivo. P-glycoprotein did not confer resistance to TGF-alpha-PE40, and toxicity was unaffected by multidrug resistance-modulating agents (cyclosporin A, tamoxifen, verapamil), indicating a role for TGF-alpha-PE40 in the clinical management of drug-resistant tumours.

Topics: Breast Neoplasms; Carcinoma, Non-Small-Cell Lung; Carcinoma, Small Cell; Cell Division; Cell Line; Cyclosporine; Dose-Response Relationship, Drug; Drug Interactions; Epidermal Growth Factor; ErbB Receptors; Exotoxins; Female; Humans; Lung Neoplasms; Recombinant Fusion Proteins; Recombinant Proteins; Tamoxifen; Transforming Growth Factor alpha; Tumor Cells, Cultured; Verapamil

The ability of a chimeric toxin containing transforming growth factor alpha (TGF alpha) and truncated Pseudomonas exotoxin A to inhibit NSCLC growth was investigated. TGF alpha-PE40 inhibited binding of 125I-EGF to NSCLC cell lines with an IC50 value of 0.5-3 micrograms/ml. Similarly, other forms of the fusion protein, TGF alpha-PE38 and TGF alpha-PE40Asp553, which have active TGF alpha binding domains, inhibited specific 125I-EGF binding to NSCLC cells with IC50 values of 0.1-2 and 0.05-05 microgram/ml respectively. TGF alpha-PE40 inhibited 35S-methionine uptake by NSCLC cells with an ED50 value of 1-30 ng/ml. TGF alpha-PE38, which has one of the two disulfide pairs of PE40, inhibited amino acid uptake with ED50 values of 3-50 ng/ml whereas TGF alpha-PE40Asp553, which lacks ADP ribosylation activity, had an ED50 > 100 ng/ml. TGF alpha-PE40 inhibited colony formation of NSCLC cells with an LD50 value of 0.008-0.1 ng/ml. Similarly, TGF alpha-PE38 inhibited NSCLC colony formation with LD50 values of 0.002-0.1 ng/ml whereas TGF alpha-PE40Asp553 had an LD50 > 10 ng/ml. Also, TGF alpha-PE40 and TGF alpha-PE38 inhibited NSCLC xenograft formation in nude mice whereas TGF alpha-PE40Asp553 was inactive. These data suggest that TGF alpha-PE40 and TGF alpha-PE38 may be useful agents to inactivate NSCLC cells.

Topics: Animals; Carcinoma, Non-Small-Cell Lung; Cell Division; Drug Screening Assays, Antitumor; Epidermal Growth Factor; ErbB Receptors; Exotoxins; Female; Humans; Lung Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Transplantation; Protein Biosynthesis; Proteins; Recombinant Fusion Proteins; Transforming Growth Factor alpha; Tumor Cells, Cultured

Topics: ABO Blood-Group System; Antigens, Tumor-Associated, Carbohydrate; Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; DNA, Neoplasm; Epidermal Growth Factor; Female; Humans; Lung Neoplasms; Male; Prognosis; Receptors, Cell Surface; Tumor Cells, Cultured

Autocrine neoplastic growth circuits are based on excess synthesis of growth factors and/or cognate membrane receptors. We analyzed by immunohistochemistry 19 typical lung carcinoids for the expression of the epidermal growth factor (EGF), transforming growth factor alpha (TGF alpha), EGF receptor (EGFr), and EGFr-related c-erbB-2 protein (p185). Thirteen tumors (68%) were positive for TGF alpha, 11 for EGFr (58%), three for EGF (16%), and four for p185 (21%). Six carcinoids (32%) were consistently negative for these gene products. The following patterns of coexpression could be documented: TGF alpha, EGFr, EGF, and p185: two cases (11%); TGF alpha, EGFr, and EGF: one case (5%); TGF alpha, EGFr, and p185: two cases (11%); TGF alpha and EGFr: six cases (32%); TGF alpha by itself: two cases (11%). Thus, EGFr was coexpressed with its ligands, TGF alpha and EGF, and the receptor encoded by c-erbB-2 was detected in carcinoids positive for EGFr and TGF alpha. Therefore, alterations of EGF/EGFr-related growth control pathways may be implicated in the pathogenesis of pulmonary carcinoids via the establishment of autocrine growth promoting circuits, as documented in adenocarcinomas and squamous cell carcinomas of the lung.

Topics: Adenocarcinoma; Carcinoma, Squamous Cell; Cell Transformation, Neoplastic; Epidermal Growth Factor; ErbB Receptors; Humans; Immunohistochemistry; Lung Neoplasms; Proto-Oncogene Proteins; Receptor, ErbB-2; Transforming Growth Factor alpha

From 1976 to 1991, 151 cases of small cell carcinoma of the lung were diagnosed by fiberoptic bronchoscopic biopsy at our institution. One hundred twenty-eight of 151 cases provided suitable material for the examination of the morphologic changes in the bronchial surface epithelium. Thirty-seven percent of the cases showed normal bronchial epithelium, 47% showed benign squamous metaplasia, 9% showed atypical squamous metaplasia, and 5% showed squamous cell carcinoma in situ. Immunohistochemical examination for bombesin and epidermal growth factor was performed on selected biopsy specimens. The biopsy specimens chosen for immunohistochemistry included 20 specimens that showed normal bronchial epithelium, 20 specimens with benign squamous metaplasia, 12 specimens with atypical squamous metaplasia, and seven specimens with squamous cell carcinoma in situ. All the specimens showed positive staining with anti-bombesin. With anti-epidermal growth factor 10% of biopsy specimens with normal epithelium showed positive staining. The positive reaction increased from 25% for biopsy specimens with benign squamous metaplasia to 58% for biopsy specimens with atypical squamous metaplasia and to 71% for biopsy specimens with carcinoma in situ. These findings suggest a connection between epidermal growth factor production by small cell carcinoma of the lung cells and changes in the bronchial surface epithelium.

Topics: Biopsy; Bombesin; Bronchi; Carcinoma in Situ; Carcinoma, Small Cell; Carcinoma, Squamous Cell; Epidermal Growth Factor; Epithelial Cells; Epithelium; Humans; Immunohistochemistry; Lung Neoplasms; Metaplasia

We report that cytosine arabinoside (Ara-C), a cytosine analogue that at low doses causes phenotypical changes on human leukemia cells in vitro and in vivo, induces growth inhibition of oropharyngeal cancer KB and lung adenocarcinoma A549 cell lines. An increase in the number of epidermal growth factor and transferrin receptors (EGFR, TrfR) is induced by Ara-C on these cells. Maximal EGFR up-regulation occurs 96 h after the beginning of Ara-C exposure while maximal TrfR up-regulation is detected 24 h later. These effects occur without changes in the affinity of EGFR and TrfR for their ligands. Two classes of EGF-binding sites with a Kd of 0.055 nM and 2.3 nM respectively, and one class of transferrin-binding sites with a Kd of about 4 nM are detected on both untreated and Ara-C-treated KB cells. [3H]Thymidine uptake is clearly stimulated on KB cells by nanomolar concentrations of EGF and transferrin, whereas in Ara-C-treated cells [3H]thymidine uptake is not increased by EGF and transferrin under conditions where maximal EGFR and TrfR up-regulation occurs. The enhanced EGF and transferrin binding is paralleled by a twofold increase of in vitro targeting of Ara-C-treated KB and A549 cells with anti-EGFR 108.1 mAb and anti-TrfR OKT9 mAb. We propose that Ara-C could provide a new approach for the improvement of the therapeutic index of anti-EGFR and anti-TrfR immunoconjugates.

Topics: Adenocarcinoma; Antibodies, Monoclonal; Cell Division; Cytarabine; Dose-Response Relationship, Drug; Epidermal Growth Factor; ErbB Receptors; Humans; Immunotherapy; Iodine Radioisotopes; KB Cells; Lung Neoplasms; Neoplasms; Radioimmunoassay; Receptors, Cell Surface; Receptors, Transferrin; Time Factors; Transferrin; Tumor Cells, Cultured; Up-Regulation

We examined factors promoting malignant progression using a weakly malignant variant cell line, ER-1, derived from c-SST-2, a rat mammary carcinoma. ER-1 cells were converted to a highly malignant phenotype (highly tumorigenic, metastatic, invasive in vitro) by the in vitro/in vivo interaction with host cells reactive to foreign body. Epidermal growth factor (EGF) and transforming growth factor-beta (TGF-beta) produced by host reactive cells, transiently enhanced the tumorigenicity and in vitro invasiveness of ER-1 cells into an endothelial cell monolayer. The host reactive cells also produced oxygen radicals and induced mutations in ER-1 cells. It is speculated that mutations induced by host reactive cells cause cellular diversification, including the emergence of highly malignant variant cells whose growth is selectively promoted by growth factors such as EGF and TGF-beta.

Topics: Animals; Cell Communication; Cell Division; Cell Movement; Epidermal Growth Factor; Lung Neoplasms; Mammary Neoplasms, Experimental; Neoplasm Invasiveness; Rats; Transforming Growth Factor beta; Tumor Cells, Cultured

The binding of 125I-epidermal growth factor (EGF) to the plasma membranes of 54 samples of human lung tumors was determined. These included 34 squamous cell carcinomas and 20 adenocarcinomas. Twenty samples of histologically normal lung excised surgically along with the tumors were used as controls. Most of the plasma membranes showed an EGF receptor level higher than that of normal tissue. A moderate increase in the amount of 125I-EGF bound (2-5 fold) was observed in the majority of the tumors. Only a few cases (5-10% of the total) showed a large increase (> than 10 fold). The binding of 125I-EGF was compared with clinical stages and grades of differentiation. No correlation between the stage of the tumor and 125I-EGF binding was observed. However, the highest levels of EGF receptor (EGF-R) were found in poorly differentiated squamous cell carcinomas. The total amount and the distribution pattern of gangliosides and phospholipids were analyzed in individual tumors. A decrease in GD1b, GD1a and sphingomyelin and an increase in GM1 and GM3 was observed. No correlation was detected when tumors with the highest or lowest levels of gangliosides or phospholipids were compared with tumors exhibiting the highest binding of 125I-EGF.

Topics: Adenocarcinoma; Adult; Aged; Carcinoma, Squamous Cell; Cell Membrane; Epidermal Growth Factor; ErbB Receptors; Gangliosides; Humans; Iodine Radioisotopes; Lung Neoplasms; Membrane Lipids; Middle Aged; Phospholipids

Lipocortin-1 mediates growth inhibition of glucocorticoids in A549 cells by suppressing the release of PGE2 necessary for their proliferation. We now show that 2 peptide fragments derived from the N-terminal portion of lipocortin-1 corresponding to amino-acids 13-25 and 21-33 also inhibited A549 cell growth and suppressed release of PGE2, whereas peptides 1-12 and 13-25 (Phe21; in which the tyrosine at position 21 was replaced by a phenylalanine residue) were inactive. Similarly, peptide 21-33 (Phe21) and a scrambled sequence of 13-25 failed to inhibit cell growth. Moreover, the EGF-induced stimulation of cell proliferation and PGE2 release in these cells was blocked by peptides 13-25 and 21-33, and also by peptides 1-12, 13-25 (Phe21) and 21-33 (Phe21), but not by a scrambled sequence of peptide 13-25.

Topics: Adenocarcinoma; Amino Acid Sequence; Annexin A1; Cell Division; Dinoprostone; Epidermal Growth Factor; Humans; Lung Neoplasms; Molecular Sequence Data; Peptide Fragments; Tumor Cells, Cultured

Epidermal growth factor (EGF) and its receptor (EGFR) were measured in 60 breast cancers (BC), 6 benign mammary tumors (BM), 8 samples of normal breast (NB), 6 endometrial carcinomas (EC) and 30 lung cancers (LC). EGF was measured in plasma, saliva and urine from 20 patients with BC, before and after tumor excision, and in 8 patients with metastatic disease. The median EGF in BM and BC was significantly higher (P < 0.05) than in NB. No significant correlation between EGF and EGFR was found in BC. Neither tumor excision nor the spreading of the disease significantly modified the EGF concentrations in biological fluids. In LC there was an inverse relationship between EGF and EGFR (rs = -0.36; P = 0.09), which disappeared in normal lung. It is concluded that EGF may play a role in malignant transformation; however, the weak correlation between EGF and EGFR lessens the importance of EGF in either autocrine or paracrine stimulation of tumor growth.

Topics: Adult; Body Fluids; Breast Neoplasms; Endometrial Neoplasms; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Lung Neoplasms; Receptors, Estradiol; Saliva

Topics: Carcinoma, Non-Small-Cell Lung; Epidermal Growth Factor; Humans; Lung Neoplasms; Plasminogen Inactivators; Tissue Plasminogen Activator; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator

Epidermal growth factor (EGF) receptor expression was evaluated in a panel of 21 small cell lung cancer cell lines with radioreceptor assay, affinity labeling, and Northern blotting. We found high-affinity receptors to be expressed in 10 cell lines. Scatchard analysis of the binding data demonstrated that the cells bound between 3 and 52 fmol/mg protein with a KD ranging from 0.5 x 10(-10) to 2.7 x 10(-10) M. EGF binding to the receptor was confirmed by affinity-labeling EGF to the EGF receptor. The cross-linked complex had a M(r) of 170,000-180,000. Northern blotting showed the expression of EGF receptor mRNA in all 10 cell lines that were found to be EGF receptor-positive and in one cell line that was found to be EGF receptor-negative in the radioreceptor assay and affinity labeling. Our results provide, for the first time, evidence that a large proportion of a broad panel of small cell lung cancer cell lines express the EGF receptor.

Topics: Blotting, Northern; Carcinoma, Small Cell; Cell Line; Epidermal Growth Factor; ErbB Receptors; Humans; Kinetics; Lung Neoplasms; Molecular Weight; Radioligand Assay; RNA, Messenger; Transforming Growth Factor alpha; Transforming Growth Factor beta

The response to growth factor stimulation was evaluated in clonally derived rat bladder carcinoma cell lines, ranging from nontumorigenic to tumorigenic and metastatic, in athymic nude mice. In the nontumorigenic cell line D44c, epidermal growth factor (EGF)/transforming growth factor (TGF) alpha weakly stimulated anchorage-dependent, but not -independent, growth. In tumorigenic/nonmetastatic cells (G1-200 Cl-17), EGF/TGF-alpha stimulated markedly anchorage-independent, but marginally anchorage-dependent growth, whereas TGF-beta 1 inhibited anchorage-independent growth and DNA synthesis. In the highly tumorigenic/metastatic cell line LMC19, EGF/TGF-alpha stimulated anchorage-dependent growth weakly and anchorage-independent growth strongly. In these cells, TGF-beta 1 did not inhibit anchorage-independent growth and DNA synthesis but increased the size of colonies irrespective of the presence of EGF, and some cells were scattered around colonies in soft agar. None of the cell lines showed evidence of TGF-alpha-specific mRNA transcription. Expression of TGF-beta 1 mRNA increased in parallel to the biological aggressiveness of the cell lines. Highly tumorigenic and metastatic cells also demonstrated gelatinase activity involving 72 kilodalton and 92 kilodalton types. Our data suggest that the growth-stimulatory effect of EGF/TGF-alpha in soft agar may be limited to cells that are already tumorigenic and that EGF/TGF-alpha is not effective in making nontumorigenic cells become tumorigenic (or in making nontumorigenic cells grow in soft agar).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Carcinoma, Transitional Cell; Cell Division; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Neoplastic; Genes, p53; Genes, ras; Lung Neoplasms; Male; Mice; Mice, Nude; Neoplasm Proteins; Neoplasm Transplantation; Proto-Oncogene Proteins; Rats; RNA, Messenger; RNA, Neoplasm; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured; Urinary Bladder Neoplasms

In this study, 3 human pancreatic adenocarcinoma cell lines (PC-1, PC-2 and PC-3) and 3 other human cancer cell lines (adenocarcinoma of lung, LETP; gastric carcinoma, SGL7901; and breast carcinoma, BCP37) were investigated by adding EGF, anti-EGF antiserum and anti-EGFR monoclonal antibody into culture medium. EGF was found to exert a mild stimulating effect on the growth of PC-1 and LETP cells, but had no effect on the other 4 cell lines. Anti-EGF and anti-EGFR antibodies inhibited the proliferation of PC-1, LETP and SGL7901 cells. No significant effect on the other 3 cell lines was seen. By using the Northern blot technique, expression of EGFR mRNA was identified in all 6 cell lines. There were 3 bands (10.5 kb, 5.8 kb and 2.8 kb) of EGFR mRNA in all cell lines except for LETP, in which the 10.5 kb band was absent. The results indicate that the effect of EGF on the growth of cancer cells is very complicated and may involve an unknown regulatory mechanism of cancer cell growth. EGF may exert stimulating or inhibiting effects on cancer cell proliferation, or it may have no effect at all, even though the EGFR gene was expressed in these cell lines.

Topics: Adenocarcinoma; Antibodies, Monoclonal; Cell Division; Epidermal Growth Factor; ErbB Receptors; Humans; Immune Sera; Lung Neoplasms; Pancreatic Neoplasms; RNA, Messenger; Stomach Neoplasms; Tumor Cells, Cultured

Although EGF receptor expression is generally elevated in human lung squamous carcinoma, the biological significance of this phenomenon and the role of EGF and TGF-alpha in this disease are poorly understood. We have investigated three human lung squamous carcinoma cell lines (NX002, CX140 and CX143) and have shown, using an antibody (EGFR1) directed against the EGF receptor, that the majority of cells in all three lines express the EGF receptor. Using a ligand binding assay, Scatchard analysis indicated high concentrations (1,300-2,700 fmol mg-1 protein) of a single low affinity binding site (Kd = 3-5 nM) within these lines. Addition of EGF or TGF-alpha at concentrations greater than 0.1 nM resulted in growth inhibition of all three lines and this was associated with an accumulation of cells in the G2/M phase of the cell cycle. Growth inhibitory effects were not explained by an enhancement of cellular differentiation as monitored by involucrin expression and the ability to form cornified envelopes. While the presence of EGF could not be detected in medium conditioned by the NX002 cell line, mRNA for TGF-alpha was detected in all three lines suggesting the possibility of an autocrine loop. These results together with reports of growth inhibition by EGF and TGF-alpha in other systems suggest that EGF and similar molecules might have a growth regulatory role in lung cancer cells and modulation of such may have therapeutic potential.

Topics: Carcinoma, Squamous Cell; Cell Cycle; Cell Division; Cell Line; Culture Media; Epidermal Growth Factor; ErbB Receptors; Humans; Immunohistochemistry; Lung Neoplasms; Protein Precursors; Tetradecanoylphorbol Acetate; Transforming Growth Factor alpha

Clonogenic cultures from 27 lung cancers were realised. For 68% of cases a clonogenic growth was observed, but no direct correlation could be done with the differentiation of the tumor or with the presence of ganglion metastases. EGF in medium increased the number of colonies obtained in 60% of cases.

Topics: Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Carcinoma, Small Cell; Cell Division; Epidermal Growth Factor; Humans; Lung; Lung Neoplasms; Prognosis; Tumor Cells, Cultured; Tumor Stem Cell Assay

Analogues of somatostatin (SS) and luteinizing hormone-releasing hormone (LH-RH) activate tyrosine phosphatases in MIA PaCa-2 human pancreatic cancer cell line membranes and inhibit growth. We compared the substrates phosphorylated by epidermal growth factor (EGF) to those dephosphorylated by the SS analogue RC-160 (D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Trp-NH2) and [D-Trp6]LH-RH in cancer cell lines such as MIA PaCa-2 (human pancreatic cancer), HCPC (hamster cheek pouch carcinoma), A-549 (human lung cancer), HT-29 (human colon cancer), and R3230AC (breast cancer). EGF phosphorylated proteins of 170, 65, and 60 kDa and analogues of SS and LH-RH promoted the dephosphorylation of these proteins in MIA PaCa-2 and HCPC cell lines. The EGF receptor is 170 kDa. pp60src (60 kDa) is known to be a substrate for EGF receptor. The LH-RH receptor is also 60 kDa. The effects of RC-160 and [D-Trp6]LH-RH were quantitatively different. Examinations of HT-29, A-549, and R3230AC cancer cell lines revealed no phosphorylation by EGF or dephosphorylation by RC-160 and [D-Trp6]LH-RH. In addition to the 170-, 65-, and 60-kDa proteins, 35-kDa proteins were also phosphorylated in some cancer cell lines. This work demonstrates that analogues of SS and LH-RH can reverse the effects of EGF biochemically as well as functionally.

Topics: Amino Acid Sequence; Animals; Antineoplastic Agents; Autoradiography; Breast Neoplasms; Cell Line; Colonic Neoplasms; Epidermal Growth Factor; Gonadotropin-Releasing Hormone; Humans; Kinetics; Lung Neoplasms; Membrane Proteins; Molecular Sequence Data; Pancreatic Neoplasms; Phosphorus Radioisotopes; Phosphorylation; Protein Kinases; Protein-Tyrosine Kinases; Somatostatin; Triptorelin Pamoate; Tyrosine

The growth of human-derived A549 lung carcinoma cells is inhibited by activators of protein kinase C (PKC) such as 12-O-tetradecanoylphorbol- 13-acetate (TPA). In this study, the effect of serum deprivation on TPA-induced growth retardation has been investigated. Cells cultured with 10% FCS and TPA (10(-8) M) stopped growing for 6 days, whereas inhibition of DNA synthesis caused by TPA in cells which were grown in medium containing the serum substitute ultraser lasted for less than 48 hr. The ability of cells to respond to the growth-inhibitory potential of TPA decreased with decreasing amounts of FCS in the cellular medium. Addition of fetuin or epidermal growth factor (EGF) to incubates with serum-deprived cells increased the ability of TPA to affect growth, but addition of platelet-derived growth factor (PDGF), transforming growth factor beta (TGF-beta) or retinoic acid (RA) was without effect. Growth arrest caused by bryostatin I, another PKC activator, was equally transitory in serum-supplemented and serum-deprived cells. Cytosol of serum-deprived cells contained only 32% of specific phorbol ester binding sites compared to cells grown with FCS; PKC enzyme activity and immunodectable protein were similarly reduced in cells grown without FCS. There was no difference in rate of TPA-induced down-regulation of PKC activity and cytosolic phorbol ester receptor sites between cells grown with or without serum.

Topics: alpha-Fetoproteins; Animals; Antineoplastic Agents; Blood; Bryostatins; Cattle; Cell Division; Culture Media; DNA Replication; Enzyme Activation; Epidermal Growth Factor; Humans; Kinetics; Lactones; Lung Neoplasms; Macrolides; Protein Kinase C; Tetradecanoylphorbol Acetate; Tretinoin

The objective of this study is to examine and characterize epidermal growth factor receptor (EGF-R) binding in inhaled plutonium-induced canine lung-tumor tissue and to compare it with that in normal canine lung tissue. Crude membrane preparations from normal and lung-tumor tissue from beagle dogs were examined in a radioreceptor assay, using 125I-labeled epidermal growth factor (EGF) as a ligand. Specific EGF receptor binding was determined in the presence of excess unlabeled EGF. We have examined EGF receptor binding in eight lung-tumor samples obtained from six dogs. Epidermal growth factor receptor binding was significantly greater in lung-tumor samples (31.38%) compared with that in normal lung tissue (3.76%). Scatchard plot analysis from the displacement assay revealed that there was no statistical difference in the binding affinity but significantly higher concentration of EGF-R sites in the lung-tumor tissue (619 fmol/mg) than in normal lung tissue (53 fmol/mg). The increase in EGF-R number in plutonium-induced dog lung tumors does not seem to correlate with increase in the initial lung burden exposure to plutonium. Our results demonstrate that there is a significant increase in EGF-R binding in inhaled plutonium-induced dog lung tumors.

Topics: Adenocarcinoma, Papillary; Administration, Inhalation; Animals; Binding, Competitive; Carcinoma, Bronchogenic; Dogs; Epidermal Growth Factor; ErbB Receptors; Female; In Vitro Techniques; Lung Neoplasms; Male; Membrane Proteins; Neoplasms, Radiation-Induced; Plutonium; Radioligand Assay

Altered and deregulated cellular oncogenes were found in many human solid tumors. Except for a few types of tumors that consistently exhibited specific altered proto-oncogenes, the majority of tumors are associated with a number of transcriptionally activated cellular oncogenes. In the heterologous group of non-small-cell lung cancer (NSCLC), nothing about a specific pattern of proto-oncogene expression is known. Therefore, we investigated the expression of a panel of cellular oncogenes in NSCLC cell lines. DNA and RNA from 11 established NSCLC cell lines (4 adenocarcinoma cell lines, 3 squamous cell carcinoma cell lines, 3 large-cell carcinoma cell lines and 1 mesothelioma cell line) were isolated and analysed using the Southern, dot blot and Northern hybridization technique. c-myc RNA expression was found in all NSCLC cell line, L-myc expression only in 1 adenocarcinoma cell line, N-myc and c-myb expression in none of the 11 cell lines examined. No c-myc amplification could be detected in the DNAs. v-sis-related mRNA was observed in 5/11 cell lines without association to a specific NSCLC subtype. v-src-related mRNA, found in all tested cells, exhibited increased levels in 1 adenocarcinoma cell line (A-549) compared to the other cell lines. Binding sites for epidermal growth factor (EGF) had been described previously in NSCL, therefore we found erbB homologue transcripts coding for the EGF receptor in all NSCLC cell lines. Also, c-raf1-, N-ras-, Ki-ras-, and H-ras-related RNA expression was observed in all lines. We conclude that L-myc, N-myc, and c-myb expression does occur less frequently in NSCLC than in SCLC. Also amplification does not appear to be an important mechanism by which the c-myc proto-oncogene is activated in NSCLC. A specific pattern of oncogene expression could not be detected in NSCLC cells; each cell line examined showed its own pattern. However, transcriptional activation of a proto-oncogene like erbB, ras, raf, src, and c-myc, which are all involved in the progression pathway of EGF, may be a common feature of NSCLC.

Topics: Carcinoma, Non-Small-Cell Lung; Epidermal Growth Factor; Gene Amplification; Gene Expression; Gene Rearrangement; Genes, ras; Humans; Lung Neoplasms; Proto-Oncogene Mas; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-myc; Proto-Oncogene Proteins pp60(c-src); Proto-Oncogenes; RNA; Tumor Cells, Cultured

Most cell lines derived from small cell lung carcinoma grow in an anchorage-independent manner; they neither possess epidermal growth factor binding activity nor express epidermal growth factor receptor (EGFR) mRNA. A variant AD320, which grew in an anchorage-dependent manner with altered morphology, was isolated from the small cell lung carcinoma cell line Lu134 by treatment with the demethylating agent 5-azacytidine. The analysis, using methylation-sensitive restriction enzymes, revealed that the methylation pattern was altered only in the structural region of the EGFR gene; EGFR mRNA and epidermal growth factor binding activity could be detected in the variant. In addition, drastic changes in gene expression including a decrease of creatine kinase B mRNA and an increase of c-myc mRNA were observed. The EGFR in the variant appeared to be an active part of the transmembrane signaling machinery since c-fos and c-jun mRNA accumulated after epidermal growth factor treatment, followed by EGFR and c-myc mRNA accumulation. A potent tumor promoter, 12-O-tetradecanoylphorbol-13-acetate, also induced EGFR mRNA. Thus, the inducible regulatory mechanism for the EGFR gene was activated in the variant even though the EGFR gene was constitutively expressed.

Topics: Blotting, Northern; Blotting, Southern; Carcinoma, Small Cell; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Neoplastic; Genes, myc; Humans; Lung Neoplasms; Methylation; Polymorphism, Restriction Fragment Length; Proto-Oncogenes; RNA, Messenger; Tumor Cells, Cultured

We observed that human epidermal growth factor (hEGF) alone prolonged the survival time of mice bearing various murine syngeneic tumors as well as athymic nude mice bearing human xenografts. No changes in the subcutaneous solid tumor mass volume were observed. Prolongation of survival time by hEGF was observed in mice bearing murine epidermoid carcinoma (BSC) and human gastric carcinoma (KATO III), but not in murine epidermoid carcinoma (KLN205) or human epidermoid carcinoma (A431). Human tumor cells such as A431, KATO III, and murine tumor cells, KLN205, BSC had roughly 2 X 10(6), 3 X 10(4), 1.3 X 10(3) and 1 X 10(3) EGF receptors/cell, respectively. Although KLN205 and BSC tumor cells maintained nearly the same number of EGF receptors, the effects of hEGF were very different. Although A431 tumor cells had nearly 100 times more receptors than KATO III cells, the prolongation of survival time of mice bearing A431 by hEGF was no better than that of mice bearing KATO III. Accordingly, it appears that this prolongation of survival time by hEGF is independent of the number of EGF receptors on tumor cells. In addition, hEGF was shown to inhibit experimental pulmonary metastasis of murine BSC tumor, but was ineffective with murine KLN205 tumor. These results suggest that prolongation of survival time by hEGF may result from the inhibition of tumor cell metastasis and EGF may play a role in preventing the metastasis of certain malignant neoplasms unrelated to its effects through the EGF receptor on tumor cells.

Topics: Adenocarcinoma; Animals; Carcinoma, Squamous Cell; Colonic Neoplasms; Epidermal Growth Factor; Humans; Lung Neoplasms; Male; Mice; Mice, Inbred C57BL; Stomach Neoplasms; Time Factors

Small cell lung cancer (SCLC) tumor progression can involve partial or complete conversion to a more treatment-resistant non-small cell (NSCLC) phenotype. In a cell culture model of this phenomenon, we have previously demonstrated that insertion of the viral Harvey ras gene (v-Ha-ras) into SCLC cell lines with amplification and overexpression of the c-myc gene induced many NSCLC phenotypic features. We now report that the v-Ha-ras gene can also induce morphologic, biochemical, and growth characteristics consistent with the NSCLC phenotype in an N-myc amplified SCLC cell line, NCI-H249. We show that v-Ha-ras has novel effects on these cells, abrogating an SCLC-specific growth requirement for gastrin-releasing peptide, and inducing mRNA expression of three NSCLC-associated growth factors and receptors, platelet-derived growth factor B chain, transforming growth factor-alpha (TGF-alpha), and epidermal growth factor receptor (EGF-R). TGF-alpha secretion and EGF-R also appear, consistent with the induction of an autocrine loop previously shown to be growth stimulatory for NSCLC in culture. These data suggest that N-myc and v-Ha-ras represent functional classes of genes that may complement each other in bringing about the phenotypic alterations seen during SCLC tumor progression, and suggest that such alterations might include the appearance of growth factors and receptors of potential importance for the growth of the tumor and its surrounding stroma.

Topics: Carcinoma, Non-Small-Cell Lung; Carcinoma, Small Cell; Cell Division; Eflornithine; Epidermal Growth Factor; ErbB Receptors; Gastrin-Releasing Peptide; Gene Expression; Genes, ras; Lung Neoplasms; Peptides; Platelet-Derived Growth Factor; Transforming Growth Factors

We immunohistochemically examined 131 primary human lung adenocarcinomas for the possible presence of autocrine factors. Transforming growth factor alpha (TGF alpha) and epidermal growth factor (EGF) were considered growth factors with epidermal growth factor receptor (EGFR) as the receptor. Of these tumors, 87 (66%) showed a high expression of TGF alpha, 66 (50%) showed a high expression of EGF, and 55 (42%) were positive for EGFR reactivity. In the EGFR-positive cases, the 5-year survival rates of patients with high TGF alpha and low TGF alpha were 36% and 85%, respectively (P less than 0.05). The 5-year survival rates of patients with high EGF and low EGF were 25% and 77%, respectively (P less than 0.05). In contrast, in the EGFR-negative cases, there was no statistical difference between the 5-year survival rates of patients with either high TGF alpha or EGF and low TGF alpha or EGF. Because autocrine growth mechanisms are present in adenocarcinoma of the human lung, these events may contribute to clarification of tumor development, and perhaps even to a better prognosis.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Cell Membrane; Cytoplasm; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Immunohistochemistry; Lung Neoplasms; Male; Middle Aged; Transforming Growth Factor alpha

Epidermal growth factor (EGF) was labelled with biotin via modification of either the amino or carboxyl groups, using suitable reagents, namely biotinyl-N-hydroxysuccinimide ester or biotinamidocaproyl hydrazide. To assure that the specific binding capacity of EGF is retained despite its chemical modification, displacement of the EGF by biotinylated derivatives in a routine binding assay was performed. The inhibitory potency compared to unmodified EGF was only slightly reduced. This result is the prerequisite for testing the usefulness of biotinylated EGF in histochemistry. The biotinylated probes were applied to sections of human tumour tissue and of monkey organs (liver, kidney, uterus of Cynomolgus and Rhesus monkey) to localize the specific binding sites for EGF. Formalin-fixed, paraffin-embedded tissue sections were deparaffinized and incubated with the probes at a concentration of 10 micrograms ml-1 at room temperature for 60 min. Specific binding of the EGF was visualized by the avidin-biotin techniques (ABC). A positive reaction in conjunction with appropriate controls by competitive inhibition was seen for all monkey tissue sections and for the following number of cancer cases: breast carcinoma: 7/10; mesothelioma: 2/4; lung carcinoma: 1/3; colon carcinoma: 1/3. The staining properties were similar for both types of probes that differed in the functional group that is involved in modification by biotin attachment. However, the batches with modification of the amino groups stained more intensely and more distinctly than the carboxyl modified EGF. Overall, the data indicate that the ligand properties of the EGF are not impaired by biotinylation of the two types of functional groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Binding Sites; Biotin; Breast Neoplasms; Colonic Neoplasms; Epidermal Growth Factor; ErbB Receptors; Female; Histocytochemistry; Humans; Kidney; Liver; Lung Neoplasms; Macaca fascicularis; Macaca mulatta; Uterus

Biotin-labeled epidermal growth factor (EGF) was applied to routinely processed sections of 64 cases of human lung carcinoma as a histochemical tool for demonstrating EGF-specific receptors. Formalin-fixed, paraffin-embedded tissue sections were deparaffinized and incubated with the labeled EGF (10 micrograms/ml) for 60 min at room temperature. The specific binding of the growth factor to its receptor was visualized by the avidin-biotin complex (ABC) technique. Positive binding capacities were obtained for the following cases: 15/16 epidermoid carcinoma; 13/15 adenocarcinoma; 2/11 large cell anaplastic carcinoma; 12/20 small cell anaplastic carcinoma; 0/11 normal lung tissue; 0/6 main bronchi; 0/1 hamartoma; 0/1 primary fibrosarcoma of lung. In addition, a strong positive reaction was seen for neutrophilic granulocytes present within the tumorous tissue. Data indicate that EGF receptors are frequently expressed in more differentiated carcinoma in comparison with anaplastic carcinoma of lung.

Topics: Aged; Cell Differentiation; Epidermal Growth Factor; ErbB Receptors; Female; Histocytochemistry; Humans; Lung Neoplasms; Male; Middle Aged

The biochemical properties of lung cancer cell lines were investigated. Bombesin-like peptides were present in three small cell lung cancer (SCLC) cell lines examined and three of four lung carcinoids but not in five non-small cell lung cancer (NSCLC) cell lines. Therefore SCLC and some lung carcinoids, but not NSCLC, are enriched in neuroendocrine properties. In contrast, 125I-EGF bound with high affinity to all five NSCLC cell lines and three of four lung carcinoids but not to the three SCLC cell lines examined. For lung carcinoid cell line NCI-H727, 125I-EGF bound with high affinity (Kd = 6 nM) to a single class of sites (Bmax = 110,000/cell). The 125I-EGF bound was rapidly internalized at 37 degrees C but not 4 degrees C. Using Western blot techniques and antiphosphotyrosine antibodies, EGF induced phosphorylation of a major 170 Kd protein. Using immunoprecipitation techniques and anti-EGF receptor antibodies a major 170 Kd protein was labeled. These data indicate that biologically active EGF receptors are present on NSCLC and lung carcinoid cell lines.

Topics: Adenocarcinoma; Bombesin; Carcinoma, Squamous Cell; Epidermal Growth Factor; ErbB Receptors; Humans; Lung Neoplasms; Peptides; Phosphorylation; Tumor Cells, Cultured

The SK-Luci-6 cell line, established from a large-cell anaplastic lung tumor of a patient with humoral hypercalcemia of malignancy (HHM), was investigated to identify osteolytic factors produced that might mediate HHM. Most HHM-associated tumors are thought to produce parathyroid hormone-related proteins or transforming growth factor (TGF) alpha. SK-Luci-6 cells formed s.c. tumors and induced hypercalcemia in athymic nude mice. Serum-free conditioned medium from SK-Luci-6 cultures induced bone resorption in neonatal mouse calvariae in vitro, and also contained TGF-beta activity and mitogenic activity. SK-Luci-6 cell conditioned medium did not displace [125I]epidermal growth factor binding to cell receptors or stimulate cyclic AMP formation in rat osteosarcoma cells, suggesting that the conditioned medium did not contain TGF-alpha or parathyroid hormone-related proteins. The osteolytic, TGF-beta, and mitogenic activities copurified in several chromatographic separations: gel filtration in acid and then in guanidine HCl; ion exchange; and reverse phase. The results suggest that in the HHM-associated SK-Luci-6 tumor, the causative osteolytic factor produced by the tumor cells is not a parathyroid hormone-related protein or TGF-alpha but, rather, may be a TGF-beta.

Topics: Animals; Calcium; Cell Line; Chick Embryo; Cyclic AMP; Epidermal Growth Factor; Female; Humans; Hypercalcemia; Lung Neoplasms; Male; Mice; Mice, Nude; Osteolysis; Osteosarcoma; Transforming Growth Factors

Membrane preparations from 36 human non-small cell lung cancers were examined for the expression of epidermal growth factor (EGF) receptors, and comparisons were made between tumor types and stage. Eight normal lung membrane preparations were also examined. The concentrations of EGF receptors were assessed by ligand binding studies using 125I-radiolabeled EGF. In two point saturation experiments using 0.3 nM 125I-EGF incubated with membranes from 35 primary lung tumors, a median of 18 fmol/mg of protein (range, 1.1 to 530) was found. This was significantly greater than binding to eight lung membranes: median, 6.1 fmol/mg of protein (range, 1.0 to 14.5) (P less than 0.02). Scatchard binding curves obtained in 21 of the 36 tumors and seven of eight of the normal lung preparations showed high and low affinity sites for EGF receptors on all but two tumor membranes. The dissociation constant of the high affinity sites was similar on tumor and normal lung membranes: range, 0.75 to 30 x 10(-10) M/liter. However, the tumors had a significantly higher concentration of these receptor sites: median, 30.4 fmol/mg of protein versus a median of 6.2 fmol/mg of protein on normal lung membranes (P less than 0.01). Likewise, there were significantly more low affinity sites on tumors: median, 237 fmol/mg compared to 60.2 fmol/mg on normal lung (P less than 0.01). No differences were found in this analysis between tumors of different histological subtypes or clinical stage. It is possible that the high level of expression of high affinity sites on lung tumors could be used as a target for ligand-complexed drugs.

Topics: Carcinoma, Non-Small-Cell Lung; Epidermal Growth Factor; ErbB Receptors; Gene Amplification; Humans; Immunohistochemistry; Kinetics; Lung Neoplasms

Lung cancer tissues from 68 patients were examined for epidermal growth factor (EGF) receptor levels and EGF receptor gene copy numbers. Histologic cell types of these lung cancer tissues included squamous-cell carcinoma (n = 30), adenocarcinoma (n = 28), large-cell carcinoma (n = 4), and small-cell carcinoma (n = 6). Tissues of squamous-cell carcinoma exhibited exceptionally high 125I-EGF binding activity, and those of small-cell carcinoma showed no EGF binding activity. Southern blot hybridization analysis revealed EGF receptor gene amplification in the squamous-cell carcinomas with high EGF binding activity. The EGF receptor levels in squamous-cell carcinomas and adenocarcinomas were compared with their pathological staging grouping and pathological findings, including degree of differentiation, diameter of tumor, and lymph node metastasis. However, unlike previous reports on breast and bladder cancers, there was no obvious correlation between these pathological characteristics and the EGF receptor levels of lung cancer.

Topics: Adenocarcinoma; Blotting, Southern; Carcinoma; Carcinoma, Small Cell; Carcinoma, Squamous Cell; Epidermal Growth Factor; ErbB Receptors; Humans; Lung Neoplasms

Tumor necrosis factor (TNF) is a cytokine which induces cytotoxicity in some but not all tumor cells. Initial studies of five tumor cell lines demonstrated that TNF was able to rapidly (within 30 min) modulate tyrosine protein kinase activity of epidermal growth factor (EGF) receptors on tumor cell lines which were sensitive to the cytotoxic effects of TNF but not alter EGF receptor kinase activity in TNF-resistant tumor cells. Two tumor cell lines (ME-180 cervical carcinoma and T24 bladder carcinoma) which have been shown to express similar TNF-binding characteristics but differ in their sensitivity to the cytotoxic actions of TNF were chosen for further characterization. Treatment of TNF-sensitive ME-180 cells with 1 nM TNF resulted in a 3-fold stimulation of EGF receptor tyrosine protein kinase activity within 10 min which correlated with increased phosphorylation of EGF receptor protein itself. In addition, dose-response studies indicate that similar concentrations of TNF modulate both ME-180 cell growth and EGF receptor kinase activity. Treatment of TNF-resistant T24 cells showed that TNF had no significant effect on their growth, EGF receptor tyrosine protein kinase activity, or phosphorylation of EGF receptor protein although EGF receptor kinase activity was stimulated by EGF. Quantitation of receptors expressed on the surface of ME-180 and T24 cells demonstrated a 3-fold difference between the number of EGF-binding sites on T24 (100,000) versus ME-180 cells (300,000), suggesting the relative abundance of EGF receptor does not solely account for differential effects of TNF on EGF receptor activation in these two cell lines. Phosphoamino acid analysis of EGF receptor from 32P-equilibrated ME-180 cells demonstrated that TNF-induced phosphorylation of amino acids which was quantitatively similar to that of EGF but distinct from the effects of phorbol ester. However, unlike EGF, TNF was unable to stimulate EGF receptor kinase activity in ME-180 cell lysates. The kinetics of EGF receptor activation and the metabolic consequence of activation of EGF receptor activity by TNF appear to be distinct from those induced by EGF. These results suggest that TNF-induced modulation of EGF receptor occurs through a unique mechanism and may play a role in the cytotoxic actions of TNF.

Topics: Carcinoma, Squamous Cell; Cell Line; Cell Survival; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Kinetics; Lung Neoplasms; Phosphorylation; Protein-Tyrosine Kinases; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Urinary Bladder Neoplasms; Uterine Cervical Neoplasms

We previously reported that two human lung adenocarcinoma cell lines (A-549 and PC-9) produce human transforming growth factor-alpha (hTGF-alpha) and express its receptors. In the present study an exogenously added monoclonal antibody against recombinant hTGF-alpha inhibited growth of these cell lines in vitro. This result indicated that endogenous hTGF-alpha produced by the cancer cells can function as an autocrine growth factor.

Topics: Adenocarcinoma; Antibodies, Monoclonal; Antibody Specificity; Cell Division; Epidermal Growth Factor; ErbB Receptors; Humans; Immunotherapy; In Vitro Techniques; Lung Neoplasms; Radioligand Assay; Recombinant Proteins; Transforming Growth Factors; Tumor Cells, Cultured

Using a rat rhabdomyosarcoma 9-4/0, we investigated the role of epidermal growth factor (EGF) in tumor dissemination. In vitro, we detected high-affinity EGF receptors on tumor cells and stimulation of their proliferation by EGF. When injected iv, EGF-pretreated cells demonstrated an increased capacity to form lung colonies and to invade lymphatic tissue. In vivo, EGF treatment led to increased metastatic spread of subcutaneous tumors. When primary tumors were ablated, and the treatment was given from the time of graft until ablation (seeding step), no effect on metastatic spread was noticed. When treatment was given from the time of ablation until death (growth step), EGF increased the number of lung metastases and of invaded lymph node sites.

Topics: Animals; Cell Division; Epidermal Growth Factor; Female; Lung Neoplasms; Neoplasm Metastasis; Rats; Rhabdomyosarcoma

Mouse lung adenoma were induced in adult female BALB/c mice by chronic i.p. urethane injection. Sialoadenectomy of the mice prior to carcinogen treatment did not alter the frequency or size of lung adenoma. However, sialoadenectomized mice exhibited a decrease in the proportion (18 versus 52%) of tumours which exhibited a low nuclear to cytoplasmic ratio and which grew along alveolar septa. Sialoadenectomy was also related to a complementary increase in the proportion (82 versus 48%) of tumours with large vesicular nuclei, prominent nucleoli and generally increased phenotypic features of malignancy. Replacement of epidermal growth factor (EGF) in sialoadenectomized mice restored the tumour-type ratio observed in non-sialoadenectomized mice. These data are discussed with respect to the possible roles of EGF in mouse lung alveologenic carcinoma formation and the cell type of origin for the tumours observed.

Topics: Adenoma; Animals; Epidermal Growth Factor; Female; Lung Neoplasms; Mice; Mice, Inbred BALB C; Submandibular Gland; Urethane

In the search for a more potent bombesin antagonist, we found [D-Arg1,D-Phe5,D-Trp7,9,Leu11]substance P to be effective in mouse fibroblasts and to inhibit the growth of small cell lung cancer, a tumor that secretes bombesin-like peptides that may act as autocrine growth factors. In murine Swiss 3T3 cells, [D-Arg1,D-Phe5,D-Trp7,9,Leu11]substance P proved to be a bombesin antagonist as judged by the following criteria: (i) inhibition of DNA synthesis induced by gastrin-releasing peptide and other bombesin-like peptides; (ii) inhibition of 125I-labeled gastrin-releasing peptide binding to the bombesin/gastrin-releasing peptide receptor; (iii) reduction in cross-linking of the Mr 75,000-85,000 protein putatively a component of the bombesin/gastrin-releasing peptide receptor; (iv) blocking of early cellular events that precede mitogenesis--calcium mobilization and inhibition of epidermal growth factor binding. [D-Arg1,D-Phe5,D-Trp7,9,Leu11]substance P was 5-fold more potent than the antagonist [D-Arg1,D-Pro2,D-Trp7,9,Leu11]substance P. [D-Arg1,D-Phe5,D-Trp7,9,Leu11]substance P also inhibits mitogenesis induced by vasopressin but not that induced by a variety of other mitogens. Both antagonists reversibly inhibited the growth of small cell lung cancer in vitro in a concentration-dependent manner. Peptide antagonists could, therefore, have far-reaching therapeutic implications.

Topics: Amino Acid Sequence; Animals; Binding, Competitive; Bombesin; Carcinoma, Small Cell; DNA Replication; Epidermal Growth Factor; Gastrin-Releasing Peptide; Lung Neoplasms; Mice; Mitosis; Molecular Weight; Peptides; Substance P; Tumor Cells, Cultured

Non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) cell lines were studied for epidermal growth factor (EGF) receptor expression. All NSCLC cell lines tested (eight of eight) had specific EGF binding sites, whereas only five of 11 SCLC cell lines bound EGF. NSCLC and SCLC cell lines expressed the same type of high affinity EGF binding sites with a Kd of 0.5 to 4.5 nM; however, NSCLC cells bound significantly more EGF than SCLC cell lines. The amount of binding sites in NSCLC cells ranged between 71 and 1,000 fmol/mg of protein and in SCLC cells, between 26 and 143 fmol/mg of protein. The two SCLC cell lines with EGF binding values within the range of NSCLC belonged to the variant subtype of SCLC. By means of an anti-erbB serum and indirect radioimmunoprecipitation, a strong Mr approximately 170,000 protein band could be detected in the NSCLC cell lines. This protein corresponds to the EGF receptor molecule. Its identity was proven by competition with excess erbB antigen for the antibody during the radioimmunoprecipitation. Furthermore, this Mr 170,000 protein exhibited protein kinase activity as evidenced by in vitro autophosphorylation. The radioactivity incorporated into the Mr 170,000 band in radioimmunoprecipitation and protein kinase assays was 10 to 100 times lower in these SCLC cell lines which were positive in the EGF binding assay compared to the NSCLC cell lines. We conclude that NSCLC in contrast to SCLC expresses high levels of EGF receptors which may be used to facilitate the differential diagnosis in some cases of lung cancer. These data suggest that EGF may play a role in growth and differentiation of NSCLC.

Topics: Carcinoma, Small Cell; Epidermal Growth Factor; ErbB Receptors; Gene Amplification; Humans; Iodine Radioisotopes; Lung Neoplasms; Molecular Weight; Protein Kinases; Temperature; Tumor Cells, Cultured

We have investigated a panel of human lung cancer cell lines representing the major groups of lung cancer, i.e., small-cell carcinoma (SCC) and the group of non-SCC, consisting of squamous-cell carcinoma (SQC), adenocarcinoma (ADC) and large-cell carcinoma (LCC), for their expression of certain growth factor genes. Messenger RNA from each cell line was hybridized with probes for platelet-derived growth factor (PDGF) A- and B-chains, insulin-like growth factor (IGF)-I and -II, transforming growth factor (TGF)-alpha and -beta, epidermal growth factor (EGF) as well as a probe for the EGF receptor. All non-SCC cell lines examined showed expression of the PDGF A-chain gene. The PDGF beta-chain and TGF-beta genes were expressed in all non-SCC cell lines but one, H-125 (ADC). TGF-alpha gene expression was demonstrated in the SQC cell line U-1752, in both ADC cell lines (H-23 and H-125) and in one of the 3 LCC cell lines, U-1810. IGF-II was only transcribed in the LCC cell line U-1810. The EGF-receptor was detected in all non-SCC cell lines but one, H-661 (LCC). Neither IGF-I nor EGF transcripts could be seen in any of the 10 cell lines examined. In contrast to the non-SCC cell lines, the 4 SCC lines were constantly negative for the probes employed in this study. The frequent and heterogeneous expression of growth factor transcripts in all non-SCC studied, but not SCC-cell lines, may contribute to the difference in biological behaviour observed in vivo and in vitro between the 2 major lung cancer entities.

Topics: Carcinoma, Non-Small-Cell Lung; Carcinoma, Small Cell; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation; Glyceraldehyde-3-Phosphate Dehydrogenases; Insulin-Like Growth Factor I; Lung Neoplasms; Peptides; Platelet-Derived Growth Factor; RNA, Messenger; Transforming Growth Factors; Tumor Cells, Cultured

The v-fps oncoprotein was expressed in a pre-neoplastic, growth factor-dependent Chinese hamster lung fibroblast line (CCL39) to study its effect on growth controls and on the induction of malignancy. Two transfectants were characterized which expressed low (39FPS-8) or high (51FPS-6) levels of P130gag-f ps protein-tyrosine kinase activity. 39FPS-8 cells still arrested in quiescence when deprived of growth factors, but developed an increased sensitivity to the mitogenic actions of epidermal growth factor (20-fold) and alpha-thrombin (50-fold), although not to insulin. In contrast, 51FPS-6 cells completely escaped growth controls, divided in serum-free medium, and were insensitive to further growth factor stimulation. Both transfectants produced rapidly growing tumors in nude mice that formed pulmonary metastases from a subcutaneous site, unlike the parental cells which are non-metastatic. 51FPS-6 cells were comparatively more efficient than 39FPS-8 cells in colonizing the lungs after intravenous inoculation. The v-fps tyrosine kinase therefore induces a partial to complete relaxation of growth factor-mediated controls on the CCL39 cell cycle, with the extent of factor independence reflecting the amount of P130gag-f ps synthesized. This reduction in growth factor requirements correlates with the capacity of v-fps to confer the attributes of metastatic tumors upon preneoplastic CCL39 fibroblasts. We speculate that increased sensitivity to growth factor stimulation represents a common mechanism by which tumor cells acquire metastatic properties.

Topics: Animals; Cell Division; Cell Line; Cricetinae; Epidermal Growth Factor; Growth Substances; Insulin; Interphase; Lung; Lung Neoplasms; Neoplasm Metastasis; Oncogene Proteins, Viral; Precancerous Conditions; Protein-Tyrosine Kinases; Thrombin

It has been shown that none of the small cell lung carcinoma (SCLC) cell lines possess epidermal growth factor (EGF) binding activity on their surface. We have examined several SCLC cell lines for the possibility that they may have EGF receptors but that the receptors are masked by an EGF-like protein factor(s), which may be produced by an autocrine mechanism. No evidence, however, was found for the production of such factors. We then used an EGF receptor complementary DNA to determine the state of the EGF receptor gene by Southern blot analysis. The receptor gene appears to be present in these cells in an intact, unrearranged form. These cells, however, were found to lack detectable levels of EGF receptor mRNA, suggesting a possible reason for the absence of EGF receptors on the cell surface. Furthermore, karyotype analysis revealed that SCLC cell lines Lu134 and H69 contained a morphologically normal chromosome 7, which carries the EGF receptor gene. Also, these SCLC cells contained the apparently normal chromosome 3 and exhibited the presence of c-raf-1 gene in an unrearranged form. Thus, the previously noted partial deletion of chromosome 3 is not necessarily common to the SCLC cells. Instead, the lack of EGF receptor is frequently found in SCLC cell lines and is distinct from the other types of lung cancer. We postulate that SCLC cells have some active regulatory mechanism which prevents the expression of EGF receptor gene.

Topics: Carcinoma, Small Cell; Cell Line; Chromosomes, Human, Pair 3; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation; Humans; Karyotyping; Kinetics; Lung Neoplasms; RNA, Messenger

Serum-free medium conditioned for 72 h by a human bronchioloalveolar carcinoma of lung, A549-1, stimulated the colony formation of normal human bronchial epithelial cells, newly cultured cells from human solid lung tumors, and established human lung tumor cell lines, including A549-1 cells themselves. This activity was concentration dependent and was stable to acid. Growth factors in A549-1 conditioned medium (CM) supported culture of solid lung tumors; primary cell cultures were obtained from nine of 10 solid lung tumors of non-small cell origin and from one small cell tumor using A549-1 CM. In addition, three cell lines have been established to date from these primary cultures. Gel filtration of concentrated A549-1 CM on Biogel P-10 separated the growth promoting activity into four regions of apparent Mr 70,000, 12,000, 8,000, and 6,000, and two broad regions of apparent Mr 3000-5000. All but the 12,000 Mr fraction contained activity which competed for specific binding of epidermal growth factor (EGF) to A431 cell membranes. CM was superior to both EGF and TGF alpha in stimulating growth of normal and neoplastic lung cells. EGF also was inhibitory to tumor cells while TGF alpha stimulated both normal and tumor cell growth. TGF beta was also found in CM but inhibited normal and neoplastic lung epithelial cell growth. Of other substances tested, ILGF-I stimulated colony formation. The results suggest that autocrine factors may be important in non-small cell lung tumor cell growth and that differences in response to EGF and TGF alpha may provide the basis for selective culturing of normal and neoplastic lung epithelial cells.

Topics: Adenocarcinoma, Bronchiolo-Alveolar; Bronchi; Cell Line; Culture Media; Epidermal Growth Factor; Epithelial Cells; ErbB Receptors; Growth Substances; Humans; Lung Neoplasms; Molecular Weight; Peptides; Transforming Growth Factors

The epidermal growth factor receptor is homologous to the oncogene erb-beta and is the receptor for a class of tumour growth factors (TGF-alpha). The clinical correlations with its expression were studied in 77 non-small cell lung cancers (NSCLC). They were stained for epidermal growth factor receptor (EGFr) by means of an indirect immunoperoxidase technique using a monoclonal antibody against the receptor. Normal lung tissue and normal bronchus were stained for comparison. Cancer tissue showed significantly increased staining compared to normal lung (P less than 0.05). Staining for EGFr in 40 squamous carcinomas was significantly stronger than in 37 specimens of other types of NSCLC (P less than 0.05), and staining in stage three NSCLC was stronger than in stage 1 and 2 (P less than 0.05). These results suggest that the presence of a high intensity of staining for EGF receptor is associated with spread of human non-small cell lung cancer and this receptor may be a suitable target for therapy.

Topics: Bronchi; Carcinoma, Non-Small-Cell Lung; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Immunoenzyme Techniques; Lung; Lung Neoplasms; Male; Middle Aged; Neoplasm Staging; Prognosis

The effect of epidermal growth factor (EGF) on the in vitro growth of 186 malignant human tumor specimens (45 melanomas, 32 sarcomas, and 56 lung, 16 gynecological, 14 breast, 12 genitourinary, and 11 gastrointestinal carcinomas) was evaluated in the cellular adhesive matrix human tumor culture system supplemented with transferrin, insulin, hydrocortisone, and estradiol. EGF increased tumor growth by at least 50% in 81% of the 186 tumors and by over 100% in 54%. The enhanced growth induced by EGF was related to an accelerated cellular division independent of tumor type and not to an increase in the actual number of clonogenic units. The drug concentrations of cell cycle-independent Adriamycin and cisplatin needed to achieve a 90% tumor cell kill were not altered by the responsiveness of the tumor to EGF.

Topics: Antineoplastic Agents; Breast Neoplasms; Carcinoma; Cell Division; Cell Line; Cell Survival; Cells, Cultured; Culture Media; Epidermal Growth Factor; Extracellular Matrix; Gastrointestinal Neoplasms; Humans; Lung Neoplasms; Melanoma; Neoplasms; Sarcoma; Urogenital Neoplasms

Topics: Animals; Antibodies, Monoclonal; Epidermal Growth Factor; Lung Neoplasms; Mice; Mice, Inbred C57BL; Neoplasm Metastasis; Nerve Growth Factors; Submandibular Gland

The mechanisms of ectopic enzyme and enzyme inhibitor-production in neoplastic tissues were investigated. No evidence was obtained to suggest any difference in the structural genes of amylase-producing tumors and normal lymphocytes. mRNA sequence coding for amylase precursor in tumor tissues was identical to that of salivary amylase, suggesting that the amylase in amylase-producing tumors was identical to, or closely resembled the amylase in salivary gland. High incidence of elevation of serum pancreatic secretory trypsin inhibitor (PSTI) was observed in patients with various malignant tumors. PSTI-positive malignant cells were also frequently found in malignant tissues. A comparison of human PSTI mRNA sequence with mouse epidermal growth factor (EGF) mRNA sequence showed that they were 46% homologous. Human PSTI stimulated [3H]thymidine incorporation into DNA in human fibroblasts at concentrations present in human serum.

Topics: Amylases; Animals; Base Sequence; Epidermal Growth Factor; Female; Humans; Lung Neoplasms; Mice; Mice, Nude; Neoplasms; Ovarian Neoplasms; RNA, Messenger; Salivary Glands; Thymoma; Thymus Neoplasms; Trypsin Inhibitor, Kazal Pancreatic; Trypsin Inhibitors

We have initiated an in-depth, comprehensive study of non-SCLC in the expectation that improvements in clinical management and diagnosis of lung cancer patients will follow. We have established and characterized over 30 such tumors in long-term culture and as xenografts in athymic nude mice. These models usually retain the morphologic and other properties of the tumors from which they were derived. With the use of fully defined medium, most SCLC and adenocarcinomas can be established as long-term cultures. The growth conditions for squamous cell carcinomas have not been fully defined, and the success rate is low. In contrast to SCLC, there is considerable heterogeneity of non-SCLC tumors, with more than 12 phenotypes identified among the first 30 lines established. In addition, there is considerable overlap of properties, both within the non-SCLC types as well as between non-SCLC and SCLC. These findings indicate a common origin for all lung cancers. Radiation and drug sensitivity studies suggest that cell line data may correlate with clinical responses. Clinical protocols utilizing our ability to culture and drug test individual tumors are currently under way. They represent the first steps in a combined clinico-laboratory approach to the management of lung cancer.

Topics: Adenocarcinoma; Animals; Carcinoma, Small Cell; Carcinoma, Squamous Cell; Cell Division; Cell Line; DNA, Neoplasm; Epidermal Growth Factor; Humans; Lung Neoplasms; Mice; Mice, Nude; Neoplasm Transplantation; Ploidies; Transplantation, Heterologous

Epidermal growth factor receptor (EGF-R) expression was assessed in 63 lung tumour samples with a monoclonal antibody (EGF-R1) by indirect immunoperoxidase staining on cryostat sections. All 15 small cell lung cancer samples were negative whereas over 80% of the 48 non small lung cancer stained positively. In 30 bronchial biopsies two monoclonal antibodies against the cytoplasmic part of the EGF-R were evaluated. These antibodies showed weaker staining than EGF-R1. No additional or enhanced staining as compared with EGF-R1 was observed, suggesting a lack of enhanced expression of a truncated EGF-R analogous to the v-erb-B oncogene product. Monoclonal antibodies against the EGF-R may be helpful diagnostically in differentiating small cell from non small cell lung cancer and may also be important in elucidating biological differences in primary lung cancer.

Topics: Adenocarcinoma; Antibodies, Monoclonal; Carcinoma, Small Cell; Carcinoma, Squamous Cell; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Immunoenzyme Techniques; Lung Neoplasms; Male; Middle Aged; Receptors, Cell Surface

Using the adjacent histologically normal tissues obtained from the same patients as controls, six human lung tumors were studied for the activities of epidermal growth factor (EGF) receptor binding, and receptor autophosphorylation. There was a 1.2- to 2.8-fold increase in EGF receptor activities in lung tumors due to an increase in the number of receptors without changes in their affinity. The increase had no direct correlation with the degree of differentiation or the type of lung tumors. The elevated expression of EGF receptor may be one of the characteristics in lung tumors. Epidermal growth factor and its receptor also may play a role in the regulatory mechanisms during tumorigenesis.

Topics: Adenocarcinoma; Carcinoma; Carcinoma, Squamous Cell; Epidermal Growth Factor; ErbB Receptors; Humans; Kinetics; Lung Neoplasms; Phosphorylation

Human squamous cell carcinoma tissue was screened to determine the epidermal growth factor (EGF) receptor level. In 9 out of 15 cases, increased EGF binding was observed relative to normal adjacent tissue. In two tissue samples (SCE1 and SCL1), amplification of the EGF receptor gene and increased mRNA level were also observed. In two other cases (SCE2 and SCL3), EGF receptor gene amplification did not occur. We propose that there is an alternative mechanism for EGF receptor hyperproduction, independent of gene content, active in these tissues. A possible role of EGF receptor hyperproduction is envisioned in human neoplasia.

Topics: Carcinoma, Squamous Cell; DNA, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Esophageal Neoplasms; Gene Amplification; Genes; Humans; Lung Neoplasms; Receptors, Cell Surface; RNA, Messenger; RNA, Neoplasm

Epidermal growth factor (EGF) promotes the growth of cultured benign and malignant cells. Recent studies have demonstrated that the amount of EGF receptor is elevated in squamous cell carcinoma cells in tissue culture when compared with normal epidermal cells. This study demonstrates that elevated levels of EGF receptor are detected in biopsy specimens of human squamous cell carcinomas of the lung with a murine monoclonal antibody, EGF-R1, which binds specifically to the receptor. The increased receptor ranged from 2.5- to 5-fold that of normal skin. These findings have been observed in 11 of 11 squamous carcinomas and two of two epidermoid head and neck cancers. Seven of eight adenocarcinomas, two of two small cell, and four of eight undifferentiated lung cancers had negligible amounts of EGF receptor. The EGF receptor antibody did not bind significantly to normal lung tissues and 35 nonepidermoid tumors. Therefore, EGF receptor may be an excellent marker for epidermoid malignancies.

Topics: Adenocarcinoma; Autoradiography; Carcinoma, Squamous Cell; Epidermal Growth Factor; ErbB Receptors; Humans; Iodine Radioisotopes; Lung Neoplasms; Radioimmunoassay; Receptors, Cell Surface; Skin

The factors controlling the growth of human tumor cells in soft agar are poorly understood. However, it has been demonstrated that serum provides factors which promote anchorage-independent growth. We tested 58 tumor specimens, which were obtained from patients with adenocarcinoma of the lung, colon, ovary or squamous cell carcinoma, for their ability to form colonies in soft agar in serum-free or serum-supplemented media. The cells were unable to replicate, and none of the hormones or growth factors tested: insulin (I), transferrin (T), selenium (S), estradiol (E), hydrocortisone (H) or epidermal growth factor (EGF) could substitute for serum. Examination of the serum dose-response curves indicated that growth factors reduced the serum concentrations needed to support anchorage-independent growth. The addition of the supplements and the lowering of serum concentrations increased cloning efficiencies (C.E.) in 38/51 trials, when cells were able to grow initially. The addition of ITS increased C.E. in 18/21 cases, HITES in 15/17 cases and EGF in 12/18 cases as compared to controls. ITS and HITES increased the number of colonies only when serum was the limiting factor. EGF, however, increased the number of colonies even when serum was not the limiting factor. The ability of the supplements to enhance growth could not be correlated to tumor type or initial cloning efficiencies. However, in only 1/25 cases were cells that were unable to form colonies under standard conditions induced to form colonies in the presence of the growth factors. Normal and tumor-derived human fibroblasts did not form colonies in soft agar in the presence of these growth factors. The results suggest that human tumor cells may require the presence of serum-derived factors for growth in soft agar.

Topics: Adenocarcinoma; Agar; Blood; Carcinoma, Squamous Cell; Cell Division; Clone Cells; Colonic Neoplasms; Epidermal Growth Factor; Estradiol; Female; Humans; Hydrocortisone; Insulin; Lung Neoplasms; Ovarian Neoplasms; Selenium; Transferrin

Seventeen well-characterized human lung cancer cell lines were examined for the presence of specific membrane receptors for epidermal growth factor (EGF) and nerve growth factor (NGF) as well as for the production of diffusible factors capable of stimulating soft agar growth. These cell lines represented all four major histological types of human lung cancer including small cell carcinoma of the lung (SCCL) and the three types of non-SCCL (epidermoid, large cell, and adenocarcinoma). The SCCL lines included three lines referred to as "converters" because they had lost SCCL morphological and biochemical properties during prolonged passage in vitro. Specific receptors for EGF and NGF were detected by measuring the binding of 125I-radiolabeled growth factor to the cell surface. These assays revealed that EGF receptors are found on five of six non-SCCL cell lines and are not found on any of the SCCL lines. In contract, NGF binding was detected at low levels on three of eight SCCL lines and on all three SCCL converters but was not observed for non-SCCL lines. Thus, SCCL and SCCL converter cell lines are distinguished from non-SCCL by the pattern of membrane receptors for EGF and NGF. Such differences may ultimately prove useful as biological markers for the different histological types of lung cancer. Moreover, the majority of SCCL cells and all of the non-SCCL cells tested were found to produce diffusible growth factors which can stimulate soft agar growth of nontransformed normal rat kidney fibroblasts. Although some correlation between soft agar growth factor production and the absence of EGF receptors may exist for SCCL cells, the production of transforming growth factors appears to be a general property of human lung cancer cells in vitro and is independent of EGF receptor expression.

Topics: Agar; Cell Line; Cell Membrane; Epidermal Growth Factor; Growth Substances; Humans; Lung Neoplasms; Nerve Growth Factors; Peptides; Protein Binding

Three different human tumor lines in culture, a rhabdomyosarcoma, a bronchogenic carcinoma and a metastatic melanoma, release proteins (transforming growth factors, TGFs) into the medium that confer the transformed phenotype on untransformed fibroblasts. These proteins are acid and heat-stable; produce profound morphologic changes in rat and human fibroblasts; and enable normal anchorage-dependent cells to grow in agar. Removal of the transforming protein results in a reversion of cell phenotype. The major activity interacts with epidermal growth factor (EGF) cell membrane receptors. The peptides from these tumor cells are similar in their action to the sarcoma growth factor (SGF) released by murine sarcoma virus-transformed rodent cells. The most anchorage-independent tumor cells released the most TGFs. EGF-related TGFs were not detectable in fluids from cultures of cells with high numbers of free EGF membrane receptors (normal human fibroblasts and human carcinomas).

Topics: Adult; Aged; Animals; Binding, Competitive; Carcinoma, Bronchogenic; Carcinoma, Squamous Cell; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; DNA; Epidermal Growth Factor; ErbB Receptors; Female; Fibroblasts; Growth Substances; Humans; Lung Neoplasms; Male; Melanoma; Peptides; Rats; Receptors, Cell Surface; Rhabdomyosarcoma