transforming-growth-factor-alpha and Lung-Neoplasms

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

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

Erlotinib is one of the standard second/third line treatments for patients with advanced non-small cell lung cancer (NSCLC). This study investigated the efficacy of erlotinib in a Chinese population with advanced NSCLC and compared the predictive value of serum vascular endothelial growth factor (VEGF) and transforming growth factor (TGF)-alpha for the efficacy of erlotinib.. Patients with advanced, previously treated NSCLC received 150 mg of erlotinib once daily orally until disease progression or intolerable toxicity. Serum levels of VEGF and TGF-alpha were measured by ELISA at baseline and 1 month after treatment commenced.. There were 112 patients enrolled during the period October 2005 to February 2008 and followed until July 2008. Serum samples were available in 50 patients. Tumour response to erlotinib was partial in 35.7% of patients and 41.1% of patients had stable disease. The severity of skin rash (P < 0.001) had a significant positive correlation with the response to erlotinib. Median progression-free survival (PFS) and overall survival were 6.3 months and 12.5 months, respectively. After erlotinib treatment, serum VEGF levels did not change significantly, while serum TGF-alpha levels increased in patients who had partial response (P = 0.075) or stable disease (P = 0.055), but not in patients with progressive disease (P = 0.155). In patients with measurable serum TGF-alpha levels at baseline the PFS and median survival were 5 and 9.9 months respectively, and in patients with no measurable TGF-alpha at baseline the PFS and median survival were 11 and 21 months, respectively. Overall survival was significantly longer in patients with negative baseline serum TGF-alpha (P = 0.002).. Oral erlotinib was effective as a second/third line treatment for patients with advanced NSCLC. Baseline serum TGF-alpha levels may be a predictor for the efficacy of erlotinib treatment.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Disease Progression; Erlotinib Hydrochloride; Female; Follow-Up Studies; Humans; Lung Neoplasms; Male; Middle Aged; Predictive Value of Tests; Protein Kinase Inhibitors; Quinazolines; Retrospective Studies; Survival Rate; Transforming Growth Factor alpha; Treatment Outcome; Vascular Endothelial Growth Factor A

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

Pneumonitis is the leading cause of death associated with the use of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (EGFR-TKIs) against non-small cell lung cancer (NSCLC). However, the risk factors and the mechanism underlying this toxicity have not been elucidated. Tumor necrosis factor (TNF) has been reported to transactivate EGFR in pulmonary epithelial cells. Hence, we aimed to test the hypothesis that EGFR tyrosine kinase activity regulates TNF-mediated bronchial epithelial cell survival, and that inhibition of EGFR activity increases TNF-induced lung epithelial cell apoptosis. We used surfactant protein C (SPC)-TNF transgenic (tg) mice which overexpress TNF in the lungs. In this model, gefitinib, an EGFR-TKI, enhanced lung epithelial cell apoptosis and lymphocytic inflammation, indicating that EGFR tyrosine kinase prevents TNF-induced lung injury. Furthermore, IL-17A was significantly upregulated by gefitinib in SPC-TNF tg mice and p38MAPK activation was observed, indicative of a pathway involved in lung epithelial cell apoptosis. Moreover, in lung epithelial cells, BEAS-2B, TNF stimulated EGFR transactivation via the TNF-α-converting enzyme in a manner that requires heparin binding (HB)-EGF and transforming growth factor (TGF)-α. These novel findings have significant implications in understanding the role of EGFR in maintaining human bronchial epithelial cell homeostasis and in NSCLC treatment.

Topics: ADAM17 Protein; Animals; Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Line; Epithelial Cells; ErbB Receptors; Gefitinib; Heparin-binding EGF-like Growth Factor; Humans; Lung; Lung Injury; Lung Neoplasms; Mice; Mice, Transgenic; Models, Animal; p38 Mitogen-Activated Protein Kinases; Pneumonia; Protein Kinase Inhibitors; Transforming Growth Factor alpha; Tumor Necrosis Factor-alpha

Topics: Antibodies, Neutralizing; Apoptosis; Cell Proliferation; Coculture Techniques; Epithelial Cells; Epithelial-Mesenchymal Transition; Humans; Leukocytes, Mononuclear; Lung; Lung Neoplasms; Nanoparticles; Silicon Dioxide; Transforming Growth Factor alpha

Lung cancer is the leading cause of cancer-related death worldwide, in large part due to its high propensity to metastasize and to develop therapy resistance. Adaptive responses to hypoxia and epithelial-mesenchymal transition (EMT) are linked to tumor metastasis and drug resistance, but little is known about how oxygen sensing and EMT intersect to control these hallmarks of cancer. Here, we show that the oxygen sensor PHD3 links hypoxic signaling and EMT regulation in the lung tumor microenvironment. PHD3 was repressed by signals that induce EMT and acted as a negative regulator of EMT, metastasis, and therapeutic resistance. PHD3 depletion in tumors, which can be caused by the EMT inducer TGFβ or by promoter methylation, enhanced EMT and spontaneous metastasis via HIF-dependent upregulation of the EGFR ligand TGFα. In turn, TGFα stimulated EGFR, which potentiated SMAD signaling, reinforcing EMT and metastasis. In clinical specimens of lung cancer, reduced PHD3 expression was linked to poor prognosis and to therapeutic resistance against EGFR inhibitors such as erlotinib. Reexpression of PHD3 in lung cancer cells suppressed EMT and metastasis and restored sensitivity to erlotinib. Taken together, our results establish a key function for PHD3 in metastasis and drug resistance and suggest opportunities to improve patient treatment by interfering with the feedforward signaling mechanisms activated by PHD3 silencing.

Topics: A549 Cells; Animals; Antineoplastic Agents; Apoptosis Regulatory Proteins; Cell Hypoxia; Cell Line, Tumor; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; ErbB Receptors; Erlotinib Hydrochloride; Female; HCT116 Cells; Humans; Hypoxia-Inducible Factor-Proline Dioxygenases; Intracellular Signaling Peptides and Proteins; Lung Neoplasms; Mice; Mice, Nude; Mitochondrial Proteins; Neoplasm Metastasis; Protein Kinase Inhibitors; Transforming Growth Factor alpha; Tumor Microenvironment; Xenograft Model Antitumor Assays

A number of complex and rare mutations in epidermal growth factor receptor (EGFR) gene have been identified and the clinical implication of serum EGFR ligands has also been reported. However, the prognostic significance of these mutations and also the serum EGFR and its ligands in Non-Small Cell Lung Carcinoma (NSCLC) has remained a challenging issue. This study is aimed at finding the prognostic importance of EGFR rare mutations and serum EGFR, amphiregulin (AR), and TGF-α (Transforming Growth Factor-alpha) in NSCLC.. NSCLC patients (n=98) with mean age of 59±10.5 were enrolled (M/F: 75/23). DNA was extracted from formalin fixed paraffin embedded tissues. Exons 19 and 21 were amplified using polymerase chain reaction followed by direct sequencing for identification of mutations. Serum EGFR, AR, and TGF-α were measured by ELISA.. EGFR mutation rate in patients was 37% (exon 19 deletions: 72.2%, exon 21 substitutions: 27.8%). The E872K in exon 21 mutation-positive cases was the most frequent rare mutation detected (90%; 9/10 samples). A significant relationship was found between EGFR exon 21mutations and serum EGFR and TGF-α (P<0.05). Increased serum AR (>3pg/ml) and TGF-α (>10.5pg/ml) were associated with shorter overall survival (P<0.05).. The data clearly show that elevation of serum TGF-α and AR are associated with poor prognosis of NSCLC. In addition to the close relationship between EGFR mutations and serum EGFR, serum TGF-α changes was associated with the gene mutations. These findings could be implicated in clinical decision making related to EGFR-TKIs.

Topics: Adenocarcinoma; Adult; Amphiregulin; Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Enzyme-Linked Immunosorbent Assay; ErbB Receptors; Female; Follow-Up Studies; Humans; Ligands; Longitudinal Studies; Lung Neoplasms; Lymphatic Metastasis; Male; Middle Aged; Mutation; Mutation Rate; Neoplasm Staging; Prognosis; Real-Time Polymerase Chain Reaction; Survival Rate; Transforming Growth Factor alpha

MiR-137 has been reported to serve as a tumor suppressor in non-small cell lung cancer (NSCLC). However, the potential mechanism remains largely unclear. The present study aimed to explore the potential molecular mechanisms by which miR-137 regulated NSCLC.. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to quantify the expression levels of miR-137 in NSCLC tissues and cell lines. Dual-luciferase reporter assay was employed to confirm the specificity of miR-137 target genes. An MTT assay and flow cytometry were used to determine the rates of cell proliferation and cell cycle distribution. Furthermore, the effect of miR-137 up-regulation on TGFA expression was examined by western blot.. miR-137 expression levels in NSCLC cell lines or tissue were significantly lower than in a normal human lung cell line or adjacent normal tissues. We further found that upregulation of miR-137 inhibited the proliferation of NSCLC cells, whereas silencing of miR-137 promoted the proliferation of NSCLC. Moreover, we identified TGFA as a direct target gene of miR-137 in NSCLC cell. Finally, Similarly, knockdown of TGFA led to the suppression of NSCLC cell proliferation.. Overall, our findings indicated that miR-137 served as a tumor suppressor in NSCLC and its suppressive effect is mediated by repressing TGFA expression.

Topics: Carcinogenesis; Carcinoma, Non-Small-Cell Lung; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Humans; Lung Neoplasms; MicroRNAs; Transforming Growth Factor alpha

Alectinib is a highly selective ALK inhibitor and shows promising efficacy in non-small cell lung cancers (NSCLC) harboring the EML4-ALK gene rearrangement. The precise mechanism of acquired resistance to alectinib is not well defined. The purpose of this study was to clarify the mechanism of acquired resistance to alectinib in ALK-translocated lung cancer cells. We established alectinib-resistant cells (H3122-AR) from the H3122 NSCLC cell line, harboring the EML4-ALK gene rearrangement, by long-term exposure to alectinib. The mechanism of acquired resistance to alectinib in H3122-AR cells was evaluated by phospho-receptor tyrosine kinase (phospho-RTK) array screening and Western blotting. No mutation of the ALK-TK domain was found. Phospho-RTK array analysis revealed that the phosphorylation level of EGFR was increased in H3122-AR cells compared with H3122. Expression of TGFα, one of the EGFR ligands, was significantly increased and knockdown of TGFα restored the sensitivity to alectinib in H3122-AR cells. We found combination therapy targeting ALK and EGFR with alectinib and afatinib showed efficacy both in vitro and in a mouse xenograft model. We propose a preclinical rationale to use the combination therapy with alectinib and afatinib in NSCLC that acquired resistance to alectinib by the activation of EGFR bypass signaling.

Topics: Anaplastic Lymphoma Kinase; Animals; Carbazoles; Cell Line, Tumor; Cell Proliferation; Disease Models, Animal; Drug Resistance, Neoplasm; ErbB Receptors; Female; Gene Expression; Humans; Lung Neoplasms; Mice; Phosphorylation; Piperidines; Receptor Protein-Tyrosine Kinases; Signal Transduction; Transforming Growth Factor alpha; Translocation, Genetic; Tumor Burden; Xenograft Model Antitumor Assays

Aberrant expression of miR-374a has been reported in several types of human cancers, including lung cancer. However, the functional significance and molecular mechanisms underlying the role of miR-374a in lung cancer remain largely unknown. We found that the expression of miR-374a was significantly downregulated in lung adenocarcinoma tissues compared to adjacent normal lung tissues in samples included in The Cancer Genome Atlas. Functional studies revealed that overexpression of miR-374a led to inhibition of lung adenocarcinoma cell proliferation, migration and invasion and that miR-374a negatively regulated transforming growth factor-alpha (TGFA) gene expression by directly targeting the 3'-UTR of TGFA mRNA. Treating lung adenocarcinoma cells with TGF-α neutralizing antibody resulted in suppression of cell proliferation and invasion, which mimicked the action of miR-374a. Additionally, TGFA gene expression was significantly higher in tumor tissues compared to adjacent normal tissue and high TGFA gene expression strongly correlated with poor survival in patients with lung adenocarcinoma. Taken together, our studies suggest that miR-374a suppresses lung adenocarcinoma cell proliferation and invasion via targeting TGFA gene expression. Our findings may provide novel treatment strategies for lung adenocarcinoma patients.

Topics: 3' Untranslated Regions; Adenocarcinoma; Adenocarcinoma of Lung; Antibodies, Neutralizing; Cell Line, Tumor; Cell Movement; Cell Proliferation; Gene Expression Regulation, Neoplastic; Humans; Lung; Lung Neoplasms; MicroRNAs; Transforming Growth Factor alpha

The distribution of omega-6 and omega-3 polyunsaturated fatty acid (PUFA) intake in Western diets is disproportionate, containing an overabundance of the omega-6 PUFA, linoleic acid (LA; C18:2). Increased enrichment with LA has been shown to contribute to the enhancement of tumorigenesis in several cancer models. Previous work has indicated that phosphatidylinositol 3-kinase (PI3K) may play a key role in LA-induced tumorigenesis. However, the modes by which LA affects carcinogenesis have not been fully elucidated. In this study, a mechanism for LA-induced upregulation of cancer cell growth is defined. LA treatment enhanced cellular proliferation in BT-474 human breast ductal carcinoma and A549 human lung adenocarcinoma cell lines. Enrichment of LA increased cyclooxygenase (COX) activity and led to increases in prostaglandin E2 (PGE2), followed by increases in matrix metalloproteinase (MMP) and transforming growth factor alpha (TGF-α) levels, which are all key elements involved in the enhancement of cancer cell growth. Further investigation revealed that LA supplementation in both BT-474 breast and A549 lung cancer cell lines greatly increased the association between the scaffolding protein GRB2-associated-binding protein 1 (Gab1) and epidermal growth factor receptor (EGFR), although Gab1 protein levels were significantly decreased. These LA-induced changes were associated with increases in activated Akt (pAkt), a downstream signaling component in the PI3K pathway. Treatment with inhibitors of EGFR, PI3K and Gab1-specific siRNAs reversed the upregulation of pAkt, as well as the observed increases in cell proliferation by LA in both cell lines. A549 xenograft assessment in athymic nude mice fed high levels of LA exhibited similar increases in EGFR-Gab1 association and increased levels of pAkt, while mice fed with high levels of the omega-3 PUFA, docosahexaenoic acid (DHA; C22:6), demonstrated an opposite response. The involvement of Gab1 in LA-induced tumorigenesis was further defined utilizing murine cell lines that express high levels of Gab1. Significant increases in cell proliferation were observed with the addition of increasing concentrations of LA. However, no changes in cell proliferation were detected in the murine paired cell lines expressing little or no Gab1 protein, establishing Gab1 as major target in LA-induced enhancement of tumorigenesis.

Topics: Adaptor Proteins, Signal Transducing; Animals; Breast Neoplasms; Carcinogenesis; Cell Line, Tumor; Cell Proliferation; Cyclooxygenase 2; Dinoprostone; Female; Humans; Linoleic Acid; Lung Neoplasms; Male; Mice; Mice, Nude; Neoplasm Transplantation; Phosphatidylinositol 3-Kinases; Transforming Growth Factor alpha

The use of biomarkers for selecting non-small cell lung cancer (NSCLC) patients for treatment with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) is essential. The aim of this study was to explore whether biomarkers detected in plasma were predictive for response to EGFR-TKIs and survival time of NSCLC patients.. Tumor tissues and paired blood were collected from 134 advanced NSCLC patients treated with EGFR-TKIs. EGFR mutations in both types of specimens, and expression of transforming growth factor-alpha and beta one (TGF-α and TGF-β1) were assessed in NSCLC patients. Concentrations of circulating free DNA were detected in plasma from both NSCLC patients and healthy subjects. The clinical significance of EGFR mutations, levels of cytokines, and circulating free DNA was assessed in advanced NSCLC patients.. EGFR mutations were detected in 68 tumor samples and 17 plasma samples of 134 NSCLC patients. The concentrations of circulating free DNA were higher in NSCLC patients than in healthy subjects. Patients with high TGF-β1 level showed shorter overall survival and worse response to EGFR-TKIs than patients with low TGF-β1 level.. Plasma levels of TGF-β1 may be a marker for predicting response to EGFR-TKIs and survival time in NSCLC patients.

Topics: Adult; Aged; Antineoplastic Agents; Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Disease Progression; ErbB Receptors; Female; Humans; Lung Neoplasms; Male; Middle Aged; Mutation; Precision Medicine; Prognosis; Protein Kinase Inhibitors; Survival Analysis; Transforming Growth Factor alpha; Transforming Growth Factor beta1

The mechanism by which SR48692 inhibits non-small cell lung cancer (NSCLC) proliferation was investigated.. The ability of SR48692 to inhibit the proliferation of NSCLC cell lines NCI-H1299 and A549 was investigated in vitro in the presence or absence of neurotensin (NTS). The ability of NTS to cause epidermal growth factor receptor (EGFR) transactivation was investigated by Western blot using NSCLC cells and various inhibitors. The growth effects and Western blot results were determined in cell lines treated with siRNA for NTSR1.. Treatment of A549 or NCI-H1299 cells with siRNA for NTSR1 reduced significantly NTSR1 protein and the ability of SR48692 to inhibit the proliferation of A549 or NCI-H1299 NSCLC cells. Treatment of A549 and NCI-H1299 cells with siRNA for NTSR1 reduced the ability of NTS to cause epidermal growth factor receptor (EGFR) transactivation. SR48692 or gefitinib (EGFR tyrosine kinase inhibitor) inhibited the ability of NTS to cause EGFR and ERK tyrosine phosphorylation. NTS transactivation of the EGFR was inhibited by GM6001 (matrix metalloprotease inhibitor), Tiron (superoxide scavenger) or U73122 (phospholipase C inhibitor) but not H89 (PKA inhibitor). NTS stimulates whereas SR48692 or gefitinib inhibits the clonal growth of NSCLC cells.. These results suggest that SR48692 may inhibit NSCLC proliferation in an EGFR-dependent mechanism.

Topics: Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Drug Screening Assays, Antitumor; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Gefitinib; Gene Knockdown Techniques; Humans; Lung Neoplasms; Phosphorylation; Protein Processing, Post-Translational; Pyrazoles; Quinazolines; Quinolines; Receptors, Neurotensin; RNA, Small Interfering; Transcriptional Activation; Transforming Growth Factor alpha

Osteosarcoma is the most common primary malignancy of bone and is characterized by a high malignant and metastatic potential. Transforming growth factor alpha (TGF-α) is classified as the EGF (epidermal growth factor)-like family, which is involved in cancer cellular activities such as proliferation, motility, migration, adhesion and invasion abilities. However, the effect of TGF-α on human osteosarcoma is largely unknown. We found that TGF-α increased the cell migration and expression of intercellular adhesion molecule-1 (ICAM-1) in human osteosarcoma cells. Transfection of cells with ICAM-1 siRNA reduced TGF-α-mediated cell migration. We also found that the phosphatidylinositol 3'-kinase (PI3K)/Akt/NF-κB pathway was activated after TGF-α treatment, and TGF-α-induced expression of ICAM-1 and cell migration was inhibited by the specific inhibitors and siRNAs of PI3K, Akt, and NF-κB cascades. In addition, knockdown of TGF-α expression markedly decreased cell metastasis in vitro and in vivo. Our results indicate that TGF-α/EGFR interaction elicits PI3K and Akt activation, which in turn activates NF-κB, resulting in the expression of ICAM-1 and contributing the migration of human osteosarcoma cells.

Topics: Animals; Antineoplastic Agents; Bone Neoplasms; Cell Line, Tumor; Cell Movement; ErbB Receptors; Humans; Intercellular Adhesion Molecule-1; Lung Neoplasms; Male; Mice; Mice, SCID; NF-kappa B; Osteosarcoma; Phosphatidylinositol 3-Kinase; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Recombinant Proteins; RNA Interference; Second Messenger Systems; Transforming Growth Factor alpha; Tumor Burden; Up-Regulation; Xenograft Model Antitumor Assays

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

A high frequency of mutations at the PTEN locus has been noticed in carcinoma of lung. However, the role of PTEN alternations and its association with outcome variables in the genesis of lung carcinoma are not understood fully. The purpose of our study was to examine the impact of EGFR, TGF-α, P-AKT and PTEN in the genesis of non-small cell lung cancer (NSCLC). Total numbers of 66 histopathologically confirmed cases of NSCLC and 10 cases of benign control samples embedded with wax were studied. We assessed EGFR, TGF-α and P-AKT by the use of specific antibody through immunohistochemistry as directed by the manufacturer, and detected PTEN expression by in situ hybridization. There were progressive loss of PTEN expression and significant increasing in EGFR, TGF-α, P-AKT expression from benign samples to NSCLC (p<0.05). The overexpression of EGFR, TGF-α, P-AKT and loss of PTEN expression were correlated to differentiation extent of cancer tissue, metastasis of lymph nodes and histological classification. Thus, alteration of EGFR, TGF-α, P-AKT and PTEN are likely important molecular events in pathogenesis and carcinogenesis of NSCLC.

Topics: Adult; Aged; Carcinoma, Non-Small-Cell Lung; Cell Differentiation; ErbB Receptors; Female; Humans; Lung; Lung Neoplasms; Male; Middle Aged; Phosphatidylinositol 3-Kinases; Phosphorylation; Prognosis; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Signal Transduction; Transforming Growth Factor alpha

Despite growing recognition of an etiologic role for inflammation in lung carcinogenesis, few prospective epidemiologic studies have comprehensively investigated the association of circulating inflammation markers with lung cancer.. We conducted a nested case-control study (n = 526 lung cancer patients and n = 592 control subjects) within the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial. Control subjects were matched to lung cancer case patients on age, sex, follow-up time (median = 2.9 years), randomization year, and smoking (pack-years and time since quitting). Serum levels of 77 inflammation markers were measured using a Luminex bead-based assay. Conditional logistic regression and weighted Cox models were used to estimate odds ratios (ORs) and cumulative risks, respectively.. Of 68 evaluable markers, 11 were statistically significantly associated with lung cancer risk (P trend across marker categories < .05), including acute-phase proteins (C-reactive protein [CRP], serum amyloid A [SAA]), proinflammatory cytokines (soluble tumor necrosis factor receptor 2 [sTNFRII]), anti-inflammatory cytokines (interleukin 1 receptor antagonist [IL-1RA]), lymphoid differentiation cytokines (interleukin 7 [IL-7]), growth factors (transforming growth factor alpha [TGF-A]), and chemokines (epithelial neutrophil-activating peptide 78 [ENA 78/CXCL5], monokine induced by gamma interferon [MIG/CXCL9], B cell-attracting chemokine 1 [BCA-1/CXCL13], thymus activation regulated chemokine [TARC/CCL17], macrophage-derived chemokine [MDC/CCL22]). Elevated marker levels were associated with increased lung cancer risk, with odds ratios comparing the highest vs the lowest group ranging from 1.47 (IL-7) to 2.27 (CRP). For IL-1RA, elevated levels were associated with decreased lung cancer risk (OR = 0.71; 95% confidence interval = 0.51 to 1.00). Associations did not differ by smoking, lung cancer histology, or latency. A cross-validated inflammation score using four independent markers (CRP, BCA-1/CXCL13, MDC/CCL22, and IL-1RA) provided good separation in 10-year lung cancer cumulative risks among former smokers (quartile [Q] 1 = 1.1% vs Q4 = 3.1%) and current smokers (Q1 = 2.3% vs Q4 = 7.9%) even after adjustment for smoking.. Some circulating inflammation marker levels are associated with prospective lung cancer risk.

Topics: Aged; Biomarkers; Biomarkers, Tumor; C-Reactive Protein; Case-Control Studies; Chemokines; Cytokines; Female; Humans; Inflammation; Logistic Models; Lung Neoplasms; Male; Middle Aged; Odds Ratio; Predictive Value of Tests; Prospective Studies; Risk Assessment; Risk Factors; Serum Amyloid A Protein; Transforming Growth Factor alpha

To observe the effects of hepatocyte growth factor (HGF) derived from tumor microenvironment and/or afatinib on the growth of human lung adenocarcinoma H1975 cells and explore the potential mechanisms by which HGF induces primary resistance to afatinib.. The effects of HGF, TGF-α and afatinib on the growth of H1975 cells were evaluated by MTT assay. The HGF concentrations of normal human fetal lung fibroblasts MRC-5 cells and human lung adenocarcinoma H1975 cells co-cultured or separately cultured were determined by ELISA assay. Western blot was used to detect the expressions of EGFR and Met signal pathway-related proteins and epithelial-mesenchymal transition (EMT) markers in H1975 cells treated with HGF and/or afatinib.. The MTT assay showed that H1975 cells were hyposensitive to afatinib in the presence of HGF. The ELISA assay showed that HGF production by H1975 cells was less than 0.1 ng/2.0×10(6) cells, but HGF production by MRC-5 cells was (151.37 ± 2.07)ng/2.0×10(6) cells incubated for 48 h. When H1975 cells and MRC-5 cells were co-cultured for 72 h, the concentration of HGF in the culture supernatant was (61.13 ± 16.21)ng/ml. In the presence of HGF, the expression of p-Met, p-Akt and p-ERK proteins in the H1975 cells was markedly up-regulated. afatinib inhibited p-EGFR, but did not affect the expression of p-Met, p-Akt and p-ERK proteins. In the presence of afatinib, HGF up-regulated the expression of vimentin and down-regulated the expression of E-cadherin.. HGF secreted by stromal cells in the tumor micro-environment may confer resistance to afatinib in H1975 cells by activation of the Met/PI3K/Akt and Met/MAPK/ERK signaling pathways, and is involved in the epithelial-mesenchymal transition process.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Afatinib; Antineoplastic Agents; Cadherins; Cell Line, Tumor; Cell Proliferation; Cells, Cultured; Coculture Techniques; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; ErbB Receptors; Fibroblasts; Hepatocyte Growth Factor; Humans; Lung; Lung Neoplasms; MAP Kinase Signaling System; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-met; Quinazolines; Signal Transduction; Transforming Growth Factor alpha; Tumor Microenvironment; Vimentin

Somatic mutations in the epidermal growth factor receptor (EGFR) gene, such as exon 19 deletion mutations, are important factors in determining therapeutic responses to gefitinib in non-small-cell lung cancer (NSCLC). However, some patients have activating mutations in EGFR and show poor responses to gefitinib. In this study, we examined three NSCLC cell lines, HCC827, PC9, and HCC2935, that expressed an EGFR exon 19 deletion mutation. All cells expressed mutant EGFR, but the PC9 and HCC2935 cells also expressed wild-type EGFR. The HCC827 cells were highly sensitive to gefitinib under both normoxia and hypoxia. However, the PC9 and HCC2935 cells were more resistant to gefitinib under hypoxic conditions compared to normoxia. Phosphorylation of EGFR and ERK was suppressed with gefitinib treatment to a lesser extent under hypoxia. The expression of transforming growth factor-α (TGFα) was dramatically upregulated under hypoxia, and the knockdown of TGFα or hypoxia-inducible factor-1α (HIF1α) reversed the resistance to gefitinib in hypoxic PC9 and HCC2935 cells. Finally, introduction of the wild-type EGFR gene into the HCC827 cells caused resistance to gefitinib under hypoxia. This phenomenon was also reversed by the knockdown of TGFα or HIF1α. Our results indicate that hypoxia causes gefitinib resistance in EGFR-mutant NSCLC through the activation of wild-type EGFR mediated by the upregulation of TGFα. The presence of wild-type and mutant EGFR along with tumor hypoxia are important factors that should be considered when treating NSCLC patients with gefitinib.

Topics: Carcinoma, Non-Small-Cell Lung; Cell Hypoxia; Cell Line, Tumor; Drug Resistance, Neoplasm; ErbB Receptors; Gefitinib; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Lung Neoplasms; MAP Kinase Signaling System; Mutation; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Quinazolines; Transforming Growth Factor alpha; Up-Regulation

Brain metastasis is an increasingly common complication for breast cancer patients; approximately 15- 30% of breast cancer patients develop brain metastasis. However, relatively little is known about how these metastases form, and what phenotypes are characteristic of cells with brain metastasizing potential. In this study, we show that the targeted knockdown of MMP-1 in breast cancer cells with enhanced brain metastatic ability not only reduced primary tumor growth, but also significantly inhibited brain metastasis.. Two variants of the MDA-MB-231 human breast cancer cell line selected for enhanced ability to form brain metastases in nude mice (231-BR and 231-BR3 cells) were found to express high levels of matrix metalloproteinase-1 (MMP-1). Short hairpin RNA-mediated stable knockdown of MMP-1 in 231-BR and 231-BR3 cells were established to analyze tumorigenic ability and metastatic ability.. Short hairpin RNA-mediated stable knockdown of MMP-1 inhibited the invasive ability of MDA-MB 231 variant cells in vitro, and inhibited breast cancer growth when the cells were injected into the mammary fat pad of nude mice. Reduction of MMP-1 expression significantly attenuated brain metastasis and lung metastasis formation following injection of cells into the left ventricle of the heart and tail vein, respectively. There were significantly fewer proliferating cells in brain metastases of cells with reduced MMP-1 expression. Furthermore, reduced MMP-1 expression was associated with decreased TGFα release and phospho-EGFR expression in 231-BR and BR3 cells.. Our results show that elevated expression of MMP-1 can promote the local growth and the formation of brain metastases by breast cancer cells.

Topics: Animals; Brain; Brain Neoplasms; Breast Neoplasms; Cell Line, Tumor; ErbB Receptors; Female; Humans; Lung Neoplasms; Matrix Metalloproteinase 1; Mice; Mice, Nude; Neoplasm Metastasis; Transforming Growth Factor alpha; Transplantation, Heterologous

Chemotherapy combined concurrently with TKIs produced a negative interaction and failed to improve survival when compared with chemotherapy or TKIs alone in the treatment of non-small cell lung cancer (NSCLC). The present study investigated the sequence-dependent interaction between paclitaxel and gefitinib and clarified the underlying mechanism.. The effects on cell proliferation, EGFR signaling pathway, and TGFα expression were evaluated in a panel of human NSCLC cell lines harboring EGFR mutations with three different combination sequences: sequential treatment with paclitaxel followed by gefitinib (T→G), sequential treatment with gefitinib followed by paclitaxel (G→T), or concomitant treatment (T + G).. The sequence-dependent anti-proliferative effects differed between EGFR-TKI-sensitive and -resistant cell lines carrying EGFR mutations. A synergistic anti-proliferative activity was obtained with paclitaxel treatment followed by gefitinib in all cell lines, with mean CI values of 0.63 in Hcc827, 0.54 in PC-9, 0.81 in PC-9/GR, and 0.77 in H1650 cells for the T→G sequence. The mean CI values for the G→T sequence were 1.29 in Hcc827, 1.16 in PC-9, 1.52 in PC-9/GR, and 1.5 in H1650 cells. The mean CI values for T+G concomitant treatment were 0.88 in Hcc827, 0.91 in PC-9, 1.05 in PC-9/GR, and 1.18 in H1650 cells. Paclitaxel produced a dose-dependent increase in EGFR phosphorylation. Paclitaxel significantly increased EGFR phosphorylation compared with that in untreated controls (mean differences: +50% in Hcc827, + 56% in PC-9, + 39% in PC-9/GR, and + 69% in H1650 cells; p < 0.05). The T→G sequence produced significantly greater inhibition of EGFR phosphorylation compared with the opposite sequence (mean differences: -58% in Hcc827, -38% in PC-9, -35% in PC-9/GR, and -30% in H1650 cells; p < 0.05). Addition of a neutralizing anti-TGFα antibody abolished paclitaxel-induced activation of the EGFR pathway in PC-9 and H1650 cells. Sequence-dependent TGFα expression and release are responsible for the sequence-dependent EGFR pathway modulation.. The data suggest that the sequence of paclitaxel followed by gefitinib is an appropriate treatment combination for NSCLC cell lines harboring EGFR mutations. Our results provide molecular evidence to support clinical treatment strategies for patients with lung cancer.

Topics: Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Drug Resistance, Neoplasm; Drug Synergism; ErbB Receptors; Gefitinib; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Paclitaxel; Quinazolines; Signal Transduction; Transforming Growth Factor alpha

Mucous hypersecretion is an important feature of obstructive airway diseases such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis. Multiple stimuli induce mucin production via activation of an epidermal growth factor receptor (EGFR) cascade, but the mechanisms that exaggerate mucin production in obstructive airway diseases remain unknown. In this study, we show that binding of CCL20, a G protein-coupled receptor (GPCR) ligand that is upregulated in the airways of subjects with obstructive airway diseases, to its unique GPCR CCR6 induces MUC5AC mucin production in human airway epithelial (NCI-H292) cells via metalloprotease TNF-α-converting enzyme (TACE)-dependent EGFR activation. We also show that EGFR activation by its potent ligand TGF-α induces reactivation of EGFR via binding of endogenously produced CCL20 to its receptor CCR6 in NCI-H292 cells but not in normal human bronchial epithelial (NHBE) cells, exaggerating mucin production in the NCI-H292 cells. In NCI-H292 cells, TGF-α stimulation induced two phases of EGFR phosphorylation (EGFR-P). The second EGFR-P was TACE-dependent and was responsible for most of the total mucin induced by TGF-α. Binding of endogenously produced CCL20 to CCR6 increased the second EGFR-P and subsequent mucin production induced by TGF-α. In NHBE cells, TGF-α-induced EGFR activation did not lead to significant CCL20 production or to EGFR rephosphorylation, and less mucin was produced. We conclude that NCI-H292 cells but not NHBE cells produce CCL20 in response to EGFR activation, which leads to a second phase of EGFR-P and subsequent exaggerated mucin production. These findings have potentially important therapeutic implications in obstructive airway diseases.

Topics: Carcinoma, Mucoepidermoid; Cell Communication; Cell Line, Tumor; Chemokine CCL20; ErbB Receptors; Feedback, Physiological; Humans; Ligands; Lung Neoplasms; Mucin 5AC; Phosphorylation; Protein Binding; Receptors, CCR6; Respiratory Mucosa; Transforming Growth Factor alpha

In the growth factor receptor gene FGFR4 the presence of the common single nucleotide polymorphism Arg388 has been associated with progression of various types of cancer including breast cancer. However, a causative relationship is not readily assigned due to genetic heterogeneity in different patient cohorts. To address this issue, we compared the effects of this allele on malignant progression in the WAP-TGFalpha transgenic mouse model of breast cancer. A knock-in strain was generated to introduce an analogous Arg385 allele into the murine FGFR4 gene. Mouse embryonic fibroblasts derived from this strain displayed accelerated cell transformation, with transformed cells exhibiting greater motility and invasive behavior. In the in vivo context of TGFalpha-induced mammary carcinogenesis, tumor development and progression was significantly advanced in tumor mass, size, and onset of pulmonary metastases. Our findings definitively identify the FGFR4 Arg388 allele as a functional prognostic marker for breast cancer progression.

Topics: 3T3 Cells; Alleles; Animals; Cell Adhesion; Cell Movement; Cell Transformation, Neoplastic; Disease Progression; Female; Gene Expression Regulation, Neoplastic; Genome; Lung Neoplasms; Mammary Neoplasms, Experimental; Mice; Mice, Transgenic; Milk Proteins; Polymorphism, Single Nucleotide; Receptor, Fibroblast Growth Factor, Type 4; Transforming Growth Factor alpha

To evaluate the prognostic and predictive significance of plasma levels of the epidermal growth factor receptor (EGFR) ligands, transforming growth factor α (TGF-α) and amphiregulin, in patients with non-small-cell lung cancer (NSCLC) enrolled in NCIC Clinical Trials Group BR.21 comparing erlotinib with placebo.. TGF-α and amphiregulin were assessed retrospectively by enzyme-linked immunosorbent assay from available prospectively collected baseline plasma samples in 565 of 731 BR.21 patients. Cutoff points were determined for both amphiregulin (low, <10 pg/mL; high, ≥10 pg/mL) and TGF-α (low, ≤12 pg/mL; high, >12 pg/mL) using a graphical method. Cox regression models were used to correlate biomarker data and baseline characteristics with outcomes including overall (OS) and progression-free survival (PFS).. High TGF-α and amphiregulin were associated with poorer performance status (P=.06 and P<.0001, respectively) and no prior platinum therapy (P=.06 and P=.02, respectively). High amphiregulin was also associated with anemia (P=.001), increased lactate dehydrogenase (P=.03), ever-smokers (P=.04), and non-Asian ethnicity (P=.001). Patients on the placebo arm with high amphiregulin had poorer OS than patients with low amphiregulin (hazard ratio [HR]=1.88; 95% CI, 1.34 to 2.64; P=.0002), which remained significant in multivariate analysis. Amphiregulin levels did not predict for benefit from erlotinib (interaction P=.87). Conversely, TGF-α levels did not have prognostic significance, but high TGF-α predicted lack of benefit from erlotinib compared with low TGF-α (TGF-α low, OS HR=0.66; 95% CI, 0.54 to 0.81; P<.0001; high, OS HR=1.32; 95% CI, 0.73 to 2.39; P=.36; interaction P=.04).. High baseline amphiregulin is a poor prognostic factor, whereas high baseline TGF-α predicts for lack of benefit from erlotinib in advanced NSCLC.

Topics: Amphiregulin; Antineoplastic Agents; Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; EGF Family of Proteins; Erlotinib Hydrochloride; Female; Glycoproteins; Humans; Intercellular Signaling Peptides and Proteins; Lung Neoplasms; Male; Middle Aged; Prognosis; Quinazolines; Retrospective Studies; Transforming Growth Factor alpha

The selection of patients with non-small cell lung cancer (NSCLC) for epidermal growth factor receptor (EGFR) inhibitor (EGFR-tyrosine kinase inhibitors [TKIs]) therapy is suboptimal as tumor tissue is often unavailable. Ligands of EGFR, transforming growth factor-alpha (TGFa) and amphiregulin (ARG), and the insulin-like growth factor (IGF) family have been associated with resistance to EGFR-TKIs. The aim of our study was to explore whether concentrations of these factors measured in serum were predictive of response to EGFR-TKIs.. We assessed serum levels of marker candidates using enzyme-linked immunosorbent (TGFa and ARG) and chemiluminescent (IGF1 and IGF-binding protein-3) assays in 61 patients with advanced NSCLC treated with EGFR-TKIs and 63 matched advanced NSCLC control patients without EGFR-TKIs treatment. We dichotomized marker levels at the 20th, 50th, or 80th percentile and evaluated whether the effect of EGFR-TKIs treatment on disease-specific survival (DSS) differed by marker level based on multivariate proportional hazards regression with an interaction term.. The effect of EGFR-TKIs treatment on DSS showed a significant difference by TGFa and ARG (interaction p = 0.046 and p = 0.004, respectively). Low concentrations of TGFa and high concentrations of ARG were associated with a better DSS in EGFR-TKIs patients compared with control patients. Patients with high concentrations of IGF-binding protein-3 had significantly longer DSS, independent of treatment (hazard ratio: 0.60 per 1 mg/liter, 95% confidence interval: 0.46-0.79).. Our results suggest that concentrations of TGFa and ARG measured in serum are predictive of EGFR-TKI response. The combination of these two biomarkers could be of value in the process of selecting patients for treatment with EGFR-TKIs.

Topics: Adult; Aged; Amphiregulin; Biomarkers; Carcinoma, Non-Small-Cell Lung; EGF Family of Proteins; ErbB Receptors; Female; Glycoproteins; Humans; Insulin-Like Growth Factor Binding Protein 3; Insulin-Like Growth Factor Binding Proteins; Insulin-Like Growth Factor I; Intercellular Signaling Peptides and Proteins; Lung Neoplasms; Male; Middle Aged; Protein Kinase Inhibitors; Retrospective Studies; Transforming Growth Factor alpha

Periostin is a secreted protein and has been shown to be frequently overexpressed in various types of human cancers. We have previously reported that periostin potently promotes metastatic growth of colon cancer by augmenting cell survival. However, little is known about the functions of periostin in non-small-cell lung cancer. Here, we revealed that increased expression of periostin in non-small-cell lung cancer A549 cells was one kind of cellular responses to the stress of chemical-mimic hypoxia, and this effect could be regulated by hypoxia inducible growth factors, such as TGF-alpha and bFGF. We further demonstrated that RTK/PI3-K pathway activated by TGF-alpha and bFGF was evoked in upregulating the expression of periostin, and then periostin promoted the survival of A549 cells under hypoxic microenvironment via activation of Akt/PKB pathway. Therefore, periostin and the pathway that it involved might provide a target for lung cancer treatment.

Topics: Apoptosis; Blotting, Western; Carcinoma, Non-Small-Cell Lung; Cell Adhesion Molecules; Cell Hypoxia; Cell Line, Tumor; Cell Survival; Fibroblast Growth Factor 2; Humans; Lung Neoplasms; Proto-Oncogene Proteins c-akt; Signal Transduction; Transfection; Transforming Growth Factor alpha; Up-Regulation

Lung cancer is the leading cause of cancer death worldwide. The spectrum of aberrations affecting signalling pathways in lung cancer pathogenesis has not been fully elucidated. Physiological expression of Vav1 is restricted to the haematopoietic system, where its best-known function is as a GDP/GTP nucleotide exchange factor for Rho/RacGTPases, an activity strictly controlled by tyrosine phosphorylation downstream of cell surface receptors. Here we find Vav1 expression in 42% of 78 lung cancer cell lines analysed. Moreover, immunohistochemical analysis of primary human lung cancer tissue samples revealed Vav1 expression in 26/59 malignant samples, including adenocarcinoma, squamous cell carcinoma and bronchioloalveolar carcinoma. Stronger Vav1 staining was associated with larger tumour size. siRNA-mediated knockdown of Vav1 in lung cancer cells reduced proliferation in agar and tumour growth in nude mice, while control siRNA had no effect, suggesting that Vav1 plays a critical role in the tumorigenicity of lung cancer cells. Vav1 is tyrosine-phosphorylated in lung cancer cells following activation by the growth factors EGF and TGFalpha, suggesting its participation in signalling events in these cells. Depletion of Vav1 reduced Rac-GTP activation and decreased expression of TGFalpha, an autocrine growth factor. These data suggest that Vav1 plays a role in the neoplastic process in lung cancer, identifying it as a potential therapeutic target for lung cancer therapy.

Topics: Animals; Carcinoma; Cell Line, Tumor; Female; Gene Expression; Gene Expression Regulation, Neoplastic; Glyceraldehyde-3-Phosphate Dehydrogenases; Hematopoiesis; Humans; Immunohistochemistry; Lung Neoplasms; Mice; Mice, Nude; Proto-Oncogene Proteins c-vav; rac GTP-Binding Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; RNA, Small Interfering; Signal Transduction; Transforming Growth Factor alpha

Circulating amphiregulin and transforming growth factor-alpha (TGF-alpha) have been found to be correlated with an unfavorable response to gefitinib based on the identification of patients with a higher probability of resistance to the drug. However, the association between an epidermal growth factor receptor (EGFR) somatic mutation and the overexpression of its ligands has not been determined. To verify the clinical significance of the two serum markers and EGFR mutation status, we determined serum amphiregulin and TGF-alpha levels by enzyme-linked immunosorbent assay in 93 patients with advanced non-squamous, non-small cell lung cancer and EGFR somatic mutation status using the peptic nucleic acid-locked nucleic acid clamp method in 46 cases. The relationship between each independent clinicopathological variable and the response to gefitinib therapy was examined. We also evaluated the risk factors associated with prognosis. Fourteen (41.0%) of 34 progressive disease cases were positive for amphiregulin (P = 0.007). Eleven (32.4%) of 34 progressive disease cases were positive for TGF-alpha (P = 0.005). The median survival time of patients with the EGFR somatic mutation was significantly longer (P = 0.01). The same was true of amphiregulin- (P = 0.046) and TGF-alpha-negative patients (P < 0.01). In multivariate analysis, serum TGF-alpha positivity (hazard ratio, 2.558; P = 0.005) and the wild type EGFR gene (hazard ratio, 1.894; P = 0.003) were significant independent prognostic factors. Our study demonstrates that the status of the serum EGFR ligand, in addition to EGFR activating mutation, is a predictive factor for response to gefitinib therapy.

Topics: Amphiregulin; Carcinoma, Non-Small-Cell Lung; EGF Family of Proteins; ErbB Receptors; Glycoproteins; Humans; Intercellular Signaling Peptides and Proteins; Lung Neoplasms; Mutation; Neoplasm Staging; Survival Analysis; Transforming Growth Factor alpha

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

Currently, pathologic stage is the main prognostic indicator of colorectal carcinoma, but the current staging system is insufficient to predict the risk of recurrence or the need for adjuvant treatment for the patients with Dukes'A and B disease. Biologic prognostic markers may supplement the staging system and provide a basis for the decision of therapeutic strategies according to individual tumor biology. This study was to investigate the correlations of c-erbB-2, epithelial growth factor receptor (EGFR), and transforming growth factor-alpha (TGF-alpha) expression to recurrence of Dukes'A and B colorectal carcinoma.. The expression of c-erbB-2, EGFR and TGF-alpha in 26 specimens of Dukes'A and 62 specimens of Dukes'B colorectal adenocarcinoma was detected by SP immunohistochemistry. Of the 88 patients underwent curative resection between 1989 and 1999, 44 had recurrence, and 44 had not. The patients were followed up for at least 5 years or till recurrence. The tumor location, Dukes staging, age, sex, and depth of bowel wall invasion matched as closely as possible between the 2 groups.. The overexpression rate of c-erbB-2 was higher in recurrence group than in non-recurrence group (43.2% vs. 25.0%, P=0.072); the overexpression rates of EGFR and TGF-alpha were significantly higher in recurrence group than in non-recurrence group (63.6% vs. 27.3%, P=0.001; 65.9% vs. 43.2%, P=0.032). The co-overexpression rate of EGFR and TGF was significantly higher in recurrence group than in non-recurrence group (36.4% vs. 11.4%, P=0.006). Multivariate analysis showed that the overexpression of EGFR was associated with postoperative recurrence of colorectal carcinoma.. The expression of EGFR may be associated with postoperative recurrence of Dukes'A and B colorectal carcinoma.

Topics: Adenocarcinoma; Adult; Aged; Colonic Neoplasms; ErbB Receptors; Female; Humans; Liver Neoplasms; Lung Neoplasms; Male; Middle Aged; Neoplasm Recurrence, Local; Neoplasm Staging; Proportional Hazards Models; Receptor, ErbB-2; Rectal Neoplasms; Transforming Growth Factor alpha

In recent years, molecular insights shed light on the role of the epidermal growth factor receptor (EGFR) in nonsmall cell lung cancer (NSCLC), and new therapeutic agents, such as the EGFR tyrosine kinase inhibitors, were tested successfully, with responsiveness to those agents more likely in those patients with specific EGFR gene alterations. The objective of the current study was to investigate the protein profiles of EGFR, c-erb-B2, transforming growth factor alpha (TGF-alpha) (one of the EGFR ligands commonly expressed in NSCLC), and some downstream molecules, potentially to detect a subset of tumors with an activated autocrine loop that is responsible for higher intracellular signaling.. One hundred twelve consecutive patients with resected NSCLC were analyzed by immunohistochemistry for EGFR, the c-erb-B2 receptor, TGF-alpha, and pivotal molecules downstream from EGFR activation. Statistical correlations between the investigated molecular expression profiles and clinicopathologic data were performed.. EGFR, c-erb-B2, TGF-alpha and downstream molecule expression, per se, was not correlated significantly with any clinicopathologic variables, with the exception of a significant correlation between squamous histology and EGFR and between adenocarcinoma and TGF-alpha. However, nearly 30% of NSCLCs demonstrated coexpression of both TGF-alpha and EGFR, and this molecular status was associated positively with a statistically significant expression of phosphatidylinositol 3 kinase and an inversely with mitogen-activated protein kinase expression.. The presence of a subgroup of NSCLCs with an activated autocrine loop may help to explain the mechanisms that lead to the relative ineffectiveness of the EGFR tyrosine kinase inhibitor and may support new clinical trials to define whether the subgroup of patients with these tumors reasonably may benefit from higher doses of such inhibitors or from the simultaneous inhibition of EGFR downstream signaling targets.

Topics: Aged; Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; ErbB Receptors; Female; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Italy; Lung Neoplasms; Male; Middle Aged; Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinases; Receptor, ErbB-2; Retrospective Studies; Signal Transduction; Transforming Growth Factor alpha

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

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

Members of the ErbB receptor family are associated with several cancers and appear to be providing useful targets for pharmacological therapeutics for tumours of the lung and breast. Further improvements of these therapies may be guided by a quantitative, dynamic integrative systems understanding of the complexities of ErbB dimerisation, trafficking and activation, for it is these complexities that render difficult intuiting how perturbations such as drug intervention will affect ErbB signalling activities. Towards this goal, we have developed a computational model implementing commonly accepted principles governing ErbB receptor interaction, trafficking, phosphorylation and dephosphorylation. Using this model, we are able to investigate several hypotheses regarding the compartmental localisation of dephosphorylation. Model results applied to experimental data on ErbB 1, ErbB2 and ErbB3 phosphorylation in H292 human lung carcinoma cells support a hypothesis that key dephosphorylation activity for these receptors occurs largely in an intracellular, endosomal compartment rather than at the cell surface plasma membrane. Thus, the endocytic trafficking-related compartmentalisation of dephosphorylation may define a critical aspect of the ErbB signalling response to ligand.

Topics: Cell Line; Computer Simulation; Dose-Response Relationship, Drug; Drug Combinations; Enzyme Activation; Humans; Lung Neoplasms; Metabolic Clearance Rate; Models, Biological; Neuregulin-1; Oncogene Proteins v-erbB; Phosphoric Monoester Hydrolases; Phosphorylation; Signal Transduction; Transforming Growth Factor alpha

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

Gastrin-releasing peptide receptor (GRPR) and the epidermal growth factor receptor (EGFR) are expressed in several cancers including non-small cell lung cancer (NSCLC). Here we demonstrate the activation of EGFR by the GRPR ligand, gastrin-releasing peptide (GRP), in NSCLC cells. GRP induced rapid activation of p44/42 MAPK in lung cancer cells through EGFR. GRP-mediated activation of MAPK in NSCLC cells was abrogated by pretreatment with the anti-EGFR-neutralizing antibody, C225. Pretreatment of NSCLC cells with neutralizing antibodies to the EGFR ligands, TGF-A or HB-EGF, also decreased GRP-mediated MAPK activation. On matrix metalloproteinase (MMP) inhibition, GRP failed to activate MAPK in NSCLC cells. EGF and GRP both stimulated NSCLC proliferation, and inhibition of either EGFR or GRPR resulted in cell death. Combining a GRPR antagonist with the EGFR tyrosine kinase inhibitor, gefitinib, resulted in additive cytotoxic effects. Additive effects were seen at gefitinib concentrations from 1 to 18 microM, encompassing the ID50 values of both gefitinib-sensitive and gefitinib-resistant NSCLC cell lines. Because a major effect of GRPR appears to be promoting the release of EGFR ligand, this study suggests that a greater inhibition of cell proliferation may occur by abrogating EGFR ligand release in consort with inhibition of EGFR.

Topics: Blotting, Western; Carcinoma, Non-Small-Cell Lung; Cell Death; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Enzyme Activation; ErbB Receptors; Gefitinib; Humans; Immunoblotting; Immunoprecipitation; Ligands; Lung Neoplasms; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Phosphorylation; Quinazolines; Receptors, Bombesin; Signal Transduction; Transforming Growth Factor alpha

In many human lung adenocarcinoma cell lines, a pathway involving epidermal growth factor receptor (EGFR), ErbB2 and ErbB3 receptors, phosphatidyl inositol 3-kinase (PI3K), Akt, glycogen synthase kinase 3-beta (GSK3-beta), and cyclin D1 controls cell growth, survival, and invasiveness. We have investigated this pathway in paired transformed/nontransformed cell lines from murine peripheral lung epithelium, E9/E10 and A5/C10. The E9 and A5 carcinoma lines expressed ErbB3 and transforming growth factor-alpha (TGF-alpha) and responded to TGF-alpha stimulation with protein complex formation including the p85 regulatory subunit of PI3K, activation of Akt, phosphorylation of GSK3-beta, and increased cyclin D1 protein and the cell cycle. ErbB3 and TGF-alpha were not detected in the nontransformed E10 and C10 cell lines. Nevertheless, exposure of E10 or C10 cells to TGF-alpha activated PI3K and Akt and increased cyclin D1 and cell growth. The effector pathway from the EGFR to PI3K in these nontransformed cells included the adaptor Grb2, the docking protein Gab1, and the phosphatase Shp2. Gab1 was highly expressed in E10 and C10 cells but not in the malignant E9 and A5 sister lines. Complexes of EGFR/Grb2/Gab1/Shp2 after TGF-alpha stimulation were prominent only in E10 and C10 cells. Thus, alternate pathways downstream of EGFR regulate mitosis in these paired malignant versus nontransformed lung cell lines.

Topics: Adaptor Proteins, Signal Transducing; Adenocarcinoma; Animals; Cell Line, Tumor; Cyclin D1; Enzyme Activation; Epithelial Cells; ErbB Receptors; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Intracellular Signaling Peptides and Proteins; Lung Neoplasms; Mice; Phosphatidylinositol 3-Kinases; Phosphoproteins; Protein Tyrosine Phosphatase, Non-Receptor Type 11; Protein Tyrosine Phosphatases; Proto-Oncogene Proteins c-akt; Receptor, ErbB-2; Receptor, ErbB-3; Transforming Growth Factor alpha

Serum levels of amphiregulin and transforming growth factor-alpha (TGF-alpha), which were identified previously to be expressed at high levels in non-small cell lung cancer (NSCLC) with poor response to gefitinib, were examined by ELISA using blood samples taken from 50 patients with advanced NSCLCs. Of 14 cases that revealed above the cutoff line for amphiregulin in serum, 12 responded poorly to gefitinib, whereas 18 of the 36 cases showing below the cutoff revealed partial response (PR) or stable disease (SD; P = 0.026). Thirteen of 15 patients who were positive for TGF-alpha responded poorly to gefitinib, whereas 18 of the 35 patients with negative TGF-alpha levels turned out to be relatively good responders (P = 0.014). Of 22 patients with positive values for either or both markers, 19 were poor responders. On the other hand, among 28 patients negative for both markers, 17 were classified into the PR or SD groups (P = 0.001). Gefitinib-treated NSCLC patients whose serum amphiregulin or TGF-alpha was positive showed a poorer tumor-specific survival (P = 0.037 and 0.002, respectively, by univariate analysis) compared with those whose serum amphiregulin or TGF-alpha concentrations were negative. Multivariate analysis showed an independent association between positivity for TGF-alpha and shorter survival times among NSCLC patients treated with gefitinib (P = 0.034). Amphiregulin or TGF-alpha positivity in NSCLC tissues was significantly higher in male, nonadenocarcinomas, and smokers. Our data suggest that the status of amphiregulin and TGF-alpha in serum can be an important predictor of the resistance to gefitinib among patients with advanced NSCLC.

Topics: Adult; Aged; Aged, 80 and over; Amphiregulin; Antineoplastic Agents; Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Drug Resistance, Neoplasm; EGF Family of Proteins; Enzyme-Linked Immunosorbent Assay; Female; Gefitinib; Glycoproteins; Humans; Intercellular Signaling Peptides and Proteins; Lung Neoplasms; Male; Middle Aged; Quinazolines; Transforming Growth Factor alpha

Despite maximal therapy, surgically treated patients with stage I non-small cell lung cancer (NSCLC) are at risk for developing metastatic disease. Histopathologic findings cannot adequately predict disease progression, so there is a need to identify molecular factors that serve this purpose. Because the ErbB receptors play an important role in lung cancer progression, we analyzed the expression of epidermal growth factor receptor (EGFR), phosphorylated EGFR, transforming growth factor alpha (TGFalpha), and HER2-neu as potential prognostic factors in stage I NSCLC.. Using immunohistochemical techniques, we retrospectively analyzed formalin-fixed, paraffin-embedded samples from 111 patients with resected pathological stage I NSCLC. Then we correlated these data with patient clinical outcome.. Median follow-up was 69.3 months. EGFR overexpression (defined as >10% membranous staining) was found in 66 tumors (59.5%). It was significantly more common in T(2) tumors than in T(1) tumors (P = 0.001), and in more squamous cell carcinomas than in adenocarcinomas (P = 0.07). HER2-neu overexpression was found in 19 tumors (17.1%) and was significantly more common in adenocarcinomas than in squamous cell carcinomas (P = 0.035). Synchronous overexpression of EGFR and HER2-neu was found in 11 tumors (9.9%). Patients with these tumors had a significantly shorter time to recurrence (P = 0.006) and a trend toward shorter overall survival (P = 0.093). Phosphorylated EGFR and transforming growth factor alpha were detected but were not related to prognosis.. Synchronous overexpression of EGFR and HER2-neu at the protein level predicts increased recurrence risk and may predict decreased survival in patients with stage I NSCLC. This suggests that important interactions take place among the different members of the ErbB family during tumor development and suggests a method for choosing targeted therapy. A prospective study is planned.

Topics: Adenocarcinoma; Adenocarcinoma, Bronchiolo-Alveolar; Adult; Aged; Aged, 80 and over; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Disease Progression; ErbB Receptors; Female; Follow-Up Studies; Humans; Immunoenzyme Techniques; Lung Neoplasms; Male; Middle Aged; Neoplasm Recurrence, Local; Neoplasm Staging; Phosphorylation; Prognosis; Receptor, ErbB-2; Retrospective Studies; Risk Factors; Survival Rate; Transforming Growth Factor alpha

We examined the effect of stable transfection of dominant negative TbetaR-II (dn TbetaR-II) cDNA in a human oral carcinoma cell line that contained normal Ras and was growth inhibited by TGF-beta1. Two clonal cell lines containing dn TbetaR-II were isolated and compared to the vector-only control and parent cell line. The treatment of cells with exogenous TGF-beta1 resulted in a decrease in ligand-induced growth inhibition and loss of c-myc downregulation in test cells compared to controls; transcriptional activation of certain genes including fra-1 and collagenase was retained. Cells containing dn TbetaR-II grew faster in monolayer culture, expressed less keratin 10 and exhibited increased motility and invasion in vitro compared to control cell lines. Endogenous TGF-beta1 production and the regulation of MMP-2 and MMP-9 by TGF-beta1 remained unchanged. After orthotopic transplantation to the floor of the mouth in athymic mice, cells containing dn TbetaR-II formed comparable numbers of primary tumours at the site of inoculation as controls but the tumours were less differentiated as demonstrated by the absence of keratin 10 immunostaining. Further, metastatic dissemination to the lungs and lymphatics was more evident in grafts of cells containing dn TbetaR-II than controls. Taken together, the results demonstrate that attenuation of TGF-beta signalling through transfection of dn TbetaR-II cDNA leads to an enhanced growth rate, a loss of tumour cell differentiation and an increase in migration and invasion, characteristics that corresponded to the development of the metastatic phenotype.

Topics: Cell Differentiation; Cell Division; Cell Line, Tumor; Cell Movement; Humans; Keratin-10; Keratinocytes; Keratins; Lung Neoplasms; Mouth Neoplasms; Neoplasm Metastasis; Phenotype; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor alpha

Cancer cells promote angiogenesis early in tumorigenesis because cancer cells require oxygen and nutrients for their survival they need to be supported by blood vessels. The angiogenic switch is driven by several proangiogenic factors, vascular endothelial growth factor (VEGF), interleukin-8 (IL-8), basic fibroblast growth factor (bFGF) and others. These proangiogenic factors are released from not only cancer cells but also various stromal cells by various stimulations, mutations or inflammation. From our experimental results using human lung cancer cell lines, survival factors in each cancer cell line are different. This makes cancer therapy difficult. Various angiogenesis inhibitors have been developed to block tumor angiogenesis. However we need to think how and when to use angiogenesis inhibitors together with conventional chemotherapeutic agents.

Topics: Angiogenesis Inhibitors; Animals; Antineoplastic Agents; Cell Line, Tumor; Cyclooxygenase 2; Drug Screening Assays, Antitumor; Gefitinib; Humans; Interleukin-1; Isoenzymes; Lung Neoplasms; Membrane Proteins; Mice; Prostaglandin-Endoperoxide Synthases; Quinazolines; Transforming Growth Factor alpha

Chronic obstructive pulmonary disease (COPD) is one of the leading causes of death in the U.S. Because cigarette smoking is so importantly implicated in the pathogenesis of COPD and because mucus hypersecretion plays such an important role in COPD, understanding of the mechanisms of smoking-induced mucus hypersecretion could lead to new therapies for COPD. Cigarette smoke causes mucin overproduction via EGF receptor (EGFR) in airway epithelial cells, but the cellular mechanism remains unknown. Airway epithelial cells contain EGFR proligands on their surfaces, which can be cleaved by metalloprotease and subsequently bind to EGFR resulting in mucin production. We hypothesize that TNF-alpha-converting enzyme (TACE) is activated by cigarette smoke, resulting in increased shedding of EGFR proligand, leading to EGFR phosphorylation and mucin induction in human airway epithelial (NCI-H292) cells. Here we show that cigarette smoke increases MUC5AC production in NCI-H292 cells, an effect that is prevented by an EGFR-neutralizing antibody and by specific knockdown of transforming growth factor-alpha (TGF-alpha) using small interfering RNA (siRNA) for TGF-alpha, implicating TGF-alpha-dependent EGFR activation in the responses. Cigarette smoke increases TGF-alpha shedding, EGFR phosphorylation, and mucin production, which are prevented by metalloprotease inhibitors (GM-6001 and TNF-alpha protease inhibitor-1) and by specific knockdown of TACE with TACE siRNA, implicating TACE in smoking-induced responses. Furthermore, pretreatment with antioxidants prevents smoking-induced TGF-alpha shedding and mucin production, suggesting that reactive oxygen species is involved in TACE activation. These results implicate TACE in smoking-induced mucin overproduction via the TACE-proligand-EGFR signal pathway in NCI-H292 cells.

Topics: ADAM Proteins; ADAM17 Protein; Carcinoma, Mucoepidermoid; Cell Line, Tumor; ErbB Receptors; Gene Expression; Humans; Ligands; Lung Neoplasms; Metalloendopeptidases; Mucin 5AC; Mucins; Phosphorylation; Protein Precursors; Pulmonary Disease, Chronic Obstructive; Reactive Oxygen Species; Respiratory Mucosa; Smoking; Transforming Growth Factor alpha; Tumor Necrosis Factor-alpha; Up-Regulation

This study was conducted to evaluate the clinical significance of the localization of epidermal growth factor receptor (EGF-r), transforming growth factor (TGF)-alpha, and erbB-2 in the development, progression and prognosis of squamous cell cancers (SCCs) of the lung.. The localization of EGF-r, TGF-alpha, and erbB-2 was evaluated immunohistochemically in 60 archival specimens of SCC of the lung and in 60 lung specimens without cancer. To clarify the patterns of expression of EGF-r in these tumors, the patterns of expression of EGF-r in cells in culture were monitored after challenging normal human bronchial epithelial and SCC cell lines with either EGF or TGF-alpha at physiological concentrations.. The expression of EGF-r, erbB-2, and TGF-alpha were significantly higher in SCC and associated precancerous lesions than in the normal bronchial epithelium and hyperplastic lesions of noncancer specimens. A statistically significant stepwise increase in expression from uninvolved bronchial epithelium to precancerous lesions to SCC was observed with EGF-r and TGF-alpha. The localization of EGF-r in the cytoplasm (P = 0.04), but not in the membrane (P = 0.20), of SCCs was significantly associated with poor overall survival of subjects. To demonstrate the biological relevance of cytoplasmic expression of EGF-r, we noted that there was a prompt reduction in the membrane expression and a concomitant increase in cytoplasmic expression of EGF-r after adding either EGF or TGF-alpha to the cell culture medium. Overall, the study identified an involvement of EGF-r and TGF-alpha in the development of SCCs. The prognostic importance of EGF-r expression in the cytoplasm of lung cancer probably is an indication of the prognostic importance of trafficking of the EGF-r receptor between the Golgi apparatus and cell membranes and of internalization of EGF-r after an interaction with one of the EGF-r ligands at the cellular membrane surface.

Topics: Carcinoma, Squamous Cell; Cell Membrane; Cytoplasm; Epithelial Cells; ErbB Receptors; Humans; Hyperplasia; Immunoenzyme Techniques; Ligands; Lung Neoplasms; Precancerous Conditions; Prognosis; Receptor, ErbB-2; Survival Rate; Transforming Growth Factor alpha; Tumor Cells, Cultured

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 interaction of epidermal growth factor receptor (EGFR) and its ligand transforming growth factor-alpha (TGF-alpha) leads to an autocrine activation of the ras signaling pathway and putatively its oncogenic activity. It is thus hypothesized that the co-overexpression of EGFR-TGFalpha will be redundant hence rare in tumors with oncogenic ras mutations. To test this hypothesis, we studied by immunohistochemistry the expression of EGFR and TGF-alpha in primary non small cell lung cancers. Such putative EGFR autocrine loop activation was found in 73% of squamous cell carcinomas that rarely develop ras mutations. In contrast, EGFR-TGFalpha co-expression occurred with equal frequency in adenocarcinomas irrespective of their ras genotype. The results indicate that EGFR autocrine loop activity in adenocarcinoma may have alternative signaling activities aside from the activation of ras-MAP kinase pathway.

Topics: Adenocarcinoma; Carcinoma, Large Cell; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; ErbB Receptors; Gene Expression Regulation, Neoplastic; Genes, ras; Humans; Immunohistochemistry; Lung Neoplasms; Mutation; Signal Transduction; Transforming Growth Factor alpha

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

Overexpression of the growth factor receptor ErbB-2/Her2/Neu has been implicated in the development of non-small-cell lung cancer. We have reported that the transformation of human lung epithelial cells by c-erbB-2 also requires an active ErbB-1 (EGF receptor) and the autocrine production of its ligand, TGF-alpha. In this report, we demonstrate that STAT 3 is constitutively activated in these cells by the TGF-alpha-stimulated ErbB-1/-2 heterodimer complex. STAT 3 activation was confirmed by mobility shift assays and nuclear localization. ErbB-1 was required, but not sufficient for the TGF-alpha-induced activation of STATs. Inhibition of ErbB-2 kinase activity by tyrphostin AG825 prevented the constitutive activation of STAT 3 in the TGF-alpha-producing, ErbB-1 expressing cell line. Our results demonstrate a requirement for ErbB-2 kinase activity to establish constitutive STAT 3 activation resulting from an autocrine ErbB-1/ TGF-alpha loop. Int. J. Cancer 83:564-570, 1999. Published 1999 Wiley-Liss, Inc.

Topics: Benzothiazoles; Cell Line; Cell Nucleus; Dimerization; DNA-Binding Proteins; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Enzyme Inhibitors; Epithelial Cells; ErbB Receptors; Humans; Immunoblotting; Lung Neoplasms; Milk Proteins; Precipitin Tests; Receptor, ErbB-2; STAT1 Transcription Factor; STAT3 Transcription Factor; STAT5 Transcription Factor; Trans-Activators; Transfection; Transforming Growth Factor alpha; Tyrphostins

We have determined the expression of transforming growth factor alpha (TGF alpha), amphiregulin (AR), CRIPTO, the epidermal growth factor receptor (EGFR), erbB-2, erbB-3, and tumor angiogenesis in a series of 195 patients with stage I-IIIA non-small cell lung cancer (NSCLC) treated with radical surgery to define their usefulness as prognostic indicators of survival. A variable degree of specific staining in cancer cells was observed for the three growth factors and for the three growth factor receptors in the majority of NSCLC patients. A statistically significant association between overexpression of TGF alpha, AR, and CRIPTO was observed. Enhanced expression of AR was significantly correlated with enhanced expression of erbB-2 and advanced T-stage. A direct association was also detected for overexpression of TGF alpha and of erbB-2 or erbB-3, respectively. Sex, tumor size, nodal status, stage, microvessel count, as a measure of neovascularization, and AR overexpression significantly correlated with overall survival at univariate analysis. In a Cox multivariate analysis, the only characteristics with an independent prognostic effect on OAS were microvessel count [relative hazard (RH), 6.61; P < 0.00001), nodal status (RH, 1.59; P = 0.0013), and AR overexpression (RH, 1.72; P = 0.02). These results suggest that evaluation of neoangiogenesis and of certain growth factors, such as AR, can be useful in addition to conventional pathological staging to select high-risk NSCLC patients who may benefit from post-surgical systemic therapies.

Topics: Adult; Aged; Aged, 80 and over; Amphiregulin; Carcinoma, Non-Small-Cell Lung; EGF Family of Proteins; Female; Glycoproteins; Growth Substances; Humans; Intercellular Signaling Peptides and Proteins; Lung Neoplasms; Male; Middle Aged; Neoplasm Staging; Neovascularization, Pathologic; Prognosis; Receptors, Growth Factor; Survival Rate; Transforming Growth Factor alpha

Over-expression of erbB-2 is associated with shortened survival of patients with lung adenocarcinomas. We demonstrated that human lung epithelial cells, overexpressing erbB-2, formed tumours in nude mice only when high levels of transforming growth factor (TGF)-alpha were produced (E6T cells). To define the role that TGF-alpha production played in induction of tumorigenicity, a non-tumorigenic TGF-alpha-negative clone of ErbB-2 overexpressing cells (E2 cells) was transfected with an expression vector for TGF-alpha (E2alpha cells). Transfected clones produced TGF-alpha at 11-25% of the level produced by the E6T cell line. Tumorigenic E6T cells transfected with a TGF-alpha antisense vector (E6TA cells) expressed only 6% of the TGF-alpha level of the parental cells. Clones of E6T, E6TA, E2 and E2alpha were inoculated into athymic nude mice to measure tumorigenic potential. E6T cells formed tumours with a 70% efficiency. E2, E6TA and E2alpha cells failed to form tumours. The levels of EGFR were similar in non-tumorigenic E2 and tumorigenic E6T cells but higher in E2alpha and E6TA cells, and ErbB-2 were greatly overexpressed in an E2alpha clone. In vitro, ErbB-2 co-immunoprecipitated with EGFR in lysates of unstimulated E6T and E2alpha TGF-alpha-producing cells, indicating that the lower TGF-alpha levels were sufficient to induce in vitro heterodimerization. These studies suggest that induction of the tumorigenic phenotype depends on achieving a threshold level of TGF-alpha sufficient to activate downstream signalling by ErbB-2 containing active heterodimers.

Topics: Animals; Cell Line, Transformed; Epithelial Cells; ErbB Receptors; Humans; Lung Neoplasms; Mice; Mice, Nude; Receptor, ErbB-2; Transfection; Transforming Growth Factor alpha

Surgical excision of liver tumors represents the only curative treatment for primary and metastatic liver malignancies. It has been suspected that hepatectomy may stimulate growth of microscopic tumors. To determine whether local or systemic factors after hepatectomy are responsible for enhancement of tumor growth, the effects of hepatectomy on the experimental growth of liver or pulmonary tumors were examined.. One hour after injection of 10(6) Morris hepatoma cells into either the portal or femoral vein, which produces isolated liver and lung tumors, respectively, animals were randomized to undergo 0%, 30%, or 70% partial hepatectomy (PH).. Animals that underwent portal injection of tumor had significantly increased liver tumor burden after PH (sham, 25 +/- 7 vs PH, 94 +/- 17; p < 0.01), whereas animals that underwent femoral injection had no change in lung tumor burden after PH. PH was associated with significantly increased levels of transforming growth factor-alpha, transforming growth factor-beta, and basic fibroblast growth factor in the liver but not in the lung.. Changes in liver cytokine-growth factor activation may contribute to enhanced tumor growth in the liver after hepatectomy.

Topics: Animals; Carcinoma, Hepatocellular; Fibroblast Growth Factor 2; Hepatectomy; Injections, Intravenous; Liver; Liver Neoplasms, Experimental; Lung Neoplasms; Male; Neoplasm Transplantation; Neoplasms, Experimental; Rats; Rats, Inbred BUF; Transforming Growth Factor alpha; Transforming Growth Factor beta

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

The epidermal growth factor receptor (EGFR) and its ligand transforming growth factor (TGF) alpha are hypothesized to form an autocrine growth loop in non-small cell lung cancer (NSCLC) and to play an important role in tumor formation and progression. We studied the association between overexpression of EGFR, TGF-alpha, or both, and overall survival of patients with resectable NSCLC. Overexpression, defined as >20% of tumor cells staining on immunohistochemistry, was examined in 96 tumor samples from consecutive patients having resection of previously untreated, well-staged NSCLC who were then followed prospectively (median follow-up, 20.7 months). The expression of three other ligands for EGFR (epidermal growth factor, cripto, and amphiregulin) was examined by Northern analysis to determine whether they might also contribute to a potential growth stimulatory loop. Overall, survival was calculated by the method of Kaplan and Meier, and prognostic factors were compared using the log-rank test. Overexpression of EGFR only was found in 32% (31 of 96), of TGF-alpha only in 10% (10 of 96), of both EGFR and TGF-alpha in 38% (37 of 96), and of neither in 19% (19 of 96) of tumors. EGFR and TGF-alpha overexpression was observed in all tumor stages and histological types but was most frequent in squamous cell carcinoma. By univariate and multivariate analyses, only tumor stage, not histology or overexpression of EGFR, TGF-alpha, or both, had a significant impact on overall survival. No expression of epidermal growth factor or cripto was observed at the total cellular RNA level of Northern analysis in tumor or benign lung, suggesting that in NSCLC these ligands may not participate in an autocrine growth stimulatory loop with EGFR. Differential overexpression of amphiregulin in malignant versus normal lung was observed, but this expression pattern did not have a prognostic impact. Thus, EGFR and TGF-alpha overexpression is frequent in early-stage NSCLC but is not associated with a survival difference. These findings suggest that this growth factor/receptor loop is more important for lung tumor formation than for tumor progression.

Topics: Adenocarcinoma; Carcinoma, Large Cell; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Disease Progression; ErbB Receptors; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Ligands; Lung Neoplasms; Neoplasm Staging; Predictive Value of Tests; Survival Rate; Time Factors; Transforming Growth Factor alpha

To study the difference in expression and response of certain growth factors between the two metastatic variants PGbE1 and PGLH7 of human pulmonary giant cell carcinoma, and the action of these growth factors on the metastasis of tumor cells.. RT-PCT was conducted to detect the expression of TGF alpha, TGF beta 1, IL-6, IL-8, bFGF and ANG, and the expression of receptors EGFR, IL-6R and IL-8R; 3H-TdR incorporation assay was used to determine the effects of recombinant TGF alpha, TGF beta 1 and IL-6 on the proliferation of the two cells.. TGF alpha, EGFR, IL-6 and IL-6R were expressed at a higher level in PGbE1 cells than in PGLH7 cells. No significant differences were found in the expression of TGF beta 1, bFGF, IL-8, IL-8R and ANG between the two cells. Recombinant TGF alpha and IL-6 stimulated the proliferation of both cells, while TGF beta 1 had dual effects.. TGF alpha, TGF beta 1, bFGF, IL-6, IL-8 and ANG may be involved in the autocrine regulation of the growth and proliferation of pulmonary giant cell carcinoma, TGF alpha and IL-6 may play an important role in the metastasis of the tumor cells.

Topics: Carcinoma, Giant Cell; Cell Division; ErbB Receptors; Humans; Interleukin-6; Lung Neoplasms; Receptors, Interleukin-6; Recombinant Proteins; Transforming Growth Factor alpha; Tumor Cells, Cultured

Transforming growth factor-alpha (TGF-alpha)-mediated autocrine regulation in human non-small-cell lung cancer (NSCLC) cells NCI-H226 and its brain metastatic variant H226Br were compared. An enhanced TGF-alpha-induced dose-dependent mitogenic responsiveness in H226Br cells was observed. Neutralising antibody that binds TGF-alpha inhibits H226Br cell growth more effectively than NCI-H226 cell growth. Binding assay with 125I-labelled epidermal growth factor (EGF) revealed that H226Br has two types of EGF receptors (EGFRs), whereas the parental cell line, NCI-H226, has only one. H226Br cells contain twice as many EGFRs as H226 cells, as proved by Scatchard analysis and immune kinase assay. Northern analysis indicated that there is more EGFR transcript in H226Br than in NCI-H226, indicating a transcriptional EGFR gene elevation during metastasis progression. The level of accumulated immunoactive TGF-alpha is lower in the conditioned medium of H226Br than in that of NCI-H226. demonstrating down-regulation of TGF-alpha transcript. The accumulated data suggest an elevated and sensitive autocrine modulation by TGF-alpha and EGFR in immortalising the brain metastatic variant cells that were derived from a human NSCLC squamous cell line.

Topics: Animals; Brain Neoplasms; Carcinoma, Non-Small-Cell Lung; Cell Division; ErbB Receptors; Humans; Lung Neoplasms; Mice; Mice, Nude; Phosphorylation; Polymerase Chain Reaction; RNA; Transforming Growth Factor alpha; Tumor Cells, Cultured

Primary non-small cell lung cancer samples were examined for the expression of p53, transforming growth factor alpha (TGF-alpha) and its receptor EGF-R by immunohistochemistry. Accumulation of p53 protein was found in 15 out of 24 carcinomas. Meanwhile, the results of TGF-alpha and p53 expression in 21 carcinomas showed that p53 protein was not detected in 2 out of 2 TGF-alpha negative and 4 out of 5 TGF-alpha low expression cases. However, there was no relationship found between EGF-R and p53 expression in these cases. The results suggest that p53 may have some effects on TGF-alpha expression.

Topics: Carcinoma, Non-Small-Cell Lung; ErbB Receptors; Gene Expression; Genes, p53; Humans; Immunohistochemistry; Lung Neoplasms; Transforming Growth Factor alpha; Tumor Suppressor Protein p53

Using ELISAs, we determined the concentrations of transforming growth factor alpha (TGF-alpha), the extracellular domain of the erbB-2 receptor (erbB-2 ECD), and mutant p53 protein in stored serum samples of asbestosis patients with and without cancer and control subjects (without asbestosis or cancer). The percentage of individuals in these three groups with increased serum concentrations of TGF-alpha, erbB-2 ECD, and mutant p53, respectively, were: asbestosis patients with cancer, 36%, 16%, 19%; asbestosis patients without cancer, 38%, 19%, 6%; control subjects, 0%, 5%, 10%. Although differences in serum positivity for these oncoproteins were apparent among these groups, the differences did not achieve statistical significance (P > 0.05). In several of the cancer cases, increased concentrations of TGF-alpha, erbB-2 ECD, and mutant p53 were also detected in the stored serum samples collected years before the clinical diagnosis of disease.

Topics: Asbestosis; Cohort Studies; Enzyme-Linked Immunosorbent Assay; Female; Humans; Lung Neoplasms; Male; Mesothelioma; Oncogene Proteins; Receptor, ErbB-2; Transforming Growth Factor alpha; Tumor Suppressor Protein p53

Inhibition of growth factor-stimulated DNA synthesis carried out in defined medium is often compared with inhibition of serum-stimulated DNA synthesis so as to assess the selectivity of growth-factor-receptor tyrosine kinase inhibitors such as tyrphostins. We investigated whether protein binding may influence the interpretation of these experiments. Protein binding of tyrphostins was determined by ultrafiltration, equilibrium dialysis or spectrophotometer, and was quantitated by high-performance liquid chromatography (HPLC). For growth factor-stimulated DNA synthesis, we used the non-small-cell lung cancer cell line L23/P stimulated by transforming growth factor alpha (TGF alpha). The epidermal growth factor (EGF)-receptor kinase was assayed by phosphorylation of a peptide substrate or by receptor autophosphorylation. Protein binding of a number of tyrphostins ranged from 64% to 98%. There was a positive correlation (r = 0.995) between the degree of protein binding and the hydrophobicity. Inhibition of the EGF-receptor tyrosine kinase activity by the highly protein-bound tyrphostin B56 [N-(4-phenylbutyl)-3,4-dihydroxybenzylidene cyanoacet-amide] was reduced by bovine serum albumin (BSA), but BSA had less of an effect on inhibition of the EGF-receptor kinase by the weakly protein-bound tyrphostin A47 (RG 50864: 3,4-dihydroxybenzylidene cyanothioacetamide). Tyrphostins B46 [N-(3-phenylpropyl)-3,4-dihydroxybenzylidene cyanoacetamide] and B56 (both highly protein-bound) inhibited DNA synthesis of L23/P cells with approximately 3-fold greater potency in 0.5% serum than in 10% serum, but the inhibition of DNA synthesis in 0.5% serum was reduced by the addition of BSA. Tyrphostins B46 and B56 inhibited DNA synthesis stimulated by TGF alpha in defined medium to a greater extent than DNA synthesis stimulated by serum. However, this apparent selectivity for inhibition of TGF alpha-stimulated DNA synthesis was lost when the protein concentration in the defined medium was made equivalent to that in the serum-containing medium. By contrast, BSA enhanced the selective inhibition of TGF alpha-stimulated DNA synthesis by tyrphostin A47. These results demonstrate that protein binding accounts for the apparent selectivity of some highly protein-bound tyrphostins for TGF alpha-stimulated DNA synthesis of L23/P cells. Therefore, protein binding should be taken into consideration in assessments of the selectivity of tyrphostins.

Topics: Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Catechols; Chromatography, High Pressure Liquid; DNA, Neoplasm; ErbB Receptors; Growth Inhibitors; Humans; Lung Neoplasms; Nitriles; Phosphorylation; Protein Binding; Protein-Tyrosine Kinases; Transforming Growth Factor alpha; Tumor Cells, Cultured; Tyrphostins

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

The aim of this study was to examine whether changes in growth factor or cytokine expression could be responsible for the growth inhibitory effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the human breast cancer MCF-7 cell line. Treatment of MCF-7 cells with 10 nM TCDD for 7 days reduced the cell growth to 60% of control; this effect was partly abolished by cotreatment of the cells with 100 nM 17 beta-estradiol (E2). The inhibition of cell growth by TCDD was accompanied by an enhanced secretion of transforming growth factor-beta (TGF-beta) and the TGF-beta content in cell culture supernatants was 2-fold higher than in controls. Using reverse transcription polymerase chain reaction (RT-PCR), the effect of TCDD on the expression of TGF-beta isoforms, transforming growth factor-alpha (TGF-alpha), tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) was investigated. It was demonstrated that incubation with 1, 10 and 100 nM TCDD for 24 h increased mRNA levels of TGF-alpha, TNF-alpha and IL-1 beta. The strongest effect was found on IL-1 beta, the mRNA level of which was dose-dependently increased. TCDD had a minor effect on TGF-alpha and TNF-alpha mRNA. The mRNA levels were significantly increased after treatment with 10 and 100 nM TCDD. The mRNA expression of TGF-beta 1 and TGF-beta 2 was unchanged, whereas the TGF-beta 3 mRNA level was enhanced 2 to 3-fold after TCDD treatment. From the results, we suggest that TCDD-induced growth inhibition in MCF-7 cells is related to the growth inhibitory action of a set of growth factors and cytokines which have a contextual action on MCF-7 cell proliferation.

Topics: Analysis of Variance; Breast Neoplasms; Cytokines; Growth Substances; Humans; Interleukin-1; Lung Neoplasms; Polychlorinated Dibenzodioxins; Polymerase Chain Reaction; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

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

New strategies are needed for the detection and treatment of lung cancer and must derive from a fuller understanding of lung carcinogenesis. Frequent molecular genetic abnormalities occur in non-small cell lung cancer (NSCLC), but little is known about which of these precede an invasive carcinoma. We examined the expression of p53, epidermal growth factor receptor (EGFR), and transforming growth factor alpha, the most common molecular genetic abnormalities in NSCLC, in preneoplastic bronchial lesions. Primary NSCLC and associated bronchial lesions were identified by retrospective review of resected tumors at this center. Expression in the invasive carcinomas, the associated bronchial lesions, and normal lung were contrasted using immunohistochemistry. Thirty-four NSCLC associated with 62 bronchial lesions were identified. The invasive tumors included 15 squamous cell carcinomas (SCCs) and 19 non-SCCs. Bronchial lesions included areas of squamous metaplasia (n = 14), inflammatory atypia (n = 19), dysplasia (n = 17), and carcinoma in situ (n = 12). Nineteen (56%) NSCLC and 10 (16%) bronchial lesions exhibited aberrant p53 immunostaining, whereas 18 (53%) NSCLC and 30 (48%) bronchial lesions showed abnormal EGFR immunostaining. Positive staining for transforming growth factor alpha was seen in 16 (47%) NSCLC but occurred inconsistently in the bronchial lesions and in normal bronchial epithelium. Only bronchial lesions associated with squamous cell carcinomas exhibited staining for p53. Aberrant EGFR expression was not associated with a specific type of invasive carcinoma or with specific preneoplastic lesions, although there was a trend toward increased expression in dysplasia and carcinoma in situ relative to metaplasia and atypia. All but one of the NSCLC simultaneously showing aberrant p53 and EGFR staining were SCC. We conclude that: (a) transforming growth factor alpha is variably expressed in normal respiratory epithelium as well as reactive and preneoplastic bronchial lesions; (b) p53 expression is seen in preneoplastic bronchial lesions but is not present in reactive or metaplastic epithelium; (c) aberrant EGFR expression occurs in both reactive and preinvasive bronchial lesions and may be an early marker of neoplastic transformation; and (d) the simultaneous aberrant expression of EGFR and p53 occurs predominantly in SCC and their associated bronchial lesions. These findings indicate that aberrant expression of p53 or the EGFR is frequent in bronchi

Topics: Bronchial Neoplasms; Carcinoma in Situ; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; ErbB Receptors; Humans; Immunohistochemistry; Lung Neoplasms; Transforming Growth Factor alpha; Tumor Suppressor Protein p53

To determine the role of a specific member of the metalloproteinase family, stromelysin-1, in mammary carcinogenesis and tumor progression, transgenic mice expressing activated rat stromelysin-1 under the control of the mouse mammary tumor virus promoter/enhancer were treated with the carcinogen 7,12-dimethylbenzanthracene (DMBA) to induce mammary tumors. Surprisingly, the expression of stromelysin-1 during the time of DMBA treatment reduced the number of mice developing mammary tumors, in particular adenoacanthomas, from 65 to 32% (P = 0.02). In contrast, when transgenic mice expressing both transforming growth factor alpha and stromelysin-1 under the control of the mouse mammary tumor virus long terminal repeat were treated with DMBA, there was no significant difference in the number of mice that developed tumors compared to transforming growth factor alpha controls. A 4-fold increase in the number of apoptotic cells was detected in stromelysin-1 transgenic mice compared to littermate controls at the time of DMBA administration, suggesting that the reduction in DMBA-induced tumorigenicity is likely to be due, at least in part, to an increased rate of cell turnover in stromelysin-1 transgenic mice. When malignant adenocarcinomas developed in the stromelysin-expressing mice, there was no detectable alteration in the extent of invasion or in the metastatic potential of these tumors compared to tumors from control mice. These results suggest that the expression of a single metalloproteinase, stromelysin-1, is insufficient for the progression of mammary adenocarcinomas to an invasive and metastatic phenotype, but that matrix degradation by metalloproteinases can alter basic processes of cell proliferation and apoptosis.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Adenocarcinoma; Adenoma; Animals; Animals, Suckling; Apoptosis; Cell Division; Female; Lung Neoplasms; Lymphoma; Mammary Glands, Animal; Mammary Neoplasms, Experimental; Matrix Metalloproteinase 3; Metalloendopeptidases; Metaplasia; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Mice, Transgenic; Neoplasm Invasiveness; Neoplasm Proteins; Time Factors; Transforming Growth Factor alpha

Ninety-two rat lung proliferative lesions and neoplasms induced by inhaled 239PuO2 were evaluated for aberrant expression of transforming growth factor alpha (TGF-alpha) and epidermal growth factor receptor (EGFR). Expression of TGF-alpha protein, measured by immunohistochemistry, was higher in 94% of the squamous cell carcinomas and 87% of the foci of alveolar epithelial squamous metaplasia than that exhibited by the normal-appearing, adjacent lung parenchyma. In contrast, only 20% of adenocarcinomas and foci of epithelial hyperplasia expressed elevated levels of TGF-alpha. Many neoplasms expressing TGF-alpha also expressed excessive levels of EGFR mRNA. Southern and DNA slot blot analyses showed that the elevated EGFR expression was not due to amplification of the EGFR gene. These data suggest that increased amounts of TGF-alpha were early alterations in the progression of plutonium-induced squamous cell carcinoma, and these increases may occur in parallel with overexpression of the receptor for this growth factor. Together, these alterations create a potential autocrine loop for sustaining clonal expansion of cells initiated by high-LET radiation.

Topics: Animals; Carcinoma, Squamous Cell; ErbB Receptors; Female; Immunohistochemistry; Lung Neoplasms; Neoplasms, Radiation-Induced; Plutonium; Rats; Rats, Inbred F344; Transforming Growth 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

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

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

The epidermal growth factor receptor (EGFR) and one of its ligands, transforming growth factor alpha (TGF-alpha), are thought to function as a potential autocrine loop in non-small cell lung cancer (NSCLC). However, the expression pattern of EGFR and the TGF-alpha-related ligands have not been fully characterized in primary NSCLC and adjacent benign lung tissue. For this reason, we comprehensively examined the coexpression and differential expression of EGFR and its ligands, TGF-alpha, epidermal growth factor (EGF), and amphiregulin (AR), by Northern analysis, in paired samples of primary tumors and uninvolved lung. For those RNA species overexpressed in malignant lung, single cell expression patterns were studied by immunohistochemistry. Specimens were obtained from 57 consecutive patients who underwent resection of carefully staged resectable NSCLC and were followed prospectively. Most (112 of 114) tissue samples yielded high-quality RNA. EGFR was expressed in 82 of 88 (93%) tissue samples, while TGF-alpha was expressed in 62 of 72 (86%) samples, and AR was expressed in 64 of 70 (92%) samples. EGF was unexpressed in total cellular RNA in both tumor and uninvolved lung. In a comparison of RNA expression patterns in tumors and uninvolved lung, overexpression of EGFR was found in 45% (22 of 44) of tumors, while overexpression of TGF-alpha was seen in 61% (22 of 36) of tumors, and decreased expression of AR was seen in 63% (22 of 35) of tumors. Cell type and stage did not influence differential expression, indicating that this is a frequent event in primary NSCLC. Simultaneous overexpression of EGFR and TGF-alpha was seen in only 38% of tumors. Simultaneous overexpression of EGFR and decreased expression of AR were seen in only 21% of tumors. Thus far, the differential expression of EGFR, TGF-alpha, and AR does not correlate with either disease-free or overall survival. These findings indicate that histologically dissimilar tumors can express similar components of autocrine or paracrine growth factor loops. Differential expression of EGFR and its ligands in tumor specimens compared to uninvolved lung is a common event in NSCLC and may participate in tumor growth without necessarily influencing tumor progression or histology.

Topics: Adenocarcinoma; Adult; Aged; Amphiregulin; Blotting, Northern; Carcinoma, Non-Small-Cell Lung; EGF Family of Proteins; ErbB Receptors; Female; Follow-Up Studies; Gene Expression; Glycoproteins; Growth Substances; Humans; Immunohistochemistry; Intercellular Signaling Peptides and Proteins; Ligands; Lung; Lung Neoplasms; Male; Middle Aged; Neoplasm Staging; Prospective Studies; RNA; Transforming Growth Factor alpha; Transforming Growth Factors

The mRNA expression of transforming growth factor-alpha (TGF-alpha), epidermal growth factor receptor (EGFR), c-erbB-2 and c-met proto-oncogenes in eight newly established cell lines and 29 primary tumors of human non-small-cell lung carcinoma (NSCLC) have been investigated. In vitro, the expressions of TGF-alpha, c-erbB-2, and c-met were consistently high in adenocarcinomas, while EGFR was expressed highest in a squamous cell carcinoma cell line. There was linear correlation between the levels of expression of TGF-alpha and EGFR or c-erbB-2, and between EGFR and c-erbB-2. The c-met expression was also correlated with those of TGF-alpha, EGFR, and c-erbB-2. In vivo, The mean mRNA levels of TGF-alpha, EGFR, and c-met, but not c-erbB-2, were higher in carcinomas than in normal lung tissues (2.8, 1.7, and 3.0 times, respectively); however, only adenocarcinomas expressed a significantly higher level of c-erbB-2 than their corresponding normal tissues (2.2 times). In 20 patients whose paired normal and tumor tissue were examined, the percentage of cases with greater than twofold increase in expression in carcinomas than normal were 55% for both TGF-alpha and EGFR, 30% for c-erbB-2, and 47% for c-met. Among the histological subtypes of NSCLC, a higher percentage of adenocarcinomas than squamous cell carcinomas over-expressed these genes, especially c-erbB-2 and c-met. Over-expression is rarely the result of gene amplification. The results suggest a differential expression of growth factor and receptor genes among the various histological subtypes of NSCLCs.

Topics: Blotting, Northern; Blotting, Southern; Carcinoma, Non-Small-Cell Lung; ErbB Receptors; Humans; Lung Neoplasms; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-met; Receptor, ErbB-2; Receptors, Cell Surface; Transforming Growth Factor alpha; 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

Humoral hypercalcemia of malignancy is a multifactorial syndrome caused by the action of tumor-produced factors on target organs of bone, kidney, and intestine to disrupt normal calcium homeostasis. Although parathyroid hormone-related protein (PTHrP) plays an integral role in the syndrome, tumors also produce other hypercalcemic factors, such as transforming growth factor-alpha (TGF-alpha), which may modulate the effects of PTHrP. In order to determine if the effects of PTHrP on calcium homeostasis can be modulated by TGF-alpha, we have used a human squamous cell carcinoma cell line (RWGT2) which produces PTHrP alone and Chinese hamster ovarian (CHO) cells expressing only transfected human TGF-alpha complementary DNA (CHO/TGF-alpha). We studied the effects of these tumors on calcium homeostasis in nude mice bearing both tumors or each tumor alone. Whole blood ionized calcium concentrations (mean +/- SEM in mmol/L) were significantly higher in mice bearing both RWGT2 and CHO/TGF-alpha tumors (3.11 +/- 0.06, P < 0.05) when compared with mice bearing either RWGT2 alone (2.02 +/- 0.06), CHO/TGF-alpha alone (1.42 +/- 0.01), or RWGT2 and nontransfected CHO tumors (1.86 +/- 0.01). This enhanced effect was also observed using continuous PTHrP-(1-34) infusion (2 micrograms/day) in mice bearing CHO/TGF-alpha tumors. In addition, tumor cell conditioned media was tested for bone resorbing activity in organ cultures of fetal rat long bones previously incorporated with 45calcium (45Ca++). Conditioned medium at 0.1% (vol/vol) from either RWGT2 or CHO/TGF-alpha had no bone resorbing activity over control (%45Ca++ release, mean +/- SEM; control 23 +/- 1, RWGT2 19 +/- 1, CHO/TGF-alpha 23 +/- 1). However, the combination of 0.1% conditioned medium from RWGT2 and CHO/TGF-alpha significantly increased bone resorption (53 +/- 2, P < 0.05). These data demonstrate that the hypercalcemic effects of tumor-produced PTHrP are enhanced by TGF-alpha and that this effect may be due to increased bone resorption.

Topics: Animals; Bone and Bones; Bone Resorption; Carcinoma, Squamous Cell; CHO Cells; Cricetinae; Culture Media, Conditioned; Humans; Hypercalcemia; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Middle Aged; Neoplasms; Parathyroid Hormone-Related Protein; Protein Biosynthesis; Proteins; Rats; Recombinant Proteins; Transfection; Transforming Growth Factor alpha; Tumor Cells, Cultured

Parathyroid hormone-related peptide (PTHrP) and transforming growth factor-alpha (TGF-alpha) were found to stimulate proliferation of human lung cancer cells (BEN-57). TGF-alpha stimulated PTHrP secretion from these cells. The polyclonal antisera raised against PTHrP significantly inhibited the growth of BEN-57 cells, and also the proliferation induced by TGF-alpha. Treatment of cells for up to 10 days with either a PTHrP receptor antagonist (PTHrP(7-34)) or PTHrP antiserum significantly inhibited the subsequent growth of these cells. We suggest that PTHrP may be a component of a complex autocrine loop involving TGF-alpha.

Topics: Cell Division; Humans; Lung Neoplasms; Neoplasm Proteins; Parathyroid Hormone-Related Protein; Proteins; Transforming Growth Factor alpha; Tumor Cells, Cultured

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

Using a xenotransplantation system in which immortalized nontumorigenic human bronchial epithelial cells (BEAS-2B cells) are grown in deepithelialized rat tracheas that are subcutaneously transplanted into athymic nude mice, we exposed BEAS-2B cells either to cigarette smoke condensate or to the tobacco-specific N-nitrosamine 4-(methylnitrosamine)-1-(3-pyridyl)-1- butanone. After 6 mo the carcinogen-exposed BEAS-2B cells were neoplastically transformed to invasive adenocarcinomas. Cell lines obtained from xenografts exposed in vivo to chemicals exhibited several features typical of malignant lung cancer cells, such as increased in vivo invasiveness that correlated well with enhanced type IV collagenolytic activity, resistance to serum-induced growth inhibition, and increased expression of transforming growth factor alpha and its cellular-membrane receptor. Invasiveness, similar to that seen after exposure to phorbol esters, was also detected after in vitro exposure of BEAS-2B cells to cigarette smoke condensate. Collectively, these data indicate that cigarette smoke condensate and N-nitrosamine 4-(methylnitrosamine)-1-(3-pyridyl)-1-butanone induce in vivo phenotypic changes in BEAS-2B cells similar to the progressive changes that occur during human lung carcinogenesis.

Topics: Animals; Biomarkers, Tumor; Bronchi; Carcinogens; Cell Differentiation; Cell Line; Cell Transformation, Neoplastic; Chemotaxis; Epithelium; ErbB Receptors; Gelatinases; Humans; Isoenzymes; Lung Neoplasms; Mice; Mice, Nude; Neoplasm Invasiveness; Nitrosamines; Pepsin A; Rats; Smoke; Smoking; Tetradecanoylphorbol Acetate; Trachea; Transforming Growth Factor alpha; Transplantation, Heterologous

The expression of transforming growth factor-alpha (TGF alpha) was assessed by immunohistochemical staining in 52 human lung tumor samples. All of the 8 small cell lung cancers were negative whereas all of the 18 adenocarcinomas and 23 of the 26 squamous cell carcinomas showed positive immunoreaction to TGF alpha. Distribution of TGF alpha stainings in the squamous cell carcinomas was weaker and more heterogeneous as compared to the adenocarcinomas. Ultrastructural localization of TGF alpha in the squamous cell lung carcinomas by indirect immunogold staining revealed that TGF alpha is present in the cytoplasm as well as the cell membrane but not in the nucleus. This suggests that the lung cancer cells are not only the producer of TGF alpha, but also the target cells of the TGF alpha action. The expression of TGF alpha in lung tumors may be useful diagnostically in differentiating small cell lung cancer from non-small cell lung cancer and may also be important in the study of the biological properties of primary lung cancers.

Topics: Adenocarcinoma; Adult; Aged; Carcinoma, Small Cell; Carcinoma, Squamous Cell; Female; Humans; Immunohistochemistry; Lung Neoplasms; Male; Middle Aged; Transforming Growth Factor alpha

We studied the biological response to and production of transforming growth factor-alpha (TGF-alpha) by the non-small cell lung carcinoma (NSCLC) clonal cell lines H226b, H322a, H460a, H596b. Each of these cell lines expressed epidermal growth factor receptor (EGFR) as determined by [125I]EGF competitive binding and Scatchard analysis and by phosphorylation. The receptors were functionally active as determined in immune complex kinase assays. H322a, H226b, H460a, and H596b cells showed stimulated [3H]thymidine (Thd) uptake in response to TGF-alpha. Exogenously added TGF-alpha increased colony formation in soft agar for three of the cell lines in media containing serum. All cell lines expressed TGF-alpha detected by immunohistochemistry and TGF-alpha mRNA, although to differing degrees. Cell lysates and spent media competed for EGFR binding with EGF, thus demonstrating production of TGF-alpha-like activity. The anti-TGF-alpha monoclonal antibody AB-3 inhibited the uptake of [3H]Thd by proliferating H322a and H226b cells but not H460a and H596b cells. No inhibition occurred with MOPC21 antibody and inhibition was completely reversed by addition of TGF-alpha to the culture. Suramin inhibited cell proliferation and [3H]Thd uptake by all cell lines. Inhibition of H460a and H596b cells was reversed with exogenous TGF-alpha but not PDGF. Our data suggests that TGF-alpha is a mediator of autocrine growth stimulation for NSCLC cells, and that for some NSCLC cells cytoplasmic binding of receptor and ligand is the primary mechanism for autocrine growth stimulation.

Topics: Animals; Carcinoma, Non-Small-Cell Lung; Dose-Response Relationship, Drug; ErbB Receptors; Humans; Lung Neoplasms; Mice; Mice, Nude; Neoplasm Transplantation; Radioligand Assay; Recombinant Proteins; RNA, Neoplasm; Suramin; Transforming Growth Factor alpha; Tumor Cells, Cultured; Tumor Stem Cell Assay

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

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

In vitro and in vivo metastatic variants derived from Lewis lung carcinoma (3LL) were examined for the level of the expression of several growth-regulated genes, oncogenes, and transforming growth factor (TGF) genes. To determine whether the proliferative advantage of metastatic cells is due to an increased growth fraction of the cell population or to a deregulated expression of some growth-regulated genes, the mRNA levels of the S-phase-specific H3 histone gene were compared with that of some cell cycle-related genes (vimentin, calcyclin, c-myc, and p53) and oncogenes (Ki-ras, Ha-ras, c-sis, c-src, c-fes, and c-erb). In addition, to evaluate whether an autocrine pattern of cell proliferation is responsible for the proliferative advantage of metastatic cells, the level of the expression of TGF genes (alpha and beta 1) was studied. Northern blot analysis demonstrated that in 3LL metastatic variants the expression of TGF-alpha as well as the expression of all growth-regulated genes and oncogenes studied are similar. Only the TGF-beta 1 gene is expressed at higher levels in highly metastatic 3LL variants maintained either in vitro or in vivo. Data suggest that the proliferative advantage of 3LL metastatic cells is not due to a deregulated expression of some growth-regulated genes and oncogenes, but more likely is acquired through the expression of genes which might interfere with the ability of the tumor cells to escape hostile microenvironmental conditions.

Topics: Animals; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Mice; Mice, Inbred C57BL; Proto-Oncogenes; RNA, Messenger; RNA, Neoplasm; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured

We have previously shown that 47% of radiation-induced lung neoplasms in dogs exhibit increased expression of epidermal growth factor receptor (EGFR). In this study, we investigated the expression of transforming growth factor alpha (TGF-alpha), a ligand for EGFR, to determine if an autocrine mechanism for growth stimulation was present in these tumors. As determined by immunohistochemistry, 59% (26/44) of the lung neoplasms examined had increased expression of TGF-alpha. Expression of TGF-alpha was not related to the etiology of the tumor, e.g., spontaneous or plutonium-induced; however, it was related to the phenotype of the tumor. Statistical analysis of the correlation of EGFR and TGF-alpha expression within the same tumor did not show a positive association; however, specific phenotypes did have statistically significant expression of EGFR or TGF-alpha, suggesting that overexpression of either the ligand or its receptor conferred a growth advantage to the neoplasm. Twenty-seven percent (32/117) of radiation-induced proliferative epithelial foci expressed TGF-alpha, and a portion of those foci (8/32) expressed both EGFR and TGF-alpha. This supports the hypothesis that these foci represent preneoplastic lesions, and suggests that those foci exhibiting increased expression of the growth factor or its receptor are at greater risk for progressing to neoplasia.

Topics: Administration, Inhalation; Animals; Cell Division; Dogs; ErbB Receptors; Immunoenzyme Techniques; Lung Neoplasms; Neoplasms, Radiation-Induced; Plutonium; Transforming Growth Factor alpha

The expression of TGF-alpha in human colon and lung carcinoma cell lines has been reported previously, but its expression in primary tumours has not been described in detail. We have used the radio-immunoassay method to measure the specific content of immunoreactive TGF-alpha in the acid ethanol extracts of normal and cancerous tissues of human colon and lung. The average TGF-alpha content of colon carcinomas is 4 times that of the normal mucosa, and for non-small cell lung carcinomas it is twice that of the normal parenchyma. Because of variability in the TGF-alpha expression among individuals and in different segments of colon and lobes of lung, the ratio of TGF-alpha content of paired tumour and normal tissue was also calculated. On average, the tumour/normal ratio for colon carcinoma is higher than that for lung carcinoma. Although 55% of colon tumours show a ratio 4 times, or greater, only 33% of lung carcinomas demonstrate this ratio. The level of TGF-alpha in both colon and lung carcinomas does not correlate with histological type stage, grade nor degree of desmoplasia of these tumours. Northern blot analysis of total cellular RNA confirms the expression of an approximately 4.8 kb TGF-alpha mRNA in normal colonic mucosa and lung parenchyma. However, in contrast to the results of radio-immunoassay, significant over-expression of TGF-alpha mRNA is uncommon in primary human colon carcinomas.

Topics: Blotting, Northern; Colonic Neoplasms; Gene Expression; Humans; Intestinal Mucosa; Lung; Lung Neoplasms; Nucleic Acid Hybridization; Radioimmunoassay; RNA; RNA, Messenger; Transforming Growth Factor alpha

Multiple transforming growth factors (TGFs) capable of conferring the neoplastic phenotype on NRK-49F cells without the addition of any other exogenous growth factor in the soft agar assay, were purified from two human solid malignant neoplasms: a squamous lung carcinoma and a pectoral rhabdomyosarcoma. In both tumors, low-molecular-weight transforming activities (4000-6000) that were not potentiated by epidermal growth factor (EGF), competed for binding to the EGF receptor, possessed mitogenic activity on NRK fibroblasts arrested in serum-deprived medium, and did not show inhibitory effects on DNA synthesis induced by EGF and insulin in NRK cells. Other TGFs with molecular weights 9000 to 48,000, were also found in the malignant tissues examined; these TGFs, were not potentiated by EGF, did not compete for binding to the EGF receptor, were not mitogenic for NRK cells, and acted as potent inhibitors of DNA synthesis induced by EGF and insulin in NRK cells. These results demonstrate that growth-promoting activities, and modulating agents that can act as either enhancers or inhibitors of cell proliferation, are present in neoplastic tissues of different embryologic origin and histologic type.

Topics: Carcinoma, Squamous Cell; Cell Transformation, Neoplastic; Cells, Cultured; Chromatography, Gel; DNA; Dose-Response Relationship, Drug; ErbB Receptors; Fibroblasts; Humans; In Vitro Techniques; Lung Neoplasms; Rhabdomyosarcoma; Thoracic Neoplasms; Transforming Growth Factor alpha

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

12 cases of human lung cancer tissue were analyzed for the presence of transforming growth factor-alpha with RIA. Histologically 9 were squamous cell carcinoma and 3 were adenocarcinoma. Except one squamous carcinoma, TGF-alpha could be detected in 11 cases. TGF-alpha level was from 0.26 ng to 1.26 ng per gram tissue. There was no significant difference in TGF-alpha level between squamous cell carcinoma and adenocarcinoma. The relationship between TGF-alpha and human lung cancer remains for further study.

Topics: Adenocarcinoma; Carcinoma, Squamous Cell; Female; Humans; Lung Neoplasms; Male; Middle Aged; Radioimmunoassay; Transforming Growth Factor alpha