transforming-growth-factor-alpha and Carcinoma--Squamous-Cell

Traditionally, squamous cell cancers of the head and neck (SCCHN) have been classified by their anatomic location and stage. This system has been unsatisfactory in that it leaves substantial heterogeneity in prognosis and inadequate definition of optimal therapy. The most promising novel marker for superior prognosis in SCCHN is human papillomavirus (HPV). Overexpression of the EGFR bears an adverse prognosis; no marker provides clear predictive power for benefit from EGFR inhibition. Low expression of the DNA repair enzymes and excision repair cross-complementing rodent repair deficiency (ERCC1) and x-ray repair complementing defective repair in Chinese hamster cells 1 (XRCC1) may predict chemotherapy and chemoradiotherapy sensitivity. Tumors expressing cyclin D1 have a poor prognosis. Genomic characterization has subdivided SCCHN into four categories with clear biologic themes. Basal cancers express high levels of TGF-α and have other perturbations of the EGFR axis. Mesenchymal cancers show evidence for epithelial to mesenchymal transition. Atypical cancers lack both EGFR amplification and deletion of 9p; they also have a higher rate of HPV positivity than the other groups. Classical tumors demonstrate gene signatures similar to those previously associated with exposure to cigarette smoke; patients with this signature had a greater smoking history than patients in the other groups.

Topics: Animals; Carcinoma, Squamous Cell; Cricetinae; Cricetulus; Epithelial-Mesenchymal Transition; ErbB Receptors; Gene Expression Regulation, Neoplastic; Head and Neck Neoplasms; Humans; Prognosis; Smoking; Squamous Cell Carcinoma of Head and Neck; Transforming Growth Factor alpha

The activation of the epidermal growth factor receptor (REGF) participates in oncogenesis by inducing cell proliferation, cell mobility and angiogenesis, and inhibiting apoptosis. This activation might be due to numerous abnormalities, including increased expression of its ligand. Although based on retrospective analyses with no standard technique of evaluation, the level of REGF expression is a prognosis factor for several tumors. It appears to be an indicator of poor prognosis which might influence treatments in head and neck tumors, cancers of the cervix, oesophagus, bladder and ovary. Its prognostic value is not observed in non-small cell lung cancer and remains to be demonstrated in adenocarcinomas such as colorectal tumors and beast cancer. Because of its role in oncogenesis and its prognostic value, REGF might in the future become a therapeutic target.

Topics: Carcinoma, Squamous Cell; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Male; Neoplasm Proteins; Neoplasms; Organ Specificity; Prognosis; Receptor, ErbB-2; Transforming Growth Factor alpha

An understanding of the molecular basis of oral carcinogenesis will alter our clinical approach to oral cancer. The nomenclature and major themes of molecular oral tumor biology are reviewed, beginning with the regulation events governing normal cellular physiology. In carcinogenesis, chromosomal or cytogenetic alterations lead to deregulation of tightly controlled stimulatory and inhibitory pathways, growth-promoting proto-oncogenes are mutated into overactive oncogenes, and growth-suppressing or tumor suppressor genes are inactivated. Recent advances in defining these fundamental mechanisms of tumor biology may allow prevention, diagnosis, and treatment of oral cancer to be approached at the molecular level.

Topics: Anticarcinogenic Agents; Carcinoma, Squamous Cell; Disease Progression; DNA, Neoplasm; ErbB Receptors; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Genetic Therapy; Humans; Mouth Neoplasms; Neoplasm Staging; Point Mutation; Proteins; Proto-Oncogenes; Signal Transduction; Transforming Growth Factor alpha; Tumor Suppressor Proteins

The mouse skin model of multistage carcinogenesis continues to serve as a major in vivo model for studying the sequential and stepwise evolution of the cancer process by chemical and physical carcinogens. The initiation stage of mouse skin carcinogenesis involves genetic damage in the form of DNA adducts or initiator-induced DNA base changes. These changes ultimately lead to mutations in critical target genes of epidermal stem cells. The rasHa gene, and to a limited extent the N-ras gene, have been identified as target genes for certain tumor initiators in this model system (reviewed in DiGiovanni 1992). The promotion stage of mouse skin carcinogenesis involves the production and maintenance of a chronic state of hyperplasia and cell proliferation and ultimately the selective clonal expansion of initiated cells. The hallmark of all tumor promoters that have been adequately tested is their ability to induce a potentiated hyperplasia after several treatments that is greater than that observed after a single application. Tumor promoters produce many effects when applied topically to mouse skin. Many of the effects that occur after a single application of phorbol esters such as TPA appear to be mediated by its interaction with PKC (Nishizuka 1989). An important question is whether the activation of PKC per se is responsible for tumor promotion by TPA. Because repetitive treatments with TPA lead to a sustained loss of PKC, it is possible that other effects not mediated by PKC but produced by phorbol esters and related compounds may play an important role in the production and maintenance of chronic hyperplasia and cell proliferation in the skin and for skin tumor promotion. More attention should be placed on studying the promoting actions of other compounds outside of the most commonly studied phorbol esters. Investigations of some of these compounds already have and will continue to provide important clues regarding possible common pathways shared by diverse promoting agents. One such pathway may involve the EGFr and its ligand TGF alpha. As discussed in this review, it is now evident that many different types of promoting agents increase production of TGF alpha (Ellem et al. 1988, Pittelkow et al. 1989, Choi et al. 1991, Imamoto et al. 1991, J. DiGiovanni unpublished studies). Although many tumor promoters initially decrease the binding of 125I-EGF to EGFr in specific cell types, including mouse epidermal cells, the long-term effects of tumor promoters, espe

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Carcinogens; Carcinoma, Squamous Cell; Cell Transformation, Neoplastic; Cocarcinogenesis; Disease Progression; ErbB Receptors; Mice; Models, Biological; Papilloma; Phorbol Esters; Precancerous Conditions; Skin Neoplasms; Transforming Growth Factor alpha

The upper aerodigestive tract mucosa of patients who develop squamous cell carcinoma of the head and neck (SCCHN) is predisposed to abnormally regulated growth, evidenced by the high incidence of synchronous and metachronous malignancies. We conducted a series of experiments to show that aberrant regulation of the epithelial growth factor, transforming growth factor alpha (TGF-alpha) and its cell surface receptor, the epidermal growth factor receptor (EGFR), contribute to this predisposition. Using molecular biological techniques, we compared the incidence and mechanism of TGF-alpha and EGFR over-production in fresh tumors and histologically normal mucosal specimens from patients with SCCHN and normal, control patients without cancer. In patients with SCCHN, TGF-alpha and EGFR mRNA levels were significantly elevated in both tumor and normal mucosal specimens as compared to levels in control mucosa from non-cancer patients. Neither an enhancement of message stability nor an increase in gene copy number alone accounted for the elevation of EGFR mRNA. Increased production of TGF-alpha and EGFR mRNAs in the histologically "normal" mucosa of patients at risk for a primary or secondary head and neck cancer may serve both as a marker for malignant transformation and as a target for chemoprevention.

Topics: Carcinoma, Squamous Cell; ErbB Receptors; Head and Neck Neoplasms; Humans; Transforming Growth Factor alpha

Topics: Animals; Carcinoma, Squamous Cell; ErbB Receptors; Head and Neck Neoplasms; Humans; Transforming Growth Factor alpha

Targeting the epidermal growth factor receptor (EGFR) using the tyrosine kinase inhibitor (TKI) erlotinib has demonstrated activity in aerodigestive tract malignancies. Co-targeting of the G-protein-coupled receptor cyclooxygenase (COX) with EGFR inhibitors has shown promise in preclinical models and early phase clinical studies.. We studied the modulation of serum proteins after neoadjuvant treatment with erlotinib with or without sulindac in head and neck cancer patients. In a prospective, randomized, double-blind clinical trial, paired serum samples were obtained before and after neoadjuvant treatment in three groups of patients (n = 23 total), who were randomized to receive 7-14 consecutive days of erlotinib alone, erlotinib plus sulindac, or placebo. Two separate multiplexed ELISA systems (SearchLight™ or Luminex™) were used to measure serum biomarkers. HGF and IL-6 levels were tested on both systems, and validated using single analyte ELISAs.. Several analytes were significantly altered (generally decreased) post-treatment, in patients who received erlotinib (with or without sulindac) as well as in the placebo groups. No single analyte was differentially altered across the three treatment groups using either multiplex platform. Single HGF ELISA suggested a nonspecific decrease in all patients.. These results demonstrate the importance of a placebo group when assessing changes in expression of serum biomarkers. While multiplex platforms can provide quantitative information on a large number of serum analytes, results should be cautiously compared across platforms due to their intrinsic features. Furthermore, the dynamic range of expression of a single analyte is constrained in multiplex versus standard ELISA.

Topics: Adult; Aged; Antineoplastic Agents; Biomarkers, Tumor; Carcinoma, Squamous Cell; Double-Blind Method; Enzyme-Linked Immunosorbent Assay; ErbB Receptors; Erlotinib Hydrochloride; Female; Head and Neck Neoplasms; Hepatocyte Growth Factor; Humans; Interleukin-6; Male; Middle Aged; Neoadjuvant Therapy; Pilot Projects; Placebos; Prospective Studies; Protein Kinase Inhibitors; Quinazolines; Sulindac; Transforming Growth Factor alpha

Epidermal growth factor receptor (EGFR) is a validated target in squamous-cell carcinoma of the head and neck, but in patients with recurrent or metastatic disease, EGFR targeting agents have displayed modest efficacy. Vascular endothelial growth factor (VEGF)-mediated angiogenesis has been implicated as a mechanism of resistance to anti-EGFR therapy. In this multi-institutional phase I/II study we combined an EGFR inhibitor, erlotinib, with an anti-VEGF antibody, bevacizumab.. Between April 15, 2003, and Jan 27, 2005, patients with recurrent or metastatic squamous-cell carcinoma of the head and neck were enrolled from seven centres in the USA and were given erlotinib (150 mg daily) and bevacizumab in escalating dose cohorts. The primary objectives in the phase I and II sections, respectively, were to establish the maximum tolerated dose and dose-limiting toxicity of bevacizumab when administered with erlotinib and to establish the proportion of objective responses and time to disease progression. Pretreatment serum and tissues were collected and analysed by enzyme-linked immunosorbent assay and immunofluorescence quantitative laser analysis, respectively. This study was registered with ClinicalTrials.gov, number NCT00055913.. In the phase I section of the trial, ten patients were enrolled in three successive cohorts with no dose-limiting toxic effects noted. 46 patients were enrolled in the phase II section of the trial (including three patients from the phase I section) on the highest dose of bevacizumab (15 mg/kg every 3 weeks). Two additional patients were accrued beyond the protocol-stipulated 46, leaving a total of 48 patients for the phase II assessment. The most common toxic effects of any grade were rash and diarrhoea (41 and 16 of 48 patients, respectively). Three patients had serious bleeding events of grade 3 or higher. Seven patients had a response, with four showing a complete response allowing rejection of the null hypothesis. Median time of overall survival and progression-free survival (PFS) were 7.1 months (95% CI 5.7-9.0) and 4.1 months (2.8-4.4), respectively. Higher ratios of tumour-cell phosphorylated VEGF receptor-2 (pVEGFR2) over total VEGFR2 and endothelial-cell pEGFR over total EGFR in pretreatment biopsies were associated with complete response (0.704 vs 0.386, p=0.036 and 0.949 vs 0.332, p=0.036, respectively) and tumour shrinkage (p=0.007 and p=0.008, respectively) in a subset of 11 patients with available tissue.. The combination of erlotinib and bevacizumab is well tolerated in recurrent or metastatic squamous-cell carcinoma of the head and neck. A few patients seem to derive a sustained benefit and complete responses were associated with expression of putative targets in pretreatment tumour tissue.

Topics: Adult; Aged; Aged, 80 and over; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Combined Chemotherapy Protocols; Bevacizumab; Carcinoma, Squamous Cell; ErbB Receptors; Erlotinib Hydrochloride; Female; Head and Neck Neoplasms; Humans; Male; Middle Aged; Multivariate Analysis; Quinazolines; Regression Analysis; Transforming Growth Factor alpha; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-2

An objective response rate of 11% was reported in patients with recurrent and/or metastatic squamous cell carcinoma of the head and neck (SCCHN) treated with 500 mg daily gefitinib although the recommended dose in lung cancer is 250 mg. This study evaluated the efficacy and toxicity of 250 mg daily gefitinib in patients with recurrent and/or metastatic SCCHN.. Phase II trial with objective response rate as the primary end point. Measurements of quality of life and levels of serum vascular endothelial growth factor and transforming growth factor-alpha were assessed before and during therapy.. In 70 patients, 1 (1.4%) partial response was observed. Median progression-free survival and overall survival were 1.8 and 5.5 months, respectively. Quality of life scores improved transiently during the first weeks of therapy before returning to baseline. Median vascular endothelial growth factor and transforming growth factor-alpha levels were above the normal range but were not predictive of outcome. Four patients experienced grade 3 drug-related adverse events. Rash of any grade was observed in 64% of subjects. Correlation between disease control (partial response + stable disease), progression-free survival, and overall survival and grade of cutaneous toxicity was observed (P = 0.001, 0.001, and 0.008 respectively).. Gefitinib monotherapy at 250 mg in recurrent and/or metastatic SCCHN seems to have less activity than was previously observed for 500 mg daily. A dose-response relationship may exist for this agent in SCCHN and grade of cutaneous toxicity attributable to gefitinib is a clinical predictor of better outcome.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Agents; Carcinoma, Squamous Cell; Disease-Free Survival; Drug Administration Schedule; Female; Gefitinib; Head and Neck Neoplasms; Humans; Male; Middle Aged; Neoplasm Recurrence, Local; Quality of Life; Quinazolines; Salvage Therapy; Survival Rate; Transforming Growth Factor alpha; Vascular Endothelial Growth Factor A

Dysplastic oral leukoplakia (DOL) has been the index lesion in prevention trials for upper aerodigestive tract squamous cell carcinoma (SCC). Vitamin A derivatives, including 13-cis retinoic acid (13-CRA), have been used to treat DOL and to reduce the risk of subsequent SCC. Results from a trial of 13-CRA in patients with DOL are presented here. Transforming growth factor-alpha (TGF-alpha) and the epidermal growth factor receptor messenger RNA (mRNA) expression were studied to validate their use as surrogate endpoint biomarkers in prevention trials for SCC.. In a prospective, randomized, double-blind trial of 13-CRA in 28 patients with DOL, TGF-alpha and epidermal growth factor receptor mRNA expression were analyzed in sequential biopsy specimens of DOL and of adjacent normal-appearing mucosa, utilizing a quantitative, competitive, reverse transcriptase polymerase chain reaction and were compared using the Wilcoxon signed-rank test for paired comparisons.. In biopsy specimens of DOL, TGF-alpha mRNA expression at baseline, but not baseline expression of epidermal growth factor receptor mRNA, was significantly elevated when compared with its expression in specimens from adjacent normal-appearing mucosa (p = 0.003). In patients randomized to 13-CRA who had > or = 50% clearance of DOL during treatment, significant modulation of TGF-alpha mRNA overexpression was seen after 6 months of treatment (p = 0.016). TGF-alpha mRNA overexpression at baseline predicted a subsequent response to 13-CRA (p 0.066).. The full extent of the association between TGF-alpha overexpression and the development of SCC is unknown. Evidence is presented in this article that TGF-alpha overexpression mediates the relationship between 13-CRA and DOL, but there is no direct evidence that it mediates the relationship between 13-CRA and the prevention of SCC. Determination of the extent to which TGF-alpha overexpression mediates this relationship and complete validation of TGF-alpha's role as a surrogate endpoint biomarker await the results of animal and human trials that utilize reduction in the incidence of SCC as their endpoint.

Topics: Aged; Biomarkers, Tumor; Carcinoma, Squamous Cell; Disease Progression; Double-Blind Method; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Humans; Isotretinoin; Leukoplakia, Oral; Male; Mouth Neoplasms; Prospective Studies; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transforming Growth Factor alpha

Liarozole is a 1-substituted imidazole derivative that inhibits cytochrome P450 activity and increases endogenous plasma concentrations of retinoid acid (RA). We have previously demonstrated that RA down-modulates transforming growth factor (TGF)-alpha and epidermal growth factor receptor (EGFR) levels in head and neck squamous cell carcinoma by decreasing the transcription rate of these two genes. Previous reports suggest that RA receptor (RAR)-beta levels are down-modulated in head and neck cancer and are restored by RA therapy. Cellular RA-binding protein (CRABP)-II is up-regulated by RA and appears to modulate intracellular RA metabolism. In conjunction with a Phase I clinical trial, total intact RNA was extracted from oral cavity mucosa biopsied from 17 patients with advanced malignancies, before and after treatment with a 4-week course of liarozole. To analyze these limited quantities of total RNA (as little as 0.6 microg/sample), a quantitative reverse transcription-PCR assay was developed using delayed dropping of the 5' beta-actin primer to amplify the highly abundant beta-actin gene as an internal control. We used this method to determine the expression levels of TGF-alpha, EGFR, RAR-beta, and CRABP-II before and after treatment. There was a trend toward elevation of RAR-beta levels in oral mucosa after liarozole therapy (P = 0.107), whereas TGF-alpha, EGFR, and CRABP-II were not modulated by systemic liarozole treatment. These results suggest that liarozole may up-regulate RAR-beta in tissues from cancer patients and that expression levels of potential intermediate biomarkers may be determined in small tissue biopsies using a quantitative reverse transcription-PCR assay.

Topics: Actins; Antineoplastic Agents, Hormonal; Biomarkers; Carcinoma, Squamous Cell; DNA Primers; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; ErbB Receptors; Humans; Imidazoles; Mouth Mucosa; Mouth Neoplasms; Receptors, Retinoic Acid; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transforming Growth Factor alpha; Tretinoin; Up-Regulation

The invasive behaviour of squamous cell carcinoma (SCC), a common malignant tumour of the mouth, is a process mediated by cell proliferation, extracellular matrix proteolysis and other factors. Studies have shown a potential relationship between growth factors, metallothionein 2A (MT2A) and matrix metalloproteinase (MMP) activation in malignant tumours. The aim of this study was to downregulate MT2A in cells (Cal27) derived from human squamous cell carcinoma.. Cal27 cells with reduced MT2A were subjected to proliferation, migration and invasion assays. Immunofluorescence and western blot confirmed MT2A depletion by siRNA. Growth curve assays assessed cell proliferation. Indirect immunofluorescence analysed the expression of MT2A, MMP-2, MMP-9, epidermal growth factor (EGF), transforming growth factor alpha (TGF-α), tumour necrosis factor alpha (TNF-α) and Ki67. Zymography evaluated the effects of MT2A silencing on MMP-2 and -9 expression. Migration and invasion activities were evaluated using migration and invasion assays.. CAL27 cells displayed MT2A, MMP-2, MMP-9, EGF, TGF-α, TNF-α and Ki67. MT2A depletion decreased MMP-9, EGF, TGF-α and Ki67 protein levels, while increasing TNF-α.. MT2A downregulation reduced cell proliferation, migration and invasion activities. Therefore, MT2A has an important role in cell proliferation, migration and invasion in human oral SCC cells.

Topics: Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Movement; Cell Proliferation; Down-Regulation; Epidermal Growth Factor; Humans; Ki-67 Antigen; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Metallothionein; Transforming Growth Factor alpha; Tumor Necrosis Factor-alpha

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

The TGFβ signaling pathway is essential to epithelial homeostasis and is often inhibited during progression of esophageal squamous cell carcinoma. Recently, an important role for TGFβ signaling has been described in the crosstalk between epithelial and stromal cells regulating squamous tumor cell invasion in mouse models of head-and-neck squamous cell carcinoma (HNSCC). Loss of TGFβ signaling, in either compartment, leads to HNSCC however, the mechanisms involved are not well understood. Using organotypic reconstruct cultures (OTC) to model the interaction between epithelial and stromal cells that occur in dysplastic lesions, we show that loss of TGFβ signaling promotes an invasive phenotype in both fibroblast and epithelial compartments. Employing immortalized esophageal keratinocytes established to reproduce common mutations of esophageal squamous cell carcinoma, we show that treatment of OTC with inhibitors of TGFβ signaling (A83-01 or SB431542) enhances invasion of epithelial cells into a fibroblast-embedded Matrigel/collagen I matrix. Invasion induced by A83-01 is independent of proliferation but relies on protease activity and expression of ADAMTS-1 and can be altered by matrix density. This invasion was associated with increased expression of pro-inflammatory cytokines, IL1 and EGFR ligands HB-EGF and TGFα. Altering EGF signaling prevented or induced epithelial cell invasion in this model. Loss of expression of the TGFβ target gene ROBO1 suggested that chemorepulsion may regulate keratinocyte invasion. Taken together, our data show increased invasion through inhibition of TGFβ signaling altered epithelial-fibroblasts interactions, repressing markers of activated fibroblasts, and altering integrin-fibronectin interactions. These results suggest that inhibition of TGFβ signaling modulates an array of pathways that combined promote multiple aspects of tumor invasion.

Topics: ADAM Proteins; ADAMTS1 Protein; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Movement; Cell Proliferation; Esophageal Neoplasms; Fibroblasts; Heparin-binding EGF-like Growth Factor; Humans; Interleukin-1; Keratinocytes; Nerve Tissue Proteins; Receptors, Immunologic; Receptors, Transforming Growth Factor beta; Roundabout Proteins; Transforming Growth Factor alpha; Transforming Growth Factor beta

Cetuximab is an epidermal growth factor receptor (EGFR)-targeting drug that has shown effects in head and neck squamous cell carcinoma (HNSCC). The effects are, however, small and have mainly been proven in a subset of patients, and the cost-effectiveness has been questioned. For this reason, we need to know more about the basic mechanisms controlling the effect of EGFR signalling on tumour growth.. We investigated the effect of the EGFR ligand transforming growth factor alpha (TGF-α) and cetuximab, alone and in combination, on HNSCC cell lines, measuring proliferation and the activity of intracellular signalling pathways.. In line with previous reports we found the majority of the cell lines to be growth-inhibited by TGF-α. Surprisingly, two of the cell lines, which were more thoroughly investigated, were either growth-inhibited or stimulated by both cetuximab and TGF-α, depending on the presence or absence of the other substance. We also present data indicating the existence of two different receptor activities emanating from the EGFR protein.. We therefore show that TGF-α can have both growth-stimulating and growth-inhibiting effects in the same cell line and that EGFR-targeting drugs can be similarly double-edged. The implication for such drugs is that the micro-environment within a tumour, and possibly within portions of a tumour, may influence the growth-inhibiting effect of the drug. There may also be important implications for our understanding of EGFR signalling and its influence on growth and development.

Topics: Antibodies, Monoclonal, Humanized; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Cetuximab; Head and Neck Neoplasms; Humans; Transforming Growth Factor alpha

Human papillomavirus (HPV)-positive and HPV-negative oropharyngeal squamous cell carcinomas (OSCCs) are two distinct entities. We defined the molecular profiles of druggable receptor tyrosine kinases (RTKs) in both groups.. E5 expression and RTK alterations were studied in 17 HPV-positive and 59 HPV-negative formalin-fixed OSCCs. RTK activation was explored in further 12 frozen OSCCs.. The HPV-positive OSCCs showed E5 expression and 33.3% expressed low level of HER2. The HPV-negative OSCCs showed HER2 expression (31.2%), increased HER2 gene copy number (46.51%, P = 0.045) and HER2 activation through HER2/EGFR heterodimerisation; HER3 (51.06%, P = 0.008) and neuregulin (65.63%; P = 0.03) expression, HER3 activation and HER3/EGFR heterodimerisation; and increased IGF-1R copy number (40.50%, P = 0.021), high IGF-1R cDNA values (P = 0.002), IGF-1R activation and expression of IGF1/2 and amphiregulin. PI3KCA mutations/expression/increased gene copy number and PTEN mutations were found in both groups, whereas PTEN gene loss was only observed in the HPV-positive cases.. Human papillomavirus-positive and HPV-negative OSCC showed different RTK profiles. In HPV-positive cases, it would be interesting to study the expression of E5, which may modulate EGFR turnover and activate VEGF and PDGFRβ. In HPV-negative cases, HER3 may be a promising druggable biomarker that deserves further investigation. PI3KCA and PTEN alterations encourage the promising clinical evaluation of PI3K/mTOR inhibitor activity in OSCC, particularly in HPV-positive/PI3KCA-mutated OSCCs because they may be driven by PI3KCA mutation alone.

Topics: Amphiregulin; Biomarkers, Tumor; Carcinoma, Squamous Cell; Humans; Mouth Neoplasms; Mutation; Oropharyngeal Neoplasms; Papillomaviridae; Papillomavirus Infections; Peptide Fragments; Phosphatidylinositol 3-Kinases; PTEN Phosphohydrolase; Real-Time Polymerase Chain Reaction; Receptor Protein-Tyrosine Kinases; Receptor, ErbB-2; Transforming Growth Factor alpha

Squamous cell carcinoma (SCC) and keratoacanthoma (KA) are skin neoplasms of epithelial origin. In contrast to clearly malignant skin neoplasm SCC, KA is an unusual cutaneous neoplasm with a tendency to regression. The distinction between these two neoplasms, on histological grounds only, is still a challenge. In order to investigate further and to assess the possible differences in transforming growth factor-alpha (TGF-alpha) expression between SCC and KA, 40 of skin tumor specimens, 20 cases of each SCC and KA were analyzed immunohystochemicaly. We have found a significant difference in staining patterns between KA and SCC. In KAs we have detected TGF-alpha staining mainly diffusely (90% of cases) and without peripheral staining of cells in 1-2 layers (60% of cases). Contrary, there was a mostly patchy staining (55% of cases) with peripheral staining of cells in 1-2 layers (100% of cases) in SCCs. Generally, differentiation between KA and SCC can be based on clinical and histological ground, but the distinction between these two skin tumors could sometimes be difficult. We have shown that these skin neoplasms could be differentiated based on staining patterns of TGF-alpha expression, thus this method could aid in differentiation between these two closely related entities in clinical practice.

Topics: Carcinoma, Squamous Cell; Cell Proliferation; Diagnosis, Differential; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Keratoacanthoma; Medical Oncology; Skin Neoplasms; Staining and Labeling; Transforming Growth Factor alpha

The transmembrane glycoprotein podoplanin (PDPN) plays an important role in cell motility. However, mechanisms regulating PDPN expression have not been fully elucidated. Here, we investigated the effect of intercellular contact on signal transduction pathways and PDPN expression in human squamous cell carcinoma (SCC) cell lines. PDPN expression was higher in confluent SCC cells than sparse cultures. This PDPN induction leads to increased SCC cell migration and invasion, which was reversed by shRNA PDPN knockdown. This cell density dependent PDPN induction required activation of epidermal growth factor receptor (EGFR) and its effector, signal transducer and activator of transcription 3 (STAT3). These observations also extend to human clinical specimens, in which PDPN expression localized to confluent basal cell layers at the invading front of in situ SCC lesions. Taken together, these results illuminate an EGFR-STAT3-PDPN pathway as a potential pharmacological opportunity to target invasive SCC cells.

Topics: Carcinoma, Squamous Cell; Cell Line; Cell Movement; ErbB Receptors; Humans; Membrane Glycoproteins; Phosphorylation; RNA Interference; RNA, Small Interfering; Signal Transduction; STAT3 Transcription Factor; Transforming Growth Factor alpha

Predictive strategies for the treatment efficacy of cetuximab are currently not available for head and neck cancer. We investigated the correlation between the expression of epidermal growth factor receptor (EGFR) ligands and EGFR expression, and the growth inhibitory activity of cetuximab in a panel of head and neck squamous cell carcinoma (HNSCC) cell lines.. The growth inhibiting effect of cetuximab was measured for eight HNSCC cell lines and correlated with the autocrine production of five EGFR ligands as measured by ELISA, and the mRNA expression of two ligands, as measured by quantitative RT-PCR. EGFR expression was assessed by western blot analysis.. There was a good correlation between the expression of four of the EGFR ligands (TGF-α, amphiregulin, epiregulin and epigen) and the growth inhibiting effect of cetuximab. TGF-α had the highest predictive potential but had to be combined with epigen for full prediction. EGFR expression also correlated with cetuximab sensitivity but less clearly.. The results indicate that the expression of several EGFR ligands has to be used to predict sensitivity to cetuximab in HNSCC. This has to be further evaluated in clinical samples.

Topics: Amphiregulin; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Blotting, Western; Carcinoma, Squamous Cell; Cell Line, Tumor; Cetuximab; EGF Family of Proteins; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; Epigen; Epiregulin; ErbB Receptors; Gene Expression Regulation, Neoplastic; Glycoproteins; Head and Neck Neoplasms; Humans; Intercellular Signaling Peptides and Proteins; Ligands; Predictive Value of Tests; Reverse Transcriptase Polymerase Chain Reaction; Squamous Cell Carcinoma of Head and Neck; Transforming Growth Factor alpha

Head and neck squamous cell carcinoma (HNSCC) is usually fatal, and innovative approaches targeting growth pathways are necessary to effectively treat this disease. Both the epidermal growth factor receptor (EGFR) and the hepatocyte growth factor (HGF)/c-Met pathways are overexpressed in HNSCC and initiate similar downstream signaling pathways. c-Met may act in consort with EGFR and/or be activated as a compensatory pathway in the presence of EGFR blockade.. Expression levels of EGFR and c-Met were determined by Western analysis in HNSCC cell lines and correlated with antitumor responses to inhibitors of these pathways.. Combining the c-Met inhibitor PF2341066 with the EGFR inhibitor gefitinib abrogated HNSCC cell proliferation, invasion, and wound healing significantly more than inhibition of each pathway alone in HNSCC cell lines. When both HGF and the EGFR ligand, TGF-α, were present in vitro, P-AKT and P-MAPK expression were maximally inhibited by targeting both EGFR and c-Met pathways, suggesting that c-Met or EGFR can compensate when phosphorylation of the other receptor is inhibited. We also showed that TGF-α can induce phosphorylation of c-Met over sixfold by 8 hours in the absence of HGF, supporting a ligand-independent mechanism. Combined targeting of c-Met and EGFR resulted in an enhanced inhibition of tumor volumes accompanied by a decreased number of proliferating cells and increased apoptosis compared with single agent treatment in vivo.. Together, these results suggest that dual blockade of c-Met and EGFR may be a promising clinical therapeutic strategy for treating HNSCC.

Topics: Animals; Antineoplastic Agents; Carcinoma; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; ErbB Receptors; Female; Gefitinib; Head and Neck Neoplasms; Hepatocyte Growth Factor; Humans; Mice; Mice, SCID; Mitogen-Activated Protein Kinases; Neoplasm Invasiveness; Neoplasms, Squamous Cell; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-met; Quinazolines; Signal Transduction; Squamous Cell Carcinoma of Head and Neck; Transforming Growth Factor alpha; Wound Healing; Xenograft Model Antitumor Assays

The monoclonal epidermal growth factor receptor (EGFR) antibody cetuximab (Erbitux) was recently approved by the European Medicines Agency for the treatment of recurrent and/or metastatic head and neck squamous cell carcinoma (HNSCC) in combination with a platinum-based chemotherapy. Since the antibody has only a limited effect as a monotherapy, possible explanations for the synergistic effect with cisplatin are enhanced antibody-dependent cytoxicity and increased sensitivity to the drug. Most of our knowledge of EGFR biology in HNSCC is based on studies using EGFR inhibitors and/or antibodies. Our study was designed to evaluate the impact of EGFR stimulation on cisplatin-induced DNA damage. Therefore, tissue cultures were produced of tumor-free oropharyngeal mucosa biopsies of HNSCC patients and controls. In a previous study, overexpression of EGFR in tissue cultures from tumor patients compared to controls was confirmed by immunohistochemical staining. Twenty-four-hour stimulation of tissue cultures with transforming growth factor alpha (TGF-alpha), a specific EGFR ligand, resulted in a reduction of cisplatin-induced DNA damage by 35% in cases, whereas in controls TGF-alpha had no effect. This reflects a statistically significant increase in cellular chemoresistance to cisplatin following TGF-alpha stimulation and helps to further understand effects of EGFR antisense therapy in combination with chemotherapy.

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Carcinoma, Squamous Cell; Cetuximab; Cisplatin; DNA Damage; Drug Resistance, Neoplasm; ErbB Receptors; Head and Neck Neoplasms; Humans; Mucous Membrane; Oropharyngeal Neoplasms; Tissue Culture Techniques; Transforming Growth Factor alpha

Activation of the transforming growth factor (TGF) α/epidermal growth factor receptor (EGFR)-mediated signaling pathway is a common mechanism for dysregulated growth of head and neck squamous cell carcinoma (HNSCC). c-Cbl-interacting protein of 85 kDa (CIN85) is an adaptor protein that facilitates EGFR internalization. Little is known, however, about a role of CIN85 in EGFR signaling as well as its relevance to tumor development and progression of HNSCC. Here, we demonstrate that CIN85 is highly expressed in HNSCC tumor samples compared with adjacent normal tissues, and this overexpression is significantly correlated with advanced clinical stage. The experiments using CIN85-overexpressing and knockdown HNSCC cell lines showed that CIN85 promotes HNSCC growth and facilitates EGFR internalization without apparently affecting phosphorylation of EGFR. Moreover, CIN85 promoted TGF-α-induced activation of Ras and phosphorylation of downstream molecules such as c-Raf, MEK, and extracellular signal-regulated kinase, leading to expression of c-Myc that is critical for sustained proliferation of HNSCC. Taken together, these findings suggest that CIN85 not only controls EGFR internalization but also promotes the EGFR-mediated tumor development and progression, and thus, CIN85 may serve as a potential therapeutic target in a subset of HNSCC.

Topics: Adaptor Proteins, Signal Transducing; Apoptosis; Blotting, Western; Carcinoma, Squamous Cell; Cell Proliferation; Disease Progression; ErbB Receptors; Female; Head and Neck Neoplasms; Humans; Immunoenzyme Techniques; Lymphatic Metastasis; Male; MAP Kinase Kinase 1; Microscopy, Fluorescence; Middle Aged; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Phosphorylation; Proto-Oncogene Proteins c-raf; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Transforming Growth Factor alpha; Tumor Cells, Cultured

Topics: beta-Defensins; Biomarkers; Carcinoma, Squamous Cell; Epidermal Growth Factor; ErbB Receptors; Erythroplasia; Humans; Leukoplakia, Oral; Mouth Neoplasms; Precancerous Conditions; Prognosis; Transforming Growth Factor alpha; Tumor Suppressor Protein p53

Calcitriol, acting through vitamin D receptors (VDR) in the parathyroid, suppresses parathyroid hormone synthesis and cell proliferation. In secondary hyperparathyroidism (SH), VDR content is reduced as hyperplasia becomes more severe, limiting the efficacy of calcitriol. In a rat model of SH, activation of the EGF receptor (EGFR) by TGF-alpha is required for the development of parathyroid hyperplasia, but the relationship between EGFR activation and reduced VDR content is unknown. With the use of the same rat model, it was found that pharmacologic inhibition of EGFR activation with erlotinib prevented the upregulation of parathyroid TGF-alpha, the progression of growth, and the reduction of VDR. Increased TGF-alpha/EGFR activation induced the synthesis of liver-enriched inhibitory protein, a potent mitogen and the dominant negative isoform of the transcription factor CCAAT enhancer binding protein-beta, in human hyperplastic parathyroid glands and in the human epidermoid carcinoma cell line A431, which mimics hyperplastic parathyroid cells. Increases in liver-enriched inhibitory protein directly correlated with proliferating activity and, in A431 cells, reduced VDR expression by antagonizing CCAAT enhancer binding protein-beta transactivation of the VDR gene. Similarly, in nodular hyperplasia, which is the most severe form of SH and the most resistant to calcitriol therapy, higher TGF-alpha activation of the EGFR was associated with an 80% reduction in VDR mRNA levels. Thus, in SH, EGFR activation is the cause of both hyperplastic growth and VDR reduction and therefore influences the efficacy of therapy with calcitriol.

Topics: Animals; Calcitriol; Carcinoma, Squamous Cell; CCAAT-Enhancer-Binding Protein-beta; Cell Line, Tumor; Disease Models, Animal; Drug Resistance; ErbB Receptors; Erlotinib Hydrochloride; Female; Genes, Reporter; Humans; Hyperparathyroidism, Secondary; Hyperplasia; Protein Kinase Inhibitors; Quinazolines; Rats; Rats, Sprague-Dawley; Receptors, Calcitriol; Renal Insufficiency, Chronic; Transforming Growth Factor alpha

To determine the effect of tyrosine-phosphorylated signal transducer and activator of transcription 3 (pSTAT3) immunoexpression on survival in two independent cohorts of patients with squamous cell carcinoma of the head and neck (SCCHN) and to evaluate pSTAT3, transforming growth factor-alpha (TGF-alpha), epidermal growth factor receptor (EGFR), and gastrin-releasing peptide receptor (GRPR) expression in matched tumor and lymph node metastases in one of these cohorts. EXPERIMENTAL TECHNIQUE: Immunostaining for pSTAT3, TGF-alpha, EGFR, and GRPR was done in two SCCHN cohorts (cohort 1, 61 tumors; cohort 2, 69 paired primary tumors and lymph node metastases). Semiquantitative scores derived from the product of staining intensity (scale 0-3) score and percentage of positive tumor cells were correlated with clinical outcome.. Immunoexpression of pSTAT3 did not correlate with clinical outcome in either cohort (cohort 1, P = 0.914; cohort 2, P = 0.312). In cohort 2, TGF-alpha and EGFR expression in the primary tumors showed some association with decreased disease-free survival (P = 0.0306 and P = 0.0985, respectively). Both pSTAT3 and EGFR showed a correlation of expression between tumor and matched lymph node metastasis (P < 0.0001 and P = 0.0046, respectively). In addition, the expression of EGFR and GRPR in the primary tumors correlated with TGF-alpha expression in paired nodal metastases (P = 0.0043 and P = 0.0268, respectively). In the nodal metastases, TGF-alpha expression correlated with EGFR expression (P = 0.0069). In primary tumors, GRPR expression correlated with TGF-alpha and EGFR expression (P = 0.0378 and P = 0.0026, respectively).. These findings support an autocrine signaling pathway involving TGF-alpha, EGFR, and pSTAT3 in metastatic SCCHN as well as transactivation of EGFR by GRPR via TGF-alpha, but fails to identify an independent prognostic role for pSTAT3 immunoexpression.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Squamous Cell; Cohort Studies; ErbB Receptors; Female; Head and Neck Neoplasms; Humans; Immunoenzyme Techniques; Lymph Nodes; Lymphatic Metastasis; Male; Middle Aged; Phosphotyrosine; Receptors, Bombesin; Signal Transduction; STAT3 Transcription Factor; Transcriptional Activation; Transforming Growth Factor alpha

Eosinophil cationic protein (ECP) is a major component of eosinophil granule protein that is used as a clinical bio-marker for asthma and allergic inflammatory diseases. Previously, it has been reported that the signal peptide of human ECP (ECPsp) inhibits the cell growth of Escherichia coli (E. coli) and Pichia pastoris (P. pastoris), but not mammalian A431 cells. The inhibitory effect is due to the lack of human signal peptide peptidase (hSPP), a protease located on the endoplasmic reticulum (ER) membrane, in the lower organisms. In this study, we show that the epidermal growth factor receptor (EGFR) is upregulated by the exogenous ECPsp-eGFP as a result of the increased expression of the transforming growth factor-alpha (TGF-alpha) at both transcriptional and translational levels in A431 and HL-60 clone 15 cell lines. Furthermore, the N-terminus of ECPsp fragment generated by the cleavage of hSPP (ECPspM1-G17) gives rise to over threefold increase of TGF-alpha protein expression, whereas another ECPsp fragment (ECPspL18-A27) and the hSPP-resistant ECPsp (ECPspG17L) do not show similar effect. Our results indicate that the ECPspM1-G17 plays a crucial role in the upregulation of TGF-alpha, suggesting that the ECPsp not only directs the secretion of mature ECP, but also involves in the autocrine system.

Topics: Aspartic Acid Endopeptidases; Carcinoma, Squamous Cell; Culture Media, Conditioned; Enzyme-Linked Immunosorbent Assay; Eosinophil Cationic Protein; ErbB Receptors; Green Fluorescent Proteins; HL-60 Cells; Humans; Protein Sorting Signals; Recombinant Fusion Proteins; Transforming Growth Factor alpha; Tumor Cells, Cultured; Up-Regulation

To evaluate the possibility of treatment with antiepidermal growth factor receptor (EGFR) mAb, Cetuximab against esophageal squamous cell carcinoma (SCC), we performed detail analysis of the antibody-dependent cellular cytotoxicity (ADCC) mediated by Cetuximab against esophageal SCC. Esophageal SCC cell lines with various levels of EGFR (n = 8) were evaluated for their Cetuximab-mediated ADCC by (51)Cr-release assay. As a result, Cetuximab was able to induce ADCC against EGFR-expressing esophageal SCC and the activities reflected the degree of EGFR expression on the esophageal SCC. The activities of Cetuximab-mediated ADCC by patients' PBMC were impaired in comparison with those by healthy donors' PBMC. Moreover, the inhibition of transforming growth factor (TGF)-beta could enhance Cetuximab-mediated ADCC against TGF-beta-producing SCC. In conclusion, Cetuximab was able to induce ADCC against EGFR-expressing esophageal SCC. Some modalities aiming at enhancing the Cetuximab-mediated ADCC may be necessary for successful Cetuximab treatment of patients with esophageal SCC.

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antibody-Dependent Cell Cytotoxicity; Antineoplastic Agents; Apoptosis; Carcinoma, Squamous Cell; Cell Proliferation; Cetuximab; Epidermal Growth Factor; ErbB Receptors; Esophageal Neoplasms; Humans; Interleukin-2; Leukocytes, Mononuclear; Transforming Growth Factor alpha; Transforming Growth Factor beta2; Tumor Cells, Cultured

In this study, we examined ErbB1 signaling in human basal and squamous cell carcinomas (BCC and SCC) of the skin in vivo. We used enzyme-linked immunosorbent assay, laser capture microdissection-coupled real-time reverse transcriptase-polymerase chain reaction, and immunohistochemistry to assess expression and activation levels of ErbB1 protein, ligands, and potential downstream effectors, in BCC and SCC tumors, stroma, and adjacent epidermis. Although total ErbB1 protein and mRNA were similar in cancerous and normal skin, we found that ErbB1 activation (phospho-Tyr(1068)) was greater in bulk SCC versus BCC or normal skin. In addition, three ErbB1 ligand transcripts (amphiregulin, heparin-binding epidermal growth factor-like growth factor, and transforming growth factor-alpha) were up-regulated in tumor cells of SCC but not BCC. Expression of these ligands was also increased in asymptomatic epidermis adjacent to both SCC and BCC, relative to normal skin. Interestingly, betacellulin transcript levels were inversely regulated compared with the other ligands. Consistently, downstream ErbB1 effectors (Erk1/2 and Akt) were activated in tumor cells of SCC but not of BCC and in adjacent epidermis of both BCC and SCC. These results demonstrate that ErbB1 signaling is hyperactive in tumor cells of SCC but not of BCC and in nearby asymptomatic epidermis of both tumor types. Our results suggest that targeting ErbB1 signaling might be of benefit in the treatment of SCC.

Topics: Amphiregulin; Animals; Betacellulin; Carcinoma, Basal Cell; Carcinoma, Squamous Cell; EGF Family of Proteins; Enzyme Activation; Epidermal Growth Factor; Epiregulin; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Glycoproteins; Heparin-binding EGF-like Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Proto-Oncogene Proteins c-akt; RNA, Messenger; Signal Transduction; Skin; Skin Neoplasms; Transforming Growth Factor alpha

Signal transducer and activator of transcription 3 (STAT3) has been reported to be activated by interleukin-6 receptor (IL-6R) or epidermal growth factor receptor (EGFR) in head and neck squamous cell carcinomas (HNSCC), which may have important implications for responsiveness to therapeutics targeted at EGFR, IL-6R, or intermediary kinases. Suppressor of cytokine signaling-1 (SOCS-1) has been implicated recently in the negative regulation of IL-6R/Janus-activated kinase (JAK)-mediated activation of STAT3, suggesting that SOCS-1 could affect alternative activation of STAT3 by EGFR, IL-6R, and associated kinases. We investigated whether epigenetic modification of SOCS-1 affects STAT3 activation in response to IL-6R-, EGFR-, JAK-, or mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK)-mediated signal activation. STAT3 was predominantly activated by IL-6R via Jak1/Jak2 in HNSCC lines UMSCC-9 and UMSCC-38 in association with transcriptional silencing of SOCS-1 by hypermethylation. In UMSCC-11A cells with unmethylated SOCS-1, STAT3 activation was regulated by both EGFR and IL-6R via a JAK-independent pathway involving MEK. Pharmacologic inhibitors of JAK and MEK and expression of SOCS-1 following demethylation or transient transfection inhibited STAT3 activation and cell proliferation and induced cell apoptosis in corresponding cell lines. Hypermethylation of SOCS-1 was found in about one-third of human HNSCC tissues, making it a potentially relevant marker for STAT-targeted therapy in HNSCC patients. We conclude that SOCS-1 methylation status can differentially affect STAT3 activation by IL-6R and EGFR through JAK or MEK in different HNSCC and response to pharmacologic antagonists. Identifying the potential factors and the regulatory pathways in STAT3 activation has important implications for the development and selection of molecularly targeted therapy in HNSCC.

Topics: Carcinoma, Squamous Cell; DNA Methylation; Enzyme Inhibitors; Epigenesis, Genetic; ErbB Receptors; Head and Neck Neoplasms; Humans; Interleukin-6; Intracellular Signaling Peptides and Proteins; Janus Kinase 1; MAP Kinase Kinase Kinase 1; Phosphorylation; Promoter Regions, Genetic; Protein-Tyrosine Kinases; Receptors, Interleukin-6; Repressor Proteins; Signal Transduction; STAT3 Transcription Factor; Suppressor of Cytokine Signaling 1 Protein; Suppressor of Cytokine Signaling Proteins; Transforming Growth Factor alpha; Tumor Cells, Cultured

To identify the molecules involved in esophageal carcinogenesis and those applicable as novel tumor markers and for the development of new molecular therapies, we performed gene expression profile analysis of 19 esophageal squamous cell carcinoma (ESCC) cells purified by laser microbeam microdissection (LMM). Using a cDNA microarray representing 32,256 genes, we identified 147 genes that were commonly up-regulated and 376 transcripts that were down-regulated in ESCC cells compared with non-cancerous esophageal epithelial cells. A comparison of clinicopathological data with the expression profiles of the 19 ESCCs identified 20 genes whose expression levels could most significantly separate cases with lymph node metastasis from those without. In addition, immunohistochemical analysis of candidate tumor markers on tissue microarrays demonstrated transactivation of a secretory protein, transforming growth factor alpha (TGFA) in the great majority of 228 ESCC cases and an association of their expression with the poor prognosis of patients. Our data provide valuable information for establishing novel diagnostic markers for early diagnosis and choice of therapy, and identifying therapeutic target molecules for the development of novel anti-cancer drugs and immunotherapy in esophageal cancer treatment.

Topics: Aged; Carcinoma, Squamous Cell; DNA, Complementary; DNA, Neoplasm; Epithelial Cells; Esophageal Neoplasms; Esophagus; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Genome, Human; Humans; Male; Middle Aged; Oligonucleotide Array Sequence Analysis; Prognosis; Reference Values; Transcription, Genetic; Transforming Growth Factor alpha

To measure HPV status, epidermal growth factor receptor (EGFR) and transforming growth factor-alpha (TGF-alpha) expression and Ki-67 index in exophytic papilloma (EP), inverted papilloma (IP) with dysplasia, IP with carcinoma and invasive squamous cell carcinoma (SCC).. Forty-four patients with sinonasal papilloma and invasive SCC were selected. The nasal tissues were stained with monoclonal antibodies to EGFR, TGF-alpha and Ki-67. The results were analysed using quantitative immunohistochemical analysis. In situ hybridization studies for HPV DNA for 6/11, 16/18 and 31/33 were also performed on the tissue.. Significant increase of EGFR and TGF-alpha was observed in IP with severe dysplasia, IP with carcinoma and invasive SCC compared to IP with mild dysplasia and control nasal mucosa. And a serial upreguration in terms of Ki-67 index in IP with dysplasia was observed. Among IP, HPV 6/11-positive was present in 42% tumour and HPV 16/18-positive was present in 31% of tumours. Among HPV 6/11 and 16/18-positive IP, significant increase of EGFR and Ki-67 index were observed.. Pre-cancerous lesions of IP exhibited elevated levels of EGFR and TGF-alpha and these expression may be associated with early events in IP carcinogenesis. HPV infection may be an early event in a multistep process of malignant formation of IP.

Topics: Antibodies, Monoclonal; Biomarkers, Tumor; Carcinoma; Carcinoma, Squamous Cell; Cell Transformation, Neoplastic; ErbB Receptors; Female; Humans; In Situ Hybridization; Ki-67 Antigen; Male; Middle Aged; Nasal Mucosa; Neoplasm Invasiveness; Nose Neoplasms; Papilloma; Papilloma, Inverted; Papillomaviridae; Paranasal Sinus Neoplasms; Precancerous Conditions; 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 described recently the growth inhibitory effects of the novel compound acyclic retinoid (ACR) in human hepatoma cell lines (M. Suzui et al., Cancer Res., 62: 3997-4006, 2002). In this study we examined the cellular and molecular effects of ACR on human squamous cell carcinoma (SCC) cells. ACR inhibited growth of the esophageal SCC cell line HCE7, and the head and neck SCC cell lines YCU-N861 and YCU-H891, with IC(50) values of approximately 10, 25, and 40 microM, respectively. Detailed studies were then done with HCE7 cells. Treatment of these cells with 10 microM ACR caused an increase of cells in G(0)-G(1) and induced apoptosis. This was associated with two phases of molecular events. During phase 1, which occurred within 6-12 h, there was an increase in the retinoic acid receptor beta (RARbeta) and p21(CIP1) proteins, and their corresponding mRNAs, and a decrease in the hyperphosphorylated form of the retinoblastoma protein. During phase 2, which occurred at approximately 24 h, there was a decrease in the cellular level of transforming growth factor alpha, and the phosphorylated (i.e., activated) forms of the epidermal growth factor receptor, Stat3, and extracellular signal-regulated kinase proteins, and a decrease in both cyclin D1 protein and mRNA. Reporter assays indicated that ACR inhibited the transcriptional activity of the cyclin D1, c-fos, and activator protein promoters. On the other hand, ACR markedly stimulated the activity of a retinoic acid response element-CAT reporter when the cells were cotransfected with a RARbeta expression vector. A hypothetical model explaining these two phases is presented. The diverse effects that we obtained with ACR suggest that this agent might be useful in the chemoprevention and/or therapy of human SCCs.

Topics: Antineoplastic Agents; Apoptosis; Blotting, Western; Carcinoma, Squamous Cell; Cell Cycle; Cell Division; Cell Line, Tumor; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; DNA-Binding Proteins; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; ErbB Receptors; Esophagus; G1 Phase; Genes, Reporter; Genetic Vectors; Humans; Inhibitory Concentration 50; Phosphorylation; Resting Phase, Cell Cycle; Retinoblastoma Protein; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; STAT3 Transcription Factor; Time Factors; Trans-Activators; Transcription, Genetic; Transforming Growth Factor alpha; Tretinoin

Head and neck cancers are characterized by a vigorous desmoplastic response, but the contribution of stromal-derived growth factors to the tumor microenvironment is poorly understood. We evaluated the expression of stromal growth factor expression in head and neck squamous cell carcinoma (HNSCC) in normal and tumor-associated stromal cells. Stromal tissue was isolated from epithelial cells with laser capture microdissection (LCMD) and analyzed by cDNA array for the expression of TGFalpha, TGF-beta1, HGF, PDGF-alpha, IGFII, bFGF, aFGF, VEGFC, and VEGF. Primary fibroblasts were isolated in vitro from HNSCC tumors, adjacent histologically normal mucosa, and skin in vitro. Fibroblast populations were assessed for TGF-beta1 expression by ELISA and luciferase reporter assay to assess protein expression. We identified TGF-beta1 and IGFII overexpression in normal and tumor-associated stromal cells; however, only TGF-beta1 was significantly overexpressed (3.4-fold) in tumor-associated stroma. Assessment of carcinoma-associated fibroblasts (CAFs), normal dermal fibroblasts (NDFs), and normal mucosal fibroblasts (NMFs) in propagated fibroblasts demonstrated persistently elevated levels of TGF-beta1 in CAFs compared to NMF and NDF populations. Elevated levels of TGF-beta1 were identified in the stromal compartment of HNSCC tumors compared to normal mucosa by immunohistochemical analysis. These results suggest that TGF-beta1 mRNA and protein is specifically upregulated in CAFs in vitro and in vivo.

Topics: Animals; Carcinoma, Squamous Cell; Cells, Cultured; Fibroblast Growth Factor 1; Fibroblast Growth Factor 2; Fibroblasts; Gene Expression Regulation; Head and Neck Neoplasms; Hepatocyte Growth Factor; Humans; Insulin-Like Growth Factor II; Mouth Mucosa; Platelet-Derived Growth Factor; Reference Values; Stromal Cells; Transforming Growth Factor alpha; Transforming Growth Factor beta; Transforming Growth Factor beta1; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor C

Head and neck squamous cell carcinomas (HNSCCs) are characterized by up-regulation of the epidermal growth factor receptor (EGFR). We previously reported that a gastrin-releasing peptide/gastrin-releasing peptide receptor (GRP/GRPR) autocrine growth pathway is activated early in HNSCC carcinogenesis. GRP can induce rapid phosphorylation of EGFR and p42/44 mitogen-activated protein kinase (MAPK) activation in part via extracellular release of transforming growth factor alpha (TGF-alpha) by matrix metalloproteinases (MMPs). It has been reported that Src family kinases are activated by G-protein-coupled receptors (GPCRs), followed by downstream EGFR and MAPK activation. To further elucidate the mechanism of activation of EGFR by GRP in HNSCC, we investigated the role of Src family kinases. Blockade of Src family kinases using an Src-specific tyrosine kinase inhibitor A-419259 decreased GRP-induced EGFR phosphorylation and MAPK activation. GRP also failed to induce MAPK activation in dominant-negative c-Src-transfected HNSCC cells. Invasion and growth assays showed that c-Src was required for GRP-induced proliferation or invasion of HNSCC cells. In addition to TGF-alpha release, GRP induced amphiregulin, but not EGF, secretion into HNSCC cell culture medium, an effect that was blocked by the MMP inhibitor marimastat. TGF-alpha and amphiregulin secretion by GRP stimulation also was inhibited by blockade of Src family kinases. These results suggest that Src family kinases contribute to GRP-mediated EGFR growth and invasion pathways by facilitating cleavage and release of TGF-alpha and amphiregulin in HNSCC.

Topics: Amphiregulin; Carcinoma, Squamous Cell; Cell Division; Cell Line, Tumor; EGF Family of Proteins; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Gastrin-Releasing Peptide; Glycoproteins; Head and Neck Neoplasms; Humans; Intercellular Signaling Peptides and Proteins; Mitogen-Activated Protein Kinases; Neoplasm Invasiveness; Phosphorylation; Receptors, Bombesin; src-Family Kinases; Transforming Growth Factor alpha

To develop an immune-competent animal model for mucosally derived squamous cell carcinoma (SCCA).. Fifteen Fischer 344 rats were inoculated with 1, 2, 5, 10, or 20 x 10(6) FAT7 cells in their flanks. The animals were observed for tumor growth and metastasis.. All animals developed tumors that grew exponentially. Pulmonary metastases developed in all animals and 13% developed lymph node metastases.. The FAT7 flank tumor in Fischer 344 rats is a new animal model that closely resembles the behavior of human mucosal head and neck cancer.. The existence of an immune-competent, mucosally derived, and reliable animal model of SCCA that somewhat resembles human head and neck SCCA gives the opportunity to perform immune-modulating experiments on head and neck cancer in these animals.. B-3.

Topics: Animals; Carcinoma, Squamous Cell; Cell Line, Tumor; ErbB Receptors; Female; Genes, p53; Head and Neck Neoplasms; Immunocompetence; Models, Animal; Mutation; Nasal Mucosa; Rats; Transforming Growth Factor alpha

Granular cell tumors (GCT) are uncommon benign neoplasms that have a predilection for the head and neck region. These tumors can frequently be associated with pseudoepitheliomatous hyperplasia (PEH), which in turn may be mistaken for squamous cell carcinoma. Although epidermal growth factors are overexpressed in squamous cell carcinomas of the head and neck, their presence in PEH, especially its relation to GCT, is unknown. We hypothesize that the expression of epidermal growth factor receptor (EGFR), epidermal growth factor (EGF), and transforming growth factor alpha (TGFalpha) in GCT have a role in the development of PEH overlying some GCT. Sections from 13 cases of GCT (five with overlying PEH) were examined histologically and evaluated immunohistochemically using monoclonal antibodies for EGFR, EGF, and TGFalpha. These were compared with nine cases of PEH independent of GCT. Two of five GCT with overlying PEH and two of six GCT without overlying PEH stained positively for TGFalpha. None of the GCT stained with EGFR or EGF. All cases of PEH, whether or not associated with GCT, were reactive for EGFR and EGF. Four of the five cases of PEH overlying GCT stained with TGFalpha. The staining pattern and intensity of all three antibodies were comparable to that of the adjacent normal squamous mucosa. Among the three antibodies, only TGFalpha in GCT appears to be related to the development of PEH. Epidermal growth factor receptor and EGF do not seem to be directly involved. The reason of PEH formation associated with GCT in the absence of growth factors is unknown.

Topics: Adolescent; Adult; Aged; Antibodies, Monoclonal; Carcinoma, Squamous Cell; Child; Diagnosis, Differential; Epidermal Growth Factor; ErbB Receptors; Female; Granular Cell Tumor; Head and Neck Neoplasms; Humans; Hyperplasia; Immunohistochemistry; Male; Middle Aged; Retrospective Studies; Transforming Growth Factor alpha

The signal transducing protein Stat3 activates gene transcription in cells in response to multiple cytokines. Constitutive activation of Stat3 has been observed in solid tumors including head and neck squamous cell carcinoma. Stat3 activation in cancer has been associated with autocrine stimulatory loops and is believed to convey a growth advantage to cells. We now demonstrate ligand-independent activation of Stat3 by high cell density in multiple cancer cell lines. Activation of Stat3 is associated with antiproliferative rather than proliferative conditions. Interference with cdk2 activity upregulates Stat3 phosphorylation and Stat3-directed DNA-binding activity. Our data supports a model in which Stat3 activity is partially suppressed by cdk2 in growing cells and derepressed upon cell confluence.

Topics: 3T3 Cells; Animals; Carcinoma, Squamous Cell; CDC2-CDC28 Kinases; Cell Communication; Cell Division; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinases; DNA-Binding Proteins; Enzyme Inhibitors; ErbB Receptors; Fibroblasts; Head and Neck Neoplasms; Humans; Mice; Mice, Knockout; Phosphorylation; Protein Serine-Threonine Kinases; Purines; rac1 GTP-Binding Protein; Roscovitine; Signal Transduction; STAT3 Transcription Factor; Trans-Activators; Transforming Growth Factor alpha; Tumor Cells, Cultured; Up-Regulation

Activation of telomerase, which stabilizes the telomere length of chromosomes, is crucial for the continued growth or progression of cancer cells. In a previous study, we showed that telomerase is frequently activated in skin tumors. Because epidermal growth factor plays an important role during the tumorigenesis of epithelial tissue, we have now examined the role of epidermal growth factor signaling in regulating telomerase activity using HSC-1 human cutaneous squamous cell carcinoma cells. Treatment of HSC-1 cells with AG 1478, an inhibitor of the epidermal growth factor receptor, or with a neutralizing antibody to the epidermal growth factor receptor, significantly suppressed their telomerase activity, in association with inhibiting their growth. The suppression of telomerase activity was obvious at day 3 and was maximal at day 5 after treatment with AG 1478. The suppression of telomerase activity correlated with the decreased expression of human telomerase catalytic subunit (hTERT) mRNA, the rate-limiting determinant of its enzyme activity. The expression of c-Myc and of Sp1 proteins, transcription factors for hTERT, were also suppressed by AG 1478 in HSC-1 cells, but the expression of Ets-2 protein, another transcription factor, was not affected. The expression of Mad-1, a competitor of c-Myc, was increased. Inhibition of ERK, Src, or Akt suppressed telomerase activity in HSC-1 cells, but to a lesser extent than did treatment with AG 1478. Serum starvation suppressed telomerase activity, but addition of epidermal growth factor or transforming growth factor alpha did not increase it, indicating the involvement of other epidermal growth factor receptor ligands in the activation of telomerase in HSC-1 cells. These data indicate that blockade of the epidermal growth factor receptor might be effective in inhibiting telomerase activity of squamous cell carcinomas, which would lead to the suppression of tumor growth.

Topics: Carcinoma, Squamous Cell; Cell Cycle Proteins; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; DNA-Binding Proteins; Dose-Response Relationship, Drug; Epidermal Growth Factor; ErbB Receptors; Humans; Mitogen-Activated Protein Kinases; Nuclear Proteins; Phosphoproteins; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-myc; Quinazolines; Repressor Proteins; RNA, Messenger; Skin Neoplasms; Telomerase; Transforming Growth Factor alpha; Tyrphostins

The aim of this review was to describe expression of epithelial growth factor receptor (EGFR) and of its ligands in a panel of tumours (which are not specified elsewhere in this special issue of Bulletin du Cancer): squamous cell carcinomas, adenocarcinomas, sarcomas, brain and germ line tumours. The role of EGFR and its ligands in the carcinogenesis and the progression of these tumours, as well as the relevance of targeting EGFR.

Topics: Adenocarcinoma; Carcinoma, Squamous Cell; ErbB Receptors; Female; Humans; Male; Neoplasm Proteins; Neoplasms; Sarcoma; Transforming Growth Factor alpha

Verrucous carcinoma (VC) is a locally invasive, nonmetastasizing variant of squamous cell carcinoma (SCC) with distinct clinical and histologic features. Molecular alterations detectable by immunohistochemical analyses in VC have not been extensively studied. This study investigates the expression of p53, epidermal growth factor receptor (EGFR), transforming growth factor-alpha (TGF-alpha), and cyclin D1 in VC, verrucous hyperplasia (VH), and classic SCC of the head and neck. Twenty-six cases of VC, 12 cases of SCC of various differentiations, and 4 cases of VH were studied. Formalin-fixed, paraffin-embedded archival material was used for immunohistochemistry (avidin-biotin immunoperoxidase technique) to study the expression of oncogenes and their tumor markers. Identification of p53 protein was found in 100% of VH, 88% of VC, and 100% of SCC. EGFR expression was noted in 25% of VH, 54% of VC, 40% of well-differentiated SCC (WDSCC), and 100% of moderately and poorly differentiated SCC (MDSCC/PDSCC). TGF-alpha was detected in 25% of VH, 88% of VC, 80% WDSCC, and 100% of MDSCC/PDSCC. Cyclin-D1 expression was seen in 75% of VH, 35% of VC, 100% of WDSCC, 67% of MDSCC, and 50% of PDSCC. Correlation between the level of expression of all markers and the grade of this group of squamous lesions revealed statistically significant correlation coefficients for p53 and EGFR but not for TGF-alpha and cyclin D1.

Topics: Biomarkers, Tumor; Carcinoma, Squamous Cell; Carcinoma, Verrucous; Cell Differentiation; Cyclin D1; Epithelium; ErbB Receptors; Head and Neck Neoplasms; Humans; Hyperplasia; Immunohistochemistry; Transforming Growth Factor alpha; Tumor Suppressor Protein p53

Squamous cell carcinomas (SCCs) of the head and neck are characterized by high tendency to invade locally and metastasize to lymph nodes. SCC cells express several matrix metalloproteinases (MMPs) and they often harbor mutations in p53 tumor suppressor gene. Collagenase-3 (MMP-13) is specifically expressed by tumor cells of SCCs and it apparently plays an important role in their invasion and metastasis. We used adenoviral gene delivery to examine the effect of wild-type p53 on MMP-13 expression in four head and neck SCC cell lines with mutated p53. Adenoviral delivery of p53 resulted in potent inhibition in production of proMMP-13 (by 71 to 92%) and collagenase-1 (MMP-1) (by 27 to 93%) by all cell lines in 24 h, whereas production of gelatinase-A (MMP-2) and gelatinase-B (MMP-9) was not altered. Adenoviral expression of p53 also suppressed invasion of SCC cells through Matrigel by 35%. Expression of cyclin-dependent kinase inhibitor p21(Waf1/Cip1) was induced 24 h after p53 gene delivery in all SCC cell lines, except one, which lacked detectable p21(Waf1/Cip1) expression. Number of viable cells was not altered and no apoptotic cells were seen 24 h after p53 delivery. These results show, that wild-type p53 potently inhibits expression of MMP-13 and MMP-1 by SCC cells independently of its pro-apoptotic effect. Together these results indicate, that p53 exerts a bi-phasic tumor suppressor effect on SCC cells: inhibition of cell invasion followed by induction of programmed cell death.

Topics: Adenoviridae; Apoptosis; Blotting, Western; Carcinoma, Squamous Cell; Cell Cycle Proteins; Cell Survival; Collagen; Collagenases; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cyclins; Drug Combinations; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Genes, p53; Humans; Laminin; Matrix Metalloproteinase 1; Matrix Metalloproteinase 13; Neoplasm Invasiveness; Proteoglycans; RNA, Messenger; Tissue Inhibitor of Metalloproteinase-1; Transforming Growth Factor alpha; Transforming Growth Factor beta; Transgenes; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Tumor Suppressor Proteins; 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

Up-regulation of the epidermal growth factor receptor (EGFR) is critical for the loss of growth control in a variety of human cancers, including squamous cell carcinoma of the head and neck (SCCHN). Stimulation of EGFR results in activation of mitogenic signaling pathways including Signal Transducers and Activators of Transcription (STATs). Stat5 activation has been primarily demonstrated in hematopoietic malignancies. Gene disruption studies suggest potentially distinct functions of the Stat5 isoforms, Stat5a and Stat5b, which are encoded by two genes closely linked on human chromosome 17. To determine the function of Stat5 in SCCHN growth control, we studied the expression and constitutive activation of Stat5a and Stat5b in normal and transformed human squamous epithelial cells. Increased constitutive activation of Stat5 was detected in transformed compared with normal squamous cells. Blockade of TGF-alpha or EGFR, abrogated Stat5 activation. Targeting of Stat5b using antisense oligonucleotides inhibited SCCHN growth. In addition, SCCHN cells stably transfected with dominant negative mutant Stat5b failed to proliferate in vitro. In contrast, targeting of Stat5a using either antisense or dominant negative strategies had no effect on cell growth. These results suggest that TGF-alpha/EGFR-mediated autocrine growth of transformed epithelial cells is dependent on activation of Stat5b but not Stat5a.

Topics: Carcinoma, Squamous Cell; Cell Division; Cell Line, Transformed; DNA-Binding Proteins; ErbB Receptors; Head and Neck Neoplasms; Humans; Milk Proteins; Protein Isoforms; Signal Transduction; STAT5 Transcription Factor; Trans-Activators; Transforming Growth Factor alpha; Tumor Cells, Cultured; Tumor Suppressor Proteins

Tamoxifen (TAM) is a well-tolerated compound in the treatment of breast cancer and is primarily considered to act by competition with estrogen receptors (ER). Here we investigated the in vitro efficacy and potentially underlying mechanisms of TAM in established cell lines of squamous cell carcinomas of the head and neck (SCCHN). Using proliferation and apoptosis assays the antitumor activity of TAM in five SCCHN and the breast carcinoma line MCF-7 (positive control) was determined. MCF-7 was more sensitive to low-dose TAM (below 1 microM), whereas SCCHN showed significant growth inhibition at higher TAM concentrations (5-10 microM). Growth curve analysis and apoptosis assays were indicative for a cytostatic effect of low-dose TAM and high-dose TAM led to cell loss by apoptosis in sensitive SCCHN. In order to further characterize the observed antitumor effects we determined the amount of steroid hormone receptors with the dextran-coated charcoal method and immunocytochemistry. In addition, production of transforming growth factor (TGF-)-alpha, -beta1 and -beta2 was measured by ELISA, and protein kinase C (PKC) activity was assessed with a radioligand assay. Except MCF-7, none of the SCCHN lines was positive for ER. TAM caused decreased TGF-alpha and increased TGF-beta levels in MCF-7, but not in SCCHN supernatants. Furthermore, the antiestrogen reduced PKC activity in MCF-7, but not in SCCHN. In the present in vitro system, the observed antitumor activity of high-dose TAM in SCCHN cannot be explained by estrogen antagonism, alterations of TGF-alpha/beta levels or decreased PKC activity.

Topics: Antineoplastic Agents, Hormonal; Apoptosis; Breast Neoplasms; Carcinoma, Squamous Cell; Cell Division; Down-Regulation; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation, Neoplastic; Head and Neck Neoplasms; Humans; Neoplasms, Hormone-Dependent; Protein Kinase C; Receptors, Estrogen; Tamoxifen; Transforming Growth Factor alpha; Transforming Growth Factor beta; Up-Regulation

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 objectives of the present study were to evaluate the expression of three proliferation markers, viz., epidermal growth factor receptor (EGFR), transforming growth factor-alpha (TGF-alpha) and proliferating cell nuclear antigen (PCNA) and one genomic marker, c-myc in OSMF. Oral tissues were stained with monoclonal antibodies to PCNA, c-myc, TGF-alpha and EGFR. The results were analyzed with Photoquant image analysis software. The expression of PCNA, c-myc, TGF-alpha and EGFR was higher in oral submucous fibrosis (OSMF) than in normal control oral mucosa. The oral epithelium was divided into a proliferative compartment (stratum germinativum) and a differentiated compartment (stratum spinosum). A differential pattern of expression of PCNA and c-myc was observed in OSMF. While the intensity of staining decreased, the percent area of expression of PCNA and c-myc increased in stratum germinativum in OSMF (P<0.05). This suggests that greater proportions of cells exhibit PCNA and c-myc immunoreactivity and are in the proliferative pool in OSMF. TGF-alpha levels increased in the proliferative layers and EGFR levels increased in the differentiated layers (P<0.05) of the oral epithelium in OSMF. Quantitative measurement of these oncoproteins substantiates the precancerous nature of OSMF and may provide intermediate end-points in prospective chemopreventive trials.

Topics: Adult; Analysis of Variance; Antibodies, Monoclonal; Biomarkers; Carcinoma, Squamous Cell; Case-Control Studies; Cell Division; Epithelial Cells; ErbB Receptors; Female; Genes, myc; Humans; Image Processing, Computer-Assisted; Male; Mouth Neoplasms; Oral Submucous Fibrosis; Proliferating Cell Nuclear Antigen; Transforming Growth Factor alpha

Clinical studies have demonstrated that retinoids, including retinol (Vitamin A) and its synthetic derivatives, can eradicate leukoplakia and suppress the formation of squamous cell carcinoma of the head and neck (SCCHN). Nonselective retinoids have been shown to abrogate transcriptional activation of transforming growth factor-alpha (TGF-alpha) and epidermal growth factor receptor (EGFR), which characterize SCCHN. LGD1069 (Targretin) is a potent RXR-selective retinoic acid agonist with a reduced toxicity profile compared with other nonselective retinoids. We examined the effect of LGD1069 (10 microm) on cellular proliferation and expression of putative intermediate biomarker genes including TGF-alpha, EGFR, and RAR-beta in seven SCCHN cell lines. A quantitative reverse transcription-PCR assay using a novel "primer dropping" method was used to determine expression levels of EGFR, TGF-alpha, and RAR-beta before and after treatment with LGD1069 (10 microM). SCCHN proliferation was reduced by a mean of 50% at 4 days in seven SCCHN cell lines after LGD1069 treatment (P < or = 0.05). EGFR expression levels were decreased by a mean of 58.4% (P = 0.007), TGF-alpha levels were decreased by a mean of 28.8% (P = 0.01), and RAR-beta levels were increased by a mean of 60% (P = 0.03). TGF-alpha stimulation of EGFR is associated with constitutive signal transducer and activator of transcription 3 (Stat3) activation in SCCHN. Abrogation of constitutive Stat3 activation was seen with LGD1069 treatment. These results suggest that an RXR-selective retinoic acid decreases SCCHN proliferation in part by interfering with TGF-alpha/EGFR autocrine signaling.

Topics: Animals; Anticarcinogenic Agents; Bexarotene; Biomarkers, Tumor; Carcinoma, Squamous Cell; Cell Division; DNA-Binding Proteins; ErbB Receptors; Female; Head and Neck Neoplasms; Humans; Mice; Mice, Nude; Receptors, Retinoic Acid; Retinoid X Receptors; Signal Transduction; STAT3 Transcription Factor; Tetrahydronaphthalenes; Trans-Activators; Transcription Factors; Transforming Growth Factor alpha; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

This study was designed to determine the prognostic value of immunohistochemical tumor marker expression in a population of patients with node-negative esophageal cancer treated with complete resection alone.. Resection specimens were collected from 61 patients with node-negative T1 (n = 31), T2 (n = 14), and T3 (n = 16) esophageal cancer. A panel of 10 tumor markers was chosen for immunohistochemical analysis, based on associations with differing oncologic mechanisms: apoptosis (p53), growth regulation (transforming growth factor-alpha, epidermal growth factor receptor, and Her2-neu), angiogenesis (factor VIII), metastatic potential (CD44), platinum resistance (p-glycoprotein and metallothionein), 5-fluorouracil resistance (thymidylate synthetase), and carcinogenic detoxification (glutathione S-transferase-pi).. Complete resection was performed in all patients (44 adenocarcinoma, 17 squamous cell carcinoma), with no operative deaths. Multivariable analysis demonstrated a significant relationship between cancer-specific death and the following variables: low-level P-gp expression (p = 0.004), high-level expression of p53 (p = 0.04), and low-level expression of transforming growth factor-alpha (p = 0.03). In addition, the number of involved tumor markers present was strongly predictive of negative outcome (p = 0.0001).. This study supports the prognostic value of immunohistochemical tumor markers, specifically the expression pattern of P-gp, p53, and transforming growth factor-alpha, in patients with esophageal carcinoma treated with complete resection alone.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; ATP Binding Cassette Transporter, Subfamily B, Member 1; Biomarkers, Tumor; Carcinoma, Squamous Cell; Esophageal Neoplasms; Female; Humans; Immunohistochemistry; Male; Middle Aged; Multivariate Analysis; Neoplasm Proteins; Predictive Value of Tests; Prognosis; Survival Analysis; Transforming Growth Factor alpha; Tumor Suppressor Protein p53

Epithelial growth factor receptor (EGFR) sends signals to the proliferation signal transduction system, receiving two ligands: epithelial growth factor (EGF) and transforming growth factor-alpha (TGF-alpha). This immunohistochemical study examined the roles of EGFR and its ligands in the proliferation of normal and neoplastic vulvar squamous cells in 25 patients with vulvar squamous cell carcinoma (VSCC), 10 patients with vulvar condyloma acuminata (VCA), 15 patients with vulvar intra-epithelial neoplasm I-II or III (VIN I-II or III), and 5 subjects with vulvar normal squamous cells (VNSC). EGFR was detected in a few basal cells in 40% of the VNSC, in highly dysplastic cells in 40% of the VIN III, in many neoplastic cells in 80% of the VCA, and in some malignant cells in 64% of the VSCC. EGF was seen in the cytoplasm in 20% of the VIN I-II, 100% of the VIN III, 100% of the VCA, and 100% of the VSCC. Diffuse TGF-alpha was weakly expressed in the cytoplasm in 100% of the VNSC, more intensely in 100% of the VIN and 100% of the VCA, and intensely in 100% of the VSCC. These findings led to the suggestion that the TGF-alpha-EGFR system maintains the growth of normal squamous cells and, in part, maintains the growth of dysplastic and neoplastic squamous cells in the vulva. EGF expression was an early sign of neoplasia. The expression of EGFR with overexpression of its two ligands contributed to the proliferation of dysplastic and neoplastic squamous cells in VIN III and VCA. EGFR expression appeared to contribute to essential neoplastic abnormalities in 64% of the VSCC.

Topics: Carcinoma in Situ; Carcinoma, Squamous Cell; Condylomata Acuminata; DNA, Viral; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Immunohistochemistry; Ligands; Papillomaviridae; Papillomavirus Infections; Transforming Growth Factor alpha; Tumor Virus Infections; Vulva; Vulvar Neoplasms

In rat liver epithelial cells constitutively expressing transforming growth factor alpha (TGFalpha), c-Met is constitutively phosphorylated in the absence of its ligand, hepatocyte growth factor. We proposed that TGFalpha and the autocrine activation of its receptor, epidermal growth factor receptor (EGFR), leads to phosphorylation and activation of c-Met. We found that there is constitutive c-Met phosphorylation in human hepatoma cell lines and the human epidermoid carcinoma cell line, A431 which express TGFalpha, but not in normal human hepatocytes. Constitutive c-Met phosphorylation in A431, HepG2, AKN-1, and HuH6 cells was inhibited by neutralizing antibodies against TGFalpha and/or EGFR. Exposure to exogenous TGFalpha or EGF increased the phosphorylation of c-Met in the human epidermoid carcinoma cell line, A431. The increase of c-Met phosphorylation by TGFalpha in A431 cells was inhibited by neutralizing antibodies against TGFalpha and/or EGFR and by the EGFR-specific inhibitor tyrphostin AG1478. These results indicate that constitutive c-Met phosphorylation, and the increase of c-Met phosphorylation by TGFalpha or EGF, in tumor cell lines is the result of the activation via EGFR. We found that c-Met in tumor cells co-immunoprecipitates with EGFR regardless of the existence of their ligands in tumor cells, but not in normal human hepatocytes. We conclude that c-Met associates with EGFR in tumor cells, and this association facilitates the phosphorylation of c-Met in the absence of hepatocyte growth factor. This cross-talk between c-Met and EGFR may have significant implications for altered growth control in tumorigenesis.

Topics: Animals; Carcinoma, Hepatocellular; Carcinoma, Squamous Cell; Cell Transformation, Neoplastic; Epidermal Growth Factor; ErbB Receptors; Hepatocyte Growth Factor; Humans; Liver Neoplasms; Phosphorylation; Protein Binding; Proto-Oncogene Proteins c-met; Quinazolines; Rats; Receptor Cross-Talk; Transforming Growth Factor alpha; Tumor Cells, Cultured; Tyrphostins

It has been demonstrated that vascular endothelial growth factor (VEGF) is associated with tumor progression as an angiogenic factor in esophageal squamous cell carcinoma (SCC)s. However, the role of other angiogenic factors such as transforming growth factor-alpha (TGF-alpha), platelet-derived endothelial cell growth factor (PD-ECGF), and basic fibroblast growth factor (bFGF) are still unknown in esophageal SCCs. In this study, we detected the expression of VEGF, TGF-alpha, PD-ECGF and bFGF in tissue specimens from 96 patients with SCC of the esophagus by immunohistochemical staining. To evaluate angiogenesis, endothelial cells were stained immunohistochemically and microvessel density (MVD) was counted in 24 cases. The positive rates for VEGF, TGF-alpha, PD-ECGF and bFGF were 65% (62/96), 67% (64/96), 66% (63/96), and 49% (47/96), respectively. Only TGF-alpha expression had a strong correlation with the average MVD (p=0.0059). However, the MVD increased as the number of positive factors for these 4 factors increased (p=0.0023). The expression of all of these factors significantly correlated to the depth of tumor invasion, and lymph node metastasis. Finally, survival analysis of the patients revealed that VEGF, TGF-alpha, and PD-ECGF were significant prognostic factors. However, multivariate analysis revealed that these factors were not prognostic. Thus, we suggest that TGF-alpha as well as VEGF, PD-ECGF and bFGF may be associated with angiogenesis, and the progression and metastasis of esophageal squamous cell carcinoma.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Squamous Cell; Endothelial Growth Factors; Esophageal Neoplasms; Female; Fibroblast Growth Factor 2; Gene Expression Regulation, Neoplastic; Humans; Lymphokines; Male; Middle Aged; Neoplasm Proteins; Neovascularization, Pathologic; Prognosis; Thymidine Phosphorylase; Transforming Growth Factor alpha; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

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

Like other types of pre-malignant lesions and carcinoma, angiogenesis is associated with high-grade cervical dysplasia and with invasive squamous carcinoma of the cervix. Vascular endothelial cell growth factor (VEGF) is known to be one of the most important inducers of angiogenesis and is upregulated in carcinoma of the cervix. Human Papilloma Virus 16 (HPV-16) has been etiologically linked to human cervical cancer, and the major oncogenic proteins encoded by the viral genome, E6 and E7, are involved in the immortalization of target cells. Because several oncogenes including mutant ras, EGF receptor, ErbB2/Her2, c-myc and v-src upregulate VEGF expression, we asked whether HVP-16 E6 oncoprotein could act in a similar fashion. We found that HPV-16 E6-positive cells generally express high levels of VEGF message. Furthermore, co-expression of the VEGF promoter-Luc (luciferase) reporter gene with E6 in both human keratinocytes and mouse fibroblast showed that E6 oncoprotein upregulates VEGF promoter activity, and does so in a p53 independent manner. An E6 responsive region which comprises four Sp-1 sites, between -194 and -50 bp of the VEGF promoter, is also necessary for constitutive VEGF transcription. Taken together, our results suggest the possibility that the HPV oncoprotein E6 may contribute to tumor angiogenesis by direct stimulation of the VEGF gene.

Topics: Autocrine Communication; Carcinoma, Squamous Cell; Endothelial Growth Factors; ErbB Receptors; Female; Genes, p53; HeLa Cells; Humans; Keratinocytes; Lymphokines; Neoplasm Proteins; Neovascularization, Pathologic; Oncogene Proteins, Viral; Papillomaviridae; Papillomavirus Infections; Promoter Regions, Genetic; Recombinant Fusion Proteins; Repressor Proteins; Transcription, Genetic; Transcriptional Activation; Transforming Growth Factor alpha; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Tumor Virus Infections; Uterine Cervical Neoplasms; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors; Vulvar Neoplasms

We recently reported that multiple c-erbB ligands differentially modulate in vitro proliferation, invasion and expression of matrix metalloproteinases in human head and neck squamous carcinoma cells (HNSCC). In order to evaluate further the importance of c-erbB ligands in tumor progression, the expression and regulation of this growth factor family in HNSCC cells was studied. We demonstrate that mRNAs for the 6 major c-erbB ligands, namely, epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), betacellulin (BTC), heparin-binding epidermal growth factor-like growth factor (HB-EGF), amphiregulin (AR) and heregulin (HRG), are expressed in a large panel of HNSCC cell lines. In addition to TGF-alpha, other ligands (notably BTC and HRG-beta1) are involved in the autocrine growth regulation of these cells. Each c-erbB ligand when applied exogenously, induced mRNA expression of both itself and the remaining family members and a differential response in the kinetics of induction was found. HB-EGF and HRG mRNAs were induced rapidly (within 1 hr) and to a greater extent (3.2-6.2- and 4.8-7. 3-fold increase) than TGF-alpha, BTC and AR mRNAs (1.6-2.7, 1.8-3.6- and 1.6-4.2-fold, respectively). This pattern was observed for all inducing ligands tested. Analysis of mRNA stability, and concurrent treatment with BTC (as an inducing ligand) and cycloheximide (to inhibit protein synthesis) suggested both transcriptional and post-transcriptional regulatory mechanisms. These results support and extend previous observations of c-erbB receptor signaling as a critical element in the pathogenesis and progression of HNSCC, and emphasize the role of autocrine ligand production.

Topics: Amphiregulin; Antibodies; Betacellulin; Carcinoma, Squamous Cell; Cell Division; EGF Family of Proteins; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Glycoproteins; Growth Substances; Head and Neck Neoplasms; Heparin-binding EGF-like Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Ligands; Neuregulin-1; Receptor Protein-Tyrosine Kinases; Receptor, ErbB-2; RNA, Messenger; Transforming Growth Factor alpha; Tumor Cells, Cultured

Dihydropyrimidine dehydrogenase (DPD) and pyrimidine nucleoside phosphorylase (PyNPase) are the first and rate-limiting enzymes that regulate 5-fluorouracil (5-FU) metabolism, and tumoral DPD activity appears to be a promising predictor of 5-FU sensitivity. However, the regulatory mechanisms determining these enzyme activities have not been fully understood. We investigated the biological effects of epidermal growth factor (EGF) and transforming growth factor (TGF)-alpha on cell growth and tumoral DPD and PyNPase activities, and the subsequent effects on 5-FU sensitivity in uterine cervical carcinoma SKG-IIIb cells. The treatment of tumor cells with EGF or TGF-alpha resulted in a concentration-dependent increase in tumor cell growth and PyNPase activity, whereas tumoral DPD activity was inhibited. Their stimulatory effects on tumor cell growth correlated well with PyNPase activity, but were inversely related to DPD activity (P < 0.01). 5-FU sensitivity of tumor cells increased in the presence of EGF or TGF-alpha. These growth factors were shown to stimulate the first, rate-limiting enzyme activity in 5-FU anabolism and to inhibit that in 5-FU catabolism, leading to enhancement of the antiproliferative action of 5-FU at achievable therapeutic levels. The tumor environmental factors, EGF and TGF-alpha, may act as intrinsic regulators of DPD and PyNPase activities that affect the 5-FU sensitivity of individual tumors.

Topics: Antimetabolites, Antineoplastic; Carcinoma, Squamous Cell; Cell Division; Dihydrouracil Dehydrogenase (NADP); Drug Synergism; Epidermal Growth Factor; Female; Fluorouracil; Humans; Oxidoreductases; Pentosyltransferases; Pyrimidine Phosphorylases; Transforming Growth Factor alpha; Tumor Cells, Cultured; Uterine Cervical Neoplasms

Unlike normal mucosal squamous epithelial cells, head and neck squamous cell carcinomas (HNSCCs) overexpress TGF-alpha mRNA and protein which is required to sustain the proliferation of HNSCC cells in vitro. To determine whether TGF-alpha expression contributes to tumor growth in vivo, cationic liposome-mediated gene transfer was used to deliver an antisense expression construct targeting the human TGF-alpha gene into human head and neck tumor cells, grown as subcutaneous xenografts in nude mice. The TGF-alpha antisense gene was immediately detected in the cytoplasm of the tumor cells, translocated to the nucleus by 12 h and remained localized to the nucleus for up to 3 days. Direct inoculation of the TGF-alpha antisense (but not the corresponding sense) construct into established HNSCC tumors resulted in inhibition of tumor growth. Sustained antitumor effects were observed for up to 1 year after the treatments were discontinued. Down-modulation of TGF-alpha was accompanied by increased apoptosis in vivo. These experiments indicate that interference with the TGF-alpha/EGFR autocrine signaling pathway may be an effective therapeutic strategy for cancers which overexpress this ligand/receptor pair.

Topics: Animals; Apoptosis; Carcinoma, Squamous Cell; Cell Division; Cell Line; Gene Expression; Genetic Therapy; Head and Neck Neoplasms; Humans; Mice; Mice, Nude; Microscopy, Fluorescence; Neoplasms, Experimental; Oligonucleotides, Antisense; Random Allocation; Transfection; Transforming Growth Factor alpha; Xenograft Model Antitumor Assays

Transforming growth factor-alpha (TGF-alpha) increased the expression of 12-lipoxygenase activity in a time-dependent manner in human epidermoid carcinoma A431 cells. The increase of 12-lipoxygenase activity was accompanied by an increase in 12-lipoxygenase mRNA. The effect of TGF-alpha on the promoter activation of 12-lipoxygenase gene was analyzed by using the luciferase fusion vectors. A dose-dependent effect of TGF-alpha on the reporter activity was observed, which paralleled with its effect on enzyme activity. Transient transfection with a series of 5'-deleted constructs showed that the 5'-flanking region spanning from -224 to -100 bp from translation starting site played an important role for TGF-alpha response. Site-directed mutagenesis and gel mobility shift assay indicated that two Sp1 binding sequences residing at -158 to -150 bp and 123 to -114 bp were responsible for the TGF-alpha in activation of human 12-lipoxygenase gene transcription. Expression of Sp1, but not Sp3, stimulated the promoter activity of 12-lipoxygenase in SL2 cells, indicating that the binding of Sp1 with Sp1 binding sequences played a significant role in the regulation of 12-lipoxygenase gene.

Topics: Arachidonate 12-Lipoxygenase; Base Sequence; Carcinoma, Squamous Cell; DNA; Gene Expression Regulation, Enzymologic; Humans; Molecular Sequence Data; Mutagenesis, Site-Directed; Promoter Regions, Genetic; Sp1 Transcription Factor; Transforming Growth Factor alpha; Tumor Cells, Cultured

Exposure of A431 squamous and MDA-MB-231 mammary carcinoma cells to ionizing radiation has been associated with short transient increases in epidermal growth factor receptor (EGFR) tyrosine phosphorylation and activation of the mitogen-activated protein kinase (MAPK) and c-Jun NH(2)-terminal kinase (JNK) pathways. Irradiation (2 Gy) of A431 and MDA-MB-231 cells caused immediate primary activations (0-10 min) of the EGFR and the MAPK and JNK pathways, which were surprisingly followed by later prolonged secondary activations (90-240 min). Primary and secondary activation of the EGFR was abolished by molecular inhibition of EGFR function. The primary and secondary activation of the MAPK pathway was abolished by molecular inhibition of either EGFR or Ras function. In contrast, molecular inhibition of EGFR function abolished the secondary but not the primary activation of the JNK pathway. Inhibition of tumor necrosis factor alpha receptor function by use of neutralizing monoclonal antibodies blunted primary activation of the JNK pathway. Addition of a neutralizing monoclonal antibody versus transforming growth factor alpha (TGFalpha) had no effect on the primary activation of either the EGFR or the MAPK and JNK pathways after irradiation but abolished the secondary activation of EGFR, MAPK, and JNK. Irradiation of cells increased pro-TGFalpha cleavage 120-180 min after exposure. In agreement with radiation-induced release of a soluble factor, activation of the EGFR and the MAPK and JNK pathways could be induced in nonirradiated cells by the transfer of media from irradiated cells 120 min after irradiation. The ability of the transferred media to cause MAPK and JNK activation was blocked when media were incubated with a neutralizing antibody to TGFalpha. Thus radiation causes primary and secondary activation of the EGFR and the MAPK and JNK pathways in autocrine-regulated carcinoma cells. Secondary activation of the EGFR and the MAPK and JNK pathways is dependent on radiation-induced cleavage and autocrine action of TGFalpha. Neutralization of TGFalpha function by an anti-TGFalpha antibody or inhibition of MAPK function by MEK1/2 inhibitors (PD98059 and U0126) radiosensitized A431 and MDA-MB-231 cells after irradiation in apoptosis, 3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), and clonogenic assays. These data demonstrate that disruption of the TGFalpha-EGFR-MAPK signaling module represents a strategy to decrease carcinoma cell growth an

Topics: Antibodies; Breast Neoplasms; Butadienes; Calcium-Calmodulin-Dependent Protein Kinases; Carcinoma; Carcinoma, Squamous Cell; Cell Death; Cell Division; DNA, Antisense; Dose-Response Relationship, Radiation; Enzyme Activation; Enzyme Inhibitors; ErbB Receptors; Humans; JNK Mitogen-Activated Protein Kinases; MAP Kinase Kinase 4; Mitogen-Activated Protein Kinase Kinases; Nitriles; Phosphorylation; Protein Kinases; Receptors, Tumor Necrosis Factor; Recombinant Proteins; Transforming Growth Factor alpha; Tumor Cells, Cultured; Tyrosine

Surrogate endpoint biomarkers (SEBs) are detectable molecular, cellular, and tissue changes that take place during tumorigenesis and can be modulated by a chemoprevention agent.. To identify candidate SEBs for invasive squamous cell carcinoma of the upper aerodigestive tract (SCC), we have studied the expression of transforming growth factor-alpha (TGF-alpha) and epidermal growth factor receptor (EGFr) in sequential biopsy specimens of dysplastic oral leukoplakia and adjacent normal-appearing mucosa. Biopsies were taken from patients before, during, and after treatment with 13-cis retinoic acid, a vitamin A derivative. Immunohistochemistry was performed using the Biogenex Super Sensitive Biotin-Streptavidin horseradish peroxidase detection system.. The pretreatment expression of TGF-alpha and EGFr in dysplastic oral leukoplakia was increased when compared with their expression in adjacent normal-appearing mucosa (p = 0.001 and p = 0.01, respectively). Eleven of 14 patients enrolled in the study (78.6%) completed 3 months of treatment with 13-cis retinoic acid (1. 0 mg/kg/day). TGF-alpha expression in dysplastic oral leukoplakia, but not in adjacent normal-appearing mucosa, decreased during treatment (p < 0.05).. TGF-alpha is a candidate SEB for future SCC chemoprevention trials.

Topics: Antineoplastic Agents; Biomarkers, Tumor; Biopsy; Carcinoma, Squamous Cell; Chemoprevention; ErbB Receptors; Follow-Up Studies; Gene Expression Regulation, Neoplastic; Humans; Immunoenzyme Techniques; Immunohistochemistry; Isotretinoin; Keratolytic Agents; Leukoplakia, Oral; Mouth Mucosa; Pilot Projects; Single-Blind Method; Transforming Growth Factor alpha

Invasive potentials of malignant cancer cells are regulated by cell motility factors. To examine the regulation of motility and invasiveness in oral squamous carcinoma, we investigated autocrine- and/or paracrine-acting cell motility factors, using a newly established human cell line (IF cells) from oral squamous cell carcinoma, which has highly invasive and metastatic characteristics. Conditioned medium derived from IF cells stimulated cell scattering and migration of GB-d1 gallbladder carcinoma cells, indicating that IF cells secreted cell motility factors. Using antibodies, IF-derived cell motility factors proved to be transforming growth factor (TGF)-alpha and TGF-beta1. Antibodies against TGF-alpha and TGF-beta1 inhibited autonomous migration of the IF cells. On the other hand, in vitro invasion of IF cells was strongly enhanced by hepatocyte growth factor (HGF) but only slightly by TGF-alpha and TGF-beta1. The conditioned medium from fibroblasts enhanced in vitro invasion of IF cells, an event abrogated by anti-HGF antibody, but not by antibodies against TGF-alpha and TGF-beta1. Importantly, IF cells secreted a factor inducing HGF production in fibroblasts and the factor was identified as interleukin-1, which means that a mutual interaction exists between tumour cells and fibroblasts, as mediated by the HGF/HGF-inducer loop. These results indicate that IF cells utilize TGF-alpha and TGF-beta1 as autocrine-acting motility factors and HGF as a paracrine-acting motility factor, and that invasiveness of IF cells is particularly stimulated by HGF derived from stromal fibroblasts. Utilization of multiple cell motility/invasion factors that act in distinct pathways may confer highly invasive and metastatic potentials in IF oral squamous carcinoma cells.

Topics: Carcinoma, Squamous Cell; Cell Division; Cell Movement; Cells, Cultured; Culture Media, Conditioned; Fibroblasts; Gallbladder Neoplasms; Gingival Neoplasms; Hepatocyte Growth Factor; Humans; Models, Biological; Mouth Neoplasms; Neoplasm Invasiveness; Skin; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured

Because regional spread to lymph nodes without systemic spread is a relatively common event in squamous cell cancer of the head and neck (SCCHN), it is possible that lymphoid-related receptors or cytokines might directly impact the growth of these tumors. In the present study, we have shown by flow cytometry and Western blotting that the central lymphoid regulatory molecule, CD40, is expressed on the surface of all seven SCCHN tumor cell lines studied. Tumor cell lines also expressed epidermal growth factor (EGF) receptor, MHC class I, and CD95 (Fas) but did not uniformly express other important lymphoid regulatory molecules such as CD80, CD86, or interleukin (IL) 2 receptor components. CD40 ligation by trimeric CD40 ligand (CD40L) resulted in a 20-45% inhibition of tumor cell growth in three of seven cell lines tested. The cytokines IL-1alpha, IL-1beta, IL-2, IL-4, IL-6, IL-10, IL-11, and IL-15 neither inhibited nor stimulated growth in any of the cell lines tested. EGF had pleiotropic effects on cell growth; it inhibited growth in two cell lines, stimulated growth in one cell line, and had no effect in four cell lines. When coligation by EGF and CD40L was studied, additive or supra-additive growth inhibition was seen in four cell lines. Three cell lines were unaffected by EGF, CD40, or coligation with both reagents. Examination of tumor tissues from 12 previously untreated patients representing a broad spectrum of patients presenting with SCCHN demonstrated CD40 expression in all 12 tumor specimens. This study supports the notion that CD40 is a regulatory molecule for the growth of SCCHN. The important role of CD40-CD40L interactions in the regulation of immune cells in the lymph node and the unique high-level expression of CD40L by these immune cells lend support to the hypothesis that this ligand/receptor pair is an important mediator of cell growth in SCCHN.

Topics: Antibodies, Monoclonal; Blotting, Western; Carcinoma, Squamous Cell; CD40 Antigens; CD40 Ligand; Cell Division; Cell Membrane; Cytokines; Dose-Response Relationship, Drug; Epidermal Growth Factor; Flow Cytometry; Head and Neck Neoplasms; Humans; Immunohistochemistry; Immunophenotyping; Membrane Glycoproteins; Receptors, Cell Surface; Transforming Growth Factor alpha; Tumor Cells, Cultured

Treatment of normal human epidermal keratinocytes (NHEK) with interferon-gamma (IFN-gamma) causes a 9-fold increase in the level of cyclooxygenase-2 (COX-2) mRNA expression. Nuclear run-off assays indicate that this induction is at least partly due to increased transcription. Activation of the epidermal growth factor receptor (EGFR) signaling pathway due to the enhanced transforming growth factor alpha (TGFalpha) expression plays an important role in the induction of COX-2 by IFN-gamma. This is supported by the ability of TGFalpha to rapidly induce COX-2 and the inhibition of the IFN-gamma-mediated COX-2 mRNA induction by an EGFR antibody and EGFR-selective kinase inhibitors. Deletion and mutation analysis indicates the importance of the proximal cAMP-response element/ATF site in the transcriptional control of this gene by TGFalpha. The increase in COX-2 mRNA by TGFalpha requires activation of both the extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) pathways. Inhibition of p38 MAPK decreases the stability of COX-2 mRNA, while inhibition of MAPK/ERK kinase (MEK) does not. These results suggest that the p38 MAPK signaling pathway controls COX-2 at the level of mRNA stability, while the ERK signaling pathway regulates COX-2 at the level of transcription. In contrast to NHEK, IFN-gamma and TGFalpha are not very effective in inducing TGFalpha or COX-2 expression in several squamous carcinoma cell lines, indicating alterations in both IFN-gamma and TGFalpha response pathways.

Topics: Amphiregulin; Carcinoma, Squamous Cell; Cyclooxygenase 1; Cyclooxygenase 2; Dinoprostone; EGF Family of Proteins; Eicosanoids; Enzyme Inhibitors; ErbB Receptors; Flavonoids; Gene Expression Regulation, Enzymologic; Glycoproteins; Growth Substances; Humans; Intercellular Signaling Peptides and Proteins; Interferon-gamma; Isoenzymes; Keratinocytes; Membrane Proteins; Mitogen-Activated Protein Kinases; Promoter Regions, Genetic; Prostaglandin-Endoperoxide Synthases; Regulatory Sequences, Nucleic Acid; RNA, Messenger; Signal Transduction; Transforming Growth Factor alpha; Tumor Cells, Cultured

The previously reported streptavidin-TGFalpha chimeric protein-based delivery system (Ohno and Meruelo, DNA Cell Biol. 15:401-406, 1996) could efficiently transfer protein molecules into A431 cells via the epidermal growth factor (EGF) receptor. We have modified this delivery system for the transfer of DNA. For this purpose, we have linked the chimeric protein ST-TGFalpha to DNA through biotinylated polylysine molecules. We show with this system, in the presence of the endosome-destabilizing reagent chloroquine, an average of 50-fold increase in reporter gene expression in comparison with polylysine DNA complexes alone. This gene expression is specific for EGF receptor-expressing cells and is blocked by EGF-binding molecules. These results suggest that the ST-TGFalpha biotinylated polylysine system could be used to deliver DNA to targeted cells.

Topics: Animals; Biotinylation; Carcinoma, Squamous Cell; Cell Line, Transformed; Chlorocebus aethiops; Chloroquine; COS Cells; DNA, Recombinant; Drug Carriers; Endocytosis; Endosomes; ErbB Receptors; Gene Expression Regulation; Genes, Reporter; Humans; Luciferases; Neoplasm Proteins; Polylysine; Recombinant Fusion Proteins; Simian virus 40; Streptavidin; Transfection; Transforming Growth Factor alpha; Tumor Cells, Cultured

We investigated the effects of epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) on migration, invasion and proteinase expression of gynecological cultured cancer cells (SKG-IIIb cervical squamous cell carcinoma, OMC-4 cervical adenocarcinoma, SNG-M endometrial adenocarcinoma and OMC-3 ovarian adenocarcinoma), and whether these growth factors affect thymidine phosphorylase/platelet-derived endothelial cell growth factor expression of tumor cells. Tumor cell migration along a gradient of substratum-bound fibronectin and invasion into reconstituted basement membrane were stimulated by 0.1-10 nM EGF and TGF-alpha in a concentration-dependent manner. The zymography of tumor-conditioned medium showed that the treatment of tumor cells with EGF and TGF-alpha resulted in the increase of type IV collagenases, stromelysin and urokinase-type plasminogen activator which was partly confirmed by immunoblot analysis. The expression of thymidine phosphorylase/platelet-derived endothelial cell growth factor which has angiogenic activity, was also upregulated by these growth factors. These results suggest that EGF and TGF-alpha act as positive regulators on the invasion process of gynecological tumor cells which may be associated with their stimulatory action on the motility of tumor cells, the expression of proteinases secreted by tumor cells and the angiogenic phenotype.

Topics: Adenocarcinoma; Carcinoma, Squamous Cell; Cell Movement; Endometrial Neoplasms; Epidermal Growth Factor; Female; Genital Neoplasms, Female; Humans; Metalloendopeptidases; Neoplasm Invasiveness; Ovarian Neoplasms; Thymidine Phosphorylase; Transforming Growth Factor alpha; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator; Uterine Cervical Neoplasms

Using immunohistochemistry, expression of p53, transforming growth factor-alpha (TGF-alpha), epidermal growth factor receptor (EGFR), c-erbB-2/neu and proliferating cell nuclear antigen (PCNA) was examined in 26 fresh frozen tissue specimens of oropharyngeal squamous cell carcinomas (SCCs). p53 gene mutations were examined by polymerase chain reaction (PCR)/DNA sequencing methods in 22 carcinomas. The findings were examined for correlations with patients' clinicopathological parameters. Expressions of p53 and PCNA were also examined in 21 formalin-fixed corresponding tissues. Of the fresh frozen tissue specimens, 77% (20/26) showed expression and 68% (15/22) showed mutations (substitutions) of the p53, with significant clustering of the mutations in exons 5 (8/22; 36%), 7 (4/22; 18%) and 8 (5/22; 23%). No mutations were found in exon 6. There was a discordance between expression of p53 protein and mutations of the gene. Parallel to expression and mutations of the p53 found in most of the specimens, expression of TGF-alpha, EGFR, c-erbB-2/neu and PCNA was found in 88% (22/25), 92% (23/25), 58% (14/24) and 91% (21/23) of the specimens, respectively. For the formalin-fixed tissue specimens, 62% (13/21) and 90% (19/21) expressed p53 and PCNA, respectively. Examining for correlations with patients' clinicopathological parameters, expression of p53, TGF-alpha, EGFR and c-erB-2/neu seemed to negatively correlate with the increase of the tumour grade. The present work suggests that: (1) lack of negative growth regulation due to inactivation of the p53 gene together with activation of other proto-oncogenes are necessary genetic events in the carcinogenesis of oropharyngeal SCCs; (2) in oropharyngeal SCCs, p53 gene mutations were clustered in exons 5 (codons 130-186), 7 (codons 230-248) and 8 (codons 271-282) which perhaps suggests that tobacco carcinogens probably affect the mutational hot spots of the p53 gene at codons 157, 175, 186, 248, 273 and 282; and (3) fresh frozen and formalin-fixed tissue specimens give similar results when an immunohistochemical method is applied. The importance of p53, TGF-alpha, EGFR, c-erbB-2/neu and PCNA as biomarkers in oropharyngeal SCCs deserves particular attention because it might offer further understanding of the development of these carcinomas.

Topics: Adult; Aged; Aged, 80 and over; Alcohol Drinking; Biomarkers, Tumor; Carcinoma, Squamous Cell; DNA, Neoplasm; ErbB Receptors; Female; Humans; Immunohistochemistry; Male; Middle Aged; Mutation; Oropharyngeal Neoplasms; Polymerase Chain Reaction; Proliferating Cell Nuclear Antigen; Receptor, ErbB-2; Sequence Analysis, DNA; Smoking; Transforming Growth Factor alpha; Tumor Suppressor Protein p53

To elucidate the stimulating role of the growth factor autocrine loop in the carcinogenesis and development of laryngeal carcinomas.. The reverse transcription-polymerase chain reaction technique was applied, and with beta-actin as internal standard, TGF-alpha and EGFR mRNA in laryngeal carcinomas, macroscopically normal laryngeal mucosas adjacent to the tumor and in vocal cord polyps were quantitatively examined.. The mRNA index of both TGF-alpha and EGFR in laryngeal carcinomas and adjacent mucosas was significantly higher than those in vocal cord polyps (P < 0.05). The index of TGF-alpha mRNA was significantly higher in laryngeal carcinomas than in mucosas adjacent to the tumor (P < 0.05).. The autocrine system consisting of TGF-alpha and EGFR may play an important role in the very early stage of laryngeal carcinoma, or even when the tissue phenotypic alteration does not occur. The elevated TGF-alpha mRNA level may trigger a switch from genotypic alteration to the phenotypic. The results are expected to be a theoretical basis for the preventive chemotherapy of laryngeal carcinomas.

Topics: Carcinoma, Squamous Cell; ErbB Receptors; Humans; Laryngeal Neoplasms; RNA, Messenger; Transforming Growth Factor alpha

Interruption of an autocrine growth pathway involving TGF-alpha and EGFR may inhibit tumor growth and improve survival in head and neck cancer patients. We previously demonstrated that biopsy specimens and established cell lines from patients with squamous cell carcinoma of the head and neck (SCCHN) overexpress TGF-alpha and its receptor, epidermal growth factor receptor (EGFR) at both the mRNA and protein levels. Protein localization studies showed that TGF-alpha and EGFR are produced by the same epithelial cells in tissues from head and neck cancer patients further supporting a role for this ligand-receptor pair in an autocrine growth pathway. To confirm that TGF-alpha contributes to autocrine growth, we examined the effect of down regulation of TGF-alpha protein on SCCHN cell proliferation. Treatment of 6 SCCHN cell lines with antisense oligodeoxynucleotides targeting the translation start site of human TGF-alpha mRNA decreased TGF-alpha protein production by up to 93% and reduced cell proliferation by a mean of 76.2% compared to a 9.7% reduction with sense oligonucleotide (range P = 0.036-0.0001). TGF-alpha antisense oligonucleotide exposure also decreased TGF-alpha protein levels in normal oropharyngeal mucosal epithelial cells, however their growth rate was not affected. These findings indicate that TGF-alpha is participating in an autocrine signaling pathway in transformed, but not in normal mucosal epithelial cells, that promotes proliferation.

Topics: Base Sequence; Carcinoma, Squamous Cell; Cell Division; Cell Transformation, Neoplastic; Down-Regulation; Epithelial Cells; Head and Neck Neoplasms; Humans; Mucous Membrane; Oligonucleotides, Antisense; Signal Transduction; Transforming Growth Factor alpha; Tumor Cells, Cultured

The development of head and neck squamous cell carcinoma occurs as a result of the accumulation of genotypic and phenotypic alterations in the upper aerodigestive tract mucosa. Up-regulation of epidermal growth factor receptor (EGFR) and its ligand, transforming growth factor alpha (TGF-alpha), have been identified previously as early events in head and neck carcinogenesis. To determine the timing of increased TGF-alpha and EGFR protein expression in the development of head and neck cancer, we examined progressive mucosal dysplasias from three distinct and complimentary patient groups: (a) samples from patients with lesions demonstrating different degrees of dysplasia (n = 22) compared with mucosa samples from gender and age-matched controls (n = 8); (b) patients with lesions demonstrating different degrees of dysplasia at a single time point (n = 3); and (c) patients who progressed over several years to invasive cancer at the site of dysplasia (n = 7). Immunohistochemical analysis with monoclonal antibodies specific for TGF-alpha and EGFR were used to detect protein expression in all specimens. Protein levels were further quantitated using a computerized image analysis system. In all three groups, we found that TGF-alpha protein levels were elevated in mild dysplasia compared with control normal mucosa and were not further modulated with increasing degrees of dysplasia. In contrast, EGFR levels were relatively low in mild dysplasia and increased with higher degrees of dysplasia. These findings indicate that up-regulation of TGF-alpha and EGFR are distinct events both chronologically and, possibly, mechanistically in the pathogenesis of head and neck squamous cell carcinoma.

Topics: Carcinoma, Squamous Cell; ErbB Receptors; Female; Head and Neck Neoplasms; Humans; Immunohistochemistry; Male; Precancerous Conditions; Transforming Growth Factor alpha

The inhibitory effect of human epidermoid cancer cells A431 caused by conjugate toxin containing transforming growth factor (TGF-alpha) and ricin A was studied. TGF-alpha is a protein with 50 amino acids that specifically binds and stimulates phosphorylation of cell surface epidermal growth factor receptor (EGFR) and, subsequently, triggers cell proliferation. TGF-alpha as a ligand for EGFR is internalized upon binding and decomposed within lysosome. Lectin ricin is contained in the castor bean plant. The lectin consists of two subunits, ricin A and B. Toxic ricin A binds to ribosome and inhibits protein synthesis of target cells. In view of the abundance of EGFR in human cancer cells, the receptor-mediated endocytosis with the conjugate toxin composed of TGF-alpha and ricin A was synthesized, purified and tested for growth inhibition in both normal and tumor cells. The cytotoxicity of the conjugate was studied within the range of 10(-12) and 10(-8) M and IC50 was found to be 10(-10) M for human A431 epidermoid cells that over-express EGFR. Compared to A431 cells, the brain metastatic variant of human Non-Small Cell Lung Cancer (NSCLC) H226Br squamous cells showed a reduced inhibitory effect. On the other hand, no inhibitory effect was found with other NSCLC cells studied and normal human lung cells because of the fewer available EGF binding sites on the surface of the cells. These results indicated that the amount of the available EGFR contributes to the cytotoxic effect on human cancer cells, thereby demonstrating involvement of the receptor-mediated endocytosis of the conjugate. The result from 12labeled EGF-mediated competition assay further demonstrated the specific inhibition activity conferred by TGF-alpha and ricin A conjugation. Due to poor recovery of the chemical conjugation, modification in the form of a recombinant toxin is needed for further in-depth studies.

Topics: Affinity Labels; Antineoplastic Agents; Carcinoma, Squamous Cell; Cell Division; Disulfides; ErbB Receptors; Humans; Recombinant Fusion Proteins; Ribosomes; Ricin; Transforming Growth Factor alpha; Tumor Cells, Cultured

The most accurate predictor of disease recurrence in patients treated for head and neck squamous cell carcinoma is, at present, the extent of regional lymph node metastasis. Since elevated levels of epidermal growth factor receptor (EGFR) and of its ligand, transforming growth factor-alpha (TGF-alpha), have been detected in primary tumors of patients with head and neck squamous cell carcinoma, we determined whether tumor levels of these proteins were of prognostic importance.. Monoclonal antibodies specific for EGFR and TGF-alpha were used for immunohistochemical detection of each protein in tissue sections of primary tumors from 91 patients who were treated by surgical resection. Levels of immunoreactive EGFR and TGF-alpha were quantified by use of a computerized image analysis system and were normalized to appropriate standards. The logrank test and proportional hazards regression analysis were used to calculate the probability that EGFR and TGF-alpha levels were associated with disease-free survival (i.e., no recurrence of cancer) and cause-specific survival (i.e., patients do not die of their disease). All P values were two-sided.. When tumor levels of EGFR or TGF-alpha were analyzed as continuous variables, disease-free survival and cause-specific survival were reduced among patients with higher levels of EGFR (both P = .0001) or TGF-alpha (both P = .0001). In a multivariate analysis, tumor site, tumor level of EGFR, and tumor level of TGF-alpha were statistically significant predictors of disease-free survival; in a similar analysis, regional lymph node stage and tumor levels of EGFR and of TGF-alpha were significant predictors of cause-specific survival.. Quantitation of EGFR and TGF-alpha protein levels in primary head and neck squamous cell carcinomas may be useful in identifying subgroups of patients at high risk of tumor recurrence and in guiding therapy.

Topics: Adult; Aged; Antibodies, Monoclonal; Biomarkers, Tumor; Carcinoma, Squamous Cell; Disease-Free Survival; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Head and Neck Neoplasms; Humans; Immunohistochemistry; Life Tables; Lymphatic Metastasis; Male; Middle Aged; Neoplasm Invasiveness; Neoplasm Proteins; Neoplasm Recurrence, Local; Proportional Hazards Models; Retrospective Studies; Survival Analysis; Transforming Growth Factor alpha

Multiple epidermal growth factor receptor (EGFr) ligands have been identified, including transforming growth factor alpha (TGFalpha), heparin-binding epidermal growth factor (HB-EGF), amphiregulin (AR), and betacellulin (BTC). Previous work from our laboratory demonstrated that TGFalpha mRNA and protein are upregulated in epidermis during tumor-promoter treatment of mouse skin and in skin tumors produced by initiation-promotion regimens. The purpose of the study described here was to explore the role of other EGFr ligands in multistage skin carcinogenesis. A single topical treatment of either 12-O-tetradecanoylphorbol-13-acetate (TPA) or chrysarobin or a single full-thickness wound induced the expression of HB-EGF and AR in mRNA samples isolated from whole mouse skin. However, only full-thickness wounding significantly elevated expression of the BTC transcript. The levels of HB-EGF and AR transcripts were significantly elevated in skin tumors (both papillomas and squamous cell carcinomas) induced by initiation-promotion protocols. BTC transcript levels were low and barely detectable in all skin tumors examined. The level of keratinocyte growth factor (KGF) mRNA was also examined as a possible mechanism for upregulation of EGFr ligands. Only full-thickness wounding significantly elevated KGF transcript levels in whole-skin RNA samples. Furthermore, no evidence for upregulation of KGF mRNA in skin tumors was obtained. The results are discussed in terms of the role of EGFr activation in skin carcinogenesis and the mechanisms for altered regulation of EGFr ligands.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Amphiregulin; Animals; Betacellulin; Carcinogens; Carcinoma, Squamous Cell; EGF Family of Proteins; Epidermal Growth Factor; ErbB Receptors; Female; Fibroblast Growth Factor 10; Fibroblast Growth Factor 7; Fibroblast Growth Factors; Fluorescent Antibody Technique; Gene Expression Regulation, Neoplastic; Glycoproteins; Growth Substances; Heparin-binding EGF-like Growth Factor; Intercellular Signaling Peptides and Proteins; Ligands; Mice; Mice, Inbred SENCAR; Papilloma; RNA, Messenger; Skin; Skin Neoplasms; Transforming Growth Factor alpha; Up-Regulation

During the last decade the key role of antimicrobial peptides in innate immunity has been argued. They were found in plants and in different phylogenic groups of animals (insects, amphibia, and even in mammals). We report the production of a human peptide antibiotic that was previously characterized as an EGF receptor tyrosine kinase inhibitor in epidermoid carcinoma A431/1522 cell subline overexpressing TGF alpha. It is a 3 kDa hydrophobic cationic peptide cytotoxic for different species of Gr+ and Gr- bacteria in micromolar concentration range, and demonstrating slight fungicidal activity.

Topics: Anti-Bacterial Agents; Carcinoma, Squamous Cell; Cell Line; Chromatography, High Pressure Liquid; Electrophoresis, Polyacrylamide Gel; Gram-Negative Bacteria; Gram-Positive Bacteria; Humans; Protein Kinase Inhibitors; Transforming Growth Factor alpha

Strategies to sensitize human tumors that are resistant to apoptosis have been clinically unsuccessful. We demonstrate that a structurally modified chimeric Pseudomonas exotoxin, PEdelta53L/TGF-alpha/KDEL, with binding specificity for the epidermal growth factor receptor, markedly enhances sensitivity of human xenografts to radiation killing. Exposure to PEdelta53L/TGF-alpha/KDEL decreases the apoptotic threshold through protein synthesis inhibition and simultaneous production of ceramide in tumor cells that lack functional p53 protein. In contrast, no increase in local or systemic toxicity was observed with the chimeric toxin and radiation. We conclude that biochemical targeting of the chimeric toxin and physical targeting of ionizing radiation may increase the therapeutic ratio in the treatment of human cancers with alterations of p53 expression. This strategy offers a high therapeutic potential for Pseudomonas exotoxin A chimeric proteins and irradiation.

Topics: ADP Ribose Transferases; Animals; Apoptosis; Bacterial Toxins; Binding Sites; Carcinoma, Squamous Cell; Ceramides; Combined Modality Therapy; Drug Synergism; ErbB Receptors; Exotoxins; Female; Head and Neck Neoplasms; Humans; Mice; Mice, Nude; Mutation; Neoplasm Transplantation; Oligopeptides; Protein Sorting Signals; Pseudomonas aeruginosa Exotoxin A; Radiation Tolerance; Recombinant Fusion Proteins; Transforming Growth Factor alpha; Transplantation, Heterologous; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Virulence Factors

Stimulation of epidermal growth factor receptor (EGFR) by ligand(s) leads to activation of signaling molecules including Stat1 and Stat3, two members of the signal transducers and activators of transcription (STAT) protein family. Activation of Stat1 and Stat3 was constitutive in transformed squamous epithelial cells, which produce elevated levels of TGF-alpha, and was enhanced by the addition of exogenous TGF-alpha. Targeting of Stat3 using antisense oligonucleotides directed against the translation initiation site, resulted in significant growth inhibition. In addition, cells stably transfected with dominant negative mutant Stat3 constructs failed to proliferate in vitro. In contrast, targeting of Stat1 using either antisense or dominant-negative strategies had no effect on cell growth. Thus, TGF-alpha/EGFR-mediated autocrine growth of transformed epithelial cells is dependent on activation of Stat3 but not Stat1.

Topics: Carcinoma, Hepatocellular; Carcinoma, Squamous Cell; Cell Division; Cells, Cultured; DNA-Binding Proteins; Epithelial Cells; ErbB Receptors; Head and Neck Neoplasms; Humans; Kinetics; Liver Neoplasms; Mouth Mucosa; Recombinant Proteins; STAT1 Transcription Factor; STAT3 Transcription Factor; Trans-Activators; Transforming Growth Factor alpha; Tumor Cells, Cultured

The roles of protein-tyrosine phosphatases (PTPs) in processes such as cell growth and adhesion are poorly understood. To explore the ability of specific PTPs to regulate cell signaling pathways initiated by stimulation of growth factor receptors, we expressed the receptor-like PTP, PTPalpha, in A431 epidermoid carcinoma cells. These cells express high levels of the epidermal growth factor (EGF) receptor and proliferate in response to the autocrine production of transforming growth factor-alpha. Conversely, EGF stimulation of A431 cells in vitro leads to growth inhibition and triggers the rapid detachment of these cells from the substratum. Although PTPalpha expression did not alter the growth characteristics of either unstimulated or EGF-stimulated cells, this phosphatase was associated with increased cell-substratum adhesion. Furthermore, PTPalpha-expressing A431 cells were strikingly resistant to EGF-induced cell rounding. Overexpression of PTPalpha in A431 cells was associated with the dephosphorylation/activation of specific Src family kinases, suggesting a potential mechanism for the observed alteration in A431 cell-substratum adhesion. Src kinase activation was dependent on the D1 catalytic subunit of PTPalpha, and there was evidence of association between PTPalpha and Src kinase(s). PTPalpha expression also led to increased association of Src kinase with the integrin-associated focal adhesion kinase, pp125(FAK). In addition, paxillin, a Src and/or pp125(FAK) substrate, displayed increased levels of tyrosine phosphorylation in PTPalpha-expressing cells and was associated with elevated amounts of Csk. In view of these alterations in focal adhesion-associated molecules in PTPalpha-expressing A431 cells, as well as the changes in adhesion demonstrated by these cells, we propose that PTPalpha may have a role in regulating cell-substratum adhesion.

Topics: Amino Acid Sequence; Carcinoma, Squamous Cell; Cell Adhesion; Cell Adhesion Molecules; Cell Division; Cell Size; Cloning, Organism; Cytoskeletal Proteins; Epidermal Growth Factor; ErbB Receptors; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Humans; Kinetics; Molecular Sequence Data; Paxillin; Peptide Fragments; Phosphopeptides; Phosphoproteins; Protein Tyrosine Phosphatases; Protein-Tyrosine Kinases; Receptor, Insulin; Recombinant Proteins; Rosaniline Dyes; src Homology Domains; Substrate Specificity; Transfection; Transforming Growth Factor alpha; Tumor Cells, Cultured

To investigate the patterns of expression of transforming growth factor alpha (TGF-alpha) and epidermal growth factor receptor (EGFR) in squamous metaplasia and squamous cell carcinomas of the urinary bladder with and without schistosomiasis.. Immunohistochemical study of the expression of TGF-alpha and EGFR in squamous metaplasias (n = 12) and various grades of squamous cell carcinomas (n = 21) of the bladder with and without schistosomiasis.. Focal cytoplasmic and membranous positivity for EGFR and TGF-alpha was seen in all cases of squamous metaplasia. The markers were diffusely coexpressed in a concordant pattern in areas of hyperplastic keratinising squamous metaplasia. A similar pattern of positivity was seen in verrucous carcinomas (n = 2) and well differentiated squamous carcinomas (n = 6). Progressive loss of differentiation was associated with increasing loss of EGFR staining while TGF-alpha staining was retained. Squamous cell carcinoma in situ (n = 2) showed focal positivity for TGF-alpha and EGFR. There were no differences in staining patterns between cases with and without schistosomiasis.. The coexpression of TGF-alpha and EGFR by well differentiated squamous cell carcinomas and hyperplastic keratinising squamous metaplasia is consistent with the active regulatory role exerted by this autocrine loop. There is regional absence of expression of EGFR but not of TGF-alpha in squamous cell carcinomas of lesser differentiation, suggesting heterogeneity of such control in these tumours. The focal expression of the two markers in squamous cell carcinomas in situ indicates a possible second pathway of oncogenesis for less differentiated tumours. These observations may have important implications for the effectiveness of putative growth factor based treatments.

Topics: Adult; Aged; Carcinoma, Squamous Cell; ErbB Receptors; Female; Humans; Male; Metaplasia; Middle Aged; Neoplasm Proteins; Precancerous Conditions; Schistosomiasis haematobia; Transforming Growth Factor alpha; Urinary Bladder; Urinary Bladder Neoplasms

The epidermal growth factor receptor (EGF-R) and its major ligands EGF and transforming growth factor alpha (TGF alpha) play an important role in the development of multiple human tumors. However, little is known of the comparative effects of each ligand on the regulation of EGF-R expression. To investigate this issue we used two similar human epidermoid cancer cell lines that overexpress EGF-Rs (KB and A431). In KB cells, EGF and TGF alpha increased EGF-R mRNA and protein levels by 2-3 fold over 8 h, associated with a greater than 4-fold stabilization of EGF-R mRNA half-life. EGF and TGF alpha also increased transcription of EGF-R mRNA 2-3-fold in KB cells. In contrast, EGF and TGF alpha only minimally increased EGF-R mRNA and protein in A431 cells, without changing EGF-R mRNA half-life. Basal EGF-R mRNA half-life was 2 fold greater in A431 cells than in KB cells (6-7 h versus 2-3 h), whilst the half-life of a mutant 2.6 kb EGF-R mRNA present in A431 cells, which lacks the 3-untranslated region (3'-UTR), was 2 fold greater than the full-length EGF-R mRNA. RNA gel-shift studies demonstrated that KB and A431 cells contain cytoplasmic proteins that bind specifically to an AU-rich sequence from the 3'-UTR of EGF-R mRNA. Taken together, these results demonstrate that in KB cells EGF and TGF alpha upregulate EGF-R expression at both transcriptional and post-transcriptional levels. The identification of AU-rich EGF-R mRNA-specific RNA-binding proteins from epidermoid cancer cells that overexpress EGF-Rs suggests that regulated RNA-protein interactions involving this region may play a central role in modulating EGF-R mRNA stability.

Topics: 3' Untranslated Regions; Carcinoma, Squamous Cell; Cycloheximide; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Neoplastic; Humans; RNA, Messenger; Transcription, Genetic; Transforming Growth Factor alpha; Tumor Cells, Cultured

The endothelial cell-specific mitogen vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) represents a central regulator of cutaneous angiogenesis. Increased VPF/VEGF expression has recently been reported in psoriatic skin and healing wounds, both conditions in which transforming growth factor-alpha (TGF alpha) and its ligand, the epidermal growth factor receptor, are markedly up-regulated. Since TGF alpha strongly induces VPF/VEGF synthesis in keratinocytes, TGF alpha-mediated VPF/VEGF expression is likely to play a significant role in the initiation and maintenance of increased vascular hyperpermeability and hyperproliferation in skin biology. The objectives of the present studies were to determine the molecular mechanisms responsible for TGF alpha-induced transcriptional activation of the VPF/VEGF gene. We have identified a GC-rich TGF alpha-responsive region between -88 bp and -65 bp of the VPF/VEGF promoter that is necessary for constitutive and TGF alpha-inducible transcriptional activation. In electrophoretic mobility shift assays, this region binds Sp1-dependent protein complexes constitutively and an additional TGF alpha-inducible protein complex that is distinct from Sp1 protein. Both AP-2 and Egr-1 transcription factors were detected as components of the TGF alpha-inducible protein complex in supershift EMSA studies. In co-transfection studies, an AP-2 but not an Egr-1 expression vector activated VPF/VEGF transcription, thus indicating that AP-2 protein is functionally important in TGF alpha-induced VPF/VEGF gene expression. By clarifying regulatory mechanisms that are critical for angiogenic processes in the skin, these studies may form the basis for new therapeutic strategies to modulate VPF/VEGF expression in cutaneous inflammation and wound healing.

Topics: Base Sequence; Carcinoma, Squamous Cell; DNA; DNA-Binding Proteins; Early Growth Response Protein 1; Endothelial Growth Factors; Humans; Immediate-Early Proteins; Lymphokines; Promoter Regions, Genetic; Recombinant Fusion Proteins; RNA, Messenger; Sequence Deletion; Sp1 Transcription Factor; Transcription Factor AP-2; Transcription Factors; Transcriptional Activation; Transforming Growth Factor alpha; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Transforming growth factor-alpha (TGF alpha) can stimulate keratinocyte proliferation and function as an autocrine tumor promoter in 7,12-dimethylbenz[a]anthracene (DMBA)-initiated TGF alpha-transgenic mouse skin. In this study, we examined the effect of ectopic TGF alpha transgene expression on skin tumor growth and progression after DMBA initiation in the presence of 12-O-tetradecanoylphorbol-13-acetate (TPA). Both the multiplicity and size of skin tumors arising in TGF alpha-transgenic mice were significantly higher than those of the nontransgenic parental CD-1 strain. There were more dysplastic papillomas and squamous cell carcinomas (SCCs) in the transgenic animals as well. ProTGF alpha protein was expressed in transgenic papillomas, but mature TGF alpha was not detected. The epidermal growth factor receptor (EGFR) appeared to be downregulated and was associated with enhanced tyrosine phosphorylation of several substrates in TGF alpha-transgenic mouse tumors. Characteristic codon 61 mutations in the Ha-ras gene were found in most of the papillomas and SCCs induced by DMBA and TPA in transgenic as well as nontransgenic mice. However, no p53 gene mutations were found in any skin tumors from either transgenic or control animals. Analysis of cellular proliferation in both DMBA-TPA-induced papillomas and in skin 48 h after TPA treatment alone revealed significantly more DNA synthesis in TGF alpha-transgenic mice relative to controls. These results demonstrate that TGF alpha, through EGFR overstimulation, can act synergistically with TPA to induce the formation, growth, and development of DMBA-initiated skin tumors containing classic Ha-ras gene mutations but not p53 gene inactivation.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Carcinogens; Carcinoma, Squamous Cell; Cloning, Molecular; ErbB Receptors; Exons; Genes, p53; Humans; Mice; Mice, Transgenic; Papilloma; Polymerase Chain Reaction; Polymorphism, Single-Stranded Conformational; Recombinant Proteins; Skin; Skin Neoplasms; Transforming Growth Factor alpha

The carotenoids beta-carotene and canthaxanthin and the retinoid 13-cis-retinoic acid (13-RA) have inhibited oral carcinogenesis in the hamster cheek pouch (16 wks, 3 times/wk at 1.4 mg/kg) induced by an 0.5% solution of 7, 12-dimethylbenz[a]anthracene (DMBA). However, 13-RA at a higher dose (> 2.0 mg/kg per treatment) increased squamous cell carcinoma growth (Eur J Cancer Clin Oncol 24, 839-850, 1988). 13-RA, beta-carotene, and canthaxanthin administered to 60 hamsters (16 wks, 3 times/wk, 10 mg/kg) altered neovascularization characterized by immunohistochemistry for transforming growth factor-alpha (TGF-alpha) and factor VIII. 13-RA + DMBA resulted in more smaller-sized tumors, with a reduced volume and tumor burden (tumor controls, 185.9; 13-RA + DMBA, 151.0). The carotenoids reduced the number and the sizes of the carcinomas formed (beta-carotene, 60 tumors, 142.3 x 10(3) mm3; canthaxanthin, 30 tumors, 116.1 x 10(3) mm3). Factor VIII and TGF-alpha were expressed in high intensity at cancer sites of the 13-RA + DMBA and DMBA groups with > 50 and > 10 cells, respectively, per x 400 field. In contrast, beta-carotene- and canthaxanthin + DMBA-treated pouches showed > 20 and 5 cells, respectively, per x 400 field for factor VIII and TGF-alpha. These results suggest that 13-RA treatment may increase vascular growth, but the carotenoids also produced enhanced levels of endothelial cell growth and TGF-alpha compared with the untreated mucosa. The carotenoids may enhance tumor growth under the appropriate carcinogenic environment.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; beta Carotene; Canthaxanthin; Carcinoma, Squamous Cell; Cheek; Cricetinae; Dose-Response Relationship, Drug; Factor VIII; Immunoenzyme Techniques; Isotretinoin; Male; Mesocricetus; Mouth Mucosa; Mouth Neoplasms; Neovascularization, Pathologic; Transforming Growth Factor alpha

We previously found that interferon alpha2 recombinant (IFNalpha) increases the expression of epidermal growth factor receptor (EGF-R) in the human epidermoid cancer KB cell line. Here we report the effects of IFNalpha and epidermal growth factor (EGF) on KB cell cycle kinetics. IFNalpha (1000 i.u./ml) for 48 h decreased the S-phase fraction and diminished the expression of Ki67 and proliferating cell nuclear antigen on KB cells. Incubation of IFNalpha-treated KB cells with 10 nM EGF for 12 h reversed these effects. We then studied several biochemical markers of cell proliferation. Ornithine decarboxylase activity was decreased to about one-tenth by IFNalpha and partly restored by EGF. Hypusine is contained only in eukaryotic initiation factor 5A and its levels are correlated with cell proliferation. IFNalpha decreased hypusine synthesis by 75%; exposure of cells to EGF for 12 h restored hypusine synthesis almost completely. We also studied the effects of IFNalpha on the cytotoxicity of the recombinant toxin TP40, which inhibits elongation factor 2 through EGF-R binding and internalization. IFNalpha greatly enhanced the TP40-induced inhibition of protein synthesis in KB cells. In conclusion, IFNalpha, which affects protein synthesis machinery and increases EGF-R expression, enhances the tumoricidal activity of TP40 and hence could be useful in the setting of anti-cancer therapy.

Topics: Antineoplastic Agents; Carcinoma, Squamous Cell; Cell Cycle; Cell Division; Epidermal Growth Factor; Exotoxins; Humans; Interferon alpha-2; Interferon-alpha; Lysine; Ornithine Decarboxylase; Oropharyngeal Neoplasms; Recombinant Proteins; Transforming Growth Factor alpha; Tumor Cells, Cultured

We investigated the effects of epidermal growth factor (EGF) and transforming growth factor (TGF)-alpha on migration, invasion and matrix metalloproteinase (MMP) expression of uterine cervical-carcinoma SKG-IIIb cells, and whether these growth factors affect pyrimidine-nucleoside-phosphorylase (PyNPase) activity and 5'-deoxy-5-fluorouridine (5'-dFUrd) sensitivity of tumor cells. Tumor-cell migration along a gradient of substratum-bound fibronectin and invasion into reconstituted basement membrane were stimulated by 0.1 to 100 ng/ml of EGF and TGF-alpha in a concentration-dependent manner. The zymography of tumor-conditioned medium showed that the treatment of tumor cells with EGF and TGF-alpha resulted in an increase of the 92-kDa type-IV collagenase (MMP-9), which was confirmed by immunoblot analysis. These growth factors also up-regulated the expression of PyNPase activity of tumor cells and consequently enhanced the anti-proliferative action of 5'-dFUrd, a cytostatic that is biotransformed to 5-fluorouracil (5-FUra) by PyNPase. However, EGF and TGF-alpha did not have significant effects on the 5-FUra sensitivity of tumor cells. These results suggest that EGF and TGF-alpha, tumor environmental factors, simultaneously up-regulate the potential of uterine cervical-carcinoma cells to invade extracellular matrices and their PyNPase activity, which are subsequently associated with the specific action of 5'-dFUrd selectively killing tumor cells of gynecological origin with high invasive and metastatic potential.

Topics: Basement Membrane; Carcinoma, Squamous Cell; Chemotaxis; Collagen; Collagenases; Dose-Response Relationship, Drug; Drug Combinations; Epidermal Growth Factor; Female; Fibronectins; Floxuridine; Gelatin; Humans; Laminin; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Pentosyltransferases; Proteoglycans; Pyrimidine Phosphorylases; Transforming Growth Factor alpha; Tumor Cells, Cultured; Uterine Cervical Neoplasms

The epidermal growth factor receptor (EGFR) is activated by a variety of ligands including EGF and transforming growth factor-alpha (TGFalpha), whereas no ligand for the homologous ErbB2 oncoprotein has yet been identified. Here we use both an ErbB2 phosphoantibody (aPY1222) and an activation-specific EGFR antibody to show that low concentrations of EGF induce more efficient tyrosine phosphorylation of ErbB2 in A431 cells than does equimolar TGFalpha, while EGFR is more potently activated by TGFalpha. Co-precipitation studies confirm that heterodimerization of activated EGFR and transphosphorylated ErbB2 is readily induced by EGF but not TGFalpha. EGFR downregulation is also more efficiently induced by EGF, suggesting that ligand-dependent modification of ErbB2 may be required to terminate EGFR signalling in cells expressing both receptor types. These findings indicate that EGF and TGFalpha differ in their abilities to induce tyrosine phosphorylation and heterodimerization of ErbB2, and raise the possibility that ErbB2 exerts its oncogenic effect in part by impairing TGFalpha-dependent EGFR downregulation.

Topics: Antibodies; Antibody Specificity; Carcinoma, Squamous Cell; Dimerization; Down-Regulation; Drug Synergism; Epidermal Growth Factor; ErbB Receptors; Humans; Immunoblotting; Ligands; Phosphorylation; Precipitin Tests; Receptor, ErbB-2; Transforming Growth Factor alpha; Tumor Cells, Cultured; Tyrosine

Transforming growth factor alpha (TGF-alpha) and its receptor (EGF-R) may regulate normal and malignant epithelial cell growth by an autocrine mechanism. We investigated the role of TGF-alpha in regulating head and neck SCC tumor growth.. TGF-alpha and EGF-R levels were measured in 7 SCC cell lines and 14 SCC biopsies by RIA, Scatchard, and Western analysis. TGF-alpha autocrine stimulation of DNA synthesis in SCC cell lines was assessed by incubation with TGF-alpha neutralizing antibodies and tyrphostin AG 1478, a selective and potent inhibitor of EGF-R kinase.. All SCC cell lines synthesized TGF-alpha and expressed elevated EGF-R levels compared to normal keratinocytes. Twelve of the 14 SCC biopsies contained TGF-alpha protein and 8 had specific EGF-R. Exogenous TGF-alpha or EGF significantly increased DNA synthesis in 4 of 5 SCC cell lines. TGF-alpha neutralizing antibodies or tyrphostin AG 1478 reduced DNA synthesis in the two SCC cell lines (FaDu and SCC9) tested.. These results indicate that SCC cell lines and tumors usually synthesize TGF-alpha, have elevated levels of EGF-R, and are mitogenically stimulated by a TGF-alpha autocrine system. Selective inhibition of the TGF-alpha system by EGF-R kinase inhibitors or TGF-alpha neutralizing antibodies may be useful strategies for treating SCC that overexpress TGF-alpha and its receptor.

Topics: Autocrine Communication; Carcinoma, Squamous Cell; DNA, Neoplasm; ErbB Receptors; Head and Neck Neoplasms; Humans; Protein-Tyrosine Kinases; Regression Analysis; Transforming Growth Factor alpha; Tumor Cells, Cultured

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

Retinoic acid (RA) has been shown to be effective in eradicating premalignant lesions and preventing second primary malignancies in patients cured of squamous cell carcinoma of the head and neck (SCCHN) in clinical trials. The basis for this effect is unclear. We have previously demonstrated that messenger RNA from tumor growth factor-alpha (TGF-alpha) and its receptor, the epidermal growth factor (EGFR), is unregulated in tumors and histologically normal mucosal samples from patients with SCCHN compared with control normal mucosa from patients without cancer, implicating this ligand-receptor pair in an autocrine growth pathway early in the molecular pathogenesis of this disease. In this report, we examined the hypothesis that the action of RA on the mucosa of the upper aerodigestive tract is mediated via downregulation of steady-state TGF-alpha and/or EGFR mRNA levels. Following exposure to all-trans-RA, a series of SCCHN cell lines demonstrated a 35.4% reduction in TGF-alpha mRNA expression (P = 0.022) and 58.5% reduction in EGFR mRNA (P = 0.0027). Nuclear run-on analysis indicated that the RA-mediated reduction of TGF-alpha and EGFR steady-state mRNA levels was a result of decreased gene transcription. These results suggest that the clinical effects of RA in SCCHN patients may be due to a downmodulation of TGF-alpha and EGFR mRNA production.

Topics: Carcinoma, Squamous Cell; Down-Regulation; Epidermal Growth Factor; Head and Neck Neoplasms; Humans; Keratolytic Agents; Mouth Mucosa; RNA, Messenger; Transcription, Genetic; Transforming Growth Factor alpha; Tretinoin; Tumor Cells, Cultured

Transforming growth factor alpha (TGF-alpha) is biosynthesized as a membrane-bound precursor protein, pro-TGF-alpha, that undergoes sequential endoproteolytic cleavages to release a soluble form of the factor. In the present study, we have analyzed the biosynthesis and regulation of TGF-alpha production in human tumor-derived cell lines that endogenously express pro-TGF-alpha and the epidermal growth factor (EGF) receptor. These cells biosynthesized membrane-anchored forms of the TGF-alpha that accumulated on the cell surface. Membrane-bound pro-TGF-alpha interacted with the EGF receptor, and complexes of receptor and pro-TGF-alpha contained tyrosine-phosphorylated receptor. Activation of the EGF receptor by soluble EGF or TGF-alpha had a dual effect on TGF-alpha production: an increase in pro-TGF-alpha mRNA levels and an increase in pro-TGF-alpha cleavage. These effects were largely prevented by preincubation with an anti-EGF receptor monoclonal antibody that blocked ligand binding. Growth factor autoinduction of cleavage could be stimulated by several second messenger pathways that are activated by the EGF receptor, including protein kinase C and intracellular calcium, and by other alternative mechanisms. EGF-stimulated cleavage of pro-TGF-alpha could be partially blocked by inhibition of these second messenger pathways. These results suggest that juxtacrine stimulation takes place in human tumor cells that coexpress both the EGF receptor and membrane-anchored TGF-alpha and that TGF-alpha is able to induce its own endoproteolytic cleavage by activating the EGF receptor.

Topics: Animals; Antibodies; Autoradiography; Calcimycin; Carcinoma, Squamous Cell; Cell Membrane; CHO Cells; Cricetinae; Cysteine; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Humans; Male; Protein Precursors; Protein Processing, Post-Translational; Rats; Recombinant Proteins; Sulfur Radioisotopes; Tetradecanoylphorbol Acetate; Transfection; Transforming Growth Factor alpha; Tumor Cells, Cultured

The purpose of this study was to determine the overexpression of both epidermal growth factor receptor (EGFR) and transforming growth factor-alpha (TGF-alpha) (a ligand of EGFR) in early laryngeal squamous cell carcinoma. In addition, we attempted to evaluate the prognostic values of our findings. Expression of EGFR and TGF-alpha in tumor tissue was examined immunohistochemically in 68 patients who had been treated with radiotherapy for early laryngeal cancer. Overexpression of the two factors was noted in 42.6% and 55.9%, respectively. No significant differences due to age, tumor size, and location or grade of cancer tissues were seen. Higher survival rates, found in patients with EGFR (-) and TGF-alpha (-) tumors as compared with those with EGFR (+) and TGF-alpha (+) (97.4%, 100% and 86.2%, 86.8%, respectively), were not statistically significant. The recurrence rates were similar between EGFR (+) and EGFR (-) (37.9% and 35.9%, respectively). However, the recurrence rate in patients with TGF-alpha (+) was significantly higher (57.9%) than in those with TGF-alpha (-) (10%; P<.01). Therefore overexpression of TGF-alpha may be an important indicator for recurrence in patients with early laryngeal squamous cell carcinoma treated by irradiation.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Squamous Cell; ErbB Receptors; Female; Humans; Immunohistochemistry; Laryngeal Neoplasms; Male; Middle Aged; Prognosis; Survival Rate; Transforming Growth Factor alpha

Transforming growth factor alpha (TGF-alpha) is a polypeptide that is structurally similar to epidermal growth factor (EGF) that binds to the epidermal growth factor receptor (EGFR) and has been implicated in the development of several types of human tumours.. The expression of TGF-alpha is examined in laryngeal squamous cell carcinoma (SCC) (n = 24) and non-neoplastic polyps (n = 7) using streptavidin-biotin immunohistochemistry and a monoclonal antibody to the TGF-alpha protein. These cases had been previously characterized for EGFR immunoreactivity. The carcinomas were classified as well differentiated (n = 2), moderately differentiated (n = 16) and poorly differentiated (n = 6). Tissues from metastatic tumour deposits in lymph nodes (n = 5) were also studied.. TGF-alpha overexpression was defined as intense immunoreactivity in more than two-thirds of tumour cells immunostained for TGF-alpha and was present in the majority of the SCC cases (n = 15; 63%) and metastatic tumour deposits (n = 4; 80%). In contrast, although some of the vocal cord polyps showed weak (n = 2) to moderate (n = 5) immunostaining, none had evidence of strong TGF-alpha immunoreactivity. The differences in TGF-alpha immunoreactivity were significant between primary laryngeal SCC and vocal cord polyps (P = 0.013; chi 2 test with continuity correction), and between metastatic laryngeal SCC and vocal cord polyps (P = 0.023; chi 2 test with continuity correction). There was no significant difference in TGF-alpha expression between the different grades of carcinomas (P = 0.92, chi 2 test) or between non-metastatic and metastatic carcinomas (P = 0.82; chi 2 test with continuity correction). No significant correlation was found between TGF-alpha expression and patient survival or tumour recurrence (r = 0.077, r2 = 0.006, P = 0.75; simple regression analysis), or between TGF-alpha expression and EGFR immunoreactivity (r = 0.325, r2 = 0.106, P = 0.0851).. In conclusion, increased TGF-alpha immunoreactivity is present in most cases of laryngeal SCC with no specific relationship to tumour grade, suggesting that it may be important in the development of laryngeal carcinomas but not in its progression. No significant correlation was found between TGF-alpha and EGFR expression in laryngeal tumours and TGF-alpha immunoreactivity is of no prognostic value.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Squamous Cell; ErbB Receptors; Female; Humans; Immunohistochemistry; Laryngeal Neoplasms; Male; Middle Aged; Polyps; Prognosis; Regression Analysis; Transforming Growth Factor alpha; Vocal Cords

Proliferative verrucous leukoplakia is a unique type of oral leukoplakia that has a high risk of malignant transformation. The aim of this study was to examine the expression of transforming growth factor-alpha in proliferative verrucous leukoplakia, oral squamous cell carcinoma, and normal mucosa. Transforming growth factor-alpha, a potent mitogen, is known to play an important role in various neoplasms including oral squamous cell carcinoma. Immunohistochemical localization of transforming growth factor-alpha in archival paraffin-embedded sections was performed with commercially available monoclonal antibodies. Ten cases each of normal mucosa, proliferative verrucous leukoplakia, and oral squamous cell carcinoma were stained. Quantification of the staining intensity, expressed as the cytoplasmic optical density, was done with the Roche Image Analysis System. The data were statistically analyzed with the one-way analysis of variance and Tukey tests. Notably, the mean cytoplasmic optical density of proliferative verrucous leukoplakia was significantly higher than the mean cytoplasmic optical density of normal mucosa (p < 0.01). The mean cytoplasmic optical density of proliferative verrucous leukoplakia was slightly higher than that of oral squamous cell carcinoma, however, this difference was not significant (p > 0.05). The mean cytoplasmic optical density values demonstrate that increased transforming growth factor-alpha immunoreactivity occurs in proliferative verrucous leukoplakia and oral squamous cell carcinoma relative to normal mucosa.

Topics: Adult; Aged; Aged, 80 and over; Analysis of Variance; Biomarkers, Tumor; Carcinoma, Squamous Cell; Case-Control Studies; Cell Count; Cell Transformation, Neoplastic; Densitometry; Female; Humans; Immunoenzyme Techniques; Leukoplakia, Oral; Male; Middle Aged; Mouth Mucosa; Mouth Neoplasms; Retrospective Studies; Sex Ratio; Transforming Growth Factor alpha

A case of synchronous squamous cell carcinomas in the soft palate, larynx and esophagus is reported, along with findings of molecular-pathological analysis. A biopsy sample from the aryngeal carcinoma revealed well differentiated squamous cell carcinoma harboring two point mutations at codons 144 and 148 of the p53 gene but not at codon 299, and more than 50% of the cancer cells showed accumulation of p53 protein immunohistochemically. The esophageal tumor, which was moderately differentiated squamous cell carcinoma, showed immunoreactivity for p53 within the nuclei of 25-50% of cancer cells with a missense mutation at codon 299 but not at codon 144 or 148. This cancer also showed immunoreactivity for transforming growth factor alpha. On the other hand, the poorly differentiated squamous cell carcinoma in the soft palate showed negative immunoreactivity for p53 and no point mutation in exons 5 to 8 of the gene. These results suggest that the three synchronous squamous cell carcinomas arose as independent events.

Topics: Aged; Carcinoma, Squamous Cell; Epidermal Growth Factor; ErbB Receptors; Esophageal Neoplasms; Genes, p53; Humans; Immunohistochemistry; Laryngeal Neoplasms; Male; Mutation; Neoplasms, Multiple Primary; Palatal Neoplasms; Palate, Soft; Polymerase Chain Reaction; Stomach Neoplasms; Transforming Growth Factor alpha; Tumor Suppressor Protein p53

Tissue-specific delivery of a variety of molecules has been a valuable technique for biological and medical research. Therefore, we have constructed a recombinant plasmid containing the coding regions for streptavidin core and mature human transforming growth factor-alpha (TGF-alpha). The recombinant plasmid has been expressed in Escherichia coli to produce a chimeric protein with both streptavidin and TGF-alpha activity. The streptavidin-TGF-alpha chimeric protein (ST-TGF-alpha) could efficiently transfer biotinylated beta-galactosidase into A431 cells via the epidermal growth factor receptor. More than 99% of the cells contained the enzyme transferred. Furthermore, ST-TGF-alpha complexed with biotinylated-glucose oxidase had a significant cytotoxic effect when incubated with A431 cells. These findings suggest that the ST-TGF-alpha chimeric protein could be used to deliver proteins of interest into target cells without the need for chemical linkage or genetic construction. Essentially, ST-TGF-alpha serves as a high-modular "molecular bridge" for the passage of a wide variety of effector molecules into target cells.

Topics: Amino Acid Sequence; Bacterial Proteins; beta-Galactosidase; Biotin; Carcinoma, Squamous Cell; Drug Carriers; Drug Delivery Systems; Epidermal Growth Factor; ErbB Receptors; Glucose Oxidase; Humans; Molecular Sequence Data; Radioligand Assay; Recombinant Fusion Proteins; Streptavidin; Transforming Growth Factor alpha; Tumor Cells, Cultured

Depletion of specific cellular proteins is a powerful tool in biological research and has many medical and agricultural benefits. In contrast to genetic methods currently available to attenuate protein levels, we describe an alternative approach that redirects the ubiquitin-dependent proteolytic pathway to facilitate specific proteolytic removal. Degradation via the ubiquitin pathway requires the prior attachment of multiple ubiquitins to the target protein. This attachment is accomplished, in part, by a family of enzymes designated E2s (or ubiquitin-conjugating enzymes), some of which use domains near their C termini for target recognition. Here, we demonstrate that E2 target recognition can be redefined by engineering E2s to contain appropriate protein-binding peptides fused to their C termini. In five dissimilar examples, chimeric E2s were created that recognized and ubiquitinated their respective binding partners with high specificity. We also show that ubiquitination of one protein targeted by this method led to its ATP-dependent degradation in vitro. Thus, by exploiting interacting domains derived from natural and synthetic ligands, it may be possible to design E2s capable of directing the selective removal of many intracellular proteins.

Topics: Amino Acid Sequence; Animals; Base Sequence; Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Cell-Free System; DNA Primers; ErbB Receptors; Humans; Ligases; Molecular Sequence Data; Mutagenesis, Site-Directed; Oligodeoxyribonucleotides; Polymerase Chain Reaction; Protein Engineering; Protein Processing, Post-Translational; Rabbits; Recombinant Fusion Proteins; Reticulocytes; Transforming Growth Factor alpha; Triticum; Tumor Cells, Cultured; Ubiquitin-Conjugating Enzymes; Ubiquitin-Protein Ligases; Ubiquitins

Mutation of the p53 tumor suppressor gene is a common event in many human cancers and has been specifically associated with invasive squamous cell carcinoma of the human skin and respiratory tract. Alterations in the p53 gene have also been identified in certain rodent tumors, including formaldehyde-induced nasal squamous cell carcinomas. Overexpression of transforming growth factor-alpha (TGF-alpha) is associated with carcinomas of the head and neck and respiratory tract in human patients and formaldehyde-induced rat nasal squamous cell carcinomas. Sections of rat noses containing tumors and other formaldehyde-induced lesions from rats exposed to 15 ppm formaldehyde vapor were examined using immunohistochemical techniques to detect and identify potential relationships between the presence and distribution of p53, proliferating cell nuclear antigen (PCNA), and TGF-alpha proteins. The five tumors that had p53 mutations were for mutant p53 protein by immunohistochemistry and three of six tumors with no detected p53 mutations were also immunoreactive for p53 protein. The presence, pattern, and distribution of p53 staining in tissue sections depended on the morphology of the lesion. PCNA immunoreactivity was strikingly similar in pattern and distribution to p53 immunoreactivity. The pattern and distribution of immunoreactivity for TGF-alpha did not directly correlate with the other markers. Mutation of the p53 tumor suppressor gene may be an important step in the progression of formaldehyde-induced nasal carcinogenesis in the rat. This study demonstrated that immunohistochemistry is a useful tool for the identification of sites within tumors that might have p53 mutations.

Topics: Animals; Carcinoma, Squamous Cell; Formaldehyde; Immunoenzyme Techniques; Male; Nose Neoplasms; Proliferating Cell Nuclear Antigen; Rats; Rats, Inbred F344; Transforming Growth Factor alpha; Tumor Cells, Cultured; Tumor Suppressor Protein p53

F.2a and B.2, cell clones of the human squamous cell carcinoma SCC-12, were examined to characterize their interactions through the expression of growth factors. F.2a was nontumorigenic yet B.2 was fully tumorigenic when injected into the flanks of athymic nude mice. Combination injections of F.2a and a subtumorigenic level of B.2 produced tumors. F.2a and B.2 overexpressed the 4.8-kb transcript for transforming growth factor-alpha (TGF-alpha) as well as the 10.5- and 5.8- kb transcripts for the epidermal growth factor receptor. Neither clone expressed the transcript for epidermal growth factor, while both expressed transcripts for insulin-like growth factor-I (IGF-I) of 8.15, 4.9, and 1.6 kb and transcripts for its receptor of 8.5 and 6.5 kb. Conditioned medium (CM) from either clone stimulated the growth of itself and the other clone in tissue culture. Both clones produced intracellular TGF-alpha detectable by immunohistochemical staining, but not detectable in CM by enzyme-linked immunosorption assay. IGF-I was detected at low levels in CM by radioimmunoassay. Neutralizing antibodies to TGF-alpha but not IGF-I partially inhibit the growth of both clones in tissue culture. These results suggest that (1) TGF-alpha is active in autocrine signaling (2) IGF-I is not directly stimulatory, and (3) additional factors, as yet unidentified, are present in CM and enhance tumor growth. It is concluded that human squamous cell carcinoma SCC-12 is composed of tumorigenic and nontumorigenic clones which interact to enhance growth.

Topics: Animals; Carcinoma, Squamous Cell; Cell Division; Epidermal Growth Factor; ErbB Receptors; Humans; Insulin-Like Growth Factor I; Mice; Mice, Nude; 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

The purpose of this study was to determine whether production of liver metastasis by human colon carcinoma (HCC) cells depends on the response of tumor cells to organ-derived growth factors. HCC cells were isolated from several surgical specimens whose malignant potential differed (Dukes' stage B or D tumors), adapted to grow in culture, and assessed for expression of the epidermal growth factor receptor (EGF-R). Northern blot analyses revealed that highly metastatic HCC cells expressed >5-fold the number of EGF-R mRNA transcripts as low metastatic cells. The level of mRNA correlated with the amount of EGF-R protein as detected by Western blotting, immunohistochemistry, and Scatchard analyses. HCC growth response in vitro to picograms of transforming growth factor alpha was associated with functional cell surface EGF-Rs as determined by receptor tyrosine kinase activity assays. The EGF-R gene was not amplified or rearranged in highly metastatic cells. However, fluorescence in situ hybridization analysis showed that the copy number of chromosome 7 was higher in the highly metastatic cells. HCC cells were selected in vitro for low or high expression of EGF-R. Subsequent to injection into nude mice, only cells with high expression of EGF-R produced a high incidence of liver metastasis. These data demonstrate that expression of EGF-R by HCC cells directly correlates with their ability to produce hepatic metastasis.

Topics: Animals; Carcinoma, Squamous Cell; Cell Division; Chromosome Mapping; Chromosomes, Human, Pair 7; Colonic Neoplasms; ErbB Receptors; Humans; Liver Neoplasms; Male; Mice; Mice, Nude; Neoplasm Metastasis; RNA, Messenger; Transcription, Genetic; Transforming Growth Factor alpha; Transplantation, Heterologous; Tumor Cells, Cultured

In the study presented here, we examined the possible role of the transforming growth factor-alpha (TGF alpha)/epidermal growth factor receptor (EGFR) system during multistage carcinogenesis in mouse skin. In this regard, the expression (mRNA and protein) of both TGF alpha and EGFR was examined in primary papillomas and squamous cell carcinomas (SCCs) obtained from SENCAR mice treated with standard initiation-promotion regimens and compared with the levels of expression in normal epidermis. The level of a 4.8-kb TGF alpha transcript was elevated in 100% of the skin tumors examined (both papillomas and SCCs), including papillomas obtained 13 wk after the start of promotion, compared with normal epidermis. Immunohistochemical analyses detected elevated levels of TGF alpha protein in these skin tumors and in papillomas as early as 10 wk after the start of promotion. The levels of EGFR transcripts were also significantly elevated in most (90%) of the skin tumors examined, including again those harvested after 13 wk of promotion. Interestingly, multiple EGFR transcripts (10.5, 5.8, 2.8, and 1.8 kb) were detected in both papillomas and SCCs. The two smaller transcripts appeared to encode truncated versions of the EGFR, and the 1.8-kb transcript appeared to be unique to RNA samples isolated from skin tumors, based on comparative analyses of several normal tissues. As with TGF alpha, immunohistochemical analyses detected elevated levels of EGFR protein in these skin tumors (both papillomas and SCCs), including papillomas harvested as early as 10 wk after the start of promotion. Southern analyses of genomic DNAs for TGF alpha and EGFR failed to detect any cases of gene rearrangements or amplification as a possible explanation for the elevated levels of the transcripts of these two genes. These results support the hypothesis that a key step in the development of autonomous growth in mouse skin papillomas generated in SENCAR mice by an initiation-promotion regimen may involve alterations in the synthesis of TGF alpha and its cognate receptor.

Topics: Animals; Carcinoma, Squamous Cell; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Mice; Mice, Inbred SENCAR; Neoplasm Proteins; Papilloma; RNA, Neoplasm; Skin Neoplasms; Transforming Growth Factor alpha

The binding of epidermal growth factor (EGF) or an EGF-like growth factor to the EGF receptor is the initial event which leads to receptor activation, and consequently the induction of cell growth. In order to study this binding interaction in detail, we produced the extracellular domain of the EGF receptor (EGFR) using the baculovirus expression system. Affinity-labeling and Western-blot analyses revealed that the baculovirus-infected insect cells secrete active EGFR extracellular domain relatively efficiently, however a significant amount of inactive EGFR extracellular domain is retained within the cells. The apparent dissociation constant (Kd) of the secreted EGFR extracellular domain for EGF and transforming growth factor alpha (TGF-alpha), as determined using an immobilized receptor binding assay, was approximately 200 nM. Interestingly, this Kd value is 30-40-fold lower than that of the full-length EGFR derived from detergent-solubilized A431 cell membranes. The stoichiometry of binding of the EGFR extracellular domain to EGF and TGF-alpha was examined by band-shift analysis on non-denaturing PAGE and was estimated to be 1:1. We have also shown, using sedimentation equilibrium analysis, that ligand binding induces significant dimerization of the EGFR extracellular domain. Finally, we carried out site-specific mutagenesis on the EGFR extracellular domain in order to define the ligand-binding region. We identified amino acid residues which are close to the binding site since they are common to the epitopes of several ligand-competitive monoclonal antibodies. However, these residues do not contribute directly to ligand binding since the affinity of the mutated EGFR extracellular domain for EGF and TGF-alpha was unaffected.

Topics: Amino Acid Sequence; Animals; Baculoviridae; Base Sequence; Binding Sites; Blotting, Western; Carcinoma, Squamous Cell; Cell Line; Chickens; Electrophoresis, Polyacrylamide Gel; Epidermal Growth Factor; ErbB Receptors; Humans; Kinetics; Macromolecular Substances; Male; Mice; Molecular Sequence Data; Mutagenesis, Site-Directed; Recombinant Proteins; Spodoptera; Transfection; Transforming Growth Factor alpha; Tumor Cells, Cultured

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

We previously reported that anti-epidermal growth factor (EGF) receptor monoclonal antibody (mAb) 225 can block receptor activation and inhibit proliferation of tumor cells bearing EGF receptors. To further explore the mechanism of mAb-mediated growth inhibition, we compared the capacities of bivalent 225 mAb and 225 F(ab')2, and monovalent 225 Fab' fragment to block ligand binding to EGF receptors, inhibit activation of receptor tyrosine kinase by exogenous and endogenous ligand, produce receptor dimerization, down-regulate receptors, and inhibit proliferation of cultured A431 squamous carcinoma cells. Unlike 225 mAb and 225 F(ab')2, 225 Fab' fragment was a poor inhibitor of A431 cell proliferation. The weak antiproliferative capacity of 225 Fab' was not due to depletion of active fragment from cultures. When cells were exposed to exogenous EGF, monovalent 225 Fab' remaining in conditioned culture medium could act as well as the bivalent forms of mAb to block binding and tyrosine kinase activation by exogenous EGF. However, unlike the bivalent forms, 225 Fab' fragment was unable to induce receptor dimerization and down-regulation, and it lacked the capacity to block autocrine activation of EGF receptors by endogenous ligand. These deficiencies were corrected by addition of rabbit anti-mouse IgG antibody, which also enabled 225 Fab' fragment to inhibit cell proliferation. We conclude that in A431 cells, inhibition of autocrine-stimulated proliferation by anti-EGF receptor mAbs requires antibody bivalency, which provides the capacity to produce EGF receptor dimerization accompanied by receptor down-regulation. These properties may explain the greater efficacy of bivalent mAb and F(ab')2, compared with monovalent Fab' fragment, in inhibiting proliferation of a variety of malignant and nonmalignant cultured cell lines.

Topics: Antibodies, Monoclonal; Carcinoma, Squamous Cell; Cell Division; Down-Regulation; Enzyme Activation; ErbB Receptors; Humans; Immunoglobulin Fab Fragments; In Vitro Techniques; Receptor Aggregation; Receptor Protein-Tyrosine Kinases; Transforming Growth Factor alpha

Ionizing radiation (XRT) is often used to treat squamous cell carcinoma of the tongue (SCCT) but little is known of its genetic effects on surviving cancer cells. The effect of XRT on p53, epidermal growth factor receptor (EGFR), and transforming growth factor alpha (TGF alpha) tumor marker expression was evaluated using immunohistochemical analysis in 79 patients with SCCT. Sixty-six patients received no radiation, while 13 received XRT before surgery. Radiation did not influence EGFR or p53 expression. TGF alpha expression, however, was significantly decreased in radiated tumors (15% versus 43%, P = 0.04). These data suggest that XRT either decreases the expression of TGF alpha in SCCT (suggesting a genetic alteration in surviving cancer cells), or does not kill cancer cells with decreased TGF alpha expression. In the latter case, diminished TGF alpha expression may serve as a marker of radioresistance.

Topics: Aged; Carcinoma, Squamous Cell; ErbB Receptors; Female; Humans; Male; Middle Aged; Radiation, Ionizing; Tongue Neoplasms; Transforming Growth Factor alpha; Tumor Suppressor Protein p53

To investigate the expression and distribution of transforming growth factor-alpha (TGF-alpha) in the normal cervix and in benign and malignant lesions of the uterine cervix.. Immuno-histochemical reactivity with a monoclonal antibody against TGF-alpha was examined in tissue specimens from 15 normal cervices, six cervical polyps, four cervical condylomata acuminata, 34 cervical intra-epithelial neoplasias, 35 invasive squamous cell carcinomas, five adenocarcinomas, and three mixed adenosquamous carcinomas.. Normal squamous cells of the exocervix were found to be negative for TGF-alpha immunoreactivity, whereas reserve cells and metaplastic squamous cells in the transformation zone were positive for TGF-alpha. Although TGF-alpha immuno-reactivity was variable in the cervical condylomas, most cases of cervical intra-epithelial neoplasia with or without koilocytotic atypia were negative for TGF-alpha. In the invasive carcinomas, however, TGF-alpha immuno-reactivity was observed in 17 out of the 35 cases of squamous carcinoma, and in all cases of adeno- and adenosquamous carcinomas. In addition, intense TGF-alpha immuno-reactivity was found in clinically advanced tumours.. These results suggest that the expression of TGF-alpha is associated with squamous metaplasia in the normal cervix, and that TGF-alpha may play an important role in cervical carcinogenesis, especially in its progression.

Topics: Adenocarcinoma; Carcinoma, Squamous Cell; Cervix Uteri; Condylomata Acuminata; Female; Humans; Immunohistochemistry; Neoplasm Staging; Polyps; Transforming Growth Factor alpha; Uterine Cervical Neoplasms

This study examined the expression of epidermal growth factor (EGF) cell-surface receptors, the response to exogenous ligand and the autocrine production of transforming growth factor alpha (TGF-alpha) in normal and carcinoma-derived human oral keratinocytes. One of eight malignant cell lines overexpressed EGF receptors, while the remainder expressed receptor numbers similar to normal cells. Exogenous EGF stimulated incorporation of tritiated thymidine in a dose-dependent manner. In keratinocytes expressing normal numbers of EGF receptors, the cellular response to exogenous EGF correlated positively with total EGF receptor number. SCC-derived keratinocytes produced more TGF-alpha than normal cells. There was no statistical correlation between the autocrine production of TGF-alpha, EGF cell-surface receptor expression and cellular response to exogenous EGF. While the growth-stimulatory effects of exogenous TGF-alpha were inhibited by the addition of a neutralising antibody, the presence of this antibody in conditioned medium failed to produce a similar decrease in growth. The results indicate that overexpression of EGF receptors is not an invariable characteristic of human oral squamous carcinoma-derived cell lines. Further, the contribution of TGF-alpha to the growth of normal and carcinoma-derived human oral keratinocytes in vitro may be less significant than previously documented.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Squamous Cell; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Immunohistochemistry; Keratinocytes; Male; Middle Aged; Mouth Neoplasms; Transforming Growth Factor alpha; Tumor Cells, Cultured

Transforming growth factor alpha (TGF alpha) expression was analyzed immunocytochemically on formalin-fixed wax-embedded sections obtained from 24 benign prostatic hyperplasia (BPH) specimens and 76 prostatic carcinoma tissues, 3 human prostatic tumor xenografts, normal kidney, and salivary gland. Low amounts of TGF alpha immunopositivity were encountered in the epithelium of BPH glandular tissues, whereas in the prostatic adenocarcinoma samples, a greater heterogeneity and intensity of TGF alpha immunostaining was observed. The most intense staining was exhibited by the least differentiated tumors, although a few of these were weakly stained. Statistical analysis of the relationship of histopathological grade of tumor with TGF alpha expression in the carcinomas showed a significant correlation of these parameters, 0.01 > P > 0.001. The expression of the proliferation markers Ki-67 and PCNA was also analyzed in the carcinoma specimens, and the relationship of these to TGF alpha expression indicated that there was no significant correlation in this series of tumors between increased growth activity and TGF alpha expression (p approximately 0.25 with both markers). The prostatic carcinoma xenografts TEN12 and TEN15 contained low levels of immunoreactive TGF alpha, which was uniformly distributed, whilst heterogeneous immunostaining was observed in the uroepithelial xenograft TEN16. In the normal human kidney, TGF alpha was concentrated in the epithelium of the distal convoluted tubules (DCT) and the collecting tubules (CT), and lower amounts were identified in the proximal convoluted tubules (PCT). As in the prostatic carcinomas, the immunostaining was eliminated by prior absorption of the antibody with pure TGF alpha and not with human or mouse EGF. No crossreactivity of the TGF alpha antibody with salivary EGF was demonstrated. This study concludes that, in prostate carcinoma, the least differentiated tumors more often expressed greater amounts immunoreactive TGF alpha; however, no relationship between TGF alpha expression and cellular proliferation markers was found.

Topics: Adenocarcinoma; Adenocarcinoma, Papillary; Animals; Antigens, Neoplasm; Carcinoma, Squamous Cell; Humans; Immunohistochemistry; Ki-67 Antigen; Kidney; Male; Mice; Mice, Nude; Neoplasm Proteins; Neoplasm Transplantation; Nuclear Proteins; Proliferating Cell Nuclear Antigen; Prostatic Hyperplasia; Prostatic Neoplasms; Submandibular Gland; Transforming Growth Factor alpha

Fifteen laryngeal squamous cell carcinomas were investigated for the presence of transforming growth factor alpha (TGF-alpha) and epidermal growth factor (EGF) using immunohistochemical methods. In a recent study the same material was characterized for epidermal growth factor receptors (EGF receptors) which were confined predominantly to the undifferentiated cells. The expression of this growth factor system in malignant cells may play a role in carcinogenesis and/or tumour growth. All carcinomas were positive for TGF-alpha and 12 were positive for EGF. In moderately-to-well differentiated carcinomas, the immunoreactivity was mainly detected in the cytologically more differentiated cells. Nine sections included both laryngeal stratified squamous epithelium of normal appearance and carcinoma. The immunoreactivity was here again localized in the cytologically more differentiated cells above the basal cell layer. The present investigation and our previous results confirm the existence of EGF receptors, TGF-alpha and EGF in laryngeal carcinomas. In addition, we conclude that the conditions do exist for growth factors to act through an autocrine system in poorly differentiated tumours and through a paracrine system in the moderately-to-well differentiated tumours.

Topics: Carcinoma, Squamous Cell; Cell Differentiation; Cytoplasm; Endothelium; Epidermal Growth Factor; Epithelium; Exocrine Glands; Female; Humans; Immunohistochemistry; Keratins; Laryngeal Muscles; Laryngeal Neoplasms; Macrophages; Male; Mucins; Neoplasm Staging; Transforming Growth Factor alpha

Growth factor overexpression has been shown to be an integral part of the growth promotion of a number of tumors, including breast carcinoma, colon carcinoma, and sarcomas. The role of peptide growth factors in the regulation of growth of head and neck cancers has not been extensively investigated, however. In this study, we present our preliminary results of the pattern of expression of four growth factors: insulin-like growth factor II, transforming growth factor-alpha, basic fibroblast growth factor, and platelet-derived growth factor A chain in human head and neck tumors.. We examined eight benign and 27 malignant human head and neck surgical specimens for the expression of growth factor messenger RNAs using the polymerase chain reaction technique.. Our preliminary data demonstrate that growth factor messenger RNA is expressed by most of our malignant head and neck cancer specimens and by all eight benign head and neck tissue specimens. However, we were not able to show any distinction in the pattern of growth factor RNA expression among the malignant head and neck surgical specimens for the four growth factors studied to date.. These findings demonstrate that the expression of specific biologically active genes can be studied in routine surgical specimens using the polymerase chain reaction. The clinical prognostic significance of the overexpression of growth factors in head and neck malignancy as well as future therapeutic implications are discussed.

Topics: Base Sequence; Carcinoma, Squamous Cell; Electrophoresis, Agar Gel; Fibroblast Growth Factor 2; Gene Expression Regulation, Neoplastic; Growth Substances; Head and Neck Neoplasms; Humans; Insulin-Like Growth Factor II; Molecular Sequence Data; Platelet-Derived Growth Factor; Polymerase Chain Reaction; RNA; RNA, Messenger; Transforming Growth Factor alpha

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

We have previously shown that four human oesophageal squamous cell carcinoma (SCC) cell lines secrete significant quantities of transforming growth factor alpha (TGF-alpha) in vitro. Three of these lines are known to produce supernumerary low-affinity epidermal growth factor receptors (EGF-Rs). Using an 125I-EGF competitive binding assay and Scatchard analysis, we show that the fourth also overproduces low-affinity receptors. According to slot blot DNA analyses, the secretion of high levels of TGF-alpha is not associated with amplification of the TGF-alpha gene, and hyperproduction of the EGF-R is correlated with receptor gene amplification. Western blot analyses show that the c-Myc protein is overexpressed in two of the cell lines; and Southern and Northern blot analyses indicate that this overexpression occurs independently of c-myc gene amplification. Our results are consistent with an autocrine role for TGF-alpha and EGF-R in oesophageal carcinogenesis and support the possibility that c-myc overexpression may be required for the in vivo tumourigenicity of cells that produce high levels of TGF-alpha and the EGF-R.

Topics: Adenocarcinoma; Carcinoma, Squamous Cell; Colonic Neoplasms; ErbB Receptors; Esophageal Neoplasms; Gene Amplification; Gene Expression Regulation, Neoplastic; Genes, myc; Humans; Neoplasm Proteins; Transforming Growth Factor alpha; Tumor Cells, Cultured

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

The squamous mucosa of patients who develop head and neck cancer is "condemned" or predisposed to disregulated growth as reflected by the high incidence of synchronous and metachronous primary tumors. We hypothesized that transformed and nontransformed mucosa from head and neck cancer patients would produce increased levels of transforming growth factor alpha (TGF-alpha) and its cell surface receptor, the epidermal growth factor receptor (EGFR), thereby contributing to this predisposition. Using molecular biological techniques, we examined the incidence and mechanism of TGF-alpha and EGFR overproduction in tumors and histologically normal mucosa excised from patients with squamous cell carcinoma of the head and neck (SCCHN) to test this hypothetical mechanism of field cancerization. Northern blot hybridization was used to evaluate the frequency of increased TGF-alpha and EGFR mRNA production in tissue excised from 24 patients with SCCHN and 10 cell lines compared with 7 control patients without cancer or a history of alcohol and tobacco use. Southern blot hybridization was used to examine for gene amplification. In patients with SCCHN, TGF-alpha mRNA was elevated by a mean of 5-fold in 95% of histologically "normal" mucosa samples (P = 0.001) and by a mean of 5-fold in 87.5% of tumors (P = 0.0001) while EGFR mRNA was elevated by a mean of 29-fold in 91% of histologically normal mucosa specimens (P = 0.0005) and by a mean of 69-fold in 92% of tumors (P = 0.0005), compared with mRNA levels in control normal mucosa. In 10 SCCHN cell lines, TGF-alpha mRNA was increased by a mean of 16-fold and EGFR mRNA levels were increased by a mean of 77-fold. Increased production of TGF-alpha and EGFR mRNA in the histologically normal mucosa of patients at risk for a primary or secondary head and neck cancer may serve both as a marker for malignant transformation and as a target for preventive therapies.

Topics: Aged; Aged, 80 and over; Biomarkers, Tumor; Carcinoma, Squamous Cell; ErbB Receptors; Female; Head and Neck Neoplasms; Humans; Male; Middle Aged; RNA, Messenger; RNA, Neoplasm; Transforming Growth Factor alpha; Tumor Cells, Cultured; Up-Regulation

We investigated the growth-regulatory mechanism of 2 esophageal squamous-cancer cell lines, TE2-NS and TE3-OS cells, both of which can grow stably in protein-free conditions in vitro. Protein-free conditioned media from TE2-NS and TE3-OS cells stimulated the growth of these cells. Exogenous epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), insulin-like growth factor (IGF)-I and -II enhanced cell proliferation by 2.2- to 3.8-fold in protein-free conditions, as compared with an untreated control. Receptor-binding assays showed that both TE2-NS and TE3-OS cells possessed a single class of high-affinity binding sites for IGF-I and 2 classes of binding sites for TGF-alpha, as confirmed on the cell membrane by immunochemistry. These results suggest that EGF, TGF-alpha and IGFs are candidates for the autocrine growth factor in cancer cells. The addition of inhibitory monoclonal antibodies against TGF-alpha and EGFR, but not those against either EGF or IGF-IR, significantly inhibited growth of the cells. Immunocytochemical staining and ELISA of the conditioned media both confirmed the production of TGF-alpha protein, but not EGF protein, in these cell lines. The data for a protein-free culture system strongly suggested that TGF-alpha, but not EGF or IGF, is biologically important as an autocrine growth factor in the growth of these cell lines in vitro.

Topics: Antibodies, Monoclonal; Carcinoma, Squamous Cell; Cell Division; Culture Media, Serum-Free; Epidermal Growth Factor; ErbB Receptors; Esophageal Neoplasms; Humans; Insulin-Like Growth Factor I; Insulin-Like Growth Factor II; Receptor, IGF Type 1; Transforming Growth Factor alpha; Tumor Cells, Cultured

Modification of proteins with monomethoxy-polyethylene glycol (mPEG) has been shown to prolong circulation time and to reduce immunogenicity. To make a mPEG-modified recombinant toxin that retained cytotoxic activity but had a longer residence time in circulation, we have constructed an altered form of TGF alpha-PE40, a recombinant toxin composed of human transforming growth factor alpha (TGF alpha) fused to a fragment of Pseudomonas exotoxin (PE38) devoid of its cell-binding domain. In the newly designed protein, termed TGF alpha R29-L2-CH2-PE38QQ delta (TCP), there are no lysine residues in the TGF alpha and PE38 portions. Human IgG4 constant region CH2 and a tetradecapeptide linker; L2, are inserted between TGF alpha and PE38. Together, L2 and CH2 contain 13 lysine residues as potential modification sites for mPEG. mPEG conjugates of TCP (PEG-TCP) were generated and the products were resolved by ion exchange chromatography. Two PEG-TCP species termed B4 and B6 retained 15 and 4% of cytotoxicity, respectively, and 26% of their receptor binding activity compared with the unmodified TCP. Both B4 and B6 had prolonged circulation times in the blood and reduced toxicity in animals. The mean residence times of B4 and B6 were 37 and 68 min, respectively, compared to 7 min for TCP. When administered i.v. to tumor bearing mice, both B4 and B6 produced marked antitumor effects whereas the unmodified TCP had none. Also, the immunogenicity of PEG-TCP was 5-10 times less than that of TCP. We suggest that the prolonged circulating time and reduced toxicity of PEG-TCP compensate for a diminished cytotoxic activity and enlarge significantly the therapeutic window of this chimeric toxin.

Topics: ADP Ribose Transferases; Amino Acid Sequence; Animals; Antibodies; Bacterial Toxins; Carcinoma, Squamous Cell; Cloning, Molecular; Escherichia coli; Exotoxins; Humans; Immunoglobulin Constant Regions; Immunoglobulin G; Immunotoxins; Mice; Mice, Inbred BALB C; Mice, Nude; Molecular Sequence Data; Polyethylene Glycols; Pseudomonas aeruginosa; Pseudomonas aeruginosa Exotoxin A; Recombinant Fusion Proteins; Transforming Growth Factor alpha; Transplantation, Heterologous; Tumor Cells, Cultured; Virulence Factors

The authors recently used immunostaining to demonstrate that patients with epidermal growth factor receptor (EGFR) overexpression have poor survival after surgery. However, the clinical significance of transforming growth factor (TGF)-alpha, one of the ligands of EGFR, has not been demonstrated in esophageal carcinoma.. Immunohistochemical study for TGF-alpha and EGFR was performed on 57 esophageal squamous cell carcinomas using monoclonal antibodies.. TGF-alpha expression was positive in 35% of the tumors, and EGFR overexpression, defined as stronger staining in cancer cells than in normal epithelium, was positive in 43% of the tumors, according to the authors' arbitrary criteria. The incidence of TGF-alpha positivity was relatively higher in patients with the EGFR overexpression (EGFR+) than in the patients with non-overexpression (EGFR-). The survival rate was significantly lower in patients with TGF-alpha(+) than in those with TGF-alpha(-) (P < 0.01) and in patients with EGFR(+) than in patients with EGFR(-) (P < 0.01), respectively. Considering TGF-alpha and EGFR expression simultaneously, the survival rate of the patients with TGF-alpha(+)/EGFR(+) tumors was the lowest of the four subgroups, with statistically significant differences noted. These relationships between the immunoreactivities and survival curves were observed in the analysis within patients with node-positive disease. In addition, a multivariate statistical analysis demonstrated that TGF-alpha was the only significant variable, whereas EGFR and nodal status provided no additional information regarding postoperative survival.. The results presented suggest that TGF-alpha may act as an autocrine growth factor through hyperproducing EGFR and that its expression and EGFR overexpression may prove useful as a valuable prognostic indicator for patients with esophageal carcinoma.

Topics: Carcinoma, Squamous Cell; Cell Division; ErbB Receptors; Esophageal Neoplasms; Female; Humans; Immunoenzyme Techniques; Male; Multivariate Analysis; Prognosis; Survival Analysis; Transforming Growth Factor alpha

The expression of mRNAs for epidermal growth factor (EGF), transforming growth factor alpha(TGF alpha), EGFR, platelet-derived growth factor (PDGF) A and B chain, PDGF receptor (PDGFR), transforming growth factor beta (TGF beta), erbB-2 and estrogen receptor (ER) genes was first examined in 6 human esophageal carcinoma cell lines, 6 xenoplanted and 15 surgically resected esophageal carcinomas. Secondly, the effect of EGF and TGF alpha on the expression of these genes by the TE-1 esophageal carcinoma cell line was investigated. The expression of EGF mRNA was detected in 8 (29.6%) of 27 tumors including the cell lines, whereas the TGF alpha and EGFR genes were expressed in 21 (77.8%) and 24 (88.9%) tumors respectively. PDGF B chain and PDGFR were detected in 18 (66.7%) and 20 (74.1%), respectively, and ER mRNA was observed in 16 (59.3%) tumors. Genes for PDGF A chain and TGF beta and the erbB-2 gene were commonly expressed. On the other hand, exogenous EGF and TGF alpha stimulated the expressions of fos and myc genes by TE-1 cells. The expression of mRNAs for TGF alpha, PDGF A and B chain and the erbB-2 genes was also increased after treatment with EGF. TGF alpha increased the accumulation of mRNAs for EGF, TGF alpha, EGFR, PDGF A and B chain and the erbB-2 gene. Moreover, the expression of mRNAs for interstitial collagenase, stromelysin and type IV collagenase was increased after EGF or TGF alpha treatment. These results indicate that EGF and TGF alpha may regulate the multi-growth-factor receptor expression and may play a central role for tumor invasion and metastasis as autocrine modulators for human esophageal carcinoma.

Topics: Adenocarcinoma; Adult; Aged; Carcinoma, Squamous Cell; Epidermal Growth Factor; ErbB Receptors; Esophageal Neoplasms; Female; Gene Expression Regulation, Neoplastic; Humans; Male; Metalloendopeptidases; Middle Aged; Platelet-Derived Growth Factor; Receptors, Estrogen; Receptors, Platelet-Derived Growth Factor; RNA, Messenger; Transforming Growth Factor alpha; Transforming Growth Factor beta

Forty oral squamous cell carcinomas have been investigated immunohistochemically for the presence of transforming growth factor alpha (TGF-alpha) and epidermal growth factor (EGF). The same cases were recently characterized for the expression of EGF-receptors. TGF-alpha was detected with a monoclonal mouse antibody and EGF with polyclonal rabbit antiserum. Thirty-five of the tumours were positive for TGF-alpha and 26 of the tumours for EGF. None of the poorly differentiated tumours was positive for EGF, but they all were for TGF-alpha. In sections including normal differentiated oral mucosa, the cells above the basal cell layer were positive for both TGF-alpha and EGF. The same staining pattern was observed in oral mucosa obtained from healthy persons. In moderately to well differentiated carcinomas, the immunoreactivity was mainly confined to the cytologically more differentiated cells, thus paralleling the situation observed in the normal differentiated oral mucosa. In four cases, material was available from both a primary tumour and a metastasis. Three of these were positive for TGF-alpha and EGF with the same staining pattern as that of the primary tumours. This investigation together with our previous results confirms the existence of TGF-alpha, EGF, and EGF-receptors in the majority of oral squamous cell carcinomas and their metastases.

Topics: Carcinoma, Squamous Cell; Epidermal Growth Factor; Female; Humans; Immunohistochemistry; Male; Mouth Mucosa; Mouth Neoplasms; Transforming Growth Factor alpha

Hyperkeratosis of the palms and soles (tylosis) is an uncommon genetic disorder. A small number of English families, however, have been described in which it is associated with carcinoma of the esophagus. The current report is of the first American family described with this condition. Members of those families affected with tylosis have at least a 90% risk of esophageal carcinoma by age 65 years. The paired conditions have an autosomal dominant mode of transmission and probably are controlled at a single genetic locus. The actual pathologic state might be mediated through an increase in epidermal growth factor receptors in the abnormal tissues.

Topics: Carcinoma, Squamous Cell; ErbB Receptors; Esophageal Neoplasms; Family Health; Humans; Keratoderma, Palmoplantar; Male; Middle Aged; Pedigree; Transforming Growth Factor alpha

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

Transforming growth factor alpha (TGF-alpha) is a single chain polypeptide which exists in a variety of forms differing in molecular weight. These forms are variously present in normal and neoplastic cells. Of particular interest are TGF-alpha's well-known mitogenic properties. The transition from a normal to a neoplastic cellular state results from signalling defects that may depend upon, inter alia, abnormal levels of expression and secretion of TGF-alpha. It is known that the secretion of TGF-alpha may be enhanced appreciably by agents such as phorbol 12-myristate 13-acetate (PMA), serum factors and epidermal growth factor (EGF). Here, we compare the efficacy of these three agents in the elevation of TGF-alpha secretion in the well studied A431 cell line with their previously undocumented efficacy in certain interesting, but little known, human oesophageal squamous cell carcinoma (SCC) lines.

Topics: Carcinoma, Squamous Cell; Epidermal Growth Factor; ErbB Receptors; Esophageal Neoplasms; Humans; In Vitro Techniques; Secretory Rate; Tetradecanoylphorbol Acetate; Transforming Growth Factor alpha; Tumor Cells, Cultured

Epidermal growth factor receptor (EGFR), transforming growth factor alpha (TGFA), and p53 are frequently overexpressed in squamous cell carcinomas (SCC) of the upper aerodigestive tract. We chose to study SCC of the tongue base, which is often advanced at presentation and fatal, to evaluate whether overexpression correlates with survival. Complete follow-up was available for 20 patients, 18 of whom had stage III or IV disease. A number of clinical (age, sex, stage of disease) and histologic (tumor grade, keratinization, mitotic rate, perineural invasion, lymphatic invasion, vascular invasion, host response) variables were analyzed. None of these variables correlated with survival. Immunohistochemical analysis was performed on paraffin-embedded tissue from each patient. Because EGFR and TGFA expression were routinely found in normal squamous epithelium, overexpression was considered present if greater uptake of the antibody was manifested by a deeper immunostain. In contrast, p53 oncoprotein was not detected in normal epithelium, so detection of the antibody was believed to indicate overexpression. EGFR was overexpressed in 60% of tumors, TGFA in 35%, and p53 in 20%. Those patients who had an overexpression of p53 had a greater mean survival than those who did not (48 versus 16 months, respectively, p = 0.06). This difference was significant for patients with clinical stage IV lesions (p = 0.03). EGFR overexpression and TGFA overexpression did not correlate with survival. p53 may serve as a biologic marker indicative of improved survival potential.

Topics: Adult; Carcinoma, Squamous Cell; ErbB Receptors; Gene Expression Regulation, Neoplastic; Genes, p53; Humans; Middle Aged; Neoplasm Staging; Prognosis; Survival Rate; Tongue Neoplasms; Transforming Growth Factor alpha

Three morphologically distinct cell lines--F.2a, V, and B.2--were isolated from a single human squamous cell carcinoma. Although all three cell lines can grow indefinitely in culture, they differ in a number of important transformation-related phenotypes. Only B.2 is strongly tumorigenic when injected into the flanks of nude mice, and only V can efficiently grow in semisolid media. The dominance of these traits was investigated by generating somatic cell hybrids among the three cell lines. F.2a was able to suppress the tumorigenicity of B.2 cells, whereas B.2 inhibited the capacity for anchorage-independent growth of V, the latter trait being a function of the ability of these epithelial cells to differentiate when deprived of support. The influence of exogenously added growth factors was also evaluated. This study indicates that the particular tumor we examined consisted of a heterogeneous population of cells with distinct growth and differentiation capacities.

Topics: Animals; Carcinoma, Squamous Cell; Cell Adhesion; Cell Differentiation; Cell Division; Cell Fusion; Cell Line; Culture Media, Serum-Free; Epidermal Growth Factor; Genetic Markers; Humans; Keratinocytes; Mice; Mice, Nude; Neoplasm Transplantation; Phenotype; Transfection; Transforming Growth Factor alpha; Transplantation, Heterologous

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

Altered expression of growth factors and growth factor receptors is frequently described in human tumors and human tumor cell lines. This further supports the hypothesis that oncogenesis is due to the subversion of mitogen-responsive pathways. The aim of this study was to investigate the expression of epidermal growth factor receptor (EGFR) and transforming growth factor alpha (TGF alpha) in 13 larynx carcinomas and 2 carcinomas of the oral cavity. We found receptor overexpression in 7 out of 15 tumors at mRNA and/or protein level but low expression in the majority of the normal adjacent tissues. TGF alpha was expressed only in 1 case, but no tyrosine kinase activity of the receptor was detected by antiphosphotyrosine antibody.

Topics: Aged; Aged, 80 and over; Biomarkers, Tumor; Carcinoma, Squamous Cell; Electrophoresis, Polyacrylamide Gel; ErbB Receptors; Humans; Immunoblotting; Laryngeal Neoplasms; Middle Aged; Mouth Neoplasms; RNA, Messenger; RNA, Neoplasm; Transforming Growth Factor alpha

Multicellular tumour spheroids are cellular aggregates that can be prepared from many types of tumour cells. These three-dimensional structures provide a model for analysing the effects of cell-cell contact and intercellular microenvironments on phenomena such as autocrine regulation of growth factor synthesis. Autoregulation of the synthesis of transforming growth factor-alpha (TGF-alpha) was investigated at the message and protein levels in spheroid and monolayer cultures prepared from the A431 human squamous carcinoma cell line. The epidermal growth factor receptor (EGF-R) of these monolayer A431 cells had an average surface density of 2.2 x 10(6)/cell. Constitutive expression of TGF-alpha mRNA was an average of 3-fold greater in A431 spheroids than in monolayers, even for densely packed, confluent monolayers. This effect did not depend on hypoxic stress within the spheroids. TGF-alpha protein synthesis was enhanced in comparison with that in monolayer culture, reaching a value of up to 2-fold greater on a per cell basis. These results are discussed in the context of a TGF-alpha/EGF-R autocrine loop operating within cells that produce high local concentrations of TGF-alpha in the three-dimensional architecture of a spheroid.

Topics: Blotting, Northern; Carcinoma, Squamous Cell; Cell Aggregation; Cell Communication; Cell Count; Epidermal Growth Factor; Humans; Radioimmunoassay; RNA, Messenger; Time Factors; Transforming Growth Factor alpha; Tumor Cells, Cultured

Keratoacanthomas may be difficult to distinguish histologically from squamous cell carcinomas. We studied 20 keratocanthomas and 22 squamous cell carcinomas immunohistochemically using an antibody directed against transforming growth factor alpha to determine if the pattern of transforming growth factor alpha expression would provide a useful method of differentiating these tumors. Ninety percent of the keratoacanthomas demonstrated a diffuse pattern within tumor lobules in which all but the most peripheral rim of cells were stained. A similar localization of transforming growth factor alpha was not identified in squamous cell carcinomas. In addition, 40% of the squamous cell carcinomas but none of the keratoacanthomas showed focal transforming growth factor alpha immunostaining. Our results suggest that transforming growth factor alpha expression may be a marker of epithelial differentiation and may help distinguish between these two tumors.

Topics: Carcinoma, Squamous Cell; Diagnosis, Differential; Humans; Keratoacanthoma; Skin Diseases; Skin Neoplasms; Transforming Growth Factor alpha

Topics: Binding, Competitive; Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Epidermal Growth Factor; ErbB Receptors; Humans; Iodine Radioisotopes; Kinetics; Radioligand Assay; Transforming Growth Factor alpha

Transforming growth factor alpha is an autocrine mitogen for nonneoplastic keratinocytes, which exerts its function by binding to the receptor for epidermal growth factor. In order to determine whether this autocrine pathway is activated in squamous carcinoma cells, we analyzed the production of transforming growth factor alpha as well as the expression and regulation of epidermal growth factor receptors in a panel of human squamous carcinoma cell lines. Immunoreactive transforming growth factor alpha was detectable in squamous carcinoma cells as well as in quiescent nonneoplastic keratinocytes. However, in the absence of exogenous mitogens, only the squamous carcinoma cells secreted the growth factor into the medium, whereas untransformed keratinocytes did not. Each of the squamous carcinoma cell lines expressed significantly greater numbers of cell surface epidermal growth factor receptors than normal keratinocytes. The epidermal growth factor receptor gene was amplified and overexpressed in three of the squamous carcinoma cell lines (A431, CaSki, SqCC/Y1). Two of the squamous carcinoma cell lines (C4-1 and CE-48) displayed a relative inability to down-regulate epidermal growth factor receptors in response to epidermal growth factor. The mechanism of receptor overexpression in the remaining three cell lines (A253, CaLu-1, FaDu) is unexplained. Thus, human squamous carcinoma cell lines frequently exhibit a combination of the constitutive secretion of transforming growth factor alpha and the overexpression of epidermal growth factor receptors. Treatment of these tumor cells with an antibody directed against the ligand-binding domain of the epidermal growth factor receptor inhibited their growth by approximately 50%. These findings suggest that designing strategies to interrupt the transforming growth factor alpha autocrine pathway might lead to new modalities to treat this class of malignant tumors.

Topics: Carcinoma, Squamous Cell; Cell Division; DNA, Neoplasm; Down-Regulation; ErbB Receptors; Gene Expression Regulation, Neoplastic; Humans; Keratinocytes; RNA, Messenger; RNA, Neoplasm; Transforming Growth Factor alpha

Transforming growth factor-alpha (TGF-alpha) is a potent mitogen for epithelial cells that is expressed at low levels in normal epidermis and overexpressed in psoriasis. Epidermal growth factor (EGF) has been shown to inhibit hair growth but stimulate the growth of sebaceous and sweat glands, suggesting a potential role for a member of the EGF/TGF-alpha family in the normal development and function of skin appendages as well as epidermis. The present work demonstrates TGF-alpha protein in eccrine ducts, and eccrine, sebaceous, and apocrine glands. The proliferative dermal hair bulb does not express TGF-alpha in contrast to the differentiated outer root sheath hair follicle epithelia. In addition, hyperproliferative skin diseases including bullous congenital ichthyosiform erythroderma, squamous cell carcinoma, and psoriasis show increased TGF-alpha expression. Thus, TGF-alpha may play a role in the morphogenesis and function of normal skin appendages and its overexpression is common in benign and malignant hyperproliferative skin diseases.

Topics: Blotting, Western; Carcinoma, Squamous Cell; Cell Differentiation; Cell Division; Humans; Immunoenzyme Techniques; Psoriasis; Skin; Skin Neoplasms; Transforming Growth Factor alpha

TGF-alpha-PE40 is a chimeric protein composed of transforming growth factor alpha (TGF-alpha) linked to a modified Pseudomonas toxin from which the cell recognition domain has been deleted (PE40). TGF-alpha-PE40 has been shown to have cytotoxic effects on human cancer cell lines that express the epidermal growth factor (EGF) receptor on their surface, and when given i.p., it prolongs the survival of nude mice bearing i.p. tumors. Because several normal tissues, including liver, express EGF receptors on their surfaces, it has not been clear that this agent can be used systemically to treat EGF receptor-bearing tumors. In this study, we have delivered TGF-alpha-PE40 for 7 days by continuous infusion through a miniosmotic pump placed in the peritoneal cavity of nude immunodeficient mice. Two different human cancer cell lines that express EGF receptors on their surface were implanted s.c. One was A431, an epidermoid carcinoma; the other was DU-145, a prostate carcinoma. By using this mode of continuous i.p. delivery, we were able to achieve a constant serum level of TGF-alpha-PE40 that was nontoxic to the mice and yet delayed the growth of both tumors implanted s.c. and caused partial regression of one. We conclude that it is possible to deliver TGF-alpha-PE40 systemically and achieve a therapeutic serum level in mice without major toxicity. Although side effects may be expected, this study establishes that there is a therapeutic window for this agent in the therapy of cancers with high numbers of EGF receptors.

Topics: Animals; Carcinoma, Squamous Cell; Drug Screening Assays, Antitumor; Drug Stability; Exotoxins; Female; Infusion Pumps; Male; Mice; Mice, Nude; Prostatic Neoplasms; Recombinant Fusion Proteins; Transforming Growth Factor alpha; Tumor Cells, Cultured

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

Monoclonal antibody 425 binds to a protein epitope of the human EGF receptor and blocks EGF dependent functions such as EGF receptor phosphorylation and mitogenesis (1). We now show that MAb 425 blocks TGF alpha-induced second messenger signals, namely inositol 1,4,5 triphosphate and Ca2+ in two carcinoma cell lines, A 431 and SW-948. In this study we have further characterized the specificity of this antibody for inhibiting TGF alpha induced mitogenesis in MRC-5, a EGF-receptor expressing fibroblast cell line.

Topics: Antibodies, Monoclonal; Calcium; Carcinoma, Squamous Cell; Cell Line; DNA Replication; ErbB Receptors; Humans; Inositol 1,4,5-Trisphosphate; Kinetics; Second Messenger Systems; 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

The expression of epidermal growth factor receptor (EGF-R), transforming growth factor alpha (TGF alpha) and the c-myc oncogene was investigated in different specimens of gynecologic carcinomas. EGF specific binding sites were detected in about 50% of adenocarcinomas (ovarian, endometrial, breast) and in over 90% of squamous carcinomas (cervical). There is a positive correlation between the EGF-R binding assay, immunohistochemistry and the relative amounts of mRNA by Northern blotting. TGF alpha was investigated by immunohistochemistry and Northern blotting. TGF alpha immunoreactivity was detected exclusively in the epithelial cells of nonmalignant tissues (skin, cervix, endometrium, large bowel, lung) as well as different ovarian carcinomas. The TGF alpha immunostaining score correlates with the TGF alpha mRNA amounts. The c-myc expression was analyzed by Northern blotting in the specimens of ovarian carcinomas. Whereas, a positive correlation between the c-myc and TGF alpha expression was noticed, no correlation existed between EGF-R and c-myc expression. Progressive disease (PD) of ovarian carcinomas after chemotherapy was mainly noticed in the group of EGF-R- tumors and those with high amounts of c-myc mRNA. EGF-R+ ovarian carcinomas responded significantly better to chemotherapy. However, similar survival times existed between the EGF-R+ and EGF-R- group and the survival times of patients having responded to the treatment was reduced in the EGF-R+ group. This indicates that EGF-R+ and those carcinomas expressing high amounts of c-myc constitute a more aggressive group of ovarian carcinomas.

Topics: Adenocarcinoma; Antineoplastic Agents; Biomarkers, Tumor; Carcinoma, Squamous Cell; ErbB Receptors; Female; Humans; Oncogenes; Ovarian Neoplasms; Prognosis; RNA, Messenger; Transforming Growth Factor alpha