transforming-growth-factor-alpha and Melanoma

E1694 tested GM2-KLH-QS21 vaccine versus high-dose interferon-α2b (HDI) as adjuvant therapy for operable stage IIB-III melanoma. We tested banked serum specimens from patients in the vaccine arm of E1694 for prognostic biomarkers.. Aushon Multiplex Platform was used to quantitate baseline serum levels of 115 analytes from 40 patients. Least absolute shrinkage and selection operator proportional hazard regression (Lasso PH) was used to select markers that are most informative for relapse-free survival (RFS) and overall survival (OS). Regular Cox PH models were then fit with the markers selected by the Lasso PH. Survival receiver operating characteristic (ROC) analysis was used to evaluate the ability of the models to predict 1-year RFS and 5-year OS.. Four markers that include Tumor Necrosis Factor alpha Receptor II (TNF-RII), Transforming Growth Factor alpha (TGF-α), Tissue Inhibitor of Metalloproteinases 1 (TIMP-1), and C-reactive protein (CRP) were found to be most informative for the prediction of OS (high levels correlate with worse prognosis). The dichotomized risk score based on the four markers could significantly separate the OS curves (p = 0.0005). When using the four-marker PH model to predict 5-year OS, we achieved an area under the curve (AUC) of 89% (cross validated AUC = 72%). High baseline TNF-RII was also significantly associated with worse RFS. The RFS with high (above median) TNF-RII was significantly lower than low TNF-RII (p = 0.01).. The biomarker signature consisting of TNFR-II, TGF-α, TIMP-1 and CRP is significantly prognostic of survival in patients with high-risk melanoma and warrants further investigation.

Topics: Biomarkers, Tumor; C-Reactive Protein; Disease-Free Survival; Humans; Kaplan-Meier Estimate; Melanoma; Prognosis; Proportional Hazards Models; Receptors, Tumor Necrosis Factor, Type II; Risk Factors; Skin Neoplasms; Tissue Inhibitor of Metalloproteinase-1; Transforming Growth Factor alpha

Epidermal growth factor receptor (EGFR) activation by transforming growth factor alpha (TGFalpha) has been implicated in autocrine growth in melanoma, but does not alter melanocyte proliferation. This raises the possibility that different signalling pathways are activated via EGFR or ErbB receptors. Here, we demonstrate that ErbB2, ErbB3 and ErbB4 are expressed in cultured human melanocytes. Western analyses with receptor-specific antisera revealed protein bands with Mr values of 185 and 160 kDa, corresponding to ErbB2 and ErbB3, respectively. Blots probed with ErbB4 antibodies showed bands with Mr values of 180, 120 and 80 kDa, corresponding to the receptor and its reported variants. Two malignant melanoma cell lines expressed ErbB2 and ErbB3, but not the full-length ErbB4 receptor. As TGFalpha binds to EGFR and the heregulins (HRG) bind to ErbB3 and ErbB4, these growth factors were examined for effects on receptor activation and on cell growth and motility in a scratch wound closure assay. In normal melanocytes, HRGbeta1 activated the phosphorylation of tyrosine residues of proteins that immunoprecipitated with EGFR and ErbB4 antisera, and significantly enhanced cell migration but not proliferation. Neither TGFalpha nor HRGalpha1 promoted migration or growth in normal melanocytes. By contrast, TGFalpha stimulated migratory activity in the MM96L cell line, but not in the MELJG line, whereas HRGbeta1 significantly enhanced cell growth, but not migration, in both malignant cell lines. The apparent transition of HRGbeta1 from a migratory to a proliferative function after malignant transformation, and the change in TGFalpha from a non-migratory to a migratory activity in one melanoma line, suggests multiple switches in ErbB signalling pathways via EGFR/ErbB heterodimer formation.

Topics: Cell Movement; Cell Proliferation; ErbB Receptors; Humans; Immunoprecipitation; Melanocytes; Melanoma; Neuregulin-1; Phosphorylation; Receptor, ErbB-2; Receptor, ErbB-3; Receptor, ErbB-4; Signal Transduction; Transforming Growth Factor alpha; Wound Healing

Transforming growth factor-alpha (TGFalpha) has been implicated in melanocyte transformation, as it is expressed in melanocytic lesions and in melanoma cells. We investigated its role in melanoma development using a transgenic mouse model. The mice were generated by microinjection of a transgene with 270 bp of the mouse tyrosinase promoter and the cDNA for human TGFalpha. No significant skin abnormalities were found, but individuals from three transgenic lines developed ocular melanocytoses (seven out of 10 transgenics), usually after a long latency period. In particular, the melanocyte component of the choroid was thicker than in non-transgenic controls, consistent with hyperplasia. The retinal pigment epithelium was unaffected. Melanocytic lesions were also present in the posterior eye, and abnormal distributions of melanocytes were found in neural tissue of the brain, skeletal muscle of the head and the Harderian glands, indicating migration from the choroid. It was concluded that mice engineered to express the normal growth factor TGFalpha from a tyrosinase promoter spontaneously developed melanocytic lesions in the eye but not the skin.

Topics: Animals; Cell Transformation, Neoplastic; Eye; Eye Neoplasms; Melanocytes; Melanoma; Mice; Mice, Transgenic; Models, Genetic; Monophenol Monooxygenase; Promoter Regions, Genetic; Tissue Distribution; Transforming Growth Factor alpha; Transgenes

Previous research in animals supports the use of tyrosine and phenylalanine (Tyr-Phe) restriction as an adjuvant to the treatment of cancer. In this regard, dietary restriction of Tyr-Phe specifically inhibits the growth of B16BL6 melanoma tumors, dramatically suppresses spontaneous hematogenous metastasis, and modulates the sensitivity of these tumor cells to growth factors. Two chimeric toxins, HB-TGF alpha-PE4EKDEL and TGF alpha-PE4EKDEL, were examined for their toxicity against the B16BL6 melanoma cell line, and the ability of Tyr-Phe limitation to modulate the potential of these toxins was examined. Tyr-Phe limitation significantly enhanced the cytotoxic effects of HB-TGF alpha-PE4EKDEL approximately 10-fold toward B16BL6 melanoma, and free heparin diminished the cytotoxicity of HB-TGF alpha-PE4EKDEL. Although TGF alpha-PE4EKDEL is cytotoxic to this cell line, Tyr-Phe limitation did not effect the cytotoxicity of this toxin. Tyr-Phe limitation inhibited the synthesis and secretion of heparin-binding proteins but did not alter the expression of surface heparan sulfate proteoglycans. These data suggest that cell surface heparan sulfate proteoglycan is a target for binding and execution of the cytotoxicity of HB-TGF alpha-PE4EKDEL and that augmentation of cytotoxicity by Tyr-Phe limitation is due to the inhibition of heparin-binding protein production.

Topics: Animals; Antineoplastic Agents; Carrier Proteins; Cell Membrane; Culture Media; Epidermal Growth Factor; Heparin; Melanoma; Mice; Phenylalanine; Proteoglycans; Recombinant Fusion Proteins; Transforming Growth Factor alpha; Tumor Cells, Cultured; Tyrosine

Prion diseases are transmissible spongiform encephalopathies of humans and animals characterized by the accumulation of a proteinase-resistant isoform of the cellular prion-related protein (PrPc) within the central nervous system. In the present report we demonstrate for the first time the presence of PrPc on squamous epithelia of normal and diseased human skin and show that inflammatory cytokines regulate PrPc expression in cultured human keratinocytes (KCs). By immunohistochemistry, only little expression of PrPc, which was mainly confined to KCs, was detected in normal skin. In contrast, in inflammatory skin diseases including psoriasis and contact dermatitis, PrPc was strongly present on both KCs and infiltrating mononuclear cells. Strong PrPc expression was also observed in squamous cell carcinomas and viral warts whereas basal cell carcinomas were mostly negative. In mucous membranes of the upper digestive tract and the genital region, distinct PrPc expression by basal squamous epithelial cells was a constant feature. In tissue culture, primary KCs constitutively expressed PrPc mRNA and protein. Exposure of these cells to transforming growth factor (TGF)-alpha or interferon (IFN)-gamma led to an increase of PrPc protein expression. The presence of PrPc on epithelial cells of skin and mucous membranes suggests that these cells represent possible first targets for peripheral infection with prions.

Topics: Blotting, Western; Cells, Cultured; Dermatitis; Flow Cytometry; Humans; Interferon-gamma; Keratinocytes; Melanoma; Mucous Membrane; PrPC Proteins; Transforming Growth Factor alpha; Tumor Cells, Cultured; Up-Regulation

Atypical (dysplastic) nevi are melanocytic lesions, which are precursors of melanoma as well as markers of increased melanoma risk. Although these lesions exhibit distinct clinical and histological features, their molecular features are largely unknown. To determine whether atypical, compared to benign nevi, from patients with a clinical history of malignant melanoma reveal molecular changes, we analyzed these lesions for the expression of two growth factors (basic fibroblast growth factor and transforming growth factor alpha), their receptors (fibroblast growth factor receptor-1 and epidermal growth factor receptor), and two cell adhesion molecules (MUC18 and alpha v beta 3 integrin), all of which are expressed in primary and metastatic melanomas. The results demonstrated a statistically significant correlation (P = 0.02) between increasing degrees of histological atypia and expression of epidermal growth factor receptor in the epidermal keratinocytes of atypical melanocytic lesions. Furthermore, both atypical and benign nevi revealed considerably high levels of overall gene activity in their dermal melanocytic and epidermal keratinocytic compartments. In contrast, the epidermal-dermal junction wherein melanoma evolves showed little gene activity, suggesting that molecular events occurring adjacent to this junction may be important for melanocytic transformation.

Topics: Antigens, CD; Biomarkers, Tumor; CD146 Antigen; Cell Adhesion Molecules; Dysplastic Nevus Syndrome; ErbB Receptors; Fibroblast Growth Factor 2; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; In Situ Hybridization; Melanoma; Membrane Glycoproteins; Neural Cell Adhesion Molecules; Receptors, Fibroblast Growth Factor; Receptors, Vitronectin; RNA, Messenger; Skin Neoplasms; Transforming Growth Factor alpha

The aromatic fatty acids phenylacetate (PA) and phenylbutyrate (PB) induce tumour cell differentiation in experimental models and both are currently in clinical trials. The purpose of this study was to determine the effect of these antitumour agents on the expression of transforming growth factor-alpha (TGF-alpha) in neoplastic cells. Treatment of human melanoma 1011 cultures with either PA or PB caused over 40-fold increase in TGF-alpha biosynthesis and secretion into the media. Whereas elevation in TGF-alpha mRNA steady-state levels became evident within 6-12 h and reached peak quantities the following day, the amounts of its coded protein increased gradually over a period of 5 days of treatment. Further molecular analysis revealed that regulation of TGF-alpha expression occurred at the transcriptional level. In contrast to TGF-alpha, expression of its receptor remained below detectable levels, indicating that an autocrine loop involving this growth factor is unlikely. Interestingly, the increase in TGF-alpha production paralleled drug-induced cytostasis and differentiation defined by morphological changes and increased melanogenesis. Like PA and PB, other differentiation inducers such as all-trans-retinoic acid, dimethyl sulfoxide, and 5-aza-2'-deoxycytidine, all induced TGF-alpha expression in the melanoma cells. The close association between enhanced TGF-alpha production and melanoma cell differentiation suggests that this growth factor, often linked to mitogenesis, may play a novel role in tumour differentiation by PA and PB.

Topics: Antineoplastic Agents; Cell Differentiation; Culture Media; Gene Expression Regulation, Neoplastic; Humans; Melanoma; Phenylacetates; Phenylbutyrates; Transcription, Genetic; Transforming Growth Factor alpha; Tumor Cells, Cultured; Up-Regulation

The purpose of this study was to determine the mRNA expression level of multiple cytokine and growth factor genes in human malignant melanoma. Melanoma cells were isolated from several surgical specimens, adapted to growth in culture, characterized for their ability to produce experimental metastases in nude mice, and assessed for cytokine and growth factor steady-state gene expression. Highly metastatic in vivo- and in vitro-derived variants isolated from a single melanoma, A375, were also analyzed. Northern blot analyses revealed that all melanomas analyzed constitutively expressed steady-state mRNA transcripts for the growth and angiogenic factors, basic fibroblast growth factor (bFGF), and transforming growth factor alpha (TGF-alpha), which correlated with metastatic propensity. Only one highly metastatic melanoma, TXM-1, originally isolated from a lymph node metastasis, expressed mRNA transcripts specific for monocyte chemotactic and activating factor (MCAF) and granulocyte-macrophage colony-stimulating factor (GM-CSF). Similarly, of the nine melanomas examined, only TXM-1 expressed interleukin (IL)-1 alpha, IL-1 beta, and IL-6, important immunomodulatory cytokines. These data demonstrate the differential and heterogeneous expression of cytokine and growth factor genes in human malignant melanoma.

Topics: Animals; Chemokine CCL2; Chemotactic Factors; Cytokines; Fibroblast Growth Factor 2; Gene Expression Regulation, Neoplastic; Genetic Heterogeneity; Granulocyte-Macrophage Colony-Stimulating Factor; Growth Hormone; Homeostasis; Humans; Interleukins; Melanoma; Mice; Mice, Nude; Neoplasm Transplantation; RNA, Messenger; Transforming Growth Factor alpha

A sensitive sandwich enzyme immunoassay (EIA) for transforming growth factor-alpha (TGF-alpha) utilizing a polyclonal antibody that recognizes limited epitopes of both human TGF-alpha and rat TGF-alpha in combination with a monoclonal anti-TGF-alpha IgG1 galactosidase conjugate was developed. This assay shows no cross-reactivity with human epidermal growth factor. We can quantify the TGF-alpha level in not only human TGF-alpha (detection limit: 1 pg/ml), but also rat TGF-alpha (detection limit: 10 pg/ml) by virtue of cross-reactivity. Employing this assay system, we demonstrated that TGF-alpha is present in both human submandibular glands and submandibular/sublingual saliva.

Topics: Animals; Humans; Immunoenzyme Techniques; Male; Melanoma; Rats; Rats, Sprague-Dawley; Saliva; Submandibular Gland; Transforming Growth Factor alpha; Tumor Cells, Cultured

Stimulation of epidermal growth factor (EGF) receptor by ligands such as transforming growth factor (TGF) alpha may be associated with cell proliferation or transformation in both nevocytes and keratinocytes. Previously, EGF receptors have been identified within a variety of pigmented lesions, suggesting a possible responsiveness to ligands such as TGF alpha. In the present study, we characterize the intralesional expression and distribution of immunoreactive TGF alpha protein by avidin-biotin immunoperoxidase localization in benign nevi, congenital nevi, dysplastic nevi, and malignant melanomas. In situ hybridization techniques with TGF alpha riboprobes confirmed the constitutive production of TGF alpha in all types of pigmented lesions. The localization of TGF alpha expression to nevocytes when coupled with the previous reports of expression in basal keratinocytes suggests the possibility of either an autocrine mechanism of action for TGF alpha or a paracrine interplay of TGF alpha between keratinocytes and nevocytes within melanocytic lesions. An increase in immunoreactive TGF alpha in nevocytes was noted in both benign and dysplastic nevi from dysplastic nevus patients, as compared to benign nevi from normal patients. Congenital nevi and malignant melanomas showed an intermediate and variable level of TGF alpha immunoreactivity. When coupled with previous studies the data suggest linkage of the TGF alpha/EGF receptor pathway in the evolution of melanocytic lesions.

Topics: Adolescent; Adult; Child; Child, Preschool; Dysplastic Nevus Syndrome; Female; Humans; Immunohistochemistry; In Situ Hybridization; Male; Melanocytes; Melanoma; Middle Aged; Nevus; RNA, Messenger; Tissue Distribution; Transforming Growth Factor alpha

Evidence indicates DNA methylation as a part of the regulatory machinery controlling mammalian gene expression. The human melanoma cell line HA-A expresses low levels of transforming growth factor alpha (TGF-alpha). TGF-alpha mRNA accumulated, however, in response to DNA demethylation induced by a nucleoside analog, 5-azacytidine (5-azaC). The importance of DNA methylation in the TGF-alpha promoter region was examined by a transient transfection assay with luciferase reporter plasmids containing a portion of the TGF-alpha promoter. 5-azaC treatment of HA-A cells before the transfection caused a significant increase in the luciferase activity. Since input plasmids were confirmed to remain unmethylated, DNA demethylation of the TGF-alpha promoter itself does not account for the observed increase in TGF-alpha mRNA. Using an electrophoretic mobility shift assay, enhanced formation of protein-TGF-alpha promoter complex was detected in response to 5-azaC treatment. This 5-azaC-induced complex was shown to contain the transcription factor Sp1 by the following criteria: the protein-DNA complex formed on the TGF-alpha promoter contained immunoreactive Sp1; the mobility of the complex in an electrophoretic mobility shift assay was similar to that formed by recombinant Sp1; and DNase I footprinting analysis demonstrated that the 5-azaC-induced complex produced a footprint on the TGF-alpha promoter identical to that of authentic Sp1. These observations suggest that 5-azaC induces TGF-alpha expression by augmenting the Sp1 activity. However, neither the Sp1 mRNA nor its protein was induced by 5-azaC. These results suggest that in HA-A cells, TGF-alpha expression is down-modulated by DNA methylation. In addition, this process may involve the specific regulation of Sp1 activity without altering the amount of the transcription factor.

Topics: Azacitidine; Base Sequence; DNA; Gene Expression Regulation; Humans; Kinetics; Melanoma; Methylation; Molecular Sequence Data; Plasmids; Promoter Regions, Genetic; RNA, Messenger; Sp1 Transcription Factor; Transfection; Transforming Growth Factor alpha; Tumor Cells, Cultured

Low, mitogenic fluences of UVC (3.7-5.6 Jm-2) have previously been shown to cause increases of radioimmunoassayable transforming growth factor alpha (TGF alpha) in the medium and cells of cultures of melanocytes, melanoma lines, and HeLa cells (Ellem, K.A.O., Cullinan, M., Baumann, K.C., Dunstan, A.: Carcinogenesis 9:797-801, 1988). Here the cellular mechanism of this increase is explored by Northern blotting to detect any changes in TGF alpha mRNA levels, and the use of inhibitors of macromolecular synthesis to attempt to block the increase in TGF alpha protein. We were unable to detect any increase in TGF alpha mRNA levels attributable to UVC between 2 and 24 hours after irradiation. Inhibition of DNA synthesis (arabinosylcytosine, 10 microM), RNA synthesis (actinomycin D, 3 micrograms/ml; DRB 93 microM), or protein synthesis (cycloheximide, 10 micrograms/ml) failed to prevent the UVC induced increase in TGF alpha. We conclude that the UVC induction of TGF alpha is by a posttranslational mechanism. There was considerable discordance between the amount of TGF alpha protein and its mRNA in cultures of 15 different melanoma cell lines, which again emphasized that posttranscriptional mechanisms modulate the release of immunodetectable TGF alpha. We also found that the inhibitors themselves were capable of inducing an increase in TGF alpha in MM229 cultures. This suggests that the inhibitors and UV may effect the increase by a common mechanism, perhaps the activation of cell surface proteases as suggested for other stimuli (e.g., Pandiella, A., and Massagué, J.: Proc. Natl. Acad. Sci., USA 88:1726-1730, 1991) and that the response may be part of a global response to perturbation of DNA synthesis.

Topics: Cell Death; Humans; Melanoma; Nucleic Acids; Protein Biosynthesis; Proteins; RNA, Messenger; Time Factors; Transforming Growth Factor alpha; Tumor Cells, Cultured; Ultraviolet Rays