[CANCER RESEARCH 59, 547–550, February 1, 1999] Advances in Brief Independent Regulation of Growth and SMAD-mediated Transcription by Transforming Growth Factor  in Human Melanoma Cells 1 Ulrich Rodeck, Takafumi Nishiyama, and Alain Mauviel 2 Department of Dermatology and Cutaneous Biology, Jefferson Medical College [U. R., T. K., A. M.], Jefferson Institute of Molecular Medicine [U. R., A. M.], and the Kimmel Cancer Center [U. R.], Thomas Jefferson University, Philadelphia, Pennsylvania 19107 Abstract Increased production of transforming growth factor  (TGF-) cou- pled with resistance to the growth-inhibitory effects of TGF- is charac- teristic of several types of neoplasia including human melanoma. In select epithelial malignancies, lack of TGF--induced growth inhibition is asso- ciated with disruptions of TGF--dependent SMAD signaling and tran- scription. In contrast, the results of the present study indicate intact SMAD-dependent transcription in human melanoma cells, regardless of their proliferative response to exogenous TGF-. Furthermore, in some melanoma cell lines constitutive SMAD-dependent transcription was ob- served, which was due in part to endogenous TGF-. These results establish that resistance of melanoma cells to TGF--induced growth inhibition occurs independently of intact TGF- receptor/SMAD-medi- ated transcriptional regulation. They also suggest that melanoma-derived TGF- may exert autocrine effects on SMAD-sensitive target genes. Introduction TGF 3 - superfamily members (activin, bone morphogenic proteins, TGF-s, decapentaplegic) are multifunctional cytokines that affect cell proliferation, interaction with the extracellular matrix, differenti- ation, and/or survival. All TGF- family members signal through multimeric serine/threonine kinase receptor complexes on the cell surface, which phosphorylate cytoplasmic mediators called SMADs. Specifically, upon phosphorylation by activated TGF- receptors, ligand/receptor-specific SMADs (SMAD2 and/or SMAD3 in the case of the three mammalian TGF- isoforms) associate with SMAD4 and translocate as a complex to the nucleus to transactivate promoters containing SBSs (reviewed in Ref. 1). Many, if not all, of the biolog- ical effects of TGF- are considered to be SMAD dependent, through transcriptional regulation of extracellular matrix, adhesion, and growth regulatory genes. Cell cycle progression of normal epithelial cells is inhibited by exogenous TGF-, whereas malignant epithelial cells are often resist- ant to the growth-inhibitory effects of TGF-. Acquired resistance to the growth inhibitory effects of TGF- is generally considered as a mechanism by which malignant tumor cells subvert normal growth controls. In some epithelial malignancies, TGF- resistance is asso- ciated with functional inactivation of either the TGF- receptors (2, 3) or of signal transducers of the SMAD family (4), suggesting that resistance to TGF--induced growth inhibition is due to disrupted TGF-/SMAD-dependent transcriptional regulation. In particular, SMAD4, also known as DPC4 (deleted in pancreatic carcinoma), has frequently been found to be nonfunctional, either by chromosomal deletion or by mutation in pancreatic and, to a lesser degree, other epithelial malignancies (5). TGF- receptor and/or SMAD activation have been shown to up-regulate mRNA expression of the cell cycle inhibitors p21 (6) and p27 (7), suggesting that TGF--mediated cell growth inhibition is due, at least in part, to up-regulation of cell cycle inhibitory genes. Taken together, these results show that disruption of TGF- receptor/SMAD signaling provides one mechanism by which tumor cells may escape TGF--induced growth inhibition. On the basis of its role in mediating the growth-inhibitory effects of TGF- in normal cells and its loss of function in some tumor types, SMAD4 is considered to represent a tumor suppressor protein (5). Melanocytes are derived from the neuroectoderm and, like epithe- lial cells, are highly sensitive to growth inhibition by TGF- (8). By contrast and similar to carcinomas, many malignant melanomas ex- hibit various degrees of resistance to the growth-inhibitory effects of TGF- (8, 9). The molecular basis of TGF- resistance in melanomas and its relationship to TGF- receptor/SMAD signaling is not under- stood. This led us to investigate whether, in human melanoma cells, TGF- resistance was associated with functional inactivation of TGF- receptor/SMAD-dependent signal transduction and transcrip- tion. We describe that, in contrast to pancreatic carcinoma cells, melanoma cells could be induced to efficiently activate SMAD3/4- mediated transcription in a TGF--dependent manner. SMAD-in- duced transcriptional activity in melanoma cells did not correlate with effects of exogenous TGF- on proliferation of these cells. In addi- tion, tumor-derived TGF- contributed to comparatively high consti- tutive activity of SMAD-dependent transcription in some melanoma cell lines. These results indicate that, in contrast to some epithelial cancers, transcriptional regulation of gene expression via SMAD signaling pathways was preserved in human melanoma cells. Materials and Methods Cells and Culture Conditions. TGF--induced effects on proliferation of the primary (WM902-B, WM983-B, and WM793) and metastatic (WM239-A, WM164, and WM852) melanoma cell lines used in the present study have been described before (10). The cell lines 451-LU and 1205-LU were derived from WM164 and WM793, respectively, by serial passage through athymic mice and selection of cells metastatic to the lungs (9). FM516SV3/3 are postcrisis, immortalized, nontumorigenic melanocytes transformed by transfection with the SV40T antigen (11). Unless otherwise noted, melanoma and FM516SV3/3 cells were grown in a composite medium (W489) consisting of three parts MCDB153 and one part L15 supplemented with 2% (by volume) FCS. Normal melanocytes (FM1085 and FM1094), kindly provided by Dr. M. Herlyn, were propagated in W489 medium supplemented with 10 g/ml insulin, 10 ng/ml EGF, 100 g/ml bovine pituitary extract, 10 ng/ml phorbol 12-myristate 13-acetate, and 2% fetal calf serum and used between passages 15 and 20. Human dermal fibroblasts were isolated and propagated from neonatal foreskin using standard procedures. Human recombinant TGF-1 and pan-TGF- neutralizing antibody were Received 10/20/98; accepted 12/14/98. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This study was supported by Grant CA25874 from the NIH (to U. R.) and a grant from the W. W. Smith Charitable Trust (to A. M.). 2 To whom requests for reprints should be addressed, at Department of Dermatology and Cutaneous Biology, Jefferson Medical College, 233 South 10th Street, Room 430, Philadelphia, PA 19107. Phone: (215) 503-5622/0113; Fax: (215) 923-9354; E-mail: alain.mauviel@mail.tju.edu. 3 The abbreviations used are: TGF, transforming growth factor; SBS, SMAD binding sequence; CAT, chloramphenicol acetyltransferase; TK, thymidine kinase; CDK, cyclin- dependent kinase. 547 Research. on August 10, 2015. © 1999 American Association for Cancer cancerres.aacrjournals.org Downloaded from