Expression of Activated N-ras in a Primary Melanoma Cell Line Counteracts Growth Inhibition by Transforming Growth Factor-β

Abstract

One critical factor in melanoma progression is the change from radial growth phase to vertical growth phase. We previously showed a high incidence of ras mutations in progressing but not early human melanomas. We also found that stable expression of activated Ras in a primary human melanoma cell line (WM35) led to enhanced proliferation, anchorage-independent survival, migration and invasion in vitro and enhanced subcutaneous tumor formation in vivo, transforming the melanoma phenotype from the radial growth phase to the vertical growth phase. Inhibitory cytokines, especially transforming growth factor-beta, are important in homeostasis of normal human melanocytes. Proliferation of early melanoma cells can be inhibited by transforming growth factor-beta, whereas more aggressive stages lose this response. Using a transforming growth factor-beta activated luciferase reporter transiently transfected into WM35, WM35N-ras, and WM35H-ras (WM35 transfected with mutant N-ras or H-ras genes), we demonstrated significant decreases (p < 0. 04) in transforming growth factor-beta induced reporter expression in both ras transfected cell lines. Transforming growth factor-beta also induced significant decreases (p < 0.002) in the proportion of WM35 cells in S-phase of the cell cycle; this effect was not observed in WM35N-ras cells. Furthermore, we demonstrated that an important controlling factor in transforming growth factor-beta inhibition of cell cycle progression, the phosphorylation of the Rb protein, was altered in WM35N-ras; transforming growth factor-beta caused a marked relative increase in hypophosphorylated pRb in WM35 cells, but not in WM35N-ras. These data suggest that activated Ras plays an important part in melanoma progression from the radial growth phase to the vertical growth phase by counteracting inhibition by cytokines such as transforming growth factor-beta, thus providing a growth advantage.

Melanoma cells secrete follistatin, an antagonist of activin-mediated growth inhibition

“…Although it is possible that melanoma cells indeed secrete only residual amounts of activin A, we cannot exclude that it is rapidly degraded, or alternatively, remains associated with the cell surface, prohibiting measurement in the CM. Therefore, although several of the melanoma cell lines had higher expression of the bA subunit, when compared to the levels found in non-PMA treated melanocytes, a role for activin in melanomagenesis, as has been proposed for TGF-b (Shellman et al, 2000;Berking et al, 2001), remains speculative. Further in vitro and in vivo experiments, using activin-transduced cells, are needed to clarify this issue.…”

“…Alternatively, activation of the MAPK pathway, either by mutation of signaling molecules (N-ras and B-raf) (Davies et al, 2002;Tuveson et al, 2003) or by autocrine loops (La´za´r-Molna´r et al, 2000;Stove et al, 2003a) was shown to be of major importance in many melanomas. Constitutive activation of this pathway was shown to result in insensitivity to growth inhibition by TGF-b, while still allowing other effects of these molecules to occur (Shellman et al, 2000).…”

cDNA array technology in melanoma: An overview

“…Among these, the data suggest that activated ras plays a crucial role in melanoma progression from RGP to VGP pattern and that ras activation is associated with the nodular type of melanoma (Shellman et al, 2000). Moreover, a crucial role in the genesis of vertical growth seems to be played by adhesion molecules, such as Mel-CAM/MUC18, aand b-integrins and b-catenin (Van Belle et al, 1999;Kageshita et al, 2001).…”

Inhibition of the Motility and Growth of B16F10 Mouse Melanoma Cells by Dominant Negative Mutants of Dok-1

“…Dok family proteins are thought to regulate in either a positive or a negative manner the activity of Ras (18,47), which is itself an important regulator of cell motility and cell growth (11,19,28,30,33). The activation state of Ras has been shown to correlate with invasiveness, proliferation, and anchorage-independent growth of melanoma cells (12,36). To investigate the possible role of Ras in the effects of the dominant negative mutants of Dok-1, we therefore examined Ras activity in the various B16F10 cell lines with the use of a binding assay in which the GTP-bound (activated) form of Ras was precipitated from cell lysates with a GST fusion protein containing the Ras-binding domain of c-Raf-1.…”