The multifunctional cytokine Transforming Growth Factor-β (TGFβ) induces cell cycle arrest in normal epithelial cells. This effect is mediated primarily through the activation of TGFβ receptor (TβR) kinases, which, in turn, phosphorylate and activate downstream target proteins called Smads. By and large, cancer cells escape from growth inhibition by TGFβ early on during malignant transformation. However, only in a small fraction of cases is this caused by direct inactivation of one of the TGFβ signaling pathway components. This observation has raised the question whether activation of mitogenic or survival pathways might overcome the ability of TGFβ to induce cell cycle arrest. For example, several studies have implicated activation of the proto-oncogenes c-H-ras and c-myc as the root cause for escape from TGFβ control. However, these observations have been difficult to confirm and remain controversial. In order to further clarify the relationships between c-H-ras and c-myc activation and TGFβ signaling, we have compared the phenotypes of TGFβ-sensitive HaCaT human epidermal keratinocytes with cells that stably over-express c-myc (HaCaT-myc) or mutant H-ras (HaCaT-ras). The population doubling time of HaCaT-ras cells was shorter than that of HaCaT cells (approximately 14 versus 20 h), while HaCaT-myc cells proliferated at a slower rate. Treatment with exogenous TGFβ inhibited growth of HaCaT, HaCaT-ras and HaCaT-myc cells to similar degrees (60-65%), with an IC50 of 10 pM. Moreover, all three cell lines expressed high basal levels of phosphorylated Smad2 (pSmad2), the primary product of TGFβ type I receptor (TβR-I) kinase activity, while the levels of the cell cycle regulator, c-myc, were low. Treatment with TGFβ dramatically increased pSmad2 levels in all three cell lines and reduced basal c-myc levels by 60%. Most importantly, treatment with a specific inhibitor of the TβR-I kinase (SD-093) in the absence of exogenous TGFβ stimulated the growth of HaCaT, HaCaT-ras and HaCaT-myc cells by 50%, 35%, and 30%, respectively. This effect was accompanied by a reduction in the level of pSmad2 (IC50: 25 nM) and a 1.5-fold increase in c-myc protein levels. Our observations demonstrate that both exogenous and endogenous TGFβ are equally effective in causing phosphorylation of Smad2, repression of c-myc, and growth inhibition of HaCaT cells in a TβR-I kinase-dependent manner. In addition, that fact that blockade of the TβR-I kinase stimulated cell proliferation indicates that cell growth is partly regulated by endogenous TGFβ signaling. While individual overexpression of activated H-ras or c-myc was insufficient to abrogate any of these physiological responses to TGFβ, the possibility remains that combined overexpression of these two (proto)oncogenes is required for human keratinocytes to escape from TGFβs tumor suppressive effects.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]