Blocakde of Smad4 in transformed keratinocytes containing a Ras oncogene leads to hyperactivation of the Ras-dependent Erk signalling pathway associated with progression to undifferentiated carcinomas

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Abstract

Smad4 functions as a transcription factor TGF-β signalling. We have investigated the role of Smad4 in the TGF-β1 cell responses of transformed PDV keratinocytes, wich contain a Ras oncogene, and of nontumorigenic MCA3D keratinocytes, by transfecting both cell lines with a dominant-negative Smad4 construct. Smad4 mediates TGF-β1-induced p21cip1 as growth factor. TGF-β1 activates Ras/Erk signalling activity in both cell lines. PD098059, as a specific inhibitor of MEK, disminishes TGF-β1-induced p21cip1 levels in PDV cells. PDV dominant-negative Smad4 cell transfectants, but not MCA3D transfectants, showed constitutive hyperactivation of the Ras/Erk signalling pathway, increased secretion of urokinase, higher motility properties, and a change to a fibroblastoid cell morphology associated in vivo with teh transition from a well differentiated to a pooly differentiated tumour phenotype. Infection of MCA3D control and dominant negative Smad4 cell transfectants with retroviruses carying a Ras oncogene led to enhaced p21cip1 and urokinase secreted levels, independently of TGF-β1 stimulation, that where reduced by PD098059. These results suggest that Smad4 acts inhibiting Ras-dependent Erk signalling activity in Ras-transformed Keratinocytes. Loss of Smad4 function in these cells results in hyperactivation of Erk signalling and progression to undifferentiated carcinomas.