Blockade 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

Smad4 functions as a transcription factor in TGF-beta signalling. We have i nvestigated the role of Smad4 in the TGF-beta(1) cell responses of transfor med PDV keratinocytes, which contain a Ras oncogene, and of non-tumorigenic MCA3D keratinocytes, by transfecting both cell lines with a dominant-negat ive Smad4 construct. Smad4 mediates TGF-beta(1)-induced up-regulation of p2 1(Cip1) and growth arrest in MCA3D cells, However, in PDV keratinocytes, Sm ad4 is only partially involved in TGF-beta(1)-induced growth inhibition, an d does not mediate enhancement of p21(Cip1) levels by the growth factor. TG F-beta(1) activates Ras/Erk signalling activity in both cell lines, PD09805 9, a specific inhibitor of MEK, disminishes TGF-beta(1)-induced p21(Cip1) l evels in PDV but not in MCA3D cells, suggesting an involvement of Erk in up regulation of p21(Cip1) by TGF-beta(1) 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 u rokinase, higher motility properties, and a change to a fibroblastoid cell morphology associated in vivo with the transition from a well differentiate d to a poorly differentiated tumour phenotype. infection of MCA3D control a nd dominant negative Smad4 cell transfectants with retroviruses carrying a Ras oncogene led to enhanced p21(Cip1) and urokinase secreted levels, indep endently of TGF-beta(1) stimulation, that were reduced by PD098059, These r esults suggest that Smad4 acts inhibiting Ras-dependent Erk signalling acti vity in Ras-transformed keratinocytes. Loss of Smad4 function in these cell s results in hyperactivation of Erk signalling and progression to undiffere ntiated carcinomas.