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  • Original Paper
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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

Oncogene volume 19, pages 4134–4145 (2000)Cite this article

Abstract

Smad4 functions as a transcription factor in TGF-β signalling. We have investigated the role of Smad4 in the TGF-β1 cell responses of transformed PDV keratinocytes, which contain a Ras oncogene, and of non-tumorigenic MCA3D keratinocytes, by transfecting both cell lines with a dominant-negative Smad4 construct. Smad4 mediates TGF-β1-induced up-regulation of p21Cip1 and growth arrest in MCA3D cells. However, in PDV keratinocytes, Smad4 is only partially involved in TGF-β1-induced growth inhibition, and does not mediate enhancement of p21Cip1 levels by the growth factor. TGF-β1 activates Ras/Erk signalling activity in both cell lines. PD098059, a specific inhibitor of MEK, disminishes TGF-β1-induced p21Cip1 levels in PDV but not in MCA3D cells, suggesting an involvement of Erk in up-regulation of p21Cip1 by TGF-β1 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 the transition from a well differentiated to a poorly differentiated tumour phenotype. Infection of MCA3D control and dominant negative Smad4 cell transfectants with retroviruses carrying a Ras oncogene led to enhanced p21Cip1 and urokinase secreted levels, independently of TGF-β1 stimulation, that were 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.

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Acknowledgements

We thank Dr Joan Massagué for kindly providing Smad4 plasmids, Dr Jaime Renart for the GST-RBD fusion construct, and Dr Carmelo Bernabeu for the 3TP-lux reporter. We also thank Drs Piero Crespo and Antonio Villalobo for critical reading of the manuscript and helpful suggestions. This work was supported by grants: SAF98-0085-CO3-02 from the Comisión Interministerial de Ciencia y Tecnología (CICYT), and 8.1/22/97 from the Comunidad Autónoma de Madrid (CAM).

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  1. Instituto de Investigaciones Biomédicas Alberto Sols CSIC-UAM, Arturo Duperier 4, Madrid, 28029, Spain

    Maite Iglesias, Pilar Frontelo & Miguel Quintanilla

  2. Departamento de Anatomía Patológica, Hospital de la Princesa, Facultad de Medicina, UAM, Madrid, 28029, Spain

    Carlos Gamallo

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Iglesias, M., Frontelo, P., Gamallo, C. et al. 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. Oncogene 19, 4134–4145 (2000). https://doi.org/10.1038/sj.onc.1203764

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