p21(WAF1) Expression induced by MEK/ERK pathway activation or inhibition correlates with growth arrest, myogenic differentiation and onco-phenotype reversal in rhabdomyosarcoma cells

Background: p21(WAF1), implicated in the cell cycle control of both normal and malignant cells, can be induced by p53-dependent and independent mechanisms. In some cells, MEKs/ERKs regulate p21(WAF1) transcriptionally, while in others they also affect the post-transcriptional processes. In myogenic differentiation, p21(WAF1) expression is also controlled by the myogenic transcription factor MyoD. We have previously demonstrated that the embryonal rhabdomyosarcoma cell line undergoes growth arrest and myogenic differentiation following treatments with TPA and the MEK inhibitor U0126, which respectively activate and inhibit the ERK pathway. In this paper we attempt to clarify the mechanism of ERK-mediated and ERK-independent growth arrest and myogenic differentiation of embryonal and alveolar rhabdomyosarcoma cell lines, particularly as regards the expression of the cell cycle inhibitor p21(WAF1). Results: p21(WAF1) expression and growth arrest are induced in both embryonal ( RD) and alveolar ( RH30) rhabdomyosarcoma cell lines following TPA or MEK/ERK inhibitor ( U0126) treatments, whereas myogenic differentiation is induced in RD cells alone. Furthermore, the TPA-mediated post-transcriptional mechanism of p21(WAF1)-enhanced expression in RD cells is due to activation of the MEK/ERK pathway, as shown by transfections with constitutively active MEK1 or MEK2, which induces p21(WAF1) expression, and with ERK1 and ERK2 siRNA, which prevents p21(WAF1) expression. By contrast, U0126-mediated p21(WAF1) expression is controlled transcriptionally by the p38 pathway. Similarly, myogenin and MyoD expression is induced both by U0126 and TPA and is prevented by p38 inhibition. Although MyoD and myogenin depletion by siRNA prevents U0126-mediated p21(WAF1) expression, the over-expression of these two transcription factors is insufficient to induce p21(WAF1). These data suggest that the transcriptional mechanism of p21(WAF1) expression in RD cells is rescued when MEK/ERK inhibition relieves the functions of myogenic transcription factors. Notably, the forced expression of p21(WAF1) in RD cells causes growth arrest and the reversion of anchorage- independent growth. Conclusion: Our data provide evidence of the key role played by the MEK/ERK pathway in the growth arrest of Rhabdomyosarcoma cells. The results of this study suggest that the targeting of MEK/ERKs to rescue p21(WAF1) expression and myogenic transcription factor functions leads to the reversal of the Rhabdomyosarcoma phenotype.[...]