p21WAF1 expression induced by MEK/ERK pathway activation or inhibition correlates with growth arrest, myogenic differentiation and onco-phenotype reversal in rhabdomyosarcoma cells.
BACKGROUND
p21WAF1, 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 p21WAF1 transcriptionally, while in others they also affect the post-transcriptional processes. In myogenic differentiation, p21WAF1 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 p21WAF1.


RESULTS
p21WAF1 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 p21WAF1-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 p21WAF1 expression, and with ERK1 and ERK2 siRNA, which prevents p21WAF1 expression. By contrast, U0126-mediated p21WAF1 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 p21WAF1 expression, the over-expression of these two transcription factors is insufficient to induce p21WAF1. These data suggest that the transcriptional mechanism of p21WAF1 expression in RD cells is rescued when MEK/ERK inhibition relieves the functions of myogenic transcription factors. Notably, the forced expression of p21WAF1 in RD cells causes growth arrest and the reversion of anchorage-independent growth.


CONCLUSIONS
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 p21WAF1 expression and myogenic transcription factor functions leads to the reversal of the Rhabdomyosarcoma phenotype.
Ciccarelli
Carmela
C
Department of Experimental Medicine, University of L'Aquila, L'Aquila, Italy.
Marampon
Francesco
F
Scoglio
Arianna
A
Mauro
Annunziata
A
Giacinti
Cristina
C
De Cesaris
Paola
P
Zani
Bianca M
BM
D015415:Biomarkers; D002070:Butadienes / Q000494:pharmacology; D002454:Cell Differentiation; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics / Q000378:metabolism*; D015536:Down-Regulation; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics; D006801:Humans; D020935:MAP Kinase Signaling System; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism*; D017570:MyoD Protein / Q000235:genetics; D018008:Myogenin / Q000235:genetics; D009570:Nitriles / Q000494:pharmacology; D010641:Phenotype; D012208:Rhabdomyosarcoma / Q000098:blood supply / Q000196:embryology / Q000378:metabolism* / Q000473:pathology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014158:Transcription, Genetic / Q000235:genetics; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
D015415:Biomarkers; D002070:Butadienes; D050759:Cyclin-Dependent Kinase Inhibitor p21; D017570:MyoD Protein; D018008:Myogenin; D009570:Nitriles; C113580:U 0126; D048049:Extracellular Signal-Regulated MAP Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D020929:Mitogen-Activated Protein Kinase Kinases; D013755:Tetradecanoylphorbol Acetate