Ciccarelli Carmela, Marampon Francesco, Scoglio Arianna, Mauro Annunziata, Giacinti Cristina, De Cesaris Paola, Zani Bianca M
Department of Experimental Medicine, University of L'Aquila, L'Aquila, Italy.
Mol Cancer. 2005 Dec 13;4:41. doi: 10.1186/1476-4598-4-41.
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.
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.
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.
p21WAF1参与正常细胞和恶性细胞的细胞周期调控，可通过p53依赖和非依赖机制诱导产生。在某些细胞中，丝裂原活化蛋白激酶/细胞外信号调节激酶（MEKs/ERKs）通过转录调控p21WAF1，而在其他细胞中，它们也影响转录后过程。在肌源性分化过程中，p21WAF1的表达也受肌源性转录因子MyoD的控制。我们之前已经证明，胚胎性横纹肌肉瘤细胞系在经佛波酯（TPA）和MEK抑制剂U0126处理后会发生生长停滞和肌源性分化，这两种处理分别激活和抑制ERK信号通路。在本文中，我们试图阐明ERK介导的和ERK非依赖性的胚胎性和肺泡性横纹肌肉瘤细胞系生长停滞及肌源性分化的机制，尤其是关于细胞周期抑制剂p21WAF1的表达情况。
TPA或MEK/ERK抑制剂（U0126）处理后，胚胎性（RD）和肺泡性（RH30）横纹肌肉瘤细胞系中均诱导了p21WAF1的表达及生长停滞，而仅在RD细胞中诱导了肌源性分化。此外，RD细胞中TPA介导的p21WAF1表达增强的转录后机制是由于MEK/ERK信号通路的激活，组成型活性MEK1或MEK2转染可诱导p21WAF1表达，而ERK1和ERK2的小干扰RNA（siRNA）转染则可阻止p21WAF1表达，这证明了上述结论。相比之下，U0126介导的p21WAF1表达受p38信号通路的转录调控。同样，U0126和TPA均可诱导生肌调节因子和MyoD的表达，而p38抑制可阻止这种诱导作用。虽然通过siRNA耗尽MyoD和生肌调节因子可阻止U0126介导的p21WAF1表达，但这两种转录因子的过表达不足以诱导p21WAF1。这些数据表明，当MEK/ERK抑制解除肌源性转录因子的功能时，RD细胞中p21WAF1表达的转录机制得以恢复。值得注意的是，在RD细胞中强制表达p21WAF1会导致生长停滞和非锚定依赖性生长的逆转。
我们的数据证明了MEK/ERK信号通路在横纹肌肉瘤细胞生长停滞中发挥的关键作用。本研究结果表明，靶向MEK/ERKs以恢复p21WAF1表达和肌源性转录因子功能可导致横纹肌肉瘤表型的逆转。
