Wu D-W, Wu T-C, Wu J-Y, Cheng Y-W, Chen Y-C, Lee M-C, Chen C-Y, Lee H
Graduate Institute of Cancer Biology and Drug Discovery, Taipei Medical University, Taipei, Taiwan, ROC.
Department of Internal Medicine, Chung Shan Medical University, Taichung, Taiwan, ROC.
Oncogene. 2014 Aug 28;33(35):4385-95. doi: 10.1038/onc.2013.389. Epub 2013 Oct 7.
Paxillin (PXN) is required for receptor tyrosine kinase-mediated ERK activation, and the activation of the Raf/MEK/ERK cascade has been linked with Bcl-2 expression. We hypothesized that phosphorylation of PXN by the EGFR/Src pathway might contribute to cisplatin resistance via increased Bcl-2 expression. We show that cisplatin resistance was dependent on PXN expression, as evidenced by PXN overexpression in TL-13 and TL-10 cells and PXN knockdown in H23 and CL1-5 cells. Specific inhibitors of signaling pathways indicated that the phosphorylation of PXN at Y118 and Y31 via the Src pathway was responsible for cisplatin resistance. We further demonstrated that ERK activation was also dependent on this PXN phosphorylation. Bcl-2 transcription was upregulated by phosphorylated PXN-mediated ERK activation via increased binding of phosphorylated CREB to the Bcl-2 promoter. A subsequent increase in Bcl-2 levels by a PXN/ERK axis was responsible for the resistance to cisplatin. Animal models further confirmed the findings of in vitro cells indicating that xenograft tumors induced by TL-13-overexpressing cells were successfully suppressed by cisplatin combined with Src or ERK inhibitor compared with treatment of cisplatin, Src inhibitor or ERK inhibitor alone. A positive correlation of phosphorylated PXN with phosphorylated ERK and Bcl-2 was observed in lung tumors from NSCLC patients. Patients with tumors positive for PXN, phosphorylated PXN, phosphorylated ERK and Bcl-2 more commonly showed a poorer response to cisplatin-based chemotherapy than did patients with negative tumors. Collectively, PXN phosphorylation might contribute to cisplatin resistance via activating ERK-mediated Bcl-2 transcription. Therefore, we suggest that Src or ERK inhibitor might be helpful to improve the sensitivity for cisplatin-based chemotherapy in NSCLC patients with PXN-positive tumors.
桩蛋白（PXN）是受体酪氨酸激酶介导的细胞外信号调节激酶（ERK）激活所必需的，而Raf/MEK/ERK级联反应的激活与Bcl-2表达有关。我们推测表皮生长因子受体（EGFR）/Src途径介导的PXN磷酸化可能通过增加Bcl-2表达而导致顺铂耐药。我们发现顺铂耐药取决于PXN表达，这在TL-13和TL-10细胞中PXN过表达以及H23和CL1-5细胞中PXN敲低得到证实。信号通路的特异性抑制剂表明，通过Src途径在Y118和Y31位点对PXN的磷酸化是顺铂耐药的原因。我们进一步证明ERK激活也依赖于这种PXN磷酸化。磷酸化的PXN介导的ERK激活通过增加磷酸化的环磷腺苷效应元件结合蛋白（CREB）与Bcl-2启动子的结合而上调Bcl-2转录。随后由PXN/ERK轴导致的Bcl-2水平升高是对顺铂耐药的原因。动物模型进一步证实了体外细胞实验的结果，表明与单独使用顺铂、Src抑制剂或ERK抑制剂相比，顺铂联合Src或ERK抑制剂成功抑制了由过表达TL-13的细胞诱导的异种移植肿瘤。在非小细胞肺癌（NSCLC）患者的肺肿瘤中观察到磷酸化的PXN与磷酸化的ERK和Bcl-2呈正相关。与肿瘤阴性的患者相比，肿瘤中PXN、磷酸化的PXN、磷酸化的ERK和Bcl-2呈阳性的患者对基于顺铂的化疗反应通常更差。总体而言，PXN磷酸化可能通过激活ERK介导的Bcl-2转录而导致顺铂耐药。因此，我们建议Src或ERK抑制剂可能有助于提高PXN阳性肿瘤的NSCLC患者对基于顺铂的化疗的敏感性。
