PYK2 mediates BRAF inhibitor vermurafenib induced invadopodia formation and metastasis in melanoma
PYK2 mediates BRAF inhibitor vermurafenib induced invadopodia formation and metastasis in melanoma
复制标题
PYK2 介导 BRAF 抑制剂维莫非尼诱导黑色素瘤侵袭伪足的形成和转移
作者：
Junling Shen;Jilong Yang;Rui Sun;Lei Sang;Weiyu Bai;Chao Wang;Yan Sun;Jianwei Sun
目的：BRAF抑制剂vemurafenib已广泛应用于BRAFV 600 E突变黑色素瘤的治疗。虽然vemurafenib的初始反应通常很好，但大多数患者最终会产生耐药性和转移性疾病。然而，潜在的分子机制仍然难以捉摸。本研究的目的是确定维罗非尼耐药的其他分子靶点。研究方法：Western blot和免疫组化检测PYK 2和p-PYK 2在培养细胞和黑色素瘤组织芯片（TMA）中的表达。统计分析p-PYK 2与临床病理参数的关系。采用侵袭伪足、细胞侵袭和Ca 2+测定来测试vemurafenib抗性诱导的p-PYK 2对黑色素瘤进展的影响。利用小鼠模型来评估PYK 2对黑素瘤转移的作用。结果：在维罗非尼耐药的黑色素瘤细胞中检测到升高的p-PYK 2水平。PYK 2显示出调节黑素瘤细胞中的侵袭伪足形成。Vemurafenib通过激活PYK 2抑制侵袭伪足的形成。用shRNA或小分子抑制剂PF 562711抑制PYK 2显著减少了维罗非尼诱导的侵袭伪足形成。此外，PYK 2的敲低显著减少体内黑色素瘤肺转移。p-PYK 2在黑色素瘤中的高表达不仅与肿瘤的晚期（P = 0.002）、转移（P <0.001）、Clark分级（P <0.001）呈正相关，而且与总生存期（HR =3.304，P=0.007）和无进展生存期（HR=2.930，P=0.001）短相关。结论：PYK 2介导维罗非尼诱导的黑色素瘤细胞迁移和侵袭。抑制PYK 2使黑素瘤细胞对维罗非尼再敏感。磷酸化PYK 2是黑色素瘤患者的预后生物标志物。
Objective: BRAF inhibitor vemurafenib has been widely used in treatment of patients with melanoma bearing BRAFV600E mutation. While the initial response to vemurafenib is usually excellent, the majority of patients eventually developed resistance and metastatic disease. However, the underlying molecular mechanism remains elusive. The objective of this study is to identify additional molecular targets in vemurafenib resistance..Methods: Western blot and immunohistochemistry analyses were used to evaluate expression of PYK2 and p-PYK2 in culture cells and melanoma tissue microarray (TMA). The relationship of p-PYK2 with clinicopathological parameters was statistically analyzed. Invadopodia, cell invasion and Ca2+ assay were employed to test the effect of vemurafenib resistance-induced p-PYK2 on melanoma progression. A mouse model was utilized to assess the effect of PYK2 on melanoma metastasis..Result: Elevated p-PYK2 level was detected in vemurafenib-resistant melanoma cells. PYK2 was shown to regulate invadopodia formation in melanoma cells. Vemurafenib triggerred invadopodia formation through activation of PYK2. Inhibition of PYK2 with either shRNA or small molecule inhibitor PF562711 dramatically reduced vemurafenib-induced invadopodia formation. Furthermore, knockdown of PYK2 significantly reduced melanoma lung metastasis in vivo. Notably, increase expression of p-PYK2 in patients with melanoma was not only positively correlated with advanced stage (P = 0.002), metastasis (P <0.001), Clark grade (P < 0.001), but also associated with short overall survival (hazard ratio [HR] =3.304, P=0.007) and progress-free survival (HR=2.930, P=0.001)...Conclusion: PYK2 mediates vemurafenib-induced melanoma cell migration and invasion. Inhibition of PYK2 resensitizes melanoma cells to vemurafenib. Phospho-PYK2 is a prognostic biomarker in patients with melanoma..