ERK5促进黑色素瘤对威罗菲尼耐药的作用和机制

Melanoma is characterized by high malignancy. Vemurafenib that targets the BRAF kinase has been widely used in treating melanoma, however, drug resistance is still the bottleneck of treatment. Using substituted cysteine accessibility method (SCAM), we described that the resistance mechanism of the RAS upstream, histidine protein kinase (HPK) by alteration of the topology structure (Biochim Biophys Acta, 2014). The pilot experiment indicated that extracellular signal regulated kinase (ERK5) pathway is universally activated in melanoma after the development of resistance to BRAF inhibitors, vemurafenib. And ERK5 inhibitor, XMD8-92, in combination with vemurafenib significantly inhibited the tumor growth. Based on these, it is hypothesized that ERK1/2 is suppressed and ERK5 is activated in vemurafenib-resistant melanoma . In this study, RNA-seq, overexpression and Cas9-mediated knock out will be applied. After the specific treatment by small molecular dugs that target the ERK1/2 and ERK5 pathways, proliferation of the non-resistant and resistant cells will be investigated. Mouse model will also be applied to study the role of ERK5 in vemurafenib-resistant melanoma. Furthermore, the gene expression map regulated by the ERK5 pathway will be built to unveil the chemo-resistant mechanism of melanoma. We hope that this study could provide a new insight into improving clinical efficacy.

黑色素瘤恶性程度高，BRAF抑制剂威罗菲尼已广泛用于其治疗，但耐药性仍是治疗瓶颈。申请人前期利用SCAM等技术，阐述了RAS上游组氨酸蛋白激酶（HPK）经拓扑结构改变产生耐药的分子机制(Biochim Biophys Acta, 2014)。预实验发现对威罗菲尼耐药的黑色素瘤细胞中ERK5通路持续激活，且ERK5抑制剂XMD8-92与威罗菲尼联合使用显著抑制肿瘤。据此提出假说，耐药黑色素瘤细胞中ERK1/2抑制，ERK5表达上调且持续激活，进而导致其对威罗菲尼耐药。本课题拟利用RNA-seq、过表达和Cas9介导基因敲除等手段，通过检测XMD8-92和威罗菲尼等处理下，非耐药和耐药细胞增殖等变化，结合小鼠移植瘤模型，研究ERK5在黑色素瘤对威罗菲尼耐药中的作用；同时构建受ERK5通路调控的基因表达图谱，进行表达检测，并结合功能分析，探讨其耐药机制和作用靶点，为改善临床化疗效果提供新思路。

黑色素瘤恶性程度高，BRAF抑制剂威罗菲尼已广泛用于其治疗，但耐药性仍是治疗瓶颈。考虑到ERK1/2通路通过磷酸化调控细胞进程，我们首先对BRAF 和MEK1/2联合抑制（CIBM）进行了SILAC磷酸化蛋白质组学分析。239个蛋白磷酸化下调，同时47个蛋白的磷酸化上调。47个上调蛋白的经siRNA筛选后结果显示，ERK5敲低后显著的抑制CIBM耐药细胞增殖的能力。后 47个上调蛋白的siRNA筛选结果显示，ERK5的下调最为明显具有显著的抑制CIBM耐药细胞增殖的能力。我们也发现ERK5磷酸化作用在耐药细胞中增强，提示ERK5与耐药相关。进一步研究表明，MEK5D对BMKI磷酸化活化增强了对CIBM抗性。相反，shRNAi或ERK5抑制剂(XMD8-92)对ERK5的抑制作用不仅使CIBM的获得性抗性受损，同时也抑制了CIBM抗性细胞的增殖。我们的研究结果不仅为黑色素瘤的CIBM提供了全局的磷酸化蛋白组学观点，同时也证明了抑制ERK5对于治疗黑素瘤具有很大的潜力。