ITGA3介导靶向BRAF突变黑色素瘤MAPK和PI3K/mTOR双通路耐药的分子机制研究

Drug resistance is a major factor affecting the effectiveness of targeted therapy in clinic. Recently, many clinical trials have been carried out by targeting MAPK and PI3K/mTOR dual pathways in BRAF mutant melanoma. Based on recent progress and our previous work, although targeting dual pathways could prolong the time of tumor tolerance to drugs, finally tumor will relapse after long-term treatment. In order to reveal the potential molecular mechanism of dual drug resistance, previously we had identified the ECM-receptor interaction pathway in dual drug-resistant cells by transcriptome sequencing and bioinformatics analysis based on the dual drug-resistant cell model. The integrins, ECM receptors, were up-regulated in dual drug-resistant cells. At the same time, preliminary data showed that ITGA3 was a key gene regulating the occurrence of dual drug resistance. Based on preliminary data, the key role of ITGA3 in regulating the development of melanoma dual drug resistance will be confirmed by in vitro and in vivo experiments. Furthermore, the upstream and downstream regulatory proteins of ITGA3 were screened and identified, and the signaling pathway and molecular mechanism of how ITGA3 activated the ECM receptor interaction pathway were elucidated. Through the implementation of this subject, it is expected to clarify the molecular mechanism of how ITGA3 regulated the dual drug resistance in melanoma, and to clarify the potential application value of ITGA3 as a dual drug resistance therapeutic target.

肿瘤耐药是影响临床靶向治疗效果的主要因素。目前靶向BRAF突变黑色素瘤MAPK和PI3K/mTOR双通路的临床试验正陆续开展，基于相关研究进展和我们前期工作基础，尽管双药治疗可以延长肿瘤对药物产生耐受的时间，但长期治疗不可避免将产生耐药。为了前瞻性揭示黑色素瘤双药耐药的分子机制，前期我们以双药耐药细胞模型为基础，通过转录组测序和生物信息学分析发现在耐药细胞中ECM受体互作通路激活，作为ECM受体的整合素家族基因在耐药细胞中表达上调。同时初步验证发现ITGA3是调控双药耐药发生的关键基因。在此基础上，通过体内外表型实验确认ITGA3在调控黑色素瘤双药耐药的发生过程中的关键作用；进而筛选验证ITGA3上下游调控蛋白，阐明ITGA3调控ECM受体互作通路激活的信号通路及分子机理。通过本课题的实施，有望阐明ITGA3调控黑色素瘤双药耐药的分子机制，明确ITGA3作为双药耐药治疗靶点的潜在应用价值。

PI3K/AKT通路的激活是BRAF突变黑色素瘤MAPKi靶向治疗最常见的耐药机制之一, MAPK和PI3K/AKT抑制剂联合治疗已经成为临床上BRAF突变黑色素瘤患者最有前景的治疗方案之一。尽管在临床前实验和早期临床试验中双药联合治疗取得了显著的效果，但长期治疗不可避免的产生双药耐药。在本课题中，为了前瞻性揭示BRAF突变黑色素瘤双药耐药的分子机制，我们系统地探讨了BRAF突变黑色素瘤对MAPKi和PI3K/mTORi的双重耐药机制。通过构建双药耐药的细胞和动物模型，结合RNA-seq和生物信息学分析，我们发现药物耐受是由整合素α3β1和α11β1信号介导的。整合素通过结合ECM激活下游激酶Src，促进肿瘤增殖。敲除整合素α3、α11和β1显著抑制双药耐药细胞系的增殖，而对亲代细胞的影响较小。尽管Srci抑制了AKT、c-JUN和p38的磷酸化，但靶向这些激酶的抑制剂均未逆转耐药细胞的耐药性。通过进一步研究发现，Srci促进了LATS1和YAP1的磷酸化，随后将YAP1从细胞核重新定位到细胞质，促进了进一步的降解。靶向YAP1或Src的小分子抑制剂和shRNA都克服了MAPKi和PI3K/mTORi双耐药细胞的耐药性。我们的数据阐明了integrin-Src-YAP1途径介导的MAPKi和PI3K/mTORi双重耐药机制。另外，我们还通过建立小鼠BRAF突变黑色素瘤单药耐药演进模型，发现血管重构是促进耐药发生的关键因素。进一步通过RNA-seq和实验验证，发现IGF1介导的血管重构是驱动BRAF突变黑色素瘤耐药发生的关键基因，联合靶向IGF1R和BRAF可以显著延缓耐药发生。