通过抑制表皮生长因子受体信号通路来防止和克服黑色素瘤对BRAF抑制剂威罗菲尼的继发性耐药

The significant clinical activity (~70% response rate) of BRAF selective inhibitor Vemurafenib represents a great success and a paradigm shift in the treatment of BRAF V600E mutated melanoma. However, its medium response duration is short (7months), due to acquired resistance. There is an urgent need to further understand the underlying molecular signaling mechanisms, which will allow the to-be-acquired knowledge pertaining to the mechanistic details of resistance to better guide the designing of rational combinational targeted therapies to overcome this resistance. . Our most recent preliminary data indicate that: i) receptor tyrosine kinase (RTK) EGFR is upregulated in melanoma cell lines and that have acquired BRAF inhibitor resistance, and ii) a unique, fully human monoclonal antibody (mAb) scFv-FcC21 (C21), targeting cell surface chondroitin sulfate proteoglycan (CSPG4) and blocking the activation of EGFR in melanoma cells.We hypothesize that we can prevent/overcome acquired Vemurafenib resistance via targeting of EGFR signaling through the use of its inhibitor, or by use of C21, which does not crossreact with normal tissues. In this proposal, the specific aims which test our hypotheses are: i) to study the mechanisms of action of the CSPG4-specifc antibody C21 in blocking the activation of EGFR, ii) to determine whether downregulation of EGFR signaling is responsible for the ability of CSPG4-specific antibody C21 to prevent/overcome Vemurafenib resistance in BRAFV600E mutant melanoma cell lines in vitro, and iii) to investigate whether C21 as well as a combination of EGFR inhibitor Gefitinib can significantly prevent and overcome Vemurafenib resistance in mice bearing BRAFV600E mutant xenografts.. The results derived from the proposed studies will build a foundation for the design and implementation of the next generation of clinical trials to improve the therapeutic efficacy of Vemurafenib.

晚期恶性黑色素瘤进展快，缺乏有效的治疗。BRAF选择性抑制剂威罗菲尼在治疗BRAFV600E突变的黑色素瘤中取得了显著性的成功。但该药面临的挑战之一是约七个月后病人对其产生耐药。目前迫切需要了解引起耐药的的机制，设计有针对性的治疗，以克服这种耐药。我们前期研究发现1)表皮生长因子受体(EGFR)在对威罗菲尼获得性耐受的黑色素瘤细胞中表达增高；2)硫酸软骨素蛋白多糖4(CSPG4)特异性抗体能够抑制EGFR的激活。我们假设CSPG4抗体能够通过抑制EGFR通路来防止/克服威罗菲尼耐药的产生。因此，本课题将通过免疫共沉淀方法探索CSPG4抗体抑制EGFR信号通路的机制。在细胞水平和小鼠动物模型两方面验证CSPG4抗体通过下调EGFR信号通路来防止/克服黑色素瘤细胞对威罗菲尼产生耐药。本课题的研究结果，将阐明威罗菲尼的耐药机制,为防止/克服威罗菲尼耐受,最大限度发挥威罗菲尼功效提供科学理论依据。

针对BRAFV600E抑制剂威罗菲尼 (vemurafenib)在治疗黑色素瘤的过程中受困于获得性耐药的问题，本项目旨在探讨建立联合治疗方案来防止/克服黑色素瘤细胞对威罗菲尼产生耐药。根据前期研究，本研究将目标分子设定为：硫酸软骨素蛋白多糖4。本项目按计划完成了以下研究：1) 体外细胞培养诱导构建威罗菲尼获得性耐药株细胞，并验证联合使用硫酸软骨素蛋白多糖4特异性抗体可有效延缓耐药株发生；2) 构建高表达EGFR的黑色素瘤细胞株，评估了EGFR与威罗菲尼获得性耐药的关系；并以此开展联合使用EGFR小分子抑制剂吉非替尼对抗威罗菲尼获得性耐药；3)基于靶向硫酸软骨素蛋白多糖4，EGFR引起的细胞传导信号通路的变化，通过western-blot, 免疫共沉淀，新型电化学免疫传感器等方法探索性的研究了硫酸软骨素蛋白多糖4与EGFR的相互结合作用；4) 利用免疫缺陷小鼠建立黑色素瘤肿瘤模型，并考察联合打击硫酸软骨素蛋白多糖4, BRAFV600E以对抗黑素瘤的疗效，尤其是防止/延缓威罗菲尼耐药性的发生。通过三年的项目执行，完成了研究计划，取得了预期结果。研究结果揭示硫酸软骨素蛋白多糖4通过抑制EGFR相关信号通路协同抑制黑色素瘤细胞生长，且可协同对抗威罗菲尼耐受发生的机制, 为最大限度发挥威罗菲尼功效提供科学理论依据。此外，在本项目执行中，注重进行肿瘤生物学研究与以微流控细胞芯片，微型生物传感器等新技术，新方法的交叉。围绕所研究的威罗菲尼耐药性问题，构建电化学传感器首次直接检测了黑素瘤细胞在迁移过程中活性氧水平的变化，探讨了活性氧分子在威罗菲尼治疗中的作用，为深入研究活性氧通过调节信号通路干扰细胞对威罗菲尼的敏感性提供基础。