Wee1蛋白激酶在胃肠间质瘤伊马替尼耐药中的作用及机制研究

Imatinib resistance is general in gastrointestinal stromal tumor patients (GIST) and restricts the overall survival. However, the effective therapeutic medicines are very limited for those not induced by the secondary mutation of KIT/PDGFRA. Therefore, seeking novel effective therapeutic targets and exploring the definite mechanisms of Imatinib resistance are imperative. Our preliminary study showed that specific overexpression of Wee1 occurred in Imatinib-resistant GIST cell lines and clinical specimen. MK-1775, a small molecular inhibitor, can enhance the function of Imatinib to suppress GIST cells. Moreover, GenMAPP results revealed the overexpression of MCL1 in Imatinib-resistant GIST was related with Wee1. Consequently, we hypothesize Wee1 mediate Imatinib resistance of GIST via targeting MCL1 and Wee1 is likely to be a potential therapeutic target. Based on the above-mentioned results, our research will be carried out with the following work: 1. To confirm the relationship between Wee1 and Imatinib-resistant GISTs. 2. To verify the mechanism of Wee1 regulate post-transcriptionally MCL1. 3. To analyze the viability of combination Imatinib and MK-1775 in the reversal of imatinib-resistance. This study results will provide possible therapeutic targets and method of drugs combination to overcome Imatinib resistance for GIST.

伊马替尼继发耐药是制约胃肠间质瘤患者（GIST）生存的关键问题。在临床治疗上，非KIT/PDGFRA二次突变的伊马替尼耐药患者往往缺乏有效的治疗药物，因此寻求有效的治疗靶点、探索明确的耐药机制是伊马替尼耐药亟待解决的问题。本课题组前期初步研究显示，Wee1蛋白激酶在伊马替尼耐药GIST中显著升高；Wee1抑制剂MK-1775能显著增强伊马替尼抗肿瘤作用。通过高通量芯片通路分析发现，MCL1在伊马替尼耐药GIST中过表达，且与Wee1高表达相关。由此推测Wee1可能通过MCL1介导抗凋亡作用进而诱导GIST对伊马替尼耐药，提示Wee1可作为治疗伊马替尼耐药GIST的潜在靶点。本课题拟进一步解析Wee1与伊马替尼耐药的临床相关性；探讨Wee1对MCL1转录后的调控机制；系统分析MK-1775逆转伊马替尼耐药的可行性。本项目顺利实施，将为突破伊马替尼继发耐药的治疗困局、联合药物治疗提供理论支持。

背景：KIT或PDGFRA的激活突变是胃肠间质瘤(GIST)的主要分子机制。虽然伊马替尼对GIST治疗有革命性的影响，但其疗效并不持久。越来越多的报道表明，细胞周期检查点在GIST中起着至关重要的作用。本课题我们探讨WEE1激酶在GIST进展中的作用。.方法：采用肿瘤学公共数据库、Western blotting和免疫组织化学方法检测WEE1在GIST中的表达。通过MTT比色法、细胞集落形成实验和流式细胞术，检测WEE1在GIST细胞中的作用以及抑制剂MK1775单独或与伊马替尼联合应用的抗肿瘤活性。放线酮菌、药物抑制自噬和蛋白酶体途径分析KIT的表达。用Western blotting检测自噬标记物Beclin1和LC3B。.结果：WEE1在GIST组织中表达上调，并与肿瘤大小、核分裂象和NIH危险度分级相关。敲除WEE1可显著抑制GIST细胞增殖，诱导细胞凋亡和细胞周期阻滞。伊马替尼与MK1775联用可显著增强其抗肿瘤活性。靶向WEE1可降低KIT的表达。此外，药物抑制自噬可以逆转WEE1稳定的KIT蛋白和抑制WEE1的KIT减少，但不能逆转蛋白酶体途径。WEE1抑制剂还可增加GIST细胞Beclin1的表达和LC3BⅡ/I比值。.结论：WEE1在GIST增殖中起关键作用。抑制WEE1可以促进KIT蛋白自噬降解，因此，靶向WEE1可能是GIST治疗的一种新策略。