TIMP1诱导c-Met信号激活促进乳腺癌对CDK4/6抑制剂耐药的机制研究

Pharmacologic inhibitors of cyclin-dependent kinases 4 and 6 (CDK4/6) have shown promising clinical activities in estrogen receptor (ER)-positive breast cancer. However, patients eventually develop clinical resistance with progression and the mechanism of drug resistance remains to be determined. Previously, through a screening from a phosphokinase array and deep sequencing, we found that the protein level of the tumor inhibitor of metalloproteinase 1 (TIMP1) was upregulated following abemaciclib treatment in ER-positive breast cancer cells. TIMP1 was also overexressed in abemaciclib-resistant cells. Loss-of-function study suggested that the expression of TIMP1 is associated with the resistance of abemaciclib in breast cancer. To further investigate the pro-survive mechanism of TIMP1, we selected six drugs that target key receptor tyrosine kinases (RTKs) to screen for effective combination therapies. Abemaciclib had a synergistic effect with c-Met inhibition with the small molecule SGX-523, whereas other drug combinations were less effective or antagonistic. The overexpression of c-Met is associated with the upregulation of TIMP1. Thus, according to the above-mentioned results, we propose the following hypotheses: TIMP1 reduces the shedding activity of c-Met and leads to the accumulation of c-Met, which leads to the abnormal activition of c-Met signaling and drives the resistance of CDK4/6 inhibitors. This applicant aims to explore the precise mechanism of CDK4/6 inhibitor resistance, and provide a pre-clinical basis for a combination therapy rationale for treating breast cancer.

细胞周期依赖性激酶4/6(CDK4/6)抑制剂近年被证明能有效治疗ER+乳腺癌，但乳腺癌患者最终仍会产生耐药，且耐药机制尚不明确。申请人前期通过激酶芯片与深度测序筛选，发现CDK4/6抑制剂诱导乳腺癌细胞特异性TIMP1（金属蛋白酶组织抑制因子1）蛋白分泌增多，而且药物诱导的耐药细胞中TIMP1基因也持续性高表达。功能缺失实验证明TIMP1表达的确与CDK4/6抑制剂耐药相关。为明确TIMP1介导的促生存机制，与多种RTK靶向药物联合用药发现c-Met抑制剂表现出良好的协同性效应，并验证了c-Met表达增多与TIMP1上调有关。根据TIMP1的生物学功能我们提出以下科学假说：TIMP1降低膜表面蛋白脱落作用从而使c-Met积聚，这种特殊的转录后调控机制导致c-Met信号异常激活从而诱导CDK4/6抑制剂耐药。本项目力求探索乳腺癌对CDK4/6抑制剂耐药的机制，为联合治疗方案提供临床前依据。

尽管CDK4/6抑制剂在乳腺癌临床上取得良好的疗效。CDK4/6抑制剂治疗的16%的激素受体阳性乳腺癌患者是无效的，且大多数病人在治疗后出现耐药而最终复发或进展。CDK4/6抑制剂耐药的具体原因有待进一步研究。我们通过对CDK4/6抑制剂耐药株进行RNA测序分析，通过对比耐药株和敏感的表达谱数据进行GSEA分析发现耐药株高表达基因富集在mTOR通路。连通性图谱分析（Connectivity Map Analysis）表明，mTOR通路抑制剂是排行第一位的潜在逆转CDK4/6抑制剂耐药的药物。这表明mTOR通路激活介导CDK4/6抑制剂耐药。应用mTORC1抑制剂依维莫司结合阿贝西利能明显降低耐药株的细胞活力。基因抑制RAPTOR和RHEB可以恢复耐药株对于阿贝西利的敏感性。结合mTORC1抑制剂依维莫司和阿贝西利能明显减缓耐药株的体内移植瘤肿瘤体积和肿瘤重量。这些结果提出克服CDK4/6抑制剂耐药的联合治疗方案。本研究的成果有良好的临床应用价值，为临床试验提供重要的证据支持，也可通过临床转化来服务广大乳腺癌患者。