RNA结合蛋白ERH介导乳腺癌CDK4/6抑制剂耐药的作用及机制研究

CDK4/6 are attractive anti-tumor targets against breast cancer and three of their inhibitors have been approved for the therapy of ER positive subtypes. However, primary or acquired resistance to CDK4/6 inhibitors emerged in some patients, making it a big challenge in clinic. In the present study, we used a systematic loss-of-function screening and identified that enhancer of rudimentary homolog (ERH)，a RNA-binding protein, whose downregulation confers drug resistance to palbociclib in ER positive breast cancer MCF-7 cells. ERH overexpressed in breast cancer cells and its knockdown increased the sensitivity of MCF-7 cells to palbociclib. Meanwhile, both the mRNA and protein level of ERH elevated in palbociclib-resistant MCF-7R cells. ERH downregulation also restored the drug sensitivity of MCF-7R cells to palbociclib. These results indicated that ERH participated in mediating drug resistance to CDK4/6 inhibitors. Next, through taking advantage of sensitive and resistant breast cancer cell lines, animal models and clinical patients samples, we plan to investigate the relationship and the underlying mechanism of ERH involved in mediating drug resistance to CDK4/6 inhibitors, explore new strategies to overcome the resistance and provide reference for the clinical application of CDK4/6 inhibitors.

CDK4/6为乳腺癌治疗的重要靶点，其抑制剂已被批准用于ER阳性乳腺癌的治疗。然部分患者存在原发或继发耐药，克服该耐药为临床亟待解决的核心问题。本项目经高通量筛选并鉴定出ER阳性乳腺癌中与CDK4/6抑制剂耐药密切相关的RNA结合蛋白ERH（enhancer of rudimentary homolog）。ERH在乳腺癌中高表达，降低ERH表达可增强细胞对CDK4/6抑制剂的敏感性；在palbociclib耐药细胞中，ERH mRNA及蛋白水平均明显增加，而干扰ERH使耐药细胞重新对palbociclib敏感，提示ERH参与CDK4/6抑制剂耐药。接下来我们将利用敏感和耐药的乳腺癌细胞株、裸小鼠原位移植瘤模型及临床乳腺癌组织标本等验证ERH与CDK4/6抑制剂耐药的关系，揭示耐药的具体分子机制并据此探索逆转耐药新策略，最终为临床上CDK4/6抑制剂应用提供新思路。

随着CDK4/6抑制剂临床应用的增多，克服该类抑制剂耐药已成为临床亟待解决的核心问题。本项目通过CRISPR/Cas9基因文库敲除技术，系统性地筛选并鉴定出ER阳性乳腺癌细胞中与CDK4/6抑制剂耐药密切相关RNA结合蛋白ERH（enhancer of rudimentary homolog）。与正常乳腺上皮细胞和乳腺组织相比，ERH在乳腺癌细胞及乳腺肿瘤组织中的表达均明显增加，在卵巢癌中也有类似趋势，但其在乳腺癌发生发展中的具体作用及其与靶向治疗耐药的关系尚不可知。本项目研究显示ERH在ER阳性乳腺癌细胞中表达升高，干扰其表达降低细胞对CDK4/6抑制剂的敏感性，而过表达ERH可促进多株ER阳性乳腺癌细胞增殖，并增加细胞对CDK4/6抑制剂的敏感性。在palbociclib获得性耐药的T47D细胞中，ERH蛋白表达下调，而回复其表达可使耐药细胞重新对CDK4/6抑制剂敏感。机制上，通过对RNAseq及RIP-seq测序结果进行联合分析，发现PITX1可能为ERH介导CDK4/6抑制剂耐药的相关蛋白，ERH干扰引起PITX1表达上调，而降低PITX1表达则增加细胞对CDK4/6抑制剂的敏感性。最后研究发现，联合NF-κB通路抑制剂可显著增强ER阳性乳腺癌细胞对palbociclib敏感性。本研究将拓宽我们对CDK4/6抑制剂耐药相关生物学机制的了解，为CDK4/6抑制剂临床用药指导奠定理论和实验基础。