YAP新型磷酸化在乳腺癌紫杉醇耐药中的作用及其机制研究

Breast cancer is the most common malignant tumor and the leading cause of cancer-related deaths in women in China. Taxol based chemotherapy is the first-line treatment of breast cancer. One of the major obstacle for successful breast cancer therapy is that mammary tumors often recur due to native or acquired resistance to Taxol, however, the mechanism of Taxol resistance in breast cancer is still unclear. ..Our previous studies demonstrated that YAP is phosphorylated at three novel sites during Taxol treatment in breast cancer cell lines. The novel YAP phosphorylation is found to induce mitotic defects and promote cell migration and invasion，which define them as the only known phosphorylation sites promoting YAP’s onocogenic activity. Based on our previous report, we hypothesize that YAP's novel phosphorylation may mediate Taxol resistance in breast cancer and maybe the biomarker of Taxol resistance. In the current proposal, we aim to uncover the mechanism of Taxol resistance in breast cancer. We will perform apoptosis assay in breast cancer cell lines and immunostaining in breast cancer tissue samples in order to examine the role of YAP novel phosporylation in Taxol treatment. Then, we will explore the downstream effector of this novel YAP phosphorylation. Verteporfin is a newly discovered small molecular which disrupts the interaction between YAP and TEAD. We next treat the breast cancer stable cell lines and orthotopic mouse model with Taxol or Taxol plus Verteporfin and then examine their response. ..Collectively, this study will uncover novel mechanism for Taxol resistance, highlight YAP's novel phosphorylation as a potential biomarker for chemotherapy resistance and also provide rationale for Vertrporfin as a novel chemosensitizer in breast cancer treatment.

乳腺癌是中国女性最常见的恶性肿瘤和最主要的癌症死因。紫杉醇作为乳腺癌的一线化疗药物，因其原发性及继发性耐药的产生，极易造成乳腺癌复发而导致治疗失败，但其耐药的分子机制尚不明确。我们前期研究首次发现乳腺癌细胞系给予紫杉醇处理后导致YAP三个新型位点的磷酸化，可引起细胞有丝分裂异常并诱导细胞迁移，为目前唯一已知的激活YAP癌基因功能的磷酸化位点。据此，我们提出假设其可能介导了紫杉醇耐药的产生并可能做为乳腺癌对紫杉醇耐药的生物标记物。为探寻紫杉醇的耐药机制，本研究拟在细胞水平及临床标本中检测YAP新型磷酸化对于乳腺癌紫杉醇处理的影响，进一步探讨其下游靶基因，并使用YAP-TEAD结合抑制剂维替泊芬，在体外及小鼠乳腺癌原位移植模型中探索维替泊芬与紫杉醇联合应用对乳腺癌的治疗效果。本研究将有助于揭示乳腺癌紫杉醇耐药新的机制，并对紫杉醇耐药标志物的选择和维替泊芬作为潜在的紫杉醇化疗增敏剂提供理论依据。

乳腺癌作为目前世界及中国女性发病率及死亡率均为第一位的恶性肿瘤。化疗是治疗乳腺癌的主要手段，多西他赛是其治疗中具有基石地位的化疗药物，然而，紫杉醇类药物治疗乳腺癌的有效率不足50%，其作为乳腺癌二、三线方案的有效率甚至不足20%，乳腺癌对紫杉醇的原发性及继发性耐药是导致其治疗无效的主要原因。目前对于紫杉醇耐药的具体机制尚不明确，同时缺乏明确的耐药标志物用于指导临床用药，一直以来是乳腺癌研究的重点和热点问题之一。本课题在稳转细胞系中发现YAP模拟磷酸化（YAP3D）对于紫杉醇有明显的耐药性，凋亡细胞数目明显减少。通过收集临床病理标本进行免疫组化染色发现，对于紫杉醇原发性耐药的乳腺癌患者中，YAP表达明显增高，预后不佳，且在三阴乳腺癌中，YAP及其特异性磷酸化与肿瘤大小及无进展生存时间 （PFS）有更明显的相关性。通过荧光素酶实验发现YAP通过与TEAD结合诱导下游LPAR3的表达来调节细胞对于Taxol处理的反应，并进一步在裸鼠模型中证实，维替泊芬与紫杉醇联用较单用紫杉醇有明显抑制肿瘤生长的作用。本课题的完成将有助于进一步揭示乳腺癌中紫杉类药物的耐药机制，YAP新型磷酸化水平有望成为判断治疗预后的检测标志物，可据此筛选敏感患者指导用药。维替泊芬通过抑制YAP与TEAD的结合增强肿瘤患者对于紫杉醇的敏感性，为乳腺癌的靶向药物研发提供了理论基础。