table1_CPEB4-Promoted Paclitaxel Resistance in Ovarian Cancer In Vitro Relies on Translational Regulation of CSAG2.docx

Background: Drug resistance is a major obstacle in chemotherapy for ovarian cancer, wherein the up regulation of drug-resistant genes plays an important role. The cytoplasmic polyadenylation element binding protein 4 (CPEB4) is an RNA binding protein that controls mRNA cytoplasmic polyadenylation and translation.
Methods: The expression of CPEB4 in paclitaxel-resistant ovarian cancer cell lines and recurrent ovarian tumors relative to counterparts was determined by qRT-PCR, Western blotting and immunohistochemistry. The response to paclitaxel treatment was evaluated by cellular viability test and colony formation assay. RNA immunoprecipitation and poly(A) tail test were applied to examine the levels of RNA binding and cytoplasmic polyadenylation.
Results: CPEB4 is elevated in paclitaxel-resistant ovarian cancer cells and recurrent ovarian tumors treated with paclitaxel-based chemotherapy. In addition, CPEB4 overexpression promotes paclitaxel resistance in ovarian cancer cells in vitro, and vice versa, CPEB4 knockdown restores paclitaxel sensitivity, indicating that CPEB4 confers paclitaxel resistance in ovarian cancer cells. Mechanistically, CPEB4 binds with the taxol (paclitaxel)-resistance-associated gene-3 (TRAG-3/CSAG2) mRNAs and induces its expression at a translational level. Moreover, CSAG2 expression is upregulated in paclitaxel-resistant ovarian carcinoma and cancer cell lines, and more importantly, siRNA-mediated CSAG2 knockdown overtly attenuates CPEB4-mediated paclitaxel resistance.
Conclusion: This study suggests that the drug-resistant protein CSAG2 is translationally induced by CPEB4, which underlies CPEB4-promoted paclitaxel resistance in ovarian cancer in vitro. Thus, interfering CPEB4/CSAG2 axis might be of benefit to overcome paclitaxel-resistant ovarian cancer.

背景：耐药性是卵巢癌化疗的主要障碍，其中耐药基因的上调发挥着关键作用。胞质多聚腺苷酸化元件结合蛋白4（CPEB4）是一类调控mRNA胞质多聚腺苷酸化与翻译过程的RNA结合蛋白。方法：采用实时定量逆转录聚合酶链反应（qRT-PCR）、蛋白质印迹法（Western blotting）与免疫组织化学法（immunohistochemistry），检测紫杉醇（taxol）耐药卵巢癌细胞系及复发性卵巢肿瘤相较于对应对照样本中CPEB4的表达水平。通过细胞活力检测与集落形成实验（colony formation assay）评估细胞对紫杉醇（taxol）的应答反应。借助RNA免疫沉淀（RNA immunoprecipitation）与poly(A)尾检测（poly(A) tail test）实验，分析RNA结合水平与胞质多聚腺苷酸化程度。结果：在紫杉醇（taxol）耐药卵巢癌细胞及接受紫杉醇（taxol）为基础化疗的复发性卵巢肿瘤中，CPEB4的表达显著升高。此外，CPEB4过表达可在体外促进卵巢癌细胞对紫杉醇（taxol）的耐药性；反之，敲低CPEB4则可恢复细胞对紫杉醇（taxol）的敏感性，表明CPEB4可赋予卵巢癌细胞紫杉醇（taxol）耐药性。从机制层面分析，CPEB4可与紫杉醇（taxol）耐药相关基因3（TRAG-3/CSAG2）的mRNA结合，并在翻译水平诱导其表达。进一步研究发现，CSAG2在紫杉醇（taxol）耐药卵巢癌组织及细胞系中表达上调，更关键的是，小干扰RNA（siRNA）介导的CSAG2敲低可显著减弱CPEB4介导的紫杉醇（taxol）耐药性。结论：本研究表明，耐药蛋白CSAG2可被CPEB4在翻译层面诱导表达，这一机制是CPEB4体外介导卵巢癌紫杉醇（taxol）耐药的核心基础。因此，靶向干预CPEB4/CSAG2轴或可为克服卵巢癌紫杉醇（taxol）耐药带来潜在临床益处。