Department of Obstetrics and Gynecology, Division of Reproductive Sciences, Anschutz Medical Campus, University of Colorado Denver, Aurora, Colorado.
Department of Obstetrics and Gynecology, Division of Gynecologic Oncology, Anschutz Medical Campus, University of Colorado Denver, Aurora, Colorado.
Mol Cancer Res. 2019 Mar;17(3):741-750. doi: 10.1158/1541-7786.MCR-18-0451. Epub 2019 Jan 3.
A significant factor contributing to poor survival rates for patients with ovarian cancer is the insensitivity of tumors to standard-of-care chemotherapy. In this study, we investigated the effect of claudin-4 expression on ovarian tumor cell apoptotic response to cisplatin and paclitaxel. We manipulated claudin-4 gene expression by silencing expression [short hairpin RNA (shRNA)] in cells with endogenously expressed claudin-4 or overexpressing claudin-4 in cells that natively do not express claudin-4. In addition, we inhibited claudin-4 activity with a claudin mimic peptide (CMP). We monitored apoptotic response by caspase-3 and Annexin V binding. We examined proliferation rate by counting the cell number over time as well as measuring the number of mitotic cells. Proximity ligation assays, immunoprecipitation (IP), and immunofluorescence were performed to examine interactions of claudin-4. Western blot analysis of tubulin in cell fractions was used to determine the changes in tubulin polymerization with changes in claudin-4 expression. Results show that claudin-4 expression reduced epithelial ovarian cancer (EOC) cell apoptotic response to paclitaxel. EOCs without claudin-4 proliferated more slowly with enhanced mitotic arrest compared with the cells expressing claudin-4. Furthermore, our results indicate that claudin-4 interacts with tubulin, having a profound effect on the structure and polymerization of the microtubule network. In conclusion, we demonstrate that claudin-4 reduces the ovarian tumor cell response to microtubule-targeting paclitaxel and disrupting claudin-4 with CMP can restore apoptotic response. IMPLICATIONS: These results suggest that claudin-4 expression may provide a biomarker for paclitaxel response and can be a target for new therapeutic strategies to improve response.
导致卵巢癌患者生存率低的一个重要因素是肿瘤对标准治疗化疗的不敏感性。在这项研究中，我们研究了 Claudin-4 表达对卵巢肿瘤细胞对顺铂和紫杉醇凋亡反应的影响。我们通过沉默内源性表达 Claudin-4 的细胞中的 Claudin-4 基因表达[短发夹 RNA（shRNA）]或在天然不表达 Claudin-4 的细胞中过表达 Claudin-4 来操纵 Claudin-4 基因表达。此外，我们用 Claudin 模拟肽（CMP）抑制 Claudin-4 活性。我们通过 caspase-3 和 Annexin V 结合来监测凋亡反应。我们通过随时间计数细胞数量以及测量有丝分裂细胞数量来检查细胞增殖率。进行接近连接测定、免疫沉淀（IP）和免疫荧光以检查 Claudin-4 的相互作用。用细胞分数中的微管蛋白进行 Western blot 分析，以确定 Claudin-4 表达变化时微管蛋白聚合的变化。结果表明 Claudin-4 表达降低了紫杉醇诱导的上皮性卵巢癌（EOC）细胞的凋亡反应。与表达 Claudin-4 的细胞相比，缺乏 Claudin-4 的 EOC 增殖更慢，有丝分裂停滞增强。此外，我们的结果表明 Claudin-4 与微管蛋白相互作用，对微管网络的结构和聚合有深远影响。总之，我们证明 Claudin-4 降低了卵巢肿瘤细胞对微管靶向紫杉醇的反应，并且用 CMP 破坏 Claudin-4 可以恢复凋亡反应。意义：这些结果表明 Claudin-4 表达可能为紫杉醇反应提供生物标志物，并且可以成为改善反应的新治疗策略的靶标。
