青蛙活性肽WL-8通过抑制TMEM16A钙激活氯通道抑制乳腺癌作用的分子机制

The Ca2+-activated chloride channel TMEM16A is overexpressed in breast cancer and becomes a novel biomarker for predicting the prognosis of breast cancer. Identification of new drugs that target TMEM16A will be a promising therapeutic strategy for treating breast cancer. The applicant found that the frog bioactive peptide WL-8 was directly bound to TMEM16A using molecular docking. In addition, WL-8 inhibited Ca2+-activated chloride currents in breast cancer cell and also inhibited breast cancer cell proliferation, suggesting that WL-8 may suppress breast cancer by inhibiting TMEM16A. Furthermore, the applicant found that epidermal growth factor (EGF) increased TMEM16A expression in breast cancer cells and TMEM16A overexpression activated the EGF receptor (EGFR). The purpose of this project is to identify the structure-activity relationship between WL-8 and TMEM16A, to investigate the cellular mechanisms by which the EGFR/STAT3 signaling pathway upregulates TMEM16A expression and TMEM16A overexpression activates the EGFR/STAT3 signaling pathway in breast cancer cells, and to explore the mechanisms by which WL-8 suppresses breast cancer via the TMEM16A/EGFR/STAT3 signaling pathway in breast cancer cells and in nude mice xenografted with breast cancer cells. We believe that achieving these goals will provide the new theoretical and experimental basis for the development of TMEM16A-targeted drugs for the treatment of breast cancer.

TMEM16A钙激活氯通道在乳腺癌中高表达，是新的乳腺癌预后标志物。发现新的靶向TMEM16A的药物有望成为治疗乳腺癌的突破口。应用分子对接方法申请者发现青蛙肽WL-8与TMEM16A直接结合。另外，WL-8抑制乳腺癌细胞钙激活氯电流及乳腺癌细胞增生，表明WL-8可能通过抑制TMEM16A抑制乳腺癌。另外，申请者发现表皮生长因子（EGF）促进TMEM16A在乳腺癌细胞上的表达，而高表达TMEM16A激活表皮生长因子受体（EGFR）。本项目拟在明确WL-8与TMEM16A构效关系基础上，阐明EGFR/STAT3信号通路促进乳腺癌细胞高表达TMEM16A而高表达TMEM16A又能激活该通路的分子机制，并在乳腺癌细胞和荷瘤鼠模型上验证WL-8通过TMEM16A/EGFR/STAT3信号通路抑制乳腺癌的作用机制。本研究为开发靶向TMEM16A的药物用于治疗乳腺癌提供新思路与实验依据。

靶向乳腺癌分子标记物是针对个体化药物治疗乳腺癌的最佳方案。发现新的肿瘤生物标记物、寻找高效低毒药物并深入研究其在乳腺癌发生发展的机制是亟待解决的科学问题。TMEM16A钙激活氯通道在乳腺癌中高表达且TMEM16A高表达与接受他莫昔芬（Tamoxifen）治疗乳腺癌患者生存期延长显著相关。然而，TMEM16A高表达的原因及其促进乳腺癌发生发展的机制尚不清楚。我们应用Western blot、免疫组织化学、膜片钳等方法，发现TMEM16A在乳腺癌中高表达，EGF促进乳腺癌细胞表达TMEM16A。进一步发现TMEM16A过表达通过激活EGFR/STAT3信号通路促进乳腺癌细胞体外增殖以及在体荷瘤生长。TMEM16A抑制剂T16Ainh-A01以及转染具有低通道活性的TMEM16A突变体阻断EGFR/STAT3信号通路激活以及细胞增殖，提示TMEM16A通道活性发挥重要作用，以上结果提示EGFR/STAT3信号通路促进乳腺癌细胞中表达TMEM16A而高表达TMEM16A又能激活该通路的正反馈机制。另外，我们还发现青蛙肽WL-8抑制TMEM16A钙激活氯通道电流并通过TMEM16A/EGFR/STAT3通路发挥抗乳腺癌作用，此结果为发现新型TMEM16A抑制剂用于治疗乳腺癌的药物提供理论基础和实验依据，为深入了解TMEM16A促进乳腺癌发生发展提供新思路，为研究靶向TMEM16A治疗乳腺癌提供新方向。本项目培养硕士研究生3名，研究成果发表相关论文6篇，其中5篇论文发表在SCI杂志，总影响因子达24.594。