Cx43抑制E-cadherin降解的分子机制及治疗学意义

Downregulation of E-cadherin is the critical event for epithelial-to-mesenchymal transition (EMT) of carcinoma. In our previous study, we found that connexin43 (Cx43) inhibited the lysosomal degradation of E-cadherin. However, the molecular mechanism, which contains important potential therapeutic significance, is remained unknown. According to scientific references and our preliminary results: (1) Cx43 can bind to the Hsc70, the core recognition molecule of chaperone-mediated autophagy (CMA); (2) Hsc70 can bind to E-cadherin; (3) Lysosomal degradation of E-cadherin decreased significantly after the knock-down of Hsc70; (4) the effect for stablizing E-cadherin, which caused by the Cx43 upregulation, was recovered after the co-overexpression of Hsc70, we presumed that Cx43 might competitive inhibit the binding between E-cadherin and Hsc70, and then decrease the degradation of E-cadherin through CMA process. In order to manifest this hypothesis, truncate mutation, GST pull-down, and co-immunoprecipitation assays will be used to assess the protein-protein interaction and to identify the core binding sequence. Then, cell-penetrating peptides targeting this sequence will be synthesized to inhibit the degradation of E-cadherin mediated by CMA. Metastasis of colorectal cancer will be analyzed in vitro by Transwell invasion assays as well as by xenograft in mice via inguinal fold, armpit, footpad, and tail vein injection.

阻遏上皮型-钙粘蛋白(E-cadherin)下调，是抑制上皮间质转化(EMT)进而减少上皮源恶性肿瘤细胞侵袭转移的努力方向。前期研究发现，间隙连接蛋白43(Cx43)能够抑制E-cadherin通过溶酶体降解(Stem Cells 2012；发明专利Z.L.201010508063.X)进而减少肿瘤细胞侵袭，但具体机制不明。探讨相关分子机制，具有重要的治疗学意义。根据文献和预实验，我们推测：Cx43通过竞争性抑制Hsc70对E-cadherin的结合，阻止Hsc70介导的分子伴侣自噬(CMA)对E-cadherin的降解。本研究拟以结直肠癌为研究模型，证实上述科学假说，并在明确E-cadherin/Hsc70/Cx43结合核心序列的基础上，获得模拟Cx43竞争Hsc70与E-cadherin结合的小分子穿膜肽段，减少E-cadherin的降解，进而深入探讨其抗结直肠癌侵袭转移的治疗学意义。

阻遏上皮型-钙粘蛋白(E-cadherin)下调，是抑制上皮间质转化(EMT)进而减少上皮源恶性肿瘤细胞侵袭转移的努力方向。前期研究发现，间隙连接蛋白43(Cx43)能够抑制E-cadherin通过溶酶体降解进而减少肿瘤细胞侵袭，但具体机制不明。探讨相关分子机制，具有重要的治疗学意义。. 其主要研究目的是：证实Cx43通过竞争性抑制Hsc70对E-cadherin的结合，阻止Hsc70与溶酶体膜相关蛋白2（LAMP2）复合体所介导的分子伴侣自噬(CMA)在溶酶体中对E-cadherin的降解。.通过研究我们发现：. 1、E-cadherin表达水平与肿瘤细胞内溶酶体数量呈负相关,溶酶体数目多的患者预后不良。. 2、溶酶体在肿瘤细胞内呈现两种类型的分布：I型细胞邻面聚集型，II型散在分布型。I型分布为主的患者E-cad水平低且生存时间显著短于以II型分布为主的患者。. 3、溶酶体指数高的结直肠癌患者淋巴结转移发生率高，AJCC分期晚，生存时间短；并且证实该指数可作为结直肠癌患者独立的预后预测因素。. 4、LAMP2的表达与结直肠癌的AJCC分期、淋巴结及远处转移率相关，LAMP2高表达的患者预后差，并可作为结直肠癌预后不良的独立预测因素。敲低LAMP2后，溶酶体数目减少，E-cadherin蛋白水平增加，而mRNA水平无明显变化，结直肠癌细胞迁移、侵袭能力降低。. 5、EMT诱导因子作用后，E-cadherin与Hsc70存在共定位. 6、免疫共沉淀则证实，E-cadherin与CMA识别分子Hsc70之间的确存在结合。. 7、敲低Hsc70，E-cadherin的降解减少。. 8、超表达Cx43后E-cadherin溶酶体降解减少的效应可被过表达Hsc70所抑制。. 9、我们还发现，Cx43-C端可影响AKT、ERK信号通路活性