E3泛素连接酶WWP2与Hippo-YAP形成正反馈环路促进胃癌侵袭转移的机制研究

Invasion and metastasis are the main causes of deaths in patients with gastric cancer. Dysregulation of Hippo-YAP pathway plays a vital role in the invasion and metastasis of gastric cancer, however, the mechanism involved remains to be further elucidated. Our preliminary data showed that WWP2, an E3 ubiquitin ligase, was upregulated in gastric cancer tissues and cell lines. Increased WWP2 expression was correlated with poor prognosis in patients with gastric cancer. Moreover, WWP2 silencing could inhibit gastric cancer cell proliferation and invasion. Mechanistically, our Co-IP data showed that LATS1 is a binding partner of WWP2. Knockdown of WWP2 increased LATS1 expression and downregulated the expression of downstream target genes of Hippo-YAP pathway. Interestingly, we found WWP2 might be a direct target gene of the YAP/TEADs complex. Therefore, we hypothesized that there exists a positive feedback loop involving WWP2 and Hippo-YAP pathway which drives gastric cancer cell invasion and metastasis. In our present study, we will confirm the biological functions of WWP2 in promoting gastric cancer cell invasion and metastasis using in vitro and in vivo assays, as well as clinical samples. Meanwhile, the Co-IP (co-immunoprecipitation), ubiquitination assay and ChIP (Chromatin immunoprecipitation) will be used to clarify if WWP2 inactivates Hippo-YAP pathway through ubiquitinating and degrading LATS1, and to validate if WWP2 is a direct target of YAP/TEADs complex. Our study will not only uncover a mechanism for constitutive WWP2-LATS1-YAP/TEADs positive feedback loop activation in gastric cancer, but also provide potential therapeutic strategies for patients with gastric cancer.

侵袭转移是导致胃癌患者死亡的重要原因，Hippo-YAP通路在胃癌侵袭转移过程中发挥重要作用，其调控机制仍待深入阐明。我们前期研究发现：（1）泛素E3连接酶WWP2在胃癌组织和细胞中表达升高，是预后不良的分子指标；（2）干扰WWP2显著抑制胃癌细胞的增殖和侵袭；（3）干扰WWP2能通过上调LATS1进而抑制Hippo通路靶基因的活性，且WWP2是YAP/TEADs的潜在靶基因。据此，我们提出WWP2与Hippo-YAP形成正反馈环路促进胃癌侵袭转移的科学假说。本项目拟采用细胞实验、裸鼠模型、临床验证明确WWP2促胃癌细胞侵袭转移的功能；利用Co-IP、泛素化实验、ChIP等探讨WWP2泛素化降解LATS1调控Hippo-YAP通路的机制，鉴定WWP2是Hippo通路新的靶基因。本项目将阐明WWP2-LATS1-YAP/TEADs正反馈环路促胃癌侵袭转移的分子机制，为胃癌的诊疗提供新策略。

WWP2( WW domain-containing E3 ubiquitin protein ligase 2)属于 HECT E3泛素连接酶家族，能通过将泛素连接到底物蛋白上而促进其被泛素化降解。多项研究表明WWP2在多种肿瘤中表达异常升高，如胶质瘤、肺癌、肝癌及口腔肿瘤等，且能通过不同的调控机制实现其促肿瘤发生进展的重要作用。本项目分析了WWP2在胃癌细胞系及组织中的表达情况并进一步探讨了WWP2通过泛素化降解LATS1从而调控Hippo-YAP信号通路促进胃癌侵袭转移的调控机制。利用蛋白质免疫共沉淀实验和免疫荧光实验鉴定WWP2是能与Hippo信号通路上游组分LATS1蛋白结合的分子。通过免疫组织化学染色实验(Immunohistochemistiy，IHC)和相关生物信息学网站分析WWP2在胃癌组织中的表达情况，发现WWP2在胃癌中高表达，且与患者不良临床病理特征及不良预后密切相关。采用细胞学功能实验发现上调WWP2后对胃癌细胞增殖、迁移和侵袭能力增强，下调WWP2则效果相反。进一步机制研究发现WWP2通过调控Hippo-YAP通路使得胃癌细胞功能发生改变；裸鼠体内成瘤实验进一步验证了WWP2对胃癌细胞体内生长能力的影响和分子机制；蛋白质半衰期实验(cycloheximide，CHX)、泛素化实验、逆转实验进一步发现WWP2通过促进LATS1蛋白泛素化降解介导Hippo-YAP通路的失活，进而促进胃癌的发生发展。我们的研究表明：WWP2表达升高可能成为胃癌患者疾病进展和生存期较差的分子指标；WWP2可能通过泛素化降解LATSI进而调控Hippo-YAP信号通路活性促进胃癌发生进展。本研究首次阐明WWP2-LATS1-YAP信号轴在胃癌发生进展过程中发挥着重要的作用，可能为胃癌患者提供潜在的诊治新靶点。