YAP1-AGK正反馈环路促进胃癌细胞转移的分子机制

Gastric cancer is the fourth most commonly diagnosed cancer and the second major cause of cancer-related death in the world. Despite the declining incidence of GC, patients have poor prognosis due to the high rate of metastasis and postsurgical recurrence. The Hippo pathway is an emerging signaling pathway that plays important roles in development and disease. Recent studies showed Hippo-YAP pathway plays crucial roles in gastric cancer cell metastasis, however, the underlying molecular mechanism of Hippo inactivation remain largely unknown. In our preliminary work, we identified AGK might be a new direct target gene of the Hippo-YAP1 signaling pathway. Knockdown of endogenous AGK inhibited gastric cancer cell proliferation and invasion, over-expression of AGK promoted cell proliferation and invasion. Moreover, analysis of TCGA and immunohistochemistry (IHC) on gastric cancer tissues revealed overexpression of AGK mRNA and protein was associated with TNM staging and lymph node metastasis, and the high cytoplasm expression of AGK predicted a poor survival time of patients. We found AGK might bind to ITCH to inactivate the Hippo signaling pathway. So, we hypothesized that YAP1-AGK positive feedback loop promoted gastric cancer cells metastasis. Therefore, in the present project, we aim to use both in vitro and in vivo systems to verify the function of YAP1/AGK on gastric cancer cells. Moreover, we will use chromatin immunoprecipitation (Chip), co-immunoprecipitation (Co-IP), GST pull-down assay and protein mass spectrometry to illuminate whether AGK is a direct target gene of YAP/TEADs complex, and how AGK stabilizes ITCH to inactivate the Hippo signaling pathway, to establish this positive feedback regulation in gastric cancer cells. Finally, we aim to use the cancer genome atlas TCGA data and clinical samples to further investigate the clinical significant of this feedback in gastric patients. Those results may provide new therapeutic targets for the treatment of gastric cancer in the future.

转移是胃癌患者主要死因，Hippo通路在胃癌转移过程中发挥重要作用，然而其调控机制尚未阐明。我们前期发现：1）酰基甘油激酶AGK可能是Hippo通路新的靶基因；2) 下调AGK显著抑制胃癌细胞增殖和侵袭，过表达则相反，AGK在胃癌组织中表达升高，高表达提示患者易转移和预后差；3）AGK可能稳定结合ITCH失活Hippp通路。据此，我们推测YAP1-AGK正反馈环路促进胃癌细胞转移。为此，我们将构建抑制或上调YAP1/AGK的细胞模型，利用细胞培养和裸鼠实验观察调节YAP1/AGK对胃癌转移的作用；通过Chip、免疫共沉淀和质谱等方法，证实AGK是Hippo通路的靶基因，探索AGK稳定ITCH失活Hippo通路的机制，解析此正反馈环路的作用；采用TCGA数据结合组织样本，明确YAP1-AGK正反馈轴在胃癌中的临床意义。本研究揭示了Hippo通路新的调控机制，为胃癌的靶向治疗提供新的治疗策略。

背景：.酰基甘油激酶 AGK 位于人类染色体 7q34 区域，是一个多功能的脂质激酶，通过催化酰基甘油磷酸化产生溶血磷脂酸（LPA），从而参与到人体众多生物学过程中。最近多项证据表明，AGK 表达失调与各种人类癌症的发生、发展有关。本课题研究了 AGK 对胃癌细胞增殖和癌变的影响，并深入研究了其中的分子作用机制。.方法:.采用免疫组织化学和蛋白质印迹分析检测了胃癌细胞系和胃癌组织中的AGK 表达情况；基于免疫组化的结果分析了 AGK 表达水平与胃癌患者临床病理特征的关系。通过检测上皮-间充质转变标志物以及 CCK-8，集落形成，transwell 侵袭和迁移实验以及异种移植模型明确 AGK 在胃癌中的生物学功能。之后，在胃癌细胞和组织中分析 AGK 和 YAP1 之间的相关性，并通过荧光素酶报告基因实验，染色质免疫共沉淀，免疫荧光和 qRT-PCR 等实验进一步研究两者之间的相互作用机制。.结果：.免疫组化和 western blot 实验均显示 AGK 的表达在胃癌细胞系和组织样品中上调，高表达 AGK 蛋白与胃癌患者组织分化不佳（P=0.009）并且与胃癌患者预后不良密切相关。过表达 AGK 能促进胃癌细胞的增殖、侵袭能力和上皮-间充质转变标志物的表达。相反，敲减 AGK 的表达则抑制胃癌细胞的增殖、侵袭以及裸鼠异种移植模型中的肿瘤形成能力。进一步研究其内在分子机制，我们发现 AGK 的表达与胃癌细胞和组织中的 YAP1 的表达呈正相关。转录共激活因子 YAP1 可通过 TEAD 与 AGK 基因启动子区域结合从而转录诱导 AGK 的表达。此外，AGK 通过抑制 Hippo 信号通路激活进而诱导 YAP1 核定位增多并增强 YAP1/TEAD 的转录活性。.结论：.本研究表明 AGK 不仅是 Hippo-YAP1 通路的新靶点，而且它还通过抑制Hippo 通路激活从而促进 YAP1/TEAD 转录活性，形成 YAP1-AGK 正反馈环路。此外，YAP1-AGK 正反馈环路与胃癌的发生、发展密切相关。因此，本研究提示双重靶向 AGK 和 YAP1 可能成为未来胃癌治疗的新策略。