CXXC4-AMOTL2-YAP轴在调控胃癌细胞凋亡中的作用及分子机制研究

The dysregulation of many essential signaling pathways is involved in the initiation and progression of human cancers,for instance gastric cancer. These abnormalities can be either attributed to genetic changes or epigenetic disorder-induced aberrant expression of relevant regulatory proteins. Hippo-YAP signaling pathway plays an critical role in the development of human cancers through regulating cell growth,cell proliferation,cell apoptosis and so on,however the regulatory mechanism in gastric cancer remains elusive.In our previous studies,we found that novel tumor suppressor gene CXXC4 was downregulated in gastric cancer cells due to epigenetic changes;CXXC4 can induce gastric cancer cell apoptosis,however the exact mechanism is not yet clear.Moreover,CXXC4 significantly attenuated YAP expression and inhibited the transcription of YAP downstream target genes,such as survivin. Furthermore,we discovered that CXXC4 was located in the nucleus and might be a novel transcription factor;CXXC4 indeed interacted with the negative regulator of Hippo signaling AMOTL2 directly and promoted its expression transcriptionally.Based on these preliminary results,we hypothesized that CXXC4 blunts YAP activity by regulating the transcription of AMOTL2 directly and eventually inhibits gastric cancer development.In summary,we plan to investigate the detailed molecular mechanisms of CXXC4-induced cell apoptosis and transcriptionally regulation of AMOTL2 to verify our hypothesis,which will be beneficial to clarify the pathogenesis of gastric cancer and provide theoretical basis for tumor prevention and treatment.

胃癌等肿瘤中存在Hippo-YAP等信号通路的异常。这些通路的异常既可以是遗传学变化的结果，也可能是表观遗传紊乱导致的相关调控蛋白表达异常。Hippo-YAP信号通路通过调控细胞凋亡等过程与肿瘤发生发展密切相关，但在胃癌中的调控机制尚未明确。我们在前期研究中发现新抑癌基因CXXC4因表观遗传变化在胃癌中表达下调；CXXC4可诱导胃癌细胞凋亡，但具体机制未明；CXXC4可抑制YAP表达及YAP下游靶基因如survivin的转录；CXXC4定位于细胞核，且可能是一个新的转录因子；CXXC4可直接结合YAP负调控因子AMOTL2基因并促进其转录表达。因此，CXXC4可能通过直接调控AMOTL2转录来抑制YAP活性，从而促进细胞凋亡并抑制胃癌发生发展。本项目拟通过分析CXXC4诱导细胞凋亡的机制以及对AMOTL2转录调控的分子机制验证该假说，从而明确胃癌发病机制，为胃癌的临床防治提供新的理论基础。

胃癌等肿瘤中存在Hippo-YAP等信号通路的异常。这些通路的异常既可以是遗传学变化的结果，也可能是表观遗传紊乱导致的相关调控蛋白表达异常。Hippo-YAP信号通路通过调控细胞凋亡等过程与肿瘤发生发展密切相关，但在胃癌中的调控机制尚未明确。我们在前期研究中发现新抑癌基因CXXC4因表观遗传变化在胃癌中表达下调；CXXC4可诱导胃癌细胞凋亡，但具体机制未明；CXXC4可抑制YAP表达及YAP下游靶基因如survivin的转录；CXXC4定位于细胞核，且可能是一个新的转录因子；CXXC4可直接结合YAP负调控因子AMOTL2基因并促进其转录表达。因此，我们提出假说：CXXC4可能通过直接调控AMOTL2转录来抑制YAP活性，从而促进细胞凋亡并抑制胃癌发生发展。但在后期实验中我们发现在Dox诱导CXXC4表达的SGC7901-WT细胞中YAP的蛋白表达不稳定，多次重复后仍不能确定YAP的表达变化，无法确定CXXC4是否真正通过调控Hippo-YAP信号通路导致细胞凋亡的机制。因此我们重新分析基因芯片筛选结果后筛选出CXXC4下游一个潜在靶基因GDF15，并在mRNA和蛋白水平均证实GDF15确实是CXXC4下游的一个靶基因，CXXC4是通过促进GDF15的转录进而发挥促进凋亡的作用。我们进一步研究发现，转录因子特殊蛋白1（Sp1）可以直接结合到GDF15的启动子区域，并通过Western blot和染色质免疫共沉淀实验证实CXXC4诱导GDF15的转录调控是通过增强Sp1对于GDF15的结合能力来实现的。因此，本项目通过分析CXXC4诱导细胞凋亡的机制以及CXXC4对GDF15转录调控的分子机制，为明确胃癌发病机制及胃癌的临床防治提供新的理论基础。