Ras-ING4-TDG-Fas轴在胰腺癌中的调控和作用机制

Cancer remains one of major health problems worldwide. Particularly, pancreatic cancer, with the worst 1 and 5 year survival rates among all cancers, is the fourth common cause of deaths due to cancer and kills more than 338,000 people each year worldwide (http://globocan.iarc.fr). Ras signaling was activated in many especially pancreatic cancers. In addition, epigenetic changes mainly aberrant DNA methylation contributes to human cancer development. Over the years, we have revealed many epigenetic changes leading to the activation of Ras signaling. Meanwhile, activated Ras signaling can also remodel epigenetic regulatory network to promote carcinogenesis. Therefore, it is important to clarify the regulation and function of the interplay between Ras signaling and DNA methylation in human carcinogenesis. In preliminary studies, we found that active Ras significantly reduced the expression of DNA demethylation related enzyme called Thymine DNA Glycosylase (TDG) and transcription regulator ING4 seemed to be involved in this process. Ras downregulated the expression of ING4 which could directly bind to TDG promoter. In addition, ING4 depletion inactivated TDG transcription. Interestingly, ectopic TDG expression inhibited tumor growth and induced Fas expression and cell apoptosis. Therefore, we proposed that activated Ras signaling suppresses Fas expression to promote pancreatic cancer development through repressing TDG transcription. In this proposed study, we will try to prove this hypothesis by clarifying the regulation and function of TDG in Ras-transformed cancer cells and pancreatic cancer cells with Ras gene mutations. By doing so, we believe our study will be helpful to understand the regulation of DNA methylation and its contribution to cancer development and facilitate the development of novel strategies for the prevention and treatment of human cancers with Ras gene mutations.

肿瘤是严重的公共卫生问题，胰腺癌更是恶性程度最高的肿瘤之一。胰腺癌中通常存在Ras基因突变；DNA甲基化紊乱也在胰腺癌中发挥重要作用，但Ras对DNA去甲基化及相关酶的调控及其机制有待进一步阐明。我们前期发现DNA去甲基化相关酶胸腺嘧啶DNA糖基化酶（TDG）的表达受转录调控因子ING4所控制；活化Ras可降低ING4蛋白水平并抑制TDG转录；TDG可上调Fas表达，进而诱导胰腺癌细胞发生凋亡，表明Ras可能通过抑制ING4-TDG-Fas轴来下调细胞对凋亡的敏感性，从而促进胰腺癌发生发展。为证实该假说，我们将在本研究中通过细胞和动物等实验来阐明Ras对ING4以及ING4对TDG的表达调控作用及其机制，明确TDG对Fas表达以及凋亡敏感性的调控作用及其机制。因此，本研究将阐明Ras对DNA去甲基化相关酶的调控机制及其在肿瘤中的作用，从而为胰腺癌等Ras相关肿瘤的有效防治提供新的理论依据。

作为第一个从人类肿瘤细胞中分离出来的癌基因, Ras基因在胰腺癌等恶性肿瘤中的突变发生率非常高。DNA主动去甲基化是近年来表观遗传学领域的研究热点。5-甲基胞嘧啶在TET双加氧酶蛋白家族的催化下被不断氧化形成5-羟甲基胞嘧啶、5-醛基胞嘧啶和5-羧基胞嘧啶，最终被胸腺嘧啶-DNA糖基化酶介导的碱基切除修复途径还原为未修饰的胞嘧啶。同DNA甲基化一样，DNA主动去甲基化过程也具有重要的生物学意义，与细胞分化、个体发育以及恶性肿瘤等生理病理过程密切相关。明确肿瘤发生过程中DNA主动去甲基化相关酶的表达调控以及作用机制，将对阐明表观遗传调控机制，认识肿瘤发病过程并探索新的肿瘤防治策略等具有重要意义。目前的 DNA 主动去甲基化调控研究大多集中在 TET 蛋白家族上，针对 TDG 的研究相对较少。我们发现DNA主动去甲基化过程关键酶TDG是Ras信号通路的下游调控基因，在Ras突变的胰腺癌细胞和胰腺癌组织中，TDG表达显著下降。抑癌基因ING4通过与TDG启动子的特异性结合促进TDG转录表达，Ras信号通路以m-Calpain依赖的方式促进ING4蛋白的降解，间接抑制TDG的转录。TDG能够结合到死亡受体Fas基因的启动子区并招募组蛋白去甲基化酶JMJD3使组蛋白H3K27Me3 去甲基化从而激活Fas的表达，在体内外诱导肿瘤细胞发生凋亡。Calpain抑制剂能够提高ING4的蛋白稳定性，使TDG、Fas的表达上调，从而提高肿瘤细胞的凋亡敏感性。通过以上研究，我们明确了Ras对TDG的表达调控作用及其机制，阐明了TDG 的肿瘤抑制作用及其机制，并在体内外初步验证了Calpain抑制剂作为一种潜在的胰腺癌等Ras突变相关肿瘤的靶向治疗药物。