Ras通过Calpain 2促进ING4降解在肿瘤发生中的调控及其作用机制

Cancer remains one of major health problems worldwide. Aberrant activation of Ras signaling is important to the development of many cancers. Loss of contact inhibition is one of fundamental features to distinguish tumor cells from normal cells. However, how Ras activation leads to the loss of contact inhibition remain largely unknown. In preliminary studies for this proposed work, we screened proteomic changes upon Ras-initiated malignant transformation and found tumor suppressor ING4 was significantly down-regulated. Transient transfection of ING4 inhibited the anchorage-independent growth of Ras-transformed cells, indicating the importance of ING4 to Ras-initiated transformation. Moreover, we found that Ras-induced ING4 down-regulation was most likely due to the stimulation of calpain 2-mediated degradation. Calpain 2 expression was increased in Ras-transformed cells and inhibition of calpain 2 could restore ING4 expression. Interestingly, calpain 2 expression was decreased accompanied with increased ING4 expression in non-transformed cells upon contact inhibition. The knock-down of RNA-binding protein KSRP significantly restored calpain 2 expression. Taken together, we hypothesized that oncogenic Ras stimulates calpain 2-mediated ING4 degradation to promote loss of contact inhibition. In this proposed work, we will try to prove this hypothesis by clarifying the function of ING4 in contact inhibition and the regulation of calpain 2-mediated ING4 degradation as well as Ras-promoted calpain 2 expression. By uncovering the relevance of Ras-KSRP-calpain 2-ING4 signaling to contact inhibition, we believe our study will be helpful to understand the molecular mechanism underlying cancer development and facilitate the development of novel strategies for the prevention and treatment of human cancers.

Ras在恶性转化过程中至关重要，而接触抑制的丢失是肿瘤细胞有别于正常细胞的基本特征之一，但Ras导致接触抑制丢失的机制尚未清楚。我们在预实验研究中通过蛋白组学筛选发现抑癌蛋白ING4在Ras转化细胞中表达下降；同时calpain 2 表达上升，抑制calpain 2则可恢复ING4表达；但在正常细胞发生接触抑制时，calpain 2 表达下降而ING4则上升，敲低RNA结合蛋白KSRP可恢复calpain 2表达，因此推测Ras可能通过抑制KSRP来上调calpain 2表达，以促进ING4发生降解，导致接触抑制丢失。为验证该假说，我们将在本项目中探索ING4在Ras诱导恶性转化中的作用，分析calpain 2对ING4的降解作用和Ras对calpain 2的表达调控，阐明Ras-KSRP-calpain 2-ING4通路在接触抑制和肿瘤发生中的作用，从而为恶性肿瘤的有效防治提供新的思路。

作为在人类肿瘤细胞中最早被鉴定发现的致癌基因之一，Ras在恶性转化过程中至关重要，而接触抑制的丢失是肿瘤细胞有别于正常细胞的基本特征之一，但Ras导致接触抑制丢失的机制尚未清楚。在本项目研究中，我们通过蛋白组学筛选发现抑癌蛋白ING4在Ras转化细胞中表达下降；同时CAPN2表达上升，抑制CAPN2表达则可恢复ING4表达；但在正常细胞发生接触抑制时，CAPN2表达下降而ING4则上升。胰腺癌是预后最差的恶性肿瘤之一。胰腺癌之中最常发生的分子事件是KRas的突变。Calpains是一类Ca 2+ 依赖的半胱氨酸蛋白酶，和多种肿瘤的发生发展关系密切，其中包括胰腺癌。但其与KRas之间的调控关系仍未明确。我们发现高表达的CAPN2和胰腺癌的差预后呈现正相关，并且CAPN2和KRas的表达呈现正相关关系。通过siRNA敲减CAPN2以及使用Calpain抑制剂PD150606发现抑制CAPN2可以抑制胰腺癌细胞增殖。在调控机制上，我们发现KRas延长了CAPN2的mRNA半衰期。N6-甲基腺嘌呤（M6A）是RNA之中最多的修饰，和肿瘤的发生以及RNA稳定性调控密切相关。通过M6A-Seq和RIP我们接着发现了CAPN2 mRNA存在M6A修饰。并且，我们发现M6A修饰关键酶METTL16可结合CAPN2的mRNA并并直接调控其M6A修饰，并且YTHDF3作为甲基识别蛋白结合CAPN2的mRNA并影响了CAPN2的mRNA的半衰期。敲减KRas可以影响METTL16和CAPN2 mRNA的结合，从而使其mRNA的M6A修饰下降。因此，我们认为活化后KRas通过METTL16介导的m6A来上调其表达，以促进ING4发生降解，导致接触抑制丢失，最终促进胰腺癌等Ras依赖性恶性肿瘤的发生发展。因此，本项目研究揭示了Ras促进肿瘤发生发展的新机制，从而为恶性肿瘤的有效防治提供了新的思路；同时，发现了Ras对RNA M6A修饰调控的新作用，拓展了新的研究方向。