基本同源物增强子在癌症的角色

Abstract

基本同源物增强子(ERH)，最初是在果蠅中發現，是一個高度演化保留的基因，但它在細胞中的功能仍未明。RAS基因是一個常見的致癌基因，在人類的大腸癌、肺癌或胰臟癌都可以發現有很高比例的突變。經由我們的研究發現與Ras正常的細胞相比，剔除ERH蛋白在Ras突變的細胞中會產生較強的毒性。 此外，ERH會與重組人小核醣核蛋白肽D3 (SNRPD3) 互相作用。由於SNRPD3蛋白負責信使核糖核酸的剪接，進一步研究發現有絲分裂驅動蛋白CENP-E的信使核糖核酸在剪接過程必須依賴ERH的存在。若失去ERH將會降低CENP-E蛋白質的形成進而導致染色體排列異常。我們也經由基因表達譜研究發現許多細胞分裂或脫氧核醣核酸損傷修復機制(DDR)相關的基因需要依賴ERH才能正常表現。這些研究結果顯示ERH在信使核糖核酸的剪接及細胞分裂的過程扮演重要的角色。臨床上，我們也發現ERH降低表現後的基因表達標記與RAS基因表達標記成反比。在Ras突變的大腸癌患者若其腫瘤呈現較高的ERH表現量則患者存活率較差。經由種種證據顯示Ras突變的腫瘤一旦ERH表現量下降，腫瘤本身的存活也會下降。目前Ras突變的癌症仍面臨治療上的困境，近年來大規模 RNA干擾篩檢發現在Ras突變的細胞中仍須依賴許多非致癌基因的存在才能存活，ERH就是其中之一。我們的研究證明Ras突變的癌細胞需依賴ERH才能存活，這也提供了這類腫瘤的治療契機. 脫氧核醣核酸損傷修復機制(DDR)的調解失衡會促進肝癌的形成且增加肝癌對化療產生抗藥性。在第二部分的論文,我們觀察到ERH的表現量與共濟失調微血管擴張及Rad3-相關 (ATR) 的信使核糖核酸表現量在肝臟組織中呈現正相關。此外ERH、ATR及檢測點激-1 (Chk-1)蛋白這三個蛋白的信使核糖核酸在肝癌的表現量皆高於正常肝組織。在HepG2細胞中剔除ERH會降低去氧核醣核酸受損後的修復功能。而剔除ERH也會導致ATR信使核糖核酸的剪接異常進而降低ATR蛋白的表現量。由於ATR磷酸化會活化下游的Chk-1蛋白，在經由紫外線或hydroxyurea誘導去氧核醣核酸受損後，剔除ERH將會導致Chk-1無法有效被活化，並進一步降低細胞的修復功能。最後, 我們證明無論是或老鼠實驗，使用Chk1 抑制劑AZD7762皆可以促進 doxorubicin針對肝癌細胞的療效。本研究顯示ERH可以調節ATR信使核糖核酸的剪接，使用Chk-1抑制劑來降低去氧核醣核酸的修復機制有助於增加化療在肝癌的療效。

Enhancer of rudimentary homolog (ERH), originally identified in Drosophila, is a highly conserved gene among metazoans, whereas its molecular function is poorly understood. The Ras family of small GTPases are mutated in a significant fraction of human cancers, with high frequencies of KRAS mutations found in colon, lung and pancreatic cancers. In our study, we identify that depletion of ERH causes greater toxicity in cancer cells with mutations in the small GTPase KRAS compared with KRAS WT cells. ERH interacts with the Sm protein SNRPD3 and is required for the mRNA splicing of the mitotic kinesin CENP-E. Loss of ERH leads to loss of CENP-E and consequently, chromosome congression defects. Gene expression profiling indicates that ERH is required for the expression of multiple cell cycle and DNA damage response (DDR) genes. These findings identify a new role of ERH in mRNA splicing and mitosis. Clinically, the gene expression signature resulting from ERH down-regulation inversely correlates with KRAS signatures. Tumor ERH expression is inversely associated with survival of colorectal cancer patients whose tumors harbor KRAS mutations. Cancers with Ras mutations represent a major therapeutic problem. Recent RNAi screens have uncovered multiple non-oncogene addiction pathways that are necessary for the survival of Ras mutant cells. Our study provided evidence that KRAS mutant cancer cells are dependent on ERH for their survival and could present one such potential avenue for therapeutic exploration. Dysregulation of DNA damage response (DDR) is often involved in the carcinogenesis of HCC and may contribute to HCC’s resistance to chemotherapies. In the following study, we observed positive correlation between ERH and ataxia telangiectasia and Rad3 related (ATR) expression in liver tissues. Expression of ERH, ATR as well as checkpoint kinase 1 (CHK1) were higher in HCCs than in normal liver tissues. Knocking-down ERH augmented ultraviolet light induced DNA damage in HepG2 cells. ATR protein level is reduced upon ERH depletion as a result of defect in the splicing of ATR mRNA. Consequently, the ATR effector kinase Chk1 failed to be phosphorylated upon ultraviolet light or hydroxyurea treatment in ERH knocked-down HepG2 cells. Finally, we observed Chk1 inhibitor AZD7762 enhanced the effect of doxorubicin on inhibiting growth of HCC cells in vitro and in vivo. This study suggested that ERH regulates the splicing of the DNA damage response proteins ATR in HCC cells, and targeting DNA damage response by Chk1 inhibitor augments chemotherapy to treat HCC cells.