定性分析PREX2在肝癌發展過程中所扮演之角色

Characterization of the role of PREX2 plays in hepatocellular carcinoma tumorigenesis

李國睿(Kuo-Jui Lee)

指導教授 ： 陳宜民

高雄醫學大學/醫學院/醫學研究所碩士班/碩士(2016年)

摘要

肝細胞癌是全球最常見的惡性疾病之一。肝細胞的癌化是多階段的過程牽涉到許多的分子機制。甘胺酸甲基轉移酶 (Glycine N-Methyltransferase, GNMT)在肝臟中含量豐富，但在肝細胞癌組織中的表現明顯降低，甚至不表現。本實驗室建立GNMT基因剔除鼠會自發性的產生肝細胞癌。然而，GNMT在肝癌發展過程中所扮演的角色仍不清楚。實驗室鑑別出一個GNMT結合蛋白PREX2 (Phosphatidylinositol-3,4,5-trisphosphate-dependent Rac exchange factor 2)。文獻指出，PREX2為PTEN的結合蛋白且會抑制PTEN活性。 GNMT與PREX2的蛋白質交互作用會誘導PREX2蛋白質被蛋白酶體降解，也進一步發現PREX2的E3接合酶為HectH9 (Homologous to E6AP carboxyl terminus homologous protein 9)。因此，本研究目的在探討GNMT、PREX2及HectH9之間的交互作用在肝細胞癌形成過程中所扮演的角色。我們發現GNMT會與PREX2結合並且透過HectH9使得PREX2蛋白質被蛋白酶體降解，進而調控肝癌細胞中AKT訊息傳遞路徑與細胞增生。此外，定序肝細胞癌檢體PREX2的基因組，發現了4個不同位點體細胞突變的PREX2。其中變異體PREX2-S1113R減弱了跟HectH9蛋白質的交互作用，此一差異泛素化的程度與延長了PREX2變異體的半衰期。更進一步的增加了活化AKT訊息傳遞路徑與細胞增生的作用。綜合以上結果，本研究證明了GNMT、PREX2及HectH9交互作用在肝癌的發展中扮演了重要的角色，也提出了一個PREX2變異體逃避HectH9泛素化的可能的機制。

關鍵字

肝癌腫瘤新生 ； GNMT ； PREX2 ； HectH9 ； 體細胞突變

並列摘要

Hepatocellular carcinoma (HCC) is one of the important cancer in the world. The pathogenesis of HCC is complicated and involved with many molecular pathways. Glycine N-methyltransferase (GNMT) was abundant in liver but reduced significantly in HCC. Previously our lab established GNMT knock-out mice and found that high percentage of both genders developed HCC spontaneously. However, the mechanisms of GNMT plays in HCC tumorigenesis remain to be elucidated. Recently, We identified PREX2 (Phosphatidylinositol-3,4,5-trisphosphate-dependent Rac exchange factor 2), a novel inhibitor which antagonist PTEN activity, as a GNMT interacting protein. Furthermore we identified the HectH9 (Homologous to E6AP carboxyl terminus homologous protein 9) as the E3-ligase for PREX2. Thus, this study was to characterize the roles of GNMT, PREX2 and HectH9 play in HCC tumorigenesis. We found that GNMT promoted degradation of PREX2 through HectH9, resulted in activation of AKT pathway and cell proliferation. In addition, we sequenced the PREX2 genome using HCC specimens and 4 non-silence somatic mutations were found. Compare to wild-type, one of the somatic mutation, PREX2-S1113R, displayed weaker interaction with HectH9, reduced ubiquitination level and prolonged the half-life of PREX2 protein. Furthermore, PREX2-S1113R enhanced AKT activation and cell proliferation. In summary, these results demonstrate the functional axis of GNMT,PREX2 and HectH9 during HCC tumorigenesis. Besides, we found a mechanism by which PREX2 mutation may be used to escape the GNMT-mediated HectH9 regulation.

並列關鍵字

HCC tumorigenesis ； GNMT ； PREX2 ； HectH9 ； somatic mutations

參考文獻

Abhijit &ha CW, Richard G. MacDonaldl, and Edward Bresnick. 1994. Rat liver cytosolic 4 s polycyclic aromatic hydrocarbon-binding protein is glycine n-methyltransferas. The Journal of Biological Chemistry 269: 5750-56

Adhikary S, Marinoni F, Hock A, Hulleman E, Popov N, et al. 2005. The ubiquitin ligase HectH9 regulates transcriptional activation by Myc and is essential for tumor cell proliferation. Cell 123: 409-21

Andrea Fusoa RAC, Luigi Orru a, Francesca R. Buttarellib,, Scarpaa S. 2001. Gene silencing by S-adenosylmethionine in muscle di¡erentiation. FEBS letters 508: 377-40

Avram Hershko and Aaron Ciechanover. 1998. The ubiquitin system. Annu Rev Biochem 67: 425-79

Barber MA, Hendrickx A, Beullens M, Ceulemans H, Oxley D, et al. 2012. The guanine-nucleotide-exchange factor P-Rex1 is activated by protein phosphatase 1alpha. The Biochemical journal 443: 173-83