研究G9a相關網絡於癌症進程之角色

Abstract

組蛋白甲基轉移酶G9a過量表現導致組蛋白3離胺酸9位點甲基化活化已在數種癌症被證實。抑制G9a可有效減緩癌症進程，顯示G9a於癌症發展扮演關鍵角色。因此，進一步研究癌細胞G9a的相關網絡將有助於發展更有效地抑制癌症的策略。於論文的第一章節，我們闡明了G9a於頭頸部鱗狀上皮細胞癌之臨床意義，並且也發現其下游所調控的致癌機制。我們發現於頭頸部鱗狀上皮細胞癌組織中，G9a與細胞增生指標Ki-67有共同表現之趨勢，且與病人預後呈現負相關性。利用RNA干擾技術抑制G9a基因表現抑或是以專一性抑制劑抑制其酵素活性顯著降低癌細胞增生，然而此現象並非起因於促進癌細胞凋亡或是壞死，而是由於活化了細胞自噬依賴型死亡機制所致。進一步發現，此機制係因抑制G9a會促進雙特異性去磷酸酶DUSP4表現，導致細胞外信號調節激酶ERK去活化，進而造成癌細胞進行細胞自噬依賴性死亡而降低其增生能力。最後利用小鼠原位腫瘤移植模式，我們證實了上述抑制G9a導致細胞自噬死亡機制的發生，並且也有效降低腫瘤生長情形。此結果提供了以G9a作為頭頸癌治療標的可能性。於論文的第二個章節，我們則探討G9a與其結合蛋白間的交互作用於癌症進程之角色。藉由液相層析串聯式質譜儀分析，我們發現甲硫胺酸腺苷轉移酶 MATIIα參與細胞核G9a蛋白複合體，且可直接與G9a結合。我們意外地發現MATIIα可維持G9a蛋白穩定性，然而此現象並非經由MATIIα酵素活性所調控，而是透過MATIIα與G9a的直接結合，進而抑制G9a經由APC/CCdh1依賴型泛素化降解所致。利用細胞及動物實驗，我們也證實了細胞核MATIIα—G9a交互作用於癌症進程之重要角色，且細胞核MATIIα與G9a之表現可作為有效評估大腸癌病患預後之指標。最後我們利用動物實驗證實阻斷癌細胞內生性G9a與MATIIα結合將有助於抑制大腸癌細胞轉移至肝臟。綜合以上，上述兩章節實驗結果說明了阻斷G9a以及其相關網絡可作為控制癌症進程之策略，期望我們所發現的G9a相關網絡於癌症發展之重要性可進一步作為G9a被異常調控之癌症治療新標的的發展。

Histone methyltransferase G9a overexpression causing hyperactive of following histone 3 lysine 9 (H3K9) methylations has been identified in diverse cancers. Inhibition of G9a shows a significant anti-tumor effect, supposing G9a plays a pivotal role in mediating cancer development. Therefore, investigation of G9a-related networks may be benefit to develop more effective therapeutic approaches for cancers. In the first chapter, we revealed the clinical significance of G9a and its downstream oncogenic mechanism in head and neck squamous cell carcinoma (HNSCC). We found that G9a is co-expressed with proliferation marker Ki-67 in HNSCC specimen and is correlated to poor prognosis of patients. Genetic or pharmacological inhibition of G9a decreased cell proliferation without inducing necrosis or apoptosis. Instead, autophagic cell death was the major consequence, and our investigation of the mechanism suggested it is mediated via induction of dual specificity phosphatase-4 (DUSP4) caused ERK inactivation that followed by G9a suppression. By using orthotopic tumor model, the growth inhibiting effect and autophagy induction that followed suppression of G9a were both confirmed, providing proof for the possibility that targeting G9a may offer an additional avenue for curing HNSCC. In the second chapter, we investigated the interplay between G9a and its associated protein during cancer progression. By liquid chromatography-tandem mass spectrometry analysis, we identified methionine adenosyltransferase IIα (MATIIα) involves in G9a complex, and may directly interact with G9a. Surprisingly, we found that nuclear MATIIα stabilizes G9a protein that is independent of the enzymatic activity of MATIIα. The further mechanistic study suggested the interaction between MATIIα and G9a might help to maintain G9a protein stability through inhibition of APC/CCdh1-dependent ubiquitin-proteasome degradation of G9a. Moreover, the cooperation between nuclear MATIIα and G9a in promoting cancer progression was also demonstrated, and both high expressions of G9a and MATIIα in nucleus of tumor specimen reflected to the worse prognosis of colon cancer patients. A further therapeutic implication showed that blocking the interplay between nuclear MATIIα and G9a provided benefits for cancer treatment. In summary, the above findings suggest that disruption of G9a and its related networks may be effective approaches to eliminate cancer. We hope that our clarification of the importance of G9a-related networks in cancer progression will allow further development of promising therapeutic strategies for cancers driven by G9a dysregulation.