探討組蛋白甲基轉移酶G9a在頭頸癌中參與腫瘤生成與細胞自噬之分子機制

Abstract

癌症的形成往往是由於關鍵性的基因突變導致正常的細胞繁衍失衡。除了DNA的鹼基序列發生突變外，表觀遺傳修飾亦扮演重要角色參與調控基因的表現。其在特定組蛋白殘基上透過不同的共價鍵修飾作用調控基因的轉錄。研究已知在胚胎發育與癌症發生的過程中，組蛋白H3上第九個胺基酸離胺酸 (Lysine 9)的甲基化對於基因的表現與否扮演關鍵性的角色。G9a為一哺乳動物的組蛋白甲基轉移酶，負責催化組蛋白H3K9的甲基化。已知G9a透過組蛋白H3K9的甲基化抑制某些腫瘤抑制基因的轉錄，研究也顯示在缺氧的情形下會增加G9a的表現與H3K9的甲基化進而調控基因的轉錄。在肺癌與攝護腺癌的相關研究中也指出G9a扮演維繫腫瘤惡化的角色。先前本實驗室研究發現 G9a高度表現在不同類型癌症病人的腫瘤組織中，其中包括頭頸部的癌症。因此，我們企圖探討G9a在頭頸癌發展過程中的重要性及其調控機轉。 在本篇研究中，我們發現相較於腫瘤周邊的正常組織，G9a高度表現在病人的腫瘤組織中，顯示G9a可能參與調控腫瘤的生成。實驗中利用干擾性核醣核酸抑制G9a的表現，結果降低了癌細胞的存活率與細胞貼附性生長的能力，在動物實驗中我們也發現當G9a的表現受到抑制會減緩腫瘤的生成。以G9a的專一性抑制劑BIX-01294阻斷G9a的組蛋白甲基轉移酶活性，也觀察到癌細胞的存活率有下降的情形，顯示G9a的酵素活性參與了G9a調控腫瘤生成的能力。有趣的是，我們意外發現G9a參與了細胞自噬 (Autophagy)作用。利用干擾性核醣核酸抑制G9a的表現，我們發現與細胞自噬相關的蛋白LC3的表現量有上升的趨勢，相同的結果也反應在加入G9a的專一性抑制劑上，我們認為G9a可能透過影響細胞自噬相關的訊息傳遞路徑來達到促進腫瘤的生成。綜合以上實驗結果，顯示G9a在頭頸癌的發生過程中扮演重要的角色，未來可進一步釐清G9a與上下游可能分子的交互作用，以便於有機會運用在癌症之臨床檢測與治療上。

Tumorigenesis is considered to progress through a multistep process from normal histologic features to carcinoma features underlying genetic instabilities including the loss of heterozygocity (LOH) of certain chromosomes and the mutations of certain key genes. Epigenetic was later defined as heritable changes in gene expression that are not due to any alteration in the DNA sequence. Methylation of specific histone residues has important regulatory functions in chromatin organization and gene transcription, which have been linked to the silencing of a number of critical tumor suppressor genes during tumor formation. G9a is a histone methyltransferase (HMTase) for histone H3 lysine 9 dimethylation (H3K9me2), and it was reported that G9a-mediated H3K9me2 aberrantly repressed tumor suppressor genes in breast cancer. Downregulation of G9a was also demonstrated markedly inhibiting cell growth in prostate cancer. Therefore, G9a might serve as a potent therapeutic target. Here, we evaluate the role of G9a in tumor growth together with the G9a enzymatic activity inhibitor BIX-01294 in HNSCC. Exposure of HNSCC cell lines to BIX-01294 resulted in inhibition of cell growth and anchorage-independent growth, and similar results were also observed in G9a knockdown cells. Moreover, in vivo animal model also showed that G9a knockdown results in growth inhibition of tumor cells. Interestingly, we found several specific features characteristic of autophagy after G9a knockdown and BIX-01294 treatment, including appearance of membranous vacuoles and recruitment of microtubule-associated protein 1 light chain 3 (LC3) to autophagosomes. In conclusion, our findings provide a potential role of G9a involved in regulation of autophagy signaling pathway that further to affect tumorigenesis in head and neck cancer.