探討肝癌中性激素路徑對於TERT基因表現之調控

Abstract

B型肝炎病毒(HBV)相關肝癌有顯著男性發生率高於女性的現象。實驗室先前研究針對HBV相關肝癌DNA進行NGS分析，探討是否存在任何男女差異的現象，結果指出HBV嵌入TERT promoter區域與TERT promoter -124, -146位點發生mutation之兩種活化TERT的機制，均有顯著好發於男性病患的現象。有鑑於實驗室先前研究發現雄激素路徑會經由受體直接結合至病毒基因體enhancer I區域，促進HBV轉錄，而雌激素路徑會藉由受體螯合HNF4α抑制HBV轉錄，造成男性病患較高之病毒量，為可能導致HBV相關肝癌男女差異的一種機制。本研究因此提出探討性激素，包括雄激素路徑及雌激素路徑，是否亦參與在調控上述兩種活化TERT的基因體變異，造成TERT於男性較高表現之可能假說。 本實驗提出利用HBV嵌入TERT promoter區域及包含TERT promoter mutation之報導基因，於細胞培養模式驗證上述假說。針對HBV-TERT promoter integration報導基因所進行之實驗結果顯示，雄激素路徑會促進而雌激素路徑則會抑制報導基因之轉錄。進一步發現將調控HBV轉錄之重要轉錄因子HNF4α敲落後，性激素造成的差異變得不明顯，顯示HNF4α為造成性激素路徑於此報導基因轉錄差異之重要因子。此外針對TERT promoter mutation報導基因所進行之實驗結果顯示，雄激素路徑會促進其轉錄活性，而雌激素路徑較無明顯之影響，而上述作用在將轉錄因子GABPα敲落後即顯著降低，顯示性激素活化此報導基因的機制是建立在GABPα之上。我們實驗結果進一步也發現HNF4α對於GABPα促進此報導基因轉錄上扮演重要的角色，且其促進轉錄作用和雄激素路徑可能為兩獨立調控機制。上述實驗結果初步支持性激素參與調控上述兩種活化TERT的基因體變異，造成TERT於男性較高表現之可能。

Hepatitis B virus (HBV) induced hepatocellular carcinoma (HCC) occurs predominantly in male patients. Aided by the capture-NGS, our recent study showed that the insertional mutagenesis to active TERT oncogene occurs preferentially in the male HCC. Meanwhile, the TERT promoter mutations at -124G>A and -146G>A, the most frequent somatic mutation identified in HCC, also occurred preferentially in male HCC. We have previously identified androgen and estrogen effects on the transcription of HBV via targeting to viral enhancer I, leading to higher viral titer in male carriers. This study proposed to investigate if sex hormones can also through the HBV integrated in the host genome or frequent TERT promoter mutations to regulate the expression of TERT oncogenes, as a mechanism contributing to male HCC. We propose to approach this by the cell culture based TERT-promoter-Luc reporter assay, containing the HBV integration or -124G>A or -146G>A mutations at promoter region. We first found a significantly higher Luc activity from HBV/TERT-Luc than that from WT/TERT-Luc construct, which can be further increased by AR but suppressed by ER pathway. Knockdown of HNF4Α abolished the difference, indicating the critical role of HNF4Α in this regulation. Next, we found a significant elevation in both G(-124)A and G(-146)A-Luc constructs relative to the WT-TERT-Luc, which is dependent on the GABPα. The effect of AR and ERα pathway was examined, showing the reporter activity to be further elevated by AR, in a ligand independent manner, which however was not affected by the ERα pathway. Interestingly, or results showed that HNF4Αalso contributed to the activation of G(-124)A-TERT-Luc. The function of AR however is independent of HNF4Α. Our study thus supported the involvement of sex hormones in regulating the transcription of TERT through HBV integration or frequent TERT promoter mutations.