探討 c-Src 磷酸激酶於B型肝炎病毒 X 蛋白質 活化男性荷爾蒙受體轉錄活性過程中之角色

Abstract

台灣為 B型肝炎病毒感染高流行地區， B型肝炎病毒感染並且被視為在台灣引起肝癌 (Hepatocellular carcinoma, HCC) 的主要危險因子。此類由Ｂ型肝炎病毒造成的 HCC 有好發於男性之特徵，依據流行病學及動物肝癌模式指出男性荷爾蒙訊息傳導路徑活性的上升，可能為造成肝癌好發於男性的主要宿主因子 (host factor) 之一。由本實驗室先前研究得知 HBV所表現的病毒蛋白 HBx具有扮演男性荷爾蒙受體正向轉錄調控因子的能力，能夠在男性荷爾蒙 ligand存在下，促進於 promoter帶有 androgen-response element (ARE) 的基因轉錄活性。 為了進一步研究 HBx促進男性荷爾蒙受體轉錄活化的分子機制，論文著重在探討是否有重要的磷酸激酶參與在此調控路徑當中，依據下列幾點論述，本論文將著重在 c-Src kinase 活性相關之訊息傳遞路徑。首先，已知 HBx會藉由提升細胞內鈣離子濃度，活化 Pyk2 kinase而間接活化 c-Src kinase；第二，已知c-Src kinase的活性在許多肝癌組織中有活化情形；第三， c-Src kinase 扮演細胞質中許多訊息傳導路徑重要的開關，包括Ras/MEK/MAPK、JNK/JAK/STAT及PI3K/Akt等路徑，這些路徑有可能參與調控男性荷爾蒙受體的活性；最後，在近年來於 androgen-independent之前列腺癌的研究指出，活化的 c-Src kinase可透過刺激 AR特定位點的磷酸化程度，促使男性荷爾蒙受體的活化，進一步引起前列腺癌產生。 因此本論文主要研究的問題包括 (1) c-Src kinase 在 HBx 所促進的男性荷爾蒙受體轉錄活化中是否扮演重要角色﹔(2) c-Src kinase 的活性如何影響男性荷爾蒙受體的轉錄活性，包括 c-Src kinase 是否影響男性荷爾蒙受體進入細胞核內所必須的 dimerization 以及是否影響受體的 N-terminal transactivation domain (NTD) 之 transactivation 能力﹔(3)另外也將探討 c-Src kinase是否可能經由影響男性荷爾蒙受體的磷酸化修飾進而調控 AR 的轉錄活性之可能。 我們選用Huh-7及Hep3B 肝癌細胞株作為研究的實驗材料，結果分述如下。首先， HBx所促使之 AR的轉錄活化，在使用特定 c-Src kinase抑制劑 PP2以及利用 c-Src si-RNA技術降低 c-Src表現時均有顯著下降情形，此結果支持c-Src kinase參與在 HBx促進 AR轉錄活化當中，並且扮演重要的角色；然而活化態的 c-Src kinase似乎並不足夠提升 AR的活性。接著我們利用 mammalian two hybrid assay及 mammalian one hybrid assay分別研究 c-Src對於 AR進行轉錄活化的兩個重要步驟之影響，分別探討 c-Src於 AR N端及 C端的相互作用以及 AR NTD的轉錄活化過程中所扮演的角色。結果顯示雖然 HBx可提高 AR N端及 C端的相互作用以及 N端的轉錄的活性，但 c-Src kinase的活性主要貢獻於 AR N端的轉錄活化，而非 N端及 C端的相互作用。最後為了進一步研究c-Src kinase的活性如何影響 AR N端的轉錄活性，著重於其是否透過對 AR磷酸化修飾影響其活性。首先利用特定磷酸化抗體測定 AR Ser81磷酸化程度，結果指出HBx的存在會提高此位點的磷酸化，並且此位點磷酸化會受到c-Src kinase抑制劑 PP2所抑制。為了進一步研究 Ser81位點的磷酸化對於 HBx促進AR轉錄活性有何影響，我們將此位點進行點突變變成 alanine，發現相較於wild-type的 AR，此 mutant AR的轉錄活性下降約 30至 50%；有趣的是，我們發現相較於 wild type AR， AR S81A mutant在 HBx存在及有荷爾蒙刺激之下，蛋白質表現量明顯減少。 以上研究提供證據支持 c-Src kinase在 HBx促進 AR轉錄活性上扮演重要角色，並提出一些 c-Src參與此過程中可能涉及的分子機制，可支持未來將 c-Src kinase 列為預防和治療此類 HCC 之標靶基因之一的可能性。

Taiwan is a hyperendemic area for hepatitis B virus (HBV) infection, which is the major risk factor for HCC in Taiwan. HBV-related HCC has a higher prevalence in men than in women. Both the epidemiological study and the animal liver cancer models suggested that elevated activity of androgen signaling pathway is one of the major host factors attributing to such male gender preference. In our study the role of viral infection in the process, our lab has previously identified that the viral protein HBx can function as a positive transcriptional regulator of AR in activating the androgen response element (ARE)-containing target genes, in a ligand dependent manner. To further investigate the molecular mechanisms underlying HBx enhanced AR activation, the specific aim of this study is to search for the critical kinase involved in the HBx-mediated AR transcriptional activation. In the current study we have focused on the c-Src kinase based on the reasons as followed. First, it was demonstrated that HBx can activate c-Src indirectly through the activation of calcium-dependent tyrosine kinase Pyk2. Secondly, c-Src activity was reported to be elevated in many primary HCC tissues. Thirdly, the c-Src kinase functions as a key switch turning on many cytoplasmic signaling cascades, including Ras/MEK/MAPK, JNK/JAK/STAT, and PI3K/Akt pathways. Some of them might regulate the AR activity. Finally, a recent study in androgen independent prostate cancers indicated that c-Src activation contributes to AR activation through stimulating specific AR phosphorylation. The specific objectives we want to address in the current study are (1) the critical role of c-Src kinase in HBx-enhanced AR transcriptional activation; (2) the effects of c-Src kinase on two critical steps of ligand stimulated AR transactivation, including its N-C dimerization and the activation of its N-termial transcriptional domain (NTD); (3) the possible role of c-Src kinase in regulating AR transcriptional activation through modifying its phosphorylation. Huh-7 and Hep3B hepatoma cell lines are used as assay system in this study and the results are summarized as followed. First, the HBx-enhanced AR transcriptional activation is significantly decreased both by specific c-Src kinase inhibitor PP2 and by the c-Src siRNA. The results support the critical role of c-Src in HBx enhanced AR transactivation. However, the active form of c-Src seems not sufficient to activate AR activity. Secondly, we have investigated the effect of c-Src both on the AR N/C interaction by mammalian two hybrid assay system, and on the transcriptional activity of AR NTD by mammalian one hybrid assay system. The results indicated that although HBx can enhance both AR N-C interaction and AR NTD activity, c-Src activity is mainly contributing to enhance the transcriptional activity of AR NTD rather than the AR N-C interaction. Finally, to study the effect of c-Src in regulating AR phosphorylation, we first used the antibody specifically against the AR phosphorylation site at Ser 81 and found HBx can increase AR phosphorylation at least for this site, which can be inhibited by the c-Src inhibitor PP2. To address the effect of this phosphorylation on HBx enhanced AR activation, we have mutated this residue to alanine as S81A AR and found that the AR reporter activity decrease in this construct (30~50%) compared to wild type AR. Interestingly, we found that the protein level of AR S81A mutant is lower than wild type AR in the presence of HBx and under ligand stimulation. The results of current study support the critical role of c-Src kinase activity in HBx-enhanced AR transcriptional activation and delineate some possible molecular mechanisms underlying such effect. Prospectively, we propose the possibility that c-Src kinase could be a potential target for HCC prevention and therapy in the future.