探討AR協同作用蛋白參與HBV活化男性荷爾蒙受體轉錄活性過程之可能機制

Abstract

肝癌為全球最常見的惡性腫瘤，B型肝炎病毒(HBV)感染為引發肝癌的危險因子之一。台灣為B型肝炎病毒(HBV)感染高流行區，HBV相關之肝癌，有好發於男性之特性，但HBV如何影響肝癌發展及造成性別上的差異，其機制尚未清楚。本實驗室先前研究發現，HBV所表現的病毒蛋白HBx可在男性荷爾蒙的存在下正向調控男性荷爾蒙受體(androgen receptor, AR)的轉錄能力，為HBV相關肝癌好發於男性提供一可能機制。有鑑於在前列腺癌研究中，發現許多AR 協同作用蛋白(co-regulator)可藉由影響AR的轉錄活性促進癌症的發展。為了瞭解HBx如何影響AR轉錄活性，本研究著眼於探討HBx是否可能藉由影響AR的co-regulator而增強AR轉錄活性。研究策略即尋找HBx存在下，與AR結合發生改變之特定蛋白質，純化之並利用蛋白質譜(Mass spectrophotometry) 方式決定其身份，並進一步探討此特定蛋白於HBx促進AR轉錄活性過程中所扮演之角色進行功能性分析。 本實驗所使用的蛋白純化方法為Tandem Affinity Purification (TAP)系統。實驗設計為製備TAP-AR融合質體，於Huh-7 hepatoma細胞株細胞中表現，再利用兩步驟的樹脂純化AR及其結合蛋白。此方法的優點為可使蛋白複合體在接近生理狀態下，被完整的分離出來，且無損蛋白的活性。首先製備pN(C)TAP-AR融合質體，將質體送入細胞中表現，觀察AR的蛋白質表現及確定HBx增強AR的活性之效果。實驗結果發現，HBx 增強NTAP-AR之轉錄活性相較於CTAP-AR 顯著許多，因此決定使用NTAP-AR進行後續純化實驗。將NTAP-AR送入Huh-7細胞後收細胞進行純化，發現HBV存在下，有幾條蛋白質band與單獨表現AR時得到的純化蛋白質結果不同，其中尤以大於170KD之蛋白質band表現差異最大。經蛋白質譜結果得知其為Acetyl-CoA carboxylase-alpha(ACCα)蛋白，為一參與脂肪酸合成過程之重要酵素。以ACCα專一性抗體分析TAP純化及共同免疫沉澱(co-immunoprecipitation)分析，證實ACCα蛋白的確會與AR結合，同時也發現ACCa與AR的交互作用可受到HBx影響。 進一步探討ACCα與AR之結合是否影響了AR的轉錄活性或蛋白表現之研究，利用核質分離方式發現兩者的交互作用位置主要於細胞質中，推測ACCα不會直接影響AR的轉錄活性。接著利用lenti-si-ACCα降低Huh-7細胞中ACCα蛋白表現量，以探討ACCα對HBV活化AR轉錄活性的影響。結果發現降低ACCα表現量，AR蛋白量也隨之下降，利用定量PCR(quantitative PCR)證實此調控不在RNA層面。接著，以蛋白水解酶(proteasome)抑制劑(MG132)發現此調控可影響proteasome的作用。 以上研究結果發現了一個新的AR結合蛋白ACCα。ACCα與AR的交互作用可受HBx調控且影響了AR蛋白表現量，為HBx促進AR轉錄活性過程提出另一可能調控機制。由於AR訊息傳遞路徑之過度活化為男性肝癌形成之一重要機制，此研究結果因此提出可針對抑制ACCα表現作為為來治療男性肝癌之可能性

Taiwan is a hyperendemic area for hepatitis B virus (HBV) infection, which significantly increase the risk of HCC. One characteristic for HBV-related HCC is the male gender preference. Our previous studies identified a possible mechanism underlying the male gender preference, showing that HBx viral protein can up-regulate the transcriptional activity of androgen receptor (AR) in an androgen concentration dependent manner. However, how does HBx enhances the AR activity still remained un-clarified. According to the findings in prostate cancers that the transcriptional activity of AR can be regulated by its associated co-regulators. We propose to test the possibility that HBx can also through affecting the AR associated co-regulators to enhance its transcriptional activity as one possible mechanism. Therefore, the specific aim of this study is to investigate the AR associated proteins involved in HBV enhanced AR activation. The strategy is to identify the specific AR coregulators changing the affinity with AR in the presence of HBx, and then further investigate their functional roles in the HBx-enhanced AR transactivation. The Tandem Affinity Purification (TAP) system was used to purify the AR associated proteins in the current study, due to its great advantages for isolation of proteins with high specificity under physiological conditions. We have constructed pNTAP-AR and pNCTAP-AR plasmids, and found that HBx can enhance the transcriptional activity of pNTAP-AR (but not pNCTAP-AR) close to the level of wild type AR without tag. We thus co-transfected pNTAP-AR and 1.5-mer HBV constructs into Huh-7 cells for the subsequent TAP purification studies. Aided by the MALDI-TOF MS analysis, we have identified one protein, Acetyl-coA carboxylase alpha (ACCα), which was enriched in the fraction of HBV+AR compared with AR only. Further study pointed out that the increased interaction is caused by HBx viral protein. Aided by co-immunoprecipitation analysis, we found that ACCαcan indeed interact with AR. ACCα is an enzyme involved in the synthesis of fatty acid through catalyzing the condensation of malonyl-CoA. To further investigate the functional effect of the interaction between ACCαand AR on AR activity, we approached by knockdown the expression of ACCαby lentivirus based si-RNA in Huh-7 cells. Intriguingly, we found that both the protein level and transcriptional activity of AR were significantly deceased. Aided by quantitative PCR, we found that the effect of si-ACCαon AR protein did not occur at the RNA level. Instead, the results from MG132 treatment suggested that the effect might be attributed by the effect ACCαon regulating the protein degradation of AR. Moreover, we have identified that the interaction of AR and ACCα might mainly occur at the cytosol rather than in the nucleus. In summary, we have identified a novel AR associated protein, ACCα, with its association with AR enhanced by HBx. It might provide a possible regulatory mechanism for HBx enhanced AR activation. Although how does the cytosolic ACCα regulate the protein amount of AR through affecting its degradation process awaits further investigation, the current study points out ACCα as one potential target for blocking the AR activation in male hepatocarcinogenesis.