C型肝炎病毒對肝細胞癌的影響

Abstract

肝細胞癌是一種相當特殊的癌症，因為大多數病人都有明確的病因。常見的病因包括B型肝炎、C型肝炎以及酒精性肝病變。不同的病因會導致不同的癌變機轉，所造成的癌症其分子標記也會有所不同。之前研究發現B型肝炎與C型肝炎在基因突變與基因表現等都不太一樣。 Sorafenib是晚期肝癌的標準第一線治療。在兩個第三期臨床試驗中，sorafenib被證實能延長病人的存活時間。在其中一個研究的子分析中，sorafenib似乎對於C型肝炎的肝癌病人有比較好的效果。因此，我試著探討肝炎病因對於肝細胞癌細胞的影響，不論是在臨床治療成效上或是癌細胞的生物特性上。 我使用PubMed及Cochrane資料庫來搜尋於2013年11月30日前已發表的第三期臨床試驗結果，且這些臨床試驗必須比較sorafenib與其他治療做為晚期肝細胞癌第一線治療的成效。我從已發表的文章中截取資料並為每一次分組病人計算統合風險比率。最後，我納入四個第三期臨床試驗於研究中，總共分析了3057位病的結果。C型肝炎的病人中，整體存活時間的統合風險比率是0.65（95%信賴區間為0.53-0.80），傾向使用sorafenib較佳，而無C型肝炎的病人中，整體存活時間的統合風險比率是0.87（95%信賴區間：0.79-0.96），雖亦傾向使用sorafenib較佳，但兩個統合風險比率的差別達到統計上顯著（p = 0.013）。相對的，類似的分析在其他子分組，例如不同地理區域、不同個人生活表現分數、不同腫瘤侵犯，都沒有統計上的顯著差別。此研究顯示sorafenib做為晚期肝細胞癌第一線治療，對於C型肝炎病人帶來的好處比無C型肝炎的病人來得大。 接著，我使用兩株過度表現C型肝炎核心蛋白的HuH7肝癌細胞株細胞（HuH7-core-high及HuH7-core-low，分別表現較高量或較低量的C型肝炎核心蛋白）進行研究。管柱形成實驗及基質膠栓實驗用來比較細胞促進血管生成的能力。在兩種不同的實驗中，HuH7-core-high及HuH7-core-low細胞都比控制組具有更強的血管新生活性，而HuH7-core-high的血管新生活性又比HuH7-core-low來得更明顯。這些表現C型肝炎核心蛋白的細胞株表現了比較多的血管內皮生長因子（VEGF），不論是在RNA或蛋白質上。若使用抗體抑制血管內皮生長因子，則HuH7-core-high細胞促進血管新生的能力會顯著下降。血管內皮生長因子的基因前方有AP-1的結合處，而HuH7-core-high細胞也的確比未表現核心蛋白的HuH7細胞有較高的AP-1活性及更多在核內表現的磷酸化c-jun。使用藥物或是基因表現抑制來降低HuH7-core-high細胞AP-1活性後，細胞的血管內皮生長因子表現下降且促進血管新生能力降低。在131位肝癌病人的檢體中，我們也發現C型肝炎病人的肝癌組織比B型肝炎病人的肝癌組織有較高的血管內皮生長因子表現。此研究發現C型肝炎核心蛋白藉由AP-1造成的血管內皮生長因子表現增強而擁有較高的促進血管新生能力。 總而言之，C型肝炎對於肝細胞癌細胞有重大的影響。臨床上，sorafenib做為晚期肝癌第一線治療，對C型肝炎病人能提供較大的幫助。而C型肝炎核心蛋白，更可藉著調控AP-1的活性，增強肝癌細胞血管內皮生長因子的表現，繼而使之有較強的促進血管新生能力。

Hepatocellular carcinoma (HCC) is a unique type of cancer with known etiologies. Common etiologies include chronic infection of hepatitis B virus (HBV) or hepatitis C virus (HCV) and alcoholic liver disease. Different etiologies lead to distinctive carcinogenesis, so the molecular signatures of HCC from various etiologies may be diverse. Prior studies found that HBV-related HCC and HCV-related HCC differ in gene mutation, gene expression, etc. For advanced HCC, sorafenib is the current standard first-line treatment because it was demonstrated to provide survival benefits in 2 phase III clinical trials. In the subgroup analysis of 1 of the 2 studies, sorafenib seemed to provide more benefits for patients with HCV-related HCC than for other patients. Therefore, I planned this study to explore the impact of hepatitis etiology on HCC, regarding the treatment efficacy and biological behaviors. PubMed and Cochrane library were searched for phase III clinical trials published before November 30, 2013 that compared sorafenib with other treatments as the first-line therapy for advanced HCC. I retrieved data from the published articles and then calculated synthesized hazard ratios (HRs) for patients of different subgroups. Four phase III clinical trials comparing sorafenib with other treatments, which enrolled 3057 patients, were included in the study. The synthesized HR of HCV (+) patients was 0.65 (95% confidence interval [CI], 0.53-0.80, favoring sorafenib), which was significantly lower than that of HCV (-) patients (0.87, 95% CI 0.79-0.96, p = 0.013). By contrast, the HRs between other subgroups, including geographic regions, performance status, and different tumor involvement, were not significantly different. With this study, I demonstrated that sorafenib provided more survival benefits to HCV (+) patients than to HCV (-) patients as the first-line therapy for advanced HCC. I used two HCC cell lines that overexpressed HCV core protein, HuH7-core-high and HuH7-core-low, with high and low core protein expression, respectively, for further experiments. I used tube formation and Matrigel plug assays to assess the proangiogenic activity. In both assays, HuH7-core-high and HuH7-core-low cells dose-dependently induced stronger angiogenesis than control cells. HuH7 cells with HCV core protein expression showed increased mRNA and protein expression of vascular endothelial growth factor (VEGF). VEGF inhibition by bevacizumab reduced the proangiogenic activity of HuH7-core-high cells. The promotor region of VEGF contains the binding site of activator protein-1 (AP-1). Compared with controls, HuH7-core-high cells had an increased AP-1 activity and nuclear localization of phospho-c-jun. AP-1 inhibition using either RNA knockdown or AP-1 inhibitors reduced the VEGF mRNA expression and the proangiogenic activity of HuH7-core-high cells. Among 131 tissue samples from HCC patients, HCV-related HCC revealed stronger VEGF expression than did hepatitis B virus-related HCC. With this study, I demonstrated that increased VEGF expression through AP-1 activation is a crucial mechanism underlying the proangiogenic activity of the HCV core protein in HCC cells. Above all, hepatitis C virus has pivotal impact on HCC cells. In clinical practice, sorafenib as first-line therapy for advanced HCC benefits HCV-positive patients more. Moreover, HCV core protein augments proangiogenic activity of HCC cells through AP-1 induced VEGF expression.