| 摘要: | 本計畫的目標，在於確認男性激素受體(AR)對於在後期肝癌疾病進展，它如何促進循環血 液中，癌細胞死亡，以及其潛在發展為肝癌復發或轉移危險因子之評估。男性激素受體相關訊 息路徑已經證實對於肝癌發生扮演重要的角色。然而，對於其在肝癌後期的角色卻不清楚。趁 著基因轉殖動物之便，我們發展了致癌物質引發肝癌的小鼠模式，並且得以研究這個困難且迫 切的問題。有趣的是，當剔除小鼠肝臟中的AR，在肝癌後期發現了肺部轉移的腫瘤，癌症存活 率變得更遭，以及在肝臟組織學上變得更惡化。 除此之外，我們發現AR具有促進細胞飄零 （Anoikis），及抑制細胞侵移(Invasion)活性，以達到抑制小鼠肝癌轉移的作用。更進一步的， 我們引進雞尾酒式治療法的觀念，將AR表現結合Sorafenib標把藥物，可以在小鼠癌轉移模式， 達到加乘的治療效果。受益於國科會上一階段的補助，這部份的結果已經整理並且投稿並被接 受於肝臟醫學期刊 (HEPATOLOGY, accepted 14, Dec, 2011)。延續同一觀念，我們將進一步探討 其現象之分子機轉。 如同初步數據所示，我們發現AR訊息促進細胞飄零可能是透過細胞骨架之重組(cytoskeleton rearrangement)，以及相關之訊息調節。此現象與臨床之發現中，循環血液癌細胞存活率，相對 於病人癌症轉移/復發呈現正相關，是完全吻合的。所以，本計畫將進一步探索AR透過促進細 胞骨架之重組，以及細胞的焦點貼附促進細胞飄零之分子機制做完整的分析。我們據此發展出 新的理論，也就是，經由調節血循中癌細胞的AR蛋白表現，我們可以達到預測，或者防止癌症 轉移/復發的目標。為了達到此目標，我們預計將完成以下三個主要目標。 第一，確認AR 在乙型肝炎病毒引發的肝癌基因轉殖小鼠之癌轉移，以及驗證AR在人 類肝癌細胞上，扮演癌轉移之抑制角色 1.a, 在乙型肝炎引發肝癌小鼠上比較缺乏AR表現之癌轉移率，轉移腫瘤病灶數，侵犯能力等 1.b，用微小核糖核酸針對抑制AR，以驗證抑制AR將促進乙型肝炎病毒引發的肝癌基因轉殖 小鼠之癌細胞侵犯，以及抑制飄零的作用 1.c，用病毒為媒介的方式，將AR互補的去氧核糖核酸送到癌細胞內，以驗證AR將抑制乙型 肝炎病毒引發的肝癌基因轉殖小鼠之癌細胞侵犯，以及抑制飄零的作用 第二，探索AR對於抑制癌轉移之分子機制 2.a，利用人類肝癌細胞株以驗證AR在肝癌轉移之細胞機制 2.b，利用乙型肝癌小鼠之癌細胞，探討細胞骨架重組對於肝細胞AR表現引發之細胞瓢零之分 子機制 2.c，利用人類肝癌細胞， 探討細胞骨架重組對於肝細胞AR表現引發之細胞瓢零之分子機制 第三，檢驗是否在血液中癌細胞AR之基因多樣性，以及其表現量是預測肝癌轉移暨復 發的因子 。 3.a， 檢驗AR在肝癌病人的血液中癌細胞的表現比例及程度，以及表現量是否與肝癌轉移及復 發相關 3.b， 檢驗血液中癌細胞之AR基因的多樣型，與肝癌病人的肝癌轉移及復發之相關性
The Study of Androgen Receptor in Circulating Cancer Cells in Cancer Recurrence/Metastasis—Hepatocellular carcinoma Summary: The goal of this proposal is to determine the roles of androgen receptor (AR) in later stage of HCC progression, how it promotes circulating cancer cell (CCC) death, and the potential applications for evaluating it as prognostic risk factor for hepatocellular carcinoma (HCC) recurrence/metastasis. Androgen/AR signals were proved to promote hepatocarcinogenesis; however, it is unknown of its role in advanced HCC progression. Using conditional knockout in liver AR (L-ARKO) of the carcinogen-HCC mouse model, we were able to examine the roles of AR in advanced HCC progression. Interestingly, we found increased lung metastasis incidence, poorer cancer survival, and poorer differentiated histological HCC patterns in the L-ARKO-HCC mice. In addition, we found AR could enhance anoikis and suppress invasion to suppress HCC metastasis in animal model. We further introduce a combination strategy that expressing AR with Sorafenib could synergistically improve prognosis and suppress metastasis in mouse model. Supported by NSC in the last funding term, we have submitted to Hepatology, and been accepted on Dec 2011. Following the concept of those findings, we further explore the detailed mechanism of this unique function of AR in advanced stage of cancer progression. As showed in the preliminary results, we found AR signals enhance detached cell death (Anoikis) through cytoskeleton rearrangement signals. This observation is perfectly fit to the clinical observations that CCC survival in the patient is highly correlates to cancer recurrence/metastasis. Therefore, in this application, we would like to further approach to the molecular mechanism that AR enhance cell Anoikis through cell adhesion signals and cytoskeleton rearrangement. We’ve established a novel hypothesis that by measuring CCC AR expression levels and AR gene polymorphism, we could predict cancer prognosis and prevent recurrence/metastasis. To achieve those goals, we would like to propose the following aims. Specific Aim 1. Using HBV-lowDEN-liver ARKO HCC mouse model and primary culture HCC cells to prove hepatic AR functions as suppressor in HCC metastasis Aim 1a. Comparison of lung metastatic rate, foci numbers, invasive capacity, of HBV-lowDEN-WT mice and HBV-lowDEN-ARKO mice Aim 1b. Using mAR-siRNA to targeting hepatic AR in metastatic HCC cells to prove the reduction of AR expression will promote the invasion and metastatic ability of HCC cells Aim 1c. Using lentiviral AR cDNA to restore hepatic AR expression to confirm the restoration of AR expression will suppress the metastasis of HCC cells. Specific Aim 2. To study the mechanisms by which hepatic AR suppresses HCC metastasis. Aim 2a. Using human HCC cell lines to prove hepatic AR suppressor role in HCC metastasis Aim 2b. Mechanism of hepatic AR enhances HCC anoikis via affecting cytoskeletal rearrangement in HBV-lowDEN-HCC mouse model Aim 2c. Mechanism of AR enhances HCC anoikis via modulation of cytoskeletal rearrangement in human HCC cells Specific Aim 3: To examine AR gene polymorphism and protein expression in CCC for predicting HCC metastasis and recurrence. Aim 3a. To examine CCC AR positive population and AR expression in HCC patients to associate with metastasis/recurrence. Aim 3b. To evaluate the association of AR gene SNP in CCC with HCC metastasis/recurrence. |
