摘要:目前攝護腺癌是台灣男性第七位死亡病因且致病率持續快速上升中。在攝護腺癌惡化過程中,癌細 胞經常轉變成對男性賀爾蒙非依賴型並同時獲得較高的生長,移動與侵襲能力,造成預後效果較差。 最近的研究指出嵌膜絲胺酸蛋白酶,經由其不正常增高的蛋白水解活性降解細胞外基質或其他相關 蛋白質,增加癌細胞移動與侵襲能力。然而,失調的男性賀爾蒙訊息與不正常細胞周圍蛋白水解作 用,是否存在連結並交互影響促進攝護腺癌惡化,仍然不清楚。我們之前研究發現第二型嵌膜絲氨 酸蛋白酶(TMPRSS2)在男性賀爾蒙所促進攝護腺癌細胞的侵襲力,腫瘤生成能力與轉移,扮演關鍵 角色。我們初步結果並發現間質蛋白酶(matriptase)是TMPRSS2其中一個受質,傳遞男性賀爾蒙所誘 發TMPRSS2的訊息。組織染色的結果進一步指出在攝護腺癌組織中,TMPRSS2蛋白的表達量,與 間質蛋白酶的活化和癌症的惡化程度,呈現正相關。因此,TMPRSS2在攝護腺癌惡化過程中扮演一 個正向的角色。最近我們研究結果並發現第一及二型肝細胞生長因子活化抑制蛋白質(HAI-1和 HAI-2)可與TMPRSS2形成複合體。純化的重組HAI-1或HAI-2蛋白質可抑制TMPRSS2蛋白水解的 活性。因此,在本研究計晝中,我們將進一步鑑選出調控TMPRSS2活性的抑制蛋白,TMPRSS2蛋 白水解酶疊層及其受質,並探討這些蛋白質在攝護腺癌惡化過程中,所扮演的角色。我們假設 TMPRSS2與其内在的抑制蛋白失去平衡時,經由活化TMPRSS2所啟動的蛋白酶疊層及其相關受 質,會促進攝護腺癌細胞移動,侵襲,腫瘤生成及轉移。此計劃主要探討分析(1)鑑選細胞内可抑制 TMPRSS2蛋白酶活性的抑制蛋白質,(2)利用可標定活性絲氨酸蛋白酶的探針及LC/MS/MS分析, 鑑選出由TMPRSS2所活化的絲氨酸蛋白酶,(3)使用胺基標定物質共價結合至在攝護腺癌細胞中由 TMPRSS2蛋白水解的受質,經純化及LC/MS/MS分析,系統性地鑑選出TMPRSS2的受質,(4)研究 這些新鑑選由TMPRSS2所活化的絲胺酸蛋白酶及其受質,是否在攝護腺腫瘤生成及轉移扮演角色, (5)偵測這些新鑑選出的蛋白質在攝護腺癌病人癌組織,血液及尿意中的表現量與分布。本計劃的研 究成果將有助於我們更瞭解TMPRSS2促進攝護腺癌惡化進展的分子機制,並有助於發展新的有效醫 療藥物或方式來避免或阻止攝護腺癌惡化與轉移。
Abstract: During the prostate cancer progression, cancer cells often transform from androgen dependence to independence, that often acquires high cell proliferation, migration and invasion, leading to poor prognosis. Recently, membrane-anchored serine proteases have been reported to play an important role in cancer cell migration and invasion by using their aberrant proteolytic activities to break down extracellular matrix components. However, the link between deregulated androgen signaling and pericellular proteolysis in prostate cancer (PCa) progression remains elusive. Our preliminary results showed that membrane-anchored serine protease TMPRSS2 (Type II transmembrane protease, serine 2) mediated androgen-induced PCa cell invasion, tumor growth and metastasis. We further demonstrated that matriptase acts as one of TMPRSS25s substrates for this androgen action and higher levels of TMPRSS2 expression are correlated with higher levels of matriptase activation and clinical stages in PCa tissues. Thus, TMPRSS2 may play a positive role in the prostate cancer progression. Moreover, our preliminary results further showed that hepatocyte growth factor activator inhibitor-1 or -2 (HAI-1 or HAI-2) could form complexes with TMPRSS2 in cells, and recombinant proteins of HAI-1 and HAI-2 could inhibit the proteolytic activity of TMPRSS2. Therefore, in this study, we will further identify TMPRSS25s endogenous inhibitor(s), proteolytic cascade and substrate(s) and their roles in prostate cancer progression. Our hypothesis is that imbalance of TMPRSS2 and its endogenous inhibitor(s) plays roles in promoting cancer cell migration, invasion, tumorigenicity and metastasis, via activating TMPRSS2-activated proteolytic cascade and substrate(s). To test this hypothesis, the following experiments will be carried out: 1) To identify the serine protease inhibitor(s) of TMPRSS2 that can inhibit the proteolytic activity of TMPRSS2, 2) To isolate TMPRSS2-activated serine protease(s) in human prostate cancer cells using activity-based probe labeling and LC/MS/MS analysis, 3) To systematically identify TMPRSS25s substrate(s) in human prostate cancer cells using amino-terminal labelling agents, pulldown assays and LC/MS/MS analysis, 4) To validate the roles of the identified serine proteases and substrates of TMPRSS2 in the tumor growth and metastasis of prostate cancer, 5) To examine the expression levels of the identified TMPRSS2-induced serine proteases and TMPRSS25s substrates in human prostate cancer archival specimens and patients’ urine and serum samples. The results of these experiments will help us to understand the molecular mechanism in which TMPRSS2 promotes prostate cancer progression. The information generated from the study may be useful for developing a novel strategy for prostate cancer therapy.