摘要:此研究計畫繼續延伸上期計畫,持續探討細胞周圍絲氨酸蛋白酶第二型間質蛋白酶(Matriptase-2) 如何抑制攝護腺癌細胞侵襲能力,腫瘤生長及轉移的分子機制。癌症轉移與病人極差的預後診斷息息相關,同時是造成攝護腺癌病人死亡的主要原因。失調的細胞周圍蛋白水解作用一直被認為可經由改變癌細胞的侵襲能力與癌組織的微環境,提升癌轉移。第二型間質蛋白酶(又名TMPRSS6)是一個最新被找到的嵌膜絲氨酸蛋白酶。在肝臟中,第二型間質蛋白酶參與鐵離子的恆定調控,但在攝護腺癌及乳癌惡化過程中,其表現量顯著的下降。然而, 第二型間質蛋白酶降低攝護腺癌細胞侵襲能力及惡化過程的分子作用機制,仍然相當不清楚。我們先前研究結果指出在人類攝護腺癌組織中,相較於正常組織,第二型間質蛋白酶的表達量下降。大量表達第二型間質蛋白酶能降低攝護腺癌細胞的侵襲移動能力;反之,剔除第二型間質蛋白酶可增高攝護腺PNT2表皮細胞, 乳癌MCF-7和肝癌HepG2細胞的侵襲力。更有趣的發現為當第二型間質蛋白酶大量表達時,可壓制由轉化生長因子-β1 (TGF-β1)或成肌纖維細胞(myofibrobloast)所誘發促進攝護腺癌PC3細胞的侵襲能力。更進一步的發現經由第二型間質蛋白酶所抑制攝護腺癌細胞的侵襲力,至少一部分經由提升第三型轉化生長因子-β受體Endoglin的表達, 降低uPA對PAI-1的比率和MMP9膠原蛋白的水解能力,增高金屬水解酶抑制蛋白TIMP4的表達,和促進類間葉表皮細胞間的轉換。經微陣列(microarray)分析,結果顯示第二型間質蛋白酶可改變一族群的基因表達。其中,核受體(NR4A3) 特別引起我們的注意,因其具有抑制血癌惡化的潛力。 進一步的結果顯示剃除NR4A3後可提升由第二型間質蛋白酶所抑制的PC3細胞侵襲力,並降低由第二型間質蛋白酶訊息所誘發的Endoglin and PAI-1表達量。Kaplan-Meier病人存活分析顯示高表達核受體NR4A3與攝護腺癌和乳癌病人的無復發生存期 (recurrence-free survival),呈正相關。因此,在此研究計畫中,我們將探討NR4A3是否參與第二型間質蛋白酶所抑制攝護腺癌細胞移動侵襲力的作用中,扮演重要角色以及第二型間質蛋白酶如何促進NR4A3表達,用以降低攝護腺癌的惡化過程的分子機制。我們假設第二型間質蛋白酶訊息能夠藉由增高核受體NR4A3的表達,進而調控增加Endoglin,PAI-1 和TIMP4的基因表達量,導致攝護腺癌細胞侵襲力,腫瘤生長和轉移能力的下降。為了釐清此問題,我們將探討下列四大研究目標:1)驗證核受體NR4A3是否在由第二型間質蛋白酶所抑制的攝護腺癌侵襲力過程中,扮演重要角色。 2)利用免疫沉降法、化合物標定及質譜分析鑑選出第二型間質蛋白酶在攝護腺癌細胞中之受質並釐清其角色。3) 探討核受體NR4A3及鑑選出的第二型間質蛋白酶受質在攝護腺腫瘤新生及轉移中所扮演的角色。 4) 分析核受體NR4A3,Endoglin,TIMP4及由2)研究中鑑定出的第二型間質蛋白酶受質在攝護腺癌病患切片組織的表現量,是否有相關性。這些實驗結果可以幫助我們更加了解第二型間質蛋白酶如何在抑制攝護腺癌症惡化過程中之角色與分子機制。進一步期望這些研究成果可提供並找出有用的標的用來發展有效方式治療攝護腺癌。
Abstract: This research proposal is continuing to address the molecular mechanisms of how pericellular seine protease matriptase-2 suppresses prostate cancer cell invasion, tumor growth and metastasis. Cancer metastasis is often associated with poor prognosis and a leading cause of cancer death in prostate cancer. Dysregulation of pericellular proteolysis has been strongly implicated in the development of metastasis via alteration of cancer cell invasion and tumor microenvironment. Matriptase-2 (MTX-2, also named as TMPRSS6) is a newly identified membrane-anchored serine protease as a regulator for iron homeostasis in liver and can decrease breast and prostate cancer progression. However, the molecular mechanisms how MTX-2 reduces prostate cancer cell invasion and progression remain largely unknown. Our preliminary study showed that MTX-2 expression was decreased in human prostate cancer. MTX-2 overexpression decreased prostate cancer cell motility, while MTX-2 knockdown increased the invasion of immortalized prostate epithelial PNT2, breast cancer MCF-7 and liver cancer HepG2 cells. Interestingly, MTX-2 overexpression suppressed TGF-β1- or myofibrobloast-induced prostate cancer PC3 cell invasion. Moreover, MTX-2-suppressed prostate cancer cell invasion was at least in part via up-regulation of Endoglin, a TGF-β co-receptor, a decreased ratio of uPA to PAI-1, a reduced MMP9 gelatinolytic activity with an increase of its inhibitor TIMP4, and a mesenchymal to epithelial-like transition. Microarray results further showed MTX-2 stimulated a profile of gene expression. Among them, nuclear receptor NR4A3 received our attention because of its potential as a tumor suppressor. Our preliminary results further showed that NR4A3 silencing could increase the invasion of MTX-2-overexpressing PC3 cells, and reduced the expression levels of the Endoglin and PAI-1 which were induced by MTX-2 signaling. The Kaplan-Meier survival plot showed that the high expression levels of NR4A3 were correlated with the recurrence-free survival of human prostate and breast cancer patients. Thus, in this study, we will explore whether NR4A3 is involved in MTX-2-inhibited prostate cancer cell motility and the molecular mechanism of how MTX-2 induces NR4A3 expression, leading to suppression of prostate cancer progression. Our hypothesis is that MTX-2 signaling can increase the expression of NR4A3, leading to up-regulation of Endoglin, PAI-1 and TIMP4, and then inhibition of prostate cancer cell invasion, tumor growth and metastasis. To answer this hypothesis, several specific aims will be carried out as follows: 1) To examine whether NR4A3 plays an important role in MTX-2-inhibited prostate cancer cell invasion, 2) To identify the in vivo substrates of MTX-2 in prostate cancer cells using pulldown and chemical-labelling/LC-MS/MS approaches, 3) To examine the role of NR4A3 and identified MTX-2’s substrate(s) in tumorigenicity and metastasis of prostate cancer, and 4) To analyze the expression levels of NR4A3, Endoglin, TIMP4 and identified MTX-2’s substrate(s) in archival specimens of prostate cancer patients. The results of these experiments will help us to understand the detailed molecular mechanism how MTX-2 plays inhibitory roles in prostate cancer progression. The information generated from the study may provide insights to find out some important targets for developing a therapeutic approach against prostate cancer.