Functional study of type II transmembrane protease, serine 2

肝癌是正常肝細胞變異所產生的惡性腫瘤，同時是國人因癌症致死的主因之一。在大部分亞洲國家中，肝癌也是常見致死的病症。在西方國家中，攝護腺癌是好發於男性的惡性腫瘤。近幾年來，由於生活方式與習慣趨於西化，在許多亞洲國家中，攝護腺癌的罹患率也有逐漸攀升的趨勢。 第二型穿膜絲胺酸蛋白酶 2（TMPRSS2）屬於第二型絲胺酸蛋白酶（TTSP）的一員。早期的文獻中指出第二型絲胺酸蛋白酶 2 大量表現於攝護腺癌病人身上，但其表現在不受賀爾蒙調控之實驗動物攝護腺癌細胞中，則有明顯下降的趨勢。 為了進一步研究第二型絲胺酸蛋白酶2在肝癌以及攝護腺癌中所扮演的角色，我們分析統計臨床上第二型絲胺酸蛋白酶 2在肝癌病人組織切片中的表現量。結果顯示，約有六成的肝癌患者，有下降的第二型絲胺酸蛋白酶 2的表現。相較於第一、二期的肝癌患者，在第三、四期的患者，此蛋白酶表現量更是大幅地降低。在癒後方面，我們發現第二型絲胺酸蛋白酶2表現量愈高的患者，其存活率也有愈高的趨勢。在肝癌細胞株及攝護腺癌細胞株中，較惡化的癌細胞株（如SK-Hep1以及DU-145細胞）表現其第二型絲胺酸蛋白酶2的量，較早期腫瘤形成能力低的癌細胞（HUH7以及LNCaP C-33細胞）少，其下降程度非常顯著。藉由大量表現外源性第二型絲胺酸蛋白酶2在肝癌細胞株（HUH7）中，結果發現癌細胞生長速度趨緩及其移動能力減弱的現象。 在非賀爾蒙依賴型攝護線癌細胞株（DU-145）中，外源性的第二型絲胺酸蛋白酶2 可降低癌細胞的移動及侵襲能力。若將癌細胞株(LNCaP C-33)中的第二型絲胺酸蛋白酶表現量壓低，發現癌細胞生長明顯加快。我們推測是由於癌細胞中的第二型絲胺酸蛋白酶表現量下降，造成某些抑癌基因，如P53，的表現量下降，進而導致癌細胞生長加速。 在共軛焦螢光顯微鏡的分析中，可以發現癌細胞因第二型絲胺酸蛋白酶2的表現量下降，促成細胞形態明顯地變圓；同時觀察到，原本位於細胞膜周邊的beta-catenin蛋白，在膜上的量也有降低的現象。因此，綜合以上實驗結果，在肝癌和攝護腺癌中，第二型絲胺酸蛋白酶2扮演一個負向調控者的角色，以抑制癌細胞生長、移動以及侵襲能力。Hepatocellular carcinoma (HCC) arises from hepatocytes and is the primary malignant lesion of liver. In some areas of Asia, HCC is the most common cause of cancer-related death. Moreover, prostate cancer is a common malignant disease of males in western countries. Recently, the incidence of prostate cancer tends to increase in most Asia countries including Taiwan, because of westernized lifestyle and diet. To further identify genes involved in cancer malignancy, our preliminary data from a microarray analysis showed that TMPRSS2 gene expression was altered. TMPRSS2 (type II transmembrane protease, serine 2) is a member of the type II transmembrane serine protease (TTSP) family. It has been shown that TMPRSS2 protein was abundantly expressed in prostate cancer patients, but down-regulated in hormone-depleted prostate cancer xenograft. o further explore the role of TMPRSS2 in HCC and prostate cancer, initially we investigated the expression of TMPRSS2 in 20 patients’ tissues of hepatocellular carcinoma (HCC) and found that approximately 61% of HCC patients had a decrease of TMPRSS2 expression. The degree of decreased TMPRSS2 expression is higher in stage3~4 patients than that in stage1~2 patients. Using a microarray analysis, a higher expression level of TMPRSS2 was related to a higher survival rate of patients. In human HCC and prostate cancer cell lines, the expression of TMPRSS2 was down-regulated in malignant cancer cell lines (SK-Hep1 and DU-145, compared to HUH7 and LNCaP C-33 cells). Overexpression of TMPRSS2 in HUH7 cells resulted in reducing their cell proliferation and cell migration. In androgen-independent prostate cancer DU-145 cells, ectopic expression of TMPRSS2 also reduced their cell migration and invasion, as shown in HCC cells. Knockdown of TMPRSS2 led to an increase of cell proliferation in LNCaP C-33 cells, at least in part due to TMPRSS2 down-regulating some of tumor suppressor genes in cell cycle including P53 gene. The results from the confocal fluorescence microscopy analysis showed that cell morphology was changed with a roundish shape and membrane-bound beta-catenin decreased in TMPRSS2 knockdown prostate cancer cells. Thus, the data taken togehter indicate that TMPRSS2 exhibits a negative role in cell proliferation, migration, and invasion in human HCC and prostate cancer cells.口試委員審定書 i錄 ii謝 iv文摘要 vbstract: viintroduction 1aterials and methods 6esults 19. Down regulation of TMPRSS2 gene expression in advanced hepatocellular carcinoma (HCC) among 20 HCC tissue pairs. 19. TMPRSS2 expression is related to the survival rate of liver cancer patients. 20. Analysis of the TMPRSS2 gene expression and cell motility in HCC cell lines 20. Establishment of a TMPRSS2 mammalian expression plasmid and stable clones of HUH7 cells expressing TMPRSS2. 21. Overexpression of TMPRSS2 could inhibit cell proliferation of a HUH7 stable clone. 22. Role of TMPRSS2 in migration of HCC cells. 23. Analysis of the TMPRSS2 expression levels in prostate cancer cell lines by a quantitative real-time PCR. 23. Ectopic expression of TMPRSS2 in DU-145 cells could reduce cell transmigration by a transwell assay. 24. Cell proliferation was enhanced in siRNA-TMPRSS2 knockdown cells. 260. Analysis of tumor suppression genes in TMPRSS2-knockdown LNCaP C-33 cells by a quantitative real-time PCR. 271. Analysis of β-catenin distribution and cell morphology in siRNA-TMPRSS2 LNCaP C-33 cells. 272. Cell proliferation was enhanced in TMPRSS2-shRNA knockdown cells. 283. Analysis of β-catenin distribution and cell morphology in shRNA-TMPRSS2 LNCaP C-33 cells. 29iscussion 30igures 35egends 50eferences 6