摘要:在台灣肺癌是造成癌症病人死亡的主因。肺癌主要分為兩種:小型與非小型肺癌細胞。相較於小型肺癌,非小型肺癌病人佔的比例最高、其癌細胞生長較快、擴散較早、病人治癒率最低及預後效果最差。其中主因是由於此種癌細胞容易發生癌細胞轉移。證據顯示在肺組織中,失調的蛋白酶水解作用在細胞周圍的微細環境中,扮演關鍵性重要角色,以增進細胞不正常生長、存活並促進細胞癌化、侵襲與轉移。膜蛋白酶和其抑制者(第一型與第二型肝臟生長因子活化抑制者(HAI-1及HAI-2))是新發現的蛋白族群;當其變異時,造成蛋白酶作用失調,誘使癌症產生和惡化。HAI-1和HAI-2最初被找到時,發現其具有抑制肝細胞生長因子活化者的絲氨酸蛋白酶水解的活性。進而阻止此蛋白酶轉化肝細胞生長因子先驅物成為有活性的肝細胞生長因子(HGF)。肝細胞生長因子擁有高能力活化其接受體(c-Met)。正常的生理上,c-Met訊息會驅動細胞的移動和組織的重新建構。然而,不正常地HGF/c-Met訊息傳遞,牽連著癌症的惡化。研究結果進一步發現HAI-1和HAI-2生理上也具有抑制膜蛋白酶的功能,如抑制間質蛋白酶(matriptase)。雖然HAI-1與HAI-2具有相似的蛋白結構,具有兩個細胞外Kunitz型態的絲氨酸蛋白酶抑制區塊,但是從兩者基因剔除研究中,發現HAI-1與HAI-2的功能是相當不同,擁有其專有的生物與生理功能。癌細胞經常降低HAIs的表現,異常地增高蛋白酶活性,使癌細胞獲得侵襲與轉移能力。為了瞭解HAI-1和HAI-2在非小型肺癌進展、侵襲和轉移所扮演的角色,我們運用楊泮池院長所建構非小型肺癌進展的細胞模組,作為研究的模式平台。我們先前的研究發現:相較於不易轉移的母株細胞,具高侵襲能力的細胞(CL1-5 cells),其HAI-2表現明顯地下降。在高侵襲能力的非小型肺癌細胞中,重新表達HAI-2,會降低細胞的生長,移動,侵襲;同時發現HAI-2量的上升,也會使c-Met和Akt磷酸化,以及β-catenin蛋白量的下降。非小型肺癌CL1細胞並不表達間質蛋白酶(matriptase)但獲得HGFA基因表現。隨著癌細胞的惡化過程,HGFA蛋白表達量會上升。因此,在本研究計畫,我們將探討HAI-2在非小型肺癌細胞生長,移動,侵襲,腫瘤形成與轉移中,所扮演的角色。我們假設在非小型肺癌細胞,下降的HAI-2表現,會活化c-Met,Akt與β-catenin的訊息,造成癌細胞生長,移動,侵襲並增進腫瘤生成與轉移。為了驗證這個假設,五大研究主題將被進一步地探討:1)研究HAI-2在非小型肺癌細胞生長,移動與侵襲過程中,所扮演的角色;2)探索HAI-2是否透過調控HGFA,以調降c-Met和Akt的活性,讓β-catenin蛋白量下降,進一步降低非小型肺癌細胞生長,移動與侵襲的能力;3) 使用實驗動物模式分析HAI-2在肺腫瘤形成與癌轉移過程中是否扮演一個抑制者的角色;4)利用Pulldown與質譜分析,在非小型肺癌細胞中,找出HAI-2相關聯的絲胺酸蛋白酶;5)使用肺癌病人組織切片及其肺積水中癌細胞,測試HAI-2、HGFA,c-Met和β-catenin表現量,以及與癌化過程的相關性。本計劃的成果將有助於我們更瞭解分子機制,肺癌細胞如何因HAI-2表現下降,提升c-Met,Akt與β-catenin訊息的活化,造成非小型肺癌細胞生長,移動、侵襲,腫瘤形成和轉移。並確認在肺癌細胞中,HAI-2,HGFA,c-Met和β-catenin的表現量,是否可作為有用的醫療診斷生物標記。這些研究成果將幫助發展有效的醫療、診斷方式來防範或阻止肺癌惡化與轉移。
Abstract: Lung cancer is the leading cause of cancer-related deaths in Taiwan. It is grossly divided into two types: small cells and non-small cells. Compared to small cell lung carcinoma, non-small cell lung carcinoma (NSCLC) grows more rapidly, spreads earlier and has a low cure rate, mainly due to occurrence of cancer metastasis. It is evidenced that deregulated proteolysis in the pericellular microenvironment has been critically important for cancer cells to enable growth, survival, invasion and metastasis. Membrane-anchored serine proteases (MASPs) and their cognate inhibitors, e.g., hepatocyte growth factor activator inhibitors-1 and -2 (HAI-1 and HAI-2) are recently emerging families with potential roles in cancer. HAI-1 and HAI-2 are initially known as inhibitors that inhibit the serine proteolytic activity of HGF activator (HGFA) and block hepatocyte growth factor (HGF) production from proHGF. HGF is a potent activator for its receptor, c-Met, whose signaling plays roles in cell motility and tissue remodeling, whereas abnormal regulation of HGF/c-Met signaling is implicated in cancer malignancies. Furthermore, HAI-1 and HAI-2 can also physiologically inhibit MASPs, including matriptase. Although HAI-1 and HAI-2 have similar protein structures with two extracellular Kunitz-type serine protease inhibitor domains, HAI-1 and HAI-2 gene knock-out studies have shown their functions are quite distinct in vivo with non-redundant biological roles. To understand the roles of HAI-1 and HAI-2 in NSCLC progression, invasion and metastasis, we employ a NSCLC progression model, established by Dean Yang. Our preliminary data showed a dramatically decrease of HAI-2 level but not HAI-1, and increased protein levels of c-Met, HGFA and β-catenin in highly invasive NSCLC, compared to their parental lowly invasive cells. Ectopic expression of HAI-2 in highly invasive NSCLC cells resulted in reduced cell proliferation, migration and invasion, decreased phosphorylation levels of c-Met and Akt, as well as a reduction of β-catenin protein level. Moreover, NSCLC CL1 cells did not express matriptase but obtained HGFA expression with an increasing trend following the cancer progression. In this proposal, we thus will explore the role of HAI-2 in NSCLC cell proliferation, migration, invasion, tumorigenecity and metastasis. Our hypothesis is that a decrease or loss of HAI-2 expression in NSCLC may up-regulate c-Met, Akt and/or β-catenin signaling, leading to cancer cell proliferation, migration, invasion, tumorigenecity and metastasis. To test this hypothesis, the experiments will be carried out: 1) To delineate the functional role of HAI-2 in NSCLC cell proliferation, migration and invasion, 2) To examine whether HAI-2 can modulate HGFA, leading to down-regulating c-Met, Akt and β-catenin signaling, and a decrease of NSCLC cell proliferation, migration and invasion. 3) To analyze whether HAI-2 plays a negative role in tumorigenicity and cancer metastasis of NSCLC in animal models, 4) To identify a HAI-2-associated serine protease(s) in NSCLC cells, 5) To test whether HAI-2, HGFA, c-Met and β-catenin expression levels are altered in pleural effusions and specimens of human lung cancer patients. The results of these experiments will help us to understand the molecular mechanisms in which down-regulation of HAI-2 in favor of proteolysis may promote c-Met, Akt and β-catenin activation, leading to NSCLC cell proliferation, migration, invasion, tumorigenecity and metastasis, and to identify if the protein levels of HAI-2, HGFA, c-Met and β-catenin in lung tumors may serve as a cancer progression biomarker for diagnosis. Such results may be useful in developing novel strategy to prevent or deter cancer cell invasion, tumorigenicity, and metastasis of NSCLC.