探討第二型肝細胞生長因子活化抑制者在抑制人類攝護腺癌細胞與肺腺癌細胞侵襲的作用機制與差異性

Differential Mechanisms of Hepatocyte Growth Factor Activator Inhibitor-2 in Repressing Cell Invasion of Human Prostate Cancer and Lung Adenocarcinoma

吳尚儒(Shang-Ru Wu)

指導教授 ： 李明學

國立臺灣大學/醫學院/生物化學暨分子生物學研究所/博士(2017年)

https://doi.org/10.6342/NTU201800283

摘要

在腫瘤侵襲和癌症惡化過程中，細胞表面蛋白降解系統的失調經常扮演重要角色。過去已報導第二型肝細胞生長因子活化抑制者HAI-2 (Hepatocyte growth factor activator inhibitor-2)在人類攝護腺癌細胞中，當其表現降低時會導致間質蛋白酶Matriptase活化，進而促使攝護腺癌細胞的侵襲與腫瘤生長。此研究中，我進一步探討HAI-2是如何抑制Matriptase，細究HAI-2是利用那個蛋白結構區域(domain)來影響Matritpase的活化以及細胞侵襲能力。結果顯示過度表達HAI-2能夠抑制Matriptase活化，且造成攝護腺癌細胞侵襲能力降低。進一步研究顯示HAI-2是利用第一個結構域(KD1)來抑制Matriptase的活化與攝護腺癌細胞的移動力，儘管酵素實驗與蛋白動力分析顯示HAI-2的第二個結構域(KD2)與其第一個結構域(KD1)對Matriptase也有同樣的抑制與結合效果。這些結果綜合指出HAI-2是Matriptase同源的抑制者，而且是透過其第一個結構域(KD1)來抑制細胞Mtriptase的活化以及人類攝護腺癌細胞的侵襲力。另一方面，HAI-2同樣也在人類肺腺癌細胞的侵襲力與轉移扮演重要角色。由於在肺腺癌細胞株中，只存在極少量的Matriptase，因此我鑑尋在肺腺癌中HAI-2的抑制標的蛋白。結果顯示HAI-2能夠抑制一個血中大量存在的重要蛋白酶，纖溶酶(plasmin)，進而抑制肺癌細胞的侵襲力。此外，當HAI-2表現量下降時，亦會導致肺腺癌細胞發生上皮間葉細胞轉化 (epithelial-mesenchymal transition; EMT)的現象，進而增進細胞移動能力。進一步研究發現，HAI-2是透過調控plasmin抑制兩種生長因子的活化，pro-HGF (pro-form hepatocyte growth factor) 和pro-TGF-β1 (pro-form transforming growth factor-β1)，來控制EMT的發生。重要的是，這些發現都能在動物實驗上觀察到。結果清楚指出在HAI-2表現下降的肺腺癌細胞中，其細胞表面plasmin的蛋白活性、EMT現象、與轉移能力都大幅提升。同時研究證實純化的重組HAI-2蛋白能有效抑制Matriptase和plasmin的活性，並且降低人類攝護腺癌細胞及肺腺癌細胞的侵襲能力。綜合以上結果，顯示HAI-2不論在未來臨床診斷以及癌症治療的研究與發展上，皆有極大的重要性。

關鍵字

第二型肝細胞生長因子活化抑制者 ； 間質蛋白酶 ； 人類攝護腺癌細胞 ； 人類肺腺癌細胞 ； 纖溶酶 ； 細胞侵襲 ； 轉移

並列摘要

Dysregulation of pericellular proteolysis is often required for tumor invasion and cancer progression. It has been shown that down-regulation of hepatocyte growth factor activator inhibitor-2 (HAI-2) results in activation of matriptase (a membrane-anchored serine protease), human prostate cancer cell motility and tumor growth. This study investigated the role of HAI-2 in two common human cancers, prostate cancer and lung adenocarcinoma. In prostate cancer, it is further characterized if HAI-2 was a cognate inhibitor for matriptase and identified which Kunitz domain of HAI-2 was required for inhibiting matriptase and human prostate cancer cell motility. The results showed that HAI-2 overexpression suppressed matriptase-induced prostate cancer cell motility. The data further demonstrate that HAI-2 interacts with matriptase on cell surface and inhibits matriptase proteolytic activity. Moreover, cellular HAI-2 harnesses its Kunitz domain 1 (KD1) to inhibit matriptase activation and prostate cancer cell motility although recombinant KD1 and KD2 of HAI-2 both show an inhibitory activity and interaction with matriptase protease domain. The results together indicate that HAI-2 is a cognate inhibitor of matriptase, and KD1 of HAI-2 plays a major role in the inhibition of cellular matritptase activation and cell invasion in human prostate cancer. On the other hand, HAI-2 also plays an important role in the cell motility, progression and tumor invasion of lung adenocarcinoma. Since there is none or little matriptase in lung adenocarcinoma cells, I then sought and identified a serum-derived serine protease plasmin to be an alternative target of HAI-2 in lung adenocarcinoma. The results revealed that HAI-2 repressed cell-surface plasmin activity and lung cancer cell motility. Down-regulation of HAI-2 promoted the epithelial-mesenchymal transition (EMT) of lung cancer cells which contributed to an increase of the cell motility. Moreover, the data showed that HAI-2 could suppress plasmin-mediated activations of pro-HGF (pro-form hepatocyte growth factor) and pro-TGF-β1 (pro-form transforming growth factor-β1), and then decrease the plasmin-induced EMT of lung adenocarcinoma cells. Most importantly, those findings were further proven in a xenografted animal model, indicating that down-regulation of HAI-2 can increase the EMT, plasmin activity, and lung metastatic ability of lung cancer cells. In addition, recombinant HAI-2 proteins exhibited a potent inhibition against matriptase, plasmin and cell invasion of human prostate cancer and lung adenocarcinoma. These results together indicate a suppression role of HAI-2 in the progression of human prostate and lung cancer and may exhibit a potential to develop a therapeutic strategy against human cancer.

並列關鍵字

HAI-2 ； human prostate cancer ； human lung adenocarcinoma ； matriptase ； plasmin ； cell invasion ； metastasis

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