第二型肝細胞生長因子活化抑制者在攝護腺癌細胞移動侵襲與調控間質蛋白酶活性的角色

Abstract

第二型肝細胞生長因子活化抑制者(HAI-2)為一絲胺酸蛋白酶抑制者，具有抑制肝細胞生長活化子(HGFA)的活性。此外，HAI-2可有效降低神經膠質瘤細胞的轉移能力。在動物體實驗中也發現，相較於正常表現HAI-2的老鼠，HAI-2缺陷的老鼠在腹壁皮下注射肺癌細胞後，細胞轉移至肺部的機率較高。且研究發現HAI-2的表現量會隨著腫瘤發展過程逐漸減少。這些結果顯示HAI-2具有對抗癌細胞侵襲及轉移的特性。 因此，我們利用一套由中研院農業生技研究中心蕭培文博士建構出可模擬攝護腺癌惡化轉移的細胞模式，包括103E、N1以及N2細胞，來探討HAI-2在攝護腺癌發展過程中所扮演的角色。 我們發現，隨著103E、N1及N2細胞的轉移能力愈來愈強，HAI-2基因在細胞中的表現逐漸降低。另外，過量表現HAI-2可抑制N2細胞的侵襲能力(invasion)，但卻可增加由肝細胞生長因子(HGF)所調控的細胞遷移能力(migration)。而這個 受到肝細胞生長因子所調控的遷移現象可能與間質蛋白酶(matriptase)相關。我們的研究結果顯示，HAI-2會經由其第一個Kuniz domain抑制間質蛋白酶的活化，而降低間質蛋白酶與其抑制者HAI-1所形成的130 kDa複合物，並使得另一70 kDa複合物的生成增加。HAI-2也會穩定原態間質蛋白酶的生成。經由免疫螢光染色分析，雖然HAI-2與間質蛋白酶共同聚集，但進一步利用免疫沉澱法後，卻發現HAI-2與間質蛋白酶之間沒有直接的交互作用。 综合以上結果，我們認為，HAI-2在人類攝護腺癌細胞的侵襲轉移以及間質蛋白酶的活化上扮演抑制的角色，但可提升細胞遷移能力。

HAI-2 (Hepatocyte Growth Factor Activator Inhibitor-2) was identified as a serine protease inhibitor, showing a potent inhibition on HGFA (Hepatocyte Growth Factor Activator) activity. HAI-2 has been proved to play anti-invasive and anti-metastatic roles in human glioma. The expression level of HAI-2 is inversely correlated with tumor progression in human glioma and breast cancer. In the study, a set of prostate cancer cell lines 103E, N1, and N2 cells with an increasing metastasis potential in mice, was established and kindly provided by Dr. Pei-Wen Hsiao, Agriculture Biotechnology Research Center, Academia Sinica. This model was used to examine whether HAI-2 also plays a role in the progression of human prostate cancer cells. We found that gene expression level of HAI-2 was inversely related to the increasing metastatic ability in 103E, N1, and N2 cells. Moreover, HAI-2 was shown to inhibit N2 cells invasion in vitro, but to promote cell migration in a HGF-mediated manner. In addition, the Kunitz domain I of HAI-2 plays an important role in enhancement of prostate cancer cell migration. We hypothesized that the involvement of HAI-2 in HGF-mediated motility is related to matriptase. Our data indicated that HAI-2 inhibited matriptase activation accompanied with a reduced formation of 130 kDa, activated matriptase/HAI-1 complex. HAI-2 also increased a formation of 70 kDa matriptase/HAI-1 complex. Furthermore, HAI-2 was found to stabilize the expression of latent matriptase. Colocalization of HAI-2 and matriptase was observed in vesicle-like structures with an immunofluorescence assay, but CoIP showed that there was no direct interaction between HAI-2 and matriptase. The results taken together indicated that HAI-2 plays an inhibitory role in cancer cell invasion and matriptase activation while HAI-2 can promote cell migration of human prostate cancer cells.