第二型肝細胞生長因子活化抑制者在非小型肺癌細胞移動侵襲中的抑制角色

Abstract

在全世界因癌症所造成的死亡中，肺癌佔了最大的部分。肺癌可分為兩種主要的型態，包括非小型細胞肺癌(約佔所有肺癌的百分之八十五)以及小型細胞肺癌(約佔所有肺癌的百分之十五)。儘管有早期預防性的健康檢查及標準的肺癌治療流程，非小型細胞肺癌通常在肺癌的後期才被診斷出，且常伴隨著高度的轉移能力以及很差的預後情況。因此，對於非小型細胞肺癌產生的機制及其惡化的過程，迫切的需要被了解，以期對此病症建立並提供有效的治療方式。第二型肝細胞生長因子活化抑制者(HAI-2)，被認為可以有效降低神經膠質瘤(glioma)以及乳癌(breast cancer)細胞的轉移能力。HAI-2具有兩個Kunitz domain，可以廣泛地抑制絲胺酸蛋白脢，例如plasmin、trypsin及kallikreins等。為了更進一步探討HAI-2在非小型肺癌細胞移動與侵襲中所扮演的角色，我們使用一套由台大醫學院楊泮池院長建構出可模擬非小型肺癌細胞惡化轉移的細胞模式進行研究，包括具低侵略性的CL1-0細胞以及高侵略性的CL1-5細胞。 我們發現，隨著非小型肺癌細胞移動侵襲能力的增強，HAI-2基因的表現就愈低。當過量表現HAI-2在CL1-5細胞時，可抑制細胞移動(migration)與侵襲(invasion)的能力。當在CL1-0細胞降低HAI-2的表現量，則會增加細胞移動及侵襲力。更進一步地，我們發現相較於CL1-0細胞，CL1-5細胞的c-Met、Akt、PDK1及GSK-3β的磷酸化和beta-catenin及FAK的蛋白質表現量都有明顯的上升。相當有趣的是，在CL1-5細胞中過量表現HAI-2時，c-Met、Akt、PDK1及GSK-3beta的磷酸化和beta-catenin及FAK的蛋白質表現量都有明顯的下降。我們更進一步地觀察到，HAI-2的每一個domain對於抑制細胞侵襲的功能都需參與。但在HAI-2抑制細胞移動的過程中，第二個Kunitz domain則扮演一個重要的角色。綜合以上，我們的結果證實肺癌細胞惡化過程，會降低HAI-2表現，以增進其移動侵襲能力。HAI-2表現則可以透過調控c-Met、PDK1、GSK-3beta/Akt的活性及beta-catenin和FAK量，以降低非小型肺癌細胞的移動侵襲與轉移能力。

Lung cancer is the leading cause of cancer deaths in the worldwide and Taiwan. The two major forms of lung cancer are non-small cell lung cancer (about 85% of all lung cancers) and small-cell lung cancer (about 15%). Despite advances in early detection and standard treatment, non-small cell lung cancer is often diagnosed at an advanced stage with a highly metastatic potential and a poor prognosis. Hepatocyte growth factor activator inhibitor type-2 (HAI-2) is proposed to be involved in anti-invasion and anti-metastasis in human glioma and breast cancer, and functions as a serine protease inhibitor with two Kunitz-domains. HAI-2 has broad inhibitory spectra against various serine proteases including hepatocyte growth factor activator, plasmin, trypsin and kallikreins. To further explore the role of HAI-2 in the progression and malignance of non-small cell lung carcinoma (NSCLC) cell migration and invasion, in the current study, we examined the role of HAI-2 in the progression of NSCLC, by using a NSCLC progression model including lowly invasive CL1-0 cells and highly invasive CL1-5 cells, established by Dean Yang. We found that the gene expression level of HAI-2 was inversely correlated with the cell migration and invasion of NSCLC. Ectopic expression of HAI-2 in CL1-5 cells reduced their cell migration and invasion, while knockdown of HAI-2 in CL1-0 cells promoted those cell migrating and invading capabilities. Moreover, the phosphorylation levels of c-Met, Akt, PDK1 and GSK-3β, as well as the protein levels of FAK and β-catenin were increased in CL1-5 cells, compared to CL1-0 cells. Interestingly, over-expression of HAI-2 in CL1-5 cells reduced the phosphorylation levels of c-Met, Akt, PDK1 and GSK-3β, as well as the protein levels of FAK and β-catenin. Furthermore, we also found that each domain of HAI-2 was important for inhibiting NSCLC cell invasion. The KD2 but not KD1 of HAI-2 played a major role in modulating NSCLC cell migration. The results taken together indicate that HAI-2 plays an inhibitory role in NSCLC cell migration and invasion, at least in part via down-regulating c-Met, PDK1 and Akt/GSK-3β activities, or the protein levels of β-catenin and FAK.