肝素第二輔助因子促進非小細胞肺癌轉移之研究

Heparin Cofactor II Promotes Metastasis in Non-Small-Cell Lung Cancer

廖唯昱(Wei-Yu Liao)

指導教授 ： 楊泮池 ； 余忠仁

國立臺灣大學/醫學院/臨床醫學研究所/博士(2014年)

https://doi.org/10.6342/NTU.2014.10233

摘要

肺癌在台灣以及世界各地是癌症死亡最常見的原因之一，主要分小細胞與非小細胞肺癌，其中大約85％的肺癌為非小細胞肺癌。初次診斷時大約只有15-20%的肺癌病患有機會接受手術治療，而有機會接受手術治療的病患也只有一半可以長期存活，其餘病患往往死於癌症轉移。如何早期診斷肺癌且給予有效的治療，並找出轉移率高的危險群施以有效的輔助性療法，是減少肺癌的死亡率的關鍵。我們利用CGAP（Cancer Genome Anatomy Project）所提供的SAGE（Series Analysis of Gene Expression）Genie工具，找到Heparin cofactor II（肝素第二輔助因子，HCII，又稱為SERPIN D1，serine protease inhibitor，clade D，member 1）在肺癌大量表現且具有分泌功能。我們進一步分析一系列人類非小細胞肺癌細胞株中HCII的表現，發現HCII在侵入性及轉移能力較高的細胞株中表現量較高。我們以75位臨床上可接受手術的非小細胞肺癌患者之腫瘤組織進行免疫組織染色以分析HCII的表現量，結果顯示HCII表現量高的患者，無疾病存活期顯著較短（P=0.011）且整體存活期縮短 （P=0.005）。多變項Cox比例危險迴歸分析顯示，HCII 表現量高、男性及病理分期第 III 期和第 IV 期，與非小細胞肺癌患者的整體存活期降低有關聯性。另外我們檢測57位非小細胞肺癌患者的HCII血漿濃度，發現治療前血漿HCII濃度高的患者，整體存活期顯著縮短（P =0.039）。進一步研究發現，外源性HCII可促進非小細胞肺癌細胞的絲狀偽足動態變化，此外利用過量表現或壓抑HCII表現的實驗證實，HCII會增加癌細胞移動力及侵入性，並在體外及體內促進癌症轉移。而且HCII對於肺癌細胞促進移動作用，與凝血酶的抑制沒有直接相關性。接下來的實驗證實HCII是透過活化Cdc42及Rac1來促進肺癌細胞的絲狀偽足動態變化與移動能力。而HCII對肺癌細胞產生的移動能力提升作用，是透過PI3K而活化下游的Cdc42及Rac1。肝素可阻斷上述HCII作用所造成的癌細胞移動與轉移，且經由HCII肝素結合位置的突變（K185M）證實HCII是透過其肝素結合位置與非小細胞肺癌細胞交互作用。我們的研究證實HCII為非小細胞肺癌的轉移促進因子之一。PI3K、Rac1 及 Cdc42 為HCII誘發肺癌細胞絲狀偽足形成和移動的下游作用因子。HCII的表現，與癌症復發增加及治療結果不佳有關聯性。HCII的表現可能做為預後因子，藉以鑑別非小細胞肺癌手術切除後具有高復發風險而需要立即接受輔助治療的患者。腫瘤組織或血漿檢體中的HCII 濃度，將有可能做為肝素治療非小細胞肺癌時的預測因子。

關鍵字

肝素第二輔助因子 ； 非小細胞肺癌 ； 細胞移動能力 ； 癌症轉移 ； 絲狀偽足動態

並列摘要

Lung cancer is the leading cause of cancer deaths worldwide. Non-small-cell lung cancer (NSCLC) accounts for 85% of all cases of lung cancer. Although there has been progress in the diagnosis and treatment of lung cancer, NSCLC caries a 5-year survival rate of only 17%. Despite the fact that early stage disease may be cured with surgery, only 15%-20% of lung cancer patients were operable at presentation, and about half of them had long-term survival. The remaining almost always died of metastatic disease. Identifying early stage NSCLC with metastatic potential and providing individualized treatment strategies will have a higher probability of truly curing the disease. By using Serial Analysis of Gene Expression (SAGE) database from Cancer Genome Anatomy Project (CGAP), we identified heprain cofactor II (HCII), also named, serine protease inhibitor D1 (SERPIN D1), which is overexpressed in NSCLC. Here, we investigate the clinical significance of HCII and provide molecular evidence to support that HCII could enhance cancer metastasis in NSCLC. HCII expression was higher in NSCLC cell lines with high metastatic ability. We found that high HCII expression in tumor tissue is associated with increased cancer recurrence (P = 0.011) and shorter overall survival times (P=0.005) in 75 clinically operable NSCLC patients. Multivariate Cox proportional hazards regression analysis showed that high HCII expression in tumor tissue, male gender and pathological stage III&IV were associated with reduced overall survival of patients with NSCLC. High pre-treatment plasma concentration of HCII is associated with reduced overall survival in 57 consecutive NSCLC patients. We overexpressed and knockdown HCII expressions in lung cancer cell lines and confirmed that HCII can promote cell motility, invasion ability and filopodium dynamics in NSCLC cells in vitro and increased metastatic colonization in an in vivo mouse model. Exogenous treatment of HCII promoted cancer cell migration, and this promigratory effect of HCII was independent of thrombin. We further showed that HCII could upregulate cancer cell migration through activation of PI3K, which acts upstream of Rac1 and Cdc42, and this effect could be blocked by heparin. We also demonstrated that the promigratory and invasion ability of HCII on lung cancer cells were abolished by mutagenesis at heparin binding site (K185M) of HCII. We suggest that HCII is a novel metastasis enhancer and may be used as a predictor for heparin treatment in NSCLC. We propose that HCII expression levels in tumor tissues or plasma samples could be regarded as a predictive factor for heparin treatment in NSCLC patients.

並列關鍵字

Heparin cofactor II (HCII) ； Non-small-cell lung cancer ； Cell motility ； Metastasis ； Filopodium dynamics

參考文獻

Afonso, P.V., Parent, C.A., 2011. PI3K and chemotaxis: a priming issue? Science Signaling 4, pe22.

Aihara, K., 2010. Heparin cofactor II attenuates vascular remodeling in humans and mice. Circulation Journal : official journal of the Japanese Circulation Society 74, 1518-1523.

Aihara, K., Azuma, H., Takamori, N., Kanagawa, Y., Akaike, M., Fujimura, M., Yoshida, T., Hashizume, S., Kato, M., Yamaguchi, H., Kato, S., Ikeda, Y., Arase, T., Kondo, A., Matsumoto, T., 2004. Heparin cofactor II is a novel protective factor against carotid atherosclerosis in elderly individuals. Circulation 109, 2761-2765.

Baglin, T.P., Carrell, R.W., Church, F.C., Esmon, C.T., Huntington, J.A., 2002. Crystal structures of native and thrombin-complexed heparin cofactor II reveal a multistep allosteric mechanism. Proceedings of the National Academy of Sciences of the United States of America 99, 11079-11084.

Bepler, G., Olaussen, K.A., Vataire, A.L., Soria, J.C., Zheng, Z., Dunant, A., Pignon, J.P., Schell, M.J., Fouret, P., Pirker, R., Filipits, M., Brambilla, E., 2011. ERCC1 and RRM1 in the international adjuvant lung trial by automated quantitative in situ analysis. The American Journal of Pathology 178, 69-78.