鼻咽癌患者Caveolin-1的表現與臨床特性之研究

Abstract

研究背景: 鼻咽癌原發於鼻咽部上皮細胞，是一種在白種人非常罕見的惡性腫瘤，然而在中國東南沿海省分、香港以及台灣卻是一個常見的疾病，顯示此疾病不同於其它頭頸部癌症，具有種族性、地域性與特殊致病因子。鼻咽癌發生之機轉乃多重因素所構成，目前認為和遺傳因子、環境因素以及Epstein-Barr病毒感染等三大項有關。潛伏期膜蛋白-1(LMP-1)與潛伏期膜蛋白2A(LMP2A)為EB病毒潛伏期表現的產物，在鼻咽癌檢體中65%-95%可測得其轉錄產物的表現，LMP-1是EB病毒使細胞轉型(transformation)的主要蛋白，而LMP2A蛋白可透過活化PI3-kinase-Akt路徑使上皮細胞株細胞轉型增生並抑制細胞分化。根據研究不論是LMP-1或LMP2A都和細胞膜上的脂筏(lipid rafts)相關，Caveolae則是具有調控多種細胞訊息傳遞的一種脂筏，主要的組成蛋白為Caveolin-1，最近有研究指出Caveolin-1可以調控PI3K/Akt路徑進而增強細胞的存活。目前已知Caveolin-1在膀胱癌、食道癌、甲狀腺乳突癌、攝護腺癌及T細胞血癌有一致性升高的趨勢，而在卵巢癌、肺癌、乳癌則為降低的現象，至於鼻咽癌則文獻尚未有報告。 研究目的: 本論文嘗試研究台灣地區鼻咽癌組織中Caveolin-1 mRNA的表現量與對照組之比較，其次想探討Caveolin-1的表現量高低和病患臨床表現如年齡、性別、病理分類、血中膽固醇、腫瘤分期、遠處轉移、腫瘤復發及存活率之相關性。最後利用In vitro鼻咽癌細胞株分析Caveolin-1的表現量是否會影響Akt及磷酸化Akt蛋白質的表現量。 研究方法: 本研究採用回溯性(retrospective)方式進行分析，收集台大及馬偕醫院鼻咽癌檢體(1990年到2002年間)，及非鼻咽癌鼻咽組織為對照檢體。萃取mRNA，利用同步定量聚合酶連鎖反應分析Caveoin-1 mRNA在不同檢體的表現量。再依上述資料統計分析Caveoin-1在鼻咽癌的表現及其對鼻咽癌患者年齡、性別、病理分類、臨床分期、腫瘤轉移、預後的影響。進而分析鼻咽癌檢體中Caveoin-1和Akt的表現量之相關性。最後以shRNA-Cav1 降低 (knock down)鼻咽癌TW01細胞株中的Caveolin-1 並探討其影響Akt及磷酸化Akt 蛋白質表現量的能力。

結果: 初步研究結果顯示，鼻咽癌患者Caveolin-1的表現量比對照組高，且具有統計上的差異(p=0.0002)。進一步分析在鼻咽癌74名患者中，臨床上晚期(AJCC stage III,IV)患者比早期(AJCC stage I,II)患者有較高的Caveolin-1的相對表現量(p=0.0002)。使用回歸分析發現只有臨床分期、原發腫瘤侵犯的範圍(T)、淋巴節轉移情形(N)等因子有顯著統計差異(臨床分期P<0.001，腫瘤侵犯的範圍P=0.017，淋巴節轉移情形 P=0.015)，而年齡、性別、病理WHO分纇、遠端轉移、顱內侵犯均無顯著差異。存活分析則用Kaplan-Meier method，以Caveolin-1的中位數為準，將其分為高、低兩組，Caveoin-1表現較高者存活率較差(Log-rank test，P=0.03)。而在體外試驗中顯示鼻咽癌TW01細胞株中，過度表現Caveolin-1可以增加磷酸化Akt蛋白量﹔而以shRNA降低Cav-1的量會減少磷酸化Akt蛋白以及Akt蛋白的表現。 結論: 鼻咽癌患者Caveolin-1的表現量比對照組高，其表現量和臨床分期、原發腫瘤侵犯的範圍(T)、淋巴節轉移情形(N)三個因子有顯著統計差異且Caveoin-1表現較高者鼻咽癌存活率較差。而本實驗亦證明在鼻咽癌TW01細胞株中，Caveolin-1可以調控Akt的表現。

Nasopharyngeal carcinoma (NPC) is a squamous-cell carcinoma occurring in the epithelial lining of the nasopharynx. The incidence is high in Southeast China, Hong Kong and Taiwan, however it is rare in western countries indicating a remarkably distinctive ethnic and geographic distribution that differing from other head and neck cancers. The tumorgenesis of NPC is a multi-factorial process, including genetic, environmental and Epstein-Barr virus. Latent membrane protein 1(LMP-1) and Latent membrane protein 2A (LMP2A) are EBV-encoded proteins expressed during latent phase, and transcripts are detected in 65% and 95% of the tumors respectively. It has been demonstrated EBV-encoded LMP-1 can cause cellular transformation, and LMP2A transforms epithelial cells, inhibits cell differentiation through activating PI3-kinase-Akt pathway. Interesting, both LMP-1 and LMP2A is targeted to lipid rafts/caveolae. Caveolin-1, an essential protein constituent of caveolae can modulate Akt signaling pathway and enhances cell survival. Caveolin-1 was over-expressed on bladder cancer, prostate cancer, esophageal cancer and thyroid papillary carcinoma. The aim of this study is to investigate the expression of caveolin-1 on NPC and its impacts on clinical manifestations of NPC patients. The effect of caveolin-1 on Akt signaling in NPC-TW01 cells will be elucidated in this study. The expression of caveolin-1 was investigated in 74 NPC specimens, 29 control tissues and some cell lines. Our data revealed the caveolin-1 is over-expressed at transcription level in NPC patients (P=0.0002). Late-stage NPC (stage III, IV) patients have higher level of caveolin-1 expression than early-stage (stage I,II) ones (P=0.0002). After multiple regression analysis, three factors including tumor staging, tumor size (T) and lymph node metastasis (N) are included in the model with significant difference. The overall survival rate is poor in patients with high level of caveolin-1 expression (Log-rank test, P=0.03). Over-expressed caveolin-1 in TW01 cells increased more than 50% phosphorylated Akt protein intensity. Knockdown of caveolin-1 by four shRNAs substantially reduced Akt protein levels and almost abolished phosphorylated Akt protein compared with that in untreated TW01 and empty vector control cells. It suggested caveolin-1 may modulate Akt signaling in NPC TW01 cell lines.