以微矩陣比較基因體雜合方法偵測東西方肺癌族群新穎致癌基因及其機制與臨床探討

Abstract

近年研究顯示，腫瘤形成基因於不同種族之間存在差異性。因 此，鑑別各人種族群之間共通及具有差異性的基因群，是近代研究腫 瘤形成的重要課題。為了進一步探究腫瘤形成相關基因的分子機制， 本研究收集了40 對來自於臺灣肺癌病人（由台北榮民總醫院胸腔外 科許瀚水醫師提供檢體）及20 對來自於美國白種肺癌病人（由美國 芝加哥大學附設醫院胸腔外科Dr. Ravi Salgia 醫師提供檢體）的東西 方肺癌族群樣本，對其進行微矩陣比較基因體(array-comparative genomic hybridization, array-CGH) 的圖譜分析。本研究發現，於東方肺癌群族偵測到17 段染色體變異區域，涵蓋註解基因數為476；並 於西方肺癌群族偵測到20 段染色體變異區域，涵蓋註解基因數為 459。進一步針對本研究室先前分析出的expression array 基因群進行 一致性比對，篩選214 個於肺癌族群中基因結構變異及基因表現異常 趨勢相符的候選基因，並對候選基因進行基因已知功能的資料庫搜 尋。這些候選基因包含位於6p22.1 參與MAPK 路徑的 ZNF322A 基 因、位於10q24.1 參與Rho GTPase 路徑的 ARHGAP19 基因、位於 10q24.1 參與Wnt 路徑的 FRAT2 基因、以及位於17p13.3 功能與 motility 相關的 PAFAH1B1 基因。本研究對這四個極具潛力的候選 基因進行即時定量聚合酶鏈鎖反應系統、chromogenic in situ hybridization 方法、反轉錄即時定量聚合酶鏈鎖反應系統及免疫組織 染色法，確認候選候選基因於臨床肺癌樣本及肺癌細胞株中的基因變 異情形，其結果顯示候選候選基因其基因體套數及其 mRNA 表現量 皆於肺癌組織中高於正常組織 (P<0.001~P=0.06)。本研究亦利用反轉 錄即時定量聚合酶鏈鎖反應 及免疫組織染色法對101 位肺癌族群檢 測 PAFAH1B1 基因其 mRNA 及蛋白層次變異情形。結果發現 PAFAH1B1 基因於 mRNA 層次的過度表現頻率為62.4%，於蛋白層 次過度表現頻率為57.4%，且此基因於mRNA 及蛋白層次的過度表 現皆與病人的晚期具有相關性 (mRNA：P=0.008，蛋白層：P=0.008)， 且屬於腺細胞癌 (P=0.020) 及男性 (P=0.049) 的病人於蛋白層次的 過度表現具有較差的預後，顯示PAFAH1B1 基因於肺癌族群具有過 度表達的變異；細胞及動物實驗顯示過度表達PAFAH1B1 且可能促 進肺癌細胞轉移能力。本研究提供了第一個東西方肺癌族群新穎基因 變異資料庫，並以細胞、動物及臨床模式探討基因變異之致癌機轉。

Cancer-related genes show racial differences. Therefore, identification and characterization of DNA copy number alteration regions in different racial groups help to dissect the mechanism of tumorigenesis. Here, DNA copy number alteration profile was analyzed by array-comparative genomic hybridization (array-CGH) for 40 Asian and 20 Caucasian lung cancer patients. We identified 20 chromosomal imbalance regions harboring 459 genes for Caucasian and 17 regions containing 476 genes for Asian lung cancer patients. Among the genes identified, the ion transport and chromatin remodeling are the two main common biological processes altered in both Asia and Caucasian populations. Interestingly, genes involved in Wnt receptor signaling pathway were unique in Asian lung cancer, whereas Caucasian lung cancer was addictive to cell surface receptor linked signal transduction and G-protein coupled receptor protein signaling pathways. Genes residing within the chromosomal imbalance regions were integrated with gene expression databases to identify 214 potential cancer-related genes. Four racial-specific candidate oncogenes were validated in 164 patients using quantitative polymerase chain reaction (q-PCR), chromogenic in situ hybridization (CISH), reverse transcriptase-q-PCR, and immunohistochemistry. These four genes were ARHGAP19 (10q24.1) functioning in Rho activity control, FRAT2 (10q24.1) involved in Wnt signaling, PAFAH1B1 (17p13.3) functioning in motility control, and ZNF322A (6p22.1) involved in MAPK signaling. Mean gene dosage and mRNA expression level of the four candidate genes in tumor tissues were significantly higher than the corresponding normal tissues (P<0.001~P=0.06). PAFAH1B1 which showed amplification and overexpression in both Asian and Caucasian lung cancer patients was further characterized in cell, animal, and clinical models. The PAFAH1B1 mRNA and protein overexpression frequency were 62.4% (63/101) and 57.4% (58/101) in 101 lung cancer patients. The results indicated that mRNA and protein overexpression level of PAFAH1B1 was significantly associated with late stage (mRNA: P=0.008, protein: P=0.008) and poor survival in lung adenocarcinoma (P=0.020) and male patients (P=0.049). Collectively, our study provides the first database revealing common and differential chromosomal imbalance regions among lung cancer from Asians and Caucasians. Four validation methods in a large patient cohort confirm our database. The cell and animal studies verify a novel metastasis-promoting oncogene, PAFAH1B1.