非小細胞肺癌之基因組缺失圖譜：新基因組缺失及新腫瘤抑制基因之精確定位和其變異分析

Abstract

中文摘要 肺癌為國人癌症死亡的首位，近年來每年約有6000人死於肺癌，但其分子致癌機制至今仍未釐清。由於癌症的形成過程中主要變異的基因為致癌基因 (Oncogenes) 活性過增或抑癌基因 (Tumor suppress genes, TSGs) 失去活性；抑癌基因經研究證明需二個基因座皆變異才導致其失去活性而致癌，其變異的方式多為一基因座產生點突變、小片段鹼基缺失、或啟動子過度甲基化 (promoter hypermethylation)，而另一基因座產生抑癌基因及鄰近區域DNA大片段的缺失，因此異質性缺失 (loss of heterozygosity, LOH) 經常可作為抑癌基因區位的指標。為了偵測肺癌組織之異質性缺失，並瞭解一些特定抑癌基因參與台灣肺癌形成之機制，本研究設計了以下三個主要目標：（1）以微切片（microdissection）取得高純度癌細胞與其配對之正常細胞進行基因體異質性缺失分析，以建立一台灣肺癌基因體異質性缺失圖譜；（2）針對缺失的高頻率區域如染色體1p36.2、10q24.3及 17q24.3所包含的三個抑癌基因ICAT、BTRCP及 AXIN2在肺癌病人中進行DNA、RNA、蛋白質變異分析；（3）針對基因體缺失區域17q24.3設計高密度之微衛星序列，以精確定出缺失區位，再進一步以肺癌細胞株及病人組織進行該區位之異常轉錄產物及啟動子甲基化分析。 目標一：本研究結果顯示以177個微衛星序列進行71位肺癌病人的基因體缺失分析，有20個染色體區位其缺失頻率可達48％以上，其中8個區位尚未有文獻報告，僅在台灣發現；卡方分析統計的結果顯示許多區位的缺失與病人的年齡、性別、抽煙狀況、癌症種類及分期有關，例如：9個微衛星序列與抽煙的病人有關（P值小於0.05）；2個微衛星序列與肺腺癌 (adenocarcinoma) 有關（P值小於0.05）。利用Cox’s多變項分析，3個區位的缺失與病人存活率顯著相關（P值小於0.05）。上述為文獻中針對肺癌最完整的全基因異質性缺失研究，這些與台灣地區非小細胞肺癌有關的異質性缺失，可能用來作為肺癌早期偵測的指標或預後的依據，同時這些區域也可用來尋找一些因為失去功能而導致非小細胞肺癌形成的新穎抑癌基因。 目標二：上述基因體異質性缺失結果顯示，染色體1p36.2、10q24.3及 17q24.3 其基因體缺失的頻率皆高於40%，這三個基因座分別包含wingless (Wnt) 訊號傳遞中的三個重要抑癌基因ICAT、BTRCP及 AXIN2；而Wnt 訊號傳遞與細胞增生、活動及癌化生成是有關連的，並與β-catenin蛋白質降解過程中扮演重要角色。為瞭解在非小細胞肺癌中，AXIN2、BTRCP及ICAT基因是否發生變異，我們利用反轉錄-聚合及免疫組織化學染色法偵測78位非小細胞肺癌及非腫瘤配對肺組織中AXIN2、BTRCP及ICAT基因之異常。研究結果顯示，分別有35%、32%及35%的病人有蛋白質表現異常之情形，其啟動子過度甲基化 (promoter hypermethylation) 亦分別有38%、50%及42%。卡方分析發現AXIN2 mRNA低表現經常發生於腫瘤分期之早期病人，其比例為53%（P值為0.028）；另外免疫組織化學染色法與卡方分析結果顯示AXIN2、BTRCP及ICAT基因蛋白低表現與啟動子過度甲基化顯著相關 (P值<0.001) 而且與β-catenin蛋白過度累積於細胞核中有顯著相關性（AXIN2，P值為0.004；BTRCP，P值為0.013）。顯示抑癌基因ICAT、BTRCP及 AXIN2變異與非小細胞肺癌形成及β-catenin蛋白過度累積有關。本研究結果亦為第一篇針對Wnt pathway之相關抑癌基因AXIN2、BTRCP及ICAT在癌症組織樣本中的完整分子及臨床研究。 目標三：基因體異質性缺失的研究顯示，染色體17q24.3 其缺失的頻率高於50%，推測此區可能有新腫瘤抑制基因。因此我們利用精確缺失定位分析17q24.3在肺癌中之情形，並且針對位於此區之新穎基因LOC51321檢測其在15株肺癌細胞株及53個肺癌組織中各有47%及36%其mRNA低表達的情形。另外，肺癌細胞株CL1-5-F4之LOC51321啟動子甲基化情形及mRNA不表達有相關性。因此推測此新穎基因LOC51321可能參與肺癌形成，且其變異與異質性缺失及啟動子過度甲基化相關。ABSTRACT Lung cancer is the leading cause of cancer deaths in Taiwan. It has been shown that alterations of tumor suppressor genes (TSGs) involve in the multi-step carcinogenesis of human cancer including lung cancer. Both copies of TSGs have to be inactivated for their function to be lost. Therefore, to search for the genomic regions that potentially contain the TSGs and to investigate the etiological association of allelic deletion in candidate TSGs in lung cancer, this study is designed to conduct (1) genome-wide loss of heterozygosity (LOH) analysis in microdissected surgically resected lung tumors to prescreen the potential chromosomal regions containing TSGs; (2) gene/protein alteration studies on high LOH regions at 1p36.2, 10q24.3, and 17q24.3 including genes encoding the ICAT, BTRCP and AXIN2 proteins respectively that involved in the β-catenin/wingless (Wnt) regulation pathway, in tumors from 78 cancer patients; and (3) high density LOH analysis at 17q24.3 chromosomal region and refined mapping of candidate TSG, LOC51321, by analyzing aberrant transcripts and promoter hypermethylation of in lung tumor tissues and cancer cell lines. Aim 1: The genome-wide LOH analysis using 177 microsatellite markers in 71 microdissected surgical lung tumors and paired normal cells indicated that 20 markers showed an LOH frequency greater than 48%, and eight of them (2p23.3, 2p24.3, 2q35, 6p22.2, 7p14.3, 7p22.2, 17q24.3 and 21q22.3) were novel in non-small cell lung cancer (NSCLC). In addition, markers specifically associated with clinicopathological parameters such as ages, sexes, smoking habits, tumor types, and tumor stages were identified. For example, there were nine markers specifically associated with patients who smoked (P<0.05). The markers D14S1426 and D20S186 were also associated with adenocarcinoma (AD) patients (P<0.05). Furthermore, three markers, D2S2968, D6S2439, and D7S1818, were significantly associated with poor prognosis of NSCLC patients using both univariate and multivariate Cox’s regression analyses (P<0.05). These markers can be potentially used for early lung cancer detection, outcome measurement, and positional cloning of new TSGs.

Aim 2: Chromosome regions at 1p36.2, 10q24.3, and 17q24.3 showed a high frequency of LOH in tumors from NSCLC patients. These frequently deleted regions included gene loci encoding the ICAT (inhibitor of β-catenin and TCF-4), BTRCP (β transducin repeat containing protein), and AXIN2 (Axis inhibition protein 2) proteins, which were putative tumor suppressor proteins involved in regulating the Wnt signaling pathway. Therefore, we further investigated the possibility of alterations of ICAT, BTRCP, and AXIN2 including loss of protein/mRNA expression and promoter hypermethylation and allelic imbalance in 78 NSCLC patients. The gene/protein alterations with clinical associations were also examined. The β-catenin deregulation was significantly attributable to low mRNA/protein expression of AXIN2 (P = 0.004) and BTRCP (P = 0.013). A high concordance was observed between low protein/mRNA expression and promoter hypermethylation (P< 0.001) for the AXIN2, BTRCP, and ICAT genes. Our data provide compelling evidence for an inverse correlation of AXIN2, BTRCP, and ICAT expression with β-catenin expression in the NSCLC tumorigenesis and suggest that promoter hypermethylation is the predominant mechanism in AXIN2, BTRCP, and ICAT alterations. Aim 3: Based on our genome-wide LOH data, chromosome region at 17q24.3 was a novel frequent LOH region in NSCLC. The refined mapping using 9 additional markers was then performed onchromosome 17q24.3. The allelic loss pattern of 48 tumors suggests that the minimal deletion region was located between markers D17S1882 and D17S2193, spanning a distance of approximately 2.7 Mb and reaching 65% LOH at locus D17S1816. A putative gene LOC51321 was speculated to be a deletion target on 17q24.3 in NSCLC. The expression analysis indicated decreased expression of LOC51321 in 47% (7/15) of the NSCLC cell lines and 36% (19/53) of the tumor tissues. In addition, 5-Aza-deoxycytosine successfully restored mRNA expression and de-methylated the putative promoter region in CL1-5-F4 cells that lacked LOC51321 expression and that harbored a methylated respective promoter. The results showed that LOC51321 may be involved in lung tumorigenesis.