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

Genome-wide Allelotyping of Non-small Cell Lung Cancer:Novel Region of Allelic Loss,Refined Mapping of Novel Tumor Suppressor Genes and Their Alteration Analyses

曾若嘉(Ruo-Chia Tseng)

指導教授 ： 王憶卿 博士

國立臺灣師範大學/理學院/生命科學研究所/博士(2004年)

摘要

無資料

關鍵字

非小細胞肺癌 ； 異質性缺失 ； AXIN2基因 ； BTRCP基因 ； ICAT基因 ； 新基因

並列摘要

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 on chromosome 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.

並列關鍵字

NSCLC ； LOH ； AXIN2 gene ； BTRCP gene ； ICAT gene ； Novel gene

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