DNA雙股斷裂修補變異參與台灣肺癌形成之機制探討

Abstract

自1982年起，癌症即為台灣地區十大死亡原因之首位，而肺癌在台灣地區分別佔女性及男性癌症死亡率的首位及第二位。儘管目前醫學已相當進步，但對於肺癌分子致癌機制仍未完全釐清。目前所知，癌症形成的原因，部分是由於基因變異造成，如致癌基因 (Oncogene)活性過度增加、腫瘤抑制基因 (Tumor suppressor gene)失去活性或DNA修補基因變異所導致。由於DNA雙股斷裂 (Double-strand breaks, DSBs)對於基因體穩定性是很嚴重的傷害，因為它會導致染色體的不正常情形，如基因異質性喪失 (Loss of heterozygosity, LOH)；因此，我們想要了解這些DNA雙股斷裂修補基因(DSBR genes)若發生變異，是否參與台灣地區非小細胞肺癌 (non-small cell lung cancer, NSCLC)病人肺癌形成。在本研究中，檢查了三個DSBs修補基因BRCA1、BRCA2和XRCC5之DNA、RNA和蛋白質層面的變異情形；BRCA1與BRCA2參與homologous recombination (HR)，而XRCC5參與non-homologous end-joining (NHEJ)。研究結果發現在BRCA1基因部分：87位NSCLC病人發生LOH頻率為26.3% (20/76)，啟動子高度甲基化頻率為31% (27/87)，而其蛋白質和RNA變異頻率分別為28.7% (25/87)和29.9% (26/87)。在BRCA2部分：44.9% (31/69)病人發生LOH，啟動子高度甲基化頻率為39.1% (34/87)，而36.8% (32/87)的病人其RNA有顯著下降的情形，並且其變異與癌症型式為肺腺癌 (lung adenocarcinoma) (P=0.017)與年齡較大 (P=0.035)的病人有關。在XRCC5部分：37.3% (25/67)病人發生LOH，啟動子高度甲基化頻率為21.8% (19/87)，18.4% (16/87)蛋白質表現降低，27.6% (24/87)病人RNA表現下降，且其變異與癌症型式為肺上皮細胞癌 (squamous carcinoma, SQ) (P=0.031)和有吸菸 (P=0.043)的病患較有關。綜觀以上，研究中的BRCA1、BRCA2和XRCC5，任一蛋白質層面 (除BRCA2以mRNA計)發生變異的有54人，佔總研究人數87人的62.1%，並且發現在87位病人中，此三基因同時發生變異的只有4位，暗示HR與NHEJ在肺癌形成的的過程中，這二個修補路徑均具有相當的重要性。另外，我們還檢查了DNA傷害反應誘發p53蛋白表現情形，發現87位NSCLC病人中，52.8% (46/87)病人的p53有不正常累積的現象，同時發現這些病人多為SQ型式 (P=0.006)，並多屬於肺癌晚期 (P=0.044)的病患。 本研究為首篇對DNA雙股斷裂修補基因：BRCA1、BRCA2和XRCC5與非小細胞肺癌形成，做一完整的DNA、RNA和蛋白質之研究，而研究結果亦顯示：DNA雙股斷裂修補基因變異，在非小細胞肺癌的形成中扮演重要角色。

BACKGROUND: Since 1982, lung cancer is the leading and second cause of cancer deaths among women and men in Taiwan, respectively. Although the medical science makes rapid progress, the molecular mechanisms involved in lung tumorigenesis in Taiwan remain poorly defined. Alterations of oncogenes, tumor suppressor genes or DNA repair genes have been shown to involve in the multi-steps carcinogenesis of human cancer. Double-strand breaks in DNA are serious threats to genome integrity because they can result in chromosomal aberrations, such as loss of heterozygosity (LOH). AIM: The purpose of this study is to identify the molecular mechanism of alterations of the DNA double-strand break repair (DSBR) genes, BRCA1, BRCA2, and XRCC5, involved in non-small cell lung cancer (NSCLC) tumorigenesis in Taiwan. RESULTS: We found that the frequency of BRCA1 LOH and promoter hypermethylation was 26.3% (20/76) and 31% (27/87), respectively. In addition, 28.7% (25/87) and 29.9% (26/87) NSCLC patients had decreased or loss of BRCA1 protein and mRNA expression, respectively. With regard to BRCA2 gene alteration analyses, we found that the frequency of BRCA2 LOH and promoter hypermethylation was 44.9% (31/69) and 39.1% (34/87), respectively. Note that 36.8% (32/87) NSCLC patients had decreased or loss of BRCA2 mRNA expression. The abnormal BRCA2 expression was found more frequently in adenocarcinoma (P=0.017) and old patients (P=0.035). In addition, we found that the frequency of XRCC5 LOH and promoter hypermethylation was 37.3% (25/67) and 21.8% (19/87), respectively. There were 18.4% (16/87) and 27.6% (24/87) of NSCLC patients had decreased or loss of XRCC5 protein and mRNA expression, respectively. The abnormal XRCC5 expression was found more frequently in squamous carcinoma (SQ, P=0.031) and smoking (P=0.043) patients. Among the 87 NSCLC patients analyzed, alterations in at least one of the DSBR genes were 62.1% (54/87). In addition, we analyzed the protein expression of the damage response gene, p53. It was found that 52.8% (46/87) NSCLC patients with p53 overexpression, and most of them were SQ (P=0.006) and late stage (P=0.044) patients. CONCLUSION: The study was the first report which comprehensively examines the alteration of the DSBR genes, BRCA1, BRCA2, and XRCC5 at DNA, RNA and protein levels in lung tumorigenesis. Our data indicate that the alterations in DSBR involve in NSCLC tumorigenesis in Taiwan.