2026-11-092026-11-09https://scholars.csmu.edu.tw/handle/123456789/38728Lung cancer is the leading cause of cancer death worldwide including Taiwan. Cigarette smoking is the major cause of lung cancer which is obtained from the observation, showing that more than 85% of lung cancer in western countries is caused by cigarette smoking. Reduced cigarette smoking is thus to be the best strategy to decreased suffering lung cancer rate in these countries. However, about 50% of lung cancer in Taiwan are life-time never nonsmokers. Therefore, it is conceivable that environmental factors other than cigarette smoking might play a critical role in lung carcinogenesis in Taiwanese nonsmokers. Lung cancer is a very progressive disease and there is no reliable clinical strategy for early diognosis. Five-year survival rate of patients is still less than 15% during past two decades. Thus, the etiological study of lung cancer is an important strategy for the prevention of this disease. Our recent report showed that the levels of nickel (Ni) and chromium (Cr) in lung tissues from lung cancer patients were significantly higher than those of non-cancer controls. Both metals levels in lung tissues were not associated with cigarette consumptions. Our preliminary data surprisingly showed that Ni or Cr levels in lung cancer patient harborted with p53 mutation were higher than those of p53 wildtype (Table 3). The difference in both heavy metals was observed in nonsmokers, not in smokers. Ni and Cr have been classified by IARC to be human lung carcinogens, but the evidence was only obtained from the studies of occupational exposure population, animal and cell models, no data from non-occupational exposure population to support the association between lung cancer and both metals exposure. Therefore, in this project, we will explore whether Ni or Cr accumulated in lung tissues could be an etiological factor to contribute to lung cancer development in Taiwanese nonsmokers. The first year study will be to determine whether both heavy metals in lung tissues could be act as an etiological factor of lung cancer among nonsmokers. (1) Two hundrends lung tissues from nonsmoking lung cancer patients will be enrolled to determine Cr and Ni levels by atomic absorption spectrometry or ICP, to further confirm our preliminary data showing that both metals exposure is associated with p53 mutation occurrence in lung cancer. (2) Real-time RT-PCR will be performed to evaluate XPC, ERCC1, ERCC5, MGMT, BRCA1, BRCA2, hOGG1, hMLH1, and hMLH2 mRNA level in lung tumors from these patients to understand which increased DNA repair gene expression is related with heavey metal exposure, (3) to verify which DNA repair gene expression is associated with p53 mutation. In the second year study, we will explore whether the decrease of DNA repair gene mRNA expression is medicated through the promoter methylation machinary. (1) Promoter methylation of these genes in lung tumors will be evaluated by methylation specific-PCR (MSP). The association between promoter methylation and mRNA expression levels of these DNA repair genes will be statistically analyzed to verify whether promoter methylation could be responsible for the decreased mRNA levels of these genes. (2) To further elucidate the possible role of Ni or Cr exposure in the promoter methylation of these genes, lung cancer cells which are established from Taiwanese lung cancer patients and habored with relatively higher expression of DNA repair gene, will be exposed with low doses of Ni or Cr for a long time to verify whether promoter hypermethylation or other mechanisms could be involved in decreased DNA repair gene transcription. In the third year study, we will investigate (1) whether loss of heterozygosity (LOH) could be linked with the reduction of these DNA repair gene expressions, LOH analysis will be performed to verify the possibility in lung tumors which have Ni or Cr levels. These results will be helpful to verify which mechanism (promoter methylation or LOH) is responsible for modulated DNA repair gene expression. (2) to verify whether LOH or promoter methylation of BRCA1, BRCA2, hMLH1, and hMSH2 in both metals high-exposed patients will be more frequently occurred than those with a low-exposed patients, because reduced the four DNA repair gene expressions by promoter methylation and/or LOH have been shown to associate with lung carcinogenesis mediated through chromosomal instability. (3) To verify whether Ni or Cr accumulation could accelete allelic imbalance of some tumor suppressor genes or oncogenes in nonsmoking lung tumors, which have been charatherized by a genome-wide study in another project funded by National Genomic Medicine Program. Collectively, the results obtained from the project will be helpful to verify whether Ni or Cr exposed from general environmental pollution could be as an aetiological factor of lung cancer in Taiwanese nonsmokers. In addition, we hope to verify whether altered DNA repair gene expression by promoter methylation and/or LOH and the allelic imbalance of certain genes could play an important role in lung tumorigenesis in Taiwanese nonsmokers who exposed relatively higher levels of Ni or Cr.二十多年，肺癌來一直是臺灣地區民眾的首要癌症死因。已知抽菸是肺 癌的主要致病因子，在歐美國家之肺癌有 85% 以上患者都是抽煙所致，但是 台灣卻有一半的肺癌患者是不抽煙者，無法以抽菸來解釋其致病原因。已知 肺癌患者大多在晚期才被診斷到，因此患者之預後通常都比較差，直至今日， 各國肺癌患者之五年存活率，都是低於 15%。這些結果顯示找出主要致病因 子，才是當前減少罹患肺癌之重要策略。尤其在台灣，肺癌之病因可能需要 由環境因子中尋找，來減少國人罹患肺癌之發生。十五年來，本研究室一直 在探討不抽菸台灣女性罹患肺癌之致病因子，主要發現 HPV16/18 感染與女 性肺癌發生有關。最近本研究室更驚訝的發現，台灣肺癌患者之肺組織中， 鉻與鎳之含量顯著高於非癌症者，並發現這兩種重金屬含量與患者抽菸無關 (Kao et al., 2006)。最近發現 p53 突變之肺癌患者鉻與鎳含量較沒有發生 p53 突變者為高，且主要是在不抽菸之患者觀察到此現象 (Table 3)。IARC 已確 認鉻與鎳是人類肺癌可能致癌物，但是證據僅止於職業暴露、動物與細胞研 究，在非職業暴露與肺癌發生之相關研究，至今尚無報告。因此本計畫擬探 討這兩種重金屬暴露，是否可能參與台灣不抽菸肺癌之形成？ 第一年計畫將增加重金屬分析之樣本至 200 位不抽菸肺癌患者，(1) 以 瞭解肺癌患者肺組織中鉻與鎳之含量，是否確實與 p53 突變具有正相關性？ 為了解重金屬污染是否是抑制DNA repair 基因表現而增加 p53 發生突變頻 率？本計畫以 real-time RT-PCR 分析(2) 肺腫瘤組織中之 XPC, ERCC1, ERCC5, MGMT, BRCA1, BRCA2, hOGG1, hMLH1, hMSH2 等 DNA repair 基因 mRNA 表現，並分析重金屬含量與DNA repair 基因表現之相關性。為 了解 DNA repair 基因表現降低之可能機轉， 將進一步以 MSP 分析這些基 因啟動子之甲基化，以瞭解(3) 這些基因的啟動子甲基化與 mRNA 表現之 相關性。最後(4) 瞭解何種 DNA 修補基因的表現與 p53 發生突變有關？ 第二年計畫將以細胞模式探討低劑量之鉻或鎳處理 DNA repair 基因相 對表現較高之肺癌細胞，主要以本研究室自行培養出的台灣肺癌細胞株進行 本研究，(1) 觀察這兩種重金屬是否會降低 DNA repair 基因的表現，尤其要 確定主要抑制何種 DNA repair 基因的表現？並探討(2) 重金屬抑制肺癌細 胞之DNA修補基因表現，是否是經由引起該基因啟動子甲基化所致？(3) 與過 去已知之啟動子甲基化之機轉是否不同？而有其他機轉參與其中？ 第三年計畫為釐清 DNA 修補基因表現降低是經由何種機制所致，將在 肺腫瘤組織上分析(1) 八種 DNA repair 基因所在位置之 LOH，以瞭解這些 基因表現降低，是否可能經由該基因位置發生 LOH？(2) 瞭解重金屬污染較 高之肺腫瘤中，BRCA1, BRCA2, hMLH1 和 hMSH2 基因發生啟動子甲基化 或 LOH 之頻率，是否高於重金屬污染較低的腫瘤？這結果可用來推測重金 屬污染，是否會經由抑制以上四種基因的表現，而造成chromosomal instability 導致肺癌之形成？(3) 將過去計畫已完成 genome-wide analysis 的肺腫瘤資 料，來瞭解重金屬污染較高之肺腫瘤，是否有較高之已知參與肺腫瘤化之基 因發生 allelic imbalance？或是有特異的基因發生 allelic imbalance? 以及這 些 allelic imbalance 是否與 BRCA1, BRCA2, hMLH1 和 hMSH2 基因發生 啟動子甲基化或 LOH 有關？ 總之，本計畫之成果將有助於了解台灣有一半的不抽菸肺癌患者，是否 是經由鉻與鎳重金屬的污染所致？並提出可能之致癌機轉是否是主要經由抑 制 DNA repair 基因的表現以及 chromosomal instability 而參與台灣肺癌之 形成。Project