鎳暴露與台灣肺癌之p53與EGFR基因突變的相關性研究

過去流行病學研究發現職業暴露鎳化合物會導致肺癌之發生。本研究室之病例-對照組研究結果發現，肺癌患者相較於非癌症患者之肺組織中有較高之鎳含量，因此鎳暴露可能參與台灣肺癌之發生。過去研究指出鎳化合物會抑制DNA修補能力，促進化學致癌物BPDE引起之基因突變。本研究假設肺組織中之鎳累積可能增加p53和EGFR發生突變之風險，進而促進肺癌之發生。本研究篩選1993 ~ 2003年之210位肺癌患者之周邊正常肺組織，利用DNA自動定序分析儀分析p53和EGFR基因之突變。其中187名肺癌患者，以石墨式原子吸收光譜儀分析肺組織中之鎳含量，以了解肺癌患者p53突變與肺組織中鎳含量是否具有相關性? 另外選取76名不抽菸肺癌患者，以感應偶和電漿質譜分析儀分析肺組織中之鎳含量，以了解EGFR突變與肺組織中鎳含量是否具有相關? 肺癌患者肺組織中之鎳含量以中位數，區分患者為高鎳與低鎳患者兩組。結果顯示高鎳患者較低鎳患者，具有較高發生p53或EGFR突變之風險，尤其女性不抽菸肺癌患者。為了解鎳抑制DNA修補能力，是否增加這兩種基因突變之頻率?本研究以氯化鎳處理A549和H1975肺癌細胞，利用host cell reactivation assay分析發現氯化鎳能抑制細胞之DNA修補能力，而增加H2O2誘發中8-oxo-deoxyguanosine (8-oxo-dG) 之含量，因此鎳化合物會經由抑制DNA修補能力，促進p53和EGFR基因發生突變之頻率。另一方面，同樣以肺癌細胞處理不同濃度之氯化鎳，結果發現氯化鎳經由EGFR/NF-κB訊息傳遞路徑，促進miR-21之表現，抑制SPRY2和RECK表現，進而促進細胞之侵襲能力，尤其是EGFR突變之肺癌細胞。在肺癌患者之鎳含量與miR-21表現量之間呈正相關性。Kaplan-Meier統計分析發現，高鎳肺癌患者較低鎳肺癌患者有較差之overall survival (OS)和relapse free survival (RFS)。具有高鎳同時又有高miR-21之肺癌患者較具有低鎳又有低miR-21之肺癌患者，有較差之OS和RFS。總之本研究結果支持過去流行病學之研究結果，指出鎳暴露不但會增加肺癌發生之風險，同時會促進肺腫瘤之侵襲能力，因此即使非職業暴露，環境中鎳化合物累積於一般人肺組織中，同樣會促進肺癌之發生與腫瘤惡化。

Occupational exposure to nickel compounds has been associated with lung cancer. The correlation of a high nickel levels with an increased risk of lung cancer has been reported in our previous case-control study. Thus, nickel exposure might participate in lung cancer development in Taiwan. Nickel-induced carcinogenesis has been reported to be mediated through increased benzo[a]pyrene 7,8-diol-epoxide (BPDE)-induced mutagenesis due to reduced DNA repair activity. We next examined whether nickel exposure could increase the occurrences of p53 and EGFR mutations. Lung tumors and adjacent normal lung tissues were surgically resected from 210 non-small cell lung cancer (NSCLC) patients. One hundred eighteen nine out of 210 lung tumors and adjacent normal lung tissues were enrolled to determine the nickel levels in adjacent normal lung tissues and p53 mutations in lung tumors using Graphite furnace atomic absorption spectrometry (AAS) and direct sequencing, respectively. Seventy six non-smoking patients among 210 patients were collected to evaluate the association between nickel level and EGFR mutations using inductively coupled plasma mass spectrometry (ICP-MS) and direct sequencing. The mean value of nickel levels in the study population was used as a cutoff point to divide patients into high- and low-nickel subgroup. The prevalence of p53 and EGFR mutations was significantly higher in the high-nickel subgroup than in the low-nickel subgroup, especially in female non-smokers. To determine whether nickel affected DNA repair capacity, we conducted the host cell reactivation assay in A549 and H1975 lung cancer cells and showed that the DNA repair activity was dose-dependently reduced by nickel chloride. This was associated with the elevated production of hydrogen peroxide-induced 8-oxo-deoxyguanosine (8-oxo-dG). These results suggest that nickel may increase p53 mutation occurrence risk via inhibiting DNA repair activity. SPRY2 and RECK expressions were decreased by nickel-induced miR-21 via activation of the EGFR/NF-κB signaling pathway, which promoted invasiveness in H1355 and H23 lung cancer cells, and particularly in the cells with EGFR L858R expression vector transfection. The patients’ nickel levels were associated with miR-21 expression levels. Kaplan-Meier analysis revealed poorer overall survival (OS) and shorter relapse free survival (RFS) in the high-nickel subgroup than in low-nickel subgroup. The high-nickel/high-miR-21 subgroup exhibited shorter OS and RFS periods when compared to the low-nickel/low-miR-21 subgroup. Our findings support previous epidemiological studies indicating that nickel exposure may not only contribute to cancer incidence but also promote tumor invasion in lung cancer. We therefore suggest that environmental nickel exposure in general population may contribute to tumor initiation and invasion in NSCLC.