2026-11-092026-11-09https://scholars.csmu.edu.tw/handle/123456789/38605Molecular mechanisms of lung carcinogenesis is predominately focused on cigarette smoke-induced lung cancer, few information is obtained from studies of nonsmoking lung cancer until now. More than 50% of lung cancer patients in Taiwan are nonsmokers, especially for over 90% of Taiwanese women to be life-time never smokers. Our previous report indicated that B[a]P-DNA adduct levels in lung tissues from smoking lung cancer patients were not higher than those of nonsmoking patients. It appears to suggest that the DNA adducts induced by environmental carcinogen exposure may be not a lower contribution than those by cigarette smoking. We also observed that the adduct levels in nonsmoking female lung cancer patients were significantly higher than in nonsmoking male patients, suggesting that female may have more susceptible to environmental carcinogen exposure than male. We unexpectedly observed a high prevalence of HPV16/18 infection in female lung cancer patients compared to male patients. Moreover, our recent report further indicated that HPV16/18 E6 oncoprotein is expressed in lung tumors and related with p53 inactivation, strongly suggesting HPV16/18 infection may be involved in lung tumor formation, at least in Taiwanese women nonsmokers. It is conceivable that only HPV infection could not induce tumor formation, it should be combined with DNA damage induced certain gene mutations to cause lung tumor development. Chromosome instability at a numerical or structural level is a hallmark of malignant tumors. Previous studies indicated that the alteration of DNA repair genes involved in mismatch repair and DNA double strand break genes, such as hMLH1, hMSH2, XRCC1, XRCC3, XRCC5, BRCA1, and BRCA2 could contribute to chromosomal instability during human carcinogenesis. Our preliminary data showed that HPV-infected lung tumors had higher frequent of chromosomal instability at some loci than in HPV-noninfected lung tumors. We thus hypothesized that the combined effects of accumulated DNA damage and HPV infection could synergistically increase chromosomal instability occurrence to cause lung tumor formation. To explore the hypothesis, the following three-year project will be performed to provide the available evidence to support the hypothesis. The first year study: To verify whether environmental carcinogen exposure could synergistically promote chromosomal instability in HPV-infected lung cancer cells compared to HPV-noninfected lung cancer cells. (1) HPV-infected and -noninfected lung cancer cells, which have been established from pleural effusions of lung cancer patients, will be treated with various doses of B[a]P for various time intervals, and then the frequency of micronuclei will be determined by FISH analysis. The micronuclei formation will be used as a marker to predict the chromosomal instability of the cells after treated with B[a]P. We will compare the micronuclei number induced by B[a]P in HPV-infected and -noninfected lung cancer cells to understand whether HPV infection could synergistically promote the occurrence of chromosomal instability in HPV-positive cells as compared to HPV-noninfected cells. (2) The above B[a]P-treated cells with HPV-infection or without HPV infection will be used to examine whether the DNA repair genes, which were involved in mismatch repair and double strand break, could be synergistically altered by B[a]P. Real-time RT-PCR and Western blot will be performed to determine the expression of these DNA repair genes. To explore possible mechanism involved in the reduction of DNA repair gene expressions, the promoter hypermethylation of these genes will be evaluated by methylation specific PCR (MSP). These results obtained from the works could be elucidated the role of DNA repair genes involved in mismatch repair and double strand break on chromosomal instability (or micronuclei) formation. (3) To explore the role of HPV16 E6 oncoprotein in chromosomal instability, E6 will be transfected into HPV-noninfected lung cancer cells, on the other hands, E6 will be knockdown by RNAi technique in HPV-infected lung cancer cells. Both cells will be treated with B[a]P as previously described to check whether micronuclei formation could be increased or decreased in E6-positive or –negative cells when compared with their parental cells. The second-year study: To characterize whether different profiles of chromosomal instability loci were observed in lung tumors with a high adduct level/HPV-infection as compared to other three categories of lung tumors. (1) ELISA and immunohistochemistry will be respectively performed to evaluate B[a]P-like DNA adduct levels in lung tumor tissues and paraffin embedded sections of tissue array from nonsmoking lung cancer patients using a specific antibody of BPDE-DNA adduct which was developed by our Lab. Additionally, HPV16/18 E6 will also be evaluated by immunohistochemistry in paraffin embedded sections of tissue array. Four categories of lung tumors will be classified by the adduct level and HPV E6 including high adduct level/HPV+, low adduct level/HPV+, high adduct level/HPV-, and low adduct level/HPVcategory. (2) To explore whether different profile of chromosomal instability loci could be existed in high adduct level/HPV+ lung tumors, the genome-wide analysis using 396 markers will be performed to verify whether higher frequency of chromosomal instability at certain loci will be observed in high adduct level/HPV+ lung tumors as compared with the other three categories. (3) To verify whether high chromosomal instability frequency occurred in high adduct level/HPV+ tumors compared tumors of other three categories could be resulted by higher frequency of loss of heterozygosity of DNA repair genes which are involved in mismatch repair and double strand break. The third-year study: To explore the candidate tumor suppressor or oncogene involved in lung tumorigenesis in Taiwanese nonsmokers with HPV infection and high DNA damage. (1) To verify whether a certain locus with a high prevalence of chromosomal instability could play an important role in lung tumorigenesis in nonsmokers, real-time RT-PCR and immunohistochemistry will be used to evaluate the expressions of the candidate gene to verify whether the chromosomal instability of these candidate genes could alter their expressions. (2) The loci of chromosome instability in HPV-positive lung cancer cell lines with B[a]P treatment for a long time interval will also be evaluated by genome-wide analysis to verify whether these candidate genes altered by chromosomal instability in lung cancer cells could be similar with those of tumor tissues. The candidate genes will be analyzed by a software package to predict the possible tumorigenic pathway of lung cancer among nonsmokers with HPV infection. (3) RNAi and transfection experiments, follow cytometry and soft agar assay will be performed to verify whether the candidate genes could really be involved in lung tumorigenesis among nonsmokers. These results obtained from the project will be helpful to explore whether synergistic effect of environmental carcinogen exposure could contribute to HPV16/18 infected lung tumorigenesis in nonsmokers. We hope to explore the novel gene(s) involved in lung tumorigenesis in nonsmokers, especially in Taiwanese women nonsmokers with HPV16/18 infection.過去對於肺癌的研究大多著重於抽菸所引起的腫瘤化機制，少有不抽菸肺癌之研究，因此 至今不抽菸引起之肺癌分子機轉仍相當不清楚。在台灣有50%以上的肺癌患者為不抽菸， 尤其是女性肺癌患者更有90%以上為不抽菸者。本研究室過去研究發現不抽菸肺癌患者的 DNA鍵結物含量與抽菸者並無統計上的差異，這結果暗示不抽煙肺癌患者暴露環境汙染 物所產生的DNA傷害並不低於抽菸者。本研究室又發現不抽菸女性肺癌之DNA鍵結物含 量，顯著高於不抽菸之男性患者，顯示女性較男性患者對環境污染物的感受性為強。本研 究室亦發現有高達 70%以上的女性肺癌患者，有人類乳突病毒16/18 型 (human papillomavirus, HPV16/18) 的感染，約有四分之ㄧ的 HPV感染之患者腫瘤組織能偵測到 HPV16/18 E6 致癌蛋白的表現，並參與抑制 p53 路徑而致肺腫瘤化。有研究指出僅有高 危險型 HPV的感染，並無法誘發人類腫瘤形成，必須同時有致癌物暴露，當無法及時修 復時，會堆積大量之 DNA 鍵結物，而引起抑癌或致癌基因發生突變，導致癌症形成。 因此大膽推測台灣不抽菸女性肺癌患者，有高含量之 DNA鍵結物堆積，又有高頻率之 HPV16/18 感染，可能會經由促進染色體不穩定 (chromosomal instability, CIN) 發生之頻 率增加導致肺癌形成。過去研究指出染色體不穩定可能與參與 mismatch repair 和 DNA double strand break的 hMLH1、hMLH2、XRCC1、XRCC3 、XRCC5、 BRCA1及 BRCA2 等基因的不正常表現有關。因此提出本三年研究計畫，期望能釐清環境汙染物暴露以及 HPV感染的交互影響，是否會促進染色體不穩定之發生，而造成不抽菸者發生肺癌？ 第一年計畫將釐清暴露環境汙染物及 HPV 感染交互影響，是否會促進CIN之發生？ (1) 利用本研究室自行由肺癌患者之胸水，建立培養出來的三株具有HPV16 E6 不同表現量以 及 12 株沒有 HPV 感染的肺腺癌細胞株，以低劑量 B[a]P長期處理後，以 FISH方法偵 測 micronuclei的發生頻率，以推測 CIN 發生的程度，此結果將用來了解 HPV感染之細 胞經過 B[a]P處理，是否會有較多的 micronuclei 的形成？並推測是否會增加 CIN 之頻 率，亦可了解 E6 表現較高的肺癌細胞，是否會有較高的 CIN 發生？(2)為了解 CIN 是 否是經由抑制 hMLH1、hMLH2、XRCC1、XRCC3、XRCC5、BRCA1和 BRCA2 等基 因的表現所致，本研究將以 Methylation Specific PCR (MSP) 分析這些基因的轉錄起始區 是否發生甲基化？並以 real-time RT-PCR 和 Western blotting分析這些基因的 mRNA 及 其蛋白之表現，以瞭解這些基因的表現是否發生異常？(3)以上肺癌細胞同樣以低劑量 B[a]P長期處理後，分別在 HPV感染與 HPV 沒有感染的肺癌細胞分別以 RNAi 和轉染 技術大量表現或剔除 E6 之表現，以 FISH 分析比較 micronuclei 的發生頻率，以及上 述各種修補基因的表現是否改變？以了解 CIN 發生是經由 HPV16/18 DNA 之嵌入或是 E6 蛋白表現所引起？ 第二年計畫(1) 將以肺腫瘤組織和本研究室已建立好的 200例不抽煙肺腺癌組織陣列切 片分別進行ELISA 和 Immunohistochemistry 分析 B[a]P-DNA 鍵結物含量、HPV16 E6 以及上述修補基因之蛋白表現。由以上之所得結果將肺癌患者分為 adduct high/HPV E6+、adduct high/HPV E6-、adduct low/HPV E6+及adduct low/HPV E6- 等四組，並與上述 修補基因的表現做一比較，以了解環境汙染物及HPV感染之交互作用，是否會造成上述修 補基因的表現改變？(2) 本研究將以 396 個 genome-wide marker全面篩檢 adduct high/HPV E6+、adduct high/HPV E6-、adduct low/HPV E6+及adduct low/HPV E6- 等四組 肺癌患者之基因體發生不穩定的位置，並將四組腫瘤所得之結果做一比較，以找出與環境 汙染物暴露、HPV感染以及兩者交互作用所產生的 CIN 頻率與位置有何不同？以找出 adduct high/HPV E6+ 肺腫瘤可能影響的標的基因。(3)同時本研究亦會探討 CIN之發生， 是否與以上 DNA 修補基因本身發生 CIN，而促進基因體不穩定有關？ 第三年計畫為瞭解環境暴露、HPV感染以及兩者交互作用所產生的 CIN 是否會影響特異 的下游基因的表現改變？本年度擬分三部分實驗 (1)由第二年計畫所得之結果選取出可 能重要之標的基因，以 Real-time RT-PCR及 Immunohistochemistry 分析其mRNA及蛋白 的表現是否改變，以確定同時有高環境汙染物暴露和HPV感染的肺腫瘤，是否可能藉由增 加基因體不穩定，而造成一些特異的抑癌基因的去活化或是致癌基因的活化？(2)以不同 劑量B[a]P長期處理 HPV感染的肺癌細胞後，比較這些細胞在B[a]P處理前後之基因體不 穩定是否不同？與第二年所發現之結果是否相符？利用分析軟體將這些可能影響之基因 作一分析，以推測出可能的致腫瘤路徑。 (3) 以siRNA或基因轉染的技術改變這些基因的 表現，並分析其細胞週期，並以soft agar assay 方式分析其致癌能力是否發生改變以了解 這些經由染色體發生不穩定而改變的基因，在肺癌形成過中是否確實扮演重要的角色？ 因此本計畫的研究成果，將有助於了解環境汙染物暴露和HPV感染之交互作用，是否會經 由增加染色體不穩定頻率而參與不抽菸者肺癌的形成，尤其是台灣不抽菸婦女的肺癌形 成。當然最重要的是能找到參與不抽菸者肺癌形成之可能致腫瘤基因和路徑，以做為未來 預防與臨床治療的重要參考。Project