2026-11-092026-11-09https://scholars.csmu.edu.tw/handle/123456789/38461Our preliminary data showed that 23 of 43 (53.5%) lung tumors were detected with Xeroderma pigmentosum group C (XPC) LOH. Our recent report indicated that 53 of 158 (33.5%) lung tumors have found to occur XPC promoter hypermethylation. We thus considered that XPC gene inactivation through LOH and promoter hypermethylation may partly be involved in lung tumorigenesis. Notably, XPC inactivation in nonsmoker was more common than in smokers with lung cancer. In animal model studies, lung and liver tumors were frequently occurred in XPC knockout mice when mice were treated with 2-AAF chemical carcinogen. Additionally, the number of lung tumor burden in XPC-/-+p53-/- double knockout mice were more frequent than those of XPC-/- mice. A recent study showed that adenomas were spontaneously occurred in XPC-/- mice without any carcinogen exposure. More interestingly, lung adenocarcinomas were developed in the XPC-/-GADD45-/- double knockout mice, spontaneously. Based above mentions, XPC inactivation could be involved in early event of lung tumorigenesis. However, whether the involvement of XPC in lung tumor aggressiveness is still unclear. In this three-year project, the specific aim of each year is addressed as follows: Fist-year: The role of the possible XPC-hHR23B-p53 complex in lung tumor progression Our previous study has indicated that p53 mutation was more common occurred in lung tumors with XPC hypermethylation than in those without XPC hypermethylation. XPC protein was incorporated with hHR23B to recognize DNA damage site. It is believed that p53 protein could be involved in the recognition. In the first-year project, XPC knockdown A549 lung cancer cells have been developed by small interference RNA (RNAi) technique. Our preliminary western blot data showed that the expression levels of hHR23B and p53 proteins were significantly reduced in XPC-knockdown cells as compared with those of A549 parental cells (Fig. 1). In this year project, immunoprecipitation (IP) will be used to verify whether hHR23B could be interacted with XPC and/or p53 protein. Whether MDM2 is involved in the proteolysis of p53 protein in XPC-knockdown cells and MDM2-hHR23B-p53 interaction in XPC-knockdown cells will be analyzed by co-IP. The p53 function will be determined by the evaluation of p53 downstream genes, such as MdM2, p21 and Bax transcripts. The cell proliferation and apoptosis will be evaluated by flow cytometer to elucidate whether aberrant p53 will be observed in XPC knockdown cells. To further explore the possible complex of XPC-hHR23B-p53, p53 and hHR23B in A549 cells will be knocked down respectively to understand whether the interaction of XPC-hHR23B and p53-hHR23B will be diminished as similar as the observation in XPC knockdown cells. The migration and invasion activity of these RNAi cells including XPC-, p53- and hHR23B-knockdown cells will be evaluated by Boyden chamber migration and matrigel invasion assay. These results will be helpful to understand whether XPC inactivation could be through the aberrant XPC-hHR23B-p53 complex to promote lung tumor aggressiveness. Second-year: The involvement of cullin 4A-DDB1 ubiquitin ligase in lung tumor aggressiveness through degradation of p53, p27, and CRMP-1 proteins Previous studies indicated that DDB2 and centrin 2, as similar as hHR23B, were directly interacted with XPC to recognize the DNA damage sites for NER process. In addition, cullin 4A-DDB1 ubiquitin ligase has been shown in involvement of XPC and p27 protein degradation. Our preliminary data indicated that p27 and CRMP-1 proteins were significantly reduced in XPC-knockdown A549 cells as compared with those of parental cells (Fig. 2). Therefore, we hypothesize that cullin 4A-DDB1 ubiquitin ligase may be involved in degradation of XPC-hHR23B-p53 complex to cause lung tumor progression. In this year-project, the proteasomal inhibitors will be first used to check whether ubiquitin/proteaosome pathway, could be partially through cullin 4A-DDB1 ubiquitin ligase to cause the degradation of XPC, p27, CRMP-1 and p53 proteins. We will establish the cullin 4A- and DDB1-knockdown cells by RNAi to verify whether cullin 4A-DDB1 ubiquitin ligase is indeed involved in the proteolysis of XPC, p53, CRMP-1 and p27 proteins. Previously, CL1-0~CL1-5 cells harbored p53 mutation were shown to have gradually increased the capabilities of lung tumor migration and invasion. Thus, we will use the cell lines to test whether XPC protein expression was correlated with tumor migration and invasion in lung cancer cells with abnormal p53 function. Our preliminary data indicated that the XPC protein expression in CL-1 series cells was as similar as CRMP-1 protein expression (Fig. 3), which was shown to associate with lung tumor aggressiveness. In this year study, XPC gene in CL1-0 will be knockdown by RNAi and CL1-5 will be overexpressed by transfected with XPC cDNA. The XPC, p27, CRMP-1 and p53 proteins in XPC-knockdown CL1-0 and XPC-overexpressed CL1-5 cells will be evaluated by western blot and migration as well as invasion assay to understand whether XPC is indeed involved in tumor aggressiveness. More importantly, we will establish cullin 4A- and DDB1-knockdown CL1-5 cells to examine whether these proteins including XPC, p27, p53 and CRMP-1 will be restored and the migration and invasion capability will be repressed. These results will be helpful to elucidate why XPC inactivation was related with tumor aggressiveness. Third-year: The role of XPC and p53 inactivation in lung tumor aggressiveness in animal model. To further elucidate the role of XPC and p53 in lung tumor progression, XPC, p53 and double knockout mice will be purchased to treat with or without an oxidative DNA damage agent 2,4-DDE because XPC has been shown to accelerate hOGG1 enzyme activity and oxidative stress was thus increased in XPC-inactivated cells and animals. The lung tumor burden and tumor metastasis will be determined by pathological examination in each organ of mice. These results might be helpful to understand the role of XPC in the development of lung adenocarcinoma in XPC-/- mice and XPC-/- + p53-/- double knockout mice.We also verify whether p53 inactivation will accelerate lung tumor aggressiveness as compared with those of p53-/- and XPC-/- knockout alone mice. The lung tumors and metastatic tumors will be isolated to evaluate p27, CRMP-1 and other related tumor metastatic protein expression by western blot to compare the findings between cell in intro and tissue in vivo. These in vivo data may be helpful to elucidate the role of oxidative stress caused by XPC and/or p53 inactivation in the development of lung adenocarcinoma and their tumor aggressiveness.本研究室初步loss of heterozygosity (LOH) 之分析結果得知，肺癌患者之 XPC 基因有一半以上發生LOH (23 of 43, 53.5%)。又發現肺腫瘤之XPC 基因經 常發生啟動子甲基化(53 of 158, 33.5%)，因此XPC 失去活性可能經由雙重缺失 而參與肺癌之形成。已知XPC knockout 小鼠會自然形成肺腺瘤，但並不會引起 腺癌，而XPC, p53 雙基因knockout 之雄鼠則會發生睪丸癌。最近我們的結果顯 示A549 肺癌細胞之XPC 基因被small interference RNA (siRNA) 剔除後，經過 benzo[a]pyrene 連續處裡，則此細胞會發生p53 基因的突變(Wu et al., 2007)。這 和肺腫瘤組織之結果相似，即甲基化之腫瘤較易發生p53 基因發生突變，而此突 變大多為G->T or C 之突變型(Wu et al., 2007)。我們觀察到有些XPC 剔除的細 胞，p53 基因既使沒有發生突變，但是該基因卻還是失去活性，但是其分子機轉 則有待釐清。本三年計畫擬首先探討為何XPC 去活化會影響p53 之活性？是否 經由ubiquitin/proteasomal pathway 將p53 以及其他參與腫瘤化之蛋白降解，而參 與肺癌之腫瘤轉移與侵入? 第一年計畫： 首先假設XPC 去活化可能會影響p53 蛋白之穩定度， 而較易被 ubiquitin-dependent proteolysis 降解。其次假設p53 蛋白功能被改變，而失去轉錄 活化下游基因之能力。推測可能XPC 會藉由hHR23 與p53 蛋白形成複合體去 執行認知DNA 傷害之位置，因此當XPC 失去時，可能會影響此複合物之蛋白 穩定，而使p53 蛋白降解，或失去做為轉錄因子之能力，而無法轉錄活化下游基 因之表現。我們初步結果發現以siRNA 技術剔除XPC 基因的A549 細胞之p53, 和hHR23 蛋白之表現量會確實隨著XPC 的剔除而下降(Fig. 1)，我們將以 real-time RT-PCR 分析三種基因之轉錄，是否受到XPC 基因剔除之影響，另外亦 需要以proteasomal inhibitor 處裡以瞭解p53 和hHR23B 經由ubiquitin-proteasmal pathway 降解，以確定其作用確實在蛋白層次。為了瞭解p53 蛋白之降解是否改 變了MDM2-hHR23B-p53 complex 之穩定以及是否可能存在XPC-hHR23B-p53 複合體，我們將以co-immunoprecipitation 技術瞭解三種蛋白間是否有交互作用？ 同時將分析p53 下游基因之表現，以印證p53 是否失去轉錄活性？並以流式細胞 儀瞭解其細胞週期是否發生改變？是否細胞增生？以及細胞之apoptosis 之路徑 受到抑制？以上結果都可瞭解XPC 失去活性，是否會促進肺癌細胞之增生與存 活，而使其有機會惡化為具有轉移和侵入之癌細胞。 第二年計畫： 將探討XPC 基因去活化造成肺腫瘤轉移是否是經由活化cullin 4A-DDB1 ubiquitin ligase 而造成參與腫瘤轉移與侵入之CRMP-1 和p27 蛋白降解所致？我 們初步結果發現XPC 剔除之細胞之CRMP-1 和p27 蛋白之表現量會顯著降低， 同時腫瘤轉移與侵入之能力則增高(Fig. 2)。為了確定此依初步發現，本年度將 以已知具有不同轉移與侵入能力之CL1 系列之肺癌細胞進行研究。首先將以 western blot 分析XPC 蛋白之表現是否會隨著轉移與侵襲之能力增強而遞減。若 是則同樣將以siRNA 技術剔除CL1-0 細胞之XPC 基因，然後分析轉移和侵襲之 能力是否增強？另外，將XPC 基因轉染入轉移和侵襲能力最強的CL1-5 細胞， 以瞭解XPC 基因是否會抑制細胞之轉移和侵襲之能力？以上結果將對照過去已 發現，會抑制腫瘤轉移和侵入能力之CRMP-1 蛋白表現是否與XPC 之表達有 關？以瞭解XPC 基因與肺腫瘤轉移和侵襲之角色。為瞭解cullin 4A-DDB1 ubiquitin ligase 在XPC 基因參與肺腫瘤轉移和侵入之角色，我們將建立cullin 4Asi 與DDB1si 的細胞，並分析其轉移和侵襲能力是否降低？ 第三年計畫： 將進入動物模式系統探討XPC knockout 小鼠和XPC, p53 雙基因knockout 之小鼠在肺腺癌之轉移與侵襲之角色。本研究將以引起氧化緊迫之2,4-DDE 分 別處理三種小鼠四週與十二週，再分析兩種處理時間，產生肺腺癌之能力是否不 同？短時間是否XPC-/-小鼠僅會產生腺瘤？而XPC-/-和p53-/-小鼠會產生腺癌？ 長時間`處理是否兩種小鼠都會產生腺癌？兩者間是否有差異，尤其是轉移與侵 襲之能力是否不同？ 總之，以上三年之研究成果，不僅能釐清XPC 在肺腫瘤形成之角色，同時還能 釐清p53 基因在XPC 去活化後，參與肺腫瘤轉移與侵襲之角色。Project