| Abstract: | 肺癌為台灣癌症死亡率很高之癌症，肺癌細胞所造成的遠端轉移為肺癌病人治療失敗之主因。在腫瘤轉移過程的初始，原位癌細胞伴隨著上皮細胞轉變成間質細胞之型態改變，此過程稱為”上皮-間質細胞轉換過程”（Epithelial-mesenchymal transition，EMT）。細胞與細胞之間的游離可以透過EMT 造成，EMT 途徑一旦活化會有一些基因標記如E-cadherin 的基因表現降低，vimentin, fibronectin 的基因表現增加等，使得細胞行為傾向易侵襲(invasion)及爬行(migration)的狀態。EMT 的誘發因子相當的多，探討肺癌細胞轉移的誘發因子以及相關基因是目前抑制腫瘤轉移的一大課題。環境污染物重金屬-氯化鎳遍佈於人類環境之中，國際癌症研究中心(Internation Agency for Research on Cancer; IARC)的致癌性物質分類中，鎳化合物為第一類具有致癌性，多篇文獻指出鎳會造成肺炎、肺氣腫及不同程度肺部細胞的損傷以及肺腫瘤的發生。本研究主要探討重金屬氯化鎳透過轉錄及轉譯層次影響E-cadherin, fibronectin 等指標蛋白的表現，經由何種重要途徑或轉錄因子如slug, snail 進而調控指標蛋白的表現啟動EMT 的發生。釐清氯化鎳誘發肺上皮及肺癌上皮細胞株EMT 表現機制。進一步開發反轉機轉與期望減緩肺癌惡化之發生。本研究建立細胞模式以及長期低劑量以呼吸途徑暴露動物模式深入探討氯化鎳造成肺腫瘤發生以及腫瘤惡化轉移的機制，並使用專一性抑制劑與臨床用藥配套以阻斷氯化鎳使腫瘤轉移的途徑以達到反轉EMT 的目的。針對這些課題我們將以三年計畫分述各個階段的目標與策略: 第一年目標:闡釋重金屬氯化鎳對肺上皮細胞以及肺癌上皮細胞誘發EMT 現象及其相關的基因表現; 了解氯化鎳影響肺上皮細胞BEAS-2B及A549 肺癌細胞的E-cadherin, fibronectin, 轉錄因子slug, snail 等與EMT 有關基因的表現? 釐清氯化鎳是否會參與或同時啟動多種誘發因子進而使EMT 發生? 釐清氯化鎳是否會造成細胞內胞器壓力,如:ER stress 或自由基的增加與誘發EMT 關係。第二年目標: 釐清氯化鎳啟動EMT 之可能途徑及其訊息傳遞，分別以抑制劑或拮抗劑或RNA interference 方式逐一探討: (1)是否透過ROS 途徑啟動EMT：以H2DCFDA 同步偵測ROS 變化量。 (2)是否透過異常甲基化使E-cadherin 基因表現喪失，釐清氯化鎳造成E-cadherin promoter 過度甲基化的專一性區位以及影響何種轉路因子結合 (3)是否透過Histone 去乙醯基化使E-cadherin 表現喪失 (4)是否透過其他途徑如TGF-β, MAPK,等調控其下游轉錄因子抑制E-cadherin，增加fibronectin 表現第三年目標: 建立動物模式釐清氯化鎳調控EMT 與肺纖維化的發生, 肺腫瘤轉移有關以裸鼠接種腫瘤細胞後再暴露氯化鎳，釐清氯化鎳造成EMT 且是否促進血管新生及相關指標基因變化，如Cap43, uPA, DNMT, vimentin, fibronectin 及GST-M2 等等的調控機制。又slug, TGF-等是否參與此機制。針對第一、二期非小細胞肺癌病患組織進行免疫染色並且持續追蹤病患是否惡化或轉移現象釐清氯化鎳的暴露是否造成腫瘤轉移的危險因子。於細胞及動物模式中使用機制阻斷劑、抑制劑搭配臨床藥物藉以減緩氯化鎳造成的肺癌轉移。
Lung cancer is the leading cause of cancer deaths in Taiwan. Metastasis is a complex multi-step process, which requires the concerted action of many genes and is the primary cause of cancer death. Epithelial-to-mesenchymal transition (EMT) is a process during which epithelial cells acquire the attributes of mesenchymal cells, including increased motility and invasiveness. The expression of differentiation markers switched from cell-cell junction proteins such as E-cadherin to mesenchymal markers including fibronectin and vimentin. The mechanism underlying EMT involves coordination of multiple signaling pathways. Research investigations on the molecular basis of lung carcinoma metastasis are helpful to identify therapeutic targets for metastasis. Nickel, the carcinogenic effects of nickel and nickel compounds have been critically evaluated by the International Agency for Research on Cancer (IARC). Exposure to nickel compounds can produce a variety of adverse effects on human health including lung inflammation, lung cancer and lung injury. It is believed that epithelial-mesenchymal transition (EMT) occurs during the development and progression of lung cancer; however, the correlation nickel and EMT remains to be elucidated. In addition, this repression of E-cadherin was strongly correlated with aberrant hypermethylation, histone deacetylation, proteasome degradation and ROS pathway. We hypothesize that there is an association between the nickel modulates multiple pathways and EMT, and we investigate multiple genes that are involved in the EMT. In the study, we will investigate that nickel down regulate E-cadherin through which mechanisms and transcription factors in lung epithelial cells. The aims of this study are (I) To confirm that nickel induce epithelial-to-mesenchymal transition phenotype: we utilized RT-PCR, real-time PCR, western blot and confocal microscope to confirm EMT phenotype by nickel in lung epithelial and cancer cells; (II) Using CHIP, EMSA assay, DNMT activity and RNA interference techniques to clarify which mechanisms of nickel effect E-cadherin in association with transcriptional factors or repressors in vivo and in vitro; (III) To determine which pathways that involve in EMT phenotype by nickel such as DNA hypermethylation, histone deacetylation, ROS pathway, TGF-, MAPK pathways or other genes such as DNMT1, Cap43, uPA, vimentin, fibronectin and GST-M2. (IV) To evaluate some specific inhibitors and blockers to alleviate nickel-induced EMT pathway in lung epithelial cells and lung cancer cells ; (V) To investigate the correlation with nickel and clinical outcomes of early stage I and II NSCLC patients and follow up the recurrence and survival. Here, we investigate which transcriptional repressors contribute to non-small cell lung cancer (NSCLC) progression, to identify a potential pathway for improved therapies. The study will indicate that nickel seems to induce EMT in lung cancer cells, and it also may drive disease progression in patients with lung cancer. |
