鎳化合物常使用於現代工業，已被認定為Group I的人類致癌物，慢性暴露鎳會提高肺癌發生的風險，且已知誘導缺氧效應。類血管生成素蛋白(angiopoietin like 4, ANGPTL4)是具有多種功能的脂肪激素，包含參與能量代謝、血管新生及癌症轉移等，但是否促進肺癌的發展及其機制仍需要釐清。本研究探討ANGPTL4是否參與氯化鎳誘導的致癌機轉，並利用具干擾癌症能量代謝能力的藥物二甲雙胍 (metformin)抑制ANGPTL4誘發之肺癌發展。首先利用美國癌症基因體圖譜資料(The Cancer Genome Atlas)和免疫組織化學染色法，分析肺癌病人中的ANGPTL4及缺氧誘導因子(hypoxia-inducible factor 1α, HIF-1α)與臨床病理表現之相關性。在細胞實驗中發現氯化鎳的刺激下ANGPTL4基因及蛋白表現會逐漸增加，ANGPTL4也促進肺癌細胞的轉移能力。由染色質免疫沉澱及螢光素報導基因分析法發現，氯化鎳刺激ROS累積，並活化HIF-1α坐落在距離轉錄起始位點上游2000 bp處調控ANGPTL4轉錄活性，並增加HIF-1α與ANGPTL4啟動子的結合能力，使用metformin、HIF-1α抑制劑及HIF-1α short hairpin RNA (shRNA)抑制內生性HIF-1α，可以減少氯化鎳誘導的ANGPTL4表現。利用甲基化專一性PCR發現氯化鎳促進ANGPTL4啟動子的去甲基化，並活化去甲基化基因ten-eleven translocation methylcytosine dioxygenase 1 (TET1)表現，而metformin能反轉鎳造成的低度甲基化並抑制TET1。最後利用TET1 shRNA可顯著抑制ANGPTL4。綜合上述結果證實，氯化鎳透過HIF-1α的累積並調控TET1促進ANGPTL4表現，這可能是鎳至肺癌形成的機轉之一。另外，metformin具有抑制鎳活化的氧化傷害、抑制腫瘤轉移及反轉表觀遺傳的能力，本研究提出了metformin可作為化學預防藥物抵抗細胞癌化及抗腫瘤發展的證明。 Nickel (Ni), a carcinogenic workplace hazard, increases the risk of lung cancer and induces hypoxia response. Angiopoietin-like protein 4 (ANGPTL4) is a multifunctional adipokine involved in energy metabolism, angiogenesis and metastasis, but its role in lung cancer progression is still not clear. Here, we assessed the role of ANGPTL4 in lung carcinogenesis under nickel exposure and investigated the effects of metformin which interfere energetic metabolism on ANGPTL4 expression and lung cancer chemoprevention. We examined the clinical relevance of ANGPTL4 and hypoxia-inducible factor 1α (HIF-1α) expression in lung cancer using the Cancer Genome Atlas and immunohistochemistry results. ANGPTL4 was increased in NiCl2-treated lung cells and promoted the ability of lung cancer cells to migrate. Chromatin immunoprecipitation and the luciferase assay revealed that NiCl2-induced HIF-1α and hypoxia response element, which located at 2k bp from the upstream of transcription start site, binding activate ANGPTL4 expression, which is then inhibited by metformin, the HIF-1α inhibitor and the HIF-1α short hairpin RNA (shRNA). NiCl2-induced ANGPTL4 was regulated by promoter demethylation, which was also reversed by metformin. Additionly, NiCl2 promoted ANGPTL4 via HIF-1α-dependent ten-eleven translocation methylcytosine dioxygenase 1 (TET1) using TET1 shRNA. In conclusion, the increased presence of ANGPTL4 due to HIF-1α accumulation and the promoter demethylation caused by nickel in lung cells may be one mechanism by which nickel exposure contributes to lung cancer progression. Furthermore, metformin has the ability to prevent NiCl2-induced ANGPTL4 and metastasis in lung cancer cells. These results provide evidence that metformin in oncology therapeutics could be a beneficial chemopreventive agent.
