低劑量DNA甲基轉移酶抑制劑於肺癌細胞敏感性差異研究

Abstract

肺癌是全球癌症致死率第一的疾病，亦是2013年國人癌症死亡統計人數的第一名。近年來，多項證據指出不正常的DNA甲基化修飾參與並促進肺癌與其他癌症的形成，包括染色體低甲基化與抑癌基因啟動子區域過甲基化，此現象造成基因組結構不穩並增加腫瘤的惡性程度。近年來，多種DNA甲基轉移酶抑制劑，例如decitabine (DAC)，已被應用在血癌及其他癌症之臨床治療，顯示表觀遺傳調控在細胞癌化過程中之重要性。臨床前之研究顯示DAC能抑制癌細胞株增生並活化抑癌基因之表現，使腫瘤生長受到抑制；在臨床試驗中，低劑量DAC能促進血癌病人之治療，然而只有極少數的肺癌病人對此藥物有反應並達到治療效果。因此，透過特定的基因標記與表觀遺傳標記來篩選出適用於此藥物的敏感族群可能是改善部分肺癌病人治療之方法。本實驗使用之DAC (10，100及200 nM) 為72小時藥物暴露下評估對不同肺癌細胞株不具急毒性但具抑癌效果之低劑量。我們首先透過細胞增生實驗分析DAC抑制不同肺癌細胞株的增生情況，並將細胞依據敏感度差異分為四組。接著透過群落形成分析與癌細胞球體形成分析 進一步觀察DAC對不同組別細胞的抑癌效果，發現細胞對DAC的反應與分組之敏感性相符；DAC還能抑制敏感族群中具有ALDH活性之癌幹細胞族群。最後我們分析DAC敏感度與癌細胞之上皮細胞生長因子受體 (Epidermal growth factor receptor, EGFR) 突變是否具相關性, 以及DNA甲基轉移酶在細胞中的表現量之關係，研究結果顯示具EGFR基因突變的肺癌細胞株 (L858R 與 del-19) 對DAC有較低的敏感性，並且DAC造成細胞早期Dnmt1表現量降低與細胞後期對DAC的敏感度有關。本研究建立對DAC不同敏感性之肺癌細胞株族群，並發現可能與DAC敏感度相關之基因指標。

Lung cancer is the leading cause of cancer death in the world. It was also elevated to the leading cause of Taiwan in 2013. Recently, aberrant modifications of DNA methylation have been found on the initiation and progression of numerous tumors including lung cancer. The genomic hypomethylation and hypermethylation in the promoter regions of tumor suppressor genes may increase the chromatin instability and promote malignancy. During the past few years, inhibitors of DNA methyltransferase (DNMT), such as decitabine (DAC), have been applied clinically to treat leukemia and other solid tumors, highlighting the epigenetic basis of cancer progression. The pre-clinical data of DAC in lung cancer shows promising effects: it can reduce cancer cell proliferation and re-activate tumor suppressor genes to inhibit tumor growth. Although low-doses DAC showed the promising responses in the treatment of leukemia, only a small portion of lung cancer patients had responses in previous clinical trials. Therefore, to identify the genetic or epigenetic markers for selection of patients more likely to benefit from hypomethylating agents may improve the treatment for lung cancer. In this study, we used low doses of DAC (10, 100, 200 nM), which were sufficient to observe the anti-tumor effects without acute cytotoxicity in NSCLC cell lines within 72 h of pre-treatments. Base on the anti-proliferation efficacy via MTT assay, we characterized the cell responses to DAC and divided NSCLC cell lines to four groups showing different DAC sensitivities under different treatment protocol. Next we further confirm the anti-tumor effects of DAC in the four groups by functional assays, including colony formation and tumorous sphere forming; moreover, DAC also decreased ALDH high activity stem-like population in sensitive cell lines. Finally, by organizing the DAC sensitivity with epidermal growth factor receptor (EGFR) status and validating the DNMT expression levels in each cell lines, we found that NSCLC cell lines with EGFR mutation (L858R and del-19) show more resistant to DAC treatment; whereas, early depletion of Dnmt1 expression may associate with DAC sensitivity. In conclusion, we found the DAC sensitive, intermediate, and resistant cell lines. We also found that the genetic markers could be associated with DAC-sensitivity. Using different DAC sensitive groups to find out the molecular mechanisms and the biomarkers for DAC treatments, and combining DAC with chemotherapy to improve the treatments of lung cancer with EGFR wild type status are the goals of our future works.