王憶卿Yi-Ching Wang林若凱Ruo-Kai Lin2019-09-05不公開2019-09-052007http://etds.lib.ntnu.edu.tw/cgi-bin/gs32/gsweb.cgi?o=dstdcdr&s=id=%22GN0891430103%22.&%22.id.&http://rportal.lib.ntnu.edu.tw:80/handle/20.500.12235/104428抑癌基因的啟動子位置上若被過度甲基化，導致該基因的不表達，往往造成腫瘤的生成。負責將啟動子甲基化的酵素是甲基轉移酵素(DNA 5’-cytosine-methyltransferase, DNMT)，包括有DNMT1、DNMT3a及DNMT3b；但其在肺癌病人或細胞模式中並未有完整之變異分子機制與臨床研究，且其在癌症病人及細胞常發生不正常活化的改變，故引發出在癌症的治療中將 DNMT 作為癌症治療標靶的想法。本研究在所檢測的100位非小細胞肺癌病人發現，其DNMT1、DNMT3a及DNMT3b mRNA及protein在腫瘤組織比正常肺組織有顯著過度表達的情形，P值分別有0.002、0.034 及0.027；特別是在鱗狀上皮細胞癌 (squamous carcinoma, SQ) 的病人統計發現DNMT1的蛋白有過度表達的情形，P值達0.04。當這些病人同時過度表達DNMT1及DNMT3b時，則顯示與p16INK4a、RARβ及FHIT 抑癌基因啟動子過度甲基化有關，P值達0.006。在病人的腫瘤組織採組織染色質免疫沉澱分析 (tissue chromatin immunoprecipitation) 的確發現DNMT1及DNMT3b蛋白是結合在過度甲基化的p16INK4a、RARβ及FHIT 基因啟動子上。 為了檢查DNMTs過度表達的原因，於是在肺癌細胞株H1299（不含p53基因）送入DNMTs的一個可能的負調控蛋白Wild-type p53，來了解p53對DNMTs啟動子的影響。本研究採用營火蟲冷光啟動子活性分析，發現Wild-type p53可抑制DNMT1、DNMT3a及DNMT3b的啟動子活性，且在H1299肺癌細胞株長期表達Wild-type p53時，發現內生性的DNMTs之mRNA與蛋白表現量有下降的情形。而突變的p53蛋白則有增加其啟動子活性的趨勢。在病人組織樣本裡發現，這些有過度表達DNMT1的病人的確有p53 基因突變的情形，P值達0.016。這些病人同時以免疫組織染色來偵測DNMT1另一個可能的負調控蛋白RB，發現有過度表達DNMT1的病人同時伴隨著RB蛋白表達過低，P值達0.014。本研究進一步在正常的肺組織中利用組織染色質免疫沉澱分析，檢測到Wild-type p53及RB蛋白可以結合在DNMT1、DNMT3a及DNMT3b的啟動子上。DNMTs除了受轉錄的調控之外，此研究更發現DNMT1的過度表達的病人與吸煙有顯著的相關，P值達0.037。在正常肺細胞IMR-90及肺癌細胞H1299測試下發現，DNMT1、3a及3b的蛋白質表現量可受香煙致癌物4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)的誘導而使其上升；此外，利用組織染色質免疫沉澱分析，發現在NNK處理細胞之後相較於控制組，檢測到DNMT1及DNMT3b蛋白結合在p16INK4a、RARβ及FHIT的啟動子上的程度顯著增加，該啟動子也的確被檢測是呈現過度甲基化。因此我們推測肺癌病人DNMTs的過度表達的原因可分為兩類：一是分子層次中的p53及RB變異有關，因其失去了負調控DNMT轉錄的能力；另一則是與吸煙有關，因香菸致癌物會誘導DNMTs的蛋白質表現量上升。而過度表達的DNMTs則使抑癌基因過度甲基化，最後導致肺癌的發生。 由於癌細胞基因體普遍出現CpG島群的甲基化異常而使抑癌基因失去活性的現象，因此抑制形成CpG島群的甲基化的藥物正可以作為恢復抑癌基因的表現及活化癌細胞中抑制癌症的重要路徑的新穎標靶藥物。而Mithramycin A (簡稱MMA)是一個會與富含GC及CG 序列的DNA 結合之藥物，因此本研究檢測癌細胞在經過MMA的處理之後，是否會抑制CpG島群甲基化的情形。我們發現當以低劑量(10 nM)的MMA處理癌細胞14天後，會減少轉移抑癌基因SLIT2及TIMP3的CpG島群過度甲基化的情形，並進而使這個具有抑制癌細胞轉移的SLIT2及TIMP3重現基因表達。同時間藉由膜穿透 (transwell) 實驗我們也發現MMA可降低具有高轉移能力的癌細胞CL1-5的穿越及移動能力。為了暸解MMA的作用途徑，我們使用西方點漬法發現MMA會使DNMT1蛋白明顯下降，但是DNMT1的基因表現不受影響。使用分子模擬 (molecular modeling) 發現DNMT的催化位置可被MMA藥劑所鍵結。總結研究結果發現，MMA具有去DNA甲基化及抑制癌細胞轉移的潛力。而抑制的機轉可能藉由抑制DNMT酵素催化能力並降低癌細胞中DNMT1的蛋白表達量，進而導致抑制癌細胞轉移的基因啟動子去甲基化且重新恢復表達。 本研究為首篇在肺癌病人或細胞模式針對三種主要甲基轉移酵素DNMT1, DNMT3a, DNMT3b完整之變異分子機制與臨床研究，且在肺癌細胞以GC DNA結合之MMA驗證其抑制 DNMT 作為癌症治療標靶的想法。Down-regulation of tumor suppressor genes (TSGs) by hypermethylation of 5’CpGs is one of the important events involved in tumor development. However, mechanism of overexpression of DNA 5’-cytosine-methyltransferase (DNMT), which is enzyme that methylates the cytosine residue of CpGs, and the clinical significance of DNMT alterations remain unclear in many cancers. In the current study, we demonstrated that the mRNA and protein of DNMT1, DNMT3a, and DNMT3b genes were expressed in a coordinate manner in most tissues and at a significantly higher level in 100 non-small cell lung cancer (NSCLC) tumors than in adjacent non-tumorous lung tissues (P = 0.002, P = 0.034 and P = 0.027, respectively). The patients with DNMT1 overexpression showed poor prognosis especially in squamous carcinoma (SQ) lung cancer (P = 0.04). The patients with overexpression of both DNMT1 and DNMT3b correlated with hypermethylation in the p16INK4a, RARβ, and FHIT promoters, especially in smoking SQ patients (P = 0.006). The physical binding of DNMT to these promoters was identified by tissue chromatin immunoprecipitation in lung tumors of NSCLC patients. To understand what the mechanisms are for DNMTs overexpression in lung cancer, we performed DNMT1 promoter luciferase assay and found that wild-type p53 could negatively regulate the DNMT1, DNMT3a and DNMT3b promoters. The mRNA expression level of DNMT1 and DNMT3b was significantly reduced when transfected with wild-type p53 construct to H1299 which is originally a p53 null cell. In normal lung tissue, the physical binding of wild-type p53 and RB to DNMT1 promoters was identified by chromatin immunoprecipitation PCR. We further analyzed the correlation between overexpression of DNMTs and alterations of p53 gene and RB protein. A significant association between DNMT overexpression with the p53 gene mutation (P = 0.016) and low RB expression (P = 0.014) was found. The results also showed that DNMT1 protein overexpression was significantly associated with those who are smoker (P = 0.037). In addition, we treated the model cells including IMR-90 (normal lung cell) and H1299 (lung cancer cell) with smoking carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1- butanone (NNK), the protein expressions of DNMT1, 3a and 3b were induced. Interestingly, DNMT1 and DNMT3b proteins were detected at p16INK4a, RARβ, and FHIT promoters showing hypermethylation after treated with NNK. These data suggest that deregulation of DNMTs was associated with smoking and the loss of transcriptional repression of the p53 and RB. DNMT overexpression results in epigenetic alteration of target TSGs and ultimately leads to NSCLC tumorigenesis and poor prognosis. Pharmacologic inhibitors of CpG island methylation provide a rational approach to reactivate the TSGs in tumor cells and restoring of critical cellular pathways in cancer cells. Mithramycin A (MMA) is known to be a GC and CG-rich DNA binding agent. We sought to determine whether MMA could inhibit CpG island methylation and DNMT expression in lung cancer cells. We found that MMA reduced CpG island methylation of anti-metastasis TSGs, including slit homolog 2 (SLIT2) and tissue inhibitor of metalloproteinase 3 (TIMP3) genes, and associated with the prevention of metastasis. When highly metastatic CL1-5 lung cancer cells were treated with low does (10 nM) of MMA for 14 days, they re-expressed mRNA levels for SLIT2 and TIMP3 genes. MMA also inhibited the invasion phenotypes of CL1-5 cells as indicated by its inhibition of cancer cell migration using wound healing and transwell assays. Molecular docking of MMA onto the DNMT1 catalytic domain revealed that MMA may interact with the catalytic pocket of DNMT1. Western blots showed that DNMT1 protein levels were depleted after MMA. These data support the idea that MMA has demethylation and anti-metastasis effects on lung cancer cells. This mechanism may be mediated by interaction of MMA with DNMT1, leading to the depletion of DNMT1 protein and reversal of the metastasis phenotype in lung cancer cells. Our data is the first comprehensive molecular and clinical study on overexpression of various DNMTs in the same serious of lung cancer cohort. The deregulation of DNMTs was associated with smoking and the loss of transcriptional repression of the p53 and RB. In addition, GC-rich binding reagent such as MMA can be potential DNMT inhibitor thus for novel therapeutic development.甲基轉移酵素p53 基因RB 蛋白香菸致癌物NNKMithramycin 藥物非小細胞肺癌DNMTp53 geneRB proteinNNKMithramycinNSCLC