HIC1基因變異參與臺灣肺癌形成及其受p53/SIRT1蛋白調控之機制研究
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2006
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研究背景：在癌症病人的研究中發現，位於染色體17p13.3的Hypermethylated in Cancer (HIC1) 抑癌基因常發生DNA啟動子過度甲基化 (promoter hypermethylation)，以及異質性缺失 (loss of heterozygosity, LOH) 情形。我們實驗室先前的研究結果也顯示，臺灣地區肺癌病人在染色體17p13區域發生LOH的頻率高達55 %，因此我們懷疑是否位於17p13.3的HIC1基因會參與肺癌形成。目前也知，當細胞受到DNA傷害時，HIC1基因/蛋白，SIRT1去乙醯酶及p53蛋白彼此之間呈現環形 (circular regulation) 的調控關係。所以，本研究便針對台灣地區的肺癌病人其HIC1基因變異情形以及HIC1基因/蛋白受p53/SIRT1蛋白調控情況做研究。材料與方法：我們分析了108個非小細胞肺癌 (non-small cell lung cancer, NSCLC) 病人的HIC1蛋白表現、mRNA表現及啟動子過度甲基化情形，並將變異結果和臨床病歷資料做相關的統計分析。我們使用西方墨點法 (Western blot)，觀察病人HIC1蛋白、乙醯化p53及SIRT1去乙醯酶表現情形，再以反轉錄--聚合酵素鏈反應 (Reverse-transcriptase polymerase chain reaction, RT-PCR) 分析組織細胞中HIC1基因mRNA轉錄是否異常，續以聚合酵素鏈反應為基礎的甲基化分析 (methylation-specific PCR, MSP) 偵測HIC1基因的啟動子過度甲基化頻率。結果：我們發現NSCLC病人HIC1蛋白低表達頻率達65.5%， HIC mRNA有75.9%呈現低表達情形，且HIC1啟動子甲基化頻率達40%；另外，蛋白質/mRNA、mRNA/啟動子甲基化表現彼此間都呈統計上顯著相關性 (P=0.045)。除此之外，HIC1 mRNA是低表達者中有87.5% (28/32) 的病人其p53蛋白為去乙醯化的病人，且統計上達顯著相關性 (P＝0.016)；此外，HIC1蛋白有表達的病人當中，有69% (20/29) 病人，其SIRT1蛋白是低表達 (P＝0.004)；再者，SIRT1蛋白低表達的病人之中，有75% (39/52) 的病人其p53蛋白有乙醯化情形 (P＝0.002)。另外，本研究利用去甲基化藥物5’-Aza-2’-dC (5’-aza -2’-deoxycytidine) 及SIRT1的抑制劑Nicotinamide (Vitamin B3) 處理肺癌細胞株A549，結果發現：A549經5’-Aza-2’-dC處理之後，HIC1基因啟動子會被去甲基化，且HIC1基因mRNA的表現量也會上升；而A549經SIRT1抑制劑Nicotinamide處理之後，SIRT1蛋白表現量在處理8小時候逐漸下漸，乙醯化p53及HIC1蛋白則在處理12小時後上升，進一步分析HIC1 mRNA表現情形，也在處理藥物12小時後上升。最後，本研究將HIC1變異情形、p53去乙醯化及SIRT1表現情況和臨床病歷資料做統計相關性分析，結果發現，在AD的病人中，HIC1啟動子過度甲基化頻率比SQ的病人來得明顯，統計上達邊緣顯著相關 (P＝0.059)，而在SQ病人中，則發現p53去乙醯化頻率比AD的病人來得明顯，且達統計上的相關 (P＝0.046)；因此我們推測：AD的病人其HIC1基因的變異可能傾向於是啟動子過度甲基化所造成，而SQ病人其HIC1基因的變異則可能傾向於是p53去乙醯化所導致。結論：本研究證實，HIC1變異情形確實在肺癌形成過程中扮演一個很重要的角色，其表現變異的機制主要透過啟動子過度甲基化及p53異常去乙醯化所致，我們的研究是第一篇在癌組織樣本中，同時探討HIC1/SIRT1/p53彼此之間分子調控關係參與癌症形成的研究。
Purpose: We have previously reported that the chromosomal regions at 17p13.3 showed a high frequency of loss of heterozygosity in tumors from non-small cell lung cancer (NSCLC) patients. HIC1 (hypermethylated in cancer 1) is a candidate tumor suppressor gene at 17p13.3 and is epigenetically inactivated in many human cancers. A circular regulation of HIC1, SIRT1 deacetylase, and p53 is proposed for modulation of cellular responses to DNA damage in cell and animal studies. However, the etiological role of HIC1 alteration and its correlation with p53 and SIRT1 deregulation have never been examined in the same series of human cancer patients. Therefore, we investigated the alterations of HIC1 at the DNA, RNA, and protein levels, and their correlation with deregulation of p53/SIRT-mediated control in lung cancer. Materials and Methods: The alterations of HIC1 including loss of protein/mRNA expression and promoter hypermethylation as well as their clinical correlations were examined in 108 NSCLC patients. In addition, the expression of acetylated p53 and SIRT1 deacetylase was examined by Western blot. We also treated A549 lung cancer cell line with demethylation reagent 5’aza 2’deoxycytidine (5’-Aza-2’-dC) and SIRT1 inhibitor nicotinamide (vitamin B3) to investigate the effects in the regulation of HIC1, p53 and SIRT1. Results: Overall, 65.5%, 75.9%, and 40% of NSCLC patients showed low protein, low mRNA, and promoter hypermethylation for HIC1 gene, respectively. A high concordance was observed between low mRNA expression and promoter hypermethylation for the HIC1 gene (P<0.05). Low mRNA expression of HIC1 was significantly associated with low acetylated p53, supporting that HIC1 is a transactivating target of p53 (P=0.016). In addition, low acetylated p53 is significantly associated with high SIRT1 protein (P=0.002), and high SIRT1 protein is significantly associated with low HIC1 protein (P=0.004). In addition, in the A549 lung cancer cells, which showed low levels of HIC1 mRNA and promoter hypermethylation, treated with demethylation reagent 5’-Aza-2’-dC, theHIC1 mRNA and protein expressions were restored along with the demethylation of HIC1 promoter. In the A549 cells treated with SIRT1 inhibitor nicotinamide, an increase of p53 acetylation and HIC1 re-expression were seen. In addition, promoter hypermethylation of HIC1 gene was tended to be associated with adenocarcinomas than with squamous carcinomas (P=0.059), whereas deacetylation of p53 was tended to be associated with squamous carcinomas than with adenocarcinomas (P=0.046). Conclusion: HIC1 alteration plays an important role in lung tumorigenesis and the predominant mechanisms of HIC inactivation were HIC1 promoter hypermethylation and p53 deacetylation. The present study shows the first clinical evidence that alteration of HIC1/SIR1/p53 pathway is involved in tumorigenesis.
Purpose: We have previously reported that the chromosomal regions at 17p13.3 showed a high frequency of loss of heterozygosity in tumors from non-small cell lung cancer (NSCLC) patients. HIC1 (hypermethylated in cancer 1) is a candidate tumor suppressor gene at 17p13.3 and is epigenetically inactivated in many human cancers. A circular regulation of HIC1, SIRT1 deacetylase, and p53 is proposed for modulation of cellular responses to DNA damage in cell and animal studies. However, the etiological role of HIC1 alteration and its correlation with p53 and SIRT1 deregulation have never been examined in the same series of human cancer patients. Therefore, we investigated the alterations of HIC1 at the DNA, RNA, and protein levels, and their correlation with deregulation of p53/SIRT-mediated control in lung cancer. Materials and Methods: The alterations of HIC1 including loss of protein/mRNA expression and promoter hypermethylation as well as their clinical correlations were examined in 108 NSCLC patients. In addition, the expression of acetylated p53 and SIRT1 deacetylase was examined by Western blot. We also treated A549 lung cancer cell line with demethylation reagent 5’aza 2’deoxycytidine (5’-Aza-2’-dC) and SIRT1 inhibitor nicotinamide (vitamin B3) to investigate the effects in the regulation of HIC1, p53 and SIRT1. Results: Overall, 65.5%, 75.9%, and 40% of NSCLC patients showed low protein, low mRNA, and promoter hypermethylation for HIC1 gene, respectively. A high concordance was observed between low mRNA expression and promoter hypermethylation for the HIC1 gene (P<0.05). Low mRNA expression of HIC1 was significantly associated with low acetylated p53, supporting that HIC1 is a transactivating target of p53 (P=0.016). In addition, low acetylated p53 is significantly associated with high SIRT1 protein (P=0.002), and high SIRT1 protein is significantly associated with low HIC1 protein (P=0.004). In addition, in the A549 lung cancer cells, which showed low levels of HIC1 mRNA and promoter hypermethylation, treated with demethylation reagent 5’-Aza-2’-dC, theHIC1 mRNA and protein expressions were restored along with the demethylation of HIC1 promoter. In the A549 cells treated with SIRT1 inhibitor nicotinamide, an increase of p53 acetylation and HIC1 re-expression were seen. In addition, promoter hypermethylation of HIC1 gene was tended to be associated with adenocarcinomas than with squamous carcinomas (P=0.059), whereas deacetylation of p53 was tended to be associated with squamous carcinomas than with adenocarcinomas (P=0.046). Conclusion: HIC1 alteration plays an important role in lung tumorigenesis and the predominant mechanisms of HIC inactivation were HIC1 promoter hypermethylation and p53 deacetylation. The present study shows the first clinical evidence that alteration of HIC1/SIR1/p53 pathway is involved in tumorigenesis.