Low expression of ITIH5 in adenocarcinoma of the lung is associated with unfavorable patients' outcome

Inter-α-trypsin inhibitor heavy chain 5 (ITIH5) is supposed to be involved in extracellular matrix stability and thus may play a key role in the inhibition of tumor progression. The current study is the first to analyze in depth ITIH5 expression and DNA methylation, as well as its potential clinical impact in non-small-cell lung carcinoma (NSCLC). We examined ITIH5 mRNA expression in tumor and adjacent normal lung tissue specimens of NSCLC patients. In addition, methylation frequency of the ITIH5 promoter was investigated using methylation-specific PCR and pyrosequencing. Significance of our data was validated by independent data sets from The Cancer Genome Atlas and the Kaplan-Meier Plotter platform. Furthermore, ITIH5 protein expression was evaluated by immunohistochemistry utilizing a tissue microarray with 385 distinct lung tissue samples. Based on our tissue collections, ITIH5 mRNA expression was significantly decreased in NSCLC compared to normal lung tissue in line with an increased methylation frequency in lung cancer tissue. Independent TCGA data confirmed significant expression loss of ITIH5 in lung cancer concordant with ITIH5 promoter hypermethylation in NSCLC. Of interest, low ITIH5 mRNA expression was particularly found in the magnoid and squamoid ADC expression subtype, concordant with an unfavorable patients'' outcome in squamoid as well as tobacco smoking ADC patients. In conclusion, ITIH5 may be a novel putative tumor suppressor gene in NSCLC with a potential molecular significance in the squamoid ADC subtype and further clinical impact for risk stratification of adenocarcinoma patients. In addition, ITIH5 may serve as a novel biomarker for prognosis of tobacco smoking ADC patients.     

Epigenetic inactivation of the novel candidate tumor suppressor gene ITIH5 in colon cancer predicts unfavorable overall survival in the CpG island methylator phenotype

Inter-α-trypsin inhibitor heavy chain 5 (ITIH5) is supposed to be involved in extracellular matrix stability and thus may play a key role in the inhibition of tumor progression. The current study is the first to analyze in depth ITIH5 expression as well as its potential clinical and functional impact in colon cancer. Based on 30 tumor and 30 adjacent normal tissues we examined ITIH5 mRNA expression and promoter methylation, whose significance was further validated by independent data sets from The Cancer Genome Atlas (TCGA) platform. In addition, ITIH5 protein expression was evaluated using immunohistochemistry. ITIH5 mRNA expression loss was significantly associated (P < 0.001) with hypermethylation of the ITIH5 promoter in primary colon tumors. In addition, treatment of tumor cell lines with demethylating (DAC) and histone acetylating (TSA) agents induced ITIH5 expression. In line, independent TCGA data revealed a significant expression loss of ITIH5, particularly in the MSI-high and CIMP-positive phenotype concordant with an increased ITIH5 hypermethylation in CIMP-positive colon tumors (P < 0.001). In proximal, i.e., right-sided tumors, abundant ITIH5 expression was associated with longer overall survival (OS, P = 0.049) and the CIMP-positive (P = 0.032) subgroup. Functionally, ITIH5 re-expression mediated a reduced proliferation in HCT116 and CaCo2 cells. In conclusion, our results indicate that ITIH5 is a novel putative tumor suppressor gene in colon cancer with a potential impact in the CIMP-related pathway. ITIH5 may serve as a novel epigenetic-based diagnostic biomarker with further clinical impact for risk stratification of CIMP-positive colon cancer patients.     

Epigenetic inactivation of the novel candidate tumor suppressor gene ITIH5 in colon cancer predicts unfavorable overall survival in the CpG island methylator phenotype

Inter-α-trypsin inhibitor heavy chain 5 (ITIH5) is supposed to be involved in extracellular matrix stability and thus may play a key role in the inhibition of tumor progression. The current study is the first to analyze in depth ITIH5 expression as well as its potential clinical and functional impact in colon cancer. Based on 30 tumor and 30 adjacent normal tissues we examined ITIH5 mRNA expression and promoter methylation, whose significance was further validated by independent data sets from The Cancer Genome Atlas (TCGA) platform. In addition, ITIH5 protein expression was evaluated using immunohistochemistry. ITIH5 mRNA expression loss was significantly associated (P < 0.001) with hypermethylation of the ITIH5 promoter in primary colon tumors. In addition, treatment of tumor cell lines with demethylating (DAC) and histone acetylating (TSA) agents induced ITIH5 expression. In line, independent TCGA data revealed a significant expression loss of ITIH5, particularly in the MSI-high and CIMP-positive phenotype concordant with an increased ITIH5 hypermethylation in CIMP-positive colon tumors (P < 0.001). In proximal, i.e., right-sided tumors, abundant ITIH5 expression was associated with longer overall survival (OS, P = 0.049) and the CIMP-positive (P = 0.032) subgroup. Functionally, ITIH5 re-expression mediated a reduced proliferation in HCT116 and CaCo2 cells. In conclusion, our results indicate that ITIH5 is a novel putative tumor suppressor gene in colon cancer with a potential impact in the CIMP-related pathway. ITIH5 may serve as a novel epigenetic-based diagnostic biomarker with further clinical impact for risk stratification of CIMP-positive colon cancer patients.     

ZIC1 is downregulated through promoter hypermethylation in gastric cancer

As one of major epigenetic changes responsible for tumor suppressor gene inactivation in the development of cancer, promoter hypermethylation was proposed as a marker to define novel tumor suppressor genes. In the current study we identified ZIC1 (Zic family member 1, odd-paired Drosophila homolog) as a novel tumor suppressor gene silenced through promoter hypermethylation in gastric cancer, the second leading cause of cancer death worldwide. In all of gastric cancer cells lines examined, ZIC1 expression was downregulated and such downregulation was accompanied with the hypermethylation of ZIC1 promoter. Demethylation treatment with 5-aza-2′-deoxycytidine (Aza) reversed ZIC1 downregulation, highlighting the importance of promoter methylation to ZIC1 downregulation in gastric cancer cells. Notably, ZIC1 expression was significantly downregulated in primary gastric carcinoma tissues in comparison with non-tumor adjacent gastric tissues (p < 0.01). Accordingly, promoter methylation of ZIC1 was frequently detected in primary gastric carcinoma tissues (94.6%, 35/37) but not normal gastric tissues, indicating that promoter hypermethylation mediated ZIC1 downregulation may play an important role in gastric carcinogenesis. Indeed, ectopic expression of ZIC1 led to the growth inhibition of gastric cancer cells through the induction of S-phase cell cycle arrest (p < 0.01). Our results revealed ZIC1 as a novel candidate tumor suppressor gene downregulated through promoter hypermethylation in gastric cancer.     

Polymerase chain reaction-based methods of DNA methylation analysis

DNA methylation is the main epigenetic modification in humans, and changes in methylation patterns play an important role in tumorigenesis. Hypermethylation of normally unmethylated CpG islands in the promoter regions often occurs in important tumor suppressor genes, DNA repair genes, and metastasis inhibitor genes. The changes of methylation status of various gene promoters seem to be a common feature of malignant cells and these changes can occur early in the progression process. Therefore detection of aberrant promoter hypermethylation of cancer-related genes may be useful for cancer diagnosis or detection of cancer recurrence. The purpose of this review is to provide a summary of the most commonly used techniques for the study of DNA methylation. Current scientific literature involving methylation detection methods was reviewed with an emphasis on polymerase chain reaction (PCR)-based detection methods. The current methodologies may be broadly classed into PCR-based methylation assays and non-PCR-based methylation assays. The problems and advantages of the different methods for detecting aberrant methylation are discussed. As the number of genes known to be hypermethylated in cancer is growing, the detection of aberrant promoter region methylation will be a promising approach for using DNA-based markers for the early detection of human cancers. Many techniques, especially PCR-based methylation assay techniques, make it practical to use these new methylation biomarkers in early cancer diagnosis.     

p16基因甲基化状态与散发性大肠癌的相关性研究

为探讨p1 6基因甲基化状态与散发性大肠癌发生发展的关系 ,用甲基化特异性的聚合酶链反应 (methylati omspecificPCR ,MSP)结合测序检测散发性大肠癌及相应癌旁组织p1 6基因甲基化状态。研究发现p1 6基因在散发性大肠癌中甲基化率为 2 8 9% (1 3 4 5 ) ,有 8例癌及癌旁组织都发生了甲基化 ;有淋巴结及远处转移的甲基化率为5 0 % (8 1 6 ) ,高于无转移的甲基化率 2 0 8(5 2 4 ) (P <0 0 5 )。p1 6基因高甲基化是散发性大肠癌中常见的分子改变之一 ,大肠癌中p1 6基因高甲基化可能发生在癌变早期并与大肠癌的恶性进展有相关性     

HOP/OB1/NECC1 promoter DNA is frequently hypermethylated and involved in tumorigenic ability in esophageal squamous cell carcinoma

Promoter DNA hypermethylation with gene silencing is a common feature of human cancer, and cancer-prone methylation is believed to be a landmark of tumor suppressor genes (TSG). Identification of novel methylated genes would not only aid in the development of tumor markers but also elucidate the biological behavior of human cancers. We identified several epigenetically silenced candidate TSGs by pharmacologic unmasking of esophageal squamous cell carcinoma (ESCC) cell lines by demethylating agents (5-aza-2'-deoxycitidine and trichostatin A) combined with ESCC expression profiles using expression microarray. HOP/OB1/NECC1 was identified as an epigenetically silenced candidate TSG and further examined for (a) expression status, (b) methylation status, and (c) functional involvement in cancer cell lines. (a) The HOP gene encodes two putative promoters (promoters A and B) associated with two open reading frames (HOPalpha and HOPbeta, respectively), and HOPalpha and HOPbeta were both down-regulated in ESCC independently. (b) Promoter B harbors dense CpG islands, in which we found dense methylation in a cancer-prone manner (55% in tumor tissues by TaqMan methylation-specific PCR), whereas promoter A does not harbor CpG islands. HOPbeta silencing was associated with DNA methylation of promoter B in nine ESCC cell lines tested, and reactivated by optimal conditions of demethylating agents, whereas HOPalpha silencing was not reactivated by such treatments. Forced expression of HOP suppressed tumorigenesis in soft agar in four different squamous cell carcinoma cell lines. More convincingly, RNA interference knockdown of HOP in TE2 cells showed drastic restoration of the oncogenic phenotype. In conclusion, HOP is a putative TSG that harbors tumor inhibitory activity, and we for the first time showed that the final shutdown process of HOP expression is linked to promoter DNA hypermethylation under the double control of the discrete promoter regions in cancer.     

Methylation of E-cadherin and hMLH1 genes in Indian sporadic breast carcinomas

Hypermethylation of promoter regions leading to inactivation of tumor suppressor genes is a common event in the progression of several tumor types. We have employed a novel restriction digestion based multiplex PCR assay to analyse the methylation status of promoter regions of tumor suppressor genes (p16, hMLH1, MGMT and E-cadherin) in sporadic breast carcinomas of Indian women. The present results indicated the absence of hypermethylation in promoter region of p16 and MGMT genes. However, 6 of the 19 (31.6%) sporadic breast carcinomas showed hypermethylation in the promoters of two of the genes analysed; three in hMLH1 and another three in E-cad. Since our earlier studies have shown lack of genetic alterations such as missense mutations and deletions in the tumor associated genes-p16, ras and p14ARF in sporadic breast tumors, the epigenetic alterations of the two genes reported in the present study could be of interest and might be among the events in the genesis/progression of sporadic breast carcinomas.     

小鼠骨髓细胞中p16和Ralb基因启动区CpG岛的甲基化位点分析

抑癌基因p16和白血病致癌因子Ralb与白血病的发生密切相关,其启动子区CpG岛的甲基化对基因表达具有重要作用.本文旨在分析p16、Ralb基因启动子区CpG岛甲基化位点信息,并比较这两个基因在小鼠骨髓细胞和原代培养的骨髓细胞中甲基化状态的差异.运用"MethPrimer"软件预测p16、Ralb基因启动子区的CpG岛,设计甲基化特异性引物.利用重亚硫酸盐测序法(BSP)检测甲基化位点信息.结果显示,p16有1个CpG岛,岛上21个CpG位点全部未发生甲基化;Ralb有2个CpG岛,CpG岛1上的5个CpG位点全部呈甲基化状态,而CpG岛2上的17个CpG位点全部呈非甲基化状态,且小鼠骨髓细胞和体外原代培养的骨髓细胞中两基因的甲基化状态一致.表明p16、Ralb基因甲基化状态未受外界培养条件的影响而改变,提示在与两基因甲基化相关的研究中体外试验可替代体内试验.