DNA methylation in hepatocellular carcinoma

As for many other tumors, development of hepatocellular carcinoma （HCC） must be understood as a multistep process with accumulation of genetic and epigenetic alterations in regulatory genes, leading to activation of oncogenes and inactivation or loss of tumor suppressor genes （TSG）. In the last decades, in addition to genetic alterations, epigenetic inactivation of （tumor suppressor） genes by promoter hypermethylation has been recognized as an important and alternative mechanism in tumorigenesis. In HCC, aberrant methylation of promoter sequences occurs not only in advanced tumors, it has been also observed in premalignant conditions just as chronic viral hepatitis B or C and cirrhotic liver. This review discusses the epigenetic alterations in hepatocellular carcinoma focusing DNA methylation.     

Epigenetic Inactivation of Tumor Suppressor Genes in Hematologic Malignancies

A number of genetic alterations are involved in the development of hematologic malignancies. These alterations include the activation of oncogenes by chromosomal translocation or gene amplification and the inactivation of tumor suppressor genes by gene deletion or mutations. Recently, epigenetic change has been proven to be another important means of inactivating tumor suppressor genes in tumor cells, and hypermethylation of promoter DNA is one of the most important mechanisms. In hematologic malignancies, many kinds of tumor suppressor genes and candidate suppressor genes are epigenetically inactivated. Inactivation of tumor suppressor genes usually occurs in a disease-specific manner and plays important roles in the development and progression of the disease. Some of these alterations have clinical effects on treatment results or the prognoses of the patients.     

Diagnosis and management of cholangiocarcinomas: a comprehensive review

First described by Maximilian deStoll in 1777, cholangiocarcinoma remains to be a diagnostic and therapeutic challenge. The incidence of cholangiocarcinoma is of 2000-3000 cases a year in the US but much higher in Southeast Asia and Eastern Europe. Sclerosing cholangitis, ulcerative colitis, liver fluke infestations, hepatolithiasis, thorotrast exposure, choledochal cysts are associated with an increased risk. Recently, substantial progress has been achieved in the understanding of the cellular and molecular mechanisms playing a role in biliary carcinogenesis including oncogenes and tumor suppressor genes. Surgery alone can be a curative treatment in only a fraction of patients with a 5-year survival rate of 0-39%. The role of radiotherapy including intensification with intraoperative radiation therapy, brachytherapy or conformal external beam radiation therapy has not been proven. Experience with chemotherapy is not widespread in cholangiocarcinoma. Chemotherapy trials have mostly included small numbers of patients and there have been no large prospective randomized trials. Observed objective response rates have been between 0% and 40% with no complete remissions. Further studies are required to find out the impact of chemotherapy in the management of cholangiocarcinoma and search for newer agents with novel mechanisms of action that can be more active in cholangiocarcinoma should continue. Meanwhile prevention and early diagnosis of cholangiocarcinoma is essential. Efforts should also continue to discover means to detect high-risk patients with less invasive methods.     

Allelic loss of chromosomes 16q and 10q in human prostate cancer.

Recent advances in understanding the molecular genetics of common adult tumors have indicated that multiple genetic alterations including the activation of oncogenes and the inactivation of tumor suppressor genes are important in the pathogenesis of these tumors. Loss of heterozygosity is a hallmark of tumor suppressor gene inactivation and has been used to identify chromosomal regions that contain these genes. We have examined allelic loss in the most common tumor in men, prostate cancer. Twenty-eight prostate cancer specimens have been examined for loss of heterozygosity at 11 different chromosomal arms including 3p, 7q, 9q, 10p, 10q, 11p, 13q, 16p, 16q, 17p, and 18q. Fifty-four percent (13/24) of clinically localized tumors and 4 of 4 metastatic tumors showed loss of heterozygosity on at least one chromosome. Chromosomes 16q and 10q exhibited the highest frequency of loss of heterozygosity with 30% of tumors showing loss at these chromosomes. These data demonstrate that allelic loss is a common event in prostate cancer and suggest that chromosomes 16q and 10q may contain the sites of tumor suppressor genes important in the pathogenesis of human prostate cancer.     

Promoter methylation and loss of coding exons of the fragile histidine triad (FHIT) gene in intrahepatic cholangiocarcinomas.

AIMS: About 10-30% of primary liver cancers represent intrahepatic cholangiocarcinomas (IHCC). Since chromosomal losses of 3p are detectable in about 40% of cholangiocarcinomas our study aimed at the identification of mechanisms leading to functional deletion of tumor suppressor genes in this region. Our efforts focussed on genomic losses and epigenetic inactivation of two tumor suppressor genes, the fragile histidine triad (FHIT) and the ras association domain family 1 (RASSF1A) genes, both located on the short arm of chromosome 3. METHODS: Methylation-specific PCR (MSP) and combined bisulfite-dependent restriction analysis (COBRA) were applied to detect epigenetic silencing of gene promoters. Genomic duplex PCR was used to identify exon losses of the FHIT gene. Nineteen paraffin-embedded samples of intrahepatic cholangiocarcinomas were studied. RESULTS: Here we report for the first time that in addition to frequent losses of the exons 5 and 6, hypermethylation of the FHIT promoter occured in a significant portion of IHCC. Methylation specific PCR (MSP) detected epigenetic inactivation of the FHIT/FRA3B locus in 8 of 19 (42%) cases. Combined bisulfite restriction analysis (COBRA) revealed that high levels of methylated FHIT promoter sequences were present in 6 of the 8 methylation positive samples. In agreement with previous reports MSP identified hypermethylation of the RASSF1A gene in 13 of 19 (68%) IHCC specimens examined. CONCLUSIONS: Epigenetic silencing of the FHIT tumor suppressor gene is a novel inactivation mechanism to be considered in the development of intrahepatic cholangiocarcinomas. However, a statistically significant inverse correlation between K-Ras activation and RASSF1A inactivation was not found.     

VHL and PTEN loss coordinate to promote mouse liver vascular lesions

Von Hippel-Lindau (VHL) inactivation develops a tumor syndrome characterized by highly vascularized tumors as a result of hypoxia inducible factors (HIF) stabilization. The most common manifestation is the development of hemangioblastomas typically located in the central nervous system and other organs including the liver. PTEN (Phosphatase and tension homologue deleted on chromosome 10) inactivation also upregulates HIF-1α and may take part in promoting vascular lesions in tumors. The coordinate effect of loss of these tumor suppressors on HIF levels, and the subsequent effect on vascular lesion formation would elucidate the potential for mechanisms to modify HIF dosage supplementally and impact tumor phenotype. We therefore employed models of somatic conditional inactivation of Vhl, Pten, or both tumor suppressor genes in individual cells of the liver by Cre-loxP recombination to study the cooperativity of these two tumor suppressors in preventing tumor formation. Nine months after tumor suppressor inactivation, Vhl conditional deletion (Vhl ^loxP/loxP) mice showed no abnormalities, Pten conditional deletion (Pten ^loxP/loxP) mice developed liver steatosis and focal nodular expansion of hepatocytes containing lipid droplet and fat. Vhl and Pten conditional deletion (Vhl ^loxP/loxP;Pten ^loxP/loxP) mice, however, developed multiple cavernous liver lesions reminiscent of hemangioblastoma. Liver hemangioblastomas in VHL disease may, therefore, require secondary mutation in addition to VHL loss of heterozygosity which is permissive for vascular lesion development or augments levels of HIF-1α.     

Genetics in secondary hyperparathyroidism of uremia

Little is known about genetic alterations in parathyroid hyperplasias in uremic patients. Although overexpression of cyclin D1 and inactivation of MEN1 gene are found in subsets of common parathyroid adenomas, specific oncogenes or tumor suppressor genes contributing to monoclonal uremic hyperparathyroidism have not yet been identified. On the other hand, some candidate gene polymorphisms have been suggested to play a key role in the pathogenesis of this disease.     

MicroRNA和DNA甲基化在肺癌诊断中的研究进展

小分子核糖核酸（MicroRNA）作为肺癌早期的一种新的生物标志物,为肿癌的诊断提供了一种新的方法。MicroRNA在肿瘤发生发展过程中发挥癌基因和抑癌基因的作用。MicroRNA与肺癌增殖、侵袭、治疗、预后和复发有着密切关系。DNA甲基化是目前最主要的一种表观遗传修饰形式,抑癌基因的高甲基化使DNA染色质构象发生改变导致转录失活,引起肿瘤发生。DNA甲基化和MicroRNA在功能上相互调控,对肺癌的发生、发展同样重要。     

Frequency of loss of heterozygosity on chromosome 17 in intrahepatic cholangiocarcinoma]

BACKGROUNDS/AIM: Intrahepatic cholangiocarcinoma is the second most common intrahepatic neoplasm. Carcinogenesis is believed to be a multistage process that occurs as a result of mutations in oncogenes and tumor suppressor genes. Loss of heterozygosity (LOH) is the phenotype of genetic instability which has been used as a tool for detecting genetic phenotype alterations. Large number of the molecular alterations have been described in human cancer. Among them, that of p53 is quite common. The aim of this study was to determine the frequency of LOH at chromosome 17p related with p53. METHODS: Twenty cases who underwent hepatic resection due to intrahepatic cholangiocarcinoma, were included. LOH was analysed with four microsatellite markers by PCR. For the clinicopathologic parameters, tumor size, differentiation, and metastasis were evaluated. RESULTS: Fifteen patients (75%) showed LOH at one of the loci at the least. Five patients were LOH-high and 10 were LOH-low. The highest frequency of LOH was observed at D17S5 by 38.9%. Those of TP53, D17S796 and D17S513 were 29.4%, 21.4% and 35.3%, respectively. In addition, LOH tended to be more frequent when the tumor is mass forming type, poorly differentiated, or has tumor emboli or vascular invasion. CONCLUSIONS: This study showed that LOH was positive in 75% on chromosome 17p in intrahepatic cholangiocarcinoma which was relatively frequent at D17S5.     

Detailed deletion mapping of loss of heterozygosity on 22q13 in sporadic colorectal cancer

AIM: Both development and progression of malignancies occur as a multistep process, requiring the activation of oncogenes and the inactivation of several tumor suppressor genes. The loss of heterozygosity (LOH) of tumor suppressor genes is believed to play a key role in carcinogenesis of colorectal cancer (CRC). In this study, we analyzed the LOH of seven loci on chromosome 22q13 in an effort to identify candidate tumor suppressor genes involved in colorectal carcinogenesis. METHODS: Matched tumor and normal tissue DNA were analyzed by PCR using fluorescence-labeled polymorphic microsatellite markers in 83 CRC patients. PCR products were eletrophoresed and LOH was determined by calculating the peak height acquired through computer software. Comparisons between LOH frequency and clinicopathological features were performed by x2 test. P<0.05 was considered as statistical significance. RESULTS: The average LOH frequency of chromosome 22q13 was 28.38%. The highest LOH frequency was 64.71% on D22S1160 locus, and the lowest was 21.43% on D22S1141 locus. We detected two obvious minimal deletion regions: one between markers D22S1171 and D22S274, the other flanked by markers D22S1160 and D22S1149, each about 2.7 and 1.8 cm, respectively. None had lost in all informative loci. LOH frequency on D22S1171 is 50% on distal colon, which was higher than that on proximal one (P= 0.020); on D22S114 locus, none LOH event occurred in patients with liver metastasis, whilst 46.94% occurred in patients without liver metastasis (P= 0.008); on D22S1160 locus, LOH frequency in lymph nodes metastasis patients was 83.33%, which was much higher than 43.75% without lymph nodes metastasis ones (P= 0.016). There was no statistical significance between clinicopathological features and other loci. CONCLUSION: This study provides evidence of two minimal deletion regions, which may harbor putative tumor suppressor genes related to progression and metastasis in sporadic colorectal carcinoma on chromosome 22q13.     

Frequent loss of chromosome 3p and hypermethylation of RASSF1A in cholangiocarcinoma

BACKGROUND/AIMS: Cholangiocarcinoma comprises 5-20% of all primary malignant tumors of the liver. However, the lack of information about the genetic basis of cholangiocarcinoma has impeded characterization and understanding of its biological behavior. METHODS: In this study, genome-wide aberrations in 13 cases of cholangiocarcinoma were examined by the molecular cytogenetic technique, comparative genomic hybridization. RESULTS: Frequent gains of 1q, 3q, 8q, 15q and 17q, and common losses on 3p, 4q, 6q, 9p, 17p and 18q were found. The finding of common chromosome 3p loss (approximately 40%) with a minimal deleted region of 3p13-p21 has prompted our further investigation on the tumor suppressor gene RASSF1A, located at 3p21.3. Using bisulphite modification followed by methyl-specific PCR, a high incidence of methylated RASSF1A promoter region was detected in our current series (9/13 cases; 69%). Further expression analysis on the nine cases with promoter hypermethylation indicated much reduced RASSF1A expression compared to normal livers. CONCLUSIONS: Our current molecular cytogenetic investigation on primary cholangiocarcinoma provided overall karyotypic information and represents the first report on the methylation status of RASSF1A in cholangiocarcinoma. The high incidence of 3p loss and RASSF1A promoter hypermethylation detected may have implications for this tumor suppressor gene in the malignant progression of cholangiocarcinoma.     

Perihilar cholangiocarcinoma arising in hepatitis C virus-related liver cirrhosis with hepatocellular carcinoma

Liver cirrhosis is reportedly one of the conditions preceding peripheral-type intrahepatic cholangiocarcinoma but not hilar/perihilar cholangiocarcinoma. Herein, we report a case of perihilar cholangiocarcinoma arising in a hepatitis C virus-related cirrhotic liver. The patient was a 69-year-old man. He was diagnosed with hepatitis C virus-related chronic hepatitis at the age of 56 years, and 9 years later, multiple hepatocellular carcinomas were detected by imaging modalities. Despite treatments, including chemotherapy, he died of hepatic failure at the age of 69 years. At autopsy, in addition to multiple nodules of hepatocellular carcinoma, we found a white mucinous and fibrous tumor spreading from the hepatic hilum to the periphery along the left lateral segmental bile ducts in the advanced cirrhotic liver. This tumor was histologically a cholangiocarcinoma that involved mainly the peribiliary glands and showed variable cystic dilation, suggesting that it might have been derived from these peribiliary glands. Immunohistochemically, the cholangiocarcinoma cells were positive for cytokeratin 7 and mucin core protein 1, and negative for cytokeratin 20 and mucin core protein 2. Hilar/perihilar cholangiocarcinoma arising in hepatitis C virus-related liver cirrhosis has rarely been reported. This case warrants further studies to clarify the possible involvement of hepatitis C virus in tumorigenesis of hilar/perihilar cholangiocarcinoma.     

Tissue factor pathway inhibitor-2 as a frequently silenced tumor suppressor gene in hepatocellular carcinoma

In HCC, inactivation of tumor suppressor genes plays a significant role in carcinogenesis. Apart from deletions and mutations, growing evidence has indicated that epigenetic alterations including aberrant promoter methylation and histone deacetylation are also implicated in inactivation of tumor suppressor genes. The goal of this study was to identify epigenetically silenced candidate tumor suppressor genes in human HCC by comparing the changes in oligonucleotide microarray gene expression profiles in HCC cell lines upon pharmacological treatment with the demethylating agent 5-Aza-2'-deoxycytidine (5-Aza-dC). By analyzing the gene expression profiles, we selected tissue factor pathway inhibitor-2 (TFPI-2), a Kunitz-type serine protease inhibitor, for validation and further characterization. Our results showed that TFPI-2 was frequently silenced in human HCC and HCC cell lines. TFPI-2 was significantly underexpressed in approximately 90% of primary HCCs when compared with their corresponding nontumorous livers. TFPI-2 promoter methylation was detected in 80% of HCC cell lines and 47% of human HCCs and was accompanied by reduced TFPI-2 messenger RNA expression. In addition, TFPI-2 expression in HCC cell lines can be robustly restored by combined treatment with 5-Aza-dC and histone deacetylase inhibitor trichostatin A. These findings indicate that TFPI-2 is frequently silenced in human HCC via epigenetic alterations, including promoter methylation and histone deacetylation. Moreover, ectopic overexpression of TFPI-2 significantly suppressed the proliferation and invasiveness of HCC cells. CONCLUSION: Our findings suggest that TFPI-2 is a candidate tumor suppressor gene in human HCC.     

Receptor protein-tyrosine phosphatase gamma is a candidate tumor suppressor gene at human chromosome region 3p21.

PTPG, the gene for protein-tyrosine phosphatase gamma (PTP gamma), maps to a region of human chromosome 3, 3p21, that is frequently deleted in renal cell carcinoma and lung carcinoma. One of the functions of protein-tyrosine phosphatases is to reverse the effect of protein-tyrosine kinases, many of which are oncogenes, suggesting that some protein-tyrosine phosphatase genes may act as tumor suppressor genes. A hallmark of tumor suppressor genes is that they are deleted in tumors in which their inactivation contributes to the malignant phenotype. In this study, one PTP gamma allele was lost in 3 of 5 renal carcinoma cell lines and 5 of 10 lung carcinoma tumor samples tested. Importantly, one PTP gamma allele was lost in three lung tumors that had not lost flanking loci. PTP gamma mRNA was expressed in kidney cell lines and lung cell lines but not expressed in several hematopoietic cell lines tested. Thus, the PTP gamma gene has characteristics that suggest it as a candidate tumor suppressor gene at 3p21.     

Transforming growth factor-beta signaling promotes hepatocarcinogenesis induced by p53 loss

Hepatocellular carcinoma (HCC) results from the accumulation of deregulated tumor suppressor genes and/or oncogenes in hepatocytes. Inactivation of TP53 and inhibition of transforming growth factor-beta (TGF-β) signaling are among the most common molecular events in human liver cancers. Thus, we assessed whether inactivation of TGF-β signaling, by deletion of the TGF-β receptor, type II (Tgfbr2), cooperates with Trp53 loss to drive HCC formation. Albumin-cre transgenic mice were crossed with floxed Trp53 and/or floxed Tgfbr2 mice to generate mice lacking p53 and/or Tgfbr2 in the liver. Deletion of Trp53 alone (Trp53(KO) ) resulted in liver tumors in approximately 41% of mice by 10 months of age, whereas inactivation of Tgfbr2 alone (Tgfbr2(KO) ) did not induce liver tumors. Surprisingly, deletion of Tgfbr2 in the setting of p53 loss (Trp53(KO) ;Tgfbr2(KO) ) decreased the frequency of mice with liver tumors to around 17% and delayed the age of tumor onset. Interestingly, Trp53(KO) and Trp53(KO) ;Tgfbr2(KO) mice develop both HCC and cholangiocarcinomas, suggesting that loss of p53, independent of TGF-β, may affect liver tumor formation through effects on a common liver stem cell population. Assessment of potential mechanisms through which TGF-β signaling may promote liver tumor formation in the setting of p53 loss revealed a subset of Trp53(KO) tumors that express increased levels of alpha-fetoprotein. Furthermore, tumors from Trp53(KO) mice express increased TGF-β1 levels compared with tumors from Trp53(KO) ;Tgfbr2(KO) mice. Increased phosphorylated Smad3 and ERK1/2 expression was also detected in the tumors from Trp53(KO) mice and correlated with increased expression of the TGF-β responsive genes, Pai1 and Ctgf. CONCLUSION: TGF-β signaling paradoxically promotes the formation of liver tumors that arise in the setting of p53 inactivation.     

Homozygous deletions localize novel tumor suppressor genes in B-cell lymphomas

Integrative genomic and gene-expression analyses have identified amplified oncogenes in B-cell non-Hodgkin lymphoma (B-NHL), but the capability of such technologies to localize tumor suppressor genes within homozygous deletions remains unexplored. Array-based comparative genomic hybridization (CGH) and gene-expression microarray analysis of 48 cell lines derived from patients with different B-NHLs delineated 20 homozygous deletions at 7 chromosome areas, all of which contained tumor suppressor gene targets. Further investigation revealed that only a fraction of primary biopsies presented inactivation of these genes by point mutation or intragenic deletion, but instead some of them were frequently silenced by epigenetic mechanisms. Notably, the pattern of genetic and epigenetic inactivation differed among B-NHL subtypes. Thus, the P53-inducible PIG7/LITAF was silenced by homozygous deletion in primary mediastinal B-cell lymphoma and by promoter hypermethylation in germinal center lymphoma, the proapoptotic BIM gene presented homozygous deletion in mantle cell lymphoma and promoter hypermethylation in Burkitt lymphoma, the proapoptotic BH3-only NOXA was mutated and preferentially silenced in diffuse large B-cell lymphoma, and INK4c/P18 was silenced by biallelic mutation in mantle-cell lymphoma. Our microarray strategy has identified novel candidate tumor suppressor genes inactivated by genetic and epigenetic mechanisms that substantially vary among the B-NHL subtypes.     

大肠癌与抑癌基因相关性的研究现状

大肠癌是常见的高危害消化系恶性肿瘤,全球每年新发病例约为120万例.近年来,随着人们生活水平的提高,饮食习惯和结构的改变,我国大肠癌的发病率和死亡率增长迅速,而且,发病年龄明显提前,目前,我国大肠癌中位发病年龄为58岁,比欧美等国家提前12-18年.大肠癌的发生是一个多阶段多步骤的、涉及多个基因改变的复杂过程.许多研究表明,结直肠癌变是一个涉及原癌基因激活、抑癌基因失活等多基因、多阶段、多步骤渐进演化的积累过程.与结直肠癌相关的抑癌基因有p53、APC、DCC、MMR等,原癌基因有k-ras、c-myc等.本文就以上基因改变与大肠癌的发生发展相关性的研究现状作一简单复习.     

Hormonal signaling and pituitary adenomas

In recent years the demonstration that human pituitary adenomas are monoclonal in origin provides further evidence that pituitary neoplasia arise from the replication of a single mutated cell in which growth advantage results from either activation of proto-oncogenes or inactivation of tumor suppressor genes. Mutations in common oncogenes and tumor suppressor genes are only exceptionally involved in pituitary tumors. Since pituicytes may proliferate in response to hypothalamic neurohormones, locally produced growth factors and peripheral hormones, it has been speculated that dysregulation of the signaling molecules that constitute these pathways may confer growth advantage to the target cell, finally resulting in tumor formation. The only mutational change so far recognized to be unequivocally associated with pituitary tumors occur in the Gs alpha gene (GNAS1) and cause constitutive activation of the cAMP-dependent pathway. However, other components of pituitary-specific pathways are frequently altered in their expression and activity. This review will focus on the possible impact of G proteins and other components of hormone signaling on pituitary tumorigenesis.     

正常肝组织与肝癌组织差异表达基因消减cDNA文库的构建

目的 构建人正常肝组织与肝癌组织差异表达基因的消减cDNA文库。方法 采用新近建立的抑制消减杂交技术,以癌旁正常肝组织及肝癌组织作为对比材料,分离肝癌组织中不表达或低表达基因的cDNA片段,将其与T载体进行T/A连接构建文库,将连接产物用电穿孔法转化大肠杆菌进行文库扩增后,随机挑取100个白色克隆进行酶切鉴定。结果 扩增消减cDNA文库获得4000余个白色阳性克隆,随机挑取的100个白色克隆进行酶切鉴定。结果 扩增消减cDNA文库获得4000余个白色阳性克隆,随机挑取的100个白色克隆经酶切后均有200-600bp的插入片段。结论 用SSH法及T/A克隆技术成功构建了正常肝组织与肝癌组织差异表达基因的消减cDNA文库,该文库的建立为进一步筛选、克隆肝癌组织中失活或低表达的新的抑癌基因奠定了基因。