Hypermethylated In Cancer 1 Research Articles

Expression and promoter methylation of the hypermethylated in cancer 1 gene in papillary thyroid carcinoma

Objective To investigate the expression and promoter methylation of hypermethylated in cancer 1(HIC1) gene in papillary thyroid carcinoma and adjacent normal thyroid tissue, and its biological significance. Methods Bisulfite sequencing PCR(BSP), real-time fluorescent quantitative polymerase chain reaction(FQ-PCR) and Western blotting were used to analyze the promoter methylation and expression differences of HIC1 gene in tissues of papillary thyroid carcinoma(40 cases)and adjacent normal thyroid(40 cases). Results Compared with adjacent normal thyroid tissue (2.12±0.104, and 0.936±0.106), the expression of HIC1 m RNA and protein in papillary thyroid carcinoma was significantly decreased(0.47±0.07, and 0.417±0.071,P< 0.01). The expression of HIC1 m RNA and protein was lower in the group with lymph node metastasis(0.21±0.02, and 0.191±0.031) than in the group without lymph node metastasis(0.68±0.09, and 0.601±0.037, P<0.05).Further clinical analysis demonstrated significant association of the expression of HIC1 mRNA and protein with:age of patients(< 45 years old: 0.64±0.07, and 0.535±0.097;≥45 years old: 0.19±0.04, and 0.221±0.007), stage of disease(Ⅰ-Ⅱstage: 0.58±0.06, and 0.494±0.051;Ⅲ-Ⅳstage:0.21±0.02, and 0.237±0.023, P< 0.05).The rate of HIC1 gene promoter methylation was significantly higher(61.7%)than that in adjacent normal tissue(42.5%, P< 0.01). Conclusion Promoter hypermethylation of HIC1 in papillary thyroid carcinoma causes decreased expression of HIC1, which may be one of major reasons for the metastasis in papillary thyroid carcinoma. Key words: Hypermethylated in cancer 1; Papillary thyroid carcinoma; Methylation

Class II transactivator (CIITA) mediates transcriptional repression of pdk4 gene by interacting with hypermethylated in cancer 1 (HIC1).

Increased accumulation and/or impaired utilization of fatty acid in extra-adipose tissues are implicated in the pathogenesis of insulin resistance and type 2 diabetes. Pyruvate dehydrogenase kinase 4 (Pdk4) is a key enzyme involved in fatty oxidation and energy expenditure, and its expression can be repressed by pro-inflammatory stimuli. Previously, we have shown that class II transactivator (CIITA) mediates the adverse effect of interferon gamma (IFN-γ) in skeletal muscle cells by cooperating with hypermethylated in cancer 1 (HIC1) to repress silent information regulator 1 (SIRT1) transcription. Building upon this finding, we report here that CIITA interacted with HIC1 via the GTP-binding domain (GBD) while HIC1 interacted with CIITA via the BTB/POZ domain. The GBD domain was required for CIITA to repress SIRT1 transcription probably acting as a bridge for CIITA to bind to HIC1 and consequently to bind to the SIRT1 promoter. IFN-γ stimulation, CIITA over-expression, or HIC1 over-expression repressed Pdk4 promoter activity while silencing either CIITA or HIC1 normalized Pdk4 expression in the presence of IFN-γ. An increase in SIRT1 expression or activity partially rescued Pdk4 expression in the presence of CIITA, but SIRT1 inhibition abrogated Pdk4 normalization even in the absence of CIITA. Taken together, our data have identified a HIC1-CIITA-SIRT1 axis that regulates Pdk4 transcription in response to IFN-γ stimulation.

Expression of the tumor suppressor gene hypermethylated in cancer 1 in laryngeal carcinoma.

Hypermethylated in cancer 1 (HIC1) is a putative suppressor gene, cooperating with TP53 in the regulation of apoptosis. The promoter site of this gene contains CpG islands susceptible to methylation. Altered methylation leads to the silencing of HIC1. Persistent loss of HIC1 function reflects the attenuation of proapoptotic characteristics of TP53 and may constitute the background for carcinogenesis. Altered methylation profiles along with diminished expression of HIC1 were documented in a number of solid neoplasms. The aim of this study was to evaluate the expression of the HIC1 gene in laryngeal carcinoma. RNA was extracted from samples of laryngeal cancer and corresponding healthy tissues of 21 patients with advanced laryngeal cancer (T3-T4). The amount of RNA (cDNA) was evaluated using reverse transcription-quantitative polymerase chain reaction with GADPH as the reference gene. Data demonstrated that HIC1 expression was significantly reduced in laryngeal cancer tissues. The relative expression of HIC1 was found to be ~40% lower in tumor samples compared to that in healthy controls. The median tumor/normal tissue ratio for HIC1 was 0.615. These results suggest that low HIC1 expression may be associated with neoplastic transformation in the larynx.

Reactivation of HIC-1 gene by saRNA inhibits clonogenicity and invasiveness in breast cancer cells.

Hypermethylated in cancer 1 (HIC-1) is a tumor suppressor gene, which is epigenetically silenced in breast cancer. It is known that the loss of HIC-1, caused by promoter hypermethylation, is associated with tumor aggression and poor survival in breast carcinoma. It has been shown that small activating RNA (saRNA) targeting promoter sequences may induce gene re-expression. In the current study, saRNA was used to restore HIC-1 expression, and the effects on colony formation, invasiveness and the cell cycle in breast cancer cells were explored. dsHIC1-2998, an saRNA, exhibited activating efficacy on MCF-7 and MDA-MB-231 cancer cell lines. A clonogenicity assay showed that evident colony inhibition was induced via saRNA-mediated re-expression of HIC-1 in the two cancer cell lines. Reactivation of HIC-1 significantly inhibited cell migration and invasion, resulting in G0/G1 cell cycle arrest in these cell lines. These findings suggest that HIC-1 may be a potential target in gene therapy for the treatment of breast cancer. saRNA may function as a therapeutic option for upregulating tumor suppressor genes in breast cancer.

Molecular cloning and expression of high GC-rich novel tumor suppressor gene HIC-1.

Hypermethylated in Cancer-1 (HIC-1) is a novel tumor suppressor plays crucial role in tumor formation through loss of function by hypermethylation. HIC-1 is known as transcriptional factor whereas little known about its structure and function. Requirement felt to clone and express full coding protein and reveal various domains and binding pattern onto promoters conducting biophysical studies which lack in current scenario. Production of sufficient amounts of protein is frequent bottleneck in structural biology. Cloning full-length HIC-1 with >73 % GC content poses a daunting task with sequencing and expression adds more to the challenge. We describe the methodology for specific amplification, cloning, sequencing, and expression of HIC-1 in E. coli. Standardization using 1.5 U pfu polymerase in (NH4)2SO4 containing buffer gave specific amplification with 10 % DMSO and 1.5 mM MgCl2. Sequencing achieved using base analog 7-de aza dGTP (0.2 mM) or denaturant like DMSO (10 %) or betaine (1 M). Expression using strains of E. coli induced by different concentrations of IPTG (0.5-5.0 mM) for time points of 4, 8, 16, 20, and 24 h at different temperatures 25, 30, and 37 °C. Full-length clone successfully expressed in BL21-Codon Plus-RP using 1 mM concentration of IPTG for 8 h at 37 °C gave prominent band of 74 kDa.

P53 induction accompanying G2/M arrest upon knockdown of tumor suppressor HIC1 in U87MG glioma cells.

Hypermethylated in cancer 1 (HIC1) is a novel tumor suppressor gene (tsg) frequently silenced by epigenetic modification, predominantly by methylation in different tumors. HIC1 functionally co-operates with p53 in cultured cells as well as in transgenic animals to suppress tumors and has binding site on its promoter. Its over expression often leads to cell cycle arrests. Although HIC1 proven to have role as tsg, its regulation to cell cycle and dependency upon p53 is grossly unknown. In this study, we investigated the role of HIC1 in cell cycle and proliferation of glioma cell line U87MG which has wild type p53, in both serum-containing and serum-deprived medium. Microscopic analysis and MTT assay showed reduced cell number and rate of proliferation upon HIC1 knock down compared to control siRNA (p=0.025) and untreated cells (p=0.03) in serum-containing medium and serum-free medium (p=0.014 vs control siRNA; p=0.018 vs untreated cells). Cell cycle analysis revealed an arrest at G2/M phase of cell cycle with no demonstrable increase in apoptosis with both medium. An increased expression of p53 concomitant with HIC1 knockdown was observed. Furthermore P21, a p53 responsive gene, along with p27 was significantly increased in comparison with controls. Our results demonstrated an important role of HIC1 for the normal progression of cell cycle, and at molecular level, it could affect the homeostasis of p53 as well as number of cell cycle-related genes, which may or may not be directly linked to p53.

Aberrant Methylation of Hypermethylated-in-Cancer-1 and Exocyclic DNA Adducts in Tobacco Smokers

Tobacco smoke has been shown to produce both DNA damage and epigenetic alterations. However, the potential role of DNA damage in generating epigenetic changes is largely underinvestigated in human studies. We examined the effects of smoking on the levels of DNA methylation in genes for tumor protein p53, cyclin-dependent kinase inhibitor2A, hypermethylated-in-cancer-1 (HIC1), interleukin-6, Long Interspersed Nuclear Element type1, and Alu retrotransposons in blood of 177 residents in Thailand using bisulfite-PCR andpyrosequencing. Then, we analyzed the relationship of this methylation with the oxidative DNA adduct, M₁dG (a malondialdehyde adduct), measured by ³²P-postlabeling. Multivariate statistical analyses showed that HIC1 methylation levels were significantly increased in smokers compared with nonsmokers (p ≤ .05). A dose response was observed, with the highest HIC1 methylation levels in smokers of ≥ 10 cigarettes/day relative to nonsmokers and intermediate values in smokers of 1-9 cigarettes/day (p for trend ≤ .001). No additional relationships were observed. We also evaluated correlations between M₁dG and the methylation changes at each HIC1 CpG site individually. The levels of this adduct in smokers showed a significant linear correlation with methylation at one of the 3 CpGs evaluated in HIC1: hypermethylation at position 1904864340 was significantly correlated with the adduct M₁dG (covariate-adjusted regression coefficient (β) = .224 ± .101 [SE], p ≤ .05). No other correlations were detected. Our study extends prior work by others associating hypermethylation of HIC1 with smoking; shows that a very specific hypermethylation event can arise from smoking; and encourages future studies that explore a possible role for M₁dG in connecting smoking to this latter hypermethylation.

The Reelin receptors ApoER2 and VLDLR are direct target genes of HIC1 (Hypermethylated In Cancer 1)

The tumor suppressor gene HIC1 (Hypermethylated In Cancer 1) is located in 17p13.3 a region frequently hypermethylated or deleted in tumors and in a contiguous-gene syndrome, the Miller-Dieker syndrome which includes classical lissencephaly (smooth brain) and severe developmental defects. HIC1 encodes a transcriptional repressor involved in the regulation of growth control, DNA damage response and cell migration properties. We previously demonstrated that the membrane-associated G-protein-coupled receptors CXCR7, ADRB2 and the tyrosine kinase receptor EphA2 are direct target genes of HIC1. Here we show that ectopic expression of HIC1 in U2OS and MDA-MB-231 cell lines decreases expression of the ApoER2 and VLDLR genes, encoding two canonical tyrosine kinase receptors for Reelin. Conversely, knock-down of endogenous HIC1 in BJ-Tert normal human fibroblasts through RNA interference results in the up-regulation of these two Reelin receptors. Finally, through chromatin immunoprecipitation (ChIP) in BJ-Tert fibroblasts, we demonstrate that HIC1 is a direct transcriptional repressor of ApoER2 and VLDLR. These data provide evidence that HIC1 is a new regulator of the Reelin pathway which is essential for the proper migration of neuronal precursors during the normal development of the cerebral cortex, of Purkinje cells in the cerebellum and of mammary epithelial cells. Deregulation of this pathway through HIC1 inactivation or deletion may contribute to its role in tumor promotion. Moreover, HIC1, through the direct transcriptional repression of ATOH1 and the Reelin receptors ApoER2 and VLDLR, could play an essential role in normal cerebellar development.

HIC1 interacts with and modulates the activity of STAT3

HIC1 (hypermethylated in cancer 1) is a tumor suppressor gene, expression of which is frequently suppressed in human cancers. Very little is known about the molecular basis of HIC1 in antagonizing oncogenic pathways. Here, we report that HIC1 forms complexes with the signal transducers and activators of transcription 3 (STAT3) and attenuates STAT3-mediated transcription. STAT3 was identified as a HIC1-interacting protein by affinity capture and followed by mass spectrometry analysis. Overexpression or depletion of HIC1 resulted in decreased or increased levels of interleukin-6 (IL-6)/oncostatin M (OSM)-induced STAT3-mediated reporter activity and expression of target genes such as VEGF and c-Myc, respectively. Furthermore, HIC1 suppressing the VEGF and c-Myc promoter activity and the colony formation of MDA-MB 231 cells were STAT3-dependent. Further studies showed that HIC1 interacts with the DNA binding domain of STAT3 and suppresses the binding of STAT3 to its target gene promoters. Domain mapping study revealed that HIC1 C-terminal domain binds to STAT3. HIC1 mutant defective in STAT3 interaction reduced its repressive effect on STAT3 DNA binding activity, the reporter activity and gene expression of the VEGF and c-Myc genes, and cell growth in MDA-MB 231 cells. Altogether, our findings not only provide a novel role of HIC1 in antagonizing STAT3-mediated activation of VEGF and c-Myc gene expression and cell growth, but also elucidate a molecular basis underlying the inhibitory effect of HIC1 on STAT3 transcriptional potential.

Methylation Pattern of THBS1, GATA-4, and HIC1 in Pediatric and Adult Patients Infected with Helicobacter pylori

Helicobacter pylori infection is usually acquired in childhood and persists into adulthood if untreated. The bacterium induces a chronic inflammatory response, which is associated with epigenetic alterations in oncogenes, tumor-suppressor genes, cell-cycle regulators, and cell-adhesion molecules. The aim of this study was to analyze the effect of H. pylori infection on the methylation status of Thrombospondin-1 (THBS1), Hypermethylated in cancer 1 (HIC1) and Gata binding protein-4 (GATA-4) in gastric biopsy samples from children and adults infected or uninfected with the bacterium and in samples obtained from gastric cancer patients. The methylation pattern was analyzed with methylation-specific PCR. Our results showed that H. pylori infection was associated with methylation of the promoter regions of the THBS1 and GATA-4 genes in pediatric and adult samples (p < 0.01). HIC1 showed the lowest level of methylation, which was not an early event during gastric carcinogenesis. The results from this study indicate that methylation of THBS1 and GATA-4 occurs in the early stages of chronic gastritis and gastric cancer in association with H. pylori infection; however, in gastric cancer samples, other mechanisms cooperate with the down-regulation of these genes. Methylation of HIC1 may not be the principal mechanism implicated in its down-regulation in gastric cancer samples.

DNA Double-strand Breaks Lead to Activation of Hypermethylated in Cancer 1 (HIC1) by SUMOylation to Regulate DNA Repair

HIC1 (hypermethylated in cancer 1) is a tumor suppressor gene frequently epigenetically silenced in human cancers. HIC1 encodes a transcriptional repressor involved in the regulation of growth control and DNA damage response. We previously demonstrated that HIC1 can be either acetylated or SUMOylated on lysine 314. This deacetylation/SUMOylation switch is governed by an unusual complex made up of SIRT1 and HDAC4 which deacetylates and thereby favors SUMOylation of HIC1 by a mechanism not yet fully deciphered. This switch regulates the interaction of HIC1 with MTA1, a component of the NuRD complex and potentiates the repressor activity of HIC1. Here, we show that HIC1 silencing in human fibroblasts impacts the repair of DNA double-strand breaks whereas ectopic expression of wild-type HIC1, but not of nonsumoylatable mutants, leads to a reduced number of γH2AX foci induced by etoposide treatment. In this way, we demonstrate that DNA damage leads to (i) an enhanced HDAC4/Ubc9 interaction, (ii) the activation of SIRT1 by SUMOylation (Lys-734), and (iii) the SUMO-dependent recruitment of HDAC4 by SIRT1 which permits the deacetylation/SUMOylation switch of HIC1. Finally, we show that this increase of HIC1 SUMOylation favors the HIC1/MTA1 interaction, thus demonstrating that HIC1 regulates DNA repair in a SUMO-dependent way. Therefore, epigenetic HIC1 inactivation, which is an early step in tumorigenesis, could contribute to the accumulation of DNA mutations through impaired DNA repair and thus favor tumorigenesis.

Decreased vitamin B12 availability induces ER stress through impaired SIRT1-deacetylation of HSF1

Vitamin B12 (cobalamin) is a key determinant of S-adenosyl methionine (SAM)-dependent epigenomic cellular regulations related to methylation/acetylation and its deficiency produces neurodegenerative disorders by elusive mechanisms. Sirtuin 1 deacetylase (SIRT1) triggers cell response to nutritional stress through endoplasmic reticulum (ER) stress. Recently, we have established a N1E115 dopaminergic cell model by stable expression of a transcobalamin–oleosin chimera (TO), which impairs cellular availability of vitamin B12, decreases methionine synthase activity and SAM level, and reduces cell proliferation. In contrast, oleosin-transcobalamin chimera (OT) does not modify the phenotype of transfected cells. Presently, the impaired cellular availability of vitamin B12 in TO cells activated irreversible ER stress pathways, with increased P-eIF-2α, P-PERK, P-IRE1α, ATF6, ATF4, decreased chaperon proteins and increased pro-apoptotic markers, CHOP and cleaved caspase 3, through reduced SIRT1 expression and consequently greater acetylation of heat-shock factor protein 1 (HSF1). Adding either B12, SIRT1, or HSF1 activators as well as overexpressing SIRT1 or HSF1 dramatically reduced the activation of ER stress pathways in TO cells. Conversely, impairing SIRT1 and HSF1 by siRNA, expressing a dominant negative form of HSF1, or adding a SIRT1 inhibitor led to B12-dependent ER stress in OT cells. Addition of B12 abolished the activation of stress transducers and apoptosis, and increased the expression of protein chaperons in OT cells subjected to thapsigargin, a strong ER stress stimulator. AdoX, an inhibitor of methyltransferase activities, produced similar effects than decreased B12 availability on SIRT1 and ER stress by a mechanism related to increased expression of hypermethylated in cancer 1 (HIC1). Taken together, these data show that cellular vitamin B12 has a strong modulating influence on ER stress in N1E115 dopaminergic cells. The impaired cellular availability in vitamin B12 induces irreversible ER stress by greater acetylation of HSF1 through decreased SIRT1 expression, whereas adding vitamin B12 produces protective effects in cells subjected to ER stress stimulation.

Hypermethylated in cancer 1 and neoplasms

HIC1 (hypermethylated in cancer 1) encodes a transcriptional repressor,and extensively resides in various kinds of normal tissue.HIC1 gene is located in chromosome 17p13.3,in which loss of heterozygote or super-methylation is frequently found in a variety of human cancers.As a new tumor marker,HIC1 has been confirmed down-regulated in a wide variety of solid cancers because of HIC1 promoter hypermethylation,and may be associated with tumor prognosis.As a tumor suppressor,HIC1 participates in the development of tumor process through various ways,and is involved in cell proliferation,tumour growth,and angiogenesis.Therefore,the abnormal hypermethylation or the loss of expression of HIC1 in tumor cells or abnormal function could be one of the important mechanisms of tumor development,and may become a new potential therapeutic targets for cancer. Key words: Neoplasms; DNA methylation; HIC1 gene

Identification of p21 (CIP1/WAF1) as a direct target gene of HIC1 (Hypermethylated In Cancer 1)

The tumor suppressor gene HIC1 (Hypermethylated In Cancer 1) encodes a transcriptional repressor involved in the regulation of growth control and DNA damage response. We previously demonstrated that p57Kip2; a member of the CIP/KIP family of CDK (cyclin dependent kinase) inhibitors (CKI); is a direct target gene of HIC1 in quiescent cells. Here we show that ectopic expression of HIC1 in MDA-MB-231 cells or its overexpression in BJ-Tert fibroblasts induces decreased mRNA and protein expression of p21 (CIP1/WAF1) another member of this CKI family that plays essential roles in the p53-mediated DNA damage response. Conversely, knock-down of endogenous HIC1 in BJ-Tert through RNA interference up-regulates p21 in basal conditions and further potentiates this CKI in response to apoptotic etoposide-induced DNA damage. Through promoter luciferase activity and chromatin immunoprecipitation (ChIP), we demonstrate that HIC1 is a direct transcriptional repressor of p21. Thus, our results further demonstrate that HIC1 is a key player in the regulation of the DNA damage response.

DNA methylation differences in exposed workers and nearby residents of the Ma Ta Phut industrial estate, Rayong, Thailand

Adverse biological effects from airborne pollutants are a primary environmental concern in highly industrialized areas. Recent studies linked air pollution exposures with altered blood Deoxyribo-nucleic acid (DNA) methylation, but effects from industrial sources and underlying biological mechanisms are still largely unexplored. The Ma Ta Phut industrial estate (MIE) in Rayong, Thailand hosts one of the largest steel, oil refinery and petrochemical complexes in south-eastern Asia. We measured a panel of blood DNA methylation markers previously associated with air pollution exposures, including repeated elements [long interspersed nuclear element-1 (LINE-1) and Alu] and genes [p53, hypermethylated-in-cancer-1 (HIC1), p16 and interleukin-6 (IL-6)], in 67 MIE workers, 65 Ma Ta Phut residents and 45 rural controls. To evaluate the role of DNA damage and oxidation, we correlated DNA methylation measures with bulky DNA and 3-(2-deoxy-β-D-erythro-pentafuranosyl)pyrimido[1,2-α]purin-10(3H)-one deoxyguanosine (M(1)dG) adducts. In covariate-adjusted models, MIE workers, compared with rural residents, showed lower LINE-1 (74.8% vs 78.0%; P < 0.001), p53 (8.0% vs 15.7%; P < 0.001) and IL-6 methylation (39.2% vs 45.0%; P = 0.027) and higher HIC1 methylation (22.2% vs 15.3%, P < 0.001). For all four markers, Ma Ta Phut residents exhibited methylation levels intermediate between MIE workers and rural controls (LINE-1, 75.7%, P < 0.001; p53, 9.0%, P < 0.001; IL-6, 39.8%, P = 0.041; HIC1, 17.8%, P = 0.05; all P-values vs rural controls). Bulky DNA adducts showed negative correlation with p53 methylation (P = 0.01). M(1)dG showed negative correlations with LINE-1 (P = 0.003) and IL-6 methylation (P = 0.05). Our findings indicate that industrial exposures may induce alterations of DNA methylation patterns detectable in blood leucocyte DNA. Correlation of DNA adducts with DNA hypomethylation suggests potential mediation by DNA damage.

Hypermethylation of HIC1 Promoter and Aberrant Expression of HIC1/SIRT1 Might Contribute to the Carcinogenesis of Pancreatic Cancer

DNA hypermethylation is proved to be involved in carcinogenesis. Because chronic pancreatitis (CP) is a consistent risk factor for pancreatic cancer, the possible alteration and tumor contribute effects of hypermethylated in cancer-1 (HIC1) promoter methylation in CP was investigated. Methylation of HIC1 promoter HIC1 and SIRT1 expression were detected in human normal pancreas (NP), CP and pancreatic adenocarcinoma tissues. Furthermore, HIC1/SIRT1 pathway was regulated by demethylating reagent and exogenous expression in PANC-1, BxPC-3 and AsPC-1 cell lines, cell biology behavior including proliferation, apoptosis, cell cycle and senescence were detected. The methylation of HIC1 promoter was demonstrated in 70.3 % pancreatic carcinoma (45 of 64), 47.5 % CP (19 of 40) and 11.4 % NP tissues (4 of 35). Moreover, hypermethylation of HIC1 promoter and deregulation of HIC1 expression in pancreatic cancer were significantly related to high-stage tumor and older patient age. HIC1 promoter hypermethylation was also observed in pancreatic cancer cell lines including PANC-1, BxPC-3 and AsPC-1. Restoration of HIC1 function with 5-aza-dC treatment or pCDNA3FlagHIC1 plasmid transfection leaded to a reduction in cell proliferation, obvious cell senescence, cell cycle arrest and apoptosis, accompanied with acetylated p53 and p21(WAF1 of Cip1) upregulation. While after further transfected with pCDNA3FlagSIRT1 plasmid, the growth inhibition, senescence and cycle arrest without apoptosis were partially rescued with deregulated acetylated p53 and p21(WAF1 of Cip1). Our results indicate that hypermethylation of HIC1 promoter in CP may contribute to the aberrant expression of HIC1/SIRT1 pathway and then involve in the pancreatic carcinogenesis.

Signification of Hypermethylated in Cancer 1 (HIC1) as Tumor Suppressor Gene in Tumor Progression.

Hypermethylated in cancer 1(HIC1) was identified as a strong suppressor gene in chromosome region 17p13.3 telomeric to TP53. This gene encodes a transcriptional repressor and is ubiquitously expressed in normal tissues but downexpressed in different tumor tissues where it is hypermethylated. The hypermethylation of this chromosomal region leads to epigenetic inactivation of HIC1, which would prompt cancer cells to alter survival and signaling pathways or specific transcription factors during the period of tumorigenesis. In vitro, HIC1 function is mainly a sequence-specific transcriptional repressor interacting with a still growing range of histone deacetylase(HDAC)-dependent and HDAC-independent corepressor complexes. Furthermore, a role for HIC1 in tumor development is firmly supported by Hic1 deficient mouse model and two double heterozygote models cooperate with p53 and Ptch1. Notably, our findings suggest that potential factors derived from tumor microenviroment may play a role in modulating HIC1 expression in tumor cells by epigenetic modification, which is responsible for tumor progression. In this review, we will describe genomic and proteinic structure of HIC1, and summary the potential role of HIC1 in human various solid tumors and leukemia, and explore the influence of tumor microenviroment on inducing HIC1 expression in tumor cells.

Molecular dissection of the interaction between HIC1 and SIRT1

HIC1 (Hypermethylated in Cancer 1) is a tumor suppressor gene frequently epigenetically silenced in human cancers. HIC1 encodes a transcriptional repressor involved in the regulation of growth control, cell survival and DNA damage response. The deacetylase SIRT1 regulates the repressive capacity of HIC1 in several fashions. First SIRT1 interacts with the BTB/POZ domain of HIC1 to form a transcriptional repression complex that prevents the transcription of SIRT1 itself. SIRT1 is also responsible of the deacetylation of the lysine 314 of HIC1 that allows its subsequent SUMOylation which in turn favors its interaction with the NuRD complex. To better understand the interplay between HIC1 and SIRT1, we performed co-immunoprecipitation experiments to define the domains essential for the HIC1/SIRT1 interaction. We demonstrated that the isolated four last zinc fingers of HIC1 were capable to interact with SIRT1 and that the amino-acids 610–677 of SIRT1 encompassing the ESA region of the deacetylase were crucial for the HIC1/SIRT1 interaction and HIC1 deacetylation. Finally we demonstrated that this interaction mainly depends on CKII-mediated phosphorylation of SIRT1 serine 659/661 which occurs upon DNA damage. Therefore, our results demonstrate that the activating acetylation to SUMOylation switch of HIC1 is favored by genotoxic stresses to regulate the DNA damage response.

Hypermethylated in Cancer 1 (HIC1) Recruits Polycomb Repressive Complex 2 (PRC2) to a Subset of Its Target Genes through Interaction with Human Polycomb-like (hPCL) Proteins

HIC1 (hypermethylated in cancer 1) is a tumor suppressor gene epigenetically silenced or deleted in many human cancers. HIC1 is involved in regulatory loops modulating p53- and E2F1-dependent cell survival, growth control, and stress responses. HIC1 is also essential for normal development because Hic1-deficient mice die perinatally and exhibit gross developmental defects throughout the second half of development. HIC1 encodes a transcriptional repressor with five C(2)H(2) zinc fingers mediating sequence-specific DNA binding and two repression domains: an N-terminal BTB/POZ domain and a central region recruiting CtBP and NuRD complexes. By yeast two-hybrid screening, we identified the Polycomb-like protein hPCL3 as a novel co-repressor for HIC1. Using multiple biochemical strategies, we demonstrated that HIC1 interacts with hPCL3 and its paralog PHF1 to form a stable complex with the PRC2 members EZH2, EED, and Suz12. Confirming the implication of HIC1 in Polycomb recruitment, we showed that HIC1 shares some of its target genes with PRC2, including ATOH1. Depletion of HIC1 by siRNA interference leads to a partial displacement of EZH2 from the ATOH1 promoter. Furthermore, in vivo, ATOH1 repression by HIC1 is associated with Polycomb activity during mouse cerebellar development. Thus, our results identify HIC1 as the first transcription factor in mammals able to recruit PRC2 to some target promoters through its interaction with Polycomb-like proteins.

Prognostic and diagnostic relevance of hypermethylated in cancer 1 (HIC1) CpG island methylation in renal cell carcinoma

The tumour suppressor gene hypermethylated in cancer 1 (HIC1) is a transcriptional repressor, which functionally cooperates with p53. Loss of HIC1 function is associated with the development of various tumor entities. The aim of this study was to elucidate the relevance of CpG island (CGI) methylation of HIC1 in renal cell carcinoma (RCC). DNA methylation of HIC1 was analysed in a total of 98 tumor and 70 tumor adjacent normal specimens. After conducting bisulfite conversion, relative methylation levels were quantitated using pyrosequencing. Relative methylation values were compared for paired tumor and normal specimen and for correlation with clinico-pathologic and follow-up data of patients. Tumor-specific hypermethylation could not be detected for the subregion of the HIC1 - CGI analyzed in this study. Comparing the level of methylation in tumors to clinicopathological data solely, patients without lymph node metastases demonstrated a higher level of methylation compared to patients with lymph node metastases (p=0.030). Patients recurrence-free survival (p=0.0074) both in univariate as well as bivariate cox regression analysis. This study identifies HIC1 hypermethylation in tumors as an independent predictor of reduced recurrence-free survival, which fits into our current understanding of hypermethylated HIC1 being a marker for poor prognosis. Therefore, HIC1 - CGI methylation could be a candidate marker to improve individualized therapy and risk stratification.