High In Normal-1 Research Articles

HIN-1: a New Epigenetic Biomarker Crucial for Therapy Selection in Glioblastoma Multiforme.

Glioblastoma multiforme (GBM) is the most common brain tumor in adults. The role of high in normal-1 (HIN-1) as a potential biomarker in combating this disease is being described for the first time in this study. A combination of O6-methylguanine DNA methyltransferase (MGMT) and HIN-1 methylation could be a possible biomarker in therapy choice. Interestingly, survival data shows a similar trend for the methylation of MGMT and for unmethylation of HIN-1 and vice versa. Eighty-eight paraffin-embedded brain tumors were analyzed to screen methylation rates of different genes and evaluate the association between genes methylation and clinicopathologic variables. Our study is the first of its kind to indicate that MGMT and HIN-1 methylation status are inverted (97.7% of methylated ones) and could be new markers in the study of GBM prognosis, especially in the therapy selection.

Increased hypermethylation of glutathione S-transferase P1, DNA-binding protein inhibitor, death associated protein kinase and paired box protein-5 genes in triple-negative breast cancer Saudi females.

Triple negative breast cancer (TNBC) is an aggressive subtype of breast cancer (BC) with higher metastatic rate and both local and systemic recurrence compared to non-TNBC. The generation of reactive oxygen species (ROS) secondary to oxidative stress is associated with DNA damage, chromosomal degradation and alterations of both hypermethylation and hypomethylation of DNA. This study concerns differential methylation of promoter regions in specific groups of genes in TNBC and non-TNBC Saudi females in an effort to understand whether epigenetic events might be involved in breast carcinogenesis, and whether they might be used as markers for Saudi BCs. Methylation of glutathione S-transferase P1 (GSTP1), T-cadherin (CDH13), Paired box protein 5 (PAX5), death associated protein kinase (DAPK), twist-related protein (TWIST), DNA-binding protein inhibitor (ID4), High In Normal-1 (HIN-1), cyclin-dependent kinase inhibitor 2A (p16), cyclin D2 and retinoic acid receptor-β (RARβ1) genes was analyzed by methylation specific polymerase chain reaction (MSP) in 200 archival formalin- fixed paraffin embedded BC tissues divided into 3 groups; benign breast tissues (20), TNBC (80) and non-TNBC (100). The relationships between methylation status, and clinical and pathological characteristics of patients and tumors were assessed. Higher frequencies of GSTP1, ID4, TWIST, DAPK, PAX5 and HIN-1 hypermethylation were found in TNBC than in non-TNBC. Hypermethylation of GSTP1, CDH13, ID4, DAPK, HIN-1 and PAX5 increased with tumor grade increasing. Other statistically significant correlations were identified with studied genes. Data from this study suggest that increased hypermethylation of GSTP1, ID4, TWIST, DAPK, PAX5 and HIN-1 genes in TNBC than in non-TNBC can act as useful biomarker for BCs in the Saudi population. The higher frequency of specific hypermethylated genes paralleling tumor grade, size and lymph node involvement suggests contributions to breast cancer initiation and progression.

Aberrant promoter methylation of HIN-1 gene may contribute to the pathogenesis of breast cancer: a meta-analysis.

We conducted the present meta-analysis of relevant cohort studies to evaluate whether promoter methylation of the high in normal-1 (HIN-1) gene contributes to breast cancer. The MEDLINE (1966 ~ 2013), Cochrane Library (Issue 12, 2013), EMBASE (1980 ~ 2013), CINAHL (1982 ~ 2013), Web of Science (1945 ~ 2013), and Chinese Biomedical (CBM) (1982 ~ 2013) databases were searched without any language restrictions. Meta-analyses were conducted using Stata software (version 12.0; Stata Corporation, College Station, TX, USA). Crude odds ratios (ORs) with their 95 % confidence interval (CI) were calculated. Nine clinical cohort studies that enrolled a total of 693 breast cancer patients were included in the meta-analysis. The results of our meta-analysis demonstrated that HIN-1 methylation frequency in cancer tissue was significantly higher than that of normal and benign tissues (cancer tissue vs. normal tissue: OR = 52.60, 95 % CI = 33.77 ~ 81.92, P < 0.001; cancer tissue vs. benign tissue: OR = 2.38, 95 % CI = 1.53 ~ 3.70, P < 0.001; respectively). Ethnicity-stratified analysis indicated that HIN-1 promoter methylation was correlated with the pathogenesis of breast cancer among both Asians and Caucasians (all P < 0.05). Our findings provide empirical evidence that aberrant HIN-1 promoter methylation may contribute to the pathogenesis of breast cancer. Thus, aberrant HIN-1 promoter methylation could be an independent and important biomarker used in predicting the prognosis and progression of breast cancer.

AKT signaling pathway activated by HIN-1 methylation in non-small cell lung cancer

The purpose of this study is to determine the epigenetic changes and function of High in Normal-1 (HIN-1) in non-small cell lung cancer (NSCLC). HIN-1 expression was examined by semiquantitative RT-PCR before and after 5-aza-2'-deoxycytidine (5-aza) treatment in NSCLC cell lines. Promoter methylation status of HIN-1 was tested by methylation-specific PCR (MSP). Effect of forced expression of HIN-1 on different key molecules of AKT signaling pathway was tested by Western Blot analysis in H157 and H23 cell lines. Promoter methylations are inversely correlated with expression of HIN-1 in eight (H23, H157, 95D, H1299, H358, H1752, H460, A549) of ten NSCLC cell lines and re-expression was observed by 5-aza treatment. We then tested promoter methylation of HIN-1 in primary NSCLC tissues. Methylation was detected in 73 out of 152 (48%) NSCLC cases. Forced expression of HIN-1 in NSCLC cell lines inhibited colony formation and induce apoptosis. Furthermore, overexpression of HIN-1 reduces the expression of phosphorated-AKT (p-AKT), c-myc, Bcl-2 and cyclinD1 while Bax was increased. Our data suggest that HIN-1 is a potential tumor suppressor gene in NSCLC, silenced by promoter hypermethylation and negatively regulate AKT signaling pathway.

Silence of HIN-1 expression through methylation of its gene promoter in gastric cancer

To clarify the role of high in normal-1 (HIN-1) gene promoter methylation during gastric cancer development. Gastric cancer cell lines and tissue specimens were analyzed for expression of HIN-1 mRNA and protein using the semi-quantitative reverse transcription polymerase chain reaction and immunohistochemistry. The methylation of the HIN-1 gene promoter was detected in gastric carcinoma cells and tissues using methylation-specific polymerase chain reaction. The 3-(4,5-dimethylthiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium cell viability assay and flow cytometry were used to assess the changes in behaviors of gastric cancer cells with or without 5-aza-2'-deoxycytidine treatment. HIN-1 was not expressed in 4 of 5 gastric cancer cell lines. The demethylation reagent 5-aza-2'-deoxycytidine was able to induce or upregulate HIN-1 expression in gastric cancer cell lines, which is associated with reduction of tumor cell viability. Furthermore, methylation of the HIN-1 gene promoter was shown in 57.8% (26/45) of the primary gastric cancer and 42.1% (17/38) of adjacent tissue samples, but was not shown in normal gastric mucosa (0/10). From the clinicopathological data of the patients, methylation of the HIN-1 gene promoter was found to be associated with tumor differentiation (P = 0.000). High methylation of HIN-1 gene promoter results in silence of HIN-1 expression in gastric cancer. 5-aza-2'-deoxycytidine reverses HIN-1 methylation and reduces viability of gastric cancer cells.

Relation between normal rectal methylation, smoking status, and the presence or absence of colorectal adenomas

Colorectal cancer (CRC) is 1 of the leading causes of death in the Western world. CRC develops from premalignant lesions, chiefly colorectal adenomas. Currently, the most accurate and recommended screening method for finding colorectal adenomas is colonoscopy performed on all individuals aged>50 years. However, the costs and risks associated with this procedure are relatively high. The objectives of the current study were to correlate epigenetic alterations that occur in normal rectal mucosa, smoking status, and age with the presence or absence of concomitant colorectal adenomas and to assess the potential clinical value of methylation in normal rectal biopsies as a screening assay for the presence of polyps and, hence, the need for a full colonoscopy. One hundred thirteen normal rectal mucosal biopsies from 113 patients were studied. DNA was extracted, modified with sodium bisulfite, and subjected to real-time quantitative, methylation-specific polymerase chain reaction analysis for the following genes: adenomatous polyposis coli (APC); cadherin 1, type 1, E-cadherin (epithelial) (CDH1); estrogen receptor 1 (ESR1); cytokine high in normal 1 (HIN1); hyperplastic polyposis protein 1 (HPP1); O-6 methylguanine-DNA methyltransferase (MGMT); neural epidermal growth factor-like 1 (NELL1); splicing factor 3B, 14-kDa subunit (p14); cyclin-dependent kinase (CDK) inhibitor 2B (inhibits CDK4) (p15); retinoic acid receptor beta (RARβ); somatostatin (SST); tachykinin, precursor 1 (TAC1); and tissue inhibitor of metalloproteinase (TIMP) metallopeptidase inhibitor 3 (TIMP3). Data were then analyzed using several proprietary software programs. By using several sets of genes, clinical characteristics, and multivariate analyses, the authors developed a prediction model for the presence of concomitant colorectal adenomas at the time of rectal biopsy. They also observed strong correlations between smoking status and rectal methylation pattern and between smoking status and the presence or risk of concomitant adenomas. A prediction model was developed for the presence of colorectal adenomas based on gene methylation patterns in the normal rectum. The results indicated that these genes may be involved in early stages of adenoma formation. The observed epigenetic alterations in these markers may be caused in part by the effects of smoking and/or age. Normal rectal methylation may be useful as a biomarker for narrowing the population in need of screening colonoscopy.

Promoter methylation of HIN-1 in the progression to esophageal squamous cancer

HIN-1 (High in normal-1) is a tumor suppressor gene that is highly expressed in many epithelial tissues, including breast lung, trachea, pancreas, prostrate, and salivary gland. Inactivation of HIN-1 expression by promoter methylation is frequent in many epithelial carcinomas and carcinoma-in-situ, including breast, lung, and nasopharyngeal. Because HIN-1 silencing commences at an early stage of malignant transformation in these tissues, it may be a useful marker for tumor presence. The status of HIN-1 regulation in esophageal cancer has not been previously reported. Thus, we analyzed 18 esophageal cancer cell lines for HIN-1 expression and methylation by reverse transcription PCR (RT-PCR) and methylation specific PCR (MSP). HIN-1 gene silencing and promoter methylation was present in 13 (72%) of the cell lines. Bisulfite-treated sequencing confirmed the methylation status in cell lines and demonstrated dense methylation of HIN-1 throughout the promoter region. Epigenetic changes of HIN-1 were examined throughout the progression of carcinogenesis in esophageal squamous lesions through analysis of archived surgical specimens from patients with normal esophageal mucosa (n=17), grade I dysplasia (n=39), grade II dysplasia (n=12), grade III dysplasia (n=9), and invasive squamous esophageal cancer (n=126). Methylation of HIN-1 was present in 0% of normal mucosa, 31% of grade I dysplasia, 33% of grade II dysplasia, 44% of grade III dysplasia, and 50% of esophageal cancer specimens analyzed. These studies demonstrate HIN-1 silencing is associated with dense promoter region hypermethylation in esophageal cancer and suggest that methylation of HIN-1 is an early event in dysplastic transformation.

Recent Patents Concerning Diagnostic and Therapeutic Applications of Aberrantly Methylated Sequences in Pancreatic Cancer

Epigenetic modifications are important events in regulation of gene expression and cancer progression. Methylation of cytosine (C) residues in CpG dinucleotides of promoters is associated with transcriptional silencing. Knowledge about the CpG island methylation status of pancreatic cancer-specific genes could support the development of earlier diagnostic assays and finding new treatment strategies. Several recent patents comprising some of these discoveries have been published. One recent patent describes a method of determining the methylation status of CpG sites in deoxyribonucleic acid (DNA) and correlating it with the presence of tumour cells in pancreas. Another one suggests the usage of the high in Normal 1 (HIN-1) gene as a tool for diagnosis of pancreatic carcinoma. In addition, therapeutic approaches are proposed by two recent patents applying modulators of DNA cytosine-5 methyltransferase such as Decitabine or C-5 methylcytosine. Additionally, a patent introduces a method that couples an early cancer-related and tissue or cell- specific gene marker detection assay, useful as a simultaneous screening test for cancers, including pancreatic cancer. These patents provide new methods in fighting pancreatic cancer by focusing on methylated CpG islands in pancreatic cancer related genes.

Gene Promoter Hypermethylation in Tumors and Plasma of Breast Cancer Patients

To measure the hypermethylation of four genes in primary tumors and paired plasma samples to determine the feasibility of gene promoter hypermethylation markers for detecting breast cancer in the plasma. DNA was extracted from the tumor tissues and peripheral blood plasma of 34 patients with invasive breast cancer, and the samples examined for aberrant hypermethylation in cyclin D2, retinoic acid receptor beta (RARbeta), twist and high in normal-1 (HIN-1) genes using methylation-specific PCR (MSP), and the results correlated with the clinicopathological parameters. Promoter hypermethylation was detected at high frequency in the primary tumors for cyclin D2 (53%), RARbeta (56%), twist (41%) and HIN-1 (77%). Thirty-three of the 34 (97%) primary tumors displayed promoter hypermethylation in at least one of the genes examined. The corresponding plasma samples showed hypermethylation of the same genes, although at lower frequencies (6% for cyclin D2, 16% for RARbeta, 36% for twist, and 54% for HIN-1). Overall, 22 of the 33 (67%) primary tumors with hypermethylation of at least one of the four genes also had abnormally hypermethylated DNA in their matched plasma samples. No significant relationship was recognized between any of the clinical or pathological parameters (tumor size, axillary lymph node metastasis, stage, or Ki-67 labeling index) with the frequency of hypermethylated DNA in the primary tumor or plasma. The detection of aberrant promoter hypermethylation of cancer-related genes in the plasma may be a useful tool for the detection of breast cancer.

Aberrant methylation of HIN-1 (high in normal-1) is a frequent event in many human malignancies.

HIN-1 (high in normal-1) is a putative cytokine with growth inhibitory activities and is downregulated by aberrant methylation in breast cancers. We studied HIN-1 methylation status in many types of adult and pediatric malignancies and cell lines. We examined the expression of HIN-1 mRNA in 52 cell lines and the promoter methylation status in the cell lines and in over 800 primary tumors representing 17 tumor types using methylation specific PCR. Promoter methylation was observed in 73% of breast cancer, 67% of nonsmall cell lung cancer (NSCLC), 30% of small cell lung cancer (SCLC) and 57% of malignant mesothelioma (MM) cell lines, and methylation was completely correlated with loss of expression. Expression negative cell lines restored HIN-1 expression after treatment with 5-aza-2'-deoxycytidine. Promoter methylation of HIN-1 was found in 90% of retinoblastomas, 73% of Wilms' tumors, 61% of rhabdomyosarcomas, 57% of breast cancers, 52% of prostate cancers, 40% of MMs, 28% of NSCLCs and 27% of lymphomas. Methylation frequencies in colorectal cancers, cervical cancers, bronchial carcinoids, SCLCs, neuroblastomas, osteosarcomas, leukemia, medulloblastomas and bladder cancers were lower (4-21%), while hepatoblastomas lacked methylation. HIN-1 methylation was rarely detected in nonmalignant tissues (8 of 165, 5%). Aberrant methylation of HIN-1 with loss of expression is a common event and may contribute to the pathogenesis of many types of human malignancies.

Expression of high in normal-1 (HIN-1) and uteroglobin related protein-1 (UGRP-1) in adult and developing tissues

We analyzed the expression of high in normal-1 (HIN-1), a putative breast tumor suppressor gene, and uteroglobin related protein-1 (UGRP-1), a homologue of HIN-1, in adult and developing mouse tissues. Highest HIN-1 and UGRP-1 expression is detected in the lung, while lower level HIN-1 expression is also detected in the stomach, heart, small intestine, uterine and mammary glands. The expression of both genes was detected only at E17.5–18.5 and the HIN-1 messenger RNA was localized to the epithelia of the trachea, bronchi, and uterine glands. The expression of HIN-1 is up-regulated during retinoic acid induced differentiation of bronchial epithelial cells. We also identified two putative Drosophila HIN-1 homologues. The expression of HIN-1 is restricted to terminally differentiated airway epithelial cells in vivo and in vitro implicating HIN-1 in the acquisition or maintenance of terminally differentiated epithelial phenotype.