Dataset Information

Characterization of NOL7 gene point mutations, promoter methylation, and protein expression in cervical cancer.

ABSTRACT: NOL7 is a putative tumor suppressor gene localized to 6p23, a region with frequent loss of heterozygosity in a number of cancers, including cervical cancer (CC). We have previously demonstrated that reintroduction of NOL7 into CC cells altered the angiogenic phenotype and suppressed tumor growth in vivo by 95%. Therefore, to understand its mechanism of inactivation in CC, we investigated the genetic and epigenetic regulation of NOL7. NOL7 mRNA and protein levels were assessed in 13 CC cell lines and 23 consecutive CC specimens by real-time quantitative polymerase chain reaction, western blotting, and immunohistochemistry. Methylation of the NOL7 promoter was analyzed by bisulfite sequencing and mutations were identified through direct sequencing. A CpG island with multiple CpG dinucleotides spanned the 5' untranslated region and first exon of NOL7. However, bisulfite sequencing failed to identify persistent sites of methylation. Mutational sequencing revealed that 40% of the CC specimens and 31% of the CC cell lines harbored somatic mutations that may affect the in vivo function of NOL7. Endogenous NOL7 mRNA and protein expression in CC cell lines were significantly decreased in 46% of the CC cell lines. Finally, immunohistochemistry demonstrated strong NOL7 nucleolar staining in normal tissues that decreased with histologic progression toward CC. NOL7 is inactivated in CC in accordance with the Knudson 2-hit hypothesis through loss of heterozygosity and mutation. Together with evidence of its in vivo tumor suppression, these data support the hypothesis that NOL7 is the legitimate tumor suppressor gene located on 6p23.

SUBMITTER: Doci CL

PROVIDER: S-EPMC3237951 | biostudies-literature | 2012 Jan

REPOSITORIES: biostudies-literature

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Characterization of NOL7 gene point mutations, promoter methylation, and protein expression in cervical cancer.

Doçi Colleen L CL Mankame Tanmayi P TP Langerman Alexander A Ostler Kelly R KR Kanteti Rajani R Best Timothy T Onel Kenan K Godley Lucy A LA Salgia Ravi R Lingen Mark W MW

International journal of gynecological pathology : official journal of the International Society of Gynecological Pathologists 20120101 1

NOL7 is a putative tumor suppressor gene localized to 6p23, a region with frequent loss of heterozygosity in a number of cancers, including cervical cancer (CC). We have previously demonstrated that reintroduction of NOL7 into CC cells altered the angiogenic phenotype and suppressed tumor growth in vivo by 95%. Therefore, to understand its mechanism of inactivation in CC, we investigated the genetic and epigenetic regulation of NOL7. NOL7 mRNA and protein levels were assessed in 13 CC cell lines ...[more]

PMID: 22123719

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Project description:DNA methylation, and consequent down-regulation, of tumour suppressor genes occurs in response to epigenetic stimuli during cancer development. Similarly, human oncoviruses, including human papillomavirus (HPV), up-regulate and augment DNA methyltransferase (DNMT) and histone deacetylase (HDAC) activities, thereby decreasing tumour suppressor genes (TSGs) expression. Ubiquitin-like containing PHD and RING finger domain 1 (UHRF1), an epigenetic regulator of DNA methylation, is overexpressed in HPV-induced cervical cancers. Here, we investigated the role of UHRF1 in cervical cancer by knocking down its expression in HeLa cells using lentiviral-encoded short hairpin (sh)RNA and performing cDNA microarrays. We detected significantly elevated expression of thioredoxin-interacting protein (TXNIP), a known TSG, in UHRF1-knockdown cells, and this gene is hypermethylated in cervical cancer tissue and cell lines, as indicated by whole-genome methylation analysis. Up-regulation of UHRF1 and decreased TXNIP were further detected in cervical cancer by western blot and immunohistochemistry and confirmed by Oncomine database analysis. Using chromatin immunoprecipitation, we identified the inverted CCAAT domain-containing UHRF1-binding site in the TXNIP promoter and demonstrated UHRF1 knockdown decreases UHRF1 promoter binding and enhances TXNIP expression through demethylation of this region. TXNIP promoter CpG methylation was further confirmed in cervical cancer tissue by pyrosequencing and methylation-specific polymerase chain reaction. Critically, down-regulation of UHRF1 by siRNA or UHRF1 antagonist (thymoquinone) induces cell cycle arrest and apoptosis, and ubiquitin-specific protease 7 (USP7), which stabilises and promotes UHRF1 function, is increased by HPV viral protein E6/E7 overexpression. These results indicate HPV might induce carcinogenesis through UHRF1-mediated TXNIP promoter methylation, thus suggesting a possible link between CpG methylation and cervical cancer.

Project description:Loss of 3p11-p14 is a frequent event in epithelial cancer and a candidate prognostic biomarker in cervical cancer. In addition to loss, promoter methylation can participate in gene silencing and promote tumor aggressiveness. We have performed a complete mapping of promoter methylation at 3p11-p14 in two independent cohorts of cervical cancer patients (n = 149, n = 121), using Illumina 450K methylation arrays. The aim was to investigate whether hyperm-ethylation was frequent and could contribute to gene silencing and disease aggressiveness either alone or combined with loss. By comparing the methylation level of individual CpG sites with corresponding data of normal cervical tissue, 26 out of 41 genes were found to be hypermethylated in both cohorts. The frequency of patients with hypermethylation of these genes was found to be higher at tumor stages of 3 and 4 than in stage 1 tumors. Seventeen of the 26 genes were transcriptionally downregulated in cancer compared to normal tissue, whereof 6 genes showed a significant correlation between methylation and expression. Integrated analysis of methylation, gene dosage, and expression of the 26 hypermethylated genes identified 3 regulation patterns encompassing 8 hypermethylated genes; a methylation driven pattern (C3orf14, GPR27, ZNF717), a gene dosage driven pattern (THOC7, PSMD6), and a combined methylation and gene dosage driven pattern (FHIT, ADAMTS9, LRIG1). In survival analysis, patients with both hypermethylation and loss of LRIG1 had a worse outcome compared to those harboring only hypermethylation or none of the events. C3orf14 emerged as a novel methylation regulated suppressor gene, for which knockdown was found to promote invasive growth in human papilloma virus (HPV)-transformed keratinocytes. In conclusion, hypermethylation at 3p11-p14 is common in cervical cancer and may exert a selection pressure during carcinogenesis alone or combined with loss. Information on both events could lead to improved prognostic markers.

Dataset Information

Characterization of NOL7 gene point mutations, promoter methylation, and protein expression in cervical cancer.

Publications

Characterization of NOL7 gene point mutations, promoter methylation, and protein expression in cervical cancer.

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