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Functional validation of putative tumor suppressor gene C13ORF18 in cervical cancer by Artificial Transcription Factors.

ABSTRACT: C13ORF18 is frequently hypermethylated in cervical cancer but not in normal cervix and might serve as a biomarker for the early detection of cervical cancer in scrapings. As hypermethylation is often observed for silenced tumor suppressor genes (TSGs), hypermethylated biomarker genes might exhibit tumor suppressive activities upon re-expression. Epigenetic drugs are successfully exploited to reverse TSG silencing, but act genome-wide. Artificial Transcription Factors (ATFs) provide a gene-specific approach for re-expression of silenced genes. Here, we investigated the potential tumor suppressive role of C13ORF18 in cervical cancer by ATF-induced re-expression. Five zinc finger proteins were engineered to bind the C13ORF18 promoter and fused to a strong transcriptional activator. C13ORF18 expression could be induced in cervical cell lines: ranging from >40-fold in positive (C13ORF18-unmethylated) cells to >110-fold in negative (C13ORF18-methylated) cells. Re-activation of C13ORF18 resulted in significant cell growth inhibition and/or induction of apoptosis. Co-treatment of cell lines with ATFs and epigenetic drugs further enhanced the ATF-induced effects. Interestingly, re-activation of C13ORF18 led to partial demethylation of the C13ORF18 promoter and decreased repressive histone methylation. These data demonstrate the potency of ATFs to re-express and potentially demethylate hypermethylated silenced genes. Concluding, we show that C13ORF18 has a TSG function in cervical cancer and may serve as a therapeutic anti-cancer target. As the amount of epimutations in cancer exceeds the number of gene mutations, ATFs provide promising tools to validate hypermethylated marker genes as therapeutic targets.

SUBMITTER: Huisman C

PROVIDER: S-EPMC5528488 | biostudies-literature | 2013 Jun

REPOSITORIES: biostudies-literature

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Functional validation of putative tumor suppressor gene C13ORF18 in cervical cancer by Artificial Transcription Factors.

Huisman Christian C Wisman G Bea A GB Kazemier Hinke G HG van Vugt Marcel A T M MA van der Zee Ate G J AG Schuuring Ed E Rots Marianne G MG

Molecular oncology 20130305 3

C13ORF18 is frequently hypermethylated in cervical cancer but not in normal cervix and might serve as a biomarker for the early detection of cervical cancer in scrapings. As hypermethylation is often observed for silenced tumor suppressor genes (TSGs), hypermethylated biomarker genes might exhibit tumor suppressive activities upon re-expression. Epigenetic drugs are successfully exploited to reverse TSG silencing, but act genome-wide. Artificial Transcription Factors (ATFs) provide a gene-specif ...[more]

PMID: 23522960

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Project description:BackgroundETS transcription factors regulate important signaling pathways involved in cell differentiation and development in many tissues and have emerged as important players in prostate cancer. However, the biological impact of ETS factors in prostate tumorigenesis is still debated.Methodology/principal findingsWe performed an analysis of the ETS gene family using microarray data and real-time PCR in normal and tumor tissues along with functional studies in normal and cancer cell lines to understand the impact in prostate tumorigenesis and identify key targets of these transcription factors. We found frequent dysregulation of ETS genes with oncogenic (i.e., ERG and ESE1) and tumor suppressor (i.e., ESE3) properties in prostate tumors compared to normal prostate. Tumor subgroups (i.e., ERG(high), ESE1(high), ESE3(low) and NoETS tumors) were identified on the basis of their ETS expression status and showed distinct transcriptional and biological features. ERG(high) and ESE3(low) tumors had the most robust gene signatures with both distinct and overlapping features. Integrating genomic data with functional studies in multiple cell lines, we demonstrated that ERG and ESE3 controlled in opposite direction transcription of the Polycomb Group protein EZH2, a key gene in development, differentiation, stem cell biology and tumorigenesis. We further demonstrated that the prostate-specific tumor suppressor gene Nkx3.1 was controlled by ERG and ESE3 both directly and through induction of EZH2.Conclusions/significanceThese findings provide new insights into the role of the ETS transcriptional network in prostate tumorigenesis and uncover previously unrecognized links between aberrant expression of ETS factors, deregulation of epigenetic effectors and silencing of tumor suppressor genes. The link between aberrant ETS activity and epigenetic gene silencing may be relevant for the clinical management of prostate cancer and design of new therapeutic strategies.

Project description:Previous microRNA (miR) microarray analysis revealed that miR‑218 is downregulated in cervical cancer tissues. The present study aimed to further evaluate the expression of miR‑218 in cervical cancer specimens, determine the association between its expression with disease progression, and investigate the roles of miR‑218 in cervical cancer cells. Tissue specimens were obtained from 80 patients with cervical squamous cell carcinoma, 30 patients with high‑grade cervical intraepithelial neoplasia [(CIN) II/III] and 15 patients with low‑grade CIN (CINI); in addition, 60 plasma samples were obtained from patients with cervical cancer, and 15 normal cervical tissue specimens and 30 plasma samples were obtained from healthy women. These samples were used for analysis of miR‑218 expression via reverse transcription‑-quantitative PCR. In addition, tumor cells were transfected with miR‑218 mimics, human papillomavirus (HPV)16 E6/E7 small interfering RNA, or their respective negative controls to determine the viability, colony formation, migration and invasion of cells using MTT, colony formation, wound healing and Transwell assays, respectively. Target genes of miR‑218 were bioinformatically predicted and analyzed using Gene Ontology (GO) terms. The results revealed that miR‑218 was downregulated in the tumor tissues and plasma of patients with cervical cancer, with expression associated with the advanced clinicopathological characteristics of patients, including HPV positivity, tumor size, blood vessel invasion and lymph node metastasis. Furthermore, miR‑218 overexpression reduced tumor cell viability and xenograft growth, and suppressed tumor cell migration and invasion. HPV was detected in 75% of the 80 patients with cervical cancer, and HPV positivity was inversely associated with miR‑218 expression. In addition, bioinformatics analysis predicted that roundabout guidance receptor 1 (ROBO1) was a target gene of miR‑218; miR‑218 overexpression significantly reduced ROBO1 levels. Furthermore, GO analysis revealed that ROBO1 was involved in regulating cell proliferation, adhesion and migration, and the cell cycle. In conclusion, the findings of the present study suggested that miR‑218 may possess antitumor activities in cervical cancer.

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Functional validation of putative tumor suppressor gene C13ORF18 in cervical cancer by Artificial Transcription Factors.

Publications

Functional validation of putative tumor suppressor gene C13ORF18 in cervical cancer by Artificial Transcription Factors.

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