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DNA methylation transcriptionally regulates the putative tumor cell growth suppressor ZNF677 in non-small cell lung cancers.

ABSTRACT: In our study, we investigated the role of ZNF677 in non-small cell lung cancers (NSCLC). By comparing ZNF677 expression in primary tumor (TU) and in the majority of cases also of corresponding non-malignant lung tissue (NL) samples from > 1,000 NSCLC patients, we found tumor-specific downregulation of ZNF677 expression (adjusted p-values < 0.001). We identified methylation as main mechanism for ZNF677 downregulation in NSCLC cells and we observed tumor-specific ZNF677 methylation in NSCLC patients (p < 0.0001). In the majority of TUs, ZNF677 methylation was associated with loss of ZNF677 expression. Moreover, ZNF677 overexpression in NSCLC cells was associated with reduced cell proliferation and cell migration. ZNF677 was identified to regulate expression of many genes mainly involved in growth hormone regulation and interferon signalling. Finally, patients with ZNF677 methylated TUs had a shorter overall survival compared to patients with ZNF677 not methylated TUs (p = 0.013). Overall, our results demonstrate that ZNF677 is trancriptionally regulated by methylation in NSCLCs, suggest that ZNF677 has tumor cell growth suppressing properties in NSCLCs and that ZNF677 methylation might serve as prognostic parameter in these patients.

SUBMITTER: Heller G

PROVIDER: S-EPMC4381603 | biostudies-literature | 2015 Jan

REPOSITORIES: biostudies-literature

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DNA methylation transcriptionally regulates the putative tumor cell growth suppressor ZNF677 in non-small cell lung cancers.

Heller Gerwin G Altenberger Corinna C Schmid Bianca B Marhold Maximilian M Tomasich Erwin E Ziegler Barbara B Müllauer Leonhard L Minichsdorfer Christoph C Lang György G End-Pfützenreuter Adelheid A Döme Balazs B Arns Britt-Madeleine BM Fong Kwun M KM Wright Casey M CM Yang Ian A IA Klepetko Walter W Zielinski Christoph C CC Zöchbauer-Müller Sabine S

Oncotarget 20150101 1

In our study, we investigated the role of ZNF677 in non-small cell lung cancers (NSCLC). By comparing ZNF677 expression in primary tumor (TU) and in the majority of cases also of corresponding non-malignant lung tissue (NL) samples from > 1,000 NSCLC patients, we found tumor-specific downregulation of ZNF677 expression (adjusted p-values < 0.001). We identified methylation as main mechanism for ZNF677 downregulation in NSCLC cells and we observed tumor-specific ZNF677 methylation in NSCLC patien ...[more]

PMID: 25504438

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Project description:BACKGROUND:DNA methylation regulates together with other epigenetic mechanisms the transcriptional activity of genes and is involved in the pathogenesis of malignant diseases including lung cancer. In non-small cell lung cancer (NSCLC) various tumor suppressor genes are already known to be tumor-specifically methylated. However, from the vast majority of a large number of genes which were identified to be tumor-specifically methylated, tumor-specific methylation was unknown so far. Thus, the major aim of this study was to investigate in detail the mechanism(s) responsible for transcriptional regulation of the genes SPAG6 and L1TD1 in NSCLCs. METHODS:We analysed publically available RNA-sequencing data and performed gene expression analyses by RT-PCR. DNA methylation analyses were done by methylation-sensitive high-resolution melt analyses and bisulfite genomic sequencing. We additionally investigated protein expression using immunohistochemistry. Cell culture experiments included tumor cell growth, proliferation, viability as well as colony formation assays. Moreover, we performed xenograft experiments using immunodeficient mice. RESULTS:We observed frequent downregulation of SPAG6 and L1TD1 mRNA expression in primary tumor ?(TU) samples compared to corresponding non-malignant lung tissue (NL) samples of NSCLC patients. We furthermore observed re-expression of both genes after treatment with epigenetically active drugs in most NSCLC cell lines with downregulated SPAG6 and L1TD1 mRNA expression. Frequent tumor-specific DNA methylation of SPAG6 and L1TD1 was detected when we analysed TU and corresponding NL samples of NSCLC patients. ROC curve analyses demonstrated that methylation of both genes is able to distinguish between TU and NL samples of these patients. Immunohistochemistry revealed a close association between SPAG6/L1TD1 methylation and downregulated protein expression of these genes. Moreover, by performing functional assays we observed reduced cell growth, proliferation and viability of pCMV6-L1TD1 transfected NSCLC cells. In addition, reduced volumes of tumors derived from pCMV6-L1TD1 compared to pCMV6-ENTRY transfected NCI-H1975 cells were seen in a xenograft tumor model. CONCLUSIONS:Overall, our results demonstrate that SPAG6 and L1TD1 are tumor-specifically methylated in NSCLCs and that DNA methylation is involved in the transcriptional regulation of these genes. Moreover, in vitro as well as in vivo experiments revealed tumor-cell growth suppressing properties of L1TD1 in NSCLC cells.

Project description:BackgroundTumor suppressor epigenetic silencing plays an important role in non-small cell lung cancer (NSCLC) development and progression. Previously, the expression of speckle-type POZ protein (SPOP) has been found to be significantly inhibited in NSCLC. Our research aimed to investigate the molecular mechanisms, clinical significance and epigenetic alteration of SPOP in NSCLC.Materials and methodsBisulfite sequencing PCR and methylation-specific PCR were performed to test gene methylation. Chromatin immunoprecipitation (ChIP) was performed to detect transcription factor C/EBP? combinations and the promoter of the SPOP gene. Furthermore, we evaluated the effects of C/EBP? siRNA on SPOP expression, tumor cell migration and proliferation via MTT and Transwell assays in vitro and tumor growth in vivo. The relationship between the methylation status of the SPOP gene and clinicopathologic characteristics was investigated.ResultsHypermethylation was found in the CpG island of the SPOP gene promoter in NSCLC tissues, and this methylation was found to be correlated with SPOP expression. SPOP promoter methylation was associated with the pathology grade. The transcriptional activities were significantly inhibited by the hypermethylation of specific CpG sites within the SPOP gene promoter, while 5-aza-2'-deoxycytidine significantly increased SPOP gene expression. C/EBP? also played a key role in SPOP regulation. Five C/EBP? binding sites in the CpG island of the SPOP gene promoter were identified by ChIP. Inhibition of C/EBP? significantly reduced SPOP expression. SPOP mediated the C/EBP?-regulated suppression of invasion, migration and proliferation in vitro and tumor growth in vivo.ConclusionsSPOP function and expression in NSCLS were regulated by DNA methylation and C/EBP? transcriptional regulation combination effects, indicating that the SPOP promoter methylation status could be utilized as an epigenetic biomarker and that the C/EBP?-SPOP signaling pathway could be a potential therapeutic target in NSCLC.

Dataset Information

DNA methylation transcriptionally regulates the putative tumor cell growth suppressor ZNF677 in non-small cell lung cancers.

Publications

DNA methylation transcriptionally regulates the putative tumor cell growth suppressor ZNF677 in non-small cell lung cancers.

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