DNA methylation transcriptionally regulates the putative tumor cell growth suppressor <i>ZNF677</i> in non-small cell lung cancers

Heller, Gerwin; Altenberger, Corinna; Schmid, Bianca; Marhold, Maximilian; Tomasich, Erwin; Ziegler, Barbara; Müllauer, Leonhard; Minichsdorfer, Christoph; Láng, György; End-Pfützenreuter, Adelheid; Döme, Balázs; Arns, Britt Madeleine; Fong, Kwun M.; Wright, Casey M.; Yang, Ian A.; Klepetko, Walter; Zielinski, Christoph; Zöchbauer‐Müller, Sabine

doi:10.18632/oncotarget.2697

Cited by 25 publications

(59 citation statements)

References 41 publications

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“…Cell lines were maintained and grown as described [11]. Detailed descriptions of NSCLC and non-lung cancer cell lines are provided in Additional file 1: Table S1 and Table S4.…”

Section: Methodsmentioning

confidence: 99%

“…Genomic DNA from NHBECs, 18 tumor cell lines and tissue samples of 146 NSCLC patients was isolated and modified by treatment with sodium bisulfite using EpiTect Bisulfite kit (Qiagen) [11]. The genomic sequences of SPAG6 and L1TD1 were obtained from ENSEMBL database (release 69).…”

Section: Methodsmentioning

confidence: 99%

“…Total RNA was isolated from NHBECs and from 5 NSCLC cell lines and reverse transcribed using OmniScript Reverse Transcriptase Kit (Qiagen) [11]. Expression of SPAG6 was determined using Taqman Gene Expression Assays Hs00542625_m1 ( SPAG6 ) and Hs03929097_g1 ( GAPDH ) in a StepOne cycler (Applied Biosystems) and expression of L1TD1 was determined using Qiagen’s QuantiTect® SYBR Green PCR Kit (primer sequences see Additional file 1: Table S3).…”

Section: Methodsmentioning

confidence: 99%

“…Besides other epigenetic and genetic factors methylation may be responsible for transcriptional inactivation of these genes [11–15]. However, from the vast majority of ~500 genes which were identified to be tumor-specifically methylated in a genome-wide approach, tumor-specific methylation was unknown so far [12].…”

Section: Introductionmentioning

confidence: 99%

“…However, from the vast majority of ~500 genes which were identified to be tumor-specifically methylated in a genome-wide approach, tumor-specific methylation was unknown so far [12]. When we additionally analysed publically available microarray data in primary tumors (TU) compared to non-malignant lung tissue (NL) samples of NSCLC patients, we observed tumor-specifically downregulated expression of many of these genes [11, 16, 17]. An extensive PubMed search revealed that many of these genes are functionally uncharacterised in NSCLCs and in other cancer types.…”

Section: Introductionmentioning

confidence: 99%

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SPAG6 and L1TD1 are transcriptionally regulated by DNA methylation in non-small cell lung cancers

et al. 2017

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BackgroundDNA 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.MethodsWe 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.ResultsWe 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.ConclusionsOverall, 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-016-0568-5) contains supplementary material, which is available to authorized users.

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“…Cell lines were maintained and grown as described [11]. Detailed descriptions of NSCLC and non-lung cancer cell lines are provided in Additional file 1: Table S1 and Table S4.…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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SPAG6 and L1TD1 are transcriptionally regulated by DNA methylation in non-small cell lung cancers

et al. 2017

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Identification of hub methylated‐CpG sites and associated genes in oral squamous cell carcinoma

Dai

et al. 2020

Cancer Medicine

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To improve personalized diagnosis and prognosis for oral squamous cell carcinoma (OSCC) by identification of hub methylated-CpG sites and associated genes, weighted gene comethylation network analysis (WGCNA) was performed to examine and identify hub modules and CpG sites correlated with OSCC. Here, WGCNA modeling yielded blue and brown comethylation modules that were significantly associated with OSCC status. Following screening of the differentially expressed genes (DEGs) from gene expression microarrays and differentially methylated-CpG sites (DCGs), integrated multiomics analysis of the DEGs, DCGs, and hub CpG sites from the modules was performed to investigate their correlations. Expression levels of 16 CpG sites-associated genes were negatively correlated with methylation patterns of promoter. Moreover, Kaplan-Meier survival analysis of the hub CpG sites and associated genes was carried out using 2 public databases, MethSurv and GEPIA.Only 5 genes, ACTA1, ACTN2, OSR1, SYNGR1, and ZNF677, had significant overall survival using GEPIA. Hypermethylated-CpG sites ACTN2-cg21376883 and OSR1-cg06509239 were found to be associated with poor survival by MethSurv.Methylation status of specific site and expression levels of associated genes were determined using clinical samples by quantitative methylation-specific PCR and real-time PCR. Pearson's correlation analysis showed that methylation levels of cg06509239 and cg18335068 were negatively related to OSR1 and ZNF677 expression levels, respectively. Our classification schema using multiomics analysis represents a screening framework for identification of hub CpG sites and associated genes. K E Y W O R D SCpG site, methylation, multiomics, oral squamous cell carcinoma, survival analysis, WGCNA | 3175 DAI et Al.

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ZNF677 suppresses renal cell carcinoma progression through N6‐methyladenosine and transcriptional repression of CDKN3

Cao

Gan

et al. 2022

Clinical & Translational Med

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Background Studies on biological functions of N6‐methyladenosine (m 6 A) modification in mRNA have sprung up in recent years. Previous studies have reported m 6 A can determine mRNA fate and play a pivotal role in tumour development and progression. The zinc finger protein 677 (ZNF677) belongs to the zinc finger protein family and possesses transcription factor activity by binding sequence‐specific DNA. Methods The expression of ZNF677 and its clinicopathological impact were evaluated in renal cell carcinoma (RCC) patients. The m 6 A level of ZNF677 was determined by m 6 A methylated RNA immunoprecipitation‐sequencing (MeRIP‐seq) and MeRIP‐qPCR in RCC tissues and adjacent normal tissues. RNA immunoprecipitation‐qPCR (RIP‐qPCR) and luciferase assays were performed to identify the targeted effect of IGF2BP2 and YTHDF1 on ZNF677. RCC cells and subcutaneous models uncovered the role of ZNF677 methylated by CRISPR/dCas13b‐METTL3 in tumour growth. ZNF677‐binding sites in the CDKN3 promoter were investigated by chromatin immunoprecipitation (ChIP) and luciferase assays. Results ZNF677 is frequently downregulated in RCC tissues and its low expression is associated with unfavourable prognosis and decreased m 6 A modification level. Further, we find the m 6 A‐modified coding sequence (CDS) of ZNF677 positively regulates its translation and mRNA stability via binding with YTHDF1 and IGF2BP2, respectively. Targeted specific methylation of ZNF677 m 6 A by CRISPR/dCas13b‐METLL3 system can significantly increase the m 6 A and expression level of ZNF677, and dramatically inhibit cell proliferation and induce cell apoptosis of RCC cells. In addition, ZNF677 exerted its tumour suppressor functions in RCC cells through transcriptional repression of CDKN3 via binding to its promoter. In vitro and clinical data confirm the negative roles of ZNF677/CDKN3 in tumour growth and progression of RCC. Conclusion ZNF677 functions as a tumour suppressor and is frequently silenced via m 6 A modification in RCC, which may highlight m 6 A methylation‐based approach for RCC diagnosis and therapy.

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

Cited by 25 publications

References 41 publications

SPAG6 and L1TD1 are transcriptionally regulated by DNA methylation in non-small cell lung cancers

SPAG6 and L1TD1 are transcriptionally regulated by DNA methylation in non-small cell lung cancers

Identification of hub methylated‐CpG sites and associated genes in oral squamous cell carcinoma

ZNF677 suppresses renal cell carcinoma progression through N6‐methyladenosine and transcriptional repression of CDKN3

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