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CpG island hypermethylation frequently silences FILIP1L isoform 2 expression in prostate cancer.

ABSTRACT: Senescence related regulatory pathways serve as barriers to cancer immortalization and progression but they are currently not well defined. FILIP1L is a growth inhibitory gene with multiple isoforms whose expression is increased in senescent prostate and prostate cancer cells, and decreased in many cancers. We investigated whether DNA methylation regulates FILIP1L in senescence and in prostate cancer development.FILIP1L mRNA expression was assessed in prostate cancer and associated normal prostate tissues using quantitative polymerase chain reaction. A tissue microarray was constructed using 95 prostate cancer specimens and 45 benign prostate specimens. Vectra™ imaging was used to quantitate nuclear and cytoplasmic FILIP1L protein expression. Bisulfite sequencing and Pyrosequencing® were used to assess methylation. Prostate cancer cell lines were treated with 2'-deoxy-5-azacytidine and mRNA expression was assessed.FILIP1L isoform 2 mRNA was increased in replicatively senescent human prostate epithelial cells and decreased in prostate cancer specimens. We verified a reduction in nuclear FILIP1L protein in prostate cancer using tissue microarrays (p = 0.006). A CpG island 5' of the isoform 2 translational start site was identified that showed hypermethylation in prostate cancer cell lines and tumors compared to normal prostate cells and tissues. Pyrosequencing confirmed FILIP1L hypermethylation in all 14 tumors compared to paired normal tissues (p <0.0001). Isoform 2 expression was induced in prostate cancer cell lines using 2'-deoxy-5-azacytidine.FILIP1L isoform 2 is one of the most commonly hypermethylated genes in prostate cancer. It may serve as an important marker of prostate cancer. Isoform 2 expression is associated with senescence and its down-regulation may represent an early important biological event in prostate cancer development.

SUBMITTER: Desotelle J

PROVIDER: S-EPMC3581042 | biostudies-literature | 2013 Jan

REPOSITORIES: biostudies-literature

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CpG island hypermethylation frequently silences FILIP1L isoform 2 expression in prostate cancer.

Desotelle Joshua J Truong Matthew M Ewald Jonathan J Weeratunga Pushpa P Yang Bing B Huang Wei W Jarrard David D

The Journal of urology 20121120 1

<h4>Purpose</h4>Senescence related regulatory pathways serve as barriers to cancer immortalization and progression but they are currently not well defined. FILIP1L is a growth inhibitory gene with multiple isoforms whose expression is increased in senescent prostate and prostate cancer cells, and decreased in many cancers. We investigated whether DNA methylation regulates FILIP1L in senescence and in prostate cancer development.<h4>Materials and methods</h4>FILIP1L mRNA expression was assessed i ...[more]

PMID: 23174249

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Project description:Much effort in cancer research has focused on the tiny part of our genome that codes for mRNA. However, it has recently been recognized that microRNAs also contribute decisively to tumorigenesis. Studies have also shown that epigenetic silencing by CpG island hypermethylation of microRNAs with tumor suppressor activities is a common feature of human cancer. The importance of other classes of non-coding RNAs, such as long intergenic ncRNAs (lincRNAs) and transcribed ultraconserved regions (T-UCRs) as altered elements in neoplasia, is also gaining recognition. Thus, we wondered whether there were other ncRNAs undergoing CpG island hypermethylation-associated inactivation in cancer cells. We focused on the small nucleolar RNAs (snoRNAs), a subset of ncRNA with a wide variety of cellular functions, such as chemical modification of RNA, pre-RNA processing and control of alternative splicing. By data mining snoRNA databases and the scientific literature, we selected 49 snoRNAs that had a CpG island within ? 2 Kb or that were processed from a host gene with a 5'-CpG island. Bisulfite genomic sequencing of multiple clones in normal colon mucosa and the colorectal cancer cell line HCT-116 showed that 46 snoRNAs were equally methylated in the two samples: completely unmethylated (n = 26) or fully methylated (n = 20). Most interestingly, the host gene-associated 5'-CpG islands of the snoRNAs SNORD123, U70C and ACA59B were hypermethylated in the cancer cells but not in the corresponding normal tissue. CpG island hypermethylation was associated with the transcriptional silencing of the respective snoRNAs. Results of a DNA methylation microarray platform in a comprehensive collection of normal tissues, cancer cell lines and primary malignancies demonstrated that the observed hypermethylation of snoRNAs was a common feature of various tumor types, particularly in leukemias. Overall, our findings indicate the existence of a new subclass of ncRNAs, snoRNAs, that are targeted by epigenetic inactivation in human cancer.

Project description:BackgroundTumor cell lines are commonly used as experimental tools in cancer research, but their relevance for the in vivo situation is debated. In a series of 11 microsatellite stable (MSS) and 9 microsatellite unstable (MSI) colon cancer cell lines and primary colon carcinomas (25 MSS and 28 MSI) with known ploidy stem line and APC, KRAS, and TP53 mutation status, we analyzed the promoter methylation of the following genes: hMLH1, MGMT, p16INK4a (CDKN2A alpha-transcript), p14ARF (CDKN2A beta-transcript), APC, and E-cadherin (CDH1). We compared the DNA methylation profiles of the cell lines with those of the primary tumors. Finally, we examined if the epigenetic changes were associated with known genetic markers and/or clinicopathological variables.ResultsThe cell lines and primary tumors generally showed similar overall distribution and frequencies of gene methylation. Among the cell lines, 15%, 50%, 75%, 65%, 20% and 15% showed promoter methylation for hMLH1, MGMT, p16INK4a, p14ARF, APC, and E-cadherin, respectively, whereas 21%, 40%, 32%, 38%, 32%, and 40% of the primary tumors were methylated for the same genes. hMLH1 and p14ARF were significantly more often methylated in MSI than in MSS primary tumors, whereas the remaining four genes showed similar methylation frequencies in the two groups. Methylation of p14ARF, which indirectly inactivates TP53, was seen more frequently in tumors with normal TP53 than in mutated samples, but the difference was not statistically significant. Methylation of p14ARF and p16INK4a was often present in the same primary tumors, but association to diploidy, MSI, right-sided location and female gender was only significant for p14ARF. E-cadherin was methylated in 14/34 tumors with altered APC further stimulating WNT signaling.ConclusionsThe present study shows that colon cancer cell lines are in general relevant in vitro models, comparable with the in vivo situation, as the cell lines display many of the same molecular alterations as do the primary carcinomas. The combined pattern of epigenetic and genetic aberrations in the primary carcinomas reveals associations between them as well as to clinicopathological variables, and may aid in the future molecular assisted classification of clinically distinct stages.

Dataset Information

CpG island hypermethylation frequently silences FILIP1L isoform 2 expression in prostate cancer.

Publications

CpG island hypermethylation frequently silences FILIP1L isoform 2 expression in prostate cancer.

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