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  • Oncogenomics
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Long-range epigenetic silencing of chromosome 5q31 protocadherins is involved in early and late stages of colorectal tumorigenesis through modulation of oncogenic pathways

Oncogene volume 31, pages 4409–4419 (2012)Cite this article

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Abstract

Loss of tumour suppressor gene function can occur as a result of epigenetic silencing of large chromosomal regions, referred to as long-range epigenetic silencing (LRES), and genome-wide analyses have revealed that LRES is present in many cancer types. Here we utilize Illumina Beadchip methylation array analysis to identify LRES across 800 kb of chromosome 5q31 in colorectal adenomas and carcinomas (n=34) relative to normal colonic epithelial DNA (n=6). This region encompasses 53 individual protocadherin (PCDH) genes divided among three gene clusters. Hypermethylation within these gene clusters is asynchronous; while most PCDH hypermethylation occurs early, and is apparent in adenomas, PCDHGC3 promoter methylation occurs later in the adenoma–carcinoma transition. PCDHGC3 was hypermethylated in 17/28 carcinomas (60.7%) according to methylation array analysis. Quantitative real-time reverse transcription–polymerase chain reaction showed that PCDHGC3 is the highest expressed PCDH in normal colonic epithelium, and that there was a strong reciprocal relationship between PCDHGC3 methylation and expression in carcinomas (R=−0.84). PCDH LRES patterns are reflected in colorectal tumour cell lines; adenoma cell lines are not methylated at PCDHGC3 and show abundant expression at the mRNA and protein level, while the expression is suppressed in hypermethylated carcinoma cell lines (R=−0.73). Short-interfering RNA-mediated reduction of PCDHGC3 led to a decrease of apoptosis in RG/C2 adenoma cells, and overexpression of PCDHGC3 in HCT116 cells resulted in the reduction of colony formation, consistent with tumour suppressor capabilities for PCDHGC3. Further functional analysis showed that PCDHGC3 can suppress Wnt and mammalian target of rapamycin signalling in colorectal cancer cell lines. Taken together, our data suggest that the PCDH LRES is an important tumour suppressor locus in colorectal cancer, and that PCDHGC3 may be a strong marker and driver for the adenoma–carcinoma transition.

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Acknowledgements

This work was supported by a Wellcome Trust VIP award, Cancer Research UK Grants C20791/A12743 and C19/A11975, Kidney Research UK and the Cancer and Leukaemia in Childhood—Sargent Trust. Further funding was from the John and Birthe Meyer Foundation, the Lundbeck Foundation and The European Union’s Seventh Framework Program (SYSCOL HEALTHF5-2010-258236). We thank Dr Greg Phillips for the antibody towards the γ-PCDH constant domain, Professor Moon for reporter plasmids and Professor Vogelstein for mutant HCT116 cells. We also thank Dr Andrea Buda, Dr Katy Petherick, Dr Zsombor Melegh, Dr Newton Wong and Professor Andrew Silver for helpful discussions and comments.

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Authors and Affiliations

  1. Cancer Epigenetics Laboratory, School of Cellular and Molecular Medicine, University of Bristol, Bristol, UK

    A R Dallosso, T J Curry, C Owen, A L Hancock, M Szemes & K Malik

  2. Department of Molecular Medicine, Aarhus University Hospital, Skejby, Denmark

    B Øster, K Thorsen & C L Andersen

  3. Colorectal Cancer Laboratory, School of Cellular and Molecular Medicine, University of Bristol, Bristol, UK

    A Greenhough & C Paraskeva

  4. Developmental Biology, Institute of Biology 1, University of Freiburg, Freiburg, Germany

    M Frank

  5. These authors contributed equally to this work.,

    A R Dallosso & B Øster

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  1. A R Dallosso
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  2. B Øster
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Correspondence to C L Andersen or K Malik.

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Dallosso, A., Øster, B., Greenhough, A. et al. Long-range epigenetic silencing of chromosome 5q31 protocadherins is involved in early and late stages of colorectal tumorigenesis through modulation of oncogenic pathways. Oncogene 31, 4409–4419 (2012). https://doi.org/10.1038/onc.2011.609

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