Oncogene (2012) 31, 4409–4419 & 2012 Macmillan Publishers Limited All rights reserved 0950-9232/12 www.nature.com/onc ONCOGENOMICS Long-range epigenetic silencing of chromosome 5q31 protocadherins is involved in early and late stages of colorectal tumorigenesis through modulation of oncogenic pathways AR Dallosso1,5, B Øster2,5, A Greenhough3, K Thorsen2, TJ Curry1, C Owen1, AL Hancock1, M Szemes1, C Paraskeva3, M Frank4, CL Andersen2 and K Malik1 1Cancer Epigenetics Laboratory, School of Cellular and Molecular Medicine, University of Bristol, Bristol, UK; 2Department of Molecular Medicine, Aarhus University Hospital, Skejby, Denmark; 3Colorectal Cancer Laboratory, School of Cellular and Molecular Medicine, University of Bristol, Bristol, UK and 4Developmental Biology, Institute of Biology 1, University of Freiburg, Freiburg, Germany Loss of tumour suppressor gene function can occur as a PCDH LRES is an important tumour suppressor locus in result of epigenetic silencing of large chromosomal colorectal cancer, and that PCDHGC3 may be a strong regions, referred to as long-range epigenetic silencing marker and driver for the adenoma–carcinoma transition. (LRES), and genome-wide analyses have revealed that Oncogene (2012) 31, 4409–4419; doi:10.1038/onc.2011.609; LRES is present in many cancer types. Here we utilize published online 16 January 2012 Illumina Beadchip methylation array analysis to identify LRES across 800 kb of chromosome 5q31 in colorectal Keywords: colorectal cancer; protocadherin; epigenetic adenomas and carcinomas (n ¼ 34) relative to normal silencing; Wnt; mTOR colonic epithelial DNA (n ¼ 6). This region encompasses 53 individual protocadherin (PCDH) genes divided among three gene clusters. Hypermethylation within these gene clusters is asynchronous; while most PCDH hyper- Introduction methylation occurs early, and is apparent in adenomas, PCDHGC3 promoter methylation occurs later in the The development of colorectal cancers is characterized adenoma–carcinoma transition. PCDHGC3 was hyper- by the accumulation of multiple genetic and epigenetic methylated in 17/28 carcinomas (60.7%) according to alterations (Cho and Vogelstein, 1992). In approxi- methylation array analysis. Quantitative real-time reverse mately 60–80% of sporadic colorectal cancers (CRCs), transcription–polymerase chain reaction showed that tumorigenesis is initiated by mutations of the adenoma- PCDHGC3 is the highest expressed PCDH in normal tous polyposis coli (APC) gene (Miyoshi et al., 1992; colonic epithelium, and that there was a strong reciprocal Powell et al., 1992). As APC is an essential component relationship between PCDHGC3 methylation and expres- of the destruction complex that regulates the turnover of sion in carcinomas (R ¼À0.84). PCDH LRES patterns b-catenin, a transcriptional co-activator of the WNT are reflected in colorectal tumour cell lines; adenoma cell signalling pathway (Klaus and Birchmeier, 2008), this lines are not methylated at PCDHGC3 and show highlights constitutive activation of the Wnt/b-catenin abundant expression at the mRNA and protein level, signalling pathway as a prerequisite for colorectal while the expression is suppressed in hypermethylated tumorigenesis. Subsequent somatic mutations that are carcinoma cell lines (R ¼À0.73). Short-interfering RNA- frequently identified in colorectal cancers (CRCs) are mediated reduction of PCDHGC3 led to a decrease of considered ‘drivers’ necessary for tumorigenesis (Wood apoptosis in RG/C2 adenoma cells, and overexpression of et al., 2007), and mutations in the BRAF, KRAS, PCDHGC3 in HCT116 cells resulted in the reduction of PIK3CA, PTEN and TP53 genes reflect the importance of the RAS-MAPK, PI(3)K and p53 pathways (Fearon, colony formation, consistent with tumour suppressor ONCOGENOMICS capabilities for PCDHGC3. Further functional analysis 2011). The majority (B80%) of sporadic CRCs exhibit showed that PCDHGC3 can suppress Wnt and mamma- chromosomal instability characterized by aneuploidy lian target of rapamycin signalling in colorectal cancer (Pino and Chung, 2010), whereas a smaller group cell lines. Taken together, our data suggest that the (B15%) with defective mismatch repair exhibit micro- satellite instability (MSI). MSI is also associated with an increased frequency of DNA hypermethylation at Correspondence: Dr CL Andersen, Department of Molecular multiple gene loci and such tumours have been defined Medicine, Aarhus University Hospital, Skejby, DK 8200, Denmark or as having a CpG island methylator phenotype (CIMP) Dr K Malik, Cancer Epigenetics Laboratory, School of Cellular and (Ahuja et al., 1997; Weisenberger et al., 2006). Molecular Medicine, University of Bristol, Bristol BS8 1TD, UK. Although tumour-specific promoter hypermethylation E-mail: [email protected] or [email protected] 5These authors contributed equally to this work. is prevalent in many cancers, including colon cancer, the Received 10 September 2011; revised 14 November 2011; accepted 23 stage specificity and consequences of diverse epigenetic November 2011; published online 16 January 2012 alterations are less well defined than genetic defects Long-range epigenetic silencing of protocadherins in colorectal cancer AR Dallosso et al 4410 (Jones and Baylin, 2007). Genome-wide DNA hypo- and proliferation. Indeed, our studies of selected g- methylation is ubiquitous even in early benign tumours PCDHs, PCDHGA2, PCDHGA6 or PCDHGA12, in and is linked to chromosomal instability (Ji et al., 1997; Wilms’ tumour demonstrated their ability to suppress Matsuzaki et al., 2005), oncogene activation (Baylin and Wnt pathway activity in vitro (Dallosso et al., 2009); this Ohm, 2006) and deregulation of imprinted loci (Cui was supported by proteomic analysis of the g-PCDH- et al., 2003). Hypermethylation of the SFRP Wnt interacting proteins, which showed interactions with antagonists (Suzuki et al., 2004) and the tumour b-catenin (Han et al., 2010), together with several other suppressor RASSF1 (Greenspan et al., 2006) is observed proteins implicated in carcinogenesis. in aberrant crypt foci; although most instances of We previously detected hypermethylation of some hypermethylation occur early in colorectal tumorigen- 5q31 PCDH genes in the colorectal cancer cell line esis, a small subset of ‘late’ silenced genes, including HCT116, prompting us to investigate whether PCDH MLH1, are carcinoma specific and may be associated LRES might also occur in primary colorectal cancers. with malignant progression (Ibrahim et al., 2011; Oster Here we show that 5q31 LRES is also frequent in et al., 2011). colorectal adenoma (CRA) and carcinoma. We establish Taken together with gene-specific silencing events, that, unlike the majority of silencing events within the epigenetic suppression can also involve transcriptional LRES region, protocadherin PCDHGC3 is largely lock-down spanning large chromosomal domains, unmethylated in normal and adenoma tissues, but resulting in the coordinate silencing of numerous genes. frequently epigenetically silenced in carcinomas. As this Such long-range epigenetic silencing (LRES) has been is also the highest expressed PCDH in normal colonic described in colon cancer at chromosome 2q14.2, at the epithelium, the role of PCDHGC3 was examined in MLH1 locus on 3p22 and, most recently, the Ikaros CRA and carcinoma models. PCDHGC3 is capable of locus on 7p12 (Frigola et al., 2006; Hitchins et al., 2007; negatively regulating Wnt and mammalian target of Javierre et al., 2011). LRES and CIMP are suggested to rapamycin (mTOR) signalling consistent with tumour share a common aetiology (Wong et al., 2011). LRES suppressor function in colorectal cancer. has also been identified at additional loci and in different adult cancer types, including lung (Rauch et al., 2007), breast (Novak et al., 2008) and prostate Results cancer (Coolen et al., 2010), and also in Wilms’ tumour- a paediatric kidney cancer (Dallosso et al., 2009). A Epigenetic alterations of multiple 5q31 protocadherins common 800 kbp LRES region identified in breast in colorectal tumorigenesis and carcinoma-associated cancer and Wilms’ tumour resides on chromosome silencing of PCDHGC3 5q31; this region contains 53 genes of the protocadherin We examined the DNA methylation profile of genes at (PCDH) superfamily in three multi-gene clusters (a-, b- 5q31 (Figure 1a) in normal colonic epithelia from 6 non- and g-PCDHs; Human Genome Organization nomen- cancer individuals, 6 CRAs (all microsatellite stable clature PCDHA@, PCDHB@ and PCDHG@, respec- (MSS)) and 28 CRCs (five MSI and 23 MSS) using tively). methylation arrays. The majority of PCDHs from PCDH functions have predominantly been studied in PCDHA@, PCDHB@ and PCDHG@ are hypermethy- neuronal cells, although PCDH expression is also lated in either adenomas, carcinomas or both, relative to observed in other cells including gut, lung and kidney normal tissue (18 of 22 PCDHs examined by the arrays, (Bass et al., 2007; Dallosso et al., 2009). PCDHs have 82%) (Figure 1b), and all adenomas and carcinomas extracellular cadherin domains involved in cell adhesion displayed hypermethylation of genes spanning the three (Obata et al., 1995; Fernandez-Monreal et al., 2009; PCDH clusters. Methylation at non-PCDH genes Schreiner and Weiner, 2010) and intracellular domains, flanking the gene cluster is stable compared with the which can translocate to the nucleus and regulate gene widespread changes observed in all three of the PCDH expression (Haas et al., 2005; Hambsch et al., 2005). clusters (Figure 1c). None