Anthony R. Dallosso

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

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.

Perilobar Nephrogenic Rests Are Nonobligate Molecular Genetic Precursor Lesions of Insulin-Like Growth Factor-II-Associated Wilms Tumors

Purpose: Perilobar nephrogenic rests (PLNRs) are abnormally persistent foci of embryonal immature blastema that have been associated with dysregulation at the 11p15 locus by genetic/epigenetic means and are thought to be precursor lesions of Wilms tumor. The precise genomic events are, however, largely unknown. Experimental Design: We used array comparative genomic hybridization to analyze a series of 50 PLNRs and 25 corresponding Wilms tumors characterized for 11p15 genetic/epigenetic alterations and insulin-like growth factor-II expression. Results: The genomic profiles of PLNRs could be subdivided into three categories: those with no copy number changes (22 of 50, 44%); those with single, whole chromosome alterations (8 of 50, 16%); and those with multiple gains/losses (20 of 50, 40%). The most frequent aberrations included 1p- (7 of 50, 14%) +18 (6 of 50, 12%), +13 (5 of 50, 10%), and +12 (3 of 50, 6%). For the majority (19 of 25, 76%) of cases, the rest harbored a subset of the copy number changes in the associated Wilms tumor. We identified a temporal order of genomic changes, which occur during the insulin-like growth factor-II/PLNR pathway of Wilms tumorigenesis, with large-scale chromosomal alterations such as 1p-, +12, +13, and +18 regarded as “early” events. In some of the cases (24%), the PLNRs harbored large-scale copy number changes not observed in the concurrent Wilms tumor, including +10p, +14q, and +18. Conclusions: These data suggest that although the evidence for PLNRs as precursors is compelling, not all lesions must necessarily undergo malignant transformation.

Genome‐wide DNA methylation analysis identifies MEGF10 as a novel epigenetically repressed candidate tumor suppressor gene in neuroblastoma

Neuroblastoma is a childhood cancer in which many children still have poor outcomes, emphasising the need to better understand its pathogenesis. Despite recent genome‐wide mutation analyses, many primary neuroblastomas do not contain recognizable driver mutations, implicating alternate molecular pathologies such as epigenetic alterations. To discover genes that become epigenetically deregulated during neuroblastoma tumorigenesis, we took the novel approach of comparing neuroblastomas to neural crest precursor cells, using genome‐wide DNA methylation analysis. We identified 93 genes that were significantly differentially methylated of which 26 (28%) were hypermethylated and 67 (72%) were hypomethylated. Concentrating on hypermethylated genes to identify candidate tumor suppressor loci, we found the cell engulfment and adhesion factor gene MEGF10 to be epigenetically repressed by DNA hypermethylation or by H3K27/K9 methylation in neuroblastoma cell lines. MEGF10 showed significantly down‐regulated expression in neuroblastoma tumor samples; furthermore patients with the lowest‐expressing tumors had reduced relapse‐free survival. Our functional studies showed that knock‐down of MEGF10 expression in neuroblastoma cell lines promoted cell growth, consistent with MEGF10 acting as a clinically relevant, epigenetically deregulated neuroblastoma tumor suppressor gene.