Forerunner genes contiguous to RB1 contribute to the development of in situ neoplasia

Abstract

We used human bladder cancer as a model system and the whole-organ histologic and genetic mapping strategy to identify clonal genetic hits associated with growth advantage, tracking the evolution of bladder cancer from intraurothelial precursor lesions. Six putative chromosomal regions critical for clonal expansion of intraurothelial neoplasia and development of bladder cancer were identified by using this approach. Focusing on one of the regions, which includes the model tumor suppressor RB1, we performed allelotyping of single-nucleotide polymorphic sites and identified a 1.34-Mb segment around RB1 characterized by a loss of polymorphism associated with the initial expansion of in situ neoplasia. This segment contains several positional candidate genes referred to by us as forerunner genes that may contribute to such expansion. We subsequently concentrated our efforts on the two neighbor genes flanking RB1, namely ITM2B and CHC1L, as well as P2RY5, which is located inside RB1. Here, we report that ITM2B and P2RY5 modulated cell survival and were silenced by methylation or point mutations, respectively, and thus by functional loss may contribute to the growth advantage of neoplasia. We also show that homozygous inactivation of P2RY5 was antecedent to the loss of RB1 during tumor development, and that nucleotide substitutions in P2RY5 represent a cancer predisposing factor.bladder cancer ͉ single-nucleotide polymorphic sites ͉ whole-organ histologic and genetic mapping M any common epithelial cancers, including those arising in the bladder, begin as clonal in situ expansion of neoplastic cells, which show no or minimal deviation from the normal phenotype (1-4). Such lesions often form plaques involving large areas of the affected mucosa, and their expansion precedes the development of microscopically recognizable dysplasia or carcinoma in situ (4, 5). Identification of chromosomal regions associated with the initial expansion of neoplasia is a requisite for more specific studies of their positional candidate genes that may drive the initial clonal expansion of neoplasia.We have used an approach referred to as whole-organ histologic and genetic mapping (WOHGM) to identify clonal hits associated with growth advantage, tracking the evolution of human bladder cancer from occult in situ lesions to invasive disease on a total genomic scale (6-8). Human bladder carcinoma was used as a model of common epithelial malignancy that develops by progression of microscopically recognizable intraurothelial preneoplastic lesions ranging from mild dysplasia to carcinoma in situ (5). Bladder cancer was also selected because it is close to an ideal model human tumor to study the early events of carcinogenesis due to simple anatomy of the organ and the ease in mapping preneoplastic conditions geographically across the entire mucosa of cystectomy specimens (9-11).In this paper, we begin by presenting the identification of the six critical chromosomal regions that may contain genes driving the development of bladder cancer. Then, we f...

Molecular biology of bladder cancer: new insights into pathogenesis and clinical diversity

“…No FGFR3 mutations are reported in CIS 146 . In morphologically 'normal' or dysplastic urothelium adjacent to MIBC, alterations found in the tumour are present, indicating spread of cells from the tumour or tumour development within a field of altered urothelium [147][148][149][150] . Detailed mapping of entire cystectomy specimens reveals that in the absence of detectable abnormality, large macroscopically 'normal' urothelial patches show LOH of specific chromosomal regions.…”

“…Thus, it is envisaged that the development of MIBC involves clonal expansion, within which sub-clones with additional alterations arise (Figure 4). Six critical regions of LOH were identified within one of which, biallelic inactivation of integral membrane protein 2B (ITM2B) and lysophosphatidic acid receptor 6 (LPAR6, also known as P2RY5) were demonstrated 149,151 .…”

The origins of bladder cancer

“…44,45 The detailed analysis of P2RY5 polymorphisms, including germ-line mutations, provides remarkable data in support of potential inherited host susceptibility to the carcinogenic effects of smoking. Mapping of the P2RY5 polymorphisms in normal bladder mucosa demonstrated that this gene, which resides within intron 17 of the RB1 gene, is affected before loss of RB expression during bladder carcinogenesis.…”

“…This is based on the concept that these genes are relevant for the development of the initial clonal expansion of in situ urothelial neoplasia. 44 …”

“…Using a whole-organ mapping strategy, Czerniak and coworkers [42][43][44] have previously examined genetic hits across the entire mucosa of bladders affected by cancer. Their construction of a genome-wide map over the entire mucosa has enabled identification not only of genetic alterations in mucosal regions of evident dysplasia, but also in morphologically normal mucosa.…”