Forerunner Genes Research Articles

Field effect and forerunner genes drive bladder cancer initiation.

Field effect and forerunner genes drive bladder cancer initiation.

Abstract 5172: Forerunner genes contribute to bladder carcinogenesis by altering the basal to luminal urothelial transition program

Abstract Background: The idea that microscopically recognizable dysplasia and carcinoma in situ act as precursor conditions for invasive cancer is the prevailing view of how many common epithelial malignancies develop. However, new evidence suggests that these are not the initiating events of carcinogenesis but represent the evolution of pre-existing occult disease, caused by the field effects of carcinogens and/or chronic inflammation, which shows minimal phenotypic deviation from normal tissue. These effects may form large plaques in the mucosa of the affected organ but the mechanisms driving their developments are unknown. Design: We developed a strategy that combines histologic and genetic mapping that permits interrogation of the chronology of genetic changes associated with cancer development on a whole-organ scale. By using this approach, we analyzed the sequence of genetic alterations contiguous to the tumor suppressor RB1 and identified a set of alternative target genes that we term “forerunner” (FR) genes (ITM2B, LPAR6, MLNR, CAB39L, and ARL11) whose silencing was associated with development of clonal plaque-like mucosal field effects initiating bladder carcinogenesis. We focused on two model FR genes, LPAR6 and CAB39L, and validated their involvement in the development of mucosal field effects by in vitro mechanistic studies and in vivo models. Results: We provide evidence that silencing of prototypic FR genes by hypermethylation (LPAR6 and CAB39L) and less frequently by mutations (LPAR6) is associated with the initial expansion of intraurothelial neoplasia and sets the stage for subsequent events of carcinogenesis. We also found that LPAR6 and CAB39L control cell survival and proliferation consistent with the loss of function being contributory to tumorigenesis. We validated their involvement in three large independent cohorts of bladder cancer, which were profiled according to a novel molecular taxonomy and show that they distinctively contribute to the development of luminal (LPAR6) and basal (CAB39L) subtypes. The in vitro studies using cell lines and their ability to form urospheres showed that these genes contribute to carcinogenesis by altering the transition from basal to luminal phenotypes. Consistent with these observations the BBN induced cancers in Lpar6−/− mice were of luminal type while the cancers induced in Lpar6+/+ mice were of basal type. Conclusion: Loss of prototypic FR genes contributes to the development of mucosal field effects initiating bladder carcinogenesis by altering the basal to luminal urothelial transition program. Citation Format: Sangkyou Lee, Jolanta Bondaruk, Sooyoung Lee, June G. Lee, Tadeusz Majewski, Woonyoung Choi, Charles Guo, Colin Dinney, Li Zhang, Keith Baggerly, Richard Behringer, David McConkey, Bogdan Czerniak. Forerunner genes contribute to bladder carcinogenesis by altering the basal to luminal urothelial transition program. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5172.

Origins of Bladder Cancer

Bladder cancer, one of the most frequently occurring human cancers, develops via two tracks referred to as papillary and nonpapillary that correspond to clinically different forms of the disease. Most bladder cancers are chemically induced, with tobacco smoking being the leading risk factor. Recent advances in bladder cancer research have enhanced our understanding of the origin of this disease from urothelial progenitor cells via field effects along papillary/luminal and nonpapillary/basal pathways. Evident from the outset of the disease, the diversity of the luminal and basal pathways, together with cell lineage tracing studies, postulates the origin of molecularly distinct subtypes from different uroprogenitor cells. The molecular mechanisms initiating field effects involve a new class of genes referred to as forerunner (FR) genes that generally map around major tumor suppressors such as RB1. These genes are silenced, predominantly by hypermethylation and less frequently by mutations, and drive the expansion of intraurothelial preneoplastic cells. Different FR genes are involved in various molecular subtypes of bladder cancer and they sensitize the uroprogenitor cells to the development of luminal and basal bladder cancers in animal models. In human bladder cancer, luminal and basal forms have dissimilar clinical behavior and response to conventional and targeted chemotherapeutic manipulations. These new research developments hold the promise of expanding our armamentarium of diagnostic and treatment options for patients with bladder cancer and improving our ability to select patients most likely to respond to a specific therapy.

Abstract 307: Germline ablation of candidate forerunner gene, P2RY5, in a mouse model.

Abstract Our prior studies have suggested that genes mapping around the tumor suppressor RB1 termed forerunner (FR) genes were silenced by methylation or mutations in in situ phases of urothelial carcinogenesis and were potentially driving the development of clonal mucosal field effects. In order to verify these correlative observations, we have developed a mouse ablation model of a candidate FR gene, P2RY5 mapping within intron 17 of RB1 and encoding G protein receptor. In order to facilitate a germline ablation of P2RY5, a knock-out construct was generated to delete the P2RY5 reading frame. The construct was inserted into mouse embryonic stem cells and the gene was ablated by mating the animals with CMV-cre mice. The effect of P2RY5 deletion on cell proliferation was assessed on cultured fibroblasts derived from embryos with P2RY5 ablation (P2RY5-/-) and from control embryos (P2RY5 +/+). The effect of P2RY5 ablation in the urothelium was assessed on bladders of P2RY5-/- and P2RY5 +/+ mice. The bladders were fixed in formalin and the proliferation status in the urothelium was assessed by immunohistochemical and immunoflourescent staining for Ki67and PCNA. The status of the urothelium was assessed by immunohistochemical and immunoflourescent staining for cytokeratins 5, 14, and 18. In addition, the effect of silencing P2RY5 on global gene expression patterns was evaluated on mouse embryonic fibroblasts using Illumina mouse cDNA expression microarrays. P2RY5-/- embryonic fibroblasts showed dramatically increased proliferation and colony formation rates as compared to P2RY5 +/+ cells. cDNA micro analysis shows that silencing P2RY5 activated several important oncogenic pathways including NFκB and STAT3. Similarly, P2RY5-/- urothelium contained nearly three times more proliferating cells as compared to P2RY5 +/+ urothelium. The increased proliferation rate was associated with the striking expansion of the peribasal layer as documented by staining for cytokeratin 5 and 14. In the P2RY5 +/+ urothelium the cytokeratin 5 and 14 positive cells formed a sparse single basal layer. In contrast, in the P2RY5-/- urothelium, these cells formed irregular mounds overcrowded with peribasal cells. Taken together, our observations show that the silencing of P2RY5 increases the growth rate of mouse embryonic fibroblasts via the activation of several oncogenic pathways and causes an increase in the proliferation rate in the urothelium resulting in urothelial hyperplasia recapitulating incipient events of urothelial carcinogenisis. Citation Format: Bogdan A. Czerniak, Sangkyou Lee, Ying Wang, Jolanta Bondaruk, Taddeusz Majewski, Woongyoung Choi, David McConkey, Richard R. Behringer. Germline ablation of candidate forerunner gene, P2RY5, in a mouse model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 307. doi:10.1158/1538-7445.AM2013-307

Abstract 3065: Loss of ARL11 function promotes growth of in situ neoplasia by activating the ras pathway

Abstract Our previously reported whole-organ genome-wide mapping studies of bladder cancer provided evidence that a new class of genes, referred to as forerunner (FR) genes (ITM2B, P2RY5, GPR38 and ARL11), located near known tumor suppressors such as RB1, may drive early clonal expansion of neoplasia. (S Lee, et al. PNAS 104: 13732-13737, 2007; T Majewski et al. Lab Inv 88: 694-712, 2008) These studies suggested three major steps for the involvement of the RB1 locus in tumor development. In the first step, one allele of FR gene and RB1 is inactivated by large deletions. In the second step, homozygous inactivation of the FR genes is accomplished by hypermethylation or less frequently by missence mutations. Homozygous inactivation of FR genes is associated with the clonal expansion of in situ lesions referred to as low-grade intraurothelial neoplasia. In the final, third step, the contiguous tumor suppressor, such as RB1, is inactivated most commonly by a mutation. This step is associated with clonal evolution into a transformed phenotype with microscopic features of carcinoma in situ progressing to invasive cancer. Recently, we concentrated our efforts on the ARL11 candidate FR gene, which encodes ADP-ribosylation factor-like 11 protein, a ras-related member of the small GTPases, that functions as GDP/GTP nucleotide interswitch for signal transduction. We have shown that it has been silenced by hypermethylation in approximately 40% of urothelial carcinomas of the bladder and the silencing was associated with in situ expansion of intraurothelial neoplasia. Moreover, loss of ARL11 expression was almost mutually exclusive with the FGFR3 mutations (<5% of bladder tumors showed a co-existence of mutant FGFR3 and methylated ARL11). Functional studies conducted on immortalized normal urothelial cells grown in vitro showed that ARL11 reduced cell proliferation, which was mediated by the inhibition of the ras pathway. This effect was associated with the down-regulation of the active ras and ERK1/2 phosphorylation as well as with the up-regulation of negative cell cycle regulator proteins such as p27kip1 and parallel down-regulation of cyclins D1 and E. Conversely, the silencing of ARL11 conferred a tumor suppressor effect promoting cell proliferation via the activation of the ras signaling pathway. In summary, our studies show that recessive events in the RB1 locus associated with the loss of ARL11 function promote clonal expansion of intraurothelial precursor lesions by activating the ras pathway in early occult phases of human bladder carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3065.

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

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.