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RNAseq expression patterns of canine invasive urothelial carcinoma reveal two distinct tumor clusters and shared regions of dysregulation with human bladder tumors.

ABSTRACT: BACKGROUND:Invasive urothelial carcinoma (iUC) is highly similar between dogs and humans in terms of pathologic presentation, molecular subtypes, response to treatment and age at onset. Thus, the dog is an established and relevant model for testing and development of targeted drugs benefiting both canine and human patients. We sought to identify gene expression patterns associated with two primary types of canine iUC tumors: those that express a common somatic mutation in the BRAF gene, and those that do not. METHODS:We performed RNAseq on tumor and normal tissues from pet dogs. Analysis of differential expression and clustering, and positional and individual expression was used to develop gene set enrichment profiles distinguishing iUC tumors with and without BRAFV595E mutations, as well as genomic regions harboring excessive numbers of dysregulated genes. RESULTS:We identified two expression clusters that are defined by the presence/absence of a BRAFV595E (BRAFV600E in humans) somatic mutation. BRAFV595E tumors shared significantly more dysregulated genes than BRAF wild-type tumors, and vice versa, with 398 genes differentiating the two clusters. Key genes fall into clades of limited function: tissue development, cell cycle regulation, immune response, and membrane transport. The genomic site with highest number of dysregulated genes overall lies in a locus corresponding to human chromosome 8q24, a region frequently amplified in human urothelial cancers. CONCLUSIONS:These data identify critical sets of genes that are differently regulated in association with an activating mutation in the MAPK/ERK pathway in canine iUC tumors. The experiments also highlight the value of the canine system in identifying expression patterns associated with a common, shared cancer.

SUBMITTER: Parker HG

PROVIDER: S-EPMC7092566 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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RNAseq expression patterns of canine invasive urothelial carcinoma reveal two distinct tumor clusters and shared regions of dysregulation with human bladder tumors.

Parker Heidi G HG Dhawan Deepika D Harris Alex C AC Ramos-Vara Jose A JA Davis Brian W BW Knapp Deborah W DW Ostrander Elaine A EA

BMC cancer 20200324 1

<h4>Background</h4>Invasive urothelial carcinoma (iUC) is highly similar between dogs and humans in terms of pathologic presentation, molecular subtypes, response to treatment and age at onset. Thus, the dog is an established and relevant model for testing and development of targeted drugs benefiting both canine and human patients. We sought to identify gene expression patterns associated with two primary types of canine iUC tumors: those that express a common somatic mutation in the BRAF gene, ...[more]

PMID: 32209086

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Project description:BackgroundInvasive urothelial carcinoma (iUC) is a major cause of death in humans, and approximately 165,000 individuals succumb to this cancer annually worldwide. Comparative oncology using relevant animal models is necessary to improve our understanding of progression, diagnosis, and treatment of iUC. Companion canines are a preferred animal model of iUC due to spontaneous tumor development and similarity to human disease in terms of histopathology, metastatic behavior, and treatment response. However, the comprehensive molecular characterization of canine iUC is not well documented. In this study, we performed transcriptome analysis of tissue samples from canine iUC and normal bladders using an RNA sequencing (RNA-Seq) approach to identify key molecular pathways in canine iUC.MethodsTotal RNA was extracted from bladder tissues of 11 dogs with iUC and five healthy dogs, and RNA-Seq was conducted. Ingenuity Pathway Analysis (IPA) was used to assign differentially expressed genes to known upstream regulators and functional networks.ResultsDifferential gene expression analysis of the RNA-Seq data revealed 2531 differentially expressed genes, comprising 1007 upregulated and 1524 downregulated genes, in canine iUC. IPA revealed that the most activated upstream regulator was PTGER2 (encoding the prostaglandin E2 receptor EP2), which is consistent with the therapeutic efficiency of cyclooxygenase inhibitors in canine iUC. Similar to human iUC, canine iUC exhibited upregulated ERBB2 and downregulated TP53 pathways. Biological functions associated with cancer, cell proliferation, and leukocyte migration were predicted to be activated, while muscle functions were predicted to be inhibited, indicating muscle-invasive tumor property.ConclusionsOur data confirmed similarities in gene expression patterns between canine and human iUC and identified potential therapeutic targets (PTGER2, ERBB2, CCND1, Vegf, and EGFR), suggesting the value of naturally occurring canine iUC as a relevant animal model for human iUC.

Project description:There is growing evidence that molecular subtypes (e.g. luminal and basal subtypes) affect the prognosis and treatment response in patients with muscle invasive urinary bladder cancer (invasive urothelial carcinoma, iUC). Modeling these subtypes in pre-clinical animal studies is essential, but it is challenging to produce these subtypes, along with other critical host and tumor features, in experimentally-induced animal models. This study was conducted to determine if luminal and basal molecular subtypes are present in naturally-occurring canine iUC, a cancer that mimics the human condition in other key aspects. RNA sequencing was performed on 29 canine treatment naive iUC tissue samples and on four normal canine bladder mucosal samples. Data were aligned to CanFam 3.1, and differentially expressed genes were identified. Unsupervised hierarchical clustering of these genes revealed two distinct groups (n = 13, n = 16). When genes that distinguish basal and luminal subtypes in human cancer (n = 2015) were used to probe genes differentially expressed between normal canine bladder and iUC, 829 enriched signature genes were identified. Unsupervised hierarchical clustering of these genes revealed two distinct groups comprised of 18 luminal subtype tumors and 11 basal subtype tumors. The enriched genes included MMP9, SERPINE2, CAV1, KRT14, and RASA3 in basal tumors, and PPARG, LY6E, CTSE, CDK3, and TBX2 in luminal tumors. In supervised clustering, additional genes of importance in human iUC were identified in canine iUC associated with claudin-low and infiltrated tumors. A smaller panel of genes (n = 60) was identified that distinguished canine luminal and basal iUC with overall 93.1% accuracy. Immune signature patterns similar to those in human iUC were also identified with the greatest enrichment of immune genes being in the basal subtype tumors. These findings provide additional compelling evidence that naturally-occurring canine iUC is a highly relevant and much needed model of human iUC for translational research.

Project description:Urothelial carcinoma (UC) comprises up to 2% of all naturally occurring neoplasia in dogs and can be challenging to diagnose. MicroRNAs (miRNAs) have been reported to be dysregulated in numerous diseases, including neoplasia. MiRNA expression has been evaluated in human UC, but there is limited information regarding the miRNA transcriptome of UC in dogs. Our study aimed to evaluate differential miRNA expression in bladder tissue collected from normal canine urothelium and canine invasive UC (iUC) to elucidate the dysregulated pathways in canine UC. Next-Generation RNA sequencing (RNA-Seq) was performed for dogs with UC (n = 29) and normal canine urothelium (n = 4). Raw RNA data were subjected to normalization, and pairwise comparison was performed using EdgeR with Benjamini-Hochberg FDR multiple testing correction (p < 0.05; >2-fold change) comparing tissue samples of normal urothelium to canine iUC samples. Principal component analysis and hierarchical cluster analysis were performed. MiRNA of FFPE tissue samples of separate iUC (n = 5) and normal urothelium (n = 5) were used to evaluate five miRNAs using RT-qPCR. Pathway analysis was performed utilizing miRWalk, STRING database, and Metascape utilizing KEGG pathways and GO terms databases. Twenty-eight miRNAs were differentially expressed (DE) by RNA-Seq. RT-qPCR confirmed that four miRNAs are significantly downregulated in UC compared to healthy urothelial samples (miR-105a, miR-143, miR-181a, and miR-214). Principal component analysis and hierarchical cluster analysis showed separation between miRNAs in iUC and the control group. The DE miRNAs are most often associated with gene silencing by miRNA, miRNAs in cancer, and miRNAs involved in DNA damage responses. Proteins involved include HRAS, KRAS, ARAF, RAF1, MAPK1, MAP2K1, MAPK3, FGFR3, EGFR, HBEGF, RASSF1, E2F2, E2F3, ERBB2, SRC, MMP1, and UP3KA. The differential expression of miRNAs in canine iUC compared to normal canine urothelial tissue indicates that these markers should be further evaluated for their potential role as diagnostic and therapeutic targets.

Dataset Information

RNAseq expression patterns of canine invasive urothelial carcinoma reveal two distinct tumor clusters and shared regions of dysregulation with human bladder tumors.

Publications

RNAseq expression patterns of canine invasive urothelial carcinoma reveal two distinct tumor clusters and shared regions of dysregulation with human bladder tumors.

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