Cross-species oncogenomics offers insight into human muscle-invasive bladder cancer

Abstract

Background: In humans, muscle-invasive bladder cancer (MIBC) is highly aggressive and associated with a poor prognosis. With a high mutation load and large number of altered genes, strategies to delineate key driver events are necessary. Dogs and cats develop urothelial carcinoma (UC) with histological and clinical similarities to human MIBC. Cattle that graze on bracken fern also develop UC, associated with exposure to the carcinogen ptaquiloside. These species may represent relevant animal models of spontaneous and carcinogen-induced UC that can provide insight into human MIBC.

Results: Whole-exome sequencing of domestic canine (n = 87) and feline (n = 23) UC, and comparative analysis with human MIBC reveals a lower mutation rate in animal cases and the absence of APOBEC mutational signatures. A convergence of driver genes (ARID1A, KDM6A, TP53, FAT1, and NRAS) is discovered, along with common focally amplified and deleted genes involved in regulation of the cell cycle and chromatin remodelling. We identify mismatch repair deficiency in a subset of canine and feline UCs with biallelic inactivation of MSH2. Bovine UC (n = 8) is distinctly different; we identify novel mutational signatures which are recapitulated in vitro in human urinary bladder UC cells treated with bracken fern extracts or purified ptaquiloside.

Conclusion: Canine and feline urinary bladder UC represent relevant models of MIBC in humans, and cross-species analysis can identify evolutionarily conserved driver genes. We characterize mutational signatures in bovine UC associated with bracken fern and ptaquiloside exposure, a human-linked cancer exposure. Our work demonstrates the relevance of cross-species comparative analysis in understanding both human and animal UC.

Keywords: Bovine; Bracken; Cancer; Canine; Cross-species comparison; Feline; Mutational signature; Ptaquiloside; Pteridium aquilinum; Urinary bladder.

Fig. 1

Somatic mutational landscape of canine urinary bladder UC. a Somatic mutations in genes mutated in five or more samples. Asterisk indicates significantly mutated gene (SMG); GPRASP1 is also a SMG but is not shown, as it is mutated in 2 samples. A summary of the canine cases analyzed in this study is provided in Additional File 1: Table S1a and a full list of variants is provided in Additional File 3: Table S2. b Single base substitution (SBS) observed mutational spectra and reconstructed spectra for samples DD0194a and DD0355a. Mutational signatures SBS1 and SBS6 were found in both samples as well as SBS21 in sample DD0194a. The reconstructed mutational spectra based on these signatures have cosine similarity > 0.93 when compared to the observed spectra

Fig. 2

Somatic mutational landscape of feline urinary bladder UC. a Somatic mutations in genes mutated in three or more samples. Asterisk indicates significantly mutated gene (SMG). A summary of feline cases analyzed in this study is provided in Additional File 1: Table S1b and a list of all variants is provided in Additional File 4: Table S3. b Single base pair (SBS) mutational spectrum for sample CATD0037a (upper panel). The activities of mutational signatures SBS1, SBS6, SBS20, and SBS21 are shown in the reconstructed mutational spectrum (lower panel). c Indel mutational spectrum for sample CATD0037a, showing a prevalence of single base pair deletions in homopolymer regions. d Penetrance plot showing somatic copy number gains and losses 5 Mb or larger in 1-Mb windows along each chromosome. Only samples with sufficient quality, based on manual inspection of Sequenza plots, are represented (n = 21; see ‘‘Methods’’)

Fig. 3

Comparative mutational landscape of human, canine, and feline urinary bladder UC. a The proportion of human MIBC cases [22] (n = 412) with somatic mutations in COSMIC Cancer Gene Census genes that have a one-to-one orthologous relationship with both a canine and a feline gene. Shown are mutations present in 4 or more canine or feline samples, which are prefixed with DD and CATD, respectively. Also shown are canine samples with MDM2 amplification, which is shown in the same row as TP53 mutations, to enable visual comparison with feline and human TP53 mutations. CDKN2A has not been included, as, although the feline cohort had 4 samples with CDKN2A mutations, Ensembl does not classify the human and feline genes as orthologs, and, in canines, human CDKN2B is designated an ortholog of canine CDKN2A. b Circos plot displaying genomic regions with recurrent somatic copy number alterations in human, feline, and canine UC. Chromosomes are represented by the outer track. Data for human chromosome X was not available. The histogram (inner track) shows the frequencies of copy number gains (purple, blue, and green) and losses (orange, red, and yellow) in human, canine, and feline, respectively. Links between chromosomes show syntenic regions within recurrently amplified/deleted chromosomes (feline and canine) or chromosome arms (human). Red links represent deletions and purple links represent amplifications. Genes shown in orange and purple text are in syntenic regions in chromosomes or chromosome arms that were recurrently deleted or amplified, respectively, in all 3 species. Genes in red and blue text are genes that were focally amplified or deleted in 2 or more species. ARHGEF10 is the only gene focally deleted in all 3 species. Shown in black text are other genes of interest

Fig. 4

Recurrently mutated Cancer Gene Census (CGC) genes in bovine urinary bladder UC. Shown are COSMIC CGC genes mutated in at least 6 bovine UC cases (left), and the proportions of human UC cases [22] with mutations in these genes (right). Genes shown are those that had a one-to-one orthologous relationship between the human and bovine gene

Fig. 5

Mutational signatures in bovine UC. a Signature extraction identified 2 novel single base substitution (SBS) signatures, designated Signatures BF-A and BF-B. The SBS mutational spectra are comprised of 96 substitution types, which are derived from six possible SBS mutations, each with 4 possible bases directly 5′ and 3′. The observed and reconstructed SBS mutation spectra of the samples with the lowest and highest mutation rates, BTAUD0029a (b) and BTAU0055a (c), respectively, are shown. d The absolute (upper panel) and relative (lower panel) proportion of mutations attributed to Signatures BF-A and BF-B in bovine UC samples. e The indel mutation spectrum of BTAUD0055a. f The doublet substitution spectrum in BTAUD0055a

Fig. 6

Mutational signatures in human bladder cancer cell lines after exposure to bracken extracts and ptaquiloside. a Signature extraction identified 2 novel single base substitution (SBS) signatures, Signatures BFA-A and BFA-B in KU-19–19 cells exposed to BF whole extract (BFA). b Similar signatures, Signature PT-A and PT-B, were identified in KU-19–19 cells exposed to purified PT. For comparison, bovine Signature BF-A is shown (lower panel). c The SBS mutation spectra after 3 days (upper panel) and 14 days (lower panel) of PT exposure at IC₅₀. d The absolute (upper panel) and relative (lower panel) proportion of mutations attributed to Signatures PT-A and PT-B in KU-19–19 cells exposed to PT. NTC is non-toxic concentration; IC₂₀ and IC₅₀ are 20 and 50% inhibitory concentration (of cell growth), respectively; d3, d7, d10, and d14 are the number of days. e The observed and reconstructed SBS spectra from KU-19–19 cells exposed to PT for 10 days (IC₅₀), showing the activity of each signature for each substitution type. f The indel mutation spectrum observed at day 14 (IC₅₀) in KU-19–19 cells exposed to PT. g The doublet substitution spectrum observed at day 14 (IC₅₀) in KU-19–19 cells exposed to PT