Identifying the ErbB/MAPK Signaling Cascade as a Therapeutic Target in Canine Bladder Cancer.

Transitional cell carcinoma (TCC) of the bladder comprises 2% of diagnosed canine cancers. TCC tumors are generally inoperable and unresponsive to traditional chemotherapy, indicating a need for more effective therapies. BRAF, a kinase in the mitogen-activated protein kinase (MAPK) pathway, is mutated in 70% of canine TCCs. In this study, we use BRAF mutant and wild-type TCC cell lines to characterize the role of BRAF mutations in TCC pathogenesis and assess the efficacy of inhibition of the MAPK pathway alone and in combination with other gene targets as a treatment for canine TCC. Analysis of MAPK target gene expression and assessment of extracellular signal-regulated kinase (ERK) 1/2 phosphorylation following serum starvation indicated constitutive MAPK activity in all TCC cell lines. BRAF mutant TCC cell lines were insensitive to the BRAF inhibitor vemurafenib, with IC50 values greater than 5 µM, but exhibited greater sensitivity to a paradox-breaking BRAF inhibitor (IC50: 0.2-1 µM). All TCC cell lines had IC50 values less than 7 nM to the mitogen-activated protein kinase kinase (MEK) 1/2 inhibitor trametinib independent of their BRAF mutation status. ERK1/2 phosphorylation decreased after 6-hour treatments with MAPK inhibitors, but rebounded by 24 hours, suggesting the presence of resistance mechanisms. Microarray analysis identified elevated expression of the ErbB family of receptors and ligands in TCC cell lines. The pan-ErbB inhibitor sapitinib synergized with BRAF inhibition in BRAF mutant Bliley TCC cells and synergized with MEK1/2 inhibition in Bliley and BRAF wild-type Kinsey cells. These findings suggest the potential for combined MAPK and ErbB receptor inhibition as a therapy for canine TCC. SIGNIFICANCE STATEMENT: The results of this study (1) identify a novel combination strategy for canine bladder cancer treatment: targeting the ErbB/MAPK signaling cascade and (2) establish the utility of canine bladder cancer as a naturally-occurring model for human MAPK-driven cancers.

Characterizing the molecular and immune landscape of canine bladder cancer.

Transitional cell carcinoma (TCC), also known as urothelial carcinoma, is the most common bladder cancer in humans and dogs. Approximately one-quarter of human TCCs are muscle-invasive and associated with a high risk of death from metastasis. Canine TCC (cTCC) tumours are typically high-grade and muscle-invasive. Shared similarities in risk factors, histopathology, and clinical presentation suggest that cTCC may serve as a model for the assessment of novel therapeutics that may inform therapies for human muscle-invasive TCC. The goal of this study was to characterize cTCC at the molecular level to identify drivers of oncogenesis and druggable targets. We performed whole exome sequencing (WES) of 11 cTCC tumours and three matched normal samples, identifying 583 variants in protein-coding genes. The most common variant was a V-to-E missense mutation in BRAF, identified in 4 out of 11 samples (36%) via WES. Sanger sequencing identified BRAF variants in 8 out of the same 11 cTCC samples, as well as in 22 out of 32 formalin-fixed paraffin embedded (FFPE) cTCC samples, suggesting an overall prevalence of 70%. RNA-Seq was performed to compare the gene expression profiles of cTCC tumours to normal bladder tissue. cTCC tumours exhibited up-regulation of genes involved in the cell cycle, DNA repair, and antiviral immunity. We also analysed the immune landscape of cTCC using immune gene signatures and immunohistochemical analysis. A subset of tumours had characteristics of a hot tumour microenvironment and exhibited high expression of signatures associated with complete response to PD-1/PD-L1 blockade in human bladder cancer.

Prevalence, Colonization, Epidemiology, and Public Health Significance of Clostridioides difficile in Companion Animals.

Clostridioides difficile, previously Clostrdium difficile, is a major cause of antibiotic-associated enteric disease in humans in hospital settings. Increased incidence of C. difficile infection (CDI) in community settings raises concerns over an alternative source of CDI for humans. The detection of genetically similar and toxigenic C. difficile isolates in companion animals, including asymptomatic pets, suggests the potential role of household pets as a source of community-associated CDI. The close association between companion animals and humans, in addition to the use of similar antibiotics in both species, could provide a selective advantage for the emergence of new C. difficile strains and thus increase the incidental transmission of CDI to humans. Therefore, screening household pets for C. difficile is becoming increasingly important from a public health standpoint and may become a part of routine testing in the future, for the benefit of susceptible or infected individuals within a household. In this review, we analyze available information on prevalence, pathophysiology, epidemiology, and molecular genetics of C. difficile infection, focusing on companion animals and evaluate the risk of pet-borne transmission of CDI as an emerging public health concern. Molecular epidemiological characterization of companion animal C. difficile strains could provide further insights into the interspecies transmission of CDI. The mosaic nature of C. difficile genomes and their susceptibility to horizontal gene transfer may facilitate the inter-mixing of genetic material, which could increase the possibility of the emergence of new community-associated CDI strains. However, detailed genome-wide characterization and comparative genome analysis are warranted to confirm this hypothesis.