Mammary cancer is highly prevalent and very aggressive in cats, with a poorly characterized pathogenesis. MiRNAs are small non-coding regulatory RNAs that function as prominent post-transcriptional regulators of gene expression, with established roles in the development and progression of malignancies, including mammary cancer. Consequently, miRNAs are intensively studied for their role in cancer pathogenesis, as well as their use as biomarkers or targets for therapeutic interventions. The role of miRNAs in feline mammary cancer, however, has not yet been explored to our knowledge.

Intriguingly, whereas mammary cancer is highly prevalent in cats, as well as dogs and humans, other mammals, such as horses, cows and pigs, rarely develop this type of cancer. Our lab has developed a unique comparative mammary stem/progenitor cell (MaSC) approach, taking advantage of this variation for mammary tumorigenesis amongst different mammals to identify mechanisms that can be linked to either mammary cancer susceptibility or resistance. In collaboration with the Sethupathy lab, we recently performed nextgeneration small RNA sequencing (smRNA-seq) to analyze micro (mi)RNA expression in samples from 3 susceptible (human, dog, rat) and 3 resistant (horse, cow, pig) species. We identified 5 cancer-related miRNAs that were differentially expressed between these two groups, and 11 annotated miRNAs that were selectively
expressed in species from only one group.

We now propose, based on our comparative miRNA data combined with our recent isolation and characterization of feline MaSC, to identify miRNAs with potential roles in feline mammary tumorigenesis using our comparative mammary stem/progenitor model. To this end, we propose following aims. In Aim 1, we will perform smRNA-seq of feline MaSC and perform comparative miRNA expression analyses with our already obtained miRNA data set from six other species. The goal is to identify miRNAs that are differentially
expressed in MaSC from mammary cancer-susceptible species (with cat included) relative to mammary cancer-resistant species, as well as selectively expressed in feline MaSC. Based on our already obtained miRNA database that provided us with 16 miRNAs of interest, we are confident that we will identify multiple feline miRNAs of interest. In Aim 2, we will select two feline miRNAs for functional follow-up studies by performing miRNA loss-of-function (LOF) and gain-of-function (GOF) studies in MaSC cultures and evaluate
the resulting phenotypes. In Aim 3, we will perform next generation RNA sequencing (RNAseq) of feline MaSC in the context of the miRNA LOF and GOF to identify candidate target genes of feline miRNAs of interest.

The overall importance and significance of this proposal is that it will increase our understanding of the role miRNAs in the initiation of feline mammary cancer, which may lead to improved diagnostics and therapeutic interventions in the future.