Mammary cancer is common and highly aggressive in cats, with limited treatment options and poor prognosis. A lack of knowledge regarding the pathogenesis of feline mammary cancer is hampering the development of new diagnostic tools and novel therapeutics. In this proposal, the goal is to investigate if microRNAs (miRNAs), small non-coding RNAs that regulate post-transcriptional gene expression and with well-established roles in breast cancer pathogenesis, could have potential as diagnostic markers and/or therapeutic targets. Specifically, my project focuses on how normal feline mammary cells respond to carcinogens that cause DNA damage, which can result in genomic instability and mammary cancer. Since mammary stem and progenitor cells (MaSCs) are proposed as targets of tumorigenic mutations that give rise to mammary cancer, I study how carcinogens target and affect MaSCs. Additionally, I propose to study these questions in mammary organoids (MaOs) as well, which are 3D mini-organs consisting of both MaSCs and differentiated mammary epithelial cells. Aim 1: Based on preliminary data, I have identified four miRNA candidates that may play a role in feline mammary cancer pathogenesis, namely miR-16, miR-543, miR-218, and miR-328. First, I will use miRNA inhibitors and mimics to knock-down or overexpress, respectively, these miRNAs in feline MaSCs. I expect that manipulating the expression of these four miRNAs, individually and/or in combination, will result in (i) decreased cell viability, (ii) increased apoptosis, and (iii) decreased proliferation, of feline MDECs in response to carcinogen exposure (Aim 1a). Then, I will identify the target genes of the miRNAs of interest using RNA-sequencing (Aim 1b). Aim 2: In this aim, I will evaluate whether responses observed in 2D feline MaSC cultures also occur in other cell types within the mammary epithelium by performing similar experiments as described in Aim 1 using feline 3D MaOs.