Abstract Ductal carcinoma in situ (DCIS), the pre-invasive form of breast cancer, counts for one out of every five new breast cancer diagnoses. About one-third to half of the DCIS patients will progress to invasive breast cancer within 10 years of initial diagnosis. The disappearance of myoepithelial layer and disruption of the basement membrane are the hallmarks of transition from DCIS to invasive ductal carcinoma (IDC). Our laboratory has previously shown that vitamin D compounds decreased the MCF10DCIS.com xenograft tumors and inhibited the invasive transition from DCIS to IDC. We have also demonstrated that vitamin D compounds inhibited mammosphere formation by targeting breast cancer stem-like population and putative stem cell markers in MCF10DCIS cells. However, the key molecular mechanisms that govern the transition from DCIS to IDC are yet to be elucidated. In this study, we have performed RNA sequencing analysis of mammospheres treated with vitamin D compounds to identify potential genes that regulate breast cancer stemness and potentially the progression of DCIS to IDC. The mammosphere culture system has been shown to enrich stemness in tumor cells grown in low-attachment tissue culture plates. Upon treatment of mammospheres with vitamin D compounds, we identified down-regulation of genes that are involved in maintenance of breast cancer stem-like cells (e.g. GDF15, XBP1 and ALDH1A3), genes that mediate epithelial-mesenchymal transition, invasion and metastasis (e.g. LCNZ and S100A4), genes that confer breast cancer chemo-resistance (e.g. NGFR, PPP1R1B1, and AGR2) and genes that serve as biomarkers of basal breast cancer (e.g. NGFR). We also observed the up-regulation of genes that are associated with basal-like phenotype (e.g. KRT6A and KRT5), genes that regulate breast tumorigenesis (e.g. EMP1) and genes that regulate breast cancer stem cell self-renewal (e.g. DICER1). Our data set encompasses over 20,000 genes, providing us with unbiased insight to understand the overall gene expression profiling in DCIS breast cancer stem cells treated with vitamin D compounds. Thus, this study helps us identify genes that are key drivers of breast cancer stemness and progression and those regulated by vitamin D compounds. Further mechanistic studies of these significant genes will elucidate the natural history of DCIS progression to IDC, and provide us with target genes to prevent breast cancer progression. (This project is funded by Busch Biomedical Grant, Rutgers University) Citation Format: Naing Lin Shan, Min Ji Bak, Li Cai, Roman Wernyj, Davit Sargsyan, David Cheng, Audrey Minden, Ah-Ng Tony Kong, Nanjoo Suh. Identification of key drivers of cancer stemness and progression regulated by vitamin D compounds in ductal carcinoma in situ breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5067.