Analysis of the Transcriptome: Regulation of Cancer Stemness in Breast Ductal Carcinoma <i>In Situ</i> by Vitamin D Compounds

Abstract

Ductal carcinoma <i>in situ</i> (DCIS), which accounts for one out of every five new breast cancer diagnoses, will progress to potentially lethal invasive ductal carcinoma (IDC) in about 50% of cases. Vitamin D compounds have been shown to inhibit progression to IDC in the MCF10DCIS model. This inhibition appears to involve a reduction in the cancer stem cell-like population in MCF10DCIS tumors. To identify genes that are involved in the vitamin D effects, a global transcriptomic analysis was undertaken of MCF10DCIS cells grown in mammosphere cultures, in which cancer stem-like cells grow preferentially and produce colonies by self-renewal and maturation, in the presence and absence of 1α25(OH)₂D₃ and a vitamin D analog, BXL0124. Using next-generation RNA-sequencing, we found that vitamin D compounds downregulated genes involved in maintenance of breast cancer stem-like cells (e.g., <i>GDF15</i>), epithelial-mesenchymal transition, invasion, and metastasis (e.g., <i>LCN2</i> and <i>S100A4</i>), and chemoresistance (e.g., <i>NGFR, PPP1R1B</i>, and <i>AGR2</i>), while upregulating genes associated with a basal-like phenotype (e.g., <i>KRT6A</i> and <i>KRT5</i>) and negative regulators of breast tumorigenesis (e.g., <i>EMP1</i>). Gene methylation status was analyzed to determine whether the changes in expression induced by vitamin D compounds occurred via this mechanism. Ingenuity pathway analysis was performed to identify upstream regulators and downstream signaling pathway genes differentially regulated by vitamin D, including TP63 and vitamin D receptor -mediated canonical pathways in particular. This study provides a global profiling of changes in the gene signature of DCIS regulated by vitamin D compounds and possible targets for chemoprevention of DCIS progression to IDC in patients.