We have determined that R-spondin3 (RSPO3), a secreted protein that potentiates Wnt signaling pathway, is a key modulator of tumor progression and stem cell behavior in triple-negative (TN) breast cancer (BC) cells. The highest RSPO3 mRNA expression levels have been detected in TN cells and TNBC tumors compared with the other BC subtypes. However, immunohistochemical analysis showed that 70% of tumor samples of patients are positive for RSPO3, regardless of its molecular classification. Pharmacological blockade of RUNX-CBFβ activity inhibited RSPO3 expression in the TN cell line MDA-MB-231. In these cells it has been determined that RUNX1 binds to its DNA motif at the end of RSPO3 first intron. Our in silico analyses suggest that this site constitutes a relevant putative regulatory region of the human RSPO3 gene (named RE4), as indicated by combined bioinformatic studies of publicly available data from ChIP-seq and ATAC-seq, as well as transcription factor (TF) binding motives in the human genome. Besides, not only RUNX1, but also STAT3, and the SWI/SNF chromatin remodeling complex bind to RE4, while the presence of estrogen receptor (ER) was detected attached to the promoter region of RSPO3 in luminal BC cells. Therefore, our goal is to analyze the regulation of RSPO3 gene expression in a subset of human BC cell lines, which includes both luminal and TN phenotypes. Our preliminary results indicate that RUNX-CBFβ regulates RSPO3 in various TNBC cell lines, while in luminal cells, expression of this oncogene is induced by estradiol treatment. We are currently analyzing the possible interaction between RUNX1 and ER in luminal cells and the putative involvement of the SWI/SNF complex in RSPO3 expression regulation. In summary, we propose that differential regulatory mechanisms would be responsible for ER+ and ER- high RSPO3 protein levels found in BC patients samples.