IDENTIFICATION OF A PUTATIVE REGULATORY REGION INVOLVED IN R-SPONDIN3 EXPRESSION MODULATION IN TRIPLE NEGATIVE BREAST CANCER CELLS

ANA LAURA ORTIZ; CARLA FELCHER; SALABERRY PEDRO; MICAELA STEDILE; ARCUSCHIN CAMILA; SCHOR IGNACIO; CASTILLA LUCIO; KORDON EDITH

Mar del Plata

Congreso; Reunión Anual SAIC 2022; 2022

SAIC

We have determined that R-spondin3 (RSPO3), a secreted proteinthat potentiates Wnt signaling pathway, is a key modulator of tumorprogression and stem cell behavior in basal breast cancer (BC).Besides, we have previously reported that blocking RUNX-CBFβtranscription factor (TF) activity inhibited RSPO3 expression in humantriple-negative BC cells MDA-MB231. Interestingly, RUNX1-KOmice also showed reduced Rspo3 levels in their mammary glands.Then, the possible direct expression regulation exerted by RUNXsin human cells was assessed by an in silico analysis that revealed aputative DNA region containing three RUNX binding motifs (namedR4) in the first intron of the human RSPO3 locus. The physical interactionbetween RUNX1 and R4 was confirmed by a ChIP-PCRassay in MDA-MB231 cells. Besides, we performed a combinedbio-informatic analysis of publicly available data (including TF andmodified histone CHIP-seq, ATAC-seq and TF binding motifs in thehuman genome), which revealed that this 363bp region may constitutea putative enhancer of the human RSPO3 in BC cells. Therefore,using Crispr-Cas9 technology, we proceeded to remove theputative regulatory region from these cells genome. Surprisingly, wedetermined that RSPO3 expression was increased at both mRNAand protein levels, by RT-qPCR and Western Blot analysis, in theedited ΔR4 cells. Therefore, we postulate that in the MDA-MB231cells the R4 region may recruit a transcription repressor that mightbe absent in BC cells with higher RSPO3 expression. However,the role played by RUNX1 binding to the R4 in the RSPO3 locusremains to be elucidated. Finally, given the relevance of RSPO3 inbasal-like mammary tumor cell behavior, we propose that the newlyfound regulatory region may be important for triple-negative BCprogression.