Identification of a putative regulatory region involved in r-spondin3 expression modulation in triple negative breast cancer cell

FELCHER, C. M.; ORTIZ, A. L.; SALABERRY, P. J.; STEDILE, M. N.; ARCUSCHIN, C. D.; SCHOR, I. E.; CASTILLA, L. H.; KORDON, E. C.

Mar del Plata

Congreso; LXVII Reunión anual de la Sociedad Argentina de Investigación Clínica (SAIC); 2022

Sociedad Argentina de Investigación Clínica

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-CBFβ transcription factor (TF) activity inhibited RSPO3 expression in hu-man triple-negative BC cells MDA-MB231. Interestingly, RUNX1-KO mice 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 (named R4) in the first intron of the human RSPO3 locus. The physical in-teraction between RUNX1 and R4 was confirmed by a ChIP-PCR assay 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 consti-tute a putative enhancer of the human RSPO3 in BC cells. There-fore, 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 new-ly found regulatory region may be important for triple-negative BC progression.