Analysis of RUNX-CBFbeta activity on basal breast cancer progression through direct control of Rspo3 expression

FELCHER, C. M.; STEDILE, M. N.; ORTIZ, A. L.; ARCUSCHIN, C. D.; TOCCI, J. M.; BOGNI, E. S.; VALLONE, S. A.; SCHOR, I. E.; BUSHWELLER, J. H.; CASTILLA, L. H.; KORDON, E. C.

Buenos Aires

Congreso; LXVI Reunión anual de la Sociedad Argentina de Investigación Clínica (SAIC); 2021

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. Previousreports indicated the potential role of RUNX1-CBFβ axis on Rspo3expression in mammary tumor cells. Besides, we found that treatingbasal breast cancer cells with small molecules that block the interaction between CBFβ and RUNX reduced Rspo3 mRNA and proteinlevels. These treatments also induced inhibition of cell migration,an ability that was recovered upon addition of recombinant RSPO3.Also, we have determined that those inhibitors enhanced the effectsof the chemotherapeutic drug Doxorubicin on MDA-MB231 cell survival and migration. To determine whether there is a direct controlof RUNX1-CBFβ on Rspo3 mRNA transcription, we performed anin silico analysis of publicly available data from two RUNX1 CHIPseq reports and an ATAC-seq study from human breast cell lines.We aligned the emerging data with the occurrences of the RUNX1DNA-recognition-motif in the Rspo3 locus. A few putative RUNX1binding sites were revealed by this analysis. Among them, a regionlocated on Rspo3 first intronic region that seems to be particularlyactive in triple negative breast cancer cells. Importantly, we have determined that RUNX1 actually binds to that site in MDA-MB231 cellsby ChIP-qPCR assay (3-fold increase compared to control). Then,to further explore the mechanisms underlying the control exertedby RUNX-CBFβ on Rspo3 expression, we proceeded to delete thatRUNX1 binding site by introducing, through electroporation, Cas9protein together with specific sg-RNAs in MDA-MB231 cells. Weare currently analyzing the phenotype of the obtained cells to verifythe relevance of the discovered RUNX1 binding site. In summary,our results confirm that RUNX-CBFβ axis may contribute to basalbreast cancer progression