Development of an hydroxymethylglutharyl-coenzyme a reductase (hmgcr) overexpression system for the study of reprogramming to stem-like states in human breast cancer.

VELLÓN L.; PEREYRA BONNET F.; ROMORINI L.; CALVO JC,; FLETCHER S.; MARKS MP. ; CHASSEING NA.; VILA AS. ; GIMÉNEZ CA.

Melbourne

Congreso; ISSCR 2018 Annual Meeting - International Society for Stem Cell Research.; 2018

ISSCR

Abstract: The rate-limiting enzyme in the mevalonic acid (MVA) pathway, hydroxymethylglutharyl-coenzyme A reductase (HMGCR), is deregulated in tumors, increasing the synthesis de novo of cholesterol, critical for cell survival and proliferation. However, the role of HMGCR in the induction and maintenance of stemness in both transformed and non-transformed cells is still unclear. Therefore, we set out to induce an HMGCR-on phenotype in the breast cancer (BC)-derived cell line MCF-7, to evaluate whether this phenotype facilitates the acquisition of stem-like traits in BC. With this purpose, we developed an HMGCR overexpression model taking advantage of a CRISPR-on system (dCas9-VP160), which includes expression plasmids for guide RNAs (pSPgRNAs) and a plasmid carrying the sequence coding for the dCAS9. Five guide RNAs (gRNAs) targeted to the promoter of the human HMGCR gene were designed with the informatics tools Genome Engineering Toolbox from the Zhang Lab (MIT, Cambridge, MA) and CRISPR-ERA. The gRNAs and the dCAS9 were then co-transfected into MCF-7 cells, and the levels of total HMGCR was assessed by qRT-PCR at 2 days post-transfection. The CRISPR-dCAS9 system increased HMGCR total levels in MCF-7 cells (MCF-7/CR) a 2,46 +/-0,4-fold (X+/-SD; p