CRISPR Knockout of CUL5 increases cell growth and affects the expression of Estrogen Receptor‐α in T47D breast cancer cells

Abstract

The CUL5/VACM‐1 protein is a component of the CRL5 E3 ligase complex responsible for tagging proteins with ubiquitin for degradation in the proteasome. Previous work has shown CUL5/VACM‐1 dependent E3 ligase decreases cellular proliferation, and lack of regulation in this ubiquitin proteasome pathway can lead to uncontrolled cell growth and tumor formation. The CRISPR/Cas9 system, which is a bacterial immune system used to edit genomic DNA, was independently designed in the lab to target and knockout the function of CUL5/VACM‐1 in T47D breast cancer cells and human umbilical vein endothelial cells (HUVEC). AlamarBlue® growth assays and Matrigel® growth imaging demonstrate that in the absence of CUL5/VACM‐1 cellular proliferation significantly increases. Subsequent immunocytochemistry analysis of estrogen receptor‐α (ERα) expression in T47D cells showed that in the absence of CUL5/VACM‐1 ERα expression was increased. Finally, q‐PCR OpenArray targeted gene expression analysis of control and CRISPR treated cells showed changes in gene regulation across signal transduction pathways. These results make CUL5/VACM‐1 a potential potent target for drug design and development in breast cancer treatment.Support or Funding InformationThis work was supported by the A. Paul Schaap Fund, Hope College, Chemistry Department.