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CRISPR Knockout of CUL5 increases cell growth and affects the expression of Estrogen Receptor‐α in T47D breast cancer cells
Author(s) -
Bonema Sarah C.
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.05698
Subject(s) - ubiquitin ligase , biology , estrogen receptor , cell growth , cancer research , microbiology and biotechnology , ubiquitin , cancer , breast cancer , gene , biochemistry , genetics
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 Information This work was supported by the A. Paul Schaap Fund, Hope College, Chemistry Department.