CRISPR-Mediated Knockout of Transcription Factor FOXC1 in Breast Cancer Cells

Breast cancer is the most commonly occurring cancer in women in the US and accounts for about 40,000 deaths each year. Through gene expression profiling, the Perou Lab has categorized breast cancer into five unique subtypes, some of which are sensitive to chemotherapy (basal-like), and some that are not (luminal A). Gene expression profiling has also revealed that certain transcription factors, such as FOXC1, are highly expressed in basal-like cancers. The goal of this project is to study the functional role of FOXC1 in basal-like breast cancers. We propose to do this by using CRISPR/Cas9 to modify the genome of basal-like breast cancer cell lines and delete the transcription factor FOXC1. We hypothesize that deletion of FOXC1 will cause changes in the expression of other genes important for maintaining cells in the basal-like phenotype. To test this hypothesis, we transfected three basal-like breast cancer cell lines with a CRISPR/Cas9 plasmid to induce a knockout at the FOXC1 locus. We validated knockout clones for modification of the FOXC1 gene by sequencing genomic DNA and showed loss of FOXC1 protein expression by western blot. We then used DNA microarrays to measure global changes in gene expression when FOXC1 is knocked out. Analysis of gene expression after inactivation of FOXC1 identified hundreds of differentially expressed genes, and a possible role for FOXC1 in maintenance of mesenchymal features. By better understanding of what genetic factors determine a given cancer subtype, we believe we can manipulate a tumor to be more basal-like and therefore possibly more sensitive to chemotherapy.Bachelor of Scienc