CRISPR deletion of MIEN1 in Triple Negative Breast Cancer

Purpose/Objective: Triple negative breast cancer (TNBC), which accounts for 15-20% of all breast cancer diagnoses, is the most aggressive subtype of breast cancer. The propensity of TNBC to metastasize to vital organs, such as lung, brain and bone, early in the disease progression is its most devastating feature, which leads to the high mortality rates seen among women diagnosed with TNBC. Efforts to identify specific factors that are able to predict disease progression to aggressive TNBC have proved to be fruitless thus far. In addition, the standard treatments for TNBC are radiation therapy, systemic chemotherapy and/or resection surgery since there are currently no target-based therapies for TNBC. In an effort to address these knowledge gaps, the objective of this project is to knock-out (KO) the oncogene Migration and Invasion Enhancer 1 (MIEN1), implicated in TNBC disease progression, in MDA-MB-231 TNBC cells using CRISPR genome editing technology in order to assess MIEN1’s ability to mediate migration and invasion. Methods: Single-guide RNAs (sgRNAs) were designed to delete the C-terminal end of exon 2, along with exon 3 and 4 of the MIEN1 gene. Following transient transfection of CRISPR plasmids containing sgRNA/Cas9/GFP, MDA-MB-231 cells were FACS sorted into 96-well plates at a concentration of 1 cell/well. Colonies were then identified, genotyped and screened via western blot to confirm MIEN1 protein KO. Downstream signaling pathways were also analyzed via western blot. Various in vitro migration assays were performed to evaluate functional consequence of MIEN1 KO in TNBC cells. Results: 22.2% of MDA-MB-231 single-cell colonies screened showed deletion of at least one MIEN1 allele. 10.3% of colonies screened showed complete KO of MIEN1 protein. In addition, western blots showed a reduction in downstream signaling through Akt/NF-kB as a result of MIEN1 KO. MIEN1 KO also resulted in reduced migratory phenotype in vitro. Conclusions: Designed CRISPR sgRNAs effectively target the MIEN1 gene. MIEN1 KO in TNBC cells results in reduction of pro-metastasis signaling and cell migration.