Exploring the role of Nesprin-2 upregulation mediated by CRISPR/Cas9 in breast cancer cell metastasis (360G-Wellcome-207305_Z_17_Z)

The purpose of the study is to elucidate the role of the nuclear membrane protein nesprin-2 expression in LINC complex formation and the subsequent effect on nuclear rigidity in breast cancer. Their invasive phenotype permits breast cancer cells to metastasize and move through collagen-rich environments. The cells are hallmarked by nuclear softening in breast cancer and it is hypothesized that decreased nuclear rigidity due to mutations in nesprins facilitates metastasis. Therefore, addressing nesprin-2 functions in cancer development by understanding novel mechanistic concepts in metastasis is the principal underpinning objective. The study involves contrasting cells exhibiting changes in nesprin-2 expression. Two breast cancer cell lines MDA-MB231 will be used one of which will be modified with a CRISPR/dCas9 activation plasmid to upregulate specifically the expression of nesprin-2. The consequent protein products will be compared by using standard immunoblotting. Effects on LINC complex formation and structure in cancer cells will be assessed by confocal microscopy. Finally, 3-D space-restrictive porous scaffolds will be used to examine the migratory behavior of nesprin-2 activated MDA-MB231 cells relative to parental cells, which will help to understand how LINC nuclear complex architecture influences metastatic cell migration. This would potentially aid in developing novel treatments for breast cancer.