RACK1 facilitates the activity and substrate specificity of PP2A in breast cancer cells

Breast cancer is a leading cause of cancer death worldwide but targeted therapies have proven to be very successful in the treatment of this disease. The aim of this study was to develop models and parameters that will help identify potential therapeutic targets and strategies for breast cancer treatment. We utilized the RTCA xCELLigence platform to develop protocols and determine the effect of novel compounds on breast cancer cells. We found that the vitamin K (VK) derivative, VK2 reduced the viability of Triple Negative Breast Cancer (TNBC) cells. Culturing the cells in media with a combination of VK2 and low glucose shows a further reduction in cell viability. This contributes to the body of knowledge about the benefits of calorie restriction (CR) in cancer patients. Using the platforms and parameters we developed, we expanded our study to investigate the effects of PRIMA1Met on cancer cell behaviour. PRIMA1Met is a small molecule that can revert mutant p53 back to Wild Type. Treatment of a panel of breast cancer cell lines with PRIMA1Met showed cell lines with mutant p53 were sensitive to the molecule. These results correlated very well with more traditional cell based assays. This is the first known analysis of breast cancer cells treated with either VK2 or PRIMA1Met conducted in real time. We next characterised the interaction between RACK1 and PP2A in order to identify a possible role for the complex in breast cancer cells. It is known that RACK1 and PP2A bind directly in an IGF-1 dependant manner and play a role in cell migration. We confirmed that RACK1 was in a complex with both the PP2A C and A subunits. Mutation of the interaction sites between RACK1 and the PP2A C subunit allowed us to determine the consequences for disruption of the complex. Using stable cell lines expressing the mutant subtype, we showed that the RACK1/PP2A complex plays a role iv in the progression of many important cellular processes including adhesion, proliferation, migration and invasion. It also plays a role in maintenance of the cancer phenotype. We hypothesized that RACK1 was scaffolding PP2A to a novel set of substrates through its role as a signalling hub. To identify novel interacting proteins of the RACK1/PP2A complex we isolated the complex from breast cancer cells and used mass spectrometry to identify 66 novel binding partners of the complex. Our aim was to characterise some of these proteins with a particular focus on those involved in cancer. We chose Tau Tubulin Kinase-1 (TTBK-1) and Metadherin for further study and we suggest that TTBK-1 has the potential to be a prognostic indicator of disease. Gene expression analysis identified Metadherin to be upregulated in a cohort of breast cancer patient samples. We confirmed the interaction between Metadherin and the RACK1/PP2A complex and mapped potential binding sites for RACK1 on Metadherin. These findings confirm the direct interaction between RACK1 and PP2A and are the first to characterise the interaction between these proteins in cancer cells. Our results show that the RACK1/PP2A complex is interacting with proteins known to be involved in the advancement of breast cancer disease and disturbance of the RACK1/PP2A complex shows anti-tumourigenic potential. We have determined that disruption of the RACK1/PP2A complex has implications for a wide range of cellular processes involved in maintenance and progression of cancer including migration and invasion and suggests that targeting the RACK1/PP2A complex may provide novel therapeutic approaches in cancer.