Poster Presentation HUPO 2019 - 18th Human Proteome Organization World Congress

Phosphoproteomics for Identification of Activated Signalling Pathways in Breast Cancers (#465)

Yanfang Chen 1 2 , Trisha Almazi 1 2 , Heather Murray 1 2 , Abdul Mannan 1 2 , Nikita Panicker 1 2 , Matthew D Dun 1 2 , Nicole Verrills 1 2
  1. School of Biomedical Sciences & Pharmacy, University of Newcastle, Callaghan, NSW, Australia
  2. Cancer Research Program, Hunter Medical Research Institute, New Lambton, NSW, Australia

Breast cancer is a major national and global health problem. Outcomes for patients with triple negative or Luminal-B subtype tumours remains poor. Activation of growth and survival signalling pathways, in large part mediated by phosphorylation cascades, is a key feature of all human tumours, including breast cancer. Low gene and proteins level of PPP2R2A, encoding for the B55α regulatory subunit of the ser/thr protein phosphatase, PP2A, is associated with aggressive breast tumours, and predicts for worse outcome in breast cancer patients. Our laboratory has found that molecular inhibition of PPP2R2A in normal mammary epithelial cells results in a tumourigenic phenotype, supporting a role for PPP2R2A as a tumour suppressor in breast cancer. However, the pathways regulated by PPP2R2A are unknown. Comparative phosphoproteomics was performed on BT474 cells (human Luminal-B-like breast cancer), transduced with either control shRNA or shRNA sequences directed toward two different regions of the PPP2R2A. Titanium dioxide enriched phosphopeptides were quantified by LC-MS/MS and differential proteins and pathways analysed using Ingenuity Pathway Analysis software. A significant increase in phosphorylation of 56 sites from 48 proteins, and decrease in phosphorylation of 24 sites from 22 proteins were identified in both of the PPP2R2A knockdown cell lines. These included YAP1, a transcriptional regulator involved in suppressing apoptotic genes, which was confirmed using phospho-PRM analysis. Follow up studies revealed increased nuclear localization of YAP1 in both knockdown lines, and this was reversed upon re-activation of PP2A. Nuclear localization of YAP1 in human breast tumours has recently been associated with poor outcome, thus functional loss of PPP2R2A may contribute to poor outcome in breast cancer at least in part by dysregulation of YAP1. this study sheds light on the pathways regulated by this important tumour suppressor, and paves the way for novel therapies for breast cancer patients.