Poster Presentation 32nd Lorne Cancer 2020

Quantitative proteomics reveals that p53 status can drive distinct matrisomal signatures in murine pancreatic tumours     (#339)

Shona Ritchie 1 2 , Brooke Pereira 1 2 , David Herrmann 1 2 , Max Nobis 1 2 , Romain Rouet 1 2 , Kendelle Murphy 1 2 , Xanthe Metcalf 1 2 , Astrid Magenau 1 2 , Young K Lee 1 2 , Benjain Parker 3 , Marina Pajic 1 2 , Jennifer Morton 4 , Anthony Gill 5 6 7 , Thomas Cox 1 2 , Paul Timpson 1 2
  1. Garvan Institute of Medical Research, Darlinghurst, NSW, Australia
  2. St Vincent’s Clinical School, Faculty of Medicine, University of New South Wales, Sydney, NSW, Australia
  3. Schools of Life and Environmental Sciences, the Charles Perkin Centre, the University of Sydney, Sydney, NSw, Australia
  4. Cancer Research UK Beatson Institute, Glasgow, Scotland, UK
  5. Sydney Medical School, University of Sydney, Sydney, NSW, Australia
  6. Department of Anatomical Pathology, Royal North Shore Hospital, St Leonards, Sydney, NSW, Australia
  7. Cancer Diagnosis and Pathology Research Group , Kolling Institute of Medical Research, St Leonards, Sydney, NSW, Australia

Pancreatic ductal adenocarcinoma (PDAC) diagnosis is accompanied by a particularly dire prognosis of 9% survival at 5 years[1]. A significant contribution to these poor outcomes lies within the extracellular matrix (ECM) surrounding the primary tumour. PDAC is characterised by a fibrotic desmoplastic response, where ECM remodelling generates a highly dense stroma, which acts as a protective niche. This desmoplasia aids in cancer progression, invasion and metastasis[2].

Considering this, we aim to investigate the matrisomal signature of murine pancreatic tumours using the genetically distinct and highly metastatic KPC (Pdx1-Cre; LSL-K-rasG12D/+; LSL-p53R172H/+) mouse, and the poorly-metastatic KPflC (Pdx1-Cre; LSL-K-rasG12D/+; LSL-p53fl/+) mouse. To enrich for fibrotic proteins, tumours from three disease timepoints will be subjected to a decellularization protocol before data-independent acquisition liquid-chromatography tandem mass spectrometry (DIA LC-MS/MS) will be used to identify differentially abundant proteins between the samples.

Our previous findings show that KPC cancer cells with gain-of-function mutant p53 expression (termed mt-CC) can educate cancer-associated fibroblasts (or mt-e-CAFs), causing them to enhance cancer cell  invasion and metastasis, compared to CAFs educated by the poorly-invasive KPflC (fl-e-CAFs)[3]. Using these cells, we will perform a number of explorative assays to investigate protein aberrations between genetically-driven CAFs, using knowledge gained from our mass spectrometry cohort. IHC staining will spatially assess protein differences within tumour samples, and immunofluorescent (IF) imaging of cancer cells and CAFs will determine the origin of these aberrations. Western blotting will also highlight protein variances in the mt-e-CAFs vs. fl-e-CAFs that could be driving enhanced invasiveness in our KPC model. Future work involves knock-down of our protein-of-interest expression using CRISPR RNA interference technology (CRISPRi) in CAF lines. Functional experiments, such as our organotypic collagen invasion assays, as well as in vivo experimentation, will address our hypothesis that proteins within the desmoplastic extracellular compartment are contributing to an aggressive and pro-metastatic behaviour observed in the mt-e-CAFs, and ultimately aiding in the progression of PDAC.

 

  1. 1. Siegel, R.L., et al., Cancer statistics, 2019. CA: A Cancer Journal for Clinicians 69, 2019. (1), 7-34. 2. Pereira, B.A., et al., CAF Subpopulations: A New Reservoir of Stromal Targets in Pancreatic Cancer. Trends Cancer, 2019. 5(11): p. 724-741. 3. Vennin, C., et al., CAF hierarchy driven by pancreatic cancer cell p53-status creates a pro-metastatic and chemoresistant environment via perlecan. Nat Commun, 2019. 10(1): p. 3637.