Poster Presentation 36th Lorne Cancer Conference 2024

Pinpointing and targeting novel drivers of pancreatic cancer progression and metastasis using TRAP-seq. (#245)

Michael Trpceski 1 2 , Chi Kin (Kenny) Ip 1 2 , Cecilia Chambers 1 2 , Shona Ritchie 1 2 , Daniel Reed 1 2 , Marjan Naeini 1 2 , Leonard Goldstein 1 2 , Grazi Vieira 1 2 , Robert Weatheritt 1 2 , Herbert Herzog 1 2 , Kendelle Murphy 1 2 , Tatyana Chtanova 1 2 , Paul Timpson 1 2 , David Herrmann 1 2
  1. The Garvan Institute of Medical Research, Sydney, New South Wales, Australia
  2. St Vincent’s Clinical School, University of New South Wales, Sydney, New South Wales, Australia

Pancreatic Cancer (PC) has a low 5-year survival rate of 12%1, which can be attributed to its’ rapid metastatic spread and resistance to chemotherapy. Therefore, there is an urgent need to identify novel candidates that become de-regulated during progression and metastasis and can be co-targeted with standard-of-care therapies to improve patient survival.

Here, we aim to use innovative translating-ribosome-affinity-purification followed by RNA-sequencing (TRAP-seq) to enrich in a cell-type-specific manner for mRNAs, which are actively being translated into proteins in metastatic PC and may therefore represent valid ‘druggable’ targets.

 

Our genetically engineered mouse models closely mimic the mutational landscape, histopathology and progression of human PC. Both models are driven by an initiating KrasG12D mutation and either loss of p53 (p53 flox mouse; poorly metastatic) or a gain-of-function mutation in p53 (p53R172H; KPC mouse; highly metastatic)2. These were crossed to the Cre-inducible Rpl10a-GFP mouse to activate expression of the GFP-tagged ribosomal protein Rpl10a (on the 60s ribosomal subunit) specifically in PC cells, which was then immunoprecipitated with associated translating mRNAs for downstream RNA-sequencing.

 

We isolated primary tumours from end-stage mice of both models, as well as matched KPC metastases. All cancer cells retain Rpl10a-GFP expression, with polysome profiling confirming that (i) GFP is incorporated onto the 60s ribosomal subunit and that (ii) subsequent GFP-tagged polysomes exhibit active translation. We also verified the genomic presence of the KrasG12D mutation and either loss of p53 or the gain-of-function mutation p53R172H. TRAP of all samples resulted in high-quality mRNA transcripts suitable for RNA-seq. This approach allowed us to assess the translatome of metastatic PC and pinpoint molecular pathways that may drive metastasis. Both canonical metastasis promotors as well as novel genes of interest were found to be translationally upregulated in the KPC TRAP tumours when compared to the p53 flox tumours.

 

Validation of deregulated genes in our libraries of human PC samples is currently ongoing and will be followed by functional assessment using our established PC in vitro and in vivo models. This will allow us to identify novel candidates to target PC progression and metastasis in conjunction with standard-of-care therapies in a pre-clinical setting.

  1. 1. Siegel, R.L et al. (2023) Cancer statistics, 2023. CA Cancer J. Cin. 73 (1), 17-48.
  2. 2. Morton, J.P et al. (2010) Mutant p53 drives metastasis and overcomes growth arrest/senescence in pancreatic cancer. PNAS. 107 (1), 246-251.