Pancreatic Cancer (PC) has a low 5-year survival rate of 13%, which can be attributed to its’ rapid metastatic spread and resistance to standard-of-care chemotherapy. Here, we aim to use innovative Translating-Ribosome-Affinity-Purification followed by RNA-sequencing (TRAP-seq) to assess the deregulated translatome of mutant p53-driven PC metastasis and identify potential novel therapeutic targets.
Our genetically engineered mouse models of PC are driven by an initiating KrasG12D mutation and either loss of p53(p53flox mouse; poorly metastatic) or a gain-of-function mutation in p53 (p53R172H; KPC mouse; highly metastatic). They also express GFP-tagged ribosomes specifically in PC cells, which can then be immunoprecipitated with associated translating mRNAs for downstream RNA-seq.
As such, we isolated primary tumours from end-stage mice of both models, as well as matched KPC metastases. All cancer cells retain Rpl10a-GFP expression and we confirmed the genomic presence of PC driver mutations. TRAP-seq of all samples resulted in a database representing a pool of over 1400 deregulated genes of the metastatic PC translatome. Four genes were subsequently identified as potential novel drivers of mutant p53-driven metastasis, with high expression of these genes being correlated with poor patient outcome. Validation confirmed increased expression of candidate genes in our discovery cohort as well as independent samples of highly metastatic PC when compared to poorly metastatic models. Functional assessment of deregulated genes using genetic inhibition and our established PC in vitro models showed a decreased ability for genetic knock-down PC cells to migrate on cancer-associated fibroblast-derived extracellular matrix as well as grow out clusters in an anchorage-independent setting. Knock-down PC cells also displayed an increased susceptibility to standard-of-care chemotherapy gemcitabine/Abraxane when compared to control. Further validation using our established in vivo PC models is underway.