Abstract C51: Targeted inhibition of DUSP1 and DUSP6 suppresses pancreatic adenocarcinoma cells’ growth and glucose metabolism via SAPK/JNK pathway activation

The highly malignant potential and aggressiveness of pancreatic adenocarcinoma cells is mainly promoted by oncogenic KRAS activation, which triggers numerous signaling pathways and controls key processes for tumor progression such as cell survival and metabolic reprograming. The major adjustments in metabolic reprogramming mediated by KRAS activity are controlled by MAP kinase activities, which are precisely regulated by protein phosphatases to coordinate complex signaling networks. The major class of phosphatases controlling MAPKs is represented by dual-specificity protein phosphatases (DUSPs). As DUSPs’ role in PDAC cells remains to be elucidated, here we investigated the impact of DUSPs on PDAC chemoresistance and metabolic reprogramming. PDAC cell lines (MIA PaCa-2 and PANC-1) stably expressing the Cas9 endonuclease were submitted to CRISPR using an Edit-R library (GE Dharmacon) of crRNAs targeting all DUSP genes with 3 crRNAs per gene. Phenotypic analysis was conducted to address PDAC cells’ glucose uptake and identify the most relevant targets. Glucose uptake and lactate production were performed by flow cytometry and colorimetric assay, respectively. DUSP1 and DUSP6 were inhibited through a pharmacologic and genetics approach. Gemcitabine response was performed in a 4-day MTT assay. In vitro assays such as cell viability, apoptosis, colony-forming assay, ROS production, glucose uptake rate, and protein expression were performed in three independent experiments. For gene expression, a PCR array with a set of 92 glucose metabolism-related genes has been performed. RNAseq data from pancreatic adenocarcinoma (data generated by the TCGA Research Network: http://cancergenome.nih.gov/) was analyzed by using the LinkedOmics platform (Vasaikar S, 2017). Among the whole DUSP family members, CRISPR screening revealed DUSP1 and DUSP6 as the major players in glucose uptake control in PDAC cell lines. BCI (inhibitor of DUSP1 and DUSP6) treatment of PDAC cells significantly modulated glucose uptake rates (p