Abstract A07: Inflammation enables pancreatic acinar cells to overcome resistance to oncogenic Kras by increasing its expression and plasma membrane localization
Authors: Assi Mohamad Nabil , Lodewyckx Jean-Nicolas , Achouri Younes , Gérard Claude , Dauguet Nicolas , Houbracken Isabelle , Heremans Yves , Tyteca Donatienne , Rooman Ilse , Bouwens Luc , Lemaigre Frédéric , Jacquemin Patrick 
Published: June 2021

Abstract
Kras mutations are not sufficient to induce precancerous lesions in the pancreas, unlike in other organs such as lungs. Pancreatic intraepithelial neoplasia (PanIN) appear when mutated Kras is associated with pancreatitis. Since acinar cells are thought to be the cell origin of PanIN and pancreatic ductal adenocarcinoma (PDAC), our aim is to understand how acinar cells initially resist to Kras mutations and how pancreatitis enables mutated Kras to promote pancreatic neoplasia. Pancreatitis and associated inflammation were induced by injections of cerulein in tamoxifen-treated Elastase-CreER/LSLKrasG12D mice, in which mutated Kras expression is induced exclusively in acinar cells. RNAseq analysis on FACS-sorted acinar cells revealed that Kras expression and several cancer-associated pathways were rapidly induced in the presence of inflammation. In situ hybridization, RTqPCR, and Western blot (WB) experiments confirmed that Kras and/or KrasG12D mRNA and protein were significantly increased in acinar cells after cerulein treatment. To further explore the control of Kras expression, we generated a novel mouse model in which a citrine gene (a variant of GFP) is fused in frame with the endogenous Kras gene, allowing to detect Kras on tissue sections using a GFP antibody. We demonstrated by WB that the protein expression of Kras, KrasG12D and citrine-Kras fusion was similarly regulated in response to cerulein treatment, suggesting the involvement of the same regulatory elements on all three alleles. The ability of citrine-Kras fusion to bind GTP and translocate to the plasma membrane in acini was not altered compared to wild-type Kras protein. Little or no citrine-Kras was visualized in acinar cells of tamoxifen-treated Elastase-CreER/LSLKrasG12D/citrine-Kras mice. However, a 30-fold increase of citrine-Kras at the cell membrane of neoplastic lesions was detected after cerulein treatment. This indicates that inflammation is required for expression and plasma membrane targeting of Kras and KrasG12D in metaplastic acini. Importantly, protein expression of FT-α/β, Rce1 and ICMT, three enzymes necessary for Kras targeting to the membrane was greatly increased in murine acinar cells during pancreatitis. Basal FT-α/β, Rce1 and ICMT levels were found to be very low in normal mouse and human pancreata, compared to other organs, and their expression increased in primary mouse and human acini grown under conditions inducing the formation of preneoplastic lesions. In addition, computational analysis of a PDAC cohort (n=178) from The Cancer Genome Atlas (TCGA) revealed a positive correlation between expression of KrasG12D and its regulating enzymes. We conclude that Kras and KrasG12D are normally not present in acinar cells in vivo and that inflammation induces a coordinated program driving their expression and translocation to plasma membrane. Our findings provide a first clue to the long-standing observation that in several organs, Kras mutations, alone, are not sufficient to affect tissue homeostasis.
Citation Format: Mohamad Nabil Assi, Jean-Nicolas Lodewyckx, Younes Achouri, Claude Gérard, Nicolas Dauguet, Isabelle Houbracken, Yves Heremans, Donatienne Tyteca, Ilse Rooman, Luc Bouwens, Frédéric Lemaigre, Patrick Jacquemin. Inflammation enables pancreatic acinar cells to overcome resistance to oncogenic Kras by increasing its expression and plasma membrane localization [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A07.