Background and aimsBesides well-defined genetic alterations, the dedifferentiation of mature acinar cells is an important prerequisite for pancreatic carcinogenesis. Acinar-specific genes controlling cell homeostasis are extensively downregulated during cancer development; however, the underlying mechanisms are poorly understood. Now, we devised a novel in vitro strategy to determine genome-wide dynamics in the epigenetic landscape in pancreatic carcinogenesis.DesignWith our in vitro carcinogenic sequence, we performed global gene expression analysis and ChIP sequencing for the histone modifications H3K4me3, H3K27me3 and H2AK119ub. Followed by a comprehensive bioinformatic approach, we captured gene clusters with extensive epigenetic and transcriptional remodelling. Relevance of Ring1b-catalysed H2AK119ub in acinar cell reprogramming was studied in an inducible Ring1b knockout mouse model. CRISPR/Cas9-mediated Ring1b ablation as well as drug-induced Ring1b inhibition were functionally characterised in pancreatic cancer cells.ResultsThe epigenome is vigorously modified during pancreatic carcinogenesis, defining cellular identity. Particularly, regulatory acinar cell transcription factors are epigenetically silenced by the Ring1b-catalysed histone modification H2AK119ub in acinar-to-ductal metaplasia and pancreatic cancer cells. Ring1b knockout mice showed greatly impaired acinar cell dedifferentiation and pancreatic tumour formation due to a retained expression of acinar differentiation genes. Depletion or drug-induced inhibition of Ring1b promoted tumour cell reprogramming towards a less aggressive phenotype.ConclusionsOur data provide substantial evidence that the epigenetic silencing of acinar cell fate genes is a mandatory event in the development and progression of pancreatic cancer. Targeting the epigenetic repressor Ring1b could offer new therapeutic options.

Acinar-to-ductal metaplasia (ADM) occurring in cerulein-mediated pancreatitis or in oncogenic Kras-driven pancreatic cancer development is accompanied by extensive changes in the transcriptional program. In this process, acinar cells shut down the expression of acinar specific differentiation genes and re-express genes usually found in embryonic pancreatic progenitor cells. Previous studies have demonstrated that a loss of acinar-specific transcription factors sensitizes the cells towards oncogenic transformation, ultimately resulting in cancer development. However, the mechanism behind the transcriptional silencing of acinar cell fate genes in ADM and pancreatic cancer is largely unknown. Here, we analyzed whether elevated levels of the polycomb repressor complex 1 (PRC1) components Bmi1 and Ring1b and their catalyzed histone modification H2AK119ub in ADMs and tumor cells, are responsible for the mediation of acinar gene silencing. Therefore, we performed chromatin-immunoprecipitation in in vitro generated ADMs and isolated murine tumor cells against the repressive histone modifications H3K27me3 and H2AK119ub. We established that the acinar transcription factor complex Ptf1-L is epigenetically silenced in ADMs as well as in pancreatic tumor cells. For the first time, this work presents a possible mechanism of acinar gene silencing, which is an important prerequisite in the initiation and maintenance of a dedifferentiated cell state in ADMs and tumor cells.

GNAT3 ablation enhances CXCL1 and CXCL2 epithelial expression and increases the number of CXCR2 expressing myeloidderived suppressor cells with altered immunomodulatory gene expression. Analysis of long-term ablation during pancreatic cancer progression found increased metastatic carcinoma.BACKGROUND & AIMS: Pancreatic ductal adenocarcinoma (PDA) initiation and progression are accompanied by an immunosuppressive inflammatory response. Here, we evaluated the immunomodulatory role of chemosensory signaling in metaplastic tuft cells (MTCs) by analyzing the role of GNAT3, a gustatory pathway G-protein expressed by MTCs, during PDA progression.METHODS: Gnat3-null (Gnat3 -/-) mice were crossbred with animals harboring a Cre-inducible Kras LSL-G12D/þ allele with either Ptf1a Cre/þ (KC) or tamoxifen-inducible Ptf1a CreERT/þ (KC ERT ) mice to drive oncogenic KRAS expression in the pancreas. Ex vivo organoid conditioned medium generated from KC and Gnat3 -/-;KC acinar cells was analyzed for cytokine secretion. Experimental pancreatitis was induced in KC ERT and Gnat3 -/-;KC ERT mice to accelerate tumorigenesis, followed by analysis using mass cytometry and single-cell RNA sequencing. To study PDA progression, KC and Gnat3 -/-;KC mice were aged to morbidity or 52 weeks.RESULTS: Ablation of Gnat3 in KC organoids increased release of tumor-promoting cytokines in conditioned media, including CXCL1 and CXCL2. Analysis of Gnat3 -/-;KC ERT pancreata found altered expression of immunomodulatory genes in Cxcr2 expressing myeloid-derived suppressor cells (MDSCs) and an increased number of granulocytic MDSCs, a subset of tumor promoting MDSCs. Importantly, expression levels of CXCL1 and CXCL2, known ligands for CXCR2, were also elevated in Gnat3 -/-;KC ERT pancreata. Consistent with the tumor-promoting role of MDSCs, aged Gnat3 -/-;KC mice progressed more rapidly to metastatic carcinoma compared with KC controls. CONCLUSIONS:Compromised gustatory sensing, achieved by Gnat3 ablation, enhanced the CXCL1/2-CXCR2 axis to alter the MDSC population and promoted the progression of metastatic PDA.

Background & AimsAlthough nearly half of pancreatic ductal adenocarcinoma (PDAC) patients have diabetes mellitus with episodes of hyperglycemia, its tumor microenvironment is hypoglycemic. Thus, it is crucial for PDAC cells to develop adaptive mechanisms dealing with oscillating glucose levels. So far, the biological impact of such glycemic variability on PDAC biology remains unknown.MethodsMurine PDAC cells were cultured in low- and high-glucose medium to investigate the molecular, biochemical, and metabolic influence of glycemic variability on tumor behavior. A set of in vivo functional assays including orthotopic implantation and portal and tail vein injection were used. Results were further confirmed on tissues from PDAC patients.ResultsGlycemic variability has no significant effect on PDAC cell proliferation. Hypoglycemia is associated with local invasion and angiogenesis, whereas hyperglycemia promotes metastatic colonization. Increased metastatic colonization under hyperglycemia is due to increased expression of runt related transcription factor 3 (Runx3), which further activates expression of collagen, type VI, alpha 1 (Col6a1), forming a glycemic pro-metastatic pathway. Through epigenetic machinery, retinoic acid receptor beta (Rarb) expression fluctuates according to glycemic variability, acting as a critical sensor relaying the glycemic signal to Runx3/Col6a1. Moreover, the signal axis of Rarb/Runx3/Col6a1 is pharmaceutically accessible to a widely used antidiabetic substance, metformin, and Rar modulator. Finally, PDAC tissues from patients with diabetes show an increased expression of COL6A1.ConclusionsGlycemic variability promotes both local invasion and metastatic colonization of PDAC. A pro-metastatic signal axis Rarb/Runx3/Col6a1 whose activity is controlled by glycemic variability is identified. The therapeutic relevance of this pathway needs to be explored in PDAC patients, especially in those with diabetes.