MECOM permits pancreatic acinar cell dedifferentiation avoiding cell death under stress conditions.

Maintenance of the pancreatic acinar cell phenotype suppresses tumor formation. Hence, repetitive acute or chronic pancreatitis, stress conditions in which the acinar cells dedifferentiate, predispose for cancer formation in the pancreas. Dedifferentiated acinar cells acquire a large panel of duct cell-specific markers. However, it remains unclear to what extent dedifferentiated acini differ from native duct cells and which genes are uniquely regulating acinar cell dedifferentiation. Moreover, most studies have been performed on mice since the availability of human cells is scarce. Here, we applied a non-genetic lineage tracing method of human pancreatic exocrine acinar and duct cells that allowed cell-type-specific gene expression profiling by RNA sequencing. Subsequent to this discovery analysis, one transcription factor that was unique for dedifferentiated acinar cells was functionally characterized. RNA sequencing analysis showed that human dedifferentiated acinar cells expressed genes in "Pathways of cancer" with a prominence of MECOM (EVI-1), a transcription factor that was not expressed by duct cells. During mouse embryonic development, pre-acinar cells also transiently expressed MECOM and in the adult mouse pancreas, MECOM was re-expressed when mice were subjected to acute and chronic pancreatitis, conditions in which acinar cells dedifferentiate. In human cells and in mice, MECOM expression correlated with and was directly regulated by SOX9. Mouse acinar cells that, by genetic manipulation, lose the ability to upregulate MECOM showed impaired cell adhesion, more prominent acinar cell death, and suppressed acinar cell dedifferentiation by limited ERK signaling. In conclusion, we transcriptionally profiled the two major human pancreatic exocrine cell types, acinar and duct cells, during experimental stress conditions. We provide insights that in dedifferentiated acinar cells, cancer pathways are upregulated in which MECOM is a critical regulator that suppresses acinar cell death by permitting cellular dedifferentiation.

Acinar phenotype is preserved in human exocrine pancreas cells cultured at low temperature: implications for lineage-tracing of ß-cell neogenesis.

The regenerative medicine field is expanding with great successes in laboratory and preclinical settings. Pancreatic acinar cells in diabetic mice were recently converted into ß-cells by treatment with ciliary neurotrophic factor (CNTF) and epidermal growth factor (EGF). This suggests that human acinar cells might become a cornerstone for diabetes cell therapy in the future, if they can also be converted into glucose-responsive insulin-producing cells. Presently, studying pancreatic acinar cell biology in vitro is limited by their high plasticity, as they rapidly lose their phenotype and spontaneously transdifferentiate to a duct-like phenotype in culture. We questioned whether human pancreatic acinar cell phenotype could be preserved in vitro by physico-chemical manipulations and whether this could be valuable in the study of ß-cell neogenesis. We found that culture at low temperature (4°C) resulted in the maintenance of morphological and molecular acinar cell characteristics. Specifically, chilled acinar cells did not form the spherical clusters observed in controls (culture at 37°C), and they maintained high levels of acinar-specific transcripts and proteins. Five-day chilled acinar cells still transdifferentiated into duct-like cells upon transfer to 37°C. Moreover, adenoviral-mediated gene transfer evidenced an active Amylase promoter in the 7-day chilled acinar cells, and transduction performed in chilled conditions improved acinar cell labelling. Together, our findings indicate the maintenance of human pancreatic acinar cell phenotype at low temperature and the possibility to efficiently label acinar cells, which opens new perspectives for the study of human acinar-to-ß-cell transdifferentiation.

Pancreas-Specific Sirt1-Deficiency in Mice Compromises Beta-Cell Function without Development of Hyperglycemia.

AIMS/HYPOTHESIS: Sirtuin 1 (Sirt1) has been reported to be a critical positive regulator of glucose-stimulated insulin secretion in pancreatic beta-cells. The effects on islet cells and blood glucose levels when Sirt1 is deleted specifically in the pancreas are still unclear. METHODS: This study examined islet glucose responsiveness, blood glucose levels, pancreatic islet histology and gene expression in Pdx1Cre; Sirt1ex4F/F mice that have loss of function and loss of expression of Sirt1 specifically in the pancreas. RESULTS: We found that in the Pdx1Cre; Sirt1ex4F/F mice, the relative insulin positive area and the islet size distribution were unchanged. However, beta-cells were functionally impaired, presenting with lower glucose-stimulated insulin secretion. This defect was not due to a reduced expression of insulin but was associated with a decreased expression of the glucose transporter Slc2a2/Glut2 and of the Glucagon like peptide-1 receptor (Glp1r) as well as a marked down regulation of endoplasmic reticulum (ER) chaperones that participate in the Unfolded Protein Response (UPR) pathway. Counter intuitively, the Sirt1-deficient mice did not develop hyperglycemia. Pancreatic polypeptide (PP) cells were the only other islet cells affected, with reduced numbers in the Sirt1-deficient pancreas. CONCLUSIONS/INTERPRETATION: This study provides new mechanistic insights showing that beta-cell function in Sirt1-deficient pancreas is affected due to altered glucose sensing and deregulation of the UPR pathway. Interestingly, we uncovered a context in which impaired beta-cell function is not accompanied by increased glycemia. This points to a unique compensatory mechanism. Given the reduction in PP, investigation of its role in the control of blood glucose is warranted.

Conversion of human pancreatic acinar cells toward a ductal-mesenchymal phenotype and the role of transforming growth factor ß and activin signaling.

OBJECTIVE: Epithelial-mesenchymal transition may interfere with the differentiation of cultured pancreatic acinar cells toward endocrine cells. Therefore, it will be important to investigate into detail the reprogramming of human pancreatic acinar cells toward a mesenchymal phenotype: the association with acinoductal transdifferentiation, the influence of cell adhesion, and the regulation behind this process. METHODS: Human exocrine cells, isolated from donor pancreata, were cultured in suspension or as monolayers. Non-genetic lineage tracing, using labeled ulex europaeus agglutinin 1 lectin, was performed, and the role of the transforming growth factor (TGF-ß) superfamily was investigated. RESULTS: After 7 days in monolayer culture, the human acinar cells coexpressed the mesenchymal marker vimentin and the ductal marker Sox9. However, when the human exocrine cells were cultured in suspension, epithelial-mesenchymal transition was not observed. The spontaneous transition of the human acinar cells toward a ductal and mesenchymal phenotype was decreased by inhibition of the TGF-ß and activin signaling pathways. CONCLUSIONS: The human acinar cells spontaneously undergo TGF-ß- regulated reprogramming in the monolayer culture. These observations are helpful to develop culture methods for the in vitro reprogramming of pancreatic exocrine to endocrine cells. They are also of potential interest for studies on exocrine acinar cells in the development of pancreatic cancer.

Sirtuin-1 regulates acinar-to-ductal metaplasia and supports cancer cell viability in pancreatic cancer.

The exocrine pancreas can undergo acinar-to-ductal metaplasia (ADM), as in the case of pancreatitis where precursor lesions of pancreatic ductal adenocarcinoma (PDAC) can arise. The NAD(+)-dependent protein deacetylase Sirtuin-1 (Sirt1) has been implicated in carcinogenesis with dual roles depending on its subcellular localization. In this study, we examined the expression and the role of Sirt1 in different stages of pancreatic carcinogenesis, i.e. ADM models and established PDAC. In addition, we analyzed the expression of KIAA1967, a key mediator of Sirt1 function, along with potential Sirt1 downstream targets. Sirt1 was co-expressed with KIAA1967 in the nuclei of normal pancreatic acinar cells. In ADM, Sirt1 underwent a transient nuclear-to-cytoplasmic shuttling. Experiments where during ADM, we enforced repression of Sirt1 shuttling, inhibition of Sirt1 activity or modulation of its expression, all underscore that the temporary decrease of nuclear and increase of cytoplasmic Sirt1 stimulate ADM. Our results further underscore that important transcriptional regulators of acinar differentiation, that is, Pancreatic transcription factor-1a and ß-catenin can be deacetylated by Sirt1. Inhibition of Sirt1 is effective in suppression of ADM and in reducing cell viability in established PDAC tumors. KIAA1967 expression is differentially downregulated in PDAC and impacts on the sensitivity of PDAC cells to the Sirt1/2 inhibitor Tenovin-6. In PDAC, acetylation of ß-catenin is not affected, unlike p53, a well-characterized Sirt1-regulated protein in tumor cells. Our results reveal that Sirt1 is an important regulator and potential therapeutic target in pancreatic carcinogenesis.