Ilse Rooman

Whole genomes redefine the mutational landscape of pancreatic cancer

Pancreatic cancer remains one of the most lethal of malignancies and a major health burden. We performed whole-genome sequencing and copy number variation (CNV) analysis of 100 pancreatic ductal adenocarcinomas (PDACs). Chromosomal rearrangements leading to gene disruption were prevalent, affecting genes known to be important in pancreatic cancer (TP53, SMAD4, CDKN2A, ARID1A and ROBO2) and new candidate drivers of pancreatic carcinogenesis (KDM6A and PREX2). Patterns of structural variation (variation in chromosomal structure) classified PDACs into 4 subtypes with potential clinical utility: the subtypes were termed stable, locally rearranged, scattered and unstable. A significant proportion harboured focal amplifications, many of which contained druggable oncogenes (ERBB2, MET, FGFR1, CDK6, PIK3R3 and PIK3CA), but at low individual patient prevalence. Genomic instability co-segregated with inactivation of DNA maintenance genes (BRCA1, BRCA2 or PALB2) and a mutational signature of DNA damage repair deficiency. Of 8 patients who received platinum therapy, 4 of 5 individuals with these measures of defective DNA maintenance responded.

Exocrine cell transdifferentiation in dexamethasone-treated rat pancreas

Injured pancreatic tissue, for example, after duct ligation, undergoes remodeling, which involves the replacement of exocrine acini by duct-like structures. This acinoductal metaplasia is probably at least partly due to transdifferentiation of amylase-positive, cytokeratin-20 (CK20)-negative acinar cells into amylase-negative, CK20-positive duct-like cells. Due to the kinetics of these phenotypic changes, however, it has not been possible to demonstrate transitional stages of differentiation, which would express both markers at the same time. We took advantage of the fact that dexamethasone treatment inhibits the loss of amylase from acinar cells to demonstrate transitional cells co-expressing amylase and CK20. This was found both in vivo, where duct-ligation induced metaplasia, and in vitro, after isolation of acini. In addition, we found evidence for an acinar-to-islet conversion under the form of transitional cells co-expressing amylase and insulin. These observations strengthen the notion that fully differentiated cells, such as exocrine pancreatic cells, retain the capacity to undergo important phenotypic switches. This finding could have applications in tissue engineering or cell replacement strategies.

The histone deacetylase SIRT2 stabilizes Myc oncoproteins.

Myc oncoproteins are commonly upregulated in human cancers of different organ origins, stabilized by Aurora A, degraded through ubiquitin–proteasome pathway-mediated proteolysis, and exert oncogenic effects by modulating gene and protein expression. Histone deacetylases are emerging as targets for cancer therapy. Here we demonstrated that the class III histone deacetylase SIRT2 was upregulated by N-Myc in neuroblastoma cells and by c-Myc in pancreatic cancer cells, and that SIRT2 enhanced N-Myc and c-Myc protein stability and promoted cancer cell proliferation. Affymetrix gene array studies revealed that the gene most significantly repressed by SIRT2 was the ubiquitin–protein ligase NEDD4. Consistent with this finding, SIRT2 repressed NEDD4 gene expression by directly binding to the NEDD4 gene core promoter and deacetylating histone H4 lysine 16. Importantly, NEDD4 directly bound to Myc oncoproteins and targeted Myc oncoproteins for ubiquitination and degradation, and small-molecule SIRT2 inhibitors reactivated NEDD4 gene expression, reduced N-Myc and c-Myc protein expression, and suppressed neuroblastoma and pancreatic cancer cell proliferation. Additionally, SIRT2 upregulated and small-molecule SIRT2 inhibitors decreased Aurora A expression. Our data reveal a novel pathway critical for Myc oncoprotein stability, and provide important evidences for potential application of SIRT2 inhibitors for the prevention and therapy of Myc-induced malignancies.

Plasticity in the adult rat pancreas: transdifferentiation of exocrine to hepatocyte-like cells in primary culture.

Under certain experimental conditions, hepatocytes can arise in the pancreas. It has been suggested that the pancreas retains a source of hepatocyte progenitor cells. However, such cells have not been yet identified in the adult pancreas. We describe here the transdifferentiation of primary rat pancreatic exocrine cells into hepatocyte‐like cells during 5 days of tissue culture in the presence of dexamethasone (DX). Using reverse‐transcription polymerase chain reaction and immunocytochemistry, it was observed that DX treatment induced albumin RNA and protein expression in the cells. Coexpression of albumin and amylase, and the absence of cell proliferation, demonstrated a direct transdifferentiation of acinar cells to hepatocytic cells. CCAAT enhancer‐binding protein‐ß protein, a liver‐enriched transcription factor that is considered to be the master switch in pancreatohepatic transdifferentiation, and α‐fetoprotein were markedly upregulated in the cells after treatment with DX. We compared transcriptional profiles of freshly isolated exocrine cells and DX‐treated cells using oligonucleotide microarrays and found that multiple liver‐specific genes are induced along with albumin, and that certain pancreatic genes are downregulated in the DX‐treated cells. In conclusion, these observations support the notion of plasticity in the adult pancreas and that exocrine cells can be reprogrammed to transdifferentiate into other cell types such as hepatocytes. (HEPATOLOGY 2004;39:1499–1507.)

Genome-wide DNA methylation patterns in pancreatic ductal adenocarcinoma reveal epigenetic deregulation of SLIT-ROBO, ITGA2 and MET signaling

The importance of epigenetic modifications such as DNA methylation in tumorigenesis is increasingly being appreciated. To define the genome‐wide pattern of DNA methylation in pancreatic ductal adenocarcinomas (PDAC), we captured the methylation profiles of 167 untreated resected PDACs and compared them to a panel of 29 adjacent nontransformed pancreata using high‐density arrays. A total of 11,634 CpG sites associated with 3,522 genes were significantly differentially methylated (DM) in PDAC and were capable of segregating PDAC from non‐malignant pancreas, regardless of tumor cellularity. As expected, PDAC hypermethylation was most prevalent in the 5′ region of genes (including the proximal promoter, 5′UTR and CpG islands). Approximately 33% DM genes showed significant inverse correlation with mRNA expression levels. Pathway analysis revealed an enrichment of aberrantly methylated genes involved in key molecular mechanisms important to PDAC: TGF‐β, WNT, integrin signaling, cell adhesion, stellate cell activation and axon guidance. Given the recent discovery that SLIT‐ROBO mutations play a clinically important role in PDAC, the role of epigenetic perturbation of axon guidance was pursued in more detail. Bisulfite amplicon deep sequencing and qRT‐PCR expression analyses confirmed recurrent perturbation of axon guidance pathway genes SLIT2, SLIT3, ROBO1, ROBO3, ITGA2 and MET and suggests epigenetic suppression of SLIT‐ROBO signaling and up‐regulation of MET and ITGA2 expression. Hypomethylation of MET and ITGA2 correlated with high gene expression, which was associated with poor survival. These data suggest that aberrant methylation plays an important role in pancreatic carcinogenesis affecting core signaling pathways with potential implications for the disease pathophysiology and therapy.

Adult pancreatic acinar cells dedifferentiate to an embryonic progenitor phenotype with concomitant activation of a senescence programme that is present in chronic pancreatitis

Objective Acinar cells display plasticity in vitro and in vivo and can activate a variety of differentiation programmes that may contribute to pancreatic diseases. The aims were to determine: (1) the differentiation potential of acinar cells under conditions which favour stem cell survival, and (2) its relationship to the phenotypes acquired by pancreatic epithelial cells in chronic pancreatitis. Design Murine acinar cells were cultured in suspension and their molecular phenotype was characterised by qRT-PCR, chromatin immunoprecipitation, immunocytochemistry and global transcriptome analysis. These findings were compared to the changes occurring in experimental chronic pancreatitis induced by pancreatic duct ligation and chronic caerulein administration. Results Acinar cells in suspension culture acquired a dedifferentiated phenotype characteristic of pancreatic embryonic progenitors, consisting of the co-expression of Ptf1a and Pdx1, presence of an embryonic-type PTF1 transcriptional complex, activation of the Notch pathway, and expression of additional pancreatic progenitor cell markers such as CpA1, Sox9 and Hnf1b. A senescence programme, associated with activation of Ras and ERK signalling, limited the proliferative capacity of the cells. A similar progenitor-like phenotype with activation of a senescence programme was observed in experimental chronic pancreatitis induced by pancreatic duct ligation or repeated caerulein administration, with the concomitant and differential activation of proliferation and senescence in distinct cell populations. Conclusions Acinar cells dedifferentiate into an embryonic progenitor-like phenotype upon suspension culture. This is associated with the activation of a senescence programme. Both processes take place in experimental chronic pancreatitis where senescence may contribute to limit tumour progression.

Pancreatic ductal adenocarcinoma and acinar cells: a matter of differentiation and development?

Pancreatic ductal adenocarcinoma (PDAC) has long been considered to arise from pancreatic ducts on the basis of its morphology, the occurrence of dysplasia in putative preneoplastic ductal lesions, and the absence of acinar dysplasia in the pancreas of patients with PDAC. However, evidence gathered through both in vitro studies and—more importantly—genetic mouse models of PDAC shows that ductal-type tumours can arise from acinar cells. These findings raise new important questions related to PDAC pathophysiology and call for in-depth studies of acinar cell differentiation in order to better understand PDAC biology. The authors review these issues and discuss how the novel findings should impact on future work aiming at early diagnosis and improved outcome of patients with PDAC.

Notch Signaling as Gatekeeper of Rat Acinar-to-β-Cell Conversion in Vitro

BACKGROUND & AIMS Exocrine acinar cells in the pancreas are highly differentiated cells that retain a remarkable degree of plasticity. After isolation and an initial phase of dedifferentiation in vitro, rodent acinar cells can convert to endocrine beta-cells when cultured in the presence of appropriate factors. The mechanisms regulating this phenotypic conversion are largely unknown. METHODS Using rat acinar cell cultures, we studied the role of Notch signaling in a model of acinar-to-beta-cell conversion. RESULTS We report a novel lectin-based cell labeling method to demonstrate the acinar origin of newly formed insulin-expressing beta-cells. This method allows for specific tracing of the acinar cells. We demonstrate that growth factor-induced conversion of adult acinar cells to beta-cells is negatively regulated by Notch1 signaling. Activated Notch1 signaling prevents the reexpression of the proendocrine transcription factor Neurogenin-3, the key regulator of endocrine development in the embryonic pancreas. Interfering with Notch1 signaling allows modulating the acinar cell susceptibility to the differentiation-inducing factors. Its inhibition significantly improves beta-cell neoformation with approximately 30% of acinar cells that convert to beta-cells. The newly formed beta-cells mature when transplanted ectopically and are capable of restoring normal blood glycemia in diabetic recipients. CONCLUSIONS We report for the first time an efficient way to reprogram one third of the acinar cells to beta-cells by adult cell type conversion. This could find application in cell replacement therapy of type 1 diabetes, provided that it can be translated from rodent to human models.

p53-dependent regulation of growth, epithelial-mesenchymal transition and stemness in normal pancreatic epithelial cells

Pancreatic acinar cells acquire in vitro a pancreatic progenitor phenotype associated with activation of p53, growth arrest and senescence. A similar program is also activated in chronic pancreatitis. To assess the mechanisms involved in this process, we cultured pancreatic acinar cells from wild-type, p53-/-, p16-/- and p21-/- mice. Cultures from p53-/- mice, but not those from p16-/- or p21-/- mice, display an enhanced proliferation and can be expanded continuously for more than 20 passages. p53-/- cells also display features of stemness such as enhanced sphere formation, increased expression of pancreatic multipotent progenitor markers (Ptf1a, Pdx1, Cpa1, c-myc, Sox9 and Hnf1b), and of the stemness regulators Bmi1 and Klf4. Upon subculture, p53-/- cells undergo an epithelial-mesenchymal transition (EMT) and express high levels of vimentin and of the transcriptional regulators Snai1, Snai2, Twist, Zeb1 and Zeb2. Genetic lineage tracing unequivocally demonstrates the epithelial origin of the cells with mesenchymal phenotype. These cells express the endodermal markers Hhex, Pdx1, Sox9, Hnf1b, Foxa2, Gata6 and Sox17, and the stem cell markers c-myc, Bmi1 and Klf4. Cultures from p53+/- mice display intermediate levels of the transcription factors involved in EMT but do not surpass the growth arrest. Our findings support the notion that p53 controls both growth and epithelial cell differentiation in the pancreas. These observations have important implications regarding the mechanisms through which p53 inactivation in tumors may be associated with aggressive biological behavior.

Chronic pancreatitis: a path to pancreatic cancer.

Chronic pancreatitis predisposes to pancreatic cancer development and both diseases share a common etiology. A central role has been proposed for the digestive enzyme-secreting acinar cell that can undergo ductal metaplasia in the inflammatory environment of pancreatitis. This metaplastic change is now a recognised precursor of pancreatic cancer. Inflammatory molecules also foster tumour growth through autocrine and paracrine effects in the epithelium and the stroma. These insights have raised new opportunities such as the manipulation of inflammation as a preventive and/or therapeutic strategy for pancreatic cancer. Finally, we address the need for an in-depth study of the pancreatic acinar cells.