ROBO2 is a stroma suppressor gene in the pancreas and acts via TGF-ß signalling.

Whereas genomic aberrations in the SLIT-ROBO pathway are frequent in pancreatic ductal adenocarcinoma (PDAC), their function in the pancreas is unclear. Here we report that in pancreatitis and PDAC mouse models, epithelial Robo2 expression is lost while Robo1 expression becomes most prominent in the stroma. Cell cultures of mice with loss of epithelial Robo2 (Pdx1Cre;Robo2F/F) show increased activation of Robo1+ myofibroblasts and induction of TGF-ß and Wnt pathways. During pancreatitis, Pdx1Cre;Robo2F/F mice present enhanced myofibroblast activation, collagen crosslinking, T-cell infiltration and tumorigenic immune markers. The TGF-ß inhibitor galunisertib suppresses these effects. In PDAC patients, ROBO2 expression is overall low while ROBO1 is variably expressed in epithelium and high in stroma. ROBO2low;ROBO1high patients present the poorest survival. In conclusion, Robo2 acts non-autonomously as a stroma suppressor gene by restraining myofibroblast activation and T-cell infiltration. ROBO1/2 expression in PDAC patients may guide therapy with TGF-ß inhibitors or other stroma /immune modulating agents.

Hypermutation In Pancreatic Cancer.

Pancreatic cancer is molecularly diverse, with few effective therapies. Increased mutation burden and defective DNA repair are associated with response to immune checkpoint inhibitors in several other cancer types. We interrogated 385 pancreatic cancer genomes to define hypermutation and its causes. Mutational signatures inferring defects in DNA repair were enriched in those with the highest mutation burdens. Mismatch repair deficiency was identified in 1% of tumors harboring different mechanisms of somatic inactivation of MLH1 and MSH2. Defining mutation load in individual pancreatic cancers and the optimal assay for patient selection may inform clinical trial design for immunotherapy in pancreatic cancer.

ROCK signaling promotes collagen remodeling to facilitate invasive pancreatic ductal adenocarcinoma tumor cell growth.

Pancreatic ductal adenocarcinoma (PDAC) is a major cause of cancer death; identifying PDAC enablers may reveal potential therapeutic targets. Expression of the actomyosin regulatory ROCK1 and ROCK2 kinases increased with tumor progression in human and mouse pancreatic tumors, while elevated ROCK1/ROCK2 expression in human patients, or conditional ROCK2 activation in a KrasG12D/p53R172H mouse PDAC model, was associated with reduced survival. Conditional ROCK1 or ROCK2 activation promoted invasive growth of mouse PDAC cells into three-dimensional collagen matrices by increasing matrix remodeling activities. RNA sequencing revealed a coordinated program of ROCK-induced genes that facilitate extracellular matrix remodeling, with greatest fold-changes for matrix metalloproteinases (MMPs) Mmp10 and Mmp13 MMP inhibition not only decreased collagen degradation and invasion, but also reduced proliferation in three-dimensional contexts. Treatment of KrasG12D/p53R172H PDAC mice with a ROCK inhibitor prolonged survival, which was associated with increased tumor-associated collagen. These findings reveal an ancillary role for increased ROCK signaling in pancreatic cancer progression to promote extracellular matrix remodeling that facilitates proliferation and invasive tumor growth.

Sirtuin 1 stimulates the proliferation and the expression of glycolysis genes in pancreatic neoplastic lesions.

Metabolic reprogramming is a feature of neoplasia and tumor growth. Sirtuin 1 (SIRT1) is a lysine deacetylase of multiple targets including metabolic regulators such as p53. SIRT1 regulates metaplasia in the pancreas. Nevertheless, it is unclear if SIRT1 affects the development of neoplastic lesions and whether metabolic gene expression is altered.To assess neoplastic lesion development, mice with a pancreas-specific loss of Sirt1 (Pdx1-Cre;Sirt1-lox) were bred into a KrasG12D mutant background (KC) that predisposes to the development of pancreatic intra-epithelial neoplasia (PanIN) and ductal adenocarcinoma (PDAC). Similar grade PanIN lesions developed in KC and KC;Sirt1-lox mice but specifically early mucinous PanINs occupied 40% less area in the KC;Sirt1-lox line, attributed to reduced proliferation. This was accompanied by reduced expression of proteins in the glycolysis pathway, such as GLUT1 and GAPDH.The stimulatory effect of SIRT1 on proliferation and glycolysis gene expression was confirmed in a human PDAC cell line. In resected PDAC samples, higher proliferation and expression of glycolysis genes correlated with poor patient survival. SIRT1 expression per se was not prognostic but low expression of Cell Cycle and Apoptosis Regulator 2 (CCAR2), a reported SIRT1 inhibitor, corresponded to poor patient survival.These findings open perspectives for novel targeted therapies in pancreatic cancer.

Emerging Drug Target In Pancreatic Cancer: Placing Sirtuin 1 on the Canvas.

Sirtuin 1 is a protein deacetylase that regulates a large number of proteins often functionally implicated in tumor development and progression. Its pleiotropic function has turned SIRT1 into an attractive chemotherapeutic target, underscored by very promising preclinical results with SIRT1 inhibitors in the treatment of chronic myeloid leukemia. Here, we revisit the studies on SIRT1 as an emerging target for therapy in pancreatic cancer, a tumor with dismal outcomes for which currently few therapeutic options are available. We highlight those potential SIRT1 target genes that are commonly affected in pancreatic cancer according to recent genomic analyses.

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.

SOX9 regulates ERBB signalling in pancreatic cancer development.

OBJECTIVE: The transcription factor SOX9 was recently shown to stimulate ductal gene expression in pancreatic acinar-to-ductal metaplasia and to accelerate development of premalignant lesions preceding pancreatic ductal adenocarcinoma (PDAC). Here, we investigate how SOX9 operates in pancreatic tumourigenesis. DESIGN: We analysed genomic and transcriptomic data from surgically resected PDAC and extended the expression analysis to xenografts from PDAC samples and to PDAC cell lines. SOX9 expression was manipulated in human cell lines and mouse models developing PDAC. RESULTS: We found genetic aberrations in the SOX9 gene in about 15% of patient tumours. Most PDAC samples strongly express SOX9 protein, and SOX9 levels are higher in classical PDAC. This tumour subtype is associated with better patient outcome, and cell lines of this subtype respond to therapy targeting epidermal growth factor receptor (EGFR/ERBB1) signalling, a pathway essential for pancreatic tumourigenesis. In human PDAC, high expression of SOX9 correlates with expression of genes belonging to the ERBB pathway. In particular, ERBB2 expression in PDAC cell lines is stimulated by SOX9. Inactivating Sox9 expression in mice confirmed its role in PDAC initiation; it demonstrated that Sox9 stimulates expression of several members of the ERBB pathway and is required for ERBB signalling activity. CONCLUSIONS: By integrating data from patient samples and mouse models, we found that SOX9 regulates the ERBB pathway throughout pancreatic tumourigenesis. Our work opens perspectives for therapy targeting tumourigenic mechanisms.

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.

Amino acid transporters expression in acinar cells is changed during acute pancreatitis.

Pancreatic acinar cells accumulate amino acids against a marked concentration gradient to synthesize digestive enzymes. Thus, the function of acinar cells depends on amino acid uptake mediated by active transport. Despite the importance of this process, pancreatic amino acid transporter expression and cellular localization is still unclear. We screened mouse pancreas for the expression of genes encoding amino acid transporters. We showed that the most highly expressed transporters, namely sodium dependent SNAT3 (Slc38a3) and SNAT5 (Slc38a5) and sodium independent neutral amino acids transporters LAT1 (Slc7a5) and LAT2 (Slc7a8), are expressed in the basolateral membrane of acinar cells. SNAT3 and SNAT5, LAT1 and LAT2 are expressed in acinar cells. Additional evidence that these transporters are expressed in mature acinar cells was gained using acinar cell culture and acute pancreatitis models. In the acute phase of pancreatic injury, when acinar cell loss occurs, and in an acinar cell culture model, which mimics changes occurring during pancreatitis, SNAT3 and SNAT5 are strongly down-regulated. LAT1 and LAT2 were down-regulated only in the in vitro model. At protein level, SNAT3 and SNAT5 expression was also reduced during pancreatitis. Expression of other amino acid transporters was also modified in both models of pancreatitis. The subset of transporters with differential expression patterns during acute pancreatitis might be involved in the injury/regeneration phases. Further expression, localization and functional studies will follow to better understand changes occurring during acute pancreatitis. These findings provide insight into pancreatic amino acid transport in healthy pancreas and during acute pancreatitis injury.

Histomolecular phenotypes and outcome in adenocarcinoma of the ampulla of vater.

PURPOSE: Individuals with adenocarcinoma of the ampulla of Vater demonstrate a broad range of outcomes, presumably because these cancers may arise from any one of the three epithelia that converge at that location. This variability poses challenges for clinical decision making and the development of novel therapeutic strategies. PATIENTS AND METHODS: We assessed the potential clinical utility of histomolecular phenotypes defined using a combination of histopathology and protein expression (CDX2 and MUC1) in 208 patients from three independent cohorts who underwent surgical resection for adenocarcinoma of the ampulla of Vater. RESULTS: Histologic subtype and CDX2 and MUC1 expression were significant prognostic variables. Patients with a histomolecular pancreaticobiliary phenotype (CDX2 negative, MUC1 positive) segregated into a poor prognostic group in the training (hazard ratio [HR], 3.34; 95% CI, 1.69 to 6.62; P < .001) and both validation cohorts (HR, 5.65; 95% CI, 2.77 to 11.5; P < .001 and HR, 2.78; 95% CI, 1.25 to 7.17; P = .0119) compared with histomolecular nonpancreaticobiliary carcinomas. Further stratification by lymph node (LN) status defined three clinically relevant subgroups: one, patients with histomolecular nonpancreaticobiliary (intestinal) carcinoma without LN metastases who had an excellent prognosis; two, those with histomolecular pancreaticobiliary carcinoma with LN metastases who had a poor outcome; and three, the remainder of patients (nonpancreaticobiliary, LN positive or pancreaticobiliary, LN negative) who had an intermediate outcome. CONCLUSION: Histopathologic and molecular criteria combine to define clinically relevant histomolecular phenotypes of adenocarcinoma of the ampulla of Vater and potentially represent distinct diseases with significant implications for current therapeutic strategies, the ability to interpret past clinical trials, and future trial design.

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.

Pancreatic cancer genomes reveal aberrations in axon guidance pathway genes.

Pancreatic cancer is a highly lethal malignancy with few effective therapies. We performed exome sequencing and copy number analysis to define genomic aberrations in a prospectively accrued clinical cohort (n = 142) of early (stage I and II) sporadic pancreatic ductal adenocarcinoma. Detailed analysis of 99 informative tumours identified substantial heterogeneity with 2,016 non-silent mutations and 1,628 copy-number variations. We define 16 significantly mutated genes, reaffirming known mutations (KRAS, TP53, CDKN2A, SMAD4, MLL3, TGFBR2, ARID1A and SF3B1), and uncover novel mutated genes including additional genes involved in chromatin modification (EPC1 and ARID2), DNA damage repair (ATM) and other mechanisms (ZIM2, MAP2K4, NALCN, SLC16A4 and MAGEA6). Integrative analysis with in vitro functional data and animal models provided supportive evidence for potential roles for these genetic aberrations in carcinogenesis. Pathway-based analysis of recurrently mutated genes recapitulated clustering in core signalling pathways in pancreatic ductal adenocarcinoma, and identified new mutated genes in each pathway. We also identified frequent and diverse somatic aberrations in genes described traditionally as embryonic regulators of axon guidance, particularly SLIT/ROBO signalling, which was also evident in murine Sleeping Beauty transposon-mediated somatic mutagenesis models of pancreatic cancer, providing further supportive evidence for the potential involvement of axon guidance genes in pancreatic carcinogenesis.

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.

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.