Loss of Pten and Activation of Kras Synergistically Induce Formation of Intraductal Papillary Mucinous Neoplasia From Pancreatic Ductal Cells in Mice.

BACKGROUND & AIMS: Intraductal papillary mucinous neoplasias (IPMNs) are precancerous cystic lesions that can develop into pancreatic ductal adenocarcinomas (PDACs). These large macroscopic lesions are frequently detected during medical imaging, but it is unclear how they form or progress to PDAC. We aimed to identify cells that form IPMNs and mutations that promote IPMN development and progression. METHODS: We generated mice with disruption of Pten specifically in ductal cells (Sox9CreERT2;Ptenflox/flox;R26RYFP or PtenΔDuct/ΔDuct mice) and used PtenΔDuct/+ and Pten+/+ mice as controls. We also generated KrasG12D;PtenΔDuct/ΔDuct and KrasG12D;PtenΔDuct/+ mice. Pancreata were collected when mice were 28 weeks to 14.5 months old and analyzed by histology, immunohistochemistry, and electron microscopy. We performed multiplexed droplet digital polymerase chain reaction to detect spontaneous Kras mutations in PtenΔDuct/ΔDuct mice and study the effects of Ras pathway activation on initiation and progression of IPMNs. We obtained 2 pancreatic sections from a patient with an invasive pancreatobiliary IPMN and analyzed the regions with and without the invasive IPMN (control tissue) by immunohistochemistry. RESULTS: Mice with ductal cell-specific disruption of Pten but not control mice developed sporadic, macroscopic, intraductal papillary lesions with histologic and molecular features of human IPMNs. PtenΔDuct/ΔDuct mice developed IPMNs of several subtypes. In PtenΔDuct/ΔDuct mice, 31.5% of IPMNs became invasive; invasion was associated with spontaneous mutations in Kras. KrasG12D;PtenΔDuct/ΔDuct mice all developed invasive IPMNs within 1 month. In KrasG12D;PtenΔDuct/+ mice, 70% developed IPMN, predominately of the pancreatobiliary subtype, and 63.3% developed PDAC. In all models, IPMNs and PDAC expressed the duct-specific lineage tracing marker yellow fluorescent protein. In immunohistochemical analyses, we found that the invasive human pancreatobiliary IPMN tissue had lower levels of PTEN and increased levels of phosphorylated (activated) ERK compared with healthy pancreatic tissue. CONCLUSIONS: In analyses of mice with ductal cell-specific disruption of Pten, with or without activated Kras, we found evidence for a ductal cell origin of IPMNs. We also showed that PTEN loss and activated Kras have synergistic effects in promoting development of IPMN and progression to PDAC.

Pancreatic ß-cell Raf-1 is required for glucose tolerance, insulin secretion, and insulin 2 transcription.

Regulation of glucose homeostasis by insulin depends on pancreatic ß-cell growth, survival, and function. Raf-1 kinase is a major downstream target of several growth factors that promote proliferation and survival of many cell types, including the pancreatic ß cells. We have previously reported that insulin protects ß cells from apoptosis and promotes proliferation by activating Raf-1 signaling in cultured human islets, mouse islets, and MIN6 cells. As Raf-1 activity is critical for basal apoptosis and insulin secretion in vitro, we hypothesized that Raf-1 may play an important role in glucose homeostasis in vivo. To test this hypothesis, we utilized the Cre-loxP recombination system to obtain a pancreatic ß-cell-specific ablation of Raf-1 kinase gene (RIPCre(+/+):Raf-1(flox/flox)) and a complete set of littermate controls (RIPCre(+/+):Raf-1(wt/wt)). RIPCre(+/+):Raf-1(flox/flox) mice were viable, and no effects on weight gain were observed. RIPCre(+/+):Raf-1(flox/flox) mice had increased fasting blood glucose levels and impaired glucose tolerance but normal insulin tolerance compared to littermate controls. Insulin secretion in vivo and in isolated islets was markedly impaired, but there was no apparent effect on the exocytosis machinery. However, islet insulin protein and insulin 2 mRNA, but not insulin 1 mRNA, were dramatically reduced in Raf-1-knockout mice. Analysis of insulin 2 knockout mice demonstrated that this reduction in mRNA was sufficient to impair in vivo insulin secretion. Our data further indicate that Raf-1 specifically and acutely regulates insulin 2 mRNA via negative action on Foxo1, which has been shown to selectively control the insulin 2 gene. This work provides the first direct evidence that Raf-1 signaling is essential for the regulation of basal insulin transcription and the supply of releasable insulin in vivo.

Synergism between vitamin D and secreted protein acidic and rich in cysteine-induced apoptosis and growth inhibition results in increased susceptibility of therapy-resistant colorectal cancer cells to chemotherapy.

Patients with advanced colorectal cancer continue to have poor outcomes because of therapy-refractory disease. We previously showed that secreted protein acidic and rich in cysteine (SPARC) gene and protein could function as a chemotherapy sensitizer by enhancing tumor regression in response to radiation and chemotherapy in tumor xenograft models of chemotherapy-resistant tumors. This function of SPARC was gleamed from a microarray analysis that also revealed down-regulation of the vitamin D receptor (VDR) in therapy-refractory colorectal cancer cells. This study examines the potential synergistic effect of SPARC and vitamin D, which up-regulates VDR, in enhancing chemotherapy response in colorectal cancer. Using MIP101 colorectal cancer cell lines and SPARC-overexpressing MIP101 cells, we were able to show that, in the presence of SPARC, exposure to low doses of 1alpha,25-dihydroxyvitamin D(3) significantly reduces cell viability, enhances chemotherapy-induced apoptosis, and inhibits the growth of colorectal cancer cells. Moreover, in tumor xenograft mouse models, up-regulation of VDR was seen in tumors that had the greatest regression following treatment that combined SPARC with chemotherapy. Therefore, our findings reveal a synergistic effect between SPARC and low doses of 1alpha,25-dihydroxyvitamin D(3) that further augments the sensitivity of tumors to chemotherapy. This combination may prove to be a useful adjunct in the treatment of colorectal cancer, especially in those patients with therapy-refractory disease.

Heparanase Overexpression Induces Glucagon Resistance and Protects Animals From Chemically Induced Diabetes.

Heparanase, a protein with enzymatic and nonenzymatic properties, contributes toward disease progression and prevention. In the current study, a fortuitous observation in transgenic mice globally overexpressing heparanase (hep-tg) was the discovery of improved glucose homeostasis. We examined the mechanisms that contribute toward this improved glucose metabolism. Heparanase overexpression was associated with enhanced glucose-stimulated insulin secretion and hyperglucagonemia, in addition to changes in islet composition and structure. Strikingly, the pancreatic islet transcriptome was greatly altered in hep-tg mice, with >2,000 genes differentially expressed versus control. The upregulated genes were enriched for diverse functions including cell death regulation, extracellular matrix component synthesis, and pancreatic hormone production. The downregulated genes were tightly linked to regulation of the cell cycle. In response to multiple low-dose streptozotocin (STZ), hep-tg animals developed less severe hyperglycemia compared with wild-type, an effect likely related to their ß-cells being more functionally efficient. In animals given a single high dose of STZ causing severe and rapid development of hyperglycemia related to the catastrophic loss of insulin, hep-tg mice continued to have significantly lower blood glucose. In these mice, protective pathways were uncovered for managing hyperglycemia and include augmentation of fibroblast growth factor 21 and glucagon-like peptide 1. This study uncovers the opportunity to use properties of heparanase in management of diabetes.

Reduced Insulin Production Relieves Endoplasmic Reticulum Stress and Induces ß Cell Proliferation.

Pancreatic ß cells are mostly post-mitotic, but it is unclear what locks them in this state. Perturbations including uncontrolled hyperglycemia can drive ß cells into more pliable states with reduced cellular insulin levels, increased ß cell proliferation, and hormone mis-expression, but it is unknown whether reduced insulin production itself plays a role. Here, we define the effects of ∼50% reduced insulin production in Ins1(-/-):Ins2(f/f):Pdx1Cre(ERT):mTmG mice prior to robust hyperglycemia. Transcriptome, proteome, and network analysis revealed alleviation of chronic endoplasmic reticulum (ER) stress, indicated by reduced Ddit3, Trib3, and Atf4 expression; reduced Xbp1 splicing; and reduced phospho-eIF2α. This state was associated with hyper-phosphorylation of Akt, which is negatively regulated by Trib3, and with cyclinD1 upregulation. Remarkably, ß cell proliferation was increased 2-fold after reduced insulin production independently of hyperglycemia. Eventually, recombined cells mis-expressed glucagon in the hyperglycemic state. We conclude that the normally high rate of insulin production suppresses ß cell proliferation in a cell-autonomous manner.

A feature analysis of lower solubility proteins in three eukaryotic systems.

Because misfolded and damaged proteins can form potentially harmful aggregates, all living organisms have evolved a wide variety of quality control mechanisms. However, the timely clearance of aggregation-prone species may not always be achieved, potentially leading to the accumulation of low solubility proteins. At the same time, promiscuity, which can be a driving force for aggregation, is also important to the functionality of certain proteins which have a large number of interaction partners. Considerable efforts have been made towards characterizing why some proteins appear to be more aggregation-prone than others. In this study, we analyze the features of proteins which precipitate following centrifugation in unstressed yeast cells, human SH-SY5Y cells and mouse brain tissue. By normalizing for protein abundance, we devised an approach whereby lower solubility proteins are reliably identified. Our findings indicate that these tend to be longer, low abundance proteins, which contain fewer hydrophobic amino acids. Furthermore, low solubility proteins also contain more low complexity and disordered regions. Overall, we observed an increase in features that link low solubility proteins to functional aggregates. Our results indicate that lower solubility proteins from three biologically distinct model systems share several common traits, shedding light on potentially universal solubility determinants. BIOLOGICAL SIGNIFICANCE: We set up a novel approach to identify lower solubility proteins in unstressed cells by comparing precipitated proteins with those that remain soluble after centrifugation. By analyzing three eukaryotic model systems in parallel, we were able to identify traits which cross the species barrier, as well as species-specific characteristics. Notably, our analyses revealed a number of primary and secondary structural features that set apart lower solubility proteins, a number of which connected them to a greater potential for promiscuity. This article is part of a Special Issue entitled: Protein dynamics in health and disease. Guest Editors: Pierre Thibault and Anne-Claude Gingras.

Bcl-2 and Bcl-xL suppress glucose signaling in pancreatic ß-cells.

B-cell lymphoma 2 (Bcl-2) family proteins are established regulators of cell survival, but their involvement in the normal function of primary cells has only recently begun to receive attention. In this study, we demonstrate that chemical and genetic loss-of-function of antiapoptotic Bcl-2 and Bcl-x(L) significantly augments glucose-dependent metabolic and Ca(2+) signals in primary pancreatic ß-cells. Antagonism of Bcl-2/Bcl-x(L) by two distinct small-molecule compounds rapidly hyperpolarized ß-cell mitochondria, increased cytosolic Ca(2+), and stimulated insulin release via the ATP-dependent pathway in ß-cell under substimulatory glucose conditions. Experiments with single and double Bax-Bak knockout ß-cells established that this occurred independently of these proapoptotic binding partners. Pancreatic ß-cells from Bcl-2(-/-) mice responded to glucose with significantly increased NAD(P)H levels and cytosolic Ca(2+) signals, as well as significantly augmented insulin secretion. Inducible deletion of Bcl-x(L) in adult mouse ß-cells also increased glucose-stimulated NAD(P)H and Ca(2+) responses and resulted in an improvement of in vivo glucose tolerance in the conditional Bcl-x(L) knockout animals. Our work suggests that prosurvival Bcl proteins normally dampen the ß-cell response to glucose and thus reveals these core apoptosis proteins as integrators of cell death and physiology in pancreatic ß-cells.

Acute insulin signaling in pancreatic beta-cells is mediated by multiple Raf-1 dependent pathways.

Insulin enhances the proliferation and survival of pancreatic beta-cells, but its mechanisms remain unclear. We hypothesized that Raf-1, a kinase upstream of both ERK and Bad, might be a critical target of insulin in beta-cells. To test this hypothesis, we treated human and mouse islets as well as MIN6 beta-cells with multiple insulin concentrations and examined putative downstream targets using immunoblotting, immunoprecipitation, quantitative fluorescent imaging, and cell death assays. Low doses of insulin rapidly activated Raf-1 by dephosphorylating serine 259 and phosphorylating serine 338 in human islets, mouse islets, and MIN6 cells. The phosphorylation of ERK by insulin was eliminated by exposure to a Raf inhibitor (GW5074) or transfection with a dominant-negative Raf-1 mutant. Insulin also enhanced the interaction between mitochondrial Raf-1 and Bcl-2 agonist of cell death (Bad), promoting Bad inactivation via its phosphorylation on serine 112. Insulin-stimulated ERK phosphorylation was abrogated by calcium chelation, calcineurin and calmodulin-dependent protein kinase II inhibitors, and Ned-19, a nicotinic acid adenine dinucleotide phosphate receptor (NAADPR) antagonist. Blocking Raf-1 and Ca(2+) signaling resulted in nonadditive beta-cell death. Autocrine insulin signaling partly accounted for the effects of glucose on ERK phosphorylation. Our results demonstrate that Raf-1 is a critical target of insulin in primary beta-cells. Activation of Raf-1 leads to both an ERK-dependent pathway that involves nicotinic acid adenine dinucleotide phosphate-sensitive Ca(2+) stores and Ca(2+)-dependent phosphorylation events, and an ERK-independent pathway that involves Bad inactivation at the mitochondria. Together our findings identify a novel insulin signaling pathway in beta-cells and shed light on insulin's antiapoptotic and mitogenic mechanisms.