Bidyut Ghosh

Notch mediates TGFα-induced changes in epithelial differentiation during pancreatic tumorigenesis

Notch signaling regulates cell fate decisions in a wide variety of adult and embryonic tissues. Here we show that Notch pathway components and Notch target genes are upregulated in invasive pancreatic cancer, as well as in pancreatic cancer precursors from both mouse and human. In mouse pancreas, ectopic Notch activation results in accumulation of nestin-positive precursor cells and expansion of metaplastic ductal epithelium, previously identified as a precursor lesion for pancreatic cancer. Notch is also activated as a direct consequence of EGF receptor activation in exocrine pancreas and is required for TGF alpha-induced changes in epithelial differentiation. These findings suggest that Notch mediates the tumor-initiating effects of TG alpha by expanding a population of undifferentiated precursor cells.

Notch inhibits Ptf1 function and acinar cell differentiation in developing mouse and zebrafish pancreas

Notch signaling regulates cell fate decisions in a variety of adult and embryonic tissues, and represents a characteristic feature of exocrine pancreatic cancer. In developing mouse pancreas, targeted inactivation of Notch pathway components has defined a role for Notch in regulating early endocrine differentiation, but has been less informative with respect to a possible role for Notch in regulating subsequent exocrine differentiation events. Here, we show that activated Notch and Notch target genes actively repress completion of an acinar cell differentiation program in developing mouse and zebrafish pancreas. In developing mouse pancreas, the Notch target gene Hes1 is co-expressed with Ptf1-P48 in exocrine precursor cells, but not in differentiated amylase-positive acinar cells. Using lentiviral delivery systems to induce ectopic Notch pathway activation in explant cultures of E10.5 mouse dorsal pancreatic buds, we found that both Hes1 and Notch1-IC repress acinar cell differentiation, but not Ptf1-P48 expression, in a cell-autonomous manner. Ectopic Notch activation also delays acinar cell differentiation in developing zebrafish pancreas. Further evidence of a role for endogenous Notch in regulating exocrine pancreatic differentiation was provided by examination of zebrafish embryos with homozygous mindbomb mutations, in which Notch signaling is disrupted. mindbomb-deficient embryos display accelerated differentiation of exocrine pancreas relative to wild-type clutchmate controls. A similar phenotype was induced by expression of a dominant-negative Suppressor of Hairless [Su(H)] construct, confirming that Notch actively represses acinar cell differentiation during zebrafish pancreatic development. Using transient transfection assays involving a Ptf1-responsive reporter gene, we further demonstrate that Notch and Notch/Su(H) target genes directly inhibit Ptf1 activity, independent of changes in expression of Ptf1 component proteins. These results define a normal inhibitory role for Notch in the regulation of exocrine pancreatic differentiation.

Interactions between hairy/enhancer of split-related proteins and the pancreatic transcription factor Ptf1-p48 modulate function of the PTF1 transcriptional complex.

In the developing pancreas, the onset of exocrine differentiation is driven by the activity of the PTF1 (pancreas transcription factor 1) transcriptional complex, which is comprised of the class II bHLH (basic helix-loop-helix) protein, Ptf1-p48 [also known as Ptf1a (pancreas specific transcription factor 1a)], and a class I E-box binding partner. Activity of the PTF1 complex is normally inhibited by the Notch signalling pathway, a process mediated by Notch effector proteins in the HES (Hairy/Enhancer of Split) family of bHLH transcriptional repressors. In the present study, we show that this inhibitory effect occurs through direct interaction between HES family members and Ptf1-p48. The HES family members Hey1 (hairy/enhancer-of-split related with YRPW motif 1) and Hey2 co-immunoprecipitate with Ptf1-p48, and Ptf1-p48 binding by Hes1 is also evident in yeast two-hybrid and GST (glutathione S-transferase) pull-down assays. The ability of Hes1 to interact with Ptf1-p48 resides within a fragment comprised of the bHLH, Orange and C-terminal domains, and does not require the N-terminal or WRPW elements. The ability of truncated versions of Hes1 to bind Ptf1-p48 correlates with their ability to down-regulate the activity of the PTF1 transcriptional complex, defining Ptf1-p48 binding as the most likely mechanism by which Notch effector proteins delay exocrine pancreatic differentiation.

Primary Explant Cultures of Adult and Embryonic Pancreas

The developmental plasticity of adult pancreas is evidenced by the ability to undergo conversion between different epithelial cell types. Specific examples of such conversions include acinar to ductal metaplasia, ductal to islet metaplasia, and generation of ductal structures within islets. Although 90% of human pancreatic cancers are classified as ductal adenocarcinoma, markers of all pancreatic epithelial cell types (acini, ductal, and endocrine) as well as markers of gastric and intestinal lineages can be detected in these tumors. In recent years considerable knowledge has been gained regarding regulation of cellular differentiation and various signaling pathways involved in normal and neoplastic pancreas through studies of pancreatic cancer and immortalized ductal cell lines. However, these studies provide little insight into the context of normal developmental cues, the disruption of which leads to organ pathology. Here we have described a detailed method for preparation, maintenance, and manipulation of adult and embryonic mouse pancreas. These methods may be utilized in studies involving normal epithelial differentiation, contributing to improved understanding of pancreatic development and disease.

Regulation of PPAR-alpha pathway by Dicer revealed through proteomic analysis.

UNLABELLED Dicer is a crucial RNase III enzyme in miRNA biogenesis pathway. Although numerous studies have been carried out to investigate the role of miRNAs and Dicer in the regulation of biological processes, few studies have examined proteomic alterations upon knockout of Dicer. We employed a Cre-loxP-based inducible knockout mouse system to investigate the proteome regulated by Dicer-dependent miRNAs. We utilized spiked liver lysates from metabolically labeled mice to quantify the subtle changes in the liver proteome upon deletion of Dicer. We identified 2137 proteins using high resolution tandem mass spectrometry analysis. The upregulated proteins included several enzymes involved in peroxisomal β-oxidation of fatty acids and a large majority of the upregulated proteins involved in lipid metabolism were known PPARα targets. MRM-based assays were carried out to confirm the upregulation of enzymes including peroxisomal bifunctional enzyme, phosphoenolpyruvate carboxykinase 1, cytochrome P450 3A13, cytochrome P450 3A41 and myristoylated alanine-rich protein kinase C substrate. Further, miRNA-124 which is predicted to regulate expression of peroxisomal bifunctional enzyme was confirmed to be downregulated in the Dicer knockout mice. Our study demonstrates the strength of coupling knockout mouse models and quantitative proteomic strategies to investigate functions of individual proteins in vivo. BIOLOGICAL SIGNIFICANCE Dicer dependent miRNA biogenesis is the major pathway for generation of mature miRNAs. We developed SILAC mouse-based proteomics screen to identify protein targets of Dicer-dependent miRNAs in liver of Dicer knockout mice. We spiked liver lysates of induced and uninduced Dicer knockout mice with liver lysate of SILAC labeled mice for identification of dysregulated proteome. We quantitated 1217 proteins of which 257 were upregulated in induced Dicer knockout mice. We observed enrichment of PPAR-α targets and proteins involved in lipid metabolism among upregulated proteins. We further carried out MRM-based validation of peroxisomal bifunctional enzyme, phosphoenolpyruvate carboxykinase 1, Cyp3A13, Cyp3A41 and myristoylated alanine-rich protein kinase C substrate. We further validated upregulation of peroxisomal bifunctional enzyme using Western blot analysis and downregulation of its predicted upstream miRNA, miR-124 using qRT-PCR. Our study demonstrates that upon ablation of Dicer, certain Dicer-dependent miRNAs are dysregulated which result in dysregulation of their target proteins such as proteins associated with lipid metabolism. Our study illustrates the use of SILAC strategy for quantitative proteomic investigations of animal model systems.

Dicer is required for maintenance of adult pancreatic acinar cell identity and plays a role in Kras-driven pancreatic neoplasia

The role of miRNA processing in the maintenance of adult pancreatic acinar cell identity and during the initiation and progression of pancreatic neoplasia has not been studied in detail. In this work, we deleted Dicer specifically in adult pancreatic acinar cells, with or without simultaneous activation of oncogenic Kras. We found that Dicer is essential for the maintenance of acinar cell identity. Acinar cells lacking Dicer showed increased plasticity, as evidenced by loss of polarity, initiation of epithelial-to-mesenchymal transition (EMT) and acinar-to-ductal metaplasia (ADM). In the context of oncogenic Kras activation, the initiation of ADM and pancreatic intraepithelial neoplasia (PanIN) were both highly sensitive to Dicer gene dosage. Homozygous Dicer deletion accelerated the formation of ADM but not PanIN. In contrast, heterozygous Dicer deletion accelerated PanIN initiation, revealing complex roles for Dicer in the regulation of both normal and neoplastic pancreatic epithelial identity.

p53: Guardian of pancreatic epithelial identity.

Comment on: Andreia V. Pinho, et al. Cell Cycle 2011; 10: In press.

GnasR201C Induces Murine Pancreatic Cystic Neoplasms through Suppression of YAP1 Signaling and Transcriptional Reprogramming

Background & Aims Somatic “hotspot” mutations of GNAS, which encodes for the alpha subunit of stimulatory G-protein, are present in ~60% of intraductal papillary mucinous neoplasms (IPMNs) of the pancreas. There are currently no cognate animal models that recapitulate the biology of mutant Gnas-induced IPMNs, and the underlying mechanisms that lead to the cystic pathway of neoplasia in the pancreas remain unknown. Methods We generated p48-Cre; LSL-KrasG12D; Rosa26R-LSL-rtTA-TetO-GnasR201C mice (Kras; Gnas mice) where pancreas-specific GnasR201C expression was induced by doxycycline administration. In this model, mutant Kras is constitutively expressed, and control mice were produced through absence of doxycycline. Separate cohorts of mice were utilized for timed necropsies and for Kaplan-Meier survival analysis. Isogenic cell lines (with doxycycline inducible mutant Gnas expression) were propagated from the resulting pancreatic ductal adenocarcinoma (PDAC). Results Co-expression of KrasG12D and GnasR201C resulted in the development of pancreatic cystic lesions resembling human IPMNs in 100% of mice, with higher grades of epithelial dysplasia observed over time. Approximately one-third of Kras; Gnas mice developed PDAC at a median of 38 weeks post doxycycline induction. GnasR201C did not accelerate oncogenic transformation with KrasG12D, but rather, reprogrammed Ras-induced neoplasms towards a well-differentiated phenotype. GnasR201C induction led to activation of the inhibitory Hippo kinase cascade and cytoplasmic sequestration of phosphorylated YAP1 protein, a phenomenon that was also observed in human IPMN with GNAS mutations. Conclusions GNASR201C functions not as a traditional oncogene, but rather as an “oncomodulator” of KRAS-mediated pancreatic neoplasia, through suppression of YAP1 and transcriptional reprogramming towards a differentiated (large ductal) phenotype.

Ablation of Dicer leads to widespread perturbation of signaling pathways.

Dicer is an essential ribonuclease involved in the biogenesis of miRNAs. Previous studies have reported downregulation of Dicer in multiple cancers including hepatocellular carcinoma. To identify signaling pathways that are altered upon Dicer depletion, we carried out quantitative phosphotyrosine profiling of liver tissue from Dicer knockout mice. We employed antibody-based enrichment of phosphotyrosine containing peptides coupled with SILAC spike-in approach for quantitation. High resolution mass spectrometry-based analysis identified 349 phosphotyrosine peptides corresponding to 306 unique phosphosites of which 75 were hyperphosphorylated and 78 were hypophosphorylated. Several receptor tyrosine kinases including MET, PDGF receptor alpha, Insulin-like growth factor 1 and Insulin receptor as well as non-receptor tyrosine kinases such as Src family kinases were found to be hyperphosphorylated upon depletion of Dicer. In addition, signaling molecules such as IRS-2 and STAT3 were hyperphosphorylated. Activation of these signaling pathways has been implicated previously in various types of cancers. Interestingly, we observed hypophosphorylation of molecules including focal adhesion kinase and paxillin. Our study profiles the perturbed signaling pathways in response to dysregulated miRNAs resulting from depletion of Dicer. Our findings warrant further studies to investigate oncogenic effects of downregulation of Dicer in cancers.