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Adeno-associated virus' (AAV) relatively simple structure makes it accommodating for engineering into controllable delivery platforms. Cancer, such as pancreatic ductal adenocarcinoma (PDAC), are often characterized by upregulation of membrane-bound proteins, such as MMP-14, that propagate survival integrin signaling. In order to target tumors, we have engineered an MMP-14 protease-activatable AAV vector that responds to both membrane-bound and extracellularly active MMPs. This "provector" was generated by inserting a tetra-aspartic acid inactivating motif flanked by the MMP-14 cleavage sequence IPESLRAG into the capsid subunits. The MMP-14 provector shows lower background transduction than previously developed provectors, leading to a 9.5-fold increase in transduction ability. In a murine model of PDAC, the MMP-14 provector shows increased delivery to an allograft tumor. This proof-of-concept study illustrates the possibilities of membrane-bound protease-activatable gene therapies to target tumors.

Genetically engineered mouse models (GEMMs) that recapitulate the major genetic drivers in pancreatic ductal adenocarcinoma (PDAC) have provided unprecedented insights into the pathogenesis of this lethal neoplasm. Nonetheless, generating an autochthonous model is an expensive, time consuming and labor intensive process, particularly when tissue specific expression or deletion of compound alleles are involved. In addition, many of the current PDAC GEMMs cause embryonic, pancreas-wide activation or loss of driver alleles, neither of which reflects the cognate human disease scenario. The advent of CRISPR/Cas9 based gene editing can potentially circumvent many of the aforementioned shortcomings of conventional breeding schema, but ensuring the efficiency of gene editingin vivoremains a challenge. Here we have developed a pipeline for generating PDAC GEMMs of complex genotypes with high efficiency using a single “workhorse” mouse strain expressing Cas9 in the adult pancreas under ap48promoter. Using adeno-associated virus (AAV) mediated delivery of multiplexed guide RNAs (sgRNAs) to the adult murine pancreas ofp48-Cre; LSL-Cas9mice, we confirm our ability to express an oncogenicKrasG12Dallele through homology-directed repair (HDR), in conjunction with CRISPR-induced disruption of cooperating alleles (Trp53, Lkb1andArid1A). The resulting GEMMs demonstrate a spectrum of precursor lesions (pancreatic intraepithelial neoplasia [PanIN] or Intraductal papillary mucinous neoplasm [IPMN] with eventual progression to PDAC. Next generation sequencing of the resulting murine PDAC confirms HDR of oncogenicKrasG12Dallele at the endogenous locus, and insertion deletion (“indel”) and frameshift mutations of targeted tumor suppressor alleles. By using a single “workhorse” mouse strain and optimal AAV serotype forin vivogene editing with combination of driver alleles, we have created a facile autochthonous platform for interrogation of the PDAC genome.

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.

In the developing pancreas, the onset of exocrine differentiation is driven by the activity of the PTF1 (pancreas transciption 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.

Mammalian studies have implicated important roles for the basic helix–loop–helix transcription factor Ptf1a-p48 in the development of both exocrine and endocrine pancreas. We have cloned the Ptf1a-p48 ortholog in Danio rerio. Early zebrafish ptf1a expression is observed in developing hindbrain and in endodermal pancreatic precursors. Analysis of ptf1a and insulin expression reveals a population of exocrine precursors that, throughout early development, are temporally and spatially segregated from endocrine elements. Morpholino-mediated knockdown of ptf1a confirms early divergence of these endocrine and exocrine lineages. Ptf1a morphants lack differentiated exocrine pancreas, but maintain normal differentiation and organization of the principal islet. In addition to the exocrine phenotype, ptf1a knockdown also reduces the prevalence of a small population of anterior endocrine cells normally found outside the principal islet. Together, these findings suggest the presence of distinct ptf1a-dependent and ptf1a-independent precursor populations in developing zebrafish pancreas.

The present study reports the effect of ceramide generated by hydrolysis of membrane sphingomyelin with bacterial sphingomyelinase (SMase) and of cell-permeable ceramide analogues on the expression of manganese superoxide dismutase (MnSOD). Incubation of the rat primary astrocytes with SMase led to a time- and dose-dependent increase in MnSOD activity. The increase in MnSOD activity was accompanied by an increase in MnSOD protein and mRNA. A similar effect on the expression of MnSOD was observed with the addition of cell-permeable ceramide analogues (C2 and C6). On the other hand, C2-dihydroceramide (N-acetylsphinganine), which lacks the functional critical double bond, was ineffective in inducing the expression of MnSOD. Nuclear run-on analysis showed that SMase and ceramide increased the rate of transcription of the MnSOD gene. Besides astrocytes, SMase was also found to induce the expression of MnSOD in rat mesangial cells, C6 glial cells, PC12 cells, and human skin fibroblasts. Markedly higher expression of mRNA, protein, and activity of MnSOD in skin fibroblasts from patients with Farber disease, a human disorder with pathognomonic accumulation of ceramide due to a deficiency of ceramidase, than in normal skin fibroblasts indicate that ceramide may act as a physiological inducer of MnSOD gene expression. However, stimulation of ceramide-mediated DNA fragmentation by antisense knockdown of MnSOD suggests that induction of MnSOD by ceramide is a protective response of the cell.

A polyclonal antibody against a glutathione S:-transferase fusion protein containing the 76 COOH-terminal amino acids of Hex, a divergent homeobox gene, was raised in rabbits. Western blot and immunofluorescence reveal that Hex is a 35-37-kD soluble protein present both in the nucleus and cytoplasm of transfected and nontransfected cultured cells as well as in whole mouse embryo. Confocal microscopy of whole mount immunostained mouse embryos at E7. 5 and E8.5 demonstrates that Hex is differentially localized in the cytoplasm and nucleus of definitive endoderm, developing blood islands, and hepatic diverticulum. In particular, in the region of the foregut that gives rise to the liver, Hex expression is nuclear in the endodermal cells of the hepatic diverticulum, whereas expression is primarily cytoplasmic in cells lateral to the liver-forming region. This suggests that nuclear localization of Hex is involved in early hepatic specification and that compartmentalization of Hex protein plays an important role in its function during mouse development.

Reactive oxygen species (ROS; O2-, H2O2, and OH), normal by-products of cellular metabolic processes, are kept in control by antioxidant enzymes, such as catalase, glutathione peroxidase (GPX) and superoxide dismutases (SODs). To understand the role of antioxidant enzymatic defenses against ROS injury following ischemia-reperfusion, we examined the effect on kidney exposed to varying periods (30, 60 or 90 min) of ischemia followed by different periods of reperfusion. The enzymatic activities and protein levels of catalase, GPX, CuZnSOD and MnSOD were relatively unaffected at 30 min of ischemia followed by 0, 2 or 24 h reperfusion. However, 60 or 90 min of ischemia followed by 0, 2 or 24 h of reperfusion resulted in a decrease in activities and protein levels which paralleled the duration of ischemic injury. MnSOD activity tended to recover towards normal during reperfusion. Examination of the mRNA levels of these antioxidant enzymes demonstrated a severe decrease in mRNA levels of catalase and GPX at a time point of minimal ischemic injury (30 min of ischemia followed by reperfusion) suggesting that loss of mRNA of catalase and GPX may be the first markers of alterations in cellular redox in ischemia-reperfusion injury. Greater loss of mRNA for catalase, GPX and CuZnSOD was observed following longer periods (60 or 90 min) of ischemia. The mRNA for MnSOD was upregulated at all time points of ischemia-reperfusion injury. Actually, the greater decrease in mRNAs for catalase, GPX and CuZnSOD in the acute phase (within 24 h) subsequently showed a further decrease in these enzyme activities in the subacute phase (72 or 120 h after ischemia). These enzyme activities in the 30 min ischemia group, (but not in the 90 min group), already showed tendencies for normalization at 120 h after ischemia. To understand the molecular basis of the loss of mRNA of these antioxidant enzymes during ischemia-reperfusion injury, we examined the rate of transcription by nuclear run-on assays. The similar rates of transcription in control and kidney exposed to ischemia-reperfusion indicates that the loss of mRNA for catalase, GPX and CuZnSOD is possibly due to the increased rate of turnover of their mRNAs. These studies suggest that expression of antioxidant genes during ischemia-reperfusion are not coordinately expressed and that the differential loss of antioxidant enzymes may be the contributing factor(s) towards the heterogeneous renal tissue damage as a result of ischemia-reperfusion induced oxidative stress.

The excessive expression of catalase protein and its activity in cultured skin fibroblast from Zellweger Syndrome (ZS), a disorder of peroxisomal biogenesis, was found to be regulated at the translational level (J. Neurochem. 67: 2373-2378, 1996). Overall there is a considerable increase in the association of catalase mRNA with polysomes in ZS cell lines as compared to control indicating translational upregulation. To investigate the possibility that RNA-protein interactions are involved in the mediation of this increase in translation, the interaction between 3′ untranslated region of human catalase mRNA and human fibroblast cytoplasmic proteins were investigated by RNA gel shift assay technique. Competition experiments demonstrated that all the 600 bases of 3′ UTR (of human catalase gene) was required for efficient binding. Catalase RNA- protein interaction was sensitive to the altered redox state in these in vitro assays and this RNA-protein interaction could be enhanced by the addition of β-mercaptoethanol in cytoplasm from control fibroblast but not in cytoplasm from ZS fibroblast. UV cross linked RNA-protein complexes on SDS polyacrylamide gel electrophoresis revealed the presence of at least four protein bands with approximate molecular masses of 38 kDa, 50 kDa, 66 kDa and 80 kDa. The potential role of these mRNA binding proteins in the regulation of catalase gene expression is discussed.

The effect of endotoxin on antioxidant gene expression and antioxidant enzyme activity in homogenates of the heart, liver, and kidney from Sprague-Dawley rats was compared by quantitation of m-RNA and enzyme activities. Alterations in the message level for Cu-Zn superoxide dismutase (SOD), Mn SOD, and catalase varied with the tissue type, length of exposure to endotoxin, and dose of endotoxin. In general, endotoxin treatment reduced Cu-Zn SOD expression in the heart and liver, but had no noticeable effect in the kidney. Mn SOD message levels were increased in the heart and kidney but decreased in the liver. Catalase expression was reduced in the kidney and increased marginally in the heart and liver. With regard to enzyme activity, endotoxin treatment reduced Cu-Zn SOD activity in the heart, liver, and kidney. Mn SOD activity showed little change in the heart, but increased in the liver and, to a lesser extent, in the kidney. Catalase activity showed little change in the heart and kidney but was decreased at 12 h in the liver. The differing responses of tissues to the oxidant stress of endotoxin exposure should be considered when evaluating the effect of endotoxin on antioxidant enzymes.

Dicer is a ribonuclease whose major role is to generate mature microRNAs, although additional functions have been proposed. Deletion of Dicer leads to embryonic lethality in mice. To study the role of Dicer in adults, we generated mice in which administration of tamoxifen induces deletion of Dicer. Surprisingly, disruption of Dicer in adult mice induced lipid accumulation in the small intestine. To dissect the underlying mechanisms, we carried out miRNA, mRNA, and proteomic profiling of the small intestine. The proteomic analysis was done using mice metabolically labeled with heavy lysine (SILAC mice) for an in vivo readout. We identified 646 proteins, of which 80 were up-regulated2-fold and 75 were down-regulated. Consistent with the accumulation of lipids, Dicer disruption caused a marked decrease of microsomal triglyceride transfer protein, long-chain fatty acyl-CoA ligase 5, fatty acid binding protein, and very-long-chain fatty acyl-CoA dehydrogenase, among others. We validated these results using multiple reaction monitoring (MRM) experiments by targeting proteotypic peptides. Our data reveal a previously unappreciated role of Dicer in lipid metabolism. These studies demonstrate that a systems biology approach by integrating mouse models, metabolic labeling, gene expression profiling, and quantitative proteomics can be a powerful tool for understanding complex biological systems.

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.

A complimentary DNA clone encoding the entire human palmitoyl-CoA ligase has been isolated from a liver cDNA library and sequenced in it's entirety. The predicted product is a 699 amino acid protein. Southern analysis utilizing the human palmitoyl-CoA ligase gene as a probe revealed varying degrees of similarity amongst various mammalian species. The palmitoyl-CoA ligase gene is highly expressed in liver, heart, skeletal muscle and kidney, and to a lesser extent in brain, lung, placenta and pancreas. The expression of palmitoyl-CoA ligase in various tissue parallels the function of this enzyme in the metabolism of fatty acids in these tissues.