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Ghanbar Mahmoodi Chalbatani,1,* Hassan Dana,1,2,* Elahe Gharagouzloo,1 Santiago Grijalvo,3,4 Ramon Eritja,3,4 Craig D Logsdon,5,6 Fereidoon Memari,1 Seyed Rouhollah Miri,1 Mahdi Rezvani Rad,7 Vahid Marmari21Cancer Research Center, Cancer Institute of Iran, Tehran University of Medical Science, Tehran, Iran; 2Department of Biology, Damghan Branch, Islamic Azad University, Damghan, Iran; 3Institute of Advanced Chemistry of Catalonia (IQAC-CSIC), Barcelona 08034, Spain; 4Biomedical Research Networking Center in Bioengineering, Biomaterials and Nanomedicine (CIBER BBN), Madrid, Spain; 5Department of Cancer Biology, University of Texas, M.D. Anderson Cancer Center, Houston, TX, USA; 6Department of GI Medical Oncology, University of Texas, M.D. Anderson Cancer Center, Houston, TX, USA; 7Department of Biology, Zabol University, Zabol, Iran*These authors contributed equally to this workAbstract: Cancer is one of the most complex diseases that has resulted in multiple genetic disorders and cellular abnormalities. Globally, cancer is the most common health concern disease that is affecting human beings. Great efforts have been made over the past decades in biology with the aim of searching novel and more efficient tools in therapy. Thus, small interfering RNAs (siRNAs) have been considered one of the most noteworthy developments which are able to regulate gene expression following a process known as RNA interference (RNAi). RNAi is a post-transcriptional mechanism that involves the inhibition of gene expression through promoting cleavage on a specific area of a target messenger RNA (mRNA). This technology has shown promising therapeutic results for a good number of diseases, especially in cancer. However, siRNA therapeutics have to face important drawbacks in therapy including stability and successful siRNA delivery in vivo. In this regard, the development of effective siRNA delivery systems has helped addressing these issues by opening novel therapeutic windows which have allowed to build up important advances in Nanomedicine. In this review, we discuss the progress of siRNA therapy as well as its medical application via nanoparticle-mediated delivery for cancer treatment.Keywords: delivery strategies, lipoplexes, nanovectors, polymeric nanoparticles, siRNA

Genetic mutations that give rise to active mutant forms of Ras are oncogenic and found in several types of tumor. However, such mutations are not clear biomarkers for disease, since they are frequently detected in healthy individuals. Instead, it has become clear that elevated levels of Ras activity are critical for Ras-induced tumorigenesis. However, the mechanisms underlying the production of pathological levels of Ras activity are unclear. Here, we show that in the presence of oncogenic Ras, inflammatory stimuli initiate a positive feedback loop involving NF-κB that further amplifies Ras activity to pathological levels. Stimulation of Ras signaling by typical inflammatory stimuli was transient and had no long-term sequelae in wild-type mice. In contrast, these stimuli generated prolonged Ras signaling and led to chronic inflammation and precancerous pancreatic lesions (PanINs) in mice expressing physiological levels of oncogenic K-Ras. These effects of inflammatory stimuli were disrupted by deletion of inhibitor of NF-κB kinase 2 (IKK2) or inhibition of Cox-2. Likewise, expression of active IKK2 or Cox-2 or treatment with LPS generated chronic inflammation and PanINs only in mice expressing oncogenic K-Ras. The data support the hypothesis that in the presence of oncogenic Ras, inflammatory stimuli trigger an NF-κB-mediated positive feedback mechanism involving Cox-2 that amplifies Ras activity to pathological levels. Because a large proportion of the adult human population possesses Ras mutations in tissues including colon, pancreas, and lung, disruption of this positive feedback loop may be an important strategy for cancer prevention. [ABSTRACT FROM AUTHOR]

Pancreatic ductal adenocarcinoma (PDAC) exhibits an immunosuppressive tumor microenvironment (TME) contributing to its therapeutic resistance. Following our previous studies, we report that mast cells infiltrating the PDAC TME foster this immunosuppression and desmoplasia. Mast cell infiltration correlated with human PDAC progression, and genetic or pharmacological mast cell depletion reduced tumor growth and desmoplasia while enhancing survival in mouse PDAC models. Mechanistically, mast cell-derived IL-10 promoted PDAC progression. Strikingly, combining an agonistic anti-OX40 immunotherapy with mast cell blockade synergistically elicited durable anti-tumor immunity, marked by increased infiltration of CD8 + T effector cells expressing granzyme B and dramatic survival benefit unachievable with either approach alone. An OX40-associated gene signature correlated with improved survival in human PDAC, supporting therapeutic translation. Our findings establish mast cells as promoters of the suppressive PDAC TME and rational targets for combination immunotherapy. Targeting this mast cell-mediated resistance mechanism could overcome immunotherapy failure in PDAC., Competing Interests: The authors disclose no potential conflicts of interest., (© 2024 The Author(s).)

The KRAS G12D mutation was believed to be locked in a GTP-bound form, rendering it fully active. However, recent studies have indicated that the presence of mutant KRAS alone is insufficient; it requires additional activation through inflammatory stimuli to effectively drive the development of pancreatic ductal adenocarcinoma (PDAC). It remains unclear to what extent RAS activation occurs during the development of PDAC in the context of inflammation. Here, in a mouse model with the concurrent expression of Kras G12D/+ and inflammation mediator IKK2 in pancreatic acinar cells, we showed that, compared to KRAS G12D alone, the cooperative interaction between KRAS G12D and IKK2 rapidly elevated both the protein level and activity of KRAS G12D and NRAS in a short term. This high level was sustained throughout the rest phase of PDAC development. These results suggest that inflammation not only rapidly augments the activity but also the protein abundance, leading to an enhanced total amount of GTP-bound RAS (KRAS G12D and NRAS) in the early stage. Notably, while KRAS G12D could be further activated by IKK2, not all KRAS G12D proteins were in the GTP-bound state. Overall, our findings suggest that although KRAS G12D is not fully active in the context of inflammation, concurrent increases in both the protein level and activity of KRAS G12D as well as NRAS at the early stage by inflammation contribute to the rise in total GTP-bound RAS., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

KRAS mutations are prevalent in patients with pancreatic ductal adenocarcinoma (PDAC) and are critical to fostering tumor growth in part by aberrantly rewiring glucose, amino acid, and lipid metabolism. Obesity is a modifiable risk factor for pancreatic cancer. Corroborating this epidemiological observation, mice harboring mutant KRAS are highly vulnerable to obesogenic high-fat diet (HFD) challenges leading to the development of PDAC with high penetrance. However, the contributions of other macronutrient diets, such as diets rich in carbohydrates that are regarded as a more direct source to fuel glycolysis for cancer cell survival and proliferation than HFD, to pancreatic tumorigenesis remain unclear. In this study, we compared the differential effects of a high-carbohydrate diet (HCD), an HFD, and a high-protein diet (HPD) in PDAC development using a mouse model expressing an endogenous level of mutant KRAS G12D specifically in pancreatic acinar cells. Our study showed that although with a lower tumorigenic capacity than chronic HFD, chronic HCD promoted acinar-to-ductal metaplasia (ADM) and pancreatic intraepithelial neoplasia (PanIN) lesions with increased inflammation, fibrosis, and cell proliferation compared to the normal diet (ND) in Kras G12D/+ mice. By contrast, chronic HPD showed no significant adverse effects compared to the ND. Furthermore, ablation of pancreatic acinar cell cyclooxygenase 2 ( Cox-2 ) in Kras G12D/+ mice abrogated the adverse effects induced by HCD, suggesting that diet-induced pancreatic inflammation is critical for promoting oncogenic KRAS-mediated neoplasia. These results indicate that diets rich in different macronutrients have differential effects on pancreatic tumorigenesis in which the ensuing inflammation exacerbates the process. Management of macronutrient intake aimed at thwarting inflammation is thus an important preventive strategy for patients harboring oncogenic KRAS.

Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related death in the United States. Despite the high prevalence of Kras mutations in pancreatic cancer patients, murine models expressing the oncogenic mutant Kras (Kras mut ) in mature pancreatic cells develop PDAC at a low frequency. Independent of cell of origin, a second genetic hit (loss of tumor suppressor TP53 or PTEN) is important for development of PDAC in mice. We hypothesized ectopic expression and elevated levels of oncogenic mutant Kras would promote PanIN arising in pancreatic ducts. To test our hypothesis, the significance of elevating levels of K-Ras and Ras activity has been explored by expression of a CAG driven LGSL-Kras G12V allele (cKras) in pancreatic ducts, which promotes ectopic Kras expression. We predicted expression of cKras in pancreatic ducts would generate neoplasia and PDAC. To test our hypothesis, we employed tamoxifen dependent CreER T2 mediated recombination. Hnf1b:CreER T2 ;Kras G12V (cKras Hnf1b/+ ) mice received 1 (Low), 5 (Mod) or 10 (High) mg per 20 g body weight to recombine cKras in low (cKras Low ), moderate (cKras Mod ), and high (cKras High ) percentages of pancreatic ducts. Our histologic analysis revealed poorly differentiated aggressive tumors in cKras High mice. cKras Mod mice had grades of Pancreatic Intraepithelial Neoplasia (PanIN), recapitulating early and advanced PanIN observed in human PDAC. Proteomics analysis revealed significant differences in PTEN/AKT and MAPK pathways between wild type, cKras Low , cKras Mod , and cKras High mice. In conclusion, in this study, we provide evidence that ectopic expression of oncogenic mutant K-Ras in pancreatic ducts generates early and late PanIN as well as PDAC. This Ras rheostat model provides evidence that AKT signaling is an important early driver of invasive ductal derived PDAC.

Background & Aims: Mutations in the trypsinogen gene (PRSS1) cause human hereditary pancreatitis. However, it is not clear how mutant forms of PRSS1 contribute to disease development. We studied the effects of expressing mutant forms of human PRSS1 in mice., Methods: We expressed forms of PRSS1 with and without the mutation encoding R122H (PRSS1 R122H ) specifically in pancreatic acinar cells under control of a full-length pancreatic elastase gene promoter. Mice that did not express these transgenes were used as controls. Mice were given injections of caerulein to induce acute pancreatitis or injections of lipopolysaccharide to induce chronic pancreatitis. Other groups of mice were fed ethanol or placed on a high-fat diet to induce pancreatitis. Pancreata were collected and analyzed by histology, immunoblots, real-time polymerase chain reaction, and immunohistochemistry. Trypsin enzymatic activity and chymotrypsin enzymatic activity were measured in pancreatic homogenates. Blood was collected and serum amylase activity was measured., Results: Pancreata from mice expressing transgenes encoding PRSS1 or PRSS1 R122H had focal areas of inflammation; these lesions were more prominent in mice that express PRSS1 R122H . Pancreata from mice that express PRSS1 or PRSS1 R122H had increased levels of heat shock protein 70 and nuclear factor (erythroid-derived 2)-like 2, and reduced levels of chymotrypsin C compared with control mice. Increased expression of PRSS1 or PRSS1 R122H increased focal damage in pancreatic tissues and increased the severity of acute pancreatitis after caerulein injection. Administration of lipopolysaccharide exacerbated inflammation in mice that express PRSS1 R122H compared to mice that express PRSS1 or control mice. Mice that express PRSS1 R122H developed more severe pancreatitis after ethanol feeding or a high-fat diet than mice that express PRSS1 or control mice. Pancreata from mice that express PRSS1 R122H had more DNA damage, apoptosis, and collagen deposition and increased trypsin activity and infiltration by inflammatory cells than mice that express PRSS1 or control mice., Conclusions: Expression of a transgene encoding PRSS1 R122H in mice promoted inflammation and increased the severity of pancreatitis compared with mice that express PRSS1 or control mice. These mice might be used as a model for human hereditary pancreatitis and can be studied to determine mechanisms of induction of pancreatitis by lipopolysaccharide, ethanol, or a high-fat diet., (Copyright © 2020 AGA Institute. Published by Elsevier Inc. All rights reserved.)

The oncogenic receptor tyrosine kinase AXL is overexpressed in cancer and plays an important role in carcinomas of multiple organs. However, the mechanisms of AXL overexpression in cancer remain unclear. In this study, using HEK293T, Panc-1, and Panc-28 cells and samples of human pancreatic intraepithelial neoplasia (PanIN), along with several biochemical approaches and immunofluorescence microscopy analyses, we sought to investigate the mechanisms that regulate AXL over-expression in pancreatic ductal adenocarcinoma (PDAC). We found that AXL interacts with hematopoietic progenitor kinase 1 (HPK1) and demonstrate that HPK1 down-regulates AXL and decreases its half-life. The HPK1-mediated AXL degradation was inhibited by the endocytic pathway inhibitors leupeptin, bafilomycin A1, and monensin. HPK1 accelerated the movement of AXL from the plasma membrane to endosomes in pancreatic cancer cells treated with the AXL ligand growth arrest-specific 6 (GAS6). Moreover, HPK1 increased the binding of AXL to the Cbl proto-oncogene (c-Cbl); promoted AXL ubiquitination; decreased AXL-mediated signaling, including phospho-AKT and phospho-ERK signaling; and decreased the invasion capability of PDAC cells. Importantly, we show that AXL expression inversely correlates with HPK1 expression in human PanINs and that patients whose tumors have low HPK1 and high AXL expression levels have shorter survival than those with low AXL or high HPK1 expression ( p < 0.001). Our results suggest that HPK1 is a tumor suppressor that targets AXL for degradation via the endocytic pathway. HPK1 loss of function may contribute to AXL overexpression and thereby enhance AXL-dependent downstream signaling and tumor invasion in PDAC., (© 2020 Song et al.)

Background & Aims: Obesity is a risk factor for pancreatic cancer. In mice, a high-fat diet (HFD) and expression of oncogenic KRAS lead to development of invasive pancreatic ductal adenocarcinoma (PDAC) by unknown mechanisms. We investigated how oncogenic KRAS regulates the expression of fibroblast growth factor 21, FGF21, a metabolic regulator that prevents obesity, and the effects of recombinant human FGF21 (rhFGF21) on pancreatic tumorigenesis., Methods: We performed immunohistochemical analyses of FGF21 levels in human pancreatic tissue arrays, comprising 59 PDAC specimens and 45 nontumor tissues. We also studied mice with tamoxifen-inducible expression of oncogenic KRAS in acinar cells (Kras G12D/+ mice) and fElas CreERT mice (controls). Kras G12D/+ mice were placed on an HFD or regular chow diet (control) and given injections of rhFGF21 or vehicle; pancreata were collected and analyzed by histology, immunoblots, quantitative polymerase chain reaction, and immunohistochemistry. We measured markers of inflammation in the pancreas, liver, and adipose tissue. Activity of RAS was measured based on the amount of bound guanosine triphosphate., Results: Pancreatic tissues of mice expressed high levels of FGF21 compared with liver tissues. FGF21 and its receptor proteins were expressed by acinar cells. Acinar cells that expressed Kras G12D/+ had significantly lower expression of Fgf21 messenger RNA compared with acinar cells from control mice, partly due to down-regulation of PPARG expression-a transcription factor that activates Fgf21 transcription. Pancreata from Kras G12D/+ mice on a control diet and given injections of rhFGF21 had reduced pancreatic inflammation, infiltration by immune cells, and acinar-to-ductal metaplasia compared with mice given injections of vehicle. HFD-fed Kras G12D/+ mice given injections of vehicle accumulated abdominal fat, developed extensive inflammation, pancreatic cysts, and high-grade pancreatic intraepithelial neoplasias (PanINs); half the mice developed PDAC with liver metastases. HFD-fed Kras G12D/+ mice given injections of rhFGF21 had reduced accumulation of abdominal fat and pancreatic triglycerides, fewer pancreatic cysts, reduced systemic and pancreatic markers of inflammation, fewer PanINs, and longer survival-only approximately 12% of the mice developed PDACs, and none of the mice had metastases. Pancreata from HFD-fed Kras G12D/+ mice given injections of rhFGF21 had lower levels of active RAS than from mice given vehicle., Conclusions: Normal acinar cells from mice and humans express high levels of FGF21. In mice, acinar expression of oncogenic KRAS significantly reduces FGF21 expression. When these mice are placed on an HFD, they develop extensive inflammation, pancreatic cysts, PanINs, and PDACs, which are reduced by injection of FGF21. FGF21 also reduces the guanosine triphosphate binding capacity of RAS. FGF21 might be used in the prevention or treatment of pancreatic cancer., (Copyright © 2019 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Oncogenic KRAS plays a vital role in controlling tumor metabolism by enhancing aerobic glycolysis. Obesity driven by chronic consumption of high-fat diet (HFD) is a major risk factor for oncogenic KRAS-mediated pancreatic ductal adenocarcinoma (PDAC). However, the role of HFD in KRAS-mediated metabolic reprogramming has been obscure. Here, by using genetically engineered mouse models expressing an endogenous level of KRAS G12D in pancreatic acinar cells, we demonstrate that hyperactivation of KRAS G12D by obesogenic HFD, as compared to carbohydrate-rich diet, is responsible for enhanced aerobic glycolysis that associates with critical pathogenic responses in the path towards PDAC. Ablation of Cox-2 attenuates KRAS hyperactivation leading to the reversal of both aggravated aerobic glycolysis and high-grade dysplasia under HFD challenge. Our data highlight a pivotal role of the cooperative interaction between obesity-ensuing HFD and oncogenic KRAS in driving the heightened aerobic glycolysis during pancreatic tumorigenesis and suggest that in addition to directly targeting KRAS and aerobic glycolysis pathway, strategies to target the upstream of KRAS hyperactivation may bear important therapeutic value.

Replacement of the exocrine parenchyma by fibrous tissue is a main characteristic of chronic pancreatitis. Understanding the mechanisms of pancreatic fibrogenesis is critical for the development of preventive and therapeutic interventions. Cyclooxygenase-2 (COX-2), a rate-limiting enzyme for prostaglandin synthesis, is expressed in patients with chronic pancreatitis. However, it is unknown whether COX-2 can cause chronic pancreatitis. To investigate the roles of pancreatic acinar COX-2 in fibrogenesis and the development of chronic pancreatitis, COX-2 was ectopically expressed specifically in pancreatic acinar cells in transgenic mice. Histopathological changes and expression levels of several profibrogenic factors related to chronic pancreatitis were evaluated. COX-2 was expressed in the pancreas of the transgenic mice, as detected by Western blot analysis. Immunohistochemical staining showed COX-2 was specifically expressed in pancreatic acinar cells. COX-2 expression led to progressive changes in the pancreas, including pancreas megaly, persistent inflammation, collagen deposition, and acinar-to-ductal metaplasia. Quantitative RT-PCR and immunostaining showed that profibrogenic factors were upregulated and pancreatic stellate cells were activated in the COX-2 transgenic mice. Expression of COX-2 in pancreatic acinar cells is sufficient to induce chronic pancreatitis. Targeting this pathway may be valuable in the prevention of chronic pancreatitis. NEW & NOTEWORTHY COX-2 expression is observed in pancreatic tissues of human chronic pancreatitis. In this study, we showed that COX-2 expression caused the development of chronic pancreatitis in transgenic mice, supporting the idea that COX-2 inhibition may be an effective preventive and therapeutic strategy.

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis, and it is unclear whether its stromal infiltrate contributes to its aggressiveness. Here, we demonstrate that Dickkopf-3 (DKK3) is produced by pancreatic stellate cells and is present in most human PDAC. DKK3 stimulates PDAC growth, metastasis, and resistance to chemotherapy with both paracrine and autocrine mechanisms through NF-κB activation. Genetic ablation of DKK3 in an autochthonous model of PDAC inhibited tumor growth, induced a peritumoral infiltration of CD8 + T cells, and more than doubled survival. Treatment with a DKK3-blocking monoclonal antibody inhibited PDAC progression and chemoresistance and prolonged survival. The combination of DKK3 inhibition with immune checkpoint inhibition was more effective in reducing tumor growth than either treatment alone and resulted in a durable improvement in survival, suggesting that DKK3 neutralization may be effective as a single targeted agent or in combination with chemotherapy or immunotherapy for PDAC., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Background & Aims: Pancreatic ductal adenocarcinoma (PDAC) is characterized by activated pancreatic stellate cells (PSCs), abundance of extracellular matrix (ECM), and production of cytokines and chemokines. Galectin 3 (GAL3), a β-galactoside-specific lectin, contributes to PDAC development but its effects on the stroma and cytokine production are unclear., Methods: The effect of recombinant human GAL3 (rGAL3) on activation of PSCs, production of cytokines, and ECM proteins was determined by proliferation, invasion, cytokine array, and quantitative polymerase chain reaction. We assessed co-cultures of PDAC cells with GAL3 genetic alterations with PSCs. Production of interleukin 8 (IL8) and activities of nuclear factor (NF)-κB were determined by enzyme-linked immunosorbent assay and luciferase reporter analyses. We studied the effects of inhibitors of NF-κB and integrin-linked kinase (ILK) on pathways activated by rGAL3., Results: In analyses of the Gene Expression Omnibus database and our dataset, we observed higher levels of GAL3, IL8, and other cytokines in PDAC than in nontumor tissues. Production of IL8, granulocyte-macrophage colony-stimulating factor, chemokine ligand 1, and C-C motif chemokine ligand 2 increased in PSCs exposed to rGAL3 compared with controls. Culture of PSCs with PDAC cells that express different levels of GAL3 resulted in proliferation and invasion of PSCs that increased with level of GAL3. GAL3 stimulated transcription of IL8 through integrin subunit beta 1 (ITGB1) on PSCs, which activates NF-κB through ILK. Inhibitors of ILK or NF-κB or a neutralizing antibody against ITGB1 blocked transcription and production of IL8 from PSCs induced by rGAL3. The GAL3 inhibitor significantly reduced growth and metastases of orthotopic tumors that formed from PDAC and PSC cells co-implanted in mice., Conclusion: GAL3 activates PSC cells to produce inflammatory cytokines via ITGB1signaling to ILK and activation of NF-κB. Inhibition of this pathway reduced growth and metastases of pancreatic orthotopic tumors in mice., (Copyright © 2018 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Oncogenic KRas activity is central to several cancer types including pancreatic ductal adenocarcinoma (PDAC) but has been determined to be "undruggable". Recent studies have indicated that oncogenic KRas is not constitutively active but relies on a feed-forward stimulatory mechanism involving NFκB mediated inflammation. In the current study, we investigated the role of the receptor for advanced glycation end-products (RAGE) in maintaining oncogenic signaling in PDAC. We observed that there was a shift in the levels of specific RAGE isoforms and altered cellular localization in PDAC. Furthermore, inhibition of RAGE using a pharmacological antagonist, FPS-ZM1, or a blocking antibody, decreased phosphorylation of IKBα and inhibited Erk activity down-stream of Kras in PDAC cell lines. In vivo, inhibition of RAGE using FPS-ZM1 reduced the growth of PDAC syngeneic orthotopic xenografts and prolonged survival. These data indicate that RAGE plays a central role in maintaining inflammatory signaling in PDAC that benefits tumor growth. These observations support the development of approaches to inhibit the carcinogenic actions of Kras indirectly by blocking the mechanisms which maintain its activity., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Metastatic disease is the leading cause of death among cancer patients and involves a complex and inefficient process. Every step of the metastatic process can be rate limiting and is influenced by non-malignant host cells interacting with the tumor cell. Over a century ago, experiments first indicated a link between the immune system and metastasis. This phenomenon, called concomitant immunity, indicates that the primary tumor induces an immune response, which may not be sufficient to destroy the primary tumor, but prevents the growth of a secondary tumor or metastases. Since that time, many different immune cells have been shown to play a role in both inhibiting and promoting metastatic disease. Here we review classic and new observations, describing the links between the immune system and metastasis that inform the development of cancer therapies.

Chronic inflammation (CI) is a risk factor for pancreatic cancer (PC) including the most common type, ductal adenocarcinoma (PDAC), but its role and the mechanisms involved are unclear. To investigate the role of CI in PC, we generated genetic mouse models with pancreatic specific CI in the presence or absence of TP53. Mice were engineered to express either cyclooxygenase-2 (COX-2) or IκB kinase-2 (IKK2), and TP53 +/+ or TP53 f/f specifically in adult pancreatic acinar cells by using a full-length pancreatic elastase promoter-driven Cre. Animals were followed for >80 weeks and pancreatic lesions were evaluated histologically and immunohistochemically. The presence of K-ras mutations was assessed by direct sequencing, locked nuclei acid (LNA)-based PCR, and immunohistochemistry. We observed that sustained COX-2/IKK2 expression caused histological abnormalities of pancreas, including increased immune cell infiltration, proliferation rate and DNA damage. A minority of animals with CI developed pre-neoplastic lesions, but cancer was not observed in any TP53 +/+ animals within 84 weeks. In contrast, all animals with CI-lacking TP53 developed various subtypes of PC, including acinar cell carcinoma, ductal adenocarcinoma, sarcomatoid carcinoma and neuroendocrine tumors, and all died within 65 weeks. No evidence of K-ras mutations was observed. Variations in the activity of the Hippo, pERK and c-Myc pathways were found in the diverse cancer subtypes. In summary, chronic inflammation is extremely inefficient at inducing PC in the presence of TP53. However, in the absence of TP53, CI leads to the development of several rare K-ras-independent forms of PC, with infrequent PDAC. This may help explain the rarity of PDAC in persons with chronic inflammatory conditions.

Lipocalin-2 (LCN2) promotes malignant development in many cancer types. LCN2 is upregulated in patients with pancreatic ductal adenocarcinoma (PDAC) and in obese individuals, but whether it contributes to PDAC development is unclear. In this study, we investigated the effects of Lcn2 depletion on diet-induced obesity, inflammation, and PDAC development. Mice with acinar cell-specific expression of Kras G12D were crossed with Lcn2-depleted animals and fed isocaloric diets with varying amounts of fat content. Pancreas were collected and analyzed for inflammation, pancreatic intraepithelial neoplasia (PanIN), and PDAC. We also used a syngeneic orthotopic PDAC mouse model to study tumor growth in the presence or absence of Lcn2 expression. In addition, to understand the mechanistic role of how LCN2 could be mediating PDAC, we studied LCN2 and its specific receptor solute carrier family 22 member 17 (SLC22A17) in human pancreatic cancer stellate cells (PSC), key mediators of the PDAC stroma. Depletion of Lcn2 diminished extracellular matrix deposition, immune cell infiltration, PanIN formation, and tumor growth. Notably, it also increased survival in both obesity-driven and syngeneic orthotopic PDAC mouse models. LCN2 modulated the secretion of proinflammatory cytokines in PSC of the PDAC tumor microenvironment, whereas downregulation of LCN2-specific receptor SLC22A17 blocked these effects. Our results reveal how LCN2 acts in the tumor microenvironment links obesity, inflammation, and PDAC development. Cancer Res; 77(10); 2647-60. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Acinar cells represent the primary target in necroinflammatory diseases of the pancreas, including pancreatitis. The signaling pathways guiding acinar cell repair and regeneration following injury remain poorly understood. The purpose of this study was to determine the importance of Hepatocyte Growth Factor Receptor/MET signaling as an intrinsic repair mechanism for acinar cells following acute damage and chronic alcohol-associated injury. Here, we generated mice with targeted deletion of MET in adult acinar cells (MET-/-). Acute and repetitive pancreatic injury was induced in MET-/- and control mice with cerulein, and chronic injury by feeding mice Lieber-DeCarli diets containing alcohol with or without enhancement of repetitive pancreatic injury. We examined the exocrine pancreas of these mice histologically for acinar death, edema, inflammation and collagen deposition and changes in the transcriptional program. We show that MET expression is relatively low in normal adult pancreas. However, MET levels were elevated in ductal and acinar cells in human pancreatitis specimens, consistent with a role for MET in an adaptive repair mechanism. We report that genetic deletion of MET in adult murine acinar cells was linked to increased acinar cell death, chronic inflammation and delayed recovery (regeneration) of pancreatic exocrine tissue. Notably, increased pancreatic collagen deposition was detected in MET knockout mice following repetitive injury as well alcohol-associated injury. Finally, we identified specific alterations of the pancreatic transcriptome associated with MET signaling during injury, involved in tissue repair, inflammation and endoplasmic reticulum stress. Together, these data demonstrate the importance of MET signaling for acinar repair and regeneration, a novel finding that could attenuate the symptomology of pancreatic injury., Competing Interests: The authors have declared that no competing interests exist

The genetic landscape of pancreatic cancer shows nearly ubiquitous mutations of K-RAS. However, oncogenic K-Ras(mt) alone is not sufficient to lead to pancreatic ductal adenocarcinoma (PDAC) in either human or in genetically modified adult mouse models. Many stimulants, such as high fat diet, CCK, LPS, PGE2 and others, have physiological effects at low concentrations that are mediated in part through modest increases in K-Ras activity. However, at high concentrations, they induce inflammation that, in the presence of oncogenic K-Ras expression, substantially accelerates PDAC formation. The mechanism involves increased activity of oncogenic K-Ras(mt). Unlike what has been proposed in the standard paradigm for the role of Ras in oncogenesis, oncogenic K-Ras(mt) is now known to not be constitutively active. Rather, it can be activated by standard mechanisms similar to wild-type K-Ras, but its activity is sustained for a prolonged period. Furthermore, if the level of K-Ras activity exceeds a threshold at which it begins to generate its own activators, then a feed-forward loop is formed between K-Ras activity and inflammation and pathological processes including oncogenesis are initiated. Oncogenic K-Ras(mt) activation, a key event in PDAC initiation and development, is subject to complex regulatory mechanisms. Reagents which inhibit inflammation, such as the Cox2 inhibitor celecoxib, block the feed-forward loop and prevent induction of PDAC in models with endogenous oncogenic K-Ras(mt). Increased understanding of the role of activating and inhibitory mechanisms on oncogenic K-Ras(mt) activity is of paramount importance for the development of preventive and therapeutic strategies to fight against this lethal disease.

Background/aims: The cell surface protein transmembrane 4 L6 family member 1 (TM4SF1) has been detected in various tumors and plays a major role in the development of cancer. We aimed to investigate the effects of TM4SF1 on the migration and invasion of pancreatic cancer in vitro and in vivo and explore its related molecular mechanisms., Methods: qRT-PCR and immunohistochemical analyses were used to measure the expression of TM4SF1 in pancreatic cancer tissues and adjacent tissues. TM4SF1 was silenced using siRNA and shRNA to investigate the role of this protein in the proliferation and metastasis of pancreatic cancer cells. MTS and Transwell assays were used to examine the effect of TM4SF1 on pancreatic cancer cell lines. The expression and activity of MMP-2 and MMP-9 were determined by qRT-PCR, western blots and gelatin zymography. In vivo, orthotopic pancreatic tumor models were used to examine the formation of metastasis., Results: qRT-PCR and immunohistochemical analyses showed that TM4SF1 was highly expressed in pancreatic cancer tissues compared with the adjacent tissues. In in vitro experiments the silencing of TM4SF1 reduced cell migration and invasion and down-regulated the expression and activity of MMP-2 and MMP-9. However, no significant difference in cell proliferation was detected after silencing TM4SF1. Additionally, knocking down TM4SF1 decreased the formation of lung and liver metastases in orthotopic pancreatic tumor models., Conclusion: Our results demonstrate that the expression of TM4SF1 is higher in pancreatic cancer tissues and pancreatic cancer cell lines than controls. Knockdown of TM4SF1 inhibited the migration and invasion of pancreatic cancer cells by regulating the expression and activity of MMP-2 and MMP-9, which suggests that TM4SF1 may play a significant role in metastasis in pancreatic cancer., (© 2016 The Author(s) Published by S. Karger AG, Basel.)

Background: TM4SF1 is overexpressed in pancreatic ductal adenocarcinoma (PDAC) and affects the development of this cancer. Also, multidrug resistance (MDR) is generally associated with tumor chemoresistance in pancreatic cancer. However, the correlation between TM4SF1 and MDR remains unknown. This research aims to investigate the effect of TM4SF1 on gemcitabine resistance in PDAC and explore the possible molecular mechanism between TM4SF1 and MDR., Methods: The expression of TM4SF1 was evaluated in pancreatic cancer cell lines and human pancreatic duct epithelial (HPDE) cell lines by quantitative RT-PCR. TM4SF1 siRNA transfection was carried out using Hiperfect transfection reagent to knock down TM4SF1. The transcripts were analyzed by quantitative RT-PCR, RT-PCR and western blotting for further study. The cell proliferation and apoptosis were obtained to investigate the sensitivity to gemcitabine of pancreatic cancer cells after silencing TM4SF1 in vitro. We demonstrated that cell signaling of TM4SF1 mediated chemoresistance in cancer cells by assessing the expression of multidrug resistance (MDR) genes using quantitative RT-PCR. In vivo, we used orthotopic pancreatic tumor models to investigate the effect of proliferation after silencing TM4SF1 by a lentivirus-mediated shRNA in MIA PaCa-2 cell lines., Results: The mRNA expression of TM4SF1 was higher in seven pancreatic cancer cell lines than in HPDE cell lines. In three gemcitabine-sensitive cell lines (L3.6pl, BxPC-3, SU86.86), the expression of TM4SF1 was lower than that in four gemcitabine-resistant cell lines (MIA PaCa-2, PANC-1, Hs766T, AsPC-1). We evaluated that TM4SF1 was a putative target for gemcitabine resistance in pancreatic cancer cells. Using AsPC-1, MIA PaCa-2 and PANC-1, we investigated that TM4SF1 silencing affected cell proliferation and increased the percentages of cell apoptosis mediated by treatment with gemcitabine compared with cells which were treated with negative control. This resistance was associated with the expression of multidrug resistance genes including ABCB1 and ABCC1. In vivo, silencing of TM4SF1 in MIA PaCa-2 cell lines increased the effectiveness of gemcitabine-based treatment in orthotopic pancreatic tumor models evaluated using noninvasive bioluminescent imaging., Conclusion: These findings suggest that TM4SF1 is a surface membrane antigen that is highly expressed in pancreatic cancer cells and increases the chemoresistance to gemcitabine. Thus, TM4SF1 may be a promising target to overcome the chemoresistance of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is relatively rare but extremely lethal. Standard cytotoxic therapeutics provide little benefit. To date, newer targeted therapeutics have also not been highly successful. Often novel therapeutics that have appeared to perform well in preclinical models have failed in the clinic. Many factors contribute to these failures, but the one most often attributed is the shortcomings of the preclinical models. A plethora of animal models now exist for PDAC, including cell line xenografts, patient-derived xenografts, a wide variety of genetic mouse models, and syngeneic xenografts. These models have generated a tremendous amount of information useful for the understanding of PDAC. Yet none seems to well predict clinical outcomes of new treatments. This review will discuss how genetic instability and cellular heterogeneity make this disease so difficult to model accurately. We will also discuss the strengths and weaknesses of many of the popular models. Ultimately we will argue that there is no perfect model and that the best approach to understanding clinical performance is the use of multiple preclinical models with an understanding of their salient features., (Copyright © 2015 the American Physiological Society.)

Salivary duct carcinoma (SDC) is an uncommon, but aggressive malignant tumor with a high mortality rate. Herein, we reported the detection of somatic KRAS A146T and Q61H mutations in 2 out of 4 (50%) sarcomatoid SDC variants. Transgenic mice carrying the human oncogenic KRAS(G12V), which spatiotemporal activation by tamoxifen (TAM)-inducible Cre recombinase Ela-CreERT in the submandibular gland (SMG) ductal cells, was established and characterized. Visible carcinoma was detected as early as day-15 following oncogenic KRAS(G12V) induction alone, and these tumors proliferate rapidly with a median survival of 28-days accompanied with histological reminiscences to human sarcomatoid SDC variants. Moreover, these tumors were resistant to cetuximab treatment despite augmented EGFR signaling, attesting its malignancy. Our findings suggest that LGL-KRas(G12V);Ela-CreERT transgenic mice could serve as a useful preclinical model for investigating underlying mechanisms and developing potential therapies.

Anterior gradient 2 (AGR2) promotes cancer growth, metastasis, and resistance to therapy via unknown mechanisms. We investigated the effects of extracellular AGR2 signaling through the orphan glycosylphosphatidylinositol-linked receptor C4.4A in pancreatic ductal adenocarcinoma (PDAC). Proliferation, migration, invasion, and apoptosis were measured using colorimetric, Boyden chamber, and FACS analyses. We developed blocking mAbs against AGR2 and C4.4A and tested their effects, along with siRNAs, on cancer cell functions and on orthotopic tumors in nude mice. Extracellular AGR2 stimulated proliferation, migration, invasion, and chemoresistance of PDAC cell lines. AGR2 interacted with C4.4A in cell lysates and mixtures of recombinant proteins. Knockdown of C4.4A reduced migration and resistance to gemcitabine. PDAC tissues, but not adjacent healthy pancreatic tissues, expressed high levels of AGR2 and C4.4A. AGR2 signaling through C4.4A required laminins 1 or 5 and integrin β1. Administration of antibodies against AGR2 and C4.4A reduced growth and metastasis and caused regression of aggressive xenograft tumors, leading to increased survival of mice. These data support a model in which AGR2 binds and signals via C4.4A in an autocrine loop and promotes the growth of pancreas tumors in mice. Blocking mAbs against AGR2 and C4.4A may have therapeutic potential against PDAC., (©2015 American Association for Cancer Research.)

Introduction: Early detection of pancreatic cancer could save many thousands of lives. Non-invasive diagnostic imaging, including PET with [(18)F]FDG, has inadequate resolution for detection of small (2-3 mm) pancreatic tumours. We demonstrated the efficacy of PET imaging with an (18)F-labelled lactose derivative, [(18)F]FEDL, that targets HIP/PAP, a biomarker that is overexpressed in the peritumoural pancreas. We developed another analogue, 1-[(18)F]fluoroethyl lactose ([(18)F]FEL), which is simpler to synthesise, for the same application. We conducted a preliminary evaluation of the new probe and its efficacy in detecting orthotopic pancreatic carcinoma xenografts in mice., Methods: Xenografts were developed in nude mice by injecting L3.6 pl/GL(+) pancreatic carcinoma cells into the pancreas of each mouse. Tumour growth was monitored by bioluminescence imaging (BLI); accuracy of BLI tumour size estimates was verified by MRI in two representative mice. When the tumour size reached approximately 2-3mm, the animals were injected with [(18)F]FEL (3.7 MBq) and underwent static PET/CT scans. Blood samples were collected at 2, 5, 10, 20 and 60 min after [(18)F]FEL injection to track blood clearance. Following imaging, animals were sacrificed and their organs and tumours/pancreatic tissue were collected and counted on a gamma counter. Pancreas, including tumour, was frozen, sliced and used for autoradiography and immunohistochemical analysis of HIP/PAP expression., Results: Tumour growth was rapid, as observed by BLI and MRI. Blood clearance of [(18)F]FEL was bi-exponential, with half-lives of approximately 3.5 min and 40 min. Mean accumulation of [(18)F]FEL in the peritumoural pancreatic tissue was 1.29±0.295 %ID/g, and that in the normal pancreas of control animals was 0.090±0.101 %ID/g. [(18)F]FEL was cleared predominantly by the kidneys. Comparative analysis of autoradiographic images and immunostaining results demonstrated a correlation between [(18)F]FEL binding and HIP/PAP expression., Conclusion: [(18)F]FEL may be useful for non-invasive imaging of early-stage pancreatic tumours by PET. The results warrant further studies., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Nanotechnology is emerging as a promising modality for cancer treatment; however, in the realm of cancer prevention, its full utility has yet to be determined. Here, we discuss the potential of integrating nanotechnology in cancer prevention to augment early diagnosis, precision targeting, and controlled release of chemopreventive agents, reduced toxicity, risk/response assessment, and personalized point-of-care monitoring. Cancer is a multistep, progressive disease; the functional and acquired characteristics of the early precancer phenotype are intrinsically different from those of a more advanced anaplastic or invasive malignancy. Therefore, applying nanotechnology to precancers is likely to be far more challenging than applying it to established disease. Frank cancers are more readily identifiable through imaging and biomarker and histopathologic assessment than their precancerous precursors. In addition, prevention subjects routinely have more rigorous intervention criteria than therapy subjects. Any nanopreventive agent developed to prevent sporadic cancers found in the general population must exhibit a very low risk of serious side effects. In contrast, a greater risk of side effects might be more acceptable in subjects at high risk for cancer. Using nanotechnology to prevent cancer is an aspirational goal, but clearly identifying the intermediate objectives and potential barriers is an essential first step in this exciting journey., (©2014 American Association for Cancer Research.)

The mechanisms that allow cancer cells to adapt to the typical tumor microenvironment of low oxygen and glucose and high lactate are not well understood. GPR81 is a lactate receptor recently identified in adipose and muscle cells that has not been investigated in cancer. In the current study, we examined GPR81 expression and function in cancer cells. We found that GPR81 was present in colon, breast, lung, hepatocellular, salivary gland, cervical, and pancreatic carcinoma cell lines. Examination of tumors resected from patients with pancreatic cancer indicated that 94% (148 of 158) expressed high levels of GPR81. Functionally, we observed that the reduction of GPR81 levels using shRNA-mediated silencing had little effect on pancreatic cancer cells cultured in high glucose, but led to the rapid death of cancer cells cultured in conditions of low glucose supplemented with lactate. We also observed that lactate addition to culture media induced the expression of genes involved in lactate metabolism, including monocarboxylase transporters in control, but not in GPR81-silenced cells. In vivo, GPR81 expression levels correlated with the rate of pancreatic cancer tumor growth and metastasis. Cells in which GPR81 was silenced showed a dramatic decrease in growth and metastasis. Implantation of cancer cells in vivo was also observed to lead to greatly elevated levels of GPR81. These data support that GPR81 is important for cancer cell regulation of lactate transport mechanisms. Furthermore, lactate transport is important for the survival of cancer cells in the tumor microenvironment. Cancer Res; 74(18); 5301-10. ©2014 AACR., (©2014 American Association for Cancer Research.)

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer death in the USA, accounting for ~40,000 deaths annually. The dismal prognosis for PDAC is largely due to its late diagnosis. Currently, the most sensitive diagnosis of PDAC requires invasive procedures, such as endoscopic ultrasonography, which has inherent risks and accuracy that is highly operator dependent. Here we took advantage of a general characteristic of solid tumors, the acidic microenvironment that is generated as a by-product of metabolism, to develop a novel approach of using pH (Low) Insertion Peptides (pHLIPs) for imaging of PDAC. We show that fluorescently labeled pHLIPs can localize and specifically detect PDAC in human xenografts as well as PDAC and PanIN lesions in genetically engineered mouse models. This novel approach may improve detection, differential diagnosis and staging of PDAC.

Oncogenic Ras mutations are widely considered to be locked in a permanent 'On' state and 'constitutively active'. Yet, many healthy people have cells possessing mutant Ras without apparent harm, and in animal models mutant Ras causes transformation only after upregulation of Ras activity. Here, we demonstrate that oncogenic K-Ras is not constitutively active but can be readily activated by upstream stimulants to lead to prolonged strong Ras activity. These data indicate that in addition to targeting K-Ras downstream effectors, interventions to reduce K-Ras activation may have important cancer-preventive value, especially in patients with oncogenic Ras mutations. As other small G proteins are regulated in a similar manner, this concept is likely to apply broadly to the entire Ras family of molecules.

Background & Aims: Obesity is a risk factor for pancreatic ductal adenocarcinoma (PDAC), but it is not clear how obesity contributes to pancreatic carcinogenesis. The oncogenic form of KRAS is expressed during early stages of PDAC development and is detected in almost all of these tumors. However, there is evidence that mutant KRAS requires an additional stimulus to activate its full oncogenic activity and that this stimulus involves the inflammatory response. We investigated whether the inflammation induced by a high-fat diet, and the accompanying up-regulation of cyclooxygenase-2 (COX2), increases Kras activity during pancreatic carcinogenesis in mice., Methods: We studied mice with acinar cell-specific expression of KrasG12D (LSL-Kras/Ela-CreERT mice) alone or crossed with COX2 conditional knockout mice (COXKO/LSL-Kras/Ela-CreERT). We also studied LSL-Kras/PDX1-Cre mice. All mice were fed isocaloric diets with different amounts of fat, and a COX2 inhibitor was administered to some LSL-Kras/Ela-CreERT mice. Pancreata were collected from mice and analyzed for Kras activity, levels of phosphorylated extracellular-regulated kinase, inflammation, fibrosis, pancreatic intraepithelial neoplasia (PanIN), and PDACs., Results: Pancreatic tissues from LSL-Kras/Ela-CreERT mice fed high-fat diets (HFDs) had increased Kras activity, fibrotic stroma, and numbers of PanINs and PDACs than LSL-Kras/Ela-CreERT mice fed control diets; the mice fed the HFDs also had shorter survival times than mice fed control diets. Administration of a COX2 inhibitor to LSL-Kras/Ela-CreERT mice prevented these effects of HFDs. We also observed a significant reduction in survival times of mice fed HFDs. COXKO/LSL-Kras/Ela-CreERT mice fed HFDs had no evidence for increased numbers of PanIN lesions, inflammation, or fibrosis, as opposed to the increases observed in LSL-Kras/Ela-CreERT mice fed HFDs., Conclusions: In mice, an HFD can activate oncogenic Kras via COX2, leading to pancreatic inflammation and fibrosis and development of PanINs and PDAC. This mechanism might be involved in the association between risk for PDAC and HFDs., (Copyright © 2013 AGA Institute. Published by Elsevier Inc. All rights reserved.)

New molecular targets and intervention strategies for breaking the obesity-pancreatic cancer link are urgently needed. Using relevant spontaneous and orthotopically transplanted murine models of pancreatic cancer, we tested the hypothesis that dietary energy balance modulation impacts pancreatic cancer development and progression through an insulin-like growth factor (IGF)-I-dependent mechanism. In LSL-Kras(G12D)/Pdx-1-Cre/Ink4a/Arf(lox/+) mice, calorie restriction versus overweight- or obesity-inducing diet regimens decreased serum IGF-I, tumoral Akt/mTOR signaling, pancreatic desmoplasia, and progression to pancreatic ductal adenocarcinoma (PDAC), and increased pancreatic tumor-free survival. Serum IGF-I, Akt/mTOR signaling, and orthotopically transplanted PDAC growth were decreased in liver-specific IGF-I-deficient mice (vs. wild-type mice), and rescued with IGF-I infusion. Thus, dietary energy balance modulation impacts spontaneous pancreatic tumorigenesis induced by mutant Kras and Ink4a deficiency, the most common genetic alterations in human pancreatic cancer. Furthermore, IGF-I and components of its downstream signaling pathway are promising mechanistic targets for breaking the obesity-pancreatic cancer link.

Changes in the intracellular levels of the essential micronutrient zinc have been implicated in multiple diseases including pancreatic cancer; however, the molecular mechanism is poorly understood. Here, we report a novel mechanism where increased zinc mediated by the zinc importer ZIP4 transcriptionally induces miR-373 in pancreatic cancer to promote tumour growth. Reporter, expression and chromatin immunoprecipitation assays demonstrate that ZIP4 activates the zinc-dependent transcription factor CREB and requires this transcription factor to increase miR-373 expression through the regulation of its promoter. miR-373 induction is necessary for efficient ZIP4-dependent enhancement of cell proliferation, invasion, and tumour growth. Further analysis of miR-373 in vivo oncogenic function reveals that it is mediated through its negative regulation of TP53INP1, LATS2 and CD44. These results define a novel ZIP4-CREB-miR-373 signalling axis promoting pancreatic cancer growth, providing mechanistic insights explaining in part how a zinc transporter functions in cancer cells and may have broader implications as inappropriate regulation of intracellular zinc levels plays an important role in many other diseases., (© 2013 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO.)

Pancreatic ductal adenocarcinoma (PDAC) exists in a complex desmoplastic microenvironment, which includes cancer-associated fibroblasts [also known as pancreatic stellate cells (PSC)] and immune cells that provide a fibrotic niche that impedes successful cancer therapy. We have found that mast cells are essential for PDAC tumorigenesis. Whether mast cells contribute to the growth of PDAC and/or PSCs is unknown. Here, we tested the hypothesis that mast cells contribute to the growth of PSCs and tumor cells, thus contributing to PDAC development. Tumor cells promoted mast cell migration. Both tumor cells and PSCs stimulated mast cell activation. Conversely, mast cell-derived interleukin (IL)-13 and tryptase stimulated PSC proliferation. Treating tumor-bearing mice with agents that block mast cell migration and function depressed PDAC growth. Our findings suggest that mast cells exacerbate the cellular and extracellular dynamics of the tumor microenvironment found in PDAC. Therefore, targeting mast cells may inhibit stromal formation and improve therapy., (©2013 AACR.)

The Kras gene is mutated to an oncogenic form in most pancreatic tumors. However, early attempts to use this molecule as a specific biomarker of the disease, or inhibit its activity as a cancer therapy, failed. This left a situation in which everyone was aware of the association between this important oncogene and pancreatic cancer, but no one knew what to do about it. Recent findings have changed this picture-many assumptions made about KRAS and its role in pancreatic cancer were found to be incorrect. Several factors have contributed to increased understanding of the activities of KRAS, including creation of genetically engineered mouse models, which have allowed for detailed analyses of pancreatic carcinogenesis in an intact animal with a competent immune system. Cancer genome sequencing projects have increased our understanding of the heterogeneity of individual tumors. We also have a better understanding of which oncogenes are important for tumor maintenance and are therefore called "drivers." We review the advances and limitations of our knowledge about the role of Kras in development of pancreatic cancers and the important areas for future research., (Copyright © 2013 AGA Institute. Published by Elsevier Inc. All rights reserved.)

The exocrine pancreas is the organ with the highest level of protein synthesis in the adult--each day the pancreas produces litres of fluid filled with enzymes that are capable of breaking down nearly all organic substances. For optimal health, the pancreas must produce sufficient enzymes of the right character to match the dietary intake. Disruption of normal pancreatic function occurs primarily as a result of dysfunction of the acinar cells that produce these digestive enzymes, and can lead to acute or chronic diseases. For many years, the prevailing dogma has been that inappropriate intracellular activation of the digestive enzymes produced by acinar cells was the key to pancreatic inflammatory diseases, as digestive enzymes themselves are potentially harmful to the cells that secrete them. However, we now know that many stressors can affect pancreatic acinar cells, and that these stressors can independently trigger pancreatic pathology through various mechanisms. This Review focuses on protein synthesis and active digestive enzymes--two key stressors faced by the acinar cell that are likely to be the major drivers of pathology encountered in the pancreas.

Pancreatic ductal adenocarcinoma (PDAC) is challenging to treat, and better means to detect and/or treat pancreatic cancer are urgently needed to save lives. Cathepsin E (Cath E) is a proteolytic enzyme highly expressed in PDAC. In this study, a novel approach using Cath E activation of a Cath E-specific prodrug was demonstrated. Specific activation of the prodrug is expected to kill pancreatic cancer cells without harming normal pancreatic cells. A novel 5-aminolevulinic acid (5-ALA) prodrug was custom-designed to be activated selectively by endogenous Cath E within the PDAC cells. The 5-ALA prodrug was incubated with Cath E-positive and -negative tumor cells and illuminated with various doses of light. In addition, mice genetically engineered to develop PDAC were injected intravenously with the 5-ALA prodrug, and the pancreas was treated with light irradiation. One day after treatment, PDAC tissue was assessed for apoptosis. The 5-ALA prodrug was activated within the Cath E-positive tumor but not in the normal pancreatic tissue. When used in combination with light treatment, it allowed delivery of selective photodynamic therapy (PDT) to the cancerous tissues, with minimal harm to the adjacent normal tissues. With this novel Cath E activation approach, it is possible to detect pancreatic cancer cells accurately and specifically impair their viability, while sparing normal cells. This treatment could result in fewer side effects than the non-specific treatments currently in use. Cath E is a specific and effective drug activator for PDAC treatment., (Copyright © 2013 Elsevier B.V. All rights reserved.)

We have previously shown that the antiallergic drug cromolyn blocks S100P interaction with its receptor receptor for advanced glycation end product (RAGE) and improves gemcitabine effectiveness in pancreatic ductal adenocarcinoma (PDAC). However, the concentration required to achieve its effectiveness was high (100 μmol/L). In this study, we designed and synthesized analogs of cromolyn and analyzed their effectiveness compared with the parent molecule. An ELISA was used to confirm the binding of S100P with RAGE and to test the effectiveness of the different analogs. Analog 5-methyl cromolyn (C5OH) blocked S100P binding as well as the increases in NF-κB activity, cell growth, and apoptosis normally caused by S100P. In vivo C5OH systemic delivery reduced NF-κB activity to a greater extent than cromolyn and at 10 times lesser dose (50 mg vs. 5 mg). Treatment of mice-bearing syngeneic PDAC tumors showed that C5OH treatment reduced both tumor growth and metastasis. C5OH treatment of nude mice bearing orthotopic highly aggressive pancreatic Mpanc96 cells increased the overall animal survival. Therefore, the cromolyn analog, C5OH, was found to be more efficient and potent than cromolyn as a therapeutic for PDAC., (©2013 AACR)

Pancreatic ductal adenocarcinoma (PDAC) is highly malignant disease that is the fourth leading cause of cancer-related death in the US. Gene therapy using AAV vectors to selectively deliver genes to PDAC cells is an attractive treatment option for pancreatic cancer. However, most AAV serotypes display a broad spectrum of tissue tropism and none of the existing serotypes specifically target PDAC cells. This study tests the hypothesis that AAV2 can be genetically re-engineered to specifically target PDAC cells by modifying the capsid surface to display a peptide that has previously been shown to bind plectin-1. Toward this end, a Plectin-1 Targeting Peptide (PTP) was inserted into the loop IV region of the AAV2 capsid, and the resulting capsid (AAV-PTP) was used in a series of in vitro and in vivo experiments. In vitro, AAV-PTP was found to target all five human PDAC cell lines tested (PANC-1, MIA PaCa-2, HPAC, MPanc-96, and BxPC-3) preferentially over two non-neoplastic human pancreatic cell lines (human pancreatic ductal epithelial and human pancreatic stellate cells). In vivo, mice bearing subcutaneous tumor xenografts were generated using the PANC-1 cell line. Once tumors reached a size of ∼1-2 mm in diameter, the mice were injected intravenously with luciferase reporter vectors packaged in the either AAV-PTP or wild type AAV2 capsids. Luciferase expression was then monitored by bioluminescence imaging on days 3, 7, and 14 after vector injection. The results indicate that the AAV-PTP capsid displays a 37-fold preference for PANC-1 tumor xenographs over liver and other tissues; whereas the wild type AAV2 capsid displays a complementary preference for liver over tumors and other tissues. Together, these results establish proof-of-principle for the ability of PTP-modified AAV capsids to selectively target gene delivery to PDAC cells in vivo, which opens promising new avenues for the early detection, diagnosis, and treatment of pancreatic cancer.

Objectives: Pancreatic stellate cells are source of dense fibrotic stroma, a constant pathological feature of chronic pancreatitis and pancreatic adenocarcinoma. We observed correlation between levels of cyclooxygenase 2 (COX-2) and its product prostaglandin E2 (PGE2) and the extent of pancreatic fibrosis. The aims of this study were to delineate the effects of PGE2 on immortalized human pancreatic stellate cells (HPSCs) and to identify the receptor involved., Methods: Immunohistochemistry, reverse transcription-polymerase chain reaction and quantitative reverse transcription-polymerase chain reaction were used to assess COX-2, extracellular matrix, and matrix metalloproteinase gene expression. Eicosanoid profile was determined by liquid chromatography-tandem mass spectrometry. Human pancreatic stellate cell proliferation was assessed by MTS assay, migration by Boyden chamber assay, and invasion using an invasion chamber. Transient silencing was obtained by small interfering RNA., Results: Human pancreatic stellate cells express COX-2 and synthesize PGE2. Prostaglandin E2 stimulated HPSC proliferation, migration, and invasion and stimulated expression of both extracellular matrix and matrix metalloproteinase genes. Human pancreatic stellate cells expressed all 4 EP receptors. Only blocking the EP4 receptor resulted in abrogation of PGE2-mediated HPSC activation. Specificity of EP4 for the effects of PGE2 on stellate cells was confirmed using specific antagonists., Conclusions: Our data indicate that PGE2 regulates pancreatic stellate cell profibrotic activities via EP4 receptor, thus suggesting EP4 receptor as useful therapeutic target for pancreatic cancer to reduce desmoplasia.

Background & Aims: Nuclear factor-κB (NF-κB) is activated during early stages of pancreatitis. This transcription factor regulates genes that control many cell activities, including inflammation and survival. There is evidence that activation of NF-κB protects against pancreatitis, and, in other cases, that it promotes this disease. We compared the effects of NF-κB in different mouse models of pancreatitis to understand these complications., Methods: To model constitutive activation of NF-κB, we expressed a transgene that encodes its p65 subunit or the inhibitor of κB kinase (IKK)2 in pancreatic acinar cells of mice. We analyzed effects on pancreatic tissues and levels of NF-κB target genes in these mice and compared them with mice that did not express transgenic p65 or IKK2 (controls)., Results: Transgenic expression of p65 led to compensatory expression of the inhibitory subunit IKB-α and, therefore, no clear phenotype. However, p65 transgenic mice given injections of cerulein, to induce acute pancreatitis, had higher levels of NF-κB activity in acinar cells, greater levels of inflammation, and more severe outcomes than control mice. In contrast, constitutive expression of IKK2 directly increased the activity of NF-κB in acinar cells and induced pancreatitis. Prolonged activity of IKK2 (3 months) resulted in activation of stellate cells, loss of acinar cells, and fibrosis, which are characteristics of chronic pancreatitis. Co-expression of IKK2 and p65 greatly increased the expression of inflammatory mediators and the severity of pancreatitis, compared with control mice., Conclusions: The level of NF-κB activation correlates with the severity of acute pancreatitis in mice. Longer periods of activation (3 months) lead to chronic pancreatitis. These findings indicate that strategies to inactivate NF-κB might be used to treat patients with acute or chronic pancreatitis., (Copyright © 2013 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Pancreatic cancer is the fourth leading cause of cancer deaths and is characterized by dismal prognosis. Xenograft and genetically engineered mouse (GEM) models have recapitulated critical elements of human pancreatic cancer, providing useful tools to probe the underlying cause of cancer etiology. In this review, we provide a brief description of the common genetic lesions that occur during the development of pancreatic cancer. Next, we describe the strengths and weaknesses of these two models and highlight key discoveries each has made. Although the relative merits of GEM and xenograft pancreatic cancer mouse models are subject to debate, both systems have and will continue to yield essential insights in understanding pancreatic cancer etiology. This information is critical for the development of new methods to screen, treat, and prevent pancreatic cancer., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background & Aims: New-onset diabetes in patients with pancreatic cancer is likely to be a paraneoplastic phenomenon caused by tumor-secreted products. We aimed to identify the diabetogenic secretory product(s) of pancreatic cancer., Methods: Using microarray analysis, we identified adrenomedullin as a potential mediator of diabetes in patients with pancreatic cancer. Adrenomedullin was up-regulated in pancreatic cancer cell lines, in which supernatants reduced insulin signaling in beta cell lines. We performed quantitative reverse-transcriptase polymerase chain reaction and immunohistochemistry on human pancreatic cancer and healthy pancreatic tissues (controls) to determine expression of adrenomedullin messenger RNA and protein, respectively. We studied the effects of adrenomedullin on insulin secretion by beta cell lines and whole islets from mice and on glucose tolerance in pancreatic xenografts in mice. We measured plasma levels of adrenomedullin in patients with pancreatic cancer, patients with type 2 diabetes mellitus, and individuals with normal fasting glucose levels (controls)., Results: Levels of adrenomedullin messenger RNA and protein were increased in human pancreatic cancer samples compared with controls. Adrenomedullin and conditioned media from pancreatic cell lines inhibited glucose-stimulated insulin secretion from beta cell lines and islets isolated from mice; the effects of conditioned media from pancreatic cancer cells were reduced by small hairpin RNA-mediated knockdown of adrenomedullin. Conversely, overexpression of adrenomedullin in mice with pancreatic cancer led to glucose intolerance. Mean plasma levels of adrenomedullin (femtomoles per liter) were higher in patients with pancreatic cancer compared with patients with diabetes or controls. Levels of adrenomedullin were higher in patients with pancreatic cancer who developed diabetes compared those who did not., Conclusions: Adrenomedullin is up-regulated in patients with pancreatic cancer and causes insulin resistance in β cells and mice., (Copyright © 2012 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Purpose: The Hedgehog (Hh) pathway has emerged as an important pathway in multiple tumor types and is thought to be dependent on a paracrine signaling mechanism. The purpose of this study was to determine the role of pancreatic cancer-associated fibroblasts (human pancreatic stellate cells, HPSCs) in Hh signaling. In addition, we evaluated the efficacy of a novel Hh antagonist, AZD8542, on tumor progression with an emphasis on the role of the stroma compartment., Experimental Design: Expression of Hh pathway members and activation of the Hh pathway were analyzed in both HPSCs and pancreatic cancer cells. We tested the effects of Smoothened (SMO) inhibition with AZD8542 on tumor growth in vivo using an orthotopic model of pancreatic cancer containing varying amounts of stroma., Results: HPSCs expressed high levels of SMO receptor and low levels of Hh ligands, whereas cancer cells showed the converse expression pattern. HPSC proliferation was stimulated by Sonic Hedgehog with upregulation of downstream GLI1 mRNA. These effects were abrogated by AZD8542 treatment. In an orthotopic model of pancreatic cancer, AZD8542 inhibited tumor growth only when HPSCs were present, implicating a paracrine signaling mechanism dependent on stroma. Further evidence of paracrine signaling of the Hh pathway in prostate and colon cancer models is provided, demonstrating the broader applicability of our findings., Conclusion: Based on the use of our novel human-derived pancreatic cancer stellate cells, our results suggest that Hh-targeted therapies primarily affect the tumor-associated stroma, rather than the epithelial compartment.

Background and Aims: Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer death in the USA. Surgical resection is the only effective treatment; however, only 20% of patients are candidates for surgery. The ability to detect early PDAC would increase the availability of surgery and improve patient survival. This study assessed the feasibility of using the enzymatic activity of cathepsin E (Cath E), a protease highly and specifically expressed in PDAC, as a novel biomarker for the detection of pancreas-bearing pancreatic intraepithelial neoplasia (PanIN) lesions and PDAC., Methods: Pancreas from normal, chronic pancreatitis and PDAC patients was assessed for Cath E expression by quantitative real-time PCR and immunohistochemistry. Human PDAC xenografts and genetically engineered mouse models (GEMM) of PDAC were injected with a Cath E activity selective fluorescent probe and imaged using an optical imaging system., Results: The specificity of Cath E expression in PDAC patients and GEMM of pancreatic cancer was confirmed by quantitative real-time PCR and immunohistochemistry. The novel probe for Cath E activity specifically detected PDAC in both human xenografts and GEMM in vivo. The Cath E sensitive probe was also able to detect pancreas with PanIN lesions in GEMM before tumour formation., Conclusions: The elevated Cath E expression in PanIN and pancreatic tumours allowed in-vivo detection of human PDAC xenografts and imaging of pancreas with PanIN and PDAC tumours in GEMM. Our results support the usefulness of Cath E activity as a potential molecular target for PDAC and early detection imaging.

Purpose: The receptor for advanced glycation end products (RAGE) contributes to multiple pathologies, including diabetes, arthritis, neurodegenerative diseases, and cancer. Despite the obvious need, no RAGE inhibitors are in common clinical use. Therefore, we developed a novel small RAGE antagonist peptide (RAP) that blocks activation by multiple ligands., Experimental Design: RAGE and its ligands were visualized by immunohistochemical analysis of human pancreatic tissues, and siRNA was used to analyze their functions. Interactions between RAGE and S100P, S100A4, and HMGB-1 were measured by ELISA. Three S100P-derived small antagonistic peptides were designed, synthesized, and tested for inhibition of RAGE binding. The effects of the peptide blockers on NFκB-luciferase reporter activity was used to assess effects on RAGE-mediated signaling. The most effective peptide was tested on glioma and pancreatic ductal adenocarcinoma (PDAC) models., Results: Immunohistochemical analysis confirmed the expression of RAGE and its ligands S100P, S100A4, and HMGB-1 in human PDAC. siRNA silencing of RAGE or its ligands reduced the growth and migration of PDAC cells in vitro. The most effective RAP inhibited the interaction of S100P, S100A4, and HMGB-1 with RAGE at micromolar concentrations. RAP also reduced the ability of the ligands to stimulate RAGE activation of NFκB in cancer cells in vitro and in vivo. Importantly, systemic in vivo administration of RAP reduced the growth and metastasis of pancreatic tumors and also inhibited glioma tumor growth., Conclusion: RAP shows promise as a tool for the investigation of RAGE function and as an in vivo treatment for RAGE-related disorders.