Pancreatic Intraepithelial Neoplasia Progression Research Articles

ARID1A Deficiency Promotes Pancreatic Tumorigenesis by Attenuating Mutant KRAS Induced Senescence

ARID1A (AT-rich interacting domain-containing protein 1A) is one of the frequently mutated epigenetic regulators in a wide spectrum of cancers. Recent studies have shown that ARID1A deficiency induces global changes in the epigenetic landscape of enhancers and gene regulations. These complex changes make it challenging to identify the driving mechanisms of ARID1A deficiency in promoting cancer progression. Using a mouse model, we identified the anti-senescence effect of ARID1A deficiency, which promotes the progression of pancreatic intraepithelial neoplasia (PanIN). To identify the underlying mechanism and the players, we profiled the transcriptomes of individual PanIN lesions. As a result, we observed not only the anti-senescence effect but also the complex trans-differentiation of neoplastic cells. Furthermore, we discovered that significantly upregulated aldehyde dehydrogenase (ALDH) help to inhibit Kras-induced reactive oxygen species (ROS) production and lead to the reduction of the cellular senescence, which results in the accelerated PanIN progression.

Low Incidence of High-Grade Pancreatic Intraepithelial Neoplasia Lesions in a Crmp4 Gene–Deficient Mouse Model of Pancreatic Cancer

Pancreatic intraepithelial neoplasia (PanIN), the most common premalignant lesion of the pancreas, is a histologically well-defined precursor to invasive pancreatic ductal adenocarcinoma (PDAC). However, the molecular mechanisms underlying the progression of PanINs have not been fully elucidated. Previously, we demonstrated that the expression of collapsin response mediator protein 4 (CRMP4) in PDAC was associated with poor prognosis. The expression of CRMP4 was also augmented in a pancreatitis mouse model. However, the role of CRMP4 in the progression of PanIN lesions remains uncertain. In the present study, we examined the relationship between CRMP4 expression and progression of PanIN lesions using genetically engineered mouse models. PanIN lesions were induced by peritoneal injection of the cholecystokinin analog caerulein in LSL-KRASG12D; Pdx1-Cre (KC-Crmp4 wild-type, WT) mice and LSL-KRASG12D; Pdx1-Cre; Crmp4−/− (KC-Crmp4 knockout, KO) mice. We analyzed pancreatic tissue sections from these mice and evaluated PanIN grade by hematoxylin and eosin staining. CRMP4 expression was examined and the cellular components assessed by immunohistochemistry using antibodies against CRMP4, CD3, and α-smooth muscle actin (SMA). The incidence of high-grade PanIN in KC-Crmp4 WT mice was higher than that in KC-Crmp4 KO animals. CRMP4 was expressed not only in epithelial cells but also in αSMA-positive cells in stromal areas of PanIN lesions. The CRMP4 expression in stromal areas correlated with PanIN grade in WT mice. These results suggested that the expression of CRMP4 in stromal cells may underlie the incidence or progression of PanIN.

BCAT2-mediated BCAA catabolism is critical for development of pancreatic ductal adenocarcinoma

Branched-chain amino acid (BCAA) metabolism is potentially linked with development of pancreatic ductal adenocarcinoma (PDAC)1-4. BCAA transaminase 2 (BCAT2) was essential for the collateral lethality conferred by deletion of malic enzymes in PDAC and the BCAA-BCAT metabolic pathway contributed to non-small-cell lung carcinomas (NSCLCs) other than PDAC3,4. However, the underlying mechanism remains undefined. Here we reveal that BCAT2 is elevated in mouse models and in human PDAC. Furthermore, pancreatic tissue-specific knockout of Bcat2 impedes progression of pancreatic intraepithelial neoplasia (PanIN) in LSL-KrasG12D/+; Pdx1-Cre (KC) mice. Functionally, BCAT2 enhances BCAA uptake to sustain BCAA catabolism and mitochondrial respiration. Notably, BCAA enhances growth of pancreatic ductal organoids from KC mice in a dose-dependent manner, whereas addition of branched-chain α-keto acid (BCKA) and nucleobases rescues growth of KC organoids that is suppressed by BCAT2 inhibitor. Moreover, KRAS stabilizes BCAT2, which is mediated by spleen tyrosine kinase (SYK) and E3 ligase tripartite-motif-containing protein 21 (TRIM21). In addition, BCAT2 inhibitor ameliorates PanIN formation in KC mice. Of note, a lower-BCAA diet also impedes PDAC development in mouse models of PDAC. Thus, BCAT2-mediated BCAA catabolism is critical for development of PDAC harbouring KRAS mutations. Targeting BCAT2 or lowering dietary BCAA may have translational significance.

Deletion of Histone Methyltransferase G9a Suppresses Mutant Kras-driven Pancreatic Carcinogenesis.

The entire mechanisms by which epigenetic modifiers contribute to the development of pancreatic cancer remain unknown. Although the histone methyltransferase G9a is a promising target in human cancers, its role in pancreatic carcinogenesis has been under-studied. The aim of the study was to examine the role of G9a in pancreatic carcinogenesis by a gene-targeting mouse model. We established pancreas-specific G9aflox/flox mice and crossed them with Ptf1aCre/; KrasG12D/+ (KC) mice, which spontaneously develop pancreatic cancer. The phenotypes of the resulting KC mice with G9a deletion were examined. We analyzed transcriptomic data by microarray and genome-wide chromatin accessibility by transposase-accessible chromatin using sequencing. We established pancreatic organoids from KC mice. G9a deficiency impaired the progression of pancreatic intraepithelial neoplasia (PanIN) and prolonged the survival of KC mice. The number of phosphorylated Erk-positive cells and Dclk1-positive cells, which are reported to be essential for the progression of PanIN, were decreased by G9a deletion. UNC0638, an inhibitor of G9a, suppressed the growth of organoids and increased global chromatin accessibility, especially around the regions including the protein phosphatase 2A genes. Thus, our study suggested the functional interaction of G9a, Dclk1 and Mapk pathway in the Kras-driven pancreatic carcinogenesis. The inhibition of G9a may suppress the initiation of oncogenic Kras-driven pancreatic carcinogenesis.

Actin-Bundling Proteins (Actinin-4 and Fascin-1) are Involved in the Development of Pancreatic Intraepithelial Neoplasia (PanIN)

Fascin-1 and actinin-4 are involved in key processes of tumor cell adhesion, migration and metastasis. Actinin-4 plays an important role in promotion of cell proliferation, whereas fascin-1 regulates cellular motility. Its over-expression leads to the loss of cell adhesion and metastasis. The aim of our study was to assess fascin-1 and actinin-4 expression in normal pancreatic ducts and in pancreatic intraepithelial neoplasia (PanIN) - precursor lesion of pancreatic ductal adenocarcinoma (PDAC). The study involved 70 patients treated surgically due to PDAC, cysts and pancreatitis, who had also been diagnosed with pancreatic intraepithelial neoplasia. Fascin-1 and actinin-4 expressions were evaluated using the immunohistochemistry method. A statistically significant relationship was observed between the expression of fascin-1 and actinin-4 (cytoplasmic) and patients' age (P=0.01, P=0.002, respectively). The expression of fascin-1 and actinin-4 was associated with the diagnosis (P<0.001,P=0.04, respectively). Statistical analysis revealed correlations of fascin-1 and actinin-4 expressions with the presence and grade of PanIN (P < 0.001, P=0.002, respectively). The expression of these proteins was observed in each degree of PanIN and increased with the pancreatic intraepithelial neoplasia progression. The expression of fascin-1 and actinin-4 is connected with the degree of PanIN advancement and depends on the type of the primary disease. Overexpression of these proteins may be linked to cytological and architectural abnormalities observed in advanced PanIN. Elevated expression of fascin-1 and actinin-4 indicates the role of these proteins in the progression from PanIN to PDAC.

Oncogenic KRAS Induces NIX-Mediated Mitophagy to Promote Pancreatic Cancer.

Activating KRAS mutations are found in nearly all cases of pancreatic ductal adenocarcinoma (PDAC), yet effective clinical targeting of oncogenic KRAS remains elusive. Understanding of KRAS-dependent PDAC-promoting pathways could lead to the identification of vulnerabilities and the development of new treatments. We show that oncogenic KRAS induces BNIP3L/NIX expression and a selective mitophagy program that restricts glucose flux to the mitochondria and enhances redox capacity. Loss of Nix restores functional mitochondria to cells, increasing demands for NADPH reducing power and decreasing proliferation in glucose-limited conditions. Nix deletion markedly delays progression of pancreatic cancer and improves survival in a murine (KPC) model of PDAC. Although conditional Nix ablation in vivo initially results in the accumulation of mitochondria, mitochondrial content eventually normalizes via increased mitochondrial clearance programs, and pancreatic intraepithelial neoplasia (PanIN) lesions progress to PDAC. We identify the KRAS-NIX mitophagy program as a novel driver of glycolysis, redox robustness, and disease progression in PDAC. SIGNIFICANCE: NIX-mediated mitophagy is a new oncogenic KRAS effector pathway that suppresses functional mitochondrial content to stimulate cell proliferation and augment redox homeostasis. This pathway promotes the progression of PanIN to PDAC and represents a new dependency in pancreatic cancer.This article is highlighted in the In This Issue feature, p. 1143.

MST1R kinase accelerates pancreatic cancer progression via effects on both epithelial cells and macrophages.

The MST1R (RON) kinase is overexpressed in >80% of human pancreatic cancers, but its role in pancreatic carcinogenesis is unknown. In this study, we examined the relevance of Mst1r kinase to Kras driven pancreatic carcinogenesis using genetically engineered mouse models. In the setting of mutant Kras, Mst1r overexpression increased acinar-ductal metaplasia (ADM), accelerated progression of pancreatic intraepithelial neoplasia (PanIN), and resulted in the accumulation of (mannose receptor C type 1) MRC1+, (arginase 1) Arg+ macrophages in the tumor microenvironment. Conversely, absence of a functional Mst1r kinase slowed PanIN initiation, resulted in smaller tumors, prolonged survival and a reduced tumor associated macrophage content. Mst1r expression was associated with increased production of its ligand Mst1, and in orthotopic models, suppression of Mst1 expression resulted in reduced tumor size, changes in macrophage polarization and enhanced T cell infiltration. This study demonstrates the functional significance of Mst1r during pancreatic cancer initiation and progression. Further, it provides proof of concept that targeting Mst1r can modulate pancreatic cancer growth and the microenvironment. This study provides further rationale for targeting Mst1r as a therapeutic strategy.

Pancreatic Ductal Deletion of Hnf1b Disrupts Exocrine Homeostasis, Leads to Pancreatitis, and Facilitates Tumorigenesis.

Background & AimsThe exocrine pancreas consists of acinar cells that produce digestive enzymes transported to the intestine through a branched ductal epithelium. Chronic pancreatitis is characterized by progressive inflammation, fibrosis, and loss of acinar tissue. These changes of the exocrine tissue are risk factors for pancreatic cancer. The cause of chronic pancreatitis cannot be identified in one quarter of patients. Here, we investigated how duct dysfunction could contribute to pancreatitis development.MethodsThe transcription factor Hnf1b, first expressed in pancreatic progenitors, is strictly restricted to ductal cells from late embryogenesis. We previously showed that Hnf1b is crucial for pancreas morphogenesis but its postnatal role still remains unelucidated. To investigate the role of pancreatic ducts in exocrine homeostasis, we inactivated the Hnf1b gene in vivo in mouse ductal cells.ResultsWe uncovered that postnatal Hnf1b inactivation in pancreatic ducts leads to chronic pancreatitis in adults. Hnf1bΔduct mutants show dilatation of ducts, loss of acinar cells, acinar-to-ductal metaplasia, and lipomatosis. We deciphered the early events involved, with down-regulation of cystic disease–associated genes, loss of primary cilia, up-regulation of signaling pathways, especially the Yap pathway, which is involved in acinar-to-ductal metaplasia. Remarkably, Hnf1bΔduct mutants developed pancreatic intraepithelial neoplasia and promote pancreatic intraepithelial neoplasia progression in concert with KRAS. We further showed that adult Hnf1b inactivation in pancreatic ducts is associated with impaired regeneration after injury, with persistent metaplasia and initiation of neoplasia.ConclusionsLoss of Hnf1b in ductal cells leads to chronic pancreatitis and neoplasia. This study shows that Hnf1b deficiency may contribute to diseases of the exocrine pancreas and gains further insight into the etiology of pancreatitis and tumorigenesis.

Progression to pancreatic ductal adenocarcinoma from pancreatic intraepithelial neoplasia: Results of a simulation model

ObjectivesTo gain insight into the natural history and carcinogenesis pathway of Pancreatic Intraepithelial Neoplasia (PanIN) lesions by building a calibrated simulation model of PanIN progression to pancreatic ductal adenocarcinoma (PDAC) MethodsWe revised a previously validated simulation model of solid PDAC, calibrating the model to fit data from the National Cancer Institute's Surveillance, Epidemiology, and End Results program and published literature on PanIN prevalence by age. We estimated the likelihood of progression from PanIN states (1, 2, and 3) to PDAC and the time between PanIN onset and PDAC (dwell time). We evaluated a hypothetical intervention to test for and treat PanIN 3 lesions to estimate the potential benefits from PanIN detection. ResultsWe estimated the lifetime probability of progressing from PanIN 1 to PDAC to be 1.5% (men), 1.3% (women). Progression from PanIN 1 to PDAC took 33.6 years and 35.3 years, respectively, and from PanIN 3 to PDAC took 11.3 years and 12.3 years. A hypothetical test for PanIN 3 detection and treatment could provide a maximum, average life expectancy gain of 40 days. ConclusionsOur modeling analysis estimates PanINs have a relatively indolent course to PDAC, supporting the feasibility of potential future early detection strategies.

Cadherin-1 and cadherin-3 cooperation determines the aggressiveness of pancreatic ductal adenocarcinoma

Background:Pancreatic ductal adenocarcinoma (PDAC) is characterised by an extensive tissue invasion and an early formation of metastasis. Alterations in the expression of cadherins have been reported in PDAC. Yet, how these changes contribute to tumour progression is poorly understood. Here, we investigated the relationship between cadherins expression and PDAC development.Methods:Cadherins expression was assessed by immunostaining in both human and murine tissue specimens. We have generated pancreatic cancer cell lines expressing both cadherin-1 and cadherin-3 or only one of these cadherins. Functional implications of such genetic alterations were analysed both in vitro and in vivo.Results:Cadherin-3 is detected early at the plasma membrane during progression of pancreatic intraepithelial neoplasia 1 (PanIN-1) to PDAC. Despite tumoural cells turn on cadherin-3, a significant amount of cadherin-1 remains expressed at the cell surface during tumourigenesis. We found that cadherin-3 regulates tumour growth, while cadherin-1 drives type I collagen organisation in the tumour. In vitro assays showed that cadherins differentially participate to PDAC aggressiveness. Cadherin-3 regulates cell migration, whereas cadherin-1 takes part in the invadopodia activity.Conclusions:Our results show differential, but complementary, roles for cadherins during PDAC carcinogenesis and illustrate how their expression conditions the PDAC aggressiveness.

The Mouse Model of Pancreatic Cancer Atlas (MMPCA) for classification of pancreatic cancer lesions: A large histological investigation of the Ptf1aCre/+;LSL-KrasG12D/+ transgenic mouse model of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is one of the leading forms of cancer related deaths in the United States. With limited treatment options and unreliable diagnostic methods, long-term survival rates following a diagnosis of pancreatic cancer remain poor. Pancreatic intraepithelial neoplasia (PanIN) are precancerous lesions that precede progression towards PDAC. PanIN occur in increasing complexity as the disease progresses and the description of PanIN plays a critical role in describing, staging and diagnosing PDAC. Inconsistencies in PanIN classifications exist even amongst leading pathologists. This has led to debate and confusion among researchers and pathologists involved in pancreatic cancer research, diagnosis and treatment. We have sought to initiate a discussion with leading pathologists with a goal of increasing consensus in the interpretation of PanIN and associated structures within the precancerous pancreas. Toward achieving this goal, we are in the process of conducting an extensive study of over 1000 male and female pancreata in varying stages of PanIN progression isolated from the Ptf1aCre/+;LSL-KrasG12D/+ transgenic mouse model of pancreatic cancer. Using this extensive database, we have established the Mouse Model of Pancreatic Cancer Atlas (MMPCA) to serve as a platform for meaningful and interactive discussion among researchers and pathologists who study pancreatic disease. We hope that the MMPCA will be an effective tool for promoting a more consistent and accurate consensus of PanIN classifications in the future.

Statins and pancreatic cancer.

Pancreatic cancer remains among the most lethal cancers, despite ongoing advances in treatment for all stages of the disease. Disease prevention represents another opportunity to improve patient outcome, with metabolic syndrome and its components, such as diabetes, obesity and dyslipidemia, having been recognized as modifiable risk factors for pancreatic cancer. In addition, statins have been shown to potentially reduce pancreatic cancer risk and to improve survival in patients with a combination of metabolic syndrome and pancreatic cancer. Furthermore, preclinical studies have demonstrated that statins exhibit antitumor effects in pancreatic cancer cell lines in vitro and animal models in vivo, in addition to delaying the progression of pancreatic intraepithelial neoplasia to pancreatic ductal adenocarcinoma (PDAC) and inhibiting PDAC formation in conditional K-Ras mutant mice. The mechanisms by which statins produce anticancer effects remain poorly understood, although appear to involve inhibition of the mevalonate/cholesterol synthesis pathway, thus blocking the synthesis of intermediates important for prenylation and activation of the Ras/mitogen-activated protein kinase 1 signaling pathway. Furthermore, statins have been identified to modulate the phosphoinositide 3-kinase/Akt serine/threonine kinase 1 and inflammation signaling pathways, and to alter the expression of genes involved in lipid metabolism, which are important for PDAC growth and proliferation. In addition, statins have been demonstrated to exhibit further antitumor mechanisms in a number of other cancer types, which are beyond the scope of the present review. In the present review, current evidence highlighting the potential of statins as chemopreventive agents in pancreatic cancer is presented, and the antitumor mechanisms of statins elucidated thus far in this disease are discussed.

Abstract A76: Influence of IL-17-secreting immune cells on pancreatic cancer stemness

Abstract Little is known about how the immune system influences pancreatic cancer stemness. Using a murine model of pancreatic intraepithelial neoplasia (PanIN), we have previously reported that KrasG12D induces expression of IL-17 receptors (IL-17RA) on emerging PanINs, as well as pancreatic infiltration by IL-17-producing CD4+ and gamma-delta-T cells. Forced IL-17 over-expression dramatically accelerates PanIN initiation and progression, while inhibition of IL-17 signaling using either genetic or pharmacologic techniques effectively prevents PanIN formation. When IL-17 signaling was blocked using monoclonal antibodies on transgenic mice harboring PanINs, transcriptional analysis of Kras-mutated epithelial cells revealed significant down-regulation of multiple stemness-related genes: doublecortin-like kinase 1 (Dclk1) [2], Trefoil factor 1 (Tff1)[3], Sonic Hedgehog (SHH) [4], and others. Dclk1 has been recently described as a marker of tumor initiating cells and its functional role in cancer initiation and development is currently being studied. We found that adenovirus mediated-IL-17 overexpression in the murine pancreas harvesting Kras mutation results in PanINs with significantly higher number of Dclk1+ cells that PanINs generated in the presence of a luciferase control virus. Reversely, the genetic ablation of IL-17 in the hematopoietic compartment of the same type of mice resulted in PanINs with significantly lower number of Dclk1+ cells when compared with PanINs from mice transplanted with wild type (WT) bone marrow. We have found that in vitro stimulation of KPC cells (obtained from KrasG12D/+; LSL- Trp53R172H/+; Pdx1Cre mice) with IL-17A results in direct induction of Dclk1, Aldh1 and SHH protein expression in a time-dependent and dose-dependent fashion, validating the results of our gene microarray in a different system and at the protein level. Furthermore, we have found that one of the mechanisms by which IL-17 induces Dclk-1 and ALDH1a1 is through activation of NF-KB canonical pathway. We found that IL-17 had no effect in KPC cells proliferation in 2D culture. However, in vitro stimulation of KPC cells with IL-17A resulted in a significant increase in tumorspheres formation. Even more important, KPC cells in vitro incubated with IL-17A for 7 days have accelerated capacity for in vivo- tumor initiation when injected subcutaneously into syngeneic immune-competent mice, confirming the functional in vivo role that IL-17-immune secreting cells have in inducing pancreatic cancer stemness. Better understanding of the regulation that IL-17-secreting immune cells can impose on pancreatic cancer- tumor initiating cells would be of high importance given the potential translational value of blocking IL-17 signaling for pancreatic cancer prevention and treatment with currently available IL-17 neutralizing monoclonal antibodies or oral small molecule inhibitors. Citation Format: Yu Zhang, Ismet Sahin, Sonal Gupta, Anirban Maitra, Jay K. Kolls, Jennifer Bailey, Steven D Leach, Florencia McAllister.{Authors}. Influence of IL-17-secreting immune cells on pancreatic cancer stemness. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr A76.

Abstract A138: Increased CD3(+) cell infiltration during pancreatic intraepithelial neoplasia progression in KrasG12D/Pdx1-Cre transgenic mouse model

Abstract This study aim to quantify the dynamic number changes of CD3(+) lymph cells in a pre-malignant stage of pancreatic cancer, namely, pancreatic intraepithelial neoplasia (PanIN). The experiment was conducted using a well-established KrasG12D/Pdx1-Cre transgenic mouse model. The animal model expressed a pancreas-specific active KrasG12D gene, resulting in PanIN progression and acinar structure loss in a time dependent manner. Experiment mice were sacrificed at 5, 7, 9, and 11 months (n = 3 for each time point). The pancreas tissues were fixed and stained by a rabbit monoclonal [EPR4517] to CD3G (Abcam Cat#: ab134096, 1:500 dilution). The CD3(+) lymph cell number were quantified by Leica optical system and the CD3(+) lymph cells were manually counted (ten fields for each mouse). All experimental conducted were approved by the ethical committee. The CD3(+) lymph cell number was calculated by averaging all field in each time point (thirty fields per time point). The number was then presented as mean ± SEM. Quantitatively, the CD3(+) cell were 11.73 ± 2.83, 14.23 ± 2.07, 14.90 ± 1.99, and 29.13 ± 2.04 per field in 5, 7, 9, and 11 month-old mice. The CD3(+) cell number was not significantly different among 5, 7, and 9 month-old mice. However, the number significantly increase in the 11 month-old mice compared with the 9-month ones (P&amp;lt; 0.0001, unpaired t-test). We observed that CD3(+) lymph cells were predominately present in PanIN area, while almost not observable in the remaining normal acinar area. The phenomenon that CD3(+) lymph cells infiltrate in PanIN area indicates that this type of immune cells may involve in PanIN progression. Citation Format: Xiafei Hong, Hongmei Dai, Xianze Wang, Feng Tian, Wenming Wu, Yupei Zhao. Increased CD3(+) cell infiltration during pancreatic intraepithelial neoplasia progression in KrasG12D/Pdx1-Cre transgenic mouse model [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A138.

RelA: a tale of a stitch in time.

Pancreatic ductal adenocarcinoma (PDAC) is a deadly cancer in which NF-κB pathways promote biological aggressiveness. In this issue of the JCI, Lesina et al. investigated the role of RelA, the p65 partner of p50 that together form the most common NF-κB complex, in the early stages of pancreatic malignant transformation and in established PDAC. By deleting Rela in the context of an oncogenic Kras-driven autochthonous model of PDAC, the authors demonstrated that RelA is a mediator of oncogene-induced senescence (OIS) and the senescence-associated secretory phenotype (SASP) that attenuates acinar-to-ductal metaplasia, pancreatic intraepithelial neoplasia (PanIN) formation, and PanIN progression to PDAC. Loss of the tumor-suppressor function of RelA in the early stages of Kras-driven pancreatic neoplastic transformation was associated with decreased OIS and SASP and a protumorigenic tumor microenvironment that harbored more M2 macrophages and myeloid-derived suppressor cells. The beneficial effects of RelA were mediated by increased expression of CXCL1 and its activation of CXCR2. By contrast, in advanced stages of Kras-driven murine PDAC, loss of p53 or p16 was associated with senescence bypass, and RelA deficiency in this context attenuated cancer cell proliferation and prolonged mouse survival, indicating that RelA enhances tumor progression in established PDAC.

Abstract 1447: Influence of IL-17-secreting immune cells in pancreatic cancer stemness

Abstract Little is known about how the immune system influences pancreatic cancer stemness. Using a murine model of pancreatic intraepithelial neoplasia (PanIN), we have previously reported that KrasG12D induces expression of IL-17 receptors (IL-17RA) on emerging PanINs, as well as pancreatic infiltration by IL-17-producing CD4+ and gamma-delta-T cells. Forced IL-17 over-expression dramatically accelerates PanIN initiation and progression, while inhibition of IL-17 signaling using either genetic or pharmacologic techniques effectively prevents PanIN formation. When IL-17 signaling was blocked using monoclonal antibodies on transgenic mice harboring PanINs, transcriptional analysis of Kras-mutated epithelial cells revealed significant down-regulation of multiple stemness-related genes: doublecortin-like kinase 1 (Dclk1) [2], Trefoil factor 1 (Tff1)[3], Sonic Hedgehog (SHH) [4], and others. Dclk1 has been recently described as a marker of tumor initiating cells and its functional role in cancer initiation and development is currently being studied. We found that adenovirus mediated-IL-17 overexpression in the murine pancreas harvesting Kras mutation results in PanINs with significantly higher number of Dclk1+ cells that PanINs generated in the presence of a luciferase control virus. Reversely, the genetic ablation of IL-17 in the hematopoietic compartment of the same type of mice resulted in PanINs with significantly lower number of Dclk1+ cells when compared with PanINs from mice transplanted with wild type (WT) bone marrow. We have found that in vitro stimulation of KPC cells (obtained from KrasG12D/+; LSL- Trp53R172H/+; Pdx1−Cre mice) with IL-17A results in direct induction of Dclk1, Aldh1 and SHH protein expression in a time-dependent and dose-dependent fashion, validating the results of our gene microarray in a different system and at the protein level. Furthermore, we have found that one of the mechanisms by which IL-17 induces Dclk-1 and ALDH1a1 is through activation of NF-KB canonical pathway. We found that IL-17 had no effect in KPC cells proliferation in 2D culture. However, stimulation of matrigel embedded- KPC cells with IL-17A resulted in significant increase in tumorspheres formation in vitro. But even more important, KPC cells in vitro incubated with IL-17A for 7 days have accelerated capacity for in vivo- tumor initiation when injected subcutaneously into syngeneic immune-competent mice, confirming the functional in vivo role that IL-17-immune secreting cells have in inducing pancreatic cancer stemness. Better understanding of the regulation that IL-17-secreting immune cells can impose on pancreatic cancer- tumor initiating cells would be of high importance given the potential translational value of blocking IL-17 signaling for pancreatic cancer prevention and treatment with the currently available IL-17 neutralizing monoclonal antibodies. Citation Format: Yu Zhang, Ismet Sahin, Sonal Gupta, Anirban Maitra, Jennifer Bailey, Steven Leach, Florencia McAllister. Influence of IL-17-secreting immune cells in pancreatic cancer stemness. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1447.

Image-based detection and targeting of therapy resistance in pancreatic adenocarcinoma

Pancreatic intraepithelial neoplasia (PanIN) is a premalignant lesion that can progress to pancreatic ductal adenocarcinoma, a highly lethal malignancy marked by its late stage at clinical presentation and profound drug resistance1. The genomic alterations that commonly occur in pancreatic cancer include activation of KRAS2 and inactivation of p53, and SMAD42-4. To date, however, it has been challenging to target these pathways therapeutically; thus the search for other key mediators of pancreatic cancer growth remains an important endeavor. Here we show that the stem cell determinant Musashi (Msi) is a critical element of pancreatic cancer progression in both genetic models and patient derived xenografts. Specifically, we developed Msi reporter mice that allowed image based tracking of stem cell signals within cancers, revealing that Msi expression rises as PanIN progresses to adenocarcinoma, and that Msi-expressing cells are key drivers of pancreatic cancer: they preferentially harbor the capacity to propagate adenocarcinoma, are enriched in circulating tumor cells, and are markedly drug resistant. This population could be effectively targeted by deletion of either Msi1 or Msi2, which led to a striking defect in PanIN progression to adenocarcinoma and an improvement in overall survival. Msi inhibition also blocked the growth of primary patient-derived tumors, suggesting that this signal is required for human disease. To define the translational potential of this work we developed antisense oligonucleotides against Msi; these showed reliable tumor penetration, uptake and target inhibition, and effectively blocked pancreatic cancer growth. Collectively, these studies highlight Msi reporters as a unique tool to identify therapy resistance, and define Msi signaling as a central regulator of pancreatic cancer.

Challenges in detecting pre-malignant pancreatic lesions during acute pancreatitis using a serum microRNA assay: a study based on KrasG12D transgenic mice.

Caerulein-induced acute pancreatitis accelerates the progression of pancreatic intraepithelial neoplasia (PanIN) lesions in a pancreas-specific KrasG12D mouse model. The purpose of this study was to explore whether serum microRNAs (miRNAs) can serve as sensitive biomarkers to detect occult PanIN in the setting of acute pancreatitis. Serum miRNA profiles were quantified by an array-based method and normalized by both Variance Stabilization Normalization (VSN) and invariant methods. Individual miRNAs were validated by TaqMan real-time PCR with synthetic spike-in C. elegans miRNAs as external controls. Serum miRNA profiles distinguished KrasG12D mice with pancreatitis from wild-type mice without pancreatitis, but failed to differentiate KrasG12D mice with pancreatitis from wild-type mice with pancreatitis. Most individual miRNAs that increased in KrasG12D mice with pancreatitis were not significantly different between KrasG12D mice without pancreatitis and wild-type mice without pancreatitis. Mechanistically, Gene Set Enrichment Analysis (GSEA) of the mRNA array data and immunohistochemical assays showed that caerulein-induced acute pancreatitis involved acinar cell loss and immune cell infiltration, which might contribute to serum miRNA profile changes. This study highlighted the challenges in using sensitive serum miRNA biomarker screening for the early detection of pancreatic malignancies during acute pancreatitis.

Hif1a Deletion Reveals Pro-Neoplastic Function of B Cells in Pancreatic Neoplasia.

Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer-related deaths worldwide, with an exceedingly low 5-year survival rate. PDAC tumors are characterized by an extensive desmoplastic stromal response and hypovascularity, suggesting that tumor hypoxia could regulate PDAC initiation and/or progression. Using a well-defined, autochthonous Kras(G12D)-driven murine model, as well as human tumors, we demonstrate that hypoxia and stabilization of hypoxia-inducible factor 1α (HIF1α), a principal mediator of hypoxic adaptation, emerge early during preinvasive stages of PDAC. Surprisingly, pancreas-specific Hif1a deletion drastically accelerated Kras(G12D)-driven pancreatic neoplasia and was accompanied by significant increases in intrapancreatic B lymphocytes, featuring prominent influx of a rare "B1b" B-cell subtype. Finally, treatment of HIF1α-deficient mice with B cell-depleting αCD20 monoclonal antibodies inhibited progression of pancreatic intraepithelial neoplasia (PanIN). Our data reveal a previously unrecognized role for B cells in promoting pancreatic tumorigenesis and implicate HIF1α as a critical regulator of PDAC development. We show here that pancreas-specific Hif1a deletion promotes PDAC initiation, coincident with increased intrapancreatic accumulation of B cells, and that B-cell depletion suppresses pancreatic tumorigenesis. We therefore demonstrate a protective role for HIF1α in pancreatic cancer initiation and uncover a previously unrecognized function of B cells.

Presence of pancreatic intraepithelial neoplasia-3 in a background of chronic pancreatitis in pancreatic cancer patients.

The clinical significance of pancreatic intraepithelial neoplasia (PanIN) lesions in non-neoplastic pancreata of pancreatic ductal adenocarcinoma (PDAC) patients remains controversial. As chronic inflammation has been recently demonstrated to promote dissemination of in situ precancerous lesions, we investigated the prognostic significance of PanINs associated with chronic pancreatitis (CP) in PDAC patients. This retrospective study analyzed 125 curatively resected PDAC specimens for the presence of PanIN and CP. Univariate and multivariate analyses were performed to identify significant predictive factors for poor disease-free survival (DFS) and overall survival (OS). Immunohistochemical staining for E-cadherin and S100A4, markers of epithelial-mesenchymal transition, was performed on resected specimens containing PanIN-3 lesions. CP was observed in 27.2% (34/125) and PanIN-3 in 25.6% (32/125) of specimens. In the presence of CP, PanIN-3 was significantly associated with decreased survival (DFS: 4.3 vs 15.5 months, P = 0.021; OS: 16.3 vs 30.9 months, P = 0.004). PanIN-3 was not a prognostic factor in the absence of CP. The presence of both PanIN-3 and CP was associated with a reduced survival compared to the other cases, in both univariate (DFS: P = 0.039; OS: P = 0.023) and multivariate (DFS: P = 0.020; OS: P = 0.076) analyses. Furthermore, E-cadherin loss and S100A4 expression were more frequently observed in PanIN-3 lesions of CP specimens than in those of non-CP specimens, although not statistically significant. PanIN-3 in association with CP is a significant prognostic factor for decreased survival in PDAC patients, suggesting that chronic inflammation may accelerate the progression of preinvasive high-grade PanIN.