Early Pancreatic Intraepithelial Neoplasia Research Articles

Regulation of ROS signaling by TIGAR induces cancer-modulating responses in the tumor microenvironment

The consequences of reactive oxygen species (ROS) in cancer cells are complex and have been shown to both promote and retard tumorigenesis in different models. In mouse models of pancreatic ductal adenocarcinoma (PDAC), loss of the antioxidant defense gene Tigar results in both a reduction in the development of early pancreatic intraepithelial neoplasia and an increase in invasive and metastatic capacity, accompanied by decreased survival of mice lacking pancreatic TIGAR. We previously demonstrated that increased ROS following loss of TIGAR promotes various cancer cell-intrinsic changes that contribute to metastatic capacity, including epithelial to mesenchymal transition, enhanced migration and invasion, and an increase in ERK signaling. In this study, we show that pancreatic overexpression of TIGAR decreases metastatic capacity and migratory phenotypes in an aggressive model of PDAC, consistent with the concept that dynamic modulation of TIGAR in PDAC contributes to the development and progression of these tumors. Using TIGAR deficient and overexpressing mouse models, we find that the impact of modulation of TIGAR and ROS in PDAC cells also has a profound effect on the normal stromal cells surrounding the tumor. Loss of TIGAR promotes the production of cytokines by cancer cells that induce changes in the surrounding fibroblasts to adopt a tumor-supportive phenotype. Furthermore, these cytokines also attract macrophages that support PDAC dissemination and metastasis. Taken together our work shows that TIGAR-modulated ROS in PDAC can control cell intrinsic and extrinsic changes to impact tumor aggression.

Abstract A081: Single-cell transcriptomic analysis of organoids derived from normal pancreata reveals epithelial cell heterogeneity and expression of PanIN-associated genes

Abstract Pancreatic intraepithelial neoplasia (PanIN) lesions are the microscopic pre-malignant precursor to pancreatic ductal adenocarcinoma (PDAC). Animal models have demonstrated that PanINs arise from acinar cells which have undergone acinar-to-ductal metaplasia (ADM), but the exact molecular mechanisms which govern this transformation remain poorly understood. Novel in vitro models of PanIN genesis are necessary to better characterize pancreatic carcinogenesis and identify clinically actionable biomarkers of early disease. Organoids cultured from normal human pancreata have generated promise in this regard, but the cellular identity and malignant potential of their component cells have not been comprehensively characterized. We performed single-cell RNA sequencing (10X Genomics) on 6 normal pancreatic organoid lines maintained in culture for 3 weeks as well as their parent pancreata obtained from donors with no history of PDAC or other exocrine pancreatic pathology. Integration of whole pancreatic and organoid data using the R package Seurat generated a UMAP with clearly defined “ductal” and “acinar” clusters based on marker gene expression. The majority of organoid cells (47.3-81.5%) colocalized to the “ductal” cluster, and expressed SOX9, KRT19, and PDX1. However, a subset of cells (1.0-33.3%) present in each organoid sample aligned with the “acinar” cluster and expressed PRSS1, PRSS3, REG3A, MUC1, and SPINK1. In accordance with prior reports, organoid cells did not meaningfully express the acinar/endocrine markers PTF1A, CPA1, CHGA, or INS. Further examination of the “acinar” organoid cells revealed heightened expression of CLDN18, TFF1, TFF2, CTSE, CA2, and LYZ, genes associated with PanIN in prior spatial RNA sequencing studies. Other genes significantly enriched in this “acinar” organoid cluster include MUC5AC, a known PanIN marker, OLFM4, a tumor suppressor implicated in intestinal carcinogenesis, and the chemokine CXCL17, which is expressed in cells undergoing ADM. Overall, this data suggests that organoids derived from normal pancreata represent a heterogeneous population of epithelial cells with both acinar and ductal features. Furthermore, these organoids may represent an in vitro model of ADM and/or early PanIN formation, though additional functional mechanistic studies are required for confirmation. Citation Format: Alexander W Bray, Eileen S Carpenter, Timothy L Frankel, Marina Pasca di Magliano. Single-cell transcriptomic analysis of organoids derived from normal pancreata reveals epithelial cell heterogeneity and expression of PanIN-associated genes [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A081.

Preventive Treatment with a CD73 Small Molecule Inhibitor Enhances Immune Surveillance in K-Ras Mutant Pancreatic Intraepithelial Neoplasia.

Immunoprevention is an emerging consideration for solid tumors, including pancreatic ductal adenocarcinoma (PDAC). We and others have shown that Kras mutations in genetic models of spontaneous pancreatic intraepithelial neoplasia (PanIN), which is a precursor to PDAC, results in CD73 expression in the neoplastic epithelium and some populations of infiltrating immune cells, including macrophages and CD8 T cells. CD73 is an ecto-enzyme that converts extracellular adenosine monophosphate to adenosine, a critical immune inhibitory molecule in PDAC. We hypothesized inhibition of CD73 would reduce the incidence of PanIN formation and alter the immune microenvironment. To test our hypothesis, we used the KrasG12D; PdxCre1 (KC) genetically engineered mouse model and tested the utility of AB-680, a small molecule inhibitor targeting CD73, to inhibit PanIN progression. AB-680, or vehicle control, was administered using oral gavage delivery 3 days/week at 10 mg/kg, beginning when the mice were 2 months old and lasting 3 months. We euthanized the mice at 5 months old. In the KC model, we quantified significantly less pancreatitis, early and advanced PanIN, and quantified a significant increase in M1 macrophages in AB-680-treated mice. Single-cell RNA sequencing (scRNA-seq) of pancreata of AB-680-treated mice revealed increased infiltration of CD4+ T cells, CD8+ T cells, and mature B cells. The scRNA-seq analysis showed that CD73 inhibition reduced M2 macrophages, acinar, and PanIN cell populations. CD73 inhibition enhanced immune surveillance and expanded unique clonotypes of TCR and BCR, indicating that inhibition of CD73 augments adaptive immunity early in the neoplastic microenvironment. Prevention Relevance: Previous studies found PanIN lesions in healthy pancreata. Not all progress to PDAC, suggesting a window for enhanced antitumor immunity through immunoprevention therapy. CD73 inhibition in our study prevents PanIN progression, reduces immune-suppressive macrophages and expands TCR and BCR unique clonotypes, highlighting an encouraging therapeutic avenue for high-risk individuals.

Reprogramming Pancreatic Ductal Adenocarcinoma to Pluripotency.

The generation of induced pluripotent stem cells (iPSCs) using transcription factors has been achieved from almost any differentiated cell type and has proved highly valuable for research and clinical applications. Interestingly, iPSC reprogramming of cancer cells, such as pancreatic ductal adenocarcinoma (PDAC), has been shown to revert the invasive PDAC phenotype and override the cancer epigenome. The differentiation of PDAC-derived iPSCs can recapitulate PDAC progression from its early pancreatic intraepithelial neoplasia (PanIN) precursor, revealing the molecular and cellular changes that occur early during PDAC progression. Therefore, PDAC-derived iPSCs can be used to model the earliest stages of PDAC for the discovery of early-detection diagnostic markers. This is particularly important for PDAC patients, who are typically diagnosed at the late metastatic stages due to a lack of reliable biomarkers for the earlier PanIN stages. However, reprogramming cancer cell lines, including PDAC, into pluripotency remains challenging, labor-intensive, and highly variable between different lines. Here, we describe a more consistent protocol for generating iPSCs from various human PDAC cell lines using bicistronic lentiviral vectors. The resulting iPSC lines are stable, showing no dependence on the exogenous expression of reprogramming factors or inducible drugs. Overall, this protocol facilitates the generation of a wide range of PDAC-derived iPSCs, which is essential for discovering early biomarkers that are more specific and representative of PDAC cases.

A phase I study of a mutant KRAS-targeted long peptide vaccine in patients at high risk of developing pancreatic cancer.

TPS758 Background: KRAS mutations are identified in the majority of premalignant lesions that precede pancreatic ductal adenocarcinoma (PDAC). Arising during tumorigenesis, mutant KRAS (mKRAS) neoantigens are less susceptible to central tolerance mechanisms and serve as ideal vaccine targets. Indeed, targeting mKRAS neoantigens with vaccines has shown promising anti-tumor activity in the preclinical setting. For instance, our group previously demonstrated that a Listeria-based vaccine targeting mKRAS G12D combined with Treg-depleting agents can prevent the progression of early pancreatic intraepithelial neoplasia to overt PDAC in a mouse model (Keenan et al, 2014). Building upon this work, the current study aims to determine the safety and immunogenicity of a pooled synthetic long peptide (SLP) mKRAS vaccine in patients identified as high risk for developing PDAC based on family history and germline mutation testing. Methods: This is a single-arm, open-label phase I trial evaluating a pooled SLP mKRAS vaccine in patients at high risk of developing PDAC ( n = 20). The vaccine consists of poly-ICLC adjuvant admixed with SLPs corresponding to six common mKRAS subtypes: G12D, G12R, G12V, G12A, G12C, and G13D. A four-dose series of the mKRAS vaccine is administered on weeks 1, 3, 4, and 17. Following completion of the treatment phase, all patients have the option to continue annual follow-up visits until study closure. Eligible patients must have radiographic evidence of a premalignant pancreatic lesion and fall under at least one of the following three high-risk groups: 1) ≥ 2 familial pancreatic cancer relatives, 2) germline mutation carriers with ≥ 10% lifetime PDAC risk and 3) germline mutation carriers with ~5% lifetime PDAC risk. The co-primary endpoints of this study are safety and T cell response. Safety will be assessed by the frequency and grading of adverse events per NCI CTCAE v5.0. T cell response will be determined by the maximal percent change in IFNγ-producing mKRAS-specific T cell density within 16 weeks post-vaccination compared to baseline. Correlative studies will explore vaccine-associated changes in T cell quality (e.g., memory, exhaustion, poly-functionality, and activation) using mass cytometry analysis of peripheral blood samples. Patient accrual began in April 2022 and is currently ongoing. Clinical trial information: NCT05013216 .

Abstract 816: Cyclin c suppresses pancreatic intraepithelial neoplasm progression and neuroendocrine neoplasm development in a murine Kras model

Abstract Cyclin c is a component of the Cdk8 kinase module that plays both a positive and negative role in transcription. In addition, cellular damage induces cyclin c re-localization to the mitochondria where it stimulates fission and recruits Bax in preparation for apoptotic initiation. Previous studies revealed that cyclin c suppresses thyroid hyperplasia in a Pten knockout mouse model. In the present study, a role for cyclin c in suppressing pancreatic cancer progression was investigated in the Pdx1-Cre LSL-KrasG12D murine pancreatic cancer model. Within eight weeks, a 6-8 fold increase in the appearance of pancreatic intraepithelial neoplasia (PanIN) and acinar ductal metaplasia (ADM) lesions was observed in the KrasG12D+Ccnc-/- animals compared to KrasG12D alone. Surprisingly, high-grade neuroendocrine neoplasms (NEN) were also observed. These aggressive NEN showed mixed morphology with areas of acinar-like differentiation. High grade NEN exhibited pronounced proliferative index (Ki67), high EZH2 expression, and elevated Notch1, suggesting a connection between aberrant Notch signaling and NEN formation. Our previous studies identified a positive role for cyclin c in autophagy gene transcription. Similarly, cell lines derived from a KrasG12D+Ccnc-/- pancreas exhibited a reduction in autophagy compared to KrasG12D+Ccnc+/+ cells. Autophagic deficiency induces pancreatic cell stress and is associated with apoptosis. Consistent with this model, high caspase 3 expression was observed in early KrasG12D+Ccnc-/- NEN but not in KrasG12D+Ccnc+/+ tissues. Taken together, these results demonstrate a role for cyclin c in suppressing both progression of early PanIN and the development of high-grade NEN and suggest a model that cyclin c loss suppresses autophagy and increases apoptosis. Acknowledgements: Support was provided by NCI CCSG grant CA06927 for the FCCC Histopathology and Cell Culture Facilities, the PA Department of Health CURE fund (KSC), the Boye Foundation (RS), and the New Jersey Health Foundation (RS). Citation Format: Kathy Q. Cai, Sara E. Hanley, Andres Klein-Szanto, Kerry S. Campbell, Randy Strich. Cyclin c suppresses pancreatic intraepithelial neoplasm progression and neuroendocrine neoplasm development in a murine Kras model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 816.

Increased neutrophil infiltration as a body-wide effect in pancreatic cancer development.

Increased neutrophil infiltration as a body-wide effect in pancreatic cancer development.

Kras mutation rate precisely orchestrates ductal derived pancreatic intraepithelial neoplasia and pancreatic cancer

AbstractPancreatic 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 (Krasmut) 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-KrasG12V 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 CreERT2 mediated recombination. Hnf1b:CreERT2;KrasG12V (cKrasHnf1b/+) mice received 1 (Low), 5 (Mod) or 10 (High) mg per 20 g body weight to recombine cKras in low (cKrasLow), moderate (cKrasMod), and high (cKrasHigh) percentages of pancreatic ducts. Our histologic analysis revealed poorly differentiated aggressive tumors in cKrasHigh mice. cKrasMod 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, cKrasLow, cKrasMod, and cKrasHigh 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. SummaryIn this study, the authors provide evidence that ectopic expression of oncogenic mutant Kras in pancreatic ducts generates early and late (PanIN) and pancreatic ductal adenocarcinoma (PDAC) . They characterized this Ras rheostat model which reveals elevated Kras mutation frequency and loss of PTEN are important drivers of PanIN and invasive ductal derived PDAC.

Secretory Mucin 5AC Promotes Neoplastic Progression by Augmenting KLF4-Mediated Pancreatic Cancer Cell Stemness.

Secreted mucin 5AC (MUC5AC) is the most abundantly overexpressed member of the mucin family during early pancreatic intraepithelial neoplasia stage I (PanIN-I) of pancreatic cancer. To comprehend the contribution of Muc5ac in pancreatic cancer pathology, we genetically ablated it in an autochthonous murine model (KrasG12D; Pdx-1cre, KC), which mirrors the early stages of pancreatic cancer development. Neoplastic onset and the PanIN lesion progression were significantly delayed in Muc5ac knockout (KrasG12D; Pdx-1 cre; Muc5ac-/-, KCM) animals with a 50% reduction in PanIN-2 and 70% reduction in PanIN-3 lesions compared with KC at 50 weeks of age. High-throughput RNA-sequencing analysis from pancreatic tissues of KCM animals revealed a significant decrease in cancer stem cell (CSC) markers Aldh1a1, Klf4, EpCAM, and CD133. Furthermore, the silencing of MUC5AC in human pancreatic cancer cells reduced their tumorigenic propensity, as indicated by a significant decline in tumor formation frequency by limiting dilution assay upon subcutaneous administration. The contribution of MUC5AC in CSC maintenance was corroborated by a significant decrease in tumor burden upon orthotopic implantation of MUC5AC-depleted pancreatic cancer cells. Mechanistically, MUC5AC potentiated oncogenic signaling through integrin αvβ5, pSrc (Y416), and pSTAT3 (Y705). Phosphorylated STAT3, in turn, upregulated Klf4 expression, thereby enriching the self-renewing CSC population. A strong positive correlation of Muc5ac with Klf4 and pSTAT3 in the PanIN lesions of KC mouse pancreas reinforces the crucial involvement of MUC5AC in bolstering the CSC-associated tumorigenic properties of Kras-induced metaplastic cells, which leads to pancreatic cancer onset and progression. SIGNIFICANCE: This study elucidates that de novo expression of MUC5AC promotes cancer cell stemness during Kras-driven pancreatic tumorigenesis and can be targeted for development of a novel therapeutic regimen.

Disruption of C1galt1 Gene Promotes Development and Metastasis of Pancreatic Adenocarcinomas in Mice

Pancreatic ductal adenocarcinomas (PDACs) produce higher levels of truncated O-glycan structures (such as Tn and sTn) than normal pancreata. Dysregulated activity of core 1 synthase glycoprotein-N-acetylgalactosamine 3-β-galactosyltransferase 1 (C1GALT1) leads to increased expression of these truncated O-glycans. We investigated whether and how truncated O-glycans contributes to the development and progression of PDAC using mice with disruption of C1galt1. We crossed C1galt1 floxed mice (C1galt1loxP/loxP) with KrasG12D/+; Trp53R172H/+; Pdx1-Cre (KPC) mice to create KPCC mice. Growth and progression of pancreatic tumors were compared between KPC and KPCC mice; pancreatic tissues were collected and analyzed by immunohistochemistry; immunofluorescence; and Sirius red, alcian blue, and lectin staining. We used the CRISPR/Cas9 system to disrupt C1GALT1 in human PDAC cells (T3M4 and CD18/HPAF) and levels of O-glycans were analyzed by lectin blotting, mass spectrometry, and lectin pulldown assay. Orthotopic studies and RNA sequencing analyses were performed with control and C1GALT1 knockout PDAC cells. C1GALT1 expression was analyzed in well-differentiated (n= 36) and poorly differentiated (n= 23) PDAC samples by immunohistochemistry. KPCC mice had significantly shorter survival times (median 102 days) than KPC mice (median 200 days) and developed early pancreatic intraepithelial neoplasias at 3 weeks, PDAC at 5 weeks, and metastasis at 10 weeks compared with KPC mice. Pancreatic tumors that developed in KPCC mice were more aggressive (more invasive and metastases) than those in KPC mice, had a decreased amount of stroma, and had increased production of Tn. Poorly differentiated PDAC specimens had significantly lower levels of C1GALT1 than well-differentiated PDACs. Human PDAC cells with knockout of C1GALT1 had aberrant glycosylation of MUC16 compared with control cells and increased expression of genes that regulate tumorigenesis and metastasis. In studies of KPC mice with disruption of C1galt1, we found that loss of C1galt1 promotes development of aggressive PDACs and increased metastasis. Knockout of C1galt1 leads to increased tumorigenicity and truncation of O-glycosylation on MUC16, which could contribute to increased aggressiveness.

ER stress protein AGR2 precedes and is involved in the regulation of pancreatic cancer initiation

The mechanisms of initiation of pancreatic ductal adenocarcinoma (PDAC) are still largely unknown. In the present study, we analysed the role of anterior gradient-2 (AGR2) in the earliest stages of pancreatic neoplasia. Immunohistochemical analysis of chronic pancreatitis (CP) and peritumoral areas in PDAC tissues showed that AGR2 was present in tubular complexes (TC) and early pancreatic intraepithelial neoplasia (PanINs). Moreover, AGR2 was also found in discrete subpopulations of non-transformed cells neighbouring these pre-neoplastic lesions. In primary cells derived from human patient-derived xenograft (PDX) model, flow-cytometry revealed that AGR2 was overexpressed in pancreatic cancer stem cells (CSC) compared with non-stem cancer cells. In LSL-KrasG12D;Pdx1-Cre (KC) mouse model Agr2 induction preceded the formation of pre-neoplastic lesions and their development was largely inhibited by Agr2 deletion in engineered LSL-KrasG12D;Pdx1-Cre; Agr2−/− mice. In vitro, AGR2 expression was stimulated by tunicamycin-induced endoplasmic reticulum (ER) stress in both KRAS wild-type normal pancreas cells, as well as in KRAS mutated pancreatic cancer cells and was essential for ER homoeostasis. The unfolded protein response proteins GRP78, ATF6 and XBP1s were found expressed in CP and PDAC peritumoral tissues, but in contrast to AGR2, their expression was switched off during TC and PanIN formation. Real-time PCR and ELISA analyses showed that ER stress induced a pro-inflammatory phenotype in pancreatic normal, cancer and stellate cells. Moreover, AGR2 expression was inducible by paracrine transfer of ER stress and pro-inflammation between different pancreatic cell types. Our findings demonstrate that AGR2 induced in ER-stressed and inflammatory pre-neoplastic pancreas is a potential marker of cancer progenitor cells with an important functional role in PDAC initiation.

Pigment Epithelium-derived Factor (PEDF) Blocks Wnt3a Protein-induced Autophagy in Pancreatic Intraepithelial Neoplasms

An increase in autophagy characterizes pancreatic carcinogenesis, but the signals that regulate this process are incompletely understood. Because canonical Wnt/β-catenin signaling is necessary for the transition from early to advanced pancreatic intraepithelial neoplasia (PanIN) lesions, we assessed whether Wnt ligands and endogenous inhibitors of Wnt signaling modulate autophagy. In this study, canonical Wnt3a ligand induced autophagy markers and vacuoles in murine PanIN cells. Furthermore, pigment epithelium-derived factor (PEDF), a secreted glycoprotein known for its anti-tumor properties, blocked Wnt3a-directed induction of autophagy proteins. Autophagy inhibition was complemented by reciprocal regulation of the oxidative stress enzymes, superoxide dismutase 2 (SOD2) and catalase. Transcriptional control of Sod2 expression was mediated by PEDF-induced NFκB nuclear translocation. PEDF-dependent SOD2 expression in PanIN lesions was recapitulated in a murine model of PanIN formation where PEDF was deleted. In human PanIN lesions, co-expression of PEDF and SOD2 was observed in the majority of early PanIN lesions (47/50, 94%), whereas PEDF and SOD2 immunolocalization in high-grade human PanIN-2/3 was uncommon (7/50, 14%). These results indicate that PEDF regulates autophagy through coordinate Wnt signaling blockade and NFκB activation.

Abstract C157: Syndecan1 is required for oncogenic Kras-driven PDAC tumorigenesis and maintenance

Abstract Although the universal presence of KRAS mutations and their critical role in human pancreatic ductal adenocarcinoma (PDAC) designates it as an ideal therapeutic target, oncogenic KRAS (KRAS*) is still regarded as ‘undruggable’ to date. Therefore, to identify therapeutic points of intervention, it is critical to understand the impact of KRAS*-mediated pathways on in vivo tumor pathogenesis. We have recently generated an inducible KrasG12D-driven (iKRAS*) mouse PDAC model and established a critical role for sustained KRAS* activity in tumor maintenance, providing a model to characterize pathways required for KRAS*-dependent tumorigenicity. Cell surface proteins are relatively accessible and can not only provide candidates for biomarker discovery, but also be utilized as therapeutic targets. To characterize the KRAS*-specific organization of cell surface proteins for the development of possible diagnostic or therapeutic tools, we conducted an unbiased surfaceome analyses of tumor cells in the presence and absence of KRAS* signaling using the iKRAS* mouse PDAC model. Syndecans (SDC), a family of heparin sulfate proteoglycans, were identified as candidates whose membrane expression is correlated with KRAS* activity. SDC1 expression is increased in premalignant and malignant pancreatic lesions of primary human PDAC and various KRAS*-driven pancreatic cancer mouse models. In addition, SDC1 expression is specifically upregulated in response to KRAS* induction in acinar-ductal metaplasia (ADM) and early pancreatic intraepithelial neoplasia (PanIN), but not in cerulin-induced chromic pancretitis. Moreover, SDC1 membrane expression was abolished upon KRAS* extinction in PDAC cells and our data indicated that MAPK pathway, but not PI3K pathway, is important for KRAS*-mediated SDC1 surface localization. To further study the role of SDC1 in KRAS*-driven pancreatic cancers, SDC1 knockout mouse model was generated and crossed with LSL-KrasG12D-PDAC models. Our preliminary data indicated that genetic ablation of SDC1 expression effectively suppressed KRAS*-driven PDAC initiation and progression. The mechanisms of KRAS*-mediated SDC1 membrane localization and its impact on PDAC initiation and progression is being further explored. SDC1 might potentially be the drug target and diagnostic marker for PDAC. Citation Format: Wantong Yao, Haoqiang Ying, Giulio Draetta. Syndecan1 is required for oncogenic Kras-driven PDAC tumorigenesis and maintenance. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C157.

IL17 Functions through the Novel REG3β-JAK2-STAT3 Inflammatory Pathway to Promote the Transition from Chronic Pancreatitis to Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) offers an optimal model for discovering "druggable" molecular pathways that participate in inflammation-associated cancer development. Chronic pancreatitis, a common prolonged inflammatory disease, behaves as a well-known premalignant condition that contributes to PDAC development. Although the mechanisms underlying the pancreatitis-to-cancer transition remain to be fully elucidated, emerging evidence supports the hypothesis that the actions of proinflammatory mediators on cells harboring Kras mutations promote neoplastic transformation. Recent elegant studies demonstrated that the IL17 pathway mediates this phenomenon and can be targeted with antibodies, but the downstream mechanisms by which IL17 functions during this transition are currently unclear. In this study, we demonstrate that IL17 induces the expression of REG3β, a well-known mediator of pancreatitis, during acinar-to-ductal metaplasia and in early pancreatic intraepithelial neoplasia (PanIN) lesions. Furthermore, we found that REG3β promotes cell growth and decreases sensitivity to cell death through activation of the gp130-JAK2-STAT3-dependent pathway. Genetic inactivation of REG3β in the context of oncogenic Kras-driven PDAC resulted in reduced PanIN formation, an effect that could be rescued by administration of exogenous REG3β. Taken together, our findings provide mechanistic insight into the pathways underlying inflammation-associated pancreatic cancer, revealing a dual and contextual pathophysiologic role for REG3β during pancreatitis and PDAC initiation.

The canonical Wnt pathway regulates the metastasis-promoting mucin MUC4 in pancreatic ductal adenocarcinoma

Aberrant Wnt signaling frequently occurs in pancreatic cancer (PC) and contributes to disease progression/metastases. Likewise, the transmembrane-mucin MUC4 is expressed de novo in early pancreatic intraepithelial neoplasia (PanINs) and incrementally increases with PC progression, contributing to metastasis. To determine the mechanism of MUC4 upregulation in PC, we examined factors deregulated in early PC progression, such as Wnt/β-catenin signaling. MUC4 promoter analysis revealed the presence of three putative TCF/LEF-binding sites, leading us to hypothesize that MUC4 can be regulated by β-catenin. Immunohistochemical (IHC) analysis of rapid autopsy PC tissues showed a correlation between MUC4 and cytosolic/nuclear β-catenin expression. Knock down (KD) of β-catenin in CD18/HPAF and T3M4 cell lines resulted in decreased MUC4 transcript and protein. Three MUC4 promoter luciferase constructs, p3778, p3000, and p2700, were generated. The construct p3778, encompassing the entire MUC4 promoter, elicited increased luciferase activity in the presence of stabilized β-catenin. Mutation of the TCF/LEF site closest to the transcription start site (i.e., −2629/−2612) and furthest from the start site (i.e., −3425/−3408) reduced MUC4 promoter luciferase activity. Transfection with dominant negative TCF4 decreased MUC4 transcript and protein levels. Chromatin immunoprecipitation confirmed enrichment of β-catenin on −2629/−2612 and −3425/−3408 of the MUC4 promoter in CD18/HPAF. Functionally, CD18/HPAF and T3M4 β-catenin KD cells showed decreased migration and decreased Vimentin, N-cadherin, and pERK1/2 expression. Tumorigenicity studies in athymic nude mice showed CD18/HPAF β-catenin KD cells significantly reduced primary tumor sizes and metastases compared to scrambled control cells. We show for the first time that β-catenin directly governs MUC4 in PC.

Abstract PR04: Clonal composition and clonal selection during PanIN progression

Abstract Pancreatic cancer is the fourth most common cause of cancer death in the United States. It remains a highly lethal malignancy despite surgical and chemotherapeutic advances. In spite of progress made delineating the mechanisms underlying pancreatic cancer, there has been little improvement in survival rates over the past fifty years. Copy number analysis of human invasive pancreatic ductal adenocarcinoma (PDAC) revealed massively rearranged cellular karyotypes as well as significant clonal complexity. Given the genetic complexity of already invasive PDAC, we believe it will be important to understand earlier events in pancreatic cancer, i.e. PanIN formation and progression. We hypothesized that stage-specific bottlenecks may lead to clonal selection during pancreatic intraepithelial neoplasia (PanIN) formation and progression. In order to test this hypothesis, we examined evolving clonal complexity during the progression of pancreatic intraepithelial neoplasia in the Mist1Tg/wt; LSL-KrasG12D (KCiMist1) mouse model of pancreatic cancer. Following KrasG12D activation in adult acinar cells, these mice develop acinar to ductal metaplasia (ADM) and preinvasive PanIN 1 - PanIN 3 lesions in a manner that faithfully recapitulates the human disease. Our strategy involves crossing MistTg/wt; LSL-KrasG12D to Brainbow2.1Tg/Tg “Confetti” mice, in which individual cells undergo stochastic recombination of the Confetti reporter to activate expression of either nuclear GFP, membrane-associated CFP, cytoplasmic RFP or cytoplasmic YFP. Following tamoxifen induction, mice were given cerulein injections to increase PanIN density, and sacrificed at four and nine weeks post-tamoxifen. Following additional immunofluorescent labeling with EpCAM to demarcate individual cells, confocal imaging was performed. Lesions were characterized by grade (ADM, early PanIN and late PanIN), and the number of clones, defined as adjacent cells sharing an identical fluorescent protein signature, was compared at 4 and 9 weeks. For AMDs, early PanIN and late PanIN, we observe both monoclonal and polyclonal lesions. Our analysis of evolving clonal complexity over time is ongoing, but preliminary data suggests a time-dependent decrease in clonal complexity. The average number of clones seen in individual ADM and late PanIN lesions decreased in the mice sacrificed at nine week post tamoxifen induction compared to those sacrificed at four weeks post tamoxifen induction (1.5 +/- 0.52 clones per lesion vs. 2.29 +/- 1.14 clones per lesion for ADM and 2.5 +/- 1.29 vs. 3.75 +/- 1.89 clones per lesion for late PanIN). In contrast, there was little difference in clonal complexity noted for early PanINs at 4 vs. 9 weeks (2.17 +/- 1.19 clones per lesion in the four week cohort vs. 2.42 +/- 1.24 clones per lesion in the nine week time point). These preliminary data suggest an overall trend towards decreased clonal complexity of PanIN lesions over time, suggesting significant selective pressure at even the earliest stages of pancreatic cancer. This abstract is also presented as Poster A62. Citation Format: Kelly J. Lafaro, Audrey Hendley, Jennifer Bailey, Steven Leach. Clonal composition and clonal selection during PanIN progression. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr PR04.

Abstract A18: Syndecan-1 is required for oncogenic Kras-driven PDAC tumorigenesis and maintenance

Abstract Although the universal presence of KRAS mutations and their critical role in human pancreatic ductal adenocarcinoma (PDAC) designates it as an ideal therapeutic target, oncogenic KRAS (KRAS*) is still regarded as ‘undruggable’ to date. Therefore, to identify therapeutic points of intervention, it is critical to understand the impact of KRAS*-mediated pathways on in vivo tumor pathogenesis. We have recently generated an inducible KrasG12D-driven (iKRAS*) mouse PDAC model and established a critical role for sustained KRAS* activity in tumor maintenance, providing a model to characterize pathways required for KRAS*-dependent tumorigenicity. Cell surface proteins are relatively accessible and can provide candidates for biomarker discovery, as well as be utilized as therapeutic targets. To characterize the KRAS*-specific organization of cell surface proteins toward the development of possible diagnostic or therapeutic tools, we conducted an unbiased surfaceome analysis of tumor cells in the presence and absence of KRAS* signaling using the iKRAS* mouse PDAC model. Syndecans (SDC), a family of heparin sulfate proteoglycans, were identified as candidates whose membrane expression is correlated with KRAS* activity. Specifically, Syndecan-1 (SDC1) expression was upregulated in response to KRAS* induction in acinar-ductal metaplasia (ADM) and early pancreatic intraepithelial neoplasia (PanIN), but not in cerulein-induced chronic pancreatitis. Moreover, SDC1 membrane expression was abolished upon KRAS* extinction, and our data indicated that the MAPK pathway, but not the PI3K pathway, drives KRAS*-mediated SDC1 surface localization. Supportive of our findings, it has been shown that SDC1 expression is increased in premalignant and malignant pancreatic lesions of primary human PDAC, as well as in various other KRAS*-driven pancreatic cancer mouse models. To further study the role of SDC1 in KRAS*-driven pancreatic cancers, we generated an SDC1-knockout mouse model and crossed with LSL-KrasG12D-PDAC models. Our preliminary data indicate that genetic ablation of SDC1 expression effectively suppressed KRAS*-driven PDAC initiation and progression. The mechanisms of KRAS*-mediated SDC1 membrane localization and its impact on PDAC initiation and progression will be further explored. Citation Format: Wantong Yao, Wei Wang, Ayumu Taguchi, Avnish Kapoor, Amin Momin, Hong Jiang, Johnathon Rose, Muge Celiktas, Angela Deem, Samir Hanash, Ronald DePinho, Haoqiang Ying, Giulio Draetta. Syndecan-1 is required for oncogenic Kras-driven PDAC tumorigenesis and maintenance. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A18.

Distinct serum metabolomics profiles associated with malignant progression in the KrasG12D mouse model of pancreatic ductal adenocarcinoma.

BackgroundPancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer deaths worldwide with less than a 6% 5-year survival rate. PDAC is associated with poor prognosis based on the late stage diagnosis of the disease. Current diagnostic tests lack the sensitivity and specificity to identify markers of early staging. Metabolomics has provided biomarkers for various diseases, stressors, and environmental exposures. In this study we utilized the p48-Cre/LSL-KrasG12D mouse model with age-matched wild type mice. This model shows malignant progression to PDAC analogous to the human disease stages via early and late pancreatic intra-epithelial neoplasia (PanIN) lesions.ResultsSerum was collected from mice with early PanIN lesions (at 3-5 months) and with late PanIN or invasive PDAC lesions (13-16 months), as determined by histopathology. Metabolomics analysis of the serum samples was conducted through UPLC-TOFMS (Ultra Performance Liquid Chromatography coupled to Time-of-flight Mass Spectrometry). Multivariate data analysis revealed distinct metabolic patterns in serum samples collected during malignant progression towards invasive PDAC. Animals with early or late stage lesions were distinguished from their respective controls with 82.1% and 81.5% accuracy, respectively. This also held up for randomly selected subgroups in the late stage lesion group that showed less variability between animals. One of the metabolites, citrate, was validated through tandem mass spectrometry and showed increased levels in serum with disease progression. Furthermore, serum metabolite signatures from animals with early stage lesions identified controls and animals with late stage lesions with 81.5% accuracy (p<0.01) and vice-versa with 73.2% accuracy (p<0.01).ConclusionsWe conclude that metabolomics analysis of serum samples can identify the presence of early and late stage pancreatic cancer.

Cholecystokinin receptor antagonist halts progression of pancreatic cancer precursor lesions and fibrosis in mice.

Exogenous administration of cholecystokinin (CCK) induces hypertrophy and hyperplasia of the pancreas with an increase in DNA content. We hypothesized that endogenous CCK is involved in the malignant progression of pancreatic intraepithelial neoplasia (PanIN) lesions and the fibrosis associated with pancreatic cancer. The presence of CCK receptors in early PanIN lesions was examined by immunohistochemistry in mouse and human pancreas. Pdx1-Cre/LSL-Kras transgenic mice were randomized to receive either untreated drinking water or water supplemented with a CCK receptor antagonist (proglumide, 0.1 mg/mL). Pancreas from the mice were removed and examined histologically for number and grade of PanINs after 1, 2, or 4 months of antagonist therapy. Both CCK-A and CCK-B receptors were identified in early stage PanINs from mouse and human pancreas. The grade of PanIN lesions was reversed, and progression to advanced lesions arrested in mice treated with proglumide compared with the controls (P = 0.004). Furthermore, pancreatic fibrosis was significantly reduced in antagonist-treated animals compared with vehicle (P < 0.001). These findings demonstrate that endogenous CCK is in part responsible for the development and progression of pancreatic cancer. The use of CCK receptor antagonists may have a role in cancer prophylaxis in high-risk subjects and may reduce fibrosis in the microenvironment.

Of autophagy and in vivo pancreatic carcinogenesis: The p53 status matters!

Autophagy is a lysosomal recycling process essential for tissue or cell homeostasis. The role of autophagy in cancer is complex with either tumor suppressive or pro-carcinogenetic activities. This question has been addressed by Kevin Ryan's laboratory by using Kras-driven genetic engineering mouse models in order to decipher the involvement of essential Atg5/7 autophagy genes and p53 status in pancreatic homeostasis and carcinogenetic progression. The authors show that combined loss of autophagy and p53 dramatically promotes progression from early Pancreatic Intraepithelial Neoplasia (PanIN) lesions towards adenocarcinoma and alters the cellular metabolism with an enrichment of anabolic pathway that can fuel the tumor growth.