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Lack of durable response to cytotoxic chemotherapy is a major contributor to the dismal outcomes seen in pancreatic ductal adenocarcinoma (PDAC). Extensive tumor desmoplasia and poor vascular supply are two predominant characteristics which hinder the delivery of chemotherapeutic drugs into PDAC tumors and mediate resistance to therapy. Previously, we have shown that STAT3 is a key biomarker of therapeutic resistance to gemcitabine treatment in PDAC, which can be overcome by combined inhibition of the Src and EGFR pathways. Although it is well-established that concurrent EGFR and Src inhibition exert these antineoplastic properties through direct inhibition of mitogenic pathways in tumor cells, the influence of this combined therapy on stromal constituents in PDAC tumors remains unknown. In this study, we demonstrate in both orthotopic tumor xenograft and Ptf1acre/+;LSL-KrasG12D/+;Tgfbr2flox/flox (PKT) mouse models that concurrent EGFR and Src inhibition abrogates STAT3 activation, increases microvessel density, and prevents tissue fibrosis in vivo. Furthermore, the stromal changes induced by parallel EGFR and Src pathway inhibition resulted in improved overall survival in PKT mice when combined with gemcitabine. As a phase I clinical trial utilizing concurrent EGFR and Src inhibition with gemcitabine has recently concluded, these data provide timely translational insight into the novel mechanism of action of this regimen and expand our understanding into the phenomenon of stromal-mediated therapeutic resistance. Implications: These findings demonstrate that Src/EGFR inhibition targets STAT3, remodels the tumor stroma, and results in enhanced delivery of gemcitabine to improve overall survival in a mouse model of PDAC.

Co-occurrent KRAS and TP53 mutations define a majority of patients with pancreatic ductal adenocarcinoma (PDAC) and define its pro-metastatic proclivity. Here, we demonstrate that KRAS-TP53 co-alteration is associated with worse survival compared with either KRAS-alone or TP53-alone altered PDAC in 245 patients with metastatic disease treated at a tertiary referral cancer center, and validate this observation in two independent molecularly annotated datasets. Compared with non-TP53 mutated KRAS-altered tumors, KRAS-TP53 co-alteration engenders disproportionately innate immune-enriched and CD8+ T-cell-excluded immune signatures. Leveraging in silico, in vitro, and in vivo models of human and murine PDAC, we discover a novel intersection between KRAS-TP53 co-altered transcriptomes, TP63-defined squamous trans-differentiation, and myeloid-cell migration into the tumor microenvironment. Comparison of single-cell transcriptomes between KRAS-TP53 co-altered and KRAS-altered/TP53WT tumors revealed cancer cell-autonomous transcriptional programs that orchestrate innate immune trafficking and function. Moreover, we uncover granulocyte-derived inflammasome activation and TNF signaling as putative paracrine mediators of innate immunoregulatory transcriptional programs in KRAS-TP53 co-altered PDAC. Immune subtyping of KRAS-TP53 co-altered PDAC reveals conflation of intratumor heterogeneity with progenitor-like stemness properties. Coalescing these distinct molecular characteristics into a KRAS-TP53 co-altered “immunoregulatory program” predicts chemoresistance in metastatic PDAC patients enrolled in the COMPASS trial, as well as worse overall survival.

A hallmark of pancreatic ductal adenocarcinoma (PDAC) is the presence of a dense, desmoplastic stroma and the consequent altered interactions between cancer cells and their surrounding tumor microenvironment (TME) that promote disease progression, metastasis, and chemoresistance. We have previously shown that IL6 secreted from pancreatic stellate cells (PSC) stimulates the activation of STAT3 signaling in tumor cells, an established mechanism of therapeutic resistance in PDAC. We have now identified the tumor cell–derived cytokine IL1α as an upstream mediator of IL6 release from PSCs that is involved in STAT3 activation within the TME. Herein, we show that IL1α is overexpressed in both murine and human PDAC tumors and engages with its cognate receptor IL1R1, which is strongly expressed on stromal cells. Further, we show that IL1R1 inhibition using anakinra (recombinant IL1 receptor antagonist) significantly reduces stromal-derived IL6, thereby suppressing IL6-dependent STAT3 activation in human PDAC cell lines. Anakinra treatment results in significant reduction in IL6 and activated STAT3 levels in pancreatic tumors from Ptf1aCre/+;LSL-KrasG12D/+; Tgfbr2flox/flox (PKT) mice. Additionally, the combination of anakinra with cytotoxic chemotherapy significantly extends overall survival compared with vehicle treatment or anakinra monotherapy in this aggressive genetic mouse model of PDAC. These data highlight the importance of IL1 in mediating tumor–stromal IL6/STAT3 cross-talk in the TME and provide a preclinical rationale for targeting IL1 signaling as a therapeutic strategy in PDAC.

Simple Summary Pancreatic cancer has a dismal 5-year survival rate of 10%, making it one of the deadliest forms of malignancy. The poor prognosis associated with this disease is because it is resistant to almost every type of chemotherapy. Another major hallmark of pancreatic cancer is the presence of several activated oncogenic signaling pathways, including PI3K/Akt/mTOR, which promotes disease aggressiveness and therapeutic resistance. Previously, we have shown that targeted inhibition of PI3K/Akt/mTOR together led to a significant reduction of tumor burden and improvement of overall survival in an aggressive mouse model of pancreatic cancer. This review article discusses the significance of targeting PI3K and its direct downstream effector components in pancreatic cancer. Additionally, we will also update on the recent studies highlighting the tumor cell-extrinsic impact of PI3K inhibition in the modulation of the immune microenvironment within the context of this malignancy. Abstract Pancreatic ductal adenocarcinoma (PDAC) remains among the deadliest solid tumors that remain treatment-refractory and show a dismal prognosis. More than 90% of PDAC tumors harbor mutations in the K-Ras that exert a strong pro-tumorigenic effect by activating several downstream effector pathways, including phosphatidylinositol-3-kinase (PI3K)-Akt. The role of frequently activated PI3K/Akt pathway in promoting PDAC aggressiveness is well established. Therapeutic approaches targeting PI3K and downstream signaling components in different cellular compartments, including tumor, stromal and immune cells, have directly impacted the tumor burden in this cancer type. Our previous work has demonstrated that targeting the PI3K/Akt/mTOR pathway reduced tumor growth and improved survival in the genetic mouse model of PDAC. Here, we discuss the significance of targeting PI3K signaling and the biological impact of PI3K inhibition in modulating the tumor–stromal immune crosstalk within the microenvironment of pancreatic cancer. Furthermore, this review updates on the current challenges involving the therapeutic implications of targeting this pathway in PDAC.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy and is highly resistant to standard treatment regimens. Targeted therapies against KRAS, a mutation present in an overwhelming majority of PDAC cases, have been largely ineffective. However, inhibition of downstream components in the KRAS signaling cascade provides promising therapeutic targets in the management of PDAC and warrants further exploration. Here, we investigated Urolithin A (Uro A), a novel natural compound derived from pomegranates, which targets numerous kinases downstream of KRAS, in particular the PI3K/AKT/mTOR signaling pathways. We showed that treatment of PDAC cells with Uro A blocked the phosphorylation of AKT and p70S6K in vitro, successfully inhibited the growth of tumor xenografts, and increased overall survival of Ptf1aCre/+;LSL-KrasG12D/+;Tgfbr2flox/flox (PKT) mice compared with vehicle or gemcitabine therapy alone. Histologic evaluation of these Uro A–treated tumor samples confirmed mechanistic actions of Uro A via decreased phosphorylation of AKT and p70S6K, reduced proliferation, and increased cellular apoptosis in both xenograft and PKT mouse models. In addition, Uro A treatment reprogrammed the tumor microenvironment, as evidenced by reduced levels of infiltrating immunosuppressive cell populations such as myeloid-derived suppressor cells, tumor-associated macrophages, and regulatory T cells. Overall, this work provides convincing preclinical evidence for the utility of Uro A as a therapeutic agent in PDAC through suppression of the PI3K/AKT/mTOR pathway.

The Src family of non-receptor tyrosine kinases are frequently activated in pancreatic ductal adenocarcinoma (PDAC), contributing to disease progression through downregulation of E-cadherin and induction of epithelial-to-mesenchymal transition (EMT). The purpose of this study was to examine the efficacy of Src kinase inhibition in restoring E-cadherin levels in PDAC. Immunohistochemical analysis of human PDAC samples showed Src activation is inversely correlated with E-cadherin levels. Protein and mRNA levels of E-cadherin, the gene expression of its various transcriptional repressors (Zeb1, Snail, Slug, LEF-1, TWIST), and changes in sub-cellular localization of E-cadherin/β-catenin in PDAC cells were characterized in response to treatment with the Src inhibitor, dasatinib (DST). DST repressed Slug mRNA expression, promoted E-cadherin transcription, and increased total and membranous E-cadherin/β-catenin levels in drug-sensitive PDAC cells (BxPC3 and SW1990), however no change was observed in drug-resistant PANC1 cells. BxPC3, PANC1, and MiaPaCa-2 flank tumor xenografts were treated with DST to examine changes in E-cadherin levels in vivo. Although DST inhibited Src phosphorylation in all xenograft models, E-cadherin levels were only restored in BxPC3 xenograft tumors. These results suggest that Src kinase inhibition reverses EMT in drug-sensitive PDAC cells through Slug-mediated repression of E-cadherin and identifies E-cadherin as potential biomarker for determining response to DST treatment.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by immune exclusion, pro-inflammatory polarization of cancer-associated fibroblasts (CAF), and resistance to immune checkpoint inhibition (ICI). We have previously demonstrated that reciprocally activated RAS/MEK/ERK and JAK/STAT3 pathways mediate therapeutic resistance, while combined MEK and STAT3 inhibition (MEKi+STAT3i) overcomes such resistance in preclinical models. We now show that combined MEKi+STAT3i not only alters stromal architecture but also uncovers stromal plasticity by revealing a substantial attenuation of Il6/Cxcl1-expressing secretory and Lrrc15-expressing myofibroblastic CAF phenotypes with a concomitant enrichment of Ly6a/Cd34-expressing CAF phenotypes exhibiting mesenchymal progenitor-like properties via single-cell RNA sequencing in Ptf1acre/+;LSL-KrasG12D/+;Tgfbr2flox/flox(PKT) mice. This remodeling of CAF heterogeneity is associated with reprogramming of immunosuppressive myeloid populations and enhanced trafficking of CD8+ T-cells which exhibit a distinct effector transcriptional program. These MEKi+STAT3i-mediated repercussions are in part CAF-dependent, since CRISPR/Cas9 genetic silencing of CAF-restricted Mek1/Stat3 mitigates inflammatory CAF polarization and myeloid infiltration in vivo. Addition of MEKi+STAT3i to PD-1 blockade overcomes ICI resistance by significantly augmenting anti-tumor responses and dramatically improving survival in PKT mice compared with anti-PD-1 monotherapy. The addition of MEKi+STAT3i to PD-1 blockade not only augments the recruitment of activated and memory T-cell populations, but also improves their degranulating capacity and functional cytotoxicity compared to PD-1 blockade alone. Importantly, treatment of a patient with chemotherapy-refractory metastatic PDAC with MEKi (Trametinib), STAT3i (Ruxolitinib), and PD-1 inhibitor (Nivolumab) was well-tolerated and yielded clinical benefit. These data uncover a novel paradigm in which combined MEKi+STAT3i reprograms stromal inflammation and immune tolerance to overcome immunotherapy resistance in PDAC.

Pancreatic adenocarcinoma is a devastating disease with an abysmal survival rate of 9%. A robust fibro-inflammatory and desmoplastic stroma, characteristic of pancreatic cancer, contribute to the challenges in developing viable therapeutic strategies in this disease. Apart from constricting blood vessels and preventing efficient drug delivery to the tumor, the stroma also contributes to the aggressive biology of cancer along with its immune-evasive microenvironment. In this study, we show that in pancreatic tumors, the developing stroma increases tumor initiation frequency in pancreatic cancer cells in vivo by enriching for CD133 + aggressive “stem-like” cells. Additionally, the stromal fibroblasts secrete IL6 as the major cytokine, increases glycolytic flux in the pancreatic tumor cells, and increases lactate efflux in the microenvironment via activation of the STAT signaling pathway. We also show that the secreted lactate favors activation of M2 macrophages in the tumor microenvironment, which excludes CD8 + T cells in the tumor. Our data additionally confirms that the treatment of pancreatic tumors with anti-IL6 antibody results in tumor regression as well as decreased CD133 + population within the tumor. Furthermore, inhibiting the lactate efflux in the microenvironment reduces M2 macrophages, and makes pancreatic tumors more responsive to anti-PD1 therapy. This suggests that stromal IL6 driven metabolic reprogramming plays a significant role in the development of an immune-evasive microenvironment. In conclusion, our study shows that targeting the metabolic pathways affected by stromal IL6 can make pancreatic tumors amenable to checkpoint inhibitor therapy.

Major contributors to therapeutic resistance in pancreatic ductal adenocarcinoma (PDAC) include Kras mutations, a dense desmoplastic stroma that prevents drug delivery to the tumor, and activation of redundant signaling pathways. We have previously identified a mechanistic rationale for targeting STAT3 signaling to overcome therapeutic resistance in PDAC. In this study, we investigate the molecular mechanisms underlying the heterogeneous response to STAT3 and RAS pathway inhibition in PDAC. Effects of JAK/STAT3 inhibition (STAT3i) or MEK inhibition (MEKi) were established in Ptf1acre/+; LSL-KrasG12D/+; and Tgfbr2flox/flox (PKT) mice and patient-derived xenografts (PDX). Amphiregulin (AREG) levels were determined in serum from human patients with PDAC, LSL-KrasG12D/+;Trp53R172H/+;Pdx1Cre/+ (KPC), and PKT mice. MEKi/STAT3i–treated tumors were analyzed for integrity of the pancreas and the presence of cancer stem cells (CSC). We observed an inverse correlation between ERK and STAT3 phosphorylation. MEKi resulted in an immediate activation of STAT3, whereas STAT3i resulted in TACE-induced, AREG-dependent activation of EGFR and ERK. Combined MEKi/STAT3i sustained blockade of ERK, EGFR, and STAT3 signaling, overcoming resistance to individual MEKi or STAT3i. This combined inhibition attenuated tumor growth in PDX and increased survival of PKT mice while reducing serum AREG levels. Furthermore, MEKi/STAT3i altered the PDAC tumor microenvironment by depleting tumor fibrosis, maintaining pancreatic integrity, and downregulating CD44+ and CD133+ CSCs. These results demonstrate that resistance to MEKi is mediated through activation of STAT3, whereas TACE-AREG-EGFR–dependent activation of RAS pathway signaling confers resistance to STAT3 inhibition. Combined MEKi/STAT3i overcomes these resistances and provides a novel therapeutic strategy to target the RAS and STAT3 pathway in PDAC. Significance: This report describes an inverse correlation between MEK and STAT3 signaling as key mechanisms of resistance in PDAC and shows that combined inhibition of MEK and STAT3 overcomes this resistance and provides an improved therapeutic strategy to target the RAS pathway in PDAC. Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/21/6235/F1.large.jpg. Cancer Res; 78(21); 6235–46. ©2018 AACR.

Activating KRAS mutations, a defining feature of pancreatic ductal adenocarcinoma (PDAC), promote tumor growth in part through the activation of cyclin-dependent kinases (CDK) that induce cell-cycle progression. p16INK4a (p16), encoded by the gene CDKN2A, is a potent inhibitor of CDK4/6 and serves as a critical checkpoint of cell proliferation. Mutations in and subsequent loss of the p16 gene occur in PDAC at a rate higher than that reported in any other tumor type and results in Rb inactivation and unrestricted cellular growth. Therefore, strategies targeting downstream RAS pathway effectors combined with CDK4/6 inhibition (CDK4/6i) may have the potential to improve outcomes in this disease. Herein, we show that expression of p16 is markedly reduced in PDAC tumors compared with normal pancreatic or pre-neoplastic tissues. Combined MEK inhibition (MEKi) and CDK4/6i results in sustained downregulation of both ERK and Rb phosphorylation and a significant reduction in cell proliferation compared with monotherapy in human PDAC cells. MEKi with CDK4/6i reduces tumor cell proliferation by promoting senescence-mediated growth arrest, independent of apoptosis in vitro. We show that combined MEKi and CDK4/6i treatment attenuates tumor growth in xenograft models of PDAC and improves overall survival over 200% compared with treatment with vehicle or individual agents alone in Ptf1acre/+;LSL-KRASG12D/+;Tgfbr2flox/flox (PKT) mice. Histologic analysis of PKT tumor lysates reveal a significant decrease in markers of cell proliferation and an increase in senescence-associated markers without any significant change in apoptosis. These results demonstrate that combined targeting of both MEK and CDK4/6 represents a novel therapeutic strategy to synergistically reduce tumor growth through induction of cellular senescence in PDAC.

// Nagaraj S. Nagathihalli 1 , Jason A. Castellanos 2 , Michael N. VanSaun 1 , Xizi Dai 1 , Mahogany Ambrose 3 , Qiaozhi Guo 4 , Yanhua Xiong 2 , Nipun B. Merchant 1 1 Division of Surgical Oncology, Department of Surgery, University of Miami Miller School of Medicine, Sylvester Comprehensive Cancer Center, Miami, Florida, USA 2 Department of Surgery, Vanderbilt University School of Medicine, Nashville, Tennessee, USA 3 Meharry Medical College, Nashville, Tennessee, USA 4 Yale School of Medicine, New Haven, Connecticut, USA Correspondence to: Nipun B. Merchant, email: nmerchant@med.miami.edu Keywords: pancreatic stellate cells, IL-6, STAT3, PanIN, pancreatic cancer Received: January 06, 2016 Accepted: August 22, 2016 Published: September 01, 2016 ABSTRACT Pancreatic ductal adenocarcinoma (PDAC) is a dynamic tumor supported by several stromal elements such as pancreatic stellate cells (PSC). Significant crosstalk exists between PSCs and tumor cells to stimulate oncogenic signaling and malignant progression of PDAC. However, how PSCs activate intercellular signaling in PDAC cells remains to be elucidated. We have previously shown that activated signal transducer and activator of transcription 3 (STAT3) signaling is a key component in the progression of pancreatic neoplasia. We hypothesize that PSC secreted IL-6 activates STAT3 signaling to promote PanIN progression to PDAC. Human PDAC and mouse PanIN cells were treated with PSC-conditioned media (PSC-CM), and phospho- and total-STAT3 levels by immunoblot analysis were determined. IL-6 was quantified in PSC-CM and cell invasion and colony formation assays were performed in the presence or absence of a neutralizing IL-6 antibody and the JAK/STAT3 inhibitor AZD1480. Serum from Ptf1a Cre/+ ;LSL-Kras G12D/+ ;Tgfbr2 flox/flox’ (PKT) and LSL-Kras G12D/+ ; Trp53 R172H/+ ; Pdx1 Cre/+ (KPC) mice demonstrated increased levels of IL-6 compared to serum from non-PDAC bearing KC and PK mice. PSC secreted IL-6 activated STAT3 signaling in noninvasive, precursor PanIN cells as well as PDAC cells, resulting in enhanced cell invasion and colony formation in both cell types. There was a significant positive linear correlation between IL-6 concentration and the ratio of phosphorylated STAT3/total STAT3. IL-6 neutralization or STAT3 inhibition attenuated PSC-CM induced activation of STAT3 signaling and tumorigenicity. These data provide evidence that PSCs are directly involved in promoting the progression of PanINs towards invasive carcinoma. This study demonstrates a novel role of PSC secreted IL-6 in transitioning noninvasive pancreatic precursor cells into invasive PDAC through the activation of STAT3 signaling.

Background Activating KRAS mutations, stromal desmoplasia, and immune exclusion are fundamental to therapeutic resistance in PDAC. We have previously shown an inverse correlation of reciprocally-activated MEK1 and STAT3 signaling, combined inhibition of which remodels the tumor stroma resulting in enhanced effector immune cell infiltration and improved survival in an aggressive genetically engineered mouse model (GEM) of PDAC. Based on these results, we sought to determine the effects of cancer-associated fibroblasts (CAF) specific silencing of MEK1 and STAT3 on the immune microenvironment and PDAC tumor growth. Methods CRISPR/Cas9 ablation of MEK1 and STAT3 was performed in CAFs derived from LSL-KrasG12D/+; Trp53fl/+; Pdx1-Cre (KPC) GEM. RNA sequencing identified transcriptomics changes in MEK1 KO / STAT3KO CAFs vs. wildtype KPC-CAFs. The effect of CAF-specific deletion of MEK1/STAT3 in modulating the immune microenvironment was assessed using multiplex immunophenotyping in orthotopic tumor implantation models of co-cultured KPC tumor cells with MEK1KO / STAT3KO CAFs. Results CAF-specific MEK1/STAT3 ablation resulted in significant retardation of growth kinetics of 3D spheroids, as well as migration and invasion, in KPC tumor cell: CAF co-culture systems. Interestingly, the transcriptome of MEK1KO/ STAT3KO CAFs was strongly enriched for antigen-presenting machinery, IL-12, and IFN signaling pathways, while revealing downregulation of Wnt/β-catenin and collagen pathways. Multiplex cytokine arrays confirmed dramatically increased IL-12p70, IL-2, CXCL10 secretion, and decreased IL-10 secretion in MEK1KO / STAT3KO vs. wildtype. Flow cytometric analysis confirmed increased expression of class I and class II MHC, but not co stimulatory molecules, expression in these double knock out CAFs. Orthotopic implantation of MEK1KO / STAT3KO vs. wildtype CAFs with KPC tumor cells in C57BL/6 pancreata revealed significantly increased infiltration of activated effector degranulating CD8+ T-cells (CD62LlowCD44highCD107low) with reduced exhaustion phenotype (PD1highCD107low)in MEK1KO /STAT3KO vs. wild type CAF tumor-bearing mice. Conclusion CAF-specific ablation of MEK1 and STAT3 generates features suggestive of augmented antigen-presenting capacity and results in adaptive immune invigoration in the PDAC tumor microenvironment. Further mechanistic studies exploring this novel CAF-specific phenotype is warranted. Citation Format: Siddharth Mehra, Xizi Dai, Anna Bianchi, Austin Dosch, Iago de Castro Silva, Prateek Sharma, Samara Singh, Supriya Srinivasan, Nagaraj Nagathihalli, Jashodeep Datta, Nipun Merchant. Targeting of cancer associated fibroblast-specific MEK1 and STAT3 to overcome immunosuppressive microenvironment in Pancreatic Ductal Adenocarcinoma (PDAC) [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-056.

INTRODUCTION: Ras-p53 cooperative mutations, which define >50% of human pancreatic ductal adenocarcinoma (PDAC), promote an immune-excluded tumor microenvironment. In Ras-p53 murine models that phenocopy human PDAC, overexpression of the myeloid chemo-attractant Cxcl1 is associated with T-cell exclusion. We sought to dissect the role of tumor-intrinsic Cxcl1 in mediating immune exclusion in Ras-p53 PDAC. METHODS: Single-cell immune deconvolution and Cxcl1 expression were queried in TCGA PDAC samples (n=150). Single-cell RNA sequencing (scRNAseq) data in KrasG12D/+;Trp53fl/+;Pdx1Cre (KPC WT) genetically engineered mice (GEM) were interrogated. CRISPR/Cas9-based genome editing was used to silence Cxcl1 in KPC tumor cells (KPC-Cxcl1KO). Multiparameter flow cytometry-based immunophenotyping of tumor-infiltrating and circulating myeloid/T-cell populations was performed in in-vivo orthotopic models utilizing KPCWT / KPC-Cxcl1KO cells. RESULTS: In human TCGA samples, Cxcl1 overexpression was strongly associated with expression of its cognate receptor CXCR2, reduced CD4+/CD8+ T-cells, and increased CD33+ myeloid-derived suppressor cells (MDSCs). In KPC GEMs, scRNAseq revealed that CXCR2 expression was exclusively present on the polymorphonuclear MDSC subset (PMN-MDSC). Exogenous Cxcl1 conditioning in murine bone marrow-derived cells induced PMN-MDSC polarization and upregulated CXCR2. Genetic ablation of Cxcl1 in KPC tumor cells disrupted transwell migration of splenocyte-enriched MDSC in-vitro; this effect was dependent on CXCR2 expression on MDSCs. There was a dramatic reduction in tumor weights and metastatic outgrowth (p Citation Format: Anna Bianchi, Siddharth Mehra, Daisy Dai, Iago de Castro Silva, Prateek Sharma, Antonio Colaprico, Austin R. Dosch, Samara Singh, Nagaraj S. Nagathihalli, Nipun B. Merchant, Jashodeep Datta. Dissecting the role of tumor-intrinsic Cxcl1 in mediating immune exclusion in Ras-p53 cooperative pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-046.

Introduction: Pancreatic ductal adenocarcinoma (PDAC) is resistant to immune checkpoint blockade due to its intrinsic immunosuppressive properties. The fibroinflammatory stroma, particularly inflammatory cancer associated fibroblasts (CAFs), are a key determinant of the immune microenvironment in PDAC, however therapies aimed at broad-based targeting of PDAC stroma have been decidedly unsuccessful. Recently, interleukin-1α (IL-1) has emerged as a critical mediator of CAF activation and orchestrator of immune suppression in PDAC. We sought to explore the effects of IL-1 blockade on reprogramming the immune microenvironment and enhancing susceptibility to checkpoint inhibition through suppression of inflammatory stromal signaling in PDAC. Methods: Ptf1acre/+;LSL-KrasG12D/+; Tgfbr2flox/flox (PKT) mice were treated with the recombinant IL-1 receptor antagonist (anakinra, 50 mg/kg IP BID) or vehicle control. Single-cell cDNA library generation was performed using the 10× Chromium System. Expression matrices were processed using the R package Seurat 3.0 and clustered using AUC-based scoring algorithm. Immunophenotyping experiments were performed on single-cell suspensions using the Cytoflex platform. For survival studies, PKT mice were treated with vehicle, anti-PD1 antibody (200 μg/twice weekly), anakinra, or combination until moribund. Tissues were further processed for downstream qPCR and histologic analyses. Human pancreatic stellate cell (hPSC) and immortalized CAF cell lines procured from patient-derived xenografts were utilized for in vitro mechanistic studies. Results: Single-cell analysis revealed that anakinra treatment in PKT mice reduced levels of numerous pro-inflammatory CAF cytokines, including Cxcl1 and Il6, within CAF subclusters. Confirmatory qPCR of bulk tumor samples revealed a significant reduction in Cxcl1 and Il6 gene transcription and a global reduction in stromal fibrosis with anakinra treatment. IL-1 inhibition selectively reduced alternatively-activated macrophage (CD11b+F480+CD206+) and PMN-MDSC (CD11b+Ly6G+) populations within PDAC tumors. Combination treatment of anakinra with anti-PD1 antibody reduced tumor weight, increased intratumoral levels of effector memory (CD8+CD44+CD62L-) and activated cytotoxic (CD8+CD107+) T cells, and improved overall survival in PKT mice. In vitro studies demonstrated that conditioned media from PDAC tumor cells significantly activated Cxcl1 and Il6 gene transcription in hPSCs and CAFs in an IL-1α-dependent manner. IL-1-mediated transcription of these cytokines was abrogated by selective inhibition of both the p38 MAPK and NFκB cascades, demonstrating a coordinated effort between these signaling pathways in modulating the IL-1-induced stress response in stromal cells. Conclusions: These findings provide important mechanistic data and compelling pre-clinical evidence to explore IL-1 inhibition in combination with immune checkpoint blockade in PDAC patients. Citation Format: Austin R. Dosch, Samara Singh, Xizi Dai, Siddharth Mehra, Anna Bianchi, Iago De Castro Silva, Supriya Srinivasan, Nagaraj Nagathihalli, Jashodeep Datta, Nipun B. Merchant. Single-cell transcriptomic analysis reveals interleukin-1 inhibition suppresses inflammatory cancer-associated fibroblast signaling and improves the immune response in pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-049.

Lack of durable response to cytotoxic chemotherapy is a major contributor to the dismal outcomes seen in pancreatic ductal adenocarcinoma (PDAC). Extensive tumor desmoplasia and poor vascular supply are two predominant characteristics that hinder the delivery of chemotherapeutic drugs into PDAC tumors and mediate resistance to therapy. Previously, we have shown that STAT3 is a key biomarker of therapeutic resistance to gemcitabine treatment in PDAC, which can be overcome by combined inhibition of the Src kinase and epidermal growth factor receptor (EGFR) pathways. Although it is well established that concurrent EGFR and Src inhibition exert these anti-eoplastic properties through inhibiting mitogenic pathways in tumor cells directly, the influence of this combined therapy on stromal constituents in PDAC tumors remains unknown. In the present study, we demonstrate in both orthotopic tumor xenografts and Ptf1acre/+;LSL-KrasG12D/+;Tgfbr2flox/flox (PKT) mouse models that concurrent EGFR and Src inhibition abrogates STAT3 activation, resulting in stromal remodeling through the depletion of tumor collagen content, increasing microvessel density, and preventing tissue fibrosis in vivo. Furthermore, these stromal changes resulting from combined pathway inhibition produce a direct increase in intratumoral gemcitabine levels as determined by MALDI-IMS and improve overall survival in PKT mice treated with combined EGFR and Src inhibition with gemcitabine. As a phase 1 clinical trial utilizing concurrent EGFR and Src inhibition in combination with gemcitabine has recently concluded, these data provide translational insight into the novel mechanism of action of this regimen and expand our understanding into the phenomenon of stromal-mediated therapeutic resistance. Citation Format: Austin R. Dosch, Xizi Dai, Michelle L. Reyzer, Deukwoo Kwon, Richard Caprioli, Nipun B. Merchant, Nagaraj S. Nagathihalli. Combined Src and EGFR inhibition targets STAT3 to induce stromal remodeling and increase drug delivery to improve survival in a mouse model of pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr A11.

Although smoking is a significant risk factor for pancreatic ductal adenocarcinoma (PDAC), the molecular mechanisms underlying PDAC development and progression in smokers are still unclear. Here, we show the role of cyclic AMP response element-binding protein (CREB) in the pathogenesis of smoking-induced PDAC. Smokers had significantly higher levels of activated CREB when compared with nonsmokers. Cell lines derived from normal pancreas and pancreatic intraepithelial neoplasm (PanIN) exhibited low baseline pCREB levels compared with PDAC cell lines. Furthermore, elevated CREB expression correlated with reduced survival in patients with PDAC. Depletion of CREB significantly reduced tumor burden after tobacco-specific nitrosamine 4-(methyl nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) treatment, suggesting a CREB-dependent contribution to PDAC growth and progression in smokers. Conversely, NNK accelerated PanIN lesion and PDAC formation via GM-CSF–mediated activation of CREB in a PDAC mouse model. CREB inhibition (CREBi) in mice more effectively reduced primary tumor burden compared with control or GM-CSF blockade alone following NNK exposure. GM-CSF played a role in the recruitment of tumor-associated macrophages (TAM) and regulatory T cell (Treg) expansion and promotion, whereas CREBi significantly reduced TAM and Treg populations in NNK-exposed mice. Overall, these results suggest that NNK exposure leads to activation of CREB through GM-CSF, promoting inflammatory and Akt pathways. Direct inhibition of CREB, but not GM-CSF, effectively abrogates these effects and inhibits tumor progression, offering a viable therapeutic strategy for patients with PDAC. Significance: These findings identify GM-CSF-induced CREB as a driver of pancreatic cancer in smokers and demonstrate the therapeutic potential of targeting CREB to reduce PDAC tumor growth. Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/21/6146/F1.large.jpg. Cancer Res; 78(21); 6146–58. ©2018 AACR.

Secondary bile acids (BA) such as deoxycholic acid (DCA) promote the development of several gastrointestinal malignancies, but how they mediate this effect is unclear. In this study, we offer evidence of a mechanism involving ectodomain shedding of the EGFR ligands amphiregulin (AREG) and TGF-α, which rely upon the cell surface protease TACE/ADAM-17. Specifically, we show that AREG participates in DCA-induced EGFR and STAT3 signaling, cell-cycle progression, and tumorigenicity in human colorectal cancer and pancreatic ductal adenocarcinoma (PDAC). TACE and AREG, but not TGF-α, were overexpressed in both colorectal cancer and PDAC tissues compared with normal tissues. Exposure of colorectal cancer and PDAC cells to DCA resulted in colocalization of Src and TACE to the cell membrane, resulting in AREG-dependent activation of EGFR, mitogen-activated protein kinase (MAPK), and STAT3 signaling. Src or TACE inhibition was sufficient to attenuate DCA-induced AREG, but not TGF-α shedding. We also examined a role for the BA transporter TGR5 in DCA-mediated EGFR and STAT3 signaling. RNA interference-mediated silencing of TGR5 or AREG inhibited DCA-induced EGFR, MAPK, and STAT3 signaling, blunted cyclin D1 expression and cell-cycle progression, and attenuated DCA-induced colorectal cancer or PDAC tumorigenicity. Together, our findings define an AREG-dependent signaling pathway that mediates the oncogenic effects of secondary BAs in gastrointestinal cancers, the targeting of which may enhance therapeutic responses in their treatment. Cancer Res; 74(7); 2062–72. ©2014 AACR.

Cellular homeostasis is dependent on a balance between DNA damage and DNA repair mechanisms. Cells are constantly assaulted by both exogenous and endogenous stimuli leading to high levels of reactive oxygen species (ROS) that cause oxidation of the nucleotide dGTP to 8-oxodGTP. If this base is incorporated into DNA and goes unrepaired, it can result in G > T transversions, leading to genomic DNA damage. MutT Homolog 1 (MTH1) is a nucleoside diphosphate X (Nudix) pyrophosphatase that can remove 8-oxodGTP from the nucleotide pool before it is incorporated into DNA by hydrolyzing it into 8-oxodGMP. MTH1 expression has been shown to be elevated in many cancer cells and is thought to be a survival mechanism by which a cancer cell can stave off the effects of high ROS that can result in cell senescence or death. It has recently become a target of interest in cancer because it is thought that inhibiting MTH1 can increase genotoxic damage and cytotoxicity. Determining the role of MTH1 in normal and cancer cells is confounded by an inability to reliably and directly measure its native enzymatic activity. We have used the chimeric A TP- r eleasing g uanine- o xidized (ARGO) probe that combines 8-oxodGTP and ATP to measure MTH1 enzymatic activity in colorectal cancer (CRC), non-small cell lung cancer (NSCLC) and pancreatic ductal adenocarcinoma (PDAC) along with patient-matched normal tissue. MTH1 8-oxodGTPase activity is significantly increased in tumors across all three tissue types, indicating that MTH1 is a marker of cancer. MTH1 activity measured by ARGO assay was compared to mRNA and protein expression measured by RT-qPCR and Western blot in the CRC tissue pairs, revealing a positive correlation between ARGO assay and Western blot, but little correlation with RT-qPCR in these samples. The adoption of the ARGO assay will help in establishing the level of MTH1 activity in model systems and in assessing the effects of MTH1 modulation in the treatment of cancer.

MutT Homolog 1 (MTH1) is a NUDIX pyrophosphorylase that hydrolyzes oxidized purine nucleoside triphosphates in the nucleotide pool, thus preventing their incorporation into DNA. Our prior work has shown that MTH1 is critical for the maintenance of multiple pro-tumorigenic phenotypes in oncogenic RAS-driven cancer cells, with its depletion leading to decreased tumor formation in vivo. Our subsequent analyses of TCGA patient datasets showed elevated MTH1 expression to be significantly associated with poorer disease-free survival in RAS-mutated cancers, such as that of the lung and pancreas. We found that MTH1 mRNA levels were positively correlated with KRAS levels even in early-stage non-small cell lung cancer patient tissues, and that the introduction of oncogenic KRAS was sufficient to upregulate MTH1 mRNA and protein levels. The aim of this study is to identify RAS-effector signaling intermediates affecting MTH1 expression and activity. Chemical inhibitors of the MAPK/ERK, PI3K/AKT and NOX pathways, plus oncogenic RASV12-effector domain mutants (RASV12- S35/ E38/ G37/ C40), were used to identify key signaling molecular mediators of MTH1 expression in the distinct RAS isoforms (H- and K-RAS). The dependencies of the different KRASG12-mutant polymorphisms (KRASG12- C/ D/ V) on MTH1 expression and activity, as well as candidate transcription factors regulating MTH1 expression, were evaluated. Our work shows MTH1 at the nexus of crosstalk between different effector pathways activated downstream of RAS. Dissecting these signaling intermediates are important in identifying alternate pathways of MTH1 regulation, which may manifest as resistance mechanisms to standard-of-care cancer treatments. Our work will also help understand how to best leverage MTH1 as a therapeutic target in oncogenic RAS-driven cancers driven by the different isoforms, and their respective mutant polymorphisms. Citation Format: Govindi J. Samaranayake, Clara I. Troccoli, Christina Jayaraj, Brittany C. Durden, Nagaraj Nagathihalli, Nipun Merchant, Priyamvada Rai. Exploring the signaling and activity interactome between oncogenic RAS and the nucleotide pool-detoxifying enzyme MTH1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 888.

Epidemiological studies have shown that most cases of lung cancers (85%–90%) are directly attributable to tobacco smoking. Although association between cigarette smoking and lung cancer is well documented, surprisingly little is known about the molecular mechanisms of how smoking is involved in epithelial-to-mesenchymal transition (EMT) through epigenetic changes. Here, we show that lung cancer patients with a smoking history have low E-cadherin levels and loss of E-cadherin is a poor prognostic factor in smokers. Moreover, the downregulation of E-cadherin correlates with the number of pack years. In an attempt to determine the role of long-term cigarette smoking on EMT, we observed that treatment of lung cell lines with cigarette smoke condensate (CSC) induces EMT through downregulation of epithelial markers, including E-cadherin and upregulation of mesenchymal markers. CSC decreases E-cadherin expression at the transcriptional level through upregulation of LEF1 and Slug, and knockdown of these two proteins increases E-cadherin expression. Importantly, chromatin immunoprecipitation assays suggest that LEF-1 and Slug binding to E-cadherin promoter is important for CSC-mediated downregulation of E-cadherin. The histone deacetylase (HDAC) inhibitor MS-275 reverses CSC-induced EMT, migration, and invasion through the restoration of E-cadherin expression. These results suggest that recruitment of HDACs by transcriptional repressors LEF-1 and Slug is responsible for E-cadherin suppression and EMT in cigarette smokers and provide a potential drug target toward the treatment of lung cancer. Mol Cancer Ther; 11(11); 2362–72. ©2012 AACR.

Epidemiologic studies have shown that most cases of lung cancers (85%–90%) are directly attributable to cigarette smoking. Although much information has been gained about the effects of cigarette smoking on various signaling pathways causing lung cancer, nothing is known about the effect of cigarette smoking on the TGF-β–induced tumor suppressor function in lung cancer. To address this issue, lung adenocarcinoma A549 and immortalized bronchial epithelial HPL1A cells were chronically treated with cigarette smoke condensate (CSC) and dimethyl sulfoxide (as a control) to mimic the conditions of long-term cigarette smoking. Prolonged exposure of these cells to CSC resulted in a decrease in Smad3 and Smad4 complex formation and TGF-β–mediated transcription due to reduced expression of Smad3. Long-term CSC treatment reduced apoptosis, increased cell viability, decreased TGF-β–mediated growth inhibition, and enhanced tumorigenicity. The decrease in apoptosis is due to the upregulation of Bcl-2, which is a downstream target of Smad3. Re-expression of Smad3 in the CSC-treated cells restored TGF-β signaling, increased apoptosis, and decreased cell viability and tumorigenicity. Withdrawal of CSC treatment resulted in the restoration of Smad3 expression, reduction in cell viability, and increased TGF-β–mediated growth inhibition. Expression of Smad3 is lower in lung tumors of current smokers than that observed in never-smokers. Collectively, these data provide evidence that cigarette smoking promotes tumorigenicity partly by abrogating TGF-β–mediated growth inhibition and apoptosis by reducing expression of Smad3. Cancer Prev Res; 5(3); 453–63. ©2012 AACR.

Background: Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related deaths in the United States. Chronic alcohol (>60 grams/day) consumption is strongly associated with the risk of PDAC development. The metabolites generated from alcohol have been shown to cause significant pancreatic injury. Although the association of alcohol with PDAC progression has been established, the details of the underlying molecular and cellular mechanisms governing this process are unknown. Our study revealed that alcohol-associated pancreatic carcinogenesis correlates with CXCL12-induced activation of cyclic AMP response element binding protein (CREB). In this study, we examined newly developed CREB knockout (CREBfl/fl) mouse in pancreas. Methods: Human pancreatic epithelial lines (HPNE), HPNE cells with KRAS (HPNE-KRAS), pancreatic stellate cells (PSCs), pancreatic intraepithelial neoplasia (PanIN) mouse cell lines (LSL-KrasG12D/+; Pdx1Cre/+) and cancer-associated fibroblasts (CAFs) cells were exposed to chronic alcohol (50 mM) and analyzed for phospho-kinases in cell lysates and cytokines in conditioned media. Inducible Ptf1aCreERTM;KRASG12D/+ (iPK) mice and CREB knockout (CREBfl/fl) with KRAS activation [Ptf1aCreERTM;KRASG12D/+;CREBfl/fl (iPKCREBKO)] mice were used to investigate the effect of alcohol on CREB activation and the role of CREB in alcohol-associated PDAC pathogenesis. iPK and iPKCREBKO mice were exposed to Lieber-DeCarli alcohol diet for up to 14 weeks with or without caerulein injections. The number of acinar cells (amylase), ducts (cytokeratin 19), PanIN lesions (alcian blue positive), fibrosis (sirius red) and activation of CREB were measured by immunohistochemistry at 6 and 14 weeks of alcohol exposure in vivo. Serum obtained from alcohol-fed iPK and iPKCREBKO mice were analyzed for significant cytokine release upon alcohol exposure in vivo. We then determined the biologic effects of pharmacologic CREB inhibition in iPK mice exposed to alcohol diet. Results: We found increased pCREB levels in HPNE and HPNE-KRAS cells upon treatment with alcohol, which was further associated with the up-regulation of the CXCL12 protein in the conditioned media. Serum CXCL12 and tissue pCREB levels were high in alcohol fed iPK mice when compared to their corresponding control diet-fed mice. Blocking CXCL12 with Plerixafor, a CXCL12-CXCR4 axis inhibitor, decreased alcohol-induced pCREB levels. Finally, exposure of iPK mice to an alcohol diet coupled with cerulein administration significantly increased the number of PanIN lesions (alcian blue+ cells), and decreased acinar cells when compared to alcohol-fed iPKCREBKO mice. Conclusion: These findings implicate CREB as a critical oncogenic driver in alcohol addiction-induced pancreatic carcinogenesis. Additionally, CXCL12 represents an important mediator of CREB activation, which can be pharmacologically targeted. Citation Format: Tulasigeri Totiger, Supriya Srinivasan, Michael VanSaun, Chandrashekhar Joshi, Chanjuan Shi, Xizi Dai, Rajinder Dawra, Alexander Gaidarski, Eric Nestler, Nipun Merchant, Nagaraj Nagathihalli. CREB transgenic mice to study alcohol-associated pancreatic carcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5042.

Background: Alcohol abuse is a major risk factor for pancreatitis. Alcohol addiction-induced molecular pathogenesis of pancreatitis remains obscure, and no current effective treatment exists. Therefore, approaches to investigate pathogenesis, prevention and cellular mechanisms by which alcohol causes pancreatitis are necessary for establishing therapeutics. Our efforts demonstrate that alcohol induces activation of a major intracellular transcriptional factor, cyclic AMP response element-binding protein (CREB). We further investigated the functional role of CREB in alcohol-induced pathogenesis of pancreatitis using cellular and genetic mouse models of pancreas. Materials and Methods: Human tissue microarrays were immunostained to determine the significance of pCREB expression among pancreatic tissues obtained from normal and chronic pancreatitis. Rat acinar cell line AR42J and mouse PSCs (mPSCs) were exposed to alcohol (50 mMol/L). Inducible Ptf1aCreERTM knockin mice and Ptf1aCreERTM;CREBfl/fl (iPC) mice were fed with Lieber Decarli diet alcohol or regular diet for 14 weeks with or without caerulein (50 µg/kg). Mice were then euthanized 24 hours after the last caerulein injection, and pancreas tissues were processed for morphometric analysis (necrosis, vacuolization, hemorrhage, edema and inflammation) and immunohistochemical analysis of amylase, trichrome blue, SMA, collagen 1, fibronectin and pCREB expression. To determine whether alcohol accelerated morbidity in mice, we evaluated pathogenesis of chronic pancreatitis by analyzing acinar atrophy and pancreatic fibrosis. Results: Expression of pCREB was significantly higher (p Conclusion: CREB is overexpressed in pancreatitis and alcohol activates CREB, which then drives pathogenesis of pancreatitis. Severity of pancreatitis in response to alcohol is diminished in the absence of CREB. Therefore, we conclude that targeting CREB represents a promising treatment for alcohol-induced pancreatitis. Citation Format: Supriya Srinivasan, Tulasigeri Totiger, Michael VanSaun, Fanuel Messaggio, Chanjuan Shi, Austin Dosch, Eric Nestler, Nipun Merchant, Nagaraj Nagathihalli. Animal model in the prevention of alcoholic pancreatitis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1249.

Pancreatic ductal adenocarcinoma (PDAC) is the 3rd leading cause of cancer-related mortality in the United States. Most patients present with an advanced disease and the majority die within five years, many surviving less than six months. Cytotoxic chemotherapy including Gemcitabine (Gem), FOLFIRINOX, nab-paclitaxel offer modest improvement in survival, albeit at the cost of increased side effects and unwanted toxicities. Therefore, developing novel chemotherapeutic agents for PDAC treatment is critical to improve survival. Ellagic acid/ellatitannins are abundantly present in the pomegranate and berries, are actively metabolized by the intestinal microflora to Urolithin A (UA). Oral administration of UA has shown to be highly bioavailable and non-toxic. UA inhibits multiple kinases that are known to be involved in PDAC progression and metastasis. Therefore, we hypothesized that UA would elicit potent anti-cancer therapeutic potential in PDAC. The effect of UA on kinase activity was assessed . Inhibition of AKT (downstream of PI3K/PDK1), p70 S6 Kinase (PS6K) and STAT3 activation was quantified in PDAC cells treated with UA in dose-dependent manner. The mechanism of action was validated for UA’s activity on PI3K/PDK1, PS6K and STAT3 activation using immunoblot analysis. MiaPaCa2 cells were treated with specific inhibitors for either AKT (MK2206) or STAT3 (AZD1480) and analyzed for tumorigenicity. UA treated PDAC cells were analyzed for cell proliferation, cell invasion and colony formation. Cell cycle analysis and cell apoptosis were measured by flow cytometry. To test the efficacy of UA in vivo, cells were implanted subcutaneously in athymic nude mice. The animals received UA daily and tumor volume was measured for 5 weeks. Next, we assessed tumor growth and overall survival (OS) in PKT (Ptf1acre/+;LSL-KrasG12D;Tgfbr2fl/fl) mice, an aggressive genetically engineered PDAC mouse model, in response to UA and/or Gem treatment. Tissues from the xenografts and PKT mice treated with vehicle or UA were analyzed for cell proliferation (Ki67) and apoptosis (cleaved Caspase 3) by immunohistochemistry. High expression levels of activated STAT3 or AKT correlate with decreased survival in PDAC. UA treated MiaPaCa2 cells showed significant dose-dependent increase in apoptosis and decrease in anchorage-independent growth. UA inhibited AKT, PS6K and STAT3 signaling. As a single agent, UA effectively reduced in vivo PDAC tumor growth. Immunohistochemistry of UA treated tissues from tumor xenografts and PKT mice showed inhibition of Ki67 positive tumor cells and increased cleaved caspase 3 staining. PKT mice treated with UA showed a decrease in tumor size and an increased OS when compared to vehicle or Gem treated mice alone. These findings show that UA is a novel inhibitor/modulator/regulator for multiple signaling pathways in PDAC. These results suggest UA has potential for pre-clinical development in pancreatic cancer. Citation Format: Supriya Srinivasan, Venkatakrishna Jala, Kumar Honnenahally, Jason Castellanos, Praveen Kumar Vermula, Michael VanSaun, Nipun Merchant, Nagaraj Nagathihalli. Urolithin A prevents pancreatic tumor growth and increases survival by inhibiting PI3K/PDK1 and STAT3 signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5259. doi:10.1158/1538-7445.AM2017-5259

Introduction: Hepatocellular carcinoma (HCC) is the third leading cause of cancer deaths worldwide. HCC typically arises in patients with chronic liver disease or cirrhosis, yet it is increasingly associated with non-alcoholic fatty liver disease (NAFLD), specifically nonalcoholic steatohepatitis (NASH) in the absence of cirrhosis. NAFLD is associated with obesity, metabolic syndrome, and/or patients with type II diabetes. Our previous studies have shown that high fat diet induced hepatic steatosis increases proliferation of hepatocytes and the growth of malignant tumors in a murine model. Glycine N-MethylTransferase (GNMT) expression is lost in over 95% of HCC, and mice deficient in GNMT develop spontaneous HCC by 6 months of age. We hypothesized that GNMT deficient mice would have an increased susceptibility for the development and growth of HCC when a fed high fat diet. Methods: Wildtype and GNMT deficient mice were placed on lean diet (LD, 13% calories from fat) or high fat diet (HFD, 42% calories from fat) at eight weeks of age. An initial cohort of mice were sacrificed after 3 months on diet (6 months of age) to assess for early tumor burden. A second cohort of mice was analyzed by magnetic resonance imaging (MRI) after 6 months on diet (9 months of age) and then sacrificed to assess for late stage disease. All mice were assessed for body weight, liver weight, pancreatic weight, and proliferative index (Ki67). Results: GNMT deficient mice failed to gain weight when placed on HFD, which remained at levels equivalent to wildtype LD mice. At three months of age, wildtype mice on HFD had significantly enlarged livers due to hepatic steatosis. HFD fed GNMT deficient mouse livers were nearly 50% the size of wildtype livers and contained only minimal fatty deposits. Further, livers from HFD and LD fed GNMT mice were equivalent after 3 months, yet they were larger than wildtype mice fed LD. After six months on diet, MRI analysis showed significantly larger livers in HFD fed GNMT mice compared to LD fed GNMT mice due to extensive tumor burden. All wildtype mice lacked any tumors after six months regardless of diet. Histological analysis revealed a heightened cellular proliferation via Ki67 staining in GNMT deficient livers compared to wildtype livers. In comparison, GNMT silencing also occurs in pancreatic cancer, yet none of the GNMT deficient mice developed pancreatic tumors. However, small focal areas of pancreatitis were detected regardless of diet. Additionally, pancreatic weight was significantly decreased in HFD fed GNMT deficient mice compared the LD GNMT deficient mice. Conclusions: While high fat diet did not induce obesity in GNMT deficient mice, it significantly increased cellular proliferation and primary tumor growth in the liver. Understanding dietary factors that impact the microenvironment of the liver and contribute to HCC development and progression is vital to finding new therapeutics for this malignancy. Citation Format: Michael N. VanSaun, Alisha Mendonsa, Fanuel Messaggio, Nagaraj Nagathihalli, Lee Gorden. High fat diet increases development of hepatocellular carcinoma in glycine N-methyltransferase deficient mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2802. doi:10.1158/1538-7445.AM2017-2802

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive solid malignancies and is characterized by poor response to current therapy and a dismal survival rate. Recent evidence indicates that PDAC is associated with frequent, altered cellular signaling pathways. We review these key pathways as well as recent investigations in the area of pancreatic cancer stem cells. Unfortunately, most phase III randomized trials of targeted therapies have not shown a survival advantage in PDAC. However, there has been extensive laboratory and clinical research in these areas which has revealed mechanisms of resistance involved in targeting PDAC and has provided insights into the ineffectiveness of current therapies. Current investigations will hopefully provide better insights into improved therapies and understanding of ways to overcome chemoresistance in PDAC. In this article, we discuss the current research exploring altered signaling pathways in PDAC and potential therapeutic targets.

Introduction: Activating KRAS mutations are commonly found in PDAC and lead to constitutive downstream activation of MEK, which results in uncontrolled proliferation. We have previously shown that MEK inhibition results in activation of STAT3 signaling which confers drug resistance and continued cancer cell growth while combined STAT3 and MEK inhibition overcomes this resistance. Since STAT3 is a critical mediator of cytokine signaling and MEK is a mediator of cytokine production, we sought to determine the effects of MEK and STAT3 inhibition on the immune tumor microenvironment (TME). Tumor infiltrating immune/inflammatory cells, such as regulatory T cells (Tregs), myeloid –derived suppressor cells (MDSCs) and macrophages support tumor growth and contribute to therapeutic resistance. We hypothesized that combined MEK and STAT3 inhibition down regulates the suppressive immune infiltrates and promotes an anti-tumor microenvironment. Experimental procedure: To understand the effect of MEK and/or STAT3 inhibition of PDAC cells, three dimensional spheroid cultures of PDAC cells (MiaPaCa-2, Panc-1, BxPC3) were prepared. Spheroid cultures were treated with inhibitors to MEK (AZD6244) and/or STAT3 (AZD1480) for 10 days. At the end of treatment, spheroids were quantified for size and metabolic activity. To determine in vivo effects, Ptf1a Cre/+ ; LSL-KrasG12D; Tgfbr2fl/fl (PKT) mice were treated with either vehicle, MEK inhibition, STAT3 inhibition, or the combination for 2 weeks. Post-treated pancreatic tissue was extracted, weighed, and examined for pancreatic integrity using immuno-histological and enzymatic analyses. Alternately, the pancreas and spleen were extracted from the mice, cells were isolated from the tissue, and subsequently labeled with antigens for macrophages (CD45, F4/80, CD86, CD80, CD206, CD204), myeloid cells (CD45, CD11b, Ly6g, Ly6c), and T cells (CD45, CD3e, CD4, CD8, CD25, FoxP3) before assessing population percentages by flow cytometric analysis. Results: Treatment with MEK inhibition resulted in slightly reduced spheroid size and metabolic activity; however, combined MEK/STAT3 treatment led to a significant decrease in spheroid size as well as metabolic activity. In PKT mice, treatment with combined inhibitors for MEK and STAT3 resulted in the enhanced suppression of tumor formation compared to either agent alone. Histological analysis of combined MEK/STAT3 significantly inhibited tumor size, maintained a higher percentage of pancreatic integrity, displaying increased percentage of normal acini, reduced CK-19 staining, reduced collagen deposition and minimal alcian blue stain. Analysis of the tumor immune infiltrates revealed a significant reduction in the immunosuppressive/tumor promoting myeloid derived suppressor cell (MDSC) population (CD45+CD11b+Ly6g+Ly6c+) and regulatory T cell population (CD45+CD3e+CD4+CD25+FoxP3+) in the pancreas of mice treated with combined MEK/STAT3 inhibition compared to control mice or mice treated with single agents. Alternately, combined MEK/STAT3 inhibition promoted an increased neutrophil population (7AAD-CD11b+Ly6c+Ly6g-), but a decreased inflammatory M1 macrophage population (CD45+F4/80+CD80+CD86+). Conclusions: Combined MEK/STAT3 inhibition downregulates the tumor promoting immune infiltrates resulting in dramatically reduced tumor burden and enhanced normal pancreatic tissue in a highly aggressive mouse model of pancreatic cancer. Citation Format: Casey Roberts, Michael N. VanSaun, Purushottam Lamichhane, Fanuel Messaggio, Krisztina Kovacs, Supriya Srinivasan, Xizi Dai, Jennifer Barretta, Nagaraj Nagathihalli, Nipun B. Merchant.{Authors}. Targeting the immune-microenvironment with combined inhibition of MEK and STAT3 in a mouse model of pancreatic cancer. [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 B75.

Introduction: Mutations in the KRAS oncogene occur in the majority of pancreatic ductal adenocarcinomas (PDAC), resulting in aberrant activation of the MAPK (RAS-RAF-MEK-ERK) pathway, driving malignant progression. Targeting KRAS has remained an elusive goal. Therefore, efforts have focused on targeting downstream effectors of RAS. The clinical efficacy of MEK inhibitors in other malignancies confirms that targeting the MAPK pathway has therapeutic potential. Unfortunately, clinical trials of MAPK-directed therapies have been unsuccessful in PDAC. Here, we report a novel mechanism of resistance to MAPK-directed therapies, which is associated with amphiregulin (AREG)-mediated activation of EGFR-STAT3 signaling. Experimental procedure: The effects of MEK inhibition on the phosphorylation of multiple signaling proteins and EGF family ligands was assessed. Activation of MAPK and STAT3 was quantified in human pancreatic tissues, and then correlated with overall survival (OS). Cell lysates from mouse PanIN lines derived from the LSL-KrasG12D/+;Pdx1Cre/+ and PDA line from LSL-KrasG12D/+; Trp53R172H/+;Pdx1Cre/+ mouse models of PDAC were immunoblotted for activated MAPK and STAT3 expression. AREG release was measured in the conditioned media of PDAC cells treated with MEK, EGFR, STAT3, and/or TACE inhibitors and TACE siRNA knock down cells treated with MEK, EGFR, or STAT3 inhibitors. Cell invasion, colony formation, spheroid formation, metabolic activity, cell cycle analysis, and apoptosis assays were performed with human PDAC cells treated with inhibitors for TACE, MEK, EGFR and STAT3 in combinations. PDAC xenografts and patient derived xenografts (PDXs) were treated either with vehicle, the MEK inhibitor AZD6244, the STAT3 inhibitor AZD1480 or both drugs and analyzed for spheroid growth. PKT mice (Ptf1acre/+;LSL-KrasG12D;Tgfbr2fl/fl) were treated with AZD6244 and AZD1480 and assessed for OS. Tissues from the xenografts and PKT mice were analyzed for cell proliferation and apoptosis markers. Plasma from the PKT mice treated with the drugs and their combinations was analyzed released AREG by ELISA. Results: Targeting the MAPK pathway is a novel approach to inhibit KRAS mutant tumor progression and improve therapeutic response in PDAC. However, our results show that MAPK inhibition leads to activation of TACE and EGFR and subsequent activation of STAT3 signaling. Combined inhibition of MEK/STAT3 or MEK/EGFR resulted in sustained blockade of MEK, EGFR and STAT3 signaling, decreased cell invasion, colony formation, reduced spheroid size, and metabolic activity in vitro. Growth of flank PDAC xenografts and human PDX tumors in vivo were significantly decreased with combined MEK and STAT3 inhibition when compared to vehicle or monotherapy treatment. OS in PKT mice was extended to a median of 85 days with combined MEK and STAT3 inhibition vs. 52 days for vehicle treated mice (p < 0.001). AREG release was significantly reduced with combined MEK/STAT3 inhibition. TACE siRNA in PDAC cells confirms the role of AREG release in mediating EGFR signaling and overcoming MEK inhibitor resistance. Evaluation of TACE activation and AREG shedding/release in response to MEK inhibition demonstrated that resistance to MEK inhibition in PDAC is mediated by reactivation of the STAT3 pathway, which is strongly influenced by increased AREG production. Conclusions: Our study provides insights into the molecular mechanisms that help explain the heterogeneous response and therapeutic resistance of PDAC to MAPK pathway inhibition and provide a strong rationale that AREG mediated EGFR-STAT3 pathway activation is a major resistance mechanism that impairs the efficacy of MEK inhibitors. Citation Format: Nagaraj Nagathihalli, Jason Castellanos, Chanjuan Shi, Casey Roberts, Michael VanSaun, Nipun Merchant.{Authors}. Resistance to MEK inhibition in pancreatic cancer is associated with amphiregulin mediated EGFR-STAT3 activation. [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 B71.

Introduction: The increasing incidence of pancreatic cancer is associated with a rising prevalence of obesity, a documented risk factor for the disease. Obesity harbors a systemic chronic inflammatory disorder characterized by increased production and secretion of pro-inflammatory adipokines leptin, TNF-α, and IL-6; while exhibiting a decrease in the anti-inflammatory adipokine; adiponectin. Dysregulation of these factors is thought to be a key mechanism of obesity associated cancers, contributing to increased activation of mitogenic pathways including PI3K and MAPK. Adiponectin represents an important negative regulator of leptin, TNF-α and IL-6. We previously demonstrated that adiponectin inhibits pancreatic cancer proliferation and tumor growth, however, the molecular mechanisms by which adiponectin regulates these processes are unknown. We hypothesize that Adiponectin Receptor (AdipoR) agonists elicit anti-tumor effects through suppression of RAS-MAPK mediated pathways and its downstream signaling components in pancreatic cancer progression. Experimental Procedures: The anti-tumor effects of AdipoRon, a novel small molecule agonist of the AdipoR, were assessed in vitro on human (MiaPaca-2 and Panc-1) and murine (P-4313 and K8484) pancreatic cancer cell lines. Cells were treated with AdipoRon in a dose-dependent manner and then assayed for cellular proliferation, apoptosis, colony formation and anchorage-independent growth. The effect of AdipoRon on activation of key RAS-MAPK signaling regulators was investigated by immunoblot analysis. To determine whether AdipoRon could inhibit the effects of obesity associated pro-tumorigenic cytokines, human and mouse pancreatic cancer cells were exposed to plasma collected from obese mice or specifically with recombinant cytokines. To determine whether AdipoRon could inhibit tumor growth in vivo, mice were orthotopically injected in the pancreas with the murine KrasG12D mutant P-4313 cell line. Tumors were allowed to establish for two weeks and treated with either vehicle or AdipoRon. Tumor size and number of Ki67 positive cells were assessed. Results: Compared to vehicle treatment, in vitro assessment confirmed that AdipoRon was highly effective at inhibiting cell proliferation, increasing apoptosis and preventing colony formation for all pancreatic cell lines tested. Anchorage independent growth was drastically reduced for both Panc1 (3.8 fold) and MiaPaca-2 (5.1 fold) cell lines in the presence of AdipoRon. Treatment of both murine and human pancreatic cancer cell lines with AdipoRon caused a significant dose dependent decrease in pSTAT3, pERK1, and pERK2 with a simultaneous increase in pAMPK. Importantly, AdipoRon completely antagonized the stimulatory effects of obese plasma or recombinant IL-6 on the activation of pSTAT3. Administration of AdipoRon to P-4313 orthotopic pancreatic tumor bearing mice resulted in four fold decrease in tumor size and a 50% reduction in tumor cell proliferation. Conclusions: AdipoRon, an adiponectin receptor agonist, suppresses KRAS signaling mediators ERK and STAT3 while simultaneously increasing AMPK resulting in inhibition of pancreatic cancer proliferation and tumor growth. Targeting of adiponectin receptors can provide a viable therapeutic strategy for the treatment of pancreatic cancer. Citation Format: Fanuel Messaggio, Alisha M. Mendonsa, Jason A. Castellanos, Casey Roberts, Nagaraj S. Nagathihalli, Nipun B. Merchant, Lee D. Gorden, Michael N. VanSaun.{Authors}. AdipoRon suppresses ERK and STAT3 to inhibit pancreatic cancer growth. [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 A46.

Pancreatic ductal adenocarcinoma is one of the most aggressive solid malignancies and is characterized by poor response to current therapy and a dismal survival rate. Recent insights regarding the role of cancer stem cells (CSCs) and epithelial-mesenchymal transition (EMT) in tumorigenesis have brought further understanding to the field and have highlighted new therapeutic targets. CSCs are a distinct subset of cancer cells, with the ability to differentiate into other cell types and self-renew in order to fuel the maintenance of tumor amplification. Transition of a cancer cell from an EMT leads to increased migratory and invasive properties, and thus facilitates initiation of metastasis. EMT is regulated by a complex network of factors that includes cytokines, growth factors, aberrant signaling pathways, transcription factors, and the tumor microenvironment. There is emerging evidence that the EMT process may give rise to CSCs, or at least cells with stem cell-like properties. We review the key pathways involved in both of these processes, the biomarkers used to identify CSCs, and new therapeutic approaches targeting CSCs and EMT in pancreatic ductal adenocarcinoma.

Introduction: Cyclic AMP (cAMP) response element binding (CREB) overexpression in pancreatic ductal adenocarcinoma (PDAC) is associated with poor outcome, however, the mechanism(s) driving CREB overexpression in PDAC are not fully understood. We investigated the association of CREB activation with oncogenic KRAS, MEK-ERK and AKT signaling pathways. Experimental procedure: Mouse lines derived from the Ptf1aCre/+; LSL-KrasG12D/+ (K518), Ptf1acre/+;LSL-KrasG12D/+;Tgfbr2flox/flox (PKT), LSL-KrasG12D/+;Pdx1Cre/+ (PanIN) and LSL-KrasG12D/+; Trp53R172H/+;Pdx1Cre/+ (PDA) mouse models of PDAC, human immortalized pancreatic ductal epithelial lines (HPDE6-E6E7 or H6c7, HPNE E6/E7 and HPNE E6/E7/KRAS) were immunoblotted for phospho-CREB (pCREB) and total CREB expression. KRAS mutant PDAC cell lines and CREB shRNA flank xenografts (PKT GEMM and nude mice) were treated with CREB (ICG-001), MEK (AZD6244) and AKT (MK-2206) inhibitors. The effects on downstream signaling targets were interrogated with cell cycle, apoptosis, survival and anchorage-independent growth analysis. Human CREB shRNA cells were treated with AZD6244 and MK-2206 to confirm the molecular mechanism of the CREB inhibition together with MEK and AKT. Results: The expression of pCREB was higher in cells with KRAS mutation. MEK inhibition resulted in activation of AKT, while combined inhibition of CREB and MEK prevented AKT reactivation. Treatment with the combination of MEK and CREB inhibitors significantly decreased tumorigenic potential and increased cell apoptosis. Combined MEK, AKT and CREB inhibition synergistically enhanced these effects further, more so in KRAS mutant cell lines. To explore the relationship of CREB, MEK and AKT signaling in vivo, PKT mice were treated with their respective inhibitors individually and in combination. Either CREB/MEK or CREB/AKT two drug combinations significantly extended the median survival compared with individual agents. Lysates from MEK and AKT inhibited tumors showed decreased phosphorylation of CREB, confirming that CREB is activated through both, MEK and AKT signaling in vivo. Conclusions: Our study demonstrates that oncogenic KRAS activation enhances the expression of CREB through MEK and AKT signaling. CREB inhibition results in increased sensitivity to MEK and AKT targeted therapy in KRAS mutant PDACs. Citation Format: Jason Castellanos, Supriya Srinivasan, Kumar Honnenahally, Chanjuan Shi, Michael VanSaun, David Robbins, Nipun Merchant, Nagaraj Nagathihalli. Oncogenic mutant KRAS modulates CREB activation through MEK-ERK and AKT signaling in pancreatic cancer. [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 2618.

Pancreatic cancer is an aggressive solid malignancy with poor response to therapy and the subsequent dismal survival rate has remained a hallmark of this disease. There is evidence to indicate that pancreatic cancer is initiated and propagated by cancer stem cell (CSC)s. The CSC population is defined by its tumor initiating capacity and has been shown to be invasive or metastatic. Loss of genome stability is a hallmark of cancer with DNA repair enzymes aiding in maintenance of stability. The potential to assess the risk of cancer development lies in careful determination of one’s capacity in nurturing genome stability. DNA repair genes are over expressed in CSCs and both pancreatic CSCs and invasive cells in turn provide greater DNA damage response and repair mechanisms. Pancreatic tumor-initiating cells as well as invasive cells have a large number of genes related to DNA repair. RAD51, the key player in the recombinational repair of damaged DNA might act as a critical mediator of efficient DNA repair mechanisms of CSCs. We update here the current research results regarding CSCs in pancreatic cancer progression, metastasis and discuss the DNA repair mechanism in pancreatic CSCs.

Background & Aims A hallmark of pancreatic ductal adenocarcinoma (PDAC) is the presence of a dense desmoplastic reaction (stroma) that impedes drug delivery to the tumor. Attempts to deplete the tumor stroma have resulted in formation of more aggressive tumors. We have identified signal transducer and activator of transcription (STAT) 3 as a biomarker of resistance to cytotoxic and molecularly targeted therapy in PDAC. The purpose of this study is to investigate the effects of targeting STAT3 on the PDAC stroma and on therapeutic resistance. Methods Activated STAT3 protein expression was determined in human pancreatic tissues and tumor cell lines. In vivo effects of AZD1480, a JAK/STAT3 inhibitor, gemcitabine or the combination were determined in Ptf1a cre/+ ;LSL-Kras G12D/+ ;Tgfbr2 flox/flox (PKT) mice and in orthotopic tumor xenografts. Drug delivery was analyzed by matrix-assisted laser desorption/ionization imaging mass spectrometry. Collagen second harmonic generation imaging quantified tumor collagen alignment and density. Results STAT3 activation correlates with decreased survival and advanced tumor stage in patients with PDAC. STAT3 inhibition combined with gemcitabine significantly inhibits tumor growth in both an orthotopic and the PKT mouse model of PDAC. This combined therapy attenuates in vivo expression of SPARC, increases microvessel density, and enhances drug delivery to the tumor without depletion of stromal collagen or hyaluronan. Instead, the PDAC tumors demonstrate vascular normalization, remodeling of the tumor stroma, and down-regulation of cytidine deaminase. Conclusions Targeted inhibition of STAT3 combined with gemcitabine enhances in vivo drug delivery and therapeutic response in PDAC. These effects occur through tumor stromal remodeling and down-regulation of cytidine deaminase without depletion of tumor stromal content.

Chemotherapy is a very important therapeutic strategy for cancer treatment. The failure of conventional and molecularly targeted chemotherapeutic regimes for the treatment of pancreatic cancer highlights a desperate need for novel therapeutic interventions. Chemotherapy often fails to eliminate all tumor cells because of intrinsic or acquired drug resistance, which is the most common cause of tumor recurrence. Overexpression of RAD51 protein, a key player in DNA repair/recombination has been observed in many cancer cells and its hyperexpression is implicated in drug resistance. Recent studies suggest that RAD51 overexpression contributes to the development, progression and drug resistance of pancreatic cancer cells. Here we provide a brief overview of the available pieces of evidence in support of the role of RAD51 in pancreatic tumorigenesis and drug resistance, and hypothesize that RAD51 could serve as a potential biomarker for diagnosis of pancreatic cancer. We discuss the possible involvement of RAD51 in the drug resistance associated with epithelial to mesenchymal transition and with cancer stem cells. Finally, we speculate that targeting RAD51 in pancreatic cancer cells may be a novel approach for the treatment of pancreatic cancer. (C) 2011 Elsevier B.V. All rights reserved.

Introduction: Nicotine and nitrosamine exposure from smoking causes pancreatic cell injury and contributes to a cascade of oncogenic events that may be contributing to the rising rate of pancreatic cancer (PDAC). Cytokines activate kinases and transcription factors including cyclic AMP response element binding (CREB) protein. CREB activation through phosphorylation regulates diverse cellular responses. We studied whether granulocyte-macrophage colony stimulating factor (GM-CSF)-dependent phosphorylated CREB plays a role in smoking-induced pathogenesis of PDAC. Experimental procedure: Human tissue microarray analysis was performed to determine the significance of pCREB expression amongst smokers and non-smokers. Total RNA extracted from immortalized human pancreatic ductal cell (H6c7) and pancreatic intraepithelial neoplasia (PanIN) mouse cell lines (LSL-KrasG12D/+; Pdx1Cre/+) were treated with tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and RNA sequencing (RNA-seq) was performed. Network, pathway and functional analyses of the transcriptome were conducted. To analyze the signaling pathway involved in NNK-induced tumorigenicity, we performed a phospho-kinase antibody array and cytokine antibody array on NNK treated H6c7 and PanIN cells or conditioned media (CM) protein. Western blot and ELISA were used to validate the array data findings. NNK-induced activation of GM-CSF, G-CSF and IL-6 were blocked using monoclonal antibodies. PKT (Ptf1acre/+;LSL-KrasG12D/+;Tgfbr2flox/flox) mice and nude mice with MiaPaCa2 flank xenografts were treated with MEK inhibition (AZD6244) with or without NNK/in vivo smoking. Tumors from these mice were immunoblotted for phosphorylation of MEK1/2, MSK1/2, RSK and CREB. PDAC cells with CREB siRNA or human rGM-CSF were treated with NNK and analyzed for in vitro functional assays and EMT characteristics. Results: Expression of pCREB was significantly higher (p Conclusions: Our study demonstrates that NNK induces pancreatic tumorigenesis through GM-CSF mediated activation of CREB. Citation Format: Jason Castellanos, Kumaraswamy Honnenahally, Chanjuan Shi, Nipun Merchant, Nagaraj Nagathihalli. GM-CSF induces CREB signaling pathways and modulates tobacco carcinogen-induced pancreatic tumorigenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 924. doi:10.1158/1538-7445.AM2015-924

Introduction: Pancreatic ductal adenocarcinoma (PDAC) is one of the most difficult human malignancies to treat due to its innate and acquired therapeutic resistance. Our hypothesis is that cyclin-dependent kinase 4 (CDK4) mediates therapeutic resistance to targeting of the KRAS-MAPK pathway in PDAC. Experimental Procedure: We characterized the expression of total and phosphorylated Retinoblastoma (Rb) and MEK protein levels in KRAS wild-type (BxPC3) and KRAS mutant (PANC1, MiaPaca2) human PDAC cell lines at baseline and with MEK and CDK4 inhibition alone or in combination. We then assessed the effects of combined therapy on cell-cycle progression and tumorigenicity (in vitro and in vivo). Finally, using the Ptf1a cre/+;LSL-Kras G12D/+;Tgfbr2 flox/flox (PKT) mouse model of PDAC, we assessed the in vivo overall tumor growth and survival after combined treatment with CDK4/6 and MEK inhibitors. Results: Rb functions as a tumor suppressor, and it is inactivated when phosphorylated by CDK4-Cyclin-D1. The CDK4/6 inhibitor (LEE011) effectively inhibits phosphorylation of Rb in cell lines regardless of KRAS mutational status. Combined inhibition of CDK4/6 and MEK (MEK162) decreased phosphorylation of RB and MAPK expression synergistically in the KRAS mutant cell lines, but not the KRAS wild-type BxPC3 cell line. Cell cycle progression was delayed effectively with MEK inhibition alone in the KRAS wild-type cell line, yet only combined CDK4/6 and MEK inhibition effectively delayed cell cycle progression in the KRAS mutant cell lines. Colony formation and invasion were also significantly decreased when Kras mutant cells were treated with combined CDK4/6 and MEK inhibition compared to all monotherapy and control groups. Finally, treatment of PKT mice resulted in a modest increase in OS with MEK inhibition alone, but mice receiving combined CDK4/6 and MEK inhibition exhibited a four-fold increase in OS. Conclusions: Combined inhibition of CDK4/6 and MEK results in significantly enhanced therapeutic efficacy and prolonged survival in the aggressive PKT mouse model of PDAC. This study suggests that concurrent inhibition of CDK4/6 and MEK may be an effective treatment for PDAC. Citation Format: Jason A. Castellanos, Nagaraj Nagathihalli, Michael N. Van Saun, Cameron Kasmai, Yanhua Xiong, Nipun Merchant. CDK4/6 inhibition synergizes with KRAS-MAPK pathway targeting in pancreatic cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2537. doi:10.1158/1538-7445.AM2015-2537

Introduction: Cigarette smoking is an important risk factor for the development pancreatic ductal adenocarcinoma (PDAC). Nicotine and nitrosamines cause pancreatic cell injury and contribute to a cascade of oncogenic events. Among multiple tobacco specific nitrosamines, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is considered to be the most carcinogenic and induces DNA double strand-breaks (DSBs). However, identification of potential prognostic biomarkers and experimental models of smoking-induced PDAC remain unknown. We have studied phosphorylated histone H2AX (γH2AX), a marker for double-stranded DNA breaks, as a new biomarker for detecting cigarette smoke-induced pathogenesis of PDAC. Experimental Procedure: We have characterized the expression of γH2AX protein in human pancreatic tissues, human pancreatic duct epithelial (HPDE)6, PDAC cell lines, and PanIn, primary PDAC (PDA) and liver metastasis (LMP) cell lines generated from a genetically engineered mice model of PDAC. Kaplan-Meier survival analysis was performed to determine the significance of γH2AX expression amongst smokers and non-smokers with PDAC. HPDE6 and PanIN cells were cultured for 4 days in the presence or absence of cigarette smoke (CS) (0-25μg/ml) or NNK (0-10μM). Cell cycle analysis and apoptosis were assessed through flow cytometry with the markers propidium iodide and annexin V respectively. HPDE6 and PanIN cells treated with CS or NNK for up to 50 days were analyzed for rapid activation of DNA damage response (DDR) pathway proteins γH2AX, RAD51, BRCA1, BRCA2, phospho-CHK2 and phospho-CHK1. Cells were treated with either CS or NNK along with a CHK2 inhibitor and analyzed for activation of γH2AX. To determine DNA integrity, accumulation of γH2AX was measured by ELISA in cells treated with CS or NNK. Immunofluorescence confocal microscopy was utilized to examine the role of γH2AX on nuclear foci formation with DNA repair complexes by quantifying the amount of co-localization. To replicate chronic smoke exposure, we treated HPDE6 and PanIn cells with 1 and 10 µg/ml CS or 10nM and 1µM NNK for 10 passages, and examined these cell lines for DSBs by comet assay, apoptosis markers by protein array and in vitro tumorigenicity by colony formation assay. Results: γH2AX expression was significantly higher (p Conclusion: This work supports the role of γH2AX as a new molecular biomarker to assess the contribution of cigarette smoking-induced PDAC. γH2AX is necessary for DNA damage repair in PDAC and its activation is dependent on CHK2. Therapies that can block γH2AX expression by inhibiting upstream kinases may be a new potential therapeutic approach to treat and/or prevent PDAC. Citation Format: Kumaraswamy Honnenahally, Chanjuan Shi, Xi Chen, Jason Castellanos, Nipun Merchant, Timothy Blackwell, Nagaraj Nagathihalli. γH2AX: A molecular marker of DNA damage response in smoking-induced pancreatic ductal adenocarcinoma. [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 A33.

Background: Pancreatic cancer is the fourth leading cause of cancer death with a five year survival rate around 5%, which has not changed in 30 years. Obesity and increased abdominal adipose tissue independently correlate with an increased relative risk for the development of pancreatic cancer. These conditions have been associated with altered levels of adipokines, or adipose secreted cytokines. Circulating serum levels of the adipokine leptin are increasesddramatically in obese patients as well as in high fat diet induced obese mice. Leptin has been shown to induce oncogenic signaling in breast and prostate cancer. We have previously shown an increase in orthotopic pancreatic tumor size in high fat diet induced obese mice compared with regular diet control mice. We hypothesize that leptin signaling mediates pancreatic tumorigenesis. Methods: Leptin receptor status was determined in human as well as murine pancreatic cell lines. Leptin stimulated cell proliferation was determined using a modified BrdU assay. Leptin receptor levels were knocked down in human and murine pancreatic tumor cells using a shRNAmir approach. Leptin receptor shRNA Panc02 knockdown cells were injected orthotopically into the pancreas of C57/Bl6J mice on regular or high fat diet to determine the contribution of leptin to pancreatic tumor growth. Results: We have detected the long form of the leptin receptor in five human and four murine pancreatic cancer cell lines. In vitro administration of leptin stimulated proliferation of Panc1 and CFPAC1 cell lines, which was abrogated with co-incubation of a leptin antagonist. To better understand the mechanism of leptin-mediated signaling, we studied downstream targets and identified a significant increase in phosphorylation of STAT3 in Panc1, BXPC3 and CFPAC1 cell lines after leptin treatment. Orhtotopic injection of leptin receptor shRNA Panc02 cells into normal and obese mice showed a markedly diminished tumor growth in obese mice when compared to the nonsilencing control Panc02 cell growth in obese mice. Conclusion: These results implicate leptin as a mediator of pancreatic tumorigenesis and suggest that leptin activation is mediated in part through STAT3 signaling. Knockdown of the leptin receptor results in inhibition of high fat diet associated tumor growth in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 299. doi:1538-7445.AM2012-299

181 Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most difficult human malignancies to treat due to its intrinsic (de novo) and extrinsic (acquired) chemoresistance. We have previously identified constitutively activated STAT3 as a mediator of treatment resistance. Src or EGFR activate STAT3 and promote STAT3 mediated tumor progression and mediate communication within the tumor microenvironment (TME). The purpose of this study was to further understand the molecular mechanisms of stromal-mediated chemoresistance in PDAC to generate new and promising targeted therapies. Methods: We characterized the expression of total and activated STAT3 and MAPK proteins in human pancreatic tissues (n=106), PDAC cell lines (n=9) and in PanIn lesions, primary PDAC and liver metastasis cell lines generated from tumors established in genetically engineered mice. Effects of STAT3 and MAPK inhibition (drug or siRNA) were assessed for phosphorylation of STAT3, Src, MAPK, EGFR and GSK3β and expression of cyclin-D1, SPARC, VEGF, fibronectin, CD31 and tumorigenicity in vitro and in vivo. Results: STAT3 activation is necessary for the malignant phenotype and affects survival in PDAC. In both human and mouse PDAC cell lines and tissues, there is an inverse correlation between activation of STAT3 on MAPK and GSK3β signaling. Inhibition of STAT3 reciprocally activated MAPK, GSK3β, Src and EGFR which led to subsequent reactivation of STAT3. Targeting both STAT3 and MAPK inhibited activation of STAT3, MAPK, GSK3β, Src, EGFR and cyclin-D1. Combined inhibition of STAT3 and MAPK overcame STAT3 mediated resistance and resulted in synergistic inhibition of tumorigenicity as well as inhibition of the tumor stroma, angiogenesis and hypoxia within the TME. Conclusions: The mechanism of STAT3-mediated treatment resistance is dependent on activation of MAPK signaling which in turn leads to reactivation of multiple oncogenic signaling pathways. Combined inhibition of STAT3 and MAPK overcomes therapeutic resistance, targets the TME and reduces tumorigenicity in PDAC. Targeting STAT3 and MAPK is a potent treatment regimen and may be a novel approach to enhance drug delivery and improve therapeutic response in PDAC.

The Src family of non receptor tyrosine kinases are integrators of divergent signal transduction pathways which regulate numerous cellular processes, including tumorigenicity and angiogenesis. In pancreatic adenocarcinoma, c-Src (Src) is frequently activated and results in increased tumor progression, invasion and metastasis. Dysfunction of the E-cadherin-mediated cell adhesion system plays an important role in tumor progression to invasive, metastatic carcinoma. Src has been shown to play a role in E-cadherin regulation and epithelial to mesenchymal transition (EMT). Increased Src activity promotes EMT while Src inhibition suppresses this process. Recent studies have focused on Src dependent regulation of E-cadherin and other tumor progression-related events such as EMT with the development of metastasis. Src has also been shown to be involved in chemoresistance of PDAC cells by promoting EMT. Although the molecular events associated with Src-dependent regulation of E-cadherin are becoming better defined, the cellular processes that trigger the onset of EMT remain unclear. Here we highlight recent work that advances our understanding of Src signaling as it relates to E-cadherin associated regulation and EMT in PDAC.