Human PDAC Cell Lines Research Articles

Cannabidiol and Oxygen-Ozone Combination Induce Cytotoxicity in Human Pancreatic Ductal Adenocarcinoma Cell Lines

Simple SummaryPancreatic cancer (PC) is related to lifestyle risks, chronic inflammation, and germline mutations. Surgical resection and adjuvant chemotherapy are the main therapeutic strategies but are less effective in patients with high-grade tumors. Oxygen-ozone (O2/O3) therapy is an emerging alternative tool for the treatment of several clinical disorders. The advantages of using cannabinoids have been evaluated in several human cancers. Regarding PC, activation of cannabinoid receptors was found to induce PC cell apoptosis without affecting the normal pancreas cells. Herein, we evaluate the anticancer effect of cannabidiol (CBD) and O2/O3, alone or in combination, on two human pancreatic ductal adenocarcinoma (PDAC) cell lines, PANC-1 and MiaPaCa-2, examining expression profiles of 92 pancreatic adenocarcinoma associated genes, cytotoxicity, migration properties, and cell death. Finally, we assess the combination effects with gemcitabine and paclitaxel. Summarizing, for the first time the antitumoral effect of combined therapy with CBD and oxygen-ozone therapy in PDAC is evidenced.Pancreatic cancer (PC) is related to lifestyle risks, chronic inflammation, and germline mutations in BRCA1/2, ATM, MLH1, TP53, or CDKN2A. Surgical resection and adjuvant chemotherapy are the main therapeutic strategies but are less effective in patients with high-grade tumors. Oxygen-ozone (O2/O3) therapy is an emerging alternative tool for the treatment of several clinical disorders. O2/O3 therapy has been found to ameliorate mechanisms promoting chronic pain and inflammation, including hypoxia, inflammatory mediators, and infection. The advantages of using cannabinoids have been evaluated in vitro and in vivo models of several human cancers. Regarding PDAC, activation of cannabinoid receptors was found to induce pancreatic cancer cell apoptosis without affecting the normal pancreas cells. In a murine model of PDAC, a combination of cannabidiol (CBD) and gemcitabine increased survival length by nearly three times. Herein, we evaluate the anticancer effect of CBD and O2/O3, alone or in combination, on two human PDAC cell lines, PANC-1 and MiaPaCa-2, examining expression profiles of 92 pancreatic adenocarcinoma associated genes, cytotoxicity, migration properties, and cell death. Finally, we assess the combination effects with gemcitabine and paclitaxel. Summarizing, for the first time the antitumoral effect of combined therapy with CBD and oxygen-ozone therapy in PDAC is evidenced.

Abstract 5108: Investigation of how extracellular matrix composition affects pancreatic cancer chemoresistance through the use of biomimetic models

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal and malignant type of cancer with few effective treatments. Despite recent advancement in knowledge of cancer treatment, median survival of PDAC patients remains within 2 years, and less than 8% patients live beyond 5 years after diagnosis. The main factor that drives PDAC lethality is the development of chemoresistance in nearly all clinical PDACs. While intrinsic factors that facilitates chemoresistance have been studied extensively, few studies have illustrated how complex and unique microenvironment surrounding the PDAC tumor affects therapeutic responses. Moreover, a physiologically relevant co-culture model of PDAC and stromal cells remained challenging. To address this issue, we have developed a Matrigel-based, orthogonally tunable 3-dimensional (3D) culture system to culture established human PDAC cell lines, cancer-associated fibroblast (CAF) cells and mouse model-derived organoids. Individual matrix parameters can be customized to mimic actual tumor matrix composition and mechanical properties. Moreover, we have optimized a co-culture medium to maintain viability of both organoid tumor and cancer associated fibroblasts. Through our 3D culture system, our data suggests that matrix-induced exosome secretion directly facilitate gemcitabine resistance in PDAC cells. By directly increasing stiffness of our 3D culture platform, we have observed that PDAC cells potentially display more invasive phenotype. Moreover, in the co-culture systems, we have seen that CAF cells cultured in different stiffness substrates affect PDAC drug response in organoid cultures. Our investigation provides insight into how matrix composition and stiffness together affect therapeutic outcome in PDAC. We believe that specific therapeutic strategies aimed at targeting the matrix microenvironment will be instrumental in order to overcome matrix-mediated chemoresistance in PDAC. Citation Format: Weikun Xiao, Chae-Young Eun, Charlene Dekalb, Kexin Zhang, Reginald Hill. Investigation of how extracellular matrix composition affects pancreatic cancer chemoresistance through the use of biomimetic models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5108.

ENO1-targeted superparamagnetic iron oxide nanoparticles for detecting pancreatic cancer by magnetic resonance imaging.

The aim of this study was to investigate in vitro magnetic resonance imaging (MRI) of PDAC using ENO1‐targeted superparamagnetic iron oxide nanoparticles and xenograft models. Expression level and location of ENO1 protein in pancreatic cancer cell lines of CFPAC‐1 and MiaPaCa‐2 were detected by Western blotting, flow cytometry and confocal microscopy. Dex‐g‐PCL/SPIO nanoparticles targeting ENO1 were constructed with ENO1 antibody and characterized by MRI. In addition, ENO1‐Dex‐g‐PCL/SPIO nanoparticles were tested to assess their efficacy on the detection of PDAC using in vitro and in vivo MRI. The results showed that ENO1 was expressed in both human PDAC cell lines of CFPAC‐1 and MiaPaCa‐2, demonstrating that the localization of cytoplasm and membrane was dominant. It was confirmed that ENO1 antibody was connected to the SPIO surface in ENO1‐Dex‐g‐PCL/SPIO nanoparticles. The nanoparticles had satisfactory superparamagnetism and significantly enhance the detection of PDAC by in vivo and in vitro MRI. In conclusion, ENO1 can serve as a membrane protein expressed on human PDAC cell lines. ENO1‐targeted SPIO nanoparticles using ENO1 antibody can increase the efficiency of detection of PDAC by in vitro and in vivo MRI.

MicroRNA-122-5p inhibits cell proliferation, migration and invasion by targeting CCNG1 in pancreatic ductal adenocarcinoma

BackgroundPancreatic ductal adenocarcinoma (PDAC) is a lethal human malignancy, and previous researches support the contribution of microRNA (miRNA) to cancer progression. MiR-122-5p is reported to participate in the regulation of various cancers, while the function of miR-122-5p in PDAC remains unclear. In this study, we investigated the precise mechanism of miR-122-5p involved in PDAC pathogenesis.MethodsThe expression levels of miR-122-5p were detected in human PDAC tissues and cell lines by miRNA RT-PCR. The effects of miR-122-5p on cell proliferation were explored by MTT assays, colony formation assays and flow cytometry assays. The ability of migration and invasion was determined by transwell assays. Dual Luciferase reporter assay was performed to validate the direct interaction between miR-122-5p and its target gene. The related molecules of cell cycle, apoptosis and epithelial–mesenchymal transition (EMT) were examined with qRT-PCR and western blot. In addition, xenograft mouse models were applied to explore the effects of miR-122-5p in vivo.ResultsMiR-122-5p was underexpressed, while CCNG1 was highly expressed in PDAC tissues and cells. MiR-122-5p was negatively correlated with TNM stage, tumor size and lymph node metastasis in PDAC patients. Overexpression of miR-122-5p suppressed the proliferation, migration and invasion in vitro and inhibited tumorigenesis in vivo. Furthermore, CCNG1 was a direct target of miR-122-5p. Upregulated CCNG1 could partially reverse the effects caused by miR-122-5p. Moreover, miR-122-5p inhibited EMT through downregulation of CCNG1.ConclusionOverexpression of miR-122-5p could inhibit cell proliferation, migration, invasion, and EMT by downregulating CCNG1 in PDAC, suggesting a potential therapeutic target for PDAC.

Abstract C11: Linoleic acid elevates sirtuin 6 expression and modulates glycolysis and epithelial-mesenchymal transition of pancreatic cancer with loss of Krüpple like factor 10

Abstract Introduction: Pancreatic adenocarcinoma (PDAC) is well known for its deregulated TGFβ signaling. TGFβ signaling exerts a contextual effect that suppresses malignant growth early in epithelial tumorigenesis but promotes metastasis at later stages. The Krüppel-like factor 10 (Klf10) is an early response gene that mediates the antiproliferative effects of TGFβ but opposes the prometastatic effects of TGFβ by limiting its ability to induce epithelial-to-mesenchymal transition (EMT). The distinction between the antiproliferative versus prometastatic functions of TGFβ makes Klf10 a potential target for the development of therapy in cancers with aberrant TGFβ pathway such as PDAC. Klf10 knock-out mice do not have abnormal pancreas development or carcinogenesis. However, ablating Klf10 in murine PDAC models increases pancreatic tumorigenesis and distant metastasis. Genome-wide comparison of mRNA profiles from wild-type and Klf10 knock-out mice revealed significant enrichment for transcripts involved in glucose metabolism. We propose that Klf10 modulates PDAC progression via enhanced glycolysis, which leads to EMT and cancer stemness phenotypes. Materials and Methods: Genetically manipulated Klf10 of human PDAC cell lines was established to measure EMT markers, glycolysis activity and stemness related proteins, cell migration, and murine liver metastasis after intrasplenic injection. Chip-Chip assay was performed to find sirtuin 6 (Sirt6), governing both cancer metabolism and progression, to be one of the candidate molecules regulated by Klf10. The interaction between Klf10 and Sirt6 was evaluated by ChIP-PCR and luciferase reporter assay. PDAC cell lines with or without Klf10 mRNA silencing were overexpressed with Sirt6 to reverse malignant phenotypes induced by Klf10 deficiency. Linoleic acid, a specific Sirt6 activator, was used in vitro and in vivo to ameliorate the aberrant glucose metabolism, EMT phenotype, and distant metastasis of PDAC with loss of Klf10 expression. Results: In vitro and in vivo studies demonstrated Klf10 deficiency in pancreatic cancer enhanced metastasis, EMT, stemness, and glycolysis. Klf10 bond to the promoter and transcriptionally regulated Sirt6. Sirt6 expression paralleled the level of Klf10 in PDAC. Modulating Sirt6 genetically or pharmacologically, by linoleic acid, reversed the EMT, glycolysis, and metastatic phenotypes of PDAC with KLF10 deficiency. Using PDK1, a glycolysis inhibitor, and BSI#38, an EMT inhibitor, we demonstrated the interaction of cancer metabolism and EMT phenotypes in PDAC. Discussion: Loss of Klf10 contributes to pancreatic cancer progression and enhances glycolysis, EMT, and cancer stemness by transcriptionally regulating Sirt6. Overexpressing Sirt6 genetically or pharmacologically by linoleic acid may ameliorate PDAC progression. Targeting Klf10/Sirt6 signaling is a potential strategy to modulate glycolysis and EMT simultaneously in PDAC for preventing malignant progression. Citation Format: Hui-Ju Ch'ang, Yi-Chih Tsai, Su-Liang Chen, Vincent H.S. Chang. Linoleic acid elevates sirtuin 6 expression and modulates glycolysis and epithelial-mesenchymal transition of pancreatic cancer with loss of Krüpple like factor 10 [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 C11.

Abstract 454: Transferrin receptor identifies a novel circulating tumor cell population in patients with pancreatic cancer with a unique metastasis-associated molecular signature

Abstract Pancreatic Ductal Adenocarcinomas (PDAC) is the fourth most common cancer, expected to be the second leading cause of cancer mortality by 2030. Most PDAC patients are diagnosed with metastatic disease, with dismal prognosis of less than 1% 5-year survival. A major challenge in PDAC treatment is the absence of actionable molecular targets and the scarcity of available tumor tissue impeding molecular understanding of PDAC progression. Circulating Tumor Cells (CTCs) provide an easily accessible source for tumor tissue. CellSearchTM is the only FDA-cleared platform for CTC isolation based on positive selection of EpCAM+ cells. However, this method performs poorly in PDAC failing to show any prognostic relevance, likely due to EpCAM down-regulation during epithelial-to-mesenchymal transition (EMT), which precedes metastasis. To overcome this limitation, we tested Transferrin Receptor 1 (TfR) in the identification and isolation of CTCs from PDAC patients. TfR is a cell surface protein that mediates iron uptake, is overexpressed in multiple tumors with sustained expression throughout EMT, making it a suitable candidate for CTC isolation. To test this hypothesis, we first assessed TfR expression in a panel of 9 human PDAC cell lines, and we observed that TfR was expressed in 9/9 (100%) in contrast to 6/9 (66.7%) being EpCAM positive. No TfR expression was detected in healthy donor and PDAC patients’ PBMCs. To assess the performance of TfR in CTC identification and isolation from the peripheral blood of PDAC patients, we collected samples from 37 PDAC patients with stage 4 disease, enriched CTCs by negative CD45 depletion (RossetteSepTM) and subsequently stained live CTCs with TfR, EpCAM, and CD45. CTCs were defined as CD45- cells, positive for TfR+ or EpCAM+. We observed that the number of TfR+ CTCs (median: 148, range 2-4182) was significantly higher than EpCAM+ CTCs (median: 68, range 0-1552) within the same patient and across different patients (P-value 0.007). In addition, serial sampling at baseline (chemotherapy-naïve) and at disease progression from 5 PDAC patients revealed that TfR+CTC enumeration correlated with disease progression radiologically and/or CA19-9 (PDAC tumor marker) levels. In contrast, there was no correlation between the number of EpCAM+CTCs and clinical outcomes. RNA-Sequencing of isolated pools of TfR+ or EpCAM+ CTCs, followed by gene set enrichment analysis, revealed significant enrichment in oncogenic pathways, such as EMT, G2M, MYC and KRAS in TfR+ CTCs as compared with EpCAM+ CTCs. Taken together, our results reveal that TfR identifies a clinically relevant CTC subpopulation which correlates with disease progression and is molecularly distinct. Ongoing studies are focused on the function of TfR in disease progression and the potential use of TfR+ CTC molecular profiles in personalized PDAC treatment. Citation Format: Ahmed Halima, Jiaren Zhang, Giuseppe Galletti, Allyson J. Ocean, Paraskevi Giannakakou. Transferrin receptor identifies a novel circulating tumor cell population in patients with pancreatic cancer with a unique metastasis-associated molecular signature [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 454.

Abstract 4836: Ly6-neurotoxin1 is a potential target in pancreatic ductal adenocarcinoma cells

Abstract Ly6/neurotoxin1 (Lynx1) is a cholinergic transmission modulator, which was described as a tumor suppressor in lung cancer. In addition, the expression of Lynx1 was downregulated in rat PDAC cells colonizing rat liver. As cholinergic signaling via the vagus nerve may slow pancreatic tumor progression, we investigated the role of Lynx1 in human PDAC cell lines. Transient gene knockdown (KD) by siRNA was used to modulate the expression of Lynx1 in BXPC3 and Miapaca cells and KD was confirmed by qRT-PCR and western blot (WB). Resulting effects were determined by respective assays for proliferation, migration and colony formation. Induction of apoptosis was assessed by Hoechst and Annexin V-FITC staining. Induction of autophagy was determined by acridine orange and immunofluorescence (IF) staining for LC3b. Finally, we performed microarrays for gene expression in cells with Lynx1 KD and analyzed by WB cancer pathways of related signaling molecules. Following successful KD of Lynx1, the response of Miapaca and BXPC3 cells was altered regarding proliferation (-60% and -10%), migration (+200% and -10%) at 72 h after transfection and colony formation (-50% and -40%) at 9 d after transfection. Hoechst staining revealed increased rates of apoptosis in both cell lines. This was confirmed by Annexin V assay in Miapaca cells and these changes were associated with significantly reduced BCL2 levels. Autophagy was increased in both cell lines as were LC3b levels detected by IF. Alterations in mRNA expression, as assessed by microarray, were analyzed by IPA software using a 1.5 fold cutoff. Miapaca cells showed significant activation of 43 pathways including phospholipase C signaling, cholecystokinin/gastrin mediated signaling, and cell cycle regulation. In addition, five pathways showed uniform downregulation, i.e estrogen mediated S phase entry, sirtuin signaling, small cell lung cancer signaling, cyclins and cell cycle regulation, and ataxia-telangiectasia-mutated (ATM) signaling. In contrast to the more sensitive Miapaca cells, BXPC3 cells showed modulation of few pathways only: activation of G protein beta gamma signaling and osteoarthritis pathways, as well as downregulation of the sirtuin signaling pathway. At protein level, the mTOR pathway was downregulated in both cell lines (including phosphorylated forms of mTOR, Rictor, Raptor, PRAS40), as were the upstream regulators PI3K and p-AKT. In conclusion, KD of Lynx1 caused changes in gene product levels, which are related to cholinergic signal transmission as well as to DNA damage and repair systems. The observed decreased colony formation in Lynx1 KD cells is also indicative of defective cholinergic signaling. Reduced mTOR pathway signaling caused induction of autophagy. These findings suggest that Lynx1 is beneficial for regular cellular functions and its lack will contribute to increased apoptosis, DNA damage and autophagy. Citation Format: Doaa M. Ali, Nevena Nedyalkova, Michael Zepp, Martin R. Berger. Ly6-neurotoxin1 is a potential target in pancreatic ductal adenocarcinoma cells [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 4836.

Abstract 2052: A novel antibody blocks anti-apoptotic activity of MUC1 in pancreatic cancer cell lines

Abstract Introduction: The third leading cause of cancer-related deaths in the United States is pancreatic cancer, >95% of which is Pancreatic Ductal Adenocarcinoma or PDAC. MUC1 is a transmembrane glycoprotein expressed on normal glandular epithelial cells. In PDAC, MUC1 is overexpressed and aberrantly glycosylated and is designated tumor associated MUC1 (tMUC1). Over 80% of human PDAC expresses tMUC1. In a NCI initiated study, out of 75 tumor antigens, MUC1 was ranked the second most targetable antigen. Furthermore, the 72 amino acid cytoplasmic tail of MUC1 (MUC1 CT) is reported to aid in oncogenic signaling leading to tumor progression and metastasis by blocking apoptosis. A novel monoclonal antibody, TAB004, has been developed specifically against tMUC1. TAB004 detects tMUC1 with a high rate of specificity and sensitivity and spares recognition of normal epithelial MUC1. HYPOTHESIS: Treatment with TAB004 curbs PDAC cell survival by blocking MUC1 CT associated oncogenic signaling and renders the cells more susceptible to standard chemotherapy drugs. Methods: Several human PDAC cell lines based on their tMUC1 expression were grown in media with heat-inactivated serum to ensure that it is devoid of complement proteins. Cells were treated with various concentrations of TAB004 antibody, control IgG, 5-FU, paclitaxel, or gemcitabine and the IC50 was determined using the MTT or SRB cell survival assays. Once IC50 was determined, cells were treated with combinations of TAB004 and the chemotherapy drugs. To determine apoptosis, treated cells were stained with Annexin V-FITC and PI, and analyzed by flow cytometer. tMUC1 expression and localization was determined by staining cells with TAB004.Cy5.5 and imaged by confocal microscopy. Data analysis was performed using GraphPad Prism 7.0 and a p value of <0.05 was considered significant. Results: By itself, TAB004 treatment had minimal effect on most of the cell lines tested except for Capan2. The IC50 of TAB004 was ~400ugs/ml for Capan2. However, when combined with Gemcitabine, PTX, or 5-FU, TAB004 significantly increased anti-tumor efficacy of the drugs, especially 5-FU in Capan2, and 5-FU and gemcitabine, and PTX in BxPC3.MUC1. Annexin V-FITC and PI staining confirmed the MTT data in Capan2 cells. Fluorescent images showed that TAB004 internalizes in MUC1-positive PDAC cell lines but not in MUC1-negative cells. Conclusions: TAB004 has a dose-dependent, complement-independent growth-inhibitory effect on Capan2 cells (IC50 is ~400ug/ml). TAB 004 internalizes and activates apoptosis in Capan2 cells. The combination of TAB004 with chemotherapy drugs increases the efficacy of drug-induced killing of PDAC cells. Future studies will determine the mechanism of TAB004 induced apoptosis and sensitization of PDAC cells to drug-induced killing. Citation Format: Mukulika Bose, Pinku Mukherjee. A novel antibody blocks anti-apoptotic activity of MUC1 in pancreatic cancer cell lines [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 2052.

Targeting Purinergic Receptor P2Y2 Prevents the Growth of Pancreatic Ductal Adenocarcinoma by Inhibiting Cancer Cell Glycolysis.

Extensive research has reported that the tumor microenvironment components play crucial roles in tumor progression. Thus, blocking the supports of tumor microenvironment is a promising approach to prevent cancer progression. We aimed to determine whether blocking extracellular ATP-P2RY2 axis could be a potential therapeutic approach for PDAC treatment. Expression of P2RY2 was determined in 264 human PDAC samples and correlated to patient survival. P2RY2 was inhibited in human PDAC cell lines by antagonist and shRNA, respectively, and cell viability, clonogenicity, and glycolysis were determined. RNA sequencing of PDAC cell line was applied to reveal underlying molecular mechanisms. Multiple PDAC mouse models were used to assess the effects of the P2RY2 inhibition on PDAC progression. P2RY2 was upregulated and associated with poor prognosis in PDAC. Activated P2RY2 by increased extracellular ATP in tumor microenvironment promoted PDAC growth and glycolysis. Further studies showed that the agonist-activated P2RY2 triggered PI3K/AKT-mTOR signaling by crosstalk with PDGFR mediated by Yes1, resulting in elevated expression of c-Myc and HIF1α, which subsequently enhanced cancer cell glycolysis. Genetic and pharmacologic inhibition of P2RY2 impaired tumor cell growth in subcutaneous and orthotopic xenograft model, as well as delayed tumor progression in inflammation-driven PDAC model. In addition, synergy was observed when AR-C118925XX, the selective antagonist of P2RY2 receptor, and gemcitabine were combined, resulting in prolonged survival of xenografted PDAC mice. These findings reveal the roles of the P2RY2 in PDAC metabolic reprogramming, suggesting that P2RY2 might be a potential metabolic therapeutic target for PDAC.

The ATR Inhibitor AZD6738 Synergizes with Gemcitabine In Vitro and In Vivo to Induce Pancreatic Ductal Adenocarcinoma Regression.

Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest cancers, and overall survival rates have barely improved over the past five decades. The antimetabolite gemcitabine remains part of the standard of care but shows very limited antitumor efficacy. Ataxia telangiectasia and Rad3-related protein (ATR), the apical kinase of the intra-S-phase DNA damage response, plays a central role in safeguarding cells from replication stress and can therefore limit the efficacy of antimetabolite drug therapies. We investigated the ability of the ATR inhibitor, AZD6738, to prevent the gemcitabine-induced intra-S-phase checkpoint activation and evaluated the antitumor potential of this combination in vitro and in vivo In PDAC cell lines, AZD6738 inhibited gemcitabine-induced Chk1 activation, prevented cell-cycle arrest, and restrained RRM2 accumulation, leading to the strong induction of replication stress markers only with the combination. Moreover, synergistic growth inhibition was identified in a panel of 5 mouse and 7 human PDAC cell lines using both Bliss Independence and Loewe models. In clonogenic assays, the combination abrogated survival at concentrations for which single agents had minor effects. In vivo, AZD6738 in combination with gemcitabine was well tolerated and induced tumor regression in a subcutaneous allograft model of a KrasG12D; Trp53R172H; Pdx-Cre (KPC) mouse cancer cell line, significantly extending survival. Remarkably, the combination also induced regression of a subgroup of KPC autochthonous tumors, which generally do not respond well to conventional chemotherapy. Altogether, our data suggest that AZD6738 in combination with gemcitabine merits evaluation in a clinical trial in patients with PDAC. Mol Cancer Ther; 17(8); 1670-82. ©2018 AACR.

Abstract A18: The integrin αvβ6 regulates PDAC cell growth and stromal cell behavior

Abstract Pancreatic adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death in the UK. Five-year survival rates are less than 3% and haven’t improved over the last 40 years. Thus novel approaches are required urgently. We have found that over 90% of PDAC express αvβ6 and high expression of the integrin β6 gene (ITGB6) correlates with a significant reduction (Hazard Ratio 2.07; Logrank p=3.17x10-8) in overall survival from PDAC. Thus we have examined the biological functions mediated by αvβ6 in PDAC cell lines. A panel of human PDAC cell lines was incubated with αvβ6-blocking antibodies in order to define the role of αvβ6 in vitro. Some cell lines were driven into G2/M cell cycle arrest (Panc0403), whilst others died through apoptosis (CfPac1). Moreover, adhesion, migration and invasion through Matrigel were suppressed by blockade of αvβ6. Treatment of subcutaneous tumors composed of human αvβ6-positive cell lines in combination with human pancreatic stellate cells, with an αvβ6-blocking antibody resulted in significantly reduced tumor volume and growth rate in vivo, via modulation of the stromal compartment. In combination, these data suggested that targeting αvβ6 may be an effective therapeutic strategy in PDAC. To investigate the biological roles and molecular bases of αvβ6 in PDAC, cell lines were generated from PDAC tumors of a novel PDAC transgenic mouse. Two primary cell populations from PDAC tumors, abbreviated as KVC1 and KPCD3, demonstrated both epithelial and mesenchymal characteristics. Thus, cells were subsequently sorted by FACS to obtain high-αvβ6 expressing monoclonal cell lines. Resultant monoclonal cell lines expressed higher levels of αvβ6 than the parental line and expressed only epithelial markers. Transduction with lentivirus particles containing shRNA to itgb6 under a tetracycline-inducible promoter (SMARTvector, Dharmacon) was optimized for four high-αvβ6 expressing monoclonal cell lines, and doxycycline-induced knock-down of β6 protein was 70-80%, confirmed using FACS and western blotting. In vitro functional assays demonstrated that knock-down of itgb6 reduced invasion of Matrigel. To assess the role of αvβ6 in an assay that better reflected the PDAC environment, we developed 3D organotypic assays with inducible knockout PDAC combined with mouse embryo fibroblasts or mouse pancreatic stellate cells. Doxycycline treatment reduced PDAC tumor cell growth and invasion, but also reduced the contraction of the collagen-Matrigel gel matrix. Since αvβ6 activates TGFβ, and as TGFβ promotes fibroblast and stellate cell contractile propensity, current studies will determine whether this is the mechanism of how αvβ6 regulates stromal cell behavior. Citation Format: Claire S. Reader, Jennifer P. Morton, Owen J. Sansom, John F. Marshall.{Authors}. The integrin αvβ6 regulates PDAC cell growth and stromal cell behavior. [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 A18.

Loss of Trefoil Factor 2 From Pancreatic Duct Glands Promotes Formation of Intraductal Papillary Mucinous Neoplasms in Mice

Little is known about the origin of pancreatic intraductal papillary mucinous neoplasms (IPMN). Pancreatic duct glands (PDGs) are gland-like outpouches budding off the main pancreatic ducts that function as a progenitor niche for the ductal epithelium; they express gastric mucins and have characteristics of side-branch IPMNs. We investigated whether PDGs are a precursor compartment for IPMNs and the role of Trefoil factor family 2 (TFF2)-a protein expressed by PDGs and the gastric mucosa that are involved in epithelial repair and tumor suppression. We obtained pancreatectomy specimens from 20 patients with chronic pancreatitis, 13 with low-grade side-branch IPMNs, and 15 patients with PDAC; histologically normal pancreata were used as controls (n= 18). Samples were analyzed by immunohistochemistry to detect TFF1 and TFF2 and cell proliferation. We performed mitochondrial DNA mutational mapping studies to determine the cell lineage and fate of PDG cells. Pdx1-Cre;LSL-KRASG12D (KC) mice were bred with TFF2-knockout mice to generate KC/Tff2-/- and KC/Tff2+/- mice. Pancreata were collected and histologically analyzed for formation of IPMN, pancreatic intraepithelial neoplasias, and PDAC, in addition to proliferation and protein expression. Human pancreatic ductal epithelial cells and PDAC cell lines were transfected with vectors to overexpress or knock down TFF2 or SMAD4. Histologic analysis of human samples revealed gastric-type IPMN to comprise 2 molecularly distinct layers: a basal crypt segment that expressed TFF2 and overlying papillary projections. Proliferation occurred predominantly in the PDG-containing basal segments. Mitochondrial mutation mapping revealed a 97% match between the profiles of proliferating PDG cells and their overlying nonproliferative IPMN cells. In contrast to KC mice, 2-month-old KC/Tff2+/- and KC/Tff2-/- mice developed prominent papillary structures in the duct epithelium with cystic metaplasia of the PDG, which resembled human IPMN; these expressed gastric mucins (MUC5AC and MUC6), but not the intestinal mucin MUC2. KC/TFF2-knockout mice developed a greater number and higher grade of pancreatic intraepithelial neoplasias than KC mice, and 1 mouse developed an invasive adenocarcinoma. Expression of TFF2 reduced proliferation of PDAC cells 3-fold; this effect required up-regulation and activation of SMAD4. We found expression of TFF2 to be down-regulated in human PDAC by hypermethylation of its promoter. In histologic analyses of human IPMNs, we found PDGs to form the basal segment and possibly serve as a progenitor compartment. TFF2 has tumor-suppressor activity in the mouse pancreas and prevents formation of mucinous neoplasms.

Abstract 754: Targeting carbonic anhydrase IX in multiple pancreatic ductal adenocarcinoma models results in tumor growth inhibition and increased survival

Abstract Pancreatic ductal adenocarcinoma (PDAC) is characterized by aggressive growth, metastatic spread and poor 5 year survival rates of less than 5%. PDAC tumors develop extensive regions of hypoxia that contribute to its aggressive behavior and pose a significant hurdle to therapeutic response. Hypoxia-mediated adaptive responses for tumor cell survival and metastasis include metabolic reprogramming and pH regulation. Carbonic anhydrase IX (CAIX) is a membrane-bound, hypoxia-inducible enzyme that is highly expressed in solid tumors and functions as a critical component of the pH regulatory machinery required by hypoxic cancer cells for survival and metastasis. We have recently developed and validated novel small molecule inhibitors of CAIX and have demonstrated that these inhibitors are effective at reducing breast tumor growth and metastasis. The lead compound, SLC-0111, is currently being assessed in a Phase 1 clinical trial (NCT02215850). In the present study, we have evaluated the efficacy of targeting CAIX in PDAC using genetic and pharmacologic strategies. CAIX expression was found to be induced by hypoxia in a panel of human PDAC cell lines and was also upregulated in cell line-derived xenografts, patient-derived xenografts as well as the KRASG12D/p53-/- KPCY transgenic mouse model of PDAC (Rhim, AD et al. Cell, 2012:148;349-361). shRNA-mediated silencing of CAIX expression in PK-8 PDAC cells significantly reduced hypoxia-mediated cell proliferation (P<0.05) and invasion (P<0.01) in vitro, and dramatically delayed tumor growth (P<0.001) in vivo, resulting in increased median survival of the mice from 36 days to 59 days (P<0.01), similar to that observed following treatment with gemcitabine (36 days to 58 days, P<0.01). Interestingly, gemcitabine treatment in this model increased the fraction of CAIX-positive tumor cells, and silencing of CAIX expression in combination with gemcitabine treatment further delayed tumor growth (P<0.01) and increased median survival to 86 days (P<0.01). Similarly, sequential administration of gemcitabine and the CAIX inhibitor, SLC-0111, significantly delayed tumor growth (P<0.01) and enhanced survival (P<0.01) in PK-8 and PK-1 xenografts, compared to gemcitabine alone. Preliminary data indicate that treatment with SLC-0111, alone and in combination with gemcitabine inhibits the growth of spontaneous PDAC tumors in the KPCY transgenic model of PDAC, as assessed by ultrasound imaging. These results demonstrate that CAIX plays an important role in PDAC progression and is a potential therapeutic target for pancreatic cancer. A focused Phase 1b clinical trial of gemcitabine and SLC-0111 in patients with metastatic PDAC is planned, based on the outcome of the Phase 1 trial. Citation Format: Paul C. McDonald, Shawn C. Chafe, Marylou Vallejo, David Schaeffer, Don Yapp, Claudiu T. Supuran, Ben Stanger, Mac Parmar, Daniel Renouf, Shoukat Dedhar. Targeting carbonic anhydrase IX in multiple pancreatic ductal adenocarcinoma models results in tumor growth inhibition and increased survival. [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 754.

The P2X7 receptor regulates cell survival, migration and invasion of pancreatic ductal adenocarcinoma cells.

BackgroundPancreatic ductal adenocarcinoma (PDAC) is presently one of the cancers with the worst survival rates and least effective treatments. Moreover, total deaths due to PDAC are predicted to increase in the next 15 years. Therefore, novel insights into basic mechanism of PDAC development and therapies are needed. PDAC is characterized by a complex microenvironment, in which cancer and stromal cells release different molecules, such as ATP. ATP can be transported and/or exocytosed from active cancer cells and released from dying cells in the necrotic core of the cancer. We hypothesized that one of the ATP receptors, the P2X7 receptor (P2X7R) could be an important player in PDAC behaviour.MethodsWe determined the expression (real time PCR and Western blot) and localization (immunofluorescence) of P2X7R in human PDAC cell lines (AsPC-1, BxPC-3, Capan-1, MiaPaCa-2, Panc-1) and a “normal” human pancreatic duct epithelial cell line (HPDE). The function of P2X7R in proliferation (BrdU assay), migration (wound assay) and invasion (Boyden chamber with matrigel) was characterized. Furthermore, we studied P2X7R-dependent pore formation (YoPro-1 assay) and cell death (caspase and annexin V / propidium iodide assays).ResultsWe found higher expression of P2X7R protein in PDAC compared to HPDE cells. P2X7R had notable disparate effects on PDAC survival. Firstly, high concentrations of ATP or the specific P2X7R agonist, BzATP, had cytotoxic effects in all cell lines, and cell death was mediated by necrosis. Moreover, the P2X7R–pore antagonist, A438079, prevented ATP-induced pore formation and cell death. Second, in basal conditions and with low concentrations of ATP/BzATP, the P2X7R allosteric inhibitor AZ10606120 reduced proliferation in all PDAC cell lines. P2X7R also affected other key characteristics of cancer cell behavior. AZ10606120 reduced cell migration and invasion in PDAC cell lines compared to that of untreated/vehicle-treated control cells, and stimulation with sub-millimolar concentrations of ATP or BzATP substantially increased cell invasion.ConclusionsPDAC cell lines overexpress P2X7R and the receptor plays crucial roles in cell survival, migration and invasion. Therefore, we propose that drugs targeting P2X7R could be exploited in therapy of pancreatic cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0472-4) contains supplementary material, which is available to authorized users.

Abstract B44: The histone deacetylase SIRT6 regulates metabolism in pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer death and one of the most aggressive and deadly malignancies, with limited therapeutic options. Sirtuin 6 (SIRT6) is a nicotinamide adenine dinucleotide (NAD+)-dependent histone deacetylase that functions as a novel tumor suppressor, which regulates aerobic glycolysis in cancer cells. Whether glycolysis plays a role in pancreatic cancer development remains a topic of intense study. Importantly, we found SIRT6 to be downregulated in human PDAC and most pancreatic cancer cell lines, resulting in increased expression of glycolytic genes. To determine the role of SIRT6 in pancreatic cancer we utilized human PDAC cell lines that demonstrated low expression of SIRT6 compared to a human pancreatic ductal epithelial (HPDE) control cell line. Restoration of SIRT6 expression decreased the levels of glycolytic gene expression, reduced glucose uptake, and inhibited cell growth. Conversely, knockdown of SIRT6 in HPDE cells increased glycolytic gene expression, glucose uptake and enhanced cell growth. To further explore the role of SIRT6 in a genetically defined PDAC model, we used an autochthonous mouse model with conditional alleles for KRASG12D, p53, and SIRT6, together with a p48-Cre allele to delete SIRT6 and p53 while expressing oncogenic Ras exclusively in the pancreas. SIRT6null, KRASG12D, p53+/- p48 tumor cells also exhibited increased expression of glycolytic genes and had enhanced glucose uptake compared to SIRT6+/+, KRASG12D, p53+/- p48 tumor cells, a phenotype which is reversed by restoration of wild-type but not catalytically inactive SIRT6 expression. Understanding how SIRT6 regulates PDAC metabolism through the modulation of chromatin dynamics will hopefully lead to new therapies to tackle aberrant metabolism in this devastating disease. Citation Format: Sita Kugel, Carlos Sebastián, Rushika M. Perera, Nabeel Bardeesy, Raul Mostoslavsky. The histone deacetylase SIRT6 regulates metabolism in 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 B44.

Abstract A06: ATM loss and KrasG12D cooperate to promote widely metastatic pancreatic ductal adenocarcinoma in mice

Abstract Invasive pancreatic adenocarcinoma (PDAC) is one of the most lethal solid malignancies. Mutational activation of Kras is nearly universal in preneoplastic pancreatic lesions (Pancreatic Intraepithelial Neoplasias-PanINs) and PDAC, whereas the progression of PanINs to PDAC is accompanied by both inactivating mutations in p16/CDKN2A, TP53, DPC4/SMAD4 and activating mutations in PI3K signaling. Although most PDAC cases represent a sporadic disease, about 5-10% of cases have a hereditary basis. Some of the loci responsible for inherited high-risk PDAC encode gene products implicated in DNA repair, such as BRCA2 and PALB2. The kinase Ataxia-Telangiectasia Mutated (ATM) plays a critical role in DNA damage responses activated by double-strand breaks, by triggering cell cycle checkpoints that repair DNA or induce p53-dependent apoptosis or senescence. ATM was recently identified as a novel predisposition gene for PDAC because deleterious ATM mutations were found in some families with hereditary PDAC, as well as in ~8% of sporadic PDAC cases. Thus far, the role of ATM as an anti-cancer barrier – especially in pancreatic cancer biology – is poorly understood. Here we sought to address this crucial issue using 3 different mouse strains expressing both an oncogenic version of Kras (KrasG12D) and variable ATM genetic dosage in the pancreas: 1) KC mice, express wild-type ATM, 2) KC;ATMcHET mice, carry deletion of one ATM-allele; and 3) KC;ATMcKO mice, carry deletion of both ATM-alleles. Our preliminary analysis of KrasG12D-induced PanIN formation uncovered that these lesions form earlier in pancreatic tissues carrying one functional ATM allele or lacking ATM in comparison to KC pancreata. In addition, we determined that the median survival due to metastatic tumor burden is considerably shorter in both KC;ATMcKO mice (~6 months) and KC;ATMcHET mice (~9 months) compared to KC mice (~15 months). We also began establishing primary cell lines from both PDAC and metastatic foci formed in KC, KC;ATMcKO and KC;ATMcHET mice. Preliminary analyses of 2 primary cell lines treated with ionizing radiation (IR) to induce DNA damage and processed for Western blot analysis showed lack of total ATM, phospho-ATM, phospho-KAP1 (an ATM-specific target), and phospho-p53 in ATM-deficient primary pancreatic cancer cells. In contrast, primary cell lines from KC;Cdkn2a+/- tumors and established human PDAC cell lines display functional ATM signaling after IR. In addition, primary cell lines from PDAC and liver metastases of KC;ATMcKO mice also exhibited genomic instability depicted by a high number of anaphase bridges. To complement the characterization of these primary cell lines, we will attempt to identify DNA repair pathway(s) compensating for the loss of ATM and exploit these findings to induce synthetic lethality. Likewise, additional signaling pathways that are activated or inactivated during PDAC progression in the context of ATM deficiency will be identified using both biased and unbiased approaches. To our knowledge, these preliminary results provide the first evidence in vivo that ATM activity poses an intrinsic barrier to oncogenic transformation in the pancreas. A comprehensive characterization of pancreatic tumor formation in the context of ATM deficiency using our generated mouse models is currently underway, together with experiments in primary cell lines attempting to identify vulnerabilities that could be exploited to develop adequate tailored treatments. Citation Format: Yiannis Drosos, Kathryn Roys, Emin Kuliyev, Peter McKinnon, Beatriz Sosa-Pineda. ATM loss and KrasG12D cooperate to promote widely metastatic pancreatic ductal adenocarcinoma in mice. [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 A06.

Inflammatory cytokines regulate the expression of glycosyltransferases involved in the biosynthesis of tumor-associated sialylated glycans in pancreatic cancer cell lines

BackgroundPancreatic ductal adenocarcinoma (PDAC) is characterized by an abundant stroma containing several pro-inflammatory cytokines, which are described to modulate the expression of important genes related to tumor promotion and progression. In the present work we have investigated the potential role of these cytokines in the biosynthesis of tumor-associated carbohydrate antigens such as sialyl-Lewisx (SLex) through the regulation of specific glycosyltransferase genes. MethodsTwo human PDAC cell lines MDAPanc-3 and MDAPanc-28 were treated with pro-inflammatory cytokines IL-1β, TNFα, IL-6 or IL-8, and the content of tumor-associated carbohydrate antigens at the cell membrane was analyzed by flow cytometry. In addition, variation in the mRNA expression of sialyltransferase (ST) and fucosyltransferase (FUT) genes, which codify for the ST and FucT enzymes involved in the carbohydrate antigens’ biosynthesis, was determined. The inflammatory microenvironment of PDAC tissues and the expression of Lewis-type antigens were analyzed by immunohistochemistry to find a possible correlation between inflammation status and the presence of tumor-associated carbohydrate antigens. ResultsIL-1β stimuli increased SLex and α2,6-sialic acid levels in MDAPanc-28 cells and enhanced the mRNA levels of ST3GAL3-4 and FUT5-7, which codify for ST and FucT enzymes related to SLex biosynthesis, and of ST6GAL1. IL-6 and TNFα treatments increased the levels of SLex and Ley antigens in MDPanc-3 cells and, similarly, the mRNA expression of ST3GAL3-4, FUT1-2 and FUT6, related to these Lewis-type antigens’ biosynthesis, were increased. Most PDAC tissues stained for SLex and SLea and tended to be expressed in the tumor samples with a higher presence of inflammatory immune cells. ConclusionsThe inflammatory microenvironment can modulate the glycosylation pattern of PDAC cells, increasing the expression of tumor-associated sialylated antigens such as SLex, which contributes to pancreatic tumor malignancy.

Abstract B10: Epidermal growth factor receptor inhibitor prolongs survival in pancreatic cancer by blocking gemcitabine-induced mitogen-activated protein kinase signal

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the most deadly cancer worldwide. Although many regimens have been tried against PDAC, an epidermal growth factor receptor (EGFR) inhibitor erlotinib in combination with gemcitabine is the only molecular target drug superior to gemcitabine alone. However, the mechanism by which PDAC with extremely frequent KRAS-mutation benefits from EGFR inhibition remains largely unknown. In this study, we evaluated the efficacy of erlotinib in combination with gemcitabine using a murine PDAC model with transforming growth factor-beta receptor II knockout plus Kras activation and investigated the mode of action. The mice were treated using the following drug doses and treatment schedules; erlotinib was administered from 3 weeks of age and gemcitabine was administered from 4 weeks of age. We isolated PDAC cells from the murine PDAC tissues. Effects of erlotinib on the proliferation and intracellular signaling of the murine PDAC cells or human PDAC cell lines were examined in vitro. We sacrificed the mice at 7 weeks of age, and excised the pancreatic tissues and processed for western blot analysis and immunohistochemistry. We evaluated the expression of EGFR ligands by real-time PCR and the heterodimer formation of EGFR with ErbB2 by immunoprecipitaiton after incubation with gemcitabine in vitro. We assessed whether the effect of gemcitabine on EGFR/ErbB2 activation is secondary to mitogen activated protein kinase (MAPK) signal activation after incubation with or without MEK inhibitor and gemcitabine by western blot analysis and real-time PCR. Gemcitabine + erlotinib inhibited PDAC progression and significantly prolonged the survival of the PDAC mice compared to gemcitabine alone. Gemcitabine or erlotinib also inhibited in vitro PDAC cell proliferation. Interestingly, Gemcitabine induced MAPK signaling, which was dramatically inhibited by adding erlotinib, even in the Kras-mutant PDAC cells. The suggested mechanisms were that gemcitabine induced EGFR ligand expression and also ErbB2 activation by increasing heterodimer formation with EGFR and maintaining high ErbB2 protein level in PDAC cells. We observed that the gemcitabine-induced MAPK signaling activation was in part due to induction of Egfr ligands(Egf, Tgf-a, Amphiregulin) expression by real-time PCR and ELISA. Using a phospho-RTK antibody array, we also observed that Gem induced Erbb2 activation in PDAC cells, and validated by western blot analysis, real-time PCR, and immunohistochemistry. Erlotinib inhibited the ErbB2 activation, partly by inhibiting heterodimer formation with EGFR and also decreasing ErbB2 protein expression in PDAC cells. We observed that gemcitabine-induced EGFR ligands up-regulation and EGFR/ErbB2 activation require intact MAPK signaling and these are secondary effects of MAPK signal activation and that gemcitabine induced the activation irrespective of KRAS status and gemcitabine sensitivity. This model helps us to evaluate an efficacy of new drugs and to investigate mechanisms of the mode of action and chemoresistance. This study provides clinical insights into potent therapeutic strategies for this difficult cancer. Citation Format: Koji Miyabayashi, Hideaki Ijichi, Ryota Takahashi, Dai Mohri, Keisuke Yamamoto, Yoshinari Asaoka, Tsuneo Ikenoue, Keisuke Tateishi, Harold L. Moses, Kazuhiko Koike. Epidermal growth factor receptor inhibitor prolongs survival in pancreatic cancer by blocking gemcitabine-induced mitogen-activated protein kinase signal. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B10. doi: 10.1158/1557-3125.RASONC14-B10

Abstract 1035: hMENA splicing program and TGF-β1-mediated EMT in pancreatic cancer

Abstract Background Epithelial to mesenchymal transition (EMT) is an early event in pancreatic cancer and has been involved in cancer invasiveness. An intense stromal reaction, peculiar to the pancreatic tumor microenvironment, includes cancer-associated fibroblasts(CAFs), abundant cells in the tumor stroma, recently linked to the induction of EMT. On the other hand, the EMT process requires a dynamic remodeling of the actin cytoskeleton, and the splicing program of hMENA, a regulator of actin, has been associated with the EMT process. We have described two alternatively expressed isoforms, hMENA11a and hMENAΔv6, with opposite functions in invasiveness in breast cancer (1). hMENA expression was detected in human pancreatic ductal adenocarcinoma samples (PDAC) (2), but no data are available on the alternative isoform expression in this neoplasia. The aim of this study is to investigate the role of hMENA splicing in TGF-β -mediated EMT in pancreatic cancer, the mechanisms involved in hMENA induction in PDAC and the role of CAFs in this process. Methods hMENA isoform expression was evaluated in PDAC tissues by immunohistochemistry using isoform-specific antibodies. Human PDAC cell lines, untreated or TGF-β treated, were characterized for the expression of hMENA isoforms and markers of EMT by qRT-PCR and WB analysis. The effects of both hMENAΔv6 knockdown or overexpression were also evaluated. Pancreatic cancer associated-fibroblasts were isolated from primary PDAC tissues. To study the role of fibroblast-cancer cell interactions on hMENA expression, a noncontact coculture system was used. Results IHC analysis of PDAC tissues revealed that epithelial hMENA11a is rarely expressed in primary pancreatic tumors that express a high level of hMENA and hMENAΔv6 isoforms. In a panel of pancreatic cancer cell lines, hMENA11a expression correlates with an epithelial phenotype, wherea hMENAΔv6 expression is associated with a mesechymal phenotype. TGF-β treatment specifically upregulated the invasive hMENAΔv6 isoform expression. Knockdown of endogenous hMENA/hMENAΔv6 isoform reduced cell invasiveness, reverted cells to an epithelial -“like” phenotype with an increased E-cadherin expression and impaired the TGF-β-mediated vimentin up-regulation. Conversely, overexpression of hMENAΔv6 increased the expression of the mesenchymal marker vimentin. Freshly explanted CAFs expressed the “mesenchymal” hMENAΔv6, and not hMENA11a and produced paracrine factors involved in the induction of hMENA isoforms in tumor cells. Conclusions These data provide new and critical insights into the role of hMENA splicing in TGF-β mediated EMT and identify the hMENA splicing program as a promising pathway for the development of new diagnostics and therapeutics in PDAC. (1) Di Modugno F. et al PNAS 2012 (2) Pino S. et al Clin Cancer Res 2008 Citation Format: Roberta Melchionna, Pierluigi Iapicca, Francesca Di Modugno, Paola Trono, Novella Gualtieri, Maria Grazia Diodoro, Sheila Spada, Giuliana Falasca, Gian Luca Grazi, Mina J Bissell, Paola Nisticò. hMENA splicing program and TGF-β1-mediated EMT in pancreatic cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1035. doi:10.1158/1538-7445.AM2014-1035

Resistance to the tyrosine kinase inhibitor axitinib is associated with increased glucose metabolism in pancreatic adenocarcinoma.

Alterations in energy (glucose) metabolism are key events in the development and progression of cancer. In pancreatic adenocarcinoma (PDAC) cells, we investigated changes in glucose metabolism induced by resistance to the receptor tyrosine kinase inhibitor (RTKI) axitinib. Here, we show that human cell lines and mouse PDAC cell lines obtained from the spontaneous pancreatic cancer mouse model (KrasG12DPdx1-cre) were sensitive to axitinib. The anti-proliferative effect was due to a G2/M block resulting in loss of 70–75% cell viability in the most sensitive PDAC cell line. However, a surviving sub-population showed a 2- to 3-fold increase in [C-14]deoxyglucose ([C-14]DG) uptake. This was sustained in axitinib-resistant cell lines, which were derived from parental PDAC. In addition to the axitinib-induced increase in [C-14]DG uptake, we observed a translocation of glucose transporter-1 (Glut-1) transporters from cytosolic pools to the cell surface membrane and a 2-fold increase in glycolysis rates measured by the extracellular acidification rate (ECAR). We demonstrated an axitinib-induced increase in phosphorylated Protein Kinase B (pAkt) and by blocking pAkt with a phosphatidylinositol-3 kinase (PI3K) inhibitor we reversed the Glut-1 translocation and restored sensitivity to axitinib treatment. Combination treatment with both axitinib and Akt inhibitor in parental pancreatic cell line resulted in a decrease in cell viability beyond that conferred by single therapy alone. Our study shows that PDAC resistance to axitinib results in increased glucose metabolism mediated by activated Akt. Combining axitinib and an Akt inhibitor may improve treatment in PDAC.