PaCa-2 Cells Research Articles

Synthesis, anticancer evaluation of novel hybrid pyrazole-based chalcones, molecular docking, DNA fragmentation, and gene expression: in vitro studies.

A unique series of pyrazolyl-chalcone derivatives was synthesized via the method of Claisen-Schmidt condensation. The desired chalcone derivatives 7a-d and 9a-f were obtained in good yields by reacting the 4-acetyl-5-thiophene-pyrazole with the appropriate heteroaryl aldehyde derivatives. The novel chalcones have undergone complete elemental analysis, 1H-NMR, 13C-NMR, mass spectrometry, and IR characterization. The three human cancer cell lines MCF7 (human Caucasian breast adenocarcinoma), PC3 (prostatic cancer) and PACA2 (pancreatic carcinoma) as well as the normal cell line BJ1 (normal skin fibroblasts) were tested in vitro for the anti-cancer properties of the newly synthesized chalcone derivatives. When compared to the reference medicine doxorubicin (IC50 = 52.1 μM), compound 9e showed the most promise derivative (IC50 = 27.6 μM) against PACA2 cells, while compound 7d demonstrated anticancer efficacy (IC50 = 42.6 μM against MCF7 cells compared to the reference drug doxorubicin (IC50 = 48 μM). Using breast and pancreatic cell lines, the gene expression, DNA damage, and DNA fragmentation percentages for compounds 7d and 9e were evaluated. Moreover, the molecular docking study of compounds 7d and 9e was assessed. The binding affinities of compound 9e toward P53 mutant Y220C was -22 kcal per mole, while those of compound 7d towards Bcl2 and CDK4 were -27.81 and -26.9 kcal per mole, respectively, compared to the standard values (-15.82, -33.96 and -29.9 kcal per mole).

Discovery of Novel Thiophene/Hydrazones: In Vitro and In Silico Studies against Pancreatic Cancer.

Cancer is the disease with the highest mortality. Drug studies contribute to promising treatments; however there is an urgent need for selective drug candidates. Pancreatic cancer is difficult to treat and the cancer progresses rapidly. Unfortunately, current treatments are ineffective. In this study, ten new diarylthiophene-2-carbohydrazide derivatives were synthesized and evaluated for their pharmacological activity. The 2D and 3D anticancer activity studies suggested the compounds 7a, 7d, and 7f were promising. Among these, 7f (4.86 µM) showed the best 2D inhibitory activity against PaCa-2 cells. Compounds 7a, 7d and 7f were also tested for their cytotoxic effects on healthy cell line but only compound 7d showed selectivity. Compounds 7a, 7d, and 7f showed the best 3D cell line inhibitory effect according to spheroid diameters. The compounds were screened for their COX-2 and 5-LOX inhibitory activity. For COX-2, the best IC50 value was observed for 7c (10.13 µM) and all compounds showed significantly lower inhibition compared to standard. In the 5-LOX inhibition study, compounds 7a (3.78 µM), 7c (2.60 µM), 7e (3.3 µM), and 7f (2.94 µM) demonstrated influential activity compared to standard. Regarding molecular docking studies, binding mode of compounds 7c, 7e, and 7f to the 5-LOX enzyme were non-redox or redox types, but not the iron-binding type. As dual inhibitors of 5-LOX and pancreatic cancer cell line, 7a and 7f were identified as the most promising compounds.

Abstract 1548: Examination of miRNA dysregulation in pancreatic cancer PDX models with parallel proteomic and transcriptomic investigations

Abstract Introduction: Pancreatic cancer is a high-fatality cancer. An estimated 57,600 new cases were diagnosed in the United States in 2020 with a corresponding 47,050 deaths. Despite improvements in the molecular understanding of pancreatic cancer, the prognosis for this disease has not improved in nearly 4 decades, largely because of the lack of early stage disease detection and limited efficiency of systemic therapies. MicroRNAs (miRNAs) are a class of non-coding RNAs approximately 22 nucleotides (nt) in length that regulate gene expression. in recent years, the dysregulation of miRNAs has been implicated in the development of various human cancers. Methods: Parallel analysis of global mRNA, miRNA and protein expression was conducted on pancreatic cancer patient-derived xenografts. The data from this analysis of these F1 tumours, was used to refine/identify miRNA targets of interest to generate 2 differential expression (DE) lists for miRNA; 1) Tumour vs Normal and 2) Tumour vs F1. Predictive software was used to identify targets and pathways of the DE priority miRNAs. These targets were further refined by integrating the targets with the global mRNA and proteomic profiling (unpublished data) performed on the same clinical samples. Alteration of cancer phenotype was investigated using overexpression and/or sponge vectors for each miRNA. Results: Analysis generated a list of priority miRNAs that had inverse interactions with predicted protein targets and were shown to be differentially expressed in our proteomic data. From 39 miRNAs investigated from the priority list, 7 miRNAs targeted 2 or more proteins across the 2 comparisons. These miRNAs - miRs-2467, -4742, -509, -615-3p, -4534, -222 and 2-06 when altered in expression MIA PaCa-2 and PANC-1 pancreatic cancer cells resulted in modulated cell proliferation, colony forming ability and anoikis resistance. Studies are ongoing on the changes to the chemotherapy and radiosensitivity of each cell line in response to changing these miRNA levels. Conclusions: The study identified miRNAs which displayed increased expression between normal and tumour and again increased between tumour and F1. From this list using predicative software and integration with additional global analysis of mRNA and Protein expression a priority list of 7 miRNAs were identified. The core concept in this study was to use differential expression of miRNAs as a tool to further unravel the biology of human pancreatic cancer, in the hope of uncovering new targets or vulnerabilities which might be exploited therapeutically or finding new bio markers which might contribute to better or earlier diagnosis. Citation Format: Taylor-Jade Allen. Examination of miRNA dysregulation in pancreatic cancer PDX models with parallel proteomic and transcriptomic investigations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1548.

Abstract 3463: Nischarin agonist rilmenidine inhibits pancreatic ductal adenocarcinoma cell migration and invasion

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive lethal malignancy due to the lack of early diagnosis and limited response to all treatment modalities. Early metastatic dissemination, rich desmoplastic stroma and immunosuppressive infiltrate are some of the features contributing to the poor prognosis of PDAC patients (5-year survival rate of less than 10%). Despite scientific progress in the development of new therapeutic regimes, PDAC patients in low- and middle-income countries have restricted access to high-cost innovative drugs. Recently, attention has been given to the concept of drug repurposing that could meet the need for novel but affordable and widely accessible therapies. Imidazoline receptor 1 Nischarin (NISCH, IR1, IRAS) is an integrin α5-interacting protein that acts as a tumor suppressor in breast cancer through modulation of cancer cell motility and survival. Importantly, NISCH has several clinically approved agonists that are used for the treatment of hypertension and are shown to modulate autophagy and reduce inflammation and fibrosis, which makes NISCH a potentially good therapeutic target for defying PDAC progression. Based on our GSEA analysis of NISCH expression in PDAC patient samples, NISCH was predicted to have an effect on cancer cell focal adhesion assembly and cytoskeletal organization. Therefore, we examined the effects of NISCH agonists on PDAC cells fitness in vitro. A panel of PDAC cell lines was tested in MTT assay to determine the effects of NISCH agonists rilmenidine, clonidine and moxonidine on cell viability. Flow cytometry, western blot and immunofluorescence were used to examine the effects of NISCH agonists on PDAC cell metabolism, migration and cytoskeletal organization. Tg(fli1:EGFP) zebrafish model was used to examine the effects of NISCH agonists on PDAC cell growth and invasion. We found that NISCH was expressed in PDAC cell lines, and that out of the three tested agonists, rilmenidine most potently inhibited cancer cell viability. Treatment with rilmenidine significantly reduced PANC-1, MIA PaCa-2 and BxPC-3 cell attachment to extracellular matrix (collagen type I and fibronectin) and migratory potential in wound healing and transwell assays. Furthermore, treatment with rilmenidine altered the organization of actin cytoskeleton and focal adhesion assembly, potentially through the reduction of intracellular reactive oxygen species (ROS) levels. Importantly, treatment with rilmenidine inhibited invasion of PANC-1 cells in Tg(fli1:EGFP) zebrafish model. With these antimigratory and potentially antimetastatic effects of rilmenidine, our study lays the ground for a more extensive examination of the biological role of NISCH in PDAC progression and implies that its agonists may be good candidates for drug repurposing in this type of cancer. Citation Format: Marijana Pavlovic, Marija Ostojic, Kristina Zivic, Aleksandar Pavic, Tatjana Srdic-Rajic, Jelena Grahovac. Nischarin agonist rilmenidine inhibits pancreatic ductal adenocarcinoma cell migration and invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3463.

Efficacy of MDX-124, a novel anti-annexin-A1 antibody, in preclinical models of pancreatic cancer.

590 Background: Pancreatic cancer is a highly fatal disease with poor survival and response to both chemotherapy and immunotherapy. Novel approaches to treat this disease are urgently required. Annexin-A1 (ANXA1) is secreted in response to several physiological stimuli where it activates formyl peptide receptors (FPR1/2) triggering multiple oncogenic processes. High ANXA1 expression in pancreatic cancer patients is associated with poor overall survival, and influences cancer progression, drug sensitivity, migration and invasion. MDX-124 is a novel humanized antibody targeting ANXA1 and we have previously presented data demonstrating its significant antiproliferative activity. Here we present further data showing the efficacy of MDX-124 in several preclinical models of pancreatic cancer. Methods: In-vitro models utilized MIA PaCa-2, PANC-1 or BxPC-3 human pancreatic cancer cell lines. Cell cycle progression was evaluated by measuring changes in DNA content via flow cytometry. Pancreatic cancer cell viability following incubation with MDX-124 (0-10 µM) and 5FU (IC50) was assessed via MTT assay. A transwell migration assay was used to evaluate the effect of MDX-124 (0-50 µM) on pancreatic cancer cell migration. In-vivo efficacy was evaluated using an orthotopic mouse model of metastatic pancreatic cancer (FC1242luc/zsGreen; KPC-derived cell line) with bioluminescent imaging used to quantify the incidence and burden of lung metastases. Results: When compared to untreated MIA PaCa-2 pancreatic cancer cells, MDX-124 treatment decreased the proportion of cells in S-phase by 29% and G2 phase by 9.1%, with a concomitant increase in G1 of 38.1%. This occurred in a dose-dependent manner and is consistent with an MDX-124 mediated increase in cell cycle arrest. MDX-124 significantly reduced the viability of MIA PaCa-2 and PANC-1 cell lines versus an IgG control in a dose-dependent manner. Additionally in these two cell lines, combination of MDX-124 with 5FU (IC50) had a significant synergistic impact reducing cancer cell viability by 99.8% and 91.2% respectively. Furthermore, MDX-124 significantly reduced the migratory ability of MIA PaCa-2 and BxPC-3 pancreatic cancer cells. In the orthotopic model of metastatic pancreatic cancer, the murine analog of MDX-124 (MDX-001), markedly reduced both the incidence and size of lung metastases. Conclusions: MDX-124 demonstrated significant anti-tumor efficacy in several preclinical models of pancreatic cancer as a single agent, with increased potency observed when used in combination with 5FU. Medannex will initiate a First-In-Human study in Q4 2021 to evaluate MDX-124 in solid malignancies, including pancreatic cancer.

Biophysical and Biomechanical Effect of Low Intensity US Treatments on Pancreatic Adenocarcinoma 3D Cultures

Current developments in medical technology have focused on therapeutic treatments that selectively and effectively address specific pathological areas, minimizing side effects on healthy tissues. In this regard, many procedures have been developed to provide non-invasive therapy, for example therapeutic ultrasound (US). In the medical field, in particular in cancer research, it has been observed how ultrasounds can cause cell death and inhibit cell proliferation of cancer cells, while preserving healthy ones with almost negligible side effects. Various studies have shown that low intensity pulse ultrasound (LIPUS) and low intensity continuous ultrasound (LICUS) regulate the proliferation, cell differentiation and cavitation phenomena. Nowadays, there are poorly known aspects of low intensity US treatment, in terms of biophysical and biomechanical effects on target cells. The aim of this study is to set up an innovative apparatus for US treatment of pancreatic ductal adenocarcinoma (PDAC) cells, monitoring parameters such as acoustic intensity, acoustic pressure, stimulation frequency and treatment protocol. To this purpose, we have developed a custom-made set up for the US stimulation at 1.2 and 3 MHz of tridimensional (3D) cultures of PDAC cells (PANC-1, Mia Paca-2 and BxPc3 cells). Images of the 3D cultures were acquired, and the Calcein/PI assay was applied to detect US-induced cell death. Overall, the setup we have presented paves the way to an innovative protocol for tumor treatment. The system can be used either alone or in combination with small molecules or recombinant antibodies in order to propose a novel combined therapeutic approach.

Abstract PO-039: Inhibition of migration, invasion and drug resistance of pancreatic adenocarcinoma cells- Role of Snail, Slug and Twist and small molecule Inhibitors

Abstract Purpose: The main purpose of this study is to demonstrate the effects of epithelial to mesenchymal transition activating transcription factor silencing (EMT-ATF silencing) on migration, invasion, drug resistance and tumor forming abilities of various pancreatic cancer cell lines. Additionally, the contribution of small molecule inhibitors of EMT (SD-208 and CX4945) to the effects of gene silencing was evaluated. Methods: EMT activating transcription factors “Snail, Slug and Twist” were silenced by short hairpins on Panc-1, MIA PaCa-2, BxPC-3, and AsPC-1 pancreatic cancer cell lines. The changes in migration, invasion, laminin attachment, cancer stem-like cell properties and tumor forming abilities were investigated. Chemo sensitivity assays and small molecule inhibitors of EMT were applied to the metastatic pancreatic cancer cell line AsPC-1. Results: EMT-ATF silencing reduce EMT and stem cell-like characteristics of pancreatic cancer cell lines. Following EMT-ATF silencing amongst the four PC cell lines, AsPC-1 showed the best response and was chosen for further chemo resistance and combinational therapy applications. EMT downregulated AsPC-1 cells showed less resistance to selected chemotherapeutics compared to the control group. Both small molecule inhibitors enhanced the outcomes of EMT-ATF silencing. Conclusion: Overall it was found that EMT-ATF silencing, either by EMT-ATF silencing or with the enhancement by small molecules, is a good candidate to treat pancreatic cancer since it simultaneously minimizes metastasis, stem cell properties, and drug resistance. Citation Format: Ezgi Kasikci, Esra Aydemir, Omer Faruk Bayrak, Fikrettin Sahin. Inhibition of migration, invasion and drug resistance of pancreatic adenocarcinoma cells- Role of Snail, Slug and Twist and small molecule Inhibitors [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-039.

The Evaluation of Gene Oct4 Expression as a New Tumor Marker in Pancreatic Tumor and Non-Tumor Cell Lines

Introduction: Oct4 gene is one of the important genes involved in stem cell self-renewal control. Recently, this gene has been proposed as a novel molecular marker for cancer detection. Therefore, in this study we examined the expression of Oct4 gene in MIA Paca-2, PA-TU-8902 and AsPC-1 cell lines as well as pancreatic cancer.

Abstract 713: BBI-608 modulates DNA repair pathways, induces apoptosis and impacts response to 5-Fluorouracil and ionizing radiation in pancreatic cancer cells

Abstract Background: BBI-608 has been shown to modulate multiple oncogenic cellular pathways which are often constitutively active in pancreatic ductal adenocarcinoma (PDAC). Among these are key mediators of DNA repair and cellular survival pathways. We hypothesized that BBI-608 would sensitize PDAC cells to chemoradiotherapy. Methods: The combined effects of BBI-608 and chemoradiotherapy (5-FU + IR) were evaluated in human (MIA PaCa-2) and murine (PANC-02) PDAC cell lines using a Br-dU cell proliferation assay. To elucidate the mechanism of action, effects on the expression of DNA damage and repair (pATM, γ-H2AX, p53; pATR, and Rad51) molecules were examined by Western blot. Apoptosis was assessed via Annexin V staining and flow cytometric analysis to evaluate mechanism of cell death following treatment of MIA PaCa-2 and PANC-02 cell lines. The in vivo activity of BBI-608, 5-FU + IR, alone and in combination, were evaluated in C57BL/6 mice bearing subcutaneous PANC-02 tumors. Results: The combination of BBI-608 and chemoradiotherapy significantly decreased (p<0.001) cell proliferation and induced Annexin V positive cells in both MIA PaCa-2 and PANC-02 cell lines. BBI-608 and chemoradiotherapy also decreased the activation of pATR, MDM2, and Rad51 and increased pATM, γ-H2AX and p53. Administration of BBI-608 potentiated the effects of chemoradiotherapy in vivo in mice bearing subcutaneous PANC-02 tumors as measured by tumor volume. Conclusion: These data identify a role for BBI-608 as a modulator of DNA damage and apoptosis. These effects enhanced the efficacy of chemoradiotherapy in cell lines and in vivo models of PDAC and provide rationale for continued investigation in the pre-clinical and clinical setting. Note: This abstract was not presented at the meeting. Citation Format: Batoul Farran, Ganji P. Nagaraju, Sneha G. Shipra, Shipra R. Bethi, Gregory B. Lesinski, Bassel El-Rayes. BBI-608 modulates DNA repair pathways, induces apoptosis and impacts response to 5-Fluorouracil and ionizing radiation in pancreatic cancer 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 713.

Abstract 5915: Overexpression of tumor suppressor p53 gene by oncolytic adenovirus boosts immune responses in human pancreatic ductal adenocarcinoma cells

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is the worst prognosis disease with an overall 5-year survival rate of less than 5%. Although immune checkpoint therapy has been emerged as a novel antitumor therapy, PDAC is less sensitive to immunotherapy due to small number of infiltrating T cells. Recently, oncolytic virotherapy has been shown to promote intratumoral T cell infiltration as an immunogenic antitumor therapy. In the current study, we explored whether overexpression of the wild-type p53 tumor suppressor gene could synergize with oncolysis for induction of immunogenic cell death in human PDAC cells. Methods: OBP-702 is a tumor-specific replication-competent oncolytic adenovirus, in which the hTERT promoter drives the expression of the viral E1A and E1B genes for tumor-specific viral replication and the Egr-1 promoter induces wild-type p53 expression. We used 4 PDAC cell lines (MIA PaCa-2, Capan-1, BxPC-3, Panc-1). Molecular mechanisms of OBP-702-mediated cell death was investigated. Migration and invasion properties were assessed using transwell chamber assay. In addition, virus-induced immunogenic cell death was assessed by analyzing the level of extracellular ATP and high-mobility group box protein B1 (HMGB1). In vivo antitumor effect of OBP-702 was assessed using an orthotopic BxPC-3 xenograft tumor model. Results: At 72 hours after virus infection, OBP-702 induced profound antitumor effect in all human PDAC cells. OBP-702 induced autophagy- and apoptosis-related cell deaths. In migration and invasion assays, MIA Paca-2 and Capan-1 cells were low-invasive type, and Panc-1 and BxPC-3 cells were high-invasive type. OBP-702 inhibited migration and invasion properties of high-invasive type PDAC cells At 24 and 48 hours after virus infection, the secretion of ATP was significantly higher in OBP-702-infected PDAC cells compared to p53-null virus-infected cells. Moreover, OBP-702 significantly induced the higher amount of extracellular HMGB1 in all PDAC cells. Finally, OBP-702 significantly suppressed tumor growth in an orthotopic BxPC-3 xenograft tumor model compared to p53-null virus. Conclusions: Our data suggest that oncolytic adenovirus-mediated p53 overexpression induces more profound immunogenic cell death to boost the immune responses in PDAC. This additional immunological function of p53 is quite attractive and warrants clinical trials combined with immune checkpoint inhibitors. Citation Format: Hiroyuki Araki, Hiroshi Tazawa, Takeshi Ieda, Takuro Fushimi, Shinji Kuroda, Ryuichi Yoshida, Hiroyuki Kishimoto, Masahiko Nishizaki, Yasuo Urata, Shunsuke Kagawa, Toshiyoshi Fujiwara. Overexpression of tumor suppressor p53 gene by oncolytic adenovirus boosts immune responses in human pancreatic ductal adenocarcinoma cells [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 5915.

Abstract 585: Exosomal EphA2 transmits chemoresistance and predicts pancreatic cancer patient responses to therapy

Abstract Exosomes are small extracellular vesicles secreted by most cells that are found in blood and other bodily fluids, and which contain cytoplasmic material and membrane factors corresponding to their cell type of origin. Exosome membrane factors and contents have been reported to alter adjacent and distant cell behavior in multiple studies, but the impact of cancer-derived exosomes on chemoresistance is less clear. We therefore analyzed exosomes from the human pancreatic cancer (PC) cell lines PANC-1, MIA PaCa-2 and BxPC-3 for their ability to transmit chemoresistance, finding that PANC-1 exosomes increased the gembicitabine (GEM) resistance of MIA PaCa-2 and BxPC-3 cell cultures. Comparative proteomics determined that PANC-1 exosomes overexpressed Ephrin type-A receptor 2 (EphA2) versus exosomes of less chemoresistant PC cell lines MIA PaCa-2 and BxPC-3. EphA2-knockdown in PANC-1 cells inhibited their ability to transmit exosome-mediated chemoresistance to MIA PaCa-2 and BxPC-3, while treatment of MIA PaCa-2 and BxPC-3 cells with soluble EphA2 did not promote chemoresistance, indicating that membrane carried EphA2 was important for the EphA2 chemoresistance effect. EphA2 levels on circulating exosomes were markedly increased in mice bearing PANC-1 tumors and PC patients who subsequently revealed poor responses to treatment. We thus concluded that exosomal EphA2 expression could transmit chemoresistance and may serve as a minimally-invasive predictive biomarker for PC treatment response. Further work should address whether additional exosomal factors regulate resistance to other cancer therapeutic agents for PC or other cancer types. Citation Format: Jia Fan, Qian Wei, Eugene J. Koay, Yang Liu, Zhen Zhao, Ye Hu. Exosomal EphA2 transmits chemoresistance and predicts pancreatic cancer patient responses to therapy [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 585.

Biodegradable zwitterionic sulfobetaine polymer and its conjugate with paclitaxel for sustained drug delivery.

The applicability of FDA-approved biodegradable aliphatic polyesters has been significantly restricted because they are hydrophobic and lack functionalities. Recently zwitterionic polymers have emerged as promising hydrophilic biomaterials, but most of the reported zwitterionic polymers are non-biodegradable. This study reports a novel aliphatic polyester-based zwitterionic polymer and the corresponding polymer-drug conjugate. Their aliphatic polyester and zwitterionic components provide them with high enzymatic degradability and low nonspecific interactions with biomolecules, respectively. While the zwitterionic polymer did not show noticeable cytotoxicity, the corresponding polymer-anticancer drug conjugate exhibited acid-sensitive sustained drug release, remarkable effectiveness in killing cancer cells, as well as the ready cellular internalization. This work lays a foundation for the further development of synthetic biodegradable zwitterionic polymer-based materials which potentially may have broad and significant biomedical applications.

Abstract A43: Activin receptor type IA in pancreatic cancer and its implications in tumor progression

Abstract Several studies have shown the importance of the Transforming Growth Factor Beta (TGFβ) pathway in pancreatic tumorigenesis and cancer progression, displaying a seemingly paradoxical role in tumor development. However, Activin, a TGFβ family member and its contributions to tumor development and progression are largely understudied in most cancers, particularly pancreatic cancer. The functions of Activin through its receptors are context and cell-type specific, as they exert different effects in various cancer types, including colon, breast and prostate. In normal pancreas, Activin A isoform levels in serum are low, while in pancreatic cancer Activin A levels positively correlate with shorter survival and metastasis. Activin, like TGFβ, signals through a complex containing Activin A type I and type II receptors. A type II homodimer comprised of Activin receptor type IIA (ACVRIIA) or IIB (ACVRIIB) binds ligand via an extracellular domain and recruits a homodimer of Activin receptor type IA (ACVRIA) or IB (ACVRIB), leading to formation of a tetrameric complex and phosphorylation of type I receptor by the type II serine/threonine kinase activity. ACVRIA and ACVRIB differ in amino acid sequence within the activation region, called the GS region, presumably giving receptor specificity and differences in regulation of canonical (SMAD-dependent) and non-canonical (SMAD-independent) downstream signaling. Human pancreatic ductal adenocarcinomas contain somatic mutations in the ACVR1B gene, and one recent study in vivo showed that pancreatic-specific loss of ACVR1B accelerates development of mutant KRAS-induced intraductal papillary mucinous neoplasms in mice. Our in silico data compiled from human pancreatic cancer patients demonstrates that there is a significant 50% loss of ACVR1B mRNA in cancer patients compared to normal tissue from the same cohort. Moreover, pancreatic cancer patients show a significant 80% upregulation of ACVR1A mRNA compared to normal. Yet, Activin effects through ACVRIA in pancreatic cancer have not yet been established. Our studies aim to characterize the effects of ACVR1A activation in pancreatic tumor development and progression. We hypothesize that the opposing effects of Activin signaling in pancreatic cancer may be due to ACVRIA activation and its subsequent pro-survival and pro-tumorigenic functions. Pancreatic cancer cell lines were analyzed for levels of ACVR1A expression, MIA PaCa-2 showed the highest level of expression followed by AsPC-1, with PANC-1 showing the lowest expression. MIA PaCa-2 and AsPC-1 cell lines showed increased migration in scratch assays compared to control following treatment with Activin A. We are currently establishing MIA PaCa-2 and AsPC-1 stable cell lines with a knockdown of ACVR1A to assess migratory and proliferative changes upon Activin treatment when ACVR1A is downregulated. Human and murine tissue immunostaining of ACVR1A showed expression of the receptor in the normal pancreas as well as tumor tissue samples. Human pancreas staining for ACVR1A was stronger in the carcinoma sections of tumor tissue samples compared to a lighter staining in normal tissue samples. Murine tissues showed staining in the neoplastic lesions of KC and EL-KRAS transgenic mice, while no strong staining was observed in the wild type mouse. We conclude that not only TGFβ exerts dual effects in regards to pro and anti-tumor promoting effects in the context of pancreatic cancer, but rather Activin signaling pathway is also important in pancreatic carcinogenesis and a novel target that can be useful for therapeutic interventions in pancreatic cancer. Determining Activin signaling through ACVR1A can be essential in deciphering the role of Activin in pancreatic tumor development and progression. Citation Format: Georgina E. Mancinelli, Jonas Staudacher, Barbara Jung, Paul J. Grippo.{Authors}. Activin receptor type IA in pancreatic cancer and its implications in tumor progression. [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 A43.

Abstract 3748: Virally enhanced p53 reactivation impairs KRAS-driven pancreatic cancer invasion

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is the worst prognosis disease with an overall 5-year survival rate of less than 5%. More than 80% of patients are not eligible for curative surgical resection due to extensive local tumor invasion and early systemic metastasis at the time of diagnosis. Moreover, even after curative surgery, PDAC patients still have poor prognosis due to local recurrence and systemic metastasis. Although activation of oncogenic KRAS is implicated in the initiation, promotion and progression of pancreatic cancer, the development of cancer treatment targeting KRAS remains unsuccessful. Moreover, tumor suppressor p53 is also frequently inactivated in PDAC patients. We recently developed two types of tumor-specific replication-competent oncolytic adenoviruses, OBP-301 and OBP-702, which is modified OBP-301 to express tumor suppressor p53 protein. In this study, we investigated whether oncolytic adenoviruses inhibit cell viability, migration, invasion and KRAS-driven signaling pathway in PDAC cells. Methods: We used 4 PDAC cells (MIA PaCa-2, Capan-1, Panc-1, BxPC-3). The cell viability was examined by XTT assay. Migration and invasion properties were assessed using transwell chamber assay. The modulation of KRAS-driven signaling pathway was investigated by Western blot analysis. Results: OBP-301 induced moderate antitumor effect in MIA Paca-2, Capan-1 and BxPC-3 cells and strong antitumor effect in Panc-1 cells in a dose-dependent manner, whereas OBP-702 induced profound antitumor effect in all human PDAC cells, suggesting the broad spectrum of OBP-702's efficacy. OBP-301 induced autophagy-related cell death, whereas OBP-702 induced autophagy- and apoptosis-related cell deaths. In migration and invasion assays, MIA Paca-2 and Capan-1 cells were low-invasive type, and Panc-1 and BxPC-3 cells were high-invasive type. OBP-301 and OBP-702 inhibited migration and invasion properties of high-invasive type PDAC cells, and the inhibitory effect of OBP-702 was stronger than that of OBP-301. We also found that oncolytic adenoviruses inhibited the expression of KRAS and KRAS-downstream target MEK/ERK proteins in high-invasive type PDAC cells. Conclusions: Our results suggest that oncolytic adenoviruses, OBP-301 and OBP-702, inhibit the survival and invasive phenotype of pancreatic cancer by suppressing KRAS-driven signaling pathway and reactivating p53-driven signaling pathway. In vivo experiments are under way to investigate the anti-tumor and anti-invasive effects of oncolytic adenoviruses using BxPC-3 xenograft tumors. The clinical trial of intratumoral administration of oncolytic adenoviruses in patients with invasive PDAC should be also warranted. Citation Format: Takeshi Koujima, Hiroshi Tazawa, Takeshi Ieda, Kazuya Kuwada, Terutaka Tanimoto, Shinji Kuroda, Hiroyuki Kishimoto, Masahiko Nishizaki, Yasuo Urata, Shunsuke Kagawa, Toshiyoshi Fujiwara. Virally enhanced p53 reactivation impairs KRAS-driven pancreatic cancer invasion. [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 3748.

Abstract 4416: Combination of HSP90 and proteasome inhibitor is effective in pancreatic cancer

Abstract Background: Heat shock protein (HSP90) and proteasome play an important role in cellular protein trafficking and degradation in pancreatic cancer (PC). Given the overlap in the mechanism of action, we investigated the effects of the combination of pharmacological inhibitors of HSP90 (Ganetespib) and proteasome (Carfilzomib) on PC cells in vitro and in vivo. Methods: The combined effects of ganetespib and carfilzomib were evaluated in MIA PaCa-2 and HPAC cell lines using a cell proliferation assay. Effects on the expression of survival (PI3K/AKT, and ERK), cell cycle (Cdc-2 and cyclin B1), angiogenesis (HIF-1α and VEGF) and autophagy (LC3 A/B, and Atg7) pathways were examined by Western blot. Cell cycle analysis was performed by FACS assay. HPAC cell lines were tested for efficacy to ganetespib, carfilzomib, alone and in combination, using an in vivo tumor xenograft model. Results: The combination of ganetespib and carfilzomib significantly decreased cell proliferation, induced G2-M cell cycle arrest and induced autophagy in both MIA PaCa-2 and HPAC cell lines. Ganetespib and carfilzomib also decreased the activation of ERK, PI3K/AKT, and autophagy signaling molecules (LC3 A/B, and Atg7). In animal models, ganetespib potentiated the effects of carfilzomib, as measured by tumor volume. Western blot analysis from tumors removed from animals confirmed the effects of ganetespib and carfilzomib on survival, cell cycle, autophagy and angiogenesis. Conclusion: These observations provide preclinical proof-of-principle that combinatorial targeting of HSP90 and proteasome is a rational approach for development in PC clinical trials. Citation Format: Ganji Purnachandra Nagaraju, Balney Rajitha, Leah Benton, Bassel F. El-Rayes. Combination of HSP90 and proteasome inhibitor is effective 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 4416.

Abstract 4009: The expression of PD-L1 on human and murine pancreatic ductal adenocarcinoma is enhanced by anticancer agents via the JAK/STAT pathway

Abstract Background: Pancreatic ductal adenocarcinoma(PDA) is the fourth most common cause of cancer-related death in Japan. Recently, new standard chemotherapies for PDA have been developed, but they are still largely unsatisfactory. Therefore, development of new treatment options has been required to improve the outcomes of patients with PDA. To break through this situation, blocking one of inhibitory immune checkpoints, Programmed death-1 (PD-1) /Programmed death-ligand 1 (PD-L1) pathway is considered to be a hopeful candidate for new treatment strategies for PDA. In this context, for the future combination therapy of anticancer agents and immune check point inhibitors, we investigated how anticancer agents influence the expression of PD-L1 on pancreatic cancer cell lines. Additionally we analyzed the molecular mechanism by which PD-L1 expression on the pancreatic cancer cell lines are regulated. Methods: Human PDA cell lines MIA PaCA-2, AsPC-1 and murine PDA cell line Pan02 were used in this study. These cells were adjusted to 1.0 × 105 / ml and incubated with anticancer agents (i.e. gemcitabine, paclitaxel and 5-fluorouracil) at 37°C for 24-72h. Then, the expression level of PD-L1 was determined using qRT-PCR and flow cytometry. For the blocking experiment of the JAK/STAT pathway, AG490 was used as a blocking agent. After 48h incubation of AsPC-1 cells at 37°C, cells were treated with various concentration of AG490 for 1h. Cells were stimulated with 5-fluorouracil, paclitaxel and gemcitabine, and then incubated in the presence or absence of AG490 for an additional 6-48h. The expression of PD-L1 was analyzed using qRT-PCR and flow cytometry. Stat1 and phospho-Stat1 protein were analyzed by western blotting. Results: In AsPC-1, MIA PaCA-2 and Pan02, the expression of PD-L1 was enhanced with all three anticancer agents in a concentration dependent manner. The phosphorylation of STAT1 and the increase of total STAT1 was observed in AsPC-1 when stimulated by each anticancer agents. After the treatment of JAK/STAT inhibitor, the phosphorylation of STAT1 was attenuated, and the PD-L1 upregulation induced by anticancer agents was cancelled in a concentration dependent manner. Conclusion: The stimulation of anticancer agents leads to an enhancement of PD-L1 expression on PDA cell lines. The JAK/STAT pathway is reported to be involved in IFN-γ mediated PD-L1 upregulation in lung cancer and hepatocellular carcinoma cell lines. In the present study, the phosphorylation of Stat1 and the expression of total Stat1 were enhanced after the anticancer agents treatment. Moreover, JAK/STAT inhibitor attenuated anticancer agents-induced PD-L1 expression. Taken together, the JAK/STAT pathway may be responsible for the anticancer agents mediated PD-L1 transcription. Citation Format: Toshifumi Doi, Takeshi Ishikawa, Tomoyo Yasuda, Tetsuya Okayama, Kaname Oka, Naoyuki Sakamoto, Yuji Naito, Yoshito Itoh. The expression of PD-L1 on human and murine pancreatic ductal adenocarcinoma is enhanced by anticancer agents via the JAK/STAT pathway. [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 4009.

Abstract 4879: Cancer cell-mediated signaling of TLR 2, 4, and 9 causes activation of PI3K/Akt/mTOR pathway and induces tumor cell proliferation in pancreatic cancer

Abstract Background: Toll like receptor (TLR) ligands are in clinical use for the immunotherapy of different cancers. They are supposed to induce an inflammatory immune response against the tumor. Interestingly, several studies showed that TLRs were expressed by cancer cells in different tumor entities and that their activation can contribute to an inflammatory microenvironment. Yet, for pancreatic cancer, TLR expression and its impact in tumor cells is only poorly understood. Therefore this study analyzed the influence of TLR2, 4, and 9 expression and activation on tumor cell signaling and proliferation in pancreatic cancer. Methods: The expression of TLR 2, 4, and 9 was analyzed in vitro in several established as well as primary human pancreatic cancer cell lines by qRT-PCR and Western Blot. TLR stimulation was then performed in BxPC-3, MIA Paca2, and PacaDD135 cells using the TLR ligands oligodeoxyribonucleotide2006 (ODN2006), lipoteichonic acid of Staphylococcus aureus (LTA-SA) and High-Mobility Group Box 1 (HMGB1). Expression of MyD88 and pAkt was then analyzed by Western Blot. Functional analysis on proliferation (ATP assay) and cytokine expression (Luminex) was additionally performed. Results: Expression of TLR2, 4 and/or 9 was demonstrated in all investigated human pancreatic cancer cell lines. Receptor activation by single or combined use of TLR ligands resulted in increased MyD88 and pAkt (Ser473) expression. Additionally, up-regulated anti-apoptotic protein Bcl-2 expression was found in stimulated cells, but not in the unstimulated cells. Furthermore, TLR activation resulted in the production of the interleukin(IL)-6, IL-8 and tumor necrosis factor á (TNF-á). Interestingly, tumor cell proliferation was increased within 24 and 48 hours of stimulation. Conclusion: Our results demonstrate TLR2, 4 and/or 9 expression in all human pancreatic cancer cell lines. Additionally, our findings on TLR activation suggest chronic inflammation-mediated TLR signaling to negatively influence tumor cell apoptosis and to shift the cytokine release in pancreatic cancer towards an inflammatory microenvironment. These findings emphasize the particular role of TLR2, 4 and 9 and their activation in pancreatic cancer, outlining their relevance as potential targets for cancer therapy. Citation Format: Jennifer Kreckel, Tanja Grimmig, Romana Moench, Christoph T. Germer, Martin Gasser, Ana Maria Waaga-Gasser. Cancer cell-mediated signaling of TLR 2, 4, and 9 causes activation of PI3K/Akt/mTOR pathway and induces tumor cell proliferation 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 4879.

Multiple Pharmacological Properties of a Novel Parthenin Analog P16 as Evident by its Cytostatic and Antiangiogenic Potential Against Pancreatic Adenocarcinoma PANC -1 Cells.

Pancreatic ductal adenocarcinoma (PDA) remains one of the deadliest types of cancers. Median survival rate is very poor with the currently available chemotherapeutical regimens. Therefore, discovery of new antineoplastic agents against PDA is one of the focused areas of contemporary research. The present study was undertaken to explore the antitumour activity of a potent parthenin analog P16. Among PANC-1, Mia PaCa-2 and AsPC-1 pancreatic cancer cells, PANC-1 showed highest sensitivity to P16 with an IC50 value of 3.4 μM. Time dependent cell cycle studies revealed that P16 suppressed the growth of PANC-1 cells by arresting the progression through the cell cycle in G2/M phase via downregulation of cyclin B1 and cyclin A. However, P16 did not alter the expressions of CDK-1 and CDC25C in PANC-1 cells. The P16 induced cell cycle arrest, which consequently, led to induction of apoptosis, which was accompanied by activation of caspase-9 and -3. Interestingly, PANC-1 cells displayed increasing loss of mitochondrial potential, which seemed to be correlated to the activation of caspase-3. Additionally, P16 was also able to down-regulate the cell migration in PANC-1 cells. Furthermore, P16 treatment of hypoxic PANC-1 cells strongly suppressed the expression of proangiogenic factors VEGFR-2, HIF1α and HIF1β. Antiangiogenic ability of P16 was also reflected in the human umbilical vascular endothelial cells (HUVECs), where it effectively suppressed the migration and inhibited the formation of the tube in a matrigel based assay. Therefore, cytostatic and antiangiogenic properties of P16 against pancreatic adenocarcinoma cells make it a suitable candidate for further investigation.

MiR-100 regulates cell growth through targeting FGFR3 in patients with pancreatic cancer

Objective To investigate the effects of miR-100 on the proliferation of MIA PaCa-2 and CFPAC-1 cells through targeting fibroblast growth factor receptor 3 (FGFR3). Methods miR-100 expression levels in 17 cancer tissues and 17 nonmalignant tissues were examined by Real-time PCR. The effect of miR-100 overexpression on cell proliferation was examined by CCK-8 assay in vitro. Luciferase assay was used to confirm that miR-100 could directly target FGFR3. Real-time PCR and Western blot were used to examine the expression of FGFR3 in miR-100 overexpressing pancreatic cancer cells. The predicted target gene of miR-100, FGFR3, was downregulated by siRNA, and its effect on cell proliferation was also examined. Cell proliferation was analyzed using CCK-8 and Edu assay. Results miR-100 was lowly expressed in pancreatic cancer tissues (P<0.05). In pancreatic cancer cells, the transfection of lv-miR-100 was able to upregulate the endogenous expression of miR-100 and inhibit the cell proliferation (P<0.05). Luciferase assay showed FGFR3 was the direct target of miR-100. FGFR3 was significantly downregulated by overexpressing miR-100 in pancreatic cancer cells (P<0.05), and FGFR3 knockdown by specific siRNA exerted the similar effect as miR-100 overexpression (P<0.05). Conclusions Our study identified a new miRNA regulator, miR-100, and clarified a novel mechanism of how miR-100 regulates cell proliferation in pancrea-tic cancer. The strategy of overexpressing the tumor suppressor miR-100 may provide a new therapeutic approach for treating patients with pancreatic cancer. Key words: Pancreatic cancer; microRNA-100; Fibroblast growth factor receptor 3; Cell proliferation

Abstract 3574: Novel roles of an axon-guidance molecule, semaphorin 3E, in pancreatic cancer

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is the 4th leading cause of cancer death in the US with a 5-year survival rate of less than 6%. There is an urgent need for a better understanding of PDAC in order to find better therapeutic targets. Two recent large-scale genomic analyses of human PDAC have uncovered increased copy numbers of an axon-guidance gene SEMA3E that codes for Semaphorin-3E (Sema3E). Several recent reports have implicated Sema3E in metastasis of breast and colon cancers, particularly in promoting tumor cell migration and invasion, as well as inducing epithelial-to-mesenchymal transition (EMT). We hypothesize a possible new role of an axon-guidance gene Sema3E in PDAC progression by promoting cell proliferation, invasion, and metastasis. Materials and Methods: To determine expression levels of Sema3E in human PDAC, qRT-PCR and IHC analysis was performed on resected PDAC samples and matched tissue controls. To determine Sema3E levels in mouse PDAC, IHC analysis was performed on pancreas tissue from a genetically engineered spontaneous PDAC mouse model, the PDX-1-Cre; KrasG12D; P53R172H (KPC) mouse. To generate Sema3E-overexpressing stable cells, human PDAC cell lines Mia PaCa-2 and Panc-48 cells were transduced with Sema3E-expressing lentivirus or vector control. QRT-PCR and western blot analyses were performed to verify the overexpression of Sema3E. Colony formation assay (CFA) and MTT assay were performed on Sema3E-transduced cells to determine the effects of Sema3E on cell survival and proliferation respectively, while a wound healing assay and boyden chamber assay were performed to determine effects of Sema3E on cell migration and invasion respectively. Results: Sema3E was found to be significantly upregulated in 46% of patient PDAC samples (n = 24), with an average fold-change of 9.54 across the samples. IHC analyses of patient and mouse PDAC revealed higher expression of Sema3E in tumor cells compared to normal cells. Interestingly, Sema3E expression was concentrated in the nuclei of tumor cells while it has been reported in current literature to be a secreted protein found in normal tissues. Sema3E-transduced PDAC cell lines Mia PaCa-2-Sema3E and Panc-48-Sema3E had higher Sema3E expression in cell lysates and culture supernatant compared to control cells. Mia PaCa-2-Sema3E had a 60.4% higher rate of colony formation than the control (p = 0.005), while both Mia PaCa-2-Sema3E and Panc-48-Sema3E had higher proliferation rates (58.8%; p&amp;lt;0.0001 and 31.0%; p&amp;lt;0.0001 respectively) compared to controls. Interestingly, both Sema3E-transduced cells did not have a significant difference in migration and invasion compared to controls. Conclusions: Our findings indicate that Sema3E is overexpressed in PDAC and it may play some pathogenic roles in PDAC progression by enhancing tumor cell proliferation. These findings indicate that Sema3E could be an attractive novel therapeutic target in PDAC treatment strategies. Citation Format: Lin-Kin Yong, Dali Li, Ethan Poteet, Zhengdong Liang, William Fisher, Changyi Chen, Qizhi Cathy Yao. Novel roles of an axon-guidance molecule, semaphorin 3E, 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 3574. doi:10.1158/1538-7445.AM2015-3574