Normal Pancreatic Cells Research Articles

Glucose-Dependent FOXM1 Promotes Epithelial-to-Mesenchymal Transition Via Cellular Metabolism and Targeting Snail in Human Pancreatic Cancer.

Transcription factor Forkhead box protein M1 (FOXM1) plays critical roles in the progression of cancer including epithelial-to-mesenchymal transition (EMT). The aim of this study is to characterize the regulatory mechanisms of FOXM1 in EMT via pancreatic cancer metabolism. We investigated the regulation of EMT via mitochondrial respiration by FOXM1 using pancreatic cancer cell lines HPAC and PANC-1 and normal human pancreatic duct epithelial cells. Forkhead box protein M1 and Snail were strongly expressed in HPAC and PANC-1. Epithelial-to-mesenchymal transition-modulated claudin-1 level was lower in PANC-1 than in HPAC. In both cell lines in low-glucose medium, FOXM1 and Snail were decreased and claudin-1 was increased. Knockdown of FOXM1 increased claudin-1 and decreased Snail in both cell lines. Low-glucose medium and downregulation of FOXM1 inhibited the cell migration in both cell lines. In both cell lines, mitochondrial respiration was at higher levels in low-glucose medium than in high-glucose medium. Downregulation of FOXM1 induced mitochondrial respiration in high-glucose medium. In normal human pancreatic duct epithelial cells, FOXM1 and Snail were low and claudin-1 was highly expressed, whereas overexpression of FOXM1 decreased claudin-1. Glucose-dependent FOXM1 promoted EMT via Snail and pancreatic cancer metabolism.

Antiplatelet Drug Ticagrelor Enhances Chemotherapeutic Efficacy by Targeting the Novel P2Y12-AKT Pathway in Pancreatic Cancer Cells.

Background: Extensive research has reported that extracellular ADP in the tumour microenvironment can stimulate platelets through interaction with the platelet receptor P2Y12. In turn, activated platelets release biological factors supporting cancer progression. Experimental data suggest that the tumour microenvironment components, of which platelets are integral, can promote chemotherapy resistance in pancreatic ductal adenocarcinoma (PDAC). Thus, overcoming chemoresistance requires combining multiple inhibitors that simultaneously target intrinsic pathways in cancer cells and extrinsic factors related to the tumour microenvironment. We aimed to determine whether ticagrelor, an inhibitor of the ADP–P2Y12 axis and a well-known antiplatelet drug, could be a therapeutic option for PDAC. Methods: We investigated a functional P2Y12 receptor and its downstream signalling in a panel of PDAC cell lines and non-cancer pancreatic cells termed hTERT-HPNE. We tested the synergistic effect of ticagrelor, a P2Y12 inhibitor, in combination with chemotherapeutic drugs (gemcitabine, paclitaxel and cisplatin), in vitro and in vivo. Results: Knockdown studies revealed that P2Y12 contributed to epidermal growth factor receptor (EGFR) activation and the expression of SLUG and ZEB1, which are transcriptional factors implicated in metastasis and chemoresistance. Studies using genetic and pharmacological inhibitors showed that the P2Y12–EGFR crosstalk enhanced cancer cell proliferation. Inhibition of P2Y12 signalling significantly reduced EGF-dependent AKT activation and promoted the anticancer activity of anti-EGFR treatment. Importantly, ticagrelor significantly decreased the proliferative capacity of cancer but not normal pancreatic cells. In vitro, synergism was observed when ticagrelor was combined with several chemodrugs. In vivo, a combination of ticagrelor with gemcitabine significantly reduced tumour growth, whereas gemcitabine or ticagrelor alone had a minimal effect. Conclusions: These findings uncover a novel effect and mechanism of action of the antiplatelet drug ticagrelor in PDAC cells and suggest a multi-functional role for ADP-P2Y12 signalling in the tumour microenvironment.

Lower Nr5a2 Level Downregulates the β-Catenin and TCF-4 Expression in Caerulein-Induced Pancreatic Inflammation.

Nuclear receptor subfamily 5 group A member 2 (Nr5a2) is widely involved in the physiological and pathological processes of the pancreas. However, the cytological and molecular evidence regarding how Nr5a2 implicated in acute pancreatitis (AP) remains insufficient. Here, we explored this problem by using cellular AP model in both normal and Nr5a2 silenced AR42J pancreatic acinar cells. An in vitro cellular model of AP was established by stimulating AR42J cells with caerulein (CAE) for 24 h. Reduced Nr5a2 expression was observed in the CAE-treated cells. Nr5a2 silencing led to AP-like inflammation, with increased interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α mRNA levels. In the cellular AP model, Nr5a2 silencing further increased IL-1β, IL-6, and TNF-α mRNA levels, as well as amylase activity. In addition, we found that Nr5a2 silencing did not affect IL-10 level under physiological conditions but inhibited the anti-inflammatory response of IL-10 in AP model. Moreover, in CAE-induced pancreatic inflammation, Nr5a2 silencing increased the apoptosis and necrosis of acinar cells and inhibited the proliferation of acinar cells, which has not been shown previously. Further experiments showed, for the first time, that Nr5a2 silencing downregulated the expression of β-catenin and its downstream target gene T-cell factor (TCF)-4 in the cellular AP model but increased the expression of nuclear factor (NF)-κB. In conclusion, in CAE-induced pancreatic inflammation, lower Nr5a2 level leads to downregulation of β-catenin and its downstream target gene TCF-4 and upregulation of NF-κB, which exacerbates the inflammatory response and cell damage and inhibits the proliferation and regeneration of acinar cells.

Upregulation of DAB2IP Inhibits Ras Activity and Tumorigenesis in Human Pancreatic Cancer Cells.

KRAS mutation-induced Ras activation plays an important role in the pathogenesis of pancreatic cancer, but the role of wild-type Ras and Ras GTPase-activating proteins remains unclear. The present study was designed to determine the expression spectra of Ras GTPase-activating proteins genes in pancreatic cancer cells, and the role of DAB2IP, a Ras GTPase-activating proteins gene, in the development and progression of pancreatic cancer. Following the analyses of the expression profiles of 16 Ras GTPase-activating proteins in 6 pancreatic cancer cell lines including Bxpc-3 (with wild-type KRAS), Capan-2, Sw1990, Aspc-1, CFPAC-1, and Panc-1 (with mutant KRAS) and 1 normal human pancreatic ductal epithelial cell line, H6C7, the expression of DAB2IP messenger RNA was further analyzed by quantitative real-time polymerase chain reaction. The role of DAB2IP in pancreatic cancer was further investigated in vitro and in vivo by upregulating DAB2IP in Bxpc-3 cells through transfection of DAB2IP into Bxpc-3 cells with recombinant lentivirus. The DAB2IP expression in pancreatic cancer cells and tissues with wild-type KRAS was significantly lower than that in cells and tissues with mutant KRAS (P < .05). In Bxpc-3 cells with wild-type KRAS, overexpression of DAB2IP decreased the expression of P-AKT and P-ERK and the Ras activity; increased the expression of P-JNK and caspase 3; inhibited cell proliferation, invasiveness, and migration; and increased the cell sensitivity to cetuximab. Overexpression of DAB2IP inhibited tumor progression in a mouse model. In conclusion, DAB2IP downregulates Ras activity in wild-type pancreatic cancer cells. Overexpression of DAB2IP decreases the Ras activity, inhibits cell proliferation, and increases sensitivity to cetuximab in wild-type pancreatic cancer cells. In conclusion, DAB2IP may serve as a potential molecular therapeutic target for the treatment of pancreatic cancer.

Nanoparticles modified by triple single chain antibodies for MRI examination and targeted therapy in pancreatic cancer.

Triple scAbs (scAbMUC4, scAbCEACAM6, scFvCD44v6, MCC triple scAbs) were conjugated to the surface of polyethylene glycol modified IONPs (IONPs-PEG), forming the IONPs-PEG-MCC triple scAbs nanocomposite. Characterization of the nanocomposite was performed, and its cytotoxicity, specificity, and apoptosis induction were evaluated. In vivo MRI study and anti-pancreatic cancer effect assessment were performed in tumor-bearing nude mice. The size of the IONPs-PEG-MCC triple scAbs nanocomposite was about 23.6 nm. The nanocomposite was non-toxic to normal pancreatic ductal epithelial cells, and could specifically bind to and be internalized by MUC4/CEACAM6/CD44v6-expressing PDAC cells. With an r2 relaxivity of 104.2 mM-1 s-1, the IONPs-PEG-MCC triple scAbs nanocomposite could significantly shorten the MRI T2-weighted signal intensity both in vitro and in vivo. The IONPs-PEG-MCC triple scAbs nanocomposite also showed a favorable anti-pancreatic cancer effect. In the present study, the IONPs-PEG-MCC triple scAbs nanocomposite was firstly confirmed as a bi-functional nanocomposite in both MRI and treatment, providing its critical clinical transformation potential in PDAC detection and treatment.

Normal Components and Contaminants.

The pancreas is a retroperitoneal organ located in the duodenal loop with the posterior wall of the stomach overlying it and the left lobe of the liver lying anteriorly to it. Tissues from these organs, in addition to the lesion of interest within the pancreas, may be sampled during fine-needle aspiration (FNA) procedures. Therefore, it is important to recognize the cytology of normal benign components of the pancreas and potential contaminants in order to render a correct diagnosis and avoid pitfalls. Normal components of the pancreas include ductal epithelial cells, acinar cells, and islet cells. In addition to the normal pancreatic cells, it is not uncommon to encounter epithelial cells from the duodenal and gastric mucosa with endoscopic ultrasound-guided fine-needle aspiration. It is important to recognize these cells as benign and to distinguish them from a well-differentiated pancreatic adenocarcinoma. Besides these, mesothelial cells and hepatocytes and bile duct cells from the liver may be sampled as well. Here, the cytological features of normal components and contaminants are described in detail.

Abstract C41: Establishment of a novel mouse model of pancreatic squamous carcinoma

Abstract Pancreatic cancer is a lethal malignancy with the highest mortality rate among all cancers and a 5-year survival rate of 9% in the US. Among all subtypes of pancreatic cancers, the molecular mechanisms underlying the development of squamous carcinomas are not well understood. While pure squamous carcinoma makes up only 0.05-5% of all pancreatic cancers, it is very aggressive and leads to the worst prognosis among all pancreatic cancers. However, animal models to study the initiation and progression of pancreatic squamous carcinoma are lacking. Genetic studies have recently revealed the molecular signature of pancreatic cancers and identified the transcription factor p63 as a candidate that contributes to the development of pancreatic carcinomas. In one study (Bailey et al., Nature 2016;47-52), upregulation of the p63-mediated transcriptional network was reported. Another genome-wide analysis has identified a SNP in the p63 gene as a risk factor for the development of pancreatic cancers (Childs et al., Nat Genet 2015;911-6). Furthermore, it has been shown that expression of TAp63, an isoform of p63 with a tumor suppressor function, is frequently suppressed in pancreatic cancers (Bailey et al., Nature 2016;47-52). While p63 is rarely mutated in human cancers, ΔNp63α, the predominant isoform of p63 in epithelia, is overexpressed in a variety of human cancers of squamous cell origin. Notably, although p63 proteins are not expressed in normal pancreatic cells, they are expressed at high levels in areas of squamous differentiation of pancreatic cancers (Basturk et al., Modern Pathol 2005;1193-8). We hypothesize that aberrant expression of ΔNp63α in the pancreatic epithelia drives the tumorigenesis of pancreatic squamous carcinomas. To test this hypothesis, we have created transgenic (Tg) mice conditionally expressing ectopic ΔNp63α upon crossing with tissue/cell type-specific Cre-Tg mice in the presence of the K-rasG12D mutation (Hingorani et al., Cancer Cell 2003;437-50), a pancreatic cancer-promoting factor. Our data show that Pdx1Cre:KrasG12D:ΔNp63α-Tg mice develop pancreatic squamous carcinoma as determined by the cellular morphology and the shift of the expression of cytokeratin 8 (K8), a pan-pancreatic epithelial cell marker, into K5, a squamous epithelial cell marker. Interestingly, the absence of the K-ras mutation did not lead to the development of pancreatic tumors, indicating that de novo expression of ΔNp63α alone is not sufficient for the generation of pancreatic squamous carcinomas. None of the mice in our control cohort of the Pdx1Cre:KrasG12D:p53+/- genotype (Bardeesy et al., PNAS 2006;5947-52) developed pancreatic squamous carcinomas or showed p63 expression, emphasizing the functional differences between the two p53 family members. We aim to further characterize our novel animal model and determine the molecular mechanisms by which p63 contributes to the squamous pathogenesis of pancreatic cancer. Citation Format: Filipa Pinto, Tachira Pichardo, Adrien Leu, Makoto Senoo. Establishment of a novel mouse model of pancreatic squamous carcinoma [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 C41.

Abstract I22: New players and unique features of cancer lysosomes

Abstract Pancreatic ductal adenocarcinoma (PDA) cells and tumors constitutively activate nutrient scavenging pathways, namely, autophagy (cellular self-catabolism) and macropinocytosis (bulk uptake of extracellular material) in order to maintain metabolic homeostasis. Importantly, both of these pathways converge on the lysosome, an acidic organelle that harbors within its lumen a complex series of enzymatic reactions that enable degradation of incoming cargo material. Our prior work has shown that the lysosome is greatly expanded and qualitatively different in PDA cells compared to normal tissue. PDA tumors are effectively “addicted” to enhanced catabolic activity as blocking lysosome function specifically inhibits in vitro and in vivo growth of PDA cell lines and tumor xenografts with minimal effects on normal cells. How key changes in lysosome composition and content contribute to the aggressive nature of PDA remains an open question. We have optimized methodologies to isolate and purify lysosomes from normal human pancreatic epithelial cells and several established patient-derived PDA cancer cell lines, using affinity-based capture techniques that isolate 50-80% intact lysosomes free of cytoplasm and other contaminating organelles. By systematically implementing lysosome purification from normal, primary, and metastatic PDA tumors combined with mass spectrometry-based proteomics, we have identified both resident and substrate proteins that are uniquely associated with PDA lysosomes. Several cancer-specific lysosomal membrane proteins emerged from this analysis that may endow cancer lysosomes with enhanced functionality, including rapid membrane repair in the face of sustained mechanical and chemical insults. Similarly, we find that PDA lysosomes degrade select cellular proteins to enable dynamic remodeling of the cellular proteome in a manner that favors tumor growth and progression. Collectively, our data provide strong evidence in support of the lysosome as a central mediator of cellular adaptation to stress and promoter of tumorigenesis. Citation Format: Rushika M Perera. New players and unique features of cancer lysosomes [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 I22.

Abstract A42: Single-cell RNA-seq analysis of human pancreatic ductal adenocarcinoma identifies a novel cell type expressing the intestinal stem cell marker OLFM4

Abstract Pancreatic ductal adenomcarcinoma (PDAC) is one of the most lethal forms of cancer with a 5-year survival rate of 8%. To determine the composition of different cell types in PDAC, we performed single-cell RNA-sequencing (scRNA-seq) analysis of two resected patient tumors. Nine distinct cell types were identified: (1) ductal carcinoma cells expressing mutant KRAS, (2) normal pancreatic islet cells, (3) B-lymphocytes, (4) macrophages, (5) mast cells, (6) nerve cells, (7-8) two types of cancer-associated fibroblasts (CAFs), and (9) a previously unidentified epithelial type with distinctly high expression of OLFM4 and CRISP3. Histologic analysis of human PDAC and chronic pancreatitis (CP) tissue sections confirmed the presence of this distinct cell type and furthermore demonstrated that it corresponded to inflamed/injured ducts and early pancreatic intraepithelial neoplasms (PanINs) but that actual PDACs did not express OLFM4. CRISP3 was not always expressed in this new cell type, but the expression of OLFM4 was always detected in these histologic structures. This distinct pancreatic epithelial cell type was also observed in mouse models for chronic pancreatitis and in mutant-KRAS induced PanINs. We hypothesize that inflammation-induced OLFM4 expression helps drive PanIN formation, and that its expression could be exploited for early PDAC detection. We are currently performing functional studies using organoids to address the role that OLFM4 might play in generation of this distinct cell type. Citation Format: Manisha Rao, Alice Nemajerova, Jinyu Li, Mei Gao, Ki Oh, Richard Moffit, Joseph Kim, George Georgakis, Aaron Sasson, Agnieszka Bialkowkska, Scott Powers. Single-cell RNA-seq analysis of human pancreatic ductal adenocarcinoma identifies a novel cell type expressing the intestinal stem cell marker OLFM4 [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 A42.

Effect of microRNA-135a-5p on proliferation, migration and chemoresistance of pancreatic cancer cell

Objective To investigate the effect of microRNA (miRNA, miR)-135a-5p on proliferation, migration and chemoresistance of pancreatic cancer. Methods Expression of miR-135a-5p in pancreatic cancer tissues and adjacent normal tissues, Panc1 and AsPc-1 cell lines as well as normal pancreatic duct epithelial cell line HPDE were detected by reverse transcription-polymerase chain reaction (RT-qPCR). The Panc1 and AsPc-1 cells were transfected with miR-135a-5p mimics, the proliferation and migration abilities of cells in each group were detected by cell counting kite-8 (CCK-8) assay and scratch assay. Panc1 and AsPc-1 cells were treated with cisplatin of different concentrations, absorbance values were detected by microplate reader and the chemosensitivity to cisplatin were compared in each group. The downstream target genes of miR-135a-5p were predicted using online software and the dual-luciferase reporter gene system was used for verification. The expression levels of branched chain amino acid aminotransferase1 (BCAT1) in each group were detected by Western blotting. Results The expression of miR-135a-5p in pancreatic cancer tissues was significantly lower than that in adjacent tissues (0.23±0.08 vs. 1.03±0.18), and the expression of miR-135a-5p in Panc1 and AsPc-1 cells was significantly lower than that in normal pancreatic duct epithelial cell line HPDE (0.30±0.13, 0.56±0.09 vs. 0.97±0.12, P<0.05). The proliferation and migration ability of Panc1 and AsPc-1 cells in the miR-135a-5p mimic group were significantly inhibited (P<0.05), and the expression levels of BCAT1 protein in the cells were significantly decreased. The dual-luciferase reporter gene system confirmed that miR-135a-5p could target and regulate the expression of BCAT1 gene. The sensitivity of Panc1 cells to cisplatin chemotherapy was significantly increased in the miR-135a-5p mimic group (P<0.05). The co-transfection of Panc1 cells with miR-135a-5p mimic and BCAT1 overexpression vector partially reversed the decreased cell proliferation, decreased migration ability and increased sensitivity to cisplatin chemotherapy mediated by the upregulation of miR-135a-5p. Conclusion MiR-135a-5p is low expressed in pancreatic cancer, and upregulation of miR-135a-5p can inhibit cell proliferation and migration of pancreatic cancer cells by targeting BCAT1 gene, and increase the sensitivity of cisplatin chemotherapy. Key words: MicroRNA-135a-5p; Branched chain amino acid aminotransferase 1; Pancreatic cancer; Drug resistance

Attenuation of hedgehog/GLI signaling by NT1721 extends survival in pancreatic cancer

BackgroundPancreatic cancer is one of the most lethal malignancies due to frequent late diagnosis, aggressive tumor growth and metastasis formation. Continuously raising incidence rates of pancreatic cancer and a lack of significant improvement in survival rates over the past 30 years highlight the need for new therapeutic agents. Thus, new therapeutic agents and strategies are urgently needed to improve the outcome for patients with pancreatic cancer. Here, we evaluated the anti-tumor activity of a new natural product-based epidithiodiketopiperazine, NT1721, against pancreatic cancer.MethodsWe characterized the anticancer efficacy of NT1721 in multiple pancreatic cancer cell lines in vitro and in two orthotopic models. We also compared the effects of NT1721 to clinically used hedgehog inhibitors and the standard-of-care drug, gemcitabine. The effect of NT1721 on hedgehog/GLI signaling was assessed by determining the expression of GLI and GLI target genes both in vitro and in vivo.ResultsNT1721 displayed IC50 values in the submicromolar range in multiple pancreatic cancer cell lines, while largely sparing normal pancreatic epithelial cells. NT1721 attenuated hedgehog/GLI signaling through downregulation of GLI1/2 transcription factors and their downstream target genes, which reduced cell proliferation and invasion in vitro and significantly decreased tumor growth and liver metastasis in two preclinical orthotopic mouse models of pancreatic cancer. Importantly, treatment with NT1721 significantly improved survival times of mice with pancreatic cancer compared to the standard-of-care drug, gemcitabine.ConclusionsFavorable therapeutics properties, i.e. 10-fold lower IC50 values than clinically used hedgehog inhibitors (vismodegib, erismodegib), a 90% reduction in liver metastasis and significantly better survival times compared to the standard-of-care drug, gemcitabine, provide a rational for testing NT1721 in the clinic either as a single agent or possibly in combination with gemcitabine or other therapeutic agents in PDAC patients overexpressing GLI1/2. This could potentially result in promising new treatment options for patients suffering from this devastating disease.

Inhibitory Effect of Oat Bran Ethanol Extract on Survival and Gemcitabine Resistance of Pancreatic Cancer Cells.

Pancreatic cancer (PC) is one of the most aggressive malignancies in the world. Gemcitabine (Gem), a nucleoside pyrimidine analogue, is a first-line chemotherapeutic drug for PC, but the tumor response rate of Gem is very low and resistance to Gem has emerged as a major problem in the treatment of PC. Oat bran, used as animal and human food, has been found to be beneficial to health. In this study, effects of oat bran ethanol extract (OBE) on PC cells and Gem-resistant PC cells were investigated in vitro. OBE decreased cell survival and colony forming ability of PC cells, without any cytotoxicity on the normal pancreatic cells. Flow cytometry analysis and TUNEL assay showed that the OBE reduced G1/S phase transition and induced death in PC cells through AMPK activation and downregulation of JNK. Additionally, OBE could overcome Gem resistance through reduction in RRM1/2 expression and showed synergistic effect by combinatorial treatment with Gem on Gem-resistant PC cells. Additionally, LC-MS data showed that avenacoside A was a component of OBE. Thus, this study elucidated the anti-proliferative effect of OBE and synergistic effect of OBE with Gem on PC cells and Gem-resistant cells.

RETRACTED ARTICLE: Silencing of Glutathione S-Transferase Pi Inhibits Cancer Cell Growth via Oxidative Stress Induced by Mitochondria Dysfunction

Antitumor drug development based on the concept of intervening in the antioxidant system of cancer cells has been gaining increased interest. In this study, we propose a promising strategy for cancer treatment using modulation of oxidative stress by suppression of glutathione S-transferases (GSTs), a typical antioxidant enzyme. siRNA which can be applied to the development of nucleic acid drugs, enabling them to eliminate unwanted side effects, increase specificity, and avoid the problem of drug resistance, was employed for GSTP-silencing at the transcriptional level. The silencing of the pi class of GST (GSTP) that displayed the most characteristic expression profile in 13 kinds of cancer cell lines has shown significant impairment in the growth of cancer cells due to oxidative stress caused by excess ROS accumulation. Comparative proteomics between normal cells and GSTP-silenced pancreatic cancer cell PANC-1 suggested that GSTP-silencing facilitated the mitochondrial dysfunction. These findings show promise for the development of strategies toward cancer therapy based on the mechanism that allows genetic silencing of GSTP to promote oxidative stress through mitochondria dysfunction.

Phospho-valproic acid (MDC-1112) reduces pancreatic cancer growth in patient-derived tumor xenografts and KPC mice: enhanced efficacy when combined with gemcitabine.

New chemotherapeutic agents are needed for pancreatic cancer (PC). We have previously shown that phospho-valproic acid (MDC-1112) is effective in cell-line xenografts of PC. Here, we explored whether MDC-1112 is effective in additional clinically relevant animal models of PC and whether MDC-1112 enhances the anticancer effect of clinically used chemotherapeutic agents. MDC-1112 alone strongly reduced patient-derived pancreatic tumor xenograft growth, and extended survival of LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx1-Cre (KPC) mice. In both models, MDC-1112 inhibited STAT3 activation and its downstream signals, including Bcl-xL and cyclin D1. In human PC cell lines, P-V enhanced the growth inhibitory effect of gemcitabine (GEM), Abraxane and 5-FU, but not that of irinotecan. Normal human pancreatic epithelial cells were more resistant to the cytotoxic effects of MDC-1112/GEM combination. Furthermore, MDC-1112 enhanced GEM's effect on colony formation, apoptosis, cell migration, and cell invasion. In vivo, MDC-1112 and GEM, given alone, reduced patient-derived pancreatic tumor xenograft growth by 58% and 87%, respectively; whereas MDC-1112/GEM combination reduced tumor growth by 94%, inducing tumor stasis. In conclusion, MDC-1112 should be further explored as a potential agent to be used in combination with GEM for treating PC.

NELFE promoted pancreatic cancer metastasis and the epithelial‑to‑mesenchymal transition by decreasing the stabilization of NDRG2 mRNA.

Negative elongation factor E (NELFE) has been demonstrated to promote cancer progression as an RNA-binding protein (RBP). However, the expression patterns, biological role and molecular mechanism of NELFE in pancreatic cancer (PC) remain largely unknown. The expression levels of NELFE in 120 pairs of PC tissues and adjacent non-tumor clinical samples collected from patients with PC were examined via reverse transcription-quantitative (RT-q) PCR and immunohistochemistry. The mRNA expression levels of NELFE, N-Myc downstream-regulated gene 2 (NDRG2), c-Myc, survivin and cyclin D1 were detected via RT-qPCR. The protein expression levels of NELFE, NDRG2, total β-catenin, nuclear β-catenin, cytosolic β-catenin, E-cadherin, N-cadherin and Vimentin were measured by western blotting. NELFE and NDRG2 were then knocked-down by short hairpin (sh)RNA. PC cell proliferation was detected by MTT and colony formation assays. Invasion and migration were detected by transwell assays. The interaction between NELFE and NDRG2 was detected by luciferase reporter assays, mRNA decay assays and RNA immunoprecipitation. NELFE expression was increased in PC tissues compared with the paired non-cancerous tissues. NELFE expression was upregulated in PC cells when compared with normal pancreatic cells (HPDE6-C7). The present study revealed that knockdown of NELFE inhibited the proliferation, invasion and migration of PC cells. In addition, transfection of the sh-NELFE vector inhibited the epithelial-to-mesenchymal transition in PC cells by suppressing the expression and nuclear accumulation of β-catenin. Further mechanistic studies revealed that NELFE activates the Wnt/β-catenin signaling pathway by decreasing the stabilization of NDRG2 mRNA in PC. To the best of our knowledge, these results revealed the promotional function of NELFE on PC tumorigenesis and metastasis for the first time, helping to provide a promising strategy for the treatment of patients with PC.

2019P - Protein tyrosine phosphatase non-receptor type 3 (PTPN3) could be a new therapeutic target for pancreatic cancer

BackgroundPancreatic cancer (PC) is a refractory disease and we have not an effective therapeutic strategy yet. Therefore, the development of a new therapeutic procedure is urgently required. Recently, protein tyrosine phosphatase (PTP) has been attracted as a new therapeutic target for some types of cancers. However, the biological significance of PTP in PC has never been elucidated yet. In the present study, to develop effective therapeutic strategy for refractory PC, the biological significance of PTP non-receptor type 3 (PTPN3) in PC is investigated. MethodsThree pancreatic ductal adenocarcinoma cell (PDAC) lines were used as target cells. Inhibition or overexpression of PTPN3 was performed using PTPN3 siRNA/shRNA and plasmid, respectively. Protein expression was analyzed by western blot and immunostaining. Proliferation assay was performed by MTT assay. Migration and invasion were estimated by time-lapse imaging and matrigel invasion assay. Mice xenograft experiments were performed using BALB/c nude mice. Twenty-three surgically resected human PC specimens were used for immunostaining. Results1) PTPN3 is highly expressed in PC cells compared to that in normal pancreatic duct cells by immunostaining. 2) Inhibition of PTPN3 significantly decreased migration and invasion in PDAC through inhibition of epithelial mesenchymal transition (EMT) and matrix metalloproteinase (MMP)-2 expression. 3) PTPN3 inhibition significantly decreased proliferation in vitro in PDAC. 4) PTPN3 overexpression led to increased proliferation, migration and invasion in PDAC. 5) Tumor volume in mice injected with PTPN3-inhibited PDAC was significantly lower than that in control mice. The expressions of Ki-67 and VEGF in PTPN3-inhibited PDAC were lower than those in control. 6) Signaling from PTPN3 was through PI3K and MAPK signaling pathways. 7) When intensity of PTPN3 expression was divided into 2 groups; weak and strong, intensity of PTPN3 expression was positively correlated with tumor size (T factor) and clinical staging. ConclusionsThese results suggest that PTPN3 contributes to the induction of malignant phenotypes of pancreatic cancer and that PTPN3 could be a new effective and specific therapeutic target for PC. Legal entity responsible for the studyKyushu University Hospital, Cancer Therapy and Research. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.

The RNA m6A methyltransferase METTL3 promotes pancreatic cancer cell proliferation and invasion

Epigenetic modifications are involved in carcinogenesis and METTL3 is involved in RNA methylation. This study aimed to explore the role of the RNA m6A methyltransferase METTL3 in pancreatic cancer cells. The m6A modification was analyzed in human pancreatic cancer and paracancerous specimens, as well as in the normal HPDE6-C7 pancreatic cell line and the MIA-PaCa-2 and BxPC-3 pancreatic cancer cell lines. Immunohistochemistry (IHC), western blotting, and RT-qPCR were used to detect the expression of METTL3. Cell lines were transfected with siRNAs against METLL3. Proliferation, invasion, and migration were examined. The functions of METTL3 were predicted by bioinformatics analysis. In the 40 patients included, high METTL3 expression was associated with high pathological stage (P = 0.02) and high N stage (P = 0.02). Survival was better in patients with low METTL3 expression compared with those with high MTTL3 expression (P < 0.01). METTL3 and CIITA expression levels were inversely correlated (r = −0.71, P < 0.01). RNA m6A content in tumor specimens was significantly higher than that in paracancerous specimens. METTL3 protein and mRNA levels were significantly higher in tumor specimens compared with paracancerous specimens, as well as in cancerous cell lines vs. normal cells. METTL3 knockdown in MIA PaCa-2 and BxPC-3 cells decreased RNA m6A modifications. Cell proliferation, invasion, and migration were decreased by METTL3 knockdown in cancerous cell lines. A total of 673 differentially expressed genes were identified by bioinformatics: 659 were upregulated and 14 were downregulated. In conclusion, METTL3 is probably involved in pancreatic carcinogenesis. It could eventually be a prognostic marker or a treatment target.

Long noncoding RNA DIO3OS interacts with miR-122 to promote proliferation and invasion of pancreatic cancer cells through upregulating ALDOA

BackgroundLong noncoding RNA (lncRNA) has been implicated in numerous tumors, including pancreatic cancer (PC). However, the precise cellular roles and molecular mechanisms of lncRNA DIO3OS on PC development remains to be fully clarified.MethodsWe performed the meta-analysis on PC samples and non-tumor samples retrieved from the TCGA database, and measured the levels of DIO3OS in PC cell lines and a normal pancreatic duct epithelial cell line HPDE6-C7. Cell proliferation was evaluated via CCK-8 assay. Cell invasion in vitro was investigated by transwell assay. The RNA immunoprecipitation assay and luciferase reporter assay was utilized to confirm the putative miR-122-binding site in DIO3OS. The effects of DIO3OS on PC progression were tested using in vivo subcutaneous xenografts.ResultsOur results showed that DIO3OS was highly expressed in human PC tissues and PC cell lines. DIO3OS exhibited oncogenic properties in stimulating PC cell proliferation and invasion in vitro and promoting cancer growth in vivo. Through online predictive tools and functional experiments, we found that DIO3OS could bind directly to microRNA-122 (miR-122) and inhibited its expression, which functioned as a tumor suppressor in PC cells. We also verified that ALDOA was the direct target of miR-122, and the tumor suppressive effects caused by DIO3OS knockdown or miR-122 overexpression could be rescued by re-expression of ALDOA in PC cells.ConclusionsOverall, our study suggested that lncRNA DIO3OS promotes PC cell growth and invasion by competing for miR-122 to modulate the expression of ALDOA. These findings yield a better understanding of the potential mechanisms by which gain of DIO3OS expression accelerates PC progression.

Abstract 3628: Comparison of CD44 expression in pancreatic cancer, pancreatic stem and normal pancreatic cells: Development of CLENs for tumor targeting and therapy using cell models

Abstract Purpose: The incidence of drug resistance in pancreatic cancer correlates with poor prognosis, increased disease progression and unfavorable treatment outcomes. CD44, a surface membrane proteoglycan has been identified to be directly involved in pancreatic cancer drug resistance and metastasis. As such, the aim of this research was to screen various pancreatic cells for their expression of CD44, and to begin the early design and development of cell membrane lipid-extracted nanoliposmes (CLENs) for enhanced targeting and therapy. Methods: Three pancreatic cancer cells- Panc-1, MiaPaCa 2, MS1 VEGF and a normal cell line- hTERT- were employed for this study. Stem cell, Mia PaCa-2, served as a positive control for screening purposes. Four liposome compositions were prepared using various ratios of DOPC, Chol, DPPE-PEG5000, and cellular membrane lipid-extracted (LE) was derived from target cell, Panc 1. To evaluate CD44 glycoprotein expression, cells were seeded in 48-well plates and incubated overnight. Fluorescence measurements were determined at excitation wavelength of 485/25 and emission wavelength at 528/20 nm. G44-26 monoclonal antibody was used for CLENs antibody conjugation studies. A Sephadex G-25 column was used for separation of bound and unbound antibody material, and fluorescence detection was used for cell culture analysis. Fluorescence detection was performed at excitation and emission wavelengths of 530/25 and 590/25, respectively. Results: The relative level of CD44 expression for each of the cell lines was as follows. Panc1&amp;gt;Mia PaCa-2 = hTERT&amp;gt;MS1-VEGF. There was a statistically significant difference between Panc-1 and Mia Paca 2 (P&amp;lt;0.001), between hTERT and MS1 VEGF (P&amp;lt;0.05), and between Panc-1 and hTERT (P&amp;lt;0.0001). The inclusion of Panc1-LE in combination with additional liposome components did not increase cellular uptake significantly. The conjugation of G44-26 monoclonal antibody to the liposomes increased average size of all 4 preparations. Average size for CLENs and G44-26 monoclonal antibody -CLENs increased from 140.75 ± 3.22 nm to 250.23 ± 15.31 nm. Conclusion: Future studies are required to demonstrate the effect of G44-26 monoclonal antibody liposome conjugates to further exploit the optimal expression of CD44 for pancreatic drug targeting. Citation Format: Dotun D. Adegunle, Eugene Boakye Ansah, Danny Nguyen, Kanchi Sanghvi, Drew Goodrich, Robert Campbell. Comparison of CD44 expression in pancreatic cancer, pancreatic stem and normal pancreatic cells: Development of CLENs for tumor targeting and therapy using cell models [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 3628.

Abstract 2038: NG1/NGL1 engagement supports PDAC development via CAF to PDAC nutrition and CAF-regulated immunosuppression

Abstract Pancreatic ductal adenocarcinoma (PDAC) is predicted to soon become the second killer cancer in the USA. One of the hallmarks of PDAC is desmoplasia, which consists of an expansion in activated stellate cells, known as cancer associated fibroblasts (CAFs), and remodeling of their extracellular matrix. Desmoplasia is a major culprit for the unique PDAC microenvironment, which both collapses blood vessels restricting nutrients and fosters immunosuppression. Crosstalk between CAFs, PDAC, and immune cells, within this microenvironment, is not fully understood. To study the functions of CAFs on PDAC development, we first performed a microarray comparing patient matched normal pancreatic stellate cells and CAFs cultured using our stromal 3D system. We identified proteins upregulated in CAFs that have not been previously implicated in desmoplasia. To understand the functional role of these proteins, CRISPR/Cas9 mediated deletion of target proteins was undertaken and cells were subjected to a number of co-culture assays, with PDAC cells and NK cells. Assays consisted of material transfer, cell engagement, proliferation, survival, NK cell activation and PDAC killing. Syngeneic/orthotopic in vivo models, RNAseq analyzes and quantitative multi-plex microscopy, on pathological samples, were also used. We uncovered the ectopic expressions of NetrinG1 (NG1), a neural pre-synaptic protein, in CAFs, and of NG1’s post-synaptic binding partner, NGL1, in PDAC cells. Using our 3D system, we observed that heterotypic CAF-to-PDAC interactions, via NG1/NGL1 engagement, are critical for providing survival advantages to nutrient deprived PDAC cells and for CAFs to protect PDAC from NK cell-driven elimination. Mechanistically, we uncovered that PDAC starvation is overcome by NG1 expressing CAFs that provide nutrition via material transfer. Further, knockout of NG1, in CAFs, significantly reduced the production of immunosuppressive cytokines, such as IL-6, IL-8, and TGF-β. Moreover, loss of NG1 from CAFs resulted in anti-tumor NK cell activation. Unexpectedly, we observed a link between glutamate metabolism in CAFs and the inactivation of anti-tumor NK cells, and this was dependent on CAFs’ NG1 expression. These results were confirmed in an orthotopic PDAC mouse model in which ablation of NGL1, in syngeneic mouse PDAC cells, significantly halted tumor growth. RNAseq comparing CON or NG1+ CAFs vs. knockout NG1- CAFs rendered changes in neural, metabolic and fibrous signature pathways. Translationally, we saw clear expression of stromal NG1 only in PDAC affected pancreata. Overall, this study identifies two novel targets for PDAC; a cancer that lacks effective therapies. Inhibition of the NG1/NGL1 axis, constitutes a potential future therapeutic approach, as it alters CAF mediated metabolism and reverts immunosuppression, stunting PDAC growth in vitro and in vivo. Citation Format: Ralph Francescone, Débora Barbosa Vendramini-Costa, Janusz Franco-Barraza, Jessica Wagner, Alex Muir, Linara Gabitova, Tatiana Pazina, Tiffany Luong, Neelima Shah, Dustin Rollins, Ruchi Malik, Sapna Gupta, Roshan Thapa, Diana Restifo, Allison Lau, Yan Zhou, Kathy Q. Cai, Harvey Harvey Hensley, Emmanuelle Nicolas, Warren D. Kruger, Karthik Devarajan, Siddharth Balachandran, Wafik S. El-Deiry, Matthew Vander Heiden, Kerry Campbell, Igor Astsaturov, Edna Cukierman. NG1/NGL1 engagement supports PDAC development via CAF to PDAC nutrition and CAF-regulated immunosuppression [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 2038.