Human Pancreatic Ductal Adenocarcinoma Cell Lines Research Articles

68477 Pancreatic cancer cell extracellular vesicles drive the unfolded protein response in recipient normal pancreatic cells.

ABSTRACT IMPACT: This study advances our understanding of potentially key drivers in the early formation of pancreatic cancer, a disease with few treatment options and poor patient outcomes. OBJECTIVES/GOALS: Patients diagnosed with pancreatic ductal adenocarcinoma (PDAC) have a 5-year survival rate of ˜9%. A key driver of poor patient outcomes is late-stage diagnosis. A better understanding of PDAC onset is needed. This study was developed to understand how extracellular vesicles may be involved in the early formation of PDAC. METHODS/STUDY POPULATION: Extracellular vesicles (EVs) were isolated from several human PDAC and normal pancreatic cell lines, using ultracentrifugation with filtration or size exclusion chromatography. We next treated normal pancreatic cell lines with cancer cell EVs (cEVs). Next generation sequencing was used to measure global gene expression changes after treatment. Validations were performed using qPCR and luciferase activity assays. Multi-omics characterization of EVs was accomplished using mass spectrometry based proteomics, metabolomics and lipidomics analysis. RESULTS/ANTICIPATED RESULTS: We found that normal cells upregulated a variety of stress response pathways in response to cEVs. Lipid synthesis was also severely downregulated in these cells. We further validated activation of the unfolded protein response (UPR) in normal cells treated with cEVs. Multi-omics characterization of cEVs identified several enriched proteins, lipids and metabolites which may play a role in the activation of the UPR. DISCUSSION/SIGNIFICANCE OF FINDINGS: Our results indicate that cEVs induce stress, and in particular the UPR, in normal pancreatic cells. Long-term UPR can impact a variety of cancer hallmarks. The UPR can mediate progression of pancreatic intraepithelial neoplasia (PanIN) to PDAC. Our results highlight a potential role for cEVs to alter the function of normal cells, aiding disease onset.

Adipose-derived mesenchymal stem cells differentiate into heterogeneous cancer-associated fibroblasts in a stroma-rich xenograft model

Cancer-associated fibroblasts (CAFs) are the key components of the densely proliferated stroma in pancreatic ductal adenocarcinoma (PDAC) and contribute to tumor progression and drug resistance. CAFs comprise heterogeneous subpopulations playing unique and vital roles. However, the commonly used mouse models have not been able to fully reproduce the histological and functional characteristics of clinical human CAF. Here, we generated a human cell-derived stroma-rich CDX (Sr-CDX) model, to reproduce the clinical tumor microenvironment. By co-transplanting human adipose-derived mesenchymal stem cells (AD-MSCs) and a human PDAC cell line (Capan-1) into mice, the Sr-CDX model recapitulated the characteristics of clinical pancreatic cancer, such as accelerated tumor growth, abundant stromal proliferation, chemoresistance, and dense stroma formed from the heterogeneous CAFs. Global RNA sequencing, single-cell based RNA sequencing, and histological analysis of CAFs in the Sr-CDX model revealed that the CAFs of the Sr-CDX mice were derived from the transplanted AD-MSCs and composed of heterogeneous subpopulations of CAF, including known and unknown subtypes. These lines of evidences suggest that our new tumor-bearing mouse model has the potential to address an open question in CAF research, that is the mechanism of CAF differentiation.

Overall survival of pancreatic ductal adenocarcinoma is doubled by Aldh7a1 deletion in the KPC mouse.

Rationale: The activity of aldehyde dehydrogenase 7A1 (ALDH7A1), an enzyme that catalyzes the lipid peroxidation of fatty aldehydes was found to be upregulated in pancreatic ductal adenocarcinoma (PDAC). ALDH7A1 knockdown significantly reduced tumor formation in PDAC. We raised a question how ALDH7A1 contributes to cancer progression.Methods: To answer the question, the role of ALDH7A1 in energy metabolism was investigated by knocking down and knockdown gene in mouse model, because the role of ALDH7A1 has been reported as a catabolic enzyme catalyzing fatty aldehyde from lipid peroxidation to fatty acid. Oxygen consumption rate (OCR), ATP production, mitochondrial membrane potential, proliferation assay and immunoblotting were performed. In in vivo study, two human PDAC cell lines were used for pre-clinical xenograft model as well as spontaneous PDAC model of KPC mice was also employed for anti-cancer therapeutic effect.Results: ALDH7A1 knockdown significantly reduced tumor formation with reduction of OCR and ATP production, which was inversely correlated with increase of 4-hydroxynonenal. This implies that ALDH7A1 is critical to process fatty aldehydes from lipid peroxidation. Overall survival of PDAC is doubled by cross breeding of KPC (KrasG12D; Trp53R172H; Pdx1-Cre) and Aldh7a1-/- mice.Conclusion: Inhibitions of ALDH7A1 and oxidative phosphorylation using gossypol and phenformin resulted in a regression of tumor formation in xenograft mice model and KPC mice model.

The short isoform of PRLR suppresses the pentose phosphate pathway and nucleotide synthesis through the NEK9-Hippo axis in pancreatic cancer.

Prolactin binding to the prolactin receptor exerts pleiotropic biological effects in vertebrates. The prolactin receptor (PRLR) has multiple isoforms due to alternative splicing. The biological roles and related signaling of the long isoform (PRLR-LF) have been fully elucidated. However, little is known about the short isoform (PRLR-SF), particularly in cancer development and metabolic reprogramming, a core hallmark of cancer. Here, we reveal the role and underlying mechanism of PRLR-SF in pancreatic ductal adenocarcinoma (PDAC).Methods: A human PDAC tissue array was used to investigate the clinical relevance of PRLR in PDAC. The in vivo implications of PRLR-SF in PDAC were examined in a subcutaneous xenograft model and an orthotopic xenograft model. Immunohistochemistry was performed on tumor tissue obtained from genetically engineered KPC (KrasG12D/+; Trp53R172H/+; Pdx1-Cre) mice with spontaneous tumors. 13C-labeled metabolite measures, LC-MS, EdU incorporation assays and seahorse analyses were used to identify the effects of PRLR-SF on the pentose phosphate pathway and glycolysis. We identified the molecular mechanisms by immunofluorescence, coimmunoprecipitation, proximity ligation assays, chromatin immunoprecipitation and promoter luciferase activity. Public databases (TCGA, GEO and GTEx) were used to analyze the expression and survival correlations of the related genes.Results: We demonstrated that PRLR-SF is predominantly expressed in spontaneously forming pancreatic tumors of genetically engineered KPC mice and human PDAC cell lines. PRLR-SF inhibits the proliferation of PDAC cells (AsPC-1 and BxPC-3) in vitro and tumor growth in vivo. We showed that PRLR-SF reduces the expression of genes in the pentose phosphate pathway (PPP) and nucleotide biosynthesis by activating Hippo signaling. TEAD1, a downstream transcription factor of Hippo signaling, directly regulates the expression of G6PD and TKT, which are PPP rate-limiting enzymes. Moreover, NEK9 directly interacts with PRLR-SF and is the intermediator between PRLR and the Hippo pathway. The PRLR expression level is negatively correlated with overall survival and TNM stage in PDAC patients. Additionally, pregnancy and lactation increase the ratio of PRLR-SF:PRLR-LF in the pancreas of wild-type mice and subcutaneous PDAC xenograft tumors.Conclusion: Our characterization of the relationship between PRLR-SF signaling, the NEK9-Hippo pathway, PPP and nucleotide synthesis explains a mechanism for the correlation between PRLR-SF and metabolic reprogramming in PDAC progression. Strategies to alter this pathway might be developed for the treatment or prevention of pancreatic cancer.

S100A16 induces epithelial-mesenchymal transition in human PDAC cells and is a new therapeutic target for pancreatic cancer treatment that synergizes with gemcitabine

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers, with a poor 5-year survival rate of approximately 6%, mostly due to poor treatment response and early progression. The S100 gene family participates in various pathophysiological processes in various malignancies. S100A16 is a member of the S100 family, which is abnormally expressed in PDAC; however, its biological functions and mechanisms of action remain unclear. We analysed the Gene Expression Omnibus (GEO) public database and the gene ChIP data collected in our previous study of human PDAC cell line PANC-1 cocultured with M2 macrophages to identify differentially expressed genes (DEGs). Twenty-three overexpressed genes were identified by screening. Then, the selected genes were analysed using The Cancer Genome Atlas (TCGA) database to assess whether they have significant impact on the overall survival (OS) of PDAC patients. Of the 14 DEGs identified, S100A16 was associated with poor prognosis and was selected for further investigation; the results indicate that S100A16 is positively correlated with epithelial-mesenchymal transition (EMT)-related genes in the TCGA dataset. Subsequent in vitro and in vivo experiments demonstrated that S100A16 induces the EMT to promote the metastasis of human PDAC cells and that the effect is mediated by the enhanced expression of TWIST1 and activation of the STAT3 signalling pathway. The antitumour effect of gemcitabine (GEM) was enhanced in combination with S100A16 downregulation. In conclusion, our findings suggest that S100A16 is a novel potential therapeutic target for human PDAC treatment.

Abstract PO-061: The role of Cathepsin E expression in pancreatic ductal adenocarcinoma tumorigenesis

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) has a dismal 5-year survival rate of 10% due to a lack of early detection and effective treatments. Aspartic protease Cathepsin E (CTSE) expression is increased early in PDAC, but its role in tumorigenesis is unknown. We hypothesize that high CTSE expression promotes proliferation, invasion, inhibition of apoptosis, and the secretion of cancer-associated cytokines in PDAC. Methods: Human PDAC cell lines (Mpanc96, Panc-1, and Miapaca2) were engineered to express high levels of CTSE. Cell proliferation, invasion, and apoptosis were analyzed and compared to control cell lines expressing low levels of CTSE. Cells were treated with aspartic protease inhibitors (Pepstatin A and Ritonavir) and monitored for proliferation (24 - 72 hours) and invasion (48 hours). Apoptosis was measured using propidium iodide and analyzed via flow cytometry. Conditioned media from cells was collected (72 hours) after treatment to assess differential secretion of cancer-associated cytokines using a cytokine array. Results: Proliferation was not affected by the overexpression of CTSE; however, inhibition of CTSE slowed proliferation in the Panc-1 and MiaPaca2, but not Mpanc96 cells. CTSE inhibitors promoted invasion of Mpanc96 and Panc-1, but not Miapaca2 cells, regardless of CTSE levels. Apoptosis was not significantly different between the cell lines treated with the aspartic protease inhibitors compared to controls. Increased expression of CTSE induced the secretion of various cytokines such as epithelial neutrophil-activating protein 78 (ENA-78), a chemokine expressed in inflammatory pancreatic disorders. Treatment with the inhibitor Pepstatin A decreased ENA-78 secretion. Conclusions: CTSE levels does not appear to affect proliferation or apoptosis; however, inhibition of CTSE increased cell invasion and resulted in altered cytokine secretion. Further studies examining how CTSE influences invasiveness, and cytokine secretion may create new opportunities for developing effective therapies for PDAC. Citation Format: Ericka Velez-Bonet, Sabrina Kaul, Corbin Pontious, Marcus Hong, Kelly Dubai, Zobeida Cruz-Monserrate. The role of Cathepsin E expression in pancreatic ductal adenocarcinoma tumorigenesis [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-061.

Abstract PO-055: Signaling regulation of epithelial-mesenchymal transition in the hypoxic tumor microenvironment of pancreas cancer

Abstract Epithelial-mesenchymal transition (EMT) is a normal developmental process by which epithelial cells lose their polarity and cell-cell adhesions to become more motile and invasive mesenchymal cells. EMT is associated with the earliest stages in metastasis and promotes chemoresistance in a variety of carcinomas, including pancreatic ductal adenocarcinoma (PDAC). While specific growth factors (e.g., transforming growth factor beta) are perhaps the most well studied inducers of EMT, the low oxygen tension (hypoxia) characteristic of the PDAC tumor microenvironment has also been reported to drive EMT. The signaling pathways that promote EMT in the hypoxic PDAC tumor microenvironment may therefore represent useful drug targets for combination therapy approaches as adjuvants to augment the efficacy of chemotherapy. However, hypoxia-driven EMT has not been well characterized in PDAC, nor have the relevant hypoxia-driven signaling pathways been identified. Our lab recently developed a statistical model of the multivariate signaling regulation of growth factor-induced EMT to identify that the ERK, JNK, and NF-kappaB pathways coordinate to drive EMT in PDAC cells. In the present study, we sought to determine the roles of those pathways in promoting EMT in PDAC cells in response to hypoxia. We created an in vitro model of hypoxia-driven EMT by culturing PDAC cells in 1% oxygen and characterizing dynamic changes in the expression of hypoxia inducible factors (HIF) 1 and 2 alpha, as well as the concomitant loss of E-cadherin protein and transcripts, increased abundance of vimentin-positive cells, and increased expression of the mesenchymal transcripts VIM, SNAI1, and SNAI2. Hypoxia-driven EMT was observed in human PDAC cell lines, patient-derived xenograft cell lines, and KPC murine cell lines. To probe for the activation of specific signaling pathways, we used a combination of immunoblotting and immunofluorescence microscopy. Automated image analysis of immunofluorescence images was used to understand the relevance of specific signaling pathways for explaining the heterogeneity of EMT within populations of cells. We further identified potential kinase targets for antagonizing EMT in the hypoxic microenvironment and confirmed their roles by pharmacological inhibition and/or siRNA-mediated knockdown. We also found that specific kinase pathways are involved in the stabilization of HIF proteins in hypoxia. The relevance of these pathways in driving EMT is being further validated through analyses of a patient-derived xenograft mouse model of PDAC and a HIF1A deletion/Kras mutant autochthonous mouse model of PDAC. Citation Format: Brooke A. McGirr, Nicholas M. Seyler, Matthew J. Lazzara. Signaling regulation of epithelial-mesenchymal transition in the hypoxic tumor microenvironment of pancreas cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-055.

Abstract PO-041: Preclinical in vivo characterization of a first-in-class humanized antibody targeting alternatively spliced Tissue Factor

Abstract Tissue Factor, the initiator of the extrinsic pathway of coagulation, and its isoform alternatively spliced Tissue Factor (asTF) are often overexpressed in cancer cells. While asTF is only minimally procoagulant, it activates both the extracellular mechanical and intracellular signaling functions of beta-integrins. This leads to activation of multiple MAPK pathways thereby promoting increases in proliferation, migration, and angiogenesis. Previously, we have shown that RabMab1, a rabbit monoclonal antibody specific for human asTF, disrupts the binding of asTF to beta-integrins and thereby inhibits the growth of breast cancer and pancreatic ductal adenocarcinoma (PDAC) cell lines in vitro and in vivo. Here, we report on humanization of RabMab1, assessment of its binding characteristics, and determination of its in vivo properties. The variable regions of the heavy and light chains of hybridoma derived RabMab1 were cloned and used to generate a rabbit/human chimera (cRabMab1). Substitutions of species-specific residues were then carried out to produce humanized RabMab1 (hRabMab1; IgG1 isotype). Antigen binding was assessed via anti-human IgG Fc capture biosensor assays. cRabMab1 was found to have a KD of 4.24 nM whereas hRabMab1 was found to have a KD in the low picomolar range, which could not be precisely measured due to an extremely slow off-rate. The in vivo half-lives of each were determined to be 280 and 908 hours, respectively. Orthotopic co-implantation of cRabMab1 with high-grade, human PDAC cell line Pt45.P1 in athymic nude mice resulted in tumors that were 82% and 92% smaller than tumors in the vehicle and IgG control group groups, respectively, with no statistical difference between vehicle and IgG isotype control groups. Intravenous administration of hRabMab1 slowed the growth of pre-formed orthotopic Pt45.P1 tumors in athymic nude mice by 64% and 61% compared to vehicle and isotype control groups, respectively; again, there was no difference between vehicle and IgG isotype control groups. Immunohistochemical analysis of tumor tissue revealed a statistically significant 68% reduction in neovascularization (CD31), a 58% reduction in M2-polarized macrophages (CD206), and a 24% reduction in proliferating cells (Ki67) in the hRabMab1 cohort. RNAseq analysis of tumor tissue revealed a significant suppression of pathways that promote mitosis and the cell cycle. By qRT-PCR, we validated a greater than 82% reduction in the expression of HBEGF, a gene encoding a cell surface mitogen, and a greater than 68% reduction in the expression of STN1, a gene encoding a component of the CST-complex, a telomere replication and maintenance structure. This is the first proof-of-concept study whereby a novel biologic that targets asTF has been used as a systemically administered single agent, with encouraging results. Because hRabMab1 has a favorable pharmacokinetic profile and is able to suppress PDAC tumor growth in vivo, it comprises an attractive candidate for further clinical development. Citation Format: Clayton S. Lewis, Aniruddha Karve, Kateryna Matiash, Timothy Stone, Pankaj Desai, Vladimir Y. Bogdanov. Preclinical in vivo characterization of a first-in-class humanized antibody targeting alternatively spliced Tissue Factor [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-041.

Suppression of pancreatic cancer liver metastasis by secretion-deficient ITIH5

BackgroundPreviously, we identified ITIH5 as a suppressor of pancreatic ductal adenocarcinoma (PDAC) metastasis in experimental models. Expression of ITIH5 correlated with decreased cell motility, invasion and metastasis without significant inhibition of primary tumour growth. Here, we tested whether secretion of ITIH5 is required to suppress liver metastasis and sought to understand the role of ITIH5 in human PDAC.MethodsWe expressed mutant ITIH5 with deletion of the N-terminal secretion sequence (ITIH5Δs) in highly metastatic human PDAC cell lines. We used a human tissue microarray (TMA) to compare ITIH5 levels in uninvolved pancreas, primary and metastatic PDAC.ResultsSecretion-deficient ITIH5Δs was sufficient to suppress liver metastasis. Similar to secreted ITIH5, expression of ITIH5Δs was associated with rounded cell morphology, reduced cell motility and reduction of liver metastasis. Expression of ITIH5 is low in both human primary PDAC and matched metastases.ConclusionsMetastasis suppression by ITIH5 may be mediated by an intracellular mechanism. In human PDAC, loss of ITIH5 may be an early event and ITIH5-low PDAC cells in primary tumours may be selected for liver metastasis. Further defining the ITIH5-mediated pathway in PDAC could establish future therapeutic exploitation of this biology and reduce morbidity and mortality associated with PDAC metastasis.

Paradoxical Role of AT-rich Interactive Domain 1A in Restraining Pancreatic Carcinogenesis.

Simple SummaryGenes with deleterious mutations in tumors are widely accepted as tumor suppressors, since, loss of their normal expression often promotes tumor development. However, most tumors develop over a long period, with gradual accumulation of tumor-promoting events. Thus, to comprehend the role of individual genes in this evolving process of tumorigenesis, it is critical to investigate their role in both early precursors and established tumor cells. Despite recurrent mutations in ARID1A in genomes of human cancer, including pancreatic cancer, its role in tumorigenesis is not clear. We aim to understand the role of Arid1a in pancreatic cancer development and maintenance by investigating its role in both early pancreatic precursor cells and established pancreatic cancer cells. Besides novel understanding of context-dependent role of Arid1a in pancreatic cancer, this study will also enable development of therapeutic strategies for pancreatic cancers patients with ARID1A mutations, which is currently a critical unmet need in clinic.Background & Aims: ARID1A is postulated to be a tumor suppressor gene owing to loss-of-function mutations in human pancreatic ductal adenocarcinomas (PDAC). However, its role in pancreatic pathogenesis is not clear despite recent studies using genetically engineered mouse (GEM) models. We aimed at further understanding of its direct functional role in PDAC, using a combination of GEM model and PDAC cell lines. Methods: Pancreas-specific mutant Arid1a-driven GEM model (Ptf1a-Cre; KrasG12D; Arid1af/f or “KAC”) was generated by crossing Ptf1a-Cre; KrasG12D (“KC”) mice with Arid1af/f mice and characterized histologically with timed necropsies. Arid1a was also deleted using CRISPR-Cas9 system in established human and murine PDAC cell lines to study the immediate effects of Arid1a loss in isogenic models. Cell lines with or without Arid1a expression were developed from respective autochthonous PDAC GEM models, compared functionally using various culture assays, and subjected to RNA-sequencing for comparative gene expression analysis. DNA damage repair was analyzed in cultured cells using immunofluorescence and COMET assay. Results: Retention of Arid1a is critical for early progression of mutant Kras-driven pre-malignant lesions into PDAC, as evident by lower Ki-67 and higher apoptosis staining in “KAC” as compared to “KC” mice. Enforced deletion of Arid1a in established PDAC cell lines caused suppression of cellular growth and migration, accompanied by compromised DNA damage repair. Despite early development of relatively indolent cystic precursor lesions called intraductal papillary mucinous neoplasms (IPMNs), a subset of “KAC” mice developed aggressive PDAC in later ages. PDAC cells obtained from older autochthonous “KAC” mice revealed various compensatory (“escaper”) mechanisms to overcome the growth suppressive effects of Arid1a loss. Conclusions: Arid1a is an essential survival gene whose loss impairs cellular growth, and thus, its expression is critical during early stages of pancreatic tumorigenesis in mouse models. In tumors that arise in the setting of ARID1A loss, a multitude of “escaper” mechanisms drive progression.

Sauchinone inhibits hypoxia-induced epithelial-mesenchymal transition in pancreatic ductal adenocarcinoma cells through the Wnt/β-catenin pathway.

The hypoxic microenvironment is commonly found in various solid tumors including pancreatic ductal adenocarcinoma (PDAC). Saururus chinensis is a medicinal Chinese herb widely used because of documented anti-inflammatory and anti-angiogenic properties. Sauchinone is special active lignin extracted from S. chinensis and its biological functions have been extensively explored. Recent studies have found that sauchinone could affect tumor initiation, metastasis and progression of some cancers. However, the specific role of sauchinone in PDAC remains to be elucidated. The main aim of this study was to elucidate the involvement of sauchinone in the progression of PDAC under the hypoxic condition. The human PDAC cell lines PANC-1 and BxPC-3 were exposed to hypoxia and various concentrations of sauchinone. The CCK-8 assay was performed to detect cytotoxic effects of sauchinone on PDAC cells. The levels of vascular endothelial growth factor, hypoxia-inducible factor-1α, E-cadherin, N-cadherin, Wnt3a and β-catenin were examined by the western blot analysis. Wound healing and transwell assays were used to assess cell migration and invasion. The results showed that the migratory and invasive abilities of PDAC cells were enhanced after exposure to hypoxia and the expression of epithelial-mesenchymal transition markers was also significantly regulated by hypoxia. All these effects induced under the hypoxic condition were terminated by sauchinone treatment. In addition, sauchinone suppressed hypoxia-induced activation of the Wnt/β-catenin signaling pathway. Our study provided important insight into understanding the mechanisms of the anti-cancer effect of sauchinone. Taken together, we suggested that sauchinone may be considered a new therapeutic agent for PDAC treatment.

Antiproliferative Effects of Monoclonal Antibodies against (Pro)Renin Receptor in Pancreatic Ductal Adenocarcinoma

We previously reported that silencing of the PRR gene, which encodes the (pro)renin receptor [(P)RR], significantly reduced Wnt/β-catenin-dependent development of pancreatic ductal adenocarcinoma (PDAC). Here, we examined the effects of a panel of blocking mAbs directed against the (P)RR extracellular domain on proliferation of the human PDAC cell lines PK-1 and PANC-1 in vitro and in vivo We observed that four rat anti-(P)RR mAbs induced accumulation of cells in the G0-G1-phase of the cell cycle and significantly reduced proliferation in vitro concomitant with an attenuation of Wnt/β-catenin signaling. Systemic administration of the anti-(P)RR mAbs to nude mice bearing subcutaneous PK-1 xenografts significantly decreased tumor expression of active β-catenin and the proliferation marker Ki-67, and reduced tumor growth. In contrast, treatment with the handle region peptide of (pro)renin did not inhibit tumor growth in vitro or in vivo, indicating that the effects of the anti-(P)RR mAbs were independent of the renin-angiotensin system. These data indicate that mAbs against human (P)RR can suppress PDAC cell proliferation by hindering activation of the Wnt/β-catenin signaling pathway. Thus, mAb-mediated (P)RR blockade could be an attractive therapeutic strategy for PDAC.

Abstract 6041: LAMC2 as a therapeutic target in pancreatic ductal adenocarcinoma

Abstract BACKGROUND. Pancreatic cancer stands as one of the deadliest tumors, in part due to the limited efficacy of currently existing therapies. Pancreatic ductal adenocarcinoma (PDAC) represents the predominant subtype (around 85% of total cases), where KRAS is the most frequently mutated oncogene. Tumor microenvironment in PDAC is characterized by a desmoplastic stroma which is composed, among other molecules, of collagens of different types, fibronectin and laminins. A member of the laminin family, LAMININ γ2 or LAMC2, was previously identified by our group as part of a cross-tumors KRAS signature whose high expression was a marker of poor survival in PDAC patients. Given the relevant functional implication of other members of the cross-tumors KRAS signature in PDAC, we aimed to investigate the role of LAMC2 in this tumor type. METHODS. A meta-analysis of several human PDAC data sets (n=6) and survival analysis of the TCGA data were performed to query the expression levels and prognosis significance of LAMC2. Human and mouse PDAC cell lines as well as a mouse model of PDAC (Kras, Tp53f/f, Ptf1aCre) were used to assess LAMC2 expression. In vitro (2D and 3D organoids) and in vivo models derived from human and mouse PDAC cell lines or primary tumors were used to define the functional role of LAMC2 in PDAC. Lastly, cellular and molecular analysis were deployed to dissect the mechanism of action of LAMC2 in PDAC. RESULTS. At the clinical level, we have observed that LAMC2 is overexpressed in human PDAC patients with regard to normal tissue and that its high levels identify the group of patients with the worst survival. The overexpression of LAMC2 was recapitulated in human and mouse cell lines. Genetic depletion of LAMC2 had an adverse effect in 2D proliferation, clonogenic efficiency, 3D organoid growth, and PDAC-based xenograft/allograft tumor development. This deleterious effect is driven by an induction in apoptosis and a decrease in S phase consistent across species. Mechanistically, LAMC2 is mainly linked to the KRAS pathway through ERK1/2 and is transcriptionally regulated by the transcription factor FOSL1/AP1, which was also a member of the cross-tumors signature. These data suggest that LAMC2 is involved in the control of different functions related to the PDAC phenotype induced by KRAS. CONCLUSION. LAMC2 is a molecular target in PDAC with clinical implications based on the potential development of new therapeutic and diagnostic strategies for PDAC patients. Citation Format: Oihane Erice, Ester Blanco, Karmele Valencia, Elizabeth Guruceaga, Haritz Moreno, Fernando Lecanda, Vincenzo Corbo, Daniel Ölund, Mariano Ponz-Sarvise, Vicent Silve. LAMC2 as a therapeutic target in pancreatic ductal adenocarcinoma [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 6041.

Abstract 1837: MUC1 confers sensitivity to STAT-3 inhibitor napabucasin in pancreatic ductal adenocarcinoma cells

Abstract Introduction: Pancreatic cancer is the third leading cause of cancer-related deaths in the US and 95% of pancreatic cancers are Pancreatic Ductal Adenocarcinomas (PDACs). Napabucasin is a small molecule inhibitor that targets the signal transducer and activator of transcription 3 (STAT-3) pathway in cancer stem cells. Napabucasin in combination with nab-paclitaxel and gemcitabine, was under phase III clinical trials for gastroenterological cancers including Pancreatic Cancer. The trials have been stopped recently due to futility. Therefore, it is important to find subpopulations of tumor cells that are likely to respond better to Napabucasin treatment alone or as a combination. Mucin1 or MUC1 is a transmembrane glycoprotein found in almost all glandular epithelial cells, and is overexpressed and aberrantly glycosylated in greater than 80% of PDACs. The tumor form of MUC1 is associated with poor prognosis. In PDAC cells, MUC1 expression is critical for tumor cell survival, oncogenic signaling, metastasis, and conferring stemness. Phosphorylated STAT-3 is known to regulate MUC1 expression. Thus, we hypothesize that 1) the STAT-3-MUC1 survival pathway is constitutively activated in MUC1-high PDAC cells and 2) Napabucasin will be more effective in MUC1-high compared to MUC1-low PDAC cells. Methods: Human PDAC cell lines with varying levels of MUC1 (CFPAC, Capan 2, Capan 1, HPAF II, HS766T, BxPc3.MUC1 and BxPC3.Neo) and a normal pancreatic ductal epithelial cell line (HPDE) were treated with increasing doses of Napabucasin (0.05 uM, 0.1uM, 0.2uM, 0.4uM, 0.8uM, 1.6uM, 3.2uM, 6.4uM, 12.8uM and 25.6uM) for 48 and 72 hours. Following treatment, MTT and colony formation assays were performed to determine survival and growth respectively. Results: The cells with high MUC1, CFPAC and Capan 1 showed increased susceptibility to Napabucasin at lower doses (IC50 - 2.088uM and 0.371uM respectively) compared to cells with low MUC1, Capan 2 and HS766T (IC50 - 6.885uM and 7.464uM respectively). MUC1-high HPAF II cells were resistant even at high doses. Data further confirmed that the IC50 of Napabucasin was significantly lower for Bx.Pc3.MUC1 (1.86uM) compared to Bx.Pc3.Neo cells (23.19uM). MUC1-high expressing cell lines lost their colony forming potential at lower doses compared to MUC1-low expressing cell lines. To test if oncogenic signaling linked to MUC1 dimerization confers sensitivity to Napabucasin, the PDA cells were pre-treated with GO-203, a small molecule that inhibits dimerization of MUC1 which renders signaling through the MUC1 cytoplasmic tail non-functional. Conclusion: MUC1 induces susceptibility to Napabucasin in PDACs. Napabucasin has shown anti-tumor activity in preclinical models and early clinical trials of gastroenterological cancers. However, lack of specificity led to increased toxicities and decreased efficacy. It is important to identify and validate biomarkers that better identify patients who have upregulated stemness pathways. MUC1 is one such specific biomarker that could be incorporated in future clinical trials to help identify patient subpopulations that may most likely benefit from combination therapies with stemness-inhibiting drugs. Further study of the crosstalk between stemness signaling pathways is necessary to understand mechanisms of synergistic effects and treatment resistance. Citation Format: Mukulika Bose, Aabha Vora, Taylor Colleton, Pinku Mukherjee. MUC1 confers sensitivity to STAT-3 inhibitor napabucasin in pancreatic ductal adenocarcinoma cells [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 1837.

Abstract 1906: Combination MEK and mTORC1/2 inhibition overcomes resistance to single agent therapy in pancreatic cancer

Abstract Activating mutations in the KRAS oncogene are found in greater than 90% of pancreatic ductal adenocarcinoma (PDAC), yet KRAS remains difficult to inhibit pharmacologically. Small molecule inhibitors of RAF/MEK/ERK (MAPK) or mTOR are first line treatments for several human malignancies, however resistance to these targeted therapies continues to be a major clinical problem. We show in vitro dose dependent inhibition of proliferation and pERK1/2 in human PDAC cell lines and a KRAS-driven genetically engineered mouse model (GEMM) of pancreatic cancer in response to Trametinib, a small molecule MEK-inhibitor (MEKi). Similarly, Trametinib elicited a dose dependent inhibition of the in vivo tumor growth of a subcutaneous human PDAC xenograft, however these tumors rapidly developed resistance and contained areas of increased activation of pAkt(Ser473). We determined that genetic depletion of KRAS using doxycycline-inducible shRNA or small molecule MEKi leads to increased pAkt(Ser473) and activation of mTORC2 by western blot, implicating activation of the Akt/mTORC1/2 pathway may be a mechanism of resistance to MEKi. Interestingly, treatment of PDAC cell lines with Torin1, a small molecule inhibitor of mTORC1/2, results in the increased activation of pERK1/2, suggesting crosstalk and compensation of these two pathways as mechanisms of resistance. The combination of Trametinib and Torin1 leads to the inhibition of both pERK1/2 and pAkt(Ser473) that is accompanied by a dose dependent, synergistic increase in cytotoxicity not observed with either single agent. In human PDAC xenografts and a syngeneic GEMM, combination treatment of Trametinib and Torin1 dramatically reduced tumor burden and increased survival, without serious toxicity. These results demonstrate that combined inhibition of MEK/ERK and mTORC1/2 is a potent and viable therapeutic strategy for PDAC. Citation Format: Wells Brown, Oksana Nemirovsky, Jordan Gillespie, Paul McDonald, Shoukat Dedhar. Combination MEK and mTORC1/2 inhibition overcomes resistance to single agent therapy in pancreatic cancer [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 1906.

Abstract 135: Increased expression of ganglioside GM2 correlates with aggressiveness of human pancreatic ductal adenocarcinoma

Abstract (a) Purpose: Gangliosides are molecules composed of glycosphingolipids linked with one or more sialic acids, and are known to be key components of lipid rafts, which act as platforms for signal transduction. Changes in ganglioside levels affect the expression of raft-associated proteins on the cell surface and lead to reduced membrane fluidity, thus, resulting in cellular dysfunction, such as impaired signal transduction. The ganglioside GM2 is one of the major series of gangliosides that has several biological functions, such as in cell adhesion and signal transduction. GM2 is highly expressed in several types of human malignant tumors, such as melanomas, gliomas, and neuroblastomas, but is absent or weakly expressed in normal tissues. However, the association between gangliosides and pancreatic ductal adenocarcinoma (PDAC) has not been elucidated yet. Thus, in this study, we aimed to clarify the expression and role of ganglioside GM2 in PDAC. (b) Experimental procedures: GM2 expression in eight human PDAC cell lines was examined by fluorescence-activated cell sorter (FACS) analysis. The morphology, growth, and stemness of GM2- and GM2+ sorted-cells were compared by transmission electron microscopy, growth assays, real-time PCR analysis of the markers of stemness, and sphere forming assays. Cell motility was evaluated by invasion assay. For comparing tumorigenicity between the GM2- and GM2+ sorted-cells, heterotopic implantations were performed. The expression levels and clinico-pathological roles of GM2 in patients with PDAC were examined by immunohistochemical analysis of 117 pancreatic tissue samples. (c) Results: The fraction of GM2+ cells was the highest (21.4%) in MIA PaCa-2 from among the eight cell lines, as revealed by FACS analysis. GM2-expressing MIA PaCa-2 cells had higher growth rates under adherent growth conditions. In 3D-culture, which enriches cancer stem cells (CSCs), most MIA PaCa-2 cells expressed GM2 and the cells responded to TGF-β1 treatment to promote invasion. Transplantation of the GM2-expressing cells into nude mice resulted in development of larger tumors as compared to that for control cells that did not express GM2. In the PDAC cases, GM2 expression was significantly associated with younger age, larger tumor size, advanced stage, and higher histological grade. (d) Conclusions: We show that a PDAC cell line overexpressing GM2 exhibits high growth rate and high tumor initiation in 2D-culture. Further, pancreatic CSC-like cells expressing GM2 exhibit responsiveness against TGF-β1, resulting in enhanced invasion in 3D-culture. Furthermore, GM2 expression is associated with the growth and advanced stage of human PDAC. Further studies will be required for the development of an early detection method for GM2-overexpressing pancreatic cancers and to develop novel therapeutic strategies targeting GM2. Citation Format: Norihiko Sasaki, Kenichi Hirabayashi, Masaki Michishita, Kimimasa Takahashi, Fumio Hasegawa, Fujiya Gomi, Yoko Itakura, Naoya Nakamura, Masashi Toyoda, Toshiyuki Ishiwata. Increased expression of ganglioside GM2 correlates with aggressiveness of human pancreatic ductal adenocarcinoma [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 135.

Abstract 5160: A GPCR candidate in pancreatic ductal adenocarcinoma: A potential repurposing opportunity

Abstract The 5-year survival of patients with pancreatic ductal adenocarcinoma (PDAC), the most common type of pancreatic cancer (90%), is very low (~9% all stages). PDAC thus requires new, effective and safe therapies. By comparing transcriptomic data for PDAC (in The Cancer Genome Atlas) with that of normal pancreas (in the GTEx database), we discovered that many G protein-coupled receptors (GPCRs) are increased in expression in PDAC. One such GPCR is the histamine receptor H1 (HRH1). GPCRs regulate metabolism, growth/death and functional activities of normal and cancer cells, including features of the malignant phenotype. GPCRs are the largest family of targets of approved drugs but have been largely ignored in cancer therapy. We found that HRH1 is prominently (~32-fold) overexpressed in PDAC tumors compared to normal pancreatic tissue and that high HRH1 expression is an unfavorable prognostic marker for PDAC patients. HRH1 mRNA is also highly expressed in multiple human PDAC cell lines. Studies with such cell lines revealed that histamine, acting via HRH1, increases intracellular Ca2+, cell proliferation and migration, effects blocked by multiple FDA-approved HRH1 antihistamines. PDAC tumors and cancer cells isolated from KPC mice also have high HRH1 expression compared to normal mouse pancreas. Similar to human PDAC cells, the KPC cells showed histamine/HRH1-promoted increase in intracellular Ca2+. Altogether, our findings identify HRH1 as a prominently overexpressed, functional GPCR in human and mouse PDAC tumors and cells. HRH1 may thus be a novel therapeutic target; its blockade by FDA-approved antihistamines represents a novel potential repurposing approach for the treatment of PDAC. Citation Format: Cristina Salmerón, Krishna Sriram, Alyssa Baird, Paul A. Insel. A GPCR candidate in pancreatic ductal adenocarcinoma: A potential repurposing opportunity [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 5160.

Abstract 554: Histone deacetylase inhibition is synthetically lethal with arginine deprivation in pancreatic cancers with low argininosuccinate synthetase 1 expression

Abstract Background: Arginine (Arg) deprivation is a promising therapeutic approach for tumors with low argininosuccinate synthetase 1 (ASS1) expression. However, its efficacy as a single agent therapy needs to be improved as resistance is frequently observed. Methods: A tissue microarray was performed to assess ASS1 expression in surgical specimens of pancreatic ductal adenocarcinoma (PDAC) and its correlation with disease prognosis. An RNA-Seq analysis examined the role of ASS1 in regulating global gene transcriptome. A high throughput screen of FDA-approved oncology drugs identified synthetic lethality between histone deacetylase (HDAC) inhibitors and Arg deprivation in PDAC cells with low ASS1 expression. We examined HDAC inhibitor panobinostat (PAN) and Arg deprivation in a panel of human PDAC cell lines, in ASS1-high and -knockdown/knockout isogenic models, in both anchorage-dependent and -independent cultures, and in multicellular complex cultures that model the PDAC tumor microenvironment. We examined the effects of combined Arg deprivation and PAN on DNA damage and the protein levels of key DNA repair enzymes. We also evaluate the efficacy of PAN and ADI-PEG20 (an Arg-degrading agent currently in Phase II clinical trials) in xenograft models with ASS1-low and -high PDAC tumors. Results: Low ASS1 protein level is a negative prognostic indicator in PDAC. Arg deprivation in ASS1-deficient PDAC cells upregulated asparagine synthetase (ASNS) which redirected aspartate (Asp) from being used for de novo nucleotide biosynthesis, thus causing nucleotide insufficiency and impairing cell cycle S-phase progression. Comprehensively validated, HDAC inhibitors and Arg deprivation showed synthetic lethality in ASS1-low PDAC cells. Mechanistically, combined Arg deprivation and HDAC inhibition triggered degradation of a key DNA repair enzyme C-terminal-binding protein interacting protein (CtIP), resulting in DNA damage and apoptosis. In addition, S-phase-retained ASS1-low PDAC cells (due to Arg deprivation) were also sensitized to DNA damage, thus yielding effective cell death. Compared to single agents, the combination of PAN and ADI-PEG20 showed better efficacy in suppressing ASS1-low PDAC tumor growth in mouse xenograft models. Conclusion: The combination of PAN and ADI-PEG20 is a rational translational therapeutic strategy for treating ASS1-low PDAC tumors through synergistic induction of DNA damage. Citation Format: Stephanie S Kim, JING CUI, Shili Xu, Soumya Poddar, Thuc M. Le, Luyi Li, Nanping Wu, Alexandra Moore, Lei Zhou, Alice Yu, Amanda M. Dann, Irmina A. Elliott, Evan R. Abt, Woosuk Kim, David W. Dawson, Caius G. Radu, Timothy R. Donahue. Histone deacetylase inhibition is synthetically lethal with arginine deprivation in pancreatic cancers with low argininosuccinate synthetase 1 expression [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 554.

Anti-Proliferative and Apoptosis-Promoting Effect of microRNA-125b on Pancreatic Cancer by Targeting NEDD9 via PI3K/AKT Signaling.

PurposeThe expression of microRNA-125b (miR-125b) is low in a variety of cancers, including gastric, lung, bladder, thyroid, and esophageal cancers. However, its specific mechanism in pancreatic ductal adenocarcinoma (PDAC) remains unclear. This study is aimed to explore the role of miR-125b in PDAC.MethodsPDAC tissues and adjacent tissues were collected for miR-125b analysis by qRT-PCR. Different PDAC cell lines were cultured for miR-125b detection by qRT-PCR, and CAPAN1 cells were selected for the downstream experiments. Cell proliferation was characterized by methyl thiazolyl tetrazolium (MTT) and 5-bromo-2-deoxyUridine (BrdU) staining. Flow cytometry was utilized for apoptosis and cell cycle changes. Cell invasion was determined by the Transwell assay and the dual-luciferase assay was utilized for validating the target gene. Western blotting was used to detect apoptosis related and PI3K/AKT signaling proteins.ResultsmiR-125b was significantly down-regulated in human PDAC tissues and cell lines (P < 0.05). miR-125b inhibited the growth and invasion of CAPAN1 cells, facilitated apoptosis, and blocked the cell cycle at the G0/G1 phase. Furthermore, miR-125 directly targeted NEDD9. The high expression of NEDD9 impaired the anti-proliferative and anti-apoptotic activity of miR-125b. miR-125b also inhibited apoptosis-related proteins and PI3K/AKT signaling pathways via NEDD9.ConclusionmiR-125b decreased cell growth and invasion, and facilitated apoptosis in CAPAN1 cells through PI3K/AKT inhibition via targeting NEDD9.

The USP22 promotes the growth of cancer cells through the DYRK1A in pancreatic ductal adenocarcinoma.

As a member of the ubiquitin-specific protease (USP) family, USP22 could remove ubiquitin moieties from its target proteins to control the function of the target proteins. Accumulating studies show that USP22 essentially participates in diverse types of cancer as an oncogene-like protein. However, the roles of USP22 in human pancreatic ductal adenocarcinoma (PDAC) and the underlying mechanism are unknown. Here we report that USP22 promotes the growth of PDAC cells by promoting the expression of dual-specificity tyrosine regulated kinase 1A (DYRK1A). Our results showed that the expression levels of USP22 were up-regulated in human PDAC tissues and cell lines (BxPC-3, AsPC-1, MIA-PaCa-2, PANC-1, and CAPAN-1). Lentivirus-mediated knockdown of USP22 repressed the rate of proliferation and capacity of colony formation of BxPC3 and CAPAN1 cancer cells and USP22 overexpression promoted the proliferation and capacity of the colony formation of BxPC3 and CAPAN1 cancer cells. The further mechanism study showed that USP22 elevated the expression of the mRNA and protein levels of DYRK1A in PDAC cancer cells. Inhibition of DYRK1A with EHT-5732 or lentivirus-mediated knockdown of DYRK1A blocked the function of USP22 overexpression in the regulation of the proliferation and colony formation of PDAC cells. Taken together, our findings demonstrated that USP22 overexpression in PDAC promoted the growth of the cancer cells partially through upregulating the expression of DYRK1A.