Aggressive Pancreatic Ductal Adenocarcinoma Research Articles

Upregulated Expression of TUBA1C Predicts Poor Prognosis and Promotes Oncogenesis in Pancreatic Ductal Adenocarcinoma via Regulating the Cell Cycle

Background: Pancreatic ductal adenocarcinoma (PDAC) is a highly malignant disease and has the worst prognosis and survival rate. TUBA1C is a microtubule component implicated in multiple cancers, however, the clinical significance and biological functions of TUBA1C in the progression of PDAC remain unexplored.Methods: The Cancer Genome Atlas (TCGA) and Gene Expression Profiling Interactive Analysis (GEPIA) data were employed to detect the TUBA1C mRNA expression and the relation between TUBA1C expression and overall survival (OS) in PDAC. Then, bioinformatic analysis was employed to determine the potential pathway and genes related to TUBA1C. Human pancreatic cancer tissue and adjacent non-tumor tissues samples were detected by immunochemistry (IHC) staining, and the correlation between TUBA1C expression and the clinicopathological features were investigated. Meanwhile, TUBA1C expression in PDAC cell lines was evaluated by western blotting. Furthermore, functional assays including cell viability, apoptosis, cell cycle, transwell assay, wound healing assay, and a xenograft tumor model were performed to determine the oncogenic role of TUBA1C in PDAC, respectively.Results: TUBA1C was overexpressed in the PDAC tissues and cells. IHC analysis showed that the TUBA1C overexpression was associated with short OS. Bioinformatic analysis indicated that TUBA1C overexpression was mainly associated with cell cycle regulation. The downregulation of TUBA1C significantly suppressed cell proliferation, induced cell apoptosis and cycle arrest, and inhibited invasion and migration in PDAC cells. Furthermore, TUBA1C downregulation also inhibited tumor growth in vivo.Conclusion: These findings suggested that TUBA1C downregulation suppressed PDAC aggressiveness via cell cycle pathway and that TUBA1C may serve as a potential prognostic marker for PDAC therapy.

Abstract C32: Therapeutic targeting of keratin 17 and nuclear export uncover therapeutic vulnerabilities of pancreatic cancer

Abstract The purpose of this study is to launch a novel biomarker-based targeted therapy that may result in improved precision and personalized medicine for pancreatic ductal adenocarcinoma (PDAC). We discovered that keratin 17 (K17), an intermediate filament (IF), is a nuclear oncoprotein found in 50% of PDAC cases, identifying the most aggressive and treatment-resistant form of PDAC and functioning as a biomarker. We set out to validate K17 as a druggable target and to identify K17-targeting mechanisms. In preclinical mouse models, animals bearing K17-expressing PDACs display the shortest survival interval, suggesting that K17 drives tumor aggression and is a potential therapeutic target. We used the following two approaches aimed to inhibit K17 nuclear shuttle functions in order to identify potential therapeutic strategies. 1) Targeting K17 functional domains: By protein-sequence modeling analyses and site-directed mutagenesis, we identified functional domains in K17 (K17-FDs) that bind to, shift the subcellular localization of, and promote the degradation of nuclear tumor suppressors (i.e., p27). Using SILAC mass spectrometry analysis of nuclear proteomes from isogenic human cells, we found that 80% of the nuclear proteome is altered by K17, that half of these proteins encode the domain targeted by the K17-FDs, and that a large proportion of these proteins are involved in gene expression. Animals harboring tumors with mutated K17-FDs lived two times longer than control animals, suggesting that the K17-FDs may be therapeutic targets. We are currently testing small-molecule and peptide inhibitors to target the K17-FDs in vitro and in vivo, as a discovery approach to design small-molecule inhibitors of K17. 2) Targeting K17 nuclear export: K17 impacts the export of ~50% of the proteome and depends on exportin-1 (XPO1) for nuclear export. We found that PDAC cells expressing K17 were more sensitive to Selective Inhibitor of Nuclear Export (SINE) therapy, leading to a 2-fold increase in cell death compared to control cells, suggesting that nuclear export is a necessary oncogenic pathway exploited by K17 in PDAC cells. SINE therapy is currently FDA approved for treatment of other cancer types, and repurposing for the treatment of K17-positive PDACs may result in enhanced therapeutic efficacy. In conclusion, these studies provide the basis for novel biomarker-based therapeutic approaches for the most aggressive PDAC cases using small-molecule inhibitors that directly target K17 active sites or the pathways altered by this protein. Citation Format: Cindy V. Leiton, Chun-Hao Pan, Ji Dong Bai, Lucia Roa-Peña, Sruthi Babu, Alex Penson, Nashaat Turkman, Richard Moffitt, Markus Seeliger, David Talmage, Kenneth R. Shroyer, Luisa F. Escobar-Hoyos. Therapeutic targeting of keratin 17 and nuclear export uncover therapeutic vulnerabilities of pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr C32.

Regulation of Heparanase in Diabetes-Associated Pancreatic Carcinoma

While at least six types of cancer have been associated with diabetes, pancreatic ductal adenocarcinoma (PDAC) and diabetes exhibit a unique bidirectional relationship. Recent reports indicate that majority of PDAC patients display hyperglycemia, and ~50% have concurrent diabetes. In turn, hyperglycemic/diabetic state in PDAC patients fosters enhanced growth and dissemination of the tumor. Heparanase enzyme (the sole mammalian endoglycosidase degrading glycosaminoglycan heparan sulfate) is tightly implicated in PDAC progression, aggressiveness, and therapy resistance. Overexpression of heparanase is a characteristic feature of PDAC, correlating with poor prognosis. However, given the lack of heparanase expression in normal pancreatic tissue, the regulatory mechanisms responsible for induction of the enzyme in PDAC have remained largely unknown. Previously reported inducibility of heparanase gene by diabetic milieu components in several non-cancerous cell types prompted us to hypothesize that in the setting of diabetes-associated PDAC, hyperglycemic state may induce heparanase overexpression. Here, utilizing a mouse model of diet-induced metabolic syndrome/diabetes, we found accelerated PDAC progression in hyperglycemic mice, occurring along with induction of heparanase in PDAC. In vitro, we demonstrated that advanced glycation end-products (AGE), which are largely thought as oxidative derivatives resulting from chronic hyperglycemia, and the receptor for AGE (RAGE) are responsible for heparanase induction in PDAC cells. These findings underscore the new mechanism underlying preferential expression of heparanase in pancreatic cancer. Moreover, taken together with the well-established causal role of the enzyme in PDAC progression, our findings indicate that heparanase may sustain (at least in part) reciprocal causality between diabetes and pancreatic tumorigenesis.

Meflin-Positive Cancer-Associated Fibroblasts Inhibit Pancreatic Carcinogenesis.

Cancer-associated fibroblasts (CAF) constitute a major component of the tumor microenvironment. Recent observations in genetically engineered mouse models and clinical studies have suggested that there may exist at least two functionally different populations of CAFs, that is, cancer-promoting CAFs (pCAF) and cancer-restraining CAFs (rCAF). Although various pCAF markers have been identified, the identity of rCAFs remains unknown because of the lack of rCAF-specific marker(s). In this study, we found that Meflin, a glycosylphosphatidylinositol-anchored protein that is a marker of mesenchymal stromal/stem cells and maintains their undifferentiated state, is expressed by pancreatic stellate cells that are a source of CAFs in pancreatic ductal adenocarcinoma (PDAC). In situ hybridization analysis of 71 human PDAC tissues revealed that the infiltration of Meflin-positive CAFs correlated with favorable patient outcome. Consistent herewith, Meflin deficiency led to significant tumor progression with poorly differentiated histology in a PDAC mouse model. Similarly, genetic ablation of Meflin-positive CAFs resulted in poor differentiation of tumors in a syngeneic transplantation model. Conversely, delivery of a Meflin-expressing lentivirus into the tumor stroma or overexpression of Meflin in CAFs suppressed the growth of xenograft tumors. Lineage tracing revealed that Meflin-positive cells gave rise to α-smooth muscle actin-positive CAFs that are positive or negative for Meflin, suggesting a mechanism for generating CAF heterogeneity. Meflin deficiency or low expression resulted in straightened stromal collagen fibers, which represent a signature for aggressive tumors, in mouse or human PDAC tissues, respectively. Together, the data suggest that Meflin is a marker of rCAFs that suppress PDAC progression. SIGNIFICANCE: Meflin marks and functionally contributes to a subset of cancer-associated fibroblasts that exert antitumoral effects.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/20/5367/F1.large.jpg.

P110γ deficiency protects against pancreatic carcinogenesis yet predisposes to diet-induced hepatotoxicity

Pancreatic ductal adenocarcinoma (PDAC) is notorious for its poor survival and resistance to conventional therapies. PI3K signaling is implicated in both disease initiation and progression, and specific inhibitors of selected PI3K p110 isoforms for managing solid tumors are emerging. We demonstrate that increased activation of PI3K signals cooperates with oncogenic Kras to promote aggressive PDAC in vivo. The p110γ isoform is overexpressed in tumor tissue and promotes carcinogenesis via canonical AKT signaling. Its selective blockade sensitizes tumor cells to gemcitabine in vitro, and genetic ablation of p110γ protects against Kras-induced tumorigenesis. Diet/obesity was identified as a crucial means of p110 subunit up-regulation, and in the setting of a high-fat diet, p110γ ablation failed to protect against tumor development, showing increased activation of pAKT and hepatic damage. These observations suggest that a careful and judicious approach should be considered when targeting p110γ for therapy, particularly in obese patients.

Abstract 2374: Non-canonical TGF-beta signaling modulates pancreatic ductal adenocarcinoma progression

Abstract Introduction: While the genomic and proteomic characterization of pancreatic ductal adenocarcinoma (PDAC) is extraordinarily complex, the transforming growth factor-beta (TGF-β) pathway is critically important. TGF-β has a context dependent role- it is tumor suppressive in early PDAC and tumor promotive in metastatic PDAC. Our hypothesis is that non-canonical TGF-β signaling induces metastatic PDAC. Non-canonical signaling may help explain the paradoxical nature of TGF-β in PDAC. Methods: Two cell lines were utilized- Panc-1, a cell line derived from primary PDAC, was compared to Capan-1, a cell line derived from metastatic PDAC- in order to model an early PDAC (Panc-1) and a more aggressive PDAC (Capan-1). We investigated the gene and protein expression of markers of epithelial to mesenchymal transition (Snail and Twist), Akt, β-catenin, and PD-L1 upon treatment with TGF-β and the Akt inhibitor MK-2206. Results: While TGF-β increased Snail gene and protein expression in Panc-1 cells, it had nil effect on Capan-1 cells where TWIST1 protein expression increased with TGF-β. TGF-β increased pAkt expression in both cell lines. TGF-β increased active and total β-catenin expression in Capan-1 but not Panc-1 cell lines. We then found that PD-L1 expression was generally diminished by Akt inhibition in both cell lines but this was reversed with TGF-β receptor inhibition and gemcitabine treatment in the Capan-1 cells. Conclusions: Our data suggests that modulation of the non-canonical TGF-β pathway may lead to increased epithelial to mesenchymal transition through multiple mechanisms. PD-L1 expression is a response to chemotherapy in metastatic PDAC when multiple proteins in that pathway are inhibited. Our data suggests that (1) personalized therapy based on the degree of non-canonical TGF-β pathway activation will be critical, (2) patients with uncontrolled non-canonical TGF-β pathway activation may have lower survival, and (3) there is an opportunity to utilize anti-PD-L1 based therapy in combination with other therapy in selected patients based on their response to chemotherapy. Citation Format: S. Mazher Hussain, Gustavo Miranda-Carboni, Marcus A. Alvarez, Abul Elahi, Leah E. Hedrick, David Shibata, Evan S. Glazer. Non-canonical TGF-beta signaling modulates pancreatic ductal adenocarcinoma progression [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 2374.

PDX1, a key factor in pancreatic embryogenesis, can exhibit antimetastatic activity in pancreatic ductal adenocarcinoma.

Background: In cancer biology, metastasizing is one of the most poorly studied processes. Pancreatic ductal adenocarcinoma (PDAC) is characterized by early metastasis, which is the leading cause of death. The PDX1 protein is crucial for the development of cancer, and its low levels are characteristic of the most aggressive PDAC tumors. The PDX1 is a mediator of initiation and progression of PDAC. However, further studies are needed to elucidate the role of PDX1 in the cancer metastasis.Purpose: To confirm the hypothesis that PDX1 in PDAC plays suppressor role of epithelial–mesenchymal transition (EMT), and to study its possible ability to inhibit metastasis.Methods: A PDX1-overexpressing PDAC cell line was obtained by lentiviral transduction of PANC-1 cells. PDX1 overexpression was confirmed by RT-PCR and Western blotting. Effects of PDX1 ectopic expression on cell proliferation and motility were determined in PANC-1 cells using MTS, cell cycle analysis, transwell and wound-healing assay. EMT genes expression was analyzed in PDX1-overexpressing and Control PANC-1. Finally, the migration potential of pancreatic cancer cells expressing PDX1 was evaluated using a zebrafish embryo model.Results: The motility of human PDAC cells PANC-1 considerably decreased at ectopic expression of PDX1. The decreased expression of ZEB1, the key factor of EMT, and almost unchanged expression of the genes that characterize the epithelial state suggest a decrease in the EMT ability. Suppression of PDX1 expression by siRNA knockdown restored the PANC1 motility.Conclusion: The results obtained suggest a possible therapeutic use of PDX1 delivery into PDAC patients with a reduced or absent expression of PDX1 in the most aggressive tumors.

TGIF1 functions as a tumor suppressor in pancreatic ductal adenocarcinoma.

A prominent function of TGIF1 is suppression of transforming growth factor beta (TGF-β) signaling, whose inactivation is deemed instrumental to the progression of pancreatic ductal adenocarcinoma (PDAC), as exemplified by the frequent loss of the tumor suppressor gene SMAD4 in this malignancy. Surprisingly, we found that genetic inactivation of Tgif1 in the context of oncogenic Kras, KrasG12D , culminated in the development of highly aggressive and metastatic PDAC despite de-repressing TGF-β signaling. Mechanistic experiments show that TGIF1 associates with Twist1 and inhibits Twist1 expression and activity, and this function is suppressed in the vast majority of human PDACs by KrasG12D /MAPK-mediated TGIF1 phosphorylation. Ablating Twist1 in KrasG12D ;Tgif1KO mice completely blunted PDAC formation, providing the proof-of-principle that TGIF1 restrains KrasG12D -driven PDAC through its ability to antagonize Twist1. Collectively, these findings pinpoint TGIF1 as a potential tumor suppressor in PDAC and further suggest that sustained activation of TGF-β signaling might act to accelerate PDAC progression rather than to suppress its initiation.

Significant neutrophil accumulation, IL-18 deposition, and active inflammasome in tumor regions of human pancreatic ductal adenocarcinoma.

e15754 Background: Obesity is an established risk factor for cancer and cancer-related mortality. Adipocytes may act as a “damage” signal resulting in the accumulation of innate immune cells, fueling an inflammatory micro-environment. Among the innate immune cells, tumor-associated neutrophils (TANs) are confirmed to accumulate inside the tumor. As obesity is a significant risk factor for a poorer prognosis in pancreatic ductal adenocarcinoma (PDAC), we hypothesize that obesity-driven adipose fuels the progression of PDAC in a TAN and inflammasome-facilitated manner. This may happen through several mechanisms, including genomic instability of aggressive PDAC clonal populations. Methods: Tissue from resected tumors of PDAC patients with a body-mass index (BMI) > 27 (obesity, n = 5) and BMI < 22 (normal, n = 5) were incubated with anti-human IL-18 and CD66b antibodies and detected using Alexa Fluor 488-conjugated donkey anti–mouse and Alexa Fluor 594-conjugated goat anti-rabbit secondary antibodies. Images were acquired in a Zeiss Axioplan microscope workstation and analyzed by ZEN and Metamorph software. Results: We identified the presence of CD66b+ neutrophils and IL-18 in all tissue sections. There is a significantly greater density of CD66b+ neutrophils and IL-18 in the tumor environment when compared to adjacent normal areas and IL-18+ signals appear to be deposited alongside the luminal area in a unique lasso-like pattern. These data suggest an association between BMI and inflammasome accumulation in the tumor environment. There does not appear to be an association between tumor stage and TAN or IL-18 accumulation in the tumor area or the adjacent normal tissue in the tissues. Conclusions: Our data uniquely shows that CD66b+ neutrophils accumulate inside PDAC and this is associated with intra-tumoral active inflammasomes. The novel periductal deposition of IL-18 in the tumor areas may be of mechanistic relevance in understanding how inflammation induces progression of PDAC. A larger cohort of tissues from PDAC obtained from low and high BMI individuals will be needed to test our hypothesis and to begin to decipher the role of obesity on inflammation and PDAC progression.

Oncogenic mechanism of soluble Keratin 17 offers potential therapeutic vulnerability in pancreatic cancer

CLINICAL NEED & OBJECTIVESOur group found that Keratin 17 (K17), an intermediate filament (IF), is a tumor cell‐specific marker of aggressive pancreatic ductal adenocarcinoma (PDAC), and that the detection of K17 by immunohistochemistry level is a more accurate prognostic indicator than transcriptomic subtyping. We determined that K17 can act as an oncoprotein after transitioning from its “insoluble” cytoskeleton form to a “soluble” form that can regulate cell cycle progression. In patients, mice, and PDAC cell lines, the soluble fraction of K17 exists as full length (flK17) and caspase‐cleaved peptide (ccK17), each with potentially unique tumorigenic capacity. Literature suggests that post‐translational modifications (PTMs) are necessary to regulate these and other IF dynamics, though the mechanisms behind K17 solubilization in PDAC are still unclear. Thus, our aims are to 1) determine the mechanisms regulating K17 dynamics, 2) demonstrate inhibited cell cycle progression and tumor growth following disruption of these mechanisms, and 3) verify soluble K17 as a robust negative prognostic biomarker of PDAC.METHODS & RESULTSIn a PDAC tumor mouse model, we show, independent of total K17 protein amount, that the percent of soluble ccK17 in tumor cells is negatively prognostic. Work is ongoing to validate this finding in human PDAC samples. We sequenced both the soluble and cytoskeletal insoluble forms of K17 from primary PDACs using liquid chromatography mass spectrometry (LC‐MS) and identified that K17 is phosphorylated on several N‐terminal serine residues. Specifically, we reproducibly identified phosphorylation of a semi‐conserved IF motif that may promote caspase cleavage and solubilization. Ser/Thr phosphatase inhibition in vitro demonstrated that phosphorylation is necessary to trigger K17 solubilization followed by nuclear translocation. Mutagenesis of serine sites is ongoing to determine the specific motifs regulating K17. Using an unbiased kinase inhibitor screen and follow‐up pharmacologic analyses, we identified that polo‐like kinase 1 (PLK1) likely phosphorylates K17. Further studies are underway to validate K17 as a key mediator of PLK1 inhibitor sensitivity in PDAC.CONCLUSIONPhosphorylation is an important regulator of K17 dynamics in PDAC, promoting solubilization and, in some cases, caspase cleavage. Our results suggest that soluble K17 may have a functional role in promoting tumor aggression. Inhibiting kinases that phosphorylate K17 could impact tumor growth by blocking K17 solubilization/cleavage and this line of research could accelerate the development of more effective treatments, coupled to soluble K17 testing as a predictive biomarker for PDAC.Support or Funding InformationThis work was partially supported by the 2018 Pancreatic Cancer Action Network Translational Research Grant (K.R.S.).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Pancreatic Ductal Adenocarcinoma Tumor Suppressors Rgs8–16

IntroductionG protein‐coupled receptors (GPCRs) are the largest family of targets for approved drugs, but rarely exploited in cancer treatment. Kras oncogenic mutations (e.g. KrasG12D) are found in over 90% of human pancreatic ductal adenocarcinoma (PDA) and can be activated by protein kinases and GPCR signaling. Regulator of G protein signaling (Rgs) proteins modulate GPCR signaling by accelerating the GTPase activity of Gq‐ and Gi‐class alpha subunits. Rgs genes are often upregulated in primary cancers. We showed that Rgs8::GFP and Rgs16::GFP reporter genes are highly expressed in an oncogenic Kras (KrasG12D)‐dependent PDA model at initiation and throughout progression. Furthermore, activating alleles of Gq that are resistant to Rgs inhibition are found in benign precursors of PDA in humans. By contrast, Rgs16 down‐regulation is associated with increased lymph node metastasis in PDA. Here we test our hypothesis that Rgs8–16 deletion will accelerate PDA initiation, progression, and metastasis.MethodsWe made the Rgs8–16 double knockout (dKO) and crossed it into pancreas‐specific KrasG12D (KC) mutant mice (termed KCR8–16). Caerulein (analog of the Gq‐coupled GPCR agonist CCK) induced acute and chronic pancreatitis, PDA initiation and progression. Response to caerulein was compared in KC and KCR8–16 mice using physiologic, histologic and immunologic assays.ResultsCaerulein induced Rgs16 expression in acute pancreatitis within 2 days in Rgs16::GFP transgenic mice, and they recovered normally within 7days. Although deletion of Rgs8–16 did not cause PDA initiation, Rgs8–16 dKO mice took longer to recover from caerulein‐induced pancreatitis than normal mice. PDA initiation and progression was greatly accelerated in KCR8–16 compared to KC mice. Caerulein treatment caused the entire pancreas to transform to aggressive PDA within 7 days in KCR8–16 mice, whereas KC pancreata showed milder acinar‐to‐ducal metaplasia and differentiated, early stage PDA. Cell proliferation and epithelial‐to‐mesenchymal transmission (EMT) was amplified in KCR8–16 mice, with increased Ki67 and EMT marker expression, suggesting a greater potential for metastasis.DiscussionWe find Rgs8 and Rgs16 are tumor suppressors in PDA initiation and progression. Little is known about GPCR expression and function in PDA. Few GPCR‐targeted therapeutics exist for PDA. The KCR8–16 and Rgs16::GFP mice we developed provide excellent models for screening ligands to identify GPCRs involved in PDA initiation and progression. KCR8–16 hypersensitivity to Gq‐coupled ligands provides a model for rapid in vivo validation of PDA therapeutics.Support or Funding InformationThis work supported by NCI CA161624 and UT Southwestern Cancer Center Pilot Project Award to RAB and TMWThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

MicroRNA-365a-3p inhibits c-Rel-mediated NF-κB signaling and the progression of pancreatic cancer.

NF-κB contributes to the aggressiveness of pancreatic ductal adenocarcinoma (PDA), which is counteracted by the bioactive agent sulforaphane. We investigated sulforaphane-induced microRNA signaling and its influence on progression features. Using established cell lines, microRNA and gene arrays, we predicted miR-365a as the top candidate for the sulforaphane-induced inhibition of the NF-κB subunit c-Rel. The lipofection of miR-365a-3p mimics inhibited the luciferase activity of a c-Rel 3'-UTR construct, as well as c-Rel expression, NF-κB activity, and tumor viability, migration, and clonogenicity, whereas apoptosis was induced. In vivo, miR-365a-3p reduced the volume of tumor xenografts and the expression of progression markers. In a tissue array, the expression of miR-365a-3p was absent in almost all 91 malignant tissues but not in 5 normal tissues, thus confirming the previous results. Our observations suggest that sulforaphane-induced miR-365a-3p expression inhibits NF-κB activity by downregulating c-Rel, which prevents the progression of PDA.

MSP-RON Signaling Is Activated in the Transition From Pancreatic Intraepithelial Neoplasia (PanIN) to Pancreatic Ductal Adenocarcinoma (PDAC).

Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest epithelial malignancies and remains difficult to treat. Pancreatic intraepithelial neoplasias (PanINs) represent the majority of the pre-cancer lesions in the pancreas. The PDAC microenvironment consists of activated pancreatic stellate cells (PSCs) and immune cells, which are thought to contribute to neoplastic transformation. However, the signaling events involved in driving the transition from the neoplastic precursor to the more advanced and aggressive forms in the pancreas are not well understood. Recepteur d’Origine Nantais (RON) is a c-MET family receptor tyrosine kinase that is implicated in playing a role in cell proliferation, migration and other aspects of tumorigenesis. Macrophage stimulating protein (MSP) is the ligand for RON and becomes activated upon proteolytic cleavage by matriptase (also known as ST14), a type II transmembrane serine protease. In the current study, by immunohistochemistry (IHC) analysis of human pancreatic tissues, we found that the expression levels MSP and matriptase are drastically increased during the transition from the preneoplastic PanIN stages to the more advanced and aggressive PDAC. Moreover, RON is highly expressed in both PDAC and in cancer-associated stellate cells. In contrast, MSP, RON, and matriptase are expressed at low levels, if any, in normal pancreas. Our study underscores an emerging role of MSP-RON autocrine and paracrine signaling events in driving malignant progression in the pancreas.

Upregulation of macrophage migration inhibitory factor promotes tumor metastasis and correlates with poor prognosis of pancreatic ductal adenocarcinoma.

Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine that serves important roles in cancer. MIF overexpression is frequently observed in numerous human cancer types, including pancreatic carcinoma. However, the prognostic value and function of MIF in pancreatic ductal adenocarcinoma (PDAC) have not been fully elucidated. In the present study, upregulation of MIF expression in PDAC tissue compared with adjacent normal tissue was observed. Furthermore, MIF overexpression was identified to be significantly associated with poor survival rates in patients with PDAC. Multivariate Cox regression analysis confirmed that MIF was an independent risk factor for poor survival. Functional analyses demonstrated that MIF knockdown significantly inhibited the proliferation and invasion of pancreatic cancer cells in vitro compared with control cells. IN addition, mechanistic investigations revealed that silencing MIF leads to inhibition of AKT serine/threonine kinase and extracellular-signal-regulated kinase activation, and suppression of cyclin D1 and matrix metalloproteinase-2 expression, which may suppress tumor proliferation and invasion. These results highlight the importance of MIF overexpression in PDAC aggressiveness, and indicate that MIF may be a potential therapeutic target for pancreatic cancer.

Disruption of C1galt1 Gene Promotes Development and Metastasis of Pancreatic Adenocarcinomas in Mice

Pancreatic ductal adenocarcinomas (PDACs) produce higher levels of truncated O-glycan structures (such as Tn and sTn) than normal pancreata. Dysregulated activity of core 1 synthase glycoprotein-N-acetylgalactosamine 3-β-galactosyltransferase 1 (C1GALT1) leads to increased expression of these truncated O-glycans. We investigated whether and how truncated O-glycans contributes to the development and progression of PDAC using mice with disruption of C1galt1. We crossed C1galt1 floxed mice (C1galt1loxP/loxP) with KrasG12D/+; Trp53R172H/+; Pdx1-Cre (KPC) mice to create KPCC mice. Growth and progression of pancreatic tumors were compared between KPC and KPCC mice; pancreatic tissues were collected and analyzed by immunohistochemistry; immunofluorescence; and Sirius red, alcian blue, and lectin staining. We used the CRISPR/Cas9 system to disrupt C1GALT1 in human PDAC cells (T3M4 and CD18/HPAF) and levels of O-glycans were analyzed by lectin blotting, mass spectrometry, and lectin pulldown assay. Orthotopic studies and RNA sequencing analyses were performed with control and C1GALT1 knockout PDAC cells. C1GALT1 expression was analyzed in well-differentiated (n= 36) and poorly differentiated (n= 23) PDAC samples by immunohistochemistry. KPCC mice had significantly shorter survival times (median 102 days) than KPC mice (median 200 days) and developed early pancreatic intraepithelial neoplasias at 3 weeks, PDAC at 5 weeks, and metastasis at 10 weeks compared with KPC mice. Pancreatic tumors that developed in KPCC mice were more aggressive (more invasive and metastases) than those in KPC mice, had a decreased amount of stroma, and had increased production of Tn. Poorly differentiated PDAC specimens had significantly lower levels of C1GALT1 than well-differentiated PDACs. Human PDAC cells with knockout of C1GALT1 had aberrant glycosylation of MUC16 compared with control cells and increased expression of genes that regulate tumorigenesis and metastasis. In studies of KPC mice with disruption of C1galt1, we found that loss of C1galt1 promotes development of aggressive PDACs and increased metastasis. Knockout of C1galt1 leads to increased tumorigenicity and truncation of O-glycosylation on MUC16, which could contribute to increased aggressiveness.

Abstract 4751: APOBEC3A inhibits tumor immune response independent of deamination in a novel genetic model of pancreatic cancer

Abstract Introduction: Apolipoprotein B mRNA Editing enzyme, Catalytic polypeptide 3A (A3A) is a C to U cytidine deaminase, preferentially deaminating the lagging strand of double-stranded DNA at specific sequence motifs. A3A is overexpressed across multiple tumor types, and in pancreatic ductal adenocarcinoma (PDA), high expression is associated with significantly decreased survival in early stage patients. Large scale sequencing studies of various human tumors (TCGA and ICGC), including PDA, have implicated A3A as a potential driver of tumor mutagenesis. However, the biological relevance of the deamination function of A3A has not yet been shown. Here, we utilize a novel genetically engineered mouse model to show that A3A supports the development of aggressive PDA independent of mutagenic capabilities. Methods: As opposed to humans, mice only contain one APOBEC3 isoform which has limited to no deaminating activity on genomic DNA. Thus, to dissect the function of A3A on PDA initiation and progression, we made mice with a germline knockin for the coding sequence for human A3A and bred to a well-established GEMM of PDA to yield LSL-KrasG12D; p53fl/+; Pdx1-Cre; RosaLSL-YFP; A3A+/- (KPCY;A3A) mice and compared to KPCY mice. We genotyped tumors from both cohorts using VarScan after exome capture sequencing. Tumor and immune cells were FACS sorted comparative RNAseq performed. The immune contexture of murine and 155 untreated and treated human PDAs was analyzed by performing 7-color multiplex staining and immune cell spatial interaction analysis. Results: All KPCY;A3A (n=11) mice developed tumors and expired significantly faster compared to KPCY (n=18) controls (3.9 v. 7.0 mo; p<0.01). One third of the KPCY; A3A mice developed macrometastatic disease compared to 3/12 in the KPCY control. Whole exome sequencing of tumors revealed no significant difference in the number of point mutations or neoantigen load between KPCY;A3A and KPCY tumors. However, histologically, KPCY;A3A tumors contained significantly more desmoplasia and altered immune cell infiltrates. Specifically, comparative RNAseq and IHC analyses revealed dramatic differences in the number and distribution of various T-cell and B-cell subsets in KPCY;A3A mice. Moreover, A3A expression led to abrogation of CD8+ T cell activation and proliferation. Concomitantly, a panel of immune checkpoint-related genes were upregulated in A3A high expressing tumors and cell lines compared to non-A3A expressing controls. This was confirmed in a panel of resected human PDA. Conclusions: Counter to our hypothesis, A3A supports the initiation and growth of PDA in vivo through effects independent of deamination or mutagenesis of cancer cell genomes but rather indirectly through suppression of the tumor immune response. Future studies will address the precise mechanisms underlying A3A-mediated tumor immune evasion. Citation Format: Sonja Woermann, Robert Cowan, Susan M. Ross, Andrew D. Rhim. APOBEC3A inhibits tumor immune response independent of deamination in a novel genetic model of pancreatic cancer [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 4751.

From RNA‐seq to Immunohistochemistry: Keratin 17 Defines Pancreatic Cancer Subtypes

BackgroundPancreatic ductal adenocarcinoma (PDAC) remains a highly lethal disease, in part because of the lack of molecular biomarkers, allowing the rationale assignment of patient subgroups to appropriate targeted therapies. As a result, almost all PDAC patients are treated with one of two chemotherapeutic regimens (Gem/Abraxane or FOLFIRINOX), with marginal improvement in overall survival. In the last two years, RNA sequencing (RNASeq) studies reported that mRNA expression from bulk tumor defines molecular subtypes of PDAC that are highly correlated with survival. The 25‐gene mRNA signature defining the clinically most aggressive subtype of PDAC has yielded a refined landscape through which biomarker and targetable therapeutics can be identified. Thus, the aim of the current study was to determine if K17, one member of this mRNA signature, is sufficient to define the most aggressive subtype of PDAC and to validate K17 by immunohistochemistry (IHC) as a negative prognostic biomarker.Methods and ResultsData‐mining of 125 cases from the University of North Carolina Medical Center (Moffitt et al., 2015) identified K17 mRNA as an independent and sufficient biomarker of the most aggressive molecular subtype of PDAC (hazard ratio [HR] for death at 6 years =1.676, p=0.012). K17 mRNA was next validated as a negative prognostic biomarker in The Cancer Genome Atlas (TCGA) database of margin‐positive (n=48, HR=2.726, p=0.005), and lymph node‐positive (n=107 HR=1.913, p=0.016) PDAC. Increased K17 mRNA was also correlated with protein expression by qRT‐PCR and Immunohistochemistry (IHC) in formalin‐fixed paraffin‐embedded tissue blocks. IHC stained demonstrated specific staining in malignant and pre‐malignant cells. Tumors were classified as high for K17 based on the proportion of cells that showed strong (2+) staining in ≥ 25% of tumor cells. K17 was validated as a powerful independent prognostic biomarker in an separate set of PDACs from Stony Brook Medicine, Memorial Sloan Kettering Cancer Center and the University of Massachusetts Memorial Medical Center (n=82, HR=2.444, p=0.002). High‐K17 cases had shorter survival among cases with positive regional lymph nodes metastasis (n=52, HR=2.542, p=0.006) and negative regional lymph nodes metastasis (n=30, HR= 2.883, p=0.058).ConclusionThis is the first study to show that the expression of a single gene, K17, can accurately define the most aggressive PDAC subtype and is an independent, clinically relevant, prognostic biomarker. K17 expression, measured at the protein level by IHC, performs better than mRNA testing to predict outcome at the time of initial diagnosis and has the potential to inform clinical decisions regarding chemotherapeutic intervention.Support or Funding InformationThe Marvin Kuschner AccountThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

MiRNA-1290 Promotes Aggressiveness in Pancreatic Ductal Adenocarcinoma by Targeting IKK1

Background/Aims: MicroRNAs (miRNAs) are a group of non-coding RNAs that play diverse roles in pancreatic carcinogenesis. In pancreatic ductal adenocarcinoma (PDAC), NF-kB is constitutively activated in most patients and is linked to a mutation in KRAS via IkB kinase complex 1 (IKK1, also known as IKKa). We investigated the link between PDAC aggressiveness and miR-1290. Methods: We used miRCURY^TM LNA Array and in situ hybridization to investigate candidate miRNAs and validated the findings with PCR. The malignant behavior of cell lines was assessed with Cell Counting Kit-8, colony formation, and Transwell assays. A dual-luciferase reporter assay was used to evaluate the interaction between miR-1290 and IKK1. Protein expression was observed by western blotting. Results: In this study, 36 miRNAs were dysregulated in high-grade pancreatic intraepithelial neoplasia (PanIN) and PDAC tissues compared with low-grade PanIN tissues. The area under the curve values of miR-1290 and miR-31-5p were 0.829 and 0.848, respectively (95% confidence interval, 0.722–0.936 and 0.749–0.948, both P < 0.001). There was a significant correlation between miR-1290 and histological differentiation (P = 0.029), pT stage (P = 0.006), and lymph node metastasis (P = 0.001). In addition, the in vitro work showed that miR-1290 promoted PDAC cell proliferation, invasion, and migration. Western blotting and the dual-luciferase reporter assay showed that miR-1290 promoted cancer aggressiveness by directly targeting IKK1. The synergist effect of miR-1290 on the proliferation and metastasis of PDAC cells was attenuated and enhanced by IKK1 overexpression and knockdown, respectively. Consistent with the in vitro results, a subcutaneous tumor mouse model showed that miR-1290 functioned as a potent promoter of PDAC in vivo. Conclusion: MiR-1290 may act as an oncogene by directly targeting the 3’-untranslated region of IKK1, and the miR-1290/IKK1 pathway may prove to be a novel diagnostic and therapeutic target for PDAC.

Apparent Diffusion Coefficient (ADC) predicts therapy response in pancreatic ductal adenocarcinoma

Recent advances in molecular subtyping of Pancreatic Ductal Adenocarcinoma (PDAC) support individualization of therapeutic strategies in this most aggressive disease. With the emergence of various novel therapeutic strategies and neoadjuvant approaches in this quickly deteriorating disease, robust approaches for fast evaluation of therapy response are urgently needed. To this aim, we designed a preclinical imaging-guided therapy trial where genetically engineered mice harboring endogenous aggressive PDAC were treated with the MEK targeting drug refametinib, which induces rapid and profound tumor regression in this model system. Multi-parametric non-invasive imaging was used for therapy response monitoring. A significant increase in the Diffusion-Weighted Magnetic Resonance Imaging derived Apparent Diffusion Coefficient (ADC) was noted already 24 hours after treatment onset. Histopathological analyses showed increased apoptosis and matrix remodeling at this time point. Our findings suggest the ADC parameter as an early predictor of therapy response in PDAC.

Cadherin-1 and cadherin-3 cooperation determines the aggressiveness of pancreatic ductal adenocarcinoma

Background:Pancreatic ductal adenocarcinoma (PDAC) is characterised by an extensive tissue invasion and an early formation of metastasis. Alterations in the expression of cadherins have been reported in PDAC. Yet, how these changes contribute to tumour progression is poorly understood. Here, we investigated the relationship between cadherins expression and PDAC development.Methods:Cadherins expression was assessed by immunostaining in both human and murine tissue specimens. We have generated pancreatic cancer cell lines expressing both cadherin-1 and cadherin-3 or only one of these cadherins. Functional implications of such genetic alterations were analysed both in vitro and in vivo.Results:Cadherin-3 is detected early at the plasma membrane during progression of pancreatic intraepithelial neoplasia 1 (PanIN-1) to PDAC. Despite tumoural cells turn on cadherin-3, a significant amount of cadherin-1 remains expressed at the cell surface during tumourigenesis. We found that cadherin-3 regulates tumour growth, while cadherin-1 drives type I collagen organisation in the tumour. In vitro assays showed that cadherins differentially participate to PDAC aggressiveness. Cadherin-3 regulates cell migration, whereas cadherin-1 takes part in the invadopodia activity.Conclusions:Our results show differential, but complementary, roles for cadherins during PDAC carcinogenesis and illustrate how their expression conditions the PDAC aggressiveness.