Primary Pancreatic Ductal Adenocarcinoma Cell Lines Research Articles

Abstract 6485: Profile guided low dose drug combination strategies and kinase activities with prognostic and therapeutic avenues in pancreatic ductal adenocarcinoma

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease, commonly characterized by multiple aberrant signaling. Phosphoproteomics provides a direct read-out of these complex signaling networks and the resultant clinically relevant phenotype, as well as a functional scaffold to identify new targets. In the absence of an oncogenic driver, low dose (LD) kinase inhibitor (KI) combinations against multiple (parallel) activated kinases might provide higher efficacy and reduce toxicity as compared to single drug treatment. Aims: 1) To identify targets and test combinations of multiple KIs at LDs for potential synergism in preclinical models. 2) To chart the phosphoproteome of 42 PDAC tumors to reveal signalling pathways that may be involved in PDAC progression. Methods: By using a two step phosphopeptide enrichment with phosphotyrosine immunoprecipitation and immobilized metal affinity chromatography, followed by label free MS analysis, we analyzed phosphoproteome data of 7 immortalized and 2 primary PDAC cell lines, and of 42 PDAC tumors. We used integrative inferred kinase activity (INKA) scoring of the maxquant output to identify hyperactive kinases. For the cell line panel, five KIs were selected based on targeting coverage of the INKA profiles. LD were set as IC20s for 2,3,4 drug combinations. Cell growth inhibition was assessed by SRB assay. Median-effect analysis was used to assess synergy. Functional testing was performed in immortalized 2D, xenoderived 2D and 3D cultures. Effective low dose 3 drug combinations were further validated using patient-derived xenografts. Results: High INKA scoring of multiple activities per cell line without clear outliers of single kinases underscores the need for combination therapies. Multiple LD combinations showed effective growth inhibition (70 to 92%) and synergism, mostly 3 drug combinations, which required targeting of several RTKs and downstream signaling. Different responses were further observed between epithelial and mesenchymal cell lines. These top performing combinations were further validated in PDAC organoids and in vivo. Clinical utility of these kinase targets was then confirmed in 42 tumor phosphoprofiles, which were characterized in different subtypes with distinct therapeutic options. Phosphoproteome signals and kinases activities with potential prognostic value and mutational associations were further described. Conclusion: Our INKA pipeline, which can rank kinase activities in individual tumors, is optimally suited to specifically prioritize actionable kinases with targeting purposes. Tailored LD combination strategies exhibited promising efficacy in preclinical models and may ultimately improve treatment outcomes. Next Steps: Multicellular patient-derived models will be used to further study and target the TME in PDAC tumors. Citation Format: Andrea Vallés Martí, Giulia Mantini, Cynthia Waasdorp, Richard R. de Goeij- de Haas, Alex A. Henneman, Sander R. Piersma, Thang V. Pham, Jaco C. Knol, Joanne Verheij, Frederike Dijk, Hans Halfwerk, Elisa Giovannetti, Connie R. Jiménez, Maarten F. Bijlsma. Profile guided low dose drug combination strategies and kinase activities with prognostic and therapeutic avenues in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6485.

Lipopolysaccharide (LPS) stimulation of Pancreatic Ductal Adenocarcinoma (PDAC) and macrophages activates the NLRP3 inflammasome that influences the levels of pro-inflammatory cytokines in a co-culture model

ABSTRACT Modified macrophages, tumor-associated macrophages (TAMs), are key contributors to the survival, growth, and metastatic behavior of pancreatic ductal adenocarcinoma (PDAC) cells. Central to the role of inflammation and TAMs lies the NLRP3 inflammasome. This study investigated the effects of LPS-stimulated inflammation on cell proliferation, levels of pro-inflammatory cytokines, and the NLRP3 inflammasome pathway in a co-culture model using PDAC cells and macrophages in the presence or absence of MCC950, a NLRP3-specific inhibitor. The effects of LPS-stimulated inflammation were tested on two PDAC cell lines (Panc 10.05 and SW 1990) co-cultured with RAW 264.7 macrophages. Cell proliferation was determined using the MTT assay. Levels of pro-inflammatory cytokines, IL-1β, and TNF-α were determined by ELISA. Western blot analyses were used to examine the expression of NLRP3 in both PDAC cells and macrophages. The co-culture and interaction between PDAC cell lines and macrophages led to pro-inflammatory microenvironment under LPS stimulation as evidenced by high levels of secreted IL-1β and TNF-α. Inhibition of the NLRP3 inflammasome by MCC950 counteracted the effects of LPS stimulation on the regulation of the NLRP3 inflammasome and pro-inflammatory cytokines in PDAC and macrophages. However, MCC950 differentially modified the viability of the metastatic vs primary PDAC cell lines. LPS stimulation increased PDAC cell viability by regulating the NLRP3 inflammasome and pro-inflammatory cytokines in the tumor microenvironment of PDAC cells/macrophages co-cultures. The specific inhibition of the NLRP inflammasome by MCC950 effectively counteracted the LPS-stimulated inflammation.

Suppressive Role of ACVR1/ALK2 in Basal and TGFβ1-Induced Cell Migration in Pancreatic Ductal Adenocarcinoma Cells and Identification of a Self-Perpetuating Autoregulatory Loop Involving the Small GTPase RAC1b.

Pancreatic ductal adenocarcinoma (PDAC) cells are known for their high invasive/metastatic potential, which is regulated in part by the transforming growth factor β1 (TGFβ1). The involvement of at least two type I receptors, ALK5 and ALK2, that transmit downstream signals of the TGFβ via different Smad proteins, SMAD2/3 and SMAD1/5, respectively, poses the issue of their relative contribution in regulating cell motility. Real-time cell migration assays revealed that the selective inhibition of ALK2 by RNAi or dominant-negative interference with a kinase-dead mutant (ALK2-K233R) strongly enhanced the cells’ migratory activity in the absence or presence of TGFβ1 stimulation. Ectopic ALK2-K233R expression was associated with an increase in the protein levels of RAC1 and its alternatively spliced isoform, RAC1b, both of which are implicated in driving cell migration and invasion. Conversely, the RNAi-mediated knockdown or CRISPR/Cas9-mediated knockout of RAC1b resulted in the upregulation of the expression of ALK2, but not that of the related BMP type I receptors, ALK3 or ALK6, and elevated the phosphorylation of SMAD1/5. PDAC is a heterogeneous disease encompassing tumors with different histomorphological subtypes, ranging from epithelial/classical to extremely mesenchymal. Upon treatment of various established and primary PDAC cell lines representing these subtypes with the ALK2 inhibitor, LDN-193189, well-differentiated, epithelial cell lines responded with a much stronger increase in the basal and TGFβ1-dependent migratory activity than poorly differentiated, mesenchymal ones. These data show that (i) ALK2 inhibits migration by suppressing RAC1/RAC1b proteins, (ii) ALK2 and RAC1b act together in a self-perpetuating the autoregulatory negative feedback loop to mutually control their expression, and (iii) the ALK2 antimigratory function appears to be particularly crucial in protecting epithelial subtype cells from becoming invasive, both spontaneously and in a TGFβ-rich tumor microenvironment.

Targeting the ubiquitin-proteasome system in a pancreatic cancer subtype with hyperactive MYC.

The myelocytomatosis oncogene (MYC) is an important driver in a subtype of pancreatic ductal adenocarcinoma (PDAC). However, MYC remains a challenging therapeutic target; therefore, identifying druggable synthetic lethal interactions in MYC-active PDAC may lead to novel precise therapies. First, to identify networks with hyperactive MYC, we profiled transcriptomes of established human cell lines, murine primary PDAC cell lines, and accessed publicly available repositories to analyze transcriptomes of primary human PDAC. Networks active in MYC-hyperactive subtypes were analyzed by gene set enrichment analysis. Next, we performed an unbiased pharmacological screen to define MYC-associated vulnerabilities. Hits were validated by analysis of drug response repositories and genetic gain- and loss-of-function experiments. In these experiments, we discovered that the proteasome inhibitor bortezomib triggers a MYC-associated vulnerability. In addition, by integrating publicly available data, we found the unfolded protein response as a signature connected to MYC. Furthermore, increased sensitivity of MYC-hyperactive PDACs to bortezomib was validated in genetically modified PDAC cells. In sum, we provide evidence that perturbing the ubiquitin-proteasome system (UPS) might be an option to target MYC-hyperactive PDAC cells. Our data provide the rationale to further develop precise targeting of the UPS as a subtype-specific therapeutic approach.

Primary and Metastatic Pancreatic Cancer Cells Exhibit Differential Migratory Potentials.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by early metastatic spread in more than 50% of patients. In this study, we sought to understand the migratory properties of (non)metastatic PDAC cells and determine whether the migration of cancer stem cell (CSC) populations accounts for the aggressive nature of this disease. The migratory abilities of primary and metastatic PDAC cell lines were investigated using a microfluidic device and time-lapse photography. The velocity, time of delay of mobilization, and number of migratory cells were analyzed. Cancer stem cell subpopulations were isolated by fluorescence-activated cell sorting and their migratory properties compared with their non-CSC counterparts. Primary cancer cells exhibited higher velocities, greater number of migratory cells, and a shorter time of delay of mobilization in comparison to metastatic cell lines. Characterization of CSC populations revealed primary PDAC cell lines were composed of fewer CD133 and CD24CD44 CSC subpopulations than metastatic cells. Moreover, migratory analysis of CSC subpopulations revealed lower velocities, fewer migratory cells, and a greater time of delay of mobilization than non-CSC. Primary cancer cells demonstrate enhanced migratory abilities in comparison to metastatic PDAC cells. Those differences may result from lower CSC subpopulations in primary cells because CSC populations demonstrated impaired migratory abilities in contrast to non-CSC.

A drug‐repositioning screen for primary pancreatic ductal adenocarcinoma cells identifies 6‐thioguanine as an effective therapeutic agent for TPMT‐low cancer cells

Pancreatic cancer is one of the most difficult cancers to cure due to the lack of early diagnostic tools and effective therapeutic agents. In this study, we aimed to isolate new bioactive compounds that effectively kill pancreatic ductal adenocarcinoma (PDAC) cells, but not untransformed, human pancreatic ductal epithelial (HPDE) cells. To this end, we established four primary PDAC cell lines and screened 4141 compounds from four bioactive‐compound libraries. Initial screening yielded 113 primary hit compounds that caused over a 50% viability reduction in all tested PDAC cells. Subsequent triplicate, dose‐dependent analysis revealed three compounds with a tumor cell‐specific cytotoxic effect. We found that these three compounds fall into a single category of thiopurine biogenesis. Among them, 6‐thioguanine (6‐TG) showed an IC50 of 0.39–1.13 μm toward PDAC cells but had no effect on HPDE cells. We propose that this cancer selectivity is due to differences in thiopurine methyltransferase (TPMT) expression between normal and cancer cells. This enzyme is responsible for methylation of thiopurine, which reduces its cytotoxicity. We found that TPMT levels were lower in all four PDAC cell lines than in HPDE or Panc1 cells, and that knockdown of TPMT in HPDE or Panc1 cells sensitized them to 6‐TG. Lastly, we used a patient‐derived xenograft model to confirm that 6‐TG has a significant antitumor effect in combination with gemcitabine. Overall, our study presents 6‐TG as a strong candidate for use as a therapeutic agent against PDAC with low levels of TPMT.

Expression of G protein‐coupled receptors in pancreatic cancer cells

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal forms of cancer and has an overall 5‐year survival rate ~7%. PDAC has poor response to chemotherapy and radiotherapy; thus new treatments for PDAC are an important, unmet medical need. G protein‐coupled receptor (GPCR) regulate many biological functions, including in cancer cells, but GPCR expression and the role of GPCRs in PDAC cells is unclear. We generated two primary PDAC cell lines (79E and 34E) and an immortalized normal pancreatic ductal epithelial cell line (HPDE6). Hypothesizing that GPCRs may regulate PDAC cells, we used Taqman GPCR array to assess GPCR expression in four PDAC cell lines, 79E, 34E, Miapaca, and AsPC. We identified 68 commonly expressed GPCRs in all four PDAC cell lines, among which 22 GPCRs were significantly up‐regulated (at least 2‐fold) in PDAC cells compared to normal control cell HPDE6; 13 GPCRs were significantly down‐regulated and 3 GPCRs were uniquely expressed in PDACs. We further tested the 13 most up‐regulated GPCRs using real time qPCR and confirmed that LPHN1, CHRM4, FZD4, HRH1 and CD97 have much higher expression in PDACs compared to HPED6, suggesting that these GPCRs might contribute to the malignant phenotype and perhaps could be novel treatment targets for pancreatic cancer, in particular for PDAC.Support or Funding InformationThis work is supported by NIH/NCI Research Grants R21 CA189477, R01 CA155620, and Padres Pedal the Cause PTC2017.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A novel chemoradiation targeting stem and nonstem pancreatic cancer cells by repurposing disulfiram

Pancreatic ductal adenocarcinoma (PDAC) has a 5-year relative survival rate of 8% and is projected to be the second leading cause of cancer death by 2030, underscoring the urgency to develop new strategies to improve current therapeutic modalities for PDAC. Targeting pancreatic cancer stem cells (PCSCs), which are resistant to radiation and chemotherapy, is a promising strategy. A novel approach which can be readily clinically translated is to repurpose disulfiram (DSF), a drug for treating alcoholism, to target PCSCs. Chemoradiation or the combination of chemotherapy agents FOLFIRINOX, currently standard care for PDAC, can increase stemness in some established or primary PDAC cell lines. However, DSF in the presence of exogenously or endogenously supplied copper (Cu), when combined with chemotherapy or chemoradiation, targets both PCSCs and nonstem PDAC cells. Previously, we demonstrated that DSF/Cu effectively targets breast cancer stem cells in the context of fractionated radiation (FIR) by inhibiting the NF-κB–stemness gene pathway. Therefore, the hypothesis that PCSCs can be effectively targeted by incorporating DSF/Cu into the standard chemoradiation regimen consisting of 5-FU and FIR was investigated and found to be effective in vitro in targeting PCSCs, identified as either ALDHbright or CD24+/CD44+/ESA+ or sphere-forming cells, as well as nonstem PDAC cells. In vivo, the combination of IR+5-FU+DSF/Cu was more effective (72.46%) than either IR+5-FU (30.32%) or IR+FOLFIRINOX therapy (43.04%) in inhibiting growth of the mouse Panc02 tumor. These encouraging results provide a solid foundation for clinical trials to improve the outcomes of the current standard chemoradiation therapy regimen for PDAC.

Abstract B29: The elucidation for functional roles of Metadherin in metastatic cascade of pancreatic cancer

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers. At present, surgical resection is the only curative treatment for PDAC, but the majority of PDAC patients show early recurrence even after curative resection. There is a possibility of having been distant micrometastasis at early stage of carcinogenesis in PDAC. The control of PDAC metastatic progression leads to improve the prognosis of PDAC patients. Recently, it is reported that a verified mediator of distant metastasis, Metadherin (MTDH), promotes metastasis in experimental models of breast cancer, and its high expression is associated with poor prognosis in a large spectrum of cancer types. Recent evidences imply that the epithelial-mesenchymal transition (EMT) and putative cancer stem cell (CSC) functions orchestrates to play crucial roles in cancer progression. In this study, we aim to investigate the function of MTDH in PDAC progression concomitant with undergoing EMT/MET plasticity or involving putative CSC functions. Experimental methods: We investigated MTDH expression in 7 human and 6 mouse PDAC cell lines and elucidated the roles of MTDH in PDAC progression by in vitro experiments for the loss of function using siMTDH and shMTDH. In immunohistochemical (IHC) staining of MTDH in resected PDAC tissues, we analyzed the correlation between MTDH expression and the clinico-pathological parameters as well as clinical outcome of the 82 PDAC patients. Results: Western blot analyses showed higher MTDH protein expression in metastatic PDAC compared to primary PDAC cell lines. In metastatic cell lines, MTDH expression has a positive correlation with E-cadherin expression. These data suggest that MTDH might be related to mesenchymal-epithelial transition (MET) and play a crucial role in metastatic cascade of PDAC. Conversely, MTDH has a negative correlation with E-cadherin in primary PDAC cell lines. It is implicated that MTDH might play inverse functions in primary site and metastatic site. Next, to examine what is the role of MTDH in the EMT/MET process, we evaluated the expression pattern of EMT markers and MTDH in response to transient TGF-β1 treatment. We found that E-cadherin expression decreases at 8 to 24 hours after treatment (EMT initiation), and then comes back to increase of the expression at 4 days after treatment (MET induction). While MTDH and E-cadherin expression shows an inverse relation during EMT initiation, these two expressions have a positive correlation during MET period (from 7 days after treatment). Additionally, we found EMT induction by TGF-β1 is attenuated by knockdown of MTDH, These results suggest MTDH might contribute to the phenotypic switch of PDAC cells via the EMT/MET process. MTDH knockdown significantly impaired the ability of pancreatosphere formation in PDAC cells and sensitized PDAC cells to gemcitabine. In FACS analyses, we also found that the percentage of CD133-positive cancer stem cell subpopulation was significantly reduced in MTDH knockdown metastatic PDAC cells. In anoikis assay, (this assay evaluate the resistance for apoptosis after losing contact from extracelluar matrix), MTDH knockdown reduced anoikis resistance in PDAC cells. These data indicate that MTDH might foster putative CSC functions in PDAC cells. In IHC staining, high MTDH expression indicated significantly higher incidence of hematogenous metastasis (p=0.02). Furthermore, High expression group showed poorer prognosis of PDAC patients compared to Low expression group in Kaplan Meier analysis (p=0.029). Conclusions: MTDH might promote PDAC metastasis to undergo EMT in primary PDAC and MET in metastatic PDAC along with.putative CSC functions. The regulation of MTDH might develop the new strategy for PDAC treatment, and lead to improve the outcome of patients. Citation Format: Kensuke Suzuki, Shigetsugu Takano, Hideyuki Yoshitomi, Shingo Kagawa, Hiroaki Shimizu, Masayuki Otsuka, Katunori Furukawa, Tukasa Takayashiki, Satoshi Kuboki, Daisuke Suzuki, Nozomu Sakai, Hiroyuki Nojima, Masaru Miyazaki.{Authors}. The elucidation for functional roles of Metadherin in metastatic cascade of pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B29.

Up-regulation of microRNA let-7c by quercetin inhibits pancreatic cancer progression by activation of Numbl.

Pancreatic Ductal Adenocarcinoma (PDA) is a highly malignant tumor with poor prognosis. MicroRNAs (miRs) may offer novel therapeutic approaches to treatment. The polyphenol quercetin, present in many fruits and vegetables, possesses anti-carcinogenic properties. To unravel the effect of quercetin to miR signaling we performed miR profiling in PDA cells before and after quercetin treatment, followed by biostatistical analysis. miR let-7c was among the top up-regulated candidates after quercetin treatment, as measured by qRT-PCR and confirmed in two established and one primary PDA cell lines. By computational analysis we identified the Notch-inhibitor Numbl as let-7c target gene. This was strengthened by luciferase assays, where lipofected let-7c mimics induced a Numbl 3-UTR wild type construct, but not the mutated counterpart. Let-7c induced Numbl mRNA and protein expression but inhibited Notch just like quercetin. It also inhibited colony formation, wound healing, and protein expression of progression markers. In vivo xenotransplantation of PDA cells and subsequent intravenous injection of let-7c resulted in a significant decrease in tumor mass without obvious toxic effects in the fertilized chick egg model. The delivery rate of the miR mimics to the tumor mass was 80%, whereas minor amounts were present in host tissue. By immunohistochemistry we demonstrated that let-7c inhibited Notch and progression markers but up-regulated Numbl. These findings show that quercetin-induced let-7c decreases tumor growth by posttranscriptional activation of Numbl and indirect inhibition of Notch.

Upstream and Downstream Co-inhibition of Mitogen-Activated Protein Kinase and PI3K/Akt/mTOR Pathways in Pancreatic Ductal Adenocarcinoma

BACKGROUND: Extensive cross talk exists between PI3K/Akt/mTOR and mitogen-activated protein kinase (MAPK) pathways, and both are upregulated in pancreatic ductal adenocarcinoma (PDAC). Our previous study suggested that epidermal growth factor receptor inhibitor erlotinib which acts upstream of these pathways acts synergistically with PI3K inhibitors in PDAC. Horizontal combined blockade upstream and downstream of these two pathways is therefore explored. METHODS: Erlotinib paired with PI3K inhibitor (BYL719) was tested against erlotinib plus dual PI3K/mTOR inhibitor BEZ-235, and MEK inhibitor (PD98059) plus BEZ235, on five primary PDAC cell lines and on two pairs of parent and erlotinib-resistant (ER) cell lines. A range of in vitro assays including cell proliferation, Western blotting, migration, clonogenic, cell cycle, and apopotic assays was used to test for the efficacy of combined blockade. RESULTS: Dual downstream blockade of the MAPK and PAM pathways was more effective in attenuating downstream molecular signals. Synergy was demonstrated for erlotinib and BEZ235 and for PD-98059 and BEZ-235. This resulted in a trend of increased growth cell cycle arrest, apoptosis, cell proliferation, and colony and migration suppression. This combination showed more efficacy in cell lines with acquired resistance to erlotinib. CONCLUSIONS: The additional mTOR blockade provided by BEZ235 in combined blockade resulted in increased anticancer effect. The hypersensitivity of ER cell lines to additional mTOR blockade suggested PAM pathway oncogenic dependence via mTOR. Dual downstream combined blockade of MAPK and PAM pathways with MEK and PI3K/mTOR inhibitor appeared most effective and represents an attractive therapeutic strategy against pancreatic cancer and its associated drug resistance.

Hexokinase 2 promotes tumor growth and metastasis by regulating lactate production in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a KRAS-driven cancer with a high incidence of metastasis and an overall poor prognosis. Previous work in a genetically engineered mouse model of PDAC showed glucose metabolism to be important for maintaining tumor growth. Multiple glycolytic enzymes, including hexokinase 2 (HK2), were upregulated in primary PDAC patient tumors, supporting a role for glycolysis in promoting human disease. HK2 was most highly expressed in PDAC metastases, suggesting a link between HK2 and aggressive tumor biology. In support of this we found HK2 expression to be associated with shorter overall survival in PDAC patients undergoing curative surgery. Transient and stable knockdown of HK2 in primary PDAC cell lines decreased lactate production, anchorage independent growth (AIG) and invasion through a reconstituted matrix. Conversely, stable overexpression of HK2 increased lactate production, cell proliferation, AIG and invasion. Pharmacologic inhibition of lactate production reduced the HK2-driven increase in invasion while addition of extracellular lactate enhanced invasion, together providing a link between glycolytic activity and metastatic potential. Stable knockdown of HK2 decreased primary tumor growth in cell line xenografts and decreased incidence of lung metastasis after tail vein injection. Gene expression analysis of tumors with decreased HK2 expression showed alterations in VEGF-A signaling, a pathway important for angiogenesis and metastasis, consistent with a requirement of HK2 in promoting metastasis. Overall our data provides strong evidence for the role of HK2 in promoting PDAC disease progression, suggesting that direct inhibition of HK2 may be a promising approach in the clinic.

Role of TFEB-driven autophagy regulation in pancreatic cancer treatment.

Autophagy pathways promote the growth of pancreatic ductal adenocarcinoma (PDAC), but the critical role is yet to be determined. Transcription factor EB (TFEB) centrally controls lysosomal and autophagy biogenesis. This study aimed to explore the role of TFEB for autophagy regulation in PDAC. We found that TFEB expression was significantly elevated in human PDAC samples (n=45), and localized to the cytoplasm and nucleus in 11 of 15 cases. In primary PDAC cell lines, TFEB nuclear expression was evident even under basal conditions, and further nuclear enrichment was achieved by starvation. Transient RNA interference reduced TFEB expression to 11-23%, but starvation-induced accumulation of the lipidated, autophagosome-associated LC3-II and the autophago-to-lysosome route was maintained after TFEB silencing. Likewise, gemcitabine treatment of the cancer cells augmented apoptosis and LC3-II as an indicator of autophagy, regardless of the TFEB expression levels. Moreover, the interplay of oncogenic KRAS with TFEB and autophagy was investigated. KRAS silencing caused PDAC cell apoptosis and a reciprocal increase in TFEB expression. This inverse correlation could be confirmed in published data sets of genetically engineered mouse models and human PDAC samples using the the Pubmed GEO and BioPortal databases, and was independent of KRAS mutation status. In conclusion, the central autophagy regulator TFEB is expressed and active in PDAC, but autophagy is sustained after TFEB knockdown, suggesting alternative bypass signaling. TFEB is dispensable for gemcitabine-induced cell death, but inversely correlated with KRAS expression.

Abstract B52: The role of extracellular superoxide dismutase activity in pancreatic cancer biology and therapy

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDA) cells are known to produce excessive amounts of reactive oxygen species (ROS), particularly superoxide (O2•-), which may contribute to the aggressive nature of this disease. Extracellular superoxide dismutase (EcSOD) is an antioxidant enzyme that catalyzes the dismutation of O2•- to hydrogen peroxide (H2O2) in the extracellular environment. By limiting O2•- concentration and producing H2O2, EcSOD is thought to regulate the redox state of the tumor microenvironment that could contribute to pancreatic cancer progression. The current work tests the hypothesis that EcSOD modulates PDA growth and invasion by modifying the balance between H2O2 and ONOO- formation. Methods: EcSOD and 3-nitrotyrosine (3-NT), a marker of protein nitration by ONOO-, were evaluated in specimens from patients with PDA. EcSOD expression was correlated with clinicopathologic and treatment-related variables and survival in a tissue microarray (TMA) of 114 patients with Stage I-IV PDA. EcSOD was constitutively overexpressed in two primary PDA cell lines and clonogenic survival and doubling time were measured. A Matrigel invasion assay was used to determine the impact of EcSOD on PDA tumor cell invasive capacity. We also utilized and GC4419, a selective SOD mimic currently in clinical development. GC4419 dismutates O2•- but does not react with H2O2 under physiologic conditions. PDA cells stably expressing EcSOD and luciferase were used to create hind leg xenografts in athymic nude mice to assess the impact of EcSOD on tumor growth and then via peritoneal injection to determine the impact on peritoneal metastasis formation using bioluminescence imaging (BLI). Results: EcSOD expression was reduced in PDA compared to normal pancreas in 11/16 specimens evaluated (69%). A decrease in expression was also seen in areas of premalignant ductal epithelium (PanIN-3) relative to adjacent pancreas. In normal pancreas tissue, there was no evidence of immunoreactive 3-NT; however, 11/16 PDA specimens (69%) had robust levels of immunoreactive 3-NT. In the TMA, EcSOD expression was absent in 41/114 of the PDA biopsies (36%). The median survival of patients with intact EcSOD expression was 11.0 months vs. 6.5 months for patients with loss of EcSOD expression. Compared to patients with intact EcSOD, patients with loss of EcSOD more often had advanced stage disease and were less likely to be treated with surgery. In an adjusted proportional hazard model of more than 10 clinical variables, the HR for death with loss of EcSOD was 1.63 (95% CI=1.02-2.58, p=0.04). EcSOD overexpression significantly reduced clonogenic survival, doubling time, and tumor cell invasion relative to controls. GC4419 also significantly reduced PDA invasiveness in the Matrigel assay. PDA cells stably expressing EcSOD showed decreased growth and doubling time in hind limb xenografts and significantly less peritoneal metastasis using BLI. Conclusions: Loss of EcSOD expression and oxidative stress as indicated by the presence of 3-NT is a common finding in PDA. Loss of EcSOD expression correlates with negative prognostic factors and shorter survival in patients with PDA. Overexpression of EcSOD resulted in reduced PDA growth, invasion, and peritoneal metastasis. The selective SOD mimic GC4419 also reduced invasion of PDA tumor cells. Together these findings support the hypothesis that modulating O2•- in the PDA microenvironment alters parameters of progression, oxidative damage, and may represent a target for limiting the spread of PDA. (Supported by The American Surgical Association Foundation Fellowship (JJM) and the American Cancer Society (JJM). Citation Format: James J. Mezhir, Brianne R. O'Leary, Andrew M. Bellizzi, Sean Altekruse, Charles F. Lynch, Brenda Y. Hernandez, Wendy Cozen, Michael D. Henry, Jeffrey Keene, Robert A. Beardsley, Douglas R. Spitz, Frederick E. Domann. The role of extracellular superoxide dismutase activity in pancreatic cancer biology and therapy. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B52.

Aspirin counteracts cancer stem cell features, desmoplasia and gemcitabine resistance in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDA) is characterized by an extremely poor prognosis. An inflammatory microenvironment triggers the pronounced desmoplasia, the selection of cancer stem-like cells (CSCs) and therapy resistance. The anti-inflammatory drug aspirin is suggested to lower the risk for PDA and to improve the treatment, although available results are conflicting and the effect of aspirin to CSC characteristics and desmoplasia in PDA has not yet been investigated. We characterized the influence of aspirin on CSC features, stromal reactions and gemcitabine resistance. Four established and 3 primary PDA cell lines, non-malignant cells, 3 patient tumor-derived CSC-enriched spheroidal cultures and tissues from patients who did or did not receive aspirin before surgery were analyzed using MTT assays, flow cytometry, colony and spheroid formation assays, Western blot analysis, antibody protein arrays, electrophoretic mobility shift assays (EMSAs), immunohistochemistry and in vivo xenotransplantation. Aspirin significantly induced apoptosis and reduced the viability, self-renewal potential, and expression of proteins involved in inflammation and stem cell signaling. Aspirin also reduced the growth and invasion of tumors in vivo, and it significantly prolonged the survival of mice with orthotopic pancreatic xenografts in combination with gemcitabine. This was associated with a decreased expression of markers for progression, inflammation and desmoplasia. These findings were confirmed in tissue samples obtained from patients who had or had not taken aspirin before surgery. Importantly, aspirin sensitized cells that were resistant to gemcitabine and thereby enhanced the therapeutic efficacy. Aspirin showed no obvious toxic effects on normal cells, chick embryos or mice. These results highlight aspirin as an effective, inexpensive and well-tolerated co-treatment to target inflammation, desmoplasia and CSC features PDA.

Abstract 182: SPARC as a regulator of collagen signaling in pancreatic cancer

Abstract The extracellular matrix (ECM) is a principal component of pancreatic ductal adenocarcinoma (PDA), which is rich in fibrillar collagens, and provides structural support that facilitates tumor cell survival and chemoresistance. Collagen expression and deposition is a complex process that is orchestrated in part by the matricellular protein SPARC. SPARC expression in human patients with PDA correlates with improved chemoresponse; however, this mechanism is unclear. We propose that SPARC controls collagen binding to the cell surface and reduces collagen-mediated signaling via Discoidin domain receptors (DDR1, DDR2). DDR1/DDR2 are homologous receptor tyrosine kinases that bind specifically to fibrillar collagens and have been implicated regulating cell proliferation, migration, adhesion, ECM remodeling, and response to growth factors. Additionally, collagen has been shown to promote chemoresistance in pancreatic tumor cells. We hypothesize that SPARC promotes chemoresponse by reducing collagen-mediated DDR signaling. Structural studies identified that SPARC and DDRs share the same collagen binding site. We have demonstrated that SPARC inhibits collagen binding to DDR1/DDR2 via in vitro binding assays and cell based activity assays. To determine the functional relevance of SPARC expression and collagen-mediated DDR activation in PDA, Sparc-null (Sparc-/-) mice were crossed with KIC (LSL-KrasG12D; Cdkn2alox/lox; p48Cre/+) mice. Survival was reduced and tumors were more aggressive in Sparc-/- KIC along with an elevation in DDR-mediated signaling in the tumor microenvironment. Primary PDA cell lines isolated from Sparc+/+ and Sparc-/- KIC animals were used to probe collagen signaling cascades revealing that collagen activated Ddr1 and downstream intermediates including protein tyrosine kinase 2 (Pyk2) and pseudopodium-enriched atypical kinase 1 (Peak1) in Sparc-/- cell lines. Furthermore, recombinant SPARC or neutralizing antibodies to Ddr1 abrogated collagen induced Ddr1 signaling in vitro implicating Ddr1 as a putative therapeutic target in PDA. Pharmacologic inhibition of Ddr1 was evaluated with nilotinib, a BCR-Abl inhibitor that shows high activity against Ddr1, and 7rh, a novel inhibitor with high specificity for Ddr1. In vitro nilotinib and 7rh suppress collagen-induced Ddr1 signaling, cell migration, and colony formation. In vivo nilotinib reduced the growth of implanted pancreatic tumors in Sparc-/- animals with little effect on tumor growth in Sparc+/+ mice. Studies with 7rh in vivo demonstrate an inhibition of Ddr1 signaling in syngeneic pancreatic tumors and is well tolerated at doses up to 30 mg/kg given daily via oral gavage. Therapy studies combining standard chemotherapy (gemcitabine + Nab-paclitaxel) with 7rh are ongoing. Our results suggest that Ddr1 enhances tumor cell survival and tumor progression and that Sparc functions to reduce PDA progression by blunting collagen-induced DDR signaling. Citation Format: Kristina Y. Aguilera, Courtney D. Goldstein, Lee B. Rivera, Amy D. Bradshaw, Ke Ding, Rolf A. Brekken. SPARC as a regulator of collagen signaling in pancreatic cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 182. doi:10.1158/1538-7445.AM2014-182

Inhibition of glucose turnover by 3-bromopyruvate counteracts pancreatic cancer stem cell features and sensitizes cells to gemcitabine.

According to the cancer stem cell (CSC) hypothesis, the aggressive growth and early metastasis of pancreatic ductal adenocarcinoma (PDA) is due to the activity of CSCs, which are not targeted by current therapies. Otto Warburg suggested that the growth of cancer cells is driven by a high glucose metabolism. Here, we investigated whether glycolysis inhibition targets CSCs and thus may enhance therapeutic efficacy. Four established and 3 primary PDA cell lines, non-malignant cells, and 3 patient-tumor-derived CSC-enriched spheroidal cultures were analyzed by glucose turnover measurements, MTT and ATP assays, flow cytometry of ALDH1 activity and annexin positivity, colony and spheroid formation, western blotting, electrophoretic mobility shift assay, xenotransplantation, and immunohistochemistry. The effect of siRNA-mediated inhibition of LDH-A and LDH-B was also investigated. The PDA cells exhibited a high glucose metabolism, and glucose withdrawal or LDH inhibition by siRNA prevented growth and colony formation. Treatment with the anti-glycolytic agent 3-bromopyruvate almost completely blocked cell viability, self-renewal potential, NF-κB binding activity, and stem cell-related signaling and reverted gemcitabine resistance. 3-bromopyruvate was less effective in weakly malignant PDA cells and did not affect non-malignant cells, predicting minimal side effects. 3-bromopyruvate inhibited in vivo tumor engraftment and growth on chicken eggs and mice and enhanced the efficacy of gemcitabine by influencing the expression of markers of proliferation, apoptosis, self-renewal, and metastasis. Most importantly, primary CSC-enriched spheroidal cultures were eliminated by 3-bromopyruvate. These findings propose that CSCs may be specifically dependent on a high glucose turnover and suggest 3-bromopyruvate for therapeutic intervention.