Human Pancreatic Tumor Cell Lines Research Articles

The urolithin B nanomicellar delivery system as an efficient selective anticancer compound.

Urolithin B (UB), the antioxidant polyphenol has a protective impact on several organs against oxidative stress. However, its bioactivity is limited by its hydrophobic structure. In the current study, UB was encapsulated into a liposomal structure to improve its bioactivities anticancer, and antimicrobial potential. The UB nano-emulsions (UB-NE) were synthesized and characterized utilizing FESEM, DLS, FTIR, and Zeta-potential analysis. The UB-NMs' selective toxicity was studied by conducting an MTT assay on MCF-7, PANC, AGS, and ASPC1 cells. The AO/PI analysis verified the UB-NMs' cytotoxicity on ASPC1 cell lines and approved the MTT results. Finally, the antibacterial activity of the UB-NMs was studied on both gram-positive (B. subtilis, S. aureus) and gram-negative (E. Coli, P. aeruginosa) bacteria by conducting MIC and MBC analysis. The 68.15nm UB-NMs did not reduce the normal HDF cells' survival. However, they reduced the cancer cells' (PANC and AGS cell lines) survival at high treatment concentrations (> 250µg/mL) compared with normal HDF and cancer MCF-7 cells. Moreover, the IC50 doses of UB-NMs for the ASPC1 and PANC cancer cells were measured at 44.87, and 221.02µg/mL, respectively. The UB-NMs selectively exhibited apoptotic-mediated cytotoxicity on the human pancreatic tumor cell line (ASPC1) by down-regulating BCL2 and NFKB gene expression. Also, the BAX gene expression was up-regulated in the ASPC1-treated cells. Moreover, they exhibited significant anti-bactericidal activity against the E. coli (MIC = 50µg/mL, MBC = 150µg/mL), P. aeruginosa (MIC = 75µg/mL, MBC = 275µg/mL), B. subtilis (MIC = 125µg/mL, MBC = 450µg/mL), and S. aureus (MIC = 50µg/mL, MBC = 200µg/mL) strains. The significant selective cytotoxic impact of the UB-NMs on the human pancreatic tumor cell line makes it an applicable anti-pancreatic cancer compound. Moreover, the antibacterial activity of UB-NMs has the potential to decrease bacterial-mediated pancreatic cancer. However, several bacterial strains and further cancer cell lines are required to verify the UB-NMs' anticancer potential.

Cannabis sativa Extract Induces Apoptosis in Human Pancreatic 3D Cancer Models: Importance of Major Antioxidant Molecules Present Therein.

In recent years, interest in Cannabis sativa L. has been rising, as legislation is moving in the right direction. This plant has been known and used for thousands of years for its many active ingredients that lead to various therapeutic effects (pain management, anti-inflammatory, antioxidant, etc.). In this report, our objective was to optimize a method for the extraction of cannabinoids from a clone of Cannabis sativa L. #138 resulting from an agronomic test (LaFleur, Angers, FR). Thus, we wished to identify compounds with anticancer activity on human pancreatic tumor cell lines. Three static maceration procedures, with different extraction parameters, were compared based on their median inhibitory concentration (IC50) values and cannabinoid extraction yield. As CBD emerged as the molecule responsible for inducing apoptosis in the human pancreatic cancer cell line, a CBD-rich cannabis strain remains attractive for therapeutic applications. Additionally, while gemcitabine, a gold standard drug in the treatment of pancreatic cancer, only triggers cell cycle arrest in G0/G1, CBD also activates the cell signaling cascade to lead to programmed cell death. Our results emphasize the potential of natural products issued from medicinal hemp for pancreatic cancer therapy, as they lead to an accumulation of intracellular superoxide ions, affect the mitochondrial membrane potential, induce G1 cell cycle arrest, and ultimately drive the pancreatic cancer cell to lethal apoptosis.

Therapeutic properties of isoliquiritigenin with molecular modeling studies: investigation of anti-pancreatic acinar cell tumor and HMG-CoA reductase inhibitor activity for treatment of hypercholesterolemia.

Isoliquiritigenin, one of the components in the root of Glycyrrhiza glabra L., is a member of the flavonoids, which are known to have anti-tumor activity in vitro and in vivo. HMG-CoA reductase inhibitors, called statins, are used to reduce the risk of heart disease by lowering blood cholesterol levels. HMG-CoA reductase activity was determined according to the method described by Takahashi et al. The structure of human HMG-COA reductase in the resolution of 2.22 Å with the X-ray diffraction method (PDB ID: 1HWK) was obtained from the PDB database. In our study, the inhibitory activity of isoliquiritigenin towards HMG-CoA reductase showed a lower value of IC50 = 193.77 ±14.85 μg/ml. For a better understanding of biological activities and interactions, the molecular docking study was performed. The results of molecular docking revealed that isoliquiritigenin with a docking score of -6.740 has a strong binding affinity to HMG-COA reductase. Therefore, this compound could be considered as a potential inhibitor for the enzyme. Also, the activity of isoliquiritigenin against common human pancreatic acinar cell tumor cell lines, i.e. 266-6, TGP49, and TGP47, was evaluated. The cells treated with isoliquiritigenin were assessed by MTT assay for 48 h as regards the cytotoxicity and anti-human pancreatic acinar cell tumor properties in normal (HUVEC) and human pancreatic acinar cell tumor cell lines, i.e. 266-6, TGP49, and TGP47. The IC50 values of isoliquiritigenin were 262, 389, and 211 μg/ml against 266-6, TGP49, and TGP47 cell lines, respectively. The viability of the human pancreatic acinar cell tumor cell line decreased dose-dependently in the presence of isoliquiritigenin. After clinical study, isoliquiritigenin can be utilized as an efficient drug in the treatment of human pancreatic acinar cell tumor in humans.

Abstract LB154: A potent human CAR NK cell therapy directed against pancreatic cancer

Abstract Pancreatic cancer (PC) is one of the most lethal forms of cancer with a 5-year overall survival rate of 9%, routinely presenting as a late-stage incurable cancer that responds only modestly to standard chemotherapy. It is the third leading cause of cancer-related deaths. Approximately 60-80% of PC express prostate stem cell antigen (PSCA), as do gastric, bladder, prostate and some lung cancers. We therefore developed a chimeric antigen receptor (CAR) directed against PSCA for transduction into human natural killer (NK) cells. NK cells spontaneously kill both liquid and solid tumors without regards to expression of MHC self-antigens. In vivo, human NK cells utilize endogenous IL-15 to develop, survive, expand and activate against tumor cells. We therefore incorporated a soluble (s), secretable form of IL-15 into the PSCA CAR construct itself, followed by transduction into human NK cells obtained from umbilical cord blood with ~50% transduction efficiency. Transduced NK cells expressed the CAR directed against PSCA and secreted measurable amounts of human IL-15 protein in vitro. The PSCA CAR NK cells could be expanded ex vivo over 1,000-fold in approximately 16 days and retain their expression and their capacity to specificity kill PSCA(+) tumor cell targets in vitro. Indeed, when directed against the PSCA(+) human pancreatic tumor cell line (Capan-1), PSCA CAR NK cells produced significantly greater TNFα (P < 0.0001), IFNγ (P < 0.0001), and CD107a (P < 0.05), when compared to PSCA CAR NK cells against the PSCA(-) human pancreatic tumor cell line PANC-1. Moreover, we showed that the co-expression of sIL-15 with PSCA CAR NK cells significantly enhances their cytotoxic function against pancreatic tumor cells compared to PSCA CAR NK cells without expression of sIL-15 (P < 0.01) determined by a real-time cytolysis assay (RTCA) over 3.5 days. Encouraged by these in vitro data, we developed a model of metastatic human pancreatic cancer in immunodeficient mice using the PSCA(+) Capan-1 cell line. Compared to NK cells only expressing sIL-15, repeated infusions of human PSCA CAR NK cells from a viably frozen source resulted in a significantly prolonged survival (P < 0.001), including clearance of metastatic disease. In summary, our in vitro and in vivo studies utilizing viably frozen human PSCA CAR NK cells co-expressing sIL-15 demonstrate significant efficacy in prolonging survival against a human pancreatic tumor cell line without evidence of systemic toxicity, providing a rationale to move this novel form of cell therapy into the clinic for PSCA(+) solid tumors. Citation Format: Kun-Yu Teng, Anthony Mansour, Zhu Zheng, Lei Tien, Yi Zheng, Zhiyao Li, Jianying Zhang, Saul J. Priceman, Michael A. Caligiuri, Jianhua Yu. A potent human CAR NK cell therapy directed against pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB154.

A Novel E2F1-EP300-VMP1 Pathway Mediates Gemcitabine-Induced Autophagy in Pancreatic Cancer Cells Carrying Oncogenic KRAS

Autophagy is an evolutionarily preserved degradation process of cytoplasmic cellular constituents, which participates in cell response to disease. We previously characterized VMP1 (Vacuole Membrane Protein 1) as an essential autophagy related protein that mediates autophagy in pancreatic diseases. We also demonstrated that VMP1-mediated autophagy is induced by HIF-1A (hypoxia inducible factor 1 subunit alpha) in colon-cancer tumor cell lines, conferring resistance to photodynamic treatment. Here we identify a new molecular pathway, mediated by VMP1, by which gemcitabine is able to trigger autophagy in human pancreatic tumor cell lines. We demonstrated that gemcitabine requires the VMP1 expression to induce autophagy in the highly resistant pancreatic cancer cells PANC-1 and MIAPaCa-2 that carry activated KRAS. E2F1 is a transcription factor that is regulated by the retinoblastoma pathway. We found that E2F1 is an effector of gemcitabine-induced autophagy and regulates the expression and promoter activity of VMP1. Chromatin immunoprecipitation assays demonstrated that E2F1 binds to the VMP1 promoter in PANC-1 cells. We have also identified the histone acetyltransferase EP300 as a modulator of VMP1 promoter activity. Our data showed that the E2F1-EP300 activator/co-activator complex is part of the regulatory pathway controlling the expression and promoter activity of VMP1 triggered by gemcitabine in PANC-1 cells. Finally, we found that neither VMP1 nor E2F1 are induced by gemcitabine treatment in BxPC-3 cells, which do not carry oncogenic KRAS and are sensitive to chemotherapy. In conclusion, we have identified the E2F1-EP300-VMP1 pathway that mediates gemcitabine-induced autophagy in pancreatic cancer cells. These results strongly support that VMP1-mediated autophagy may integrate the complex network of events involved in pancreatic ductal adenocarcinoma chemo-resistance. Our experimental findings point at E2F1 and VMP1 as novel potential therapeutic targets in precise treatment strategies for pancreatic cancer.

Abstract PR15: A novel NK cell-targeted therapeutic strategy against pancreatic cancer

Abstract Recent years have witnessed an increased incidence of pancreatic adenocarcinoma (PDAC). Pancreatic tumors are poorly immunogenic; current immunotherapeutic strategies largely focus on boosting adaptive immunity while ignoring immunosuppressed innate immune players such as natural killer cells (NKs). NK cells respond to tumor insults by generating IFN-γ for T-cell activation. Multiple studies suggest that tumor cells evade NK cell-mediated killing by 1) developing “escape variants” and 2) regulating inhibitory and activating receptors on NK cells, imparting anergic/exhaustive phenotype inside the pancreatic tumor microenvironment. We posit that a therapeutic combination that not only overturns tumor-mediated NKs dysfunction but also boosts cytotoxic CD8 T cells will provide long-lasting therapeutic benefits in PDAC. Given this, we explored the efficacy of a unique therapeutic combination, tumor antigen-targeted IgE antibody (humanized anti-MUC1.IgE) in combination of anti-PD-L1 (for relieving T-cell exhaustion) and PolyICLC (for dendritic cell maturation) in a preclinical model of pancreatic cancer using mice transgenic for human MUC1 and FcϵRI (hMUC1/hFcϵRI). This therapeutic combination induced MUC1 specific rejection of two different human MUC1-expressing pancreatic tumor cell lines (Panc02.MUC1, KPC.MUC1) and prolonged the overall survival of mice challenged with subcutaneous and orthotopic tumors as compared to control counterparts. Additionally, this combination generated CD8 T-cell memory response as evidenced by MUC1 specific rejection/delays of tumors in mice rechallenged with MUC1-expressing tumors. Cytokine/chemokine profiling of anti-MUC1.IgE+anti-PD-L1+PolyICLC treated tumors further demonstrates a reduction in proinflammatory cytokines as compared to control counterparts. Most importantly, NK and CD8 T cells were involved in cell-mediated antitumor responses, as in vivo depletion of these subtypes abrogated the tumor-protective benefits in mice bearing orthotopic tumors. Anti-MUC1.IgE+anti-PD-L1+PolyICLC combination appears to increase circulating NKs and reverse NK cell exhaustion inside pancreatic tumor microenvironment. Additional data suggest that this therapeutic combination boosts tumor cell killing by NK cells in antibody-dependent cell cytotoxicity assays (ADCC). In sum, this is the first study to show that specific stimulation of IgE/FcϵRI axis in combination with PolyICLC and anti-PD-L1 can activate both CD8 T and NK cell effector pathways and provide long-lasting tumor-protective benefits against pancreatic cancer. This abstract is also being presented as Poster A48. Citation Format: Kamiya Mehla, Thomas C. Caffrey, Kelly A. O'Connel, Raghupathy Madiyalakan, Christopher F. Nicodemus, Michael A. Hollingsworth. A novel NK cell-targeted therapeutic strategy against pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr PR15.

The Histone Demethylase KDM3A, Increased in Human Pancreatic Tumors, Regulates Expression of DCLK1 and Promotes Tumorigenesis in Mice

The histone lysine demethylase 3A (KDM3A) demethylates H3K9me1 and H3K9Me2 to increase gene transcription and is upregulated in tumors, including pancreatic tumors. We investigated its activities in pancreatic cancer cell lines and its regulation of the gene encoding doublecortin calmodulin-like kinase 1 (DCLK1), a marker of cancer stem cells. We knocked down KDM3A in MiaPaCa-2 and S2-007 pancreatic cancer cell lines and overexpressed KDM3A in HPNE cells (human noncancerous pancreatic ductal cell line); we evaluated cell migration, invasion, and spheroid formation under hypoxic and normoxic conditions. Nude mice were given orthotopic injections of S2-007 cells, with or without (control) knockdown of KDM3A, and HPNE cells, with or without (control) overexpression of KDM3A; tumor growth was assessed. We analyzed pancreatic tumor tissues from mice and pancreatic cancer cell lines by immunohistochemistry and immunoblotting. We performed RNA-sequencing analysis of MiaPaCa-2 and S2-007 cells with knockdown of KDM3A and evaluated localization of DCLK1 and KDM3A by immunofluorescence. We analyzed the cancer genome atlas for levels of KDM3A and DCLK1 messenger RNA in human pancreatic ductal adenocarcinoma (PDAC) tissues and association with patient survival time. Levels of KDM3A were increased in human pancreatic tumor tissues and cell lines, compared with adjacent nontumor pancreatic tissues, such as islet and acinar cells. Knockdown of KDM3A in S2-007 cells significantly reduced colony formation, invasion, migration, and spheroid formation, compared with control cells, and slowed growth of orthotopic tumors in mice. We identified KDM3A-binding sites in the DCLK1 promoter; S2-007 cells with knockdown of KDM3A had reduced levels of DCLK1. HPNE cells that overexpressed KDM3A formed foci and spheres in culture and formed tumors and metastases in mice, whereas control HPNE cells did not. Hypoxia induced sphere formation and increased levels of KDM3A in S2-007 cells and in HPNE cells that overexpressed DCLK1, but not control HPNE cells. Levels of KDM3A and DCLK1 messenger RNA were higher in human PDAC than nontumor pancreatic tissues and correlated with shorter survival times of patients. We found human PDAC samples and pancreatic cancer cell lines to overexpress KDM3A. KDM3A increases expression of DCLK1, and levels of both proteins are increased in human PDAC samples. Knockdown of KDM3A in pancreatic cancer cell lines reduced their invasive and sphere-forming activities in culture and formation of orthotopic tumors in mice. Hypoxia increased expression of KDM3A in pancreatic cancer cells. Strategies to disrupt this pathway might be developed for treatment of pancreatic cancer.

Comparison of Radiosensitization by HDAC Inhibitors CUDC-101 and SAHA in Pancreatic Cancer Cells

Pancreatic cancer has a poor prognosis. New treatment options are urgently required to improve patient outcomes. One promising new class of anticancer drugs are synthetic histone deacetylase inhibitors (HDACi) which modulate chromatin structure and gene expression by blocking histone deacetylation. In this study, we aimed at comparing the in vitro capacities of the HDACi SAHA and CUDC-101 to increase radiosensitivity of human pancreatic tumor cell lines. Therefore, three pancreatic cancer cell lines (Su.86.86, MIA Paca-2, T3M-4) were treated with SAHA (1.5–5 µM) or CUDC-101 (0.25–3 µM) and after 24 h irradiated. Cell proliferation, clonogenic survival and apoptosis was determined. Additionally, cell lysates were investigated for the expression of apoptosis-related proteins. CUDC-101 and SAHA increased the radiation sensitivity of pancreatic tumor cell lines in a dose-dependent manner. This was evidenced by cell proliferation and clonogenic survival. Furthermore, enhanced radiation sensitivity after CUDC-101 or SAHA treatment was confirmed for Su.86.86 and T3M-4 cells in a 3-D microtissue approach. Increased amounts of subG1 cells and diminished full length PARP-1 suggest increased radiation-induced apoptosis after SAHA or CUDC-101 treatment. The comparison of both inhibitors in these assays manifested CUDC-101 as more potent radiosensitizer than SAHA. In line, western blot quantification of the apoptosis-inhibitory proteins XIAP and survivin showed a stronger down-regulation in response to CUDC-101 treatment than after SAHA application. These proteins may contribute to the synergy between HDAC inhibition and radiation response. In conclusion, these preclinical results suggest that treatment with the HDAC inhibitors CUDC-101 or SAHA can enhance radiation-induced cytotoxicity in human pancreatic cells. However, comparison of both inhibitors identified the multi target inhibitor CUDC-101 as more potent radiosensitizer than the HDAC inhibitor SAHA.

Usp9x Promotes Survival in Human Pancreatic Cancer and Its Inhibition Suppresses Pancreatic Ductal Adenocarcinoma In Vivo Tumor Growth

Usp9x has emerged as a potential therapeutic target in some hematologic malignancies and a broad range of solid tumors including brain, breast, and prostate. To examine Usp9x tumorigenicity and consequence of Usp9x inhibition in human pancreatic tumor models, we carried out gain- and loss-of-function studies using established human pancreatic tumor cell lines (PANC1 and MIAPACA2) and four spontaneously immortalized human pancreatic patient-derived tumor (PDX) cell lines. The effect of Usp9x activity inhibition by small molecule deubiquitinase inhibitor G9 was assessed in 2D and 3D culture, and its efficacy was tested in human tumor xenografts. Overexpression of Usp9x increased 3D growth and invasion in PANC1 cells and up-regulated the expression of known Usp9x substrates Mcl-1 and ITCH. Usp9x inhibition by shRNA-knockdown or by G9 treatment reduced 3D colony formation in PANC1 and PDX cell lines, induced rapid apoptosis in MIAPACA2 cells, and associated with reduced Mcl-1 and ITCH protein levels. Although G9 treatment reduced human MIAPACA2 tumor burden in vivo, in mouse pancreatic cancer cell lines established from constitutive (8041) and doxycycline-inducible (4668) KrasG12D/Tp53R172H mouse pancreatic tumors, Usp9x inhibition increased and sustained the 3D colony growth and showed no significant effect on tumor growth in 8041-xenografts. Thus, Usp9x inhibition may be therapeutically active in human PDAC, but this activity was not predicted from studies of genetically engineered mouse pancreatic tumor models.

Abstract B72: Environmental temperature-induced chronic stress drives therapeutic resistance in murine tumor models through β2-adrenergic receptor activation

Abstract Pre-clinical evaluation of novel therapies primarily relies on mouse models for predicting efficacy and toxicity. Unfortunately, when taken into the clinic, promising therapies often fail to achieve expected results in patients or unexpected toxicities are encountered. Emerging evidence now suggests that laboratory housing conditions, including ad libitum feeding and limited physical activity, can lead to metabolic abnormalities which in turn directly affect experimental outcomes in models of diseases such as obesity. We are particularly interested in the ramifications of housing temperature in tumor-bearing mice. IACUC guidelines dictate that all animal facilities must select a single temperature within the range of 20-26˚C (standard temperature, ST) to maintain mice, even though the thermoneutral temperature of mice ranges from 29-31˚C (TT). Because mice are able to maintain a normal body temperature of 37˚C, investigators have not been concerned, but in fact mice must generate a significant amount of heat through adaptive thermogenesis, and this is a norepinephrine (NE) driven process. The tumor promoting effects of catecholamines (epinephrine and norepinephrine) are currently under investigation in several labs. We have previously reported that at ST, the anti-tumor immune response is severely suppressed and when cold stress is alleviated by housing at TT, significantly higher numbers of tumor specific CD8 T-cells and fewer suppressor cells (MDSC, Tregs) are seen, correlating with significantly improved tumor control. Now we have observed that housing mice at TT instead of ST also significantly improves the response of pancreatic tumors to apoptosis inducing therapies in both immunocompetant and SCID mouse models. We found that a syngeneic tumor, Pan02, in C57BL/6 mice showed increased sensitivity to cisplatin when mice were placed at 30˚C vs. 22˚C. Similarly, human cell lines (MiaPaca2, BxPC3) and patient derived xenografts also demonstrated improved responses to both cisplatin and a second therapy, Apo2L/TRAIL, when mice were placed at thermoneutrality. Examination of the tumors revealed that the norepinephrine levels and the numbers of tyrosine hydroxylase-positive macrophages, a major source of norepinephrine, decreased in the tumors of mice housed at TT compared with those at ST. Thus, we hypothesized that tumor cells respond to the elevated levels of norepinephrine in the microenvironment in chronically cold stressed animals by increasing the expression of key survival molecules. Subsequent analysis of tumors from mice housed at TT revealed that the expression of anti-apoptotic molecules Bcl-2, Bcl-XL, Mcl-1 and phosphorylated BAD112 were decreased compared to those housed at ST. Tumor cells were found to express β1,2 adrenergic receptors and in vitro treatment demonstrated that adrenergic signaling can directly induce the expression of Bcl-2 and Bcl-XL in a time dependent manner in human pancreatic tumor cell lines, and that this increased protein expression correlates with increased resistance to both chemotherapeutic and death receptor-mediated apoptosis. In vivo, treatment of tumor-bearing mice with the β1, 2-blocker, propranolol, increased the response of tumors in mice housed at 22˚C to Apo2L/TRAIL, but not mice housed at 30˚C. Resistance to apoptotic therapy at ST was lost by shRNA knock down of β2-AR (MiaPaca2). Altogether, these findings have important implications for the study of immunotherapies which depend on both activation of the anti-tumor immune response and the sensitivity of tumors to induction of apoptosis. Our results support the idea that adrenergic signaling in the tumor microenvironment (at ST) can greatly impact the testing of novel therapies and suggests strategies for improving their efficacy both in mouse models and in the clinic where patients experience a variety of stresses. Supported by the Roswell Park Alliance Foundation and NIH R01 CA135368. Citation Format: Jason W-L Eng, Chelsey B. Reed, Kathleen Kokolus, Mark Bucsek, Rose Pitoniak, Adam T. Utley, WenWee Ma, Elizabeth A. Repasky, Bonnie L. Hylander. Environmental temperature-induced chronic stress drives therapeutic resistance in murine tumor models through β2-adrenergic receptor activation. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B72.

Signal Transducer and Activator of Transcription 3, Mediated Remodeling of the Tumor Microenvironment Results in Enhanced Tumor Drug Delivery in a Mouse Model of Pancreatic Cancer

A hallmark of pancreatic ductal adenocarcinoma (PDAC) is the presence of a dense desmoplastic reaction (stroma) that impedes drug delivery to the tumor. Attempts to deplete the tumor stroma have resulted in formation of more aggressive tumors. We have identified signal transducer and activator of transcription (STAT) 3 as a biomarker of resistance to cytotoxic and molecularly targeted therapy in PDAC. The purpose of this study is to investigate the effects of targeting STAT3 on the PDAC stroma and on therapeutic resistance. Activated STAT3 protein expression was determined in human pancreatic tissues and tumor cell lines. Invivo effects of AZD1480, a JAK/STAT3 inhibitor, gemcitabine or the combination were determined in Ptf1a(cre/+);LSL-Kras(G12D/+);Tgfbr2(flox/flox) (PKT) mice and in orthotopic tumor xenografts. Drug delivery was analyzed by matrix-assisted laser desorption/ionization imaging mass spectrometry. Collagen second harmonic generation imaging quantified tumor collagen alignment and density. STAT3 activation correlates with decreased survival and advanced tumor stage in patients with PDAC. STAT3 inhibition combined with gemcitabine significantly inhibits tumor growth in both an orthotopic and the PKT mouse model of PDAC. This combined therapy attenuates invivo expression of SPARC, increases microvessel density, and enhances drug delivery to the tumor without depletion of stromal collagen or hyaluronan. Instead, the PDAC tumors demonstrate vascular normalization, remodeling of the tumor stroma, and down-regulation of cytidine deaminase. Targeted inhibition of STAT3 combined with gemcitabine enhances invivo drug delivery and therapeutic response in PDAC. These effects occur through tumor stromal remodeling and down-regulation of cytidine deaminase without depletion of tumor stromal content.

Abstract 1748: Usp9x as a novel therapeutic target in human pancreatic cancer

Abstract Usp9x regulates tumor cell survival and responsiveness to targeted-, chemo- and radio-therapy in a broad number of tumors through effects on multiple proteins. The role of Usp9x in human pancreatic cancer is largely unknown, although a recent study demonstrated that Usp9x co-operated in promoting KrasG12D tumorigenesis in mice. To examine Usp9x tumorigenicity and therapeutic potential of Usp9x inhibition in human pancreatic cancer vs. mouse pancreatic tumor models we developed 3D cultures of cell lines established from constitutive (8041) and doxycycline-inducible (4668) KrasG12D/Tp53R172H mouse pancreatic tumors, established human cell lines (BxPC3, PANC1 and MIAPACA2) and spontaneously immortalized human pancreatic patient tumor derived cell lines (UM2, UM6, UM16 and UM76). The effect of Usp9x knockdown (KD), overexpression (OE) or Usp9x activity inhibition by our small molecule deubiquitinase inhibitor EOAI3402143 (G9) were assessed by growth in 2D and 3D culture and anti-tumor efficacy studies in mouse (8041) and human (MIAPACA2) xenograft models. Usp9x KD in constitutive KrasG12D/Tp53R172H 8041 cells led to a 3-fold increase in the number of colonies formed in 3D and Usp9x KD sustained 3D colony growth even after withdrawal of mutant Kras support in KrasG12D/Tp53R172H inducible 4668 cells. Usp9x-OE in 8041 cells decreased 3D colony growth by 2.5-fold. These results contrast with outcomes in human pancreatic tumors where Usp9x KD induced either rapid apoptosis (in MIAPACA2) or reduced 3D colony formation by >50% (in PANC1). Usp9x-OE in PANC1 cells enhanced 3D colony growth (2-fold) and increased invasion activity in Boyden chambers (3.5-fold). Mechanistically, we found differential effects of Usp9x on mutant Kras protein expression levels in mouse vs human cells. In vivo G9 treatment (15 mg/kg, every other day) of mouse 8041 xenografts showed a statistically insignificant trend towards tumor growth inhibition but effectively inhibited MIAPACA2 tumor growth in mice. Usp9x KD in human pancreatic patient tumor cell lines inhibited 3D colony growth by >75% and this activity was phenocopied by G9 with nM efficacy. We conclude that Usp9x acts as a tumor promoter in established human pancreatic cancer and a tumor suppressor in murine cells from genetically engineered pancreatic tumors, possibly through differential regulation of mutant Kras. Overall, these results suggest that Usp9x may be a novel therapeutic target in pancreatic cancer. Note: This abstract was not presented at the meeting. Citation Format: Anupama Pal, Marina Pasca Di Magliano, Diane Simeone, Luke Peterson, Harish Potu, Moshe Talpaz, Nicholas Donato. Usp9x as a novel therapeutic target in human pancreatic cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1748. doi:10.1158/1538-7445.AM2015-1748

Targeted nuclear factor-kappaB suppression enhances gemcitabine response in human pancreatic tumor cell line murine xenografts

Pancreatic ductal adenocarcinoma (PDAC) is an almost uniformly fatal malignancy characterized by resistance to chemotherapy. Currently, gemcitabine is the agent used most commonly but demonstrates only a partial response. The transcription factor nuclear factor-kappaB (NF-κB), known to be involved in the inflammatory response, is constitutively activated in PDAC and further activated by gemcitabine. Our aim was to examine the effects of targeted NF-κB suppression on gemcitabine resistance using an in vivo tumor growth model. To suppress the NF-κB pathway, the mutant IκBα super-repressor protein was stably expressed in PaCa-2 human PDAC cells. Athymic mice were injected subcutaneously with IκBα-super-repressor (SR) or vector-expressing PaCa-2 cells and randomized to receive phosphate-buffered saline (PBS) or 100 mg/kg gemcitabine(gem) for 4 weeks. The mean increase in tumor volume was 47 mm(3) (89%) and 196 mm(3) (326%) in gem/SR and gem/vector groups, respectively (P = .03). The PBS-treated groups demonstrated greater tumor growth, ∼340 mm(3) (850%) increase, in both PBS/vector and PBS/SR groups. Intratumoral NF-κB activity was decreased in gem/SR compared with the gem/vector group (P = .04). Decreased Ki-67 positivity was noted in gem/SR (49%) versus gem/vector tumors (73%) (P = .04), with no difference in apoptosis (apoptag, P = .3) or angiogenesis (CD31+, P = .9). Stable IκBα-SR expression in vivo potentiated the antitumor effects of gemcitabine, resulting in decreased tumor growth in association with decreased cell proliferation. Molecular suppression of the NF-κB pathway decreases successfully gemcitabine resistance in a relatively chemoresistant PDAC line. Thus, NF-κB-targeted agents may complement gemcitabine-based therapies and decrease chemoresistance in patients with PDAC.

Effect of Newcastle disease virus on different human and murine pancreatic tumour cell lines in vitro

Effect of Newcastle disease virus on different human and murine pancreatic tumour cell lines in vitro

Tumor radiosensitization by monomethyl auristatin E: mechanism of action and targeted delivery.

Intrinsic tumor resistance to radiotherapy limits the efficacy of ionizing radiation (IR). Sensitizing cancer cells specifically to IR would improve tumor control and decrease normal tissue toxicity. The development of tumor-targeting technologies allows for developing potent radiosensitizing drugs. We hypothesized that the anti-tubulin agent monomethyl auristatin E (MMAE), a component of a clinically approved antibody-directed conjugate, could function as a potent radiosensitizer and be selectively delivered to tumors using an activatable cell-penetrating peptide targeting matrix metalloproteinases and RGD-binding integrins (ACPP-cRGD-MMAE). We evaluated the ability of MMAE to radiosensitize both established cancer cells and a low-passage cultured human pancreatic tumor cell line using clonogenic and DNA damage assays. MMAE sensitized colorectal and pancreatic cancer cells to IR in a schedule- and dose-dependent manner, correlating with mitotic arrest. Radiosensitization was evidenced by decreased clonogenic survival and increased DNA double-strand breaks in irradiated cells treated with MMAE. MMAE in combination with IR resulted in increased DNA damage signaling and activation of CHK1. To test a therapeutic strategy of MMAE and IR, PANC-1 or HCT-116 murine tumor xenografts were treated with nontargeted free MMAE or tumor-targeted MMAE (ACPP-cRGD-MMAE). While free MMAE in combination with IR resulted in tumor growth delay, tumor-targeted ACPP-cRGD-MMAE with IR produced a more robust and significantly prolonged tumor regression in xenograft models. Our studies identify MMAE as a potent radiosensitizer. Importantly, MMAE radiosensitization can be localized to tumors by targeted activatable cell-penetrating peptides.

In vivo biodistribution of no-carrier-added 6-18F-fluoro-3,4-dihydroxy-L-phenylalanine (18F-DOPA), produced by a new nucleophilic substitution approach, compared with carrier-added 18F-DOPA, prepared by conventional electrophilic substitution.

A novel synthetic approach to 6-(18)F-fluoro-3,4-dihydroxy-L-phenylalanine ((18)F-DOPA), involving the nucleophilic substitution of a diaryliodonium salt precursor with non-carrier-added (18)F-fluoride, yielded a product with a specific activity that was 3 orders of magnitude higher than the product of the conventional synthesis method, involving an electrophilic substitution of a trialkylstannane precursor with (18)F2. We performed a direct comparison of high- and low-specific-activity (18)F-DOPA in a neuroendocrine tumor model to determine whether this difference in specific activity has implications for the biologic behavior and imaging properties of (18)F-DOPA. (18)F-DOPA was produced via the novel synthesis method, yielding (18)F-DOPA-H with a high specific activity (35,050 ± 4,000 GBq/mmol). This product was compared in several experiments with conventional (18)F-DOPA-L with a low specific activity (11 ± 2 GBq/mmol). In vitro accumulation experiments with the human pancreatic neuroendocrine tumor cell line BON-1 were performed at both 0 °C and 37 °C and at 37 °C in the presence of pharmacologic inhibitors of proteins involved in the uptake mechanism of (18)F-DOPA. Small-animal PET experiments were performed in athymic nude mice bearing a BON-1 tumor xenograft. At 37 °C, the uptake of both (18)F-DOPA-H and (18)F-DOPA-L did not differ significantly during a 60-min accumulation experiment in BON-1 cells. At 0 °C, the uptake of (18)F-DOPA-L was significantly decreased, whereas the lower temperature did not alter the uptake of (18)F-DOPA-H. The pharmacologic inhibitors carbidopa and tetrabenazine also revealed differential effects between the 2 types of (18)F-DOPA in the 60-min accumulation experiment. The small-animal PET experiments did not show any significant differences in distribution and metabolism of (18)F-DOPA-H and (18)F-DOPA-L in carbidopa-pretreated mice. The advantages of the novel synthesis of (18)F-DOPA, which relies on nucleophilic fluorination of a diaryliodonium salt precursor, lie in the simplicity of the synthesis method, compared with the conventional, electrophilic approach and in the reduced mass of administered, pharmacologically active (19)F-DOPA. (18)F-DOPA-H demonstrated comparable imaging properties in an in vivo model for neuroendocrine tumors, despite the fact that the injected mass of material was 3 orders of magnitude less than (18)F-DOPA-L.

Further characterization of HDAC and SIRT gene expression patterns in pancreatic cancer and their relation to disease outcome.

Ductal adenocarcinoma of the pancreas is ranking 4 for patient' death from malignant disease in Western countries, with no satisfactory treatment. We re-examined more precisely the histone deacetylases (HDAC) and Sirtuin (SIRT) gene expression patterns in pancreatic cancer with more pancreatic tumors and normal tissues. We also examinedthe possible relationship between HDAC gene expression levels and long term disease outcome. Moreover, we have evaluated by using an in vitro model system of human pancreatic tumor cell line whether HDAC7 knockdown may affect the cell behavior. We analyzed 29 pancreatic adenocarcinoma (PA), 9 chronic pancreatitis (CP), 8 benign pancreatic (BP) and 11 normal pancreatic tissues. Concerning pancreatic adenocarcinoma, we were able to collect biopsies at the tumor periphery. To assess the possible involvement of HDAC7 in cell proliferation capacity, we have generated recombinant human Panc-1 tumor which underexpressed or overexpressed HDAC7. The expression of HDAC1,2,3,4,7 and Nur77 increased in PA samples at levels significantly higher than those observed in the CP group (p = 0.0160; 0.0114; 0.0227; 0.0440; 0.0136; 0.0004, respectively). The expression of HDAC7, was significantly greater in the PA compared with BP tissue samples (p = 0.05). Mean mRNA transcription levels of PA for HDAC7 and HDAC2 were higher when compared to their counterpart biopsies taken at the tumor periphery (p = 0.0346, 0.0053, respectively). Moreover, the data obtained using confocal microscopy and a quantitative method of immunofluorescence staining strongly support the HDAC7 overexpression in PA surgical specimens. The number of deaths and recurrences at the end of follow up were significantly greater in patients with overexpression of HDAC7. Interestingly, the rate of growth was significantly reduced in the case of cell carrying shRNA construct targeting HDAC7 encoding gene when compared to the parental Panc-1 tumor cells (p = 0.0015) at 48 h and 96 h (p = 0.0021). This study strongly support the notion that HDAC7play a role in pancreatic adenocarcinoma progression.

Abstract 4833: The eicosanoid 20-HETE triggers Ras activation, pancreatic cancer growth and metastasis through inflammatory macrophage signaling

Abstract Prolonged, unresolving inflammation is increasingly implicated in the pathogenesis of pancreatic cancer, but the critical inflammatory mediators remain largely unknown. Most efforts to elucidate these mediators have focused on protein cytokines rather than lipid autacoids. Lipid autacoids are metabolites of arachidonic acid and other related fatty acids that regulate inflammation and tumor growth. Among these, the metabolites of the cytochrome P450 (CYP) epoxygenases and ω-hydroxylases, including EETs and 20-HETE, respectively, have been largely neglected in cancer. Recently, we showed that epoxyeicosatrienoic acids (EETs) stimulate multi-organ metastasis and tumor dormancy escape. We report for the first time, that elevated levels of 20-hydroxyeicosatetraenoic acid (20-HETE), a product of the ω-hydroxylases CYP4A/F, stimulate primary pancreatic tumor growth and metastasis. Using genetic and pharmacological manipulation of endogenous 20-HETE, we demonstrate that systemic administration of 20-HETE promotes primary human and murine pancreatic cancer in subcutaneous and orthotopic models. Furthermore, a transgenic murine model of endogenously high 20-HETE level (Tie2-CYP4F2 Tr), exhibited spontaneous liver and mediastinal lymph node metastasis after resection of subcutaneous primary pancreatic tumors. In contrast, wild-type mice did not develop liver and mediastinal lymph node metastasis after primary tumor resection. We show that 20-HETE activated Ras in human pancreatic tumor cell lines and the 20-HETE antagonist, HET0016, decreased Ras activity. The p65 subunit of NF-κB also increased in a 20-HETE-dependent manner in these tumor cell lines. 20-HETE induced migration of human macrophages in vitro, and tumor-associated macrophages expressed the 20-HETE generating enzyme CYP4F2. Depletion of macrophages with clodronate liposomes impaired CYP-metabolite-stimulated tumor growth. In addition, we show that 14,15-EET promotes pancreatic tumor growth in vivo. Moreover, inhibitors of soluble epoxide hydrolase, the enzyme that metabolizes EETs, elevated EET levels and promoted pancreatic tumor growth. Either the 20-HETE antagonist HET0016, or the EET antagonist 14,15-EEZE alone significantly inhibited primary tumor growth. Remarkably, systemic administration of both antagonists together resulted in sustained regression of established pancreatic tumors. This combination of 20-HETE and EET antagonists also suppressed ascites formation and prolonged survival in an orthotopic pancreatic cancer model without toxic effects. Thus, 20-HETE and EET production by macrophages in the tumor microenvironment critically regulates Ras activation, pancreatic tumor growth and metastasis. Our studies offer a mechanistic rationale for using 20-HETE and EET antagonists as a novel approach and anti-cancer therapeutics for pancreatic cancer. Citation Format: Yael Gus-Brautbar, Jessica Casper, Diane R. Bielenberg, Jennifer Cheng, Birgitta Schmidt, Bruce D. Hammock, Darryl C. Zeldin, Mark W. Kieran, Dipak Panigrahy. The eicosanoid 20-HETE triggers Ras activation, pancreatic cancer growth and metastasis through inflammatory macrophage signaling. [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 4833. doi:10.1158/1538-7445.AM2014-4833

Abstract 4434: Role of Deubiquitinase Usp9x in pancreatic cancers: Tumor promoter or tumor suppressor

Abstract Deubiquitinase Usp9x has been shown to promote tumor cell survival and resistance to chemo- and radiotherapy through effects on the Mcl-1 pro-survival protein. It is predicted that a small molecule Usp9x inhibitor could have a major impact in cancer therapy. However, recent data has raised caution about therapeutic targeting of Usp9X in cancer. In a study to identify genes that reduce the latency of pancreatic tumor formation in a mutant Kras model, Pérez-Mancera et al. 2012 reported that the Usp9x locus was frequently inactivated in pancreatic cancer cells, resulting in protection from anoikis and enhanced transformation. In order to clarify the role of Usp9X in pancreatic cancer, we used a newly created cell line 4668 established from mouse pancreatic tumors with doxycycline (DOX) inducible expression of KrasG12D and Tp53R172H. Stable Usp9x knockdown (KD) in 4668 cells had no effect on their proliferation or phenotype in 2D cultures. However, withdrawal of DOX from DOX-treated Usp9x KD 4668 cells resulted in 4-fold increase in colony formation, unlike DOX depleted control KD 4668 cells which undergo apoptosis resulting in colony disintegration. DOX treated control KD and Usp9x KD 4668 cells did not show any difference in basal 3D colony formation. These results suggest that Usp9x loss in the absence of continuous mutant Kras/p53 expression is sufficient to sustain 3D cancer cell growth, confirming a tumor suppressor role for Usp9x in a Kras mouse model of pancreatic cancer. To determine if Usp9x also plays a tumor suppressor role in human pancreatic tumors, we did Usp9x KD in three human pancreatic cancer cell lines with varied Kras mutational status; BxPC3 (wild type), PANC1 (heterozygous) and MIA-PACA2 (homozygous) which expressed high basal levels of Usp9x. MIA-PACA2-Usp9x KD cells undergo apoptosis and caspase activation. In contrast, BxPC3-Usp9x KD cells showed no effect on growth in 2D or in 3D culture. However, PANC1-Usp9x KD cells showed more rapid (38% p=0.004) 2D growth, but 3D colony formation was consistently reduced by >50%, (p=0.03). These results suggest that the effect of Usp9x loss in established human pancreatic tumor cells is distinct from that detected in a murine pancreatic tumor model. Distinctions may be partially dependent on their Kras mutational status or Usp9x substrate utilization in human vs. murine cells. To further assess the clinical relevance of our observations, Usp9x KD was evaluated in four recently established, low passage number human pancreatic patient tumor cell lines (UM2, UM6, UM16, UM76). Usp9x KD did not affect 2D growth but fully suppressed 3D colony growth. This activity was phenocopied by our small molecule Usp9x inhibitor, EOAI3402143 (G9), with nM efficacy. Together, these results suggest that possible species distinctions exist in the tumorigenic functions of Usp9x and necessitate further assessment of its role in pancreatic tumor development and treatment. Citation Format: Anupama Pal, Marina Pasca di Magliano, Moshe Talpaz, Michelle Dziubinski, Diane Simeone, Nicholas J. Donato. Role of Deubiquitinase Usp9x in pancreatic cancers: Tumor promoter or tumor suppressor. [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 4434. doi:10.1158/1538-7445.AM2014-4434

A role for immunotherapy: preventing macrophage anergy in the tumour environment

The tumour microenvironment is shaped by the interaction of tumour cells with a variety of “non” malignant cellular and acellular components. It is the secretion of cytokines, chemokines, metabolites and other mediators in the tumour stroma that shapes the natural evolution of cancer. Whereas the tumour strives to promote an immunosuppressive environment to limit anti-tumour immune response, infiltrating immune cells aim to control tumour growth. The balance between these two conflicting interests shapes the immune contexture of cancer and ultimately patient prognosis. In an effort to characterize the role played by macrophages, we have developed an in vitro system using human pancreatic tumour cell lines and a human monocyte-macrophage leukemic cell line (ThP1). We have investigated the immunosuppressive properties of pancreatic cancer cell lines and how the immune properties of macrophages are influenced by the tumour supernatant in vitro. We have applied our findings to a clinically relevant murine model of pancreatic cancer. We found that, despite some heterogeneity, the profile of MIAPACA-2, BxPC3 and PANC-1, included IL-8, MCP-1, SCF and VEGF secretion. Not surprisingly, when ThP1 cell were cultured in the presence of increasing concentrations of tumour supernatant and stimulated with LPS, we observed a significant downregulation of their ability to produce cytokines associated with an M1 profile (e.g. IL-6, IL-12p70, IL-23, TNF-α). However, ThP1 also failed to display an M2 profile; IL-10 and IL-1ra were similarly downregulated. ThP1 stimulated with LPS in the presence of tumour supernatant also had impaired secretion of a number of other cytokines such as IL-18, MIP-1α and MIP-1β and MCP-1. In contrast, they appeared to maintain secretion of VEGF and IL-7. Next we investigated how the immune stimulant IMM-101, an immunotherapeutic agent comprising heat-killed whole cell Mycobacterium obuense, might be able to shift this anergic phenotype. IMM-101 is currently under clinical evaluation in two Phase II clinical trials in pancreatic (EudraCT number 2010-022757-42) and colorectal cancer (EudraCT number 2011-003958-85). We found that despite the presence of tumour supernatant, macrophages stimulated with IMM-101 retained the ability to secrete the full range of cytokines and we did not observe the impairment reported after LPS stimulation. We believe the immunological properties of IMM-101 make this a potential candidate for successful immunotherapy.