PANC-1 Xenograft Model Research Articles

Combined PET Radiotracer Approach Reveals Insights into Stromal Cell-Induced Metabolic Changes in Pancreatic Cancer In Vitro and In Vivo.

Background/Objective Pancreatic stellate cells (PSCs) in pancreatic adenocarcinoma (PDAC) are producing extracellular matrix, which promotes the formation of a dense fibrotic microenvironment. This makes PDAC a highly heterogeneous tumor-stroma-driven entity, associated with reduced perfusion, limited oxygen supply, high interstitial fluid pressure, and limited bioavailability of therapeutic agents. Methods In this study, spheroid and tumor xenograft models of human PSCs and PanC-1 cells were characterized radiopharmacologically using a combined positron emission tomography (PET) radiotracer approach. [18F]FDG, [18F]FMISO, and [18F]FAPI-74 were employed to monitor metabolic activity, hypoxic metabolic state, and functional expression of fibroblast activation protein alpha (FAPα), a marker of activated PSCs. Results In vitro, PanC-1 and multi-cellular tumor spheroids demonstrated comparable glucose uptake and hypoxia, whereas FAPα expression was significantly higher in PSC spheroids. In vivo, glucose uptake as well as the transition to hypoxia were comparable in PanC-1 and multi-cellular xenograft models. In mice injected with PSCs, FAPα expression decreased over a period of four weeks post-injection, which was attributed to the successive death of PSCs. In contrast, FAPα expression increased in both PanC-1 and multi-cellular xenograft models over time due to invasion of mouse fibroblasts. Conclusion The presented models are suitable for subsequently characterizing stromal cell-induced metabolic changes in tumors using noninvasive molecular imaging techniques.

Discovery of a Highly Potent Lysine Methyltransferases G9a/NSD2 Dual Inhibitor to Treat Solid Tumors.

Both G9a and NSD2 have been recognized as promising therapeutic targets for cancer treatment. However, G9a inhibitors only showed moderate inhibitory activity against solid tumors and NSD2 inhibitors were limited to the treatment of hematological malignancies. Inspired by the advantages of dual-target inhibitors that show great potential in enhancing efficiency, we developed a series of highly potent G9a/NSD2 dual inhibitors to treat solid tumors. The candidate 16 demonstrated much enhanced antiproliferative activity compared to the selective G9a inhibitor 3 and NSD2 inhibitor 15. In addition, it exhibited superior potency in inhibiting colony formation, inducing cell apoptosis, and blocking cancer cell metastasis. Furthermore, it effectively inhibited the catalytic functions of both G9a and NSD2 in cells and exhibited significant antitumor efficacy in the PANC-1 xenograft model with good safety. Therefore, compound 16 as a highly potent G9a/NSD2 dual inhibitor presents an attractive anticancer drug candidate for the treatment of solid tumors.

Structure-Based Design and Characterization of the Highly Potent and Selective Covalent Inhibitors Targeting the Lysine Methyltransferases G9a/GLP.

Protein lysine methyltransferases G9a and GLP, which catalyze mono- and di-methylation of histone H3K9 and nonhistone proteins, play important roles in diverse cellular processes. Overexpression or dysregulation of G9a and GLP has been identified in various types of cancer. Here, we report the discovery of a highly potent and selective covalent inhibitor 27 of G9a/GLP via the structure-based drug design approach following structure-activity relationship exploration and cellular potency optimization. Mass spectrometry assays and washout experiments confirmed its covalent inhibition mechanism. Compound 27 displayed improved potency in inhibiting the proliferation and colony formation of PANC-1 and MDA-MB-231 cell lines and exhibited enhanced potency in reducing the levels of H3K9me2 in cells compared to noncovalent inhibitor 26. In vivo, 27 showed significant antitumor efficacy in the PANC-1 xenograft model with good safety. These results clearly indicate that 27 is a highly potent and selective covalent inhibitor of G9a/GLP.

Design, synthesis and anticancer evaluation of selective 2,4-disubstituted pyrimidine CDK9 inhibitors

Specific inhibition on CDK9 has been proven to be a promising targeted cancer therapy. In this work, fourteen novel 2,4-disubstituted pyrimidine derivatives were designed and synthesized as potent and selective CDK9 inhibitors. These compounds showed broad anti-proliferative activities in various tumor cell lines, especially for PANC-1 cells with IC50 values as low as 0.08 μM. The most selective compound 8d was 84-fold selective for CDK9 over CDK2. Mechanism study indicated that 8d induced apoptosis of PANC-1 cells and arrested the cell cycle at G2/M phase in a dose-dependent manner. Decreased phosphorylation of the CTD of RNAPII at Ser-2 and downregulation of CDK9 were confirmed in PANC-1 cells. Besides, Molecular docking was also performed to gain insights into the ligand-binding interactions of 8d inside CDK9 and CDK2 binding sites. In vivo studies indicated that 8d exhibited potent anti-tumor effects in PANC-1 xenograft models without causing obvious loss of body weight. Our research suggests that compound 8d, as a potent CDK9 inhibitor, can be considered as a good lead-candidate for further development.

MiRNA-193b-5p Suppresses Pancreatic Cancer Cell Proliferation, Invasion, Epithelial Mesenchymal Transition, and Tumor Growth by Inhibiting eEF2K.

Pancreatic ductal adenocarcinoma (PDAC) is the 4th leading cause of cancer deaths in the US due to the lack of effective targeted therapeutics and extremely poor prognosis. The study aims to investigate the role of miR-193b and related signaling mechanisms in PDAC cell proliferation, invasion, and tumor growth. Using PDAC cell lines, we performed cell viability, colony formation, in vitro wound healing, and matrigel invasion assays following transfection with miR-193b mimic or control-miR. To identify potential downstream targets of miR-193b, we utilized miRNA-target prediction algorithms and investigated the regulation of eukaryotic elongation factor-2 kinase (eEF2K) and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathways and mediators of epithelial mesenchymal transition (EMT). The role of miR-193b in PDAC tumorigenesis was evaluated in in vivo tumor growth of Panc-1 xenograft model in nude mice. We found that miR-193b is under expressed in PDAC cells compared to corresponding normal pancreatic epithelial cells and demonstrated that ectopic expression of miR-193b reduced cell proliferation, migration, invasion, and EMT through downregulation of eEF2K signaling in PDAC cells. miR-193b expression led to increased expression of E-Cadherin and Claudin-1 while decreasing Snail and TCF8/ZEB1 expressions via eEF2K and MAPK/ERK axis. In vivo systemic injection of miR-193b using lipid-nanoparticles twice a week reduced tumor growth of Panc-1 xenografts and eEF2K expression in nude mice. Our findings suggest that miR-193b expression suppresses PDAC cell proliferation, migration, invasion, and EMT through inhibition of eEF2K/MAPK-ERK oncogenic axis and that miR-193b-based RNA therapy might be an effective therapeutic strategy to control the growth of PDAC.

VHH212 nanobody targeting the hypoxia-inducible factor 1α suppresses angiogenesis and potentiates gemcitabine therapy in pancreatic cancer in vivo.

Objective:We aimed to develop a novel anti-HIF-1α intrabody to decrease gemcitabine resistance in pancreatic cancer patients.Methods:Surface plasmon resonance and glutathione S-transferase pull-down assays were conducted to identify the binding affinity and specificity of anti-HIF-1α VHH212 [a single-domain antibody (nanobody)]. Molecular dynamics simulation was used to determine the protein-protein interactions between hypoxia-inducible factor-1α (HIF-1α) and VHH212. The real-time polymerase chain reaction (PCR) and Western blot analyses were performed to identify the expressions of HIF-1α and VEGF-A in pancreatic ductal adenocarcinoma cell lines. The efficiency of the VHH212 nanobody in inhibiting the HIF-1 signaling pathway was measured using a dual-luciferase reporter assay. Finally, a PANC-1 xenograft model was developed to evaluate the anti-tumor efficiency of combined treatment. Immunohistochemistry analysis was conducted to detect the expressions of HIF-1α and VEGF-A in tumor tissues.Results:VHH212 was stably expressed in tumor cells with low cytotoxicity, high affinity, specific subcellular localization, and neutralization of HIF-1α in the cytoplasm or nucleus. The binding affinity between VHH212 and the HIF-1α PAS-B domain was 42.7 nM. Intrabody competitive inhibition of the HIF-1α heterodimer with an aryl hydrocarbon receptor nuclear translocator was used to inhibit the HIF-1/VEGF pathway in vitro. Compared with single agent gemcitabine, co-treatment with gemcitabine and a VHH212-encoding adenovirus significantly suppressed tumor growth in the xenograft model with 80.44% tumor inhibition.Conclusions:We developed an anti-HIF-1α nanobody and showed the function of VHH212 in a preclinical murine model of PANC-1 pancreatic cancer. The combination of VHH212 and gemcitabine significantly inhibited tumor development. These results suggested that combined use of anti-HIF-1α nanobodies with first-line treatment may in the future be an effective treatment for pancreatic cancer.

Abstract 4060: Discovery and preclinical development of KRAS-targeting siRNA therapeutic modality for the treatment of pancreatic cancer

Abstract Pancreatic cancer is the third most common cause of death from cancer in the US. The disease has a poor prognosis with a five-year survival rate under 10%. Mutations in KRAS are driving tumor growth in about 95% of patients with pancreatic cancer. Currently there are no approved drugs that target KRAS. Recently there has been progress targeting a specific mutation, KRAS G12C with small molecules in lung cancer, but this mutation accounts for only 1% of patients with Pancreatic Ductal Adenocarcinoma (PDAC). RNA interference has the potential to directly target KRAS, but delivery to pancreas has been challenging. We have developed a lipid nanoparticle formulation that is able to distribute to pancreatic tumor tissue. The proprietary lipid nanoparticle (LNP) is comprised of an ionizable, non-immunogenic, biodegradable lipid and the composition has been tuned to favor enrichment in pancreatic tumor by screening in orthotopic pancreatic tumor mouse models. A potent siRNA with IC50 in the range of 10pM targeting KRAS directly has been identified and the activity on downstream pathways and kinetics have been demonstrated. Transfection of ND-05-1192 led to more impeded cell growth in pancreatic cancer cells (PANC-1, AsPC-1, Capan-1, SW1990 and BxPC-3) than normal cells (human hepatocyte, pancreatic cells and HUVEC cells). Western blot results demonstrated effective downregulations of key proteins involved in MAPK and PI3K signaling pathways and upregulated activities of proteins involved in apoptotic signaling pathways, such as BIM, caspase 3/7 and cleaved-PARP following inhibition of KRAS protein expression. In vivo biodistribution and tumor cell uptake study results demonstrated that LNP transported ND-05-1192 siRNA molecules to the pancreas site and pancreatic orthotopic tumor tissues with the majority of tumor cells showing LNP product uptake. The application of ND-05-1192/LNP product significantly suppressed tumor growth in PANC-1 and AsPC-1 bioluminescent orthotopic pancreatic xenograft models. Inhibition efficiency revealed by tumor weight and bioluminescent response (BLR) as the treatment end points reached up to 50% in the PANC-1 orthotopic xenografts following once weekly intravenous administration. In the orthotopic AsPC-1 model, the average BLR was significantly reduced in a dose-dependent manner. Additionally, in AsPC-1 orthotopic xenografts, the survival rate of the ND-05-1192/LNP treatment group was significantly prolonged at a 4 mg/kg dose compared to the vehicle control group. In summary, we conclude that ND-05-1192/LNP is delivered to the orthotopic pancreatic tumor and its considerable uptake by tumor cells results in KRAS target downregulation, tumor growth inhibition and overall survival benefit as demonstrated in our orthotopic xenograft models. Citation Format: Zhihong (Julie) OBrien, Li Wang, Cima Cina, Bharat Majeti, James Chu, Xiaoen Wang, Robiel Baclig, James Lin, Jose Ibarra, Dhrubamitra Chatterjee, Jung-Kang Jin, Jean-Pierre Clamme, Jennifer Daniels, Hai Wang, Jens Harborth, Wenbin Ying. Discovery and preclinical development of KRAS-targeting siRNA therapeutic modality for the treatment of pancreatic cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4060.

Chemosensitivity to HM90822, a novel synthetic IAP antagonist, is determined by p-AKT-inducible XIAP phosphorylation in human pancreatic cancer cells.

Inhibitor of apoptosis proteins (IAPs) are overexpressed in the majority of cancers and prevent apoptosis by inhibiting caspases. IAPs have therefore attracted considerable attention as potential targets for anticancer therapy. Here, we demonstrated that HM90822 (abbreviated HM822; a new synthetic IAP antagonist) induced apoptotic cell death via proteasome-dependent degradation of BIR2/3 domain-containing IAPs in human pancreatic cancer cells. HM822 inhibited the expression of XIAP and cIAP1/2 proteins in Panc-1 and BxPC-3 cells, which are sensitive to HM822. HM822 also induced IAP ubiquitination and promoted proteasome-dependent IAP degradation. However, cells expressing phospho-XIAP (Ser87) and AKT exhibited resistance to HM822. In other words, the overexpression of AKT-CA (constitutive active form for AKT) or AKT-WT induced resistance to HM822. In addition, in Panc-1 xenograft and orthotopic mouse models, we revealed that tumor growth was suppressed by the administration of HM822. Taken together, these results suggest that HM822 induces apoptosis through ubiquitin/proteasome-dependent degradation of BIR3 domain-containing IAPs. These findings suggest that phospho-XIAP and phospho-AKT may be used as biomarkers for predicting the efficacy of HM822 in pancreatic cancer patients.

Abstract 5078: Modulation of glucose metabolism and lactate efflux in tumors by bitter melon juice in its efficacy against pancreatic cancer

Abstract Pancreatic cancer (PanC) is currently ranked as the fourth leading cause of cancer-related deaths across the United States. The dismal statistics for the year 2018 estimates about 55,440 new incidences and 44,330 PanC-associated fatalities in men and women combined, with a 5-year survival rate of less than 10%. PanCs possess a very intricately designed metabolic profile favoring excess aerobic glycolysis in addition to altered glutamine metabolism, contributing to tumor cell proliferation and PanC progression. Targeting metabolic phenotype in PanC with natural/diet-derived agents has immense scope for better non-toxic intervention, improved overall survival and quality of life; the major concerns with the use of frontline PanC chemotherapeutics. Herein, we utilized bitter melon juice (BMJ), a natural agent, which we recently reported to possess anticancer potential via targeting PanC stem cell and bulk cell populations and exerting a role in elevating drug sensitivity of gemcitabine resistant PanC cells. The established role of BMJ in activating master metabolic regulator APMK in PanC cells served as a basis for pursuing deeper investigation into the underlying metabolic alterations leading to BMJ efficacy in PanC. We investigated the comparative metabolic profiles of PanC cells with differential KRAS mutational status (PANC1 - mutated KRAS and BxPC3 -; wild type KRAS), on BMJ exposure. Specifically, we employed Nuclear magnetic resonance (NMR) metabolomics and in vivo imaging platforms to assess whether BMJ modulates PanC cell metabolome, together with establishing the molecular metabolomic targets to better understand the relevance of altered metabolism in PanC management by BMJ. Multinuclear NMR metabolomics was performed post 72 hours of BMJ treatment followed by PLS-DA (partial least square discriminant analysis) assessments on the quantitative metabolic data sets to visualize the treatment group clustering; altered glucose uptake, lactate export and energy state were identified as the key components responsible for cell death induction. We next employed PANC1 xenograft model for assessing in vivo BMJ efficacy against PanC. Positron Emission Tomography ([18FDG]-PET) and Magnetic Resonance Imaging (MRI) on PANC1 tumor-bearing animals reiterated the in vitro results, with BMJ-associated significant changes in tumor volumes, tumor cellularity and glucose uptake. Additional studies in BMJ-treated PanC cells and xenografts displayed a strong decrease in the expression of glucose and lactate transporters GLUT1 and MCT4, respectively, supporting their role in metabolic changes by BMJ. Collectively, these results highlight BMJ-induced modification in PanC metabolomics phenotype and establish primarily lactate efflux and glucose metabolism, specifically GLUT1 and MCT4 transporters, as the potential metabolic targets underlying BMJ efficacy in PanC. Citation Format: Deepanshi Dhar, Rama Kant, Komal Raina, Michael F. Wempe, Natalie J. Serkova, Chapla Agarwal, Rajesh Agarwal. Modulation of glucose metabolism and lactate efflux in tumors by bitter melon juice in its efficacy against pancreatic cancer [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 5078.

Bitter melon juice-intake modulates glucose metabolism and lactate efflux in tumors in its efficacy against pancreatic cancer.

The established role of bitter melon juice (BMJ), a natural product, in activating master metabolic regulator adenosine monophosphate-activated protein kinase in pancreatic cancer (PanC) cells served as a basis for pursuing deeper investigation into the underlying metabolic alterations leading to BMJ efficacy in PanC. We investigated the comparative metabolic profiles of PanC cells with differential KRAS mutational status on BMJ exposure. Specifically, we employed nuclear magnetic resonance (NMR) metabolomics and in vivo imaging platforms to understand the relevance of altered metabolism in PanC management by BMJ. Multinuclear NMR metabolomics was performed, as a function of time, post-BMJ treatment followed by partial least square discriminant analysis assessments on the quantitative metabolic data sets to visualize the treatment group clustering; altered glucose uptake, lactate export and energy state were identified as the key components responsible for cell death induction. We next employed PANC1 xenograft model for assessing in vivo BMJ efficacy against PanC. Positron emission tomography ([18FDG]-PET) and magnetic resonance imaging on PANC1 tumor-bearing animals reiterated the in vitro results, with BMJ-associated significant changes in tumor volumes, tumor cellularity and glucose uptake. Additional studies in BMJ-treated PanC cells and xenografts displayed a strong decrease in the expression of glucose and lactate transporters GLUT1 and MCT4, respectively, supporting their role in metabolic changes by BMJ. Collectively, these results highlight BMJ-induced modification in PanC metabolomics phenotype and establish primarily lactate efflux and glucose metabolism, specifically GLUT1 and MCT4 transporters, as the potential metabolic targets underlying BMJ efficacy in PanC.

Rhein sensitizes human pancreatic cancer cells to EGFR inhibitors by inhibiting STAT3 pathway

BackgroundRhein is a lipophilic anthraquinone extensively found in medicinal herbs. Emerging evidence suggests that rhein has significant antitumor effects, supporting its potential use as an antitumor agent. The IL6/STAT3 signaling pathway has been suggested as an attractive target for the discovery of novel cancer therapeutics.MethodsThe human pancreatic cancer cell lines AsPC-1, Patu8988T, BxPC-3 and PANC-1, and immunodeficient mice were chosen as models to study the effects of rhein. The potent antiproliferative and proapoptotic effects of rhein were examined by cell viability, cellular morphology, apoptosis and colony formation assays. The STAT3 luciferase report assay, immunostaining analysis and Western blot analysis revealed the inhibition of the IL6/STAT3 signaling axis.ResultsApoptosis was induced by adjunctive use of rhein with epidermal growth factor receptor (EGFR) inhibitors in pancreatic cancer cells as verified by cell apoptosis analysis and changes in the expression level of apoptotic/anti-apoptotic proteins BCL-2, BAX, Caspase 3 and Cl-PARP. Suppression of the phosphorylation of STAT3 and EGFR were also observed as a result of the treatment with a combination of rhein and EGFR inhibitors. Most interestingly, it was found that rhein considerably sensitized cells to erlotinib, thus suppressing tumor growth in PANC-1 and BxPC-3 xenograft models. The in vivo anti-tumor effect was associated with increased apoptosis and combined inhibition of the STAT3 and EGFR pathways in tumor remnants.ConclusionsRhein sensitizes human pancreatic cancer cells to EGFR inhibitors through inhibition of STAT3. Taken together, the results indicate that rhein offers a novel blueprint for pancreatic cancer therapy, particularly when combined with EGFR inhibitors.

Induction of endoplasmic reticulum stress by aminosteroid derivative RM-581 leads to tumor regression in PANC-1 xenograft model.

The high fatality and morbidity of pancreatic cancer have remained almost unchanged over the last decades and new clinical therapeutic tools are urgently needed. We determined the cytotoxic activity of aminosteroid derivatives RM-133 (androstane) and RM-581 (estrane) in three human pancreatic cancer cell lines (BxPC3, Hs766T and PANC-1). In PANC-1, a similar level of antiproliferative activity was observed for RM-581 and RM-133 (IC50 = 3.9 and 4.3μM, respectively), but RM-581 provided a higher selectivity index (SI = 12.8) for cancer cells over normal pancreatic cells than RM-133 (SI = 2.8). We also confirmed that RM-581 induces the same ER stress-apoptosis markers (BIP, CHOP and HERP) than RM-133 in PANC-1 cells, pointing out to a similar mechanism of action. Finally, these relevant in vitro results have been successfully translated in vivo by testing RM-581 using different doses (10-60mg/kg/day) and modes of administration in PANC-1 xenograft models, which have led to tumor regression without any sign of toxicity in mice (animal weight, behavior and histology). Interestingly, RM-581 fully reduced the pancreatic tumor growth when administered orally in mice.

Abstract LB-009: Intracellular delivery with SOCS3 suppresses angiogenesis in tumors

Abstract Introduction: Suppressor of cytokine signaling 3 (SOCS3) is a negative feedback regulator in the Janus kinase/signal transducer and activator of transduction (JAK/STAT) signaling pathway, in which it suppresses inflammation and cancer. The JAK/STAT pathway is activated in some tumors thus enhancing the expression of genes involved in proliferation and angiogenesis, notably, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), angiopoietin-1 and phospoinositide 3-kinase (PI3K)-protein kinase B (AKT). Objective: The aim of this study is to develop an improved cell-permeable (iCP)-SOCS3 and determine its applicability as an anti-angiogenic agent to treat cancer. Methods: The iCP-SOCS3 was expressed in E. coli as a 37 kDa recombinant protein fused to an advanced macromolecule transduction domain (aMTD) and humanized solubilization domain (SD). Uptake of a fluorescently tagged iCP-SOCS3 by cells and tissues was monitored by flow cytometry and confocal microscopy. Biological and biochemical activities of iCP-SOCS3 were determined by in vitro angiogenesis assays and changes in biomarker expression and anti-tumor activities were analyzed in mice bearing human tumor xenografts. Results: The iCP-SOCS3 improved on an earlier generation of CP-SOCS3 fused to FGF4-derived membrane translocating motif (MTM) with regard to solubility, yield, delivery into cells and tissues and biological activity. The iCP-SOCS3 suppressed proliferation of human umbilical vein endothelial cells (HUVECs) by 60% and migration by 54% via inhibition of AKT/mTOR signaling pathway. Moreover, iCP-SOCS3 inhibited invasion of EC cells in PANC-1 and U87MG conditioned medium by 38% and 95%, respectively. In tube formation assay, iCP-SOCS3 reduced the number of branch points by 79% and tube length by 60% compared to the vehicle. In chick chorioallantoic membrane (CAM) assay, iCP-SOCS3 significantly inhibited angiogenesis by 83% in dose dependent manner compared to the reference drug, retinoic acid. The anti-angiogenic activity of iCP-SOCS3 was also shown in matrigel plug assay. The new vessel formation induced by U87-MG glioblastoma cells was significantly reduced by the treatment of iCP-SOCS3. In PANC-1, U87-MG and HepG2 cell-derived xenograft models, iCP-SOCS3 suppressed expression of pro-angiogenic factors including VEGF, ERK and angiopoietin 1. Conclusion: These results validate iCP-SOCS3 targets in promoting angiogenesis and suggest ways to reduce angiogenesis in various types of tumors. Citation Format: Yukdong Jung, Kuysook Lee, Youngsil Choi, Daewoong Jo. Intracellular delivery with SOCS3 suppresses angiogenesis in tumors [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 LB-009.

Bioengineered microRNA-1291 effects on pancreatic cancer cell proliferation and xenograft tumor growth.

307 Background: MicroRNAs (miR) have proved to be vital regulators in the control of tumor progression. Our recent studies have revealed miR-1291 is downregulated in patient pancreatic cancer (PC) specimens and re-introduction of miR-1291 suppresses tumorigenesis of PC cells. We have developed a novel ncRNA bioengineering technology to produce a miR-1291 prodrug. In this study, we aimed to assess the effectiveness of this miR-1291 prodrug as a monotherapy, as well as in combination with chemotherapy, for treatment of PC. Methods: Sensitivity of PC cells to miR-1291 prodrug alone, gemcitabine plus nab-paclitaxel (Gem-nP) alone, and their combination was evaluated by CellTiter-Glo assay. Mature miR-1291 and ARID3B mRNA levels were determined by quantitative real-time PCR (q-PCR) assay. A luciferase reporter gene assay was used to validate interaction between miR-1291 and ARID3B 3’UTR. Target protein expression was examined by Western blot and immunofluorescence analyses. PANC-1 and PC patient-derived xenograft (PDX) mouse models were established and used to assess anti-tumor effects of miR-1291 monotherapy and combination therapy with Gem-nP. Results: Cytotoxicity assays showed that miR-1291 prodrug enhanced the sensitivity of PANC-1 and AsPC-1 cells to Gem-nP. Luciferase assays confirmed ARID3B as a target for miR-1291 as predicted by computational analysis. qPCR analysis demonstrated that miR-1291 prodrug was readily processed to mature miR-1291 and subsequently upregulated ARID3B mRNA levels. miR-1291 prodrug also elevated the protein levels of ARID3B. Co-administration of miR-1291 prodrug and Gem-nP increased caspase-3/7 and γH2AX levels in PC cells, compared to miR-1291 or Gem-nP treatment alone. In addition, systemic administration of in vivo-jet PEI formulated miR-1291 prodrug suppressed tumor growth in both a PANC-1 xenograft model and three PDX models, and largely enhanced the efficacy of Gem-nP. All treatments were well tolerated in mice in vivo. Conclusions: Our bioengineered miR-1291 prodrug has therapeutic potential as a monotherapy but also can act as a sensitizing agent to chemotherapy. This novel treatment approach should be further explored for PC.

Design, synthesis and biological evaluation of N-arylsulfonyl carbazoles as novel anticancer agents.

In this work, a set of structurally diverse synthetic carbazoles was screened for their anticancer activities. According to structure–activity relationship studies, carbazoles with an N-substituted sulfonyl group exhibited better anticancer activity. Moreover, compound 8h was discovered to show the most potent anticancer effects on Capan-2 cells by inducing apoptosis and cell cycle arrest in G2/M phase. Finally, the in vivo study demonstrated that 8h prevented the tumor growth in PANC-1 and Capan-2 xenograft models without apparent toxicity.

RGD-modified oncolytic adenovirus-harboring shPKM2 exhibits a potent cytotoxic effect in pancreatic cancer via autophagy inhibition and apoptosis promotion

The M2 isoform of pyruvate kinase (PKM2) is a key driver of glycolysis in cancer cells and has critical ‘non-metabolic’ functions in some cancers; however, the role of PKM2 in pancreatic cancer remains unclear. The aim of the current study was to elucidate the role of PKM2 in pancreatic cancer progression and the potential of PKM2 as a therapeutic target. In this study, we observed that PKM2 is highly expressed in patients with pancreatic cancer and is correlated to survival. Elevated PKM2 expression promoted cell proliferation, migration and tumor formation. The inhibition of cell growth by silencing PKM2 is caused by impairment of the autophagy process. To test the potential effects of downregulating PKM2 as a clinical therapy, we constructed an RGD-modified oncolytic adenovirus containing shPKM2 (OAd.R.shPKM2) to knock down PKM2 in pancreatic cancer cells. Cells transduced with OAd.R.shPKM2 exhibited decreased cell viability, and, in a PANC-1 xenograft model, intratumoral injection of OAd.R.shPKM2 resulted in reduced tumor growth. Furthermore, OAd.R.shPKM2 induced apoptosis and impaired autophagy in PANC-1 cells. Our results suggested that targeting PKM2 with an oncolytic adenovirus produced a strong antitumor effect, and that this strategy could broaden the therapeutic options for treating pancreatic cancer.

Phospho-Aspirin (MDC-22) Prevents Pancreatic Carcinogenesis in Mice.

Pancreatic cancer is a deadly disease with a dismal 5-year survival rate of <6%. The currently limited treatment options for pancreatic cancer underscore the need for novel chemopreventive and therapeutic agents. Accumulating evidence indicates that aspirin use is associated with a decreased risk of pancreatic cancer. However, the anticancer properties of aspirin are restricted by its gastrointestinal toxicity and its limited efficacy. Therefore, we developed phospho-aspirin (MDC-22), a novel derivative of aspirin, and evaluated its chemopreventive efficacy in preclinical models of pancreatic cancer. Phospho-aspirin inhibited the growth of human pancreatic cancer cell lines 8- to 12-fold more potently than aspirin; based on the 24-hour IC50 values. In a Panc-1 xenograft model, phospho-aspirin, at a dose of 100 mg/kg/d 5 times per week for 30 days, reduced tumor growth by 78% (P < 0.01 vs. vehicle control). Furthermore, phospho-aspirin prevented pancreatitis-accelerated acinar-to-ductal metaplasia in mice with activated Kras. In p48-Cre;Kras(G12D) mice, cerulein treatment (6 hourly injections two times per week for 3 weeks) led to a significant increase in ductal metaplasia, replacing the majority of the exocrine compartment. Administration of phospho-aspirin 100 mg/kg/day five times per week for 21 days (starting on the first day of cerulein injection) inhibited the acinar-to-ductal metaplasia, reducing it by 87% (P < 0.01, vs. cerulein-treated control). Phospho-aspirin appeared to be safe, with the animals showing no signs of toxicity during treatment. Mechanistically, phospho-aspirin inhibited EGFR activation in pancreatic cancer, an effect consistently observed in pancreatic cancer cells, primary acinar explants and in vivo In conclusion, our findings indicate that phospho-aspirin has strong anticancer efficacy in preclinical models of pancreatic cancer, warranting its further evaluation. Cancer Prev Res; 9(7); 624-34. ©2016 AACR.

The JNK Signaling Pathway Is a Novel Molecular Target for S-Propargyl- L-Cysteine, a Naturally-Occurring Garlic Derivatives: Link to Its Anticancer Activity in Pancreatic Cancer In Vitro and In Vivo

Pancreatic ductal adenocarcinoma (PDA) remains a devastating disease worldwide. Although significant improvement has been made in understanding its pathophysiology, only small portion of patients with PDA are likely to benefit from curative surgery and current chemotherapy. Thus, there is an urgent need for developing novel effective therapeutic approaches to the treatment of PDA. Today, garlic products have become an important source of effective compounds for the treatment of cancer. We have recently identified a novel garlic active component, S-propargyl-L-cysteine (SPRC), an analogue of S-Allyl Cysteine (SAC). Although its anticancer activity has been shown against several cancers, the mechanism of action is not fully understood. The present study was first designed to determine the anticancer activity of SPRC in PDA and the underlying mechanisms of action in vitro and in vivo. Our results demonstrated that SPRC reduced cell viability and colony formation, inhibited cell proliferation, induced G2/M phase cell cycle arrest and apoptosis in human PDA cells with various p53 statuses (HPAC, p53 wt; Panc-1, p53 mt). Furthermore, SPRC inhibited tumor growth in Panc-1 xenograft models. We also demonstrated that SPRC achieved its anticancer effects by regulation of the JNK protein levels through increasing its phosphorylation and decreasing its polyubiquitination-mediated degradation. In conclusion, SPRC has significant anti-PDA activity and the effects do not depend on p53 status, presumably through activating the JNK signaling pathway, providing a basis for the development of this compound as a novel target anticancer therapeutic agent for PDA.

Blockade of dual-specificity phosphatase 28 decreases chemo-resistance and migration in human pancreatic cancer cells.

Pancreatic cancer remains one of the most deadly cancers, with a grave prognosis. Despite numerous endeavors to improve treatment of the neoplasm, limited progress has been made. In the present study, we investigated the role of dual specificity phosphatase 28 (DUSP28) in relation to anti-cancer drug sensitivity and migratory activity in human pancreatic cancer cells for the first time. Analysis using Universal exPress Codes (UPCs) with the GEO database showed significantly higher DUSP28 mRNA expression in pancreatic cancers. We found that DUSP28 was highly expressed in several human pancreatic cancer cell lines that showed resistance to anti-cancer drugs. Overexpression of DUSP28 decreased anti-cancer drug-sensitivity and enhanced cellular migration via the ERK1/2 pathway in DUSP28-negative cell lines. Knockdown of DUSP28 re-sensitized cells to anti-cancer drugs even at sublethal doses by inducing an apoptotic pathway and significantly reduced migration in DUSP28-positive human pancreatic cancer cell lines. Furthermore, DUSP28-positive cell line (Panc-1) xenograft models were more resistant to gemcitabine treatment than DUSP28-negative cell line (SNU-213) xenograft models. Collectively, these results indicate that DUSP28 plays a key role in drug resistance and migratory activity in human pancreatic cells, and suggest that targeting DUSP28 might have clinical relevance in eradicating malignant pancreatic cancers.

Efficacy and safety of the hypoxia-activated prodrug TH-302 in combination with gemcitabine and nab-paclitaxel in human tumor xenograft models of pancreatic cancer

Tumors often contain hypoxic regions resistant to chemo- and radiotherapy. TH-302 (T) is an investigational hypoxia-activated prodrug that selectively releases the DNA cross-linker bromo-isophosphoramide mustard under hypoxic conditions. This study evaluated the efficacy and safety profile of combining T with gemcitabine (G) and nab-paclitaxel (nP) in human pancreatic ductal adenocarcinoma (PDAC) xenograft models in mice. Antitumor activity of the G + nP + T triplet was assessed and compared with T-alone or the G + nP doublet in the Hs766t, MIA PaCa-2, PANC-1, and BxPC-3 PDAC xenograft models. Efficacy was assessed by tumor growth kinetic analysis. Body weight, blood cell counts, blood chemistry, and the von Frey neuropathy assay were analyzed to evaluate safety profiles. Pharmacodynamic changes after the treatment were determined by immunohistochemistry of cell proliferation, DNA damage, apoptosis, hypoxia, and tumor stroma density. The G + nP + T triplet exhibited enhanced efficacy compared with T-alone or the G + nP doublet. Compared with vehicle (V), G + nP induced body weight loss, reduced neutrophil and lymphocyte counts, increased the levels of liver function parameters, and induced neurotoxicity. However, when T was added to G + nP, there was no statistically increased impairment compared to G + nP. The triplet significantly increased DNA damage, apoptosis, and tumor necrosis. Furthermore, the triplet further inhibited cell proliferation and reduced stroma density and intratumoral hypoxia. The triplet combination of G + nP + T exhibited superior efficacy but additive toxicity was not evident compared to the G + nP doublet in this study. This study provides a translational rationale for combining G, nP, and T in the clinical setting to assess efficacy and safety. A Phase I clinical trial of the triplet combination is currently underway (NCT02047500).