Antitumor Activity In Pancreatic Cancer Research Articles

SUMO3 inhibition by butyric acid suppresses cell viability and glycolysis and promotes gemcitabine antitumor activity in pancreatic cancer

BackgroundExcavation of key molecules can help identify therapeutic targets and improve the prognosis of pancreatic cancer. This study evaluated the roles of SUMO3 in cell viability, glycolysis, gemcitabine (GEM) sensitivity, and the antitumor activity of butyric acid (BA) in pancreatic cancer.MethodsThe mRNA and protein levels of SUMO3 were detected by qRT-PCR, Western blot, and immunohistochemical assay. SUMO3 was silenced or overexpressed in pancreatic cancer cells with or without Wnt/β-catenin pathway inhibitor, glycolysis inhibitor, GEM, or BA treatment. Cell viability was measured using the Cell Counting Kit-8 assay. Glycolysis was measured by determining the extracellular acidification rate, ATP level, and lactate content. Apoptosis was measured by flow cytometry, and TUNEL staining was used to examine in vitro and in vivo sensitivity to GEM chemotherapy. Luciferase reporter and chromatin immunoprecipitation assays were conducted to detect the binding of the SUMO3 promoter and NF-κB p65.ResultsSUMO3 was increased and associated with poor survival in pancreatic cancer. SUMO3 knockdown decreased cell viability and glycolysis in vitro and inhibited tumor growth in vivo. SUMO3 overexpression increased cell viability and glycolysis in vitro through the β-catenin pathway. SUMO3 knockdown increased GEM sensitivity, whereas SUMO3 overexpression decreased GEM sensitivity and inhibited the antitumor activity of BA. BA promoted histone acetylation and p-IκBα expression to inhibit NF-κB p65-mediated SUMO3 transcription.ConclusionSUMO3 acted as an active molecule in cell survival and growth by enhancing glycolysis in response to either GEM or BA. The mechanism was related to the constitutive IκBα/NF-κB/SUMO3/β-catenin signaling pathway.

Targeting minimal residual disease (MRD) in resected RAS mutated pancreatic cancer with vaccine TG01/QS-21 +/- PD-1 inhibitor, balstilimab: A randomized phase II study (TESLA).

TPS4205 Background: MRD detected by presence of circulating tumor DNA (ctDNA) after intended curative treatment is associated with high risk of relapse in pancreatic cancer. Early treatment of patients with presence of ctDNA after completion of surgery +/- adjuvant therapy may offer an opportunity to clear ctDNA and improve outcomes. TG01 is a RAS-neoantigen peptide vaccine adjuvanted by QS-21 (Stimulon) targeting the seven most frequent codon 12-13 RAS mutations. TG01 has previously demonstrated ability to activate mutant RAS specific CD4+ and CD8+ T-cell responses in vaccinated patients and repeated TG01 dosing in resected pancreatic cancer was found to be well tolerated and associated with a median OS of 33.4 months (95% CI 24.0, 45.8)1,2. Checkpoint inhibitors as single agents have not shown anti-tumor activity in pancreatic cancer, suggesting that a priming agent inducing tumor-specific T-cells may be required to support efficacy. Balstilimab is a human monoclonal antibody targeting programmed cell death protein 1 (PD-1) which is intended to reverse the immunosuppressive effects of this signaling pathway in the context of tumor immuno-surveillance by T-cells. Methods: Design: A two-arm, open-label, phase II randomized trial of TG01/QS-21 vaccine or TG01/QS-21 vaccine plus balstilimab (n=12 per arm, N=24) with surgically resected Stage 1-3 RAS mutant PDAC who are MRD+ following completion of standard adjuvant chemotherapy. Assay: MRD is detected by a commercially available ctDNA assay (Signatera, Natera). Somatic variants are identified by whole–exome sequencing of the primary tumor and the matched normal (whole blood) sample and a bespoke assay of up to 16 clonal, somatic variants are generated for each patient. This “tumor signature” will be monitored in plasma throughout the study. Treatment schedule: A priming phase of six vaccine administrations once every two weeks followed by a maintenance phase of administrations once every 8 weeks for up to 51 weeks. Balstilimab will be administered every 2 weeks for up to 51 weeks beginning at week 3. Imaging assessment will be done every 12 weeks. Eligibility: Surgically resected pancreatic adenocarcinoma with pathogenic RAS mutation, and no evidence of recurrent disease on baseline imaging. Inclusion criteria also include ECOG PS 0-1, and positive Signatera ctDNA MRD. Objectives: The primary objective is to assess the 6-month molecular disease control rate as defined by ctDNA stable, decreased or cleared. Secondary objectives include safety of TG01/QS-21 with or without balstilimab, 6 and 12-month DFS rate in each cohort, as well as correlation between the depth of molecular response and DFS. Exploratory: changes in clonality of RAS mutations and assess immune response. Enrollment is ongoing. 1) Gjertsen MK et al. Int J Cancer 1997, 72(5) 784-90. 2) Palmer DH et al. Br J Cancer 2020 122:971-77. Clinical trial information: NCT05638698 .

L-asparaginase anti-tumor activity in pancreatic cancer is dependent on its glutaminase activity and resistance is mediated by glutamine synthetase

l-Asparaginase is a cornerstone of acute lymphoblastic leukemia (ALL) therapy since lymphoblasts lack asparagine synthetase (ASNS) and rely on extracellular asparagine availability for survival. Resistance mechanisms are associated with increased ASNS expression in ALL. However, the association between ASNS and l-Asparaginase efficacy in solid tumors remains unclear, thus limiting clinical development. Interestingly, l-Asparaginase also has a glutaminase co-activity that is crucial in pancreatic cancer where KRAS mutations activate glutamine metabolism. By developing l-Asparaginase-resistant pancreatic cancer cells and using OMICS approaches, we identified glutamine synthetase (GS) as a marker of resistance to l-Asparaginase. GS is the only enzyme able to synthesize glutamine, and its expression also correlates with l-Asparaginase efficacy in 27 human cell lines from 11 cancer indications. Finally, we further demonstrated that GS inhibition prevents cancer cell adaptation to l-Asparaginase-induced glutamine starvation. These findings could pave the way to the development of promising drug combinations to overcome l-Asparaginase resistance.

A Carabrane-Type Sesquiterpenolide Carabrone from Carpesium cernuum Inhibits SW1990 Pancreatic Cancer Cells by Inducing Ferroptosis.

Pancreatic cancer has an extremely poor prognosis, and the clinical drugs for the treatment of pancreatic cancer are usually multi-drug combinations. Therefore, it is necessary to search for and find specific new bioactive agents against pancreatic cancer. Carabrone is a carabrane-type sesquiterpenolide extracted from Carpesium cernuum L., and this natural compound has been reported to be a potential anti-tumor agent. However, there are few reports on the function of carabrone related to anti-tumor activity in pancreatic cancer. Herein, cell experiments indicated that carabrone had anti-proliferation inhibition and anti-migration and anti-invasion activity against SW1990 cells. Furthermore, the tandem mass spectrometry and network pharmacology analysis showed that this activity may be related to the ferroptosis and Hippo signaling pathway. Taken together, our results demonstrated that carabrone exhibited prominent anti-pancreatic cancer activity and could be a promising agent against pancreatic cancer.

The HDL particle composition determines its antitumor activity in pancreatic cancer.

Despite enormous efforts to improve therapeutic options, pancreatic cancer remains a fatal disease and is expected to become the second leading cause of cancer-related deaths in the next decade. Previous research identified lipid metabolic pathways to be highly enriched in pancreatic ductal adenocarcinoma (PDAC) cells. Thereby, cholesterol uptake and synthesis promotes growth advantage to and chemotherapy resistance for PDAC tumor cells. Here, we demonstrate that high-density lipoprotein (HDL)-mediated efficient cholesterol removal from cancer cells results in PDAC cell growth reduction and induction of apoptosis in vitro. This effect is driven by an HDL particle composition-dependent interaction with SR-B1 and ABCA1 on cancer cells. AAV-mediated overexpression of APOA1 and rHDL injections decreased PDAC tumor development in vivo. Interestingly, plasma samples from pancreatic-cancer patients displayed a significantly reduced APOA1-to-SAA1 ratio and a reduced cholesterol efflux capacity compared with healthy donors. We conclude that efficient, HDL-mediated cholesterol depletion represents an interesting strategy to interfere with the aggressive growth characteristics of PDAC.

1550P Combination therapy of low molecular weight heparins, chemotherapy and immunotherapy induce antitumor activity in pancreatic cancer

1550P Combination therapy of low molecular weight heparins, chemotherapy and immunotherapy induce antitumor activity in pancreatic cancer

Exosomes-Coated miR-34a Displays Potent Antitumor Activity in Pancreatic Cancer Both in vitro and in vivo.

PurposeMiR-34a, which acts as an important tumor suppressor gene, plays an important role in pancreatic cancer. However, the therapeutic application of miR-34a is limited by the lack of an effective delivery system. In the present study, we synthesize exosomes-coated miR-34a (exomiR-34a), and the anticancer effect of exomiR-34a was evaluated in pancreatic cancer.Materials and MethodsAn ultrasound approach was used to synthesize exomiR-34a, and its transfection efficiency was examined by confocal microscopy and flow cytometry. The level of miR-34a and its targeted gene Bcl-2 was detected by real-time quantitative PCR (qRT-PCR). MTT analysis was performed to determine the effect of exomiR-34a on the growth of pancreatic cancer cells. Annexin-V/PI double staining and Western blot analysis were carried out to determine the apoptosis of the pancreatic cancer cells. The xenograft nude mice model bearing human pancreatic cancer Panc28 cells was used to determine the antitumor effect of exomiR-34a in vivo.ResultsThe exomiR-34a could cross the cell membrane efficiently, and downregulated the expression of the targeted gene Bcl-2. Treatment with exomiR-34a inhibited the growth of the pancreatic cancer cells significantly and the nanoparticles also induced apoptosis in cancer cells via affecting the expression of apoptotic-related genes. In vivo study using xenograft nude mice bearing Panc28 cancer cells revealed that exomiR-34a suppressed the growth of tumors significantly.ConclusionExomiR-34a can inhibit the growth of pancreatic cancer both in vitro and in vivo. Targeting miR-34a is a promising strategy for the treatment of pancreatic cancer. ExomiR-34a has the potential to be developed as a novel anticancer agent for the treatment of human pancreatic malignancy.

FBP1 loss contributes to BET inhibitors resistance by undermining c-Myc expression in pancreatic ductal adenocarcinoma

BackgroundPancreatic ductal adenocarcinoma (PDAC) is one of the most lethal tumor types worldwide. BET inhibitors display anti-tumor activity in pancreatic cancer, however the cells often develop resistance after a long-term treatment and the underlying molecular basis is not fully understood.MethodsDrug screening assay in Fructose-1, 6-biphosphatase (FBP1) knockdown or overexpressing pancreatic cancer cells was performed. Tumor cell motility, FBP1 protein and mRNA changes were investigated after BET inhibitors treatment. The interaction between TRIM28 and FBP1 after BET inhibitors treatment was examined by Co-immunoprecipitation (IP) and GST pull-down. The relationship between FBP1 and c-Myc was examined by western blot, RT-qPCR and immunohistochemistry (IHC).ResultsThe expression of FBP1 protein increased the sensitivity of pancreatic cancer cells to JQ1. Furthermore, we showed that JQ1 stabilized FBP1 protein level by disrupting the interaction between FBP1 and TRIM28 in pancreatic cancer cells. Moreover, we demonstrated that FBP1 promoted c-Myc degradation through disrupting the ERK-c-Myc axis.ConclusionsFBP1 modulates the sensitivity of pancreatic cancer cells to BET inhibitors by decreasing the expression of c-Myc. These findings highlight FBP1 could be used as a therapeutic niche for patient-tailored therapies.

Inhibition of Cell Survival by Curcumin Is Associated with Downregulation of Cell Division Cycle 20 (Cdc20) in Pancreatic Cancer Cells

Pancreatic cancer is one of the most aggressive human tumors in the United States. Curcumin, a polyphenol derived from the Curcuma longa plant, has been reported to exert its antitumor activity in pancreatic cancer. However, the molecular mechanisms of curcumin-mediated tumor suppressive function have not been fully elucidated. In the current study, we explore whether curcumin exhibits its anti-cancer function through inhibition of oncoprotein cell division cycle 20 (Cdc20) in pancreatic cancer cells. We found that curcumin inhibited cell growth, enhanced apoptosis, induced cell cycle arrest and retarded cell invasion in pancreatic cancer cells. Moreover, we observed that curcumin significantly inhibited the expression of Cdc20 in pancreatic cancer cells. Furthermore, our results demonstrated that overexpression of Cdc20 enhanced cell proliferation and invasion, and abrogated the cytotoxic effects induced by curcumin in pancreatic cancer cells. Consistently, downregulation of Cdc20 promoted curcumin-mediated anti-tumor activity. Therefore, our findings indicated that inhibition of Cdc20 by curcumin could be useful for the treatment of pancreatic cancer patients.

Retraction: "Concurrent inhibition of NF-κB, cyclooxygenase-2, and epidermal growth factor receptor leads to greater anti-tumor activity in pancreatic cancer" by Ali et al.

The above article, published online on March 8, 2010 in Wiley Online Library (wileyonlinelibrary.com), has been retracted by agreement between the journal Editor in Chief, Gary S. Stein, and Wiley Periodicals, Inc. The retraction has been agreed following an investigation from Wayne State University involving the first author and the corresponding author that found Figures 2A, 4, 6A, and 6C to be inappropriately manipulated. REFERENCE Ali S, Banerjee S, Schaffert JM, El-Rayes BF, Philip PA, Sarkar FH. 2010. Concurrent inhibition of NF-κB, cyclooxygenase-2, and epidermal growth factor receptor leads to greater anti-tumor activity in pancreatic cancer. J Cell Biochem 110:171-181; doi: 10.1002/jcb.22523.

Inhibition of renalase expression and signaling has antitumor activity in pancreatic cancer.

An essential feature of cancer is dysregulation of cell senescence and death. Renalase, a recently discovered secreted flavoprotein, provides cytoprotection against ischemic and toxic cellular injury by signaling through the PI3K-AKT and MAPK pathways. Here we show that renalase expression is increased in pancreatic cancer tissue and that it functions as a growth factor. In a cohort of patients with pancreatic ductal adenocarcinoma, overall survival was inversely correlated with renalase expression in the tumor mass, suggesting a pathogenic role for renalase. Inhibition of renalase signaling using siRNA or inhibitory anti-renalase antibodies decreased the viability of cultured pancreatic ductal adenocarcinoma cells. In two xenograft mouse models, either the renalase monoclonal antibody m28-RNLS or shRNA knockdown of renalase inhibited pancreatic ductal adenocarcinoma growth. Inhibition of renalase caused tumor cell apoptosis and cell cycle arrest. These results reveal a previously unrecognized role for the renalase in cancer: its expression may serve as a prognostic maker and its inhibition may provide an attractive therapeutic target in pancreatic cancer.

LB-1 Exerts Antitumor Activity in Pancreatic Cancer by Inhibiting HIF-1α and Stat3 Signaling.

Hypoxia is widely present in pancreatic cancer and subsequently causes the overexpression of hypoxia-inducible factor-1α (HIF-1α) and signal transducer and activator of transcription-3 (Stat3). HIF-1α and Stat3 function cooperatively to regulate a number of downstream genes that are implicated in tumorigenesis. Thus, inhibition of HIF-1α and Stat3 is a potential therapeutic strategy for pancreatic cancer. In this study, we explored how LB-1, a novel triptolide (LA) derivative, exerted its antitumor effect through blockade of HIF-1α and Stat3 signaling. Our data showed that LB-1 was able to inhibit the proliferation and colony formation of Mia-PaCa2 and SW1990 cells. LB-1 suppressed HIF-1α protein accumulation by promoting its proteasome degradation and reducing transactivation. Moreover, the silence of HIF-1α by shRNA partially prevented the proliferation inhibition triggered by LB-1. As expected, LB-1 also decreased Stat3 protein accumulation and blocked the physical interactions between HIF-1α/p300/phosphor-Stat3 (p-Stat3) at the pharmacological concentration to reduce VEGF expression, thereby hypoxia-induced angiogenesis. In the Mia-PaCa2 nude xenograft model, therapeutic treatment with LB-1 significantly inhibited tumor growth and had minimal systemic toxicity compared to the mother drug LA. Furthermore, in accordance with in vitro results, HIF-1α activation and Stat3 expression in tumors were blocked by LB-1 through mTOR-dependent pathway. Taken together, these results illustrate that, as a potent inhibitor of HIF-1α and Stat3 signaling, LB-1 exhibits antitumor effect and could be potentially used to treat pancreatic cancer.

Abstract 4505: Anti-tumor activity in pancreatic cancer of a low immunogenic and clinically optimized anti-mesothelin immunotoxin RG7787

Abstract Background Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis and new therapies are needed. SS1P is a recombinant immunotoxin (RIT) containing an anti-mesothelin dsFv fused to a cytotoxic fragment of Pseudomonas Exotoxin A (PE). In clinical trials, patients developed dose-limiting neutralizing anti-drug antibodies within the first cycle of treatment. RG7787 is a next generation, clinically optimized RIT with decreased immunogenicity and increased resistance to lysosomal proteases. In this study, we evaluated the in vitro and in vivo activity of RG7787 in PDAC. Method Five established PDAC cell lines (KLM-1, AsPC-1, BxPC-1, BxpC3, Panc 3.014, and PK-1) and one primary PDAC line directly obtained from a patient tumor (GUMC108) were used. Cell viability and proliferation were calculated by measuring ATP levels with Cell Titer-Glo assays. In vivo activity was evaluated in a subcutaneous KLM-1 mouse tumor model. Delivery of fluor-labeled RG7787 to mouse tumors was assessed by flow cytometry. Results RG7787 had significant in vitro activity in the PDAC cell lines tested with most IC50 values in the subnanomolar range. It was significantly more active than SS1P in KLM-1, Panc 3.014 and GUMC108, with similar activity in the other cell lines. GUMC108 (IC50 = 18.9 pM) was the most sensitive cell line. In KLM-1 cells, SS1P and RG7787 had similar internalization rates. RG7787 could be safely administered to mice at a dose of 2.5 mg/ kg IV QOD x3, which is more than five times the maximum tolerated murine dose of SS1P. In vivo uptake studies in a KLM-1 subcutaneous mouse xenograft model demonstrated intracellular delivery of RG7787-Alexa647 to 50% of tumor cells after a single 2.5 mg/kg IV dose. Tumor volume was reduced by 13% after a single cycle of RG7787 treatment (n=6). Two cycles of combination treatment with paclitaxel (50 mg/kg IP) and RG7787 shrank tumors to <10 mm3 in 100% of mice by 30 days (n = 11 over 2 experiments). Two single doses of paclitaxel alone (n=6) induced stable disease without significant tumor regression. Complete remission persisted through day 100 in 2/11 combination treated animals. Conclusions RG7787 has superior or similar activity compared to SS1P in established PDAC cell lines and a primary PDAC cell line in vitro. In a KLM-1 mouse xenograft model, the combination of paclitaxel with RG7787 cured 18.1% of mice and reduced tumor size by more than 90% in all other mice treated. RG7787 merits further development for the treatment of pancreatic cancer. Citation Format: Ira Pastan, Kevin Hollevoet, Emily Mason-Osann, Christine Alewine, Xiu-Fen Liu. Anti-tumor activity in pancreatic cancer of a low immunogenic and clinically optimized anti-mesothelin immunotoxin RG7787. [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 4505. doi:10.1158/1538-7445.AM2014-4505

Curcumin inhibits cell growth and invasion through up-regulation of miR-7 in pancreatic cancer cells

Accumulating evidence has revealed that a natural compound curcumin exerts its anti-tumor activity in pancreatic cancer. However, the underlying molecular mechanism remains elusive. Recently, miRNAs have been demonstrated to play a crucial role in tumorigenesis, suggesting that targeting miRNAs could be a promising approach for the treatment of human cancers. In this study, we explored whether curcumin regulates miR-7, leading to the inhibition of cell growth, migration and invasion in pancreatic cancer cells. We observed that curcumin suppressed cell growth, migration and invasion, and induced cell apoptosis, which is associated with increased expression of miR-7 and subsequently decreased expression of SET8, one of the miR-7 targets. These findings demonstrated that targeting miR-7 by curcumin could be a novel strategy for the treatment of pancreatic cancer.

Abstract 1257: Investigating the protective role of HLA-B35 in pancreatic cancer.

Abstract HLA-B35 has recently been reported as a protective marker for pancreatic cancer development. The goal of the current study was to investigate whether this link between B35 and cancer protection was related to cellular immunity and correlated with the presence of HLA-B35-restricted cytotoxic T cell activity. As a model antigen we chose mesothelin (MSLN), a tumor antigen expressed by >90% of pancreatic tumors and known to be immunogenic to T cells. To activate MSLN-specific T cells we generated autologous DCs from 15 HLA-B35 positive and 12 HLA-B35 negative donors and loaded them with a pepmix (15mers overlapping by 11aas) spanning MSLN in order to use an HLA unbiased approach for antigen stimulation. These were used to stimulate PBMCs in the presence of a cytokine cocktail (IL7, 12, 15, 6), which we have optimized for activating tumor-directed T cells in vitro. We were successfully able to amplify an immune response against MSLN in 12 of 15 (80%) B35+ donors based on IFNg ELIspot analysis (range 23-10108 SFC/2x10e5). In contrast we were successful in only 1 of 12 (8%) B35-ve donors. Since the CTL lines were polyclonal (CD8+, median 69%, range 38-97%; CD4+, median 30%, range 2-55%) we next investigated, using ICS, whether this anti-tumor activity was in the helper or cytotoxic T cell compartments. Eight of 13 MSLN responders had activity exclusively in CD8+ T cells (range 3-64% IFNg+ cells) and 2 had activity in both CD8 and CD4 T cells, while in the only non-B35 responder activity was detected exclusively in the CD4+ T cell compartment. We next performed epitope mapping studies using peptide minipools and detected activity against 3 published CD8+ epitopes [HLA-24 FYP (aa435-443) and LYP (aa475-483); HLA-A2 VLP (aa530-538)] as well as 5 novel B35-restricted epitopes, one of which was detected in 7 of our 12 B35 responders. Finally, to assess whether these novel epitopes were recognized in the context of HLA-B35 we loaded autologous and partially HLA-matched APCs with the individual peptides and assessed cytolytic activity. Autologous peptide-loaded targets were killed (range 52-98% killing, E:T 40:1) as were allogeneic targets exclusively matched at HLA-B35 (range 32-92% killing), confirming that all epitopes were presented by the HLA-B35 allele. These B35-restricted CTLs were also able to kill HLA-matched tumors expressing MSLN - we saw specific lysis of CFPAC-1 (B35+MSLN+) (36% killing E:T 40:1) with no recognition of CAPAN-1 (B35-MSLN+) (3% killing). Thus, our results support a contribution of HLA-B35-restricted T cells and anti-tumor activity in pancreatic cancer and highlight the importance of cellular immunity in protection against cancer. Citation Format: Ryu Yanagisawa, Jacqueline M. Keirnan, Usha L. Katari, Usanarat Anurathapan, Malcolm K. Brenner, Cliona M. Rooney, Juan F. Vera, Ann M. Leen. Investigating the protective role of HLA-B35 in pancreatic cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1257. doi:10.1158/1538-7445.AM2013-1257

Abstract 2362: Targeted suppression of TGF-beta2 by trabedersen (AP 12009) in an orthotopic xenograft melanoma mouse model

Abstract Background: TGF-β2 is a mediator of carcinogenesis by regulating key mechanisms such as cell proliferation, metastasis, and immunosuppression. The antisense oligonucleotide trabedersen (AP 12009) specifically inhibits TGF-β2 expression. Clinical studies already demonstrated its antitumor activity in pancreatic cancer, high-grade glioma, and malignant melanoma. Here we examined the anti-tumorigenic and anti-metastatic effects of trabedersen in an orthotopic xenograft melanoma mouse model. Methods: BALB/c nu/nu mice were intradermally injected with 0.5x10E6 cells of the human TGF-β2 expressing melanoma cell line C8161. Intraperitoneal treatment with different doses of trabedersen or vehicle (controls) started 2 days after tumor implantation thrice weekly. Doses of trabedersen were 50/16 mg/kg (i.e. initial dose of 50 mg/kg and subsequent 16 mg/kg), 16 mg/kg, 4 mg/kg, 1 mg/kg, and 0.25 mg/kg. Treatment continued until first tumor reached 2000 mm3 (approx. 32-35 days after inoculation). The incidence of liver and lung metastases as well as growth and weight of the dermal tumors were determined. To evaluate the immunomodulatory effect of trabedersen, NK cells and MDSCs prepared from spleens were analyzed of trabedersen-treated or control mice (flow cytometry; MDSCs: anti-CD11b+, anti-Ly6G, anti-Ly6C; NK cells: CD49+, CD314+). Cellular uptake was examined by using fluorescently labeled trabedersen (5′-FAM-AP 12009) which was intra-tumorally injected 4 h prior to termination of the study. Cryosections were semi-quantitatively evaluated for the presence of fluorescence. Results: Tumor weight was significantly reduced for trabedersen-treated mice (50/16 mg/kg) compared to control (0.395 g vs. 0.715 g, p<0.05). Moreover, a dose dependent response for trabedersen treatment with 1 mg/kg, 4 mg/kg, and 16 mg/kg regarding tumor weight and tumor growth was observed. Trabedersen treatment significantly inhibited lung metastasis. There was no difference for liver metastasis between trabedersen and control. Analysis of spleen cell preparations of trabedersen-treated and control mice revealed a trend to less monocytic MDSCs under trabedersen treatment. There was no difference in the amount of granulocytic MDSCs and NK cells between trabedersen-treated and control mice. Intra-cellular and intra-nuclear fluorescence was clearly detectable in all tumor sections of all mice receiving the intratumoral injection of 5′-FAM-AP 12009, indicating uptake of trabedersen in the tumor cells and nuclear localisation. Conclusions: In an orthotopic xenograft melanoma mouse model trabedersen demonstrated potent antitumor activity as it inhibits the tumor promoting and metastatic effects of TGF-β2 such as tumor cell proliferation and immunosuppression. This is in line with promising results from clinical studies in patients with advanced pancreatic cancer or malignant melanoma (Phase I/II). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2362. doi:1538-7445.AM2012-2362

A phase II study of the halichondrin B analog eribulin mesylate in gemcitabine refractory advanced pancreatic cancer

Eribulin mesylate is a halichondrin B analog that inhibits microtubule dynamics. Pre-clinical studies have suggested anti-tumor activity in pancreatic cancer. This phase II study of eribulin in patients with advanced pancreatic cancer previously treated with gemcitabine was conducted by the Princess Margaret Hospital Phase II consortium. Eligibility criteria included locally advanced or metastatic pancreatic adenocarcinoma and previous treatment with gemcitabine. The study was a single arm phase II trial using a Simon 2-stage design. The primary endpoint was response rate, secondary endpoints included time to progression and overall survival. Fifteen patients were enrolled, 14 received treatment, and 12 were evaluable for response. The median age was 61, and the majority of patients were ECOG performance status 1. Grade 3 or greater adverse events included neutropenia (29%), fatigue (14%), peripheral neuropathy (7%) and thrombosis (7%). There were no complete or partial responses and therefore the study was closed after the first stage. The best response was stable disease in 5/12 (42%) of patients. Of these five patients, three had stable disease for 9 months or greater. Median time to progression was 1.4 months, and median overall survival was 6.1 months. Eribulin was well tolerated but did not result in any objective responses in gemcitabine refractory pancreatic cancer. However, several patients had prolonged stable disease, suggesting that further studies of eribulin in pancreatic cancer may be warranted.

Abstract LB-374: The Hh inhibitor IPI-926 enhances tumor perfusion and nab-paclitaxel activity in a pancreatic xenograft model

Abstract Malignant activation of the Hedgehog (Hh) pathway is associated with multiple tumor types. In certain cancers, such as pancreatic, a paracrine role for the Hh ligand has been described, wherein cancer cells produce Hh ligand that activates the Hh pathway in the surrounding stroma. Consistent with this model, IPI-926, a potent and selective Smoothened (Smo) inhibitor, blocks Hh signaling in the mouse stroma -but not in the cancer cells- of several pancreatic xenograft models. We recently published (Olive, Science 2009) that IPI-926 increases vascular perfusion and enhances gemcitabine drug delivery to tumors in a genetically engineered mouse model of pancreatic cancer (KPC) leading to an increase in overall survival. To determine if similar effects could be observed in xenograft models of human pancreatic cancer, experiments were designed to study the combination of IPI-926 with nab-paclitaxel, an agent that has recently demonstrated anti-tumor activity in pancreatic cancer (Van Hoff, ASCO 2009). While IPI-926 had no single agent activity in the L3.6pl human pancreatic xenograft model, it enhanced the activity of nab-paclitaxel from 61% tumor growth inhibition (nab-paclitaxel alone) to 83% tumor growth inhibition (nab-paclitaxel plus IPI-926, p=0.0048). Tumor IHC analysis of phosphohistone 3 showed a higher frequency of cells arrested at the late G2/M phase in the IPI-926 plus nab-paclitaxel group versus nab-paclitaxel alone (p=0.02). One possible explanation for the synergistic effect of a combination of IPI-926 and nab-paclitaxel is that IPI-926 affects the mouse stroma and increases tumor perfusion and nab-paclitaxel accessibility to the tumor. Tumor perfusion was directly measured in IPI-926 treated and untreated animals using contrast enhanced ultrasound. In tumor bearing animals treated with IPI-926 for 7 days, the ultrasound data showed greater tumor perfusion with IPI-926. On average, the peak time for contrast agent levels decreased from 11.0 seconds to 4.75 seconds in the vehicle versus IPI-926 treated animals, respectively, (p=0.0321). These data suggest that the mechanism of synergy between IPI-926 and nab-paclitaxel is likely enhanced drug delivery to the tumor through the effect of IPI-926 on the stroma. Studies are ongoing to measure nab-paclitaxel and paclitaxel levels in IPI-926 treated and untreated tumors, and to investigate these findings with the KPC in situ mouse model of pancreatic cancer. These preclinical data provide a strong rationale for evaluating the Hh inhibitor IPI-926 not only with the current standard of care, gemcitabine, but with emerging new potential therapies like nab-paclitaxel in pancreatic cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-374.

Effects of Combination of EMAP II with Doxorubicin, Docetaxel, Oxaliplatin and Gemcitabine to Enhance Antitumor Activity in Pancreatic Cancer

Background: Pancreatic ductal adenocarcinoma (PDAC) remains a highly lethal malignancy, and is highly resistant to conventional chemotherapy. Gemcitabine (G) is the standard therapy for PDAC, with a limited clinical response. Doxorubicin (Dox), docetaxel (DT) and oxaliplatin (OX) have been tested as second line chemotherapy for treatment of PDAC without significant clinical benefit. Endothelial monocyte activating polypeptide II (EMAP, E) is a proinflammatory cytokine with antiendothelial and antiangiogenic properties. It has been shown to inhibit primary and metastatic tumor growth in animal models, and has been able to enhance G effects against PDAC. We tested the combination benefits of EMAP with doxorubicin, docetaxel, oxaliplatin and gemcitabine for enhanced antitumor effects on PaCa. Methods: Human PDAC ASPC cells were grown in RPMI growth medium, and human umbilical vein endothelial cells (HUVECs) were grown in E-stim growth medium. Cell proliferation assay was performed by WST-1 assay. In vivo survival was tested using ASPC cells in intraperitoneal murine xenograft model. Results: In vivo, animal survival study revealed that compared to controls (median survival: 21 days), EMAP alone (20 d) or oxaliplatin (23 d) had no significant effect on survival, while doxorubicin (31 d), docetaxel (35 d) and gemcitabine (32 d) extended the overall survival. Combination treatment of EMAP with docetaxel (44 d) or gemcitabine (38 d) enhanced the resulting survival, but the combination of EMAP with doxorubicin or oxaliplatin had no obvious benefits (31 and 23 days, respectively; p=NS). An in vitro cell proliferation assay showed that in ASPC cells, EMAP had no effect, while doxorubicin and gemcitabine provided a minor (IC50>10 μM), and docetaxel a major inhibitory effect (IC50: 8 μM); at 10 μM concentrations, inhibition in cell proliferation (in %) was 1.4, 26.5, 40 and 60, respectively. Addition of EMAP had no increased antiproliferative effects. In HUVECs, EMAP, doxorubicin, docetaxel and gemcitabine induced significant antiproliferative effects (59%, 79%, 96% and 85% at 10 μM, respectively). These inhibitory effects were significantly increased by EMAP addition to the three cytotoxic agents. Conclusions: In experimental PDAC, there is a differential effect to monotherapy with various cytotoxic agents. Addition of EMAP enhanced docetaxel and gemcitabine effects, but not those after doxorubicin or oxaliplatin. In vitro, EMAP addition increased the antiproliferative effects of doxorubicin, docetaxel and gemcitabine in HUVECs but not in ASPC PDAC cells. This antiendothelial combination therapy may represent an avenue to enhance clinical PDAC therapy with cell-cycle sensitive cytotoxic agents other than gemcitabine.

Rottlerin stimulates apoptosis in pancreatic cancer cells through interactions with proteins of the Bcl-2 family

Rottlerin is a polyphenolic compound derived from Mallotus philipinensis. In the present study, we show that rottlerin decreased tumor size and stimulated apoptosis in an orthotopic model of pancreatic cancer with no effect on normal tissues in vivo. Rottlerin also induced apoptosis in pancreatic cancer (PaCa) cell lines by interacting with mitochondria and stimulating cytochrome c release. Immunoprecipitation results indicated that rottlerin disrupts complexes of prosurvival Bcl-xL with Bim and Puma. Furthermore, siRNA knockdown showed that Bim and Puma are necessary for rottlerin to stimulate apoptosis. We also showed that rottlerin and Bcl-2 and Bcl-xL inhibitor BH3I-2' stimulate apoptosis through a common mechanism. They both directly interact with mitochondria, causing increased cytochrome c release and mitochondrial depolarization, and both decrease sequestration of BH3-only proteins by Bcl-xL. However, the effects of rottlerin and BH3I-2' on the complex formation between Bcl-xL and BH3-only proteins are different. BH3I-2' disrupts complexes of Bcl-xL with Bad but not with Bim or Puma, whereas rottlerin had no effect on the Bcl-xL interaction with Bad. Also BH3I-2', but not rottlerin, required Bad to stimulate apoptosis. In conclusion, our results demonstrate that rottlerin has a potent proapoptotic and antitumor activity in pancreatic cancer, which is mediated by disrupting the interaction between prosurvival Bcl-2 proteins and proapoptotic BH3-only proteins. Thus rottlerin represents a promising novel agent for pancreatic cancer treatment.