Hedgehog (HH) and ErbB signaling as mediators of tumor-stroma interactions in pancreatic ductal adenocarcinoma (PDAC)

Hedgehog (HH) and ErbB signaling as mediators of tumor-stroma interactions in pancreatic ductal adenocarcinoma (PDAC)

S134 Abstracts METHODS: CICs were identified as aldehyde-dehydrogenase1A1 bright (ALDHbright) cells on flow cytometry. The MIAPaCa-2 human pancreati...

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S134

Abstracts

METHODS: CICs were identified as aldehyde-dehydrogenase1A1 bright (ALDHbright) cells on flow cytometry. The MIAPaCa-2 human pancreatic cancer cell line was incubated with mAb W9 (10␮g/ mL) and/or cyclopamine (20␮M) for 72 hours at 37 °C, and treated with 20Gy of XRT, then stained with ALDEFLUOR. The % of ALDHbright cells was determined. RESULTS: ALDHbright cells composed approximately 12% of the entire cell population. When MIAPaCa-2 cells were treated with mAb W9 or cyclopamine the percentage of ALDHbrigh cells decreased to 6.0% and 5.3%, respectively (p⫽ ns). However, when mAb W9 was combined with cyclopamine the % of CICs was significantly reduced to 2.2% (p⬍0.05). CICs were reduced to 0.8% (p⬍0.001) by combining cyclopamine, mAbW9 and radiation. CONCLUSIONS: Our in vitro experiments demonstrate that combinatorial mAb W9-based immunotherapy effectively targets pancreatic adenocarcinoma CICs. These results open the horizon to preclinical models and future clinical trials for patients with pancreatic adenocarcinoma.

The novel ability of a tyrosine kinase inhibitor to alter myeloid-derived suppressor cell and t-regulatory cell (Treg) function Brian A Coakley, MD, David Z Kalir, MS, Ge Ma, MD, Junko Ozao-Choy, MD, Ping Y Pan, PhD, Shu-Hsia Chen, PhD, Celia Divino, MD, FACS The Mount Sinai Medical Center, New York, NY INTRODUCTION: Myeloid-derived suppressor cells (MDSCs) have been shown to promote tumor progression and correlate with tumor prognosis. Our previous studies demonstrate that the tyrosine-kinase inhibitor sunitinib, currently FDA-approved for treatment of renal cell carcinoma and imatinib-resistant GIST, inhibits MDSC development and impairs the MDSC-mediated activation of Tregs, favoring Th1-type responses in tumor-bearing mice. Herein, we further investigate the regulatory effects of sunitinib on MDSCs and Tregs. METHODS: Cells purified via magnetic separation from tumorbearing mice were cultured with or without sunitinib and analyzed via ELISA, FACS, suppression assays and qPCR. In vivo assays were performed using sorted RFP cells from OT-II X FoxP3-RFP mice and transferred into OVA-B16 tumor-bearing mice, which were then administered sunitinib daily. RESULTS: Sunitinib inhibited STAT3 phosphorylation (p⫽0.02) in MDSCs and induced apoptosis of M2-functional phenotype type macrophages (p⫽0.002), resulting in selection for MDSCs differentiating into classical, M1-type macrophages (p⬍0.005). Sunitinib treatment dramatically inhibited Treg proliferation (p⬍0.001) and FoxP3 expression (p⬍0.001) and significantly converted Tregs into RORgammaT and T-bet expressing cells, as measured by IL-17 and IFN-gamma (both p⬍0.001). Thus, sunitinib abrogated FoxP3 expression and induced Treg conversion, indicating multi-targeted RTKIs like sunitinib can regulate MDSCs and Tregs through different regulatory mechanisms.

J Am Coll Surg

CONCLUSIONS: Although sunitinib has traditionally been used to target cancer directly, its mechanism remains poorly described. Our data suggests that sunitinib controls MDSC differentiation into M1type macrophages and Treg functional switching into Th17/Th1 cells. This represents a novel role for sunitinib in modulating immunosuppressive function in tumor-bearing hosts, which enhances our understanding of its activity on oncologic disease.

Enhanced therapeutic effects of a novel oncolytic and anti-angiogenic vaccinia virus against triple-negative breast cancer Sepideh Gholami, MD, Andrew A Marano, BA, Nanhai G Chen, PhD, Alexa Frentzen, PhD, Chun-Hao Chen, MD, Clarisse Eveno, MD, Emil Lou, MD, PhD, Laurence Belin, MD, MPH, Aladar A Szalay, PhD, Yuman Fong, MD, FACS Memorial Sloan-Kettering Cancer Center, New York, NY INTRODUCTION: Vascular endothelial growth factor (VEGF) expression is higher in triple-negative breast cancers (TNBC) compared to other breast cancer subtypes and is reported to predict distant metastases and shorter overall survival. We investigated the therapeutic impact of a novel oncolytic vaccinia virus (VACV) GLV1h164, encoding a single-chain antibody (scAb) against VEGF (GLAF-2) in an orthotopic TNBC murine model. METHODS: GLV-1h164, a replication-competent VACV expressing the anti-VEGF antibody (derived from its parent virus GLV1h100), was tested against multiple TNBC cell lines. Viral infectivity, cytotoxicity, and replication were determined. Mammary fat pad tumors were generated in athymic nude mice using MDA-MB-231 cells. Xenografts were treated with GLV-1h164, GLV-1h100, or PBS and followed for tumor growth. RESULTS: Green fluorescent protein expression, a surrogate for viral infectivity, was time- and concentration-dependent. GLV-1h164 killed TNBC cell lines in a dose-dependent fashion with greater than 90% cytotoxicity within 4 days at a multiplicity of infection of 5 (plaqueforming units/cell). In vitro, cytotoxicity of GLV-1h164 was identical to the parent virus. GLV-1h164 replicated efficiently in all cell lines with an over 400-fold increase in viral copy numbers from the initial viral dose within 4 days. In vivo, mean tumor volumes after 3 weeks of treatment were 73, 191, and 422mm3 (GLV-1h164, GLV-1h100, and PBS, respectively)(p⬍0.05). CONCLUSIONS: This is the first study to demonstrate efficient combination of oncolytic and anti-angiogenic activity of a novel vaccinia virus on TNBC xenografts. Our results suggest that GLV1h164 is a promising therapeutic agent that warrants testing for patients with TNBC.

Hedgehog (HH) and ErbB signaling as mediators of tumor-stroma interactions in pancreatic ductal adenocarcinoma (PDAC) Brett L Broussard, MD, Alevtina Mikhaylina, MS, Martin J Heslin, MD, FACS, Juan P Arnoletti, MD, FACS,

Vol. 215, No. 3S, September 2012

Andrey Frolov, MD, PhD University of Alabama at Birmingham, Birmingham, AL INTRODUCTION: HH and ErbB signaling play important roles in PDAC tumor progression through mechanisms that are poorly understood. We have demonstrated the significance of Epidermal Growth Factor Receptor (EGFR) heterodimerization with ErbB3 as an escape mechanism to EGFR-targeted therapies. In an effort to identify additional valid therapeutic targets, we hypothesize that the HH pathway is involved in bi-directional interplay with ErbB signaling and promotes interaction between stromal and PDAC tumor cells. METHODS: Cancer-associated fibroblasts (CAF) were isolated from resected PDAC surgical specimens. AsPC-1, Panc-1 (ErbB3-deficient), Panc-1⫹ErbB3 (stable ErbB3 transfected cell line) and BxPC-3 PDAC cells were co-cultured with CAF. Cells were then treated with cyclopamine (HH pathway inhibitor) and PF-299 (a pan-ErbB inhibitor) either as single agent or in combination. RESULTS: The co-culture of PDAC cells and CAF induced secretion of Sonic HH in cell-conditioned-media. The presence of CAF translated into activation of the HH pathway as evidenced by GLI-1 transcript expression in PDAC cells evaluated by qRT-PCR. PanErbB inhibition with PF-299 resulted in increased GLI-1 expression by AsPC-1 and Panc-1⫹ErbB3 cells, suggesting reactive promotion of Sonic HH-mediated signaling. GLI-1 expression did not change upon treatment with PF-299 in ErbB3-deficient Panc-1 cells, further confirming a role for ErbB3 as mediator of stromal-epithelial interactions in PDAC. CONCLUSIONS: Stromal CAF promote ligand-dependent HH pathway activation by increasing Sonic HH secretion. Ongoing HH signaling may provide an escape mechanism to the anti-proliferative effects of ErbB inhibitors in PDAC cells. Combined targeting of ErbB receptors and HH signaling may be necessary for effective therapeutic approaches in PDAC.

JAK-2 downregulation sensitizes pancreatic cancer cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced cell death Vikas Dudeja, MD, Amanda R Oliveira, DVM, MS, Steven J Skube, MS, Sulagna Banerjee, PhD, Veena Sangwan, PhD, Rajinder K Dawra, PhD, Selwyn M Vickers, MD, FACS, Ashok K Saluja, PhD University of Minnesota, Minneapolis, MN INTRODUCTION: TRAIL (Tumor-Necrosis-Factor-Related-ApoptosisInducing-Ligand) is emerging as a promising anti-cancer therapy. Unfortunately, pancreatic cancer is resistant to TRAIL. Developing strategies to overcome this resistance will lead to emergence of TRAIL as an effective treatment for this challenging disease. JAK-2/ STAT-3 is a pro-survival pathway which is up-regulated in a variety of cancers. The aim of the current study was to evaluate role of JAK-2/ STAT-3 pathway in resistance to TRAIL mediated apoptosis and to evaluate the combination of TRAIL and JAK-2 downregulation as a novel treatment for pancreatic cancer.

Abstracts S135

METHODS: Highly aggressive metastatic pancreatic cancer cell lines (S2013, S2VP10) were treated with JAK-2 inhibitor FLLL31(0-5␮M), TRAIL(0-40ng/mL) or a combination of JAK-2 inhibition and TRAIL. The effect on viability and parameters of apoptosis (annexin-V, caspase-3, -8 and -9 activation) was measured. RESULTS: Even though TRAIL in itself was ineffective in inducing cell death in pancreatic cancer cells, combination of TRAIL and JAK2 inhibitor FLLL-31 induced markedly decreased viability and increased annexin positivity and increased caspase-3, -9 and -8 activation as compared to TRAIL and FLLL-31 alone, suggesting that TRAIL and JAK-2 inhibition synergize in inducing caspasedependent apoptosis in pancreatic cancer cells. CONCLUSIONS: Inhibition of JAK-2 pathway sensitizes pancreatic cancer cells to TRAIL induced apoptosis and cell death. Combination of JAK-2 silencing and TRAIL has immense potential to emerge as novel therapeutic strategy against pancreatic cancer. Furthermore, elucidation of the mechanism by which JAK-2 downregulation sensitizes pancreatic cancer cells to TRAIL induced cell death will lead to discovery of novel drug development targets.

Triptolide and sorafenib combination therapy in hepatocellular carcinoma results in superior cell death in vitro and prolonged survival in vivo Tara CK Krosch, MD, Veena Sangwan, PhD, Sulagna Banerjee, PhD, Ashok K Saluja, PhD, Selwyn M Vickers, MD, FACS, Eric H Jensen, MD, FACS University of Minnesota, Minneapolis, MN INTRODUCTION: Treatment of advanced hepatocellular carcinoma (HCC) is limited to sorafenib, with suboptimal outcomes. We investigated triptolide as a potential chemotherapeutic option, with and without sorafenib therapy. METHODS: HuH-7 HCC cells were treated in vitro with triptolide and/or sorafenib. Cell viability, caspase activation, and Annexin-V positivity were assessed. Real-time PCR was utilized for mRNA evaluation, and Western blots for protein expression. Sixteen nude mice were subcutaneously xenografted with HuH-7 tumors, with treatment groups (n⫽4) of 0.21 mg/kg/day IP Minnelide, 4 mg/kg/day OG sorafenib, combined therapies, or equivalent control. Mice were sacrificed when tumors reached 1 cm3, and survival compared. RESULTS: Triptolide treatment (100 nM) resulted in 40% cell death within 72 hours in vitro. Increased caspase-3 activation and Annexin-V positivity confirmed apoptosis. Triptolide resulted in downregulation of the heat shock protein (HSP) cascade, with decreased mRNA and protein levels of HSF-1, as well as downstream HSP70 and HSP27. Combination therapy, using low concentrations of each drug, resulted in further apoptotic cell death (80%). Preliminary studies of combination therapy in a mouse model showed increased survival (28.3 ⫹ 2.4 days) in comparison to Minnelide (19 ⫹ 8.8 days) or sorafenib (12.5 ⫹ 3.1 days) treatment alone. CONCLUSIONS: Triptolide treatment in vitro induces HuH-7 HCC cell death by apoptosis, with decreased HSP expression. In a preliminary mouse model, combination therapy with low doses of