Inhibitor of Nuclear Factor κB Activation Enhances the Antitumor Effect of Radiation Therapy for Pancreatic Cancer

Vol. 219, No. 3S, September 2014

Surgical Forum Abstracts

Table 1. The anti-SIAH molecule (SIAHPD 2 ) is effective in inhibiting established human pancreatic (MiaPaCa) and human triple negative breast (MDA-MB-231) tumor growth in athymic nude mice Untreated MiaPaCa tumor volume (mm3  SEM)

Treated MiaPaCa tumor volume (mm3  SEM)

95  31

110.5  27

59

384  107

58  20

66

770  215

54  22

1445  366

55  21

Day after cancer cell injection 52 e induction of SIAHPD 2 expression for MiaPaCa

72

Untreated MDA-MB-231 Treated MDA-MB-231 tumor volume tumor volume (mm3  SEM) (mm3  SEM) 88 e induction of SIAHPD 2 expression for MDA-MB-231

114  24

106  21

92

168  32

62  13

98

299  56

46  13

101

425  72

43  14

cancer lines were established. One million MiaPaCa-SIAH2-PD or MDA-MB-SIAH2-PD cells were injected subcutaneously bilaterally in the dorsal scapular areas or mammary fat pads, respectively, in 4-week-old female athymic nude mice. Once the implanted tumors reached certain volumes, SIAH2-PD expression was induced via doxycycline treatment. Western blot, immunohistochemistry and immunofluorescence were used to determine endogenous SIAH and exogenous SIAH2-PD expression in treated and untreated tumors. RESULTS: Expression of SIAH2-PD blocks RAS-mediated tumor growth in established pancreatic and breast tumor xenografts in mice (table). Immunohistochemistry and immunofluorescence results show a marked reduction in endogenous SIAH and upregulated expression of SIAH2-PD in doxycycline-treated tumors compared to untreated tumors. CONCLUSIONS: We have identified a novel way to block well-established and oncogenic K-RAS-driven pancreatic and breast tumors in a xenograft model. We hope to develop small molecule inhibitors against SIAH and translate these findings to the cancer clinics. LL-37 Enhances Homing and Long-Term Engraftment after In Utero Hematopoietic Cell Transplantation (IUHCT) Matthew M Boelig, MD, Aimee G Kim, MD, Michael A Conner, BS, Stavros P Loukogeorgakis, MD, PhD, Alan W Flake, MD, FACS, William H Peranteau, MD Children’s Hospital of Philadelphia, Philadelphia PA INTRODUCTION: IUHCT can induce donor-specific tolerance (DST), allowing for non-myeloablative postnatal cellular and organ transplantation. Manipulation of donor cells to enhance trafficking to the fetal hematopoietic niche is a promising strategy to increase

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engraftment and frequency of DST. A potential target is the SDF1a/CXCR4 pathway, which plays a critical role in the trafficking of hematopoietic stem/progenitor cells (HSPCs) to hematopoietic niches. LL-37, an endogenous antimicrobial peptide, enhances HSPC migration toward an SDF-1a gradient. We hypothesized that LL-37 would augment homing and long-term engraftment of allogeneic donor cells after IUHCT. METHODS: 10 million BM mononuclear cells from B6-GFP transgenic mice were injected intravenously into embryonic day 14 Balb/c fetuses with or without pre-treatment with 2.5 mg/mL LL-37. Fetal liver (FL) homing and early engraftment were observed with fluorescence stereoscopic microscopy and quantified by flow cytometry. Long-term hematopoietic chimerism was quantified monthly in peripheral blood (PB). Statistical analysis was performed using 2-way ANOVA with Bonferroni correction. RESULTS: Donor cells pre-treated with LL-37 demonstrated a significant homing advantage to the FL up to 72 hours postIUHCT (p< 0.05). Long-term hematopoietic chimerism in PB after IUHCT was significantly increased in LL-37-treated animals (n¼15) vs untreated animals (n¼13) at all postnatal time-points. No significant toxicity or growth impairment was observed with LL-37 treatment (Table).

Age, mo

1 2 3

LL-37 treated (% PB chimerism  SEM)

Untreated (% PB chimerism  SEM)

p Value

29.98  6.56 21.32  5.05 16.47  3.88

8.00  1.74 3.89  0.94 2.71  0.72

< 0.001 < 0.01 < 0.05

CONCLUSIONS: We observed a significant treatment effect with LL-37 with regards to homing and long-term engraftment in our allogeneic murine model of IUHCT. LL-37 may facilitate the establishment of DST, thereby increasing the clinical applicability of IUHCT to enable non-myeloablative postnatal cellular or organ transplantation. Inhibitor of Nuclear Factor kB Activation Enhances the Antitumor Effect of Radiation Therapy for Pancreatic Cancer Yoshihiro Shirai, MD, Hiroaki Shiba, MD, PhD, Ryota Iwase, MD, Koichiro Haruki, MD, PhD, Yuki Fujiwara, MD, PhD, Kenei Furukawa, MD, PhD, Tadashi Uwagawa, MD, PhD, Takeyuki Misawa, MD, PhD, FACS, Toya Ohashi, MD, PhD, Katsuhiko Yanaga, MD, PhD, FACS The Jikei University School of Medicine, Tokyo, Japan INTRODUCTION: Radiation therapy, alone or in combination with chemotherapy, is effective for patients with locally advanced and recurrent pancreatic cancer. However, the therapeutic efficacy is attenuated by radiation-induced activation of nuclear factorkappa B (NF-kB). We previously reported that nafamostat mesilate (NM), a synthetic serine protease inhibitor, inhibited NF-kB

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Surgical Forum Abstracts

activation and induced antitumor effects in patients with pancreatic cancer. Therefore, we hypothesized that NM downregulates the radiation-induced activation of NF-kB and improves therapeutic outcome of pancreatic cancer. METHODS: In vitro, we assessed NF-kB activity, induction of caspase cascade, and cell proliferation using human pancreatic cancer cell lines (PANC-1, MIA PaCa-2) treated with radiation (6Gy) alone, NM alone, and combination of radiation and NM. In vivo, we established xenograft pancreatic cancer in mice by subcutaneous injection of MIA PaCa-2 cells. At 5 weeks after injection, the tumor was irradiated (6Gy) once (radiation group), with i.p. injection of NM 3 times a week (NM group), or with combination of radiation once and NM 3 times a week (combination group). RESULTS: In combination group, NF-kB activation was significantly inhibited in comparison with that of radiation group both in vitro and in vivo (p<0.05). Cleaved caspase-3, 8, and 9 levels were the highest in the combination group both in vitro and in vivo. In combination group, cell proliferation and tumor growth were significantly slower than those in other groups (p<0.05) both in vitro and in vivo. CONCLUSIONS: Combination therapy of radiation with NM exertrs enhanced anti-tumor effect against human pancreatic cancer. Combination of Small RNAs Enhances Muscle Development John R Jackson, PhD, NaJung Kim, PhD, James Yoo, MD, PhD, Sang Jin Lee, Anthony Atala, MD, FACS Wake Forest School of Medicine, Winston-Salem, NC INTRODUCTION: Selectively controlling the expression of the target genes through small RNAs has significant potential for treatment of numerous injuries and diseases. We introduced combinations of multiple small RNAs to direct the production of various cellular factors, to promote in situ muscle tissue regeneration in a targeted site. We expect this novel combination of small RNAs would enhance muscle recovery and regeneration. METHODS: To evaluate the myogenic potentials of small RNAs in vitro, we used three different small RNAs, siGDF-8, miR-1, and miR-206. Murine myoblasts were transfected with individual or combinations of the RNAs, and incubated in differentiation media until used for gene expression, proliferation, and differentiation analyses. Chemically injured Lewis rat tibialis anterior (TA) muscles were treated with RNAs and analyzed for functional and structural recovery. RESULTS: Adding siGDF-8 to these miRNAs significantly increased gene expressions of all the myogenic regulatory factors (MRFs) tested. This combination of small RNAs also enhanced myosin protein expression by miR-1 and miR-206. It also resulted in acceleration of myoblast proliferation by the action of siGDF-8. Structural and

J Am Coll Surg

functional recovery of hind leg was significantly accelerated by this combination delivery of siGDF-8, miR-1, and miR-206. CONCLUSIONS: Combinations of small RNAs enhanced myogenic activation by overexpressing MRFs such as MyoD, myogenin, Pax7, and MyHC1. The combination delivery also accelerated in vivo regenerative efficiency compared with the effects of any single- or two-factor mixture. This novel combination of siGDF-8, and miR-1 and miR-206 is expected to have a great therapeutic potential to fine-tune skeletal muscle recovery from traumatic injury. Multiple Injections of Oncolytic Adenoviruses Suppressed the Growth of Human Papilloma Virus (HPV)-Positive Head and Neck Squamous Cell Carcinomas in an In Vivo Model Christopher J LaRocca, MD, Amanda R Oliveira, DVM, MS, Julia Davydova, MD, PhD, Mark Herzberg, DDS, PhD, Rajaram Gopalakrishnan, BDS, PhD, Masato Yamamoto, MD, PhD University of Minnesota, Minneapolis, MN INTRODUCTION: The incidence of HPV-positive head and neck squamous cell carcinomas (HNSCC) has been steadily increasing. Oncolytic viruses are an emerging class of anti-cancer agents with great therapeutic potential. Specifically, conditionally replicative oncolytic adenoviruses (CRAds) targeted to the HPV E6 and E7 oncogenes have the ability to become a novel treatment modality for this disease. METHODS: Two CRAds (5/3 D24 and 5/3 CB016) were designed with an Ad5/Ad3 chimeric fiber to maximize infectivity and overexpression of the adenovirus death protein to increase oncolysis. Each CRAd possesses a distinct deletion (D24 or CB016) in the E1a region of the adenoviral genome which allows for selective replication in HPV-positive HNSCC cells. The viral vectors were generated and thoroughly tested in an in vitro setting. Thereafter, subcutaneous tumors were generated in the flanks of athymic nude mice using a HPV-positive HNSCC cell line (UPCI SCC 090). The tumors were then injected with one of the adenoviruses or a saline control. Three subsequent injections were performed at four-day intervals. RESULTS: Both groups (5/3 D24 and 5/3 CB016) treated with multiple injections of the oncolytic adenoviruses (3.5x1011 vp/injection) demonstrated significant reductions in tumor volume compared to the saline control group (p¼0.000001 for 5/3 D24, p¼0.0003 for 5/3 CB016) by day 23 after the initial viral inoculation. Notably, the 5/3 D24 virus’ oncolytic effect was strong enough to cause tumor regression. CONCLUSIONS: Multiple injections of oncolytic adenoviruses designed to target HPV-positive HNSCC tumors demonstrated significant anti-tumor effect in vivo and show excellent potential for clinical translation.