Brianne R. O'Leary

Loss of SOD3 (EcSOD) Expression Promotes an Aggressive Phenotype in Human Pancreatic Ductal Adenocarcinoma

Purpose: Pancreatic ductal adenocarcinoma (PDA) cells are known to produce excessive amounts of reactive oxygen species (ROS), particularly superoxide, which may contribute to the aggressive and refractory nature of this disease. Extracellular superoxide dismutase (EcSOD) is an antioxidant enzyme that catalyzes the dismutation of superoxide in the extracellular environment. This study tests the hypothesis that EcSOD modulates PDA growth and invasion by modifying the redox balance in PDA. Experimental Design: We evaluated the prognostic significance of EcSOD in a human tissue microarray (TMA) of patients with PDA. EcSOD overexpression was performed in PDA cell lines and animal models of disease. The impact of EcSOD on PDA cell lines was evaluated with Matrigel invasion in combination with a superoxide-specific SOD mimic and a nitric oxide synthase (NOS) inhibitor to determine the mechanism of action of EcSOD in PDA. Results: Loss of EcSOD expression is a common event in PDA, which correlated with worse disease biology. Overexpression of EcSOD in PDA cell lines resulted in decreased invasiveness that appeared to be related to reactions of superoxide with nitric oxide. Pancreatic cancer xenografts overexpressing EcSOD also demonstrated slower growth and peritoneal metastasis. Overexpression of EcSOD or treatment with a superoxide-specific SOD mimic caused significant decreases in PDA cell invasive capacity. Conclusions: These results support the hypothesis that loss of EcSOD leads to increased reactions of superoxide with nitric oxide, which contributes to the invasive phenotype. These results allow for the speculation that superoxide dismutase mimetics might inhibit PDA progression in human clinical disease. Clin Cancer Res; 21(7); 1741–51. ©2015 AACR.

Rational design of a secreted enzymatically inactive mutant of extracellular superoxide dismutase

Abstract Extracellular superoxide dismutase (SOD3) is a secreted enzyme that regulates levels of extracellular superoxide and protects the extracellular matrix from degradation by reactive species. The SOD3 protein contains a heparin-binding domain and resides in a microenvironment rich in other heparin-bound growth factors, raising the possibility that SOD3 may have some biological role independent of its catalytic activity. To begin to address this, we designed and created enzymatically inactive mutant constructs targeting either the copper coordinating (i.e. H96 and H98) or superoxide channeling (i.e. N180 and R186) amino acid residues of SOD3. All constructs expressed equal quantities of immature intracellular SOD proteins, but only the N180A, R186A, and combination N180A/R186A mutants produced fully processed and secreted extracellular protein. Furthermore, while SOD activity was significantly inhibited in the single N180A and R186A mutants, the activity was completely abrogated in the N180A/R186A double mutant. Overall, the use of this novel tool may have broad reaching impacts into various fields of biology and medicine, and will aid in the delineation of cellular processes that are regulated by solely the SOD3 protein, its reactive oxygen species substrates and products, or the combination of both.

Pharmacological Ascorbate as an Adjuvant for Enhancing Radiation-Chemotherapy Responses in Gastric Adenocarcinoma

Gastric adenocarcinoma most often presents at an advanced stage and overall five-year survival of ∼30%. Pharmacological ascorbate (high-dose IV ascorbate) has been proposed as a promising nontoxic adjuvant to standard radio-chemotherapies in several cancer types. In the current study, pharmacological ascorbate (0.5–2 mM) caused a dose-dependent decrease (70–85% at 2 mM) in clonogenic survival of gastric adenocarcinoma cells (AGS and MNK-45), but was relatively nontoxic to a small intestinal epithelial nonimmortalized human cell isolate (FHs 74 Int). The addition of pharmacological ascorbate (1 mM) to standard radio-chemotherapies [i.e., 5-FU (5 μM); cisplatin (0.5 μM); irinotecan (2.5 μM); carboplatin (5 μM); paclitaxel (2–4 nM); and X rays (1.8 Gy)] also potentiated gastric cancer clonogenic cell killing [additional decreases were noted with: ascorbate plus 5-FU/radiation (1%); ascorbate plus cisplatin/irinotecan (9–19%); and ascorbate plus paclitaxel/carboplatin (6–7%)]. The gastric cancer cell toxicity and chemosensitization seen with pharmacological ascorbate was dependent on H2O2 and the presence of catalytic metal ions. In addition, pharmacological ascorbate dosing resulted in a concentration-dependent decrease (64% at 20 mM, P ≤ 0.0001) in cancer cell invasion and migration that was inhibited by catalase. Finally, pharmacological ascorbate significantly increased the overall survival of mice with gastric cancer xenografts when used in combination with paclitaxel, carboplatin and radiation (P = 0.019). These results demonstrate that pharmacological ascorbate is selectively cytotoxic to gastric adenocarcinoma cells (relative to normal intestinal epithelial cells) by a mechanism involving H2O2 and redox active metal ions. Furthermore, pharmacological ascorbate significantly enhances gastric cancer xenograft responses to radio-chemotherapy as well as inhibiting tumor cell migration and invasiveness. Overall, these results support the hypothesis that pharmacological ascorbate can be used as an adjuvant with standard-of-care radio-chemotherapies for the treatment of gastric adenocarcinomas.

A model for the detection of pancreatic ductal adenocarcinoma circulating tumor cells

Metastatic disease is the leading cause of pancreatic ductal adenocarcinoma (PDAC) associated death. PDAC cells invade and enter the bloodstream early, before frank malignancy can be detected. Our objective was to develop an in vivo assay enabling the identification and quantification of circulating tumor cells (CTCs) from primary orthotopic PDAC tumors. Human PDAC cells expressing luciferase and green fluorescent protein were orthotopically injected into the pancreas of mice utilizing ultrasound guidance. Bioluminescent imaging was conducted to identify and track tumor growth. CTCs were then isolated and analyzed by flow cytometry to detect GFP-expressing cancer cells. Tumor growth as measured by bioluminescent imaging increased over time. The concentration of CTCs correlated with the strength of bioluminescent imaging signal. In addition, livers bearing macroscopic disease were harvested for further imaging under fluorescence stereomicroscopy and confocal microscopy, which confirmed the presence of metastases. This study represents an orthotopic animal model that reliably detects the presence of CTCs from PDAC. There is a positive correlation between the concentrations of CTCs with overall tumor burden.

Abstract B52: The role of extracellular superoxide dismutase activity in pancreatic cancer biology and therapy

Introduction: Pancreatic ductal adenocarcinoma (PDA) cells are known to produce excessive amounts of reactive oxygen species (ROS), particularly superoxide (O2•-), which may contribute to the aggressive nature of this disease. Extracellular superoxide dismutase (EcSOD) is an antioxidant enzyme that catalyzes the dismutation of O2•- to hydrogen peroxide (H2O2) in the extracellular environment. By limiting O2•- concentration and producing H2O2, EcSOD is thought to regulate the redox state of the tumor microenvironment that could contribute to pancreatic cancer progression. The current work tests the hypothesis that EcSOD modulates PDA growth and invasion by modifying the balance between H2O2 and ONOO- formation. Methods: EcSOD and 3-nitrotyrosine (3-NT), a marker of protein nitration by ONOO-, were evaluated in specimens from patients with PDA. EcSOD expression was correlated with clinicopathologic and treatment-related variables and survival in a tissue microarray (TMA) of 114 patients with Stage I-IV PDA. EcSOD was constitutively overexpressed in two primary PDA cell lines and clonogenic survival and doubling time were measured. A Matrigel invasion assay was used to determine the impact of EcSOD on PDA tumor cell invasive capacity. We also utilized and GC4419, a selective SOD mimic currently in clinical development. GC4419 dismutates O2•- but does not react with H2O2 under physiologic conditions. PDA cells stably expressing EcSOD and luciferase were used to create hind leg xenografts in athymic nude mice to assess the impact of EcSOD on tumor growth and then via peritoneal injection to determine the impact on peritoneal metastasis formation using bioluminescence imaging (BLI). Results: EcSOD expression was reduced in PDA compared to normal pancreas in 11/16 specimens evaluated (69%). A decrease in expression was also seen in areas of premalignant ductal epithelium (PanIN-3) relative to adjacent pancreas. In normal pancreas tissue, there was no evidence of immunoreactive 3-NT; however, 11/16 PDA specimens (69%) had robust levels of immunoreactive 3-NT. In the TMA, EcSOD expression was absent in 41/114 of the PDA biopsies (36%). The median survival of patients with intact EcSOD expression was 11.0 months vs. 6.5 months for patients with loss of EcSOD expression. Compared to patients with intact EcSOD, patients with loss of EcSOD more often had advanced stage disease and were less likely to be treated with surgery. In an adjusted proportional hazard model of more than 10 clinical variables, the HR for death with loss of EcSOD was 1.63 (95% CI=1.02-2.58, p=0.04). EcSOD overexpression significantly reduced clonogenic survival, doubling time, and tumor cell invasion relative to controls. GC4419 also significantly reduced PDA invasiveness in the Matrigel assay. PDA cells stably expressing EcSOD showed decreased growth and doubling time in hind limb xenografts and significantly less peritoneal metastasis using BLI. Conclusions: Loss of EcSOD expression and oxidative stress as indicated by the presence of 3-NT is a common finding in PDA. Loss of EcSOD expression correlates with negative prognostic factors and shorter survival in patients with PDA. Overexpression of EcSOD resulted in reduced PDA growth, invasion, and peritoneal metastasis. The selective SOD mimic GC4419 also reduced invasion of PDA tumor cells. Together these findings support the hypothesis that modulating O2•- in the PDA microenvironment alters parameters of progression, oxidative damage, and may represent a target for limiting the spread of PDA. (Supported by The American Surgical Association Foundation Fellowship (JJM) and the American Cancer Society (JJM). Citation Format: James J. Mezhir, Brianne R. O9Leary, Andrew M. Bellizzi, Sean Altekruse, Charles F. Lynch, Brenda Y. Hernandez, Wendy Cozen, Michael D. Henry, Jeffrey Keene, Robert A. Beardsley, Douglas R. Spitz, Frederick E. Domann. The role of extracellular superoxide dismutase activity in pancreatic cancer biology and therapy. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B52.