Extracellular superoxide dismutase inhibits hepatocyte growth factor-mediated breast cancer-fibroblast interactions

// Briana Ormsbee Golden 1, * , Brandon Griess 1, * , Shakeel Mir 1 , Matthew Fitzgerald 2 , Charlotte Kuperwasser 3 , Frederick Domann 4 and Melissa Teoh-Fitzgerald 1 1 Department of Biochemistry and Molecular Biology, Fred and Pamela Buffett Cancer Center, College of Medicine, University of Nebraska Medical Center, Omaha, NE 68198, USA 2 Department of Surgery-General Surgery, University of Nebraska Medical Center, Omaha, NE 68198, USA 3 Developmental, Molecular and Chemical Biology, Tufts University School of Medicine, Boston, MA 02111, USA 4 Free Radical and Radiation Biology Program, Radiation Oncology, University of Iowa, Iowa City, IA 52241, USA * These authors contributed equally to this work Correspondence to: Melissa Teoh-Fitzgerald, email: m.teohfitzgerald@unmc.edu Keywords: tumor-stroma interactions; EcSOD; breast cancer; c-Met; cancer-associated fibroblasts Received: June 01, 2017      Accepted: October 25, 2017      Published: November 10, 2017 ABSTRACT We have previously shown tumor suppressive effects of extracellular superoxide dismutase, EcSOD in breast cancer cells. In this study, an RTK signaling array revealed an inhibitory effect of EcSOD on c-Met phosphorylation and its downstream kinase c-Abl in MDA-MB231 cells. Moreover, an extracellular protein array showed that thrombospondin 1 (TSP-1), a scavenger of the c-Met ligand, hepatocyte growth factor (HGF) is significantly up-regulated in EcSOD overexpressing cells (Ec.20). We further determined the effects of EcSOD on HGF/c-Met-mediated cancer-fibroblast interactions by co-culturing normal fibroblasts (RMF) or RMF which overexpresses HGF (RMF-HGF) with MDA-MB231 cells. We observed that while RMF-HGF significantly promoted Matrigel growth of MDA-MB231, overexpression of EcSOD inhibited the HGF-stimulated growth. Similarly, a SOD mimetic, MnTE-2-PyP, inhibited HGF-induced growth and invasion of MDA-MB231. In addition, a long-term heterotypic co-culture study not only showed that Ec.20 cells are resistant to RMF-HGF-induced invasive stimulation but RMF-HGF that were co-cultured with Ec.20 cells showed an attenuated phenotype, suggesting an oxidative-mediated reciprocal interaction between the two cell types. In addition, we demonstrated that RMF-HGF showed an up-regulation of an ROS-generating enzyme, NADPH oxidase 4 (Nox4). Targeting this pro-oxidant significantly suppressed the activated phenotype of RMF-HGF in a collagen contraction assay, suggesting that RMF-HGF contributes to the oxidative tumor microenvironment. We have further shown that scavenging ROS with EcSOD significantly inhibited RMF-HGF-stimulated orthotopic tumor growth of MDA-MB231. This study suggests the loss of EcSOD in breast cancer plays a pivotal role in promoting the HGF/c-Met-mediated cancer-fibroblast interactions.