MMP-9, uPAR and Cathepsin B Silencing Downregulate Integrins in Human Glioma Xenograft Cells In Vitro and In Vivo in Nude Mice Krishna Kumar Veeravalli1, Chandramu Chetty1, Shivani Ponnala1, Christopher S. Gondi1, Sajani S. Lakka1, Daniel Fassett2, Jeffrey D. Klopfenstein2, Dzung H. Dinh 2 , Meena Gujrati3, Jasti S. Rao1,2* 1 Department of Cancer Biology and Pharmacology, University of Illinois College of Medicine at Peoria, Peoria, Illinois, United States of America, 2 Department Neurosurgery, University of Illinois College of Medicine at Peoria, Peoria, Illinois, United States of America, 3 Department Pathology, University of Illinois College of Medicine at Peoria, Peoria, Illinois, United States of America Abstract Background: Involvement of MMP-9, uPAR and cathepsin B in adhesion, migration, invasion, proliferation, metastasis and tumor growth has been well established. In the present study, MMP-9, uPAR and cathepsin B genes were downregulated in glioma xenograft cells using shRNA plasmid constructs and we evaluated the involvement of integrins and changes in their adhesion, migration and invasive potential. Methodology/Principal Findings: MMP-9, uPAR and cathepsin B single shRNA plasmid constructs were used to downregulate these molecules in xenograft cells. We also used MMP-9/uPAR and MMP-9/cathepsin B bicistronic constructs to evaluate the cumulative effects. MMP-9, uPAR and cathepsin B downregulation significantly inhibits xenograft cell adhesion to several extracellular matrix proteins. Treatment with MMP-9, uPAR and cathepsin B shRNA of xenografts led to the downregulation of several alpha and beta integrins. In all the assays, we noticed more prominent effects with the bicistronic plasmid constructs when compared to the single plasmid shRNA constructs. FACS analysis demonstrated the expression of aVb3, a6b1 and a9b1 integrins in xenograft cells. Treatment with bicistronic constructs reduced aVb3, a6b1 and a9b1 integrin expressions in xenograft injected nude mice. Migration and invasion were also inhibited by MMP-9, uPAR and cathepsin B shRNA treatments as assessed by spheroid migration, wound healing, and Matrigel invasion assays. As expected, bicistronic constructs further inhibited the adhesion, migration and invasive potential of the xenograft cells as compared to individual treatments. Conclusions/Significance: Downregulation of MMP-9, uPAR and cathespin B alone and in combination inhibits adhesion, migration and invasive potential of glioma xenografts by downregulating integrins and associated signaling molecules. Considering the existence of integrin inhibitor-resistant cancer cells, our study provides a novel and effective approach to inhibiting integrins by downregulating MMP-9, uPAR and cathepsin B in the treatment of glioma. Citation: Veeravalli KK, Chetty C, Ponnala S, Gondi CS, Lakka SS, et al. (2010) MMP-9, uPAR and Cathepsin B Silencing Downregulate Integrins in Human Glioma Xenograft Cells In Vitro and In Vivo in Nude Mice. PLoS ONE 5(7): e11583. doi:10.1371/journal.pone.0011583 Editor: Nils Cordes, Dresden University of Technology, Germany Received February 22, 2010; Accepted June 22, 2010; Published July 15, 2010 Copyright: 2010 Veeravalli et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was supported by a grant from the National Institute of Neurological Disorders and Stroke (NINDS NS047699). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: jsrao@uic.edu Introduction Integrins are a family of adhesion molecules involved in interactions between the cell and the surrounding extracellular matrix (ECM). Although the classic role of integrins is to anchor cells to the ECM, they are known to participate in a variety of signaling pathways and to play important roles in fetal development, morphogenesis, cell migration, wound healing, and malignant transformation [1–6]. The heterodimerization of 19a- integrin and 8b-integrin subunits is thought to yield 25 integrin ab heterodimers, which form receptors for an overlapping group of ECM molecules in almost every cell type [7]. Through interactions with the ECM, integrins control many cellular processes that occur in the progression of diseases such as cancer. Integrin signaling regulates diverse functions in tumor cells, including migration, invasion, proliferation and survival. In several tumor types, the expression of particular integrins correlates with increased disease progression and decreased patient survival [8]. Glioblastoma multiforme (GBM) is a highly malignant neo- plasm of central nervous system. Strategies to treat infiltrating gliomas, such as chemotherapy and gene therapy, have remained largely unsuccessful and the property that makes glioma resistant to treatment is the tendency of the tumor cells to invade normal brain tissue [9]. Invasiveness is thus considered to be a major determinant of the malignant behavior of human gliomas. Integrins, the family of adhesive receptors promote the invasive- ness of glioma. The role of integrins in cell migration and invasion is one of their most studied functions in tumor biology [10,11]. PLoS ONE | www.plosone.org 1 July 2010 | Volume 5 | Issue 7 | e11583