MMP-9, uPAR and Cathepsin B Silencing Downregulate Integrins in Human Glioma Xenograft Cells In Vitro and In Vivo in Nude Mice

Abstract

BackgroundInvolvement 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 FindingsMMP-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 αVβ3, α6β1 and α9β1 integrins in xenograft cells. Treatment with bicistronic constructs reduced αVβ3, α6β1 and α9β1 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/SignificanceDownregulation 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.

Human matrix metalloproteinases: An ubiquitarian class of enzymes involved in several pathological processes

“…MMP-9 in turn can readily activate integrin promoting cell motility. Invadopodia of tumor cells show strong MMP-9 gelatinolytic activity and down-regulation of MMP-9 expression results in decreased invasion and decreased integrin expression on the cell surface (Lagarrigue et al, 2010;Veeravalli et al, 2010).…”

Hyaluronan Regulation of Endothelial Barrier Function in Cancer

“…For intravasation, the tumor cell must invade through the tumor stroma (Chiang & Massague, 2008; Joyce & Pollard, 2009). HA, such as that found in the HA-rich stroma of tumors, can induce expression of several ECM degrading enzymes on tumor cells including the matrix metalloproteinases MT1-MMP, MMP-2, MMP-7, and MMP-9 as well as EMMPRIN and the cysteine proteases cathepsin D and cathepsin K (Abecassis, Olofsson, Schmid, Zalcman, & Karniguian, 2003; Bourguignon et al, 2004; Chetty et al, 2012; Droller, 2003; Kim et al, 2008; Kosunen et al, 2007; Marrero-Diaz et al, 2009; Mitchell & King, 2014; Murray, Morrin, & McDonnell, 2004; Nalla, Gorantla, Gondi, Lakka, & Rao, 2010; Veeravalli et al, 2010). Once at the blood vessel, the tumor cell can secrete a host of factors to induce disruption of EC junctions and EC retraction including MMP1, ADAM12, and TNF1α to allow for paracellular transendothelial migration (Mierke, 2011).…”

Regulation of DNA Repair Mechanism in Human Glioma Xenograft Cells both In Vitro and In Vivo in Nude Mice

“…Further, Hematoxylin and Eosin staining performed on mouse brains revealed a prominent reduction of tumor growth in pMU- and pMC-treated mouse brains when compared to the controls [22], [26]. We next pursued to analyze the expression of DDR related proteins in control, pMU and pMC-treated brain sections.…”

“…Furthermore, uPAR interaction with uPA has been shown to activate a downstream signal cascade involving plasminogen and MMP, which leads to ECM degradation, the release of growth factors, and ultimately tumor growth [20], [21]. Our earlier studies clearly demonstrated the efficacy of simultaneous downregulation of these bicistronic plasmid shRNA constructs in treating tumors by reducing adhesion, migration and invasion potential and inducing apoptosis in cancer cells [22]–[26]. However, none of our earlier studies have addressed the effect of these bicistronic constructs on DNA damage response (DDR) and the related molecular mechanisms involved.…”