Abstract 511: Integrin-mediated inhibition of adhesion, migration and invasion in glioma xenografts by MMP-9, uPAR and cathepsin B gene silencing
Authors: Veeravalli Krishna Kumar , Vanamala Sravan K. , Chetty Chandramu , Fassett Daniel , Klopfenstein Jeffrey D. , Dinh Dzung H. , Gujrati Meena , Rao Jasti S. 
Published: February 2011

Involvement of MMP-9, cathepsin B and uPAR in glioma adhesion, migration, invasion, proliferation, metastasis and tumor growth has been well studied and reported. In the present study, we have downregulated MMP-9, uPAR and cathepsin B genes by using siRNA plasmid constructs in glioma xenografts (4910 and 5310) to study their effects on adhesion, migration and invasion. We hypothesized that downregulation of these genes in combination will have additive/synergistic effect in inhibiting adhesion, migration and invasion of glioma xenografts and hence, treated these xenografts with MMP-9-uPAR and MMP-9-cathepsin B bicistronic plasmid constructs. MMP-9 (pM), uPAR (pU) and cathepsin B (pC) downregulation in glioma xenografts significantly inhibited their adhesion to several extracellular matrix proteins, such as collagen I (pM-39%, pU-51%, pC-57% inhibition in 4910 cells and pM-40%, pU-44%, pC-59% inhibition in 5310 cells), vitronectin (pM-51%, pU-45%, pC-57% inhibition in 4910 cells and pM-56%, pU-58%, pC-58% inhibition in 5310 cells), fibronectin (pM-36%, pU-48%, pC-48% inhibition in 4910 cells and pM-30%, pU-37%, pC-41% inhibition in 5310 cells), and laminin (pM-50%, pU-34%, pC-45% inhibition in 4910 cells and pM-48%, pU-52%, pC-65% inhibition in 5310 cells). Migration and invasion were also inhibited by MMP-9, uPAR and cathepsin B siRNA treatments as assessed by spheroid migration, migration through 8 μm transwell inserts, wound healing and matrigel invasion assays. As expected, downregulation of MMP-9 in combination either with uPAR (pMU) and cathepsin B (pMC) further inhibited the adhesion, migration and invasion of 4910 and 5310 glioma xenografts as compared to individual treatments. Furthermore, MMP-9, uPAR and cathepsin B siRNA treatments of these xenografts led to the downregulation of several alpha and beta integrins (such as α6, α9, αV, β1 and β3) as evidenced by reverse transcriptase PCR analysis. Immunofluorescence analysis revealed the reduced expression of these integrins in the glioma xenografts when treated with MMP-9, uPAR and cathepsin B siRNA either alone or in combination. Based on these findings, we conclude that downregulation of MMP-9, uPAR and cathespin B using siRNA plasmid constructs inhibits adhesion, migration and invasion of human glioma xenografts via the downregulation of integrins. Considering the recent clinical failures with integrin inhibitors and the existence of integrin inhibitor resistant cancer cells, the present study provides a novel and effective approach for cancer treatment by inhibiting the integrins while downregulating MMP-9, cathepsin B and uPAR.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 511.