MMP-9, uPAR and cathepsin B silencing downregulate integrins in human glioma xenograft cells in vitro and in vivo in nude mice.
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.


RESULTS
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 alphaVbeta3, alpha6beta1 and alpha9beta1 integrins in xenograft cells. Treatment with bicistronic constructs reduced alphaVbeta3, alpha6beta1 and alpha9beta1 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
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.
Veeravalli
Krishna Kumar
KK
Department of Cancer Biology and Pharmacology, University of Illinois College of Medicine at Peoria, Peoria, Illinois, United States of America.
Chetty
Chandramu
C
Ponnala
Shivani
S
Gondi
Christopher S
CS
Lakka
Sajani S
SS
Fassett
Daniel
D
Klopfenstein
Jeffrey D
JD
Dinh
Dzung H
DH
Gujrati
Meena
M
Rao
Jasti S
JS
D000818:Animals; D015153:Blotting, Western; D002401:Cathepsin B / Q000235:genetics / Q000378:metabolism*; D002448:Cell Adhesion / Q000235:genetics / Q000502:physiology; D002465:Cell Movement / Q000235:genetics / Q000502:physiology; D015536:Down-Regulation / Q000235:genetics / Q000502:physiology; D005434:Flow Cytometry; D005910:Glioma / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D039001:Integrin alpha Chains / Q000235:genetics / Q000378:metabolism; D039121:Integrin alpha6beta1 / Q000235:genetics / Q000378:metabolism; D039302:Integrin alphaVbeta3 / Q000235:genetics / Q000378:metabolism; D016023:Integrins / Q000235:genetics / Q000378:metabolism*; D020780:Matrix Metalloproteinase 9 / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008819:Mice, Nude; D034622:RNA Interference; D055293:Receptors, Urokinase Plasminogen Activator / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D018874:Spheroids, Cellular / Q000378:metabolism; D014945:Wound Healing / Q000235:genetics / Q000502:physiology
D039001:Integrin alpha Chains; D039121:Integrin alpha6beta1; D039302:Integrin alphaVbeta3; D016023:Integrins; D055293:Receptors, Urokinase Plasminogen Activator; C084613:integrin alpha9; D002401:Cathepsin B; D020780:Matrix Metalloproteinase 9