title: MMP-9 silencing regulates hTERT expression via β1 integrin-mediated FAK signaling and induces senescence in glioma xenograft cells.
abstract: In more than 90% of cancers including glioma, telomere elongation reverse transcriptase (hTERT) is overexpressed. In the present study, we sought to explore whether matrix metalloproteinase-9 (MMP-9) shRNA could alter hTERT-mediated proliferation in glioma cells. MMP-9 shRNA induced senescence and apoptosis in glioma cells by inhibiting hTERT expression and telomere activity. MMP-9 silencing decreased oncogenic c-Myc expression (hTERT activator), whereas the expression of the c-Myc antagonist MAD increased drastically (hTERT repressor); both c-Myc and MAD are transcription factors for hTERT. In addition, MMP-9 suppression turns the switch from c-Myc/MAX to MAD/MAX heterodimer binding to the hTERT promoter as determined by chromatin immunoprecipitation assay. We also show that silencing MAD via siRNA restored hTERT expression and inhibited senescence in glioma cells. MMP-9 transcriptional suppression decreased the expression of FAK, phospho FAK and β1 integrin in glioma xenograft cells. Further, MMP-9 suppression decreased the interaction of β1 integrin/FAK and also MMP-9/β1 integrin as confirmed by immunoprecipitation analysis. Studies with either function blocking β1 integrin or FAK shRNA indicate that suppression of MMP-9 decreased β1 integrin-mediated induction of FAK, which led to decreased hTERT expression. Moreover, 4910 and 5310 glioma xenograft tissue sections from mice treated with MMP-9 shRNA showed reduced expression of FAK/c-Myc and elevated MAD levels. Decreased co-localization of β1 integrin and MMP-9 was associated with MMP-9-suppressed tumor sections. Further, immunoprecipitation analysis showed decreased association of proteins involved in telomere end repair in MMP-9 shRNA-treated glioma cells. Elevated levels of p73 and TRAIL and the results of the FACS analysis show induction of apoptosis in MMP-9-silenced glioma cells. Taken together, these data provide new insights into the mechanisms underlying MMP-9-mediated hTERT expression in glioma proliferation.
authors.affiliation: Department of Cancer Biology and Pharmacology, University of Illinois College of Medicine at Peoria, Peoria, IL 61656, USA.
authors.forename: Shivani
authors.initials: S
authors.lastname: Ponnala
authors.forename: Chandramu
authors.initials: C
authors.lastname: Chetty
authors.forename: Krishna Kumar
authors.initials: KK
authors.lastname: Veeravalli
authors.forename: Dzung H
authors.initials: DH
authors.lastname: Dinh
authors.forename: Jeffrey D
authors.initials: JD
authors.lastname: Klopfenstein
authors.forename: Jasti S
authors.initials: JS
authors.lastname: Rao
mesh_terms: D000818:Animals; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors / Q000235:genetics / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D057075:Enzyme Assays; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000378:metabolism; D055785:Gene Knockdown Techniques; D005910:Glioma / Q000378:metabolism / Q000473:pathology; D006801:Humans; D047468:Immunoprecipitation; D019012:Integrin beta1 / Q000378:metabolism*; D020780:Matrix Metalloproteinase 9 / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008819:Mice, Nude; D009368:Neoplasm Transplantation; D011485:Protein Binding; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D019098:Telomerase / Q000235:genetics / Q000378:metabolism*; D016615:Telomere / Q000378:metabolism; D014158:Transcription, Genetic; D014183:Transplantation, Heterologous
chemical_list: D051017:Apoptosis Regulatory Proteins; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D018797:Cell Cycle Proteins; D019012:Integrin beta1; C079439:MXD1 protein, human; C489427:MYC protein, human; D016271:Proto-Oncogene Proteins c-myc; D012097:Repressor Proteins; D051416:Focal Adhesion Protein-Tyrosine Kinases; C509186:TERT protein, human; D019098:Telomerase; D020780:Matrix Metalloproteinase 9