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A MT1-MMP/NF-kappaB signaling axis as a checkpoint controller of COX-2 expression in CD133+ U87 glioblastoma cells.

Annabi B, Laflamme C, Sina A, Lachambre MP, Béliveau R - J Neuroinflammation (2009)

Bottom Line: The CD133(+) stem cell population in recurrent gliomas is associated with clinical features such as therapy resistance, blood-brain barrier disruption and, hence, tumor infiltration.MT1-MMP gene silencing antagonized COX-2 expression in neurospheres, while overexpression of recombinant MT1-MMP directly triggered COX-2 expression in U87 cells independent from MT1-MMP's catalytic function.COX-2 induction by MT1-MMP was also validated in wild-type and in NF-kappaB p65-/- mutant mouse embryonic fibroblasts, but was abrogated in NF-kappaB 1 (p50-/-) mutant cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratoire d'Oncologie Moléculaire, Département de Chimie, Centre de Recherche BIOMED, Université du Québec à Montréal, Quebec, Canada. annabi.borhane@uqam.ca

ABSTRACT

Background: The CD133(+) stem cell population in recurrent gliomas is associated with clinical features such as therapy resistance, blood-brain barrier disruption and, hence, tumor infiltration. Screening of a large panel of glioma samples increasing histological grade demonstrated frequencies of CD133(+) cells which correlated with high expression of cyclooxygenase (COX)-2 and of membrane type-1 matrix metalloproteinase (MT1-MMP).

Methods: We used qRT-PCR and immunoblotting to examine the molecular interplay between MT1-MMP and COX-2 gene and protein expression in parental, CD133(+), and neurospheres U87 glioma cell cultures.

Results: We found that CD133, COX-2 and MT1-MMP expression were enhanced when glioma cells were cultured in neurosphere conditions. A CD133(+)-enriched U87 glioma cell population, isolated from parental U87 cells with magnetic cell sorting technology, also grew as neurospheres and showed enhanced COX-2 expression. MT1-MMP gene silencing antagonized COX-2 expression in neurospheres, while overexpression of recombinant MT1-MMP directly triggered COX-2 expression in U87 cells independent from MT1-MMP's catalytic function. COX-2 induction by MT1-MMP was also validated in wild-type and in NF-kappaB p65-/- mutant mouse embryonic fibroblasts, but was abrogated in NF-kappaB 1 (p50-/-) mutant cells.

Conclusion: We provide evidence for enhanced COX-2 expression in CD133(+) glioma cells, and direct cell-based evidence of NF-kappaB-mediated COX-2 regulation by MT1-MMP. The biological significance of such checkpoint control may account for COX-2-dependent mechanisms of inflammatory balance responsible of therapy resistance phenotype of cancer stem cells.

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COX-2 induction by MT1-MMP occurs through NF-κB-mediated mechanisms. Wild-type (Wt), p65-/-, and p50-/- mouse embryonic fibroblasts (MEF) were Mock-transfected or transfected with a cDNA plasmid encoding MT1-MMP. Gelatin zymography was used to monitor the extent of latent proMMP-2 and active MMP-2 expression from the conditioned media of the serum-starved cells (upper panel). Cell lysates were isolated and SDS-PAGE performed (20 μg protein/well), followed by Western blotting and COX-2 immunodetection.
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Figure 5: COX-2 induction by MT1-MMP occurs through NF-κB-mediated mechanisms. Wild-type (Wt), p65-/-, and p50-/- mouse embryonic fibroblasts (MEF) were Mock-transfected or transfected with a cDNA plasmid encoding MT1-MMP. Gelatin zymography was used to monitor the extent of latent proMMP-2 and active MMP-2 expression from the conditioned media of the serum-starved cells (upper panel). Cell lysates were isolated and SDS-PAGE performed (20 μg protein/well), followed by Western blotting and COX-2 immunodetection.

Mentions: MT1-MMP was previously demonstrated to possess the ability to trigger intracellular signaling through its 20 amino acid intracellular domain [20-22]. Moreover, COX-2 transcriptional expression is thought to be regulated, in part, through NF-κB-mediated signaling involving nuclear translocation of the NF-κB heterodimer p50:p65 [36]. Wild-type mouse embryonic fibroblasts (MEF) as well as p50-/- and p65-/- NF-κB mutants were used to assess MT1-MMP involvement in COX-2 expression. Cell lysates as well as conditioned media were isolated from Mock-transfected and MT1-MMP-transfected cells. Expression and cell surface activity of the recombinant MT1-MMP were confirmed in transfected cells as Wt, p50-/- and p65-/- cells all exhibited increased proMMP-2 activation into its active MMP-2 form as judged by gelatin zymography (Figure 5, upper panel). When COX-2 protein expression was assessed, we observed the induction of COX-2 by MT1-MMP in Wt-MEF (Figure 5, middle panel) confirming the results observed in U87 glioma cells (Figure 4a). Similar MT1-MMP-mediated COX-2 induction was also observed in p65-/- mutant MEF but COX-2 expression was completely abrogated in p50-/- mutant MEF (Figure 5, middle panel). This cell-based evidence directly demonstrates the specific involvement of p50 in NF-κB-mediated MT1-MMP regulation of COX-2 expression.


A MT1-MMP/NF-kappaB signaling axis as a checkpoint controller of COX-2 expression in CD133+ U87 glioblastoma cells.

Annabi B, Laflamme C, Sina A, Lachambre MP, Béliveau R - J Neuroinflammation (2009)

COX-2 induction by MT1-MMP occurs through NF-κB-mediated mechanisms. Wild-type (Wt), p65-/-, and p50-/- mouse embryonic fibroblasts (MEF) were Mock-transfected or transfected with a cDNA plasmid encoding MT1-MMP. Gelatin zymography was used to monitor the extent of latent proMMP-2 and active MMP-2 expression from the conditioned media of the serum-starved cells (upper panel). Cell lysates were isolated and SDS-PAGE performed (20 μg protein/well), followed by Western blotting and COX-2 immunodetection.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2655289&req=5

Figure 5: COX-2 induction by MT1-MMP occurs through NF-κB-mediated mechanisms. Wild-type (Wt), p65-/-, and p50-/- mouse embryonic fibroblasts (MEF) were Mock-transfected or transfected with a cDNA plasmid encoding MT1-MMP. Gelatin zymography was used to monitor the extent of latent proMMP-2 and active MMP-2 expression from the conditioned media of the serum-starved cells (upper panel). Cell lysates were isolated and SDS-PAGE performed (20 μg protein/well), followed by Western blotting and COX-2 immunodetection.
Mentions: MT1-MMP was previously demonstrated to possess the ability to trigger intracellular signaling through its 20 amino acid intracellular domain [20-22]. Moreover, COX-2 transcriptional expression is thought to be regulated, in part, through NF-κB-mediated signaling involving nuclear translocation of the NF-κB heterodimer p50:p65 [36]. Wild-type mouse embryonic fibroblasts (MEF) as well as p50-/- and p65-/- NF-κB mutants were used to assess MT1-MMP involvement in COX-2 expression. Cell lysates as well as conditioned media were isolated from Mock-transfected and MT1-MMP-transfected cells. Expression and cell surface activity of the recombinant MT1-MMP were confirmed in transfected cells as Wt, p50-/- and p65-/- cells all exhibited increased proMMP-2 activation into its active MMP-2 form as judged by gelatin zymography (Figure 5, upper panel). When COX-2 protein expression was assessed, we observed the induction of COX-2 by MT1-MMP in Wt-MEF (Figure 5, middle panel) confirming the results observed in U87 glioma cells (Figure 4a). Similar MT1-MMP-mediated COX-2 induction was also observed in p65-/- mutant MEF but COX-2 expression was completely abrogated in p50-/- mutant MEF (Figure 5, middle panel). This cell-based evidence directly demonstrates the specific involvement of p50 in NF-κB-mediated MT1-MMP regulation of COX-2 expression.

Bottom Line: The CD133(+) stem cell population in recurrent gliomas is associated with clinical features such as therapy resistance, blood-brain barrier disruption and, hence, tumor infiltration.MT1-MMP gene silencing antagonized COX-2 expression in neurospheres, while overexpression of recombinant MT1-MMP directly triggered COX-2 expression in U87 cells independent from MT1-MMP's catalytic function.COX-2 induction by MT1-MMP was also validated in wild-type and in NF-kappaB p65-/- mutant mouse embryonic fibroblasts, but was abrogated in NF-kappaB 1 (p50-/-) mutant cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratoire d'Oncologie Moléculaire, Département de Chimie, Centre de Recherche BIOMED, Université du Québec à Montréal, Quebec, Canada. annabi.borhane@uqam.ca

ABSTRACT

Background: The CD133(+) stem cell population in recurrent gliomas is associated with clinical features such as therapy resistance, blood-brain barrier disruption and, hence, tumor infiltration. Screening of a large panel of glioma samples increasing histological grade demonstrated frequencies of CD133(+) cells which correlated with high expression of cyclooxygenase (COX)-2 and of membrane type-1 matrix metalloproteinase (MT1-MMP).

Methods: We used qRT-PCR and immunoblotting to examine the molecular interplay between MT1-MMP and COX-2 gene and protein expression in parental, CD133(+), and neurospheres U87 glioma cell cultures.

Results: We found that CD133, COX-2 and MT1-MMP expression were enhanced when glioma cells were cultured in neurosphere conditions. A CD133(+)-enriched U87 glioma cell population, isolated from parental U87 cells with magnetic cell sorting technology, also grew as neurospheres and showed enhanced COX-2 expression. MT1-MMP gene silencing antagonized COX-2 expression in neurospheres, while overexpression of recombinant MT1-MMP directly triggered COX-2 expression in U87 cells independent from MT1-MMP's catalytic function. COX-2 induction by MT1-MMP was also validated in wild-type and in NF-kappaB p65-/- mutant mouse embryonic fibroblasts, but was abrogated in NF-kappaB 1 (p50-/-) mutant cells.

Conclusion: We provide evidence for enhanced COX-2 expression in CD133(+) glioma cells, and direct cell-based evidence of NF-kappaB-mediated COX-2 regulation by MT1-MMP. The biological significance of such checkpoint control may account for COX-2-dependent mechanisms of inflammatory balance responsible of therapy resistance phenotype of cancer stem cells.

Show MeSH
Related in: MedlinePlus