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Role of MMP-2 in the regulation of IL-6/Stat3 survival signaling via interaction with α5β1 integrin in glioma.

Kesanakurti D, Chetty C, Dinh DH, Gujrati M, Rao JS - Oncogene (2012)

Bottom Line: MMP-2/α5β1 binding is enhanced in human recombinant MMP-2 treatments, resulting in elevated Stat3 DNA-binding activity and recruitment on CyclinD1 and c-Myc promoters.In vivo experiments with orthotropic tumor model revealed the decreased tumor size in pM treatment compared with mock or pSV treatments.Immunofluorescence studies in tumor sections corroborated our in vitro findings evidencing high expression and co-localization of MMP-2/α5β1, which is decreased upon pM treatment along with significantly reduced IL-6, phospho-Stat3, CyclinD1, c-Myc, Ki-67 and PCNA expression levels.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Biology and Pharmacology, University of Illinois College of Medicine at Peoria, Peoria, IL 61605, USA.

ABSTRACT
Matrix metalloproteinase-2 (MMP-2) has pivotal role in the degradation of extracellular matrix, and thereby enhances the invasive, proliferative and metastatic potential in cancer. Knockdown of MMP-2 using MMP-2 small interfering RNA (pM) in human glioma xenograft cell lines 4910 and 5310 decreased cell proliferation compared with mock and pSV (scrambled vector) treatments, as determined by 5-bromo-2'-deoxyuridine incorporation, Ki-67 staining and clonogenic survival assay. Cytokine array and western blotting using tumor-conditioned media displayed modulated secretory levels of various cytokines including granulocyte-macrophage colony-stimulating factor, interleukin-6 (IL-6), IL-8, IL-10, tumor necrosis factor-α, angiogenin, vascular endothelial growth factor and PDGF-BB in MMP-2 knockdown cells. Further, cDNA PCR array indicated potential negative regulation of Janus kinase/Stat3 pathway in pM-treated cells. Mechanistically, MMP-2 is involved in complex formation with α5 and β1 integrins and MMP-2 downregulation inhibited α5β1 integrin-mediated Stat3 phosphorylation and nuclear translocation. Electrophoretic mobility shift assay and chromatin immunoprecipitation assays showed inhibited Stat3 DNA-binding activity and recruitment at CyclinD1 and c-Myc promoters in pM-treated cells. In individual experiments, IL-6 or siRNA-insensitive MMP-2 overexpression by pM-FL-A141G counteracted and restored the pM-inhibited Stat3 DNA-binding activity, suggesting IL-6/Stat3 signaling suppression in pM-treated 4910 and 5310 cells. MMP-2/α5β1 binding is enhanced in human recombinant MMP-2 treatments, resulting in elevated Stat3 DNA-binding activity and recruitment on CyclinD1 and c-Myc promoters. Activation of α5β1 signaling by Fibronectin adhesion elevated pM-inhibited Stat3 phosphorylation whereas blocking α5β1 abrogated constitutive Stat3 activation. In vivo experiments with orthotropic tumor model revealed the decreased tumor size in pM treatment compared with mock or pSV treatments. Immunofluorescence studies in tumor sections corroborated our in vitro findings evidencing high expression and co-localization of MMP-2/α5β1, which is decreased upon pM treatment along with significantly reduced IL-6, phospho-Stat3, CyclinD1, c-Myc, Ki-67 and PCNA expression levels. Our data indicate the possible role of MMP-2/α5β1 interaction in the regulation of α5β1-mediated IL-6/Stat3 signaling activation and signifies the therapeutic potential of blocking MMP-2/α5β1 interaction in glioma treatment.

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Effect of MMP-2 knockdown on in vivo tumor growth. Tumor growth was established by intracranial injection of 4910 and 5310 cells into athymic nude mice (nu/nu) and pSV and pM plasmids were injected into the tumors as described in Materials and Methods while sterile 1×PBS injection served as mock treatment. A, Hematoxylin and eosin staining of brain sections displaying the neoplastic growth of tumors. The relative brain tumor size was plotted as mean±SE obtained from three sets of treatment groups (n=10) and the significance is represented by ** at p<0.01. B, Immunoflorescence showing co-localization of MMP-2 and α5β1 integrin in tumor sections. The deparaffinized mock, pSV-, and pM-treated tumor sections were passed through graded ethanol series, blocked with 1% BSA, and incubated with anti-MMP-2 and anti-α5β1 integrin antibodies (1:100) overnight at 4°C. Sections were washed and incubated with specific Alexa Fluor® antibodies and counterstained with DAPI and observed for cellular MMP-2 and α5β1 expression. Depicted pictures are representative of 20 different sections from each set of treatments (n=10). C, Immunocytochemical DAB staining showing IL-6, phospho-Stat3 (Tyr 705) expression in tumor sections. Hematoxylin was used for nuclear counter staining. Negative control staining was performed by incubation with non-specific IgG (Insets).
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Figure 8: Effect of MMP-2 knockdown on in vivo tumor growth. Tumor growth was established by intracranial injection of 4910 and 5310 cells into athymic nude mice (nu/nu) and pSV and pM plasmids were injected into the tumors as described in Materials and Methods while sterile 1×PBS injection served as mock treatment. A, Hematoxylin and eosin staining of brain sections displaying the neoplastic growth of tumors. The relative brain tumor size was plotted as mean±SE obtained from three sets of treatment groups (n=10) and the significance is represented by ** at p<0.01. B, Immunoflorescence showing co-localization of MMP-2 and α5β1 integrin in tumor sections. The deparaffinized mock, pSV-, and pM-treated tumor sections were passed through graded ethanol series, blocked with 1% BSA, and incubated with anti-MMP-2 and anti-α5β1 integrin antibodies (1:100) overnight at 4°C. Sections were washed and incubated with specific Alexa Fluor® antibodies and counterstained with DAPI and observed for cellular MMP-2 and α5β1 expression. Depicted pictures are representative of 20 different sections from each set of treatments (n=10). C, Immunocytochemical DAB staining showing IL-6, phospho-Stat3 (Tyr 705) expression in tumor sections. Hematoxylin was used for nuclear counter staining. Negative control staining was performed by incubation with non-specific IgG (Insets).

Mentions: In vivo experiments showing the effect of pM-treatment on intracranial tumor growth by orthotropic injection of 4910 and 5310 cells showed remarkable decrease in the tumor size when compared to mock- and pSV-treated tumors (Figure 8A). High expression and predominant co-localization MMP-2 and α5β1 integrin at membrane periphery were identified in both mock- and pSV-treated tumors (Figure 8B). In contrast, pM-treated tumors showed substantial decrease in MMP-2 and α5β1 expression, which subsequently led to decreased MMP-2/α5β1 co-localization correlating with reduced IL-6 and phospho-Stat3 expression levels (Figure 8C). Further expression levels of proliferation markers Ki-67 and PCNA were studied to check the number of proliferating cells in sections which revealed the severely inhibition Ki-67 and PCNA positivity in pM-treated tumor sections whereas the mock- and pSV- treated control tumors showed high proliferating cells with intense nuclear Ki-67 and PCNA staining (Supplementary Figure S6). The CyclinD1 and c-Myc expression was significantly decreased in pM-treated tumor sections when compared to pSV-treated control counterparts (Supplementary Figure S7). These in vivo observations corroborate the in vitro findings confirming the role of MMP-2 in α5β1 mediated IL-6/Stat3 signaling activation in glioma.


Role of MMP-2 in the regulation of IL-6/Stat3 survival signaling via interaction with α5β1 integrin in glioma.

Kesanakurti D, Chetty C, Dinh DH, Gujrati M, Rao JS - Oncogene (2012)

Effect of MMP-2 knockdown on in vivo tumor growth. Tumor growth was established by intracranial injection of 4910 and 5310 cells into athymic nude mice (nu/nu) and pSV and pM plasmids were injected into the tumors as described in Materials and Methods while sterile 1×PBS injection served as mock treatment. A, Hematoxylin and eosin staining of brain sections displaying the neoplastic growth of tumors. The relative brain tumor size was plotted as mean±SE obtained from three sets of treatment groups (n=10) and the significance is represented by ** at p<0.01. B, Immunoflorescence showing co-localization of MMP-2 and α5β1 integrin in tumor sections. The deparaffinized mock, pSV-, and pM-treated tumor sections were passed through graded ethanol series, blocked with 1% BSA, and incubated with anti-MMP-2 and anti-α5β1 integrin antibodies (1:100) overnight at 4°C. Sections were washed and incubated with specific Alexa Fluor® antibodies and counterstained with DAPI and observed for cellular MMP-2 and α5β1 expression. Depicted pictures are representative of 20 different sections from each set of treatments (n=10). C, Immunocytochemical DAB staining showing IL-6, phospho-Stat3 (Tyr 705) expression in tumor sections. Hematoxylin was used for nuclear counter staining. Negative control staining was performed by incubation with non-specific IgG (Insets).
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Related In: Results  -  Collection

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Figure 8: Effect of MMP-2 knockdown on in vivo tumor growth. Tumor growth was established by intracranial injection of 4910 and 5310 cells into athymic nude mice (nu/nu) and pSV and pM plasmids were injected into the tumors as described in Materials and Methods while sterile 1×PBS injection served as mock treatment. A, Hematoxylin and eosin staining of brain sections displaying the neoplastic growth of tumors. The relative brain tumor size was plotted as mean±SE obtained from three sets of treatment groups (n=10) and the significance is represented by ** at p<0.01. B, Immunoflorescence showing co-localization of MMP-2 and α5β1 integrin in tumor sections. The deparaffinized mock, pSV-, and pM-treated tumor sections were passed through graded ethanol series, blocked with 1% BSA, and incubated with anti-MMP-2 and anti-α5β1 integrin antibodies (1:100) overnight at 4°C. Sections were washed and incubated with specific Alexa Fluor® antibodies and counterstained with DAPI and observed for cellular MMP-2 and α5β1 expression. Depicted pictures are representative of 20 different sections from each set of treatments (n=10). C, Immunocytochemical DAB staining showing IL-6, phospho-Stat3 (Tyr 705) expression in tumor sections. Hematoxylin was used for nuclear counter staining. Negative control staining was performed by incubation with non-specific IgG (Insets).
Mentions: In vivo experiments showing the effect of pM-treatment on intracranial tumor growth by orthotropic injection of 4910 and 5310 cells showed remarkable decrease in the tumor size when compared to mock- and pSV-treated tumors (Figure 8A). High expression and predominant co-localization MMP-2 and α5β1 integrin at membrane periphery were identified in both mock- and pSV-treated tumors (Figure 8B). In contrast, pM-treated tumors showed substantial decrease in MMP-2 and α5β1 expression, which subsequently led to decreased MMP-2/α5β1 co-localization correlating with reduced IL-6 and phospho-Stat3 expression levels (Figure 8C). Further expression levels of proliferation markers Ki-67 and PCNA were studied to check the number of proliferating cells in sections which revealed the severely inhibition Ki-67 and PCNA positivity in pM-treated tumor sections whereas the mock- and pSV- treated control tumors showed high proliferating cells with intense nuclear Ki-67 and PCNA staining (Supplementary Figure S6). The CyclinD1 and c-Myc expression was significantly decreased in pM-treated tumor sections when compared to pSV-treated control counterparts (Supplementary Figure S7). These in vivo observations corroborate the in vitro findings confirming the role of MMP-2 in α5β1 mediated IL-6/Stat3 signaling activation in glioma.

Bottom Line: MMP-2/α5β1 binding is enhanced in human recombinant MMP-2 treatments, resulting in elevated Stat3 DNA-binding activity and recruitment on CyclinD1 and c-Myc promoters.In vivo experiments with orthotropic tumor model revealed the decreased tumor size in pM treatment compared with mock or pSV treatments.Immunofluorescence studies in tumor sections corroborated our in vitro findings evidencing high expression and co-localization of MMP-2/α5β1, which is decreased upon pM treatment along with significantly reduced IL-6, phospho-Stat3, CyclinD1, c-Myc, Ki-67 and PCNA expression levels.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Biology and Pharmacology, University of Illinois College of Medicine at Peoria, Peoria, IL 61605, USA.

ABSTRACT
Matrix metalloproteinase-2 (MMP-2) has pivotal role in the degradation of extracellular matrix, and thereby enhances the invasive, proliferative and metastatic potential in cancer. Knockdown of MMP-2 using MMP-2 small interfering RNA (pM) in human glioma xenograft cell lines 4910 and 5310 decreased cell proliferation compared with mock and pSV (scrambled vector) treatments, as determined by 5-bromo-2'-deoxyuridine incorporation, Ki-67 staining and clonogenic survival assay. Cytokine array and western blotting using tumor-conditioned media displayed modulated secretory levels of various cytokines including granulocyte-macrophage colony-stimulating factor, interleukin-6 (IL-6), IL-8, IL-10, tumor necrosis factor-α, angiogenin, vascular endothelial growth factor and PDGF-BB in MMP-2 knockdown cells. Further, cDNA PCR array indicated potential negative regulation of Janus kinase/Stat3 pathway in pM-treated cells. Mechanistically, MMP-2 is involved in complex formation with α5 and β1 integrins and MMP-2 downregulation inhibited α5β1 integrin-mediated Stat3 phosphorylation and nuclear translocation. Electrophoretic mobility shift assay and chromatin immunoprecipitation assays showed inhibited Stat3 DNA-binding activity and recruitment at CyclinD1 and c-Myc promoters in pM-treated cells. In individual experiments, IL-6 or siRNA-insensitive MMP-2 overexpression by pM-FL-A141G counteracted and restored the pM-inhibited Stat3 DNA-binding activity, suggesting IL-6/Stat3 signaling suppression in pM-treated 4910 and 5310 cells. MMP-2/α5β1 binding is enhanced in human recombinant MMP-2 treatments, resulting in elevated Stat3 DNA-binding activity and recruitment on CyclinD1 and c-Myc promoters. Activation of α5β1 signaling by Fibronectin adhesion elevated pM-inhibited Stat3 phosphorylation whereas blocking α5β1 abrogated constitutive Stat3 activation. In vivo experiments with orthotropic tumor model revealed the decreased tumor size in pM treatment compared with mock or pSV treatments. Immunofluorescence studies in tumor sections corroborated our in vitro findings evidencing high expression and co-localization of MMP-2/α5β1, which is decreased upon pM treatment along with significantly reduced IL-6, phospho-Stat3, CyclinD1, c-Myc, Ki-67 and PCNA expression levels. Our data indicate the possible role of MMP-2/α5β1 interaction in the regulation of α5β1-mediated IL-6/Stat3 signaling activation and signifies the therapeutic potential of blocking MMP-2/α5β1 interaction in glioma treatment.

Show MeSH
Related in: MedlinePlus