Effect of brain- and tumor-derived connective tissue growth factor on glioma invasion

Lincoln A. Edwards, Kevin D Woolard, Myung Jin Son, Aiguo Li, Jeongwu Lee, Chibawanye Ene, Samuel A. Mantey, Dragan Maric, Hua Song, Galina Belova, Robert T. Jensen, Wei Zhang, Howard A. Fine

Research output: Contribution to journalArticle

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Abstract

Background Tumor cell invasion is the principal cause of treatment failure and death among patients with malignant gliomas. Connective tissue growth factor (CTGF) has been previously implicated in cancer metastasis and invasion in various tumors. We explored the mechanism of CTGF-mediated glioma cell infiltration and examined potential therapeutic targets. Methods Highly infiltrative patient-derived glioma tumor-initiating or tumor stem cells (TIC/TSCs) were harvested and used to explore a CTGF-induced signal transduction pathway via luciferase reporter assays, chromatin immunoprecipitation (ChIP), real-time polymerase chain reaction, and immunoblotting. Treatment of TIC/TSCs with small-molecule inhibitors targeting integrin b1 (ITGB1) and the tyrosine kinase receptor type A (TrkA), and short hairpin RNAs targeting CTGF directly were used to reduce the levels of key protein components of CTGF-induced cancer infiltration. TIC/TSC infiltration was examined in real-time cell migration and invasion assays in vitro and by immunohistochemistry and in situ hybridization in TIC/TSC orthotopic xenograft mouse models (n = 30; six mice per group). All statistical tests were two-sided. Results Treatment of TIC/TSCs with CTGF resulted in CTGF binding to ITGB1-TrkA receptor complexes and nuclear factor kappa B (NF-kB) transcriptional activation as measured by luciferase reporter assays (mean relative luciferase activity, untreated vs CTGF 200 ng/mL: 0.53 vs 1.87, difference = 1.34, 95% confidence interval [CI] = 0.69 to 2, P < .001). NF-kB activation resulted in binding of ZEB-1 to the E-cadherin promoter as demonstrated by ChIP analysis with subsequent E-cadherin suppression (fold increase in ZEB-1 binding to the E-cadherin promoter region: untreated + ZEB-1 antibody vs CTGF200 ng/mL + ZEB-1 antibody: 1.5 vs 6.4, difference = 4.9, 95% CI = 4.8 to 5.0, P < .001). Immunohistochemistry and in situ hybridization revealed that TrkA is selectively expressed in the most infiltrative glioma cells in situ and that the surrounding reactive astrocytes secrete CTGF. Conclusion A CTGF-rich microenvironment facilitates CTGF-ITGB1-TrkA complex activation in TIC/TSCs, thereby increasing the invasiveness of malignant gliomas.

Original languageEnglish (US)
Pages (from-to)1162-1178
Number of pages17
JournalJournal of the National Cancer Institute
Volume103
Issue number15
DOIs
StatePublished - Aug 3 2011
Externally publishedYes

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Connective Tissue Growth Factor
Brain Neoplasms
Glioma
Cadherins
Luciferases
Integrins
NF-kappa B
Chromatin Immunoprecipitation
Neoplasms
In Situ Hybridization
trkA Receptor
Immunohistochemistry
Cell Migration Assays
Confidence Intervals
Neoplastic Stem Cells
Antibodies
Receptor Protein-Tyrosine Kinases
Treatment Failure
Immunoblotting
Heterografts

ASJC Scopus subject areas

  • Cancer Research
  • Oncology

Cite this

Effect of brain- and tumor-derived connective tissue growth factor on glioma invasion. / Edwards, Lincoln A.; Woolard, Kevin D; Son, Myung Jin; Li, Aiguo; Lee, Jeongwu; Ene, Chibawanye; Mantey, Samuel A.; Maric, Dragan; Song, Hua; Belova, Galina; Jensen, Robert T.; Zhang, Wei; Fine, Howard A.

In: Journal of the National Cancer Institute, Vol. 103, No. 15, 03.08.2011, p. 1162-1178.

Research output: Contribution to journalArticle

Edwards, LA, Woolard, KD, Son, MJ, Li, A, Lee, J, Ene, C, Mantey, SA, Maric, D, Song, H, Belova, G, Jensen, RT, Zhang, W & Fine, HA 2011, 'Effect of brain- and tumor-derived connective tissue growth factor on glioma invasion', Journal of the National Cancer Institute, vol. 103, no. 15, pp. 1162-1178. https://doi.org/10.1093/jnci/djr224
Edwards, Lincoln A. ; Woolard, Kevin D ; Son, Myung Jin ; Li, Aiguo ; Lee, Jeongwu ; Ene, Chibawanye ; Mantey, Samuel A. ; Maric, Dragan ; Song, Hua ; Belova, Galina ; Jensen, Robert T. ; Zhang, Wei ; Fine, Howard A. / Effect of brain- and tumor-derived connective tissue growth factor on glioma invasion. In: Journal of the National Cancer Institute. 2011 ; Vol. 103, No. 15. pp. 1162-1178.
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abstract = "Background Tumor cell invasion is the principal cause of treatment failure and death among patients with malignant gliomas. Connective tissue growth factor (CTGF) has been previously implicated in cancer metastasis and invasion in various tumors. We explored the mechanism of CTGF-mediated glioma cell infiltration and examined potential therapeutic targets. Methods Highly infiltrative patient-derived glioma tumor-initiating or tumor stem cells (TIC/TSCs) were harvested and used to explore a CTGF-induced signal transduction pathway via luciferase reporter assays, chromatin immunoprecipitation (ChIP), real-time polymerase chain reaction, and immunoblotting. Treatment of TIC/TSCs with small-molecule inhibitors targeting integrin b1 (ITGB1) and the tyrosine kinase receptor type A (TrkA), and short hairpin RNAs targeting CTGF directly were used to reduce the levels of key protein components of CTGF-induced cancer infiltration. TIC/TSC infiltration was examined in real-time cell migration and invasion assays in vitro and by immunohistochemistry and in situ hybridization in TIC/TSC orthotopic xenograft mouse models (n = 30; six mice per group). All statistical tests were two-sided. Results Treatment of TIC/TSCs with CTGF resulted in CTGF binding to ITGB1-TrkA receptor complexes and nuclear factor kappa B (NF-kB) transcriptional activation as measured by luciferase reporter assays (mean relative luciferase activity, untreated vs CTGF 200 ng/mL: 0.53 vs 1.87, difference = 1.34, 95{\%} confidence interval [CI] = 0.69 to 2, P < .001). NF-kB activation resulted in binding of ZEB-1 to the E-cadherin promoter as demonstrated by ChIP analysis with subsequent E-cadherin suppression (fold increase in ZEB-1 binding to the E-cadherin promoter region: untreated + ZEB-1 antibody vs CTGF200 ng/mL + ZEB-1 antibody: 1.5 vs 6.4, difference = 4.9, 95{\%} CI = 4.8 to 5.0, P < .001). Immunohistochemistry and in situ hybridization revealed that TrkA is selectively expressed in the most infiltrative glioma cells in situ and that the surrounding reactive astrocytes secrete CTGF. Conclusion A CTGF-rich microenvironment facilitates CTGF-ITGB1-TrkA complex activation in TIC/TSCs, thereby increasing the invasiveness of malignant gliomas.",
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T1 - Effect of brain- and tumor-derived connective tissue growth factor on glioma invasion

AU - Edwards, Lincoln A.

AU - Woolard, Kevin D

AU - Son, Myung Jin

AU - Li, Aiguo

AU - Lee, Jeongwu

AU - Ene, Chibawanye

AU - Mantey, Samuel A.

AU - Maric, Dragan

AU - Song, Hua

AU - Belova, Galina

AU - Jensen, Robert T.

AU - Zhang, Wei

AU - Fine, Howard A.

PY - 2011/8/3

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N2 - Background Tumor cell invasion is the principal cause of treatment failure and death among patients with malignant gliomas. Connective tissue growth factor (CTGF) has been previously implicated in cancer metastasis and invasion in various tumors. We explored the mechanism of CTGF-mediated glioma cell infiltration and examined potential therapeutic targets. Methods Highly infiltrative patient-derived glioma tumor-initiating or tumor stem cells (TIC/TSCs) were harvested and used to explore a CTGF-induced signal transduction pathway via luciferase reporter assays, chromatin immunoprecipitation (ChIP), real-time polymerase chain reaction, and immunoblotting. Treatment of TIC/TSCs with small-molecule inhibitors targeting integrin b1 (ITGB1) and the tyrosine kinase receptor type A (TrkA), and short hairpin RNAs targeting CTGF directly were used to reduce the levels of key protein components of CTGF-induced cancer infiltration. TIC/TSC infiltration was examined in real-time cell migration and invasion assays in vitro and by immunohistochemistry and in situ hybridization in TIC/TSC orthotopic xenograft mouse models (n = 30; six mice per group). All statistical tests were two-sided. Results Treatment of TIC/TSCs with CTGF resulted in CTGF binding to ITGB1-TrkA receptor complexes and nuclear factor kappa B (NF-kB) transcriptional activation as measured by luciferase reporter assays (mean relative luciferase activity, untreated vs CTGF 200 ng/mL: 0.53 vs 1.87, difference = 1.34, 95% confidence interval [CI] = 0.69 to 2, P < .001). NF-kB activation resulted in binding of ZEB-1 to the E-cadherin promoter as demonstrated by ChIP analysis with subsequent E-cadherin suppression (fold increase in ZEB-1 binding to the E-cadherin promoter region: untreated + ZEB-1 antibody vs CTGF200 ng/mL + ZEB-1 antibody: 1.5 vs 6.4, difference = 4.9, 95% CI = 4.8 to 5.0, P < .001). Immunohistochemistry and in situ hybridization revealed that TrkA is selectively expressed in the most infiltrative glioma cells in situ and that the surrounding reactive astrocytes secrete CTGF. Conclusion A CTGF-rich microenvironment facilitates CTGF-ITGB1-TrkA complex activation in TIC/TSCs, thereby increasing the invasiveness of malignant gliomas.

AB - Background Tumor cell invasion is the principal cause of treatment failure and death among patients with malignant gliomas. Connective tissue growth factor (CTGF) has been previously implicated in cancer metastasis and invasion in various tumors. We explored the mechanism of CTGF-mediated glioma cell infiltration and examined potential therapeutic targets. Methods Highly infiltrative patient-derived glioma tumor-initiating or tumor stem cells (TIC/TSCs) were harvested and used to explore a CTGF-induced signal transduction pathway via luciferase reporter assays, chromatin immunoprecipitation (ChIP), real-time polymerase chain reaction, and immunoblotting. Treatment of TIC/TSCs with small-molecule inhibitors targeting integrin b1 (ITGB1) and the tyrosine kinase receptor type A (TrkA), and short hairpin RNAs targeting CTGF directly were used to reduce the levels of key protein components of CTGF-induced cancer infiltration. TIC/TSC infiltration was examined in real-time cell migration and invasion assays in vitro and by immunohistochemistry and in situ hybridization in TIC/TSC orthotopic xenograft mouse models (n = 30; six mice per group). All statistical tests were two-sided. Results Treatment of TIC/TSCs with CTGF resulted in CTGF binding to ITGB1-TrkA receptor complexes and nuclear factor kappa B (NF-kB) transcriptional activation as measured by luciferase reporter assays (mean relative luciferase activity, untreated vs CTGF 200 ng/mL: 0.53 vs 1.87, difference = 1.34, 95% confidence interval [CI] = 0.69 to 2, P < .001). NF-kB activation resulted in binding of ZEB-1 to the E-cadherin promoter as demonstrated by ChIP analysis with subsequent E-cadherin suppression (fold increase in ZEB-1 binding to the E-cadherin promoter region: untreated + ZEB-1 antibody vs CTGF200 ng/mL + ZEB-1 antibody: 1.5 vs 6.4, difference = 4.9, 95% CI = 4.8 to 5.0, P < .001). Immunohistochemistry and in situ hybridization revealed that TrkA is selectively expressed in the most infiltrative glioma cells in situ and that the surrounding reactive astrocytes secrete CTGF. Conclusion A CTGF-rich microenvironment facilitates CTGF-ITGB1-TrkA complex activation in TIC/TSCs, thereby increasing the invasiveness of malignant gliomas.

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