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Six1 promotes glioblastoma cell proliferation and invasion by upregulation of connective tissue growth factor.

ABSTRACT: Glioblastoma multiforme (GBM) is the deadliest and most common form of malignant primary brain tumor in humans. However, until now, little is known about the glioma genesis and progression at the molecular level. Here we report that overexpression of sine oculis homeobox homolog 1 (Six1), a developmental transcription factor implicated in tumor onset and progression, can significantly promote glioblastoma cell proliferation and invasion by upregulating connective tissue growth factor (CTGF). Our results revealed that expression of Six1 mRNA was increased and small hairpin RNAi silencing of Six1 could dramatically inhibit cell proliferation and invasion in GBM. Moreover, it was found that CTGF gene could be transcriptionally regulated by Six1. Its overexpression induced CTGF up-regulation in GBM at both the mRNA and protein level, and significantly enhanced the activity of CTGF promoter in these tumor cells, while decreasing CTGF expression impeded Six1-induced cell proliferation and invasion, revealing that CTGF is required for Six1-mediated GBM growth and metastasis. Collectively, these findings suggest that Six1 overexpression may contribute to cell proliferation and invasion via upregulation of CTGF in GBM. Our study provides new insights into the important roles of Six1 and CTGF in tumor regulation, suggesting that Six1 might be a potential therapeutic target for preventing proliferation and metastasis of GBM.

SUBMITTER: Tian T

PROVIDER: S-EPMC4497448 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Six1 promotes glioblastoma cell proliferation and invasion by upregulation of connective tissue growth factor.

Tian Tian T Li Aimin A Lu Hong H Luo Ran R Zhang Mingzhi M Li Zhaoming Z

American journal of cancer research 20150415 5

Glioblastoma multiforme (GBM) is the deadliest and most common form of malignant primary brain tumor in humans. However, until now, little is known about the glioma genesis and progression at the molecular level. Here we report that overexpression of sine oculis homeobox homolog 1 (Six1), a developmental transcription factor implicated in tumor onset and progression, can significantly promote glioblastoma cell proliferation and invasion by upregulating connective tissue growth factor (CTGF). Our ...[more]

PMID: 26175950

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Project description:Connective-tissue growth factor (CTGF/CCN2) is a matricellular-secreted protein involved in complex processes such as wound healing, angiogenesis, fibrosis and metastasis, in the regulation of cell proliferation, migration and extracellular matrix remodeling. Glioblastoma (GBM) is the major malignant primary brain tumor and its adaptation to the central nervous system microenvironment requires the production and remodeling of the extracellular matrix. Previously, we published an in vitro approach to test if neurons can influence the expression of the GBM extracellular matrix. We demonstrated that neurons remodeled glioma cell laminin. The present study shows that neurons are also able to modulate CTGF expression in GBM. CTGF immnoreactivity and mRNA levels in GBM cells are dramatically decreased when these cells are co-cultured with neonatal neurons. As proof of particular neuron effects, neonatal neurons co-cultured onto GBM cells also inhibit the reporter luciferase activity under control of the CTGF promoter, suggesting inhibition at the transcription level. This inhibition seems to be contact-mediated, since conditioned media from embryonic or neonatal neurons do not affect CTGF expression in GBM cells. Furthermore, the inhibition of CTGF expression in GBM/neuronal co-cultures seems to affect the two main signaling pathways related to CTGF. We observed inhibition of TGF? luciferase reporter assay; however phopho-SMAD2 levels did not change in these co-cultures. In addition levels of phospho-p44/42 MAPK were decreased in co-cultured GBM cells. Finally, in transwell migration assay, CTGF siRNA transfected GBM cells or GBM cells co-cultured with neurons showed a decrease in the migration rate compared to controls. Previous data regarding laminin and these results demonstrating that CTGF is down-regulated in GBM cells co-cultured with neonatal neurons points out an interesting view in the understanding of the tumor and cerebral microenvironment interactions and could open up new strategies as well as suggest a new target in GBM control.

Project description:BackgroundTumor 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.MethodsHighly 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 β1 (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.ResultsTreatment of TIC/TSCs with CTGF resulted in CTGF binding to ITGB1-TrkA receptor complexes and nuclear factor kappa B (NF-κB) 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-κB 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 CTGF(200 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.ConclusionA CTGF-rich microenvironment facilitates CTGF-ITGB1-TrkA complex activation in TIC/TSCs, thereby increasing the invasiveness of malignant gliomas.

Dataset Information

Six1 promotes glioblastoma cell proliferation and invasion by upregulation of connective tissue growth factor.

Publications

Six1 promotes glioblastoma cell proliferation and invasion by upregulation of connective tissue growth factor.

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