Expression of the constitutively activated mutant EGFR variant III (
EGFRvIII), the most common mutation in
glioblastoma multiforme (GBMs), has been clinically correlated with
tumor proliferation, invasion, and angiogenesis. In this study, we examined the role of
EGFRvIII on the tumor microenvironment, especially on angiogenesis.
METHODS: To study the role of
EGFRvIII in
tumor angiogenesis, we prepared LN229
glioblastoma transfected with
enhanced green fluorescent protein (EGFP), wild-type EGFR, or
EGFRvIII (LN229-WT or -vIII), and examined
tumor growth and microvessel density in the
tumors. Additionally, the potential angiogenic factors were identified by real-time PCR analysis, and the functions in LN229-vIII cells were examined.
RESULTS: LN229-vIII cells showed more aggressive
tumor growth and higher vascularity as compared to LN229-WT cells in vivo, although there was no significant difference in the cell growth rates in vitro. We next investigated the expression of 60 angiogenesis-related factors to clarify the mechanisms underlying the difference in vascularity between
tumor xenografts of LN229-vIII and LN229-WT. We found that the
mRNA and
protein expressions of
angiopoietin-like 4 (Angptl4), a secreted
protein involved in angiogenesis and metabolism regulation, were significantly induced by
EGFRvIII overexpression, both in vitro and in vivo. Constitutive knockdown of Angptl4 in LN229-vIII using
shRNA significantly decreased the microvessel density in the
tumor xenografts and suppressed
tumor growth. To clarify the regulatory mechanisms of Angptl4 by
EGFRvIII, we analyzed the signaling pathways and
transcription factors by pharmacological inhibition and RNA interference.
U0126, an ERK signal inhibitor dramatically suppressed Angptl4 expression. The
transcription factor c-Myc, which is regulated by ERK, was activated in the LN229-vIII cells and knockdown of c-Myc using
siRNA also attenuated Angptl4 expression in the LN229-vIII cells. Furthermore,
chromatin immunoprecipitation (ChIP) assay revealed increased recruitment of c-Myc to the promoter region of Angptl4 in the LN229-vIII cells.
CONCLUSIONS: