Adenosine diphosphate-ribosylation factor 6 is required for epidermal growth factor-induced glioblastoma cell proliferation

Cancer. 2009 Nov 1;115(21):4959-72. doi: 10.1002/cncr.24550.


Background: : Epidermal growth factor (EGF) signaling plays a pivotal role in gliomagenesis. The authors previously demonstrated that adenosine diphospate-ribosylation factor 6 (ARF6), a member of the Ras-related small guanosine-5'-triphospate-binding protein family, is required for EFA6A-induced glioma cell migration and invasion. However, the role of ARF6 in EGF signaling is unknown.

Methods: : The authors analyzed messenger RNA (mRNA) levels of ARF6 and EGF receptor (EGFR) in 16 high-grade glioma samples and in 6 low-grade glioma samples by reverse transcriptase-polymerase chain reaction analysis. To determine whether EGF induces ARF6 expression in human glioblastoma U87 cells through transcriptional regulation and EGFR activation, the levels of ARF6 were assayed in EGF-treated U87 cells that were preincubated with a transcriptional inhibitor (actinomycin D) and an EGFR tyrosine kinase inhibitor (PD153035), respectively. The downstream signaling of EGFR-mediated ARF6 up-regulation also was investigated using specific inhibitors of mitogen-activated protein kinase (MEK), phosphatidylinositol 3' kinase (PI3K), and Janus kinase 2. The involvement of SP1 in the downstream signaling was studied by using an SP1 inhibitor (mithramycin A). Small-interfering RNAs (siRNAs) targeting ARF6 were used to investigate the effects of ARF6 on EGF-mediated glioma cell proliferation.

Results: : The results demonstrated that ARF6 and EGFR mRNA levels were elevated in glioma tissues. Furthermore, EGF stimulated ARF6 expression in U87 cells in a dose-dependent and time-dependant manner. This stimulation was caused by increased transcription of ARF6 and by activation of the MEK/extracellular signal-regulated kinase 1 and 2 (ERK1/2) and PI3K signaling pathways. It is noteworthy that SP1 was essential for EGF-induced ARF6 up-regulation. Finally, EGF-induced glioblastoma cell proliferation depended on ARF6, because the suppression of ARF6 by siRNA or by a dominant-negative mutant significantly inhibited EGF-induced cell proliferation.

Conclusions: : The results of the current study suggested that EGF-induced ARF6 expression plays a significant role in glioma cell proliferation. Cancer 2009. (c) 2009 American Cancer Society.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • ADP-Ribosylation Factors / metabolism*
  • Cell Proliferation
  • Dose-Response Relationship, Drug
  • Epidermal Growth Factor / pharmacology
  • ErbB Receptors / metabolism*
  • Glioblastoma / metabolism
  • RNA, Messenger / metabolism
  • Signal Transduction
  • Sp1 Transcription Factor / metabolism


  • RNA, Messenger
  • Sp1 Transcription Factor
  • Epidermal Growth Factor
  • ErbB Receptors
  • ADP-Ribosylation Factors
  • ADP-ribosylation factor 6