Efa6a/Arf6 Signaling and Functions in Glioblastoma Carcinogenesis

This dissertation, "EFA6A/ARF6 signaling and functions in glioblastoma carcinogenesis" by Ming, Li, 李明, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract of thesis entitled EFA6A/ARF6 SIGNALING AND FUNCTIONS IN GLIOBLASTOMA CARCINOGENESIS Submitted by Ming LI for the degree of Doctor of Philosophy at The University of Hong Kong in August 2006 Human gliomas are the most common primary tumors in the brain. They account for more than 40% of all neoplasms of the central nervous system, and patients diagnosed with grade IV glioma (glioblastoma mulfiforme) have a median survival rate of less than 12 months. The highly lethal nature of this tumor results from the acquisition of an invasive phenotype that allows the tumor cells to infiltrate the surrounding brain tissues. The mechanisms for this invasive process, however, are poorly understood. In this study, I characterized the role of a guanine nucleotide exchange factors (GEFs) for ADP-ribosylation factor 6 (ARF6), EFA6A (also termed Pleckstrin and Sec7 domain protein), in glioma cell invasion. I first showed that elevated EFA6A expression was detectable in both low-grade and high-grade human glioma tissues samples. To investigate the role of EFA6A in glioma carcinogenesis, I generated a human glioblastoma cell line (U373-EFA6A) which conditionally overexpressed EFA6A. I showed that overexpression of EFA6A had no effect on cell proliferation, apoptosis, or cell cycle control. However, as shown by wound healing and in vitro cell invasion assays, overexpression of EFA6A significantly enhanced the cell motility and invasiveness whereas silencing EFA6A by its dominant negative mutant EFA6A(E242K) produced opposite effects. I further demonstrated that ARF6/ERK signaling was required for the EFA6A-mediated cell invasion because both EFA6A(E242K) and ARF6 dominant negative mutant ARF6(T27N) markedly reduced the phosphorylated extracellular signal-regulated kinase (ERK) level and EFA6A- mediated invasive capacity. Consistently, MAPK/ERK kinase (MEK) inhibitor U0126 could abolish the EFA6A-induced cell invasion. These results reveal an important role of EFA6A/ARF6/ERK signal cascade in glioma cell migration and invasion. Epidermal growth factor (EGF)/EGF receptor (EGFR) signal cascade plays pivotal roles in glioma carcinogenesis through multiple mechanisms. In this study, I also have identified EGF/EGFR as a novel determinant of ARF6 expression in human glioblastoma cell lines, and ARF6 induction is associated with EGF/EGFR signaling- induced glioblastoma cell proliferation. I showed that ARF6 was overexpressed in approximately 60% of human glioblastoma cancers, whereas EGFR was upregulated both in low- and high-grade glioma tissues as compared to that of normal brain tissues. I demonstrated that the treatment with EGF increased ARF6 expression in a time and dose-dependent manner in vitro. This upregulation of ARF6 was due to increased transcription but independent of de novo protein synthesis. I further demonstrated that both MEK/ERK and PI3K signaling pathways were required for ARF6 induction because EGF-mediated ARF6 upregulation was prevented when both pathways were blocked by specific chemical inhibitors. Furthermore, I showed that SP1 was a downstream target of the MEK/ERK and PI3K pathways, and finally, EGF induced glioblastoma cell proliferation was dependent on ARF6 expression as suppression of ARF6 by siRNA or ARF6 dominant negative mutant significantly i