Abstract
The chemokine receptor CXCR4 is expressed in many cancers where it may regulate tumor cell growth and migration. The role of CXCR4 in cancer will depend on it being in an activated, signaling state. To better define the significance of CXCR4 expression in cancer, we developed an antibody that can distinguish CXCR4 phosphorylated on serine 339, a residue previously identified as a site for ligand-induced phosphorylation. With this antibody, we investigated the mechanisms of CXCR4 phosphorylation and evaluated the phosphorylation status of CXCR4 in human astrocytomas. In vitro, phosphorylation of serine 339 occurred in response to CXCL12 or epidermal growth factor (EGF) treatment and was increased by protein kinase C activation. In all grades of astrocytomas, CXCR4 was expressed in tumor cells and some endothelial cells, whereas CXCL12 was present in endothelial cells and infiltrating microglia. We found that CXCR4 phosphorylated on serine 339 was present in tumor cells and vascular endothelial cells in all grades of astrocytoma. These data indicate that CXCR4 is expressed and activated in astrocytomas and that phosphorylation of CXCR4 can occur through ligand activation or transactivation via the EGF receptor. These studies extend the potential roles of CXCR4 in cancer to include functions associated with benign (grade 1) tumors. The ability to distinguish phosphorylated CXCR4 will be invaluable for the continued analysis of the role of CXCR4 in cancer and the development of CXCR4 antagonist therapy for patients suffering with primary tumors of the brain and other sites.
Highlights
Identification of novel therapeutic targets is critical to the advancement of cancer treatment
Phosphopeptide corresponding to amino acids 332 to 346 [332-46] of human CXCR4 phosphorylated on S339 (P339) was injected into rabbits and phosphospecific antibody was isolated from serum by sequential column chromatography
The activity of the antipeptide antibody was evaluated by Western blot–based assays using lysates from the glioblastoma cell line LN428, which is known to express high levels of CXCR4 [26]
Summary
Identification of novel therapeutic targets is critical to the advancement of cancer treatment. The chemokine receptor CXCR4, which is expressed in as many as 23 different tumor types, is a exciting new target [1]. Most models of CXCR4 function in cancer focus on its potential role as a mediator of motility, invasiveness, and metastatic behavior [2]. CXCR4 activation, is necessary for growth in intracranial models of primary brain tumors [3] as well as in models of primary breast.
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