Linking γ-aminobutyric acid A receptor to epidermal growth factor receptor pathways activation in human prostate cancer
Introduction
γ-Aminobutyric acid (GABA) is an amino acid synthesized from glutamate via glutamic acid decarboxylase (GAD; GAD65 and GAD67). GABA acts as a neurotransmitter regulating neuronal excitability in the central nervous system (CNS) (Soghomonian and Martin, 1998); and neurons that produce GABA as their output are called GABAergic neurons. Although GABA is believed to inhibit nerve transmission in the brain (Miles et al., 1996), recent data suggest that it has both excitatory and inhibitory functions depending upon development stages of the brain (Li and Xu, 2008). GABA regulates excitability of the nervous system via two classes of receptors: GABAA receptors (GABAARs), ligand-gated ion channels or ionotropic receptors, and GABAB receptors (GABABRs), G-protein coupled receptors. Postsynaptic GABAARs are heteropentamers composed of subunits from 7 different families (α1–6, β1–3, γ1–3, δ, ε, θ, and ρ1–3) to form a GABA-activated chloride channel. Composition of GABAAR subunits determines functional property of the receptor. In addition to GABA, allosteric modulators can bind to different allosteric binding sites and indirectly modulate the activity of the receptor (Johnstone et al., 2011, Sancar and Czajkowski, 2011).
GABA has emerged as a tumor promoting molecule that controls tumor cell growth (Takehara et al., 2007); and a positive correlation between GABAergic system and oncogenesis has been reported (Hirano et al., 2004, Watanabe et al., 2006). For example, significantly increased GABA contents with elevated GAD and GABAAR expression have been detected in neoplastic tissues including colorectal (Kleinrok et al., 1998), gastric (Matuszek et al., 2001), pancreatic (Johnson and Haun, 2005), and breast cancers (Zafrakas et al., 2006).
The GABAergic signaling has been implicated in progression and metastasis of prostate cancer (PCa). In a rodent model, the GAD–GABA–GABAAR pathway is associated with a more aggressive neuroendocrine (NE) differentiation of PCa (Hu et al., 2002). GABA promotes the progression of prostate NE tumors via the ionotropic GABAAR; and inhibition of the GABA pathway by flumazenil administration suppresses NE tumor growth in a xenograft mouse model (Ippolito et al., 2006). In human cases, GABA significantly enhances PCa cell invasion in cultures; and levels of GAD67 are significantly elevated in cancerous prostate with metastasis versus those without metastasis (Azuma et al., 2003). In addition, more frequent and elevated expressions of GABAAR are positively associated with human PCa (Abdul et al., 2008). However, the precise action of GABAergic signaling in nonneuronal cells and role of this pathway in controlling PCa growth and metastasis remains unknown.
Epidermal growth factor receptor (EGFR) is strongly implicated in PCa progression to a castration-resistant state (Di Lorenzo et al., 2002), and has been proposed as a therapeutic target for treating PCa based on results from experimental models (Bianco et al., 2004, Sirotnak et al., 2002) and human trials (Vuky et al., 2009). Src, a nonreceptor tyrosine kinase (NRTK), is one of many molecules transducing signals from the EGFR (Kraus et al., 2003). Following phosphorylation, Src can activate mitogen-activated protein kinase (MAPK) and signal transducers and activators of transcription (STAT) signaling molecules (Olayioye et al., 1999). Activation of Src tyrosine kinase has also been associated with PCa progression in a murine model (Drake et al., 2012). In human cases, Src kinase activity is elevated in approximately 30% of cases with castration-resistant disease, and correlated with distant metastasis and shorter patient survival (Tatarov et al., 2009). Src inhibitors, including dasatinib, have been developed as therapeutic agents to control metastatic, castration-resistant PCa (CRPC) (Yu et al., 2011).
In this communication, we sought the mechanism of GABAAR-mediated proliferation of PCa cells. For the first time, we demonstrated that stimulation of the GAGAAR leads to EGFR signaling activation, including elevated expression of EGFR ligands, trans-phosphorytion of the EGFR, and tyrosyl phosphorylation of Src in human PCa cells. Immunohistochemical staining of human prostate tissues demonstrated that GABAAR subunit α1 and phospho-Src are differentially expressed between normal and cancerous prostates; and these two molecules are co-localized in cancerous areas. These results suggest that metabolic abnormality that accumulates GABA or other GABAAR positive allosteric modulators can activate GABAergic signaling with subsequent activation of the EGFR-Src pathway. Therapeutic interventions targeting GABAergic signaling might be a viable strategy to suppress CRPC progression.
Section snippets
Reagents and chemicals
RPMI 1640, F-12 nutrient mixture, OPTI-MEM, fetal bovine serum (FBS), penicillin–streptomycin, Trizol® reagent, CyQUANT® cell proliferation assay kit, and DNA-freeTM kit were purchased from Life Technologies (Grand Island, NY). Charcoal-dextran (CD) treated fetal bovine serum (FBS) with testosterone <10−10 M was obtained from HyClone (Logan, UT). Isoguvacine hydrochloride and picrotoxin was obtained from Tocris (Elisville, MO). Gefitinib (ZD1839 or Iressa) was a gift from Astra Zeneca
Isoguvacine and GABA-stimulated human PCa cell proliferation
GABAAR-mediated PCa cell growth was performed in the presence of isoguvacine (a GABAAR agonist) and GABA, under a serum deprived condition. Both PC-3 and LNCaP cells responded to isogucacine and GABA treatments with elevated growth in concentration-dependent manners (Fig. 1A and C). Significant increases in cell numbers were observed in PC-3 cells when 10−9 M isoguvacine and 10−12 M GABA were used versus untreated cells at day 2 day after treatment (Fig. 1B). Maximal LNCaP cell growth was observed
Discussion
In addition to the CNS, GABA and GABAAR have been identified in hormone-dependent organs, including the prostate. In rats, GABA contents are lower in the prostate than the brain (Erdo et al., 1983). Using a 3H-labled potent and selective GABAAR agonist, muscimol, GABAAR expression is limited to prostatic granular tissues in the rat (Napoleone et al., 1990). Ionotropic GABAAR is pentameric and generally consists of 2α, 2β and 1γ subunits, with α and β subunits being required for activation by
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