Linking γ-aminobutyric acid A receptor to epidermal growth factor receptor pathways activation in human prostate cancer

Abstract

Neuroendocrine (NE) differentiation has been attributed to the progression of castration-resistant prostate cancer (CRPC). Growth factor pathways including the epidermal growth factor receptor (EGFR) signaling have been implicated in the development of NE features and progression to a castration-resistant phenotype. However, upstream molecules that regulate the growth factor pathway remain largely unknown. Using androgen-insensitive bone metastasis PC-3 cells and androgen-sensitive lymph node metastasis LNCaP cells derived from human prostate cancer (PCa) patients, we demonstrated that γ-aminobutyric acid A receptor (GABA_AR) ligand (GABA) and agonist (isoguvacine) stimulate cell proliferation, enhance EGF family members expression, and activate EGFR and a downstream signaling molecule, Src, in both PC-3 and LNCaP cells. Inclusion of a GABA_AR antagonist, picrotoxin, or an EGFR tyrosine kinase inhibitor, Gefitinib (ZD1839 or Iressa), blocked isoguvacine and GABA-stimulated cell growth, trans-phospohorylation of EGFR, and tyrosyl phosphorylation of Src in both PCa cell lines. Spatial distributions of GABA_AR α₁ and phosphorylated Src (Tyr416) were studied in human prostate tissues by immunohistochemistry. In contrast to extremely low or absence of GABA_AR α₁-positive immunoreactivity in normal prostate epithelium, elevated GABA_AR α₁ immunoreactivity was detected in prostate carcinomatous glands. Similarly, immunoreactivity of phospho-Src (Tyr416) was specifically localized and limited to the nucleoli of all invasive prostate carcinoma cells, but negative in normal tissues. Strong GABA_AR α₁ immunoreactivity was spatially adjacent to the neoplastic glands where strong phospho-Src (Tyr416)-positive immunoreactivity was demonstrated, but not in adjacent to normal glands. These results suggest that the GABA signaling is linked to the EGFR pathway and may work through autocrine or paracine mechanism to promote CRPC progression.

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: GABA_A receptors (GABA_ARs), ligand-gated ion channels or ionotropic receptors, and GABA_B receptors (GABA_BRs), G-protein coupled receptors. Postsynaptic GABA_ARs are heteropentamers composed of subunits from 7 different families (α_1–6, β_1–3, γ_1–3, δ, ε, θ, and ρ_1–3) to form a GABA-activated chloride channel. Composition of GABA_AR 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 GABA_AR 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–GABA_AR 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 GABA_AR; 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 GABA_AR 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 GABA_AR-mediated proliferation of PCa cells. For the first time, we demonstrated that stimulation of the GAGA_AR 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 GABA_AR 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 GABA_AR 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.