GABA_B Receptor Complex as a Potential Target for Tumor Therapy

Abstract

γ-Aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the vertebrate central nervous system. Metabotropic GABA_B receptors are heterodimeric G-protein-coupled receptors (GPCRs) consisting of GABA_B1 and GABA_B2 subunits. The intracellular C-terminal domains of GABA_B receptors are involved in heterodimerization, oligomerization, and association with other proteins, which results in a large receptor complex. Multiple splice variants of the GABA_B1 subunit have been identified in which GABA_B1a and GABA_B1b are the most abundant isoforms in the nervous system. Isoforms GABA_B1c through GABA_B1n are minor isoforms and are detectable only at mRNA levels. Some of the minor isoforms have been detected in peripheral tissues and encode putative soluble proteins with C-terminal truncations. Interestingly, increased expression of GABA_B receptors has been detected in several human cancer cells and tissues. Moreover, GABA_B receptor agonist baclofen inhibited tumor growth in rat models. GABA_B receptor activation not only induces suppressing the proliferation and migration of various human tumor cells but also results in inactivation of CREB (cAMP-responsive element binding protein) and ERK in tumor cells. Their structural complexity makes it possible to disrupt the functions of GABA_B receptors in various ways, raising GABA_B receptor diversity as a potential therapeutic target in some human cancers.

γ-Aminobutyric acid (GABA), the main inhibitory neurotransmitter in the vertebrate brain, acts on ionotropic (GABA_A or GABA_C) and metabotropic (GABA_B) receptors. Ionotropic GABA receptors are ligand-gated chloride channels that mediate fast GABA response, whereas metabotropic GABA_B receptors, belonging to the C family of G-protein-coupled receptors (GPCRs), mediate slow GABA response by activating G-proteins and their downstream effectors (Pinard et al. 2010). GABA_B receptors were discovered in the mammalian central nervous system by Bowery and colleagues (1980) and cloned by Bettler’s research team (Kaupmann et al. 1997) with similarity to other metabotropic receptors in sequence. Being distributed in most regions of the brain, GABA_B receptors have been implicated in a variety of neurological and psychiatric disorders, including epilepsy, spasticity, schizophrenia, anxiety, depression, cognitive impairment, and drug abuse (Bettler et al. 2004; Filip and Frankowska 2008). GABA_B receptors, therefore, have become key therapeutic targets in neurological diseases. It is of particular interest that GABA_B receptors are also involved in tumor development and tumor cell proliferation and migration. This review summarizes the molecular complexity of GABA_B receptors and the influences of GABA_B receptors in tumor cells.

Molecular Organization of GABA_B Receptors

GABA_B receptors are heterodimers composed of GABA_B1 and GABA_B2 subunits, which are encoded by two different genes (Kaupmann et al. 1997; Jones et al. 1998; Kaupmann, Malitschek, et al. 1998; White et al. 1998; Ng et al. 1999). Both subunits contain a large extracellular N-terminal domain, seven-transmembrane domains, and a short intracellular C-terminal domain. Importantly, the C-terminal domains of both subunits contain a coiled-coil structure involved in the heterodimerization of GABA_B receptors (Kaupmann, Malitschek, et al. 1998; Bettler et al. 2004; Benke 2010) (Fig. 1).

Figure 1.

The schematic structure of heterodimeric GABA_B receptors and their downstream effector systems. The extracellular domain (ECD) of GABA_B1 can bind endogenous neurotransmitter (GABA) and receptor ligands (agonists and antagonists), whereas the ECD of GABA_B2 has no ligand-binding activity. The endoplasmic reticulum (ER) retention motif RSRR locates at the C-terminus of GABA_B1, proximal to the coiled-coil domain. Allosteric modulators such as CGP7930 bind to the transmembrane domains of GABA_B2. The intracellular loops of GABA_B2 are coupled to G_i/o-type G-proteins. Activation of GABA_B receptors regulates the opening of Ca²⁺ and K⁺ channels and the activities of adenylyl cyclase (AC) and phospholipase C (PLC). GABA_B receptors may also activate phosphoinositide 3-kinase (PI3K) and classical mitogen-activated protein kinase (MAPK) signaling pathways. The dashed lines indicate unconfirmed signaling cascades following GABA_B receptor activation. RTK, receptor tyrosine kinase.

Despite their similar three-dimensional structures, GABA_B1 and GABA_B2 subunits have different ligand-binding capabilities, G-protein coupling, and cell surface trafficking (Fig. 1). First, the extracellular domain of GABA_B1 is capable of binding endogenous neurotransmitter (i.e., GABA), agonists (i.e., baclofen), and antagonists (i.e., CGP54626 or CGP54626) (Kaupmann et al. 1997; Filip and Frankowska 2008). The extracellular domain of GABA_B2 has no ligand-binding activity, whereas the transmembrane domain of GABA_B2 can bind allosteric modulators (i.e., CGP7930), which have no intrinsic activity but modulate the affinity of the ligand for GABA_B1 and signal transduction efficacy following ligand binding (Kaupmann, Malitschek, et al. 1998; Galvez et al. 2001; Kniazeff et al. 2002; Urwyler et al. 2005; Filip and Frankowska 2008). Second, the intracellular domain of GABA_B2 but not GABA_B1 is coupled to G-proteins and regulates the activities of the Ca²⁺ channel, K⁺ channel, adenylyl cyclase (AC), or phospholipase C (PLC) (Galvez et al. 2001; Margeta-Mitrovic et al. 2001; Robbins et al. 2001; Duthey et al. 2002; Havlickova et al. 2002; Bettler et al. 2004). Third, GABA_B1 is prevented from reaching the cell surface due to an endoplasmic reticulum (ER) retention signal (RSRR) in its intracellular tail. However, the coiled-coil interaction between C-terminal tails of GABA_B1 and GABA_B2 subunits can mask the ER retention signal, resulting in cell surface trafficking of heterodimeric GABA_B receptors (Calver et al. 2001; Margeta-Mitrovic et al. 2001; Pagano et al. 2001). As a result, GABA_B receptors are obligatory heterodimers, and each subunit is functionally impaired when expressed alone in heterologous cells. Moreover, mice deficient in GABA_B1 or GABA_B2, with decreased protein expression of GABA_B2 or GABA_B1, respectively, have lost typical GABA_B responses such as the inhibition of glutamate release, suggesting that both subunits are required for normal receptor function in vivo (Prosser et al. 2001; Schuler et al. 2001; Gassmann et al. 2004).

Recent studies have been revealed that heterodimeric GABA_B receptors can form higher-ordered oligomers. Using snap-tag technology to specifically label cell surface GABA_B1 and GABA_B2 with time-resolved fluorescence resonance energy transfer (TR-FRET)–compatible fluorophores, Pin’s results identified that GABA_B receptors can spontaneously form dimers of heterodimers through the interactions between GABA_B1 subunits in a heterologous system (Maurel et al. 2008). Further study using specific antibodies labeled with TR-FRET-compatible fluorophores demonstrated the existence of dimers of dimers in the mouse brain. A lower G-protein coupling efficacy per GABA_B heterodimer was observed in the tetramers, suggesting a negative functional cooperativity between the heterodimers (Comps-Agar et al. 2011).

Several GABA_B receptor–associated proteins have been identified since 1999. The extracellular domains of GABA_B receptors interact with the extracellular matrix protein fibulin, which binds to the Sushi domains (SDs) of the GABA_B1 subunit (Fig. 2). The C-termini of GABA_B receptors interact with a variety of proteins, including transcription factors ATF4 (activating transcription factor 4)/CREB2 (cAMP-responsive element binding protein 2) and CHOP, scaffold proteins 14-3-3, NEM-sensitive factor (NSF) and tamalin, PDZ domain-containing protein MUPP1, and actin-binding protein β-filamin (reviewed in Bettler et al. 2004). Recently, potassium channel tetramerization domain-containing (KCTD) proteins KCTD-8, 12, 12b, and 16 were identified as auxiliary GABA_B receptor subunits. The KCTDs exist as tetramers, and each tetramer offers one binding site for the C-terminus of GABA_B2, resulting in heteromultimers composed of GABA_B1, GABA_B2, and KCTD tetramers. KCTD proteins increase the agonist potency and significantly alter the G-protein signaling of GABA_B receptors (Schwenk et al. 2010).

Figure 2.

Schematic structures of GABA_B1 isoforms. (A) GABA_B1a is encoded by 23 exons. The N-terminal Sushi domains (SDs) (purple box) are encoded by exons 3 and 4, the ligand-binding domain (white box) is encoded by exons 5 to 14, the seven-transmembrane domains (blue box) are encoded by exons 15 to 21, and the C-terminal domain is encoded by exons 22 and 23, with the coiled-coil domain being yellow and the C-terminal tail being orange. The endoplasmic reticulum (ER) retention signal is indicated by the black bar. (B) Alignment of GABA_B1 isoforms. Compared with GABA_B1a, GABA_B1b lacks the N-terminal SDs. Human GABA_B1c lacks one SD encoded by exon 4 (Martin et al. 2001). Rat GABA_B1(c-a) and GABA_B1(c-b) are similar to GABA_B1a and GABA_B1b, respectively, except that the former two contain an in-frame insertion of 93 bp between exons 4 and 5 (light blue box) (Isomoto et al. 1998). GABA_B1d has no SDs at the N-terminus, but it contains an insertion of 566 bp at the 3′-end, resulting in a divergent C-terminus with half of the coiled-coil domain being deleted and the loss of the ER retention signal (light orange box) (Isomoto et al. 1998). Due to the skipping of exon 15, GABA_B1e encodes the extracellular ligand binding domain with a C-terminal truncation (only 9 amino acid residues of the transmembrane domain are retained; indicated by the dark blue box) (Schwarz et al. 2000). GABA_B1f contains an in-frame deletion of exon 5, resulting in a 21-bp deletion in the extracellular N-terminal domain and a 93-bp in-frame insertion between exons 4 and 5 (light blue box) (Wei, Eubanks, et al. 2001). GABA_B1g contains exons 1 to 4 followed by 124 bp of intron 4, and the predicted protein is a C-terminal-truncated GABA_B1a with the N-terminal 157 amino acids identical to those of GABA_B1a and a unique C-terminal sequence of 82 amino acids (Wei, Jia, et al. 2001). GABA_B1h is characterized by an insertion of 80 bp at the 3′-end of exon 5, and the predicted protein is a C-terminal-truncated GABA_B1a with the first 164 amino acids identical to the corresponding sequence in GABA_B1a and a unique C-terminal sequence of 39 amino acids. GABA_B1i contains the first 4 exons, an insertion of 124 bp of intron 4, and an 80-bp insertion at the 3′-end of exon 5, with the two insertions separated by exon 5 (21 bp). The predicted GABA_B1i protein is a C-terminal-truncated GABA_B1a with the N-terminal 157 amino acids identical to those of GABA_B1a and a unique C-terminal sequence of 49 amino acids (Holter et al. 2005). Rodent GABA_B1j contains exons 1 to 4 followed by 870 bp of intron 4 as 3′-UTR. Similar to GABA_B1g/1i, the N-terminal 157 amino acids of the predicted rodent GABA_B1j protein are identical to those of GABA_B1a, but the C-terminal 72 residues have no significant homology to known proteins. Human GABA_B1j is similar to rodent GABA_B1j but lacks the C-terminal 39 amino acids of rodent GABA_B1j (Tiao et al. 2008; Lee et al. 2010). The unique C-termini of GABA_B1g/h/i/j are indicated by the light yellow box. GABA_B1k contains the 3′ part of intron 4 as 5′-UTR and exons 5 to 23; the predicted GABA_B1k protein (784 amino acids) is an N-terminal-truncated GABA_B1a containing a ligand-binding site, seven-transmembrane domains, and an intracellular C-terminal domain. GABA_B1l is similar to GABA_B1k but has additional skipping of exon 15. Compared with GABA_B1l, GABA_B1m has additional deletion of the 5′ part of exon 6. The predicted GABA_B1l and GABA_B1m proteins are both N-terminal and C-terminal truncated, with the C-terminal truncation identical to GABA_B1e. GABA_B1n has a single-nucleotide polymorphism (SNP) at exon 4, and the predicted GABA_B1n protein (107 amino acids) does not have any functional domains except a partial ligand-binding site (Lee et al. 2010). The letters in parentheses indicate the species from which the cDNAs were cloned (h for human, r for rat, m for mouse). Adapted from Bettler et al. (2004) and Lee et al. (2010).

GABA_B receptors may exist as a large complex due to oligomerization and association with other proteins. To investigate the localization, oligomerization, dynamics, and functions of GABA_B receptors in living cells, Liu and his collaborators developed a three-part probe consisting of GABA_B1 high-affinity antagonist CGP64213, a photo–cross-linking group, and a fluorophore or biotin tag. The probe has been shown to bind the GABA_B1 subunit specifically on the cell surface in living Chinese hamster ovary (CHO) cells, providing the first line of evidence for its future use in studying GABA_B receptors in living systems (Li et al. 2008).

Molecular Diversity of GABA_B1 Subunits

GABA_B1 is distinguished by its molecular diversity. To date, 14 splice variants, designated GABA_B1a through GABA_B1n, have been reported (Fig. 2). However, most of the isoforms have been detectable only at mRNA levels, and some exist only in certain species. The most abundant GABA_B1 isoforms are GABA_B1a and GABA_B1b. GABA_B1a is the longest isoform and encoded by 23 exons. A tandem pair of SDs, encoded by exons 3 and 4, respectively, is present at the N-terminus of GABA_B1a SD, consisting of ~60 amino acids, also known as the short consensus repeat or complement control module, which exists in a variety of complement and adhesion proteins that are involved in protein-protein interactions. However, the function of GABA_B1a SDs is unclear. Compared with GABA_B1a, GABA_B1b has no SDs at its N-terminus due to an alternative transcription initiation site within intron 5. As a result, the N-terminal 147 amino acids of mature GABA_B1a are replaced by 18 different residues in GABA_B1b (Fig. 2). GABA_B1a and GABA_B1b are the only isoforms that are highly conserved in human, rat, mouse, chicken, frog, and zebrafish (Kaupmann et al. 1997; Kaupmann, Schuler, et al. 1998; Bettler et al. 2004), but they have distinct temporal and spatial expression patterns. GABA_B1a protein expression in the brain of the newborn mouse was maintained at a high level until postnatal day 7, after which the expression decreased, with a significantly lower level in the adult brain. By contrast, GABA_B1b protein was generally expressed at higher levels in the adult brain compared with the fetal brain. In the cerebellum, GABA_B1a mRNAs were predominantly in the granule cell layer, whereas GABA_B1b transcripts were mostly expressed in Purkinje cells (Bettler et al. 2004). Further studies using mice with mutations in the initiation codon of GABA_B1a or GABA_B1b, referred to as GABA_B1a^–/– or GABA_B1b^–/– mice, respectively, showed differential compartmentalization and distinct functions of these two isoforms. In the CA1 region of the hippocampus, GABA_B1a was predominantly localized at glutamatergic terminals, whereas GABA_B1b was mostly localized in dendritic spines. Similar results were obtained from the organotypic hippocampal slice cultures, in which transfected GABA_B1a–green fluorescence protein (GFP) was selectively expressed in distal axons, whereas GABA_B1b-GFP was expressed in the majority of dendritic spines, implying that SDs might be responsible for retaining GABA_B1a at distal axons. At hippocampal CA3-to-CA1 synapses, GABA_B1a heteroreceptors inhibited presynaptic glutamate release, whereas GABA_B1b mediated postsynaptic inhibition. Of note, synaptic plasticity and hippocampus-dependent memory were impaired in GABA_B1a^–/– mice rather than GABA_B1b^–/– mice, suggesting the functional specialization of GABA_B1a and GABA_B1b isoforms (Vigot et al. 2006; Pinard et al. 2010).

Compared with GABA_B1a and GABA_B1b, reports regarding the minor isoforms GABA_B1c through GABA_B1n are quite limited, with only one or two publications for each isoform. GABA_B1c transcripts were detected in human and rat (Fig. 2). The expression pattern of human GABA_B1c mRNA parallels that of GABA_B1a (Martin et al. 2001). It would be interesting to know whether a single SD is enough to guide GABA_B1c to its appropriate destinations. Rat GABA_B1c transcripts were widely distributed in the brain, with the highest expression levels in cerebellar Purkinje cells, cerebral cortex, thalamus, and hippocampal CA1 and CA3 regions. When coexpressed with GABA_B2, GABA_B1c forms a functional receptor in Xenopus oocytes and HEK-293 cells, suggesting that GABA_B1c might be functional in the rat brain (Isomoto et al. 1998; Pfaff et al. 1999).

Rat GABA_B1d transcript (Fig. 2) was detected in the forebrain, cerebellum, eye, kidney, and urinary bladder, indicating that the protein may function in peripheral tissues in addition to the central nervous system (CNS) (Isomoto et al. 1998).

GABA_B1e transcript (Fig. 2) is a minor component in the CNS of both humans and rats, but it is the primary isoform in a variety of peripheral tissues (Schwarz et al. 2000). Interestingly, the GABA_B1e transcript was detected in human lung epithelial adenocarcinoma cells H441 (Mizuta et al. 2008). The broad mRNA expression of GABA_B1e indicates that it may have important functions in peripheral tissues and even tumor cells, which is unrelated to synaptic regulation. When expressed in HEK293 cells, GABA_B1e was both secreted and membrane associated. In addition, GABA_B1e heterodimerized with coexpressed GABA_B2 and impaired the normal GABA_B1a/GABA_B2 association. However, GABA_B1e lacked the ability to bind antagonist CGP54626A and had no influence on the activity of K⁺ channel and cAMP production when it was expressed alone or together with GABA_B2 in heterologous systems (Schwarz et al. 2000). GABA_B1e may regulate other molecular events, which are not typical of GABA_B receptors.

GABA_B1f/1g/1h/1i were identified from rat cDNA library screens (Fig. 2). GABA_B1f and GABA_B1g transcripts were prevalently expressed in peripheral tissues (heart, liver, lung, kidney, colon, stomach, spleen, and testis) when compared with brain tissues (Wei, Eubanks, et al. 2001; Wei, Jia, et al. 2001), whereas GABA_B1h and GABA_B1i transcripts were widely expressed in the brain and peripheral tissues with variable expression levels (Holter et al. 2005).

GABA_B1j transcripts were detected in human, rat, and mouse (Fig. 2). When compared with GABA_B1a, the relative expression level of the GABA_B1j transcript was remarkably lower in the human brain than in rat and mouse brains (Tiao et al. 2008; Lee et al. 2010). It is unknown whether the GABA_B1j transcript is similarly expressed in peripheral tissues as GABA_{B1e/1f/1g/1h/1i}. GABA_B1j was expressed as a secreted glycoprotein in HEK293 cells. An anti-SD monoclonal antibody made by Bettler’s laboratory could immunoprecipitate a protein corresponding to GABA_B1j in molecular mass from rat cortical neurons, providing indirect evidence for the expression of GABA_B1j protein in vivo. Recombinant SD protein, lacking the C-terminal 72 amino acids of rat GABA_B1j, impaired the inhibitory effect of GABA_B heteroreceptors on glutamate release, but the function of full-length GABA_B1j was not studied (Tiao et al. 2008).

GABA_B1k/l/m/n were recently cloned in human and/or mouse (Fig. 2). It is reported that GABA_B1l and GABA_B1m disrupted the function of GABA_B1a/GABA_B2 when expressed in Xenopus oocytes (Lee et al. 2010).

In contrast to the molecular diversity of GABA_B1, there are no confirmed isoforms of GABA_B2 so far (Bettler et al. 2004). Taken together, GABA_B1 has multiple isoforms. Of note, there is no direct evidence for the in vivo protein expressions of minor isoforms 1c through 1n. Because isoforms 1e and 1j lacking transmembrane domains are expressed as secreted proteins in heterologous systems, isoforms 1g, 1h, 1i, 1l, and 1m, all of which have C-terminal truncations, may also be expressed as secreted proteins in vivo. Are these proteins able to disrupt the association of GABA_B1a/GABA_B2 as well as impair the function of GABA_B receptors? Based on the ubiquitous mRNA expressions of isoforms 1d through 1i in peripheral tissues, could the transcripts of minor isoforms 1j through 1n also be detected in peripheral tissues? What are the functions of GABA_B1 isoforms, particularly the truncated isoforms in the peripheral tissues in vivo? All these questions need to be addressed.

GABA_B Receptor Expression in Tumors

A growing body of evidence showed that neurotransmitters have modulatory roles in tumor cells, therefore influencing tumor development and progression. Accordingly, the potential roles of neurotransmitter receptors in tumors have attracted more and more research interest (Schuller 2008). GABA_B receptors have been reported to be involved in tumor development and the proliferation and migration of tumor cells.

GABA_B Receptors and Tumor Development

The expression of GABA_B receptors was detected in human hepatocellular carcinoma (HCC) cell lines (HepG2, Bel-7402, Huh-7) and colon cancer cell lines (KM12SM, HT29, RKO) by immunoblotting (Thaker et al. 2005; Wang et al. 2008). Importantly, the expression level of GABA_B receptors was upregulated in some human tumor tissues when compared with the corresponding normal tissues. Roberts et al. (2009) assessed the expression of GABA_B receptors by immunohistochemistry in 70 human thyroid tumor samples, including 13 follicular adenomas, 14 follicular carcinomas, and 43 papillary carcinomas. No significant staining of GABA_B2 was detected in normal thyroid tissues, whereas 4 of the 13 follicular adenomas and 41 of the 57 thyroid carcinomas showed high intensity of GABA_B2 staining, indicating a positive correlation between GABA_B2 expression and the malignancy of thyroid tumors. Another study examined GABA_B receptor expression in 30 human gastric cancer samples. The immunostaining intensities of both GABA_B receptor subunits were significantly higher in gastric cancer tissues compared with the adjacent normal tissues. Furthermore, GABA_B receptors had different subcellular localization in cancer versus normal cells. Although GABA_B receptors were mainly distributed in the cytoplasm of normal cells, the majority of GABA_B receptors were localized on the cell surface of cancer cells (Zhu et al. 2004), suggesting an altered cell surface trafficking of GABA_B receptors in gastric cancer cells. Our group examined the expression of GABA_B1 in human ductal breast cancer (six cases) by immunohistochemistry, and the preliminary data (Fig. 3) showed that GABA_B1 expression was significantly higher in malignant tissues than in nonmalignant tissues (three cases). Surprisingly, GABA_B1 was distributed in the cytosol and/or nucleus instead of cell surface; the reason is unclear.

Figure 3.

GABA_B1 expression in human ductal breast cancer tissues. (A) Control staining in the absence of primary antibody. (B) Immunostaining of a fibroadenoma tissue. Weak staining of GABA_B1 was detected in the cytosol and occasionally in the nucleus. (C) and (D) are representative GABA_B1 immunostaining of human ductal breast cancer tissues (from patients 117909 and 117911, respectively). Intensive staining was observed in the cytosol and less frequently in the nucleus. Six cases in total were examined, and more specimens are being collected. Scale bar: 20 µm.

The increased expression of GABA_B receptors in tumors suggested a potential role of GABA_B receptors in tumor growth and development. Tatsuta et al. (1990) investigated the effects of GABA_B receptor activation on gastric carcinogenesis induced by N-methyl-N′-nitro-N- nitrosoguanidine in Wistar rats and found that systemic administration of GABA_B receptor agonist baclofen significantly reduced the incidence and number of gastric cancers. Historically, baclofen remarkably decreased the number of BrdU-labeled cells in the gastric mucosa, indicating an inhibitory role of GABA_B receptors in cell proliferation. Later on, Tatsuta et al. (1992) examined the effects of baclofen on colon carcinogenesis induced by azoxymethane in Wistar rats. The results showed that baclofen had no influence on the incidence but instead attenuated the malignancy of colon tumors. Although colon adenocarcinomas were developed in most rats of the control group, almost all the tumors developed in baclofen-treated rats were colon adenomas. In other words, baclofen treatment reduced the malignancy of colon tumors. Both studies suggested an inhibitory role of GABA_B receptor activation in tumor development.

GABA_B Receptors and Tumor Cell Proliferation and Migration

Several studies revealed that activation of GABA_B receptors suppresses tumor cell proliferation. Schuller et al. (2008a, 2008b) examined the effect of baclofen on the proliferation of human pancreatic ductal adenocarcinoma (PDAC) cells PANC-1 and BXPC-3, human pulmonary adenocarcinoma (PAC) cells NCI-H322, immortalized human pancreatic duct epithelial cells HPDE6-C7, and small airway epithelial cells HPL1D. The β-adrenergic receptor (β-AR) agonist isoproterenol significantly promoted the proliferation of these five cell lines, but baclofen completely abrogated such response. Wang et al. (2008) reported that the proliferation of hepatocarcinoma cells Bel-7402 and Huh-7 was inhibited by baclofen in a dose-dependent manner. Systemic administration of baclofen significantly suppressed the growth of Bel-7402 xenograft induced in nude mice. In addition, a remarkable growth inhibition of experimental mammary cancer 16/C was observed in mice treated with baclofen (Opolski et al. 2000). These observations suggest that GABA_B receptor activation inhibits tumor cell proliferation both in vivo and in vitro. However, when human prostate cancer cells LNCaP, MDA-PCA-2b, DU145, and PC-3 were treated with baclofen, no inhibitory effect on BrdU incorporation was observed (Abdul et al. 2008).

GABA_B receptor activation also inhibits tumor cell migration. The β-AR agonist norepinephrine strongly increased the migration of human colon carcinoma cells SW480 and breast carcinoma cells MDA-MB-468, but baclofen completely blocked the effect of norepinephrine (Drell et al. 2003; Joseph et al. 2002). Similarly, the stimulatory effect of isoproterenol on the migration of human PAC cells NCI-H322 and human PDAC cells PANC-1 and BXPC-3 was also abrogated by baclofen (Schuller et al. 2008a, 2008b). However, the invasion of C4-2 human prostate cancer cells as well as their expression of matrix metalloproteinase–3 (MMP-3) was significantly enhanced by baclofen but inhibited by GABA_B receptor antagonist CGP 35348, suggesting that activated GABA_B receptors may increase the invasive ability of tumor cells by promoting MMP-3 production (Azuma et al. 2003).

Collectively, GABA_B receptor activation has inhibitory effects on the proliferation and migration of most human tumor cell types but has no influence on the proliferation of human prostate cancer cells and even promotes the invasion of C4-2 cells. The underlying mechanism remains unclear. It is possible that human prostate cancer cells express high levels of soluble GABA_B1 isoforms, which compete for baclofen binding with GABA_B1a/1b, resulting in different responses to baclofen.

Signaling Events Downstream of GABA_B Receptor Activation in Tumor Cells

The classical mitogen-activated protein kinase (MAPK) pathway (Raf/MEK/ERK), activated by cell surface receptor tyrosine kinases (RTKs) following growth factor binding, regulates cell proliferation. Activation of the Raf/MEK/ERK pathway is a frequent event in different human cancers (Pratilas and Solit 2010). In human PAC cells NCI-H322 and immortalized human small airway epithelial cells HPL1D, β-AR agonist significantly increased the phosphorylation of ERK1/2 via dual signaling pathways involving the activation of protein kinase A (PKA) and transactivation of epithelial growth factor receptor (EGFR). Such response was completely abrogated by preexposure of the cells to baclofen (Laag et al. 2006; Schuller et al. 2008b). However, baclofen strongly induced the phosphorylation of ERK1/2 in the CA1 area of the mouse hippocampus as well as in cultured mouse cerebellar granular neurons (CGNs) (Vanhoose et al. 2002; Tu et al. 2007). The reason for the opposing effects of baclofen on cell proliferation remains unknown but may relate to differential expression of GABA_B1 isoforms in neurons and tumor cells. As mentioned previously, transcripts of the C-terminal-truncated GABA_B1e and GABA_B1g were mainly or prevalently expressed in peripheral tissues even in tumor cells (Schwarz et al. 2000; Wei, Jia, et al. 2001). The expression of these soluble GABA_B1 isoforms may impair the normal function of heterodimeric GABA_B1a/b/GABA_B2, leading to a different cellular response upon ligand binding.

Published data showed that cAMP production in a variety of human cancer cell lines induced by the β-AR agonist was completely blocked by baclofen (Joseph et al. 2002; Schuller et al. 2008a, 2008b; Wang et al. 2008). Similar results were obtained from rat CGNs, in which cAMP production stimulated by forskolin was reduced by baclofen in a dose-dependent manner (Barthel et al. 1996). CREB, a classic downstream effector of PKA, was strongly phosphorylated in NCI-H322 and HPL1D cells treated with β-AR agonist, whereas pretreatment of the cells with baclofen completely blocked CREB phosphorylation (Laag et al. 2006; Schuller et al. 2008b). Again, opposing effects were observed in mouse CGNs; briefly, baclofen induced ERK-dependent CREB phosphorylation (Tu et al. 2007).

CREB belongs to the ATF/CREB family of transcription factors, which is grouped into several subgroups, including CREB/CREM, CRE-BP1/ATF2, ATF3, ATF4/CREB2, ATF6, and B-ATF subgroups. ATF/CREB family members contain the consensus ATF/CRE binding site and share conserved C-terminal basic leucine zipper domains but vary in their N-termini, which contain phosphorylation sites for PKA, protein kinase C (PKC), or MAPK. Phosphorylation of ATF/CREB family members regulates gene transcription positively or negatively (Karpinski et al. 1992; Hai and Hartman 2001). ATF4/CREB2, a ubiquitous expressed transcription factor, contains phosphorylation sites for MAPK instead of PKA at the N-terminus (Karpinski et al. 1992). Colocalization of ATF4/CREB2 with GABA_B receptors has been observed in primary neuron cultures, and the interaction involves the leucine zipper of ATF4/CREB2 and the coiled-coil domain of GABA_B1a (Nehring et al. 2000; White et al. 2000; Vernon et al. 2001). The activation of the GABA_B receptor in mouse CGNs may transactivate an RTK, which has been observed in mouse CGNs (Tu et al. 2010), resulting in MAPK activation and subsequent CREB phosphorylation. The interaction between ATF4/CREB2 and GABA_B receptors provides a spatial advantage for the phosphorylation of ATF4/CREB2 by MAPK, which is also recruited to the vicinity of the cell membrane upon RTK activation. It would be interesting to know whether disrupting the interaction between ATF4/CREB2 and GABA_B receptors will suppress the stimulatory effect of baclofen on CREB phosphorylation in CGNs. Compared with ATF4/CREB2 in CGNs, the ATF/CREB family members expressed in tumor cells may contain phosphorylation sites for PKA, which are inactivated by the decreased cAMP level triggered by baclofen, leading to reduced phosphorylation of CREB. Identification of the predominant ATF/CREB family member(s) expressed in different tumor cell types will help researchers understand the mechanism regulating ATF/CREB phosphorylation by GABA_B receptors.

The phosphoinositide 3-kinase (PI3K) pathway regulates cell growth, survival, metabolism, and motility, and it is frequently activated in a variety of human cancers (Courtney et al. 2010; Wong et al. 2010). Currently, no published data show the activation of the PI3K pathway by the GABA_B receptor agonist in tumor cells, but a recent report revealed that activation of GABA_B receptors by baclofen in mouse CGNs transactivated insulin-like growth factor 1 receptor (IGF-1R), leading to Akt activation and protection of neurons from apoptosis (Tu et al. 2010). Probably, the PI3K pathway is also regulated by GABA_B receptors in tumor cells, which results in altered cell proliferation and migration.

As described above, the expression of GABA_B receptors in human gastric and thyroid cancer samples is upregulated compared with normal tissues, which may contribute to tumor development in vivo. The inhibitory effects of GABA_B receptor activation on tumor cell proliferation may result from the inactivation of the Raf/MEK/ERK pathway and CREB-dependent gene transcription. The mechanism of GABA_B receptor activation suppressing tumor cell migration remains unknown.

Conclusions

Heterodimeric GABA_B receptors may exist as a large complex due to oligomerization and association with a variety of other proteins, mainly through the C-termini of both subunits. Molecular diversity of the GABA_B1 subunit has resulted from alternative splicing, making the GABA_B receptor complex even more complicated. The expression of multiple GABA_B1 isoform transcripts in peripheral tissues suggests that GABA_B receptors have additional functions to that of synaptic regulation in neurons. Of interest, the expression of GABA_B receptors is increased in human gastric and thyroid cancers. GABA_B receptor activation inhibits tumor development in vivo and suppresses the proliferation and migration of a variety of human cancer cells in vitro, including pancreatic ductal adenocarcinoma, pulmonary adenocarcinoma, hepatocellular carcinoma, colon carcinoma, and breast carcinoma cells. It is necessary to further examine whether the expression of GABA_B receptors is upregulated in other human cancer types. Meanwhile, it is important to study the effects of GABA_B receptor activation on the proliferation and migration of other human cancer cell lines.

Because of the complexity of GABA_B receptors, there are multiple options to block the functions of GABA_B receptors in human cancers. The most time-saving and economic way is to test the antitumor effects of marketed drugs targeting GABA_B receptors, which were developed for clinical treatment of neurological and psychiatric disorders. The second option is to target GABA_B receptor–associated proteins to decrease the G-protein coupling efficacy or block the signaling cascades in tumors. KCTD, CREB, and ERK are good candidates for this purpose. If the protein expression of soluble GABA_B1 isoforms can be confirmed in vivo, it will then be conceivable to examine whether the serum concentration of soluble GABA_B1 proteins in cancer patients is significantly higher than that in healthy individuals. If this is the case, soluble GABA_B1 proteins could be used as tumor markers. In addition, antibodies made against soluble GABA_B1 proteins could be considered for clinical use. Putting all these together, GABA_B receptor complexes may be promising therapeutic targets in human cancers.