- Email: [email protected]
2-dependent phosphorylation
Cancer Letters 348 (2014) 100–108
Contents lists available at ScienceDirect
Cancer Letters journal homepage: www.elsevier.com/locate/canlet
GABAergic signaling facilitates breast cancer metastasis by promoting ERK1/2-dependent phosphorylation Depu Zhang 1, Xiaowei Li 1, Ziming Yao, Chuanfei Wei, Nannan Ning, Jingxin Li ⇑ Department of Physiology, School of Medicine, Shandong University, Jinan 250012, China
a r t i c l e
i n f o
Article history: Received 17 October 2013 Received in revised form 23 January 2014 Accepted 7 March 2014
Keywords: c-Aminobutyric acid GABA receptor Breast cancer Metastasis ERK
a b s t r a c t The present study aims to determine the role of c-aminobutyric acid (GABA) signaling molecules in breast cancer metastasis. Our results reveal that GABAergic system exists in breast cancer cells. Both the GABA synthetic enzyme. (GAD65/67) and GABAB receptor are expressed in 4T1 mouse breast cancer cells, MCF-7 human breast cancer cells and human breast cancer tissue. Baclofen, a GABABR agonist, significantly promoted 4T1 cells invasion and migration in vitro and metastasis in vivo, an event that was attenuated by GABABR antagonist CGP55845. Baclofen-induced breast cancer metastasis was mediated by ERK1/2 pathway. Ó 2014 Elsevier Ireland Ltd. All rights reserved.
Introduction Breast cancer is the most prevalent cancer in women worldwide, comprising 16% of all female cancers. Although much has been done about diagnosis and treatment, mortality of breast cancer is still high, expecially in economically developing countries. What is more serious is that the mortality of breast cancer has been increasing in many places [1]. c-Aminobutyric acid (GABA), is synthesized predominantly from glutamate by glutamate decarboxylase (GAD) and exerts its effects via ionotropic GABAA receptors and/or metabotropic GABAB receptors [2]. GABA is the main inhibitory neurotransmitter in the vertebrate central nervous system (CNS). However, out of the CNS, a growing amount of researches have been producing evidence that GABA and GABA receptor are present in peripheral tissues [3,4]. Our previous studies have also shown that GABAergic system exists in lung [5], small intestine [6] and oviduct [7] and plays an essential role in these organ. In addition to its role in the adult mammalian nervous system as an inhibitory neurotransmitter,
Abbreviations: GABA, c-aminobutyric acid; GABAAR, type A GABA receptors; GABABR, type B GABA receptors; GAD, glutamate decarboxylase; MMP, matrix metalloproteinase; CNS, central nervous system; FV, field of view; ERK, extracellular signal-regulated protein kinases; MAPK, mitogen-activated protein kinases. ⇑ Corresponding author. Tel.: +86 0531 88383902. E-mail address: [email protected] (J. Li). 1 These authors contributed equally as first author. http://dx.doi.org/10.1016/j.canlet.2014.03.006 0304-3835/Ó 2014 Elsevier Ireland Ltd. All rights reserved.
GABA takes part in the proliferation, differentiation, and migration of several kinds of cells including cancer cells [4,8]. Several recent reports have suggested a relationship between the GABAergic system and oncogenesis. It has been confirmed that both GABA content and GAD activity are increased in material from breast [9], colon [10], stomach [11], thyroid [12] and ovarian cancer [13]. Although studies have demonstrated that GABA and GAD are detectable both in normal mammary gland and in neoplastic alterations, the function of this molecules in breast cancer metastasis is unclear. In our present research, we found a marked expression of GABA signaling molecules GABAA receptor, GABAB receptor and GAD65/67 in the 4T1 mouse breast cancer cells. Importantly, we found that GABAB receptor pathway mediates breast cancer cell invasion/migration in vitro and metastasis in vivo. Previous studies have revealed that activation of GABAB receptor mediates the phosphorylation of extracellular signal-regulated protein kinases 1/2 (ERK1/2) [14,15]. We therefore investigated the underlying mechanisms, focusing on ERK pathway. Our results indicate that GABAB R pathway facilitates breast cancer cell invasion and migration by promoting the phosphorylaton of ERK1/2 and subsequently increasing the expression of matrix metalloproteinase-2 (MMP-2). Intriguingly, our studies revealed that baclofen elicited a biphasic effect on the phosphorylaton of ERK1/2 via differential signaling pathway in breast cancer cells. The present finding hint that the GABAB R may be a potential target for the treatment of breast cancer, which should be of great value clinically.
D. Zhang et al. / Cancer Letters 348 (2014) 100–108 Materials and methods Cell culture Two breast cancer cell lines (4T1 and MCF-7) were obtained from the American Type Culture Collection (ATCC). Cells were cultured in RPMI 1640 (Hyclone) with 2.05 mM L-glutamine and 10% fetal bovine serum (Hyclone and incubated at 37 °C under 5% CO2 atmosphere. Immunofluorescence Cells were seeded into dishes containing a few sterile coverslips on the bottom. Cells were used after 12 h in culture. Specifically, the cells were fixed to the coverslips with 4% paraformaldehyde for 20 min and washed three times with PBS and were blocked by 10% goat serum (goat serum in PBS) for 1 h at room temperature. In order, the cells were incubated with anti-GABAB R1 (abcam, ab55051, 1:200), rabbit anti-GAD65/67 (Sigma, 1:2000) diluted in 10% goat serum overnight at 4 °C. Next, the cells were incubated with FITC labled secondary antibodies (Invitrogen, 1:400 for GABAB R1 receptor; 1:2000 for GAD) for 1 h at room temperature, followed by washes with PBS. DAPI (Beyotime, c1002, 1 lg/ml) was used to stain the nucleus. The cover slips were mounted 75% glycerol. Negative controls were done with the omission of primary antibodies. Images were taken on a Nikon H550L microscope with a Penguin 600CL digital camera. The merged images were accomplished with the help of Adobo Photoshop software.
101
10 lL CCK-8 solution was added into each well. Cells were then incubated for another 4 h at 37 °C. The absorbance was measured by a microplate reader at the wave length of 450 nm. In each experiment, six parallel wells were set up. 4T1-breast cancer xenograft model pulmonary metastasis Assay Female BALB/c mice (8 weeks of age) weighing approximately 20 g, were inoculated with 2 105 4T1 tumor cells in the right mammary fat pad by gently penetrating the skin. After one week of inoculation, mice with approximate tumor size were divided into four groups and administered intraperitoneally with normal saline, baclofen (10 mg/kg), baclofen (30 mg/kg) and CGP55845 (30 mg/kg) respectively for 4 weeks. Every 3 days, measure tumor longitudinal (L) and transverse (T) diameters with vernier calipers and record the general condition of the mice expecially weight. Tumor volumes were calculated according to the formula V = L T2/2[16]. Sacrifice mice, get orthotopic neoplasm and weight. Then expose the chest cavity and trachea. The number of lung metastases was determined by staining with Bouin’s solution (saturated picric acid:formalin:acetic acid = 15:5:1) after washing the lungs with PBS. White tumor nodules against a yellow lung background were counted by two independent observers. All experimental procedures were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals of Shandong University, and the study was approved by the Medical Ethics Committee for Experimental Animals, Shandong University, China (number ECAESDUSM 2012029). Statistical analysis
Western blot assays Human breast cancer tissues and matched non-tumor tissues (n = 3) were obtain from the Provincial Hospital of Shandong University, China. For all participants in this study, written informed consent was obtained. The protocol was approved by the Ethical Committee of Shandong University. The lysates were obtained using RIPA Lysis Buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, sodium orthovanadate, sodium fluoride, EDTA, leupeptin and so on) supplemented with PMSF. Protein concentrations were determined with a BCA Protein Assay Kit (Beyotime, P0012). Appropriate amounts of protein lysates were separated on SDS–PAGE and then transferred onto polyvinylidene difluoride membranes. The membranes were blocked in TBST buffer (Tris-buffered saline with 0.1% Tween-20) that contained of 5% skim milk. The membranes were incubated overnight at 4 °C in primary antibodies (GABAB R1 receptor antibodies, solarbio, a8100, 1:200; rabbit anti-GAD65/67, Sigma, 1:10,000; p-ERK antibodies, CST, 9101S, 1:1000; t-ERK antibodies, CST, 9102s, 1:1000; MMP-2 antibodies, Immunoway, B9801, 1:1000; b-actin antibodies, Santa Cruz, B0211, 1:2000) diluted in primary antibody dilution buffer (Beyotime, P0023A). The next day, membranes were washed in TBST, and incubated with horseradish peroxidase-conjugated secondary antibody (ZSGB-BIO, ZB-2301,1:400 for GABAB R1 receptor, 1:10,000 for p-ERK, 1:15,000 for t-ERK, 1:2000 for MMP-2, 1:2000 for b-actin; Beyotime, 90126, 1:400 for GAD). If necessary, membranes were subsequently stripped and reprobed to evaluate the expression of t-ERK. Immunoreactive proteins were detected using enhanced chemiluminescence (Millipore). Anti-b-actin and antit-ERK were used as controls of MMP-2 and p-ERK for the Western blots, respectively. The levels of p-ERK or MMP-2 were quantified using ImageJ software and all bands were normalized to percentages of control values.
Transwell invasion and migration assays Assays were performed using a modified Boyden chamber (Corning Costar, Rochester, NY, USA) containing a gelatin coated polycarbonate membrane filter (6.5 mm diameter, 8 lm pore size). The upper surface of the filter was coated with 50 ll Matrigel (0.17 mg ml–1; BD Biosciences, Bedford, MA, USA) for invasion assay. 8 104 cells were seeded into the transwell insert chamber with RPMI 1640 that contained of 1% FBS and then the inserts were placed in the lower chambers filled with 500 ll of RPMI 1640 that contained of 10% FBS. Chambers were incubated at37 °C under 5% CO2 atmosphere for different time (baclofen invasion for 24 h, baclofen invasion with U0126 preincubation and CGP invasion for 36 h, migration for 12 h). Thereafter, non-migrated cells were scraped from the upper surface of the membrane with a cotton swab. Migrated cells remaining on the bottom surface were viewed under microscope and counted in >10 fields of view (FV) at 100 magnification after staining with crystal violet.
Cell proliferation studies The proliferation of 4T1 cells was assessed by Cell Counting Kit-8 (CCK-8, Beyotime, C0038) assay according to the protocol recommended by the manufacturer. This method is an optical density colorimetric assay that quantifies the number of viable cells based on the activity of cellular dehydrogenases. To estimate the number of viable cells, approximately 2000 cells were seeded in each well of 96-well plates with 100 lL medium. Baclofen was added to each well with final concentrations of 0 lM, 10 lM and 100 lM for 12 h, 24 h and 48 h. After treatment,
Data was expressed as mean ± s.e.m. Means were compared with One-way analysis of variance (ANOVA) followed by Dunnett’s test or t-tests. The differences between groups were analyzed using the Sigma Stat 3.5 software (SPSS,Chicago,IL). A P-value < 0.05 was considered as significant. (⁄: p < 0.05, ⁄⁄: p < 0.01).
3. Results GABAergic system existed in breast cancer cells We performed immunoblot assays and immunofluorescence staining to examine the expressions of GABABR1, GAD65/67 and GABAAR subunits in breast cancer cells. Our results showed that GABABR1 and GAD65/67 were expressed distinctively in breast cancer cell line 4T1 cells, MCF-7 cells and human breast cancer tissue. (Fig. 1), and several types of GABAAR subunits including a1, b2 or b3 were also observed (Supplement Fig. 1A and B). These results suggest that GABAergic signaling-related molecules are expressed in breast cancer cells. GABAB pathway involved in the invasion and migration of breast cancer cells Next, we investigate the role of GABAergic signaling in breast cancer cell invasion and migration using transwell invasion and migration assays. Both of the upper and lower chambers contained different concentration of GABABR agonist baclofen. Chambers were incubated at37 °C under 5% CO2 atmosphere for 24 h (invasion assay) or 8 h (migration assay). Fig. 2A showed that baclofen significantly increased the invasive ablity of 4T1 breast cancer cells with dose-dependence. Similar effects were observed for migration assay without Matrigel-coated transwells (Fig. 2A, low lane). The baclofen-induced increase in cell migration was further confirmed by wound-healing experiment (Supplemental Fig. 2). To assess the effect of baclofen on breast cancer cell proliferation, approximately 2,000 cells were seeded in each well of 96-well plates with 100 lL medium. Baclofen was added to each well with final concentrations of 0 lM, 10 lM and 100 lM. After incubation for 12 h, 24 h and 48 h, the number of viable cells were estimated by Cell Counting Kit-8 (CCK-8) assay. However, no effect on proliferation stimulated by baclofen was observed (Fig. 2B). In our studies, 4T1 cells also expressed GAD65/67, key GABA-synthesizing enzymes, thus they may release GABA. A new study had been carried to investigate the role of endogenous GABA in the invasion and migration of 4T1 cells. Fig. 2C showed that, after incubation of GABAB
102
D. Zhang et al. / Cancer Letters 348 (2014) 100–108
Fig. 1. GABAergic system existed in breast cancer cells. (A) Immunoblot of breast cancer cells and human breast cancer tissue for GABABR1 and GAD65/67 subunits. (B and C) Immunostaining of GABABR1 (green) and GAD65/67 (red). Nuclei were identified by DAPI staining (blue). Original magnification of immunohistochemical pictures, 400 in B and C. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
receptor antagonist CGP55845 for 36 h (invasion assay) or 12 h (migration assay), remarkaly inhibitory effects on the invasion and migration of 4T1 cells were observed (Fig. 2C). Several GABAA receptor subunits were also distributed in 4T1 cells (Supplemental Fig. 1A and B). To test whether GABAARs are involved in breast cancer metastasis, we carried out wound-healing assay with GABAAR agonist muscimol. However, our results showed that muscimol had no effect on the migration of 4T1 cells (Supplemental Fig. 1C). The involvement of GABAB receptors in breast cancer invasion and migration is mediated by ERK pathway ERK1/2 is a member of mitogen-activated protein kinases (MAPK) family. We detected the expression of p-ERK and t-ERK stimulated by baclofen with time gradient or concentration gradient by western blot assay. 4T1 cells were lysed after incubation for 0 s, 30 s, 1 min, 5 min, 10 min, 30 min, 1 h, 2 h, 4 h and 24 h. Fig. 3A
showed that baclofen activated ERK1/2 in a dose- and time-dependent fashion. The ERK phosphorylation levels was rapidly increased after incubation in 5 min, and then declined to basal levels of ERK phosphorylation 10 min later. Interestingly, after incubation of 1 h, the levels of ERK phosphorylation were upregulated again, then gradually lowered down to the basal level on 4 h. We also evaluated the levels of p-ERK stimulated by different concentration of baclofen. With the increasing of concentration, a gradually increase of p-ERK was observed. Conversely, CGP55845 significantly decreased the level of p-ERK (Fig. 3B). To further determine if the activation of ERK1/2 was required for the invasion and migration signaling triggered by baclofen, U0126, a highly specific MAPK kinase inhibitor to block ERK1/2 activation, was used to verify the effect of the ERK1/2 pathway on baclofen-induced cell invasion and migration. Data showed that pre-incubation of U0126 reversed the baclofen-induced increase in ERK phosphorylation (Fig. 3C).
D. Zhang et al. / Cancer Letters 348 (2014) 100–108
103
Fig. 2. GABAB pathway involved in the invasion and migration of 4T1 breast cancer cells. Baclofen significantly increased the invasive and migration ablity of 4T1 cells with time- and dose-dependence (n = 10) (A). Baclofen had no effect on proliferation (n = 6) (B). GABAB receptor antagonist CGP55845 remarkaly inhibited the invasion and migration of 4T1 cells (C). The invasive or migrative ability of 4T1 cells was expressed as mean ± s.e.m of cells/FV (n = 12) .The proliferation ability of 4T1 cell was expressed as mean ± s.e.m of absorbance/well (n = 6). Original magnification, 200 in A and C. ⁄; p < 0.05 compared with 0 or control; #; p < 0.05 compared with 10. FV, field of view.
Intriguingly, after the pretreatment of U0126, an opposite effect of baclofen on ERK phosphorylation was found (Fig. 3C), which was markedly attenuated by H-89, a selective PKA inhibitor (Fig. 4). Consistant with the change of ERK phosphorylation levels, baclofen significantly depressed the invasion and migration of 4T1 cells in the presence of U0126 (Fig. 3 D and E). These results indicated that GABAB receptor participates in breast cancer cells migration and invasion via ERK signaling.
Involvement of the GABAB receptor pathway on MMP production in cancer cells In order to further prove the effects of GABAB receptor on invasion and migration, the expression of MMP-2 in 4T1 cells was detected by western blot. Fig. 5A showed that baclofen
dose-dependently increased the expression of MMP-2. We also detected the effect of baclofen on the expression of MMP-2 in the pre-incubation of U0126. Conversely, baclofen decreased the expression of MMP-2 (Fig. 5B). This finding was consistant with the results in Fig. 3C–E. In addition, GABAB receptors antagonist CGP55845 decreased the expression of MMP-2 (Fig. 5C). These data demonstrate that MMP-2, as downstream of p-ERK, also take part in breast cancer cells migration and invasion mediated by GABAB receptor.
GABAB receptor pathway in vivo facilitated pulmonary metastasis of breast cancer To determine whether GABAB pathway can promote breast cance rmetastasis, we examined the efects of GABAB receptor
104
D. Zhang et al. / Cancer Letters 348 (2014) 100–108
Fig. 3. GABAB receptor-induced breast cancer invasion and migration was mediated by the MAPK/ERK pathway. Baclofen activated ERK in a dose-(A, right, n = 5) and time-(A, left, n = 3) dependent fashion in 4T1 cells. In contrast with baclofen, CGP55845 significantly decreased the level of p-ERK compared with control (B, n = 3). Pre-incubation of U0126 reversed the baclofen-induced increase in the levels of ERK phosphorylation (C, n = 3). Baclofen significantly depressed the invasion (D, n = 10) and migration (E, n = 10) of 4T1 cells in the presence of U0126. t-ERK1/2 was used as a loading control for A, B, C. Data are presented as mean ± s.e.m. ⁄; p < 0.05 and ⁄⁄; p < 0.01 compared with 0; # ; p < 0.05 compared with 10 (A) or control (C).
agonist baclofen or its antagonist CGP55845 on spontaneous lung metastasis using 4T1-breast cancer xenograts in BALB/c mice. The average number of tumor nodules was 20.6 in the salinetreated group and 71 in the baclofen-treated group. These results
indicated that baclofen-treatment (B10 and B30) significantly increased tumor colonization in the lung compared with the saline group (<0.01; Fig. 6A). Conversely, CGP55845 decreased the number of lung nodules. However, the primary tumor weight or size
D. Zhang et al. / Cancer Letters 348 (2014) 100–108
105
was no statistical difference among the four experimental groups (<0.05) (Fig. 6B and C). We measured the body weight of mice every week. As shown in Fig. 6D, The body weight of mice in the baclofen-treated group (B30) was significantly decreased compared with that in control group. Together, these results strongly suggested that GABAB receptor pathway facilitated pulmonary metastasis of breast cancer, but didn’t show obvious impact on orthotopic neoplasm.
Discussion
Fig. 4. The selective PKA inhibitor H-89 largerly alleviate the inhibitory effect of baclofen on ERK phosphorylation after preincubation of U0126 in 4T1 cells. t-ERK1/2 was used as a loading control. Data are presented as mean ± s.e.m (n = 3).⁄⁄; p < 0.01 compared with 0; #; p < 0.05 compared with control group (Ctr).
Our research found that breast cancer cells remarkablely expressed GABA receptor (GABAAR and GABABR) and GAD65/67, the key enzyme in GABA synthesis. This finding proved that breast cancer cells possess a GABAerigc signaling system. Then we detected the function of GABAergic system in breast cancer and found that GABAB receptor pathway facilitates breast cancer cell invasion/migration in vitro and metastasis in vivo, which was mediated by promoting the phosphorylaton of ERK1/2 and subsequently increasing the expression of MMP-2. Although many researches demonstrated activation of GABAB receptor is associated with the proliferation of some cancer cells like pancreatic cancer [17], lung adenocarcinoma [18], in our present studies, we found that baclofen had no effect on proliferation in breast cancer cells in vitro and tumor growth in vivo.
Fig. 5. GABAB receptor pathway mediated the MMP-2 production in 4T1 breast cancer cells. Baclofen dose-dependently increased the the expression of MMP-2 (A, n = 3). However, baclofen decreased the expression of MMP-2 in the U0126 pre-incubation (B, n = 3). GABAB receptors antagonist CGP5584 decreased the expression of MMP-2 (C, n = 3). b-actin was used as a loading control for A, B, C. Data are presented as mean ± s.e.m. ⁄; p < 0.05 compared with 0 or control; #; p < 0.05 compared with 10.
106
D. Zhang et al. / Cancer Letters 348 (2014) 100–108
Fig. 6. GABAB receptor pathway in vivo facilitated pulmonary metastasis of breast cancer. Baclofen-treatment (B10 and B30) significantly increased tumor colonization in the lung compared with the saline group. Conversely, CGP55845 decreased the number of lung nodules (A). However, the primary tumor weight or size was no statistical difference among the four experimental groups (B and C). The body weight of mice in the baclofen-treated group (B30) was significantly decreased compared with that in control group (D). Data are presented as mean ± s.e.m (n = 6).⁄; p < 0.05 and ⁄⁄; p < 0.01 compared with control.
Metabotropic GABAB receptors are heterodimeric G-proteincoupled receptors (GPCRs) consisting of GABABR1 and GABABR2 subunits. The extracellular domain of GABABR1 not GABABR2 has ligand-binding activity, but the intracellular domain of GABAB R2 not GABABR1 has regulating activity due to coupled to G-protein. However, GABA might result in the different effects on cancer growth/metastasis in cancer-type or GABA receptor-type dependent manner. Several reports indicate that GABAB receptor activation can inhibit the migration of colon carcinoma [19], pancreatic cancer [17], lung adenocarcinoma [18], hepatocarcinoma [20] and proliferation of pancreatic cancer [17], lung adenocarcinoma [18]. But an opposite effect of GABA or GABAB agonist was found in prostate [21] and renal cancer cells [22]. GABA or GABAB agonist baclofen can significantly promote the invasive ability of prostate and renal cancer cells through the production of matrix metalloproteinases (MMPs). On the other hand, different types of GABA receptors may mediate different effect on cancer growth. Activition of GABAA receptors can increase pancreatic cancer cell proliferation [23], but stimulation of the GABABR show an inhibitory role in human pancreatic cancer cells [17]. In our experiment, we found that GABAB receptor pathway promoted breast cancer cell invasion and migration, but activation of GABAA receptors had no such effect. Our in vivo studies further confirmed that GABAB receptor pathway involved in breast cancer cell metastasis. ERK1/2 is a member of MAPK family. After phosphorylation through different ways, p-ERK can transfer to the nucleus and phosphorylate transcription factors. Aberrant activation of the ERK pathway has been shown to be a key contributing factor to many types of human cancer [24–27]. It has been shown that
GABAB receptor activates ERK1/2 in neurons [28] and renal cancer cells [22]. For reason above, we detected the effects of activation of GABAB receptor on the levels of p-ERK. Our results showed that baclofen increased the levels of p-ERK in a time- and dose-dependent manner. Interestingly, two peaks of ERK activation occurred somewhere between 0 s–10 min for the first peak and 1–4 h for the second one, in which the first one was much shorter. A paradigm GPCR pathway is mediated by G protein-dependent and G protein-independent signaling, in which b-arrestins seems to be the most important is widely accepted. Although both signaling pathways can trigger MEK/ERK activation, it was reported they had distinct activation kinetics: the rapid and transient G protein-dependent activation and the slower but persistent b-arrestin-mediated activation [29], in accordance with our results-two peaks and different durations. Both of two peaks disappeared when MEK, the common pathway, was blocked. Futhermore, U0126 completely depressed the baclofen-induced increase in the levels of p-ERK. However, it is worthwhile to note that the opposite effect of baclofen on ERK phosphorylation, cell invasion and migration were observed after pre-incubation of U0126. Owing to the complexity of signal pathway, we have to consider other pathways induced by baclofen. Those, focusing on inhibiting MEK1/2 or its upstream such as ras, raf, b-arrestin, cannot explain the inhibitory effect of baclofen on ERK phosphorylation, so appropriate reasons should be settled in other signaling pathway. Fava G discovered activaton of GABAB receptor down-regulated ERK1/2 phosphorylation via cAMP/PKA dependent pathway [30]. The selective PKA inhibitor H-89 was chosen to investigate the inhibitory effect of baclofen on ERK phosphorylation after pre-incubation of U0126.
D. Zhang et al. / Cancer Letters 348 (2014) 100–108
Our result show that the opposite effect was largerly alleviated. Obviously, PKA pathway, one of GABAB receptor downstream signaling, involved in baclofen-induced down-regulation of ERK1/2 phosphorylation, which was much weaker than G protein and b-arrestin signaling, so its inhibitory effect cannot display without U0126. Certainly a detail of modulatory mechanism is still to be clarified and further studies are required. Cancer metastasis involves a complex cascade of events including detaching from the primary tumor, invading the stromal tissue, entering the circulation, extravasating and invading the target organ. During this process, proteolytic degradation of the extracellular matrix has been considered a rate-limiting step. MMPs are believed to be vitally important protease which enable tumor cells to penetrate the basement membrane. The expression of MMP-2 immunoreactive protein has been strongly associated with invasive and metastatic tumours in previous in vitro studies [31–33]. In breast cancer, MMP-2 immunoreactive protein is an independent prognostic marker of survival [34–36], but MMP-9 levels do not correlate with survival [36]. In the present study, we investigated the influence of activation of GABAB receptor on the expression of MMP-2 in 4T1 cells. Western blot analysis demonstrated that baclofen stimulation significantly increased the expression of MMP-2 with a concentration dependent manner, however, CGP55845 decreased the expression of MMP-2. Consistent with our findings from the measurement of the ERK phosphorylation and cell metastasis, baclofen also decreased the the expression of MMP-2 in the presence of U0126. In conclusion, this study provides clear evidence that GABA signaling molecules GABAA receptor, GABAB receptor and GAD65/67 are expressed in breast cancer cells, and the activation of GABAB receptor promotes breast cancer cell metastasis by enhancing p-ERK levels and subsequently increasing the expression of MMP-2. Authors’ contributions D.Z., X.L., Z.Y., C.W. and N.N. carried out all the experiments and wrote the manuscript. J.L. is the principal investigator of the laboratory in which the research was performed, designed all the experiments the contributed to the interpretation of the data and wrote the manuscript. All of the authors read and approved the final manuscript. Conflict of Interest None declared. Acknowledgement This work was funded by grant to J. Li from the National Natural Science Foundation of China, China (Grant No. 31171108). Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.canlet.2014. 03.006. References [1] M.H. Forouzanfar, K.J. Foreman, A.M. Delossantos, R. Lozano, A.D. Lopez, C.J. Murray, M. Naghavi, Breast and cervical cancer in 187 countries between 1980 and 2010: a systematic analysis, Lancet 378 (2011) 1461–1484. [2] J.T. Kittler, S.J. Moss, Modulation of GABAA receptor activity by phosphorylation and receptor trafficking: implications for the efficacy of synaptic inhibition, Curr. Opin. Neurobiol. 13 (2003) 341–347.
107
[3] M.K. Akinci, P.R. Schofield, Widespread expression of GABA(A) receptor subunits in peripheral tissues, Neurosci. Res. 35 (1999) 145–153. [4] S.Z. Young, A. Bordey, GABA’s control of stem and cancer cell proliferation in adult neural and peripheral niches, Physiology (Bethesda) 24 (2009) 171– 185. [5] Y.Y. Xiang, S. Wang, M. Liu, J.A. Hirota, J. Li, W. Ju, Y. Fan, M.M. Kelly, B. Ye, B. Orser, P.M. O’Byrne, M.D. Inman, X. Yang, W.Y. Lu, A GABAergic system in airway epithelium is essential for mucus overproduction in asthma, Nat. Med. 13 (2007) 862–867. [6] Y. Li, Y.Y. Xiang, W.Y. Lu, C. Liu, J. Li, A novel role of intestine epithelial GABAergic signaling in regulating intestinal fluid secretion, Am. J. Physiol. Gastrointest. Liver Physiol. 303 (2012) G453–460. [7] Z. Zhou, H. Sun, X. Li, Y. Li, S. Zhao, D. Zhang, Z. Yao, J. Li, A local GABAergic system is functionally expressed in human fallopian tube, Biochem. Biophys. Res. Commun. 398 (2010) 237–241. [8] M. Watanabe, K. Maemura, K. Oki, N. Shiraishi, Y. Shibayama, K. Katsu, Gamma-aminobutyric acid (GABA) and cell proliferation: focus on cancer cells, Histol. Histopathol. 21 (2006) 1135–1141. [9] M. Mazurkiewicz, A. Opolski, J. Wietrzyk, C. Radzikowski, Z. Kleinrok, GABA level and GAD activity in human and mouse normal and neoplastic mammary gland, J. Exp. Clin. Cancer Res. 18 (1999) 247–253. [10] K. Maemura, H. Yamauchi, H. Hayasaki, K. Kanbara, T. Tamayama, I. Hirata, M. Watanabe, Gamma-amino-butyric acid immunoreactivity in intramucosal colonic tumors, J. Gastroenterol. Hepatol. 18 (2003) 1089–1094. [11] M. Matuszek, M. Jesipowicz, Z. Kleinrok, GABA content and GAD activity in gastric cancer, Med. Sci. Monit. 7 (2001) 377–381. [12] S.S. Roberts, M.C. Mendonca-Torres, K. Jensen, G.L. Francis, V. Vasko, GABA receptor expression in benign and malignant thyroid tumors, Pathol. Oncol. Res. 15 (2009) 645–650. [13] C.S. Nicholson-Guthrie, G.D. Guthrie, G.P. Sutton, J.C. Baenziger, Urine GABA levels in ovarian cancer patients: elevated GABA in malignancy, Cancer Lett. 162 (2001) 27–30. [14] B.H. Im, H. Rhim, GABA(B) receptor-mediated ERK1/2 phosphorylation via a direct interaction with Ca(V)1.3 channels, Neurosci. Lett. 513 (2012) 89–94. [15] H. Tu, P. Rondard, C. Xu, F. Bertaso, F. Cao, X. Zhang, J.P. Pin, J. Liu, Dominant role of GABAB2 and Gbetagamma for GABAB receptor-mediated-ERK1/2/CREB pathway in cerebellar neurons, Cell Signal 19 (2007) 1996–2002. [16] C.G. Roehrborn, S. Sech, J. Montoya, T. Rhodes, C.J. Girman, Interexaminer reliability and validity of a three-dimensional model to assess prostate volume by digital rectal examination, Urology 57 (2001) 1087–1092. [17] H.M. Schuller, H.A. Al-Wadei, M. Majidi, GABA B receptor is a novel drug target for pancreatic cancer, Cancer 112 (2008) 767–778. [18] H.M. Schuller, H.A. Al-Wadei, M. Majidi, Gamma-aminobutyric acid, a potential tumor suppressor for small airway-derived lung adenocarcinoma, Carcinogenesis 29 (2008) 1979–1985. [19] J. Joseph, B. Niggemann, K.S. Zaenker, F. Entschladen, The neurotransmitter gamma-aminobutyric acid is an inhibitory regulator for the migration of SW 480 colon carcinoma cells, Cancer Res. 62 (2002) 6467–6469. [20] K. Szczaurska, M. Mazurkiewicz, A. Opolski, The role of GABA-ergic system in carcinogenesis, Postepy Hig. Med. Dosw. 57 (2003) 485–500. [21] H. Azuma, T. Inamoto, T. Sakamoto, S. Kiyama, T. Ubai, Y. Shinohara, K. Maemura, M. Tsuji, N. Segawa, H. Masuda, K. Takahara, Y. Katsuoka, M. Watanabe, Gamma-aminobutyric acid as a promoting factor of cancer metastasis; induction of matrix metalloproteinase production is potentially its underlying mechanism, Cancer Res. 63 (2003) 8090–8096. [22] T. Inamoto, H. Azuma, T. Sakamoto, S. Kiyama, T. Ubai, Y. Kotake, M. Watanabe, Y. Katsuoka, Invasive ability of human renal cell carcinoma cell line Caki-2 is accelerated by gamma-aminobutyric acid, via sustained activation of ERK1/2 inducible matrix metalloproteinases, Cancer Invest. 25 (2007) 574–583. [23] A. Takehara, M. Hosokawa, H. Eguchi, H. Ohigashi, O. Ishikawa, Y. Nakamura, H. Nakagawa, Gamma-aminobutyric acid (GABA) stimulates pancreatic cancer growth through overexpressing GABAA receptor pi subunit, Cancer Res. 67 (2007) 9704–9712. [24] H.A. Kim, K.J. Kim, K.H. Seo, H.K. Lee, S.Y. Im, PTEN/MAPK pathways play a key role in platelet-activating factor-induced experimental pulmonary tumor metastasis, FEBS Lett. 586 (2012) 4296–4302. [25] M.A. Chetram, C.V. Hinton, PTEN regulation of ERK1/2 signaling in cancer, J. Recept. Signal Transduct. Res. 32 (2012) 190–195. [26] H. Rubinfeld, I. Shimon, PI3K/Akt/mTOR and Raf/MEK/ERK signaling pathways perturbations in non-functioning pituitary adenomas, Endocrine 42 (2012) 285–291. [27] M. Kohno, J. Pouyssegur, Targeting the ERK signaling pathway in cancer therapy, Ann. Med. 38 (2006) 200–211. [28] A.M. Vanhoose, M. Emery, L. Jimenez, D.G. Winder, ERK activation by Gprotein-coupled receptors in mouse brain is receptor identity-specific, J. Biol. Chem. 277 (2002) 9049–9053. [29] S. Ahn, S.K. Shenoy, H. Wei, R.J. Lefkowitz, Differential kinetic and spatial patterns of beta-arrestin and G protein-mediated ERK activation by the angiotensin II receptor, J. Biol. Chem. 279 (2004) 35518–35525. [30] G. Fava, L. Marucci, S. Glaser, H. Francis, S. De Morrow, A. Benedetti, D. Alvaro, J. Venter, C. Meininger, T. Patel, S. Taffetani, M. Marzioni, R. Summers, R. Reichenbach, G. Alpini, Gamma-aminobutyric acid inhibits cholangiocarcinoma growth by cyclic AMP-dependent regulation of the protein kinase A/extracellular signal-regulated kinase 1/2 pathway, Cancer Res. 65 (2005) 11437–11446.
108
D. Zhang et al. / Cancer Letters 348 (2014) 100–108
[31] L.A. Liotta, K. Tryggvason, S. Garbisa, I. Hart, C.M. Foltz, S. Shafie, Metastatic potential correlates with enzymatic degradation of basement membrane collagen, Nature 284 (1980) 67–68. [32] S. Garbisa, R. Pozzatti, R.J. Muschel, U. Saffiotti, M. Ballin, R.H. Goldfarb, G. Khoury, L.A. Liotta, Secretion of type IV collagenolytic protease and metastatic phenotype: induction by transfection with c-Ha-ras but not c-Ha-ras plus Ad2E1a, Cancer Res. 47 (1987) 1523–1528. [33] M. Nakajima, D.R. Welch, P.N. Belloni, G.L. Nicolson, Degradation of basement membrane type IV collagen and lung subendothelial matrix by rat mammary adenocarcinoma cell clones of differing metastatic potentials, Cancer Res. 47 (1987) 4869–4876.
[34] A. Talvensaari-Mattila, P. Paakko, M. Hoyhtya, G. Blanco-Sequeiros, T. Turpeenniemi-Hujanen, Matrix metalloproteinase-2 immunoreactive protein: a marker of aggressiveness in breast carcinoma, Cancer 83 (1998) 1153–1162. [35] A. Talvensaari-Mattila, P. Paakko, T. Turpeenniemi-Hujanen, Matrix metalloproteinase-2 (MMP-2) is associated with survival in breast carcinoma, Br. J. Cancer 89 (2003) 1270–1275. [36] S. Leppa, T. Saarto, L. Vehmanen, C. Blomqvist, I. Elomaa, A high serum matrix metalloproteinase-2 level is associated with an adverse prognosis in nodepositive breast carcinoma, Clin. Cancer Res. 10 (2004) 1057–1063.
Contents lists available at ScienceDirect
Cancer Letters journal homepage: www.elsevier.com/locate/canlet
GABAergic signaling facilitates breast cancer metastasis by promoting ERK1/2-dependent phosphorylation Depu Zhang 1, Xiaowei Li 1, Ziming Yao, Chuanfei Wei, Nannan Ning, Jingxin Li ⇑ Department of Physiology, School of Medicine, Shandong University, Jinan 250012, China
a r t i c l e
i n f o
Article history: Received 17 October 2013 Received in revised form 23 January 2014 Accepted 7 March 2014
Keywords: c-Aminobutyric acid GABA receptor Breast cancer Metastasis ERK
a b s t r a c t The present study aims to determine the role of c-aminobutyric acid (GABA) signaling molecules in breast cancer metastasis. Our results reveal that GABAergic system exists in breast cancer cells. Both the GABA synthetic enzyme. (GAD65/67) and GABAB receptor are expressed in 4T1 mouse breast cancer cells, MCF-7 human breast cancer cells and human breast cancer tissue. Baclofen, a GABABR agonist, significantly promoted 4T1 cells invasion and migration in vitro and metastasis in vivo, an event that was attenuated by GABABR antagonist CGP55845. Baclofen-induced breast cancer metastasis was mediated by ERK1/2 pathway. Ó 2014 Elsevier Ireland Ltd. All rights reserved.
Introduction Breast cancer is the most prevalent cancer in women worldwide, comprising 16% of all female cancers. Although much has been done about diagnosis and treatment, mortality of breast cancer is still high, expecially in economically developing countries. What is more serious is that the mortality of breast cancer has been increasing in many places [1]. c-Aminobutyric acid (GABA), is synthesized predominantly from glutamate by glutamate decarboxylase (GAD) and exerts its effects via ionotropic GABAA receptors and/or metabotropic GABAB receptors [2]. GABA is the main inhibitory neurotransmitter in the vertebrate central nervous system (CNS). However, out of the CNS, a growing amount of researches have been producing evidence that GABA and GABA receptor are present in peripheral tissues [3,4]. Our previous studies have also shown that GABAergic system exists in lung [5], small intestine [6] and oviduct [7] and plays an essential role in these organ. In addition to its role in the adult mammalian nervous system as an inhibitory neurotransmitter,
Abbreviations: GABA, c-aminobutyric acid; GABAAR, type A GABA receptors; GABABR, type B GABA receptors; GAD, glutamate decarboxylase; MMP, matrix metalloproteinase; CNS, central nervous system; FV, field of view; ERK, extracellular signal-regulated protein kinases; MAPK, mitogen-activated protein kinases. ⇑ Corresponding author. Tel.: +86 0531 88383902. E-mail address: [email protected] (J. Li). 1 These authors contributed equally as first author. http://dx.doi.org/10.1016/j.canlet.2014.03.006 0304-3835/Ó 2014 Elsevier Ireland Ltd. All rights reserved.
GABA takes part in the proliferation, differentiation, and migration of several kinds of cells including cancer cells [4,8]. Several recent reports have suggested a relationship between the GABAergic system and oncogenesis. It has been confirmed that both GABA content and GAD activity are increased in material from breast [9], colon [10], stomach [11], thyroid [12] and ovarian cancer [13]. Although studies have demonstrated that GABA and GAD are detectable both in normal mammary gland and in neoplastic alterations, the function of this molecules in breast cancer metastasis is unclear. In our present research, we found a marked expression of GABA signaling molecules GABAA receptor, GABAB receptor and GAD65/67 in the 4T1 mouse breast cancer cells. Importantly, we found that GABAB receptor pathway mediates breast cancer cell invasion/migration in vitro and metastasis in vivo. Previous studies have revealed that activation of GABAB receptor mediates the phosphorylation of extracellular signal-regulated protein kinases 1/2 (ERK1/2) [14,15]. We therefore investigated the underlying mechanisms, focusing on ERK pathway. Our results indicate that GABAB R pathway facilitates breast cancer cell invasion and migration by promoting the phosphorylaton of ERK1/2 and subsequently increasing the expression of matrix metalloproteinase-2 (MMP-2). Intriguingly, our studies revealed that baclofen elicited a biphasic effect on the phosphorylaton of ERK1/2 via differential signaling pathway in breast cancer cells. The present finding hint that the GABAB R may be a potential target for the treatment of breast cancer, which should be of great value clinically.
D. Zhang et al. / Cancer Letters 348 (2014) 100–108 Materials and methods Cell culture Two breast cancer cell lines (4T1 and MCF-7) were obtained from the American Type Culture Collection (ATCC). Cells were cultured in RPMI 1640 (Hyclone) with 2.05 mM L-glutamine and 10% fetal bovine serum (Hyclone and incubated at 37 °C under 5% CO2 atmosphere. Immunofluorescence Cells were seeded into dishes containing a few sterile coverslips on the bottom. Cells were used after 12 h in culture. Specifically, the cells were fixed to the coverslips with 4% paraformaldehyde for 20 min and washed three times with PBS and were blocked by 10% goat serum (goat serum in PBS) for 1 h at room temperature. In order, the cells were incubated with anti-GABAB R1 (abcam, ab55051, 1:200), rabbit anti-GAD65/67 (Sigma, 1:2000) diluted in 10% goat serum overnight at 4 °C. Next, the cells were incubated with FITC labled secondary antibodies (Invitrogen, 1:400 for GABAB R1 receptor; 1:2000 for GAD) for 1 h at room temperature, followed by washes with PBS. DAPI (Beyotime, c1002, 1 lg/ml) was used to stain the nucleus. The cover slips were mounted 75% glycerol. Negative controls were done with the omission of primary antibodies. Images were taken on a Nikon H550L microscope with a Penguin 600CL digital camera. The merged images were accomplished with the help of Adobo Photoshop software.
101
10 lL CCK-8 solution was added into each well. Cells were then incubated for another 4 h at 37 °C. The absorbance was measured by a microplate reader at the wave length of 450 nm. In each experiment, six parallel wells were set up. 4T1-breast cancer xenograft model pulmonary metastasis Assay Female BALB/c mice (8 weeks of age) weighing approximately 20 g, were inoculated with 2 105 4T1 tumor cells in the right mammary fat pad by gently penetrating the skin. After one week of inoculation, mice with approximate tumor size were divided into four groups and administered intraperitoneally with normal saline, baclofen (10 mg/kg), baclofen (30 mg/kg) and CGP55845 (30 mg/kg) respectively for 4 weeks. Every 3 days, measure tumor longitudinal (L) and transverse (T) diameters with vernier calipers and record the general condition of the mice expecially weight. Tumor volumes were calculated according to the formula V = L T2/2[16]. Sacrifice mice, get orthotopic neoplasm and weight. Then expose the chest cavity and trachea. The number of lung metastases was determined by staining with Bouin’s solution (saturated picric acid:formalin:acetic acid = 15:5:1) after washing the lungs with PBS. White tumor nodules against a yellow lung background were counted by two independent observers. All experimental procedures were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals of Shandong University, and the study was approved by the Medical Ethics Committee for Experimental Animals, Shandong University, China (number ECAESDUSM 2012029). Statistical analysis
Western blot assays Human breast cancer tissues and matched non-tumor tissues (n = 3) were obtain from the Provincial Hospital of Shandong University, China. For all participants in this study, written informed consent was obtained. The protocol was approved by the Ethical Committee of Shandong University. The lysates were obtained using RIPA Lysis Buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, sodium orthovanadate, sodium fluoride, EDTA, leupeptin and so on) supplemented with PMSF. Protein concentrations were determined with a BCA Protein Assay Kit (Beyotime, P0012). Appropriate amounts of protein lysates were separated on SDS–PAGE and then transferred onto polyvinylidene difluoride membranes. The membranes were blocked in TBST buffer (Tris-buffered saline with 0.1% Tween-20) that contained of 5% skim milk. The membranes were incubated overnight at 4 °C in primary antibodies (GABAB R1 receptor antibodies, solarbio, a8100, 1:200; rabbit anti-GAD65/67, Sigma, 1:10,000; p-ERK antibodies, CST, 9101S, 1:1000; t-ERK antibodies, CST, 9102s, 1:1000; MMP-2 antibodies, Immunoway, B9801, 1:1000; b-actin antibodies, Santa Cruz, B0211, 1:2000) diluted in primary antibody dilution buffer (Beyotime, P0023A). The next day, membranes were washed in TBST, and incubated with horseradish peroxidase-conjugated secondary antibody (ZSGB-BIO, ZB-2301,1:400 for GABAB R1 receptor, 1:10,000 for p-ERK, 1:15,000 for t-ERK, 1:2000 for MMP-2, 1:2000 for b-actin; Beyotime, 90126, 1:400 for GAD). If necessary, membranes were subsequently stripped and reprobed to evaluate the expression of t-ERK. Immunoreactive proteins were detected using enhanced chemiluminescence (Millipore). Anti-b-actin and antit-ERK were used as controls of MMP-2 and p-ERK for the Western blots, respectively. The levels of p-ERK or MMP-2 were quantified using ImageJ software and all bands were normalized to percentages of control values.
Transwell invasion and migration assays Assays were performed using a modified Boyden chamber (Corning Costar, Rochester, NY, USA) containing a gelatin coated polycarbonate membrane filter (6.5 mm diameter, 8 lm pore size). The upper surface of the filter was coated with 50 ll Matrigel (0.17 mg ml–1; BD Biosciences, Bedford, MA, USA) for invasion assay. 8 104 cells were seeded into the transwell insert chamber with RPMI 1640 that contained of 1% FBS and then the inserts were placed in the lower chambers filled with 500 ll of RPMI 1640 that contained of 10% FBS. Chambers were incubated at37 °C under 5% CO2 atmosphere for different time (baclofen invasion for 24 h, baclofen invasion with U0126 preincubation and CGP invasion for 36 h, migration for 12 h). Thereafter, non-migrated cells were scraped from the upper surface of the membrane with a cotton swab. Migrated cells remaining on the bottom surface were viewed under microscope and counted in >10 fields of view (FV) at 100 magnification after staining with crystal violet.
Cell proliferation studies The proliferation of 4T1 cells was assessed by Cell Counting Kit-8 (CCK-8, Beyotime, C0038) assay according to the protocol recommended by the manufacturer. This method is an optical density colorimetric assay that quantifies the number of viable cells based on the activity of cellular dehydrogenases. To estimate the number of viable cells, approximately 2000 cells were seeded in each well of 96-well plates with 100 lL medium. Baclofen was added to each well with final concentrations of 0 lM, 10 lM and 100 lM for 12 h, 24 h and 48 h. After treatment,
Data was expressed as mean ± s.e.m. Means were compared with One-way analysis of variance (ANOVA) followed by Dunnett’s test or t-tests. The differences between groups were analyzed using the Sigma Stat 3.5 software (SPSS,Chicago,IL). A P-value < 0.05 was considered as significant. (⁄: p < 0.05, ⁄⁄: p < 0.01).
3. Results GABAergic system existed in breast cancer cells We performed immunoblot assays and immunofluorescence staining to examine the expressions of GABABR1, GAD65/67 and GABAAR subunits in breast cancer cells. Our results showed that GABABR1 and GAD65/67 were expressed distinctively in breast cancer cell line 4T1 cells, MCF-7 cells and human breast cancer tissue. (Fig. 1), and several types of GABAAR subunits including a1, b2 or b3 were also observed (Supplement Fig. 1A and B). These results suggest that GABAergic signaling-related molecules are expressed in breast cancer cells. GABAB pathway involved in the invasion and migration of breast cancer cells Next, we investigate the role of GABAergic signaling in breast cancer cell invasion and migration using transwell invasion and migration assays. Both of the upper and lower chambers contained different concentration of GABABR agonist baclofen. Chambers were incubated at37 °C under 5% CO2 atmosphere for 24 h (invasion assay) or 8 h (migration assay). Fig. 2A showed that baclofen significantly increased the invasive ablity of 4T1 breast cancer cells with dose-dependence. Similar effects were observed for migration assay without Matrigel-coated transwells (Fig. 2A, low lane). The baclofen-induced increase in cell migration was further confirmed by wound-healing experiment (Supplemental Fig. 2). To assess the effect of baclofen on breast cancer cell proliferation, approximately 2,000 cells were seeded in each well of 96-well plates with 100 lL medium. Baclofen was added to each well with final concentrations of 0 lM, 10 lM and 100 lM. After incubation for 12 h, 24 h and 48 h, the number of viable cells were estimated by Cell Counting Kit-8 (CCK-8) assay. However, no effect on proliferation stimulated by baclofen was observed (Fig. 2B). In our studies, 4T1 cells also expressed GAD65/67, key GABA-synthesizing enzymes, thus they may release GABA. A new study had been carried to investigate the role of endogenous GABA in the invasion and migration of 4T1 cells. Fig. 2C showed that, after incubation of GABAB
102
D. Zhang et al. / Cancer Letters 348 (2014) 100–108
Fig. 1. GABAergic system existed in breast cancer cells. (A) Immunoblot of breast cancer cells and human breast cancer tissue for GABABR1 and GAD65/67 subunits. (B and C) Immunostaining of GABABR1 (green) and GAD65/67 (red). Nuclei were identified by DAPI staining (blue). Original magnification of immunohistochemical pictures, 400 in B and C. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
receptor antagonist CGP55845 for 36 h (invasion assay) or 12 h (migration assay), remarkaly inhibitory effects on the invasion and migration of 4T1 cells were observed (Fig. 2C). Several GABAA receptor subunits were also distributed in 4T1 cells (Supplemental Fig. 1A and B). To test whether GABAARs are involved in breast cancer metastasis, we carried out wound-healing assay with GABAAR agonist muscimol. However, our results showed that muscimol had no effect on the migration of 4T1 cells (Supplemental Fig. 1C). The involvement of GABAB receptors in breast cancer invasion and migration is mediated by ERK pathway ERK1/2 is a member of mitogen-activated protein kinases (MAPK) family. We detected the expression of p-ERK and t-ERK stimulated by baclofen with time gradient or concentration gradient by western blot assay. 4T1 cells were lysed after incubation for 0 s, 30 s, 1 min, 5 min, 10 min, 30 min, 1 h, 2 h, 4 h and 24 h. Fig. 3A
showed that baclofen activated ERK1/2 in a dose- and time-dependent fashion. The ERK phosphorylation levels was rapidly increased after incubation in 5 min, and then declined to basal levels of ERK phosphorylation 10 min later. Interestingly, after incubation of 1 h, the levels of ERK phosphorylation were upregulated again, then gradually lowered down to the basal level on 4 h. We also evaluated the levels of p-ERK stimulated by different concentration of baclofen. With the increasing of concentration, a gradually increase of p-ERK was observed. Conversely, CGP55845 significantly decreased the level of p-ERK (Fig. 3B). To further determine if the activation of ERK1/2 was required for the invasion and migration signaling triggered by baclofen, U0126, a highly specific MAPK kinase inhibitor to block ERK1/2 activation, was used to verify the effect of the ERK1/2 pathway on baclofen-induced cell invasion and migration. Data showed that pre-incubation of U0126 reversed the baclofen-induced increase in ERK phosphorylation (Fig. 3C).
D. Zhang et al. / Cancer Letters 348 (2014) 100–108
103
Fig. 2. GABAB pathway involved in the invasion and migration of 4T1 breast cancer cells. Baclofen significantly increased the invasive and migration ablity of 4T1 cells with time- and dose-dependence (n = 10) (A). Baclofen had no effect on proliferation (n = 6) (B). GABAB receptor antagonist CGP55845 remarkaly inhibited the invasion and migration of 4T1 cells (C). The invasive or migrative ability of 4T1 cells was expressed as mean ± s.e.m of cells/FV (n = 12) .The proliferation ability of 4T1 cell was expressed as mean ± s.e.m of absorbance/well (n = 6). Original magnification, 200 in A and C. ⁄; p < 0.05 compared with 0 or control; #; p < 0.05 compared with 10. FV, field of view.
Intriguingly, after the pretreatment of U0126, an opposite effect of baclofen on ERK phosphorylation was found (Fig. 3C), which was markedly attenuated by H-89, a selective PKA inhibitor (Fig. 4). Consistant with the change of ERK phosphorylation levels, baclofen significantly depressed the invasion and migration of 4T1 cells in the presence of U0126 (Fig. 3 D and E). These results indicated that GABAB receptor participates in breast cancer cells migration and invasion via ERK signaling.
Involvement of the GABAB receptor pathway on MMP production in cancer cells In order to further prove the effects of GABAB receptor on invasion and migration, the expression of MMP-2 in 4T1 cells was detected by western blot. Fig. 5A showed that baclofen
dose-dependently increased the expression of MMP-2. We also detected the effect of baclofen on the expression of MMP-2 in the pre-incubation of U0126. Conversely, baclofen decreased the expression of MMP-2 (Fig. 5B). This finding was consistant with the results in Fig. 3C–E. In addition, GABAB receptors antagonist CGP55845 decreased the expression of MMP-2 (Fig. 5C). These data demonstrate that MMP-2, as downstream of p-ERK, also take part in breast cancer cells migration and invasion mediated by GABAB receptor.
GABAB receptor pathway in vivo facilitated pulmonary metastasis of breast cancer To determine whether GABAB pathway can promote breast cance rmetastasis, we examined the efects of GABAB receptor
104
D. Zhang et al. / Cancer Letters 348 (2014) 100–108
Fig. 3. GABAB receptor-induced breast cancer invasion and migration was mediated by the MAPK/ERK pathway. Baclofen activated ERK in a dose-(A, right, n = 5) and time-(A, left, n = 3) dependent fashion in 4T1 cells. In contrast with baclofen, CGP55845 significantly decreased the level of p-ERK compared with control (B, n = 3). Pre-incubation of U0126 reversed the baclofen-induced increase in the levels of ERK phosphorylation (C, n = 3). Baclofen significantly depressed the invasion (D, n = 10) and migration (E, n = 10) of 4T1 cells in the presence of U0126. t-ERK1/2 was used as a loading control for A, B, C. Data are presented as mean ± s.e.m. ⁄; p < 0.05 and ⁄⁄; p < 0.01 compared with 0; # ; p < 0.05 compared with 10 (A) or control (C).
agonist baclofen or its antagonist CGP55845 on spontaneous lung metastasis using 4T1-breast cancer xenograts in BALB/c mice. The average number of tumor nodules was 20.6 in the salinetreated group and 71 in the baclofen-treated group. These results
indicated that baclofen-treatment (B10 and B30) significantly increased tumor colonization in the lung compared with the saline group (<0.01; Fig. 6A). Conversely, CGP55845 decreased the number of lung nodules. However, the primary tumor weight or size
D. Zhang et al. / Cancer Letters 348 (2014) 100–108
105
was no statistical difference among the four experimental groups (<0.05) (Fig. 6B and C). We measured the body weight of mice every week. As shown in Fig. 6D, The body weight of mice in the baclofen-treated group (B30) was significantly decreased compared with that in control group. Together, these results strongly suggested that GABAB receptor pathway facilitated pulmonary metastasis of breast cancer, but didn’t show obvious impact on orthotopic neoplasm.
Discussion
Fig. 4. The selective PKA inhibitor H-89 largerly alleviate the inhibitory effect of baclofen on ERK phosphorylation after preincubation of U0126 in 4T1 cells. t-ERK1/2 was used as a loading control. Data are presented as mean ± s.e.m (n = 3).⁄⁄; p < 0.01 compared with 0; #; p < 0.05 compared with control group (Ctr).
Our research found that breast cancer cells remarkablely expressed GABA receptor (GABAAR and GABABR) and GAD65/67, the key enzyme in GABA synthesis. This finding proved that breast cancer cells possess a GABAerigc signaling system. Then we detected the function of GABAergic system in breast cancer and found that GABAB receptor pathway facilitates breast cancer cell invasion/migration in vitro and metastasis in vivo, which was mediated by promoting the phosphorylaton of ERK1/2 and subsequently increasing the expression of MMP-2. Although many researches demonstrated activation of GABAB receptor is associated with the proliferation of some cancer cells like pancreatic cancer [17], lung adenocarcinoma [18], in our present studies, we found that baclofen had no effect on proliferation in breast cancer cells in vitro and tumor growth in vivo.
Fig. 5. GABAB receptor pathway mediated the MMP-2 production in 4T1 breast cancer cells. Baclofen dose-dependently increased the the expression of MMP-2 (A, n = 3). However, baclofen decreased the expression of MMP-2 in the U0126 pre-incubation (B, n = 3). GABAB receptors antagonist CGP5584 decreased the expression of MMP-2 (C, n = 3). b-actin was used as a loading control for A, B, C. Data are presented as mean ± s.e.m. ⁄; p < 0.05 compared with 0 or control; #; p < 0.05 compared with 10.
106
D. Zhang et al. / Cancer Letters 348 (2014) 100–108
Fig. 6. GABAB receptor pathway in vivo facilitated pulmonary metastasis of breast cancer. Baclofen-treatment (B10 and B30) significantly increased tumor colonization in the lung compared with the saline group. Conversely, CGP55845 decreased the number of lung nodules (A). However, the primary tumor weight or size was no statistical difference among the four experimental groups (B and C). The body weight of mice in the baclofen-treated group (B30) was significantly decreased compared with that in control group (D). Data are presented as mean ± s.e.m (n = 6).⁄; p < 0.05 and ⁄⁄; p < 0.01 compared with control.
Metabotropic GABAB receptors are heterodimeric G-proteincoupled receptors (GPCRs) consisting of GABABR1 and GABABR2 subunits. The extracellular domain of GABABR1 not GABABR2 has ligand-binding activity, but the intracellular domain of GABAB R2 not GABABR1 has regulating activity due to coupled to G-protein. However, GABA might result in the different effects on cancer growth/metastasis in cancer-type or GABA receptor-type dependent manner. Several reports indicate that GABAB receptor activation can inhibit the migration of colon carcinoma [19], pancreatic cancer [17], lung adenocarcinoma [18], hepatocarcinoma [20] and proliferation of pancreatic cancer [17], lung adenocarcinoma [18]. But an opposite effect of GABA or GABAB agonist was found in prostate [21] and renal cancer cells [22]. GABA or GABAB agonist baclofen can significantly promote the invasive ability of prostate and renal cancer cells through the production of matrix metalloproteinases (MMPs). On the other hand, different types of GABA receptors may mediate different effect on cancer growth. Activition of GABAA receptors can increase pancreatic cancer cell proliferation [23], but stimulation of the GABABR show an inhibitory role in human pancreatic cancer cells [17]. In our experiment, we found that GABAB receptor pathway promoted breast cancer cell invasion and migration, but activation of GABAA receptors had no such effect. Our in vivo studies further confirmed that GABAB receptor pathway involved in breast cancer cell metastasis. ERK1/2 is a member of MAPK family. After phosphorylation through different ways, p-ERK can transfer to the nucleus and phosphorylate transcription factors. Aberrant activation of the ERK pathway has been shown to be a key contributing factor to many types of human cancer [24–27]. It has been shown that
GABAB receptor activates ERK1/2 in neurons [28] and renal cancer cells [22]. For reason above, we detected the effects of activation of GABAB receptor on the levels of p-ERK. Our results showed that baclofen increased the levels of p-ERK in a time- and dose-dependent manner. Interestingly, two peaks of ERK activation occurred somewhere between 0 s–10 min for the first peak and 1–4 h for the second one, in which the first one was much shorter. A paradigm GPCR pathway is mediated by G protein-dependent and G protein-independent signaling, in which b-arrestins seems to be the most important is widely accepted. Although both signaling pathways can trigger MEK/ERK activation, it was reported they had distinct activation kinetics: the rapid and transient G protein-dependent activation and the slower but persistent b-arrestin-mediated activation [29], in accordance with our results-two peaks and different durations. Both of two peaks disappeared when MEK, the common pathway, was blocked. Futhermore, U0126 completely depressed the baclofen-induced increase in the levels of p-ERK. However, it is worthwhile to note that the opposite effect of baclofen on ERK phosphorylation, cell invasion and migration were observed after pre-incubation of U0126. Owing to the complexity of signal pathway, we have to consider other pathways induced by baclofen. Those, focusing on inhibiting MEK1/2 or its upstream such as ras, raf, b-arrestin, cannot explain the inhibitory effect of baclofen on ERK phosphorylation, so appropriate reasons should be settled in other signaling pathway. Fava G discovered activaton of GABAB receptor down-regulated ERK1/2 phosphorylation via cAMP/PKA dependent pathway [30]. The selective PKA inhibitor H-89 was chosen to investigate the inhibitory effect of baclofen on ERK phosphorylation after pre-incubation of U0126.
D. Zhang et al. / Cancer Letters 348 (2014) 100–108
Our result show that the opposite effect was largerly alleviated. Obviously, PKA pathway, one of GABAB receptor downstream signaling, involved in baclofen-induced down-regulation of ERK1/2 phosphorylation, which was much weaker than G protein and b-arrestin signaling, so its inhibitory effect cannot display without U0126. Certainly a detail of modulatory mechanism is still to be clarified and further studies are required. Cancer metastasis involves a complex cascade of events including detaching from the primary tumor, invading the stromal tissue, entering the circulation, extravasating and invading the target organ. During this process, proteolytic degradation of the extracellular matrix has been considered a rate-limiting step. MMPs are believed to be vitally important protease which enable tumor cells to penetrate the basement membrane. The expression of MMP-2 immunoreactive protein has been strongly associated with invasive and metastatic tumours in previous in vitro studies [31–33]. In breast cancer, MMP-2 immunoreactive protein is an independent prognostic marker of survival [34–36], but MMP-9 levels do not correlate with survival [36]. In the present study, we investigated the influence of activation of GABAB receptor on the expression of MMP-2 in 4T1 cells. Western blot analysis demonstrated that baclofen stimulation significantly increased the expression of MMP-2 with a concentration dependent manner, however, CGP55845 decreased the expression of MMP-2. Consistent with our findings from the measurement of the ERK phosphorylation and cell metastasis, baclofen also decreased the the expression of MMP-2 in the presence of U0126. In conclusion, this study provides clear evidence that GABA signaling molecules GABAA receptor, GABAB receptor and GAD65/67 are expressed in breast cancer cells, and the activation of GABAB receptor promotes breast cancer cell metastasis by enhancing p-ERK levels and subsequently increasing the expression of MMP-2. Authors’ contributions D.Z., X.L., Z.Y., C.W. and N.N. carried out all the experiments and wrote the manuscript. J.L. is the principal investigator of the laboratory in which the research was performed, designed all the experiments the contributed to the interpretation of the data and wrote the manuscript. All of the authors read and approved the final manuscript. Conflict of Interest None declared. Acknowledgement This work was funded by grant to J. Li from the National Natural Science Foundation of China, China (Grant No. 31171108). Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.canlet.2014. 03.006. References [1] M.H. Forouzanfar, K.J. Foreman, A.M. Delossantos, R. Lozano, A.D. Lopez, C.J. Murray, M. Naghavi, Breast and cervical cancer in 187 countries between 1980 and 2010: a systematic analysis, Lancet 378 (2011) 1461–1484. [2] J.T. Kittler, S.J. Moss, Modulation of GABAA receptor activity by phosphorylation and receptor trafficking: implications for the efficacy of synaptic inhibition, Curr. Opin. Neurobiol. 13 (2003) 341–347.
107
[3] M.K. Akinci, P.R. Schofield, Widespread expression of GABA(A) receptor subunits in peripheral tissues, Neurosci. Res. 35 (1999) 145–153. [4] S.Z. Young, A. Bordey, GABA’s control of stem and cancer cell proliferation in adult neural and peripheral niches, Physiology (Bethesda) 24 (2009) 171– 185. [5] Y.Y. Xiang, S. Wang, M. Liu, J.A. Hirota, J. Li, W. Ju, Y. Fan, M.M. Kelly, B. Ye, B. Orser, P.M. O’Byrne, M.D. Inman, X. Yang, W.Y. Lu, A GABAergic system in airway epithelium is essential for mucus overproduction in asthma, Nat. Med. 13 (2007) 862–867. [6] Y. Li, Y.Y. Xiang, W.Y. Lu, C. Liu, J. Li, A novel role of intestine epithelial GABAergic signaling in regulating intestinal fluid secretion, Am. J. Physiol. Gastrointest. Liver Physiol. 303 (2012) G453–460. [7] Z. Zhou, H. Sun, X. Li, Y. Li, S. Zhao, D. Zhang, Z. Yao, J. Li, A local GABAergic system is functionally expressed in human fallopian tube, Biochem. Biophys. Res. Commun. 398 (2010) 237–241. [8] M. Watanabe, K. Maemura, K. Oki, N. Shiraishi, Y. Shibayama, K. Katsu, Gamma-aminobutyric acid (GABA) and cell proliferation: focus on cancer cells, Histol. Histopathol. 21 (2006) 1135–1141. [9] M. Mazurkiewicz, A. Opolski, J. Wietrzyk, C. Radzikowski, Z. Kleinrok, GABA level and GAD activity in human and mouse normal and neoplastic mammary gland, J. Exp. Clin. Cancer Res. 18 (1999) 247–253. [10] K. Maemura, H. Yamauchi, H. Hayasaki, K. Kanbara, T. Tamayama, I. Hirata, M. Watanabe, Gamma-amino-butyric acid immunoreactivity in intramucosal colonic tumors, J. Gastroenterol. Hepatol. 18 (2003) 1089–1094. [11] M. Matuszek, M. Jesipowicz, Z. Kleinrok, GABA content and GAD activity in gastric cancer, Med. Sci. Monit. 7 (2001) 377–381. [12] S.S. Roberts, M.C. Mendonca-Torres, K. Jensen, G.L. Francis, V. Vasko, GABA receptor expression in benign and malignant thyroid tumors, Pathol. Oncol. Res. 15 (2009) 645–650. [13] C.S. Nicholson-Guthrie, G.D. Guthrie, G.P. Sutton, J.C. Baenziger, Urine GABA levels in ovarian cancer patients: elevated GABA in malignancy, Cancer Lett. 162 (2001) 27–30. [14] B.H. Im, H. Rhim, GABA(B) receptor-mediated ERK1/2 phosphorylation via a direct interaction with Ca(V)1.3 channels, Neurosci. Lett. 513 (2012) 89–94. [15] H. Tu, P. Rondard, C. Xu, F. Bertaso, F. Cao, X. Zhang, J.P. Pin, J. Liu, Dominant role of GABAB2 and Gbetagamma for GABAB receptor-mediated-ERK1/2/CREB pathway in cerebellar neurons, Cell Signal 19 (2007) 1996–2002. [16] C.G. Roehrborn, S. Sech, J. Montoya, T. Rhodes, C.J. Girman, Interexaminer reliability and validity of a three-dimensional model to assess prostate volume by digital rectal examination, Urology 57 (2001) 1087–1092. [17] H.M. Schuller, H.A. Al-Wadei, M. Majidi, GABA B receptor is a novel drug target for pancreatic cancer, Cancer 112 (2008) 767–778. [18] H.M. Schuller, H.A. Al-Wadei, M. Majidi, Gamma-aminobutyric acid, a potential tumor suppressor for small airway-derived lung adenocarcinoma, Carcinogenesis 29 (2008) 1979–1985. [19] J. Joseph, B. Niggemann, K.S. Zaenker, F. Entschladen, The neurotransmitter gamma-aminobutyric acid is an inhibitory regulator for the migration of SW 480 colon carcinoma cells, Cancer Res. 62 (2002) 6467–6469. [20] K. Szczaurska, M. Mazurkiewicz, A. Opolski, The role of GABA-ergic system in carcinogenesis, Postepy Hig. Med. Dosw. 57 (2003) 485–500. [21] H. Azuma, T. Inamoto, T. Sakamoto, S. Kiyama, T. Ubai, Y. Shinohara, K. Maemura, M. Tsuji, N. Segawa, H. Masuda, K. Takahara, Y. Katsuoka, M. Watanabe, Gamma-aminobutyric acid as a promoting factor of cancer metastasis; induction of matrix metalloproteinase production is potentially its underlying mechanism, Cancer Res. 63 (2003) 8090–8096. [22] T. Inamoto, H. Azuma, T. Sakamoto, S. Kiyama, T. Ubai, Y. Kotake, M. Watanabe, Y. Katsuoka, Invasive ability of human renal cell carcinoma cell line Caki-2 is accelerated by gamma-aminobutyric acid, via sustained activation of ERK1/2 inducible matrix metalloproteinases, Cancer Invest. 25 (2007) 574–583. [23] A. Takehara, M. Hosokawa, H. Eguchi, H. Ohigashi, O. Ishikawa, Y. Nakamura, H. Nakagawa, Gamma-aminobutyric acid (GABA) stimulates pancreatic cancer growth through overexpressing GABAA receptor pi subunit, Cancer Res. 67 (2007) 9704–9712. [24] H.A. Kim, K.J. Kim, K.H. Seo, H.K. Lee, S.Y. Im, PTEN/MAPK pathways play a key role in platelet-activating factor-induced experimental pulmonary tumor metastasis, FEBS Lett. 586 (2012) 4296–4302. [25] M.A. Chetram, C.V. Hinton, PTEN regulation of ERK1/2 signaling in cancer, J. Recept. Signal Transduct. Res. 32 (2012) 190–195. [26] H. Rubinfeld, I. Shimon, PI3K/Akt/mTOR and Raf/MEK/ERK signaling pathways perturbations in non-functioning pituitary adenomas, Endocrine 42 (2012) 285–291. [27] M. Kohno, J. Pouyssegur, Targeting the ERK signaling pathway in cancer therapy, Ann. Med. 38 (2006) 200–211. [28] A.M. Vanhoose, M. Emery, L. Jimenez, D.G. Winder, ERK activation by Gprotein-coupled receptors in mouse brain is receptor identity-specific, J. Biol. Chem. 277 (2002) 9049–9053. [29] S. Ahn, S.K. Shenoy, H. Wei, R.J. Lefkowitz, Differential kinetic and spatial patterns of beta-arrestin and G protein-mediated ERK activation by the angiotensin II receptor, J. Biol. Chem. 279 (2004) 35518–35525. [30] G. Fava, L. Marucci, S. Glaser, H. Francis, S. De Morrow, A. Benedetti, D. Alvaro, J. Venter, C. Meininger, T. Patel, S. Taffetani, M. Marzioni, R. Summers, R. Reichenbach, G. Alpini, Gamma-aminobutyric acid inhibits cholangiocarcinoma growth by cyclic AMP-dependent regulation of the protein kinase A/extracellular signal-regulated kinase 1/2 pathway, Cancer Res. 65 (2005) 11437–11446.
108
D. Zhang et al. / Cancer Letters 348 (2014) 100–108
[31] L.A. Liotta, K. Tryggvason, S. Garbisa, I. Hart, C.M. Foltz, S. Shafie, Metastatic potential correlates with enzymatic degradation of basement membrane collagen, Nature 284 (1980) 67–68. [32] S. Garbisa, R. Pozzatti, R.J. Muschel, U. Saffiotti, M. Ballin, R.H. Goldfarb, G. Khoury, L.A. Liotta, Secretion of type IV collagenolytic protease and metastatic phenotype: induction by transfection with c-Ha-ras but not c-Ha-ras plus Ad2E1a, Cancer Res. 47 (1987) 1523–1528. [33] M. Nakajima, D.R. Welch, P.N. Belloni, G.L. Nicolson, Degradation of basement membrane type IV collagen and lung subendothelial matrix by rat mammary adenocarcinoma cell clones of differing metastatic potentials, Cancer Res. 47 (1987) 4869–4876.
[34] A. Talvensaari-Mattila, P. Paakko, M. Hoyhtya, G. Blanco-Sequeiros, T. Turpeenniemi-Hujanen, Matrix metalloproteinase-2 immunoreactive protein: a marker of aggressiveness in breast carcinoma, Cancer 83 (1998) 1153–1162. [35] A. Talvensaari-Mattila, P. Paakko, T. Turpeenniemi-Hujanen, Matrix metalloproteinase-2 (MMP-2) is associated with survival in breast carcinoma, Br. J. Cancer 89 (2003) 1270–1275. [36] S. Leppa, T. Saarto, L. Vehmanen, C. Blomqvist, I. Elomaa, A high serum matrix metalloproteinase-2 level is associated with an adverse prognosis in nodepositive breast carcinoma, Clin. Cancer Res. 10 (2004) 1057–1063.