Elevated expression of GNAS promotes breast cancer cell proliferation and migration via the PI3K/AKT/Snail1/E-cadherin axis

We studied 150 breast tumor tissues from a cohort of 217 patients diagnosed with breast cancer who underwent tumor removal at the First People’s Hospital of Yibin between 2006 and 2009. Overall survival (OS) was defined as the time between initial surgery and death. We prepared tissue microarray (TMA) cores (1.5 mm diameter) from formalin-fixed, paraffin-embedded samples. IHC staining was performed on all of the TMA slides, and the results were interpreted by two pathologists using a blinded method. GNAS staining were scored according to the cytoplasmic staining intensity: 0–2 indicated low staining and 3–4 high staining. The mean score was the final score. Classical core clinical characteristics, such as the WHO grade, clinical stage, tumor size, nodal status, distal metastasis, ER stage, PR stage and Her-2 stage, were included to analyze the correlation of GNAS with breast cancer. Moreover, univariate and multivariate analyses of different prognostic variables of GNAS with overall survival were performed. Approval for this study was granted by the Ethics Committee of the First People’s Hospital of Yibin.

The human breast cancer cell lines, including MCF-7, MDA-MB-231, MDA-MB-468, BT-474 and SK-BR-3, were purchased from the ATCC. The cells were cultured in RMPI Medium 1640 supplemented with 10% fetal bovine serum (FBS), 1% penicillin and 1% streptomycin at 37 °C in a 5% CO2 humidified incubator. To specifically inhibit PI3Ks, 0.2 μM NVP-BKM120 hydrochloride (BKM120) was used.

For transfection, cells were washed with serum-free medium once and then incubated with serum-free medium for 4 h. The siGNAS (5′-TGCATGTTAATGGGTTTAA-3′ and 5′-ACTACTGCTACCCTCATTT-3′), siControl (RiBio, Guangzhou, China) and lipofectamine 2000 (Invitrogen, CA, USA) were separately mixed with 500 μl of Opti-MEM I Reduced Serum Medium (Gibco, Grand Island, NY) for 5 min. Then, the two mixtures were combined and incubated at room temperature for 20 min. The lipofectamine: siRNA mixture was added to the cells and incubated at 37 °C for 6 h. Subsequently, fresh medium containing 10% FBS was added, and the cells were maintained in culture until the following experiments.

CCK8 (Sigma-Aldrich Co., St Louis, MO, USA) and EdU incorporation (RiboBio, Guangzhou, China) assays were carried out to evaluate cell viability and proliferation according to the manufacturers’ instructions.

Cell cycle and apoptosis were measured using the Cell Cycle and Apoptosis Analysis Kit according to the manufacturer’s instruction (Beyotime Biotechnology, Shanghai, China). The results were analyzed with FlowJo software.

Approximately 2 × 10⁵ cells were plated in 6-well plates after the different treatments. A linear scratch was generated on the cell monolayer with a sterile pipette. Photomicrographs of live cells were obtained at 40× magnification, and the distance migrated was observed after 24 h or 48 h. The remaining wound area was measured using ImageJ software.

The Matrigel invasion assay was performed in 24-well transwell culture plates. Cells were resuspended and then seeded in 24-well transwell plates containing FBS-free medium in the upper chamber and complete growth medium supplemented with 10% FBS in the lower chamber for 24 h at 37 °C. Noninvading cells were removed from the upper surfaces of the invasion membranes, and the cells on the lower surface were stained with hematoxylin. The average number of cells per field was determined by counting the cells in six random fields per well. Cells were counted in four separate fields in three independent experiments.

A 6-well plate was coated with a 1:1 ratio of 1.2% agarose and 2 × complete phenol red-free RIPM1640, and it was solidified for 30 min. The top portion was prepared with 0.6% agarose and 2 × medium with cells were plated at a density of 3000 cells/ml; a total of 1000 cells were used. Images were photographed after culturing for 14 days. Colonies were counted and statistically analyzed. Assays were performed three times using triplicate wells.

Cells and tissues were lysed with RIPA buffer according to the manufacturer’s instructions (Beyotime Biotechnology, Shanghai, China). Proteins were separated with 12% SDS-PAGE gels and transferred to PVDF membranes. Membranes were blocked with PBS containing 0.05% tween and 5% nonfat milk and probed with antibodies against GNAS, p-PKA, p85α, AKT, p-AKT, vimentin, E-cadherin, snail 1, slug, Cyclin D1, CDK4 and GAPDH (Huabio, Hangzhou, China). These antibodies were purchased from Abcam or CST if not mentioned. Signal intensities were quantified and normalized to the GAPDH intensity using ImageJ.

BALB/c nude mice aged 4–6 weeks were purchased from the Animal Center at the Cancer Institute at the Chinese Academy of Medical Science (Beijing, China). Next, 1 × 10⁶ MDA-MB-231 cells transfected with siGNAS or siControl were subcutaneously injected into the abdomen of each nude mouse. In addition, cholesterol-modified siGNAS or control siRNA (RiboBio, 5 nmol/kg) dissolved in saline buffer were intratumorally injected every 3 days for 5 weeks. The tumors were measured weekly and harvested 5 weeks later.

Associations between the expression of GNAS and clinical variables as well as breast cancer were assessed using t tests. The Kaplan–Meier method was used to estimate disease-free survival (DFS) and overall survival (OS), and the log-rank test was used to compare survival between two strata. The significance of different prognostic variables of GNAS for OS was analyzed in univariate and multivariate analyses. All tests were two-sided, and p < 0.05 was considered statistically significant. All statistical analyses were performed using the SPSS version 19.0 software program (SPSS Inc., Chicago, USA).

To identify the function of GNAS in breast cancer, we first examined the expression of GNAS in 150 breast cancer TMA samples with complete follow-up information. As a housekeeping protein, all the clinical samples were GNAS positive, and finally 97 samples were identified as the high expression and the other 53 as the low expression subtype (Fig. 1a). Western blot analysis revealed an elevated expression of GNAS in tumor tissues (Fig. 1b). The association of GNAS expression and the baseline clinical characteristics is presented in Tables 1 and 2. The impact of the clinicopathologic characteristics and prognostic factors was calculated by Kaplan–Meier analysis, and survival curves were delineated by the log-rank test. The results showed that high expression of GNAS was more likely to be observed in patient with distal metastasis (p = 0.026), a more advanced clinical stage (p = 0.001), and poor survival (p = 0.021, Fig. 1c). Moreover, we also examined ki-67 expression in GNAS -high and -low samples. The representative images and statistical results revealed that samples with high GNAS expression displayed more active proliferative activity (p = 0.035, Fig. 1d).

To further examine whether GNAS is important for the proliferation of breast cancer cells, we firstly evaluated the expression of GNAS in five different breast cancer cell lines (Fig. 2a). According to the results, the MDA-MB-231 cell line possessed the highest GNAS expression, while there was no obvious difference in MCF-7, BT-474, SK-BR-3 and MDA-MB-468. Therefore, MDA-MB-231 and MCF-7 were chosen for further study. To evaluate the role of GNAS in cell proliferation, specific siRNAs targeting GNAS were used, and both siRNAs dramatically decreased the protein level of GNAS after 72 h of transfection (Fig. 2b). The CCK8 assay results demonstrated that knockdown of GNAS markedly inhibited MCF-7 cell viability (Fig. 2c). Additionally, as shown in Fig. 2d, EdU incorporation assay revealed a reduced proliferation rate of MDA-MB-231 cells in the siGNAS-treated group (70.883 ± 11.37% vs 45.85 ± 8.68%).

Furthermore, siGNAS decreased the percentage of MDA-MB-231 cells in S phase and MDA-MB-231 cell proliferation rate. An increase in G1 phase was observed, which indicated that the cell cycle was arrested in G1 phase, which was further confirmed by the reduced expression of the G1-S transition marker Cyclin D1 and CDK4 (Fig. 3a, b). In contrast, no significant differences in apoptosis were observed in siGNAS- and siControl-treated cells (Fig. 3C). To obtain further evidence that GNAS facilitated the proliferation of breast cancer cell lines, a colony formation assay was performed. As shown in Fig. 3d, the number of colonies was lower in the siGNAS-treated group than the control group. The colony-forming ability of MDA-MB-231 and MCF-7 cells was significantly inhibited by 49% and 71%, respectively (p < 0.01, Fig. 3d).

As a critical component of G protein, GNAS plays pivotal role in the process of intracellular and extracellular signal transduction regardless of the physiological or pathological condition [13]. It has been shown that high expression of GNAS is tightly associated with distal metastasis in breast cancer patients, but whether this phenomenon in fact occurs requires further confirmation. Knockdown of GNAS suppressed wound closure at 48 h to 54.4% and 69.7%, respectively, in siControl-treated MDA-MB-231 and MCF-7 cells (p < 0.05, Fig. 4a, c), indicating that GNAS was important for breast cancer cell migration.

Moreover, consistent with the clinical analysis and wound healing results, knockdown of GNAS reduced to almost half the number of migrated and invaded cells (209 ± 13 vs 108 ± 7 and 102 ± 6 vs 52 ± 7 migrated cells; 221 ± 14 vs 114 ± 11, 116 ± 4 vs 56 ± 7 invaded cells, respectively, in MDA-MB-231 and MCF-7) (Fig. 4b, d).

According to our results, GNAS accelerates breast cancer cell proliferation and migration, but the underlying molecular mechanism remains unknown. To gain further insight into the molecular events responsible for this phenomenon, we examined key proteins in signaling pathways that are highly related to GNAS. In siGNAS-treated cells, protein kinase A (PKA), a critical downstream effector of cyclic AMP signaling, was dramatically reduced, which further confirmed the siRNA efficiency (Fig. 5a, b). The PI3K/AKT signaling pathway is indispensable for cell growth, proliferation, survival, etc., and increasing evidence has documented that an interaction may exist between PI3K/AKT and cyclic AMP signaling pathway [14‐16]. We noticed that although the total AKT level was not altered, the p85α subunit of PI3K and p-AKT was significantly reduced after siGNAS treatment, indicating that the function of GNAS in breast cancer might be partly dependent on the PI3K/AKT signaling pathway.

In addition, it has been well documented that epithelial mesenchymal transition (EMT) is a critical process for cell migration and invasion, as well as scilicet cancer cell metastasis [17]. Given the correlation between high GNAS expression and the presence of distal tumor metastases in breast cancer patients, we examined several key proteins associated with the EMT process. As a result, the expression of E-cadherin was increased 2.08-fold in siGNAS-treated cells, indicating an impairment of cell migration and invasion. Although both snail1 and snail2 (slug) are upstream transcription factors of E-cadherin, only the expression of snail1 was reduced by 60% in siGNAS-treated cells (Fig. 5a, b).

To further investigate whether GNAS regulates cell proliferation and EMT through the PI3K/AKT signaling pathway, we selectively inhibited the PI3K/AKT signaling pathway by treating MDA-MB-231 cell with the pan-class I PI3K inhibitor BKM120 [18, 19]. As a result, p-AKT was severely reduced, and the alteration of E-cadherin and snail1 was consistent with that in siGNAS-treated cells (Fig. 5c). Additionally, the proliferation of MDA-MB-231 cells was dramatically reduced, as assessed by the EdU and colony formation assay (Fig. 5d, e). Moreover, the migration and invasion abilities were also impaired according to the transwell assay results (Fig. 5f). Although the expression of GNAS was unchanged, the inhibition of PI3K/AKT signaling pathway mimicked the phenotype caused by GNAS silencing, which suggested that GNAS was an upstream factor in the PI3K/AKT signaling pathway.

To investigate the role of GNAS in tumor growth and metastasis in vivo, an MDA-MB-231 subcutaneous tumor transplantation model was established in nude mice. The tumor volume was measured weekly. Although all the mice vaccinated with MDA-MB-231 cells possessed tumors, the tumor volume in the siGNAS-treated group was significantly reduced, indicating that knockdown of GNAS dramatically delayed tumor growth (Fig. 6a–c). This phenomenon may be partly due to the lower proliferation rate of the siGNAS-treated group, as measured by Ki-67 staining (Fig. 6d). To further analyze the EMT status of the newly generated tumor cells, an immunoblotting assay was carried out, and the results were similar to the in vitro situation with an increase in E-cadherin and decrease in Snail1 in siGNAS-treated tumors (Fig. 6e, f). Collectively, silencing of GNAS severely affected tumor growth and EMT in vivo.