GABARAP is downregulated in primary breast cancer specimens as well as in breast cancer cell lines
To investigate the potential role of GABARAP in breast cancer, we first analyzed GABARAP mRNA expression via breast cancer RNA-seq data from the TCGA database. We found that GABARAP expression was significantly downregulated in breast cancers compared with normal tissues (P < 0.0001, Fig. 1a). GABARAP expression was lower in all subtypes of breast cancer tissues than in normal tissues (Fig. 1b) (P < 0.0001). Moreover, the results of the TCGA data analysis were validated in 87 IDC samples, 48 DCIS samples and 24 noncancerous tissue samples via IHC (Fig. 1e). Positive staining (brown) was detected in the majority of noncancerous tissues (16/24) and in some DCIS tissues (17/48) but was detected less frequently in IDC tissues (27/87) (P < 0.01, Fig. 1f, Additional file 1: Table S1). This suggests that the low expression of GABARAP is related to the occurrence and development of breast cancer.
To test whether GABARAP was also decreased in cultured breast cancer cell lines, we conducted western blot analysis on eight breast cancer cell lines and nontransformed MCF-10A cells. As shown in Fig. 1g and h, GABARAP displayed high expression levels in the nontransformed MCF-10A cells but only weak expression in T47D, UACC-812, and MCF-7 cells; furthermore, GABARAP had low expression in MDA-MB-453, SKBR3, HCC70, MDA-MB-231 and MDA-MB-468 cells (P < 0.05). These results indicated that GABARAP is downregulated in breast cancer cells.
The clinical significance of GABARAP in patients with breast cancer
To further investigate whether low GABARAP expression is involved in breast cancer progression, we analyzed GABARAP mRNA expression based on RNA-seq data of individual cancer stages in breast cancer from the TCGA database (Fig. 1c). GABARAP downregulation was strongly associated with pTNM stage (P < 0.01). Then, we analyzed the correlation between GABARAP levels and the clinicopathological features of 87 patients with histopathologically confirmed IDC at Harbin Medical University Cancer Center (HMUCC). As shown in Additional file 1: Table S2, statistical analyses using the IHC results revealed that low levels of GABARAP in patients correlated with advanced pT classification (p = 0.025), axillary lymph node metastasis (P = 0.023), advanced pTNM stage (p = 0.001), histological grade (p = 0.019) and ER status (p = 0.039). However, no significant association was found between GABARAP and age, Her-2 status, Ki-67 levels or P53 status. Therefore, we hypothesize that low expression of GABARAP is involved in tumor cell proliferation and plays an important role in breast cancer development. Additionally, survival analysis showed that patients with low levels of GABARAP expression had lower overall survival (p = 0.0047) than those with high GABARAP expression levels (TCGA, Fig. 1d). These results demonstrate that GABARAP is expressed at low levels in breast cancer tissue and that low expression is associated with a poor prognosis for these patients.
Low GABARAP levels enhance the malignant behavior of breast cancer cells
We performed western blotting analysis to compare the expression levels of GABARAP in breast cancer cell lines and nontransformed MCF-10A cells. As shown in Fig. 1g, T47D and UACC-812 cells were used as GABARAP “loss-of-function” models, while MDA-MB-453 cells served as a GABARAP “gain-of-function” model. We detected the protein expression level of GABARAP in these target cells by western blot. As shown in Fig. 2a and b, compared with control cells, T47D and UACC-812 cells exhibited significant knockdown of GABARAP while MDA-MB-453 cells presented increased GABARAP expression was increased in (Fig. 3a).
To test whether GABARAP affects the proliferation of breast cancer cell lines, we conducted CCK8 assays and measured cell viability. Knockdown of GABARAP significantly accelerated cell growth in T47D and UACC-812 cells compared to the respective control cells (Fig. 2c and d), whereas overexpression of GABARAP in MDA-MB-453 cells attenuated cell growth (Fig. 3b). Accordingly, we observed similar patterns in the colony forming efficiency of GABARAP-overexpressing MDA-MB-453 cells (Fig. 3c) and GABARAP-shRNA-transfected T47D and UACC-812 cells (Fig. 2e and f), suggesting that GABARAP negatively regulates cell proliferation in vitro.
We further studied the potential effects of GABARAP on cell migration and invasion using wound healing and transwell assays, respectively. As shown in Fig. 2g - j, both migration and invasion were significantly enhanced in T47D and UACC-812 cells with knockdown of GABARAP. However, overexpression of GABARAP in MDA-MB-453 cells markedly inhibited cell migration and invasion compared to MDA-MB-453 cells transfected with vector controls (Fig. 3d and e) These results suggest that GABARAP inhibits cell migration and invasion in vitro.
GABARAP Regulates Breast Cancer Progression Via EMT
EMT plays an important role in tumor metastasis, and we hypothesized that GABARAP affects the EMT process and inhibits breast cancer progression. Then, we analyzed the expression profile data of breast cancer patients clinical cohort from TCGA database. Pearson correlation was calculated among genes related to GABARAP, CDH1, MMP2, MMP14 and Akt/ mTOR signaling pathway. In result, the expression correlation of GABARAP and Akt/mTOR signaling pathway was identified in the clinical cohort of TCGA breast cancer patients, revealed the potential influence of GABARAP and Akt/mTOR signaling pathway. Furthermore, Akt/mTOR signaling pathway mediates genes related to epithelial mesenchymal transformation, such as CDH1, MMP2 and MMP14 (Fig. 4a). Here, we show that downregulation of GABARAP was associated with high levels of Vimentin, N-cadherin, MMP2 and MMP14 and low levels of E-cadherin (Fig. 4b). Conversely, GABARAP upregulation inhibited Vimentin, N-cadherin, MMP2 and MMP14 expression and upregulated E-cadherin expression (Fig. 4c). Taken together, these results indicate that GABARAP inhibits EMT and thereby suppresses breast cancer progression.
Low levels of GABARAP induce EMT by activating the AKT/mTOR pathway
Multiple pathways, including mTOR, PI3K/AKT and NF-κB signaling, are involved in GABARAP-regulated autophagy, inflammation and angiogenic activity [8–13]. To further verify how GABARAP regulates EMT, we measured AKT/mTOR, NF-κB and ERK/MAPK signaling in breast cancer cells. Knockdown of GABARAP in T47D and UACC-812 cells increased the levels of phosphorylated AKT, mTOR and p70s6k (Fig. 4b) but had no effect on p-ERK, p-MEK, p-IKK-β and p-IκBα levels (Additional file 3: Fig. S1). By contrast, overexpression of GABARAP suppressed the phosphorylation of AKT, mTOR and p70s6k (Fig. 4c), and showed no effect on p-ERK, p-MEK, p-IKK- β and p-IκBα levels (Additional file 3: Fig. S1). These results indicate that GABARAP levels are negatively correlated with the activation of AKT/mTOR pathways. Using pathway inhibitors, we found that GABARAP-mediated inhibition of invasion, migration, and EMT was reversed by the AKT pathway inhibitor LY-294002 (50 µM, 24 h; Fig. 5a, b, c, d and e). Taken together, these data indicate that downregulation of GABARAP activates the AKT/mTOR pathway, which in turn promotes EMT of breast cancer cells.
GABARAP Suppresses Breast Cancer Progression In Vivo
To evaluate the role GABARAP plays in regulating breast cancer progression in vivo, we established a xenograft tumor model in nude mice using UACC-812 cell lines stably transfected with vector control or GABARAP-shRNA and treated the animals with LY294002 (75 mg/kg) or sterile water. Mice were randomly assigned to the following experimental groups: vector control group, GABARAP-shRNA group, vector control + LY294002 group and GABARAP-shRNA + LY294002 group (n = 5 per subgroup). (Fig. 6a). As expected, silencing GABARAP significantly increased tumor volumes and weight in mice compared with those in the control group. However, GABARAP-mediated inhibition of tumor formation was reversed by LY-294002 (Fig. 6b and c). Furthermore, tumors with GABARAP knockdown had a higher metastasis capacity, as evidenced by the increased MMP2 and p-mTOR staining and reduced E-cadherin staining (Fig. 6d). These results indicate that knocking down GABARAP suppresses breast cancer progression in vivo.
GABARAP Suppresses Tumor Progression In Clinical Breast Cancer Specimens
To further examine the relationship between GABARAP and metastasis in human breast cancer, we performed IHC staining of GABARAP, MMP2 and MMP14 in 87 IDC specimens (Fig. 7a and c). Consistent with our observations in the tumor cell lines and xenograft models, the distribution and intensity of GABARAP were negatively correlated with MMP2 (P = 0.0013, Fig. 7b) and MMP14 (P = 0.019, Fig. 7d) in breast cancer tissue specimens. Which clearly indicated that low GABARAP expression was associated with elevated metastasis in breast cancer patients.