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. 2016 May 31;7(22):32341-50.
doi: 10.18632/onco_target.8700.

GABRA3 promotes lymphatic metastasis in lung adenocarcinoma by mediating upregulation of matrix metalloproteinases

Liping Liu  1 , Chenglin Yang  2   3 , Jianfei Shen  3 , Liyan Huang  1 , Weixuan Lin  1 , Hailing Tang  1 , Wenhua Liang  3 , Wenlong Shao  3 , Haibo Zhang  1   4 , Jianxing He  2   3
Affiliations

GABRA3 promotes lymphatic metastasis in lung adenocarcinoma by mediating upregulation of matrix metalloproteinases

Liping Liu et al. Onco_target. .

Abstract

Tumor metastasis is the main reason for the poor prognosis of lung cancer patients. The GABAA receptor subunit GABRA3 is reportedly upregulated in lung cancer. Herein, we show that high GABRA3 protein expression in lung adenocarcinoma correlated positively with disease stage, lymphatic metastasis status and poor patient survival. In addition, GABRA3 induced MMP-2 and MMP-9 expression through activation of the JNK/AP-1 signaling pathway, which enhanced lymphatic metastasis by lung adenocarcinoma both in vitro and in vivo. These results indicate that GABRA3 promotes lymph node metastasis and may thus be an effective therapeutic _target for anticancer treatment.

Keywords: GABRA3; JNK/AP-1; MMP; lymphatic metastasis; non-small cell lung cancer.

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Conflict of interest statement

No potential conflicts of interest were disclosed.

Figures

Figure 1
Figure 1. Upregulation of GABRA3 correlates with poor prognosis in LUAD
(A) Western blots showing expression of GABRA3 protein in lung epithelial cells and LUAD cells. a-Tubulin was used as a loading control. (B–C)Comparison of GABRA3 protein expression between primary LUAD tissues (T) and matched adjacent non-tumor tissues (ANT) using Western blot analysis (B) and IHC (C). Scale bars, 50 μm. (D). Kaplan-Meier survival curves for LUAD patients showing low and high levels of GABRA3 expression. (E). Correlation of GABRA3 expression patients with lymphatic metastasis and those without metastasis. Left panel: micrographs of two representative cases. Scale bars, 50 mm; insets, 10 mm. Right panel: Chi-square analysis of the relation between low or high GABRA3 expression and lymphatic metastasis.
Figure 2
Figure 2. GABRA3 promotes the invasiveness of LUAD cells
(A–B) Western blot analysis of the effect of overexpressing (A) or silencing (B) GABRA3 in the A549 and NCI-H1650 cell lines. a-Tubulin was used as a loading control. (C–D) Representative micrographs (left panel) and quantification (right panel) of invaded cells in a Transwell matrix penetration assay. Scale bars, 50 mm. Error bars depict the mean ± SD from three independent experiments, *P < 0.05.
Figure 3
Figure 3. GABRA3 promotes lymph node metastasis in vivo
(A) Representative micrographs of the popliteal lymph node metastasis model. The indicated cells stably expressing firefly luciferase were inoculated into the footpads of mice. (B) Representative micrographs of popliteal lymph nodes immunostained with anti-luciferase antibody. Scale bars: upper panel, 200 mm; lower panel, 20 mm. (C) Ratios of metastatic to total lymph notes in the indicated cells. *P < 0.05.
Figure 4
Figure 4. GABRA3 induces MMP-2 and MMP-9 expression in LUAD cells
(A) Real-time PCR analysis of MMP-2 and MMP-9 mRNA expression in the indicated cells. Transcript levels were normalized to GAPDH expression. (B) Levels of MMP-2 and MMP-9 protein in supernatants from the indicated cell cultures assessed using ELISAs. (C) Representative micrographs (upper panel) and quantification (lower panel) of invaded cells with or without treatment with MMP-2/MMP-9 inhibitor (200 nM). Scale bars, 50 mm. Error bars depict the mean ± SD of three independent experiments, *P < 0.05.
Figure 5
Figure 5. Clinical relevance between GABRA3 and MMP-2/MMP-9 expression in human LUAD
(A) Relation between GABRA3 and MMP-2/MMP-9 expression. Shown are representative micrographs of two cases. Scale bars, 50 mm. (B) Percentages of LUAD specimens showing low- or high- GABRA3 expression in relation to MMP-2/MMP-9 expression.
Figure 6
Figure 6. GABRA3 activates the JNK/AP-1 signaling pathway
(A) Transcriptional activities of an AP-1 luciferase reporter plasmid in the indicated cells. (B) Western blot analysis of p-JNK1/2(T183/Y185), total JNK1/2, p-c-Jun (S73), and total c-Jun expression in the indicated cells. a-Tubulin was used as a loading control. (C and D) MMP-2 and MMP-9 mRNA and protein expression in the indicated cells with or without treatment with a JNK inhibitor (150 nM) (C) or c-Jun siRNA (D). E and F. Representative micrographs (left panel) and quantification (right panel) of invaded cells with or without treatment with a JNK inhibitor (E) or c-Jun siRNA (F). Scale bars, 50 mm. Error bars depict the mean ± SD of three independent experiments, *P < 0.05.
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