The mRNA-edited form of GABRA3 suppresses GABRA3-mediated Akt activation and breast cancer metastasis

Abstract

Metastasis is a critical event affecting breast cancer patient survival. To identify molecules contributing to the metastatic process, we analysed The Cancer Genome Atlas (TCGA) breast cancer data and identified 41 genes whose expression is inversely correlated with survival. Here we show that GABAA receptor alpha3 (Gabra3), normally exclusively expressed in adult brain, is also expressed in breast cancer, with high expression of Gabra3 being inversely correlated with breast cancer survival. We demonstrate that Gabra3 activates the AKT pathway to promote breast cancer cell migration, invasion and metastasis. Importantly, we find an A-to-I RNA-edited form of Gabra3 only in non-invasive breast cancers and show that edited Gabra3 suppresses breast cancer cell invasion and metastasis. A-to-I-edited Gabra3 has reduced cell surface expression and suppresses the activation of AKT required for cell migration and invasion. Our study demonstrates a significant role for mRNA-edited Gabra3 in breast cancer metastasis.

Figure 1. Gabra3 is highly expressed in human breast cancer cells and tissues but not in normal human breast tissues and cells.

High Gabra3 expression is inversely correlated with survival in breast cancer. (a) Expression of Gabra3 transcript in normal human tissues. Error bars represent mean±s.d.; n=3. (b) Gabra3 protein expression in human breast cancer cell lines SKBR3, MDA-MB-453, MDA-MB-436, MDA-MB-231 and normal human breast epithelial cell HMEL. (c) Expression of Gabra3 transcript in paired metastatic and primary human breast cancer tissues. P value was determined using Student's t-test (P<0.01). Error bars represent mean±s.d.; n=3. (d) Association of high Gabra3 expression with poor survival in breast cancer (Cox regression P=0.001, hazard ratio=2.85).

Figure 2. Gabra3 promotes tumour cell migration, invasion and metastasis in breast cancer.

(a,b) Human breast cancer MCF7 cells stably expressing Gabra3 were subjected to migration (a) and invasion (b) assays. The numbers of migrated and invaded cells were quantified. P value was determined using Student's t-test (*P<0.001). Error bars represent mean±s.d.; n=3. (c) Luciferase-tagged human breast cancer MCF7 cells stably expressing Gabra3 or a vector control were transplanted in mouse mammary fat pads. Primary tumour and metastasis were imaged using Xenogen bioluminescence system. P value was determined using Fisher's exact test (P<0.0001).

Figure 3. Suppression of Gabra3 expression inhibits tumour cell invasion and metastasis in breast cancer.

(a,b) Human breast cancer MDA-MB-436 cells stably expressing Gabra3 shRNAs were subjected to migration (a) and invasion (b) assays. The numbers of migrated and invaded cells were quantified. P value was determined using Student's t-test (*P<0.01). Error bars represent mean±s.d.; n=3. (c) Luciferase-tagged human breast cancer MDA-MB-436 cells stably expressing Gabra3 shRNA or control shRNA were transplanted in mice. Metastasis was imaged using Xenogen bioluminescence system. P value was determined using Fisher's exact test (P=0.035).

Figure 4. A-to-I RNA-edited Gabra3 is expressed in non-invasive human breast cancer cells.

(a) Percentage of A-to-I-edited Gabra3 expressed in human breast cancer cell lines were determined by sequencing. (b) Sequencing of Gabra3 expressed in non-invasive human breast cancer MCF7. Arrow indicates the A-to-I-edited nucleotide of Gabra3. (c) Sequencing of Gabra3 expressed in invasive human breast cancer MDA-MB-436 cells. Arrow indicates the nucleotide of adenosine of the unedited Gabra3. (d) Percentage of A-to-I-edited Gabra3 expressed in paired human breast cancer primary and metastatic samples was determined by sequencing. (e) Expression of RNA-editing enzyme ADAR1p110 in human breast cancer cells and normal human breast epithelial cells. All error bars in the figure represent the mean±s.d. of three independent experiments.

Figure 5. A-to-I RNA-edited Gabra3 suppresses tumour cell migration, invasion and metastasis in breast cancer.

(a,b) Human breast cancer MDA-MB-436 cells stably expressing RNA-edited Gabra3 were subjected to migration (a) and invasion (b) assays. The numbers of migrated and invaded cells were quantified. P value was determined using Student's t-test (*P<0.001). (c,d) Human breast cancer MCF7 cells stably expressing a control vector, or unedited Gabra3, or unedited Gabra3 and RNA-edited Gabra3, were subjected to migration (c) and invasion (d) assays. The numbers of migrated and invaded cells were quantified. P value was determined using Student's t-test (*P<0.001, **P<0.01). (e) Luciferase-tagged human breast cancer MDA-MB-436 cells stably expressing RNA-edited Gabra3 or a vector control were transplanted in mice. Luciferase signals of metastasis were quantified using Xenogen bioluminescence system. P value was determined using Student's t-test (P<0.05). All error bars in the figure represent the mean±s.d. of three independent experiments.

Figure 6. A-to-I RNA-edited Gabra3 reduces Gabra3 expression on cell surface and suppresses AKT activation.

(a) Representative flow cytometry histogram overlay of Gabra3 surface expression in MDA-MB-436. Human breast cancer MDA-MB-436 cells expressing RNA-edited Gabra3 (blue), or a control vector (grey), were subjected to FACS analysis using a Gabra3 antibody or control IgG. (b) Representative flow cytometry histogram overlay of Gabra3 surface expression in MCF7 cells. Human breast cancer MCF7 cells stably expressing a control vector (blue), or unedited Gabra3 (grey), or both unedited Gabra3 and RNA-edited Gabra3 (red), were subjected to FACS analysis using a Gabra3 antibody or control IgG. The expression of RNA-edited Gabra3 reverses the phenotypes of wild-type Gabra3. (c) Phosphorylated and total AKT in human breast cancer MDA-MB-436 cells expressing RNA-edited Gabra3, or a control vector, were determined by immunoblotting. (d) Phosphorylated and total AKT in human breast cancer MCF7 cells stably expressing a control vector, or unedited Gabra3, or both unedited Gabra3 and RNA-edited Gabra3, were determined by immunoblotting.