Glutamate Ionotropic Receptor Kainate Type Subunit 3 (GRIK3) promotes epithelial‐mesenchymal transition in breast cancer cells by regulating SPDEF/CDH1 signaling

Abstract Glutamate Ionotropic Receptor Kainate Type Subunit 3 (GRIK3) is an important excitatory neurotransmitter receptor that plays a significant role in various neurodegenerative diseases. However, the biological functions of GRIK3 in malignancies are largely unknown because of limited related studies. Here, we primarily reported that the expression of GRIK3 was higher in breast cancer tissues than in adjacent noncancerous tissues. GRIK3 expression was also positively correlated with the prognosis of patients with breast cancer. GRIK3 promoted the proliferation and migration abilities of breast cancer cells and enhanced the growth of orthotopically implanted tumors. Mechanically, GRIK3 influenced a range of signaling pathways and key signal transducers, including two epithelial‐mesenchymal transition regulators, SPDEF and CDH1. Heterogenous expression of SPDEF and CDH1 counteracted the migration and invasion abilities, respectively, of breast cancer cells induced by GRIK3. Moreover, overexpression of GRIK3 increased the expression of mesenchymal markers and decreased the expression of epithelial markers, resulting in the translocation of β‐catenin into the nucleus and the increased β‐catenin transcriptional activity. In conclusion, the present study reported a novel oncogenic role of GRIK3. Meanwhile, GRIK3, as a membrane receptor, may also serve as a potential therapeutic target for the treatment of breast cancer.


| INTRODUCTION
Breast cancer is one of the most serious threats to the health of women and is the leading disease in gynecological oncology. However, current conventional therapeutic targets for breast cancer treatment are still limited and drug resistance has become a growing problem. Therefore, developing novel therapeutic targets and designing targeted drugs will greatly improve the clinical treatment of breast cancer.
Ionotropic glutamate receptors (iGluRs) serve as the first messenger of glutamic acid through binding to glutamic acid and mediating the signal transmission. iGluRs are divided into three subfamilies on the basis of the structural similarity: N-methyl-d-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-iso-xazolepropionic acid and kainate receptors (KA-R). 1 Glutamate receptor ionotropic, kainate 3 (GRIK3) is the member of the ionotropic glutamate KA-R subfamily, a branch of glutamate receptor family, which plays a critical role in synaptic potentiation-an essential process for learning and memory. [2][3][4][5][6] To our knowledge, few studies have reported on the association of GRIK3 with cancer. The expression of GRIK3 was found in rhabdosarcoma, neuroblastoma, thyroid tumor, lung cancer, breast cancer, astrocytoma, multiple myeloma, glioma, and colorectal cancer. 7 Moreover, the GRIK3 gene was found to be methylated across all stages of lung adenocarcinoma, indicating that GRIK3 might be an epigenetic marker for diagnosis. 8 Recently, Gong et al 9 showed that GRIK3 expression could serve as an independent prognostic biomarker and a novel treatment target for patients with gastric cancer. However, the precise mechanism by which GRIK3 expression influences cancer progression remains unclear.
It has been shown that GRIK3 is enriched in the neuroactive ligand receptor interaction pathway and haploinsufficiency of GRIK3 may be responsible for the severe developmental delay. 2,9,10 Interestingly, a previous study indicated that, in breast cancer, multiple estradiol (E2) stimulated or inhibited genes also enriched in the neuroactive ligand receptor interaction pathway were able to affect the cell proliferation. 11 On the basis of the current studies, we proposed that GRIK3 might function in the development of breast cancer.
In the present study, we sought to gain new insights into the role of GRIK3 in the progression and development of breast cancer. Our study aimed to reveal the expression and clinical significance of GRIK3 in breast cancer, explore further whether the overexpression of GRIK3 protein is a key step in the growth and metastasis of breast cancer, and explore the specific molecular mechanism GRIK3 affects breast cancer development.  Triple-negative breast cancer (TNBC) and 76 adjacent nontumor tissues. The messenger RNA (mRNA) expression data of five GRIK family members, from GRIK1 to GRIK5, was extracted and the heat map was drawn using R software pheatmap (https://cran.r-project.org/ web/packages/pheatmap/index.html). The relative expression levels of these genes were shown with a log2-transformed value.

| Statistical analysis
The data were presented as mean ± standard error of the mean. All experimental data were analyzed using the SPSS 13.0 (SPSS Inc, Chicago, IL) and Graphpad Prism 6 software. Kaplan-Meier plots and the log-rank test were used to construct the survival curve. Paired or independent the Student t test was used to compare two groups with Gaussian data. Differences were considered significant when the P value was less than 0.05. (*P < 0.05, **P < 0.01 and ***P < 0.001)

| Immunofluorescence assay
The cells were seeded on coverslips laid in six-well plates and cultured at 37°C in 5% CO 2

| Colony formation assay
The colony formation assay was performed in a six-well plate. The cells were cultured at 37°C in 5% CO 2 for 15 to 20 days until colonies formed. The cells (colonies) were fixed with methanol and stained with 0.5% crystal violet. The number of colonies was counted three times by three independent laboratory technicians.  week. The masses of tumors and the lifetime of mice in the different groups were compared using the two-tailed paired the Student t test.

| Quantitative Real-Time polymerase chain reaction
RNA was extracted from tissue or cells using TRIzol reagent (Invitrogen, Carlsbad, CA) and complementary DNA was obtained by reverse transcription using the PrimeScrip RT-PCR kit (Takara, Tokyo), following the manufacturers' protocols. Real-time PCR was performed on an ABI 7500 RT-PCR instrument (Applied Biosystems, Singapore). The amplification parameters were as follows: 30 seconds at 95°C, followed by 40 cycles at 95°C for 5 seconds and 65°C for 34 seconds. The melt curve procedure was as follows: 15 seconds at 95°C, followed by 60°C Values of P < 0.05 were considered statistically significant. Gene ontology (GO) enrichment (www.geneontology.org) and KEGG (www.genome.jp/kegg/) pathway analyses were performed to analyze and classify the differentially expressed genes.   Figure 1F). Taken together, these results suggested that the mechanism underlying how GRIK3 was transcribed into mRNA and further translated into protein is complicated and requires further study, however GRIK3 expression at protein level could be served as a biomarker for breast cancer.

| GRIK3 promotes breast cancer cell proliferation and migration
Given the high expression level of GRIK3 in breast cancer tissues, we hypothesized that GRIK3 could be an oncogene. Heterogenous

| GRIK3 promotes the growth of subcutaneously transplanted tumors in nude mice
To investigate whether GRIK3 could promote tumor growth in nude mice, 1 × 10 6 MDA-MB-231 GRIK3 overexpressing cells and corresponding control cells were injected subcutaneously. The tumor size and the weight of each nude mouse were measured every week.
After 5 weeks, the tumors were surgically removed, photographed, and weighed. The results showed that, compared with the control group, overexpression of GRIK3 significantly promoted the growth of tumors ( Figure 3A and 3B). The mice injected with GRIK3 overexpressing cells had survival times much shorter than the control group ( Figure 3D). However, there were no significant differences in the weight between the two groups ( Figure 3C). On the basis of these results, we concluded that GRIK3 is involved in the tumorigenesis of breast cancer.

| The transcriptomic analysis and the genes expressed differentially between the GRIK3 overexpressing cells and the control cells
To explore further the molecular mechanisms underlying GRIK3 related signaling, we compared the transcriptional landscape between GRIK3 overexpressing cells and the control cells using RNA-sequencing ( Figure   4A). Total RNA were extracted from GRIK3 overexpressing cells and control cells and subsequently sequenced. Comparing with the control group (fold change >2), a total of 21 genes showed higher expression levels in GRIK3 overexpressing cells than in the control cells. Meanwhile, 68 genes were downregulated and the expression of 12 735 genes did not change significantly. The GO analysis of the differentially expressed genes showed that GRIK3 mainly influenced the biological process "regulation of biological quality" and neuronal part of the molecular function ( Figure 4B). Interestingly, GRIK3 was also involved in actin cytoskeleton regulation, indicating a possible role of GRIK3 in the morphologic change of tumor cells. The KEGG analysis revealed that GRIK3 affected several key signaling pathways in breast cancer, such as the Notch signaling pathway and Jak-STAT signaling pathway ( Figure 4C).
We then verified the mRNA expression of 18 genes with 丨fold change丨  Figure 6A). Immunoblot assays demonstrated that E-cadherin, the epithelial markers, were inhibited, whereas mesenchymal markers, such as Vimentin, Slug, and N-cadherin were upregulated in GRIK3-overexpressing cells ( Figure 6B). In contrast, knockdown of GRIK3 reduced the expression of Vimentin, Slug, and N-cadherin and increased Ecadherin expression ( Figure 6B). Interestingly, the same tendency of protein expression changes of E-cadherin, Vimentin, Slug, and N-cadherin accorded with their mRNA expression changes, according to the RNA-seq data of GRIK3 overexpressing cells and control cells. Using immunofluorescence to visualize intracellular β-catenin, we observed an increase in β-catenin levels and the translocation of β-catenin from cell membrane to nucleus ( Figure 6C). To validate whether the translocation of β-catenin is the key step for its transcriptional activation, we examined several downstream target genes of β-catenin. As shown in Figure 6D SAM pointed domain containing ETS transcription factor (SPDEF), also known as prostate-derived ETS factor, belongs to the ETS (E26 transformation specific) transcription factor family and is a specific transcription factor which preferentially binds a GGAT DNA motif rather than the GGAA ETS family consensus sequence. The expression of SPDEF could be detected in multiple organs, including the airway, breast, gastric, prostate, and small and large intestinal epithelia, and is able to inhibit the cell proliferation. 17 It has also been demonstrated that SPDEF acts as a tumor and metastasis suppressor in multiple epithelium-derived cancers. 18   Firfly-luc/Renilla-luc F I G U R E 6 Overexpression of GRIK3 promoted EMT. A, Phase-contrast microscopic images of GRIK3 overexpressing cells and the control cells. B, Western blot showing the effect of GRIK3 overexpression and knockdown on the expression of epithelial and mesenchymal markers in GRIK3 overexpressing cells and the control cells. This assay was repeated in triplicate. C, The expression of β-catenin levels between GRIK3 overexpressing cells and the control cells was observed under a confocal microscope. **P < 0.01 D, qPCR assay measuring the mRNA expression of β-catenin downstream target gene in response to GRIK3 overexpression. *P < 0.05, **P < 0.01, and ***P < 0.001. E, Relative luciferase activity promoted by β-catenin promoter was measured in control cells, GRIK3 overexpressing cells and GRIK3 overexpressing cells that transfected with siGRIK3. Luc: luciferase. *P < 0.05 by one-way ANOVA. ANOVA, analysis of variance; EMT, epithelial-mesenchymal transition; GRIK3, glutamate receptor ionotropic, kainate 3; mRNA, messenger RNA [Color figure can be viewed at wileyonlinelibrary.com] The present study is the first study suggesting that GRIK3 is an oncogene in breast cancer and is involved in the EMT pathway.
Although the biological functions of GRIK3 in breast cancer have been exposed only slightly, additional research should be conducted into the mechanics of its oncogenic effects, such as how GRIK3 inhibits the expression of CDH1 and is the structure of the GRIK3centric protein interaction network. A greater understanding of membrane protein GRIK3 offers new opportunities for the design of anti-GRIK3 therapeutic strategies.