ELK3 Controls Gastric Cancer Cell Migration and Invasion by Regulating ECM Remodeling-Related Genes

Current therapeutic strategies for gastric cancer, including surgery and chemotherapy improve patient survival; however, the survival rate of patients with metastatic gastric cancer is very low. The molecular mechanisms underlying the dissemination of gastric cancer cells to distant organs are currently unknown. Here, we demonstrate that the E26 transformation-specific (ETS) transcription factor ELK3 (ELK3) gene is required for the migration and invasion of gastric cancer cells. The ELK3 gene modulates the expression of extracellular matrix (ECM) remodeling-related genes, such as bone morphogenetic protein (BMP1), lysyl oxidase like 2 (LOXL2), Snail family transcriptional repressor 1 (SNAI1), serpin family F member 1 (SERPINF1), decorin (DCN), and nidogen 1 (NID1) to facilitate cancer cell dissemination. Our in silico analyses indicated that ELK3 expression was positively associated with these ECM remodeling-related genes in gastric cancer cells and patient samples. The high expressions of ELK3 and other ECM remodeling-related genes were also closely associated with a worse prognosis of patients with gastric cancer. Collectively, these findings suggest that ELK3 acts as an important regulator of gastric cancer cell dissemination by regulating ECM remodeling.


Introduction
Gastric cancer is one of the most common types of cancer worldwide and a leading cause of cancer-related mortality [1,2]. The five-year survival rate of patients with gastric cancer with early-stage disease is 70%, whereas that of patients with invasive and distant metastasis is less than one third [3]. It is well-known that metastasis is closely associated with patient mortality. Several reports suggest that microRNAs, Wnt signaling, and extracellular matrix (ECM)-related factors are possible mechanisms of gastric cancer metastasis [4][5][6][7][8][9][10][11]. However, the molecular mechanisms underlying gastric cancer invasion and metastasis remain to be elucidated.
ETS transcription factor ELK3 (ELK3) plays a pivotal role in promoting the progression and metastasis of a number of types of cancer, including breast, prostate, bladder and liver cancer [12][13][14][15][16]. ELK3 is an E26 transformation-specific (ETS) transcription factor family member that regulates various genes, including zinc finger E-box binding homeobox 1 (Zeb1) and hypoxia-inducible factor-1α (HIF1α), to control cell migration and metastasis [14,15]. ELK3 also modulates cell migration by regulating the expression of extracellular matrix (ECM)-related genes, such as bone morphogenetic protein (BMP1), serpin family E member 1 (SERPINE1), and membrane type 1-matrix metalloproteinase (MT1-MMP) [17][18][19][20]. Of note, ECM remodeling-related proteins, including BMP1 and serpin family members, are involved in gastric cancer invasion and metastasis [7,21,22]. However, the functional link between the ELK3 gene and ECM on gastric cancer cell migration is not yet fully understood, although the high expression of ELK3 in patients with gastric cancer is closely associated with cancer progression [23].
Here, we investigated whether ELK3 expression is functionally associated with the metastatic phenotype of gastric cancer. We found that ELK3 regulated the migration and invasion of gastric cancer cells. RNA sequencing data revealed that ELK3 regulated other ECM remodeling-related genes, particularly bone morphogenetic protein (BMP1), lysyl oxidase like 2 (LOXL2), snail family transcriptional repressor 1 (SNAI1), serpin family F member 1 (SERPINF1), decorin (DCN), and nidogen 1 (NID1) for controlling cell dissemination. The analysis of various gastric cancer cell lines and gastric cancer patient samples also revealed a positive association between ELK3 and BMP1, LOXL2, SNAI1, SERPINF1, DCN, and NID1, and these genes were associated with a poor prognosis. Our finding demonstrated that the ELK3-mediated genetic network in ECM remodeling contributed to the migration and invasion of gastric cancer cells.

ELK3 Controls Gastric Cancer Cell Migration and Invasion in E-Cadherin Independent Manner
To examine the effect of ETS transcription factor ELK3 (ELK3) on the cell dissemination capacity of gastric cancer cells, we examined ELK3 mRNA expression in various gastric cancer cell lines using the Cancer Dependency Map (DepMap) database ( Figure 1A). We also analyzed ELK3 mRNA level and protein level with five gastric cancer cell lines ( Figure 1B,C). From these analyses and a previous report [24], we selected gastric cancer cell lines exhibiting a relatively low and high expression of the ELK3 gene (SNU484 and SNU638 cells, respectively) and further examined the molecular link between ELK3 and cell migration. yet fully understood, although the high expression of ELK3 in patients with gastric cancer is closely associated with cancer progression [23].
Here, we investigated whether ELK3 expression is functionally associated with the metastatic phenotype of gastric cancer. We found that ELK3 regulated the migration and invasion of gastric cancer cells. RNA sequencing data revealed that ELK3 regulated other ECM remodeling-related genes, particularly bone morphogenetic protein (BMP1), lysyl oxidase like 2 (LOXL2), snail family transcriptional repressor 1 (SNAI1), serpin family F member 1 (SERPINF1), decorin (DCN), and nidogen 1 (NID1) for controlling cell dissemination. The analysis of various gastric cancer cell lines and gastric cancer patient samples also revealed a positive association between ELK3 and BMP1, LOXL2, SNAI1, SERPINF1, DCN, and NID1, and these genes were associated with a poor prognosis. Our finding demonstrated that the ELK3-mediated genetic network in ECM remodeling contributed to the migration and invasion of gastric cancer cells.

ELK3 Controls Gastric Cancer Cell Migration and Invasion in E-Cadherin Independent Manner
To examine the effect of ETS transcription factor ELK3 (ELK3) on the cell dissemination capacity of gastric cancer cells, we examined ELK3 mRNA expression in various gastric cancer cell lines using the Cancer Dependency Map (DepMap) database ( Figure 1A). We also analyzed ELK3 mRNA level and protein level with five gastric cancer cell lines ( Figure 1B,C). From these analyses and a previous report [24], we selected gastric cancer cell lines exhibiting a relatively low and high expression of the ELK3 gene (SNU484 and SNU638 cells, respectively) and further examined the molecular link between ELK3 and cell migration. First, we determined whether the overexpression of ELK3 regulated cell migration and invasion. The SNU484 cells exhibited overexpression of ELK3 mRNA and protein after transfection with pLenti-cMyc-DDK-ELK3 plasmid DNA (Figure 2A,B). The SNU484 cells overexpressing the ELK3 gene exhibited a marked increase in cell migration and invasion ( Figure 2C). Our previous study demonstrated that ELK3 repressed the expression of E-cadherin, a marker of epithelial cell, to trigger breast cancer metastasis [14]. Therefore, we examined the expression of epithelial-mesenchymal transition (EMT) markers in First, we determined whether the overexpression of ELK3 regulated cell migration and invasion. The SNU484 cells exhibited overexpression of ELK3 mRNA and protein after transfection with pLenti-cMyc-DDK-ELK3 plasmid DNA (Figure 2A,B). The SNU484 cells overexpressing the ELK3 gene exhibited a marked increase in cell migration and invasion ( Figure 2C). Our previous study demonstrated that ELK3 repressed the expression of E-cadherin, a marker of epithelial cell, to trigger breast cancer metastasis [14]. Therefore, we examined the expression of epithelial-mesenchymal transition (EMT) markers in ELK3 overexpressed SNU484 cells. Unlike the data from the previous study on breast cancer, E-cadherin expression was no different between control and ELK3 overexpressed SNU484 cells ( Figure 2D). However, the Fibronectin protein levels were significantly increased by ELK3 in SNU484 cells. To confirm our finding, we transfected SNU638 cells with siELK3 to deplete the ELK3 gene ( Figure 3A,B). The depletion of ELK3 significantly inhibited cell migration and invasion ( Figure 3C). However, E-cadherin and Fibronectin expression were no different between control and ELK3-depleted SNU638 cells ( Figure 3D), suggesting ELK3mediated cell migration and invasion in gastric cancer cells is unnecessary for E-cadherin.
23, x FOR PEER REVIEW 3 of 15 ELK3 overexpressed SNU484 cells. Unlike the data from the previous study on breast cancer, E-cadherin expression was no different between control and ELK3 overexpressed SNU484 cells ( Figure 2D). However, the Fibronectin protein levels were significantly increased by ELK3 in SNU484 cells. To confirm our finding, we transfected SNU638 cells with siELK3 to deplete the ELK3 gene ( Figure 3A,B). The depletion of ELK3 significantly inhibited cell migration and invasion ( Figure 3C). However, E-cadherin and Fibronectin expression were no different between control and ELK3-depleted SNU638 cells ( Figure  3D), suggesting ELK3-mediated cell migration and invasion in gastric cancer cells is unnecessary for E-cadherin.  Bar graphs indicate the number of migrated and invaded cells, respectively. Error bars represent the standard deviation; * p < 0.05 and ** p < 0.01 (student's t-test). (D) Epithelial marker (E-cadherin), mesenchymal marker (Fibronectin), and ELK3 protein expression in SNU638 cells transfected with siCon or siELK3. Bar graphs indicate the quantified levels of each protein, in relative scales. Error bars represent the standard deviation; ** p < 0.01 (student's t-test). ELK3, ETS transcription factor ELK3; si, small interfering RNA; Con, control.

ELK3 Regulates extracellular matrix (ECM) Remodeling-Related Genes to Control Cell Migration and Invasion
To further investigate the molecular mechanisms underlying ELK3-mediated cell migration and invasion, RNA-Seq analysis of the control and ELK3-depleted SNU638 cells was performed. Among 25,000 genes, 262 genes were selected exhibiting a >1.5-fold change in expression, and a p-value < 0.05 was selected (Table S1). Notably, the GSEA (Gene Set Enrichment Analysis) plot revealed that the ELK3 depletion downregulated the expression of genes associated with cell junction disassembly and upregulated the genes Bar graphs indicate the number of migrated and invaded cells, respectively. Error bars represent the standard deviation; * p < 0.05 and ** p < 0.01 (student's t-test). (D) Epithelial marker (E-cadherin), mesenchymal marker (Fibronectin), and ELK3 protein expression in SNU638 cells transfected with siCon or siELK3. Bar graphs indicate the quantified levels of each protein, in relative scales. Error bars represent the standard deviation; ** p < 0.01 (student's t-test). ELK3, ETS transcription factor ELK3; si, small interfering RNA; Con, control.

ELK3 Regulates Extracellular Matrix (ECM) Remodeling-Related Genes to Control Cell Migration and Invasion
To further investigate the molecular mechanisms underlying ELK3-mediated cell migration and invasion, RNA-Seq analysis of the control and ELK3-depleted SNU638 cells was performed. Among 25,000 genes, 262 genes were selected exhibiting a >1.5-fold change in expression, and a p-value < 0.05 was selected (Table S1). Notably, the GSEA (Gene Set Enrichment Analysis) plot revealed that the ELK3 depletion downregulated the expression of genes associated with cell junction disassembly and upregulated the genes involved in cell adhesion ( Figure 4A). Figure 4B 262 selected genes. The pie graph showed the categories of the top five of the biological pathway in the ELK3-depleted SNU638 cells compared with the control SNU638 cells ( Figure 4B). Among them, we focused on the genes related to cell migration and adhesion. Heatmap showed 39 genes belonging to the category of cell migration and adhesion ( Figure 4C). In particular, we were interested in 26 downregulated genes and these genes were very closely associated with cell migration and locomotion ( Figure 4D). To further analyze the molecular network, we used STRING-based analysis using the 262 selected genes. BMP1-mediated ECM remodeling was one of the main molecular networks by ELK3induced cell migration and adhesion ( Figure 4E). Our bioinformatics analyses indicated that ELK3 regulated cell migration by modulating ECM remodeling-associated genes. After two different analyses, we selected six genes such as bone morphogenetic protein (BMP1), lysyl oxidase like 2 (LOXL2), Snail family transcriptional repressor 1 (SNAI1), serpin family F member 1 (SERPINF1), decorin (DCN), and nidogen 1 (NID1) for further studies and performed RT-qPCR for validation of ELK3-related cell migration via those genes. The ELK3depleted SNU638 cells exhibited a significant downregulation in the mRNA expression of BMP1, LOXL2, SNAI1, SERPINF1, DCN, and NID1. The overexpression of ELK3 in ELK3-depleted SNU638 cells markedly increased the expression of these genes ( Figure 5A), indicating that the ELK3 gene positively regulates the expression of BMP1-mediated ECM remodeling-related genes in SNU638 cells. Finally, we determined whether ELK3 is essential for gastric cancer cell migration. Transwell assays revealed the inhibition of the migration of ELK3-depleted SNU638 cells; however, the cell migratory ability recovered after the ELK3-depleted SNU638 cells were transfected with the ELK3 overexpression ( Figure 5B). We performed an adhesion assay using collagen type I pre-coated plates to test the ability of binding of cancer cells to extracellular matrix (ECM) component after cell seeding. Notably, adhesion assay also revealed the markedly reduced adhesive ability of the ELK3-depleted SNU638 cells compared with the controls and ELK3-overexpressing cells ( Figure 5C). To further study the molecular link between ELK3 and BMP1-mediated ECM remodelingrelated genes, we analyzed ELK3 mRNA level after transfection with siBMP1 in ELK3depleted SNU638 with ELK3 overexpression. In Figure 6A, BMP1 depletion was insufficient for affecting the ELK3 mRNA level although BMP1 level was depleted, suggesting ELK3 was an upstream regulator of the BMP1 gene. Indeed, BMP1 depletion significantly suppressed LOXL2, SNAI1, SERPINF1, DCN, and NID1 genes after transfection with siBMP1 in ELK3-depleted SNU638 with ELK3 overexpression ( Figure 6B). In addition, adhesion assay indicated significantly reduced cell adhesive ability in BMP1 depleted SNU638 cells compared with ELK3-depleted SNU638 with ELK3 overexpression ( Figure 6C). Transwell and invasion assays also showed that BMP1 depletion significantly decreased cell migration and invasion ability in ELK3-depleted SNU638 cells with ELK3 overexpression ( Figure 6D). These results clearly indicate that ELK3 is a key regulator of the migration and adhesion of gastric cancer cells and that it functions by regulating the expression of BMP1-mediated ECM remodeling-related genes.    Expression of mRNA encoding the ELK3 and BMP1 genes in SNU638 cells transfected with shCon, shELK3, shELK3 plus pLenti-cMyc-DDK-ELK3 plasmid DNA (ELK3 rescue), or ELK3 rescue plus siBMP1. Error bars represent the standard deviation; * p < 0.05, *** p < 0.001, and **** p < 0.0001 (one-way ANOVA). (B) Expression of mRNA encoding the LOXL2, SNAI1, SERPINF1, DCN, and NID1 genes in SNU638 cells transfected with shCon, shELK3, ELK3 rescue, or ELK3 rescue plus siBMP1. Error bars represent the standard deviation; ** p < 0.01, *** p < 0.001, and **** p < 0.0001 (one-way ANOVA). (C) Representative images showing cell adhesion of SNU638 cells transfected with shCon, shELK3, ELK3 rescue, or ELK3 rescue plus siBMP1. Scale bar, 200 μm. Bar graphs indicate the absorbance value of adherent cells. Error bars represent the standard deviation; ** p < 0.01 and *** p < 0.001 (one-way ANOVA). (D) Representative images showing cell migration and invasion of SNU638 cells transfected shCon, shELK3, ELK3 rescue, or ELK3 rescue plus siBMP1. Scale bar, 200 μm. Bar graphs indicate the number of migrated cells. Error bars represent the standard deviation; * p < 0.05 and ** p < 0.01 (one-way ANOVA). ELK3, ETS transcription factor ELK3; sh, short hairpin RNA; Con, control.

ELK3 Positively Correlates with ECM Remodeling-Related Genes in Gastric Cancer Cells and Patient Samples
To confirm the positive correlation between ELK3 and ECM remodeling-related genes in various gastric cancer cells and patient samples, in silico analyses were performed using databases from DepMap and TCGA. First, the correlation between ELK3 expression and BMP1, LOXL2, SNAI1, SERPINF1, DCN, and NID1 expression was analyzed in 19 different gastric cancer cell lines. The dot plots revealed a significant positive correlation between the expression of the ELK3 and LOXL2 genes; however, this positive correlation was not significant for the other genes ( Figure 7A). Notably, the analysis of patients with gastric cancer indicated the marked positive regulation of ELK3 and BMP1, LOXL2, SNAI1, SERPINF1, DCN, and NID1 gene expression (Figure 7B), suggesting a significant positive correlation between ELK3 and ECM remodeling-associated genes in gastric cancer. To further examine the role of these genes in gastric cancer progression, their expression levels were compared between healthy tissue and gastric cancer tissue. The analysis of data from TCGA and GTEx revealed that ELK3 and BMP1, and LOXL2 were highly expressed in the gastric cancer samples compared with the healthy samples ( Figure 7C). The SNAI1 gene also exhibited a higher expression in the gastric cancer samples, although this increase was not statistically significant. No differences were observed in SERPINF1, DCN, and NID1 expression between the healthy samples and cancer patient samples. Subsequently, the clinical relevance between the ELK3 gene and ECM remodeling-related genes and the prognosis of patients with gastric cancer were examined using Kaplan-Meier survival plots. The overall survival of patients with gastric cancer with a high expression of ELK3 was significantly lower than that of patients with a low expression. A similar pattern was observed in patients with a high expression of ECM remodeling-related genes. Patients with gastric cancer with a high expression of BMP1, LOXL2, SNAI1, SERPINF1, DCN, and NID1 had a poor prognosis ( Figure 7D). The data from in silico analyses indicate that ELK3 positively regulate the expression of ECM remodeling-related genes, resulting in a poor prognosis of patients with gastric cancer. Thus, this gene signature might be a surrogate marker for gastric cancer progression. correlation was not significant for the other genes ( Figure 7A). Notably, the analysis of patients with gastric cancer indicated the marked positive regulation of ELK3 and BMP1, LOXL2, SNAI1, SERPINF1, DCN, and NID1 gene expression ( Figure 7B), suggesting a significant positive correlation between ELK3 and ECM remodeling-associated genes in gastric cancer. To further examine the role of these genes in gastric cancer progression, their expression levels were compared between healthy tissue and gastric cancer tissue. The analysis of data from TCGA and GTEx revealed that ELK3 and BMP1, and LOXL2 were highly expressed in the gastric cancer samples compared with the healthy samples ( Figure  7C). The SNAI1 gene also exhibited a higher expression in the gastric cancer samples, although this increase was not statistically significant. No differences were observed in SER-PINF1, DCN, and NID1 expression between the healthy samples and cancer patient samples. Subsequently, the clinical relevance between the ELK3 gene and ECM remodelingrelated genes and the prognosis of patients with gastric cancer were examined using Kaplan-Meier survival plots. The overall survival of patients with gastric cancer with a high expression of ELK3 was significantly lower than that of patients with a low expression. A similar pattern was observed in patients with a high expression of ECM remodeling-related genes. Patients with gastric cancer with a high expression of BMP1, LOXL2, SNAI1, SERPINF1, DCN, and NID1 had a poor prognosis ( Figure 7D). The data from in silico analyses indicate that ELK3 positively regulate the expression of ECM remodelingrelated genes, resulting in a poor prognosis of patients with gastric cancer. Thus, this gene signature might be a surrogate marker for gastric cancer progression.

Discussion
ELK3 is highly expressed in patients with gastric cancer; however, the association between ELK3 expression and gastric cancer progression remains unknown. Our findings demonstrated that ELK3 promoted gastric cancer cell migration and invasion. ELK3 also regulated the expression of extracellular matrix (ECM) remodeling-related genes, thereby enhancing cancer cell dissemination. The gene analysis of samples from patients with gastric cancer indicated that a high ELK3 expression is positively correlated with BMP1, LOXL2, SNAI1, SERPINF1, DCN, and NID1 expression, all of which are closely associated with a poor prognosis.
ELK3 is a transcription factor that can cooperate with numerous partners to control cancer metastasis. Previous studies have demonstrated that ELK3 regulates breast cancer metastasis through Zeb-1, MT1-MMP, and GATA3 gene-mediated signaling pathways [14,25,26]. Another study reported that a blocking agent suppressed Ras/Erk-ELK3 signaling and also inhibited the progression of prostate cancer [16], suggesting that ELK3 is a promising target molecule for preventing cancer metastasis. However, evidence of ELK3-mediated metastasis in numerous cancer types is lacking. Our study identified a molecular network of ELK3-mediated gastric cancer cell migration and invasion, the underlying mechanism of which is the regulation of ECM remodeling.
The ECM, which comprises numerous proteins, including collagens, glycoproteins, and secreted proteins, interacts with cells and delivers extracellular signals that can alter the cellular phenotype [27][28][29]. ECM remodeling leads to a change in the microenvironment, which contributes to cancer progression and metastasis [30]. The LOX family of oxidases controls ECM structural components and accelerates cancer metastasis by altering the tumor microenvironment [31,32]. LOXL2 also promotes EMT-mediated metastasis by stabilizing the SNAI1 protein [33]. NID1 is a basement membrane glycoprotein that expedites breast lung metastasis [34]. Several groups suggest that genes associated with ECM remodeling are involved in promoting metastasis; however, the detailed molecular mechanisms that regulate gene expression remain unclear. The present study demonstrated that ELK3 regulated the expression of certain ECM remodeling-related genes to induce gastric cancer cell migration and invasion.
In conclusion, our finding suggests that ELK3 contributes to gastric cancer cell dissemination by regulating the expression of ECM remodeling-related genes. The ELK3 expression in gastric cancer cells and human gastric cancer samples positively correlated with that of BMP1, LOXL2, SNAI1, SERPINF1, DCN, and NID1, suggesting that this gene signature may be predictive molecular markers for aggressive gastric cancer progression.

Reverse Transcription-Quantitative PCR (RT-qPCR)
Total RNA was extracted from the cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Reverse transcription was conducted using SuperScript™ II Reverse Transcriptase (Invitrogen; Thermo Fisher Scientific, Inc.) and temperature protocol was as follows; 25 • C for 10 min, 42 • C for 60 min, 72 • C for 10 min. qPCR was performed using TOPreal™ qPCR 2X PreMIX (Enzynomics, Inc., Daejeon, South Korea) and temperature protocol is as follows; 95 • C for 10 min and 15 s, 60 • C for 30 s, 72 • C for 30 s. The mRNA expression levels were analyzed using the 2-∆∆Cq [35] method and normalized against GAPDH. The sequences of the primers are listed in Table 1.

Cell Migration and Invasion Assays
Cell migration and invasion were analyzed using a 24-well Transwell insert (a poly carbonate membrane with a pore size of 8.0 µm; Corning, Inc., Corning, NY, USA). For the invasion assay, the upper surface of the membrane was coated with Matrigel (BD Biosciences, San Jose, CA, USA) and 2 × 10 4 SNU638 cells were seeded into the top chamber, which was then filled with 100 µL serum-free RPMI-1640 medium. Subsequently, 700 µL complete medium were placed into the bottom chamber, and the Transwell assay kit was incubated for 24 h at 37 • C. Migrated or invaded cells on the lower surface of the insert filter were fixed with 4% paraformaldehyde and stained with crystal violet (Sigma-Aldrich;