Serine/threonine-protein kinase 24 is an inhibitor of gastric cancer metastasis

Background: Gastric cancer patients often present with distant metastasis and advanced stages. Suppressing serine/threonine-protein kinase 24 (STK24, also known as MST3) is known to promote gastric tumorigenesis. Here, we investigated the association between STK24 and the metastasis of gastric cancer. Methods: CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 technology was used for genetic knockout of STK24 at the genomic DNA level in human MKN45 and mouse M12 gastric cancer cells. To assess the effects of STK24 knockdown, western blot, cell migration, and wound healing assays were conducted in vitro. An in vivo mouse model of liver metastasis was established and tested, and bioinformatics analyses were performed. Results: The knockdown of the STK24 gene enhanced cell migration and increased liver metastasis in the mouse model of gastric cancer. STK24-silenced tumors suppressed CD4 + T cells and induced the expansion of CD11b + Ly6C + myeloid-derived suppressor cells and F4/80 + macrophages in the spleen of the mice. In MKN45 cells, STK24 silencing resulted in downregulation of E-cadherin (CDH1, Cadherin-1, or epithelial cadherin). In 38 matched specimens of gastric adenocarcinomas and normal tissues, we examined STK24 and CDH1 expression levels via western blot; a signicant positive correlation was found between the expression levels of STK24 and CDH1 (R 2 = 0.5507, P = 9.72 × 10 −8 ). Furthermore, in Oncomine database and Kaplan-Meier plotter analysis, the loss of CDH1, increase in CCL2, and upregulation of CD44 were correlated with poor prognosis in gastric cancer patients. Conclusions: Our results demonstrate that knockdown of STK24 increases cell migration and metastasis. STK24


Background
Serine/threonine-protein kinase 24 (STK24) belongs to the germinal center kinase III (GCKIII) subfamily and is expressed in normal and gastric cancer tissues [1,2]. In a previous study, the STK24 protein was found in normal tissues at signi cantly greater levels than in gastric cancer samples and, according to bioinformatics analyses of Kaplan-Meier Plotter and Oncomine data, the STK24 gene was associated with the poor prognosis of gastric cancer patients; importantly, STK24 knockdown was shown to promote the tumorigenicity of gastric cancer [2]. In a cell-based study, suppression of endogenous STK24 by small interference RNA was found to enhance cellular migration in MCF-7 breast cancer cells, whereas overexpression of STK24 inhibited the migration of these cells [3]. To date, however, the effects of STK24 on gastric cancer metastasis are yet to be studied in detail; therefore, we investigate the association between the two in the present study.
Escape from immune surveillance is a critical element of metastasis. Tumor evasion mechanisms include the expansion of immunosuppressive myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment [4]. MDSCs represent a heterogeneous population of myeloid cells; they consist of two major groups of cells: mononuclear and polymorphonuclear MDSCs (M-MDSCs and PMN-MDSCs, respectively) [5][6][7]. Mouse M-MDSCs and PMN-MDSCs are respectively de ned as CD11b + Ly6C high Ly6G − and CD11b + Ly6C l°w Ly6G + in ammatory monocytes [6]. MDSCs play an important role in immune suppression during tumor growth and in the formation of the premetastatic niche [7]. The accumulation of circulating MDSCs is correlated with the advanced stage of gastric cancer in patients [8]. The accumulation of M-MDSCs is associated with tumor metastasis and poor response to chemotherapy in advanced non-small cell lung cancer [9]. Both M-MDSCs and PMN-MDSCs are associated with the development of metastases and poor survival in melanoma cases [10]. Moreover, in an orthotopic immunocompetent gastric cancer model, STK24 silencing in tumors induces an expansion of CD11b + Ly6C + cells and F4/80 + macrophages cells [2].
In the present study, we hypothesized that STK24 plays an important role in tumor metastasis. To test this hypothesis, we examined the changes in the metastatic abilities of gastric cancer cell lines after knockdown of STK24. In addition, a mouse model of liver metastasis was used to explore the effect of STK24 in gastric cancer and tumor-in ltrating MDSCs.
Cell culture MKN45 cells were kindly provided by Professor Ming-Derg Lai (National Cheng Kung University, Tainan, Taiwan). The MKN45 cell lines were authenticated in 2013 by DNA short tandem repeat pro ling at Bioresource Collection and Research Center. These cell lines were maintained in RPMI 1640 medium containing 10% fetal bovine serum (FBS) (Gibco, Life Technologies, Grand Island, NY) and 1% penicillin/streptomycin. M12 cells were maintained in Dulbecco's modi ed Eagle's medium/high glucose supplemented with 10% FBS and 1% penicillin/streptomycin.

Mice
For use in all animal experiments, 8-week-old C578BL/6 mice were purchased from the Laboratory Animal Center of National Cheng Kung University (Tainan, Taiwan) and maintained under pathogen-free conditions.

Metastatic model of gastric cancer
The metastatic abilities of M12 cells were evaluated in vivo using a hepatic metastasis model in immunocompetent C57BL/6 mice [11]. To establish this model, mice were rst anesthetized by an intraperitoneal injection of Zoletil (50 mg/kg; Parnell Laboratories, Alexandria, Australia) and xylazine (10 mg/kg; Troy Laboratories, Glendenning, Australia). A small midline incision was then made in the abdomen. The spleen was exteriorized and 5 × 10 5 tumor cells in 0.05 mL of PBS were injected into the spleen using a 1-cm 3 U-100 disposable insulin syringe (Becton-Dickinson, Franklin Lakes, NJ). Fourteen days after this injection, the mice were sacri ced. Hepatic and splenic masses were examined macroscopically and histologically. Formalin-xed/para n-embedded sections of the stomach, liver, and spleen were subjected to hematoxylin and eosin staining. Each animal experiment was performed at least twice.

Western blot analysis
Total cell lysates were prepared and analyzed by SDS-PAGE as previously described [11]. For quanti cation, the bands were measured using the AlphaImager 2200 system (Alpha Innotech, San Leandro, CA) and normalized using the density of β-actin. The expression of STK24 was quanti ed and given as the STK24-to-β-actin ratio. These experiments were repeated three times using independent batches of cell clones or cell lysates. Quantitative data are presented as values relative to those in control cells.
Cell migration and wound healing assays Cell migration was evaluated in modi ed Boyden chambers (NeuroProbe, Inc., Gaithersburg, MD) for 8 h, as previously described [11]. MKN45 cells (70 μL of 1 × 10 6 cells per mL) or M12 cells (70 μL of 2 × 10 5 cells per mL) were seeded in an ibidi culture insert (Applied BioPhysics, Inc., Martinsried, Germany) on top of a 24-well plate. After overnight incubation, the insert was carefully removed to form a cell-free gap in the attached cells. The time of incubation was dependent on the tumor cells used. The number of migrating cells was calculated and analyzed. Six elds were randomly selected for analysis.

Flow cytometry analysis
To characterize the immune cells from the spleens of tumor-bearing mice, individual spleens were isolated and subjected to ow cytometry as previously described [12].

Patients
For patient studies, fresh specimens were collected from 38 patients with gastric adenocarcinoma who underwent radical resection at the National Cheng Kung University Hospital between August 2003 and August 2008. In total, 38 pairs of cancerous tissues and matched adjacent normal gastric mucosa were collected and analyzed as previously described [13].

Bioinformatics
A search was conducted in the Oncomine database (http://www.oncomine.com) [14] to systematically assess the expression level of CDH1 genes in gastric cancer. For differential analyses, we compared normal tissues and cancer tissues, speci cally via analysis of P values, fold changes, and cancer subtypes. The prognostic value of CDH1 genes in gastric cancer was also analyzed using the Kaplan-Meier Plotter (http://kmplot.com/analysis/) as previously described [15]. The overall survival (OS), progression-free survival (PFS), and postprogression survival (PPS) were recorded, and the cut-off points for gene expression were automatically selected using the default setting. The probe of CDH1 gene was "201131_s_at." The hazard ratio (HR), 95% con dence intervals, and log rank P values were displayed. Data from the Oncomine database and Kaplan-Meier Plotter were extracted between July and August 2020. Finally, the association between CDH1 protein expression (CDH1-to-b-actin ratio) and the Lauren classi cation (intestinal, diffuse, and mixed) of patients with gastric adenocarcinoma was assessed in the fresh specimens. The statistical differences between each two groups were analyzed.
Epithelial mesenchymal transition (EMT)-related genes were de ned according to a meta-analysis of 14 gene expression studies [16]. The gene lists were applied to the raw data of gastric cancer in The Cancer Genome Atlas (TCGA). Hierarchical clustering was performed in R to produce a heatmap. Gene expression data were also obtained from GSE112369, a dataset of gastric organoids for which the raw data was publicly available [17]. Expression levels of STK24 and CDH1 were extracted. Gastric organoids forming from CDH1-single-knockout and parental cells were selected for further comparison.

Statistical analysis
Data were expressed as means ± standard deviations (SDs). Statistical analyses were performed in Prism (Graphpad Software, San Diego, CA). Student's t-test was used for two-group comparisons, whereas oneway ANOVA followed by Tukey's test was used for multiple-group comparisons. P values <0.05 were considered statistically signi cant.

Results
Suppression of STK24 expression in the gastric cancer cells To examine the effect of STK24 in cancer metastasis, we knocked down STK24 gene expression using two different sgRNAs in gastric cancer cell lines (human MKN45 and mouse M12). We established four clones of STK24-sgRNA constructs (sgSTK24-1.1, sgSTK24-1.2, sgSTK24-2.1, and sgSTK24-2.2) and one clone of a pEGFP (enhanced green uorescent protein) control in each cell line. The successful suppression of the STK24 protein in MKN45 (Fig. 1a) and M12 (Fig. 1b) cells was validated by western blotting. The cell proliferation rates of the pEGFP control (EGFP-Ctrl) and sgSTK24-expressing cells were similar in MKN45 cells (Fig. 1c). In a previous study, the knockout of STK24 expression did not affect the cell growth rates of mouse M12 cancer cells [2]. Therefore, the suppression of STK24 did not affect the cell growth rates of gastric cancer. respectively. MKN45-and M12-sgSTK24 cells each exhibited stronger potential for cell migration. In addition, M12-sgSTK24 cells exhibited a relatively higher potential for migration in a Transwell migration assay performed for 8 h using 10% FBS as a chemoattractant (see Fig. S1 in Additional le 1). This association between cell migration and STK24 expression in gastric cancer cell lines suggests STK24 plays important role in mediating metastasis.

Effect of STK24 suppression on liver metastasis in a mouse model of gastric cancer
To test the hypothesis that STK24 plays an important role in tumor metastasis, we examined the changes of metastatic ability in the in vivo orthotopic intrasplenic implantation model of gastric cancer established in C57BL/6 mice [11]. Injection of M12 parental cells resulted in macroscopic nodules in the liver (Figure 3a). The weights of the livers (Fig. 3a and c) and spleens ( Fig. 3b and d) of mice injected with sgSTK24-1.1 and sgSTK24-2.1 cells were signi cantly higher than the weights of equivalent organs in mice injected with EGFP-Ctrl cells. Moreover, the nodules were con rmed as liver metastasis by histopathologic analyses of liver sections (Fig. 3e). In the M12 mouse model, we demonstrated that the metastatic burden was increased in STK24-knockdown cells. Thus, in vitro and in vivo results showed that STK24 plays a signi cant role in the metastasis of mouse gastric cancer.
Immune regulation in tumor-bearing mice M12 mouse gastric cancer cells were transfected with EGFP-Ctrl or two types of STK24-sgRNA. Liver metastases developed after intrasplenic injection of cancer cells in immunocompetent mice; subtypes of splenocytes were then investigated to assess STK24-mediated immunity in liver metastasis of gastric cancer. The proportion of CD4 + cells was signi cantly higher in the spleens of EGFP-Ctrl-tumor-bearing mice than in those of both types of sgSTK24-tumor-bearing mice ( Fig. 4a and b). The proportion of the CD8 + T cells in splenocytes signi cantly decreased in sgSTK24-1.1-tumor-bearing mice but not in sgSTK24-2.1-bearing mice (Fig. 4a and c). The proportion of F4/80 + macrophages signi cantly increased in the spleens of sgSTK24-tumor-bearing mice (Fig. 4a and d). Considering the two major MDSC subtypes, i.e., the CD11b + Ly6C + or CD11b + Ly6G + phenotypes, the CD11b + Ly6C + subtype signi cantly increased in the spleens of sgSTK24-tumor-bearing mice ( Fig. 4e and f). In addition, the subpopulations of in ltrating monocytes were assessed: accumulations of CD11b + Ly6C high (CD11b + Ly6C hi ) cells (in ammatory monocytes) and CD11b + Ly6C l°w (CD11b + Ly6C l°) cells (reparative monocytes) were con rmed by gating on CD11b + Ly6C + cells (Fig. 4e). In ammatory CD11b + Ly6C hi and reparative CD11b + Ly6C l° cells were markedly increased in the spleens of sgSTK24-tumor-bearing mice ( Fig. 4e and   f). These results indicate that STK24 silencing in tumors induces the expansion of F4/80 + macrophages, CD11b + Ly6C hi , and CD11b + Ly6C l° monocytes in vivo; thus, an increase in these types of monocytes/macrophages may play an important role in gastric metastasis.
The recruitment of immune cells relies on cancer-secreted cytokines. Because CCL2 is associated with metastatic behavior in cancer cells [18], we explored the transcript expression of CCL2 genes in gastric cancer patients using the Oncomine database. We focused on datasets in which cancer patients and normal patients were compared [19][20][21][22]. The histological type of gastric adenocarcinoma was divided into gastric intestinal adenocarcinoma (GITA), diffuse gastric adenocarcinoma (DGA), and gastric mixed adenocarcinoma (GMA), all of which showed upregulation of CCL2 (see Fig. S2a-c in Additional le 1). Compared to the other subtypes of gastric cancer, the expression of CCL2 was signi cantly increased in DGA (see Fig. S2d-f in Additional le 1). Therefore, analysis of the Oncomine cancer microarray database revealed that CCL2 gene expression was signi cantly increased in gastric cancer, especially in DGA.

Regulation of the EMT process by STK24
We hypothesized that STK24 silencing induced migration and metastasis of gastric cancer through the EMT process; hence, we examined the EMT proteins of MKN45 parental cells and knockdown clones in vitro. The knockout of STK24 expression did not affect the AKT1 protein of MKN45 cells (Fig. 5a). Ecadherin and b-catenin are key proteins in EMT; expression of each protein was suppressed by STK24 silencing in MKN45 cells (Fig. 5b and c). To further investigate the relationship between STK24 and CDH1 in gastric cancer, we compared the expression of STK24 and CDH1 in 38 matched specimens of gastric adenocarcinoma (which included 11 DGA, 22 GITA, and 5 GMA) and normal tissues by western blot analysis (see Table S1 in Additional le 1). Fig. 5d shows the relative expression of STK24 and CDH1 in these tissues. The relative ratio of CDH1-to-b-actin was higher in normal gastric tissues than in gastric cancer tissues (Fig. 5e). Furthermore, a signi cant positive correlation was identi ed between the expression levels of STK24 and CDH1 (R 2 = 0.5507; Fig. 5f). This positive correlation was validated using TCGA database. The mRNA expression levels of STK24 and CDH1 showed a signi cant positive correlation in clinical data of stomach adenocarcinoma (Fig. S3a in Additional le 1).
Gene expression in gastric cancer from TCGA was hierarchically clustered as EMT-related and EMTunrelated (see Fig. S3b in Additional le 1). The upper half was correlated with mesenchymal phenotypes and the lower half with epithelial phenotypes, while CDH1 and STK24 were strongly related. These correlations from our patients' cancer specimens and TCGA con rmed the positive association between CDH1 and STK24; however, such correlations do not prove causal relationships. Currently, highthroughput datasets including STK24-knockout cells are unavailable; however, we identi ed a dataset of gastric organoids for which the raw data was publicly available (i.e., GSE112369) [17]. The expression level of STK24 was similar in CDH1-single-knockout and parental cells (see Fig. S3c in Additional le 1). Thus, knockdown of CDH1 was directly shown to have no effect on the expression of STK24; furthermore, STK24 was indirectly shown to be upstream of CDH1.

Association of CDH1 expression with the survival of gastric cancer patients
Because E-cadherin is associated with metastatic behavior in cancer cells, we explored the expression of CDH1 genes in gastric cancer patients using the Oncomine database to compare data from cancer and normal patients [19,20,22]. The statistical signi cance and fold change of CDH1 expression were comparatively analyzed in normal and cancer tissues. Downregulation of CDH1 genes occurred in DGA than normal gastric mucosa ( Fig. 6a and b). CDH1 expression signi cantly decreased in DGA than other subtypes of gastric cancer (Fig. 6c-f). Thus, analysis of the Oncomine cancer microarray database revealed that CDH1 gene expression was signi cantly downregulated in DGA.
According to the Kaplan-Meier Plotter (Fig. 7), a signi cant relationship existed between CDH1 mRNA expression and patient survival: low expression of CDH1 was correlated with worse OS, PFS, and PPS ( Fig. 7a-c, respectively). Analysis of the Kaplan-Meier survival curves revealed that CDH1 gene expression signi cantly reduced OS, PFS, and PPS in DGA and GITA patients speci cally (Fig. 7a-c,  respectively). In summary, the downregulation of CDH1 in DGA and GITA was associated with poor patient prognosis.

Prediction of protein-protein interactions in gastric cancer
The Search Tool for the Retrieval of Interacting Genes (STRING) database was used to identify relevant protein-protein interactions (Fig. 8a). A network including STK24, CDH1, AKT1, and CTNNB1 was constructed and linked to CCND1, IL6, STAT3, and CD44 via this database. As STK24 knockdown was associated with enhanced cell migration (Fig. 2), we considered the upregulation of other migrationassociated molecules. Speci cally, we analyzed the expression of stem cell marker CD44 in MKN45 cells. Flow cytometry revealed that increased membranous expression of CD44 occurred in MKN45-STK24-sgRNA cells (see Fig. S4 in Additional le 1). Western blotting showed that MKN45-sgSTK24-expressing cells exhibited relatively high CD44 expression (see Fig. S5 in Additional le 1). We also extracted expression data of CD44 transcripts from the Oncomine database for gastric cancer, focusing on comparisons between cancer and patient datasets [19,20,23]. Our analysis included comparisons of statistical signi cance and fold change of CD44 expression between normal and cancer tissues. Upregulation of CD44 was determined in DGA, GITA, and GMA than normal gastric mucosa (see Fig.  S6a-i in Additional le 1), with CD44 expression being signi cantly increasing in DGA relative to the other subtypes (see Fig. S6j).
A scheme of STK24 signaling in gastric cancer is shown in Fig. 8B. STK24 suppression effectively enhances the migration and metastatic potential of human MKN45 and mouse M12 gastric cancer cells in vitro and in vivo. Our data suggest that the STK24 mediates stemness and immunosuppression in gastric cancer through CD44 and via interactions with macrophages and CD11b + Ly6C + MDSCs.

Discussion
In our previous studies, we demonstrate that suppression of STK24 in M12 gastric cancer cells promotes tumorigenesis in an animal model [2] and is a predictor of poor prognosis in gastric cancer patients [13]. In the present study, we demonstrate the effects of STK24 on migration and metastasis in human and mouse gastric cancer cells. STK24 is constitutively expressed in MKN45, AGS, and NCI-N87 cells, and the relative expression of the STK24 protein in normal tissues is signi cantly greater than that in gastric cancer samples [2]. We also nd that the suppression of STK24 does not affect the proliferation of MKN45 cancer cells in vitro; however, STK24 suppression effectively enhances the migration and metastatic potential of human MKN45 and mouse M12 gastric cancer cell lines in vitro and in vivo. Our data suggest that STK24 suppression is associated with the high metastatic potential of gastric cancer through stemness and immunosuppression.
The human MKN45 gastric cancer cell line is derived from a metastatic lesion in the liver; these cells exhibit a poorly differentiated primary gastric cancer of diffuse histology [24]. Gastric adenocarcinomas can be approximately subgrouped into 50% GITA, 30% DGA, and 15%-20% GMA [25]. DGA is an aggressive, in ltrating carcinoma with poor prognosis [26][27][28][29]. DGA is associated with decreased responsiveness to chemotherapy and chemoradiation [30][31][32]. The possible mechanism of progression in gastric adenocarcinomas has been studied. For example, tumor suppressor p53 (TP53) is known to be altered in ~50% of gastric cancer [33], and DGA is characterized by the loss of E-cadherin due to mutation or hypermethylation [34]. The expression levels of the STK24/MST3 protein are examined in the tumor and adjacent normal gastric tissues of DGA, GITA, and GMA [2]. Low STK24 gene expression is correlated with poor OS and rst progression in both GITA and DGA. We successfully establish an orthotopic gastric cancer model and mutated p53 in C57BL/6 mice. Further knockdown of STK24 in this model enhances tumorigenesis [2]. Another study shows that the loss of CDH1 and TP53 promotes gastric tumorigenesis and metastases [35]. Our present data indicate that high STK24 and CDH1 expression in gastric cancer are protective factors; thus, they are apparently advantageous to survival. We also nd that upregulation of CD44 and proliferation of F4/80 + macrophages or CD11b + Ly6C + MDSCs occur after STK24 knockdown. Therefore, targeting CD44 or immunotherapy against macrophages/MDSCs could be used as potential treatments for selected gastric cancer patients with low STK24.
A heterogeneous population of MDSCs promotes tumor progression, metastasis, and resistance to immunotherapy [36]. As previously shown, high levels of MDSCs in gastric cancer patients are associated with advanced cancer stages as well as lower OS [8]. In addition, increased M-MDSCs are correlated with poor differentiation, lymph node metastasis, and lower OS in these patients [37]. Wang et al. [30] detect higher levels of CD4 + memory T cells and lower levels of CD8 + T cells, monocytes, NK cells, myeloid dendritic cells, and normal peritoneal broblasts in tumor specimens of peritoneal carcinomatosis from patients with DGA. Therefore, we suggest that different subsets of MDSCs are associated with the metastases of gastric cancer types. MDSCs are increased in the spleens of human cancer patients, with M-MDSCs known to be most prominent in the spleen and peripheral blood [38]. M-MDSCs are characterized by CD14 + CD33 + HLA -DR -/l° expression (CD11b + GR1 + Ly6C + Ly6Gcells in mice) [39]. They are recruited to primary and metastatic tumor sites through chemokine secretion by tumors, primarily CCL2 and CCL5 [40,41]. In addition, splenic M-MDSCs suppress T-cell function in an ROS-dependent manner [39,42]. The spleen reportedly acts as the local reservoir of Ly6C hi monocytes, which migrate toward the tumor and differentiate into tumor-associated macrophages [43]. Thus, the targeting of MDSCs represents a promising immunotherapy for cancer patients [36]. In the present study, spleen in ammatory CD11b + Ly6C hi and reparative CD11b + Ly6C l° cells are markedly increased in mice with sgSTK24 tumors; thus, reduced STK24 expression in gastric cancer seems to cause an accumulation of M-MDSCs in the spleen. Moreover, in ammatory CD11b + Ly6C hi and reparative CD11b + Ly6C l° MDSCs may be biomarkers for liver metastasis of gastric cancer and targets for future treatment.
In our previous study, STK24 expression is signi cantly decreased in DGA and GITA according to bioinformatics analyses [2]; in particular, downregulation of the STK24 gene is associated with the poor prognosis of gastric cancer patients. In the present study, STK24 is revealed as participant in cancer metastasis and immune regulation. In our constructed protein-protein interaction network, CDH1, CTNNB1, CD44, and CCND1 are all linked with STK24. Previously, decreased expression of CDH1 protein (known as E-cadherin) has been correlated with the in ltrating and metastatic abilities of gastric cancer [44]. Additionally, loss of CTNNB1 protein (known as b-catenin) has been detected in DGA and metastatic lesions of gastric cancer [45]. Patients with E-cadherin-expressing gastric cancer are known to have signi cantly better survival rates than those with E-cadherin-negative tumors [46]. E-cadherin is the primary mediator of cell-cell adhesion and loss of this molecule is associated with the metastatic potential of tumor cells [47,48]. Furthermore, downregulation of E-cadherin is associated with invasion and metastasis of DGA [29,49,50]. Mutations of the CDH1 gene have been reported in DGA [51,52]. Here, we nd a signi cant decrease in CDH1 expression in DGA relative to CDH1 expression in normal gastric mucosa, GITA, and GMA (according to the Oncomine database). We also show that low expression of STK24 is correlated with downregulated CDH1 protein expression in the tumors of gastric cancer patients. CD44 is a known stem cell marker in gastric cancer [53]; its expression has been positively correlated with distant metastasis [54]. Oncomine database analysis of cancer vs. normal tissues showed that CD44 mRNA was signi cantly upregulated in DGA, GITA, and GMA. Moreover, increased expression of CD44 was detected in sgSTK24 gastric cancer cells. Therefore, our results suggest that STK24 suppression, which is apparently upstream of CDH1, is associated with the loss of b-catenin and activation of CD44 cancer stemness in metastasis of gastric cancer.

Conclusion
In conclusion, STK24 silencing induces overexpression of CD44 and stemness in gastric cancer, while suppression of CDH1 (E-cadherin) promotes gastric cancer metastasis. Furthermore, reduced expression of STK24 induces CCL2 secretion and the recruitment of M-MDSCs to promote metastasis. Overall, decreased STK24 expression apparently promotes gastric metastasis through stemness and immunosuppression. The ndings of this study further reveal the mechanisms of gastric cancer metastasis and provide a potential therapeutic target for the development of gastric cancer treatments.

Declarations
Ethics approval and consent to participate The animal study was approved by the institutional animal care and use committee of National Cheng Kung University (approval number: NCKU-IACUC-106-288). All procedures were conducted in accordance with approved guidelines. All patients involved in this study provided written informed consent, and the patient study was approved by the Institutional Review Board of National Cheng Kung University Hospital (IRB number: ER-97-148).

Consent for publication
Not applicable.

Availability of data and materials
All data generated or analyzed during this study are included in this article.

Competing interests
The authors declare that they have no competing interests.

Funding
The present study was supported by grants from the Ministry of Science and Technology, Taiwan (MOST 107-2314-B-006-037, 108-2314-B-006-082, and 109-2314-B-006-018-MY3), and National Cheng Kung University Hospital (grant NCKUH-11002013). This research was supported in part by Higher Education Sprout Project, Ministry of Education to the Headquarters of University Advancement at National Cheng Kung University.
Author contributions YLC performed most of the experiments and wrote the rst draft. HPH contributed to the experimental design and edited the manuscript. CYW contributed to the experimental design and bioinformatics analysis. JHF assisted in the animal experiments. All authors read and approved the nal manuscript.