RPS15A promotes gastric cancer progression via activation of the Akt/IKK‐β/NF‐κB signalling pathway

Abstract This study aimed to investigate the clinical significance, potential biological function and underlying mechanism of RPS15A in gastric cancer (GC) progression. RPS15A expression was detected in 40 pairs of GC tissues and matched normal gastric mucosae (MNGM) using qRT‐PCR analysis. Immunohistochemistry assay was conducted using a tissue microarray including 186 primary GC samples to characterize the clinical significance of RPS15A. A series of in vitro and in vivo assays were performed to elucidate the biological function of RPS15A in GC development and underlying molecular mechanisms. The expression of RPS15A was significantly up‐regulated in GC samples compared to MNGM, and its expression was closely related to TNM stage, tumour size, differentiation, lymph node metastasis and poor patient survival. Ectopic expression of RPS15A markedly enhanced the proliferation and metastasis of GC cells both in vitro and in vivo. RPS15A overexpression also promoted the epithelial‐mesenchymal transition (EMT) phenotype formation of GC cells. Investigations of underlying mechanisms found that RPS15A activated the NF‐κB signalling pathway by inducing the nuclear translocation and phosphorylation of the p65 NF‐κB subunit, transactivation of NF‐κB reporter and up‐regulating target genes of this pathway. In addition, RPS15A overexpression activated, while RPS15A knockdown inhibited the Akt/IKK‐β signalling axis in GC cells. And both Akt inhibitor LY294002 and IKK inhibitor Bay117082 neutralized the p65 and p‐p65 nuclear translocation induced by RPS15A overexpression. Collectively, our findings suggest that RPS15A activates the NF‐κB pathway through Akt/IKK‐β signalling axis, and consequently promotes EMT and GC metastasis. This newly identified RPS15A/Akt/IKK‐β/NF‐κB signalling pathway may be a potential therapeutic target to prevent GC progression.


| INTRODUCTION
Gastric cancer (GC) is one of the most aggressive malignancies with a high incidence and metastasis rate, accounting for an estimated annual 720 000 deaths worldwide. 1 Despite important advances in diagnosis and therapeutic strategies, the 5-year survival rate of GC, especially patients with metastatic GC, is still low. 2 Therefore, identification and better understanding of novel biomolecules and signalling pathways involved in GC progression remain of great importance.
Like nearly all cancer types, metastasis represents the main cause of death in GC patients. The initial stage of metastatic progression is essentially dependent on the prominent biological event referred to as epithelial-mesenchymal transition (EMT). 3 During the EMT process, epithelial cells lose their junctions and apical-basal polarity, reorganize their cytoskeletons, undergo a change in the signalling programs that define cell shape and reprogram gene expression. 4 Biologically, EMT is a complex process that is typically driven by aberrant activation of transcription factors such as Slug, Snail, ZEB1, ZEB2 and Twist, as well as various signalling pathways, including NF-κB, Wnt, Notch, Hedgehog, TGF-β and others. 5 The NF-κB signalling pathway has been widely demonstrated as one of the most commonly activated and essential pathways for EMT and GC metastasis. [5][6][7] The core of this pathway, transcription factor NF-κB, consisting of two distinct subunits, p50 and p65, is subject to multiple levels of control. In non-stimulated cells, NF-κB is normally sequestered in the cytoplasm through interaction with the general inhibitors of NF-κB (IκBs), the best characterized of which is IκB-α. Upon activation of the pathway, IκB-α is phosphorylated by IκB kinases (IKK). 8 Such phosphorylation triggers IκB-α degradation by ubiquitin-mediated proteolysis, and promotes the release and relocalization of NF-κB (p65/p50) to the nucleus, where it exerts its role in gene expression. 9 Despite these multiple levels of tight regulation, however, molecular mechanisms causing constitutive activation of NF-κB signalling are still largely unknown.
Ribosomal protein S15A (RPS15A), a member of the RPS family, maps to human chromosome 16p12.3 locus and encodes a highly conserved 40S ribosomal protein. RPS15A promotes the binding of capped mRNA to the small ribosomal subunit at the early stages of translation. 10 RPS15A has also been identified as Ca2+/CaM binding partner modulating ribosome assembly and translation. 11 Meanwhile, increasing evidence indicates that RPS15A, like other RPS family proteins, exhibits various extra-ribosomal functions, such as cell division, tumourigenesis and progression. 12 In response to induction of transforming growth factor-β, RPS15A enhances cell growth of lung cancer. 13,14 Down-regulation of RPS15A induces apoptosis and inhibits proliferation of human glioblastoma cells in vivo and in vitro via AKT pathway. 15,16 In addition, RPS15A promotes malignant transformation of colorectal cancer through misregulation of p53 signalling pathway. 17 However, the clinical significance, potential biological function of RPS15A in GC progression and underlying mechanisms remain unclear.
In this study, we investigated the role of RPS15A in GC development. Our data revealed that RPS15A is significantly up-regulated in GC tissues and associated with poor prognosis. By gain-and loss-of-function studies, we demonstrated that RPS15A promotes the proliferation, migration and invasion of GC cells both in vitro and in vivo.
Mechanically, RPS15A activates the Akt/IKK-β/NF-κB signalling pathway to enhance EMT and GC progression.  were obtained from the Shanghai Cell Bank, Chinese Academy of Sciences (Shanghai, China) and maintained in the recommended growth medium. All medium contained 10% foetal bovine serum (FBS) and 1% penicillin-streptomycin. All cell lines were cultured in a humidified atmosphere of 5% CO 2 at 37°C.

Western blotting, immunofluorescence assays
Quantitative real-time PCR (qRT-PCR), Western blotting and immunofluorescence assays were performed as previously described. 4 The primers were described in Table S1.

| Cell proliferation assay
Indicated cells were seeded into 96-well plates at a density of 2 × 10 3 /well for culture, and cell proliferation was measured using CCK-8 reagents (Dojindo, Kumamoto, Japan). The staining intensity in the medium was documented every 24 hours for 5 days. Each experiment was repeated three times and five wells were used for each time-point per group.

| Colony formation assay
Cells were seeded into six-well plates at a density of 1×10 3 cells/well and cultured in complete medium at 37°C in 5% CO 2 for 14 days.
Growth medium was refreshed every three days. At the end of the experiment, the cells were fixed with 4% paraformaldehyde for 15 minutes, stained with Giemsa solution for 10 minutes and then photographed and calculated. A group of 50 cells or more was counted as a colony.

| Wound-healing assay
Cells were seeded in six-well plates with complete medium and cultured until they reached confluence. Then a linear wound about 300-500 μm wide was generated with a standard 200 μL pipette tip.
Wounded monolayers were washed twice with 1xPBS to remove non-adherent cells. Wound closure was examined and photographed at pre-determined time-points (0, and 48 hours) in five random microscopic regions.

| In vivo tumour growth assay
The transduced cells (2×10 6 ) were injected subcutaneously into the groin of 4-to 6-week-old male nude mice (n = 5 for each group) (Institute of Zoology, Chinese Academy of Sciences, Shanghai, China). Tumours were measured every 4 days, and tumour volumes were calculated using the following formula: Volume (mm 3 ) = 4π/ 3 × (width/2) 2 × (length/2). One month after inoculation, all mice were killed. Tumour xenografts were collected, photographed and weighed.

| Lung metastasis model
A lung metastasis model was generated to validate the effect of RPS15A on GC metastatic ability in vivo by injecting the transduced cells (5 × 10 6 ) into the tail veins of nude mice (n = 5 for each group).
All mice were killed 6 weeks after operation, and the lungs were then removed for pathologic examination, H&E staining and count of the lung metastatic nodules. The animal study was performed according to the Animal Care Guidelines of FUSCC, Shanghai, China.

| RNA-seq and computational analysis
RNA-seq was performed using Hiseq3000 (Illumina, USA). LifeScope v2.5.1 was used to align the reads to the genome, generate raw counts corresponding to each known gene and calculate the RPKM (reads per kilobase per million) values. KEGG enrichment was used for the pathway analysis.

| Luciferase reporter assay
The response element of NF-κB was subcloned into the pGM-CMV-Luc vector (Yeasen, Shanghai, China). The final constructs were confirmed by DNA sequencing. Firefly luciferase activity was normalized to that of Renilla luciferase. Luciferase activity was detected using the dual-luciferase reporter assay system (Promega) according to the manufacturer's instructions.

| Statistical analyses
Data were analysed using one-way analysis of variance or Student's t test for comparison between groups. The protein expression levels and clinicopathological parameters were compared by χ 2 test. Survival curves were plotted with the Kaplan-Meier method and compared using the log-rank test. P < 0.05 with a two-sided test was considered to be statistically significant. Statistical analyses were performed with GraphPad Prism software Version 5.0 (San Diego, CA, USA).

| RPS15A is up-regulated in GC patients and associated with poor prognosis
We first examined RPS15A mRNA levels in 40 pairs of GC and matched normal gastric mucosae (MNGM). The results showed that RPS15A mRNA was significantly increased in GC tissues compared with MNGM ( Figure 1A). Similarly, up-regulation of RPS15A in GC LIU ET AL.
| 2209 tissues was also confirmed at the translational level using Western blotting analysis ( Figure 1B). In addition, we evaluated the RPS15A expression levels in normal gastric epithelial cell GES-1 and a panel of GC cell lines. According to Western blotting results, elevated expression of RPS15A was observed in all seven GC cell lines compared to GES-1 ( Figure 1C). Taken together, these findings indicated that RPS15A is significantly up-regulated in GC patients.
In order to determine the clinical significance of RPS15A overexpression in GC, immunohistochemistry analysis was performed using tissue microarrays including 186 primary GC samples. The immunostaining score of RPS15A was evaluated on the basis of staining intensity and extent, and all patients were categorized into high or low RPS15A expression group to simplify data analysis ( Figure 1D).
We then analysed the relationship between RPS15A expression and clinicopathological characteristics in GC patients, and found that RPS15A expression was closely correlated with aggressive phenotypes of GC, including TNM stage, tumour size, differentiation and lymph node metastasis (Table 1) (Table 2). Collectively, RPS15A expression is significantly up-regulated in GC patients and contributes to poor prognosis.

| RPS15A enhances the malignant phenotypes of GC cells in vitro
To assess the role of RPS15A in GC development, we performed loss-and gain-of-function studies in GC cells. We constructed a len-  To further investigate the in vivo effect of RPS15A on metastasis, we established a lung metastasis model by injecting stable MKN-28-knockdown and control cells into the tail veins of nude mice (n = 5 for each group). All mice were sacrificed 6 weeks after injection, and the lungs were then dissected out for histological analysis.
As shown in Figure 3D,E, lung metastasis was found in 20.0% (1/5) of mice in the RPS15A-silencing group compared with 100% (5/5) in the control group. In addition, a marked less number of lung metastatic nodules was observed in the RPS15A-silencing group compared with the control group. These results indicate functional significance of RPS15A in GC metastasis.

| RPS15A induces EMT in GC cells
Given that epithelial-mesenchymal transition (EMT) is considered a striking feature of most cancers and plays a crucial role in cancer metastasis and invasion, 5 we then examined whether EMT might be an underlying mechanism for RPS15A-induced GC metastasis. As shown in Figure 4A Figure 4A,B). In addition, the involvement of EMT was further supported by immunofluorescence assay (Figure 4C). Taken together, these findings demonstrated that RPS15A induces EMT in GC cells.

| RPS15A promotes EMT through NF-κB signalling
To further explore the molecular mechanism by which RPS15A promotes GC development, RNA-seq analysis was introduced to obtain the transcriptional profiles of RPS15A-depleted MKN-28 and control cells. Subsequent KEGG enrichment analysis identified the NF-κB as the top-ranked signalling pathway ( Figure 5A). We then performed luciferase reporter assay to validate above findings, and found that the NF-κB transcriptional activity was significantly activated by RPS15A overexpression, whereas markedly inhibited by RPS15A depletion ( Figure 5B). Activation of NF-κB pathway is known to   (Figure 5H,I). Collectively, these data suggested that RPS15A promotes EMT through NF-κB signalling.

IKK-β pathway
NF-κB activation is known to be mediated through the Akt/IKK-β pathway. 20,21 Previous study has demonstrated that RPS15A is involved in the activation of the Akt pathway in glioblastoma. 15 Thus, we hypothesized that RPS15A might activate NF-κB through the Akt/IKK-β pathway in GC cells. To test this, we first examined the effect of modified RPS15A on p-Akt and total Akt protein. The results revealed that a significant decrease of p-Akt, but not total Akt, was observed in RPS15A-depleted MKN-28 compared to control cells ( Figure 6A). In contrast, RPS15A overexpression significantly increased p-Akt expression levels ( Figure 6A). A key kinase for NF-κB activation is IKK-β, which releases subunit p65 from IkB-α and facilitates its nuclear translocation. 19 We then investigated whether RPS15A affects the status of IKK-β and IkB-α in GC cells.
To further validate the involvement of Akt and IKK-β in RPS15Ainduced NF-κB activation, we adopted a pharmacologic approach.

| DISCUSSION
The promotive role of RPS15A in tumour progression has been increasingly recognized recent years, whereas the underlying molecular mechanism remains largely unclear. In this study, we highlighted the clinical significance and oncogenic role of RPS15A in GC evolution. Furthermore, we provided evidence that RPS15A activates the NF-κB pathway through Akt/IKK-β signalling axis, and consequently promotes EMT and GC metastasis (Figure 7).
Distant metastasis and invasion of cancer are responsible for more than 90% of cancer-related deaths. 22 Metastasis is a complex process that involves multiple sequential steps, including invasion of cancer cells into surrounding tissue, intravasation, survival in the The initial stage of metastatic progression is essentially dependent on EMT. 3 RPS15A expressed low levels of E-cadherin, and high levels of vimentin and slug, suggesting that RPS15A may be a potent inducer of EMT, which may result in more invasive and metastatic GC cells.
Tumour invasion and metastasis are complex, multistep processes underlain by genetic and/or epigenetic changes within probably a fraction of malignant cells in the tumour. 29 The systematic control of the NF-κB pathway relies on the unique property of its major inhibitor, IκB-α. 33 Upon activation of the pathway, the IKK kinase complex phosphorylates IκB-α, inducing its degradation and promoting the release and relocalization of NF-κB to the nucleus, where it exerts its role in gene expression. 34,35 Akt/IKK-β axis, known as the upstream signalling of NF-κB pathway, plays vital roles in promoting tumour cell survival, invasive behaviour and chemosensitivity in various malignancies. 9,21,36 A previous study has shown that RPS15A depletion leads to decrease in p-Akt level in glioblastoma cells. 15 In this study, to investigate the mechanism by which RPS15A induces p65 nuclear translocation, we adopted Akt inhibitor LY294002 or IKK inhibitor Bay117082. Our data suggested that both LY294002 and Bay117082 significantly interfered with the activation of Akt/IKK-β signalling induced by RPS15A overexpression. Thus we speculated that RPS15A activates NF-κB through the Akt/IKK-β pathway.
In conclusion, our study demonstrated that elevated expression of RPS15A is closely correlated with poor prognosis of GC patients and promotes EMT and GC progression via Akt/IKK-β/NF-κB signalling pathway, thus possibly providing a promising candidate for treatment against GC metastasis.