A‐kinase‐interacting protein 1 facilitates growth and metastasis of gastric cancer cells via Slug‐induced epithelial‐mesenchymal transition

Abstract A‐kinase‐interacting protein 1 (AKIP1) has previously been reported to act as a potential oncogenic protein in various cancers. The clinical significance and biological role of AKIP1 in gastric cancer (GC) is, however, still elusive. Herein, this study aimed to investigate the functional and molecular mechanism by which AKIP1 influences GC. AKIP1 mRNA and protein expressions in GC tissues were examined by quantitative real‐time PCR (qRT‐PCR), Western blot and immunohistochemistry. Other methods including stably transfected against AKIP1 into gastric cancer cells, wound healing, transwell assays, CCK‐8, colony formation, qRT‐PCR and Western blot in vitro and tumorigenesis in vivo were also performed. The up‐regulated expression of AKIP1 in GC specimens significantly correlated with clinical metastasis and poor prognosis in patients with GC. AKIP1 knockdown markedly suppressed GC cells proliferation, invasion and metastasis both in vitro and in vivo. In contrast, AKIP1 overexpression resulted in the opposite effects. Moreover, mechanistic analyses indicated that Slug‐induced epithelial‐mesenchymal transition (EMT) might be responsible for AKIP1‐influenced GC cells behaviour. Our findings demonstrated that high AKIP1 expression significantly correlated with clinical metastasis and unfavourable prognosis in patients with GC. Additionally, AKIP1 promoted GC cells proliferation, migration and invasion by activating Slug‐induced EMT.

correlates with aberrant expression of usually repressed transcriptional factors such as Snail, Slug, ZEB1, SIP1 and Twist. Besides, several signalling pathways, including TGFβ, Notch, Wnt and PI3K/ AKT signalling cascade, activated in the EMT evolution are essential regulators of EMT. 4 Since accumulating evidences have unveiled the potential clinical value of targeting EMT in cancer treatment, 7 we focus here on the molecular mechanisms by which certain genes control EMT in GC progression.
A-kinase-interacting protein 1 (AKIP1), a small 23-kDa protein, is also named breast cancer-associated gene 3, which encodes an alternatively spliced proline-rich protein. 8 Recent studies have indicated that AKIP1, identified as a differentially expressed gene, was elevated in many human malignancies, and might be involved in tumorigenesis and invasiveness, such as non-small-cell lung cancer and breast cancer, suggesting that AKIP1 could be exploited as a target for cancer treatment. 9,10 However, to the best of our knowledge, the clinicopathological and biological roles of AKIP1 in GC has not yet been elucidated.
In this study, we therefore investigated the clinical significance of AKIP1 in GC tissues, as well as the function and the underlying mechanism of AKIP1 in GC cells growth, migration and invasion.

| Patients, specimens and cell lines
A total of 96 patients with GC who underwent radical resection at the Fifth Affiliated Hospital of Nantong University were enrolled in this study. None of these participants was exposed to preoperative chemotherapy or radiotherapy. Among them, fresh tissues of 50 cases were performed for the detection of AKIP1 mRNA and protein expressions. In addition, another cohort of 96 cases was used for immunohistochemistry (IHC) evaluation. Written informed consent was obtained from all patients. The study was approved by the Ethics Committee of the

| Lentivirus infection
Stable cell lines expressing shAKIP1 or AKIP1 were generated by transfection of pSuper-shAKIP1 or pMSCV-AKIP1. 11 Transfections were carried out using Lipofectamine 2000 (Invitrogen, USA) in accordance with the manufacturer's protocols. The transfection efficiency was determined by Western blot analysis.

| RNA isolation and quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was extracted from tissues or cells using Trizol reagent (Invitrogen, USA). The complementary DNA (cDNA) was synthesized using the reverse transcription kit (TaKaRa, Japan) following the manufacturer's description. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis was conducted using SYBR Green assay kit (Takara, China) on a 7500 RT-PCR system (Applied Biosystems, USA). The 2 −ΔΔCt method was performed for relative quantification.
The primers are listed in Supplementary Material.

| Western blot
The approach for Western blot was conducted as described previously. 12 Primary antibody against AKIP1 was bought from Abcam (UK). Primary antibodies against E-cadherin, Snail, Slug, ZEB1, SIP1, Twist and N-cadherin were obtained from CST (USA). GAPDH antibody, employed as the loading control, was purchased from Bioworld Technology (USA). Proteins were visualized with an enhanced chemiluminescence detection reagent (Thermo scientific, USA).

| Immunohistochemistry
IHC was performed using standard procedures as described previously. 13 Antibodies used for IHC analysis were AKIP1 (Abcam, UK), Slug (CST, USA) and E-cadherin (CST, USA). Quantification of the IHC scores was performed by two independent pathologists (double-blinded). The final staining scores were graded as reported previously. 13

| Wound healing assay
Cells were added to a six-well plate, and then cultured to a confluent monolayer. A linear scratch was gently made using a sterile 10µl plastic tip, followed by an observation of the distance migrated by the cells at the indicated time (0 hour and 48 hour) to monitor the wound healing process. The percentage recovering of the wound = (0 hour width − 48 hour width)/0 hour width × 100%.

| Cells migration and invasion assays
For migration assay, cells were plated into a 24-well 8-µm pore size transwell chamber (Corning, USA). For invasion assay, transwell chamber was coated with Matrigel (BD Biosciences, USA). For cells migration and invasion assays, the detailed conditions were described previously. 14

| Cell proliferation assay
Cells were seeded in a 96-well plate. Ten microlitres of cell counting kit-8 (CCK-8, Dojindo, Japan) reagent was added to each well at the appropriate times (0, 24, 48, 72 and 96h after culture), followed by a detection of the absorbance value at 450 nm to analyse cell viability.

| Colony formation assay
Cells were added in a six-well plate. Following incubation at 37°C in a 5% CO 2 humidified incubator for 2 weeks, cells were stained with crystal violet solution, and imaged under a microscope. Clone formation was defined as those containing ≥50 cells.

| Animal experiments
BALB/c nude mice (six-week-old, male) were performed to assess GC cells tumorigenicity and metastasis in vivo. To characterize the effect of AKIP1 on tumour formation, cells were subcutaneously implanted into nude mice followed by measurement of tumour volume at 5-day intervals using the formula: volume = (short diameter) 2 × (long diameter)/2. 15 At 30 days after inoculation, the tumours were weighed and sectioned for Western blot analysis. To figure out the role of AKIP1 in tumour metastasis, cells were injected intravenously into the tail vein of nude mice. The mice were killed at 30 days after injection, when the lungs were harvested for the count of metastasis nodules, and then processed for IHC and Western blot analysis. All animal work was carried out following the guidelines of the Ethics Committee of the Fifth Affiliated Hospital of Nantong University.

| Statistical analysis
A chi-square test was performed to analyse the association of AKIP1 expression with clinicopathological features. Kaplan-Meier plot and Log-rank test were used for survival analysis. All the data were presented as the means ± SD, and were analysed by Student's t test. All statistical analyses were performed using SPSS 21.0 software (SPSS Inc, USA). A value of P < 0.05 was considered statistically significant.

| Clinical significance of AKIP1 in patients with GC
To determine the role of AKIP1 in GC tissues, the mRNA and protein tissues were detected by qRT-PCR and Western blot. As shown in Figure 1A,B, compared with that in matched normal tissues, AKIP1 mRNA was significantly increased in GC tissues. The AKIP1 protein analysis indicated the similar result ( Figure 1C). Besides, the IHC analysis of 96 cases further revealed that AKIP1 expression was markedly elevated in tumour tissues compared with that in corresponding normal tissues ( Figure 1D,E), especially in metastatic tumour tissues ( Figure 1D,F).
Next, the relationship between AKIP1 and clinicopathological variables demonstrated that AKIP1 was closely related with tumour size, T stage, pTNM stage and lymph node metastasis respectively (P < 0.05) ( Table 1), suggesting that AKIP1 might function importantly in GC progression and metastasis.
Additionally, Table 2 showed that the expression level of AKIP1 was positively related to that of Slug ( Figure 1D; P < 0.001, contingency coefficient = 0.512) in GC tissues. Kaplan-Meier survival analysis turned out that patients with AKIP1 positive had poorer prognosis than those with AKIP1 negative (P < 0.05) ( Figure 1E).

| AKIP1 promotes GC cells growth, migration and invasion in vitro
To understand the function of AKIP1 in GC cells, we first selected the appropriate cells for a further study. As shown in Figure 2A,

| Slug-mediated EMT signalling activation is responsible for AKIP1-induced GC cells growth, migration and invasion
Emerging evidence suggested that EMT functions importantly in cancer initiation, progression and metastasis. 16 In this study, qRT-  Figure 3B). Based on these results, we hypothesized that AKIP1 might facilitate EMT by interacting with transcription factor Slug in GC cells.
Next, to test the hypothesis, the Slug expression was suppressed in GC cells. According to the results, we found that Slug silencing in AGS cells resulted in an obvious reversal of AKIP1-induced EMT ( Figure 4A). Moreover, Slug knockdown in AGS cells notably suppressed AKIP1-facilitated cell migration and invasion ( Figure 4B), as well as cells growth ( Figure 4C). In summary, these results implied that AKIP1 promoted GC cells growth, migration and invasion, at least in part, via the activation of Slug-mediated EMT.

| AKIP1 promotes tumour formation and metastatic potential in vivo
To make a further verification of the role of AKIP1 in vivo, we  Figure 5B). On the contrary, AKIP1 overexpression resulted in the opposite effects ( Figure 5A,B).
In addition, the lung metastasis model demonstrated that

| D ISCUSS I ON
Since recurrence and distant metastases remain the major reasons for death in cancer patients, 17  In this study, we demonstrated that AKIP1 was highly ex- Moreover, we investigated the biological roles of AKIP1 in GC cells. The results showed that AKIP1 depletion suppressed GC

cells growth, migration and invasion both in vitro and in vivo,
whereas AKIP1 overexpression resulted in the opposite effects.
Mechanistic investigation revealed that AKIP1 promoted EMT in GC cells via activation of transcription factor Slug.
It is widely accepted that the initial step, acquisition of migration and invasion capability, from a relatively immobile type to a more invasive phenotype, is the rate-limiting step in metastatic cascade. 16,20 As mentioned in the introduction, EMT is generally considered as a key event during the initial steps in cancer metastasis and progression. In consideration of the clin- However, it is worth further exploring the indirect or direct relationship between AKIP1 and Slug. Additionally, as reported, the NF-κB signalling, Akt/GSK-3β/Snail signalling or transcription factor ZEB1 was also demonstrated to function importantly in AKIP1-mediated tumour progression and metastasis. [9][10][11] Consequently, we will further assess whether other genes or certain signalling pathways are involved in AKIP1-induced EMT in subsequent studies.
Taken together, these results revealed that high AKIP1 expression significantly correlated with clinical metastasis and unfavourable prognosis in patients with GC, and AKIP1 facilitated GC cells growth, invasion and metastasis by activating Slug-induced EMT.
Thus, repressing AKIP1 and EMT inducers such as Slug have potential clinical applications for GC treatment.

ACK N OWLED G EM ENTS
This work was supported in part by funding from Taizhou Science and Technology Support Plan (Social Development) Project (TS201732),

Science Foundation of Taizhou People's Hospital (ZL201818) and
Medical Science and Technology Development Foundation of Jiangsu University (JLY20160148 and JLY20160149). Chen dehu performed the research. Chen dehu and Cao gan analysed the data and wrote the paper. Liu qinghong and Chen dehu designed the research study.

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.