RBM10 inhibits cell proliferation of lung adenocarcinoma via RAP1/AKT/CREB signalling pathway

Abstract Initial functional studies have demonstrated that RNA‐binding motif protein 10 (RBM10) can promote apoptosis and suppress cell proliferation; however, the results of several studies suggest a tumour‐promoting role for RBM10. Herein, we assessed the involvement of RBM10 in lung adenocarcinoma cell proliferation and explored the potential molecular mechanism. We found that, both in vitro and in vivo, RBM10 overexpression suppresses lung adenocarcinoma cell proliferation, while its knockdown enhances cell proliferation. Using complementary DNA microarray analysis, we previously found that RBM10 overexpression induces significant down‐regulation of RAP1A expression. In this study, we have confirmed that RBM10 decreases the activation of RAP1 and found that EPAC stimulation and inhibition can abolish the effects of RBM10 knockdown and overexpression, respectively, and regulate cell growth. This effect of RBM10 on proliferation was independent of the MAPK/ERK and P38/MAPK signalling pathways. We found that RBM10 reduces the phosphorylation of CREB via the AKT signalling pathway, suggesting that RBM10 exhibits its effect on lung adenocarcinoma cell proliferation via the RAP1/AKT/CREB signalling pathway.

that RBM10 can promote apoptosis 9 and suppress cell proliferation. 10,11 Furthermore, its expression was found to be correlated with increased expression of the proapoptotic protein BAX and the tumour suppressor protein TP53 in breast cancer. 13 These findings suggest RBM10 as a potential tumour suppressor. However, insufficient research has focused on RBM10-related signalling pathways, which would explain the mechanism of its tumour-suppressing effect.
To elucidate the underlying molecular mechanism, we previously performed an Affymetrix GeneChip Primeview Human cDNA microarray analysis. A total of 690 genes that were regulated by RBM10 expression in A549 cells were identified, of which 304 were up-regulated and 386 were down-regulated. Among these, RAP1A expression was the most down-regulated (63.5%) 14 ; thus, we explored its downstream signalling pathway. RAP1A is a type of Ras-associated protein 1 (RAP1), with 95% sequence homology to RAP1B. 15 It is a significant regulator and mediator of Ras functions, and its activation has been linked to a variety of cancers. 16,17 EPAC is a guanine nucleotide exchange factor (GEF) for the RAP1, which activates RAP1 by GDP-GTP exchange. 21,22 Agonists and antagonists selective for EPAC have been developed and can be used for further studies on the activation of RAP1, which will increase our understanding on the signalling pathway. 23 In this study, we aimed to explore the effect of RBM10 overexpression and knockdown on the proliferation of lung adenocarcinoma cells as well as the affected downstream pathways by assessing the expression of several regulatory proteins that have previously been identified as being modulated by RBM10. 14 We then sought to verify the effect of RBM10 overexpression and inhibition in vivo in BALB/c mice.

| Cell culture and reagents
The human NSCLC cell lines A549 and H1299 were obtained from the Chinese Academy of Medical Sciences (Beijing, China) and cultured in RPMI1640 (Invitrogen, Carlsbad, CA) supplemented with 10% foetal bovine serum and 100 U/mL penicillin and 0.1 mg/mL streptomycin (Invitrogen) at 37°C, in a humidified atmosphere containing 5% CO 2 . Dimethylsulfoxide (DMSO) was purchased from Sigma-Aldrich (St. Louis, MO) 0.8-pCPT-2'-OMe-cAMP and ESI-09 were obtained from the Biological Life Science Institute (Bremen, Germany).

| Colony formation assay
A549 and H1299 cells were seeded in six-well plates at a density of 500 cells per well and cultured in RPMI 1640 containing 10% of FBS for 2 weeks. Cells were then fixed with 4% paraformaldehyde for 10 min, stained with Giemsa for 10 min and washed three times with ddH 2 O. The cells were photographed with a digital camera, and colonies of more than 50 cells were counted. The assay was performed in triplicate and repeated at least twice.

| Western blotting assay
Protein concentrations in the supernatants were quantified using the DC Protein Assay (Bio-Rad), and 50 μg aliquots of each protein sample were added to the sample buffer (10% glycerol, 0.7 mol/L beta-mercaptoethanol, 3% sodium dodecyl sulfate, 62 mmol/L Tris-HCl, pH 6.8), boiled for 10 min, separated using 12% sodium

| RAP1-GTP pull-down assay
Cells were lysed with radioimmunoprecipitation assay (RIPA) buffer with protease and phosphatase inhibitors. An equal amount of protein (approximately 150 mg) was incubated with GST-RAP-binding domain (RBD) of RalGDS conjugated to glutathione beads for 2 hour at 4°C and centrifuged for 30 s. Beads were washed with RIPA buffer and mixed with loading dye containing β-mercaptoethanol. RAP1-GTP levels were detected by Western blotting.

| Xenograft experiment
The use of animals in this study was in accordance with animal care guidelines, and the protocol was approved by the Jilin University

| Statistical analysis
Data were analysed by SPSS 24 software (IBM, Chicago, IL) and are expressed as means ± SD. Analysis of significant differences between groups was performed using unpaired Student's t test or one-way ANOVA P < 0.05 was considered as a statistically significant difference.

| RBM10 overexpression suppresses cell proliferation, while RBM10 knockdown enhances cell proliferation in vitro
We first performed functional in vitro assays to explore the involvement of RBM10 in NSCLC progression. RBM10 was either overexpressed or silenced in A549 and H1299 cells through lentiviral transduction. The expression efficiency was determined by Western blotting ( Figure 1A). Results of CCK-8 and colony formation assays indicated that RBM10 overexpression significantly attenuated the cell proliferation ability, whereas its knockdown markedly enhanced cell proliferation ( Figure 1B,C).These results indicate that RBM10 overexpression could suppress and RBM10 knockdown could enhance cell growth in vitro.

| RBM10 inhibits cell proliferation by decreasing the activation of RAP1, and EPAC stimulation or inhibition can reverse it
The activation of RAP1, a well-known EPAC effector, previously selected by the microarray analysis, was examined by Western blotting

| RBM10-mediated cell proliferation is independent of MAPK/ERK and P38 MAPK signalling pathways
To determine whether the effects of RBM10 on cell proliferation were associated with the MAPK/ERK or P38 MAPK pathways, we assessed the expression of key markers of these pathways that are closely associated with GTP-bound RAP1 expression. Compared to F I G U R E 2 RBM10 decreases the activation of RAP1. A, A549 cells were transfected with GV358-RBM10 and with its corresponding control vectors (NC) and subjected to pull-down assay using GST-RBD, followed by immunoblotting with antibodies against RAP1; GV358-RBM10 and NC were treated with PBS (P) or 100 μmol/L of the EPAC stimulator 8-pCPT-2'-O-Me-cAMP (8-pCPT) for 30 min, and GTP-RAP1 was detected by western blotting. B: The effect of RBM10 overexpression on cell viability was measured by the CCK-8 assay. C: A549 cells were transfected with GV248-RBM10-RNAi and with its corresponding control vector (V) subjected to pull-down assay using GST-RBD followed by immunoblotting with antibodies to RAP1; GV248-RBM10-RNAi and Vector were treated with DMSO (D) or 10 μmol/L of the EPAC inhibitor ESI-09 for 30 min, and GTP-RAP1 was detected by Western blotting. D: The effect of RBM10 knockdown on cell viability was measured by the CCK-8 assay. *P < 0.05, **P < 0.01, ***P < 0.001. Data represent mean values ± SD

| RBM10 reduces the phosphorylation of CREB via AKT signalling pathway
Overexpression of RBM10 resulted in decreased phospho-AKT and phospho-CREB in A549 cells ( Figure 3B). In contrast, the expression of phospho-AKT and phospho-CREB in RBM10-knockdown A549 cells was increased greatly compared to the control group

| D ISCUSS I ON
According to a recent review, results suggesting a tumour-promoting role for RBM10 have emerged. 24  To elucidate the molecular mechanism behind this finding, we previously discovered that RAP1A expression was the most down-regulated in the cDNA microarray analysis and we explored its downstream signalling pathways. Among them, the synergistic activation of the ERK signalling pathway is the most common related pathway. 27 The CREB protein is a crucial transcription factor that regulates a wide range of biological processes that orchestrate cell differentiation and cell growth. 31 CREB enhances cell proliferation, reduces apoptosis sensitivity and increases angiogenesis and radiation-induced differentiation. 32 Overexpression of CREB was reported in many solid tumour types like breast cancer, liver cancer and melanoma when compared to adjacent normal tissues. 33,34 CREB is an important factor in cAMP signalling, which regulates protein levels by controlling gene transcription. cAMP signalling commonly activates three major cAMP effector molecules: PKA, EPAC and cyclic-nucleotide-gated ion channels. 36 Herein, we revealed that RBM10 mediates an increase in CREB expression and found that EPAC/RAP1, but not PKA, mediates CREB expression. This result is further supported by the observation that CREB expression increased after EPAC-selective stimulation and decreased after EPAC-selective inhibition. In conclusion, RBM10 inhibited the expression of CREB via EPAC/RAP1-specific signalling.
To conclude, this study found that RBM10 inhibits lung adenocarcinoma cell growth in vitro and in vivo. Specifically, it inhibits cell proliferation via the RAP1/AKT/CREB signalling pathway. Our observations may help to better understand how deregulation of RBM10 contributes to lung adenocarcinoma progression.

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