RN181 is a tumour suppressor in gastric cancer by regulation of the ERK/MAPK–cyclin D1/CDK4 pathway

Abstract RN181, a RING finger domain‐containing protein, is an E3 ubiquitin ligase. However, its biological function and clinical significance in cancer biology are obscure. Here, we report that RN181 expression is significantly down‐regulated in 165 tumour tissues of gastric carcinoma (GC) versus adjacent non‐tumour tissues, and inversely associated with tumour differentiation, tumour size, clinical stage, and patient's overall survival. Alterations of RN181 expression in GC cells by retrovirus‐transduced up‐regulation and down‐regulation demonstrated that RN181 functions as a tumour suppressor to inhibit growth of GC in both in vitro culture and in vivo animal models by decreasing tumour cell proliferation and increasing tumour cell apoptosis. Cell cycle analysis revealed that RN181 controls the cell cycle transition from G1 to S phase. Mechanistic studies demonstrated that RN181 inhibits ERK/MAPK signalling, thereby regulating the activity of cyclin D1–CDK4, and consequently controlling progression in the cell cycle from G1 to S phase. Restoring CDK4 in GC cells rescued the inhibitory phenotype produced by RN181 in vitro and in vivo, suggesting a dominant role of CDK4 in control of the tumour growth by RN181. Importantly, RN181 expression is inversely correlated with the expression of cyclin D1 and CDK4 in GC clinical samples, substantiating the role of the RN181–cyclin D1/CDK4 pathway in control of the tumour growth of GC. Our results provide new insights into the pathogenesis and development of GC and rationale for developing novel intervention strategies against GC by disruption of ERK/MAPK–cyclin D1/CDK4 signalling. In addition, RN181 may serve as a novel biomarker for predicting clinical outcome of GC. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.


Introduction
Gastric adenocarcinoma (gastric cancer, GC) is the fifth most common cancer and the third leading cause of cancer mortality worldwide [1]. In China, however, GC is the second most common cancer and the second leading cause of cancer death, having estimated numbers of 679 100 new cases and 498 000 deaths in 2015 [2]. Most GC patients present with advanced or metastatic disease in clinics and are treated only by palliative chemotherapy, with an overall 5-year survival around 5-20% and a median overall survival of 11-12 months [3]. In addition to standard perioperative chemotherapy or postoperative chemoradiation [4][5][6], targeted therapy, which targets specific molecules and disrupts the activity of a specific oncogenic signalling pathway, has emerged as a promising therapeutic strategy. Recently, a randomised phase 3 trial for the first-line treatment of HER2-positive advanced GC showed that trastuzumab, a humanised monoclonal antibody targeting HER2, when combined with standard chemotherapy, significantly improves an overall survival of GC patients from 11.1 months to 13.8 months [3]. Unfortunately, only 7-34% of GC patients are HER2-positive [3,7] and thus clinical application of trastuzumab is highly restricted. Therefore, it is imperative that identification of new targets will help Role of RN181 in tumour growth of gastric cancer 205 to understand the molecular mechanisms of pathogenesis and develop a novel targeted therapy or biomarker for evaluating the therapeutic efficacy or predicting the prognosis of GC patients [8].
RN181 (also named RNF181 or HSPC238) is a member of the really interesting new gene (RING) finger protein family whose members are well recognised in protein dimerisation and protein-protein interaction and possess ubiquitin ligase activities [9]. It was reported that in platelets, RN181 interacts with the cytoplasmic regulatory domain of integrin αIIbβ3 and exhibits E3 ubiquitin ligase activity for auto-ubiquitination [10]. However, the physiological substrates and biological functions of RN181 in cancer biology are obscure. We have been interested in RING finger ubiquitin ligases in cancer biology and clinical significance. In this paper, we report that RN181 is significantly down-regulated in GC clinical samples and is closely associated with some clinicopathological features and patient's overall survival. Up-regulation and down-regulation of RN181 demonstrated that RN181 functions as a tumour suppressor that controls the tumour growth of GC. Mechanistic studies revealed that RN181 controls tumour growth through induction of cell cycle arrest at the G1-S phase transition by inhibition of ERK/MAPK signalling and thereby regulation of the cyclin D1-CDK4 activity in GC.

Cell culture
The lentivirus packaging cell line (GP2-293) and GC cell lines (AGS, MKN28, and MKN45) were purchased from and authenticated by the Shanghai Institutes for Biological Sciences (Shanghai, PR China). All cell lines were cultured in Dulbecco's modified Eagle's medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin, and maintained at 37 ∘ C with 5% CO 2 .

Patients and tissue samples
GC clinical samples (HStm-Ade167Sur and HStm-Ade180Sur) containing clinicopathological information were purchased from Shanghai Outdo Biotech Co, Ltd (http://www.superchip.com.cn/). One hundred and sixty-five GC tissue specimens paired with adjacent non-tumour gastric tissues were used for immunohistochemical (IHC) studies. Patients' consent and approval from local Ethics Committee were obtained for research in use of clinical materials. The patients included 118 males and 47 females, with a median age of 65 years (range 34-84 years) (supplementary material, Table S1). Clinical stages were according to the 7th edition of the AJCC Cancer Staging Manual [11]. Another cohort (HStm-Ade150CS-01) of clinical samples, consisting of 75 pairs of GC and adjacent non-tumour tissues, was used for analysis of the expression correlation between RN181, cyclin D1, and CDK4.

Tumour xenografts
BALB/c female nude mice 4-5 weeks of age were purchased from the Experimental Animal Center, Southern Medical University (Guangzhou, PR China) and maintained under standard pathogen-free conditions. Tumour xenografts were performed as described previously [12]. The left flank of each mouse was implanted with control tumour cells, while the right side was injected with the test tumour cells. Each group contained six mice. Tumour growth was monitored by measuring tumour length and width with callipers. Tumour volume was calculated with the formula [length × width 2 × (π/6)] [13]. All experimental procedures were approved by the Ethical Committee of Southern Medical University.
Immunohistochemical staining and scoring IHC staining was performed as described previously [12]. Histopathological features of stained tumour were assessed by two researchers who were blinded to the patient's clinical characteristics. The intensity of RN181 immunostaining of GC tissues was scored as negative (0), weak (1), medium (2) or strong (3). The extent of staining, defined as the per cent of positive staining cells, was scored as 1 (≤ 10%), 2 (11-50%), 3 (51-75%) or 4 (> 75%). An overall expression score for statistical analysis, ranging from 0 to 12, was calculated by multiplying the score of intensity and that of extent. The final staining score was presented as low (overall score of ≤ 3) or high (overall score of > 3). Nuclear localisation of cyclin D1 or CDK4 was quantified by counting approximately 500 stained cells under 400× magnification.
Statistics SPSS 13.0 software (SPSS Inc, Chicago, IL, USA) was used. The analysis of variance (ANOVA) test was used to compare mean values among three or more groups, whereas independent-sample Student's t-test was used to compare two groups with normal distribution data. The data normality was verified using the Kolmogorov-Smirnov test. For non-normal distribution data, the Mann-Whitney test was used for two-group comparisons, while the Jonckheere-Terpstra test was used to compare more than two groups. Sample-paired t-tests were employed to analyse the expression of RN181 in paired clinical samples and xenograft tumour sizes. Kaplan-Meier plots and the log-rank test were 206 S Wang, X Wang, Y Gao et al used for analysis of overall survival data. Univariate and multivariate survival analyses were conducted using the Cox proportional hazards regression model. The Pearson correlation test was used to analyse the correlation between the expression of two proteins. Statistical significance is indicated in the figures by an asterisk where p < 0.05 and by two asterisks where p < 0.01.

RN181 is down-regulated and associated with overall survival of GC patients
We examined the expression of RN181 in a cohort of 165 pairs of GC and adjacent non-tumour tissues by IHC ( Figure 1A and supplementary material, Figure S1). Quantitative analysis by scoring the staining showed that RN181 was significantly down-regulated in tumour tissues versus adjacent tissues (2.19 ± 2.10 versus 5.94 ± 4.22) (p < 0.01) ( Figure 1B). Clinicopathological analyses demonstrated that the expression of RN181 was significantly associated with tumour differentiation (Z = −2.045, p = 0.041), tumour size (Z = −2.484, p = 0.013), and clinical stage (Z = −2.765, p = 0.006) but not with patient's gender or age (supplementary material, Table S1). Decreased expression of RN181 was frequently observed in tumours that were poorly differentiated, had a larger tumour size, and were in a late clinical stage. Kaplan-Meier analysis revealed that expression of RN181 was significantly associated with overall survival of GC patients (p = 0.004), showing a median survival of more than 70.0 months in the RN181-high expression group versus 31.9 months in the RN181-low expression group ( Figure 1C). Univariate Cox regression analysis showed that many parameters including age, tumour size, metastasis, and clinical stage were significantly correlated with patient overall survival (Table 1). Multivariate analysis demonstrated that RN181 expression has a prognostic role (HR = 2.337, 95% CI 1.238-4.422, p = 0.009). Thus, the results suggest that down-regulation of RN181 may be involved in the tumour development of GC and serve as an independent poor prognostic biomarker for GC patients.

RN181 suppresses tumour growth of GC in vitro and in vivo
We up-regulated the expression of RN181 in GC cells by retrovirus-mediated transduction ( Figure 2A). Cell proliferation assays demonstrated that up-regulation of RN181 significantly inhibited the AGS growth (AGS-RN181 versus AGS-RV control, p < 0.01) ( Figure 2B). Colony formation assays showed that up-regulation of RN181 significantly reduced the number of colonies formed by AGS cells (AGS-RN181 versus AGS-RV control, p < 0.01) ( Figure 2C). Consistently, down-regulation of endogenous RN181 by transduction of shRNA-containing viruses significantly increased the proliferation and colony formation of AGS cells (AGS-KD versus AGS-NC control, p < 0.01). Similar results were obtained from MKN28 and MKN45 cell lines in which RN181 expression was down-regulated (supplementary material, Figure S2A-C).
We then implanted the GC cells into nude mice. Up-regulation of RN181 significantly inhibited the tumour growth (AGS-RN181 versus AGS-RV, p < 0.01), while down-regulation of RN181 dramatically promoted the tumour growth of AGS (AGS-KD versus AGS-NC, p < 0.01) ( Figure 2D). Similar results were obtained from the xenografted tumour of MKN28 cells in which RN181 expression was modulated (supplementary material, Figure S2D).
IHC staining of tumour sections confirmed the up-regulation (AGS-RN181 versus AGS-RV, p < 0.01) and down-regulation of RN181 (AGS-KD versus AGS-NC, p < 0.01) ( Figure 2E) in GC tumours. Tumour cell proliferation in xenograft tumours was significantly decreased, as revealed by IHC for Ki67 (AGS-RN181 versus AGS-RV, p < 0.05), whereas tumour cell apoptosis was significantly increased, as demonstrated by anti-activated caspase 3 staining (AGS-RN181 versus AGS-RV, p < 0.01). In agreement with the up-regulation results, down-regulation of RN181 increased tumour cell proliferation and decreased tumour cell apoptosis in xenograft tumours (AGS-KD versus AGS-NC, p < 0.05) and in AGS cells cultured in vitro (supplementary material, Figure S3). Taken together, the results suggest that RN181 functions as a tumour suppressor in the stomach to control the tumour growth of GC by decreasing tumour cell proliferation and increasing tumour cell apoptosis.

RN181 controls cell cycle progression of GC
We performed cell cycle analyses by flow cytometry and western blots after synchronising the cells at the G1 phase by double-thymidine block (supplementary material, Figure S4A,B). After release from the blockade, AGS-RV cells progressed from the G1 to the S phase at 4-6 h, whereas AGS-RN181 cells transited from the G1 to the S phase at 6-8 h (supplementary material, Figure S4A). Cell cycle analyses at the time point of 6 h (supplementary material, Figure S4C) showed 67.0% ± 5.6% of AGS-RN181 cells in the G1 phase versus 13.0% ± 2.2% of AGS-RV control cells in the G1 phase (p < 0.01). In contrast, 16.7% ± 3.0% in the S phase and 16.2% ± 1.8% in the G2 phase were observed for AGS-RN181 versus 79.3% ± 6.6% in the S phase and 7.1% ± 1.8% in the G2 phase for AGS-RV, respectively (both p < 0.01). Thus, the results indicate that RN181 controls the cell cycle transition from G1 to S phase and compels the GC cells to reside in the G1 phase for a prolonged period of 2 h.
Cell cycle analyses were also performed for AGS cells after knockdown of RN181 (supplementary material, Figure S4B). Compared with up-regulation, down-regulation of RN181 did not affect the G1-S phase transition but accelerated DNA synthesis, shortened the S phase, and promoted the cell cycle   Figure   S4D) showed 20.7% ± 1.7% of AGS-KD in S phase versus 61.4% ± 4.4% of AGS-NC in S phase (p < 0.01).
In contrast, 73.0%± 1.7% in G2 phase was observed for AGS-KD compared with 32.8% ± 4.7% for AGS-NC (p < 0.01). Thus, the results indicate that knockdown of  which knockdown of RN181 decreased the expression of p21 but increased the expression of cyclin D1 and CDK4 (supplementary material, Figure S5A). To verify the in vitro results, we further investigated the correlation of RN181 expression with the activities of p21, cyclin D1, and CDK4 in vivo. IHC staining revealed that up-regulation of RN181 significantly increased the expression of p21 but dramatically reduced the expression of cyclin D1 and CDK4 in xenograft tumours of AGS cells (AGS-RN181 versus AGS-RV, all p < 0.01) ( Figure 3B). In agreement with up-regulation, down-regulation of RN181 significantly decreased the expression of p21 but increased the expression of cyclin D1 and CDK4 in xenograft tumours of AGS cells (AGS-KD versus AGS-NC control, all p < 0.05) ( Figure 3C). Similar results were obtained from xenograft tumours of MKN28 cells in which RN181 was modulated (supplementary material, Figure S5B,C).
We also examined the cellular localisation of cyclin D1 and CDK4. Up-regulation of RN181 dramatically reduced the percentage of tumour cells that have nuclear staining for cyclin D1 and CDK4 (AGS-RN181 versus AGS-RV, p < 0.01) ( Figure 3D), while down-regulation of RN181 significantly increased the percentage of tumour cells showing nuclear accumulation of cyclin D1 and CDK4 (AGS-KD versus AGS-NC, p < 0.05) ( Figure 3E). Similar results were obtained from AGS cells cultured in vitro (supplementary material, Figure  S6). Taken together, the results indicate that RN181 controls the G1-S phase transition by inhibiting the activity of cyclin D1-CDK4.

Reconstitution of CDK4 rescues the inhibitory phenotype of GC cells by RN181
We reconstituted the expression of CDK4 in AGS-RN181 cells by adenovirus transductions. Western blots ( Figure 4A) showed that transduction with pAD-Empty adenovirus did not affect the expression of either RN181 (RV + Empty versus RV-Empty; RN181 + Empty versus RN181-Empty) or CDK4 (RV + Empty versus RN181 + Empty) in AGS cells, while transduction with pAD-CDK4-His increased the expression of CDK4 in both AGS-RV control (RV-CDK4 versus RV + CDK4) and AGS-RN181 cells (RN181-CDK4 versus RN181 + CDK4). Cell proliferation assays ( Figure 4B) confirmed the inhibitory effect on AGS growth by RN181 (RN181 + Empty versus RV + Empty, p < 0.01). Interestingly, CDK4 reconstitution not only abolished the growth inhibition by RN181 (RN181 + CDK4 versus RN181 + Empty, p < 0.01) but also further increased the cell proliferation of AGS (RN181 + CDK4 versus RV + Empty, p < 0.05), highlighting a dominant role of CDK4 in control of the GC growth. However, the increased growth of AGS-RV cells by CDK4 was much stronger than that of AGS-RN181 (RV + CDK4 versus RN181 + CDK4, p < 0.01), suggesting that RN181 may also affect other molecules that regulate GC growth. Similar results were obtained from colony formation assays ( Figure 4C).

RN181 suppresses tumour growth by inhibition of ERK/MAPK in GC cells
We performed RNA-Seq to compare gene expression profiles in AGS-RN181 cells versus those in AGS-RV cells. Ingenuity pathway analysis revealed that many differentially expressed genes regulated by RN181 were integrated in the pathways of ERK1/2-MAPK and cyclin D ( Figure 5A), suggesting that RN181 may regulate ERK1/2-MAPK signalling which affects the cyclin D1-CDK4 activity that controls the cell cycle transition from G1 to S phase.
To address this hypothesis, we first evaluated the phosphorylation of pERK1/2 in GC cells. Down-regulation of RN181 significantly increased the phosphorylation of pERK1/2 in AGS cells both cultured in vitro (KD versus NC) ( Figure 5B) and xenografted in vivo (KD versus NC, p < 0.01) ( Figure 5C). Consistent with the down-regulation results, up-regulation of RN181 dramatically decreased the phosphorylation of pERK1/2 (AGS-RN181 versus AGS-RV, p < 0.01). Similar results were observed in MKN28 cells in which RN181 was modulated (supplementary material, Figure  S7A,B).
We then treated GC cells with U0126, a highly selective inhibitor of MEK1/MEK2. Cell proliferation ( Figure 5D) and colony formation ( Figure 5E  (NC + U0126 versus NC-U0126) ( Figure 5F), suggesting that both cyclin D1 and CDK4 are downstream targets of ERK/MAPK signalling. Similar results were observed in MKN28 cells in which RN181 was modulated (supplementary material, Figure S7C-E).
Taken together, the results indicate that RN181 suppresses the tumour growth of GC by inhibition of the ERK/MAPK pathway and consequently control of the activity of cyclin D1-CDK4 that regulates the cell cycle transition from G1 to S phase.

Correlation between RN181 and cyclin D1/CDK4 in GC clinical samples
We further investigated the expression of RN181, cyclin D1, and CDK4 in GC clinical specimens by IHC ( Figure 6A). Quantitative analysis by scoring the staining showed that the expression of RN181 was significantly down-regulated in tumour tissues versus adjacent non-tumour tissues, while the expression of cyclin D1 and CDK4 was significantly up-regulated in tumour tissues versus adjacent tissues, respectively (all p < 0.001) ( Figure 6B). Pearson correlation analyses revealed that the expression of RN181 was reversely associated with the levels of cyclin D1 and CDK4, respectively (all p < 0.001) ( Figure 6C). In contrast, the expression of cyclin D1 was positively correlated with the expression of CDK4 (p < 0.001) ( Figure 6D). Altogether, the results substantiate the role of the RN181-cyclin D1/CDK4 pathway in control of the tumour development of GC.

Discussion
The activation of oncogenes and inactivation of tumour suppressor genes play major roles in the pathogenesis of GC [14,15]. Recently, several novel putative tumour suppressors such as BCL6B [16], CPEB1 [17], ZNF331 [18], ZNF545 [19] and CHIP [20] have been identified in GC. In this study, we demonstrated that RN181 was significantly down-regulated in tumour tissues versus adjacent non-tumour tissues. Remarkably, RN181 expression was inversely associated with tumour differentiation, tumour size, clinical stage, and patient's overall survival. These results suggest that RN181 may be involved in the pathogenesis and development of GC and may serve as a biomarker for predicting the outcome of GC patients. To study the possibility that RN181 could affect the carcinogenesis and development of GC, we modulated the expression of RN181 in GC cells. We found that up-regulation of RN181 significantly inhibited tumour growth, while down-regulation of RN181 promoted tumour growth by regulation of tumour cell proliferation and apoptosis in vitro and in vivo. Therefore, we conclude that RN181 is a novel tumour suppressor in the stomach and controls the tumour growth of GC.

S Wang, X Wang, Y Gao et al
Many tumour suppressors constrain cell growth and proliferation by affecting a variety of signalling pathways that impinge on the core cell-cycle machinery [14,21]. To explore underlying cellular mechanisms, we performed cell cycle analyses. Up-regulation of RN181 compelled GC cells to reside in the G1 phase for an extra 2 h, whereas down-regulation of RN181 accelerated DNA synthesis and increased the cell cycle progression from the S to the G2 phase for 2 h. Thus, we conclude that suppression of tumour cell proliferation by RN181 is attributed to the delay of the cell cycle transition from the G1 to the S phase of GC. Deregulation of signalling networks during the G1 phase of the cell cycle represents major driving forces in the tumourigenesis and development of cancer [21]. Many oncogenes promote cell proliferation by accelerating the G1-S phase transition. Cyclin D1, CDK4, and CDK6 are among the core players for such G1-S phase transition [21][22][23][24]. We found that up-regulation of RN181 significantly decreased the expression of cyclin D1 and CDK4, while down-regulation of RN181 increased the expression of cyclin D1 and CDK4 in GC cells. Strikingly, knockdown of RN181 significantly increased nuclear localisation of cyclin D1 and CDK4 in tumour cells, suggesting an increase of cyclin D1-CDK4 activity [22,25,26]. Interestingly, CDK4-deficient MEF cells were reported to stay in the G1 phase for a prolonged period [27], suggesting that postponement of the G1-S phase transition may be mediated by decreasing CDK4 expression by RN181. To corroborate the role of CDK4 in the inhibition of tumour growth by RN181, we reconstituted the expression of CDK4 in RN181-overexpressing GC cells. Indeed, CDK4 reconstitution not only rescued the growth inhibitory phenotype conferred by RN181 both in vitro and in vivo but also further increased the cell growth and colony formation of GC, highlighting a dominant role of CDK4 in the control of GC growth by RN181.
To further investigate underlying mechanisms, we profiled gene expression by RNA-Seq. We found that many differentially expressed genes regulated by RN181 were tightly integrated in the pathways of ERK/MAPK and cyclin D1. In many cell types, both transcription of cyclin D1 and CDK4 and assembly of cyclin D1 with CDK4 rely on activation of RAS-RAF-MEK-ERK signalling [28]. In GC, several factors including the RAF/MEK/ERK pathway [29] are involved in cyclin D1 up-regulation. We previously found that RN181 could inhibit the ERK/MAPK pathway in hepatocellular carcinoma [12]. Therefore, we hypothesised that the suppression of tumour growth of GC by RN181 may undergo by reducing the expression and assembly of cyclin D1 with CDK4 mediated by inhibition of ERK/MAPK signalling in the stomach. Indeed, up-regulation of RN181 significantly inhibited the phosphorylation of pERK1/2 and down-regulation of RN181 greatly increased the phosphorylation of pERK1/2 in GC cells in vitro and in vivo. Remarkably, inhibition of MEK/ERK/MAPK signalling by U0126 not only eliminated the increased growth of tumour cells by down-regulation of RN181 but also equally decreased the abilities of proliferation and colony formation of GC cells. More importantly, U0126 dramatically reduced the expression of cyclin D1 and CDK4, implying that both cyclin D1 and CDK4 are downstream targets of ERK/MAPK signalling in GC. Tob1, which is known to repress the expression of cyclin D1 and CDK4 in GC [30], is also phosphorylated by ERK/MAPK kinases, which relieves the transcriptional repression of cyclin D1 [31]. ASK1, which was known to participate in colon and skin tumourigenesis, could increase the expression of cyclin D1 through AP-1 activation and cyclin D1 could up-regulate ASK1 via the Rb-E2F pathway in GC to stimulate tumour growth [32]. Thus, either Tob1 or ASK1 may also be involved in the suppression of tumour growth of GC by RN181.
To verify the results from in vitro and in vivo models, we further investigated the activity of cyclin D1 and CDK4 in another cohort of GC clinical specimens. We found that the expression of cyclin D1 and CDK4 was significantly elevated in GC tissues versus adjacent non-tumour tissues. Indeed, up-regulation of cyclin D1 and CDK4 was observed in a wide spectrum of tumours [22,24,26]. Remarkably, about 37-56% of GC patients expressed abnormally high levels of cyclin D1 [32][33][34]. More importantly, the expression of RN181 was reversely correlated with the expression of cyclin D1 and CDK4, respectively, while the expression level of cyclin D1 was positively associated with the expression of CDK4, highlighting the role of the RN181-cyclin D1/CDK4 pathway in the tumour development of GC. Accordingly, we speculate that specific inhibition of cyclin D1/CDK4 activity may produce clinical benefits for GC patients who have elevated expression of cyclin D1 and CDK4 in their tumours. Importantly, palbociclib, a small-molecule inhibitor of CDK4 and CDK6, combined with letrozole has recently been approved to treat postmenopausal women with ER-positive, HER2-negative advanced breast cancer as a first-line therapy [35].
In conclusion, we have demonstrated that RN181 is down-regulated in GC and is inversely associated with some clinicopathological features and patient's overall survival. RN181 functions as a tumour suppressor in the stomach to inhibit tumour growth in vitro and in vivo by control of the cell cycle progression from G1 to S phase. Mechanistically, RN181 inhibited ERK/MAPK signalling, thereby regulating cyclin D1-CDK4 activity and consequently controlling the cell cycle progression. Reconstitution of CDK4 rescued the inhibitory phenotype conferred by RN181. Importantly, in clinical tumour samples, RN181 was reversely correlated with the expression of cyclin D1 and CDK4, highlighting the role of the RN181-cyclin D1/CDK4 pathway in the control of tumour growth. Our results provide new insights into the carcinogenesis and development of GC and facilitate the development of novel intervention strategies against GC by disrupting the ERK/MAPK-cyclin D1/CDK4 pathway. RN181 may become a prognostic biomarker for predicting the outcome of GC patients.