TY - JOUR
AU - Yang,, Huawei
AB - Abstract Doxorubicin (DOX) is one of the most effective chemotherapy drugs for the treatment of metastatic breast cancer (BC), but drug resistance becomes an obstacle to treatment. This study aims to investigate the role of Ribosomal S6 protein kinase 4 (RSK4) in regulating BC resistance to DOX. We first used Kaplan–Meier Plotter to identify the prognostic roles of RSK4 in BC. DOX-resistant BC cells (MCF-7/DOX) were constructed and the expression of RSK4 was determined by reverse transcript polymerase chain reaction and western blot. Subsequently, we overexpressed the RSK4 in MCF-7/DOX cells, and measured drug resistance, colony formation, cell migration, invasion ability and cell apoptosis after transfection. In addition, western blot was used to explore the expression of apoptosis-related proteins and BC-resistance protein. Effects of RSK4 on activation of the PI3K/AKT signalling pathway were also tested. Furthermore, tumour xenograft in nude mice was constructed to observe the effect of RSK4 overexpression on tumour growth in vivo. In conclusion, RSK4 was positively correlated with survival rate in BC patients, which is lowly expressed in MCF-7/DOX. Meanwhile, the overexpression of RSK4 may inhibit drug resistance, cell migration, invasion, apoptosis and tumour growth. RSK4 may effectively attenuate DOX resistance in BC by inhibiting the PI3K/AKT signalling pathway. breast cancer, doxorubicin, drug-resistance, PI3K/AKT signalling pathway, RSK4 Breast cancer (BC) is one of the most common diagnosed cancers and the leading cause of cancer-related deaths in women worldwide. Every year, more than 1.2 million people are diagnosed with BC worldwide and ∼500,000 people die of this disease (1). Currently, BC treatment is based on a comprehensive treatment model, including surgery, chemotherapy, hormone therapy, targeted drugs or combination therapy, which greatly improves the long-term survival rate of BC patients (2). Although chemotherapy plays a key role in the comprehensive treatment of BC, drug-resistance is a vital barrier in BC treatment. Doxorubicin (DOX) is a natural anthracycline antibiotic that induces cancer cell death through a variety of mechanisms (3). It is one of the most effective drugs for first-line treatment of metastatic BC, and the acquired chemoresistance to DOX results in poor prognosis (4). Therefore, there is an urgent need to better understand the molecular mechanism of drug resistance so as to provide appropriate treatment methods for improving the clinical outcome of BC patients. Ribosomal S6 protein kinase (RSK4), a serine-threonine kinase of the p90 ribosomal S6 kinase family, is located on the X chromosome (Xq21.1) and has been shown to be an important effector downstream of the Ras-MAPKs signalling pathway (5). RSK4 directly accepts signal regulation from ERK and is involved in cell growth, survival and senescence (6). In recent years, it has been determined that RSK4 is closely related to tumorigenesis and development, and it is used as a tumour suppressor gene in various tumours such as endometrial cancer, colorectal cancer, ovarian cancer and BC (7). Studies have found that exogenous expression of RSK4 leads to increased accumulation of cells in the G0/G1 phase of the cell cycle, which inhibited the proliferation of BC cells (8). In addition, RSK4 negatively regulates the receptor tyrosine kinase signalling pathway and induces cellular senescence by regulating p21 (9,10). Multiple studies have shown that RSK4 plays an important role in the development and progression of cancer. Recent studies have shown that RSK4 is associated with sunitinib and cisplatin resistance in chemotherapy and mediates resistance to PI3K inhibitors in BC cells (11, 12). We speculate that RSK4 may regulate the sensitivity of BC to chemotherapy. There are still few studies on the relationship between RSK4 and chemotherapy in BC. This study aims to investigate the effect of RSK4 on the DOX resistance in BC by upregulating the expression of RSK4, and provide a reference for improving the chemotherapy effect of BC. Materials and Methods Cell culture and transfection Human BC cell lines MCF-7 were purchased from Shanghai Cell Biology Institute (Shanghai, China). DOX-resistant MCF-7 cells (MCF-7/DOX) were successfully established by exposing MCF-7 cells to gradually increased concentrations of DOX from 0.05 to 1 µg/ml. The cells were incubated in complete Dulbecco’s modified Eagle’s medium (DMEM; Gibco, BRL, USA) with 10% foetal bovine serum (FBS; BI, USA) at 37°C in a humidified 5% CO2 incubator. To maintain the DOX-resistant phenotype, 1 µg/ml DOX was added to the medium of MCF-7/DOX cells. The recombinant lentivirus vectors GV358 with green fluorescent protein (GFP) carrying LV-RSK4-overexpression (NM_014496.5), negative control vectors CON238 (NC) were synthesized by GeneChem Co., Ltd (Shanghai, China). The lentivirus was transfected into MCF-7/DOX cells according to the manufacturer’s instructions. The transfection efficiency was evaluated by reverse transcript polymerase chain reaction (RT-PCR) and western blot. Reverse transcript polymerase chain reaction Total RNAs were obtained from cultured cells with Trizol (Takara, Japan) and then reverse-transcribed into cDNA using the PrimeScript™ RT reagent Kit (TaKaRa, Shiga, Japan). Real-time PCR was conducted using SYBR Premix Ex Taq II under the following conditions: pre-denaturation at 95°C for 30 s, 40 amplification cycles at 95°C for 5 s, 60°C for 30 s and at 72°C for 10 min. β-Actin was used to normalize the relative mRNA level of RSK4. The primers were synthesized by Sangon Biomart (Shanghai, China): RSK4 forward: 5′-CAGCCAGTGCAAATGCTCATC-3′, reverse: 5′-GGAGCCAACACCAATATCCTC-3′ and β-Actin forward: 5′-CATGTACGTTGCTATCCAGGC-3′, reverse: 5′-CTCCTTAATGTCACGCACGAT-3′. The relative expression was analysed using 2-△△Ct method in triplicate. Western blot assay Cells were collected and lysed in RIPA Buffer (Solarbio, Beijing, China) with 1% phenylmethanesulfonyl fluoride. The protein concentration was measured by bicinchoninic acid (BCA) method. After mixing with Laemmli’s sample buffer, the proteins were isolated by 10% sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE) for 90 min and transferred to a polyvinylidene fluoride (PVDF) membrane. The membranes were incubated in 5% milk for 1 h, and then incubated with primary antibody RSK4 (1:1000, ab76117, Abcam), Bcl-2-associated X protein (Bax, 1:1,000, #5023, CST), B-cell lymphoma-2 (Bcl-2, 1:1,000, #4223, CST), cleaved-caspase3 (1:1,000, #9664, CST), anti-PI3K (1:1,000, #4249, CST), anti-AKT (1:500, #4691, CST), anti-p-PI3K (tyr458, 1:1,000, CST), anti-p-AKT (ser473, 1:500, #4060, CST), anti-BCRP(ATP-binding cassette superfamily G member 2, ABCG2, 1:1,000, # 42078 T, CST), anti-P-gp (ATP binding cassette subfamily B member 1, ABCB1, 1:500, #22336-1-AP, Proteintech), anti-MRP2 (ATP binding cassette subfamily C member 2, ABCC2, 1:500, #24893-1-AP, Proteintech) and anti-GAPDH (1:1,000, #2118, CST), respectively, at 4°C overnight. After that, the blots were washed with tris-buffered saline tween (TBST) and exposed to IgG secondary antibody (1:5,000, ab186696, Abcam) at room temperature for 1 h. The proteins were visualized by chemiluminescence detection reagents and results were analysed by Image J software. GAPDH was used to normalize the levels of protein. Cell viability assay For cell proliferation assay, each group of cells was digested and seeded in 96‐well plate (5 × 103 cells/well) and incubated at 37°C for 12, 24, 48 and 72 h, respectively. Afterward, 10 μl Cell Counting Kit-8 reagent (CCK-8; Dojindo, Tokyo, Japan) was added to cells incubated for 2 h. For cytotoxicity assessment, cells were seeded in 96-well plates and cultured for 24 h. Then, cells were treated with different doses of DOX (0.25, 0.5, 1, 2, 4, 8 and 16 µg/ml). Cells were cultured for 48 h, and then the number of viable cells was detected using a CCK-8. Finally, the optical density values were detected at 450 nm using a microplate reader. Half maximal inhibitory concentration (IC50) value was calculated using SPSS software. Resistance index = IC50 (MCF-7/DOX)/IC50 (MCF-7). Colony-formation assay Cells (500 cells/well) were inoculated into six-well plates and cultured at 37°C with 5% CO2 for 14 days. After that, terminate the cultivation and the plates were washed three times with phosphate-buffered saline (PBS). Cells were fixed with 4% paraformaldehyde for 15 min at room temperature and stained with 1% crystal violet for 30 min. After the crystal violet was gradually washed away, the plates were dried in the air. Cell colony number was observed under a microscope. Colony-formation rate was calculated according to the formula (cell colony number/number of inoculated cells) × 100%. Transwell assay Matrigel (BD Biosciences) was diluted with serum-free DMEM at 1:6. About 100 μl of diluted matrigel was added into the upper chamber of each Transwell (Corning, USA) and placed at 37°C for 2 h. About 200 μl of cells suspension (5 × 104/well) was seeded into the upper chamber and 600 μl DMEM (0.5 ml) containing 10% FBS was added into the lower chamber. Furthermore, cells were incubated at 37°C with 5% CO2 for 36 h. Afterwards, the non-invasive cells on the upper surface were removed with a cotton swab and the invasive cells on the lower surface of the membrane were fixed with 4% paraformaldehyde for 15 min and stained with 1% crystal violet for 30 min. Invasive cells were observed under an inverted microscope, and five random fields from each sample were photographed and counted. Scratch assays After 72 h transfection, three groups of cells (4 × 105/well) were plated into six-well plate and cultured at 37°C with 5% CO2 until cells confluence reached ∼90%. About 100 μl micropipette tip was used to vertically scratch the plates and the suspension cells were washed away with PBS. The cells were then further cultured for 24 h with serum-free medium. Cells were photographed at 0 and 24 h after scratching under a microscope, and the scratched area was measured by Image J software. Flow cytometry assay Annexin V-APC/7-AAD apoptosis kit (BD, USA) was applied to detect cell apoptosis. The cells were digestion with trypsin without ethylenediaminetetraacetic acid and washed twice with PBS. After gently resuspending in 100 μl of 1× binding buffer, cells were incubated with 5 μl Annexin V-APC and 5 μl 7-AAD for 15 min at room temperature in the dark. Then, 400 μl of 1× binding buffer was added into the each sample, and cell apoptosis was performed by a flow cytometer (BD). Cells that showed both APC Annexin V and 7-AAD negative were healthy cells. Cells in early apoptosis were APC Annexin V +/7-AAD and cells in late apoptosis or already death were both APC Annexin V and 7-AAD positive. Nude mice experiments Animal experiments were carried out in accordance with the approved animal protocols and guidelines established by Ethical Committee for animal Management and Use of the Affiliated Tumor Hospital of Guangxi Medical University. A total of 16 female BALB/C nude mice (4–5 weeks, 15–20 g) were purchased from Guangxi Medical University Experimental Animal Center (Guangxi, China), which were fed in SPF animal room with constant temperature and humidity. Food, water and padding were replaced regularly and aseptically. The nude mice were randomly grouped into four groups (with four mice in each group): MCF-7/DOX, MCF-7/DOX/LV-RSK4, MCF-7/DOX+DOX and MCF-7/DOX/LV-RSK4+DOX. And then, ∼0.2 ml suspension (5 × 106 MCF-7/DOX or transfected MCF-7/DOX cells) was subcutaneously injected to the forelimb axils of each nude mouse, respectively. Subsequently, mice in the MCF-7/DOX+DOX and MCF-7/DOX/LV-RSK4+DOX groups were intraperitoneally injected with DOX 10 (mg/kg) per week, while the mice in other two groups were treated with the equal amount of saline. Tumour dimensions were observed and measured every third day. After 30 days, the mice were sacrificed by cervical dislocation, and the tumour samples were collected. Tumour volume (mm3) was calculated according to the formula (length × width2)/2. Survival analysis Kaplan–Meier Plotter (http://kmplot.com/analysis/) was used to analyse the effect of RSK4 mRNA level on the relapse-free survival (RFS) in BC patients. The clinical data such as lymph node status of BC patients were also obtained in this database. We only chose the JetSet best probe of RSK4 to obtain Kaplan–Meier survival plots, and the significance difference of curves was compared by log-rank test. In addition, RSK4 expression and therapy response were compared using ROC Plotter (http://www.rocplot.org/). In ROC Plotter database, all patients were divided into two cohorts, including patients with no residual histological evidence of the tumour remains after chemotherapy (responder) and all other patients with residual tumour tissue (non-responder). Statistical analysis All analyses were analysed by SPSS 22.0 software (SPSS Inc.). Measurement data were documented as mean ± standard deviation. One-way analysis of variance (ANOVA) and Dunnett’s test were used for group comparison. P < 0.05 was considered statistically significant. Results The effect of RSK4 expression on the prognosis of BC At the beginning of the study, we performed a bioinformatics analysis to examine the effect of RSK4 levels on the prognosis and therapy of BC patients using Kaplan–Meier Plotter and ROC Plotter databases (13, 14). It was found that RSK4 high mRNA expression was significantly associated with longer RFS (HR = 0.56, P = 3.6e−13) (Fig. 1A). In particular, high level of RSK4 was significantly associated with better RFS in BC patients with lymph node-positive tumours (HR = 0.68, P = 0.0024), but not in node-negative tumours (HR = 0.78, P = 0.2) (Fig. 1B–C). In BC patients treated with anthracyclines, the chemotherapy effect of the RSK4 high-expression group was better than that of the RSK4 low-expression group (P = 2.8e−02, AUC = 0.589) (Fig. 1D), suggesting a potential role of RSK4 in contribution to chemosensitivity in BC. Fig. 1. Open in new tabDownload slide RSK4 was associated with prognosis in patients with BC and downregulated in DOX-resistant MCF-7 cells. (A) Kaplan–Meier curves showed that BC patients with high level of RSK4 had better RFS. (B, C) The expression of RSK4 was positively correlated with RFS in BC patients with lymph node positive, but not in node-negative tumours. (D) ROC plotter showed that in BC patients treated with anthracyclines, RSK4 expression in the responder group was higher than that in the non-responder group (Responder: patients with no residual histological evidence of the tumour remains after chemotherapy. Non-responder: patients with residual tumour tissue). (E) The mRNA and protein expression of RSK4 in MCF-7 and MCF-7/DOX cells. **P < 0.01 versus the MCF-7 group. Fig. 1. Open in new tabDownload slide RSK4 was associated with prognosis in patients with BC and downregulated in DOX-resistant MCF-7 cells. (A) Kaplan–Meier curves showed that BC patients with high level of RSK4 had better RFS. (B, C) The expression of RSK4 was positively correlated with RFS in BC patients with lymph node positive, but not in node-negative tumours. (D) ROC plotter showed that in BC patients treated with anthracyclines, RSK4 expression in the responder group was higher than that in the non-responder group (Responder: patients with no residual histological evidence of the tumour remains after chemotherapy. Non-responder: patients with residual tumour tissue). (E) The mRNA and protein expression of RSK4 in MCF-7 and MCF-7/DOX cells. **P < 0.01 versus the MCF-7 group. Expression of RSK4 in MCF-7 and MCF-7/DOX cell lines To analyse the involvement of RSK4 during the acquisition of DOX resistance, RT-PCR and western blot were performed to detect the expression of RSK4 in DOX-sensitive and DOX-resistant cell lines. We found that RSK4 was significantly decreased in MCF-7/DOX cells, compared with MCF-7 cells (P < 0.01, Fig. 1E), indicating that RSK4 may have certain effect on the chemical resistance of MCF-7 cells to DOX. Overexpressed RSK4 reduced DOX resistance and proliferation of BC cells For further research, MCF-7/DOX cells were transfected with lentivirus particles (LV-RSK4, NC-vector, respectively). The expression of GFP was observed under a fluorescence microscope (Fig. 2A). RT-PCR and western blot showed that the mRNA and protein levels of RSK4 were significantly upregulated in MCF-7/DOX/RSK4 group, compared with the MCF-7/DOX/NC and blank control groups (P < 0.001, Fig. 2B). These results indicated a successful transfection. To investigate how RSK4 overexpression is involved in DOX-resistance in BC, we detected the toxicity of DOX and proliferation ability in each group by CCK-8 assay and colony-formation assay. The results showed that the DOX-sensitive MCF-7 cells and MCF-7/DOX/RSK4 cells suppressed cell viability under different DOX concentrations (P < 0.001, Fig. 2D). The upregulated RSK4 decreased the IC50 value in MCF-7/DOX cells (P < 0.001, Table I). We also detected reduced cell viability in MCF-7 cells and MCF-7/DOX/RSK4 cells within 96 h (P < 0.01, Fig. 2C). Colony-formation results indicated that cell colony formation was significantly reduced in the MCF-7/DOX/RSK4 group (P < 0.001, Fig. 2E). Those results indicated that RSK4 might serve a key role in DOX resistance. Fig. 2. Open in new tabDownload slide Overexpression of RSK4 decreased drug resistance and proliferation of MCF-7/DOX cells. (A) GFP in MCF-7/DOX cells after transfection was observed and imaged under a fluorescence microscope (images magnification ×40). (B) The mRNA and protein levels of RSK4 in MCF-7/DOX cells in each group after transfection. (C) The OD values of transfected cells were measured at 0, 24, 48, 72 and 96 h. (D) The drug resistance of transfected cells was determined by CCK-8 under the treatment of different DOX concentrations and the IC50 values of different groups were tested. (E) The colony formation of transfected MCF-7/DOX cells was performed to assess cell proliferation. *P < 0.05, ***P < 0.001 versus the MCF-7/DOX and MCF-7/DOX/NC groups. #P < 0.05, ###P < 0.001 versus the MCF-7/DOX group. Fig. 2. Open in new tabDownload slide Overexpression of RSK4 decreased drug resistance and proliferation of MCF-7/DOX cells. (A) GFP in MCF-7/DOX cells after transfection was observed and imaged under a fluorescence microscope (images magnification ×40). (B) The mRNA and protein levels of RSK4 in MCF-7/DOX cells in each group after transfection. (C) The OD values of transfected cells were measured at 0, 24, 48, 72 and 96 h. (D) The drug resistance of transfected cells was determined by CCK-8 under the treatment of different DOX concentrations and the IC50 values of different groups were tested. (E) The colony formation of transfected MCF-7/DOX cells was performed to assess cell proliferation. *P < 0.05, ***P < 0.001 versus the MCF-7/DOX and MCF-7/DOX/NC groups. #P < 0.05, ###P < 0.001 versus the MCF-7/DOX group. Table I. IC50 levels of DOX are reduced in MCF-7/DOX/RSK4 group Group . IC50 (µg/ml) . Resistance index (RI) . MCF-7 1.07 ± 0.8 – MCF-7/DOX 8.75 ± 0.72 8.18 MCF-7/DOX/NC 8.25 ± 0.7 7.71 MCF-7/DOX/RSK4 2.72 ± 0.53 2.54 Group . IC50 (µg/ml) . Resistance index (RI) . MCF-7 1.07 ± 0.8 – MCF-7/DOX 8.75 ± 0.72 8.18 MCF-7/DOX/NC 8.25 ± 0.7 7.71 MCF-7/DOX/RSK4 2.72 ± 0.53 2.54 Open in new tab Table I. IC50 levels of DOX are reduced in MCF-7/DOX/RSK4 group Group . IC50 (µg/ml) . Resistance index (RI) . MCF-7 1.07 ± 0.8 – MCF-7/DOX 8.75 ± 0.72 8.18 MCF-7/DOX/NC 8.25 ± 0.7 7.71 MCF-7/DOX/RSK4 2.72 ± 0.53 2.54 Group . IC50 (µg/ml) . Resistance index (RI) . MCF-7 1.07 ± 0.8 – MCF-7/DOX 8.75 ± 0.72 8.18 MCF-7/DOX/NC 8.25 ± 0.7 7.71 MCF-7/DOX/RSK4 2.72 ± 0.53 2.54 Open in new tab Overexpressed RSK4 inhibited migration, invasion abilities and promoted apoptosis of drug-resistance BC cells In order to understand the influence of RSK4 on migration and invasion abilities of drug-resistant BC cells, we conducted Scratch test, transwell assay and flow cytometry (FCM). Results showed that cell migration and invasion abilities of MCF-7/DOX/RSK4 were markedly inhibited compared with the other two groups (P < 0.05, Fig. 2C;P < 0.05, Fig. 3A–B). Additionally, FCM results indicated that the cell apoptosis rate was increased by RSK4 overexpression (P < 0.01, Fig. 3C). Fig. 3. Open in new tabDownload slide Overexpression of RSK4 inhibited migration, invasion and promoted apoptosis of MCF-7/DOX cells. (A, B) The migration and invasion ability of MCF-7/DOX cells after transfection were analysed by scratch assay (×40) and transwell assay (×200). (C) The apoptosis of MCF-7/DOX cells after transfection were analysed by FCM. *P < 0.05, **P < 0.01, ***P < 0.001 versus the MCF-7/DOX and MCF-7/DOX/NC groups. Fig. 3. Open in new tabDownload slide Overexpression of RSK4 inhibited migration, invasion and promoted apoptosis of MCF-7/DOX cells. (A, B) The migration and invasion ability of MCF-7/DOX cells after transfection were analysed by scratch assay (×40) and transwell assay (×200). (C) The apoptosis of MCF-7/DOX cells after transfection were analysed by FCM. *P < 0.05, **P < 0.01, ***P < 0.001 versus the MCF-7/DOX and MCF-7/DOX/NC groups. Overexpression of RSK4 promoted cell apoptosis by regulating apoptosis-associated proteins To further investigate the effect of RSK4 on cell apoptosis, the expression of apoptosis-associated proteins (Bax, cleaved-caspase3 and Bcl-2) were determined via western blot analysis. As demonstrated in Fig. 4A, in contrast to MCF-7/DOX group, the levels of cleaved-caspase3 and Bax in the MCF-7 and MCF-7/DOX/RSK4 groups were significantly increased, while the Bcl-2 levels were significantly decreased (P < 0.05). There was no significant difference in the expression of apoptosis-related proteins between the MCF-7/DOX and MCF-7/DOX/NC groups (P > 0.05). Altogether, these results showed that RSK4 overexpression may promote cells apoptosis by regulating the expression of apoptosis-related proteins. Fig. 4. Open in new tabDownload slide Overexpression of RSK4 decreased drug resistance of MCF-7/DOX by regulating apoptosis-related proteins, multidrug-resistant proteins and PI3K/AKT signalling pathway. The expressions of cell apoptosis-related proteins (cleaved-caspase3, Bax and Bcl-2) (A), multidrug resistant proteins (BCRP, p-gp and MRP2) (B), p-PI3K and p-AKT (C) in each group were detected by western blot. *P < 0.05, **P < 0.01 versus the MCF-7/DOX and MCF-7/DOX/NC groups. #P < 0.05, ##P < 0.01 versus the MCF-7/DOX group. Fig. 4. Open in new tabDownload slide Overexpression of RSK4 decreased drug resistance of MCF-7/DOX by regulating apoptosis-related proteins, multidrug-resistant proteins and PI3K/AKT signalling pathway. The expressions of cell apoptosis-related proteins (cleaved-caspase3, Bax and Bcl-2) (A), multidrug resistant proteins (BCRP, p-gp and MRP2) (B), p-PI3K and p-AKT (C) in each group were detected by western blot. *P < 0.05, **P < 0.01 versus the MCF-7/DOX and MCF-7/DOX/NC groups. #P < 0.05, ##P < 0.01 versus the MCF-7/DOX group. Overexpression of RSK4 affected drug resistance via inhibiting drug-resistant proteins and PI3K/AKT signalling pathway The PI3K/AKT pathway plays a significant role in cancer progression and cancer drug resistance. To elucidate the mechanism by which RSK4 regulates drug resistance, we examined the expression of classical multidrug-resistant proteins and core proteins in the PI3K/AKT signalling pathway by western blot. The results showed that the levels of multidrug-resistant protein (P-gp, MRP2 and BCRP), p-PI3K/PI3K ratio and p-AKT/AKT ratio were effectively decreased in MCF-7 and MCF-7/DOX/RSK4 cells compared with MCF-7/DOX cells (P < 0.05, Fig. 4B–C). These results indicate that the overexpression of RSK4 might alter the resistance of MCF-7 cells to DOX by inhibiting multidrug-resistant proteins and PI3K/AKT pathway. Overexpression of RSK4 inhibited drug-resistance and growth of nude mice xenografts To further clarify the effect of RSK4 on the growth of BC in vivo, drug resistance BC cells were transplanted into BALB/c female mice. The tumour growth curve showed that the mean volume of the tumours in the MCF-7/DOX/RSK4 group was significantly smaller than that in the MCF-7/DOX group (P < 0.05, Fig. 5A–B). In addition, the tumour volume of DOX-treated mice was smaller than that of the untreated group, while the MCF-7/DOX/RSK4 + DOX group had the smallest tumour volume (P < 0.05). These results indicated that RSK4 overexpression reversed DOX resistance and inhibited tumour growth. Fig. 5. Open in new tabDownload slide Overexpressed RSK4 inhibited proliferation of MCF-7/DOX cells in vivo. (A) Tumour size in the nude mice in different groups. (B) The tumour volume in nude mice of each group. *P < 0.05 versus the MCF-7/DOX group; #P < 0.05 versus the MCF-7/DOX+DOX group. Fig. 5. Open in new tabDownload slide Overexpressed RSK4 inhibited proliferation of MCF-7/DOX cells in vivo. (A) Tumour size in the nude mice in different groups. (B) The tumour volume in nude mice of each group. *P < 0.05 versus the MCF-7/DOX group; #P < 0.05 versus the MCF-7/DOX+DOX group. Discussion So far, anticancer drug resistance is the leading cause of failure in most cancer treatments (15). Drug resistance is a complex process involving multiple mechanisms, such as metabolic pathways, drug efflux, drug target alternation, tumour heterogeneity, tumour microenvironment, aberrant signalling, apoptotic pathways, DNA repair pathways and so on (16, 17). Therefore, finding ways to reverse the drug resistance of tumour cells is of great significance for improving the survival and prognosis of cancer patients. RSK4 is downregulated in many human tumours, and its expression levels are associated with clinical pathologic classifications and survival in a variety of cancers, such as breast carcinoma, colorectal cancer, ovarian cancer, acute myeloid leukaemia and renal cell carcinoma (18–20). Arechavleta-Velasco et al. (21) found that RSK4 expression was increased after ovarian cancer SKOV3 and TOV-112D cells were treated with cisplatin and vorinostat. Bender et al. (12) found that the expression of RSK4 in renal cancer and melanoma cell lines was significantly related to the resistance of sunitinib. However, whether RSK4 is involved in drug resistance in BC and the definite mechanism of RSK4 in chemotherapy resistance have not been reported. We analysed the prognosis of BC patients and found that the expression of RSK4 was significant correlated with the RFS of BC patients, especially those with positive lymph nodes, and it was of great significance in the treatment of anthracyclines. Subsequently, we found that RSK4 expression in DOX-resistant BC cells was lower than that in DOX-sensitive cells. Those results indicated that RSK4, as a tumour suppressor gene, improved the prognosis of BC patients and may be involved in regulating DOX sensitivity, which was consistent with previous research reports. To confirm the effect of RSK4 on DOX resistance, we upregulated the level of RSK4 in the DOX-resistant cell line. Our data suggested that upregulation of RSK4 suppressed cell proliferation, invasion and migration, as well as promoted the cell apoptosis and the sensitivity of DOX to drug-resistant cells. What’s more, RSK4 overexpression was also found to inhibit tumour growth and enhance the sensitivity of BC to DOX in a nude mouse xenograft tumour model, indicating that the antitumour activity of DOX increased in vivo after upregulating RSK4. Studies shown that RSK4 could disrupt mesoderm formation induced by the RAS-ERK pathway and proposed as an inhibitor of growth factor signalling (22). RSK4 not only inhibits cell proliferation by arresting cells in G0/G1 phase, but also regulates claudin-2 to alter cell adhesion and inhibit tumour invasion and metastasis (23, 24). These findings indicated that RSK4 inhibited cell transformation and excessive proliferation, and increased the chemosensitivity of MCF-7/DOX cells to a certain extent. The molecular mechanisms of chemotherapeutic resistance in tumours are intricate, and the abnormal expression of efflux proteins, such as transporter proteins P-gp, BCRP and MRP2, is considered to be the main cause of drug resistance (25). As drug discharge pumps, multidrug-resistant proteins are overexpressed in drug-resistant cells, which can reduce the concentration of intracellular drugs and protect cells from toxic substances (26–28). Furthermore, the abnormal expression of apoptosis-related genes is also an important cause of chemotherapy resistance. Since most chemotherapeutic therapies induce cell death by promoting apoptosis, the inhibition of the apoptotic pathway provides favourable conditions for the survival of tumour cells under high pressure, so it is believed that this change provides the possibility of intrinsic drug resistance for tumour cells (29). In our study, the overexpression of RSK4 reduced the levels of multidrug-resistant proteins, promoted the expression of apoptosis-related genes (Bax/Bcl-2, cleaved caspase3) in drug-resistant cell lines. Therefore, the mechanism of RSK4 reduced DOX resistance may be dependent on the regulation of multidrug-resistant proteins and apoptosis-related factors. Accumulating evidence showed that PI3K/AKT activation leaded to the increased transcription of downstream target genes such as Bax, CDK4, cyclin D1, ABCB1 and ABCG2, which were involved in the regulation of cell cycle, proliferation, apoptosis and other cellular processes, and its disrupted expression was frequently found in human cancers (30, 31). It was proved that the PI3K/AKT pathway is closely related to chemotherapy resistance. For instance, it is reported that epidermal growth factor receptor (EGFR) in BC can produce endocrine resistance by activating downstream PI3K/AKT and MAPK signalling pathways (32). Prostate cancer and gastric cancer show resistance to paclitaxel due to upregulation of the PI3K/AKT pathway and MAPK pathway (33, 34). In addition, in colorectal cancer, activation of the PI3K/AKT pathway triggers chemical resistance to 5-FU (35). Recent studies have confirmed that the inhibition of PI3K/AKT restored sensitivity to DOX by repressing BCRP expression (36). RSK4 acts as an important endogenous inhibitor of the MAPK pathway and inhibit the phosphorylation of AKT, which promotes cell apoptosis and reverses drug-resistance (10, 37). In our study, the phosphorylation of PI3K and AKT in the RSK4 overexpression group was significantly inhibited, suggesting that RSK4 may reduce DOX resistance in BC cells by inhibiting the PI3K/AKT pathway. In conclusion, the results of this study indicate that RSK4 overexpression inhibits cell proliferation, migration, invasion and promotes apoptosis in MCF-7/DOX cell lines. Furthermore, RSK4 overexpression reduces DOX resistance in BC cells by inhibiting PI3K/AKT signalling pathway, suggesting that RSK4 may be a therapeutic target for the treatment of BC. Finally, the exact role of RSK4 in BC remains to be further studied since this research only proposed a possible mechanism. 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Med . 40 , 883 – 890 Google Scholar Crossref Search ADS PubMed WorldCat Abbreviations Abbreviations ABCG2 ATP-binding cassette superfamily G member 2 BC breast cancer DMEM Dulbecco’s modified Eagle’s medium DOX doxorubicin FBS foetal bovine serum FCM flow cytometry GFP green fluorescent protein PBS phosphate-buffered saline RFS relapse-free survival RI resistance index RSK4 ribosomal S6 protein kinase 4 RTK receptor tyrosine kinase RT-PCR reverse transcript polymerase chain reaction. Author notes " Yan Mei and Xiaoming Liao contributed equally to this work. © The Author(s) 2020. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Overexpression of RSK4 reverses doxorubicin resistance in human breast cancer cells via PI3K/AKT signalling pathway
JO - The Journal of Biochemistry
DO - 10.1093/jb/mvaa009
DA - 2020-06-01
UR - https://www.deepdyve.com/lp/oxford-university-press/overexpression-of-rsk4-reverses-doxorubicin-resistance-in-human-breast-lWF4rcGZDF
SP - 603
VL - 167
IS - 6
DP - DeepDyve
ER -