The transcription factor FOXO4 is down-regulated and inhibits tumor proliferation and metastasis in gastric cancer
- Linna Su†1,
- Xiangqiang Liu†1,
- Na Chai†2,
- Lifen Lv1,
- Rui Wang1,
- Xiaosa Li1,
- Yongzhan Nie1,
- Yongquan Shi1Email author and
- Daiming Fan1Email author
© Su et al.; licensee BioMed Central Ltd. 2014
Received: 14 January 2014
Accepted: 20 May 2014
Published: 28 May 2014
FOXO4, a member of the FOXO family of transcription factors, is currently the focus of intense study. Its role and function in gastric cancer have not been fully elucidated. The present study was aimed to investigate the expression profile of FOXO4 in gastric cancer and the effect of FOXO4 on cancer cell growth and metastasis.
Immunohistochemistry, Western blotting and qRT-PCR were performed to detect the FOXO4 expression in gastric cancer cells and tissues. Cell biological assays, subcutaneous tumorigenicity and tail vein metastatic assay in combination with lentivirus construction were performed to detect the impact of FOXO4 to gastric cancer in proliferation and metastasis in vitro and in vivo. Confocal and qRT-PCR were performed to explore the mechanisms.
We found that the expression of FOXO4 was decreased significantly in most gastric cancer tissues and in various human gastric cancer cell lines. Up-regulating FOXO4 inhibited the growth and metastasis of gastric cancer cell lines in vitro and led to dramatic attenuation of tumor growth, and liver and lung metastasis in vivo, whereas down-regulating FOXO4 with specific siRNAs promoted the growth and metastasis of gastric cancer cell lines. Furthermore, we found that up-regulating FOXO4 could induce significant G1 arrest and S phase reduction and down-regulation of the expression of vimentin.
Our data suggest that loss of FOXO4 expression contributes to gastric cancer growth and metastasis, and it may serve as a potential therapeutic target for gastric cancer.
KeywordsFOXO4 Gastric cancer Proliferation Metastasis EMT
Although the incidence of gastric cancer(GC) is declining, it remains the fourth most common cancer and second leading cause of cancer-related death worldwide . The key molecules involved in cell proliferation and metastasis in GC progression may aid in clinical diagnosing or predicting the progression of this disease.
Tumor growth and metastasis depend on various factors, including transcription factors [2–5]. The FOXO transcription factors family comprises four highly related members: FOXO1, FOXO3, FOXO4, and FOXO6 [6–8]. In recent years, FOXO have been shown to play crucial roles in a plethora of cellular processes, including proliferation, apoptosis, differentiation, stress resistance, and metabolic responses , and may therefore be promising targets for new medications in the field of oncology [10, 11].
Our previous results demonstrated that the FOXO4 mRNA expression level was dramatically down-regulated in lymph node-positive colorectal carcinoma tissues compared to lymph node-negative tissues, suggested it may function as a negative regulator of the metastasis of colorectal carcinoma . However, the expression and function of FOXO4 in gastric cancer were not known yet. The aim of our work has been to investigate the possible role of FOXO4 in gastric cancer carcinogenesis. Here, we report that FOXO4 repress cell proliferation and metastasis in gastric cancer by the regulation G1 cell-cycle arrest and vimentin.
For tissue specimens, all patients provided informed consent to use excess pathological specimens for research purposes. The protocols used in this study were approved by the hospital’s Protection of Human Subjects Committee. The use of human tissues was approved by the institutional review board of the Fourth Military Medical University and conformed to the Helsinki Declaration, as well as local legislation. Patients providing samples for the study signed informed consent forms.
Immunohistochemical staining was performed using the the avidin-biotin complex immunoperoxidase method. The primary antibody against FOXO4 (1:100, ab63254, Abcam) diluted in PBS containing 1% (wt/vol) bovine serum albumin (BSA). Negative controls were performed by replacing the primary antibody with pre-immune mouse serum. Images were obtained under a light microscope (Olympus BX51, Olympus, Japan) equipped with a DP70 digital camera. The observer was blinded to the identity of the samples when scoring immunoreactivity.
Evaluation of staining
For evaluation of the cell staining, the sections were examined by two independent pathologists without prior knowledge of the clinic-pathological status of the specimens. Cells that were stained brown were considered to be positive. The expression of FOXO4 was evaluated according to the ratio of positive cells per specimen (R) and staining intensity (I). The ratio of positive cells per specimen was scored as follows: 0 for staining of < 1%, 1 for staining of 2% to 25%, 2 for staining of 26% to 50%, 3 for staining of 51% to 75%, and 4 for staining of > 75% of the cells examined. The intensity was graded as follows: 0, no signal; 1, weak staining; 2, moderate staining; and 3, strong staining. A total score (R × I) of 0 to 12 was finally calculated and graded as negative (−score: 0–2) or positive (+, 3–12).
Tissue arrays were purchased from the Aomei company(Aomei C0124H,AM01C09,Aomei Biotechnology Co. Ltd., Xi’an, China) (Additional file 1: Table S1 and Additional file 2: Table S2). For the western blot analysis, GC tissues and adjacent nontumorous tissues were obtained from eight patients who had undergone surgery at the Department of General Surgery in our hospital. All cases of GC and normal gastric mucosa were clinically and pathologically proven. The protocols used in the studies were approved by the Hospital’s Protection of Human Subjects Committee. Patients who contributed fresh surgical tissue for the study had signed informed consent forms.
RNA extraction and real-time PCR
Total RNA from the cells was extracted using Trizol (Invitrogen, Carlsbad, CA), and cDNA was synthesized using the Prime Script RT reagent kit (TaKaRa Biotechnology, Dalian, China) according to the manufacturer’s recommendations. A Light Cycler Fast Start DNA Master SYBR Green I System (Roche, Basel, Switzerland) was used for the real-time PCR. GAPDH mRNA was used as the internal control, and the reaction mix without the template DNA was used as the negative control. All of the samples were measured independently three times. The primer sequences were as follows: GAPDH: (forward) 5′-TGGTGAAGACGCCAGTGGA-3′ and (reverse)5′-GCACCGTCAAGGCTGAGAAC-3′; FOXO4: (forward) 5′-CTTTCTGAAGACTGGCAGGAATGTG-3′ and (reverse) 5′-GATCTAGGTCTATGATCGCGGCAG-3′; E-cadherin: (forward) 5′-GAGTGCCAACTGGACCATTCAGTA-3′and (reverse) 5′- AGTCACCCACCTCTAAGGCCATC-3′; and Vimentin: (forward) 5′-CAGGCAAAGCAGGAGTCCAC -3′and (reverse) 5′-GCAGCTTCAACGGCAAAGTTC -3′. All real-time PCR reactions were performed in triplicate.
Oligonucleotide construction and lentivirus production
Three pairs of siRNA oligonucleotides targeting FOXO4 were synthesized by GenePharma Co., Ltd. The GAPDH sequences were used as a positive control. An unrelated sequence was used as a negative control (provided by GenePharma). The sequences were as follows: FOXO4 siRNA oligo-1: 5′-CGCGAUCAUAGACCUAGAUTTAUCUAGGUCUAUGAUCGCGTT-3′ (sense); FOXO4 siRNA oligo-2: 5′-CAGCUUCAGUCAGCAGUUATTUAACUGCUGACUGAAGCUGTT-3′ (sense); FOXO4 siRNA oligo-3: 5′-GUGACAUGGAUAACAUCAUTTAUGAUGUUAUCCAUGUCACTT-3′ (sense); GAPDH siRNA oligo (positive control): 5′-GUAUGACAACAGCCUCAAGTT-3′ (sense); and negative control: 5′-UUCUCCGAACGUGUCACGUTT-3′ (sense).
According to the manufacturers’ instructions, FOXO4 siRNA oligos were transfected into cells using the siRNA-Mate™ reagent (GenePharma Ltd., Shanghai, China). After cultured for 2 to 3 days, total RNA and protein were extracted. For stable transfection, a lentiviral overexpression vector (Lenti-FOXO4) was constructed (Shanghai GeneChem Co., Ltd., Shanghai, China). Using a GV166-puro Vector (GeneChem Co., Ltd., Shanghai, China), a lentiviral vector that expressed GFP alone (LV-control) was used as a negative control (NC).
Equal amounts of proteins were separated using sodium dodecyl sulfate–polyacrylamide gel(SDS-PAGE) electrophoresis and transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA). FOXO4 rabbit polyclonal antibody (Abcam, 1:500), CyclinD1 rabbit polyclonal antibody (ImmunoWay, 1:1000), β-actin mouse monoclonal antibody (Sigma,1:2,000), E-cadherin and Vimentin rabbit polyclonal antibody (Santa Cruz, CA, 1:1000) antibodies were used for the western blot experiments.
Cell proliferation assay
The 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay was performed to evaluate the speed of cell proliferation,and was performed according to standard procedures. Each cell line was detected in triplicate.
Migration and invasion assay
Transwell migration assays were performed in modified Boyden chambers (Transwell; Corning Inc. Lowell, MA, USA) at a density of 5 × 103 cells per well. After 24 h of incubation at 37°C, the cells on the lower surface of the wells were fixed with 4% paraformaldehyde, stained with 1% crystal violet, and counted.
High-content screening assay
Cell motility was surveyed using a Cellomics Array Scan VTI 1700 plus (Thermo Scientific, USA). In brief, cells in the log phase were harvested and plated into 96-well plates (5 × 103 cells/well). After overnight culture at 37°C for adhesion, the culture medium was replaced with serum-free RPMI1640 medium, and the culture was continued for an additional 24 h. Then, cells were washed twice with ice-cold PBS and stained with Hoechst 33342 for 15 min in an incubator. Subsequently, the cells were again washed twice with ice-cold PBS and exposed to different treatments. Cell motility was detected using the Cellomics Array Scan VTI 1700 plus (Thermo Scientific) according to the manufacturer’s protocol (each group included five repeated wells).
For confocal microscopy experiments, cells were grown on Lab-Tek 24-well chamber slides (Thermo Fisher Scientific, USA). After overnight culturing, the cells were fixed, washed, and permeabilized with 0.3% Triton X- 100 in PBS for 10 min. Then,the cells were incubated with primary antibodies against E-cadherin and vimentin (dilution 1:300, Abcam) overnight at 4°C. The cells were also incubated with Cy3-conjugated anti-rabbit IgG (dilution 1:200 (Jackson Immuno Research, West Grove, PA, USA) for 1 h at room temperature in the dark. The cell nucleus was counterstained using DAPI for 5 min. Fluorescence was monitored and photographed with a confocal microscope (Thermo Fisher Scientific, USA).
For animal research, nude mice 4 to 6 weeks of age were purchased from the Animal Center of the Chinese Academy of Science (Shanghai, China) and maintained in laminar flow cabinets under specific pathogen-free conditions. All procedures for animal experimentation were performed in accordance with the Institutional Animal Care and Use Committee guidelines of the Experiment Animal Center of the Fourth Military Medical University.
Tumorigenicity in nude mice
Logarithmically growing cells were harvested using trypsin and washed twice with PBS. Then, 2 × 106 cells in 0.2 ml were injected subcutaneously into the right upper back region of the mice. Four weeks after inoculation, tumor-bearing mice were sacrificed, and the size of the tumor was determined by caliper measurement of the subcutaneous tumor mass. Each experimental group contained 6 mice. Two independent experiments were performed, and they yielded similar results.
Tail vein metastatic assay
Approximately 2 × 106 cells were suspended in 0.2 ml of sterile PBS and injected into the tail veins of 10 mice. The mice were then monitored for tumor volume and overall health, and their lungs and livers were regularly observed using imaging microscopy.
All statistical analyses were performed using SPSS 17.0 statistical software (SPSS, Inc., Chicago, Illinois). Variables with a P value less than 0.05 were considered to be statistically significant. χ 2 tests were used to evaluate the significance of differences in FOXO4 expression frequency between GC tissues and adjacent nontumorous gastric tissues. The t-test (a one-way ANOVA test) was performed to evaluate the significance of the difference between cell proliferation, plate clones, and migration assays. Overall survival curves were plotted using the Kaplan-Meier method and were evaluated for statistical significance using a log-rank test(the Mann–Whitney U test and Kruskal-Wallis H test were adopted for other data).
Expression of FOXO4 is down-regulated in GC tissues and cell lines
FOXO4 inhibits GC proliferation in vitro and induces cell cycle arrest in the G0/G1 phase
FOXO4 inhibits the migration and invasion of GC cells in vitro
FOXO4 up-regulation inhibits tumorigenesis and metastasis of GC cells in vivo
Molecular mechanisms of FOXO4 in the metastasis of GC
These data indicate that FOXO4 may partially influence GC cell metastasis by regulating EMT process, and additional molecular mechanisms will be studied in future work.
The forkhead box class O (FOXO) family of transcription factors is evolutionarily conserved and characterized by the so-called forkhead box DNA-binding domain. In mammals, the FOXO gene family consists of four members: FOXO1, FOXO3A, FOXO4, and FOXO6. Numerous studies have shown that FOXO proteins play an important role in a wide range of normal biological processes, including cellular proliferation, cell cycle arrest, stress response, and apoptosis [10, 13, 14], as well as in diseases such as cancer and diabetes mellitus . However, there is little study reported about the role of FOXO4 plays in GC.
In the present study, we found the FOXO4 expression in non-tumorous tissues was consistently stronger than that of the GC samples, and GCs showed a lower expression level of FOXO4 in metastatic lesions compared to the corresponding primary tumor samples. The FOXO4 mRNA and protein expression levels were both reduced in various types of GC cell lines compared to the normal gastric mucosal epithelial cell line, suggesting that FOXO4 might serve as a negative regulator for GC. Additionally, elevated expression of FOXO4 expression inhibited tumor cell growth, invasion, and metastasis in vitro and in vivo, indicating that FOXO4 may play a role in GC progression and metastasis.
The mechanisms responsible for the impact of FOXO4 alterations on GC development and progression remain unclear. Several recent studies have indicated that FOXO regulates many aspects of cancer biology. For example, FOXO is normally restrained by the PI3K/Akt signaling pathway, which prevents FOXO translocation into the nucleus, and FOXO regulate transcriptional responses independently of direct DNA binding via association with a variety of unrelated transcription factors . Our findings showed that FOXO4 induced significant G1 arrest and S phase reduction in GC cells, which indicated that FOXO4 inhibited GC proliferation may at least partly by the result of G1 cell-cycle arrest.
One critical step in the metastatic cascade is the process of epithelial to mesenchymal transition (EMT) [17, 18]. During the EMT process, the expression of E-cadherin was often down-regulated, while which of vimentin often shows up-regulated . FOXO4 may regulate EMT in gastric cancer. To test this hypothesis, we assessed the expressions of E-cadherin and vimentin in the cell models above. Although no obvious alteration was observed for E-cadherin, a dramatic decrease of vimentin expression was displayed in FOXO4 overexpression cells compared to the control cells, as indicated by immunofluorescent assay and qRT-PCR. These studies strongly suggest that FOXO4 might inhibit gastric cancer metastasis by regulating EMT.
In conclusion, our study demonstrates a critical function of FOXO4 in the inhibition of GC proliferation and metastasis via the regulation of G1 cell-cycle arrest and EMT, suggests it may serve as a potential therapeutic target for gastric cancer.
Forkhead box O4
Bovine serum albumin
Real-time quantitative PCR
Phosphate buffered saline
This work was supported by National Natural Science Foundation of China (grant number 81172062, 81270445). We thank Prof. Zengshan Li (Department of Pathology at Xijing Hospital)for his help in pathological analysis. We also thank Mrs. Zuhong Tian for the help with animal imaging experiments. The authors disclose no potential conflicts of interest.
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