Introduction
Gastric cancer is one of the most common causes of cancer death worldwide [
1]. Additionally, most patients are diagnosed with advanced metastatic disease; the 5-year survival rate is approximately 10–15% [
2]. Although chemotherapy, radiotherapy, and targeted therapy have improved the response rate, patients with metastatic gastric cancer remain have a poor prognosis [
2,
3]. Contributing to this problem is the lack of effective biomarkers for metastasis prediction. Therefore, it is necessary and urgent to explore the mechanisms of metastasis in gastric cancer.
Tumor metastasis is a multi-step dynamic process involving multiple factors and genes. Recent evidence indicates that epithelial-to-mesenchymal transition (EMT) is a key driver of progression and metastasis in tumors, including gastric cancer, breast cancer, hepatocellular carcinoma, and prostate cancer [
4‐
7]. In this process, epithelial cells lose cell-cell adhesions and acquire properties of mesenchymal cells, namely enhanced migratory and invasive abilities [
8]. Many growth factors are involved in the initiation of EMT, including the insulin-like growth factor-I receptor (IGF-IR)/ligand system that has been reported to increase the metastatic potential of prostate and breast cancer cells [
5,
6]. Consistently, clinical studies have observed increased baseline IGF-I serum levels in patients with gastric cancer and overexpression of IGF-IR is a significant predictive value for poor survival in such patients [
9,
10]. However, whether IGF-I promotes gastric cancer metastasis by EMT, and the mechanisms by which this may occur remain unclear.
Ubiquitination is a post-translational modification that targets cellular proteins for degradation [
11]. Almost all cellular processes are regulated by the ubiquitin proteasome system, including EMT [
12]. Cbl-b is the second member of the E3 ubiquitin ligase Cbl family, and our group and others have revealed that Cbl-b regulates cancer cell proliferation, drug sensitivity, and migration [
13‐
15]. Knock-down of Cbl-b enhances epidermal growth factor-induced disruption of human mammary epithelial cell adherens junctions (AJs) and cell motility [
16]. The inducible up-regulation of c-Cbl and Cbl-b affects cell adhesion through regulation of the adhesion-related kinases Pyk2 and Paxillin in HL-60 cell differentiation [
17]. Moreover, Cbl-b can also degrade the IGF-I signaling intermediate IRS-1 and reduce protein synthesis in unloading-induced muscle atrophy [
18]. Our recent published data demonstrated that Cbl-b suppressed TRAIL-induced IGF-IR activation by regulating its distribution in the lipid raft [
19]. However, whether Cbl-b can target IGF-IR for degradation and if this process is involved in IGF-I-induced EMT require further investigations.
Here, we reveal the existence of an Akt/ERK-miR-200c-ZEB2 axis in IGF-I-induced EMT in gastric cancer cells. Furthermore, the ubiquitin ligase Cbl-b ubiquitinated IGF-IR and repressed IGF-I-induced EMT through negative regulation of this Akt/ERK-miR-200c-ZEB2 axis.
Materials and methods
Cell cultures
Human gastric cell lines MGC803, SGC-7901 were obtained from the Type Culture Collection of the Chinese Academy of Sciences (China). The cells were maintained in RPMI-1640 medium (Gibco) with 10% heat-inactivated fetal bovine serum (FBS), penicillin (100 U/mL) and streptomycin (100 mg/mL) in an atmosphere of 95% air and 5% CO2 at 37°C. The cells were sub-cultured every 2–3 days and harvested in their logarithmic phase of growth.
Reagents and antibodies
Recombinant human IGF-I was purchased from R&D System (Wiesbaden, Germany). The dual IGF-IR/IR inhibitor OSI-906 was purchased from SelleckBio (USA). Specific PI3K/Akt inhibitor LY294002 was purchased from Sigma (St. Louis, MO), and specific ERK1/2 inhibitor PD98059 was purchased from Promega (Madison, WI). Proteasome inhibitor bortezomib (PS-341) was purchased from Millenium Pharmaceuticals Inc (Cambridge, MA, USA). Anti-E-cadherin, anti-Vimentin, anti-ZEB1, anti-IGF-IR, anti-phospho-IGF-IR (Tyr1131), anti-phospho-GSK-3β, anti-GSK-3β and anti-phospho-P53 (Ser15) were purchased from Cell Signaling Technology (Beverly, MA). Anti-Snail and anti-Twist2 were purchased from Abcam (Cambridge, MA). All the other antibodies were purchased from Santa Cruz Biotechnology (USA).
Patients and tissue samples
A total of 50 lymph node metastasis and 50 non-lymph node metastasis surgically resected primary gastric adenocarcinoma patient specimens were obtained from the First Hospital of China Medical University between Jan 1st 2007 and Dec 31st 2008. Age, sex, pTNM stage and Lauren grade were evaluated following medical charts and pathology records. pTNM stage was examined according to the 7th edition of AJCC cancer staging manual. Lauren grade was reference to WHO classification. No patients had received any neoadjuvant chemotherapy and radiotherapy. All research involving human participants were approved by the Ethics Committee of China Medical University. Written informed consents were obtained from all the participants in accordance with the Helsinki Declaration.
Immunohistochemistry
One hundred of formalin-fixed, paraffin-embedded primary gastric cancer tissues were cut into 3-mm sections. All sections were de-paraffinized in xylene and dehydrated through a graduated alcohol series followed by the standard procedure for the S-P immunohistochemical kit (Fuzhou Maixin Biological Technology Ltd., Fujian, China). Sections were incubated with anti-Cbl-b or anti-IGF-IR in PBS at 4°C overnight in a moist box. 3, 30-diamino-benzidine tetrahydrochloride (DAB kit; Fuzhou Maixin Biological Technology Ltd., Fujian, China) was used for immune complex visualization. The staining was evaluated by scanning the entire tissue specimen under low magnification (×10) and confirmed under high magnification (×20 and × 40). The protein expression was visualized and classified based on the percentage of positive cells and the intensity of staining. Tumors with less than 10% Cbl-b or IGF-IR expression were regarded as negative. Immunostaining was considered as positive when more than 10% of the neoplastic cells were stained. Final scores were assigned by two independent pathologists.
Western blot and immunoprecipitation assay
Western blot analysis was performed as described in our previous studies [
15]. Briefly, samples were solubilized in 1% Triton lysis buffer on ice or in RIPA buffer. For immunoprecipitation, the collected cell lysates were incubated with the indicated antibodies (1–4 μg) or immunoglobulin-G (Cell Signaling Technology, Beverly, MA) and precleared protein G-agarose beads overnight at 4°C. On the other day, the immunoprecipitates were washed extensively with lysis buffer for four times. For the preparation of total cell lysates, the monolayers were lysed directly as described in western blot analysis. Both immunoprecipitated proteins and cell lysates were then eluted by boiling water at 100°C for 5 min with 3 × sampling buffer. Total proteins were subjected to SDS-polyacrylamide gel electrophoresis and electronically transferred to nitrocellulose membranes. After blocking with 5% skim milk in TBST buffer, the blots were incubated in the primary antibodies followed by secondary antibodies as indicated time. Proteins were detected using an enhanced chemiluminescence reagent (SuperSignal Western Pico Chemiluminescent Substrate; Pierce, USA). The final result was analyzed by NIH Image J software.
Reverse-transcription-polymerase chain reaction (RT-PCR)
The cells were cultured and harvested at the indicated times. Total RNA was isolated with the RNeasy mini kit (Qiagen, Carlsbad, CA, USA). RT-PCR was performed with primer pairs for ZEB2: forward (5′-CGCTTGACATCACTGAAGGA-3′) and reverse (5′-CTTGCCACACTC TGTGCATT-3′). For actin as control: forward (5′-GTGGGG CGCCCCAGGCACCA-3′) and reverse (5′-CTCCTTAATGTCACGCACGATTTC- 3′). PCR conditions were 95°C for 5 min; 31 cycles of 95°C for 30 s, 55°C for 45 s, 72°C for 40 s; one cycle of 72°C for 10 min. Then the amplified products were separated on 1.5% agarose gels, and stained with ethidium bromide and visualized under UV illumination.
Quantitative reverse transcription real-time PCR (qRT-PCR)
Total RNA was extracted as mentioned above. For microRNAs, The One Step PrimeScript® miRNA cDNA Synthesis Kit (Takara, Japan) was used for RNA reverse transcription. Relative expression of microRNA was calculated via the comparative cycle threshold (Ct) method, and the expression of U6 small nuclear RNA was used as reference. The sequence-specific forward primers for mature miR-200c was: 5′-ACACTCCAGCTGGGTAATACTG CCGGGTAA-3′ and for U6 internal control was: forward (5′-GCTTCGGCAGCACATAT ACTAAAAT-3′) and reverse (5′-CGCTTCACGAATTTGCGTGTCAT-3′), respectively. The Uni-miR qPCR Primer was included in the kit. SYBR® Premix Ex Taq™ II (Perfect Real Time) (Takara, Japan) was used for monitoring the amount of miRNA. The PCR conditions were 30 s at 95°C, followed by 45 cycles at 95°C for 5 s and 58°C for 25 s. The threshold cycle and 2-ΔΔCt method were used for calculating the relative amount of the target RNA.
MicroRNA microarray
The expression levels of 847 human and 609 mouse microRNAs were quantitated using a GeneChip miRNA Array (Affymetrix, Santa Clara, CA) according to the manufacturer’s instructions by Gene Tech Biotechnology Company (Shanghai, China). In brief, total RNA (1 μg) was extracted with miRNeasy Mini Kit (Qiagen) and labeled with a FlashTag Biotin RNA Labeling kit (Genisphere, Hatfield, PA). Then the labeled RNA was injected onto the microarrays and incubated at 48°C for 16 hours. After washing and staining, the signals were obtained using a GeneChip Scanner 3000 (Affymetrix, Santa Clara, CA). Data were normalized using the RMA algorithm.
Migration assay
Migration assay was performed using Boyden chambers and polycarbonate inserts with 8-um pore size membranes. The cells (1 × 104 cells/well) were seeded into the upper chamber with 200 μL serum-free RPMI 1640 medium with or without IGF-I (100 ng/mL). Then the upper chamber was inserted into the lower chamber of 24-well culture dishes with 500 μL of RPMI 1640 containing 2.5% FBS. After incubation for 48 hours, the culture media in the upper chamber and non-migrated cells on the inner side of the membrane were carefully removed with a cotton swab. After dried for 1 hour at room temperature, the migrated cells onto the outer side of the membrane were fixed with 4% formaldehyde for 1 min and stained with 0.1% Giemsa stain solution for 2 hours. Then the migrated cells were counted in five different fields at × 10 magnification under the microscope.
Immunofluorescence
The cells were seeded in Lab-Tek chamber slides (Nunc S/A, Polylabo, Strasbourg, France). After starved overnight, the cells were treated with or without IGF-I (100 ng/mL) for 48 hours and fixed in 3.3% paraformaldehyde for 15 min, permeabilized with 0.2% Triton X-100 for 5 min, blocked with 5% bovine serum albumin (BSA) for 1 hour and then incubated with anti-E-cadherin and anti-Vimentin antibody overnight at 4°C. The next day, Alexa Fluor 546-conjugated goat anti-rabbit IgG or Alexa Fluor 488-conjugated goat anti-rabbit IgG (Molecular Probes) were added in blocking solution for 1 hour at room temperature in the dark. 4′6′-diamidino-2- phenylindole was used to stain nuclei for 5 min. After mounted with the Slow Fade Antifade Kit (Molecular Probes, Eugene, OR, USA), the cells were visualized by fluorescence microscopy (BX61, Olympus, Japan).
Small interfering RNA transfections
Cells were seeded at a density of 3 × 105 cells/well in 6-well plates. The cells were transfected with siRNAs using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. Two siRNA sequences (Qiagen Inc., Valencia, CA) each for ERK1 and ERK2 were as follows: ERK1: 5′-CGUCUAAUAUAUAAAUAUA dTdT-3′ (sense), 5′-UAUAUUUAUAUAUUAGACGdGdG-3′ (antisense); ERK2: 5′-CACU UGUCAAGAAGCGUUAdTdT-3′ (sense), 55′-UAACGCUUCUUGACAAGUGdTdT-3′ (anti-sense). The Akt siRNA was obtained from Sigma (MO, USA) and siRNA sequence was: 5′-GAGACUGACACCAGGUAUUdT dT-3′ (sense), 5′-AAUACCUGGUGUCAGUCUCdT dT-3′ (anti-sense). The control sequence was: AATTCTCCGAACGTGTCACGT. Western blot analysis was used to verify gene-silencing efficiency.
Plasmid construction and stable cell lines establishment
The method of plasmid construction is performed as described previously [
20].
Statistical analysis
All the presented data were expressed as the mean ± SD and representative results were from at least three independent experiments. Statistical comparisons were calculated by Student’s two-tailed t-test. The correlation between Cbl-b and IGF-IR expression was assessed using Spearman rank correlation for continuous variables. The effect of Cbl-b and IGF-IR on metastasis was analyzed by Fisher exact test for tables. p < 0.05 was considered statistically significant. Statistical analysis was carried out using SPSS 18.0 software package (SPSS, Inc., Chicago, IL, USA).
Discussion
The IGF-I/IGF-IR signaling pathway has been reported to induce EMT through activation of the MEK/MAPK and PI3K/Akt pathways in immortalized mammary epithelial, breast, and prostate cancer cells [
5,
6,
25,
26]. Furthermore, increased expression of the transcription factor ZEB1 is essential for IGF-I-induced EMT [
6]. However, whether IGF-I can induce gastric cancer cell EMT or up-regulate expression of ZEB2, another ZEB transcription factor family member, is unknown. Here, we demonstrated that IGF-I initiated EMT in gastric cancer cells and increased their migration potential through up-regulation of ZEB2. This process was at least partially dependent on Akt/ERK downstream signaling pathways, which were upstream factors of ZEB2 activation in gastric cancer cells in vitro.
ZEB2 is an important transcriptional factor in EMT and functions as a metastasis regulator via direct binding to the promoter site of the cell adhesion molecule E-cadherin in several cancer types [
27‐
29]. Previous studies have identified that an increased ZEB2/E-cadherin ratio positively correlates with invasive disease and poor prognosis in breast and ovarian cancers [
30]. Here, we observed that ZEB2 protein levels were up-regulated after IGF-I treatment without an increase in ZEB2 mRNA, suggesting that IGF-I-induced ZEB2 up-regulation might be post-transcriptionally regulated. Recent studies have shown that reduced levels of MicroRNA-200 (miR-200) family members are associated with tumor metastasis and poor disease outcomes [
31]. MiR-200c is able to suppress EMT through targeting of ZEB1/2 in some tumor cells [
32‐
34]. Furthermore, the existence of an Akt-miR-200c-E-cadherin axis in the EMT process in renal cell carcinoma has been identified [
35]. Here, we found reduced expression levels of miR-200c in both MGC-803 and SGC-7901 gastric cancer cells following IGF-I stimulation. PI3K/Akt inhibitor LY294002, ERK inhibitor PD98059 and transient knockdown of ERK or Akt gene partially reversed the down-regulation of miR-200c by IGF-I. These results support the existence of an Akt/ERK-miR-200c-ZEB2 axis in IGF-induced EMT in gastric cancer cells.
Cbl ubiquitin ligase is reported to maintain AJ dynamics and suppress cell migration through down-regulation of epidermal growth factor receptor-Vav2 signaling in human mammary epithelial cells [
16]. Additionally, expression of 70z-Cbl in Madin-Darby canine kidney epithelial cells results in breakdown of cell-cell junctions in a manner characteristic of EMT [
22]. Our previous study reported that up-regulation of c-Cbl and Cbl-b was involved in all-trans retinoic acid and bufalin-induced cell adhesion in human promyelocytic cells [
17]. This new report revealed that knockdown of Cbl-b facilitated the initiation and progression of EMT in MGC-803 cells. Meanwhile, IGF-induced EMT and migration potential were increased in Cbl-b-knockdown cells. This process was accompanied by prolonged activation time for the Akt/ERK downstream signaling pathways, inhibition of miRNA-200c expression, and up-regulation of the transcriptional repressor ZEB2. Our microRNA array and real-time PCR data reveal a decreased expression level of miRNA-200c in Cbl-b knockdown cells. Together, these findings suggested that Cbl-b repressed IGF-I-induced EMT and migration ability through negative regulation of the Akt/ERK-miR-200c-ZEB2 axis.
To understand how exactly Cbl-b regulated the Akt/ERK-miR-200c-ZEB2 axis in IGF-I-induced EMT, we further investigated the relationship between the Cbl-b and IGF-IR signaling pathways. We found that IGF-IR was first phosphorylated and degraded by IGF-I through the proteasome system. Phosphorylation is the premise of ubiquitination. Phosphorylation of the substrate makes proteins more susceptible to be recognized by the appropriate ligase and formed the ligase complex [
36]. Previous study has implicated that EGFR recognition by c-Cbl probably depends on the phosphorylation of a specific receptor tyrosine residue [
37]. Consistently, a recent study has reported that c-Cbl combines with IGF-IR and mediates receptor polyubiquitination in response to IGF-I ligand in human osteosarcoma cells [
24]. Furthermore, ubiquitination of activated EGFR by c-Cbl complexes is involved in ERβ-1-mediated repression of EMT in basal-like breast cancer cells [
38]. In the present study, we found that IGF-IR combined with Cbl-b and initiated IGF-IR ubiquitination after IGF-IR phosphorylation and activation in MGC-803 gastric cancer cells. Combination with Cbl-b initiated receptor degradation of IGF-IR following IGF-I stimulation. Knockdown of Cbl-b significantly inhibited this process. These results suggest that Cbl-b likely ubiquitinates and degrades IGF-IR, and that this is necessary to repress the Akt/ERK-miR-200c-ZEB2 axis and the process of IGF-I-induced EMT. By examining 100 clinical gastric adenocarcinoma tissues, we found that IGF-IR positive expression was significantly associated with late-stage pTNM and positive lymph node metastasis, which was consistent with previous reports [
10]. More importantly, the expression of IGF-IR was negatively correlated with the expression of Cbl-b. Cbl-b positive expression was associated with early-stage pTNM and negative lymph node metastasis. These newly reported results further strengthened the possibility that Cbl-b could repress IGF-IR and decrease the risk of developing lymph node metastasis in patients with gastric cancer.
Acknowledgements
This work was supported by National Science and Technology Major Project (No. 2013ZX09303002). Chinese National Foundation of National Sciences grants (No.81201802, No.81172369, No.81172198, No.81270036), Specialized Research Fund for the Doctoral Program of Higher Education (No.20102104120008, No.20112104110005).
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
YL and XQ designed research; HL and LX performed the data acquisition; CL and LZ supervised the data and algorithms; CL and YM performed data analysis and interpretation; HZ and ZL carried out the statistical analysis; YZ and HL performed immunohistochemistry. RW and ZL performed clinical cases collecting and follow-up. HL performed manuscript preparation; YL and XQ participated in manuscript editing and review. All authors read and approved the final manuscript.