Background
Esophageal carcinoma, a malignant tumor of gastrointestinal tract, contains two major subtypes of adenocarcinoma and squamous cell carcinoma [
1]. Esophageal carcinoma is the eighth prevalent tumor in all malignancies and ranks the sixth in cancer-related death worldwide [
2]. In 2016, there was an estimated 16,940 new cases (1% of all new carcinoma cases) and 15,690 deaths (2.6% of all carcinoma-associated deaths) of esophageal carcinoma occurred in USA [
3]. Although much progress has been made in the diagnose and therapy of esophageal carcinoma, its 5-year survival rate is still unsatisfactory [
3,
4]. Therefore, further exploring the molecular basis underlying esophageal carcinoma tumorigenesis and figuring out more effective therapeutic strategies are imperative.
Human tumor necrosis factor-α-induced protein-8 like-2 (TIPE2, TNFAIP8L2), located on chromosome 1q21.2–1q21.3, shared 94% identical amino acid sequence with murine TIPE2 [
5]. TIPE2 is a vital negative regulator of immune and inflammation homeostasis, which is closely associated with the development and progression of cancer [
5‐
8]. Recent studies showed that TIPE2 was downregulated and acted as a tumor suppressor in non-small cell lung cancer [
9], glioma [
10], prostate carcinoma [
11] and gastric carcinoma [
12,
13]. However, its roles and molecular mechanisms underlying esophageal carcinoma progression are still undefined till now.
In the present study, we firstly demonstrated that TIPE2 expression was downregulated in esophageal carcinoma tissues and cells. Functional and mechanistic analyses revealed that TIPE2 overexpression repressed the progression of esophageal carcinoma in vitro and in vivo by inhibiting the Wnt/β-catenin pathway.
Methods
Tissues samples
Tumor tissue specimens and the corresponding adjacent normal tissues were obtained from 29 patients diagnosed with esophageal carcinoma (19 males, 10 females; 50–70 years old) at the First Affiliated Hospital of Zhengzhou University between August 2014 and January 2016. Before surgery resection, none of the patients had received any radiotherapy or chemotherapy. The surgically resected specimens were immediately snap-frozen in liquid nitrogen and stored at − 80 °C for the subsequent assays. Our study was performed with the approval of Ethics Committee of the First Affiliated Hospital of Zhengzhou University and written informed consent was obtained from every patients.
Cell lines and culture
Human esophageal carcinoma cell lines (EC9076, KYSE410, KYSE150, TE-1 and EC109) and human normal esophageal epithelial cells HEEC were purchased from Cell Bank of Chinese Academy of Science (Shanghai, China). These cells were grown in RPMI-1640 medium (Hyclone, Logan, UT, USA) containing 10% heat-inactivated fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA) and 1% penicillin/streptomycin (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37 °C in a humidified chamber with 5% CO2.
Adenovirus preparation and transduction
The full length coding sequences of TIPE2 were amplified and constructed into pAdTrack-CMV shuttle plasmid with the name of pAdTrack-CMV-TIPE2, followed by the recombination of pAdTrack-CMV-TIPE2 and pAdEasy-1 backbone vector to generate pAd-TIPE2 recombination plasmids. Meanwhile, the control pAd-V recombination plasmid was also generated using the same method. After transfection for 8–10 days, pAd-V and pAd-TIPE recombination plasmids were transfected into HEK 293 cells using Lipofectamine 2000 (Invitrogen) to generate Ad-V and Ad-TIPE2 adenovirus, respectively. The titer of adenovirus was determined using plaque formation assays.
After EC109 and EC9706 cells seeded in 24-well plates were grown to 80% confluence, they were infected with recombinant adenovirus Ad-V and Ad-TIPE2 at multiplicity of infection (MOI) of 50.
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assay
Total RNA was extracted from tissues and cells using the Trizol reagent (Invitrogen) following the instructions of manufacturer. Then 1 μg RNA was reversely transcribed into cDNA using M-MLV reverse transcriptase (Invitrogen) and random primers. Fast SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA) was used to detect expression of TIPE2 mRNA on the Applied Biosystems 7500 Fast Real-Time PCR System (Applied Biosystems), with β-actin as an endogenous control. The primers sequences of TIPE2 and β-actin were listed as follows: TIPE2, 5′-ACT GAG TAA GAT GGC GGG TCG-3′ (forward) and 5′-TTC TGG CGA AAG CGG GTA G-3′ (reverse); β-actin, 5′-AAA TCG TGC GTG ACA TCA AAG A-3′ (forward) and 5′-GGC CAT CTC CTG CTC GAA-3′ (reverse).
Western blot assay
Total proteins were extracted from cells and tissues using RIPA buffer (Beyotime, Shanghai, China) containing protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland) and quantified using a Pierce™ BCA protein assay kit (Invitrogen; Thermo Scientific). Then equal amount of proteins (50 µg) were separated using 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose filter membrane (NC membrane; Millipore, Billerica, MA, USA). Next, the membranes were blocked in 5% skim milk for 1 h at room temperature and probed with primary antibodies against TIPE2 (ab110389, 1:1000, Abcam, Cambridge, MA, USA), β-actin (ab8227, 1:5000, Abcam), β-catenin (ab32572, 1:5000, Abcam), c-Myc (ab32072, 1:1000, Abcam), and cyclinD1 (ab134175, 1:1000, Abcam) overnight at 4 °C, followed by the incubation with horseradish peroxidase (HRP)-conjugated goat-anti rabbit second antibodies (ab6721, 1:1000, Abcam) for 1 h at room temperature. At last, specific protein signal was visualized using a ECL western blotting substrate (Promega, Madison, WI, USA) and quantified by A Image J software (National Institutes of Health, Bethesda, Maryland, USA).
Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay
EC9076 and EC109 cells (104/well) were seeded into 96-well plates and incubated overnight in a humidified atmosphere with 5% CO2 at 37 °C. Then, the cells were infected with Ad-V or Ad-TIPE adenovirus at a dose of 50 multiplicity of infection (MOI). At the indicated time points (day 0, 1, 2, 3) following infection, 20 μl MTT solution (5 mg/ml, Sigma-Aldrich, St. Louris, MO, USA) was added into each well. After incubation for another 4 h at 37 °C, the medium was discarded and 150 μl dimethyl sulfoxide (DMSO, Sigma-Aldrich) was carefully added into each well to dissolve formed formazan precipitates. Optical density (OD) values were measured at the wavelength of 490 nm on a microplate reader (Molecular Devices, Sunnyvale, CA, USA).
5-Ethynyl-2′-deoxyuridine (EdU) cell proliferation assay
Baseclick EdU-Click 488 kit (Sigma-Aldrich) was used to detect cell proliferation. Briefly, EC9076 and EC109 cells were seeded on cover slips and then infected with Ad-V or Ad-TIPE2 adenovirus for 3 days. Next, the cells were maintained for 3 h in serum-medium containing 50 μM final concentration of Edu, followed by fixation with 4% formaldehyde in PBS for 15 min and permeabilization using 0.5% Triton® X-100 in PBS for 20 min at room temperature. Then, permeabilization solution was removed and 500 μl of the reaction cocktail was added to each well with a coverslip for 30 min in the dark. Subsequently, cells were processed by 0.1 μg/ml of DAPI for 10 min to stain cell nucleus in the dark. At last, cells were imaged with a confocal laser-scanning microscope (LSCM, Leica Microsystems, Solms, Germany). The relative proliferation rate was calculated following the formula: relative proliferation rate = Edu-incorporated cell numbers/DAPI-stained cell numbers. The relative proliferation rate was normalized by Ad-V-infected group.
Transwell invasion assay
Cell invasion capability was assessed using an invasion chamber (BD Bioscience, San Diego, CA, USA) with 8 μm pore-size membrane (BD Bioscience). Briefly, EC9076 and EC109 cells (105/well) infected with Ad-V or Ad-TIPE2 were resuspended in serum-free RPMI-1640 medium (100 μl) and seeded into the upper chamber pre-coated with 40 µl Matrigel (BD Biosciences), while the lower chamber was filled with 700 μl RPMI-1640 medium containing 10% FBS. Following a 48 h incubation at 37 °C, the cells on the upper sides of membranes were removed by a sterile cotton swab. The cells on the bottom sides of membranes were fixed with methanol for 30 min, stained with 0.1% crystal violet for 20 min, and counted in 6 randomly selected fields using the inverted microscope (Nikon Eclipse TE300, Tokyo, Japan) at ×200 magnification.
Flow cytometry for apoptosis detection
Cell apoptosis rates were determined using a Annexin V-FITC/propidium iodide (PI) apoptosis detection kit (Nanjing Kaiji Biotechnology Development Co., Ltd., Nanjing, China) following the protocols of manufacturer. Briefly, EC9076 and EC109 cells were infected with Ad-V or Ad-TIPE2 adenovirus for 3 days. Then the cells were collected and resuspended in 500 μl 1 × Binding Buffer at a concentration of 1 × 106 cells/ml, followed by the addition of 5 μl Annexin V-FITC and 5 μl PI. Then the treated cells were incubated for 5 min in the dark at room temperature. Finally, a FACS Calibur Flow Cytometer (Beckman Coulter, Atlanta, GA, USA) was used to measure the apoptosis rate of cells.
In vivo experiment
BALB/c nude mice (6–7 weeks old, male) were obtained from Chinese Academy of Science (Shanghai, China) and grown in specific pathogen-free conditions. All animal studies were performed according to the Guide for the Care and Use of Laboratory Animals of Zhengzhou University. To explore the effect of TIPE2 overexpression on tumor growth in vivo, EC9706 cells infected with Ad-V or Ad-TIPE2 at a concentration of 1 × 107 cells/ml were injected subcutaneously into the flank region of nude mice. Tumor volume was measured every 3 days for 9 times with a caliper following the formula of 0.5 × length × width2. At day 24 after injection, the tumors of all mice were obtained, weighted and fixed in formalin for the following assays.
Immunohistochemical (IHC) assay
Surgically excised xenograft tumor specimens were fixed with 10% formalin and embedded in paraffin, followed by deparaffinization, rehydration, endogenous peroxidase blocking, and antigen retrieval. Then the specimens were blocked with 1% bovine serum albumin (BSA) for 10 min at room temperature and incubated with proliferating cell nuclear antigen (PCNA) antibody (ab18197, 1:1000, Abcam) overnight at 4 °C, followed by incubation with HRP-labeled secondary antibody (ab6721, 1:1000, Abcam) for 1 h at 37 °C. Next, DAB Peroxidase Substrate kit (General bioscience, Brisbane, California, USA) was used to visualize the immunoreactivity. For hematoxylin–eosin (H&E) staining, tissues were counterstained with hematoxylin–eosin and then photographed using a microscope (Nikon Eclipse TE300, Tokyo, Japan). Blue stands for cell nucleus and red or pink is an indicator of cytoplasm in H&E staining. Positive PCNA displays brown in IHC.
Statistical analysis
All data were obtained from three independent experiments and presented as mean ± standard deviation (mean ± SD). Student’s t-test or one-way ANOVA was employed to explore the difference of data in different groups. Differences were statistically significant when P < 0.05.
Discussion
TIPE2 is a member of tumor necrosis factor (TNF)-α-induced protein 8 (TNFAIP8) family, which also includes TNFAIP8, TIPE1 and TIPE3 members [
6]. Previous studies showed that TNFAIP8 family was implicated in the development and progression of various cancers such as gastric adenocarcinoma and ovarian carcinoma [
15,
16]. Moreover, TNFAIP8 family has been verified to be closely associated with clinicopathological characteristics and prognosis of esophageal carcinoma patients [
17]. As mentioned above, TIPE2 has been identified as a tumor suppressor in multiple malignancies. For instance, it was reported that TIPE2 overexpression hampered proliferation, epithelial–mesenchymal transition (EMT) process, migration, as well as invasion in prostate carcinoma [
11]. Additionally, enforced expression of TIPE2 was revealed to suppress cell proliferation, colony formation and invasion in lung carcinoma cells, and suppressed lung carcinoma tumor growth in vivo [
9]. Notably, it was also manifested that TIPE2 suppressed activation of oncogenic gene Ras, indicating that TIPE2 played a critical role in the development of carcinoma [
18]. The present study illustrated the roles and molecular mechanisms of TIPE2 in esophageal carcinoma.
Firstly, we demonstrated that TIPE2 expression was downregulated in esophageal carcinoma tissues and cells compared with normal counterparts, which was in accordance with the studies in other cancers such as non-small cell lung cancer [
9]. Previous studies manifested that abnormal expression of TIPE2 was closely correlated with pathological processes of cancer including proliferation, migration, invasion and apoptosis [
11,
19]. Furthermore, it was documented that adenovirus-mediated TIPE2 overexpression inhibited the growth of gastric cancer xenografted tumors [
20]. Hence, we further investigated the effect of TIPE2 overexpression on the progression of esophageal carcinoma in vitro and in vivo. The results showed that adenovirus-directed TIPE2 overexpression strikingly suppressed proliferation and invasion, as well as induced apoptosis in esophageal carcinoma cells in vitro. In vivo assay further demonstrated that adenovirus-mediated TIPE2 overexpression markedly blocked tumor growth in xenograft tumors of esophageal carcinoma. Therefore, we concluded that TIPE2 acted as a tumor suppressor in the development and progression of esophageal carcinoma.
Subsequently, the molecular mechanism of TIPE2 was further explored in esophageal carcinoma. TIPE2 has been demonstrated to be closely correlated with the Wnt/β-catenin pathway in cancer. For instance, TIPE2 suppressed hypoxia-triggered the Wnt/β-catenin pathway activation in glioma [
8]. Moreover, TIPE2 overexpression hampered metastasis by promoting β-catenin degradation and inhibiting β-catenin signaling pathway in gastric cancer [
10]. In the present study, we demonstrated that adenovirus-mediated TIPE2 overexpression suppressed the expression of β-catenin in EC9706 cells and xenograft tumors of esophageal carcinoma, indicating that TIPE2 acted as a tumor suppressor by inactivating the Wnt/β-catenin pathway in esophageal carcinoma. β-catenin, a critical component of the Wnt signaling cascade, has been demonstrated to be implicated in the progression of various carcinomas including esophageal carcinoma [
20‐
24]. For instance, depletion of β-catenin reversed HIF-1α-induced EMT process in human prostate carcinoma cells [
25]. Additionally, β-catenin downregulation hindered proliferation of esophageal carcinoma cells [
22,
26].
Moreover, β-catenin as a transcriptional activator can induce the transcription of multiple genes such as c-myc, cyclinD1, c-jun and survivin [
27]. Wang et al. reported that β-catenin knockdown suppressed the expression of downstream effectors such as c-myc and cyclinD1 in esophageal carcinoma [
28]. Additionally, the expressions of β-catenin and c-myc was closely correlated with invasion depth and lymph node metastasis together with prognosis of esophageal carcinoma [
28,
29]. C-myc, a downstream effector of β-catenin, also has been demonstrated to be implicated in the development of various tumors including esophageal carcinoma [
29‐
32]. Moreover, as a downstream effector of β-catenin, cyclinD1 has been identified as a vital mediator of cell cycle and cancer progression [
33,
34]. In esophageal carcinoma, cyclinD1 suppression was associated with a decreased cell proliferation, a reduced tumorigenicity, as well as a good prognosis [
35,
36]. CyclinD1 acted as an oncogene and was frequently overexpressed in esophageal carcinoma [
37]. Hence, the effects of TIPE2 on expressions of c-myc and cyclinD1 in esophageal carcinoma were further explored. In accordance with these data, our results showed that adenovirus-mediated TIPE2 overexpression suppressed the expressions of c-myc and cyclinD1 in esophageal carcinoma in vitro and in vivo.
Authors’ contributions
LZ, XZ, MZ and XF assisted in the design of study, performed experiments, analyzed/interpreted data, and drafted the manuscript; ZL, ZS, and JW made substantial contributions to study conception and design, data analysis and interpretation, and drafting and revising the manuscript; XW, FW, XL and SN provided technical support and helped to revise the manuscript; MD, ZY, WY, MY, LZ was involved in designing and performing some experiments. All authors read and approved the final manuscript.