TES functions as a Mena-dependent tumor suppressor in gastric cancer carcinogenesis and metastasis

Cell lines

All cell lines were authenticated by short-tandem repeat analysis. The human embryonic kidney cell line HEK293A (obtained in November 2009, authenticated in June 2015) and GC cell lines MKN45, SGC7901, MGC803, AGS, and HGC27 (obtained in July 2011, authenticated in June 2015) were obtained from the Committee of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). All cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) at 37 °C in a humidified chamber containing 5% CO₂.

Patients and tissue samples

The medical records of 172 GC patients treated at Sun Yat-sen University Cancer Center (Guangzhou, China) between January 2003 and December 2005 were reviewed. The patient selection criteria were as follows: (1) the patient was pathologically diagnosed with gastric adenocarcinoma; (2) the patient had received gastrectomy with limited or extended lymphadenectomy; (3) the patient did not receive any anticancer treatment before surgery; (4) the patient had complete clinical information, including follow-up data; (5) the patient had no other synchronous malignancies or familial malignancy; (6) the patient had no recurrent or remnant GC; and (7) the patient survived at least 3 months after surgery. Follow-up data were obtained through on-site interview, telephone calling or medical chart review. Overall survival (OS) was defined as the time from surgery to death from any cause or last follow-up. The study was approved by the Ethics Committee of Sun Yat-sen University Cancer Center (Guangzhou, China), and written informed consent was obtained from all participants.

Recombinant adenoviral expression vector construction and transfection

The TES recombinant adenoviral expression vector (Ad-TES) and control vector (Ad-Control) were constructed using the Gateway cloning system (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s protocol. After linearization by PacI enzyme, Ad-TES and Ad-Control were transfected into HEK293A cells using Lipofectamine 2000 (Invitrogen). After 10–13 days, when an approximately 80% cytopathic effect was observed, cells and medium were collected. After lysing the cells by three freeze–thaw cycles, the adenoviral supernatant was harvested by centrifugation (1000×g) at 4 °C for 15 min, tittered using Adenovirus Titer Immunoassay Kit (Innogent, Shenzhen, Guangdong, China) and stored at − 80 °C. To increase the transfection efficiency, HEK293A cells were re-infected with the initial harvested viral supernatant. Three to 4 days later, the cell lysates were collected after three freeze–thaw cycles. MKN45 or SGC7901 cells were transfected with Ad-TES at a multiplicity of infection (MOI) of 200. The transfection efficiency was calculated by dividing the amount of cells presenting green fluorescence by the total number of attached cells in 10 fields randomly selected for each sample under a fluorescence microscope with 100× magnification.

Extraction of total RNA and reverse transcription-polymerase chain reaction (RT-PCR)

Total RNA was extracted using TRIzol (Invitrogen) according to the manufacturer’s protocol. The concentration of total RNA was assessed by measuring absorbance at 260 nm using a NANO DROP spectrophotometer (ND-1000, Thermo Scientific, Waltham, MA, USA). Two mg of total RNA was reversely transcribed into cDNA using M-MLV reverse transcriptase (Promega, Madison, Wisconsin, USA) according to the manufacturer’s recommendation. The cDNA templates were amplified using the specific primer set for TES. The samples amplified with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primer set were used as an internal control. Primers used in this study were as follows: the forward primer 5′-CATGGACCTGGAAAACAAAGTG-3′ and the reverse primer 5′-CTAAGACATCCTCTTCTTACATTCCAC-3′ for TES; the forward primer 5′-CGGGAAGCTTGTCATCAATGG-3′ and the reverse primer 5′-G GCAGTGATGGCATGGACTG-3′ for GAPDH. The corresponding PCR products were 1267 bp for TES and 358 bp for GAPDH.

Protein extraction and western blotting

Forty-eight h after adenoviral transfection, protein expression was examined by Western blotting. GC cells (MKN45, SGC7901, MGC803, AGS, and HGC27) were lysed in RIPA lysis buffer (Beyotime, Shanghai, China), and lysates were harvested by centrifugation (13,000×g at 4 °C for 30 min. Western blotting was carried out as we previously described [3], using GAPDH as an internal control. The following primary antibodies and secondary antibodies were used:

A mouse monoclonal antibody against TES (1:500 dilution; Santa Cruz, Dallas, TX, USA), a rabbit monoclonal antibody against Mena (1:1000 dilution; Cell Signaling Technology, Boston, MA, USA), a rabbit polyclonal antibody against Lpd (1:1000 dilution; Sigma, St.Louis, MI, USA), HRP-conjugated rabbit anti-mouse IgG antibody (1:2000 dilution; Santa Cruz) and HRP-conjugated goat anti-rabbit IgG antibody (1:2000 dilution; Epitomics, Burlingame, CA, USA), a HRP-conjugated mouse anti-human GAPDH monoclonal antibody (1:5000 dilution; Shanghai Kangchen, Shanghai, China).

Proliferation assay

MKN45 or SGC7901 cells were seeded in RPMI-1640 medium supplemented with 10% FBS in 96-well plates (500 cells per well). CellTiter 96^® AQueous One Solution Cell Proliferation Assay (Promega, Beijing, China) was used to assess cell viability according to the manufacturer’s instruction for 5 consecutive days. Triplicate independent experiments were performed. The proliferation rate was calculated as follows: proliferation rate = (OD_n− OD₁)/OD₁ × 100% (n: days after transfection).

Colony formation assay

MKN45 or SGC7901 cells were seeded onto 6-well plates (500 cells per well). After cultured in RPMI-1640 medium supplemented with 10% FBS for 12 days, surviving colonies (> 50 cells per colony) were counted after 0.5% (m/v) crystal violet staining. Colony-forming efficiency (CFE %) was defined as the ratio of the number of colonies formed to the number of cells inoculated. Triplicate independent experiments were performed.

Cell cycle analysis

Forty-eight h after adenoviral transfection, GC cells were collected, washed twice with precooled phosphate buffer sodium (PBS) and fixed with ice-cold 75% ethanol at − 20 °C for 1 h. After washing with PBS, cells were resuspended in ice-cold PBS (500 µL) containing RNAase (100 µg) and incubated at 37 °C for 30 min, followed by propidium iodide (PI) staining at 4 °C for 30–60 min. Cell cycles of MKN45 and SGC7901 cells were analyzed on a FC500 flow cytometer (Beckman, Brea, CA, USA) using the Cylchred software (University Wales College Medicine, Cardiff, UK).

Cell invasion and migration assay

Cell invasion and migration assay were performed using MKN45 and SGC7901 cells transfected with Ad-TES or Ad-Control. The invasion assay was performed in a Transwell comprising a polycarbonate membrane with 8-μm pores (Corning, Shanghai, China) placed in a 24-well plate. The Transwell insert was coated with 50 µL Matrigel Basement Membrane Matrix (BD Biosciences, Bedford, MA, USA). Cells in 100 μL of RPMI-1640 medium without FBS were added to the Transwell insert, and 0.5 mL of RPMI-1640 medium containing 20% FBS was placed in the lower chamber. The cells were incubated at 37 °C and allowed to invade through the Matrigel layer. After 48 h, cells on the lower surface of the Transwell insert were fixed with 75% methanol and stained with 0.5% (m/v) crystal violet. The stained cells were counted in 10 random fields with 200× magnification. The migration assay was similar to the invasion assay, except that the Transwell insert was uncoated. Each experiment was performed in triplicate.

Apoptosis assay

MKN45 and SGC7901 cells were washed twice with ice-cold PBS and resuspended in 400 µL 1× Binding Buffer (Bestbio, Shanghai, China), according to the manufacturer’s protocol. After incubation with 5 µL Annexin V-FITC (Bestbio) for 15 min at room temperature in the dark, 10 µL PI (Bestbio) was added, and the cells stained with PI and Annexin V were counted using flow cytometer (Beckman).

Small interfering RNA (siRNA) transfection

siRNA targeting Mena (siMena, synthesized by GenePharma Company, Shanghai, China) were used to knockdown Mena expression in MKN45 and SGC7901 cells. The cells were seeded in 6-well plates (1 × 10⁶ cells per well). When an approximately 50% cell confluence was observed, the cells were transfected with 400 pmol of siMena or siNC (negative control) at 37 °C for 48 h using Lipofectamine RNAi MAX reagent (Invitrogen), according to the manufacturer’s instructions. The expression level of Mena was examined 48 h after the transfection. siRNA sequences used in this study were as follows:

Sense sequence 5′-GGUCCUAUGAUUCAUUACATT-3′ and antisense sequence 5′-UGUAAUGAAUCAUAGGACCTT-3′ for siMena 1;

Sense sequence: 5′-GCGAGAAAGAAUGGAAAGATT-3′ and antisense sequence 5′-UCUUUCCAUUCUUUCUCGCTT-3′ for siMena 2;

Sense sequence: 5′-UUCUCCGAACGUGUCACGUTT-3′ and antisense sequence 5′-ACGUGACACGUUCGGAGAATT-3′ for siNC.

Immunoprecipitation-based mass spectrometry (IP-based MS)

A Pierce^® Classic IP Kit (Thermo Scientific) was used to perform the protein interaction assay, according to the manufacturer’s protocol. In brief, after lysing in IP Lysis/Wash Buffer (0.025 mol/L Tris, 0.15 mol/L NaCl, 0.001 mol/L EDTA, 1% NP-40, and 1% protease inhibitor cocktail) at 4 °C for 5 min, cell lysates were collected by centrifugation (13,000×g) at 4 °C for 10 min. The supernatants were transferred to new tubes immediately. Total proteins were quantified with BCA Protein Quantification Kit (Beyotime) and adjusted to 3.5 μg/μL with PBS. An amout of 10 μg primary antibody against the target protein were added and incubated at 4 °C overnight. The mixtures were then incubated with 20 μL Protein A/G Plus Agarose for 6 h at 4 °C. After incubation, the Protein A/G Plus Agarose were washed three times with IP Lysis/Wash Buffer. Collected precipitates were eluted with 50 μL elution buffer at 100 °C for 10 min. Eluted proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and the protein bands were stained with silver solution (1×; Beyotime). The bands of interest were analyzed using MS (Institutes of Life and Health Engineering, Jinan University, Guangzhou, Guangdong, China). The antibodies used in immunoprecipitation were as follows: a mouse monoclonal antibody against TES (1:100 dilution; Sigma) and a rabbit monoclonal antibody against Mena (1:50 dilution; Cell Signaling Technology).

Tumorigenicity assays in nude mice

Five-week-old BALB/c nude mice (Guangdong Medical Experimental Animal Center, Guangzhou, Guangdong, China) were randomly assigned to four groups (7 mice per group) before inoculation. At 48 h after adenoviral transfection, MKN45 and SGC7901 cells were injected subcutaneously into the groin of mice respectively (5 × 10⁶ cells suspended in 200 μL PBS per mouse). The length (L) and width (W) of the tumor was measured with calipers every 3 days. The tumor volume was calculated as (L × W²)/2. Thirty-two days after tumor inoculation, the mice were sacrificed by cervical dislocation, and tumor weight was assessed.

Metastasis assay in nude mice

To investigate the suppressive effect of TES on tumor metastasis, 1 × 10⁶ cells (MKN45 cells transfected with Ad-TES or Ad-Control) in 200 μL PBS were injected intravenously through the lateral tail vein into 5-week-old nude mice (8 mice per group). After 6 weeks, the mice were necropsied after anesthesia. Their lungs were fixed in 3.7% formaldehyde, 5% glacial acetic acid, and 72% ethanol for 24 h before proceeding to paraffin embedding. Serial 5-μm sections were stained with hematoxylin and eosin (H&E) for histopathological examination. Metastasis lesions from 10 random high-power fields were counted. All animal experiments were conducted in compliance with the guidelines of the laboratory animal ethics committee of Sun Yat-sen University (Guangzhou, Guangdong, China).

Immunohistochemistry (IHC) and semi-quantitative analysis

IHC was performed on GC tissue sections as previously described [3]. The antibodies used in IHC were as follows: a mouse monoclonal antibody against TES (1:300 dilution; Santa Cruz) and a rabbit monoclonal antibody against Mena (1:500 dilution, Cell Signaling Technology). The intensity and extent of immunostaining were evaluated for all tumor samples by three pathologists under double-blinded conditions. In brief, the percentage of positive staining was scored as 0 (0–9%), 1 (10–25%), 2 (26–50%), or 3 (51–100%), and the intensity as 0 (no staining), 1 (weak staining), 2 (moderate staining) or 3 (dark staining). The total immunostaining score was calculated as the product of extent and intensity, ranging from 0 to 9.

Based on IHC scores, expression levels of Mena and TES were defined as low (score 0–3) or high (score 4–9) in subgroups.

Statistical analysis

Student t test was performed for comparison of continuous variables between groups. Repeated measurement analysis of variance (ANOVA) analysis was used to compare curves of tumor growth. Chi square test was used to examine differences of categorical variables between subgroups. Kaplan–Meier survival curve was plotted and compared by log-rank test. The patients did not have an event during the observation time were described as censored. Multivariate Cox proportional hazards regression model was used to calculate the hazard ratio (HR) and 95% confidence interval (CI) for prognosis evaluation. All statistical analyses were performed using the SPSS Statistics 22.0 software (IBM, Armonk, NY, United States), and P < 0.05 was considered statistically significant.

TES inhibits GC cell proliferation in vitro

To evaluate the function of TES in GC cells, the SGC7901 and MKN45 cells with low TES expression were transfected with Ad-TES and Ad-Control. The transfection efficiency of Ad-TES in SGC7901 and MKN45cells were 81.9% and 98.0%, respectively (Additional file 1: Figure S1). The RT-PCR and Western blotting results confirmed that the mRNA and protein levels of TES in the cells transfected with Ad-TES were markedly higher than those in the cells transfected with Ad-Control (Additional file 1: Figure S2). As shown in Fig. 1a, the proliferation rates of both SGC7901 and MKN45 cells were significantly suppressed after Ad-TES transfection. Similarly, colony formation assay revealed that Ad-TES transfection significantly inhibited colony formation (Fig. 1b). To further investigate the suppressive effect of TES on GC cell growth, we determined cell cycle distributions of Ad-TES and Ad-Control transfectants using flow cytometry based on the DNA content. As shown in Fig. 1c, TES overexpression significantly increased the proportion of cells at G₁ phase and decreased the proportion of cells at S phase, suggesting that TES inhibited cell cycle proceeding in GC cells. However, TES overexpression did not observably influence the apoptosis of GC cells (Additional file 1: Figure S3).

TES suppresses carcinogenesis of GC cells in vivo

We next analyzed the effects of TES on GC carcinogenesis in vivo. As shown in Fig. 2a, Ad-TES transfection remarkably delayed the tumor formation of SGC7901 and MKN45 cells in nude mice as compared with Ad-Control transfection. The mean tumor volume of the Ad-TES group was significantly smaller than that of the Ad-Control group (329.88 mm³ vs. 4026.26 mm³ for SGC7901, P = 0.018; 404.22 mm³ vs. 1884.46 mm³ for MKN45, P < 0.001; Fig. 2b) at the end of the experiment. Similarly, the mean tumor weight of the Ad-TES group was markedly lower than that of the Ad-Control group (0.42 g vs. 2.30 g for SGC7901, P = 0.018; 0.46 g vs. 1.79 g for MKN45, P < 0.001; Fig. 2c).

TES inhibits migration and invasion of GC cells

The invasion assays revealed that the invasion ability of SGC7901 and MKN45 cells overexpressing TES were significantly decreased as compare with their control counterparts (Fig. 3a, b). Similarly, significantly fewer SGC7901 and MKN45 cells transfected with Ad-TES migrated into the lower compartment of the migration chamber than those transfected with Ad-Control (Fig. 3c, d). Thus, these results suggest that TES overexpression can inhibit GC cell migration and invasion.

TES inhibits pulmonary metastasis in vivo

To determine the impact of TES on GC metastasis, nude mice were injected with MKN45 and SGC7901 cells transfected with Ad-TES, whereas the cells transfected with Ad-Control were used as controls. Six weeks after injection, the body weights of the mice did not differ between the Ad-TES group and the Ad-Control group. However, there were fewer metastatic nodules on the surface of excised lungs in the Ad-TES group than in the Ad-Control group (Fig. 3e). Lung sections stained with H&E showed that the Ad-TES group had significantly fewer lung metastasis lesions than the Ad-Control group (10 vs. 3, P = 0.019; Fig. 3f, g), indicating a suppressive effect of TES on GC metastasis.

TES interacts with Mena

To further investigate the potential molecular mechanism of TES in cell cycle arrest, cell migration and invasion, we performed IP-based MS study to identify TES-interacting proteins. Among the top listed protein candidates that were identified as putative binding partners of TES (Additional file 1: Table S1), Mena, encoded by the enabled homolog gene (ENAH), was of primary interest. Western blotting of the immunoprecipitates confirmed the interaction between TES and Mena (Fig. 4a, b).

TES inhibits the interaction between Mena and Lpd

Previous studies have implicated the interaction between Mena and Lpd [15]. We therefore explored the impact of TES on the interaction between Mena and Lpd. The protein expression of Mena was detected in GC cell lines (Fig. 4c). MKN45 cells with high expression of Mena were chosen for the subsequent experiments. Overexpression of TES had no effect on Lpd expression in MKN45 cells (Fig. 4d). However, our immunoprecipitation assay revealed that TES overexpression significantly reduced the binding of Lpd to Mena (P = 0.005; Fig. 4e, f), indicating that TES may inhibit the interaction between Lpd and Mena.

TES inhibits migration and invasion of GC cells in a Mena-dependent fashion

To test whether down-regulation of Mena might function in TES-induced inhibition of GC cell migration and invasion, we inhibited Mena expression with siRNA in Ad-TES transfected cells (Fig. 5a). The results of migration and invasion assays revealed that Mena silencing significantly promoted migration and invasion of Ad-TES-transfected cells (Fig. 5b, c), suggesting that TES inhibits migration and invasion of GC cells in a Mena-dependent fashion.

TES expression is associated with OS of GC patients in a Mena-dependent fashion

Since TES inhibited the migration and invasion of GC cells in a Mena-dependent manner, we investigated the impact of Mena on the association between TES expression and the prognosis of 172 GC patients. Representative images of IHC staining using antibodies against Mena are shown in Fig. 6. Similar to previous findings [16], high Mena expression was significantly associated with high rate of lymph node metastasis (P = 0.024, Table 1) and worse prognosis (P = 0.017, Fig. 7a). We next analyzed the association of TES with clinicopathological characteristics and OS of patients in different subgroups divided by Mena expression. Interestingly, in the group with high Mena expression, TES expression was negatively associated with tumor infiltration (P = 0.005), local lymph node metastasis (P = 0.003), and TNM stage (P = 0.003) (Additional file 1: Figure S4, Table 2). Moreover, high TES expression was significantly associated with long OS of patients with high Mena expression (P = 0.010; Fig. 7b). However, in the group with low Mena expression, there was no significant association between TES expression and clinicopathological characteristics (Table 2) or prognosis (P = 0.158, Fig. 7c).

Characteristic	Total (cases)	Mena expression (cases)		χ2	P value
		High	Low
All	172	99	73
Age (years)				0.401	0.527
< 55	80	44	36
≥ 55	92	55	37
Gender				0.351	0.553
Male	115	68	47
Female	57	31	26
Tumor infiltration				13.786	0.008
T1	27	10	17
T2	24	9	15
T3	3	1	2
T4a	82	56	26
T4b	36	23	13
Local lymph node metastasis				5.115	0.024
N0	59	27	32
N1–N3	113	72	41
Distant metastasis				0.061	0.806
M0	152	88	64
M1	20	11	9
TNM stage				16.565	0.001
0–I	30	9	21
II	69	40	29
III	50	38	12
IV	23	12	11

Characteristic	High Mena expression group (cases)					Low Mena expression group (cases)
	Total (cases)	High TES expression	Low TES expression	χ2	P value	Total (cases)	High TES expression	Low TES expression	χ2	P value
Tumor infiltration				8.141	0.005				1.486	0.854
T1–T2	19	14	5			32	16	16
T3–T4	80	30	50			41	18	23
Local lymph node metastasis				10.106	0.003				0.269	0.643
N0	27	19	8			32	16	16
N1–N3	72	25	47			41	18	23
Distant metastasis				3.457	0.105				0.333	0.725
M0	88	42	46			64	29	35
M1	11	2	9			9	5	4
TNM stage				13.109	0.003				2.560	0.489
0–I	9	7	2			21	9	12
II	40	23	17			29	13	16
III	38	12	26			12	8	4
IV	12	2	10			11	4	7