J Cancer Res Clin Oncol (2015) 141:2109–2120 DOI 10.1007/s00432-015-1973-7

ORIGINAL ARTICLE – CANCER RESEARCH

Significances of contactin‑1 expression in human gastric cancer and knockdown of contactin‑1 expression inhibits invasion and metastasis of MKN45 gastric cancer cells De‑Hu Chen1 · Ji‑Wei Yu1 · Ju‑Gang Wu1 · Shou‑Lian Wang1 · Bo‑Jian Jiang1 

Received: 30 December 2014 / Accepted: 13 April 2015 / Published online: 8 May 2015 © The Author(s) 2015. This article is published with open access at Springerlink.com

Abstract  Purpose  Contactin-1 (CNTN-1) has been shown to promote cancer metastasis. Previously, we have reported that the expression of CNTN-1 was upregulated in gastric cancer tissues compared with adjacent normal tissues. Here, we investigated the significance of CNTN-1 expression and its underlying mechanism of metastasis mediated by epithelial–mesenchymal transition (EMT) in gastric cancer. Methods  The expressions of CNTN-1 and EMT-related proteins were assayed through immunohistochemical staining of pathological specimens from patients with gastric cancer. Other methods including reverse transcriptase polymerase chain reaction, Western blotting, stably transfected against CNTN-1 into MKN45 cells, migration and invasion assays in vitro and nude mouse tumorigenicity in vivo were also utilized. Results  The results revealed that CNTN-1 expression was elevated and positively correlated with metastasis, EMT-related markers and poor prognosis in patients with gastric cancer. Moreover, CNTN-1 expression might associate with invasive ability to some extent in gastric cancer cell lines KATO-Ш, SGC7901 and MKN45. Knockdown of CNTN-1 expression in MKN45 cells using short hairpin RNA (shRNA) had notable effects on cell migration and invasion, rather than proliferation in vitro and in vivo. Furthermore, suppression of CNTN-1 expression altered EMT

De-Hu Chen and Ji-Wei Yu have contributed equally to this work. * Bo‑Jian Jiang jiang‑[email protected] 1



First Department of General Surgery, 3rd People’s Hospital, School of Medicine, Shanghai Jiao Tong University, No 280, Mohe Road, Shanghai 201900, China

through inhibition of transcription factor Slug, rather than Snail. Conclusion  Our study demonstrated that the elevated CNTN-1 expression closely correlated with cancer metastasis and patient survival, and its functions seemed to be important in migration and invasion of gastric cancer cells via EMT alteration probably mediated by inhibition of Slug. CNTN-1 may be a potential therapeutic target for gastric cancer. Keywords  Contactin-1 · Gastric cancer · Epithelial– mesenchymal transition · Tumorigenicity

Introduction Gastric cancer, as the fourth most common malignancy (Jemal et al. 2011) and the second leading cause of cancerrelated death in the world (Nagini 2012), remains highly prevalent and accounts for a considerable amount of morbidity and mortality (Ferlay et al. 2015). Metastasis is still a major impediment to the successful treatment of gastric cancer despite advancements in surgical technique and chemotherapy over the years (Catalano et al. 2009). Therefore, identification of novel biomarkers involved in metastatic progression of gastric cancer may provide potential therapeutic targets. The neuronal cell adhesion molecule contactin-1 (CNTN-1), as a member of the immunoglobulin gene family and a glycosylphosphatidylinositol (GPI)-anchored neuronal membrane protein (Falk et al. 2002), mediates cell surface interactions in the nervous system and participates in various signal transduction pathways such as a functional ligand for Notch (Berglund and Ranscht 1994; Hu et al. 2003). Recent studies have shown that CNTN-1 is involved

13

2110

in tumor invasion and metastasis, for example in non-small cell lung cancer and so on (Lehembre et al. 2008; Van Kilsdonk et al. 2008; Gavert et al. 2007; Su et al. 2006a, b). Notably, Su et al. (2006a, b) first determined CNTN-1 as a metastasis-promoting oncogene. Knockdown of CNTN-1 expression suppressed invasion and metastasis of lung adenocarcinoma cells in vitro and inhibited the metastasis dramatically but not the formation of transplanted tumor in an animal model. CNTN-1-mediated RhoA (not Cdc42 or Rac1) activation suggested that CNTN-1 was a critical regulator for the invasion and the metastasis of lung adenocarcinoma cells through RhoA-dependent pathway (Su et al. 2006a). Moreover, CNTN-1 had also been found to correlate with invasion and metastasis in esophageal cancer (Liu et al. 2011) and oral squamous cell carcinoma (Liu et al. 2012). The vascular endothelial growth factorc/vascular endothelial growth factor receptor 3 (VEGF-C/ VEGFR-3)-mediated invasion and metastasis of cancer cells was reported to elevate CNTN-1 expression through Src/p38 MAPK-C/EBP-dependent pathway, suggesting that CNTN-1 was a downstream effector of the VEGF-C/ VEGFR-3 signal pathway in cancer cells (Su et al. 2006b). Knockdown of VEGFR-3 resulted in downregulation of CNTN-1 expression in the gastric cancer cell line MKN45 (Qin et al. 2011). Additionally, the fact that CNTN-1 correlated with VEGF-C and VEGFR-3 expression in gastric cancer indicated that CNTN-1 could play a key role in the gastric cancer progression (Yu et al. 2013). However, further trials demonstrating whether CNTN-1 promotes invasion and metastasis of gastric cancer and the underlying mechanism of CNTN-1-mediated metastasis of gastric cancer seem to be required. Recent research revealing that the neuronal cell adhesion molecule (NCAM) induced epithelial cell migration and correlated with tumor invasion indicated a link between NCAM function, the loss of E-cadherin expression and cell invasion (Lehembre et al. 2008). Furthermore, CNTN-1 has been shown to advance cancer metastasis. CNTN-1 ablation resulted in upregulation of E-cadherin expression via activating AKT pathway in lung cancer (Yan et al. 2013), consistent with E-cadherin being suppressive of cancer cell invasion. Despite mounting evidences in favor of CNTN-1-mediated cancer metastasis, the role of CNTN-1 in gastric cancer invasion and metastasis and the underlying mechanisms responsible for this process should be further investigated. Thus, the objective of the present study was to demonstrate the association of CNTN-1 expression with clinicopathological features and the possibility of CNTN-1 being a prognostic marker in the patients with gastric cancer, the potential regulation of CNTN-1-induced invasion and metastasis of gastric cancer cells in vitro and in vivo and the relationship between CNTN-1 expression and expression of EMT-related proteins.

13

J Cancer Res Clin Oncol (2015) 141:2109–2120

Materials and methods Patients selection and tissues preparation A total of 72 patients who underwent radical gastrectomy for primary gastric carcinoma at our hospital from January 2009 to December 2010 were selected. None of them accepted any preoperative chemotherapy or radiotherapy. Preoperative informed consent was obtained from each patient registered in this study in accordance with institutional guidance, and this study was also approved by the ethics committee of our hospital before its start. Primary lesion and the adjacent normal gastric mucosas at the places of 5 cm far from the edge of primary lesion, which were identified pathologically, were excised and fixed in 10 % neutral-buffered formalin and then embedded in paraffin blocks for HE staining and various immunohistochemical stainings. Regarding prognostic analysis of these patients, the deadline of follow-up for patients was until October 2014, and a total of nine cases (12.5 % patients) lost to follow-up. Immunohistochemistry Serial tissue sections were cut at 4 μm thick and then deparaffinized, rehydrated and heat-treated for 30 min in citrate buffer (pH 6.0) for antigen retrieval. Endogenous peroxidase activity was blocked by 3 % hydrogen peroxide for 10 min. Sections were blocked with 5 % normal blocking serum. Afterward, sections were incubated overnight with the following antibodies: CNTN-1 mAb (1:500, Abcam, UK), E-cadherin mAb (1:200, Cell Signaling Technology, USA), Snail pAb (1:200, Abcam, UK), Slug mAb (1:200, Cell Signaling Technology, USA) and N-cadherin mAb (1:100, Cell Signaling Technology, USA). The next day, sections were incubated for 40 min with biotin-conjugated anti-IgG serum (Boster, China). Sections were then incubated with an SABC solution according to the manufacturer’s instruction. The primary antibody was visualized using diaminobenzidine tetrachloride kit (Boster, China) for 10 min. Finally, sections were observed under a light microscopy. All sections were evaluated and scored by two independent pathologists blind to each patient’s status. The evaluation of staining result was graded as described previously (Yu et al. 2013), and the percentage of staining cell scores (0 point = 0–5 %; 1 point = 6–25 %; 2 points = 26–50 %; 3 points = more than 50 %) and the staining intensity scores (0 point = negative; 1 point = weak intensity; 2 points  = moderate intensity; 3 points = strong intensity) were summed. The sum scores with ≥3 points were regarded as positive, while the sum scores with <3 points were considered as negative.

J Cancer Res Clin Oncol (2015) 141:2109–2120

Cell culture The cell lines KATO-III, SGC7901 and MKN45 of human gastric cancer were purchased from American Type Culture Collection (ATCC, USA). According to the manufacturer’s instruction, both of SGC7901 and MKN45 cells were cultured in 90 % RPMI-1640 (Gibco) supplemented with 10 % fetal bovine serum (Gibco), and KATO-III cells were cultured in 80 % IMDM (ATCC, USA) supplemented with 20 % fetal bovine serum (Gibco). All cell lines were maintained in a humidified atmosphere of 37 °C at 5 % CO2. RT‑PCR Total RNA was extracted from gastric cancer cells with Trizol reagent (Invitrogen, USA) following the manufacturer’s protocol. Reverse transcriptase polymerase chain reaction (RT-PCR) was done using the isolated RNA with the primer pairs as follows: the primer sequences for CNTN-1 were 5′-TGTTCAGCAAATTCATCCCA-3′ (forward) and 5′-TCTACCCACTCAGGGAATGC-3′ (reverse), for E-cadherin were 5′-TGCCCAGAAAATGAAAAAGG-3′ (forward) and 5′-GTGTATGTGGCAATGCGTTC-3′ (reverse), for Slug were 5′-GGGGAGAAGCCTTTTTCTTG-3′ (forward) and 5′-TCCTCATGTTTGTGCAGGAG-3′ (reverse), for Snail were 5′-CCTCCCTGTCAGATGAGGAC-3′ (forward) and 5′-CCAGGCTGAGGTATTCCTTG-3′ (reverse), for N-cadherin were 5′-ACAGTGGCCACCTACAAAGG-3′ (forward) and 5′-CCGAGATGGGGTTGATAATG-3′ (reverse), and for GAPDH, as the internal control, were 5′-ACGGATTTGG TCGTATTGGGCG-3′ (forward) and 5′-CTCCTGGAAG ATGGTGATGG-3′ (reverse). PCR products were visualized by ethidium bromide staining after separation by agarose gel electrophoresis. Western blotting assay Cells were lysed with lysis buffer radioimmunoprecipitation assay (RIPA) (Beyotime Institute of Biotechnology, China) and a mixture of protease inhibitors phenylmethanesulfonyl fluoride (PMSF) (Beyotime Institute of Biotechnology, China). The protein concentration was estimated by bicinchoninic acid (BCA) protein assay kit (Beyotime Institute of Biotechnology, China). Equal amounts of protein were separated on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). After being transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, USA), membranes were blocked with 5 % non-fat milk and then incubated with primary antibodies at 4 °C overnight. Secondary antibodies were incubated for 2 h at room temperature. Protein bands were visualized using the enhanced chemiluminescence detection kit (Thermo scientific, USA). The primary antibodies were: CNTN-1 mAb (1:1000,

2111

Abcam, UK), E-cadherin rabbit mAb (1:1000, Cell Signaling Technology, USA), Slug rabbit mAb (1:1000, Cell Signaling Technology, USA), Snail rabbit pAb (1:1000, Abcam, UK), N-cadherin rabbit mAb (1:1000, Cell Signaling Technology, USA) and GAPDH mAb-HRP (1:5000, Bioworld Technology, USA). RNA interference and stably transfected clone selection Four shRNA oligonucleotide sequences for targeting human CNTN-1 gene mRNA were designed using the RNAi designer. A scrambled sequence of targeting CNTN-1 sequences was used as a control. The control shRNA (con sh) targeting sequence is 5′-TTCTCCGAACGTGTC ACGTAA-3′; the shRNA1 (sh#1) targeting sequence is 5′-GCCGTGGTTCAGACAATCATA-3′; the shRNA2 (sh#2) targeting sequence is 5′-CCCGGTTTACAAATGG AGAAT-3′; and the shRNA3 (sh#3) targeting sequence is GCCGGAATGTATCAGTGCATA-3′. The recombinant lentivirus with CNTN-1-gene was produced by co-transfection of 293T cells with helper plasmids (psPAX2 and pMD2G) with Lipofiter (Hanbio, China). After 48 h, the cultured supernatant was harvested, filtered and concentrated by ultracentrifugation. To establish stable CNTN1-gene-knockdown cell lines, MKN45 cells were transduced with lentiviral RNAi vector. After 72 h, cells were treated with 2 μg/ml puromycin. Puromycin-resistant clones were selected and expanded for additional studies. Cell proliferation assay Cells were seeded in 96-well plates at a density 5 × 103 cells per well. At the indicated times (0, 24, 48, 72 and 96 h after culture), 10 µl of cell counting kit-8 (CCK-8, Dojindo, Japan) solution was added to each well and incubated for another 80 min at 37 °C, and then the absorbance at 450 nm was measured to calculate cell growth rates. Immunofluorescence staining Cells were fixed with 4 % paraformaldehyde for 15 min and then blocked by incubation in normal goat serum for 30 min. The primary antibody was applied to the samples at a dilution of 1:100 and incubated at 4 °C overnight. The slides were incubated with Texas Red-conjugated secondary antibody (Sigma, USA) for 1 h and counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Finally, the Texas Red-labeled cells were analyzed under a fluorescence microscope. Cell scratch‑wound assay Cells were cultured at 80–90 % confluence in six-well plates Wound areas were scraped using 10-µl plastic tips

13

2112

to produce a straight line. Images were taken every 24 h to monitor the wound healing process, using an inverted research microscope eclipse TS100/100-F (Nikon, Japan). The healing rate = (0–72 h width)/0 h width. Cell migration and invasion assays Migration assay was performed using 24-well 8-µm pore size transwell plates (Corning, USA) without Matrigel Matrix Basement Membrane coated on the bottom of transwell chamber. To measure cell invasion, the bottom of transwell chamber was coated with BD Matrigel™ Matrix Basement Membrane (BD Biosciences, USA). The upper chamber was filled with 2 × 105 cells in 200-μl serumfree medium. The lower chamber was filled with 600-μl medium containing 20 % fetal bovine serum as a chemoattractant. After 24 h of incubation, non-invading cells on the upper surface of the membrane were removed by scraping. Invading cells on the lower side of the membrane were fixed in 4 % paraformaldehyde and stained with 0.05 % crystal violet. The number of invasive cells was counted under a microscope (Zhang et al. 2013). In vivo tumorigenesis Six-week-old male nude athymic BALB/c mice were used to examine tumorigenicity. To determine the function of CNTN-1 in tumor formation, the con sh or sh#2 group cells (4 × 106 cells in 0.2 ml volume) were injected subcutaneously into the right flanks of nude mice followed by measurements of the tumors size at 5-day intervals. Tumor size was measured by the formula: volume = (short diameter)2  × (long diameter)/2 (Su et al. 2006a; Yang et al. 2014). At 30 days after inoculation, the mice were killed, and the tumor mass were weighted. To ascertain the role of CNTN-1 in tumor metastasis, the above two group cells (3 × 106 cells in 0.2 ml volume) were intravenously injected into the lateral tail vein of nude mice, respectively. At 4 weeks after injection, the lungs of nude mice were observed by Siemens Inveon Micro-PET/CT (Inveon MM platform, Siemens Preclinical Solutions, USA). Mice were killed, and lungs were removed for counting the number of metastasis nodules and then being fixed in 10 % neutralbuffered formalin for additional examination. The assays were done using six nude mice per group, and all animal work was carried out according to institutional guidelines. Statistical analysis The data are presented using mean ± SD. Statistical analyses of clinicopathological parameters were performed by Chi-square. Bivariate relations between study variables were analyzed by Spearman’s rank correlation coefficient.

13

J Cancer Res Clin Oncol (2015) 141:2109–2120

Survival curves were obtained using the Kaplan–Meier method, and statistic assess for survival was analyzed by log-rank method. The multivariate evaluation was performed by logistic analysis for these correlations to explain the more important significances of the observed features. The Cox regression model was used for multivariate analysis of prognostic factors. For cell and animal experiments, data were analyzed by one-way ANOVA or Student’s t test. All the analyses were performed using SPSS 17.0 software (SPSS Inc, USA). Statistical significance was defined as p < 0.05.

Results Increased expression of CNTN‑1 is associated with EMT‑related proteins in gastric cancer The expression levels of CNTN-1, E-cadherin, Snail, Slug and N-cadherin were compared in primary lesion and in adjacent normal gastric mucosas of 72 patients with gastric cancer by immunohistochemical staining (Fig. 1a). As shown in Table 1, CNTN-1, Snail, Slug and N-cadherin shared significantly higher expression in primary lesion, respectively, compared with those in adjacent normal gastric mucosa (p < 0.001, p = 0.001, p = 0.029 and p = 0.011, respectively). E-cadherin showed significantly lower expression in primary lesion (p < 0.001). Then, the correlations between clinicopathological features of patients with gastric cancer and expression levels of CNTN-1 and EMTrelated proteins in primary lesion were evaluated (Table 2). The expression levels of CNTN-1, E-cadherin, Slug and N-cadherin were closely correlated with T stage, pTNM stage and lymph node metastasis, respectively (p < 0.05). Snail was found to be significantly correlated with T stage and pTNM (p = 0.036 and p = 0.005, respectively), rather than with lymph node metastasis (p  = 0.260) (Table 2). Table 3 shows that the expression levels of CNTN-1 were positively related to those of Slug (r = 0.324, p = 0.005) and N-cadherin (r  = 0.244, p  = 0.039), but negatively related to E-cadherin expression (r  =  −0.430, p < 0.001) in primary lesion. Furthermore, the multivariate evaluation by logistic analysis revealed that lymph node metastasis (p < 0.001) was the independent risk factor for CNTN-1 expression. The expression levels of CNTN‑1 and EMT‑related proteins predict overall survival time of patients with gastric cancer The correlation of the expression of CNTN-1 or EMTrelated proteins with the prognosis of patients suffering from gastric cancer was also investigated (Fig. 1b–f), which

J Cancer Res Clin Oncol (2015) 141:2109–2120

2113

Fig. 1  Immunohistochemical expressions of CNTN-1 and EMTrelated markers and survival curves. a Immunohistochemical stainings of CNTN-1, E-cadherin, Snail, Slug and N-cadherin in primary

lesion and adjacent normal gastric mucosas. b–f The overall survival matching to the different expressions of CNTN-1 and EMT-related markers in the patients with gastric cancer. Scale bar = 100 µm

Table 1  Expressions of CNTN-1, E-cadherin, Snail, Slug and N-cadherin (Chi-square test) [n (%)]

those with CNTN-1 negative, Slug negative, or N-cadherin negative but E-cadherin positive, while there were no significant difference between patients with Snail positive and patients with Snail negative. Further investigation by multivariate analysis showed that CNTN-1 positive (p = 0.001) and Slug positive (p  = 0.002) were the independent risk factors for survival, respectively.

Proteins

Gastric cancer tissues Gastric normal mucosa p value tissues

CNTN-1 + –

43 (59.7) 29 (40.3)

22 (30.6) 50 (69.4)

<0.001

E-cadherin + –

31 (43.1) 41 (56.9)

52 (72.2) 20 (27.8)

<0.001

Snail + –

38 (52.8) 34 (47.2)

18 (25.0) 54 (75.0)

0.001

Slug + –

39 (54.2) 33 (45.8)

26 (36.1) 46 (63.9)

0.029

38 (52.8)

23 (31.9)

0.011

34 (47.2)

49 (68.1)

N-cadherin + –

showed that patients with gastric cancer, respectively, with CNTN-1 positive, Slug positive, or N-cadherin positive but E-cadherin negative survived significantly shorter than

CNTN‑1 expression might associate with in vitro invasive ability to some extent The expressions of CNTN-1 mRNA and CNTN-1 protein were detected by RT-PCR and Western blotting, respectively (Fig. 2a, b). Then, the cell invasive ability was demonstrated in vitro based on the principle of transwell invasion assay. Cells migrating through the Matrigel Matrix are shown in Fig. 2c. Obviously, the expressions of CNTN-1 mRNA and CNTN-1 protein were proved to increase in highly invasive SGC7901 and MKN45 cells but to reduce in poorly invasive KATO-III cells, suggesting that CNTN-1 expression was positively associated with gastric cancer cell invasiveness. Furthermore, the above-mentioned clinical results in this investigation demonstrated that the elevated expression of CNTN-1 was associated

13

2114

J Cancer Res Clin Oncol (2015) 141:2109–2120

Table 2  Correlation of CNTN1, E-cadherin, Snail, Slug and N-cadherin immunostaining with clinicopathological features in gastric cancer tissues (Chi-square) (n)

Features

n

CNTN-1 +

–

E-cadherin p

+

–

p

+

–

p

+

–

p

29 20 0.112 25 24 0.434 27 22 0.564 9 14 14 9 11 12

Gender Male Female

48 25 23 0.062 24 18 6

23 25 0.239 8 16

23 25 0.243 26 22 1.000 24 24 0.504 15 9 13 11 14 10

Tumor size (cm) ≥5 41 26 15 0.463 <5 31 17 14

16 25 0.427 15 16

24 17 0.260 25 16 0.182 22 19 0.863 14 17 14 17 18 15

Lauren’s classification Diffuse 21 15 6 0.194 Intestinal 51 28 23

8 13 0.585 23 28

13 8 0.320 12 9 0.745 9 12 0.279 25 26 27 24 29 22

Vascular invasion With 21 16 5 0.068 Without 51 27 24

6 15 0.111 25 26

11 10 0.965 13 8 0.398 13 8 0.320 27 24 26 25 25 26

Lymphatic vessel invasion With 31 22 9 0.091 Without 41 21 20

10 21 0.108 21 20

19 12 0.208 19 12 0.291 19 12 0.208 19 22 20 21 19 22

T stage T 1 + T2 T3 + T4

33 13 20 0.001 39 30 9

19 14 0.022 12 27

13 20 0.036 27 21 0.005 12 21 0.010 25 14 12 12 26 13

pTNM stage I + II 34 14 20 0.002 III + IV 38 29 9

20 14 0.011 11 27

12 22 0.005 12 22 0.002 13 21 0.019 26 12 27 11 25 13

<0.001 10 31 <0.001 24 17 0.260 28 13 0.006 26 15 0.038

31 11 20

21 10

Table 3  Spearman relation analysis among expressions of CNTN-1, E-cadherin, Snail, Slug and N-cadherin in gastric cancer tissues by immunohistochemistry (n) CNTN-1 +

–

r

p value

E-cadherin + –

11 32

20 9

−0.430

<0.001

Snail + –

25 18

13 16

0.131

0.274

Slug + –

29 14

10 19

0.324

0.005

27

11

0.244

0.039

16

18

13

p

N-cadherin

22 27 0.645 9 14

Without

–

–

Slug

Age (years) ≥60 49 30 19 0.704 <60 23 13 10

Lymph node metastasis With 41 32 9

N-cadherin +

+

Snail

14 17

11 20

12 19

with EMT-related proteins in gastric cancer. As shown in Fig.  2a, b, the expressions of E-cadherin mRNA and protein increased in poorly invasive gastric cancer cells but declined in highly invasive gastric cancer cells, and the expressions of Snail mRNA and protein were almost identical. The mRNA and protein levels of Slug and N-cadherin showed the opposite results of E-cadherin expression. All of these results implied that CNTN-1 might have a correlation with gastric cancer cells metastasis to some extent by inducing the EMT process via activating Slug, but probably not Snail. Transfection efficiency of lentiviruses containing CNTN‑1 shRNA on CNTN‑1 expression in MKN45 cells To further ascertain the function of CNTN-1 in gastric cancer metastasis, three human CNTN-1-targeted RNA interference expression vectors (siRNA) as well as a control

J Cancer Res Clin Oncol (2015) 141:2109–2120

2115

Fig. 2  Correlation of CNTN-1 expression and invasive ability in vitro. a, b The mRNA and protein expressions of CNTN-1 and EMTrelated markers in various gastric cancer cell lines. GAPDH mRNA

and protein were used as internal control. c Relative invasion abilities of three different gastric cancer cell lines. *p < 0.05 vs lower invasive KATO-III cells. Scale bar = 100 µm

Fig. 3  Knockdown of CNTN-1 expression with lentiviral expression vector in MKN45 cells. a The interfering efficiency of CNTN-1 in MKN45 cells transfected with lentiviral expression vectors (con sh, sh#1, sh#2 and sh#3) was determined by Western blotting, and GAPDH was used as internal control. b Immunofluorescence stain-

ing to demonstrate the interfering efficiency of CNTN-1 in different groups. Targeted protein was stained in red color, and the nuclei were stained in blue color. GFP meant green fluorescent protein. **p < 0.001, *p < 0.05 vs group con sh

vector to knock down CNTN-1 expression were used in MKN45 cells. Compared with those in the con sh group, the CNTN-1 protein expressions dramatically reduced

in the sh#2 group (Fig. 3a). Similarly, the most widely decreased staining of the sh#2 group was observed by immunofluorescence staining (Fig. 3b). These results

13

2116

J Cancer Res Clin Oncol (2015) 141:2109–2120

Fig. 4  Suppression effects of CNTN-1 expression on EMT-related markers and the invasive ability of MKN45 cells. a After stable knockdown of CNTN-1, EMT-related markers were detected by Western blotting, and GAPDH was used as internal control. b, d Effects of inhibition of CNTN-1 expression on the migration capacity of MKN45 cells by transwell assay and cell scratch-wound assay, respectively. The account of migratory cells and the measurement

of the gap were taken under a microscope. c Silencing effects of CNTN-1 expression on the invasive ability of MKN45 cells detected by transwell assay. The invasive cells were counted under a microscope. e Effects of knockdown of CNTN-1 expression on the proliferation capacity of MKN45 cells by CCK-8 assay. **p < 0.001 vs group parental. Scale bar = 100 µm

indicated that the sh#2 group had the highest interfering efficiency in MKN45 cells. Therefore, the MKN45 sh#2 group cells were used for follow-up experiments.

CNTN‑1 modulates cell migration and invasion in MKN45 cells

CNTN‑1 promotes EMT in MKN45 cells To prove whether CNTN-1 knockdown induces EMT in MKN45 cells, the quantitative expression levels of these biomarkers were measured by Western blotting in the parental MKN45 cells, the con sh MKN45 cells and the sh#2 MKN45 cells. Compared with those in the parental and those in the con sh groups, the protein levels of CNTN-1, Slug and N-cadherin in the sh#2 group significantly decreased. Inversely, the protein levels of E-cadherin in the sh#2 group dramatically increased (Fig. 4a). However, knockdown of CNTN-1 in the sh#2 group did not affect Snail expression (Fig. 4a), which suggested that EMT process mediated by CNTN-1 was due to Slug rather than Snail, based on the above results of this investigation.

13

The migration and invasion capacities of the parental MKN45 cells, the con sh MKN45 cells and the sh#2 MKN45 cells were compared using transwell chambers. The migratory and invasive activities were strongly impaired in the sh#2 group cells (Fig. 4b, c). On the other hand, the cell scratch-wound assay was used in order to provide further evidence for the effect of CNTN-1 expression on the ability of cell migration (Fig. 4d). As revealed, the sh#2 group cells migrated observably slower compared with those in the parental group or in the con sh group. Taken together, these above results indicated that knockdown of CNTN-1 expression played a critical role in migration and invasion of MKN45 cells. To rule out the possibility that the effects of CNTN-1 on cell migration and invasion were attributable to different proliferation rates, the cellular growth rates in three groups were compared. As shown in Fig. 4e, all cells exhibited similar

J Cancer Res Clin Oncol (2015) 141:2109–2120

proliferation rates under the same conditions, suggesting that the impaired MKN45 cells migration and invasion via knockdown of CNTN-1 expression were not associated with cellular proliferation rate.

2117

To characterize whether CNTN-1 could affect tumor formation in vivo, cells from the con sh group or sh#2 group were injected subcutaneously into the right flanks of nude mice. The weight and the volume of tumor were almost identical between the two groups (Fig. 5a), and expression of CNTN-1 was further demonstrated to be downregulated,

while E-cadherin expression upregulated in the sh#2 group in xenograft tumors (Fig. 5b), suggesting that CNTN-1 was not essential for tumor formation by these cells. To explore whether loss of CNTN-1 expression could inhibit cell metastasis in vivo, cells from above-mentioned groups were injected into the tail vein of nude mice, respectively. The lungs of nude mice were scanned via Micro-PET/CT exposure, and the number of lung metastatic nodules was calculated 4 weeks later. Obviously, compared with those in the sh#2 group, images showed that the lung metastatic foci in the con sh group were much more (Fig. 5c). Consistent with the result of Micro-PET/CT imaging, a significantly less number of lung metastatic nodules could be counted

Fig. 5  Influences of CNTN-1 knockdown on the proliferation and the metastasis of cancer cells in vivo. a Tumor in group con sh and group sh#2 in nude mice. Tumor volume was measured every 5 days. At 30 days after inoculation, nude mice were killed and tumors were weighed. b Expressions of CNTN-1 and E-cadherin in xenograft tumors of group con sh and group sh#2 were determined by Western blotting. c, d Tumor metastasis in the lung of nude mice using lung metastasis model. c Observation on the lungs of nude mice by

Micro-PET/CT scanning. d At 4 weeks after injection of cancer cells, nude mice were killed, and the average metastasis nodules in lungs were counted. e Metastatic tumor nodules (shown by red arrows) could be histologically identified by HE staining in the lungs of nude mice. f Expressions of CNTN-1 and E-cadherin in metastasis nodules in lungs of nude mice were determined by immunohistochemistry. n = 6 in each group. **p < 0.001 vs group con sh. Scale bar = 100 µm

CNTN‑1 is essential for MKN45 cells metastasis in vivo

13

2118

in the sh#2 group compared with those in the con sh group (Fig.  5d, e). Additionally, expression of CNTN-1 was decreased, while E-cadherin expression was increased in the sh#2 group in metastasis nodules in the lungs (Fig. 5f), which was consistent with the results in vitro. Therefore, knockdown of CNTN-1 expression in MKN45 cells could be concluded to inhibit the formation and the development of metastatic nodules in the lungs.

Discussion Identification of novel targets in the metastatic process of gastric cancer is promising. The present study reveals that CNTN-1 plays a significant role in the invasion and the metastasis of gastric cancer. CNTN-1, highly expressed in gastric cancer compared with that in adjacent normal gastric mucosa, was found to be correlated with clinical metastasis and patient survival. It was revealed that the suppression of CNTN-1 expression in gastric cancer cell could inhibit the migration and the invasion of gastric cancer cells in vitro and gastric cancer cell metastasis in vivo. Furthermore, this study demonstrated that CNTN-1 overexpression was closely correlated with EMT markers (E-cadherin, Slug and N-cadherin) changes proved by immunohistochemistry in primary lesion of patients with gastric cancer and that knockdown of CNTN-1 could reverse EMT markers alterations via silencing of Slug expression (not Snail) in gastric cancer MKN45 cells. Based on these observations, this investigation of ours not only contributed to describe a role for CNTN-1 in gastric cancer but also explained, at least partially, the mechanism through which CNTN-1 facilitated gastric cancer metastasis. CNTN-1, located in the 12q11–q12 chromosomal region (Berglund and Ranscht 1994) and composed of six Ig repeats and four fibronectin type III domains, is the first identified member of the CNTN family of NCAMs (Shimoda and Watanabe 2009). NCAM has been observed to be upregulated coupled with loss of E-cadherin, and a subpopulation of NCAM protein translocates to lipid rafts to bind to p59Fyn (Fyn, a member of the Src kinase family regulating cell mobility). Subsequent activation of p59Fyn and its effector substrate focal adhesion kinase (FAK) leads to increased cell spreading and migration by β1-integrindependent focal adhesions (Lehembre et al. 2008). Notably, CNTN-1 has been found to interact with other cell surface proteins deemed to participate in various signal transduction pathways. This finding is consistent with previous investigations suggesting that CNTN-1 binds to receptor protein tyrosine phosphatase β (RPTP-β) to facilitate neurite outgrowth (Sakurai et al. 1997) and RPTP-α to transduce extracellular signals to Fyn kinase (Umemori et al. 1994) that regulates cell mobility. Such findings as above

13

J Cancer Res Clin Oncol (2015) 141:2109–2120

mentioned and demonstrations in this study of ours are in line with the fact that CNTN-1 plays a critical role in cancer metastasis. However, the studies involving the mechanism by which CNTN-1 promotes cancer metastasis remain insufficient to deduce the mechanism of gastric cancer metastasis. Here, we demonstrated, for the first time according to our knowledge, that one of the unclear mechanisms is how to inhibit E-cadherin expression in gastric cancer. The idea that is now commonly known as EMT arose in the early 1980s from observations by Elizabeth Hay (Hay 1995), who delineated the process of from epithelial to mesenchymal phenotype in the primitive streak of chick embryos. Moreover, subsequent studies revealed that loss of E-cadherin was a crucial promoter of invasion and metastasis of epithelial-origin cancers (Onder et al. 2008; Lim et al. 2000). The acquisition of mesenchymal cell markers (N-cadherin and vimentin) and loss of epithelial cell markers (E-cadherin) are mainly due to the modulation of such transcription factors as Slug, Snail, ZEB1, SIP1 and Twist (Peinado et al. 2004; Wu and Zhou 2010). In line with the loss of E-cadherin principally on account of transcription activation, knockdown of CNTN-1 enhanced E-cadherin expression through inhibition of Slug and SIP1 in lung cancer (Yan et al. 2013). Considering CNTN-1 as a cell surface protein, CNTN-1 may indirectly affect E-cadherin expression. Here, we ascertained a reduction in transcription factor such as Slug rather than Snail, which might contribute to the decrease in E-cadherin expression due to the indirect mediation of CNTN-1. This deduction is based on our research of the suppression of CNTN-1 expression in MKN45 cells as well as the investigation regarding the correlation of CNTN-1 with EMT-related proteins in the primary lesion and the adjacent normal gastric mucosas in 72 patients with gastric cancer. Nonetheless, further insights into the pathways involved in the process whereby CNTN-1 activates transcription factor Slug are needed to improve an in-depth understanding of the full view to cancerous metastatic mechanism. Additional investigations that whether other transcription factors are related to the process of CNTN-1-mediated reduction in E-cadherin are also required. How to achieve the most effective silencing of CNTN-1 gene expression in cancer cells? As reported, RNAi had been used as a therapeutic approach (Elbashir et al. 2002). To make the function of siRNA more effective, the present study used the shRNA-based recombinant lentiviral vectors to produce siRNA. Results in this investigation of ours showed that the sh#2 group could effectively suppress CNTN-1 expression in MKN45 cells. Following this foundational outcomes, knockdown of CNTN-1 expression in the sh#2 group was identified to inhibit cell migration and invasion but not affect cell proliferation in vitro.

J Cancer Res Clin Oncol (2015) 141:2109–2120

Furthermore, two in vivo models were used in this study in which one is subcutaneous of transplanted cancer cells in mice and another is intravenous of injection of cancer cells into tail vein of mice. Consistent with the results in vitro, the outcomes in vivo revealed that CNTN-1 significantly affected the metastasis rather than the proliferation in gastric cancer cells. Although the construction of nude mice metastatic models in the research does not follow the principle of physiologic metastatic model, it is able to imitate the overflow of cancer cells from blood vessels into the target organ which is deemed to be a key step in the metastatic process (Elkin and Vlodavsky 2001). Thus, as in previous researches (Su et al. 2006a; Huang et al. 2014; Johnson et al. 2012; Yang et al. 2011), this model has been used to compare the metastatic potential among different groups. Furthermore, CT has been developed specifically for high anatomical resolution imaging of small animals (Berger et al. 2002; Paulus et al. 2001). To find the metastatic nodules in lung of nude mice in due course, Micro-PET/CT was employed in non-invasive in vivo imaging to monitor tumor growth in the present study. In summary, CNTN-1 was selectively expressed in cancer tissues, and its expression was correlated with tumor stage, lymph node metastasis and patient survival. Silencing of CNTN-1 expression suppressed the invasive ability of MKN45 gastric cancer cells. Regarding the mechanisms through which CNTN-1 promoted cancer cell metastasis, EMT alteration was observed through CNTN-1-induced activation of Slug. The above findings suggest that CNTN-1 is involved in invasion and metastasis of MKN45 gastric cancer cells and seems to be therefore an outstanding candidate for a targeted therapy for gastric cancer. However, the phenomenon was determined only in MKN45 gastric cancer cells. Therefore, to deepen the understanding of the real role of CNTN-1-mediated migration and invasion of gastric cancer cells, it is worth investigating whether the phenomenon in MKN45 cells exists in other gastric cancer cells in the future. Acknowledgments  This work was supported by Grants of the National Natural Science Foundation (81101850), the Shanghai Municipal Health Bureau Foundation of China (20134393) and Science Research Foundations of Shanghai Jiao Tong University School of Medicine (13XJ10028). The excellent pathological and immunohistochemical assistance from Dr. Hong-Xiu Han is highly appreciated. Conflict of interest  We declare that we have no conflict of interest. Ethical standards  The study has been approved by Ethics Committee of 3rd People’s Hospital, School of Medicine, Shanghai Jiao Tong University. Preoperative informed consent was obtained from each patient registered in this study in accordance with institutional guidance. The pathological samples were taken from the surgical resection specimens, which would not result in any disadvantages to health and prognosis of patients. We are committed to maintain the privacy

2119 of patients’ information. Furthermore, the care and use of laboratory animals was in accordance with the principles and standards set forth in the Principles for Use of Animals (National Guide for Grants and Contracts). Open Access  This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

References Berger F, Lee YP, Loening AM, Chatziioannou A, Freedland SJ, Leahy R, Lieberman JR, Belldeqrun AS, Sawyers CL, Gambhir SS (2002) Whole-body skeletal imaging in mice utilizing microPET: optimization of reproducibility and applications in animal models of bone disease. Eur J Nucl Med Mol Imaging 29:1225– 1236. doi:10.1007/s00259-002-0850-1 Berglund EO, Ranscht B (1994) Molecular cloning and in situ localization of the human contactin gene (CNTN1) on chromosome 12q11-q12. Genomics 21:571–582. doi:10.1006/geno.1994.1316 Catalano V, Labianca R, Beretta GD, Gatta G, De Braud F, Van Cutsem E (2009) Gastric cancer. Crit Rev Oncol Hematol 71:127– 164. doi:10.1016/j.critrevonc.2009.01.004 Elbashir SM, Harborth J, Weber K, Tuschl T (2002) Analysis of gene function in somatic mammalian cells using small interfering RNAs. Methods 26:199–213. doi:10.1016/S1046-2023(02)00023-3 Elkin M, Vlodavsky I (2001) Tail vein assay of cancer metastasis. Curr Protoc Cell Biol Chapter 19. Unit 19.2. doi:10.1002/0471143030.cb1902s12 Falk J, Bonnon C, Girault JA, Faivre-Sarrailh C (2002) F3/contactin, a neuronal cell adhesion molecule implicated in axogenesis and myelination. Biol Cell 94:327–334. doi:10.1016/ S0248-4900(02)00006-0 Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M, Parkin DM, Forman D, Bray F (2015) Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer 136:E359–E386. doi:10.1002/ijc.29210 Gavert N, Sheffer M, Raveh S, Spaderna S, Shtutman M, Brabletz T, Barany F, Paty P, Notterman D, Domany E, Ben-Ze’ev A (2007) Expression of L1-CAM and ADAM10 in human colon cancer cells induces metastasis. Cancer Res 67:7703–7712. doi:10.1158/0008-5472.CAN-07-0991 Hay ED (1995) An overview of epithelio-mesenchymal transformation. Acta Anat 154:8–20 Hu QD, Ang BT, Karsak M et al (2003) F3/contactin acts as a functional ligand for Notch during oligodendrocyte maturation. Cell 115:163–175. doi:10.1016/S0092-8674(03)00810-9 Huang J, Xiao D, Li G, Ma J, Chen P, Yuan W, Hou F, Ge J, Zhong M, Tang Y, Xia X, Chen Z (2014) EphA2 promotes epithelial– mesenchymal transition through the Wnt/β-catenin pathway in gastric cancer cells. Oncogene 33:2737–2747. doi:10.1038/ onc.2013.238 Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D (2011) Global cancer statistics. CA Cancer J Clin 61:69–90. doi:10.3322/caac.20107 Johnson JL, Pillai S, Pernazza D, Sebti SM, Lawrence NJ, Chellappan SP (2012) Regulation of matrix metalloproteinase genes by E2F transcription factors: Rb-Raf-1 interaction as a novel target for metastatic disease. Cancer Res 72:516–526. doi:10.1158/00085472.CAN-11-2647

13

2120 Lehembre F, Yilmaz M, Wicki A, Schomber T, Strittmatter K, Ziegler D, Kren A, Went P, Derksen PWB, Berns A, Jonkers J, Christofori G (2008) NCAM-induced focal adhesion assembly: a functional switch upon loss of E-cadherin. EMBO J 27:2603–2615. doi:10.1038/emboj.2008.178 Lim SC, Jang IG, Kim YC, Park KO (2000) The role of E-cadherin expression in non-small cell lung cancer. J Korean Med Sci 15:501–506 Liu PF, Zhou JD, Zhu H, Xie L, Wang FJ, Liu BT, Shen WD, Ye W, Xiang B, Zhu XZ, Shi RH, Zhang SY (2011) VEGF-C promotes the development of esophageal cancer via regulating CNTN-1 expression. Cytokine 55:8–17. doi:10.1016/j.cyto.2011.03.008 Liu PF, Chen SM, Wu WT, Liu BT, Shen WD, Wang FJ, He XW, Zhang SY (2012) Contactin-1 (CNTN-1) overexpression is correlated with advanced clinical stage and lymph node metastasis in oesophageal squamous cell carcinomas. Jpn J Clin Oncol 42:612–618. doi:10.1093/jjco/hys066 Nagini S (2012) Carcinoma of the stomach: a review of epidemiology, pathogenesis, molecular genetics and chemoprevention. World J Gastrointest Oncol 4:156–169. doi:10.4251/wjgo.v4.i7.156 Onder TT, Gupta PB, Mani SA, Yang J, Lander ES, Weinberg RA (2008) Loss of E-cadherin promotes metastasis via multiple downstream transcriptional pathways. Cancer Res 68:3645– 3654. doi:10.1158/0008-5472.CAN-07-2938 Paulus MJ, Gleason SS, Easterly ME, Foltz CJ (2001) A review of high-resolution X-ray computed tomography and other imaging modalities for small animal research. Lab Anim 30:36–45 Peinado H, Ballestar E, Esteller M, Cano A (2004) Snail mediates E-cadherin repression by the recruitment of the Sin3A/histone deacetylase 1 (HDAC1)/HDAC2 complex. Mol Cell Biol 24:306–319. doi:10.1128/MCB.24.1.306-319.2004 Qin XJ, Dai DJ, Gao ZG, Huan JL, Zhu L (2011) Effect of lentivirusmediated shRNA targeting VEGFR-3 on proliferation, apoptosis and invasion of gastric cancer cells. Int J Mol Med 28:761–768. doi:10.3892/ijmm.2011.758 Sakurai T, Lustig M, Nativ M, Hemperly JJ, Schlessinger J, Peles E, Grumet M (1997) Induction of neurite outgrowth through contactin and Nr-CAM by extracellular regions of glial receptor tyrosine phosphatase beta. J Cell Biol 136:907–918. doi:10.1083/jcb.136.4.907 Shimoda Y, Watanabe K (2009) Contactins: emerging key roles in the development and function of the nervous system. Cell Adh Migr 3:64–70. doi:10.4161/cam.3.1.7764

13

J Cancer Res Clin Oncol (2015) 141:2109–2120 Su JL, Yang CY, Shih JY, Wei LH, Hsieh CY, Jeng YN, Wang NY, Yang PC, Kuo ML (2006a) Knockdown of contactin-1 expression suppresses invasion and metastasis of lung adenocarcinoma. Cancer Res 66:2553–2561. doi:10.1158/0008-5472.CAN-05-2645 Su JL, Yang PC, Shih JY, Yang CY, Wei LH, Hsieh CY, Chou CH, Jeng YM, Wang MY, Chang KJ, Hung MC, Kuo ML (2006b) The VEGF-C/Flt-4 axis promotes invasion and metastasis of cancer cells. Cancer Cell 9:209–223. doi:10.1016/j.ccr.2006.02.018 Umemori H, Sato S, Yagi T, Aizawa S, Yamamoto T (1994) Initial events of myelination involve Fyn tyrosine kinase signalling. Nature 367:572–576. doi:10.1038/367572a0 Van Kilsdonk JWJ, Wilting RH, Bergers M, van Muijen GNP, Schalkwijk J, van kempen LCLT, Swart GWM (2008) Attenuation of melanoma invasion by a secreted variant of activated leukocyte cell adhesion molecule. Cancer Res 68:3671–3679. doi:10.1158/0008-5472.CAN-07-5767 Wu Y, Zhou BP (2010) TNF-alpha/NF-kappaB/Snail pathway in cancer cell migration and invasion. Br J Cancer 102:639–644. doi:10.1038/sj.bjc.6605530 Yan J, Wong N, Hung C, Chen WXY, Tang DM (2013) Contactin-1 reduces E-cadherin expression via activating AKT in lung cancer. PLoS One 8:e65463. doi:10.1371/journal.pone.0065463 Yang CH, Yue JM, Pfeffer SR, Handorf CR, Pfeffer LM (2011) MicroRNA miR-21 regulates the metastatic behavior of B16 melanoma cells. J Biol Chem 286:39172–39178. doi:10.1074/ jbc.M111.285098 Yang M, Gu YY, Peng H, Zhao M, Wang J, Huang SK, Yuan XH, Li J, Sang JL, Luo Q, Huang CZ (2014) NAIF1 inhibits gastric cancer cells migration and invasion via the MAPK pathways. J Cancer Res Clin Oncol. doi:10.1007/s00432-014-1865-2 Yu JW, Wu SH, Lu RQ, Wu JG, Ni XC, Zhou GC, Jiang HG, Zheng LH, Li XQ, Du GY, Jiang BJ (2013) Expression and significances of contactin-1 in human gastric cancer. Gastroenterol Res Pract 2013:210205. doi:10.1155/2013/210205 Zhang H, Liu LL, Wang YF, Zhao GH, Xie RG, Liu CH, Xiao X, Wu KC, Nie YZ, Zhang HB, Fan DM (2013) KLF8 involves in TGF-beta-induced EMT and promotes invasion and migration in gastric cancer cells. J Cancer Res Clin Oncol 139:1033–1042. doi:10.1007/s00432-012-1363-3