MFAP2 Promotes Epithelial–Mesenchymal Transition in Gastric Cancer Cells by Activating TGF-Β/SMAD2/3 Signaling Pathway

Journal name: OncoTargets and Therapy Article Designation: Original Research Year: 2018 Volume: 11 OncoTargets and Therapy Dovepress Running head verso: Wang et al Running head recto: MFAP2 supports EMT of GC cells by activating TGF-β signaling pathways open access to scientific and medical research DOI: 160831

Open Access Full Text Article Original Research MFAP2 promotes epithelial–mesenchymal transition in gastric cancer cells by activating TGF-β/SMAD2/3 signaling pathway

Jian-Kai Wang1 Introduction: Microfibril-associated protein 2 (MFAP2) is an extracellular matrix protein that Wen-Juan Wang2 interacts with fibrillin to modulate the function of microfibrils. MFAP2 has been reported to play Hong-Yi Cai1 a significant role in obesity, diabetes, and osteopenia, and has been shown to be upregulated in Bin-Bin Du3 head and neck squamous cell carcinoma. However, the molecular function and prognostic value Ping Mai4 of MFAP2 have never been reported in gastric cancer (GC) or any other tumors. Li-Juan Zhang1 Methods: The current study investigated the expression patterns, prognostic significance, functional role, and possible mechanisms of MFAP2 in GC. Wen Ma1 Results: We demonstrated that MFAP2 was overexpressed in GC tissues, and its overexpres- Yong-Guo Hu1 sion was significantly correlated with poor overall and disease-free survival in patients with 1 Shi-Fang Feng GC. Moreover, we found that MFAP2 promoted the proliferation, migration, invasion, and 1 Guo-Ying Miao epithelial–mesenchymal transition (EMT) phenotype in GC cells. MFAP2 might modulate 1Department of Radiotherapy, EMT of GC cells by activating the TGF-β/SMAD2/3 signaling pathway. Gansu Provincial Hospital, Conclusion: These findings provide novel evidence that MFAP2 plays a crucial role in the Lanzhou, Gansu 730000, China; 2Physical Examination Center, progression of GC. Therefore, MFAP2 may be a promising prognostic marker and a potent The Third People’s Hospital of anticancer agent. Gansu, Lanzhou, Gansu 730000, gastric cancer, MFAP2, prognosis, epithelial–mesenchymal transition, TGF-β China; 3Department of Anorectal Keywords: Surgery, Gansu Provincial Hospital, Lanzhou, Gansu 730000, China; 4Department of Gastroenterology, Introduction Gansu Provincial Hospital, Lanzhou, Gastric cancer (GC) is the fourth most common cancer and the second most common Gansu 730000, China cause of cancer mortality worldwide.1,2 Surgical treatment can cure 90% of patients with early GC,3 but most patients are diagnosed with GC in its late stages.4 Approximately 60% of patients with GC have locally advanced and metastatic tumors at the time of surgery, resulting in a relatively low therapeutic efficacy.5 Although the clinical treatment of GC has been significantly enhanced in the recent years due to improvements in surgical techniques and chemotherapy, the long-term survival rate of patients with GC remains poor.6 At present, the valid therapeutic methods for advanced GC with invasion and metastasis remain poor and limited.7 As a result, investigation of the molecular mechanisms underlying GC invasion and metastasis will provide a strong theoretical basis for its diagnosis and treatment. Correspondence: Guo-Ying Miao Microfibril-associated protein 2 (MFAP2), also known as microfibril-associated Department of Radiotherapy, Gansu glycoprotein 1 (MAGP1), is an abundant component of microfibrils.8,9 There is a Provincial Hospital, 204 Donggangxi Road, Lanzhou, Gansu 730000, China fibrillin-binding domain in the carboxyl-terminal region of MFAP2, which contains Tel +86 153 3986 6618 13 cysteine residues and binds MFAP2 to the extracellular matrix and an acidic amino- Fax +86 931 8281 408 Email [email protected] terminal region containing a growth factor interaction motif capable of interacting with submit your manuscript | www.dovepress.com OncoTargets and Therapy 2018:11 4001–4017 4001 Dovepress © 2018 Wang et al. This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms.php and incorporate the Creative Commons Attribution – Non Commercial (unported, v3.0) License (http://creativecommons.org/licenses/by-nc/3.0/). By accessing the work you http://dx.doi.org/10.2147/OTT.S160831 hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms (https://www.dovepress.com/terms.php). Wang et al Dovepress the active form of transforming growth factor beta (TGF-β), histopathologically diagnosed with primary GC by the Depart- but not with the latent form of the growth factor.10,11 MFAP2 ment of Pathology, and the patients had not been treated with plays an important role in the extracellular deposition of chemotherapy or radiotherapy prior to surgery. All cases were fibrillin-1 during development of the human ciliary zonule,12 followed up for at least 5 years. All patients were treated and an in vivo study found that MFAP2 plays a key role in with radical gastrectomy and D2 lymphadenectomy followed hemostasis and thrombosis because MFAP2-deficient mice with postoperative chemotherapy. The cases were staged and showed delayed thrombotic occlusion of the carotid artery graded according to the Cancer Staging Manual (Seventh following injury, prolonged bleeding, and lower platelet Edition) of the American Joint Committee on Cancer.17 The number.13 normal tissues adjacent to the tumors were used as controls. Thus far, studies of MFAP2 have mainly focused on its This study was approved by the Ethics Committee of Gansu function in modulating tropoelastin deposition onto micro- Provincial Hospital. Written informed consent was obtained fibrils for formation of elastic fibers,14 and few studies have from all study subjects. The clinical parameters of patients been conducted concerning the role of MFAP2 in cancers. involved in the study are summarized in Table S1. MFAP2 was identified as one of the most co-expressed genes of the NF-κB/Snail/YY1/RKIP circuitry in multiple Cell lines and culture myeloma.15 In head and neck squamous cell carcinoma, Human GC cell lines BGC823, MKN-45, MGC803, MFAP2 has been shown to be significantly upregulated SGC7901, and AGS were purchased from the Shanghai in tumor tissues, especially in lymph node metastasis.16 Institute of Biochemistry and Cell Biology, Chinese Academy However, the function and prognostic role of MFAP2 were of Sciences (Shanghai, China). All cells were maintained in not investigated in these two studies. To the best of our Roswell Park Memorial Institute (RPMI)-1640 medium knowledge, no studies have reported the molecular role and supplemented with 10% fetal bovine serum (FBS; Gibco, clinicopathological significance of MFAP2 in GC or any Grand Island, NY, USA), 100 µg/mL streptomycin, and other type of malignancy. As a result, the elucidation of the 100 U/mL penicillin (Invitrogen, Carlsbad, CA, USA). expression and function of MFAP2 in GC will promote the Cells were cultured at 37°C in a humidified atmosphere with knowledge of the function of MFAP2 in the progression 5% CO2. For transforming growth factor-beta 1 (TGF-β1) of cancer. (Sigma-Aldrich Co., St Louis, MO, USA) treatment, cells In the current study, we report that MFAP2 is overex- were cultured in RPMI-1640 medium supplemented with pressed in human GC tissues and its overexpression cor- 10% FBS containing 5 ng/mL TGF-β1 for 24 h. relates with tumor invasion depth, lymph node metastasis, distant metastasis, and tumor node metastasis (TNM) stage. Immunohistochemistry (IHC) Patients with GC with high MFAP2 expression are associated For the immunohistochemical detection of MFAP2, 4 μm- with poor overall survival (OS) and disease-free survival thick sections from the formalin-fixed, paraffin-embedded (DFS). Knockdown of MFAP2 significantly abrogated the tissues were used. The samples were deparaffinized in xylene proliferative, migratory, and invasive properties as well as and rehydrated through a graded series of ethanol washes. epithelial–mesenchymal transition (EMT) phenotype of GC After the endogenous peroxidase was inhibited by expo- cells. Moreover, our results demonstrated that MFAP2 func- sure to 3% hydrogen peroxide for 10 min and the antigen tions upstream of the TGF-β/SMAD2/3 signaling pathway was retrieved, the sections were incubated with primary as an activator. Our results suggest that MFAP2 may be antibody against MFAP2 (Sigma-Aldrich Co.; 1:100) at considered as a promising prognostic marker and a potential 4°C overnight. Sections were then incubated with horse- therapeutic target for human GC. radish peroxidase (HRP)-conjugated secondary antibodies (Dako, Glostrup, Denmark) according to the instructions Materials and methods of the Dako REAL EnVision Detection System. For the Tissue specimens from patients with GC evaluation of MFAP2 staining, a staining score value was Paraffin-embedded sections of GC tissues and correspond- calculated as the intensity staining (0: negative, 1: weak, ing adjacent normal tissues were obtained from 168 patients 2: moderate, 3: strong) multiplied by the percentage of posi- who underwent surgical treatment in Gansu Provincial tive tumor cells (1: 1%–25%; 2: 26%–50%; 3: 51%–75%; Hospital between August 2009 and December 2010. The 4: .75%). An IHC score of 3 was determined as the optimal samples collected from all the patients were clinically and cutoff value for the 168 patients with GC as calculated by

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X-tile software version 3.6.1 (Yale University School of real-time polymerase chain reaction (qRT-PCR) and Medicine, New Haven, CT, USA)18 and SPSS 19.0 soft- Western blotting analysis. ware (IBM Corporation, Armonk, NY, USA) (Figure S1). As a result, the final staining scores #3 were regarded as Western blotting analysis low expression and the scores $4 were regarded as high Total proteins from cell samples were prepared using radio- expression. immunoprecipitation lysis buffer (Beyotime Biotechnology Co., Shanghai, China) containing phenylmethanesulfonyl Bioinformatics analysis fluoride and phosphatase inhibitors. The protein lysates were The Gene Expression Omnibus (GEO) database (www. quantified using a BCA (bicinchoninic acid) protein assay ncbi.nlm.nih.gov/geo) was explored to analyze the mRNA kit (Thermo Fisher Scientific, San Jose, CA, USA). Equal expression of MFAP2 and survival in patients with GC. amounts of protein lysates were separated using a 10% sodium The accession numbers of the GEO database involved in dodecyl sulfate-polyacrylamide electrophoresis gel and trans- the study were GSE29272, GSE27342, and GSE54129. ferred onto polyvinylidene difluoride membranes (Millipore, Expression data were log2 transformed and quantile normal- Bedford, MA, USA) for 2 h. The membranes were blocked ized using the R software (version 3.2.5; R Foundation for with tris-buffered saline with 0.05% Tween containing 5% Statistical Computing, Vienna, Austria) with packages of the non-fat milk for 2 h at room temperature and then incubated BioConductor project (http://www.bioconductor.org/)19 as overnight at 4°C with targeted primary antibodies: anti- previously described. For The Cancer Genome Atlas (TCGA) MFAP2 (1:1,000; Abcam, Cambridge, UK) anti-Vimentin database analysis, an online tool Gene Expression Profiling (1:1,000; Cell Signaling Technology (CST), Beverly, MA, Interactive Analysis (GEPIA) (http://gepia.cancer-pku.cn/)20 USA), anti-E-cadherin (1:1,000; CST), anti-Snail (1:1,000; was explored to compare the mRNA levels of MFAP2 in CST), anti-SMAD3 (1:1,000; CST), anti-p-SMAD3 (1:1,000; 408 GC tissues and 211 normal tissues (TCGA-STAD). In CST), anti-SMAD2 (1:1,000; CST), anti-p-SMAD2 (1:1,000; addition, the OS and DFS analyses of patients with GC in the CST), anti-TGF-β1 (1:1,500; Abcam), and anti-β-actin TCGA-STAD database were conducted using the online tool (1:1,000; CST). The membranes were incubated with HRP- GEPIA and results were downloaded from the website. conjugated secondary antibody (1:2,000; CST) at room Another online tool KM plotter database (http://www. temperature for 1 h. The bands were then visualized using kmplot.com) was explored to conduct the survival analyses an enhanced chemiluminescence kit (Bio-Rad, Hercules, of patients with GC, which contains updated gene expression CA, USA) and analyzed using Image Lab 4.0 (Bio-Rad) data and survival information from a total of 876 patients imaging software. β-actin was used as an internal control. with GC.21 The association between OS/DFS and MFAP2 mRNA expression levels was calculated by allocating the qRT-PCR assay patients to high- and low-expression groups, according to Total cellular RNA was extracted using TRIzol reagent the best performing threshold. Processed data from the KM (Invitrogen) according to the manufacturer’s instructions. plotter were then downloaded and survival curves were Total RNA (500 ng) was reverse-transcribed into comple- plotted using GraphPad Prism 5.0 (GraphPad Software, mentary DNA using the PrimeScript RT Master Mix (Takara La Jolla, CA, USA). Bio, Shiga, Japan). qRT-PCR was then performed in a 10 μL reaction volume using the SYBR PremixEx Taq™ (TaKaRa) Lentivirus construction and transfection with appropriate primers (Table S3) according to the manu- The construction of short-hairpin RNA (shRNA) targeting facturer’s instructions. β-actin was used to normalize the MFAP2 (shMFAP2) and control shRNA (mock) were relative expression levels of other target genes. The n-fold designed and synthesized by GeneChem (Shanghai, China; change in mRNA expression was analyzed according to 2−∆∆Ct Table S2). Transfections were performed in accordance method to calculate the relative expression of the targeted with the manufacturer’s protocol of Lipofectamine 2000 genes. Each experiment was performed in triplicates. (Invitrogen) in BGC823 and MKN-45 GC cells due to their relatively high MFAP2 expression. Stably transfected Colony-formation assay cells were selected in media with 5 μg/mL puromycin Cells were suspended and seeded into six-well plates (Sigma-Aldrich Co.) for 2 weeks, and the knockdown (500 cells/well) and incubated at 37°C in RPMI-1640 of MFAP2 in GC cells was confirmed by quantitative medium containing 10% FBS for 14 days. The medium was

OncoTargets and Therapy 2018:11 submit your manuscript | www.dovepress.com 4003 Dovepress Wang et al Dovepress changed every 3 days. When most colonies contained more The mean number of cells was determined by selecting five than 50 cells, the cells were fixed with 4% paraformaldehyde random mid-power fields and the mean was calculated. All and stained using 0.1% crystal violet. Only colonies contain- experiments were performed in triplicates. ing more than 50 cells were counted under light microscopy. Experiments were performed in triplicates. Statistical analysis Data were analyzed using the SPSS 19.0 software (SPSS Inc., Cell proliferation assay Chicago, IL, USA). Comparisons between two groups were Cell proliferation was examined using a Cell Counting Kit-8 performed using Student’s t-test (two-sided). The relation- (CCK-8; Dojindo, Kumamoto, Japan) according to the manu- ship between MFAP2 expression and the clinicopathological facturer’s instructions, and GC cells were seeded in 96-well characteristics of the patients with GC was evaluated using plates at a density of 3×103 cells per well. After 1, 2, 3, and the chi-squared test. Ranked data were displayed by the rank sum test. In addition, OS and DFS were analyzed using the 4 days of culture at 37°C with 5% CO2, CCK-8 solution was added to each well and incubated for 1 h. The absorbance at Kaplan–Meier method and the P-value was assessed by the 450 nm was measured with a plate reader. The experiments log-rank test. A Cox proportional hazards regression analysis were performed in triplicates. was used for univariate and multivariate analyses of prognostic values to investigate the differences in all possible Wound healing assay death risk factors. Values were presented as mean ± standard deviation. P,0.05 was considered to indicate a statistically BGC823 and MKN-45 GC cells were seeded into six-well significant difference. plates and cultured in RPMI-1640 containing 10% FBS until they reached 80% confluence and then serum starved for 12 h Results before wounding. Then wounds were made across the mono- MFAP2 expression is significantly layer using a 200-μL plastic pipette tip. The remaining cells were washed twice with PBS buffer to remove cell debris and upregulated in human GC tissues The expression of MFAP2 protein was examined by immuno- incubated at 37°C in RPMI-1640 without FBS. Subsequently, the wound closures were visualized at 0 and 24 h, and images histochemical staining in 168 paired tumor tissues and corre- were then captured by inverted microscopy (Olympus IX50; sponding normal (non-tumorous) tissues. As shown in Table 1 Olympus, Tokyo, Japan) to detect the migratory ability. The and Figure 1A, 56.55% (95/168) of GC specimens demon- migration rate was determined by the percentage of the recov- strated high expression of MFAP2, which was significantly ered gap distance at each time point compared with the gap dis- higher than that of normal tissues (39.88%, 67/168) (P=0.003, tance at 0 h. The experiments were performed in triplicates. Table 1). We then analyzed the IHC score of MFAP2 in 168 paired samples. The IHC score of MFAP2 in GC tissues was significantly higher than that in the corresponding normal tis- Transwell invasion assay sues (P,0.001, Figure 1B). To further investigate the mRNA BGC823 and MKN-45 cells were cultured in serum-free expression of MFAP2 in larger cohorts of GC samples, we RPMI-1640 medium overnight and then suspended in serum- analyzed the mRNA level of MFAP2 in National Center free RPMI-1640 medium. Cells (4×104/well) were seeded for Biotechnology Information Gene Expression Omnibus into the upper chamber of transwell inserts (pore size, 8 µm; (NCBI GEO) and TCGA-STAD databases. The datasets Costar; Corning Incorporated, Corning, NY, USA) pre- GSE29272 and GSE27342 included the expression data of 134 coated with Matrigel in 24-well plates. The lower chambers and 80 pairs of GC tissues and normal tissues, respectively. were filled with 500 µL RPMI-1640 supplemented with 10% FBS as a chemoattractant. The cells were then treated Table 1 MFAP2 expression in gastric cancer and adjacent normal with or without TGF-β1 (5 ng/mL) for 24 h. After incuba- mucosa tion for 24 h, the cells invading into the lower chambers 2 were fixed in 4% paraformaldehyde for 30 min and stained Group MFAP2 χ P-value expression with 1% crystal violet for 10 min, and non-migrating cells Low High remaining in the top surface of the insert were removed using Carcinoma 73 95 9.345 0.003 cotton swabs. The invaded cells in the bottom chamber were Adjacent normal mucosa 101 67 counted and photographed under the inverted microscope. Abbreviation: MFAP2, microfibril-associated protein 2.

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Figure 1 MFAP2 is highly expressed in gastric cancer (GC) tissues. (A) Immunohistochemical staining of MFAP2 protein expression in 168 gastric cancer tissues and their corresponding non-cancerous tissues; two representative cases are shown (original magnification: 200×; scale bar, 50 µm). (B) The IHC score of MFAP2 was significantly higher in gastric cancer tissues than in adjacent normal tissues. Analysis of GSE29272 database (C), GSE27342 database (D), and GSE54129 database (E) showing a significantly higher LTBP2 expression in GC tissues than in adjacent normal tissues. (F) Analysis of TCGA-STAD database by an online tool GEPIA indicating that MFAP2 expression was higher in gastric cancer tissues than in adjacent normal tissues; *P,0.05. Abbreviations: IHC, immunohistochemistry; MFAP2, microfibril-associated protein 2; TCGA, The Cancer Genome Atlas.

The results showed that the mRNA levels of MFAP2 were Moreover, in the TCGA-STAD database, MFAP2 expression significantly upregulated in GSE29272 and GSE27342 was also overexpressed in 408 GC tissues compared with that (P,0.001, Figure 1C and D). In GSE54129, the MFAP2 in 211 normal tissues (P,0.05, Figure 1F). Collectively, these mRNA levels were significantly higher in 111 GC samples data suggested that the expression of MFAP2 was aberrantly compared with the 21 normal samples (P,0.001, Figure 1E). increased in GC at both mRNA and protein levels.

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High MFAP2 expression is associated associated variables in the univariate log-rank test into a with clinical features and predicts poor multivariate Cox regression analysis. The results of the prognosis in patients with GC multivariate analysis demonstrated that N stage (P=0.035) In order to investigate the clinical role of MFAP2 in GC, we and MFAP2 (P=0.044, Table 3) were independent prognostic analyzed the relationship between MFAP2 expression and factors for OS. We then examined the correlation between the the clinicopathological factors of the 168 patients with GC. mRNA levels of MFAP2 and the survival rate in the NCBI As shown in Table 2, MFAP2 expression was positively cor- GEO and TCGA-STAD databases by using online tools KM related with tumor (T) stage (P=0.019, Table 2), node (N) plotter (available at http://www.kmplot.com) and GEPIA stage (P=0.031, Table 2), metastasis (M) stage (P=0.036, (available at http://www.gepia.cancer-pku.cn). A total of Table 2), and TNM stage (P=0.001, Table 2) but not with 875 patients with GC were involved in the analysis of OS. high sex (P=0.609), age (P=0.106), tumor location (P=0.184), or The results demonstrated that patients with MFAP2 tumors histological differentiation (P=0.110) in patients with GC. had significantly lower OS (P,0.0001, Figure 2B) rate than low To further elucidate the importance of the role of MFAP2 those with MFAP2 tumors. In the TCGA-STAD database, in the survival of patients with GC, we examined the correla- we also found that high MFAP2 expression was correlated tion between MFAP2 and OS and DFS of patients with GC. with markedly lower OS (P=0.0056, Figure 2C). The results from our cohort of 168 patients with GC showed The results from our cohort of 168 patients with GC dem- that higher expression level of MFAP2 was associated with onstrated that high MFAP2 expression was correlated with a significantly lower OS P( =0.0007, Figure 2A). Univariate a significantly lower DFS (P=0.0004, Figure 2D). T stage analysis showed that T stage (P,0.001), N stage (P,0.001), (P,0.001), N stage (P,0.001), TNM stage (P,0.001), M stage (P=0.001), TNM stage (P,0.001), and high MFAP2 and high MFAP2 expression (P=0.001) were the significant expression (P=0.001) correlated with the OS of patients with univariate variables incorporated into multivariate analysis. GC patients (Table 3). Then, we incorporated the significantly The multivariate Cox regression analysis for DFS showed that T stage (P=0.007) and MFAP2 expression (P=0.028, Table 4) were independent prognostic factors. Moreover, Table 2 The relationship between MFAP2 expression and a total of 641 patients were included into the analysis of clinicopathological features of patients with gastric cancer DFS in the KM plotter database and the results demon- Clinicopathological N MFAP2 expression strated that patients with MFAP2high tumors had significantly features 2 Low High χ P-value lower DFS (P,0.0001, Figure 2E) rate than those with Age (years) 73 95 2.612 0.106 MFAP2low tumors. The results from TCGA-STAD database $60 74 27 47 ,60 94 46 48 also revealed the same trend (P=0.0042, Figure 2F). Taken Gender 0.262 0.609 together, our results indicated that MFAP2 contributes to Male 114 48 66 poor OS and DFS of patients with GC and might be used as Female 54 25 29 a promising prognostic predictor. Histological grade 2.561 0.110 G1+G2 45 15 30 G3 123 58 65 MFAP2 promotes the proliferation of T stage 5.548 0.019 T1–T2 55 31 24 GC cells T3–T4 113 42 71 To explore the function of MFAP2 in GC cells, we first N stage 4.661 0.031 detected the expression of MFAP2 in SGC7901, AGS, N0 65 35 30 MGC803, BGC823, and MKN-45 by qRT-PCR and Western N1–N3 103 38 65 M stage 4.375 0.036 blotting analyses. Our results demonstrated that BGC823 and M0 149 69 80 MKN-45 had the highest expression of MFAP2 (Figure S2A). M1 19 4 15 As a result, these two GC cell lines were used in our further TNM stage 10.334 0.001 work. Knockdown of MFAP2 was verified by qRT-PCR I/II 101 54 47 III/IV 67 19 48 and Western blotting analysis in BGC823 and MKN-45 Tumor site 1.766 0.184 (Figure S2B and C) GC cells. Distal gastric 120 56 64 First, we examined the functional roles of MFAP2 on the Proximal gastric 48 17 31 proliferation of GC cells. The colony formation assay was Abbreviations: M, metastasis; MFAP2, microfibril-associated protein 2; N, node; T, tumor; TNM, tumor node metastasis. conducted, and the results demonstrated that knockdown of

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Figure 2 High MFAP2 expression is associated with poor overall survival and disease-free survival of patients with gastric cancer. (A) Kaplan–Meier estimation of 168 patients with gastric cancer indicating lower overall survival rate in patients with MFAP2high tumors. (B) Kaplan–Meier analysis of 875 patients with gastric cancer in KM plotter database indicating that patients with MFAP2high tumors were associated with a lower overall survival rate. (C) Kaplan–Meier survival curve for overall survival of 424 patients with gastric cancer from TCGA-STAD database generated using an online tool GEPIA. (D) Kaplan–Meier analysis of 168 patients with gastric cancer indicating a lower disease-free survival rate in MFAP2high patients. (E) Kaplan–Meier analysis of disease-free survival in 641 patients with gastric cancer generated using KM plotter database. (F) Kaplan–Meier analysis of disease-free survival in 424 patients with gastric cancer from the TCGA-STAD database generated using an online tool GEPIA. Note: n(high) and n(low) refer to patient numbers. Abbreviations: HR, hazard ratio; MFAP2, microfibril-associated protein 2; TCGA, The Cancer Genome Atlas; TPM, transcripts per million.

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Table 3 Univariate and multivariate analyses of overall survival in patients with gastric cancer Prognostic variables Univariate analysis Multivariate analysis HR (95% CI) P-value HR (95% CI) P-value Age (years) 1.275 (0.874–1.859) 0.207 – – Gender 0.932 (0.624–1.394) 0.733 – – Histological grade 1.061 (0.690–1.630) 0.788 – – T stage 2.425 (1.558–3.775) 0.000 1.536 (0.915–2.577) 0.104 N stage 2.523 (1.659–3.837) 0.000 1.717 (1.039–2.838) 0.035 M stage 2.521 (1.477–4.306) 0.001 1.481 (0.822–2.672) 0.191 TNM stage 2.745 (1.876–4.016) 0.000 1.326 (0.770–2.285) 0.309 Tumor site 1.031 (0.680–1.564) 0.885 – – High MFAP2 expression 1.943 (1.308–2.887) 0.001 1.528 (1.012–2.307) 0.044 Notes: The endashes indicate that only the significant variables in the univariate log-rank test were incorporated into a multivariate Cox regression analysis. If theP -value of a variable in univariate analysis was 0.05, then the variable was not calculated in the multivariate analysis. Abbreviations: CI, confidence interval; HR, hazard ratio; M, metastasis; MFAP2, microfibril-associated protein 2; N, node; T, tumor; TNM, tumor node metastasis.

MFAP2 significantly decreased the colony number in both tumor cell invasion and metastasis,22 we detected EMT BGC823 and MKN-45 cells (P,0.001, Figure 3A and B). markers in MFAP2-knockdown BGC823 and MKN-45 Moreover, the results of our CCK-8 assay indicated that the cells. E-cadherin, Vimentin, and Snail were selected as the proliferation ability was significantly inhibited by knockdown EMT markers and were then detected by Western blotting of MFAP2 in BGC823 (P,0.01, Figure 3C) and MKN-45 and qRT-PCR analysis. The results demonstrated that the (P,0.05, Figure 3C) cells. These results indicated that mRNA and protein levels of mesenchymal markers Snail and MFAP2 played an important role in promoting the prolifera- Vimentin were downregulated in MFAP2-knockdown GC tion of GC cells. cells, while the epithelial marker E-cadherin was significantly upregulated (Figure 4E and F). Interestingly, we observed a MFAP2 enhances the migratory and markedly significant downregulation of Snail compared with invasive abilities of GC cells by promoting the downregulation of Vimentin at both mRNA and protein TGF-β-induced EMT levels (Figure 4E and F). We further explored the functional role of MFAP2 in Previous reports have suggested that MFAP2 inhibits regulating the migration and invasion of GC cells. The latent transforming growth factor beta binding protein 1 scratch-wound healing assay demonstrated that MFAP2- (LTBP1) binding to fibrillin microfibrils, resulting in ele- knockdown BGC823 and MKN-45 cells exhibited sig- vated TGF-β signaling.23 To investigate whether MFAP2 nificantly decreased migratory capabilities compared with influences the expression of TGF-β in GC cells, Western mock cells (Figure 4A and C). In addition, our transwell blotting analysis was conducted to detect TGF-β expres- assay showed that knockdown of MFAP2 in BGC823 and sion in MFAP2-knockdown and mock cells. Interestingly, MKN-45 cells significantly inhibited their invasive capabili- our Western blotting analysis found that the protein level of ties (Figure 4B and D). Because EMT has been elucidated TGF-β1 was markedly decreased in MFAP2-knockdown as a crucial mechanism in GC progression, especially during BGC823 and MKN-45 cells (Figure 4E). Considering that

Table 4 Univariate and multivariate analyses of disease-free survival in patients with gastric cancer Prognostic variables Univariate analysis Multivariate analysis HR (95% CI) P-value HR (95% CI) P-value Age (years) 1.487 (0.971–2.278) 0.068 – – Gender 0.793 (0.497–1.265) 0.330 – – Histological grade 1.319 (0.798–2.179) 0.280 – – T stage 3.248 (1.952–5.404) 0.000 2.202 (1.238–3.917) 0.007 N stage 2.459 (1.560–3.874) 0.000 1.673 (0.972–2.879) 0.063 M stage 1.729 (0.830–3.602) 0.144 – – TNM stage 3.075 (1.967–4.807) 0.000 1.336 (0.748–2.387) 0.328 Tumor site 0.981 (0.615–1.566) 0.937 – – High MFAP2 expression 2.180 (1.397–3.404) 0.001 1.391 (1.059–2.702) 0.028 Notes: The endashes indicate that only the significant variables in the univariate log-rank test were incorporated into a multivariate Cox regression analysis. If theP -value of a variable in univariate analysis was 0.05, then the variable was not calculated in the multivariate analysis. Abbreviations: CI, confidence interval; HR, hazard ratio; M, metastasis; MFAP2, microfibril-associated protein 2; N, node; T, tumor; TNM, tumor node metastasis.

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Figure 3 MFAP2 promotes the proliferation ability of gastric cancer cells. (A) Representative images of colony formation test in mock, shMFAP2-1, and shMFAP2-2 gastric cancer cells (BGC823 and MKN-45). (B) Quantification of the colony formation assay results showing decreased colony formation capability of gastric cancer cells with knockdown of MFAP2; ***P,0.001. (C) CCK-8 assays showing decreased proliferation ability of gastric cancer cells with knockdown of MFAP2. Mock: cells infected with mock lentivirus. Abbreviations: CCK-8, Cell Counting Kit-8; MFAP2, microfibril-associated protein 2; shMFAP2-1, #1 short-hairpin RNA targeting MFAP2; shMFAP2-2, #2 short-hairpin RNA targeting MFAP2.

TGF-β signaling pathway plays an important role in promot- markers in MFAP2-knockdown BGC823 and MKN-45 cells. ing EMT in GC cells, our data indicate that MFAP2 promotes Interestingly, the Western blotting analysis showed that the the migration and invasion of GC cells by modulating expression of MFAP2 was not significantly changed after TGF-β-induced EMT. TGF-β1 treatment (Figure 5B). However, the expression of E-cadherin was significantly downregulated together with MFAP2 promotes EMT of GC cells by upregulation of Vimentin and Snail after TGF-β1 treat- activating the TGF-β/SMAD2/3 signaling ment (Figure 5B). The qRT-PCR also showed the same pathway results (Figure 5C). Considering that MFAP2 knockdown The key event in EMT is the switch of E-cadherin to decreased the expression of TGF-β1 with inhibition of N-cadherin, which renders the single cell more motile and EMT, and that exogenous TGF-β1 treatment restored the invasive.24,25 TGF-β is a major inducer of EMT, and TGF-β- expression of EMT markers and invasive capabilities without induced EMT has been suggested to be associated with the changing the expression of MFAP2 in GC cells, our data development and progression of GC.26 Considering that suggested that MFAP2 might be an important upstream knockdown of MFAP2 decreased the expression of TGF-β1, regulator of TGF-β1. Because SMAD2 and SMAD3 are we decided to explore whether the TGF-β/SMAD2/3 signal- critical downstream regulators in the TGF-β pathway,27 we ing pathway was involved in MFAP2-modulated EMT in further examined the expression of SMAD2 and SMAD3 GC cells. First, we investigated whether TGF-β1 treatment and their phosphorylation levels. The results of Western could restore the impaired invasive abilities due to knock- blotting analysis showed that the total expression levels down of MFAP2. Results of our transwell assays showed of SMAD2 and SMAD3 were not significantly changed that the impaired invasive abilities of MFAP2-knockdown in MFAP2-knockdown GC cells. However, the levels of GC cells were significantly restored after TGF-β1 treatment phosphorylated-SMAD2 (p-SMAD2) and p-SMAD3 were (Figure 5A). Then, we explored whether exogenous TGF-β1 all significantly decreased in MFAP2-knockdown BGC823 treatment could change the expression of MFAP2 and EMT and MKN-45 cells (Figure 5D). Collectively, our data

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Figure 4 MFAP2 promotes the migration, invasion, and epithelial–mesenchymal transition (EMT) phenotype in gastric cancer cells. (A) Representative images of the wound- healing assay in mock and MFAP2-knockdown BGC823 and MKN-45 cells. (B) Representative images of transwell assay in mock and MFAP2-knockdown BGC823 and MKN-45 cells (original magnifications: 200×). (C) Quantification of the wound-healing assay results; ***P,0.001. (D) Quantification of the transwell invasion assay results; ***P,0.001. (E) Western blotting analyses of the protein levels of EMT-related markers (E-cadherin, Snail, and Vimentin) and TGF-β1 in mock and MFAP2-knockdown BGC823 and MKN-45 cells. (F) qRT-PCR analyses of E-cadherin, Snail, and Vimentin in mock and MFAP2-knockdown BGC823 and MKN-45 cells; *P,0.05, **P,0.01, ***P,0.001. Mock: cells infected with mock lentivirus. Abbreviations: MFAP2, microfibril-associated protein 2; qRT-PCR, quantitative real-time polymerase chain reaction; shMFAP2-1, #1 short-hairpin RNA targeting MFAP2; shMFAP2-2, #2 short-hairpin RNA targeting MFAP2; TGF-β, transforming growth factor beta. suggest that MFAP2 promotes the EMT of GC cells partially progression of GC.30,31 There are three isoforms of TGF-β in by activating the TGF-β/SMAD2/3 signaling pathway as an mammalian cells, including the most abundant TGF-β1, and upstream regulator. the uncommon isoforms: TGF-β2 and TGF-β3.32 The TGF-β/ SMAD pathway is initiated by TGF-β1 binding to its type II Discussion receptor (TβRII). Then TβRII forms a heterodimer with The tumorigenesis of GC is complex and is associated TGF-β type I receptor (TβRI or ALK5), thereby activating with multiple disorders including dysregulation of cancer- the intracellular kinase domain of ALK5 to phosphorylate related signaling pathways.28,29 Among the known pathways, SMAD2/3 complex.33 The p-SMAD2/3 and p-SMAD4 form TGF-β signaling plays a central role in the development and a transcription complex, which translocates to the nucleus

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Figure 5 MFAP2 enhances epithelial–mesenchymal transition (EMT) of GC cells by activating the TGF-β/SMAD2/3 signaling pathway. (A) Representative images and quantification of transwell assay in mock and MFAP2-knockdown BGC823 and MKN-45 cells with or without TGF-β treatment (original magnifications: 200×); **P,0.01, ***P,0.001. (B) Western blotting analyses of the protein levels of EMT-related markers (E-cadherin, Snail, and Vimentin) and MFAP2 in MFAP2-knockdown BGC823 and MKN-45 cells with or without TGF-β treatment. (C) qRT-PCR analyses of EMT-related markers (E-cadherin, Snail, and Vimentin) and MFAP2 in MFAP2-knockdown BGC823 and MKN-45 cells with or without TGF-β treatment; **P,0.01. (D) Knockdown of MFAP2 attenuated phosphorylation of SMAD2 and SMAD3 without changing the total levels of the proteins as evaluated by Western blotting analysis. Mock: cells infected with mock lentivirus. Abbreviations: GC, gastric cancer; MFAP2, microfibril-associated protein 2; qRT-PCR, quantitative real-time polymerase chain reaction; shMFAP2-1, #1 short-hairpin RNA targeting MFAP2; shMFAP2-2, #2 short-hairpin RNA targeting MFAP2; TGF-β, transforming growth factor beta. and binds to SMAD binding elements in the promoter region. such as MFAP2 that had an inhibitory function for TGF-β This complex then regulates the transcription of TGF-β target activity, many other molecules are involved in releasing genes.34 Previous findings indicated that TGF-β signaling has TGF-β from the extracellular matrix.43,44 A study of human both tumor-promoting and tumor-suppressive roles in differ- dermal fibroblasts revealed that MFAP2 strongly inhibited ent kinds of tumors.35,36 Activation of the TGF-β signaling the interaction between LTBP1 and fibrillin-1 because their pathway results in potent cell-cycle arrest in healthy cells binding affinities to fibrillin-1 were mutually exclusive. As and inhibits tumor formation in early stage tumor cells.37,38 a result, LTBP1 was released from assembled microfibrils, However, the upregulation of TGF-β and overactivation of thereby strongly increasing the activity of TGF-β.23 Consider- TGF-β receptor-initiated intracellular signaling to promote ing that paradoxical roles have been proposed for TGF-β in the progression in advanced-stage tumors have been observed early stage and advanced-stage tumors, it is not surprising in many cancers including GC.39,40 As a result, understanding that different modulatory roles of MFAP2 in the regulation of the regulation mechanism of TGF-β in GC is extremely of TGF-β have been reported. As a result, the elucidation of important for understanding the role of TGF-β as well as the the role of MFAP2 in regulating TGF-β in cancer will help us discovery of new drugs targeting TGF-β. to understand TGF-β signaling in malignancy. Intriguingly, MFAP2 was found to regulate the function of TGF-β we found that knockdown of MFAP2 significantly decreased in several kinds of healthy cells by binding the active form the expression of TGF-β and its activity, as evaluated by of TGF-β instead of the latent form.41 In MFAP2-deficient SMAD-2 and SMAD-3 phosphorylation, and that TGF-β C57BL/6 mice, loss of MAGP1 significantly increased treatment reversed the impaired migratory and invasive the activity of TGF-β in epididymal white adipose tissue, abilities in MFAP2-knockdown GC cells without changing as evaluated by SMAD-2 phosphorylation.41 In rat lung the expression of MFAP2. Our results were comparable with fibroblast line-6 (RFL-6) cells, which express no MFAP2 the study in human dermal fibroblasts that MFAP2 promotes naturally, MFAP2 expression was induced by stable the activity of TGF-β. Additionally, we demonstrated that transfection with expression vectors and the expression of MFAP2 was significantly upregulated in GC tissues and its MFAP2 reduced SMAD-2 phosphorylation.42 Although the overexpression correlated with lower OS and DFS rates. results of tissues or cell lines from rat and mice indicated Knockdown of MFAP2 impaired the migratory and invasive that MFAP2 suppressed the activity of TGF-β, we believe capabilities of GC cells. To the best of our knowledge, the that the regulation of TGF-β is very sophisticated and is current study of MFAP2 in GC is the first one to illustrate significantly different in species and tissue types such as its expression, prognostic significance, and function as well healthy and tumor tissues. Moreover, in contrast to proteins as its role in the regulation of TGF-β in tumors.

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Accumulating studies have indicated that EMT can be Disclosure initiated and maintained by growth factors or inflammatory The authors report no conflicts of interest in this work. cytokines.45 Among these factors, TGF-β has been reported to effectively induce EMT in gastric cells, increasing their References migratory and invasive properties and resulting in enhanced 1. Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J, Jemal A. Global cancer statistics, 2012. CA Cancer J Clin. 2015;65(2):87–108. 45 metastasis. It is known that EMT plays an important role 2. Ferlay J, Soerjomataram I, Dikshit R, et al. Cancer incidence and mor- in the invasion and metastasis of GC cells and is one of the tality worldwide: sources, methods and major patterns in GLOBOCAN earliest processes during tumor cell metastasis.46 To increase 2012. Int J Cancer. 2015;136(5):E359–E386. 3. Zong L, Abe M, Seto Y, Ji J. The challenge of screening for early gastric motility and invasiveness, cancer cells lose apical–basal cancer in China. Lancet. 2016;388(10060):2606. polarity and cell adhesion, remodel the cytoskeleton, and 4. Lordick F, Allum W, Carneiro F, et al. Unmet needs and challenges in gastric cancer: the way forward. Cancer Treat Rev. 2014;40(6): develop a mesenchymal phenotype; then the cancer cells 692–700. detach from one another and migrate to new locations.47 5. Shen L, Shan YS, Hu HM, et al. Management of gastric cancer in Asia: In addition, an EMT phenotype is often accompanied by the resource-stratified guidelines.Lancet Oncol. 2013;14(12):e535–e547. 6. Cidon EU, Ellis SG, Inam Y, Adeleke S, Zarif S, Geldart T. Molecular downregulation of epithelial markers E-cadherin and cytok- targeted agents for gastric cancer: a step forward towards personalized eratin, and upregulation of mesenchymal markers such as therapy. Cancers (Basel). 2013;5(1):64–91. 7. Wadhwa R, Song S, Lee JS, Yao Y, Wei Q, Ajani JA. Gastric cancer- 48,49 N-cadherin, snail, Vimentin and fibronectin. In the current molecular and clinical dimensions. Nat Rev Clin Oncol. 2013;10(11): study, we found that knockdown of MFAP2 significantly 643–655. decreased the expression of mesenchymal markers Vimentin 8. Corson GM, Charbonneau NL, Keene DR, Sakai LY. Differential expression of fibrillin-3 adds to microfibril variety in human and avian, and Snail, along with the upregulation of the epithelial marker but not rodent, connective tissues. Genomics. 2004;83(3):461–472. E-cadherin. Moreover, we found that TGF-β treatment effec- 9. Sakai LY, Keene DR, Engvall E. Fibrillin. A new 350-kD glycoprotein is a component of extracellular microfibrils.J Cell Biol. 1986;103(6 Pt 1): tively induced the expression of Vimentin and Snail and sup- 2499–2509. pressed the expression of E-cadherin in MFAP2-knockdown 10. Finnis ML, Gibson MA. Microfibril-associated glycoprotein-1 GC cells without changing the expression of MFAP2, indicat- (MAGP-1) binds to the pepsin-resistant domain of the alpha3(VI) chain of type VI collagen. J Biol Chem. 1997;272(36):22817–22823. ing that MFAP2 might be an important upstream regulator 11. Brown-Augsburger P, Broekelmann T, Mecham L, et al. Microfibril- of TGF-β/SMAD2/3 signaling. associated glycoprotein binds to the carboxyl-terminal domain of tropoelastin and is a substrate for transglutaminase. J Biol Chem. 1994; In summary, our results demonstrate that the overexpres- 269(45):28443–28449. sion of MFAP2 predicts poor outcome in patients with GC. 12. Fujita T, Tsuruga E, Yamanouchi K, Sawa Y, Ishikawa H. Microfibril- In addition, MFAP2 functions upstream of TGF- and pro- associated glycoprotein-1 controls human ciliary zonule development β in vitro. Acta Histochem Cytochem. 2014;47(1):11–17. motes the proliferation, migration, invasion, and EMT phe- 13. Werneck CC, Vicente CP, Weinberg JS, et al. Mice lacking the extra- notype by activating the TGF-β/SMAD2/3 signaling. These cellular matrix protein MAGP1 display delayed thrombotic occlusion following vessel injury. Blood. 2008;111(8):4137–4144. data highlight the importance of MFAP2 in the progression 14. Mecham RP, Gibson MA. The microfibril-associated glycoproteins of GC and revealed a novel MFAP2-mediated mechanism (MAGPs) and the microfibrillar niche.Matrix Biol. 2015;47:13–33. that may be used as a target for TGF- . 15. Zaravinos A, Kanellou P, Lambrou GI, Spandidos DA. Gene set enrich- β ment analysis of the NF-kappaB/Snail/YY1/RKIP circuitry in multiple myeloma. Tumour Biol. 2014;35(5):4987–5005. Conclusion 16. Silveira NJ, Varuzza L, Machado-Lima A, et al. Searching for molecular markers in head and neck squamous cell carcinomas (HNSCC) by Our results indicated that MFAP2 was significantly upregu- statistical and bioinformatic analysis of larynx-derived SAGE libraries. lated in human GC tissues and its overexpression was cor- BMC Med Genomics. 2008;1:56. related with poor OS and DFS rates in patients with GC. 17. Edge SB, Compton CC. The American Joint Committee on Cancer: the 7th edition of the AJCC cancer staging manual and the future of TNM. MFAP2 promotes the proliferation, migration, invasion, Ann Surg Oncol. 2010;17(6):1471–1474. and EMT phenotype in GC cells by activating the TGF-β/ 18. Camp RL, Dolled-Filhart M, Rimm DL. X-tile: a new bio-informatics tool for biomarker assessment and outcome-based cut-point optimiza- SMAD2/3 signaling pathway as an upstream regulator. tion. Clin Cancer Res. 2004;10(21):7252–7259. Thus, MFAP2 might be a promising prognostic marker, and 19. Gentleman RC, Carey VJ, Bates DM, et al. Bioconductor: open software targeting the MFAP2/TGF-β/SMAD2/3 axis might be a development for computational biology and bioinformatics. Genome Biol. 2004;5(10):R80. potential therapeutic strategy for GC treatment. 20. Tang Z, Li C, Kang B, Gao G, Li C, Zhang Z. GEPIA: a web server for cancer and normal gene expression profiling and interactive analyses. Acknowledgment Nucleic Acids Res. 2017;45(W1):W98–W102. 21. Szasz AM, Lanczky A, Nagy A, et al. Cross-validation of survival The study was supported by a grant from the National Natural associated biomarkers in gastric cancer using transcriptomic data of Science Foundation of China (grant number 81560093). 1,065 patients. Oncotarget. 2016;7(31):49322–49333.

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Table S1 Clinical parameters of GC patients involved in the study Clinical characteristics Number Percentage Age (years) $60 74 44.05 ,60 94 55.95 Gender Male 114 67.86 Female 54 32.14 Histology grade G1 6 3.57 G2 G2 39 23.21 G3 123 73.21 T stage T1 31 18.45 T2 24 14.29 T3 82 48.81 T4 31 18.45 N stage N0 65 38.69 N1 48 28.57 N2 44 26.19 N3 11 6.55 M stage M0 149 88.69 M1 19 11.31 TNM stage I 42 25.00 II 59 35.12 III 48 28.57 IV 19 11.31 Tumor site Proximal gastric 48 28.57 Distal gastric 120 71.43 Abbreviations: GC, gastric cancer; M, metastasis; N, node; T, tumor; TNM, tumor node metastasis.

A B 1.0

Pt no % total Events Rate Rank Range 73 43.45 38 52.05 0 to 3 0.00 thru 3.00 95 56.55 71 74.74 4 to 8 4.00 thru 12.00 0.8 168 100.00 109 64.88 0 to 8 0.00 thru 12.00

Relative risk plot 1.90 0.6

0.4 Sensitivit y

0.2

0.53 0.0 0.0 0.2 0.40.6 0.81.0 1 – specificity

Figure S1 (Continued)

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C D Positive if Sensitivity1 – specificity Youden index greater than or equal toa –1.000011.000 .000 0.000 0.5000 0.982 0.983 –0.001 Area Std errora Asymptotic Asymptotic 95% CI 1.5000 0.954 0.932 0.022 sigb Lower bound Upper bound 2.5000 0.917 0.847 0.070 3.5000 0.651 0.407 0.245 0.626 0.045 0.007 0.537 0.714 5.0000 0.174 0.1190.056 7.0000 0.138 0.068 0.070 8.5000 0.092 0.000 0.092 10.5000 0.028 0.000 0.028 13.0000 0.000 0.000 0.000

Figure S1 The determination of the optimal cutoff value for 168 patients with gastric cancer. (A) The cutoff value of 4 as calculated by X-tile software. (B) The ROC curve generated using SPSS 19.0 software (the blue line shows sensitivity and the green line shows specificity). (C) The area under the curve was 0.626 as calculated by SPSS 19.0 software. aUnder the nonparametric assumption. bNull hypothesis: true area =0.5. (D) The Youden index was calculated by SPSS 19.0 software. The red box highlights that the best Youden index is 0.245 when the cutoff value is 3.5. As the maximum value of Youden index was 0.245 when the immunohistochemical score was 3.5, a score $4 was defined as MFAP2high and a score #3 as MFAP2low. The test result variable(s): MFAP2 has at least one tie between the positive actual state group and the negative actual state group. Statistics may be biased. Abbreviations: CI, confidence interval; MFAP2, microfibril-associated protein 2; Pt no, patient number; ROC, receiver operating characteristic; sig, significance; std, standard.

Table S2 Sequences of MFAP2 knockdown and control shRNAs Name Sequence shMFAP2-1 Forward: 5′-CACCGGAACAGTTCCAGTTCCAGTCCGAAGACTGGAACTGGAACTGTTCC-3′ Reverse: 5′-AAAAGGAACAGTTCCAGTTCCAGTCTTCGGACTGGAACTGGAACTGTTCC-3′ shMFAP2-2 Forward: 5′-CACCGGCCTTGCAAACAGTGTCTCACGAATGAGACACTGTTTGCAAGGCC-3′ Reverse: 5′-AAAAGGCCTTGCAAACAGTGTCTCATTCGTGAGACACTGTTTGCAAGGCC-3′ shMFAP2-3 Forward: 5′-CACCGCCGTGTGTACGTCATTAACACGAATGTTAATGACGTACACACGGC-3′ Reverse: 5′-AAAAGCCGTGTGTACGTCATTAACATTCGTGTTAATGACGTACACACGGC-3′ Control Forward: 5′-CCGGTTCTCCGAACGTGTCACGTTTCAAGAGAACGTGACACGTTCGGAGAATTTTTG-3′ Reverse: 5′-AATTCAAAAATTCTCCGAACGTGTCACGTTCTCTTGAAACGTGACACGTTCGGAGAA-3′ Abbreviations: MFAP2, microfibril-associated protein 2; shMFAP2-1, #1 short-hairpin RNA targeting MFAP2; shMFAP2-2, #2 short-hairpin RNA targeting MFAP2; shMFAP2-3, #3 short-hairpin RNA targeting MFAP2; shRNAs, short-hairpin RNAs.

Table S3 Sequences of primers used for qRT-PCR in this study Gene Primer Sequence MFAP2 Forward 5′-CCCAAGCTTGTGAGGAACAGTACCCGT-3′ Reverse 5′-CGGAATTCGATACTCCCCCAACCCGA-3′ E-cadherin Forward 5′-CGAGAGCTACACGTTCACGG-3′ Reverse 5′-GGGTGTCGAGGGAAAAATAGG-3′ Snail Forward 5′-AGATGAGGACAGTGGGAAAGG-3′ Reverse 5′-TGAAGTAGAGGAGAAGGACGAAGGAG-3′ Vimentin Forward 5′-GACGCCATCAACACCGAGTT-3′ Reverse 5′-CTTTGTCGTTGGTTAGCTGGT-3′ TGF-β Forward 5′-CTTTGGTATCGTGGAAGGACTC-3′ Reverse 5′-AGCTGTACCAGAAATACAGCAACA-3′ β-actin Forward 5′-ACTGGAACGGTGAAGGTGAC-3′ Reverse 5′-AGAGAAGTGGGGTGGCTTTT-3′

Abbreviations: MFAP2, microfibril-associated protein 2; qRT-PCR, quantitative real-time polymerase chain reaction; TGF-β, transforming growth factor beta.

4016 submit your manuscript | www.dovepress.com OncoTargets and Therapy 2018:11 Dovepress Dovepress MFAP2 supports EMT of GC cells by activating TGF-β signaling pathways

A 2.0

SGC7901 AGS MKN-45 MGC803 BGC823 1.5 MFAP2 1.0

β-actin SGC7901) 0.5 AP2 expression (normalized to

MF 0.0

AGS MKN-45 BGC823 SGC7901 MGC803

B BGC823 BGC823 1.5 WT Mock shMFAP2-1 shMFAP2-2 1.0 MFAP2 0.5

β-actin AP2 expression MF (normalized to WT) 0.0

WT MOCK AP2-1 AP2-2

shMF shMF

C MKN-45 MKN-45 1.5 WT Mock shMFAP2-1 shMFAP2-2 1.0 MFAP2 0.5 AP2 expression β-actin MF (normalized to WT) 0.0

WT MOCK AP2-1 AP2-2

shMF shMF

Figure S2 The selection of gastric cancer cell lines and knockdown of MFAP2. (A) Five gastric cancer cells lines analyzed using Western blotting and qRT-PCR analyses indicating that BGC823 and MKN-45 had the highest expression of MFAP2. Knockdown efficiency of MFAP2 was confirmed by Western blotting and qRT-PCR analyses in BGC823 (B) and MKN-45 (C) cells. Abbreviations: MFAP2, microfibril-associated protein 2; qRT-PCR, quantitative real-time polymerase chain reaction; shMFAP2, short-hairpin RNA targeting MFAP2; WT, wild type.

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