SHCBP1 Promotes Synovial Sarcoma Cell Metastasis Via Targeting TGF-Β1

Peng et al. Journal of Experimental & Clinical Cancer Research (2017) 36:141 DOI 10.1186/s13046-017-0616-z

RESEARCH Open Access SHCBP1 promotes synovial sarcoma cell metastasis via targeting TGF-β1/Smad signaling pathway and is associated with poor prognosis Changliang Peng1, Hui Zhao2, Yan Song3, Wei Chen4, Xiaoying Wang5, Xiaoli Liu6, Cheng Zhang1, Jie Zhao1,JiLi1, Guanghui Cheng7, Dongjin Wu1, Chunzheng Gao1 and Xiuwen Wang1*

Abstract Background: Our previous studies reported that SHC SH2-domain binding protein 1 (SHCBP1) functions as an oncogene via promoting cell proliferations in synovial sarcoma (SS) cells. However, whether SHCBP1 has any effect on tumor metastasis remains unexplored. Methods: The expression of SHCBP1 was analyzed in 76 SS tissues and two SS cell lines by immunohistochemistry and real-time RT-PCR. The relationship between SHCBP1 expression and the clinicopathological features of SS was investigated. The role of SHCBP1 in SS cell adhesion, migration, invasion and angiogenesis was explored by adhesion, Wound healing, Transwell, and Matrigel tube formation assays. Western blotting was conducted to detect the protein expressions of TGF-β1/Smad signaling pathway and EMT-related markers. The key molecules associated with migration, invasion and EMT were evaluated by immunohistochemistry in tumor specimens. Results: In current study, we demonstrated that SHCBP1 overexpression significantly enhanced adhesion, migration, invasion and angiogenesis of SS cells. In contrast, SHCBP1 knockdown elicited the opposite effects on these phenotypes in vitro. SHCBP1 promoted tumor metastasis through inducing epithelial-mesenchymal transition (EMT) in SS cells. SHCBP1 knockdown could block the incidence of metastasis and EMT in SS cells. Furthermore, transforming growth factor-β1 (TGF-β1) induced SHCBP1 expression in a time-dependent pattern and SHCBP1 knockdown inhibited TGF-β1-induced EMT. The activation of the TGF-β1/Smad signaling pathway was involved in the oncogenic functions of SHCBP1 in SS. In addition, high expression of SHCBP1 in SS patients was associated with tumor progression and decreased survival as well as poor prognosis. Conclusions: Taken together, our results indicate that SHCBP1 may promote the metastasis of SS by inducing EMT through targeting TGF-β1/Smad signaling pathway and can be a potential molecular target for SS therapy. Keywords: SHCBP1, EMT, Synovial sarcoma, Invasion, Metastasis, TGF-β1

* Correspondence: [email protected] 1Department of Orthopaedics, The Second Hospital of Shandong University, Shandong University, Jinan, China Full list of author information is available at the end of the article

© The Author(s). 2017 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. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Peng et al. Journal of Experimental & Clinical Cancer Research (2017) 36:141 Page 2 of 12

Background of breast cancer cells [20]. Tao HC et al. [19] also found As a high-grade aggressive soft tissue tumor, synovial that SHCBP1 is remarkably upregulated in samples of sarcoma (SS) accounts almost 10% in soft tissue sarco- human hepatocellular carcinoma (HCC), and decreasing mas. Although SS may occur at all ages, the morbidity the expression of SHCBP1 can significantly inhibit the in adolescent and young adults is more higher [1]. Char- proliferation and colony formation of HCC cells [19]. acterized by local recurrence and distant metastasis Meanwhile, several lines of studies also confirmed (found in more than 80% of SS patients), SS can occur upregulation of SHCBP1 in certain leukemia/lymphoma almost anywhere in the body [2, 3]. However, the 5-year in both rodents and humans [21–23]. Moreover, survival rate is estimated to be only around 27% to 55% research on the mechanism of SHCBP1 in growth of with current available therapies including surgery, adju- tumor showed that the upregulation of SHCBP1 might vant chemotherapy and radiotherapy [4, 5]. Even more, be correlated with the activation of MEK/ERK signaling no effective treatment is available currently to patients pathway [19]. SHCBP1 overexpression can increase with metastasis, which often have a more poorer prog- cyclin-dependent kinase (CDK) inhibitor p21, and nosis, and the 5-year overall survival rate usually lowers decrease the Cyclin B1 and CDK1 [20]. These findings than 10% [6]. Therefore, elucidating the molecular collectively signified that SHCBP1 functions as an onco- mechanism involved in the metastasis of SS cell is des- gene and represents as an attractive anti-cancer target. perately needed, which undoubtedly will greatly contrib- Our previous study indicated that as a novel down- ute to the development of novel therapeutic strategies stream target gene of SS18-SSX1, SHCBP1 is overex- for the treatment of SS. pressed in SS and can promote the proliferation of SS The metastasis of tumor cell is a complex and multi- cells, thus promoting its tumorigenesis [24]. However, stage process, which includes adhesion, migration, whether SHCBP1 is involved in SS cell’s invasion and invasion, angiogenesis, and eventually unremitting pro- metastasis, and the related regulating mechanisms are liferation in distant target organs [7–9]. During this still not clear. Thus, the expression of SHCBP1 in SS malignant process, tumor cells express mesenchymal specimens was examined, and the correlation between markers, including vimentin, N-cadherin, and fibronec- its expression levels with SS patients’ survival was tin, as well as lose the expression of epithelial markers, assayed in current study. Furthermore, the promotive including E-cadherin and a-catenin, which results in effect of SHCBP1 on the invasion and migration was epithelial-mesenchymal transition (EMT), and subse- also investigated. Additionally, since EMT is a key quent local and distant metastases occur. The occur- process associated with the progression of SS, we hence rence and development of EMT are stimulated by some investigated the mechanism of SHCBP1 on the process cytokines and growth factors, such as TGF-β1, hepato- of TGF-β1-induced EMT in SS cells. cyte growth factor (HGF) and epidermal growth factor (EGF) etc. TGF-β1 is the strongest inducer among them and is overexpressed in many types of human cancers, Methods including SS [10]. However, the regulatory mechanisms Patients, specimens, and cell lines of TGF-β1 signaling pathway in EMT are largely Investigation has been conducted in accordance with the unknown, although EMT is considered to take part in ethical standards and according to the Declaration of SS metastasis [11, 12]. Helsinki and according to national and international SHCBP1(initially designated as murine protein of acti- guidelines and has been approved by the ethics commit- vated lymphocytes (mPAL)) that mapped on a region of tee of the Second Hospital of Shandong University. Each chromosome 16q11.2, is first identified by its direct patient had signed an informed consent. A total of interaction with the adapter protein p52Shc [13, 14]. seventy-six SS tissue specimens were obtained from Evolutionarily SHCBP1 is conserved in eukaryotes, patients who underwent surgical treatment without prior human SHCBP1 sequence shares 78% identity with radiotherapy or chemotherapy from the years between mouse’s and 23% with the Drosophila melanogaster 2005 and 2015 at the Department of Orthopaedics, the homolog Nessun Dorma’s [15]. Recent studies Second Hospital of Shandong University (Shandong, demonstrated that SHCBP1 plays critical roles in cell China). All tissue specimens were diagnosed clinically proliferation, migration, adhesion, and cell cycle and pathologically and routinely fixed in 10% formalin progression, especially in the carcinogenesis [16–20]. and embedded in paraffin for immunohistochemical Colak D et al. [18] found that SHCBP1 is highly staining. Histological grade was used by the Federation expressed in breast cancer and significantly correlated Natinale des centres de Lutte Cotre le Cancer with metastatic potential, advanced stage, and poor (FNCLCC) grading system, and disease stage according prognosis. Whereas silencing the expression of SHCBP1 to the International Union Against Cancer (UICC) through RNA interference can inhibit the proliferation staging system. Peng et al. Journal of Experimental & Clinical Cancer Research (2017) 36:141 Page 3 of 12

The human SS cell line HS-SY-II was kindly provided PCR assay kit (TAKARA, Otsu, Japan) on an ABI by Dr. Yi Guo (University of California, Irvine, USA), PRISM 7300 Sequence Detection System (Applied and cultured in Dulbecco’s modified Eagle’s medium Biosystems, Foster City, California, USA). The level of (DMEM, Gibco, NY, USA). Human SS cell line SW982 GAPDH was used as an internal control. Fold changes ΔΔ (HTB-93) was obtained from the ATCC (American Type in expression were calculated using the 2- Ct method. Culture Collection, Manassas, VA, USA), and maintained in L-15 medium (Gibco). Human umbilical vascu- Vector construction and lentivirus infection lar endothelial cells (HUVECs, ATCC) were cultured in The lentiviral vector containing SHCBP1 DNA sequence RPMI-1640 medium. Human breast cancer cell line (LV-SHCBP1), lentiviral vector containing SHCBP1 MCF-7 and human hepatocarcinoma cell line HepG2 siRNA sequence (LV-shSHCBP1), and non-effective were purchased from ATCC and both maintained in scrambled shRNA (LV-Control) were constructed by Eagle’s Minimum Essential Medium (EMEM, Gibco). Genechem (Shanghai, China). Constructs containing the Human osteosarcoma cell line Saos2 was also from ATCC sequences of SHCBP1 and SHCBP1 shRNA were cloned and cultured in McCoy’s 5A Medium (Gibco). All media into the pGCSIL-GFP (green fluorescent protein) vector were supplemented with 10% fetal bovine serum (FBS, (Genechem). The shSHCBP1 target sequence was 5′- Gibco), and all the cell lines were cultured at 37 °C in a TGGTGAAACCTACAATCTT-3′. Lentiviruses produc- humidified atmosphere with 5% CO2. tion and infection were performed as previously reported [24]. Stable cell lines expressing SHCBP1 or SHCBP1 Immunohistochemistry (IHC) shRNAs were selected for 10 days with 0.5 μg/ml IHC was performed as previously described [24, 25]. puromycin. Antibody against SHCBP1 (12672–1-AP) was purchased from Proteintech (Wuhan, China). Antibodies against Western blot analysis phosphorylated (p)-Smad2 (ab53100), p-Smad3 Standard western blot analysis was carried out as (ab51177) and TGF-β1 (ab25121) were purchased from described previously [26]. Antibodies against E-cadherin Abcam (Cambridge, MA). All sections were assessed (ab76055), vimentin (ab92547), VEGF (vascular endothe- and scored by two independent pathologists (Xiaoying lial growth factor, ab46154) and TGF-β1(ab25121) were Wang and Chengjun Zhou) in a blind manner. Ten purchased from Abcam (Cambridge, MA). The following high-power fields were selected randomly for each slide. antibodies were purchased from Cell Signaling The expression levels of each marker in tumor cells were Technology (Beverly, MA, USA): Smad2/3 (#3102), p- independently evaluated. The percentage of positive- Smad2 (#3108), p-Smad3 (#9520), MMP-2 (matrix staining cells were graded on a scale of 0 to 3, with less metalloproteinase-2, #4022) and MMP-9 (#3852). than 5% positive-staining cells as grade 0, 5 to 25% as SHCBP1 antibody (12672–1-AP) was purchased from grade 1, 26 to 50% as grade 2, and more than 50% as Proteintech (Wuhan, China). β-actin (sc-8432) antibody grade 3. The intensity of staining was also graded on a was purchased from Santa Cruz Biotechnology (Santa scale of 0 to 2, with negative to weak intensity as grade Cruz, CA). 0, weak to moderate intensity as grade 1, and moderate to strong intensity as grade 2. After that, the percentage Endothelial adhesion assay score was multiplied by the intensity score. A final score Endothelial adhesion assay was performed as described between 0 and 2 was defined as low expression, and a previously [27]. Briefly, HUVECs were grown to score higher than 2 was defined as high expression. confluency in a 6-well plate. 2 × 104 tumor cells infected with LV-SHCBP1, LV-shSHCBP1, or LV-Control were RNA extraction and real-time RT-PCR then delivered into the wells pre-incubated HUVEC cells Total RNA was extracted using Trizol reagent (Invitro- for 30 min at 37 °C. The wells were gently washed with gen, Carlsbad, CA, USA), and reversely transcripted to PBS twice to remove non-adherent cells. For quantifica- cDNA with M-MLV reverse transcriptase kit (Promega, tion, at least five random images were taken. Tumor USA) in accordance to the manufacturer’s instructions. cells adhering to the endothelial monolayer were then The PCR primers for SHCBP1 and glyceraldehyde-3- quantified and photographed using a fluorescent phosphate dehydrogenase (GAPDH) were as follows: microscope system. SHCBP1 forward, 5′-GCTACCGTGATAAAC- CAGGTTC-3′; and SHCBP1 reverse, 5′-AGGCTCT- Wound healing assay GAATCGCTCATAGA-3′; GAPDH forward, 5′- The wound healing assay was used to measure the TGACTTCAACAGCGACACCCA-3′; GAPDH reverse migration ability of SS cells. Cells were plated in six-well 5′-CACCCTGTTGCTGTAGCCAAA-3′. Then real time plates at a cell density of 5 × 104 /ml. After 24 h culture, RT-PCR was performed with the SYBR Green Real-Time cells were infected with LV-SHCBP1, LV-shSHCBP1, or Peng et al. Journal of Experimental & Clinical Cancer Research (2017) 36:141 Page 4 of 12

LV-Control and cultured to confluence. A wound was groups. Data were represented as the mean ± SD. A made by scratching with a plastic pipette tip. Plates were value of P < 0.05 was considered statistically significant. washed twice with phosphate buffer saline (PBS) to remove any cellular debris and detached cells. Then the cells were incubated with the complete growth medium. Results Cells migrated into the wounded empty space, and pho- SHCBP1 overexpression correlated with progression and tographs were taken at 0 and 24 h after wounding, poor prognosis of SS patients respectively. The clinical and pathologic characteristics of the 76 SS patients participating in the study are summarized in Cell migration and invasion assays Table 1. The SHCBP1 expression of SS samples was examined by immunohistochemically. Representative Cell migration assay was performed using Transwell SHCBP1 staining patterns in SS tissues of the defined chambers with 8.0 μm pore membrane as described pre- scoring criteria are shown in Fig. 1a. SHCBP1 expression viously [28]. Briefly, after infection with LV-SHCBP1, was strong in 56 (74%) samples and weak in other 20 LV-shSHCBP1, or LV-Control for 72 h, cells were har- samples (26%). In subsequent analysis, patients were vested and resuspended in serum-free medium. Then thus divided into SHCBP1-low (weak expression; n = 20) cells were added to the upper chambers at a density of 5 and SHCBP1-high (strong expression; n = 56) groups. In 1×10 cells/ml (200 μl per chamber) with 600 μl corresponding with this, SHCBP1 gene expression in the medium containing 10% FBS in the lower chambers. two tested SS cell lines, human osteosarcoma cell line Cells were allowed to migrate for 24 h at 37 °C in a CO 2 (Saos2), human hepatocarcinoma cell line (HepG2) and incubator. After that, non-migratory cells on the upper human breast cancer cell line (MCF-7) were significant surface of the membrane were carefully scraped off with upregulated (Fig. 1b), which is consistent with previous a cotton swab. The migratory cells on the bottom sur- studies [19, 20]. face were fixed in methanol and stained in 0.1% crystal To evaluate the relationship between SHCBP1 violet, and countered in 5 randomly selected micro- expression and tumor prognosis, the correlation scopic fields per well. between high SHCBP1 expression and clinicopathologi- For the invasion assay, the same procedures were per- cal features of SS was analyzed. The results indicated formed as those for the migration assay described above that SHCBP1 expression was significantly correlated except that the upper chamber membrane was coated with tumor size (P = 0.003), phosphor-Smad2 (P = 0.017) with 100 μl of 200 μg/ml Matrigel (BD Biosciences, or phosphor-Smad3 expression (P = 0.022), FNCLCC Franklin Lakes, NJ, USA) for 60 min at room grade (P = 0.003), UICC stage (P = 0.000), recurrence temperature before experiment. (P = 0.024) and distant metastasis (P = 0.000), whereas there was no significant correlation between SHCBP1 Matrigel tube formation assay expression and the age (P = 0.453), the gender of the SS Cells were seeded in 60 mm plates with fresh 1% serum patient (P = 0.655) as well as pathologic type (P = 0.246) medium for 24 h, and 2 ml of conditioned medium was (Table 1). μ harvested. The 96-well plates were pre-coated with 50 l The comparison of the prognostic effect of per well of Matrigel (10 mg/ml; BD Biosciences) and in- SHCBP1 on overall survival in SS patients with high 4 cubated at 37 °C for 1 h. Then, 5× 10 HUVECs were and low SHCBP1 protein levels revealed that μ suspended in 100 l of conditioned medium of SS cells, patients with high SHCBP1 expression displayed a and then gently added to the precoated 96-well plate. significantly lower 10-year survival rate compared to The plates were then incubated for 8 h at 37 °C, and those with low SHCBP1 expression, according to capillarylike tube formation was photographed with a Kaplan-Meier curve assessment (P =0.018, log-rank digital microscope system. test;Fig.1c).Notably,SHCBP1expressionalso significantly correlated with overall survival in both Statistical analysis the FNCLCC grade 2 (n =32, P =0.028; Fig. 1d) and The experimental data were performed using the UICC Stage 1+2 (n=32, P =0.03; Fig. 1f), whereas GraphPad Prism software. The relationship between the prognostic value of SHCBP1 expression in SHCBP1 and the survival of patients was assessed by the selective patient subgroups stratified according to Kaplan–Meier analysis, and the association between FNCLCC grade 3 and UICC Stage 3 + 4 was not SHCBP1 and clinicopathological variables was analyzed evident (Fig. 1e and g). Collectively, these results by Chi-square test. The differences were evaluated by indicated that SHCBP1 upregulation was significantly the log-rank test and the χ2, Fisher exact probability, associated with progression, metastasis, and poor and Student’s t tests were used for comparison between outcome in SS patients. Peng et al. Journal of Experimental & Clinical Cancer Research (2017) 36:141 Page 5 of 12

Table 1 Association of SHCBP1 expression with the lentivirus cells relative to the negative control lentivirus clinicopathological characteristics of SS cells (Fig. 2). Conversely, cells infected with LV- Variables All patients Expression of SHCBP1 P shSHCBP1 showed reduced adhesion capability diagnosed values Low High (P < 0.01) (Fig. 2). Further analysis the effect of SHCBP1 (n = 76) expression expression on the migration of SS cells by wound healing and trans- Gender 0.655 well migration assays showed that SHCBP1 overex- Male 45 11 34 pressed SS cells exhibited a remarkable increased cell Female 31 9 22 migration (Fig. 3a-3d). By contrast, SHCBP1 inhibition led to a significant decreased migration compared to Age 0.453 that of control cells (Fig. 3a-3d). Matrigel-coated Trans- ≤ 35 44 13 31 well chamber was further used to examine the effect of >35 32 7 25 SHCBP1 on tumor cell invasion at in vitro level. Corre- Tumor size(cm) 0.003 sponding to the results of migration assays, SHCBP1 ≤ 5351520 overexpression enhanced cell’s invasion (Fig. 3b-3d), > 5 41 5 36 whereas SHCBP1 inhibition suppressed that (Fig. 3b-3d). Furthermore, the expressions of MMP-2 and MMP-9 Pathologic type 0.246 that are closely associated with tumor’s invasion and MSS 36 15 21 migration were found clearly increased in LV-SHCBP1 BSS 40 22 18 lentivirus cells. By contrast, the results were opposite Expression of p-smad2 0.017 when SS cells were infected with LV-shSHCBP1 (Fig. 3e). 0–1462521 These findings suggested that SHCBP1 could promote SS 2–330822 cell adhesion, metastasis and invasion. Expression of p-smad3 0.022 SHCBP1 promotes HUVECs tube formation and the – 0 1372017 expression of VEGF in SS cells 2–3391128 In the later stages of angiogenesis, endothelial cells self- FNCLCC grade 0.003 assemble into tubes to form new blood vessels. To inves- 2321418 tigate the effect of SHCBP1 on neovascularization, 344638 HUVECs were cultured on Matrigel-coated plates in the conditioned media. After an 8 h incubation, cells UICC stage 0.000 infected with LV-SHCBP1 lentivirus showed significantly 1 or 2 32 16 16 increased HUVECs tube formation compared with the 3 or 4 44 4 40 negative control lentivirus cells (p < 0.01) (Fig. 4). How- Recurrence 0.024 ever, cells infected with LV-shSHCBP1 exhibited oppos- No 50 27 23 ite effect (Fig. 4). VEGF has been shown to be the most Yes 26 7 19 potent angiogenic factor in tumor angiogenesis [29]. VEGF protein levels assay in conditioned media from Distant metastasis 0.000 cells infected with LV-SHCBP1, LV-shSHCBP1, or LV- M0 52 28 24 Control showed that VEGF expression was significantly M1 24 2 22 decreased in LV-shSHCBP1 lentivirus cells, whereas it Abbreviations: MSS monophasic synovial sarcoma, BSS biphasic synovial was dramatically increased in LV-SHCBP1 lentivirus sarcoma, FNCLCC Federation Natinale des centres de Lutte Cotre le Cancer, UICC International Union Against Cancer cells (Fig. 4). Thus it can be concluded that SHCBP1 could markedly promote angiogenesis of SS cells. SHCBP1 promotes SS cell adhesion, metastasis and invasion in vitro SHCBP1 induces EMT of SS cells in vitro To examine whether SHCBP1 affects SS cell adhesion, EMT that characterized by a process in which epithelial metastasis and invasion, we constructed lentiviral cells obtain the migratory and invasive ability of mesen- SHCBP1 (LV-SHCBP1) and SHCBP1 shRNA (LV- chymal cells plays a key role in tumor progression and shSHCBP1) vectors and measured the in vitro migration metastasis [30].To determine whether SHCBP1 induces and invasion abilities of transduced cells. As known, EMT, the expression of EMT markers was evaluated. As tumor cell adhesion to the extracellular matrix is impli- expected, SHCBP1 overexpression decreased the levels cated in tumor cell’s motility, invasion and metastasis. of epithelial markers (E-cadherin) and increased the The cell adhesion assay indicated that cell adhesion levels of mesenchymal markers (vimentin) in both SS capability was significantly increased in LV-SHCBP1 cell lines (Fig. 5a). Conversely, silencing SHCBP1 Peng et al. Journal of Experimental & Clinical Cancer Research (2017) 36:141 Page 6 of 12

Fig. 1 Strong SHCBP1 expression in SS correlates with poorer patient overall survival. a Scores indicating SHCBP1 levels in representative SS tissues. Scale bar = 100 μm. b SHCBP1 expression was determined by real-time RT-PCR on total RNA extracted from human HS-SY-II and SW982 synovial sarcoma cell lines, Saos2 osteosarcoma cell line, HepG2 hepatocarcinoma cell line, MCF-7 breast cancer cell line and normal synovialcytes. c Kaplan-Meier survival analysis of overall survival in all patients. The curves of overall survival according to (d) FNCLCC grade 2, P = 0.028; (e) FNCLCC grade 3, P = 0.564; (f) UICC stage 1/2, P = 0.03; (g) UICC stage 3/4, P = 0.818 in 76 patients with synovial sarcoma

Fig. 2 SHCBP1 promotes the SS cell adhesion. Representative images of GFP transfected tumor cells adhering to the endothelial monolayer (magnification ×100). The data (mean ± SD) are statistically significant of three separated experiments, ** p < 0.01 Peng et al. Journal of Experimental & Clinical Cancer Research (2017) 36:141 Page 7 of 12

Fig. 3 SHCBP1 was associated with the invasive and metastatic potential of SS. a Representative micrographs of the motility of SHCBP1- overexpressing or SHCBP1-silencing cells in the wound healing assay at 0 h and 24 h compared to untreated and vector control cells. b, c and d Representative micrographs and quantification of the invasiveness of SHCBP1-overexpressing or SHCBP1-silencing cells in the transwell migration and invasion assays compared to untreated and vector control cells. e SHCBP1 increased the expression of MMP-2 and MMP-9 in SS cells. Error bars represent mean ± SD from three independent experiments, ** p < 0.01 increased the levels of epithelial markers, and lowered EMT and cell invasiveness. As showed in Fig. 6b, an the levels of mesenchymal markers (Fig. 5a). Similar increased expression of epithelial markers and a results were obtained when we performed real time RT- decreased expression of mesenchymal markers were PCR analysis for these genes (Fig. 5b). These findings apparent after LV-shSHCBP1 infection treated with together indicated that SHCBP1 could induce EMT in TGF-β1 for 48 h. Moreover, in accordance with the SS cells. above changes of EMT, silencing SHCBP1 decreased the invasive ability of tumor cells (Fig. 6c). These data dem- SHCBP1 participates in TGF-β1-induced EMT onstrated that SHCBP1 played a critical role in TGF-β1- With the most potent EMT inducer, TGF-β1, we then induced EMT in SS. assessed the impact of SHCBP1 on EMT of SS cells. Fig. 6a showed that TGF-β1 time-dependently induced SHCBP1 activates TGF-β1 signaling pathway SHCBP1 expression. This induction was accompanied Considering that the TGF-β1/Smad signaling is a key by higher expression of the mesenchymal marker vimen- pathway leading the high invasion and migration ability tin and lower expression of E-cadherin, an epithelial of many cancer cells, we consequently investigated the marker (Fig. 6a). LV-shSHCBP1 and LV-control were mechanisms by which SHCBP1 promotes SS cell migra- transfected into HS-SY-II and SW982 cells to further tion and invasion by examining the TGF-β1/Smad investigate the influence of SHCBP1 on TGF-β1-induced signaling pathway. As shown in Fig. 7a, the expression Peng et al. Journal of Experimental & Clinical Cancer Research (2017) 36:141 Page 8 of 12

Fig. 4 SHCBP1 promotes HUVECs tube formation and upregulates the expression of VEGF of SS cells. Representative phase-contrast images of the tubes that formed (magnification ×100). SHCBP1 enhanced the expression of VEGF protein levels of TGF-β1 significantly increased in SHCBP1- regulating TGF-β1/Smad signaling pathway. Moreover, overexpressing cells and decreased in SHCBP1-silenced TGF-β1, phospho-smad 2, and phospho-smad3 expres- cells. Activated TGF-β1 is known to induce the phos- sions were found to be upregulated in SHCBP1-high SS phorylation of the downstream targets Smad2 and samples (Fig. 7b). Altogether, our data suggest that Smad3, which then form hetero-oligomeric complexes SHCBP1 may promote the invasive capability of SS cells with Smad4 and translocate to the nucleus to regulate via activation of the TGF-β1/Smad signaling pathway. gene expression through transcription and thus mediate the biological effects of TGF-β1 such as cell growth, Discussion invasion and migration [31]. As expected, SHCBP1 over- Our previous studies uncovered that SHCBP1 is a novel expression markedly increased, but silencing SHCBP1 downstream target gene of SS18-SSX1, who acts as an reduced, the levels of phospho-smad2, and phospho- oncogene by promoting proliferation and tumorigenicity smad3 (Fig. 7a), suggesting that SHCBP1 contributes to of SS cells [24]. Here, we disclosed a significant

Fig. 5 SHCBP1 regulated EMT in vitro. a Western blot analysis of E-cadherin and vimentin proteins in SHCBP1-overexpressing or SHCBP1-silencing cells. b Representative micrographs of E-cadherin and vimentin mRNA expressions in SHCBP1-overexpressing or SHCBP1-silencing cells as determined by real-time RT-PCR. Bars represent the mean ± SD of three independent experiments, ** p < 0.01 Peng et al. Journal of Experimental & Clinical Cancer Research (2017) 36:141 Page 9 of 12

Fig. 6 SHCBP1 participates in TGF-β1-induced EMT. a Western blot of SHCBP1, vimentin and E-cadherin in the indicated cells in response to treatment with 10 ng/mL TGF-β1 for 0, 24, and 48 h. b Twenty-four hours post-transfection of LV-Control or LV-shSHCBP1 lentivirus, the cells were treated with TGF-β1 (2 ng/ml) for an additional 48 h. The expressions of SHCBP1, E-cadherin and vimentin were detected by Western blot. c Representative images and data of a transwell invasion assay for HS-SY-II and SW982 cells. TGF-β1 stimulation significantly increased the invasiveness of both tumor cell lines compared with that in the absence of TGF-β1 (LV-Control) or LV-shSHCBP1 lentivirus cells. Each bar represents the mean ± SD, **P < 0.01. All images are representative of three independent experiments with similar findings association between elevated SHCBP1 expression and with lower SHCBP1 expression, indicating that SHCBP1 tumor size, FNCLCC grade, UICC stage, and distant seems represent an independent prognostic factor for SS metastasis. Similar to breast cancer [20] and hepatocel- patients. Most importantly, SHCBP1 overexpression lular carcinoma [19], patients with higher SHCBP1 increased adhesion, migration and invasion of SS cells expression had shorter overall survival time than those through inducing EMT, activated TGF-β1/Smad

Fig. 7 SHCBP1 activates the TGF-β1/Smad signaling pathway. a Western blot analysis of TGF-β1, p-Smad2, p-Smad3, and total Smad2/3 in SHCBP1-overexpressing or SHCBP1-silencing cells. b Immunostaining revealed the expressions of TGF-β1, p-Smad2, and p-Smad3 in SHCBP1-high and SHCBP1-low SS tissues (magnification ×200). Scale bar = 100 μm Peng et al. Journal of Experimental & Clinical Cancer Research (2017) 36:141 Page 10 of 12

signaling pathway and significantly correlated with SS whereas it promotes tumor invasion and metastasis progression, through EMT at later stage of cancer [36]. The present SHCBP1 contributes to enhanced aggressive behavior study showed that induction of EMT in SS cells by of SS cells, overexpression of SHCBP1 increased adhe- TGF-β1 was associated with a significant elevation in sion, angiogenesis, migration and invasion in vitro, while SHCBP1 expression. These changes included signifi- silencing it inhibited these malignant behaviors, signify- cantly decreased expression of E-cadherin, concomi- ing prominent positive correlation between the expres- tantly increased expression of vimentin, and enhanced sion of SHCBP1 and the metastasis and invasion of SS. the ability of invasion of SS cells. These results indicated Tumor metastasis is a complex and multistage process. that SHCBP1 serves as a co-stimulator in TGF-β1- During this, tumor cells need to express a variety of dis- induced EMT in SS. tinct properties including altered adhesiveness, increased In order to better understand the molecular mecha- motility, invasive and angiogenic capacity to finish the nisms by which SHCBP1 regulated the metastasis of SS metastatic process [32]. Meanwhile, degradation of stro- cells, we further investigated the TGF-β/Smad signaling mal extracellular matrix (ECM) is a crucial step for pathway. TGF-β1 binds to TGF-β type I receptor, a tumor invasion and metastasis. Accumulating evidences transmembrane protein with Ser/Thr kinase domain, had demonstrated that MMP-2 and MMP-9 play key and then phosphorylates Smad2 and Smad3, which regu- roles in invasion and metastasis of various cancers [33], lates the expression of downstream target genes and including SS [34], due to their specificity for degrading subsequently contributes to promoting EMT and metas- the basement membrane. Additionally, VEGF, the most tasis of many cancer cells [37]. In accordance with previ- potent tumor angiogenic factor, closely involved in ous reports, the levels of phospho-smad2 and phospho- tumor cell invasion and metastasis. The proliferation of smad3 were found to be increased in SHCBP1- endothelial cells in many human cancers can be stimu- overexpressing cells and decreased in SHCBP1-silenced lated by VEGF. Indeed, VEGF expression is frequently cells. Therefore, the effects of SHCBP1 on inducing of upregulated in many tumors, including SS [34]. In EMT and promoting the invasion of SS cells are at least current study, the expressions of MMP-2, MMP-9 and partly mediated by activating the TGF-β1/Smad VEGF were found conformably decreased in SHCBP1- signaling. How SHCBP1 activates the TGF-β/Smad silencing cells and enhanced in SHCBP1-overexpressing signaling pathway is under investigation currently in our cells, suggesting that SHCBP1-induced metastasis and laboratory. invasion at least partially realized through modulating the expressions of MMP-2, MMP-9 and VEGF. Conclusions Previous research suggests that cancer cells obtain the SHCBP1 was found highly expressed in SS cell lines and ability of invasion principally through the EMT, a process tissues, which was linked to the poor clinical prognosis that epithelial cells lose their cell-cell adhesion and obtain and was implicated in SS aggressive progression. More- mesenchymal feature, which plays critical roles in the over, SHCBP1 regulated invasion and metastasis by development and progression, the invasion and migration modulating the TGF-β1/Smad signaling pathway and of diverse human tumors, including SS [11, 12]. Our data promoting the EMT in SS. Most important, this is the revealed that SHCBP1 overexpression increased adhesion, first study that demonstrating the contribution of EMT migration and invasion, which was accompanied with the to the metastasis and invasion of SS cells. Thus SHCBP1 elevated expression of mesenchymal markers vimentin might serve as a promising target for SS treatment. and the decreased expression of epithelial markers E- cadherin. Conversely, silencing SHCBP1 had the opposite Abbreviations effects. These data suggested that SHCBP1 plays a key role ECM: Extracellular matrix; EGF: Epidermal growth factor; EMT: Epithelial- in the acquisition of cell motility and invasiveness of SS emesenchymal transition; FBS: Fetal bovine serum; FNCLCC: Federation Natinale cells through induction of EMT. des centres de Lutte Cotre le Cancer; GAPDH: Glyceraldehyde-3-phosphate β dehydrogenase; GFP: Green fluorescent portein; HCC: Hepatocellular carcinoma; TGF- 1 is a multifunctional cytokine that takes part in HGF: Hepatocyte growth factor; HUVECs: Human umbilical vascular endothelial regulating multiple cellular processes, such as cell prolif- cells; IHC: Immunohistochemistry; LV: Lentiviruses; MMP: Matrix eration, differentiation, motility, angiogenesis, invasion metalloproteinase; mPAL: Murine protein of activated lymphocytes; β PBS: Phosphate buffer saline; SHCBP1: SHC SH2-domain binding protein 1; and metastasis. Previous studies had shown that TGF- 1 shRNA: Short-hairpin RNA; SS: Synovial sarcoma; TGF-β1: Transforming growth is overexpressed in many kinds human malignant factor-β1; UICC: International Union Against Cancer; VEGF: Vascular endothelial tumors, including SS [11]. TGF-β1 can function in either growth factor inhibiting tumors or promoting tumors, depending on the course of tumor progression [35]. At early stage of Acknowledgements β We express our sincere gratitude to Dr. Yang Jiang for the helpful discussion tumorigenesis, TGF- 1 signaling impedes tumor prolif- and comments. We also thank Dr. Yun Luan for her assistance in English eration via inducing cell cycle arrest and apoptosis, language editing. Peng et al. Journal of Experimental & Clinical Cancer Research (2017) 36:141 Page 11 of 12

Funding 8. Pantel K, Otte M. Occult micrometastasis: enrichment, identification and This work was supported by the National Natural Science Foundation of characterization of single disseminated tumour cells. Semin Cancer Biol. China (81301727). 2001;11:327–37. 9. Gemoll T, Epping F, Heinrich L, Fritzsche B, Roblick UJ, Szymczak S, et al. Availability of data and materials Increased cathepsin D protein expression is a biomarker for osteosarcomas, Data sharing not applicable to this article as no datasets were generated or pulmonary metastases and other bone malignancies. Oncotarget. 2015;6: – analysed during the current study. 16517 26. 10. Fan Y, Shen B, Tan M, Mu X, Qin Y, Zhang F, et al. TGF-beta-induced upregulation of malat1 promotes bladder cancer metastasis by associating Authors’ contributions with suz12. Clin Cancer Res. 2014;20:1531–41. CLP, XWW and CZG conceived and designed the study. CLP, HZ, YS, XYW 11. Qi Y, Wang CC, He YL, Zou H, Liu CX, Pang LJ, et al. The correlation and XLL performed the experiments. CZ, JZ, JL, GHC and DJW analyzed and between morphology and the expression of TGF-beta signaling pathway interpreted the data. CLP wrote the manuscript. All authors read and proteins and epithelial-mesenchymal transition-related proteins in synovial approved the final manuscript. sarcomas. Int J Clin Exp Pathol. 2013;6:2787–99. 12. Saito T. The SYT-SSX fusion protein and histological epithelial differentiation Ethics approval and consent to participate in synovial sarcoma: relationship with extracellular matrix remodeling. Int J This study was performed in accordance with the ethical standards and Clin Exp Pathol. 2013;6:2272–9. according to the Declaration of Helsinki and according to national and 13. Schmandt R, Liu SK, McGlade CJ. Cloning and characterization of mPAL, a international guidelines and has been approved by the ethics committee of novel Shc SH2 domain-binding protein expressed in proliferating cells. the Second Hospital of Shandong University. Informed consents (written in Oncogene. 1999;18:1867–79. the light of the ethical guidelines) were obtained from all the patients. 14. Zhang L, Camerini V, Bender TP, Ravichandran KS. A nonredundant role for the adapter protein Shc in thymic T cell development. Nat Immunol. 2002;3: Consent for publication 749–55. Not applicable. 15. Montembault E, Zhang W, Przewloka MR, Archambault V, Sevin EW, Laue ED, et al. Nessun Dorma, a novel centralspindlin partner, is Competing interests required for cytokinesis in drosophila spermatocytes. J Cell Biol. 2010; The authors declare that they have no competing interests. 191:1351–65. 16. Zheng Y, Zhang C, Croucher DR, Soliman MA, St-Denis N, Pasculescu A, et al. Temporal regulation of EGF signalling networks by the scaffold protein Publisher’sNote Shc1. Nature. 2013;499:166–71. Springer Nature remains neutral with regard to jurisdictional claims in 17. Asano E, Hasegawa H, Hyodo T, Ito S, Maeda M, Takahashi M, et al. The published maps and institutional affiliations. aurora-B-mediated phosphorylation of SHCBP1 regulates cytokinetic furrow ingression. J Cell Sci. 2013;126:3263–70. Author details 18. Colak D, Nofal A, Albakheet A, Nirmal M, Jeprel H, Eldali A, et al. Age-specific 1Department of Orthopaedics, The Second Hospital of Shandong University, gene expression signatures for breast tumors and cross-species conserved Shandong University, Jinan, China. 2Department of Orthopedics, Beijing potential cancer progression markers in young women. PLoS One. 2013;8: Chaoyang Hospital, Capital Medical University, Beijing, China. 3Department of e63204. Nephrology, The Second Hospital of Shandong University, Shandong 19. Tao HC, Wang HX, Dai M, Gu CY, Wang Q, Han ZG, et al. Targeting SHCBP1 University, Jinan, China. 4Beijing Institute of Pharmacology and Toxicology, inhibits cell proliferation in human hepatocellular carcinoma cells. Asian Pac Beijing, China. 5Department of Pathology, The Second Hospital of Shandong J Cancer Prev. 2013;14:5645–50. University, Shandong University, Jinan, China. 6Department of Hematology, 20. Feng W, Li HC, Xu K, Chen YF, Pan LY, Mei Y, et al. SHCBP1 is over- The Second Hospital of Shandong University, Shandong University, Jinan, expressed in breast cancer and is important in the proliferation and China. 7Central Research Laboratory, The Second Hospital of Shandong apoptosis of the human malignant breast cancer cell line. Gene. 2016;587: University, Shandong University, Jinan, China. 91–7. 21. Eckerle S, Brune V, Doring C, Tiacci E, Bohle V, Sundstrom C, et al. Gene Received: 15 March 2017 Accepted: 9 October 2017 expression profiling of isolated tumour cells from anaplastic large cell lymphomas: insights into its cellular origin, pathogenesis and relation to Hodgkin lymphoma. Leukemia. 2009;23:2129–38. References 22. Piccaluga PP, Agostinelli C, Califano A, Rossi M, Basso K, Zupo S, et al. Gene 1. Lagarde P, Przybyl J, Brulard C, Perot G, Pierron G, Delattre O, et al. expression analysis of peripheral T cell lymphoma, unspecified, reveals Chromosome instability accounts for reverse metastatic outcomes of distinct profiles and new potential therapeutic targets. J Clin Invest. 2007; pediatric and adult synovial sarcomas. J Clin Oncol. 2013;31:608–15. 117:823–34. 2. Yasui H, Naka N, Imura Y, Outani H, Kaneko K, Hamada K, et al. Tailored 23. Truffinet V, Pinaud E, Cogne N, Petit B, Guglielmi L, Cogne M, et al. The 3′ therapeutic strategies for synovial sarcoma: receptor tyrosine kinase IgH locus control region is sufficient to deregulate a c-myc transgene and pathway analyses predict sensitivity to the mTOR inhibitor RAD001. Cancer promote mature B cell malignancies with a predominant Burkitt-like Lett. 2014;347:114–22. phenotype. J Immunol. 2007;179:6033–42. 3. Fricke A, Ullrich PV, Heinz J, Pfeifer D, Scholber J, Herget GW, et al. 24. Peng C, Zhao H, Chen W, Song Y, Wang X, Li J, et al. Identification of Identification of a blood-borne miRNA signature of synovial sarcoma. Mol SHCBP1 as a novel downstream target gene of SS18-SSX1 and its functional Cancer. 2015;14:151. analysis in progression of synovial sarcoma. Oncotarget. 2016;7:66822–34. 4. Spillane AJ, A'Hern R, Judson IR, Fisher C, Thomas JM. Synovial sarcoma: a 25. Fletcher SJ, Sacca PA, Pistone-Creydt M, Colo FA, Serra MF, Santino FE, clinicopathologic, staging, and prognostic assessment. J Clin Oncol. 2000;18: et al. Human breast adipose tissue: characterization of factors that 3794–803. change during tumor progression in human breast cancer. J Exp Clin 5. Lewis JJ, Antonescu CR, Leung DH, Blumberg D, Healey JH, Woodruff JM, et Cancer Res. 2017;36:26. al. Synovial sarcoma: a multivariate analysis of prognostic factors in 112 26. Wang D, Hu G, Du Y, Zhang C, Lu Q, Lv N, et al. Aberrant activation of patients with primary localized tumors of the extremity. J Clin Oncol. 2000; hedgehog signaling promotes cell proliferation via the transcriptional 18:2087–94. activation of forkhead box M1 in colorectal cancer cells. J Exp Clin Cancer 6. Palmerini E, Staals EL, Alberghini M, Zanella L, Ferrari C, Benassi MS, et al. Res. 2017;36:23. Synovial sarcoma: retrospective analysis of 250 patients treated at a single 27. Ji T, Guo Y, Kim K, McQueen P, Ghaffar S, Christ A, et al. Neuropilin-2 institution. Cancer. 2009;115:2988–98. expression is inhibited by secreted Wnt antagonists and its down-regulation 7. Bogenrieder T, Herlyn M. Axis of evil: molecular mechanisms of cancer is associated with reduced tumor growth and metastasis in osteosarcoma. metastasis. Oncogene. 2003;22:6524–36. Mol Cancer. 2015;14:86. Peng et al. Journal of Experimental & Clinical Cancer Research (2017) 36:141 Page 12 of 12

28. Wang Z, Zhou Y, Hu X, Chen W, Lin X, Sun L, et al. RILP suppresses invasion of breast cancer cells by modulating the activity of RalA through interaction with RalGDS. Cell Death Dis. 2015;6:e1923. 29. Hu J, Cheng Y, Li Y, Jin Z, Pan Y, Liu G, et al. microRNA-128 plays a critical role in human non-small cell lung cancer tumourigenesis, angiogenesis and lymphangiogenesis by directly targeting vascular endothelial growth factor- C. Eur J Cancer. 2014;50:2336–50. 30. Thiery JP, Acloque H, Huang RY, Nieto MA. Epithelial-mesenchymal transitions in development and disease. Cell. 2009;139:871–90. 31. Javelaud D, Alexaki VI, Dennler S, Mohammad KS, Guise TA, Mauviel A. TGF- beta/SMAD/GLI2 signaling axis in cancer progression and metastasis. Cancer Res. 2011;71:5606–10. 32. Li C, Li F, Zhao K, Yao J, Cheng Y, Zhao L, et al. LFG-500 inhibits the invasion of cancer cells via down-regulation of PI3K/AKT/NF-kappaB signaling pathway. PLoS One. 2014;9:e91332. 33. Che YL, Luo SJ, Li G, Cheng M, Gao YM, Li XM, et al. The C3G/Rap1 pathway promotes secretion of MMP-2 and MMP-9 and is involved in serous ovarian cancer metastasis. Cancer Lett. 2015;359:241–9. 34. Arai R, Tsuda M, Watanabe T, Ose T, Obuse C, Maenaka K, et al. Simultaneous inhibition of Src and aurora kinases by SU6656 induces therapeutic synergy in human synovial sarcoma growth, invasion and angiogenesis in vivo. Eur J Cancer. 2012;48:2417–30. 35. Massague J. TGFbeta in cancer. Cell. 2008;134:215–30. 36. Wu YY, Peck K, Chang YL, Pan SH, Cheng YF, Lin JC, et al. SCUBE3 is an endogenous TGF-beta receptor ligand and regulates the epithelial- mesenchymal transition in lung cancer. Oncogene. 2011;30:3682–93. 37. Xu M, He J, Li J, Feng W, Zhou H, Wei H, et al. Methyl-CpG-binding domain 3 inhibits epithelial-mesenchymal transition in pancreatic cancer cells via TGF-beta/Smad signalling. Br J Cancer. 2017;116:91–9.

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