Silencing Formin-like 2 inhibits growth and metastasis of gastric cancer cells through suppressing internalization of integrins

Silencing Formin-like 2 inhibits growth and metastasis of gastric cancer cells through... Background: Formin-like 2 (FMNL2) is a member of Formin family which governs cytokinesis, cellular polarity and morphogenesis. Dysregulation of FMNL2 has been discovered in cancers and is closely related to cancers. However, the role of FMNL2 in gastric cancer remains unclear. In this study, we aimed to investigate the role of FMNL2 in gastric cancer cells. Methods: A FMNL2-specific shRNA was employed to decrease the endogenous expression of FMNL2. Then the degree of proliferation, apoptosis, migration and invasion of gastric cancer cells was assessed by MTT assay, flow cytometry, wound healing assay and transwell assay, respectively. The expression and distribution of FMNL2 and pro- tein kinase C (PKC) α was detected by immunofluorescence. The internalization of integrins was detected by enzyme- linked immunosorbent assay. Results: Our results showed that silencing FMNL2 suppressed proliferation, migration and invasion, and induced apoptosis of gastric cancer cells. The integrin internalization induced by PKC was declined by FMNL2 silencing. Conclusions: Our study reveals that silencing FMNL2 suppresses growth and metastasis of gastric cancer cells. Modulation on integrin internalization may be implicated in the role of FMNL2 in growth and migration of gastric cancer cells. Our study indicates that FMNL2 may become a potential therapeutic target for gastric cancer. Keywords: Apoptosis, Formin-like 2, Gastric cancer, Integrin internalization, Invasion, Migration, Proliferation Background and recycling of integrins emerges as a major player in Gastric cancer, which threatens human health, is the fifth controlling integrin action [3]. Integrins also contribute most common cancer worldwide with approximately one to tumor progression and are implicated in cell survival, million cases in 2012 [1]. Surgery is the curative modal- metastasis, angiogenesis and drug resistance [4–8]. They ity for gastric cancer treatment. However, the recurrence also influence the response of host cells to cancers [ 8]. rate is still high, and the 5-year survival rate is low [2]. Integrins are regarded as appealing targets for tumor Integrins are a group of αβ heterodimers. Integrins therapy [8]. are parts of the major receptors spanning through the Formins are a group of Rho guanosine triphosphatase lipid bilayer of cells. Integrins bind directly to extracel- (GTPase) effectors, governing cytokinesis, polarity, adhe - lular matrix components, transduce signals outside-in sion and morphogenesis [9]. Diaphanous-related Formins and activate intracellular signals in context of cytokine (DRFs) are a conserved subgroup of Formins. They are receptors or growth factor receptors. Internalization involved in a wide range of cellular processes, including filopodium formation, cell migration and cell polarity [ 9– 11], and contribute to many physiological or pathological *Correspondence: fanminkong@126.com processes [12–14]. Formin-like 2 (FMNL2), a member of Department of Gastric, Intestine and Hernia Surgery, The First Affiliated DRFs, locates on chromosome 2q23.3. FMNL2 contains Hospital of China Medical University, Shenyang 110001, People’s Republic a GTPase-binding domain and an autoregulatory domain of China © The Author(s) 2018. 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. Zhong et al. Cancer Cell Int (2018) 18:79 Page 2 of 10 [15], and acts as both a downstream effector and an Oligo(dT) and Super M-MLV Reverse Transcriptase upstream modulator of Rho family GTPases [11]. FMNL2 (BioTeke) according to the manufacturers’ instructions. plays a key role in actin filament nucleation or elongation mRNA level of FMNL2 in each group was measured by which influences cell morphology [9]. FMNL2 also has qRT-PCR (SYBR Green method) with cDNA as the tem- a close relationship with cancer. For example, FMNL2 plate. The following primers were used: forward primer enhances the growth and metastasis of colon cancer for FMNL2, 5′-CCC GCT CTG GAA GAC ATT -3′; reverse in which FMNL2 is highly expressed [16–18]. Moreo- primer for FMNL2, 5′-CTG CCA ACA GTT CTA AGA ver, FMNL2 performs an inhibitory effect on motility CAAG-3′; forward primer for β-actin, 5′-CTT AGT TGC of hepatocarcinoma which shows a lower FMNL2 level GTT ACA CCC TTT CTT G-3′; reverse primer for β-actin, than normal livers [19]. In addition, patients with aber- 5′-CTG TCA CCT TCA CCG TTC CAG TTT -3′. SYBR rant FMNL2 expression show a lower 5-year survival rate Green Reagent was obtained from Solarbio (Beijing, [19]. Dysregulation of FMNL2 has been discovered in China). The relative mRNA level of FMNL2 was normal - −ΔΔCt several types of cancers and is associated with the devel- ized to β-actin and calculated using 2 method. opment of aggressive tumors [16–20]. However, the role of FMNL2 in gastric cancer remains unclear. Western blot In the present study, we aimed to investigate the effect Proteins in each group were extracted using radio of FMNL2 silencing on gastric cancer cells. The results of immunoprecipitation assay lysis buffer (Beyotime Bio - our study indicate that FMNL2 may act as an oncogene technology, Haimen, China) containing 1% phenylmeth- in gastric cancer cells and has the potential to become a anesulfonyl fluoride (Beyotime Biotechnology). The therapeutic target for gastric cancer. concentration of proteins in each group was measured using a BCA Protein Assay Kit (Beyotime Biotechnol- Materials asnd methods ogy). Forth microgramme proteins in each group were Cells separated by sodium dodecyl sulfate polyacrylamide Gastric cancer cell lines BGC-823, MGC-803, HGC- gel electrophoresis. After electrophoresis, the proteins 27 were obtained from Zhongqiaoxinzhou Biotechnol- were transferred onto polyvinylidene fluoride mem - ogy Co., Ltd (Shanghai, China). SGC-7901 cell line was branes (Millipore, Bedford, MA, USA). The membranes obtained from CHI Scientific (Jiangyin, China). BGC-823 were blocked with 5% skim milk, followed by incubating and MGC-803 cells were grown in Dulbecco’s Modified with primary antibodies against FMNL2 (1:500; Novus, Eagle’s Medium (Gibco, Grand Island, NY, USA) with Oakville, ON, Canada), E-cadherin (1:2000, Protein- 10% fetal bovine serum (FBS; Hyclone, Logan, UT, USA). tech, Wuhan, China), Vimentin (1:2000, Proteintech), SGC-7901 and HGC-27 cells were grown in RPMI 1640 N-cadherin (1:2000, Proteintech) or β-actin (1:500; medium (Gibco) with 10% FBS. All these cells were cul- Bioss, Beijing, China) at 4  °C overnight. After rinsing in tured in a humid atmosphere at 37 °C with 5% CO . Tris buffered saline with Tween (TBST), the membranes were incubated with horseradish peroxidase (HRP)-con- Transfection jugated goat-anti-rabbit IgG(H+L) or HRP-conjugated HGC-27 cells were seeded in a 6-well plate (2 × 10 cells/ goat-anti-mouse IgG(H+L) (1:5000; Beyotime Biotech- well) and cultured in a cell incubator. Twenty-four hours nology) at 37  °C for 45  min. After rinsing in TBST, the later, the cells were transfected with FMNL2 shRNA (tar- targeted bands were visualized using an ECL Kit (Beyo- get sequence: 5′-GTG GTA GCA GGT AAC TCT G-3′) or time Biotechnology). negative control using Lipofectamin 2000 Reagent (Inv- itrogen, Carlsbad, CA, USA) according to the manufac- turer’s protocol. Then the cells were maintained in cell 3‑(4,5‑Dimethyl‑2‑thiazolyl)‑2,5‑diphenyl‑2‑H‑tetrazolium medium with 800  μg/ml G418 (Invitrogen) for 7  days bromide (MTT) assay to select stably transfected cells. Cells stably transfected Cells in each group were seeded into 96-well plates with FMNL2 shRNA were named as FMNL2 silenc- (4 × 10 cells/well) and cultured in a cell incubator for ing. Cells stably transfected with negative control were 0, 24, 48 and 72  h, respectively. Then MTT (Sigma, St. named as negative control. Cells without transfection Louis, MO, USA) at a final concentration of 0.5  mg/ were named as cells only. ml was added into each well. After incubating for addi- tional 4 h, 150 μl dimethyl sulfoxide (DMSO, Sigma) was Quantitative real‑time PCR (qRT‑PCR) added into each well to dissolve the crystals after removal Total RNA in cells from each group was extracted using of supernate. Thereafter, the absorbance at 490  nm was a High-purity Total RNA Fast Extraction kit (BioTeke, measured with a microplate reader (BIOTEK, Winooski, Beijing, China) and reverse transcribed to cDNA using VT, USA). Zhong et al. Cancer Cell Int (2018) 18:79 Page 3 of 10 Flow cytometry Wuhan, China) at 4  °C overnight. Thereafter, the cells Degree of apoptosis in each group was analyzed by flow were rinsed with PBS and incubated Cy3-conjugated cytometry with a Cell Apoptosis Detection Kit (Key- goat-anti-rabbit IgG(H+L) or FITC-conjugated goat- Gen, Nanjing, China). Cells in each group were collected, anti-mouse IgG(H+L) (1:200; Beyotime Biotechnology). rinsed in phosphate buffered saline (PBS) and resus - The cells were then rinsed with PBS and observed under pended in 500  μl binding buffer. Then 5  μl Annexin– a fluorescence microscope (600×; OLYMPUS, Tokyo, Fluorescein Isothiocyanate (FITC) and 5  μl Propidium Japan). Iodide (PI) were added into cells for incubation for addi- tional 15 min at room temperature in dark. The cells were Integrin internalization assay then analyzed with a flow cytometer (BD, Franklin Lakes, Cells were labeled with EZ-Link Sulfo-NHS-SS-Biotin NJ, USA). (0.5 mg/ml; Thermo Fisher Scientific, Rockford, IL, USA) on ice for 1  h. Then the cells were treated with DMSO Wound healing assay or TPA for 30 min. The cells were kept on ice and rinsed Cells in each group were seeded into a 6-well plate. with ice-cold PBS. Biotin residual on cell surface was When the confluence reached 90%, the cell medium was removed by incubating in Tris buffer containing 20  mM changed to fresh serum-free medium. Then the cells MesNa (Aladdin, Shanghai, China) at 4  °C for 15  min. were treated with mitomycin C (1  μg/ml; Sigma) for Superfluous MesNa was quenched by 20 mM iodoaceta - 1  h. Thereafter, scratches were made on single-cell sur - mide (Aladdin). Then the cells were collected and lysed face with 200 μl pipette tips. The cells were washed with in lysis solution (containing 200 mM NaCl, 15 mM NaF, serum-free medium to remove cell debris, and allowed to 75  mM Tris, 7.5  mM EDTA, 7.5  mM EGTA, 1.5  mM migrate for 24  h in serum-free medium. Images of cells Na3VO4, and 1.5% TritonX-100). The supernatant was were captured with a microscope at 0  h and 24  h. The collected by centrifugation at 1500×g at 4 °C for 10 min. relative migrate rate was calculated using the following After measuring the concentration with a BCA Protein formula: relative migrate rate = (distance between gap at Assay Kit, the level of biotinylated integrin was analyzed 0 h − distance between gap at 24 h)/distance between gap by enzyme-linked immunosorbent assay (ELISA). Briefly, at 0 h × 100%. the ELISA plate was precoated with antibodies against integrin-β1 (Proteintech), integrin-α2 (Thermo Fisher Scientific), or integrin-α5 (Proteintech) at 4 °C overnight, Transwell assay and then blocked with 5% bovine serum albumin. Equiv- Matrigel (BD) was thawed at 4 °C and diluted at 1:3 with alent sample from each group was added into each well serum-free medium. Transwell inserts (Corning, Tewks- and incubated at 4 °C overnight. Streptavidin-conjugated bury, MA, USA) were put into a 24-well plate and pre- HRP (Beyotime Biotechnology) was added into each well coated with 40 μl of diluted Matrigel. Cells in each group after rinsing and incubated at 4  °C for 1  h. Thereafter, were digested with trypsin (Sigma) and made into single- the plate was rinsed, and OPD Color-Substrate Solution cell suspension. 200  μl single-cell suspension contain- (Sigma) was added into each well for incubation at 37 °C ing 1 × 10 cells was added into the upper chamber and for 25 min. After adding of stop solution, the absorbance 800  μl of RPMI 1640 medium containing 30% FBS was was measured at 492 nm. added into the lower chamber. The cells were allowed to invade for 24  h. Then the cells were rinsed with PBS, Statistical analysis fixed with 4% paraformaldehyde (Sinopharm, Shanghai, All experiments were carried out in triplicate. The results China) at room temperature for 20 min, and stained with were shown as mean ± SD. Differences between groups 0.5% crystal violet dye (Amresco, Solon, OH, USA) for were analyzed with one-way analysis of variance. p < 0.05 5 min. Images of cells were captured under a microscope was considered as significant. at 200× magnification. Results Immunofluorescence assay FMNL2 expression level in gastric cancer cell lines Cells were treated with DMSO or 12-O-tetradecanoyl- To select a gastric cancer cell line for our study, the phorbol-13-acetate (TPA; 200  nM; Cell Signaling Tech- FMNL2 level in gastric cancer cell lines BGC-823, nology, Danvers, MA, USA) for 5  min. The cells were MGC-803, SGC-7901 and HGC-27 was detected by rinsed with PBS and permeabilized with 0.1% Tri- Western blot. As shown in Fig.  1, the FMNL2 level was tonX-100. After blocking with goat serum, the cells were 2.64 ± 0.33-fold in MGC-803 cells, 3.82 ± 0.41-fold in incubated with FMNL2 antibody (1: 100; Novus) or SGC-7901 cells, and 5.96 ± 0.75-fold in HGC-27 cells, protein kinase C (PKC) α antibody (1: 50; Proteintech, compared with that in BGC-823 cells. Thus, HGC-27 cell Zhong et al. Cancer Cell Int (2018) 18:79 Page 4 of 10 Fig. 1 FMNL2 level in gastric cancer cell lines. a Protein level of FMNL2 in gastric cancer cell lines BGC-823, MGC-803, SGC-7901 and HGC-27 was detected by western blot. β-Actin served as the internal control. b Relative FMNL2 level in each cell line was calculated. All experiments were repeated three times. The results were shown as mean ± SD line, with the highest FMNL2 expression, was selected for subsequent experiments. FMNL2 shRNA decreases the FMNL2 level in HGC‑27 To explore the role of FMNL2 in gastric cancer, a FMNL2-specific shRNA was employed in our study. Then the efficiency of FMNL2 shRNA was verified by Fig. 2 FMNL2 shRNA decreases FMNL2 level in HGC-27 cells. a qRT-PCR and western blot. After transfection with mRNA level of FMNL2 in HGC-27 cells was measured by quantitative FMNL2 shRNA, the relative mRNA level of FMNL2 was real-time PCR after FMNL2 silencing. mRNA level of FMNL2 was decreased to 25 ± 4% (Fig.  2a), and the relative protein normalized to β-actin, and relative mRNA level was calculated using −ΔΔCt level of FMNL2 was decreased to 22 ± 4% (Fig.  2b, c). 2 method. b, c After FMNL2 silencing, protein level of FMNL2 These results demonstrate that FMNL2 shRNA declines was assessed by western blot with β-actin as internal control. Each experiment was repeated three times. The results were shown as FMNL2level effectively, both at mRNA level and protein mean ± SD. ***p < 0.001 compared with negative control cells level. FMNL2 silencing inhibits proliferation and induces apoptosis of HGC‑27 cells After silencing FMNL2, proliferation of HGC-27 cells Apoptosis plays a crucial role in cell growth. In this was assessed by MTT assay. As shown in Fig.  3a, the study, effect of FMNL2 silencing on apoptosis of HGC- growth of FMNL2 silencing cells was much slower than 27 cells was assessed by flow cytometry. Results of flow that of negative control cells (Fig. 3a). These results dem - cytometry showed that there was no obvious difference onstrate that FMNL2 silencing inhibits proliferation of between cells only and negative control cells. FMNL2 HGC-27 cells. silencing cells showed a significant increase in the Zhong et al. Cancer Cell Int (2018) 18:79 Page 5 of 10 Fig. 3 FMNL2 silencing inhibits growth of HGC-27 cells. a Cell viability of HGC-27 in each group was assessed by MTT assay. b, c After silencing FMNL2, cell apoptosis in each group was detected by flow cytometry. All experiments were performed three times. The results were shown as mean ± SD. ***p < 0.001 when compared with negative control cells percentage of apoptotic cells when compared with nega- (Fig.  4c, d). These results reveal that silencing FMNL2 tive control cells (Fig.  3b, c). These results illustrate that suppresses invasion of HGC-27 cells. FMNL2 silencing induces apoptosis of HGC-27 cells. Also, proteins associated with epithelial-to-mesen- chymal transition (EMT) were also assessed by western blot. After silencing FMNL2, the protein level of E-cad- FMNL2 silencing retards migration and invasion of HGC‑27 herin, which was an epithelial marker, was increased to cells 2.11 ± 0.29-fold (Fig. 5a, b). While the levels of Vimentin Effect of FMNL2 silencing on migration of gastric can - and N-cadherin, which were mesenchymal markers, were cer cells was evaluated by wound healing assay. Results decreased to 54 ± 8 and 41 ± 6%, respectively (Fig. 5c–f ). of our study showed that there was no striking differ - These results indicate that silencing FMNL2 inhibits ence between cells only and negative control cells. After EMT of HGC-27 cells. silencing FMNL2, the migration rate of FMNL2 silencing cells was 25.45 ± 1.81%, which was significantly declined compared with negative control cells (34.84 ± 0.99%) FMNL2 silencing inhibits integrin internalization induced (Fig.  4a, b). These results show that silencing FMNL2 by PKC suppresses migration of HGC-27 cells. TPA was employed to activate PKC in our study. Upon To explore the effect of FMNL2 silencing on invasion TPA treatment, more PKCα was concentrated on plas- of gastric cancer cells, a transwell assay was carried out. malemma compared with cells treated with DMSO, indi- As shown in Fig. 4, negative control cells showed no sig- cating the activation of PKC. Moreover, more FMNL2 nificant difference when compared with that of cells only. was concentrated in cytoplasm upon TPA treatment While, the percentage of invasive cells of FMNL2 silenc- (Fig. 6). ing cells was 40 ± 6%, which was decreased significantly Additionally, internalization of integrins was also compared with that of negative control cells (94 ± 9%) detected in this study. As shown in Fig.  7, activation Zhong et al. Cancer Cell Int (2018) 18:79 Page 6 of 10 Fig. 4 FMNL2 silencing inhibits migration and invasion of HGC-27 cells. a, b Migration capability of HGC-27 cells in each group was evaluated by wound healing assay. Relative migration rate was calculated. c, d After silencing FMNL2, invasion capability was assessed by transwell assay. Each experiment was repeated three times. The results were presented as mean ± SD. **p < 0.01, ***p < 0.001 compared with negative control cells of PKC by TPA treatment enhanced the internali- invasion of gastric cancer cells. Further study showed zation of integrin-β1, integrin-α2 and integrin-α5. that internalization of integrins induced by PKC was Compared with negative control cells, silencing rescued by FMNL2 silencing. These results of our study FMNL2 significantly decreased the internalization indicate that inhibition of integrin internalization may be of integrin-β1, integrin-α2 and integrin-α5. In TPA involved in the effect of FMNL2 silencing on growth and treated cells, silencing FMNL2 also decreased the PKC- metastasis of gastric cancer cells. induced internalization of integrin-β1, integrin-α2 MicroRNAs have close relationships with cancer biol- and integrin-α5. There was no significant difference ogy. Clinical trials using microRNA profiling as markers between FMNL2 silencing + TPA group and cells of prognosis and clinical responses were underway [21]. only + DMSO or negative control + DMSO group. Several microRNAs were reported to suppress growth of These results demonstrate that silencing FMNL2 colon cancer through targeting FMNL2 [22–24]. Circu- reduces PKC-induced internalization of integrins to a lar RNAs, as emerging biomarkers and targets for can- nearly normal level. cer [25], recently catch the eyes of researchers. It was reported that circRNA_001569, as a sponge of miR-145, promoted proliferation and invasion of colon cancer Discussion through up-regulating FMNL2, which was a functional In the present study, we explored the role of FMNL2 in target of miR-145 [26]. As dysregulation of FMNL2 gastric cancer cells. Silencing FMNL2 suppressed pro- has been revealed in human cancers and is associated liferation of gastric cancer cells and induced their apop- with tumor progression and poor outcomes [19, 20], we tosis. FMNL2 silencing also suppressed migration and speculate that FMNL2 may perform a boosting role in Zhong et al. Cancer Cell Int (2018) 18:79 Page 7 of 10 Fig. 5 FMNL2 silencing inhibits epithelial-to-mesenchymal transition of HGC-27 cells. a, b After FMNL2 silencing, protein level of E-cadherin was assessed by western blot. β-actin served as internal control. c, d Western blot was performed to detect the protein level of Vimentin with β-actin as internal control. e, f Protein level of N-cadherin was assessed by western blot after silencing FMNL2. Each experiment was repeated three times. The results were shown as mean ± SD. **p < 0.01, ***p < 0.001 compared with negative control cells colon cancer growth based on the above indirect clues. cancer cells, there is no direct or indirect evidence. In And Zhu et al. show that FMNL2 boosts proliferation of our study, FMNL2 silencing suppressed proliferation of colon cancer cells [18]. To our knowledge, only the report gastric cancer cells and induced their apoptosis, indi- of Zhu et  al. [18] showed direct evidence for the role of cating that FMNL2 may contribute to gastric cancer FMNL2 in cancer cell growth. When it comes to gastric growth. As far as we know, our study is the first report Zhong et al. Cancer Cell Int (2018) 18:79 Page 8 of 10 Fig. 6 TPA influences the distribution of FMNL2 and PKCα. Before and after TPA or DMSO treatment, the levels and distribution of FMNL2 and PKCα were detected by immunofluorescence. Red fluorescence, FMNL2. Green fluorescence, PKCα Fig. 7 TPA suppresses internalization of integrins induced by PKC. After treatment with TPA, internalization of integrin-β1 (a), integrin-α2 (b) and integrin-α5 (c) was detected. All experiments were repeated three times. The results were presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 showing direct evidence for the role of FMNL2 in gastric suppressed migration and invasion of gastric cancer cells, cancer growth. However, how exactly FMNL2 performs indicating that FMNL2 also contributes to metastasis of its growth-boosting role in gastric cancer needs to be gastric cancer cells. Consistently, the FMNL2 expression revealed. in colon cancer, which has a high FMNL2 level, is corre- Migration and invasion are crucial initial steps of lated with tumor invasion and lymphatic metastasis [16]. tumor metastasis. In our study, we also investigated the FMNL2 is also found to boost invasion and migration of effect of FMNL2 silencing on migration and invasion colon cancer [16, 18]. Conversely, in hepatocarcinoma of gastric cancer cells. We found that FMNL2 silencing which has a low FMNL2 level, overexpression of FMNL2 Zhong et al. Cancer Cell Int (2018) 18:79 Page 9 of 10 suppresses motility and invasion of hepatocarcinoma declined integrin internalization induced by PKC in gas- cells [19]. Literature research shows that FMNL2 has a tric cancer cells. We speculate that integrin internaliza- close relationship with tumor metastasis. First, FMNL2 is tion may be implicated in the role of FMNL2 in gastric a catalyst for polymerization of linear actin. FMNL2 gov- cancer cells. erns many processes, including cytokinesis, morphogen- esis, invasion and migration, depended on remodeling of actin [9]. FMNL2 accumulating at lamellipodia and filo - Conclusion podia tips contributes to actin filament nucleation and Our study demonstrates for the first time that silenc - elongation, and boosts actin assembly [27, 28]. Second, ing FMNL2 suppresses proliferation, invasion and FMNL2 also modulates EMT which is an important phe- migration and induced apoptosis of gastric cancer cells. nomenon contributing to tumor metastasis. The expres - Additionally, integrin internalization may be implicated sion of FMNL2 is negatively correlated with epithelial in the role of FMNL2 in gastric cancer cells. Our study marker E-cadherin and positively correlated with Vimen- indicates that FMNL2 may become a potential thera- tin. And knockdown FMNL2 leads to EMT, with elevated peutic target for gastric cancer. E-cadherin and declined Vimentin and Snail [17]. Lossing FMNL2 also decreases TGF-β-induced EMT [17]. In this Abbreviations study, we also confirmed a similar effect of FMNL2 on FMNL2: Formin-like 2; PKC: protein kinase C; GTPase: guanosine triphos- the protein levels of EMT markers in gastric cancer cells. phatase; DRF: diaphanous-related Formin; qRT-PCR: quantitative real-time PCR; TBST: Tris buffered saline with Tween; HRP: horseradish peroxidase; MTT: The integrin signal has a close relationship with tumor 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide; PBS: progression, including cell proliferation, migration, inva- phosphate buffered saline; FITC: fluorescein isothiocyanate; PI: propidium sion and differentiation. Integrins interact with extra - iodide; TPA: 12-O-tetradecanoylphorbol-13-acetate; ELISA: enzyme-linked immunosorbent assay; EMT: epithelial-to-mesenchymal transition; MMP: cellular matrix to provide traction which is required for matrix metalloprotease; EGF: epidermal growth factor; PDGF: platelet-derived tumor cell invasion [8]. Integrins also contribute to tumor growth factor. invasion through regulating matrix metalloproteases Authors’ contributions (MMPs) which are critical to the proteolysis of matrix BZ and FK conceived and designed the study. BZ, KW and HX carried out the proteins [8, 29]. The ability of cell proliferation is depend - experiments and analyzed the data. BZ and FK drafted the manuscript. All ing on collagenous matrix status [30]. Through regulat - authors read and approved the final manuscript. ing MMPs and collagenous matrix status, integrins may also influence tumor cell proliferation. Moreover, integ - Acknowledgements rins are revealed to control the expression of cyclins and Not applicable. cyclin-dependent kinase inhibitors [31, 32], which are Competing interests key regulators of cell cycle progress, thus contributing to The authors declare that they have no competing interests. cancer cell proliferation. Integrins are also implicated in Availability of data and materials the process of EMT [33, 34], a critical process triggered Not applicable. during tumor metastasis, and contribute to tumor angio- genesis and chemoresistance [30]. Content of publication Not applicable. Interestingly, integrin trafficking (internalization and recycling) is very important in controlling integ- Ethics approval and consent to participate rin actions [3]. Tissue factors, such as epidermal growth Not applicable. factor (EGF), platelet-derived growth factor (PDGF) Funding and protein kinase C (PKC), have been shown to induce This study was supported by a Grant from the Foundation of Department of integrin internalization [35–37]. Meanwhile, dysregula- Science and Technology, Liaoning Province (No. 2015020512). tion of integrin trafficking is implied in tumorigenesis [38], and integrin internalization is reported to boost Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- cell migration [3]. Due to the important role of integrin lished maps and institutional affiliations. internalization, targeting integrin trafficking is regarded as a potential cancer therapy [38]. FMNL2 is required Received: 8 January 2018 Accepted: 26 May 2018 for integrin internalization downstream of PKC. 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MicroRNA-613 targets FMNL2 and suppresses promotes beta1-integrin trafficking and invasive motility downstream of progression of colorectal cancer. Am J Transl Res. 2016;8:5475–84. PKCalpha. Dev Cell. 2015;34:475–83. Ready to submit your research ? Choose BMC and benefit from: fast, convenient online submission thorough peer review by experienced researchers in your field rapid publication on acceptance support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year At BMC, research is always in progress. Learn more biomedcentral.com/submissions http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Cancer Cell International Springer Journals

Silencing Formin-like 2 inhibits growth and metastasis of gastric cancer cells through suppressing internalization of integrins

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Biomedicine; Cancer Research; Cell Biology
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Abstract

Background: Formin-like 2 (FMNL2) is a member of Formin family which governs cytokinesis, cellular polarity and morphogenesis. Dysregulation of FMNL2 has been discovered in cancers and is closely related to cancers. However, the role of FMNL2 in gastric cancer remains unclear. In this study, we aimed to investigate the role of FMNL2 in gastric cancer cells. Methods: A FMNL2-specific shRNA was employed to decrease the endogenous expression of FMNL2. Then the degree of proliferation, apoptosis, migration and invasion of gastric cancer cells was assessed by MTT assay, flow cytometry, wound healing assay and transwell assay, respectively. The expression and distribution of FMNL2 and pro- tein kinase C (PKC) α was detected by immunofluorescence. The internalization of integrins was detected by enzyme- linked immunosorbent assay. Results: Our results showed that silencing FMNL2 suppressed proliferation, migration and invasion, and induced apoptosis of gastric cancer cells. The integrin internalization induced by PKC was declined by FMNL2 silencing. Conclusions: Our study reveals that silencing FMNL2 suppresses growth and metastasis of gastric cancer cells. Modulation on integrin internalization may be implicated in the role of FMNL2 in growth and migration of gastric cancer cells. Our study indicates that FMNL2 may become a potential therapeutic target for gastric cancer. Keywords: Apoptosis, Formin-like 2, Gastric cancer, Integrin internalization, Invasion, Migration, Proliferation Background and recycling of integrins emerges as a major player in Gastric cancer, which threatens human health, is the fifth controlling integrin action [3]. Integrins also contribute most common cancer worldwide with approximately one to tumor progression and are implicated in cell survival, million cases in 2012 [1]. Surgery is the curative modal- metastasis, angiogenesis and drug resistance [4–8]. They ity for gastric cancer treatment. However, the recurrence also influence the response of host cells to cancers [ 8]. rate is still high, and the 5-year survival rate is low [2]. Integrins are regarded as appealing targets for tumor Integrins are a group of αβ heterodimers. Integrins therapy [8]. are parts of the major receptors spanning through the Formins are a group of Rho guanosine triphosphatase lipid bilayer of cells. Integrins bind directly to extracel- (GTPase) effectors, governing cytokinesis, polarity, adhe - lular matrix components, transduce signals outside-in sion and morphogenesis [9]. Diaphanous-related Formins and activate intracellular signals in context of cytokine (DRFs) are a conserved subgroup of Formins. They are receptors or growth factor receptors. Internalization involved in a wide range of cellular processes, including filopodium formation, cell migration and cell polarity [ 9– 11], and contribute to many physiological or pathological *Correspondence: fanminkong@126.com processes [12–14]. Formin-like 2 (FMNL2), a member of Department of Gastric, Intestine and Hernia Surgery, The First Affiliated DRFs, locates on chromosome 2q23.3. FMNL2 contains Hospital of China Medical University, Shenyang 110001, People’s Republic a GTPase-binding domain and an autoregulatory domain of China © The Author(s) 2018. 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. Zhong et al. Cancer Cell Int (2018) 18:79 Page 2 of 10 [15], and acts as both a downstream effector and an Oligo(dT) and Super M-MLV Reverse Transcriptase upstream modulator of Rho family GTPases [11]. FMNL2 (BioTeke) according to the manufacturers’ instructions. plays a key role in actin filament nucleation or elongation mRNA level of FMNL2 in each group was measured by which influences cell morphology [9]. FMNL2 also has qRT-PCR (SYBR Green method) with cDNA as the tem- a close relationship with cancer. For example, FMNL2 plate. The following primers were used: forward primer enhances the growth and metastasis of colon cancer for FMNL2, 5′-CCC GCT CTG GAA GAC ATT -3′; reverse in which FMNL2 is highly expressed [16–18]. Moreo- primer for FMNL2, 5′-CTG CCA ACA GTT CTA AGA ver, FMNL2 performs an inhibitory effect on motility CAAG-3′; forward primer for β-actin, 5′-CTT AGT TGC of hepatocarcinoma which shows a lower FMNL2 level GTT ACA CCC TTT CTT G-3′; reverse primer for β-actin, than normal livers [19]. In addition, patients with aber- 5′-CTG TCA CCT TCA CCG TTC CAG TTT -3′. SYBR rant FMNL2 expression show a lower 5-year survival rate Green Reagent was obtained from Solarbio (Beijing, [19]. Dysregulation of FMNL2 has been discovered in China). The relative mRNA level of FMNL2 was normal - −ΔΔCt several types of cancers and is associated with the devel- ized to β-actin and calculated using 2 method. opment of aggressive tumors [16–20]. However, the role of FMNL2 in gastric cancer remains unclear. Western blot In the present study, we aimed to investigate the effect Proteins in each group were extracted using radio of FMNL2 silencing on gastric cancer cells. The results of immunoprecipitation assay lysis buffer (Beyotime Bio - our study indicate that FMNL2 may act as an oncogene technology, Haimen, China) containing 1% phenylmeth- in gastric cancer cells and has the potential to become a anesulfonyl fluoride (Beyotime Biotechnology). The therapeutic target for gastric cancer. concentration of proteins in each group was measured using a BCA Protein Assay Kit (Beyotime Biotechnol- Materials asnd methods ogy). Forth microgramme proteins in each group were Cells separated by sodium dodecyl sulfate polyacrylamide Gastric cancer cell lines BGC-823, MGC-803, HGC- gel electrophoresis. After electrophoresis, the proteins 27 were obtained from Zhongqiaoxinzhou Biotechnol- were transferred onto polyvinylidene fluoride mem - ogy Co., Ltd (Shanghai, China). SGC-7901 cell line was branes (Millipore, Bedford, MA, USA). The membranes obtained from CHI Scientific (Jiangyin, China). BGC-823 were blocked with 5% skim milk, followed by incubating and MGC-803 cells were grown in Dulbecco’s Modified with primary antibodies against FMNL2 (1:500; Novus, Eagle’s Medium (Gibco, Grand Island, NY, USA) with Oakville, ON, Canada), E-cadherin (1:2000, Protein- 10% fetal bovine serum (FBS; Hyclone, Logan, UT, USA). tech, Wuhan, China), Vimentin (1:2000, Proteintech), SGC-7901 and HGC-27 cells were grown in RPMI 1640 N-cadherin (1:2000, Proteintech) or β-actin (1:500; medium (Gibco) with 10% FBS. All these cells were cul- Bioss, Beijing, China) at 4  °C overnight. After rinsing in tured in a humid atmosphere at 37 °C with 5% CO . Tris buffered saline with Tween (TBST), the membranes were incubated with horseradish peroxidase (HRP)-con- Transfection jugated goat-anti-rabbit IgG(H+L) or HRP-conjugated HGC-27 cells were seeded in a 6-well plate (2 × 10 cells/ goat-anti-mouse IgG(H+L) (1:5000; Beyotime Biotech- well) and cultured in a cell incubator. Twenty-four hours nology) at 37  °C for 45  min. After rinsing in TBST, the later, the cells were transfected with FMNL2 shRNA (tar- targeted bands were visualized using an ECL Kit (Beyo- get sequence: 5′-GTG GTA GCA GGT AAC TCT G-3′) or time Biotechnology). negative control using Lipofectamin 2000 Reagent (Inv- itrogen, Carlsbad, CA, USA) according to the manufac- turer’s protocol. Then the cells were maintained in cell 3‑(4,5‑Dimethyl‑2‑thiazolyl)‑2,5‑diphenyl‑2‑H‑tetrazolium medium with 800  μg/ml G418 (Invitrogen) for 7  days bromide (MTT) assay to select stably transfected cells. Cells stably transfected Cells in each group were seeded into 96-well plates with FMNL2 shRNA were named as FMNL2 silenc- (4 × 10 cells/well) and cultured in a cell incubator for ing. Cells stably transfected with negative control were 0, 24, 48 and 72  h, respectively. Then MTT (Sigma, St. named as negative control. Cells without transfection Louis, MO, USA) at a final concentration of 0.5  mg/ were named as cells only. ml was added into each well. After incubating for addi- tional 4 h, 150 μl dimethyl sulfoxide (DMSO, Sigma) was Quantitative real‑time PCR (qRT‑PCR) added into each well to dissolve the crystals after removal Total RNA in cells from each group was extracted using of supernate. Thereafter, the absorbance at 490  nm was a High-purity Total RNA Fast Extraction kit (BioTeke, measured with a microplate reader (BIOTEK, Winooski, Beijing, China) and reverse transcribed to cDNA using VT, USA). Zhong et al. Cancer Cell Int (2018) 18:79 Page 3 of 10 Flow cytometry Wuhan, China) at 4  °C overnight. Thereafter, the cells Degree of apoptosis in each group was analyzed by flow were rinsed with PBS and incubated Cy3-conjugated cytometry with a Cell Apoptosis Detection Kit (Key- goat-anti-rabbit IgG(H+L) or FITC-conjugated goat- Gen, Nanjing, China). Cells in each group were collected, anti-mouse IgG(H+L) (1:200; Beyotime Biotechnology). rinsed in phosphate buffered saline (PBS) and resus - The cells were then rinsed with PBS and observed under pended in 500  μl binding buffer. Then 5  μl Annexin– a fluorescence microscope (600×; OLYMPUS, Tokyo, Fluorescein Isothiocyanate (FITC) and 5  μl Propidium Japan). Iodide (PI) were added into cells for incubation for addi- tional 15 min at room temperature in dark. The cells were Integrin internalization assay then analyzed with a flow cytometer (BD, Franklin Lakes, Cells were labeled with EZ-Link Sulfo-NHS-SS-Biotin NJ, USA). (0.5 mg/ml; Thermo Fisher Scientific, Rockford, IL, USA) on ice for 1  h. Then the cells were treated with DMSO Wound healing assay or TPA for 30 min. The cells were kept on ice and rinsed Cells in each group were seeded into a 6-well plate. with ice-cold PBS. Biotin residual on cell surface was When the confluence reached 90%, the cell medium was removed by incubating in Tris buffer containing 20  mM changed to fresh serum-free medium. Then the cells MesNa (Aladdin, Shanghai, China) at 4  °C for 15  min. were treated with mitomycin C (1  μg/ml; Sigma) for Superfluous MesNa was quenched by 20 mM iodoaceta - 1  h. Thereafter, scratches were made on single-cell sur - mide (Aladdin). Then the cells were collected and lysed face with 200 μl pipette tips. The cells were washed with in lysis solution (containing 200 mM NaCl, 15 mM NaF, serum-free medium to remove cell debris, and allowed to 75  mM Tris, 7.5  mM EDTA, 7.5  mM EGTA, 1.5  mM migrate for 24  h in serum-free medium. Images of cells Na3VO4, and 1.5% TritonX-100). The supernatant was were captured with a microscope at 0  h and 24  h. The collected by centrifugation at 1500×g at 4 °C for 10 min. relative migrate rate was calculated using the following After measuring the concentration with a BCA Protein formula: relative migrate rate = (distance between gap at Assay Kit, the level of biotinylated integrin was analyzed 0 h − distance between gap at 24 h)/distance between gap by enzyme-linked immunosorbent assay (ELISA). Briefly, at 0 h × 100%. the ELISA plate was precoated with antibodies against integrin-β1 (Proteintech), integrin-α2 (Thermo Fisher Scientific), or integrin-α5 (Proteintech) at 4 °C overnight, Transwell assay and then blocked with 5% bovine serum albumin. Equiv- Matrigel (BD) was thawed at 4 °C and diluted at 1:3 with alent sample from each group was added into each well serum-free medium. Transwell inserts (Corning, Tewks- and incubated at 4 °C overnight. Streptavidin-conjugated bury, MA, USA) were put into a 24-well plate and pre- HRP (Beyotime Biotechnology) was added into each well coated with 40 μl of diluted Matrigel. Cells in each group after rinsing and incubated at 4  °C for 1  h. Thereafter, were digested with trypsin (Sigma) and made into single- the plate was rinsed, and OPD Color-Substrate Solution cell suspension. 200  μl single-cell suspension contain- (Sigma) was added into each well for incubation at 37 °C ing 1 × 10 cells was added into the upper chamber and for 25 min. After adding of stop solution, the absorbance 800  μl of RPMI 1640 medium containing 30% FBS was was measured at 492 nm. added into the lower chamber. The cells were allowed to invade for 24  h. Then the cells were rinsed with PBS, Statistical analysis fixed with 4% paraformaldehyde (Sinopharm, Shanghai, All experiments were carried out in triplicate. The results China) at room temperature for 20 min, and stained with were shown as mean ± SD. Differences between groups 0.5% crystal violet dye (Amresco, Solon, OH, USA) for were analyzed with one-way analysis of variance. p < 0.05 5 min. Images of cells were captured under a microscope was considered as significant. at 200× magnification. Results Immunofluorescence assay FMNL2 expression level in gastric cancer cell lines Cells were treated with DMSO or 12-O-tetradecanoyl- To select a gastric cancer cell line for our study, the phorbol-13-acetate (TPA; 200  nM; Cell Signaling Tech- FMNL2 level in gastric cancer cell lines BGC-823, nology, Danvers, MA, USA) for 5  min. The cells were MGC-803, SGC-7901 and HGC-27 was detected by rinsed with PBS and permeabilized with 0.1% Tri- Western blot. As shown in Fig.  1, the FMNL2 level was tonX-100. After blocking with goat serum, the cells were 2.64 ± 0.33-fold in MGC-803 cells, 3.82 ± 0.41-fold in incubated with FMNL2 antibody (1: 100; Novus) or SGC-7901 cells, and 5.96 ± 0.75-fold in HGC-27 cells, protein kinase C (PKC) α antibody (1: 50; Proteintech, compared with that in BGC-823 cells. Thus, HGC-27 cell Zhong et al. Cancer Cell Int (2018) 18:79 Page 4 of 10 Fig. 1 FMNL2 level in gastric cancer cell lines. a Protein level of FMNL2 in gastric cancer cell lines BGC-823, MGC-803, SGC-7901 and HGC-27 was detected by western blot. β-Actin served as the internal control. b Relative FMNL2 level in each cell line was calculated. All experiments were repeated three times. The results were shown as mean ± SD line, with the highest FMNL2 expression, was selected for subsequent experiments. FMNL2 shRNA decreases the FMNL2 level in HGC‑27 To explore the role of FMNL2 in gastric cancer, a FMNL2-specific shRNA was employed in our study. Then the efficiency of FMNL2 shRNA was verified by Fig. 2 FMNL2 shRNA decreases FMNL2 level in HGC-27 cells. a qRT-PCR and western blot. After transfection with mRNA level of FMNL2 in HGC-27 cells was measured by quantitative FMNL2 shRNA, the relative mRNA level of FMNL2 was real-time PCR after FMNL2 silencing. mRNA level of FMNL2 was decreased to 25 ± 4% (Fig.  2a), and the relative protein normalized to β-actin, and relative mRNA level was calculated using −ΔΔCt level of FMNL2 was decreased to 22 ± 4% (Fig.  2b, c). 2 method. b, c After FMNL2 silencing, protein level of FMNL2 These results demonstrate that FMNL2 shRNA declines was assessed by western blot with β-actin as internal control. Each experiment was repeated three times. The results were shown as FMNL2level effectively, both at mRNA level and protein mean ± SD. ***p < 0.001 compared with negative control cells level. FMNL2 silencing inhibits proliferation and induces apoptosis of HGC‑27 cells After silencing FMNL2, proliferation of HGC-27 cells Apoptosis plays a crucial role in cell growth. In this was assessed by MTT assay. As shown in Fig.  3a, the study, effect of FMNL2 silencing on apoptosis of HGC- growth of FMNL2 silencing cells was much slower than 27 cells was assessed by flow cytometry. Results of flow that of negative control cells (Fig. 3a). These results dem - cytometry showed that there was no obvious difference onstrate that FMNL2 silencing inhibits proliferation of between cells only and negative control cells. FMNL2 HGC-27 cells. silencing cells showed a significant increase in the Zhong et al. Cancer Cell Int (2018) 18:79 Page 5 of 10 Fig. 3 FMNL2 silencing inhibits growth of HGC-27 cells. a Cell viability of HGC-27 in each group was assessed by MTT assay. b, c After silencing FMNL2, cell apoptosis in each group was detected by flow cytometry. All experiments were performed three times. The results were shown as mean ± SD. ***p < 0.001 when compared with negative control cells percentage of apoptotic cells when compared with nega- (Fig.  4c, d). These results reveal that silencing FMNL2 tive control cells (Fig.  3b, c). These results illustrate that suppresses invasion of HGC-27 cells. FMNL2 silencing induces apoptosis of HGC-27 cells. Also, proteins associated with epithelial-to-mesen- chymal transition (EMT) were also assessed by western blot. After silencing FMNL2, the protein level of E-cad- FMNL2 silencing retards migration and invasion of HGC‑27 herin, which was an epithelial marker, was increased to cells 2.11 ± 0.29-fold (Fig. 5a, b). While the levels of Vimentin Effect of FMNL2 silencing on migration of gastric can - and N-cadherin, which were mesenchymal markers, were cer cells was evaluated by wound healing assay. Results decreased to 54 ± 8 and 41 ± 6%, respectively (Fig. 5c–f ). of our study showed that there was no striking differ - These results indicate that silencing FMNL2 inhibits ence between cells only and negative control cells. After EMT of HGC-27 cells. silencing FMNL2, the migration rate of FMNL2 silencing cells was 25.45 ± 1.81%, which was significantly declined compared with negative control cells (34.84 ± 0.99%) FMNL2 silencing inhibits integrin internalization induced (Fig.  4a, b). These results show that silencing FMNL2 by PKC suppresses migration of HGC-27 cells. TPA was employed to activate PKC in our study. Upon To explore the effect of FMNL2 silencing on invasion TPA treatment, more PKCα was concentrated on plas- of gastric cancer cells, a transwell assay was carried out. malemma compared with cells treated with DMSO, indi- As shown in Fig. 4, negative control cells showed no sig- cating the activation of PKC. Moreover, more FMNL2 nificant difference when compared with that of cells only. was concentrated in cytoplasm upon TPA treatment While, the percentage of invasive cells of FMNL2 silenc- (Fig. 6). ing cells was 40 ± 6%, which was decreased significantly Additionally, internalization of integrins was also compared with that of negative control cells (94 ± 9%) detected in this study. As shown in Fig.  7, activation Zhong et al. Cancer Cell Int (2018) 18:79 Page 6 of 10 Fig. 4 FMNL2 silencing inhibits migration and invasion of HGC-27 cells. a, b Migration capability of HGC-27 cells in each group was evaluated by wound healing assay. Relative migration rate was calculated. c, d After silencing FMNL2, invasion capability was assessed by transwell assay. Each experiment was repeated three times. The results were presented as mean ± SD. **p < 0.01, ***p < 0.001 compared with negative control cells of PKC by TPA treatment enhanced the internali- invasion of gastric cancer cells. Further study showed zation of integrin-β1, integrin-α2 and integrin-α5. that internalization of integrins induced by PKC was Compared with negative control cells, silencing rescued by FMNL2 silencing. These results of our study FMNL2 significantly decreased the internalization indicate that inhibition of integrin internalization may be of integrin-β1, integrin-α2 and integrin-α5. In TPA involved in the effect of FMNL2 silencing on growth and treated cells, silencing FMNL2 also decreased the PKC- metastasis of gastric cancer cells. induced internalization of integrin-β1, integrin-α2 MicroRNAs have close relationships with cancer biol- and integrin-α5. There was no significant difference ogy. Clinical trials using microRNA profiling as markers between FMNL2 silencing + TPA group and cells of prognosis and clinical responses were underway [21]. only + DMSO or negative control + DMSO group. Several microRNAs were reported to suppress growth of These results demonstrate that silencing FMNL2 colon cancer through targeting FMNL2 [22–24]. Circu- reduces PKC-induced internalization of integrins to a lar RNAs, as emerging biomarkers and targets for can- nearly normal level. cer [25], recently catch the eyes of researchers. It was reported that circRNA_001569, as a sponge of miR-145, promoted proliferation and invasion of colon cancer Discussion through up-regulating FMNL2, which was a functional In the present study, we explored the role of FMNL2 in target of miR-145 [26]. As dysregulation of FMNL2 gastric cancer cells. Silencing FMNL2 suppressed pro- has been revealed in human cancers and is associated liferation of gastric cancer cells and induced their apop- with tumor progression and poor outcomes [19, 20], we tosis. FMNL2 silencing also suppressed migration and speculate that FMNL2 may perform a boosting role in Zhong et al. Cancer Cell Int (2018) 18:79 Page 7 of 10 Fig. 5 FMNL2 silencing inhibits epithelial-to-mesenchymal transition of HGC-27 cells. a, b After FMNL2 silencing, protein level of E-cadherin was assessed by western blot. β-actin served as internal control. c, d Western blot was performed to detect the protein level of Vimentin with β-actin as internal control. e, f Protein level of N-cadherin was assessed by western blot after silencing FMNL2. Each experiment was repeated three times. The results were shown as mean ± SD. **p < 0.01, ***p < 0.001 compared with negative control cells colon cancer growth based on the above indirect clues. cancer cells, there is no direct or indirect evidence. In And Zhu et al. show that FMNL2 boosts proliferation of our study, FMNL2 silencing suppressed proliferation of colon cancer cells [18]. To our knowledge, only the report gastric cancer cells and induced their apoptosis, indi- of Zhu et  al. [18] showed direct evidence for the role of cating that FMNL2 may contribute to gastric cancer FMNL2 in cancer cell growth. When it comes to gastric growth. As far as we know, our study is the first report Zhong et al. Cancer Cell Int (2018) 18:79 Page 8 of 10 Fig. 6 TPA influences the distribution of FMNL2 and PKCα. Before and after TPA or DMSO treatment, the levels and distribution of FMNL2 and PKCα were detected by immunofluorescence. Red fluorescence, FMNL2. Green fluorescence, PKCα Fig. 7 TPA suppresses internalization of integrins induced by PKC. After treatment with TPA, internalization of integrin-β1 (a), integrin-α2 (b) and integrin-α5 (c) was detected. All experiments were repeated three times. The results were presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 showing direct evidence for the role of FMNL2 in gastric suppressed migration and invasion of gastric cancer cells, cancer growth. However, how exactly FMNL2 performs indicating that FMNL2 also contributes to metastasis of its growth-boosting role in gastric cancer needs to be gastric cancer cells. Consistently, the FMNL2 expression revealed. in colon cancer, which has a high FMNL2 level, is corre- Migration and invasion are crucial initial steps of lated with tumor invasion and lymphatic metastasis [16]. tumor metastasis. In our study, we also investigated the FMNL2 is also found to boost invasion and migration of effect of FMNL2 silencing on migration and invasion colon cancer [16, 18]. Conversely, in hepatocarcinoma of gastric cancer cells. We found that FMNL2 silencing which has a low FMNL2 level, overexpression of FMNL2 Zhong et al. Cancer Cell Int (2018) 18:79 Page 9 of 10 suppresses motility and invasion of hepatocarcinoma declined integrin internalization induced by PKC in gas- cells [19]. Literature research shows that FMNL2 has a tric cancer cells. We speculate that integrin internaliza- close relationship with tumor metastasis. First, FMNL2 is tion may be implicated in the role of FMNL2 in gastric a catalyst for polymerization of linear actin. FMNL2 gov- cancer cells. erns many processes, including cytokinesis, morphogen- esis, invasion and migration, depended on remodeling of actin [9]. FMNL2 accumulating at lamellipodia and filo - Conclusion podia tips contributes to actin filament nucleation and Our study demonstrates for the first time that silenc - elongation, and boosts actin assembly [27, 28]. Second, ing FMNL2 suppresses proliferation, invasion and FMNL2 also modulates EMT which is an important phe- migration and induced apoptosis of gastric cancer cells. nomenon contributing to tumor metastasis. The expres - Additionally, integrin internalization may be implicated sion of FMNL2 is negatively correlated with epithelial in the role of FMNL2 in gastric cancer cells. Our study marker E-cadherin and positively correlated with Vimen- indicates that FMNL2 may become a potential thera- tin. And knockdown FMNL2 leads to EMT, with elevated peutic target for gastric cancer. E-cadherin and declined Vimentin and Snail [17]. Lossing FMNL2 also decreases TGF-β-induced EMT [17]. In this Abbreviations study, we also confirmed a similar effect of FMNL2 on FMNL2: Formin-like 2; PKC: protein kinase C; GTPase: guanosine triphos- the protein levels of EMT markers in gastric cancer cells. phatase; DRF: diaphanous-related Formin; qRT-PCR: quantitative real-time PCR; TBST: Tris buffered saline with Tween; HRP: horseradish peroxidase; MTT: The integrin signal has a close relationship with tumor 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide; PBS: progression, including cell proliferation, migration, inva- phosphate buffered saline; FITC: fluorescein isothiocyanate; PI: propidium sion and differentiation. Integrins interact with extra - iodide; TPA: 12-O-tetradecanoylphorbol-13-acetate; ELISA: enzyme-linked immunosorbent assay; EMT: epithelial-to-mesenchymal transition; MMP: cellular matrix to provide traction which is required for matrix metalloprotease; EGF: epidermal growth factor; PDGF: platelet-derived tumor cell invasion [8]. Integrins also contribute to tumor growth factor. invasion through regulating matrix metalloproteases Authors’ contributions (MMPs) which are critical to the proteolysis of matrix BZ and FK conceived and designed the study. BZ, KW and HX carried out the proteins [8, 29]. The ability of cell proliferation is depend - experiments and analyzed the data. BZ and FK drafted the manuscript. All ing on collagenous matrix status [30]. Through regulat - authors read and approved the final manuscript. ing MMPs and collagenous matrix status, integrins may also influence tumor cell proliferation. Moreover, integ - Acknowledgements rins are revealed to control the expression of cyclins and Not applicable. cyclin-dependent kinase inhibitors [31, 32], which are Competing interests key regulators of cell cycle progress, thus contributing to The authors declare that they have no competing interests. cancer cell proliferation. Integrins are also implicated in Availability of data and materials the process of EMT [33, 34], a critical process triggered Not applicable. during tumor metastasis, and contribute to tumor angio- genesis and chemoresistance [30]. Content of publication Not applicable. Interestingly, integrin trafficking (internalization and recycling) is very important in controlling integ- Ethics approval and consent to participate rin actions [3]. Tissue factors, such as epidermal growth Not applicable. factor (EGF), platelet-derived growth factor (PDGF) Funding and protein kinase C (PKC), have been shown to induce This study was supported by a Grant from the Foundation of Department of integrin internalization [35–37]. Meanwhile, dysregula- Science and Technology, Liaoning Province (No. 2015020512). tion of integrin trafficking is implied in tumorigenesis [38], and integrin internalization is reported to boost Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- cell migration [3]. Due to the important role of integrin lished maps and institutional affiliations. internalization, targeting integrin trafficking is regarded as a potential cancer therapy [38]. FMNL2 is required Received: 8 January 2018 Accepted: 26 May 2018 for integrin internalization downstream of PKC. 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Cancer Cell InternationalSpringer Journals

Published: Jun 1, 2018

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