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Experimental evidence of Migfilin as a new therapeutic target of hepatocellular carcinoma metastasis
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Research Article
Experimental evidence of Migfilin as a new therapeutic target of hepatocellular carcinoma metastasis Vasiliki Gkretsia,n, Dimitrios P. Bogdanosa,b,c a
Department of Biomedical Research and Technology, Institute for Research and Technology-Thessaly, Centre for Research and TechnologyHellas (CE.R.T.H.), Larissa 41222, Greece b Department of Rheumatology, School of Medicine, University of Thessaly, University Hospital of Larissa, 41110 Larissa, Greece c Institute of Liver Studies, King's College Hospital, Denmark Hill, London SE5 9RS, UK
article information
abstract
Article Chronology:
Migfilin is a novel cell–matrix adhesion protein known to interact with Vasodilator Stimulated
Received 15 December 2014
Phosphoprotein (VASP) and be localized both at cell–matrix and cell–cell adhesions. To date there is
Received in revised form
nothing known about its role in hepatocellular carcinoma (HCC). As matrix is important in
1 March 2015
metastasis, we aimed to investigate the Migfilin's role in HCC metastasis using two human HCC
Accepted 4 March 2015
cell lines that differ in their metastatic potential; non-invasive Alexander cells and the highly invasive HepG2 cells. We silenced Migfilin by siRNA and studied its effect on signaling and metastasis-related
Keywords: Hepatocellular carcinoma Migfilin VASP Invasion Adhesion Metastasis Fascin-1
cellular properties. We show that Migfilin's expression is elevated in HepG2 cells and its silencing leads to upregulation of actin reorganization-related proteins, namely phosphor-VASP (Ser157 and Ser239), Fascin-1 and Rho-kinase-1, promoting actin polymerization and inhibiting cell invasion. Phosphor-Akt (Ser473) is decreased contributing to the upregulation of free and phosphor-β-catenin (Ser33/37Thr41) and inducing proliferation. Migfilin elimination upregulates Extracellular Signal– regulated kinase, which increases cell adhesion in HepG2 and reduces invasiveness. This is the first study to reveal that Migfilin inhibition can halt HCC metastasis in vitro, providing the molecular mechanism involved and presenting Migfilin as potential therapeutic target against HCC metastasis.
Introduction Hepatocellular carcinoma (HCC) accounts for 5.5% of all cancer cases worldwide being the fifth leading cause of cancer mortality in the world [1]. The fact that, in most cases, there are no symptoms in the early stages along with the high metastatic potential and the rapid
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growth rate of the tumors justifies the mortality rates observed. In fact, it has been estimated that the 5-year fatality rate for individuals diagnosed with HCC is greater than 95% [1]. Metastasis (both intrahepatic and extrahepatic) is a fundamental biological behavior of HCC and the main cause of treatment failure [2]. It occurs as the last stage of a series of events which
Abbreviations list in order of appearance: lHCC, hepatocellular carcinoma; ECM, extracellular matrix; FBLP-1, filamin binding LIM protein -1 (or Migfilin); VASP, vasodilator stimulated phosphoprotein; PBGD, porphobilinogen deaminase; PKA, cAMP-dependent kinase; PKG, cGMP-dependent protein kinase; NSC, non-specific control; ROCK-1, rho kinase-1; ERK1/2, extracellular signal –regulated kinase n
Corresponding author. Fax: þ30 2410 258889. E-mail address: [email protected] (V. Gkretsi).
http://dx.doi.org/10.1016/j.yexcr.2015.03.002 0014-4827/& 2015 Elsevier Inc. All rights reserved.
Please cite this article as: V. Gkretsi, D.P. Bogdanos, Experimental evidence of Migfilin as a new therapeutic target of hepatocellular carcinoma metastasis, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.03.002
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include replacement of the injured or impaired liver tissue by fibrotic, scar tissue and regenerative nodules (cirrhosis), generation of phenotypically altered hepatocytes and generation of dysplastic hepatocytes which serve as precursors to HCC [1]. Since scarring is involved, integrins, extracellular matrix (ECM) and ECM-proximal proteins play a fundamental role in HCC metastasis. Although normally cells have excellent communication both with the ECM through integrins, and with neighboring cells through cell– cell adhesion proteins, this communication is severely disrupted in cancer. Thus, cell dissociation from the original tumor mass is facilitated and migration towards a new environment occurs, leading to the formation of a metastatic lesion [3]. The significance of cell adhesion proteins in cancer cell metastasis is even more apparent in the case of HCC, as hepatocytes are greatly depended on the communication with the surrounding matrix [4]. Migfilin (also known as Filamin Binding LIM-protein-1 or FBLP-1) is a novel LIM domain-containing protein present both at cell-ECM [5] and cell–cell adhesions [6]. It interacts with Vasodilator Stimulated Phosphoprotein (VASP) [7] which is known to regulate actin polymerization in lamellipodia [8–10], the cellular protrusions responsible for migration and invasion of the cell. VASP is also a substrate of cAMP-dependent kinases (PKA), and cGMP-dependent protein kinases (PKG) that primarily phosphorylate the sites Ser157, and Ser239 respectively of the VASP protein [11]. A few recent studies have implicated Migfilin in cancer cell metastasis, although its exact role seems to depend on the type of cancer [12–15]. Interestingly, Migfilin's role in the liver and HCC is completely unknown. Hence, the aim of the present study was to investigate the in vitro role of Migfilin in two liver cell lines that differ in terms of their metastatic potential; the non-invasive hepatoma cell line PLC/PRF/5 (Alexander cells herein) and the highly invasive HCC HepG2 cell line. We utilized an siRNA-mediated silencing approach to knockdown the Migfilin gene from both cell lines and study the effect of gene silencing on basic signaling pathways and functional cellular properties related to the metastatic potential of the cells.
Materials and methods Antibodies and reagents Antibodies against phosphor-VASP (Ser157), phosphor-VASP (Ser239), VASP, Fascin-1, phosphor-β-catenin (Ser33/37/Thr41), β-catenin and Extracellular Signal-regulated kinase (ERK1/2) were purchased from Cell Signaling. The antibody against phosphor-Akt (Ser473) was purchased from Abcam while the antibody against ROCK-1 was purchased from R&D systems. The monocloncal antibody against Migfilin was kindly provided by Dr. Chuanyue Wu (Professor at the University of Pittsburgh Medical School, Pittsburgh, PA, USA) while anti-β-actin antibody (Sigma-Aldrich) was used as loading control for all western blot experiments. Alamar Blue reagent was obtained from Invitrogen.
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the HCC cell line HepG2. Both cell lines were purchased from ATCC.
RNA isolation Total cellular RNA was extracted from cells using Trizol reagent (Invitrogen). RNA was further purified using an RNeasy mini kit (Qiagen). Preservation of 28S and 18S ribosomal RNA (rRNA) species was used to assess RNA integrity. All samples included in the study were with prominent 28S and 18S rRNA components. The yield was quantified spectrophotometrically.
Real time PCR Transcription of 1 mg RNA to complementary DNA (cDNA) was performed using Superscript (Invitrogen). Quantification of Migfilin was performed by real-time PCR using Platinum SYBR-green reagen (Invitrogen) in a MiniOpticon MJ Thermal Cycler Real Time PCR machine (BioRad). Porphobilinogen deaminase (PBGD) was used as a housekeeping gene. Reactions were always performed in triplicate and at least 4 independent experiments were performed. The primers for Migfilin were as follows; forward: 50 CGA ATG CAT GGG AAG AAA CT 30 , reverse 50 GCA GGT TAG GAA GGG AAA CC 30 . To quantify the relative expression of each gene, Ct values were normalized against the endogenous reference (ΔCt¼Ct targetCt PBGD) and were compared with a calibrator using the ΔΔCt method (ΔΔCt¼ΔCt sampleΔCt calibrator). Alexander cells were used as calibrators for the HepG2 cells.
Protein extraction and western blot analysis Total cell lysates were obtained using 1% sodium dodecyl sulfate in RIPA buffer (20mMTris/Cl pH7.5, 150 mM NaCl, 0.5% NP-40, 1% TX-100, 0.25% sodium deoxycholated, 0.6–2 μg/ml aprotinin, 10 μMleupeptin, 1 μM pepstatin). Protein concentrations in the samples were determined by the BCA protein assay kit (Pierce) using bovine serum albumin as standard. Equal amount of protein was loaded on each lane of a 10–12% acrylamide gel and transferred to a PVDF membrane (Millipore) using the BioRad semi-dry transfer system (BioRad). Signals were detected using suitable secondary immunoglobulin IgG conjugated with horseradish peroxidase (Invitrogen). Antibody detection was performed using super-signal ECL detection system (PIERCE).
Transfection with siRNAs Both Alexander and HepG2 cells were treated for 48 h with 100 nM siRNA non-specific control (NSC) siRNA or siRNA against Migfilin using the Lipofectamine 2000 transfection reagent (Invitrogen) according to the company's guidelines. The siRNA sequence used to silence Migfilin, was as follows: 50 - AAA GGG GCA UCC ACA GAC AUC-30 , while the sequence 50 AAA CUC UAU CUG CAC GCU GAC30 was used as NSC. Silencing efficiency prior to every experiment performed was tested by western blot and/or real time PCR, as specified in each experiment.
Liver cell lines
Quantification of protein expression in western blots
Two liver cell lines of different invasive capacity were used in the present study; the hepatoma cell line PLC/PRF/5 (Alexander) and
Quantification of protein expression in western blots was performed using Image J analysis according to the software's guidelines.
Please cite this article as: V. Gkretsi, D.P. Bogdanos, Experimental evidence of Migfilin as a new therapeutic target of hepatocellular carcinoma metastasis, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.03.002
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Fig. 1 – Migfilin expression at the protein and mRNA level in Alexander and HepG2 cells. (Α) Representative western blot showing Migfilin protein expression in the two HCC cell lines tested; the low invasiveness Alexander and the highly invasive HepG2 cells. (B) Diagram showing the relative mRNA expression of Migfilin using Real Time Polymerase Chain Reaction. Relative expression was calculated using the ΔΔCt method as described in the materials and methods section. Porphobilinogen deaminase (PBGD) was used as the housekeeping gene and Alexander cells were used as calibrator for the analysis of the results. Reactions were performed in triplicate and three independent experiments were conducted. (C) Relative protein expression of migfilin quantified using the Image J software according to the software's guidelines. The graph represents mean value of band intensity from 3 different western blots. A p Value of o0.05 was considered as statistically significant. The mean intensity from relative protein bands from three different western blots corresponding to three independent experiments was used for the quantification. A p Value of o0.05 was considered as statistically significant.
Alamar blue proliferation assay Twenty four hours following siRNA treatment, cells were subjected to Alamar blue assay (Invitrogen) [16] according to the company's guidelines. Briefly, cells were seeded in 96 well plates at a concentration of 104cells/ml and Alamar blue was added at a volume of 1/10 of the volume of medium in the well. Following incubation at 37oC for 2, 4 and 24 h post-plating, fluorescence was measured using Perkin-Elmer Enspire plate reader at 560/590 nm. Three independent experiments were performed.
Cell adhesion assay Cell adhesion assay was performed as described previously [7]. Briefly, cells were transfected with a control NSC siRNA or siRNA against Migfilin. Forty eight hours (48 h) post-transfection, 104cells/ well were seeded in six (6) wells of a 96-well plate pre-coated with 0.1% gelatin. After 60 min incubation at 37 1C, three of the wells were washed three times with phosphate-buffered saline (PBS) while the remaining three were fixed with 4% paraformaldehyde (PFA). Washed wells were also fixed with PFA and then cells in all wells were quantified using crystal violet [7]. Crystal violet was washed using ddH20 and cells were solubilized using acetic acid.
Absorbance was measured at 570 nm using Perkin-Elmer Enspire plate reader. Adhesion was presented as the ratio of the absorbance at 570 nm of adhered cells (washed) divided by the absorbance at 570 nm of the total seeded cells (not washed). The data from two independent experiments were analyzed using the student's T-test. p Values o0.05 were considered as statistically significant.
Cell invasion Cell invasion was assessed using QCM™ Collagen Cell Invasion Assay, 24-well invasion chambers (8 μm) (Cat. #ECM551, Merck-Millipore) following the manufacturer's protocol. Briefly, twenty four hours (24 h) post-transfection cells were trypsinized and a suspension of 0.5 106 cells/ml was prepared in serum-free DMEM medium. Equal volume (0.25 ml/chamber) of the cell suspensions were seeded on the invasion chambers while 0.5 ml of DMEM supplemented with 10% Fetal Bovine Serum (chemoattractant) was added to the bottom of the transwell. Cells were incubated at 37 1C for an additional 24 h period (48 h post-trasfection). At the end of the incubation period, the transwells were placed on 0.4 ml of the provided staining solution and incubated for 20 min. Transwells were then washed several times with distilled water. The non-invaded cells that remained on the upside of the filter were removed using a cotton swab while the stained insert with invaded cells was transferred to a well containing 0.2 ml of extraction buffer. After 15 min incubation at room temperature, the transwell was removed and the dye mixture was transferred to three wells of a 96-well plate. Optical density (OD) was measured at 560 nm using an automated Enspire
Please cite this article as: V. Gkretsi, D.P. Bogdanos, Experimental evidence of Migfilin as a new therapeutic target of hepatocellular carcinoma metastasis, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.03.002
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Perkin-Elmer spectrophotometer. Three independent experiments were performed.
Statistical analysis Comparison of means using Statgraphics software was used for the statistical analysis. T-test was performed and a p-Value o0.05 was considered statistically significant.
Results Migfilin is upregulated in HepG2 compared to Alexander cells both at the protein and mRNA level Up to date, Migfilin's expression has not been assessed in liver cells. Thus, we first set out to examine the expression of Migfilin in the two selected liver cell lines both at the mRNA and protein level. To that regard, western blot analysis and real time PCR experiments were performed, respectively. Results showed that Migfilin is significantly upregulated in HepG2 cells compared to Alexander cells both at the protein (Fig. 1A, compare lane 2 with lane 1 and Fig. 1C) and the mRNA level (Fig. 1B).
Migfilin gene is effectively silenced In order to investigate the role of Migfilin in the two cell lines, we first inhibited its expression by using siRNA-mediated gene silencing. As shown in Figs. 2–4A, , Migfilin is effectively silenced in both cell lines (compare lane 2 with lane 1, and lane 4 with lane 3).
Migfilin silencing leads to upregulation of phosphor-VASP (Ser 157 and Ser239) in HepG2 cells As cell migration and invasion, which are vital processes taking place during cancer cell metastasis, are coordinately regulated by cell–matrix adhesion and actin cytoskeleton, we first investigated the effect of Migfilin knock-down on actin-regulating molecules. Thus, following Migfilin silencing, we examined the protein expression of its binding partner VASP, which is also known as a regulator of actin polymerization at lamellipodia [8,17]. As shown in Fig. 2A, the expression of VASP is slightly reduced in the Migfilin-depleted cells but that difference is not statistically significant in all experiments performed, as shown by the quantification of protein expression using the Image J software (Fig. 2D). Interestingly, though, when we examined the two main phosphorylated forms of VASP, we found that both phosphorVASP (Ser 157) (Fig. 2(B) and (D)) and phosphor-VASP (Ser239) (Fig. 2(C) and (D)) are significantly upregulated in the Migfilindepleted HepG2 cells but not in the Alexander cells.
Migfilin silencing leads to upregulation of phosphor-βcatenin in both cell lines We then examined the effect of Migfilin depletion on β-catenin, which is known to be deregulated in cancer and has been previously shown to be inversely correlated with Migfilin expression [14,15]. Consistent with these findings, we also showed that β-catenin expression is dramatically increased in HepG2 cells
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lacking Migfilin (Fig. 2(A) and (D)). We then tested the expression of the phosphorylated form of β-catenin at sites Ser33/37/Thr41, which is known to result in its degradation and promotion of cell proliferation [18,19], and found that it is significantly increased in Migfilin depleted cells in both cell lines (Fig. 2(C) and (D)).
Migfilin depletion leads to dramatic upregulation of Fascin-1 and ROCK-1 in both cell lines Intrigued by the finding that Migfilin depletion inversely correlates with phosphorylated VASP, and phosphorylated β-catenin, we proceeded further to investigate the implicated molecular mechanism. As the process of reorganization of actin cytoskeleton during cell migration is governed predominantly by actin binding proteins, VASP and Fascin-1 which cooperate with each other [17,20], we proceeded to test the expression level of Fascin-1 in the two HCC cell lines after Migfilin silencing. Interestingly, the fact that Fascin-1 is known to be greatly upregulated in HCC samples [21,22] and associated with poorer prognosis, made our investigation more stimulating. Indeed, as shown in Fig. 2(C) and (D), Fascin-1 is dramatically upregulated following Migfilin silencing in both cell lines. Furthermore, we tested the expression of Rho kinase (ROCK-1) which is upstream of Fascin-1 and has been shown to phosphorylate VASP at Ser157 [23]. As shown in Fig. 2(C) and (D), ROCK-1 is also significantly upregulated following Migfilin depletion in both HCC cell lines.
Migfilin silencing leads to elevated expression of extracellular signal-regulated kinase (ERK1/2) in both cell lines and reduced expression of phosphor-Akt (Ser 473) in HepG2 cells As Migfilin was recently shown to bind to oncogene Src which is also recruited to cell-ECM adhesions [24] and contributes to cellECM survival signaling, we tested whether the ERK1/2 pathway is affected by Migfilin depletion. Consistent with previous data in Migfilin null bone marrow monocytes [25], we found that Extracellular Signal-regulated kinase (ERK1/2) expression level is dramatically increased in both cell lines after Migfilin depletion (Fig. 3(A) and (C)). In an attempt to further dissect the molecular mechanism and since Migfilin has been connected to the Akt pro-survival signaling [14] that is known to inhibit β-catenin, we tested the expression level of phosphorylated Akt (Ser473). Migfilin depletion led to a significant decrease in phosphorylated Akt (S473) in HepG2 cells only (Fig. 3(B) and (C)) which means that Migfilin induces Akt phosphorylation in the more invasive HCC cell line.
Migfilin silencing induces cell proliferation in both cell lines Intrigued by our findings that Migfilin depletion has an effect on important pro-survival signaling pathways, we wanted to further elucidate its functional role in the two HCC cell lines, and, if possible, correlate it with the metastatic properties of the cells. Thus, we first assessed the effect of Migfilin depletion on cell proliferation. Our results showed that following Migfilin silencing, cell proliferation is significantly increased in both cell lines (Fig. 4B). In fact, the proliferation of the more aggressive HepG2
Please cite this article as: V. Gkretsi, D.P. Bogdanos, Experimental evidence of Migfilin as a new therapeutic target of hepatocellular carcinoma metastasis, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.03.002
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Fig. 2 – Effect of Migfilin depletion on the protein expression of molecules involved in actin organization. Alexander and HepG2 cells were transfected with either non specific control siRNA or siRNA targeting Migfilin and 48 h post-transfection, whole cell lysates were obtained in order to perform western blot analysis for: (A) Migfilin, Vasodilator Stimulated phosphoprotein, β-catenin, (B) phosphor-Vasodilator Stimulated phopshoprotein (Ser 157), (C) phosphor-Vasodilator Stimulated phosphoprotein (Ser 239), phosphor-β-catenin (Ser33/37/Thr41), Fascin-1, and ROCK-1. B-actin was used as loading control, (D) relative protein expression for all proteins examined in this figure quantified using the Image J software according to the software's guidelines. The graph represents mean value of band intensity from 2 or 3 different western blots. A p Value of o0.05 was considered as statistically significant.
cells is more dramatically affected by Migfilin silencing, compared to the less invasive Alexander cells (compare gray lines with black lines in Fig. 4B).
HepG2 cells whereas this is not the case for the less invasive Alexander cells.
Migfilin elimination inhibits cell invasion in HepG2 cells Migfilin depletion leads to increased cell adhesion in HepG2 cells We next sought to find out whether Migfilin silencing affects the property of cells to adhere to the ECM and influence migratory status. Thus, we performed a series of adhesion assays on gelatin in both cell lines using cells that were transfected with NSC or Migfilin siRNA. As shown in Fig. 4C, inhibition of Migfilin expression by siRNA induces an increase in cell adhesion ability of
To complete our study on the effect of Migfilin silencing in the metastatic status of the HCC cells, we proceeded to test the cells' invasive properties. As invasion of surrounding tissues is the most important characteristic that differentiates malignant tumors from benign neoplasms, evaluation of cell invasion following Migfilin silencing would provide us with the answer of whether Migfilin facilitates or hinders metastasis. In accordance with the notion that Alexander cells are low-invasive cells, we found in all cell invasion experiments in Alexander cells that there was no
Please cite this article as: V. Gkretsi, D.P. Bogdanos, Experimental evidence of Migfilin as a new therapeutic target of hepatocellular carcinoma metastasis, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.03.002
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Fig. 3 – Effect of Migfilin depletion on the protein expression on Extracellular Signal-regulated kinase 1/2 and phosphor-Akt. Alexander and HepG2 cells were transfected with either non specific control siRNA or siRNA targeting Migfilin and 48 h posttransfection whole cell lysates were obtained to perform western blot analysis for: (A) Migfilin, Extracellular Signal –regulated kinase1/2, and (B) phosphor-Akt (Ser 473). B-actin was used as loading control. (C) Relative protein expression for all proteins examined in this figure quantified using the Image J software according to the software's guidelines. The graph represents mean value of band intensity from 3 different western blots. A p Value of o0.05 was considered as statistically significant. significant difference between control-transfected cells and Migfilin siRNA-transfected cells (Fig. 4D). Interestingly though, HepG2 cells lacking Migfilin are less invasive than their controltransfected counterparts (Fig. 4D), indicating that Migfilin inhibition reduces cell invasion and thus Migfilin itself promotes invasiveness.
Discussion Cell adhesion proteins connecting cells to the ECM and/or neighboring cells are fundamental for tissue homeostasis and are known to be deregulated in cancer cell metastasis. Migfilin is a relatively novel LIM domain-containing protein localized both at cell-ECM and cell– cell adhesion sites [6] that has been implicated in regulation of cellECM adhesion and motility although the underlying mechanisms of its action are not fully elucidated. The role of Migfilin in cancer is also not very well defined while, to date, there is no study on the role or the expression of Migfilin in HCC. Interestingly, there are a few studies implicating Migfilin in cancer cell metastasis, although the exact effect depends on the type of cancer. More specifically, Migfilin's expression in human glioma samples correlates with pathological tumor grade and poor prognosis [26] while it was shown to promote migration and invasion in glioma cells [26]. Similarly, while Migfilin was barely detectable in normal smooth muscle cells, increased levels were observed in the majority of leiomyosarcomas and the cytoplasmic
level of migfilin was strongly associated with higher tumour grades [27]. On the other hand, Migfilin's expression level was upregulated in esophageal cancer samples [14] while being inversely correlated with clinical metastasis status [15]. Moreover, Migfilin was shown to inhibit esophageal cancer cell invasion through decreased free β-catenin level and thus promotion of β-catenin degradation [15] in an Akt dependent manner. Migfilin was also examined in advanced-stage serous ovarian carcinoma [12] and was found to have significantly lower expression in primary carcinomas and solid metastases compared to effusions [12]. Finally, its expression was significantly reduced in the majority of the breast cancer tissues compared to normal tissues regardless of metastatic status and disease stage [13]. Given that Migfilin's role in HCC and the liver is completely unknown our aim in the present study was to investigate the role of Migfilin in vitro in two human liver cell lines that differ in terms of their metastatic potential. Although these human cell lines cannot substitute patient samples, they do offer some advantages such as reproducibility of results, easier handling, and survival in culture for longer time periods than primary hepatocytes cultures so that experiments including siRNA transfection, invasion and adhesion assays can be performed effectively. Our results show that Migfilin is elevated in the more invasive HepG2 cells compared to the less invasive Alexander cells both at the mRNA and protein level (Fig. 1), indicating a possible contribution of the protein to the aggressive phenotype of HepG2 cells.
Please cite this article as: V. Gkretsi, D.P. Bogdanos, Experimental evidence of Migfilin as a new therapeutic target of hepatocellular carcinoma metastasis, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.03.002
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Fig. 4 – Migfilin silencing leads to increased proliferation, increased cell adhesion and decreased cell invasion. Alexander and HepG2 cells transfected with non-specific control siRNA or siRNA targeting Migfilin were subjected to: (A) western blotting 48 h post-transfection. (B) Cell proliferation assay using Alamar Blue reagent at 2, 4 and 24 h post-replating (or 26, 28 and 48 h posttransfection). Three independent experiments were performed. (C) Cell adhesion assay on 0.1% gelatin performed 48 h posttransfection. The data from two (2) independent experiments were analyzed. (D) Cell invasion assay using QCM™ Collagen Cell Invasion Assay, 24-well invasion chambers (8 μm). Three independent experiments were performed. Gene silencing of Migfilin leads to upregulation of proteins involved in actin reorganization such as the two main forms of phosphorylated VASP (Ser157 and Ser239) in HepG2 cells, and Fascin-1 and Rho kinase (ROCK-1) in both cell lines. Increased expression of these molecules leads to increased actin polymerization and stabilization (Fig. 5), thus less available monomeric actin for cell migration or invasion which ultimately contributes to a reduction of cell migration [28,29]. Indeed, Migfilin depletion leads to reduced cell invasion, indicating that Migfilin promotes the metastatic phenotype of HepG2 cells (Fig. 4D). In fact, this is in accordance with previous studies showing Migfilin to be crucial for cell migration in a variety of cell types (HeLa, HT-1080, and MDA-MB-231 cells), where it was shown that its depletion impairs cell migration [7]. As Migfilin has been previously shown to link the cell–matrix adhesions to the actin cytoskeleton [5], the migratory defect induced by the loss of Migfilin is probably caused, at least in part, by the impaired connection between cell–matrix adhesions and the actin cytoskeleton. Our data also show that Migfilin silencing leads to downregulation of phosphor-Akt (Ser473) (Fig. 3(B) and (C)) while at the same time leading to upregulation of phosphorylated β-catenin (Ser33/37Thr41), as well as free β-catenin (Fig. 2A, C, and D ) resulting in increased cell proliferation (Fig. 4B), which is another characteristic of tumor aggressiveness. Finally, the fact that depletion of Migfilin results in elevated ERK1/2 expression levels (Fig. 3(A) and (C)) along with increased cell adhesion in HepG2 cells (Fig. 4C) also indicates an
involvement of Migfilin in the aggressiveness of HepG2 cells. Moreover, the fact that Migfilin interacts with VASP at cell-ECM adhesion sites and enhances cell-ECM adhesion further explains the observed suppression in cell invasion (Fig. 4D), as it has been previously shown that increased adhesion is associated with decreased migration and invasion [7]. Notably, the fact that elimination of Migfilin severely impairs HepG2 cell invasion, while at the same time increases cell proliferation, both through elevation of ERK1/2 (Fig. 3(A) and (C)) and phosphor-β-catenin (Fig. 2(C) and (D)), indicates that Migfilin has a dual function in HCC cells activating different signaling pathways (Fig. 5) perhaps through interaction with different binding partners. This could provide an explanation as to why its role in cancer is not strictly defined but greatly depends upon the type of cancer, as mentioned above. This is the first study to examine the expression and role of Migfilin in two human HCC cell lines with different invasiveness. Our findings demonstrate for the first time that Migfilin is associated with a more aggressive HCC phenotype and we also provide solid evidence for the molecular mechanism involved. A diagrammatic representation of the molecular mechanisms being activated following Migfilin silencing in HepG2 highly invasive cells is shown in Fig. 5. Finally, this study provides the first line of evidence showing that Migfilin could be a potential therapeutic target against HCC metastasis. However, its evaluation as a therapeutic target should
Please cite this article as: V. Gkretsi, D.P. Bogdanos, Experimental evidence of Migfilin as a new therapeutic target of hepatocellular carcinoma metastasis, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.03.002
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Fig. 5 – Diagrammatic representation of the molecular mechanism that defines the role of Migfilin in HCC. Migfilin silencing regulates three main cellular functions: (a) reduces cell invasion through upregulation of Vasodilator Stimulated Phosphoprotein, phopsho-Vasolidator Stimulated Phosphoprotein (Ser157), phosphor-Vasodilator Stimulated Phosphoprotein (Ser 239), Rho kinase-1 and Fascin-1, all of which lead to increased actin polymerization and thus invasion and migration inhibition, (b) increases proliferation, through phosphor-Akt (Ser 473) and phosphor-β-catenin (Ser33/37/Thr41), and (c) increases cell adhesion through Extracellular Signal-regulated kinase1/2 upregulation, which is also known to lead to decreased migration and invasion. Bold red arrows indicate decrease while bold green arrows indicate increase following Migfilin silencing.
be preceded by a thorough investigation of its expression in human HCC samples and correlation with prognosis and metastatic status to determine its exact role in human HCC metastasis.
Acknowledgements This study was supported by the European Association for the Study of the Liver (EASL) Sheila Sherlock fellowship 2012.We are also grateful to Dr. Chuanyue Wu, Professor at the University of Pittsburgh Medical School, Pittsburgh, PA, USA for providing us with the anti-Migfilin monoclonal antibody.
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Please cite this article as: V. Gkretsi, D.P. Bogdanos, Experimental evidence of Migfilin as a new therapeutic target of hepatocellular carcinoma metastasis, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.03.002
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[19] I.R. Cho, S.S. Koh, H.J. Min, et al., Pancreatic adenocarcinoma upregulated factor (PAUF) enhances the expression of beta-catenin, leading to a rapid proliferation of pancreatic cells, Exp. Mol. Med. 43 (2011) 82–90. [20] J.D. Winkelman, C.G. Bilancia, M. Peifer, et al., Ena/VASP Enabled is a highly processive actin polymerase tailored to self-assemble parallel-bundled F-actin networks with Fascin, Proc. Natl. Acad. Sci. USA 111 (2014) 4121–4126. [21] X. Huang, J. Ji, H. Xue, et al., Fascin and cortactin expression is correlated with a poor prognosis in hepatocellular carcinoma, Eur. J. Gastroenterol. Hepatol. 24 (2012) 633–639. [22] T. Iguchi, S. Aishima, K. Umeda, et al., Fascin expression in progression and prognosis of hepatocellular carcinoma, J. Surg. Oncol. 100 (2009) 575–579. [23] J.K. Wentworth, G. Pula, A.W. Poole, Vasodilator-stimulated phosphoprotein (VASP) is phosphorylated on Ser157 by protein kinase C-dependent and independent mechanisms in thrombinstimulated human platelets, Biochem. J. 393 (2006) 555–564.
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[24] J. Zhao, Y. Zhang, S.S. Ithychanda, et al., Migfilin interacts with Src and contributes to cell–matrix adhesion-mediated survival signaling, J. Biol. Chem. 284 (2009) 34308–34320. [25] G. Xiao, H. Cheng, H. Cao, et al., Critical role of filamin-binding LIM protein 1 (FBLP-1)/migfilin in regulation of bone remodeling, J. Biol. Chem. 287 (2012) 21450–21460. [26] Y. Ou, L. Ma, L. Dong, et al., Migfilin protein promotes migration and invasion in human glioma through epidermal growth factor receptor-mediated phospholipase C-gamma and STAT3 protein signaling pathways, J. Biol. Chem. 287 (2012) 32394–32405. [27] D.J. Papachristou, V. Gkretsi, Y. Tu, et al., Increased cytoplasmic level of migfilin is associated with higher grades of human leiomyosarcoma, Histopathology 51 (2007) 499–508. [28] M. Maekawa, T. Ishizaki, S. Boku, et al., Signaling from Rho to the actin cytoskeleton through protein kinases ROCK and LIM-kinase, Science 285 (1999) 895–898. [29] K. Riento, A.J. Ridley, Rocks: multifunctional kinases in cell behaviour, Nat. Rev. Mol. Cell Biol. 4 (2003) 446–456.
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Available online at www.sciencedirect.com
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Research Article
Experimental evidence of Migfilin as a new therapeutic target of hepatocellular carcinoma metastasis Vasiliki Gkretsia,n, Dimitrios P. Bogdanosa,b,c a
Department of Biomedical Research and Technology, Institute for Research and Technology-Thessaly, Centre for Research and TechnologyHellas (CE.R.T.H.), Larissa 41222, Greece b Department of Rheumatology, School of Medicine, University of Thessaly, University Hospital of Larissa, 41110 Larissa, Greece c Institute of Liver Studies, King's College Hospital, Denmark Hill, London SE5 9RS, UK
article information
abstract
Article Chronology:
Migfilin is a novel cell–matrix adhesion protein known to interact with Vasodilator Stimulated
Received 15 December 2014
Phosphoprotein (VASP) and be localized both at cell–matrix and cell–cell adhesions. To date there is
Received in revised form
nothing known about its role in hepatocellular carcinoma (HCC). As matrix is important in
1 March 2015
metastasis, we aimed to investigate the Migfilin's role in HCC metastasis using two human HCC
Accepted 4 March 2015
cell lines that differ in their metastatic potential; non-invasive Alexander cells and the highly invasive HepG2 cells. We silenced Migfilin by siRNA and studied its effect on signaling and metastasis-related
Keywords: Hepatocellular carcinoma Migfilin VASP Invasion Adhesion Metastasis Fascin-1
cellular properties. We show that Migfilin's expression is elevated in HepG2 cells and its silencing leads to upregulation of actin reorganization-related proteins, namely phosphor-VASP (Ser157 and Ser239), Fascin-1 and Rho-kinase-1, promoting actin polymerization and inhibiting cell invasion. Phosphor-Akt (Ser473) is decreased contributing to the upregulation of free and phosphor-β-catenin (Ser33/37Thr41) and inducing proliferation. Migfilin elimination upregulates Extracellular Signal– regulated kinase, which increases cell adhesion in HepG2 and reduces invasiveness. This is the first study to reveal that Migfilin inhibition can halt HCC metastasis in vitro, providing the molecular mechanism involved and presenting Migfilin as potential therapeutic target against HCC metastasis.
Introduction Hepatocellular carcinoma (HCC) accounts for 5.5% of all cancer cases worldwide being the fifth leading cause of cancer mortality in the world [1]. The fact that, in most cases, there are no symptoms in the early stages along with the high metastatic potential and the rapid
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growth rate of the tumors justifies the mortality rates observed. In fact, it has been estimated that the 5-year fatality rate for individuals diagnosed with HCC is greater than 95% [1]. Metastasis (both intrahepatic and extrahepatic) is a fundamental biological behavior of HCC and the main cause of treatment failure [2]. It occurs as the last stage of a series of events which
Abbreviations list in order of appearance: lHCC, hepatocellular carcinoma; ECM, extracellular matrix; FBLP-1, filamin binding LIM protein -1 (or Migfilin); VASP, vasodilator stimulated phosphoprotein; PBGD, porphobilinogen deaminase; PKA, cAMP-dependent kinase; PKG, cGMP-dependent protein kinase; NSC, non-specific control; ROCK-1, rho kinase-1; ERK1/2, extracellular signal –regulated kinase n
Corresponding author. Fax: þ30 2410 258889. E-mail address: [email protected] (V. Gkretsi).
http://dx.doi.org/10.1016/j.yexcr.2015.03.002 0014-4827/& 2015 Elsevier Inc. All rights reserved.
Please cite this article as: V. Gkretsi, D.P. Bogdanos, Experimental evidence of Migfilin as a new therapeutic target of hepatocellular carcinoma metastasis, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.03.002
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include replacement of the injured or impaired liver tissue by fibrotic, scar tissue and regenerative nodules (cirrhosis), generation of phenotypically altered hepatocytes and generation of dysplastic hepatocytes which serve as precursors to HCC [1]. Since scarring is involved, integrins, extracellular matrix (ECM) and ECM-proximal proteins play a fundamental role in HCC metastasis. Although normally cells have excellent communication both with the ECM through integrins, and with neighboring cells through cell– cell adhesion proteins, this communication is severely disrupted in cancer. Thus, cell dissociation from the original tumor mass is facilitated and migration towards a new environment occurs, leading to the formation of a metastatic lesion [3]. The significance of cell adhesion proteins in cancer cell metastasis is even more apparent in the case of HCC, as hepatocytes are greatly depended on the communication with the surrounding matrix [4]. Migfilin (also known as Filamin Binding LIM-protein-1 or FBLP-1) is a novel LIM domain-containing protein present both at cell-ECM [5] and cell–cell adhesions [6]. It interacts with Vasodilator Stimulated Phosphoprotein (VASP) [7] which is known to regulate actin polymerization in lamellipodia [8–10], the cellular protrusions responsible for migration and invasion of the cell. VASP is also a substrate of cAMP-dependent kinases (PKA), and cGMP-dependent protein kinases (PKG) that primarily phosphorylate the sites Ser157, and Ser239 respectively of the VASP protein [11]. A few recent studies have implicated Migfilin in cancer cell metastasis, although its exact role seems to depend on the type of cancer [12–15]. Interestingly, Migfilin's role in the liver and HCC is completely unknown. Hence, the aim of the present study was to investigate the in vitro role of Migfilin in two liver cell lines that differ in terms of their metastatic potential; the non-invasive hepatoma cell line PLC/PRF/5 (Alexander cells herein) and the highly invasive HCC HepG2 cell line. We utilized an siRNA-mediated silencing approach to knockdown the Migfilin gene from both cell lines and study the effect of gene silencing on basic signaling pathways and functional cellular properties related to the metastatic potential of the cells.
Materials and methods Antibodies and reagents Antibodies against phosphor-VASP (Ser157), phosphor-VASP (Ser239), VASP, Fascin-1, phosphor-β-catenin (Ser33/37/Thr41), β-catenin and Extracellular Signal-regulated kinase (ERK1/2) were purchased from Cell Signaling. The antibody against phosphor-Akt (Ser473) was purchased from Abcam while the antibody against ROCK-1 was purchased from R&D systems. The monocloncal antibody against Migfilin was kindly provided by Dr. Chuanyue Wu (Professor at the University of Pittsburgh Medical School, Pittsburgh, PA, USA) while anti-β-actin antibody (Sigma-Aldrich) was used as loading control for all western blot experiments. Alamar Blue reagent was obtained from Invitrogen.
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the HCC cell line HepG2. Both cell lines were purchased from ATCC.
RNA isolation Total cellular RNA was extracted from cells using Trizol reagent (Invitrogen). RNA was further purified using an RNeasy mini kit (Qiagen). Preservation of 28S and 18S ribosomal RNA (rRNA) species was used to assess RNA integrity. All samples included in the study were with prominent 28S and 18S rRNA components. The yield was quantified spectrophotometrically.
Real time PCR Transcription of 1 mg RNA to complementary DNA (cDNA) was performed using Superscript (Invitrogen). Quantification of Migfilin was performed by real-time PCR using Platinum SYBR-green reagen (Invitrogen) in a MiniOpticon MJ Thermal Cycler Real Time PCR machine (BioRad). Porphobilinogen deaminase (PBGD) was used as a housekeeping gene. Reactions were always performed in triplicate and at least 4 independent experiments were performed. The primers for Migfilin were as follows; forward: 50 CGA ATG CAT GGG AAG AAA CT 30 , reverse 50 GCA GGT TAG GAA GGG AAA CC 30 . To quantify the relative expression of each gene, Ct values were normalized against the endogenous reference (ΔCt¼Ct targetCt PBGD) and were compared with a calibrator using the ΔΔCt method (ΔΔCt¼ΔCt sampleΔCt calibrator). Alexander cells were used as calibrators for the HepG2 cells.
Protein extraction and western blot analysis Total cell lysates were obtained using 1% sodium dodecyl sulfate in RIPA buffer (20mMTris/Cl pH7.5, 150 mM NaCl, 0.5% NP-40, 1% TX-100, 0.25% sodium deoxycholated, 0.6–2 μg/ml aprotinin, 10 μMleupeptin, 1 μM pepstatin). Protein concentrations in the samples were determined by the BCA protein assay kit (Pierce) using bovine serum albumin as standard. Equal amount of protein was loaded on each lane of a 10–12% acrylamide gel and transferred to a PVDF membrane (Millipore) using the BioRad semi-dry transfer system (BioRad). Signals were detected using suitable secondary immunoglobulin IgG conjugated with horseradish peroxidase (Invitrogen). Antibody detection was performed using super-signal ECL detection system (PIERCE).
Transfection with siRNAs Both Alexander and HepG2 cells were treated for 48 h with 100 nM siRNA non-specific control (NSC) siRNA or siRNA against Migfilin using the Lipofectamine 2000 transfection reagent (Invitrogen) according to the company's guidelines. The siRNA sequence used to silence Migfilin, was as follows: 50 - AAA GGG GCA UCC ACA GAC AUC-30 , while the sequence 50 AAA CUC UAU CUG CAC GCU GAC30 was used as NSC. Silencing efficiency prior to every experiment performed was tested by western blot and/or real time PCR, as specified in each experiment.
Liver cell lines
Quantification of protein expression in western blots
Two liver cell lines of different invasive capacity were used in the present study; the hepatoma cell line PLC/PRF/5 (Alexander) and
Quantification of protein expression in western blots was performed using Image J analysis according to the software's guidelines.
Please cite this article as: V. Gkretsi, D.P. Bogdanos, Experimental evidence of Migfilin as a new therapeutic target of hepatocellular carcinoma metastasis, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.03.002
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Fig. 1 – Migfilin expression at the protein and mRNA level in Alexander and HepG2 cells. (Α) Representative western blot showing Migfilin protein expression in the two HCC cell lines tested; the low invasiveness Alexander and the highly invasive HepG2 cells. (B) Diagram showing the relative mRNA expression of Migfilin using Real Time Polymerase Chain Reaction. Relative expression was calculated using the ΔΔCt method as described in the materials and methods section. Porphobilinogen deaminase (PBGD) was used as the housekeeping gene and Alexander cells were used as calibrator for the analysis of the results. Reactions were performed in triplicate and three independent experiments were conducted. (C) Relative protein expression of migfilin quantified using the Image J software according to the software's guidelines. The graph represents mean value of band intensity from 3 different western blots. A p Value of o0.05 was considered as statistically significant. The mean intensity from relative protein bands from three different western blots corresponding to three independent experiments was used for the quantification. A p Value of o0.05 was considered as statistically significant.
Alamar blue proliferation assay Twenty four hours following siRNA treatment, cells were subjected to Alamar blue assay (Invitrogen) [16] according to the company's guidelines. Briefly, cells were seeded in 96 well plates at a concentration of 104cells/ml and Alamar blue was added at a volume of 1/10 of the volume of medium in the well. Following incubation at 37oC for 2, 4 and 24 h post-plating, fluorescence was measured using Perkin-Elmer Enspire plate reader at 560/590 nm. Three independent experiments were performed.
Cell adhesion assay Cell adhesion assay was performed as described previously [7]. Briefly, cells were transfected with a control NSC siRNA or siRNA against Migfilin. Forty eight hours (48 h) post-transfection, 104cells/ well were seeded in six (6) wells of a 96-well plate pre-coated with 0.1% gelatin. After 60 min incubation at 37 1C, three of the wells were washed three times with phosphate-buffered saline (PBS) while the remaining three were fixed with 4% paraformaldehyde (PFA). Washed wells were also fixed with PFA and then cells in all wells were quantified using crystal violet [7]. Crystal violet was washed using ddH20 and cells were solubilized using acetic acid.
Absorbance was measured at 570 nm using Perkin-Elmer Enspire plate reader. Adhesion was presented as the ratio of the absorbance at 570 nm of adhered cells (washed) divided by the absorbance at 570 nm of the total seeded cells (not washed). The data from two independent experiments were analyzed using the student's T-test. p Values o0.05 were considered as statistically significant.
Cell invasion Cell invasion was assessed using QCM™ Collagen Cell Invasion Assay, 24-well invasion chambers (8 μm) (Cat. #ECM551, Merck-Millipore) following the manufacturer's protocol. Briefly, twenty four hours (24 h) post-transfection cells were trypsinized and a suspension of 0.5 106 cells/ml was prepared in serum-free DMEM medium. Equal volume (0.25 ml/chamber) of the cell suspensions were seeded on the invasion chambers while 0.5 ml of DMEM supplemented with 10% Fetal Bovine Serum (chemoattractant) was added to the bottom of the transwell. Cells were incubated at 37 1C for an additional 24 h period (48 h post-trasfection). At the end of the incubation period, the transwells were placed on 0.4 ml of the provided staining solution and incubated for 20 min. Transwells were then washed several times with distilled water. The non-invaded cells that remained on the upside of the filter were removed using a cotton swab while the stained insert with invaded cells was transferred to a well containing 0.2 ml of extraction buffer. After 15 min incubation at room temperature, the transwell was removed and the dye mixture was transferred to three wells of a 96-well plate. Optical density (OD) was measured at 560 nm using an automated Enspire
Please cite this article as: V. Gkretsi, D.P. Bogdanos, Experimental evidence of Migfilin as a new therapeutic target of hepatocellular carcinoma metastasis, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.03.002
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Perkin-Elmer spectrophotometer. Three independent experiments were performed.
Statistical analysis Comparison of means using Statgraphics software was used for the statistical analysis. T-test was performed and a p-Value o0.05 was considered statistically significant.
Results Migfilin is upregulated in HepG2 compared to Alexander cells both at the protein and mRNA level Up to date, Migfilin's expression has not been assessed in liver cells. Thus, we first set out to examine the expression of Migfilin in the two selected liver cell lines both at the mRNA and protein level. To that regard, western blot analysis and real time PCR experiments were performed, respectively. Results showed that Migfilin is significantly upregulated in HepG2 cells compared to Alexander cells both at the protein (Fig. 1A, compare lane 2 with lane 1 and Fig. 1C) and the mRNA level (Fig. 1B).
Migfilin gene is effectively silenced In order to investigate the role of Migfilin in the two cell lines, we first inhibited its expression by using siRNA-mediated gene silencing. As shown in Figs. 2–4A, , Migfilin is effectively silenced in both cell lines (compare lane 2 with lane 1, and lane 4 with lane 3).
Migfilin silencing leads to upregulation of phosphor-VASP (Ser 157 and Ser239) in HepG2 cells As cell migration and invasion, which are vital processes taking place during cancer cell metastasis, are coordinately regulated by cell–matrix adhesion and actin cytoskeleton, we first investigated the effect of Migfilin knock-down on actin-regulating molecules. Thus, following Migfilin silencing, we examined the protein expression of its binding partner VASP, which is also known as a regulator of actin polymerization at lamellipodia [8,17]. As shown in Fig. 2A, the expression of VASP is slightly reduced in the Migfilin-depleted cells but that difference is not statistically significant in all experiments performed, as shown by the quantification of protein expression using the Image J software (Fig. 2D). Interestingly, though, when we examined the two main phosphorylated forms of VASP, we found that both phosphorVASP (Ser 157) (Fig. 2(B) and (D)) and phosphor-VASP (Ser239) (Fig. 2(C) and (D)) are significantly upregulated in the Migfilindepleted HepG2 cells but not in the Alexander cells.
Migfilin silencing leads to upregulation of phosphor-βcatenin in both cell lines We then examined the effect of Migfilin depletion on β-catenin, which is known to be deregulated in cancer and has been previously shown to be inversely correlated with Migfilin expression [14,15]. Consistent with these findings, we also showed that β-catenin expression is dramatically increased in HepG2 cells
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lacking Migfilin (Fig. 2(A) and (D)). We then tested the expression of the phosphorylated form of β-catenin at sites Ser33/37/Thr41, which is known to result in its degradation and promotion of cell proliferation [18,19], and found that it is significantly increased in Migfilin depleted cells in both cell lines (Fig. 2(C) and (D)).
Migfilin depletion leads to dramatic upregulation of Fascin-1 and ROCK-1 in both cell lines Intrigued by the finding that Migfilin depletion inversely correlates with phosphorylated VASP, and phosphorylated β-catenin, we proceeded further to investigate the implicated molecular mechanism. As the process of reorganization of actin cytoskeleton during cell migration is governed predominantly by actin binding proteins, VASP and Fascin-1 which cooperate with each other [17,20], we proceeded to test the expression level of Fascin-1 in the two HCC cell lines after Migfilin silencing. Interestingly, the fact that Fascin-1 is known to be greatly upregulated in HCC samples [21,22] and associated with poorer prognosis, made our investigation more stimulating. Indeed, as shown in Fig. 2(C) and (D), Fascin-1 is dramatically upregulated following Migfilin silencing in both cell lines. Furthermore, we tested the expression of Rho kinase (ROCK-1) which is upstream of Fascin-1 and has been shown to phosphorylate VASP at Ser157 [23]. As shown in Fig. 2(C) and (D), ROCK-1 is also significantly upregulated following Migfilin depletion in both HCC cell lines.
Migfilin silencing leads to elevated expression of extracellular signal-regulated kinase (ERK1/2) in both cell lines and reduced expression of phosphor-Akt (Ser 473) in HepG2 cells As Migfilin was recently shown to bind to oncogene Src which is also recruited to cell-ECM adhesions [24] and contributes to cellECM survival signaling, we tested whether the ERK1/2 pathway is affected by Migfilin depletion. Consistent with previous data in Migfilin null bone marrow monocytes [25], we found that Extracellular Signal-regulated kinase (ERK1/2) expression level is dramatically increased in both cell lines after Migfilin depletion (Fig. 3(A) and (C)). In an attempt to further dissect the molecular mechanism and since Migfilin has been connected to the Akt pro-survival signaling [14] that is known to inhibit β-catenin, we tested the expression level of phosphorylated Akt (Ser473). Migfilin depletion led to a significant decrease in phosphorylated Akt (S473) in HepG2 cells only (Fig. 3(B) and (C)) which means that Migfilin induces Akt phosphorylation in the more invasive HCC cell line.
Migfilin silencing induces cell proliferation in both cell lines Intrigued by our findings that Migfilin depletion has an effect on important pro-survival signaling pathways, we wanted to further elucidate its functional role in the two HCC cell lines, and, if possible, correlate it with the metastatic properties of the cells. Thus, we first assessed the effect of Migfilin depletion on cell proliferation. Our results showed that following Migfilin silencing, cell proliferation is significantly increased in both cell lines (Fig. 4B). In fact, the proliferation of the more aggressive HepG2
Please cite this article as: V. Gkretsi, D.P. Bogdanos, Experimental evidence of Migfilin as a new therapeutic target of hepatocellular carcinoma metastasis, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.03.002
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Fig. 2 – Effect of Migfilin depletion on the protein expression of molecules involved in actin organization. Alexander and HepG2 cells were transfected with either non specific control siRNA or siRNA targeting Migfilin and 48 h post-transfection, whole cell lysates were obtained in order to perform western blot analysis for: (A) Migfilin, Vasodilator Stimulated phosphoprotein, β-catenin, (B) phosphor-Vasodilator Stimulated phopshoprotein (Ser 157), (C) phosphor-Vasodilator Stimulated phosphoprotein (Ser 239), phosphor-β-catenin (Ser33/37/Thr41), Fascin-1, and ROCK-1. B-actin was used as loading control, (D) relative protein expression for all proteins examined in this figure quantified using the Image J software according to the software's guidelines. The graph represents mean value of band intensity from 2 or 3 different western blots. A p Value of o0.05 was considered as statistically significant.
cells is more dramatically affected by Migfilin silencing, compared to the less invasive Alexander cells (compare gray lines with black lines in Fig. 4B).
HepG2 cells whereas this is not the case for the less invasive Alexander cells.
Migfilin elimination inhibits cell invasion in HepG2 cells Migfilin depletion leads to increased cell adhesion in HepG2 cells We next sought to find out whether Migfilin silencing affects the property of cells to adhere to the ECM and influence migratory status. Thus, we performed a series of adhesion assays on gelatin in both cell lines using cells that were transfected with NSC or Migfilin siRNA. As shown in Fig. 4C, inhibition of Migfilin expression by siRNA induces an increase in cell adhesion ability of
To complete our study on the effect of Migfilin silencing in the metastatic status of the HCC cells, we proceeded to test the cells' invasive properties. As invasion of surrounding tissues is the most important characteristic that differentiates malignant tumors from benign neoplasms, evaluation of cell invasion following Migfilin silencing would provide us with the answer of whether Migfilin facilitates or hinders metastasis. In accordance with the notion that Alexander cells are low-invasive cells, we found in all cell invasion experiments in Alexander cells that there was no
Please cite this article as: V. Gkretsi, D.P. Bogdanos, Experimental evidence of Migfilin as a new therapeutic target of hepatocellular carcinoma metastasis, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.03.002
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Fig. 3 – Effect of Migfilin depletion on the protein expression on Extracellular Signal-regulated kinase 1/2 and phosphor-Akt. Alexander and HepG2 cells were transfected with either non specific control siRNA or siRNA targeting Migfilin and 48 h posttransfection whole cell lysates were obtained to perform western blot analysis for: (A) Migfilin, Extracellular Signal –regulated kinase1/2, and (B) phosphor-Akt (Ser 473). B-actin was used as loading control. (C) Relative protein expression for all proteins examined in this figure quantified using the Image J software according to the software's guidelines. The graph represents mean value of band intensity from 3 different western blots. A p Value of o0.05 was considered as statistically significant. significant difference between control-transfected cells and Migfilin siRNA-transfected cells (Fig. 4D). Interestingly though, HepG2 cells lacking Migfilin are less invasive than their controltransfected counterparts (Fig. 4D), indicating that Migfilin inhibition reduces cell invasion and thus Migfilin itself promotes invasiveness.
Discussion Cell adhesion proteins connecting cells to the ECM and/or neighboring cells are fundamental for tissue homeostasis and are known to be deregulated in cancer cell metastasis. Migfilin is a relatively novel LIM domain-containing protein localized both at cell-ECM and cell– cell adhesion sites [6] that has been implicated in regulation of cellECM adhesion and motility although the underlying mechanisms of its action are not fully elucidated. The role of Migfilin in cancer is also not very well defined while, to date, there is no study on the role or the expression of Migfilin in HCC. Interestingly, there are a few studies implicating Migfilin in cancer cell metastasis, although the exact effect depends on the type of cancer. More specifically, Migfilin's expression in human glioma samples correlates with pathological tumor grade and poor prognosis [26] while it was shown to promote migration and invasion in glioma cells [26]. Similarly, while Migfilin was barely detectable in normal smooth muscle cells, increased levels were observed in the majority of leiomyosarcomas and the cytoplasmic
level of migfilin was strongly associated with higher tumour grades [27]. On the other hand, Migfilin's expression level was upregulated in esophageal cancer samples [14] while being inversely correlated with clinical metastasis status [15]. Moreover, Migfilin was shown to inhibit esophageal cancer cell invasion through decreased free β-catenin level and thus promotion of β-catenin degradation [15] in an Akt dependent manner. Migfilin was also examined in advanced-stage serous ovarian carcinoma [12] and was found to have significantly lower expression in primary carcinomas and solid metastases compared to effusions [12]. Finally, its expression was significantly reduced in the majority of the breast cancer tissues compared to normal tissues regardless of metastatic status and disease stage [13]. Given that Migfilin's role in HCC and the liver is completely unknown our aim in the present study was to investigate the role of Migfilin in vitro in two human liver cell lines that differ in terms of their metastatic potential. Although these human cell lines cannot substitute patient samples, they do offer some advantages such as reproducibility of results, easier handling, and survival in culture for longer time periods than primary hepatocytes cultures so that experiments including siRNA transfection, invasion and adhesion assays can be performed effectively. Our results show that Migfilin is elevated in the more invasive HepG2 cells compared to the less invasive Alexander cells both at the mRNA and protein level (Fig. 1), indicating a possible contribution of the protein to the aggressive phenotype of HepG2 cells.
Please cite this article as: V. Gkretsi, D.P. Bogdanos, Experimental evidence of Migfilin as a new therapeutic target of hepatocellular carcinoma metastasis, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.03.002
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Fig. 4 – Migfilin silencing leads to increased proliferation, increased cell adhesion and decreased cell invasion. Alexander and HepG2 cells transfected with non-specific control siRNA or siRNA targeting Migfilin were subjected to: (A) western blotting 48 h post-transfection. (B) Cell proliferation assay using Alamar Blue reagent at 2, 4 and 24 h post-replating (or 26, 28 and 48 h posttransfection). Three independent experiments were performed. (C) Cell adhesion assay on 0.1% gelatin performed 48 h posttransfection. The data from two (2) independent experiments were analyzed. (D) Cell invasion assay using QCM™ Collagen Cell Invasion Assay, 24-well invasion chambers (8 μm). Three independent experiments were performed. Gene silencing of Migfilin leads to upregulation of proteins involved in actin reorganization such as the two main forms of phosphorylated VASP (Ser157 and Ser239) in HepG2 cells, and Fascin-1 and Rho kinase (ROCK-1) in both cell lines. Increased expression of these molecules leads to increased actin polymerization and stabilization (Fig. 5), thus less available monomeric actin for cell migration or invasion which ultimately contributes to a reduction of cell migration [28,29]. Indeed, Migfilin depletion leads to reduced cell invasion, indicating that Migfilin promotes the metastatic phenotype of HepG2 cells (Fig. 4D). In fact, this is in accordance with previous studies showing Migfilin to be crucial for cell migration in a variety of cell types (HeLa, HT-1080, and MDA-MB-231 cells), where it was shown that its depletion impairs cell migration [7]. As Migfilin has been previously shown to link the cell–matrix adhesions to the actin cytoskeleton [5], the migratory defect induced by the loss of Migfilin is probably caused, at least in part, by the impaired connection between cell–matrix adhesions and the actin cytoskeleton. Our data also show that Migfilin silencing leads to downregulation of phosphor-Akt (Ser473) (Fig. 3(B) and (C)) while at the same time leading to upregulation of phosphorylated β-catenin (Ser33/37Thr41), as well as free β-catenin (Fig. 2A, C, and D ) resulting in increased cell proliferation (Fig. 4B), which is another characteristic of tumor aggressiveness. Finally, the fact that depletion of Migfilin results in elevated ERK1/2 expression levels (Fig. 3(A) and (C)) along with increased cell adhesion in HepG2 cells (Fig. 4C) also indicates an
involvement of Migfilin in the aggressiveness of HepG2 cells. Moreover, the fact that Migfilin interacts with VASP at cell-ECM adhesion sites and enhances cell-ECM adhesion further explains the observed suppression in cell invasion (Fig. 4D), as it has been previously shown that increased adhesion is associated with decreased migration and invasion [7]. Notably, the fact that elimination of Migfilin severely impairs HepG2 cell invasion, while at the same time increases cell proliferation, both through elevation of ERK1/2 (Fig. 3(A) and (C)) and phosphor-β-catenin (Fig. 2(C) and (D)), indicates that Migfilin has a dual function in HCC cells activating different signaling pathways (Fig. 5) perhaps through interaction with different binding partners. This could provide an explanation as to why its role in cancer is not strictly defined but greatly depends upon the type of cancer, as mentioned above. This is the first study to examine the expression and role of Migfilin in two human HCC cell lines with different invasiveness. Our findings demonstrate for the first time that Migfilin is associated with a more aggressive HCC phenotype and we also provide solid evidence for the molecular mechanism involved. A diagrammatic representation of the molecular mechanisms being activated following Migfilin silencing in HepG2 highly invasive cells is shown in Fig. 5. Finally, this study provides the first line of evidence showing that Migfilin could be a potential therapeutic target against HCC metastasis. However, its evaluation as a therapeutic target should
Please cite this article as: V. Gkretsi, D.P. Bogdanos, Experimental evidence of Migfilin as a new therapeutic target of hepatocellular carcinoma metastasis, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.03.002
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Fig. 5 – Diagrammatic representation of the molecular mechanism that defines the role of Migfilin in HCC. Migfilin silencing regulates three main cellular functions: (a) reduces cell invasion through upregulation of Vasodilator Stimulated Phosphoprotein, phopsho-Vasolidator Stimulated Phosphoprotein (Ser157), phosphor-Vasodilator Stimulated Phosphoprotein (Ser 239), Rho kinase-1 and Fascin-1, all of which lead to increased actin polymerization and thus invasion and migration inhibition, (b) increases proliferation, through phosphor-Akt (Ser 473) and phosphor-β-catenin (Ser33/37/Thr41), and (c) increases cell adhesion through Extracellular Signal-regulated kinase1/2 upregulation, which is also known to lead to decreased migration and invasion. Bold red arrows indicate decrease while bold green arrows indicate increase following Migfilin silencing.
be preceded by a thorough investigation of its expression in human HCC samples and correlation with prognosis and metastatic status to determine its exact role in human HCC metastasis.
Acknowledgements This study was supported by the European Association for the Study of the Liver (EASL) Sheila Sherlock fellowship 2012.We are also grateful to Dr. Chuanyue Wu, Professor at the University of Pittsburgh Medical School, Pittsburgh, PA, USA for providing us with the anti-Migfilin monoclonal antibody.
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Please cite this article as: V. Gkretsi, D.P. Bogdanos, Experimental evidence of Migfilin as a new therapeutic target of hepatocellular carcinoma metastasis, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.03.002