G Model
BCP-11938; No. of Pages 11 Biochemical Pharmacology xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Biochemical Pharmacology journal homepage: www.elsevier.com/locate/biochempharm
Transforming growth factor alpha promotes osteosarcoma metastasis by ICAM-1 and PI3K/Akt signaling pathway Chun-Han Hou a, Feng-Ling Lin b, Kai-Biao Tong c, Sheng-Mon Hou d,*, Ju-Fang Liu e,* a
Department of Orthopedic Surgery, National Taiwan University Hospital, Taipei, Taiwan Department of Dermatology, Sijhih Cathay General Hospital, Taipei, Taiwan c Veterinarian Central Laboratory, Shin-Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan d Department of Orthopedic Surgery, Shin-Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan e Central Laboratory, Shin-Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 4 December 2013 Accepted 20 March 2014 Available online xxx
Osteosarcoma is the most common primary malignancy of bone and is characterized by a high malignant and metastatic potential. Transforming growth factor alpha (TGF-a) is classified as the EGF (epidermal growth factor)-like family, which is involved in cancer cellular activities such as proliferation, motility, migration, adhesion and invasion abilities. However, the effect of TGF-a on human osteosarcoma is largely unknown. We found that TGF-a increased the cell migration and expression of intercellular adhesion molecule-1 (ICAM-1) in human osteosarcoma cells. Transfection of cells with ICAM-1 siRNA reduced TGF-a-mediated cell migration. We also found that the phosphatidylinositol 30 -kinase (PI3K)/ Akt/NF-kB pathway was activated after TGF-a treatment, and TGF-a-induced expression of ICAM-1 and cell migration was inhibited by the specific inhibitors and siRNAs of PI3K, Akt, and NF-kB cascades. In addition, knockdown of TGF-a expression markedly decreased cell metastasis in vitro and in vivo. Our results indicate that TGF-a/EGFR interaction elicits PI3K and Akt activation, which in turn activates NFkB, resulting in the expression of ICAM-1 and contributing the migration of human osteosarcoma cells. ß 2014 Elsevier Inc. All rights reserved.
Keywords: TGF-a Osteosarcoma Migration
1. Introduction Osteosarcoma is the most common primary bone cancer in adolescents and young adults [1–3]. Approximately 2400 new cases of osteosarcoma are diagnosed per year in the United States [4]. Nearly 2/3 of patients with osteosarcoma can be expected to be cured with routine chemotherapy and surgery, and approximately 1/3 of patients have metastasis detectable at presentation, especially pulmonary metastasis [5]. Unfortunately, the main cause of the death in osteosarcoma is lung metastasis [6]. Hence, chemotherapy is usually employed in an adjuvant situation to improve the prognosis and long-term survival. Therefore, the novel treatment strategies that would effectively inhibit metastasis, especially to the lung, from the primary osteosarcoma site are highly desirable. Tumor invasion and metastasis are the complicated process, which is influenced by multiple regulatory genes [7,8]. However,
* Corresponding authors. E-mail addresses: [email protected] (S.-M. Hou), [email protected], [email protected] (J.-F. Liu).
metastatic cascade involves the reducing of the cell–cell adhesion, the disruption of the extracellular matrix (ECM), and the rearrangement of the cytoskeletons, eventually tumor cells separate from the primary lesion and slowly proliferate at distant organs [7–13]. The cell adhesion molecules (CAMs), for example the integrin, immunoglobulin, selectins, and cadherin superfamilies, play an important role in regulating cell–cell and cell– matrix adhesion [14–16]. Intercellular adhesion molecule-1 (ICAM-1, also called CD54) is important in inflammation, immune responses, and intracellular signaling [17]. Moreover, many growth factors such as interlukin-1b (IL-1b), interlukin-6 (IL-6), and tumor necrosis factor alpha (TNF-a), could induce ICAM-1 expression [18,19]. It has been reported that ICAM-1 expression is associated with aggressive neoplasm such as breast cancer, lung cancer, gastric cancer, colorectal cancer, and melanoma cancer [20–25], and further, the up-regulation expression of the ICAM-1 can facilitate cancer cell invasion [24]. Therefore, decreased or disrupted expression of the ICAM-1 may prevent metastasis. Transforming growth factor-alpha (TGF-a), a member of the epidermal growth factors (EGF), exhibits multiple biological activities among various types of tissues and cells. TGF-a has been shown to stimulate cell growth in some cancer types [26–29].
http://dx.doi.org/10.1016/j.bcp.2014.03.010 0006-2952/ß 2014 Elsevier Inc. All rights reserved.
Please cite this article in press as: Hou C-H, et al. Transforming growth factor alpha promotes osteosarcoma metastasis by ICAM-1 and PI3K/Akt signaling pathway. Biochem Pharmacol (2014), http://dx.doi.org/10.1016/j.bcp.2014.03.010
G Model
BCP-11938; No. of Pages 11 2
C.-H. Hou et al. / Biochemical Pharmacology xxx (2014) xxx–xxx
On the other hand, it has been reported that high TGF-a expression is found in variety of malignant tumors, including kidney cancer, pancreatic cancer, colon cancer, and breast cancer [30–33]. Furthermore, it has been demonstrated that TGF-a is overexpressed in liver cancer and promotes carcinogenesis through TGF-a-EGFR-RAS-MAPK signaling pathway [34–39]. In ovarian cancer, TGF-a regulates cell growth by ERK and PI3K/Akt signaling pathways. On the other hand, TGF-a-induced cell migration is associated with PLCg pathway [29]. TGF-a is significantly correlated with the development and progression of pancreatic cancer and liver cancer [40,41]. Previous studies have shown that TGF-a regulates cell migration and invasion in human cancer cells [29,42–44]. However, the effect of TGF-a expression in human osteosarcoma cells is rarely reported. In this investigation, we demonstrated that TGF-a and EGFR interaction increased the migration and expression of ICAM-1 in human osteosarcoma cells. In addition, phosphatidylinositol 3kinase (PI3K)/Akt and NF-kB signaling pathways were involved. The elucidation of this TGF-a-responsive signaling pathway provides valuable clues to understand the mechanisms of human osteosarcoma metastasis and offered the opportunity to develop more effective clinical therapies in the future. 2. Materials and methods
temperature for the expression of the large T antigen. All experiments with hFOB 1.19 cells were carried out at the permissive temperature of 33.5 8C. 2.3. Migration assay The migration assay was performed using Transwell inserts (Costar, NY; 8 mm pore size) in 24-well dishes. Before performing the migration assay, cells were pretreated for 30 min with different concentrations of inhibitors (PD158780, BIBX1382, LY294002, Wortmannin, Akti, PDTC or TPCK) or vehicle control (0.1% dimethyl sulfoxide, DMSO). Approximately 1 104 cells in 200 mL of serumfree medium were placed in the upper chamber, and 300 mL of the same medium containing different concentrations of TGF-a were placed in the lower chamber. The cells were incubated for 24 h at 37 8C in 5% CO2, then fixed in 3.7% formaldehyde for 15 min and stained with 0.05% crystal violet in phosphate-buffered saline (PBS) for 60 min. Cells on the upper side of the filters were removed with cotton-tipped swabs and the filters were washed with PBS. Cells on the underside of the filters were examined and counted under a microscope. Each experiment was performed in triplicate and repeated at least three times. The number of migrating cells in each experiment was corrected for proliferation effects using a cell viability assay (corrected migrating cell number 5 counted migrating cell number/percentage of viable cells) [45].
2.1. Materials 2.4. Wound-healing migration assay Protein A/G beads, anti-mouse and anti-rabbit IgG conjugated horseradish peroxidase, polyclonal antibodies specific for PI3K, Akt, IKKa/b, IkB, p65, EGFR, ICAM-1, VCAM-1 and b-Actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Polyclonal antibody specific for EGFR phosphorylated at PY1068 and PY992, PI3K phosphorylated at Tyr458/199, Akt phosphorylated at Ser473, IKKa/b phosphorylated at Ser176/180, IkBa phosphorylated at Ser32/36 and p65 phosphorylated at Ser536 were purchased from Cell Signaling and Neuroscience (Danvers, MA, USA). PD158780, BIBX1382, Pyrrolidine-dithiocarbamate (PDTC), L-1-tosylamido-2phenylenylethyl chloromethyl ketone (TPCK), LY294002, Wortmannin, and Akti were purchased from Calbiochem (San Diego, CA, USA). The recombinant human TGF-a was purchased from PeproTech (Rocky Hill, NJ, USA). The NF-kB luciferase plasmid was purchased from Stratagene (La Jolla, CA, USA). The p85 and Akt (Akt K179A) dominant-negative mutants were gifts from Dr. W. M. Fu (National Taiwan University, Taipei, Taiwan). The pSV-bgalactosidase vector and the luciferase assay kit were purchased from Promega (Madison, MA, USA). The human siRNAs for control, ICAM-1 and EGFR were as pools sequences and were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All other chemicals were purchased from Sigma–Aldrich (St. Louis, MO, USA).
For the wound-healing migration assay, cells were seeded on 12-well plates at a density of 1 105 cells/well in culture medium. At 24 h after seeding, the confluent monolayer of culture was scratched with a fine pipette tip, and migration was visualized by microscope and magnification. The rate of wound closure was observed at the indicated time [46]. 2.5. Western blotting analysis The cellular lysates were prepared as described previously [47,48]. Proteins were resolved on SDS–PAGE and transferred to Immobilon polyvinyldifluoride (PVDF) membranes. The blots were blocked with 5% BSA for 1 h at room temperature and then probed with rabbit anti-human antibodies against PI3K, Akt, IKKa/b, IkB, p65, EGFR, ICAM-1 and VCAM-1 (1:1000) for 1 h at room temperature. After being washed three times in TBST, the blots were subsequently incubated with a donkey anti-rabbit peroxidaseconjugated secondary antibody (1:1000) for 1 h at room temperature. The blots were visualized by enhanced chemiluminescence using Kodak X-OMAT LS film (Eastman Kodak, Rochester, NY). Quantitative data were obtained using a computing densitometer and Image Quant software (Molecular Dynamics, Sunnyvale, CA, USA).
2.2. Cell culture 2.6. Quantitative real time PCR The human osteosarcoma cell lines (U2OS and MG63) and human fetal osteoblastic cell line (hFOB 1.19) were purchased from the American Type Cell Culture Collection (Manassas, VA, USA). MG63 cells were maintained at 37 8C with 5% CO2 and cultured in DMEM supplemented with 20 mM HEPES, 10% heat-inactivated FBS, 2 mM-glutamine and antibiotics (100 U/mL of penicillin and 100 mg/mL of streptomycin). U2OS cells were maintained at 37 8C with 5% CO2 and culture in McCoy’s 5A medium supplemented with 10% heat-inactivated FBS, 1.5 mM L-glutamine and antibiotics. hFOB 1.19 cells were maintained in 1:1 mixture of phenolfree DMEM/Ham’s F12 medium (GIBCO-BRL, Gaithersburg, MD, USA) containing 10% FBS supplemented with 2.5 mM L-glutamine, 0.3 mg/mL G418 and antibiotics at 33.5 8C, the permissive
Total RNA was extracted from osteosarcoma cells by using a TRIzol kit (MDBio Inc., Taipei, Taiwan). Two mg of total RNA was reverse transcribed into cDNA by using oligo (dT) primer [49,50]. The quantitative real time PCR (qPCR) analysis was carried out using Taqman1 one-step PCR Master Mix (Applied Biosystems, CA, USA). Two mg of cDNA were added per 25-mL reaction with sequence-specific primers and Taqman1 probes. Sequences for all target gene primers and probes were purchased commercially (GAPDH was used as internal control) (Applied Biosystems, CA, USA). qPCR assays were carried out in triplicate on an Step One Plus sequence detection system. The cycling conditions were 10-min polymerase activation at 95 8C followed by 40 cycles at 95 8C for
Please cite this article in press as: Hou C-H, et al. Transforming growth factor alpha promotes osteosarcoma metastasis by ICAM-1 and PI3K/Akt signaling pathway. Biochem Pharmacol (2014), http://dx.doi.org/10.1016/j.bcp.2014.03.010
G Model
BCP-11938; No. of Pages 11 C.-H. Hou et al. / Biochemical Pharmacology xxx (2014) xxx–xxx
15 s and 60 8C for 60 s. The threshold was set above the nontemplate control background and within the linear phase of target gene amplification to calculate the cycle number at which the transcript was detected (denoted CT). 2.7. Transfection and reporter gene assay Human osteosarcoma cells were co-transfected with 0.8 mg kB driven luciferase plasmid, 0.4 mg b-galactosidase expression vector. Cells were grown to 80% confluence in 12 well plates and were transfected on the following day by Lipofectamine 2000 (LF2000; Invitrogen). DNA and LF2000 were premixed for 30 min and then applied to the cells (the transfection efficiency is more than 45% confirmed by transfection with GFP-expressing plasmid). DMEM containing 20% FBS was added 4 h later. After 24 h of transfection, the cells were incubated with the indicated agents. After further 24 h incubation, the media were removed, and cells were washed once with cold PBS. To prepare lysates, 100 mL reporter lysis buffer (Promega, Madison, WI) were added to each well, and cells were scraped from dishes. The supernatant was collected after centrifugation at 11,000 g for 2 min. Aliquots of cell lysates (20 mL) containing equal amounts of protein (20–30 mg) were placed into wells of an opaque black 96-well microplate. An equal volume of luciferase substrate was added to all samples, and luminescence was measured in a microplate luminometer. The value of luciferase activity was normalized to transfection efficiency monitored by the co-transfected b-galactosidase expression vector.
3
understand its effect on osteosarcoma cell, we first examined the levels of TGF-a in human fetal osteoblastic cell line (hFOB 1.19) and osteosarcoma cell lines (MG63 and U2OS). TGF-a was significantly elevated in MG63 and U2OS cell lines compared with hFOB 1.19 (Fig. 1A). The TGF-a messenger RNA (mRNA) in MG63 and U2OS cells were also higher than in hFOB 1.19 (Fig. 1B). In addition, MG63 and U2OS cells were more migratory than hFOB 1.19 (Fig. 1C). Moreover, treatment of cells with TGF-a recombinant protein (5–100 ng/mL) dramatically increased migration in MG63 and U2OS cell lines (Fig. 1D). However, TGF-a treatment did not affect cell viability in MG63 and U2OS cells by using MTT assay (data not shown). On the other hand, TGF-a also increased woundhealing activity in human osteosarcoma cells (Fig. 1E). Finally, comparison of EGF and EGFR status between hFOB1.19 and osteosarcoma is important. We examined the levels of EGF and EGFR between hFOB 1.19 and osteosarcoma (MG63 and U2OS). The level of EGF was significantly elevated in MG63 and U2OS cell lines compared with hFOB 1.19 (Fig. 1F). The level of EGFR was significantly elevated in normal osteoblastic cell line compared with malignant cell (Fig. 1G). Furthermore, tumor necrosis factoralpha converting enzyme (TACE/ADAM17) plays an important role in the regulated shedding of EGFR ligands [52]. We examined the level of TACE between hFOB 1.19 and osteosarcoma (MG63 and U2OS). The level of TACE was significantly elevated in MG63 and U2OS cell lines compared with hFOB 1.19 (Fig. 1F). These results indicate that the elevated TGF-a in metastatic osteosarcoma cells increased their cell migration in vitro. 3.2. ICAM-1 is involved in TGF-a-induced cell migration
2.8. Establishment of stably transfected cells TGF-a shRNA or control shRNA plasmids were transfected into cancer cells with Lipofetamine 2000 transfection reagent. Twentyfour hours after transfection, stable transfectants were selected in puromycin at a concentration of 10 mg/mL. Thereafter, the selection medium was replaced every 3 days. After 2 weeks of selection in puromycin (10 mg/mL), clones of resistant cells were isolated. 2.9. In vivo tumor xenograft study All animal experiments were performed in accordance with a protocol approved by the Shin-Kong Wu Ho-Su Memorial Hospital (Taipei, Taiwan) institutional animal care and use committees. Male CB17-SCID mice (4 weeks old) were used. For experimental metastasis assays, 5 106 cells were resuspended in 0.1 mL of saline and injected into the tail vein. After 4 weeks, the mice were euthanized by an overdose of the anesthetic agent. The lungs were removed and fixed in 10% formalin. The number of lung tumor metastases was counted under a dissecting microscope.
ICAM-1 plays a key role in cancer cell migration and invasion [53,54]. However, the expression of ICAM-1 in human osteosarcoma cells is largely unknown. First, we examined hFOB 1.19 and osteosarcoma cells for the expression of ICAM-1 by using western blot and qPCR. Expression levels of ICAM-1 in osteosarcoma cells were significantly higher than in hFOB 1.19 (Fig. 2A and B). Next, to examine whether ICAM-1 is involved in TGF-a-induced migration of osteosarcoma cells, we measured the level of ICAM-1 mRNA and protein in TGF-a treated osteosarcoma cells. Indeed, TGF-a increased ICAM-1 mRNA expression and protein level in a dosedependent and time-dependent manner, but TGF-a treatment did not have any effect on the mRNA or protein level of VCAM-1 (Fig. 2C–F). To confirm this finding, MG63 and U2OS cells were transfected with control and ICAM-1 small interfering RNA (siRNA) for 24 h, and the western blot analysis showed that the expression of protein levels of ICAM-1 was suppressed by transfection with ICAM-1 siRNA (Fig. 2G). Transfected cells with ICAM-1 siRNA markedly reduced cell migration and inhibited TGF-a-induced cell migration (Fig. 2H and I). These data clearly showed that TGF-ainduced cancer cell migration may occur via activation of ICAM-1.
2.10. Statistical analysis Data are presented as mean standard error of the mean (SEM). Statistical analysis between two samples was performed using the Student’s t-test. Statistical comparisons of more than two groups were performed using one-way analysis of variance with Bonferroni’s post hoc test. In all cases, a p < 0.05 was considered to be statistically significant and the experiments were repeated four times. 3. Results 3.1. TGF-a increases the migration and expression of ICAM-1 in human osteosarcoma cells TGF-a has been shown to increase cell migration and metastasis in a variety of human cancer cells [29,51]. To
3.3. Involvement of EGFR in TGF-a-mediated migration of osteosarcoma Previous study has shown that TGF-a affects cellular function by specifically bind with EGFR and then activates EGFR [55,56]. Therefore, we next examined whether EGFR was involved in TGFa-mediated cell migration in human osteosarcoma cells. We hypothesized that EGFR signaling pathway may be involved in TGF-a-induced osteosarcoma cell migration. Pretreatment of cells with EGFR tyrosine kinase inhibitors: BIBX1382 (10 mM) and PD158780 (5 mM) for 30 min markedly inhibited TGF-a-induced cancer cell migration, ICAM-1 mRNA expression and ICAM-1 protein production (Fig. 3A–C). Additionally, Stimulation of cells with TGF-a led to a significant increase in phosphorylation of EGFR (Fig. 3D). Following transfection of cells with control and EGFR
Please cite this article in press as: Hou C-H, et al. Transforming growth factor alpha promotes osteosarcoma metastasis by ICAM-1 and PI3K/Akt signaling pathway. Biochem Pharmacol (2014), http://dx.doi.org/10.1016/j.bcp.2014.03.010
G Model
BCP-11938; No. of Pages 11 4
C.-H. Hou et al. / Biochemical Pharmacology xxx (2014) xxx–xxx
Fig. 1. TGF-a induces cell migration of human osteosarcoma cells. (A and B) Total protein and mRNA were extracted form hFOB 1.19, MG63, and U2OS cells, the TGF-a expression was examined by western blot analysis and qPCR. (C) In vitro migration activity of hFOB 1.19, MG63, and U2OS cells was measured using the Transwell migration assay. (D and E) Cells were incubated with various concentrations of TGF-a for 16 h, and in vitro migration was measured using the Transwell migration assay and Woundhealing assay (F and G) Total protein and mRNA were extracted form hFOB 1.19, MG63, and U2OS cells, the EGF, EGFR and TACE expression were examined by western blot analysis and qPCR. Results are expressed as the mean SEM. *, p < 0.05 compared with control.
siRNA, the expression of EGFR is reduced (Fig. 3E), and also inhibited the migration activity and ICAM-1 mRNA expression of osteosarcoma cells (Fig. 3F and G). On the other hand, we hypothesis that used EGFR inhibitor alone or combined with cancer chemotherapy drugs (e.g. Cisplatin and Doxorubicin) could abolish TGF-a effects. First, we found that Cisplatin and Doxorubicin in low dose did not affect cell survival (Fig. 3H and I). Next, pretreatment of cells with EGFR inhibitors alone, cancer chemotherapy drugs alone or EGFR inhibitor combined with cancer chemotherapy drug for 30 min, followed by treatment with TGF-a for 24 h, we found EGFR inhibitor combined with cancer chemotherapy drug markedly inhibited TGF-a-induced cancer cell migration (Fig. 3J). These data suggested that TGF-a/EGFR interaction played a key role in the cell migration and ICAM-1 expression of osteosarcoma. 3.4. PI3K and Akt signaling pathways are involved in TGF-a-mediated ICAM-1 up-regulation and cell migration of osteosarcoma cells The PI3K-Akt pathway is activated by EGFR [57]. We examined whether TGF-a enhanced the activation of PI3K. TGF-a-induced
cell migration and ICAM-1 expression of osteosarcoma cells were greatly reduced by treatment with PI3K inhibitors Ly294002 and Wortmannin (Fig. 4A–C). On the other hand, stimulation of cells with TGF-a led to a significant increasing in phosphorylation of p85 (Fig. 4D). In addition, transfection of cells with pI3K mutant also inhibited TGF-a induced cell migration and ICAM-1 expression of osteosarcoma cells (Fig. 4E and F). In order to explore whether TGF-a-induced PI3K signaling is mediated by EGFR activation; we used EGFR inhibitors PD158780 and BIBX1382. Pretreated cells with PD158780 and BIBX1382 for 30 min, and then treated TGF-a for 15 min. Western blot analysis indicated that both PD158780 and BIBX1382 significantly inhibited TGF-ainduced PI3K activation (Fig. 4G). The Akt Ser473 residue phosphorylation by a PI3K-dependent signaling pathway causes enzymatic activation [58]. To examine the important role of PI3K/Akt in cancer migration and ICAM-1 upregulation, we next determined Akt Ser473 phosphorylation in response to TGF-a treatment. Treatment of cells with TGF-a resulted in time-dependent phosphorylation of Akt Ser473 (Fig. 5A). Pretreatment of cells with Akti antagonized TGF-ainduced cell migration and ICAM-1 expression of osteosarcoma
Please cite this article in press as: Hou C-H, et al. Transforming growth factor alpha promotes osteosarcoma metastasis by ICAM-1 and PI3K/Akt signaling pathway. Biochem Pharmacol (2014), http://dx.doi.org/10.1016/j.bcp.2014.03.010
G Model
BCP-11938; No. of Pages 11 C.-H. Hou et al. / Biochemical Pharmacology xxx (2014) xxx–xxx
5
Fig. 2. TGF-a directed cell migration of human osteosarcoma cells involves upregulation of ICAM-1. (A and B) Total protein and mRNA were extracted form hFOB 1.19, MG63, and U2OS cells, the ICAM-1 expression was examined by western blot and qPCR analysis. (C and D) Cells were incubated with various concentrations of TGF-a for 24 h. The mRNA and protein expression of ICAM-1 were measured by qPCR and western blot. (E and F) Cells were incubated with TGF-a (10 ng/mL) for 6, 12 or 24 h. The mRNA and protein expression of ICAM-1 were measured by qPCR and western blot. (G and H) MG63 and U2OS cells were transfected with ICAM-1 or negative control siRNA for 24 h, the cell migration was analyzed using Transwell migration assay. (I) Cells were transfected with ICAM-1 or control siRNA for 24 h, followed by treatment with TGF-a for 24 h, and cell migration was analyzed using the Transwell migration assay. Results are expressed as the mean SEM. *, p < 0.05 compared with control; #, p < 0.05 compared with TGFa-treated group.
cells (Fig. 5B–D). On the other hand, transfection of cells with Akt mutant also inhibited TGF-a-induced cell migration and ICAM-1 expression of osteosarcoma cells (Fig. 5E and F). Based on these results, it appeared that TGF-a/EGFR can enhance ICAM-1 expression and cell migration through the PI3K and Akt-dependent signaling pathway to in human osteosarcoma cells. 3.5. NF-kB is involved in TGF-a-induced cell migration and ICAM-1 expression As previously mentioned, NF-kB is an important transcription factor whose activity is often correlated with cancer cell migration and invasion [59]. Therefore, we examined whether NF-kB activation was involved in the signal transduction pathway which leading TGF-a-promoted cell migration and ICAM-1 expression. Pretreatment of cells with the NF-kB inhibitor PDTC (10 mM) and the IkB protease inhibitor TPCK (10 mM) for 30 min markedly
inhibited TGF-a-induced cancer cell migration, ICAM-1 mRNA expression and ICAM-1 protein production (Fig. 6A–C). We next examined the levels of IKKa/b, IkBa and, NF-kB subunit p65 expression. The expression of phosphorylated IKKa/b, IkBa and NF-kB subunit p65 were higher after 30 min of TGF-a treatment and diminished after 60 min (Fig. 6D). Furthermore, Dominantnegative mutant of IKKa or IKKb markedly inhibited TGF-ainduced cell migration, ICAM-1 mRNA expression and ICAM-1 protein production (Fig. 6E and F). In addition, Pretreatment of cells with Ly294002, Wortmannin, or Akti reduced TGF-a-increased IKKa/b, IkB and p65 phosphorylation (Fig. 6G). Therefore, TGF-ainduced cell migration and ICAM-1 expression were mediated by the NF-kB signaling pathway. Next, we directly determined kB-luciferase activation after TGF-a treatment, the cells were transiently transfected with kBluciferase plasmid as an indicator of NF-kB activation. As shown in Fig. 6H, treated cells with TGF-a for 24 h resulted in increased
Please cite this article in press as: Hou C-H, et al. Transforming growth factor alpha promotes osteosarcoma metastasis by ICAM-1 and PI3K/Akt signaling pathway. Biochem Pharmacol (2014), http://dx.doi.org/10.1016/j.bcp.2014.03.010
G Model
BCP-11938; No. of Pages 11 6
C.-H. Hou et al. / Biochemical Pharmacology xxx (2014) xxx–xxx
Fig. 3. EGFR is involved in TGF-a-mediated migration of human osteosarcoma cells. (A) Cells were pretreated for 30 min with PD158780 (5 mM) or BIBX1382 (10 mM) followed by stimulation with TGF-a (10 ng/mL) for 24 h. In vitro migration was measured after 24 h using the Transwell migration assay. (B and C) Cells were pretreated for 30 min with PD158780 (5 mM) or BIBX1382 (10 mM) followed by stimulation with TGF-a (10 ng/mL) for 24 h. The mRNA and protein expression of ICAM-1 were measured by qPCR and Western blotting. (D) Cells were incubated with TGF-a (10 ng/mL) for indicated time intervals, and EGFR phosphorylation was examined by western blot. (E and F) Cells were transfected with EGFR siRNA for 24 h, followed by treatment with TGF-a (10 ng/mL) for 24 h, and cell migration was analyzed using the Transwell migration assay. (G) Cells were transfected with control and EGFR siRNA for 24 h, the ICAM-1 expression was examined by qPCR. (H-I) MG63 and U2OS cells were incubated with various concentrations of Cisplatin and Doxorubicin for 24 h, and the cell viability was examined by MTT assay. (J) Cells were pretreated for 30 min with PD158780 (5 mM), BIBX1382 (10 mM), Cisplatin (50 nM) or Doxorubicin (50 nM) followed by stimulation with TGF-a (10 ng/mL) for 24 h. In vitro migration was measured after 24 h using the Transwell migration assay. Results are expressed as the mean SEM. *, p < 0.05 compared with control; #, p < 0.05 compared with TGF-a-treated group.
kB-luciferase activity. (TNF-a acts as a positive control). In addition, BIBX1382, PD158780, LY294002, Wortmannin, Akti, PDTC and TPCK antagonized the TGF-a-induced kB-luciferase activity. Co-transfection of cells with PI3K, Akt, IKKa and IKKb mutant also reduced TGF-a-increased kB-luciferase activity (Fig. 6I). Based on these results, activation of the PI3K and Akt signaling pathways were required for the TGF-a-induced NF-kB activity in human osteosarcoma cells. 3.6. Knockdown of TGF-a expression inhibits cell migration in a mouse model of osteosarcoma To confirm that TGF-a mediated ICAM-1-dependent cell migration in human osteosarcoma cells, we took advantage of MG63 cells that stably expressed TGF-a shRNA. Following puromycin (10 mg/mL) selection, individual stable clones (TGF-a sh1, TGF-a sh2, TGF-a sh3 and TGF-a sh4) were collected for analysis. Empty vector plasmid was used as a negative control. The
expression levels of both TGF-a and ICAM-1 were decreased in all four TGF-a shRNA stable clones, and TGF-a sh4 showed the most knockdown efficiency (Fig. 7A). TGF-a knockdown did not affect the rate of cell proliferation (Fig. 7B), but significantly reduced cell migration (Fig. 7C). To further confirm that TGF-a mediated ICAM1-dependent cell migration in human osteosarcoma cells by EGFRPI3K-Akt pathway, the expression levels of EGFR, PI3K and Akt were decreased in TGF-a sh4 cells (Fig. 7D). Furthermore, to examine the effect of TGF-a on osteosarcoma growth in vivo, mouse tail vein was injected with TGF-a-knockdown cell (5 106), then sacrificed after 28 days later, and the results showed lung metastatic nodules. Furthermore, to investigate the metastatic potential of Control-shRNA and TGF-a-shRNA cells, we counted the number of metastatic nodules in tumor-bearing mice. The mice which were injected with TGF-a shRNA4 cells showed significantly decreased in mean number of lung metastatic nodules (Fig. 7E–G). Therefore, knockdown of TGF-a reduced cell migration in vitro and lung metastasis in vivo.
Please cite this article in press as: Hou C-H, et al. Transforming growth factor alpha promotes osteosarcoma metastasis by ICAM-1 and PI3K/Akt signaling pathway. Biochem Pharmacol (2014), http://dx.doi.org/10.1016/j.bcp.2014.03.010
G Model
BCP-11938; No. of Pages 11 C.-H. Hou et al. / Biochemical Pharmacology xxx (2014) xxx–xxx
7
Fig. 4. PI3K signaling affects the TGF-a response in human osteosarcoma. (A–C) Cells were pretreated with the control (0.1% DMSO), LY294002 (10 mM) and Wortmannin (1 mM) for 30 min followed by stimulation with TGF-a for 24 h the cell migration and ICAM-1 expression were examined by Transwell, qPCR and western blot. (D) Cells were incubated with TGF-a (10 ng/mL) for the indicated times, and PI3K phosphorylation was examined by western blot. (E and F) Cells were transfected with control and PI3K mutant for 24 h followed by stimulation with TGF-a for 24 h the cell migration and ICAM-1 expression were examined by Transwell migration and qPCR assay. (G) Cells were pretreated with the control (0.1% DMSO) and EGFR inhibitor PD158780 (5 mM) or BIBX1382 (10 mM) followed by stimulation with TGF-a for 15 min, and PI3K phosphorylation was examined by western blot. Results are expressed as the mean SEM. *, p < 0.05 compared with control; #, p < 0.05 compared with TGF-a-treated group.
Fig. 5. Akt is involved in TGF-a-mediated migration in human osteosarcoma cells. (A) Cells were incubated with TGF-a (10 ng/mL) for the indicated times and p-Akt or Akt levels were determined by western blot. (B–D) Cells were pretreated for 30 min with the Akti (1 mM) followed by treatment with TGF-a for 24 h, the cell migration and ICAM1 expression was examined by Transwell migration, qPCR and western blot. (E and F) Cells were transfected with Akt mutant for 24 h followed by treatment with TGF-a for 24 h, the cell migration and ICAM-1 expression was examined by Transwell migration and qPCR assay. Results are expressed as the mean SEM. *, p < 0.05 compared with control; #, p < 0.05 compared with TGF-a-treated group.
Please cite this article in press as: Hou C-H, et al. Transforming growth factor alpha promotes osteosarcoma metastasis by ICAM-1 and PI3K/Akt signaling pathway. Biochem Pharmacol (2014), http://dx.doi.org/10.1016/j.bcp.2014.03.010
G Model
BCP-11938; No. of Pages 11 8
C.-H. Hou et al. / Biochemical Pharmacology xxx (2014) xxx–xxx
Fig. 6. NF-kB mediates the response of human osteosarcoma cells to TGF-a stimulation. (A–C) Cells were pretreated with the control (0.1% DMSO), PDTC (10 mM) and TPCK (10 mM) for 30 min followed by stimulation with TGF-a for 24 h the cell migration and ICAM-1 expression was examined by Transwell migration, qPCR and western blot. (D) Cells were incubated with TGF-a (10 ng/mL) for the indicated times, and IKKa/b, IkBa, and p65 phosphorylation was examined by western blot. (E and F) Cells were transfected with control and IKKa and IKKb mutant for 24 h followed by stimulation with TGF-a (10 ng/mL) for 24 h the cell migration and ICAM-1 expression was examined by Transwell migration and qPCR assay. (G) Cells were pretreated with the control (0.1% DMSO), LY294002 (10 mM), Wortmannin (1 mM) and Akti (1 mM) followed by stimulation with TGF-a (10 ng/mL) for 30 min, and IKKa/b, IkBa, and p65 phosphorylation was examined by western blot. (H and I) Cells were transfected with an NF-kB promoter reporter plasmid for 24 h, then incubated with TGF-a for 24 h and luciferase activity was measured. Results are expressed as the mean SEM. *, p < 0.05 compared with control; #, p < 0.05 compared with TGF-a-treated group.
4. Discussion Osteosarcoma is the most common bone cancer, particularly among children and adolescents, with chronic symptoms of bone pain and swelling in leg or arm [60]. Osteosarcoma has a high propensity for local aggression and a tendency to metastasize to the lung and distant bones, which is the leading cause of mortality. Therefore, it is important to develop effective adjuvant therapy for preventing osteosarcoma metastasis. Here, we found that the expression of TGF-a correlated strongly with the tumor stage in human osteosarcoma patients. We also found that TGF-a increased cell migration both in human osteosarcoma cell lines
and in vivo models. One mechanism underlying TGF-a-directed migration was the transcriptional up-regulation of ICAM-1 and the activation of the EGFR, PI3K, Akt and NF-kB pathways. The ligands of EGFR, comprising 6 members (EGF, TGF-a, heparin-binding EGF-like growth factor (HB-EGF), amphiregulin (AR), epiregulin (EREG), and betacellulin (BTC)) are involved in the stimulation of cell proliferation, inflammation, migration, metastasis, angiogenesis and tumorigenesis [61–63]. TGF-a has been demonstrated capacity to promote tumor development, progression, invasion in the breast, lung, colon, ovary, prostate, and skin cancers [29,64]. In addition, TGF-a is believed to stimulate cell motility and promote aggressive behavior. However, a correlation
Please cite this article in press as: Hou C-H, et al. Transforming growth factor alpha promotes osteosarcoma metastasis by ICAM-1 and PI3K/Akt signaling pathway. Biochem Pharmacol (2014), http://dx.doi.org/10.1016/j.bcp.2014.03.010
G Model
BCP-11938; No. of Pages 11 C.-H. Hou et al. / Biochemical Pharmacology xxx (2014) xxx–xxx
9
Fig. 7. Knockdown of TGF-a reduces the migratory ability in vitro and in vivo. (A) The protein levels of TGF-a and ICAM-1 in control-shRNA and TGF-a-shRNA cells was examined by western blot. (B) MG63 cells stably expressing shRNA constructs were seeded as monolayers and counted daily. Cells (1 104) were reseeded after each count, and the cell numbers were plotted. (C) In vitro migration of MG-63 cells stably expressing shRNA constructs was measured using the Transwell migration assay. (D) The protein levels of p-EGFR, EGFR, p-PI3K, PI3K, p-Akt and Akt in control-shRNA and TGF-a-shRNA cells was examined by western blot. (E) MG63 cells were injected into the mouse tail vein. Mice were sacrificed after 28 days later developed lung metastasis nodules. Lungs were removed and inflated with 10% formalin, and the number of metastatic nodules on the lungs was counted with the aid of a dissecting microscope. (F) H&E staining of metastatic nodules in the lung of mice. (G) Quantification of metastatic nodules following tail vein injection. Results are expressed as mean SEM. #, p < 0.05 compared with control.
between osteosarcoma and TGF-a still remains unclear. Here, we found that the expression of TGF-a in osteosarcoma cell lines were significantly higher than that in the normal bone cell line. Treatment with exogenous TGF-a increased the migration of osteosarcoma cells. In contrast, over-expression of TGF-a shRNA inhibited the migratory ability. We also observed reduced effects with increasing concentration of TGF-a, we consider the cell surface has a limited number of receptor binding sites; therefore, it is saturated at high ligand concentration. The concentration of bound ligand versus that of total ligand is curvilinear. When all the binding sites are occupied, there is no further binding as increasing ligand concentration. Another reason is that the relationship between ligand concentration at the receptor and the response might be described with a sigmoid curve. As a result, reduced effects with increasing conc. of TGF-a were observed. Taken together, these data suggest that TGF-a is a novel marker for cancer progression and metastasis of bone sarcomas. ICAM-1 plays a pivotal role in leukocyte adhesion and cancer cell invasion [65,66]. In breast cancer, ICAM-1 can mediate adhesion-dependent cellular interactions and has a potential role in invasion. Moreover, its high expression induced tumor malignancy and poor prognosis [67]. A correlation between ICAM-1 and breast cancer has been reported, but the biological functions between ICAM-1 and osteosarcoma is unclear. Here, we show that ICAM-1 plays an important role in TGF-a-mediated osteosarcoma cell motility. First, TGF-a increased ICAM-1 mRNA and protein expression. Second, TGF-a-induced cell migration was inhibited by specific ICAM-1 siRNA. Third, TGF-a-shRNA cells showed a greater reduction in migration and ICAM-1 expression than control-shRNA cells. We provide evidence here that ICAM-1 is required for osteosarcoma metastasis. Although ICAM-1 was
reportedly associated with the cell migratory of osteosarcoma, we demonstrated that ICAM-1 is a downstream effector in TGF-aincreased metastasis of osteosarcoma. It has been reported that activation of EGFR and subsequent enhancement of PI3K/Akt activation mediated the signaling in response to TGF-a [68]. EGFR family plays an important role in inducing cell proliferation, survival, migration, and invasion [69,70]. In this study, we found that EGFR siRNA reduced TGFa-induced cell migration, through the blocking of TGF-a-increased ICAM-1 expression. Therefore, the interaction between TGF-a and EGFR is very important for cancer migration and ICAM-1 expression in human osteosarcoma cells. Recent studies indicate that overexpression of EGFR could promote cancer cell survival, proliferation, migration and invasion through up-regulated PI3K and Akt signaling pathway [71–73]. PI3K is a heterodimer consisting of an 85 kDa regulatory subunit and an 110 kDa catalytic subunit. The autophosphorylation of EGFR tyrosine residues can elicit PI3K and Akt signaling pathway through the interaction between the phosphotyrosine-binding SH2 domains on p85 subunit and tyrosine residues [74]. Phosphorylation of the p85 subunit is required for activation of the p110 catalytic subunit of PI3K [75]. We found TGF-a enhanced the p85 subunit and Akt phosphorylation in human osteosarcoma cells. Pretreatment of cells with PI3K and Akt inhibitor or transfection of cells with PI3K and Akt mutation plasmids could reduce the TGF-a-mediated ICAM-1 expression. These results suggest that TGF-a induced ICAM-1 production through EGFR activation and via the PI3K/Akt signaling pathway in osteosarcoma. The transcriptional factor NF-kB is associated with cancer cell invasion and migration [76]. Occasionally, NF-kB transcription factors exist in the cytoplasm and bind to the inhibitory protein
Please cite this article in press as: Hou C-H, et al. Transforming growth factor alpha promotes osteosarcoma metastasis by ICAM-1 and PI3K/Akt signaling pathway. Biochem Pharmacol (2014), http://dx.doi.org/10.1016/j.bcp.2014.03.010
G Model
BCP-11938; No. of Pages 11 C.-H. Hou et al. / Biochemical Pharmacology xxx (2014) xxx–xxx
10
IkB, which inhibits NF-kB binding and prevent nuclear uptake. Upon stimulation of cells, IkB proteins lead to rapid phosphorylation by the multisubunit IKK complex, then this targets IkB for ubiquitination and subsequent degradation by the 26S proteasome. The NF-kB pathway has also been linked to TGF-a signaling, and free NF-kB translocate to the nucleus, where it regulates gene transcription [77]. Treatment of cells with TGF-a led to increase levels of phosphorylated IKK, IkBa and p65. Using transient transfection of a kB-luciferase reporter construction that indicates NF-kB activity, we found that TGF-a increased NF-kB activity. Taken together, these results indicate that TGF-a acted through the EGFR, PI3K, Akt and NF-kB pathways to induce ICAM-1 expression in osteosarcoma. Finally, to directly determine the effect of TGF-a on osteosarcoma, we inhibited TGF-a expression in MG63 cells by using shRNA transfection. TGF-a knockdown significantly reduced ICAM-1 expression and inhibited cell migration ability in MG63 cells. These data indicated that TGF-a play an important role in osteosarcoma metastasis. Our study presents that TGF-a and EGFR interaction increases the expression of ICAM-1 via PI3K, Akt and NF-kB dependent pathway and increasing migration of human osteosarcoma cells. To the best of our knowledge, this study is the first time to attempt to examine the migratory activity of TGF-a in human osteosarcoma. Whether TGF-ainduces cell motility through same pathways in clinic should be examined further. Furthermore, the discovery of TGF-a-mediated pathway helps us to understand the mechanism of human osteosarcoma metastasis and may help us to develop effective therapy in the future. Author’s contributions JFL and SMH conceived and designed the experiments. CHH, FLL, KBT and JFL performed the experiments. CHH, KBT, SMH and JFL analyzed the data. CHH, FLL, KBT, SMH and JFL contributed reagents/materials/analysis tools. JFL and SMH wrote the paper. All authors read and approved the final manuscript. Conflicts of interest The authors state no conflict of interest. Acknowledgments This work was supported by grants from the National Science Council of Taiwan (NSC99-2320-B-039-003-MY3; NSC101-2314B-341-001-MY2) and Shin-Kong Wu Ho-Su Memorial Hospital (SKH-8302-102-0301; SKH-8302-102-0302). We thank the staff of the Eighth Core Lab, Department of Medical Research, National Taiwan University Hospital for technical support during the study. We thank Dr. W.M. Fu for kindly providing Akt dominant negative mutant. References [1] Damron TA, Ward WG, Stewart A. Osteosarcoma, chondrosarcoma, and Ewing’s sarcoma: National Cancer Data Base Report. Clin Orthop Relat Res 2007;459:40–7. [2] Dorfman HD, Czerniak B. Bone cancers. Cancer 1995;75:203–10. [3] Sweetnam R. Osteosarcoma. Br J Hosp Med 1982;28:112. 116–121. [4] Ottaviani G, Jaffe N. The epidemiology of osteosarcoma. Cancer Treat Res 2009;152:3–13. [5] Gill J, Ahluwalia MK, Geller D, Gorlick R. New targets and approaches in osteosarcoma. Pharmacol Ther 2012. [6] Munajat I, Zulmi W, Norazman MZ, Wan Faisham WI. Tumour volume and lung metastasis in patients with osteosarcoma. J Orthop Surg (Hong Kong) 2008;16:182–5. [7] Tumbar T, Guasch G, Greco V, Blanpain C, Lowry WE, Rendl M, et al. Defining the epithelial stem cell niche in skin. Science 2004;303:359–63.
[8] Spradling A, Drummond-Barbosa D, Kai T. Stem cells find their niche. Nature 2001;414:98–104. [9] Shen A, Zhang Y, Yang H, Xu R, Huang G. Overexpression of ZEB1 relates to metastasis and invasion in osteosarcoma. J Surg Oncol 2012;105:830–4. [10] Yi C, Li X, Xu W, Chen A. Relationship between the expression of MTA-1 gene and the metastasis and invasion in human osteosarcoma. J Huazhong Univ Sci Technol Med Sci 2005;25:445–7. [11] Tsubaki M, Satou T, Itoh T, Imano M, Ogaki M, Yanae M, et al. Reduction of metastasis, cell invasion, and adhesion in mouse osteosarcoma by YM529/ ONO-5920-induced blockade of the Ras/MEK/ERK and Ras/PI3K/Akt pathway. Toxicol Appl Pharmacol 2012;259:402–10. [12] Desgrosellier JS, Cheresh DA. Integrins in cancer: biological implications and therapeutic opportunities. Nat Rev Cancer 2010;10:9–22. [13] Lin YM, Chang ZL, Liao YY, Chou MC, Tang CH. IL-6 promotes ICAM-1 expression and cell motility in human osteosarcoma. Cancer Lett 2012. [14] Bruijn JA, de Heer E. Adhesion molecules in renal diseases. Lab Invest 1995;72:387–94. [15] Carlos TM, Schwartz BR, Kovach NL, Yee E, Rosa M, Osborn L, et al. Vascular cell adhesion molecule-1 mediates lymphocyte adherence to cytokine-activated cultured human endothelial cells. Blood 1990;76:965–70. [16] Garcia-Barcina M, Lukomska B, Gawron W, Winnock M, Vidal-Vanaclocha F, Bioulac-Sage P, et al. Expression of cell adhesion molecules on liver-associated lymphocytes and their ligands on sinusoidal lining cells in patients with benign or malignant liver disease. Am J Pathol 1995;146:1406–13. [17] Gahmberg CG, Tolvanen M, Kotovuori P. Leukocyte adhesion–structure and function of human leukocyte beta2-integrins and their cellular ligands. Eur J Biochem 1997;245:215–32. [18] Kurokouchi K, Kambe F, Yasukawa K, Izumi R, Ishiguro N, Iwata H, et al. TNFalpha increases expression of IL-6 and ICAM-1 genes through activation of NFkappaB in osteoblast-like ROS17/2.8 cells. J Bone Miner Res 1998;13:1290–9. [19] Kohka H, Yoshino T, Iwagaki H, Sakuma I, Tanimoto T, Matsuo Y, et al. Interleukin-18/interferon-gamma-inducing factor, a novel cytokine, up-regulates ICAM-1 (CD54) expression in KG-1 cells. J Leukoc Biol 1998;64:519–27. [20] Yu JA, Sadaria MR, Meng X, Mitra S, Ao L, Fullerton DA, et al. Lung cancer cell invasion and expression of intercellular adhesion molecule-1 (ICAM-1) are attenuated by secretory phospholipase A(2) inhibition. J Thorac Cardiovasc Surg 2012;143:405–11. [21] Tian MM, Sun Y, Li ZW, Wu Y, Zhao AL, Li JY. Polymorphisms of ICAM-1 are associated with gastric cancer risk and prognosis. World J Gastroenterol 2012;18:368–74. [22] Fong YC, Lin CY, Su YC, Chen WC, Tsai FJ, Tsai CH, et al. CCN6 enhances ICAM-1 expression and cell motility in human chondrosarcoma cells. J Cell Physiol 2012;227:223–32. [23] Ohchi T, Akagi Y, Kinugasa T, Kakuma T, Kawahara A, Sasatomi T, et al. Amphiregulin is a prognostic factor in colorectal cancer. Anticancer Res 2012;32:2315–21. [24] Makrilia N, Kollias A, Manolopoulos L, Syrigos K. Cell adhesion molecules: role and clinical significance in cancer. Cancer Invest 2009;27:1023–37. [25] Roland CL, Harken AH, Sarr MG, Barnett Jr CC. ICAM-1 expression determines malignant potential of cancer. Surgery 2007;141:705–7. [26] Stromberg K, Collins TJt, Gordon AW, Jackson CL, Johnson GR. Transforming growth factor-alpha acts as an autocrine growth factor in ovarian carcinoma cell lines. Cancer Res 1992;52:341–7. [27] Morishige K, Kurachi H, Amemiya K, Fujita Y, Yamamoto T, Miyake A, et al. Evidence for the involvement of transforming growth factor alpha and epidermal growth factor receptor autocrine growth mechanism in primary human ovarian cancers in vitro. Cancer Res 1991;51:5322–8. [28] Crew AJ, Langdon SP, Miller EP, Miller WR. Mitogenic effects of epidermal growth factor and transforming growth factor-alpha on EGF-receptor positive human ovarian carcinoma cell lines. Eur J Cancer 1992;28:337–41. [29] Sewell JM, Smyth JF, Langdon SP. Role of TGF alpha stimulation of the ERK, PI3 kinase and PLC gamma pathways in ovarian cancer growth and migration. Exp Cell Res 2005;304:305–16. [30] Pelletier S, Tanguay S, Lee S, Gunaratnam L, Arbour N, Lapointe R. TGF-alpha as a candidate tumor antigen for renal cell carcinomas. Cancer Immunol Immunother 2009;58:1207–18. [31] Ahmed SR, Badger B, Wright C, Manni A. Role of transforming growth factoralpha (TGF-alpha) in basal and hormone-stimulated growth by estradiol, prolactin and progesterone in human and rat mammary tumor cells: studies using TGF-alpha and EGF receptor antibodies. J Steroid Biochem Mol Biol 1991;38:687–93. [32] Liu D, Gagliardi G, Nasim MM, Alison MR, Oates T, Lalani EN, et al. TGF-alpha can act as morphogen and/or mitogen in a colon-cancer cell line. Int J Cancer 1994;56:603–8. [33] Kullenberg B, Jansen C, Fredang N, Ohlsson B, Axelson J. Transforming growth factor alpha (TGF-alpha) increases cell number in a human pancreatic cancer cell line but not in normal mouse pancreas. Int J Pancreatol 2000;28:199–205. [34] Baek JY, Morris SM, Campbell J, Fausto N, Yeh MM, Grady WM. TGF-beta inactivation and TGF-alpha overexpression cooperate in an in vivo mouse model to induce hepatocellular carcinoma that recapitulates molecular features of human liver cancer. Int J Cancer 2010;127:1060–71. [35] Breuhahn K, Longerich T, Schirmacher P. Dysregulation of growth factor signaling in human hepatocellular carcinoma. Oncogene 2006;25:3787–800. [36] Calvisi DF, Ladu S, Gorden A, Farina M, Conner EA, Lee JS, et al. Ubiquitous activation of Ras and Jak/Stat pathways in human HCC. Gastroenterology 2006;130:1117–28.
Please cite this article in press as: Hou C-H, et al. Transforming growth factor alpha promotes osteosarcoma metastasis by ICAM-1 and PI3K/Akt signaling pathway. Biochem Pharmacol (2014), http://dx.doi.org/10.1016/j.bcp.2014.03.010
G Model
BCP-11938; No. of Pages 11 C.-H. Hou et al. / Biochemical Pharmacology xxx (2014) xxx–xxx [37] Jhappan C, Stahle C, Harkins RN, Fausto N, Smith GH, Merlino GT. TGF alpha overexpression in transgenic mice induces liver neoplasia and abnormal development of the mammary gland and pancreas. Cell 1990;61:1137–46. [38] Santoni-Rugiu E, Jensen MR, Factor VM, Thorgeirsson SS. Acceleration of cmyc-induced hepatocarcinogenesis by co-expression of transforming growth factor (TGF)-alpha in transgenic mice is associated with TGF-beta1 signaling disruption. Am J Pathol 1999;154:1693–700. [39] Yeh MM, Larson AM, Campbell JS, Fausto N, Rulyak SJ, Swanson PE. The expression of transforming growth factor-alpha in cirrhosis, dysplastic nodules, and hepatocellular carcinoma: an immunohistochemical study of 70 cases. Am J Surg Pathol 2007;31:681–9. [40] Greten FR, Wagner M, Weber CK, Zechner U, Adler G, Schmid RM. TGF alpha transgenic mice. A model of pancreatic cancer development. Pancreatology 2001;1:363–8. [41] Kitano M, Wada J, Ariki Y, Kato M, Wanibuchi H, Morimura K, et al. Possible tumor development from double positive foci for TGF-alpha and GST-P observed in early stages on rat hepatocarcinogenesis. Cancer Sci 2006;97:478–83. [42] Pedersen PH, Ness GO, Engebraaten O, Bjerkvig R, Lillehaug JR, Laerum OD. Heterogeneous response to the growth factors [EGF, PDGF (bb), TGF-alpha, bFGF, IL-2] on glioma spheroid growth, migration and invasion. Int J Cancer 1994;56:255–61. [43] Kikuchi K, Li X, Zheng Y, Takano Y. Invasion of breast cancer cells into collagen matrix requires TGF-alpha and Cdc42 signaling. FEBS Lett 2011;585:286–90. [44] Ueda M, Fujii H, Yoshizawa K, Terai Y, Kumagai K, Ueki K, et al. Effects of EGF and TGF-alpha on invasion and proteinase expression of uterine cervical adenocarcinoma OMC-4 cells. Invasion Metastasis 1998;18:176–83. [45] Liu JF, Fong YC, Chang CS, Huang CY, Chen HT, Yang WH, et al. Cyclooxygenase2 enhances alpha2beta1 integrin expression and cell migration via EP1 dependent signaling pathway in human chondrosarcoma cells. Mol Cancer 2010;9:43. [46] Lin YM, Chang ZL, Liao YY, Chou MC, Tang CH. IL-6 promotes ICAM-1 expression and cell motility in human osteosarcoma. Cancer Lett 2013;328:135–43. [47] Hou CH, Tang CH, Hsu CJ, Hou SM, Liu JF. CCN4 induces IL-6 production through alphavbeta5 receptor, PI3K, Akt, and NF-kappaB singling pathway in human synovial fibroblasts. Arthritis Res Ther 2013;15:R19. [48] Liu JF, Yang WH, Fong YC, Kuo SC, Chang CS, Tang CH. BFPP, a phloroglucinol derivative, induces cell apoptosis in human chondrosarcoma cells through endoplasmic reticulum stress. Biochem Pharmacol 2010;79:1410–7. [49] Liu JF, Fong YC, Chang KW, Kuo SC, Chang CS, Tang CH. FPTB, a novel CA-4 derivative, induces cell apoptosis of human chondrosarcoma cells through mitochondrial dysfunction and endoplasmic reticulum stress pathways. J Cell Biochem 2011;112:453–62. [50] Liu JF, Huang YL, Yang WH, Chang CS, Tang CH. 1-Benzyl-2-phenylbenzimidazole (BPB), a benzimidazole derivative, induces cell apoptosis in human chondrosarcoma through intrinsic and extrinsic pathways. Int J Mol Sci 2012;13:16472–88. [51] Wang C, Lv X, Jiang C, Cordes CM, Fu L, Lele SM, et al. Transforming growth factor alpha (TGFalpha) regulates granulosa cell tumor (GCT) cell proliferation and migration through activation of multiple pathways. PLoS One 2012;7:e48299. [52] Kenny PA, Bissell MJ. Targeting TACE-dependent EGFR ligand shedding in breast cancer. J Clin Invest 2007;117:337–45. [53] Katerinaki E, Haycock JW, Lalla R, Carlson KE, Yang Y, Hill RP, et al. Sodium salicylate inhibits TNF-alpha-induced NF-kappaB activation, cell migration, invasion and ICAM-1 expression in human melanoma cells. Melanoma Res 2006;16:11–22. [54] Kesanakurti D, Chetty C, Rajasekhar Maddirela D, Gujrati M, Rao JS. Essential role of cooperative NF-kappaB and Stat3 recruitment to ICAM-1 intronic consensus elements in the regulation of radiation-induced invasion and migration in glioma. Oncogene 2012. [55] Kneissl J, Keller S, Lorber T, Heindl S, Keller G, Drexler I, et al. Association of amphiregulin with the cetuximab sensitivity of gastric cancer cell lines. Int J Oncol 2012;41:733–44.
11
[56] Higginbotham JN, Demory Beckler M, Gephart JD, Franklin JL, Bogatcheva G, Kremers GJ, et al. Amphiregulin exosomes increase cancer cell invasion. Curr Biol 2011;21:779–86. [57] Ivanov VN, Hei TK. Combined treatment with EGFR inhibitors and arsenite upregulated apoptosis in human EGFR-positive melanomas: a role of suppression of the PI3K-AKT pathway. Oncogene 2005;24:616–26. [58] Qiao M, Sheng S, Pardee AB. Metastasis and AKT activation. Cell Cycle 2008;7:2991–6. [59] Tang CH, Tan TW, Fu WM, Yang RS. Involvement of matrix metalloproteinase-9 in stromal cell-derived factor-1/CXCR4 pathway of lung cancer metastasis. Carcinogenesis 2008;29:35–43. [60] Goldsmith JR, Lee TH. High-grade intramedullary osteosarcoma of the talus: case report. J Foot Ankle Surg 2007;46:480–3. [61] Zhou ZN, Sharma VP, Beaty BT, Roh-Johnson M, Peterson EA, Van Rooijen N, et al. Autocrine HBEGF expression promotes breast cancer intravasation, metastasis and macrophage-independent invasion in vivo. Oncogene 2013. [62] Wang Z, Jin C, Li H, Li C, Hou Q, Liu M, et al. GPR48-induced keratinocyte proliferation occurs through HB-EGF mediated EGFR transactivation. FEBS Lett 2010;584:4057–62. [63] Sagmeister S, Drucker C, Losert A, Grusch M, Daryabeigi A, Parzefall W, et al. HB-EGF is a paracrine growth stimulator for early tumor prestages in inflammation-associated hepatocarcinogenesis. J Hepatol 2008;49:955–64. [64] Etindi R, Manni A. TGF-alpha and IGF-I effects on calcium ion transients in human breast cancer cells. Cancer Lett 1992;66:131–7. [65] Maruo Y, Gochi A, Kaihara A, Shimamura H, Yamada T, Tanaka N, et al. ICAM-1 expression and the soluble ICAM-1 level for evaluating the metastatic potential of gastric cancer. Int J Cancer 2002;100:486–90. [66] Liu G, Place AT, Chen Z, Brovkovych VM, Vogel SM, Muller WA, et al. ICAM-1activated Src and eNOS signaling increase endothelial cell surface PECAM-1 adhesivity and neutrophil transmigration. Blood 2012;120:1942–52. [67] Schroder C, Witzel I, Muller V, Krenkel S, Wirtz RM, Janicke F, et al. Prognostic value of intercellular adhesion molecule (ICAM)-1 expression in breast cancer. J Cancer Res Clin Oncol 2011;137:1193–201. [68] De Luca A, Gallo M, Aldinucci D, Ribatti D, Lamura L, D’Alessio A, et al. Role of the EGFR ligand/receptor system in the secretion of angiogenic factors in mesenchymal stem cells. J Cell Physiol 2011;226:2131–8. [69] Minder P, Bayha E, Becker-Pauly C, Sterchi EE. Meprinalpha transactivates the epidermal growth factor receptor (EGFR) via ligand shedding, thereby enhancing colorectal cancer cell proliferation and migration. J Biol Chem 2012;287:35201–11. [70] Xu JW, Li QQ, Tao LL, Cheng YY, Yu J, Chen Q, et al. Involvement of EGFR in the promotion of malignant properties in multidrug resistant breast cancer cells. Int J Oncol 2011;39:1501–9. [71] Lo HW, Hsu SC, Hung MC. EGFR signaling pathway in breast cancers: from traditional signal transduction to direct nuclear translocalization. Breast Cancer Res Treat 2006;95:211–8. [72] Diaz ME, Gonzalez L, Miquet JG, Martinez CS, Sotelo AI, Bartke A, et al. Growth hormone modulation of EGF-induced PI3K-Akt pathway in mice liver. Cell Signal 2012;24:514–23. [73] Berg M, Soreide K. EGFR and downstream genetic alterations in KRAS/BRAF and PI3K/AKT pathways in colorectal cancer: implications for targeted therapy. Discov Med 2012;14:207–14. [74] Heeg S, Hirt N, Queisser A, Schmieg H, Thaler M, Kunert H, et al. EGFR overexpression induces activation of telomerase via PI3K/AKT-mediated phosphorylation and transcriptional regulation through Hif1-alpha in a cellular model of oral-esophageal carcinogenesis. Cancer Sci 2011;102:351–60. [75] Chen PC, Lin TH, Cheng HC, Tang CH. CCN3 increases cell motility and ICAM-1 expression in prostate cancer cells. Carcinogenesis 2012;33:937–45. [76] Tang CH, Tsai CC. CCL2 increases MMP-9 expression and cell motility in human chondrosarcoma cells via the Ras/Raf/MEK/ERK/NF-kappaB signaling pathway. Biochem Pharmacol 2012;83:335–44. [77] Streicher KL, Willmarth NE, Garcia J, Boerner JL, Dewey TG, Ethier SP. Activation of a nuclear factor kappaB/interleukin-1 positive feedback loop by amphiregulin in human breast cancer cells. Mol Cancer Res 2007;5:847–61.
Please cite this article in press as: Hou C-H, et al. Transforming growth factor alpha promotes osteosarcoma metastasis by ICAM-1 and PI3K/Akt signaling pathway. Biochem Pharmacol (2014), http://dx.doi.org/10.1016/j.bcp.2014.03.010
BCP-11938; No. of Pages 11 Biochemical Pharmacology xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Biochemical Pharmacology journal homepage: www.elsevier.com/locate/biochempharm
Transforming growth factor alpha promotes osteosarcoma metastasis by ICAM-1 and PI3K/Akt signaling pathway Chun-Han Hou a, Feng-Ling Lin b, Kai-Biao Tong c, Sheng-Mon Hou d,*, Ju-Fang Liu e,* a
Department of Orthopedic Surgery, National Taiwan University Hospital, Taipei, Taiwan Department of Dermatology, Sijhih Cathay General Hospital, Taipei, Taiwan c Veterinarian Central Laboratory, Shin-Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan d Department of Orthopedic Surgery, Shin-Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan e Central Laboratory, Shin-Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 4 December 2013 Accepted 20 March 2014 Available online xxx
Osteosarcoma is the most common primary malignancy of bone and is characterized by a high malignant and metastatic potential. Transforming growth factor alpha (TGF-a) is classified as the EGF (epidermal growth factor)-like family, which is involved in cancer cellular activities such as proliferation, motility, migration, adhesion and invasion abilities. However, the effect of TGF-a on human osteosarcoma is largely unknown. We found that TGF-a increased the cell migration and expression of intercellular adhesion molecule-1 (ICAM-1) in human osteosarcoma cells. Transfection of cells with ICAM-1 siRNA reduced TGF-a-mediated cell migration. We also found that the phosphatidylinositol 30 -kinase (PI3K)/ Akt/NF-kB pathway was activated after TGF-a treatment, and TGF-a-induced expression of ICAM-1 and cell migration was inhibited by the specific inhibitors and siRNAs of PI3K, Akt, and NF-kB cascades. In addition, knockdown of TGF-a expression markedly decreased cell metastasis in vitro and in vivo. Our results indicate that TGF-a/EGFR interaction elicits PI3K and Akt activation, which in turn activates NFkB, resulting in the expression of ICAM-1 and contributing the migration of human osteosarcoma cells. ß 2014 Elsevier Inc. All rights reserved.
Keywords: TGF-a Osteosarcoma Migration
1. Introduction Osteosarcoma is the most common primary bone cancer in adolescents and young adults [1–3]. Approximately 2400 new cases of osteosarcoma are diagnosed per year in the United States [4]. Nearly 2/3 of patients with osteosarcoma can be expected to be cured with routine chemotherapy and surgery, and approximately 1/3 of patients have metastasis detectable at presentation, especially pulmonary metastasis [5]. Unfortunately, the main cause of the death in osteosarcoma is lung metastasis [6]. Hence, chemotherapy is usually employed in an adjuvant situation to improve the prognosis and long-term survival. Therefore, the novel treatment strategies that would effectively inhibit metastasis, especially to the lung, from the primary osteosarcoma site are highly desirable. Tumor invasion and metastasis are the complicated process, which is influenced by multiple regulatory genes [7,8]. However,
* Corresponding authors. E-mail addresses: [email protected] (S.-M. Hou), [email protected], [email protected] (J.-F. Liu).
metastatic cascade involves the reducing of the cell–cell adhesion, the disruption of the extracellular matrix (ECM), and the rearrangement of the cytoskeletons, eventually tumor cells separate from the primary lesion and slowly proliferate at distant organs [7–13]. The cell adhesion molecules (CAMs), for example the integrin, immunoglobulin, selectins, and cadherin superfamilies, play an important role in regulating cell–cell and cell– matrix adhesion [14–16]. Intercellular adhesion molecule-1 (ICAM-1, also called CD54) is important in inflammation, immune responses, and intracellular signaling [17]. Moreover, many growth factors such as interlukin-1b (IL-1b), interlukin-6 (IL-6), and tumor necrosis factor alpha (TNF-a), could induce ICAM-1 expression [18,19]. It has been reported that ICAM-1 expression is associated with aggressive neoplasm such as breast cancer, lung cancer, gastric cancer, colorectal cancer, and melanoma cancer [20–25], and further, the up-regulation expression of the ICAM-1 can facilitate cancer cell invasion [24]. Therefore, decreased or disrupted expression of the ICAM-1 may prevent metastasis. Transforming growth factor-alpha (TGF-a), a member of the epidermal growth factors (EGF), exhibits multiple biological activities among various types of tissues and cells. TGF-a has been shown to stimulate cell growth in some cancer types [26–29].
http://dx.doi.org/10.1016/j.bcp.2014.03.010 0006-2952/ß 2014 Elsevier Inc. All rights reserved.
Please cite this article in press as: Hou C-H, et al. Transforming growth factor alpha promotes osteosarcoma metastasis by ICAM-1 and PI3K/Akt signaling pathway. Biochem Pharmacol (2014), http://dx.doi.org/10.1016/j.bcp.2014.03.010
G Model
BCP-11938; No. of Pages 11 2
C.-H. Hou et al. / Biochemical Pharmacology xxx (2014) xxx–xxx
On the other hand, it has been reported that high TGF-a expression is found in variety of malignant tumors, including kidney cancer, pancreatic cancer, colon cancer, and breast cancer [30–33]. Furthermore, it has been demonstrated that TGF-a is overexpressed in liver cancer and promotes carcinogenesis through TGF-a-EGFR-RAS-MAPK signaling pathway [34–39]. In ovarian cancer, TGF-a regulates cell growth by ERK and PI3K/Akt signaling pathways. On the other hand, TGF-a-induced cell migration is associated with PLCg pathway [29]. TGF-a is significantly correlated with the development and progression of pancreatic cancer and liver cancer [40,41]. Previous studies have shown that TGF-a regulates cell migration and invasion in human cancer cells [29,42–44]. However, the effect of TGF-a expression in human osteosarcoma cells is rarely reported. In this investigation, we demonstrated that TGF-a and EGFR interaction increased the migration and expression of ICAM-1 in human osteosarcoma cells. In addition, phosphatidylinositol 3kinase (PI3K)/Akt and NF-kB signaling pathways were involved. The elucidation of this TGF-a-responsive signaling pathway provides valuable clues to understand the mechanisms of human osteosarcoma metastasis and offered the opportunity to develop more effective clinical therapies in the future. 2. Materials and methods
temperature for the expression of the large T antigen. All experiments with hFOB 1.19 cells were carried out at the permissive temperature of 33.5 8C. 2.3. Migration assay The migration assay was performed using Transwell inserts (Costar, NY; 8 mm pore size) in 24-well dishes. Before performing the migration assay, cells were pretreated for 30 min with different concentrations of inhibitors (PD158780, BIBX1382, LY294002, Wortmannin, Akti, PDTC or TPCK) or vehicle control (0.1% dimethyl sulfoxide, DMSO). Approximately 1 104 cells in 200 mL of serumfree medium were placed in the upper chamber, and 300 mL of the same medium containing different concentrations of TGF-a were placed in the lower chamber. The cells were incubated for 24 h at 37 8C in 5% CO2, then fixed in 3.7% formaldehyde for 15 min and stained with 0.05% crystal violet in phosphate-buffered saline (PBS) for 60 min. Cells on the upper side of the filters were removed with cotton-tipped swabs and the filters were washed with PBS. Cells on the underside of the filters were examined and counted under a microscope. Each experiment was performed in triplicate and repeated at least three times. The number of migrating cells in each experiment was corrected for proliferation effects using a cell viability assay (corrected migrating cell number 5 counted migrating cell number/percentage of viable cells) [45].
2.1. Materials 2.4. Wound-healing migration assay Protein A/G beads, anti-mouse and anti-rabbit IgG conjugated horseradish peroxidase, polyclonal antibodies specific for PI3K, Akt, IKKa/b, IkB, p65, EGFR, ICAM-1, VCAM-1 and b-Actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Polyclonal antibody specific for EGFR phosphorylated at PY1068 and PY992, PI3K phosphorylated at Tyr458/199, Akt phosphorylated at Ser473, IKKa/b phosphorylated at Ser176/180, IkBa phosphorylated at Ser32/36 and p65 phosphorylated at Ser536 were purchased from Cell Signaling and Neuroscience (Danvers, MA, USA). PD158780, BIBX1382, Pyrrolidine-dithiocarbamate (PDTC), L-1-tosylamido-2phenylenylethyl chloromethyl ketone (TPCK), LY294002, Wortmannin, and Akti were purchased from Calbiochem (San Diego, CA, USA). The recombinant human TGF-a was purchased from PeproTech (Rocky Hill, NJ, USA). The NF-kB luciferase plasmid was purchased from Stratagene (La Jolla, CA, USA). The p85 and Akt (Akt K179A) dominant-negative mutants were gifts from Dr. W. M. Fu (National Taiwan University, Taipei, Taiwan). The pSV-bgalactosidase vector and the luciferase assay kit were purchased from Promega (Madison, MA, USA). The human siRNAs for control, ICAM-1 and EGFR were as pools sequences and were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All other chemicals were purchased from Sigma–Aldrich (St. Louis, MO, USA).
For the wound-healing migration assay, cells were seeded on 12-well plates at a density of 1 105 cells/well in culture medium. At 24 h after seeding, the confluent monolayer of culture was scratched with a fine pipette tip, and migration was visualized by microscope and magnification. The rate of wound closure was observed at the indicated time [46]. 2.5. Western blotting analysis The cellular lysates were prepared as described previously [47,48]. Proteins were resolved on SDS–PAGE and transferred to Immobilon polyvinyldifluoride (PVDF) membranes. The blots were blocked with 5% BSA for 1 h at room temperature and then probed with rabbit anti-human antibodies against PI3K, Akt, IKKa/b, IkB, p65, EGFR, ICAM-1 and VCAM-1 (1:1000) for 1 h at room temperature. After being washed three times in TBST, the blots were subsequently incubated with a donkey anti-rabbit peroxidaseconjugated secondary antibody (1:1000) for 1 h at room temperature. The blots were visualized by enhanced chemiluminescence using Kodak X-OMAT LS film (Eastman Kodak, Rochester, NY). Quantitative data were obtained using a computing densitometer and Image Quant software (Molecular Dynamics, Sunnyvale, CA, USA).
2.2. Cell culture 2.6. Quantitative real time PCR The human osteosarcoma cell lines (U2OS and MG63) and human fetal osteoblastic cell line (hFOB 1.19) were purchased from the American Type Cell Culture Collection (Manassas, VA, USA). MG63 cells were maintained at 37 8C with 5% CO2 and cultured in DMEM supplemented with 20 mM HEPES, 10% heat-inactivated FBS, 2 mM-glutamine and antibiotics (100 U/mL of penicillin and 100 mg/mL of streptomycin). U2OS cells were maintained at 37 8C with 5% CO2 and culture in McCoy’s 5A medium supplemented with 10% heat-inactivated FBS, 1.5 mM L-glutamine and antibiotics. hFOB 1.19 cells were maintained in 1:1 mixture of phenolfree DMEM/Ham’s F12 medium (GIBCO-BRL, Gaithersburg, MD, USA) containing 10% FBS supplemented with 2.5 mM L-glutamine, 0.3 mg/mL G418 and antibiotics at 33.5 8C, the permissive
Total RNA was extracted from osteosarcoma cells by using a TRIzol kit (MDBio Inc., Taipei, Taiwan). Two mg of total RNA was reverse transcribed into cDNA by using oligo (dT) primer [49,50]. The quantitative real time PCR (qPCR) analysis was carried out using Taqman1 one-step PCR Master Mix (Applied Biosystems, CA, USA). Two mg of cDNA were added per 25-mL reaction with sequence-specific primers and Taqman1 probes. Sequences for all target gene primers and probes were purchased commercially (GAPDH was used as internal control) (Applied Biosystems, CA, USA). qPCR assays were carried out in triplicate on an Step One Plus sequence detection system. The cycling conditions were 10-min polymerase activation at 95 8C followed by 40 cycles at 95 8C for
Please cite this article in press as: Hou C-H, et al. Transforming growth factor alpha promotes osteosarcoma metastasis by ICAM-1 and PI3K/Akt signaling pathway. Biochem Pharmacol (2014), http://dx.doi.org/10.1016/j.bcp.2014.03.010
G Model
BCP-11938; No. of Pages 11 C.-H. Hou et al. / Biochemical Pharmacology xxx (2014) xxx–xxx
15 s and 60 8C for 60 s. The threshold was set above the nontemplate control background and within the linear phase of target gene amplification to calculate the cycle number at which the transcript was detected (denoted CT). 2.7. Transfection and reporter gene assay Human osteosarcoma cells were co-transfected with 0.8 mg kB driven luciferase plasmid, 0.4 mg b-galactosidase expression vector. Cells were grown to 80% confluence in 12 well plates and were transfected on the following day by Lipofectamine 2000 (LF2000; Invitrogen). DNA and LF2000 were premixed for 30 min and then applied to the cells (the transfection efficiency is more than 45% confirmed by transfection with GFP-expressing plasmid). DMEM containing 20% FBS was added 4 h later. After 24 h of transfection, the cells were incubated with the indicated agents. After further 24 h incubation, the media were removed, and cells were washed once with cold PBS. To prepare lysates, 100 mL reporter lysis buffer (Promega, Madison, WI) were added to each well, and cells were scraped from dishes. The supernatant was collected after centrifugation at 11,000 g for 2 min. Aliquots of cell lysates (20 mL) containing equal amounts of protein (20–30 mg) were placed into wells of an opaque black 96-well microplate. An equal volume of luciferase substrate was added to all samples, and luminescence was measured in a microplate luminometer. The value of luciferase activity was normalized to transfection efficiency monitored by the co-transfected b-galactosidase expression vector.
3
understand its effect on osteosarcoma cell, we first examined the levels of TGF-a in human fetal osteoblastic cell line (hFOB 1.19) and osteosarcoma cell lines (MG63 and U2OS). TGF-a was significantly elevated in MG63 and U2OS cell lines compared with hFOB 1.19 (Fig. 1A). The TGF-a messenger RNA (mRNA) in MG63 and U2OS cells were also higher than in hFOB 1.19 (Fig. 1B). In addition, MG63 and U2OS cells were more migratory than hFOB 1.19 (Fig. 1C). Moreover, treatment of cells with TGF-a recombinant protein (5–100 ng/mL) dramatically increased migration in MG63 and U2OS cell lines (Fig. 1D). However, TGF-a treatment did not affect cell viability in MG63 and U2OS cells by using MTT assay (data not shown). On the other hand, TGF-a also increased woundhealing activity in human osteosarcoma cells (Fig. 1E). Finally, comparison of EGF and EGFR status between hFOB1.19 and osteosarcoma is important. We examined the levels of EGF and EGFR between hFOB 1.19 and osteosarcoma (MG63 and U2OS). The level of EGF was significantly elevated in MG63 and U2OS cell lines compared with hFOB 1.19 (Fig. 1F). The level of EGFR was significantly elevated in normal osteoblastic cell line compared with malignant cell (Fig. 1G). Furthermore, tumor necrosis factoralpha converting enzyme (TACE/ADAM17) plays an important role in the regulated shedding of EGFR ligands [52]. We examined the level of TACE between hFOB 1.19 and osteosarcoma (MG63 and U2OS). The level of TACE was significantly elevated in MG63 and U2OS cell lines compared with hFOB 1.19 (Fig. 1F). These results indicate that the elevated TGF-a in metastatic osteosarcoma cells increased their cell migration in vitro. 3.2. ICAM-1 is involved in TGF-a-induced cell migration
2.8. Establishment of stably transfected cells TGF-a shRNA or control shRNA plasmids were transfected into cancer cells with Lipofetamine 2000 transfection reagent. Twentyfour hours after transfection, stable transfectants were selected in puromycin at a concentration of 10 mg/mL. Thereafter, the selection medium was replaced every 3 days. After 2 weeks of selection in puromycin (10 mg/mL), clones of resistant cells were isolated. 2.9. In vivo tumor xenograft study All animal experiments were performed in accordance with a protocol approved by the Shin-Kong Wu Ho-Su Memorial Hospital (Taipei, Taiwan) institutional animal care and use committees. Male CB17-SCID mice (4 weeks old) were used. For experimental metastasis assays, 5 106 cells were resuspended in 0.1 mL of saline and injected into the tail vein. After 4 weeks, the mice were euthanized by an overdose of the anesthetic agent. The lungs were removed and fixed in 10% formalin. The number of lung tumor metastases was counted under a dissecting microscope.
ICAM-1 plays a key role in cancer cell migration and invasion [53,54]. However, the expression of ICAM-1 in human osteosarcoma cells is largely unknown. First, we examined hFOB 1.19 and osteosarcoma cells for the expression of ICAM-1 by using western blot and qPCR. Expression levels of ICAM-1 in osteosarcoma cells were significantly higher than in hFOB 1.19 (Fig. 2A and B). Next, to examine whether ICAM-1 is involved in TGF-a-induced migration of osteosarcoma cells, we measured the level of ICAM-1 mRNA and protein in TGF-a treated osteosarcoma cells. Indeed, TGF-a increased ICAM-1 mRNA expression and protein level in a dosedependent and time-dependent manner, but TGF-a treatment did not have any effect on the mRNA or protein level of VCAM-1 (Fig. 2C–F). To confirm this finding, MG63 and U2OS cells were transfected with control and ICAM-1 small interfering RNA (siRNA) for 24 h, and the western blot analysis showed that the expression of protein levels of ICAM-1 was suppressed by transfection with ICAM-1 siRNA (Fig. 2G). Transfected cells with ICAM-1 siRNA markedly reduced cell migration and inhibited TGF-a-induced cell migration (Fig. 2H and I). These data clearly showed that TGF-ainduced cancer cell migration may occur via activation of ICAM-1.
2.10. Statistical analysis Data are presented as mean standard error of the mean (SEM). Statistical analysis between two samples was performed using the Student’s t-test. Statistical comparisons of more than two groups were performed using one-way analysis of variance with Bonferroni’s post hoc test. In all cases, a p < 0.05 was considered to be statistically significant and the experiments were repeated four times. 3. Results 3.1. TGF-a increases the migration and expression of ICAM-1 in human osteosarcoma cells TGF-a has been shown to increase cell migration and metastasis in a variety of human cancer cells [29,51]. To
3.3. Involvement of EGFR in TGF-a-mediated migration of osteosarcoma Previous study has shown that TGF-a affects cellular function by specifically bind with EGFR and then activates EGFR [55,56]. Therefore, we next examined whether EGFR was involved in TGFa-mediated cell migration in human osteosarcoma cells. We hypothesized that EGFR signaling pathway may be involved in TGF-a-induced osteosarcoma cell migration. Pretreatment of cells with EGFR tyrosine kinase inhibitors: BIBX1382 (10 mM) and PD158780 (5 mM) for 30 min markedly inhibited TGF-a-induced cancer cell migration, ICAM-1 mRNA expression and ICAM-1 protein production (Fig. 3A–C). Additionally, Stimulation of cells with TGF-a led to a significant increase in phosphorylation of EGFR (Fig. 3D). Following transfection of cells with control and EGFR
Please cite this article in press as: Hou C-H, et al. Transforming growth factor alpha promotes osteosarcoma metastasis by ICAM-1 and PI3K/Akt signaling pathway. Biochem Pharmacol (2014), http://dx.doi.org/10.1016/j.bcp.2014.03.010
G Model
BCP-11938; No. of Pages 11 4
C.-H. Hou et al. / Biochemical Pharmacology xxx (2014) xxx–xxx
Fig. 1. TGF-a induces cell migration of human osteosarcoma cells. (A and B) Total protein and mRNA were extracted form hFOB 1.19, MG63, and U2OS cells, the TGF-a expression was examined by western blot analysis and qPCR. (C) In vitro migration activity of hFOB 1.19, MG63, and U2OS cells was measured using the Transwell migration assay. (D and E) Cells were incubated with various concentrations of TGF-a for 16 h, and in vitro migration was measured using the Transwell migration assay and Woundhealing assay (F and G) Total protein and mRNA were extracted form hFOB 1.19, MG63, and U2OS cells, the EGF, EGFR and TACE expression were examined by western blot analysis and qPCR. Results are expressed as the mean SEM. *, p < 0.05 compared with control.
siRNA, the expression of EGFR is reduced (Fig. 3E), and also inhibited the migration activity and ICAM-1 mRNA expression of osteosarcoma cells (Fig. 3F and G). On the other hand, we hypothesis that used EGFR inhibitor alone or combined with cancer chemotherapy drugs (e.g. Cisplatin and Doxorubicin) could abolish TGF-a effects. First, we found that Cisplatin and Doxorubicin in low dose did not affect cell survival (Fig. 3H and I). Next, pretreatment of cells with EGFR inhibitors alone, cancer chemotherapy drugs alone or EGFR inhibitor combined with cancer chemotherapy drug for 30 min, followed by treatment with TGF-a for 24 h, we found EGFR inhibitor combined with cancer chemotherapy drug markedly inhibited TGF-a-induced cancer cell migration (Fig. 3J). These data suggested that TGF-a/EGFR interaction played a key role in the cell migration and ICAM-1 expression of osteosarcoma. 3.4. PI3K and Akt signaling pathways are involved in TGF-a-mediated ICAM-1 up-regulation and cell migration of osteosarcoma cells The PI3K-Akt pathway is activated by EGFR [57]. We examined whether TGF-a enhanced the activation of PI3K. TGF-a-induced
cell migration and ICAM-1 expression of osteosarcoma cells were greatly reduced by treatment with PI3K inhibitors Ly294002 and Wortmannin (Fig. 4A–C). On the other hand, stimulation of cells with TGF-a led to a significant increasing in phosphorylation of p85 (Fig. 4D). In addition, transfection of cells with pI3K mutant also inhibited TGF-a induced cell migration and ICAM-1 expression of osteosarcoma cells (Fig. 4E and F). In order to explore whether TGF-a-induced PI3K signaling is mediated by EGFR activation; we used EGFR inhibitors PD158780 and BIBX1382. Pretreated cells with PD158780 and BIBX1382 for 30 min, and then treated TGF-a for 15 min. Western blot analysis indicated that both PD158780 and BIBX1382 significantly inhibited TGF-ainduced PI3K activation (Fig. 4G). The Akt Ser473 residue phosphorylation by a PI3K-dependent signaling pathway causes enzymatic activation [58]. To examine the important role of PI3K/Akt in cancer migration and ICAM-1 upregulation, we next determined Akt Ser473 phosphorylation in response to TGF-a treatment. Treatment of cells with TGF-a resulted in time-dependent phosphorylation of Akt Ser473 (Fig. 5A). Pretreatment of cells with Akti antagonized TGF-ainduced cell migration and ICAM-1 expression of osteosarcoma
Please cite this article in press as: Hou C-H, et al. Transforming growth factor alpha promotes osteosarcoma metastasis by ICAM-1 and PI3K/Akt signaling pathway. Biochem Pharmacol (2014), http://dx.doi.org/10.1016/j.bcp.2014.03.010
G Model
BCP-11938; No. of Pages 11 C.-H. Hou et al. / Biochemical Pharmacology xxx (2014) xxx–xxx
5
Fig. 2. TGF-a directed cell migration of human osteosarcoma cells involves upregulation of ICAM-1. (A and B) Total protein and mRNA were extracted form hFOB 1.19, MG63, and U2OS cells, the ICAM-1 expression was examined by western blot and qPCR analysis. (C and D) Cells were incubated with various concentrations of TGF-a for 24 h. The mRNA and protein expression of ICAM-1 were measured by qPCR and western blot. (E and F) Cells were incubated with TGF-a (10 ng/mL) for 6, 12 or 24 h. The mRNA and protein expression of ICAM-1 were measured by qPCR and western blot. (G and H) MG63 and U2OS cells were transfected with ICAM-1 or negative control siRNA for 24 h, the cell migration was analyzed using Transwell migration assay. (I) Cells were transfected with ICAM-1 or control siRNA for 24 h, followed by treatment with TGF-a for 24 h, and cell migration was analyzed using the Transwell migration assay. Results are expressed as the mean SEM. *, p < 0.05 compared with control; #, p < 0.05 compared with TGFa-treated group.
cells (Fig. 5B–D). On the other hand, transfection of cells with Akt mutant also inhibited TGF-a-induced cell migration and ICAM-1 expression of osteosarcoma cells (Fig. 5E and F). Based on these results, it appeared that TGF-a/EGFR can enhance ICAM-1 expression and cell migration through the PI3K and Akt-dependent signaling pathway to in human osteosarcoma cells. 3.5. NF-kB is involved in TGF-a-induced cell migration and ICAM-1 expression As previously mentioned, NF-kB is an important transcription factor whose activity is often correlated with cancer cell migration and invasion [59]. Therefore, we examined whether NF-kB activation was involved in the signal transduction pathway which leading TGF-a-promoted cell migration and ICAM-1 expression. Pretreatment of cells with the NF-kB inhibitor PDTC (10 mM) and the IkB protease inhibitor TPCK (10 mM) for 30 min markedly
inhibited TGF-a-induced cancer cell migration, ICAM-1 mRNA expression and ICAM-1 protein production (Fig. 6A–C). We next examined the levels of IKKa/b, IkBa and, NF-kB subunit p65 expression. The expression of phosphorylated IKKa/b, IkBa and NF-kB subunit p65 were higher after 30 min of TGF-a treatment and diminished after 60 min (Fig. 6D). Furthermore, Dominantnegative mutant of IKKa or IKKb markedly inhibited TGF-ainduced cell migration, ICAM-1 mRNA expression and ICAM-1 protein production (Fig. 6E and F). In addition, Pretreatment of cells with Ly294002, Wortmannin, or Akti reduced TGF-a-increased IKKa/b, IkB and p65 phosphorylation (Fig. 6G). Therefore, TGF-ainduced cell migration and ICAM-1 expression were mediated by the NF-kB signaling pathway. Next, we directly determined kB-luciferase activation after TGF-a treatment, the cells were transiently transfected with kBluciferase plasmid as an indicator of NF-kB activation. As shown in Fig. 6H, treated cells with TGF-a for 24 h resulted in increased
Please cite this article in press as: Hou C-H, et al. Transforming growth factor alpha promotes osteosarcoma metastasis by ICAM-1 and PI3K/Akt signaling pathway. Biochem Pharmacol (2014), http://dx.doi.org/10.1016/j.bcp.2014.03.010
G Model
BCP-11938; No. of Pages 11 6
C.-H. Hou et al. / Biochemical Pharmacology xxx (2014) xxx–xxx
Fig. 3. EGFR is involved in TGF-a-mediated migration of human osteosarcoma cells. (A) Cells were pretreated for 30 min with PD158780 (5 mM) or BIBX1382 (10 mM) followed by stimulation with TGF-a (10 ng/mL) for 24 h. In vitro migration was measured after 24 h using the Transwell migration assay. (B and C) Cells were pretreated for 30 min with PD158780 (5 mM) or BIBX1382 (10 mM) followed by stimulation with TGF-a (10 ng/mL) for 24 h. The mRNA and protein expression of ICAM-1 were measured by qPCR and Western blotting. (D) Cells were incubated with TGF-a (10 ng/mL) for indicated time intervals, and EGFR phosphorylation was examined by western blot. (E and F) Cells were transfected with EGFR siRNA for 24 h, followed by treatment with TGF-a (10 ng/mL) for 24 h, and cell migration was analyzed using the Transwell migration assay. (G) Cells were transfected with control and EGFR siRNA for 24 h, the ICAM-1 expression was examined by qPCR. (H-I) MG63 and U2OS cells were incubated with various concentrations of Cisplatin and Doxorubicin for 24 h, and the cell viability was examined by MTT assay. (J) Cells were pretreated for 30 min with PD158780 (5 mM), BIBX1382 (10 mM), Cisplatin (50 nM) or Doxorubicin (50 nM) followed by stimulation with TGF-a (10 ng/mL) for 24 h. In vitro migration was measured after 24 h using the Transwell migration assay. Results are expressed as the mean SEM. *, p < 0.05 compared with control; #, p < 0.05 compared with TGF-a-treated group.
kB-luciferase activity. (TNF-a acts as a positive control). In addition, BIBX1382, PD158780, LY294002, Wortmannin, Akti, PDTC and TPCK antagonized the TGF-a-induced kB-luciferase activity. Co-transfection of cells with PI3K, Akt, IKKa and IKKb mutant also reduced TGF-a-increased kB-luciferase activity (Fig. 6I). Based on these results, activation of the PI3K and Akt signaling pathways were required for the TGF-a-induced NF-kB activity in human osteosarcoma cells. 3.6. Knockdown of TGF-a expression inhibits cell migration in a mouse model of osteosarcoma To confirm that TGF-a mediated ICAM-1-dependent cell migration in human osteosarcoma cells, we took advantage of MG63 cells that stably expressed TGF-a shRNA. Following puromycin (10 mg/mL) selection, individual stable clones (TGF-a sh1, TGF-a sh2, TGF-a sh3 and TGF-a sh4) were collected for analysis. Empty vector plasmid was used as a negative control. The
expression levels of both TGF-a and ICAM-1 were decreased in all four TGF-a shRNA stable clones, and TGF-a sh4 showed the most knockdown efficiency (Fig. 7A). TGF-a knockdown did not affect the rate of cell proliferation (Fig. 7B), but significantly reduced cell migration (Fig. 7C). To further confirm that TGF-a mediated ICAM1-dependent cell migration in human osteosarcoma cells by EGFRPI3K-Akt pathway, the expression levels of EGFR, PI3K and Akt were decreased in TGF-a sh4 cells (Fig. 7D). Furthermore, to examine the effect of TGF-a on osteosarcoma growth in vivo, mouse tail vein was injected with TGF-a-knockdown cell (5 106), then sacrificed after 28 days later, and the results showed lung metastatic nodules. Furthermore, to investigate the metastatic potential of Control-shRNA and TGF-a-shRNA cells, we counted the number of metastatic nodules in tumor-bearing mice. The mice which were injected with TGF-a shRNA4 cells showed significantly decreased in mean number of lung metastatic nodules (Fig. 7E–G). Therefore, knockdown of TGF-a reduced cell migration in vitro and lung metastasis in vivo.
Please cite this article in press as: Hou C-H, et al. Transforming growth factor alpha promotes osteosarcoma metastasis by ICAM-1 and PI3K/Akt signaling pathway. Biochem Pharmacol (2014), http://dx.doi.org/10.1016/j.bcp.2014.03.010
G Model
BCP-11938; No. of Pages 11 C.-H. Hou et al. / Biochemical Pharmacology xxx (2014) xxx–xxx
7
Fig. 4. PI3K signaling affects the TGF-a response in human osteosarcoma. (A–C) Cells were pretreated with the control (0.1% DMSO), LY294002 (10 mM) and Wortmannin (1 mM) for 30 min followed by stimulation with TGF-a for 24 h the cell migration and ICAM-1 expression were examined by Transwell, qPCR and western blot. (D) Cells were incubated with TGF-a (10 ng/mL) for the indicated times, and PI3K phosphorylation was examined by western blot. (E and F) Cells were transfected with control and PI3K mutant for 24 h followed by stimulation with TGF-a for 24 h the cell migration and ICAM-1 expression were examined by Transwell migration and qPCR assay. (G) Cells were pretreated with the control (0.1% DMSO) and EGFR inhibitor PD158780 (5 mM) or BIBX1382 (10 mM) followed by stimulation with TGF-a for 15 min, and PI3K phosphorylation was examined by western blot. Results are expressed as the mean SEM. *, p < 0.05 compared with control; #, p < 0.05 compared with TGF-a-treated group.
Fig. 5. Akt is involved in TGF-a-mediated migration in human osteosarcoma cells. (A) Cells were incubated with TGF-a (10 ng/mL) for the indicated times and p-Akt or Akt levels were determined by western blot. (B–D) Cells were pretreated for 30 min with the Akti (1 mM) followed by treatment with TGF-a for 24 h, the cell migration and ICAM1 expression was examined by Transwell migration, qPCR and western blot. (E and F) Cells were transfected with Akt mutant for 24 h followed by treatment with TGF-a for 24 h, the cell migration and ICAM-1 expression was examined by Transwell migration and qPCR assay. Results are expressed as the mean SEM. *, p < 0.05 compared with control; #, p < 0.05 compared with TGF-a-treated group.
Please cite this article in press as: Hou C-H, et al. Transforming growth factor alpha promotes osteosarcoma metastasis by ICAM-1 and PI3K/Akt signaling pathway. Biochem Pharmacol (2014), http://dx.doi.org/10.1016/j.bcp.2014.03.010
G Model
BCP-11938; No. of Pages 11 8
C.-H. Hou et al. / Biochemical Pharmacology xxx (2014) xxx–xxx
Fig. 6. NF-kB mediates the response of human osteosarcoma cells to TGF-a stimulation. (A–C) Cells were pretreated with the control (0.1% DMSO), PDTC (10 mM) and TPCK (10 mM) for 30 min followed by stimulation with TGF-a for 24 h the cell migration and ICAM-1 expression was examined by Transwell migration, qPCR and western blot. (D) Cells were incubated with TGF-a (10 ng/mL) for the indicated times, and IKKa/b, IkBa, and p65 phosphorylation was examined by western blot. (E and F) Cells were transfected with control and IKKa and IKKb mutant for 24 h followed by stimulation with TGF-a (10 ng/mL) for 24 h the cell migration and ICAM-1 expression was examined by Transwell migration and qPCR assay. (G) Cells were pretreated with the control (0.1% DMSO), LY294002 (10 mM), Wortmannin (1 mM) and Akti (1 mM) followed by stimulation with TGF-a (10 ng/mL) for 30 min, and IKKa/b, IkBa, and p65 phosphorylation was examined by western blot. (H and I) Cells were transfected with an NF-kB promoter reporter plasmid for 24 h, then incubated with TGF-a for 24 h and luciferase activity was measured. Results are expressed as the mean SEM. *, p < 0.05 compared with control; #, p < 0.05 compared with TGF-a-treated group.
4. Discussion Osteosarcoma is the most common bone cancer, particularly among children and adolescents, with chronic symptoms of bone pain and swelling in leg or arm [60]. Osteosarcoma has a high propensity for local aggression and a tendency to metastasize to the lung and distant bones, which is the leading cause of mortality. Therefore, it is important to develop effective adjuvant therapy for preventing osteosarcoma metastasis. Here, we found that the expression of TGF-a correlated strongly with the tumor stage in human osteosarcoma patients. We also found that TGF-a increased cell migration both in human osteosarcoma cell lines
and in vivo models. One mechanism underlying TGF-a-directed migration was the transcriptional up-regulation of ICAM-1 and the activation of the EGFR, PI3K, Akt and NF-kB pathways. The ligands of EGFR, comprising 6 members (EGF, TGF-a, heparin-binding EGF-like growth factor (HB-EGF), amphiregulin (AR), epiregulin (EREG), and betacellulin (BTC)) are involved in the stimulation of cell proliferation, inflammation, migration, metastasis, angiogenesis and tumorigenesis [61–63]. TGF-a has been demonstrated capacity to promote tumor development, progression, invasion in the breast, lung, colon, ovary, prostate, and skin cancers [29,64]. In addition, TGF-a is believed to stimulate cell motility and promote aggressive behavior. However, a correlation
Please cite this article in press as: Hou C-H, et al. Transforming growth factor alpha promotes osteosarcoma metastasis by ICAM-1 and PI3K/Akt signaling pathway. Biochem Pharmacol (2014), http://dx.doi.org/10.1016/j.bcp.2014.03.010
G Model
BCP-11938; No. of Pages 11 C.-H. Hou et al. / Biochemical Pharmacology xxx (2014) xxx–xxx
9
Fig. 7. Knockdown of TGF-a reduces the migratory ability in vitro and in vivo. (A) The protein levels of TGF-a and ICAM-1 in control-shRNA and TGF-a-shRNA cells was examined by western blot. (B) MG63 cells stably expressing shRNA constructs were seeded as monolayers and counted daily. Cells (1 104) were reseeded after each count, and the cell numbers were plotted. (C) In vitro migration of MG-63 cells stably expressing shRNA constructs was measured using the Transwell migration assay. (D) The protein levels of p-EGFR, EGFR, p-PI3K, PI3K, p-Akt and Akt in control-shRNA and TGF-a-shRNA cells was examined by western blot. (E) MG63 cells were injected into the mouse tail vein. Mice were sacrificed after 28 days later developed lung metastasis nodules. Lungs were removed and inflated with 10% formalin, and the number of metastatic nodules on the lungs was counted with the aid of a dissecting microscope. (F) H&E staining of metastatic nodules in the lung of mice. (G) Quantification of metastatic nodules following tail vein injection. Results are expressed as mean SEM. #, p < 0.05 compared with control.
between osteosarcoma and TGF-a still remains unclear. Here, we found that the expression of TGF-a in osteosarcoma cell lines were significantly higher than that in the normal bone cell line. Treatment with exogenous TGF-a increased the migration of osteosarcoma cells. In contrast, over-expression of TGF-a shRNA inhibited the migratory ability. We also observed reduced effects with increasing concentration of TGF-a, we consider the cell surface has a limited number of receptor binding sites; therefore, it is saturated at high ligand concentration. The concentration of bound ligand versus that of total ligand is curvilinear. When all the binding sites are occupied, there is no further binding as increasing ligand concentration. Another reason is that the relationship between ligand concentration at the receptor and the response might be described with a sigmoid curve. As a result, reduced effects with increasing conc. of TGF-a were observed. Taken together, these data suggest that TGF-a is a novel marker for cancer progression and metastasis of bone sarcomas. ICAM-1 plays a pivotal role in leukocyte adhesion and cancer cell invasion [65,66]. In breast cancer, ICAM-1 can mediate adhesion-dependent cellular interactions and has a potential role in invasion. Moreover, its high expression induced tumor malignancy and poor prognosis [67]. A correlation between ICAM-1 and breast cancer has been reported, but the biological functions between ICAM-1 and osteosarcoma is unclear. Here, we show that ICAM-1 plays an important role in TGF-a-mediated osteosarcoma cell motility. First, TGF-a increased ICAM-1 mRNA and protein expression. Second, TGF-a-induced cell migration was inhibited by specific ICAM-1 siRNA. Third, TGF-a-shRNA cells showed a greater reduction in migration and ICAM-1 expression than control-shRNA cells. We provide evidence here that ICAM-1 is required for osteosarcoma metastasis. Although ICAM-1 was
reportedly associated with the cell migratory of osteosarcoma, we demonstrated that ICAM-1 is a downstream effector in TGF-aincreased metastasis of osteosarcoma. It has been reported that activation of EGFR and subsequent enhancement of PI3K/Akt activation mediated the signaling in response to TGF-a [68]. EGFR family plays an important role in inducing cell proliferation, survival, migration, and invasion [69,70]. In this study, we found that EGFR siRNA reduced TGFa-induced cell migration, through the blocking of TGF-a-increased ICAM-1 expression. Therefore, the interaction between TGF-a and EGFR is very important for cancer migration and ICAM-1 expression in human osteosarcoma cells. Recent studies indicate that overexpression of EGFR could promote cancer cell survival, proliferation, migration and invasion through up-regulated PI3K and Akt signaling pathway [71–73]. PI3K is a heterodimer consisting of an 85 kDa regulatory subunit and an 110 kDa catalytic subunit. The autophosphorylation of EGFR tyrosine residues can elicit PI3K and Akt signaling pathway through the interaction between the phosphotyrosine-binding SH2 domains on p85 subunit and tyrosine residues [74]. Phosphorylation of the p85 subunit is required for activation of the p110 catalytic subunit of PI3K [75]. We found TGF-a enhanced the p85 subunit and Akt phosphorylation in human osteosarcoma cells. Pretreatment of cells with PI3K and Akt inhibitor or transfection of cells with PI3K and Akt mutation plasmids could reduce the TGF-a-mediated ICAM-1 expression. These results suggest that TGF-a induced ICAM-1 production through EGFR activation and via the PI3K/Akt signaling pathway in osteosarcoma. The transcriptional factor NF-kB is associated with cancer cell invasion and migration [76]. Occasionally, NF-kB transcription factors exist in the cytoplasm and bind to the inhibitory protein
Please cite this article in press as: Hou C-H, et al. Transforming growth factor alpha promotes osteosarcoma metastasis by ICAM-1 and PI3K/Akt signaling pathway. Biochem Pharmacol (2014), http://dx.doi.org/10.1016/j.bcp.2014.03.010
G Model
BCP-11938; No. of Pages 11 C.-H. Hou et al. / Biochemical Pharmacology xxx (2014) xxx–xxx
10
IkB, which inhibits NF-kB binding and prevent nuclear uptake. Upon stimulation of cells, IkB proteins lead to rapid phosphorylation by the multisubunit IKK complex, then this targets IkB for ubiquitination and subsequent degradation by the 26S proteasome. The NF-kB pathway has also been linked to TGF-a signaling, and free NF-kB translocate to the nucleus, where it regulates gene transcription [77]. Treatment of cells with TGF-a led to increase levels of phosphorylated IKK, IkBa and p65. Using transient transfection of a kB-luciferase reporter construction that indicates NF-kB activity, we found that TGF-a increased NF-kB activity. Taken together, these results indicate that TGF-a acted through the EGFR, PI3K, Akt and NF-kB pathways to induce ICAM-1 expression in osteosarcoma. Finally, to directly determine the effect of TGF-a on osteosarcoma, we inhibited TGF-a expression in MG63 cells by using shRNA transfection. TGF-a knockdown significantly reduced ICAM-1 expression and inhibited cell migration ability in MG63 cells. These data indicated that TGF-a play an important role in osteosarcoma metastasis. Our study presents that TGF-a and EGFR interaction increases the expression of ICAM-1 via PI3K, Akt and NF-kB dependent pathway and increasing migration of human osteosarcoma cells. To the best of our knowledge, this study is the first time to attempt to examine the migratory activity of TGF-a in human osteosarcoma. Whether TGF-ainduces cell motility through same pathways in clinic should be examined further. Furthermore, the discovery of TGF-a-mediated pathway helps us to understand the mechanism of human osteosarcoma metastasis and may help us to develop effective therapy in the future. Author’s contributions JFL and SMH conceived and designed the experiments. CHH, FLL, KBT and JFL performed the experiments. CHH, KBT, SMH and JFL analyzed the data. CHH, FLL, KBT, SMH and JFL contributed reagents/materials/analysis tools. JFL and SMH wrote the paper. All authors read and approved the final manuscript. Conflicts of interest The authors state no conflict of interest. Acknowledgments This work was supported by grants from the National Science Council of Taiwan (NSC99-2320-B-039-003-MY3; NSC101-2314B-341-001-MY2) and Shin-Kong Wu Ho-Su Memorial Hospital (SKH-8302-102-0301; SKH-8302-102-0302). We thank the staff of the Eighth Core Lab, Department of Medical Research, National Taiwan University Hospital for technical support during the study. We thank Dr. W.M. Fu for kindly providing Akt dominant negative mutant. References [1] Damron TA, Ward WG, Stewart A. Osteosarcoma, chondrosarcoma, and Ewing’s sarcoma: National Cancer Data Base Report. Clin Orthop Relat Res 2007;459:40–7. [2] Dorfman HD, Czerniak B. Bone cancers. Cancer 1995;75:203–10. [3] Sweetnam R. Osteosarcoma. Br J Hosp Med 1982;28:112. 116–121. [4] Ottaviani G, Jaffe N. The epidemiology of osteosarcoma. Cancer Treat Res 2009;152:3–13. [5] Gill J, Ahluwalia MK, Geller D, Gorlick R. New targets and approaches in osteosarcoma. Pharmacol Ther 2012. [6] Munajat I, Zulmi W, Norazman MZ, Wan Faisham WI. Tumour volume and lung metastasis in patients with osteosarcoma. J Orthop Surg (Hong Kong) 2008;16:182–5. [7] Tumbar T, Guasch G, Greco V, Blanpain C, Lowry WE, Rendl M, et al. Defining the epithelial stem cell niche in skin. Science 2004;303:359–63.
[8] Spradling A, Drummond-Barbosa D, Kai T. Stem cells find their niche. Nature 2001;414:98–104. [9] Shen A, Zhang Y, Yang H, Xu R, Huang G. Overexpression of ZEB1 relates to metastasis and invasion in osteosarcoma. J Surg Oncol 2012;105:830–4. [10] Yi C, Li X, Xu W, Chen A. Relationship between the expression of MTA-1 gene and the metastasis and invasion in human osteosarcoma. J Huazhong Univ Sci Technol Med Sci 2005;25:445–7. [11] Tsubaki M, Satou T, Itoh T, Imano M, Ogaki M, Yanae M, et al. Reduction of metastasis, cell invasion, and adhesion in mouse osteosarcoma by YM529/ ONO-5920-induced blockade of the Ras/MEK/ERK and Ras/PI3K/Akt pathway. Toxicol Appl Pharmacol 2012;259:402–10. [12] Desgrosellier JS, Cheresh DA. Integrins in cancer: biological implications and therapeutic opportunities. Nat Rev Cancer 2010;10:9–22. [13] Lin YM, Chang ZL, Liao YY, Chou MC, Tang CH. IL-6 promotes ICAM-1 expression and cell motility in human osteosarcoma. Cancer Lett 2012. [14] Bruijn JA, de Heer E. Adhesion molecules in renal diseases. Lab Invest 1995;72:387–94. [15] Carlos TM, Schwartz BR, Kovach NL, Yee E, Rosa M, Osborn L, et al. Vascular cell adhesion molecule-1 mediates lymphocyte adherence to cytokine-activated cultured human endothelial cells. Blood 1990;76:965–70. [16] Garcia-Barcina M, Lukomska B, Gawron W, Winnock M, Vidal-Vanaclocha F, Bioulac-Sage P, et al. Expression of cell adhesion molecules on liver-associated lymphocytes and their ligands on sinusoidal lining cells in patients with benign or malignant liver disease. Am J Pathol 1995;146:1406–13. [17] Gahmberg CG, Tolvanen M, Kotovuori P. Leukocyte adhesion–structure and function of human leukocyte beta2-integrins and their cellular ligands. Eur J Biochem 1997;245:215–32. [18] Kurokouchi K, Kambe F, Yasukawa K, Izumi R, Ishiguro N, Iwata H, et al. TNFalpha increases expression of IL-6 and ICAM-1 genes through activation of NFkappaB in osteoblast-like ROS17/2.8 cells. J Bone Miner Res 1998;13:1290–9. [19] Kohka H, Yoshino T, Iwagaki H, Sakuma I, Tanimoto T, Matsuo Y, et al. Interleukin-18/interferon-gamma-inducing factor, a novel cytokine, up-regulates ICAM-1 (CD54) expression in KG-1 cells. J Leukoc Biol 1998;64:519–27. [20] Yu JA, Sadaria MR, Meng X, Mitra S, Ao L, Fullerton DA, et al. Lung cancer cell invasion and expression of intercellular adhesion molecule-1 (ICAM-1) are attenuated by secretory phospholipase A(2) inhibition. J Thorac Cardiovasc Surg 2012;143:405–11. [21] Tian MM, Sun Y, Li ZW, Wu Y, Zhao AL, Li JY. Polymorphisms of ICAM-1 are associated with gastric cancer risk and prognosis. World J Gastroenterol 2012;18:368–74. [22] Fong YC, Lin CY, Su YC, Chen WC, Tsai FJ, Tsai CH, et al. CCN6 enhances ICAM-1 expression and cell motility in human chondrosarcoma cells. J Cell Physiol 2012;227:223–32. [23] Ohchi T, Akagi Y, Kinugasa T, Kakuma T, Kawahara A, Sasatomi T, et al. Amphiregulin is a prognostic factor in colorectal cancer. Anticancer Res 2012;32:2315–21. [24] Makrilia N, Kollias A, Manolopoulos L, Syrigos K. Cell adhesion molecules: role and clinical significance in cancer. Cancer Invest 2009;27:1023–37. [25] Roland CL, Harken AH, Sarr MG, Barnett Jr CC. ICAM-1 expression determines malignant potential of cancer. Surgery 2007;141:705–7. [26] Stromberg K, Collins TJt, Gordon AW, Jackson CL, Johnson GR. Transforming growth factor-alpha acts as an autocrine growth factor in ovarian carcinoma cell lines. Cancer Res 1992;52:341–7. [27] Morishige K, Kurachi H, Amemiya K, Fujita Y, Yamamoto T, Miyake A, et al. Evidence for the involvement of transforming growth factor alpha and epidermal growth factor receptor autocrine growth mechanism in primary human ovarian cancers in vitro. Cancer Res 1991;51:5322–8. [28] Crew AJ, Langdon SP, Miller EP, Miller WR. Mitogenic effects of epidermal growth factor and transforming growth factor-alpha on EGF-receptor positive human ovarian carcinoma cell lines. Eur J Cancer 1992;28:337–41. [29] Sewell JM, Smyth JF, Langdon SP. Role of TGF alpha stimulation of the ERK, PI3 kinase and PLC gamma pathways in ovarian cancer growth and migration. Exp Cell Res 2005;304:305–16. [30] Pelletier S, Tanguay S, Lee S, Gunaratnam L, Arbour N, Lapointe R. TGF-alpha as a candidate tumor antigen for renal cell carcinomas. Cancer Immunol Immunother 2009;58:1207–18. [31] Ahmed SR, Badger B, Wright C, Manni A. Role of transforming growth factoralpha (TGF-alpha) in basal and hormone-stimulated growth by estradiol, prolactin and progesterone in human and rat mammary tumor cells: studies using TGF-alpha and EGF receptor antibodies. J Steroid Biochem Mol Biol 1991;38:687–93. [32] Liu D, Gagliardi G, Nasim MM, Alison MR, Oates T, Lalani EN, et al. TGF-alpha can act as morphogen and/or mitogen in a colon-cancer cell line. Int J Cancer 1994;56:603–8. [33] Kullenberg B, Jansen C, Fredang N, Ohlsson B, Axelson J. Transforming growth factor alpha (TGF-alpha) increases cell number in a human pancreatic cancer cell line but not in normal mouse pancreas. Int J Pancreatol 2000;28:199–205. [34] Baek JY, Morris SM, Campbell J, Fausto N, Yeh MM, Grady WM. TGF-beta inactivation and TGF-alpha overexpression cooperate in an in vivo mouse model to induce hepatocellular carcinoma that recapitulates molecular features of human liver cancer. Int J Cancer 2010;127:1060–71. [35] Breuhahn K, Longerich T, Schirmacher P. Dysregulation of growth factor signaling in human hepatocellular carcinoma. Oncogene 2006;25:3787–800. [36] Calvisi DF, Ladu S, Gorden A, Farina M, Conner EA, Lee JS, et al. Ubiquitous activation of Ras and Jak/Stat pathways in human HCC. Gastroenterology 2006;130:1117–28.
Please cite this article in press as: Hou C-H, et al. Transforming growth factor alpha promotes osteosarcoma metastasis by ICAM-1 and PI3K/Akt signaling pathway. Biochem Pharmacol (2014), http://dx.doi.org/10.1016/j.bcp.2014.03.010
G Model
BCP-11938; No. of Pages 11 C.-H. Hou et al. / Biochemical Pharmacology xxx (2014) xxx–xxx [37] Jhappan C, Stahle C, Harkins RN, Fausto N, Smith GH, Merlino GT. TGF alpha overexpression in transgenic mice induces liver neoplasia and abnormal development of the mammary gland and pancreas. Cell 1990;61:1137–46. [38] Santoni-Rugiu E, Jensen MR, Factor VM, Thorgeirsson SS. Acceleration of cmyc-induced hepatocarcinogenesis by co-expression of transforming growth factor (TGF)-alpha in transgenic mice is associated with TGF-beta1 signaling disruption. Am J Pathol 1999;154:1693–700. [39] Yeh MM, Larson AM, Campbell JS, Fausto N, Rulyak SJ, Swanson PE. The expression of transforming growth factor-alpha in cirrhosis, dysplastic nodules, and hepatocellular carcinoma: an immunohistochemical study of 70 cases. Am J Surg Pathol 2007;31:681–9. [40] Greten FR, Wagner M, Weber CK, Zechner U, Adler G, Schmid RM. TGF alpha transgenic mice. A model of pancreatic cancer development. Pancreatology 2001;1:363–8. [41] Kitano M, Wada J, Ariki Y, Kato M, Wanibuchi H, Morimura K, et al. Possible tumor development from double positive foci for TGF-alpha and GST-P observed in early stages on rat hepatocarcinogenesis. Cancer Sci 2006;97:478–83. [42] Pedersen PH, Ness GO, Engebraaten O, Bjerkvig R, Lillehaug JR, Laerum OD. Heterogeneous response to the growth factors [EGF, PDGF (bb), TGF-alpha, bFGF, IL-2] on glioma spheroid growth, migration and invasion. Int J Cancer 1994;56:255–61. [43] Kikuchi K, Li X, Zheng Y, Takano Y. Invasion of breast cancer cells into collagen matrix requires TGF-alpha and Cdc42 signaling. FEBS Lett 2011;585:286–90. [44] Ueda M, Fujii H, Yoshizawa K, Terai Y, Kumagai K, Ueki K, et al. Effects of EGF and TGF-alpha on invasion and proteinase expression of uterine cervical adenocarcinoma OMC-4 cells. Invasion Metastasis 1998;18:176–83. [45] Liu JF, Fong YC, Chang CS, Huang CY, Chen HT, Yang WH, et al. Cyclooxygenase2 enhances alpha2beta1 integrin expression and cell migration via EP1 dependent signaling pathway in human chondrosarcoma cells. Mol Cancer 2010;9:43. [46] Lin YM, Chang ZL, Liao YY, Chou MC, Tang CH. IL-6 promotes ICAM-1 expression and cell motility in human osteosarcoma. Cancer Lett 2013;328:135–43. [47] Hou CH, Tang CH, Hsu CJ, Hou SM, Liu JF. CCN4 induces IL-6 production through alphavbeta5 receptor, PI3K, Akt, and NF-kappaB singling pathway in human synovial fibroblasts. Arthritis Res Ther 2013;15:R19. [48] Liu JF, Yang WH, Fong YC, Kuo SC, Chang CS, Tang CH. BFPP, a phloroglucinol derivative, induces cell apoptosis in human chondrosarcoma cells through endoplasmic reticulum stress. Biochem Pharmacol 2010;79:1410–7. [49] Liu JF, Fong YC, Chang KW, Kuo SC, Chang CS, Tang CH. FPTB, a novel CA-4 derivative, induces cell apoptosis of human chondrosarcoma cells through mitochondrial dysfunction and endoplasmic reticulum stress pathways. J Cell Biochem 2011;112:453–62. [50] Liu JF, Huang YL, Yang WH, Chang CS, Tang CH. 1-Benzyl-2-phenylbenzimidazole (BPB), a benzimidazole derivative, induces cell apoptosis in human chondrosarcoma through intrinsic and extrinsic pathways. Int J Mol Sci 2012;13:16472–88. [51] Wang C, Lv X, Jiang C, Cordes CM, Fu L, Lele SM, et al. Transforming growth factor alpha (TGFalpha) regulates granulosa cell tumor (GCT) cell proliferation and migration through activation of multiple pathways. PLoS One 2012;7:e48299. [52] Kenny PA, Bissell MJ. Targeting TACE-dependent EGFR ligand shedding in breast cancer. J Clin Invest 2007;117:337–45. [53] Katerinaki E, Haycock JW, Lalla R, Carlson KE, Yang Y, Hill RP, et al. Sodium salicylate inhibits TNF-alpha-induced NF-kappaB activation, cell migration, invasion and ICAM-1 expression in human melanoma cells. Melanoma Res 2006;16:11–22. [54] Kesanakurti D, Chetty C, Rajasekhar Maddirela D, Gujrati M, Rao JS. Essential role of cooperative NF-kappaB and Stat3 recruitment to ICAM-1 intronic consensus elements in the regulation of radiation-induced invasion and migration in glioma. Oncogene 2012. [55] Kneissl J, Keller S, Lorber T, Heindl S, Keller G, Drexler I, et al. Association of amphiregulin with the cetuximab sensitivity of gastric cancer cell lines. Int J Oncol 2012;41:733–44.
11
[56] Higginbotham JN, Demory Beckler M, Gephart JD, Franklin JL, Bogatcheva G, Kremers GJ, et al. Amphiregulin exosomes increase cancer cell invasion. Curr Biol 2011;21:779–86. [57] Ivanov VN, Hei TK. Combined treatment with EGFR inhibitors and arsenite upregulated apoptosis in human EGFR-positive melanomas: a role of suppression of the PI3K-AKT pathway. Oncogene 2005;24:616–26. [58] Qiao M, Sheng S, Pardee AB. Metastasis and AKT activation. Cell Cycle 2008;7:2991–6. [59] Tang CH, Tan TW, Fu WM, Yang RS. Involvement of matrix metalloproteinase-9 in stromal cell-derived factor-1/CXCR4 pathway of lung cancer metastasis. Carcinogenesis 2008;29:35–43. [60] Goldsmith JR, Lee TH. High-grade intramedullary osteosarcoma of the talus: case report. J Foot Ankle Surg 2007;46:480–3. [61] Zhou ZN, Sharma VP, Beaty BT, Roh-Johnson M, Peterson EA, Van Rooijen N, et al. Autocrine HBEGF expression promotes breast cancer intravasation, metastasis and macrophage-independent invasion in vivo. Oncogene 2013. [62] Wang Z, Jin C, Li H, Li C, Hou Q, Liu M, et al. GPR48-induced keratinocyte proliferation occurs through HB-EGF mediated EGFR transactivation. FEBS Lett 2010;584:4057–62. [63] Sagmeister S, Drucker C, Losert A, Grusch M, Daryabeigi A, Parzefall W, et al. HB-EGF is a paracrine growth stimulator for early tumor prestages in inflammation-associated hepatocarcinogenesis. J Hepatol 2008;49:955–64. [64] Etindi R, Manni A. TGF-alpha and IGF-I effects on calcium ion transients in human breast cancer cells. Cancer Lett 1992;66:131–7. [65] Maruo Y, Gochi A, Kaihara A, Shimamura H, Yamada T, Tanaka N, et al. ICAM-1 expression and the soluble ICAM-1 level for evaluating the metastatic potential of gastric cancer. Int J Cancer 2002;100:486–90. [66] Liu G, Place AT, Chen Z, Brovkovych VM, Vogel SM, Muller WA, et al. ICAM-1activated Src and eNOS signaling increase endothelial cell surface PECAM-1 adhesivity and neutrophil transmigration. Blood 2012;120:1942–52. [67] Schroder C, Witzel I, Muller V, Krenkel S, Wirtz RM, Janicke F, et al. Prognostic value of intercellular adhesion molecule (ICAM)-1 expression in breast cancer. J Cancer Res Clin Oncol 2011;137:1193–201. [68] De Luca A, Gallo M, Aldinucci D, Ribatti D, Lamura L, D’Alessio A, et al. Role of the EGFR ligand/receptor system in the secretion of angiogenic factors in mesenchymal stem cells. J Cell Physiol 2011;226:2131–8. [69] Minder P, Bayha E, Becker-Pauly C, Sterchi EE. Meprinalpha transactivates the epidermal growth factor receptor (EGFR) via ligand shedding, thereby enhancing colorectal cancer cell proliferation and migration. J Biol Chem 2012;287:35201–11. [70] Xu JW, Li QQ, Tao LL, Cheng YY, Yu J, Chen Q, et al. Involvement of EGFR in the promotion of malignant properties in multidrug resistant breast cancer cells. Int J Oncol 2011;39:1501–9. [71] Lo HW, Hsu SC, Hung MC. EGFR signaling pathway in breast cancers: from traditional signal transduction to direct nuclear translocalization. Breast Cancer Res Treat 2006;95:211–8. [72] Diaz ME, Gonzalez L, Miquet JG, Martinez CS, Sotelo AI, Bartke A, et al. Growth hormone modulation of EGF-induced PI3K-Akt pathway in mice liver. Cell Signal 2012;24:514–23. [73] Berg M, Soreide K. EGFR and downstream genetic alterations in KRAS/BRAF and PI3K/AKT pathways in colorectal cancer: implications for targeted therapy. Discov Med 2012;14:207–14. [74] Heeg S, Hirt N, Queisser A, Schmieg H, Thaler M, Kunert H, et al. EGFR overexpression induces activation of telomerase via PI3K/AKT-mediated phosphorylation and transcriptional regulation through Hif1-alpha in a cellular model of oral-esophageal carcinogenesis. Cancer Sci 2011;102:351–60. [75] Chen PC, Lin TH, Cheng HC, Tang CH. CCN3 increases cell motility and ICAM-1 expression in prostate cancer cells. Carcinogenesis 2012;33:937–45. [76] Tang CH, Tsai CC. CCL2 increases MMP-9 expression and cell motility in human chondrosarcoma cells via the Ras/Raf/MEK/ERK/NF-kappaB signaling pathway. Biochem Pharmacol 2012;83:335–44. [77] Streicher KL, Willmarth NE, Garcia J, Boerner JL, Dewey TG, Ethier SP. Activation of a nuclear factor kappaB/interleukin-1 positive feedback loop by amphiregulin in human breast cancer cells. Mol Cancer Res 2007;5:847–61.
Please cite this article in press as: Hou C-H, et al. Transforming growth factor alpha promotes osteosarcoma metastasis by ICAM-1 and PI3K/Akt signaling pathway. Biochem Pharmacol (2014), http://dx.doi.org/10.1016/j.bcp.2014.03.010
