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TWEAK promotes ovarian cancer cell metastasis via NF-κB pathway activation and VEGF expression
Cancer Letters 283 (2009) 159–167
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TWEAK promotes ovarian cancer cell metastasis via NF-jB pathway activation and VEGF expression Lan Dai, Liying Gu, Chuanwei Ding, Lihua Qiu *, Wen Di * Department of OB/GYN, Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, PR China
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Article history: Received 7 January 2009 Received in revised form 6 March 2009 Accepted 25 March 2009
Keywords: TWEAK NF-jB VEGF Ovarian cancer Metastasis
a b s t r a c t The poor prognosis of human ovarian cancer is partly due to its metastasis and recurrence. It has been demonstrated that tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK)-fibroblast growth factor inducible-14 (Fn14) signaling system may be a potential regulator of human tumorigenesis. The objective of this study was to understand the effect of TWEAK on ovarian cancer metastasis. We recently showed that activation of Fn14 signaling by TWEAK promoted cell migration and invasion in human HO-8910PM cells. Treating HO-8910PM cells with TWEAK resulted in the activation of nuclear factor-kappa B (NFjB) and subsequently the translocation of NF-jB from cytoplasm to nucleus. In addition, TWEAK promoted vascular endothelial growth factor (VEGF) protein expression, and this effect was dependent upon NF-jB transcriptional activity. Blocking the NF-jB pathway with PDTC suppressed TWEAK-induced up-regulation of VEGF protein expression and cell metastasis. Our results suggest that TWEAK-Fn14 functions, in part, through the NF-jB signaling pathway to up-regulate VEGF expression to foster ovarian cancer cell metastasis. Targeted therapy against TWEAK-Fn14 signaling system as an adjuvant to surgery may improve clinical management of invasive ovarian cancer cells and advance the outcome of this devastating cancer. Ó 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Most deaths from ovarian cancer are due to metastases that are resistant to therapy. An understanding of ovarian cancer oncogenesis has steadily improved, but the molecular mechanisms mediating ovarian cancer metastasis are still nascent. Gene discovery strategies have been used with the anticipation of identifying molecular pathways that regulate ovarian cancer cell migration and invasion [1]. Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK), a member of the TNF superfamily, is a multifunctional cytokine that regulates cellular proliferation, angiogenesis, inflammation, and apoptosis. TWEAK acts on responsive cells via binding to a small cell-surface
* Corresponding authors. Tel.: +86 21 58752345 3616; fax: +86 21 58752345 3073 (L. Qiu), tel./fax: +86 21 58752345 3073 (W. Di). E-mail addresses: [email protected] (L. Dai), [email protected] (L. Qiu), [email protected] (W. Di). 0304-3835/$ - see front matter Ó 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2009.03.036
receptor named fibroblast growth factor-inducible-14 (Fn14). Fn14 is a type I transmembrane protein of 102 amino acids in length after removal of signal peptide, making it the smallest member of TNF superfamily of receptors [2,3]. It has been shown that Fn14 expression is elevated in several cancerous tissues, including hepatocellular carcinomas, glioblastoma multiforme, and pancreatic cancer. TWEAK-Fn14 signaling contributes to various malignant processes, such as tumor invasion, metastasis, and angiogenesis [4]. Furthermore, activation of the Fn14 receptor by addition of TWEAK promotes nuclear factorkappa B (NF-jB) pathway activation, which may drive the expression of several genes related to malignant transformation [5]. Numerous in vitro and in vivo studies have suggested that NF-jB plays an important role in regulating cell proliferation, angiogenesis, adhesion, invasion and metastasis. NF-jB functions as a dimer composed of the RelA (p65) and NF-jB1 (p50) or NF-jB2 (p52) subunits. In normal
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resting cells, NF-jB is sequestered in the cytoplasm by virtue of binding to IjB. NF-jB activation involves its release from its inhibitor, IjB, and its subsequent translocation from the cytoplasm to the nucleus, where it binds to cognate sequences in the promoter region of multiple genes. Regulating gene expression by NF-jB is controlled mainly by the inhibitory IjB proteins, which include IjBa. Upon stimulation, IjB-a is rapidly phosphorylated and degraded via the ubiquitin–proteasome pathway, permitting activation and nuclear import of NF-jB [6]. Vascular endothelial growth factor (VEGF) is NF-jB inducible protein, which functions as pro-angiogenic factors. It has been shown that VEGF induce the proliferation and survival of endothelial cells, increase vascular permeability, and promote the production of plasminogen activator by these cells. VEGF is one of the most potent and specific angiogenic factors of tumor-induced angiogenesis, which is crucial for primary tumor growth and metastasis [7,8]. VEGF overexpression has been observed in several ovarian cancer cell lines and ovarian cancer biopsies of various histological grades [9]. In addition, numerous reports have demonstrated that in ovarian cancer as well as in other tumor types, the metastatic potential of tumor cells directly correlates with the expression level of VEGF [10]. Ovarian cancer exhibits highly invasive behavior constituting major obstacles for successful therapy and patient outcome. To elaborate the role of Fn14 in ovarian cancer pathobiology, we examined Fn14 activation as a potential mechanism by which cell invasion is fostered. We showed that capacities of migration and invasion of TWEAK-stimulated ovarian cancer cells were increased. In addition, we demonstrated that activation of NF-jB by the TWEAK-Fn14 ligand-receptor system underlies the molecular basis of ovarian cancer cell invasion. Moreover our data indicated that NF-jB promoted ovarian cancer cell invasion, in part, by up-regulating the expression of VEGF protein. 2. Materials and methods
2.2. Cellular proliferation assay Cell proliferation was assessed 24, 48 and 72 h after TWEAK (PeproTech) treatment by measuring the conversion of the trazolium salt WST-1 (Roche) to formazan according to the manufacturer’s instructions. Briefly, cells were seeded into 96-well plates and incubated with TWEAK at various concentrations (0, 1, 10, 100, 1000 ng/ ml). At each time point, 10 ll WST-1 were added to each well and cultured at 37 °C for 2 h. The supernatant from each plate was collected for measurement of absorbance at 450 nm. Under the experimental conditions of the current studies, there is a direct correlation between the absorbance at 450 nm and cell number. 2.3. Adhesion assay Nintey-six-well plates were dry-coated with Matrigel (BD Biosciences) and blocked with 1% bovine serum albumin for 1 h. TWEAK-pretreated cells (2 104) were allowed to adhere to the Matrigel-coated well at 37 °C for 1 h. After incubation, each well was washed five times with PBS. The number of cells in each well was measured by using WST1 assay according to the manufacturer’s instructions. 2.4. Cell migration assay Cell motility was examined using a Boyden chamber apparatus. HO-8910PM cells were collected by trypsinization and suspended in serum-free medium in the presence of 0, 1, 10, 100, 1000 ng/ml of TWEAK at 5 105/ml. Cells (5 104) were placed into the upper wells of the Boyden chamber (Millicell, 8-lm pore size, 12-mm diameter; Millipore). The medium containing 20% fetal bovine serum was added to the lower chamber. Cells were allowed to migrate for 8 h at 37 °C in a humidified atmosphere containing 5% CO2. Then cells on the upper side of the membrane were removed by cotton swabs and the membrane was fixed in methanol and stained with haematoxylin and eosin. Cells on the lower side of the membrane were counted using a light microscope at 200 magnification.
2.1. Cell culture 2.5. Matrigel invasion assay Human HO-8910PM cell line was obtained from Cell Bank, Chinese Academy of Sciences. Parental HO-8910 cell line was established from ascites of a patient with a malignant papillary serous adenocarcinoma of the ovary. HO-8910PM is a highly metastatic clone obtained by limiting dilution cloning of parental HO-8910 and has been used in many studies due to its highly metastatic activity [11–13]. It was cultured in Roswell Park Memorial Institute 1640 medium (RPMI1640) (Gibco) supplemented with 10% fetal bovine serum (Gibco) and penicillin/streptomycin (1:100, Sigma) in a humid atmosphere incubator with 5% CO2 at 37 °C. Unless otherwise indicated, cells were grown to 70–80% confluence, then serum-starved overnight in serum-free RPMI1640 medium prior to treatment. When inhibitors were used, cells were pretreated for 1 h to stimulation with the indicated concentration of inhibitor, which remained in the medium for the remainder of the experiment.
Human HO-8910PM cells were collected by trypsinization. Resuspended cells (5 105/ml) in serum-free medium in the presence of 0, 1, 10, 100, 1000 ng/ml of TWEAK were then added to an inner cup of the 48-well Boyden chamber that had been coated with 50 ll of Matrigel (1:3 dilution in serum-free medium). Medium supplemented with 20% serum was added to the outer cup. After 24 h of incubation, cells that had invaded through the Matrigel and the 8 lm pore size membrane were fixed, stained, and counted under a light microscope at 200 magnification. 2.6. Preparation of total cell lysates, cytoplasmic and nuclear protein extracts Cells with and without treatment were washed with cold PBS and harvested by scraping into 0.2 ml RIPA buffer.
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Cell lysates were incubated on ice for 30 min. After centrifugation at 14,000 rpm for 10 min at 4° C, total cell lysates were collected as supernatant. Cytoplasmic and nuclear protein extracts were prepared using the Nuclear and Cytoplasmic Extraction Reagents Kit (Pierce) according to the supplier’s instructions. Protein concentration was determined using the Bradford method. 2.7. Western blot analysis Proteins were isolated as described above and separated by 12%, 10% or 8% SDS–PAGE and electro-transferred to Immobilon-P membrane (Millipore) for 2 h at 4 °C. Nonspecific binding was blocked with 10% dry milk in TBST for 1 h at room temperature. With constant shaking, the membranes were incubated with primary antibodies against NF-jB (p65), IjB-a, IjB-a-Ser32P (Cell Signaling) and VEGF (Santa Cruz) in dilution buffer overnight at 4 °C and then in secondary antibodies at room temperature for 1 h with constant shaking. The expression of targeted proteins was detected by an ECL Kit (Amersham) following the manufacturer’s instructions and visualized by autoradiography with hyper film. 2.8. ELISA assay After the supernatants were harvested from cell cultures, probes were centrifuged at 3000g for 15 min to remove cells and other large debris, aliquoted, and immediately stored at 70 °C. To determine the level of VEGF secretion under the different experimental conditions, cell culture supernatants were collected and subject to a specific enzyme-linked immunosorbent assay (ELISA) from R&D systems according to the manufacturer’s instructions. 2.9. Statistics For statistical analyzes we used SPSS software. Values are presented as means ± SD. We used ANOVA to compare mean values. A P value of less than 0.05 was defined as statistically significant.
Fig. 2. TWEAK enhanced cell adhesion in human HO-8910PM cells. Human HO-8910PM cells were pretreated with TWEAK for 6 h. Then cells were added to a precoated 96-well plate containing Matrigel and allowed to adhere for 1 h at 37 °C. The number of attached cells was determined by using WST-1 assay. The results are shown as the mean ± SD from triplicate cultures (*p < 0.05 vs. untreated group).
3. Results 3.1. TWEAK had no effect on the proliferation of human HO-8910PM cells It was previously reported that TWEAK could stimulate human hepatocellular carcinoma cell growth in a dose-dependent manner [14]. Here we investigated the effect of TWEAK on ovarian cancer cell proliferation. Human HO-8910PM cells were cultured in 96-well cell culture plates and treated with TWEAK (0, 1, 10, 100, 1000 ng/ml) for 24, 48 and 72 h. Growth curve showed that HO-8910PM cells treated with different concentrations of TWEAK exhibited no significant difference in proliferation rate over the period tested (Fig. 1). 3.2. TWEAK enhanced cell adhesion to Matrigel in human HO-8910PM cells Matrigel is a soluble basement membrane preparation extracted from Engelbreth–Holm–Swarm (EHS) mouse sarcoma, and contains almost all of the extra-cellular matrix (ECM) components. We assessed the potential effect of TWEAK on cell adhesion to ECM by using Matrigel. Human HO8910PM cells were pretreated with TWEAK for 6 h, harvested with nonenzymatic dissociation buffer and plated to wells precoated with Matrigel. We found that the number of adherent HO-8910PM cells was significantly increased in a dose-dependent manner (Fig. 2). 3.3. TWEAK promoted cell migration and invasion in human HO-8910PM cells Invasion and metastasis are the most significant biological characteristics of malignant tumor. Human HO-8910PM cells were treated with 0, 1, 10, 100, 1000 ng/ml of TWEAK. Cell migration assay was used to evaluate the effect of TWEAK treatment on HO-8910PM cell migration, and it revealed that TWEAK treatment resulted in a remarkable promotion of ovarian cancer cell migration in a dose-dependent manner (Fig. 3). Matrigel invasion assay was used to evaluate the change in invasive ability of HO-8910PM cells treated with 0, 1, 10, 100, 1000 ng/ml of TWEAK. The invasive potential was determined on the basis of the ability of cells to invade a matrix barrier containing mainly laminin and type IV collagen, the major components of the basement membrane. The result showed that TWEAK significantly increased the invasive activity of HO-8910PM cells in a dose-dependent manner (Fig. 4). 3.4. TWEAK promoted NF-jB activation and IjB-a phosphorylation in human HO-8910PM cells
Fig. 1. TWEAK had no effect on the proliferation of human HO-8910PM cells. Human HO-8910PM cells were cultured in 96-well cell culture plates, and treated with 0, 1, 10, 100, 1000 ng/ml of TWEAK. Cell proliferation was assessed 24, 48 and 72 h after TWEAK treatment by cellular proliferation assay. The results are shown as the mean ± SD from triplicate cultures (p > 0.05 vs. untreated group).
Previous studies have shown that TWEAK treatment of various cell types stimulated NF-jB activation [15]. To determine whether TWEAK promotes NF-jB activation in human HO-8910PM cells, we isolated the nuclear fraction of cells stimulated with 100 ng/ml of TWEAK and immunoblotted for the NF-jB p65 protein subunit. Densitometric analysis revealed an increased level of p65 protein in the nuclear lysates of
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Fig. 3. TWEAK promoted cell migration in human HO-8910PM cells. Human HO-8910PM cells (5 104) in serum-free medium in the presence of 0, 1, 10, 100, 1000 ng/ml of TWEAK were placed into the upper wells of the Boyden chamber. The medium containing 20% fetal bovine serum was added to the lower chamber. Cells were then allowed to migrate for 8 h. Cell migration was assessed by the number of cells migrating into the lower surface of the polycarbonic membrane. The results are shown as the mean ± SD from triplicate cultures (p < 0.05 vs. untreated group). cells exposed to TWEAK (Fig. 5a). NF-jB translocation and transcriptional activity are inhibited by its association with IjB-a proteins [16]. For NF-jB nuclear translocation and transcriptional activation to occur, phosphorylation of IjB has to occur at serine residues 32 and 36, which results in proteosome-mediated IjB-a degradation and liberation of NF-jB. Western Blot analysis of whole cellular lysates of HO8910PM cells after TWEAK treatment using an anti-IjB-a antibody showed an induction of IjB-a phosphorylation over the indicated time (Fig. 5b). Concomitantly the level of endogenous IjB-a protein decreased, as expected, upon TWEAK treatment, corresponding to IjB-a phosphorylation. 3.5. TWEAK promoted VEGF expression through NF-jB pathway activation Angiogenesis is crucial for tumor growth and metastasis. VEGF is one of the most potent and specific angiogenic factors of tumor-induced angiogenesis. To explore whether increased invasive capability of ovarian cancer cells is attributed to increased expression of VEGF, we treated HO8910PM cells with TWEAK for various time and concentrations. Western Blot analysis and ELISA assay shown in Figs. 6a, b and 7a, b indicated that VEGF protein levels increased in a time- and dose-dependent manner upon TWEAK treatment. It’s known that VEGF is NF-jB inducible protein. To determine whether VEGF expression induced by TWEAK was mediated by NF-jB pathway, we pretreated HO-8910PM cells with pyrrolidine dithiocarbamate (PDTC) (Sigma), which is a potent anti-oxidant inhibitor of NF-jB, and has been extensively used in studies for inhibition of NF-jB activation [17]. Cells were then treated with 100 ng/ml of TWEAK for 24 h. Western Blot analysis and ELISA assay showed that PDTC remark-
ably inhibited TWEAK-induced VEGF expression in HO-8910PM cells (Figs. 6c and 7c). 3.6. NF-jB inhibitor suppressed TWEAK-induced cell adhesion, migration and invasion To investigate the role of NF-jB in TWEAK-induced ovarian cancer cell adhesion, migration and invasion, we antagonized NF-jB function by using the NF-jB inhibitor PDTC. HO-8910PM cells were treated with TWEAK (100 ng/ml) alone or in combination with PDTC (20 lM). The abilities of adhesion, migration and invasion were detected by cell adhesion assay, cell migration assay and Matrigel invasion assay, respectively. Our data indicated that the enhancement of cell invasive behavior induced by TWEAK treatment was significantly inhibited by PDTC (Figs. 8–10).
4. Discussion It has been demonstrated that TWEAK is expressed in tumor cell lines and tissues and can induce human tumor cell migration and invasion. Tran et al. [18] have reported that TWEAK binding to the Fn14 receptor stimulates human SF767 and U118 glioma cell migration. In addition, TWEAK treatment of human T98G glioma cells induces MMP-9 mRNA expression which is crucial for glioma cell invasive activity. To the best of our knowledge, there’s no
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Fig. 4. TWEAK promoted ovarian cancer cell invasion in human HO-8910PM cells. Human HO-8910PM cells (5 105/ml) in serum-free medium in the presence of 0, 1, 10, 100, 1000 ng/ml of TWEAK were added to an inner cup of the 48-well Boyden chamber that had been coated with 50 ll of Matrigel. Medium supplemented with 20% serum was added to the outer cup. After 24 h of incubation, cell invasion was assessed by the number of cells invading into the outer cup via the Matrigel and the 8 lm pore size membrane. The results are shown as the mean ± SD from triplicate cultures (p < 0.05 vs. untreated group).
published data on the effect of TWEAK on ovarian cancer. In this report, we described that TWEAK treatment may be involved in stimulating ovarian cancer cell adhesion to extra-cellular matrix, which suggested that TWEAK might play a key role in regulating tumor growth at the secondary site. In vitro invasion and migration assay revealed that treating HO-8910PM cells with TWEAK significantly promoted both cell invasion and cell migration in a dosedependent manner. Our results provide evidence for the involvement of TWEAK in inducing metastasis of ovarian cancer cells. As we know, the ability to metastasize makes ovarian cancer a fatal disease. Metastasis is also one of the major obstacles to the treatment of ovarian cancer. However, the molecular mechanisms and/or intrinsic factors controlling ovarian cancer invasion are not well understood. Thus, exploring the function of TWEAK-Fn14 may lead to the development of effective therapies against invasive ovarian cancer. Elevated NF-jB activity is observed in various cancers including ovarian cancer. NF-jB activation may contribute to cellular resistance to cytotoxic interventions and promote cell invasion by up-regulating genes involved in cell survival
(i.e., BCL-XL, A1), cell–cell adhesion (i.e., intercellular adhesion molecule-1) and cell–extra-cellular matrix (ECM) interactions (i.e., tenacin-C, laminin B2 chain) [6]. We speculate that inhibition of NF-jB activity may suppress ovarian cancer cell migration and invasion. TWEAK has been shown to induce NF-jB activation via the Fn14 receptor, which results in a rapid [5] and long lasting NF-jB activation via IjB-a and p100 regulation [15]. In the present study, both IjB-a phosphorylation and NF-jB nuclear translocation were observed in TWEAK-stimulated HO-8910PM cells. Our results are in agreement with previous studies, which revealed that TWEAK-Fn14 signaling operates via activation of NF-jB pathways. Furthermore, we noted that inhibition of NF-jB activity by its pharmacologic inhibitor PDTC suppressed TWEAK-induced cell migration and invasion. It is possible that the pathophysiological roles of TWEAK-Fn14 signaling may contribute to constitutive NF-jB activity in invasive ovarian cancer cells and hence lead to stimulation of cell migration and invasion. Angiogenesis is defined as the formation of new blood vessels from preexisting vasculature. Preclinical studies have shown the major role of angiogenesis in tumor
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Fig. 5. TWEAK induced NF-jB activation and IjB-a phosphorylation in human HO-8910PM cells. HO-8910PM cells were treated with 100 ng/ml of TWEAK for 0, 5, 15, 30 and 60 min. (a) Cells were lysed after TWEAK treatment, and nuclear extracts were prepared and immunoblotted for either the p65 subunit of NF-jB or nucleolin. (b) Whole cell lysates of TWEAK-treated cells were immunoblotted for phospho-IjB-a, total IjB-a and GAPDH. Nucleolin and GAPDH were used as the loading control. Quantification of the target protein bands relative to nucleolin or GAPDH is shown in the right panel (*p < 0.05 vs. untreated group).
Fig. 6. TWEAK promoted VEGF expression through NF-jB pathway activation in human HO-8910PM cells. (a) Cells were treated with TWEAK for various time as indicated. (b) Cells were treated with TWEAK with various concentrations as indicated for 24 h. (c) Cells were pretreated with NF-jB inhibitor PDTC (10 lM and 20 lM) for 1 h and then were treated with TWEAK (100 ng/ml) for 24 h. VEGF and GAPDH expression were analyzed by Western Blot analysis. GAPDH was used as the loading control. Quantification of the target protein bands relative to GAPDH is shown in the right panel (p < 0.05 vs. untreated group, #p < 0.05 vs. TWEAK-treated group).
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Fig. 8. NF-jB inhibitor suppressed TWEAK-induced cell adhesion in human HO-8910PM cells. Human HO-8910PM cells were pretreated with TWEAK (100 ng/ml) alone or in combination with PDTC (20 lM) for 6 h. Then cells were added to a precoated 96-well plate containing Matrigel and allowed to adhere for 1 h at 37 °C. The number of attached cells was determined by using WST-1 assay. The results are shown as the mean ± SD from triplicate cultures (*p < 0.05 vs. TWEAK-treated group).
Fig. 7. TWEAK induced secretion of VEGF through NF-jB pathway activation in human HO-8910PM cells. (a) Cells were treated with TWEAK (100 ng/ml) for various time as indicated. (b) Cells were treated with TWEAK with various concentrations as indicated for 24 h. (c) Cells were pretreated with NF-jB inhibitor PDTC (10 lM and 20 lM) for 1 h and then were treated with TWEAK (100 ng/ml) for 24 h. VEGF secretion was analyzed by ELISA. Results are presented as mean ± SD in pg/ml in relation to 1 105 cells (*p < 0.05 vs. untreated group, #p < 0.05 vs. TWEAK-treated group).
growth and metastasis formation, and therefore, inhibiting tumor angiogenesis may be a promising therapeutic modality. VEGF is one of the most potent and specific angiogenic factors of tumor-induced angiogenesis. The clinical importance of VEGF for tumor growth and invasion is supported by the fact that most tumors produce VEGF and that inhibition of VEGF-induced angiogenesis significantly suppresses tumor growth and invasion in vitro and in vivo [19,20]. In addition, molecular cross-talk between TWEAK, a potential tumor angiogenesis factor, and VEGF may stimulate angiogenesis. It has been demonstrated that VEGF-A can induce Fn14 gene expression in endothelial cells (EC) and VEGF-A-stimulated EC proliferation is potentiated by TWEAK [21]. In this study, we presented evidence that TWEAK can induce VEGF expression in HO-8910PM cells in a time- and dose-dependent manner. Moreover, blocking NF-jB pathway with PDTC sup-
Fig. 9. NF-jB inhibitor suppressed TWEAK-induced cell migration in human HO-8910PM cells. Human HO-8910PM cells in serum-free medium in the presence of TWEAK (100 ng/ml) alone or in combination with PDTC (20 lM) were placed into the upper wells of the Boyden chamber. The medium containing 20% fetal bovine serum was added to the lower chamber. Cells were then allowed to migrate for 8 h. Cell migration was assessed by the number of cells migrating into the lower surface of the polycarbonic membrane. The results are shown as the mean ± SD from triplicate cultures (*p < 0.05 vs. TWEAK-treated group).
pressed TWEAK-induced VEGF protein expression and cell invasion. These results suggest that TWEAK-Fn14 func-
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suggest that TWEAK-Fn14/NF-jB pathway may be a potential regulator of ovarian cancer cell metastasis. Targeted therapy against TWEAK-Fn14/NF-jB pathway as an adjuvant to surgery may improve management of invasive ovarian cancer cells and advance the outcome of this devastating cancer. Conflicts of interest statement None declared. Acknowledgement This project was supported by the Shanghai ‘‘Phosphor” Science Foundation, China (Grant No. 06QA14032). References
Fig. 10. NF-jB inhibitor suppressed TWEAK-induced cell invasion in human HO-8910PM cells. Human HO-8910PM cells in serum-free medium in the presence of TWEAK (100 ng/ml) alone or in combination with PDTC (20 lM) were added to an inner cup of the 48-well Boyden chamber that had been coated with 50 ll of Matrigel. Medium supplemented with 20% serum was added to the outer cup. After 24 h of incubation, cell invasion was assessed by the number of cells invading into the outer cup via the Matrigel and the 8 lm pore size membrane. The results are shown as the mean ± SD from triplicate cultures (*p < 0.05 vs. TWEAK-treated group).
tions, in part, through the NF-jB signaling pathway to upregulate VEGF expression to foster ovarian cancer cell metastasis. Targeting this pathway may result in novel approaches for ovarian cancer therapeutics. However, the exact mechanism of TWEAK-induced VEGF expression is still not clear. There have been only a few studies investigating the TWEAK intracellular signal transduction pathway. TWEAK has been shown to promote the activation of numerous intracellular signal transduction cascades, including those involving the IjB kinase responsible for NF-jB activation and the mitogen-activated protein kinases JNK, p38, and ERK responsible for c-Jun, ATF-2 and Elk-1 phosphorylation [22]. We speculate that TWEAK may also induce VEGF expression through above NF-jB independent pathways and some unknown signaling pathways. Further in-depth investigations are needed to reveal the novel mechanisms. In summary, we present here that TWEAK promoted human HO-8910PM cell metastasis through activation of NF-jB and up-regulation of VEGF expression. Inhibition of TWEAK-activated NF-jB pathway suppressed cell adhesion, migration and invasion in HO-8910PM cells. Our data
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Cancer Letters journal homepage: www.elsevier.com/locate/canlet
TWEAK promotes ovarian cancer cell metastasis via NF-jB pathway activation and VEGF expression Lan Dai, Liying Gu, Chuanwei Ding, Lihua Qiu *, Wen Di * Department of OB/GYN, Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, PR China
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Article history: Received 7 January 2009 Received in revised form 6 March 2009 Accepted 25 March 2009
Keywords: TWEAK NF-jB VEGF Ovarian cancer Metastasis
a b s t r a c t The poor prognosis of human ovarian cancer is partly due to its metastasis and recurrence. It has been demonstrated that tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK)-fibroblast growth factor inducible-14 (Fn14) signaling system may be a potential regulator of human tumorigenesis. The objective of this study was to understand the effect of TWEAK on ovarian cancer metastasis. We recently showed that activation of Fn14 signaling by TWEAK promoted cell migration and invasion in human HO-8910PM cells. Treating HO-8910PM cells with TWEAK resulted in the activation of nuclear factor-kappa B (NFjB) and subsequently the translocation of NF-jB from cytoplasm to nucleus. In addition, TWEAK promoted vascular endothelial growth factor (VEGF) protein expression, and this effect was dependent upon NF-jB transcriptional activity. Blocking the NF-jB pathway with PDTC suppressed TWEAK-induced up-regulation of VEGF protein expression and cell metastasis. Our results suggest that TWEAK-Fn14 functions, in part, through the NF-jB signaling pathway to up-regulate VEGF expression to foster ovarian cancer cell metastasis. Targeted therapy against TWEAK-Fn14 signaling system as an adjuvant to surgery may improve clinical management of invasive ovarian cancer cells and advance the outcome of this devastating cancer. Ó 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Most deaths from ovarian cancer are due to metastases that are resistant to therapy. An understanding of ovarian cancer oncogenesis has steadily improved, but the molecular mechanisms mediating ovarian cancer metastasis are still nascent. Gene discovery strategies have been used with the anticipation of identifying molecular pathways that regulate ovarian cancer cell migration and invasion [1]. Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK), a member of the TNF superfamily, is a multifunctional cytokine that regulates cellular proliferation, angiogenesis, inflammation, and apoptosis. TWEAK acts on responsive cells via binding to a small cell-surface
* Corresponding authors. Tel.: +86 21 58752345 3616; fax: +86 21 58752345 3073 (L. Qiu), tel./fax: +86 21 58752345 3073 (W. Di). E-mail addresses: [email protected] (L. Dai), [email protected] (L. Qiu), [email protected] (W. Di). 0304-3835/$ - see front matter Ó 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2009.03.036
receptor named fibroblast growth factor-inducible-14 (Fn14). Fn14 is a type I transmembrane protein of 102 amino acids in length after removal of signal peptide, making it the smallest member of TNF superfamily of receptors [2,3]. It has been shown that Fn14 expression is elevated in several cancerous tissues, including hepatocellular carcinomas, glioblastoma multiforme, and pancreatic cancer. TWEAK-Fn14 signaling contributes to various malignant processes, such as tumor invasion, metastasis, and angiogenesis [4]. Furthermore, activation of the Fn14 receptor by addition of TWEAK promotes nuclear factorkappa B (NF-jB) pathway activation, which may drive the expression of several genes related to malignant transformation [5]. Numerous in vitro and in vivo studies have suggested that NF-jB plays an important role in regulating cell proliferation, angiogenesis, adhesion, invasion and metastasis. NF-jB functions as a dimer composed of the RelA (p65) and NF-jB1 (p50) or NF-jB2 (p52) subunits. In normal
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resting cells, NF-jB is sequestered in the cytoplasm by virtue of binding to IjB. NF-jB activation involves its release from its inhibitor, IjB, and its subsequent translocation from the cytoplasm to the nucleus, where it binds to cognate sequences in the promoter region of multiple genes. Regulating gene expression by NF-jB is controlled mainly by the inhibitory IjB proteins, which include IjBa. Upon stimulation, IjB-a is rapidly phosphorylated and degraded via the ubiquitin–proteasome pathway, permitting activation and nuclear import of NF-jB [6]. Vascular endothelial growth factor (VEGF) is NF-jB inducible protein, which functions as pro-angiogenic factors. It has been shown that VEGF induce the proliferation and survival of endothelial cells, increase vascular permeability, and promote the production of plasminogen activator by these cells. VEGF is one of the most potent and specific angiogenic factors of tumor-induced angiogenesis, which is crucial for primary tumor growth and metastasis [7,8]. VEGF overexpression has been observed in several ovarian cancer cell lines and ovarian cancer biopsies of various histological grades [9]. In addition, numerous reports have demonstrated that in ovarian cancer as well as in other tumor types, the metastatic potential of tumor cells directly correlates with the expression level of VEGF [10]. Ovarian cancer exhibits highly invasive behavior constituting major obstacles for successful therapy and patient outcome. To elaborate the role of Fn14 in ovarian cancer pathobiology, we examined Fn14 activation as a potential mechanism by which cell invasion is fostered. We showed that capacities of migration and invasion of TWEAK-stimulated ovarian cancer cells were increased. In addition, we demonstrated that activation of NF-jB by the TWEAK-Fn14 ligand-receptor system underlies the molecular basis of ovarian cancer cell invasion. Moreover our data indicated that NF-jB promoted ovarian cancer cell invasion, in part, by up-regulating the expression of VEGF protein. 2. Materials and methods
2.2. Cellular proliferation assay Cell proliferation was assessed 24, 48 and 72 h after TWEAK (PeproTech) treatment by measuring the conversion of the trazolium salt WST-1 (Roche) to formazan according to the manufacturer’s instructions. Briefly, cells were seeded into 96-well plates and incubated with TWEAK at various concentrations (0, 1, 10, 100, 1000 ng/ ml). At each time point, 10 ll WST-1 were added to each well and cultured at 37 °C for 2 h. The supernatant from each plate was collected for measurement of absorbance at 450 nm. Under the experimental conditions of the current studies, there is a direct correlation between the absorbance at 450 nm and cell number. 2.3. Adhesion assay Nintey-six-well plates were dry-coated with Matrigel (BD Biosciences) and blocked with 1% bovine serum albumin for 1 h. TWEAK-pretreated cells (2 104) were allowed to adhere to the Matrigel-coated well at 37 °C for 1 h. After incubation, each well was washed five times with PBS. The number of cells in each well was measured by using WST1 assay according to the manufacturer’s instructions. 2.4. Cell migration assay Cell motility was examined using a Boyden chamber apparatus. HO-8910PM cells were collected by trypsinization and suspended in serum-free medium in the presence of 0, 1, 10, 100, 1000 ng/ml of TWEAK at 5 105/ml. Cells (5 104) were placed into the upper wells of the Boyden chamber (Millicell, 8-lm pore size, 12-mm diameter; Millipore). The medium containing 20% fetal bovine serum was added to the lower chamber. Cells were allowed to migrate for 8 h at 37 °C in a humidified atmosphere containing 5% CO2. Then cells on the upper side of the membrane were removed by cotton swabs and the membrane was fixed in methanol and stained with haematoxylin and eosin. Cells on the lower side of the membrane were counted using a light microscope at 200 magnification.
2.1. Cell culture 2.5. Matrigel invasion assay Human HO-8910PM cell line was obtained from Cell Bank, Chinese Academy of Sciences. Parental HO-8910 cell line was established from ascites of a patient with a malignant papillary serous adenocarcinoma of the ovary. HO-8910PM is a highly metastatic clone obtained by limiting dilution cloning of parental HO-8910 and has been used in many studies due to its highly metastatic activity [11–13]. It was cultured in Roswell Park Memorial Institute 1640 medium (RPMI1640) (Gibco) supplemented with 10% fetal bovine serum (Gibco) and penicillin/streptomycin (1:100, Sigma) in a humid atmosphere incubator with 5% CO2 at 37 °C. Unless otherwise indicated, cells were grown to 70–80% confluence, then serum-starved overnight in serum-free RPMI1640 medium prior to treatment. When inhibitors were used, cells were pretreated for 1 h to stimulation with the indicated concentration of inhibitor, which remained in the medium for the remainder of the experiment.
Human HO-8910PM cells were collected by trypsinization. Resuspended cells (5 105/ml) in serum-free medium in the presence of 0, 1, 10, 100, 1000 ng/ml of TWEAK were then added to an inner cup of the 48-well Boyden chamber that had been coated with 50 ll of Matrigel (1:3 dilution in serum-free medium). Medium supplemented with 20% serum was added to the outer cup. After 24 h of incubation, cells that had invaded through the Matrigel and the 8 lm pore size membrane were fixed, stained, and counted under a light microscope at 200 magnification. 2.6. Preparation of total cell lysates, cytoplasmic and nuclear protein extracts Cells with and without treatment were washed with cold PBS and harvested by scraping into 0.2 ml RIPA buffer.
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Cell lysates were incubated on ice for 30 min. After centrifugation at 14,000 rpm for 10 min at 4° C, total cell lysates were collected as supernatant. Cytoplasmic and nuclear protein extracts were prepared using the Nuclear and Cytoplasmic Extraction Reagents Kit (Pierce) according to the supplier’s instructions. Protein concentration was determined using the Bradford method. 2.7. Western blot analysis Proteins were isolated as described above and separated by 12%, 10% or 8% SDS–PAGE and electro-transferred to Immobilon-P membrane (Millipore) for 2 h at 4 °C. Nonspecific binding was blocked with 10% dry milk in TBST for 1 h at room temperature. With constant shaking, the membranes were incubated with primary antibodies against NF-jB (p65), IjB-a, IjB-a-Ser32P (Cell Signaling) and VEGF (Santa Cruz) in dilution buffer overnight at 4 °C and then in secondary antibodies at room temperature for 1 h with constant shaking. The expression of targeted proteins was detected by an ECL Kit (Amersham) following the manufacturer’s instructions and visualized by autoradiography with hyper film. 2.8. ELISA assay After the supernatants were harvested from cell cultures, probes were centrifuged at 3000g for 15 min to remove cells and other large debris, aliquoted, and immediately stored at 70 °C. To determine the level of VEGF secretion under the different experimental conditions, cell culture supernatants were collected and subject to a specific enzyme-linked immunosorbent assay (ELISA) from R&D systems according to the manufacturer’s instructions. 2.9. Statistics For statistical analyzes we used SPSS software. Values are presented as means ± SD. We used ANOVA to compare mean values. A P value of less than 0.05 was defined as statistically significant.
Fig. 2. TWEAK enhanced cell adhesion in human HO-8910PM cells. Human HO-8910PM cells were pretreated with TWEAK for 6 h. Then cells were added to a precoated 96-well plate containing Matrigel and allowed to adhere for 1 h at 37 °C. The number of attached cells was determined by using WST-1 assay. The results are shown as the mean ± SD from triplicate cultures (*p < 0.05 vs. untreated group).
3. Results 3.1. TWEAK had no effect on the proliferation of human HO-8910PM cells It was previously reported that TWEAK could stimulate human hepatocellular carcinoma cell growth in a dose-dependent manner [14]. Here we investigated the effect of TWEAK on ovarian cancer cell proliferation. Human HO-8910PM cells were cultured in 96-well cell culture plates and treated with TWEAK (0, 1, 10, 100, 1000 ng/ml) for 24, 48 and 72 h. Growth curve showed that HO-8910PM cells treated with different concentrations of TWEAK exhibited no significant difference in proliferation rate over the period tested (Fig. 1). 3.2. TWEAK enhanced cell adhesion to Matrigel in human HO-8910PM cells Matrigel is a soluble basement membrane preparation extracted from Engelbreth–Holm–Swarm (EHS) mouse sarcoma, and contains almost all of the extra-cellular matrix (ECM) components. We assessed the potential effect of TWEAK on cell adhesion to ECM by using Matrigel. Human HO8910PM cells were pretreated with TWEAK for 6 h, harvested with nonenzymatic dissociation buffer and plated to wells precoated with Matrigel. We found that the number of adherent HO-8910PM cells was significantly increased in a dose-dependent manner (Fig. 2). 3.3. TWEAK promoted cell migration and invasion in human HO-8910PM cells Invasion and metastasis are the most significant biological characteristics of malignant tumor. Human HO-8910PM cells were treated with 0, 1, 10, 100, 1000 ng/ml of TWEAK. Cell migration assay was used to evaluate the effect of TWEAK treatment on HO-8910PM cell migration, and it revealed that TWEAK treatment resulted in a remarkable promotion of ovarian cancer cell migration in a dose-dependent manner (Fig. 3). Matrigel invasion assay was used to evaluate the change in invasive ability of HO-8910PM cells treated with 0, 1, 10, 100, 1000 ng/ml of TWEAK. The invasive potential was determined on the basis of the ability of cells to invade a matrix barrier containing mainly laminin and type IV collagen, the major components of the basement membrane. The result showed that TWEAK significantly increased the invasive activity of HO-8910PM cells in a dose-dependent manner (Fig. 4). 3.4. TWEAK promoted NF-jB activation and IjB-a phosphorylation in human HO-8910PM cells
Fig. 1. TWEAK had no effect on the proliferation of human HO-8910PM cells. Human HO-8910PM cells were cultured in 96-well cell culture plates, and treated with 0, 1, 10, 100, 1000 ng/ml of TWEAK. Cell proliferation was assessed 24, 48 and 72 h after TWEAK treatment by cellular proliferation assay. The results are shown as the mean ± SD from triplicate cultures (p > 0.05 vs. untreated group).
Previous studies have shown that TWEAK treatment of various cell types stimulated NF-jB activation [15]. To determine whether TWEAK promotes NF-jB activation in human HO-8910PM cells, we isolated the nuclear fraction of cells stimulated with 100 ng/ml of TWEAK and immunoblotted for the NF-jB p65 protein subunit. Densitometric analysis revealed an increased level of p65 protein in the nuclear lysates of
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Fig. 3. TWEAK promoted cell migration in human HO-8910PM cells. Human HO-8910PM cells (5 104) in serum-free medium in the presence of 0, 1, 10, 100, 1000 ng/ml of TWEAK were placed into the upper wells of the Boyden chamber. The medium containing 20% fetal bovine serum was added to the lower chamber. Cells were then allowed to migrate for 8 h. Cell migration was assessed by the number of cells migrating into the lower surface of the polycarbonic membrane. The results are shown as the mean ± SD from triplicate cultures (p < 0.05 vs. untreated group). cells exposed to TWEAK (Fig. 5a). NF-jB translocation and transcriptional activity are inhibited by its association with IjB-a proteins [16]. For NF-jB nuclear translocation and transcriptional activation to occur, phosphorylation of IjB has to occur at serine residues 32 and 36, which results in proteosome-mediated IjB-a degradation and liberation of NF-jB. Western Blot analysis of whole cellular lysates of HO8910PM cells after TWEAK treatment using an anti-IjB-a antibody showed an induction of IjB-a phosphorylation over the indicated time (Fig. 5b). Concomitantly the level of endogenous IjB-a protein decreased, as expected, upon TWEAK treatment, corresponding to IjB-a phosphorylation. 3.5. TWEAK promoted VEGF expression through NF-jB pathway activation Angiogenesis is crucial for tumor growth and metastasis. VEGF is one of the most potent and specific angiogenic factors of tumor-induced angiogenesis. To explore whether increased invasive capability of ovarian cancer cells is attributed to increased expression of VEGF, we treated HO8910PM cells with TWEAK for various time and concentrations. Western Blot analysis and ELISA assay shown in Figs. 6a, b and 7a, b indicated that VEGF protein levels increased in a time- and dose-dependent manner upon TWEAK treatment. It’s known that VEGF is NF-jB inducible protein. To determine whether VEGF expression induced by TWEAK was mediated by NF-jB pathway, we pretreated HO-8910PM cells with pyrrolidine dithiocarbamate (PDTC) (Sigma), which is a potent anti-oxidant inhibitor of NF-jB, and has been extensively used in studies for inhibition of NF-jB activation [17]. Cells were then treated with 100 ng/ml of TWEAK for 24 h. Western Blot analysis and ELISA assay showed that PDTC remark-
ably inhibited TWEAK-induced VEGF expression in HO-8910PM cells (Figs. 6c and 7c). 3.6. NF-jB inhibitor suppressed TWEAK-induced cell adhesion, migration and invasion To investigate the role of NF-jB in TWEAK-induced ovarian cancer cell adhesion, migration and invasion, we antagonized NF-jB function by using the NF-jB inhibitor PDTC. HO-8910PM cells were treated with TWEAK (100 ng/ml) alone or in combination with PDTC (20 lM). The abilities of adhesion, migration and invasion were detected by cell adhesion assay, cell migration assay and Matrigel invasion assay, respectively. Our data indicated that the enhancement of cell invasive behavior induced by TWEAK treatment was significantly inhibited by PDTC (Figs. 8–10).
4. Discussion It has been demonstrated that TWEAK is expressed in tumor cell lines and tissues and can induce human tumor cell migration and invasion. Tran et al. [18] have reported that TWEAK binding to the Fn14 receptor stimulates human SF767 and U118 glioma cell migration. In addition, TWEAK treatment of human T98G glioma cells induces MMP-9 mRNA expression which is crucial for glioma cell invasive activity. To the best of our knowledge, there’s no
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Fig. 4. TWEAK promoted ovarian cancer cell invasion in human HO-8910PM cells. Human HO-8910PM cells (5 105/ml) in serum-free medium in the presence of 0, 1, 10, 100, 1000 ng/ml of TWEAK were added to an inner cup of the 48-well Boyden chamber that had been coated with 50 ll of Matrigel. Medium supplemented with 20% serum was added to the outer cup. After 24 h of incubation, cell invasion was assessed by the number of cells invading into the outer cup via the Matrigel and the 8 lm pore size membrane. The results are shown as the mean ± SD from triplicate cultures (p < 0.05 vs. untreated group).
published data on the effect of TWEAK on ovarian cancer. In this report, we described that TWEAK treatment may be involved in stimulating ovarian cancer cell adhesion to extra-cellular matrix, which suggested that TWEAK might play a key role in regulating tumor growth at the secondary site. In vitro invasion and migration assay revealed that treating HO-8910PM cells with TWEAK significantly promoted both cell invasion and cell migration in a dosedependent manner. Our results provide evidence for the involvement of TWEAK in inducing metastasis of ovarian cancer cells. As we know, the ability to metastasize makes ovarian cancer a fatal disease. Metastasis is also one of the major obstacles to the treatment of ovarian cancer. However, the molecular mechanisms and/or intrinsic factors controlling ovarian cancer invasion are not well understood. Thus, exploring the function of TWEAK-Fn14 may lead to the development of effective therapies against invasive ovarian cancer. Elevated NF-jB activity is observed in various cancers including ovarian cancer. NF-jB activation may contribute to cellular resistance to cytotoxic interventions and promote cell invasion by up-regulating genes involved in cell survival
(i.e., BCL-XL, A1), cell–cell adhesion (i.e., intercellular adhesion molecule-1) and cell–extra-cellular matrix (ECM) interactions (i.e., tenacin-C, laminin B2 chain) [6]. We speculate that inhibition of NF-jB activity may suppress ovarian cancer cell migration and invasion. TWEAK has been shown to induce NF-jB activation via the Fn14 receptor, which results in a rapid [5] and long lasting NF-jB activation via IjB-a and p100 regulation [15]. In the present study, both IjB-a phosphorylation and NF-jB nuclear translocation were observed in TWEAK-stimulated HO-8910PM cells. Our results are in agreement with previous studies, which revealed that TWEAK-Fn14 signaling operates via activation of NF-jB pathways. Furthermore, we noted that inhibition of NF-jB activity by its pharmacologic inhibitor PDTC suppressed TWEAK-induced cell migration and invasion. It is possible that the pathophysiological roles of TWEAK-Fn14 signaling may contribute to constitutive NF-jB activity in invasive ovarian cancer cells and hence lead to stimulation of cell migration and invasion. Angiogenesis is defined as the formation of new blood vessels from preexisting vasculature. Preclinical studies have shown the major role of angiogenesis in tumor
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Fig. 5. TWEAK induced NF-jB activation and IjB-a phosphorylation in human HO-8910PM cells. HO-8910PM cells were treated with 100 ng/ml of TWEAK for 0, 5, 15, 30 and 60 min. (a) Cells were lysed after TWEAK treatment, and nuclear extracts were prepared and immunoblotted for either the p65 subunit of NF-jB or nucleolin. (b) Whole cell lysates of TWEAK-treated cells were immunoblotted for phospho-IjB-a, total IjB-a and GAPDH. Nucleolin and GAPDH were used as the loading control. Quantification of the target protein bands relative to nucleolin or GAPDH is shown in the right panel (*p < 0.05 vs. untreated group).
Fig. 6. TWEAK promoted VEGF expression through NF-jB pathway activation in human HO-8910PM cells. (a) Cells were treated with TWEAK for various time as indicated. (b) Cells were treated with TWEAK with various concentrations as indicated for 24 h. (c) Cells were pretreated with NF-jB inhibitor PDTC (10 lM and 20 lM) for 1 h and then were treated with TWEAK (100 ng/ml) for 24 h. VEGF and GAPDH expression were analyzed by Western Blot analysis. GAPDH was used as the loading control. Quantification of the target protein bands relative to GAPDH is shown in the right panel (p < 0.05 vs. untreated group, #p < 0.05 vs. TWEAK-treated group).
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Fig. 8. NF-jB inhibitor suppressed TWEAK-induced cell adhesion in human HO-8910PM cells. Human HO-8910PM cells were pretreated with TWEAK (100 ng/ml) alone or in combination with PDTC (20 lM) for 6 h. Then cells were added to a precoated 96-well plate containing Matrigel and allowed to adhere for 1 h at 37 °C. The number of attached cells was determined by using WST-1 assay. The results are shown as the mean ± SD from triplicate cultures (*p < 0.05 vs. TWEAK-treated group).
Fig. 7. TWEAK induced secretion of VEGF through NF-jB pathway activation in human HO-8910PM cells. (a) Cells were treated with TWEAK (100 ng/ml) for various time as indicated. (b) Cells were treated with TWEAK with various concentrations as indicated for 24 h. (c) Cells were pretreated with NF-jB inhibitor PDTC (10 lM and 20 lM) for 1 h and then were treated with TWEAK (100 ng/ml) for 24 h. VEGF secretion was analyzed by ELISA. Results are presented as mean ± SD in pg/ml in relation to 1 105 cells (*p < 0.05 vs. untreated group, #p < 0.05 vs. TWEAK-treated group).
growth and metastasis formation, and therefore, inhibiting tumor angiogenesis may be a promising therapeutic modality. VEGF is one of the most potent and specific angiogenic factors of tumor-induced angiogenesis. The clinical importance of VEGF for tumor growth and invasion is supported by the fact that most tumors produce VEGF and that inhibition of VEGF-induced angiogenesis significantly suppresses tumor growth and invasion in vitro and in vivo [19,20]. In addition, molecular cross-talk between TWEAK, a potential tumor angiogenesis factor, and VEGF may stimulate angiogenesis. It has been demonstrated that VEGF-A can induce Fn14 gene expression in endothelial cells (EC) and VEGF-A-stimulated EC proliferation is potentiated by TWEAK [21]. In this study, we presented evidence that TWEAK can induce VEGF expression in HO-8910PM cells in a time- and dose-dependent manner. Moreover, blocking NF-jB pathway with PDTC sup-
Fig. 9. NF-jB inhibitor suppressed TWEAK-induced cell migration in human HO-8910PM cells. Human HO-8910PM cells in serum-free medium in the presence of TWEAK (100 ng/ml) alone or in combination with PDTC (20 lM) were placed into the upper wells of the Boyden chamber. The medium containing 20% fetal bovine serum was added to the lower chamber. Cells were then allowed to migrate for 8 h. Cell migration was assessed by the number of cells migrating into the lower surface of the polycarbonic membrane. The results are shown as the mean ± SD from triplicate cultures (*p < 0.05 vs. TWEAK-treated group).
pressed TWEAK-induced VEGF protein expression and cell invasion. These results suggest that TWEAK-Fn14 func-
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suggest that TWEAK-Fn14/NF-jB pathway may be a potential regulator of ovarian cancer cell metastasis. Targeted therapy against TWEAK-Fn14/NF-jB pathway as an adjuvant to surgery may improve management of invasive ovarian cancer cells and advance the outcome of this devastating cancer. Conflicts of interest statement None declared. Acknowledgement This project was supported by the Shanghai ‘‘Phosphor” Science Foundation, China (Grant No. 06QA14032). References
Fig. 10. NF-jB inhibitor suppressed TWEAK-induced cell invasion in human HO-8910PM cells. Human HO-8910PM cells in serum-free medium in the presence of TWEAK (100 ng/ml) alone or in combination with PDTC (20 lM) were added to an inner cup of the 48-well Boyden chamber that had been coated with 50 ll of Matrigel. Medium supplemented with 20% serum was added to the outer cup. After 24 h of incubation, cell invasion was assessed by the number of cells invading into the outer cup via the Matrigel and the 8 lm pore size membrane. The results are shown as the mean ± SD from triplicate cultures (*p < 0.05 vs. TWEAK-treated group).
tions, in part, through the NF-jB signaling pathway to upregulate VEGF expression to foster ovarian cancer cell metastasis. Targeting this pathway may result in novel approaches for ovarian cancer therapeutics. However, the exact mechanism of TWEAK-induced VEGF expression is still not clear. There have been only a few studies investigating the TWEAK intracellular signal transduction pathway. TWEAK has been shown to promote the activation of numerous intracellular signal transduction cascades, including those involving the IjB kinase responsible for NF-jB activation and the mitogen-activated protein kinases JNK, p38, and ERK responsible for c-Jun, ATF-2 and Elk-1 phosphorylation [22]. We speculate that TWEAK may also induce VEGF expression through above NF-jB independent pathways and some unknown signaling pathways. Further in-depth investigations are needed to reveal the novel mechanisms. In summary, we present here that TWEAK promoted human HO-8910PM cell metastasis through activation of NF-jB and up-regulation of VEGF expression. Inhibition of TWEAK-activated NF-jB pathway suppressed cell adhesion, migration and invasion in HO-8910PM cells. Our data
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