Synergistic effects of combined platelet-activating factor receptor and epidermal growth factor receptor targeting in ovarian cancer cells
© Yu et al.; licensee BioMed Central Ltd. 2014
Received: 14 March 2014
Accepted: 30 April 2014
Published: 6 May 2014
Genetic alterations, including the overexpression of epidermal growth factor receptor (EGFR), play a crucial role in ovarian carcinogenesis. To date, EGFR targeting has shown limited antitumor effects in ovarian cancer when administered as monotherapy. We previously identified platelet-activating factor receptor (PAFR) as being overexpressed in ovarian cancer and found that its ligand PAF evoked EGFR phosphorylation. To determine whether PAFR targeting can enhance the antitumor efficacy of EGFR inhibition, we investigated the effects of a PAFR antagonist (WEB2086) in conjunction with an EGFR inhibitor (AG1478).
The expression of EGFR and PAFR in CAOV-3 and SKOV-3 ovarian cancer cell lines was measured by Western blot and immunocytochemistry. Synergy was determined using isobologram analysis. The effects of combined PAFR and EGFR targeting on both cells were assessed by using CCK-8, transwell, flow cytometry, western blot analysis. In vivo studies were conducted using CAOV-3 cells xenografted in nu/nu mice.
Treatment with combination WEB2086 and AG1478 resulted in significantly greater inhibition of proliferation and invasion compared to either drug alone. When examining equipotent combinations of WEB2086 and AG1478 to determine potential synergy, a combination index (CI) of 0.49 was identified for CAOV-3 cells and a CI of 0.58 for SKOV-3 cells indicating synergy. This co-inhibition induced significantly more apoptosis and arrested the cells at G0/G1 phase in both cell lines. The activation of PAFR and/or EGFR induced phosphorylation of the mTOR, AKT, and MAPK pathways. Combined PAFR and EGFR targeting synergistically diminished the expression of PAFR and EGFR phosphorylation and downstream signaling. In vivo studies further verified the antitumor effects of combined PAFR and EGFR targeting in a CAOV-3 xenograft model.
These results suggest that WEB2086 and AG1478 are synergistic in ovarian cancer cells with high expression of both PAFR and EGFR. The presented approach may have important therapeutic implications in the treatment of ovarian cancer patients.
KeywordsPlatelet-activating factor receptor (PAFR) Epidermal growth factor receptor (EGFR) Ovarian cancer Combined-targeting Signal pathway
Ovarian cancer is the fifth most common cause of death from all cancers among women in the world and has the highest mortality rate of gynecological cancers. Overall, ovarian cancer has the worst prognosis of all gynecological cancers, with a 5-year survival rate of less than 40%. Surgical resection and platinum-based combination regimens offer a modest but significant survival advantage in ovarian cancer patients with advanced or metastatic disease, though most patients eventually experience disease progression. Advances in the understanding of the molecular biology of cancer have enabled the discovery of several potential molecular targets and the development of novel targeted therapies.
Epidermal growth factor receptor (EGFR) is involved in the development and progression of several human cancers, including ovarian cancer. The most common type of ovarian cancer arises from ovarian surface epithelium, tissue that commonly expresses EGFR. Approximately 70% of ovarian tumors express activated EGFR. EGFR is a transmembrane receptor that plays a significant role in neural development and the formation of skin. EGFR also plays a role in various pro-survival and anti-apoptotic pathways in cancer cells[5–7]. Furthermore, EGFR is also involved in cell migration, metastasis, angiogenesis, and the epithelial mesenchymal transition (EMT)[8–10]. However, recent clinical trials targeting EGFR with cetuximab[11–13], matuzumab[14, 15], gefitinib, and erlotinib[17, 18] in epithelial ovarian cancer patients have shown only modest clinical responsiveness. The modest responses of EGFR blockade when monoclonal antibodies or tyrosine kinase inhibitors are administered as single agents could be attributed to compensation by other signaling pathways.
Various ligands such as epidermal growth factor (EGF) and transforming growth factor (TGF) can activate EGFR. Our previous studies have demonstrated that platelet-activating factor (PAF) also induced increased EGFR phosphorylation. PAF is one of major phospholipid mediators functioning in many different biological pathways in inflammatory diseases and cancers. PAF induces diverse biological effects through its specific receptor, PAFR, which belongs to the G-protein coupled receptor (GPCR) family[21–23]. We have demonstrated that the PAFR gene and protein are overexpressed in ovarian cancer tissues and cells and that PAF can promote the proliferation and invasion of ovarian cancer cells in a PAFR-dependent manner. These results suggest that activated EGFR and PAFR may synergistically promote the progression of ovarian cancer and that the constitutive activation of EGFR and downstream signaling pathways by PAFR may contribute to the inefficacy of EGFR inhibitors in ovarian cancer.
The aim of the present work was to determine whether the addition of PAFR targeting can enhance the antitumor efficacy of EGFR tyrosine kinase inhibitors. The PAFR antagonist WEB2086 was combined with the EGFR inhibitor AG1478 in ovarian cancer in vitro and in vivo. The effects of the two agents, alone and in combination, were determined in vitro and in vivo and the underlying molecular mechanisms were assessed.
Materials and methods
Cell culture and chemical reagents
The ovarian cancer cell lines CAOV-3 and SKOV-3 (purchased from the Cell Bank of the Chinese Academy of Science, Shanghai, China) were cultured at 37°C in a humidified 5% CO2 atmosphere in RPMI-1640 medium with 10% fetal calf serum (Gibco, Invitrogen, Carlsbad, CA), 100 IU/ml penicillin G, and 100 mg/ml streptomycin sulfate (Sigma-Aldrich, St. Louis, MO). AG1478 (EGFR inhibitor) and WEB2086 (PAFR antagonist) were purchased from Sigma-Aldrich (St Louis, MO). A Cell Counting Kit 8 (CCK8) was purchased from Dojindo Molecular Technologies, Inc. (Kumamoto, Japan), and the Alexa Fluor 488 annexin V/Dead Cell Apoptosis Kit was obtained Invitrogen (Carlsbad, CA). Rabbit polyclonal antibodies directed against PAFR, cleaved-caspase3, cleaved-PARP, phospho/total- EGFR, phospho/total- β-arrestin2, phospho/total- P70S6K, phospho/total- AKT, phospho/total- 4EBP1, and phospho/total- ERK were used in this study. All of these antibodies were purchased from Cell Signaling Technology Co. Mouse monoclonal antibodies directed against actin were also used (Sigma, Missouri, USA).
Western blot analysis
Cellular extracts were prepared in modified radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris–HCl pH 7.4, 1% NP-40, 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, and protease inhibitor cocktail). The protein concentrations of the cellular extracts were measured using a Bio-Rad protein assay kit. The cellular extracts were subjected to SDS-PAGE, and the proteins were transferred to PVDF membranes. After blocking for 1 h at room temperature in 5% BSA, the blots were probed with the primary antibody at a 1:1000 dilution and incubated overnight at 4°C. Subsequently, the blots were washed three times and incubated for 1 h at room temperature with a 1:10000 dilution of the secondary peroxidase-conjugated antibodies. Following three washes, the immunoreactive bands were detected using electrochemiluminescence (ECL).
Immunocytochemistry was used to detect the expression of EGFR and PAFR. After fixation with 4% paraformaldehyde, cells were incubated with an anti-EGFR (1:50) or -PAFR (1:50) antibody overnight, and then incubated with peroxidase-conjugated anti-rabbit IgG for 30 min. The staining reaction was performed with diaminobenzidine. The cells were counter-stained with hematoxylin to detect nuclei, and imaged using light microscopy (Olympus, Tokyo, Japan).
Cell proliferation and invasion assay
Cells were seeded in 96-well plates and treated with different doses of WEB2086, AG1478, or both for 72 hours. Cell proliferation was measured with the CCK-8 assay. A 10-μL aliquot of CCK-8 solution was added to 100 μL medium in each well for 1–4 hours, and the absorbance was measured at 450 nm. The percentage of cell viability was determined relative to the control. Each experiment was performed in six replicate wells for each drug concentration. The IC50 values were calculated with the SPSS software using the bliss method.
Cell invasion activity assay were conducted with an 8 μm matrigel invasion chamber (Corning, NY). Experiments were carried out according to the manufacture’s protocal. Briefly, each well insert was coated with 100 μl mixture matrigel: serum-free medium, followed by incubation at 37°C for 4 hours. Cells were then trypsin digested and transferred into top matrigel wells (105 cancer cells per well) with WEB2086 (0.1 mM), AG1478 (10 μM) or both. 600 μl of 10% FBS media was added to the bottom of the chamber and incubated for 48 hours in the invasion assay. Invasion activity was assessed by the number of cells that crossed the matrigel and filter membrane. Cell numbers were counted under a light microscope and presented as percentage compared to the controls. Experiments were performed twice, at least three repeats for each treatment.
Assessment of apoptosis and cell cycle
Apoptosis was detected by flow cytometry via the examination of altered plasma membrane phospholipid packing by the lipophilic dye Annexin V as described elsewhere. Briefly, cells were treated with inhibitors and harvested at 24 hours after treatment. The cells were washed twice with PBS and then resuspended in binding buffer at a concentration of 1 × 106 cells/mL according to the manufacturer’s instructions. Thereafter, 5 μL of Annexin V-FITC and 1 μL of propidium iodide were added to 100 μL of cell suspension and incubated for 20 min at room temperature in the dark. After adding 400 μL of binding buffer, the labeled cells were counted by flow cytometry within 1 hour. All early apoptotic cells (Annexin V-positive, propidium iodide -negative), necrotic/late apoptotic cells (double positive), and living cells (double negative) were detected using a FACSCalibur flow cytometer and subsequently analyzed by Cell Quest software. For the cell cycle analyses, treated cells were fixed in 70% ethanol and stored at -20°C overnight; the cells were labeled with propidium iodide (50 μg/ml) and RNase (100 μg/ml) for 30 min before flow cytometry analysis.
Animals and treatments
Female athymic nu/nu mice (4 to 6 weeks old) were obtained from the Laboratory Animal Center of the Shanghai Institutes For Biological Sciences of the Chinese Academy of Sciences. All animal studies were conducted in strict accordance with protocols approved by the Ethics Committee for Animal Experimentation of Fudan University. A total of 2 × 106 CAOV-3 cells was injected into the flanks of Female athymic nu/nu mice. When established tumors of approximately 75 mm3 in Diameter were detected, the mice were randomly divided into four groups (8 mice/group), and subjected to various treatments. The PAFR antagonist WEB2086 was intraperitoneally injected at 5 mg/kg every three days for 2 weeks, and the EGFR inhibitor AG1478 was intraperitoneally injected at 10 mg/kg every three days for 2 weeks. After 3 weeks, all of the mice were killed, and the tumors were excised and weighed. The tumor size for the xenografts was determined using a caliper, and the volume was calculated as = length × width2/2, where the width is the smallest measurement and the length is the longest measurement.
Synergy between WEB2086 and AG1478 was determined by isobologram analysis using CalcuSyn software (Biosoft, Cambridge, UK), which analyzes the data from proliferation assays to determine the interaction between equipotent drug combinations. The combination index values <1, =1 and >1 indicate synergy, additivity and antagonism, respectively.
All experiments were performed at least three times. The data are expressed as the “mean ± SD”. Wherever appropriate, the data were also subjected to unpaired two-tailed Student’s t-tests. Differences were considered significant when P < 0.05.
Synergistic interaction between the PAFR antagonist WEB2086 and the EGFR inhibitor AG1478
Combined targeting of PAFR and EGFR inhibits ovarian cancer cell growth and invasion
We previously reported that PAF promots ovarian cancer cell invasion. In addition to cell growth, we examined the effects on cell invasion of combined PAFR and EGFR targeting in ovarian cancer cells. As shown in Figure 2C and D, although WEB2086 or AG1478 alone decreased ovarian cancer cell invasion, combined targeting significantly enhanced the effect when compared with either treatment alone.
Combined targeting of PAFR and EGFR increases ovarian cancer cell apoptosis and G0/G1 arrest
Identification of intracellular signaling pathways following the activation of PAFR and EGFR
Combined targeting of PAFR and EGFR enhances intracellular signaling pathway inhibition
Co-inhibition of PAFR and EGFR significantly inhibits CAOV-3 tumor xenografts
The observation that elevated levels of growth factor receptors are associated with adverse cancer outcomes has led to the development of approaches that specifically interrupt these autocrine pathways. The constitutive activation of EGFR has been reported in various cancers, including breast, prostate, and ovarian cancers[27–29]. EGFR monoclonal antibodies and EGFR tyrosine kinase inhibitors have been approved for use in cancer patients. Despite these promising preclinical results, the inhibition of EGFR, has resulted in limited antitumor effects when tested as a monotherapy in clinical settings.
The PAF/PAFR signaling axis has emerged as an important determinant of aggressive phenotypes in several malignancies. PAF has been associated with early malignant transformation in BRCA1 – mutant epithelial ovarian cells, and melanocytic tumorigenesis has been observed in transgenic mice overexpressing PAFR. The many effects of PAF in tumors, such as increased vascular permeability, the induction of neoangiogenesis, and the activation of metalloproteinases, have reinforced the concept that PAF promotes tumor metastasis[33, 34]. Recent experiments have shown that the PAFR antagonist WEB2086 inhibits tumor growth in a murine melanoma model, improving overall survival when combined with chemotherapy.
Studies on WEB2086 have primarily been performed with leukemia cells that were induced to undergo differentiation and/or apoptosis. WEB2086 has been proven to possess the ability to abrogate PAF-mediated signals and exerts a wide anticancer activity capable of significantly decreasing proliferation in human solid tumor cells of different histogeneses and with a much higher efficacy than in normal cells. In addition, earlier experiments from our group have shown that the activation of PAFR has pleiotropic effects on tyrosine phospho-EGFR/Src/Paxillin in ovarian cancer. Therefore, we hypothesized that there would be crosstalk between the PAFR and EGFR pathways, which may be one of reasons for the resistance of cancer cells to drugs, and that the combined targeting of PAFR and EGFR would synergistically inhibit ovarian cancer progression.
In this study, we evaluated for the first time the antitumor effects of PAFR and EGFR targeting strategies in ovarian cancer cell lines using the PAFR antagonist WEB2086 and EGFR inhibitor AG1478. In our in vitro and in vivo studies, we demonstrated that EGFR and PAFR were overexpressed in ovarian cancer cell lines, which led us to speculate that simultaneously targeting PAFR and EGFR may be a more effective therapeutic strategy than targeting either signaling pathway alone. Our results show that the combined inhibition of PAFR and EGFR additively inhibited ovarian cancer progression. The decrease in viable tumor cells resulted from the induction of apoptosis, G0/G1 cell cycle arrest, and the reduction of cells in S phase.
However, the mechanisms responsible for the synergistic effects of targeting both PAFR and EGFR are not completely understood. If activated PAFR signaling acts through the EGFR signaling pathway, then EGFR targeting alone should achieve the same effect as combined targeting. The enhanced antitumor effects observed when targeting both receptors in combination suggest that EGFR-independent signaling pathways are also activated by PAF. Our results show that phosphorylation levels of P70S6K, 4EBP1, AKT, and MAPK were increased when cells were stimulated with either PAF or EGF in different doses (as shown in Figure 5). These results suggest that crosstalk exists between intracullelar signaling pathways following activation of PAFR and EGFR. With the combined treatment of WEB2086 and AG1478, the phosphorylation levels of these proteins were more reduced than with either treatment alone. Taken together, the expression of these proteins was affected by both EGFR- dependent and EGFR- independent pathways. It has been reported that after specific agonist stimulation, G protein-coupled receptors (GPCRs) use multifunctional adaptor proteins such as β-arrestins to activate many substrates in cellular pathways[36, 37]. Thus β-arrestin acts as a bifunctional cellular mediator; that is, it not only terminates G protein signaling but also functions as a scaffold for transduction of the G protein signal. We also observed β-arrestin2 protein was modulated by PAFR, but not EGFR, suggesting that the inhibition of the EGFR pathway alone cannot effectively suppress ovarian cancer progression.
Our previous study reported that the PAFR ligand PAF can activate phospho-EGFR and induce proliferation and invasion in ovarian cancer, and the results of the present study show that the PAFR antagonist WEB2086 can inhibit EGFR activation. It is apparent that the persistent activation of PAFR in the face of EGFR blockade still contributes to tumor growth and resistance. Identification of the proteins that are induced by PAF, in the presence or absence of EGFR inhibition, will determine the critical pathways to be targeted in combination with EGFR blockade. Further research is underway to elucidate the exact mechanism involved in this process to optimize ovarian cancer treatment regimens.
Taken together, both in vitro and in vivo analyse suggest that PAFR and EGFR play an important role in the sustained growth, survival, and invasion of ovarian cancer cells. The combined usage of selective inhibitors of PAFR and EGFR, such as WEB2086 and AG1478, represents a promising strategy for the treatment of ovarian cancer. This novel combination of drugs offers a new choice for the current platinum- based regimens, but it is critical to evaluate the profile of PAFR and EGFR expression in ovarian cancer patients before the strategy is applied in the clinical setting.
Epidermal growth factor
Transforming growth factor
Epithelial mesenchymal transition
G-protein coupled receptor (GPCR) family
Poly-ADP-ribose polymerase (PARP)
4E-binding protein 1
Ribosomal protein S6 kinase
Extracellular-regulated protein kinase
mammalian target of rapamycin
Mitogen-activated protein kinases
An EGFR-specific tyrosine kinase inhibitor
A specific PAFR antagonist.
This work was supported by National Natural Sciences Foundation of China (81202052) awarded to WJ, and the Medical Pilot Project of the Shanghai Municipal Science and Technology Commission (114119a2300) awarded to WJ.
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