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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Acta Pharmacol Sin. Author manuscript; available in PMC Feb 28, 2014.
Published in final edited form as:
PMCID: PMC3938287
NIHMSID: NIHMS555302
Epoxyeicosatrienoic acid-stimulated proliferation in cancer cells involves EGF-R phosphorylation mediated by activation of metalloproteinases and release of HB-EGF
Liming Cheng,1 Ziyong Sun,1 Chen Chen,1 Ding Ma,1 Jianfeng Zhou,1 Ryan T. Dackor,2 Darryl C. Zeldin,2 and Dao Wen Wang1
1The Institute of Hypertension and Department of Internal Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, People's Republic of China
2Division of Intramural Research, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC 27709
Corresponding Author: Dao Wen Wang, MD, PhD Department of Internal Medicine Tongji Hospital, Tongji Medical College Huazhong University of Science & Technology 1095# Jiefang Ave., Wuhan 430030 PRC Tel. & Fax: 86-27-8366-3280 ; dwwang/at/tjh.tjmu.edu.cn
Arachidonic acid is metabolized to biologically active epoxyeicosatrienoic acids (EETs) by cytochrome P450 (CYP) epoxygenases. Previous studies showed that CYP epoxygenases promote neoplastic growth and induce potent mitogenic effects in human carcinoma cells; however, the exact molecular mechanisms involved in EET-induced tumor cell proliferation remain unclear. Exogenous 14,15-EET was added or a mutant CYP epoxygenase (CYP102 F87V, an active 14,15-epoxygenase) was transfected into three human derived cancer cell lines; Tca-8113, A549, HepG2 and MDA-MB-231. The effects of elevated EETs on tyrosine phosphorylation of the EGF receptor and ERK1/2 activation were then assessed. In this study, we found that addition of 14,15-EET and CYP102 F87V transfection markedly increased tyrosine phosphorylation of EGF-R and ERK1/2, an effect that was blocked by a selective EGF-R tyrosine kinase inhibitor (tyrphostin AG1478), a broad-spectrum metalloproteinase inhibitor (1,10-phenanthroline) and an inhibitor of HB-EGF release (CRM197) in Tca-8113 cells. In addition, AG1478, 1,10-phenanthroline and CRM197 also inhibited the tyrosine phosphorylation of EGF-R and ERK1/2 induced by 14,15-EET in A549, HepG2 and MDA-MB-231 cancer cell lines. These data suggest that EET-induced transactivation of EGF-R depends on activation of metalloproteinases and subsequent release of HB-EGF in cancer cells.
Keywords: arachidonic acid, cytochrome P450 epoxygenase, epoxyeicosatrienoic acids, tumor cell proliferation, EGF-R, ERK1/2, AG1478, phenanthroline, CRM197
Arachidonic acid is a polyunsaturated fatty acid component of membrane phospholipids and is released acutely in response to a number of agonists, including growth factors, cytokines, and hormones in various cell types. Upon release, it is either metabolized via the cyclooxygenase, lipoxygenase, or cytochrome P450 (CYP) monooxygenase pathways, producing prostaglandins, leukotrienes, hydroperoxyeicosatetraenoic acids (HETEs) and cis-epoxyeicosatrienoic acids (EETs), respectively1, 2. Metabolism of arachidonic acid by CYP epoxygenases results in the generation of four regioisomeric EETs (5,6-, 8,9-, 11,12- and 14,15-EET)3, 4. We and others have demonstrated that CYP-derived EETs possess several protective effects in endothelial cells including upregulation of endothelial nitric oxide synthase (eNOS) expression and activity, enhancement of angiogenesis, fibrolytic activity via production of tissue plasminogen activator (tPA), inhibition of TNF-α induced apoptosis, and anti-inflammatory properties57. Recently, EETs have been implicated in a variety of physiologic processes that are relevant to cancer pathogenesis including regulation of intracellular signaling pathways, gene expression, tumor cellular proliferation and metastasis via activation of MAPK and PI3 kinase/Akt pathways, as well as phosphorylation of EGF-R8, 9.
In renal proximal tubular epithelial cells, it was found that stable CYP102 F87V expression induced 14,15-EET production and enhanced cell proliferation10, 11. Further studies in these cells demonstrated that 14,15-EET induced activation of metalloproteinases (MMPs) which led to the release of the potent mitogenic EGF-R ligand, heparin-binding epidermal growth factor-like growth factor (HB-EGF)11. In human cancer cells, however, whether EGF-R phosphorylation and malignant proliferation induced by CYP epoxygenase-derived EETs involves activation of MMPs and/or HB-EGF release remains unknown. A better understanding of the mitogenic signaling events that are initiated by CYP epoxygenase-EET signaling may lead to the development of new therapeutic, anti-cancer strategies. Thus, we investigated the effects of exogenously added 14,15-EET or CYP epoxygenase overexpression on MMP activation and EGF-R phosphorylation in the human tongue squamous cell line, Tca-8113.
Materials
Tca-8113 (a human tongue squamous carcinoma cell line), A549 (a human lung cancer cell line), HepG2 (a human liver cancer cell line) and MDA-MB-231 (a human breast cancer cell line) cells were obtained from the American Type Culture Collection (Manassas, VA). Chemicals and reagents were obtained as follows: cell culture medium from Hyclone (Logan, UT); antibodies against epidermal growth factor receptor (EGF-R) and phospho-EGF-R from Cell Signaling Technology (Beverly, MA); anti-extracellular signal-regulated kinase (ERK) and anti-phospho-ERK antibodies from New England Biolabs, Inc. (Beverly, MA); antibodies against β-actin from Neomarkers (Fremont, CA); horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit immunoglobulin G and rabbit anti-mouse immunoglobulin G) from KPL (Gaithersburg, MD); enhanced chemiluminescence reagents from Pierce, Inc.(Rockford, IL); 14,15-EET from Cayman Chemical Co. (Ann Arbor, Michigan); AG1478 (EGF-R-selective tyrosine kinase inhibitor) from Calbiochem (San Diego, CA); EGF, 1,10-phenanthroline (a non-specific MMP inhibitor) and CRM197 (an inhibitor of HB-EGF release) from Sigma Chemical Co. (St. Louis, MO). All other reagents were purchased from standard commercial suppliers.
Adeno-associated virus (AAV)-mediated CYP epoxygenase overexpression
The recombinant adeno-associated virus (rAAV) vector pXXUF1, packaging plasmid pXX2, adenovirus helper plasmid pXX6, and a rAAV plasmid containing the GFP cDNA (GFP-pUF1) were a generous gift from Dr. Xiao Xiao (University of Pittsburgh, Pittsburgh, PA). CYP102 F87V is a mutant P450 from Bacillus megaterium (P450BM3) in which phenylalanine 87 is replaced with valine, converting it to a highly regio- and stereoselective epoxygenase that biosynthesizes 14(S),15(R)-EET from arachidonic acid. The coding region of the CYP102 F87V mutant was subcloned into pXXUF1 downstream from the cytomegalovirus promoter to produce the construct CYP102 F87V-pUF1. The rAAVs were produced and purified as previously described5, 12, 13 and their titers were determined by dot blot hybridization. The eluted rAAV was aliquoted and stored at −80°C. The resultant rAAVs were designated rAAV-CYP102 F87V and rAAV-GFP, respectively. Tca8113 cells were infected with rAAV-CYP102 F87V or rAAV-GFP (~50 virions/cell) and cultured for one week to obtain maximal expression. These cells were used for further experiments as detailed below.
Cell culture and cell proliferation assays
Tca-8113 cells were cultured in DMEM containing 10% (vol/vol) bovine serum, 100 mg/ml streptomycin, 100 IU/ml penicillin in a humidified atmosphere of 5% CO2 in air at 37 °C. The cells were seeded into 96-well plates (~1×104/well) in DMEM containing 10% bovine serum in a final volume of 0.2 ml. Six parallel wells were set up for each group. Once the cells were grown up to 60% confluence, the medium was changed with serum-free DMEM and the cells were incubated with serum-free DMEM at 37°C for 12 hours to allow for synchronization. 14,15-EET (250nM) and AG1478 (100nmol/L) were added into the medium (ethanol was used as vehicle control). After 24 hrs, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added (20μL/well of 5mg/ml in PBS) and the cells were incubated at 37°C for 4 more hrs at which point the reaction was stopped by addition of 100 μL DMSO. The reaction product was quantified by measuring the absorbance at 490 nm using an ELISA reader (Elx800, China).
Immunoblot analysis
Tca-8113, A549, HepG2 and MDA-MB-231 cells were treated with 14,15-EET (250nmol/L) or EGF (20ng/ml) with/without inhibitors including AG1478 (100nmol/L), CRM197 (10μg/ml), and 1,10-phenanthroline (100μmol/l). The rAAV-infected cells were treated with the inhibitors as described. Cells were then harvested for western blots to detect signaling molecules. Cells were placed on ice, washed twice with ice-cold PBS and then lysed in RIPA lysis buffer containing 50 mM Tris (pH 8.0), 150 mM NaCl, 0.1% sodium dodecyl sulfate (SDS), 1% Nonidet P-40 (NP-40), 0.5% sodium deoxycholate, 0.02% sodium azide (NaN3), 1 μg/ml aprotonin and 1 mM phenylmethylsulfonyl fluoride. Solubilized lysates were centrifuged at 10,000g for 15 min. Total protein concentrations were determined by Bradford assay. After heating at 95°C for 10 min, the supernatants were electrophoresed on SDS-PAGE (8–12%) gradient gels and transferred to PVDF membranes. Blots were incubated overnight at 4°C with primary antibodies and washed 4 times with TBST before probing with horseradish peroxidase-conjugated secondary antibodies for 2 hr at room temperature. Blots were then visualized with enhanced chemiluminescence reagent and the optical densities of the bands were semi-quantified by a Gene Genius Bio Imaging System (SynGene, USA). In some cases, blots were stripped and reprobed with other antibodies.
Statistical analysis
Data were expressed as mean ± S.E. for at least three separate experiments, and statistical evaluation was performed using the Student's t-test or ANOVA as appropriate. Values of p< 0.05 were considered statistically significant.
14,15-EET induces phosphorylation of EGF-R and ERK1/2
In a dose-dependent manner, 14,15-EET incubation led to increased phosphorylation of EGF-R and ERK1/2 (Figure 1), as previously observed by our group8. We treated cells with the EGF-R-selective tyrosine kinase inhibitor, tyrphostin AG1478, to examine its effect on EET-induced phosphorylation of EGF-R and ERK1/2. Results show that AG1478 attenuated EET-induced phosphorylation of EGF-R and ERK1/2 (Figure 2). As expected, AG1478 also attenuated EGF-induced phosphorylation of EGF-R and ERK1/2 (Figure 2). These results demonstrate that 14,15-EET enhances phosphorylation and activation of ERK1/2 in Tca8113 cells through transactivation of EGF-R.
Figure 1
Figure 1
14,15-EET increases tyrosine phosphorylation of EGF-R and ERK1/2 in a dose-dependent manner. Serum-deprived Tca8113 cells were treated with vehicle (ethanol) or different concentrations of 14,15-EET (50nM, 100nM, 250nM, 500nM, 1μM and 5μM (more ...)
Figure 2
Figure 2
Effect of tyrphostin AG1478 on EGF-R and ERK1/2 phosphorylation. Serum-deprived Tca8113 cells were pretreated with or without 100nmol/L AG1478 for 60 minutes. They were then stimulated for 30 min with 250 nM 14,15-EET or 20ng/ml EGF. Phosphorylated EGF-R, (more ...)
14,15-EET promotes cancer cell proliferation via EGF-R activation
We next examined whether 14,15-EET-stimulated proliferation of Tca8113 cells occurs through transactivation of EGF-R. The MTT assays showed that 14,15-EET (250nM) significantly increased Tca-8113 cell proliferation by 26% compared to the vehicle control (p<0.05). AG1478 completely abolished the proliferative effect of 14,15-EET (p<0.05) (Figure 3). These results suggest that 14,15-EET promotes proliferation of Tca8113 cells through transactivation of EGF-R.
Figure 3
Figure 3
Proliferative effect of 14,15-EET in Tca-8113 cells is abrogated by AG1478. 14,15-EET (250 nM) stimulated Tca-8113 cell proliferation by 26% compared to vehicle control group (*, p<0.05). Pretreatment with AG1478 completely abolished the proliferative (more ...)
Effects of 1,10-phenanthroline and diphtheria toxin/CRM197 on 14,15-EET-induced EGF-R activation
To evaluate whether EGF-R activation in Tca8113 cells results from the release of HB-EGF through proteolytic processing by MMPs, we investigated the effects of 1,10-phenanthroline (a non-specific MMP inhibitor) and diphtheria toxin/CRM197 (an inhibitor of HB-EGF release) on EGF-R and ERK1/2 phosphorylation. Results show that treatment of Tca8113 cells with 1,10-phenanthroline (100μmol/L) completely blocked 14,15-EET-induced EGF-R and ERK1/2 phosphorylation, but did not inhibit EGF-induced EGF-R and ERK1/2 phosphorylation (Figure 4). Furthemore, pretreatment of Tca8113 cells with CRM197 (10μg/ml) caused a significant inhibition of the phosphorylation of EGF-R and ERK1/2 by 14,15-EET, with no effect on EGF-induced phosphorylation of these molecules (Figure 5). These results are consistent with those obtained with tyrphostin AG1478 and show that activation of MMPs and subsequent release of HB-EGF is an important step in the phosphorylation of EGF-R and ERK1/2 by 14,15-EET.
Figure 4
Figure 4
Activation of EGF-R and ERK1/2 depends on matrix metalloproteinase activity. Serum-deprived Tca8113 cells were incubated with or without 100 μmol/L 1,10-phenanthroline for 60 min. They were then stimulated for 30 min with 250 nM 14,15-EET or 20 (more ...)
Figure 5
Figure 5
Effects of the non-toxic mutant of diphtheria toxin CRM197 on 14,15-EET-induced tyrosine phosphorylation of EGF-R and ERK1/2. Serum-deprived Tca8113 cells were incubated with or without 10 μg/ml of CRM197 for 60 min. They were then stimulated (more ...)
To further investigate the potential mechanism through which 14,15-EET transactivates EGFR, we examined the effects of AG1478, 1,10-phenanthroline and CRM197 on 14,15-EET-induced EGF-R activation in three other tumor cell lines; A549, HepG2 and MDA-MB-231. As expected, we observed a similar profile as above; 14,15-EET increased levels of phosphorylated EGFR and ERK1/2 in these tumor cells, whereas 14,15-EET induced-phosphorylation of EGF-R and ERK1/2 was dramatically attenuated by AG1478, 1,10-phenanthroline and CRM197 (Figure 6). These data further indicate that the release of HB-EGF and activation of MMPs exert regulatory effects on the phosphorylation of EGF-R and ERK1/2 induced by 14,15-EET in tumor cells.
Figure 6
Figure 6
Effects of AG1478, CRM197 and 1,10-phenanthroline on 14,15-EET-induced tyrosine phosphorylation of EGF-R and ERK1/2 in A549, HepG2 and MDA-MB-231 cell lines. Upper panel is representative of three separate experiments with similar results. Lower panel (more ...)
Effects of 14,15-epoxygenase CYP102 F87V on transactivation of EGF-R
We and others have demonstrated that CYP102 F87V is a 14,15-epoxygenase that efficiently metabolizes arachidonic acid to 14,15-EET8. In this study, we infected Tca8113 cells with rAAV-CYP102 F87V to determine if CYP epoxygenase overexpression could transactivate EGF-R by activation of the HB-EGF-shedding mechanism. The efficiency of gene transfer was evaluated 1 week after cell infection. Western blotting revealed robust CYP102 F87V expression in Tca-8113 cells infected with rAAV-CYP102 F87V (Figure 7A). Moreover, we examined the activity of CYP102 F87V in rAAV-CYP102 F87V infected cells. Given the instability of 14,15-EET, we determined the concentration of its stable metabolite, 14,15-DHET, using an ELISA assay. The result showed a dramatic increase in 14,15-DHET levels in rAAV-CYP102 F87V infected cells, compared with untreated cells (332 ±117 pg/250 μg protein vs. 122 ± 4 pg/250 μg protein, p<0.05) (Figure 7B). rAAV-CYP102 F87V infection significantly promoted phosphorylation of EGF-R and ERK1/2, as we observed previously8. Importantly, induction of phosphorylation by CYP102 F87V overexpression was abolished by the addition of AG1478 2 hours before harvest of cells (Figure 8). Furthermore, EGF-R and ERK1/2 phosphorylation was also blocked when CYP102 F87V-infected Tca8113 cells were treated with 1,10-phenanthroline or CRM107 (Figure 8).
Figure 7
Figure 7
Expression of CYP102 in tumor cells after rAAV- CYP102 F87V gene transfer. (A) CYP102 F87V expression in rAAV-CYP102 F87V infected cells. (B) CYP102 F87V activity (14,15-DHET production) in rAAV-CYP102 F87V infected cells. Data are means ± S.E. (more ...)
Figure 8
Figure 8
Effects of tyrphostin AG1478, 1,10-P and the nontoxic mutant of diphtheria toxin CRM197 on the activation of EGF-R and ERK1/2 in rAAV-CYP102 F87V-transfected Tca8113 cells. Tca-8113 cells were transfected with rAAV-CYP102 F87V or rAAV-GFP (GFP) and cultured (more ...)
Multiple studies have shown EETs to be potent mitogens that activate EGF-R and ERKs in various cell types8, 10. Previous studies by our group also demonstrated that EETs elevate expression and activity of MMPs in different human cancer cell lines9. However, the role of EETs in EGF-R activation in human cancer cells remains unclear. In the present study, we found that addition of 14,15-EET or overexpression of a selective 14,15-EET epoxygenase can induce activation of EGF-R and ERK1/2 in multiple human derived cancer cell lines; Tca-8113, A549, HepG2 and MDA-MB-23. These signaling events are abolished by the tyrosine kinase inhibitor of EGF-R, AG1478. Interestingly, addition of 1,10-phenanthroline (a non-specific MMP inhibitor) or diphtheria toxin/CRM197 (an inhibitor of HB-EGF release) also blocked EET-induced activation of EGF-R and ERK1/2. As expected, inhibition of MMPs or HB-EGF cleavage did not block EGF induced EGF-R phosphorylation and its downstream activation of ERK1/2 in the cancer cells. Together, these results demonstrate that MMP activation, followed by HB-EGF cleavage and release, is essential for EET-induced EGF-R activation in human cancer cells.
HB-EGF is synthesized as a type I transmembrane protein, similar to other members of the epidermal growth factor (EGF) family. Pro-HB-EGF can be enzymatically shed within the juxtamembrane region to release a soluble 14–22 kDa growth factor14, 15. The ectodomain shedding of pro-HB-EGF is induced by various stimuli such as phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA)16, 17, calcium ionophore18, and various growth factors and cytokines19. G-protein coupled receptor (GPCR) agonists also stimulate pro-HB-EGF shedding, which mediates EGF-R transactivation by GPCR signaling20, 21. Metalloproteinases are responsible for the proteolytic cleavage of pro-HB-EGF since the ectodomain shedding of pro-HB-EGF is efficiently inhibited by various metalloproteinase inhibitors. Protein kinase C (PKC) and mitogen-activated protein (MAP) kinase may be required for the activation of appropriate metalloproteinases since they are known to be involved in the intracellular signaling pathway for pro-HB-EGF shedding22, 23,.
In human colon carcinoma cells, IL-8 promotes cell proliferation and migration through metalloproteinase-cleavage of pro-HB-EGF 24. Lysophosphatidic acid (LPA)-induced ectodomain shedding of pro-HB-EGF is critical for tumor formation in ovarian cancer25. Deoxycholyltaurine (DCT)-induced transactivation of EGF-R is mediated by MMP-7-catalyzed release of the EGF-R ligand HB-EGF in H508 human colon cancer cells26. In this study, we demonstrate that addition of 14,15-EET or overexpression of a 14,15-EET-specific epoxygenase leads to EGF-R transactivation via MMP activation and release of HB-EGF in four cancer cells. However, the identification of the specific metalloproteinase responsible for 14,15-EET-induced cleavage of the pro-HB-EGF requires further study.
Transactivation of EGF-R and the subsequent activation of downstream ERKs by metalloproteinase-mediated release of soluble HB-EGF play an important role in EET-stimulated mitogenic signaling. The tyrosine kinase inhibitor of EGF-R, AG1478, may provide significant therapeutic value in controlling the malignant growth of carcinomas. However, as demonstrated in previous studies, EETs activate the PI3K/Akt signaling pathway in different cell lines and tissues, which can also lead to mitogenic effects6, 8. The relative importance of PI3K/Akt and EGF-R/ERK/MMP signaling pathways in mediating the mitogenic effects of CYP epoxygenase products remain to be determined.
In summary, this study reveals that induction of EGF-R transactivation is a crucial event in the mitogenic signaling transmission of EETs in cancer cells. In addition, EET-induced transactivation of EGF-R is mediated by activation of metalloproteinases that cleave pro-HB-EGF from the cell membrane and release active HB-EGF, which subsequently binds to EGF-R and activates downstream ERKs. Thus, CYP epoxygenase-derived EETs lead to malignant proliferation of cancer cells and growth of carcinomas via transactivation of EGF-R via a MMP-HB-EGF pathway. Further studies will be required to identify the precise metalloproteinases that are activated by EETs in cancer cells and to elucide the relative importance of EGF-R/ERK/MMP and other signaling pathways in mediating the mitogenic effects of CYP epoxygenase products.
Acknowledgments
Sources of funding This work was supported by the International Collaboration Project (No. 2005DFA30880) and 863 project (2006AA02A406). This work was also funded, in part, by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences (Z01 ES025034).
Footnotes
Disclosures None
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