Abstract
Purpose
Under physiologic conditions, the binding of erythropoietin-producing hepatocellular (Eph) A2 receptor and its ligand ephrinA1 results in decreased EphA2 level and tumor suppression. However, EphA2 and ephrinA1 are highly expressed in human cancers including gastric adenocarcinoma. In this study, we tested our hypothesis that cancer-associated fibroblasts (CAFs) promote gastric tumorigenesis through EphA2 signaling in a ligand-independent manner.
Methods
Expression of EphA2 protein in primary tumor tissues of 91 patients who underwent curative surgery for gastric adenocarcinoma was evaluated by immunohistochemistry and western blotting. Conditioned medium of cancer-associated fibroblasts (CAF-CM) was used to evaluate the tumorigenic effect of CAFs on gastric cancer cell lines. Epithelial–mesenchymal transition (EMT), cell proliferation, migration, and invasion were assessed. EphrinA1-Fc ligand was used to determine the suppressor role of EphA2 receptor-ligand binding.
Results
CAF-CM-induced EMT and promoted cancer cell motility even without cell–cell interaction. Treatment with a selective EphA2 inhibitor (ALW-II-41-27) or EphA2-targeted siRNA markedly reduced CAF-CM-induced gastric tumorigenesis. EphrinA1-Fc ligand treatment showing ligand-dependent tumor suppression diminished the EphA2 expression and EMT progression. In contrast, ephrinA1-targeted siRNA did not significantly affect CAF-CM-mediated increases in EphA2 expression and EMT progression. Treatment with VEGF showed effects like CAF-CM in terms of EphA2 activation and EMT progression.
Conclusion
CAFs may contribute to gastric tumorigenesis by activating EphA2 signaling pathway in a ligand-independent manner. Our results suggest that ligand-independent activation of EphA2 was triggered by VEGF released from CAF-CM. Our result may partially explain why ligand-dependent tumor suppressor roles of EphA2 are not evident in gastric cancer despite the prominent level of ephrinA1.
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Abbreviations
- EphA2:
-
Erythropoietin-producing hepatocellular A2
- CAFs:
-
Cancer-associated fibroblasts
- CAF-CM:
-
Conditioned medium of cancer-associated fibroblasts
- NF-CM:
-
Conditioned medium from normal gastric fibroblasts
- EMT:
-
Epithelial–mesenchymal transition
- siRNA:
-
Small interfering RNA
- TNM:
-
Tumor, lymph node and metastasis
- VEGF:
-
Vascular endothelial growth factor
References
Amato KR, Wang S, Hastings AK, Youngblood VM, Santapuram PR, Chen H, Cates JM, Colvin DC, Ye F, Brantley-Sieders DM, Cook RS, Tan L, Gray NS, Chen J (2014) Genetic and pharmacologic inhibition of EPHA2 promotes apoptosis in NSCLC. J Clin Invest 124:2037–2049. https://doi.org/10.1172/jci72522
Binda E, Visioli A, Giani F, Lamorte G, Copetti M, Pitter KL, Huse JT, Cajola L, Zanetti N, DiMeco F, De Filippis L, Mangiola A, Maira G, Anile C, De Bonis P, Reynolds BA, Pasquale EB, Vescovi AL (2012) The EphA2 receptor drives self-renewal and tumorigenicity in stem-like tumor-propagating cells from human glioblastomas. Cancer Cell 22:765–780. https://doi.org/10.1016/j.ccr.2012.11.005
Cui XD, Lee MJ, Yu GR, Kim IH, Yu HC, Song EY, Kim DG (2010) EFNA1 ligand and its receptor EphA2: potential biomarkers for hepatocellular carcinoma. Int J Cancer 126:940–949. https://doi.org/10.1002/ijc.24798
Davis S, Gale NW, Aldrich TH, Maisonpierre PC, Lhotak V, Pawson T, Goldfarb M, Yancopoulos GD (1994) Ligands for EPH-related receptor tyrosine kinases that require membrane attachment or clustering for activity. Science 266:816–819
Dicken BJ, Bigam DL, Cass C, Mackey JR, Joy AA, Hamilton SM (2005) Gastric adenocarcinoma: review and considerations for future directions. Ann Surg 241:27–39
Dunne PD, Dasgupta S, Blayney JK, McArt DG, Redmond KL, Weir JA, Bradley CA, Sasazuki T, Shirasawa S, Wang T, Srivastava S, Ong CW, Arthur K, Salto-Tellez M, Wilson RH, Johnston PG, Van Schaeybroeck S (2016) EphA2 expression is a key driver of migration and invasion and a poor prognostic marker in colorectal cancer. Clin Cancer Res 22:230–242. https://doi.org/10.1158/1078-0432.CCR-15-0603
Fantozzi A, Gruber DC, Pisarsky L, Heck C, Kunita A, Yilmaz M, Meyer-Schaller N, Cornille K, Hopfer U, Bentires-Alj M, Christofori G (2014) VEGF-mediated angiogenesis links EMT-induced cancer stemness to tumor initiation. Cancer Res 74:1566–1575. https://doi.org/10.1158/0008-5472.can-13-1641
Fuyuhiro Y, Yashiro M, Noda S, Matsuoka J, Hasegawa T, Kato Y, Sawada T, Hirakawa K (2012) Cancer-associated orthotopic myofibroblasts stimulates the motility of gastric carcinoma cells. Cancer Sci 103:797–805. https://doi.org/10.1111/j.1349-7006.2012.02209.x
Hou F, Yuan W, Huang J, Qian L, Chen Z, Ge J, Wu S, Chen J, Wang J, Chen Z (2012) Overexpression of EphA2 correlates with epithelial-mesenchymal transition-related proteins in gastric cancer and their prognostic importance for postoperative patients. Med Oncol 29:2691–2700. https://doi.org/10.1007/s12032-011-0127-2
Kania A, Klein R (2016) Mechanisms of ephrin-Eph signalling in development, physiology and disease. Nat Rev Mol Cell Biol 17:240–256. https://doi.org/10.1038/nrm.2015.16
Lisabeth EM, Falivelli G, Pasquale EB (2013) Eph receptor signaling and ephrins. Cold Spring Harb Perspect Biol. https://doi.org/10.1101/cshperspect.a009159
Macrae M, Neve RM, Rodriguez-Viciana P, Haqq C, Yeh J, Chen C, Gray JW, McCormick F (2005) A conditional feedback loop regulates Ras activity through EphA2. Cancer Cell 8:111–118. https://doi.org/10.1016/j.ccr.2005.07.005
Marquardt T, Shirasaki R, Ghosh S, Andrews SE, Carter N, Hunter T, Pfaff SL (2005) Coexpressed EphA receptors and ephrin-A ligands mediate opposing actions on growth cone navigation from distinct membrane domains. Cell 121:127–139. https://doi.org/10.1016/j.cell.2005.01.020
Miao H, Wang B (2009) Eph/ephrin signaling in epithelial development and homeostasis. Int J Biochem Cell Biol 41:762–770. https://doi.org/10.1016/j.biocel.2008.07.019
Miao H, Li DQ, Mukherjee A, Guo H, Petty A, Cutter J, Basilion JP, Sedor J, Wu J, Danielpour D, Sloan AE, Cohen ML, Wang B (2009) EphA2 mediates ligand-dependent inhibition and ligand-independent promotion of cell migration and invasion via a reciprocal regulatory loop with Akt. Cancer Cell 16:9–20. https://doi.org/10.1016/j.ccr.2009.04.009
Miao H, Gale NW, Guo H, Qian J, Petty A, Kaspar J, Murphy AJ, Valenzuela DM, Yancopoulos G, Hambardzumyan D, Lathia JD, Rich JN, Lee J, Wang B (2015) EphA2 promotes infiltrative invasion of glioma stem cells in vivo through cross-talk with Akt and regulates stem cell properties. Oncogene 34:558–567. https://doi.org/10.1038/onc.2013.590
Moccia M, Liu Q, Guida T, Federico G, Brescia A, Zhao Z, Choi HG, Deng X, Tan L, Wang J, Billaud M, Gray NS, Carlomagno F, Santoro M (2015) Identification of novel small molecule inhibitors of oncogenic RET kinase. PLoS One 10:e0128364. https://doi.org/10.1371/journal.pone.0128364
Nakada M, Hayashi Y, Hamada J (2011) Role of Eph/ephrin tyrosine kinase in malignant glioma. Neuro Oncol 13:1163–1170. https://doi.org/10.1093/neuonc/nor102
Nakamura R, Kataoka H, Sato N, Kanamori M, Ihara M, Igarashi H, Ravshanov S, Wang YJ, Li ZY, Shimamura T, Kobayashi T, Konno H, Shinmura K, Tanaka M, Sugimura H (2005) EPHA2/EFNA1 expression in human gastric cancer. Cancer Sci 96:42–47. https://doi.org/10.1111/j.1349-7006.2005.00007.x
Nasreen N, Mohammed KA, Lai Y, Antony VB (2007) Receptor EphA2 activation with ephrinA1 suppresses growth of malignant mesothelioma (MM). Cancer Lett 258:215–222. https://doi.org/10.1016/j.canlet.2007.09.005
Noblitt LW, Bangari DS, Shukla S, Knapp DW, Mohammed S, Kinch MS, Mittal SK (2004) Decreased tumorigenic potential of EphA2-overexpressing breast cancer cells following treatment with adenoviral vectors that express EphrinA1. Cancer Gene Ther 11:757–766. https://doi.org/10.1038/sj.cgt.7700761
O’Malley Y, Lal G, Howe JR, Weigel RJ, Komorowski RA, Shilyansky J, Sugg SL (2012) Invasion in follicular thyroid cancer cell lines is mediated by EphA2 and pAkt. Surgery 152:1218–1224. https://doi.org/10.1016/j.surg.2012.08.041
Paraiso KH, Das Thakur M, Fang B, Koomen JM, Fedorenko IV, John JK, Tsao H, Flaherty KT, Sondak VK, Messina JL, Pasquale EB, Villagra A, Rao UN, Kirkwood JM, Meier F, Sloot S, Gibney GT, Stuart D, Tawbi H, Smalley KS (2015) Ligand-independent EPHA2 signaling drives the adoption of a targeted therapy-mediated metastatic melanoma phenotype. Cancer Discov 5:264–273. https://doi.org/10.1158/2159-8290.CD-14-0293
Pasquale EB (2010) Eph receptors and ephrins in cancer: bidirectional signalling and beyond. Nat Rev Cancer 10:165–180. https://doi.org/10.1038/nrc2806
Sabet O, Stockert R, Xouri G, Bruggemann Y, Stanoev A, Bastiaens PI (2015) Ubiquitination switches EphA2 vesicular traffic from a continuous safeguard to a finite signalling mode. Nat Commun 6:8047. https://doi.org/10.1038/ncomms9047
Saito T, Masuda N, Miyazaki T, Kanoh K, Suzuki H, Shimura T, Asao T, Kuwano H (2004) Expression of EphA2 and E-cadherin in colorectal cancer: correlation with cancer metastasis. Oncol Rep 11:605–611
Sharfe N, Freywald A, Toro A, Roifman CM (2003) Ephrin-A1 induces c-Cbl phosphorylation and EphA receptor down-regulation in T cells. J Immunol 170:6024–6032
Singh DR, Ahmed F, King C, Gupta N, Salotto M, Pasquale EB, Hristova K (2015) EphA2 Receptor Unliganded Dimers Suppress EphA2 Pro-tumorigenic Signaling. J Biol Chem 290:27271–27279. https://doi.org/10.1074/jbc.M115.676866
Toma MI, Erdmann K, Diezel M, Meinhardt M, Zastrow S, Fuessel S, Wirth MP, Baretton GB (2014) Lack of ephrin receptor A1 is a favorable independent prognostic factor in clear cell renal cell carcinoma. PLoS One 9:e102262. https://doi.org/10.1371/journal.pone.0102262
Voulgari A, Pintzas A (2009) Epithelial-mesenchymal transition in cancer metastasis: mechanisms, markers and strategies to overcome drug resistance in the clinic. Biochim Biophys Acta 1796:75–90. https://doi.org/10.1016/j.bbcan.2009.03.002
Wu L, Zhang YS, Ye ML, Shen F, Liu W, Hu HS, Li SW, Wu HW, Chen QH, Zhou WB (2017) Overexpression and correlation of HIF-2alpha, VEGFA and EphA2 in residual hepatocellular carcinoma following high-intensity focused ultrasound treatment: Implications for tumor recurrence and progression. Exp Ther Med 13:3529–3534. https://doi.org/10.3892/etm.2017.4428
Yu B, Chen X, Li J, Qu Y, Su L, Peng Y, Huang J, Yan J, Yu Y, Gu Q, Zhu Z, Liu B (2013) Stromal fibroblasts in the microenvironment of gastric carcinomas promote tumor metastasis via upregulating TAGLN expression. BMC Cell Biol 14:17. https://doi.org/10.1186/1471-2121-14-17
Yuan W, Chen Z, Wu S, Ge J, Chang S, Wang X, Chen J, Chen Z (2009a) Expression of EphA2 and E-cadherin in gastric cancer: correlated with tumor progression and lymphogenous metastasis. Pathol Oncol Res 15:473–478. https://doi.org/10.1007/s12253-008-9132-y
Yuan WJ, Ge J, Chen ZK, Wu SB, Shen H, Yang P, Hu B, Zhang GW, Chen ZH (2009b) Over-expression of EphA2 and EphrinA-1 in human gastric adenocarcinoma and its prognostic value for postoperative patients. Dig Dis Sci 54:2410–2417. https://doi.org/10.1007/s10620-008-0649-4
Zhi K, Shen X, Zhang H, Bi J (2010) Cancer-associated fibroblasts are positively correlated with metastatic potential of human gastric cancers. J Exp Clin Cancer Res 29:66. https://doi.org/10.1186/1756-9966-29-66
Zhou Z, Yuan X, Li Z, Tu H, Li D, Qing J, Wang H, Zhang L (2008) RNA interference targeting EphA2 inhibits proliferation, induces apoptosis, and cooperates with cytotoxic drugs in human glioma cells. Surg Neurol 70:562–568. https://doi.org/10.1016/j.surneu.2008.04.031 (discussion 568–569)
Zhuang G, Hunter S, Hwang Y, Chen J (2007) Regulation of EphA2 receptor endocytosis by SHIP2 lipid phosphatase via phosphatidylinositol 3-Kinase-dependent Rac1 activation. J Biol Chem 282:2683–2694. https://doi.org/10.1074/jbc.M608509200
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This study was supported by a grant from Asan Institute for Life Sciences and Corporate Relations of Asan Medical Center, Seoul, Korea.
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Hea Nam Hong declares that she has no conflict of interest. You Jin Won declares that she has no conflict of interest. Ju Hee Shim declares that she has no conflict of interest. Seung Hee Han declares that she has no conflict of interest. Hyun Ji Kim declares that she has no conflict of interest. Byung Sik Kim declares that he has no conflict of interest. Hee Sung Kim declares that she has no conflict of interest.
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All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.
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This article does not contain any studies with animals performed by any of the authors.
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Hong, H.N., Won, Y.J., Shim, J.H. et al. Cancer-associated fibroblasts promote gastric tumorigenesis through EphA2 activation in a ligand-independent manner. J Cancer Res Clin Oncol 144, 1649–1663 (2018). https://doi.org/10.1007/s00432-018-2683-8
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DOI: https://doi.org/10.1007/s00432-018-2683-8