Constitutively Active Soluble Form of Erythropoietin Receptor Suppresses Growth and Angiogenesis of Xenografts of Transfected Cancer Cell Lines

DOI: 10.4236/jct.2011.21005   PDF   HTML     4,378 Downloads   8,471 Views   Citations


We examined the effect of blocking the erythropoietin (Epo) signaling using an anti-Epo antibody, soluble form of Epo receptor (sEpoR) capable of binding to Epo or EpoR antagonist, and proved to be effective against xenografts of female reproductive organ malignancies and of cancer cell lines in nude mice. We transfected seven cancer cell lines of various origins to express constitutively active sEpoR, and examined their tumorigenesis in nude mice. Suppression of the tumor growth, decrease in viable and proliferating cells and reduction of vascular density were seen individually in all xenografts of transfected cell lines compared with the controls. Quantitative RT-PCR analyses showed that expression levels of Epo, EpoR, ?1A-adrenaline receptor (?1A-ADR) and muscalinic acetylcholine receptor subunit 3 mRNAs (m3-AchR) were higher in the majority of the wild-type xenografts than in the corresponding cell lines except for A549. In some of the transfected xenografts, EpoR, ?1A-ADR and m3-AchR mRNAs were down-regulated. Western blot analyses revealed that the constitutively activated ERK1/2MAPK was discernible in the majority of non-transfected cell lines and was reduced in the transfected cell lines. However, it was regained after exposure to acetylcholine and/or noradrenaline. These findings suggest that constitutively active sEpoR can effectively destroy the xenografts but signals from the autonomic neurotransmitters of the host produced under stress may interfere with this antitumor activity.

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Y. Yasuda, Y. Maeda, S. Hara, M. Tanaka, E. Koike, Y. Watanabe, S. Masuda, H. Yamasaki, K. Okumoto, H. Konishi, Y. Horiuchi and H. Hoshiai, "Constitutively Active Soluble Form of Erythropoietin Receptor Suppresses Growth and Angiogenesis of Xenografts of Transfected Cancer Cell Lines," Journal of Cancer Therapy, Vol. 2 No. 1, 2011, pp. 40-53. doi: 10.4236/jct.2011.21005.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] Y. Yasuda, T. Musha, H. Tanaka, Y. Fujita, H. Utsumi, T. Matsuo, S. Masuda, M. Nagao, R. Sasaki and Y. Nakamura, “Inhibition of Erythropoietin Signalling Destroys Xenografts of Ovarian and Uterine Cancers in Nude Mice,” British Journal of Cancer, Vol. 84, No. 6, 2001, pp. 836-843. doi:10.1054/bjoc.2000.1666
[2] Y. Yasuda, Y. Fujita, T. Matsuo, S. Koinuma, S. Hara, A. Tazaki, M. Onozaki, M. Hashimoto, T. Musha, K. Ogawa, H. Fujita, Y. Nakamura, H. Shiozaki and H. Utsumi, “Erythropoietin Regulates Tumour Growth of Human Malignancies,” Carcinogenesis, Vol. 24, No. 6, 2003, pp. 1021-1029. doi:10.1093/carcin/bgg060
[3] G. Acs, P. Acs, S. M. Beckwith, R. L. Pitts, E. Clements, K. Wong and A. Verma, “Erythropoietin and Erythropoietin Receptor Expression in Human Cancer,” Cancer Research, Vol. 61, No. 9, 2001, pp. 3561-3565.
[4] M. O. Arcasoy, K. Amin, R. T. Vollmer, X. Jiang, W. Demark-Wahnefried and Z. A. Haroon, “Erythropoietin and Erythropoietin Receptor Expression in Human Prostate Cancer,” Modern Pathology, Vol. 18, No. 3, 2005, pp. 421-430. doi:10.1038/modpathol.3800288
[5] A. Anagnostou, Z. Liu, M. Steiner, K. Chin, E. S Lee, N. Kessimian and C. T. Noguchi, “Erythropoietin Receptor mRNA Expression in Human Endothelial Cells,” Proceedings of the National Academy of Sciences of the United States of America, Vol. 91, No. 9, 1994, pp. 3974- 3978. doi:10.1073/pnas.91.9.3974
[6] Y. Yasuda, Y. Fujita, S. Masuda, T. Musha, K. Ueda, H. Tanaka, H. Fujita, T. Matsuo, M. Nagao, R. Sasaki and Y. Nakamura, “Erythropoietin is Involved in Growth and Angiogenesis in Malignant Tumours of Female Reproductive Organs,” Carcinogenesis, Vol. 23, No. 11, 2002, pp. 1797-1805. doi:10.1093/carcin/23.11.1797
[7] M. O. Arcasoy, K. Amin, A. F. Karayal, S. C. Chou, J. A. Raleigh, M. A Varia and Z. A. Haroon, “Functional Significance of Erythropoietin Receptor Expression in Breast Cancer,” Laboratory Investigation, Vol. 82, No. 7, 2002, pp. 911-918.
[8] M. E. Hardee, Y. Cao, P. Fu, X. Jiang, Y. Zhans, Z. N. Rabbani, Z. Vujaskovic, M. W. Dewhirst and M. O. Arcasoy, “Erythropoietin Blockade Inhibits the Induction of Tumor Angiogenesis and Progression,” PLoS One, Vol. 2, No. 6, 2007, p. e549. doi:10.1371/journal.pone.0000549
[9] P. Fu, X. Jiang and M. O. Arcasoy, “Constitutively Active Erythropoietin Receptor Expression in Breast Cancer Cells Promotes Cellular Proliferation and Migration through a MAP-kinase Dependent Pathway,” Biochemical and Biophysical Research Communications, Vol. 379, No. 3, 2009, pp. 696-701. doi:10.1016/j.bbrc.2008.12.146
[10] M. Socolovsky, A. E. Fallon, S. Wang, C. Brugnara and H. F. Lodish, “Fetal Anemia and Apoptosis of Red Cell Progenitors in Stat5a-/-5b-/-mice: A Direct Role for Stat5 in Bcl-X(L) Induction,” Cell, Vol. 98, No. 2, 1999, pp. 181-191. doi:10.1016/S0092-8674(00)81013-2
[11] E. Parganas, D. Wang, D. Stravopodis, D. J. Topham, J. C. Marine, S. Teglund, E. F. Vanin, S. Bodner, O. R. Colamonici, J. M. VanDeursen, G. Grosveld and J. N. Ihle, “Jak2 is Essential for Signaling through a Variety of Cytokine Receptors,” Cell, Vol. 93, No. 3, 1998, pp. 385- 395. doi:10.1016/S0092-8674(00)81167-8
[12] H. Neubauer, A. Cumano, M. Müller, H. Wo, U. Huffstadt and K. Dfeffer, “Jak2 Deficiency Defines an Essential Developmental Checkpoint in Definitive Hematopoiesis,” Cell, Vol. 93, No. 3, 1998, pp. 397-409. doi:10.1016/S0092-8674(00)81168-X
[13] J. E. Damen, A. L. Mui, L. Puil, T. Pawson and G. Krystal, “Phosphatidylinositol 3-kinase Associates, via Its Src homology 2 Domains, with the Activated Erythropoietin Receptor,” Blood, Vol. 81, No. 12, 1993, pp. 3204-3210.
[14] S. Uddin, S. Kottegoda, D. Stigger, L. C. Platanias and A. Wickrema, “Activation of the Akt/FKHRL1 Pathway Mediates the Antiapoptotic Effects of Erythropoietin in Primary Human Eryrhroid Progenitors,” Biochemical and Biophysical Research Communications, Vol. 275, No. 1, 2000, pp. 16-19. doi:10.1006/bbrc.2000.3266
[15] J. H. Myklebust, H. K. Blomhoff, L. S. Rusten, T. Stokke and E. B. Smeland, “Activation of Phosphatidylinositol 3-kinase is Important for Erythropoietin-induced Erythropoiesis from CD34(+) Hematopoietic Progenitor Cells,” Experimental Hematology, Vol. 30, No. 9, 2002, pp. 990-1000. doi:10.1016/S0301-472X(02)00868-8
[16] S. R. Datta, H. Dudek, X. Tao, S. Masters, H. Fu, Y. Gotoh and M. E. Greenberg, “Akt phosphorylation of BAD Couples Survival Signals to the Cell-intrinsic Death Machinery,” Cell, Vol. 91, No. 2, 1997, pp. 231-241. doi:10.1016/S0092-8674(00)80405-5
[17] Y. Miura, O. Miura, J. N. Ihle and N. Aoki, “Activation of the Mitogen-activated Protein Kinase Pathway by the Erythropoietin Receptor,” Journal of Biological Chemistry, Vol. 269, No. 47, 1994, pp. 29962-29969.
[18] L. Feldman, Y. Wang, J. S. Rhim, N. Bhattacharya, M. Loda and A. J. Sytokowski, “Erythropoietin Stimulates Growth and STAT5 Phosphorylation in Human Prostate Epithelial and Prostate Cancer Cells,” Prostate, Vol. 66, No. 2, 2006, 135-145. doi:10.1002/pros.20310
[19] T. Forshew, R. G. Tatevossian, A. R. Lawson, J. Ma, G. Neale, B. W. Ogunkolade, T. A. Jones, J. Aarum, J. Dalton, S. Bailey, T. Chaplin, R. L. Carter, A. Gajjar, A. Broniscer, B. D. Young, D. W. Ellison and D. Sheer, “Activation of the ERK/MAPK Pathway: A Signature Genetic Defect in Posterior Fossa Pilocytic Astrocytomas,” Journal of Pathology, Vol. 218, No. 2, 2009, pp. 172-181.
[20] M. Um and H. F. Lodish, “Antiapoptotic Effects of Erythropoietin in Differentiated Neuroblastoma SH-SY5Y Cells Require Activation of both the STAT5 and AKT Signaling Pathways,” Journal of Biological Chemistry, Vol. 281, No. 9, 2006, pp. 5648-5656.
[21] P. H. Thaker, L. Y. Han, A. A. Kamat, J. M. Arevalo, R. Takahashi, C. Lu, NB. Jennings, G. Armaiz-Pene, J. A. Bankson, M. Ravoori, W. M. Merritt, Y. G. Lin, L. S. Mangala, T. J. Kim, R. L. Coleman, C. N. Landen, Y. Li, E. Felix, A. M. Sanguino, R. A. Neuman, M. Lloyd, D. M. Gershenson, V. Kundra, G. Lopez-Berestein, S. K. Lutgendorf, S. W. Cole and A. K. Sood, “Chronic Stress Promotes Tumour Growth and Angiogenesis in a Mouse Model of Ovarian Carcinoma,” Nature Medicine, Vol. 12, No. 8, 2006, pp. 939-944.
[22] C. N. Jr. Landen, Y. G. Lin, G. N. Armaiz Pena, P. D. Das, J. M. Arevalo, A. A. Kamat, L. Y. Han, N. B. Jennings, W. A. Spannuth, P. H. Thaker, S. K. Lutgendorf, C. A. Savary, A. M. Sangiono, G. Lopez-Berestein, S. W. Cole and A. K. Sood, “Neuroendocrine Modulation of Signal Transducer and Activator of Transcription-3 in Ovarian Cancer,” Cancer Research, Vol. 67, No. 21, 2007, pp. 10389-10396.
[23] W. Rayford, M. J. Noble, M. A. Austenfeld, J. Weigel, W. K. Mebust and G. V. Shah, “Muscarinic Cholinergic ReCeptors Promote Growth of Human Prostate Cancer Cells,” Prostate, Vo. 30, No. 3, 1997, pp. 160-166. doi:10.1002/(SICI)1097-0045(19970215)30:3<160::AID-PROS3>3.0.CO;2-Q
[24] H. Frucht, R. T. Jensen, D. Dexter, W. L. Yang and Y. Xiao, “Human Colon Cancer Cell Proliferation Mediated by the M3 Muscarinic Cholinergic Receptor,” Clinical Cancer Research,” Vol. 5, No. 9, 1999, pp. 2532-2539.
[25] L. E. Rimmaudo, E. de la Torre, E. Sacerdote de Lustig and M. E. Sales, “Muscarinic Receptors are Involved in LMM3 Tumor Cells Proliferation and Angiogenesis,” Biochemical and Biophysical Research Communications, Vol.334, No.4,2005, pp.1359-1364. doi:10.1016/j.bbrc.2005.07.031
[26] G. L. Fiszman, M. C. Middonno, E. de la Torre, M. Farima, A. J. Espa?ol and M. E. Sales, “Activation of Muscarinic Cholinergic Receptors Induces MCF-7 Cells Proliferation and Angiogenesis by Stimulating Nitric Oxide Synthase Activity,” Cancer Biology & Therapy, Vol. 6, No. 7, 2007, pp. 1106-1113. doi:10.4161/cbt.6.7.4330
[27] P. Song, H. S. Sekhon, Y. Jia, J. A. Keller, J. K. Blusztajn, G. P. Mark and E. R. Spindel, “Acetylcholine is Synthesized by and Acts as an Autocrine Growth Factor for Small Cell Lung Carcinoma,” Cancer Research, Vol. 63, No. 1, 2003, pp. 214-221.
[28] P. Song, H. S. Sekhon, A. Lu, J. Arredondo, D. Sauer, C. Gravett, G. P. Mark, S. A. Grando and E. R. Spindel, “M3 Muscarinic Receptor Antagonists Inhibit Small Cell Lung Carcinoma Growth and Mitogen-activated Protein Kinase Phosphorylation Induced by Acetylcholine Secretion” Cancer Research, Vol. 67, No. 8, 2007, pp. 3936- 3944.
[29] Y. Yasuda, M. Nagao, M. Okano, S. Masuda, R. Sasaki, H. Konishi and T. Tanimura, “Localization of Erythropoietin and Erythropoietin-receptor in Postimplantation Mouse Embryos,” Development, Growth & Differentiation, Vol. 35, No. 6, 1993, pp. 711-722.
[30] S. T. Sawyer and W. D. Hankins, “The Functional Form of the Erythropoietin Receptor is a 78-kDa Protein: Correlation with Cell Surface Expression, Endocytosis, and Phosphorylation,” Proceedings of the National Academy of Sciences of the United States of America, Vol. 90, No. 14, 1993, pp. 6849-6853.
[31] H. M. Sculler, “Neurotransmitter Receptor-mediated Signaling Pathways as Modulators of Carcinogenesis,” Progress in Experimental Tumor Research, Vol. 39, Review, 2007, pp. 45-63.
[32] N. Shah, S. Khurana, K. Cheng and J. P. Raufman, “Muscarinic Receptors and Ligands in Cancer,” American Journal of Physiology. Cell Physiology, Vol. 296, No. 2, 2009, pp. C221-232.
[33] P. Song, H. S. Sekhon, X. W. Fu, M. Maier, Y. Jia, J. Duan, B. J. Proskosil, C. Gravett, J. Lindstrom, G. P. Mark, S. Saha and E. R. Spindel, “Activated Cholinergic signaling Provides a Target in Squamous Cell lung Carcinoma,” Cancer Research, Vol. 68, No. 12, 2008, pp. 4693-4700.
[34] H. M. Sculler and M. Cekanova, “NNK-induced Hamster Lung Adenocarcinomas over Express Beta2-Adrenergic and EGFR Signaling Pathways,” Lung Cancer, Vol. 49, No. 1, 2005, pp. 35-45.
[35] H. M. Sculler, “Mechanisms of Smoking-related Lung and Pancreatic Adenocarcinoma Development,” Nature Reviews Cancer, Vol. 2, No. 6, 2002, pp. 455-463.

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