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RNA Interference-Mediated Inhibition of Erythropoietin Receptor Expression Suppresses Tumor Growth and Invasiveness in A2780 Human Ovarian Carcinoma Cells

2009; Elsevier BV; Volume: 174; Issue: 4 Linguagem: Inglês

10.2353/ajpath.2009.080592

ISSN

1525-2191

Autores

György Paragh, Suresh Kumar, Zsuzsa Rákosy, Soek-Choel Choi, Xiaowei Xu, Géza Ács,

Tópico(s)

CRISPR and Genetic Engineering

Resumo

Although recombinant human erythropoietin (rHuEpo) has revolutionized the treatment of anemia, recent clinical trials suggested that rHuEpo use may be associated with decreased survival in cancer patients. Although the expression of erythropoietin (Epo) receptor (EpoR) has been demonstrated in various human cancers, the effect of exogenous Epo on the growth and therapy resistance of EpoR-bearing tumor cells is unclear at present. In the current study, we examined the hypothesis that EpoR may contribute to tumor growth independent of Epo in A2780 human ovarian carcinoma cells. A2780 human ovarian carcinoma cells showed high levels of EpoR expression, but lacked expression of Epo mRNA and biologically active Epo protein under both normoxic and hypoxic conditions. Exogenous Epo did not stimulate EpoR-mediated signaling, proliferation, invasiveness, or resistance to cytotoxic drugs in A2780 cells. In contrast, specific inhibition of EpoR expression using a short hairpin RNA (shRNA) expression plasmid resulted in markedly reduced proliferation and invasiveness in vitro. In addition, inhibition of EpoR expression led to abrogated in vivo ovarian cancer cell growth in a tumor xenograft system and resulted in decreased EpoR signaling. Our findings suggest that EpoR may be constitutively active in some cancer cells in the absence of Epo and provide the first evidence for a potential role of an Epo-independent, EpoR-mediated pathway in the growth of some human cancers. Although recombinant human erythropoietin (rHuEpo) has revolutionized the treatment of anemia, recent clinical trials suggested that rHuEpo use may be associated with decreased survival in cancer patients. Although the expression of erythropoietin (Epo) receptor (EpoR) has been demonstrated in various human cancers, the effect of exogenous Epo on the growth and therapy resistance of EpoR-bearing tumor cells is unclear at present. In the current study, we examined the hypothesis that EpoR may contribute to tumor growth independent of Epo in A2780 human ovarian carcinoma cells. A2780 human ovarian carcinoma cells showed high levels of EpoR expression, but lacked expression of Epo mRNA and biologically active Epo protein under both normoxic and hypoxic conditions. Exogenous Epo did not stimulate EpoR-mediated signaling, proliferation, invasiveness, or resistance to cytotoxic drugs in A2780 cells. In contrast, specific inhibition of EpoR expression using a short hairpin RNA (shRNA) expression plasmid resulted in markedly reduced proliferation and invasiveness in vitro. In addition, inhibition of EpoR expression led to abrogated in vivo ovarian cancer cell growth in a tumor xenograft system and resulted in decreased EpoR signaling. Our findings suggest that EpoR may be constitutively active in some cancer cells in the absence of Epo and provide the first evidence for a potential role of an Epo-independent, EpoR-mediated pathway in the growth of some human cancers. Erythropoietin (Epo), a glycoprotein hormone produced by the kidney in response to hypoxia,1Jelkmann W Molecular biology of erythropoietin.Intern Med. 2004; 43: 649-659Crossref PubMed Scopus (288) Google Scholar, 2Ebert BL Bunn HF Regulation of the erythropoietin gene.Blood. 1999; 94: 1864-1877Crossref PubMed Google Scholar has been considered to be a specific stimulator of erythropoiesis.1Jelkmann W Molecular biology of erythropoietin.Intern Med. 2004; 43: 649-659Crossref PubMed Scopus (288) Google Scholar Epo acts via its receptor (EpoR), a member of the cytokine receptor type I superfamily. Signal transduction on Epo binding takes place because of conformational change leading to activation of EpoR-bound Janus kinase 2 (JAK2)3Witthuhn BA Quelle FW Silvennoinen O Yi T Tang B Miura O Ihle JN JAK2 associates with the erythropoietin receptor and is tyrosine phosphorylated and activated following stimulation with erythropoietin.Cell. 1993; 74: 227-236Abstract Full Text PDF PubMed Scopus (1007) Google Scholar and via recruitment and activation of signal transducer-activator of transcription-5 (STAT5), leads to activation of mitogen-activated protein kinases (MAPK), phosphatidylinositol 3-kinase (PI3K), and Akt. Recent evidence suggests that JAK2-independent Epo signaling may also exist.4Bittorf T Buchse T Sasse T Jaster R Brock J Activation of the transcription factor NF-kappaB by the erythropoietin receptor: structural requirements and biological significance.Cell Signal. 2001; 13: 673-681Crossref PubMed Scopus (64) Google Scholar EpoR stimulation in erythrocytic progenitors results in the stimulation of proliferation and differentiation, and inhibition of apoptosis.1Jelkmann W Molecular biology of erythropoietin.Intern Med. 2004; 43: 649-659Crossref PubMed Scopus (288) Google Scholar, 5Miura Y Miura O Ihle JN Aoki N Activation of the mitogen-activated protein kinase pathway by the erythropoietin receptor.J Biol Chem. 1994; 269: 29962-29969Abstract Full Text PDF PubMed Google Scholar Until recently, the action of Epo was considered to be restricted to erythropoietic cells. In recent years, however, it has become clear that EpoR is expressed by several other cell types, and various biological effects of Epo, including neuro- and cardioprotection under hypoxia, stimulation of angiogenesis, cell proliferation, and migration, have been demonstrated.6Brines M Cerami A Discovering erythropoietin's extra-hematopoietic functions: biology and clinical promise.Kidney Int. 2006; 70: 246-250Crossref PubMed Scopus (254) Google Scholar We and others have recently described that various cancer cells express Epo and EpoR at the gene and protein levels and display functional EpoR signaling.7Acs G Chen M Xu X Acs P Verma A Koch CJ Autocrine erythropoietin signaling inhibits hypoxia-induced apoptosis in human breast carcinoma cells.Cancer Lett. 2004; 214: 243-251Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 8Acs G Zhang PJ McGrath CM Acs P McBroom J Mohyeldin A Liu S Lu H Verma A Hypoxia-inducible erythropoietin signaling in squamous dysplasia and squamous cell carcinoma of the uterine cervix and its potential role in cervical carcinogenesis and tumor progression.Am J Pathol. 2003; 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24: 4442-4449Crossref PubMed Scopus (150) Google Scholar, 14Feldman L Wang YX Rhim JS Bhattacharya N Loda M Sytkowski AJ Erythropoietin stimulates growth and STAT5 phosphorylation in human prostate epithelial and prostate cancer cells.Prostate. 2006; 66: 135-145Crossref PubMed Scopus (87) Google Scholar, 15Mohyeldin A Dalgard CL Lu H Mcfate T Tait AS Patel VC Wong K Rushing E Roy S Acs G Verma A Survival and invasiveness of astrocytomas promoted by erythropoietin.J Neurosurg. 2007; 106: 338-350Crossref PubMed Scopus (33) Google Scholar, 16Arcasoy MO Amin K Karayal AF Chou SC Raleigh JA Varia MA Haroon ZA Functional significance of erythropoietin receptor expression in breast cancer.Lab Invest. 2002; 82: 911-918Crossref PubMed Scopus (161) Google Scholar Although several studies suggested that Epo may enhance tumor angiogenesis,17Ribatti D Presta M Vacca A Ria R Giuliani R Dell'Era P Nico B Roncali L Dammacco F Human erythropoietin induces a pro-angiogenic phenotype in cultured endothelial cells and stimulates neovascularization in vivo.Blood. 1999; 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24: 4442-4449Crossref PubMed Scopus (150) Google Scholar, 15Mohyeldin A Dalgard CL Lu H Mcfate T Tait AS Patel VC Wong K Rushing E Roy S Acs G Verma A Survival and invasiveness of astrocytomas promoted by erythropoietin.J Neurosurg. 2007; 106: 338-350Crossref PubMed Scopus (33) Google Scholar survival,7Acs G Chen M Xu X Acs P Verma A Koch CJ Autocrine erythropoietin signaling inhibits hypoxia-induced apoptosis in human breast carcinoma cells.Cancer Lett. 2004; 214: 243-251Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 8Acs G Zhang PJ McGrath CM Acs P McBroom J Mohyeldin A Liu S Lu H Verma A Hypoxia-inducible erythropoietin signaling in squamous dysplasia and squamous cell carcinoma of the uterine cervix and its potential role in cervical carcinogenesis and tumor progression.Am J Pathol. 2003; 162: 1789-1806Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 15Mohyeldin A Dalgard CL Lu H Mcfate T Tait AS Patel VC Wong K Rushing E Roy S Acs G Verma A Survival and invasiveness of astrocytomas promoted by erythropoietin.J Neurosurg. 2007; 106: 338-350Crossref PubMed Scopus (33) Google Scholar, 20Pajonk F Weil A Sommer A Suwinski R Henke M The erythropoietin-receptor pathway modulates survival of cancer cells.Oncogene. 2004; 23: 8987-8991Crossref PubMed Scopus (47) Google Scholar and proliferation,9Acs G Acs P Beckwith SM Pitts RL Clements E Wong K Verma A Erythropoietin and erythropoietin receptor expression in human cancer.Cancer Res. 2001; 61: 3561-3565PubMed Google Scholar, 14Feldman L Wang YX Rhim JS Bhattacharya N Loda M Sytkowski AJ Erythropoietin stimulates growth and STAT5 phosphorylation in human prostate epithelial and prostate cancer cells.Prostate. 2006; 66: 135-145Crossref PubMed Scopus (87) Google Scholar, 21Westenfelder C Baranowski RL Erythropoietin stimulates proliferation of human renal carcinoma cells.Kidney Int. 2000; 58: 647-657Crossref PubMed Scopus (200) Google Scholar other reports contradicted these findings22Belda-Iniesta C Perona R Carpeno Jde C Cejas P Casado E Manguan-Garcia C Ibanez de Caceres I Sanchez-Perez I Andreu FB Ferreira JA Aguilera A Dela PJ Perez-Sanchez E Madero R Feliu J Sereno M Gonzalez-Baron M Human recombinant erythropoietin does not promote cancer growth in presence of functional receptors expressed in cancer cells.Cancer Biol Ther. 2007; 6: 1600-1605Crossref PubMed Scopus (12) Google Scholar, 23Liu WM Powles T Shamash J Propper D Oliver T Joel S Effect of haemopoietic growth factors on cancer cell lines and their role in chemosensitivity.Oncogene. 2004; 23: 981-990Crossref PubMed Scopus (50) Google Scholar, 24LaMontagne KR Butler J Marshall DJ Tullai J Gechtman Z Hall C Meshaw A Farrell FX Recombinant epoetins do not stimulate tumor growth in erythropoietin receptor-positive breast carcinoma models.Mol Cancer Ther. 2006; 5: 347-355Crossref PubMed Scopus (69) Google Scholar and thus, currently the role for EpoR in cancer biology is unclear. Recombinant human erythropoietin (rHuEpo) has revolutionized the treatment of anemia in chronic renal failure.25Singh AK Szczech L Tang KL Barnhart H Sapp S Wolfson M Reddan D Correction of anemia with epoetin alfa in chronic kidney disease.N Engl J Med. 2006; 355: 2085-2098Crossref PubMed Scopus (2283) Google Scholar Anemia is present in many cancer patients at the time of diagnosis and/or as the result of cancer therapy.26Bohlius J Weingart O Trelle S Engert A Cancer-related anemia and recombinant human erythropoietin—an updated overview.Nat Clin Pract Oncol. 2006; 3: 152-164Crossref PubMed Scopus (97) Google Scholar Anemia not only impairs the quality of life of patients, but it leads to tumor hypoxia, resistance to chemo- and radiotherapy,27Shannon AM Bouchier-Hayes DJ Condron CM Toomey D Tumour hypoxia, chemotherapeutic resistance and hypoxia-related therapies.Cancer Treat Rev. 2003; 29: 297-307Abstract Full Text Full Text PDF PubMed Scopus (458) Google Scholar and reduces survival.26Bohlius J Weingart O Trelle S Engert A Cancer-related anemia and recombinant human erythropoietin—an updated overview.Nat Clin Pract Oncol. 2006; 3: 152-164Crossref PubMed Scopus (97) Google Scholar Because of the apparent interconnections among anemia, hypoxia, tumor responsiveness to therapy, and outcomes, clinical studies have been conducted in the assumption that correction of anemia will not only alleviate anemia-related symptoms but also improve tumor response to therapy and increase survival.26Bohlius J Weingart O Trelle S Engert A Cancer-related anemia and recombinant human erythropoietin—an updated overview.Nat Clin Pract Oncol. 2006; 3: 152-164Crossref PubMed Scopus (97) Google Scholar, 28Hudis CA Van BS Chang J Muenstedt K rHuEPO and treatment outcomes: the clinical experience.Oncologist. 2004; 9: 55-69Crossref PubMed Scopus (19) Google Scholar, 29Bohlius J Langensiepen S Schwarzer G Seidenfeld J Piper M Bennett C Engert A Recombinant human erythropoietin and overall survival in cancer patients: results of a comprehensive meta-analysis.J Natl Cancer Inst. 2005; 97: 489-498Crossref PubMed Scopus (218) Google Scholar Although rHuEpo treatment was clearly shown to be effective in reducing transfusion requirements and improve quality of life,26Bohlius J Weingart O Trelle S Engert A Cancer-related anemia and recombinant human erythropoietin—an updated overview.Nat Clin Pract Oncol. 2006; 3: 152-164Crossref PubMed Scopus (97) Google Scholar, 28Hudis CA Van BS Chang J Muenstedt K rHuEPO and treatment outcomes: the clinical experience.Oncologist. 2004; 9: 55-69Crossref PubMed Scopus (19) Google Scholar, 29Bohlius J Langensiepen S Schwarzer G Seidenfeld J Piper M Bennett C Engert A Recombinant human erythropoietin and overall survival in cancer patients: results of a comprehensive meta-analysis.J Natl Cancer Inst. 2005; 97: 489-498Crossref PubMed Scopus (218) Google Scholar its effects on survival are controversial. Although a recent meta-analysis of clinical trials indicated no adverse effect,29Bohlius J Langensiepen S Schwarzer G Seidenfeld J Piper M Bennett C Engert A Recombinant human erythropoietin and overall survival in cancer patients: results of a comprehensive meta-analysis.J Natl Cancer Inst. 2005; 97: 489-498Crossref PubMed Scopus (218) Google Scholar the results of several recent clinical trials in patients with breast,30Leyland-Jones B Semiglazov V Pawlicki M Pienkowski T Tjulandin S Manikhas G Makhson A Roth A Dodwell D Baselga J Biakhov M Valuckas K Voznyi E Liu XY Vercammen E Maintaining normal hemoglobin levels with epoetin alfa in mainly nonanemic patients with metastatic breast cancer receiving first-line chemotherapy: a survival study.J Clin Oncol. 2005; 23: 5960-5972Crossref PubMed Scopus (606) Google Scholar, 31Leyland-Jones B Breast cancer trial with erythropoietin terminated unexpectedly.Lancet Oncol. 2003; 4: 459-460Abstract Full Text Full Text PDF PubMed Scopus (471) Google Scholar (PREPARE study, http://wwwext.amgen.com/media/media_pr_detail.jsp?year=2007&releaseID=1083091, accessed 01/2009), head and neck,32Henke M Laszig R Rube C Schafer U Haase KD Schilcher B Mose S Beer KT Burger U Dougherty C Frommhold H Erythropoietin to treat head and neck cancer patients with anaemia undergoing radiotherapy: randomised, double-blind, placebo-controlled trial.Lancet. 2003; 362: 1255-1260Abstract Full Text Full Text PDF PubMed Scopus (1161) Google Scholar, 33Overgaard J Hoff C Sand Hansen H Specht L Overgaard M Grau C Andersen E Johansen J Andersen L Evensen J Randomized study of the importance of novel erythropoiesis stimulating protein (Aranesp) for the effect of radiotherapy in patients with primary squamous cell carcinoma of the head and neck (HNSCC)—the Danish Head and Neck Cancer Group DAHANCA 10 randomized trial.Eur J Cancer Suppl. 2007; 5 ([Abstract 6LB]): 7Abstract Full Text PDF Google Scholar lung,34Wright JR Ung YC Julian JA Pritchard KI Whelan TJ Smith C Szechtman B Roa W Mulroy L Rudinskas L Gagnon B Okawara GS Levine MN Randomized, double-blind, placebo-controlled trial of erythropoietin in non-small-cell lung cancer with disease-related anemia.J Clin Oncol. 2007; 25: 1027-1032Crossref PubMed Scopus (377) Google Scholar and cervix35Thomas G Ali S Hoebers FJ Darcy KM Rodgers WH Patel M Abulafia O Lucci III, JA Begg AC Phase III trial to evaluate the efficacy of maintaining hemoglobin levels above 12.0 g/dL with erythropoietin vs above 10.0 g/dL without erythropoietin in anemic patients receiving concurrent radiation and cisplatin for cervical cancer.Gynecol Oncol. 2008; 108: 317-325Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar cancers suggested a potential adverse outcome in rHuEpo-treated patients compared with placebo-treated groups, partly because of earlier tumor progression/recurrence, prompting the United States Food and Drug Administration to issue a black box warning for Epo products.36Juneja V Keegan P Gootenberg JE Rothmann MD Shen YL Lee KY Weiss KD Pazdur R Continuing reassessment of the risks of erythropoiesis-stimulating agents in patients with cancer.Clin Cancer Res. 2008; 14: 3242-3247Crossref PubMed Scopus (36) Google Scholar The hypothesis currently put forward suggests that EpoR may play a role in tumor progression by stimulating proliferation and/or inhibiting apoptosis of cancer cells on Epo binding, and some investigators suggested that the adverse effects are likely attributable to stimulation of EpoR present on cancers cells by rHuEpo treatment.37Henke M Mattern D Pepe M Bezay C Weissenberger C Werner M Pajonk F Do erythropoietin receptors on cancer cells explain unexpected clinical findings?.J Clin Oncol. 2006; 24: 4708-4713Crossref PubMed Scopus (222) Google Scholar In contrast, others, based on in vitro and in vivo data showing lack of a proliferative effect of exogenous Epo despite EpoR expression, argue that whereas EpoR is present in cancer cells, it is not biologically active and not essential for tumor growth.22Belda-Iniesta C Perona R Carpeno Jde C Cejas P Casado E Manguan-Garcia C Ibanez de Caceres I Sanchez-Perez I Andreu FB Ferreira JA Aguilera A Dela PJ Perez-Sanchez E Madero R Feliu J Sereno M Gonzalez-Baron M Human recombinant erythropoietin does not promote cancer growth in presence of functional receptors expressed in cancer cells.Cancer Biol Ther. 2007; 6: 1600-1605Crossref PubMed Scopus (12) Google Scholar, 23Liu WM Powles T Shamash J Propper D Oliver T Joel S Effect of haemopoietic growth factors on cancer cell lines and their role in chemosensitivity.Oncogene. 2004; 23: 981-990Crossref PubMed Scopus (50) Google Scholar, 24LaMontagne KR Butler J Marshall DJ Tullai J Gechtman Z Hall C Meshaw A Farrell FX Recombinant epoetins do not stimulate tumor growth in erythropoietin receptor-positive breast carcinoma models.Mol Cancer Ther. 2006; 5: 347-355Crossref PubMed Scopus (69) Google Scholar, 38Gewirtz DA Di X Walker TD Sawyer ST Erythropoietin fails to interfere with the antiproliferative and cytotoxic effects of antitumor drugs.Clin Cancer Res. 2006; 12: 2232-2238Crossref PubMed Scopus (48) Google Scholar However, cancer cells are thought to undergo a continuous cycle of natural selection during which better adapted cells are favored to pass on their genetic information, and they are unlikely to retain biologically inactive metabolic and regulatory pathways. In the present study we studied the role and significance of EpoR expressed in A2780 human ovarian carcinoma cells that are not responsive to exogenous Epo. The A2780 human ovarian carcinoma cells were provided by Dr. George Coukos (University of Pennsylvania, Philadelphia, PA). CHO cells were purchased from Invitrogen (Carlsbad, CA). UT-7 cells were a gift from Dr. Elaine Dunlop (Belfast City Hospital, Belfast, UK). HepG2 and K562 cells were received from Drs. Jiandong Chen and Mei Huang, respectively (Moffitt Cancer Center, Tampa, FL). Cells were grown in RPMI (GIBCO Invitrogen, Carlsbad, CA), supplemented with 10% fetal bovine serum (FBS) (Hyclone, Logan, UT), penicillin (50 IU/ml), and streptomycin sulfate (50 μg/ml) (GIBCO Invitrogen) at 37°C in a humidified atmosphere containing 5% CO2, unless otherwise specified. Media of UT-7 cells were supplemented with 10 U/ml rHuEpo (Epogen, Epoetin α; Amgen Pharmaceuticals, Thousand Oaks, CA) after each passage. To test the short-term effect of rHuEpo treatment on cell lines, subconfluent cultures of A2780 and 1 × 106 UT-7 cells were treated for 5 minutes with rHuEpo in six-well plates after overnight serum (1% FBS) and, in case of UT-7 cells, Epo starvation. Twenty-four hours before hypoxia treatments cells were switched to serum-free medium. Hypoxia treatment of cells was performed in an enclosed chamber (Billups-Rothenberg Inc., Del Mar, CA) flushed with premixed gas mixture (2% O2, 5% CO2, 93% N2) for 6 hours. To test the ability of Epo neutralization to effect the proliferation and survival of A2780 and UT-7 cells, the AB-286-NA Epo neutralization rabbit total IgG (R&D Systems, Minneapolis, MN) was used at a concentration of 30 μg/ml (corresponding to 10 × 50% neutralization dose). A2780 cells were plated in wells of 96-well plates (2000 cells/well) and allowed to adhere overnight. UT-7 cells, growing in suspension culture, were washed three times in culture media lacking Epo and suspended in treatment medium at a density of 2000 cells/100 μl. The medium of adhered A2780 cells was changed to 100-μl treatment medium consisting of 10% FBS in RPMI, 10% FBS in RPMI with 0.4 U/ml Epo, 10% FBS in RPMI with 30 μg/ml neutralizing antibody, or 10% FBS in RPMI with both 0.4 U/ml Epo and 30 μg/ml neutralizing antibody. Oligonucleotide cDNA inserts encoding short hairpin RNA (shRNA) specific for EpoR were designed using the Insert Design Tool available at the Ambion (Austin, TX) website. The oligonucleotides were annealed and ligated into the pSilencer 4.1-CMV hygro vector (Ambion) containing a modified cytomegalovirus (CMV) promoter for RNA polymerase II-mediated transcription. EpoR shRNA oligonucleotides (top strand, 5′-GATCCCTACAGCTTCTCCTACCAGTTCAAGAGACTGGTAGGAGAAGCTGTAGTTA-3′; bottom strand, 5′-AGCTTAACTACAGCTTCTCCTACCAGTCTCTTGAACTGGTAGGAGAAGCTGTAGG-3′) contained a region specific to bases 362 to 382 of EpoR mRNA (bold), a hairpin loop region, and 5′ and 3′ linker sequences for subcloning into the HindIII and BamHI sites of the pSilencer 4.1-CMV hygro vector. The scrambled negative control shRNA oligonucleotides contained identical hairpin loop and linker sequences but a sequence of DNA not complementary to any known human gene (5′-GACCAGCTTCTCCACAATCAT-3′). The newly created pSilencer 4.1-CMV hygro-EpoR shRNA (pS-EpoR) and pSilencer 4.1-CMV hygro-scrambled shRNA (pS-Neg) vectors were prepared from individual bacterial colonies. Correct orientation and location of the oligonucleotide cloning were confirmed by sequencing. The pBCMGS-Neo expression vector containing human wild-type full-length EpoR was generously provided by Dr. Ajay Verma (Uniformed Services University of the Health Sciences, Bethesda, MD). The sequence validated EpoR coding region was incorporated into pcDNA6.2 D-TOPO vector (Invitrogen) according to the manufacturer's protocol and used for full-length human EpoR overexpression in A2780 cells. For stable transfection, A2780 cells were plated in six-well plates at a density of 5 × 105 cells per well and incubated overnight. Cells were transfected with Lipofectamine 2000 (Invitrogen) using 4 μg of the pSilencer 4.1-CMV hygro plasmid containing EpoR shRNA or the scrambled negative control sequence. Stable transfectant cell lines were selected by growth in the presence of 200 μg/ml of hygromycin (Invitrogen). Individual cells were isolated with cloning disks. Thirty-five stable clones expressing the EpoR shRNA were screened for EpoR knockdown using quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting. Full-length EpoR-overexpressing pcDNA6.2 plasmids were nucleofected into A2780 cells using an Amaxa Nucleofector and Cell Line Nucleofector Kit V (Amaxa Inc., Gaithersburg, MD) according to the manufacturer's protocol. Stable EpoR-expressing cells were selected with 10 μg/ml of Blasticidin (Invivogen, San Diego, CA) and were used for experiments after at least 2 weeks of selection. Total RNA was extracted using the RNeasy mini kit (Qiagen, Valencia, CA). For RT-PCR 1 μg of total RNA per sample was reverse-transcribed to cDNA using the SuperScript first-strand synthesis system (Invitrogen). Quantitative real-time PCR was performed on the iCycler real-time detection system (Bio-Rad Laboratories, Hercules, CA) using a total reaction volume of 20 μl containing 5 μl of cDNA template, sense and antisense primers, and 1× iQ SYBR Green Supermix reagents (Bio-Rad Laboratories). Amplifications were performed at 95°C for 3 minutes and for 40 cycles of 30 seconds at 95°C and 30 seconds at 60°C, and SYBR Green melting curve was measured from 55 to 90°C. Gene expression was normalized to either the geometric mean of three endogenous control genes, including β-actin (BACT), succinate dehydrogenase (SDHA), and hypoxanthine phosphoribosyltransferase 1 (HPRT1), or in case of measurements from the same cell lines to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The primers used were the following: EpoR sense 5′-CGGGCAACTACAGCTTCTCC-3′, antisense 5′-GTAGGCAGCGAACACCAGAA-3′; Epo sense 5′-CTGGAAGAGGATGGAGGTCGG-3′, antisense: 5′-GCTGGGAAGAGTTGACCAACAG-3′; BACT sense 5′-CTACCTCATGAAGATCCTCACCGA-3′, antisense 5′-ACGTAGCACAGCTTCTCCTTAATG-3′; SDHA sense 5′-GGACAACTGGAGGTGGCATTT-3′, antisense 5′-TGTAGTGGATGGCATCCTGGT-3′; HPRT1 sense 5′-CTCCTCCTGAGCAGTCAGCC-3′, antisense 5′-CATCATCACTAATCACGACGCC-3′; GAPDH sense 5′-AACCTGCCAAATATGATGACATCA-3′, antisense 5′-TAGCCCAGGATGCCCTTGAG-3′. Primers were designed using the Beacon Designer software (version 3; Premier Biosoft International, Palo Alto, CA). Cells were washed twice with ice cold phosphate-buffered saline (PBS) and lysed in RIPA buffer containing 1× Halt phosphatase and protease inhibitor cocktails (Pierce, Rockford, IL). Tumor xenograft samples were lysed in Tissue Protein Extraction Reagent (T-PER, Pierce) with phosphatase and protease inhibitor cocktails. Whole cell lysates were normalized for protein. Twenty μg of proteins from each sample were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. Proteins were detected using antibodies against EpoR (goat polyclonal, 1:500 dilution; R&D Systems), phosphorylated-EpoR (p-EpoR) (rabbit polyclonal, 1:500; Santa Cruz Biotechnology, Santa Cruz, CA), and JAK2 (rabbit polyclonal, 1:1000; Santa Cruz). p-JAK2, STAT5, p-STAT5, Akt, p-Akt, MAPK, p-MAPK rabbit polyclonal antibodies were also obtained from Cell Signaling Technology (Danvers, MA) and used in 1:1000 dilution. As a loading control, horseradish peroxidase-conjugated polyclonal antibodies to β-actin (1:2000) and GAPDH (1:4000) (both from Santa Cruz) were used. Membranes were incubated with the primary antibodies overnight at 4°C; horseradish peroxidase-conjugated bovine anti-rabbit, anti-goat, or anti-mouse pre-absorbed antibodies for secondary staining were purchased from Santa Cruz and used in 1:5000 dilution. Immunoreactive bands were visualized using chemiluminescence (ECL Advanced Western blotting detection system; Amersham-GE Healthcare Bio-Sciences Co., Piscataway, NJ). Cell surface EpoR expression was assessed by flow cytometry using phycoerythrin-conjugated mouse monoclonal EpoR antibody (FAB307P) and matching isotype-negative control (IC003P) (both from R&D Systems). Cell staining was performed according to the manufacturer's protocol and measured using a FACScan flow cytometer (Becton Dickinson, Mountain View, CA). Results were analyzed using the FlowJo software (Tree Star Inc., Ashland, OR). Cell viability was assessed using a modified MTT assay (CellTiter 96; Promega, Madison, WI) according to the manufacturer's recommendations. Briefly, for determination of growth rate cells were plated in 96-well plates at a density of 1000 cells per well in 100 μl of medium containing rHuEpo (0 to 100 U/ml). The amount of viable cells was determined every 24 hours using eight wells per time point. For cytotoxic drug treatment experiments cells were plated at a density of 10,000 cells

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