Autocrine/paracrine erythropoietin signalling promotes JAK/ STAT-dependent proliferation of human cervical cancer cells Tania V. Lopez 1 , Terence R.J. Lappin 2 , Perry Maxwell 2 , Zhanzhong Shi 2 , Rebeca Lopez-Marure 3 , Cecilia Aguilar 1 and Leticia Rocha-Zavaleta 1 1 Instituto de Investigaciones Biomedicas, Departamento de Biologia Molecular y Biotecnologia, UNAM, Mexico City, Mexico 2 Centre for Cancer Research and Cell Biology, Queen’s University Belfast, Northern Ireland, United Kingdom 3 Instituto Nacional de Cardiologia ‘‘Ignacio Chavez,’’ Departamento de Biologı ´a Celular, Mexico City, Mexico Erythropoietin (Epo) regulates erythropoiesis by binding to its receptor (EpoR) and promoting cell proliferation, differentiation and inhibition of apoptosis. Epo is widely used to treat cervical cancer-related anaemia. However, there are data suggesting that administration of Epo is associated with an increment in recurrence rate, and decreased disease-free and overall survival. In the present study, we investigated the expression of Epo and EpoR on cervical cancer cell lines. We observed that both EpoR and extracellular Epo are constitutively expressed in cervical cancer cells. Inhibition of either Epo or EpoR expression with siRNA attenuated cell proliferation, whereas addition of exogenous Epo led to a significant increase in cell growth, both in vitro and in vivo. Epo-induced proliferation was associated with the activation of JAK2, JAK3, STAT3 and STAT5 but not JAK1 and STAT1. Our results are consistent with the existence of a functional, endogenous Epo/EpoR system in cervical cancer with the capacity to activate the transduction of signals resulting in an increased proliferation potential. Erythropoietin (Epo) is a glycoprotein hormone that binds to the homodimer Epo receptor (EpoR) on the surface of the erythroid precursor cells promoting proliferation, differentia- tion and protecting erythroid progenitors from apoptosis. 1 Binding of Epo to its receptor induces a conformational change of the intracellular domains resulting in activation of the EpoR-associated janus kinase 2 (JAK2) by reciprocal tyro- sine phosphorylation. Activated JAK2 mediates phosphoryla- tion of tyrosine residues of the EpoR, which function as dock- ing sites for intracellular signal molecules, such as the signal transducer and activator of transcription 5 (STAT5). 2 EpoR- mediated activation of STAT5 leads to cell proliferation. 3 In addition, it has been demonstrated that STAT1 and STAT3 are also downstream molecules of EpoR, and their activation induces a proliferative response of human leukaemia cells. 4 Expression of Epo and EpoR has been documented in many non-haematopoietic cell types and tumours. 5 Moreover, functional autocrine/paracrine Epo/EpoR systems have been identified on human breast carcinoma, 6 melanoma 7 and pros- tate cancer cells, 8 suggesting that the Epo/EpoR axis may contribute to tumour growth and progression. In the human female reproductive tract, expression of Epo has been detected in cells of the cervix, endometrium and ovary, whereas EpoR has been found in glandular and surface epi- thelial cells, follicles and endometrial cells. 9 Likewise, Epo and EpoR have been identified in dysplastic cervical epithe- lium, and cervical carcinoma specimens. 10–12 Furthermore, the expression of Epo and EpoR has been shown to correlate with the severity of dysplasia, 10 suggesting that endogenous Epo/EpoR system may play a crucial role in cervical carcino- genesis. However, the biological relevance of Epo/EpoR expression in cervical cancer remains unclear, since recent studies have provided contrasting results regarding the func- tionality of the receptor in cervical cancer-derived cell lines. 13 In this study, we investigated the expression of Epo and EpoR on cervical cancer cell lines. We present evidence show- ing the expression of EpoR at the mRNA and protein levels, Key words: cervical cancer, erythropoietin, JAK-STAT, proliferation Abbreviations: DMEM: Dulbecco’s modified Eagle’s medium; ECL: enhanced chemiluminescence; ELISA: enzyme-linked immunosorbent assay; Epo: erythropoietin; EpoR: erythropoietin receptor; FBS: foetal bovine serum; JAK: janus kinase; PBS: phosphate-buffered saline; RT-PCR: reverse transcription-polymerase chain reaction; siRNA: small interfering RNA; STAT: signal transducer and activator of transcription Additional Supporting Information may be found in the online version of this article Grant sponsor: Consejo Nacional de Ciencia y Tecnologı ´a (CONACyT); Grant numbers: CB-2005-01-49167, 195193; Grant sponsor: Universidad Nacional Auto ´noma de Me ´xico (PAPIIT); Grant number: IN210507 and IN216810; Grant sponsor: PAEP DOI: 10.1002/ijc.25935 History: Received 18 May 2010; Accepted 29 Nov 2010; Online 20 Jan 2011 Zhanzhong Shi’s present address is: School of Life Sciences, Kingston University, London, UK Correspondence to: Leticia Rocha-Zavaleta, Instituto de Investigaciones Biomedicas, Departamento de Biologia Molecular y Biotecnologia, UNAM, Circuito Escolar S/N, Ciudad Universitaria, Coyoacan, Mexico D.F. C.P. 04510, Mexico, Tel.: (5255)-5622-9218, Fax: (5255)-5622-9212, E-mail: lrochaz@correo.biomedicas.unam.mx Cancer Cell Biology Int. J. Cancer: 129, 2566–2576 (2011) V C 2011 UICC International Journal of Cancer IJC