Erythropoietin regulates tumour growth of human malignancies

Abstract

In addition to the chief function of erythropoietin (Epo) in promoting erythropoiesis, some other roles have been found in the brain and uterus. We have reported that signalling pathways of Epo and Epo receptor (EpoR) are involved in the tumourigenesis of ovarian and uterine cancers. To determine whether Epo plays a similar role in other malignancies, we studied the expression of Epo in several malignant human cell lines. We found that 24 malignant human cell lines examined express Epo and EpoR regardless of their origins, types, genetic characteristics and biological properties and secrete a very small amount of Epo individually and that most of them respond to hypoxic stimuli by enhanced secretion of Epo. To determine whether the Epo-EpoR pathway operates in tumours of these cell lines, we transplanted several cell lines into nude mice and confirmed the presence of Epo-responsive sites in xenografts in which the phosphorylation of the STAT5 (signal transducer and activator of transcription) is detectable. Furthermore, in nude mice we blocked the Epo signalling in xenografts of two representative cell lines, stomach choriocarcinoma and melanoma, by i.p. injections of EpoR antagonist and found inhibition of angiogenesis and survival of tumour cells leading to destruction of tumour masses and disturbances of phosphorylation of STAT5. In contrast, Epo mimetic peptide promotes angiogenesis and tumour cell survival. These findings suggest that Epo is indispensable for the growth and viability of malignant tumour and also that the deprivation of Epo signalling may be a promising therapy for human malignancy.

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

“…The resulting plasmid, sEpoR-(His) 6 /pcDNA3.1, produces a soluble (secreted) form of EpoR (aa 3 to 226) with five additional aa Ala-Arg-Val-Val-Ala at the N-terminal and Arg-Ser-(His) 6 at the C-terminal ends. Seven cell lines, SCH, HepG2, A549, SBC3, HeLa, P39 and PC-3, were maintained in the conditions described previously [2] and were transfected with the expression vector for sEpoR-(His) 6 (typically 0.67 μg for 3.5 cm dish) using Lipofectamine Plus (Life Technologies, Rockville, Maryland, U.S.A.) according to the manufacturer's recommendations. After selection with 500 μg/ml G418 (Life Technologies) for 10-14 days, the resistant clones were isolated and expanded.…”

“…Erythropoietin (Epo), a principal cytokine involved in erythropoiesis, has been reported to promote the growth of malignant tumors by binding to its receptor (EpoR) through both autocrine stimulation of malignant cells themselves and paracrine route to the feeding microvessels due to the expression of both transcripts for Epo and EpoR mRNA in very many cancers [1][2][3][4] and for EpoR mRNA in the human endothelial cells of vein and artery [5]. However, there is controversy over the EpoR in cancer patients to be functional to develop their malignancies.…”

“…Preclinical studies clarified that blockade of the Epo signal in resected specimens [6], xenografts [2] or tumor chamber [7] using anti-Epo antibody, soluble form of EpoR (sEpoR) capable of binding to Epo, EpoR antagonists [6] or pharmacologic JAK2 inhibitor [7] led to their destruction. The involvement of Epo in malignancies has been shown by transfection of Epo agonist R103A-Epo [8] or constitutively active EpoR-R129 [9] into rat mammary carcinoma cells.…”

“…The major signaling pathways involved in hematopoiesis are the JAK2 and STAT5 [10][11][12], the PI3K/AKT [13][14][15][16] and the ERK/MAPK [17] routes for survival and/or proliferation. The JAK2 and STAT5 route is detected in female reproductive organ malignancies [6], in xenografts of melanoma and stomach choriocarcinoma cell lines [2] and in a prostate cancer cell line [18]; and, the ERK/MAPK route is seen in mammary carcinoma [8,9] and astrocytoma [19]. Both the STAT5 and the AKT routes are in operation in neuroblastoma cells [20].…”

“…To develop more effective treatment [1,2,6], we transfected seven cancer cell lines, SCH, HepG2, A549, SBC3, HeLa, P39 and PC-3 to express constitutively active sEpoR and implanted them in nude mice. We report here that constitutive secretion of sEpoR suppressed the tumor size of all xenografts except for those of P39 and that the present device revealed the blockade of promotion pathways in all xenografts; less numerous viable and proliferating cells led to severe reduction of vascular densities in the transfected xenografts compared with those in controls.…”