Biological Significance of Erythropoietin and Erythropoietin Receptor Expression by Human Ovarian Cancer Cells.

Erythropoietin (Epo) is the primary regulator of erythroid cell proliferation and differentiation mediated through its specific binding to the Epo receptor (EpoR). Epo also is known to have non-hematopoietic actions, including the promotion of wound healing, tissue protection and others. Accumulating evidence demonstrating both Epo and EpoR expression in many types of human cancers has raised concern about the clinical use of Epo and other erythropoiesis stimulating agents (ESAs) in cancer-related anemia, since the presence of functional EpoR on the tumor cells suggests the potential for an adverse effect of Epo treatment by enhancing cancer growth and progression. Previously, we demonstrated that A2780 ovarian cancer cells express the EpoR gene and that long-term Epo treatment of these cells renders them resistant to paclitaxel. We now report the characterization and biological significance of Epo and EpoR expression by four ovarian cancer cell lines. Using semi-quantitative RT-PCR, restriction digestion of the PCR products and DNA sequence analysis, we demonstrated that A2780, CaOV, SKOV, and OVCAR-3 ovarian cancer cell lines examined express Epo and EpoR at the mRNA level. We demonstrated EpoR protein expression both by western blotting and by immunofluorescence, and demonstrated biologically active Epo protein expression by quantitative in vitro bioassay of the cell culture supernatants. The EpoR on A2780 cells are functional, since Epo stimulation of the cells resulted in a time-dependent 5-fold increase in phosphorylation of Erk1/2 that reached a peak in 10 min and then returned to the basal level. None of the cell lines exhibited a short-term (3-day) growth response in culture to exogenous Epo. However, addition of a neutralizing anti-Epo antibody to the cell culture resulted in partial growth inhibition of A2780 cells that was reversed by addition of excess Epo, consistent with an autocrine/paracrine mechanism of Epo growth enhancement of these cells. We also found that long-term Epo treatment of A2780 cells resulted in the development of a phenotype exhibiting enhanced Epo signaling, evidenced by a 20-fold increase in phosphorylation levels of Erk1/2. This phenotype was sustained even after the removal of Epo, suggesting a mechanism underlying the paclitaxel resistance developed after long-term Epo treatment. Our findings have implications for the clinical use of recombinant human Epo and other ESAs to correct and/or prevent anemia in ovarian and, potentially, other cancer patients. They also suggest that endogenous Epo and the EpoR may serve as novel therapeutic targets in the treatment of cancer patients.