Expression and Function of Erythropoietin Receptor (EpoR) Variant Forms in Human Breast Cancer.
Zsuzsa Rakosy, Gyorgy Paragh, Geza Acs. Moffitt Cancer Center, Tampa, FL
Background: Recombinant human erythropoietin (rHuEpo) is widely used in the management of cancer and therapy related anemia in patients with breast cancer. Although rHuEpo is effective in correcting anemia and improving quality of life, recent clinical trials suggested a potential adverse effect on patient outcome. Currently the role of EpoR and the effect of exogenous Epo in breast cancer is unclear. We have recently identified variant EpoR forms lacking the N-terminal ligand-binding domain in ovarian carcinoma cells. In this study we investigated the expression and function of EpoR and its variant forms in breast cancer cell lines and clinical samples of human breast cancer.
Design: To identify full length and variant EpoR mRNA forms we used a SMART RACE PCR assay. DNA purified from individual bands was subjected to sequence analysis. To identify variant EpoR forms in human breast cancers, 180 macrodissected formalin-fixed paraffin-embedded invasive breast carcinomas were used for quantitative RT-PCR analysis applying EpoR exon 2-3 and 6-7 specific Taqman assays. The activation of EpoR signaling mechisms, and in vitro and in vivo tumor growth of the cells were examined in MDA-MB-436 cells stably transfected with full length, variant or both EpoR forms.
Results: Using quantitative RT-PCR we found that mRNA regions coding for the extracellular domain of EpoR were expressed at significantly lower levels compared to the C-terminal region in breast cancer cells. Among 180 archived human breast cancer samples, 28.9% of tumors showed more than 10-fold difference between the expression levels of the C and N-terminal regions of EpoR. Using SMART RACE PCR, in breast cancer cells and human breast cancer samples we identified a common 900 bp length EpoR sequence found in most samples. Sequence analysis showed that this band contains only exons 5-8 of EpoR, with exons 1-4, coding for the N-terminal extracellular ligand binding domain of the receptor, completely missing. Breast cancer cells expressing both full length and variant EpoR forms showed increased EpoR signaling and increased tumor growth both in vitro and in vivo in a tumor xenograft model.
Conclusions: Our results indicate that variant forms of EpoR lacking the ligand binding domain are present in human breast cancers and appear to be biologically active, affecting EpoR signaling and tumor growth. Characterization of EpoR variant forms will lead to our better understanding of the mechanisms and role of EpoR signaling in breast cancer and may provide means for the selection of patients for safe, individualized supportive treatment by rHuEpo.
Tuesday, March 1, 2011 2:00 PM
Platform Session: Section H, Tuesday Afternoon