EPO Receptor Overexpression in Breast Cancer Cells Enhances EPO Sensitivity, Induces ERK and AKT Phosphorylation and Promotes Cellular Proliferation and Migration through a JNK-Dependent Pathway.

The ubiquitous expression of the EPO receptor (EpoR) in various non-hematopoietic tissues is associated with broad EPO-mediated tissue-protective effects. The role of EpoR expression in tumor cells and its potential for contribution to tumor biology and progression requires further characterization. We investigated whether EpoR expression levels and activation in tumor cells may modulate EPO sensitivity, proliferation and cell migration. MCF-7 breast cancer cells which express endogenous EpoR were engineered to stably express R129C-EpoR, a constitutively active EpoR that confers cytokine-independent hematopoietic cell growth but does not transform fibroblasts. Empty vector-transfected cells were negative controls. In untransfected cells, phosphotyrosine immunoblots showed EPO-induced tyrosine phosphorylation of several cellular proteins. EPO resulted in dose- and time-dependent phosphorylation of ERK1/2, but not JAK2, STAT5 or AKT. Increased EPO-induced ERK phosphorylation was associated with 2.3±0.3-fold increase in ERK kinase activity (P<0.05, n=3). EPO exhibited either a minor effect or no effect on proliferation of MCF-7 cells whereas cell migration in Transwell chambers was significantly stimulated by EPO (P<0.05, n=9). R129C-EpoR expression resulted in 2-fold increase in cellular proliferation (P<0.01, 2 clones, n=6) and 1.5±0.2-fold increase in cellular migration compared to vector controls (P<0.05, 3 clones, n=12). EPO significantly stimulated the migration of empty vector-transfected cells by 1.9±0.2-fold (P<0.001, n=12). Exogenous EPO did not further enhance the increased proliferation and migration of R129C-EpoR cells. We investigated the mechanisms involved in the increased migration of R129C-EpoR cells. Basal phosphorylation of ERK1/2 (2.6±0.4-fold, 3 clones, P<0.05) and AKT (1.6±0.1-fold, P<0.05) were significantly increased compared to vector controls. No changes in the phosphorylation status of JAK2, STAT5, JAK1 or p38 MAP kinases were observed under basal conditions or in response to EPO. Whereas EPO did not induce AKT phosphorylation in untransfected or vector-transfected cells, in R129C-EpoR cells, there was a significant, dose-dependent increase in AKT phosphorylation in response to EPO (3.29±0.5-fold, n=3 clones, P<0.05). In R129C-EpoR cells, EPO significantly enhanced ERK1/2 phosphorylation 2.7±0.3 fold compared to vector controls (P<0.001). Because c-Jun-NH2-kinase (JNK) is involved in cell migration, we determined the effect of EPO on JNK phosphorylation. EPO markedly increased JNK phosphorylation in R129C-EpoR cells by 5.38±1.6 fold (P<0.001) compared to vector controls in a dose-dependent manner. JNK phosphorylation and increased EPO-induced migration of vector-transfected cells were blocked by treatment with JNK kinase inhibitor SP600125. The increased migration of R129C-EpoR cells in the absence of exogenous EPO was also blocked by SP600126. These data indicate that

1. -EPO promotes the migration of breast cancer cells through a JNK-kinase dependent signaling pathway

2. -Expression of R129C-EpoR promotes the proliferation and migration of breast cancer cells

3. -EPO sensitivity is enhanced by R129C-EpoR expression leading to restoration of EPO-dependent AKT phosphorylation and increased ERK1/2 and JNK kinase phosphorylation

4. -The biologic effects of EPO in breast cancer cells are mediated through JAK2-STAT5 axis-independent signaling pathways

5. -EpoR expression levels in tumor cells may modulate tumor cell proliferation and migration responses.