Erythropoietin is a JAK2 and ERK1/2 effector that can promote renal tumor cell proliferation under hypoxic conditions

Abstract

Background: Erythropoietin (EPO) provides an alternative to transfusion for increasing red blood cell mass and treating anemia in cancer patients. However, recent studies have reported increased adverse events and/or reduced survival in patients receiving both EPO and chemotherapy, potentially related to EPO-induced cancer progression. Additional preclinical studies that elucidate the possible mechanism underlying EPO cellular growth stimulation are needed.

Methods: Using commercial tissue microarray (TMA) of a variety of cancers and benign tissues, EPO and EPO receptor immunohistochemical staining was performed. Furthermore using a panel of human renal cells (Caki-1, 786-O, 769-P, RPTEC), in vitro and in vivo experiments were performed with the addition of EPO in normoxic and hypoxic states to note phenotypic and genotypic changes.

Results: EPO expression score was significantly elevated in lung cancer and lymphoma (compared to benign tissues), while EPOR expression score was significantly elevated in lymphoma, thyroid, uterine, lung and prostate cancers (compared to benign tissues). EPO and EPOR expression scores in RCC and benign renal tissue were not significantly different. Experimentally, we show that exposure of human renal cells to recombinant EPO (rhEPO) induces cellular proliferation, which we report for the first time, is further enhanced in a hypoxic state. Mechanistic investigations revealed that EPO stimulates the expression of cyclin D1 while inhibiting the expression of p21cip1 and p27kip1 through the phosphorylation of JAK2 and ERK1/2, leading to a more rapid progression through the cell cycle. We also demonstrate an increase in the growth of renal cell carcinoma xenograft tumors when systemic rhEPO is administered.

Conclusions: In summary, we elucidated a previously unidentified mechanism by which EPO administration regulates progression through the cell cycle, and show that EPO effects are significantly enhanced under hypoxic conditions.

Figure 1

Erythropoietin (EPO) and erythropoietin receptor (EPOR) expression in human malignancies. The expression scores of EPO (A) and EPOR (C) in 20 different malignancies are shown in a bar graph. The expression score (0 to 3) was quantified by combining the proportion and intensity scores. Asterisks indicate malignancies having a significant difference (p < 0.05) between malignant tissues and corresponding benign tissue. Representative images of EPO (B, right panels) and EPOR (D, right panels) immunostaining of normal lung, lymph node and kidney are illustrated with lung cancer, lymphoma and kidney cancer. Lung cancer and lymphoma, but not renal cell carcinoma, were noted to have an increase expression of EPO (B, left panels) and EPOR (D, left panels).

Figure 2

Effect of recombinant human erythropoietin and hypoxia on the proliferative potential of human renal cell lines. A, Western blot analysis of four human renal cell lines was done to confirm EPO and EPOR status. Furthermore, other key molecules (e.g., VHL, HIF-1α, HIF-2α and VEGF) related to clear cell RCC were noted. Cells were grown in complete media in normoxic condition and total cellular protein lysate in the exponential phase were collected for analysis. B, Western blot analysis of four human renal cell lines exposed to hypoxia for 6 and 24 hrs was perform to note any change in the molecular status evident from normoxic conditions. β-actin is used as a loading control. C, Proliferation rate was measured in four human renal cell lines cells exposed to normoxia or hypoxia and grown in the indicated doses of recombinant human EPO (0–50 units/mL) at 48 hrs. Data were represented as mean ± SD relative to untreated cells, which are set to 100%. Three independent experiments were performed in triplicate. Significance compared to untreated cells is denoted by *, p < 0.05; **, p < 0.01, ***, p < 0.001.

Figure 3

The effects of erythropoietin on cell cycle. Cells, which were starved for 18 hrs in normoxic or hypoxic conditions then treated with or without rhEPO for additional 10 hrs in normoxic or hypoxic condition, were analyzed. Specifically, the percentage of population in G₀/G₁, S, and G₂/M phase of the cell cycle were analyzed by flow cytometry after propidium iodide staining of cellular DNA. Arrows indicate the major changes in EPO-treated cells compared to untreated cells. Data are representatives from three independent experiments.

Figure 4

Erythropoietin promotes S phase progression. A, Cells were synchronized in G₀/G₁-phase by using a double thymidine block and S-phase entry was monitored by the EDU incorporation following thymidine release. The percentage of proliferating cells at the indicated time after release was determined. The result of normoxia and hypoxia are shown in upper panels and lower panels, respectively. Asterisks indicate the significant difference (p < 0.05) between untreated cells (solid blue line) and rhEPO-treated cells (dashed red line). Data were represented as mean ± SD from three independent experiments. B, Cyclins, cyclin-dependent kinases and cyclin-dependent kinase inhibitors which are known to be keys for G₁/S transition were analyzed by Western blot to monitor the association of the stimulation of EPO and transduction of cell cycle proteins. Renal cells were treated with the indicated concentrations of rhEPO for 24 hrs in normoxic and hypoxic condition. Cell lysates were subjected to Western blot analysis. β-actin was used as a loading control.

Figure 5

Erythropoeitin activates the JAK and MAPK/ERK pathways. A, Four renal cell lines were starved in serum/growth factor-free media containing 0.1% BSA in normoxia or hypoxia. Cells were stimulated with the indicated concentration of rhEPO. Immunoblotting of protein extracts with indicated antibodies (at left of panel) shows JAK2 and MAPK/ERK pathway components in renal cell lines stimulated with rhEPO in the presence of hypoxia. β-actin is used as a loading control. B, Four renal cell lines were starved in serum/growth factor-free media containing 0.1% BSA in normoxia or hypoxia. Cells were subjected to 1 μM of TG10348 (a JAK2 inhibitor) or 1 μM of U0126 (a MEC inhibitor) for 60 mins prior to the addition of 10 units/mL rhEPO. Ten minutes after exposure of rhEPO, cell lysates were collected and subjected to Western blot analysis with the indicated antibodies. β-actin is used as a loading control. C, Cells are treated with the indicated concentrations of rhEPO in media containing 2% FBS for 24 hrs in normoxic or hypoxic condition. Cell lysates are subjected to Western blot analysis. Western blot analysis shows cyclin D1 was induced and p27 ^kip1 and p21 ^cip1 were down-regulated in renal cells stimulated with rhEPO in the presence of hypoxia. β-actin served as loading control.

Figure 6

Erythropoietin increases xenograft tumor growth of human 786-O renal cell carcinoma cells. Xenograft tumors were established by subcutaneous injection of Caki-1 and 786-O cells into athymic nude mice (nu/nu). One day after cell injection, administration of rhEPO was initiated as described in Material and Methods. A, The tumor size was monitored over 10 wks and plotted as mean ± SEM from the two treatment groups per cell line (n = 10 per group). Treatment with rhEPO was associated with an increase in tumor burden among 786-O xenograft tumors. B, Representative pictures of 786-O tumors of H&E staining and IHC staining for EPO, phospho-EPOR, cyclin D1, p21^cip1 and p27^kip1 are shown. Original magnification, 200 ×. Scale bars, 100 μm. C, Proliferative index (%) was quantified based on Ki-67 staining of tumor xenografts of Caki-1 and 786-O. *, p < 0.05. D, Immunohistochemical localization of pimonidazole hydrochloride (Hypoxyprobe-1) adducts in subcutaneous tumors of Caki-1 and 786-O. Original magnification in right panels, 50 ×. Scale bars, 500 μm.