Epo Signaling Research Articles

Evaluation of Functional Erythropoietin Receptor Status in Skeletal Muscle In Vivo: Acute and Prolonged Studies in Healthy Human Subjects

BackgroundErythropoietin receptors have been identified in human skeletal muscle tissue, but downstream signal transduction has not been investigated. We therefore studied in vivo effects of systemic erythropoietin exposure in human skeletal muscle.Methodology/Principal FindingsThe protocols involved 1) acute effects of a single bolus injection of erythropoietin followed by consecutive muscle biopsies for 1–10 hours, and 2) a separate study with prolonged administration for 16 days with biopsies obtained before and after. The presence of erythropoietin receptors in muscle tissue as well as activation of Epo signalling pathways (STAT5, MAPK, Akt, IKK) were analysed by western blotting. Changes in muscle protein profiles after prolonged erythropoietin treatment were evaluated by 2D gel-electrophoresis and mass spectrometry. The presence of the erythropoietin receptor in skeletal muscle was confirmed, by the M20 but not the C20 antibody. However, no significant changes in phosphorylation of the Epo-R, STAT5, MAPK, Akt, Lyn, IKK, and p70S6K after erythropoietin administration were detected. The level of 8 protein spots were significantly altered after 16 days of rHuEpo treatment; one isoform of myosin light chain 3 and one of desmin/actin were decreased, while three isoforms of creatine kinase and two of glyceraldehyd-3-phosphate dehydrogenase were increased.Conclusions/SignificanceAcute exposure to recombinant human erythropoietin is not associated by detectable activation of the Epo-R or downstream signalling targets in human skeletal muscle in the resting situation, whereas more prolonged exposure induces significant changes in the skeletal muscle proteome. The absence of functional Epo receptor activity in human skeletal muscle indicates that the long-term effects are indirect and probably related to an increased oxidative capacity in this tissue.

Downregulation of IRS2 in Myelodysplastic Syndrome (MDS) May Be Related to Impaired Cell Differentiation,

Abstract 3814Myelodysplastic syndromes (MDS) encompass a heterogeneous group of clonal hematopoietic stem cell disorders, characterized by ineffective hematopoiesis, refractory cytopenia and a tendency to progress towards acute myeloid leukemia. Abnormalities in cell differentiation are probably involved in the pathogenesis of MDS. Insulin receptor substrates (IRS) are adaptor proteins that link signaling from upstream activators to downstream effectors to modulate normal growth, metabolism, survival and differentiation. IRS2, a member of the IRS family, binds to Insulin Grow Factor 1 receptor (IGF1R), the Erythropoietin receptor (EPOR) and Thrombopoietin receptor (MPL). Recently, we reported that IRS2 was downregulated in MDS cells compared to normal cells, and in high risk compared to low-risk MDS. Additionally, IRS2 was significantly upregulated during erythroid differentiation of normal CD34+ cells, but not in MDS CD34+ cells. In view of the role of IRS2 in EPO, IGF1 and TPO signaling, and possibly in erythroid, granulocytic, and megakaryocytic differentiation, we hypothesized that IRS2 might be related to the impaired hematopoiesis of MDS cells. The aim of the present study was to correlate the mRNA expression levels of IRS2 in bone marrow from MDS patients with laboratorial and clinical data. We also elucidated the expression levels of IRS2 during megakaryocytic, granulocytic and erythroid differentiation of established leukemia cell line models. We studied 12 healthy donors and 32 patients with MDS at the time of diagnosis (16 low-risk [RA/RARS] and 13 high-risk [RAEB/RAEBt] according to FAB classification). Gene expression was evaluated by Q-PCR in total cells from bone marrow samples and cell lines. IRS2 protein expression and phosphorylation was evaluated by Western blot and immunoprecipitation. K562 cells were induced to megakaryocytic differentiation by treatment with 20nM PMA for 4 days. NB4 cells were induced to granulocytic differentiation by treatment with 10−6 M ATRA for 4 days. KU812 cells were induced to erythroid differentiation by treatment with 50μM HE and 100 μM HU for 4 days. Spearman correlation analysis showed that IRS2 expression demonstrated a significant positive correlation with peripheral blood neutrophils (r =0.43, P =0.01) and platelets counts (r =0.36, P =0.04), and bone marrow granulocytes percentages (r =0.40, P =0.02). In contrast, a significant negative correlation was observed between IRS2 expression with bone marrow erythroblasts (r =-0.360, P =0.04) and myeloid precursor percentages (grouping myeloblast, promyelocytes, myelocytes, metamyelocytes and blasts) (r =-0.60, P <0.01). There was no correlation between IRS2 expression with age, hemoglobin, number of erythrocytes in the peripheral blood, and number of dysplasias in bone marrow. Finally, since IRS2 is involved with TPO, IGF1 and EPO signaling, and IRS2 expression was correlated with platelet, granulocyte and erythroblast numbers in MDS patients, we monitored IRS2 expression during hematopoietic cell differentiation. Interestingly, a significant increase in IRS2 expression and phosphorylation was observed in K562, NB4 and KU812 after megakaryocytic, granulocytic and erythroid differentiation, respectively. In summary, the lower expression of IRS2 in MDS and its upregulation during differentiation may suggest that defective IRS2 expression in MDS contributes to disease progression and impaired hematopoietic cell differentiation, a hallmark of the disease. Disclosures:No relevant conflicts of interest to declare.

Effect of Lenalidomide Treatment on Lipid Signalling Pathways in Low-Risk MDS Patients

Abstract 4635Nuclear inositide signalling pathways are involved in the MDS progression to AML. Indeed, in the last few years our group demonstrated not only that MDS cells can show alterations on PI-PLCbeta1 and Akt pathways, but also that Akt is inversely correlated with PI-PLCbeta1, therefore affecting MDS cell survival and differentiation. Lenalidomide has proven effectiveness in 70–80% low-risk MDS cases with del(5q), resulting in transfusion-independence with a rise in hemoglobin levels, suppression of the 5q clone, and improvement of bone marrow morphologic features. In particular, in del(5q) MDS, Lenalidomide probably acts by directly suppressing the dysplastic clone, while in non-del(5q) it might enhance an effective erythropoiesis, possibly via activation of the EPO signalling, which in turn is associated with PI-PLCgamma1 pathways. However, the exact molecular mechanisms underlying the effect of Lenalidomide in MDS cells are still not completely clarified. Interestingly, Lenalidomide might inhibit the phosphatase PP2A, whose gene is located in the common deleted region and which usually targets Akt. Indeed, Akt-dependent pathways are critical in low-risk MDS cells, which display a marked apoptosis and a low proliferation rate. In this study we examined four MDS patients treated with Lenalidomide, and compared them with four low-risk MDS patients (IPSS: Low or Int-1) who only received best supportive care. In our study, all of the patients treated with Lenalidomide were affected by del(5q) low-risk MDS (IPSS: Low or Int-1), with transfusion-dependent anemia, and had only received supportive care before undergoing Lenalidomide treatment. Clinically, all of the patients responded to Lenalidomide: one patient reached Complete Remission, whilst the other three patients showed erythroid Hematologic Improvement. In contrast, all of the patients who were treated only with best supportive care maintained a Stable Disease. As for the molecular effects of Lenalidomide on lipid signalling pathways, we analyzed the expression of critical molecules involved in both cell proliferation and differentiation, that is PI-PLCbeta1 and its downstream target Cyclin D3, as well as PI-PLCgamma1, which is linked with EPO signalling. Ongoing analyses are also trying to examine the effect of Lenalidomide on Akt phosphorylation and Globin genes, which are specifically associated with erythropoiesis. So far, our results indicate that, in our responder patients, both PI-PLCbeta1 and Cyclin D3 are not significantly affected by Lenalidomide, whereas PI-PLCgamma1 is specifically induced, as compared with both healthy subjects and low-risk MDS patients treated with supportive care. Overall, these findings hint at a specific activation of PI-PLCgamma1 signalling following Lenalidomide treatment, and possibly pave the way to larger investigations aiming to better understand the role of these pathways in the mechanism of action of Lenalidomide in del(5q) MDS. Disclosures:No relevant conflicts of interest to declare.

Single Cell Network Profiling (SCNP) Reveals Age-and Disease-Based Heterogeneity in Healthy Individuals and in Patients with Low Risk (LR) Myelodysplastic Syndrome (MDS)

Abstract 2788 Background:Normal hematopoiesis changes with age through unknown mechanisms. Low risk (LR) myelodysplasia (MDS) is characterized by cytopenias arising through inefficient hematopoiesis. We hypothesized that both of these differences might result from changes in responsiveness to external signaling. To test this hypothesis, we utilized SCNP, a multiparametric flow cytometry-based assay that simultaneously measures both extracellular surface marker levels and changes in intracellular signaling proteins in response to extracellular modulators, quantitatively at the single cell level (Kornblau et al. Clin Cancer Res 2010). Methods/Objective:SCNP was applied to examine baseline and intracellular signaling responses induced by the extracellular modulators EPO and GCSF in bone marrow (BM) mononuclear cells (BMMC) derived from healthy donors (n=15) and MDS (n=9) patients. The effects of donor age on signaling profiles in healthy BMMC were compared between samples collected by BM aspirate from 6 subjects aged 23–43 years (“younger”) and from the BM present in hip replacement samples from 9 subjects aged 54–82 years (“older”). Signaling profiles were also determined for 9 LR MDS patients aged 53–83 years and compared to the age-matched healthy “older” control. Metrics used for analysis included fold change, total phosphorylation levels, and the Mann-Whitney U statistic model. Results:There were no differences in the frequency of CD34+ cells (R2= 0.006, p= 0.78) between “younger” and “older” healthy donor samples, suggesting any differences observed in signaling would likely be due to donor age rather than sample source. There was no age-related difference in functional signaling ability in response to GCSF-induced p-STAT1, p-STAT3, & p-STAT5 levels; however, early erythroblasts and normoblasts from healthy “older” donors were significantly less responsive to EPO, as measured by induced phospho (p)-STAT5 levels than those derived from healthy “younger” donors (e.g. R2= 0.654 p=0.008 for erythroblasts and R2=0.628 p=0.0004 for normoblasts). Signaling profiles classified Refractory Anemia with Excess Blasts (RAEB) patients into 2 categories based on differences in EPO- and GCSF-induced signaling (Table 1). Compared to age-matched healthy “older” controls, one subset was characterized by a high % of RBC precursors (CD45lo nRBC) and increased p-STAT5 levels in response to EPO and the other subset by a high % of myeloid cells with robust GCSF-induced p-STAT3 & p-STAT5 responses in both total myeloid and CD34+ cells. By contrast, patient samples with Refractory Anemia with Ringed Sideroblasts (RARS) had a high % of CD45lo nRBC but lacked robust p-STAT5-induced signaling after modulation with EPO. Conclusions:Overall, these data show the feasibility of using the SCNP assay in BM samples to functionally characterize signaling pathways simultaneously in different cell subsets of healthy donors and patients with MDS. In healthy individuals, age-related differences in EPO signaling were discovered. In LR MDS, differences in signaling were observed between cases and in comparison to the data from healthy controls. Deciphering signaling profiles in healthy donor versus MDS patient samples may result in improved, biologically-based disease classification that informs more effective patient management. The clinical relevance of these findings in terms of disease course and treatment is currently under investigation.Table 1Healthy Younger (n=6)Healthy Older (n=9)RAEB (n=5)RARS (n=2)MPD/MDS (n=2)Median (Range)Pt 003Pt 008Pt 004Pt 006Pt 013Pt 005Pt 010Pt 011Pt 014CD45lo nRBC%14 (8–22)31 (24–53)62697576055188Myeloid38 (26–45)24 (20–42)2021848092721747EPO→p-STAT5 nRBCUu0.68 (0.60–0.73)0.57 (0.53–0.62)0.770.680.540.610.520.620.510.660.54GCSF→p-STAT3Myeloid0.58 (0.55–0.61)0.56 (0.50–0.61)0.600.550.890.810.860.500.530.640.51CD340.78 (0.73–0.79)0.70 (0.64–0.80)NDND0.920.880.910.510.680.900.64GCSF→p-STAT5Myeloid0.60 (0.57–0.62)0.58 (0.55–0.64)0.630.560.840.820.870.510.530.620.50CD340.85 (0.80–0.86)0.76 (0.72–0.86)NDND0.860.900.940.530.740.930.68 Disclosures:Cleary Cohen:Nodality Inc.: Consultancy, Equity Ownership. Huang:Nodality Inc.: Consultancy, Equity Ownership. Cesano:Nodality: Employment, Equity Ownership. Hawtin:Nodality: Employment, Equity Ownership. Ware:Nodality Inc.: Employment, Equity Ownership.

Evaluating Clonal Dominance in a Murine Knock-in Model of Jak2V617F MPN

Abstract 614Myeloproliferative neoplasms (MPN) are clonal disorders of hematopoiesis but the mechanisms of clonal dominance in these diseases are poorly understood. The JAK2V617F mutation is found in the majority of patients with MPN and is sufficient to confer the MPN phenotype. We recently described a Jak2V617F knock-in MPN model in which the mutation was expressed from the endogenous murine Jak2 promoter and the disease phenotype closely recapitulated human polycythemia vera (PV). In the model we found that the MPN-initiating population is contained within the CD150+CD48− LineagelowSca1+cKithigh(LSK) long-term hematopoietic stem cell (HSC) compartment, and showed that the MPN is cell autonomous and serially transplantable. In long-term competitive transplantation experiments we found that the Jak2V617F CD150+CD48− LSK population demonstrates gradual clonal expansion over time and we are now investigating the regulation of this primitive HSC population in vivo.Erythropoietin (EPO) signaling is reported to be the fundamental defect in polycythemia vera (PV). The JAK2V617F mutation is present in 95% PV patients and can be detected in the HSC compartment. We evaluated the role of EPO signaling in the JAK2V617F mutant HSC compartment using a conditional Jak2V617F knock-in murine model. Floxed Jak2+/VF mice were crossed with Vav Cre or erythropoietin receptor GFP Cre (ErGFPcre) mice resulting in Jak2V617F expression in all hematopoietic lineages or in erythroid restricted Jak2V617F expression respectively. Jak2V617F-ErGFPcre mice demonstrated elevated hematocrit, expanded committed erythroid progenitors and suppressed EPO levels but had an attenuated MPN phenotype as compared with Jak2V617F-Vavcre mice. Notably, the hematopoietic stem and progenitor cell (HSPC) compartment was not expanded in Jak2V617F-ErGFPcre mice and HSCs from both Jak2V617F-Vavcre and Jak2V617F-ErGFPcre mice did not activate phosphoStat5 signaling in response to EPO stimulation. These results indicate that expression of Jak2V617F in the HSC compartment is required for development of a full MPN phenotype and suggest that cytokine receptors other than the EPO receptor are important in mediating clonal dominance within the HSC compartment in JAK2V617F mediated MPN.TET2 is one of three TET gene family members that appear to play a role in DNA demethylation. Acquired TET2 deletions and loss-of-function mutations have been found across a broad spectrum of myeloid malignancies indicating that TET2 may drive a common pathogenic step in myeloid cancers, such as the establishment and/or enhancement of clonal dominance. Tet2 null HSCs have recently been shown to have a competitive repopulating advantage over wild-type HSCs in murine transplantation assays. TET2 loss-of-function mutations are found in approximately 12% of MPN patients, are often found co-mutated with JAK2V617F and have been associated with leukemic transformation of MPN. To evaluate the effects of loss of Tet2 function on the self-renewal and differentiation of Jak2V617F mutant HSCs, we crossed Jak2V617F knock-in mice with Tet2 conditional knockout mice. At 6 weeks of age, Jak2V617F/Tet2 +/− Vav Cre mice do not demonstrate significant differences in MPN phenotype as compared with Jak2V617F/Tet2+/+ Vav Cre mice, in terms of peripheral blood counts, hematopoietic stem and progenitor cell numbers or in colony formation. Additional studies using older mice and Jak2V617F/Tet2−/− Vav Cre animals are underway to further investigate the impact of loss of Tet2 function on Jak2V617F mutant HSCs.Understanding the mechanisms that contribute to clonal dominance in MPN will help facilitate the development of strategies to selectively target MPN stem cells therapeutically, and thereby advance the treatment of MPN patients. Disclosures:No relevant conflicts of interest to declare.

The Role of the Distal Erythropoietin Receptor in Iron Deficiency Anemia

Abstract 1312Anemia affects the quality of life and the life expectancy of millions of people in the U.S. Many patients are either intolerant or unresponsive to available treatments, so alternative strategies are needed. Red blood cell production requires the action of erythropoietin (Epo) on red blood cell precursors in the bone marrow. Iron restriction results in loss of Epo-responsiveness and anemia, despite increased serum Epo levels. Iron infusion restores Epo-responsiveness suggesting that iron dominantly regulates Epo-receptor (EpoR) signaling. Understanding how iron restriction regulates EpoR signaling pathways has major clinical significance. Agonists could offer an iron-free approach that enhances the response to Epo in anemia due to iron deficiency or chronic diseases. In addition, antagonists could be used to treat polycythemia vera or other myeloproliferative disorders. We have discovered that the aconitases, multifunctional iron-sulfur cluster proteins that convert citrate into isocitrate are key in connecting iron to Epo-signaling in early erythroid progenitors (GC Bullock, et. al. Blood 2010;116:97). We also discovered that isocitrate, the downstream product of aconitase, can enhance the effectiveness of Epo during iron deficiency in vitro and in vivo in mice with IDA. These observations suggest that isocitrate or derivatives of isocitrate that synergize with erythropoiesis stimulating agents (ESAs) have important therapeutic application in the treatment of anemia. Deletion of EpoR in mice is incompatible with life, however mice and humans that express truncated EpoR show increased production of red blood cells. These observations suggest that the distal cytoplasmic domain of the EpoR inhibits production of red cells and may play a critical role in iron deficiency anemia. EpoR mutant mice lacking the distal half of the cytoplasmic domain of the EpoR (EpoR-H mice) and mice with the same EpoR truncation mutation plus an additional mutation of tyrosine 343 (EpoR-HM mice) show near normal levels of steady state erythropoiesis. To determine the role of the distal domain in erythroid suppression during iron deficiency, EpoR-H, EpoR-HM and EpoR-wildtype mice were fed a low iron diet and compared by weekly CBCs and flow cytometry. EpoR-H mutant mice continue to efficiently produce red blood cells during iron deficiency. And this occurs despite a decrease in hemoglobin. EpoR-HM mice produce fewer rbcs than EpoR-H mice, however rbc production by EpoR-HM mice resists the suppressive effects of iron restriction. Similar experiments also suggest that the distal EpoR is necessary for the isocitrate-mediated enhancement of Epo-driven erythropoiesis. In addition to aconitase/isocitrate and the distal EpoR other candidate key signaling components of this Epo-dependent, iron-responsive pathway have been identified in our recent preliminary experiments. These components include specific protein kinase C (PKC) isozymes, AKT1 and ERK1/2. These findings support a new model of iron sensing by aconitase/isocitrate that alters EpoR signaling to decrease red blood cell production and conserve iron when supplies are low. This model fits better than older “heme-deficiency” models because disorders in heme synthesis block red cell differentiation at a later stage. This model also has potential to explain changes seen in other tissues during chronic iron deficiency. Nutritional iron restriction may have unmasked a new role for the distal EpoR in red cell development and implicated new iron-responsive Epo signaling pathways that can be used to develop new therapeutic agonists and antagonists of Epo. Disclosures:No relevant conflicts of interest to declare.

Lenalidomide Upregulates Erythropoietin Receptor Expression in Hematopoietic Progenitors by Modulating Receptor Turnover

Abstract 2382The erythropoietin receptor (EpoR) is a type 1 cytokine receptor and key effector of Epo signaling. Maturation and shuttling of receptor to the plasma membrane is dependent on its association with Janus Kinase 2 (JAK2). Upon ligand binding, EpoR homo-dimerizes to activate JAK2, which in turn phosphorylates tyrosine residues on the receptor cytoplasmic tail permitting recruitment and phosphorylation of the signal transduction and activator of transcription (STAT)-5. Lenalidomide (LEN), a second generation immunomodulatory drug (IMiD), promotes the expansion of primitive erythroid progenitors in vitro and induces the expression of genes involved in erythroid lineage commitment (JCI 2008;118(1):248-58; PLoS medicine 2008;5(2):e35). In del(5q) clones, LEN is cytotoxic by inhibiting the haplodeficient serine/threonine phosphatase PP2A (Wei S, et. al PNAS 2009). To investigate the mechanism by which LEN promotes erythropoiesis, we investigated its action on EpoR cellular dynamics. Using the human erythroleukemia cell line, UT-7, treatment with LEN elicited concentration dependent upregulation of EpoR in whole cell lysates demonstrable by immunoblot (IB) analysis at concentrations ranging from 0.1–10μM, reaching a 2-fold increase vs. controls. Treatment with pomalidomide, an IMiD with superior erythropoietic activity, displayed greater potency, increasing cellular EpoR at concentrations as low as 1nM; whereas thalidomide was ineffective. Upregulation of EpoR was apparent as early as 1 hour (h) after LEN exposure, reaching maximal receptor induction at 8h. Isolation of membrane and cystosolic fractions followed by IB showed that EpoR was upregulated predominantly in the membrane fraction, increasing 4.3-fold. IB of detergent insoluble membrane raft (MR) fractions showed that LEN promoted lipid raft assembly and F-actin polymerization accompanied by translocation of EpoR into MR microdomains with JAK2, STAT-5 and Lyn signal intermediates. Pretreatment with the Rho-GTPase inhibitor, Y27632, suppressed LEN effects on MR and F-actin. Immunoprecipitation (IP) of EpoR showed that JAK2 co-precipitated with the receptor in LEN treated cells, suggesting drug induced release of pre-assembled EpoR/JAK2 complexes from golgi and endosomal compartments. Q-PCR showed no changes in EpoR gene mRNA expression up to 24 hours after drug exposure, supporting post translational drug effects. To confirm that the observed changes in EpoR dynamics in UT-7 cells extends to normal erythroid progenitors, we assessed EpoR expression and MR assembly (cholera toxin B labeled GM1) by fluorescence microscopy in CD71+ erythroid precursors from three normal bone marrow donors. Expression of EpoR increased a mean of 17% (range:0-68%) after 1h LEN [1μM] exposure (p=0.003), accompanied by an increase in MR aggregates. Because the LEN inhibitable target PP2A is a key regulator of Rho-GTPase guided receptor trafficking and AP-1 directed formation of MR targeted clathrin-coated vesicles, we examined its effects on EpoR induction. Pretreatment of UT-7 cells with the PP2A activator FTY720 antagonized the upregulation of EpoR by LEN, whereas treatment with the PP2A inhibitor cantharadin [0.5μM] induced EpoR protein expression. Inhibition of dynamin activity, a PP2A sensitive GTPase involved in type 1 receptor recycling from membrane to endosomes, recapitulated the effects of cantharadin, inducing EpoR expression. In summary, these findings indicate that LEN modulates EpoR equilibrium, mediated in part through its inhibitory effect on PP2A. LEN induces rapid translocation of mature EpoR/JAK2 complexes to the plasma membrane while promoting the assembly of MRs with signaling competent receptor platforms to optimize cytokine signal integrity. Disclosures:List:Celgene: Consultancy.

Abstract 1089: Two nonselective calcium channels, TRPC3 and TRPC6, play roles in erythropoietin signaling

Abstract The signal transduction pathways of erythropoietin (Epo), a glycoprotein regulating production of red blood cells, have been a model for understanding growth factor stimulated cell proliferation and differentiation. Erythropoietin receptors (Epo-R) are also expressed on non-hematopoietic cells, and recently Epo was reported to have antiapoptotic effects on these cells including a potential role in specific cancers. Two calcium channels were identified which are involved in Epo signaling, TRPC3 and TRPC6. These are homologous, nonselective calcium channels and are members of the canonical transient receptor potential (TRPC) subfamily expressed on human erythroblasts. Both channels have also been implicated in stimulating proliferation of cancer cells; TRPC3 in ovarian cancer, TRPC6 in gliomas, and both channels in breast cancer. Here TRPC3 and TRPC6 function was studied in transfected human embryonic kidney (HEK)-293T cells, and in the human leukemia UT-7/Epo cell line. Measurement of [Ca2+]i was performed on single cells with Digital Video Imaging. TRPC3 is activated by erythropoietin, whereas TRPC6 is not. In fact, TRPC6 can inhibit Epo-induced activation of TRPC3. Domains regulating channel activation were studied here. The TRP domain of TRPC3 is required for its activation, and substitution of five amino acids in the TRPC3 TRP domain with those of TRPC6 significantly reduced the Epo-stimulated increase in [Ca2+]i in TRPC3-expressing cells. Substitution of the same amino acids in the TRPC6 TRP with those of TRPC3 did not enhance the response of TRPC6-expressing cells to Epo stimulation. However, substitution of the distal TRPC6 C-terminus (C2) with that of TRPC3 in addition to substitution of TRPC6 TRP with TRPC3 TRP resulted in a TRPC6 channel with Epo responsiveness similar to TRPC3. In contrast, substitution of the TRPC3 C-terminal leucine zipper motif or TRPC3 phosphorylation sites S681, S708, or S764 with corresponding TRPC6 sequence did not affect TRPC3 Epo-responsiveness. TRPC3, but not TRPC6, and chimeras expressing the distal TRPC3 C terminus showed significantly increased membrane insertion following Epo stimulation. This was associated with substantial cytosketetal localization of TRPC3 and chimeras expressing TRPC3C2 but not TRPC6. These data demonstrate the important role of the TRP domain in specificity of channel responsiveness to agonist stimulation, and the requirement for other sites for optimal response to agonists, such as those found in the distal C-terminus of TRPC3. A few mutations in critical channel sites could have an important effect on proliferation and survival of cells expressing Epo-R. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1089. doi:10.1158/1538-7445.AM2011-1089

Expression of erythropoietin and its receptor increases in colonic neoplastic progression: The role of hypoxia in tumorigenesis

Tissue hypoxia is a characteristic patho-physiologic property of colorectal cancer. This process may also add to a therapeutic problem of solid tumor resistance to chemo- and radiation therapy. Erythropoietin (Epo) expression is induced by tissue hypoxia. Acting via its receptor (EpoR), Epo inhibits apoptosis of erythroid cells and has been shown to rescue neurons from hypoxic damage. Increased Epo and EpoR expression has been recently described in human breast, renal and cervical carcinoma. Given the characteristic tumor diathesis present in majority of colorectal cancers, we examined whether Epo signaling may play a role in colonic neoplastic progression. Expression of Epo and EpoR was examined using immunohistochemistry in 24 cases of primary colorectal and metastatic adenocarcinomas versus adenomas and normal colonic mucosa. Immunohistochemical stains were evaluated semiquantitatively based on a four-tiered scale. Based on the combination of extent and intensity of immunoreactivity, an immunostaining score (0-300) was determined for each sample. Expression of Epo and EpoR protein and mRNA was examined using Western blot and reverse transcriptase-polymerase chain reaction (RT-PCR), respectively, in both normal colonic tissue and carcinoma specimens in five cases. Epo expression was sequentially increased in normal colonic mucosa (8.3 ± 5.6, mean ± SEM), adenoma (26.4 ± 9.1), primary carcinoma (96.1 ± 12.8) and metastatic carcinoma (122 ± 51.3). EpoR expression was also sequentially increased in normal colonic mucosa (22.3 ± 11.8), adenoma (108.7 ± 24.2), primary carcinoma (178.7 ± 16.6) and metastatic carcinoma (220 ± 58.3) (P< 0.05 for all results). Epo and EpoR showed enhanced expression in the areas adjacent to ischemia/necrosis. Western blot and RT-PCR analysis revealed increased EpoR protein and mRNA levels in carcinoma compared to normal mucosal colon specimens. Focal stromal Epo and EpoR immunoreactivity was present in 10 and 12 cases, respectively. The uniform increase in the expression of Epo and EpoR along the colonic neoplastic sequence and further increase in ischemic/necrotic areas indicates that the Epo signaling pathway is an important component in colon carcinogenesis including possible epithelial-stromal interactions.

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

We examined the effect of blocking the erythropoietin (Epo) signaling using an anti-Epo antibody, soluble form of Epo receptor (sEpoR) capable of binding to Epo or EpoR antagonist, and proved to be effective against xenografts of female reproductive organ malignancies and of cancer cell lines in nude mice. We transfected seven cancer cell lines of various origins to express constitutively active sEpoR, and examined their tumorigenesis in nude mice. Suppression of the tumor growth, decrease in viable and proliferating cells and reduction of vascular density were seen individually in all xenografts of transfected cell lines compared with the controls. Quantitative RT-PCR analyses showed that expression levels of Epo, EpoR, ?1A-adrenaline receptor (?1A-ADR) and muscalinic acetylcholine receptor subunit 3 mRNAs (m3-AchR) were higher in the majority of the wild-type xenografts than in the corresponding cell lines except for A549. In some of the transfected xenografts, EpoR, ?1A-ADR and m3-AchR mRNAs were down-regulated. Western blot analyses revealed that the constitutively activated ERK1/2MAPK was discernible in the majority of non-transfected cell lines and was reduced in the transfected cell lines. However, it was regained after exposure to acetylcholine and/or noradrenaline. These findings suggest that constitutively active sEpoR can effectively destroy the xenografts but signals from the autonomic neurotransmitters of the host produced under stress may interfere with this antitumor activity.

Erythropoietin Regulates GATA-1 Function and Erythroid Differentiation by a PKD-HDAC5 Signal Transduction Pathway

AbstractAbstract 85How erythropoietin (Epo) signaling promotes erythroid differentiation remains unclear. Epo is known to regulate the function of the master regulator of erythroid gene transcription, GATA-1. Using murine proerythroblasts engineered with a conditional GATA-1-ER fusion, G1ER cells, Gregory et al. showed that GATA-1 induction of erythroid differentiation required signaling by Epo; in cultures lacking Epo, activation of GATA-1 caused cell death without differentiation (Blood 94:87-96, 1999). Although several models have been proposed for Epo regulation of GATA-1, no mechanism has been established. While analyzing PKC isozymes regulated by Epo and iron in primary human erythroblasts, our lab identified PKCμ, aka PKD, as activated in a dosage-dependent manner by Epo. Subsequent studies in Epo-starved G1ER cells, and in primary human progenitors, confirmed direct Epo induction of PKD phosphorylation on Ser744/748. A major PKD pathway involves its phosphorylation of class II HDACs (4, 5, 7 and 9), leading to release of HDAC-bound transcription factors (e.g. MEF2) from tonic repression. Watamoto et al. previously identified GATA-1 as a class II HDAC regulated factor, displaying physical and functional interaction with HDAC5 (Oncogene 22:9176-9184, 2003). Accordingly, we employed G1ER cells to determine whether Epo signaling to GATA-1 involved the PKD-HDAC pathway. In G1ER cells cultured in stem cell factor (SCF) minus Epo, endogenous HDAC5, but not HDAC4, co-immunoprecipitated with GATA-1. Epo stimulation induced dissociation of the HDAC5-GATA-1 complex, without affecting levels of either factor. The function of erythroid PKD-HDAC signaling was addressed by pharmacologic and shRNA approaches. Initial experiments tested whether inhibition of HDAC activity could substitute for Epo signaling in G1ER cells undergoing estradiol activation of GATA-1-ER. As described, GATA-1 activation in cells grown in SCF without Epo caused >90% cell death with minimal hemoglobinization after 48 hours. Addition of the HDAC inhibitor SAHA completely rescued viability and partially restored hemoglobinization of cells undergoing GATA-1 activation in the absence of Epo. Along similar lines, shRNA knockdown of HDAC5, but not HDAC4, significantly enhanced viability and hemoglobinization of G1ER cells undergoing GATA-1 activation under limiting Epo concentrations (0.05 U/ml). Conversely, shRNA knockdown of PKD3 impaired hemoglobinization of cells undergoing GATA-1 activation in the presence of Epo. Further implicating PKD in erythropoiesis, differentiation of G1ER cells was blocked by the kinase inhibitor Gö6976, which targets PKC and PKD, but not by the related compound Gö6983, which targets only PKC. Similar results were obtained with primary human progenitors, in which the PKD inhibitor Gö6976 but not the classical PKC inhibitor Gö6983 inhibited erythroid differentiation. In vivo roles of PKD-HDAC signaling in erythropoiesis were addressed by studying HDAC5-/- mice. In contrast to wild type counterparts, adult HDAC5-/- mice showed elevated steady state hematocrits (56.2 ± 0.8 vs 39.3 ± 1.4, P < 0.0001), resulting from increased MCVs (68.8 ± 0.7 vs 48.2 ± 0.1, P < 0.0001). In response to PHZ-induced hemolytic anemia, HDAC5-/- mice showed higher nadir RBC counts (5.0 ± 0.3 vs 3.2 ± 0.2, P < 0.001) and displayed an unexpected increase in Hb levels at the nadir point (15.9 ± 0.8 vs 8.6 ± 0.5, P < 0.0001). Spleens and livers obtained post recovery showed increased iron deposition in HDAC5-/- mice, consistent with increased net red cell turnover. To determine whether the observed abnormalities were cell-intrinsic, we sorted CD71++ Ter119- erythroid progenitors from the marrows of adult HDAC5-/- and wild type mice. The HDAC5-/- progenitors, but not wild type counterparts, displayed erythroid differentiation, manifested by Ter119 upregulation, in medium with no or low Epo (0.01-0.05 U/ml). Our data thus implicate PKD-HDAC signaling in Epo regulation of GATA-1 function and thereby provide a mechanistic basis for an instructional function of the Epo receptor. In addition, activation of PKD in non-erythroid cells could potentially explain some of the poorly-understood clinical complications of Epo therapy. Disclosures:No relevant conflicts of interest to declare.

Role of Erythropoietin Receptor Signaling in Parvovirus B19 Replication in Human Erythroid Progenitor Cells

Parvovirus B19 (B19V) infection is highly restricted to human erythroid progenitor cells. Although previous studies have led to the theory that the basis of this tropism is receptor expression, this has been questioned by more recent observation. In the study reported here, we have investigated the basis of this tropism, and a potential role of erythropoietin (Epo) signaling, in erythroid progenitor cells (EPCs) expanded ex vivo from CD34(+) hematopoietic cells in the absence of Epo (CD36(+)/Epo(-) EPCs). We show, first, that CD36(+)/Epo(-) EPCs do not support B19V replication, in spite of B19V entry, but Epo exposure either prior to infection or after virus entry enabled active B19V replication. Second, when Janus kinase 2 (Jak2) phosphorylation was inhibited using the inhibitor AG490, phosphorylation of the Epo receptor (EpoR) was also inhibited, and B19V replication in ex vivo-expanded erythroid progenitor cells exposed to Epo (CD36(+)/Epo(+) EPCs) was abolished. Third, expression of constitutively active EpoR in CD36(+)/Epo(-) EPCs led to efficient B19V replication. Finally, B19V replication in CD36(+)/Epo(+) EPCs required Epo, and the replication response was dose dependent. Our findings demonstrate that EpoR signaling is absolutely required for B19V replication in ex vivo-expanded erythroid progenitor cells after initial virus entry and at least partly accounts for the remarkable tropism of B19V infection for human erythroid progenitors.

Up‐regulation of erythropoietin receptor by nitric oxide mediates hypoxia preconditioning

Erythropoietin (Epo), known to stimulate erythroid progenitor cell survival, proliferation, and differentiation, has been shown to be neuroprotective against brain ischemia in animal models. Both Epo and Epo receptor (EpoR) are expressed in the brain and are up-regulated by hypoxia. Brain Epo signaling can stimulate neural cell survival and prevent neuron apoptosis. Neurons from EpoR null mice exhibit marked increased sensitivity to hypoxia. In endothelial cells, Epo has been shown to stimulate nitric oxide (NO) production, particularly at low pO(2). We found here that the EpoR expression on neural cells and Epo's neuroprotective effect were regulated by NO. Hypoxia increased NO production as well as EpoR expression, and inhibition of NOS activity reduced the proportion of EpoR-expressing neurons induced at low pO(2). Conversely, addition of NO donor to cultures grown under normoxia induced EpoR. Similarly, NO donor increased EpoR promoter activity in a reporter gene assay, suggesting that NO regulates EpoR at the transcription level. Preincubation of neurons with NO results in induction of EpoR, which gives rise to protection against hypoxia even in the absence of exogenous Epo, although at high concentration NO is toxic. These data provide evidence of a role for NO in Epo activity in brain and suggest links between NO production, EpoR expression, and Epo signaling in neuroprotection.

Iron and Isocitrate Calibrate Erythropoietin Responsiveness of Erythroid Progenitors Via Aconitase Tuning of Protein Kinase C Activity.

Abstract 627Our studies have focused on signaling alterations associated with the erythroid iron deprivation response, based on the hypothesis that a discrete pathway may calibrate erythropoietin (Epo) responsiveness according to iron availability. Previously we identified aconitase enzymatic activity as a critical target in the erythroid iron restriction response and provided evidence for a non-metabolic role for its product isocitrate in coupling iron and erythropoietin signaling. The striking ability of isocitrate to reverse the erythroid iron restriction response in vitro and in vivo raised the possibility for a novel therapeutic approach to Epo-resistant anemias. New data, presented below, further support a non-metabolic mechanism of action for isocitrate rescue of erythropoiesis and identify a specific signal transduction pathway mediating cross-talk among iron, aconitase/isocitrate, and erythropoietin signaling. Two approaches addressed the significance of isocitrate metabolism by isocitrate dehydrogenase (IDH) to yield NADH, NADPH, and αa-ketoglutarate. Firstly, direct measurement of cellular NADH, NADPH, and NADP showed no significant effects due to erythroid iron deprivation or isocitrate. Secondly, we synthesized a metabolically inactive enantiomer of isocitrate, L-(+)-isocitrate, which rescued erythroid viability under iron restriction with equivalent efficiency to the metabolically active D-(−)-isocitrate. L-(+)-isocitrate, also significantly rescued erythroid differentiation under iron restriction albeit somewhat less efficiently than D-(−)-isocitrate. Interestingly, high levels of L-(+)-isocitrate occur naturally in blackberries, which have been described as a folk remedy for anemia. Further evidence for non-metabolic signaling by isocitrate arose from identification of a specific signal transduction pathway dually regulated by iron and erythropoietin. In particular, erythroid deprivation of either iron or Epo induced hyperactivation of protein kinase Cαa (PKCαa), the relevance of which was supported by multiple findings. Firstly, the hyperactivation of PKCαa during erythroid iron deprivation preceded the onset of growth and differentiation defects. Secondly, PKC inhibitors significantly reversed both viability and differentiation defects associated with iron restriction. Thirdly, isocitrate reversed PKCαa hyperactivation in cells cultured with high Epo but failed to do so in cells cultured with low Epo, correlating with the previously described inability of isocitrate to reverse the iron restriction response in low Epo. Fourthly, we and others have demonstrated direct physical interaction between mitochondrial aconitase and active forms of PKCαa/β. To assess potential relevance of iron-sulfur clusters within aconitase, IRE binding assays were conducted on progenitors −/+ iron restriction −/+ isocitrate. The patterns of IRP1 and IRP2 activation suggested that aconitase iron-sulfur clusters in the erythroid lineage are uniquely sensitive to iron deprivation and that exogenous isocitrate may act to stabilize erythroid iron-sulfur clusters in the face of iron deprivation or stress. Taken together, our results suggest a role for aconitase and isocitrate in sensing the degree of erythroid iron restriction and transducing that signal to the Epo signaling pathway. A key target appears to be PKCαa. One possible mechanism involves aconitase acting as a highly dynamic scaffold that bridges iron metabolism and Epo signaling in early erythroid development. Isocitrate may act to stabilize the active site and prevent critical conformational changes during iron restriction. The resultant conformational stability may enable tonic suppression of PKC hyperactivation and thereby permit erythropoiesis in the face of iron deprivation. Novel compounds that stabilize the activity of aconitases may offer novel therapeutic avenues for Epo-refractory anemias. Disclosures:No relevant conflicts of interest to declare.

Genome-Wide ChIP-Seq Reveals a Dramatic Shift in the EKLF Binding Profile Between Erythroid Progenitors and Erythroblasts.

Abstract 565Erythroid Kruppel-Like Factor (EKLF; KLF1) is the founding member of the Kruppel family of C2H2 zinc finger transcription factors. First identified as an activator of the beta-globin locus, EKLF facilitates chromatin remodeling and transcriptional activation of target genes, at least in part through recognition of a 9-base consensus motif (NCNCNCCCN). By comparing the transcriptional profiles of E13.5 wild type and Eklf-/- mice, we demonstrated that the lethal failure to complete definitive erythropoiesis in the fetal liver (FL) was due in part to dysregulation of an EKLF target gene, the cell cycle control factor, E2F2 (Pilon et al. 2008). To identify further direct targets of EKLF activation that affect erythropoiesis, we are coupling chromatin immunoprecipitation with ultra high-throughput massively parallel sequencing (ChIP-seq). ChIP-seq is increasingly being used to map protein-DNA interactions in vivo, allowing simultaneous genome-wide analysis of transcription factor occupancy, defining an ‘interactome‘. Using mice whose endogenous Eklf gene was replaced with a fully functional HA-tagged form of EKLF, chromatin was isolated at E13.5 from immature erythroid progenitors and maturing erythroblasts by ChIP. Using a highly specific high-affinity anti-HA antibody, libraries of HA-EKLF-bound chromatin were subjected to fluorescent in situ sequencing on a Solexa 1G platform, providing 36-base signature tags that were mapped to the mouse genome using the Eland software package. A control library was derived from E13.5 FL chromatin that was not enriched for HA-EKLF occupancy. For both progenitors and erythroblasts, >1.1×107tags were obtained. 72.5% and 78.7% of progenitor and erythroblast tags mapped to unique sites within the genome, respectively. The tags were highly enriched in the ∼10% of the genome within genes (genic; 42% of tags), sites ≤10 kb from the nearest gene (adjacent; 15%), as opposed to the ∼90% of the genome that is >10 kb from the nearest gene (intergenic; 22%) or in repetitive DNA (21%) p=2.2 ×10-16. Using the MACS software package clustered peaks of EKLF occupancy were identified throughout the genome, defining the EKLF ‘interactome‘. The vast majority of peaks were mapped to non-repetitive regions of the genome (98% in progenitors; 95% in erythroblasts). Progenitors contained 4,383 peaks of EKLF occupancy, while erythroblasts contained 15,396 peaks. Only 100 peaks were common between populations. This >3.5-fold increase in genomic EKLF occupancy between progenitors and erythroblasts (p=1×10-5) reflects the shift in the expression and activity of EKLF protein in erythropoiesis described previously (Bouilloux et al. 2008; Lohmann & Bieker 2008). To identify potential EKLF target genes, we partitioned the genome into 3 categories, relative to annotated RefSeq coordinates (genic) as well as adjacent and intergenic. In progenitors, the majority of EKLF binding (54%) occurred in intergenic regions, with a minority within (38%) or adjacent (7%) to genes. By contrast, the EKLF binding profile in erythroblasts was reversed, with 62% of the peaks in genic regions, and a minority at intergenic (26%) or adjacent (12%) sites.To assess the effect of this shift in EKLF binding on gene transcription, we used publicly availabel data from the inducible G1E model of erythroid maturation (GEO: GSE628) to correlate our ChIP-seq data with mRNA expression. Informatic analyses using MetaCore demonstrated that >2,200 EKLF-associated genes were differentially expressed during maturation (949 increasing expression; 1,298 decreasing expression, all p<0.05). Among progenitors, control of cell cycle S-phase entry and progression was a significantly represented network, highlighted by focal EKLF target genes like Cdk2, Cdk4, and p107, in agreement with our previous observations. Among erythroblasts, the erythropoietin (Epo) signaling pathway was most significantly represented, highlighted by focal EKLF target genes like Stat3 and Bcl-XL, reflecting the well-established importance of the Epo axis for erythroblast survival. These data indicate that shifts in EKLF occupancy during erythropoiesis correlate with distinct functional effects on gene expression. Further, these observations support a model in which transcriptional regulators (e.g., EKLF) may collect at intergenic locations when their activity is not required, but where they remain poised for rapid recruitment. Disclosures:No relevant conflicts of interest to declare.

Erythropoietin receptor signaling regulates both erythropoiesis and megakaryopoiesis in vivo

Transgenic expression of a gain-of-function truncated mouse erythropoietin receptor gene ( EpoR) leads to expansion of the HSC pool in response to human erythropoietin (Epo). We have re-examined this observation using a knock-in mouse model, wherein the mouse EpoR gene was replaced in its proper genetic locus by a single copy of either a wild-type human or a polycythemia-inducing truncated human EPOR gene. Bone marrow cells obtained from knock-in mice were transplanted together with competitor bone marrow cells in a model that allows tracking of erythroid, platelet, and leukocyte contributions by each genotype. Secondary transplants were also performed. Stem/progenitor cells were identified phenotypically and isolated for colony-forming assays to evaluate cytokine responsiveness by cells with the wild-type human or truncated human EPOR gene. Augmented Epo signaling increased erythroid repopulation post-transplant as expected, but had no effect on short-term or long-term leukocyte repopulation. However, the wild-type human EPOR knock-in mouse showed decreases in both erythroid and platelet repopulation compared to marrow cells from the mutant human EPOR knock-in mouse or normal B6 animals. These results provide evidence supporting a role for Epo signaling in megakaryopoiesis in vivo and suggest a role for Epo signaling early in hematopoietic development.

Evaluating Erythropoietin-Associated Tumor Progression Using Archival Tissues from a Phase III Clinical Trial

Despite the prevalence of anemia in cancer, recombinant erythropoietin (Epo) has declined in use because of recent Phase III trials showing more rapid cancer progression and reduced survival in subjects randomized to Epo. Since Epo receptor (EpoR), Jak2, and Hsp70 are well-characterized mediators of Epo signaling in erythroid cells, we hypothesized that Epo might be especially harmful in patients whose tumors express high levels of these effectors. Because of the insensitivity of immunohistochemistry for detecting low level EpoR protein, we developed assays to measure levels of EpoR, Jak2 and Hsp70 mRNA in formalin-fixed paraffin-embedded (FFPE) tumors. We tested 23 archival breast tumors as well as 136 archival head and neck cancers from ENHANCE, a Phase III trial of 351 patients randomized to Epo versus placebo concomitant with radiotherapy following complete resection, partial resection, or no resection of tumor. EpoR, Jak2, and Hsp70 mRNA levels varied >30-fold, >12-fold, and >13-fold across the breast cancers, and >30-fold, >40-fold, and >30-fold across the head and neck cancers, respectively. Locoregional progression-free survival (LPFS) did not differ among patients whose head and neck cancers expressed above- versus below-median levels of EpoR, Jak2 or Hsp70, except in the subgroup of patients with unresected tumors (n = 28), where above-median EpoR, above-median Jak2, and below-median Hsp70 mRNA levels were all associated with significantly poorer LPFS. Our results provide a framework for exploring the relationship between Epo, cancer progression, and survival using archival tumors from other Phase III clinical trials.

No evidence for protective erythropoietin alpha signalling in rat hepatocytes

BackgroundRecombinant human erythropoietin alpha (rHu-EPO) has been reported to protect the liver of rats and mice from ischemia-reperfusion injury. However, direct protective effects of rHu-EPO on hepatocytes and the responsible signalling pathways have not yet been described. The aim of the present work was to study the protective effect of rHu-EPO on warm hypoxia-reoxygenation and cold-induced injury to hepatocytes and the rHu-EPO-dependent signalling involved.MethodsLoss of viability of isolated rat hepatocytes subjected to hypoxia/reoxygenation or incubated at 4°C followed by rewarming was determined from released lactate dehydrogenase activity in the absence and presence of rHu-EPO (0.2–100 U/ml). Apoptotic nuclear morphology was assessed by fluorescence microscopy using the nuclear fluorophores H33342 and propidium iodide. Erythropoietin receptor (EPOR), EPO and Bcl-2 mRNAs were quantified by real time PCR. Activation of JAK-2, STAT-3 and STAT-5 in hepatocytes and rat livers perfused in situ was assessed by Western blotting.ResultsIn contrast to previous in vivo studies on ischemia-reperfusion injury to the liver, rHu-EPO was without any protective effect on hypoxic injury, hypoxia-reoxygenation injury and cold-induced apoptosis to isolated cultured rat hepatocytes. EPOR mRNA was identified in these cells but specific detection of the EPO receptor protein was not possible due to the lack of antibody specificity. Both, in the cultured rat hepatocytes (10 U/ml for 15 minutes) and in the rat liver perfused in situ with rHu-EPO (8.9 U/ml for 15 minutes) no evidence for EPO-dependent signalling was found as indicated by missing effects of rHu-EPO on phosphorylation of JAK-2, STAT-3 and STAT-5 and on the induction of Bcl-2 mRNA.ConclusionTogether, these results indicate the absence of any protective EPO signalling in rat hepatocytes. This implies that the protection provided by rHu-EPO in vivo against ischemia-reperfusion and other causes of liver injury is most likely indirect and does not result from a direct effect on hepatocytes.

Erythropoietin expression and myoblasts survival

Erythropoietin (Epo) is mainly produced in the kidney and regulates erythroid differentiation. We previously reported that myoblasts express Epo receptor (EpoR) and Epo increases their proliferation. Myoblast isolated from transgenic mice (Tg6) overexpressing a human Epo transgene showed increased proliferation and reduced apoptosis at 5% O2 compared with WT myoblasts, due partly to Epo transgene expression in myoblasts. Epo treatment further increased proliferation and protection from apoptosis. Murine Epo (mEpo) mRNA expression was also higher in Tg6 myoblasts, suggesting that Epo may further induce Epo expression in myoblasts. Epo treatment increased mEpo mRNA in both Tg6 and WT myoblasts to a similar level. Reduced O2 further increased mEpo mRNA but not greater than Epo treatment alone, suggesting that mEpo was maximally expressed with Epo treatment. Although EpoR expression decreased with myoblast differentiation, mEpo mRNA remained unchanged. Similarly, mEpo mRNA expressed in the gastrocnemius muscles in vivo was higher level in Tg6 mice and mEpo mRNA in muscle increased after mice muscle injury in both Tg6 and WT mice. These results suggest that Epo signaling from endogenous Epo production in muscle from myoblasts and/or mature muscle fibers can promote myoblasts proliferation and survival and that Epo production in muscle can be further induced by exogenous Epo.

RNA Interference-Mediated Inhibition of Erythropoietin Receptor Expression Suppresses Tumor Growth and Invasiveness in A2780 Human Ovarian Carcinoma Cells

Although recombinant human erythropoietin (rHuEpo) has revolutionized the treatment of anemia, recent clinical trials suggested that rHuEpo use may be associated with decreased survival in cancer patients. Although the expression of erythropoietin (Epo) receptor (EpoR) has been demonstrated in various human cancers, the effect of exogenous Epo on the growth and therapy resistance of EpoR-bearing tumor cells is unclear at present. In the current study, we examined the hypothesis that EpoR may contribute to tumor growth independent of Epo in A2780 human ovarian carcinoma cells. A2780 human ovarian carcinoma cells showed high levels of EpoR expression, but lacked expression of Epo mRNA and biologically active Epo protein under both normoxic and hypoxic conditions. Exogenous Epo did not stimulate EpoR-mediated signaling, proliferation, invasiveness, or resistance to cytotoxic drugs in A2780 cells. In contrast, specific inhibition of EpoR expression using a short hairpin RNA (shRNA) expression plasmid resulted in markedly reduced proliferation and invasiveness in vitro. In addition, inhibition of EpoR expression led to abrogated in vivo ovarian cancer cell growth in a tumor xenograft system and resulted in decreased EpoR signaling. Our findings suggest that EpoR may be constitutively active in some cancer cells in the absence of Epo and provide the first evidence for a potential role of an Epo-independent, EpoR-mediated pathway in the growth of some human cancers.