EPO-independent functional EPO receptor in breast cancer enhances estrogen receptor activity and promotes cell proliferation

The main function of Erythropoietin (EPO) and its receptor (EPOR) is the stimulation of erythropoiesis. Recombinant human EPO (rhEPO) is therefore used to treat anemia in cancer patients. However, clinical trials have indicated that rhEPO treatment might promote tumor progression and has a negative effect on patient survival. In addition, EPOR expression has been detected in several cancer forms. Using a newly produced anti-EPOR antibody that reliably detects the full-length isoform of the EPOR we show that breast cancer tissue and cells express the EPOR protein. rhEPO stimulation of cultured EPOR expressing breast cancer cells did not result in increased proliferation, overt activation of EPOR (receptor phosphorylation) or a consistent activation of canonical EPOR signaling pathway mediators such as JAK2, STAT3, STAT5, or AKT. However, EPOR knockdown experiments suggested functional EPO receptors in estrogen receptor positive (ERα⁺) breast cancer cells, as reduced EPOR expression resulted in decreased proliferation. This effect on proliferation was not seen in ERα negative cells. EPOR knockdown decreased ERα activity further supports a mechanism by which EPOR affects proliferation via ERα-mediated mechanisms. We show that EPOR protein is expressed in breast cancer cells, where it appears to promote proliferation by an EPO-independent mechanism in ERα expressing breast cancer cells.     

STAT3, HIF-1alpha, EPO and EPOR - signaling proteins in human primary ductal breast cancers

STAT3 upregulates expression of HIF-1 induced EPO. Receptor EPOR was reported to activate STAT3. Our study was aimed at demonstration of tissue immunoreactivities of those proteins and determination of their relationships in reference to clinicopathological variables of breast cancers. We detected STAT3, HIF-1alpha, EPO and EPOR in specimens of 76 human, female, ductal breast cancers by immunohistochemistry. STAT3 was detected in 38 of 76 cancers (50%). HIF-1alpha was found in 55 cases (72%). EPO positive tumors comprised 89% of all the cancers (68 cases). EPOR was also visualized in 55 cases (72%). Anti-HIF-1alpha and anti-STAT3 stained nuclei and cytoplasm of breast cancer cells in diffuse and finely granular fashion. Strong membranous expressions of EPO and EPOR were distributed in cytoplasmic and membranous granularity or diffuse staining. STAT3 correlated with HIF-1 in general (r=0.4012, p<0.0001) and in different patients' subgroups. STAT3 was significantly associated with EPO and EPOR in all the cancers (r=0.2370, p=0.039 and r=0.3336, p=0.003, respectively). Besides a correlation between STAT3 and EPOR in node negative ones, STAT3 wasn't related to EPO and EPOR in remaining subgroups. HIF-1alpha correlated with EPO and EPOR in most of analyzed groups. Immunoreactivity to EPO generally was associated with EPOR (r=0.3520, p=0.002). Statistically analyzed distributions of the proteins reflected functional dependences among STAT3, HIF-1alpha, EPO and EPOR in cellular signal conduction.     

The mechanisms underlying the enrichment and action of glypican-1-positive exosomes in colorectal cancer cells

BackgroundGlypican-1 (GPC1) is overexpressed in several tumors, and GPC1⁺ exosomes have shown the potential to predict early colorectal cancer (CRC). However, the mechanisms underlying the enrichment and action of GPC1⁺ exosomes in CRC remain unknown.MethodsThe expression of slit guidance ligand 2 (SLIT2), hypoxia-inducible factor (HIF)-1α/2α, and GPC1 in clinical CRC tissues was detected using immunohistochemistry and western blot. Exosomes were isolated from the supernatants of CRC cell cultures. The effects of SLIT2, hypoxia, heparin, and phospholipase C (PLC) on exosomal GPC1 expression and GPC1⁺ exosome enrichment in CRC cells were analyzed with western blot and flow cytometry. CRC cell proliferation was assessed with MTT and colony formation assays. Co-immunoprecipitation was used to detect the binding of GPC1 and SLIT2 in SW480 cells. Nude mice were subcutaneously inoculated with SW480 cells with different treatments. The Wnt signaling was detected.ResultsSLIT2 was poorly expressed and GPC1, HIF-1α, and HIF-2α were highly expressed in human CRC tissues. SLIT2 in CRC cells inhibited GPC1⁺ exosome enrichment and exosomal GPC1 expression. PLC and heparin increased GPC1⁺ exosome enrichment in CRC cells in a concentration-dependent manner. Hypoxia increased the enrichment of GPC1⁺ exosomes in CRC cells depending on HIF-2α expression. GPC1⁺ exosomes stimulated CRC cell proliferation and xenograft tumor growth through activation of Wnt signaling.ConclusionsGPC1⁺ exosome enrichment is related to PLC and heparin. Hypoxia increases the enrichment of GPC1⁺ exosomes in CRC cells by activating HIF-2α and downregulating SLIT2. GPC1⁺ exosomes further drive CRC progression by activating Wnt signaling.     

Erythropoietin Ameliorates Rat Experimental Autoimmune Neuritis by Inducing Transforming Growth Factor-Beta in Macrophages

Erythropoietin (EPO) is a pleiotropic cytokine originally identified for its role in erythropoiesis. In addition, in various preclinical models EPO exhibited protective activity against tissue injury. There is an urgent need for potent treatments of autoimmune driven disorders of the peripheral nervous system (PNS), such as the Guillain-Barré syndrome (GBS), a disabling autoimmune disease associated with relevant morbidity and mortality. To test the therapeutic potential of EPO in experimental autoimmune neuritis (EAN) - an animal model of human GBS – immunological and clinical effects were investigated in a preventive and a therapeutic paradigm. Treatment with EPO reduced clinical disease severity and if given therapeutically also shortened the recovery phase of EAN. Clinical findings were mirrored by decreased inflammation within the peripheral nerve, and myelin was well maintained in treated animals. In contrast, EPO increased the number of macrophages especially in later stages of the experimental disease phase. Furthermore, the anti-inflammatory cytokine transforming growth factor (TGF)-beta was upregulated in the treated cohorts. In vitro experiments revealed less proliferation of T cells in the presence of EPO and TGF-beta was moderately induced, while the secretion of other cytokines was almost not altered by EPO. Our data suggest that EPO revealed its beneficial properties by the induction of beneficial macrophages and the modulation of the immune system towards anti-inflammatory responses in the PNS. Further studies are warranted to elaborate the clinical usefulness of EPO for treating immune-mediated neuropathies in affected patients.     

Atorvastatin ameliorates experimental autoimmune neuritis by decreased Th1/Th17 cytokines and up-regulated T regulatory cells

Statins have anti-inflammatory and immune-regulating properties. To investigate the effects of atorvastatin on experimental autoimmune neuritis (EAN), an animal model of Guillain–Barré syndrome (GBS), atorvastatin was administered to Lewis rats immunized with bovine peripheral myelin in complete Freund’s adjuvant. We found that atorvastatin ameliorated the clinical symptoms of EAN, decreased the numbers of inflammatory cells as well as IFN-γ⁺ and IL-17⁺ cells in sciatic nerves, decreased the CD80 expression and increased the number of CD25⁺Foxp3⁺ cells in mononuclear cells (MNC), and decreased the levels of IFN-γ in MNC culture supernatants. These data provide strong evidence that atorvastatin can act as an inhibitor in EAN by inhibiting the immune response of Th1 and Th17, decreasing the expression of co-stimulatory molecule, and up-regulating the number of T regulatory cells. These data demonstrated that statins could be used as a therapeutic strategy in human GBS in future.     

Up-regulated Endogenous Erythropoietin/Erythropoietin Receptor System and Exogenous Erythropoietin Rescue Retinal Ganglion Cells after Chronic Ocular Hypertension

Aims Recent studies have showed that erythropoietin (EPO) is a neuroprotectant for central nerve system neurons in addition to being a hematopoietic cytokine in response to hypoxia. In this study, we investigate the role of the EPO/EPO receptor (EPOR) system in the rat retina after ocular hypertension injury that mimics glaucoma. Methods Elevated intraocular pressure was induced by laser coagulation of the episcleral and limbal veins. Expression of EPO and EPOR in the normal and glaucomous retinas was investigated by immunohistochemistry and Western blot. To examine the effects of endogenous EPO on the survival of retinal ganglion cells (RGCs) subjected to hypertensive injury, soluble EPOR was directly injected into the vitreous body. Recombinant human EPO was both intravitreally and systemically administrated to study the effect of exogenous EPO on the survival of RGCs after ocular hypertension injury. Results Immunohistochemistry studies identified Müller cells as the main source of EPO in the normal retina. Expression of EPO and EPOR proteins was increased significantly 2 weeks after ocular hypertension. RGCs, amacrine and bipolar cells all demonstrated an increased expression of EPOR after ocular hypertension. Neutralization of endogenous EPO with soluble EPOR exacerbated ocular hypertensive injury, suggesting a role of the EPO/EPOR system in the survival of RGCs after injury. Similarly, topical and systemic administration of recombinant human EPO rescues RGCs after chronic ocular hypertension. Conclusions These results indicate that an endogenous EPO/EPOR system participates in intrinsic recovery mechanisms after retina injury and RGCs might be rescued by exogenous administration of EPO.     

Erythropoietin (EPO) Increases Myelin Gene Expression in CG4 Oligodendrocyte Cells through the Classical EPO Receptor

Erythropoietin (EPO) has protective effects in neurodegenerative and neuroinflammatory diseases, including in animal models of multiple sclerosis, where EPO decreases disease severity. EPO also promotes neurogenesis and is protective in models of toxic demyelination. In this study, we asked whether EPO could promote neurorepair by also inducing remyelination. In addition, we investigated whether the effect of EPO could be mediated by the classical erythropoietic EPO receptor (EPOR), since it is still questioned if EPOR is functional in nonhematopoietic cells. Using CG4 cells, a line of rat oligodendrocyte precursor cells, we found that EPO increases the expression of myelin genes (myelin oligodendrocyte glycoprotein [MOG] and myelin basic protein [MBP]). EPO had no effect in wild-type CG4 cells, which do not express EPOR, whereas it increased MOG and MBP expression in cells engineered to overexpress EPOR (CG4-EPOR). This was reflected in a marked increase in MOG protein levels, as detected by Western blot. In these cells, EPO induced by 10-fold the early growth response gene 2 (Egr2), which is required for peripheral myelination. However, Egr2 silencing with a siRNA did not reverse the effect of EPO, indicating that EPO acts through other pathways. In conclusion, EPO induces the expression of myelin genes in oligodendrocytes and this effect requires the presence of EPOR. This study demonstrates that EPOR can mediate neuroreparative effects.     

Erythropoietin promoted the proliferation of hepatocellular carcinoma through hypoxia induced translocation of its specific receptor

Background

Erythropoietin (EPO) is a hypoxia-inducible stimulator of erythropoiesis. Besides its traditional application in anemia therapy, it offers an effective treatment in the cancer patients, especially those who receive chemotherapy. Several reports indicated that it could promote the tumor cell proliferation through its specific receptor (EPOR). Unfortunately, the role of EPO/EPOR in hepatocellular carcinoma (HCC) progressing is still uncertain.

Methods

Protein in tumor tissue from HCC patients or H22 tumor-bearing mice was detected with immunohistochemistry. Cells were cultured under 1% oxygen to establish hypoxia. RT-PCR and western blotting were used to measure mRNA and protein of EPO/EPOR, respectively. MTT, flow cytometry and PCNA staining were used to detect cell proliferation. Immunofluorescence staining was applied to study the expression and location of cellular EPOR. The EPOR binding studies were performed with ¹²⁵I-EPO radiolabeling assay.

Results

EPO and EPOR protein were up-regulated in HCC tissue of patients and H22-bearing mice. These were positively correlated with hypoxia-inducible factor -1 α and ki-67. Hypoxia up-regulated the expression of EPO and EPOR in HepG2 cells. It also induced the proliferation and increased the percentage of divided cells after 24, 48 and 72 h treatment. These were inhibited in cells pre-treated with 0.5 μg/mL soluble-EPOR. Immunofluorescence staining presented that EPOR was obviously translocated from nucleus to cytoplasm and membrane under hypoxia. EPOR binding activity was also increased after exposure to hypoxia. Recombinant human erythropoietin obviously elevated cell proliferation rate and the percentage of divided under hypoxia but not normoxia, which were also inhibited by soluble-EPOR.

Conclusions

Our result indicated for the first time that EPO promoted the proliferation of HCC cells through hypoxia induced translocation of it specific receptor. Trial registration TJC20141113, retrospectively registered

     

Erythropoietin ameliorates rat experimental autoimmune neuritis by inducing transforming growth factor-β in macrophages

Erythropoietin (EPO) is a pleiotropic cytokine originally identified for its role in erythropoiesis. In addition, in various preclinical models EPO exhibited protective activity against tissue injury. There is an urgent need for potent treatments of autoimmune driven disorders of the peripheral nervous system (PNS), such as the Guillain-Barré syndrome (GBS), a disabling autoimmune disease associated with relevant morbidity and mortality. To test the therapeutic potential of EPO in experimental autoimmune neuritis (EAN) - an animal model of human GBS--immunological and clinical effects were investigated in a preventive and a therapeutic paradigm. Treatment with EPO reduced clinical disease severity and if given therapeutically also shortened the recovery phase of EAN. Clinical findings were mirrored by decreased inflammation within the peripheral nerve, and myelin was well maintained in treated animals. In contrast, EPO increased the number of macrophages especially in later stages of the experimental disease phase. Furthermore, the anti-inflammatory cytokine transforming growth factor (TGF)-beta was upregulated in the treated cohorts. In vitro experiments revealed less proliferation of T cells in the presence of EPO and TGF-beta was moderately induced, while the secretion of other cytokines was almost not altered by EPO. Our data suggest that EPO revealed its beneficial properties by the induction of beneficial macrophages and the modulation of the immune system towards anti-inflammatory responses in the PNS. Further studies are warranted to elaborate the clinical usefulness of EPO for treating immune-mediated neuropathies in affected patients.     

CHBP induces stronger immunosuppressive CD127+ M-MDSC via erythropoietin receptor

Erythropoietin (EPO) is not only an erythropoiesis hormone but also an immune-regulatory cytokine. The receptors of EPO (EPOR)₂ and tissue-protective receptor (TPR), mediate EPO’s immune regulation. Our group firstly reported a non-erythropoietic peptide derivant of EPO, cyclic helix B peptide (CHBP), which could inhibit macrophages inflammation and dendritic cells (DCs) maturation. As a kind of innate immune regulatory cell, myeloid-derived suppressor cells (MDSCs) share a common myeloid progenitor with macrophages and DCs. In this study, we investigated the effects on MDSCs differentiation and immunosuppressive function via CHBP induction. CHBP promoted MDSCs differentiate toward M-MDSCs with enhanced immunosuppressive capability. Infusion of CHBP-induced M-MDSCs significantly prolonged murine skin allograft survival compared to its counterpart without CHBP stimulation. In addition, we found CHBP increased the proportion of CD11b⁺Ly6G⁻Ly6C^high CD127⁺ M-MDSCs, which exerted a stronger immunosuppressive function compared to CD11b⁺Ly6G⁻Ly6C^high CD127⁻ M-MDSCs. In CHBP induced M-MDSCs, we found that EPOR downstream signal proteins Jak2 and STAT3 were upregulated, which had a strong relationship with MDSC function. In addition, CHBP upregulated GATA-binding protein 3 (GATA-3) protein translation level, which was an upstream signal of CD127 and regulator of STAT3. These effects of CHBP could be reversed if Epor was deficient. Our novel findings identified a new subset of M-MDSCs with better immunosuppressive capability, which was induced by the EPOR-mediated Jak2/GATA3/STAT3 pathway. These results are beneficial for CHBP clinical translation and MDSC cell therapy in the future.Subject terms: Allotransplantation, Inflammatory diseases     

EPO Promotes Bone Repair through Enhanced Cartilaginous Callus Formation and Angiogenesis

Erythropoietin (EPO)/erythropoietin receptor (EPOR) signaling is involved in the development and regeneration of several non-hematopoietic tissues including the skeleton. EPO is identified as a downstream target of the hypoxia inducible factor-α (HIF-α) pathway. It is shown that EPO exerts a positive role in bone repair, however, the underlying cellular and molecular mechanisms remain unclear. In the present study we show that EPO and EPOR are expressed in the proliferating, pre-hypertrophic and hypertrophic zone of the developing mouse growth plates as well as in the cartilaginous callus of the healing bone. The proliferation rate of chondrocytes is increased under EPO treatment, while this effect is decreased following siRNA mediated knockdown of EPOR in chondrocytes. EPO treatment increases biosynthesis of proteoglycan, accompanied by up-regulation of chondrogenic marker genes including SOX9, SOX5, SOX6, collagen type 2, and aggrecan. The effects are inhibited by knockdown of EPOR. Blockage of the endogenous EPO in chondrocytes also impaired the chondrogenic differentiation. In addition, EPO promotes metatarsal endothelial sprouting in vitro. This coincides with the in vivo data that local delivery of EPO increases vascularity at the mid-stage of bone healing (day 14). In a mouse femoral fracture model, EPO promotes cartilaginous callus formation at days 7 and 14, and enhances bone healing at day 28 indexed by improved X-ray score and micro-CT analysis of microstructure of new bone regenerates, which results in improved biomechanical properties. Our results indicate that EPO enhances chondrogenic and angiogenic responses during bone repair. EPO''s function on chondrocyte proliferation and differentiation is at least partially mediated by its receptor EPOR. EPO may serve as a therapeutic agent to facilitate skeletal regeneration.     

Quantification and localization of erythropoietin-receptor-expressing cells in the liver of Xenopus laevis

Erythropoiesis occurs in the African clawed frog, Xenopus laevis and is mediated by erythropoietin (xlEPO), a primary regulator of this process. Previously, we have shown that the xlEPO receptor (xlEPOR), which is expressed by erythroid progenitors that respond to xlEPO, is found predominantly in the liver. The aim of the present study was to determine the dynamics of erythropoiesis in the livers of normal and anemic X. laevis by identifying the number and precise location of mature and immature erythrocytes. We quantified mature and immature erythrocyte numbers by o-dianisidine staining or immunohistochemistry and investigated the dynamics of erythropoiesis in normal, acute hemolytic and blood-loss states by in vivo cell proliferation assays with 5-bromo-2′-deoxyuridine (BrdU). We detected 0.12×10⁸ xlEPOR⁺ BrdU⁺ cells in the liver of the normal X. laevis at 24 h after BrdU injection. Frogs presenting with acute hemolytic anemia and pancytopenia show a 10-fold increase in the number of xlEPOR⁺/BrdU⁺ cells (approximately 1.30×10⁸ cells) in the liver. The xlEPOR⁺ cells are found predominantly on the inner wall of hepatic sinusoids. Hematopoietic progenitors that undergo slow cell cycling were also observed in the hepatic sinusoids. This study clarifies the rate of production of mature and immature erythrocytes per day in the liver of X. laevis and the way that these cell numbers change in response to anemia.     

[(4-Hydroxyl-benzo[4,5]thieno[3,2-c]pyridine-3-carbonyl)-amino]-acetic acid derivatives; HIF prolyl 4-hydroxylase inhibitors as oral erythropoietin secretagogues

A new series of PHD (HIF prolyl 4-hydroxylase) inhibitors was designed based on the X-ray co-crystal structure of FG-2216. Using a lead generation process, a series of [(4-Hydroxyl-benzo[4,5]thieno[3,2-c]pyridine-3-carbonyl)-amino]-acetic acid derivatives was developed as potent PHD2 inhibitors. This class of compounds also showed the ability to stabilize HIF-α, to stimulate EPO secretion in in vitro studies, and to increase hematocrit, red blood cell count, and hemoglobin levels in an animal efficacy study.     

Granulocyte Colony-Stimulating Factor Activating HIF-1α Acts Synergistically with Erythropoietin to Promote Tissue Plasticity

Stroke and peripheral limb ischemia are serious clinical problems with poor prognosis and limited treatment. The cytokines erythropoietin (EPO) and granulocyte-colony stimulating factor (G-CSF) have been used to induce endogenous cell repair and angiogenesis. Here, we demonstrated that the combination therapy of EPO and G-CSF exerted synergistic effects on cell survival and functional recovery from cerebral and peripheral limbs ischemia. We observed that even under normoxic conditions, G-CSF activates hypoxia-inducible factor-1α (HIF-1α), which then binds to the EPO promoter and enhances EPO expression. Serum EPO level was significantly increased by G-CSF injection, with the exception of Tg-HIF-1α^+f/+f mice. The neuroplastic mechanisms exerted by EPO combined with G-CSF included enhanced expression of the antiapoptotic protein of Bcl-2, augmented neurotrophic factors synthesis, and promoted neovascularization. Further, the combination therapy significantly increased homing and differentiation of bone marrow stem cells (BMSCs) and intrinsic neural progenitor cells (INPCs) into the ischemic area. In summary, EPO in combination with G-CSF synergistically enhanced angiogenesis and tissue plasticity in ischemic animal models, leading to greater functional recovery than either agent alone.     

Histological Characterization of the Tumorigenic “Peri-Necrotic Niche” Harboring Quiescent Stem-Like Tumor Cells in Glioblastoma

Background

Characterization of the niches for stem-like tumor cells is important to understand and control the behavior of glioblastomas. Cell-cycle quiescence might be a common mechanism underlying the long-term maintenance of stem-cell function in normal and neoplastic stem cells, and our previous study demonstrated that quiescence induced by hypoxia-inducible factor (HIF)-1α is associated with a high long-term repopulation capacity of hematopoietic stem cells. Based on this, we examined human astrocytoma tissues for HIF-1α-regulated quiescent stem-like tumor cells as a candidate for long-term tumorigenic cells and characterized their niche histologically.

Methods

Multi-color immunohistochemistry was used to visualize HIF-1α-expressing (HIF-1α⁺) quiescent stem-like tumor cells and their niche in astrocytoma (WHO grade II–IV) tissues. This niche was modeled using spheroids of cultured glioblastoma cells and its contribution to tumorigenicity was evaluated by sphere formation assay.

Results

A small subpopulation of HIF-1α⁺ quiescent stem-like tumor cells was found in glioblastomas but not in lower-grade astrocytomas. These cells were concentrated in the zone between large ischemic necroses and blood vessels and were closer to the necrotic tissues than to the blood vessels, which suggested that a moderately hypoxic microenvironment is their niche. We successfully modeled this niche containing cells of HIF-1α⁺ quiescent stem-like phenotype by incubating glioblastoma cell spheroids under an appropriately hypoxic condition, and the emergence of HIF-1α⁺ quiescent stem-like cells was shown to be associated with an enhanced sphere-forming activity.

Conclusions

These data suggest that the “peri-necrotic niche” harboring HIF-1α⁺ quiescent stem-like cells confers a higher tumorigenic potential on glioblastoma cells and therefore may be a therapeutic target to control the behavior of glioblastomas.