Erythropoietin Receptor is a Risk Factor for Prognosis: A Potential Biomarker in Lung Adenocarcinoma

Background: Lung cancer has the highest mortality rate of all cancers, and LUAD's survival rate is particularly poor. Erythropoietin receptor (EPOR) is a member of the cytokine class I receptor family and can be detected in cancers such as lung adenocarcinoma (LUAD), however, the expression levels and prognostic value of EPOR in LUAD are still unclear.Methods: Multiple bioinformatics databases such as TIMER, Kaplan-Meier Plotter and TCGA databases, immunohistochemical method, and clinicopathological data of 92 LUADpatients between January 2008 and June 2016 were used to explore the EPOR expression, gene mutations affecting EPOR expression, EPOR-interacting or coexpressed genes, potential biological functions and the correlation of EPOR expression with prognosis, immune microenvironment and so on.All statistical analyses were performed in the R version 4.1.1.Results: In this study, the EPOR mRNA expression in LUAD tissues was possibly downregulated compared with that in normal lung tissues, but the EPOR protein expression in LUAD tissues was higher than that in paired normal lung tissues. Mutations in five genes, DDX60L, LGR6, POTEB3, RIF1 and SOX5, resulted in downregulation of EPOR expression, mutations in 10 genes includingC1orf168, DBX2 and EIF5B, resulted in upregulation of EPOR expression. Erichment analyses showed that EPOR is involved in neural tissue ligand-receptor interactions, MAPK and PI3K/Akt signaling pathways and cancer pathways. The KM Plotter and PrognoScan databases consistently concluded that EPOR was associated with prognosis in LUAD patients. Our clinicopathological data showed that high EPOR expression was associated with poorer OS (29.5 vs 46 months) and had a good predictive ability for 5-year survival probability. Conclusions: EPOR expression might be downregulated at the mRNA levels and significantly upregulated at the protein levels in LUAD, which showed that the mRNA and protein levels of EPOR are inconsistent.The high expression of EPOR was associated with poor prognosis and is expected to be a potential new prognostic marker for LUAD.

Erythropoietin Non-hematopoietic Tissue Response and Regulation of Metabolism During Diet Induced Obesity

Erythropoietin (EPO) receptor (EPOR) determines EPO response. High level EPOR on erythroid progenitor cells gives rise to EPO regulated production of red blood cells. Animal models provide evidence for EPO activity in non-hematopoietic tissue mediated by EPOR expression. Beyond erythropoiesis, EPO activity includes neuroprotection in brain ischemia and trauma, endothelial nitric oxide production and cardioprotection, skeletal muscle wound healing, and context dependent bone remodeling affecting bone repair or bone loss. This review highlights examples of EPO protective activity in select non-hematopoietic tissue with emphasis on metabolic response mediated by EPOR expression in fat and brain and sex-specific regulation of fat mass and inflammation associated with diet induced obesity. Endogenous EPO maintains glucose and insulin tolerance and protects against fat mass accumulation and inflammation. Accompanying the increase in erythropoiesis with EPO treatment is improved glucose tolerance and insulin response. During high fat diet feeding, EPO also decreases fat mass accumulation in male mice. The increased white adipose tissue inflammation and macrophage infiltration associated with diet induced obesity are also reduced with EPO treatment with a shift toward an anti-inflammatory state and decreased inflammatory cytokine production. In female mice the protective effect of estrogen against obesity supersedes EPO regulation of fat mass and inflammation, and requires estrogen receptor alpha activity. In brain, EPOR expression in the hypothalamus localizes to proopiomelanocortin neurons in the arcuate nucleus that promotes a lean phenotype. EPO stimulation of proopiomelanocortin neurons increases STAT3 signaling and production of proopiomelanocortin. Cerebral EPO contributes to metabolic response, and elevated brain EPO reduces fat mass and hypothalamus inflammation during diet induced obesity in male mice without affecting EPO stimulated erythropoiesis. Ovariectomy abrogates the sex-specific metabolic response of brain EPO. The sex-dimorphic EPO metabolic response associated with fat mass accumulation and inflammation during diet induced obesity provide evidence for crosstalk between estrogen and EPO in their anti-obesity potential in female mice mediated in part via tissue specific response in brain and white adipose tissue. Endogenous and exogenous EPO response in non-hematopoietic tissue demonstrated in animal models suggests additional activity by which EPO treatment may affect human health beyond increased erythropoiesis.

EpoR-tdTomato-Cre mice enable identification of EpoR expression in subsets of tissue macrophages and hematopoietic cells

The erythropoietin receptor (EpoR) has been traditionally thought as an erythroid specific gene. Notably, accumulating evidence suggests that EpoR is expressed well beyond erythroid cells. However, the expression of EpoR in non-erythroid cells has been controversial. In the present study we generated EpoR-tdTomato-Cre mice and used them to examine the expression of EpoR in tissue macrophages and hematopoietic cells. We show that in marked contrast to the previously available EpoR-eGFPcre mice in which very weak eGFP signal was detected in erythroid cells, tdTomato was readily detectable in both fetal liver (FL) and bone marrow (BM) erythroid cells at all developmental stages and exhibited dynamic changes during erythropoiesis. Consistent with our recent finding that erythroblastic island (EBI) macrophages are characterized by the expression of EpoR, tdTomato was readily detected in both FL and BM EBI macrophages. Moreover, tdTomato was also detected in subsets of hematopoietic stem cells, progenitors, megakaryocytes and B cells in BM as well as in spleen red pulp macrophages and liver Kupffer cells. The expression of EpoR was further demonstrated by the EpoR-tdTomato-Cre mediated excision of the floxed STOP sequence. Importantly, EPO injection selectively promoted proliferation of the EpoR-expressing cells and induced erythroid lineage bias during hematopoiesis. Our findings imply broad roles of EPO/EpoR in hematopoiesis which warrant further investigation. The EpoR-tdTomato-Cre mouse line provides a powerful tool to facilitate future studies on EpoR expression and regulation in various non-hematopoietic cells and to conditionally manipulate gene expression in EpoR-expressing cells for functional studies.

Metabolic Burden of Erythropoietin Stimulated Erythropoiesis in Mice

Erythropoietin (EPO) promotes erythroid differentiation and increases glucose uptake in erythroid progenitor cells in culture. The metabolic burden associated with EPO treatment in adult mice is suggested by a decrease in body weight concomitant with increased hematocrit. Wild type male mice (C57Bl/6, age 8 months) treated with EPO showed the expected increase in hematocrit accompanied by a fall in blood glucose level and a decrease in body weight and fat mass. However, the decrease in body weight is even more evident in obese mice on a high fat diet and has also been linked to non-hematopoietic response, particularly with EPO receptor (EpoR) expression in white adipose tissue. We examined the metabolic burden of EPO treatment (3000U/kg for 3 weeks) in young, lean male mice (3 months) placed on high fat diet at the time of EPO administration. The increase in hematocrit was accompanied by decreased blood glucose level and improved glucose tolerance. However, no difference in body weight was observed between mice treated with EPO and the saline treated group, suggesting that the EPO stimulated decrease in body weight is evident primarily in older animals with greater fat mass. To determine the contribution of EpoR expression in non-hematopoietic tissue to the metabolic EPO response, young male mice with EpoR restricted to erythroid tissue (TgEpoR) were placed on high fat diet and treated with EPO. The expected increased hematocrit was also accompanied by decreased blood glucose level and improved glucose tolerance, and no change in body weight between EPO and saline treatment. The similar responses observed in young wild type and TgEpoR mice suggest that the EPO stimulated increase in glucose metabolism is associated with increased erythropoiesis rather than a direct EPO response in non-hematopoietic tissue. TgEpoR mice exhibit an age dependent increase in fat mass even greater than that observed in wild type mice, and by 8 months TgEpoR mice are obese, glucose intolerant and insulin resistant compared with wild type mice. At 8 months, TgEpoR mice treated with EPO show the increase in hematocrit and, despite the increase in fat mass, there is only a minimal decrease in body weight compared with wild type mice. These data provide evidence that in addition to the age dependent association of EPO stimulated decrease in body weight and fat mass, this decrease in body weight is due largely to EPO response related to EpoR expression in non-hematopoietic tissue. Interestingly, young male mice with targeted deletion of EpoR in adipose tissue placed on a high fat diet and treated with EPO show the increase in hematocrit and improvement in glucose tolerance, and at 8 months, the increase in hematocrit with EPO treatment is accompanied by minimal change in body weight. The similar metabolic response of these mice with targeted deletion of EpoR in adipose tissue to TgEpoR mice indicate the contribution of EpoR expression in adipose tissue to the loss of body weight and fat mass. Therefore, the metabolic burden associated with EPO stimulated erythropoiesis appears to be reflected in improved glucose metabolism and glucose tolerance with minimal or no effect on body weight, is evident in young, lean mice, and is independent of EpoR expression in non-hematopoietic tissue. In older mice, non-hematopoietic metabolic EPO response is more readily apparent as reflected in loss of body weight/fat mass, which overshadows the erythropoietic metabolic response. In combination, the metabolic response to EPO treatment results from EPO stimulated increased erythropoiesis, improved glucose metabolism and glucose tolerance, and an age dependent decrease in body weight and fat mass associated with EpoR expression in non-hematopoietic tissue, particularly in white adipose tissue. Disclosures No relevant conflicts of interest to declare.

Salmonella Infection Enhances Erythropoietin Production by the Kidney and Liver, Which Correlates with Elevated Bacterial Burdens

Salmonellainfection profoundly affects host erythroid development, but the mechanisms responsible for this effect remain poorly understood. We monitored the impact ofSalmonellainfection on erythroid development and found that systemic infection induced anemia, splenomegaly, elevated erythropoietin (EPO) levels, and extramedullary erythropoiesis in a process independent ofSalmonellapathogenicity island 2 (SPI2) or flagellin. The circulating EPO level was also constitutively higher in mice lacking the expression of signal-regulatory protein α (SIRPα). The expression level of EPO mRNA was elevated in the kidney and liver but not increased in the spleens of infected mice despite the presence of extramedullary erythropoiesis in this tissue. In contrast to data from a previous report, mice lacking EPO receptor (EPOR) expression on nonerythroid cells (EPOR rescued) had bacterial loads similar to those of wild-type mice followingSalmonellainfection. Indeed, treatment to reduce splenic erythroblasts and mature red blood cells correlated with elevated bacterial burdens, implying that extramedullary erythropoiesis benefits the host. Together, these findings emphasize the profound effect ofSalmonellainfection on erythroid development and suggest that the modulation of erythroid development has both positive and negative consequences for host immunity.

Impact of Erythropoietin Modulation Using Hyperbaric Oxygen on Umbilical Cord Blood CD34+ Cell Homing

Background: Previously, we demonstrated that host pre-treatment with hyperbaric oxygen (HBO) improves engraftment of umbilical cord blood (UCB) CD34+ cells in a transplant murine model. Recent data suggest that erythropoietin (EPO) determines the fate of hematopoietic progenitor cells in favor of erythroid differentiation. Herein, we evaluated mechanisms underlying the favorable effect of HBO therapy on UCB CD34+ cell engraftment. We hypothesized that HBO modulates EPO which in turn enhances UCB CD34 homing to the marrow, thereby enhancing engraftment. Materials/methods: We examined EPO receptor (EPOR) expression on UCB CD34+ cells by flow cytometry and western blot. Transmigration assays were used to evaluate EPO effects on UCB CD34+ cell migration toward Stromal Cell-Derived Factor 1 (SDF-1; CXCL12) which has been shown to mediate CD34+ chemotaxis to the marrow. Using an NSG transplant murine model, we evaluated serum EPO levels by ELISA and early UCB bone marrow/spleen homing by flow cytometry. Bone marrow UCB cell differentiation was examined 1 and 2 weeks post-transplant using CFU-assay. Results: We found that on average 17.6 (4.9-38.7) % of CD34+ enriched UCB cells express EPOR. EPOR expression was confirmed by western blot. The percentage of EPOR positive cells was significantly higher in hematopoietic stem cells (HSC) defined as Lin- CD34+ CD38- CD45RA- CD90+ CD49f+ (45.7+/-1.4) compared to hematopoietic progenitor cells defined as Lin- CD34+ CD38+ (25.1+/-0.7) and multipotent progenitors defined as Lin- CD34+ CD38- CD45RA- CD90- CD49f- (27.2+/-0.3). Also, exposure of UCB CD34+ cells to EPO during cell culture inhibited their migration toward SDF-1 (Figure-1). HBO treated mice demonstrated significantly lower serum EPO blood levels compared to control mice (727.4+/- 42.02 versus1576 +/- 80.90 pg/mL, p <0.0001) measured 3-hours post-transplant. A higher percentage of human CD34+ was seen in the bone marrow of HBO treated mice 3-hours post-transplant (6.8+/-1.0 versus 3.4+/-0.6), p=0.01). HBO treated mice demonstrated significantly lower numbers of BFU-E ( 26.7+/-8.7 versus 75.3+/-19.3, p =0.043) and increasing numbers of CFU-G/M (166.0+/-27.9 versus 74.8+/-32.6, p =0.05). Conclusions: EPOR is enriched in UCB HSC. HBO lowers host blood EPO and as a result improves UCB CD34+ cell early bone marrow homing and enhances myeloid differentiation. Figure 1. EPO effects on UCB CD34+ cell transmigration toward SDF-1 gradient. Figure 1. EPO effects on UCB CD34+ cell transmigration toward SDF-1 gradient. Disclosures No relevant conflicts of interest to declare.

Gfi-1 regulates the erythroid transcription factor network through Id2 repression in murine hematopoietic progenitor cells

Key Points Reducing Id2 in Gfi-1−/− mice restores radioprotective function of hematopoietic progenitors and partially rescues erythroid development. Rescue of erythroid development in Gfi-1−/− mice by Id2 reduction directly correlates with an increase of Gata1, Eklf, and EpoR expression.

Mutation Of The Divalent Metal Transporter (Dmt1) Gene Results In Inefficient Induction Of The Erythroid Transcriptional Program Due To Latter Onset Of GATA-1 and Epor Expression

Introduction Divalent metal transporter 1 (DMT1; SLC11A2) encodes trans-membrane protein variants that execute either non-heme iron absorption through apical duodenal membrane of enterocytes or transferrin-bound iron uptake through endosomal membrane of erythroid (and other) cells. Since 2005 several DMT1 mutations affecting accurate protein folding and localization were identified in patients with defective iron uptake leading to microcytic anemia, abnormal growth of erythroid progenitors in vitro, and iron overload in the liver. We have previously demonstrated that defective growth of DMT-1-mutatnt BFU-Es in vitro and anemia associated with ineffective erythropoiesis in vivo can be improved with high-dose erythropoietin (EPO) supplementation (Horvathova et al., 2012). Data from Dmt1-mutant mk/mk mice (Gunshin et al., 2005) suggested that the anemic phenotype is a result of ineffective erythropoiesis within bone marrow and spleen. Hypothesis DMT1-mutant erythropoiesis inhibits EPO receptor (EPOR) signaling whose end-point target as well as upstream activator is the key transcription factor GATA-1. This results in defective erythroid development characterized by impaired survival capacity of erythroid progenitors, increased apoptosis of erythroblasts, and dysregulation of erythroid gene expression leading to ineffective erythropoiesis. Materials Bone marrow cells of murine mk/mk mice were sorted using flow cytometry to obtain differentiating erythroid fractions based on antigens Cd71 and Ter119 (Sokolovsky et al., 2001) which were then subjected to gene expression analysis. Results Firstly, four consecutive fractions (proerythroblasts and early basophilic erythroblasts - stage I, late basophilic erythroblasts – stage II, chromathophillic and orthochromathophillic erythroblasts – stage III, and late chromathophillic erythroblasts and reticulocytes – stage IV) were isolated from mk/mk mice and control littermates. Flow cytometry showed enrichment in stages I and II and depletion in stage IV in the mk/mk bone marrow when compared to wild type controls, consistent with our previous data. In the spleen the major cellular enrichment was seen in stage III accompanied by cell depletion in stage IV. Gene expression of GATA-1 was markedly decreased at the onset (in stages I and II) of erythropoiesis while it was increased in terminal stage IV. The expression pattern of the GATA-1 target gene Epor was similar to that of GATA-1 expression, while expression of b-maj globin was significantly reduced indicating developmental delay of the erythroid Dmt1-mutant compartment (compared to wild type). Whereas GATA-1 and Epor expression is low in early erythropoiesis, a compensatory increase in their expression at later stages is not capable to efficiently upregulate b-maj globin. These data together with flow cytometry analysis identify a developmental blockade of erythropoiesis between stages II(III) and IV. To better understand whether Dmt1 levels regulate GATA-1 and Epor expression we used murine erythroleukemia (MEL) cells containing conditional (estrogen-regulated) transgene encoding GATA-1 fused with estrogen receptor ligand-binding domain (GER). MEL cells are cytologically characterized as proerythroblasts and early basophilic erythroblasts, stage I. Firstly, we established that GATA-1 upregulates Epor expression and directly binds to the Epor gene using RT-PCR, Immunoblotting, and chromatin immunoprecipitation (ChIP) in activated GER cells. Using ChIP-sequencing analysis of GATA-1 (and a panel of histone modifications) the GATA-1 enrichment was clearly identified at three distinct Epor regions in murine erythroblasts and differentiating MEL cells. Next, we downregulated Dmt1 using siRNA and observed that GATA-1-mediated upregulation of Epor in activated GER cells became inhibited. In addition, the knockdown of Dmt1 also inhibited steady state levels of GATA-1 in MEL cells by 25%. Conclusions Ineffective erythropoiesis in Dmt1-mutant mice is blocked at stages II (III) and display deregulation of the Epor signaling cascade involving GATA-1 and its targets. Our data thus interconnect iron uptake and the Epor/GATA-1 pathways and suggest their roles during erythroid pathogenesis upon DMT1 mutations. Grants: P305/11/1745, P301/12/P380, P305/12/1033, UNCE 204021, PRVOUK-P24/LF1/3, SVV-2012-264507, GAUK 251135 82210 Disclosures: No relevant conflicts of interest to declare.

Ligand-Regulated and Developmental Stage-Specific Expression Of The EPO Receptor During Human Erythroid Progenitor Cell Formation

Upon engagement of its cell surface receptor, EPO sharply regulates erythroid progenitor cell (EPC) development. Little is known concerning the dynamics of EPOR expression, however, due to its rarity in primary cells. Using EPO dosing and response studies together with a highly specific and sensitive hEPOR antibody, we provide new insight into stage and ligand dose-dependent regulation of cell surface EPOR expression among human bone marrow derived primary EPCs. Developmentally, EPOR levels peaked within a KITposCD36posGPAneg proerythroblast cohort. In contrast to prior studies using transfected murine myeloid cell lines, the limiting of EPO substantially increased EPOR levels, demonstrating ligand dependent modulation of endogenous EPOR in hEPCs. EPO dosing and withdrawal studies further showed that in early EPCs EPO efficiently supported survival but not proliferation. In GPApos erythroblasts EPOR levels were diminished, but GPApos cells nonetheless exhibited strong EPO dependency for both survival and growth. Investigations provide novel insight into regulated EPOR surface expression among developing primary EPCs, and may also aid in understanding the action modes and effects of emerging EPOR agonists and erythropoiesis stimulating agents. Disclosures: No relevant conflicts of interest to declare.

Abstract 123: Erythropoietin Enhances Abundance of Cardiomyogenic Precursors in vitro and in vivo

Recent data suggest that erythropoietin (Epo) can improve cardiac function following ischemia reperfusion injury. We found EpoR to be prominently expressed in embryonic rather than adult mouse heart. In the latter EpoR expression was confined to interstitial cells. We isolated the non-myocyte fraction of adult mouse hearts by enzymatic digestion and straining (pore size: 30 µm) and found a 4-fold higher EpoR expression compared to unselected cardiac cells (n=3). Flow cytometry (FC) revealed that 24±3% of this heterogeneous cell pool expressed EpoR (n=3) and 11±2% co-expressed αMHC. Half of the αMHC+ cells stained positive for Ki67, consistent with FC showing that 49±7% αMHC+/EpoR+ cells were in S and G2 phase (n=3; vs. 13±3% in αMHC-/EpoR-; n=3). Interestingly, co-culture with neonatal rat cardiomyocytes yielded EGFP+ cardiomyocyte-like cells. Since αMHC+ proliferated and differentiated in vitro we termed them cardiomyogenic precursors (CMPs). Epo treatment of the non-myocyte pool enhanced Akt phosphorylation (n=6/group) and increased CMP abundance in vitro (2-fold; n=3). Cell cycle arrest abrogated the aforementioned effect, suggesting that Epo influenced CMP proliferation. Finally, we tested the potential of Epo to protect against ischemia by enhancing CMP numbers in a model of myocardial infarction (MI). Following MI mice were treated twice with 2,000 U/kg Epo (i.p.). Ten weeks post MI echocardiography revealed blunted myocardial deterioration in mice receiving Epo (ΔEF=-11.18%, n=5) compared to control (ΔEF=-20.82%, n=6). FC revealed an enhanced αMHC+/cTnT+ cell pool in the Epo treated group (20.3±1.9% vs. 10.6±2.3% in control, n=8/6) 4 weeks post MI. In conclusion, we found that EpoR is expressed in a putative cardiomyogenic precursor cell pool in the adult heart. CMPs appear to proliferate in vitro and in vivo; with further enhanced proliferation under Epo administration. This may contribute to Epo mediated protection after MI and highlights the therapeutic effect of Epo upon ischemic cardiac injury. Whether these cells remain from embryogenesis or just constitute an immature myocyte population in the adult heart deriving from cardiomyocyte dedifferentiation needs to be further investigated.