Erythropoietin and cancer: the unintended consequences of anemia correction.

Until 1990, erythropoietin (EPO) was considered to have a single biological purpose and action, the stimulation of red blood cell growth and differentiation. Slowly, scientific and medical opinion evolved, beginning with the discovery of an effect on endothelial cell growth in vitro and the identification of EPO receptors (EPORs) on neuronal cells. We now know that EPO is a pleiotropic growth factor that exhibits an anti-apoptotic action on numerous cells and tissues, including malignant ones. In this article, we present a short discussion of EPO, receptors involved in EPO signal transduction, and their action on non-hematopoietic cells. This is followed by a more detailed presentation of both pre-clinical and clinical data that demonstrate EPO's action on cancer cells, as well as tumor angiogenesis and lymphangiogenesis. Clinical trials with reported adverse effects of chronic erythropoiesis-stimulating agents (ESAs) treatment as well as clinical studies exploring the prognostic significance of EPO and EPOR expression in cancer patients are reviewed. Finally, we address the use of EPO and other ESAs in cancer patients.

Human selenium binding protein-1 (hSP56) is a negative regulator of HIF-1α and suppresses the malignant characteristics of prostate cancer cells.

In the present study, we demonstrate that ectopic expression of 56-kDa human selenium binding protein-1 (hSP56) in PC-3 cells that do not normally express hSP56 results in a marked inhibition of cell growth in vitro and in vivo. Down-regulation of hSP56 in LNCaP cells that normally express hSP56 results in enhanced anchorage-independent growth. PC-3 cells expressing hSP56 exhibit a significant reduction of hypoxia inducible protein (HIF)-1α protein levels under hypoxic conditions without altering HIF-1α mRNA (HIF1A) levels. Taken together, our findings strongly suggest that hSP56 plays a critical role in prostate cells by mechanisms including negative regulation of HIF-1α, thus identifying hSP56 as a candidate anti-oncogene product.

Human erythropoietin increases the pro-angiogenic potential of A2780 ovarian adenocarcinoma cells under hypoxic conditions.

Erythropoietin (Epo) is a key regulator of erythroid cell proliferation, differentiation and apoptosis. In the form of the recombinant protein, it is widely used to treat various types of anemias, including that associated with cancer and with the myelosuppressive effects of chemotherapy, particularly platinum-based regimens. Our previous studies confirmed the presence of Epo receptors (EpoRs) in ovarian adenocarcinoma cell lines and demonstrated that long-term Epo treatment of A2780 cells resulted in the development of a phenotype exhibiting both enhanced Epo signaling and increased paclitaxel resistance. In the present study, we carried out a series of experiments to analyze the pro-angiogenic potential of Epo-treated A2780 and SKOV-3 cells. Our studies revealed that conditioned media of Epo-treated A2780 cells had a stimulative effect on human umbilical vein endothelial cells (HUVECs). This effect was only seen when A2780 cells were incubated under hypoxic conditions. Furthermore, Epo increased the secretion of interleukin (IL)-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, GM-CSF and interferon-γ by A2780 cells that grew in hypoxic conditions. In this regard, conditioned media of hypoxic and Epo-treated A2780 cells induced a significant phosphorylation of STAT-5 in HUVECs. Our results may have important implications for ovarian cancer patients receiving Epo.

Location and the functionality of erythropoietin receptor(s) in A2780 cells.

Erythropoietin (Epo) is a critical regulator of erythroid cell proliferation, differentiation and apoptosis. In the form of a recombinant protein, it is widely used to treat various forms of anemia, including that associated with cancer and with the myelosuppressive effects of chemotherapy. Studies of ovarian cancer cell lines have demonstrated the presence of the Epo receptor (EpoR), but there are disagreements regarding its localization and functionality in these cells. Using fluorescence microscopy, we were not able to identify the EpoR on the surface of A2780 cells, in contrast to the positive control K562 cells. Flow cytometry did reveal a weak surface EpoR signal in A2780 cells. Interestingly, most of the EpoR in A2780 cells was found in the cytoplasm, more abundantly as an intracellular membrane-associated protein than a soluble one. Silencing EpoR expression by lentiviral-mediated shRNA resulted in reduced A2780 proliferation as well as reduction in Epo-induced phosphorylation of Erk1/2. Our findings provide important insights into the biology of the EpoR in ovarian cancer cells.

Erythropoietin and erythropoiesis stimulating agents.

Erythropoietin (EPO) is the main hormonal regulator of red blood cell production. Recombinant EPO has become the leading drug for treatment of anaemia from a variety of causes; however, it is sometimes misused in sport with the aim of improving performance and endurance. This paper presents an introductory overview of EPO, its receptor, and a variety of recombinant human EPOs/erythropoiesis stimulating agents (ESAs) available on the market (e.g. epoetins and their long acting analogs--darbepoetin alfa and continuous erythropoiesis receptor activator). Recent efforts to improve on EPO's pharmaceutical properties and to develop novel replacement products are also presented. In most cases, these efforts have emphasized a reduction in frequency of injections or complete elimination of intravenous or subcutaneous injections of the hormone (biosimilars, EPO mimetic peptides, fusion proteins, endogenous EPO gene activators and gene doping). Isoelectric focusing (IEF) combined with double immunoblotting can detect the subtle differences in glycosylation/sialylation, enabling differentiation among endogenous and recombinant EPO analogues. This method, using the highly sensitive anti-EPO monoclonal antibody AE7A5, has been accepted internationally as one of the methods for detecting misuse of ESAs in sport.

Differentially expressed genes associated with cisplatin resistance in human ovarian adenocarcinoma cell line A2780.

Ovarian cancer cells are usually initially sensitive to platinum-based chemotherapy, such as cisplatin (CDDP), but typically become resistant over time. Such drug resistance is a serious impediment to successful disease treatment, and the molecular mechanisms responsible for resistance are not fully understood. In search of novel mechanisms that may lead to the development of CDDP chemoresistance, we used subtractive hybridization to identify differentially expressed genes in CDDP resistant CP70 and C200 cells vs. CDDP sensitive A2780 human ovarian adenocarcinoma cells. We analyzed 256 randomly selected clones. Subtraction efficiency was determined by dot blot and DNA sequencing. Confirmation of differentially expressed cDNAs was done by virtual northern blot analysis, and 17 genes that were differentially expressed in CDDP resistant cell lines vs. CDDP sensitive A2780 cells were identified. The expression of 10 of these genes was low or undetectable in sensitive A2780 cells in comparison to resistant cells and an additional seven genes were more highly expressed in resistant CP70 and C200 vs. A2780 cells. Our identified genes are involved in numerous and diverse cellular processes, such as inhibition of apoptosis (ARHGDIB), stress response (HSPCA, TRA1), chromatin condensation (CNAP1, RanBP2), invasiveness of cells (MMP10), alteration of Ca(2+) homeostasis (ASPH, ATP2B1) and others. Further characterization of these genes and gene products should yield important insights into the biology of CDDP resistance in ovarian carcinoma.

Report on isoelectric focusing trial of erythropoietin profiling in two cancer patients during chemotherapy and darbepoetin treatment.

The level of erythropoietin, main regulator of erythropoiesis, is affected by hypoxia, anaemia, application of recombinant erythropoietin, chemotherapy and others. Isoelectric focusing (IEF) combined with double immunobloting is a method that enables distinct analysis of endogenous and recombinant erythropoietin isoforms. Aim of our study was to set up analysis of treatment effects on the pattern of endogenous erythropoietin in anaemic breast cancer patient. Urine and blood samples were collected during and after termination of the treatment and analysed by isoelectric focusing. Endogenous erythropoietin was found lower, but still detectable during darbepoetin treatment. Normal shift of erythropoietin isoforms between serum vs. urine, ordinary seen in healthy volunteers, was not observed indicating kidney damage. The patient was suffering from heavy proteinuria and had low Glomerular filtration rate indicating acute renal failure, probably caused by clinical status or cisplatin chemotherapy. IEF has not yet been used for follow up of erythropoietin profile in cancer patients. It enables to monitor the effects of treatment on the level of endogenous erythropoietin and indirectly indicates kidney function.

An erythropoietin autocrine/paracrine axis modulates the growth and survival of human prostate cancer cells.

Erythropoietin receptors have been identified on a variety of cancer-derived cell lines and primary cancer cells, including those of prostate cancer. The functional status of these extrahematopoietic erythropoietin receptors remains a matter of some dispute. The publication of several important clinical trials suggesting a direct effect of erythropoietin on the growth and survival of primary tumors adds further importance to the question of whether erythropoietin receptors on cancer cells are functional. We have reported previously that human prostate cancer cell lines and primary prostate cancer cells express functional erythropoietin receptors that respond to exogenous erythropoietin by increased cell proliferation and STAT5 phosphorylation. We now show that prostate cancer cell lines express both the EPO gene and the biologically active erythropoietin. The coexpression of functional receptor and biologically active ligand in the cells has led us to hypothesize an autocrine/paracrine mechanism, driven by endogenous erythropoietin, which may modulate the growth and progression of prostate cancer. To test our hypothesis, we have knocked down, independently, erythropoietin receptor and erythropoietin on prostate cancer cells by transfection with short hairpin RNAs. Erythropoietin receptor knockdown cells grow significantly more slowly than their erythropoietin receptor-bearing counterparts in monolayer culture, produce fewer, smaller colonies in soft agar, and do not exhibit erythropoietin-induced signaling. Erythropoietin knockdown cells exhibit dramatically slower rates of growth, which could be restored by transfecting the cells with a murine erythropoietin gene. Taken together, our data suggest that the coordinated regulation of a functional erythropoietin/erythropoietin receptor axis in prostate cancer cells may be integral to the growth and progression of prostate cancer.

Human selenium binding protein-1 (hSP56) interacts with VDU1 in a selenium-dependent manner.

Reduced expression of the 56-kDa human selenium binding protein-1 (hSP56) has been reported in many types of human malignancies, including prostate, lung, ovarian, thyroid and colorectal cancers. hSP56 also has been implicated in selenium-dependent cell growth inhibition. However, the molecular basis of hSP56's function has not been elucidated. In the present study, we identified von Hippel-Lindau protein (pVHL)-interacting deubiquitinating enzyme 1 (VDU1) as a protein partner of hSP56 using a yeast two-hybrid screen. The interaction between hSP56 and VDU1 was confirmed by yeast two-hybrid analysis and in vitro binding experiments. hSP56 and VDU1 co-localized in the perinuclear region of LNCaP human prostate cancer cells. The full-length VDU1 specifically interacted with a selenium-replete form of hSP56. We also demonstrate stable incorporation of selenium into hSP56, in a mode distinct from conventional selenocysteine-containing selenoproteins. These findings suggest that hSP56 may play a role in ubiquitination/deubiquitination-mediated protein degradation pathways in a selenium-dependent manner.

Erythropoietin inhibits apoptosis induced by photodynamic therapy in ovarian cancer cells.

Recombinant human erythropoietin is widely used to treat anemia associated with cancer and with the myelosuppressive effects of chemotherapy, particularly platinum-based regimens. Erythropoietin is the principal regulator of erythroid cell proliferation, differentiation, and apoptosis. Recently, the antiapoptotic and proliferative effects of erythropoietin on nonhematopoietic cells were also established. We now show the effect of erythropoietin treatment on the response of A2780 and SKOV3 ovarian carcinoma cell lines to photodynamic therapy (PDT) using hypericin. SKOV3 exhibited an increased resistance to hypericin when cells were treated with erythropoietin. This resistance was reversed by treatment of SKOV3 cells with the specific Janus kinase 2 kinase inhibitor AG490 or the tyrosine kinase inhibitor genistein. These results support a role for the specific erythropoietin-induced Janus kinase 2/STAT signal transduction pathway in PDT resistance. Evidence of erythropoietin signaling was obtained by the demonstration of Akt phosphorylation in both A2780 and SKOV3 cells. Erythropoietin-treated SKOV3 cells exhibited decreased apoptosis induced by hypericin, an effect that was blocked by the phosphoinositide 3-kinase/Akt inhibitor wortmannin. These results may have important implications for ovarian cancer patients undergoing PDT and receiving erythropoietin.

The erythropoietin receptor in normal and cancer tissues.

The hormone erythropoietin (EPO) is essential for the survival, proliferation and differentiation of the erythrocytic progenitors. The EPO receptor (EPO-R) of erythrocytic cells belongs to the cytokine class I receptor family and signals through various protein kinases and STAT transcription factors. The EPO-R is also expressed in many organs outside the bone marrow, suggesting that EPO is a pleiotropic anti-apoptotic factor. The controversial issue as to whether the EPO-R is functional in tumor tissue is critically reviewed. Importantly, most studies of EPO-R detection in tumor tissue have provided falsely positive results because of the lack of EPO-R specific antibodies. However, endogenous EPO appears to be necessary to maintain the viability of endothelial cells and to promote tumor angiogenesis. Although there is no clinical proof that the administration of erythropoiesis stimulating agents (ESAs) promotes tumor growth and mortality, present recommendations are that (i) ESAs should be administered at the lowest dose sufficient to avoid the need for red blood cell transfusions, (ii) ESAs should not be used in patients with active malignant disease not receiving chemotherapy or radiotherapy, (iii) ESAs should be discontinued following the completion of a chemotherapy course, (iv) the target Hb should be 12 g/dL and not higher and (v) the risks of shortened survival and tumor progression have not been excluded when ESAs are dosed to target Hb <12 g/dL.

Erythropoietin treatment of human ovarian cancer cells results in enhanced signaling and a paclitaxel-resistant phenotype.

Erythropoietin (Epo), a glycoprotein hormone that is the principal regulator of erythropoiesis, is known to act also on nonhematopoietic cell types. Epo receptors have been reported on several normal and neoplastic human cells and tissues, including ovarian cancer cells. We found that long-term Epo treatment of A2780 cells resulted in the development of a phenotype exhibiting both enhanced Epo signaling, evidenced by increased peak levels of phospho-Erk1/2 and increased paclitaxel resistance. This phenotypic effect was specific for paclitaxel, since no change in cisplatin or carboplatin sensitivity was observed. In addition, the change in phenotype was stable, even after the removal of Epo. Measurement of mono- and oligonucleosome formation revealed that long-term Epo treated A2780 cells exhibited markedly less apoptosis than nonerythropoietin treated cells at essentially all concentrations of paclitaxel tested. Western blot analyses revealed that the long-term Epo treated cells had significantly reduced expression of apoptosis-related proteins Bcl-2 and Bcl-10. These findings may have implications for the clinical use of recombinant human Epo and other erythropoiesis stimulating agents to correct anemia in paclitaxel-treated cancer patients.

Characterization of erythropoietin receptor and erythropoietin expression and function in human ovarian cancer cells.

The identification of erythropoietin receptors (EpoR) on cancer cells has caused concern, since it implies the possibility that treatment of cancer patients with erythropoietin (Epo) and related agents with demonstrable antiapoptotic activity could enhance cancer growth and progression. However, the function and even the validity of the identification of these receptors have been called into question. We now report the characterization of EpoR and Epo expression by 4 human ovarian cancer cell lines: A2780, CaOV, SKOV and OVCAR-3. Using semiquantitative RT-PCR, restriction digestion of the PCR products and DNA sequence analysis, we determined that each of the lines expresses the EpoR and Epo at the mRNA level. A2780 cells were the highest expressers of both genes. We demonstrated EpoR protein both by western blotting and by immunofluorescence and biologically active Epo protein by quantitative in vitro bioassay. The EpoR on A2780 cells was shown to be functional, since Epo stimulation resulted in phosphorylation of Erk1/2, an important EpoR mitogenic signaling intermediate. None of the cell lines exhibited a growth response in culture to exogenous Epo. However, addition of a neutralizing anti-Epo antibody to A2780 cells resulted in partial growth inhibition that was reversed by the addition of excess Epo, providing evidence for an autocrine/paracrine mechanism of growth enhancement in these cells.

Does erythropoietin have a dark side? Epo signaling and cancer cells.

Erythropoietin (Epo) stimulates red blood cell production by docking with its cognate receptor on the erythroid progenitor cell and triggering an array of signaling pathways that inhibit apoptosis and promote cell proliferation and differentiation. In its pharmaceutical forms, epoetin and darbepoetin, Epo is widely used to treat various anemias, including those associated with cancer. The Epo receptor is also expressed by nonhematopoietic cells, including cancer cells, and Epo exhibits a "tissue-protective" effect on nonhematopoietic tissues, possibly mediated through a novel heteroreceptor, blocking apoptosis induced by a variety of insults. The unexpected results of several clinical studies in which Epo was used to treat cancer patients have now raised the question of a potential direct growth-promoting action of Epo on cancer cells.

Variability in the immunodetection of His-tagged recombinant proteins.

Labeling of recombinant proteins with polypeptide fusion partners, or affinity tagging, is a useful method to facilitate subsequent protein purification and detection. Poly-histidine tags (His-tags) are among the most commonly used affinity tags. We report strikingly variable immunodetection of two His-tagged recombinant human erythropoietins (Epo): wild type Epo (Epo(wt)) and Epo containing an R103A mutation (Epo(R103A)). Both were engineered to contain a C-terminal six residue His-tag. The cDNA constructs were stably transfected into Chinese hamster ovary (CHO) cells and COS-7 cells. Clones from the CHO cell transfections were selected for further characterization and larger-scale protein expression. Three chromatographic steps were utilized to achieve pharmacologically pure Epo. Conditioned media from the Epo-expressing cell lines and protein-containing samples from each step of purification were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and dot blot, using both monoclonal anti-human Epo antibody (AE7A5) and anti-His antibodies. While the successful incorporation of the His-tag into our constructs was confirmed by Epo binding to Ni(2+)- nitrilotriacetic acid resin and by microcapillary reverse-phase high-performance liquid chromatography nano-electrospray tandem mass spectrometery amino acid sequencing, the levels of immunodetection of His-tagged protein varied markedly depending on the particular anti-His-tag antibody used. Such variability in His-tag immunorecognition can lead to critical adverse effects on several analytical methods.

Erythropoietin stimulates growth and STAT5 phosphorylation in human prostate epithelial and prostate cancer cells.

BACKGROUND: Erythropoietin (Epo), the principal regulator of erythroid progenitor survival, growth, and differentiation, initiates its action by binding to its cognate cell surface receptor (EpoR). EpoR have been identified on a variety of non-hematopoietic cells, both normal and malignant, however, little is known about the function of EpoR on malignant cells. METHODS: RT-PCR, Western blotting, and immunohistochemistry were used to demonstrate that prostate cancer cells express EpoR at both the gene and protein level. Cell proliferation assays and STAT5 phosphorylation were used to demonstrate Epo's mitogenic action and intracellular signaling, respectively. RESULTS: We have demonstrated that transformed prostate epithelial and prostate cancer cell lines, as well as primary prostate tissue, express the EpoR. Importantly, the EpoR on prostate cells are functional, as demonstrated by the observation that each of the cell lines exhibited a dose-dependent proliferative response to Epo, and that Epo triggered STAT5b phosphorylation in the cells. CONCLUSION: Human prostatic epithelial cells and prostate cancer cells express functional EpoR, and Epo serves as a growth factor for these cells. These results have implications for our understanding of normal prostatic growth and development and of the pathobiology of human prostate cancer.

Apoptosis-linked gene-2 connects the Raf-1 and ASK1 signalings.

Raf-1 plays important roles in cell proliferation, differentiation, and survival. However, the unique and essential function of Raf-1 is anti-apoptotic. The molecules that mediate Raf-1's anti-apoptotic function are not known. In the course of identifying new substrates of Raf-1, we found that the Raf-1 kinase domain interacted with apoptosis-linked gene-2 (ALG-2) in yeast two-hybrid system. Our further studies showed that Raf-1 phosphorylated ALG-2 in an in vitro kinase assay. We also found that apoptosis signal-regulating kinase 1 (ASK1) strongly phosphorylated ALG-2. Importantly, Raf-1 blocks the ASK1-dependent ALG-2 phosphorylation. Since ALG-2 associates with ASK1, and both ASK1 and ALG-2 are involved in apoptosis, our observations indicate that Raf-1 may mediate its anti-apoptotic function by interrupting ASK1-dependent phosphorylation of ALG-2.

Erythropoietin regulation of Raf-1 and MEK: evidence for a Ras-independent mechanism.

Stimulation of the erythropoietin (EPO) receptor triggers a cascade of signaling events. We reported that EPO upregulates c-myc expression through 2 pathways in BaF3-EpoR cells--a phosphatidylinositol 3-kinase (PI3K) pathway operating on transcriptional initiation and a Raf-1-mitogen-activated protein kinase (MAPK) pathway affecting elongation. We now show that EPO induces phosphorylation of Raf-1 at serine 338 and within the carboxy-terminal domain, resulting in an electrophoretic mobility change (hyperphosphorylation). Importantly, MEK 1 inhibitor PD98059 blocked only the hyperphosphorylation of Raf-1 but not the phosphorylation at serine 338. This inhibition of Raf-1 hyperphosphorylation resulted in increased kinase activity of Raf-1 and increased phosphorylation of MEK, suggesting that the hyperphosphorylation of Raf-1 inhibits its MEK kinase activity. Deletion of the first 184 amino acids of Raf-1, which are involved in its interaction with Ras, had no effect on EPO-induced phosphorylation. Introducing the dominant-negative N17Ras or GAP had no effect on EPO-induced kinase activity of Raf-1 and ELK activation. N17Ras failed to inhibit ELK activation in another cell line-Rauscher murine erythroleukemia- which expresses the EPO receptor endogenously and differentiates in response to the hormone. These results indicate the presence of a Ras-independent mechanism for Raf-1 and MEK activation in these cells.