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First Published Online July 2, 2008
The Oncologist, Vol. 13, No. 7, 761-768, July 2008; doi:10.1634/theoncologist.2008-0110
© 2008 AlphaMed Press

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Breast Cancer

Erythropoietin and Its Receptor in Breast Cancer: Putting Together the Pieces of the Puzzle

Ferdinando Mannello, Gaetana A. M. Tonti

Department of Biomolecular Sciences, Section of Clinical Biochemistry, University "Carlo Bo," Urbino, Italy

Key Words. Erythropoietin • Erythropoietin receptor • Breast cancer • JAK2 signaling • Epo–EpoR homo- and heterodimeric complex • Cancer-related anemia

Correspondence: Ferdinando Mannello, Ph.D., D.Sc., Sezione di Biochimica Clinica del Dipartimento di Scienze Biomolecolari, Università Studi "Carlo Bo," Via O. Ubaldini 7, 61029 Urbino (PU), Italy. Telephone:39-0722-351479; Fax: 39-0722-322370; e-mail: ferdinando.mannello{at}uniurb.it

Received May 5, 2008; accepted for publication June 5, 2008; first published online in THE ONCOLOGIST Express on July 2, 2008.

Disclosure: The content of this article has been reviewed by independent peer reviewers to ensure that it is balanced, objective, and free from commercial bias. No financial relationships relevant to the content of this article have been disclosed by the authors, planners, independent peer reviewers, or staff managers.


   ABSTRACT
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 Abstract
State of the Art
 Contrasting Findings on the...
 Translational and Clinical...
 Trying to Assemble the...
 Future Expectations
 Author Contributions
 Acknowledgments
 References
 
The expression of erythropoietin (Epo) and the Epo receptor (EpoR) has been detected in healthy tissue as well as in a variety of human cancers, including breast. Functional Epo/EpoR signaling in cancer cells, which contributes to disease initiation/progression, is not completely straightforward and is difficult to reconcile with the clinical practice of preventing/treating anemia in cancer patients with recombinant Epo. Preclinical and clinical investigations have provided contrasting results, ranging from a beneficial role that improves the patient's overall survival to a negative impact that promotes tumor growth progression. A careful gathering of Epo/EpoR biomolecular information enabled us to assemble an unexpected jigsaw puzzle which, via distinct JAK-dependent and JAK-independent mechanisms and different internalization/recycling as well as ubiquitination/degradation pathways, could explain most of the controversies of preclinical and clinical studies. However, until the mechanisms of the contrasting literature data are resolved, this new point of view may shed light on the Epo/EpoR paracrine/autocrine system and function, providing a basis for further studies in order to achieve the highest possible benefit for cancer patients.


   STATE OF THE ART
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 Abstract
 State of the Art
Contrasting Findings on the...
 Translational and Clinical...
 Trying to Assemble the...
 Future Expectations
 Author Contributions
 Acknowledgments
 References
 
Erythropoietin (Epo) is a glycoprotein hormone that serves as the primary regulator of erythropoiesis (by stimulating growth, preventing apoptosis, and inducing the differentiation of RBC precursors) [1]; it also has been localized in several nonhematopoietic tissues and cells. In humans Epo mRNA encodes a protein with 193 amino acids (aa). After cleavage of the signal peptide and post-translation modification, the mature protein consists of a 165-aa structure; while the O-linked sugar has no important function, the N-linked sugars are necessary for stability of the Epo molecule in the circulation. Erythropoietin receptor (EpoR) is a type-1, single-transmembrane receptor that is expressed in several forms in erythropoietic progenitor cells, including a full-length form (484 aa), a truncated form (303 aa), and a soluble form (115 aa). The truncated and soluble forms contain the extracellular Epo-binding domain, but alternative splicing of transcripts truncates the cytoplasmic or transmembrane domains. While truncated EpoR has sustained erythropoiesis with attenuated function, soluble EpoR may act as an antagonist by competing with the full-length EpoR form for binding to Epo. However, the physiological roles for these EpoR variants have not been established [2]. Epo exerts its effects via a hormonal mode, by inducing homodimerization of two molecules of the EpoR on the cell surface (Fig. 1A), which initiates the Janus kinase (JAK)2/signal transducer and activator of transcription 5 (STAT5) signal transduction cascade. After Epo–EpoR complex formation, activated JAK2 phosphorylates the EpoR and several proteins leading to their activation, translocation to the nucleus, binding to specific regulatory sequences, and activation of the transcription of target genes, resulting in erythroid proliferation and differentiation [3]. The EpoR belongs to the cytokine receptor superfamily [4]; it is present in many tissues as a novel cell surface receptor complex, comprising one class of EpoR subunit and a pair of β-common receptor (β-CR) subunits [3]. The heterodimer receptor hypothesis was first described in 1996 [5], suggesting that the EpoR is switched on and transduces signals to the interior of the cell by the formation of homo- or hetero-oligomers (dimers or trimers) sharing a common receptor subunit. The EpoR heterocomplex has been found in several mouse tissues and in the neural-like P19 cell line [6, 7], demonstrating the presence of a tissue-protective heteroreceptor; on the other hand, in the neuroblastoma SH-SY5Y and pheochromocytoma PC12 cell lines [8], Epo exerts its function through the "classical" homodimeric EpoR with no evidence of heterocomplex. The heterocomplex is involved in the Epo autocrine/paracrine signaling arising from local injury or disease processes (including cancer) (Fig. 1B). Following the formation of the heterocomplex, activated JAK2 molecules trigger a phosphorylation cascade leading to the activation of the transcription of not yet fully identified target genes. While phosphorylation activates EpoR signaling, dephosphorylation downregulates this activity, inhibiting proliferative signaling of the EpoR, reducing the STAT5 transduction signal, and finally promoting EpoR degradation. The interaction between Epo and the EpoR–β-CR heterocomplex confers tissue protection through pleiotropic functions in many nonhematopoietic tissues, even though further studies have reported contrasting findings [7]. The direct in vivo effects of Epo–EpoR signaling on cellular proliferation, tumor oxygenation, apoptosis, tumor angiogenesis, metastasis, and sensitivity to chemoradiation therapy remain to be identified [9]. The biology of the EpoR is further complicated by several processes; in particular, the translocation of EpoR to the cell surface is an inefficient mechanism, resulting in <1% of total cellular full-length EpoR molecules reaching the cell surface of hematopoietic cells. This is a consequence of the short half-life of the EpoR protein (1–2 hours), inefficient processing for surface expression, and protein degradation within the endoplasmic reticulum, proteasome, and lysosomes. Moreover, accessory factors that are required for EpoR trafficking to the surface also may be at limiting concentrations (e.g., only Epo-dependent hematopoietic cell lines express surface receptors through the accessory protein JAK2, which binds EpoR in the endoplasmic reticulum, induces correct protein folding, promotes surface expression, and is essential for EpoR signaling) [10].


Figure 1
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  		Figure 1. Model for the assembly and activation of the Epo–EpoR complex. Multiple lines of evidence indicate that the hormonal and autocrine/paracrine modes of Epo action (erythropoietic and tissue functions, respectively) are mediated through different receptors with nonoverlapping activities. Two modes of action of Epo and its binding to homo- and heterodimers of EpoR are depicted. (A): Epo regulates the RBC mass in a negative feedback control fashion that is characteristic of an endocrine hormone. The erythropoietic response to Epo is fostered through activation of the EpoR and downstream signal transduction pathways. Because EpoR does not possess endogenous tyrosine kinase activity, the Epo-induced phosphorylation cascade is initiated by JAK2, which is associated with the intracellular domain of the EpoR. Upon binding to preformed EpoR homodimer, Epo causes activation of JAK2 kinases through transphosphorylation. Activated JAK2 induces phosphorylation of tyrosine sites of the EpoR, providing docking sites for multiple intracellular signaling proteins. The major pathways that facilitate the hematopoietic response to Epo are the STAT5, MAPK, and PI3K pathways. The first of the signaling proteins activated is STAT5, which dissociates from the EpoR, translocates into the nucleus where it activates its target genes; in this way, activation of STAT5 appears to mediate the Epo antiapoptotic effects and hemoglobin synthesis. (B): Unlike erythropoiesis, the Epo in human tissues operates via an autocrine/paracrine mode. Tissue injury, local inflammation, hypoxia, and metabolic stress induce the synthesis of HIF, which increases the production of Epo locally; the local synthesis of Epo is also enhanced in cancer conditions. The tissue autocrine/paracrine mode of action does not depend on activation of the EpoR homodimer, but uses a distinct receptor complex that includes the EpoR and the dimer of the β-CR (also known as CD131). Activation of the tissue EpoR heterocomplex requires high concentrations of, but brief exposure to, Epo (which may be achieved locally after tissue damage or inflammation or cancer), because its binding affinity is much lower than the blood circulating concentration of Epo. The interaction between the intracellular domain of the EpoR and β-CR subunit leads to activation of multiple downstream signaling pathways with subsequent transcriptional activation of numerous genes (some of which are associated with antiapoptotic and mitogenic endpoints, while others are to be discovered). The signaling cascades, JAK2/STAT5/MAPK/PI3K/Akt, that are involved in erythropoiesis are also implicated in tissue Epo function through the EpoR–β-CR complex, but further signaling pathways (known only in part) are also activated (e.g., NF-{kappa}B, IAP, HSP70). The relative contributions of the two receptors, that is, the EpoR homodimer and the EpoR–β-CR heterodimer, in the stimulation of the growth of different tumor types by Epo remain to be defined.

Abbreviations: β-CR, β-common receptor; Epo, erythropoietin; EpoR, erythropoietin receptor; HIF, hypoxia-inducible factor; HSP70, heat shock protein 70; IAP, inhibitor of apoptosis; JAK2, Janus kinase 2; MAPK, mitogen-activated protein kinase; NF-{kappa}B, nuclear factor kappa B; PI3K, phosphoinositide-3 kinase; STAT5, signaling transducer and activator of transcription 5.

 
Although literature data seem apparently in contrast, the presence of two conformational models (the classical homodimer and the hetero-oligomer) may explain the pleiotropic multifaceted functions of the Epo–EpoR complex.


   CONTRASTING FINDINGS ON THE EPO–EPOR COMPLEX
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 State of the Art
 Contrasting Findings on the...
Translational and Clinical...
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Recent evidence raised the suspicion that Epo used to treat anemia during cancer [11] might reduce, via EpoR, patient survival, promoting tumor proliferation and angiogenesis [12, 13].

Following the recent debate on the evidence of the negative impact of Epo (adversely affecting prognosis in cancer patients with EpoR-positive cancer cells) [14–16], it is crucial to focus attention on Epo-modulating mechanism(s) via the EpoR, trying to explain, through an alternative biomolecular point of view, the "apparently" contradictory results obtained in breast cancer (BC).

Tumor cell lines and tumor tissues derived from neoplasia of the breast are capable of transcribing the EpoR gene, indicating that this expression has a potential role in tumor progression and showing, in some cases, a correlation between EpoR transcripts and immunostaining of the EpoR protein [17, 18]. However, several literature data highlight the discrepancies between no EpoR transcripts and the presence of the EpoR protein; the methods used in most molecular studies do not distinguish between the different splice variants of the EpoR, thus not specifying whether functional EpoR is expressed in cells. This is a crucial matter because high levels of alternatively spliced transcripts that encode attenuated (truncated form) or antagonistic (soluble form) EpoR form have been reported in breast tumor cells [19]. Moreover, the discrepancies between negative molecular and positive histochemical results may also be a result of the lack of specificity of the anti-EpoR antibody used in such studies. While the reported sizes of the EpoR using commercial anti-EpoR antibodies are in the range of 66–78 kDa, the calculated size of the EpoR protein is 53 kDa, and maturation with the addition of carbohydrate could increase the size to approximately 57–59 kDa (size was also determined and confirmed by protein microsequencing) [20]. The finding of crossreactivity to non-EpoR proteins was not surprising, because the various antibody preparations contain polyclonal antipeptide antibodies.

Epo, the principal hematopoietic growth factor regulating cellular proliferation and differentiation along the erythroid lineage [1], was recently recognized as a pleiotropic cytokine exerting broad effects in both physiologic and pathologic conditions in diverse nonhematopoietic tissues [9, 21]. Human breast tissues have been found to express both Epo and the EpoR, at the protein and mRNA levels. In particular, in the physiologic condition, both ductal and lobular epithelial cells contain both Epo and EpoR showing weak granular cytoplasmic localization [17, 22–24]. Epo was increased in lobules with secretory changes [17] and was constitutively found in milk [22, 25] and in nonlactating breast secretions (i.e., nipple aspirate fluid) [25]; Epo labeling was also found in nontumoral cells of peritumoral hyperplastic ducts [24] and in biosynthetically active apocrine cells sloughed from ducts [25]. In the BC condition, contrasting results about EpoR cell localization were found; in fact, diffuse, moderate-to-strong cytoplasmic and membrane-bound EpoR protein expression was found in BC tissues [16–18, 24, 26], whereas in BC cell lines it showed mainly a cytosolic distribution [9, 25, 27]. Contrasting findings of the cytoplasmic and membrane-bound localization of EpoR have recently raised a strong literature debate [28]; the high batch-to-batch variability and the low specificity/affinity of polyclonal antibody for the EpoR protein generated serious doubts on the histochemical identification of EpoR in cancer cells, making tricky the understanding of the Epo–EpoR signaling mechanism with endogeneous and exogenous Epo [29, 30]. Opposite opinions about the use of a polyclonal EpoR antibody (from a lack of specificity/sensitivity [20, 28] to an excellent sensitivity [31]) require the identification of the EpoR protein by new specific antibodies [32].

In addition to the debate on Epo and EpoR characterization, an alternative biomolecular point of view might help to explain why EpoR is found both in the cytoplasm and bound to membrane [28], and to clinically explain why Epo may predict a negative impact on cancer control [16, 30], suggesting physicians take into consideration the balance of the potential beneficial effects of Epo against a possible negative impact of this cytokine on a case-by-case basis [15, 16].


   TRANSLATIONAL AND CLINICAL RESEARCH: STARTING PIECES OF THE PUZZLE
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 State of the Art
 Contrasting Findings on the...
 Translational and Clinical...
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 Author Contributions
 Acknowledgments
 References
 
A more careful examination of experimental/clinical data from hematopoietic cells and their translation into nonhematopoietic tissue pathophysiology may help to explain the mechanism(s) involving the Epo–EpoR signaling axis, providing an explanation for the "apparently" contrasting results reported in the literature, in particular with respect to BC.

As starting pieces of the jigsaw, two EpoR types have been identified: a "classic" homodimeric protein complex mainly characterized in the erythroid lineage [33] (but also recently found in nonhematopoietic cells [8]) and a "novel" (not as well known) heterodimer, composed of one EpoR monomer combined with the β-CR subunit (also known as CD131) [2, 3, 7], which has up to now been identified only in extrahematopoietic tissues [7, 34]. The existence of diverse EpoR forms may be responsible for the different (in some cases contrasting) extrahematopoietic biological activities of Epo [1, 9, 30, 34, 35]. Another biological factor increasing the complexity of Epo–EpoR axis signaling is the different protein glycosylation; in fact, the presence of Epo variants (including asialo- and carbamylated Epo, which retain protective effects in nonhematopoietic tissues while exhibiting no effect on hematopoietic cells) [36, 37] has suggested a mechanistic difference in Epo-mediated cellular signaling through N-linked carbohydrates [38] in several tissues, including breast [39].

Particular attention has been focused on the expression and localization of Epo and its receptor in breast cells and tissues during physiologic and pathologic conditions. In fact, the Epo protein was characterized as an intracellular glycoprotein in BC cell lines and tissues [17–19, 23–26, 40], and additionally found in breast secretions (i.e., milk and nipple aspirate fluid) [22, 25]. Furthermore, biomolecular studies report that EpoR mRNA and protein were identified in BC cell lines and tumor tissues [18, 19, 23, 27, 40], whereas immunohistochemical studies localized EpoR in the cell cytoplasm [18, 23, 24, 26] and also in tumor tissues as membrane-bound protein [16, 17, 24, 26]. Interestingly, it has been demonstrated that BC cell lines may secrete the soluble fragment of EpoR peptide in conditioned medium, which may compete with membrane-bound receptor for ligand binding [19].


   TRYING TO ASSEMBLE THE JIGSAW PUZZLE PIECES FOR BREAST TISSUE
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 Contrasting Findings on the...
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Analyzing the ubiquitination, internalization, and degradation model of Epo and its receptor in hematopoietic cells [41–43], at least two mechanisms may justify the confounding results obtained in breast cells and tissues. Let's put together the "scattered" pieces of Epo and EpoR findings in breast through (a) JAK-dependent ubiquitination/degradation and (b) JAK-independent internalization-recycling mechanisms.

In the first JAK-dependent mechanism, the localization of the EpoR on the cell surface is regulated by physiologic gene transcription, Golgi trafficking, and heterodimeric complex assembly (Fig. 2). After Epo binding, the main processes are linked to the ubiquitination of EpoR and the lysosomal degradation of the complex that do not allow EpoR and Epo to recycle back to the cell surface, leading to a transient downregulation of EpoR as a result of Epo binding, according to the literature data [41, 42]. In fact, after Epo binding, the EpoR undergoes dimerization (in both homo- and heterodimeric complexes) and auto- or transphosphorylation of the Janus family kinase. In conjunction with other kinases, JAK2 phosphorylates several tyrosine residues in the EpoR, creating docking sites for the SH2 domains of several signal transduction proteins (e.g., STAT5 and other signaling proteins become phosphorylated and activated, promote intracellular signaling, move to the nucleus, and activate gene expression) [33]. The JAK2-induced tyrosine-phosphorylated form of EpoR is rapidly ubiquitinated [44], an important step both for the proteasomal degradation of its intracellular domain (preventing further signal transduction) and for the targeting and routing of Epo and EpoR to lysosomes [41, 43]. The proteasomal cleaved EpoR (still complexed with Epo protein) may be internalized (probably by a clathrin/caveolae-dependent mechanism) and may follow two fates: (a) lysosome targeting for the final proteolytic degradation of both Epo and cleaved EpoR [42] and (b) endosome formation for the extracellular secretion of the soluble form of cleaved EpoR and Epo. This possible JAK2-dependent mechanism is in agreement with several literature data on breast tissues and cell lines: (a) Epo treatment strongly enhances intracellular phosphotyrosine levels [23], (b) Epo binding activates EpoR phosphorylation [45], and (c) Epo induces phosphorylation of Akt, mitogen-activated protein kinase (MAPK), and extracellular signal–related kinase (ERK) [46]. Moreover, the possibility that ubiquitinated and proteosomal-degraded EpoR may maintain Epo-binding capability but not activate further intracellular signal transduction is in agreement with literature data described in EpoR-positive BC cells and tissues: (a) ubiquitinated EpoR bound to Epo does not activate MAPK, Akt, or STAT5 signaling [27, 47] and (b) proteosomal-cleaved EpoR may be secreted in the extracellular milieu as a 26-kDa soluble fragment of EpoR, still able to compete with membrane-bound receptor for ligand binding, decreasing receptor-mediated signal generation [19].


Figure 2
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  		Figure 2. Model for JAK2-dependent mechanism of EpoR–β-CR heterodimer complex in breast tissue autocrine/paracrine Epo function. Binding of Epo (produced in high amounts by inflamed or cancer breast tissue) induces the formation of the heterodimer receptor complex and auto- or transphosphorylation of JAK2 that, in conjunction with other kinases, phosphorylates several tyrosine residues in the EpoR heterocomplex, creating docking sites for several signal transduction proteins, including STAT5. Once STAT5 and other signaling proteins become phosphorylated and activated, they dissociate from the heteroreceptor and move to the nucleus, activating gene expression. After Epo binding and EpoR tyrosine phosphorylation, the EpoR–β-CR heterodimer complex is rapidly ubiquitinated at the cell surface; this process necessarily requires JAK2 activation, but up to now the E3 ligase proteins responsible for Epo heterodimer ubiquitination have not been identified. Polyubiquitination of the EpoR–β-CR heterodimer complex is probably responsible for its proteolysis by the proteasomes, which removes a significant part (more than half) of the intracellular domain. Because tyrosine residues are located in the cytoplasmic tail of the EpoR, this mechanism could hasten the desensitization process, blocking the downstream signaling cascade. As known in the literature, the proteasomal degradation of the intracellular domain of β-CR is necessary for its internalization and lysosome routing, suggesting ubiquitination as a possible control for Epo and EpoR targeting to the lysosome. After internalization (which probably occurs via clathrin- or caveolae-dependent mechanism), both Epo and the EpoR–β-CR complex are degraded by the lysosomes; and the few heterodimer EpoR complexes are disassembled and recycled back to the membrane, allowing extracellular secretion of soluble fragments of Epo and EpoR, which are still able to compete with membrane-bound receptor for ligand binding, decreasing receptor-mediated signal generation.

Abbreviations: β-CR, β-common receptor; Epo, erythropoietin; EpoR, erythropoietin receptor; JAK2, Janus kinase 2; STAT5, signaling transducer and activator of transcription 5; Ub, ubiquitination.

 
For what concerns the second JAK-independent mechanism (Fig. 3), after Epo binding, the heterodimeric EpoR complex may be efficiently internalized without either the binding/activation/phosphorylation of JAK2 or the ubiquitination process, thus not involving the protein kinase cascade/network [41, 48]. The internalization process of the Epo–EpoR complex probably occurs either by conformational changes induced by Epo binding or by caveolae/clathrin-dependent pathway(s). The internalization of the Epo–EpoR complex has two main hypothetical fates: (a) recycling back to the cell surface of Epo–EpoR (in a homo- or heterodimeric complex) and (b) endosome formation, which may, in turn, lead to (i) Epo extracellular secretion with no glycolytic or proteolytic processing, (ii) accumulation of intracellular Epo and EpoR, or (iii) Epo and EpoR degradation in lysosomes. This JAK2-independent internalization/trafficking/recycling mechanism is in agreement with the literature about the concomitant localization of both intracellular and membrane-bound EpoR [17–19, 23, 24, 26, 27, 40], the intracellular localization of the Epo protein [17–19,23–26,49,50], the lack of Akt and STAT5 activation after Epo binding [27, 47], Epo secretion in extracellular fluids (like in milk [22, 25] and nipple aspirate fluid [25]), and the identification of Epo as a protein not undergoing glycolytic processing [9, 17, 18, 22, 25, 51].


Figure 3
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  		Figure 3. Model for JAK2-independent mechanism of EpoR–β-CR heterodimer complex in breast tissue autocrine/paracrine Epo function. Evidence in the literature shows that the internalization and subsequent intracellular fates of the EpoR heterocomplex are strongly increased by ligand binding; EpoR–β-CR heterodimer complex unable to bind and activate JAK2 was efficiently internalized after Epo binding, indicating that JAK2 activation was dispensable for EpoR heterocomplex internalization. Because EpoR heterocomplex ubiquitination was inhibited when JAK2 activity was blocked, EpoR–β-CR heterodimer complex ubiquitination is not required for internalization, suggesting that Epo binding to its heteroreceptor induces a conformational change that is sufficient to promote EpoR–β-CR heterodimer complex internalization. No data are available yet to exclude a clathrin- or caveolae-dependent mechanism of internalization. The Epo–EpoR–β-CR complex, efficiently internalized and neither phosphorylated nor ubiquitinated, may follow two different routes: the complexes may either easily recycle to the cell surface in a nondegraded form or be included in endosomes, which may guide the release of soluble Epo and EpoR fragments into the extracellular milieu, their intracellular accumulation, or the lysosomal degradation by several cathepsins; in fact, lysosome inhibitors protected internalized Epo from degradation, indicating that Epo is degraded in the lysosomes mainly by cathepsin B, whereas blocking proteasome activity did not inhibit Epo lysosomal degradation.

Abbreviations: β-CR, β-common receptor; Epo, erythropoietin; EpoR, erythropoietin receptor; JAK2, Janus kinase 2.

 

   FUTURE EXPECTATIONS
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Several studies have described EpoR expression in tumors and have assumed a negative impact on tumor progression and survival. At best, the poorer overall survival linked to the use of Epo in some clinical trials or in preclinical studies is only a theory that has not yet been well established, so perhaps the issue of tumor progression associated with erythropoiesis-stimulating agents should not be definitive and should be cautiously considered. The recent debate about the Epo–EpoR signaling axis in cancer (in particular in BC patients treated with Epo for cancer-related anemia) [12, 13] has raised the need on one side to understand the presence and function of different EpoR forms on cancer cells [2, 3, 52] and on the other side to take into consideration that Epo may impair, not improve, cancer survival [16, 30, 52]. Even though many of the findings and conclusions of this matter are questionable because of problems with the methods used to detect the EpoR protein and to identify the spliced variants of the EpoR gene, the lack of appropriate controls, and the lack of detection of physiologically relevant surface EpoR on tumor cells, the fact that the erythropoietic and tissue effects of Epo are fostered through two distinct receptors (a homo- and heterodimer, respectively) with nonoverlapping functions raises the possibility of selective targeting of these activities by appropriate modulation of the two receptor systems. In fact, there is evidence that seems paradoxical: for tumor cells responding to erythropoiesis-stimulating agents, either the response was marginal and/or high levels were required to evoke a biological response; in contrast, many tumor cells expressing EpoR do not respond to erythropoiesis-stimulating agents. These results may reflect a possible absence of intracellular signaling after ligand–receptor interaction, low EpoR density, or nonfunctional EpoR at the cell surface in tumor cells. Concerning human breast tissue, besides the importance of the discovery of agents with selective tissue-protective and health-enhancing properties, the characterization of separate regions of the Epo molecule as well as the study of modified Epo protein (e.g., carbamylated, asialic, and ipersialic Epo) in conjunction with a deeper knowledge of the EpoR–β-CR heteroreceptor may help us to understand the real beneficial abilities of Epo therapeutically. Moreover, the pharmacodynamics differences between homodimer EpoR and EpoR–β-CR heterocomplex (the low-dose but sustained presence of Epo for EpoR in the erythropoietic response and the high-dose but brief exposure of Epo for tissue autocrine/paracrine functionality) can also be used to design or identify agents that selectively modulate these two responses. Although the hypothetical mechanisms proposed here need to be supported/validated, they may project a different glimpse on all of the jigsaw pieces accumulated in the BC field, without either over- or underestimating any literature data. The two proposed biomolecular mechanisms (JAK-dependent and JAK-independent Epo–EpoR processing), in conjunction with the diverse biological availability of activated or cleaved EpoR, could provide an explanation why either endogenously produced (such as in inflammation and in cancer) or exogenously administered (such as in cancer-related anemia) Epo may promote, in some instances, tumor cell proliferation in BC-initiating cells [16], whereas, in other instances, it leads to better survival in BC patients [27]. Although our hypothesis actually provides more questions than answers, until the mechanisms of the contrasting literature data are resolved, the use and risks of Epo therapy should be carefully weighed, balancing the potential beneficial functions of Epo protecting tissues and ameliorating various anemias (including those associated with cancer) [11] against its detrimental effects, mainly linked to the tumor-promoting activity of Epo, a centenarian molecule yet to be fully disclosed [1, 30, 53, 54].


   AUTHOR CONTRIBUTIONS
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Conception/design: Ferdinando Mannello

Collection/assembly of data: Gaetana A. M. Tonti

Data analysis and interpretation: Ferdinando Mannello, Gaetana A. M. Tonti

Manuscript writing: Ferdinando Mannello, Gaetana A. M. Tonti

Final approval of manuscript: Ferdinando Mannello, Gaetana A. M. Tonti


   ACKNOWLEDGMENTS
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This work was supported in part by Research Grant Award 2007 to F. M. from the Dr. Susan Love Research Foundation, Pacific Palisades, CA.


   REFERENCES
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  1. Jelkmann W. Erythropoietin after a century of research: Younger than ever. Eur J Haematol 2007;78:183–205.[CrossRef][Medline]
  2. Sinclair AM, Todd MD, Forsythe K et al. Expression and function of erythropoietin receptors in tumors: Implications for the use of erythropoiesis-stimulating agents in cancer patients. Cancer 2007;110:477–488.[CrossRef][Medline]
  3. Konstantinopoulos PA, Karamouzis MV, Papavassiliou AG. Selective modulation of the erythropoietic and tissue-protective effects of erythropoietin: Time to reach the full therapeutic potential of erythropoietin. Biochim Biophys Acta 2007;1776:1–9.[Medline]
  4. Farrell F, Lee A. The erythropoietin receptor and its expression in tumour cells and other tissues. The Oncologist 2004;9(suppl 5):18–30.[Abstract/Free Full Text]
  5. Yoshimura A, Arai K. Physician education: The erythropoietin receptor and signal transduction. The Oncologist 1996;1:337–339.[Free Full Text]
  6. Jubinsky PT, Krijanovski OI, Nathan DG et al. The beta chain of the interleukin-3 receptor functionally associates with the erythropoietin receptor. Blood 1997;90:1867–1873.[Abstract/Free Full Text]
  7. Brines M, Grasso G, Fiordaliso F et al. Erythropoietin mediates tissue protection through an erythropoietin and common β-subunit heteroreceptor. Proc Natl Acad Sci U S A 2004;101:14907–14912.[Abstract/Free Full Text]
  8. Um M, Gross AW, Lodish HF. A "classical" homodimeric erythropoietin receptor is essential for the antiapoptotic effects of erythropoietin on differentiated neuroblastoma SH-SY5Y and pheochromocytoma PC-12 cells. Cell Signal 2007;19:634–645.[CrossRef][Medline]
  9. Hardee ME, Arcasoy MO, Blackwell KL et al. Erythropoietin biology in cancer. Clin Cancer Res 2006;12:332–339.[Abstract/Free Full Text]
  10. Huang LJ, Constantinescu SN, Lodish HF. The N-terminal domain of Janus kinase 2 is required for Golgi processing and cell surface expression of erythropoietin receptor. Mol Cell 2001;8:1327–1338.[CrossRef][Medline]
  11. Goldrich A, Olivotto IA, Alexander CS et al. Anemia is a common but neglected complication of adjuvant chemotherapy for early breast cancer. Curr Oncol 2007;14:227–233.[CrossRef][Medline]
  12. Lappin TR, Maxwell AP, Johnston PG. Warning flags for erythropoiesis-stimulating agents and cancer-associated anemia. The Oncologist 2007;12:362–365.[Free Full Text]
  13. Blau CA. Erythropoietin in cancer: Presumption of innocence? STEM CELLS 2007;25:2094–2097.[CrossRef][Medline]
  14. Henke M, Mattern D, Pepe M et al. Do erythropoietin receptors on cancer cells explain unexpected clinical findings? J Clin Oncol 2006;24:4708–4713.[Abstract/Free Full Text]
  15. Henke M, Mattern D, Pajonk F. In reply [letter]. J Clin Oncol 2007;25:1815.[Free Full Text]
  16. Phillips TM, Kim K, Vlashi E et al. Effects of recombinant erythropoietin on breast cancer-initiating cells. Neoplasia 2007;9:1122–1129.[CrossRef][Medline]
  17. Acs G, Zhang PJ, Rebbeck TR et al. Immunohistochemical expression of erythropoietin and erythropoietin receptor in breast carcinoma. Cancer 2002;95:969–981.[CrossRef][Medline]
  18. Arcasoy MO, Amin K, Karayal AF et al. Functional significance of erythropoietin receptor expression in breast cancer. Lab Invest 2002;82:911–918.[Medline]
  19. Arcasoy MO, Jiang X, Haroon ZA. Expression of erythropoietin receptor splice variants in human cancer. Biochem Biophys Res Commun 2003;307:999–1007.[CrossRef][Medline]
  20. Elliott S, Busse L, Bass MB et al. Anti-Epo receptor antibodies do not predict Epo receptor expression. Blood 2006;107:1892–1895.[Abstract/Free Full Text]
  21. Lappin TR, Maxwell AP, Johnston PG. EPO's alter ego: Erythropoietin has multiple actions. STEM CELLS 2002;20:485–492.[CrossRef][Medline]
  22. Juul SE, Zhao Y, Dame JB et al. Origin and fate of erythropoietin in human milk. Pediatr Res 2000;48:660–667.[Medline]
  23. Acs G, Acs P, Beckwith SM et al. Erythropoietin and erythropoietin receptor expression in human cancer. Cancer Res 2001;61:3561–3565.[Abstract/Free Full Text]
  24. Pelekanou V, Kampa M, Kafousi M et al. Erythropoietin and its receptor in breast cancer: Correlation with steroid receptors and outcome. Cancer Epidemiol Biomarkers Prev 2007;16:2016–2023.[Abstract/Free Full Text]
  25. Mannello F, Fabbri L, Ciandrini E et al. Enhanced levels of erythropoietin in nipple aspirate fluid and in ductal cells from breast cancer patients. Cell Oncol 2008;30:51–61.[Medline]
  26. Wincewicz A, Sulkowska M, Koda M et al. STAT3, HIF-1{alpha}, EPO and EPOR-signaling proteins in human primary ductal breast cancers. Folia Histochem Cytobiol 2007;45:81–86.[Medline]
  27. LaMontagne KR, Butler J, Marshall DJ et al. Recombinant epoetins do not stimulate tumor growth in erythropoietin receptor-positive breast carcinoma models. Mol Cancer Ther 2006;5:347–355.[Abstract/Free Full Text]
  28. Della Ragione F, Cucciolla V, Borriello A et al. Erythropoietin receptors on cancer cells: A still open question [letter]. J Clin Oncol 2007;25:1812–1813; author reply 1815.[Free Full Text]
  29. Österborg A, Aapro M, Cornes P et al. Preclinical studies of erythropoietin receptor expression in tumour cells: Impact on clinical use of erythropoietic proteins to correct cancer-related anaemia. Eur J Cancer 2007;43:510–519.[CrossRef][Medline]
  30. Sytkowski AJ. Does erythropoietin have a dark side? Epo signaling and cancer cells. Sci STKE 2007;(395):pe38.
  31. Pajonk F, Weil A, Sommer A et al. The erythropoietin-receptor pathway modulates survival of cancer cells. Oncogene 2004;23:8987–8991.[CrossRef][Medline]
  32. Agarwal N, Gordeuk VR, Prchal JT. Are erythropoietin receptors expressed in tumors? Facts and fiction—more careful studies are needed [letter]. J Clin Oncol 2007;25:1813–1814.[Free Full Text]
  33. Constantinescu SN, Ghaffari S, Lodish HF. The erythropoietin receptor: Structure, activation and intracellular signal transduction. Trends Endocrinol Metab 1999;10:18–23.[CrossRef][Medline]
  34. Rossert J, Eckardt KU. Erythropoietin receptors: Their role beyond erythropoiesis. Nephrol Dial Transplant 2005;20:1025–1028.[Free Full Text]
  35. Brines M, Cerami A. Discovering erythropoietin's extra-hematopoietic functions: Biology and clinical promise. Kidney Int 2006;70:246–250.[CrossRef][Medline]
  36. Leist M, Ghezzi P, Grasso G et al. Derivatives of erythropoietin that are tissue protective but not erythropoietic. Science 2004;305:239–242.[Abstract/Free Full Text]
  37. Kennedy J, Buchan AM. C-EPO: Ready for prime-time preconditioning [letter]? Cerebrovasc Dis 2005;19:272–273.[CrossRef][Medline]
  38. Elliott S, Egrie J, Browne J et al. Control of rHuEPO biological activity: The role of carbohydrate. Exp Hematol 2004;32:1146–1155.[CrossRef][Medline]
  39. Kwon DN, Song H, Park JY et al. Dynamic control of oligosaccharide modification in the mammary gland: Linking recombinant human erythropoietin functional analysis of transgenic mouse milk-derived hEPO. Transgenic Res 2006;15:37–55.[CrossRef][Medline]
  40. Acs G, Chen M, Xu X et al. Autocrine erythropoietin signaling inhibits hypoxia-induced apoptosis in human breast carcinoma cells. Cancer Lett 2004;214:243–251.[CrossRef][Medline]
  41. Verdier F, Walrafen P, Hubert N et al. Proteasomes regulate the duration of erythropoietin receptor activation by controlling down-regulation of cell surface receptors. J Biol Chem 2000;275:18375–18381.[Abstract/Free Full Text]
  42. Walrafen P, Verdier F, Kadri Z et al. Both proteasomes and lysosomes degrade the activated erythropoietin receptor. Blood 2005;105:600–608.[Abstract/Free Full Text]
  43. Meyer L, Deau B, Forejtnikova H et al. beta-Trcp mediates ubiquitination and degradation of erythropoietin receptor and controls cell proliferation. Blood 2007;109:5215–5222.[Abstract/Free Full Text]
  44. Beckman DL, Lin LL, Quinones ME et al. Activation of the erythropoietin receptor is not required for internalization of bound erythropoietin. Blood 1999;94:2667–2675.[Abstract/Free Full Text]
  45. Gewirtz DA, Di X, Walker TD et al. Erythropoietin fails to interfere with the antiproliferative and cytotoxic effects of antitumour drugs. Clin Cancer Res 2006;12:2232–2238.[Abstract/Free Full Text]
  46. Bohnen RF, Banisadre M, Gulbrandson RN et al. Erythrocytosis caused by an erythropoietin-producing breast adenocarcinoma. West J Med 1990;152:417–418.[Medline]
  47. Yasuda Y, Fujita Y, Masuda S et al. Erythropoietin is involved in growth and angiogenesis in malignant tumours of female reproductive organs. Carcinogenesis 2002;23:1797–1805.[Abstract/Free Full Text]
  48. Kling PJ. Roles of erythropoietin in human milk. Acta Paediatr Suppl 2002;91:31–35.[CrossRef][Medline]
  49. Friedman AD, Nimbalkar D, Quelle FW. Erythropoietin receptors associate with a ubiquitin ligase, p33RUL, and require its activity for erythropoietin-induced proliferation. J Biol Chem 2003;278:26851–26861.[Abstract/Free Full Text]
  50. Lester RD, Jo M, Campana WM et al. Erythropoietin promotes MCF-7 breast cancer cell migration by an ERK/mitogen-activated protein kinase-dependent pathway and is primarily responsible for the increase in the migration observed in hypoxia. J Biol Chem 2005;280:39273–39277.[Abstract/Free Full Text]
  51. Hardee ME, Rabbani ZN, Arcasoy MO et al. Erythropoietin inhibits apoptosis in breast cancer cells via an Akt-dependent pathway without modulating in vivo chemosensitivity. Mol Cancer Ther 2006;5:356–361.[Abstract/Free Full Text]
  52. Lai S, Grandis JR. Understanding the presence and function of erythropoietin receptor on cancer cells [letter]. J Clin Oncol 2006;24:4675–4676.[Free Full Text]
  53. Burton A. Is it all over for erythropoietin? [letter]. Lancet Oncol 2007;8:285.[CrossRef][Medline]
  54. Guan X, Chen L. Role of erythropoietin in cancer-related anaemia: A double-edged sword? J Int Med Res 2008;36:1–8.[Medline]



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G. Lewis and A. P. Maxwell
A cautionary tale of a man with haemochromatosis, chronic kidney disease and anaemia referred for erythropoietin therapy
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