Erythropoietin and its receptor in breast cancer: putting together the pieces of the puzzle.
ABSTRACT 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.
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ABSTRACT: Erythropoietin (EPO) provides an alternative to transfusion for increasing red blood cell mass and treating anemia in cancer patients. However, recent studies have reported increased adverse events and/or reduced survival in patients receiving both EPO and chemotherapy, potentially related to EPO-induced cancer progression. Additional preclinical studies that elucidate the possible mechanism underlying EPO cellular growth stimulation are needed. Using commercial tissue microarray (TMA) of a variety of cancers and benign tissues, EPO and EPO receptor immunohistochemical staining was performed. Furthermore using a panel of human renal cells (Caki-1, 786-O, 769-P, RPTEC), in vitro and in vivo experiments were performed with the addition of EPO in normoxic and hypoxic states to note phenotypic and genotypic changes. EPO expression score was significantly elevated in lung cancer and lymphoma (compared to benign tissues), while EPOR expression score was significantly elevated in lymphoma, thyroid, uterine, lung and prostate cancers (compared to benign tissues). EPO and EPOR expression scores in RCC and benign renal tissue were not significantly different. Experimentally, we show that exposure of human renal cells to recombinant EPO (rhEPO) induces cellular proliferation, which we report for the first time, is further enhanced in a hypoxic state. Mechanistic investigations revealed that EPO stimulates the expression of cyclin D1 while inhibiting the expression of p21cip1 and p27kip1 through the phosphorylation of JAK2 and ERK1/2, leading to a more rapid progression through the cell cycle. We also demonstrate an increase in the growth of renal cell carcinoma xenograft tumors when systemic rhEPO is administered. In summary, we elucidated a previously unidentified mechanism by which EPO administration regulates progression through the cell cycle, and show that EPO effects are significantly enhanced under hypoxic conditions.Journal of Hematology & Oncology 09/2013; 6(1):65. · 4.46 Impact Factor
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ABSTRACT: Objective: The aim of this research is to investigate the relationship between anemia and erythropoietin (Epo) and erythropoietin receptor (EpoR) expression. This study also investigated the relationship between Epo and EpoR expression level and the proliferation rate of cancer cells. Methods: 20 samples of breast cancer tissues were divided into two groups; anemic group (from patiens with Hb level < 12) and non-anemic group (from patients with Hb level > 12). All samples were analyzed by using immunofluorescence staining in order to examine Epo and EpoR expression. Proliferation of cancer cells were analyzed by using Hematoxylin-Eosin staining. Results: Anemic breast cancer group represented higher Epo and EpoR expression than the non-anemic group. The results also indicated that in anemic samples expression levels of Epo and EpoR were negatively correlated with the number of cancer cells. In contrast, Epo and EpoR expression levels from non-anemic samples were positively correlated with the number of cancer cells. Conclusion: These results conducted that anemia is a crucial factor of hypoxic condition. Hypoxia led by anemia cause a different control mechanism of Epo and EpoR expression and cancer cell proliferation.
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ABSTRACT: Objective: The aim of this research is to investigate the relationship between anemia and erythropoietin (Epo) and erythropoietin receptor (EpoR) expression. This study also investigated the relationship between Epo and EpoR expression level and the proliferation rate of cancer cells. Methods: 20 samples of breast cancer tissues were divided into two groups; anemic group (from patiens with Hb level < 12) and non-anemic group (from patients with Hb level > 12). All samples were analyzed by using immunofluorescence staining in order to examine Epo and EpoR expression. Proliferation of cancer cells were analyzed by using Hematoxylin-Eosin staining. Results: Anemic breast cancer group represented higher Epo and EpoR expression than the non-anemic group. The results also indicated that in anemic samples expression levels of Epo and EpoR were negatively correlated with the number of cancer cells. In contrast, Epo and EpoR expression levels from non-anemic samples were positively correlated with the number of cancer cells. Conclusion: These results conducted that anemia is a crucial factor of hypoxic condition. Hypoxia led by anemia cause a different control mechanism of Epo and EpoR expression and cancer cell proliferation.Journal of Experimental and Integrative Medicine. 07/2013; 3(3):199-204.
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
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.
The expression of erythropoietin (Epo) and the Epo re-
as in a variety of human cancers, including breast.
Functional Epo/EpoR signaling in cancer cells, which
pletely 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 re-
sults, ranging from a beneficial role that improves the
patient’s overall survival to a negative impact that pro-
Epo/EpoR biomolecular information enabled us to as-
semble an unexpected jigsaw puzzle which, via distinct
JAK-dependent and JAK-independent mechanisms
and different internalization/recycling as well as ubiq-
uitination/degradation pathways, could explain most of
the controversies of preclinical and clinical studies.
However, until the mechanisms of the contrasting liter-
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 pa-
STATE OF THE ART
Erythropoietin (Epo) is a glycoprotein hormone that serves
as the primary regulator of erythropoiesis (by stimulating
growth, preventing apoptosis, and inducing the differentia-
tion of RBC precursors) ; 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 modifi-
cation, the mature protein consists of a 165-aa structure;
while the O-linked sugar has no important function, the N-
in the circulation. Erythropoietin receptor (EpoR) is a
type-1, single-transmembrane receptor that is expressed in
full-length form (484 aa), a truncated form (303 aa), and a
tain the extracellular Epo-binding domain, but alternative
splicing of transcripts truncates the cytoplasmic or trans-
Correspondence: Ferdinando Mannello, Ph.D., D.Sc., Sezione di Biochimica Clinica del Dipartimento di Scienze Biomolecolari, Uni-
versità Studi “Carlo Bo,” Via O. Ubaldini 7, 61029 Urbino (PU), Italy. Telephone: 39-0722-351479; Fax: 39-0722-322370; e-mail:
firstname.lastname@example.orgReceived May 5, 2008; accepted for publication June 5, 2008; first published online in THE ONCOL-
OGIST Express on July 2, 2008. ©AlphaMed Press 1083-7159/2008/$30.00/0 doi: 10.1634/theoncologist.2008-0110
by on August 18, 2008
membrane domains. While truncated EpoR has sustained
erythropoiesis with attenuated function, soluble EpoR may
form for binding to Epo. However, the physiological roles
for these EpoR variants have not been established . Epo
exerts its effects via a hormonal mode, by inducing ho-
modimerization 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 regula-
tory sequences, and activation of the transcription of tar-
get genes, resulting in erythroid proliferation and
differentiation . The EpoR belongs to the cytokine re-
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,
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 auto-
crine/paracrine mode. Tissue injury, local inflammation, hypoxia, and metabolic stress induce the synthesis of HIF, which in-
creases the production of Epo locally; the local synthesis of Epo is also enhanced in cancer conditions. The tissue autocrine/
the EpoR and the dimer of the ?-CR (also known as CD131). Activation of the tissue EpoR heterocomplex requires high concen-
trations 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 ?-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
NF-?B, IAP, HSP70). The relative contributions of the two receptors, that is, the EpoR homodimer and the EpoR–?-CR het-
erodimer, in the stimulation of the growth of different tumor types by Epo remain to be defined.
HSP70, heat shock protein 70; IAP, inhibitor of apoptosis; JAK2, Janus kinase 2; MAPK, mitogen-activated protein kinase; NF-
?B, nuclear factor kappa B; PI3K, phosphoinositide-3 kinase; STAT5, signaling transducer and activator of transcription 5.
The Jigsaw Puzzle of Epo in Breast Cancer
by on August 18, 2008
ceptor superfamily ; it is present in many tissues as a
novel cell surface receptor complex, comprising one
class of EpoR subunit and a pair of ?-common receptor
(?-CR) subunits . The heterodimer receptor hypothe-
sis was first described in 1996 , suggesting that the
EpoR is switched on and transduces signals to the inte-
rior of the cell by the formation of homo- or hetero-
subunit. The EpoR heterocomplex has been found in sev-
eral mouse tissues and in the neural-like P19 cell line [6,
7], demonstrating the presence of a tissue-protective het-
eroreceptor; on the other hand, in the neuroblastoma SH-
SY5Y and pheochromocytoma PC12 cell lines , Epo
exerts its function through the “classical” homodimeric
EpoR with no evidence of heterocomplex. The hetero-
complex is involved in the Epo autocrine/paracrine sig-
naling 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, de-
phosphorylation downregulates this activity, inhibiting
proliferative signaling of the EpoR, reducing the STAT5
transduction signal, and finally promoting EpoR degra-
heterocomplex confers tissue protection through pleio-
tropic functions in many nonhematopoietic tissues, even
though further studies have reported contrasting findings
. The direct in vivo effects of Epo–EpoR signaling on
cellular proliferation, tumor oxygenation, apoptosis, tu-
mor angiogenesis, metastasis, and sensitivity to chemo-
radiation therapy remain to be identified . 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), inef-
ficient processing for surface expression, and protein
degradation within the endoplasmic reticulum, protea-
some, 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) .
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
CONTRASTING FINDINGS ON THE
Recent evidence raised the suspicion that Epo used to treat
anemia during cancer  might reduce, via EpoR, patient
survival, promoting tumor proliferation and angiogenesis
Following the recent debate on the evidence of the neg-
ative impact of Epo (adversely affecting prognosis in can-
cer patients with EpoR-positive cancer cells) [14–16], it is
via the EpoR, trying to explain, through an alternative bio-
molecular point of view, the “apparently” contradictory re-
sults obtained in breast cancer (BC).
sia 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 be-
tween EpoR transcripts and immunostaining of the EpoR
protein [17, 18]. However, several literature data highlight
ence of the EpoR protein; the methods used in most molec-
ular studies do not distinguish between the different splice
variants of the EpoR, thus not specifying whether func-
tional 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
ular 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
kDa, the calculated size of the EpoR protein is 53 kDa, and
the size to approximately 57–59 kDa (size was also deter-
mined and confirmed by protein microsequencing) .
surprising, because the various antibody preparations con-
tain polyclonal antipeptide antibodies.
Epo, the principal hematopoietic growth factor regulat-
ing cellular proliferation and differentiation along the ery-
throid lineage , 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
by on August 18, 2008
ular epithelial cells contain both Epo and EpoR showing
weak granular cytoplasmic localization [17, 22–24]. Epo
was increased in lobules with secretory changes  and
was constitutively found in milk [22, 25] and in nonlactat-
ing breast secretions (i.e., nipple aspirate fluid) ; Epo
labeling was also found in nontumoral cells of peritumoral
hyperplastic ducts  and in biosynthetically active apo-
crine cells sloughed from ducts . In the BC condition,
in fact, diffuse, moderate-to-strong cytoplasmic and mem-
brane-bound EpoR protein expression was found in BC tis-
sues [16–18, 24, 26], whereas in BC cell lines it showed
ings of the cytoplasmic and membrane-bound localization
the high batch-to-batch variability and the low specificity/
affinity of polyclonal antibody for the EpoR protein gener-
ated serious doubts on the histochemical identification of
EpoR in cancer cells, making tricky the understanding of
a polyclonal EpoR antibody (from a lack of specificity/
sensitivity [20, 28] to an excellent sensitivity ) require
the identification of the EpoR protein by new specific anti-
tion, an alternative biomolecular point of view might help
to explain why EpoR is found both in the cytoplasm and
may predict a negative impact on cancer control [16, 30],
the potential beneficial effects of Epo against a possible
negative impact of this cytokine on a case-by-case basis
TRANSLATIONAL AND CLINICAL RESEARCH:
STARTING PIECES OF THE PUZZLE
A more careful examination of experimental/clinical data
from hematopoietic cells and their translation into nonhe-
viding an explanation for the “apparently” contrasting re-
sults reported in the literature, in particular with respect
As starting pieces of the jigsaw, two EpoR types have
been identified: a “classic” homodimeric protein complex
mainly characterized in the erythroid lineage  (but also
(not as well known) heterodimer, composed of one EpoR
monomer combined with the ?-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 di-
verse EpoR forms may be responsible for the different (in
some cases contrasting) extrahematopoietic biological ac-
tivities 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  in
several tissues, including breast .
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, biomolec-
tified 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 li-
gand binding .
TRYING TO ASSEMBLE THE JIGSAW PUZZLE PIECES
FOR BREAST TISSUE
Analyzing the ubiquitination, internalization, and degrada-
tion model of Epo and its receptor in hematopoietic cells
[41–43], at least two mechanisms may justify the con-
founding results obtained in breast cells and tissues. Let’s
put together the “scattered” pieces of Epo and EpoR find-
ings in breast through (a) JAK-dependent ubiquitination/
degradation and (b) JAK-independent internalization-
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 bind-
ing, according to the literature data [41, 42]. In fact, after
Epo binding, the EpoR undergoes dimerization (in both ho-
mo- and heterodimeric complexes) and auto- or transphos-
phorylation of the Janus family kinase. In conjunction with
The Jigsaw Puzzle of Epo in Breast Cancer
by on August 18, 2008
other kinases, JAK2 phosphorylates several tyrosine resi-
dues in the EpoR, creating docking sites for the SH2 do-
mains of several signal transduction proteins (e.g., STAT5
and other signaling proteins become phosphorylated and
activated, promote intracellular signaling, move to the nu-
cleus, and activate gene expression) . The JAK2-in-
duced tyrosine-phosphorylated form of EpoR is rapidly
ubiquitinated , an important step both for the proteaso-
mal degradation of its intracellular domain (preventing fur-
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
ing for the final proteolytic degradation of both Epo and
cleaved EpoR  and (b) endosome formation for the ex-
tracellular 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 , (b) Epo binding activates
EpoR phosphorylation , and (c) Epo induces phosphor-
ylation of Akt, mitogen-activated protein kinase (MAPK),
and extracellular signal–related kinase (ERK) . More-
over, 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]
Figure 2. Model for JAK2-dependent mechanism of EpoR–?-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 ty-
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–?-CR heterodimer com-
plex is probably responsible for its proteolysis by the proteasomes, which removes a significant part (more than half) of the in-
tracellular domain. Because tyrosine residues are located in the cytoplasmic tail of the EpoR, this mechanism could hasten the
intracellular domain of ?-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–?-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
signaling transducer and activator of transcription 5; Ub, ubiquitination.
by on August 18, 2008
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 li-
gand binding, decreasing receptor-mediated signal genera-
For what concerns the second JAK-independent mech-
anism (Fig. 3), after Epo binding, the heterodimeric EpoR
complex may be efficiently internalized without either the
binding/activation/phosphorylation of JAK2 or the ubiq-
uitination process, thus not involving the protein kinase
Epo–EpoR complex probably occurs either by conforma-
tional changes induced by Epo binding or by caveolae/
clathrin-dependent pathway(s). The internalization of the
heterodimeric complex) and (b) endosome formation,
which may, in turn, lead to (i) Epo extracellular secretion
with no glycolytic or proteolytic processing, (ii) accumula-
tion of intracellular Epo and EpoR, or (iii) Epo and EpoR
degradation in lysosomes. This JAK2-independent inter-
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 pro-
tein [17–19,23–26,49,50], the lack of Akt and STAT5 acti-
vation after Epo binding [27, 47], Epo secretion in
dergoing glycolytic processing [9, 17, 18, 22, 25, 51].
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
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 consider-
ation that Epo may impair, not improve, cancer survival
Figure 3. Model for JAK2-independent mechanism of EpoR–?-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 heterocom-
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–?-CR heterodimer complex ubiquiti-
nation is not required for internalization, suggesting that Epo binding to its heteroreceptor induces a conformational change that is
sufficient to promote EpoR–?-CR heterodimer complex internalization. No data are available yet to exclude a clathrin- or caveo-
lae-dependent mechanism of internalization. The Epo–EpoR–?-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 inter-
nalized Epo from degradation, indicating that Epo is degraded in the lysosomes mainly by cathepsin B, whereas blocking protea-
some activity did not inhibit Epo lysosomal degradation.
Abbreviations: ?-CR, ?-common receptor; Epo, erythropoietin; EpoR, erythropoietin receptor; JAK2, Janus kinase 2.
The Jigsaw Puzzle of Epo in Breast Cancer
by on August 18, 2008
[16, 30, 52]. Even though many of the findings and conclu-
sions 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
two distinct receptors (a homo- and heterodimer, respec-
tively) with nonoverlapping functions raises the possibility
ulation of the two receptor systems. In fact, there is evi-
erythropoiesis-stimulating agents, either the response was
marginal and/or high levels were required to evoke a bio-
logical response; in contrast, many tumor cells expressing
EpoR do not respond to erythropoiesis-stimulating agents.
signaling after ligand–receptor interaction, low EpoR den-
sity, or nonfunctional EpoR at the cell surface in tumor
cells. Concerning human breast tissue, besides the impor-
tance of the discovery of agents with selective tissue-
protective and health-enhancing properties, the character-
ization 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–?-CR heteroreceptor may help us
to understand the real beneficial abilities of Epo therapeu-
tically. Moreover, the pharmacodynamics differences be-
tween homodimer EpoR and EpoR–?-CR heterocomplex
erythropoietic response and the high-dose but brief expo-
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 bio-
dent Epo–EpoR processing), in conjunction with the
diverse biological availability of activated or cleaved
EpoR, could provide an explanation why either endog-
enously produced (such as in inflammation and in cancer)
mia) Epo may promote, in some instances, tumor cell pro-
liferation in BC-initiating cells , whereas, in other
instances, it leads to better survival in BC patients . Al-
though our hypothesis actually provides more questions
than answers, until the mechanisms of the contrasting liter-
ature data are resolved, the use and risks of Epo therapy
should be carefully weighed, balancing the potential bene-
ficial functions of Epo protecting tissues and ameliorating
various anemias (including those associated with cancer)
 against its detrimental effects, mainly linked to the tu-
mor-promoting activity of Epo, a centenarian molecule yet
to be fully disclosed [1, 30, 53, 54].
This work was supported in part by Research Grant Award
2007 to F. M. from the Dr. Susan Love Research Founda-
tion, Pacific Palisades, CA.
Conception/design: Ferdinando Mannello
Collection/assembly of data: Gaetana A. M. Tonti
Manuscript writing: Ferdinando Mannello, Gaetana A. M. Tonti
Final approval of manuscript: Ferdinando Mannello, Gaetana A. M. Tonti
Eur J Haematol 2007;78:183–205.
ropoietin receptors in tumors: Implications for the use of erythropoiesis-
stimulating agents in cancer patients. Cancer 2007;110:477–488.
3Konstantinopoulos PA, Karamouzis MV, Papavassiliou AG. Selective
modulation of the erythropoietic and tissue-protective effects of erythro-
poietin: Time to reach the full therapeutic potential of erythropoietin. Bio-
chim Biophys Acta 2007;1776:1–9.
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.
5Yoshimura A, Arai K. Physician education: The erythropoietin receptor
and signal transduction. The Oncologist 1996;1:337–339.
6Jubinsky PT, Krijanovski OI, Nathan DG et al. The beta chain of the inter-
leukin-3 receptor functionally associates with the erythropoietin receptor.
7Brines M, Grasso G, Fiordaliso F et al. Erythropoietin mediates tissue pro-
tection through an erythropoietin and common ?-subunit heteroreceptor.
Proc Natl Acad Sci U S A 2004;101:14907–14912.
8Um M, Gross AW, Lodish HF. A “classical” homodimeric erythropoietin
receptor is essential for the antiapoptotic effects of erythropoietin on dif-
ferentiated neuroblastoma SH-SY5Y and pheochromocytoma PC-12 cells.
Cell Signal 2007;19:634–645.
9Hardee ME, Arcasoy MO, Blackwell KL et al. Erythropoietin biology in
cancer. Clin Cancer Res 2006;12:332–339.
kinase 2 is required for Golgi processing and cell surface expression of
erythropoietin receptor. Mol Cell 2001;8:1327–1338.
11 Goldrich A, Olivotto IA, Alexander CS et al. Anemia is a common but ne-
glected complication of adjuvant chemotherapy for early breast cancer.
Curr Oncol 2007;14:227–233.
12 Lappin TR, Maxwell AP, Johnston PG. Warning flags for erythropoiesis-
by on August 18, 2008
13 Blau CA. Erythropoietin in cancer: Presumption of innocence? STEM
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–
15 Henke M, Mattern D, Pajonk F. In reply [letter]. J Clin Oncol 2007;25:
16 Phillips TM, Kim K, Vlashi E et al. Effects of recombinant erythropoietin
on breast cancer-initiating cells. Neoplasia 2007;9:1122–1129.
17 Acs G, Zhang PJ, Rebbeck TR et al. Immunohistochemical expression of
erythropoietin and erythropoietin receptor in breast carcinoma. Cancer
18 Arcasoy MO, Amin K, Karayal AF et al. Functional significance of eryth-
ropoietin receptor expression in breast cancer. Lab Invest 2002;82:911–
19 Arcasoy MO, Jiang X, Haroon ZA. Expression of erythropoietin receptor
20 Elliott S, Busse L, Bass MB et al. Anti-Epo receptor antibodies do not pre-
dict Epo receptor expression. Blood 2006;107:1892–1895.
multiple actions. STEM CELLS 2002;20:485–492.
22 Juul SE, Zhao Y, Dame JB et al. Origin and fate of erythropoietin in human
milk. Pediatr Res 2000;48:660–667.
23 Acs G, Acs P, Beckwith SM et al. Erythropoietin and erythropoietin recep-
tor expression in human cancer. Cancer Res 2001;61:3561–3565.
24 Pelekanou V, Kampa M, Kafousi M et al. Erythropoietin and its receptor in
breast cancer: Correlation with steroid receptors and outcome. Cancer Epi-
demiol Biomarkers Prev 2007;16:2016–2023.
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
26 Wincewicz A, Sulkowska M, Koda M et al. STAT3, HIF-1?, EPO and
EPOR-signaling proteins in human primary ductal breast cancers. Folia
Histochem Cytobiol 2007;45:81–86.
27 LaMontagne KR, Butler J, Marshall DJ et al. Recombinant epoetins do not
stimulate tumor growth in erythropoietin receptor-positive breast carci-
noma models. Mol Cancer Ther 2006;5:347–355.
cancer cells: A still open question [letter]. J Clin Oncol 2007;25:1812–
1813; author reply 1815.
29 Österborg A, Aapro M, Cornes P et al. Preclinical studies of erythropoietin
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.
32 Agarwal N, Gordeuk VR, Prchal JT. Are erythropoietin receptors ex-
pressed in tumors? Facts and fiction—more careful studies are needed [let-
ter]. J Clin Oncol 2007;25:1813–1814.
33 Constantinescu SN, Ghaffari S, Lodish HF. The erythropoietin receptor:
Structure, activation and intracellular signal transduction. Trends Endocri-
nol Metab 1999;10:18–23.
34 Rossert J, Eckardt KU. Erythropoietin receptors: Their role beyond eryth-
ropoiesis. Nephrol Dial Transplant 2005;20:1025–1028.
35 Brines M, Cerami A. Discovering erythropoietin’s extra-hematopoietic
functions: Biology and clinical promise. Kidney Int 2006;70:246–250.
36 Leist M, Ghezzi P, Grasso G et al. Derivatives of erythropoietin that are
tissue protective but not erythropoietic. Science 2004;305:239–242.
37 Kennedy J, Buchan AM. C-EPO: Ready for prime-time preconditioning
[letter]? Cerebrovasc Dis 2005;19:272–273.
38 Elliott S, Egrie J, Browne J et al. Control of rHuEPO biological activity:
The role of carbohydrate. Exp Hematol 2004;32:1146–1155.
ification in the mammary gland: Linking recombinant human erythropoie-
40 Acs G, Chen M, Xu X et al. Autocrine erythropoietin signaling inhibits hy-
poxia-induced apoptosis in human breast carcinoma cells. Cancer Lett
erythropoietin receptor activation by controlling down-regulation of cell
surface receptors. J Biol Chem 2000;275:18375–18381.
42 Walrafen P, Verdier F, Kadri Z et al. Both proteasomes and lysosomes de-
grade the activated erythropoietin receptor. Blood 2005;105:600–608.
43 Meyer L, Deau B, Forejtnikova H et al. beta-Trcp mediates ubiquitination
and degradation of erythropoietin receptor and controls cell proliferation.
44 Beckman DL, Lin LL, Quinones ME et al. Activation of the erythropoietin
receptor is not required for internalization of bound erythropoietin. Blood
45 Gewirtz DA, Di X, Walker TD et al. Erythropoietin fails to interfere with
the antiproliferative and cytotoxic effects of antitumour drugs. Clin Cancer
46 Bohnen RF, Banisadre M, Gulbrandson RN et al. Erythrocytosis caused by
an erythropoietin-producing breast adenocarcinoma. West J Med 1990;
47 Yasuda Y, Fujita Y, Masuda S et al. Erythropoietin is involved in growth
49 Friedman AD, Nimbalkar D, Quelle FW. Erythropoietin receptors associ-
ate with a ubiquitin ligase, p33RUL, and require its activity for erythropoi-
etin-induced proliferation. J Biol Chem 2003;278:26851–26861.
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.
51 Hardee ME, Rabbani ZN, Arcasoy MO et al. Erythropoietin inhibits apo-
ptosis in breast cancer cells via an Akt-dependent pathway without modu-
lating in vivo chemosensitivity. Mol Cancer Ther 2006;5:356–361.
52 Lai S, Grandis JR. Understanding the presence and function of erythropoi-
etin receptor on cancer cells [letter]. J Clin Oncol 2006;24:4675–4676.
53 Burton A. Is it all over for erythropoietin? [letter]. Lancet Oncol 2007;8:
54 Guan X, Chen L. Role of erythropoietin in cancer-related anaemia: A dou-
ble-edged sword? J Int Med Res 2008;36:1–8.
The Jigsaw Puzzle of Epo in Breast Cancer
by on August 18, 2008
2008;13;761-768; originally published online Jul 2, 2008;
Ferdinando Mannello and Gaetana A. M. Tonti
Erythropoietin and Its Receptor in Breast Cancer: Putting Together the Pieces of
This information is current as of August 18, 2008
including high-resolution figures, can be found at:
by on August 18, 2008