Abstract
Erythropoietin (Epo) may be considered as an endogenous stimulator of vessel growth during tumor progression through an autocrine and/or paracrine loop. The vascular effects of Epo would be relevant in tumor angiogenesis and the negative effect of Epo on tumor growth may be aggravated by its angiogenic activity. The mechanism of tumor growth in the context of Epo is not completely clarified, and it is still not clear whether there is a direct effect of Epo in tumor cells as opposed to exogenous effect on angiogenesis. It is also possible that the effect of Epo is multifactorial depending on the type of tumor and level of functionality of Epo receptor expression in tumor cells, as well other variables such as hypoxic stress, degree of anemia, chemotherapy, radiotherapy of surgical intervention.
S
Tumor growth and expansion are often characterized by the inability of the local vasculature to supply sufficient oxygen and nutrients to the rapidly dividing neoplastic cells. The resulting hypoxic state causes changes in the tumor cells that can lead to cell stasis or apoptosis or, alternatively, to responses aimed at improving tissue oxygenation. Epo and EpoR expression in tumors improve the hypoxic survival of cancer cells [16]. Hypoxia represents an important mechanism of tumor progression because it selects neoplastic cell clones with lower apoptotic potential, thus providing a growth advantage to cells with genetic alterations that impair apoptosis [7]. Moreover, hypoxia may also contribute to metastasis [17] and treatment resistance [18]. Hypoxia-inducible factor-1 (HIF-1) regulates the expression of several genes that give a growth advantage to hypoxic cells [19], such as vascular endothelial growth factor (VEGF), promoting angiogenesis; moreover, HIF-1-mediated Epo expression is thus unlikely to be an exclusive mechanism for hypoxic cell survival [19]. Each of the HIF family members—HIF-1α, HIF-1β, and HIF-3α—have a significant role in regulating the expression of Epo and EpoR. Winter et al. [20] showed a significant correlation between Epo and HIF-1 expression in head and neck squamous cell carcinoma and intratumoral hypoxia plays a key role in the Epo/EpoR autocrine/paracrine loop as both Epo and EpoR are up-regulated in hypoxic tumor cell lines [21]. Leo et al. [22] analyzed Epo/EpoR expression and their relationship with intratumoral pO2 levels as well as with survival in patients with cervical cancer and showed that cancers with higher Epo expression showed a significantly reduced overall survival and Epo/EpoR expression correlated significantly with apoptosis. Furthermore, no correlation was observed between Epo/EpoR expression and intratumoral hypoxia, although in well-oxygenated tumors, EpoR localized significantly more often to the invasion front.
The role of Epo has been demonstrated in tissues outside the hematopoietic system, including the cardiovascular system, where Epo promotes various effects in endothelial cells (Table 1). Epo stimulates both proliferation and migration of human and bovine endothelial cells in vitro and of endothelial cells isolated from rat mesentery [23 –26], as well as in the rat aortic ring model [27]. Moreover, Epo induces endothelin-1 (ET-1) expression in endothelial cell cultures [28,29]. rHuEpo induces an increased cell proliferation, matrix metalloproteinase-2 expression, and differentiation into vascular tubes of human endothelial cells in vitro [23]. In vivo, in the chorioallantoic membrane (CAM) assay, rHuEpo exerted an angiogenic activity comparable to that of fibroblast growth factor-2 (FGF-2), and CAM’s endothelial cells expressed both EpoR and factor VIII-related antigen [23]. EpoR mRNA is expressed in human umbilical vein endothelial cells (HUVEC) [30], bovine adrenal capillaries [24], and rat brain capillaries [25].
Abbreviations: MMP-2, matrix metalloproteinase-2; ET-1, endothelin-1; CAM, chorioallantoic membrane.
Kertesz et al. [31] demonstrated that Epo and EpoR are expressed in the vasculature during embryogenesis and that deletion of Epo and EpoR in null embryos leads to angiogenic defects, whereas de novo vasculogenesis was not affected, consistent with the differential expression of Epo and EpoR during the early stages of embryonic development.
RhuEpo has also been shown to increase the number of circulating endothelial cells and endothelial precursor cells (EPC) in preclinical models [32] and human breast cancer and lymphoma models [33]. Moreover, Epo stimulates postnatal neovascularization by increasing EPC mobilization from the bone marrow [34].
Epo favors tumor progression through effects on angiogenesis and it may be considered as an endogenous stimulator of vessel growth during tumor progression through an autocrine/paracrine loop [35]. The vascular effects of Epo would be relevant in tumor angiogenesis and the negative effect of Epo on tumor growth may be further aggravated by its known angiogenic activity [7]. Tumor cell can directly release increasing amounts of VEGF and placental growth factor (PlGF) in response to Epo [36]. In cerebral endothelial cell cultures, rhuEPO enhanced angiogenesis by increasing VEGF levels and this effect was inhibited by anti-Epo antibody and a specific VEGF receptor (VEGFR) antagonist [37]. In an experimental model of femoral artery legation using EpoR-null mice, blood flow recovery, activation of VEGF/VEGFR system, and mobilization of EPC were all impaired in EpoR-null mice as compared with wild-type mice [38]. On the contrary, exogenous Epo decreased both the host- and tumor-derived VEGF expression suggesting that the proliferation-promoting effect of rhuEpo on tumoral endothelial cells is independent of VEGF production [39].
Okazaki et al. [40] showed in a Lewis lung carcinoma xenograft model in mice that administration of subcutaneous Epo promote tumor growth through enhancement of angiogenesis because these tumor cells do not express EpoR and Epo treatment in vitro did not affect proliferation of these cells. Hardee et al. [41] demonstrated by using the dorsal skin-fold window chambers that co-injection of Epo with labeled rodent mammary carcinoma cells or expression of EpoR in tumor cells stimulated tumor neovascularization and growth. Co-injection of Epo antagonists proteins with tumor cells or stable expression of Epo antagonist inhibited angiogenesis and impaired tumor growth. These data have been confirmed by using orthotopic tumor xenograft model, where EpoR expression promoted tumor growth, increased Ki67 proliferation antigen expression, enhanced microvessel density, and decreased tumor hypoxia. On the contrary, Epo antagonist expression in tumor cells was associated with disruption of primary tumors.
Epo administration to patients with hematological malignancies, namely multiple myeloma and myelodysplastic syndrome, induced bone marrow angiogenesis and further malignant transformation in plasma cell leukemia and acute monoblastic leukemia, respectively [42 –44]. On the contrary, Mittelman et al. [45] demonstrated that rHuEpo treatment induced complete tumor regression in 30%–60% of mice with a syngeneic multiple myeloma.
The administration of anti-Epo antibody, soluble EpoR, or an inhibitor of JAK2 resulted in a delay in tumor growth with 45% reduction in maximal tumor depth in a tumor Z chambers model with rat mammary adenocarcinoma cells [3]. On the contrary, Yasuda et al. [46] reported that the injection of an antibody anti-Epo or the soluble form of EpoR into malignant tumors of the female reproductive organs reduces capillaries and causes tumor destruction. They also found that blockade of Epo signaling on xenografts of uterine and ovarian cancer leads to the destruction of tumors in nude mice.
The systemic administration of putative antiangiogenic agents targeting Epo and EpoR may be limited by the development of anemia due to the suppression of erythropoiesis. Otherwise, alleviation of anemia by systemic rHuEpo treatment has different effects on tumors: it can decrease hypoxia; it enhances proliferation or survival of cancer cells; it increases endothelial cell proliferation causing enhanced radiosensitivity of the vessels and tumor perfusion by oxygen and chemotherapeutic agents, favoring their delivery [47]. However, improved systemic perfusion may not translate in improved intratumoral blood flow, as tumors often have aberrant vasculature.
The mechanism of tumor growth in the context of Epo is not completely clarified, and it is still not clear whether there is a direct effect of Epo in tumor cells as opposed to exogenous effect on angiogenesis (Table 2). It is also possible that the effect of Epo is multifactorial depending on the type of tumor and level or functionality of EpoR expression in tumor cells as well as other variables such as hypoxic stress, degree of anemia, chemotherapy, radiotherapy, or surgical intervention. It should be mentioned that current EpoR antibodies are not specific and there is not compelling evidence for functional EpoR expression in tumor cells [48].
Abbreviations: EPC; endothelial precursor cells; VEGF, vascular endothelial growth factor; PlGF, placental growth factor.
The effect of Epo on the survival rate of cancer patients is variable. A meta-analysis of 60 relevant studies found that anemia increased the relative risk of death by 19% in lung cancer, 75% in head and neck cancer, 47% in prostate cancer, and 67% in lymphoma, and was associated with an overall estimated increased risk of death by 65% [49]. Epo-stimulating agents (ESA) have been approved for the treatment of anemia in patients with non-myeloid malignancies whose anemia is due to the effect of concomitantly administered chemotherapy and the risk of thromboembolic events, decreased survival, and poorer tumor control as a consequence of treatment with ESA has been recognized [50]. In head and neck cancer, metastatic breast cancer, lymphoproliferative malignancies, and advanced non-small-cell lung cancer, Epo has an unfavorable effect on survival rate [51 –53]. However, in Epo-treated patients the bulk of increased deaths was attributable to accelerated cancer progression, independently of the increased cardiovascular and thromboembolic events.
Footnotes
Acknowledgments
Supported in part by MIUR (PRIN 2007), Rome, and Fondazione Cassa di Risparmio di Puglia, Bari, Italy.