Matthew E. Hardee

Erythropoietin Biology in Cancer

Erythropoietin (Epo) has long been known to be the principal hematopoietic growth factor that regulates cellular proliferation and differentiation along the erythroid lineage. Recent studies have shown that Epo is a pleiotropic cytokine that is proangiogenic and exerts broad tissue-protective effects in diverse nonhematopoietic organs. Recombinant Epo (rEpo) has been widely used in the clinic to prevent or treat malignancy-associated anemia. A series of clinical trials have documented the efficacy of rEpo in reducing RBC transfusion requirements and improving quality of life in cancer patients, and a recent meta-analysis suggested a positive effect on survival. However, two randomized trials reported negative outcomes with rEpo, as patients in the rEpo arm fared worse than their placebo-treated counterparts with respect to progression-free survival. The expression of Epo receptor (EpoR) in cancer cells has raised the possibility that exogenous rEpo may exert direct effects on tumor cells associated with the potential for stimulation of proliferation, inhibition of apoptosis, or modulation of sensitivity to chemoradiation therapy. The presence of an autocrine-paracrine Epo-EpoR system in tumors and potential effects of Epo on tumor microenvironment and angiogenesis are consistent with a complex biology for Epo-EpoR signaling in cancer that requires further research. This review describes Epo and EpoR biology, focusing on the pleiotropic effects of Epo on nonhematopoietic tissues as well as the expression and function of EpoR in cancer cells.

Resistance of Glioblastoma-Initiating Cells to Radiation Mediated by the Tumor Microenvironment Can Be Abolished by Inhibiting Transforming Growth Factor-β

The poor prognosis of glioblastoma (GBM) routinely treated with ionizing radiation (IR) has been attributed to the relative radioresistance of glioma-initiating cells (GIC). Other studies indicate that although GIC are sensitive, the response is mediated by undefined factors in the microenvironment. GBM produce abundant transforming growth factor-β (TGF-β), a pleotropic cytokine that promotes effective DNA damage response. Consistent with this, radiation sensitivity, as measured by clonogenic assay of cultured murine (GL261) and human (U251, U87MG) glioma cell lines, increased by approximately 25% when treated with LY364947, a small-molecule inhibitor of TGF-β type I receptor kinase, before irradiation. Mice bearing GL261 flank tumors treated with 1D11, a pan-isoform TGF-β neutralizing antibody, exhibited significantly increased tumor growth delay following IR. GL261 neurosphere cultures were used to evaluate GIC. LY364947 had no effect on the primary or secondary neurosphere-forming capacity. IR decreased primary neurosphere formation by 28%, but did not reduce secondary neurosphere formation. In contrast, LY364947 treatment before IR decreased primary neurosphere formation by 75% and secondary neurosphere formation by 68%. Notably, GL261 neurospheres produced 3.7-fold more TGF-β per cell compared with conventional culture, suggesting that TGF-β production by GIC promotes effective DNA damage response and self-renewal, which creates microenvironment-mediated resistance. Consistent with this, LY364947 treatment in irradiated GL261 neurosphere-derived cells decreased DNA damage responses, H2AX and p53 phosphorylation, and induction of self-renewal signals, Notch1 and CXCR4. These data motivate the use of TGF-β inhibitors with radiation to improve therapeutic response in patients with GBM.

Erythropoietin Blockade Inhibits the Induction of Tumor Angiogenesis and Progression

Background The induction of tumor angiogenesis, a pathologic process critical for tumor progression, is mediated by multiple regulatory factors released by tumor and host cells. We investigated the role of the hematopoietic cytokine erythropoietin as an angiogenic factor that modulates tumor progression. Methodology/Principal Findings Fluorescently-labeled rodent mammary carcinoma cells were injected into dorsal skin-fold window chambers in mice, an angiogenesis model that allows direct, non-invasive, serial visualization and real-time assessment of tumor cells and neovascularization simultaneously using intravital microscopy and computerized image analysis during the initial stages of tumorigenesis. Erythropoietin or its antagonist proteins were co-injected with tumor cells into window chambers. In vivo growth of cells engineered to stably express a constitutively active erythropoietin receptor EPOR-R129C or the erythropoietin antagonist R103A-EPO were analyzed in window chambers and in the mammary fat pads of athymic nude mice. Co-injection of erythropoietin with tumor cells or expression of EPOR-R129C in tumor cells significantly stimulated tumor neovascularization and growth in window chambers. Co-injection of erythropoietin antagonist proteins (soluble EPOR or anti-EPO antibody) with tumor cells or stable expression of antagonist R103A-EPO protein secreted from tumor cells inhibited angiogenesis and impaired tumor growth. In orthotopic tumor xenograft studies, EPOR-R129C expression significantly promoted tumor growth associated with increased expression of Ki67 proliferation antigen, enhanced microvessel density, decreased tumor hypoxia, and increased phosphorylation of extracellular-regulated kinases ERK1/2. R103A-EPO antagonist expression in mammary carcinoma cells was associated with near-complete disruption of primary tumor formation in the mammary fat pad. Conclusions/Significance These data indicate that erythropoietin is an important angiogenic factor that regulates the induction of tumor cell-induced neovascularization and growth during the initial stages of tumorigenesis. The suppression of tumor angiogenesis and progression by erythropoietin blockade suggests that erythropoietin may constitute a potential target for the therapeutic modulation of angiogenesis in cancer.

Erythropoietin inhibits apoptosis in breast cancer cells via an Akt-dependent pathway without modulating in vivo chemosensitivity

Evidence for erythropoietin signaling has been shown in several nonhematopoietic tissues, including many tumor types. Clinically, recombinant erythropoietin treatment of malignancy-related anemia has yet to be definitively associated with any modulation of chemotherapy or radiotherapy efficacy. Preclinically, recombinant erythropoietin has been shown to increase tumor oxygenation, but the direct effects of recombinant erythropoietin on tumor cells that express erythropoietin receptor are not yet fully characterized. This study examined the effects of exogenous recombinant erythropoietin on rodent mammary adenocarcinoma cells (R3230) in vitro and in vivo, and determined the effects of systemic recombinant erythropoietin on tumor growth delay in Taxol treatment. We showed that systemic recombinant erythropoietin treatment of rats bearing R3230 mammary carcinomas induced an increase in phospho-Akt levels within tumor cells. This was associated with a decrease in the frequency of apoptotic cells in tumors from recombinant erythropoietin–treated animals, but did not noticeably affect tumor growth rate. In vitro studies revealed that not only does recombinant erythropoietin induce Akt phosphorylation, but it also stimulates phosphorylation of p44/42 mitogen-activated protein kinases, Erk1 and Erk2. Activation of erythropoietin-mediated signaling in R3230 cells was associated with dose-dependent inhibition of apoptosis in response to Taxol treatment and serum starvation, an effect that was blocked by the addition of a phosphatidylinositol-3-kinase inhibitor. Despite its cytoprotective effects in vitro, recombinant erythropoietin did not significantly affect tumor growth delay in Taxol treatment. This study shows direct recombinant erythropoietin–mediated activation of specific intracellular signaling pathways in mammary adenocarcinoma cells in vivo and in vitro. Modulation of tumor apoptosis pathways by recombinant erythropoietin may have negative consequences by decreasing the chemosensitivity and radiosensitivity of erythropoietin receptor–positive breast tumors, although it did not have any obvious effects on growth with or without chemotherapy in this model. [Mol Cancer Ther 2006;5(2):356–61]

Human recombinant erythropoietin (rEpo) has no effect on tumour growth or angiogenesis

Tumour hypoxia has been shown to increase mutation rate, angiogenesis, and metastatic potential, and decrease response to conventional therapeutics. Improved tumour oxygenation should translate into increased treatment response. Exogenous recombinant erythropoietin (rEpo) has been recently shown to increase tumour oxygenation in a mammary carcinoma model. The mechanism of this action is not yet understood completely. The presence of Epo and its receptor (EpoR) have been demonstrated on several normal and neoplastic tissues, including blood vessels and various solid tumours. In addition, rEpo has been shown in two recent prospective, randomized clinical trials to negatively impact treatment outcome. In this study, we attempt to characterize the direct effects of rEpo on tumour growth and angiogenesis in two separate rodent carcinomas. The effect of rEpo on R3230 rat mammary adenocarcinomas, CT-26 mouse colon carcinomas, HCT-116 human colon carcinomas, and FaDu human head and neck tumours, all of which express EpoR, was examined. There were no differences in tumour growth or proliferation (measured by Ki-67) between placebo-treated and rEpo-treated tumours. In the mammary window chamber, vascular length density (VLD) measurements in serial images of both placebo-treated and Epo-treated rats revealed no difference in angiogenesis between the Epo-treated tumours and placebo-treated tumours at any time point. These experiments are important because they suggest that the recent clinical detriment seen with the use of Epo is not due to its tumour growth effects or angiogenesis. These studies also suggest that further preclinical studies need to examine rEpos direct tumour effects in efforts to improve the therapeutic benefits of Epo in solid tumour patients.

Spectral imaging facilitates visualization and measurements of unstable and abnormal microvascular oxygen transport in tumors

Abnormal microvasculature contributes to the pathophysiologic microenvironment of tumors. Understanding microvascular tumor oxygen transport is necessary to comprehend the factors that influence tumor biology, physiology, and therapy. Previously, we described an in vivo spectral imaging microscopy system for measurements of microvessel hemoglobin saturation (HbSat). We measure temporal fluctuations and spatial gradients in tumor microvessel oxygenation and identify instances of anastomoses between vessels with significantly different oxygenations. Slow periodic fluctuations in HbSat <0.2 cycles per minute were observed. These measurements are consistent with microelectrode measurements of fluctuating tumor oxygenation. Gradients in HbSat along individual tumor microvessels were measured that were larger in magnitude than normal tissue microvessels. Images were captured of anastomoses of tumor microvessels with diameters <or=100 microm and significantly different HbSat values (>20%). Shunting of inspired oxygen, presumably due to arteriovenous anastomoses, from tumor feeding arterioles to adjacent venules was imaged. This effect was confined to a region around the tumor and was not observed in nearby normal microvessels. Imaging measurements of tumor microvessel oxygen transport may offer insight to current questions regarding oxygen-related tumor biology and treatment responses, and spectral imaging may be a useful research tool in this regard.

Novel Imaging Provides New Insights into Mechanisms of Oxygen Transport in Tumors

Hypoxia is a common feature of solid tumors, and abnormal tumor oxygen transport is a key factor in the imbalance between tumor oxygen supply and demand. Novel advanced imaging techniques can enable new insights into the complexities of tumor oxygen transport and hypoxia that were not previously known or fully appreciated. In this paper, we document new insights into tumor oxygen transport enabled by spectral imaging of microvascular hemoglobin saturation.

Carbonic anhydrase IX is a predictive marker of doxorubicin resistance in early-stage breast cancer independent of HER2 and TOP2A amplification

Background:In early-stage breast cancer, adjuvant chemotherapy is associated with significant systemic toxicity with only a modest survival benefit. Therefore, there is considerable interest in identifying predictive markers of response to therapy. Doxorubicin, one of the most common drugs used to treat breast cancer, is an anthracycline chemotherapeutic agent, a class of drugs known to be affected by hypoxia. Accordingly, we examined whether expression of the endogenous hypoxia marker carbonic anhydrase IX (CA IX) is predictive of outcome in early-stage breast cancer patients treated with doxorubicin.Methods:We obtained 209 early-stage pre-treatment surgically-resected breast tumours from patients, who received doxorubicin in their chemotherapeutic regimen and had >10 years of follow-up. Immunohistochemistry was used to detect CA IX, and we used fluorescence in situ hybridisation to detect both human epidermal growth factor receptor (HER2) and DNA topoisomerase II-alpha (TOP2A) gene amplification.Results:Carbonic anhydrase IX intensity was significantly correlated with progression-free survival (PFS) and overall survival (OS) in patients receiving 300 mg m−2 of doxorubicin (HR=1.82 and 3.77; P=0.0014 and 0.010, respectively). There was a significant, inverse correlation between CA IX score and oestrogen receptor expression, but no significant correlations were seen with either HER2 or TOP2A ratio.Conclusion:We demonstrate that CA IX expression is correlated with worse PFS and OS for breast cancer patients treated with doxorubicin, independent of HER2 or TOP2A gene amplification. This study provides evidence that using CA IX to detect hypoxia in surgically-resected breast tumours may be of clinical use in choosing an appropriate chemotherapy regimen.

Her2/neu signaling blockade improves tumor oxygenation in a multifactorial fashion in Her2/neu+ tumors

PurposeTumor hypoxia reduces the efficacy of radiation and chemotherapy as well as altering gene expression that promotes cell survival and metastasis. The growth factor receptor, Her2/neu, is overexpressed in 25–30% of breast tumors. Tumors that are Her2+ may have an altered state of oxygenation, relative to Her2− tumors, due to differences in tumor growth rate and angiogenesis.MethodsHer2 blockade was accomplished using an antibody to the receptor (trastuzumab; Herceptin). This study examined the effects of Her2 blockade on tumor angiogenesis, vascular architecture, and hypoxia in Her2+ and Her2− MCF7 xenograft tumors.ResultsTreatment with trastuzumab in Her2+ tumors significantly improved tumor oxygenation, increased microvessel density, and improved vascular architecture compared with the control-treated Her2+ tumors. The Her2+ xenografts treated with trastuzumab also demonstrated decreased proliferation indices when compared with control-treated xenografts. These results indicate that Her2 blockade can improve tumor oxygenation by decreasing oxygen consumption (reducing tumor cell proliferation and inducing necrosis) and increasing oxygen delivery (vascular density and architecture).ConclusionsThese results support the use of trastuzumab as an adjunct in the treatment of breast tumors with chemotherapy or radiotherapy, as improvements in tumor oxygenation should translate into improved treatment response.

Management of High-Risk Localized Prostate Cancer

Traditionally, patients with high-risk localized prostate cancer have been an extremely challenging group to manage due to a significant likelihood of treatment failure and prostate cancer-specific mortality (PCSM). The results of multiple large, prospective, randomized trials have demonstrated that men with high-risk features who are treated in a multimodal fashion at the time of initial diagnosis have improved overall survival. Advances in local treatments such as dose-escalated radiotherapy in conjunction with androgen suppression and postprostatectomy adjuvant radiotherapy have also demonstrated benefits to this subset of patients. However, therapeutic enhancement with the addition of chemotherapy to the primary treatment regimen may help achieve optimal disease control.