Elsevier

Vitamins and Hormones

Volume 105, 2017, Pages 249-271
Vitamins and Hormones

Chapter Thirteen - Erythropoietin Promotes Glioblastoma via miR-451 Suppression

https://doi.org/10.1016/bs.vh.2017.03.002Get rights and content

Abstract

Erythropoietin (EPO) is an erythropoiesis stimulating growth factor and hormone. EPO has been widely used in the treatment of chronic renal failure, cancer, and chemotherapy-related anemia for three decades. However, many clinical trials showed that EPO treatment may be associated with tumorigenesis and cancer progression. EPO is able to cross blood–brain barriers, and this may lead to an increased possibility of central nervous system tumors such as glioblastoma. Indeed, EPO promotes glioblastoma growth and invasion in animal studies. Additionally, EPO increases glioblastoma cell survival, proliferation, migration, invasion, and chemoresistancy in vitro. However, the exact mechanisms of cancer progression induced by EPO treatment are not fully understood. Posttranscriptional gene regulation through microRNAs may contribute to EPO's cellular and biological effects in tumor progression. Here, we aimed to study whether tumor suppressive microRNA, miR-451, counteracts the positive effects of EPO on U87 human glioblastoma cell line. Migration and invasion were evaluated by scratch assay and transwell invasion assay, respectively. We found that EPO decreased basal miR-451 expression and increased cell proliferation, migration, invasion, and cisplatin chemoresistancy in vitro. miR-451 overexpression by transfection of its mimic significantly reversed these effects. Furthermore, ectopic expression of miR-451 inhibited expression of its own target genes, such as metalloproteinases-2 and -9, which are stimulated by EPO treatment and involved in carcinogenesis processes, especially invasion. These findings suggest that miR-451 mimic delivery may be useful as adjuvant therapy in addition to chemotherapy and anemia treatment by EPO and should be tested in experimental glioblastoma models.

Introduction

Erythropoietin (EPO) is a 34-kDa glycoprotein growth factor and hormone, which controls erythropoiesis through the promotion of proliferation, differentiation, and survival of erythrocytes progenitor cells and survival of mature erythrocytes (Jelkmann, 2013). EPO is initially synthesized in the liver during fetal development, but shortly after birth, production site of EPO subsequently shifts to the kidney. Peritubular fibroblast-like cells in the renal cortex are the major site of EPO production (Suzuki & Yamamoto, 2016). Expression of the EPO and EPO receptor (EPOR) is low in normal adult tissues and mainly induced by hypoxia. Several lines of evidences suggest that the central nervous system (CNS) expresses EPO and EPOR both at the mRNA and protein levels (Maiese, 2016).

EPO was purified from the urine of patients with aplastic anemia, and the therapeutic use of EPO was approved by the US Food and Drug Administration for treatment of anemia in patients with chronic renal failure 30 years ago (Jelkmann, 2013). EPO is now widely used for the treatment of anemia associated with renal failure, cancer, cancer chemotherapy, prematurity, chronic inflammatory diseases, and human immunodeficiency virus infection (Bennett et al., 2016, Debeljak et al., 2014). In the last 20 years, many in vitro and in vivo studies showed that EPO has cytoprotective and tissue protective, cell-proliferative, antiapoptotic, antiinflammatory, vascular protective, angiogenic, antiedema, antioxidant, cell migration promoting, neurogenesis stimulation, and metabolism regulation effects (Maiese, 2016). It has in vitro cytoprotective effect against various insults include neurotoxic agents, irradiation, trauma, chemotherapy, hypoxia, ischemia, oxygen glucose deprivation in many cell types (Maiese, 2016). These effects have also been shown in experimental models of various acute CNS injuries, such as stroke and chronic neurodegenerative diseases including Alzheimer's disease and Parkinson's disease (Sargin, Friedrichs, El-Kordi, & Ehrenreich, 2010).

Transport of EPO via the blood–brain barrier to CNS after systemic administration has been observed both in experimental animals and humans at high doses (Brines et al., 2000, Xenocostas et al., 2005). Cerebrospinal fluid concentration of EPO highly increases following intravenous administration (Ehrenreich et al., 2007). In spite of strong preclinical evidences, many clinical studies have failed in small unrandomized and retrospective patient studies. Thus, well-designed, randomized, prospective, placebo-controlled, and larger high quality clinical trials are still needed (Kochanek and Clark, 2016, Pearl, 2014, Sargin et al., 2010). Although several recent studies in stroke, bipolar disorder, schizophrenia patients have not reported negative results, sample sizes are relatively small (Miskowiak et al., 2014, Tsai et al., 2015, Wustenberg et al., 2011). EPO is already used routinely in prematurity anemia for a long time (Juul & Pet, 2015). Use of EPO for neuroprotection in very early term infants and neonates with hypoxic-ischemic encephalopathy seems a promising strategy (Juul & Pet, 2015). However, neurodevelopmental deficits and progression of any existing CNS tumor such as pediatric glioblastoma (GB) should be considered. The results of animal studies in the models of age-related chronic neurodegenerative diseases are currently not conclusive. Furthermore, EPO's serious side effects including hypertension, procoagulant, thromboembolic events, and tumor promotion can limit EPO therapy especially in elderly patients (Chong et al., 2013).

GB is the most aggressive and most prevalent primary brain tumor in adults. The World Health Organization classification system groups GB into four histological grades (Louis et al., 2016). Glioblastoma multiforme, also known as grade IV GB, is the most common and aggressive form of GB (Bush, Chang, & Berger, 2016). The standard therapy for GB is maximal surgical resection followed by radiotherapy and adjuvant temozolomide (TMZ) chemotherapy. In spite of intensive treatment, GB is associated with poor clinical outcome and currently not curable. Recent genome-wide association studies have contributed to the elucidation of the pathogenesis of GB (Bush et al., 2016). Cell surface receptors of growth factors such as epidermal growth factor receptor, signaling pathways, and transcription factors play central roles in the pathobiology of GB. Generally, almost all cancers, in this case GB, are characterized with biological and pathological processes including self-renewal, immortality, unlimited cell proliferation, hypoxia, invasiveness, and metastasis.

Many clinical and experimental studies showed that EPO stimulates tumor growth and progression in many types of cancers (Debeljak et al., 2014). Relatively rare in situ and in vitro studies reported that high EPO and EPOR expression in patients GB tissue samples (Brunotte et al., 2011, Mittelbronn et al., 2007, Mohyeldin et al., 2007, Nico et al., 2011, Said et al., 2007). In vitro studies with GB cell lines showed that these cells express EPO and EPOR (Belenkov et al., 2004, Hassouna et al., 2008, Mohyeldin et al., 2007, Peres et al., 2011, Said et al., 2007). In situ studies searched a possible concordance between EPOR expression levels with patient survival and histopathological grade of tumor. Mohyeldin et al. found that expression of EPOR correlated with the stage of the tumor (Mohyeldin et al., 2007). Unexpectedly, an inverse correlation was found between EPOR and GB grade of malignancy (Mittelbronn et al., 2007). However, EPOR expression level is directly associated with survival (Brunotte et al., 2011, Mittelbronn et al., 2007). EPO stimulates cell growth and proliferation in cultured GB cell lines (Hassouna et al., 2008, Yin et al., 2007). EPOR may mediate these effects both in vitro and in vivo experiments (Peres et al., 2015, Peres et al., 2011). EPOR signaling pathways are also involved in responsiveness to EPO (Belenkov et al., 2004, Cao et al., 2010, Kwon et al., 2014). EPO–EPOR signaling may also increase resistance of GB cells to chemotherapy and radiotherapy (Belenkov et al., 2004, Mohyeldin et al., 2007, Peres et al., 2015). Unexpectedly, Hassouna have reported that EPO treatment increased sensitivity to radiation and TMZ (Hassouna et al., 2008). Another interesting finding was that prevention of anemia with EPO-enhanced radiosensitivity of xenografted GB cells (Stuben et al., 2003).

Epigenetic mechanisms and posttranscriptional gene regulation by noncoding RNAs (ncRNAs) can also mediate initiation and progression of GB. MicroRNAs (miRNAs) are short, single-stranded, ncRNAs molecules that regulate gene expression at the posttranscriptional level (Hammond, 2015). miRNAs can control multiple cellular and biological processes including development, proliferation, differentiation, migration, apoptosis, and growth. Association of miRNAs with many human diseases has been extensively studied. Several functional groups of miRNAs such as oncomirs, tumor suppressor miRNAs, angiomirs, and hypoxamirs contribute to GB etiopathogenesis (Costa, Cardoso, Mano, & de Lima, 2015). miR-451 is a widely dysregulated miRNA in several human cancers including GB (Godlewski et al., 2010a, Godlewski et al., 2010b, Pan et al., 2013, Tian et al., 2012). Different studies shown that miR-451 inhibited cell proliferation, migration, and invasion and induced apoptosis in GB cell lines (Godlewski et al., 2010a, Godlewski et al., 2010b, Nan et al., 2010). Recently, we have determined that EPO downregulates the expression of miR-451 in SH-SY5Y neuroblastoma cell line and biological effects of EPO such as survival, proliferation, and migration depend on miR-451 suppression (Alural et al., 2014).

In the present study we examined the GB promoting effect of EPO in GB cells and the role of miR-451 in this GB promoting effect of EPO.

Section snippets

Cell Culture and Treatment

Human U87-MG glioblastoma cells (American Type Culture Collection) were cultured in Dulbecco's Modified Eagle Medium (DMEM) (Biochrom GbmH, Berlin, Germany) and supplemented with heat-inactivated fetal bovine serum (10% v/v), l-glutamine (1% v/v), and penicillin streptomycin (1% v/v). Cells were incubated at 37°C in 5% CO2.

Presto Blue Assay

Cell viability was assessed by the reducing environment of viable cells which upon entry, converts nonfluorescent resazurin-based solution into highly fluorescents resofurin.

EPO Increased Viability of Glioblastoma Cells

To evaluate EPO effect on cell viability, U87-MG cells were stimulated with recombinant human erythropoietin (rhEPO) at different concentrations (0.1–10 U/mL). EPO increased cell viability in a time- and dose-dependent manner as shown by Presto Blue assay (Fig. 1A–C). There was no significant difference between 1 and 10 mL EPO (23% vs 26%) on cell viability at 72 h. Thus, we performed all experiments using only 1 U/mL concentration of EPO.

miR-451 Overexpression Reversed Cell Viability Promoting Effect of EPO

Using qPCR, we demonstrated that 1 U/mL EPO treatment of

Discussion

The aim of present study was to examine the influences of EPO on cell survival, proliferation, invasiveness, and migration in U87 glioblastoma cell line. In addition, we searched whether EPO restores cell survival against cisplatin treatment. Finally, functional experiments were performed to examine the possible reversal effect of miR-451 ectopic expression upon EPO exposure.

We found that 1 U/mL EPO treatment increased basal U87-MG cell survival in a dose- and time-dependent manner. However, EPO

Acknowledgment

The authors wish to thank Dr. William Gault for his careful and critical reading of the manuscript. This study was supported by Dokuz Eylul University (Grant Number: 2016.KB.SAG.008).

Conflict of interest disclosure: The authors declare no competing financial interests.

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