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ANTICANCER RESEARCH 32: 3473-3478 (2012)

The Functional Polymorphism of rs1617640 G>T Is Not Associated with Susceptibility and Clinical Outcome of Early-stage Breast

JOANNA SZKANDERA1,2, GUDRUN ABSENGER1,2, MICHAEL STOTZ1,2, MELANIE WEISSMUELLER1,2, THOMAS WINDER3, TANJA LANGSENLEHNER4, HELLMUT SAMONIGG1, WILFRIED RENNER2,5, WALTER SCHIPPINGER6 and ARMIN GERGER1,2

1Division of Clinical Oncology, Department of Internal Medicine, 2Research Unit Genetic Epidemiology and Pharmacogenetics, 4Department of Therapeutic Radiology and Oncology, 5Clinical Institute of Medical and Laboratory Diagnostics, Medical University of Graz, Graz, Austria; 3Department of Internal Medicine, Academic Teaching Hospital Feldkirch, Feldkirch, Austria; 6Department of Internal Medicine, Albert Schweitzer Clinic, Graz, Austria

Abstract. Recent data suggest that erythropoietin (EPO) transcription factors (2). The EPO gene is activated under plays a substantial role in cancer development and clinical hypoxic conditions through binding of the HIF1α and HIF1β outcome by stimulating cell proliferation, invasion and to the promoter, respectively (3). Its main function is to . A functional polymorphism (rs1617640 G>T) stimulate by promoting proliferation and in the promoter region of the EPO gene increases EPO differentiation, or by inhibiting of erythroid expression. In the present study, we investigated the progenitors (4). Its biological effects are mediated through association of EPO rs1617640 G>T with susceptibility and the binding to the erythropoietin (EPOR), which clinical outcome of early-stage breast cancer. Genomic DNA belongs to the receptor type I superfamily (1, 5). of 539 female patients with histologically confirmed early- EPO was long considered to be a specific stimulator of stage breast cancer and 804 controls was genotyped for EPO erythropoiesis, but experimental findings have shown that its rs1617640 G>T. No association was found between EPO expression is not restricted to the hematopoietic system. rs1617640 G>T and early-stage breast cancer susceptibility Differential expression of EPO has been shown in other non- and clinical outcome (hazard ratio=1.24, 95% confidence hematopoietic tissues, such as astrocytes, breast interval=1.82-1.90, p=0.31). In conclusion, our findings epithelial cells and human reproductive organs, suggesting a suggest a lack of influence of EPO rs1617640 G>T on early- pleiotropic mechanism of action, extending well beyond the stage breast carcinogenesis and clinical outcome. maintenance of red cell mass (6-8). Furthermore, multiple investigators have documented EPO production in various Erythropoietin (EPO) is a mainly tumor cell lines and cancer entities, including breast cancer produced in the adult and fetal (1). EPO gene (9-13). Major signal-transduction pathways activated by EPO expression is regulated by inhibitory [GATA binding protein involve the (JAK)/signal transducer and 2 (GATA2), nuclear factor kappa--chain-enhancer of activator of transduction (STAT) and rat sarcoma activated B cells (NF-κB)] and stimulatory [- (RAS)/mitogen-activated protein kinase pathways, which are inducible factors (HIFs), hepatocyte nuclear factor] essential for the inhibition of apoptosis and the stimulation of cell proliferation in response to this hormone (5, 14). Moreover, it has been demonstrated that exposure of tumor cells to EPO-stimulated tyrosine phosphorylation, DNA Correspondence to: Armin Gerger, MD, Priv.-Doz., Associate Prof., synthesis and proliferation, suggesting that EPO signaling is Research Unit: Genetic Epidemiology and Pharmacogenetics, biologically active in malignant cells (9, 10). Additionally, Division of Clinical Oncology, Department of Internal Medicine, recent data indicate that EPO is involved in angiogenesis and Medical University Graz, Auenbruggerplatz 15, 8036 Graz, Austria. Tel: +43 31638580625, Fax: +43 31638513355, e-mail: invasion of neoplastic cells and may be important in breast [email protected] tumorigenesis (12, 15, 16). Mohyeldin et al. reported an EPO-mediated invasion through the JAK/STAT pathway in Key Words: Breast cancer, germline polymorphism, erythropoietin. head and neck squamous carcinoma cells (17). Furthermore,

0250-7005/2012 $2.00+.40 3473 ANTICANCER RESEARCH 32: 3473-3478 (2012) it has been demonstrated that EPO stimulates proliferation Vienna, Austria). Polymerase chain reaction (PCR) and evaluation in renal cancer cells (18). In endometrial carcinoma, EPO of fluorescence data were carried out as described elsewhere (23). signaling was shown to contribute to tumor progression and For each sample, one negative control containing water instead of DNA was added to check for contamination. For purposes of quality increased aggressiveness, identifying increased EPO control of genotyping, a total of 10% of the analyzed samples were expression as an independent prognostic factor (19). In re-analyzed. The investigator responsible for genotyping was prostate cancer, EPO was shown to regulate an blinded to the clinical dataset. autocrine/ pathway that influences growth and survival of cancer cells (20). In a recent study, Acs et al. Statistics. The primary endpoint of the study was disease-free demonstrated that EPO signaling inhibits hypoxia-induced survival (DFS). DFS was calculated from the date of diagnosis of apoptosis in human breast carcinoma cells and was early-stage breast cancer to the date of the first observation of tumor recurrence. DFS was censored at the last follow-up if the patient associated with poor prognosis (21). The TT genotype of a remained tumor-free at that time. Allelic distribution of the common gene variant in EPO, rs1617640 G>T, located in the polymorphism was tested for deviation from Hardy-Weinberg promoter region, leads to increased EPO protein expression equilibrium using χ2-test. The true mode of inheritance of the through an ecotopic viral integration site 1 (EVI1)/ polymorphism tested has not been established yet and we assumed 1 (MEL1) or activator protein 1 (AP1) enhancer binding site a co-dominant, additive, dominant, or recessive genetic model was and may be associated with cancer susceptibility and clinical appropriate. The association of the polymorphism with DFS was outcome (22). In the present study, we investigated the analyzed using Kaplan Meier curves and log- test. In the multivariate Cox regression analysis, the model was adjusted for association of EPO rs1617640 G>T with susceptibility and menopausal status, stage, histopathological grading, receptor status, clinical outcome in patients with early-stage breast cancer. human epidermal receptor 2 (HER2)/neu status and adjuvant treatment modalities. Case-wise deletion for missing Materials and Methods genotypes was used in univariate and multivariate analyses. Genotype frequencies in patients and controls were compared using χ2 tests. All analyses were performed using the SPSS statistical Study participants. A total of 539 female patients with histologically software package (SPSS Inc., Sunnyvale, CA, USA). confirmed breast cancer without distant metastases were included in this retrospective study. All patients were included in a breast Results cancer surveillance program between 2001 and 2009 at the Division of Clinical Oncology, Department of Internal Medicine, Medical Baseline patient characteristics, tumor biological factors, University of Graz, Austria, providing follow-up care at regular treatment modalities and their association with clinical outcome intervals (3-month intervals in years 1-3, 6-month intervals in years 4-5, and 12-month intervals in years 6-10 after curative surgery). are shown in Table I. The median age at time of diagnosis was Follow-up investigations included clinical check-up, laboratory, 57 years (range 29 to 84 years). The median follow-up duration radiological (bone scan, liver scan, chest X-ray, and mammograms), was 61.1 months (range 12 to 107 months). Follow-up data was and gynecological examination. A total of 804 healthy female missing for 38 (7.1%) patients. Genotyping was successful in controls were recruited from local health screening studies, the 520 (96.5%) patients and 799 (99.4%) controls. In failed cases, Salzburger Atherosklerose Präventionsprogramm bei Personen mit genotyping was not successful due to limited quantity and/or hohem Infarkt Risiko and the Grazer Diabetes Screening Programm. quality of extracted genomic DNA. The genotyping quality Controls with previous malignant disease were excluded from the present study. Blood samples were taken from all patients and control by subsample re-analysis provided a genotype controls, and stored at −20˚C. The present study was performed concordance of >99%. The allelic frequencies for the according to the Austrian Gene Technology Act, and has been polymorphism were within the probability limits of Hardy- approved by the Institutional Reviewer Board of the Medical Weinberg equilibrium. A statistically significant correlation was University of Graz. All participants gave written informed consent found between clinical outcome and tumor size, lymph node and were Caucasian. involvement and tumor stage (Table I). There was no significant association between EPO rs1617640 G>T and Candidate polymorphisms. The following pre-defined criteria for the breast cancer susceptibility (Table II). When EPO rs1617640 polymorphism selection within the EPO gene were used: (i) polymorphisms that modulate the function of the gene (based on G>T was analyzed for predicting clinical outcome, no published data); (ii) minor allele frequency ≥10% in Caucasians association was found with DFS (hazard ratio (HR)=1.24, 95% (based on the population genetics section in the Ensembl Genome confidence interval (CI)=1.82-1.90, p=0.31; multivariate Browser: http://uswest.ensembl.org/index.html); (iii) reported in analysis: HR=1.30, 95% CI=0.85-1.98, p=0.23) (Figure 1). published clinical association studies. Discussion Genotyping. DNA was extracted using the GenElute™ Blood Genomic DNA Kit from SIGMA (Saint Louis, Missouri, USA). Genotyping was performed using the 5’-exonuclease (TaqMan™) In the present study, we investigated the clinical impact of a technology with primers and probes designed and manufactured by functional promoter polymorphism of the EPO gene on the Applera’s Assay by-Design custom service (Applied Biosystems, risk and outcome of early-stage breast cancer. Our findings

3474 Szkandera et al: Erythropoietin Gene Variant in Breast Cancer Susceptibility and Prognosis

Table I. Baseline patient characteristics, tumor biological factors and Table II. Erythropoietin genotypes in early-stage breast cancer patients therapy modalities and the association with clinical outcome and controls. (Univariate Cox’s model). Polymorphism Breast cancer Controls p-Value n % HR (95% CI) p-Value patients (n=799) (n=520) Menopausal status Premenopausal 182 33.8 1 (Reference) EPO rs1617640 G>T Postmenopausal 356 66 1.15 (0.623-2.08) 0.639 GG 180 (34.6%) 269 (33.7) 0.79 Unknown 1 0.2 GT 265 (51.0%) 422 (52.8) Tumor size TT 75 (14.4%) 108 (13.5) T1 361 67 1 (Reference) T2 151 28 3.15 (1.77-5.61) 0.001 EPO rs1617640 T T3 20 3.7 1.80 (0.42-7.68) 0.427 allelic frequency 0.399 0.399 0.99 T4 6 1.1 3.61 (0.49-26.93) 0.210 Unknown 1 0.2 Lymph node involvement N0 308 57.1 1 (Reference) N1 172 31.9 1.19 (0.62-2.28) 0.613 N2 41 7.6 3.73 (1.81-7.70) 0.001 have been found in patients with various tumor entities, N3 18 3.3 2.93 (0.88-9.80) 0.081 including breast carcinomas, and numerous reports link Grading G1 61 11.3 1 (Reference) EPO signaling to the modulation of tumor cell proliferation, G2 284 52.7 1.21 (0.42-3.48) 0.725 apoptosis and tumorigenesis (9, 10, 12, 18, 21). Acs et al. G3 191 35.4 1.58 (0.54-4.58) 0.402 showed that EPO promotes tyrosine phosphorylation and Unknown 4 0.6 proliferation of breast cancer cells in vitro (9). However, in Stage other studies, no biological effect to EPO was observed, as I 239 44.3 1 (Reference) II 234 43.4 1.31 (0.68-2.51) 0.419 measured by proliferation or clonogenic growth in different III 66 12.2 3.34 (1.63-6.84) 0.001 tumor cell lines (25-27). Another report suggested that Receptor status increased autocrine EPO signaling induced by moderate ER and PR negative 83 15.4 1 (Reference) levels of hypoxia inhibits hypoxia-induced apoptosis and ER or PR positive 63 11.7 1.30 (0.57-2.94) 0.533 promotes survival in MCF-7 human breast cancer cells (28). ER and PR positive 388 72 0.559 (0.28-1.12) 0.102 Unknown 1 0.2 In a study by Arcasoy et al., EPO expression was found in HER2/neu status 60% of breast cancer cells and EPO and EPOR co- Negative 417 77.4 1 (Reference) localization in tumor cells was present in many cases. Positive 82 15.2 1.11 (0.81-1.52) 0.53 Moreover, local administration of a neutralizing anti-EPO Unknown 40 7.4 antibody, soluble EPOR, or an inhibitor of JAK2, resulted Adjuvant No 383 71.1 1 (Reference) in a delay in tumor growth, demonstrating functional EPO Yes 156 28.9 0.67 (0.35-1.31) 0.246 signaling in breast cancer cells (12). In contrast, Westphal Adjuvant hormone therapy et al. showed that even though various cancer cell lines, No 94 17.4 1 (Reference) including breast cancer cells, expressed EPOR, EPO Yes 444 82.4 0.65 (0.34-1.25) 0.201 signaling was not biologically active, and EPO was not Unknown 1 0.2 Adjuvant essential for growth of these tumor cells in culture (29). No 499 92.6 1 (Reference) This observation was confirmed by LaMontagne et al., who Yes 40 7.4 1.46 (0.58-3.68) 0.423 revealed in two well-established breast carcinoma models that EPOR was predominately cytosolic and not present as n: Number; ER: receptor; PR: progesteron receptor; HER2/neu: an active surface receptor on these cell lines, supported by human epidermal 2; HR: hazard ratio; CI: confidence interval. the findings that there was no significant measurable EPO- specific binding activity on the surface of either cell line (30). In vitro, EPO reportedly stimulated angiogenesis by induction of proliferation of primary endothelial cells and indicated no association between EPO rs1617640 G>T and endothelial cell lines (31-33). Moreover, it was reported that early-stage breast cancer susceptibility and outcome. EPO high doses of EPO induced the release of vascular signaling is known to activate several intracellular kinase endothelial growth factor (VEGF) in some cultured tumor pathways (5). These pathways were investigated particularly cell lines (34). In vivo, using both rat mammary in hematopoietic cells and may also contribute to its non- adenocarcinoma and mouse colon carcinoma models, no hematopoietic/tumor cell action (24). Elevated EPO levels difference was observed in angiogenesis between EPO-

3475 ANTICANCER RESEARCH 32: 3473-3478 (2012)

compared to those with the GG genotype (22). In vitro experiments showed a 25-fold higher expression of EPO in constructs containing the T allele in the promoter region of the EPO gene (22). It has been suggested that the increase in promoter activity might be due to an EVI1/MEL1 or AP1 binding site created by the T allele. Ma et al. demonstrated a strong association between the GG genotype of EPO rs1617640 G>T with (MDS), relative to the control group and other types of acute and chronic leukemia, implicating low levels of EPO in the development of MDS. In addition, the MDS group with the GG genotype displayed significantly shorter complete remission duration compared to patients with the TT genotype, but there was no correlation between genotypes and survival (40). A limitation of our study is its retrospective design, therefore a selection bias cannot be excluded. Furthermore, the control group was not age matched; however, single nucleotide polymorphisms (SNPs) are constant during life. Frequencies of polymorphisms have been shown to vary Figure 1. Disease-free survival of early-stage breast cancer patients harboring the TT, GT and GG genotype of erythropoietin rs1617640 between different ethnic cultures; therefore our results may G>T polymorphism. not be attributable to ethnicities other than Caucasian. In conclusion, we found no association between EPO rs1617640 G>T and early-stage breast cancer risk and outcome, but as the current knowledge on the impact of EPO on carcinogenesis and cancer progression is inconclusive, treated and placebo-treated tumors (35). The effect of EPO further studies designed to specifically evaluate the effects on tumor vascular density was investigated in two different of EPO on tumor susceptibility and survival in cancer glioma xenograft models with no increased density reported patients are warranted. (36). Biological disorders that alter EPO levels in humans offer additional insights into the potential role of EPO in Grant Support tumor induction and progression. Primary congenital disorders associated with increased EPO production and This work was funded by a Styrian Cancer Aid research grant. mutations in EPOR that result in hypersensitivity to EPO are associated with erythrocytosis in humans (37, 38). References However, a higher cancer incidence has not been observed in patients with familial and congenital . In a 1 Moritz KM, Lim GB and Wintour EM: Developmental study carried out on transgenic mice that express high levels regulation of erythropoietin and erythropoiesis. Am J Physiol of EPO, no incidence of erythroleukemia was observed 273(6 Pt 2): R1829-1844, 1997. during the course of two years, indicating that EPO 2 Jelkmann W: Control of erythropoietin and its stimulation alone does not induce tumorigenesis (39). The use in medicine. Methods Enzymol 435: 179-197, 2007. 3 Chan WK, Yao G, Gu YZ and Bradfield CA: Cross-talk between conflicting results in these studies might be due to variable the aryl hydrocarbon receptor and hypoxia inducible factor methodological approaches, with some limited to signaling pathways. Demonstration of competition and histopathological and biochemical evaluation, or differences compensation. J Biol Chem 274(17): 12115-12123, 1999. in the cell lines used. Furthermore, most of these studies 4 Lacombe C and Mayeux P: The molecular of were performed in vitro and may not reflect the affection of erythropoietin. Nephrol Dial Transplant 14(Suppl 2): 22-28, EPO on tumor cell function in vivo. A recent study 1999. demonstrated that the TT genotype of EPO rs1617640 G>T 5 Wojchowski DM, Gregory RC, Miller CP, Pandit AK and Pircher TJ: Signal transduction in the system. is associated with severe diabetic microvascular Exp Cell Res 253(1): 143-156, 1999. complications, such as diabetic retinopathy and end-stage 6 Masuda S, Okano M, Yamagishi K, Nagao M, Ueda M and renal disease (22). It has been reported that in non-diabetic Sasaki R: A novel site of erythropoietin production. Oxygen- individuals with the TT genotype, EPO protein dependent production in cultured rat astrocytes. J Biol Chem concentration was 7.5-fold higher in vitreous samples as 269(30): 19488-19493, 1994.

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37 Arcasoy MO, Karayal AF, Segal HM, Sinning JG and Forget 40 Ma W, Kantarjian H, Zhang K, Zhang X, Wang X, Chen C, BG: A novel mutation in the erythropoietin receptor gene is Donahue AC, Zhang Z, Yeh CH, O’Brien S, Garcia-Manero G, associated with familial erythrocytosis. Blood 99(8): 3066-3069, Caporaso N, Landgren O and Albitar M: Significant association 2002. between polymorphism of the erythropoietin gene promoter and 38 Gordeuk VR, Sergueeva AI, Miasnikova GY, Okhotin D, myelodysplastic syndrome. BMC Med Genet 11: 163, 2010. Voloshin Y, Choyke PL, Butman JA, Jedlickova K, Prchal JT and Polyakova LA: Congenital disorder of oxygen sensing: Association of the homozygous Chuvash VHL mutation with thrombosis and vascular abnormalities but not tumors. Blood 103(10): 3924-3932, 2004. 39 Madan A, Lin C, Wang Z and Curtin PT: Autocrine stimulation by erythropoietin in transgenic mice results in erythroid Received April 15, 2012 proliferation without neoplastic transformation. Blood Cells Mol Revised June 25, 2012 Dis 30(1): 82-89, 2003. Accepted June 26, 2012

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