Erythropoietin suppresses the activation of pro-apoptotic genes in head and neck squamous cell carcinoma xenografts exposed to surgical trauma
© Lindgren et al.; licensee BioMed Central Ltd. 2014
Received: 19 June 2013
Accepted: 31 July 2014
Published: 2 September 2014
Several studies on the use of erythropoietin (Epo) to treat anaemia in patients undergoing cancer treatment have shown adverse effects on tumour control and survival. Experimental studies indicate that this could be linked to an interaction with wound healing processes and not an effect on tumour cells per se. We have previously shown that erythropoietin in combination with surgical trauma stimulates tumour growth. In the present study, we investigated the effect of surgery and Epo on gene expression.
Human tumours from oral squamous cell cancer were xenotransplanted to nude mice treated with Epo. The tumours were then transected in a standardised procedure to mimic surgical trauma and the change in gene expression of the tumours was investigated by microarray analysis. qRT-PCR was used to measure the levels of mRNAs of pro-apoptotic genes. The frequency of apoptosis in the tumours was assessed using immunohistochemistry for caspase-3.
There was little change in the expression of genes involved in tumour growth and angiogenesis but a significant down-regulation of the expression of genes involved in apoptosis. This effect on apoptosis was confirmed by a general decrease in the expression of mRNA for selected pro-apoptotic genes. Epo-treated tumours had a significantly lower frequency of apoptosis as measured by immunohistochemistry for caspase 3.
Our results suggest that the increased tumour growth during erythropoietin treatment might be due to inhibition of apoptosis, an effect that becomes significant during tissue damage such as surgery.
This further suggests that the decreased survival during erythropoietin treatment might be due to inhibition of apoptosis.
KeywordsErythropoietin Head and neck cancer Surgery Apoptosis Wound healing Xenograft
Squamous cell carcinoma of the head and neck (HNSCC) is globally a common disease. Annually, more than 147,500 cases and 63,300 attributed deaths are reported in Europe [1, 2] and the prognosis for clinically advanced cancer is still very poor. It often affects patients with severe co-morbidity and both the cancer and the treatment, such as surgery, radiotherapy, chemotherapy and combinations thereof, have strong adverse effects on the patient’s general condition and nutritional status. Weight loss and anaemia are common. It has been argued that increased blood flow and oxygenation in the tumours would make them more accessible to radiotherapy and chemotherapy [3–5]. Erythropoietin (Epo) has been advocated to increase haemoglobin concentrations with the intent of improving the effect of radiotherapy and the quality of life.
Early studies of Epo treatment in cancer patients primarily investigated the effects on haemoglobin level [3–6] and quality of life . Few studies had tumour growth, disease free survival and overall survival as primary endpoints. In 2003, a study  revealed significantly worse outcome for HNSCC patients treated with Epo. Other studies involving Epo administration during treatment of non-small-cell carcinoma of the lung (NSCLC)  and breast cancer  also showed lower survival rates for Epo treated patients. These results raised the concern that Epo might stimulate tumour growth. Epo has also been implicated in tumour invasiveness [10–12]. Several studies on the use of Epo to ameliorate anaemia in patients undergoing cancer treatment have shown adverse effects on tumour control and survival.
We have previously shown that Epo in combination with surgical trauma can stimulate growth of xenotransplanted tumours , while there was no growth stimulating effect of Epo alone. Later, we showed that the combination effect of Epo and surgery did not involve a direct interaction between Epo and the tumour cells .
In the present work, we analysed xenografted tumours using DNA microarrays in order to establish which cellular pathways that might be affected by Epo when combined with surgery.
The tumour line LU-HNSCC-7 was originally established from a moderately differentiated squamous cell carcinoma of the bucca (T2N0M0). It is aneuploid and without p53 mutation or cyclin D1 gene amplification .
Establishment of xenograft
Where V = volume. L = length, and W = width
The mice were also weighed regularly. Tumours with a volume of smaller than 40 mm3 or greater than 300 mm3 at the time of surgery were excluded from the analysis, so were animals showing weight loss in order to ensure undisturbed logarithmic growth.
Administration of erythropoietin
Recombinant human Epo (NeoRecormone, Roche; 400 IU/kg body weight) or physiological saline (placebo) was administered by subcutaneous injection (10 μL/g body weight) every third day starting from the day of transplantation.
Surgical procedure and sampling of tumours
The establishment of the solid malignant xenografts was confirmed using histological examination with hematoxylin and eosin staining performed in conjunction with the harvesting of tumours.
RNA was extracted from the tumour samples and microarray hybridisation was performed using the Illumina Human-6 Expression BeadChip KitVersion-2 (Illumina Inc., San Diego, CA, USA). The scanning was performed on Illumina Bead Array Reader (Illumina Inc., San Diego, CA, USA). The analysis of the fluorescent signals was performed using Multiexperiment Viewer software (MeV, Dana-Farber Cancer Institute, Boston, MA).
Quantification of mRNA by qRT-PCR
Extraction of RNA was done with the AllPrep DNA/RNA Mini kit from Qiagen (Hilden, Germany) according to the manufacturer’s instructions. The expression of mRNA was measured by TaqMan gene expression assays from Applied Biosystems (Carlsbad, CA, USA) (DIP, BCL2L13, CASP1, MIF, CARD10, AIFM1, BIK, and BID with FAM labelled probes, ID: Hs00209789_m1, Hs00354836_m1, Hs00354836_m1, Hs00236988_g1, Hs00367225_m1, Hs00377585_m1, Hs00609635_m1, and Hs00609632_m1 respectively, and GAPDH with a VIC labelled probe, cat. no: 4326317E) with the Rotor-Gene Multiplex RT-PCR kit (QIAGEN, Hilden, Germany) in a Rotor-Gene RG-3000 (Corbette Research, St. Neots, UK) with the following program: reverse transcription 15 min, 50°C followed by 5 min at 95°C and then 15 s at 95°C and 15 s at 60°C in 40 cycles.
Immunohistochemical analysis of apoptosis
Tumours were cut in 4-μm sections and stained using the TechMate 500 autostainer (Ventana Biotek, Tucson, AZ, USA). The primary antibody was anti-active caspase-3 antibody (cat. no. AF835, R&D Systems, Minneapolis, MN, USA). ChemMate EnVision Detection Kit (DakoCytomation, Glostrup, Denmark) was used for detection. In each of the tumour samples, the number of stained apoptotic cells was counted in three fields with a 40× objective.
The qRT-PCR results were analysed by 2-way ANOVA and in the apoptosis analysis the groups were compared using an independent samples Mann–Whitney test.
For the DNA microarray analysis, five tumours were set up in a total of six groups – three receiving Epo and three placebo (Figure 1). Of the tumours, three were excluded as the tumour sizes were outside the set limits and one due to failed hybridization. The tumours were analysed at two time points, 24 and 48 hours after surgery, since our previous results showed that the main effect of Epo ended within 48 hours after surgery and we were interested in early processes within this interval . A total of 13,461 genes were analyzed. The microarray data was deposited at the Gene Expression Omnibus (GEO) at the National Center for Biotechnology Information (NCBI) (see Additional file 1).
In line with previous results, showing that the cell line LU-HNSCC-7 does not express the Epo receptor (EpoR) , this receptor was not significantly expressed in the microarray samples. This was also true for Epo.
A combined 2-way ANOVA analysis of all groups having undergone surgery (group 3–6) showed 1371 Epo-significant genes.
Analysis of the gene expression data using the DAVID functional annotation tool
24 and 48 h
Biological theme (pathway)
6.0 × 10-4
3.0 × 10-2
1.5 × 10-4
Programmed cell death
6.0 × 10-4
6.0 × 10-4
3.7 × 10-4
5.5 × 10-1
2.5 × 10-1
9.7 × 10-1
Blood vessel development
8.3 × 10-1
Response to hypoxia
Quantification of mRNA by qRT-PCR
To verify the down-regulation of pro-apoptotic genes seen in the microarray analysis we selected 7 genes involved in apoptosis (DIP, BCL2L13, CASP1, CARD10, AIFM1, BIK and BID), all displaying decreased expression in the DNA microarray analysis, and performed qRT-PCR to measure the gene expression. This analysis was performed on a separate set of tumours that had not been included in the microarray analysis. Twelve groups of tumours were analysed: two groups each (+/-Epo) 1, 12, 24, 48, and 72 hours after surgical transection.
We chose to collect tumours 12 hours after surgery presuming that the effect on transcription would precede the response at protein and cellular levels. There was no effect after 72 hours (data not shown).We analysed the mRNA level for the following genes, which all displayed decreased expression in the DNA microarray analysis: DIP, BCL2L13, CASP1, CARD10, AIFM1, BIK and BID. The expression of the set of pro-apoptotic genes was significantly decreased at the 1 and 12 hour time points (P < 0.0001 and P = 0.0005 respectively). At 24 hours there was an increase in expression (P = 0.0003), while there was no significant difference at 48 and 72 hours (Figure 2). This indicates an early decrease in the expression of pro-apoptotic genes, followed by a transient up-regulation 24 hours after surgery and then returning to basal expression after 48 hours.
Immunohistochemical assessment of apoptosis
Originally, the stimulating effect of erythropoietin on HNSCC has been assumed to involve angiogenesis and tumour hypoxia. Therefore, an initial assumption in this work was that pathways involved in these activities would be affected. However, we found little change in the expression of genes involved in growth and angiogenesis but, on the other hand, we found an interesting decrease in the expression of pro-apoptotic genes. These results were further verified by qRT-PCR and immunohistochemical analysis of apoptosis.
In normal erythropoiesis, Epo has important apoptosis-inhibiting effects [20, 21] and it has also been shown to protect hypoxic neurons from apoptosis . Earlier data suggest that this effect does not become apparent when Epo is given alone but when the tumour is subject to some kind of concurrent stress, for example cisplatinum treatment  or irradiation .
Surgical trauma can also stimulate proliferation through wound healing . Many mechanisms in wound healing, e.g. paracrine growth factor signalling , angiogenesis  and DNA-replication initiation  are also disturbed in tumorigenesis, showing a close connection between the mechanisms of wound healing and tumour development and growth. We have previously seen increased tumour growth in vivo for this cell line after surgery while under Epo-treatment  but not at a cellular level in vitro, and the cell line was shown not to express the Epo receptor . This suggests that Epo exerts its effect through interaction with stroma cells.
A hypothesis derived from the present study is that the early wound healing response (resulting from the surgical procedure) in combination with a secondary effect of Epo, mediated by stromal cells, suppresses the apoptotic potential within the tumour. The apoptosis-inhibiting effect of erythropoietin can be the common mechanism for the increased tumour survival when it is combined with any treatment – surgery, radiation or chemotherapy.
Wound healing is a long multi-stage process involving inflammatory, proliferative and proliferative phases. In this study, we focused on the early inflammatory phase since it was during the first 48 hours that we previously had seen a growth delay in this model .
Our findings have important clinical consequences since this model of surgical trauma can be applicable to minimal residual disease after surgery. The remaining tumour tissue remains susceptible to the wound healing response in which Epo signalling plays a role . It is particularly interesting considering the result of the study by Henke et al. since they found a particularly worse prognosis in their stratum 2, i.e. those patients who underwent incomplete surgery and received erythropoietin during postoperative radiotherapy . It must be pointed out that a diagnostic biopsy also induces a surgical trauma and a subsequent wound healing process. The result also underlines the possibility of an anti-apoptotic approach in future cancer treatment.
For the understanding of tumour survival and growth, we must not only consider the innate properties of the tumour cells. We must also take into account the almost parasitic approach with which the tumour interacts with the surrounding stroma.
Surgery damages tissue and triggers a stressful wound healing response. The use of antiapoptotic substances, such as Epo, increases tumour cell survival when the tissue is under stress. The use of Epo to patients undergoing tumour treatment, including surgery, is therefore counterproductive and possibly hazardous.
Our work was supported by the Swedish Cancer Society, the King Gustaf V Jubilee Fund, Governmental funding of clinical research within the National Health System Region of Scandia R&D funding, the Foundations of the Lund University Hospital, the Gunnar Nilsson Cancer Foundation, the Crafoord Foundation, the Berta Kamprad Foundation for Investigation and Control of Cancer Diseases and the Laryngology Fund.
- Ferlay J, Parkin DM: Estimates of worldwide burden of cancer in 2008: GLOBOCAN. Int J Cancer. 2008, 46: 765-781.Google Scholar
- Ferley J, Autier P, Boniol M, Heanue M, Colombet M, Boyle P: Estimates of the cancer incidence and mortality in Europe in 2006. Ann Oncol. 2007, 18: 582-592.Google Scholar
- Grogan M, Thomas GM, Melamed I, Wong FL, Pearcey RG, Joseph PK, Portelance L, Crook J, Jones KD: The importance of hemoglobin levels during radiotherapy for carcinoma of the cervix. Cancer. 1999, 86 (8): 1528-1536. 10.1002/(SICI)1097-0142(19991015)86:8<1528::AID-CNCR20>3.0.CO;2-E.View ArticlePubMedGoogle Scholar
- Frommhold H, Guttenberger R, Henke M: The impact of blood hemoglobin content on the outcome of radiotherapy. The Freiburg experience. Strahlenther Onkol. 1998, 174 (Suppl 4): 31-34.PubMedGoogle Scholar
- Silver DF, Piver MS: Effects of recombinant human erythropoietin on the antitumor effect of cisplatin in SCID mice bearing human ovarian cancer: a possible oxygen effect. Gynecol Oncol. 1999, 73 (2): 280-284. 10.1006/gyno.1999.5368.View ArticlePubMedGoogle Scholar
- Glaser CM, Millesi W, Kornek GV, Lang S, Schull B, Watzinger F, Selzer E, Lavey RS: Impact of hemoglobin level and use of recombinant erythropoietin on efficacy of preoperative chemoradiation therapy for squamous cell carcinoma of the oral cavity and oropharynx. Int J Radiat Oncol Biol Phys. 2001, 50 (3): 705-715. 10.1016/S0360-3016(01)01488-2.View ArticlePubMedGoogle Scholar
- Wright JR, Ung YC, Julian JA, Pritchard KI, Whelan TJ, Smith C, Szechtman B, Roa W, Mulroy L, Rudinskas L, Gagnon B, Okawara GS, Levine MN: Randomized, double-blind, placebo-controlled trial of erythropoietin in non-small-cell lung cancer with disease-related anemia. J Clin Oncol. 2007, 25 (9): 1027-1032. 10.1200/JCO.2006.07.1514.View ArticlePubMedGoogle Scholar
- Henke M, Laszig R, Rube C, Schafer U, Haase KD, Schilcher B, Mose S, Beer KT, Burger U, Dougherty C, Frommhold H: Erythropoietin to treat head and neck cancer patients with anaemia undergoing radiotherapy: randomised, double-blind, placebo-controlled trial. Lancet. 2003, 362 (9392): 1255-1260. 10.1016/S0140-6736(03)14567-9.View ArticlePubMedGoogle Scholar
- Leyland-Jones B, Semiglazov V, Pawlicki M, Pienkowski T, Tjulandin S, Manikhas G, Makhson A, Roth A, Dodwell D, Baselga J, Biakhov M, Valuckas K, Voznyi E, Liu X, Vercammen: Maintaining normal hemoglobin levels with epoetin alfa in mainly nonanemic patients with metastatic breast cancer receiving first-line chemotherapy: a survival study. J Clin Oncol. 2005, 23 (25): 5960-5972. 10.1200/JCO.2005.06.150.View ArticlePubMedGoogle Scholar
- Lai SY, Lai SY, Childs EE, Xi S, Coppelli FM, Gooding WE, Wells A, Ferris RL, Grandis JR: Erythropoietin-mediated activation of JAK-STAT signaling contributes to cellular invasion in head and neck squamous cell carcinoma. Oncogene. 2005, 24 (27): 4442-4449. 10.1038/sj.onc.1208635.View ArticlePubMedGoogle Scholar
- Mohyeldin A, Lu H, Dalgard C, Lai SY, Cohen N, Acs G, Verma A: Erythropoietin signaling promotes invasiveness of human head and neck squamous cell carcinoma. Neoplasia. 2005, 7 (5): 537-543. 10.1593/neo.04685.View ArticlePubMedPubMed CentralGoogle Scholar
- Abhold E, Rahimy E, Wang-Rodriguez J, Blair KJ, Yu MA, Brumund KT, Weisman RA, Ongkeko WM: Recombinant human erythropoietin promotes the acquisition of a malignant phenotype in head and neck squamous cell carcinoma cell lines in vitro. BMC Res Notes. 4: 553-Google Scholar
- Kjellen E, Sasaki Y, Kjellstrom J, Zackrisson B, Wennerberg J: Recombinant erythropoietin beta enhances growth of xenografted human squamous cell carcinoma of the head and neck after surgical trauma. Acta Otolaryngol. 2006, 126 (5): 545-547. 10.1080/00016480500437427.View ArticlePubMedGoogle Scholar
- Sasaki Y, Kjellen E, Mineta H, Wennerberg J, Ekblad L: No direct effects of erythropoietin beta on a head and neck squamous cell carcinoma cell line which is growth stimulated in vivo. Acta Oncol. 2009, 48 (7): 1062-1069. 10.1080/02841860902913553.View ArticlePubMedGoogle Scholar
- Henriksson E, Baldetorp B, Borg A, Kjellen E, Akervall J, Wennerberg J, Wahlberg P: p53 mutation and cyclin D1 amplification correlate with cisplatin sensitivity in xenografted human squamous cell carcinomas from head and neck. Acta Oncol. 2006, 45 (3): 300-305. 10.1080/02841860600547380.View ArticlePubMedGoogle Scholar
- Wennerberg J: Changes in growth pattern of human squamous-cell carcinomas of the head and neck during serial passages in nude mice. Int J Cancer. 1984, 33 (2): 245-250. 10.1002/ijc.2910330213.View ArticlePubMedGoogle Scholar
- da Huang W, Sherman BT, Lempicki RA: Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc. 2009, 4 (1): 44-57.View ArticlePubMedGoogle Scholar
- da Huang W, Sherman BT, Lempicki RA: Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res. 2009, 37 (1): 1-13. 10.1093/nar/gkn923.View ArticlePubMedGoogle Scholar
- Hosack DA, Dennis G, Sherman BT, Lane HC, Lempicki RA: Identifying biological themes within lists of genes with EASE. Genome Biol. 2003, 4 (10): R70-10.1186/gb-2003-4-10-r70.View ArticlePubMedPubMed CentralGoogle Scholar
- Silva M, Benito A, Sanz C, Prosper F, Ekhterae D, Nuñez G, Fernandez-Luna JL: Erythropoietin can induce the expression of bcl-x(L) through Stat5 in erythropoietin-dependent progenitor cell lines. J Biol Chem. 1999, 274 (32): 22165-22169. 10.1074/jbc.274.32.22165.View ArticlePubMedGoogle Scholar
- Dolznig H, Habermann B, Stangl K, Deiner EM, Moriggl R, Beug H, Mullner EW: Apoptosis protection by the Epo target Bcl-X(L) allows factor-independent differentiation of primary erythroblasts. Curr Biol. 2002, 12 (13): 1076-1085. 10.1016/S0960-9822(02)00930-2.View ArticlePubMedGoogle Scholar
- Kawakami M, Sekiguchi M, Sato K, Kozaki S, Takahashi M: Erythropoietin Receptor-madiated Inhibition of Exocytotic Glutamate Release Confers Neuroprotection during Chemical Ischemia. J Biol Chem. 2001, 276: 39469-39475. 10.1074/jbc.M105832200.View ArticlePubMedGoogle Scholar
- Hardee ME, Rabbani ZN, Arcasoy MO, Kirkpatrick JP, Vujaskovic Z, Dewhirst MW, Blackwell KL: Erythropoietin inhibits apoptosis in breast cancer cells via an Akt-dependent pathway without modulating in vivo chemosensitivity. Mol Cancer Ther. 2006, 5 (2): 356-361. 10.1158/1535-7163.MCT-05-0196.View ArticlePubMedGoogle Scholar
- Ekblad L, Lindgren G, Persson E, Kjellen E, Wennerberg J: Cell-line-specific stimulation of tumor cell aggressiveness by wound healing factors – a central role for STAT3. BMC Cancer. 2013, 13 (1): 33-10.1186/1471-2407-13-33.View ArticlePubMedPubMed CentralGoogle Scholar
- Elenbaas B, Weinberg RA: Heterotypic signaling between epithelial tumor cells and fibroblasts in carcinoma formation. Exp Cell Res. 2001, 264 (1): 169-184. 10.1006/excr.2000.5133.View ArticlePubMedGoogle Scholar
- Haroon ZA, Amin K, Jiang X, Arcasoy MO: A novel role for erythropoietin during fibrin-induced wound-healing response. Am J Pathol. 2003, 163 (3): 993-1000. 10.1016/S0002-9440(10)63459-1.View ArticlePubMedPubMed CentralGoogle Scholar
- Riss J, Khanna C, Koo S, Chandramouli GV, Yang HH, Hu Y, Kleiner DE, Rosenwald A, Schaefer CF, Ben-Sasson SA, Yang L, Powell J, Kane DW, Star RA, Aprelikova O, Bauer K, Vasselli JR, Maranchie JK, Kohn KW, Buetow KH, Linehan WM, Weinstein JN, Lee MP, Klausner RD, Barrett JC: Cancers as wounds that do not heal: differences and similarities between renal regeneration/repair and renal cell carcinoma. Cancer Res. 2006, 66 (14): 7216-7224. 10.1158/0008-5472.CAN-06-0040.View ArticlePubMedGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/14/648/prepub
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