Erythropoietin receptor transcription is neither elevated nor predictive of surface expression in human tumour cells

Abstract

Erythropoietin receptor (EpoR) has been reported to be overexpressed in tumours and has raised safety concerns regarding the use of erythropoiesis-stimulating agents (ESAs) to treat anaemia in cancer patients. To investigate the potential for EpoR to be overexpressed in tumours, we have evaluated human tumours for amplification of the EPOR locus, levels of EPOR transcripts, and expression of surface EpoR protein. Gene amplification analysis of 1083 solid tumours found that amplification of the EPOR locus was rare with frequencies similar to other non-oncogenes. EPOR transcript levels in tumours and tumour cell lines were low in comparison with bone marrow and were equivalent to, or lower than, levels in normal tissues of tumour origin. Although EpoR mRNA was detected in some tumour lines, no EpoR could be detected on the cell surface using (125)I-Epo binding studies. This may be due to the lack of EpoR protein expression or lack of cell-surface-trafficking factors, such as Jak2. Taken together, we have found no evidence that EpoR is overexpressed in tumours or gets to the surface of tumour cells. This suggests that there is no selective advantage for tumours to overexpress EpoR and questions the functional relevance of EpoR gene transcription in tumours.

Figure 1

Genomic organisation of the EPOR locus showing alternatively spliced transcripts and location of primers and probes. Light grey boxes represent coding regions of exons 1–8, open boxes represent untranslated 5′ and 3′ regions, and black boxes represent the transmembrane coding sequences. The three major EPOR transcripts are shown with dashed lines representing normal splicing sites. The hatched boxes represent intronic regions contained in some alternatively spliced forms. Primer/probe sets A, B, C, and D were designed to amplify EPOR fragments within exons 3, 6–7, 8, and 5–8, respectively.

Figure 2

Erythropoietin receptor genomic amplification in tumour samples. Quantitative genomic microarray analysis was performed on 1083 tumours from 15 different tumour types. (A) Per cent of tumours demonstrating genomic amplification of oncogenes cyclin D1 (CCND1), EGFR, and HER2; non-oncogenes β-actin (ACTB), GUSB, and GAPDH; and test locus EPOR. The numbers of tumours with amplicons are shown below the x axis. (B) Per cent of tumours with genomic amplification of genes from panel A present in amplicons <10 Mb plotted against gene copy numbers. (C) Quantitative genomic PCR analysis of the EPOR locus in 68 breast tumour samples. The EPOR-specific primer/probe sets A and C were used to amplify EPOR fragments from exons 3 and 8, respectively. Breast tumour no. 29 had a gain in EPOR copy number (1.6-fold) and no. 50 had a deletion of one EPOR locus (0.4-fold).

Figure 3

Levels of EPOR transcripts in normal vs tumour tissues. Quantitative RT-PCR was used to determine levels of EPOR transcripts in normal and tumour tissues relative to levels of cyclophilin B transcripts. (A) Levels of EPOR transcripts in a panel of normal tissues obtained using primer/probe set B (corroborated with primer/probe set A). EPOR transcript levels relative to cyclophilin were analysed: (B) normal tissues vs tumour tissues and cell lines; (C) brain tumours and normal brain (not patient-matched samples); (D) patient-matched normal vs colon and lung tumour samples; (E) patient-matched head and neck tumour and stroma (A–C indicate different preparations from the same tumour). Bone marrow samples were included as a positive haematopoietic control in all analyses (clear bars). Results obtained using primer/probe set B (Figure 1) are shown for panels A, B, and D and primer set D for panels C and E.

Figure 4

Microarray analysis of levels of EPOR transcripts in normal vs oncogenic samples. Comparative microarray analysis of 121 tumour and 170 normal tissues from breast, colon, kidney, lung, lymph node, ovary, pancreas, prostate, and skin samples. Closed circles represent transcript levels from individual samples using EPOR probe 396_F_AT (EPOR exon 8). Other EPOR probe sets yielded similar intensity profiles. Horizontal, double-headed arrows indicate no statistical difference in EPOR levels between normal and tumour tissues. A single-headed arrow indicates a significant (P<0.05) reduction in levels of EPOR transcripts in tumour tissues compared with normal tissues. No statistical analyses were performed on pancreatic samples because of the lack of a normal control, or on ovary and melanoma samples because of their small sample sizes.

Figure 5

Lack of correlation between levels of EPOR transcripts and EpoR surface expression in tumour cell lines. (A) Quantitative RT-PCR of EPOR in tumour cell lines using primer/probe set C (Figure 1). Similar data were obtained with primer/probe sets A and B (Figure 1; data not shown). (B) Specific binding of ¹²⁵I-rHuEpo to cell lines (combined data from three to four experiments with n=11–17 per bar). Error bars represent s.e.m. (C) Scatchard analysis of rHuEpo binding to UT7/Epo cells. Each point represents the average of three samples. (D) Western blot analysis of Jak2 expression levels. Cyclophilin B is shown as a loading control for western blot analysis.