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Review
. 2007 Dec;9(12):1122-9.
doi: 10.1593/neo.07694.

Effects of recombinant erythropoietin on breast cancer-initiating cells

Tiffany M Phillips  1 , Kwanghee Kim, Erina Vlashi, William H McBride, Frank Pajonk
Affiliations
Review

Effects of recombinant erythropoietin on breast cancer-initiating cells

Tiffany M Phillips et al. Neoplasia. 2007 Dec.

Abstract

Background: Cancer anemia causes fatigue and correlates with poor treatment outcome. Erythropoietin has been introduced in an attempt to correct these defects. However, five recent clinical trials reported a negative impact of erythropoietin on survival and/or tumor control, indicating that experimental evaluation of a possible direct effect of erythropoietin on cancer cells is required. Cancer recurrence is thought to rely on the proliferation of cancer initiating cells (CICs). In breast cancer, CICs can be identified by phenotypic markers and their fate is controlled by the Notch pathway.

Methods: In this study, we investigated the effect of erythropoietin on CICs in breast cancer cell lines. Levels of erythropoietin receptor (EpoR), CD24, CD44, Jagged-1 expression, and activation of Notch-1 were assessed by flow cytometry. Self-renewing capacity of CICs was investigated in sphere formation assays.

Results: EpoR expression was found on the surface of CICs. Recombinant human Epo (rhEpo) increased the numbers of CICs and self-renewing capacity in a Notch-dependent fashion by induction of Jagged-1. Inhibitors of the Notch pathway and PI3-kinase blocked both effects.

Conclusions: Erythropoietin functionally affects CICs directly. Our observation may explain the negative impact of recombinant Epo on local control and survival of cancer patients with EpoR-positive tumors.

Keywords: CD24-/low/CD44+ breast cancer cells; Epor; Notch; breast cancer-initiating cells; rhEpo.

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Figures

Figure 1
Figure 1
MCF-7, T47D, and MDA-MB-231 monolayer cells were serum-starved for 5 hours, trypsinized, and analyzed for EpoR-expression on the cell surface by flow cytometry. EpoR was expressed on CD44+/CD24-/low cells, the putative stem cell population (gray) as well as on unselected cells (black).
Figure 2
Figure 2
(A) FACS analysis detecting the size of the CD44+/CD24-/low cell population in monolayers and supernatant of MCF-7 cells. Treatment with rhEpo (3 x 1 IU/ml) increased the size of this population 4.8-fold from 2 ± 0.08% to 9.5 ± 1.8% (P < .01, Student's t test). Data from four independent experiments are shown. (B) Representative FACS analysis of MCF-7 monolayer cultures treated with rhEpo (3 x 1 IU/ml) for 3 consecutive days. About 0.9% of the cell in the adherent cell population was CD44+/CD24-/low (left panel). Treatment with rhEpo increased the frequency of CD44+/CD24-/low cells in the supernatant from 2% (middle panel) to 14.9% (right panel). (C and D) Primary sphere formation assay of MCF-7 (C) or MDA-MB-231 (D) cells treated with rhEpo (3 x 1 IU/ml) for 3 consecutive days. RhEpo increased the rate of primary spheres formation (MCF-7: 1 ± 0.4% for untreated cells, 2.9 ± 0.6% for Epo-treated cells; MDA-MB-231: 1.2 ± 0.6 for untreated cells, 4.5 ± 1.1% for Epo-treated cells, P < .01, two-sided Student's t test; means ± SEM). Preincubation (30 minutes) with GSI (5 µM) treatment prevented the rhEpo-induced increase in primary sphere formation (MCF-7: GSI-treated cells, 1 ± 0.4%; Epo + GSI-treated cells, 1.5 ± 0.4%; MDA-MB-231: GSI-treated cells, 1.5 ± 0.7%; Epo + GSI - treated cells, 1.1 ± 0.8%).
Figure 2
Figure 2
(A) FACS analysis detecting the size of the CD44+/CD24-/low cell population in monolayers and supernatant of MCF-7 cells. Treatment with rhEpo (3 x 1 IU/ml) increased the size of this population 4.8-fold from 2 ± 0.08% to 9.5 ± 1.8% (P < .01, Student's t test). Data from four independent experiments are shown. (B) Representative FACS analysis of MCF-7 monolayer cultures treated with rhEpo (3 x 1 IU/ml) for 3 consecutive days. About 0.9% of the cell in the adherent cell population was CD44+/CD24-/low (left panel). Treatment with rhEpo increased the frequency of CD44+/CD24-/low cells in the supernatant from 2% (middle panel) to 14.9% (right panel). (C and D) Primary sphere formation assay of MCF-7 (C) or MDA-MB-231 (D) cells treated with rhEpo (3 x 1 IU/ml) for 3 consecutive days. RhEpo increased the rate of primary spheres formation (MCF-7: 1 ± 0.4% for untreated cells, 2.9 ± 0.6% for Epo-treated cells; MDA-MB-231: 1.2 ± 0.6 for untreated cells, 4.5 ± 1.1% for Epo-treated cells, P < .01, two-sided Student's t test; means ± SEM). Preincubation (30 minutes) with GSI (5 µM) treatment prevented the rhEpo-induced increase in primary sphere formation (MCF-7: GSI-treated cells, 1 ± 0.4%; Epo + GSI-treated cells, 1.5 ± 0.4%; MDA-MB-231: GSI-treated cells, 1.5 ± 0.7%; Epo + GSI - treated cells, 1.1 ± 0.8%).
Figure 3
Figure 3
(A) Treatment of MCF-7 monolayer culture with 1 IU/ml rhEpo for 2 hours caused a 1.4 ± 0.16-fold (mean ± SEM, P = .041, n = 3, two-sided paired Student's t test) induction of Jagged-1 expression. (B) Treatment of MCF-7 monolayer culture with 1 IU/ml rhEpo for 2 hours caused a 1.5 ± 0.19-fold (mean ± SEM; P = .049, n = 3, two-sided paired Student's t test) activation of Notch-1 after 2 hours. Treatment with the PI3K inhibitor LY294002 but not the JAK2 inhibitor genistein prevented rhEpo-induced activation of Notch-1.
Figure 4
Figure 4
Representative FACS analysis of MCF-7 cells, stable transfected with an expression vector coding for constitutive active Notch, or an empty vector (n = 2). Expression of constitutive active Notch increased the population of BCICs.
Figure 5
Figure 5
Representative FACS analysis of Notch-1 activation in MCF-7 cells. Treatment with 1 IU/ml rhEpo for 2 or 4 hours (upper panel: gray line and black line; filled histogram, control) caused activation of Notch-1. This activation was inhibited by pretreatment with a GSI for 30 minutes at 2 and 4 hours (middle and lower panels: gray line, rhEpo; dashed line, rhEpo + GSI; filled histogram, control).
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References

    1. Henke M, Sindlinger F, Ikenberg H, Gerds T, Schumacher M. Blood hemoglobin level and treatment outcome of early breast cancer. Strahlenther Onkol. 2004;180:45–51. - PubMed
    1. Fein DA, Lee WR, Hanlon AL, Ridge JA, Langer CJ, Curran WJ, Jr, Coia LR. Pretreatment hemoglobin level influences local control and survival of T1–T2 squamous cell carcinomas of the glottic larynx. J Clin Oncol. 1995;13:2077–2083. - PubMed
    1. Lee WR, Berkey B, Marcial V, Fu KK, Cooper JS, Vikram B, Coia LR, Rotman M, Ortiz H. Anemia is associated with decreased survival and increased locoregional failure in patients with locally advanced head and neck carcinoma: a secondary analysis of RTOG 85-27. Int J Radiat Oncol Biol Phys. 1998;42:1069–1075. - PubMed
    1. Dubray B, Mosseri V, Brunin F, Jaulerry C, Poncet P, Rodriguez J, Brugere J, Point D, Giraud P, Cosset JM. Anemia is associated with lower local-regional control and survival after radiation therapy for head and neck cancer: a prospective study. Radiology. 1996;201:553–558. - PubMed
    1. 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. - PubMed

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