Erythropoietin promotes breast tumorigenesis through tumor-initiating cell self-renewal

Abstract

Erythropoietin (EPO) is a hormone that induces red blood cell production. In its recombinant form, EPO is the one of most prescribed drugs to treat anemia, including that arising in cancer patients. In randomized trials, EPO administration to cancer patients has been associated with decreased survival. Here, we investigated the impact of EPO modulation on tumorigenesis. Using genetically engineered mouse models of breast cancer, we found that EPO promoted tumorigenesis by activating JAK/STAT signaling in breast tumor-initiating cells (TICs) and promoted TIC self renewal. We determined that EPO was induced by hypoxia in breast cancer cell lines, but not in human mammary epithelial cells. Additionally, we demonstrated that high levels of endogenous EPO gene expression correlated with shortened relapse-free survival and that pharmacologic JAK2 inhibition was synergistic with chemotherapy for tumor growth inhibition in vivo. These data define an active role for endogenous EPO in breast cancer progression and breast TIC self-renewal and reveal a potential application of EPO pathway inhibition in breast cancer therapy.

Figure 1. EPO does not affect human breast cancer cell lines in vitro.

(A) Indicated cell lines were cultured in the presence of increasing concentrations of EPO (1, 5, and 10 IU/ml, replenished every other day), and proliferation was detected by MTT assay. (B) Cell lines were cultured in the presence of PBS or EPO (1 IU/ml) for 16 hours and EdU for 1 hour. They were then analyzed for EdU incorporation by flow cytometry. (C) Indicated cell lines were treated with vehicle, etoposide (50 μM), or etoposide (50 μM) and EPO (10 IU/ml) for 24 hours. Whole-cell extracts were Western blotted with the indicated antibodies. (D) Indicated cell lines were treated with DMSO, etoposide (50 μM), or etoposide (50 μM) and EPO (10 IU/ml) for 24 hours, then stained with PI and antibodies against annexin V. The percentages of cells that were annexin V positive and PI negative were quantified by flow cytometry. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

Figure 2. EPO decreases the percentage of breast cancer GEMMs living over time yet does not affect breast cancer GEMM cell lines in vitro.

(A) Kaplan-Meier survival curves of MMTV-Neu and C3-Tag mice randomized to saline or EPO (500 IU/kg BIW) injections. (MMTV-Neu: P = 0.05, C3-Tag: P = 0.04). Tumors were sectioned and H&E stained. (B) Indicated cell lines were cultured in the presence of increasing concentrations of EPO (1, 5, and 10 IU/ml, replenished to the culture every other day). and proliferation was detected by MTT assay. (C) Cell lines were cultured in the presence of PBS or EPO (1 IU/ml) for 16 hours and EdU for 1 hour. They were then analyzed for EdU incorporation by flow cytometry. (D) Indicated cell lines were treated with vehicle, etoposide (50 μM), or etoposide (50 μM) and EPO (10 IU/ml) for 24 hours. Whole cell extracts were western blotted with the indicated antibodies. (E) Indicated cell lines were treated with DMSO, etoposide (50 μM), or etoposide (50 μM) and EPO (10 IU/ml) for 24 hours. Percentage of apoptotic cells was then determined by flow analysis using Alexa Fluor 488 annexin V/PI Cell Apoptosis Kit (Invitrogen). ***P ≤ 0.001.

Figure 3. EPO promotes progression of orthotopic C3-Tag and MMTV-Neu tumors.

(A) 1 × 10⁵ luciferase-expressing C3-Tag cells were implanted into the mammary fat pads of FVB/N mice. The mice were randomized to receive PBS or EPO (500 IU/kg BIW). Representative Xenogen images of mice on day 1 and day 21 after injection. (B) Living Image software (Caliper Life Sciences) was used to quantify luciferase activity of orthotopic C3-Tag tumors. Mean photons/s/cm² is graphed with error bars representing SEM. *P ≤ 0.05 on day 21. (C) Tumor volume of orthotopically implanted NT2 cells as measured by calipers. ***P ≤ 0.001. (D and E) Kaplan-Meier curves of percentage of C3-Tag tail vein–injected mice alive in the indicated treatment groups.

Figure 4. EPO promotes mammosphere formation, TIC self renewal, and expansion of TICs in vivo.

(A) Mammosphere formation of FACS-sorted SUM149 cells. 20,000 cells from each subpopulation were plated in triplicate in 6-well plates. PBS or EPO (1 IU/ml) were added every other day, and colonies (≥ 50 μm) were counted on day 5. (B) Representative photos of mammospheres from PBS- or EPO-treated SUM149 cells. Scale bars: 200 mm. (C) Primary mammospheres from SUM149 TICs (CD44⁺CD24^–EpCAM⁺) were dissociated and serial passaged by replating 20,000 cells for each passage. TICs cultured in PBS showed a significant exhaustion of self-renewal capacity after passage 3. (D) Mammosphere formation of Lin^–Thy1⁺CD24⁺ MMTV-Wnt1 cells cultured with PBS or EPO under 21% O₂. Non-Lin^–Thy⁺CD24⁺ cells did not grow spheres. (E) Representative photos of spheres from Lin-Thy1⁺CD24⁺ MMTV-Wnt1 cells treated with PBS or EPO. Original magnification, ×1. (F) Schematic of orthotopically implanted MMTV-Wnt1 tumors treated with PBS or EPO (500 IU/kg i.p. twice a week) for 4 weeks then assessed for TIC frequency by flow cytometry and LDA. (G) MMTV-Wnt1 orthotopic tumors were harvested and stained with antibodies specific to Thy1, CD24, a lineage cocktail, and DAPI. There were a significantly higher percentage of Lin^–Thy⁺CD24⁺ cells in tumors of mice treated with EPO. **P ≤ 0.01; ***P ≤ 0.001.

Figure 5. EPO activates JAK/STAT signaling in breast TICs, and JAK inhibition is a potential therapeutic target in breast cancer.

(A) SUM149 cells were FACS sorted into TIC (CD44⁺CD24^–EpCAM⁺) and non-TIC (not CD44⁺CD24^–EpCAM⁺) populations, treated with PBS or EPO (1 IU/ml) for 10 minutes, and immunoblotted with the indicated antibodies. (B) Gene-expression profiles were generated from total RNA of sorted SUM149 TICs (CD44⁺CD24^–EpCAM⁺), treated with PBS or EPO (1IU/ml) for 16 hours,and subjected to ssGSEA for the indicated JAK/STAT and cancer stem cell gene signatures. (C) Mammosphere formation of CD44⁺CD24^–EpCAM⁺ SUM149 cells cultured at hypoxia (2% O₂) in the presence of control antibody (anti-HA) or anti-EPO antibody at the indicated dilutions. (D) Mammosphere formation of CD44⁺CD24^–EpCAM⁺ SUM149 cells cultured at hypoxia (2% O₂) in the presence of control antibody (anti-HA) or 2 indicated anti-EPOR antibodies (1:200). (E) 20,000 (CD44⁺CD24^–EpCAM⁺) SUM149 cells were plated in duplicate in the presence of EPO (10 IU/ml) and vehicle or EPO and the JAK inhibitor TG101348 at the indicated concentrations. Spheres were counted on day 5. (F) Orthotopic MMTV-Wnt1 tumors were grown to 5 mm in length and width (average tumor volume of all groups = 60 mm³) and treated with vehicle (NT), carboplatin (Carbo), TG101348 (TG), or the combination of carboplatin and TG101348. Caliper measurements were taken weekly. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. (G) Hematocrit (HCT) measurements were done at baseline and at end point in the indicated treatment groups.

Figure 6. Endogenous EPO expression correlates negatively with relapse-free survival and is produced by breast cancer cell lines and tumor endothelial cells.

(A) Breast tumors (337 patients [GSE18229] and 855 patients [GSE26338] were rank ordered based on their EPO expression and divided into those with high and low EPO expression (high: top 2 tertiles, low: bottom tertile). P = 0.0088 for UNC337, P = 0.0012 for GSE26338, log rank test. (B) EPO mRNA levels from HMECs and the indicated breast cancer cell lines cultured in 21% and 1% O₂ using TaqMan quantitative RT-PCR. (C) EPO protein as measured by ELISA on conditioned medium from the indicated cell lines after culture under 21% O₂ or 1% O₂ for 16 hours. (D) Microarray gene expression for EPO was abstracted from a publically available data set (GSE7413) in which breast cancer–associated endothelial cells or endothelial cells from adjacent normal were laser capture microdissected. Relative gene expression Cy5 (tumor)/Cy3(reference) is shown. (E) EPO mRNA levels from HUVECs cultured in 21% and 1% O₂ were measured by TaqMan quantitative PCR. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

Figure 7. Sources of EPO and proposed mechanism of EPO-promoting breast TIC self renewal.

Intratumoral EPO may be derived from several intratumoral cells including stromal cells, cancer-associated endothelial cells, and breast cancer cells as well as exogenous administration. Intratumoral EPO activates EPO-R present on breast TICs that subsequently activates JAK/STAT signaling promoting TIC self renewal and stemness. Potential effects of EPO on the non-TIC fraction will need to be investigated. The JAK inhibitor TG101348 inhibits JAK/STAT signaling and EPO-dependent breast TIC self renewal.