Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Mar 1;109(3):1-14.
doi: 10.1093/jnci/djw236.

Estrogen Receptor β as a Therapeutic Target in Breast Cancer Stem Cells

Ran Ma  1   2 , Govindasamy-Muralidharan Karthik  1   2 , John Lövrot  1   2 , Felix Haglund  1   2   3 , Gustaf Rosin  2   4 , Anne Katchy  5 , Xiaonan Zhang  1   2 , Lisa Viberg  1   2 , Jan Frisell  6   7 , Cecilia Williams  4   5   8 , Stig Linder  9 , Irma Fredriksson  6   7 , Johan Hartman  1   2   3
Affiliations

Estrogen Receptor β as a Therapeutic Target in Breast Cancer Stem Cells

Ran Ma et al. J Natl Cancer Inst. .

Abstract

Background: Breast cancer cells with tumor-initiating capabilities (BSCs) are considered to maintain tumor growth and govern metastasis. Hence, targeting BSCs will be crucial to achieve successful treatment of breast cancer.

Methods: We characterized mammospheres derived from more than 40 cancer patients and two breast cancer cell lines for the expression of estrogen receptors (ERs) and stem cell markers. Mammosphere formation and proliferation assays were performed on cells from 19 cancer patients and five healthy individuals after incubation with ER-subtype selective ligands. Transcriptional analysis was performed to identify pathways activated in ERβ-stimulated mammospheres and verified using in vitro experiments. Xenograft models (n = 4 or 5 per group) were used to study the role of ERs during tumorigenesis.

Results: We identified an absence of ERα but upregulation of ERβ in BSCs associated with phenotypic stem cell markers and responsible for the proliferative role of estrogens. Knockdown of ERβ caused a reduction of mammosphere formation in cell lines and in patient-derived cancer cells (40.7%, 26.8%, and 39.1%, respectively). Gene set enrichment analysis identified glycolysis-related pathways (false discovery rate < 0.001) upregulated in ERβ-activated mammospheres. We observed that tamoxifen or fulvestrant alone was insufficient to block proliferation of patient-derived BSCs while this could be accomplished by a selective inhibitor of ERβ (PHTPP; 53.7% in luminal and 45.5% in triple-negative breast cancers). Furthermore, PHTPP reduced tumor initiation in two patient-derived xenografts (75.9% and 59.1% reduction in tumor volume, respectively) and potentiated tamoxifen-mediated inhibition of tumor growth in MCF7 xenografts.

Conclusion: We identify ERβ as a mediator of estrogen action in BSCs and a novel target for endocrine therapy.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
Evaluation of estrogen receptor (ER)β levels in breast cancer stem cells. A) Correlation between ERβ protein expression (PPG5/10) and molecular subclass category analyzed in 187 patients. The mean grading of ERβ was not statistically significantly different between subtypes (Kruskal-Wallis, P < .49). Mean expression scores are presented. Error bars represent SD. B) Relative number of CD44-positive (CD44+) cells that also express ERβ in breast cancer tumors, based upon immunohistochemistry. In 40.0% of the tumors, 100.0% of the CD44+ cells were also positive for ERβ (red). In 15.0% of the tumors, 50.0%–99.0% of the CD44+ cells also expressed ERβ (green). In 16.0% of the tumors, 1.0%–50.0% of the CD44+ cells also expressed ERβ (purple), whereas 29.0% of the tumors did not express any ERβ in the CD44+ cells (blue). In total, 71.0% of the patients showed ERβ/CD44 co-expression. Representative dual immunohistochemistry image is shown in the right corner (ERβ: brown, CD44: red). Scale bar = 100 µm. C) Dual immunofluorescence imaging of patient-derived breast cancer cells with tumor-initiating capabilities in the following order: ERα (green) and CD24 (red), ERβ (red) and CD44 (green), ERβ (red) and ALDH1 (green), ERβ (green) and PKH26-staining (yellow/brown). All counterstaining with DAPI (blue). Scale bar = 10 µm. D) Left panel: quantitative polymerase chain reaction (qPCR) analysis of ERα and ERβ (Student’s t test, mean ± SD, 3 replicates) in MCF7-adherent cells (white bars) and spheres (black bars). Right panel: Immunofluorescence imaging of MCF7 adherent cells and spheres: ERα (green) and ERβ (red) counterstained with DAPI (blue). Scale bar = 10 µm. E) Left panel: qPCR analysis of ERβ (Student’s t test, mean ± SD, 3 replicates) in MDA-MB-231-adherent cells and MDA-MB-231-derived spheres. Right panel: Immunofluorescence imaging of MDA-MB-231-adherent cells and MDA-MB-231 spheres: ERβ (red) counterstained with DAPI (blue). Scale bar = 10 µm. All statistical tests were two-sided. DAPI = 4’,6-diamidino-2-phenylindole; ER = estrogen receptor; HER2 = human epidermal growth factor receptor.
Figure 2.
Figure 2.
Estrogen receptor (ER)β and the cancer stem cell phenotype. A) Average number of mammospheres in control vs shRNA-ERβ knockdown clones generated from 2000 MCF7 cells after seven days of incubation in nonadherent conditions, with representative images presented below each bar (Student’s t test, mean ± SD, 3 replicates). Scale bar = 100 µm. B) Number of mammospheres formed by MCF7 cells, with transduced ERβ expression (black bar) and without (white bar) over seven passages. An equal number of cells was seeded for each passage (Student’s t test, mean ± SD, 3 replicates). C) Forced differentiation of breast cancer cells with tumor-initiating capabilities by incubation in selective medium supplemented with 5% fetal bovine serum induced a switch from nonadherent to a spindle-like, adherent cell phenotype and stained for ERβ and ALDH1 (n = 8 patients). Scale bar = 10 µm. D) Numbers of patient-derived mammospheres after lentiviral shRNA-mediated knockdown of ERβ compared with treatment with the nontargeted scrambled shRNA construct as control. Following lentiviral transduction, cells were incubated in selective medium for seven days (Student’s t test, mean ± SD, n = 4 patients, 4 replicates). E) Immunofluorescence imaging of patient-derived cells after lentiviral shRNA-mediated knockdown of ERβ, with antibodies for ERβ (red) and ALDH1 (green) counterstained with DAPI (blue). Scale bar = 10 µm. All statistical tests were two-sided. ALDH1 = Aldehyde dehydrogenase 1; BSC = breast cancer cells with tumor-initiating capabilities; DAPI = 4’,6-diamidino-2-phenylindole; ER = estrogen receptor; sh = small hairpin RNA.
Figure 3.
Figure 3.
Regulation of breast cancer cells with tumor-initiating capabilities, proliferation by selective estrogen receptor modulators. A) WST-1 assay was performed on MCF7- and MDA-MB-231-adherent cells (5000 cells, Student’s t test, mean ± SD, 3 replicates) during four days of treatment with estradiol (E2) and diarylproprionitrile (DPN) at 10 nM concentration. B) Mammosphere formation assay was carried out using MCF7 and MDA-MB-231 cells (2000 cells, Student’s t test, mean ± SD, 3 replicates) during seven days with E2 and DPN treatment at 10 nM concentration. Mammospheres were manually counted using a brightfield microscope. C) Mammosphere formation from patient-derived cancer cells; 500–1000 cells from dissociated primary breast cancer mammospheres were seeded and incubated together with 10 nM E2, 100 nM 4-hydroxytamoxifen (4OHT), 10 nM ICI-182,780 (fulvestrant), 10 nM DPN, 10 nM propylpyrazoletrisphenol (PPT), or vehicle control for 12 days. (Student’s t test, mean ± SD, n = 12 patients, 4 replicates). D) Single cells from dissociated primary mammospheres treated as described above were incubated with 10 µM BrdU in conditional medium for 72 hours. Absolute number of BrdU-positive cells was estimated under fluorescent microscope. Representative immunofluorescent staining and bar plot representing the relative percentage of positive BrdU cells from one patient. Scale bar = 10 µm. E) Formation of mammospheres from mammary stem cells (MSCs); 4–5000 primary mammary epithelial cells plated onto 48-well plates and incubated with 10 nM DPN, 10 nM PPT, and vehicle control for 12 days (Student’s t test, mean ± SD, n = 5 patients, 4 replicates). F) Hypothetical model of estrogen receptor (ER)α and ERβ action in CSCs vs differentiated cancer cells. All statistical tests were two-sided. 4OHT = 4-hydroxytamoxifen; CSC = cancer stem cells; DPN = diarylproprionitrile; ER = estrogen receptor; PPT = propylpyrazoletrisphenol. ICI-182,780 = 7α,17β-[9-[(4,4,5,5,5-Pentafluoropentyl)sulfinyl]nonyl]estra-1,3,5(10)-triene-3,17-diol.
Figure 4.
Figure 4.
Glycolytic metabolism regulated by estrogen receptor (ER)β. A) Gene set enrichment plot of statistically significantly upregulated pathways in diarylproprionitrile (DPN) stimulated MCF7S (false discovery rate < 0.001). B) MCF7 spheres were seeded into selective medium as single cells at 10 000 cells/well in the presence of 10 nM DPN, 100 nM PHTPP, combined treatment, and vehicle control. After 24-hour incubation, secreted L-lactate concentrations in the culture supernatants were determined (Student’s t test, mean ± SD, 5 replicates). C) MCF7 scramble shRNA cells and ERβ knockdown clones were seeded into selective medium at 10 000 cells/well, supplied with 10 nM DPN over 24 hours. Secreted L-lactate concentrations in the culture supernatants were determined (Student’s t test, mean ± SD, 5 replicates) afterward. D) Stable spheres from two patients were seeded as single cells into selective medium at 5000 cells/well. Treatments were given as 10 nM DPN, 100 nM PHTPP, combined treatment, and vehicle control. Secreted L-lactate concentrations in the culture supernatants were determined (Student’s t test, mean ± SD, n = 2 patients, 5 replicates) after 24 hours of incubation. E) Upper panel: Time course of oxygen consumption rate (OCR) changes were measured during different incubations: 10 nM DPN, 100 nM PHTPP, combined treatment, and vehicle control in first-generation MCF7 spheres. Statistical significance was calculated between PHTPP or DPN vs control group, respectively (Student’s t test, mean ± SD, 4 replicates). Arrows represent the following reagents used during the time course of measurements. A: glucose; B: oligomycin-ATP coupler; C: FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone)–electron transport chain accelerator; D: rotenone+antimycin–mitochondria inhibitors. Lower panel: Quantification of maximal rate (FCCP OCR–rotenone+antimypcin OCR) (Student’s t test, mean ± SD, 4 replicates). F) Relative mRNA expression levels of mitochondrial ND-1 and glycolytic enzymes in first-generation spheres from ERβ knockdown MCF7 as compared with scrambled vehicle control MCF7 (Student’s t test, mean ± SD, 6 replicates). G) Left panel: relative mRNA expression levels of mitochondrial ND-1 and glycolytic enzymes in first-generation spheres from scrambled control shRNA MCF7 treated with DPN as compared with scrambled vehicle control (Student’s t test, mean ± SD, 6 replicates). Right panel: The relative mRNA expression levels of the same genes in first-generation spheres from ERβ knockdown MCF7 treated with DPN as compared with vehicle control (Student’s t test, mean ± SD, 6 replicates). All statistical tests were two-sided. DPN = diarylproprionitrile; ENO1 = enolase 1; ER = estrogen receptor; HK2 = hexokinase 2-HK2; LDHA = lactate dehydrogenase A; MT-ND1 = mitochondrial NADH:ubiquinone oxidoreductase core subunit 1; NES = normalized enrichment score; PKM2 = pyruvate kinase muscle 2; sh = small hairpin RNA.
Figure 4.
Figure 4.
Glycolytic metabolism regulated by estrogen receptor (ER)β. A) Gene set enrichment plot of statistically significantly upregulated pathways in diarylproprionitrile (DPN) stimulated MCF7S (false discovery rate < 0.001). B) MCF7 spheres were seeded into selective medium as single cells at 10 000 cells/well in the presence of 10 nM DPN, 100 nM PHTPP, combined treatment, and vehicle control. After 24-hour incubation, secreted L-lactate concentrations in the culture supernatants were determined (Student’s t test, mean ± SD, 5 replicates). C) MCF7 scramble shRNA cells and ERβ knockdown clones were seeded into selective medium at 10 000 cells/well, supplied with 10 nM DPN over 24 hours. Secreted L-lactate concentrations in the culture supernatants were determined (Student’s t test, mean ± SD, 5 replicates) afterward. D) Stable spheres from two patients were seeded as single cells into selective medium at 5000 cells/well. Treatments were given as 10 nM DPN, 100 nM PHTPP, combined treatment, and vehicle control. Secreted L-lactate concentrations in the culture supernatants were determined (Student’s t test, mean ± SD, n = 2 patients, 5 replicates) after 24 hours of incubation. E) Upper panel: Time course of oxygen consumption rate (OCR) changes were measured during different incubations: 10 nM DPN, 100 nM PHTPP, combined treatment, and vehicle control in first-generation MCF7 spheres. Statistical significance was calculated between PHTPP or DPN vs control group, respectively (Student’s t test, mean ± SD, 4 replicates). Arrows represent the following reagents used during the time course of measurements. A: glucose; B: oligomycin-ATP coupler; C: FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone)–electron transport chain accelerator; D: rotenone+antimycin–mitochondria inhibitors. Lower panel: Quantification of maximal rate (FCCP OCR–rotenone+antimypcin OCR) (Student’s t test, mean ± SD, 4 replicates). F) Relative mRNA expression levels of mitochondrial ND-1 and glycolytic enzymes in first-generation spheres from ERβ knockdown MCF7 as compared with scrambled vehicle control MCF7 (Student’s t test, mean ± SD, 6 replicates). G) Left panel: relative mRNA expression levels of mitochondrial ND-1 and glycolytic enzymes in first-generation spheres from scrambled control shRNA MCF7 treated with DPN as compared with scrambled vehicle control (Student’s t test, mean ± SD, 6 replicates). Right panel: The relative mRNA expression levels of the same genes in first-generation spheres from ERβ knockdown MCF7 treated with DPN as compared with vehicle control (Student’s t test, mean ± SD, 6 replicates). All statistical tests were two-sided. DPN = diarylproprionitrile; ENO1 = enolase 1; ER = estrogen receptor; HK2 = hexokinase 2-HK2; LDHA = lactate dehydrogenase A; MT-ND1 = mitochondrial NADH:ubiquinone oxidoreductase core subunit 1; NES = normalized enrichment score; PKM2 = pyruvate kinase muscle 2; sh = small hairpin RNA.
Figure 5.
Figure 5.
Tumor growth and mammosphere formation in response to estrogen receptor (ER)β inhibition. Ovariectomized NOD/SCID mice were implanted with either (A) 1x106 adherent MCF7 cells (n = 5 mice/group, tumor take rate = 77.5%) or (B) 5x105 MCF7-derived mammospheres (n = 4 mice/group, tumor take rate = 85.0%) into the fourth mammary gland fat pads and randomly assigned to control or treatment groups (control DMSO: diarylproprionitrile [DPN; 4 mg/kg]; PPT [10 mg/kg]; E2 [0.1 mg/kg]; PHTPP [4 mg/kg]). Treatments were given by daily injections. The final tumor volume (mm3) was determined (Student’s t test, mean ± SD). C) Intact NOD/SCID mice were transplanted with 2 x106 doxycycline inducible shRNA-ERβ MDA231 cell or scrambled control shRNA transcended MDA-MB-231 cells (Student’s t test, mean± SD, n = 4 mice/group, tumor take rate = 100.0%). Doxycycline (2 mg/mL) was provided in the drinking water. D) Intact NOD/SCID mice were transplanted with 2 x106 MDA231 cells for control (vehicle) and PHTPP treatment groups (n = 4 mice/group, tumor take rate = 100.0%). The treatments were given by injections every second day. Tumors were resected at end point and tumor volume was calculated (Student’s t test, mean ± SD). E) Immunohistochemical staining of ALDH1 in MCF7 xenografts from Figure 5B. Because of the very small size of PHTPP-treated tumors, ALDH1-staining could not be performed. The left panel represents average values (0, 0.0%; 1, 1.0%–10.0%; 2, 10.0%–50.0%; 3, 50.0%–100.0%, positive cytoplasmic stained cells) (Student’s t test, mean± SD, n = 3 counts). Right panel: representative images for ALDH1-staining. Scale bar = 10 µm. F) Average number of mitoses/high-power field (40x) ± SD. Experiments were performed in three tumors per group (Student’s t test, mean ± SD). Mammosphere formation from (G) luminal A (n = 4 patients, 4 replicates) or (H) triple-negative (n = 3 patients, 4 replicates) patient-derived cancer cells. Five hundred to 1000 cells from dissociated primary breast cancer mammospheres were plated onto 48-well plates incubated with 10 nM E2, 100 nM PHTPP, 10 nM DPN, 10 nM E2 + 100 nM PHTPP combination, or vehicle control for 12 days (Student’s t test, mean ± SD). Four-week-old NOD/SCID mice were transplanted with 2 mm3 tumor pieces from PDX HCI001 (n = 5 mice/group, tumor take rate = 100%) (I) or HCI002 (n = 5 mice/group, tumor take rate = 90%) (J) and supplemented with DPN (4 mg/kg), PHTPP (4 mg/kg), or combination treatment every other day. Treatments started when tumor initiation reached at least 3 mm3, and tumor volume was measured at the same time of injection. I and J) Tumor volume (mm3) after end point was calculated (Student’s t test, mean ± SD). K and L) Effect of ERβ agonist and antagonist on triple-negative patient-derived xenografts. Lines show mean of tumor volumes from each treatment group; error bars represent SD. All statistical tests were two-sided. DPN = diarylproprionitrile; HCI001/002 = patient-derived xenograft models; PPT = propylpyrazoletrisphenol.
Figure 6.
Figure 6.
Effects on tumor growth by a combination of tamoxifen and an estrogen receptor (ER)β antagonist. Eight-week-old NOD/SCID mice were injected with 1x106 MCF7 cells and supplemented with tamoxifen citrate (1 mg/kg), or combined with different concentrations of PHTPP by injections (n = 4 mice/group, tumor take rate = 75%). Treatment started on day 14, and MCF7 tumor volume was measured every third day. A) Tumor volume (mm3) was determined after mice were killed at day 39 (Student’s t test, mean ± SD). B) The number after each curve indicates the final measurement of in vivo tumor volume. Lines show mean of tumor volumes from each treatment group; error bars represent SD. All statistical tests were two-sided. Tam = tamoxifen (1 mg/kg); Tam + PHTPP 25 = tamoxifen (1 mg/kg) + PHTPP (1 mg/kg); Tam + PHTPP 50 = tamoxifen (1 mg/kg) + PHTPP (2 mg/kg); Tam + PHTPP 100 = tamoxifen (1 mg/kg) + PHTPP (4 mg/kg).
Figure 7.
Figure 7.
Hypothetical overview of estrogen receptor (ER)β function during breast carcinogenesis. We propose a dual function of ERβ in the human mammary gland depending on a “neoplastic switch,” from a potential inhibitor of mammary stem cell proliferation to an activator in the malignant counterpart. 1) Stimulation of ERβ in normal mammary stem cells decreases proliferation. 2) ERβ drives proliferation in breast cancer cells with tumor-initiating capabilities in the presence of ERβ agonist by inducing glycolytic metabolism, which is essential for CSCs maintenance. 3) In the differentiated primary tumor that is mainly ERα-positive, estrogen induces proliferation as described elsewhere. 4) We propose that by combining standard endocrine therapy (tamoxifen) with an ERβ-selective antagonist (PHTPP) a more profound tumor regression can be achieved through targeting cancer cells with both differentiated and stem-cell phenotypes. CSC = cancer stem cells; ER = estrogen receptor; MSC = mammary stem cells.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Dontu G, Abdallah WM, Foley JM, et al. In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells. Genes Dev. 2003;1710:1253–1270. - PMC - PubMed
    1. Reynolds BA, Tetzlaff W, Weiss S.. A multipotent EGF-responsive striatal embryonic progenitor cell produces neurons and astrocytes. J Neurosci. 1992;1211:4565–4574. - PMC - PubMed
    1. Al-Hajj M, Wicha MS, Benito-Hernandez A, et al. Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci U S A. 2003;1007:3983–3988. - PMC - PubMed
    1. Ma R, Fredriksson I, Karthik GM, et al. Superficial scrapings from breast tumors is a source for biobanking and research purposes. Lab Invest. 2014;947:796–805. - PubMed
    1. Ginestier C, Hur MH, Charafe-Jauffret E, et al. ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome. Cell Stem Cell. 2007;15:555–567. - PMC - PubMed

MeSH terms