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01.12.2017 | Research article | Ausgabe 1/2017 Open Access

BMC Cancer 1/2017

Genome-independent hypoxic repression of estrogen receptor alpha in breast cancer cells

Zeitschrift:
BMC Cancer > Ausgabe 1/2017
Autoren:
Mercè Padró, Raymond J. Louie, Brian V. Lananna, Adam J. Krieg, Luika A. Timmerman, Denise A. Chan
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​s12885-017-3140-9) contains supplementary material, which is available to authorized users.

Abstract

Background

About 75–80% of breast tumors express the estrogen receptor alpha (ER-α) and are treated with endocrine-target therapeutics, making this the premier therapeutic modality in the breast cancer clinic. However, acquired resistance is common and about 20% of resistant tumors loose ER-α expression via unknown mechanisms. Inhibition of ER-α loss could improve endocrine therapeutic efficacy, benefiting a significant number of patients. Here we test whether tumor hypoxia might commonly produce ER-α loss.

Methods

Using standard molecular and cellular biological assays and a work station/incubator with controllable oxygen levels, we analyze the effects of hypoxia on ER-α protein, mRNA, and transcriptional activity in a panel of independently-derived ER-α positive cell lines. These lines were chosen to represent the diverse genetic backgrounds and mutations commonly present in ER-α positive tumors. Using shRNA-mediated knockdown and overexpression studies we also elucidate the role of hypoxia-inducible factor 1-alpha (HIF-1α) in the hypoxia-induced decrease in ER-α abundance.

Results

We present the first comprehensive overview of the effects of bona fide low environmental oxygen (hypoxia) and HIF-1α activity on ER-α abundance and transcriptional activity. We find that stabilized HIF-1α induces rapid loss of ER-α protein in all members of our diverse panel of breast cancer cell lines, which involves proteolysis rather than transcriptional repression. Reduced ER-α severely attenuates ER-α directed transcription, and inhibits cell proliferation without overt signs of cell death in the cell lines tested, despite their varying genomic backgrounds.

Conclusions

These studies reveal a common hypoxia response that produces reduced ER-α expression and cell cycle stalling, and demonstrate a common role for HIF-1α in ER-α loss. We hypothesize that inhibitors of HIF-1α or the proteasome might stabilize ER-α expression in breast tumors in vivo, and work in combination with endocrine therapies to reduce resistance. Our data also suggests that disease re-occurrence in patients with ER-α positive tumors may arise from tumor cells chronically resident in hypoxic environments. We hypothesize that these non-proliferating cells may survive undetected until conditions change to oxygenate the environment, or cells eventually switch to proliferation via other signaling pathways.
Zusatzmaterial
Additional file 1: Cell line characteristics. Data assembled from the citations listed in the table. nd, not determined; wt, wildtype; hom, homozygous; het, heterozygous; mis, missense; bal, balanced. Numbers from Neve 2006 represent relative signal intensity on Western blot. (DOCX 20 kb)
12885_2017_3140_MOESM1_ESM.docx
Additional file 2: Standard culture media has estrogenic effects. ER activity analyzed by a reporter assay at normoxia in estradiol-free media, E2 (−); standard culture RPMI or DMEM; or defined estrogen media, E2 (+). (A) MCF7 cell lines and (B) T47D cell lines. See cell culture methods section for media compositions. (PDF 362 kb)
12885_2017_3140_MOESM2_ESM.pdf
Additional file 3: (A) qPCR analysis of mRNA levels of ESR1 and HIF1A at normoxia (N) or hypoxia (H) (1% O2, 24 h) treated with DMSO or MG132. Relative mRNA levels normalized to TBP. (B-D) Averages and standard deviations of band intensities calculated for all repeats of each western blot in Fig.  3. Specific band intensities normalized to the loading control bands (β-actin). (B) HIF-1α and ER-α protein from Fig.  3b. (C) phospho-p70-S6K and phospho-4E-BP1 protein from Fig.  3c. (D) HIF-1α and ER-α protein from Fig.  3d. The ER-α graphs represent ER-α normalized to compare the increase in ER-α protein levels generated by MG132 treatment in normoxic versus hypoxic conditions. * MCF7 ER-α levels are significantly different, alpha = 0.05, p = 0.044). (PDF 1787 kb)
Additional file 4: Averages of ER-α band intensities for all repeats of each western blot in Fig.  1. (A) from all experimental replicates of Fig.  1a. (B) for Fig.  1b. (C) for Fig.  1d. Specific band intensities normalized to the loading control bands (β-actin). (PDF 551 kb)
Additional file 5: Averages and standard deviations of ER-α band intensities calculated for all repeats of each western blot in Fig.  1a. Specific band intensities normalized to the loading control bands (β-actin). Calculations derived from at least three independent experiments. (DOCX 15 kb)
Additional file 6: Averages and standard deviations of band intensities calculated for all repeats of each western blot in Fig.  1b. Specific band intensities normalized to the loading control bands (β-actin). Calculations derived from at least three independent experiments. (DOCX 15 kb)
Additional file 8: Averages and standard deviations of band intensities calculated for all repeats of each western blot in Fig.  2a. Specific band intensities normalized to the loading control bands (β-actin). Calculations derived from at least three independent experiments. (DOCX 17 kb)
Additional file 9: Averages and standard deviations of band intensities calculated for all repeats of each western blot in Fig.  2c. Specific band intensities normalized to the loading control bands (β-actin). Calculations derived from at least three independent experiments. (DOCX 17 kb)
Additional file 10: Averages and standard deviations of band intensities calculated for all repeats of each western blot in Additional file 7B. Specific band intensities normalized to the loading control bands (β-actin). Calculations derived from at least three independent experiments. (DOCX 16 kb)
Additional file 11: Averages and standard deviations of band intensities calculated for all repeats of each western blot in Fig.  2d. Specific band intensities normalized to the loading control bands (β-actin). Calculations derived from at least three independent experiments. (DOCX 15 kb)
Additional file 12: Averages and standard deviations of band intensities calculated for all repeats of each western blot in Fig.  3c. Specific band intensities normalized to the loading control bands (β-actin). Calculations derived from at least three independent experiments. (DOCX 15 kb)
Additional file 13: Normalized western blot values for ER-α protein levels from blots used to generate Fig.  3d, and the graphs in Additional file 3D. These values were used to test the relative increase in ER-α levels induced by MG132 treatment in normoxic versus hypoxic conditions for statistical significance. (DOCX 15 kb)
Additional file 14: Averages and standard deviations of band intensities calculated for all repeats of each western blot in Fig.  3d for HIF-1 alpha. Specific band intensities normalized to the loading control bands (β-actin). Calculations derived from at least three independent experiments. (DOCX 15 kb)
Additional file 15: Averages and standard deviations of band intensities calculated for all repeats of each western blot in Fig.  3b. Specific band intensities normalized to the loading control bands (β-actin). Calculations derived from at least three independent experiments. (DOCX 15 kb)
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