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The orphan nuclear receptor estrogen-related receptor beta (ERRβ) in triple-negative breast cancer

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Abstract

Purpose

Triple-negative breast cancer (TNBC)/basal-like breast cancer (BLBC) is a highly aggressive form of breast cancer. We previously reported that a small molecule agonist ligand for the orphan nuclear receptor estrogen-related receptor beta (ERRβ or ESRRB) has growth inhibitory and anti-mitotic activity in TNBC cell lines. In this study, we evaluate the association of ESRRB mRNA, copy number levels, and protein expression with demographic, clinicopathological, and gene expression features in breast tumor clinical specimens.

Methods

ESRRB mRNA-level expression and clinical associations were analyzed using RNAseq data. Array-based comparative genomic hybridization determined ESRRB copy number in African-American and Caucasian women. Transcription factor activity was measured using promoter–reporter luciferase assays in TNBC cell lines. Semi-automatic quantification of immunohistochemistry measured ERRβ protein expression on a 150-patient tissue microarray series.

Results

ESRRB mRNA expression is significantly lower in TNBC/BLBC versus other breast cancer subtypes. There is no evidence of ESRRB copy number loss. ESRRB mRNA expression is correlated with the expression of genes associated with neuroactive ligand–receptor interaction, metabolic pathways, and deafness. These genes contain G/C-rich transcription factor binding motifs. The ESRRB message is alternatively spliced into three isoforms, which we show have different transcription factor activity in basal-like versus other TNBC cell lines. We further show that the ERRβ2 and ERRβsf isoforms are broadly expressed in breast tumors at the protein level.

Conclusions

Decreased ESRRB mRNA expression and distinct patterns of ERRβ isoform subcellular localization and transcription factor activity are key features in TNBC/BLBC.

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Abbreviations

TNBC:

Triple-negative breast cancer

BLBC:

Basal-like breast cancer

AA:

African-American

ESRRB:

Estrogen-related receptor beta

IHC:

Immunohistochemistry

ER:

Estrogen receptor

PR:

Progesterone receptor

HER2:

Human epidermal growth factor two

CW:

Caucasian/White

NR:

Nuclear receptor(s)

ONR:

Orphan nuclear receptor

ERR:

Estrogen-related receptors

OS:

Overall survival

SCAN-B:

Sweden Cancerome Analysis Network-Breast

FPKM:

Fragments per kilobase of transcript per million mapped reads

ESR1:

Estrogen receptor

NHG:

Nottingham grade

NTN:

Non-triple-negative breast cancer

aCGH:

Array comparative genomic hybridization

AFR:

African descent

AMR:

Ad mixed American

DEGs:

Differentially expressed genes

BL2:

Basal-like 2

LAR:

Luminal androgen receptor

ML:

Mesenchymal-like

ERRE:

Estrogen-related response element

SP1:

Specificity-protein-1

TMA:

Tissue microarray

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Acknowledgements

We are grateful to Allison Fitzgerald, Dr. Hillary Stires, Ayodeji Olukoya, and Sonali Persaud for their insights and/or critical reading of the manuscript. We would like to thank Drs. Bassem Haddad, Filipa Lynce, and Michael Johnson for their guidance in developing the HTSR’s invasive ductal carcinoma breast cancer tissue microarray series. We thank Henry Cho and Gaelle Palmer for their contributions to the TMA-associated REDCap database. Thank you to the Survey, Recruitment, and Biospecimen collection Shared Resource (SRBSR) for their support of research recruitment at MedStar Georgetown University Hospital. Thank you to Dr. Lao Saal of Lund University, Sweden for kindly providing the clinical information of SCAN-B data. We thank Garrett Graham for his guidance on all computational studies and Dr. Max Kushner for his aid with the DREME analysis. The results shown here are based in part upon data generated by the TCGA Research Network: https://www.cancer.gov/tcga.

Funding

These studies were supported in part by Department of Defense Breast Cancer Research Program Award W81XWH-17-1-0615 to RBR. Georgetown University Medical Center Shared Resources are supported in part by P30 CA051008 (Lombardi Comprehensive Cancer Center Support Grant; Principal Investigator Dr. Louis Weiner). Fellowship funding for AIF was provided by the LCCC’s Graduate Training in Breast Cancer Health Disparities Research Grant from Susan G. Komen for the Cure (GTDR15330383; Principal Investigator Lucile L. Adams-Campbell).

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Authors and Affiliations

  1. Department of Oncology, Georgetown University, Washington, DC, 22209, USA

    Aileen I. Fernandez, Xue Geng, Krysta Chaldekas, Brent Harris, Anju Duttargi, V. Layne Berry, Deborah L. Berry, Akanksha Mahajan, Luciane R. Cavalli, Ming Tan & Rebecca B. Riggins

  2. Research Institute Pelé Pequeno Príncipe Faculdades Pequeno Príncipe, Curitiba, PR, Brazil

    Luciane R. Cavalli

  3. MTA TTK Lendület Cancer Biomarker Research Group and Semmelweis University 2nd Department of Pediatrics, Budapest, Hungary

    Balázs Győrffy

  4. Department of Oncology, Georgetown University, 3970 Reservoir Rd NW, E412 Research Bldg., Washington, DC, 20057, USA

    Aileen I. Fernandez & Rebecca B. Riggins

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  1. Aileen I. Fernandez
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Correspondence to Aileen I. Fernandez or Rebecca B. Riggins.

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10549_2019_5485_MOESM1_ESM.pdf

Supplementary material 1 (PDF 4240 kb) Supplemental Fig. 1. Age at diagnosis and association of ESRRB with OS in SCAN-B and TCGA datasets. a, b Average age of patients in SCAN-B dataset by Pam50 (a) and IHC (b) subtypes. ANOVA with multiple comparisons ***p < 0.001, ****p < 0.0001. c–h KM plots showing overall survival of all breast cancer patients (c, f), BLBC patients (d, g), and TNBC patients (e, h) in SCAN-B (c–e) and TCGA (f–h) data Supplemental Fig. 2. Demographics of aCGH cohort. a–e Distribution of a age, b tumor size and ESRRB copy number, c pathology, d lymph node status and e metastasis Supplemental Fig. 3. Overlap of BLBC patients and TNBC patients. a, b Heat map representing differential expression of overlapping genes in SCAN-B versus TCGA datasets. c, d Venn diagram of patients in SCAN-B (c) and TCGA (d) data. e List of overrepresented motifs in promoter region of DEGs in SCAN-B datasets Supplemental Fig. 4. Protein and mRNA levels of ERRβ/ESRRB. Densitometry quantifying ERRβ splice variants ERRβ2 (a) and ERRβsf (b) protein levels in cell lines. cESRRB mRNA levels in SCAN-B patients, sorted into TNBC subtypes. d, eESRRB mRNA levels in cell lines representing the TNBC cell lines Supplemental Fig. 5. IHC optimization and localization. a Negative and positive controls used for antibody optimization of ERRβsf ERRβ2. Scale bar = 100 μm. b High magnification view representing subcellular staining of tumor cores staining for ERRβsf or ERRβ2. Selected panels for ERRβsf from tumor cores identified by Vectra3 as > 90% nuclear and cytoplasmic staining, or > 90% cytoplasmic staining. Arrows in left panels show positive nuclei Supplemental Table 1. DEGs in ESRRB high and low patients. List of DEGs found in BLBC and TNBC patients from SCAN-B and TCGA datasets Supplemental Table 2. ERRβ isoform expression and subcellular localization. Mean (sd) and median (IQR) expression of ERRβ2 receptor, ERRβsf receptor, and total ERRβ2:total ERRβsf expression (S2.1) and nuclear/cytoplasmic localization of ERRβ2 receptor and ERRβsf (S2.2) in three IHC breast cancer subtypes Supplemental Table 3. ERRβ isoform expression and clinical features. Analysis of ERRβ2 and ERRβsf receptor expression in the IHC subtypes and lymph node status, race, and age, with and without interaction

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Fernandez, A.I., Geng, X., Chaldekas, K. et al. The orphan nuclear receptor estrogen-related receptor beta (ERRβ) in triple-negative breast cancer. Breast Cancer Res Treat 179, 585–604 (2020). https://doi.org/10.1007/s10549-019-05485-5

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