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Insufficient antibody validation challenges oestrogen receptor beta research.

ABSTRACT: The discovery of oestrogen receptor ? (ER?/ESR2) was a landmark discovery. Its reported expression and homology with breast cancer pharmacological target ER? (ESR1) raised hopes for improved endocrine therapies. After 20 years of intense research, this has not materialized. We here perform a rigorous validation of 13 anti-ER? antibodies, using well-characterized controls and a panel of validation methods. We conclude that only one antibody, the rarely used monoclonal PPZ0506, specifically targets ER? in immunohistochemistry. Applying this antibody for protein expression profiling in 44 normal and 21 malignant human tissues, we detect ER? protein in testis, ovary, lymphoid cells, granulosa cell tumours, and a subset of malignant melanoma and thyroid cancers. We do not find evidence of expression in normal or cancerous human breast. This expression pattern aligns well with RNA-seq data, but contradicts a multitude of studies. Our study highlights how inadequately validated antibodies can lead an exciting field astray.

SUBMITTER: Andersson S

PROVIDER: S-EPMC5501969 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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Insufficient antibody validation challenges oestrogen receptor beta research.

Andersson Sandra S Sundberg Mårten M Pristovsek Nusa N Ibrahim Ahmed A Jonsson Philip P Katona Borbala B Clausson Carl-Magnus CM Zieba Agata A Ramström Margareta M Söderberg Ola O Williams Cecilia C Asplund Anna A

Nature communications 20170615

The discovery of oestrogen receptor β (ERβ/ESR2) was a landmark discovery. Its reported expression and homology with breast cancer pharmacological target ERα (ESR1) raised hopes for improved endocrine therapies. After 20 years of intense research, this has not materialized. We here perform a rigorous validation of 13 anti-ERβ antibodies, using well-characterized controls and a panel of validation methods. We conclude that only one antibody, the rarely used monoclonal PPZ0506, specifically target ...[more]

PMID: 28643774

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Project description:Aims: We investigate sex differences and the role of oestrogen receptor beta (ERbeta) in a mouse model of pressure overload-induced myocardial hypertrophy. Methods and results: We performed transverse aortic constriction (TAC) or sham surgery in male and female wild-type (WT) and ER knockout (ERbeta-/-) C57Bl6 mice. All mice were characterised by echocardiography and haemodynamic measurements and were sacrificed nine weeks after surgery. Left ventricular (LV) samples were analysed by microarray profiling, real-time RT-PCR and histology. After nine weeks, WT males showed more hypertrophy and heart failure signs than WT females. Notably, WT females developed a concentric form of hypertrophy, while males developed eccentric hypertrophy. These sex differences were abolished in ERbeta-/- mice. ERbeta deletion augmented the TAC-induced increase in cardiomyocyte diameter in both sexes. Gene expression profiling revealed that male WT hearts had a stronger induction of matrix-related genes and a stronger repression of mitochondrial genes than female hearts. ERbeta-/- mice exhibited a different transcriptome. Induction of pro-apoptotic genes after TAC occurred in ERbeta-/- mice of both sexes with a stronger expression in ERbeta-/- males. Histological analysis revealed, that cardiac fibrosis was more pronounced in male WT TAC than in female mice. This was abolished in ERbeta-/- mice. Apoptosis was significantly induced in both sexes of ERbeta-/- TAC mice, but it was most prominent in males. Conclusion: Female sex offers protection against ventricular chamber dilation in the TAC model. Both the female sex and ER attenuate the development of fibrosis and apoptosis; thus slowing the progression to heart failure. The influence of sex (male/female) and estrogen receptor beta expression (ERbeta knockout/wildtype) on cardiac hypertrophy (transverse aortic constriction/sham operated) was investigated. The left ventricular transcriptome of four individual mice for each combination of the three factors (sex, genotype, surgery) was detected with Affymetrix RAE 430 2.0 GeneChip arrays.

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Insufficient antibody validation challenges oestrogen receptor beta research.

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Insufficient antibody validation challenges oestrogen receptor beta research.

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