Abstract
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 targets ERβ in immunohistochemistry. Applying this antibody for protein expression profiling in 44 normal and 21 malignant human tissues, we detect ERβ 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.
Highlights
The discovery of oestrogen receptor b (ERb/ESR2) was a landmark discovery
We first performed Immunohistochemical analysis (IHC) on a validation tissue microarray (TMA) that included a panel of Formalin-fixed and paraffin-embedded (FFPE) tissues and control cell lines (Supplementary Table 2)
The remaining 11 anti-ERb antibodies, including the widely used monoclonal antibodies (mAbs) PPG5/10 (Fig. 2a), all failed the IHC validation step since they generated distinct positive IHC staining in ERb-negative cell lines
Summary
The discovery of oestrogen receptor b (ERb/ESR2) was a landmark discovery. Its reported expression and homology with breast cancer pharmacological target ERa (ESR1) raised hopes for improved endocrine therapies. We conclude that only one antibody, the rarely used monoclonal PPZ0506, targets ERb in immunohistochemistry Applying this antibody for protein expression profiling in 44 normal and 21 malignant human tissues, we detect ERb protein in testis, ovary, lymphoid cells, granulosa cell tumours, and a subset of malignant melanoma and thyroid cancers. Most cell lines have been reported to lack ERb mRNA22,24 (see 45 cell lines analysed within the HPA project, http://www.proteinatlas.org/ENSG00000140009-ESR2/ cell), while antibody-based applications report its protein expression[25,26]. This raises the pertinent question of antibody specificity. This is reflected in recent guidelines for application-dependent validation of antibodies presented by the ad hoc International Working Group for Antibody Validation[30]
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