In 1996 a breakthrough discovery in the field of hormone signaling was made at Karolinska Institutet. Before this, we knew that the female hormone estrogen bound to the estrogen receptor (ER), a receptor often overexpressed in breast cancer. We also knew that inhibition of this receptor could cure many of these patients, however, we did not know that there were two estrogen receptors. Upon this discovery, the receptors were renamed estrogen receptor alpha (ERalpha) and estrogen receptor beta (ERbeta), a paradigm shift that changed the way we understand estrogen signaling.
Early on, an appealing idea was that targeting ERbeta could provide the basis for better cancer treatments. As it appeared, this new receptor needed to be activated, whereas ERalpha needed to be inactivated. A critical part of this theory was, of course, that ERbeta was expressed in breast cancer.
To determine this, researchers turned to antibodies. Several different antibodies towards ERbeta were generated, and many of those did indeed detect ERbeta in breast and breast cancer. For the past 20 years, intense research and large resources have been dedicated to understand the role of ERbeta in breast cancer and other diseases, and how targeting ERbeta could improve treatment. Many results were however contradictory and suspicion was raised that not all antibodies were functioning properly, but it was very unclear which antibodies could be trusted.
In a recent study performed in collaboration with the Human Protein Atlas, 13 antibodies directed towards ERbeta were thoroughly validated using positive and negative controls, different antibody-based methodologies, mass spectrometry, and determination of ERbeta protein and mRNA expression across 44 normal human tissues and 20 different cancer types. Astonishingly, NONE of the antibodies previously used to determine ERbeta expression in human tissues turned out to be specific, but instead bound to other unrelated proteins, not ERbeta. Only one antibody, not used in studies previously, was verified binding to ERbeta alone. However, it neither found ERbeta in breast or breast tumors, nor many other organs reported to express this receptor. Confirmed expression of ERbeta was only found in reproductive organs, immune cells and subsets of ovarian tumors, malignant melanomas and thyroid cancers. Thus, while this antibody did not detect the receptor in breast, and thereby removed the hopes of being used in breast cancer therapies, it indicated an opportunity being used as a biomarker in other diseases.
The consequences of working with reagents less specific than previously thought can, as this study indicates, be both large and costly. All research and literature produced during the past 20 years related to estrogen research now needs to be reevaluated and questioned, including major current research efforts such as on-going clinical trials.
Highlighting problems caused by unverified antibodies should encourage researchers and antibody vendors to thoroughly validate their antibodies, applying new guidelines recenty published by the International Working Group for Antibody Validation (IWGAV). The current work identifying the true expression of ERbeta can aid related research in the right direction, and speed up its pharmaceutical applications for relevant diseases.
Immunohistochemical stanining indicated that no expression in breast or breast cancer was observed for the only antibody specifically binding to ERbeta (left panel). Positive staining (brown cells) is shown for two different antibodies not specific for ERbeta (midde and right panel).