Physiologically Relevant Estrogen Receptor Alpha Pathway Reporters for Single-Cell Imaging-Based Carcinogenic Hazard Assessment of Estrogenic Compounds

Abstract

Estrogen receptor alpha (ERα) belongs to the nuclear hormone receptor family of ligand-inducible transcription factors and regulates gene networks in biological processes such as cell growth and proliferation. Disruption of these networks by chemical compounds with estrogenic activity, can result in adverse outcomes such as unscheduled cell proliferation, ultimately culminating in tumor formation. To distinguish disruptive activation from normal physiological responses, it is essential to quantify relationships between different key events leading to a particular adverse outcome. For this purpose, we established fluorescent protein MCF7 reporter cell lines for ERα-induced proliferation by bacterial artificial chromosome-based tagging of three ERα target genes: GREB1, PGR and TFF1. These target genes are inducible by the non-genotoxic carcinogen and ERα agonist 17β-estradiol in an ERα-dependent manner and are essential for ERα-dependent cell cycle progression and proliferation. The three GFP reporter cell lines were characterized in detail and showed different activation dynamics upon exposure to 17β-estradiol. In addition, they demonstrated specific activation in response to other established reference estrogenic compounds of different potencies, with similar sensitivities as validated OECD test methods. This study shows that these fluorescent reporter cell lines can be used to monitor the spatial and temporal dynamics of ERα pathway activation at the single cell level for more mechanistic insight, thereby allowing a detailed assessment of the potential carcinogenic activity of estrogenic compounds in humans.

EMA commentary on the ICH guideline for testing for carcinogenicity of pharmaceuticals

“…Considerable efforts have been made on identifying these biomarkers in recent years, for example, those involved in the formation of several hormonal related pathways 37 and liver tumours 38 . Duijndam et al 39 have developed a single‐cell imaging technique using fluorescent reporter human breast cancer cell lines as an approach for carcinogenic hazard assessment of estrogenic compounds. For the genomics approach to be successful and be a standard and reliable part of the WoE, a set of validated biomarkers and pathway analysis that represent a wide variety of carcinogenic mechanisms in several species including man (in vitro) is required.…”

The p21CIP1-CDK4-DREAM axis is a master regulator of genotoxic stress-induced cellular senescence

“…MCF7 FUCCI-H2B reporter cells were kindly provided by Bob van de Water (Division of Drug Discovery & Safety, Leiden Academic Centre for Drug Research, Leiden University, the Netherlands). They express mClover-Geminin( 1 -110) and mKO2-Cdt1(30–120) from a bicistronic lentiviral vector as well as H2B-iRFP670 as a nuclear marker ( 42 ). MCF7 cells were cultivated in Dulbecco's minimal essential medium (DMEM)-F12 containing 5% fetal bovine serum (FBS) under normal atmosphere (5% CO 2 ) at 37°C.…”

ICH S1 prospective evaluation study and weight of evidence assessments: commentary from industry representatives

“…Second tier screenings, conducted in a later phase of development, often as a part of the data to support Phase I clinical studies, may be more comprehensive and include targets with known carcinogenic risk, in particular kinases and nuclear hormone receptors. Examples may include the estrogen receptor ( Duijndam et al, 2021 ), Glycogen Synthase Kinase 3 beta receptor ( Heinemann et al, 2022 ) or the aryl hydrocarbon receptor ( Podtelezhnikov et al, 2020 ). Under the auspices of the DruSafe leadership group of the Innovation and Quality (IQ) consortium there is ongoing work to comprehensively review current practices.…”