Real-Time Challenging of ERα Y537S Mutant Transcriptional Activity in Living Cells

Manuela Cipolletti,Filippo Acconcia,Sara Pescatori

doi:10.3390/endocrines2010006

Manuela Cipolletti, Filippo Acconcia + Show 1 more

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https://doi.org/10.3390/endocrines2010006

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Journal: Endocrines	Publication Date: Mar 10, 2021
Citations: 7	License type: CC BY 4.0

Affiliation: Marconi University

Abstract

Metastatic estrogen receptor α (ERα)-expressing breast cancer (BC) occurs after prolonged patient treatment with endocrine therapy (ET) (e.g., aromatase inhibitors—AI; 4OH-tamoxifen—4OH-Tam). Often these metastatic BCs express a mutated ERα variant (e.g., Y537S), which is transcriptionally hyperactive, sustains uncontrolled proliferation, and renders tumor cells insensitive to ET drugs. Therefore, new molecules blocking hyperactive Y537S ERα mutation transcriptional activity are requested. Here we generated an MCF-7 cell line expressing the Y537S ERα mutation stably expressing an estrogen-responsive element (ERE) promoter, which activity can be monitored in living cells. Characterization of this cell line shows both hyperactive basal transcriptional activity with respect to normal MCF-7 cells, which stably express the same ERE-based promoter and a decreased effect of selective ER downregulators (SERDs) in reducing Y537S ERα mutant transcriptional activity with respect to wild type ERα transcriptional activity. Kinetic profiles of Y537S ERα mutant-based transcription produced by both drugs inducing receptor degradation and siRNA-mediated depletion of specific proteins (e.g., FOXA1 and caveolin1) reveals biphasic dynamics of the inhibition of the receptor-regulated transcriptional effects. Overall, we report a new model where to study the behavior of the Y537S ERα mutant that can be used for the identification of new targets and pathways regulating the Y537S ERα transcriptional activity.

Highlights

Seventy percent of all diagnosed breast cancers (BC) are estrogen receptor α-positive (ERα+)
Our results indicate that the produced Y537S-NLuc cells are sensitive stable cell lines where to study the kinetic of the Y537S ERα mutant transcriptional signaling and suggest this cellular model can be used to identify new potential “anti-estrogen”
Characterization of the generated Y537S estrogen-responsive element (ERE)-NLuc cells was performed by evaluating the expression levels of pS2/TFF and cathepsin D (CatD), two proteins which gene contain the ERE sequence in their promoters [15] both in non-transfected MCF-7 and

Summary

Introduction

Seventy percent of all diagnosed breast cancers (BC) are estrogen receptor α-positive (ERα+). ERα+ BC drugs target either the ERα or the 17β-estradiol (E2) signaling. This endocrine therapy (ET) aims to inhibit proliferative E2:ERα signaling by reducing the E2 availability through the use of aromatase inhibitors (AIs) or to prevent ERα signaling using specific receptor ligands that either inactivate ERα transcriptional activity (i.e., 4OH-tamoxifen-4OH-Tam), or that induce receptor degradation and prevent receptor transcriptional functions (i.e., fulvestrant, known as ICI182,780-ICI) [1]. Despite the proven efficacy of the ET, a considerable number of women treated for a prolonged period of time with AI and/or 4OH-Tam develop a relapsing disease, which results in a tumor recurrence [1].

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