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In silico screening for ERα down modulators identifies thioridazine as an anti-proliferative agent in primary, 4OH-tamoxifen-resistant and Y537S ERα-expressing breast cancer cells

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Abstract

Purpose

Most breast cancers (BCs) express estrogen receptor α (ERα) and are treated with the endocrine therapy (ET) drugs 4OH-tamoxifen (Tam) and fulvestrant (ICI 182,780; ICI). Unfortunately, a high fraction of ET treated women relapses and becomes resistant to ET. Therefore, additional anti-BC drugs are needed. Recently, we proposed that the identification of novel anti-BC drugs can be achieved using modulation of the intracellular ERα content in BC cells as a pharmacological target. Here, we searched for Food and Drug Administration (FDA)-approved drugs that potentially modify the ERα content in BC cells.

Methods

We screened in silico more than 60,000 compounds to identify FDA-approved drugs with a gene signature similar to that of ICI. We identified mitoxantrone and thioridazine and tested them in primary, Tam-resistant and genome-edited Y537S ERα-expressing BC cells.

Results

We found that mitoxantrone and thioridazine induced ERα downmodulation and prevented MCF-7 BC cell proliferation. Interestingly, while mitoxantrone was found to be toxic for normal breast epithelial cells, thioridazine showed a preferential activity towards BC cells. Thioridazine also reduced the ERα content and prevented cell proliferation in primary, Tam-resistant and genome-edited Y537S ERα expressing BC cells.

Conclusions

We suggest that modulation of the intracellular ERα concentration in BC cells can be exploited in in silico screens to identify anti-BC drugs and uncover a re-purposing opportunity for thioridazine in the treatment of primary and metastatic ET resistant BCs.

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Acknowledgments

This study was supported by grants from Ateneo Roma Tre to FA and from MIUR (Ricerca Corrente) to FB. The Grant of Excellence Departments, MIUR (ARTICOLO 1, COMMI 314 – 337 LEGGE 232/2016) to the Department of Science, University Roma TRE is also gratefully acknowledged. The authors thank also Prof. Simak Ali, University of London Imperial College, UK for gift of the Y537S-MCF-7 cells and Dr. Carol Dutkowski, University of Cardiff, UK for gift of the 4OH-tamoxifen resistant MCF-7 cells (Tam-Res). We thank Dr. Tommaso Colangelo and Orazio Palumbo, IRCCS Casa Sollievo della Sofferenza, Italy, for technical support (Affymetrix experiment).

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Author notes

  1. Claudia Busonero and Stefano Leone contributed equally to this work.

Authors and Affiliations

  1. Department of Sciences, Section Biomedical Sciences and Technology, University Roma Tre, Viale Guglielmo Marconi, 446, I-00146, Rome, Italy

    Claudia Busonero, Stefano Leone & Filippo Acconcia

  2. ISBREMIT, Institute for Stem-cell Biology, Regenerative Medicine and Innovative Therapies, IRCCS Casa Sollievo della Sofferenza, Viale Padre Pio, 7, 71013, San Giovanni Rotondo (FG), Italy

    Fabrizio Bianchi

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  1. Claudia Busonero
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Correspondence to Filippo Acconcia.

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Supplementary Fig. 1

Characterization of metastatic breast cancer cell lines. (a) Number of MCF-7, ZR-75-1, Tam-Res MCF-7 and Y537S ERα-expressing MCF-7 (Y537S) cells treated with the indicated doses of 4OH-Tamoxifen (4OH-Tam) for 7 days. (b) Venn diagram depicting the genes upregulated in Y537S ERα-expressing MCF-7 cells in the dataset available in literature and in our affimetrix analysis. (c) Western blotting analyses of cathepsin D (Cat D) and presenilin 2 (pS2) expression levels in wild type MCF-7 (MCF-7) and Y537S ERα-expressing MCF-7 (Y537S) cells treated for 24 h with the indicated doses of 17β-estradiol (E2). The loading control was done by evaluating vinculin expression in the same filter. (d) Number of wild type MCF-7 (MCF-7) and Y537S ERα-expressing MCF-7 (Y537S) cells treated with 17β-estradiol (E2–10 nM) both in the presence and in the absence of 4OH-Tamoxifen (4OH-Tam – 1 μM) for 48 h. * indicates significant differences with respect to the control (DMSO, −) sample. ° indicates significant differences with respect to the E2 samples. (PPTX 76 kb)

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Busonero, C., Leone, S., Bianchi, F. et al. In silico screening for ERα down modulators identifies thioridazine as an anti-proliferative agent in primary, 4OH-tamoxifen-resistant and Y537S ERα-expressing breast cancer cells. Cell Oncol. 41, 677–686 (2018). https://doi.org/10.1007/s13402-018-0400-x

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