A functional drug re-purposing screening identifies carfilzomib as a drug preventing 17β-estradiol: ERα signaling and cell proliferation in breast cancer cells

Abstract

Abstract.Most cases of breast cancer (BC) are estrogen receptor α -positive (ERα+) at diagnosis.

A high throughput method to study the physiology of E2:ERα signaling in breast cancer cells

“…Moreover, the fact that both everolimus and bortezomib (Velcade®), a 26S proteasome inhibitor, have been previously shown to induce ERα degradation and to prevent MCF‐7 cell proliferation (Lui, New, Ogony, Thomas, & Lewis‐Wambi, ; Thaler et al, ) strongly support the notion that our procedure can efficiently recognize compounds modulating ERα levels and cell proliferation. Remarkably, it is important to note here that present discoveries additionally support the concept that the selective modulation of ERα intracellular levels can be used as a pharmacological target to identify novel molecules targeting E2:ERα signaling in BC cells (Busonero, Leone, Acconcia, et al, ; Busonero, Leone, Klemm, et al, ).…”

“…In‐cell WB and in‐cell PI staining were next used to measure ERα levels and DNA content in a 4OH‐tamoxifen resistant (Tam‐Res) MCF‐7 cell line. Because Tam‐Res tumors are mainly treated with ICI (i.e., fulvestrant—faslodex) in order to eliminate ERα and to block BC cell proliferation (Tryfonidis et al, ), this cell line, which represents a model of acquired resistance to Tam and has been previously characterized (Busonero, Leone, Klemm, et al, ; Knowlden et al, ), was challenged with a library (80 compounds) of FDA approved anti‐cancer drugs according to the protocol described in Figure a with the aim to identify one or more compounds that contemporarily induce ERα degradation and prevent cell proliferation (Figure b).…”

“…In this respect, multiple high throughput methods (e.g., in silico and in vitro ERα binding assays; screens either for drugs inhibiting receptor transcriptional activity or for anti‐proliferative drugs) have been employed either to discover ERα ligands, which could work as ERα degraders, to identify inhibitors of ERα transcriptional activity or to select specific molecules that directly inhibit E2‐induced cell proliferation (Campbell et al, ; Giamas et al, ; Overk et al, ; Singh et al, ). We have recently added to this repertoire of methods the evaluation of the effects of drugs on ERα levels by assuming that drug‐induced deregulation of ERα content in BC cells would block cell proliferation (Acconcia et al, ; Busonero, Leone, Acconcia, et al, ; Busonero, Leone, Klemm, et al, ; Totta et al, ).…”

“…Although such technique would also fit for the analysis of the physiological or pathophysiological involvement of a specific protein in the modulation of E2‐dependent effects (e.g., by applying a library of siRNA oligonucleotides), we decided to apply a small‐scale library of about 80 anti‐cancer FDA‐approved drugs to 4OH‐tamoxifen resistant (Tam‐Res) MCF‐7 cells (Busonero, Leone, Klemm, et al, ; Knowlden et al, ) in in‐cell WB and PI staining to identify one or more compounds that could induce ERα degradation and prevent cell proliferation in a manner similar to fulvestrant (i.e., ICI). 4OH‐tamoxifen resistance is likely the most critical issue in BC management as a great proportion of women treated with this ERα inhibitor relapse with an emerging neoplasm still expressing ERα but being insensitive to 4OH‐tamoxifen treatment (Angus et al, ; Tryfonidis et al, ).…”

“…Recently, we approached this task by administering about 900 molecules to BC cells with the aim of identifying novel drugs changing ERα intracellular levels. We found that emetine, an alkaloid extracted from the ipecac root with anti‐parasitic and anti‐contraceptive effects and carfilzomib, a third generation 26S proteasome inhibitor used for the treatment of multiple myeloma, induce ERα degradation and prevent BC cell proliferation by affecting cellular pathways unrelated to E2:ERα signaling (Busonero, Leone, & Acconcia, ; Busonero, Leone, Klemm, & Acconcia, ). In turn, we proposed that the measurement of ERα intracellular levels can be used to fish out molecules that interfere with E2:ERα signaling and/or to identify novel and potentially druggable pathways affecting it (Acconcia et al, ).…”