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

Abstract

17β-estradiol (E2) regulates diverse physiological effects including cell proliferation through the estrogen receptor α (ERα), which as a transcription factor drives gene transcription and as an extra-nuclear localized receptor triggers the membrane-dependent activation of diverse kinase cascades. E2 also modifies ERα intracellular levels via diverse intracellular mechanisms. In this way, the E2-acivated ERα integrates signaling cascades with the modulation of receptor intracellular concentration and with the induction of DNA synthesis and ultimately drives cell proliferation. In turn, E2 signaling deregulation can cause many diseases including breast cancer (BC). Recently, we performed a Western blotting (WB)-based screen to identify novel pathways affecting ERα intracellular levels and BC cell proliferation. However, because WB lacks high throughput potential, a high-content method to detect all aspects of E2:ERα signaling (nuclear and extra-nuclear receptor activity, ERα levels, E2-induced DNA synthesis) is desirable. Here, we set up a rapid way to measure E2:ERα signaling in 96-well plate format. To demonstrate its robustness, we also challenged 4OH-tamoxifen resistant (Tam-Res) BC cells with a library of anti-cancer drugs and identified methotrexate (MTX) as a molecule inducing ERα degradation and preventing BC cell proliferation. Overall, our research provides a high-content technique to study the physiology of E2:ERα signaling in cells and further suggests a possible anti-ERα and anti-proliferative use for MTX in Tam-Res BCs.

<p>ERα, A Key Target for Cancer Therapy: A Review</p>

“…ERα is critical to the development of ER+ breast cancer, 84 which accounts for approximately 70% of all breast cancers. 7,85 Overexpression of ERα frequently sensitizes tumors to endocrine therapy. 84 When exposed to E2, ERα activation stimulates downstream signaling pathways, 86 and leads to EMT and ECM remodeling (Figure 3).…”

“…ERs can be activated when cells are exposed to estrogen. [7][8][9] Emerging evidence shows that the activation of ERs is highly associated with cancer formation and metastasis, [10][11][12] extracellular matrix (ECM) remodeling 2,13 and drug resistance. [14][15][16][17] Here, we focus on providing a comprehensive understanding of ERα.…”

The Network of Angiotensin Receptors in Breast Cancer

“…In this respect, different strategies are being used to identify novel BC treatments [ 14 ]. Our research group has recently introduced the concept of targeting the selective modulation of ERα levels as a promising approach to discover new anti-BC compounds [ 14 , 59 , 60 , 61 ]. We have indeed demonstrated that drugs that do not necessarily bind to ERα but instead change the amount of ERα protein in BC cells can prevent BC cell proliferation and subsequent tumor growth [ 14 ].…”

“…We have indeed demonstrated that drugs that do not necessarily bind to ERα but instead change the amount of ERα protein in BC cells can prevent BC cell proliferation and subsequent tumor growth [ 14 ]. In turn, we demonstrated this by screening a library of 1018 Food and Drug Administration (FDA)-approved drugs in BC cells to search for molecules that modify ERα content and prevent BC cell growth [ 14 , 59 , 61 , 62 ]. This line of research led to the discovery that more than 50 FDA-approved compounds affected ERα content in MCF-7 cells and, among them, 11 also inhibited cell proliferation [ 59 ].…”

“…This line of research led to the discovery that more than 50 FDA-approved compounds affected ERα content in MCF-7 cells and, among them, 11 also inhibited cell proliferation [ 59 ]. We further studied emetine, carfilzomib, and methotrexate, and showed that they block the proliferation of cellular models of primary BC and MBC more effectively than classic ET drugs (e.g., fulvestrant) [ 59 , 61 , 62 ]. Thus, this approach allowed us to identify molecules with “anti-estrogen-like” effects that we previously called EMERALDs (i.e., selective modulators of ERα levels and degradation) [ 14 ].…”