Continuous Exposure of Breast Cancer Cells to Tamoxifen Upregulates GPER-1 and Increases Cell Proliferation

Abstract

GPER-1 is a novel membrane sited G protein-coupled estrogen receptor. Clinical studies have shown that patients suffering an estrogen receptor a (ERa)/GPER-1 positive, breast cancer have a lower survival rate than those who have developed ERa-positive/GPER-1 negative tumors. Moreover, absence of GPER-1 improves the prognosis of patients treated with tamoxifen, the most used selective estrogen receptor modulator to treat ERapositive breast cancer. MCF-7 breast cancer cells were continuously treated with 1,000 nM tamoxifen for 7 days to investigate its effect on GPER-1 protein expression, cell proliferation and intracellular [Ca 2+ ]i mobilization, a key signaling pathway. Breast cancer cells continuously treated with tamoxifen, exhibited a robust [Ca 2+ ]i mobilization after stimulation with 1,000 nM tamoxifen, a response that was blunted by preincubation of cells with G15, a commercial GPER-1 antagonist. Continuously treated cells also displayed a high [Ca 2+ ]i mobilization in response to a commercial GPER-1 agonist (G1) and to estrogen, in a magnitude that doubled the response observed in untreated cells and was almost completely abolished by G15. Proliferation of cells continuously treated with tamoxifen and stimulated with 2,000 nM tamoxifen, was also higher than that observed in untreated cells in a degree that was approximately 90% attributable to GPER-1. Finally, prolonged tamoxifen treatment did not increase ERa expression, but did overexpress the kinin B1 receptor, another GPCR, which we have previously shown is highly expressed in breast tumors and increases proliferation of breast cancer cells. Although we cannot fully extrapolate the results obtained in vitro to the patients, our results shed some light on the occurrence of drug resistance in breast cancer patients who are ERa/GPER-1 positive, have been treated with tamoxifen and display low survival rate. Overexpression of kinin B1 receptor may explain the increased proliferative response observed in breast tumors under continuous treatment with tamoxifen.

Molecular Characterization of Membrane Steroid Receptors in Hormone-Sensitive Cancers

“…Moreover, GPER appears to be required for stemness maintenance in cancer stem cells via PKA/Bcl-2 associated agonist of cell death (BAD) pathway [ 96 ] and the development of chemoresistance via EGFR/ERK/Akt-mediated ATP Binding Cassette, Subfamily G, Member 2 (ABCG2) expression [ 97 ]. This is in line with the increasing evidence that GPER expression and activation is correlated with a poor response to chemotherapy with selective estrogen receptor modulators [ 98 , 99 ]. However, GPER role in breast cancer still needs to be fully clarified due to contrasting literature data on its significance as prognosis predictor [ 100 , 101 , 102 ].…”

Investigation of the Antitumor Effects of Tamoxifen and Its Ferrocene-Linked Derivatives on Pancreatic and Breast Cancer Cell Lines

“…This observation can be explained by the estrogen receptor expression profile of our cell lines. It is a well-described phenomenon that GPER1 signaling promotes tumor progression [ 85 , 86 ], helps cancer cells to obtain stem cell-like properties [ 87 ], and can mediate therapy resistance [ 88 , 89 ]. The presence of GPER1 poses a clinical problem as well, as patients with GPER1 expressing tumors have a worse survival rate, compared to patients with non-GPER1 expressing tumors, when receiving tamoxifen therapy [ 90 , 91 , 92 ], mostly because tamoxifen was described to be an agonist of GPER1 [ 36 , 37 , 38 ], inducing its previously mentioned tumor-promoting capability.…”

The Impact of Estrogen and Estrogen-Like Molecules in Neurogenesis and Neurodegeneration: Beneficial or Harmful?

“…Estrogens are recognized as a significant steroidal mitogen for epithelial cells, commonly related to oncogenesis ( Anderson et al, 1998 ). In this sense, experiments carried out by our group on breast cancer cell lines support that continuous pharmacological blockade of classical ERs leads to the overexpression of GPER-1 (non-classical ER) as an alternative pathway to promote proliferation ( Molina et al, 2020 ). Also, studies carried out in cancer-associated fibroblasts derived from breast cancer patients show that the GPER-1/EGFR signaling axis increases the expression of several cell cycle regulatory genes ( Pisano et al, 2017 ).…”

“…Additionally, some exogenous estrogen-like molecules, such as phytoestrogens and xenoestrogens, also have ER-binding capabilities ( Nadal et al, 2018 ). By binding to these receptors, estrogens and estrogen-like molecules modulate cell proliferation ( Gompel et al, 2004 ; Pisano et al, 2017 ; Molina et al, 2020 ), differentiation ( Bassler, 1970 ; Grubbs et al, 1985 ; Imanishi et al, 2005 ), and survival ( Weldon et al, 2004 ; Kishi et al, 2005 ; Yu et al, 2012 ) processes, by different downstream pathways. Furthermore, since estrogen receptors are ubiquitously located, estrogen-mediated signals modulate the biology of several tissues and organs, including the central nervous system.…”