Tamoxifen through GPER upregulates aromatase expression: a novel mechanism sustaining tamoxifen-resistant breast cancer cell growth

Abstract

Tamoxifen resistance is a major clinical challenge in breast cancer treatment. Aromatase inhibitors are effective in women who progressed or recurred on tamoxifen, suggesting a role of local estrogen production by aromatase in driving tamoxifen-resistant phenotype. However, the link between aromatase activity and tamoxifen resistance has not yet been reported. We investigated whether long-term tamoxifen exposure may affect aromatase activity and/or expression, which may then sustain tamoxifen-resistant breast cancer cell growth. We employed MCF-7 breast cancer cells, tamoxifen-resistant MCF-7 cells (MCF-7 TR1 and TR2), SKBR-3 breast cancer cells, cancer-associated fibroblasts (CAFs1 and CAFs2). We used tritiated-water release assay, realtime-RT-PCR, and immunoblotting analysis for evaluating aromatase activity and expression; anchorage-independent assays for growth; reporter-gene, electrophoretic-mobility-shift, and chromatin-immunoprecipitation assays for promoter activity studies. We demonstrated an increased aromatase activity and expression, which supports proliferation in tamoxifen-resistant breast cancer cells. This is mediated by the G-protein-coupled receptor GPR30/GPER, since knocking-down GPER expression or treatment with a GPER antagonist reversed the enhanced aromatase levels induced by long-term tamoxifen exposure. The molecular mechanism was investigated in ER-negative, GPER/aromatase-positive SKBR3 cells, in which tamoxifen acts as a GPER agonist. Tamoxifen treatment increased aromatase promoter activity through an enhanced recruitment of c-fos/c-jun complex to AP-1 responsive elements located within the promoter region. As tamoxifen via GPER induced aromatase expression also in CAFs, this pathway may be involved in promoting aggressive behavior of breast tumors in response to tamoxifen treatment. Blocking estrogen production and/or GPER signaling activation may represent a valid option to overcome tamoxifen-resistance in breast cancers.

International Union of Basic and Clinical Pharmacology. XCVII. G Protein–Coupled Estrogen Receptor and Its Pharmacologic Modulators

“…Multiple studies now reveal that long-term E2 deprivation of the E2-dependent human breast cancer cell line MCF-7, mimicking treatment of women with antiestrogens or aromatase inhibitors, increased expression of GPER (Craig Jordan et al, 2007), with tamoxifen treatment of such resistant cells stimulating proliferation via GPER (Ignatov et al, 2010). Prolonged tamoxifen treatment also leads to increased aromatase activity and expression via GPER signaling, suggesting a possible mechanism involved in the development of tamoxifen resistance (Catalano et al, 2014). Despite the generally stimulatory effects of GPER stimulation on cancer cell line growth, G-1, particularly at high doses (generally $1mM), has also been shown to inhibit the proliferation of certain cancer cell lines in vitro (Ariazi et al, 2010;Chimento et al, 2014a;Weißenborn et al, 2014a,b), which may be a result of reported effects on microtubules at high concentrations (Holm et al, 2012;Wang et al, 2013), or possibly protein kinase C«-mediated destabilization of microtubules (Goswami et al, 2011).…”

“…However, in contrast to its actions as an antagonist in breast cancer cells, 4-OHT acts as a GPER agonist in breast and other cancer cells (Revankar et al, 2005;Petrie et al, 2013). Consistent with this agonist activity toward GPER, increasing numbers of studies are finding roles for GPER in tamoxifen resistance in breast cancer and the increased incidence of endometrial cancer after tamoxifen treatment (Craig Jordan et al, 2007;Ignatov et al, 2010Ignatov et al, , 2011aMo et al, 2013;Petrie et al, 2013;Catalano et al, 2014).…”

The G-protein coupled estrogen receptor, GPER: The inside and inside-out story

“…Poised as an alternative estrogen receptor that is activated by ER antagonists to promote EGFR transactivation, GPER, in part, may provide a rational explanation for this observation. Particularly intriguing is the observation that GPER stimulation has been linked to increased aromatase activity [Catalano et al, 2014] perhaps suggesting a direct role for this newly appreciated estrogen receptor in advanced disease and responsiveness to endocrine therapy.…”

GPER1-mediated IGFBP-1 induction modulates IGF-1-dependent signaling in tamoxifen-treated breast cancer cells

“…Taken together, these results suggest that CREB activation downstream of GPER1 may play an inhibitory role in ERα-positive breast cancer cells. In contrast, CREB activation induces proliferation in tamoxifen-resistant breast cancer cells and other cancer cell types (Catalano et al, 2014; Daniel et al, 2014; Nguyen et al, 2014; Sofi et al, 2003). Furthermore, increased CREB expression in breast tumors is associated with poor prognosis, shorter survival and higher risk of metastasis (Chhabra et al, 2007; Fan et al, 2012).…”