The estrogen receptor α (ERα) is a transcription factor that mediates the biological effects of 17β-estradiol (E(2)). ERα transcriptional activity is also regulated by cytoplasmic signaling cascades. Here, several Gα protein subunits were tested for their ability to regulate ERα activity. Reporter assays revealed that overexpression of a constitutively active Gα(o) protein subunit potentiated ERα activity in the absence and presence of E(2). Transient transfection of the human breast cancer cell line MCF-7 showed that Gα(o) augments the transcription of several ERα-regulated genes. Western blots of HEK293T cells transfected with ER±Gα(o) revealed that Gα(o) stimulated phosphorylation of ERK 1/2 and subsequently increased the phosphorylation of ERα on serine 118. In summary, our results show that Gα(o), through activation of the MAPK pathway, plays a role in the regulation of ERα activity.

The estrogen receptor a (ERa) is a transcription factor that mediates the biological effects of 17b-estradiol (E2). ERa transcriptional activity is also regulated by cytoplasmic signaling cascades. Here, several Ga protein subunits were tested for their ability to regulate ERa activity. Reporter assays revealed that overexpression of a constitutively active Gao protein subunit potentiated ERa activity in the absence and presence of E2. Transient transfection of the human breast Journal of Endocrinology (2012) 214, 45–54 0022–0795/12/0214–045 q 2012 Society for Endocrinology Printed in Great cancer cell line MCF-7 showed that Gao augments the transcription of several ERa-regulated genes. Western blots of HEK293T cells transfected with ERGGao revealed that Gao stimulated phosphorylation of ERK 1/2 and subsequently increased the phosphorylation of ERa on serine 118. In summary, our results show that Gao, through activation of the MAPK pathway, plays a role in the regulation of ERa activity. Journal of Endocrinology (2012) 214, 45–54

Melatonin has been shown to inhibit the proliferation of estrogen receptor alpha (ER alpha)-positive human breast cancer cells in vitro and suppress the growth of carcinogen-induced mammary tumors in rats. Melatonin's antiproliferative effect is mediated, at least in part, through the MT1 melatonin receptor and mechanisms involving modulation of the estrogen-signaling pathway. To develop melatonin analogs with greater therapeutic effects, we have examined the in vitro and in vivo antimitotic activity of two MT1/MT2 melatonin receptor agonists, S23219-1 and S23478-1. In our studies, both agonists are quite effective at suppressing the growth of MCF-7 human breast cancer cells. At a concentration of 10-6 m, S23219-1 and S23478-1 inhibited the growth of MCF-7 cells by 60% and 73%, respectively. However, S23478-1 is more effective than melatonin and S23219-1 at repressing the expression and transactivation of the ER alpha, and modulating the expression of pancreatic spasmolytic polypeptide (pS2), an estrogen-regulated gene. The melatonin agonist S23478-1 exhibited enhanced antitumor potency in the subsequent studies in our animal model. At a dosage of 25 mg/kg/day, S23478-1 is more efficacious than melatonin at inducing regression of the established N-nitroso-N-methyl-urea-induced rat mammary tumors. This dose of S23478-1 (25 mg/kg/day) generated a significant (P < 0.05) overall regression response of 52%. Furthermore, at this dosage, S23478-1 is more effective than melatonin at suppressing the estrogen-signaling pathway and promoting tumor cell apoptosis, significantly increasing the expression of the pro-apoptotic protein Bax, while decreasing the expression of ER alpha and the anti-apoptotic protein Bcl-2.

Luciferase reporter constructs and transient co-transfection approaches demonstrate that elevated expression of RORα1 augments 17-β-estradiol (E2)-induced transcriptional activation of the full-length ERα, but not truncated ERα constructs (ABCD or CDEF), in MCF-7 breast cancer and HEK293 embryonic kidney cells, and that physiologic concentrations of MLT inhibit the individual and combined transcriptional activity of ERα by RORα1 and E2. Gel mobility shift and co-immunoprecipitation (IP)/pull-down assays demonstrate that RORα1 and ERα do not interact directly at the DNA-binding level or as heterodimers, however, RORα1 augments E2-induced pS2 and cyclin D1 mRNA expression while MLT inhibits RORα1/E2-induced expression of pS2 and cyclin D1 in MCF-7 cells.

The MT1 melatonin receptor is bound and activated by the pineal hormone melatonin. This G protein-coupled melatonin receptor is expressed in human breast tumor cell lines, and when activated, mediates the growth-suppressive and steroid hormone/nuclear receptor modulatory actions of melatonin on breast tumor cells. In the current studies, we have examined the expression of the MT1 receptor in breast cancer cell lines and primary human breast tumors and correlated MT1 receptor expression with the deletion, rearrangement and amplification of the MT1 gene and established markers of breast cancer such as tumor size, stage, estrogen receptor alpha (ERα) and progesterone receptor (PR) expression. Theses studies suggest amplification of the MT1 gene in some breast tumors and an inverse correlation with ERα, PR and MT1 protein expression. Furthermore, these approaches employing immunohistochemical and immunofluorescent/confocal microscopic studies demonstrate that the MT1 receptor is localized to the caveoli and that MT1 expression in MCF-7 breast cancer cells can be repressed by estradiol and melatonin.

Melatonin, via its MT1 receptor, but not the MT2 receptor, can modulate the transcriptional activity of various nuclear receptors - estrogen receptor alpha (ER alpha) and retinoic acid receptor alpha (RAR alpha), but not ER beta- in MCF-7, T47D, and ZR-75-1 human breast cancer cell lines. The anti-proliferative and nuclear receptor modulatory actions of melatonin are mediated via the MT1 G protein-coupled receptor expressed in human breast cancer cells. However, the specific G proteins and associated pathways involved in the nuclear receptor transcriptional regulation by melatonin are not yet clear. Upon activation, the MT1 receptor specifically couples to the G(alpha i2), G(alpha i3), G(alpha q), and G(alpha ll) proteins, and via activation of G(alpha i2) proteins, melatonin suppresses forskolin-induced 3',5'-cyclic adenosine monophosphate production, while melatonin activation of G(alpha q), is able to inhibit phospholipid hydrolysis and ATP's induction of inositol triphosphate production in MCF-7 breast cancer cells. Employing dominant-negative and dominant-positive) forms of these G proteins, we demonstrate that G(alpha i2) proteins mediate the suppression of estrogen-induced ER alpha transcriptional activity by melatonin, while the G(q) protein mediates the enhancement of retinoid-induced RAR alpha transcriptional activity by melatonin. However, the growth-inhibitory actions of melatonin are mediated via both G(alpha i2) and G(alpha q) proteins.