The molecular mechanisms by which ovarian hormones stimulate growth of breast tumors are unclear. It has been reported previously that estrogens activate the signal-transducing Src/p21 ras /Erk pathway in human breast cancer cells via an interaction of estrogen receptor (ER) with c-Src. We now show that progestins stimulate human breast cancer T47D cell proliferation and induce a similar rapid and transient activation of the pathway which, surprisingly, is blocked not only by anti-progestins but also by anti-estrogens. In Cos-7 cells transfected with the B isoform of progesterone receptor (PR B ), progestin activation of the MAP kinase pathway depends on co-transfection of ER. A transcriptionally inactive PR B mutant also activates the signaling pathway, demonstrating that this activity is independent of transcriptional effects. PR B does not interact with c-Src but associates via the N-terminal 168 amino acids with ER. This association is required for the signaling pathway activation by progestins. We propose that ER transmits to the Src/p21 ras /Erk pathway signals received from the agonist-activated PR B . These findings reveal a hitherto unrecognized cross-talk between ovarian hormones which could be crucial for their growth-promoting effects on cancer cells.

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Steroid hormones influence a plethora of cellular functions, depending on the nature of the target cell and the constellation of signals impinging on the cell at a given time. To achieve the necessary coordination with other signaling pathways in the complex intracellular space, steroid hormones likely use a variety of mechanisms. Until very recently, attention has mainly been focused on the transcriptional effects of steroid hormones. These responses are mediated by the intracellular hormone receptors, which participate in multiple interactions with DNA, other sequence-specific transcription factors, transcriptional coregulators, and the general transcriptional machinery (3). In the last few years, a great effort has been devoted to understanding the nature of the transcriptional coregulators and how they mediate the interaction of the hormone receptors with chromatin remodelling complexes and the transcriptional apparatus (18). Considerable progress has been achieved, leading to the recognition of covalent and conformational chromatin changes as key steps in transcriptional regulation by steroid hormone receptors and other transcription factors (5).In addition to their direct transcriptional effects, steroid hormones have been found to influence the activity of many other signaling pathways by so-called "nongenomic mechanisms" (25,34,38). These effects are mediated by interactions at the membrane or cytoplasmic level and offer a possibility for integration of the steroid hormone signals at the entry site of many other physiological signals acting via membrane receptors (39). Very often, these nongenomic effects have been attributed to poorly characterized receptors, whose relationship with the conventional nuclear receptors remains unclear (38). In the case of the ovarian hormones estrogens and progestins, cross talk with a number of other signaling pathways has been described, including cyclic AMP (1, 16), Ca-calmodulin (15), the G protein-coupled receptors (20), and the mitogen-activated protein (MAP) kinase pathway (25,26). In breast cancer cells, estrogens stimulate cell proliferation, and this effect can be blocked by inhibitors of the MAP kinase signaling pathway (30) or by intracellular calcium chelators (22). This pathway is activated by estrogens through an interaction of the classical estrogen receptor alpha (ER␣) with c-Src, which can be detected by coimmunoprecipitation (30). c-Src activity is enhanced 2 min after addition of 17␤-estradiol, reaches a peak after 5 min, and returns to basal levels after 15 to 30 min. Activation of c-Src can be inhibited by steroidal and nonsteroidal antiestrogens and is followed by transient activation of Ras, Raf, and Erk1/2 (7, 31). The participation of the classical ER␣ in activation of the mitogenic pathway was demonstrated in gene transfection studies in COS-7 cells (31). Activation of the cascade in these cells by estrogens was strictly dependent on transfection of ER␣, was inhibited by antiestrogens, and led to the formation of a complex of ER␣ and c-Src. Simi...

Under conditions of short-term hormone deprivation, epidermal growth factor (EGF) induces DNA synthesis, cytoskeletal changes, and Src activation in MCF-7 and LNCaP cells. These effects are drastically inhibited by pure estradiol or androgen antagonists, implicating a role of the steroid receptors in these findings. Interestingly, EGF triggers rapid association of Src with androgen receptor (AR) and estradiol receptor A (ERA) in MCF-7 cells or ERB in LNCaP cells. Here, we show that, through EGF receptor (EGFR) and erb-B2, EGF induces tyrosine phosphorylation of ER preassociated with AR, thereby triggering the assembly of ER/AR with Src and EGFR. Remarkably, experiments in Cos cells show that this complex stimulates EGF-triggered EGFR tyrosine phosphorylation. In turn, estradiol and androgen antagonists, through the Src-associated receptors, prevent Src activation by EGF and heavily reduce EGFR tyrosine phosphorylation and the subsequent multiple effects, including DNA synthesis and cytoskeletal changes in MCF-7 cells. In addition, knockdown of ERa or AR gene by small interfering RNA (siRNA) almost abolishes EGFR tyrosine phosphorylation and DNA synthesis in EGF-treated MCF-7 cells. The present findings reveal that steroid receptors have a key role in EGF signaling. EGFR tyrosine phosphorylation, depending on Src, is a part of this mechanism. Understanding of EGF-triggered growth and invasiveness of mammary and prostate cancer cells expressing steroid receptors is enhanced by this report, which reveals novel aspects of steroid receptor action. (Cancer Res 2005; 65(22): 10585-93)

In NIH3T3 cells, 0.001 nM of the synthetic androgen R1881 induces and stimulates association of androgen receptor (AR) with Src and phosphatidylinositol 3-kinase (Pl3-kinase), respectively, thereby triggering S-phase entry. 10 nM R1881 stimulates Rac activity and membrane ruffling in the absence of the receptor–Src–PI3-kinase complex assembly. The antiandrogen Casodex and specific inhibitors of Src and PI3-kinase prevent both hormonal effects, DNA synthesis and cytoskeletal changes. Neither low nor high R1881 concentration allows receptor nuclear translocation and receptor-dependent transcriptional activity in fibroblasts, although they harbor the classical murine AR. The very low amount of AR in NIH3T3 cells (7% of that present in LNCaP cells) activates the signaling pathways, but apparently is not sufficient to stimulate gene transcription. This view is supported by the appearance of receptor nuclear translocation as well as receptor-mediated transcriptional activity after overexpression of AR in fibroblasts. In addition, AR-negative Cos cells transiently transfected with a very low amount of hAR cDNA respond to low and high R1881 concentrations with signaling activation. Interestingly, they do not show significant transcriptional activation under the same experimental conditions. Fibroblasts are the first example of cells that respond to steroid hormones with activation of signaling pathways in the absence of endogenous receptor transcriptional activity. The data reported also show that hormone concentration can be crucial in determining the type of cell responsiveness.