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 (PRB), progestin activation of the MAP kinase pathway depends on co‐transfection of ER. A transcriptionally inactive PRB mutant also activates the signaling pathway, demonstrating that this activity is independent of transcriptional effects. PRB 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 PRB. These findings reveal a hitherto unrecognized cross‐talk between ovarian hormones which could be crucial for their growth‐promoting effects on cancer cells.

The p85‐associated phosphatidylinositol (PI) 3‐kinase/Akt pathway mediates the oestradiol‐induced S‐phase entry and cyclin D1 promoter activity in MCF‐7 cells. Experiments with Src, p85α and Akt dominant‐negative forms indicate that in oestradiol‐treated cells these signalling effectors target the cyclin D1 promoter. Oestradiol acutely increases PI3‐kinase and Akt activities in MCF‐7 cells. In NIH 3T3 cells expressing ERα, a dominant‐negative p85 suppresses hormone stimulation of Akt. The Src inhibitor, PP1, prevents hormone stimulation of Akt and PI3‐kinase activities in MCF‐7 cells. In turn, stimulation of Src activity is abolished in ERα‐expressing NIH 3T3 fibroblasts by co‐transfection of the dominant‐negative p85α and in MCF‐7 cells by the PI3‐kinase inhibitor, LY294002. These findings indicate a novel reciprocal cross‐talk between PI3‐kinase and Src. Hormone stimulation of MCF‐7 cells rapidly triggers association of ERα with Src and p85. In vitro these proteins are assembled in a ternary complex with a stronger association than that of the binary complexes composed by the same partners. The ternary complex probably favours hormone activation of Src‐ and PI3‐kinase‐dependent pathways, which converge on cell cycle progression.

The recent findings that oestradiol and progestins activate the Src/Ras/Erks signalling pathway raise the question of the role of this stimulation. Microinjection experiments of human mammary cancer‐derived cells (MCF‐7 and T47D) with cDNA of catalytically inactive Src or anti‐Ras antibody prove that Src and Ras are required for oestradiol and progestin‐dependent progression of cells through the cell cycle. The antitumoral ansamycin antibiotic, geldanamycin, disrupts the steroid‐induced Ras–Raf‐1 association and prevents Raf‐1 activation and steroid‐induced DNA synthesis. Furthermore, the selective MEK 1 inhibitor, PD 98059, inhibits oestradiol and progestin stimulation of Erk‐2 and the steroid‐dependent S‐phase entry. The MDA‐MB231 cells, which do not express oestradiol receptor, fail to respond to oestradiol in terms of Erk‐2 activation and S‐phase entry. Fibroblasts are made equally oestradiol‐responsive in terms of DNA synthesis by transient transfection with either the wild‐type or the transcriptionally inactive mutant oestradiol receptor (HE241G). Co‐transfection of catalytically inactive Src as well as treatment with PD98059 inhibit the oestradiol‐dependent S‐phase entry of fibroblasts expressing either the wild‐type oestrogen receptor or its transcriptionally inactive mutant. The data presented support the view that non‐transcriptional action of the two steroids plays a major role in cell cycle progression.

In breast cancer cells, estrogens activate the Src/Erk pathway through an interaction of the estrogen receptor alpha (ERα) with the SH2 domain of c-Src. Progestins have been reported to activate also this pathway either via an interaction of the progesterone receptor isoform B (PRB) with ERα, which itself activates c-Src, or by direct interaction of PRB with the SH3 domain of c-Src. Here we identify two domains of PRB, ERID-I and -II, mediating a direct interaction with the ligand-binding domain of ERα. ERID-I and ERID-II flank a proline cluster responsible for binding of PRB to c-Src. In mammalian cells, the interaction of PRB with ERα and the progestin activation of the Src/Erk cascade are abolished by deletion of either ERID-I or ERID-II. These regions are not required for transactivation of a progesterone-responsive reporter gene. Mutations in the proline cluster of PRB that prevent a direct interaction with c-Src do not affect the strong activation of c-Src by progestins in the presence of ERα. Thus, in cells with ERα, ERID-I and ERID-II are necessary and sufficient for progestin activation of the endogenous Src/Erk pathway.

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 α (ERα) in MCF-7 cells or ERβ 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 ERα 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.