Antimo Migliaccio

Tyrosine kinase/p21ras/MAP-kinase pathway activation by estradiol-receptor complex in MCF-7 cells.

The mechanism by which estradiol acts on cell multiplication is still unclear. Under conditions of estradiol‐dependent growth, estradiol treatment of human mammary cancer MCF‐7 cells triggers rapid and transient activation of the mitogen‐activated (MAP) kinases, erk‐1 and erk‐2, increases the active form of p21ras, tyrosine phosphorylation of Shc and p190 protein and induces association of p190 to p21ras‐GAP. Both Shc and p190 are substrates of activated src and once phosphorylated, they interact with other proteins and upregulate p21ras. Estradiol activates the tyrosine kinase/p21ras/MAP‐kinase pathway in MCF‐7 cells with kinetics which are similar to those of peptide mitogens. It is only after introduction of the human wild‐type 67 kDa estradiol receptor cDNA that Cos cells become estradiol‐responsive in terms of erk‐2 activity. This finding, together with the inhibition by the pure anti‐estrogen ICI 182 780 of the stimulatory effect of estradiol on each step of the pathway in MCF‐7 cells proves that the classic estradiol receptor is responsible for the transduction pathway activation. Transfection experiments of Cos cells with the estradiol receptor cDNA and in vitro experiments with c‐src show that the estradiol receptor activates c‐src and this activation requires occupancy of the receptor by hormone. Our experiments suggest that c‐src is an initial and integral part of the signaling events mediated by the estradiol receptor.

Steroid‐induced androgen receptor–oestradiol receptor β–Src complex triggers prostate cancer cell proliferation

Treatment of human prostate carcinoma‐derived LNCaP cells with androgen or oestradiol triggers simultaneous association of androgen receptor and oestradiol receptor β with Src, activates the Src/Raf‐1/Erk‐2 pathway and stimulates cell proliferation. Surprisingly, either androgen or oestradiol action on each of these steps is inhibited by both anti‐androgens and anti‐oestrogens. Similar findings for oestradiol receptor α were observed in MCF‐7 or T47D cells stimulated by either oestradiol or androgens. Microinjection of LNCaP, MCF‐7 and T47D cells with SrcK− abolishes steroid‐stimulated S‐phase entry. Data from transfected Cos cells confirm and extend the findings from these cells. Hormone‐stimulated Src interaction with the androgen receptor and oestradiol receptor α or β is detected using glutathione S‐transferase fusion constructs. Src SH2 interacts with phosphotyrosine 537 of oestradiol receptor α and the Src SH3 domain with a proline‐rich stretch of the androgen receptor. The role of this phosphotyrosine is stressed by its requirement for association of oestradiol receptor α with Src and consequent activation of Src in intact Cos cells.

Activation of the Src/p21ras/Erk pathway by progesterone receptor via cross‐talk with estrogen receptor

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/p21ras/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/p21ras/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.

PI3-kinase in concert with Src promotes the S-phase entry of oestradiol-stimulated MCF-7 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.

Non-transcriptional action of oestradiol and progestin triggers DNA synthesis.

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.

Two Domains of the Progesterone Receptor Interact with the Estrogen Receptor and Are Required for Progesterone Activation of the c-Src/Erk Pathway in Mammalian Cells

ABSTRACT 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.

Steroid receptor regulation of epidermal growth factor signaling through Src in breast and prostate cancer cells: steroid antagonist action.

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 alpha (ERalpha) in MCF-7 cells or ERbeta 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 ERalpha 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.

Androgen-stimulated DNA synthesis and cytoskeletal changes in fibroblasts by a nontranscriptional receptor action

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.

Sex steroid hormones act as growth factors

We observed that sex steroid hormones, like growth factors, stimulate the Src/Ras/erk pathway of cell lines derived from human mammary or prostate cancers. In addition, hormone-dependent pathway activation can be induced in Cos cells, upon transfection of classic steroid receptors. Cross-talks between sex steroid receptors regulate their association with Src and consequent pathway activation. Oestradiol treatment of MCF-7 cells triggers simultaneous association of ER with Src and p85, the regulatory subunit of phosphatidylinositol-3-kinase (PI3-kinase) and activation of Src- and PI3-K-dependent pathways. Activation of the latter pathway triggers cyclin D1 transcription, that is unaffected by Mek-1 activation. This suggests that simultaneous activation of different signalling effectors is required to target different cell cycle components. Thus, a novel reciprocal cross-talk between the two pathways appears to be mediated by the ER. In all tested cells, activation of the signalling pathways has a proliferative role. Transcriptionally inactive ER expressed in NIH 3T3 cells responds to hormone causing Src/Ras/Erk pathway activation and DNA synthesis. This suggests that in these cells genomic activity is required for later events of cell growth.

cAMP signaling selectively influences Ras effectors pathways

Thyrotropin (TSH) stimulates survival and growth of thyroid cells via a seven transmembrane G protein-coupled receptor. TSH elevates the intracellular cyclic AMP (cAMP) levels activating protein kinase A (PKA). Recent evidence indicates that p21 Ras is required for TSH-induced mitogenesis, but the molecular mechanism(s) is not known. Here we report that Ras p21 activity is necessary for the Go- G1 transition in TSH induced cycle and that the downstream effector of Ras upon TSH signaling is p85-p110 PI3K. We show that PI3K inhibitors block TSH-induced DNA synthesis, cAMP-PKA stimulate the formation of the complex PI3K-p21 Ras and reduce the complex Ras-Raf1 in thyroid and other cells types. Moreover, PKA phosphorylates immunoprecipitated p85 and PKA phosphorylation of cell extracts significantly stimulates the formation of the complex PI3K-Ras. We suggest that PKA phosphorylates p85 and stabilizes the complex p110-p85, enhancing the interaction PI3K and p21 Ras. Simultaneously, cAMP inhibits Raf-1-ERK signaling by decreasing Raf1 availability to Ras. Under these circumstances PI3K signaling is favored. These results indicate that PI3K is an important mediator of Ras effects in cAMP-induced proliferation and illustrates how cAMP can selectively influence Ras effector pathways.