Antonio Bilancio

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.

Essential role for the p110δ phosphoinositide 3-kinase in the allergic response

Inflammatory substances released by mast cells induce and maintain the allergic response. Mast cell differentiation and activation are regulated, respectively, by stem cell factor (SCF; also known as Kit ligand) and by allergen in complex with allergen-specific immunoglobulin E (IgE). Activated SCF receptors and high-affinity receptors for IgE (FcɛRI) engage phosphoinositide 3-kinases (PI(3)Ks) to generate intracellular lipid second messenger signals. Here, we report that genetic or pharmacological inactivation of the p110δ isoform of PI(3)K in mast cells leads to defective SCF-mediated in vitro proliferation, adhesion and migration, and to impaired allergen–IgE-induced degranulation and cytokine release. Inactivation of p110δ protects mice against anaphylactic allergic responses. These results identify p110δ as a new target for therapeutic intervention in allergy and mast-cell-related pathologies.

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.

The p110δ Isoform of Phosphoinositide 3-Kinase Controls Clonal Expansion and Differentiation of Th Cells

The role of PI3K in T cell activation and costimulation has been controversial. We previously reported that a kinase-inactivating mutation (D910A) in the p110δ isoform of PI3K results in normal T cell development, but impaired TCR-stimulated cell proliferation in vitro. This proliferative defect can be overcome by providing CD28 costimulation, which raises the question as to whether p110δ activity plays a role in T cell activation in vivo, which occurs primarily in the context of costimulation. In this study, we show that the PI3K signaling pathway in CD28-costimulated p110δD910A/D910A T cells is impaired, but that ERK phosphorylation and NF-κB nuclear translocation are unaffected. Under in vitro conditions of physiological Ag presentation and costimulation, p110δD910A/D910A T cells showed normal survival, but underwent fewer divisions. Differentiation along the Th1 and Th2 lineages was impaired in p110δD910A/D910A T cells and could not be rescued by exogenous cytokines in vitro. Adoptive transfer and immunization experiments in mice revealed that clonal expansion and differentiation in response to Ag and physiological costimulation were also compromised. Thus, p110δ contributes significantly to Th cell expansion and differentiation in vitro and in vivo, also in the context of CD28 costimulation.

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.

Stat3-induced apoptosis requires a molecular switch in PI(3)K subunit composition.

Physiological apoptosis is induced by a switch from survival to death signalling. Dysregulation of this process is frequently associated with cancer. A powerful model for this apoptotic switch is mammary gland involution, during which redundant milk-producing epithelial cells undergo apoptosis. Signal transducer and activator of transcription 3 (Stat3) is an essential mediator of this switch but the mechanism has not yet been defined. Stat3-dependent cell death during involution can be blocked by activation of Akt/protein kinase B (PKB), a downstream effector of the phosphoinositide-3-OH kinase (PI(3)K) pathway. Here we show that expression of the PI(3)K regulatory subunits p55α and p50α is induced by Stat3 during involution. In the absence of Stat3 in vivo, upregulation of p55α and p50α is abrogated, levels of activated Akt are sustained and apoptosis is prevented. Chromatin immunoprecipitation assays show that Stat3 binds directly to the p55α and p50α promoters in vivo. Overexpression of either p55α or p50α reduces levels of activated Akt. We propose a novel mechanism in which Stat3 regulates apoptosis by inducing expression of distinct PI(3)K regulatory subunits to downregulate PI(3)K-Akt-mediated survival signalling.

Class I Phosphoinositide 3-Kinase p110β Is Required for Apoptotic Cell and Fcγ Receptor-mediated Phagocytosis by Macrophages

Phosphoinositide 3-kinases (PI3Ks) play an important role in a variety of cellular functions, including phagocytosis. PI3Ks are activated during phagocytosis induced by several receptors and have been shown to be required for phagocytosis through the use of inhibitors such as wortmannin and LY294002. Mammalian cells have multiple isoforms of PI3K, and the role of the individual isoforms during phagocytosis has not been addressed. The class I PI3Ks consist of a catalytic p110 isoform associated with a regulatory subunit. Mammals have three genes for the class IA p110 subunits encoding p110α, p110β, and p110δ and one gene for the class IB p110 subunit encoding p110γ. Here we report a specific recruitment of p110β and p110δ (but not p110α) isoforms to the nascent phagosome during apoptotic cell phagocytosis by fibroblasts. By microinjecting inhibitory antibodies specific to class IA p110 subunits, we have shown that p110β is the major isoform required for apoptotic cell and Fcγ receptor-mediated phagocytosis by primary mouse macrophages. Macrophages from mice expressing a catalytically inactive form of p110δ showed no defect in the phagocytosis of apoptotic cells and IgG-opsonized particles, confirming the lack of a major role for p110δ in this process. Similarly, p110γ-deficient macrophages phagocytosed apoptotic cells normally. Our findings demonstrate that p110β is the major class I catalytic isoform required for apoptotic cell and Fcγ receptor-mediated phagocytosis by primary macrophages.