mTOR intersects antibody-inducing signals from TACI in marginal zone B cells.

Mechanistic target of rapamycin (mTOR) enhances immunity in addition to orchestrating metabolism. Here we show that mTOR coordinates immunometabolic reconfiguration of marginal zone (MZ) B cells, a pre-activated lymphocyte subset that mounts antibody responses to T-cell-independent antigens through a Toll-like receptor (TLR)-amplified pathway involving transmembrane activator and CAML interactor (TACI). This receptor interacts with mTOR via the TLR adapter MyD88. The resulting mTOR activation instigates MZ B-cell proliferation, immunoglobulin G (IgG) class switching, and plasmablast differentiation through a rapamycin-sensitive pathway that integrates metabolic and antibody-inducing transcription programs, including NF-κB. Disruption of TACI-mTOR interaction by rapamycin, truncation of the MyD88-binding domain of TACI, or B-cell-conditional mTOR deficiency interrupts TACI signaling via NF-κB and cooperation with TLRs, thereby hampering IgG production to T-cell-independent antigens but not B-cell survival. Thus, mTOR drives innate-like antibody responses by linking proximal TACI signaling events with distal immunometabolic transcription programs.

Androgen-induced cell migration: role of androgen receptor/filamin A association.

BACKGROUND: Androgen receptor (AR) controls male morphogenesis, gametogenesis and prostate growth as well as development of prostate cancer. These findings support a role for AR in cell migration and invasiveness. However, the molecular mechanism involved in AR-mediated cell migration still remains elusive. METHODOLOGY/PRINCIPAL FINDINGS: Mouse embryo NIH3T3 fibroblasts and highly metastatic human fibrosarcoma HT1080 cells harbor low levels of transcriptionally incompetent AR. We now report that, through extra nuclear action, AR triggers migration of both cell types upon stimulation with physiological concentrations of the androgen R1881. We analyzed the initial events leading to androgen-induced cell migration and observed that challenging NIH3T3 cells with 10 nM R1881 rapidly induces interaction of AR with filamin A (FlnA) at cytoskeleton. AR/FlnA complex recruits integrin beta 1, thus activating its dependent cascade. Silencing of AR, FlnA and integrin beta 1 shows that this ternary complex controls focal adhesion kinase (FAK), paxillin and Rac, thereby driving cell migration. FAK-null fibroblasts migrate poorly and Rac inhibition by EHT impairs motility of androgen-treated NIH3T3 cells. Interestingly, FAK and Rac activation by androgens are independent of each other. Findings in human fibrosarcoma HT1080 cells strengthen the role of Rac in androgen signaling. The Rac inhibitor significantly impairs androgen-induced migration in these cells. A mutant AR, deleted of the sequence interacting with FlnA, fails to mediate FAK activation and paxillin tyrosine phosphorylation in androgen-stimulated cells, further reinforcing the role of AR/FlnA interaction in androgen-mediated motility. CONCLUSIONS/SIGNIFICANCE: The present report, for the first time, indicates that the extra nuclear AR/FlnA/integrin beta 1 complex is the key by which androgen activates signaling leading to cell migration. Assembly of this ternary complex may control organ development and prostate cancer metastasis.

Hormone-dependent nuclear export of estradiol receptor and DNA synthesis in breast cancer cells.

In breast cancer cells, cytoplasmic localization of the estradiol receptor alpha (ERalpha) regulates estradiol-dependent S phase entry. We identified a nuclear export sequence (NES) in ERalpha and show that its export is dependent on both estradiol-mediated phosphatidylinositol-3-kinase (PI3K)/AKT activation and chromosome region maintenance 1 (CRM1). A Tat peptide containing the ERalpha NES disrupts ERalpha-CRM1 interaction and prevents nuclear export of ERalpha- and estradiol-induced DNA synthesis. NES-ERalpha mutants do not exit the nucleus and inhibit estradiol-induced S phase entry; ERalpha-dependent transcription is normal. ERalpha is associated with Forkhead proteins in the nucleus, and estradiol stimulates nuclear exit of both proteins. ERalpha knockdown or ERalpha NES mutations prevent ERalpha and Forkhead nuclear export. A mutant of forkhead in rhabdomyosarcoma (FKHR), which cannot be phosphorylated by estradiol-activated AKT, does not associate with ERalpha and is trapped in the nucleus, blocking S phase entry. In conclusion, estradiol-induced AKT-dependent phosphorylation of FKHR drives its association with ERalpha, thereby triggering complex export from the nucleus necessary for initiation of DNA synthesis and S phase entry.

Integrating signals between cAMP and MAPK pathways in breast cancer.

Breast cancer is one of the most common malignancies in Western society. Localized breast cancer, before it spreads, can be cured by surgery. However, the high mortality rate associated with breast cancer is due to a propensity of the tumor to metastasize when the primary tumor is small or undetectable. Although steroid receptor status has been recognized as the most precise predictor of response to hormone therapy, a significant number of tumors expressing these receptors metastasize and patients do not respond to the antihormone therapy. The mechanism leading to breast cancer progression and resistance to the hormone therapy is not completely understood at the present time. Compelling evidence shows that hormone-bound steroid receptors in breast cancer cells activate complex signaling networks, which include MAPK- and G protein-dependent pathways. These responses, which occur within seconds or minutes after steroid administration, are not due to changes in gene expression. Depending on cell systems, steroid activation of these networks leads to different and profound effects on extra nuclear and nuclear events. In such a way steroids foster cell cycle, reduce apoptosis and stimulate cell migration of target cells. All these processes are deregulated in breast cancer. In this review we will discuss new aspects of signaling pathways activated by steroids and their integration with other pathways in breast cancer. Recent findings on the discovery of compounds specifically interfering in such a complex network will be presented.

Crosstalk between EGFR and extranuclear steroid receptors.

Epidermal growth factor (EGF) stimulates DNA synthesis and cytoskeletal rearrangement in human breast cancer (MCF-7) and human prostate cancer (LNCaP) cells. Both effects are inhibited by estrogen (ICI 182,780) and androgen (Casodex) antagonists. This supports the view that crosstalk exists between EGF and estradiol (ER) and androgen (AR) receptors and suggests that these receptors are directly involved in the EGF action. Our recent work shows that EGF stimulates ER phosphorylation on tyrosine and promotes the association of a complex between EGFR, AR/ER, and the kinase Src. The complex assembly triggers Src activity, epidermal growth factor receptor (EGFR) phosphorylation on tyrosine, and the EGF-dependent signaling pathway activation. In these cells, the AR/ER/Src complex is required for the EGF action, as the growth factor effects are abolished upon receptor silencing by specific SiRNAs and steroid antagonists or Src inhibition by the kinase inhibitor PP2.

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.