Maria Lombardi

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.

Crosstalk between EGFR and Extranuclear Steroid Receptors

Abstract:u2002 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.

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

In breast cancer cells, cytoplasmic localization of the estradiol receptor α (ERα) regulates estradiol-dependent S phase entry. We identified a nuclear export sequence (NES) in ERα 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 ERα NES disrupts ERα–CRM1 interaction and prevents nuclear export of ERα- and estradiol-induced DNA synthesis. NES-ERα mutants do not exit the nucleus and inhibit estradiol-induced S phase entry; ERα-dependent transcription is normal. ERα is associated with Forkhead proteins in the nucleus, and estradiol stimulates nuclear exit of both proteins. ERα knockdown or ERα NES mutations prevent ERα and Forkhead nuclear export. A mutant of forkhead in rhabdomyosarcoma (FKHR), which cannot be phosphorylated by estradiol-activated AKT, does not associate with ERα and is trapped in the nucleus, blocking S phase entry. In conclusion, estradiol-induced AKT-dependent phosphorylation of FKHR drives its association with ERα, thereby triggering complex export from the nucleus necessary for initiation of DNA synthesis and S phase entry.

Role of Atypical Protein Kinase C in Estradiol-Triggered G1/S Progression of MCF-7 Cells

ABSTRACT Expression of a dominant negative atypical protein kinase C (aPKC), PKCζ, prevents nuclear translocation of extracellular regulated kinase 2 (ERK-2), p27 nuclear reduction, and DNA synthesis induced by estradiol in human mammary cancer-derived MCF-7 cells. aPKC action upstream of these events has been analyzed. In hormone-stimulated NIH 3T3 and Cos cells ectopically expressing human estrogen receptor alpha (hERα), aPKC is activated by phosphatidylinositol 3-kinase (PI 3-kinase) and, in turn, controls the Ras/MEK-1/ERK cascade. In MCF-7 and Cos cells stimulated by hormone, PI 3-kinase activates PKCζ by Thr410 phosphorylation. Serine phosphorylation of PKCζ is simultaneously induced. PKCζ activation leads to recruitment of Ras to a multimolecular complex that also includes hERα, Src, PI 3-kinase, and aPKC. We propose that PKCζ pushes Ras and the signaling complex close together in such a way that it facilitates the Src-dependent Ras activation. This activation is crucial for the interplay between estradiol-triggered signaling and cell cycle machinery.

Rapid signalling pathway activation by androgens in epithelial and stromal cells

Estradiol rapidly activates Src as well as the Src-dependent pathway in human mammary cancer-derived MCF-7 cells, in human prostate cancer-derived LNCaP cells and in Cos cells transiently expressing hERs [EMBO J. 15 (1996) 1292; EMBO J. 17 (1998) 2008]. In addition, estradiol immediately stimulates, yes, an ubiquitous member of the Src kinase family, in human colon carcinoma-derived Caco-2 cells [Cancer Res. 56 (1996) 4516]. Progestins and androgens activate the same pathway in human mammary and prostate cancer-derived cells [EMBO J. 17 (1998) 2008; EMBO J. 19 (2000) 5406]. We observed that estradiol also stimulates the phosphatidylinositol-3-kinase (PI3K)/AKT pathway in MCF-7 cells [EMBO J. 20 (2001) 6050]. In these cells, activation of the Src- and the PI3 K-dependent pathways is simultaneous and mediated by direct interactions of the two kinases with ERalpha. The signalling pathway activation by sex-steroid hormones leads to DNA synthesis and cell growth in human mammary and prostate cancer-derived cells [EMBO J. 19 (2000) 5406; EMBO J. 20 (2001) 6050; EMBO J. 18 (1999) 2500]. Furthermore, androgen stimulation of NIH3T3 fibroblasts activates the same pathways triggered by this hormone in LNCaP cells and promotes the S-phase entry or cytoskeleton changes in these cells [J. Cell Biol. 161 (2003) 547]. All the described effects are rapid and require classic steroid receptors, but, surprisingly, not their transcriptional activity. Indeed, a transcriptionally inactive mutant of hER mediates the estrogen-stimulated DNA synthesis of NIH3T3 fibroblasts [EMBO J. 18 (1999) 2500]. Furthermore, AR in NIH3T3 cells does not enter nuclei and is unable to respond to the hormone with transcription stimulation, whereas it activates signaling pathways and triggers important biological responses. Signaling pathway activation by steroids has also been described by other groups under different experimental conditions and/or in different cell types. In these cells, steroid stimulation triggers various effects, such as neuroprotection, vasorelaxation or bone protection [J. Neurosci. Res. 60 (2000) 321; Nature 407 (2000) 538; J. Cell Biochem. 76 (1999) 206]. Analysis of the mechanisms responsible for the hormone-dependent and steroid receptor-mediated pathway activation in epithelial as well as stromal cells reveals immediate association of steroid receptors with extranuclear signaling effectors [EMBO J. 17 (1998) 2008; Cancer Res. 56 (1996) 4516; EMBO J. 19 (2000) 5406; EMBO J. 20 (2001) 6050; J. Cell Biol. 161 (2003) 547]. These results further highlight the central role of the hormone-regulated protein-protein interactions in the steroid action. They also offer the possibility of interfering with important activities of hormones, such as proliferation or survival, cytoskeleton changes as well as invasiveness and vasorelaxation, without affecting the steroid effects that depend on receptor transcriptional activity.

Immunoglobulin G from Breast Cancer Patients Regulates MCF-7 Cells Migration and MMP-9 Activity by Stimulating Muscarinic Acetylcholine Receptors

PurposeWe have previously reported the expression of muscarinic acetylcholine receptors (mAChR) in human breast tumors. The activation of these receptors triggered tumor cell proliferation. Considering that invasion and metastasis is the major cause of death in cancer, we investigated the action of autoantibodies against mAChR derived from breast cancer patients in stage I (T1N0Mx-IgG) on MCF-7 cells migration and metalloproteinase-9 (MMP-9) activity. We also analyzed the participation of phospholipase C/nitric oxide synthase/protein kinase C pathway.MethodsImmunoglobulin G (IgG) was purified by chromatography in protein G-agarose from blood samples of breast cancer patients obtained under informed consent. Migration was assayed by an in vitro wound assay. MMP-9 activity was quantified by zymography.ResultsT1N0Mx-IgG promoted tumor cell migration and increased MMP9 activity mimicking the action of the muscarinic agonist carbachol. This effect was reduced not only by the presence of atropine but also by 4-DAMP or tropicamide, antagonists for M3 and M4 mAChR subtypes respectively. The actions of T1N0Mx-IgG and carbachol on MCF-7 cells, involved the participation of phospholipase C/nitric oxide synthase/protein kinase C pathway.ConclusionsIgG from breast cancer patients in stage I could be promoting tumor progression by regulating migration and MMP-9 activity in tumor cells via mAChR activation. The presence of these autoantibodies could be determining the prognosis of breast cancer in these patients.

Autoantibodies against muscarinic receptors in breast cancer: their role in tumor angiogenesis.

The presence of autoantibodies in cancer has become relevant in recent years. We demonstrated that autoantibodies purified from the sera of breast cancer patients activate muscarinic acetylcholine receptors in tumor cells. Immunoglobulin G (IgG) from breast cancer patients in T1N0Mx stage (tumor size≤2 cm, without lymph node metastasis) mimics the action of the muscarinic agonist carbachol stimulating MCF-7 cell proliferation, migration and invasion. Angiogenesis is a central step in tumor progression because it promotes tumor invasion and metastatic spread. Vascular endothelial growth factor-A (VEGF-A) is the main angiogenic mediator, and its levels have been correlated with poor prognosis in cancer. The aim of the present work was to investigate the effect of T1N0Mx-IgG on the expression of VEGF-A, and the in vivo neovascular response triggered by MCF-7 cells, via muscarinic receptor activation. We demonstrated that T1N0Mx-IgG (10−8 M) and carbachol (10−9 M) increased the constitutive expression of VEGF-A in tumor cells, effect that was reverted by the muscarinic antagonist atropine. We also observed that T1N0Mx-IgG and carbachol enhanced the neovascular response produced by MCF-7 cells in the skin of NUDE mice. The action of IgG or carbachol was reduced in the presence of atropine. In conclusion, T1N0Mx-IgG and carbachol may promote VEGF-A production and neovascularization induced by breast tumor cells via muscarinic receptors activation. These effects may be accelerating breast tumor progression.

A natural antiviral and immunomodulatory compound with antiangiogenic properties

Meliacine (MA), an antiviral principle present in partially purified leaf extracts of Melia azedarach L., reduces viral load and abolishes the inflammatory reaction and neovascularization during the development of herpetic stromal keratitis in mice. 1-cinnamoyl-3,11-dihydroxymeliacarpin (CDM), obtained from MA, displays anti-herpetic and immunomodulatory activities in vitro. We investigated whether CDM interferes with the angiogenic process. CDM impeded VEGF transcription in LPS-stimulated and HSV-1-infected cells. It proved to have neither cytotoxic nor antiproliferative effect in HUVEC and to restrain HUVEC migration and formation of capillary-like tubes. Moreover, MA inhibits LMM3 tumor-induced neovascularization in vivo. We postulate that the antiangiogenic activity of CDM displayed in vitro as a consequence of their immunomodulatory properties is responsible for the antiangiogenic activity of MA in vivo, which would be associated with the lack of neovascularization in murine HSV-1-induced ocular disease.

Synthetic stigmasterol derivatives inhibit capillary tube formation, herpetic corneal neovascularization and tumor induced angiogenesis: Antiangiogenic stigmasterol derivatives.

Angiogenesis plays a critical role in initiating and promoting several diseases, such as cancer and herpetic stromal keratitis (HSK). Herein, we studied the inhibitory effect of two synthetic stigmasterol derivatives on capillary tube-like structures and on cell migration in human umbilical vein endothelial cells (HUVEC): (22S,23S)-22,23-dihydroxystigmast-4-en-3-one (compound 1) and (22S,23S)-3β-bromo-5α,22,23-trihydroxystigmastan-6-one (compound 2). We also studied their effect on VEGF expression in IL-6 stimulated macrophages and in LMM3 breast cancer cells. Furthermore, we investigated the antiangiogenic activity of the compounds on corneal neovascularization in the murine model of HSK and in an experimental model of tumor-induced angiogenesis in mice. Both compounds inhibited capillary tube-like formation, but only compound 1 restrained cell migration. Compound 1, unlike compound 2, was able to reduce VEGF expression. Only compound 1 not only reduced the incidence and severity of corneal neovascularization, when administered at the onset of HSK, but it also restrained the development of neovascular response induced by tumor cells in mice skin. Our results show that compound 1 inhibits angiogenesis in vitro and in vivo. Therefore, compound 1 would be a promising drug in the treatment of those diseases where angiogenesis represents one of the main pathogenic events.

Nonneuronal Cholinergic System in Breast Tumors and Dendritic Cells: Does It Improve or Worsen the Response to Tumor?

Besides being the main neurotransmitter in the parasympathetic nervous system, acetylcholine (ACh) can act as a signaling molecule in nonneuronal tissues. For this reason, ACh and the enzymes that synthesize and degrade it (choline acetyltransferase and acetylcholinesterase) as well as muscarinic (mAChRs) and nicotinic receptors conform the non-neuronal cholinergic system (nNCS). It has been reported that nNCS regulates basal cellular functions including survival, proliferation, adhesion, and migration. Moreover, nNCS is broadly expressed in tumors and in different components of the immune system. In this review, we summarize the role of nNCS in tumors and in different immune cell types focusing on the expression and function of mAChRs in breast tumors and dendritic cells (DCs) and discussing the role of DCs in breast cancer.