Chiara Baldacci

Extra-nuclear signalling of estrogen receptor to breast cancer cytoskeletal remodelling, migration and invasion.

Background Estrogen is an established enhancer of breast cancer development, but less is known on its effect on local progression or metastasis. We studied the effect of estrogen receptor recruitment on actin cytoskeleton remodeling and breast cancer cell movement and invasion. Moreover, we characterized the signaling steps through which these actions are enacted. Methodology/Principal Findings In estrogen receptor (ER) positive T47-D breast cancer cells ER activation with 17β-estradiol induces rapid and dynamic actin cytoskeleton remodeling with the formation of specialized cell membrane structures like ruffles and pseudopodia. These effects depend on the rapid recruitment of the actin-binding protein moesin. Moesin activation by estradiol depends on the interaction of ERα with the G protein Gα13, which results in the recruitment of the small GTPase RhoA and in the subsequent activation of its downstream effector Rho-associated kinase-2 (ROCK-2). ROCK-2 is responsible for moesin phosphorylation. The Gα13/RhoA/ROCK/moesin cascade is necessary for the cytoskeletal remodeling and for the enhancement of breast cancer cell horizontal migration and invasion of three-dimensional matrices induced by estrogen. In addition, human samples of normal breast tissue, fibroadenomas and invasive ductal carcinomas show that the expression of wild-type moesin as well as of its active form is deranged in cancers, with increased protein amounts and a loss of association with the cell membrane. Conclusions/Significance These results provide an original mechanism through which estrogen can facilitate breast cancer local and distant progression, identifying the extra-nuclear Gα13/RhoA/ROCK/moesin signaling cascade as a target of ERα in breast cancer cells. This information helps to understand the effects of estrogen on breast cancer metastasis and may provide new targets for therapeutic interventions.

Estrogen Receptor-α Promotes Breast Cancer Cell Motility and Invasion via Focal Adhesion Kinase and N-WASP

The ability of cancer cells to move and invade the surrounding environment is the basis of local and distant metastasis. Cancer cell movement requires dynamic remodeling of the cytoskeleton and cell membrane and is controlled by multiprotein complexes including focal adhesion kinase (FAK) or the Neural Wiskott-Aldrich Syndrome Protein (N-WASP). We show that 17β-estradiol induces phosphorylation of FAK and its translocation toward membrane sites where focal adhesion complexes are assembled. This process is triggered via a Gα/Gβ protein-dependent, rapid extranuclear signaling of estrogen receptor α interacts in a multiprotein complex with c-Src, phosphatidylinositol 3-OH kinase, and FAK. Within this complex FAK autophosphorylation ensues, and activated FAK recruits the small GTPase cdc42, which, in turn, triggers N-WASP phosphorylation. This results in the translocation of Arp2/3 complexes at sites where membrane structures related to cell movement are formed. Recruitment of FAK and N-WASP is necessary for cell migration and invasion induced by 17β-estradiol in breast cancer cells. Our findings identify an original mechanism through which estrogen promotes breast cancer cell motility and invasion. This information helps to understand the effects of estrogen on breast cancer metastasis and may provide new targets for therapeutic interventions.

Extra-nuclear signaling of progesterone receptor to breast cancer cell movement and invasion through the actin cytoskeleton.

Progesterone plays a role in breast cancer development and progression but the effects on breast cancer cell movement or invasion have not been fully explored. In this study, we investigate the actions of natural progesterone and of the synthetic progestin medroxyprogesterone acetate (MPA) on actin cytoskeleton remodeling and on breast cancer cell movement and invasion. In particular, we characterize the nongenomic signaling cascades implicated in these actions. T47-D breast cancer cells display enhanced horizontal migration and invasion of three-dimensional matrices in the presence of both progestins. Exposure to the hormones triggers a rapid remodeling of the actin cytoskeleton and the formation of membrane ruffles required for cell movement, which are dependent on the rapid phosphorylation of the actin-regulatory protein moesin. The extra-cellular small GTPase RhoA/Rho-associated kinase (ROCK-2) cascade plays central role in progesterone- and MPA-induced moesin activation, cell migration and invasion. In the presence of progesterone, progesterone receptor A (PRA) interacts with the G protein Gα13, while MPA drives PR to interact with tyrosine kinase c-Src and to activate phosphatidylinositol-3 kinase, leading to the activation of RhoA/ROCK-2. In conclusion, our findings manifest that progesterone and MPA promote breast cancer cell movement via rapid actin cytoskeleton remodeling, which are mediated by moesin activation. These events are triggered by RhoA/ROCK-2 cascade through partially differing pathways by the two compounds. These results provide original mechanistic explanations for the effects of progestins on breast cancer progression and highlight potential targets to treat endocrine-sensitive breast cancers.

Activation of nitric oxide synthesis in human endothelial cells by red clover extracts

Objective: The unexpected findings of the Womens Health Initiative trial, where surrogate cardiovascular risk markers have failed to predict the cardiovascular performance of hormone therapy, showing no reduction of cardiovascular disease in postmenopausal women receiving hormonal preparations inducing a favorable lipid profile, raise the interest on how molecules with hormone-like activity used for the treatment of menopausal symptoms act on vascular cells. This is particularly important for estrogen-like compounds such as phytoestrogens, whose mechanisms of action may significantly differ from those of other estrogenic compounds. Design: Because endothelial-derived nitric oxide (NO) is a key regulator of vascular tone and atherogenesis as well as a well-characterized estrogen-regulated molecule, we studied the regulation of NO synthesis in cultured human endothelial cells by phytoestrogens contained in red clover extracts. Results: We show that red clover extracts activate NO synthesis in endothelial cells by recruiting transcriptional pathways but are not capable of inducing rapid NO synthesis through nongenomic mechanisms. During prolonged exposures, red clover extracts enhance the expression as well as the activity of endothelial nitric oxide synthase. These effects are mediated by a recruitment of estrogen receptor-β. Moreover, we show that red clover-derived isoflavones synergize with 17β-estradiol in increasing endothelial nitric oxide synthase activity and expression, therefore being devoid of antiestrogenic effects in human endothelial cells. Conclusions: These results help to understand the mechanisms of action of phytoestrogens on the cardiovascular system and have relevant clinical implications.

Comparative actions of progesterone, medroxyprogesterone acetate, drospirenone and nestorone on breast cancer cell migration and invasion

BackgroundLimited information is available on the effects of progestins on breast cancer progression and metastasis. Cell migration and invasion are central for these processes, and require dynamic cytoskeletal and cell membrane rearrangements for cell motility to be enacted.MethodsWe investigated the effects of progesterone (P), medroxyprogesterone acetate (MPA), drospirenone (DRSP) and nestorone (NES) alone or with 17β-estradiol (E2) on T47-D breast cancer cell migration and invasion and we linked some of these actions to the regulation of the actin-regulatory protein, moesin and to cytoskeletal remodeling.ResultsBreast cancer cell horizontal migration and invasion of three-dimensional matrices are enhanced by all the progestins, but differences are found in terms of potency, with MPA being the most effective and DRSP being the least. This is related to the differential ability of the progestins to activate the actin-binding protein moesin, leading to distinct effects on actin cytoskeleton remodeling and on the formation of cell membrane structures that mediate cell movement. E2 also induces actin remodeling through moesin activation. However, the addition of some progestins partially offsets the action of estradiol on cell migration and invasion of breast cancer cells.ConclusionThese results imply that P, MPA, DRSP and NES alone or in combination with E2 enhance the ability of breast cancer cells to move in the surrounding environment. However, these progestins show different potencies and to some extent use distinct intracellular intermediates to drive moesin activation and actin remodeling. These findings support the concept that each progestin acts differently on breast cancer cells, which may have relevant clinical implications.

Effects of phytoestrogens derived from red clover on atherogenic adhesion molecules in human endothelial cells.

Objective: In the search for safer approaches to address menopausal symptoms, the administration of plant-derived estrogens has gained popularity. Recent evidence suggests that these compounds may act neutrally or even beneficially on surrogate cardiovascular risk markers in postmenopausal women. However, little is known of the effects of phytoestrogens on vascular cells. Design: Endothelial expression of leukocyte adhesion molecules plays a critical role in the development of atherosclerosis and in plaque destabilization, and estrogen reduces the expression of these proatherogenic molecules. We studied the regulation of the expression of intercellular adhesion molecule-1 (ICAM-1) and of vascular cell adhesion molecule-1 (VCAM-1) in cultured human endothelial cells by phytoestrogens contained in red clover extracts. Moreover, we characterized the mechanistic basis for these actions. Results: Red clover extracts, particularly genistein and daidzein, inhibit the endothelial expression of ICAM-1 and VCAM-1 induced by bacterial lipopolysaccharide. The addition of red clover extracts to reproductive life or menopausal concentrations of 17&bgr;-estradiol results in an additive decrease in expression of endothelial adhesion molecules. The reduction of ICAM-1 and VCAM-1 expression in the presence of red clover extracts is paralleled by a cytoplasmic stabilization of the proinflammatory transcription factor nuclear factor-&kgr;B. Conclusions: Red clover extracts act as anti-inflammatory and antiatherogenic agents on human endothelial cells by reducing the expression of the leukocyte adhesion molecules ICAM-1 and VCAM-1. On the basis of these results, red clover extracts may induce beneficial actions on human vessels.

Activation of nitric oxide synthesis in human endothelial cells using nomegestrol acetate

OBJECTIVE: Recent clinical trials indicate that synthetic progestins may be unexpectedly relevant for the development of cardiovascular disease. The aim of this study was to establish whether nomegestrol acetate induces signaling events in human endothelial cells that differ from those of other progestins, such as natural progesterone or medroxyprogesterone acetate. METHODS: We used human endothelial cells to study the action of nomegestrol acetate (either alone or in the presence of estradiol [E2]) on the synthesis of nitric oxide (NO) and on the activity or expression of endothelial nitric oxide synthase (eNOS). We compared the effects of nomegestrol acetate with those of progesterone or medroxyprogesterone acetate. In addition, we characterized the signaling events recruited by these compounds. RESULTS: Progesterone and nomegestrol acetate increase NO synthesis by transcriptional and nontranscriptional mechanisms, whereas medroxyprogesterone acetate lacks such effects. When used together with physiological E2 concentrations, progesterone and nomegestrol acetate do not interfere with (or even enhance) E2 effects, whereas medroxyprogesterone acetate impairs E2 signaling. A marked difference in the recruitment of mitogen-activated protein kinase and phosphatidylinositol-3 kinase explains the divergent effects of the three gestagens. CONCLUSION: Our findings show significant differences in the signal transduction pathways recruited by progesterone, nomegestrol acetate, and medroxyprogesterone acetate in human endothelial cells that may have relevant clinical implications.

Laparoscopic Adnexectomy of Suspect Ovarian Masses: Surgical Technique Used To Avert Spillage

Herein is described and evaluated a safe laparoscopic adnexectomy technique for retroperitoneal dissection of suspect ovarian masses including the underlying peritoneum fixed to the ovary. Adopting this technique in cases of suspect adnexal masses enables the reduction of spilling and ensures an intact specimen. Twenty-two consecutive patients with suspect adnexal masses 10 cm or smaller underwent laparoscopic adnexectomy. Patients with bilateral suspect ovarian masses that required bilateral adnexectomy were enrolled only if they were no longer of childbearing age. Laparoscopy was feasible in all patients. No tumor spillage occurred. In 5 patients (23.6%), minilaparotomy was required to extract the specimen. Mean (SD) operating time was 80 (35-160) minutes, and estimated blood loss was 50 (10-100) mL. No major intraoperative complications occurred. Median (range) postoperative stay was 1 (1-3) day. Definitive pathologic analysis revealed benign pathologic conditions in 18 patients (81.8%), an ovarian tumor with low malignant potential in 3 patients (13.7%), and ovarian cancer in 1 patient (4.5%) in whom findings at frozen-section analysis were inconclusive. Median (range) follow-up of malignant ovarian tumors and of tumors with low malignant potential was 27 (21-29) months. No recurrence or port-site metastasis developed during follow-up. The data are encouraging for adoption of this technique to avert spillage during laparoscopic management of suspect adnexal masses, especially those firmly adherent to the peritoneum. However, the procedure must be validated in a larger series of patients to standardize the technique.

Laparoscopic approach for peripartum hysterectomy.

Abnormal placentation is the most common indication for peripartum hysterectomy. To date, the approach described in the literature is laparotomy, which is associated with high morbidity and mortality. A 30-year-old gravida 4 para 3 had a postpartum diagnosis of placenta percreta. She was first treated conservatively. On day 3 after delivery, because of persistent vaginal bleeding, she underwent a laparoscopic hysterectomy. No postoperative complications occurred, and the patient was discharged on postoperative day 3. Laparoscopic peripartum hysterectomy could become the approach of choice in selected patients with abnormal placentation to avoid complications associated with laparotomy.

Robotic Management of Major Vessel Injury During Pelvic Lymphadenectomy

Laparoscopic management of major vessel lesion is a challenging task during pelvic lymphadenectomy, and conversion is frequently necessary. Robotic surgery overcomes the limits of laparoscopy in vascular suturing. We describe a case of a 79-year-old woman with stage IB G3 endometrial adenocarcinoma, where an external iliac vein injury occurred during pelvic lymphadenectomy. This is the first case report that describes robotic management of a major vascular injury during pelvic lymphadenectomy by use of endoscopic bulldog clamps and robotic intracorporeal vascular sutures.