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

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

<p>Abstract</p> <p>Background</p> <p>Limited 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.</p> <p>Methods</p> <p>We 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.</p> <p>Results</p> <p>Breast 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.</p> <p>Conclusion</p> <p>These 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.</p

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 17beta-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 ER alpha with the G protein G alpha(13), 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 G alpha(13)/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 G alpha(13)/RhoA/ROCK/moesin signaling cascade as a target of ER alpha 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

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

Intracellular signaling mechanisms involved in PR-enhanced T47-D cell migration.

<p>(A) and (B) Cells were treated with progesterone or MPA (both 100 nM) for 48 h, in the presence or absence of ORG 31710 (ORG - 1 µM), of PD98059 (PD - 5 µM), of wortmannin (WM - 30 nM), of PTX (100 ng/mL), of Y-27632 (Y - 10 µM) or of 17β - estradiol (E2 - 1 nM). Other cells were transfected with moesin antisense phosphorotioate oligonucleotides (PON) (antisense - 2 µM) or sense PON (sense - 2 µM). Cell migration distances were measured and values are presented as % of control. * = P<0.01 vs. control; ** = P<0.05 vs. progesterone or MPA. The experiments were performed in triplicates and data representing the migration distance of cells from the starting line are expressed as mean±SD. Representative images are shown. The arrows indicate the direction of migration. The upper black lines indicate the starting line and the lower black lines indicate the mean migration distance.</p

PR activation increases T47-D cell migration and invasion.

<p>(A) Cells were treated with progesterone or MPA (both 100 nM) for 48 h and cell migration was assayed. T47-D cells were scraped out of the cell culture dish and the extent of migration of the remaining cells was assayed in the presence of Ara-C (see text). Cell migration distances were measured and values are presented as % of control. * = P<0.01 vs. control. The experiments were performed in triplicates and data representing the migration distance of cells from the starting line are expressed as mean±SD. The arrows indicate the direction of migration. The upper black lines indicate the starting line and the lower black lines indicate the mean migration distance. (B) Cells were treated with progesterone or MPA (both 100 nM) for 48 h. Cell invasion was assayed using invasion chambers. Invading cells were counted in three different central fields of triplicate membranes. The experiments were performed in triplicates. Invasion indexes and representative images are shown. * = P<0.01 vs. control.</p

Extra-nuclear signaling of PR to moesin in T47-D cells: Gα₁₃ and c-Src.

<p>(A) T47-D cells were treated for 15 minutes with P or MPA (both 100 nM), in the presence or absence of ORG 31710 (ORG - 1 µM) or of PTX (100 ng/mL). Protein extracts were immunoprecipitated with an Ab <i>vs</i>. Gα₁₃ and the IPs were assayed for co-immunoprecipitation of PRs. The cell extract (30 µg) was used as input and normal rabbit IgG was used as the control antibody. (B) T47-D cells were exposed to 100 nM P-BSA (membrane-impermeable) for 15 min, in the presence or absence of ORG 31710 (ORG - 1 µM). Moesin and phosphorylated moesin are shown. (C) and (D) T47-D cells were treated with 100 nM MPA for 15 min, with or without the Src kinase inhibitor, PP2 (10 µM) and wild-type or active Akt or moesin are shown. (E) and (F) T47-D cells were treated for 15 minutes with 100 nM MPA, in the presence or absence of ORG 31710 (ORG - 1 µM). Protein extracts were immunoprecipitated with an Ab vs. PR (E) or c-Src (F) and the IPs were assayed for co-immunoprecipitation of PR or c-Src as indicated. Cell extract (30 µg) was used as input. Normal rabbit IgG and normal mouse IgG were used as the control antibodies in (E) and (F), respectively. (G) T47-D cells were transfected with scrambled siRNA or c-Src targeted siRNAs for 48 h. c-Src protein expression was detected by western blot as indicated. (H) T47-D cells were exposed to 100 nM P or MPA for 15 min after transfection with c-Src siRNA or non-specific control siRNAs for 48 h. Total moesin or P-moesin cell amounts are shown. (I) PR-negative MDA-MB-231 cells were transiently transfected with empty pcDNA3.1+ plasmid (vector) or plasmids encoding full length of human PRA or PRB for 48 h, then cells were exposed to 100 nM P or MPA for 15 min. Protein extracts were immunoprecipitated with an Ab vs. PR, and the IPs were assayed for co-immunoprecipitation of Gα₁₃ or c-Src as indicated. Total moesin and phosphorylated moesin were also analyzed using western blot. The experiments were performed in triplicates and representative images are shown.</p

PR activation induces a rapid rearrangement of the actin cytoskeleton in T47-D cells.

<p>(A) T47-D cells were treated with P or MPA (both 100 nM) for 10, 15 or 30 minutes, in the presence or absence of the pure PR antagonist ORG 31710 (1 µM). Immunofluorescent analysis of Texas Red-phalloidin (in red) reveals the spatial modifications of actin fibres through the time-course and the formation of specialized cell membrane structures. Green, yellow and light blue arrows indicate lamellipodia, pseudopodia and ruffles, respectively. Nuclei are counterstained in blue. Rectangles indicate the area sampled in the corresponding upper graph. In the graph, the longitudinal axis displays the gray level and the horizontal axis shows the pixels. Light yellow, light red and light blue areas indicate the parts of the graph indicating the extracellular, plasma membrane and cytoplasmic areas. (B) Analytic results obtained by using Leica QWin image analysis and processing software showing the mean thickness of the cell membrane after treatment with P or MPA (both 100 nM). The results are derived from the sampling of five areas of the cell membrane of thirty different random cells. The areas of minimum and maximum cell membrane thickness were always included. The results are the mean±SD of the measurements. (C) shows the amount of filamentous actin (F-actin, F) versus free globular-actin (G-actin, G) content in T47-D cells after treatment with P or MPA (both 100 nM) for 15 min, in the presence or absence of PR antagonist ORG 31710 (1 µM). Positive (Pos) and negative (Neg) controls were set by adding F-actin enhancing solution (phalloidin, 1 µM) or F-actin depolymerization solution (10 µM cytochalasin-D) to the lysates, respectively. All the experiments were repeated three times with consistent results, and a representative result is shown. * = P<0.05 vs control.</p

Extra-nuclear signaling of PR to moesin in T47-D cells: ERK1/2 and PI3K.

<p>(A) shows wild-type (ERK1/ERK2) or phosphorylated ERK 1/2 (P-ERK1/ERK2) during exposure to progesterone (100 nM). (B) Shows wild-type (Akt) and phosphorylated Akt (P-Akt) in the presence of 100 nM P. (C) T47-D cells were transfected with scrambled siRNA or ERK1/2 targeted siRNAs for 48 h. After that level of ERK1/2 protein expression was detected by western blot as indicated. (D) Cells were exposed to 100 nM P or MPA for 15 min after transfection with 100 nM targeted siRNA for ERK1/2 or scrambled siRNA for 48 h. Cell contents of wild-type or phosphorylated moesin are shown. (E) Cells were exposed to 100 nM progesterone or MPA for 15 min after transfection with constitutively active p85α (WT p85α) or dominant-negative p85α (Δp85α) for 48 h. Cell contents of p85α, wild-type or phosphorylated moesin are shown. The experiments were performed in triplicates and representative images are shown.</p