RhoB modifies estrogen responses in breast cancer cells by influencing expression of the estrogen receptor.

INTRODUCTION
RhoB has been reported to exert positive and negative effects on cancer pathophysiology but an understanding of its role in breast cancer remains incomplete. Analysis of data from the Oncomine database showed a positive correlation between RhoB expression and positivity for both estrogen receptor alpha (ERα) and progesterone receptor (PR).


METHODS
This finding was validated by our analysis of a tissue microarray constructed from a cohort of 113 patients and then investigated in human cell models.


RESULTS
We found that RhoB expression in tissue was strongly correlated with ERα and PR expression and inversely correlated with tumor grade, tumor size and count of mitosis. In human breast cancer cell lines, RhoB attenuation was associated with reduced expression of both ERα and PR, whereas elevation of RhoB was found to be associated with ERα overexpression. Mechanistic investigations suggested that RhoB modulates ERα expression, controlling both its protein and mRNA levels, and that RhoB modulates PR expression by accentuating the recruitment of ERα and other major co-regulators to the promoter of PR gene. A major consequence of RhoB modulation was that RhoB differentially regulated the proliferation of breast cancer cell lines. Interestingly, we documented crosstalk between RhoB and ERα, with estrogen treatment leading to RhoB activation.


CONCLUSION
Taken together, our findings offer evidence that in human breast cancer RhoB acts as a positive function to promote expression of ERα and PR in a manner correlated with cell proliferation.


Introduction
Hormone therapy is recommended in breast cancers that express estrogen receptor alpha (ERα) and/or progesterone receptor (PR). This therapy is largely effective but there are nevertheless many cases of systemic resistance. A number of studies have addressed the question of the mechanisms of resistance to hormone therapy [1,2]. ERα transcriptional effects are not only determined by ligands as estradiol (E 2 ), but also by crosstalk between ERα and growth factor signaling [3]. The hierarchy among these associations is not known and various growth factor receptors are likely to be required [3].
Prenylated proteins such as Rho GTPases are key elements in growth factor signal transduction pathways [4]. A variety of growth factors present in the tumor microenvironment activate Rho proteins [5], especially RhoB [6][7][8]. As a Rho protein, RhoB cycles between GTP and GDP bound states, forming interactions with a variety of effectors that modulate activity and influence important processes in cancer [9]. RhoB, in contrast to its relatives RhoA and RhoC, has been shown to function as a tumor suppressor gene on the basis of investigations of genetically RhoB-deficient strains [10] and in human cancer cells [11][12][13]. RhoB is an immediate early response gene that is induced by a variety of stimuli, including growth 1 factors [6,[14][15][16][17][18][19][20][21]. Although no mutation of Rho GTPases have been detected in human tumors, a correlation has been demonstrated between Rho protein overexpression and poor clinical outcome in breast cancers [22]. RhoB overexpression has been correlated to disease progression [23], although this is a controversial issue [24], and overexpression of guanine exchange factors (GEF) for Rho GTPases have been correlated to prognosis in breast cancers [25]. Indeed, breast tumor progression is accompanied by a decrease in expression of the pro-oncogenic RhoGEF Tiam1 [26]. Moreover, expression of Rho-GDI α, a negative regulator of Rho proteins, is reported to correlate with the outcome of patients with breast cancer treated by adjuvant chemotherapy [27]. From a molecular point of view, scaffold proteins involved in Rho functions, such as Rho-GDI or Dblx, have been observed as part of ERα-containing complexes [28,29] with direct interaction between ERα and Rho-GDI [30].
Besides the suggested specific involvement of RhoB in ER signaling, there have been no detailed investigations in breast cancer cells, including the assessment of any correlation with the expression of hormone receptors in tumors. A major goal of the present study was therefore to determine the involvement of RhoB in hormonedependent breast cancers and to investigate hypothesized crosstalks between RhoB and ERα signaling.

Immunohistochemical analysis of tissue microarrays
A tissue microarray was constructed from the 113 patients described in Table S1 in Additional file 1. As detailed in Additional file 2, the clinical trial was conducted about 30 years ago with no consent required at that time. Cores (600 μm diameter) of histologically confirmed invasive breast carcinomas were extracted from the original paraffin blocks and re-embedded into a gridded recipient paraffin block using a tissue arrayer (Alphelys; Beecher Inc., Plaisir, France). For each case, three tumor cores and one normal breast core were taken from the original block.
Tissue microarray immunostaining was performed on a Techmate Horizon™ slide processor (Dako, Trappes, France { }). Sections were incubated with antibodies to ERα (NCL-L-ER-6F11; Novocastra, Nanterre, France), PR (PgR636 clone; Dako) and RhoB (sc-180; Santa Cruz Biotechnology, Heidelberg, Germany). The ERα and PR status was classified by immunohistochemistry expression as positive (≥ 10% immunoreactive cells) or negative (< 10% immunoreactive cells), according to the standards applied in France, as recommended by the Groupe d'Evaluation des Facteurs Pronostiques par Immunohistochimie dans les Cancers du Sein. RhoB immunostaining was analyzed by evaluation of the percentage of tumor-stained cells and staining intensity, allowing assessment of an ImmunoReactive Score: Correlation of RhoB expression with clinical variables was assessed using Mann-Whitney and Spearman's rank tests. Univariate survival analysis was performed for disease-free survival by applying the log-rank test to RhoB expression levels stratified by median value. The Kaplan-Meier method was used to link the disease-free survival according to RhoB expression in the tumors.
Where indicated, cells were deprived of E 2 by growing them in phenol red-free medium containing 5% DCCtreated FBS. Cells were treated with 50 nM E 2 or 50 ng/ ml epidermal growth factor or 2 mM ICI-182, 780 (Sigma-Aldrich Chimie, Lyon, France).

Adenoviral constructs expressing RhoB and transduction protocol
Replication-defective (ΔE1, E3) adenoviral vectors expressing RhoB under the transcriptional control of the CMV promoter were constructed with the Adeasy System (Qbiogen, Illkirch, France) as described previously [31]. Cells were transduced with adenoviral vectors (control empty vector or expressing RhoB) at a multiplicity of infection of 300:1.

Analysis of mouse embryonic fibroblasts generated from RhoB-deficient mice
Heterozygous (+/-) and homozygous (-/-) mutant strains of RhoB-deficient mice [10] were kindly provided by G Prendergast (Lankenau Institute for Medical Research, Wynnewood, PA, USA). Claudius Regaud Institute animal ethics committee approval was obtained (# ICR-2012-001-A) for the use of the animal model and the study protocols. Mice were housed in polycarbonate cages in controlled conditions. MEFs were generated [10] before lysis [18]. Protein were extracted and analyzed as described above with antibody against murine ERα (sc-542) or RhoB (sc-180; Santa Cruz Biotechnology).

GST pull-down assay
The level of activated RhoB and GTP-bound RhoB protein was measured using the GST fusion protein containing the Rho binding domain of Rhotekin [8]. The amount of GTPbound RhoB and the total amount of RhoB in cell lysates were determined by western blot as described above [32].

Immunocytochemistry
For each condition, 5 × 10 5 cells were seeded onto glass slides dishes and were grown for 3 days in phenol red-free DMEM, containing 5% DCC-treated FBS. Cells were then treated with E 2 during 16 hours, washed in PBS and fixed in RCL2 [33] for 2 minutes. Staining was performed by a Techmate Horizon™ slide processor as described for Figure 1. The primary antibody used was a monoclonal anti-ERα antibody (HC-20, sc-543, dilution 1:50; Santa Cruz Biotechnology) or an anti-PR antibody (PgR636 clone, dilution 1:50; Dako).
The level of ERα and PR staining was determined by evaluation of the percentage of tumor-stained cells and staining intensity, allowing assessment of an IRS.

Luciferase assay
Development of stable transfectants of MCF-7 cells (MELN cells) has been described previously [34]. These cells, kindly provided by P Balaguer (INSERM 540, France), were established by transfecting MCF-7 cells with ERE-β-globin-luc-SV-Neo plasmid and thus express luciferase in an estrogen-dependent manner.
Three days after siRNA transfection, MELN cells were seeded in DMEM-DCC-treated FBS during 3 days. They were then treated with E 2 or ethanol for 16 hours. Cells were then lysed in reporter lysis buffer (Promega, Charbonnières Les Bains, France). The luciferase activity was measured with luciferase assay reagent (Promega) according to the manufacturer's instructions. Protein concentration was measured to normalize the luciferase activity data.

Cell growth determination
Forty-eight hours after siRNA transfection or transduction, cells were seeded in DMEM 5% FBS with ethanol or E 2 and counted daily during the next 4 days with a coulter counter (Beckman Coulter, Grenoble, France).

RhoB expression correlates positively with receptor status and negatively with grade in human breast tumors
Using the Oncomine microarray database, a correlation between RhoB and either ERα or PR expression was documented in datasets from 19 breast cancer studies. Moreover, four studies showed an inverse correlation between RhoB expression and tumor grade. To confirm the clinical relevance of RhoB expression levels in breast cancer oncogenesis and outcome, we compared the expression levels of RhoB in breast carcinomas from a cohort of 113 patients treated or not by tamoxifen in an adjuvant setting in a randomized prospective study. Patient characteristics are described in Table S1 in Additional file 1. ERα and PR assessments were initially performed with biochemistry techniques at the time of diagnosis (between 1980 and 1983). They have all been performed again at the time of analysis, in parallel with RhoB assessment. After new pathological analysis, 65 tumors were classified as grade I and II, 74 were ERαpositive, and 59 were PR-positive. Among these tumors, 23 presented a lymphovascular invasion and 39 cases presented with positive lymph nodes. After randomization, we determined that age, ERα and PR status, histological grade, and type and lymphovascular invasion status were similar in the two groups of patients, whether treated or not. Nonetheless, patients treated with tamoxifen (n = 62) had less favorable prognostic factors regarding positive lymph nodes, pathological tumor size and number of mitoses. Figure 1A shows the RhoB immunohistochemistry stainings of two representative tumors of patients displaying opposite hormone receptor status (ERα/PR-negative and ERα/PR-positive), illustrating the direct correlation between RhoB and both ERα and PR expression. Enlargement of the photographs ( Figure 1B) illustrates in the tumor cells of the ERα/PR-negative patient that RhoB is present and specifically cytoplasmic, although the staining is weak. For the ERα/PR-positive patient, in addition to cytoplasmic staining, a strong staining was also observed at nucleus level.
Statistical analysis of the results indicated that RhoB expression in tumors is strongly correlated with the percentage of ERα (Spearman's ρ = 0.3659, P = 0.001) and PR (ρ = 0.2544, P = 0.0076) expression, but inversely correlated with histological tumor size (ρ = -0.2344, P = 0.0166) and number of mitoses (ρ = -0.2009, P = 0.0362). We divided the patients with ERα-positive tumors into two groups with either low or high RhoB expression, and in each group we further divided the patients into groups that did or did not receive tamoxifen. This analysis argued that the level of RhoB expression was not correlated with disease-free survival of patients with ERα-positive tumors, regardless of tamoxifen treatment or not (Kaplan-Meier curve shown in Figure S1 in Additional file 3).
The RhoB IRS (see Materials and methods) integrating both the percentage and intensity of the staining ( Figure 1C) was significantly higher in ERα-positive tumors (median 10.5 (3 to 12)) as compared with ERα-negative tumors (median 8 (1 to 12)), in PR-positive tumors (median 9 (3 to 12)) as compared with PR-negative tumors (median 8 (1 to 12)), and in patients with tumor grade I and II (median 9 (3 to 12)) as compared with grade III (median 8 (1 to 12)). The line in the center of each box represents the median value of the distribution, and the upper and lower ends of the box are the upper and lower quartiles, respectively. The RhoB level of expression was also higher in the smaller tumor size (≤ 2 cm, median 12 (2 to 12)) as compared with larger tumors (> 2 cm, median 8 (1 to 12)). The RhoB IRS score was not correlated with the presence of lymphovascular invasion (P = 0.26), nor with the presence of lymph node invasion (P = 0.74).

RhoB regulates ERa expression in MCF-7, in vivo and in other breast cancer cell lines
We confirmed the expectation of an effect of RhoB on ERα expression in MCF-7 cells in the presence of E 2 , using two different siRNA sequences to target RhoB mRNA (siB1 and siB2) (Figure 2A, left panel). We observed associated decreases in ERα expression of 60% and 62% (siB1 and siB2, respectively). This result was confirmed using two other independent siRNA sequences targeting RhoB (data not shown).
To rule out the possibility of off-target effects of the siRNA approach, we transduced MCF-7 cells with an adenoviral vector expressing RhoB (Figure 2A, right panel). The observed 4.5-fold overexpression of RhoB increased the expression of ERα (186%), further supporting the hypothesized regulatory relationship.
We extended the study of RhoB downregulation on ERα expression to additional cell lines ( Figure S2 in Additional file 4), confirming a decrease of ERα expression with RhoB depletion using siB1 and siB2 in T47D and ZR75 cells (hormone-dependent cells) or in LCC2 cells (hormone-resistant cells). We confirmed the involvement of RhoB on ERα expression in vivo using mice that are genetically deficient in RhoB ( Figure 2B). A major decrease of ERα expression was visualized in MEFs collected from RhoB +/mice with an even more dramatic decrease in MEFs collected from RhoB -/mice. These results extended the support for a hypothesized regulatory relationship between RhoB and ERα.
We also studied the levels of ERα phosphorylated forms whose involvement as prognostic markers is discussed below ( Figure 2C). We observed that the decrease of ERα expression observed when RhoB expression is downregulated is not associated with any significant specific change of P-Ser 118 or P-Ser 167 ERα level. Indeed, the ratio phosphorylated/total ERα is not modified after RhoB inhibition.
We then demonstrated that ERα expression is dramatically decreased in the presence of the pure anti-estrogen ICI-182, 780, even when RhoB is overexpressed (Figure 2D).

Estrogen induces RhoB activation
In examining the effect of estrogen on RhoB expression and activity and given that the activation of RhoB is important for its physiological effect [8,18,20], we investigated the role of estrogen stimulation on the RhoB guanine nucleotide binding status ( Figure 3A) to analyze rapid effects of E 2 stimulation on RhoB activation as well as expression. We observed that RhoB is quickly activated at 30 minutes without any change of total RhoB expression, as described with epidermal growth factor stimulation [8,20]. At 1 hour and 2 hours of treatment, E 2 rapidly increased the total RhoB expression with a second peak of GTP-bound RhoB occurring at 2 hours associated with a parallel increase of total RhoB expression.
Together these results suggested that RhoB and estrogen signaling are integrated into a feed-forward loop that may positively modify the biological effects of estrogen treatment.

RhoB regulates ERa, but not ERb, expression in the absence and presence of estradiol in MCF-7 cells
We analyzed further the effect of RhoB downregulation on ERα and ERβ expression in the absence and presence of E 2 ( Figure 3B). As expected, E 2 treatment induced a major significant decrease of ERα expression and concomitantly of RhoB expression. In the presence of siB2, a 36 to 41% decrease of ERα expression was observed in the absence of E 2 and 48 to 52% in the presence of E 2 . In contrast, ERβ expression was not clearly altered by RhoB downregulation ( Figure 3B). Using immunocytochemistry, we confirmed these results suggesting that RhoB regulates ERα expression and observed no dramatic changes of RhoB subcellular localization within MCF-7 cells ( Figure 3C). ERα was highly concentrated in the nucleus of the untreated control with a significant staining of the related cytoplasm. In the presence of E 2 , the staining intensities of both nuclei and cytoplasms were clearly decreased with no more detectable staining in the cytoplasm, confirmed by IRS assessment. For siB2-treated cells, a similar major decrease of the labeling intensity was observed in the cytoplasm and the nucleus, both in the presence and the absence of E 2 . In the presence of E 2 , the IRS score was maintained at a value of 2 because, in spite of the difference from 30 to 20% of stained cells induced by siB2 treatment, the intensity is kept very low in both cases (value of 1).
We then investigated whether RhoB downregulation modulates ERα mRNA expression ( Figure 3D). We first confirmed the expected decrease of ERα mRNA expression in control cells treated by E 2 alone (to 0.49). In the cells treated with siB2, a dramatic decrease of ERα mRNA was observed in the absence of E 2 (from 1 to 0.53) and much lighter in the presence of E 2 (from 0.49 to 0.37).

RhoB promotes ERa transcriptional activation function induced by estrogen
We hypothesized that the ability of RhoB to modulate ERα expression could control the major transcriptional functions of ERα. To examine this hypothesis, we used MELN cells that express luciferase in an estrogen-dependent manner. After siB1 or siB2 transfection and E 2 deprivation, cells were treated or not with E 2 and luciferase activity was quantified ( Figure 4A). In the absence of E 2 (upper-right corner), the transfection of cells by both siB1 and siB2 induced a significant decrease of the luciferase activity (0.5-fold for B1 and 0.7-fold for B2). In the control cells, E 2 treatment led to a ninefold induction of the luciferase activity. In the presence of E 2 , luciferase expression was significantly decreased for cells treated by the siB1 and siB2 sequences (respectively 3.12 and 4.07 compared with 9 AU). Nonetheless, E 2 induction was still observed for all cells transfected by either siB1 or siB2. We extended the analysis of RhoB inhibition on two known estrogenregulated genes, ERa itself described above ( Figure 3D) and PR, using quantitative RT-PCR ( Figure 4B).
Regarding PR, a major target gene of ERα, PR mRNA expression was induced in the presence of E 2 (7.7-fold) in the siRNA control cells ( Figure 4B). In cells treated with siB1, there was a significant decrease of PR mRNA levels as compared with their respective control, both without and with E 2 (rates 0.5 and 0.6, respectively). Consequently, E 2 induction was maintained for cells transfected by both siRNA control and siB1 (respectively 7.7-fold and ninefold).
Using immunocytochemistry, we confirmed that RhoB regulates ERα ( Figure 3C) plus PR ( Figure 4C) expression, with no dramatic changes of their subcellular localization within MCF-7 cells. PR is mainly detected in the nuclei of the untreated control. Besides, the presence of siB2 again significantly decreases IRS scores both in the absence and presence of E 2 .
Cells treated with siB2 exhibit low levels of ERα protein and mRNA in both the absence and presence of E 2 ( Figure 3B, C, D). Nonetheless, the effects of RhoB on PR expression may be supported in part by other mechanisms. We used chromatin immunoprecipitation analysis to study the effect of RhoB inhibition on the recruitment of ERα, ER transcriptional co-factors and RNA polymerase II onto PR gene promoter 1 hour after E 2 stimulation ( Figure 4D). Following siB1 transfection, the recruitment of RNA polymerase II to the PR gene promoter dramatically decreased (rate 0.3 compared with siControl). Recruitment of acetylated H3 and H4 histones were also clearly decreased by RhoB downregulation (rates 0.36 and 0.33, respectively). Moreover, RhoB downregulation induced a significant decrease in the recruitment of ER to the PR gene promoter (rate 0.52) paralleled by an increased in recruitment of the ERα corepressor HDAC1 (rate 1.67). Together, these results indicate that RhoB may support to some extent ERα transcriptional activation function by interfering with its cofactor recruitment, besides the direct effect on ERα expression itself.

RhoB induces proliferation in ER-positive but not in ER-negative breast cancer cell lines
The effect of RhoB on cell proliferation was evaluated in three cell lines exhibiting variable levels of expression of ERα, including MCF-7 (ERα/PR-positive), SK-BR-3 (ERα/ PR-negative and p185 erbB2 overexpressed) and MDA-MB-231 (ERα/PR-negative and p185 erbB2 -negative). As shown in Figure 5A, B, RhoB positively regulated the proliferation of MCF-7 cells both in the absence or presence of E 2 . siRNA-mediated inhibition of RhoB expression produced a 30 to 35% decrease in MCF-7 cell proliferation as soon as 1 day after transfection, with a 40 to 46% decrease by day 4 ( Figure 5A). Conversely, a significant increase in cell proliferation was observed in MCF-7 cells transduced with an adenoviral vector expressing RhoB ( Figure 5B), with an increase of 15 to 28% in relative cell proliferation at day 1 that reached 22 to 49% by day 4. In contrast to these observations, under similar conditions for infection of SK-BR-3 or MDA-MB-231 cells, the adenoviral RhoB vector either slightly decreased or had no significant biological effect on cell proliferation ( Figure 5C, D). The effect of RhoB downregulation was also analyzed in LCC2 cells, an E 2 -independent, tamoxifen-resistant subline of the MCF-7 cells. As for the MCF-7 cells, a significant decrease of proliferation was observed at day 4, in parallel to ERα and RhoB downregulation (see Figures S2 and S3 in Additional files 4 and 5).
In conclusion, we documented that RhoB had stimulatory effects on proliferation via ERα signaling that paralleled its effects on hormone receptor expression.

Discussion
Although the tumor suppressor function of RhoB has been documented in many human cancers [11,12], RhoB overexpression was suggested to be associated with tumor progression in breast cancers. Using cellular and human breast tumor analytical approaches, we have shown a positive crosstalk between RhoB and ERα expression and the critical role of RhoB in regulation of the proliferation of ERα-expressing breast cancer cells.
Our data bring together three lines of evidence to support the crosstalk between ERα and RhoB. First, in human breast cancer tissues we clearly showed a strong correlation between RhoB expression and the expression of ERα and PR. Moreover RhoB expression was associated with a Representative, respectively, of four and three independent experiments. (D) MCF-7 cells were treated for 1 hour with E 2 and lysed for chromatin immunoprecipitation (ChIp) experiments using indicated antibodies. Specificity of the immunoprecipitation was controlled using nonspecific rabbit IgG. Recruitment of these proteins on the PR promoter was quantified. The fold induction for the recruitment of siB1 versus siC conditions was calculated. Error bars represent the mean values ± SD from three independent experiments. Differences were considered statistically significant at *P < 0.01 and **P < 0.001, Student's t test.
low tumor grade and size, suggesting that RhoB expression is correlated with good prognosis markers. The second strand of evidence came from cellular results showing that the level of RhoB controls the expression of ERα in ERαpositive breast cancer cell in the presence or absence of E 2 . The positive modulation of ERα expression by RhoB was evidenced both at the protein and mRNA levels. We showed that the ERα mRNA level is controlled by RhoB, suggesting that transcriptional regulation could play a key role in this regulation. The phosphorylation of Ser 167 of ERα in tumors has been related to longer overall survival whereas the phosphorylation of Ser 118 could be a good According to the Kruskal-Wallis test, differences were considered statistically significant at *P < 0.01 and **P < 0.001 by comparing siB1 or adenoviral vector expressing RhoB (AdB) conditions with the related control condition (either in the presence or absence of E 2 ). The significance threshold was determined at 0.0125 using Bonferroni correction. AdC, adenoviral control empty vector.
prognostic marker [38,39]. We found a dramatically decreased level of ERα phosphorylation at both serine sites that can be attributed to the major decrease in total ERα expression (RhoB downregulation induced no significant change in the phosphorylated/total ERα ratio). Third, we clearly demonstrated in vivo the RhoB control of ERα expression in MEF cells derived from a RhoB -/mouse model. This regulation is of interest in the context of the epithelial-stromal interactions, particularly given that breast adipose fibroblasts determine the expression of aromatase [40]. Further, we demonstrated that ERα controls the activation and expression of RhoB. Notably, E 2 treatment induces an increase of active GTP-RhoB within 30 minutes, without changing RhoB expression at that time.
ERα upregulates or downregulates the transcription of hundreds of genes [41] and PR is a well-known ERα target gene with a major physiological role in cell proliferation. Both PR mRNA and protein expression were significantly decreased as a consequence of RhoB and related ERα downregulations. Further findings evidenced a clear decrease of the recruitment of RNA polymerase II, acetylated H3/H4 histones and ERα onto the promoter of the PR gene. In parallel, we described the recruitment of the major transcriptional co-repressor HDAC1, which is known to repress RhoB expression [42]. These results provide the demonstration of a regulatory role for RhoB in ERα expression and in the balance of the associated coregulators of ERα to control transcription of its target genes. The possibility of a direct interaction of RhoB with the ERα-dependent transcriptional machinery should not be excluded since a direct interaction between ERα and RhoGDIα in breast cancer cells has been demonstrated [30] and we have shown here a strong nuclear localization of RhoB in ERα-positive tumor tissues.
In contrast to other cancer models, RhoB is critical for the proliferation of ERα-expressing breast cancer cells, suggesting its role as a positive regulator in this model. It is noteworthy that the RhoB effect is also observed on the proliferation of the ERα-positive, tamoxifen-resistant LCC2 model cell line. Inversely, in ERα-negative cell lines (SK-BR-3 and MDA-MB-231), RhoB has no effect on proliferation -thus reinforcing the idea that RhoB promotes cell proliferation through ERα expression. These results suggest that RhoB downregulation in breast cancer cells could be associated with tumor progression in parallel to ERα extinction, with a chronology that remains to be elucidated.

Conclusion
We have demonstrated that RhoB GTPase is a key inducer of ERα and a key regulator of PR expression. RhoB acts through various complex mechanisms underlying a feed-forward loop that may control estrogen effects, including cell proliferation. Our new findings shed light on the role of RhoB in tumorigenesis involving a dual effect conferred by cellular context with a potential prooncogenic function in hormone-dependent breast cancer cells.