RhoB modifies estrogen responses in breast cancer cells by influencing expression of the estrogen receptor
© Médale-Giamarchi et al.; licensee BioMed Central Ltd. 2013
Received: 6 August 2012
Accepted: 10 January 2013
Published: 22 January 2013
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).
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.
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.
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.
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 (E2), but also by crosstalk between ERα and growth factor signaling . The hierarchy among these associations is not known and various growth factor receptors are likely to be required .
Prenylated proteins such as Rho GTPases are key elements in growth factor signal transduction pathways . A variety of growth factors present in the tumor microenvironment activate Rho proteins , especially RhoB [6–8]. 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 . 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  and in human cancer cells [11–13]. RhoB is an immediate early response gene that is induced by a variety of stimuli, including growth factors [6, 14–21]. 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 . RhoB overexpression has been correlated to disease progression , although this is a controversial issue , and overexpression of guanine exchange factors (GEF) for Rho GTPases have been correlated to prognosis in breast cancers . Indeed, breast tumor progression is accompanied by a decrease in expression of the pro-oncogenic RhoGEF Tiam1 . 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 . 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 .
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 hormone-dependent breast cancers and to investigate hypothesized crosstalks between RhoB and ERα signaling.
Materials and methods
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.
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.
Cell culture and reagents
The human breast adenocarcinoma cell lines MCF-7, ZR75, T47D, SK-BR-3 and MDA-MB-231 were obtained from the ATCC (Molsheim, France). Cells were grown routinely in DMEM (Lonza Levallois-Perret, France), supplemented with 5% fetal bovine serum (FBS; Pan-Biotech, Aidenbach, Germany). Hormonoresistant LCC2 cells (R Clarke, Karmanos Cancer Center, provided the parent MCF-7) were grown in F-12 (Lonza) phenol red-free medium, containing 5% dextran-coated charcoal (DCC)-treated FBS. Cryopreservation of cell cultures ranged from passage 1 to 10. Cells were used for experiments during up to 20 passages. Mouse embryonic fibroblasts (MEFs) were grown in DMEM with 10% FBS.
Where indicated, cells were deprived of E2 by growing them in phenol red-free medium containing 5% DCC-treated FBS. Cells were treated with 50 nM E2 or 50 ng/ml epidermal growth factor or 2 mM ICI-182, 780 (Sigma-Aldrich Chimie, Lyon, France).
Transient transfection of 40 nM siRNA was performed using Oligofectamine® (Life Technologies - Invitrogen, Saint Aubin, France) according to the manufacturer's instructions. The siRNA sequences of the oligonucleotide duplexes (Eurogentec, Angers, France) were siB1 (5'-CCGUCUUCGAGAACUAUGU-dTdT-3') and siB2 (5'-UGAUAUCCCUUGUCUGUAA-dTdT-3'), siER (5'-GGGAGAAUGUUGAAACACA-dTdT-3') and the nonspecific sequence siControl (5'-GACGUGGGACUGAAGGGGU-dTdT-3').
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 . Cells were transduced with adenoviral vectors (control empty vector or expressing RhoB) at a multiplicity of infection of 300:1.
Western blot analysis of human cell lines
Cells were lysed and protein analyzed by SDS-PAGE . Antibodies were used against RhoB (sc-180), ERα (sc-543) and, ERβ (sc-53494) from Santa Cruz Biotechnology, phospho-ERα (Ser118, 2511 and Ser167, 2514) from Cell Signaling (Saint Quentin Yvelines, France), β-actin (MAB1501) from Chemicon (Merck Millipore; Darmstadt, Germany), and secondary antibodies were anti-mouse (MP21120) and anti-rabbit (MP23145) horseradish peroxidase from Interchim (Montluçon, France) using a chemiluminescence detection kit (ECL; Pierce, Thermo Fisher Scientific, Courtaboeuf, France). Protein abundance was quantified by Image Quant TL analysis (GE Healthcare, Velizy-Villacoublay, France).
Analysis of mouse embryonic fibroblasts generated from RhoB-deficient mice
Heterozygous (+/-) and homozygous (-/-) mutant strains of RhoB-deficient mice  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  before lysis . 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 . The amount of GTP-bound RhoB and the total amount of RhoB in cell lysates were determined by western blot as described above .
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.
Quantitative reverse-transcribed PCR
Total RNA was isolated 16 hours after stimulation by E2, extracted using the RNeasy kit following the manufacturer's instructions (Qiagen, Courtaboeuf, France) and reverse-transcribed using the iScript™ cDNA Synthesis Kit (Biorad, Marnes la Coquette, France). Quantitative PCR was performed with an iQreal-time PCR detection system (Biorad). The specific human primer pairs used were: for GAPDH, 5'-TGCACCACCAACTGCTTAGC-3' and 5'-GGCATGGACTGTGGTCATGAG-3'; for 28s, 5'-TCGCTGGGTCTTGGATGTC-3' and 5'-AGCAGATTGTGACAGACCATTCC-3'; for PR, 5'-CGCGCTCTACCCTGCACTC-3' and 5'-TGAATCCGGCCTCAGGTAGTT-3'; and for ERα, 5'-CCACCAACCAGTGCACCATT-3' and 5'-GGT CTTTTCGTATCCCACCTTTC-3' (Eurogentec).
Development of stable transfectants of MCF-7 cells (MELN cells) has been described previously . 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 E2 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.
Chromatin immunoprecipitation assay
Chromatin immunoprecipitation assays were performed with modifications of the procedure of Metivier and colleagues  as described elsewhere . Briefly, 106 cells were synchronized by 3 days of culture in DMEM 5% DCC-treated FCS and treated during 1 hour with E2. Immunoprecipitation antibodies were ERα (sc-543), HDAC1 (sc-6299), and polII (sc-899) from Santa Cruz Biotechnology, and acetylated histone H3 (ab1791) and H4 (ab193) from Abcam (Paris, France). Quantitative PCR were performed on an iCycler (Biorad) using the primers 5'-GGCGACACAGCAGTGGGGAT-3' and 5'-TCTCCTCCCTCTGCCCCTATATTC-3' (Eurogentec) to amplify the fragment of the human PR promoter flanking the +745 AP-1 site .
Cell growth determination
Forty-eight hours after siRNA transfection or transduction, cells were seeded in DMEM 5% FBS with ethanol or E2 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 ERα-positive, 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 ERα expression in MCF-7, in vivo and in other breast cancer cell lines
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-Ser118 or P-Ser167 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
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 ERα, but not ERβ, 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 E2 (Figure 3B). As expected, E2 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 E2 and 48 to 52% in the presence of E2. 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 E2, 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 E2. In the presence of E2, 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 E2 alone (to 0.49). In the cells treated with siB2, a dramatic decrease of ERα mRNA was observed in the absence of E2 (from 1 to 0.53) and much lighter in the presence of E2 (from 0.49 to 0.37).
RhoB promotes ERα transcriptional activation function induced by estrogen
Regarding PR, a major target gene of ERα, PR mRNA expression was induced in the presence of E2 (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 E2 (rates 0.5 and 0.6, respectively). Consequently, E2 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 E2.
Cells treated with siB2 exhibit low levels of ERα protein and mRNA in both the absence and presence of E2 (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 E2 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 co-repressor 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
In conclusion, we documented that RhoB had stimulatory effects on proliferation via ERα signaling that paralleled its effects on hormone receptor expression.
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 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 ERα-positive breast cancer cell in the presence or absence of E2. 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 Ser167 of ERα in tumors has been related to longer overall survival whereas the phosphorylation of Ser118 could be a good 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 . Further, we demonstrated that ERα controls the activation and expression of RhoB. Notably, E2 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  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 . These results provide the demonstration of a regulatory role for RhoB in ERα expression and in the balance of the associated co-regulators 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  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.
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 pro-oncogenic function in hormone-dependent breast cancer cells.
Dulbecco's modified Eagle's medium
fetal bovine serum
mouse embryonic fibroblast
polymerase chain reaction
small interfering RNA.
This work was supported by the National Institute of Cancer, Canceropole Grand Sud-Ouest, Fondation RITC (C Silvagni), INSERM, Claudius Regaud Institute, University of Toulouse (UPS) and the French Ministry of Research (MRES). Thanks are due to GC Prendergast for fruitful discussion and reviewing the manuscript.
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