Coordinate Regulation of Estrogen Receptor &Beta

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ORIGINAL ARTICLE Coordinate regulation of estrogen receptor b degradation by Mdm2 and CREB-binding protein in response to growth signals

M Sanchez1, N Picard1, K Sauve´ 1 and A Tremblay1,2

The biological actions of estrogen are mediated via estrogen receptors ERa and ERb. Yet, other cellular signaling events that also impact ER functions have an important role in breast carcinogenesis. Here, we show that activation of ErbB2/ErbB3 tyrosine kinase receptors with growth factor heregulin-b prompts ERb degradation by the 26S proteasome, a mechanism that requires the coactivator cAMP response element-binding (CREB)-binding protein (CBP). We found that CBP promotes ERb ubiquitination and degradation through enhancement of the PI3-K/Akt pathway by heregulin-b, an effect potentiated by a negatively charged hinge region of ERb. Activated Akt triggered the recruitment of E3 ubiquitin ligase Mdm2 to ERb, which was further stabilized by CBP, resulting in ERb poly-ubiquitination. Mutation of CBP Thr-1872 or Mdm2 Ser-186/188 Akt sites resulted in a dissociation of the ERb-CBP-Mdm2 complex and reduced ERb turnover. We found that the decrease in ERb induced by heregulin-b was associated with reduced target gene promoter occupancy and enhanced proliferation of breast cancer cells. However, knockdown of Mdm2 restored endogenous ERb levels resulting in reduction of breast cancer cell growth. These studies identify a tripartite Akt-regulated phosphorylation mechanism that functions to hamper normal ERb activity and turnover through the concerted actions of CBP and Mdm2 in response to growth factor signaling pathways in breast cancer cells.

Oncogene (2013) 32, 117--126; doi:10.1038/onc.2012.19; published online 20 February 2012 Keywords: ErbB2/Her-2/Neu; ErbB3; PI3-K/Akt pathway; 26S proteasome-ubiquitin system; CBP/p300; breast cancer

promoting constitutive activation of the AF-1 (ref. 9) or by INTRODUCTION mutating an Akt site located within the hinge region of ERb,10 Estrogen regulates many aspects of reproductive physiology, indicating that phosphorylation of ERb by the PI3-K/Akt pathway development and metabolism, but is also mitogenic in hormone- was implicated. regulated cancers, such as breast and endometrial cancers. Its effects are mediated by the actions of estrogen receptors ERa and Several coactivators have been described to enhance the ERb. Although encoded by unique genes, both isoforms recognize activity of ERs, notably CBP and p300 among others, which palindromic estrogen-responsive elements in target gene promo- facilitate transcription by mediating acetylation of transcription ter/enhancer regions, share similar estradiol-binding affinity, and factors and histones.11 The versatile role of CBP/p300 in undergo post-translational modifications in response to hormone transcription allows it to act as an integrative regulator for diverse and to various cellular kinase pathways.1--3 Although an increased factors bridging components of the basal transcriptional machin- expression of ERa strongly correlates with the development and ery allowing CBP/p300 to act as a scaffold for the formation of proliferation of breast carcinomas, therefore providing a basis for multi-component complexes.12 The array of transcription factors endocrine adjuvant therapy, the predictive value of ERb remains that can interact with CBP/p300 suggests that competition for the uncertain. limited intracellular pool of CBP/p300 must be achieved in a very Primarily recognized as ligand-activated receptors, a diversity of dynamic process and through preferred recruiting signals. potential activation signals and mechanisms have been revealed Studies on ERa have integrated the transcriptional response to to regulate ERa and ERb function, of which most implicate estrogen with the 26S proteasome-directed degradation of the receptor phosphorylation.3,4 In that respect, the outcome of ER receptor,13,14 thus supporting a mean by which target cells can phosphorylation not only differs according to the identity of the sustain or limit a hormonal response through a continuous activator and the selectivity of targeted site(s), but also differently receptor turnover. Given the potent ability of ERb to respond to impacts the transcription potential of ERs. Phosphorylation of the kinase signaling pathways, we recently reported that ERb is N-terminal activation function (AF-1) by MAPK/Erk was shown to ubiquitinated in response to activation of the Mek1/Erk pathway, induce ERa activity.5,6 Likewise, oncogenic ras or activation of an effect involving a specific AF-1 phosphorylation that promotes EGFR/ErbB1 resulted in potent ERb AF-1 phosphorylation, recruit- the recruitment of E3 ubiquitin ligase E6-AP.15 These findings ment of steroid receptor coactivator SRC-1 and cAMP response suggest that mechanisms other than estrogen binding regulate element-binding (CREB)-binding protein (CBP), and subsequent the activation--degradation process of ERb in order to integrate a receptor activation.7,8 Conversely, activation of ErbB2/ErbB3 gene-specific response to changes in kinase signaling pathways. receptors by growth factor heregulin-b repressed the response Here, we describe how ErbB2/ErbB3 activation selectively of ERb to estrogen in breast cancer cells, an effect alleviated by commits ERb towards degradation by the PI3-K/Akt pathway.

1Ste-Justine Hospital Research Center, Department of Biochemistry, University of Montreal, Montreál, Que´bec, Canada and 2Department of Obstetrics & Gynecology, University of Montreal, Montreál, Que´bec, Canada. Correspondence: Dr A Tremblay, Ste-Justine Hospital Research Center, University of Montreal, 3175 Coˆte Ste-Catherine, Montreál (Que´bec), Canada H3 T 1C5. E-mail: [email protected] Received 26 June 2011; revised 16 December 2011; accepted 21 December 2011; published online 20 February 2012 Concerted degradation of ERb by CBP and Mdm2 M Sanchez et al 118 We identify the E3 ubiquitin ligase Mdm2 and coactivator CBP as the transcriptional repression of ERb by the ErbB2/Her-2 path- being phosphorylated and recruited to ERb in a hormone- way.10 As opposed to the wild-type receptor, the levels of ERb independent manner to elicit its ubiquitination and degradation, S255A mutant remained elevated in the presence of CBP and with an increase in breast cancer cell growth. Therefore, this under constitutive p110a activation (Figure 1d) or heregulin-b mechanism supports an anti-proliferative role to ERb, and treatment (Supplementary Figure 1c), indicating that Ser-255 is illustrates the capacity of Mdm2 and CBP to integrate growth critical in the downregulation of ERb in these conditions. To signaling pathways in breast cancer cells. determine whether the decrease in ERb steady-state levels was consequent of its degradation, we performed cycloheximide chase experiments, which showed that the half life of ERb was RESULTS increased from 5 to 8 h in the presence of CBP, revealing that CBP is able to stabilize ERb (Figure 1e). However, activation of Akt Activation of the PI3-K/Akt pathway by heregulin-b directs ERb 1 degradation greatly reduced the turnover rate of ERb (t2 ¼ 2 h) under these conditions, correlating with the observed decrease in ERb steady- We previously reported that the transcriptional response of ERb was state levels. Although basal turnover rates remained mostly impaired in response to activation of ErbB2/ErbB3 receptor 1 10 unchanged between wild-type and S255A ERb (t ¼ B5 h), the heterodimer. In order to elucidate the mechanisms involved, we 2 S255A mutation protected ERb from degradation under Akt found that the steady-state levels of ERb were markedly decreased activation (Figure 1f). This is also demonstrated on fractionated following activation of ErbB2/ErbB3 receptors with heregulin-b,and cell compartments, which revealed that the nuclear content of expression of CBP in 293T cells (Figure 1a). The reduction in ERb ERb was markedly decreased by CBP and Akt activation, whereas was not observed in cells when treated with heregulin-b alone or Ser-255 mutation largely prevented such response (Supplemen- transfected with CBP, which resulted in increased ERb levels, tary Figures 2a and b). These findings demonstrate that induction indicating that both conditions were needed to achieve maximal of the Akt pathway by ErbB2/ErbB3 receptors induced a targeted ERb downregulation. The decrease in ERb was dependent upon the degradation of ERb by the 26S proteasome and identify Ser-255 as 26S proteasome as shown with the use of MG-132 inhibitor, and an important regulatory site in this process. involves the PI3-K/Akt pathway, as the use of a dominant negative p85 regulatory subunit of PI3-K abolished the effect of heregulin-b in the context of ErbB2/ErbB3 activation (Figure 1a and Supple- The E3 ubiquitin-ligase Mdm2 regulates ERb protein levels mentary Figure 1a). To ascertain the role of Akt, we used a p110a and activity active form of PI3-K to constitutively activate Akt, which also Degradation through the 26S proteasome implicates the action of decreased ERb content in a CBP-dependent manner (Figure 1b). a family of E3 ubiquitin ligases, which covalently attach small This effect does not seem to require the hormone as ERb was ubiquitin moieties at specific lysine residues of target proteins. reduced by the PI3-K/Akt pathway both in absence (Figure 1b) and Interestingly, the E3 ligase Mdm2, which has a key role in tumor presence of estrogen (Supplementary Figure 1b). In addition, this suppressor p53 degradation, is tightly controlled by Akt activity.16 mechanism appears to be selective for ERb,asnodecreasewas In line with the regulation of ERb by the Akt pathway, we sought observed for ERa (Figure 1c), consistent with their different to investigate the involvement of Mdm2 in mediating ERb transcriptional responses under these conditions.10 These results degradation. We found that ERb levels were reduced upon support a role of the Akt pathway and of CBP in eliciting ERb expression of Mdm2 in 293T cells, an effect that was dependent downregulation in response to activation of ErbB2/ErbB3 receptors. on the E3 ligase activity of Mdm2 as the inactive C462A mutant We next tested the role of Ser-255 located within the hinge was inefficient (Figure 2a). Mdm2 also greatly impaired the region of ERb, based on our findings that this site was required in transcriptional activation of ERb by CBP in absence or presence of

Figure 1. ErbB2/ErbB3 receptors signal ERb degradation by the 26S proteasome through the PI3-K/Akt pathway. (a) 293T cells were transiently transfected with mouse ERb in the presence or absence of plasmids encoding ErbB2, ErbB3, CBP and p85a dominant negative form of PI3-K. Cells were treated with 50 ng/ml heregulin-b1 or left untreated for 16 h and then harvested for western analysis using an anti-ERb antibody. Increasing concentrations of 26S proteasome inhibitor MG-132 (1 and 10 mM) were also used to treat cells. Loading was monitored with b-actin. (b) Similar as in (a) except that constitutive p110a PI3-K and Akt plasmids were used to trigger Akt activation. MG-132 was used at 1 mM.(c) Cells were transfected with an ERa expression plasmid in the same conditions as in (a), using an anti-ERa antibody. (d) Similar as in (b) except that a ERb S255A mutant plasmid was used. (e, f) Cycloheximide chase experiment in 293T cells expressing ERb (e) or S255A (f)in absence or presence of CBP and p110a/Akt plasmids. Cells were treated with 50 mM cycloheximide and lysed at the indicated time points for western analysis. Results are normalized to b-actin content and expressed as a percentage of change of time zero, which was set at 100%.

Oncogene (2013) 117 --126 & 2013 Macmillan Publishers Limited Concerted degradation of ERb by CBP and Mdm2 M Sanchez et al 119 9 *** control ** 8 Mdm2 - - + ++ - Mdm2 7 Mdm2 C462A *** - - - - ++ Mdm2 C462A shMdm2 6 5 ** * *** 4 3 2

Luciferase activity (fold) 1 0 - CBPCBP - CBP CBP Akt Akt

Figure 2. Activation of E3 ubiquitin ligase Mdm2 by Akt promotes ERb downregulation and transcriptional repression. (a) 293T cells transfected with increasing concentrations of Mdm2 exhibit a decrease in ERb levels as determined by western analysis. The E3 ligase-deﬁcient C462A mutant of Mdm2 was also tested. b-actin was monitored for control loading. (b) The transcriptional activity of ERb was determined by luciferase reporter assay using an EREtkLuc reporter. 293T cells were transfected with or without the indicated plasmids and treated with vehicle or 10 nM E2 for 16 h. PI3-K p110a and Akt plasmids were used to constitutively activate the Akt pathway. Cells were then harvested for luciferase activity measurements, and values normalized to b-galactosidase activity and expressed as fold response compared with untreated cells set at 1.0. *Po0.05, **Po0.01, ***Po0.001. (c) Western analysis of myc-tagged ERb in response to expression of wild type, S166A (A1) and S186/188A (A2) mutated HA-tagged Mdm2. The p110a/Akt plasmids were used to activate Akt. Mdm2 knockdown was performed using a shRNA. Samples were analyzed by western blot with anti-myc and anti-HA antibodies and protein loading was monitored with b-actin. (d) CBP contributes to reduce ERb levels by Mdm2. Western analysis of myc-tagged ERb in response to Mdm2 expression and activation of Akt as in (c). Knockdown of CBP was achieved by lentiviral infection using shCBP RNA. The shLuc (luciferase) was used as a negative control. estrogen (Figure 2b), demonstrating a repressive function of with ERb in the presence of CBP or in response to the activation of Mdm2 on ERb activity. In addition, the observed decrease in ERb the PI3-K/Akt pathway (Figure 3a). An even stronger recruitment activity to combined CBP and Akt activation10 was completely was achieved when both CBP and constitutive p110a were added, prevented and even reversed with the expression of Mdm2 C462A suggesting a coordinate action of CBP and Akt to stabilize the ligase dead mutant or shRNA-mediated knockdown of endogen- ERb/Mdm2 complex. ERb Ser-255 appears to be essential to ous Mdm2 (Figure 2b), suggesting a role for Mdm2 in restraining promote this maximal interaction with Mdm2 (Figure 3a and ERb transcriptional potential. Supplementary Figure 3). We then tested the contribution of Interestingly, maximal ERb downregulation was achieved when Thr-1872, a phosphorylation site located in CBP CH3 region Mdm2 and p110a/Akt were combined as opposed to either targeted by Akt and described to enhance CBP transactivation conditions alone, revealing that activation of Mdm2 was required potential.10 Disruption of Thr-1872 using a T1872A CBP mutant (Figure 2c). Hence, we analyzed the contribution of Mdm2 completely abolished the ability of CBP to promote the recruit- phosphorylation sites Ser-166, 186 and 188, described to ment of Mdm2 to ERb (Figure 3a). Also consistent with these participate in Mdm2 response to mitogenic/survival signals.17,18 results, CBP Thr-1872 was found essential in mediating maximal We found that Ser-166 (A1) mutation still allowed Mdm2 to ERb downregulation to Akt activation in total cell extracts, as well decrease ERb levels in response to Akt activation, whereas the Ser- as in fractionated cell compartments in which the T1872A mutant 186 and 188 (A2) mutation abolished this effect (Figure 2c), prevented the decrease in both cytoplasmic and nuclear ERb suggesting that Mdm2 Ser-186 and -188 residues are required to levels (Supplementary Figures 2b and c). These results illustrate mediate ERb downregulation by Akt. The essential role of CBP in the prominent role of CBP to enhance Mdm2 recruitment to ERb regulating ERb levels in response to Akt activation (Figure 1) was causing receptor downregulation. demonstrated using shRNA-mediated knockdown of endogenous With the predominant role of Mdm2 Ser-186/188 compared CBP, which abolished the decrease in ERb promoted by Mdm2 with Ser-166 in promoting ERb reduction (Figure 2c), we found and Akt (Figure 2d). This suggests a coordinate action of CBP and that the ERb/Mdm2 complex was stabilized by the integrity of Ser- Mdm2 in downregulating ERb protein levels. 186/188 in whole cells (Figure 3b), providing a mechanistic role of these residues in ERb regulation. As cytoplasmic-nuclear shuttling of Mdm2 was reported to involve Ser-166 and -186,17 we Mdm2 interacts with and promotes ERb ubiquitination in a performed co-immunoprecipitation assays on isolated nuclei, concerted manner with CBP which showed that wild-type and mutants A1 of Mdm2 were As Mdm2 is able to promote ERb degradation and transcriptional able to maintain an interaction with nuclear ERb, whereas when repression, we tested the possibility that it could interact with ERb normalized with total amounts, the A2 mutant was less efﬁcient in cells. We found that Mdm2 was able to co-immunoprecipitate (Figure 3c). These results suggest that the activation of Akt not

& 2013 Macmillan Publishers Limited Oncogene (2013) 117 --126 Concerted degradation of ERb by CBP and Mdm2 M Sanchez et al 120 S255A ER - + + + + + C462A + Mdm2 - - + - + - + + CBP - + + + A2 Mdm2 - - - - - + - - CBP T1872A - - + + + CBP - - - + + + + + Akt - - - + + Akt

IB: Mdm2 IB: Mdm2

- + + + + + + + HA-Ubi - - + + + + + + Akt - - - - + T/A + + CBP + + A1 A2 Mdm2 - - - CBP - - Luc Mdm2 shRNA - + + + CBP/Akt IP:Mdm2

IB: HA 118K IB: Mdm2 87K

Figure 3. Mdm2 interacts with ERb and promotes its ubiquitination under Akt activation. (a) Recruitment of Mdm2 to ERb is promoted by Akt and CBP. 293T cells were transfected with myc-tagged ERb or S255A mutant in absence or presence of HA-tagged Mdm2 or C462A ligase dead mutant, CBP or T1872A Akt-defective mutant, and p110a/Akt constructs as indicated. Immunoprecipitation (IP) was carried out with an antibody speciﬁc to ERb, and Mdm2 was detected by immunoblotting analysis (IB) using an anti-HA antibody. ERb was monitored in each sample using an anti-myc antibody. Mdm2 input levels are also shown. (b) Ser-186/188 are required for Mdm2 recruitment to ERb. Immunoprecipitation assay similar as in (a) using Mdm2 S186/188A (A2) mutant compared with wild type. (c) Nuclear co-immunoprecipitation assay on isolated nuclei of cells expressing wild-type, S166A (A1) and S186/188A (A2) Mdm2 in the presence of ERb, CBP and p110a/Akt. Immunoprecipitation was carried out using an Mdm2 antibody and immunoblot with an ERb antibody. Mdm2 content was normalized in each nuclear fraction. ERb input levels are also shown. (d) Mdm2 mediates the ubiquitination of ERb. Cells were transfected with myc-ERb in the presence of HA-ubiquitin, and ubiquitination of ERb was detected by immunoblotting using an HA antibody on immunoprecipitated ERb extracts. CBP, wild type and T1872A (T/A), and p110a/Akt were added as indicated. Speciﬁc knockdown was achieved by lentiviral infection using shCBP or shMdm2 RNAs, with shLuc (luciferase) used as a negative control. ERb content was normalized in each sample using a Myc antibody.

only targets ERb to complex with Mdm2, but also facilitates the Thr-1872. However, the Thr-1872 containing-CH3 domain of entry of Mdm2 into the nucleus, thereby enhancing its interaction CBP does not appear to directly establish contact with Mdm2 as with ERb. determined by domain-mapping two-hydrid assay, but seems to Following its role in reducing ERb levels, we next determined involve Tyr-658 of the CREB interacting KIX domain (Supplemen- whether Mdm2 was able to promote ERb ubiquitination. We tary Figure 4). Tyr-658 is known to be a recruiting site of CREB to observed that activation of Akt induced the poly-ubiquitination of CBP,19 and our results suggest that it might behave as such for ERb, an effect promoted by the presence of CBP (Figure 3d). Both Mdm2 as well. However, the observation that Thr-1872 is also CBP knockdown and expression of the Akt-defective T1872A CBP involved points towards a CBP inter-domain communication to mutant led to a decrease in ERb ubiquitination. Consistent with efﬁciently recruit phosphorylated Mdm2. This is consistent with this, knockdown of Mdm2 also markedly impaired ERb ubiquitina- our results obtained in co-immunoprecipitation assay, which tion induced by Akt and CBP. These results demonstrate a showed that Thr-1872 contributes to elicit Mdm2 recruitment to coordinate contribution of CBP and Mdm2 to regulate ERb CBP (Figure 4b). Such recruitment correlates with an enhanced degradation, and establish ERb as a suitable target of Mdm2. degradation of ERb as determined in cycloheximide chase experiments, which was prevented by mutation of CBP Thr- 1872, again supporting its prominent role in regulating ERb CBP promotes the degradation of ERb by Mdm2 turnover in Akt-activated cells (Figure 4c). Phosphorylation of With the essential role for CBP in promoting ERb ubiquitination Mdm2 Ser-186/188 was also required to achieve maximal and degradation by Mdm2, we next addressed whether CBP can recruitment of Mdm2 to CBP, reﬂecting the importance of recruit Mdm2 in conditions where ERb ubiquitination was favored. these sites in mediating Mdm2 effects on ERb (Figure 4b). Using a one-hybrid reporter assay with a Gal4-Mdm2 fusion Interestingly, of the other nuclear receptor coactivators tested for construct, we found that activation of Akt enhanced by 1.5 fold their possible implication to promote ERb downregulation in the reporter activity, suggesting that phosphorylation of Mdm2 by response to Akt, none were able to mimic the effect of CBP, Akt promoted a transcriptional response (Figure 4a). Interestingly, including p300 and coactivators such as SRC-1/NCoA1, pCIP/ addition of CBP further increased by near 4-fold such response, SRC-3/NCoA3 and PCAF/KAT2B, known to share to different extent whereas the T1872A mutant did not, indicating that Akt promotes HAT enzymatic activity and coactivation of ERb (Supplementary the recruitment of CBP to Mdm2 in a manner dependent on Figure 5).

Oncogene (2013) 117 --126 & 2013 Macmillan Publishers Limited Concerted degradation of ERb by CBP and Mdm2 M Sanchez et al 121 A negatively charged hinge region dictates human ERb degradation to the Akt pathway Ser-255 of mouse ERb aligns with Asp-236 of human ERb and of other vertebrates in their respective hinge region, suggesting the interesting possibility that the negative charge provided by Asp- 236, and possibly also by the adjacent and conserved Glu-237 in human ERb, could mimic a phosphorylated state such as in the rodent isoforms (Figure 5a). We thus tested whether human ERb was subjected to downregulation by heregulin-b in a manner similar to mouse ERb. We found that indeed human ERb levels were decreased in response to ErbB2/ErbB3 activation by heregulin-b and CBP expression (Figure 5b). The reduction in human ERb levels was dependent on the Akt pathway and the 26S proteasome, as also observed with mouse ERb. The ubiquitination of human ERb was strongly induced by CBP and Akt, an effect dependent upon Mdm2 ligase activity and CBP Thr-1872 Mdm2 - + + + A2 phosphorylation (Figure 5c). To establish the role of the two CBP + + + T/A + negative sites in human ERb hinge region, we observed that Akt - - + + + replacing either Asp-236 (D/A) or in combination with Glu-237 (DE/AA) with alanine residues increased the steady-state levels of IP: CBP ERb, suggesting that the negatively charged nature of these sites might affect the stability of the receptor (Figure 5d). Furthermore, IB: Mdm2 mutation at these positions also prevented the downregulation of ERb by CBP and Akt, with a stronger effect with the DE/AA mutation (Figure 5d). These results identify Asp-236 and Glu-237 IB: CBP as important sites in the regulation of human ERb turnover by the Akt pathway, and support a role for a negatively charged hinge region in that process. IB: Mdm2 Mdm2 counteracts the anti proliferative effect of ERb in breast cancer cells The potential of Mdm2 to regulate cell growth in response to heregulin-b was addressed using human ER-positive MCF-7 breast cancer cells on the basis of the role of ErbB2/ErbB3 activation to restrain hormone responsiveness.9 Maintaining MCF-7 cells in the presence of 50 ng/ml heregulin-b markedly increased their proliferation rate over a period of 4 days without the prior need of estrogen (Figure 6a and Supplementary Figure 6a). However, Mdm2 knockdown caused a signiﬁcant and sustained reduction on the potential of heregulin-b to induce MCF-7 cell growth over that period compared with shLuc control, which had no effect, suggesting an important contribution of Mdm2 in the proliferative response of MCF-7 cells to heregulin-b (Figure 6a). To address the possibility that cell growth inhibition induced by shMdm2 might result from p53 stabilization, we measured p53 levels in MCF-7 cells treated as above and found that p53 was increased by heregulin-b, possibly ruling out an inhibitory role of p53 on the growth response of MCF-7 cells (Supplementary Figure 6b). In addition, knockdown of Mdm2 did not prevent the increase in p53 Figure 4. CBP Thr-1872 is required to mediate Mdm2 recruitment levels by heregulin-b, suggesting that in these conditions, p53 and ERb degradation. (a) CBP and Mdm2 interact in cells in response to Akt. One-hybrid assay using Gal4-Mdm2 fusion in 293T cells might have a minor role in the growth response to heregulin. A similar observation has recently been reported for estrogen- transfected with a UAStkLuc reporter. CBP (wild type and T1872A) 20 and PI3-K/Akt plasmids were added to measure their effect on elicited growth of MCF-7 cells. However, under identical luciferase reporter activity. Values are normalized to b-galactosidase conditions, there was a marked decrease in ERb content following activity and expressed as fold response compared with control treatment with heregulin-b at day 4, which was prevented with cells set at 1.0. (b) Immunoprecipitation of CBP and CBP T1872A Mdm2 knockdown (Figure 6b), supporting a role of endogenous (T/A) was carried out with an antibody speciﬁc to CBP, and samples Mdm2 to target ERb towards degradation in heregulin-activated were immunoblotted for the presence of myc-Mdm2 or -S186/188A MCF-7 cells. In parallel, the levels of ERa were not decreased by (A2) mutant using a myc antibody. CBP content was normalized heregulin-b neither following Mdm2 knockdown, indicating a by western analysis in each sample. Mdm2 input levels are also shown. (c) Thr-1872 is required in CBP-dependent ERb degradation. different regulation of ERa and ERb under these conditions Cycloheximide chase experiment in 293T cells expressing ERb. (Figure 6b). As with Mdm2, knockdown of CBP also prevented the The effect of wild-type and T1872A CBP were monitored on ERb reduction of ERb by heregulin-b in MCF-7 cells (Figure 6c), levels. Results are normalized to b-actin content and expressed supporting a coordinate effort of endogenous CBP and Mdm2 to as a percentage of change of time zero, which was set at 100%. regulate ERb cellular levels. To further address the functional response of ERb in the context of growth signals and as MCF-7 cells are both ERa and ERb positive, we performed chromatin immunoprecipitation (ChIP) assay using ER-negative Hs578t breast

& 2013 Macmillan Publishers Limited Oncogene (2013) 117 --126 Concerted degradation of ERb by CBP and Mdm2 M Sanchez et al 122 - + + - + + + + + + + ErbB2/ErbB3 - - + - - + + + p85 + + - - - + + + - + + + + CBP ------+ + - - - E2 ------+ ++ MG-132

HA-Ubi - - + - + + + - - CBP - - - + + + + + + Akt - - - - + - - - + Mdm2 CBP CBPT1872A ------+ + - Mdm2 C462A - CBP Akt Akt ------+ + CBP T1872A + - - + - - + - - + - - - + - - + - - + - - + - D/A 118K - - + - - + - - + - - + DE/AA ER 87K IB:HA

Figure 5. A negatively charged hinge region is needed to promote human ERb degradation by the Akt pathway. (a) Sequence alignment of the predicted Akt phosphorylation site of mouse ERb with corresponding hinge regions in different species. Shown are the conserved aspartic and glutamic residues found in other vertebrates that correspond to Ser-255 of rodent ERb.(b) Western analysis of human ERb in 293T cells transfected with ErbB2, ErbB3, CBP and p85a PI3-K constructs as indicated. Cells were treated with vehicle or 50 ng/ml heregulin-b in presence or absence of increasing concentrations (1 and 10 mM) of MG-132. b-actin was used to monitor loading. (c) CBP and the PI3-K/Akt pathway promotes human ERb ubiquitination by Mdm2. Cells were transfected with myc-ERb in the presence of HA-ubiquitin, and ubiquitination of ERb was detected by immunoblotting using an HA antibody on immunoprecipitated ERb extracts. CBP (wild type and T1872A) and p110a/Akt were added as indicated. ERb content was normalized in each sample using a Myc antibody. The ligase-defective C462A mutant of Mdm2 was also tested. (d) Asp-236 and Glu-237 are required to promote human ERb downregulation by Akt and CBP. Western analysis was performed on 293T cells expressing wild-type, D236A (D/A) or D236A/E237A (DE/AA) mutants of ERb. CBP (wt or 1872A) and p110a/Akt plasmids were also added as indicated. b-actin was used to control loading. h, human; m, mouse; r, rat; rh, rhesus monkey; ov, ovis aries;x,xenopus laevis; bi, sturnus vulgaris.

cancer cells transfected with ERb. We found that treatment with residing mostly within the AF-1 region, have been identiﬁed to heregulin-b strongly impaired the hormone-induced recruitment mediate ERb response to estrogen and growth factors.3 Here, we of ERb to the promoter regions of estrogen-responsive genes pS2/ identify two novel sites, Asp-236 and Glu-237, which have a crucial TFF1, PgR, Fos and Cyclin D1 (Figure 6d), suggesting that activation role in regulating ERb turnover following PI3-K/Akt activation. Both of ErbB3 receptor might disrupt a proper response to hormone. these sites are located within the hinge region of ERb and confer a These effects were strongly abrogated with Mdm2 knockdown, negatively charged nature analogous to the phosphorylation of indicating that Mdm2 was required to achieve ERb downregula- corresponding Ser-255 in mouse ERb.10 Interestingly, these sites tion to responsive promoters by heregulin-b in breast cancer cells. are part of an Arg-X-X-Ser/Asp-Glu hinge motif of ERb, which is The response to heregulin-b was also addressed in the context of conserved in higher vertebrates, including cervidae, birds and ERb expression using stably expressing Hs578t (Hs-ERb) cells primates, suggesting a shared mechanism in ERb regulation. (Supplementary Figure 7a). The Hs-ERb cells exhibited an impaired However, such motif is not present in ERa hinge region, which growth response when compared with parental Hs578t cells might explain that unlike ERb,ERa is not subjected to a similar (Supplementary Figure 7b), suggesting that cell growth is limited downregulation, although other mechanisms such as lysine by ERb expression. Similar to MCF-7 breast cancer cells, ERb modiﬁcations have implied the hinge region in ERa degrada- expression also resulted in a reduced growth response to tion.21 --23 Among the other nuclear receptors that share the motif heregulin-b treatment in Hs578t cells (Supplementary Figure 7c). with ERb, the estrogen-related receptor ERRb also exhibits a In the context of hormone, as the Hs-ERb cells were reported to conserved Arg-X-X-Ser-Glu sequence in the hinge region and respond to estrogen because of ERb expression,10 we found that similar to ERb, its activity was found inhibited by CBP and Akt.10 heregulin-b disrupted the hormonal-dependent growth of Hs-ERb Interestingly, with the emerging role of ERR isoforms in breast cells (Figure 6e), correlating with results obtained in estrogen- cancer progression, the clinical status of ERRb has been shown to treated MCF-7 cells.9 ERb responsiveness to hormone was restored correlate with that of ERb in human primary breast tumors,24 with Mdm2 knockdown (Figure 6e), indicating the inhibitory role raising the possibility that ERRb might serve as a phosphorylation of Mdm2 on ERb under these conditions. Taken together, these target of Akt and be regulated in a similar fashion as ERb by CBP results support the role of heregulin-b and Mdm2 in down- and Mdm2. regulating ERb in breast cancer cells. We demonstrate that the inhibitory response of ERb in terms of transcriptional potential to ErbB2/ErbB3 receptor activation is closely associated to its degradation, implicating CBP as an DISCUSSION important determinant in ERb turnover. This is in contrast with the This study demonstrates the coordinate action of E3 ligase Mdm2 ability of CBP to act as a transcriptional coactivator of ERb,asit and coactivator CBP in promoting the poly-ubiquitination and was shown in response to EGF and Erk activation.7 By regulating degradation of ERb in response to activated ErbB receptors. ERb ERb response to the Akt pathway and by being itself subjected to activity is tightly controlled by phosphorylation and several sites, phosphorylation at Thr-1872 in that process, CBP acts as a scaffold

Oncogene (2013) 117 --126 & 2013 Macmillan Publishers Limited Concerted degradation of ERb by CBP and Mdm2 M Sanchez et al 123

- shLuc shMdm2 - shLuc shCBP - H - H - H - H - H - H Mdm2 CBP *** * ER

-actin

TFF1 promoter PgR promoter 3.0 2.5 300 *** * *** * control 2.5 2.0 2.0 E2 1.5 250 E2 + Hrg- 1.5 E2 + Hrg- + shLuc 1.0 1.0 E2 + Hrg- + shMdm2 ** 0.5 0.5 200 0.0 0.0 150 Fos promoter CcnD1 promoter 3.0 2.5 *** * *** * 100 2.5 2.0 2.0 1.5 50

1.5 Cell growth (% of control) 1.0 1.0 ChiP signal enrichment (fold) 0 0.5 0.5 1 2 3 4 0.0 0.0 - HE E+H E+H - HE E+H E+H Treatments (days) shMdm2 shMdm2 Figure 6. Regulation of ERb degradation, target gene promoter occupancy, and proliferation of breast cancer cells by Mdm2. (a) Heregulin-b induces the growth of MCF-7 cells through Mdm2. MTT proliferation assay was performed on human breast cancer MCF-7 cells treated or not with 50 ng/ml heregulin-b starting at day 0 and maintained over a period of 4 days. Lentiviral infections with shLuc or shMdm2 were carried out 24 h before treatments (day 1). Cells were harvested each day for MTT reduction colorimetric assay. Results are expressed as percent change from untreated control cells at day 1, which is set at 100% and represent mean values of triplicate measurements from three to four independent experiments. (b) Heregulin-b promotes ERb degradation by Mdm2 in MCF-7 cells. Western analysis was performed in MCF-7 cells treated or not with 50 ng/ml heregulin-b as in (a). Samples were collected at day 4 of treatments and analyzed for ERa,ERb and Mdm2. b-actin was used to control loading. Lentiviral infections of shLuc and shMdm2 constructs were carried out as in (a). (c) MCF-7 cells were treated as in (b), except that shCBP knockdown was performed. Total CBP content was monitored and b-actin was used to control loading. (d) Promoter occupancy by ERb is impaired in breast cancer cells treated with heregulin-b. ChIP analysis of ER-negative Hs578t cells transfected with ERb and treated with 10 nM estradiol and/or 50 ng/ml heregulin-b. Cells were also infected with lentiviral shMdm2. Fold-changes of ChIP signals over input DNA are expressed relative to untreated cells for each target. (e) MTT proliferation assay was performed as in (a) except that Hs578t cells stably expressing ERb (Hs-ERb) were used. Cells were treated with 10 nM estadiol (E2) in presence or absence of 50 ng/ml heregulin-b. Lentiviral infections with shLuc and shMdm2 were carried out 24 h before treatments. Results are expressed as percent change from untreated control cells at day 1, which is set at 100% and represent mean values of triplicate measurements from three to four independent experiments. One-way ANOVA was performed with Bonferroni multiple comparison post-hoc tests to determine statistical significance (*Po0.05, **Po0.01, ***Po0.001). protein not only restricted to transcriptional regulation but to also such an intrinsic versatility to regulate transcription in seemingly favor ERb poly-ubiquitination through the potent recruitment of opposite ways. Mdm2. Such dual effect has been reported for other nuclear The primary role Akt had in ERb degradation induced by ErbB2/ receptor coactivators in situation of agonist-induced transcription. ErbB3 receptor signaling led us to identify the E3 ligase Mdm2 as Notably, AIB1/NCoA3 was reported to mediate the activation and an important component in mediating ERb ubiquitination and degradation of ERa by estrogen,25 both processes being closely degradation and to achieve ERb transcriptional repression. Our associated with each other.13,14 The E3 ligase E6-AP was also identification of ERb as a novel target of Mdm2 is in line with the shown to exhibit dual activity toward ERa by inducing both its emerging concept that Mdm2 contributes to tumor development activity and degradation in response to hormone.26 However, through additional p53-independent mechanisms. Other Mdm2 coactivators of the SRC family, such as SRC-1 and pCIP/SRC-3, were substrates such as Rb, E2F1, FoxO3a and androgen receptor have unable to mediate ERb degradation in response to Akt activation recently been described and associated to cell proliferation and in our study, possibly because of their inability to interact with tumorigenesis.29 With the ability of Mdm2 to respond to growth Mdm2.27 Our findings are in agreement with the reported role of factors and other mitogenic signals,16 our observation that Ser-186 CBP and p300 to participate in tumor suppressor p53 ubiquitina- and 188 are essential in mediating the recruitment of Mdm2 to tion and on their apparent ability to serve as E4 polyubiquitin ERb in response to Akt activating signals is consistent with a ligases.28 In the case of ERb, the reason why p300 remained favored relocalization of Mdm2 towards the nucleus.17 This inefficient is unclear, but is certainly a specific departure from the recruitment was potentiated by CBP, revealing a stabilizing effect findings with p53, and is potentially novel in suggesting a of CBP to incite ERb/Mdm2 interaction, and also promote its own biochemical difference between the p300 and CBP paralogs. The recruitment to Mdm2 through the prominent role of Thr-1872 in attractive idea that CBP may behave as an E4 ligase of ERb, ERb/CBP/Mdm2 complex assembly. However, we found that Tyr- promoting its poly-ubiquitination under cellular anti-apoptotic 658 of the KIX domain, as opposed to Thr-1872 containing CH3 signals, warrants further investigation to explain how CBP exhibits domain of CBP, was directly implicated in contacting Mdm2 under

& 2013 Macmillan Publishers Limited Oncogene (2013) 117 --126 Concerted degradation of ERb by CBP and Mdm2 M Sanchez et al 124 Heregulin- mediate protein degradation. The indispensable use of specific Akt sites of each component in order to maximize ERb/CBP/Mdm2 assembly leads us to propose a coordinate mechanism by which an intersite phosphorylation switch regulates ERb turnover in breast cancer cells. This novel mechanism is depicted in Figure 7 where heregulin-b provides the incoming Akt activating signal ErbB2 ErbB3 Cell membrane that mediates, in a concerted manner, Mdm2 nuclear translocation and activation, CBP phosphorylation and recruitment of Mdm2, PI3-K and their interaction with ERb through its negatively charged Mdm2 hinge region. Such assembly then promotes ERb poly-ubiquitination and degradation by the 26S proteasome pathway, resulting in a marked decrease in ERb and enhanced proliferation of breast Akt cancer cells. Overexpression of ErbB receptors is a frequent event in breast Mdm2 Ser-186 cancer and downstream signaling events that crosstalk with the 4,31,32 Ser-188 ER pathway are strongly associated to endocrine resistance, a major obstacle in the treatment of breast cancer. Recent studies have added to the clinical value of ERb in hormone-dependent 33 34 35 Tyr-658 ER Nucleus cancers such as breast, ovarian and prostate cancers, providing ERb with antiproliferative functions. Our findings that Asp-236 increasing ERb levels by Mdm2 and CBP knockdown resulted in CBP Glu-237 breast cancer cell growth retardation in the presence of heregulin- Thr-1872 b are consistent with such activity of ERb under pro-apoptotic conditions and bring important implications for the pathophysio- Mdm2 logical assessment of breast tumors and possibly others as stated degradation above, giving a similar role yet to be determined. Indeed, as ERa CBP ER levels were less affected compared with ERb in such setting, this raises the interesting possibility that Mdm2 activity can modulate the ERa/ERb ratio in positive breast cancer cells. Mdm2 overexpression or amplification is a common event in breast cancer, and a higher ERa/ERb ratio was observed in breast tumors 26S compared with normal tissues, and was associated with an 36 --38 Proteasome invasive phenotype. In addition, increasing ERb induced Cell growth expression of adhesion proteins resulting in a reduced migration potential of breast cancer cells39 and ERb knockdown was shown to increase the growth of MCF-7 cells.40 More recently, agonist- Figure 7. A schematic model of ERb degradation by the 26S specific activation of ERb was shown to reduce ERa/ERb ratio, proteasome involving an Akt-concerted tripartite phosphorylation 41 switch. Activation of ErbB2/ErbB3 receptors by heregulin-b signals resulting in decreased growth of resistant breast cancer cells. the PI3-K/Akt pathway to promote ERb/Mdm2/CBP assembly and Our findings define a selective and tightly controlled mechanism targets ERb to the 26S proteasome system for degradation. The E3 that associates ERb degradation with Mdm2 and CBP activity, and ligase Mdm2 translocates to the nucleus in response to Akt suggest that approaches to upregulate ERb might confer activation and interacts with ERb in a process involving Ser-186 and insensitivity to growth-initiated signals of the ErbB family. These -188 Akt sites of Mdm2. CBP acts as a scaffold protein to further results are consistent with the possibility that ERb may possess enhance and stabilize the Mdm2/ERb complex through tumor suppressor-like activity and illustrate an unmet medical phosphorylation of Thr-1872, an Akt-targeted site essential for CBP need to develop novel therapeutic strategies and tools to clarify intrinsic response and recruiting ability toward ERb and Mdm2. The negatively charged environment provided by Asp-236 and Glu-237 the role of ERb in cancer cell biology. (phosphorylated Ser-255 in mouse ERb) within the hinge region of ERb facilitates Mdm2 recruitment in Akt-activated cells. Such assembly then promotes ERb ubiquitination and degradation by the MATERIALS AND METHODS 26S proteasome pathway, resulting in a marked decrease in ERb and Plasmid constructs enhanced proliferation of breast cancer cells in response to mitogenic/survival signals. Expression plasmids for ERa,ERb, CBP, ErbB2, ErbB3 and luciferase reporter 10 constructs vitA2-ERE-tkLuc and UAS-tkLuc have been described. The mouse ERb Ser-255, human ERb Asp-236 and Glu-237 and CBP Thr-1872 these conditions, suggesting that an interdomain communication substitutions to alanine residue were generated by PCR mutagenesis using triggered by Akt is needed to confer a proper conformation of CBP Pfu polymerase (Agilent Technologies, Cedar Creek, TX, USA) or Pwo DNA in order to recruit phosphorylated Mdm2. Structural studies of CBP polymerase (Roche Diagnostics, Laval, QC, Canada), and verified by KIX domain have revealed a shallow hydrophobic groove automated sequencing. Plasmids coding for the constitutively active stabilized by Tyr-658, which mediates interaction with phosphory- p110a catalytic subunit and the dominant negative p85aDi-SH2-N subunit lated CREB.19 Whether such mechanism can also be envisioned for of PI3-K were a kind gift from J Downward. Mdm2, generously provided by the recruitment of phosphorylated Mdm2 remains to be G Ferbeyre, was inserted into pCMX-HA and pCMX-Gal4, respectively, to determined, but our data are consistent with the reported produce N-terminal-tagged constructs. The Mdm2 C462A ligase-deficient association of Mdm2 to p300 in p53 degradation, although the mutant, and the S166A (A1) and S186, 188A (A2) mutants were generated structural determinants may differ.30 With our findings that by mutagenesis as above. Thr-1872 was identified as an Akt-targeted site, which drives CBP intrinsic transcriptional activation potential in response to the Cell culture, DNA transfection and luciferase assay 10 PI3-K/Akt pathway, this study provides a functional relationship Human breast cancer MCF-7 cells and Hs578t cells were maintained in between the transcriptional activity of CBP and its ability to Dulbecco0s modified Eagle0s medium (DMEM) supplemented with 10%

Oncogene (2013) 117 --126 & 2013 Macmillan Publishers Limited Concerted degradation of ERb by CBP and Mdm2 M Sanchez et al 125 fetal bovine serum (FBS). Human embryonic kidney 293T cells were in pLenti vector, a kind gift of C Beause´jour. The shCBP-encoding plasmid cultured in DMEM in the presence of 5% FBS. For transient transfection, was kindly provided by C Massaad. Viral particles were produced in 293T cells were seeded in 24-well plates in phenol red-free DMEM supplemen- cells as described,42,43 and used to infect MCF-7 and Hs578t cells. Mdm2 ted with 5% charcoal dextran-treated FBS and transfected as described.10 and CBP-efficient knockdown was monitored by western analysis. An Typically, cells were transfected with 1.5 mg of DNA per well, containing shRNA targeting luciferase (shLuc) was used as a negative control. 400 ng of reporter plasmid, 50--100 ng of receptor expression vector, 200 ng of CMX-bgal, 100 ng each of PI3-K, Akt and Mdm2 expression ChIP assay vectors, and 30 ng of CBP plasmid when indicated. After 5--8 h, the ChIP assays were done as previously described.42,44 Hs578t cells were medium was changed and cells were treated with 10 nM estradiol (E ; 2 transfected with ERb and treated with 10 nM estradiol and/or 50 ng/ml Sigma, Oakville, ON, Canada) for 16--20 h or left untreated. Cells were then heregulin-b for 40 min before harvest. Primer pairs were designed to harvested and luciferase assays were performed in duplicate from at least encompass ER-binding sites of pS2/TFF1, PgR, Fos and Cyclin D1 three independent experiments, and values were expressed as relative promoters,42 and DNA fragments obtained from immunoprecipitated light units normalized to the b-galactosidase activity of each sample as samples and inputs were quantitated by PCR from at least three previously described.10 independent experiments. Western analysis and immunoprecipitation assay Hs-ERb stable cells Western analysis was carried out in MCF-7 cells, or by transfecting 293T ER-negative Hs578t breast cancer cells were maintained in DMEM cells with HA/myc-tagged or untagged ERa or ERb (wt or mutants). Cells containing 10% FBS and transfected with pcDNA3.1-Neo encoding human were then treated with 50 ng/ml heregulin-b1 (R&D Systems, Minneapolis, ERb as described.10 Resistant clones were selected for G418 resistance and MN, USA), 10 nM E and/or 1 mM proteasome inhibitor MG-132 (Enzo Life 2 validated for their respective expression of ERb by western analysis and Sciences, Farmingdale, NY, USA) for 16 h and harvested as described.10 their estrogenic response by luciferase assay, compared with mock- Immunoblotting was performed using specific antibodies to ERa and ERb transfected Hs578t cells. (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or anti-HA (12CA5) or anti- myc (9E10) antibodies, and signals revealed by chemiluminescence using appropriate horseradish peroxidase-conjugated secondary antibodies. In CONFLICT OF INTEREST each experiment, total protein loading was normalized using an anti-b- The authors declare no conflict of interest. actin antibody (Novus Biologicals, Oakville, ON, Canada). Western analysis of CBP and Mdm2 was monitored using respective antibodies (Santa Cruz Biotechnology). For ERb/Mdm2 interaction, co-immunoprecipitation assays ACKNOWLEDGEMENTS 15 were performed in the presence of MG-132 as described, except that We thank members of the laboratory for critical reading and useful comments. MS is NaCl concentration was raised to 0.7 M and no SDS was added in the lysis supported by a doctoral award from the FHSJ (Fondation de l’Hoˆpital Ste-Justine), NP buffer. by the FRSQ (Fonds de la Recherche en Sante´ du Que´bec) and the FHSJ, and KS by the GRUM (Groupe de Recherche sur le Me´dicament de l’Universite´ de Montreál), the Ubiquitination of ERb FHSJ and the CIHR (Canadian Institutes of Health Research). AT is a New Investigator of the CIHR. This work was supported by grants from the CIHR, the NSERC (Natural To analyze the ubiquitination of ERb, 293T cells were transfected with Sciences and Engineering Research Council of Canada), the Cancer Research Society human or mouse ERb (wild-type or mutated) in the presence or absence Inc. and the Canadian Foundation for Innovation. (negative control) of a HA-tagged ubiquitin-encoding plasmid.15 Plasmids for CBP (wt or T1872A), Mdm2 (wt or C462A) and PI3-K p110a/Akt were also added as indicated. Immunoprecipitation of ERb and blotting with an REFERENCES 15 anti-HA antibody were as described. 1 Heldring N, Pike A, Andersson S, Matthews J, Cheng G, Hartman J et al. Estrogen receptors: how do they signal and what are their targets. Physiol Rev 2007; 87: Cycloheximide chase 905 --931. 2 Weigel NL, Moore NL. Steroid receptor phosphorylation: a key modulator of 293T cells were transfected with HA-tagged wild-type or S255A ERb in the multiple receptor functions. 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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

Oncogene (2013) 117 --126 & 2013 Macmillan Publishers Limited