Decreases Breast Cancer Cell Survival by Regulating the IRE1&Sol

ORIGINAL ARTICLE ERβ decreases breast cancer cell survival by regulating the IRE1/XBP-1 pathway

G Rajapaksa1, F Nikolos1, I Bado1, R Clarke2, J-Å Gustafsson1 and C Thomas1

Unfolded protein response (UPR) is an adaptive reaction that allows cancer cells to survive endoplasmic reticulum (EnR) stress that is often induced in the tumor microenvironment because of inadequate vascularization. Previous studies report an association between activation of the UPR and reduced sensitivity to antiestrogens and chemotherapeutics in estrogen receptor α (ERα)- positive and triple-negative breast cancers, respectively. ERα has been shown to regulate the expression of a key mediator of the EnR stress response, the X-box-binding protein-1 (XBP-1). Although network prediction models have associated ERβ with the EnR stress response, its role as regulator of the UPR has not been experimentally tested. Here, upregulation of wild-type ERβ (ERβ1) or treatment with ERβ agonists enhanced apoptosis in breast cancer cells in the presence of pharmacological inducers of EnR stress. Targeting the BCL-2 to the EnR of the ERβ1-expressing cells prevented the apoptosis induced by EnR stress but not by non-EnR stress apoptotic stimuli indicating that ERβ1 promotes EnR stress-regulated apoptosis. Downregulation of inositol-requiring kinase 1α (IRE1α) and decreased splicing of XBP-1 were associated with the decreased survival of the EnR-stressed ERβ1-expressing cells. ERβ1 was found to repress the IRE1 pathway of the UPR by inducing degradation of IRE1α. These results suggest that the ability of ERβ1 to target the UPR may offer alternative treatment strategies for breast cancer.

Oncogene (2015) 34, 4130–4141; doi:10.1038/onc.2014.343; published online 27 October 2014

INTRODUCTION interaction with the chaperone glucose-regulated protein 78-kDa Implementation of screening programs and novel treatment (GRP78, also known as BIP or HSPA5). In response to EnR stress, approaches have led to a decrease in breast cancer mortality. GRP78 dissociates from the above substrates to interact with the 8,9 Nonetheless, approximately 40 000 breast cancer-associated unfolded proteins to facilitate their proper folding. Upon deaths are estimated to occur in the United States in 2014.1–3 dissociation from GRP78, IRE1α, which is an EnR transmembrane Despite the heterogeneity of breast cancer with respect to protein with functional kinase and endonuclease domain, under- molecular alterations and treatment sensitivity, there are only a goes dimerization, autophosphorylation and activation of its few well-validated prognostic markers and therapeutic targets for endonuclease activity. Active IRE1α splices a 26-nucleotide intron the clinical management of the disease. Treatment with the form X-box-binding protein-1 (XBP-1) mRNA, which, through a antiestrogen tamoxifen, which binds to estrogen receptor α (ERα) translational frame-shift, results in the expression of a strong and alters conformation of helix 12 relative to that seen with 17β- transcription factor—the activated/spliced form of XBP-1 (XBP- estradiol (E2), is a standard treatment option for the nearly 70% of -1s).10,11 XBP-1s binds to EnR stress response element to induce breast tumors that express this receptor. Although this treatment the expression of genes that are involved in the EnR-associated has been shown to reduce breast cancer mortality by one-third, degradation as well as those promoting protein folding.12,13 not all breast cancers respond to endocrine therapy.4 Adaptive Activation of the UPR serves to protect the EnR and the entire cell mechanisms that provide tumor cells with alternative survival from the accumulation of unfolded proteins. Downregulation of possibilities can confer therapeutic resistance. One mechanism of the UPR under conditions that promote EnR stress can initiate resistance is the unfolded protein response (UPR).5 UPR is an apoptosis. The apoptotic response during the EnR stress utilizes adaptive response that is activated in tumor cells because of the both intrinsic and extrinsic pathways. Upregulation of the C/EBP- limited capacity of the tumor microenvironment to supply the homologous protein induces apoptosis in response to EnR stress tumor with adequate levels of nutrients and oxygen. This leads to by decreasing the levels of the anti-apoptotic protein BCL-2. the accumulation of unfolded proteins in the endoplasmic Downregulation of BCL-2 results in mitochondrial release of reticulum (EnR), a condition known as EnR stress. Activation of cytochrome c and the subsequent activation of the cytosolic the UPR restores EnR homeostasis by increasing protein folding, caspase-dependent pro-apoptotic cascade.14 enhancing degradation of misfolded proteins and inhibiting Activation of the UPR has been associated with therapeutic protein synthesis, thus adjusting the protein folding capacity of resistance and recurrence in several types of cancer including the EnR to the folding demand.6,7 breast cancer. GRP78 has been shown to confer resistance to In the absence of EnR stress, the major transducers of the UPR, estrogen starvation-induced apoptosis15 and to antiestrogens16 in PKR-like EnR localized kinase, activating transcription factor 6 and breast cancer cells and its expression predicts response to taxane- inositol-requiring kinase 1 (IRE1) are inactive because of their based adjuvant chemotherapy in breast cancer patients.17

1Center for Nuclear Receptors and Cell Signaling, Department of Biology and Biochemistry, University of Houston, Houston, TX, USA and 2Lombardi Comprehensive Cancer Center, Department of Oncology, Georgetown University, Washington, DC, USA. Correspondence: Dr C Thomas, Center for Nuclear Receptors and Cell Signaling, Department of Biology and Biochemistry, University of Houston, 3605 Cullen Boulevard, Houston, TX 77204, USA. E-mail: [email protected] Received 7 May 2014; revised 7 August 2014; accepted 4 September 2014; published online 27 October 2014 Regulation of the UPR by ERβ in breast cancer G Rajapaksa et al 4131 Similarly, XBP-1 is overexpressed in endocrine-resistant breast upregulation of ERβ1 in the presence of EnR stress resulted in a cancer cells,18 confers resistance to antiestrogens19 and correlates progressive increase of the sub-G1 cell population (Supplementary with responses to tamoxifen in breast cancer patients.20 High Figure 1A). Quantification of ERβ1 expression shows that about expression of ERα and XBP-1 is part of the gene expression 50% of the amount of the receptor that is expressed in our ERβ1- signature in luminal breast cancers and ERα is known to bind the expressing cell model is sufficient to sensitize the cells to EnR promoter of the XBP-1 gene inducing its transcription.1,21 XBP-1s stressors. In addition to a serial increase of ERβ1 expression, MCF-7 also induces an estrogen-independent transcriptional activation of cells were transiently transfected with control or ERβ small ERα.22 In addition to ERα-positive breast cancers, activation of interfering RNA (siRNA) and the sub-G1 cell fraction was measured XBP-1 has recently been associated with tumor progression and following treatment with a concentration of Tg that significantly chemotherapy response in triple-negative breast cancer.23 enhanced apoptosis in control cells. Downregulation of ERβ1 Although the association of ERα with the activation of the EnR rescued the cells from the Tg-induced apoptosis confirming the stress response has been established, the role of ERβ in regulating pro-apoptotic function of the receptor in EnR-stressed breast the UPR is currently unknown. Wild-type ERβ (ERβ1) has been cancer cells (Figure 1e). To examine whether the ERβ splice variant shown to inhibit the growth and decrease the invasiveness of ERβ2, which differs from ERβ1 in 26 C-terminal amino acids, breast cancer cells.4,24 Further, clinical studies have reported an sensitizes breast cancer cells to EnR stress, we assessed apoptosis association between ERβ1 expression and better overall survival in in EnR-stressed control and ERβ2-expressing MDA-MB-231 cells. As breast cancer. Interestingly, patients with triple-negative breast shown in Supplementary Figure 1B, in contrast to ERβ1, cancer that were treated with adjuvant tamoxifen have signifi- upregulation of ERβ2 failed to significantly induce apoptosis in 25 cantly better survival when their tumors are positive for ERβ1. In response to EnR stress in breast cancer cells. To test whether ERβ1 the present study, we investigated whether, by regulating the sensitizes breast cancer cells to other cell death inducers, we UPR, ERβ1 alters the resistance of breast cancer cells to EnR stress. analyzed control and ERβ1-expressing MDA-MB-231 cells for ERβ1 was found to sensitize breast cancer cells to EnR stress- apoptosis following treatment with staurosporine, a kinase regulated apoptosis by repressing the IRE1 pathway of the UPR. inhibitor that induces apoptosis.30 Under staurosporine treatment, the sub-G1 fraction of ERβ1-expressing cells was significantly RESULTS higher compared with that of the control cells, suggesting that ERβ1 increases the cytotoxic effect of staurosporine in breast ERβ1 decreases the survival of breast cancer cells in response to cancer cells (Supplementary Figure 1C). This stress-dependent pro- EnR stress apoptotic effect of ERβ1 is consistent with our previous findings Activation of the UPR in breast cancer cells has been showing increased sensitivity of breast cancer cells to DNA- associated with reduced sensitivity to antiestrogens26 and damaging agent cisplatin following upregulation of ERβ1.31 chemotherapeutics.5,17,23 ERα, the principal biomarker for direct- 4,21 ing endocrine therapies, also regulates the expression of XBP-1. ERβ1 sensitizes breast cancer cells to EnR stress-induced apoptosis Although network modeling approaches predict a potential β 27 Defective UPR activation upon cell exposure to EnR stress can relationship of ER with XBP-1 in breast cancer, the role of the trigger apoptosis through activation of mitochondrial pathways second estrogen receptor in the context of UPR-regulated cell that stimulate caspase-dependent and -independent cascades.14 survival has not been experimentally tested. We hypothesized that To investigate whether ERβ1 activated mitochondria-dependent the wild-type form of the receptor affects the sensitivity of breast pro-apoptotic pathways in EnR-stressed breast cancer cells, cancer cells to EnR stress-induced apoptosis by regulating the β β control and ER 1-expressing MDA-MB-231 cells were left UPR. To test this hypothesis, we stably expressed ER 1 in two untreated or treated with Tg and the cleavage of caspase-3 was α breast cancer cell lines, the ER -positive MCF-7 cells and the triple- evaluated by immunoblotting. Consistent with the ERβ1-induced negative MDA-MB-231 cells (Figure 1a). MCF-7 cells express high apoptosis observed under EnR stress in flow cytometry histo- α β β levels of ER and relatively low levels of ER 1. The levels of ER 1in grams, the cleavage of caspase-3 was markedly accelerated in MCF-7 cells are higher compared with those in MDA-MB-231 ERβ1-expressing cells, yielding high levels of cleaved caspase-3 cells.24,28 Cells were analyzed for cell survival following exposure 2+ after 18 h of Tg treatment. In contrast, 18 h post EnR stress, to two pharmacological inducers of EnR stress, the Ca -ATPase cleaved caspase-3 was not detected in control cells (Figure 2a). In inhibitor thapsigargin (Tg) and the proteosomal inhibitor borte- addition, we assessed the activation of the pro-apoptotic marker zomib (BZ). BZ has received approval from the US Food and Drug poly-ADP ribose polymerase (PARP) in MCF-7 cells that express Administration for use in the treatment of patients with multiple caspase 7 but do not express caspase-3.32 As shown in Figure 2b, 29 myeloma and mantle cell lymphoma. As shown in Figures 1b the kinetics of PARP activation in EnR-stressed MCF-7 cells were and c, treatment with Tg or BZ for 30 h decreased the number of similar to those of caspase-3 in MDA-MB-231 cells further both control and ERβ1-expressing cells. However, the effect of the supporting our initial hypothesis that ERβ1 induces apoptosis in drugs was significantly more potent in ERβ1-expressing cells response to EnR stress. compared with the control cells, suggesting the involvement of To ascertain the effect of activating endogenous ERβ1in ERβ1 in the mechanisms that regulate cell survival in response to apoptosis, we treated wild-type and ERβ1-overexpressing MCF-7 EnR stress. Interestingly, the ERβ1-mediated decrease of survival cells with ER subtype-selective ligands together with EnR stress was much greater in triple-negative as compared with ERα- inducer. Apoptosis was then measured by monitoring the cleaved positive breast cancer cells. form of PARP by immunoblotting. As shown in Figure 2c, the ERβ- To elucidate the mechanism through which ERβ1 reduced specific agonist diarylpropionitrile (DPN) was the only ER subtype- breast cancer cell survival in response to EnR stress, breast cancer selective ligand that significantly increased the levels of cleaved cells with different ERβ1 levels were treated with BZ or Tg for 24 h PARP in both cell types. Consistent with these results, following and analyzed for apoptosis by flow cytometry. Upregulation of downregulation of ERβ1, the levels of cleaved PARP were ERβ1 dramatically enhanced apoptosis in response to either BZ or significantly lower in DPN-treated MCF-7 cells as compared with Tg treatment as shown by the higher percentage of BZ- or Tg- the cells treated with E2 or propyl pyrazole triol (PPT; Figure 2d), treated, ERβ1-expressing MDA-MB-231 cells in sub-G1 fraction that supporting a specific pro-apoptotic role for ERβ in EnR- is indicative of apoptosis (Figure 1d, left and right). To strengthen stressed cells. the pro-apoptotic effect of ERβ1, we assessed apoptosis in To confirm that ERβ1 induces EnR stress-regulated apoptosis, MDA-MB-231 cells with differential expression of ERβ1. Gradual we targeted the anti-apoptotic factor BCL-2 to EnR in

Figure 1. ERβ1 decreases breast cancer cell survival in response to EnR stress. (a) Left: ERβ1 levels in MDA-MB-231 cells stably transduced with lentivirus carrying pLenti vector or pLenti-flag-ERβ1 construct. Middle: ERβ1 levels in control and ERβ1 overexpressing MCF-7 cells. Endogenous and stably transduced flag-ERβ1 with slightly retarded electrophoretic mobility are shown. Right: ERβ1 levels in MCF-7 cells transiently transfected with control or two ERβ siRNAs (#1, #2). Recombinant full-length ERβ1 protein (rERβ1) was loaded as positive control. MDA-MB-231 (b) and MCF-7 (c) cells stably infected with lentivirus carrying an empty vector (Lenti) or ERβ1 plasmid were either mock treated (EtOH) or treated with the EnR stress inducers thapsigargin (Tg; 250 nM) or bortezomib (Bz; 100 nM) in 5% DCC-containing media for 30 h. Top: The images are representative of three independent experiments (scale bar = 100 μM). Bottom: Following incubation of the cells with trypan blue, live cells were counted in a microscope. Graph shows the mean ± s.e.m. of three independent experiments normalized to the untreated control cells. *P-value ⩽ 0.05. (d) Control and ERβ1-expressing MDA-MB-231 cells were left untreated (UT) or treated with 100 nM Bz (left) or 250 nM Tg (right) for 24 h. Cells were stained with propidium iodide and the DNA content was analyzed by flow cytometry. The percentage of the cells in sub-G1 fraction, which is indicative of apoptosis, is shown as mean of three independent experiments. (e) FACS analysis of MCF-7 cells that were transiently transfected with control or ERβ siRNA and left untreated or treated with 300 nM Tg for 24 h.

Figure 2. ERβ1 regulates EnR stress-induced apoptosis. (a) MDA-MB-231 cells stably expressing ERβ1 and control cells (Lenti) were left untreated or treated with 250 nM Tg for the indicated times and analyzed for the expression of caspase-3. The band intensity of cleaved caspase-3 was analyzed by densitometry and normalized to that of actin-β. The numbers under the immunoblot show the fold change compared with the untreated control cells that was given a value of 1. Results are representative of three independent experiments. (b) Cleaved PARP levels in control and ERβ1-overexpressing MCF-7 cells after treatment with 250 nM Tg for the indicated times. (c) Following incubation in 5% DCC-FCS media for 48 h, wild-type (left) and ERβ1-overexpressing (right) MCF-7 cells were treated with 10 nM of each one of the ligands E2, DPN, PPT and ICI 182780 (ICI) for 24 h in the presence of 250 nM Tg and cleaved PARP expression was assessed by immunoblotting. (d) MCF-7 cells were transiently transfected with ERβ siRNA and treated with ligands and Tg as in c. Cleaved PARP levels were assessed by immunoblotting. Results are representative of three independent experiments. The band intensities of cleaved PARP in b–d were analyzed as described above. (e) Top: ERβ1-expressing MDA-MB-231 cells following stable transfection with pIRES empty vector or pIRES-BCL-2 recombinant construct with BCL-2 containing the EnR-targeted signal sequence of cytochrome b5 (BCL-2cb5) were analyzed for BCL-2 expression (green) using confocal immunoﬂuorescence microscopy. DsRed ﬂuorescent protein containing the EnR-targeting sequence of calreticulin and the EnR retention sequence KDEL was transiently transfected in cells and used as an EnR marker (red). Arrows indicate colocalization in the perinuclear EnR (scale bar, 20 μM). Bottom: BCL-2 levels in ERβ1-expressing MDA-MB-231 cells stably transfected with the pIRES vector or pIRES-BCL-2cb5 plasmid. (f)ERβ1-expressing MDA-MB-231 cells stably transfected with a pIRES vector or pIRES-BCL-2cb5 plasmid were treated with 250 nM Tg or 1 μM cisplatin for 24 h. Top: cells were analyzed for their DNA content by FACS. The percentage of cells in sub-G1 fraction is shown as mean of three experiments. Bottom: cells were analyzed for cleaved caspase-3 by immunoblotting.

© 2015 Macmillan Publishers Limited Oncogene (2015) 4130 – 4141 Regulation of the UPR by ERβ in breast cancer G Rajapaksa et al 4134 ERβ1-expressing MDA-MB-231 cells by stably expressing BCL-2 absence of Tg, an increase in the spliced form of XBP-1 was again containing the EnR-targeting sequence of cytochrome b5 (BCL- observed in the cells treated with E2 and PPT (Supplementary -2cb5). BCL-2 targeted exclusively to the EnR has been shown to Figure 2C). In addition to XBP-1s, E2 was found to induce suppress cell death induced by EnR stress agents.14 BCL-2 family expression of the unspliced form of XBP-1 in the absence of EnR members are known to be key determinants of cell survival in stress, data consistent with previous studies showing E2-induced response to both antiestrogens33 and taxanes.34 ERβ1-expressing transcription of XBP-1 in breast and endometrial cancer cells cells stably transfected with BCL-2cb5 plasmid or an empty control (Supplementary Figure 2D).21 Although E2 binds both ER subtypes vector were treated with Tg or the non-EnR genotoxic stressor with a similar affinity, the higher expression of ERα may determine cisplatin and apoptosis was evaluated by fluorescence-activated the effect of the ligand on the regulation of UPR in MCF-7 cells. To cell sorting (FACS) analysis and by assessing the cleavage of strengthen the association of DPN with the downregulation of caspase-3. The stably transfected BCL-2cb5 showed EnR localiza- XBP-1s, we assessed the expression of XBP-1s in EnR-stressed tion, as determined by its similar localization pattern with that of MCF-7 cells that were transiently transfected with control or ERβ the DsRed fluorescent protein containing the EnR-targeted siRNA and treated with DPN. Importantly, DPN was found to fully sequence of calreticulin and the EnR retention sequence KDEL reverse the Tg-mediated upregulation of XBP-1s in cells that (Figure 2e). Upregulation of EnR-related BCL-2 did not rescue the express ERβ1 but not in the cells in which ERβ1 was down- ERβ1-expressing cells from the cisplatin-induced apoptosis as regulated (Figure 4b). To examine whether the ERα antagonist ICI shown by a similar increase in the sub-G1 fraction and of cleaved 182780 alters the DPN-mediated downregulation of XBP-1s, we caspase-3 in the cells carrying the empty vector and those measured the expression of XBP-1s in EnR-stressed MCF-7 cells expressing BCL-2cb5. In contrast, expression of BCL-2cb5 reversed after treatment with DPN and ICI 182780. As shown in Figure 4c, the Tg-induced apoptosis in ERβ1-expressing cells (Figure 2f), ICI 182780 did not reverse the effect of DPN on XBP-1s expression, suggesting that the apoptosis observed in ERβ1-expressing breast suggesting that ICI 182780 may act through a mechanism that cancer cells was EnR stress-regulated apoptosis. involves inhibition of ERα and/or activation of ERβ1. To further corroborate the role of ERβ1 on the regulation of XBP-1 splicing, β ERβ1 downregulates EnR stress-induced XBP-1 splicing in breast we assessed UPR downstream responses in control and ER 1- cancer cells expressing breast cancer cells in time-course experiments. Treatment of control MDA-MB-231 and MCF-7 cells with Tg led XBP-1 can activate ERα22 and confer both estrogen-independence to a more pronounced upregulation of the XBP-1s target genes and antiestrogen resistance on breast cancer cells.19,20 The ERdj4 and P58IPK when compared with ERβ1-expressing cells survival of XBP-1-deficient cells is compromised under conditions (Figures 4d and e, left and right). Taken together, these results that induce EnR-stress.6 Given that EnR stress induces splicing of suggest that ERβ1 may repress the UPR by reducing the splicing of XBP-1 and ERβ1 decreased the survival of EnR-stressed breast XBP-1. cancer cells, we explored the putative role of ERβ1 in the regulation of XBP-1 processing. We treated control and ERβ1- expressing breast cancer cells with Tg or BZ and assessed XBP-1 ERβ1 regulates the expression of IRE1α in breast cancer cells splicing by immunoblotting, real-time PCR and RT–PCR in time- To study possible involvement of IRE1α in the ERβ1-mediated course experiments. XBP-1 splicing is activated early (within a few regulation of XBP-1 splicing under EnR stress, we assessed IRE1α hours) in the course of the EnR stress response.35 As shown in expression in control and ERβ1-expressing breast cancer cells Figures 3a and b and Supplementary Figure 2A, the mRNA and following treatment with Tg. Immunoblotting revealed a strong protein levels of XBP-1s were markedly reduced in Tg-treated decrease in IRE1α protein levels in ERβ1-expressing MDA-MB-231 ERβ1-expressing MDA-MB-231 and MCF-7 cells compared with cells both in the absence and presence of Tg when compared with those of the Tg-treated control cells. In contrast, upregulation of the control cells (Figure 5a, left). In contrast to IRE1α protein levels, ERα or ERβ2 in triple-negative MDA-MB-231 cells did not induction of ERβ1 expression caused a reduction in the IRE1α significantly affect the splicing of XBP-1 (Figures 3a and c, top). mRNA only in EnR-stressed cells (Figure 5a, right). Decreased IRE1α To strengthen the association of ERβ1 upregulation with the protein levels were also detected in the MCF-7 cells following decreased XBP-1 splicing, we measured XBP-1s in MDA-MB-231 upregulation of ERβ1 (Figure 5b). cells with a serial increase of ERβ1 expression. Progressive increase As downregulation of IRE1α in unstressed ERβ1-expressing cells of ERβ1 expression in the presence of EnR stress resulted in a was observed only at the protein level, we set out to investigate gradual decrease of XBP-1 splicing (Figure 3c, bottom). Similar to whether ERβ1 regulates degradation of the IRE1α protein. In chase the Tg-treated cells, induction of ERβ1 expression inhibited the experiments, induction of ERβ1 expression in MDA-MB-231 cells BZ-mediated activation of XBP-1 splicing in MDA-MB-231 and reduced the half-life of IRE1α, suggesting that IRE1α protein MCF-7 cells (Figure 3d). In contrast to XBP-1s, ERβ1 did not alter turnover was enhanced by ERβ1 (Figure 5c, left and right). the expression of the unspliced form of XBP-1 in the absence of Treatment of the cells with the proteasome inhibitor MG-132 EnR stress, indicating that ERβ1 may not regulate the transcription inhibited the ERβ1-mediated decrease in IRE1α protein abundance of the XBP-1 gene (Figure 3e). confirming that IRE1α downregulation in ERβ1-expressing cells We then investigated the effect of endogenous ERβ1 down- was due to increased degradation (Figure 5d, left and right). IRE1α regulation on XBP-1 splicing in EnR-stressed MCF-7 cells. Real-time degradation occurs through a process that involves ubiquitylation PCR revealed a notable increase of the XBP-1s mRNA in the cells by the E3 ubiquitin ligase synoviolin 1 (SYVN1) and dissociation transfected with ERβ-specific siRNA compared with the control from the molecular chaperone HSP90.36,37 Interestingly, elevated cells in response to Tg treatment (Figure 3f). To explore the impact SYVN1 expression correlates with decreased levels of IRE1α36 and of endogenous ERβ1 activation on splicing of XBP-1, we treated E2 regulates the expression of Syvn1 gene in mouse uterus.38 wild-type and ERβ1-overexpressing MCF-7 cells with estrogen Induction of ERβ1 expression caused a significant increase in the receptor subtype-selective ligands for 24 h with or without Tg for levels of SYVN1 in MDA-MB-231 cells (Figure 5e). In addition to 4 h and assessed the levels of XBP-1s by immunoblotting and real- upregulation of SYVN1, immunoprecipitation of HSP90 under time PCR. As shown in Figure 4a and Supplementary Figure 2B, in nondenaturing conditions showed a decreased recruitment of the presence of EnR stress, the highest levels of XBP-1s were IRE1α to HSP90 in ERβ1-expressing MDA-MB-231 cells as observed in the cells treated with E2 and the ERα-specific ligand compared with the control cells (Figure 5f), implying the PPT; the lowest levels were seen in cells treated with the ERβ- involvement of SYVN1 and HSP90 in the ERβ1-mediated regula- specific agonist DPN and the ERα antagonist ICI 182780. In the tion of IRE1α pathway.

Figure 3. ERβ1 inhibits EnR stress-induced XBP-1 splicing. Control, ERα- and ERβ1-expressing MDA-MB-231 cells (a) as well as control and ERβ1- overexpressing MCF-7 cells (b) were left untreated or treated with 250 nM Tg for the indicated times. The expression of spliced form of XBP-1 (XBP-1s) was analyzed by immunoblotting (top). The mRNA levels of spliced (S) and unspliced (U) forms of XBP-1s were assessed by RT–PCR (bottom). Data are representative of three independent experiments. The band intensities of XBP-1s were quantiﬁed and normalized to actin- β.(c) Top: XBP-1s levels in control and ERβ2-expressing cells following treatment with 250 nM Tg for 4 h. Bottom: MDA-MB-231 cells with differential stable expression of ERβ1 were treated with 250 nM Tg for 4 h and analyzed for XBP-1s expression by immunobloting and ERβ1 expression by real-time PCR. The bands in two vertically sliced images correspond to protein samples run in the same gel. (d) Control and ERβ1-expressing MDA-MB-231 cells (left) as well as control and ERβ1-overexpressing MCF-7 cells (right) were analyzed for the expression of XBP-1s by immunoblotting after treatment with 100 nM Bz for 4 h (top) and real-time PCR following treatment with Bz for the indicated times (bottom). PCR values were normalized to those of the untreated control cells and shown as mean ± s.d. of three experiments. *P-value ⩽ 0.05. (e) Control and ERβ1-expressing MDA-MB-231 cells (left) as well as control and ERβ1-overexpressing MCF-7 cells (right) were analyzed for the expression of unspliced form of XBP-1 in the absence of EnR stress. PCR values were normalized to those of the control cells and shown as mean ± s.d. of three experiments. (f) MCF-7 cells were transiently transfected with control or ERβ siRNA. The graphs show the expression of ERβ1 (left) and XBP-1s (right) after treatment of the cells with 250 nM Tg for 3 h. Values were normalized to control Tg-treated cells and represent the mean of three independent experiments with s.d. and *P-value ⩽ 0.05 indicated.

ERβ1 sensitizes tamoxifen-resistant breast cancer cells to EnR cells as compared with the sensitive cells suggesting its stress and tamoxifen by downregulating the UPR involvement in the mechanisms that regulate therapeutic Tamoxifen-resistant breast cancer cells overexpress both XBP-118 resistance in breast cancer (Figure 6a). Following stable expression and the master regulator of the EnR stress response GRP78; of ERβ1 by lentiviral transduction and mass selection, we treated β downregulation of GRP78 was found to restore antiestrogen control and ER 1-expressing cells with Tg and BZ for 30 h or the 16 active metabolite of tamoxifen, 4-hydroxytamoxifen, for 3 days. sensitivity in resistant cells. Given the downregulation of the β IRE1α branch of the UPR in ERβ1-expressing breast cancer cells, we Induction of ER 1 expression decreased viability of the resistant cells in the presence of the EnR stress inducers and 4- hypothesized that ERβ1 can decrease the survival of the hydroxytamoxifen (Figure 6b). Finally, time-course experiments antiestrogen-resistant cells in response to EnR stress. To test this, showed elevated cleaved PARP and decreased expression of IRE1α we used as a model of endocrine resistance the MCF-7-RR cells and splicing of XBP-1 in Tg-treated ERβ1-expressing cells as that are estrogen-independent and tamoxifen-resistant. These compared with the Tg-treated control cells suggesting that ERβ1 cells were derived from MCF-7 cells following selection in low sensitizes antiestrogen-resistant cells to EnR stress-induced 39 serum and tamoxifen. Analysis of ERβ1 expression revealed apoptosis by downregulating the UPR (Figures 6c and d and signiﬁcantly lower levels of the receptor in the tamoxifen-resistant Supplementary Figures 3A and B).

Figure 4. Ligand-dependent ERβ1-mediated regulation of XBP-1 splicing. (a) Wild-type (left) and ERβ1-overexpressing (right) MCF-7 cells were treated with 10 nM of each one of the ligands E2, DPN, PPT and ICI for 24 h. During the last 4 h of the ligand treatment, cells were treated with Tg (250 nM) and the expression of XBP-1s was analyzed by immunoblotting. (b) MCF-7 cells were transiently transfected with control or ERβ siRNA and treated with 10 nM DPN for 24 h. Cells were treated with Tg (250 nM) together with DPN for 4 h and the expression of XBP-1s was analyzed by immunoblotting (left) and real-time PCR (right). The band intensities of XBP-1s were quantiﬁed and normalized to actin-β or p84. The graph shows the mean of three experiments with s.d. and *P-value ⩽ 0.05 indicated. (c) XBP-1s levels in MCF-7 cells that were treated with DPN or DPN together with ICI (10 nM) for 24 h. During the last 4 h of the ligand treatment, cells were treated with Tg (250 nM). (d) Control and ERβ1-expressing MDA-MB-231 cells were left untreated or treated with 250 nM Tg for 8 h. The expression of XBP-1 target genes ERdj4 (left) and P58IPK (right) was analyzed by real-time PCR. (e) ERdj4 and P58IPK mRNA levels in control and ERβ1-overexpressing MCF-7 cells following treatment with 250 nM Tg for 8 h.

DISCUSSION both antiestrogen-sensitive and -resistant breast cancer cells. Previous studies report activation of the UPR in breast tumors. The Further analysis of the same cells revealed that their increased expression of GRP78 and the increased activity of the IRE1/XBP-1 sensitivity was due to ERβ1-mediated induction of apoptosis. pathway have been associated with progression and therapeutic Importantly, in addition to the EnR stress inducers, re-expression β resistance in breast cancer.16,17,20,23 Regulation of XBP-1 expres- of ER 1 in the tamoxifen-resistant cells increased their sensitivity sion has been shown to be critical in sustaining hormone-induced to tamoxifen treatment. This observation is consistent with growth in breast cancer cells. The expression of XBP-1 is regulated previous studies showing induction of apoptosis in tamoxifen- α treated antiestrogen-sensitive breast cancer cells following by estrogen and ER is recruited at the promoter and/or enhancer β 40 β region of the XBP-1 gene.21 In addition to ERα,ERβ mediates upregulation of ER 1. In contrast to ER 1, upregulation of ERβ2 did not induce apoptosis in EnR-stressed breast cancer cells. estrogen signaling and is also expressed in many breast cancers. Thus, the ERβ isoforms perform different roles in apoptosis and The expression of ERβ has been associated with sensitivity to 4,25 survival in breast cancer cells that may explain their different tamoxifen treatment and clinical outcome in breast cancer. 41 prognostic value in breast cancer patients. However, the molecular mechanism that accounts for this Expression of ERβ1 in cancer cells that express very low levels of association is not fully understood. We initially observed that endogenous ERβ1, such as MDA-MB-231 cells, has been reported β ER 1 is expressed at lower levels in tamoxifen-resistant breast to elicit tumor repressive actions in a ligand-independent manner. cancer cells compared with sensitive cells. As activation of the UPR Assessment of estrogen response element-dependent transcrip- has been detected in resistant cells, we hypothesized that the tional activity by estrogen response element-luciferase-based downregulation of ERβ1 may be associated with the activation of reporter assay has shown increased activity in ERβ1-transfected the EnR stress response. This hypothesis was further supported by cells following treatment with ER ligands. However, the expression previous studies proposing a predicted association of ERβ with of cancer-related genes was not altered signiﬁcantly in the 5,27 markers of the UPR. Based on this information, we investigated presence of ligands, implying the involvement of other transcrip- whether ERβ affects cell growth in ERα-positive and triple-negative tion factors in ERβ1 action.24,31,42,43 In contrast, ligand-dependent breast cancer cells by regulating the EnR stress response. effects of ERβ have been observed in cancer cells that express Assessment of cell viability under conditions that induce EnR endogenous ERβ1 including MCF-7 cells.28,44 The expression levels stress led to the conclusion that ERβ1 decreases the survival of of the receptor, ERα and coregulatory proteins in transfected cells

Figure 5. ERβ1 suppresses the activation of IRE1α branch of the UPR. (a) IRE1α protein (left) and mRNA (right) levels in control and ERβ1- expressing MDA-MB-231 cells following treatment with 250 nM Tg for the indicated times. The immunoblot is representative of three independent experiments. The band intensities of IRE1α were quantified and normalized to actin-β. The values in the graph were adjusted to those of the untreated control cells and the mean of three experiments is shown with ± s.d. and *P-value ⩽ 0.05 indicated. (b) IRE1α protein levels in control and ERβ1-overexpressing MCF-7 cells. (c) Left: IRE1α protein levels in control (Lenti) and ERβ1-expressing MDA-MB-231 cells after treatment with 100 μM cycloheximide for the indicated times. Right: The graph represents the quantification of IRE1α protein abundance from three independent experiments with s.d. indicated. (d) Left: Control and ERβ1-expressing MDA-MB-231 cells were incubated in absence or presence of 1 μM MG-132 for 6 h and analyzed for IRE1α expression by immunoblotting. Right: The bar graph represents the quantification of IRE1α protein levels with s.d. and *P-value ⩽ 0.05 indicated. (e) Synoviolin 1 mRNA levels in control and ERβ1-expressing MDA-MB-231 cells. (f) Lysates from control (Lenti) and ERβ1-expressing MDA-MB-231 cells were immunoprecipitated with anti-HSP90 antibody, followed by immunoblotting with the indicated antibodies. The bottom panel is the input control of cell lysates.

and in cells that express endogenous ERβ1 may be important of ERβ1 provides the molecular basis for a novel strategy based on factors in determining the type of effect (ligand-independent or combined use of ERβ agonists and BZ to treat patients with –dependent) that ERβ1 elicits in cancer cells. Expecting ligand- metastatic breast cancer. dependent ERβ1-mediated regulation of the EnR stress response Downregulation of the UPR in cells that experience EnR stress is in MCF-7 cells that express both ERα and ERβ1, we measured known to trigger apoptosis.14 Our results suggest that the apoptosis in EnR-stressed MCF-7 cells following treatment with apoptosis induced in ERβ1-expressing EnR-stressed cells was estrogen receptor subtype-selective ligands. As expected, only the due to repression of the EnR stress response. Importantly, ERβ-specific agonist significantly upregulated markers of apopto- upregulation of ERβ1 or treatment with its specific agonist DPN sis. Ligand-dependent ERβ1-mediated upregulation of apoptotic were found to decrease the expression of the EnR stress factors in response to EnR stress suggests the use of specificERβ1 transducer XBP-1s and that of its target genes. Downregulation agonists together with EnR stress inducers as a potential of XBP-1s was also observed when the EnR-stressed cells were treatment modality for the clinical management of the therapeutic treated with ICI 182780 that is known to act as an antagonist for resistance in breast cancer. The EnR stress inducer BZ has been ERα and to induce the ERβ1-mediated tumor repressive used for the treatment of patients with metastatic breast cancer in actions.47,48 A dual role of this ligand as antagonist for ERα and a phase II study.45 Interestingly, BZ inhibited bone tumor growth agonist of ERβ1 may account for its effects on UPR in EnR-stressed of the metastatic triple-negative MDA-MB-231 cells in a mouse breast cancer cells that express both receptors. In contrast, ERα xenograft model.46 Our finding showing robust induction of agonists upregulated XBP-1s in ERα-positive breast cancer cells, apoptosis in BZ-treated MDA-MB-231 cells following upregulation which is consistent with previous studies demonstrating that

Figure 6. ERβ1 alters the EnR stress response in antiestrogen-resistant breast cancer cells. (a) Top: Immunoblot of ERβ1 in lysates from wild- type and tamoxifen-resistant MCF-7 (MCF-7-RR) cells. Lysates from H1299 lung cancer cells stably expressing untagged ERβ1 in the presence or absence of ICI were loaded as positive control. ICI is an estrogen receptor ligand that downregulates the receptor. Bottom: ERβ1 levels in control and ERβ1-expressing MCF-7-RR cells. Recombinant (rERβ1) and stably transduced ﬂag-ERβ1 with slightly retarded electrophoretic mobility are shown. (b) Top: Control (Lenti) and ERβ1-expressing MCF-7-RR cells were mock-treated (EtOH) or treated with Tg (250 nM)orBz (100 nM) for 30 h. Following incubation with trypan blue, live cells were counted in a microscope. Bottom: Control (Lenti) and ERβ1-expressing MCF-7-RR cells were treated with the indicated concentrations of 4-hydroxytamoxifen (OH-tam) for 3 days and analyzed for cell viability by a ﬂuorometric assay. Graphs show the mean ± s.e.m. of three independent experiments normalized to the untreated (UT) control cells. *P-value ⩽ 0.05. (c) Cleaved PARP levels in control and ERβ1-expressing MCF-7-RR cells after treatment with 250 nM Tg for the indicated times. The band intensities of cleaved PARP were analyzed as described above. (d) Control and ERβ1-expressing MCF-7-RR cells were treated with 250 nM Tg for the indicated times. The expression of XBP-1s and IRE1α was analyzed by immunoblotting. (e) Proposed mechanism illustrating the regulation of the UPR by ERβ1 in breast cancer cells. Induction of EnR stress leads to activation of IRE1α that promotes splicing of XBP-1 mRNA. This results in the generation of the transcription factor XBP-1s that triggers the expression of UPR genes that promote cell survival and inhibit apoptosis. By increasing the expression of Synoviolin 1, ERβ1 induces degradation of IRE1α, downregulates the pro-survival XBP-1s and enhances EnR stress-regulated apoptosis.

XBP-1 transcription is stimulated by E2.21 These ﬁndings mechanism that does not necessarily involve regulation of ERα strengthen the idea that XBP-1 is an estrogen-regulated gene. In activity. Interestingly, increased expression of XBP-1s has also addition, our observation that ERβ1 represses the expression of been associated with tumorigenicity and recurrence in triple- XBP-1s in both ERα-positive and triple-negative breast cancer cells negative breast cancer. Depletion of XBP-1 decreased cancer cell suggests that ERβ1 regulates the EnR stress response through a invasiveness and increased tumor chemosensitivity in a triple-

Oncogene (2015) 4130 – 4141 © 2015 Macmillan Publishers Limited Regulation of the UPR by ERβ in breast cancer G Rajapaksa et al 4139 negative breast cancer xenograft model.23 We have previously Bristol, UK) and the antiestrogen 4-hydroxytamoxifen in phenol red-free shown that ERβ1 decreases the invasiveness of triple-negative media containing 5% DCC-FCS. breast cancer cells and increases their sensitivity to treatment with 24,31 chemotherapeutic drugs. Our finding that ERβ1 downregu- Cell survival assay lates XBP-1s in triple-negative breast cancer cells may account for Cells were plated at a density of 7.5 × 105 cells per 35-mm dish. After 24 h, β the phenotypes observed in ER 1-expressing triple-negative cells were treated with vehicle or EnR stress inducers in 5% DCC-FCS media breast cancer cells and provides a rational for the use of ERβ- for 30 h. Cells were initially imaged using x20 objective in an OLYMPUS specific agonists as therapeutic agents to treat patients with this IX51 microscope equipped with an OLYMPUS XM10 camera (OLYMPUS, aggressive subtype of breast cancer. Center Valley, PA, USA) and then were harvested by trypsinization and The spliced form of XBP-1 is produced upon activation of the stained with 0.4% trypan blue solution (Sigma-Aldrich). Viable cells were EnR stress transducer IRE1α.6 We investigated whether inactiva- counted using Hausser Levy Hemocytometer (ThermoFisher Scientific, tion of IRE1α is associated with decreased XBP-1s in ERβ1- Waltham, MA, USA). In some of the experiments, cell viability was assessed expressing cells. Indeed, decreased protein levels of IRE1α were in cell lysates following nucleic acid staining with blue-fluorescent Hoechst detected in ERβ1-expressing cells both in the absence and the 33258 using the FluoReporter Blue Fluorometric dsDNA Quantification Kit presence of EnR stress. Further analysis of IRE1α expression (Life Technologies). Fluorescence was measured in a PerkinElmer 2030 revealed downregulation of IRE1α mRNA in ERβ1-expressing cells multilabel reader (Perkin Elmer, Waltham, MA, USA). only in the presence of EnR stress, indicating a transcriptional regulation of IRE1α by ERβ1. However, the decreased protein FACS analysis levels of IRE1α in the same cells in the absence of EnR Cells were harvested by trypsinization, washed with PBS, fixed in 70% stress supports a model that involves post-transcriptional ethanol and incubated with propidium iodide as previously described.50 regulation of IRE1α by ERβ1. IRE1α expression is regulated at Cells were analyzed for DNA content in a FACS Aria II cell sorter (BD post-transcriptional level through increased ubiquitylation and Biosciences, San Jose, CA, USA). Histogram resolutions were analyzed with degradation. Upregulation of the E3 ubiquitin ligase SYVN1 and FlowJo.10 cell cycle analysis software (Tree Star, Ashland, OR, USA). IRE1α-HSP90 dissociation have been shown to promote IRE1α 36,37 degradation. The enhanced protein turnover of IRE1α, the Immunoblotting-immunoprecipitation α decreased IRE1 -HSP90 association and the increased expression Whole-cell lysates were prepared in radioimmunoprecipitation assay buffer β β of SYVN1 detected in ER 1-expressing cells suggest that ER 1 (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.1% SDS, 0.5% deoxycholate and α induces degradation of IRE1 . 1% NP-40) containing protease (1 mM EDTA, Roche protease inhibitor This is the first demonstration that ERβ1 decreases the survival mixture (Roche, Branchburg, NJ, USA), 2 mM PMSF) and phosphatase of EnR-stressed breast cancer cells by downregulating the UPR inhibitors (1 mM NaF, 1 mM Na3VO4, Sigma phosphatase inhibitor mixture). (Figure 6e). These results may shed more light into the The lysates were subjected to SDS–polyacrylamide gel electrophoresis and mechanisms that regulate therapeutic resistance in breast cancer transferred onto nitrocellulose membrane (Amersham Biosciences, Pitts- and explain the association between the expression of ERβ1 and burgh, PA, USA). Membranes were blocked with 5% blotting-grade blocker sensitivity to endocrine therapy observed in clinical studies with (Bio-Rad, Hercules, CA, USA) in TBST (0.05% Tween-20) for 3 h at room breast cancer patients. Further understanding of the mechanisms temperature and probed with the primary antibodies overnight at 4 °C. that repress the expression of IRE1α, downregulate the UPR and Proteins were visualized using ECL detection kit (Amersham Biosciences) decrease cell survival in ERβ1-expressing cells in response to EnR following incubation of the membranes with secondary antibodies for 2 h stress should establish ERβ1 as an important regulator of the stress at room temperature. Primary antibodies against XBP-1 and BCL-2 were response in breast cancer and the clinical management of the purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). A second disease. antibody against XBP-1 (clone: Poly6195) was from BioLegend (San Diego, CA, USA). The antibodies against IRE1α,ERα, caspase-3 and PARP were from Cell Signaling (Danvers, MA, USA), the anti-actin-β from Sigma-Aldrich MATERIALS AND METHODS and the antibodies against P84 and ERβ1 (clone 14C8) were from GeneTex (Irvine, CA, USA). The 14C8 antibody, which is against the N-terminus of Cells and reagents ERβ, detects both ERβ1 and ERβ2 and has been validated for its Breast cancer cell lines (MDA-MB-231 and MCF-7) and H1299 lung cancer specificity.24,43 In addition to recombinant ERβ1 protein, as a positive cells were obtained from American Type Culture Collection (Manassas, VA, control of untagged ERβ1 expression, lysates from H1299 lung cancer cells USA). The cells were cultured in RPMI-1640 (MDA-MB-231, H1299) and that had been stably transfected with pIRES-ERβ1 plasmid and validated fi Dulbecco's modi ed Eagle's medium (MCF-7) media supplemented with for ERβ1 expression using different ERβ antibodies were loaded in some of 10% fetal calf serum (FCS). Tamoxifen-resistant MCF-7 cells (MCF-7-RR) the gels.43 For the IRE1α-HSP90 co-immunoprecipitations, cells were lysed were kindly provided by Dr R Clarke (Georgetown University). These cells in a buffer containing 50 mM HEPES, pH 7.4, 150 mM NaCI, 1 mM EDTA, 1 mM were grown in phenol red-free Iscove's modified Eagle medium media ethylene glycol tetraacetic acid, 1% Nonidet P-40, 1% glycerol including (Invitrogen, Carlsbad, CA, USA) supplemented with 5% dextran-coated protease and phosphatase inhibitors. Lysates were cleared by centrifuga- charcoal (DCC)-treated FCS (DCC-FCS) and 4-hydroxytamoxifen (Sigma- Aldrich, St Louis, MO, USA).39,49 Cells were infected with lentivirus tion and the supernatants were incubated with anti-HSP90 antibody – fi containing the empty pLenti6/V5 vector or the recombinant pLenti6/V5- (MA3 011, ThermoFisher Scienti c) overnight at 4 °C and A/G agarose D-FLAG-ERβ1, -ERβ2 and -ERα plasmids as previously described.42 Serial beads for 2 h at 4 °C. The immunocomplexes were washed and α increase of ERβ1 expression in MDA-MB-231 cells was achieved by single immunoblotted with anti-IRE1 antibody. and repeated lentiviral transduction. MCF-7 cells were transiently transfected twice with ERβ-specific siRNAs (Invitrogen), target sequences RNA extraction, real-time quantitative reverse transcription (RT)- ′ ′ ′ 1# 5 -TTAGCGACGTCTGTCGCGTCTTCAC-3 and 2# 5 -TTACGACATTAAGTA PCR and RT–PCR GTGTCGTCCC-3′. An siRNA-targeting luciferase was used as a control (12935-146, Invitrogen). For the expression of EnR-targeted BCL-2, ERβ1- Total RNA was isolated using Aurum total RNA mini kit (Bio-Rad) and expressing MDA-MB-231 cells were stably transfected with the pIRESpuro- reverse transcribed to cDNA using the iScript cDNA synthesis kit (Bio-Rad). BCL-2cb5 expression plasmid that contains the full-length human BCL-2 Real-time PCR was performed using the iTaq Universal SYBr Green cDNA with a deletion at the C-terminal 658–720 base pairs (corresponding Supermix (Bio-Rad). All quantitative data were normalized to 36b4. For the to amino acids 210–239) ligated to the EnR-targeting signal sequence of analysis of the XBP-1 splicing, RT–PCR was carried out with specific primers human cytochrome b5. Cells were treated with the EnR-stress inducers Tg to amplify a 600-bp cDNA product that was analyzed in 3% agarose gel by and BZ, the genotoxic agent cisplatin, the kinase inhibitor staurosporine electrophoresis. ACTIN-β was used as the endogenous loading control. (Sigma-Aldrich), the estrogen receptor subtype-selective ligands E2, DPN, Primer sequences for the real-time PCR and RT–PCR experiments are listed PPT, the estrogen receptor antagonist ICI 182780 (Tocris Biosciences, in Supplementary Table 1 (Supplementary Data).

© 2015 Macmillan Publishers Limited Oncogene (2015) 4130 – 4141 Regulation of the UPR by ERβ in breast cancer G Rajapaksa et al 4140 Immunofluorescence and confocal microscopy antiestrogen resistance in breast cancer cell lines. FASEB J Biology 2007; 21: Cells were plated onto 18 mm2 coverslips, fixed, permeabilized and 4013–4027. blocked as previously described.31 Slides were stained with an anti-BCL-2 20 Davies MP, Barraclough DL, Stewart C, Joyce KA, Eccles RM, Barraclough R et al. antibody (Santa Cruz Biotechnology) at 4 °C overnight, washed, incubated Expression and splicing of the unfolded protein response gene XBP-1 are fi with secondary antibody and images were collected on an Olympus signi cantly associated with clinical outcome of endocrine-treated breast cancer. 123 – FV1000 inverted confocal 1X81 microscope. International J Cancer 2008; :85 88. 21 Sengupta S, Sharma CG, Jordan VC. 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Supplementary Information accompanies this paper on the Oncogene website (http://www.nature.com/onc)