Tamoxifen is one of the most widely used endocrine agents for the treatment of estrogen receptor α (ERα)–positive breast cancer. Although effective in most patients, resistance to tamoxifen is a clinically significant problem and the mechanisms responsible remain elusive. To address this problem, we performed a large scale loss-of-function genetic screen in ZR-75-1 luminal breast cancer cells to identify candidate resistance genes. In this manner, we found that loss of function in the deubiquitinase USP9X prevented proliferation arrest by tamoxifen, but not by the ER downregulator fulvestrant. RNAi-mediated attenuation of USP9X was sufficient to stabilize ERα on chromatin in the presence of tamoxifen, causing a global tamoxifen-driven activation of ERα-responsive genes. Using a gene signature defined by their differential expression after USP9X attenuation in the presence of tamoxifen, we were able to define patients with ERα-positive breast cancer experiencing a poor outcome after adjuvant treatment with tamoxifen. The signature was specific in its lack of correlation with survival in patients with breast cancer who did not receive endocrine therapy. Overall, our findings identify a gene signature as a candidate biomarker of response to tamoxifen in breast cancer. Cancer Res; 74(14); 3810–20. ©2014 AACR.

About 70% of human breast cancers are estrogen receptor α (ERα)–positive and depend on this hormone receptor for their proliferation (1), rendering ERα an ideal target for endocrine treatment. Tamoxifen is one of the most commonly used drugs in the management of ERα-positive breast cancer. In early breast cancer, 5 years of adjuvant treatment with tamoxifen almost halves the rate of disease recurrence and reduces the annual breast cancer-related death rate by one-third (2). Despite this adjuvant treatment with tamoxifen, one-third of women still develop recurrent disease in the next 15 years (2), illustrating that endocrine resistance is a major problem in the management of breast cancer.

Several mechanisms may contribute to tamoxifen resistance. At presentation, not all ERα-positive tumors are sensitive to tamoxifen. This intrinsic endocrine resistance can be the result of ERα phosphorylation (3–5). In addition, intrinsic resistance is found to correlate with increased levels or activity of ERα coactivators (AIB1), growth factor receptors (EGFR, HER2, and IGF1R), kinases (AKT and ERK1/2) or adaptor proteins (BCAR1, c-SRC, and PAK1; refs. 3, 6). Loss of CDK10 expression (7) and loss of insulin-like growth factor–binding protein 5 (IGFBP5) expression (8) can also lead to tamoxifen resistance. Furthermore, high levels of lemur tyrosine kinase-3 (LMTK3) or CUEDC2 protein are associated with tamoxifen resistance (9, 10). ERα-independent mechanisms can play a role in endocrine therapy resistance, including the NOTCH pathway (11). Acquired endocrine resistance develops in a certain proportion of metastatic ERα-positive breast cancer that was initially sensitive to palliative tamoxifen treatment, but can also occur in the adjuvant setting when a patient relapses while on hormonal therapy. Possible mechanisms of this resistance are upregulation of the PI3K–mTOR pathway (12–14) and acquiring activating mutations in ESR1 (15). It is nevertheless likely that additional mechanisms contribute to unresponsiveness to endocrine treatment, which remains to be identified.

Ubiquitination serves a role in both protein degradation and regulation of protein function (16). The level of protein ubiquitination is highly regulated by two families of enzymes with opposing activities: the ubiquitin ligases, which add ubiquitin moieties to proteins and deubiquitinating enzymes (DUB) that remove them (17). The X-linked deubiquitinase USP9X is a member of the family of DUB enzymes and regulates multiple cellular functions by deubiquitinating and stabilizing its substrates. USP9X is involved in a number of key cellular processes, as the knockout of this gene in the mouse is embryonic lethal (18). USP9X has been shown to regulate, among others, cell adhesion molecules like β-catenin and E-cadherin, cell polarity, chromosome segregation, NOTCH, mTOR, and TGFβ signaling as well as apoptosis (18–27).

To elucidate novel mechanisms of tamoxifen resistance in breast cancer, we performed an shRNA screen in the hormone-dependent human luminal breast cancer cell line ZR-75-1 to identify genes in which knockdown could induce tamoxifen resistance. We report here an unexpected role for USP9X in ERα signaling: loss of USP9X enhances ERα/chromatin interactions in the presence of tamoxifen, leading to tamoxifen-stimulated gene expression of ERα target genes and cell proliferation.

Cell lines and culture conditions

Phoenix cells were cultured in DMEM supplemented with 10% FCS, 2 mmol/L glutamine and 100 μg/mL penicillin/streptomycin. ZR-75-1 and T47D cells were cultured under the same conditions in the presence of 1 nmol/L estradiol. Cell lines were obtained from the ATCC (www.ATCC.org) and used at low passage after receipt from the vendor.

Transfection and retroviral infection

Phoenix cells were transfected using calcium phosphate precipitation. Viral supernatant was cleared through a 0.45-μm filter. Target cells were infected twice with the viral supernatant using polybrene (8 μg/mL). For transient transfection of ZR-75-1 cells Lipofectamine 2000 (Life Technologies) was used.

The shRNA screen and recovery of shRNA inserts

Ecotropic receptor–containing ZR-75-1 cells were infected with the retroviral NKi pRetroSuper-shRNA library (12,540 shRNA vectors targeting 4,180 genes; ref. 28) or pRS as control. After puromycin selection, cells were cultured in DMEM with 1 μmol/L 4OH-tamoxifen for 6 weeks. Genomic DNA of individual colonies was isolated using DNAzol (Life Technologies). PCR amplification of the shRNA cassettes was performed using the Expand Long Template PCR System (Roche). Products were digested with EcoRI/XhoI and recloned into pRS and sequenced with Big Dye Terminator (PerkinElmer). Primers are in Supplementary Table S4.

Cell proliferation analyses

For colony formation assays, infected cells were cultured in DMEM containing 1 μmol/L 4OH-tamoxifen, 1 nmol/L estradiol, or 10−7 mol/L fulvestrant. After 2 to 6 weeks, cells were photographed, fixed with 4% formaldehyde, and stained with 0.1% Crystal violet. Plate confluence was assessed by measuring the percentage of cell-covered surface area. Alternatively, Crystal violet was extracted with 10% acetic acid and absorbance was measured at 600 nm.

For dynamic cell proliferation analyses, cells were seeded in a clear-bottom 96-well plate and proliferation was determined by plate confluency using an IncuCyte life cell imaging device (Essen BioScience). Three wells were measured per condition. Bars indicate SD.

Constructs

For retroviral transductions, ZR-75-1 and T47D cells were stably infected with supernatant of the Phoenix amphotrophic virus packaging cell line transfected with pBabeHygro-Ecotropic Receptor or pLZRS-Ecotropic Receptor-IRES-Neo. Short hairpin sequences targeting USP9X are in Supplementary Table S4.

Immunoprecipitation and immunoblotting

For coimmunoprecipitation, cells were lysed in ELB (250 mmol/L NaCl, 0.1% NP-40, 50 mmol/L Hepes pH 7.3) containing protease (Roche) and phosphatase inhibitors (Sigma-Aldrich). Supernatants were incubated with antibodies for USP9X (ab99343; Abcam), or ERα (D-12; Santa Cruz Biotechnology) coupled to protein G Dynabeads (Life Technologies). Mixed normal mouse and rabbit serum (Santa Cruz Biotechnology) was used as control. For Western blotting the following antibodies were used: USP9X (ab99343; Abcam), ERα (clone 1D5; Dako), progesterone receptor (PR; clone 1A6; Novocastra), and β-actin (clone AC-74; Sigma-Aldrich).

Luciferase assay

Monoclonal ZR-75-1 cells expressing pRS-USP9X or pRS–GFP, were plated in triplicate in 6-well plates and were transiently transfected with 1.75 μg ERE-TATA-luciferase reporter and 0.5 μg pRL-CMV Renilla luciferase (Promega) per well. 24 hours after transfection, ligand was added. Luciferase activity was determined 48 hours after transfection using the Dual Luciferase Reporter Assay System (Promega), with Renilla luciferase as control, with vehicle set at 1.

Quantitative real-time PCR

Total RNA was isolated using TRIzol (Life Technologies) or using the Quick RNA MiniPrep Kit (Zymo Research). cDNA was generated using Superscript II with random hexamer primers (Life Technologies). The qRT-PCR reaction was performed using FastStart Universal SYBR Green Master Mix (Roche) on an AB7500 Fast Real Time PCR system (Applied Biosystems). All reactions were run in parallel for GAPDH to control for the amount of cDNA input.

PCR primer sequences are shown in Supplementary Table S4.

RNA expression analysis

RNA-seq reads (14–30 million 50-bp single-end) were mapped to the human reference genome (hg19) using TopHat (29), supplied with a known set of gene models (Ensembl version 64). HTSeq count was used to obtain gene expressions. To identify differentially expressed genes, DEGseq (30) was used, P < 0.05. Levels of expressed genes were increased by 1 to avoid negative values after log2 transformation.

Chromatin immunoprecipitations

Chromatin immunoprecipitations (ChIP) were performed as described before (31). For each ChIP, 10 μg ERα antibody (HC-20; Santa Cruz Biotechnology) and 100 μL of Protein A Dynabeads (Life Technologies) were used. For qPCR, primers are in Supplementary Table S4.

Next-generation sequencing and enrichment analysis

ChIP DNA was amplified as described previously (31). Sequences were generated by the Illumina Hiseq 2000 genome analyzer (using 50 bp reads), and aligned to the Human Reference Genome (assembly hg19; February 2009). Enriched regions of the genome were identified by comparing the ChIP samples with input using MACS (32) version 1.3.7.1. Details on sequence reads are Supplementary Table S1.

Motif analysis, heatmaps, and genomic distributions of binding events

ChIP-seq data snapshots were generated using the Integrative Genome Viewer IGV 2.1 (www.broadinstitute.org/igv/). Motif analyses were performed through the Cistrome (cistrome.org), applying the SeqPos motif tool (33). The genomic distributions of binding sites were analyzed using the cis-regulatory element annotation system (34). If the binding region is within a gene, 5′ untranslated region (UTR), 3′UTR, coding exon, or intron are determined. Promoter is defined as 3 kb around RefSeq 5′ start. If a binding site is >3 kb away from the RefSeq transcription start site (TSS), it is considered distal intergenic.

Survival analyses

Normalized mRNA expression for four patient series was downloaded from GEO: GSE6532 (35), GSE22219 (36), GSE2034 (37), and GSE11121 (38). From these, two sets of ER-positive, tamoxifen-treated patients (n = 250, ref. 35; n = 134, ref. 36), and two sets of ER-positive–untreated patients (n = 209, ref. 37; n = 158, ref. 38) were extracted, for which follow-up was available. ER status for the Schmidt and colleagues data (38) is absent in the publicly available data and was determined by fitting a two-component Gaussian mixture to the expression of the ESR1 gene, which was verified to follow a bimodal distribution. Probes in the Buffa and colleagues (36), Wang and colleagues (37), and Schmidt and colleagues (38) data were median-centered before further processing; the Loi and colleagues data had already been median-centered. The 526 genes of the USP9X knockdown tamoxifen signature were mapped to the corresponding microarray platforms by selecting all probes for matching genes, and ignoring genes not present on the array. For the Loi and colleagues (35) data, these selected 949 probe sets represent 488 different genes. For the Buffa and colleagues (36) data, 363 probes were selected representing 295 genes and for both the Wang and colleagues (37) and the Schmidt and colleagues (38) datasets, 653 probe sets representing 391 genes were available. Of note, 254 of the signature genes were present on all three array platforms. Patients were stratified into two groups by applying a hierarchical complete-linkage clustering using Pearson correlation distance, and dividing by the first split of the clustering. Significant differences in distant metastasis-free survival (DMFS) time between these two groups were tested for using the log-rank test. Survival times longer than 10 years were right-censored. The array platform used for the untreated Wang and colleagues (37) and Schmidt and colleagues (38) datasets provides a subset of the probes available for the treated Loi and colleagues data (653 of 949; ref. 35). To verify that this difference does not affect the comparison between treated and untreated, the Loi and colleagues (35) samples were additionally clustered on the basis of this subset only. This clustering was still able to stratify patients according to prognosis (log-rank P = 1.3 × 10−5). The directionality of USP9X knockdown tamoxifen classification genes in the good and poor outcome patient groups in the Loi and colleagues (35) cohort is shown in Supplementary Table S2. Association of the USP9X knockdown tamoxifen signature with outcome after chemotherapy was tested on the subset of the 295 patients in the van de Vijver and colleagues cohort (39) with ER-positive breast tumors, treated with adjuvant chemotherapy, but not with tamoxifen (n = 56). Patients were stratified in two groups and the difference in survival was tested for as described above. The gene signature mapped to 758 probes representing 461 unique genes. PAM50 (40) subtyping by genefu package was applied to a pooled set of breast cancer samples (n = 1,570; GEO accession: GSE47561; ref. 41), including the Loi and colleagues study, ensuring correct assignment of molecular subtypes.

An shRNA screen identifies USP9X as a tamoxifen resistance gene

To identify new genes involved in tamoxifen resistance, a loss-of-function genetic screen was performed in the human luminal breast cancer cell line ZR-75-1 that expressed the murine ecotropic receptor. Cells were infected with retroviral supernatants containing a selection of the NKi pRS–shRNA library (12,540 shRNA vectors targeting 4,180 genes) or pRS as control (Fig. 1A; ref. 28). Library-infected cells and control cells were plated at low density and cultured in DMEM with 1 μmol/L 4OH-tamoxifen for 6 weeks. Individual colonies that grew out in the presence of tamoxifen were collected, genomic DNA was isolated and shRNA cassettes were recovered by PCR. These shRNA cassettes were subsequently recloned and sequenced. This led to the identification of USP9X. To confirm that knockdown of USP9X was responsible for the rescue of the tamoxifen-induced proliferation arrest, ZR-75-1 cells were infected with high titer shUSP9X and control virus. Proliferation in the presence of estradiol (E2) or tamoxifen (4-OHT) was determined in a colony formation assay (Fig. 1B). To exclude that the escape from tamoxifen-induced proliferation arrest was the result of an “off-target effect,” five additional shRNAs targeting different regions of the USP9X gene were designed. Figure 1C shows that three shRNAs had the identical phenotype: Infected cells grew out despite tamoxifen treatment. Importantly, only the vectors that suppressed USP9X mRNA (Fig. 1D) and protein levels (Fig. 1E) induced tamoxifen resistance. To ask whether the rescue from tamoxifen-induced proliferation arrest is independent of cellular context, we tested two USP9X shRNA vectors for their ability to confer tamoxifen resistance in a second luminal breast cancer cell line: T47D. Knocking down USP9X in T47D cells enabled cell growth in the presence of tamoxifen as well, suggesting that USP9X suppression leads to tamoxifen resistance independent of the cellular context (Supplementary Fig. S1). Importantly, knockdown of USP9X did not rescue cells from a proliferation arrest induced by the ER downregulator fulvestrant, illustrating that shUSP9X effects on cell proliferation are ERα-dependent (Fig. 1F).

Figure 1.

The shRNA screen identifies USP9X involvement in tamoxifen resistance. A, schematic outline of the screen. ZR-75-1 cells stably expressing murine ecotropic receptor were infected with retroviral supernatants containing a selection of the NKi pRS–shRNA library or pRS as control. After selection cells were cultured in DMEM with 1 μmol/L 4OH-tamoxifen for 6 weeks. Tamoxifen-resistant individual colonies were isolated, identifying an shRNA-targeting USP9X. B, knockdown of USP9X rescues tamoxifen-induced growth arrest. ZR-75-1 cells were infected with the USP9X shRNA recovered from the initial screen or pRS–GFP as control. Cells were cultured for 6 weeks in the presence of 1 μmol/L 4OH-tamoxifen or 1 nmol/L estradiol, photographed, fixed, and stained. Plate confluency was determined by calculating the number of cell-covered pixels related to the total surface area. C, USP9X hit validation. ZR-75-1 cells were infected with five independent shRNAs, targeting different regions of the USP9X gene and grown in the presence of 4OH-tamoxifen. Rescue from tamoxifen-induced growth arrest by USP9X knockdown was validated by three independent shRNAs. D, knockdown of USP9X decreases USP9X mRNA levels. E, knockdown of USP9X decreases USP9X protein levels. F, knockdown of USP9X does not rescue fulvestrant-induced growth arrest. ZR-75-1 cells were infected with shUSP9X or shGFP as control. Cells were cultured for 3 weeks in the presence of 1 μmol/L 4OH-tamoxifen or 10−7 mol/L fulvestrant. When colonies appeared, cells were photographed, fixed, and stained. Cell number was measured by crystal violet extraction.

Figure 1.

The shRNA screen identifies USP9X involvement in tamoxifen resistance. A, schematic outline of the screen. ZR-75-1 cells stably expressing murine ecotropic receptor were infected with retroviral supernatants containing a selection of the NKi pRS–shRNA library or pRS as control. After selection cells were cultured in DMEM with 1 μmol/L 4OH-tamoxifen for 6 weeks. Tamoxifen-resistant individual colonies were isolated, identifying an shRNA-targeting USP9X. B, knockdown of USP9X rescues tamoxifen-induced growth arrest. ZR-75-1 cells were infected with the USP9X shRNA recovered from the initial screen or pRS–GFP as control. Cells were cultured for 6 weeks in the presence of 1 μmol/L 4OH-tamoxifen or 1 nmol/L estradiol, photographed, fixed, and stained. Plate confluency was determined by calculating the number of cell-covered pixels related to the total surface area. C, USP9X hit validation. ZR-75-1 cells were infected with five independent shRNAs, targeting different regions of the USP9X gene and grown in the presence of 4OH-tamoxifen. Rescue from tamoxifen-induced growth arrest by USP9X knockdown was validated by three independent shRNAs. D, knockdown of USP9X decreases USP9X mRNA levels. E, knockdown of USP9X decreases USP9X protein levels. F, knockdown of USP9X does not rescue fulvestrant-induced growth arrest. ZR-75-1 cells were infected with shUSP9X or shGFP as control. Cells were cultured for 3 weeks in the presence of 1 μmol/L 4OH-tamoxifen or 10−7 mol/L fulvestrant. When colonies appeared, cells were photographed, fixed, and stained. Cell number was measured by crystal violet extraction.

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Knockdown of USP9X increases ERα activity through direct binding

Next, we examined whether the rescue from tamoxifen-induced proliferation arrest was the result of increased ERα signaling. ZR-75-1 cells stably expressing pRS–USP9X or control pRS–GFP were transiently transfected with an estrogen-responsive luciferase reporter (ERE-luciferase). First, we tested whether knockdown of USP9X increased ERα activity under the conditions used in the shRNA screen. Figure 2A shows that USP9X knockdown (USP9XKD) cells have increased luciferase activity, both when cultured in normal culture media (DMEM/FCS) and when cultured in the presence of 4OH-tamoxifen. Next, we performed luciferase assays on cells grown in phenol red-free DMEM, supplemented with 10% charcoal-stripped (and hence steroid-free) serum in the presence of vehicle, estradiol, 4OH-tamoxifen, or a combination of estradiol + 4OH-tamoxifen. Figure 2B shows that under all these conditions ERα signaling is about 2.5 times higher in the USP9XKD cell line as compared with the control cell line. To confirm the stimulating effect of USP9X knockdown on ERα signaling, ZR-75-1 cells, stably expressing pRS–USP9X or pRS–GFP, cultured in the presence of indicated ligand, were analyzed for ERα target gene expression. As shown in Fig. 2C and D, knockdown of USP9X resulted in increased mRNA (Fig. 2C) and protein levels (Fig. 2D) of PR, Trefoil factor 1 (TFF1/PS2), and of ERα itself (42).

Figure 2.

Knockdown of USP9X increases ERα activity. A, USP9XKD cells show increased ERE luciferase reporter activity in serum-supplemented DMEM in the absence and presence of 4OH-tamoxifen. Data are representative of three independent experiments. B, knockdown of USP9X increases ERE luciferase activity in hormone-deprived, estradiol- and 4OH-tamoxifen–treated cells. Data are representative of three independent experiments. C, USP9X knockdown in the presence of estradiol increases mRNA levels of the ERα target genes PGR, TFF1, and ERα. Data are representative of three independent experiments. D, knockdown of USP9X increases ERα and PR protein levels in hormone-deprived, estradiol- or 4OH-tamoxifen–treated cells.

Figure 2.

Knockdown of USP9X increases ERα activity. A, USP9XKD cells show increased ERE luciferase reporter activity in serum-supplemented DMEM in the absence and presence of 4OH-tamoxifen. Data are representative of three independent experiments. B, knockdown of USP9X increases ERE luciferase activity in hormone-deprived, estradiol- and 4OH-tamoxifen–treated cells. Data are representative of three independent experiments. C, USP9X knockdown in the presence of estradiol increases mRNA levels of the ERα target genes PGR, TFF1, and ERα. Data are representative of three independent experiments. D, knockdown of USP9X increases ERα and PR protein levels in hormone-deprived, estradiol- or 4OH-tamoxifen–treated cells.

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Given the functional interaction between USP9X and ERα, we next tested whether ERα and USP9X physically interact. Figure 3 shows that ERα coimmunoprecipitates with USP9X in estradiol- and tamoxifen-treated ZR-75-1 cells, demonstrating the existence of a physical complex of these proteins under physiologic conditions, which was recently also shown using mass spectrometry by Stanisic and colleagues (43).

Figure 3.

Physical interaction between USP9X and ERα. Hormone-deprived ZR-75-1 cells were treated with either E2 or 4-OHT. Immunoprecipitations were performed with nonimmune serum (ni, lanes 3 and 6), anti-ERα (lanes 4 and 7), or anti-USP9X (lanes 5 and 8) antibodies. Western blots were incubated with ERα and USP9X antibodies. Lanes 1 and 2 show 5% input of the whole-cell lysate.

Figure 3.

Physical interaction between USP9X and ERα. Hormone-deprived ZR-75-1 cells were treated with either E2 or 4-OHT. Immunoprecipitations were performed with nonimmune serum (ni, lanes 3 and 6), anti-ERα (lanes 4 and 7), or anti-USP9X (lanes 5 and 8) antibodies. Western blots were incubated with ERα and USP9X antibodies. Lanes 1 and 2 show 5% input of the whole-cell lysate.

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USP9X loss selectively enhances ERα/chromatin interactions upon 4OH-tamoxifen treatment

Knockdown of USP9X gave rise to both tamoxifen resistance and ERα-responsive gene activation. Next, the effects of USP9X knockdown on ERα/chromatin interactions were tested for hormone-depleted (vehicle), estradiol and tamoxifen conditions, using ChIP, followed by high-throughput sequencing (ChIP-seq). ZR-75-1 cells stably expressing pRS–USP9X or pRS–GFP (control) were plated in hormone-depleted medium for 72 hours. Typically, ERα ChIP-seq experiments are performed after a treatment for 45 minutes with ligand (44, 45), even though 3 hours of treatment is also used (46, 47). Because USP9X suppression causes long-term resistance to tamoxifen, we studied ERα biology after continued ligand treatment and the effects of USP9X knockdown thereon. Therefore, the cells were treated with vehicle, estradiol or 4OH-tamoxifen for 48 hours before the ChIP assay. In control cells, estradiol treatment greatly enhanced ERα/chromatin interactions, although this was far less pronounced when treating the cells with 4OH-tamoxifen. USP9X knockdown had no effect on ERα/chromatin interactions in vehicle- and estradiol-treated cells, but significantly increased chromatin binding upon 4OH-tamoxifen treatment, as exemplified in Fig. 4A. Enhanced ERα/chromatin interactions under tamoxifen conditions were also observed when cells were treated for 45 minutes (Supplementary Fig. S2). The stabilization of ERα/chromatin interactions in the presence of 4OH-tamoxifen could be generalized throughout the genome, as illustrated by visualizing the union of all peaks found for shGFP (5,855 sites) and shUSP9X (6,828 sites) in a heatmap (Fig. 4B) and expressed in a quantified format in a 2D graph (Fig. 4C). This increased intensity of ERα/chromatin interactions in 4OH-tamoxifen–treated cells also translated into a significant increase in the number of chromatin-binding events, representing a subset of the estradiol-induced binding patterns under the same conditions (Fig. 4D). The subgroups of binding sites for control cells and USP9XKD cells, either or not shared between ligand conditions (Fig. 4D), were separately analyzed in a heatmap visualization and quantified (Supplementary Fig. S3).

Figure 4.

USP9X loss selectively enhances ERα/chromatin interactions upon 4OH-tamoxifen treatment. Hormone-deprived monoclonal ZR-75-1 cells, stably expressing pRS–USP9X or pRS–GFP as control, were treated with vehicle (veh), estradiol (E2), or 4OH-tamoxifen (4-OHT), after which ChIP-seq analysis was performed on ERα. A, ERα ChIP-seq signal in shGFP control cells (blue) and shUSP9X cells (red) in the presence of indicated ligand. Tag counts (y-axis) and genomic locations (x-axis) are indicated. B, heatmap visualization, depicting a vertical alignment of all identified peaks of control (shGFP, 5,855 sites, blue) and USP9XKD (shUSP9X, 6,828 sites, red) raw read counts of veh, E2, or 4-OHT–treated cells. Arrowhead, top of the peak and scale bar is indicated. C, read-count quantification of data presented in B showing enrichment of ERα/chromatin interactions in the presence of 4-OHT in the shUSP9X cells compared with the control (shGFP) cells. y-axis, average tag count (arbitrary units). x-axis, distance from center of the peak (−2.5 kb, +2.5 kb). D, Venn diagrams showing a significant increase in the number of ERα/chromatin–binding events in the shUSP9X compared with control shGFP cells in the presence of 4-OHT, representing a subset of the E2-induced binding patterns. Numbers, binding events in each subgroup (veh, blue; E2, red; 4-OHT, green). E, Venn diagrams showing shared and unique peaks for control cells (blue) and shUSP9X cells (red) under vehicle, E2, and 4-OHT conditions. Numbers, binding events in each subgroup. F, genomic distributions of peaks under all tested conditions. Locations are indicated relative to the most proximal genes. 4-OHT shUSP9X unique, unique binding sites in tamoxifen-treated shUSP9X cells compared with shGFP control cells. G, de novo motif enrichment analysis identified ESR motifs enriched for 4-OHT shUSP9X unique peaks and peaks shared between 4-OHT–treated shGFP and shUSP9X cells. A P value for ESR1 motif enrichment was 690.77 (−10 log) for both situations.

Figure 4.

USP9X loss selectively enhances ERα/chromatin interactions upon 4OH-tamoxifen treatment. Hormone-deprived monoclonal ZR-75-1 cells, stably expressing pRS–USP9X or pRS–GFP as control, were treated with vehicle (veh), estradiol (E2), or 4OH-tamoxifen (4-OHT), after which ChIP-seq analysis was performed on ERα. A, ERα ChIP-seq signal in shGFP control cells (blue) and shUSP9X cells (red) in the presence of indicated ligand. Tag counts (y-axis) and genomic locations (x-axis) are indicated. B, heatmap visualization, depicting a vertical alignment of all identified peaks of control (shGFP, 5,855 sites, blue) and USP9XKD (shUSP9X, 6,828 sites, red) raw read counts of veh, E2, or 4-OHT–treated cells. Arrowhead, top of the peak and scale bar is indicated. C, read-count quantification of data presented in B showing enrichment of ERα/chromatin interactions in the presence of 4-OHT in the shUSP9X cells compared with the control (shGFP) cells. y-axis, average tag count (arbitrary units). x-axis, distance from center of the peak (−2.5 kb, +2.5 kb). D, Venn diagrams showing a significant increase in the number of ERα/chromatin–binding events in the shUSP9X compared with control shGFP cells in the presence of 4-OHT, representing a subset of the E2-induced binding patterns. Numbers, binding events in each subgroup (veh, blue; E2, red; 4-OHT, green). E, Venn diagrams showing shared and unique peaks for control cells (blue) and shUSP9X cells (red) under vehicle, E2, and 4-OHT conditions. Numbers, binding events in each subgroup. F, genomic distributions of peaks under all tested conditions. Locations are indicated relative to the most proximal genes. 4-OHT shUSP9X unique, unique binding sites in tamoxifen-treated shUSP9X cells compared with shGFP control cells. G, de novo motif enrichment analysis identified ESR motifs enriched for 4-OHT shUSP9X unique peaks and peaks shared between 4-OHT–treated shGFP and shUSP9X cells. A P value for ESR1 motif enrichment was 690.77 (−10 log) for both situations.

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Comparing control with USP9XKD under the same ligand conditions showed gained sites both for estradiol and 4OH-tamoxifen conditions, whereas this was not the case for vehicle-treated cells (Fig. 4E). ERα rarely binds promoters (5%), and the vast majority of ERα-binding events is found at distal enhancers (44). We could confirm these data for estradiol and 4OH-tamoxifen conditions, both in control and USP9XKD cells (Fig. 4F). Vehicle-treated cells showed enrichment of ERα binding to promoters as was found before (46), which was not influenced by knockdown of USP9X. The gained ERα-binding events for USP9XKD cells under tamoxifen conditions showed identical distributions as found for estradiol- and tamoxifen-treated control cells. De novo DNA motif enrichment analyses for ERα-binding sites in 4OH-tamoxifen–treated cells provided ESR motifs, both for sites shared between control cells and USP9XKD cells as well as for the sites selectively induced by USP9X knockdown (Fig. 4G).

Collectively, these data show that USP9X knockdown induces ERα-binding events, selectively in the presence of 4OH-tamoxifen, that represent a subset of estradiol-induced sites and do not deviate from normal ERα behavior with respect to genomic distributions and DNA motif enrichment.

USP9X and global gene-expression analyses

Our ChIP-seq analyses indicate that USP9X knockdown selectively enhances ERα/chromatin interactions in the presence of tamoxifen that are normally found enriched for estradiol conditions. We, therefore, tested whether USP9X knockdown in tamoxifen-treated cells would also give rise to a typical estradiol-responsive gene set. To address this, we performed RNA-seq on ZR-75-1 cells stably expressing pRS–USP9X or pRS–GFP (control) that—after hormone depletion for 72 hours—were treated for 48 hours with vehicle, estradiol, or 4OH-tamoxifen. Estradiol treatment resulted in altered expression of 8,794 genes as compared with vehicle, whereas after 4OH-tamoxifen treatment 1,906 genes were differentially expressed. All altered transcripts under 4OH-tamoxifen conditions represented a subset of the estradiol-responsive genes (Fig. 5A, left). 4OH-tamoxifen treatment in USP9XKD cells as compared with 4OH-tamoxifen–treated control cells resulted in an altered expression of 6,210 transcripts, 4,336 of which were shared with estradiol induction in control cells (Fig. 5A, right). Furthermore, integrating these differentially expressed genes in 4OH-tamoxifen–treated USP9XKD cells with the ChIP-seq data showed that a subgroup of these genes, 526 of 4,336 genes (Fig. 5B), is enriched for proximal ERα-binding events, with a chromatin-binding event within 20 kb from the TSS. This window of 20 kb represents the optimal window to identify ERα-responsive genes (48). This is a selective enrichment over the total genomic background, in which 3,001 of all 45,054 RefSeq genes were found to have a proximal ERα-binding event (Fisher exact test; P = 8.099E−30). Analyzing the raw read-count of the ChIP-seq experiments under 4OH-tamoxifen showed that also the ChIP-seq signal intensity of proximal ERα-binding events was selectively increased after USP9X knockdown (Fig. 5C).

Figure 5.

USP9X and global gene expression analyses. Hormone-deprived monoclonal ZR-75-1 cells stably expressing pRS–USP9X or pRS–GFP as control were treated with vehicle (veh), estradiol (E2), or 4OH-tamoxifen (4-OHT), after which RNA-seq analysis was performed. A, left, Venn diagram showing differentially expressed genes in control cells after treatment with E2 (blue) or 4-OHT (green) compared with vehicle control (P < 0.05). Right, Venn diagram showing differentially expressed genes of E2- versus vehicle-treated control cells (blue) and differentially expressed genes of 4-OHT–treated shUSP9X cells compared with 4-OHT–treated shGFP control cells (red). B, proximal ERα-binding events for the 4,436 differentially expressed, estradiol-regulated genes in 4-OHT–treated shUSP9X cells. ERα-binding events found in 4-OHT–treated control cells (left), 4-OHT–treated shUSP9X cells (middle), or shared between both conditions (right) were analyzed for proximal binding (<20 kb) to TSSs of differentially expressed genes in 4-OHT–treated shUSP9X cells. The y-axis shows absolute number of differentially expressed genes. C, average ERα read-count intensity of ERα chromatin–binding sites in 4-OHT–treated shUSP9X cells compared with shGFP control cells, proximal to (<20 kb) TSS regions of 526 genes, differential expressed between 4-OHT–treated shUSP9X cells and 4-OHT–treated control cells. y-axis, average read-count (a.u.). x-axis, distance from center of the peak (−2.5 kb, +2.5 kb). D, heatmap showing differentially expressed genes between 250 patients with primary ERα-positive breast cancer who received adjuvant tamoxifen. E, A USP9X knockdown tamoxifen gene signature identifies primary ERα-positive breast cancer patients with poor outcome after adjuvant tamoxifen treatment. Kaplan–Meier survival curves for DMFS of patients with primary ERα-positive breast cancer treated with adjuvant tamoxifen in the Loi and colleagues cohort (n = 250; first; ref. 35) and in the Buffa and colleagues cohort (n = 134; second; ref. 36). Kaplan–Meier survival curves for DMFS of patients with primary ERα-positive breast cancer that did not receive adjuvant endocrine treatment in the Wang and colleagues cohort (n = 209; third; ref. 37) and in the Schmidt and colleagues cohort (n = 158; fourth; ref. 38). F, distribution of the PAM50 molecular subtypes over the USP9X knockdown tamoxifen gene-expression signature-sensitive and -resistant groups.

Figure 5.

USP9X and global gene expression analyses. Hormone-deprived monoclonal ZR-75-1 cells stably expressing pRS–USP9X or pRS–GFP as control were treated with vehicle (veh), estradiol (E2), or 4OH-tamoxifen (4-OHT), after which RNA-seq analysis was performed. A, left, Venn diagram showing differentially expressed genes in control cells after treatment with E2 (blue) or 4-OHT (green) compared with vehicle control (P < 0.05). Right, Venn diagram showing differentially expressed genes of E2- versus vehicle-treated control cells (blue) and differentially expressed genes of 4-OHT–treated shUSP9X cells compared with 4-OHT–treated shGFP control cells (red). B, proximal ERα-binding events for the 4,436 differentially expressed, estradiol-regulated genes in 4-OHT–treated shUSP9X cells. ERα-binding events found in 4-OHT–treated control cells (left), 4-OHT–treated shUSP9X cells (middle), or shared between both conditions (right) were analyzed for proximal binding (<20 kb) to TSSs of differentially expressed genes in 4-OHT–treated shUSP9X cells. The y-axis shows absolute number of differentially expressed genes. C, average ERα read-count intensity of ERα chromatin–binding sites in 4-OHT–treated shUSP9X cells compared with shGFP control cells, proximal to (<20 kb) TSS regions of 526 genes, differential expressed between 4-OHT–treated shUSP9X cells and 4-OHT–treated control cells. y-axis, average read-count (a.u.). x-axis, distance from center of the peak (−2.5 kb, +2.5 kb). D, heatmap showing differentially expressed genes between 250 patients with primary ERα-positive breast cancer who received adjuvant tamoxifen. E, A USP9X knockdown tamoxifen gene signature identifies primary ERα-positive breast cancer patients with poor outcome after adjuvant tamoxifen treatment. Kaplan–Meier survival curves for DMFS of patients with primary ERα-positive breast cancer treated with adjuvant tamoxifen in the Loi and colleagues cohort (n = 250; first; ref. 35) and in the Buffa and colleagues cohort (n = 134; second; ref. 36). Kaplan–Meier survival curves for DMFS of patients with primary ERα-positive breast cancer that did not receive adjuvant endocrine treatment in the Wang and colleagues cohort (n = 209; third; ref. 37) and in the Schmidt and colleagues cohort (n = 158; fourth; ref. 38). F, distribution of the PAM50 molecular subtypes over the USP9X knockdown tamoxifen gene-expression signature-sensitive and -resistant groups.

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A USP9X knockdown tamoxifen gene-expression signature identifies patients with breast cancer with a poor outcome after adjuvant tamoxifen treatment

The RNA-seq analyses revealed that the majority of genes that were differentially expressed upon tamoxifen treatment in the USP9XKD cells represented a subgroup of estradiol-induced genes (4,336 of 8,794 genes; see Fig. 5A, right), of which 526 of 4,336 genes with a proximal ERα-binding site (Fig. 5B) are expected to be under the direct control of ERα under 4OH-tamoxifen conditions. This particular subgroup of genes most likely represents a direct ERα target gene signature in contrast with the (potentially indirectly regulated) genes that were not enriched for ERα binding. Because these directly ERα-regulated genes would also be the genes that are directly affected under tamoxifen-resistant conditions, differential expression of these particular genes in breast tumors could hallmark tamoxifen unresponsiveness. To test this hypothesis, we investigated whether these genes were differentially expressed in a publically available dataset of 250 predominantly postmenopausal patients with primary ERα-positive breast cancer with known outcome (35). All these patients received adjuvant tamoxifen. For all clinicopathologic parameters, see Supplementary Table S3. As visualized in a heatmap (Fig. 5D), unsupervised clustering on the basis of our gene signature resulted in the identification of two distinct subgroups of patients. These subgroups of patients were subsequently analyzed for differential distant metastasis-free survival (DMFS) after adjuvant tamoxifen treatment. Figure 5E, first, shows that this gene set identifies a subgroup of patients with breast cancer with a poor response to tamoxifen treatment (P = 9.4 × 10−5). These data could be validated using a second cohort of 134 mostly postmenopausal patients with ERα-positive breast cancer treated with adjuvant tamoxifen (Fig. 5E, second; P = 7.3 × 10−3; ref. 36). We then tested our signature on two cohorts of mainly postmenopausal patients with ERα-positive breast cancer (37, 38) who did not receive any adjuvant endocrine treatment. Importantly, in these patients the USP9X knockdown tamoxifen gene-expression signature did not correlate with outcome, indicating that the gene signature is not prognostic (Fig. 5E, right). Patients identified as responsive, based on our USP9X-based gene signature were mostly of the luminal A subtype, whereas resistant-classified patients were more often of the luminal B subtype, as identified by PAM50 (Fig. 5F; refs. 40, 41). However, still approximately 30% of luminal B tumors were classified as tamoxifen sensitive and approximately 30% of luminal A tumors were classified as tamoxifen resistant, suggesting that our gene signature can be used to identify subgroups of luminal A and luminal B tumors that benefit most from adjuvant tamoxifen treatment. The gene signature was selective for endocrine treatment, because our signature was not able to classify patients with ERα-positive tumors treated with adjuvant chemotherapy only, albeit this group of patients was small (Supplementary Fig. S6; ref. 39).

The majority of genes that are differentially expressed upon tamoxifen treatment in the USP9XKD cells were shared with estradiol induction. However, 1,874 of the differentially expressed genes were not estrogen affected (Fig. 5A, right). These 1,874 genes were not linked with estrogen function or endocrine resistance, but ingenuity pathway analysis enrichment and gene ontology analysis showed enrichment for mitochondrial function (Supplementary Fig. S4A and S4B). Expression-based gene signatures for these 1,874 genes did not classify patients in the Buffa and colleagues cohort [P = 0.25; HR, 1.4; 95% confidence interval (CI), 0.8–2.4; ref. 36], whereas our shUSP9X-based gene signature did (P = 6.5 ×10−4; HR, 4.0; 95% CI, 1.7–9.3; Supplementary Fig. S5A and S5B, right). Significance was reached with the Loi and colleagues series (P = 0.035; HR, 2.0; 95% CI, 1.2–3.5; Supplementary Fig. S5A, left; ref. 35), which was outperformed by our shUSP9X-based gene signature using the same cohort, even though the confidence intervals did overlap (P = 9.4 × 10−5; HR, 3.1; 95% CI, 1.9–5.2; Supplementary Fig. S5B, left).

Cumulatively, we found USP9X knockdown to induce tamoxifen resistance in cell lines, and could apply a downstream gene signature to identify tamoxifen-treated patients with breast cancer with a poor response to therapy.

We identify here a role for USP9X in regulation of the response to tamoxifen in ERα-positive breast cancer. USP9X affects the stability and activity of numerous regulatory proteins that influence cell survival. Earlier publications had attributed a prosurvival role to USP9X in B- and mantle cell lymphomas, chronic myeloid leukemia, and multiple myeloma, through stabilization of MCL1 (49). USP9X can also act as a tumor suppressor and plays a role in oxidative stress–induced cell death through ASK1 (apoptosis signal–regulating kinase 1; ref. 18). In addition, USP9X was identified as a tumor-suppressor gene in pancreatic cancer (50).

The findings presented here establish a novel role for USP9X acting as a mediator of the response to tamoxifen in ERα-positive breast cancer. USP9X knockdown stabilizes ERα–chromatin interactions, enables tamoxifen-induced ERα activation, and stimulates ERα-responsive cell proliferation in the presence of tamoxifen. A gene-expression signature composed of tamoxifen-responsive genes in shRNA USP9X cells can identify patients with breast cancer with a poor response to tamoxifen treatment. The tumors identified as tamoxifen-responsive through the USP9X signature are more often of the luminal A type, whereas the group with the resistant signature shows enrichment for luminal B-type tumors. Still our gene signature is not merely providing a distinction on the basis of the molecular subtype, because approximately 30% of the tumors classified as tamoxifen-resistant were luminal A and a comparable percentage of the tamoxifen-sensitive tumors were luminal B type. This finding is potentially of clinical relevance, as the data indicate that our gene signature can identify luminal A-type tumors that respond poorly to tamoxifen treatment and might benefit from alternative treatment regimes. Importantly, USP9X knockdown did not induce fulvestrant resistance, thus providing an alternative treatment option for patients classified as tamoxifen-resistant through our signature.

Although USP9X physically interacts with ERα, as was shown before (43), we were not able to show any deubiquitinating activity of USP9X toward ERα. We hypothesize that the role of USP9X in ERα signaling lies in regulating its interactions with chromatin, potentially facilitating cofactor recruitment and/or deubiquitination of such ERα cofactors.

The USP9X knockdown tamoxifen gene-expression signature we identify here enables the identification of patients with breast cancer with a poor response to adjuvant tamoxifen treatment. Because USP9X knockdown directly affects the biology of ERα, other modes of tamoxifen resistance might be recapitulated by the same gene signature, broadening the potential applicability of our findings. Importantly, evaluation of USP9X levels alone did not enable such a patient stratification (data not shown). This is not an unexpected finding given that USP9X has many cellular functions, and the identification of our distinct gene-expression signature enabled us to exclusively study the consequences of USP9X loss on ERα function and tamoxifen response.

Our shUSP9X-based gene signature was generated on tamoxifen-treated cells in vitro, and was subsequently applied on gene-expression data from tumor samples obtained before endocrine therapy. This demonstrated that the signature is capable of identifying intrinsic tamoxifen resistance in vivo. Future clinical studies on tumor specimens collected pre- and post-neoadjuvant tamoxifen treatment are required to further investigate directionality of the differentially expressed genes before and after tamoxifen treatment in relation to treatment response.

In summary, we report here an unexpected role for USP9X in ERα-positive breast cancer, as USP9X loss induces tamoxifen-stimulatory effects on ERα action, leading to tamoxifen resistance. Furthermore, we show that a USP9X knockdown tamoxifen gene expression signature can be used as a potential biomarker to identify patients with ERα-positive breast cancer with a poor outcome after tamoxifen treatment.

No potential conflicts of interest were disclosed.

Conception and design: H.M. Oosterkamp, E.M. Hijmans, W. Zwart, R. Bernards

Development of methodology: H.M. Oosterkamp, E.M. Hijmans, R. Bernards

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): H.M. Oosterkamp, E.M. Hijmans, T.R. Brummelkamp, W. Zwart

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): H.M. Oosterkamp, E.M. Hijmans, T.R. Brummelkamp, S. Canisius, W. Zwart, R. Bernards

Writing, review, and/or revision of the manuscript: H.M. Oosterkamp, E.M. Hijmans, L.F.A. Wessels, W. Zwart, R. Bernards

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): H.M. Oosterkamp, E.M. Hijmans

Study supervision: E.M. Hijmans, L.F.A. Wessels

The authors thank Ron Kerkhoven, Iris de Rink, and Mandy Madiredjo for their assistance.

This work was supported by grants from the Dutch Cancer Society (KWF).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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Supplementary data