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• W2887861528 abstract "•ESR1 fusions drive ligand-independent growth and endocrine therapy resistance•ESR1 fusions reprogram the ER cistrome to drive EMT and metastasis•CDK4/6 inhibition suppresses ESR1 fusion-induced growth RNA sequencing (RNA-seq) detects estrogen receptor alpha gene (ESR1) fusion transcripts in estrogen receptor-positive (ER+) breast cancer, but their role in disease pathogenesis remains unclear. We examined multiple ESR1 fusions and found that two, both identified in advanced endocrine treatment-resistant disease, encoded stable and functional fusion proteins. In both examples, ESR1-e6>YAP1 and ESR1-e6>PCDH11X, ESR1 exons 1–6 were fused in frame to C-terminal sequences from the partner gene. Functional properties include estrogen-independent growth, constitutive expression of ER target genes, and anti-estrogen resistance. Both fusions activate a metastasis-associated transcriptional program, induce cellular motility, and promote the development of lung metastasis. ESR1-e6>YAP1- and ESR1-e6>PCDH11X-induced growth remained sensitive to a CDK4/6 inhibitor, and a patient-derived xenograft (PDX) naturally expressing the ESR1-e6>YAP1 fusion was also responsive. Transcriptionally active ESR1 fusions therefore trigger both endocrine therapy resistance and metastatic progression, explaining the association with fatal disease progression, although CDK4/6 inhibitor treatment is predicted to be effective. RNA sequencing (RNA-seq) detects estrogen receptor alpha gene (ESR1) fusion transcripts in estrogen receptor-positive (ER+) breast cancer, but their role in disease pathogenesis remains unclear. We examined multiple ESR1 fusions and found that two, both identified in advanced endocrine treatment-resistant disease, encoded stable and functional fusion proteins. In both examples, ESR1-e6>YAP1 and ESR1-e6>PCDH11X, ESR1 exons 1–6 were fused in frame to C-terminal sequences from the partner gene. Functional properties include estrogen-independent growth, constitutive expression of ER target genes, and anti-estrogen resistance. Both fusions activate a metastasis-associated transcriptional program, induce cellular motility, and promote the development of lung metastasis. ESR1-e6>YAP1- and ESR1-e6>PCDH11X-induced growth remained sensitive to a CDK4/6 inhibitor, and a patient-derived xenograft (PDX) naturally expressing the ESR1-e6>YAP1 fusion was also responsive. Transcriptionally active ESR1 fusions therefore trigger both endocrine therapy resistance and metastatic progression, explaining the association with fatal disease progression, although CDK4/6 inhibitor treatment is predicted to be effective. The etiology of endocrine therapy resistance in estrogen receptor-positive (ER+) breast cancer is complex (Ma et al., 2015Ma C.X. Reinert T. Chmielewska I. Ellis M.J. Mechanisms of aromatase inhibitor resistance.Nat. Rev. Cancer. 2015; 15: 261-275Crossref PubMed Scopus (209) Google Scholar) but includes acquired somatic mutations within the ligand-binding domain (LBD) of the estrogen receptor gene (ESR1) causing ligand-independent activation (Pejerrey et al., 2018Pejerrey S.M. Dustin D. Kim J.A. Gu G. Rechoum Y. Fuqua S.A.W. The Impact of ESR1 mutations on the treatment of metastatic breast cancer.Horm. Cancer. 2018; 43: 413Google Scholar). RNA sequencing (RNA-seq) has also identified multiple ESR1 gene fusion events, but their role in endocrine therapy resistance and how they might be targeted therapeutically is unclear (Giltnane et al., 2017Giltnane J.M. Hutchinson K.E. Stricker T.P. Formisano L. Young C.D. Estrada M.V. Nixon M.J. Du L. Sanchez V. Ericsson P.G. et al.Genomic profiling of ER(+) breast cancers after short-term estrogen suppression reveals alterations associated with endocrine resistance.Sci. Transl. Med. 2017; 9: eaai7993Crossref PubMed Scopus (49) Google Scholar). The majority of ESR1 fusion transcripts have been identified in primary breast cancer, and in some of these instances patients have high-grade disease and/or resistance to endocrine therapy (Giltnane et al., 2017Giltnane J.M. Hutchinson K.E. Stricker T.P. Formisano L. Young C.D. Estrada M.V. Nixon M.J. Du L. Sanchez V. Ericsson P.G. et al.Genomic profiling of ER(+) breast cancers after short-term estrogen suppression reveals alterations associated with endocrine resistance.Sci. Transl. Med. 2017; 9: eaai7993Crossref PubMed Scopus (49) Google Scholar, Veeraraghavan et al., 2014Veeraraghavan J. Tan Y. Cao X.-X. Kim J.A. Wang X. Chamness G.C. Maiti S.N. Cooper L.J.N. Edwards D.P. Contreras A. et al.Recurrent ESR1-CCDC170 rearrangements in an aggressive subset of oestrogen receptor-positive breast cancers.Nat. Commun. 2014; 5: 4577Crossref PubMed Scopus (82) Google Scholar), implying some functionality. In some cases, up to five ESR1 coding exons are included (exons 3–7), mostly fused out of frame but occasionally, and more interestingly, in frame. However, detailed characterization of the predicted chimeric proteins and a clear demonstration of a causal role for ESR1 fusions in endocrine therapy resistance have been largely lacking. Several years ago, our group described an unequivocal stable and functional ESR1 fusion protein (Li et al., 2013Li S. Shen D. Shao J. Crowder R. Liu W. Prat A. He X. Liu S. Hoog J. Lu C. et al.Endocrine-therapy-resistant ESR1 variants revealed by genomic characterization of breast-cancer-derived xenografts.Cell Rep. 2013; 4: 1116-1130Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar). This was an in-frame fusion gene consisting of exons 1–6 of ESR1 fused to C-terminal sequences from the Hippo pathway coactivator YAP1 (ESR1-e6>YAP1), identified in a metastatic sample and matched patient-derived xenograft (PDX) from a patient with endocrine therapy-resistant disease. Limited functional characterization of ESR1-e6>YAP1 showed that the fusion protein drove resistance to endocrine therapy and estradiol-independent proliferation. Herein we build on our original report by contrasting the functional, transcriptional, and pharmacological properties of the ESR1-e6>YAP1 fusion with additional ESR1 gene fusion events identified by RNA-seq of both early-stage and metastatic ER+ breast cancers. A systematic screen was conducted to identify ESR1 translocations in three datasets: 728 primary breast tumors from The Cancer Genome Atlas (TCGA) (Ciriello et al., 2015Ciriello G. Gatza M.L. Beck A.H. Wilkerson M.D. Rhie S.K. Pastore A. Zhang H. McLellan M. Yau C. Kandoth C. et al.TCGA Research NetworkComprehensive molecular portraits of invasive lobular breast cancer.Cell. 2015; 163: 506-519Abstract Full Text Full Text PDF PubMed Scopus (900) Google Scholar), 81 primary breast cancers from two neoadjuvant aromatase inhibitor (AI) clinical trials (Ellis et al., 2011Ellis M.J. Suman V.J. Hoog J. Lin L. Snider J. Prat A. Parker J.S. Luo J. DeSchryver K. Allred D.C. et al.Randomized phase II neoadjuvant comparison between letrozole, anastrozole, and exemestane for postmenopausal women with estrogen receptor-rich stage 2 to 3 breast cancer: clinical and biomarker outcomes and predictive value of the baseline PAM50-based intrinsic subtype—ACOSOG Z1031.J. Clin. Oncol. 2011; 29: 2342-2349Crossref PubMed Scopus (344) Google Scholar, Olson et al., 2009Olson J.A. Budd G.T. Carey L.A. Harris L.A. Esserman L.J. Fleming G.F. Marcom P.K. Leight G.S. Giuntoli T. Commean P. et al.Improved surgical outcomes for breast cancer patients receiving neoadjuvant aromatase inhibitor therapy: results from a multicenter phase II trial.J. Am. Coll. Surg. 2009; 208: 906-914Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar), and 25 biopsy samples from patients with late-stage ER+ breast cancer (Figure 1A). From these analyses, 13 high-confidence ESR1 fusion transcripts were identified in 10 ER+ samples from the TCGA dataset (Table S1). Five of these fusion events were between ESR1 and CCDC170 and were recently reported (Veeraraghavan et al., 2014Veeraraghavan J. Tan Y. Cao X.-X. Kim J.A. Wang X. Chamness G.C. Maiti S.N. Cooper L.J.N. Edwards D.P. Contreras A. et al.Recurrent ESR1-CCDC170 rearrangements in an aggressive subset of oestrogen receptor-positive breast cancers.Nat. Commun. 2014; 5: 4577Crossref PubMed Scopus (82) Google Scholar). Of these, only 1 CCDC170 out-of-frame fusion included exon 5 (e5) of ESR1 (ESR1-e5>CCDC170), thereby preserving sufficient ESR1 sequence to bind DNA. A single TCGA case displayed evidence for three ESR1 gene fusions: (1) a PCR-validated ESR1-e6 fused in frame to C-terminal sequences from AKAP12 (ESR1-e6>AKAP12) (Figure S1); (2) a PCR-validated in-frame ESR1-e7 fusion involving the entire coding sequence of POLH, a DNA polymerase in the xeroderma pigmentosum gene family (ESR1-e7>POLH), and (3) an out-of-frame ESR1-e4>CCDC170 fusion. From an RNA-seq screen of 81 primary, treatment-naive, ER+ breast cancers from two neoadjuvant AI clinical trials (Table S1, NeoAI Trials), two PCR-validated ESR1 fusions were identified. The first was an in-frame fusion retaining the first six exons of ESR1 (ESR1-e6) fused to C-terminal sequences of NOP2, a nucleolar protein (ESR1-e6>NOP2). The second fusion identified involved ESR1-e6 fused out of frame to AKR1D1, an aldo-keto reductase family member (ESR1-e6>AKR1D1). In the datasets of primary ER+ breast cancer examined, ESR1 fusion events are relatively rare, occurring at ∼2% frequency. The majority of these fusions are out of frame, and 42% of these fusion events (8 of 19) include sufficient ESR1 exons to allow ESR1-specific nuclear binding. To investigate ESR1 fusion events in late-stage ER+ disease, RNA-seq data from 25 biopsy samples obtained from patients with advanced endocrine therapy refractory disease were examined (Table S1, Late Stage, and Table S2). These samples included the ESR1-e6>YAP1 sample we originally described, as it was drawn from this series (Li et al., 2013Li S. Shen D. Shao J. Crowder R. Liu W. Prat A. He X. Liu S. Hoog J. Lu C. et al.Endocrine-therapy-resistant ESR1 variants revealed by genomic characterization of breast-cancer-derived xenografts.Cell Rep. 2013; 4: 1116-1130Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar), and of these 25 samples, 2 harbored in-frame ESR1 fusion events. The ESR1-e6>PCDH11X fusion was caused by ESR1-e6 fusion in frame with C-terminal sequences of protocadherin 11X. PCDH11X encodes for an atypical cell surface cadherin family member. The sample was a chest wall recurrence from a 49-year-old man who presented with locally advanced ER+ breast cancer and experienced progression on tamoxifen, letrozole/leuprolide, and fulvestrant before the sample was accrued. Of the eight identified ESR1 fusions from all datasets that were PCR validated (Figure S1), only three in-frame fusions, ESR1-e6>YAP1 and ESR1-e6>PCDH11X from advanced disease and ESR1-e6>NOP2 from a primary tumor that showed subsequent resistance to endocrine therapy, produced stable proteins when expressed as cDNA, allowing further study (Figure 1B). Expression of all three fusion partner genes were highly expressed in patient tumors, as shown by expression rank plots for YAP1, PCDH11X, and NOP2 translocation- bearing tumors relative to the expression of these genes among TCGA breast samples (Figure 1C). Relative RNA levels of transcripts were analyzed for each fusion partner, which showed increases in transcript levels beyond the fusion breakpoint for each gene examined, confirming that the fusion partner was disproportionately expressed versus the non-translocated allele (Figure 2A). To test whether examples of ESR1 in-frame gene fusions were drivers of endocrine therapy resistance, each fusion was individually expressed in two ER+ breast cancer cell line models: T47D and MCF7. Expression of fusion ER proteins in T47D cells was similar or lower than that observed in the WHIM18 PDX bearing the ESR1-e6>YAP1 fusion, indicating that phenotypic conclusions are not based on excess expression (Figure 2B). In addition, several out-of-frame CCDC170 and an AKR1D1 fusion event identified in this study (Table S1) were also engineered into T47D cells. Growth of ESR1 fusion-expressing T47D was monitored in estradiol (E2)-deprived media and after addition of E2. Both in-frame fusions from advanced disease, ESR1-e6>YAP1 and ESR1-e6>PCDH11X, promoted estrogen-independent growth (Figure 2C, −E2), but the primary tumor fusion event, ESR1-e6>NOP2, had no growth-promoting properties. The out-of-frame events tested were also inactive (Figure S2A). E2 could stimulate growth in all conditions of fusion construct expression (Figure 2C, compare +E2 and −E2), suggesting that neither the ESR1 in-frame active fusions (ESR1-e6>YAP1 and ESR1-e6>PCDH11X) nor the ESR1-e6 truncation, and not even the in-frame but inactive ESR1-e6>NOP2 fusion, could function as a dominant-negative on endogenous ER. Cells were treated with fulvestrant to degrade endogenous ER, while retaining expression of intact ESR1 fusions that cannot bind drug or ligand, to test the specific contribution of the fusions to E2-independent growth. As expected, endogenous ER was degraded by fulvestrant, whereas levels of ESR1 fusion proteins, as well as an ESR1-e6 truncation construct, were unaffected (Figure S2B), and growth promoted by ESR1-e6>YAP1 and ESR1-e6>PCDH11X was resistant to fulvestrant treatment (Figure 2C, −E2, +Fulvestrant). There was lack of additional growth promotion by the fusions when E2 was added in the presence of fulvestrant (Figure 2C, compare +E2, +Fulvestrant and −E2, +Fulvestrant). However, under these same conditions (Figure 2C, +E2, +Fulvestrant), growth induced by the YAP1 and PCDH11X fusions remains significantly greater than controls (YFP and ESR1-WT [wild-type]). These results were confirmed in a second ER+ breast cancer cell line, MCF7 (Figures S2C–S2D). The NOP2 fusion was highly expressed in the MCF7 cell line, in contrast to NOP2 fusion-expressing T47D, but still lacked growth-promoting activity in hormone-deprived conditions, confirming that absence of functional effects was not due to inadequate expression of the NOP2 fusion. The ability of the three ESR1-e6-containing in-frame fusions to induce estrogen-independent growth was further tested in vivo in a xenograft study with stable T47D cells without supplementary E2. As controls, T47D YFP cells were used with supplementary E2. Results showed that control YFP −E2 cells produced negligible tumor growth compared with YFP cells +E2 (Figure 2D). However, T47D cells expressing YAP1 and PCDH11X in-frame ESR1 fusions formed tumors significantly larger than YFP −E2, while the cells expressing the NOP2 fusion did not (Figure 2D). To explore transcriptional properties associated with the ESR1 fusion proteins described above, genome-wide binding of HA-tagged ESR1 fusions was examined by HA chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) in hormone-deprived stable T47D. ChIP-seq identified 445 binding regions shared by ESR1-WT, ESR1-e6>YAP1, and ESR1-e6>PCDH11X (Figure 3A). Very few sites were bound by ESR1-e6>NOP2 fusion despite high expression of HA-tagged NOP2 fusion (Figure S3E), supporting earlier observations of inactivity in functional studies (Figures 2C, 2D, and S2C). ChIP-qPCR confirmed recruitment of ER to regulatory regions of known estrogen-responsive genes in a ligand-dependent manner in cells expressing WT-ER (Figure 3B). Additionally, both YAP1 and PCDH11X fusions showed estrogen-independent enrichment at regulatory regions of established estrogen-responsive genes. For example, both fusions were enriched at the promoter of a canonical ER-regulated gene, GREB1, and the PDCH11X fusion was also enriched at enhancer estrogen response elements (EREs) of TFF1 and PGR (Figure 3B). To investigate whether expression from genes bound by ESR1 fusions was modulated, RNA-seq was performed. Hierarchical clustering was conducted on differentially expressed genes near 445 shared sites bound by ESR1-WT, YAP1, and PCDH11X fusions, as indicated by the ChIP-seq data (Figure 3C). Upon stimulation with E2, the expression pattern of YFP control cells clustered away from unstimulated YFP cells, with enrichment for differential expression of estrogen-responsive genes. The YAP1 and PCDH11X fusion-expressing cells had expression patterns that clustered together under estrogen-deprived and stimulated conditions and with E2-stimulated YFP cells. The transcriptionally active ESR1 fusions maintained expression of estrogen-regulated genes in low-estrogen conditions at levels observed in YFP control cells in the presence of E2, demonstrating strong estrogen-independent gene activation. mRNA-qPCR validation of GREB1, TFF1, and PGR expression confirmed estrogen-independent and fulvestrant-resistant gene regulation (Figures 3D and S3F), suggesting that the active ESR1 fusions drive endocrine resistance in a canonical manner through ERE-dependent activation. Moreover, the estrogen-independent activity of the YAP1 and PCDH11X fusions was also independent of endogenous WT-ER, as transcriptional activity was maintained after cells were treated with fulvestrant to degrade endogenous ER. Thus, functionally important heterodimer formation between ESR1 fusion protein and WT-ER is not likely. This conclusion was also supported by the lack of ESR1 fusion association with WT-ER in a co-immunoprecipitation assay (Figure S3D). In contrast, the ESR1-e6 truncation mutant and NOP2 fusion clustered together with YFP control cells displaying similar patterns of ligand-dependent ER gene expression, supporting our earlier observations that the NOP2 fusion lacks ability to bind a large repertoire of EREs but whose inactivity is not due to mis-localization outside the nucleus, as staining for HA-tagged ESR1 fusions constructs demonstrated nuclear localization (Figure S3A). These data were further supported by ERE-luciferase reporter experiments in HEK293T cells (Figure S3B). ESR1-WT drove estrogen-dependent expression of the ERE-luciferase reporter. In contrast, both ESR1-e6>YAP1 and ESR1-e6>PCDH11X as well as the ESR1-Y537S activating mutant drove estrogen-independent expression of the ERE-luciferase reporter. The level of activation by ESR1-e6>YAP1 was substantially higher than ESR1-e6> PCDH11X, which had activity intermediate to that achieved by the constitutively active ESR1-Y537S mutant and ESR1-e6>YAP1 (Li et al., 2013Li S. Shen D. Shao J. Crowder R. Liu W. Prat A. He X. Liu S. Hoog J. Lu C. et al.Endocrine-therapy-resistant ESR1 variants revealed by genomic characterization of breast-cancer-derived xenografts.Cell Rep. 2013; 4: 1116-1130Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar). In contrast to the ESR1-e6>YAP1 and ESR1-e6>PCDH11X fusions, neither the ESR1-e6 truncation mutation nor the ESR1-e6>NOP2 fusion drove expression of the ERE reporter. The transcriptional inactivity of the NOP2 fusion was not due to abrogation of ERE binding, as pull-down experiments with a biotinylated concatenated ERE probe with a mutant ERE as a control demonstrated sequence-specific binding for all in-frame fusions (Figure S3C). In summary, our observations suggest that the inactivity of the NOP2 fusion may be due to a failure to access chromatin in the nucleus of intact cells, rather than an inability to bind DNA per se. A cluster of genes was identified that was selectively upregulated by the active YAP1 and PCDH11X fusions (Figures 3C and 4A ). Gene set enrichment analysis (GSEA) was used to examine pathway enrichment in this cluster, which indicated significant enrichment of estrogen response pathways as well as an epithelial-to-mesenchymal transition (EMT)-like signature (Figure 4B). The EMT signature included TGM2, COL3A1, INHBA, and VCAN. One of the best-described EMT genes, SNAI1, was also selectively upregulated by both active fusions. Analysis of binding site distances to transcription start sites (TSSs) of genes in this cluster demonstrated that the majority of binding occurs at distances >50 kb from the TSS (Table S3). This suggests a propensity of the active YAP1 and PCDH11X fusions to bind in enhancer regions upstream and downstream of these genes, characteristic of the ER cistrome reported in the literature (Carroll et al., 2006Carroll J.S. Meyer C.A. Song J. Li W. Geistlinger T.R. Eeckhoute J. Brodsky A.S. Keeton E.K. Fertuck K.C. Hall G.F. et al.Genome-wide analysis of estrogen receptor binding sites.Nat. Genet. 2006; 38: 1289-1297Crossref PubMed Scopus (1046) Google Scholar). Motif analysis of these binding sites showed enrichment for the ERE motif (Figure S4A), suggesting that the direct regulation of EMT genes by the active YAP1 and PCDH11X fusions is mediated by enhancer and more distant range interactions. Upregulation of VCAN and SNAI1 transcripts (Figures 4C and S4B) and Snail protein (Figure 4D) was orthogonally validated. In MCF7 cells, whose basal levels of Snail were higher in YFP controls compared with T47D YFP, showed an induction of Snail by ESR1-e6>YAP1, but not by ESR1-e6>PCDH11X, suggesting a degree of cell context-dependent effects (Figure 4D). Upregulation of Snail protein was also confirmed in T47D xenograft tumors and in a PDX model naturally harboring the ESR1-e6>YAP1 fusion (WHIM18) (Figure 4G). Expression of SNAI1 was unaffected by fulvestrant treatment in T47D cells, consistent with the conclusion that upregulation of EMT genes by the active fusions is independent of endogenous WT-ER (Figures 4C and S4B). ChIP-seq also identified 71 selectively bound sites by ESR1-e6>YAP1 and ESR1-e6>PCDH11X not bound by ESR1-WT nor ESR1-e6>NOP2 (Figure S4C). GSEA pathway analysis of differentially expressed genes near these sites showed enrichment for UV radiation response genes, as well as enrichment for EMT genes, with TGFBR3 and GJA1 contributing to EMT pathway enrichment (Figure S4C). TGFBR3 encodes for transforming growth factor-β receptor III and has roles in migration and invasion (Gatza et al., 2010Gatza C.E. Oh S.Y. Blobe G.C. Roles for the type III TGF-beta receptor in human cancer.Cell. Signal. 2010; 22: 1163-1174Crossref PubMed Scopus (140) Google Scholar). GJA1 encodes for connexin-43, a gap junction protein whose expression in breast cancer cells has been implicated in pulmonary metastasis (Elzarrad et al., 2008Elzarrad M.K. Haroon A. Willecke K. Dobrowolski R. Gillespie M.N. Al-Mehdi A.-B. Connexin-43 upregulation in micrometastases and tumor vasculature and its role in tumor cell attachment to pulmonary endothelium.BMC Med. 2008; 6: 20Crossref PubMed Scopus (110) Google Scholar), consistent with observed lung metastasis in both patients from which the ESR1-e6>YAP1 and ESR1-e6>PCDH11X fusions were identified. A decrease in E-cadherin levels from YAP1 and PCDH11X fusion-expressing cells was observed relative to YFP control and NOP2 fusion-expressing cells (Figure 4D), and a decrease in cell surface E-cadherin was also observed, consistent with an EMT-like transition (Figures S4E and S4F). However, there was no detectable increase in vimentin levels, suggesting that the YAP1 and PCDH11X fusions drive a partial EMT gene expression pattern that nonetheless can be metastasis associated (Jolly et al., 2015Jolly M.K. Boareto M. Huang B. Jia D. Lu M. Ben-Jacob E. Onuchic J.N. Levine H. Implications of the hybrid epithelial/mesenchymal phenotype in metastasis.Front. Oncol. 2015; 5: 155Crossref PubMed Scopus (347) Google Scholar). To examine the functional consequences of the active fusions with respect to the metastatic process, cell motility was examined. The YAP1 and PCDH11X fusions induced significantly greater wound recovery and motility than YFP controls and NOP2 fusion-expressing cells (Figure 4E, quantified in Figure S4D). To exclude the possibility that EMT-associated gene expression was due to phenotypic drift of cells under long-term selection, small interfering RNA (siRNA)-mediated knockdown of ESR1-e6>YAP1 fusion was examined to determine whether EMT-associated features could be reversed. Estrogen-deprived stable T47D YFP control or ESR1-e6>YAP1-expressing cells were pre-treated with fulvestrant to degrade endogenous WT-ER, before transfecting with negative control siRNA (siESR1−) or siESR1 against the N terminus of ESR1 (siESR1+). Forty-eight hours post-transfection, Snail protein levels were markedly reduced in ESR1-e6>YAP1 cells after siESR1 transfection with or without fulvestrant pre-treatment compared with siESR1− with or without fulvestrant (Figure 4F, compare lanes 5 and 7 with lanes 6 and 8). In addition, cells with decreased Snail as a result of ER-YAP1 fusion protein knockdown tended to have higher levels of E-cadherin, suggesting that knockdown of the ESR1-e6>YAP1 fusion transcript restores these aspects of a typical epithelial gene expression pattern. Similar effects were confirmed in stable MCF7 cells expressing ESR1-e6>YAP1 (Figure S4G, compare lanes 5 and 7 with lanes 6 and 8), although Snail levels were more affected by fulvestrant pre-treatment alone, showing that higher basal levels of Snail in MCF7 cells can also be driven by WT ESR1 (Figure S4G, compare lanes 1 and 3 for YFP-expressing cells and lanes 5 and 7 for ESR1-e6>YAP1-expressing cells). However, Snail expression is resistant to fulvestrant suppression in the presence of the ESR1-e6>YAP1 fusion (Figure S4G, compare lanes 3 and 7). The metastatic potential of fusion-expressing cells in vivo was measured by ER immunohistochemistry from the lungs, liver, and bones of mice bearing T47D xenografts from Figure 2D. The number of micrometastatic ER+ cells in the lungs of YAP1 and PCDH11X fusion bearing mice was significantly greater than that in the lungs of mice bearing tumors generated from YFP control cells upon estrogen deprivation (Figure 4H). YFP control tumors grown with E2 supplementation were much larger (Figure 2D), but pulmonary micrometastasis was not significantly different from YFP controls −E2, demonstrating that differences in pulmonary metastasis potential associated with the active fusions were not due simply to differences in disease burden. Bone and hepatic micrometastases were not observed. Pulmonary metastasis in this model was not a feature of YFP control cells, even when disease burden was increased markedly with E2 supplementation. Taken together, these results suggest a role for active YAP1 and PCDH11X fusions in driving pulmonary metastasis in association with the expression of genes known to contribute to EMT biology and metastatic behavior. The loss of the LBD renders the function of ESR1 fusion genes resistant to all endocrine treatments, and therefore alternative therapies will be necessary to treat patients who present with active ESR1 fusions. Palbociclib, a selective CDK4/6 inhibitor was chosen for study because of our recent report that this agent can antagonize the growth of tumors expressing ESR1 mutations as long as phospho-Rb (pRb) is present (Wardell et al., 2015Wardell S.E. Ellis M.J. Alley H.M. Eisele K. VanArsdale T. Dann S.G. Arndt K.T. Primeau T. Griffin E. Shao J. et al.Efficacy of SERD/SERM Hybrid-CDK4/6 inhibitor combinations in models of endocrine therapy-resistant breast cancer.Clin. Cancer Res. 2015; 21: 5121-5130Crossref PubMed Scopus (87) Google Scholar). Because the target of activated CDK4/6 is Rb, pRb levels were examined by immunohistochemistry (IHC) in ESR1 fusion-expressing T47D xenograft tumor sections (Figure S5A). pRb levels in YAP1 and PCDH11X fusion xenograft tumors grown without E2 supplementation were comparable with YFP controls +E2 and were elevated relative to YFP −E2 and NOP2 fusion-containing tumors. T47D stable cells expressing YFP and the three in-frame ESR1 fusions were treated with palbociclib under hormone-deprived conditions and growth-inhibitory effects were assessed (Figure 5A). Palbociclib inhibited T47D cell growth driven by the YAP1 and PCDH11X fusions in a dose-dependent manner. A similar palbociclib effect was observed in ESR1 fusion-expressing MCF7 stable cells (Figure S5B). To test palbociclib sensitivity in vivo, a PDX model naturally harboring the ESR1-e6>YAP1 fusion (WHIM18) was exposed to palbociclib. Consistent with in vitro results, tumor growth in the PDX model was inhibited in mice treated with palbociclib compared with vehicle-treated mice (Figure 5B; tumor growth rates shown in Figure S5C). Palbociclib-treated WHIM18 tumors also showed significant reduction in pRb and marked decrease in Ki-67 levels, without altering levels of ER (Figure 5C) or progesterone receptor (PR) (Figure S5D). Areas containing micrometastatic ER+ cells observed in the lungs of vehicle chow-treated WHIM18 mice were not seen in palbociclib-treated mice (Figure 5D), suggesting that pulmonary metastatic frequency could also be downregulated by CDK4/6 inhibition. This study demonstrated that two in-frame ESR1 fusions in a small late-stage cohort of metastatic ER+ cases drive not only endocrine therapy resistance but also metastatic disease progression. The functional characterization of ESR1 fusions’ properties described herein should drive efforts to identify and further characterize additional ESR1 fusions in early- and late-stage ER+ breast cancer. The ability to block active ESR1 fusion-induced growth with a CDK4/6 inhibitor has important implications for clinical practice. Patients with active ESR1 fusions may present with a clinical pattern of rapidly progressing disease despite adjuvant or metastatic endocrine therapy treatment and therefore be offered chemotherapy instead of a CDK4/6 inhibitor-containing regimen. Because therapeutically resistant disease is infrequently re-biopsied and even more rarely analyzed using RNA-seq, a prospective study of ESR1 in-frame fusion-expressing ER+ tumors will be required to establish an effective approach for these tumors. Although ESR1 fusions are challenging to diagnose because of variable 3′ fu" @default.
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