Targeting bromodomain and extraterminal proteins in breast cancer

Breast cancer is a collection of distinct tumor subtypes that are driven by unique gene expression profiles. These transcriptomes are controlled by various epigenetic marks that dictate which genes are expressed and suppressed. During carcinogenesis, extensive restructuring of the epigenome occurs, including aber- rant acetylation, alteration of methylation patterns, and accumulation of epigenetic readers at oncogenes. As epigenetic alterations are reversible, epigenome-modulating drugs could provide a mechanism to silence numerous oncogenes simultaneously. Here, we review the impact of inhibitors of the Bromod- omain and Extraterminal (BET) family of epigenetic readers in breast cancer. These agents, including the prototypical BET inhibitor JQ1, have been shown to suppress a variety of oncogenic pathways while inducing minimal, if any, toxicity in models of several subtypes of breast cancer. BET inhibitors also syn- ergize with multiple approved anti-cancer drugs, providing a greater response in breast cancer cell lines and mouse models than either single agent. The combined findings of the studies discussed here provide an excellent rationale for the continued investigation of the utility of BET inhibitors in breast cancer.

1.Introduction
Breast cancer is a heterogeneous disease, and multiple subtyp- ing methods have been developed to group these diverse tumors. The most common and clinically relevant classification system is based on the expression of estrogen receptor (ER) and progesterone receptor (PR) and the amplification status of human epidermal growth factor receptor 2 (HER2). The status of these three recep- tors governs the first line of treatment for breast cancer patients. For example, patients with tumors expressing ER and/or PR are eligible for endocrine therapy while patients with tumors with HER2 ampli- fication receive HER2-targeted therapies. Triple-negative breast cancer (TNBC) patients lack expression of ER/PR and amplification of HER2. There are currently no FDA-approved targeted therapies available for this disease, and the only treatment option is tradi- tional cytotoxic chemotherapy.
Gene expression profiling led to the subdivision of breast cancers into six intrinsic molecular subtypes: luminal A, luminal B, HER2-enriched, basal-like, claudin-low, and normal-like [1–3]. These subtypes vary in terms of phenotype, response to treatment, and clinical outcome [4–6]. The vast majority of breast cancers fall into the luminal A and B subtypes. These tumors are charac- terized by expression of ER and/or PR with a low (luminal A) or high (luminal B) Ki67 index.

They typically respond to ER-targeting agents including tamoxifen and are associated with good progno- sis [7]. HER2-enriched tumors overexpress the ERBB2 gene and, as a result, can be treated with anti-HER2 agents such as trastuzumab [8]. Most basal-like and claudin-low tumors can be categorized as TNBC, with basal-like tumors accounting for the majority of TNBCs. Basal-like tumors express a basal epithelial gene cluster which includes cytokeratins 5 and 17, laminin, and integrin-β4 [1]. Claudin-low cancers express low levels of the tight junction pro- teins E-cadherin and claudins 3, 4, and 7; are poorly differentiated; have a large cancer stem cell population; are enriched in epithelial- to-mesenchymal transition (EMT) markers; and express high levels of immune response genes [9,10]. Tumors of both TNBC subtypes respond well to cytotoxic chemotherapies such as doxorubicin and taxanes [7]. However, the incidence of metastatic recurrence for these cancers is high. Once metastasis occurs, the disease pro- gresses quickly, with patients exhibiting a median survival of 13 months [4,5,11,12].Breast cancers are driven by numerous oncogenic pathways which can be subtype-specific. As such, the various subtypes must be treated with different agents. To target a diverse array of breast tumors and to prevent recurrence, it should also be useful to develop therapies that can target multiple pathways simultane- ously and have broad implications for this group of diseases as a whole. Here, we discuss targeting Bromodomain and Extratermi- nal (BET) proteins, an approach already in clinical trials that has the potential to provide benefits across all subtypes of breast cancer.

2.BET protein structure and function
Various posttranslational modifications are added to nucle- osomes that impact their association with chromatin and the recruitment of proteins to DNA. One such modification is lysine acetylation, which marks areas of chromatin for active transcription and is recognized by bromodomains (BRDs) in various proteins [13]. The BRD is a conserved 110 amino acid structural motif composed of four α-helices (αZ, αA, αB, and αC) that comprise a left-handed bundle [14]. Two loop regions (ZA and BC) connect the α-helices and form a surface that interacts with acetylated lysines in nucleo- somal histones [15]. In humans, there are 61 BRDs found within 42 multi-domain proteins that regulate transcription, including ATP-dependent chromatin remodeling complexes, transcriptional co-activators, histone acetyltransferases (HATs), and BET proteins [16].The BET protein family consists of four members (BRD2, BRD3,BRD4, and BRDT) that reside in the nucleus and play critical roles in transcription [17]. BET proteins act as epigenetic readers and are characterized by two tandem N-terminal BRD regions fol- lowed by an extraterminal domain. The BRD regions recognize and bind acetylated lysines in histone tails (histones H3 and H4) and transcription factors. The extraterminal domain is involvedin protein–protein interactions with proteins such as E2Fs and latent nuclear antigen of Kaposi’s sarcoma-associated herpes virus [18,19]. BRD4 and BRDT have an additional C-terminal motif that links their reader function to transcriptional elongation: following the binding of BRD4/T to acetylated histones, the C-terminal motif interacts with P-TEFb [20], a complex of cyclin T and CDK9. This localizes P-TEFb to target promoters where it phosphorylates RNA Polymerase II (RNAPII) and releases it from pausing.Alternative splicing generates three isoforms of BRD4: the long- form isoform A (13622 aa), isoform B (796 aa), and isoform C (722 aa) [21]. Isoforms B and C lack the C-terminal domain and are dis- tinguishable from each other by the presence of an additional 76 amino acid peptide at the C terminus of isoform B. Isoform B has only been identified in U2OS cells, and its activity is not well char- acterized [21].

A separate set of studies identified two isoforms, a long-form (BRD4-LF) that corresponds to isoform A and a short- form (BRD4-SF) that most likely corresponds to isoform C [22,23]. BRD4-LF and BRD4-SF have differing effects on metastasis: BRD4- LF reduces metastasis while BRD4-SF promotes metastasis of the mouse mammary tumor Mvt-1 model of breast cancer [22,24]. It is possible that the ratio between the long and short isoforms of BRD4 dictate the oncogenic potential of BRD4, and differences in the ratio of these isoforms could explain why only a few studies have generated data suggesting that BRD4 acts as a breast tumor and metastasis suppressor [24–26] while the majority of studies demonstrate that BRD4 is an oncogenic driver.Binding of BET proteins to acetylated histones recruitsBET proteins to the enhancer and promoter regions of genes marked for active transcription. Here, they interact with co- activators/repressors, transcription factors, and the transcriptional machinery, forming protein complexes that influence target gene transcription [27]. While they have a similar structure and usually enhance transcription, BET proteins regulate different processes based on their binding partners, which are often tissue-specific.When BET proteins bind acetylated histones, they recruit several regulatory complexes that influence various aspects of chromatin structure and transcription. For example, BRD4 is important for transcription initiation. BRD4 regulates monoubiquitination of his- tone H2B (H2Bub1) and interacts with histone modifying proteins, such as the arginine demethylase JMJD6, and chromatin remod- eling enzymes, such as CHD8 [28–31]. This leads to a more open conformation of the chromatin and allows BRD4 to recruit key transcriptional regulators, such as Mediator and transcription elon- gation factors, to the DNA [32–35]. BRD4 additionally enhances transcription of genes with ERα-bound estrogen response ele- ments (EREs) that are also enriched for FOXA1 binding. BRD4 binds acetylated histones, including histone H4 acetylated at lysine 12 (H4K12ac) at these elements and recruits RNAPII which initiates the transcription of eRNAs [28,36].In addition, BET proteins are involved in transcriptional elongation.

BRD4 directly promotes elongation by phosphorylating RNAPII at Ser2 of the C-terminal domain [37] and recruiting P-TEFb [30,38,39]. P-TEFb in turn directly phosphorylates the carboxy-terminal heptad repeats of paused RNAPII and disrupts the interaction between RNAPII and the regulatory complexes DSIF and NELF [40,41]. Both events release RNAPII from the paused state at the promoter and thereby induce transcriptional elongation. Dur- ing elongation, both BRD2 and BRD3 act as histone chaperones, remodeling histones in order for RNAPII to move along the DNA [42]. BRD3 also recruits the polymerase-associated factor complex (PAFc), which coordinates several events during transcription, and the super elongation complex (SEC), which also regulates elonga- tion [43]. Based on the activity of BET proteins during transcriptioninitiation and elongation, the disruption of BET protein function should have an enormous impact on the production of RNA tran- scripts in varied cell contexts.BET proteins play diverse roles in multiple phases of the cell cycle and control expression of cell cycle and proliferation genes [19,42,44–46]. During mitosis, BRD4 ensures proper chromosomal segregation and cytokinesis by regulating expression of Aurora kinase B [47]. Both BRD2 and BRD4 facilitate mitotic memory by remaining bound to M/G1 genes during mitosis and recruit P-TEFb to the DNA late in mitosis. This marks G1 genes for immediate transcription following mitotic exit, ensuring cell cycle progres- sion [48–50]. BRD4 also promotes G1-S and G2-M phase transitions [51,52]. As a result of their role in the regulation of the cell cycle, loss of BET expression induces cell cycle arrest [53–59].

BET proteins also control inflammation. BRD2 binds to genes regulated by STAT5, an important mediator of cytokine signal- ing, and pan-BET inhibition suppresses expression of STAT5-target genes [60]. Furthermore, mice that express half the amount of Brd2 as wild-type mice (brd2 lo) develop severe obesity but are protected against insulin resistance and the obesity-induced inflammatory response [61]. siRNA-mediated knockdown of BRD2 suppresses NF-nB transcriptional activity [62]. In addition to regulating pro- inflammatory gene expression though NF-nB, BRD2 directly binds the promoter regions of pro-inflammatory genes, especially follow- ing LPS stimulation, and macrophages from brd2 lo mice produce significantly less pro-inflammatory cytokines such as TNF-α, IL1β, and IL6 [62]. NF-nB activity is also regulated by BRD4; BRD4 binds acetylated RelA, a subunit of NF-nB, and enhances transcrip- tion of NF-nB-dependent inflammatory genes [63]. Suppression of BET proteins induces anti-inflammatory responses [62,64], and inhibitors of BET proteins are currently being investigated as poten- tial therapeutic options for the treatment of inflammatory diseases [65].BET proteins are crucial during development, and loss of either BRD2 or BRD4 results in embryonic lethality. BRD2 controls neu- ronal differentiation [66], with Brd2 null embryos displaying deficient neural tube formation and dying during mid-gestation [67]. BRD4 maintains the self-renewal capability of stem cells by stimulating expression of genes involved in pluripotency. Over 20% of pluripotency genes, including NANOG, OCT4, SOX2, and PRDM14, are bound by BRD4 in embryonic stem cells [68–72]. BRD4 also localizes to a number of stem cell genes in preimplantation embryos and maintains the inner cell mass, with BRD4 null embryos dying shortly after implantation [70,73].Unlike the other three BET proteins which are ubiquitously expressed, BRDT expression is normally testis-specific [74]. It is expressed in pachytene and diplotene spermatocytes and round spermatids, and its expression decreases during spermatid differ- entiation [75]. BRDT is essential for spermatogenesis, and BRDT knockdown leads to sterility in male mice [75]. Pharmacological inhibition of BRDT also confers reversible infertility in male mice without impacting testosterone levels, and offspring produced bythese mice once treatment is removed are normal [76]. As a result, BRDT is seen as a potential target for reversible male contraception.

3.BET proteins in cancer
BET proteins are involved in various diseases, including inflam- mation, viral infection, heart failure, and cancer [77,78]. It is thought that BET proteins primarily mediate their effects in disease pathogenesis and progression primarily by localizing to super- enhancers (SEs) at pathology-associated genes and driving their expression [43,79,80]. An SE is a large contiguous cluster of enhancers within a locus that is associated with increased gene expression, DNase I sensitivity, histone tail acetylation, and tran- scription factor and co-activator binding [80,81]. SEs are frequently identified by ChIP-seq analysis using antibodies against histone marks such as H3K27ac or the transcriptional regulators Media- tor and BRD4, as these proteins are enriched at SEs [80–82]. SEs vary by cell type and the different binding sites in distinct cells underlie the expression of cell identity genes that specify that cell type [81,82]. In addition, SEs drive expression of disease-associated genes in numerous diseases, including Alzheimer’s disease, type 1 diabetes, and cancer [82,83]. In cancer, SEs are enriched at onco- genes known to play a role in specific cancer types, including MYC and IRF4 in multiple myeloma, RUNX1 and FOSL2 in glioblastoma, and CD79 B in diffuse large B cell lymphoma [79,80]. They are also associated with many oncogenes that are linked to general cancer pathogenesis, including CCND1, MCL1, and BCL2L1 [80].Only a fraction of enhancer regions are classified as SEs. For example, in multiple myeloma there are 308 putative SEs com- pared to nearly 8000 typical enhancers [80]. Despite the relatively small number of SEs, BRD4 disproportionately accumulates at these regions, with up to 40% of all bound BRD4 being localized to SEs [79,80]. BRD4, like other co-activators, exhibits cooperative bind- ing. Thus, loss of BRD4 leads to a greater disruption of transcription at SE-associated genes compared to typical-enhancer associated genes, providing a mechanism to preferentially silence multiple SE-associated oncogenes at once [79,80,84].

BRD2, BRD3, and BRD4 are expressed in breast tumors while BRDT is rarely expressed. When examining all breast cancers, regardless of subtype, the genes encoding the BET proteins are amplified and/or overexpressed in less than 10% of tumors in the TCGA [85] and METABRIC [86,87] datasets. However, when focus- ing on breast cancer subtypes, BRD2 and BRD4 are amplified and/or overexpressed in 12.1% and 20.6% of basal-like breast cancers, respectively [85]. Analysis of copy number aberrations of 9445 tumors representing 20 cancer types in the TCGA dataset confirmed that BRD4 is more commonly amplified in breast cancer as well as ovarian, liver, and endometrial cancers compared to cancers from other organs [88]. In addition, BRD4 was found to be amplified in a study evaluating focal amplification events in a panel of 10 DCIS and 151 invasive breast tumors representing all subtypes of breast can- cer [89]. Lastly, BRD4 mRNA was more highly expressed in tumors compared to normal breast tissue [89]. Together, these data suggest an association between BRD4 levels and breast tumorigenesis.BRD4 is expressed in non-transformed breast epithelial, luminalbreast cancer, and TNBC cell lines [90]. By interrogating a microar- ray dataset of 477 breast cancer samples, Shi et al. found there was no difference in BRD4 mRNA expression between ER+ and ER- tumors [90]. However, BET expression is less consistent at the pro- tein level. When comparing BRD4 protein expression in two TNBC(MDA-MB-231 and BT549) and two ER+ (MCF7 and T47D) cell lines, T47D cells clearly express higher levels of BRD4 compared to the other three lines [91].

In TNBC cell lines (MDA-MB-231, MDA-MB- 468, and HCC1937), BRD4 is expressed at similar levels in all three lines while BRD2 and BRD3 expression was higher in HCC1937 cells [54]. In addition, a larger study examining basal protein expression of BRD2, BRD3, and BRD4 in an 18 cell line panel representing non- transformed mammary, luminal, HER2+, and TNBC cell lines found that expression of BET proteins was variable and did not correlate with breast cancer subtype [53]. Thus, despite relatively constant expression at the RNA level, BET protein expression varies by cell line, suggesting post-transcriptional or post-translational mecha- nisms are responsible for the observed differences in BET protein expression.In an effort to identify subtype-specific and pan-disease essen- tial genes in breast cancer, Marcotte, et al. utilized pooled lentiviral shRNA dropout screens in 78 breast cancer and four non-transformed mammary epithelial cell lines and analyzed the results using an algorithm they developed termed “the siRNA/shRNA mixed effect model” (siMEM) [92]. This process successfully detected known drivers of breast cancer and breast cancer subtypes, affirming its utility. In addition, this study revealed new candidates for essential breast cancer genes, includ- ing BRD4. Silencing of BRD4 gene expression using two shRNAs in SUM159, BT474, and T47D cells resulted in decreased pro- liferation, and this response was prevented by restoring BRD4 expression using an shRNA-resistant BRD4 cDNA. However, sen- sitivity to BRD4 depletion does not always translate into sensitivity to BET inhibitors, suggesting that BRD4 has both BRD-dependent and BRD-independent roles in breast cancer.

This concept is sup- ported by the finding that SUM159 cells that are resistant to BETi are still sensitive to genetic silencing of BRD4, indicating BRD4 may be recruited to the chromatin through a BRD-independent mech- anism or that BRD4 has a role that is independent of chromatin binding [53].Evidence that BRD4 may be essential for growth of estrogen-dependent breast cancer cells was first provided by a group that applied a novel triclustering algorithm to a publicly available microarray dataset corresponding to a time course of estrogen response of MCF7 cells [93]. This approach revealed that BRD4 may be a hub-gene in estrogen receptor-driven breast cancer [94]. Later functional studies in four TNBC cell lines (SUM159, MDA-MB-231, MDA-MB-468, and MDA-MB-436) and one luminal line (ZR-75-1) using RNAi-mediated silencing of BET proteins demonstrated that suppression of either BRD2 or BRD4 reduces the growth of four of the cell lines with only siBRD4 inhibiting growth of MDA-MB-436 cells [53]. Subsequently, we reported that simultaneously silenc- ing BRD2 and BRD4 reduces expression of BRD3 as well, suggesting a complex interplay in the regulation of these factors. We further showed that combined BRD2/4 silencing reduces the expression of key mitotic regulators that play a crucial role in the BETi response of TNBC cells [44]. Together, these findings further revealed a role for BET proteins in breast cancer growth and pathogenesis.

4.Targeting BET proteins in breast cancer
The first BETi to be developed were JQ1 [95] and I-BET [64]. Since then, multiple derivatives have been utilized in preclini- cal settings, including I-BET151 [43], I-BET762 [96], MS417 [97], and OTX015 [98], and at least 11 have moved on to be assessedin early phase clinical trials for a wide variety of hematologic cancers and solid tumors. BETi belong to varied chemical classes based on their core scaffolds, such as azepines (JQ1, OTX-015, CPI- 0160), 3,5-dimethyl isoxazoles (I-BET151), pyridones (ABBV-075), and tetrahydroquinolones (I-BET762) [99].While there are 42 BRD-containing proteins in humans [16], BETi selectively target the BET family of proteins [64,95,100]. Most BETi inhibit all four members of this family, although BETi that selectively bind a specific BET protein, particularly BRD4, are cur- rently being developed. BETi inhibit BET proteins by competing with acetylated lysines for binding to both BRD regions, prevent- ing BET proteins from binding histones and thus from localizing to the chromatin [95]. The specific BET protein(s) that must be suppressed for BETi to elicit their effects differs depending on the context. For example, in breast cancer models, we found that loss of BRD2 and BRD4 expression together was necessary for the induc- tion of mitotic catastrophe in response to BETi in TNBC [44] while other studies have identified BRD4 as the sole critical target of BETi for controlling other phenotypic responses [53,55,101].Multiple studies have examined the utility of BETi in vari-ous models of breast cancer (Table 1).

Depending on the subtype studied and cell lines used, BETi can impact tumor formation, pro- liferation, the response to hypoxia, angiogenesis, cancer stem cells, metastasis, and metabolism by repressing the expression of genes that drive these critical oncogenic pathways (Fig. 1). The effects of BETi in breast cancer reported thus far are reviewed below. It is important to keep in mind that the activity of BETi in vivo most likely depends on a convergence of several of these BETi-induced outcomes as opposed to a singular response.Many cell lines representing the luminal (ER+), HER2+, and TNBC subtypes of breast cancer undergo growth inhibition in response to BETi treatment. Marcotte, et al. found luminal and HER2+ cell lines were more dependent on BRD4 expression than TNBC cells, as siRNA-mediated knockdown of BRD4 led to a greater suppression of growth in these lines compared to basal cell lines [92]. However, when comparing the IC50 of five BETi in a panel of 41 cell lines that represent luminal, HER2+, and TNBCs as well as non-transformed mammary epithelial cells, Polyak and colleagues found that TNBC cell lines were generally more sensitive to BETi than HER2+ and luminal cell lines [53]. The discrepancy between these two stud- ies could be due to the genetic silencing of a single BET protein compared to inhibition of the entire family with a small molecule. Several studies have specifically assessed BETi activity in lumi- nal breast cancer. BETi suppressed growth of MCF7, ZR75-1, and T47D cells with or without estrogen stimulation in 2D and 3D culture, and JQ1 inhibited expression of canonical estrogen- target genes [28,56,91,102,103]. Tamoxifen-resistant (Tam-R) and estrogen-deprivation-resistant versions of ER+ cell lines were more sensitive to BETi than the parental lines [104].

In addition, within two days, BETi treatment induced apoptosis of Tam-R MCF7 cells but not of parental MCF7 cells [104], suggesting BETi may be an effective treatment option for hormone therapy-resistant tumors. The difference in sensitivity of parental and Tam-R breast cancer cells to BETi likely stems from differences in the expression pat- tern of critical transcription factors. For example, parental MCF7 cells had higher expression of GATA3, which is necessary for sus- tained ESR1 (ERα) gene expression, compared to Tam-R cells [105]. Supporting this potential mechanism of BETi sensitization, silenc- ing GATA3 in parental cells rendered them more sensitive to BETitreatment [104].Affirming the relative resistance of parental MCF-7 cells to BETi in vitro, JQ1 failed to impact MCF7-derived tumor growth in mice [102]. MCF-7 cells are a model of luminal A breast cancer. In contrastto studies with these xenografts, JQ1 was reported to be effective in the MMTV-PyMT mouse model of luminal B breast cancer [106]. Precursor lesions in this model are ER+ while established tumors are ER- but maintain a luminal gene expression signature [9,107]. Pérez-Salvia, et al. discovered that not only could JQ1 suppress growth of established tumors in MMTV-PyMT mice, but the drug could also slow the development of spontaneous mammary tumors when administered to four week old mice prior to the detection of palpable tumors [103]. In addition, JQ1 improved overall survival in MMTV-PyMT mice. Based on these data, the authors suggested that BETi could be utilized as a preventative agent in women who have a high risk of developing breast cancer. BETi are quickly cleared from the target tissue, hence it would be necessary to treat these patients daily. Importantly, it is not yet known whether long-term exposure to BETi will lead to intolerable toxic side effects and increase the chances of developing BETi resistance.Information on BETi treatment alone in HER2+ breast cancergrowth is limited. In one study, both JQ1 and I-BET762 inhibited the in vitro growth of four HER2+ cell lines within five days in a dose- dependent manner [101].

However, in a four-week clonogenic assay, BETi-resistant colonies still formed. Addition of lapatinib toBETi dramatically reduced the number of colonies formed, thereby inhibiting acquired BETi resistance.The response of TNBC to BETi has been more thoroughly doc- umented. When Shu, et al. treated 26 TNBC cell lines with BETi, the majority were highly sensitive to this drug class [53]. Simi- larly, we found JQ1, I-BET151, and I-BET762 treatment suppressed growth of a panel of seven TNBC cell lines representing five of the six TNBC subtypes described by Lehmann, et al. [108] as well as both the claudin-low and basal subtypes in a dose-dependent manner [109]. Multiple other studies have also shown that BETi suppressed 2D and 3D growth, wound-healing capacity, and colony formation of diverse TNBC cell lines [53,54,57,58,91,110], indicat- ing BETi could be an effective therapy across diverse TNBC tumor types. Our studies and others also revealed that sustained inhibi- tion of BET proteins induced two terminal responses, apoptosis and senescence, and these responses did not correlate with the extent of BETi-induced growth inhibition, impact on c-Myc expression, or TNBC subtype [109]. These effects were recapitulated in vivo. Tumors derived from MDA-MB-231 cells, which senesced in vitro, grew significantly slower when treated with JQ1, while MDA- MB-468 tumors, which died in vitro, partially regressed [109]. In addition, JQ1 suppressed growth of tumors formed from a TNBC patient-derived xenograft (PDX). Three other studies confirmed the in vivo efficacy of BETi in TNBC: JQ1 and MS417 inhibited growth of MDA-MB-231, SUM1315, and SUM159 xenografted tumors as well as two PDX models [53,57,90]. Together, these studies indicate that models of TNBC are highly responsive to BET inhibition both in vitro and in vivo.

It has been suggested that BETi induce subtype switching inTNBC, where TNBC cells lose basal markers and gain luminal mark- ers, due to differential expression of luminal and basal cytokeratins following JQ1 treatment of the MDA-MB-231 and SUM159 cell lines [53]. Differentiation of TNBC models following BETi treatment has also been assessed in vivo. Treatment of a PDX model with vehi- cle or JQ1 and staining for low molecular weight (luminal) and high molecular weight (basal) cytokeratins revealed that vehicle- treated tumors had very little expression of low molecular weight cytokeratins while their expression increased significantly with JQ1 treatment. These data, coupled with the in vitro analysis, sug-gested that BETi may induce differentiation of basal tumors to a more luminal phenotype. However, this conclusion was based on the restricted analysis of cytokeratin gene expression. In contrast, when vehicle- and JQ1-treated SUM159 xenografts were stained for diverse luminal (luminal cytokeratin, CK18, and CD24) and basal (basal cytokeratin, CK17, pSTAT, and CD44) markers, JQ1- treated tumors had variable responses, and there was no consistent loss of basal and simultaneous gain of luminal markers. Similarly, we performed GSEA using Neve [111] and Charafe-Jauffret [112] breast cancer subtype classifiers on gene expression array data from MDA-MB-231 and HCC70 cells treated with vehicle or JQ1 [44]. While both cell lines lost expression of a subset of basal-signature genes, there was no consistent gain of expression of luminal genes, indicating TNBCs do not undergo extensive BETi-mediated differ- entiation to a characteristic luminal expression signature.Severe intratumoral hypoxia is common in breast cancer [113]. Hypoxia in solid tumors occurs due to increased metabolism and proliferation as well as poor vascular structure.

It is linked to metastatic progression, resistance to radiation and chemother- apy, and poor prognosis [114]. As in many other types of cancer, regions of hypoxia in breast tumors are associated with EMT and the acquisition of cancer stem cell properties via signaling through hypoxia-inducible factors (HIFs) which stimulate EMT, promote self-renewal, and inhibit differentiation [115]. The phenotypes associated with hypoxia are regulated by HIF-1α and HIF-2α which heterodimerize with HIF-1β in low oxygen conditions. This com- plex then localizes to hypoxia response elements in the promoters of target genes to induce transcription. Compared to the other sub- types of breast cancer, TNBC is particularly associated with hypoxia, and HIF target genes are upregulated in TNBC patient tumors [116]. OTX015 suppressed growth of three TNBC cell lines in both nor- moxic and hypoxic conditions, and GSEA following gene expression profiling revealed this drug downregulated hypoxia-responsive genes [54]. In a second gene expression analysis study, when MCF7 and MDA-MB-231 cells were treated with JQ1 in normoxic and hypoxic conditions, JQ1 also altered expression of hypoxia-relatedgenes and prevented the hypoxia-mediated upregulation of sev- eral gene sets, including those involved in angiogenesis and the hypoxic pathway [110]. In MDA-MB-231 cells in particular, JQ1 altered expression of 44% of hypoxia-responsive genes, the major- ity of which were suppressed with drug exposure. While expression of HIF-1α and HIF-2α remained unchanged, JQ1 reduced expres- sion of carbonic anhydrase 9 (CA9), a known hypoxia-responsive gene that helps to maintain a neutral intracellular pH [117,118], in MCF7 cells, two TNBC cell lines (MDA-MB-231 and HCC1806), and HCC1806 xenografts.

Notably, high expression of CA9 has been associated with poor overall survival and a higher rate of dis- tant metastases in a cohort of over 3600 breast cancer patients, and inhibition of CA9 suppresses metastasis [119]. Thus, BETi inhibition of CA9 expression may provide an approach to limit metastatic progression. Mechanistically, the JQ1-induced reduc- tion in CA9 expression was accompanied by the loss of HIF-1β binding at the CA9 promoter following JQ1 treatment in hypoxic conditions [110]. Thus, BETi prevented the localization of the HIF heterodimer to HIF target genes. Exposure to hypoxia can induce radio- and chemo-resistance, and inhibiting CA9 in combination with radiotherapy or chemotherapy is effective in preclinical mod- els [120,121]. Together, these data suggest that BETi may be useful for sensitizing cancers to radiotherapy and/or chemotherapy, mak- ing it an effective approach for the treatment of solid tumors, including breast tumors.In addition to disrupting hypoxia-regulated pathways, BETi also appear to suppress angiogenesis, one of the hallmarks of can- cer [122]. Hypoxia and angiogenesis are inherently linked, with hypoxia inducing expression of VEGF that then stimulates the pro- duction of new blood vessels [123]. Angiogenesis is critical for tumor growth and metastasis, making it a useful therapeutic tar- get, particularly in renal cell carcinoma. Monoclonal antibodies against VEGFA or the VEGF receptor (VEGFR) and tyrosine kinase inhibitors that target VEGFR have been developed, but clinical trials in breast cancer have yielded mixed results, leading to the revoca- tion of FDA approval of the anti-VEGFA antibody, bevacizumab, in breast cancer patients in 2011 [124].

Therefore, it is essential to develop additional strategies to effectively disrupt angiogenesis. BETi have been shown to suppress angiogenesis in rhabdomyosar- coma, Ewing sarcoma, and testicular germ cell tumors [125,126], suggesting they may also display anti-angiogenic activity in other tumor types, including breast cancer.Only one study has directly assessed the impact of BETi onangiogenesis in breast cancer. In MCF7 and MDA-MB-231 cells, JQ1 prevented the upregulation of angiogenic signature genes under hypoxic conditions [110]. BRD4 bound the promoter of VEGFA in MDA-MB-231 cells, and this binding increased in hypoxia. Both treatment with JQ1 and gene silencing of BRD4 suppressed expres- sion of VEGFA in hypoxia in MCF7, MDA-MB-231, and HCC1806 cells. In HCC1806 xenografted tumors, JQ1 also suppressed expres- sion of VEGFA, as well as the TIE2 and NRP genes that are critical for angiogenesis. Immunostaining revealed these tumors had lower expression of the blood vessel marker CD31. These data indicate BETi may impair angiogenesis. This could be due to a double hit: direct loss of BRD4 at the promoter regions of genes involved in angiogenesis and the suppression of the hypoxic response leading to the inhibition of hypoxia-induced angiogenic pathways.Anti-angiogenic therapies are already used to treat numer-ous solid tumor types. However, drug-induced hypoxia occurs in the tumors of about half of these patients, leading to therapeu- tic resistance [114]. Combining bevacizumab with CA9 knockdown suppressed colon cancer and glioblastoma xenografts growth bet- ter than bevacizumab alone [127]. As mentioned above, BETidownregulated CA9 in breast cancer cell lines and xenografts, sug- gesting that combining BETi with anti-angiogenic agents could be a beneficial treatment strategy and revive the use of drugs such as bevacizumab in breast cancer.Cancer stem cells (CSCs) are involved in numerous processes during tumor initiation and progression, are resistant to tradi- tional cytotoxic chemotherapies, and play a role in metastasis and recurrence [128], making them a desirable target for anti-cancer therapies.

A role for BET proteins in the maintenance of stem genes is now well established [68–71]. In embryonic stem cells, inhibi- tion of BET proteins suppress expression of critical stem cell factors and induce differentiation [71,72]. Extending to cancer, BETi induce apoptosis in progenitor and stem cells in acute myeloid leukemia (AML) and glioblastoma [129,130]. In MYC-driven medullablas- toma, BETi reduce stem cell signaling and the self-renewal capacity of tumor cells [131].The only subtype of breast cancer that has been investigatedthus far for the impact of BETi on CSCs is TNBC. Expression of WNT5A, which plays crucial roles in maintaining stem cell pluripo- tency, was suppressed by JQ1 treatment due to reduced binding of BRD4 at its promoter [90]. JQ1 also inhibited activity of the JAK/STAT pathway that promotes stem cell renewal as well as the pro-inflammatory response and EMT [53]. Similarly, a more extensive study utilizing OTX015 found that BETi altered stem cell- related gene expression patterns. GSEA of OTX015-treated TNBC cells showed an overall loss of expression of CSC genes [54]. In general, OTX015 downregulated CSC genes in three TNBC cell lines within 24 h, although some of the genes that changed and the direc- tion in which they were altered were cell line-specific. NANOG and OCT4, transcription factors that promote stemness, were sup- pressed as were two additional stem cell markers, CD133 and Musashi-1. Breast CSCs are often defined by high expression of CD44 and low expression of CD24. OTX015 treatment reduced CD44 expression in three cell lines while the impact on CD24 was variable. The suppression of stem cell markers by OTX015 was confirmed in vivo in mice bearing MDA-MB-231 tumors.

However, expression of the epithelial marker EpCAM did not increase in any of the cell lines assessed. No other epithelial markers were exam- ined, so it is unclear if OTX015 is capable of initiating differentiation in CSCs. As mentioned above, our studies indicated that TNBC cells do not undergo a basal to luminal transdifferentiation in response to JQ1 [44]. Thus, while the loss of stem cell markers that occurs with BETi indicates a loss of the stem cell phenotype in TNBC, it is not accompanied by the acquisition of a luminal breast cancer profile.In a separate study, JQ1 did not alter expression of three stemcell markers (CD44, CD49, and CD133) in MDA-MB-231 cells, yet it did significantly decrease the formation of primary and secondary tumorspheres [59]. It will be important in the future to perform additional functional tests in vitro and in vivo to determine if and how BETi directly impact the population of stem cells in breast cancers.The vast majority of breast cancer patients do not die from their primary tumor. Instead, they succumb to metastatic lesions that develop in vital organs. Metastasis is a multi-step process, which includes invasion of surrounding tissue, intravasation and survival within the bloodstream, extravasation, and colonization of a distant organ [132]. An early event during this cascade is EMT, a process that enables epithelial cells to adopt a more mesenchymal, motile phenotype [133]. Not only do cells that undergo EMT become moremigratory but they also acquire stem cell characteristics. BRD4 has been shown to regulate EMT in cancer. In several types of cancer, overexpression of BRD4 triggered metastasis while BET inhibition altered expression of key EMT genes, thereby preventing metastasis [134–137]. Additionally, high expression of BRD4 correlated with lymph node metastasis in non-small cell lung cancer and renal cell carcinoma [138,139].In breast cancer, changes in BRD4 expression or treatment with BETi impact expression of genes linked to EMT and metastasis. Another set of proteins, those belonging to the extracellular matrix (ECM), can also modulate the EMT response.

This has been demon- strated in mammary epithelial cells, with laminin inhibiting EMT and fibronectin promoting EMT following the addition of the EMT- inducing enzyme matrix metalloproteinase-3 [140]. In addition, multiple mouse and human studies have revealed that expres- sion of ECM genes are frequently dysregulated in tumors that are likely to metastasize [141–145]. Modulating BRD4 levels by ectopic overexpression or gene silencing altered expression of ECM reg- ulatory genes in breast cancer cell lines [24,55]. Overexpression of BRD4 also changed the activity of genes involved in other pro- cesses important for EMT and metastasis, including cytoskeletal remodeling and cellular adhesion [24]. In addition, BRD4 regulates the expression of the HOX transcript anti-sense RNA (HOTAIR), a long non-coding RNA that promotes metastasis, regulates breast CSC properties, and is a biomarker for breast cancer diagnosis and metastasis [146–149]. When claudin-low cells were grown in laminin rich ECM 3D cultures, BRD4 maintained HOTAIR expres- sion by binding its promoter [150]. As expected, treatment with JQ1 or gene silencing of BRD4 decreased HOTAIR expression. Together, these data indicate that BET proteins, and specifically BRD4, con- trol the production and sensing of the extracellular matrix, a key regulator of cellular motility.In addition to modulating the ECM, BET proteins directly regu-late EMT-modulating transcription factors. Inhibiting BET proteins with JQ1 reduces the binding of the transcription factor, activating enhancer binding protein 4 (AP4), to the MYC promoter, leading to the downregulation of MYC [58]. AP4 expression is linked to EMT, metastasis, and poor prognosis in several cancers, including breast [151–153], suggesting that BETi may suppress metastatic progression, at least in part, by modulating AP4 activity.In another study by Andrieu and colleagues, JQ1 suppressed migration and invasion in MDA-MB-231 and SUM149T cells [55]. In this case, JAG1 was identified as a key target of BRD4.

This gene encodes Jagged, a Notch receptor family ligand involved in EMT, metastasis, proliferation, survival, and resistance to therapy [154]. The clinical relevance of this finding is demonstrated by Kaplan- Meier analysis of 664 breast cancer patients [155] revealing that high co-expression of BRD4 and JAG1 is associated with shorter dis- tant metastasis-free survival [55]. BRD4 binding to the promoter of JAG1 is enhanced by the pro-inflammatory cytokine interleukin 6 (IL6), which is linked to EMT, migration, invasion, and metas- tasis and is secreted by the tumor microenvironment [156–158] and leads to increased Jagged1 protein levels. Increased Jagged1 in turn activates Notch1 to promote migration and invasion. Con- sistently, treatment with JQ1 prevents the recruitment of BRD4 to the JAG1 promoter, reducing Notch1 activation. BETi have pre- viously been shown to have anti-inflammatory activity [62,64], and these findings are further enhanced by the discovery that BETi can combat the pro-metastatic effects of IL6 from the tumor microenvironment by modulating Jagged1/Notch signaling. These data further support the notion that BETi can inhibit the secretion of pro-metastatic factors into the tumor microenvironment and may improve patient outcomes. However, the specific impact of BETi on metastatic spread was not assessed.Another mechanism by which BRD4 can directly modulatemotility and invasion of breast cancer cells involves its interac-tion with Twist, a transcription factor that plays an essential role in the activation of EMT [159].

When Twist is di-acetylated, it interacts with the second BRD of BRD4 [90]. This Twist-BRD4 com- plex drives expression of a set of EMT genes and thus maintains mesenchymal characteristics of TNBC cells. One of the gene tar- gets of this complex is WNT5A. As mentioned above, WNT5A is a secreted factor that regulates various aspects of cancer cell proper- ties, including proliferation, self-renewal, migration, and invasion [160]. The WNT5A gene has a putative super-enhancer, and bind- ing of Twist to this locus is important for the recruitment of BRD4, P-TEFb, and RNAPII to the WNT5A enhancer and promoter [90]. BETi disrupts the interaction between BRD4 and Twist, leading to the suppression of invasiveness in TNBC cells. Both JQ1 treat- ment and gene silencing of BRD4 in five basal-like breast cancer cell lines suppressed the expression of WNT5A as well as invasion and tumorsphere formation. These data suggested that BRD4 may modulate metastatic outgrowth. However, similar to the analyses by Andrieu and colleagues, this was not directly tested. Rather, the authors reported that two BETi, JQ1 and MS417, inhibited growth of primary SUM1315 tumors partially via the suppression of WNT5A expression.Only two studies have directly assessed the ability of BETi toimpact the breast cancer metastatic cascade in vivo. We found that JQ1 treatment reduces the number of liver macrometastases in mice with tumors derived from the metastatic TNBC cell line, MDA-MB-231 [109]. However, a second study using two highly metastatic cell lines (Mvt-1 and 6DT1) revealed that while I-BET151 lowered primary tumor weight, it did not suppress the incidence of pulmonary metastasis [23]. Several differences between the models used may explain this discrepancy. MDA-MB-231 cells are a claudin-low human breast cancer cell line that was examined in immune-compromised mice whereas Mvt-1 and 6DT1 murine mammary cancer cells were studied as allografts in immune- competent mice. In addition, the molecular classification of Mvt-1 and 6DT1 cell lines is mixed with both having elements of lumi- nal and claudin-low gene expression signatures [161].

The use of cell lines that have their own unique transcriptomes could account for the apparent differences in the impact of BETi on metastasis in these two studies. Lastly, these studies interrogated metastatic potential to different organs. The impact of I-BET151 on metastasis of Mvt-1 or 6DT1 to the liver or organs other than the lungs was not assessed, and it is possible that BETi modulate microenvironmental sensing in one tissue context but not another. Additional analysis of a broader spectrum of tumor models will be necessary to elucidate the molecular drivers that define the impact of BETi on metastasis.Deregulated cellular metabolism is a hallmark of cancer, with cancer cells having different energy needs than normal cells due to increased cell division and proliferation as well as altered access to nutrients [122]. Very little is known regarding the impact of BETi on cellular metabolism in breast cancer. One study found the BETi, XD14, significantly altered the expression of 67 metabolites in the ER+ MCF7 cell line [162]. These metabolites included amino acids, fatty acids, lipids, and phospholipids and could be grouped into 12 pathways including those that regulate amino acid lev- els. Eight amino acids as well as glucose were elevated following XD14 treatment, suggesting that XD14-treated MFC7 cells con- sumed less energy than vehicle-treated cells. Similarly, we found that “metabolism” was one of the top Reactome pathways altered in HCC70 cells after treatment with JQ1 [44]. To complement these descriptive reports, mechanistic and functional studies are neces- sary to clarify the effect of BETi on metabolism and how this impacts breast cancer pathogenesis.BET proteins act as co-activators or co-repressors depend- ing on their binding partners and cellular context. Considering their critical roles in modulating transcription, it is not sur- prising that gene expression analyses have revealed that BETi alter expression of hundreds of genes in breast cancer cells [28,44,54,57,91,101,103,104,163]. The genes impacted by BETi vary depending on breast cancer subtype and the cell lines used. How- ever, one of the most consistent findings among many of these reports is that BET inhibition induces cell cycle arrest. The majority of studies that performed cell cycle analysis on BETi-treated breast cancer cells found that multiple ER+ and TNBC cell lines arrest in the G1 phase as early as 24 h after drug addition [53–59]. This occurred following the suppression of cell cycle genes [44,104]. Nonethe- less, the mechanism by which cell cycle arrest occurs seems to be subtype, and even cell line, dependent.A major regulator of proliferation in varied cell types is MYC.

In numerous non-breast cancer models, BETi dramatically sup- press expression of c-MYC, and overexpression of c-MYC can reduce sensitivity to BETi [164,165]. Thus, BETi have been touted as “Myc inhibitors.” However, multiple studies have found that nei- ther MYC amplification nor BETi-mediated suppression of c-Myc were involved in the response of TNBC to BETi, because BETi had an inconsistent impact on c-Myc expression in various cell lines representing this subtype of breast cancer [53,54,58,109,110]. Fur- thermore, overexpression of MYC in SUM149, HCC38, and EVSAT cells did not induce JQ1 resistance [92], indicating that modulation of MYC likely contributes only modestly to the BETi responsive- ness of TNBC cells. In contrast, c-Myc suppression may play a more substantial role in the BETi response of ER+ and HER2+ breast cancers. Several studies reported downregulation of c-Myc in cell lines representing both of these subtypes, including those with acquired drug resistance [58,91,101–104]. Furthermore, MYC- overexpressing BT474 cells were less sensitive to JQ1 treatment [101]. It is possible these results could be partially driven by the time point selected, as at least one study showed c-Myc expres- sion initially decreased but then rebounded in BETi-treated MCF7 cells [104]. Another caveat of these studies is that they utilized a limited number of cell lines which may not fully represent the response of the luminal and HER2+ subtypes as a whole. Indeed, qRT-PCR analysis of 24 breast cancer cell lines representing the luminal, HER2+, and TNBC subtypes treated with JQ1 revealed vari- able regulation of MYC mRNA, and the suppression or upregulation of MYC did not correlate with growth sensitivity to JQ1 [92]. Inter- estingly, GSEA of MDA-MB-231 and MDA-MB-468 cells treated with vehicle or OTX015 revealed that MYC target genes were down- regulated following OTX015 treatment even though MYC itself was not suppressed [54]. This could indicate that BETi can regulate MYC- responsive genes through a mechanism other than by the direct transcriptional repression of the MYC gene itself.

In contrast to MYC, we found that BETi profoundly disrupted mitosis in TNBC, partially due to the downregulation of Aurora kinases A and B (AURKA/B) [109]. AURKA and AURKB play crit- ical roles in multiple steps of mitosis, and altered expression of these genes induces polyploidy [166–169]. Indeed, treatment with JQ1 suppresses expression of AURKA and AURKB, leading to polyploidy in MDA-MB-231 cells and multi-nucleation in several TNBC cell lines. The impact of BETi on AURKA and AURKB gene expression is direct. BRD4 binds to the promoter regions of both genes and JQ1 reduces BRD4 recruitment to these loci. In addi- tion, treatment of TNBC cells with an AURKA-selective (MLN8237) or an AURKB-selective (AZD1152) inhibitor phenocopies JQ1; both induced multi-nucleation followed by either senescence or apopto- sis. These data suggest Aurora kinases could potentially be used as biomarkers to predict response to BETi therapy.We further reported that mitotic dysfunction induced by BETi results in mitotic catastrophe, as indicated by the suppressed expression of mitosis/cytokinesis-associated genes, increased mitotic timing, and acquisition of multi-nucleation or induction of apoptosis in or immediately following mitotic exit [44]. The global downregulation of genes involved in mitosis/cytokinesis was mediated, at least in part, by the suppression of the critical mito- sis regulator LIN9 by JQ1. Silencing LIN9 alone suppressed similar cell cycle genes as JQ1 and led to multi-nucleation, indicating that LIN9 is an important regulator of mitotic progression in TNBC that is also a key target of BETi. In addition, genes that were highly cor- related with LIN9 in breast cancer or had a LIN9 binding site in HeLa cells were more likely to be downregulated by JQ1 than those that were not correlated. Interrogation of publically available datasets revealed that LIN9 is amplified and/or overexpressed in two-thirds of basal breast cancers, and its expression is linked to poor progno- sis, underscoring its clinical impact on this disease and its potential utility as a biomarker for predicting BETi responsiveness of TNBCs.

As in other cancers, SEs have been detected in several oncogenes in breast cancer cell lines [44,53,170–172]. Shu, et al. identified 219 SEs in SUM159 cells and 159 SEs in SUM149 cells, and SEs were found at known gene drivers of TNBC, including MYC and HIF1A [53]. Treatment of SUM159 and SUM149 cells with JQ1 led to a rapid (within 12 h) suppression of transcription of SE-associated genes, and the number of SEs increased when cells developed BETi resis- tance. These studies suggested that SE modulation may play a key role in mediating the effects of BETi in breast cancers. However, we found that the impact of BETi on mitosis in TNBC was not related to the suppression of SE-associated genes, as LIN9 and four other JQ1- regulated master mitosis transcription factors were not associated with SEs [44]. Cancer cells are particularly sensitive not only to SE disruption but also to mitotic catastrophe [173]. We thus hypoth- esize that the selectivity of BETi for cancer cells, at least in TNBC, is due to a combination of loss of SEs and mitotic dysregulation via the induction of mitotic catastrophe.

The observed downregulation of critical genes in breast cancer cells following BETi treatment can also result from the prevention of transcriptional elongation rather than disruption of SEs. In a study using ER+ breast cancer cells, Sengupta, et al. found that while BETi did not impact ERα expression or recruitment to promoters and EREs, it does alter transcription of estrogen-target genes [56]. JQ1 only slightly suppressed the recruitment of RNAPII to the promoters of the estrogen-target genes TFF1, GREB1, and XBP1. In contrast, there was a large reduction in RNAPII binding to the bodies of these gene following JQ1 treatment. This indicates that the transcription complex was able to form and initiate transcription in the presence of BETi but transcription elongation was inhibited. BRD4 recruits P- TEFb to the chromatin which releases RNAPII from its paused state at promoters to stimulate elongation [39]. It is likely that the impact of BETi on elongation is due to the loss of P-TEFb presence at gene promoters, thus restraining RNAPII at the promoter and preventing its progression to the gene body.Thus far, two mechanisms of resistance to BETi have been identi- fied in breast cancer. One of these is activation of the PI3K pathway (Fig. 2A). JQ1 did not inhibit proliferation of cell lines derived from mouse mammary tumors with amplified Myc in conjunction with either an activating mutation in PI3K (MCCL-278) or deletion of Pten (MCCL-357), despite suppressing MYC protein expression at higher ( 2 µM) doses [174]. A second BETi, MS417, did suppress growth in both cell lines by at least 50% but only at high ( 4 µM) doses. Similar observations were made using the TNBC cell line SUM159 which has an activating mutation in PIK3CA and ampli- fied MYC [175]. These results indicate that tumors with activated PI3K pathway and high MYC expression could be intrinsically resis- tant to BETi. Supporting this conclusion, analysis of shRNA dropout screens in 82 breast cancer and mammary epithelial cell lines using the algorithm siMEM also identified PIK3CA mutation as a poten- tial BETi resistance mechanism [92]. Cell lines that did not die in response to BETi were more likely to have a mutated form of PIK3CA, and SKBR3 cells overexpressing wild type or mutated PIK3CA were less susceptible to JQ1-induced growth inhibition. Although tumors with altered PIK3CA are less sensitive than their wild type counter- parts, they can still respond to BETi to some degree.