Epigenetic mechanisms in breast cancer treatment and treatment resistance

Cancer is a group of diseases that are both genetic and epigenetic in nature (see what is cancer). Epigenetic mechanisms regulate many aspects of cancer biology, from carcinogenesis to modulation of the immune response in the tumor microenvironment. Unlike genetic mutations, which are difficult to correct, dysregulated epigenetic mechanisms can be conveniently targeted by small-molecule compounds. Moreover, modulation of the epigenome in various solid (organ) cancers exposes cancer cells to immune system attacks, increasing sensitivity to immunotherapy. These advantages have contributed to the development of epigenetic strategies in cancer treatment over the past decade.

Epigenetic treatments in cancer

Epigenetics-based diagnostic and prognostic tools make a major contribution to oncology. In particular, several "DNA methylation assays" and diagnostic imaging methods are undergoing clinical trials or are already in use. In addition, oncology’s efforts to combat dysregulated epigenetic mechanisms have led to the development of epi-drugs, also defined as drugs targeting epigenetic modulations.

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There are currently treatments including histone deacetylases (HDACis), DNA methyltransferases (DNMTs), EZH2 and IDH inhibitors for solid and hematological tumors: 8 FDA-approved epigenetic treatments, as well as many epi-drugs being tested in clinical trials.

In ER+ (estrogen receptor-positive) breast cancer, the epi-drugs being tested have now reached phase II. In combination with conventional treatments, ER+ breast cancer growth, metastasis and treatment resistance are governed by epigenetic mechanisms; these epi-drugs reflect recent advances in our understanding of those mechanisms.

The incidence (frequency) of invasive breast cancer has continued to rise since 2004. More than 2 million cases were reported worldwide in 2018. Approximately 80% of all breast cancer cases are categorized as ER+ because of ERα receptor expression, and the 5-year overall survival rate of these patients is approximately 90%.

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Because ERα is the primary oncogenic driver of most ER+ cancers, current endocrine-based treatment options include ERα blockade, inhibition of estrogen synthesis and selective ERα degradation. Although endocrine treatments prolong overall survival, one-third of all patients with early-stage ER+ breast cancer show treatment resistance. Targeting chromatin regulators with small compounds to reconfigure the cancer epigenome may resensitize resistant cells to endocrine therapy or to new treatments.

Estrogen subtypes and mechanisms of ERα signaling

Estrogen stimulates many developmental processes, including reproductive maturation and bone growth, as well as energy homeostasis in the body, by modulating insulin sensitivity, food intake and energy expenditure through thermoregulation. Estrogen also coordinates mitogenic and epigenetic mechanisms to regulate mammary gland development.

There are five main estrogen subtypes:

• estrone (E1),
• 17-β estradiol (E2),
• estriol (E3),
• estetrol (E4),
• estrone sulfate (E1s)

E1 is reversibly converted to E2, the biologically more active form, and both represent the main estrogens in the body. However, E3 and E4 can only be detected during pregnancy, with E3 being more predominant. E1 mainly serves as an estrogen reservoir, as it is readily converted in its natural state into its active forms E1 and E2 by steroid sulfatases.

Figure 1: ERα mediates epigenetic changes by interacting and communicating with pioneer factors and coregulators.

a. Schematic representation of the various structural domains in ERα. The LBD (ligand-binding domain) contains a surface for dimerization and coactivator binding. After binding E2, helix-12 within the LBD adopts an active conformation that promotes ERα interactions with coactivators.

b. Pioneer factors such as FOXA1, GATA3, PBX1 and AP-2γ preferentially bind to hypomethylated genomic regions carrying their corresponding motifs. Importantly, these regions are generally marked by low levels of H3K4me1/2, which increase with recruitment of pioneer factors such as FOXA1. Following stimulation with the E2 ligand, pioneer factors facilitate the localization of liganded Erα to chromatin. This leads to activation of gene expression as ERα recruits epigenetic activators such as P300 / CBP, the SWI / SNF complex, PRMTs and EZH2 (through direct contact or via coactivators such as SRC-1/2/3 and the mediator (Mediator)) to deposit activating epigenetic marks such as H4R3me1 and H3K27Ac (solid colors). However, through interactions with corepressors such as LCOR and NCoR1/2, liganded ERα and tamoxifen-bound ERα can also recruit epigenetic repressors, including HDACs and the NuRD complex, to mediate gene repression by removing active epigenetic marks (faded colors).

ERα also accompanies non-genomic E2 signaling pathways. It directly supports the proliferation of ER+ breast cancer cells. Genomically mediated mechanisms following E2 stimulation begin with ERα homodimerization or recruitment to chromatin by binding directly to EREs (estrogen response elements) or indirectly to transcription factors (TFs) through AF (activation function) domains. It is estimated that up to 75% of estrogen-responsive genes require ERα binding to EREs or ERE-like sequences for their expression. In ER+ breast cancer, pioneer TFs such as FOXA1, GATA3, PBX1 and AP-2γ bind specific DNA target sequences in condensed chromatin and facilitate ERα chromatin binding in response to E2 stimulation. Activated ERα can also recruit a group of coactivators or corepressors to mediate gene transcription or repression (Figure 1b).

Epigenetic mechanisms underlying ERα signaling

Important corepressors (substances that inhibit enzyme synthesis) of ERα transcriptional activity include NCoR1, NCoR2 and LCoR (Figure 1b). BRCA1is the best-known ERα corepressor. After binding to the AF2 domain, BRCA1 targets Erα for degradation, thereby downregulating ERα transcriptional activity.

Pioneer TFs are required for E2-dependent recruitment of ERα to chromatin. They bind to chromatin independently of E2, and their depletion significantly reduces E2-induced ERα chromatin binding. The epigenetic and oncogenic roles of pioneer TFs in ER+ breast cancer have been studied. Although not yet recognized as a pioneer TF, GRHL2 shows many functional similarities to FOXA1 and GATA3, such as E2-independent recruitment to chromatin and regulation of ERα target genes, suggesting a potential role as a pioneer TF. Finally, components of PRC1 and PRC2 have been found to exhibit E2-dependent chromatin recruitment and to promote E2-induced ERα target gene expression in breast cancer cells.

Epigenetic processes in normal mammary gland development are derailed in breast cancer

Numerous signaling pathways and chromatin regulators, as well as hormonal factors coordinating the balance between self-renewal, differentiation and tissue integrity, are involved in mammary gland development. The mammary gland develops in three main stages: embryonic, pubertal and reproductive. Embryonic mammary gland development is coordinated by signaling pathways such as WNT and Hedgehog (HH), while the pubertal and reproductive stages are under hormonal control.

Reactivation of developmental pathways is a common feature of different cancer types and, in breast cancer, is closely associated with maintenance of the mammary gland stem cell population (Figure 2). Research over the past decade, with the emergence of technological advances such as next-generation sequencing, has revealed that derailment of epigenetic processes important during mammary development also plays a significant role in breast cancer progression. Functional crosstalk between epigenetic processes and developmental signaling pathways that cause breast cancer has therefore been examined.

Figure 2: Development of embryonic mammary glands

In normal mammary epithelium, DKK3 binds to LRP, a WNT pathway coactivator of Frizzled, preventing pathway activation in the presence of the WNT ligand. E-cadherin binds to cytoplasmic β-catenin, which is degraded by GSK3β in the absence of WNT activation. The SHH promoter (encoding the Hedgehog ligand SHH) is hypermethylated and the Hedgehog pathway is silenced. In breast cancer, however, the DKK3 promoter becomes hypermethylated, leading to its downregulation. In the absence of DKK3, LRP coactivates Frizzled in the presence of the WNT ligand, leading to DSH phosphorylation, which inhibits catenin degradation by GSK3β. E-cadherin is also downregulated through promoter methylation. In addition, the SHH promoter becomes hypomethylated, thereby upregulating SHH expression and activating the Hedgehog pathway through GLI1. Activation of the WNT and Hedgehog pathways leads to stem cell renewal, EMT, metastasis and tamoxifen resistance.

Epigenetic modulation of WNT signaling in ER+ breast cancer

Abnormal activation of WNT signaling leads to the development and progression of various cancer types, including breast cancer. Epigenetic silencing of WNT antagonist genes, including SFRP and DKK, causes mammary tumor formation. Mechanistically, silencing of these genes through DNA methylation is the main cause of persistent WNT signaling in breast cancer and is associated with poor prognosis. These changes lead to constitutive activation of β-catenin, resulting in increased proliferation associated with stem cell renewal and disease recurrence (Figure 2).

Interestingly, in a study of 96 patients with breast cancer, promoter methylation of DKK3, a member of the DKK family, was increased in tumors from patients with advanced disease, patients with lymph node metastases and patients positive for ERα (42 of 47 patients were ER+). Given that the WNT and ERα signaling pathways are linked particularly through the Polycomb protein EZH2, it is thought that a lack of WNT inhibition by DKK may proceed to the ERα signaling pathway to trigger growth and survival, thereby linking DKK3 promoter methylation with ERα positivity. Efforts to restore ERα expression clinically using hypomethylating agents have nevertheless been unsuccessful. However, studies on targeting derailed epigenetic regulatory circuits that activate developmental programs such as WNT signaling are ongoing.

HH signaling and Polycomb complexes

HH signaling is another important developmental pathway dysregulated by epigenetic mechanisms in mammary tumor formation. Promoter hypomethylation of the HH ligand SHH or its downstream receptor leads to increased ligand-dependent activation of the pathway and uncontrolled cell division driving cancer progression (Figure 2). HH signaling also induces expression of PCGF4 (BMI1), a component of the PRC1 complex, to promote self-renewal of normal and tumorigenic mammary stem cells. Breast cancer stem cells are linked to endocrine treatment resistance, but whether the emergence of stem-like properties in resistant cells results from expansion of pre-existing embedded tumor cells or dynamic reprogramming mediated by epigenetic changes remains debated. It is believed that targeting epigenetic agents such as DNMTs to restore HH antagonistic regulation, in combination with drugs that directly target HH signaling, could potentially modulate cancer stem cell survival and differentiation.

Recent data show that inhibitors of EZH2, the enzymatic core of the PRC2 complex, which are in advanced clinical trials in many tumor types including triple-negative breast cancer, mediate repression of the GATA3- ERα signaling pathway, inducing a luminal-like phenotype sensitive to endocrine treatment agents such as fulvestrant. These results suggest that targeting Polycomb proteins, particularly PCGF4, has a therapeutic analogue for directing a phenotypic shift in endocrine-resistant breast cancer stem cells toward an endocrine treatment-sensitive state.

In summary, these observations suggest that epigenetic mechanisms playing vital roles in normal development are altered in neoplastic tissues and are therefore attractive candidates as biomarkers and therapeutic targets.

Endocrine treatments target the oncogenic E2-ERα pathway

Steroid hormone signaling was first linked to breast cancer progression in 1896, when surgical removal of both ovaries in patients with breast cancer resulted in tumor regression. Endocrine treatment, a standard in ER+ breast cancer, refers to interventions that suppress estrogen production as well as strategies directly targeting ERα and includes three main categories: selective estrogen receptor modulators (SERMs), selective estrogen receptor degraders (SERDs) and aromatase inhibitors (AIs).

Tamoxifen, the first clinically approved ERα-targeting agent, has been a mainstay treatment option for patients with both early and advanced breast cancer for over thirty years. Tamoxifen is a SERM that competes with E2 for ERα binding and prevents coactivator recruitment mediated by the LBD of ERα. Despite the success of tamoxifen treatment, one-third of women treated with tamoxifen for 5 years are expected to experience disease recurrence within 15 years. However, because most of these patients retain ERα expression, they remain sensitive to SERDs such as fulvestrant.

In postmenopausal women, E2 is no longer synthesized in the ovaries. Instead, it is produced by aromatization of testosterone and androstenedione in various tissues, including the liver, subcutaneous fat and stroma surrounding normal breast cells, as well as mammary epithelial cells and fibroblasts of primary breast tumors. AIs act to reduce high E2 levels in breast cancer tissue by inhibiting aromatase activity and can be classified as steroidal or nonsteroidal. Steroidal AIs bind irreversibly to aromatase, whereas nonsteroidal AIs bind competitively and reversibly. Compared with tamoxifen, which is generally prescribed for premenopausal breast cancer patients, fulvestrant and AIs are primarily for postmenopausal cases, alone or in combination with other endocrine or targeted agents such as CDK4/6 inhibitors.

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ERα is the primary oncogenic driver in ER+ breast cancers. Other genetic alterations, such as cyclin D1 overexpression in 50% of breast cancers (Figure 3a) and loss of CDKN2A, cause disease progression and therapeutic response. For example, cyclin D1 overexpression triggers cell cycle progression through G1 / S, leading to increased activation of CDK4/6 and phosphorylation of RB. After years of endocrine monotherapy, approval of targeted treatments against mTOR (everolimus), PI3K (alpelisib) and CDK4/6 (palbociclib, ribociclib, abemaciclib) has led to significant progress in disease management. Numerous clinical studies have demonstrated the efficacy of CDK4/6 inhibition, and as a result, CDK4/6 inhibitors, alone or combined with AIs or fulvestrant, have been established as standard treatment options for both endocrine-sensitive and endocrine-resistant ER+/HER2- metastatic breast cancers.

Figure 3: Selected genomic alterations mediating therapeutic resistance in ER+ breast cancer

a. NF1 is a GTPase that inhibits RAS activation of the MAPK pathway (1) and functions as an ERα corepressor at the CCND1 gene encoding cyclin D1 (2). NF1 loss in treatment-resistant cells causes increased activation of the MAPK signaling pathway and overexpression of cyclin D1, which promotes the G1 / S transition by activating CDK4/6. MEK inhibitors and CDK4/6 inhibitors can overcome overexpression of MAPK and CDK4/6, respectively. Fulvestrant may also be used in cases of NF1 loss.

b. In treatment-sensitive ER+ breast cancer, ERα dimerizes on binding E2 and is phosphorylated by CDK7. ESR1 mutations frequently occur in the LBD of resistant cells and lead to constitutively active ERα mutations that mediate gene expression through E2-independent coactivator interactions. Proposed therapeutic strategies include inhibition of CDK7 with THZ1 and inhibition of SRC coactivators.

c. CYP19A1 encodes aromatase, the enzyme that converts testosterone to estrogen. Patients who relapse after AI treatment frequently acquire CYP19A1 overexpression, leading to increased estrogen and treatment resistance. Patients with CYP19A1 overexpression may be treated with irreversible steroidal AIs rather than reversible AIs, as well as fulvestrant.

d. FOXA1 is often amplified or mutated in treatment-resistant ER+ breast cancer, leading to increased FOXA1 activity and redistribution of FOXA1 to repeat enhancers. These repeat regions are enhanced near TF motifs such as AP-1, STAT5 and SOX9 and promote a metastatic transcription. Directly targeting FOXA1 is difficult. However, specific inhibitors can target FOXA1 downstream genes that mediate metastasis, such as HIF-2α.

Mechanisms of endocrine treatment resistance and potential alternative strategies

Despite the efficacy of endocrine treatment in ER+ breast cancer, resistance develops in approximately 25% of early-stage patients and nearly all patients with metastases, leading to poor clinical outcomes. Endocrine treatment resistance can be categorized as intrinsic (de novo – anew) or acquired. Patients with advanced breast cancer generally show progression at different sites that are clonally distinct and result from selection of genetic alterations under therapeutic pressure. This selective pressure leads to expansion of clones harboring mutations in the drug target itself, as well as in mitogenic signaling pathways and genes encoding epigenetic factors. In addition, microenvironmental conditions such as hypoxia can alter the epigenetic landscape and contribute to disease evolution, particularly because epigenetic enzymes are also nutrient and oxygen sensors. Specifically, clones with mutations in epigenetic machinery exhibit defects in transcription and DNA repair and replication, leading to malignant tumor self-renewal, blocked differentiation and evasion of cell death, all of which increase tissue invasion. Overcoming these outcomes is a major challenge in the therapeutic field of ER+ breast cancer.

Cell cycle changes in endocrine-resistant breast cancer

Alterations in cell cycle control are frequently linked to drug resistance. In addition, CCND1 amplification and high CDK4 levels in tumors are associated with endocrine resistance. Despite the efficacy of CDK4/6 inhibition, 10-20% of cancers are not sensitive, and a large percentage (70-80%) become resistant after 12-36 months of treatment.

Resistance to CDK4/6 inhibition is characterized by loss of tumor suppressors such as RB and FAT1. In particular, loss-of-function mutations in FAT1, a Hippo pathway receptor, are observed in 2% and 6% of primary and metastatic tumors, respectively. In addition, hyperactivation of RTK-RAS signaling and aberrant activation of CCNE1-CDK2, a downstream effector of CDK4/6, restore RB phosphorylation, drive resistance and also reduce the response to palbociclib.

There are more than 100 active clinical studies testing the efficacy of CDK4/6 inhibitors across many cancer types and treatment strategies, and they are regarded as a candidate for future cancer treatment. More specifically, in breast cancer, administration of CDK4/6 inhibitors, alone or combined with endocrine therapy or immunotherapy agents, is being actively investigated.

Changes in ERα coregulators

Genetic alterations can disrupt the balance between ERα coactivators and corepressors and are associated with poor prognosis and endocrine treatment resistance. Tamoxifen causes an ERα conformational change that blocks coactivator recruitment and promotes recruitment of corepressors such as NCoR1 and NF1 (neurofibromin). Inactivating mutations or deletions of NCoR1 and NF1 are among the most frequent genetic alterations in metastatic ER+ breast cancer. For example, NF1 drives endocrine treatment resistance through the combined effect of loss of GTPase activity and its ERα transcriptional corepressor role, and their levels are associated with response to endocrine treatment agents alone or combined with CDK4/6 or MEK inhibitors (Figure 3a).

Epigenetic factors causing endocrine-resistant breast cancer

Whole-genome sequencing studies have shown that epigenetic factors are among the most commonly mutated genes in human cancers. Among these, inactivating mutations and loss of SWI / SNF subunits are the most frequent genetic alterations in many cancer types. In breast cancer, loss-of-function ARID1A mutations are increased in the endocrine-resistant metastatic setting, suggesting that they may mediate endocrine resistance, as reflected by poor response to SERDs in patients with ARID1A mutations. Mechanistically, ARID1A loss reduces chromatin accessibility and binding of TFs controlling luminal cell fate, as well as binding of ERα and FOXA1 to chromatin (Figure 4). It is hypothesized that prolonged ERα suppression may cause the emergence of clones with ARID1A-inactivating mutations to trigger a luminal-to-basal phenotypic transition. Consequently, the increased frequency of ARID1A mutations in endocrine-resistant breast cancer and their prevalence in other cancers increase the need for therapeutic strategies targeting ARID1A-mutant cancers.

In ovarian cancer, ARID1A mutations and EZH2 inhibition are synthetically lethal, an effect further enhanced by HDAC2 inhibition (Figure 4). In ARID1A-deficient cells, HDAC2 is recruited to ARID1A / EZH2 cotarget genes, an inhibitor of PI3K/AKT signaling, leading to aberrant activation of this mitogenic pathway. These two events, namely ARID1A loss of function and activated PI3K/AKT signaling, are commonly observed in endocrine-resistant breast cancer cells. It is therefore suggested that targeting EZH2 in ARID1A-mutant breast cancer may be a valid therapeutic option to investigate (Figure 4).

Figure 4: Various dysregulated epigenetic pathways in treatment-resistant ER+ breast cancer that are potential targets for new epigenetic therapeutics

Blue and yellow panels: The PI3K/AKT pathway can phosphorylate DNMT1, stabilizing it on chromatin and leading to maintenance of DNA hypermethylation. EZH2 is also phosphorylated by PI3K/AKT, depleting the genome of H3K27me3. In addition, KMT2D phosphorylation by PI3K/AKT depletes H3K4me1 / 2, reducing FOXA1 (and therefore ERα) chromatin binding and leading to hormone treatment resistance. These aberrant epigenetic pathways can be targeted by PI3K inhibitors. Furthermore, DNMT1 stabilization and EZH2 inhibition can be targeted by DNA hypomethylating agents and HDAC inhibitors, respectively.

Purple panel: Recruitment of HDAC to the ESR1 promoter leads to reduced H3K27ac, causing DNMT1-mediated promoter hypermethylation and drug resistance through ESR1 downregulation. HDAC inhibition (entinostat) can be used to reverse HDAC-mediated ESR1 downregulation.

Orange panel: KDM5 (KDM5A / B) is a family of histone H3 lysine 4 demethylases associated with therapeutic resistance in different cancer types. Increased activity of KDM5 enzymes leads to reduced H3K4me3 levels and consequently greater transcriptional heterogeneity. In particular, high KDM5B expression levels are associated with poor prognosis in ER+ breast cancer. Inhibitors that modulate the activity of KDM5 family members may improve response to endocrine therapy agents such as fulvestrant.

Green panel: KMT2C loss redistributes ERα to AP-1-regulated genes, promoting hormone-independent but Erα -dependent transcription, suggesting that SERDs (fulvestrant) and CDK4/6 inhibition may be viable therapeutic options.

Red panel: ARID1A is a component of the SWI / SNF complex, and its mutation leads to restricted chromatin accessibility for ERαα and FOXA1 at genes regulating luminal cell fate, while also promoting a shift from a luminal to a basal-like phenotype. ARID1A mutations can be targeted with EZH2, HDAC, BET and PI3K/AKT inhibitors.

Epigenetic pathways in endocrine treatment

Epigenetic dysregulation is an important factor causing tumorigenesis and drug resistance. To date, epi-drugs have largely been limited to hematological malignancies, with little success in solid tumors. Plasticity associated with epigenetic reprogramming increases the overall fitness of cancer cells and makes personalized cancer treatment more complex. Various preclinical and clinical evidence supports synergistic effects of epi-drugs with different therapeutic modalities, including immunotherapy, radiotherapy and endocrine treatment. In fact, the development of small molecules targeting chromatin regulators is one of the most active areas of current drug discovery efforts. Interestingly, recent efforts suggest that RNA modification machinery is important for cancer progression and therapeutic resistance, and its potential as candidate targets for new treatments has been revealed.

Epigenetic dysregulation is also common in endocrine-resistant breast cancer. For example, promoter hypermethylation of ESR1 causes loss of ERα expression in approximately 20% of patients who progress on tamoxifen. Entinostat (HDACi) and letrozole (AI) can restore ERα and aromatase expression in ER− breast cancer cell lines, resulting in growth inhibition and demonstrating the efficacy of epigenetic intervention to induce endocrine treatment sensitivity (Figure 4). However, restored ERα expression has never been observed in the clinical setting, suggesting that in ESR1 silencing other repressive mechanisms play a role.

Tamoxifen-resistant cells are also characterized by increased acetylation of histones, TFs and heat shock proteins catalyzed by HATs such as P300 / CBP. Interestingly, in castration-resistant prostate cancer, A-485, a selective catalytic p300 / CBP inhibitor, inhibits the androgen receptor transcription program. Whether targeting the balance between HAT and HDAC activities is similarly effective against ERα-mediated transcription in breast cancer should be tested.

Epigenetic agents also activate cell death mechanisms in response to endocrine treatment. Tamoxifen treatment induces autophagy in ER+ breast cancer cells, promoting survival and the emergence of tamoxifen-resistant breast cancer. This phenotype can be combated with a combination of HDACi and tamoxifen, which redirects these cells toward apoptosis by downregulating BCL2 and inducing expression of the proapoptotic proteins BAX and BAK. Furthermore, epigenetic treatments such as HDACis show promising results in combination with tamoxifen to restore endocrine sensitivity and are currently in clinical trials combined with CDK4/6 inhibitors and AIs.

Emerging evidence also indicates that mutations in epigenetic factors such as histone methyltransferases are common events and that interest in other epi-drugs targeting a wide variety of chromatin regulators is growing. For example, histone methyltransferase KMT2C is considered one of the nine most commonly mutated driver genes in hormone receptor-positive metastatic breast cancer. KMT2C loss causes downregulation of ERa-dependent gene expression and relocalization of ERα to AP-1-regulated genes to maintain hormone-independent growth (Figure 4).

Oncogenic signaling through PI3K/AKT has a direct effect on the epigenetic balance, suggesting that combining PI3K/AKT inhibitors with epigenetic drugs is a candidate therapeutic strategy.

Recent advances in cellular technologies enable important questions to be addressed, such as the contribution of cell-to-cell variability to resistance. In ER+ breast cancer, KDM5B, an H3K4me3 demethylase, regulates cellular transcriptomic heterogeneity. KDM5B is commonly amplified and overexpressed in luminal ER+ breast tumors, leading to increased transcriptomic heterogeneity that causes endocrine treatment resistance. Inhibitors of this enzyme have also been shown to increase sensitivity to fulvestrant in hormone-sensitive and endocrine-resistant cell lines, suggesting their potential efficacy in the clinical setting.

Endocrine treatment reduces ER+ breast cancer mortality and recurrence, but unfortunately, in most cases the disease progresses to an incurable state. Increased genetic alterations after endocrine treatment support the use of combination strategies with agents such as CDK4/6 or mTOR inhibitors, which results in resistance. Epi-drugs provide an opportunity to reconnect dormant cells to a proliferative and therapeutically sensitive state. Although there is much excitement about using these agents, several obstacles remain on the path to their clinical use. Unfortunately, clinical results do not meet the expectations generated in the preclinical setting in terms of efficacy and toxicity. These observations may be attributed to epigenetic regulators having many substrates, including histone and nonhistone proteins. Detailed investigation of epi-drug scheduling and dosing is required to increase the benefits of combined therapeutic approaches and reduce toxicity levels. The reversibility of epigenetic changes also remains a primary concern because tumor suppressor genes may be re-expressed in the absence of epi-drugs or through an excess of mechanisms.

Summary and unanswered questions

Endocrine treatment has indisputably been established as an indispensable option for treating hormone-sensitive breast cancers. However, a serious need remains to develop approaches targeting the seemingly inevitable resistant phenotype. Recent advances in the epi-drug field are evidence of epigenetics-based treatments entering a new era for screening and treating multiple diseases, including breast cancer. As with every new field of research, studies in this area have raised new questions as well as new discoveries. Here are several important points awaiting answers:

1. What are the characteristics of the cell from which a breast tumor originates?
2. Do epigenetic mechanisms mediate clonal selection at different metastatic sites?
3. What is the role of the mesenchymal space in breast cancer progression? Which epigenetic mechanisms maintain the breast tumor microenvironment?
4. Can we exploit the dynamic nature of epigenetic changes to design short-term therapeutic strategies in efforts to avoid selection toward a resistant phenotype, or do the mechanisms underlying epi-drugs also cause resistance to emerge?
5. Can we use epigenetic signatures to monitor disease progression and response to treatment?