Epigenetics in Breast Cancer

What's New?

Yi Huang; Shweta Nayak; Rachel Jankowitz; Nancy E Davidson; Steffi Oesterreich

Disclosures

Breast Cancer Res. 2011;13(6):225 

In This Article

Acetylation and Methylation of Histones in Breast Cancer

For many years it has been known that post-translational modifications of histone tails determine, in part, which regions of the genome are in an open and thus transcriptionally active conformation, and which are closed and thus transcriptionally inactive. The modifications of histone tails include acetylation, methylation, ubiquitylation, phosphorylation, sumoylation, and ribosylation, each of which can significantly affect the expression of genes.[1] The most studied histone modifications are histone acetylation/deacetylation, and more recently methylation/demethylation. In breast cancer, abnormal histone modification in combination with DNA hypermethylation is frequently associated with epigenetic silencing of tumor suppressor genes and genomic instability.[2,3] Understanding the mechanisms of dysregulation of histone tail post-translational modifications and their contribution to breast tumorigenesis is critically important in the development of novel targeted therapy for breast cancer patients.

Inhibition of Histone Deacetylases as a Therapeutic Approach in Breast Cancer

The dynamic nature of histone acetylation is determined by the counterbalancing activity of histone acetyltransferases and histone deacetylases (HDACs). The HDAC family is divided into zinc-dependent enzymes (classes I, IIa, IIb, and IV, of which there are 11 subtype enzymes) and zinc-independent enzymes (class III, also called sirtuins), which require NAD+ for their catalytic activity.[4] Over the past decade, a number of HDAC inhibitors have been rationally designed and synthesized based on their chemical structures and divided into four groups: hydroxamic acids, cyclic tetrapeptides, short-chain fatty acids, and benzamides.[5] Most of the HDAC inhibitors developed so far are nonselective, and among the most potent inhibitors are those that have been designed to target primarily the zinc cofactor at the active site of the HDACs and to exhibit their effects in the nanomolar or micromolar range.[6,7] Some of these HDAC inhibitors were shown to change the chromatin structure and cause re-expression of aberrantly silenced genes, which in turn is associated with growth inhibition and apoptosis in cancer cells.[8,9] In estrogen receptor (ER)-negative breast cancer cells, inhibition of HDAC activity by specific HDAC inhibitors reactivates ERα and progesterone receptor (PR) gene expression, which are known to be aberrantly silenced.[10–14] Pruitt and colleagues demonstrated that inhibition of class III HDAC SIRT1 using a pharmacologic inhibitor, splitomicin, or siRNA reactivates epigenetically silenced SFRP1, SFRP2, E-cadherin, and CRBP1 genes in human breast cancer cells.[15]

The study of HDAC inhibitors is moving rapidly into a new stage of development that has now started to produce encouraging results in the clinic, particularly in the field of cancer therapy. Vorinostat (SAHA) and romidepsin (FK228) have already been approved by the US Food and Drug Administration for the clinical treatment of cutaneous T-cell lymphoma. Vorinostat is currently under evaluation in several phase II trials in breast cancer,[16,17] including combination therapy of vorinostat with standard cytotoxic agents (for example, paclitaxel), endocrine therapy (tamoxifen), or novel targeted therapy (trastuzumab, bevacizumab).[3,16,17] Other HDAC inhibitors such as MS-275 (entinostat) and LBH-589 (panobinostat) are in phase I/II studies in combination with other agents, such as trastuzumab, in women with metastatic HER2-positive breast cancer.[16]

In addition, increasing evidence suggests that combination treatment with inhibitors of HDAC and DNA methyltransferase (DNMT) results in synergy at clinically tolerable doses that may translate not only into changes in methylation but also to disease response. Preclinical studies of HDAC inhibitors in combination with DNMT inhibitors have shown superior re-expression of silenced genes and increased apoptosis in colon/lung cancer cell lines,[18] reduced tumorigenesis in lung cancer models,[19,20] superior ER re-expression compared with HDAC inhibitor alone in breast cancer cell lines,[12] and restoration of tamoxifen responsiveness.[13,21] In a phase I clinical trial of phenylbutyrate in combination with the DNMT inhibitor 5-azacitidine in myelodysplasia, response was highly correlated with reversal of aberrantly methylated genes.[22] In another phase I trial in nonsmall-cell lung cancer, the combination of a DNMT inhibitor and an HDAC inhibitor was safe and tolerable, and was associated with clinical activity.[23]

An ongoing phase II trial is testing the HDAC inhibitor entinostat (also known as SNDX-275 and MS-275), in combination with 5-azacitidine, in patients with hormonerefractory or triple-negative metastatic breast cancer. The primary endpoint will be the objective response rate; secondary endpoints will be progression-free survival, overall survival, and clinical benefit rate, as well as safety and tolerability. Other analyses will include the pharmacokinetics of 5-azacitidine and entinostat, cytidine deaminase activity, pharmacogenetics, and baseline and change in gene methylation in circulating DNA prior to/following combination therapy (quantitative multiplex methylation specific PCR). The study will also aim to evaluate baseline and change in malignant tissue via mandatory biopsies prior to/following combination therapy of gene methylation of candidate genes (by quantitative methylation specific PCR) and of genomewide methylome, coupled with the study of candidate gene re-expression (RT-PCR). We are at a critical turning point, because results from these critical studies will guide future trials with HDAC inhibitors.

Targeting Histone Lysine Methylation and Demethylation in Breast Cancer

Histone lysine methylation is a reversible process, dynamically regulated by both lysine methyltransferases and demethylases (Figure 2). In general, methylation of histone H3 lysine 4 (H3K4me), H3K36, or H3K79 is associated with active transcription, whereas methylation of H3K9, H3K27, or H4K20 is associated with gene silencing.[1] Histone methylation is regulated in breast cancer in an even more complicated manner than histone acetylation via a large number of chromosomal remodeling regulatory complexes.

Figure 2.

Model of dynamic interplay of enzymes mediating methylation of histone lysines. Methylases are shown in pink and demethylases are shown in brown.

Modification of H3K4 methylation is catalyzed by the Trithorax group of histone methyltransferases, including SET1 and MLL.[24] The activity of Trithorax proteins is balanced by the opposing effects of the Polycomb group factors, another important histone methyltransferase family that mediates methylation usually associated with epigenetic gene silencing.[25] Polycomb group proteins form at least four different complexes, including the maintenance complex PRC1 - composed of RING, HPC, HPH, and BMI1 - and three different initiation complexes, PRC2 through PRC4, which are formed by core component of zeste homolog 2 (EZH2), suppressor of zeste 12 (SUZ12), and Nurf-55.[26,27] EZH2 is a highly conserved histone methyltransferase that specifically targets H3K27 and functions as transcriptional repressor.[28] Tissue microarray analysis of breast cancers identified consistent overexpression of EZH2, which was strongly associated with tumor aggressiveness.[29] Studies from several groups demonstrated that expression of EZH2 is significantly associated with increased proliferation and other features of aggressive breast cancer, such as p53 alterations, c-erbB-2 expression, markers of the basal-like subtype, and glomeruloid microvascular proliferation.[30,31] Finally, in a recent report by Chang and colleagues, EZH2 was shown to repress DNA repair in breast-tumor-initiating cells, potentially leading to expansion of stem-cell-like cells, and finally to breast cancer progression.[32] Collectively, these results suggest that EZH2 might function as a prognostic bio-marker in breast cancer, and might also be a promising treatment target.

Histone lysine-specific demethylase 1 (LSD1, also known as BHC110, AOF2, or KDM1) is the first identified histone lysine demethylase capable of specifically demethylating monoethylated and dimethylated lysine 4 of histone H3 (H3K4me1 and H3K4me2).[33,34] The discovery of LSD1 has revolutionized the concept of histone methylation as a dynamically regulated process under enzymatic control, rather than chromatin marks that could only be changed by histone replacement. The activity of the LSD1-CoREST-HDAC complex has been implicated in tumorigenesis. A recent study using ELISA determined that LSD1 is highly expressed in ER-negative breast tumors, and hence LSD1 was suggested to serve as a predictive marker for aggressive breast tumor biology and a novel attractive therapeutic target for treatment of ER-negative breast cancers.[35] In ER-positive human breast cancer MCF-7 cells, 42% and 58% of all Pol II and ERα-bound promoters, respectively, were found to be bound by LSD1, and the recruitment of LSD1 to the promoters of LSD1+/ERα+ target genes was stimulated by estradiol.[36]

Intriguingly, Perillo and colleagues reported that LSD1-mediated demethylation produces H2O2, which subsequently modifies the surrounding DNA and recruits 8-oxoguanine-DNA glycosylase 1 and topoisomerase IIβ, triggering conformational changes in DNA and chroma-tin that are essential for estrogen-induced transcription.[37] Our recent study demonstrated that LSD1 interacts closely with HDACs in human breast cancer cells. Importantly, inhibitors of histone demethylation and deacetylation exhibit cooperation and synergy in regulating gene expression and growth inhibition, and may represent a promising and novel approach for epigenetic therapy of breast cancer.[38] Recent studies also revealed that LSD1 is able to demethylate nonhistone substrates such as p53 and DNMT1, indicating broader biological functions for LSD1.[39,40]

Subsequent to the discovery of LSD1, other Jumonji C (JmjC) domain-containing proteins were proposed to function as human histone demethylases. These enzymes use α-ketoglutarate and iron as cofactors to demethylate histone lysine residues through a hydroxylation reaction.[41–44] Little is known about the role of JmjC domaincontaining histone demethylase in breast cancer, but recent studies found that PLU-1 (also known as JARID1B or KDM5B) contributes to MCF-7 cell proliferation by facilitating G1 progression. Further, knockdown of PLU-1 led to a significant reduction of MCF-7 cell proliferation and upregulation of expression of certain tumor suppressor genes, including 14-3-3σ, BRCA1, CAV1, and HOXA5.[45] Sharma and colleagues reported that the development of drug-tolerant cancer cells was at least in part mediated by activities of the histone demethylase JARID1A/KDM5A. While these studies focused on epidermal growth factor receptor-targeting small-molecule inhibitors in lung cancer cells, the authors showed that a similar mechanism for resistance existed for other therapies, such as cis-platin. One could thus rationalize that activation of this pathway might be a more widespread phenomenon for the development of drug resistance in JARID1A/KDM5A-expressing tumors.[46,47]

Emerging Therapeutic Potential of Histone Methyltransferase and Demethylase Inhibitors in Breast Cancer

As depicted in Figure 2, histone methylation is the result of a dynamic equilibrium between activities of a number of histone methyltransferases and demethylases. Given the increasing evidence for their role in tumorigenesis, it is no surprise they are being developed and tested as novel treatment targets.

Enhanced activity of histone-modifying enzymes such as LSD1 and EZH2 leads to epigenetic silencing of critical genes, such as tumor suppressor genes, that have been shown to play an important role in breast tumor tumorigenesis. A series of novel compounds function as powerful inhibitors of histone methylation or demethylation and are capable of inducing re-expression of aberrantly silenced genes important in breast tumorigenesis. A list of identified histone methyltransferase and demethylase inhibitors is presented in Table 1. One of the first histone methyltransferase inhibitors developed is chaetocin, which exhibits some selectivity for the SUV39 class of histone methyltransferases.[48] The EZH2 inhibitor DZNep induces robust apoptosis in breast cancer cells, at least in part by including a novel apoptosis effector, FBXO32.[49] SMYD3 is a H3K4-specific methyltransferase that is frequently overexpressed in a variety of cancers, including breast cancer.[50] Novobiocin, known as a HSP90 inhibitor, decreases the expression of SMYD3 and inhibits the proliferation and migration of MDA-MB-231 cells in a dose-dependent fashion.[51]

The structural and catalytic similarities of LSD1 and monoamine oxidase or polyamine oxidase provided the rationale to investigate whether existing monoamine oxidases or polyamine oxidase inhibitors might also act as inhibitors of LSD1. Subsequently, the monoamine oxidase inhibitors tranylcypromine, clorgyline, and pargyline were shown to inhibit LSD1 activity and inhibit growth of breast cancer and prostate cancer cells.[35,52,53] Interestingly, pargyline (Eutonyl; Sigma-Aldrich, St Louis, MO, USA) has already been clinically used for the treatment of vascular hypertension, and tranylcypromine (Parnate; Sigma-Aldrich, St Louis, MO, USA) is a drug used as an antidepressant and anxiolytic agent in the clinical treatment of mood and anxiety disorders. Unless there are toxicities due to the high doses that might be required to inhibit LSD1, one might expect this drug to be tested in the cancer arena soon.

More recently, polyamine-based LSD1 inhibitors were identified and demonstrated to reactivate epigeneticsilenced tumor suppressor genes in cancer cells.[54,55] Treatment with the LSD1-inhibiting polyamine analogues 2d or PG-11144 significantly enhanced global H3K4me2 and altered gene expression in breast cancer MDA-MB-231 cells.[56] Treatment with the LSD1 inhibitor PG-11144 and the DNMT inhibitor 5-aza-2-deoxycytidine resulted in significant inhibition of the growth of established tumors in a xenograft model of human colon cancer in nude mice.[55] N-oxalylglycine, an analog of α-ketoglutarate, has been shown to be an inhibitor of the JmjC domain-containing histone demethylases JMJD2A and JMJD2C.[57] These advances show the promise of using novel compounds that target the histone methylation/demethylation pathway as an innovative approach to breast cancer treatment, and are anticipated to lead to the development of a new generation of therapeutically effective epigenetically-active drugs with considerable clinical potential.

Comments

3090D553-9492-4563-8681-AD288FA52ACE
Comments on Medscape are moderated and should be professional in tone and on topic. You must declare any conflicts of interest related to your comments and responses. Please see our Commenting Guide for further information. We reserve the right to remove posts at our sole discretion.
Post as:

processing....