Histone modifying enzymes involved in metastasis.
Metastasis represents one of the most devastating aspects of cancer. Epithelial to mesenchymal transition (EMT) has been shown to play a critical role in tumorigenic metastasis. During metastatic progression, both genetic and epigenetic modifications endow cancer cells with properties that modulate the capacity for metastatic success. Histone modification is profoundly altered in cancer cells and contributes to cancer metastasis by controlling different metastatic phenotypes. Here, we first review histone modifications and discuss their roles in EMT and metastasis, with a particular focus on histone methylation and acetylation. Second, we review the major histone modification enzymes that control chromatin in cancer metastasis. Third, we discuss the transcriptional regulation concerted by these enzymes with EMT transcription factors at different molecular layers. Finally, we discuss pharmacologic manipulation of histone modification enzymes for metastasis treatment. A comprehensive understanding of histone modification in metastasis will not only provide new insights into our knowledge of cancer progression and metastasis, but also offer a novel approach for the development of innovative therapeutic strategies.
- histone modification
Approximately 90% of cancer deaths are caused by metastasis . Cancer metastasis is an exceedingly complex process involving tumor cell motility, intravasation, and circulation in the blood or lymph system, extravasation, and growth in new tissues and organs [2, 3]. During invasion, tumor cells lose cell–cell adhesion, gain mobility and leave the site of the primary tumor to invade adjacent tissues. In intravasation, tumor cells penetrate through the endothelial barrier and enter the systemic circulation through blood and lymphatic vessels. In extravasation, cells that survive the anchorage-independent growth conditions in the bloodstream attach to vessels at distant sites and leave the bloodstream. Finally, in metastatic colonization, tumor cells form macrometastases in the new host environment [2, 3]. All of these steps, from initial breakdown of tissue structure, through increased invasiveness, and ultimately distribution and colonization throughout the body, are developmental characteristics of the processes, epithelial to mesenchymal transition (EMT) and mesenchymal to epithelial transition (MET). EMT is a distinctive morphogenic process that occurs during embryonic development, chronic degeneration and fibrosis of organs, and tumor invasion and metastasis [4, 5, 6]. The similarity of genetic controls and biochemical mechanisms that underlie the acquisition of an invasive phenotype and the subsequent systemic spread of cancer cells highlights the concept that tumor cells usurp this developmental pathway for metastatic dissemination. In total, EMT provides tumor cells with the proclivity for early metastasis, renders them resistance to therapeutics and endows cells with cancer stem cell (CSC)-like traits .
The hallmark of EMT is the loss of E-cadherin expression, an important caretaker of the epithelial phenotype. Loss of E-cadherin expression is often correlated with the tumor grade and stage because it results in the disruption of the cell–cell adhesion and an increase in the nuclear β-catenin. Several transcription factors have been implicated in the regulation of EMT, including the zinc finger proteins of the SNAIL family (SNAIL1/2/3), the basic helix–loop–helix (HLH) factor TWIST (TWIST1/2, E12/E47), and two double zinc finger and homeodomain ZEB family (ZEB1/ZEB2). These factors act as a molecular switch for the EMT program by repressing a subset of common genes that encode cadherins, claudins, integrins, mucins, plakophilin, occludin and ZO1, and thereby induce EMT.
EMT is a dynamic process that preserves plasticity . In this instance, the reprogramming of gene expression provides a rapid and dynamic regulatory mechanism to switch between the epithelial and mesenchymal conditions during cancer progression. Consistent with this, these EMT-activating transcriptional factors (EMT-TFs) are liable proteins that turn over rapidly and do not have long residence times at their binding sites. Interestingly, disseminating cells orchestrate a metastatic cascade without a concomitant need for genomic mutations, which indicates that this dissemination is epigenetically templated. Both EMT and epigenetic modification (DNA methylation and histone modifications) are dynamic and efficient processes during development, differentiation and carcinogenesis. These studies indicate that the epigenetic mechanism plays an important role in modulating the induction of EMT and tumor metastasis.
2. Epigenetics and histone modification
2.1. Epigenetic and chromatin structure
The term “epigenetics” was first coined by Conrad H. Waddington in his Principles of Embryology textbook in 1942 to designate a process in which gene regulation modulated development. The final definition of epigenetics was confirmed in the Epigenetic Meeting held by the Banbury Conference Center and Cold Spring Harbor Laboratory in 2008 as “a stably heritable phenotype resulting from changes in a chromosome without alterations in the DNA sequence.” In general, epigenetic regulation includes changes that impact histone modification, DNA methylation, histone variants, chromatin looping, noncoding RNAs and nucleosomal occupancy and remodeling.
Genomic DNA is tightly packaged in chromatin by both histone and nonhistone proteins in the nucleus of eukaryotic cells. The basic chromatin subunits, nucleosomes, are formed by wrapping 146 base pairs (bp) of DNA around an octamer of four core histones: H2A, H2B, H3, and H4. Whereas the nucleosomal core is compact, eight flexible lysine-rich histone tails protrude from the nucleosome that modulate internucleosomal contacts and provide binding sites for nonhistone proteins. From the perspective of gene transcription, chromatin structure can be divided into two distinct categories: euchromatin and heterochromatin. “Euchromatin” is an open chromatin structure that affords accessibility of transcription factors to DNA, resulting in gene activation. In contrast, “heterochromatin” is a closed chromatin structure with a low interaction between transcription factors and the genome, leading to gene repression.
2.2. Histone modifications and histone code hypothesis
The histone code hypothesis was first proposed by Strahl and Allis in 2000. They suggested that “multiple histone modifications, acting in a combinatorial or sequential fashion on one or multiple histone tails, specify unique downstream functions” . The histone “language,” based on this “histone code,” is encoded in these modifications and read by chromatin-associated proteins. So far, several histone post-translational modifications (PTMs) have been identified, including acetylation, methylation, phosphorylation, ubiquitination, sumoylation, ADP ribosylation, proline isomerization, biotinylation, citrullination and their various combinations . These modifications constitute a unique “code” to regulate histone interactions with other proteins and thereby allow modification (either overcoming or solidifying) of the intrinsic histone barrier to transcription. Accordingly, with these modifications, the various proteins that add, recognize and remove these PTMs, termed writers, readers and erasers, respectively, have been identified and structurally characterized. While “writer” and “eraser” enzymes modify histones by catalyzing the addition and removal of histone PTMs, respectively; “reader” proteins recognize these modified histones and ‘translate’ the PTMs by executing distinct cellular programs. In addition, numerous core histone chaperones also facilitate core histone deposition or removal from chromatin. Histone modifications control dynamic transitions between transcriptionally active or silent chromatin states, and regulate the transcription of genetic information encoded in DNA (the “genetic code”) . Analyses of genome-wide profiles of histone modifications and gene expression identified three distinct types of configurations: repressed, active and bivalent. First, the closed chromatin configuration is linked with suppression of gene transcription, the repressed state. Second, an open chromatin configuration is associated with active gene transcription, the active state. Third, bivalent chromatin consists of domains that have both repressive and active histone markers, predominately on developmental genes, which allows phenotypic plasticity before committing to a specific cell fate.
During EMT, histone modifications provide a regulatory platform to orchestrate the repression or activation between epithelial and mesenchymal genes. Here, we only focus on the well-studied histone acetylation and methylation, and discuss their diverse regulation and role in transcriptional reprogramming of tumor metastasis (Table 1).
3. Histone acetylation
Evidence has established that histone acetylation is associated with gene activation. A genome-wide study demonstrated that all forms of histone acetylation are positively correlated with gene expression . Histones contain amino acids with basic side chains that are positively charged and attracted to the negatively charged genomic DNA. Ultimately, histone acetylation reduces the positive charge on histones and decreases the interaction between nucleosomes and DNA. Generally, histone acetylation is greater in the promoters of active genes and influences both the initiation and elongation of gene transcription. Histone acetylation also stabilizes the binding of chromatin remodeling factors at promoter regions and induces nucleosomes unfolding as well as reduces nucleosome occupancy. The acetylation state of a chromatin leads to the structural modification of the nucleosome. Acetylated (or hyperacetylated) chromatin is in a relaxed confirmation and associated with active transcription. In contrast, deacetylated (or hypoacetylated) chromatin is condensed and supercoiled, and is associated with transcriptional silencing (and, in the context of cancer, the inhibition of tumor suppressor genes).
Histone acetylation is a rapid and reversible process controlled by histone acetyltransferases (HATs) and histone deacetylases (HDAC)s. The HATs transfer acetyl groups from acetyl-coenzyme A (CoA) to the ε-amino groups of lysine residues in histone tails, which results in gene activation. HATs contain a bromodomain that recognizes and binds to acetylated histones, categorized into three major families, GNAT (GCN5 and PCAF), MYST (Tip60 and MOF), and CBP/p300. The HDACs remove acetyl groups from lysine residues, leading to gene silencing. Sequence homology, subcellular location, and the features of the catalytic site have been used to classify the 18 members of the human HDAC family into 4 groups: class I (HDACs 1, 2, 3, and 8), class II (HDACs 4, 5, 6, 7, 9, and 10), class III (SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, and SIRT7), and class IV (HDAC11) . Class I HDACs have sequence homology to class II HDACs and class IV HDACs but not class III HDACs. Class I, II, and IV HDACs are zinc-dependent, whereas class III HDACs are nicotinamide adenine dinucleotide (NAD)+-dependent. Genome-wide mapping of the binding of HATs and HDACs to the human genome demonstrate that these enzymes regulate the activation and repression of transcription, respectively. The dysfunctional balance between acetylation and deacetylation is clearly associated with human disease and tumorigenesis.
p300 cooperates in an epigenetic manner with a DOT1L-c-Myc complex to induce EMT in breast metastasis . The elevated level of p300-DOT1L-c-Myc is associated with the acquisition of CSC-like properties during breast carcinogenesis, which implies that p300 functions as a potential oncogene to influence the clinical outcome of breast cancer. In addition, transforming growth factor-beta (TGF-β) and WNT co-operated to mediate EMT. TGF-β induces the translocation of β-catenin to the nucleus where it binds to T- cell Factor (TCF); this complex recruits p300/CBP to assemble a transcriptional complex on target gene promoters that promotes EMT signaling. Intriguingly, over-expression of SNAIL/SLUG up-regulates TGF-β-receptor 2 (TGFBR2) expression with an increase of H3K9 acetylation on TGFBR2 promoter to increase TGF-β signaling . In contrast, however, p300 was reportedly recruited by the hepatocyte nuclear factor (HNF) 3 to the E-cadherin promoter, increasing expression, and thus reducing the metastatic potential of breast cancer cells . Similarly, the p300-CBP-associated factor (PCAF) has functions that can differ among cancer types. PCAF is an anti-oncogene and its expression is down-regulated and negatively correlated with tumor metastasis in hepatocellular carcinoma (HCC) . This complex plays an important role in suppressing EMT and HCC metastasis and by targeting Gli1 . However, it was also reported that PCAF acetylates the enhancer of zeste homolog 2 (EZH2) at K348 to augment EZH2 stability, and thus promotes lung cancer cell migration and invasion . These reports indicate that the role of PCAF is context-dependent. In several breast cancer cell lines, hMOF catalyzes promoter H4K16 acetylation, which is critical to maintain expression of EMT-related tumor suppressor genes . Consistent with this, MOF also acetylates the histone demethylase lysine-specific histone demethylase 1 (LSD1), to suppress EMT, indicating that MOF is a critical suppressor of EMT and tumor progression . Recently, we found that Tip60 appears to be an important regulator of TWIST activity by acetylating at H3K73 and H3K76 of the GK-X-GK motif, resulting in an interaction between BRD4 and TWIST, hence promoting the aggressiveness of basal-like breast cancer (BLBC) .
Dysfunctional class I HDAC expression and activity is associated with cancer metastasis. HDAC1 regulates invasiveness by increasing matrix metalloproteinase (MMP) expression. Furthermore, HIF-2α is a transcriptional regulator of the
Recently, the clinical relevance of HDACs and the therapeutic potential of HDAC inhibitors (HDACi) have been reported. HDACi can generally be classified into hydroximates, cyclic peptides, aliphatic acids, and benzamides , and grouped according to their specificity. Thus far three HDACi: vorinostat (SAHA), romidepsin (Istodax) and PTCL (Belinostat or Beleodaq) are approved by the FDA for some T-cell lymphomas . However, these molecules have not produced favorable and expected outcomes in solid tumors. Currently, a number of small molecules HDACi were investigated in clinical trials with variety of solid neoplasms, including breast cancer, either alone or in combination with hormonal treatments. Entinostat (MS-275), a benzamide with high specificity for the class I HDACs, is currently in a phase II/III trial for advanced ER+ breast cancer [34, 35]. Vorinostat exerts EMT reversal effects by restoring the expression of E-cadherin. An expanded screen on 41 HDACi further identified 28 HDACi compounds, such as the class I-specific inhibitors Mocetinosat, Entinostat and CI994, that restore E-cadherin and ErbB3 expressions in ovarian, pancreatic and bladder carcinoma cells . Mocetinostat, but not other HDACi, specifically interferes with ZEB1 function, restores miR-203 expression, represses stemness properties, and induces sensitivity against chemotherapy by restoring histone acetylation on the E-cadherin promoter . Given that persistent genes activation may require targeting of multiple epigenetic silencing machineries, a combination of HDACi with anticancer drugs and/or radiotherapy demonstrate synergistic or additive effects in clinical trials. For example, HDACi have been utilized in combination with 5 Aza-dC as a synergistic strategy . However, recent reports also found that HDACi could promote EMT in prostate and nasopharyngeal cancer cells [39, 40], indicating the application of HDACi in anti-cancer therapy is cancer-context dependent and may limit application.
4. Histone methylation
Histone methylation occurs at specific lysine or arginine residues on the histone tails. This modification is associated with either transcriptional activation or repression. Histone methylation does not change the electrostatic charge of histones or affect the chromatin structure. The functional effects of histone methylation are affected by both the position of the modified residues and number of methyl groups. Histone methyltransferases (HMTs) transfer methyl groups from S-adenosylmethionine (SAM) to either lysine or arginine residues, whereas histone demethylases (HDMs) remove methyl groups. The HMTs and HDMs specifically catalyze particular lysine or arginine residues.
4.1. Lysine methylation
Methylation of lysine residues on histones was first identified in the 1960s. Histone lysines can have four states of methylation at different lysine sites. Histones H2B lysine 5 (H2BK5), H3K4, H3K9, H4K20, H3K27, H3K36, and H3K79 are subject to unmethylated, mono-methylation (me1), di-methylation (me2), or tri-methylation (me3) on the ε-amino groups of lysine residues. These lysine methylations change the chromatin structure and regulate gene transcription. Histone lysine methylation is a reversible modification and is maintained by the balance lysine methyltransferases (KMTs) and lysine demethylases (KDMs). The KMTs recruit SAM as a cofactor and catalyze the addition of methyl groups to lysine residues through the SET domain. The KMTs are grouped into the SET domain-containing enzyme families (KMT1–3 and KMT5–7), the KMT4/DOT1 family, and others. The KDMs include the flavin adenine dinucleotide- (FAD-) dependent monoamine oxidase family (KDM1/LSD), the Jumonji C domain-containing demethylase (JMJD) families (KDM2–6), and others. Methylation of H3K4, H3K36, and H3K79 usually correlate with gene activation, whereas methylation of H3K9, H3K20, H3K27, and H3K56 are associated with transcriptional silencing.
4.1.1. Transcriptional activation and lysine methylation
Histone lysine methylation is a reversible process. H3K4 is demethylated by the KDM1 family (LSD1 and LSD2), the KDM2 family (FBXL10 and FBXL11), and the KDM5 family (JARID1A, JARID1B, JARID1C, and JARID1D) as well as JARID2 and NO66. The LSD subgroup of KDMs specifically targets the mono- and dimethylated lysines. This group demethylates substrates through a flavin adenine dinucleotide-dependent oxidative reaction, producing lysine and formaldehyde. KDM1A/LSD1 was the first H3K4 lysine-specific demethylase to be identified. We and others demonstrated that SNAIL recruits LSD1 to epithelial gene promoters with demethylation of H3K4me2 and subsequent silencing of target genes to enhance tumor metastasis . SLUG also interacts with LSD1 to facilitate tumor metastasis . In addition, both SNAIL and SLUG recruit LSD1 and bind to a series of E-boxes located within the BRCA1 promoter to repress BRCA1 expression. LSD1 overexpression promoted metastasis whereas knockdown of LSD1 inhibited tumor spread, suggesting that LSD1 is a key regulator of ESCC metastasis . LSD1 and LSD2 act differently in the regulation of gene transcription and chromatin remodeling. However, both of KDM1A and KDM1B are overexpressed in invasive breast carcinoma, and depletion results in high levels of H3K4me1–2. The KDM5/JARID1 family is frequently found in the promoter region of transcriptionally active genes, and results in repressed expression of the target genes. KDM5A is highly expressed in ovarian cancer tissues and facilitates EMT and metastasis . KDM5A promotes an increase in TNC expression, which augments breast cancer cell invasion and metastasis . Reports indicate that, in gastric cancer cell, KDM5A is induced by TGF-β1 and recruited by p-SMAD3 to silence the
4.1.2. Transcriptional repression and lysine methylation
In summary, histone lysine methylation modulates chromatin accessibility, transcriptional status, and control of tumor suppressor and oncogene expression in aberrant cell metastasis. Dynamic regulation of the either permissive or repressive histone methylation at different genomic loci and through different molecular mechanisms facilitates the dynamic EMT process.
4.2. Arginine methylation
Histone arginine methylation also occurs in many arginine sites, histone H3 arginine 2 (H3R2), H3R8, H3R17, H3R26, and H4R3 undergo monomethylation (me1), symmetrical dimethylation (me2s), or asymmetrical dimethylation (me2a) on the guanidinyl groups of arginine residues. The N-arginine methyltransferases (PRMTs) are a class of enzymes that transfer a methyl group from SAM to the guanidino nitrogen of arginine. PRMTs generate three arginine methylation forms: monomethylarginine (MMA), asymmetric dimethylarginine (aDMA), and symmetric dimethylarginine (sDMA). Human PRMTs are composed of nine members that are categorized into three groups based on the type of arginine methylation reaction each member catalyzes. Type I is comprised of PRMT1, PRMT2, PRMT3, CARM1/PRMT4, PRMT6, and PRMT8; these catalyze both mono-methyl and asymmetric dimethyl arginine reactions. The type II group is made up of two members, PRMT5 and PRMT9, which catalyze both mono-methyl arginine and symmetric dimethyl arginine. Finally, PRMT7 is, at this point, considered the only bona fide type III methyltransferase and can generate only mono-methyl arginines. Many studies demonstrated that PRMTs regulate a wide range of genetic programs and cellular processes including cell cycle, RNA splicing and differentiation. Although the consequence of lysine methylation is relatively well studied, the role of PRMT action in tumorgenesis is poorly understood. Here, we provide a description of these PRMTs regarding tumor metastasis.
Many HMTs and HDMs inhibitors have been developed and evaluated in clinical trials, such as chaetocin, BIX-01294, BIX-01338, UNC0638 and DZNep. Chaetocin, a natural fungal substance, is the first inhibitor of an HMT, which targets SUV39H1 without high selectively . Treatment with Chaetocin induces expression of E-cadherin while reducing H3K9me3 but does not produce a global H3K9 methylation on its promoter in multiple tumor cells . By the contrast, BIX-01294 specifically reduces the dimethylation of H3K9me2 through an inhibition of the enzymatic activities of G9a and GLP . Treatment of BIX-01294 activates E-cadherin expression and reverse EMT phenotypes in a variety of cancer cells, and is accompanied by reduced H3K9me2 and increased H3K9 acetylation on the E-cadherin promoter . Another G9a/GLP inhibitor, UNC0638, was developed with higher potency and selectively . UNC0638 treatment not only resulted in lower global H3K9me2 levels but also markedly reduced the abundance of H3K9me2 marks at promoters of known G9a-regulated endogenous genes. UNC0638 treatment activates E-cadherin expression and reverses EMT in PANC-1 pancreatic cancer cells and triple negative breast cancer (TNBC) and suppresses migration and invasion . Because of the importance of H3K27 methylation in cancer, several highly specific EZH2 inhibitors have been developed, such as GSK2816126 and EPZ-6438, which are currently being evaluated in clinical trials for lymphoma and solid tumor/lymphoma respectively . Another EZH2 inhibitor, 3-deazaneplanocin A (DZNep), selectively inhibits H3K27me3 and H4K20me3 . DZNep dampens TGF-β-induced EMT signals and reduces tumor metastasis in pancreatic cancer and colon cancer [136, 137]. We found that Parnate, an LSD1 inhibitor, activates E-cadherin expression and suppresses motility and invasiveness in breast cancer cells . Two highly specific LSD1 inhibitors, GSK2879552 and ORY-1001 are employed to clinical trials for the treatment of small cell lung cancer and acute leukemia . Several inhibitors targeting HDMs also have been developed as well. For example, JIB-04, a specific inhibitor targeting the JMJC-domain, inhibits the activity of H3K4 and H4K9 and attenuates lung cancer cell proliferation . The first reported small molecule PRMT inhibitors, including AMI-1 and AMI-5 were identified through virtual screening and high throughput screening . AMI-1 was reported as type I PRMT and PRMT5 inhibitor . AMI-1 inhibits proliferation and decreases cell migratory activity of CRC cells
5. Histone modification readers
Sometimes, histone modifications can directly regulate the chromatin dynamic. However, in most cases, the modifications are recognized by proteins containing distinct recognition domains, which act as “readers” and bind to different histone modifications. For example, bromodomain acts as lysine acetylation “readers” of modified histones that mediate signaling transduction changes in gene regulatory networks. In the human genome, there are 61 bromodomains found within 46 proteins that can be divided into eight families based on structure/sequence similarity. Among them, bromodomain and the extra-terminal domain (BET) family recognize acetylated lysine residues in histones H3 and H4. BRD4 is a member of the BET family that carries two bromodomains. Recently, our studies revealed that the di-acetylated TWIST, mediated by Tip60, recruits BRD4 and related transcriptional components to the super-enhancer of its targeted genes during tumor progression in BLBC . In addition, pharmacologic inhibition of BRD4 with the BET-specific bromodomain inhibitors, JQ1 and MS417, effectively reduces WNT5A expression and suppresses invasion, CSC-like properties and tumorigenicity of breast cancer cells
Histone methylation provides docking sites and is recognized by specific reader proteins that contain a methyllysine binding protein, which has emerged as a focus of epigenetic research due to its critical role in gene regulation and oncogenesis. This reader harbors specific motifs, including Chromodomain (CD), MBT, WD40 repeat, PHD finger, PWWP, Tudor and Ankyrin repeat. Methyllysine binding proteins distinguish methylation marks on different residues as well as different methylation states on the same residue and in turn mediate distinct downstream functions . CD-containing HP1 proteins were the first identified methyl-lysine binding proteins and recognize methylated-H3K9 (methyl-H3K9) . HP1α was down-regulated in metastatic cells of colon cancer and thyroid carcinomas relative to non-metastatic cells, indicating HP1α may be directly involved in the silencing of genes that potentiate cancer cell invasive potential and metastasis. Recent evidence implicate HP1α in EMT. The association of HP1α to major satellite repeat sequences located in pericentric heterochromatin decreased during the initial steps of TGF-β-induced EMT in a SNAIL/LOXL2-dependent manner . In addition, HP1α post-translational modifications could participate in the heterochromatin dynamics associated with EMT. In a different set of modifications, four MBT-repeats domain of SFMBT1 recognize H3K4me2/3 and form a stable complex with LSD1. SFMBT1 is essential for SNAIL-dependent recruitment of LSD1 to chromatin, demethylation of H3K4me2, transcriptional repression of epithelial markers, and induction of EMT by TGF-β . H3K4me2/3 is also recognized by the WD40 repeat domain of WDR5, which is also important for the assembly and activity of the SET1 protein complex catalyzing H3K4me3 . Under hypoxic conditions, WDR5 is induced, interacts with HDAC3 and further recruits SET1 complex to activate mesenchymal gene expression to promote EMT . Furthermore, the PRC2 component, EED, also contains a WD40 repeat that recognizes H3K27me3. EED recruits PRC2 to chromatin with pre-existing H3K27me3 to spread the same methylation into adjacent regions . Intriguingly, G9a and GLP itself contain a methyl-lysine binding module (the ankyrin repeat domains), which generates and reads the same epigenetic mark . Several small molecule compounds targeting the lysine methylation reader domain have been developed, including UNC1215 and UNC3866 that block the methyl-lysine binding mediated by the MBT domain-containing protein L3MBTL3, and the CD-containing protein CBX4/7 respectively [152, 153]. However, whether these inhibitors reverse EMT and tumor progression remains unknown.
6. Coordinated histone modification regulation
Because different chromatin modifying enzymes coexist in the same protein complex, and because diverse catalyzed modifications have been implicated in regulating the same set of genes, it is likely that these processes act in concert to orchestrate transcriptional regulation during EMT. For example, HDAC1/2, G9a/GLP, LSD1, HP1 and ZEB1/2 were co-purified in the CtBP1 co-repressor complex [154, 155]. ZEB1/2 could first target the complex to E-cadherin promoter to initiate repression. Next, HADC1/2 would deacetylate histones while the primed H3K9 was methylated by G9a/GLP. Meanwhile, LSD1, which removes H3K4me1/2, whereby the un-methylated H3K4 could also prevent H3K9 from re-acetylation [156, 157]. An affinity purification of Flag-TWIST identified several components of the NuRD chromatin remodeling complex. Among them, TWIST directly interacts with Mi2β, MTA2 and RbAp46 and likely targets the NuRD complex for histone deacetylation and chromatin remodeling on E-cadherin promoter. Together, these epigenetic events lead to gene silencing and promote EMT and breast cancer metastasis . In addition, TWIST was also co-purified with SET8, BRCA1-associated protein (BRAP), NF-kB subunit RelA, PPP2CA and HES6 in MCF7 breast cancer cells . SET8 interacts with TWIST. However, SET8 and TWIST are functionally interdependent in promoting EMT. SET8 mediates E-cadherin repression and N-cadherin activation simultaneously via its H4K20 monomethylation to promote cell invasion and EMT. However, the molecular mechanism that underlies the same repressive protein complex that contributes to opposite functions on different genomic loci remains an open question. Our recent study found that TWIST is diacetylated by Tip60, which was further recognized by BRD4, thereby constructing an activated TWIST/BRD4/P-TEFβ/RNA-Pol II complex at the WNT5A promoter and enhancer to promote EMT and breast cancer cell metastasis . In breast cancer cells, the UTX-MLL4 forms a complex with LSD1/HDAC1/DNMT1 on the promoter of several EMT-TFs and decreases H3K4mes and H3 acetylation. UTX facilitates epigenetic silencing of EMT-TFs by inducing competition between MLL4 and the H3K4 demethylase LSD1, which results in inhibition of EMT and CSC-like properties .
MPP8, another methy-H3K9 binding protein, bridges DNMT3A and G9a/GLP to assemble a repressive trimeric protein complex on chromatin by binding to different methyl-lysines. MPP8 also couples H3K9 methylation and DNA methylation to silence epithelial genes and EMT [159, 160]. Interestingly, MPP8 also cooperates with the SIRT1 in this process through a physical interaction . SIRT1 and MPP8 reciprocally promote each other’s function and coordinate epithelial gene silencing and EMT. SIRT1 antagonizes PCAF-catalyzed MPP8-K439 acetylation to protect MPP8 from ubiquitin-proteasome-mediated proteolysis. Conversely, MPP8 recruits SIRT1 for H4K16 deacetylation after binding to methyl-H3K9 on target promoters. Therefore, MPP8 not only promote DNA-methylation but also H4K16 deacetylation to fine-tune the transcriptional regulation of EMT.
7. Conclusions and perspectives
Increasing evidences show that aberrant profiles of histone modifications contribute to a dysregulation those results in the metastatic cascade. The biochemically reversible nature of histone modifications provides a platform for rapid changes in a variety of epithelia and mesenchymal genes during EMT and MET. In concert with different ETM-TFs and oncogenic signaling, pleiotropic histone modifications form a sophisticated and regulated network to coordinate the plasticity and dynamic change required for EMT.
Recent research identifies the critical role of histone modifications in metastasis, but leaves many important, open questions. First, do tumor microenvironmental signals trigger the formation of histone modification enzyme complexes present on different EMT-TFs? Whether these extrinsic signals affect enzyme activity indirectly through intracellular signaling pathways or directly through the EMT-TFs remains to be determined. Second, how do these EMT-TFs form distinct complexes that coordinate the epigenetic regulation of gene expression programs during EMT? Third, EMT is usually activated only transiently and partially. Therefore, which and how do different histone modifying enzymes and the catalyzed modifications contribute to these dynamic changes? Finally, what consequences do epigenetic instabilities have on cancer cell fitness? Do these activities increase plasticity and/or lead to vulnerabilities that it could influence the metastasis?
We know that histone modification enzymes are highly correlated with tumor progression and a poor clinical outcome. Therefore, these enzymes can serve not only as effective biomarkers for earlier diagnosis, but also present multiple therapeutic opportunities. Over the last decade, considerable progress has been made in the discovery and development of potent and selective small molecule inhibitors targeting specific histone modifiers. Many of these molecules are currently under extensive preclinical testing or being evaluated in clinical trials. These inhibitors show great potential as clinically useful drugs. Additionally, inhibitors to specific histone modifying enzymes could serve as useful chemical probes to characterize the function of different epigenetic pathways in EMT
In all, advances in our understanding of the landscape of histone modifications in metastasis will provide a better sense of the molecular mechanisms associated with metastasis and thus help speed the development of new therapeutic strategies and biomarkers for metastasis.
We thank Dr. Cathy Anthony for the critical editing of this manuscript. Our research was supported by the Shared Resources of the University of Kentucky Markey Cancer Center (P30CA177558). Our research was also supported by grants from American Cancer Society Research Scholar Award (RSG13187) and NIH (P20GM121327 and CA230758) (to Y Wu).
Conflict of interest
The authors have declared that no conflict of interests exists.