|Year : 2021 | Volume
| Issue : 2 | Page : 311-326
Structural assembly of Polycomb group protein and Insight of EZH2 in cancer progression: A review
Nisha Gautam1, Mandeep Kaur2, Satbir Kaur1
1 Department of Human Genetics, Punjabi University, Patiala, India
2 Department of Human Genetics, Punjabi University, Patiala; Department of Human Genetics and Molecular Medicine, Central University of Punjab, Bathinda, Punjab, India
|Date of Submission||07-Dec-2019|
|Date of Decision||19-Jan-2020|
|Date of Acceptance||12-Apr-2020|
|Date of Web Publication||13-Oct-2020|
Department of Human Genetics, Punjabi University, Patiala - 147 002, Punjab
Source of Support: None, Conflict of Interest: None
Polycomb group proteins (PcG) are multi-subunit structure, consisting of polycomb repressive complex 1 (PRC1), PRC2/3, and pleiohomeotic repressive complex. PRC1 is made up of PHC, BMI-1, CBX, and Ring 1A/B. PRC2 protein complex included embryonic ectoderm development, PCL, SUZ12, SET domain, enhancer of zeste homolog-2 protein (EZH2), and Nurf55. The third subunit PhoRC consists of Pho and DSFMBT subunits. One of the important subunits of PcG group of protein is EZH2 (a histone methyltransferase), which catalyzes tri-methylation of histone H3 at Lys 27 (H3K27me3) to regulate gene expression through epigenetic machinery and induces silencing of specific gene transcription. In case of breast cancer and prostate cancer, EZH2 is very well studied. Evidence shows that EZH2 is overexpressed and mutated in a variety of human cancers, rendering EZH2 an attractive target for the design of new chemotherapeutic drugs in cancer. EZH2 also functions both as a transcriptional suppressor and a transcriptional co-activator, depending on H3K27me3 or its absence. In this review, we summarized various studies reported till date, elucidating the structure of PRC2 complex, various mechanisms involved with this, and highlighting the role of EZH2 in breast cancer and prostate cancer progression. An increased understanding of the mechanisms that underlie the pathogenesis of wide spectrum of cancers is therefore needed to develop novel therapeutic approaches for this disease and to improve the quality of life in patients.
Keywords: Breast cancer, EZH2, H3K27, methyltransferase, PRC2, prostate cancer, UTX
|How to cite this article:|
Gautam N, Kaur M, Kaur S. Structural assembly of Polycomb group protein and Insight of EZH2 in cancer progression: A review. J Can Res Ther 2021;17:311-26
| > Role of Epigenetics in Cancer|| |
In this postgenomic era, the role of epigenetics is clearly established in contributing to the pathogenesis of various complex diseases such as diabetes, hypertension, neurodegenerative conditions, and cancers. Epigenetics is the study of heritable phenotype changes that do not involve alterations in the DNA sequence. These epigenetic changes alter the activity and expression of genes. The epigenetic changes influence the regulation of expression of genes by multiple enzymatic modifications on chromatin complex. These enzymatic modifications include methylation, phosphorylation, and sulfonylation. There are various epigenetic factors responsible for the activation and inactivation of chromatin complex, which include writers (DNA methyltransferases, histone lysine methyltransferases, and histone deacetylases), readers (methyl CpG binding domain), and erasers (histone de-methylases and histone deacetylases) [Figure 1], [Figure 2], [Figure 3]. Writers are the proteins which modify the histone and CpG islands, thereby altering the chromatin accessibility for the binding of other proteins, for example, polymerase, in order to influence the gene expression. The readers identify the writers such as methyltransferase or acetylase and facilitate the chromatin change.
|Figure 3: Major categories of epigenetic enzymes regulating the chromatin structure|
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Another factor eraser, i.e., demethylase and de-acetylase, removes these chemical groups from histone to relieve the chromatin complex. The basic function of epigenetic regulation is maintaining the homeostasis of central dogma via regulating the expression of genes at various levels, i.e., cellular differentiation, cell division, and cell death. The importance of epigenetic dysregulation is more evident in cancer progression, posing this field as a new frontier in cancer research. The abnormal hypo- or hypermethylation and hypo- or hyperacetylation may lead to transformation from proto-oncogenes to oncogenes and the repression of tumor suppressor genes, which can further cause tumorigenesis [Figure 2]. Due to epigenetic alteration, there can be modification in chromatin complex and can affect transcription and translation. The epigenetically induced methylation of histone is balanced by the activity of histone methylase and histone demethylase enzyme. The abnormal activity of these enzymes results in dysregulation of expression of various genes involved in cell proliferation, cell differentiation, cell survival, and apoptosis. The hypermethylation of chromatin results in the repression of various genes such as tumor suppressor genes, cell–cell adhesion genes, DNA repair genes, and angiogenesis genes, and this type of pathogenesis has been clearly reported in prostate cancer, colorectal cancer, and breast cancer. Such changes of DNA and histone are preferably targeted for treatment because of their reversible nature. The histone deacetylases (HDACs) are critical enzymes in the regulation of expression of genes important for cell homeostasis. They also act as members of a protein complex responsible for the inhibition in the recruitment of transcription factors to the promoter region of genes, including those of tumor suppressors and DNA repair genes. High HDAC expression and histone hypoacetylation have been observed in cancer with associated transcriptional repression of genes, thus providing a rationale for the investigation of HDAC inhibitors in cancer therapeutics.
| > Polycomb Protein: the Key Players of Epigenetics|| |
Genes encoding polycomb group (PcG) protein were originally found in fruit flies and were known to have an inhibitory effect on HOX genes. It is well known that fruit flies are the best model for animal research PcG/trithorax group (TrXG) system. The PcG protein majorly plays a role in the suppression of cell–cell adhesion and gap junction proteins by inhibiting the HOX genes expression, while TrxG protein ensures the active expression of HOX genes. PcG protein is made up of multi-subunit structure which functions collectively in maintaining the active and repressive state of chromatin, thereby influencing the nucleoprotein structure; this multi-subunit structure exhibits as PcG [Figure 4]. Analytical and computational studies have revealed that three unique PcG protein complexes have been purified, namely polycomb repressive complex 1 (PRC1), PRC2/3, and pleiohomeotic repressive complex. PRC1 protein complex, which is made up of four subunits, consists of PHC, BMI-1, CBX, and Ring 1A/B. The PRC2 protein complex includes embryonic ectoderm development (EED), PCL, SUZ12, SET domain, enhancer of zeste homolog-2 protein (EZH2), and Nurf55. The third protein complex, i.e., PhoRC, is made up of Pho and DSFMBT subunits. The PRC2-dependent methylation of histone H3 lysine residue 27 is catalyzed by EZH2 subunit (an enzyme). The PRC1 component assists the PRC2 in transcriptional repression of chromatin by recognizing the methylated H3K27 (by CBX subunit of PRC1) and ubiquitination of H2AK119 [Figure 5]. The significance of H2AK119 ubiquitination is to facilitate the transcriptional inhibition and nuclear small weight [Figure 6]. The Pho subunit of third polycomb protein complex has the DNA-binding activity, while the other subunit of PhoRC, i.e., DSFMBT, acts as a modifier for histone protein and is known to methylate the modified histone activity. The fluorescent polarization of H3 and H4 reveals three to four methylation sites at their N terminus. The earlier discussed modifier, i.e., DSFMBT protein, influences the histonic methylation by interacting with N terminus of the H3 and H4. Through CHIP technology, it has been observed that PcG protein has specificity for certain DNA sequences, and these sequences are termed as polycomb response elements (PREs); the transcriptional activation of downstream genes has shown lack of binding of PcG with PREs. It has been observed experimentally that when PcG protein binds at the proximal of PREs, the gene expression decreased significantly. The polycomb protein is known to regulate the expression pattern of HOX gene at every developmental stage of cells, and this HOX gene family also influences the cellular proliferation and differentiation. Various numbers of pathological phenotypes were observed due to abnormal cell proliferation and differentiation. In such cases, HOX gene expression dysregulation was observed. It has been studied that chromatin complex is formed by interaction with polycomb protein, which plays an indispensable role in embryo differentiation and stem cell self-renewability. It has also been observed with the help of GWAS study that PcG protein along with HOX gene family is important for embryo development and in cell fate determination. Mutation in EZH2 subunit of PRC2 complex was observed to dysregulate the 1–40 genes in case of human embryonic fibroblast. In addition to the regulation of embryonic development, PcG proteins are also known to regulate the adult stem cell function such as regeneration of neural stem cells, maintenance of the homeostasis of hematopoietic cells, and also prevention of the hematopoietic stem cell failure. The process of stem cell differentiation during embryonic development in return also regulates (on/off) the expression of PcG protein coding genes. It has been observed that overexpression of PcG proteins results in repression of tumor suppressor genes, hence causing various types of tumors. A phylogenetic study has revealed that other mammals also have PcG homologous protein, which is related to human PcG protein. Positional cloning has revealed another subunit of PRC1 subunit, i.e., BMI-1, located on 10p13 chromosome. Mutation in BMI-1 has been reported to be associated with B-cell non-Hodgkin lymphoma (NHL). The mutation in BMI-1 gene encoding protein plays role in promoting B-cell lymphoma in transgenic mice and also associated with acute leukemia in mice. The BMI-1 mutation has significant impact in causing abnormal phenotype in case of mice and fruit flies, which indicates its conservative role during the course of evolution. The results further showed that the BMI-1 subunit of polycomb protein has an important role during adulteration, especially in case of providing the spectrum of specificities to the hematopoietic cells and their differentiation when compared with its role during embryogenesis. It has been observed that BMI-1 gene expression is found to be more in prehematopoietic cells and in few CD342 mature cells. There are certain homologous genes of PcG such as ZNF144 and MEL18, which have maximum similarity with BMI-1 gene and known to have similar functions. This was known to be highly expressed in placenta, lungs, and kidneys in comparison to liver, pancreas, and skeletal muscle, where the expression is comparatively low. The MEL18 gene was known to be located at 12q22. It has been studied through transgenic mice that MEL18 is responsible for T and B lymphocyte differentiation. However, when studied under normal condition, it has been observed that MEL18 regulates the tumor suppressor genes activity via interacting with PRC2 pathway. This homologous gene (MEL18) of BMI-1 was further found to be responsible for taking interaction with DNA and hence allowing the whole PcG group to get assemble on the DNA. It has also been observed that in various types of cancers, MEL18 or BMI-1 gene expression gets dysregulated in conjunction with c-myc gene, HOX gene, and BCL-2 genes. According to Kanno study, MEL18 protein controls two different biochemical pathways, i.e., transcriptional regulation of HOX gene family and cell growth/death control system. It has been observed that PcG protein also inactivates the X chromosome which can increase the risk of certain types of tumors. Another subunit of PcG protein, i.e., EZH2, is known to methylate histone 3 at the 27th residue of lysine amino acid when interact with PRC1 complex. It helps in the methylation of lysine residue at N terminus of histone 3, which indicates the PcG protein-mediated silencing of genes by regulating histone activity. The EZH2-mediated H3-K27 methylation is promoted due to transition of PcG protein into PRC1 complex, which further mediates signal for H3-K27 activity. The EED-EZH2 complex pf PcG protein complex is also known to mark the H3-K27 repression on X-inactivation during X imprinting during embryogenesis. In an experiment, when embryonic cells were cultured in vitro, it was found that X-inactivation was also induced by H3-K27 mark in the presence of EZH2-EED complex, and this experiment was termed as X-inactivation or Xi experiment. This type of Xi activity further revealed the role of XIST RNA in H3-K27 activity other than its conventional X chromosomal silencing role. Further studies revealed that this H3-K27 mark of repression is also random during X inactivation. The dysregulation of EZH2 or its overexpression is very well known in carcinogenesis of various cancers such as prostate cancers and breast cancer. The EZH2 gene is located on 7q35, and this gene in the whole genome covers approximately 40 KB region. This gene has open reading frame which consists of 20 exons. In fruit flies, the detailed structure of EZH2 gene was studied by gene sequence alignment, which revealed three conservative sequences at N terminus and C terminus codes for highly conservative subunit, i.e., SET domain E (z) domain and trithorax domain; all these regions are evolutionary conserved. In case of mutation in any subunit of PcG complex may cause loss of chromatin binding ability. Therefore c-terminus and EZH2 subunit of PcG are the most crucial for loss of chromatin function. The mutation at C terminus of PcG group protein may also cause loss of inhibitory activity. The SET domain is known to have highly conserved structure, where specific amino acids of EZH2 interact with SET domain. In case of mutation in any subunit of PcG complex may cause loss of chromatin binding ability. Therefore c-terminus and EZH2 subunit of PcG are the most crucial for loss of chromatin function. EZH2-mediated repression may get inhibited if SET domain is mutated. In several studies, the EZH2 protein mutation can cause the inhibition of chromatin repression, which can further promote unnecessary cell proliferation and stimulate carcinogenesis. Such studies have clearly shown that EZH2 could be a new prognostic or diagnostic tumor marker and with gradual research, its clinical significance has been proved in various types of cancers (Adrian et al., 2003). The microarray analysis has shown that EZH2 expression is significantly upregulated in case of metastatic prostate cancer in comparison to localized or benign prostate tumor. In another study, EZH2 was overexpressed among metastasized prostate cancer, but when prostate cancer sample was injected with EZH2-specific siRNA, it was observed that EZH2 expression got downregulated and improved inhibitory effects in excessive cell proliferation of prostate cancer cells. Another study also reported the high EZH2 expression in metastatic prostate cancer than benign, lesion and localized stage of prostate cancer. The EZH2 expression gradually increases from primary prostate cancer to advanced prostate cancer. Due to the overexpression of EZH2 gene, there are multiple sets of genes whose transcriptional repression was observed which mainly include DNA repair genes and tumor suppressor genes. The pathogenic pathways of these repressed genes were clearly known in prostate cancer progression. The dysregulated EZH2 expression is known to disrupt the mechanism of transcriptional regulation, and this influences the transition of cancer cells from epithelial to mesenchymal tissues. In addition to prostate cancer, EZH2 expression was also observed to be as upregulated in breast cancer. It was studied that invasive breast cancer has higher level of EZH2 expression when compared with benign breast tumor. The expression of EZH2The expression of EZH2 gene is regulated by Rb-E2F pathways [Figure 7]. E2F functions as transcription factor and has specificity for EZH2 promoter region. Upon the phosphorylation of retinoblastoma protein, E2F gets dissociated from pRb and becomes activated by getting recruited over EZH2 promoter at S phase and leads to the excessive transcription and production of EZH2 mRNA, hence increasing the expression of EZH2 protein during breast cancer progression. There were different models proposed for the recruitment of PcG protein at PRE on the promoter region of DNA. According to PRC1 dependent model, EZH2 interacts with Pho protein which further recruits whole PRC2 complex at PREs and further PRC2 methylates H3-K27 histones. Another model proposed that PcG proteins can get recruited via PRC1 model if Pho protein interacts with PRC1 protein, but the mechanism is not clearly understood. Other than transcriptional repression, polycomb protein group has many functions such as PcG protein which also regulate embryogenesis along with HOX gene family. The PcG protein also plays an important role in chromatin packaging, especially during heterochromatin structural assembly. The stability of nucleosome and nucleosome remodeling is also assisted by the PcG group protein. PcG group protein also helps in the recruitment of transcription-associated protein or transcription-associated factor in chromatin modification. The PcG protein also creates interference between promoters and enhances interaction, which also helps in chromatin repression. All the activities discussed above are assisted by PcG protein, only when it interacts with core promoter located upstream of PREs. The alteration in chromatin structure controlling the transcription of genes has been studied extensively in variety of cell models, and such modifications of histones are called as histone password, which is recognized by various protein complexes. These protein complexes identify the specific histones for modification and cause a series of biological effects. The histone modifications such as H3K27, H3K4, and H4K20me3 are catalyzed by PcG by interacting upstream of the open reading frame of downstream genes. When this trimethylation gets aborted, it leads to the inhibition of PcG group activity. As we know that due to extremely complex regulatory network of embryogenesis, the research is being rigid. Hence, fruit flies are the best animal models for embryogenic studies. PcG group gene family were known to influence the orientation of development in mammals, cell proliferation, differentiation, apoptosis etc. The protein complex of PcG group regulates the expression of target genes, and this enables the increased understanding of complex gene expression regulation. The deep understanding of PcG protein complex and its relative interaction with other complexes in different biological processes helps in exploring its application in treatment and diagnosis. In understanding the basis of interaction of multiple pathways such as apoptosis, cellular differentiation, and cell proliferation in tumor formation, PcG group is found to play a very important role. At the level of structural genomics and functional genomics research strategy focusing on the mechanism of malignancy, considering PcG protein complexes as a new target in developing treatment strategies for a prognosis and diagnosis of the cancer opens a new way in oncology therapeutics field. As we know according to various clinical studies, prostate cancer and breast cancer have higher mortality rate and the level of expression of EZH2 is also higher among them. Similarly, localized prostate cancer and breast cancer also show EZH2 expression, hence it can also be used as a prognostic marker in monitoring the prostate cancer and breast cancer progression.
|Figure 4: Emergence of role of polycomb repressive group proteins in carcinogenesis|
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|Figure 5: PCR1- and PCR2-dependent biological interaction of EZH2 protein|
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|Figure 6: Molecular mechanism of action of EZH2 protein in breast and prostate cancer progression|
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| > Role of EZH2 Protein in Breast Cancer and Prostate Cancer Tumorigenesis|| |
Besides promoter DNA methylation, EZH2-mediated H3K27me3 is a potential independent mechanism of epigenetic silencing of tumor suppressor genes in cancer., Recent findings implicate that EZH2 is overexpressed in a wide range of cancer types, especially in breast cancer [Figure 8].
|Figure 8: (a) Molecular insight of EZH2 in breast cancer tumorigenesis. (b) Molecular insight of EZH2 in prostate cancer tumorigenesis|
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Molecular insight of EZH2 in breast cancer and prostate cancer tumorigenesis. (a) In breast cancer cells, EZH2 expression is regulated by several factors such as hypoxia-induced HIFα, pRB-E2F, and MEK-ER K-Elk1 pathways. Genomic loss of miR101 and miR214 also upregulates EZH2 expression. Elevated EZH2 levels lead to the transcriptional repression of several tumor suppressor genes such as FOXC1, RAD51, RKIP, CDKIC, RUNX3, and CII TA, by PRC2-mediated H3K27 trimethylation [Figure 9]. High EZH2 protein levels are associated with increased expression of phospho-Akt1 (Ser473) and decreased nuclear localization of phospho-BRCA1 (Ser1423). EZH2-mediated nuclear shuttling of BRCA-1 protein in ER-negative basal-like breast cancer cells is one of its PRC2-independent functions. Nuclear retention of BRCA-1 protein leads to aneuploidy, aberrant mitosis, and genomic instability, which ultimately promotes tumorigenesis. Metastasis is responsible for the majority of cancer-related deaths, and the invasiveness or metastatic potential of breast cancer is inversely correlated with the degree of tumor differentiation. Highly undifferentiated tumors represented clinically by nuclear pleomorphism and the formation of glandular structures, have greater probability to develop metastasis. Several studies proposed EZH2 as a promising novel biomarker for aggressive breast cancer associated with poor prognosis., EZH2 protein levels have been reported to increase steadily through successive stages of neoplastic transformation from normal epithelium to epithelial hyperplasia, ductal carcinoma in situ, invasive carcinoma, and distant metastasis. In fact, EZH2 has emerged as an independent predictor of recurrence and death in patients with breast cancer. Several experimental studies have established that elevated EZH2 levels in human breast carcinomas are associated with the aggressive ER-negative basal-like phenotype characterized by lack of ER expression, nuclear polymorphism, and lack of BRCA1 protein expression, which play an important role in DNA repair and ER modulation so leading to the poor survival (Collet et al., 2006). EZH2 knockdown in breast cancer cells caused decreased proliferation and delayed the G2/M cell cycle transition. In vivo studies demonstrated that EZH2 downregulation significantly decreased breast xenograft growth and improved survival. EZH2 contributes to MEK/ERK pathway activation via RAS/RAF and ERBB2 gene amplification, which is a known pathway for cellular division and further differentiation and thus get altered, leading to cancerous condition. In breast tumor-initiating cells (BTICs), there is identified a consensus sequence for HIF response element (HRE) within the EZH2 promoter region. Molecular insights into EZH2-driven breast and prostate tumorigenesis. (a) In breast cancer cells, EZH2 expression is regulated by several factors such as hypoxia-induced HIFα, pRB-E2F, and MEK-ER K-Elk1 pathways. Genomic loss of miR101 and miR214 also upregulates EZH2 expression. Elevated EZH2 levels lead to the transcriptional repression of several tumor suppressor genes such as FOXC1, RAD51, RKIP, CDKIC, RUNX3, and CII TA, by PRC2-mediated H3K27 trimethylation. High EZH2 protein levels are associated with increased expression of phospho-Akt1 (Ser473) and decreased nuclear localization of phospho-BRCA1 (Ser1423). EZH2-mediated nuclear shuttling of BRCA-1 protein in ER negative basal-like breast cancer cells is one of its PRC2-independent functions. Nuclear retention of BRCA-1 protein leads to aneuploidy, aberrant mitosis, and genomic instability, which ultimately promotes tumorigenesis. (b) In prostate cancer cells, four molecular mechanisms are reported to be responsible for EZH2 amplification or overexpression of the EZH2 gene including deletion of its negative regulator miR-101, transcriptional regulation by MYC and ETS gene family members. MYC binds upstream of EZH2 promoter and induces EZH2 expression. Interestingly, MYC represses the transcription of CTDSPL, CTDSP2, and CTDSP1, which harbor miR-26a and miR-26b. Repression of miR-26a and miR-26b contributes additionally to EZH2 overexpression as miR-26a and miR-26b would be unavailable to destabilize EZH2 mRNA by binding specifically to the EZH2 3'-UTR in RISC complex. Furthermore, ETS transcriptional network also regulates the expression of EZH2. Epithelial-specific ETS factor ESE3 represses EZH2 expression, whereas ER G binds to the promoter of EZH2 and competes with ESE3 for promoter occupancy, opposing its effects. EZH2 overexpression leads to H3K27 methylation-associated silencing of critical tumor suppressor genes such as DAB2IP, MSMB, SLIT, TIMP-28, and TIMP-3, which contribute to increased growth, proliferation, and invasive phenotype of prostate cancer cells. The authors reported that HIF transcription factor (HIF1α) binds to the HRE-containing f promoter region in a hypoxic microenvironment and transactivates EZH2 expression. Increased EZH2 expression was found to be inversely related to double-strand break repair protein RAD51 expression, which ultimately causes genomic instability due to impaired DNA damage repair. RAF1 gene amplification was also reported in BTICs due to EZH2-mediated downregulation of DNA repair, which further activates p-ERK-β-catenin signaling. The overall functional effect is enhanced self-renewal and expansion of BTIC population, ultimately facilitating breast cancer progression. EZH2 also affects the pRB-E2F pathway. The E2F gets transactivated and binds to the promoter regions of EZH2, thus leading the silencing or downregulation of cell cycle controlling genes specially which are functional at S-phase. [Figure 9] depicts the breast cancer progression by EZH2 overexpression induced by upstream pathways such as RAF/ERK (as discussed above), there are number of genes (DNA repair genes, i.e., RAD gene family, tumor suppresser genes, i.e., RUNX3, CDKNIC, p57, p21, p53, and cell–cell adhesion genes, i.e., FOXC1 AND CDH1), which were known to be suppressed during hypoxic condition and leads to the change in the basic cellular function which helps them to escape the cell cycle control or programmed cell death such as loss of cell–cell contact, damaged DNA, and entering of cell into M-phase.
| > Epigenetic Role of EZH2 Protein in Tamoxifen Drug Treatment|| |
As we know that a histone methyltransferase enzyme, i.e., EZH2, regulates gene expression which determines the cancer cell fate by directly methylating the H3K27 at the promoters of downstream target genes, another study reported that H3K27me3 is not remarkably detectable at GREB1 promoter. EZH2 modulated the DNA methylation levels at specific CpG locations of the promoter region of downstream genes and concurrent with GREB1 expression [Figure 10]. However, it is still uncertain that whether EZH2-mediated regulation of DNA methylation affects GREB1. Researchers found that expression of DNMT1 and 3B was significantly decreased upon EZH2 silencing in tamoxifen-resistant cells (Tam R). These results showed the methylation activity at GREB1 promoter by EZH2 protein. Global DNA hypermethylation has been repeatedly observed in endocrine-resistant cells, which further decreases the expression of estrogen receptor-α (ER-α) gene activity., These observations were in agreement with the findings of other studies, which also report that EZH2 is overexpressed in breast cancer and it is known to positively regulate the levels and activities of DNMT. Another work revealed epigenetic program that determines ER-α activity as well as cell fate in response to tamoxifen treatment. Upon short-term exposure of the anti-estrogen in tamoxifen-sensitive cells, the presence of abundant GREB1 protein fails to recruit essential co-activators to ER-α-binding sites and therefore induces a rapid inactivation of downstream target genes. These findings, long-term tamoxifen treatment results in altered activities of epigenetic enzymes such as EZH2 and DNMTs, which causes hypermethylation of GREB1 promoter. Maintenance of GREB1 protein at low levels reprograms ER-α dependent transcriptional machinery and induces a distinct transcriptome that renders refractory phenotypes in breast cancer cells. Taken together, our findings provide a compelling foundation for the clinical utility of selective EZH2 inhibitors for the treatment of ER-þ, Tam R breast cancer that expresses active epigenetic regulator EZH2 and harbors DNA hypermethylation at the specific CpG locus of GREB1 promoter region.
|Figure 10: Influence of EZH2 protein in tamoxifen resistance and tamoxifen-sensitive breast cancer cell|
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| > Plethora of Studies on EZH2 Protein Insight in Cancer Progression|| |
Over expression of EZH2 is known to be associated with tumor progression and poor prognosis in several human malignancies. A study has reported the role of Akt-mediated pathway in case of breast cancer. Akt phosphorylates at the 21st position of serine residue in EZH2 protein, which inhibits the K27 methylation of histone H3. The phosphorylated serine residue has no impact on EZH2-mediated PRC2 complex, but it is known to reduce the affinity of EZH2 toward histone 3 which causes decrease in H3K27, trimethylation activity of EZH2 over downstream targeted genes. This study reported that breast cancer with elevated level of phosphorylated Akt has lower level of methyltransferase enzymatic activity of EZH2 protein. One study reported that women with inherited mutation in BRCA1 allele develop basal-like breast cancer or TNBC (triple-negative breast cancer), and such breast cancer is observed to have poor phenotype and aggressive behavior. Recent reports suggest that level of EZH2 expression in case of breast tumor having BRCA1-mutation has poor survival., Both in vitro and in vivo studies suggest that the Akt-mediated regulation of EZH2 protein (phosphorylation of serine 21) may regulate the shuttling of BRCA1 protein between nucleus and cytoplasm and if BRCA-1 protein get restored in nucleus, it may lead to mitotic defects, which ultimately enhances the risk of breast cancer progression. Various studies have reported the influence of epigenetic regulation in hormonally induced breast cancer. The EZH2 protein is known to act as link between the ER-α and ß-catenin of Wnt signaling pathway and thus promotes cell cycle progression. Another study suggests the independent role of EZH2 protein (without SET domain) in transactivating the transcription of c-myc and cyclin-D1, thereby promoting cell proliferation. One of the novel studies on the hormonal aspect of breast cancer revealed that EZH2 expression specificity for repression of estrogen receptor activity sequence (REA) and their interaction leads to the recruitment of EZH2 on estrogen receptor element (ERE) containing genes promotors, thus resulting in the repression of downstream targeted genes. A mechanistic study on EZH2 protein showed that the overexpression of EZH2 protein promotes prostate cancer progression by repressing tumor suppressor genes and developmental regulators, which further helps in the maintenance of stem cell-like state of prostate cells.,, In case of prostate cancer, overexpression of EZH2 gene is also associated with the deletion of negative regulator of miRNA-101114 and overexpression of c-myc gene. There are number of studies which report the interaction of androgen signaling in prostate cancer and its epigenetic regulation via EZH2 protein. Androgens or the AR (androgen receptor) signaling pathway was studied and known to play a crucial role in the normal development and functioning of the prostate cells as well as in the proliferation and survival of prostate cancer cells. In case of normal prostate cells, androgen deprivation may cause the involution of prostate tissue due to apoptosis in epithelium. Similarly, androgen deprivation therapy (ADT) or castration is being recommended to prostate cancer patients in order to halt the prostate cancer progression by inhibiting their growth and survival. However, it has been reported that after treatment of prostate cancer with ADT therapy, the chances of disease relapses get more lethal, and this condition is termed as androgen-refractory or castrate resistant prostate cancer. The molecular mechanism by which androgens promote prostate tumorigenesis has not been comprehensively deciphered. Because EZH2 levels rise during advanced stages of prostate cancer, it is important to investigate the role of androgens and AR signaling in regulating EZH2 expression. One study has clarified the link between EZH2 protein and AR signaling and revealed the antagonistic behavior of EZH2 and AR, the EZH2 gene expression is repressed by androgens and such type of repression requires a proper signaling of AR in prostate cancer where AR signaling pathway is also known to be mediated by RB and p130-dependent pathways. This may provide a reason for elevated EZH2 expression in hormone-refractory and metastatic prostate cancer after giving ADT therapy to prostate cancer [Figure 11]. Another recent study has highlighted the previously overlooked role of AR as a global transcriptional repressor of several genes regulating cell differentiation and tumor suppression using a systematic approach. The authors reported that AR deprivation is causing the activation of EZH2 protein, hence the chances of relapse of prostate cancer and frequency of metastasis in prostate cancer become higher after ADT therapy. AR may play a dual role by inducing genes that promote prostatic differentiation while concomitantly repressing developmental regulators involved in nonprostatic pathways. Further, they also demonstrated the EZH2 conjunction with AR in the process of prostate progression. Hence, EZH2 is frequently overexpressed in metastatic prostate cancers and may also suppress AR-repressed genes in an androgen-deprived environment. Once EZH2 is recruited to its specific ARE (androgen receptor element), motif-containing promoter sequences of the downstream genes, leads to the silencing of those genes, such repression is also called as androgen receptor-dependent transcriptional silencing subsequent chromatin remodeling.
|Figure 11: Prostate cancer relapse in ADT-treated prostate cancer patients with increased aggressiveness of the disease caused by EZH2 expression alteration induced by androgen receptor deprivation (The presence of testosterone (T) or dihydrotestosterone causes dissociation of HSP, dimerization, and phosphorylation (P) of the AR and translocation to the nucleus where the AR binds to an ARE, causing recruitment of DNA transcription factor)|
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Such studies support the importance of administration of EZH2 targeted drug along-with conventional prostate therapy, i.e., ADT in advanced malignancies. Studies reported that the synergy between AR and KRAS signaling could elevate EZH2 levels, thereby promoting prostate tumorigenesis using a prostate tissue regeneration system. The crosstalk among EZH2, AR, ERG, and HDACs upon androgen signaling was reported to promote prostate cancer progression. ERG, together with HDACs and EZH2, modulate the transcriptional output of AR, thereby promoting tumorigenesis. A group of study also investigated the role of EZH2 in other endocrine-related cancers. EZH2 has been implicated in a number of other endocrine-related cancers, such as sporadic parathyroid adenoma, anaplastic thyroid carcinoma (ATC), and pancreatic cancer. In pancreatic carcinogenesis, RAS pathway is known to upregulate the EZH2 expression via MEK-ERK signaling which further cause downregulation of tumor suppressor genes, including RUNX3. In thyroid carcinoma, EZH2 overexpression affects ATC and directly controls differentiation of ATC cells by causing transcriptional repression of the thyroid specific transcription factor paired-box gene 8 (PAX8). With the help of exome sequencing analysis of tumors of patients with sporadic parathyroid adenomas, it was found that the Y641N mutation in EZH2 was involved in molecular alterations resulting in sporadic parathyroid adenomas that cause primary hyperthyroidism [Figure 12]. Various mutations such as gain of function have been reported in case of EZH2, which contribute to its oncogenic activity. Recurrent silent mutations such as deletion, nonsense, frameshift, and missense mutations were observed in EZH2, and these mutations lead to myelodysplastic syndromes (MDS), myeloproliferative neoplasm (MPN), and MDS/MPN overlap disorders., Truncated mutations are known to be dispersed throughout the gene, while the missense mutations are known to exhibit majorly in the conserved region of the gene or the residues having specificity for the interaction space between SUZ12 of PRC2 and CXC region of SET domain, and such interaction is proved to be an indispensable step for enzymatic activity of EZH2 protein. Such mutations were also observed in homozygotic as well as in heterozygotic conditions. One of the studies on leukemia reported that if such mutation exhibited in biallelic condition among patients with leukemia, they showed to have reduced survival when compared to those leukemic patients who had heterozygous mutation. Another study revealed that EZH2 knockdown in mice leads to the progression in the initiation and proliferation of RunX-1-mutant MDS, which supports the evidence of repressive activity of EZH2 gene over tumor suppresser gene. Loss-of-function mutations and deletions of EZH2 also occur in human T cell acute lymphoblastic leukemia (T-ALL)., Other than EZH2 enzymatic subunit, mutations of other subunits such as SUZ12 have also been reported. The EZH2 gene was known to be mutated in 11 leukemic biopsies samples when compared with chances of mutation in SUZ12, i.e., 3 out of 68 leukemic cases. The frequency of mutation in PRC2 dependent subunits was observed to be comparatively higher in pediatric subtypes of T-ALL and early T-cell precursor ALL leukemia. In such cases, deletion and other types of mutations were observed in PRC2 subunits such as EED, EZH2, and SUZ12 (include 42% of ETPALL type) and 12% of non ETP pediatric T-ALL. The interaction of JAZF1 and SUZ12 creates a fusion when PRC2 gets mutated. Mutation of genes encoding PRC2 subunits other than EZH2 also occurs in other cancers. One of the studies reports that EED, another subunit of PcG protein, takes interaction with EZH2 and facilitates the H3K27 me activity, when get mutated affect EED stability in patients with MDS and MDS-related diseased conditions. The alteration in EED sequence also reported to cause myeloproliferative disorder in mice., The substrates of PRC2 such as lysine residue at the 27th position of histone H3 and other variants of histones are prone to missense mutation and out of these mutations, missense mutations are the most common especially in certain types of cancers which include 31% of pediatric glioblastoma multiforme (GBM), 78% of diffuse intrinsic pontine gliomas (DIPG), and 50% of pediatric high-grade glioma (pHGG).,, In vitro studies showed that mutation in H3K27 induces the blockage in PRC2 activity, hence affecting its protein-binding affinity by creating the alteration in the binding region of EZH2 protein and thus inhibiting methylation at other H3 site. Expression of H3.3 K27M also increases H3K27ac, a modification mutually exclusive to H3K27me3 that correlates with transcriptional activation rather than repression. Various studies have reported that mutations in histone H3 might contribute to tumorigenesis via aberrant activation of certain missense mutation and were observed to transit H3K27 methylation activity to H3K27 acetylation. Frequent mutation in H3K27 is associated with pediatric brain cancers, conferred the role of such mutation in promoting/carcinogenesis of brain cancer, this also reflects the role of EZH2/PRC2 in the regulation of neural stem cell fate. PRC2 gene mutation is not only determined in pediatric GBM or DIPG, so it has to be revealed that whether H3K27 mutations are alone contributing to EZH2/PRC2 loss of function mutation or the above discussed mutations cause other additional consequences such as defects in other non-polycomb mechanism. Conclusively, the findings about EZH2/PRC2 mutations or loss of function may lead to oncogenesis indicating the clinical applications of EZH2 inhibitors. As we know that EZH2 is the enzymatically active core subunit of the PRC2 complex, which also includes EED, SUZ12, and RbAp46/48, PRC2 methylates the lysine residue at position 27 of histone 3 (H3K27), which facilitates chromatin compaction and gene silencing. Several evidences implicated the role of EZH2 in progression and development of a variety of cancers. As discussed in previous section that EZH2 upregulation is linked to the worst aggressive nature of prostate cancer, similar findings highlighted the role of EZH2 in other human cancer which include endometrial cancer, melanoma, bladder cancer, and breast cancer, these studies indicate the level of EZH2 in relation to the aggressiveness of diseases with poor prognosis and advanced stages of disease in most types of cancers (Varambully et al., 2002)., EZH2 protein is proved to be essential for the proliferation of various cancer cell lines. Under normal circumstances, EZH2 helps in maintaining the proliferative advantage during embryogenesis. Study based on cancer cell line revealed that in an immortalized breast epithelial cell line exhibit increased level of EZH2 expression, which further induces transition from normal epithelial cell to neoplastic breast epithelial cells. The recurrent heterozygous point mutation at tyrosine residue (Y641) at C terminus of catalytic domain was found, where EZH2 binds to the SET domain in 22% of germinal center B-cell and in diffuse large cell/B-cell lymphomas (DLBCL) and in 7%–12% of follicular lymphomas., Additional findings to support an oncogenic role for EZH2 have more recently emerged. The Y641 mutation was studied as a loss-of-function mutation. A study revealed that with the help of in vitro biochemical enzymatic assays, Y641 mutation was also exhibit gain-of-function of enzyme activity.
Reduced tri-methylation activity was observed in case of Y641 mutants (Y641F, Y641N, Y641S, Y641C, and Y641H) of EZH2 gene H3K27, while enhanced di-methylated activity was observed in H3K27. Thus, it has been observed that in heterozygous condition of EZH2 variants (containing one mutant allele taking cooperation with wild type allele) shifts the trimethylation activity to di-methylation activity, hence such shift also favors overall methylation of H3K27, hence represses the expression of polycomb targeted downstream genes. The point mutation was reported at alanine 677 and 687 position (A677 and A687) of EZH2 gene found to be associated with NHL, the impact of these mutations was known to increase the rate of methylation (hypermethylation of H3K27)., One of another type of demethylase enzymes, i.e., ubiquitously transcribed tetratricopeptide repeat gene on X chromosome (UTX) is studied extensively and known to inhibit EZH2 methylation by reversal of methylation reaction [Figure 13]. But when observed, loss-of-function mutation in UTX causes several types of cancers such as multiple myeloma, bladder cancer, medulloblastoma, esophageal cancer, pancreatic cancer, and renal cancers.,, The various types of mutations were reported in case of UTX such as deletion of larger portion of X chromosome which include hemizygous male and homozygous female X-chromosome, deletion nonsense mutation, insertion/deletion type, consensus splice site mutation, and frame shift mutations that further lead to premature termination of codon. With the help of computational analysis, all the above-discussed mutations were predicted to have loss of function at C terminus of jmc domain of UTX, which is essential for demethylase activity of chromatin regulator, i.e., UTX, which is further responsible for increased H3K27 activity of EZH2 protein. In conclusion, it has been postulated that there could be analogy in relation to loss of function in UTX at C terminus of jmc domain and gain of function in trimethylation activity of EZH2 gene. SWI/SNF, the other chromatin regulatory protein, was known to regulate the chromatin complex with the help of energy driven by the hydrolysis of ATP into ADP, which was also observed to act antagonistically in relation to polycomb EZH2 protein activity and such relationship was found to be conserved on the basis of evolution, revealed by evolutionary studies.,,, SWI/SNF complexes consist of 12–15 subunits, and these subunits were found to be mutated almost in 20% of all types of human cancers.,
Evidences also reports that the cancers exhibiting mutations in SWI/SNF complexes especially in subunit containing SNF5/SMARCB1 complex also promote the abnormal activity of EZH2 hypermethylation in malignant rhabdoid tumor, which is a highly aggressive type of pediatric cancer., Various studies further extended this antagonistic relationship and revealed that in case of various cancers, it links the inactivation of other SWI/SNF subunits with EZH2 activity and this is found to be highly lethal in ovarian cancer when studied in xenografts mutant of SWI/SNF subunit i.e., ARID1A. This also sensitizes lung cancer xenografts mutant for the SWI/SNF ATPase core subunit BRG1/SMARCA4-to chemotherapy in mice., Through recent cell line based and in vivo model based studies, an extensive role of EZH2 in promotion and progression of various types of cancers have been reported and these studies also suggest that such cancer observed to exhibit mutations in SWI/SNF complexes containing tumor suppressor subunits, i.e., ARID1A, PBRM1, and SMARCA4. Notably, a noncatalytic role for EZH2 was identified in this context, indicating that dependency upon EZH2 for cancer progression can be derived from both catalytic and noncatalytic functions of EZH2.
| > EZH2 as Potential Cancer Therapeutic Target|| |
By knowing the significance of EZH2 in cancer progression, it has become a need of targeting EZH2 as a therapeutic in order to improvise the effect of conventional treatment strategies over life quality and disease management. Pharmacological and natural EZH2 inhibitors were made in context of this are explained under this section. The experimental based evidences claiming the major role of EZH2 in cancer progression and metastasis demands the search for novel and specific pharmacological inhibitors for EZH2 protein and has yielded few interesting molecules. One of the molecules is 3-dezaneplanocin-A (DZNep), which is the most widely used EZH2 inhibitor and exhibits anti-tumor activity against a variety of cancers, including brain, prostate, lung, breast, and liver cancer cells. As we know that EZH2-dependent histone methylation requires S-adenosyl-Lhomocysteine (SAH) cofactor, hence it is convenient to target SAH hydrolase enzyme with great specificity and thus inhibit the EZH2 function. This molecule was found to have great success as an anti-tumor agent where it targets histone methylation and reactivate the PRC2-repressed tumor suppressor genes studied in in-vitro models, but there found certain issues, such as its higher level of toxicity profile in animal models and selectivity issues while combating EZH2 protein. Another molecule named as DZNep is a global methyltransferase inhibitor and therefore could be used as EZH2-specific inhibitors. It has been shown in various studies that DZNep upregulate the several anti-metastatic genes and thereby prevent the cancer invasion, except in oral and ovarian cancers where genes upregulated by DZNep has no effect on metastatic nature of proliferative cancer cell. Therefore, targeting only EZH2 inhibition may not be an optimal universal treatment strategy for all tumor types. More recently, some EZH2 inhibitors that work on the basis of competitive inhibition, for example, there are few inhibiters developed that inhibit EZH2 enzymatic activity by directly binding to the enzyme and competing with the methyl group donor S-adenosyl methionine (SAM) and are highly specific and can sufficiently discriminate between EZH1 protein (isoform of EZH2) over EZH2 protein and other types of histone methyl transferases, i.e., HMTs. One of the important EZH2 inhibitor EPZ005687 is known to have greater selectivity and specificity which has been tested on lymphoma cells exhibiting tyrosine 641 or alanine 677 mutations in EZH2 gene sequence. This compound significantly reduces the H3K27 trimethylation activity which further results in apoptosis of mutant cancer cells along with minimal effect on normal cells. EPZ005687 inhibiter reveal dose-dependent inhibition of H3K27me3 in EZH2–wild-type and Y641- and A677-mutant lymphoma cells as well as in cell lines of other cancer types, including breast and prostate cancer. Another study reported that the small molecule inhibitor of EZH2, i.e., El1, which also acts in a competitive inhibition with competitive molecule SAM observed to reduce the H3K27me3 levels. There are some dietary ingredients which are also act as chemo-preventive agents have been described as potent EZH2 inhibitors and can block PRC2-mediated gene silencing. One of the dietary fatty acids, i.e., dietary omega-3 (v-3) polyunsaturated fatty acids (PUFAs), has the potential to downregulate the EZH2 expression and its enzymatic activity in breast cancer cells by inducing the ubiquitination via proteasomal degradation of EZH2. This PUFA-mediated inhibition leads to the re-activation, hence the expression of known EZH2-targeted tumor suppressor genes such as insulin-like growth factor binding protein and E cadherin, which ultimately downregulate the invasiveness nature of breast cancer cells. Earlier studies have reported the effect of curcumin, a natural ingredient present in turmeric, as ayurvedic medicine in decreasing the breast cancer cell proliferation. The recent study added that curcumin inhibits breast cancer cell proliferation by modulating EZH2 levels and inducing G1 arrest in MDA-MB-435 breast cancer cells. Mitogen-activated protein kinase pathway was reported to be involved in the downregulation of EZH2 which mediate the effect of curcumin-induced anti-proliferative effect in breast cancer cells. A recent study evidenced that epigallocatechin-3-gallate (EGCG), which is a major green tea polyphenol, reduces the EZH2 expression and inactivates the other PcG protein subunits such as EED, SUZ12, Mel18, and Bmi-1 independently or in combination with DZNep, reported in case of skin cancer cell. Recent study reports the mechanistic procedure of proteasome-dependent degradation of EZH2 and BMI-1 by EGCG and DZNep alone but there was a marked suppression in cells treated with both agents, suggesting that combined treatment could be more effective. There are certain inhibiters that disrupt PRC2 stability, the discovery of nonenzymatic functions for EZH2 and the implication of these in SWI/SNF-mutant cancers raises the possibility that the enzymatic inhibitors currently in clinical trials may not fully suppress the transformation promoting activity of EZH2. EZH2 can also be inhibited by disrupting its interaction with other PRC2 subunits with a peptide known as stabilized alpha-helix of EZH2 (SAH-EZH2) that is derived from the domain of EZH2 that interacts with EED. SAH-EZH2 disrupts the EZH2–EED complex, reduces EZH2 protein levels, and selectively inhibits H3K27 trimethylation in a dose-dependent manner. This peptide is efficacious against EZH2-dependent MLL–AF9 leukemia and EZH2-mutant lymphoma cells but has no effect on non-transformed and EZH2–wild-type controls. Notably, whereas the anti-proliferative effect of the SAH-EZH2 correlated with the reduction in H3K27me3 levels, the effect seemed to correlate even more strongly with reduction of EZH2 protein levels, consistent with the findings of dependence on nonenzymatic roles of EZH2 in SWI/SNF-mutant cancers. Developing strategies to consider EZH2 as a therapeutic target is due to the evidence for EZH2 enzymatic gain-of-function as a major cancer driver, so the development of EZH2-specific inhibitors is now an active area of research and multiple pharmaceutical and biotech companies now started developing EZH2 inhibiting compounds. Through such investigation promising preclinical results were successfully generated and human phase I trials are now underway, with preclinical results suggesting potential disease combating activity. The most extensively used EZH2 inhibitor that was widely applied for experimental work (3-deazaneplanocin) or (DZNep). Chemically, it resembles the cyclo-pentenyl analogous of 3-deazaadenosine which potentially interferes with the activity of S-adenosyl-L -homocysteine hydrolase (SAH), a component of the methionine cycle further causes cellular SAH levels to increase, thereby repressing the activity of S-adenosyl-L-methionine-dependent H3K27 activity. Hence, the effect of DZNep on the inhibition of histone methylation has a complex mechanism of action and just not specific to EZH2. Research performed on the treatment of cancers with DZnep induces significant anti-tumor activity in various types of cancers leads to inhibition of PRC2, and removal of mark H3K27me3. Despite of potentially promising results in vitro and in vivo of DZNep molecule, it has certain side effects which include very short plasma half-life may confers non-specific inhibition of histone methylation, hence can be lethal in animal models. In order to improve anti-tumor activity and reduce toxicity, significant efforts have been directed toward developing compounds that are potent and selective inhibitors of EZH2. High-throughput analysis and biochemical assays produced several potent inhibitors specific to the conserved SET domain structure which facilitate the prediction of two essential binding for the S-adenosyl-L-methionine (SAM) methyl and the H3K27 substrate. In 2012, several research groups announced independent development of SAM-competitive inhibiting compounds derived from high-throughput screening. Earlier discussed EZH2 inhibitor EPZ005687 binds to wild-type and Y641-mutant EZH2 and displays greater than 500-fold selectivity for EZH2 compared with 15 other human protein methyltransferases and express 50-fold selectivity over EZH1. The development of small sized-molecules such as EZH2 inhibitor, for examplw, GSK126, inhibits both wild-type and mutant EZH2 and has >1000-fold selectivity for EZH2 methyltransferase when compared with other methyltransferases and 150-fold selectivity over EZH1. GSK126 remarkably inhibits the growth of lymphomas having mutated EZH2 when studied in-vivo condition. One of the independent SAM-competitive inhibitors, i.e., EI1, block wild-type and mutant type EZH2 with an IC50 value of 15 nm, express >10,000-fold selectivity for EZH2 over other methyltransferases and 90-fold selectivity over EZH1. EI1 blocks the H3K27 me2/3 levels without affecting EZH2 protein levels in EZH2-mutant DLBCL cells and also known to inhibit cell growth and causes cell cycle arrest and apoptosis in SMARCB1-mutant rhabdoid tumor cell line carrying EZH2 mutations. These effects were accompanied by downregulation of a proliferation gene expression signature, i.e., EZH2 and increased expression of PRC2 targets. Another inhibiter UNC1999 was synthesized as the first orally bioavailable inhibitor that was highly selective for wild-type EZH2 and the EZH2 Y641 mutant. This compound is also relatively active against EZH1 with minimal (<10-fold) potency in comparison to EZH2, hence offering the potent to target both EZH2 and EZH1. Similarly, another compound, i.e., EPZ-6438 was subsequently developed and is found to be more competent compared to EPZ005687 along-with improved pharmacokinetic properties including good oral bioavailability. In June 2013, a Phase I/II clinical trial of EPZ-6438 in patients with advanced solid tumors or with B-cell lymphomas was launched (NCT01897571). EPZ-6438 reports the decreased levels of H3K27me3 and dose-dependent tumor regression when treatment was given to mice carrying SMARCB1-mutant malignant rhabdoid tumors. Preliminary reports from the clinical trial study have been presented at scientific meetings (http://www.epizyme.com/wp-content/uploads/2014/11/Ribrag-ENA-FINAL.pdf, http://www.epizyme.com/wp-content/uploads/2015/07/ICMLSlides -Presented-062015).v2.pdf). The data generated in this study show potentially encouraging activity of the drug by partial or complete responses in 9 out of 15 NHL patients, including a partial response in the one patient who had an EZH2 mutation at position Y646H and one complete and partial responses in malignant rhabdoid tumor patients. Two other clinical trials (NCT02082977 and NCT02395601) are actively known to enrol the patients and performing further investigation in EZH2-mutant B-cell NHL and SMARCB1-deficient tumors. Therapy resistance typically facilitates cancer cells to escape the effects of any single agent and mechanisms of resistance to EZH2 inhibitors are beginning to emerge. In a cell line model, two novel secondary mutations of EZH2 (Y111L and Y661D) were identified in resistant cells following prolonged exposure to EZH2 inhibitors and were found to cooperate to confer resistance to the EZH2 inhibiters. It had earlier been reported that loss of PRC2 subunits can amplify Ras-driven transcription in PNS tumors, high-grade gliomas, and melanomas and co-occurrence of a Ras pathway mutation with mutations in SWI/SNF correlated with resistance to EZH2 inhibition., Consequently, there become a research goal in identifying therapies that have the potential to cooperate with EZH2 inhibitors. In preclinical models of EZH2-mutant NHL, combining EPZ-6438 with conventional NHL-directed chemotherapy was synergistic in preventing tumor growth. The combination of EPZ-6438 and a glucocorticoid receptor agonist (GRag) also enhanced inhibition of proliferation both in cells harboring EZH2 mutations and in germinal center NHL. In prostate cancer models, combination of the chemotherapeutic agent etoposide witGSK126 significantly increased death of murine and human prostate cancer cell lines. Finally, in the preclinical study of non-small cell lung cancers, EZH2 inhibition was found to have different effects in subsets of these cancers as defined by their differing mutations. In tumors that carried either BRG1/SMARCA4 loss-of-function mutations or EGFR gain-of-function mutations, EZH2 inhibition sensitized the malignancies to Top-II inhibitors. In contrast, in tumors that lacked these mutations, EZH2 inhibition conversely promoted resistance to Top-II inhibitors. Collectively, while development of EZH2 inhibition as a therapeutic is only in early stages, evidence of resistance mechanisms is beginning to emerge as are potential approaches for combination therapy in both areas of ongoing study.,,
| > Conclusions, Questions, and Future Directions|| |
There is an approach to develop EZH2 inhibitors simultaneous to the conventional chemotherapy given to the cancer patients, and few drugs have already been moved to clinical trials, as we discussed in the present review study. This comprehensive review also showed that patients containing mutated EZH2 protein have improved chemotherapy response. In this review, we also discussed about non-PRC2-based/independent roles of EZH2 especially in breast cancer and prostate cancer, encourage a new perspective in relation to the understanding of cancer progression such as role of EZH2 in triple-negative breast cancer and in aggressive form of prostate cancer, which further could be helpful in improvising the treatment via targeting EZH2. By targeting EZH2 could also develop certain effective anti-cancer therapies, which can also activate the cancer combating genes such as tumor suppresser genes, cell–cell adhesion genes, and apoptosis-causing genes.
we are sincerely thankful to the computer department of Punjabi University, Patiala, for providing internet facility.
Financial support and sponsorship
University grant commission- Basic Science Research, India.
Conflicts of interest
There are no conflicts of interest.
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