Polycomb Group Proteins and Cancer

The Polycomb-group proteins (PcGs) are a family of proteins that use epigenetic mechanisms to maintain or repress expression of their target genes. They were originally discovered in Drosophila (fruit flies), though they've been shown to be conserved in many species due to their vital roles in embryonic development. These proteins' ability to alter gene expression has made them targets of investigation for research groups seeking to understand disease pathology and oncology.

Overview of the Polycomb Group Proteins

PcG Proteins

PcG proteins function as multiprotein complexes. Biochemical purification and functional genetic studies have assigned the various PcG genes into two distinct subsets, namely Polycomb Repressive Complex 1 (PRC1) and Polycomb Repressive Complex 2 (PRC2). The exact composition of these complexes varies but their core components are maintained across numerous species.

• PRC1

PRC1 is involved in the maintenance of gene repression; it carries out this function by binding to a trimethylated lysine 27 on histone 3 (H3K27me3) and subsequently marking lysine 119 of histone H2A with a single ubiquitin group (H2AK119ub). The Drosophila PRC1 core complex is formed by the Polycomb (Pc), Polyhomeotic (Ph), Posterior sex combs (Psc), and Sex combs extra (Sce, also known as Ring) subunits. In mammals, the composition of PRC1 is much more diverse and varies depending on the cellular context. All PRC1 complexes contain homologs of the Drosophila Ring protein. Ring1A and Ring1B (which are also known as Rnf1 and Rnf2, respectively) are E3 ubiquitin ligases that mark lysine 119 of histone H2A with a single ubiquitin group (H2AK119ub). Mammalian homologs of the Drosophila Psc protein, such as Mel18 (Pcgf2) or Bmi-1 (Pcgf4), regulate PRC1 enzymatic activity. PRC1 complexes can be divided into at least two classes according to the presence or absence of Cbx proteins, which are homologs of Drosophila Pc. Canonical PRC1 complexes contain Cbx proteins that recognize and bind H3K27me3, the mark deposited by PRC2. Therefore, canonical PRC1 complexes and PRC2 can act together to repress gene transcription and maintain this repression through cell division (i.e. the repressed state is also present in daughter cells). Non-canonical PRC1 complexes, which contain Rybp (together with additional proteins, such as L3mbtl2 or Kdm2b) rather than the Cbx proteins have recently been described in mammals.

• PRC2

The PRC2 core complex initiates repression by tagging genes with methyl groups (a process known as histone methylation). In Drosophila, this complex is formed by Enhancer of zeste [E(z)], Suppressor of zeste [Su(z)] and extra sexcombs (Esc). In mammals, EZH1 and EZH2, homologs of E(z), are histone methyltransferases responsible for the enzymatic activity of PRC2; EZH2 is often referred to as the 'catalytic subunit' of this complex. The other core PRC2 components, which comprise a homolog of Su(z), SUZ12, and a homolog of Esc, Eed, are necessary for complex assembly and for proper enzymatic activity. It is still not clear how PRC2 is recruited to DNA in mammals. One hypothesis is that the Jumonji/ARID domain-containing protein JARID2, and the members of the Polycomb-like family Pcl proteins, are responsible for PRC2 recruitment to target genes in mammals. The ARID domain of Jarid2 binds directly to DNA enriched in GC and GA dinucleotides, whereas the Tudor domain of Pcl proteins recognizes methylated H3K36, a histone mark that is associated with transcriptional elongation. This suggests that the Pcl family of proteins facilitates PcG-mediated silencing of previously active genes. Moreover, the fact that Jarid2 and the Pcl proteins are thought not to be present in the same complexes means that, in mammalian cells, distinct PRC2 complexes target different genes.[1]

Gene Silencing Through Chromatin Modification

PcG proteins were proposed to alter chromatin structure to maintain gene repression, but it had been very difficult to get direct evidence of this mechanism until electron microscopy studies were conducted. These showed that PRC1 was able to transform arrays of nucleosomes into highly compact chromatin structures in which the individual nucleosomes could not be distinguished.[2] With the increasing use and availability of genome-wide sequencing techniques, such as Hi-C, researchers will be able to further characterize how alterations in chromatin structure/architecture affects the expression/silencing of genes.

Hox Genes

Polycomb group proteins control nucleosome interactions and were first discovered in Drosophila melanogaster, where PcG genes maintain repression of the homeobox (Hox) genes that establish and preserve the anterior-posterior axis of the insect body plan during development. The Hox genes encode transcription factors that give a specific identity to each segment along the body axis of the animal.[3]

Maintenance of Embryonic and Adult Stem Cells

Maintenance of embryonic stem cells (ES)

Lineage-specific genes are genes that will define the final identity of the differentiated cell. These genes are primed for expression (also known as existing in a bivalent state in embryonic stem cells but are kept in a repressed state by chromatin modifications. The importance of PcG during embryogenesis is evidenced by the fact that targeted disruption of either the PRC2 members EZH2 or EED, or the PRC1 component NF2 results in early embryonic lethality.

Maintenance of adult stem cells

PcG proteins are also key players in the maintenance of adult stem cell populations. Several PcG proteins have been implicated in the regulation of the self-renewal capacity of specific stem cell types. For example, overexpression of the EZH2 prevents haematopoietic stem cell exhaustion and can block the differentiation of muscle myoblasts.[4] Stem cells are also tightly regulated by their respective cellular microenvironment or niche; PcG function can be inhibited by the JNK signaling pathway, which is inactivated in response to wounding. PcG suppression leads to an increased frequency of transdetermination, a process in which precursor cells switch their predetermined identity.[5]

Implication in Tumor Development

Traditionally, cancer has been viewed as a genetic disease that is driven by sequential acquisition of mutations, leading to the constitutive activation of proto-oncogenes and the loss of function of tumor suppressor genes. However, it has become increasingly evident that tumor development also involves epigenetic changes. These epigenetic changes include both genome-wide losses and regional gains of DNA methylation, as well as altered patterns of histone modification. The state of compaction of the chromatin fiber governs DNA accessibility and therefore has a crucial function establishing, maintaining, and propagating distinct patterns of gene expression. Perturbations of chromatin structure can cause inappropriate gene expression and genomic instability, resulting in cellular transformation and malignant outgrowth. Polycomb group proteins (PcG) function as transcriptional repressors that silence specific sets of genes through chromatin modification. Although they are primarily known for their role in maintaining cell identity during the establishment of the body plan, several mammalian PcG members are implicated in the control of cellular proliferation and neoplastic development.[4]

Proposed Mechanisms Linked to Carcinogenesis

Polycomb group proteins have been studied quite intensely and have been shown to play a role in the formation and/or maintenance of certain types of cancer. PcG target genes have been shown to be more likely to be hypermethylated in aged somatic cells,[6] and found to be 12 times more likely to be hypermethylated in cancers than non-PcG targets.[7][8] A vast majority of PcG targets are lineage and differentiation determinants. Studies have suggested that uncontrolled methylation by PcGs will lock cells in an undifferentiated or immature state, which could prime them for malignant transformation.[9] Polycomb group proteins have also been shown to affect DNA damage and apoptosis pathways preventing cells from entering senescence; this is a state in which the cell ceases to replicate.[9][10]

Bmi-1

Bmi-1 is a subunit of the Polycomb Repressive Complex 1 (PRC1) and assists in preventing differentiation of stem cells. Though PRC1 isn't as well-studied as PRC2, Bmi-1 has had a great deal of focus for its involvement in numerous cancers.[10] It has been found to regulate cell senescence and proliferation through repressing cell cycle regulating genes such as p16 and p19 (sometimes referred to as Ink4A/Arf locus).[11][12] Normally, this function allows it to assist stem cells in maintaining their self-renewing capacity. However, modulation of these cell cycle inhibitor genes also allows Bmi-1 to malignantly transform cells (both mature and stem cells) into cancer stem cells.[10] Bmi-1 is thus considered an oncogene. Expression of Bmi-1 has been found to be elevated or otherwise deregulated in numerous cancer types including squamous cell carcinoma, neuroblastoma, bladder tumors and leukemia.[13][14][15][16] Since it is known to be associated with metastasis and malignant transformation, Bmi-1 makes for a good marker of cancer and may hold prognostic or diagnostic value.[10] Silencing Bmi-1 has shown to enhance activity of chemotherapeutic agents,[17][18] and it is known for its association with chemoresistance to common chemotherapeutics.[10] For example, one study has demonstrated that reduction of Bmi-1 is capable of restoring sensitivity to the chemotherapeutic drug Gemcitabine.[19] Researchers found that Bmi-1 ubiquitinates ribonucleotide reductase M1 RRM1 for degradation. Gemcitabine binds to RRM1 and irreversibly inactivates ribonucleotide reductase, ultimately preventing the synthesis of DNA.[19] Thus, the chemotherapeutic Gemcitabine needs to bind to RRM1 to prevent cancer cells from replicating and/or repairing their DNA.

Taken together, the data on Bmi-1's functions and binding partners of Bmi-1 could aid the development of better treatment options for future cancer patients.

EZH2

EZH2 is a subunit of PRC2 and functions to mark genes for silencing. This protein is likely the most studied subunit of either Polycomb Repressive Complex (PRC1/PRC2). It is the catalytic subunit of the PRC2 complex that trimethylates the twenty-seventh lysine on histone 3 (H3K27me3). Genes containing this mark often have decreased expression or are completely repressed. It is this function that allows EZH2 to modulate gene expression without altering the DNA nucleotide sequence. EZH2 is often overexpressed in various cancers.[20] EZH2 has also been shown to play a role in regulating the apoptotic processes of cells through its gene silencing capabilities (H3K27me3). One study has shown that EZH2 performs this function by repressing DAB2-interacting protein (a tumor necrosis factor),[21] and another has demonstrated that EZH2 also accomplishes this via repression of the E2F1 target Bim.[22]

Cullin 3-SPOP-RBX1

E3 ubiquitin ligases are proteins that assist in tagging their targets with an epigenetic mark known as ubiquitin. This mark can serve several functions including marking proteins for degradation, signaling proteins to change their cellular location, affecting protein activity, and promoting or preventing protein interactions. An E3 uibiquitin ligase complex composed of Cullin 3 (CUL3), speckle-type POZ protein (SPOP), and RING-box protein 1 (RBX1) has been shown to mark the Bmi-1 with ubiquitin (a process known as ubiquitination).

Researchers initially discovered this interaction when yeast two-hybrid screens demonstrated Bmi-1 specifically bound to the SPOP subunit. This led researchers to speculate on the significance of this interaction, and they concluded that SPOP must serve to tether Bmi-1 to Cullin3 for ubiquitination.[23] Though the purpose of the ubiquitin mark is unclear, a model has been proposed that links this complex to transcriptional repression and deposition of variant histones.[23]

While the epigenetic marks from ubiquitination can have several purposes, one very well-known role is the marking of proteins for degradation.[24] Logically, one could reason that marking Bmi-1 with ubiquitin might serve to tag it for degradation, potentially reducing its contribution to carcinogenesis, though future work will be required to fully understand the role of this mark and confirm its effects.

Associations with Oncogenes

Oncogenes are genes that can cause cancer when they are mutated or if they have drastically abnormal expression levels. PcG proteins have been found to associate with such genes, serving to either directly or indirectly alter their levels of expression through epigenetic modifications. c-Myc is a canonical oncogene that has been shown to associate with members of the PcG proteins. Normally, c-Myc is highly expressed in immature cells but has almost no perceivable expression in mature/differentiated cells.[25] Its roles in the cell cycle and apoptosis help cells maintain an immature state, and its expression wanes as cells begin to differentiate. Bmi-1 and Myc were found to be partners within the cell nucleus.[26][27] Bmi-1 and c-Myc seem to function in tandem in multiple ways. Studies have found that together c-Myc and Bmi-1 possess the ability to alter tumor suppressor genes. Hypoactive c-Myc was shown to alter p16 via Bmi-1, while hyperactive c-Myc was capable of altering the p16 promoter itself [28]. Normally, p16 functions to prevent cells from progressing through the G1 phase to the S phase of the cell cycle too quickly. Altering this function helps drive cells to proliferate uncontrollably making them more tumorigenic in nature. Hence, these data present a model in which c-Myc and Bmi-1 alter cellular apoptosis via cell cycle regulator genes. Conversely, another protein has been shown to alter Bmi-1 in such a way that negates its association with c-Myc and ultimately reduces its tumorigenic capacity. Researchers found that Akt phosphorylate Bmi-1 at Serine 316 (Ser316), thus inhibiting its chromatin-modifying function, suppressing its growth-promoting potential, promoting the derepression of the Ink4a-Arf locus, and decreasing cellular transformation activities with c-Myc.[28]

c-Myc has also shown association with the catalytic subunit of PRC2, EZH2. c-Myc has been shown to repress other genes using the H3K27me3 mark laid down by EZH2.[29] This allows c-Myc to take advantage of EZH2's silencing capabilities to prevent regulatory genes from acting upon it. EZH2 has also been shown to activate c-Myc directly in primary glioblastoma cancer stem cells,[30] as well as through the ERα and Wnt pathways in breast cancer cells.[31]

Links to Specific Types of Cancer

PcG proteins have been implicated in numerous types of cancers, though they are often deregulated differently according to the type of cancer under investigation. The following table shows specific types of cancer that PcG proteins have been known to be deregulated in, the identifier that links PcGs to this cancer (usually a PRC subunit or histone mark), how it is deregulated, and references for further details.

Type of Cancer	PcG Association	Characteristic	References
Hepatocellular Carcinoma (HCC)	EZH2	Upregulation	[29][30][31][32]
	H3K27me3	Increase	[33]
	Wnt/Beta-catenin	Upregulation	[34]
	miR-125b	Downregulation	[32]
	miR-1395p	Downregulation	[32]
	Bmi-1	Upregulation	[30][35][36]
Liver Neoplasia	Cbx7	Knock-Out	[37]
Lung Neoplasia	Cbx7	Knock-Out	[37]
Breast	EZH2	Upregulation	[38][39]
Ovarian	PcG Targets	Increased Methylation	[3]
Follicular lymphoma	PcG Targets	Increased Methylation	[40]
Glioblastoma Multiforme	PcG Targets	Increased Methylation	[41]

Polycomb Group Proteins in X Chromosome Inactivation

X Chromosome Inactivation

The polycomb group proteins influence X chromosome inactivation via epigenetic marks, such as histone methylation, and these modifications of chromatin structure have been implicated in oncogenesis. X chromosome inactivation is a random process by which one of two copies of the X chromosome is inactivated in female mammals.[42] The inactive X chromosome is packed via DNA condensation into a heterochromatic Barr body formation. Once inactivated, the condensed X chromosome will remain inactive throughout the lifetime of the cell and in its descendants in the organism. This inactivation process relies on the X-inactivation center (XIC) and its two transcripts, Xist and Tsix, with overlapping DNA.[42] Xist coats one X chromosome, and this X will become inactivated except for a small number of pseudoautosomal or escape gene regions.[42]

Polycomb Group Proteins in X Chromosome Inactivation

After the coating of Xist, the Polycomb group proteins bind to the future inactive X chromosome. Xist first triggers inactivation with Xist RNA binding in cis across the chromosome.[42] Proteins then bind the Xist RNA, modifying the histones. PRC2 inserts a histone 3 lysine 27 trimethylation mark, indicative of inactive chromatin. This Xist RNA is also probably bound by EHMT2 which inserts a histone 3 lysine 9 trimethylation mark, another indicator of repression. EeD (embryonic ectoderm development: a core subunit of PRC2) specifically recognizes and binds to the repressive trimethylated lysine marks, contributing to the affinity of PRC2 for nucleosomes.[43] PRC2 recruits DNMT3, which can add the 5 methyl DNA mark to CpG islands. Histone 3 lysine 27 trimethylation is then bound by PRC1 to trigger H2A ubiqination. Condensation continues with these marks as histone 3 lysine 4 is demethylated and histone 3 lysine 9 is deacetylated. These marks promote heterochromatin formation.[10] Analysis of the spread of X chromosome inactivation into autosomal material in one study showed that genes that were subject to (or escaped from) X chromosome inactivation clustered within topologically associating domains, and these genes were more likely to be found in regions that have PRC2 and histone 3 lysine 27 trimethylation marks normally on non-rearranged chromosomes.[44] MACROH2A is a replacement for histone H2A that also supports heterochromatin formation. In particular, one subtype of MACROH2A, macroH2A1.2, is concentrated in the inactive X chromosome in adult females. In fact, in some mammals macroH2A1 appears to be the earliest marker of the inactive X chromosome and is the only change that has been shown to occur during the period when transcriptional silencing is initiated.[45]

It is proposed that the PRC1 complex is involved in the maintenance of X chromosome inactivation in somatic cells via regulation of methylation. MACROH2A deposition has been suggested to be regulated by the CULLIN3 - SPOP - RBX1 ligase complex and is actively involved in stable X inactivation, likely through the formation of an additional layer of epigenetic silencing. E3 ubiquitin ligase, consisting of SPOP and CULLIN3, is able to ubiquitinate the Polycomb group protein BMI1 and the variant histone MACROH2A. PRC1 is also recruited to the inactivated X chromosome in somatic cells in a highly dynamic, cell cycle-regulated manner.[23] Recent study has indicated that knockdown of CULLIN3 or SPOP results in the loss of MACROH2A from the inactivated X chromosome, leading to reactivation even in the presence of methylation and deacetylase inhibitors.[23] SPOP mutations have been implicated in endometrial cancer through the SPOP-CUL3-RBX1 E3 ubiquitin ligase complex.[46] Thus, the PRC1 complex is involved in the maintenance of X chromosome inactivation in somatic cells. Another study has shown that alternative splicing of the histone variant MACROH2A1 regulates cancer cell proliferation via QKI splicing factor through RNA interference.[47] MacroH2A1 splicing is perturbed in several types of cancer including lung cancer.[47] The accumulating body of evidence demonstrates that changes in chromatin structure occur in oncogenesis, and changes in the expression of histone variants are beginning to be observed in cancer due to the changes in chromatin structure and function.[47] Polycomb group proteins have been implicated in this path.

Clinical Applications

EZH2, a histone-lysine N-methyltransferase and the functional enzymatic component of PRC2, encoded by the EZH2 gene, is a popular point of study in the treatment of B cell lymphoma. As this enzyme continues to be studied, research suggests its implication in the proliferation of other cancers.[48]

One study is investigating the safety and clinical activity of GSK2816126, a histone-lysine N-methyltransferase EZH2 inhibitor with potential antineoplastic activity in subjects with relapsed/refractory diffuse large B cell and transformed follicular lymphoma.[49] Non-Hodgkin lymphoma (NHL) is the seventh most common malignancy. Diffuse large B cell lymphomas are the most common subtype of NHL, constituting about 30 to 40% of adult NHLs.[50] This selective, competitive inhibitor molecule inhibits the activity of EZH2 and prevents the methylation of histone 3 lysine 27. This decrease in histone methylation alters gene expression patterns associated with cancer pathways and results in decreased tumor cell proliferation in cancer cells that overexpress this enzyme. EZH2, included in the class of histone methyltransferases (HMTs), is overexpressed or mutated in a variety of cancers and plays a key role in tumor cell proliferation.[50] In Phase I, this study has recruited participants for testing. The treatment regimen includes the administration of GSK2816126 twice weekly by intravenous infusion.

Another proposed study is examining E7438, another EZH2 histone methyltransferase inhibitor.[51] The preclinical drug characterization identifies this agent as a potent, selective inhibitor of EZH2 with antitumor activity in EZH2 mutations. Through Phase 2, the study has recruited participants. Phase 1 consisted of administering escalating doses of the EZH2 inhibitor E7438 orally twice per day to determine the maximum tolerable dosage (MTD). The second phase will determine the safety and activity of E7438p in EZH2 mutation positive subjects with histologically confirmed diffuse large B cell lymphoma Grade 3 follicular lymphomas with relapsed or refractory disease.

A third study investigates CPI-1205, another small molecule EZH2 enzyme inhibitor, as an interventional treatment of B cell lymphoma. This Phase 1 study consists of administering escalating doses of CPI-1205 to determine the frequency of dose-limiting toxicities (DLTs).[48] Subsequent phases will seek to further characterize the safe levels of drug administration as well as characterize the pharmacodynamic effects and disease response to CPI-1205 treatment/intervention.

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