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Abstract: Chromatin deregulation is an emerging theme in cancer pathogenesis, and bladder cancer stands out among many other cancer types with frequent mutations of genes involved in epigenetic regulation. Defects in chromatin-level regulation can be manifested at multiple levels such as changes in DNA methylation, histone methylation patterns, and non-coding RNAs. Chromatin modifiers mutated in bladder cancer, such as KDM6A, KMT2D, KMT2C, ARID1A, EP300, have been studied in bladder cell line models. Also, there are studies that mapped the active regulatory landscape of bladder cancer and histone modification profiles. Collectively, existing literature emphasizes the importance of a thorough understanding of epigenetic deregulation in bladder cancer. The epigenetic signatures of bladder cancer can be targeted via epigenetic drugs or other genome editing tools, ultimately bringing specific treatment options for this cancer. This chapter provides an overview of the epigenetic modifications in bladder cancer, and the potential of epidrugs for the treatment of bladder cancer.

Keywords: chromatin modifiers in bladder cancer; epidrugs for bladder cancer; epigenetics in bladder cancer; histone methylation in bladder cancer; mutations in bladder cancer

INTRODUCTION

Cancer is a complex disease with many hallmarks (1). During the last decade, there has been a tremendous effort to characterize the genomic landscape and to identify molecular subgroups of diverse cancer types (2–4). All these molecular studies made it clear that epigenetic deregulation was a common theme implicated in tumorigenesis. It became apparent that proper epigenetic regulation is essential for normal cellular homeostasis and any deviation from this tightly regulated balance disrupts the cellular states and may result in tumor formation (5, 6). Among all the other cancers, bladder cancer has an exceptionally high rate of chromatin modifier mutations (7), and thus considered as a disease where epigenetic deregulatory mechanisms play a fundamental role. Bladder cancer mostly originates from the urothelium and causes over 200,000 deaths each year (8). Its main classification is done based on histopathology as non-muscle invasive bladder cancer (NMIBC) and muscle invasive bladder cancer (MIBC). Recent studies characterized the mutational landscape of both MIBC and NMIBC and further identified the consensus molecular subgroups, providing fundamental insights about the pathogenesis of bladder cancer (9–12). However, there is still need for further studies to characterize the epigenetic deregulation of bladder cancer in detail and use this information for specific diagnosis and treatment of bladder cancer. This chapter mainly focuses on the chromatin modifiers frequently mutated in bladder cancer, the major regulatory mechanisms disrupted, and the potential use of epigenetic therapies in bladder cancer.

EPIGENETIC REGULATION AND CANCER

To understand and explain the origin and characteristics of the cancer, several theories have been proposed throughout the years. “Hallmarks of cancer” proposed by Hanahan and Weinberg conceptualizes and organizes the principles in a logical framework (1). All hallmarks and characteristics define functional properties acquired by normal cells in the way of progressive transformation from normal state to neoplastic state (6, 13). Acquisition of hallmarks depends on alterations in the genome, epigenetic reprogramming, and microenvironmental remodeling. In addition to genetic alterations, epigenetic modifications contribute to gene expression deregulation in cancer. Aberrations in epigenetic mechanisms, such as DNA methylation, histone modifications, deregulation in non-coding RNAs (miRNA, lncRNA), play an important role in tumorigenesis contributing to the different hallmarks of cancer (14, 15). These epigenetic deregulations may result in inappropriate activation or inhibition of gene expression.

DNA methylation occurs at cytosine residues at CpG dinucleotides. While CpG dinucleotides spread throughout the genome, CpG islands (CGIs) are located at 5’ regulatory regions, such as promoter of genes. Promoter DNA methylation is associated with repression of transcription (16, 17). In human cells, 3 different DNA methyltransferases (DNMT1, DNMT3A, and DMT3B) catalyze the transfer of methyl group to cytosine residue (18). Aberrations in the maintenance of the DNA methylation are critical for tumorigenesis. Global hypomethylation and the promoter hypermethylation are the characteristics of the cancer epigenome and contribute to the overexpression of protooncogenes and the silencing of tumor suppressor genes, respectively (19, 20). Oncogenic signaling pathways also direct the activity of global methyltransferases which contributes to the shift from normal to cancer-specific methylation profile (15). Alterations in the DNA methylation have been known as early event in bladder cancer development and are considered as a hallmark of cancer (21).

In a eukaryotic nucleus, DNA is wrapped around the histone octamers forming nucleosome structure. N-terminal tails of core histone protein (H2A, H2B, H3, and H4) are largely targeted for the posttranslational modifications (PTMs), such as methylation, acetylation, and phosphorylation (16). Modifications in histone tails affect the chromatin structure which is critical for the gene regulation (22). Chromatin structure is highly dynamic, and orchestrated by chromatin remodeling complexes, and histone modifying enzymes. Aberrations in histone modification caused by defects in activity of histone modifying enzymes and chromatin remodeling complexes may contribute to the neoplastic transformation (23). Mutations in histone genes or chromatin modifier proteins are frequently detected in many cancer types, resulting in impairments in gene expression programs and genomic integrity (24) (Figure 1).

Figure 1. The landscape of epigenetic deregulations in cancer. During the tumorigenesis, DNA methylation in promoter regions is induced, while genome-wide DNA methylation is downregulated. Cancer cells exhibit disrupted histone tail modifications and chromatin organization.

CHROMATIN MODIFIERS FREQUENTLY MUTATED IN BLADDER CANCER

To advance our understanding on the molecular landscape of cancer, large-scale genome wide studies, especially the TCGA project, collected data on gene expression, transcript splice variation, protein expression, DNA copy number alterations, somatic mutation, DNA methylation, and gene fusion, and also clinicopathological data from many cancer types, including bladder cancer (11, 25). Integrated omics studies have revealed that, with five or more mutations per megabase, bladder cancer has a higher mutational burden compared to the other cancer types (10, 11, 25, 26). The most common mutations in bladder cancer occur in genes functioning in histone modification and chromatin remodeling genes. These include ARID1A (25%), KDM6A (24%), KMT2D (27%), EP300 (15%) (27). Globally, almost 80% of all bladder cancer patients have mutations in genes involved in epigenetic regulation, demonstrating the high degree of epigenetic dysregulation in this cancer (28). It is also important to the notice that chromatin modifiers mutated in bladder cancer mostly function in active chromatin organization and activation of gene expression. In this context, it might be speculated that chromatin modifier mutations in bladder cancer results in a closed chromatin configuration, likely prohibiting the expression of genes required for urothelial differentiation while resulting in gene expression programs supporting proliferation and tumorigenesis (Figure 2).

Figure 2. Chromatin modifier mutations contribute to neoplastic transformation of bladder cells. Mutations in chromatin modifying genes direct the shift from active chromatin organization to repressive state. DNMT, DNA methyltransferase; HAT, histone acetyltransferase; HDAC, histone deacetylase; HDM, histone demethylase; HMT, histone methyltransferase; RNA pol II, RNA polymerase II; TF, transcription factor.

EPIGENETIC LANDSCAPE OF BLADDER CANCER

As already mentioned, aberrations in the epigenetic landscape are one of the hallmarks of cancer and abnormalities in DNA methylation, chromatin modifier mutations, and altered gene expression of chromatin modifiers and non-coding RNAs result in changes in cellular characteristics and promote the unfavorable prognosis. The association between epigenetic landscape and gene expression in bladder cancer has been addressed in several studies (10, 11, 29). One study defined the genome-wide chromatin accessibility profiles and cancer-specific DNA regulatory elements across 23 cancer types from TCGA (including bladder cancer) and identified significant correlations between gene expression and chromatin accessibility, integrating transcriptomics data and ATAC-seq (the assay for transposase-accessible chromatin using sequencing). Besides, this study demonstrated impressive similarity between ATAC-seq based clustering and previously established mRNA, miRNA, DNA methylation or copy number variation (CNV) profile-based classifications. In this study, it has been also revealed that a somatic mutation observed in one regulatory region of bladder cancer increases chromatin accessibility and changes gene expression in mutant bladder cancer (29).

To further extend the knowledge on epigenomic landscape of bladder cancer, van der Vos et al. (10) conducted a study on genome-wide histone methylation profiling of MIBC (10). Integrated analysis of the H3K27me3, a repressive histone mark, and H3K4me1 and H3K4me3 (gene-activating histone marks) ChIP-seq and RNA-seq data indicated that different enhancer regions play critical role in the characterization of luminal and basal subtypes of MIBC.

Non-coding RNAs, such as microRNAs (miRNA), long non-coding RNAs (lncRNA), circular RNAs (circRNA), piwi-interacting RNAs (piRNA), small nuclear RNA (snRNA), and small nucleolar RNAs (snoRNA), are not translated into proteins, but they have still significant functions in every cellular process. NcRNAs also contribute to the epigenetic alterations that promote bladder cancer development and progression (30).

Prognostic biomarker potential of DNA hypermethylation has been widely investigated in bladder cancer (31, 32). Yet, further investigation is necessary to obtain more sensitive and specific biomarkers. It has been identified that CpG-rich transposons, such as LINE1, are hypomethylated in bladder cancer types. This leads to retrotranspositions inducing genomic instability (33). A recent study investigated global histone acetylation levels and its prognostic value in bladder cancer patients and reported decreased H3 acetylation level in both NMIBC and MIBC patients compared to normal urothelial control group (34).

Histone deacetylases (HDACs) are divided into different classes based on their similarity to yeast HDACs (35). Another study reported that HDAC-1, HDAC-2, and HDAC-3 expression levels are elevated in urothelial carcinoma. Notably, increased HDAC-1 and HDAC-2 levels were associated with high grade tumors. Moreover, high grade tumors with high HDAC-1 expression correlated with worse prognosis compared to low grade tumors (36). This finding supports the therapeutic target potential of HDACs. Another study identified the chromatin interactions by Hi-C, integrating it with transcriptome and enhancer profiles in luminal and basal types of bladder cancer. Even though the study implicated the association between epigenomic landscape and 3D genome structure in a subtype-specific manner, further studies are needed to comprehensively unveil the molecular basis and involved factors (37).

FUNCTIONAL OUTCOMES OF CHROMATIN MODIFIER MUTATIONS IN BLADDER CANCER

Given the high rate of chromatin modifier mutations in bladder cancer, there have been many studies investigating the functional impact of the mutations in different model systems (Table 1) (38–46). Polycomb repressive complex 2 (PRC2)-dependent epigenetic regulation is critical for cell differentiation and proliferation in bladder urothelium (47). SWI/SNF complex acts as an antagonist of PRC2 complex promoting the expression of genes which are silenced by PRC2 (48). ARID1A belongs to SWI/SNF complex proteins (49), and it is frequently mutated in primary human bladder carcinoma (25). ARID1A has role in the tumor suppressor mechanisms regulating cell cycle progression and maintaining genomic stability (38).

TABLE 1 Key functional outcomes associated with the chromatin modifier mutations in bladder cancer

Mutation/loss of function	Functional outcome	Reference
ARID1A	Impairments in cell cycle Genomic stability Induced cell proliferation	(38, 39)
KDM6A	Induced tumor immune escape Activation of proinflammatory pathways Induced proliferation Deregulation in the expression of cell identity related genes	(40–43)
KMT2C	Increased chromatin instability Impairments in DNA replication and repair Misregulation of apoptosis, and cell cycle control	(44)
KMT2D	Impairments in DNA replication and cell cycle Induced invasion, migration, and viability	(45, 68)
CBP/EP300	Impairments in histone acetylation Increased anti-tumor immunity	(46)

ARID1A protein loss is predominantly observed in high grade and high stages of bladder tumors that indicates association with poorer prognosis (39, 50). The potential functions of ARID1A have been investigated in urothelial cells of ARID1A knockout mice. It has been shown that loss-of-function mutation in ARID1A upregulates urothelial cell proliferation, emphasizing the tumor suppressor role of ARID1A in bladder cancer development (39). Additionally, findings implicated an antagonistic relationship between ARID1A and PRC2 complex in bladder (51). However, function of ARID1A might be context-dependent since different studies addressed opposing roles for ARID1A in different cellular processes and cancer types (52).

KDM6A (UTX), lysine histone demethylase, physically interacts with chromatin modifying enzymes, such as KMT2C (MLL3) and KMT2D (MLL4) (53). KDM6A protein contains tetratricopeptide repeat (TPR) domains and Jumonji C (JmjC) domain. JmjC domain catalyzes the removal of the methyl group from H3K27me2 and H3K27me3 (53, 54). TPR domain conducts interaction with components of MLL3 and MLL4 complexes (55). The function of KDM6A has been the subject of numerous studies. These studies reported that KDM6A regulates gene expression and cellular processes. As a component of the COMPASS complex, KDM6A is involved in regulation of gene activation (56–58). Loss-of-function and inactivating mutations frequently occur in several neoplasms, including bladder tumors (59–62). Reduced KDM6A expression and KDM6A mutations is correlated with poor prognosis in bladder cancer (40). Furthermore, potential roles of KDM6A in immune response have been shown via TIMER and CIBERSORT algorithms. Gene set enrichment analyses have indicated that the signaling pathways involved in immunity have been repressed in patients with mutated KDM6A. These findings imply the relationship between KDM6A mutations and anti-tumor immunity (40). In another study, Kobatake et al. showed that decreased expression of KDM6A is associated with the activation of proinflammatory pathways (41). Increased proliferation has been observed in two different KDM6A knock-out bladder cell lines (42). Notably, KDM6A has a role in safeguarding luminal gene expression program in bladder cancer cell lines (43).

Studies focused on the function of KMT2C (MLL3, histone lysine methyltransferase 2C) in normal cells defined its role in regulation of enhancer activity, focusing on the profile of H3K4me1 mark (63, 64). Independent from its H3K4me activity, the roles of KMT2C in transcription regulation have been shown in recent reports (65, 66). Tumor suppressor role of KMT2C has been reported for urothelial carcinoma. KMT2C silencing in 2 different bladder cancer cell lines has been shown to directly or indirectly affect the expression of genes involved in cell cycle control, DNA repair, DNA replication, and apoptosis (44). To investigate its further effects, genome-wide binding profile of KMT2C has been mapped via ChIP-seq (44). To evaluate the effects of KMT2C on epigenetic landscape of bladder cancer, Rampias et al. (44) also studied the changes in H3K4me3, H3K27ac, and H3K9ac histone modifications upon KMT2C silencing. Knockdown of KMT2C influences the enhancer activity in bladder cancer cell lines. In parallel with its well-established role in deposition of H3K4me1, co-localization of KMT2C with active enhancer mark H3K27ac points out the increased enhancer activity (44). Additionally, KMT2C loss affects expression of genes critical for cell adherence, extracellular organization, and epithelial differentiation (44).

KMT2D (also known as MLL4) is one of the histone methyltransferases that may play a critical role in tumorigenesis and progression of bladder cancer (63). KMT2D regulates the activity of H3K4 methylation (67). KMT2D has high mutation rates in bladder cancer. Low levels of KMT2D are associated with lymph node metastasis (68). KMT2D mRNA and protein expression is decreased in 4 bladder cancer cell lines (T24, J82, UM-UC-3, and HTB-9) compared to normal bladder cell line. Silencing of KMT2D induces invasion in T24, and HTB-9 cell lines, while its overexpression suppresses. It has been demonstrated that KMT2D regulates level of H3K4me1 in bladder cell lines (68). Interestingly, while Sun et al (68) showed association between higher KMT2D expression and higher survival rate, Ding et al (45) implied that KMT2D mutations are associated with better prognosis in bladder tumors. Gene set enrichment analysis has indicated that KMT2D mutations are also significantly associated with cell cycle and DNA replication processes (45).

CREB-binding protein (CREBBP or CBP) and E1A binding protein (EP300 or P300) are transcriptional coactivators which also have ubiquitin ligase activity and histone acetyltransferase activity (69). CBP and EP300 are frequently mutated in a variety of human tumors (70). These inactivating alterations resulting in deregulation of acetylation and neoplastic transformation have been investigated in tumor models and bladder cancer lines (71, 72). Duex et al. (71) defined that those mutations are largely enriched at histone acetyltransferase domains of EP300 and CBP, implying potential significance of the domain activity on tumorigenesis. They also postulated that impairments in histone acetyltransferase activity are more likely to be linked with aggressive, MIBC cases (71). It was also identified that mutations in EP300 promote the signaling pathways involved in anti-tumor response in bladder cancer (46).

MANIPULATING CHROMATIN MODIFIER MUTATIONS FOR TREATMENT OF BLADDER CANCER

New strategies and options for diagnosis and treatment of bladder cancer are needed to augment pharmacological outcome. The utilization of epigenetics for diagnostic markers and therapy targets is a rapidly developing and promising area. The reversibility of epigenetic changes serves a great potential as a therapeutic target in bladder cancer. Improvement of epidrugs has great advantage for cancers or disease in which epigenetic dysregulation plays a key role (Figure 3) (73).

Figure 3. Epigenetic therapy options for cancer treatment. Epidrugs can be used alone or in combination with the other treatments. Epigenome editing technologies are also emerging alternatives for cancer therapy. DNMTi, DNA methyltransferase inhibitors; EZH2i, EZH2 inhibitors; HATi, histone acetyltransferase inhibitors; HDACi, histone deacetylase inhibitors; HDMi, histone demethylase inhibitors; HMTi, histone methyltransferase inhibitors.

Inhibiting the DNMT enzymes, gene silencing can be reversed, and in turn expression of tumor suppressor genes is recovered. It has been revealed that 5-Aza-2′-Deoxycytidine (DAC), DNMT inhibitor, induces cell cycle arrest, and increases the susceptibility to chemotherapy in bladder tumors (74). 5-aza-2′-deoxycytidine and 5-azacytidine are approved for treatment of myelodysplastic syndrome and myeloid leukemia by the FDA. There are ongoing clinical trials for use in bladder cancer therapy (75).

Several HDAC inhibitors show promise in urological cancers (76). It has been demonstrated that cellular growth and proliferation is inhibited upon the treatment of bladder cancer cells with HDAC inhibitors Vorinostat, Romidepsin, and Trichostatin A (77, 78). Further analyses showed that changes in the protein expression are mostly associated with apoptosis, regulation of cell cycle, and DNA damage repair mechanisms in response to treatment with these HDAC inhibitors (78). HDAC inhibitors Romidepsin, have been approved by FDA for treatment of cutaneous T cell lymphoma (CTCL), while Belinostat and Panobinostat approved for the treatment of T cell lymphoma (79). In combination with the other chemotherapy agents, HDAC inhibitors synergistically affect the cell cycle arrest, apoptosis, and differentiation of malignant cells (30, 80). It has been shown that combination of DNMT inhibitor and HDAC inhibitors has also synergistic effect on cancer cells (76).

Increased expression levels of G9a, H3K9 methyltransferase, have been detected in bladder cancer. Inhibition of G9a in bladder cancer suppresses the proliferation inducing autophagic cell death in bladder cancer cells (81). Treatment of bladder cancer cell lines with small molecule UNC064, G9a inhibitor, decreases the cell viability, while inducing the apoptosis (82).

As a catalytic subunit of PRC2 complex, histone methyltransferase EZH2 regulates trimethylation of H3K27 (H3K27me3) (83). This histone mark is critical for repression of gene expression. An increasing number of evidence demonstrated that EZH2 dictates both development and progression of different types of tumors. Dysregulation in EZH2 expression has been associated with increased cell proliferation, invasion, and metastasis (84). Notably, it has been reported that EZH2 is also linked with the chemotherapy resistance (85). Upregulated expression of EZH2 plays oncogenic roles in bladder cancer. Since it affects the gene expression and regulates the several cellular mechanisms, EZH2 serves a great potential as target for treatment (86). Currently, EZH2 inhibitor Tazemetostat is being investigated in ongoing clinical trials for treatment of urothelial carcinoma, in addition to lymphomas, and other solid tumors (87–89).

Therapeutic targeting of epigenetic modifiers which are currently in clinical trials (https://www.clinicaltrials.gov/) is summarized in Table 2. In a review, Ozgun et al. (28) evaluated the combination of EZH2 inhibitors or HDAC inhibitors with retinoids in bladder carcinoma. They pointed out the potential therapeutic options with retinoic acid and its derivatives and emphasized the clinical trials investigating combinatorial use of retinoids with epidrugs (28).

TABLE 2 Potential epidrugs for bladder cancer

Epidrug	Biological effect	Clinical trial	Combinatory therapies
5-Aza-2′-Deoxycytidine	DNMT inhibitor	Yes (Phase 2)
5-azacytidine	DNMT inhibitor	Yes (Phase 1)	Carboplatin Paclitaxel Pembrolizumab Docetaxel
Vorinostat	HDAC inhibitor	Yes (Phase 2)
Romidepsin	HDAC inhibitor	Yes (Phase 1, 2)	Carboplatin Paclitaxel 5-Fluorouracil (5-FU) Pembrolizumab
Belinostat	HDAC inhibitor	Yes (Phase 2)
Tazemetostat	EZH2 inhibitor	Yes (Phase 1, 2)

Another type of epigenetic therapy is based on miRNA manipulation. Strategies basically focus on regulation of the miRNA expression and activity in cancer cells (75, 90). This manipulation is managed via mimicking the specific miRNAs (91) or administration of epidrugs, such as EZH inhibitors (92, 93).

CRISPR/Cas9 system has become widely used technology for genome targeting. Due to the technical improvements, targeted epigenome editing can be achieved using CRISPR platform (94). The fusion of dCas to chromatin-modifying domains represents a powerful tool for chromatin editing (95). Epigenome editing is achieved in human cells through CRISPR activation and inhibition systems (CRISPRa/CRISPRi). These approaches can be applied for epigenetic reprograming both in vivo and ex vivo, disease modeling, therapeutic targeting, and cellular therapies (94). Recent clinical studies have been performed CRISPR-based epigenome editing in human hematopoietic progenitor and stem cells for the treatment of an immune disease (96). Yet, there is a need for more effort to establish routine clinical use.

CONCLUSION

Mutations in chromatin modifying genes are highly frequent in bladder cancer (11). There have been plenty of studies which emphasize that epigenetic deregulations are critical for understanding of bladder cancer pathogenesis, characterization of phenotype, determination of disease outcome, and also for directing the treatment options. Clinical adoption of bladder cancer epigenetics is still in a developmental phase. Expanding our limited knowledge on epigenetics of urothelial malignancies will contribute to the improvements in diagnosis, and development of more precise targeted therapies. Drugs targeting epigenetic machinery, epigenetic biomarkers for diagnosis, and tailoring the epigenome state of cancer cells are the emerging fields in personalized medicine. Yet, implementation of epidrugs and epigenome editing to clinic still needs to overcome many challenges.

Acknowledgment: This work was funded by The Scientific and Technological Research Council of Turkey (TÜBİTAK), and the EMBO Installation Grant (No:4148). Burcu Akman is a recipient of scholarship from the Scientific and Technological Research Council of Turkey (TUBITAK) project (#120C129).

Conflict of Interest: The authors declare no potential conflict of interest with respect to research, authorship and/or publication of this chapter.

Copyright and Permission Statement: The authors confirm that the materials included in this chapter do not violate copyright laws. Where relevant, appropriate permissions have been obtained from the original copyright holder(s), and all original sources have been appropriately acknowledged or referenced.

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