DNA Methylation and Flavonoids in Genitourinary Cancers

Abstract

Malignancies of the genitourinary system have some of the highest cancer incidence and mortality rates. For example prostate cancer is the second most common cancer in men and ovarian cancer mortality and incidence are near equal. In addition to genetic changes modulation of the epigenome is critical to cancer development and progression. In this regard epigenetic changes in DNA methylation state and DNA hypermethylation in particular has garnered a great deal of attention. While hypomethylation occurs mostly in repeated sequence such as tandem and interspersed repeats and segment duplications, hypermethylation is associated with CpG islands. Hypomethylation leads to activation of cancer-causing genes with global DNA hypomethylation being commonly associated with metastatic disease. Hypermethylation-mediated silencing of tumor suppressive genes is commonly associated with cancer development. Bioactive phytochemicals such as flavonoids present in fruits, vegetables, beverages etc. have the ability to modulate DNA methylation status and are therefore very valuable agents for cancer prevention. In this review we discuss several commonly methylated genes and flavonoids used to modulate DNA methylation in the prevention of genitourinary cancers.

Introduction

Epigenetic modifications of DNA or associated proteins cause changes in gene expression without altering DNA sequence. This includes various pathways and mechanisms that maintain alternate states of chromatin structure by affecting protein composition and transcriptional activity through DNA methylation, histone modifications, nucleosome positioning and non-coding RNAs (1). Collectively these processes are critical players that regulate accessibility of the transcription machinery to gene promoters (2). Epigenetic changes that modify the contact between transcription machinery and gene promoters can lead to aberrant activation or inhibition of numerous signaling cascades resulting in different diseases including cancer (3). Therefore understanding epigenetic alterations involved in cancer development, progression, therapeutic response and resistance hold great significance. Phytochemicals are plant-derived substances that can affect disease development and have been used for the prevention of chronic and degenerative diseases (4). Flavonoids represent a class of low molecular weight polyphenolic phytochemicals found universally in plants (5). Based on their chemical structure they are categorized as flavonols, flavones, flavonones, isoflavones, catechins, anthocyanidins and chalcones (see fig.1). They are characterized by a flavan nucleus and C6-C8-C6 carbon-skeleton (5). The antioxidant, anti-inflammatory, anti-thrombogenic, antiangiogenic and anti-cancer activities and their ubiquitous presence in fruits, vegetables, beverages etc. underscore the importance of flavonoids in human diet and diseases such as cancer (6). Cancers of the genitourinary organ system include prostate, testicular, ovarian, cervical, urinary bladder and kidney cancers. Genitourinary cancers contribute to more than a fourth of the total cancer incidence in the US and approximately 15% of cancer related deaths. An estimated 480,000 people are expected to be diagnosed with these collective cancers and more than 89,000 deaths are expected to occur in 2014 (7). Prostate cancer is the second most commonly diagnosed cancer in men and is generally associated with aging men. Testicular germ cell tumors on the other hand are a common solid malignancy of young male adults that are either seminomas or non-seminomas and occur with almost equal frequency. Ovarian cancer is the most deadly form of gynecological cancer in women. Urinary bladder cancer has the infamous distinction of being within the top ten most common cancers and the most common urinary system cancer that affects both men and women. Kidney cancer is the second most common urinary system cancer affecting both men and women. In this chapter we discuss DNA methylation changes in different genitourinary cancers (summarized in table 1), their ability to modulate expression of genes involved in cancer development and progression and the role of flavonoids in regulating epigenetic modifications in these malignancies.

Figure 1.

Chemical structure of the flavan nucleus (adapted from www.mdpi.com)

Table 1.

List of epigenetically silenced genes in GU malignancies with the target organs

Name of genes	Type of cancer	Reference
AR	Prostate and Cervical	(35),(19)
ERα-A, ERα-B and ERα-C	Prostate	(35)
PR	Prostate and Cervical	(35),(19)
Cyclin D2	Prostate	(37)
GSTP1	Prostate	(43)
GSTM1, GPX3, MGMT	Prostate	(44–47)
APC, CAV1, Cadherin 1, CDH1, LAMA 3, γ2 Laminin, TIMP3	Prostate	(39,45,46,52)
DKK3, EDNRB, RUNX3, SFRP1	Prostate	(39,44,45,47)
DAPK	Prostate and Cervical	(39–42)
RARbeta2	Cervical	(36)
PTEN	Cervical	(19)
RASSF1A	Cervical and Renal	(20,21),(29)
POU2F3	Cervical	(22)
E-cadherin	Cervical	(55)
MGMT	Cervical and Testicular	(20,48)
Testisin	Testicular	(23,24)
p16INK4a	Testicular and Bladder	(23–25)
HOXA9 and HOXB5	Testicular	(58)
PIWIL1, PIWIL2, PIWIL4, and TDRD1	Testicular	(23)
p14	Bladder	(26)
LAMA3, LAMB3 and LAMC2	Bladder	(50)
VHL	Renal	(27,28)
TU3A	Renal	(31)
BRCA1	Ovarian	(49)
OPCML, ICAM-1and CDH1	Ovarian	(56,57)

DNA hypermethylation

Archetypal CpG islands are devoid of methylation to allow transcription of required genes in the presence of essential co-factors and appropriate chromatin structure. DNA methylation at palindromic CpG sites by DNA methyltransferases (DNMTs) generates a pattern of methylated CpG dinucleotides at the 5′ end of the promoter of human genes. Methylation is a mechanism to control embryonic development, silence the X-chromosome, and prevent unwanted transcription etc. DNA methylation can also occur in the ‘shore regions’ of CpG islands, which have comparatively lower CpG density (8,9). DNA promoter hypermethylation-mediated transcriptional repression of genes prevents the binding of transcriptional activators and helps in the recruitment of methylcytosine-binding proteins (MBPs). These proteins help load DNMTs and histone deacetylases (HDACs) participating in chromatin remodeling and transcriptional silencing (10,11). DNMT1 shows a preference for hemimethylated DNA and is considered to be a maintenance enzyme that keeps the methylation pattern intact after DNA replication (12,13). DNMT3a and DNMT3b on the other hand do not distinguish between methylated substrates and are involved in de novo DNA methylation (13). Loss of genome-wide DNA methylation and lethality of Dnmt knockout mice demonstrates the importance of these enzymes in mammalian development (14,15). DNA methylation is a critical process during germ cell development that relies heavily on DNMTs. Among them, the de novo DNMTs mainly exert their function during pre-natal germ cell development while the maintenance DNMTs become critical in proliferating spermatogonia shortly after birth in the male (16). Changes in DNA methylation profile and heritable decrease in DNA-5-MeC due to reduced DNA methylation fidelity maintenance was initially considered to be solely a hypomethylation process that resulted in overexpression of oncogenes (17). However, the theory of demethylation of oncogenes leading to their activation has been replaced by the growing popularity of hypermethylation of tumor suppressor genes. These CpG islands become hypermethylated in malignant cells thus inactivating certain tumor suppressor genes through progressive process of numerous 'waves' of dysregulated methylation unlike a gene mutation. There are two hypotheses by which hypermethylation takes place. First is that methylation spreads from normal methylation centers to CpG islands devoid of methylation and the second involves 'seeding' of methylation that is already present and certain single CpG dinucleotides become methylated inducing more cooperative methylation in the surrounding to finally lead to hypermethylation (18). As discussed below DNA methylation plays an important role in genitourinary cancers through the modulation of numerous genes that play critical roles in cancer cell biology (1). Genes involved in regulation of cellular processes such as hormone response, cell cycle progression, DNA damage and repair, signal transduction, tumor invasion and architecture have deregulated hypermethylation providing the needed advantage to the sustenance of cancer cells.

Tumor suppressors

The tumor suppressor, PTEN is commonly silenced by promoter methylation in many of the genitourinary cancers. PTEN hypermethylation is an early event seen in patients with recurrent or fatal cervical cancer (19). Several other potential tumor suppressors are atypically methylated in cervical cancer. Loss of Ras association domain family 1 isoform A (RASSF1A) leads to tumor progression suggesting a tumor suppressive role for this protein. In cervical cancer cells hypermethylation of RASSF1A is a mechanism through which cervical cancer cells extinguish death receptor mediated cell death (20,21). Aberrant DNA methylation of POU2F3 promoter, which is a transcription factor with putative tumor suppressive function involved in cell type-specific differentiation, is common in cervical cancer (22). Testisin, a putative tumor suppressor and testicular protease involved in sperm cell maturation and the CDK inhibitor, p16^INK4a are hypermethylated in testicular cancer (23,24). Methylation of p16^INK4a promoter and subsequent inactivation is involved in the initiation of bladder cancer (25). Hypermethylation of the other gene product of CDKN2A, p14 in normal bladder samples after resection has been found to be a predictor of bladder cancer recurrence (26). The most important genetic event in ccRCC is the hypermethylation-mediated inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene, which stabilizes hypoxia-inducible transcription factors HIF-1 and HIF-2 and the induction of a multitude of hypoxia inducible genes (27,28). RASSF1 is frequently methylated in sporadic RCC (either biallelically or as a second hit following 3p deletion) (29). RASSF1A methylation was detected in normal kidney tissues adjacent to the tumor but not in distant normal tissues indicating that the TSG methylation is part of a ‘field effect’ and an early event in cancer development causing epigenetic alterations in a large number of cells which is then accompanied by additional genetic and epigenetic changes (30). Another tumor suppressor gene TU3A (located at 3p21.1) is also found to be methylated in 42% of ccRCC and 25% of pRCC (31). The FHIT gene that encodes a diadenosine triphosphate hydrolase is an important player in purine metabolism and its promoter methylation has been seen in both ccRCC and pRCC (32). Methylation of RARβ, which regulates cell proliferation and differentiation, is also reported in less than 20% of RCC cases (32–34).

Steroid receptors

Steroid receptors are critically involved in the regulation proliferation, differentiation, and development of prostate cells with the androgen receptor (AR) being a key player in prostate cancer. Interestingly late-stage prostate cancer tissues have methylated AR promoters implicating methylation of AR promoter as a late event in prostate cancer (35). Estrogens, which are important in female sexual development, also affect benign prostatic hyperplasia and the development of prostate cancer with estrogen receptor (ER) expression being inversely correlated with histologic grade or pathologic stage of prostate cancer. The expression of ERα isoforms was found to be dependent on the methylation status of their respective promoters with ERα-C being unmethylated whereas ERα-A and ERα-B being methylated in prostate cancer tissue samples (35). Methylation of progesterone receptor (PR) is found in some prostate cancer cell lines and is believed to also be a late stage event in prostate cancer (35). Estrogen and progesterone receptor genes are also methylated in cervical cancer (19). Retinoic acid receptor beta2 (RARbeta2) is methylated in invasive cervical cancer, and methylation levels were found to correlate with pathological state of prostate cancer (36).

Cell cycle regulation and apoptosis

D-type cyclins play a very important part in cell cycle regulation and high cyclin D2 methylation levels has been associated with invasive prostatic carcinoma and clinically aggressive prostate cancer (37). The Human papillomavirus (HPV), a cervical cancer causative agent can exist either in the integrated or episomal state in the cancer cells. The integrated form of HPV has been reported to be associated with methylation of the cell cycle regulatory protein Cyclin A1 (CCNA1), which is inversely correlated with p53 mutation status in head and neck cancers (38). Transcriptional silencing of apoptosis-related genes such as Death-associated Kinase (DAPK) due to aberrant promoter methylation leads to the impairment of the apoptotic machinery and disrupts cell growth and death homeostasis in prostate and cervical cancer cells (39–42).

DNA repair

The glutathione S-transferases (GSTs) consist of a family of enzymes that convert electrophilic and hydrophobic compounds into more soluble and easily excreted forms by catalyzing their conjugation to glutathione. They participate in the detoxification of several potentially carcinogenic compounds thereby contributing to cancer prevention. Glutathione Stransferase P1 (GSTP1) was one of the earliest genes identified to be hypermethylated in prostate cancer (43). Subsequently others such as Glutathione S-transferase M1 (GSTM1), Glutathione peroxidase 3 (GPX3) and O-6-methylguanine DNA methyltransferases (MGMT) have been identified as having hypermethylated promoters in prostate cancer (44–47). MGMT encodes an enzyme involved in DNA repair and removes alkylated guanine in DNA through stoichiometric transfer of the alkyl group at the O-6 position to a cysteine residue. Methylation of the MGMT promoter leads to its inactivation and the development of non-seminomatous testicular cancer and is also seen in cervical cancer (20,48). Given the importance of oxidative stress in cancer, loss of function of gene products involved in neutralizing oxidative DNA damage is of great significance. Promoter hypermethylation of BRCA1 observed in ovarian cancer may be an alternative mechanism in silencing the tumor suppressive function of this gene in ovarian cancer (49).

Migration, invasion and metastasis

Laminins are components of the extracellular matrix (ECM) that maintain the architecture of the basal lamina surrounding the epithelial cells and modulate cell migration and invasion whereas E-cadherin-catenin complex plays a key part in epithelial cell-cell adhesion and tissue architecture. Methylation of LAMA3 and LAMB3 has been found to be associated with poor grade and stage in bladder cancer while the methylation of LAMC2 has been linked to a drop in survival time in patients (50). Aberrant methylation LN5 gene and E-cadherin has been found to correlate with clinicopathological features and invasion in prostate cancer (51,52). Other genes implicated in tumor invasion such as Familial adenomatous polyposis (APC), (Caveolin 1CAV 1), Cadherin 1CD44 Cluster differentiation antigen (CDH1), α-3 Laminin (LAMA 3), γ2 Laminin (LAM C2), TIMP3 have also been found to undergo hypermethylation in prostate cancer (39,45,46,52). In bladder cancer, down regulation of the CDH1 gene through promoter hypermethylation is frequently correlated with staging and grading of bladder cancer but the relation with methylation patterns is somewhat inconsistent with other studies showing high to low level of methylation at the promoter of CDH1 in bladder cancer (46,53,54). Promoter methylation of E-cadherin is also seen in cervical cancer (55). Other cell adhesion genes including OPCML, ICAM-1and CDH1 are hypermethylated in ovarian cancer (56,57).

Miscellaneous

Genes involved in normal development such as the homeobox genes (HOXA9 and HOXB5) are variably methylated in testicular cancer (58). Global methylation at long interspersed nuclear elements-1 (LINE-1) is inherited in familial testicular cancer kindred (59). Interestingly, hypermethylation mediated silencing of the small regulatory PIWI-interacting RNAs (piRNAs) including PIWIL1, PIWIL2, PIWIL4, and TDRD1, which regulate male germ line development has been observed in primary seminoma and non-seminoma testicular cancer (23). Several genes involved in signal transduction including Dickkopf (DKK3), Endothelin receptor type B (EDNRB), RASSF1A, Runt-related transcription factor 3 (RUNX3) and Secreted frizzled-related protein 1 (SFRP1) have been found to be hypermethylated in prostate cancer (39,44,45,47).

Hypomethylation

Loss of DNA methylation was the first reported epigenetic change in human cancer (60). Approximately half of the genome comprises of highly repeated DNA sequences, which contribute to the global DNA hypomethylation commonly observed in cancers (61–63). Most commonly studied DNA hypomethylated repeats in cancer include tandem centromeric satellite a, juxta-centromeric satellite 2, interspersed Alu and long interspersed elements (LINE)-1 repeats (62–66). Association between loss of methylation in mice lacking DNMT or fed methyl-deficient diet and cancer development provide evidence for the involvement of DNA hypomethylation in cancer development (67,68). In general unlike hypermethylation, hypomethylation of transcriptional regulatory sites in cancer appears to occur infrequently (69). Hypomethylation of gene promoters in cancer are associated with decreased overall genomic methylation or satellite DNA that correlates with increased transcription (70–73). Evidence for the contribution of gene-region hypomethylation to oncogenesis suggests a greater involvement in activating genes involved with tumor invasion and metastasis as well as drug-resistance (74,75).

DNA hypomethylation in genitourinary cancers

DNA hypomethylation appears to be a late event in prostate cancer contrary to other cancers. Although some LINE1 promoter hypomethylation is seen in primary prostate cancer the extent of hypomethylation is much greater when there is lymph node involvement (76). Hypomethylation of wingless-related MMTV integration site 5A (WNT5A), S100 calcium-binding protein P (S100P) and cysteine-rich protein 1(CRIP1) has been observed in prostate cancer (77). Hypomethylation and overexpression of the protease, Urokinase is associated with prostate cancer progression (78).Testicular germ-cell seminomas display a large amount of genomic hypomethylation, although this may be linked to the cell type from which this tumor originates (79). Hypomethylation has been found to cause re-expression of previously hypermethylated genes, such as the HRAS, BCL2, ABCB1, S100A4, SNCG, and CCND2 (40), and numerous cancer/testis associated genes (41) in testicular cancer. Global DNA hypomethylation has been observed in cervical cancer and increased DNA hypomethylation is involved in progression and appears to correlate with grade (80). Hypomethylation of satellite DNA has been observed in ovarian cancer and found to increase with stage and grade of ovarian cancer. Further, hypomethylation of Satellite 2 DNA correlates with poor prognosis (81). Genomic DNA hypomethylation is considered to be a biomarker for bladder cancer with increased LINE-1 hypomethylation and greater number of hypomethylated loci in cells derived from blood and urine of bladder cancer patients (82). Heparanase, an enzyme that acts both on cell-surface and within the extracellular matrix to degrade polymeric heparan sulfate molecules into shorter chain length oligosaccharides is also regulated by hypomethylation in bladder cancer (83).

Flavonoids as modulators of DNA methylation

Fruits, vegetables and other plant products used in beverages are a rich source of polyphenols with flavonoids accounting for a large proportion of the polyphenolic compounds. Almost all categories of flavonoids have cancer prevention properties due either to their antioxidant or anti-inflammatory properties. However, a comprehensive analysis of the effect of this class of polyphenolic compounds on DNA methylation-mediated effects in cancer still remains to be carried out. Table 2 shows the flavonoids that have been tested in GU malignancies.

Table 2.

List of commonly tested flavonoids in GU malignancies

Flavonoid	Targeted Cancer	Reference
Quercetin	Bladder	(84)
(−)-epigallocatechin-3-Ogallate (EGCG)	Bladder, Renal	(86–91)
Genistein	Prostate and cervical	(94,96,97)
Curcumin	Prostate, cervical and ovarian	(103,106,107)

Flavonols include quercetin, myricetin, catechins etc. Quercetin is present in red onions, buckwheat, red grapes, apple skin, green tea etc. It also forms the structural backbone of the flavonones Hesperitin. Despite its ability to demethylate p16^INK4a gene promoter in colon cancer cells, its ability to modulate DNA methylation in genitourinary cancers remains to be vigorously tested. Quercetin was shown to decrease bladder cancer cell growth and to induce apoptosis by decreasing the DNA methylation of the estrogen receptor (ER-β), p16^INK4a and RASSF1A (84). Myricetin, which is found in many grapes, berries, fruits, vegetables, herbs can also decrease DNA methylation by inhibiting SssI DNMT (85).

The green tea polyphenol, (−)-epigallocatechin-3-O-gallate (EGCG) is the most studied catechin. It inhibits tumorigenesis partially by affecting DNA methylation through inhibition of DNMTs (85). EGCG can re-express many transcriptionally silenced genes through inhibition of DNMT1 enzymatic activity in prostate cancer cell lines (86–90). EGCG was shown to decrease growth and induce apoptosis in renal cell carcinoma by re-expressing tissue factor pathway inhibitor-2 (TFPI-2), a member of the Kunitz-type serine proteinase inhibitor family and decreasing its promoter hypermethylation (91). ECGC also decreases the promoter methylation of other genes such as hTERT and CDX2 thereby helping in tumor suppression (92,93).

Isoflavones present in legumes, pomegranate seeds etc. are another important group of dietary flavonoids, which includes the soybean isoflavone, genistein. Genistein has been most widely studied for its effect on DNA methylation in cancer including genitourinary cancers. Genistein has also been shown to exert its anticancer effect through modulation of DNA methylation (94,95). Genistein can change DNA methylation at gene promoters in the prostate of C57BL/6J mice (96). Genistein was also found to decrease DNMT activity resulting in transcriptional activation of genes such as p16^INK4a, RAR β, MGMT, phosphatase and tensin homolog (PTEN) and CYLD in prostate cancer and RAR β2 in cervical cancer (94,97). Genistein-mediated modulation of methylation of pro-tumorigenic miRNA-1260b, its targets sFRP1 and Smad4 inhibits prostate cancer cell proliferation, invasion and TCF reporter activity (98). Genistein upregulates the GTP binding RAS like gene (ARHI), which is an imprinted tumor suppressor gene that is downregulated in prostate cancer (99).

Other common dietary phenolic compounds found in many fruits and vegetables including hesperetin, naringin, apigenin and luteolin can also modulate DNA methylation. Apigenin, a flavone found in parsley, artichoke, basil, celery and other plants has been shown to inhibit the hypermethylation of various tumor suppressor genes. It has also been shown to reduce MssI enzyme-mediated hypermethylation in prostate epithelial cells (100). Although these compounds are not as efficient as ECGC in directly inhibiting DNMT activity they can indirectly regulate DNMT activity by regulating the ratio of S-adenosyl methionine (SAM) and S-adenosyl homocysteine (SAH) during their metabolic methylation by catechol-O-methyltransferase (COMT) (85,101). Curcumin was found to bring about global hypomethylation in MV4–11 leukemia cell line by inhibiting DNMT (102). It was also found to negatively regulate DNMT1 in ovarian and melanoma (103,104). Curcumin was also observed to decrease CpG promoter methylation of Neurog1, a highly hypermethylated marker in prostate cancer (105) and also the hypermethylation at the promoter region of tumor suppressor retinoic acid receptor 2 gene in cervical cancer (106). It was also found to be involved in the re expression of Nrf2, a critical regulator of the antioxidant response by reducing promoter hypermethylation in TRAMP prostate cancer cells (107). Curcumin also reduces hypermethylation of Fanconi anemia (FANCF) promoter, thereby regulating growth and proliferation in cervical cancer (108).

Future directions

Clearly dietary components including flavonoids affect DNA methylation patterns. Most reports in the literature have used in vitro model systems to test the demethylation effects of bioactive dietary compounds to restore functions associated with tumor suppression. It remains unclear how demethylation of methylated cytosines in the gene promoter regions would affect hypomethylation given the suggestion that there is crosstalk between demethylation and de novo methylation pathways during tumorigenesis that can make one pathway dependent on the other. Future work is needed to systematically analyze the influence of these bioactive components on the distribution of DNA methylation and demethylation patterns in cancer and normal tissues in order to predict the usefulness of these agents as clinical epigenetic modulators. Further, more attention should be exercised with regards to dietary consumption of bioactive foods in clinical trials when DNMT inhibitors are being tested. Future studies should include more thorough investigation of specific flavonoids to determine their global epigenetic effects and how they influence outcomes in normal and cancer states. Differential effects on each flavonoid on specific genes and their functional outcomes need further elucidation. Obtaining comprehensive understanding of the capabilities of flavonoids can be result in their development as novel, natural epigenetic modulators for cancer prevention.