DNA Methylation for Bladder Cancer: Comparison
View Latest Version
Please note this is a comparison between Version 2 by Catherine Yang and Version 1 by Sandra P Nunes.

Bladder cancer (BC) is the tenth most frequent cancer worldwide and is associated with high mortality when diagnosed in its most aggressive form, which is not reverted by the current treatment options. The disruption of normal epigenetic mechanisms, namely, DNA methylation, is a known early event in cancer development. Consequently, DNA methyltransferase (DNMT) inhibitors constitute a promising therapeutic target for the treatment of BC.

  • bladder cancer
  • DNA methylation
  • DNA methyltransferases

1. DNA Methylation

DNA methylation is the covalent addition of a methyl group at the 5-position carbon of cytosine in a cytosine–phosphatidyl–guanine (CpG) dinucleotide [17][1]. Methylation occurs mainly at CpG islands—CpG-rich regions (at least 50% of cytosines and guanines) in the genome with a size larger than 200 bp [26,27][2][3]. Moreover, methylation can also be found in repetitive sequences such as retrotransposon elements and centromeres, in the X chromosome (leading to its inactivation) and genomic imprinting [28][4]. Approximately 29,000 CpG islands can be found in the human genome, most commonly in the promoter regions, close to the transcription starting site (TSS) or first exons [27][3]. Remarkably, 75% of cytosines in CpG dinucleotides dispersed throughout the genome are methylated, whereas cytosines of CpG islands located within gene promoters remain mostly hypomethylated [27,29][3][5]. Promoter DNA methylation is classically associated with transcription repression by inducing binding of transcriptional repressors or hampering binding of transcriptional factors [15,17][1][6]. In fact, a family of methyl-CpG-binding proteins (MBPs) intervenes in gene silencing by binding to methylated CpGs and recruiting histone modifier enzymes to establish histone post-translation modifications, which further sustain transcriptional repression [30][7]. DNA methylation can also be found in CpG island shores, 2-kb areas upstream of a CpG island, displaying lower CpG dinucleotide density. CpG island shore methylation is also associated with transcriptional repression [31][8]. On the other hand, methylation in the gene body was shown to stimulate transcription elongation and to have an impact on splicing, with exons disclosing higher methylation levels than introns [28,32][4][9]. Furthermore, tissue-specific methylation seems to be more frequent in intragenic CpG islands [33][10]. CpG islands in enhancers also influence gene regulation, i.e., hypermethylation is associated with loss of enhancer marks resulting in gene silencing [34][11].

2. DNA Methylation as a Therapeutic Target in BC

2.1. Preclinical Studies

A summary of the preclinical studies testing DNMT inhibitors in BC is depicted in Table 1. The effects of 5-aza were evaluated in cell lines in vitro and in a tumor xenograph model. 5-Aza was shown to inhibit cell proliferation and arrest cells at G0/G1, whereas volume and weight of tumor xenografts in mice were reduced. Interestingly, DNMT3a and DNMT3b expressions were also reduced after 5-aza treatment, causing the re-expression of hepaCAM, a TSG [56][12]. DAC treatment also led to an increase in hepaCAM expression in T24 and BIU87 cells, associated with arrest at G0/G1 phase [123][13]. In a canine model of invasive urothelial carcinoma, 5-aza disclosed anti-tumor effects, with 22.2% of the dogs demonstrating partial response and 50% depicting stable disease [124][14].
Table 1.
 Summary of pre-clinical studies targeting DNMTs in bladder cancer (BC).
Drug Model Concentration Treatment Scheme Effects Year Reference
DAC T24 1 µM 1 day ↑ Gene expression related to IFN pathway 2002 [132][15]
DAC T24 3 µM 1 day No remethylation in CpG islands in the absence of cell division 2002 [133][16]
Hydralazine and procainamide T24 10 µM 5 days ↓ p16 and RARβ methylation levels

↑ p16 and RARβ expression		 2003 [100][17]
DAC TCC and UMUC 5 µM n.a. ↑ MSH3 mRNA levels 2004 [135][18]
Zebularine T24 100 µM Every 3 days for 40 days ↓ Global methylation levels

↑ p16 expression		 2004 [130][19]
DAC J82C, T24C, TCC, and UMUC 5 µM 3 days ↑ Wif-1 mRNA expression levels 2006 [131][20]
DAC

Zebularine		 RT4 and T24 2 µM

100 µM		 2 days

Every 3 days for 7 days		 ↑ Cells doubling time

↑ APAF-1 and DAPK-1 expression

2006		 3–4 cycles of 21 days each for both arms. 3–4 cycles of 21 days each.[129 Not available][21]
2015–present [172][61] S110 T24 0.1–10 µM Every 3 days for 6 days ↓ Global methylation levels

↑ p16 expression		 2007 [137][22]
DAC
75 approved agents II

(NCT02788201)		 Completed Patients with a diagnosis of metastatic, progressive urothelial carcinoma of the bladder, urethra, ureter, or renal pelvis (n = 8) COXEN algorithm was used to determine the best therapy from among 75 FDA-approved agents (single agent or combination). Patients had regular visits for blood, urine, and tumor scans. Not available 2017–2019 [173][62] BIU87 0.1–5 µM 3 days Re-expression of RASSF1A 2009 [143][23]
Azacitidine, pembrolizumab, and epacadostat I

(NCT02959437)		 Completed Subjects with advanced or metastatic solid tumors including BC patients (n = 70) Five doses of azacitidine were administered by subcutaneous injection or intravenously (IV) over days 1 to 7 in cycles 1 through 6.

Pembrolizumab was administered in a 30-min IV infusion every 3 weeks on day 1 of each 21-day cycle.

Epacadostat tablets were administered orally twice daily.		 Not available 2017–2020 [166][63] DAC BOY 1 µM 4 days ↑ COL1A2 expression 2009 [144][24]
Atezolizumab and guadecitabine II DAC BOY, T24, and UMUC 10 µM 7 days ↑ FHL1 mRNA expression levels 2010 [145][25]
S110 Mouse tumor xenograft 10 mg/kg Daily injection for 6 days ↓ Tumor growth rate

↑ p16 expression		 2010 [138][26]
5-Aza Dogs with

naturally occurring invasive urothelial carcinoma		 0.1–0.3 mg/kg Two doses schedules:

Everyday days 1 to 5 or

days 1 to 5 and 15 to 19

Each cycle 28 days		 22.2% Tumor partial response

50% Stable disease

22.2% Progressive disease		 2012 [124][14]


(NCT03179943) Suspended Recurrent/advanced urothelial carcinoma (stage IV) patients who previously progressed on checkpoint inhibitor therapy with anti- PD-1 or PD-L1 therapy (n = 53) Atezolizumab is administered intravenously on day 1 and day 22 of a 6-week cycle for a period of 8 cycles. Guadecitabine is administered subcutaneously on days 1 through 5 of the 6-week cycle for a period of 4 cycles. Not available 2017–estimated end 2022 [167][64] DAC BIU87 and T24 0.1–10 µM 3 days Cell arrest at G0/G1

↑ hepaCAM expression		 2013 [123][13]
DAC T24 0.25–2 µM 2 days 1 ↑ Maspin expression levels

↓ Cell proliferation, migration and invasion		 2013 [127][27]
DAC 5637 1–3 µM 6 days ↑ GSTM1 expression 2014 [134][28]
DAC EJ 1 µM Every day for 3 days ↓ Cell tumorigenesis and invasiveness

Cell arrest at G2/M

↑ BTG2 expression		 2014 [128][29]
DNAzyme T24 n.a. n.a. ↓ Cell proliferation

↑ p16 expression		 2015 [142][30]
5-Aza BIU87, EJ, and T24

Mouse tumor xenograft		 0.5–7 µM

n.a.		 1–4 days

Every 3 days for 18 days		 ↓ Cell proliferation

↓ Tumor volume and weight		 2016 [56][12]
DAC T24 and J82 0.3 µM 1 day ↓ RSPH9 methylation levels 2016 [146][31]
DAC BLCAb001 (B01), BLCAb002 (B02), HT1376, and T24 0.1–1 µM Every 2 days for 5 days ↑ NOTCH1 expression

↓ CK5 positive cells

↑ IL-6 release		 2017 [125][32]
DAC T24 1 µM 1 day ↓ Methylation of 590 CpGs

↑ Methylation of 616 CpGs		 2019 [136][33]
1 For migration and invasiveness assays. Abbreviations: 5-aza—5-azacytidine; APAF-1—apoptotic peptidase activating factor 1; BTG2-BTG anti-proliferation factor 2; CK5—Cytokeratin 5; CpG—Cytosine–phosphatidyl–guanine; COL1A2—collagen type I alpha 2 chain; DAC—decitabine; DAPK-1—death associated protein kinase 1; FHL1—four and a half LIM domains 1; GSTM1—glutathione S-transferase mu 1; IFN—Interferon; IL-6—Interleukin 6; MSH3—mutS homolog 3; n.a.—not available; NOTCH1—Notch receptor 1; RARβ—retinoic acid receptor beta; RASSF1A—Ras association domain family 1 isoform A; ↑—increase, ↓—decrease.
Non-toxic concentrations of DAC were used to treat four BC cell lines in order to evaluate the impact of hypomethylation in the BC cell transcriptome. Notch receptor 1 (NOTCH1) expression increased after treatment with DAC, in parallel with 50% demethylation of the promoter and enhancer regions. Interestingly, DAC-treated cells displayed morphological changes, i.e., cell enlargement compared to the controls. To explore these effects, the active intracellular domain of NOTCH1, ICN1, was overexpressed in cell lines and a decrease in cell proliferation was observed. Moreover, there was an increase of interleukin (IL)-6, probably due to a rise in ICN1 expression and double-stranded RNA levels [125][32]. ICN1 overexpression also led to a decrease in basal stem-like cells, as assessed by the decreased cytokeratin 5 (CK5) levels, contributing to a more differentiated cell state [125][32], which may prevent BC progression [126][34]. DAC was also shown to inhibit cell proliferation, migration, and invasiveness, inducing apoptosis in T24 cells while increasing the expression of the TSG Maspin [127][27]. Another gene that appears to be regulated by methylation in BC is BTG2. After treating EJ cells with DAC, BTG anti-proliferation factor 2 (BTG2) expression increased mainly due to DNMT1 inhibition, and cell growth decreased. Furthermore, H3K9me2 levels decreased, whereas H3K4me3 increased, leading to an open chromatin state in the promoter and intronic region of BTG2 [128][29]. Apoptotic peptidase activating factor 1 (APAF-1) and death associated protein kinase 1 (DAPK-1) expressions also increased after DAC treatment in both RT4 and T24 cells, whereas zebularine caused an increased expression of those genes in cell line T24, which is p53 mutated [129][21]. The epigenetic regulation of Wnt inhibitory factor 1 (Wif-1), an antagonist of the Wnt pathway, important for carcinogenesis, was explored in four BC cell lines. DAC treatment led to increased Wif-1 mRNA levels with a simultaneous decrease in promoter methylation levels. Furthermore, Wif-1 expression was primarily regulated by DNA methylation and not genetic alterations [131][20]. DAC treatment in T24 cells induced the expression of several genes related to the IFN pathway, which could theoretically lead to inhibition of tumor cell growth [132][15]. Interestingly, Velicescu et al. showed that de novo methylation does not occur in non-dividing BC cells. Treating T24 cells with DAC allows for cell arrest at G0/G1 and determines how much time is necessary for re-methylation to occur. Indeed, no re-methylation was found in CpG islands, whereas various degrees of methylation reappeared in CpG poor regions. Furthermore, DNMT1 and 3b3 protein levels were not detected, whereas DNMT3a mRNA levels were maintained after day 10 of the experiment. This result demonstrates that DNMT3a might catalyze a de novo methylation in CpG poor regions outside the S phase of the cell cycle [133][16]. In another study, the carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine) (BBN) was used to induce bladder tumors in mice. These tumors showed downregulation of several genes including GSTM1, which seems to be regulated, in part, by DNA methylation, since treatment with DAC in 5637 cells increased GSTM1 expression [134][28]. Kawakami et al. reported for the first time that MSH3 epigenetic regulation by means of DNA methylation might contribute to gene silencing, being implicated in BC carcinogenesis [135][18]. Recently, a study comprising a wide range of different cancer cell lines, containing BC, showed that DNMT inhibitors, including DAC and 5-aza, increase methylation levels throughout the cancer epigenome. Specifically, in BC cell lines, DAC treatment increased methylation levels of 616 common CpGs and decreased methylation levels of 590 different CpGs, demonstrating that the DNMT inhibitor mechanism of action is complex and requires further exploration [136][33]. After treatment with S110, global methylation levels decreased, concomitantly with increased p16 expression [137][22]. The effects of S110 in vivo were also assessed in a tumor xenograft mouse model using EJ6 cells. Tumor-free animals tolerated S110 better than DAC, with less weight loss and mortality. S110 failed to cause reduction in tumor sizes, yet their growth rate was lower and p16 expression was induced [138][26]. The development of DAC and 5-aza variants could potentially increase their half-life, improving bioavailability and therapeutic efficacy. Procainamide and hydralazine inhibitory effects on DNA methylation were studied in T24 cells. Both compounds decreased the methylation levels of TSGs p16 and RARβ, which associated with their re-expression, both at the transcript and at the protein levels. Interestingly, hydralazine treatment maintained p16 reactivation for longer than DAC [100][17]. A novel strategy to inhibit DNMT1, using an essential enzyme for cancer cell viability [139][35] highly expressed in BC [140][36], was proposed using DNAzymes. A DNAzyme is a stable DNA molecule with catalytic activity that targets specific RNA molecules leading to their destruction [141,142][30][37]. The DNAzyme DT433, constructed and selected to target DNMT1, displayed effects that were similar to those of 5-aza. Additionally, DT433 led to an increase in p16 expression and inhibition of cell proliferation [142][30]. Novel strategies applying repurposed drugs or DNAzymes to achieve DNMT inhibition constitute interesting alternatives to the demethylating drugs already approved. On the one hand, DNAzymes showed that increasing specificity toward the target may be achieved, whereas repurposed drugs, with negligible toxicity and known safety profiles, may be administered for longer periods and be considered as the next step for DNA methylation inhibition as anti-cancer therapy.

2.2. Combination Studies

The anti-tumor effects of 5-aza, trichostatin A (TSA), and FK228 (the latter is a class I histone deacetylase (HDAC) inhibitor) were evaluated individually and in combination in a set of BC cell lines, as well as xenograft and orthotopic mouse models. The combination 5-aza and FK228 was shown to be toxic for 90% of the BC cells, inducing apoptosis and decreasing the G2/M cell population. The same combination in in vivo models led to a decrease in tumor size, mainly due to the effect of FK228 alone [147][38]. Combination treatments using 5-aza and TSA also reduced the cell number and a shift in the expression of proteins that regulate the cell cycle in canine BC cell lines [148][39]. More recently, combination of DAC and entinostat, another HDAC1 inhibitor, was tested in bladder cell lines, including two cisplatin-sensitive (J82 and RT112) and one cisplatin-resistant (J82CisR) cell lines, as well as one urothelial cell line isolate from normal tissue (HBLAK). The combination treatment did not revert cisplatin resistance of J82CisR, although treatment with both drugs promoted cell growth arrest. Additionally, increased apoptosis was observed, related to caspases 3 and 7, prompting cell arrest at G2/M transition. Forkhead box O1 (FoxO1) expression increased after the combination treatment, as well as BIM and p21, along with a decrease in survivin expression [149][40]. Several strategies using epigenetic drugs were devised to overcome chemoresistance and increase therapy success. Ramachandran et al. showed that pre-treatment with 5-aza followed by cisplatin or docetaxel exposure increased cytotoxicity in BC cells. Moreover, in UMUC3 cells that developed resistance to cisplatin or docetaxel in vitro, pre-treatment with 5-aza resulted in 44% and 55% of cytotoxicity in cisplatin- and docetaxel-resistant cells, respectively, in opposition to 2% with no pre-treatment [150][41]. Pre-treatment with DAC also enhanced cytotoxicity of cisplatin and doxorubicin in BC cells. Furthermore, RASSF1A expression was observed concomitantly with activation of the Hippo pathway [151][42]. Interestingly, UMUC14, RT4, 96-1, and 97-1 cell lines displayed low sensitivity to cisplatin, being associated with high HOXA9 methylation levels. This was also verified in MIBC patients in which high HOXA9 methylation levels were associated with resistance to chemotherapy. In line with other studies, DAC led to a 4–5-fold decrease in half maximal inhibitory concentration (IC50) for cisplatin, vinblastine, doxorubicin, and etoposide in BC cells [152][43]. Wu et al. demonstrated for the first time that DAC treatment reduced the cancer stem-cell population in mice. Specifically, in a BNN-induced mouse model of BC, DAC alone or in combination with cisplatin or gemcitabine led to a decline in the keratin 14 (KRT14)+ cell population, which originates from the bladder urothelium [153][44]. Consistently, the percentage of SRY-box transcription factor 2 (SOX2)+ cell population assumed to be responsible for the spread of the tumor [154][45] was also lower. Curiously, the percentage of such cells increased in mice treated with chemotherapy only [155][46]. These results were replicated in patient sample-derived xenografts, demonstrating that a combination treatment of DAC with chemotherapeutic agents might constitute a valuable option for BC therapy [155][46]. Another study showed that a quadruple combination therapy comprising gemcitabine, cisplatin, DAC, and TSA increased apoptosis via cyclin D1 (CCND1) downregulation, DNA fragmentation, and caspase 3 expression in T24 cells. On the other hand, a cell proliferation reduction and lower BCL2L1 mRNA levels were observed after treatment [156][47]. A novel dual inhibitor, CM-272, targeting G9a, a histone methyltransferase that catalyzes H3K9me2, and DNMTs demonstrated activity against a wide range of cancer cells [157][48]. Specifically, treatment of hematologic malignancies cell lines with concentrations in the nanomolar range led to a global decrease in H3K9me2 and 5-methylcytosine levels, a reduction in cell proliferation, induction of cell-cycle arrest and apoptosis, and a decrease in TSG promoter methylation. Interestingly, CM-272 also induced an IFN-γ type I response and immunogenic cell death. In an in vivo mouse model, CM-272 was safe to administer, with an increased overall survival of the treated mice [157][48]. Furthermore, CM-272 was also active against hepatocellular carcinoma cell lines [158][49]. Recently, the effects of CM-272 were evaluated in in vitro and in vivo models of BC. CM-272 in combination with cisplatin led to inhibition of cell proliferation, which was also verified in a BC xenograft mouse model, leading to a decreased tumor growth, whereas apoptosis and autophagy were increased [159][50]. These effects were also observed in a quadruple-knockout transgenic mouse model of advanced BC. Remarkably, BC cells treated with CM-272 showed upregulation of genes linked to the immune response, including IFN-α and γ, and tumor necrosis factor (TNF)-α, probably through induction of an endogenous retrovirus response [159][50]. Taking into account these results, the combination of CM-272 with anti-PD-L1 in the quadruple-knockout mouse model was explored. A sustained response to the combination treatment was observed, with the number of animals developing tumors or metastases being lower in the combination group when compared with the group treated with anti-PD-L1 monotherapy [159][50].

2.3. Clinical Studies

A non-randomized multicenter phase II study evaluated the effects of FdCyd in THU [80,162][51][52]. The safety, maximum tolerated dose (MTD), pharmacokinetics, and pharmacodynamics of this combination were determined in a previous phase I clinical trial (FyCyd 100 mg/m2/day and THU 350 mg/m2/day) [81][53]. Patients with metastatic or unresectable breast cancer (n = 29), head and neck cancer (n = 21), non-small-cell lung cancer (n = 25), and urothelial cell carcinoma (n = 18) which endured progression after at least one line of standard therapy were included in this study. The combination was well tolerated, and urothelial carcinoma patients showed some clinical responses, with an objective response rate (ORR) of 5.6%, a progression-free survival (PFS) of 3.6 months, and a four-month PFS probability of 42%. Furthermore, increased p16 expression was observed in cytokeratin-positive circulating tumor cells (CTCs) of some patients, although it did not associate with clinical response (Table 32) [162][52].
Table 32.
 Clinical trials in BC using DNMT inhibitors.
Drug Phase (ID) Status Enrollment Schedule Results Period Reference
5-Aza and sodium phenylbutyrate I

(NCT00005639)		 Completed Patients with diagnosis of a refractory solid tumor malignancy with no curative options including BC (n = 34) Regimen A: Low-dose of 5-aza with intermittent phenylbutyrate 400 mg/m2/day over 24 h on days 6 and 13.

Regiment B: 5-aza 75 mg/m2/day for 7 days, followed by two different doses of phenylbutyrate starting on day 8 and continuing for 7 days. Each cycle lasts 35 days for A and B.

Regiment C: 2 different daily doses of 5-AC for 21 days and phenylbutyrate 400 mg/m2/day over 24 h once per week. Each cycle lasts 42 days.		 Three doses were well tolerated. Common toxicities included bone marrow suppression-related neutropenia and anemia. One patient showed stable disease; the remaining did not show any clinical response. 2000–2005 [163][54]
DAC I

(NCT00030615)		 Completed Advanced metastatic solid tumor patients after other standard therapies fail including BC (n = 24) DAC intravenous (IV) over 30 min on days 1–5 weekly for 4 weeks. Course repeated every 6 weeks in the absence of disease progression or unacceptable toxicity. Not available 2001–2008 [168][55]
FdCyd and THU II

(NCT00978250)		 Completed Metastatic or unresectable solid tumors including urothelial transitional cell carcinoma (n = 18), whose disease progressed after at least one line of standard therapy. FdCyd (100 mg/m2/day) by 3 h intravenous infusion and THU (350 mg/m2/day) 20% as a bolus, with the remaining co-administered with FdCyd over 3-h infusion on days 1–5 and 8–12 of each 28-day cycle. Co-administration with THU was shown to increase the area under the curve of FdCyd more than 4-fold.

Combination was well tolerated.

ORR of 5.6%, PFS of 3.6 months, and 42% of 4-month PFS probability for urothelial cancer patients.		 2009–2019 [162,169][52][56]
CC-486, carboplatin, and paclitaxel protein-bound particles (ABI-007) I

(NCT01478685)		 Completed Patients with relapsed or refractory solid tumors including urinary bladder neoplasms (n = 169) Arm A: CC-486 (doses between 100–300 mg) was administered orally daily either 14 or 21 days. Carboplatin was given by intravenous (IV) infusion once every 21 days

Arm B: CC-486 (doses between 100–300 mg) was administered orally daily for either 14 or 21 days

ABI-007 was administered by intravenous (IV) infusion on two of every three weeks

Arm C: CC-486 (doses between 100–300 mg) was administered orally daily for either 14 or 21 days.		 CC-486 dosed 14/21 days was tolerated as a priming agent with carboplatin and ABI-007. Both combinations show evidence of clinical activity. 2011–2015 [164,165][57][58]
RX-3317 I

(NCT02030067)		 Completed Patients with advanced or metastatic solid tumors including advanced BC (n = 124) A cycle was 4 weeks, with up to 8 cycles. RX-3117 dosing was given 3 times each week for 3 weeks followed by 1 week off treatment. All subjects were followed for at least 30 days after the last dose of RX-3117. Not available 2013–2019 [170][59]
CC-486 I

(NCT02223052)		 Completed Subjects with hematologic or solid tumor malignancies including BC patients (n = 89) Arm 1: Two 150-mg tablets of CC-486 on day 1 and 1 × 300 mg CC-486 on day 2 Arm 2: 1 × 300 mg tablet of CC-486 on day 1 and 2 × 150 mg CC-486 on day 2. Not available 2014–2018 [171][60]
SGI-110, gemcitabine, and cisplatin Ib/IIa

(2015-004062-29)		 Recruiting Urothelial BC patients with stages T2-4aN0M0 (n = 20) Arm 1: SGI-110 days 1–5 at the determined dose, gemcitabine 1000 mg/m2 days 8 + 15, cisplatin 70 mg/m2 day 8. 3–4 cycles of 21 days each.

Arm 2: Gemcitabine 1000 mg/m2 days 8 + 15, cisplatin 70 mg/m2 day 8
Abbreviations: 5-aza—5-azacytidine; DAC—decitabine.
The pharmacokinetics, toxicity, and clinical relevance of combining 5-aza and sodium phenylbutyrate, a first-generation HDAC inhibitor, was assessed in 34 patients with refractory solid tumors with no curative options, including two BC patients. Twenty-seven patients were eligible to proceed with treatment in three different regiments. The treatment was well tolerated with the most common toxicity effects including neutropenia, anemia, nausea, vomiting, transaminase elevation, and edema. The results of the study were mostly disappointing, with only one leiomyosarcoma patient displaying stable disease for 4.5 months and the rest disclosing disease progression. Although inhibition of DNMT activity was observed in two patients, this drug combination failed to show clinical benefit (Table 32) [163][54]. In a phase Ib clinical trial, the effects of CC-486 to potentiate carboplatin or paclitaxel protein-bound particles (ABI-007) in 169 patients with relapsed or refractory solid tumors including BC were evaluated. Two arms were defined: arm A comprising treatment with CC-486 and carboplatin and arm B including combination treatment of CC-486 and ABI-007 [164][57]. Preliminary results showed that CC-486 (200 and 300 mg) was well tolerated, with treatment-emergent adverse events (TEAEs) including anemia and neutropenia in about 50% of the patients in arm A and nausea/vomiting and peripheral neuropathy in patients of arm B. Five patients in arm A showed stable disease whereas three patients displayed partial response, with both combinations disclosing clinical value. Interestingly, peripheral blood mononuclear cells (PBMCs) were found to be hypomethylated. A recommended phase II dose (RP2D) study will comprise an expansion of cohorts and the combinations of 300 mg of CC-486 with carboplatin and 200 mg of CC-486 with ABI-007 [165][58]. Other clinical trials are at this date assessing the clinical benefit of combining immune checkpoint inhibitors with epigenetic drugs. In a recently completed study, the combination of pembrolizumab, epacadostat [indoleamine 2,3-dioxygenase 1 (IDO1)-selective inhibitor], and 5-aza was assessed in a phase I and II trial enrolling 70 patients, including urothelial cancer patients [166][63]. Furthermore, in a currently recruiting phase II clinical trial, the biological and clinical efficacy of atezolizumab (which targets PD-L1) in combination with guadecitabine will be evaluated in 53 patients with checkpoint inhibitor-refractory or -resistant urothelial carcinoma (Table 32) [167][64].

References

  1. Inbar-Feigenberg, M.; Choufani, S.; Butcher, D.T.; Roifman, M.; Weksberg, R. Basic concepts of epigenetics. Fertil. Steril. 2013, 99, 607–615.
  2. Wu, H.; Caffo, B.; Jaffee, H.A.; Irizarry, R.A.; Feinberg, A.P. Redefining CpG islands using hidden Markov models. Biostatistics 2010, 11, 499–514.
  3. Ferreira, H.J.; Esteller, M. CpG Islands in Cancer: Heads, Tails, and Sides. Methods Mol. Biol. 2018, 1766, 49–80.
  4. Rodríguez-Paredes, M.; Esteller, M. Cancer epigenetics reaches mainstream oncology. Nat. Med. 2011, 17, 330–339.
  5. Kanwal, R.; Gupta, S. Epigenetic modifications in cancer. Clin. Genet. 2012, 81, 303–311.
  6. Bennett, R.L.; Licht, J.D. Targeting epigenetics in cancer. Annu. Rev. Pharmacol. Toxicol. 2018, 58, 187–207.
  7. Pasculli, B.; Barbano, R.; Parrella, P. Epigenetics of breast cancer: Biology and clinical implication in the era of precision medicine. Semin. Cancer Biol. 2018, 51, 22–35.
  8. Lao, V.V.; Grady, W.M. Epigenetics and colorectal cancer. Nat. Rev. Gastroenterol. Hepatol. 2011, 8, 686–700.
  9. Maunakea, A.K.; Chepelev, I.; Cui, K.; Zhao, K. Intragenic DNA methylation modulates alternative splicing by recruiting MeCP2 to promote exon recognition. Cell Res. 2013, 23, 1256–1269.
  10. Maunakea, A.K.; Nagarajan, R.P.; Bilenky, M.; Ballinger, T.J.; D’Souza, C.; Fouse, S.D.; Johnson, B.E.; Hong, C.; Nielsen, C.; Zhao, Y.; et al. Conserved role of intragenic DNA methylation in regulating alternative promoters. Nature 2010, 466, 253–257.
  11. Bae, M.G.; Kim, J.Y.; Choi, J.K. Frequent hypermethylation of orphan CpG islands with enhancer activity in cancer. BMC Med. Genom. 2016, 9 (Suppl. S1), 38.
  12. Wang, X.; Chen, E.; Yang, X.; Wang, Y.; Quan, Z.; Wu, X.; Luo, C. 5-azacytidine inhibits the proliferation of bladder cancer cells via reversal of the aberrant hypermethylation of the hepaCAM gene. Oncol. Rep. 2016, 35, 1375–1384.
  13. Tao, J.; Liu, Q.; Wu, X.; Xu, X.; Zhang, Y.; Wang, Q.; Luo, C. Identification of hypermethylation in hepatocyte cell adhesion molecule gene promoter region in bladder carcinoma. Int. J. Med. Sci. 2013, 10, 1860–1867.
  14. Hahn, N.M.; Bonney, P.L.; Dhawan, D.; Jones, D.R.; Balch, C.; Guo, Z.; Hartman-Frey, C.; Fang, F.; Parker, H.G.; Kwon, E.M.; et al. Subcutaneous 5-azacitidine treatment of naturally occurring canine urothelial carcinoma: A novel epigenetic approach to human urothelial carcinoma drug development. J. Urol. 2012, 187, 302–309.
  15. Liang, G.; Gonzales, F.A.; Jones, P.A.; Orntoft, T.F.; Thykjaer, T. Analysis of gene induction in human fibroblasts and bladder cancer cells exposed to the methylation inhibitor 5-aza-2′-deoxycytidine. Cancer Res. 2002, 62, 961–966.
  16. Velicescu, M.; Weisenberger, D.J.; Gonzales, F.A.; Tsai, Y.C.; Nguyen, C.T.; Jones, P.A. Cell division is required for de novo methylation of CpG islands in bladder cancer cells. Cancer Res. 2002, 62, 2378–2384.
  17. Segura-Pacheco, B.; Trejo-Becerril, C.; Perez-Cardenas, E.; Taja-Chayeb, L.; Mariscal, I.; Chavez, A.; Acuña, C.; Salazar, A.M.; Lizano, M.; Dueñas-Gonzalez, A. Reactivation of tumor suppressor genes by the cardiovascular drugs hydralazine and procainamide and their potential use in cancer therapy. Clin. Cancer Res. 2003, 9, 1596–1603.
  18. Kawakami, T.; Shiina, H.; Igawa, M.; Deguchi, M.; Nakajima, K.; Ogishima, T.; Tokizane, T.; Urakami, S.; Enokida, H.; Miura, K.; et al. Inactivation of the hMSH3 mismatch repair gene in bladder cancer. Biochem. Biophys. Res. Commun. 2004, 325, 934–942.
  19. Cheng, J.C.; Weisenberger, D.J.; Gonzales, F.A.; Liang, G.; Xu, G.L.; Hu, Y.G.; Marquez, V.E.; Jones, P.A. Continuous zebularine treatment effectively sustains demethylation in human bladder cancer cells. Mol. Cell Biol. 2004, 24, 1270–1278.
  20. Urakami, S.; Shiina, H.; Enokida, H.; Kawakami, T.; Tokizane, T.; Ogishima, T.; Tanaka, Y.; Li, L.C.; Ribeiro-Filho, L.A.; Terashima, M.; et al. Epigenetic inactivation of Wnt inhibitory factor-1 plays an important role in bladder cancer through aberrant canonical Wnt/beta-catenin signaling pathway. Clin. Cancer Res. 2006, 12, 383–391.
  21. Christoph, F.; Kempkensteffen, C.; Weikert, S.; Köllermann, J.; Krause, H.; Miller, K.; Schostak, M.; Schrader, M. Methylation of tumour suppressor genes APAF-1 and DAPK-1 and in vitro effects of demethylating agents in bladder and kidney cancer. Br. J. Cancer 2006, 95, 1701–1707.
  22. Yoo, C.B.; Jeong, S.; Egger, G.; Liang, G.; Phiasivongsa, P.; Tang, C.; Redkar, S.; Jones, P.A. Delivery of 5-aza-2′-deoxycytidine to cells using oligodeoxynucleotides. Cancer Res. 2007, 67, 6400–6408.
  23. Liu, X.; Dai, X.; Wu, B. Study of 5-Aza-CdR on transcription regulation of RASSF1A gene in the BIU87 cell line. Urol. Int. 2009, 82, 108–112.
  24. Mori, K.; Enokida, H.; Kagara, I.; Kawakami, K.; Chiyomaru, T.; Tatarano, S.; Kawahara, K.; Nishiyama, K.; Seki, N.; Nakagawa, M. CpG hypermethylation of collagen type I alpha 2 contributes to proliferation and migration activity of human bladder cancer. Int. J. Oncol. 2009, 34, 1593–1602.
  25. Matsumoto, M.; Kawakami, K.; Enokida, H.; Toki, K.; Matsuda, R.; Chiyomaru, T.; Nishiyama, K.; Kawahara, K.; Seki, N.; Nakagawa, M. CpG hypermethylation of human four-and-a-half LIM domains 1 contributes to migration and invasion activity of human bladder cancer. Int. J. Mol. Med. 2010, 26, 241–247.
  26. Chuang, J.C.; Warner, S.L.; Vollmer, D.; Vankayalapati, H.; Redkar, S.; Bearss, D.J.; Qiu, X.; Yoo, C.B.; Jones, P.A. S110, a 5-Aza-2′-deoxycytidine–containing dinucleotide, is an effective DNA methylation inhibitor in vivo and can reduce tumor growth. Mol. Cancer Ther. 2010, 9, 1443–1450.
  27. Zhang, H.; Qi, F.; Cao, Y.; Zu, X.; Chen, M.; Li, Z.; Qi, L. 5-Aza-2′-deoxycytidine enhances maspin expression and inhibits proliferation, migration, and invasion of the bladder cancer T24 cell line. Cancer Biother. Radiopharm. 2013, 28, 343–350.
  28. Chuang, J.J.; Dai, Y.C.; Lin, Y.L.; Chen, Y.Y.; Lin, W.H.; Chan, H.L.; Liu, Y.W. Downregulation of glutathione S-transferase M1 protein in N-butyl-N-(4-hydroxybutyl)nitrosamine-induced mouse bladder carcinogenesis. Toxicol. Appl. Pharmacol. 2014, 279, 322–330.
  29. Devanand, P.; Kim, S.I.; Choi, Y.W.; Sheen, S.S.; Yim, H.; Ryu, M.S.; Kim, S.J.; Kim, W.J.; Lim, I.K. Inhibition of bladder cancer invasion by Sp1-mediated BTG2 expression via inhibition of DNA methyltransferase 1. FEBS J. 2014, 281, 5581–5601.
  30. Wang, X.; Zhang, L.; Ding, N.; Yang, X.; Zhang, J.; He, J.; Li, Z.; Sun, L.Q. Identification and characterization of DNAzymes targeting DNA methyltransferase I for suppressing bladder cancer proliferation. Biochem Biophys. Res. Commun. 2015, 461, 329–333.
  31. Yoon, H.Y.; Kim, Y.J.; Kim, J.S.; Kim, Y.W.; Kang, H.W.; Kim, W.T.; Yun, S.J.; Ryu, K.H.; Lee, S.C.; Kim, W.J. RSPH9 methylation pattern as a prognostic indicator in patients with non-muscle invasive bladder cancer. Oncol. Rep. 2016, 35, 1195–1203.
  32. Ramakrishnan, S.; Hu, Q.; Krishnan, N.; Wang, D.; Smit, E.; Granger, V.; Rak, M.; Attwood, K.; Johnson, C.; Morrison, C.; et al. Decitabine, a DNA-demethylating agent, promotes differentiation via NOTCH1 signaling and alters immune-related pathways in muscle-invasive bladder cancer. Cell Death Dis. 2017, 8, 3217.
  33. Giri, A.K.; Aittokallio, T. DNMT Inhibitors increase methylation in the cancer genome. Front. Pharmacol. 2019, 10, 385.
  34. Maraver, A.; Fernandez-Marcos, P.J.; Cash, T.P.; Mendez-Pertuz, M.; Dueñas, M.; Maietta, P.; Martinelli, P.; Muñoz-Martin, M.; Martínez-Fernández, M.; Cañamero, M.; et al. NOTCH pathway inactivation promotes bladder cancer progression. J. Clin. Investig. 2015, 125, 824–830.
  35. Chen, T.; Hevi, S.; Gay, F.; Tsujimoto, N.; He, T.; Zhang, B.; Ueda, Y.; Li, E. Complete inactivation of DNMT1 leads to mitotic catastrophe in human cancer cells. Nat. Genet. 2007, 39, 391–396.
  36. Wu, C.T.; Wu, C.F.; Lu, C.H.; Lin, C.C.; Chen, W.C.; Lin, P.Y.; Chen, M.F. Expression and function role of DNA methyltransferase 1 in human bladder cancer. Cancer 2011, 117, 5221–5233.
  37. Santoro, S.W.; Joyce, G.F. A general purpose RNA-cleaving DNA enzyme. Proc. Natl. Acad. Sci. USA 1997, 94, 4262–4266.
  38. Karam, J.A.; Fan, J.; Stanfield, J.; Richer, E.; Benaim, E.A.; Frenkel, E.; Antich, P.; Sagalowsky, A.I.; Mason, R.P.; Hsieh, J.T. The use of histone deacetylase inhibitor FK228 and DNA hypomethylation agent 5-azacytidine in human bladder cancer therapy. Int. J. Cancer 2007, 120, 1795–1802.
  39. Dhawan, D.; Ramos-Vara, J.A.; Hahn, N.M.; Waddell, J.; Olbricht, G.R.; Zheng, R.; Stewart, J.C.; Knapp, D.W. DNMT1: An emerging target in the treatment of invasive urinary bladder cancer. Urol. Oncol. 2013, 31, 1761–1769.
  40. Wang, C.; Hamacher, A.; Petzsch, P.; Köhrer, K.; Niegisch, G.; Hoffmann, M.J.; Schulz, W.A.; Kassack, M.U. Combination of decitabine and entinostat synergistically inhibits urothelial bladder cancer cells via activation of FoxO1. Cancers (Basel) 2020, 12, 337.
  41. Ramachandran, K.; Gordian, E.; Singal, R. 5-azacytidine reverses drug resistance in bladder cancer cells. Anticancer Res. 2011, 31, 3757–3766.
  42. Khandelwal, M.; Anand, V.; Appunni, S.; Seth, A.; Singh, P.; Mathur, S.; Sharma, A. Decitabine augments cytotoxicity of cisplatin and doxorubicin to bladder cancer cells by activating hippo pathway through RASSF1A. Mol. Cell Biochem. 2018, 446, 105–114.
  43. Xylinas, E.; Hassler, M.R.; Zhuang, D.; Krzywinski, M.; Erdem, Z.; Robinson, B.D.; Elemento, O.; Clozel, T.; Shariat, S.F. An epigenomic approach to improving response to neoadjuvant cisplatin chemotherapy in bladder cancer. Biomolecules 2016, 6, 37.
  44. Papafotiou, G.; Paraskevopoulou, V.; Vasilaki, E.; Kanaki, Z.; Paschalidis, N.; Klinakis, A. KRT14 marks a subpopulation of bladder basal cells with pivotal role in regeneration and tumorigenesis. Nat. Commun. 2016, 7, 11914.
  45. Zhu, F.; Qian, W.; Zhang, H.; Liang, Y.; Wu, M.; Zhang, Y.; Zhang, X.; Gao, Q.; Li, Y. SOX2 Is a marker for stem-like tumor cells in bladder cancer. Stem Cell Rep. 2017, 9, 429–437.
  46. Wu, M.; Sheng, L.; Cheng, M.; Zhang, H.; Jiang, Y.; Lin, S.; Liang, Y.; Zhu, F.; Liu, Z.; Zhang, Y.; et al. Low doses of decitabine improve the chemotherapy efficacy against basal-like bladder cancer by targeting cancer stem cells. Oncogene 2019, 38, 5425–5439.
  47. Varol, N.; Konac, E.; Onen, I.H.; Gurocak, S.; Alp, E.; Yilmaz, A.; Menevse, S.; Sozen, S. The epigenetically regulated effects of Wnt antagonists on the expression of genes in the apoptosis pathway in human bladder cancer cell line (T24). DNA Cell Biol. 2014, 33, 408–417.
  48. San José-Enériz, E.; Agirre, X.; Rabal, O.; Vilas-Zornoza, A.; Sanchez-Arias, J.A.; Miranda, E.; Ugarte, A.; Roa, S.; Paiva, B.; Estella-Hermoso de Mendoza, A.; et al. Discovery of first-in-class reversible dual small molecule inhibitors against G9a and DNMTs in hematological malignancies. Nat. Commun. 2017, 8, 15424.
  49. Bárcena-Varela, M.; Caruso, S.; Llerena, S.; Álvarez-Sola, G.; Uriarte, I.; Latasa, M.U.; Urtasun, R.; Rebouissou, S.; Alvarez, L.; Jimenez, M.; et al. Dual targeting of histone methyltransferase G9a and DNA-methyltransferase 1 for the treatment of experimental hepatocellular carcinoma. Hepatology 2019, 69, 587–603.
  50. Segovia, C.; San José-Enériz, E.; Munera-Maravilla, E.; Martínez-Fernández, M.; Garate, L.; Miranda, E.; Vilas-Zornoza, A.; Lodewijk, I.; Rubio, C.; Segrelles, C.; et al. Inhibition of a G9a/DNMT network triggers immune-mediated bladder cancer regression. Nat. Med. 2019, 25, 1073–1081.
  51. Beumer, J.H.; Parise, R.A.; Newman, E.M.; Doroshow, J.H.; Synold, T.W.; Lenz, H.J.; Egorin, M.J. Concentrations of the DNA methyltransferase inhibitor 5-fluoro-2′-deoxycytidine (FdCyd) and its cytotoxic metabolites in plasma of patients treated with FdCyd and tetrahydrouridine (THU). Cancer Chemother. Pharmacol. 2008, 62, 363–368.
  52. Coyne, G.O.; Wang, L.; Zlott, J.; Juwara, L.; Covey, J.M.; Beumer, J.H.; Cristea, M.C.; Newman, E.M.; Koehler, S.; Nieva, J.J.; et al. Intravenous 5-fluoro-2′-deoxycytidine administered with tetrahydrouridine increases the proportion of p16-expressing circulating tumor cells in patients with advanced solid tumors. Cancer Chemother. Pharmacol. 2020, 85, 979–993.
  53. Newman, E.M.; Morgan, R.J.; Kummar, S.; Beumer, J.H.; Blanchard, M.S.; Ruel, C.; El-Khoueiry, A.B.; Carroll, M.I.; Hou, J.M.; Li, C.; et al. A phase I, pharmacokinetic, and pharmacodynamic evaluation of the DNA methyltransferase inhibitor 5-fluoro-2′-deoxycytidine, administered with tetrahydrouridine. Cancer Chemother. Pharmacol. 2015, 75, 537–546.
  54. Lin, J.; Gilbert, J.; Rudek, M.A.; Zwiebel, J.A.; Gore, S.; Jiemjit, A.; Zhao, M.; Baker, S.D.; Ambinder, R.F.; Herman, J.G.; et al. A phase I dose-finding study of 5-azacytidine in combination with sodium phenylbutyrate in patients with refractory solid tumors. Clin. Cancer Res. 2009, 15, 6241–6249.
  55. Decitabine in Treating Patients with Advanced Solid Tumors. Available online: https://clinicaltrials.gov/ct2/show/study/NCT00030615 (accessed on 28 April 2020).
  56. A Multi-Histology Phase II Study of 5-Fluoro-2′-Deoxycytidine with Tetrahydrouridine (FdCyd + THU). Available online: https://clinicaltrials.gov/ct2/show/results/NCT00978250 (accessed on 28 April 2020).
  57. A Phase 1 Study of CC-486 as a Single Agent and in Combination with Carboplatin or ABI-007 in Subjects with Relapsed or Refractory Solid Tumors. Available online: https://clinicaltrials.gov/ct2/show/results/NCT01478685 (accessed on 28 April 2020).
  58. LoRusso, P.; Rasco, D.; Bendell, J.; Sachdev, J.; Ramanathan, R.; Weiss, G.; Munster, P.; Edenfield, W.J.; Liu, K.; Blackwood-Chirchir, A.; et al. Abstract A120: A phase Ib study of CC-486 (oral azacitidine) as a priming agent for carboplatin or NAB-paclitaxel in subjects with relapsed and refractory solid tumors. Mol. Cancer Ther. 2013, 12, A120.
  59. Dose-Finding and Safety Study for Oral Single-Agent to Treat Advanced Malignancies. Available online: https://clinicaltrials.gov/ct2/show/results/NCT02030067 (accessed on 28 April 2020).
  60. Bioequivalence & Food Effect Study in Patients with Solid Tumor or Hematologic Malignancies. Available online: https://clinicaltrials.gov/ct2/show/study/NCT02223052 (accessed on 28 April 2020).
  61. Crabb, S.; Danson, S.J.; Catto, J.W.F.; McDowell, C.; Lowder, J.N.; Caddy, J.; Dunkley, D.; Rajaram, J.; Ellis, D.; Hill, S.; et al. SPIRE—Combining SGI-110 with cisplatin and gemcitabine chemotherapy for solid malignancies including bladder cancer: Study protocol for a phase Ib/randomised IIa open label clinical trial. Trials 2018, 19, 216.
  62. Genomic Based Assignment of Therapy in Advanced Urothelial Carcinoma. Available online: https://clinicaltrials.gov/ct2/show/study/NCT02788201 (accessed on 28 April 2020).
  63. Azacitidine Combined with Pembrolizumab and Epacadostat in Subjects with Advanced Solid Tumors (ECHO-206). Available online: https://clinicaltrials.gov/ct2/show/results/NCT02959437 (accessed on 28 April 2020).
  64. Atezolizumab + Guadecitabine in Patients with Checkpoint Inhibitor Refractory or Resistant Urothelial Carcinoma. Available online: https://clinicaltrials.gov/ct2/show/results/NCT03179943 (accessed on 28 April 2020).
More
© Text is available under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/)
Video Production Service
Feedback