Yvonne C. Tsai

Cooperativity between DNA Methyltransferases in the Maintenance Methylation of Repetitive Elements

ABSTRACT We used mouse embryonic stem (ES) cells with systematic gene knockouts for DNA methyltransferases to delineate the roles of DNA methyltransferase 1 (Dnmt1) and Dnmt3a and -3b in maintaining methylation patterns in the mouse genome. Dnmt1 alone was able to maintain methylation of most CpG-poor regions analyzed. In contrast, both Dnmt1 and Dnmt3a and/or Dnmt3b were required for methylation of a select class of sequences which included abundant murine LINE-1 promoters. We used a novel hemimethylation assay to show that even in wild-type cells these sequences contain high levels of hemimethylated DNA, suggestive of poor maintenance methylation. We showed that Dnmt3a and/or -3b could restore methylation of these sequences to pretreatment levels following transient exposure of cells to 5-aza-CdR, whereas Dnmt1 by itself could not. We conclude that ongoing de novo methylation by Dnmt3a and/or Dnmt3b compensates for inefficient maintenance methylation by Dnmt1 of these endogenous repetitive sequences. Our results reveal a previously unrecognized degree of cooperativity among mammalian DNA methyltransferases in ES cells.

Detection of methylated apoptosis-associated genes in urine sediments of bladder cancer patients.

Purpose: There is increasing evidence for a fundamental role for epigenetic silencing of apoptotic pathways in cancer. Changes in DNA methylation can be detected with a high degree of sensitivity, so we used the MethyLight assay to determine how methylation patterns of apoptosis-associated genes change during bladder carcinogenesis and whether DNA methylation could be detected in urine sediments. Experimental Design: We analyzed the methylation status of the 5′ regions of 12 apoptosis-associated genes (ARF, FADD, TNFRSF21, BAX, LITAF, DAPK, TMS-1, BCL2, RASSF1A, TERT, TNFRSF25, and EDNRB) in 18 bladder cancer cell lines, 127 bladder cancer samples, and 37 samples of adjacent normal bladder mucosa using the quantitative MethyLight assay. We also analyzed the methylation status in urine sediments of 20 cancer-free volunteers and 37 bladder cancer patients. Results: The 5′ regions of DAPK, BCL2, TERT, RASSFIA, and TNFRSF25 showed significant increases in methylation levels when compared with nonmalignant adjacent tissue (P ≤ 0.01). Methylation levels of BCL2 were significantly associated with tumor staging and grading (P ≤ 0.01), whereas methylation levels of RASSF1A and ARF were only associated with tumor stage (P ≤ 0.04), and TERT methylation and EDNRB methylation were predictors of tumor grade (P ≤ 0.02). To investigate clinical usefulness for noninvasive bladder cancer detection, we further analyzed the methylation status of the markers in urine samples of patients with bladder cancer. Methylation of DAPK, BCL2, and TERT in urine sediment DNA from bladder cancer patients was detected in the majority of samples (78%), whereas they were unmethylated in the urine sediment DNA from age-matched cancer-free individuals. Conclusions: Our results indicate that methylation of the 5′ region of apoptosis-associated genes is a common finding in patients with bladder carcinoma. The ability to detect methylation not only in bladder tissue, but also in urine sediments, suggests that methylation markers are promising tools for noninvasive detection of bladder cancers. Our results also indicate that some methylation markers, such as those in regions of RASSF1A and TNFRSF25, might be of limited use for detection because they are also methylated in normal bladder tissues.

DAP-kinase loss of expression in various carcinoma and B-cell lymphoma cell lines : possible implications for role as tumor suppressor gene

DAP-kinase is a novel calmodulin dependent serine/threonine kinase that carries ankyrin repeats and the death domain. It was recently isolated, by a functional selection approach of gene cloning, as a positive mediator of programmed cell death. In this study the expression of DAP-kinase was examined in the cell lines derived from various human neoplasms. DAP-kinase mRNA and protein expression were below the limit of detection in eight out of ten neoplastic derived B-cell lines. In six out of 14 examined bladder carcinoma, in three out of five renal cell carcinoma, and in four out of ten tested breast carcinoma cell lines, the DAP-kinase protein levels were below detection limits or lower than 1% compared to the positive cell lines. Interestingly, DAP-kinase expression could be restored in some of the negative bladder carcinoma and B-cell lines by treatment of cells with 5′-azadeoxycytidine that causes DNA demethylation. The high frequency of loss of DAP-kinase expression in human tumor cell lines, and the occasional involvement of methylation in this process raise the possibility that this novel mediator of cell death may function as a tumor suppressor gene.

A Panel of Three Markers Hyper- and Hypomethylated in Urine Sediments Accurately Predicts Bladder Cancer Recurrence

Purpose: The high risk of recurrence after transurethral resection of bladder tumor of nonmuscle invasive disease requires lifelong treatment and surveillance. Changes in DNA methylation are chemically stable, occur early during tumorigenesis, and can be quantified in bladder tumors and in cells shed into the urine. Some urine markers have been used to help detect bladder tumors; however, their use in longitudinal tumor recurrence surveillance has yet to be established. Experimental Design: We analyzed the DNA methylation levels of six markers in 368 urine sediment samples serially collected from 90 patients with noninvasive urothelial carcinoma (Tis, Ta, T1; grade low-high). The optimum marker combination was identified using logistic regression with 5-fold cross-validation, and validated in separate samples. Results: A panel of three markers discriminated between patients with and without recurrence with the area under the curve of 0.90 [95% confidence interval (CI), 0.86–0.92] and 0.95 (95% CI, 0.90–1.00), sensitivity and specificity of 86%/89% (95% CI, 74%–99% and 81%–97%) and 80%/97% (95% CI, 60%–96% and 91%–100%) in the testing and validation sets, respectively. The three-marker DNA methylation test reliably predicted tumor recurrence in 80% of patients superior to cytology (35%) and cystoscopy (15%) while accurately forecasting no recurrence in 74% of patients that scored negative in the test. Conclusions: Given their superior sensitivity and specificity in urine sediments, a combination of hyper- and hypomethylated markers may help avoid unnecessary invasive exams and reveal the importance of DNA methylation in bladder tumorigenesis. Clin Cancer Res; 20(7); 1978–89. ©2014 AACR.

Early acquisition of homozygous deletions of p16/p19 during squamous cell carcinogenesis and genetic mosaicism in bladder cancer.

We looked for p16/p19 deletion and p16 promoter methylation, as well as loss of 9p21 heterozygosity in pure squamous cell carcinomas (SCC), and in transitional cell carcinomas (TCC) of the bladder with SCC components. Homozygous deletion of p16/p19 was detected in 11 of 21 (52%) cases of pure SCCs and in three of ten (30%) cases of TCC with SCC. Three cases of TCC with SCC had p16/p19 deletion, hypermethylation of the p16 promoter, or LOH on 9p21 only in the SCC components, suggesting that these molecular alterations occurred preferentially in SCC. Interestingly, homozygous deletion of p16/p19 was observed in squamous metaplasia from bladder cancer patients (five of 11, 45%), showing that this change occurred in preneoplastic cells. On the other hand, p16/p19 deletions were not found in squamous metaplasias from non cancerous patients. Hypermethylation of the p16 promoter was observed in two of 14 tumors (14%) and none of seven metaplasias examined. These data suggest that: (a) p16/p19 deletion is associated with early carcinogenesis of SCC of the bladder, and squamous metaplasia of the bladder cancer patient has already sustained genetic changes found in cancer, and (b) genetic mosaicism occurs in cases of TCC with SCC, with the SCC component showing more frequent 9p21 alterations than the TCC component.

DNA methylator and mismatch repair phenotypes are not mutually exclusive in colorectal cancer cell lines

A potential link between DNA repair and de novo methylation of exogenous sequences in colorectal cancer cell lines suggested that cells deficient in mismatch repair (MMR−) had an increased ability to silence the introduced virus promoter by DNA methylation due to the presence of a methylator phenotype (MET+) (Lengauer et al., 1997a). We explored this relationship in more detail and found that although there was a clear difference in the abilities of MMR+ cells to express the viral promoter compared to their MMR− counterparts, this difference was not consistently explained by levels of methylation in the viral promoter. Furthermore, we were unable to distinguish differences between the levels of methylation of six endogenous known CpG islands or 100 random DNA fragments containing CCGG sites within the cells. No consistent differences between the abilities of the cells to methylate the CpG island in exon 2 of the p16 gene were observed after transient demethylation by 5-aza-2′-deoxycytidine nor in the levels of expression of three human methyltransferase enzymes. Our results do not therefore support the existence of mutually exclusive DNA methylation (MET) and DNA repair (MMR) phenotypes.

Reprogramming of the human intestinal epigenome by surgical tissue transposition

Extracellular cues play critical roles in the establishment of the epigenome during development and may also contribute to epigenetic perturbations found in disease states. The direct role of the local tissue environment on the post-development human epigenome, however, remains unclear due to limitations in studies of human subjects. Here, we use an isogenic human ileal neobladder surgical model and compare global DNA methylation levels of intestinal epithelial cells pre- and post-neobladder construction using the Infinium HumanMethylation450 BeadChip. Our study is the first to quantify the effect of environmental cues on the human epigenome and show that the local tissue environment directly modulates DNA methylation patterns in normal differentiated cells in vivo. In the neobladder, the intestinal epithelial cells lose their tissue-specific epigenetic landscape in a time-dependent manner following the tissues exposure to a bladder environment. We find that de novo methylation of many intestine-specific enhancers occurs at the rate of 0.41% per month (P < 0.01, Pearson = 0.71), while demethylation of primarily non-intestine-specific transcribed regions occurs at the rate of -0.37% per month (P < 0.01, Pearson = -0.57). The dynamic resetting of the DNA methylome in the neobladder not only implicates local environmental cues in the shaping and maintenance of the epigenome but also illustrates an unexpected cross-talk between the epigenome and the cellular environment.

Genetic aspects of bladder cancer progression

SummaryNeoplasia is the result of cumulative genetic damage. Cytogenetic analysis identifies abnormalities of chromosome number and structure. Abnormal mode, wide variation in chromosome number, and aneuploidy are associated with loss of differentiation and advanced stage in bladder cancer. Marker chromosomes have been associated with increased risk of recurrence and progression of superficial, low-grade tumors. With the use of banding techniques, aberrations can be assigned to specific chromosomes and significant nonrandom abnormalities have been identified on chromosomes 1, 3, 5, 7, 9, 11, and 17. With the use of molecular biology techniques, allelic deletions, proto-oncogene activation, and mutations can be detected and correlated to the pathophysiologic events that determine the biologic behavior of a particular tumor. Allelic deletions of chromosome 9q are independent of stage and grade and suggest that loss of a tumor suppressor gene on chromosome 9q may be an important primary event in the genesis of bladder cancer. Deletions of chromosomes 11p and 17p, evident only in high-grade tumors, may be important in bladder cancer progression.

1699 IDENTIFYING NOVEL DNA METHYLATION MARKERS TO MONITOR BLADDER CANCER RECURRENCE IN URINE SEDIMENTS FROM TURBT PATIENTS

logic subtype, carcinoma in situ (i.e., Tis), is present in 40% of total bladder cancers diagnosed. However, there is currently no ability at the time of initial diagnosis to identify which of these patients will respond to standard-of-care treatment, intravesical administration of Bacillus Calmette-Guerin (BCG). Thus, Tis bladder cancer patients are often treated subjectively using a “one size fits all” approach with only about a 60% success rate (i.e., tumor-free) among treated patients. The aim of the present study was is to correlate the BCG responsiveness of patients with Tis bladder cancer with measures of the tumor immune microenvironment at the time of initial diagnosis. METHODS: The immune microenvironments of tumor biopsies of 20 Tis bladder cancer patients responsive to BCG therapy (BCG ) were assessed relative to biopsies derived from 18 non-responsive patients (BCG-). Each tumor immune microenvironment was determined as a function of two immunohistochemical metrics: (i) The level of tumor eosinophil infiltration as well as the extent of eosinophil degranulation (i.e. Th2 effector arm) and (ii) The relative number of tumor-infiltrating GATA-3 (i.e., Th2-polarized) vs. T-bet (i.e., Th1 polarized) lymphocytes. RESULTS: The bladder biopsies of “normal” subjects displayed only a nominal eosinophil infiltrate with no evidence of degranulation or infiltrating lymphocytes. In contrast, Tis bladder tumors often displayed a robust tissue eosinophilia accompanied by degranulation. The tumor immune microenvironments were decidedly Th2 polarized with 3-fold more GATA-3 relative to T-bet lymphocytes. More importantly, each of these immune biomarkers had prognostic value in the evaluation of bladder cancer patients. Specifically, the data showed that the levels of each immune biomarker were statistically higher in patients subsequently shown to be BCG relative to BCGsubjects. In addition, an algorithm integrating these immune metrics (i.e., Th2 Signature) provided an unambiguous biomarker that stratifies bladder cancer patients prior to treatment decisions with a high degree of specificity. CONCLUSIONS: The immunohistochemical assessments of the Tis immune tumor microenvironment represents a clinically-relevant screening strategy of cancer patients as to their subsequent responsiveness to the standard-of-care treatment.

Abstract 4624: Identification of novel DNA methylation markers to track patient's response to DNA demethylation agents.

The successful use of DNA methyltransferase inhibitors (DNMTis) in Myelodysplastic Syndromes (MDS) therapy has brought epigenetic drugs to the forefront of cancer management. However it has been difficult to find a consistent association between patients’ outcome and DNA methylation changes. This could be attributed to a variety of factors including the choice of DNA methylation markers to track the response to treatment. LINE-1 methylation changes are widely studied as a marker predictive of genome-wide DNA methylation changes; however recent reports have shown that its methylation levels are variable across tissues. In addition, we have found that LINE-1 remethylation after DNMTis withdrawal occurs faster than that of other regions, suggesting that LINE-1 methylation changes might not reflect the overall demethylation effects of DNMTis. Thus, it is imperative to find more sensitive DNA methylation markers to accurately track the methylation change that occurs after DNMTi treatment. Ideally, such markers could be applied to various tumor types and to samples collected by invasive and non-invasive methods. To find improved DNA methylation markers, we took advantage of the well-established Infinium DNA methylation platform and found 1429 probes, which were consistently methylated in both normal/tumor bladder tissue samples as well as white blood cells from healthy donors. The remethylation pattern of those 1439 probes was investigated in the T24 bladder cancer and HL60 leukemia cell lines treated with 5-Aza-CdR for 24 hours. Probe consensus clustering yielded a group of 79 probes that significantly responded to the demethylation treatment and remained demethylated beyond 30 days. In silico analysis of the DNA methylation patterns of the top two probes show consistent hypermethylation in both normal and tumor samples. As a proof of principle, we tested the DNA demethylation levels of these two markers in urine sediments from 7 MDS patients treated with Azacitidine using pyrosequencing. Our results showed that the two markers were significantly demethylated; in contrast, LINE-1 methylation showed no clear decreasing trend. Demethylation of these two markers was also observed in peripheral blood samples from MDS patients treated with Azacitidine. We are in the process of testing more samples to find the association between marker demethylation and patient9s outcome. In summary, we have identified and validated two DNA methylation markers, which unlike LINE-1, show consistent demethylation in response to the DNMTi treatment irrespective of the type of sample tested. The wider range of demethylation provided by these markers may offer a more accurate representation of the patient9s response to treatment. Citation Format: Xiaojing Yang, Han Han, Marianne B. Treppendahl, Yvonne C. Tsai, Casey O9Connell, Dan Weisenberger, Fides Lay, Kirsten Gronbaek, Gangning Liang, Peter A. Jones. Identification of novel DNA methylation markers to track patient9s response to DNA demethylation agents. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4624. doi:10.1158/1538-7445.AM2013-4624