Vitamin D3 enhances the response to cisplatin in bladder cancer through VDR and TAp73 signaling crosstalk.

BACKGROUND: Vitamin D3 (VitD) deficiency is linked to increased incidence and worse survival in bladder cancer (BCa). In addition to cystectomy, patients are treated with cisplatin-based chemotherapy, however 30%-50% of patients do not benefit from this treatment. The effects of VitD deficiency on response to chemotherapy remain unknown. METHODS: To test effects of VitD supplementation on the response to cisplatin we analyzed patient serum VitD levels and correlated that with survival. In vivo, VitD deficient mice were treated with cisplatin, with or without pretreatment with the active VitD metabolite, 1,25 dihydroxyvitamin D3 (1,25D3 ). Lastly, using BCa cell lines, T24 and RT-112, the mechanism of action of 1,25D3 and cisplatin combination treatment was determined by apoptosis assays, as well as western blot and RT-PCR. RESULTS: In this study, we determined that low serum 25 hydroxyvitamin D3 (25D3 ) levels was significantly associated with worse response to cisplatin. Pretreating deficient mice with 1,25D3 , reduced tumor volume compared to cisplatin monotherapy. In vitro, 1,25D3 pretreatment increased the apoptotic response to cisplatin. 1,25D3 pretreatment increased expression of TAp73 and its pro-apoptotic targets, in a VDR dependent manner. VDR and its transcriptional targets were induced after 1,25D3 treatment and further increased after the combination of 1,25D3 and cisplatin in a TAp73 dependent manner. CONCLUSIONS: Our data suggest that VitD deficiency could be a biomarker for poor response to cisplatin, and pretreating with VitD can increase the apoptotic response to cisplatin through VDR and TAp73 signaling crosstalk.

Transcriptional changes associated with in vivo growth of muscle-invasive bladder cancer cell lines in nude mice.

Cancer cells set in motion transcriptomic programs allowing for adaptation and growth in immunocompromised mice to form xenografts, a frequently used tool in cancer research. 2D cultures may not be representative of tumors growing in a complex host microenvironment. This can result in different responses to the same agent tested in vitro and in vivo which impedes the process of developing novel therapeutics. Understanding the transition cells undergo from 2D cell culture to a 3D host microenvironment will help in developing and choosing appropriate models for pre-clinical studies. Our study characterized the transcriptome of a three frequently used muscle-invasive bladder cancer cell lines HT1376, T24 and UM-UC-3 grown in culture and xenografts in nude mice. We found that bladder cancer cells undergo few transcriptomic changes when transitioned from 2D cell culture to xenografts in nude mice. UM-UC-3 cells have the least transcriptomic alterations followed by T24 and HT1376 cells. Respective xenografts cluster with their parental cell lines rather than other xenografts or cell lines. We applied established bladder cancer molecular subtypes to our data and found that UM-UC-3, containing the least transcriptomic alterations, most closely resembled the basal-like molecular subtype of bladder cancer. HT1376 and T24 have mixed basal and luminal molecular signatures. Our studies suggest this subset of bladder cancer cell lines and derived xenografts maintain similar transcriptomic profiles in both 2D culture and 3D xenografts and can be used interchangeably in pre-clinical studies.

Calcium, magnesium, and whole-milk intakes and high-aggressive prostate cancer in the North Carolina-Louisiana Prostate Cancer Project (PCaP).

Background: Calcium and dairy product intakes have been positively associated with prostate cancer risk. An imbalance in concentrations of calcium and magnesium has been associated with multiple chronic diseases, although few studies have examined the relation with prostate cancer aggressiveness. Objective: The goal of this study was to examine the association between dietary intakes of calcium and magnesium, the calcium-to-magnesium ratio (Ca:Mg), and dairy products and prostate cancer aggressiveness. Design: Dietary intake was assessed with the use of an interviewer-administered modified National Cancer Institute Diet History Questionnaire in 996 African American and 1064 European American men with a recent histologically confirmed diagnosis of prostate cancer from the North Carolina-Louisiana Prostate Cancer Project (PCaP). High-aggressive disease was defined as Gleason sum ≥8, or prostate-specific antigen (PSA) >20 ng/mL, or Gleason score ≥7 and clinical stage T3-T4. The comparison group was all other prostate cancer cases. Logistic regression was used to determine the adjusted ORs and 95% CIs for high-aggressive prostate cancer by tertile of diet and supplement exposures. Results: There was a positive association across tertiles of dietary Ca:Mg intake, with odds of high-aggressive prostate cancer in the upper tertiles as follows-OR for tertile 2 compared with tertile 1: 1.38 (95% CI: 1.01, 1.88); OR for tertile 3 compared with tertile 1: 1.46 (95% CI: 1.06, 2.02). When stratified by race, the positive association was more pronounced in African American men (OR for tertile 3 compared with tertile 2: 1.62; 95% CI: 1.04, 2.53). Men who reported the highest daily consumption of whole-fat milk had a 74% increased odds of high-aggressive prostate cancer compared with non-whole-fat milk drinkers, which was attenuated after adjustment for potential mediating factors, such as saturated fat and Ca:Mg intake. Conclusions: Among both African American and European American men diagnosed with prostate cancer, a higher Ca:Mg and whole-milk intake were associated with higher odds of high-aggressive prostate cancer. This study was registered at www.clinicaltrials.gov as NCT03289130.

The role of STAG2 in bladder cancer.

Stromal Antigen 2 (STAG2) is one of four components of the cohesin complex and predominantly functions in sister chromatid cohesion and segregation. STAG2 is the most frequently mutated cohesin subunit and was recently identified as a gene that is commonly altered in bladder cancer. The significance of these mutations remains controversial. Some studies associate loss of STAG2 expression with low stage and low grade bladder tumors, as well as with improved clinical outcomes. In other cases, STAG2 inactivation has been shown to be a predictor of worse outcome for these patients. The role of STAG2 in aneuploidy also remains controversial. Loss of STAG2 is associated with significant changes in chromosome number in certain cell lines, while in others, aneuploidy is not induced or results remain inconclusive. At this time, little is known about the influence of STAG2 on cellular migration, invasion, proliferation, and cell death, and such studies are required to determine the role of STAG2 in bladder cancer and other malignancies.

Game-changing restraint of Ros-damaged phenylalanine, upon tumor metastasis.

An abrupt increase in metastatic growth as a consequence of the removal of primary tumors suggests that the concomitant resistance (CR) phenomenon might occur in human cancer. CR occurs in murine tumors and ROS-damaged phenylalanine, meta-tyrosine (m-Tyr), was proposed as the serum anti-tumor factor primarily responsible for CR. Herein, we demonstrate for the first time that CR happens in different experimental human solid tumors (prostate, lung anaplastic, and nasopharyngeal carcinoma). Moreover, m-Tyr was detected in the serum of mice bearing prostate cancer (PCa) xenografts. Primary tumor growth was inhibited in animals injected with m-Tyr. Further, the CR phenomenon was reversed when secondary implants were injected into mice with phenylalanine (Phe), a protective amino acid highly present in primary tumors. PCa cells exposed to m-Tyr in vitro showed reduced cell viability, downregulated NFκB/STAT3/Notch axis, and induced autophagy; effects reversed by Phe. Strikingly, m-Tyr administration also impaired both, spontaneous metastasis derived from murine mammary carcinomas (4T1, C7HI, and LMM3) and PCa experimental metastases. Altogether, our findings propose m-Tyr delivery as a novel approach to boost the therapeutic efficacy of the current treatment for metastasis preventing the escape from tumor dormancy.

Decitabine, a DNA-demethylating agent, promotes differentiation via NOTCH1 signaling and alters immune-related pathways in muscle-invasive bladder cancer.

Aberrant DNA methylation observed in cancer can provide survival benefits to cells by silencing genes essential for anti-tumor activity. DNA-demethylating agents such as Decitabine (DAC)/Azacitidine (AZA) activate otherwise silenced tumor suppressor genes, alter immune response and epigenetically reprogram tumor cells. In this study, we show that non-cytotoxic nanomolar DAC concentrations modify the bladder cancer transcriptome to activate NOTCH1 at the mRNA and protein level, increase double-stranded RNA sensors and CK5-dependent differentiation. Importantly, DAC treatment increases ICN1 expression (the active intracellular domain of NOTCH1) significantly inhibiting cell proliferation and causing changes in cell size inducing morphological alterations reminiscent of senescence. These changes were not associated with ß-galactosidase activity or increased p16 levels, but instead were associated with substantial IL-6 release. Increased IL-6 release was observed in both DAC-treated and ICN1 overexpressing cells as compared to control cells. Exogenous IL-6 expression was associated with a similar enlarged cell morphology that was rescued by the addition of a monoclonal antibody against IL-6. Treatment with DAC, overexpression with ICN1 or addition of exogenous IL-6 showed CK5 reduction, a surrogate marker of differentiation. Overall this study suggests that in MIBC cells, DNA hypomethylation increases NOTCH1 expression and IL-6 release to induce CK5-related differentiation.

Exome Sequencing of African-American Prostate Cancer Reveals Loss-of-Function ERF Mutations.

African-American men have the highest incidence of and mortality from prostate cancer. Whether a biological basis exists for this disparity remains unclear. Exome sequencing (n = 102) and targeted validation (n = 90) of localized primary hormone-naïve prostate cancer in African-American men identified several gene mutations not previously observed in this context, including recurrent loss-of-function mutations in ERF, an ETS transcriptional repressor, in 5% of cases. Analysis of existing prostate cancer cohorts revealed ERF deletions in 3% of primary prostate cancers and mutations or deletions in ERF in 3% to 5% of lethal castration-resistant prostate cancers. Knockdown of ERF confers increased anchorage-independent growth and generates a gene expression signature associated with oncogenic ETS activation and androgen signaling. Together, these results suggest that ERF is a prostate cancer tumor-suppressor gene. More generally, our findings support the application of systematic cancer genomic characterization in settings of broader ancestral diversity to enhance discovery and, eventually, therapeutic applications.Significance: Systematic genomic sequencing of prostate cancer in African-American men revealed new insights into prostate cancer, including the identification of ERF as a prostate cancer gene; somatic copy-number alteration differences; and uncommon PIK3CA and PTEN alterations. This study highlights the importance of inclusion of underrepresented minorities in cancer sequencing studies. Cancer Discov; 7(9); 973-83. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 920.

Heme oxygenase-1 in the forefront of a multi-molecular network that governs cell-cell contacts and filopodia-induced zippering in prostate cancer.

Prostate cancer (PCa) cells display abnormal expression of cytoskeletal proteins resulting in an augmented capacity to resist chemotherapy and colonize distant organs. We have previously shown that heme oxygenase 1 (HO-1) is implicated in cell morphology regulation in PCa. Here, through a multi 'omics' approach we define the HO-1 interactome in PCa, identifying HO-1 molecular partners associated with the integrity of the cellular cytoskeleton. The bioinformatics screening for these cytoskeletal-related partners reveal that they are highly misregulated in prostate adenocarcinoma compared with normal prostate tissue. Under HO-1 induction, PCa cells present reduced frequency in migration events, trajectory and cell velocity and, a significant higher proportion of filopodia-like protrusions favoring zippering among neighboring cells. Moreover forced expression of HO-1 was also capable of altering cell protrusions in transwell co-culture systems of PCa cells with MC3T3 cells (pre-osteoblastic cell line). Accordingly, these effects were reversed under siHO. Transcriptomics profiling evidenced significant modulation of key markers related to cell adhesion and cell-cell communication under HO-1 induction. The integration from our omics-based research provides a four molecular pathway foundation (ANXA2/HMGA1/POU3F1; NFRSF13/GSN; TMOD3/RAI14/VWF; and PLAT/PLAU) behind HO-1 regulation of tumor cytoskeletal cell compartments. The complementary proteomics and transcriptomics approaches presented here promise to move us closer to unravel the molecular framework underpinning HO-1 involvement in the modulation of cytoskeleton pathways, pushing toward a less aggressive phenotype in PCa.

Genomic profiling is predictive of response to cisplatin treatment but not to PI3K inhibition in bladder cancer patient-derived xenografts.

PURPOSE: Effective systemic therapeutic options are limited for bladder cancer. In this preclinical study we tested whether bladder cancer gene alterations may be predictive of treatment response. EXPERIMENTAL DESIGN: We performed genomic profiling of two bladder cancer patient derived tumor xenografts (PDX). We optimized the exome sequence analysis method to overcome the mouse genome interference. RESULTS: We identified a number of somatic mutations, mostly shared by the primary tumors and PDX. In particular, BLCAb001, which is less responsive to cisplatin than BLCAb002, carried non-sense mutations in several genes associated with cisplatin resistance, including MLH1, BRCA2, and CASP8. Furthermore, RNA-Seq analysis revealed the overexpression of cisplatin resistance associated genes such as SLC7A11, TLE4, and IL1A in BLCAb001. Two different PIK3CA mutations, E542K and E545K, were identified in BLCAb001 and BLCAb002, respectively. Thus, we tested whether the genomic profiling was predictive of response to a dual PI3K/mTOR targeting agent, LY3023414. Despite harboring similar PIK3CA mutations, BLCAb001 and BLCAb002 exhibited differential response, both in vitro and in vivo. Sustained target modulation was observed in the sensitive model BLCAb002 but not in BLCAb001, as well as decreased autophagy. Interestingly, computational modelling of mutant structures and affinity binding to PI3K revealed that E542K mutation was associated with weaker drug binding than E545K. CONCLUSIONS: Our results suggest that the presence of activating PIK3CA mutations may not necessarily predict in vivo treatment response to PI3K targeted therapies, while specific gene alterations may be predictive for cisplatin response in bladder cancer models and, potentially, in patients as well.

Association between Plasma 25-Hydroxyvitamin D, Ancestry and Aggressive Prostate Cancer among African Americans and European Americans in PCaP.

BACKGROUND: African Americans (AAs) have lower circulating 25-hydroxyvitamin D3 [25(OH)D3] concentrations and higher prostate cancer (CaP) aggressiveness than other racial/ethnic groups. The purpose of the current study was to examine the relationship between plasma 25(OH)D3, African ancestry and CaP aggressiveness among AAs and European Americans (EAs). METHODS: Plasma 25(OH)D3 was measured using LC-MS/MS (Liquid Chromatography Tandem Mass Spectrometry) in 537 AA and 663 EA newly-diagnosed CaP patients from the North Carolina-Louisiana Prostate Cancer Project (PCaP) classified as having either 'high' or 'low' aggressive disease based on clinical stage, Gleason grade and prostate specific antigen at diagnosis. Mean plasma 25(OH)D3 concentrations were compared by proportion of African ancestry. Logistic regression was used to calculate multivariable adjusted odds ratios (OR) and 95% confidence intervals (95%CI) for high aggressive CaP by tertile of plasma 25(OH)D3. RESULTS: AAs with highest percent African ancestry (>95%) had the lowest mean plasma 25(OH)D3 concentrations. Overall, plasma 25(OH)D3 was associated positively with aggressiveness among AA men, an association that was modified by calcium intake (ORT 3vs.T1: 2.23, 95%CI: 1.26-3.95 among men with low calcium intake, and ORT 3vs.T1: 0.19, 95%CI: 0.05-0.70 among men with high calcium intake). Among EAs, the point estimates of the ORs were <1.0 for the upper tertiles with CIs that included the null. CONCLUSIONS: Among AAs, plasma 25(OH)D3 was associated positively with CaP aggressiveness among men with low calcium intake and inversely among men with high calcium intake. The clinical significance of circulating concentrations of 25(OH)D3 and interactions with calcium intake in the AA population warrants further study.

Whole-genome sequencing identifies genomic heterogeneity at a nucleotide and chromosomal level in bladder cancer.

Using complete genome analysis, we sequenced five bladder tumors accrued from patients with muscle-invasive transitional cell carcinoma of the urinary bladder (TCC-UB) and identified a spectrum of genomic aberrations. In three tumors, complex genotype changes were noted. All three had tumor protein p53 mutations and a relatively large number of single-nucleotide variants (SNVs; average of 11.2 per megabase), structural variants (SVs; average of 46), or both. This group was best characterized by chromothripsis and the presence of subclonal populations of neoplastic cells or intratumoral mutational heterogeneity. Here, we provide evidence that the process of chromothripsis in TCC-UB is mediated by nonhomologous end-joining using kilobase, rather than megabase, fragments of DNA, which we refer to as "stitchers," to repair this process. We postulate that a potential unifying theme among tumors with the more complex genotype group is a defective replication-licensing complex. A second group (two bladder tumors) had no chromothripsis, and a simpler genotype, WT tumor protein p53, had relatively few SNVs (average of 5.9 per megabase) and only a single SV. There was no evidence of a subclonal population of neoplastic cells. In this group, we used a preclinical model of bladder carcinoma cell lines to study a unique SV (translocation and amplification) of the gene glutamate receptor ionotropic N-methyl D-aspertate as a potential new therapeutic target in bladder cancer.

6p22.3 amplification as a biomarker and potential therapeutic target of advanced stage bladder cancer.

Genetic and epigenetic alterations have been identified as to contribute directly or indirectly to the generation of transitional cell carcinoma of the urinary bladder (TCC-UB). In a comparative fashion much less is known about copy number alterations in TCC-UB, but it appears that amplification of chromosome 6p22 is one of the most frequent changes. Using fluorescence in situ hybridization (FISH) analyses, we evaluated chromosomal 6p22 amplification in a large cohort of bladder cancer patients with complete surgical staging and outcome data. We have also used shRNA knockdown candidate oncogenes in the cell based study. We found that amplification of chromosome 6p22.3 is significantly associated with the muscle-invasive transitional cell carcinoma of the urinary bladder (TCC-UB) (22%) in contrast to superficial TCC-UB (9%) (p=7.2-04). The rate of 6p22.3 amplification in pN>1 patients (32%) is more than twice that in pN0 (16%) patients (p=0.05). Interestingly, we found that 6p22.3 amplification is as twice as high (p=0.0201) in African American (AA) than European American (EA) TCC-UB patients. Moreover, we showed that the expression of some candidate genes (E2F3, CDKAL1 and Sox4) in the 6p22.3 region is highly correlated with the chromosomal amplification. In particular, knockdown of E2F3 inhibits cell proliferation in a 6p22.3-dependent manner, whereas knockdown of CDKAL1 and Sox4 has no effect on cell proliferation. Using gene expression profiling, we further identified some common as well as distinctive subset targets of the E2F3 family members. In summary, our data indicate that E2F3 is a key regulator of cell proliferation in a subset of bladder cancer and the 6p22.3 amplicon is a biomarker of aggressive phenotype in this tumor type.

Vitamin D and cancer: clinical aspects.

There are substantial preclinical and epidemiologic data that suggest that vitamin D plays a role in the prevention and treatment of cancer. Numerous observational studies have shown that low blood levels of 25(OH) vitamin D (cholecalciferol), estimated by geographical location, diet and activity assessment or measured serum levels are associated with a higher risk of cancer and worse cancer-specific survival as well as numerous morbidities to e.g. cardiovascular disease, stroke, infection, autoimmune disease, and neuromuscular dysfunction among large populations. A considerable number of in vitro and in vivo studies indicate that the most active metabolite of vitamin D--1,25-dihydroxycholecalciferol or calcitriol--has anti-proliferative, pro-apoptotic, pro-differentiating, and anti-angiogenic properties. Combined treatment of calcitriol and many types of cytotoxic agents has synergistic or at least additive effects. However, clinical trials testing these hypotheses have been less encouraging, though a number of methodological, pharmacological, and pharmaceutical issues confound all trials ever conducted. In order to properly assess the clinical value of vitamin D, its metabolites and analogs in cancer prevention and treatment, more studies are needed.

Coordinated cancer germline antigen promoter and global DNA hypomethylation in ovarian cancer: association with the BORIS/CTCF expression ratio and advanced stage.

PURPOSE: Cancer germline (CG) antigens are frequently expressed and hypomethylated in epithelial ovarian cancer (EOC), but the relationship of this phenomenon to global DNA hypomethylation is unknown. In addition, the potential mechanisms leading to DNA hypomethylation, and its clinicopathologic significance in EOC, have not been determined. EXPERIMENTAL DESIGN: We used quantitative mRNA expression and DNA methylation analyses to determine the relationship between expression and methylation of X-linked (MAGE-A1, NY-ESO-1, XAGE-1) and autosomal (BORIS, SOHLH2) CG genes, global DNA methylation (5mdC levels, LINE-1, Alu, and Sat-α methylation), and clinicopathology, using 75 EOC samples. In addition, we examined the association between these parameters and a number of mechanisms proposed to contribute to DNA hypomethylation in cancer. RESULTS: CG genes were coordinately expressed in EOC and this was associated with promoter DNA hypomethylation. Hypomethylation of CG promoters was highly correlated and strongly associated with LINE-1 and Alu methylation, moderately with 5mdC levels, and rarely with Sat-α methylation. BORIS and LINE-1 hypomethylation, and BORIS expression, were associated with advanced stage. GADD45A expression, MTHFR genotype, DNMT3B isoform expression, and BORIS mRNA expression did not associate with methylation parameters. In contrast, the BORIS/CTCF expression ratio was associated with DNA hypomethylation, and furthermore correlated with advanced stage and decreased survival. CONCLUSIONS: DNA hypomethylation coordinately affects CG antigen gene promoters and specific repetitive DNA elements in EOC, and correlates with advanced stage disease. The BORIS/CTCF mRNA expression ratio is closely associated with DNA hypomethylation and confers poor prognosis in EOC.

BORIS/CTCFL expression is insufficient for cancer-germline antigen gene expression and DNA hypomethylation in ovarian cell lines.

Expression of the cancer-germline (CG) (or cancer-testis) antigen gene BORIS/CTCFL has been proposed to mediate activation of CG antigen genes in cancer. Consistent with this idea, we have observed that BORIS is frequently expressed in ovarian cancer, often in conjunction with other CG genes. Here we assessed the role of BORIS in CG antigen gene regulation and DNA methylation using normal and cancerous ovarian cell lines, and the CG genes MAGE-A1, NY-ESO-1, and XAGE-1 as models. Adenoviral vectored BORIS was expressed at robust levels and exhibited predominant nuclear localization in ovarian cells. However, BORIS expression in immortalized ovarian surface epithelial cells or ovarian cancer cell lines did not induce CG antigen gene expression or lead to CG antigen promoter DNA hypomethylation. BORIS overexpression also did not alter global DNA methylation, as assessed by genomic 5-methyl-deoxycytidine levels and LINE-1 methylation. We used decitabine to further assess the role of BORIS in CG gene activation and found that decitabine treatment induced BORIS and other CG genes with similar kinetics, suggesting that BORIS induction does not account for the induction of other CG genes by decitabine in ovarian cancer cells. In agreement, siRNA knockdown of BORIS did not block decitabine-mediated induction of CG genes or DNA hypomethylation in ovarian cancer cells treated with this agent. We conclude that BORIS is insufficient for CG antigen gene expression and DNA hypomethylation in ovarian cell lines, and that additional factors are likely required for CG antigen expression in ovarian cancer.

Distinct roles for histone methyltransferases G9a and GLP in cancer germ-line antigen gene regulation in human cancer cells and murine embryonic stem cells.

The H3K9me2 histone methyltransferases G9a and GLP repress Mage-a class cancer germ-line (CG) antigen gene expression in murine embryonic stem (ES) cells, but the role of these enzymes in CG antigen gene regulation in human cancer cells is unknown. Here we show that whereas independent or dual knockdown of G9a and GLP in human cancer cells leads to reduced global and CG antigen promoter-associated H3K9me2 levels, it does not activate CG antigen gene expression. Moreover, CG antigen gene repression is maintained following pharmacologic targeting of G9a or treatment of G9a knockdown cells with the histone deacetylase inhibitor trichostatin A. However, G9a knockdown cells display increased sensitivity to CG antigen gene activation mediated by the DNA methyltransferase inhibitor decitabine. To account for these findings, we examined DNA methylation at CG antigen gene promoters in both cell types. We found robust DNA hypomethylation in G9a/GLP targeted murine ES cells but a lack of DNA methylation changes in G9a/GLP targeted human cancer cells; intriguingly, this distinction also extended to markers of global DNA methylation. These data reveal that G9a/GLP is required for DNA methylation of CG antigen genes and genomic DNA in murine ES cells, but not human cancer cells, and implicate DNA methylation status as the key epigenetic mechanism involved in CG antigen gene repression.

Intertumor and intratumor NY-ESO-1 expression heterogeneity is associated with promoter-specific and global DNA methylation status in ovarian cancer.

PURPOSE: The cancer/germline antigen NY-ESO-1 is variably expressed in epithelial ovarian cancer (EOC), with most tumors showing low or heterogeneous expression, which limits patient responses to NY-ESO-1 vaccine therapy. We tested the hypothesis that promoter and global genomic DNA methylation status correlates with intertumor and intratumor NY-ESO-1 expression status in EOC. EXPERIMENTAL DESIGN: We utilized 78 EOC tumors and 10 normal ovary controls for quantitative DNA methylation analyses and NY-ESO-1 expression analysis by immunohistochemistry (IHC) and quantitative reverse transcriptase PCR. A subset of EOC tumors were used to perform microdissections of NY-ESO-1 IHC-positive and NY-ESO-1 IHC-negative tissue regions, followed by DNA methylation analyses. EOC cell lines were treated in vitro with decitabine to determine the functional contribution of DNA methylation to NY-ESO-1 gene regulation in EOC. RESULTS: Compared with normal ovary, bulk EOC tissues display increased NY-ESO-1 expression, reduced NY-ESO-1 promoter methylation, and reduced LINE-1 DNA methylation. However, NY-ESO-1 expression is not significantly associated with NY-ESO-1 promoter methylation status in bulk tumors. We hypothesized that this resulted from heterogeneous intratumor NY-ESO-1 expression. Supporting this idea, experiments using microdissected material revealed that intertumor and intratumor NY-ESO-1 expression heterogeneity is significantly correlated with promoter and global DNA methylation status in EOC. Moreover, decitabine treatment functionally restored NY-ESO-1 expression in nonexpressing EOC cell lines. CONCLUSION: DNA methylation status is associated with both intertumor and intratumor NY-ESO-1 expression status in EOC. These findings support a novel chemoimmunotherapy approach using decitabine to augment NY-ESO-1 vaccine therapy for treatment of recurrent EOC.

DNA methylation-dependent regulation of BORIS/CTCFL expression in ovarian cancer.

Brother of the Regulator of Imprinted Sites (BORIS/CTCFL) is an autosomal cancer germline (CG) or cancer-testis antigen gene and paralog of CTCF that has been proposed to function as an oncogene in human cancer via dysregulation of the cancer epigenome. Here we show that genetic disruption of DNA methylation in human cancer cells induces BORIS expression, coincident with DNA hypomethylation and an altered histone H3 modification pattern at the BORIS promoter. Rapid amplification of cDNA ends (RACE) mapping revealed that the transcriptional start site of BORIS in human testis, DNMT deficient human cancer cells, and human epithelial ovarian cancer (EOC) tissues, is similar and lies within the 5' CpG island. The BORIS promoter is repressed by CpG methylation in a dose-dependent fashion, indicating a direct role for DNA methylation in BORIS transcriptional regulation. In human ovarian cancer cell lines, 5-aza-2'-deoxycytidine treatment activates BORIS expression and reduces BORIS promoter DNA methylation. We quantitatively measured BORIS mRNA expression and promoter DNA methylation in normal ovary (NO; n = 10) and epithelial ovarian cancer (EOC; n = 77) and found that, compared to NO, EOC tumors show increased BORIS expression and decreased BORIS methylation. Importantly, BORIS promoter DNA methylation shows a significant inverse correlation with BORIS mRNA expression in EOC (Kendall's Tau = -0.235, P = 0.007, n = 63). These data establish promoter DNA hypomethylation as a mechanism leading to BORIS expression in human ovarian cancer.

An improved synthesis of psammaplin A.

The marine natural product, psammaplin A, was first isolated from the Psammaplinaplysilla sponge in 1987. Since that time, psammaplin A has shown a wide spectrum of biological activities that include enzyme inhibitory activities resulting in antibacterial and antitumor effects. An improved synthesis of psammaplin A has been developed, making the compound more easily accessible for further biological evaluations. In this context, we find that psammaplin A is an effective DNA methyltransferase inhibitor in vitro but fails to alter genomic DNA methylation levels in treated human cancer cells.