Ewa Dudziec

Distinct MicroRNA Alterations Characterize High- and Low-Grade Bladder Cancer

Urothelial carcinoma of the bladder (UCC) is a common disease that arises by at least two different molecular pathways. The biology of UCC is incompletely understood, making the management of this disease difficult. Recent evidence implicates a regulatory role for microRNA in cancer. We hypothesized that altered microRNA expression contributes to UCC carcinogenesis. To test this hypothesis, we examined the expression of 322 microRNAs and their processing machinery in 78 normal and malignant urothelial samples using real-time rtPCR. Genes targeted by differentially expressed microRNA were investigated using real-time quantification and microRNA knockdown. We also examined the role of aberrant DNA hypermethylation in microRNA downregulation. We found that altered microRNA expression is common in UCC and occurs early in tumorogenesis. In normal urothelium from patients with UCC, 11% of microRNAs had altered expression when compared with disease-free controls. This was associated with upregulation of Dicer, Drosha, and Exportin 5. In UCC, microRNA alterations occur in a tumor phenotype-specific manner and can predict disease progression. High-grade UCC were characterized by microRNA upregulation, including microRNA-21 that suppresses p53 function. In low-grade UCC, there was downregulation of many microRNA molecules. In particular, loss of microRNAs-99a/100 leads to upregulation of FGFR3 before its mutation. Promoter hypermethylation is partly responsible for microRNA downregulation. In conclusion, distinct microRNA alterations characterize UCC and target genes in a pathway-specific manner. These data reveal new insights into the disease biology and have implications regarding tumor diagnosis, prognosis and therapy.

An evaluation of urinary microRNA reveals a high sensitivity for bladder cancer.

Background:Urinary biomarkers are needed to improve the care and reduce the cost of managing bladder cancer. Current biomarkers struggle to identify both high and low-grade cancers due to differing molecular pathways. Changes in microRNA (miR) expression are seen in urothelial carcinogenesis in a phenotype-specific manner. We hypothesised that urinary miRs reflecting low- and high-grade pathways could detect bladder cancers and overcome differences in genetic events seen within the disease.Methods:We investigated urinary samples (n=121) from patients with bladder cancer (n=68) and age-matched controls (n=53). Fifteen miRs were quantified using real-time PCR.Results:We found that miR is stable within urinary cells despite adverse handling and detected differential expression of 10 miRs from patients with cancer and controls (miRs−15a/15b/24-1/27b/100/135b/203/212/328/1224, ANOVA P<0.05). Individually, miR-1224-3p had the best individual performance with specificity, positive and negative predictive values and concordance of 83%, 83%, 75% and 77%, respectively. The combination of miRs-135b/15b/1224-3p detected bladder cancer with a high sensitivity (94.1%), sufficient specificity (51%) and was correct in 86% of patients (concordance).Conclusion:The use of this panel in patients with haematuria would have found 94% of urothelial cell carcinoma, while reducing cystoscopy rates by 26%. However, two invasive cancers (3%) would have been missed.

Hypermethylation of CpG Islands and Shores around Specific MicroRNAs and Mirtrons Is Associated with the Phenotype and Presence of Bladder Cancer

Purpose: To analyze the role and translational potential for hypermethylation of CpG islands and shores in the regulation of small RNAs within urothelial cell carcinoma (UCC). To examine microRNAs (miR) and mirtrons, a new class of RNA located within gene introns and processed in a Drosha-independent manner. Experimental design: The methylation status of 865 small RNAs was evaluated in normal and malignant cell lines by using 5-azacytidine and microarrays. Bisulfite sequencing was used for CpG regions around selected RNAs. Prognostic and diagnostic associations for epigenetically regulated RNAs were examined by using material from 359 patients, including 216 tumors and 121 urinary samples (68 cases and 53 controls). Functional analyses examined the effect of silencing susceptible RNAs in normal urothelial cells. Results: Exonic/UTR-located miRs and mirtons are most susceptible to epigenetic regulation. We identified 4 mirtrons and 16 miRs with CpG hypermethylation across 35 regions in normal and malignant urothelium. For several miRs, hypermethylation was more frequent and dense in CpG shores than islands (e.g., miRs-9/149/210/212/328/503/1224/1227/1229), and was associated with tumor grade, stage, and prognosis (e.g., miR-1224 multivariate analysis OR = 2.5; 95% CI, 1.3–5.0; P = 0.006). The urinary expression of epigenetically silenced RNAs (miRs-152/328/1224) was associated with the presence of UCC (concordance index, 0.86; 95% CI, 0.80–0.93; ANOVA P < 0.016). Conclusions: Hypermethylation of mirtrons and miRs is common in UCC. Mirtrons appear particularly susceptible to epigenetic regulation. Aberrant hypermethylation of small RNAs is associated with the presence and behavior of UCC, suggesting potential roles as diagnostic and prognostic biomarkers. Clin Cancer Res; 17(6); 1287–96. ©2010 AACR.

Reduced Expression of miRNA-27a Modulates Cisplatin Resistance in Bladder Cancer by Targeting the Cystine/Glutamate Exchanger SLC7A11

Purpose: Resistance to cisplatin-based chemotherapy is a major obstacle to bladder cancer treatment. We aimed to identify microRNAs (miRNA) that are dysregulated in cisplatin-resistant disease, ascertain how these contribute to a drug-resistant phenotype, and how this resistance might be overcome. Experimental Design: miRNA expression in paired cisplatin-resistant and -sensitive cell lines was measured. Dysregulated miRNAs were further studied for their ability to mediate resistance. The nature of the cisplatin-resistant phenotype was established by measurement of cisplatin/DNA adducts and intracellular glutathione (GSH). Candidate miRNAs were examined for their ability to (i) mediate resistance and (ii) alter the expression of a candidate target protein (SLC7A11); direct regulation of SLC7A11 was confirmed using a luciferase assay. SLC7A11 protein and mRNA, and miRNA-27a were quantified in patient tumor material. Results: A panel of miRNAs were found to be dysregulated in cisplatin-resistant cells. miRNA-27a was found to target the cystine/glutamate exchanger SLC7A11 and to contribute to cisplatin resistance through modulation of GSH biosynthesis. In patients, SLC7A11 expression was inversely related to miRNA-27a expression, and those tumors with high mRNA expression or high membrane staining for SLC7A11 experienced poorer clinical outcomes. Resistant cell lines were resensitized by restoring miRNA-27a expression or reducing SLC7A11 activity with siRNA or with sulfasalazine. Conclusion: Our findings indicate that miRNA-27a negatively regulates SLC7A11 in cisplatin-resistant bladder cancer, and shows promise as a marker for patients likely to benefit from cisplatin-based chemotherapy. SLC7A11 inhibition with sulfasalazine may be a promising therapeutic approach to the treatment of cisplatin-resistant disease. Clin Cancer Res; 20(7); 1990–2000. ©2014 AACR.

Global epigenetic profiling in bladder cancer

Urothelial carcinoma of the bladder is a common disease that arises from two distinct molecular pathways, and is one of the most expensive malignancies to manage. Accurate biomarkers that could detect tumor recurrence or predict future progression would improve the care of patients and reduce the cost of managing the disease. DNA methylation, histone modification and ncRNA expression are important epigenetic mechanisms that regulate the expression of genes. These regulatory mechanisms are altered with bladder cancer, and therefore, represent potential biomarkers and therapeutic targets owing to the reversible nature of their modification. In this article, we will discuss these epigenetic changes in bladder cancer and assess their clinical potential.

Integrated Epigenome Profiling of Repressive Histone Modifications, DNA Methylation and Gene Expression in Normal and Malignant Urothelial Cells

Epigenetic regulation of gene expression is commonly altered in human cancer. We have observed alterations of DNA methylation and microRNA expression that reflect the biology of bladder cancer. This common disease arises by distinct pathways with low and high-grade differentiation. We hypothesized that epigenetic gene regulation reflects an interaction between histone and DNA modifications, and differences between normal and malignant urothelial cells represent carcinogenic events within bladder cancer. To test this we profiled two repressive histone modifications (H3K9m3 and H3K27m3) using ChIP-Seq, cytosine methylation using MeDIP and mRNA expression in normal and malignant urothelial cell lines. In genes with low expression we identified H3K27m3 and DNA methylation each in 20–30% of genes and both marks in 5% of genes. H3K9m3 was detected in 5–10% of genes but was not associated with overall expression. DNA methylation was more closely related to gene expression in malignant than normal cells. H3K27m3 was the epigenetic mark most specifically correlated to gene silencing. Our data suggest that urothelial carcinogenesis is accompanied by a loss of control of both DNA methylation and H3k27 methylation. From our observations we identified a panel of genes with cancer specific-epigenetic mediated aberrant expression including those with reported carcinogenic functions and members potentially mediating a positive epigenetic feedback loop. Pathway enrichment analysis revealed genes marked by H3K9m3 were involved with cell homeostasis, those marked by H3K27m3 mediated pro-carcinogenic processes and those marked with cytosine methylation were mixed in function. In 150 normal and malignant urothelial samples, our gene panel correctly estimated expression in 65% of its members. Hierarchical clustering revealed that this gene panel stratified samples according to the presence and phenotype of bladder cancer.

Reduced FANCD2 influences spontaneous SCE and RAD51 foci formation in uveal melanoma and Fanconi anaemia.

Uveal melanoma (UM) is unique among cancers in displaying reduced endogenous levels of sister chromatid exchange (SCE). Here we demonstrate that FANCD2 expression is reduced in UM and that ectopic expression of FANCD2 increased SCE. Similarly, FANCD2-deficient fibroblasts (PD20) derived from Fanconi anaemia patients displayed reduced spontaneous SCE formation relative to their FANCD2-complemented counterparts, suggesting that this observation is not specific to UM. In addition, spontaneous RAD51 foci were reduced in UM and PD20 cells compared with FANCD2-proficient cells. This is consistent with a model where spontaneous SCEs are the end product of endogenous recombination events and implicates FANCD2 in the promotion of recombination-mediated repair of endogenous DNA damage and in SCE formation during normal DNA replication. In both UM and PD20 cells, low SCE was reversed by inhibiting DNA-PKcs (DNA-dependent protein kinase, catalytic subunit). Finally, we demonstrate that both PD20 and UM are sensitive to acetaldehyde, supporting a role for FANCD2 in repair of lesions induced by such endogenous metabolites. Together, these data suggest FANCD2 may promote spontaneous SCE by influencing which double-strand break repair pathway predominates during normal S-phase progression.

Abstract 5014: Differences in DNA methylation pattern of primary bladder tumours correlate with their metastatic potential

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, ILnnIntroduction: The prognosis for patients with metastatic bladder cancer is poor with a 5-year survival rate of only 6%. Early prediction of the metastatic risk in primary tumours can lead to improvements in prognosis and therapy. Currently, there are no parameters available that enable an individual risk assessment for patients with metastatic bladder cancer. Therefore, identification of new reliable prognostic markers for early prediction of tumour spread is required. The aim of this project is to determine whether changes in DNA methylation correlate with the metastasis risk and to identify a specific DNA methylation pattern that provides a reliable tool for prognosis assessment. Materials & methods: Genomic DNA was isolated from 23 invasive bladder tumour tissues with and without lymph node metastases. For enrichment of methylated fragments genomic DNA was incubated with 5-Methylcytosin antibody. Input and IP were labelled with Cy3 and Cy5. Labelled DNA was hybridizes onto CpG island microarrays. Cluster analysis enabled selection of differentially methylated candidate genes. The actual methylation state was confirmed by cleavage of genomic DNA with methylation dependent restriction enzyme (McrBc) followed by quantitative PCR. Results: In total, 201 promoter CpG islands show highly significant differences in DNA methylation between metastasised and non-metastasised primary bladder tumours (p<0,01). In metastasised tumours 74 CpG islands are hypermethylated and 127 CpG islands are hypomethylated. Data base research for identification of candidate genes resulted in 61 hypermethylated and 58 hypomethylated genes that can clearly assigned to the identified promoter associated CpG islands. Validation of methylation level of candidate gene RASSF1 resulted in higher methylation in metastatic tumours (mean methylation: 38%) than in non-metastatic tumours (mean methylation: 26%). RASSF1 methylation shows strong correlation to T stage (r=0,65). Methylation of candidate gene KISS1R shows significant differences between patient groups (p=0,001). Mean methylation in metastatic tumours is 48% and in non-metastatic tumours 22%. Conclusion: Primary bladder tumours show significant differences in their DNA methylation pattern in correlation to their metastatic potential. KISS1R methylation is strongly associated with the metastatic risk of primary tumours. Further analysis will demonstrate the correlation between changes in DNA methyaltion and expression of corresponding genes. The impact of DNA methylation on tumour behaviour shall be determined in functional analysis.nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5014. doi:1538-7445.AM2012-5014

Abstract A10: MicroRNA 27a is downreguated in cisplatin resistant bladder cancer cells, and contributes to resistance through the targeting of xCT, a cystine transporter involved with glutathione production

Abstract Cisplatin resistance represents a serious obstacle to the chemotherapeutic treatment of many tumors. The purpose of this study was to create a cell culture model of cisplatin resistant bladder cancer (CRBC), then use this model to identify changes in the expression of microRNAs (miRs) that may influence resistance. Single cell clones resistant to cisplatin were generated from parental bladder cancer cell lines by continuous culture in increasing concentrations of cisplatin for several months. The resultant CRBC cell lines were able to tolerate high doses of cisplatin (typically > 10 μM). MicroRNA expression in parental and CRBC cell lines was measured using q-pcr based low-density tiling arrays and a panel of miRs which were consistently altered in all CRBC cell lines was established. In addition the induction and repair of specific cisplatin-DNA adducts in parental and CRBC cell lines was assessed using antibodies against the guanine–guanine (Pt-[GG]) intrastrand crosslink. Four fold more cisplatin was required to produce the equivalent damage in CRBC cell lines compared to the parental control, while the rate of repair, as assessed by measurement of the removal of cisplatin-DNA adducts over time, remained broadly similar between parental and resistant lines. This demonstrated that resistance was due to reduced adduct formation rather than any defect in cross-link repair. Glutathione (GSH) inactivates cisplatin by binding with it irreversibly to form Pt(SG)2 adducts, thus preventing the drug from forming cytotoxic adducts with DNA. One cause of decreased adduct formation is therefore deregulation of the GSH biosynthesis pathway. Upon examination intracellular levels of GSH and its oxidized form glutathione disulfide (GSSG) were found to be increased in resistant cells. The expression of a number of genes involved with the synthesis of GSH was then measured by rt-pcr and western blotting, and several key proteins involved with GSH biosynthesis were found to be upregulated in resistant cells, suggesting increased levels of intracelluar GSH could be responsible for the observed decrease in the levels of cisplatin-DNA adducts observed in resistant cells. The 3′ untranslated regions (3′ UTRs) of these mRNAs were examined using TargetScan, an online miR target prediction program, to find potential sites of miR regulation. The most upregulated protein, xCT (SLC7A11), responsible for cystine import (known to be the rate-limiting step in GSH synthesis) was found to contain target sites for three miRs which had been shown to be significantly downregulated in our model of CRBC, namely miRs 25, 27a and 32. Upregulation of miRs 25, 27a and 32 by transfection of resistant cells with the pri-miR precursor molecules revealed that restoring expression of miR-27a resensitized CRBC cells to the cytotoxic effects of cisplatin. Restoration of miR-27a expression in CRBC cells was also shown to result in a downregulation of the expression of xCT at both the protein and mRNA level. In summary, we have identified a panel of microRNAs that are consistently dysregulated in CRBC, and implicated one of these (miR-27a) in the modulation of cisplatin resistance via increased glutathione production. We also provide evidence that reversal of this change in miR expression results in a reversal of the effects on downstream target, and critically, a reversal of the cisplatin resistant phenotype. The contribution made by miR-27a to the development of cisplatin resistance might represent a potential site for therapeutic intervention in the treatment of CRBC. Measurement of miR-27a expression in tumor material or urine may also serve as a predictive biomarker for patients likely response to cisplatin chemotherapy. Citation Format: Ross M. Drayton, Ewa Dudziec, James W.F. Catto, Helen E. Bryant. MicroRNA 27a is downreguated in cisplatin resistant bladder cancer cells, and contributes to resistance through the targeting of xCT, a cystine transporter involved with glutathione production [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer; 2012 Jan 8-11; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(2 Suppl):Abstract nr A10.