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.

Of the non-viral vectors, cationic lipid (CL) formulations are the most widely studied for the delivery of genes, antisense oligonucleotides and gene silencing nucleic acids such as small interfering RNAs. However, little is known about the impact of these delivery systems on global gene expression in target cells. In an attempt to study the geno-compatibility of CL formulations in target cells, we have used microarrays to examine the effect of Lipofectin and Oligofectamine on the gene expression profiles of human A431 epithelial cells. Using the manufacturer's recommended CL concentrations routinely used for gene delivery, cDNA microarray expression profiling revealed marked changes in the expression of several genes for both Lipofectin- and Oligofectamine-treated cells. Data from the 200 spot arrays housing 160 different genes indicated that Lipofectin or Oligofectamine treatment of A431 cells resulted in more than 2-fold altered expression of 10 and 27 genes, respectively. The downstream functional consequences of CL-induced gene expression alterations led to an increased tendency of cells to enter early apoptosis as assessed by annexin V-FITC flow cytometry analyses. This effect was greater for Oligofectamine than Lipofectin. Observed gene expression changes were not sufficient to induce any significant DNA damage as assessed by single cell gel electrophoresis (COMET) assay. These data highlight the fact that inadvertent gene expression changes can be induced by the delivery formulation alone and that these may, ultimately, have important safety implications for the use of these non-viral vectors in gene-based therapies. Also, the induced non-target gene changes should be taken into consideration in gene therapy or gene silencing experiments using CL formulations where they may potentially mask or interfere with the desired genotype and/or phenotype end-points.

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.

Poly(ADP-ribose) glycohydrolase (PARG), removes poly(ADP-ribose) subunits from proteins that have previously been modified by poly(ADP-ribose) polymerse. This ensures that modification is transient, and it is suggested that removal of poly(ADP-ribose) is essential for some types of DNA repair. Here we show increased γH2AX foci formation and increased homologous recombination when PARG is inhibited. These effects are reduced when replication is inhibited, suggesting that in the absence of PARG activity, replication forks collapse, and homologous recombination is induced for repair. Consistent with this, we show that cells deficient in the homologous recombination protein BRCA2 are sensitive to PARG depletion or inhibition. These data raise the exciting possibility that PARG inhibitors may be used to specifically kill BRCA2 and other homologous recombination-deficient tumors.

Metastatic disease is the most common mechanism of death in patients with advanced bladder cancer. As for most solid tumors, chemotherapy remains the only realistic option for palliating or curing metastatic disease. However, bladder cancer is characterized by chemoresistance. Only modest response rates are obtained using multiagent regimens including cisplatin. These low response rates and the toxicity of these regimens limit their use to patients at highest risk. Here, we review the molecular mechanisms of cisplatin resistance. These include methods to reduce cisplatin bioavailability within a cell, and defects in the machinery that produces cell death following cisplatin-induced DNA damage. While overcoming these mechanisms is a potential therapeutic approach that can increase response rates, in the short term this knowledge could be used to predict response in individual tumors.

Polypropylenimine (PPI) dendrimers appear attractive non-viral vectors for the delivery of genes, antisense oligonucleotides, and small interfering RNA (siRNA). However, the effects of these synthetic gene delivery vectors on global gene expression are poorly understood. Here we have examined the toxicogenomics of generation 2 (DAB-8) and generation 3 (DAB-16) PPI dendrimers in two human cell lines. At concentrations and treatment protocols routinely used for gene and oligonucleotide transfection, PPI dendrimers alone elicited marked changes in endogenous gene expression in A431 epithelial cells. The extent of PPI-induced gene changes appeared to be dependent on the dendrimer generation as the number of genes affected was greater with G3 compared to G2 PPI dendrimers in A431 cells. The signature of DAB16-induced gene changes in A549 cells was different to those elicited in A431 cells implying a strong dependence on cell type. The DAB-16 polymer complexed with DNA (dendriplexes) also elicited marked gene expression changes in A549 cells but with a signature that was different from the polymer alone implying that dendriplexes are "recognised" by cells as chemical entities that are distinct from the polymer alone. Alterations in expression of a variety of gene ontologies were observed including those involved in defence responses, cell proliferation and apoptosis. Although there was a tendency for increased DNA damage in cells treated with DAB16 alone or its DNA dendriplexes as detected by the COMET assay, these differences were not statistically significant. These data show for the first time that PPI-dendrimers, separate from their capability as transfection reagents, can intrinsically alter the expression of many endogenous genes that could potentially lead to them exerting multiple biological effects in cells. The impact and consequences of polymer-induced gene changes should guide their rational use as delivery systems for gene-based therapeutics.

Purpose: Loss of epigenetic gene regulation through altered long noncoding RNA (lncRNA) expression seems important in human cancer. LncRNAs have diagnostic and therapeutic potential, and offer insights into the biology disease, but little is known of their expression in urothelial cancer. Here, we identify differentially expressed lncRNAs with potential regulatory functions in urothelial cancer.Experimental Design: The expression of 17,112 lncRNAs and 22,074 mRNAs was determined using microarrays in 83 normal and malignant urothelial (discovery) samples and selected RNAs with qPCR in 138 samples for validation. Significantly differentially expressed RNAs were identified and stratified according to tumor phenotype. siRNA knockdown, functional assays, and whole-genome transcriptomic profiling were used to identify potential roles of selected lncRNAs.Results: We observed upregulation of many lncRNAs in urothelial cancer that was distinct to corresponding, more balanced changes for mRNAs. In general, lncRNA expression reflected disease phenotype. We identified 32 lncRNAs with potential roles in disease progression. Focusing upon a promising candidate, we implicate upregulation of AB074278 in apoptosis avoidance and the maintenance of a proproliferative state in cancer through a potential interaction with EMP1, a tumor suppressor and a negative regulator of cell proliferation.Conclusions: We report differential expression profiles for numerous lncRNA in urothelial cancer. We identify phenotype-specific expression and a potential mechanistic target to explain this observation.

Resistance to the cytotoxic effects of cisplatin can be mediated through changes in a wide variety of cellular processes and signalling pathways. The discovery of microRNAs as regulators of protein expression through the targeting of mRNA has led to a number of studies on the effect of cisplatin treatment on microRNA expression, and the ability of microRNAs to modulate cisplatin resistance.