The role of microRNA in the response to cisplatin treatment.

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.

Identification and diagnostic performance of a small RNA within the PCA3 and BMCC1 gene locus that potentially targets mRNA.

BACKGROUND: PCA3 is a long noncoding RNA (lncRNA) with unknown function, upregulated in prostate cancer. LncRNAs may be processed into smaller active species. We hypothesized this for PCA3. METHODS: We computed feasible RNA hairpins within the BMCC1 gene (encompassing PCA3) and searched a prostate transcriptome for these. We measured expression using qRT-PCR in three cohorts of prostate cancer tissues (n = 60), exfoliated urinary cells (n = 484 with cancer and n = 166 controls), and in cell lines (n = 22). We used in silico predictions and RNA knockup to identify potential mRNA targets of short transcribed RNAs. RESULTS: We predicted 13 hairpins, of which PCA3-shRNA2 was most abundant within the prostate transcriptome. PCA3-shRNA2 is located within intron 1 of PCA3 and appears regulated by androgens. Expression of PCA3-shRNA2 was upregulated in malignant prostatic tissues, exfoliated urinary cells from men with prostate cancer (13-273 fold change; t test P < 0.003), and closely correlated to PCA3 expression (r = 0.84-0.93; P < 0.001). Urinary PCA3-shRNA2 (C-index, 0.75-0.81) and PCA3 (C-index, 0.78) could predict the presence of cancer in most men. PCA3-shRNA2 knockup altered the expression of predicted target mRNAs, including COPS2, SOX11, WDR48, TEAD1, and Noggin. PCA3-shRNA2 expression was negatively correlated with COPS2 in patient samples (r = -0.32; P < 0.001). CONCLUSION: We identified a short RNA within PCA3, whose expression is correlated to PCA3, which may target mRNAs implicated in prostate biology. IMPACT: This short RNA is stable ex vivo, suggesting a role as a robust biomarker. We identify cytoplasmic enrichment of this RNA and potential targeting of mRNAs implicated in prostate carcinogenesis.

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.

Identification of differentially expressed long noncoding RNAs in bladder cancer.

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. Further studies are required to validate lncRNAs as prognostic biomarkers in this disease.

MicroRNA-99a and 100 mediated upregulation of FOXA1 in bladder cancer.

Urothelial cell carcinoma of the bladder (UCC) is a common disease often characterized by FGFR3 dysregulation. Whilst upregulation of this oncogene occurs most frequently in low-grade non-invasive tumors, recent data reveal increased FGFR3 expression characterizes a common sub-type of invasive UCC sharing molecular similarities with breast cancer. These similarities include upregulation of the FOXA1 transcription factor and reduced expression of microRNAs-99a/100. We have previously identified direct regulation of FGFR3 by these two microRNAs and now search for further targets. Using a microarray meta-database we find potential FOXA1 regulation by microRNAs-99a/100. We confirm direct targeting of the FOXA1 3'UTR by microRNAs-99a/100 and also potential indirect regulation through microRNA-485-5p/SOX5/JUN-D/FOXL1 and microRNA-486/FOXO1a. In 292 benign and malignant urothelial samples, we find an inverse correlation between the expression of FOXA1 and microRNAs-99a/100 (r=-0.33 to -0.43, p<0.05). As for FGFR3 in UCC, tumors with high FOXA1 expression have lower rates of progression than those with low expression (Log rank p=0.009). Using global gene expression and CpG methylation profiling we find genotypic consequences of FOXA1 upregulation in UCC. Genetic changes are associated with regional hypomethylation, occur near FOXA1 binding sites, and mirror gene expression changes previously reported in FGFR3 mutant-UCC. These include gene silencing through aberrant hypermethylation (e.g. IGFBP3) and affect genes characterizing breast cancer sub-types (e.g. ERBB2). In conclusion, we have identified microRNAs-99a/100 mediate a direct relationship between FGFR3 and FOXA1 and potentially facilitate cross talk between these pathways in UCC.

Molecular mechanisms of cisplatin resistance in bladder cancer.

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.

Inhibition of poly(ADP-ribose) glycohydrolase (PARG) specifically kills BRCA2-deficient tumor cells.

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.

The Fanconi anemia pathway is downregulated upon macrophage differentiation through two distinct mechanisms.

The Fanconi anaemia (FA) pathway is a DNA-damage inducible pathway critical for genomic stability. FA patients typically display high cancer susceptibility and hypersensitivity to DNA-damaging agents such as cross-linkers and ionizing radiation. A key step in the activation of the FA pathway is monoubiquitination of the FancD2 protein. Here we report that the FA pathway is downregulated by two distinct mechanisms upon differentiation of THP-1 and HL-60 leukaemia cells into macrophages. Firstly, qRT-PCR analysis revealed a transcriptional downregulation of most components of the FA complex, including FancD2. Secondly, DNA damage-induced monoubiquitination of the remaining FancD2 became deficient at various stages of differentiation depending on the type of damage. This was attributed to the differentiation-induced downregulation of Chk1, which phosphorylates FancD2 as a prelude to its ubiquitination. Although Western blotting revealed that levels of FancD2 were greatly reduced in terminally differentiated macrophages and that FancD2 ubiquitination was abolished, double-strand breaks were proficiently repaired, likely through an increase in non-homologous end joining (NHEJ). It has been suggested that the FA pathway promotes repair of double-strand breaks via homologous recombination rather than NHEJ. Its downregulation in macrophages may thus be required to avoid promoting a repair mechanism that is inefficient in post-mitotic cells.