Laoighse Mulrane

miRNA Dysregulation in Breast Cancer

miRNAs have emerged, in the last decade, as key players in the carcinogenic process, with many candidates identified as playing important roles in many aspects of tumor development, growth, metastasis, and drug resistance. More recently, polymorphisms in miRNAs themselves or in their binding sites in target genes have been identified to incur increased risk of breast cancer in certain populations. In addition, epigenetic regulation and differential expression of processing enzymes has been shown to contribute to the aberrant expression of miRNAs in breast cancer. This review focuses on the area of miRNA dysregulation in breast cancer through both genetic and epigenetic mechanisms, and the impact of this dysregulation on breast cancer risk and resistance to therapies.

Automated image analysis in histopathology: a valuable tool in medical diagnostics

Virtual pathology, the process of assessing digital images of histological slides, is gaining momentum in today’s laboratory environment. Indeed, digital image acquisition systems are becoming commonplace, and associated image analysis solutions are viewed by most as the next critical step in automated histological analysis. Here, we document the advances in the technology, with reference to past and current techniques in histological assessment. In addition, the demand for these technologies is analyzed with major players profiled. As there are several image analysis software programs focusing on the quantification of immunohistochemical staining, particular attention is paid to this application in this review. Oncology has been a primary target area for these approaches, with example studies in this therapeutic area being covered here. Toxicology-based image analysis solutions are also profiled as these are steadily increasing in popularity, especially within the pharmaceutical industry. Reinforced by the phenomenal growth of the virtual pathology field, it is envisioned that the market for automated image analysis tools will greatly expand over the next 10 years.

Characterisation and manipulation of docetaxel resistant prostate cancer cell lines

BackgroundThere is no effective treatment strategy for advanced castration-resistant prostate cancer. Although Docetaxel (Taxotere®) represents the most active chemotherapeutic agent it only gives a modest survival advantage with most patients eventually progressing because of inherent or acquired drug resistance. The aims of this study were to further investigate the mechanisms of resistance to Docetaxel. Three Docetaxel resistant sub-lines were generated and confirmed to be resistant to the apoptotic and anti-proliferative effects of increasing concentrations of Docetaxel.ResultsThe resistant DU-145 R and 22RV1 R had expression of P-glycoprotein and its inhibition with Elacridar partially and totally reversed the resistant phenotype in the two cell lines respectively, which was not seen in the PC-3 resistant sublines. Resistance was also not mediated in the PC-3 cells by cellular senescence or autophagy but multiple changes in pro- and anti-apoptotic genes and proteins were demonstrated. Even though there were lower basal levels of NF-κB activity in the PC-3 D12 cells compared to the Parental PC-3, docetaxel induced higher NF-κB activity and IκB phosphorylation at 3 and 6 hours with only minor changes in the DU-145 cells. Inhibition of NF-κB with the BAY 11-7082 inhibitor reversed the resistance to Docetaxel.ConclusionThis study confirms that multiple mechanisms contribute to Docetaxel resistance and the central transcription factor NF-κB plays an immensely important role in determining docetaxel-resistance which may represent an appropriate therapeutic target.

Performance of Novel Kidney Biomarkers in Preclinical Toxicity Studies

The kidney is one of the main targets of drug toxicity, but early detection of renal damage is often difficult. As part of the InnoMed PredTox project, a collaborative effort aimed at assessing the value of combining omics technologies with conventional toxicology methods for improved preclinical safety assessment, we evaluated the performance of a panel of novel kidney biomarkers in preclinical toxicity studies. Rats were treated with a reference nephrotoxin or one of several proprietary compounds that were dropped from drug development in part due to renal toxicity. Animals were dosed at two dose levels for 1, 3, and 14 days. Putative kidney markers, including kidney injury molecule-1 (Kim-1), lipocalin-2 (Lcn2), clusterin, and tissue inhibitor of metalloproteinases-1, were analyzed in kidney and urine using quantitative real-time PCR, ELISA, and immunohistochemistry. Changes in gene/protein expression generally correlated well with renal histopathological alterations and were frequently detected at earlier time points or at lower doses than the traditional clinical parameters blood urea nitrogen and serum creatinine. Urinary Kim-1 and clusterin reflected changes in gene/protein expression and histopathological alterations in the target organ in the absence of functional changes. This confirms clusterin and Kim-1 as early and sensitive, noninvasive markers of renal injury. Although Lcn2 did not appear to be specific for kidney toxicity, its rapid response to inflammation and tissue damage in general may suggest its utility in routine toxicity testing.

miR-187 Is an Independent Prognostic Factor in Breast Cancer and Confers Increased Invasive Potential In Vitro

Purpose: Here, we describe an integrated bioinformatics, functional analysis, and translational pathology approach to identify novel miRNAs involved in breast cancer progression. Experimental Design: Coinertia analysis (CIA) was used to combine a database of predicted miRNA target sites and gene expression data. Using two independent breast cancer cohorts, CIA was combined with correspondence analysis and between group analysis to produce a ranked list of miRNAs associated with disease progression. Ectopic expression studies were carried out in MCF7 cells and miRNA expression evaluated in two additional cohorts of patients with breast cancer by in situ hybridization on tissue microarrays. Results: CIA identified miR-187 as a key miRNA associated with poor outcome in breast cancer. Ectopic expression of miR-187 in breast cancer cells resulted in a more aggressive phenotype. In a test cohort (n = 117), high expression of miR-187 was associated with a trend toward reduced breast cancer–specific survival (BCSS; P = 0.058), and a significant association with reduced BCSS in lymph node–positive patients (P = 0.036). In a validation cohort (n = 470), high miR-187 was significantly associated with reduced BCSS in the entire cohort (P = 0.021) and in lymph node–positive patients (P = 0.012). Multivariate Cox regression analysis revealed that miR-187 is an independent prognostic factor in both cohorts [cohort 1: HR, 7.37; 95% confidence interval (CI), 2.05–26.51; P = 0.002; cohort 2: HR, 2.80; 95% CI, 1.52–5.16; P = 0.001] and in lymph node–positive patients in both cohorts (cohort 1: HR, 13.74; 95% CI, 2.62–72.03; P = 0.002; cohort 2: HR, 2.77; 95% CI, 1.32–5.81; P = 0.007). Conclusions: miR-187 expression in breast cancer leads to a more aggressive, invasive phenotype and acts as an independent predictor of outcome. Clin Cancer Res; 18(24); 6702–13. ©2012 AACR.

Assessment of candidate biomarkers of drug-induced hepatobiliary injury in preclinical toxicity studies.

This study was designed to assess the value of a set of potential markers for improved detection of liver injury in preclinical toxicity studies. Male Wistar rats were treated with drug candidates (BAY16, EMD335823, BI-3) that previously failed during development, in part due to hepatotoxicity, at two dose levels for 1, 3 and 14 days. Concentrations of lipocalin-2/NGAL and clusterin, which are frequently overexpressed and released from damaged tissues, and thiostatin, recently identified within PredTox as being elevated in urine in response to liver injury, were determined in rat urine and serum by ELISA. This was supplemented by confirmatory qRT-PCR and immunohistochemical analyses in the target organ. Serum paraoxonase-1 activity (PON1), which has been suggested as a marker of hepatotoxicity, was determined using a fluorometric assay. Clusterin and PON1 were not consistently altered in response to liver injury. In contrast, thiostatin and NGAL were increased in serum and urine of treated animals in a time- and dose-dependent manner. These changes correlated well with mRNA expression in the target organ and generally reflected the onset and degree of drug-induced liver injury. Receiver-operating characteristics (ROC) analyses supported serum thiostatin, but not NGAL, as a better indicator of drug-induced hepatobiliary injury than conventional clinical chemistry parameters, i.e. ALP, ALT and AST. Although thiostatin, an acute phase protein expressed in a range of tissues, may not be specific for liver injury, our results indicate that thiostatin may serve as a sensitive, minimally-invasive diagnostic marker of inflammation and tissue damage in preclinical safety assessment.

microRNAs: a new class of breast cancer biomarkers

MicroRNAs (miRNAs) are regulatory molecules known to be aberrantly expressed in cancer and contribute to numerous aspects of tumor biology including the initiation, growth and spread of the tumor. With such diverse roles, it is becoming apparent that some may also provide valuable information which may be of use in a clinical setting, demonstrating the potential to act as both screening tools for the stratification of high-risk patients, while informing the treatment decision-making process. There is mounting evidence to suggest that some miRNAs may even provide assistance in the diagnosis of patients with breast cancer. In addition, miRNAs may themselves be considered therapeutic targets, with inhibition or reintroduction of a particular miRNA capable of inducing a response in vivo. This review focuses on miRNAs that have prognostic, diagnostic or predictive potential in breast cancer as well as the possible challenges in the translation of such observations to the clinic.

A novel mechanism of regulation of the anti-metastatic miR-31 by EMSY in breast cancer

AbstractmiR-31 is well known as an anti-metastatic microRNA (miRNA) in the context of breast cancer. However, to date the mechanism of regulation of this miRNA has yet to be elucidated. The recent publication by Viré et al. in Molecular Cell [1] demonstrates for the first time that one mechanism of regulation of miR-31 is through the putative oncogene EMSY, whose amplification in breast cancer patients correlates with reduced expression of the miRNA. This regulation is dependent on the DNA-binding transcription factor ETS-1 which recruits EMSY and the histone demethylase KDM5B to the miR-31 promoter, thus repressing its transcription.

Tissue Microarrays and Digital Image Analysis

Tissue microarrays (TMAs) have recently emerged as very valuable tools for high-throughput pathological assessment, especially in the cancer research arena. This important technology, however, has yet to fully penetrate into the area of toxicology. Here, we describe the creation of TMAs representative of samples produced from conventional toxicology studies within a large-scale, multi-institutional pan-European project, PredTox. PredTox, short for Predictive Toxicology, formed part of an EU FP6 Integrated Project, Innovative Medicines for Europe (InnoMed), and aimed to study pre-clinically 16 compounds of known liver and/or kidney toxicity. In more detail, TMAs were constructed from materials corresponding to the full face sections of liver and kidney from rats treated with different drug candidates by members of the consortium. We also describe the process of digital slide scanning of kidney and liver sections, in the context of creating an online resource of histopathological data.

PO-345 The role of the deubiquitinase USP11 in endocrine-driven breast cancer

Introduction Approximately 80% of breast cancers overexpress the oestrogen receptor α (ERα) and depend on this key transcriptional regulator for growth. The discovery of novel mechanisms controlling ERα function represent major advances in our understanding of breast cancer progression and potentially offer new therapeutic opportunities. Here, we investigated the role of deubiquitinating enzymes (DUBs), which remove ubiquitin moieties from proteins, in regulating ERα activity in breast cancer. Material and methods To identify DUBs involved in ERα transcriptional activity, we performed an RNAi loss-of-function screen using a library of shRNA vectors targeting all 108 known or putative human DUB genes. We found that suppression of the BRCA-associated DUB, USP11, downregulated ERα transcriptional activity in ZR-75–1 cells. Dual luciferase reporter assays and qRT-PCR were used to determine ERα activity in stable ZR-75–1 USP11 knockdown cell lines. PTMScan technology, which allows for enrichment of ubiquitinated proteins and quantitative profiling by mass spectrometry, was used to reveal the ubiquitinome in these cells and results are pending. RNA sequencing (RNA-seq) technology was carried out in estrogen-independent LCC1 USP11 knockdown cells, were USP11 was hypothesised to play a vital role. Finally, immunohistochemistry (IHC) was used to investigate the prognostic relevance of USP11 in breast cancer patients. Results and discussions Knockdown of USP11 in ZR-75–1 cells decreased ERα transcriptional activity and mRNA expression of ERα target genes. These results were further validated in a HEK293T USP11 CRISPR knockout model with ectopic ERα expression. Interestingly, USP11 expression was upregulated in LCC1 cells, an isogenic, estrogen-independent model derived from MCF-7 cells. Knockdown of USP11 in LCC1 cells decreased the expression of multiple ERα target genes and cell cycle-associated genes, as determined by RNA-seq. IHC staining of a breast cancer tissue microarray (103 ER +patients) and subsequent Kaplan-Meier analysis of this cohort revealed a significant association between high USP11 expression and poor overall (p=0.030) and breast cancer-specific survival (p=0.041). In silico analysis of publically available breast cancer gene expression datasets further supported an association between high USP11 expression and poor prognosis. Conclusion These results suggest a role for USP11 in ERα transcriptional activity and reveal a novel mechanism as to how this receptor is regulated in breast cancer.