Graeme Doran

Dysregulation of microRNA-204 mediates migration and invasion of endometrial cancer by regulating FOXC1

MicroRNAs (miRNAs) regulate mRNA stability and protein expression, and certain miRNAs have been demonstrated to act either as oncogenes or tumor suppressors. Differential miRNA expression signatures have been documented in many human cancers but the role of miRNAs in endometrioid endometrial cancer (EEC) remains poorly understood. This study identifies significantly dysregulated miRNAs of EEC cells, and characterizes their impact on the malignant phenotype. We studied the expression of 365 human miRNAs using Taqman low density arrays in EECs and normal endometriums. Candidate differentially expressed miRNAs were validated by quantitative real‐time PCR. Expression of highly dysregulated miRNAs was examined in vitro through the effect of anti‐/pre‐miRNA transfection on the malignant phenotype. We identified 16 significantly dysregulated miRNAs in EEC and 7 of these are novel findings with respect to EEC. Antagonizing the function of miR‐7, miR‐194 and miR‐449b, or overexpressing miR‐204, repressed migration, invasion and extracellular matrix‐adhesion in HEC1A endometrial cancer cells. FOXC1 was determined as a target gene of miR‐204, and two binding sites in the 3′‐untranslated region were validated by dual luciferase reporter assay. FOXC1 expression was inversely related to miR‐204 expression in EEC. Functional analysis revealed the involvement of FOXC1 in migration and invasion of HEC1A cells. Our results present dysfunctional miRNAs in endometrial cancer and identify a crucial role for miR‐204‐FOXC1 interaction in endometrial cancer progression. This miRNA signature offers a potential biomarker for predicting EEC outcomes, and targeting of these cancer progression‐ and metastasis‐related miRNAs offers a novel potential therapeutic strategy for the disease.

Dysregulated microRNAs and their predicted targets associated with endometrioid endometrial adenocarcinoma in Hong Kong women

The objective of this study, a parallel study to global gene expression profiling, was to identify dysregulated microRNAs (miRNAs) associated with endometrioid endometrial adenocarcinoma (EEC), examine their correlation with clinico‐pathological characteristics and identify predicted target genes of the dysregulated miRNAs. Using real‐time quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR), profiling of miRNA expression was performed in 30 EECs and 22 normal counterparts in which genome‐wide gene expression had been previously profiled and reported. Clustering analysis identified 30 miRNAs which were significantly dysregulated in EEC. The expression of a sub‐group of miRNAs was significantly correlated with clinico‐pathological characteristics including stage, myometrial invasion, recurrence and lymph node involvement. By searching for predicted miRNA targets that were linked to the dysregulated genes previously identified, 68 genes were predicted as candidate targets of these 30 dysregulated miRNAs. miR‐205 was significantly overexpressed in EECs compared with normal controls. After transfection of a miR‐205 inhibitor, the expression of miR‐205 in endometrial cancer cell line RL95‐2 cells decreased whereas its predicted target gene, JPH4, showed increased protein expression. JPH4 seems to be a real miR‐205 target in vitro and in vivo, and a candidate tumor suppressor gene in EEC. Based on this study in EEC, miRNAs predicted to be involved in tumorigenesis and tumor progression have been identified and placed in the context of the transcriptome of EEC. This work provides a framework on which further research into novel diagnosis and treatment of EEC can be focused.

Dysregulated microRNAs in the pathogenesis and progression of cervical neoplasm.

MicroRNAs (miRNAs) play an important role in a variety of physiological as well as pathophysiological processes, including carcinogenesis. The aim of this study is to identify a distinct miRNA expression signature for cervical intraepithelial neoplasia (CIN) and to unveil individual miRNAs that may be involved in the development of cervical carcinoma. Expression profiling using quantitative real-time RT-PCR of 202 miRNAs was performed on micro-dissected high-grade CIN (CIN 2/3) tissues and compared to normal cervical epithelium. Unsupervised hierarchical clustering of the miRNA expression pattern displayed a distinct separation between the CIN and normal cervical epithelium samples. Supervised analysis identified 12 highly differentially regulated miRNAs, including miR-518a, miR-34b, miR-34c, miR-20b, miR-338, miR-9, miR-512-5p, miR-424, miR-345, miR-10a, miR-193b and miR-203, which distinguished the high-grade CIN specimens from normal cervical epithelium. This miRNA signature was further validated by an independent set of high-grade CIN cases. The same characteristic signature can also be used to distinguish cervical squamous cell carcinoma from normal controls. Target prediction analysis revealed that these dysregulated miRNAs mainly control apoptosis signaling pathways and cell cycle regulation. These findings contribute to understanding the role of microRNAs in the pathogenesis and progression of cervical neoplasm at the molecular level.

Differential splicing across immune system lineages

Alternative splicing (AS) allows increased diversity and orthogonal regulation of the transcriptional products of mammalian genomes. To assess the distribution and variation of alternative splicing across cell lineages of the immune system, we comprehensively analyzed RNA sequencing and microarray data generated by the Immunological Genome Project Consortium. AS is pervasive: 60% of genes showed frequent AS isoforms in T or B lymphocytes, with 7,599 previously unreported isoforms. Distinct cell specificity was observed, with differential exon skipping in 5% of genes otherwise coexpressed in both B and T cells. The distribution of isoforms was mostly all or none, suggesting on/off switching as a frequent mode of AS regulation in lymphocytes. From the identification of differential exon use in the microarray data, clustering of exon inclusion/exclusion patterns across all Immunological Genome Project cell types showed that ∼70% of AS exons are distributed along a common pattern linked to lineage differentiation and cell cycling. Other AS events distinguished myeloid from lymphoid cells or affected only a small set of exons without clear lineage specificity (e.g., Ptprc). Computational analysis predicted specific associations between AS exons and splicing regulators, which were verified by detection of the hnRPLL/Ptprc connection.

Genomic aberrations in cervical adenocarcinomas in Hong Kong Chinese women.

Although the rates of cervical squamous cell carcinoma have been declining, the rates of cervical adenocarcinoma are increasing in some countries. Outcomes for advanced cervical adenocarcinoma remain poor. Precision mapping of genetic alterations in cervical adenocarcinoma may enable better selection of therapies and deliver improved outcomes when combined with new sequencing diagnostics. We present whole‐exome sequencing results from 15 cervical adenocarcinomas and paired normal samples from Hong Kong Chinese women. These data revealed a heterogeneous mutation spectrum and identified several frequently altered genes including FAT1, ARID1A, ERBB2 and PIK3CA. Exome sequencing identified human papillomavirus (HPV) sequences in 13 tumors in which the HPV genome might have integrated into and hence disrupted the functions of certain exons, raising the possibility that HPV integration can alter pathways other than p53 and pRb. Together, these provisionary data suggest the potential for individualized therapies for cervical adenocarcinoma based on genomic information.

Abstract 2756: Multiplexing done differently: flexible, sensitive, and accurate detection of miRNA and protein analytes using Firefly particle technology

Detection of molecular biomarkers in biological fluids is used as an indicator of normal biological processes, pathogenic processes, and response to therapeutic intervention1,2. Using a combined signature of multiple biomarkers can better account for patient and epidemiological heterogeneity, and provide a more accurate indication of patient health2. This has necessitated the development of technologies that can be used for multiplex biomarker profiling directly from biofluids such as plasma, serum, and urine. Conventional assays consume excessive quantities of precious clinical samples, time and budget when used for discovery and validation of multi-analyte biomarker signatures. To address this need, we developed the Firefly Technology platform, which allows for sensitive and accurate detection of up to 75 protein analytes or 68 miRNAs directly from a biological sample. The Firefly platform uses patented Firefly® hydrogel particles and a three-region encoding design that allows for true, in-well multiplexing, providing flexibility over which analytes can be quantified in the same well and allowing for custom panel design. For the detection of protein analytes, the Firefly immunoassays use high-performance matched antibody pairs that reduce cross reactivity between individual analytes, provide up to 5 logs dynamic range, and typically demonstrate single-digit pg/ml sensitivity, while requiring only 12.5 µl biofluid input. Similarly, the Firefly microRNA assay can reliably detect as few as 1000 microRNA copies per sample with a linear dynamic range of ~5 logs, and without the need of prior RNA purification. This assay utilizes single step RT-PCR signal amplification using universal primers, thus leveraging PCR sensitivity while eliminating the need for separate reverse transcription reactions and mitigating amplification biases introduced by target-specific qPCR. The Firefly assays have been validated in a wide range of biological samples including plasma, serum, urine and cell culture supernatant, providing experimental flexibility. The 96well plate assay format enables high-throughput screening of samples, with readout conducted on standard flow cytometers, thereby omitting the need for complex and expensive dedicated instrumentation. Finally, the integrated Firefly Analysis Workbench software enables easy and rapid data analysis, visualization, and export in under ten minutes, and includes key features such as standard curve analysis and publication-quality heatmaps and graphs. Here we present data from several studies investigating cytokine profiling in human and rodent samples, and circulating and tumor microRNA profiles, using the Firefly Platform. Together, this novel combination of bioinformatics tools and multiplex, high-sensitivity assays enables rapid discovery and validation of biomarker signatures from fluid samples. Citation Format: Elnaz Atabakhsh, Graeme Doran, Conor Rafferty, Jennifer Heath, Sarah Albertson, Peter Gillis, Jonathan Pinney, Russell Neuner, Michael Chipchase, James Murray, Daniel Pregibon. Multiplexing done differently: flexible, sensitive, and accurate detection of miRNA and protein analytes using Firefly particle technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2756. doi:10.1158/1538-7445.AM2017-2756

Abstract 1082: High-throughput,purification-free, multiplexed profiling of circulating miRNA for discovery,validation, and diagnostics

To address the needs for circulating miRNA biomarker validation, we developed the Multiplexed Circulating microRNA assay. This assay enables the detection of up to 68 microRNA targets per sample in 96-well format with readout on standard flow cytometers and analysis with an included bioinformatics software package. The Circulating microRNA assay combines particle-based multiplexing, using patented Firefly hydrogel particles, with single-step RT-PCR signal amplification using universal primers. Thus, the Circulating microRNA assay leverages PCR sensitivity while eliminating the need for separate reverse transcription reactions and mitigating amplification biases introduced by target-specific qPCR. Furthermore, the ability to multiplex targets in each well eliminates the need to split valuable samples into multiple reactions. Results from the Circulating microRNA assay are displayed and interpreted using our included Firefly Analysis Workbench, which allows visualization, normalization, and export of experimental data with only a few mouse clicks. To aid discovery and validation of biomarkers, we have generated fixed panels for Oncology, Cardiology, Neurology, Immunology, and Liver Toxicology. These carefully curated panels include hemolysis markers to assess sample quality, as well as critical normalization factors. Here we present the data from several studies investigating circulating and tumor microRNA profiles using the Firefly Circulating microRNA Assay Fixed Panels. Together, this novel combination of bioinformatics tools and multiplexed, high-sensitivity assays enables rapid discovery and validation of microRNA biomarker signatures from fluid specimens. Citation Format: Jessica Tytell, Issac Stoner, Michael Tackett, Graeme Doran, Conor Rafferty, Andreas Windemuth, Daniel Pregibon. High-throughput,purification-free, multiplexed profiling of circulating miRNA for discovery,validation, and diagnostics. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1082.

Abstract 5047: Dysregulated microRNAs in cervical neoplasm

MicroRNAs (miRNAs) play an important role in a variety of physiological processes as well as pathophysiological processes, including cancinogenesis. This study was to identify a distinct miRNA expression signature for cervical intraepithelial neoplasia (CIN) and to reveal individual miRNAs that may be involved in the development of cervical carcinoma. Expression profiling using quantitative real-time RT-PCR of 202 miRNAs was performed on micro-dissected high-grade CIN (CIN 2/3) tissues as compared to normal cervical epithelium. The expression of 12 miRNAs including miR-518a, miR-34b, miR-34c, miR-20b, miR-338, miR-9, miR-512-5p, miR-424, miR-345, miR-10a, miR-193b and miR-203 were significantly different between high-grade CIN and normal epithelium. This miRNA signature was further validated by an independent set of high-grade CIN tissue. The same characteristic signature can also be used to distinguish cervical squamous cell carcinoma (SCC) from normal controls. Target prediction analysis revealed that these dysregulated miRNAs control the apoptosis signaling pathway and cell cycle regulation. These findings contribute to understanding of the pathogenesis of cervical carcinoma at the molecular level. Citation 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 5047. doi:1538-7445.AM2012-5047