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MicroRNAs (miRNAs) are a novel class of small noncoding RNAs that modulate the expression of genes at the posttranscriptional level. These small molecules have been shown to be involved in cancer, apoptosis, and cell metabolism. In the present study we provide an informative profile of the expression of miRNAs in primary chronic lymphocytic leukemia (CLL) cells using 2 independent and quantitative methods: miRNA cloning and quantitative real-time–polymerase chain reaction (qRT-PCR) of mature miRNAs. Both approaches show that miR-21 and miR-155 are dramatically overexpressed in patients with CLL, although the corresponding genomic loci are not amplified. miR-150 and miR-92 are also significantly deregulated in patients with CLL. In addition, we detected a marked miR-15a and miR-16 decrease in about 11% of cases. Finally, we identified a set of miRNAs whose expression correlates with biologic parameters of prognostic relevance, particularly with the mutational status of the IgVH genes. In summary, the results of this study offer for the first time a comprehensive and quantitative profile of miRNA expression in CLL and their healthy counterpart, suggesting that miRNAs could play a primary role in the disease itself.

Background DNA damage transactivates tumour protein p53 (TP53) -regulated surveillance, crucial in suppressing tumorigenesis. TP53 mediates this process directly by transcriptionally modulating gene and microRNA (miRNA) expression and indirectly by regulating miRNA biogenesis. However, the role of TP53 in regulating miRNA-AGO2 loading and global changes in AGO2 binding to its gene targets in response to DNA damage are unknown. These processes might be novel mechanisms by which TP53 regulates miRNAs in response to DNA damage. Methods To show the network of miRNA–mRNA interactions that occur in response to DNA damage, we stimulated TP53 wild-type and null cell-lines with doxorubicin and performed RNA sequencing from total RNA (RNA-Seq) and AGO2-immunoprecipitated RNA (AGO2-RIP-Seq). We used a combined AGO2 RIP-seq and AGO2 PAR-CLIP-seq (photoactivatable-ribonucleoside-enhanced cross-linking and immunoprecipitation) approach to determine the exact sites of interaction between the AGO2-bound miRNAs and their mRNA targets. Findings TP53 directly associated with AGO2, and induced and reduced loading of a subset of miRNAs, including the lethal 7 (let-7) miRNA family members, onto AGO2 in response to DNA damage. Although mutated TP53 maintained its capacity to interact with AGO2, it mediated unloading instead of loading of let-7 family miRNAs, thereby reducing their activity. We determined the miRNA–mRNA interaction networks involved in the response to DNA damage both in the presence and absence of TP53 . Furthermore, we showed that miRNAs whose cellular abundance or differential loading onto AGO2 was regulated by TP53 were involved in an intricate network of regulatory feedback and feedforward circuits that fine-tuned gene expression levels in response to DNA damage to permit the repair of DNA damage or initiation of programmed cell death. Interpretation Control of AGO2 loading by TP53 is a new mechanism of miRNA regulation in carcinogenesis. Funding UK Medical Research Council, Action Against Cancer.

Background MicroRNAs (miRNAs) are small non-coding RNAs involved in the post-transcriptional regulation of mRNAs and are aberrantly expressed in cancer with important roles in tumorigenesis. A broad analysis of the combined effects of altered activities of miRNAs in pancreatic ductal adenocarcinoma (PDAC) has not been done, and how miRNAs might affect tumour progression or patient outcomes is unclear. Methods We combined data from miRNA and mRNA expression profiles from PDAC and normal pancreas samples (each n=9) and used bioinformatic analyses to identify a miRNA–mRNA regulatory network in PDAC. We validated our findings in PDAC cell-lines (PANC-1, MIA PaCa-2, LPc006, and LPc167), subcutaneous PDAC xenografts in mice, and laser capture microdissected PDACs from patients (n=91). We used this information to identify miRNAs that contributed most to tumorigenesis. Findings We identified three miRNAs (miR-21, miR-23a, and miR-27a) that acted as cooperative repressors of a network of tumour suppressor genes that included PDCD4, BTG2 , and NEDD4L . Inhibition of miR-21, miR-23a, and miR-27a had synergistic effects in reducing proliferation of PDAC cells in culture and the growth of xenograft tumours. The level of inhibition was greater than that of silencing oncomiR-21 alone. In PDACs from patients, high levels of the combination of miR-21, miR-23a, and miR-27a was a strong independent predictor of short overall survival after surgical resection (hazard ratio 3·21, 95% CI 1·78–5·78). High expression of this combination was also associated with a more aggressive tumour phenotype: more microscopic tumour infiltration at resection margin and increased perineural invasion. Interpretation In an integrated data analysis, we identified functional miRNA–mRNA interactions that contribute to PDAC growth. These findings indicate that miRNAs act together to promote tumour progression and that future therapeutic strategies might require inhibition of several miRNAs. Furthermore, high tumour expression of the miR-21, miR-23a, and miR-27a combination could have potential use in the future as a prognostic signature for patients with PDAC. Funding Peel Medical Research Trust, Alliance Family Foundation, Action Against Cancer, National Institute for Health Research, Association for International Cancer Research, Jason Boas Fellowship, Imperial Biomedical Research Centre, Rosetrees Trust, Joseph Ettedgui Charitable Foundation.

INTRODUCTION: LC-MS/MS proteomics is an essential technology to help unravel the complex molecular events that lead to and propagate cancer. We have developed an analytical workflow to quantify the expression and phosphorylation status of thousands of proteins simultaneously in frozen resected human pancreatic tumor (T), relative to matched non-tumor (NT) tissue. These measurements enabled us to determine activity of anti-neoplastic drug targets and other signaling proteins. METHODS: Peptides resulting from tryptic digestion of proteins extracted from frozen tissue of pancreatic ductal adenocarcinoma and background pancreas (n=12), were labelled with tandem mass tags (TMT 8-plex), separated by strong cation exchange chromatography, then were analysed by LC-MS/MS directly or first enriched for phosphopeptides using Fe3+ and TiO2, prior to analysis. In-house, commercial and freeware bio-informatics platforms were used to identify relevant biological events from the complex dataset. RESULTS: Of 2,101 proteins identified, 152 demonstrated significant difference in expression between tumor and non-tumor tissue. They included proteins that are known to be up-regulated in pancreatic cancer (e.g. Mucin-1), but the majority were new candidate markers such as homeodomain interacting protein kinase 1 & Myosin light chain kinase. Of the 6,543 unique phosphopeptides identified (6,284 unique phosphorylation sites), 635 showed significant regulation, particularly those from proteins involved in cell migration (Rho GTPase signaling proteins such as Rho guanine nucleotide exchange factors & Serine/threonine-protein kinase MRCKα) as well as proteins involved in disassembly of cell-cell junctions (tight junction, adherens junction) and formation of cell-extracellular matrix (ECM) junctions (focal adhesions). We quantified activator & inhibitory phosphorylation sites on FYN, AKT1, HDAC1&2, GSK3α&β, RAF kinases, MAPKs (p38, ERK1&2), PKCs, Casein Kinases and >20 others, as well as their downstream substrates some of which were often found to be highly modulated (≥ 2 fold) in T versus NT in different cases. CONCLUSION: Application of our LC-MS/MS proteomic workflow to frozen resected human pancreatic T versus NT tissue elucidated molecular events likely contributing to pancreatic cancer in each case, particularly those contributing to cell migration, and in future may help clinicians predict the best targeted anti-cancer therapy bespoke for an individual patient. Citation Format: David Britton, Yoh Zen, Stefan Selzer, Vikram Mitra, Alberto Quaglia, Debashis Sarker, Leandro Castellano, Justin Stebbing, Julia Gee, Rob Nicholson, Nigel Heaton, Ian Pike. Quantification of pancreatic cancer proteome & phosphorylome: Indicates molecular events likely contributing to cancer & activation status of drug targets. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1617. doi:10.1158/1538-7445.AM2014-1617

Aim: Novel molecular biomarkers are needed in neuroendocrine tumours (NET) which can better stratify patients based on factors such as disease aggressiveness and treatment response. Our study was performed on small bowel neuroendocrine tumours (SBNET). Aim 1: determine the global microRNA (miRNA) profile of SBNET. Aim 2: identify miRNA involved in tumour progression for use used as potential biomarkers. Methods: Forty four samples were studied with matched primary/metastasis/normal formalin fixed paraffin embedded tissue from 15 SBNET patients. Included were primary SBNET (n = 15), lymph node metastases (n = 9), adjacent normal small bowel (n = 13) and normal lymph nodes (n = 7). RNA was extracted and 800 miRNA quantified using nCounter® technology (NanoString Technologies, Seattle, USA). Normalisation was performed using the geometric mean of the 100 highest expressed miRNA followed by quantile normalisation. Two-sample t test (p value Results: Out of 800 miRNA, 178 were significantly up or downregulated in SBNET compared to adjacent non-neoplastic tissue. The most up regulated miRNA (-204-5p, -7-5p, -375) and the most downregulated miRNA (-215, -451a -378a-3p, -378i) were chosen for future validation as biomarkers of progression. There were 3 miRNA (-1, -1233, 143-3p) significantly downregulated in lymph node metastasis compared to the primary. One miRNA (-2117) was significantly downregulated in lymph node metastasis compared to normal lymph nodes. Conclusions: These preliminary results provide information on the global miRNA profile of SBNET and show that some miRNA are differentially regulated during tumour progression. Following further validation, these miRNA have the potential to act as biomarkers for patient stratification based on disease aggressiveness, which could inform patient treatment. Disclosure: All authors have declared no conflicts of interest.

Rationale: The use of anthracyclines such as doxorubicin (DOX) has improved mortality and morbidity in cancer patients, yet associated risks of cardiomyopathy have limited their clinical application. DOX-associated cardiotoxicity is frequently irreversible and typically progresses to heart failure (HF). However, our understanding of molecular mechanisms essential for development of cardioprotective strategies and to predict HF remain largely obscure. Objective: As microRNAs (miRNAs) have been shown to play potent regulatory roles in both cardiovascular disease and cancer, we investigated miRNA changes in chemotherapy-induced HF and the alteration of cellular processes downstream. Methods and results: Myocardial miRNA profiling was performed after DOX-induced injury, either via acute application to isolated cardiomyocytes in vitro or chronic exposure in vivo, and also compared with miRNA profiles from remodeled hearts following myocardial infarction; the miR-30 family was down-regulated in all three models. We confirmed β1- and β2-adrenoceptors (β1AR, β2AR), Gi alpha 2 (Giα-2) and the pro-apoptotic gene BNIP3L/NIX as miR-30 targets. We describe regulation of the β-adrenergic pathway by miR-30, where a preferential βAR inhibition results in a β-blocker like effect. We demonstrate that high miR-30 levels are protective against DOX insult and correlate with lower reactive oxygen species (ROS) generation. Moreover, we identify GATA-6 as a mediator of the DOX-associated reduction in miR-30 expression. Conclusion: DOX causes acute and sustained miR-30 down-regulation in cardiomyocytes via GATA-6. miR-30 overexpression protected cardiac cells from DOX-induced apoptosis, and its maintenance represents a potential cardioprotective strategy for anthracycline cardiomyopathy. Additionally, intriguing synergic anti-cancer effects were observed for miR-30.

s / Pancreatolog S84 Background: The circulating tumour cells (CTCs) responsible for metastasis in pancreatic cancer (PDAC) are heterogeneous; tailoring therapy based on their molecular profile could permit personalised therapy and improve survival. Aims: Optimise whole blood enrichment to allow next generation sequencing (NGS) of CTCs in PDAC. Materials & methods: An experimental sample, 10 Panc-1 cells spiked into 7.5mls of whole blood, and a clinical sample, 7.5mls of whole blood from a patient with advanced PDAC were used. Streptavidin coated magnetic beads were chemically bonded with CD45 antibodies specific to leucocytes and thoroughly mix with the samples. Leukocytes were removed with a magnet. DNA was isolated from the supernatant using Roche Magnupure. Libraries were diluted to 10 genomes and multiple independent barcoded assays were run per analysis. Barcoded primers for exons 5,6,7 and 8 of Tp53 were used for PCR amplification. Deep sequencing of Tp53 was performed with ion torrent NGS. 10% mutation sequence in one of 100 barcodes was taken as evidence of 0.1% mutation frequency. The limiting dilution removes the possibility of low level PCR error being confused with genuine mutation. Results: The known p.R273H mutation in Panc-1 cells was identified at a frequency 2% in the spiked sample. Amutation p.E271Vwas identified at a frequency 3.4% in the processed sample but was undetectable (

Circulating tumor cells (CTCs) disseminate from the primary tumor and travel through the bloodstream and lymphatic system. The detection of and/or increase in the number of CTCs during a patient’s clinical course may be a harbinger of forthcoming overt metastasis. We aimed to examine the impact of 2 different surgical techniques, standard (ST) pancreaticoduodenectomy (PD) and no-touch isolation (NT) PD, on tumor behavior and outcome in patients with pancreatic cancer by using CTCs as biomarkers. In this pilot study, patients were randomized to either ST-PD (n = 6) or NT-PD (n = 6). Intraoperatively, blood samples were taken from the portal vein for measurement of CTCs before and immediately after removal of the tumor. An increase in CTCs was seen in 5 of 6 patients (83%) with ST-PD but no patients with NT-PD (P = .003). In the ST-PD and NT-PD groups, median overall survival was 13.0 and 16.7 months, respectively (P = .33); there was no difference in disease-free survival (P = .42). The use of NT-PD significantly reduced the number of CTCs in the portal vein with no benefit in survival outcomes compared with ST-PD, although more extensive studies are required.

Introduction MicroRNAs (miRNAs) are short, non-coding RNAs with a key role in post-transcriptional regulation, and regulate multiple pathways of tumorogenesis. Expression profiling of malignant cell lines, fresh and formalin-fixed tumour have identified potential miRNA signatures for pancreatic ductal adenocarcinoma (PDAC), and RNA deep sequencing has demonstrated that miRNAs can be quantified from bile, in bilary tract cancers 1 . Here, we report the first study of biliary miRNAs in PDAC. Methods 3 target miRNAs known to be overexpressed in PDAC were selected. Bile was collected at endoscopic retrograde cholangiopancreatography (ERCP) from patients with PDAC (n = 10) or gallstones (n = 8; used as a benign control). Bile was prepared as described1 and total RNA isolated using TRIzol (Invitrogen, Paisley, UK). Quantitative real-time reverse-transcription polymerase chain reaction (RT-qPCR) was performed using Taqman mature miRNA primers and probes (Applied Biosystems, Cheshire, UK). Expression of oncomiR-21, miR-155 and miR-106a was measured. Cycle passing threshold (Ct) was recorded and normalised to RNU6B expression. Relative expression was calculated as 2Ct_miRNA-Ct_RNU6B. PCR reactions were carried out in duplicate. Results miR-106a and oncomiR-21 were both highly expressed in the bile of patients with PDAC, compared to those with gallstones. miR-155 was not significantly upregulated in PDAC bile. Using miR-106a (cut-off level > 8.02), we discriminated PDAC from benign disease with a sensitivity and specificity of 80% [95% CI 44–98] and 100% [95% CI 63–100], respectively with an area under the curve (AUC) value of 0.88. OncomiR-21 was also upregulated in bile, but was not as reliable in discriminating diagnosis. Conclusion This study demonstrates that bile miRNAs are well-suited analytes for the development of sensitive and specific tests in PDAC. Although tumour breach of the biliary epithelium was not always suspected in our cohort from clinical investigations, malignancy-specific miRNAs were found intraluminally. Of the 3 miRNAs tested, ROC analysis identified miR-106a as the best potential PDAC biomarker, with reliable diagnostic specificity and sensitivity. However, further study may also identify miRNAs that also discriminate prognoses and response to therapy, guiding individualised treatment. Disclosure of Interest None Declared Reference Shigehara K, Yokomuro S, Ishibashi O, et al. Real-time PCR-based analysis of the human bile microRNAome identifies miR-9 as a potential diagnostic biomarker for biliary tract cancer. PloS one. 2011; 6(8):e23584.

Introduction Cholangiocarcinoma (CCA) is a primary biliary ductal cancer which is often difficult to diagnose and which carries a poor prognosis. New diagnostic tests are urgently needed to improve patient outcome. Bile is a rich source of potential novel biomarkers for CCA, due to its intimate proximity to the malignant lesion. However, there are no biliary biomarkers for CCA currently available. MicroRNAs (miRNAs) are key post-transcriptional regulators, and influence tumorogenesis. Studies on cell lines and tissue have identified several potential miRNA signatures for CCA, and recently miR-9 was identified as elevated in bile from CCA compared with benign disease 1 . In this study we aimed to measure specific miRNA expression in bile from patients with CCA, gallstone disease and pancreatic adenocarcinoma (PA) and to assess their performance in differentiating these causes of biliary obstruction. Methods Bile was collected at endoscopic retrograde cholangiopancreatography (ERCP) from patients with CCA (n = 6), PA (n = 10) and gallstones (n = 8; benign control). Bile was prepared as previously described1 and total RNA was isolated using TRIzol (Invitrogen, Paisley, UK). Quantitative real-time reverse-transcription polymerase chain reaction (RT-qPCR) was performed using Taqman mature miRNA primers and probes (Applied Biosystems, Cheshire, UK). Expression of oncomiR-21, miR-155 and miR-106a was measured. Cycle passing threshold (Ct) was recorded and normalised to RNU6B expression. Relative expression was calculated as 2Ct_miRNA-Ct_RNU6B. PCR reactions were carried out in duplicate. Results MiR-106a and oncomiR-21 were highly expressed in bile from patients with CCA compared to those with gallstones. MiR-155 was not significantly upregulated in malignancy. In discriminating CCA from benign disease, miR-106a had sensitivity and specificity of 83.3% [95% CI 36–100] and 88% [95% CI 47–100] respectively, with an AUC of 0.83 (cut-off level > 6.05). OncomiR-21 was also upregulated in the bile of both tumour types, but was not as reliable. Neither miRNA discriminated significantly between PA and CCA. Conclusion MiRNA can be reliably isolated from bile pellets. Both miR-106a and oncomiR-21 are potential novel biliary biomarkers for CCA, and could improve differentiation of benign from malignant disease at ERCP. Further work is required to validate these findings in a larger cohort and against other disease groups, and to investigate possible correlations between miRNA profile and disease course. Disclosure of Interest None Declared Reference Shigehara K, Yokomuro S, Ishibashi O, et al. Real-time PCR-based analysis of the human bile microRNAome identifies miR-9 as a potential diagnostic biomarker for biliary tract cancer. PloS one . 2011; 6(8):e23584.

Introduction: The translation of genomic pre-disposition into a disease phenotype is driven primarily by protein expression and a complex network of post-translational modifications (PTM). One such PTM, phosphorylation, affords exquisite regulation of protein function by modulating protein folding, substrate affinity, stability, and activity and is proven to modulate the activity of thousands of onco- and tumor suppressor proteins. Phosphorylation-induced dysregulation of key checkpoints in signaling pathways that drive cell division, proliferation, differentiation, and apoptosis contributes to the cancer phenotype. A priori knowledge of which phosphorylation events are driving a particular tumor will assist in drug target validation, compound selection and ultimately treatment optimization for individual patients. We have developed a liquid chromatography mass spectrometry proteomics approach to monitor the activity of these signaling pathways in cancer cells. Experimental procedure: Peptides resulting from tryptic digestion of proteins from a human breast cancer cell line (BT474 +/− lapatinib, heregulin, lapatinib+heregulin), were labeled with tandem mass tags (TMT®) and passed through phospho-peptide enrichment columns (IMAC or TiO2). The enriched phospho-peptides were then analyzed by LC-MS/MS to identify and quantify the level of protein phosphorylation in each sample relative to a spiked internal reference sample. Data summary: In total we identified 4046 phosphorylation sites on 1054 phospho-proteins in BT474 cells under the various treatment conditions stated above. Lapatinib and heregulin were shown to significantly modulate phosphorylation on key proteins involved in 32 KEGG signaling pathways, including: the pancreatic cancer pathway (11 phospho-proteins), cell cycle pathway (18 phospho-proteins) and the ERBB signaling pathway, where we observed modulation of phosphorylation sites on 11 phospho-proteins including ERBB2 (pT701, pS703, pY877, pS1050/1051, pS1054, pS1083, pS1107, pS1166, pY1248 and 4 additional novel sites), AKT1 (pS124, pS126, pS129, pS137), and GSK3B (pS9, pY216 and an additional novel site). Conclusion: The global analysis of site-specific phosphorylation on key signaling pathways relevant to a specific cancer phenotype has been developed. Our initial data provide further insight into the on- and off-target effects of the ERBB1/ERBB2 tyrosine kinase inhibitor lapatinib. These data may help define optimal combination treatments in ERBB1/ERBB2 positive tumors. The application of this method to other tumor types and therapeutic agents is widely recommended. 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 LB-8. doi:1538-7445.AM2012-LB-8

e14579 Background: Short non-coding microRNAs (miRNAs) play a role in post-transcriptional gene regulation and can contribute to cancer development and progression. Expression profiles can distingu...

Uncontrolled cell proliferation and cytoskeletal remodeling are responsible for tumor development and ultimately metastasis. A number of studies have implicated microRNAs in the regulation of cancer cell invasion and migration. Here, we show that miR-23b regulates focal adhesion, cell spreading, cell-cell junctions and the formation of lamellipodia in breast cancer (BC), implicating a central role for it in cytoskeletal dynamics. Inhibition of miR-23b, using a specific sponge construct, leads to an increase of cell migration and metastatic spread in vivo, indicating it as a metastatic suppressor microRNA. Clinically, low miR-23b expression correlates with the development of metastases in BC patients. Mechanistically, miR-23b is able to directly inhibit a number of genes implicated in cytoskeletal remodeling in BC cells. Through intracellular signal transduction, growth factors activate the transcription factor AP-1, and we show that this in turn reduces miR-23b levels by direct binding to its promoter, releasing the pro-invasive genes from translational inhibition. In aggregate, miR-23b expression invokes a sophisticated interaction network that co-ordinates a wide range of cellular responses required to alter the cytoskeleton during cancer cell motility.

There has been very little progress in improving outcomes for patients with pancreatic ductal adenocarcinoma (PDAC) over the past few decades. High-throughput array profiling has made it possible to discover new assays to diagnose or prognose PDAC more accurately based on the genetic profile of an individual tumor. To improve patient survival, there is a need to extract the most practical data to define tumor subgroups and personalize anticancer therapy. In the evaluated study, a multiplatform, survival-based analysis of molecular changes was performed for PDAC to discover clinically useful biomarkers. A composite score predictive for survival was calculated for individual genes, taking into account the DNA copy-number and any regulation by miRNAs. Several genes involved in the PI3K/AKT and SRC signaling pathways were identified and further investigated.