Micro RNA-124a Regulates Lipolysis via Adipose Triglyceride Lipase and Comparative Gene Identification 58

Lipolysis is the biochemical pathway responsible for the catabolism of cellular triacylglycerol (TG). Lipolytic TG breakdown is a central metabolic process leading to the generation of free fatty acids (FA) and glycerol, thereby regulating lipid, as well as energy homeostasis. The precise tuning of lipolysis is imperative to prevent lipotoxicity, obesity, diabetes and other related metabolic disorders. Here, we present our finding that miR-124a attenuates RNA and protein expression of the major TG hydrolase, adipose triglyceride lipase (ATGL/PNPLA2) and its co-activator comparative gene identification 58 (CGI-58/ABHD5). Ectopic expression of miR-124a in adipocytes leads to reduced lipolysis and increased cellular TG accumulation. This phenotype, however, can be rescued by overexpression of truncated Atgl lacking its 3'UTR, which harbors the identified miR-124a target site. In addition, we observe a strong negative correlation between miR-124a and Atgl expression in various murine tissues. Moreover, miR-124a regulates the expression of Atgl and Cgi-58 in murine white adipose tissue during fasting as well as the expression of Atgl in murine liver, during fasting and re-feeding. Together, these results point to an instrumental role of miR-124a in the regulation of TG catabolism. Therefore, we suggest that miR-124a may be involved in the regulation of several cellular and organismal metabolic parameters, including lipid storage and plasma FA concentration

KRAS mutation analysis using blood derived cell free tumor DNA for treatment monitoring of metastatic colorectal cancer patients [Poster]

Abstract Currently used non-invasive diagnostic methods for monitoring patients undergoing anti-cancer treatment are limited. The applicability of diagnostic imaging is limited and lack of sensitivity and specificity of routinely used tumor markers hampers clinical application. Circulating cell free DNA (ccfDNA) has been shown to contain minute fractions of tumor-derived DNA. We tested in a proof of principal study the applicability of circulating mutant DNA as a tool to (1) evaluate the clinical sensitivity of cancer detection by analyzing KRAS2 mutations and (2) monitor therapy response in metastatic colorectal cancer (mCRC) patients. In our retrospective study we analyzed 50 serum samples of 18 patients with mCRC and confirmed KRAS2 mutation in the resected primary tumor (G12D, G12V, or G13D). Samples were collected at therapy start (t0), after four cycles of therapy (t1), which was on average after 8 weeks and if available, additional samples at later time points (t2, t3) to assess the longitudinal outcome (median follow up 23 weeks, range 8-248weeks). Therapy response and course of disease were assessed using RECIST criteria at all analyzed time points. The three most commonly occurring KRAS2 (G12D, G12V, or G13D) mutations were examined by Allele-specific PCR. The assay was established using defined tumor cell line DNA and its sensitivity proofed to be up to 0.1%. In 15/18 patients (83.3%), we could detect a KRAS2 mutation at start of therapy (t0) and could confirm diagnosis of mCRC by analysis of ccfDNA. In contrast only 8/18 (44.4%) patients and 7/18 (38.9%) patients showed increased levels of the tumor markers CEA and CA19-9, respectively. Therefore, the analysis of the KRAS2 status identified 5 additional patients as positive for colorectal cancer. To assess therapy response we quantified mutant KRAS2 levels in ccfDNA of 15 patients with detectable KRAS2 mutations. The changes of KRAS2 level were compared to the information from imaging, evaluated by RECIST criteria. A concordance in the assessment of therapy response after 4 cycles (t1) could be observed in 10/15 (66.7%) analyzed cases. In 12/15 patients the longitudinal outcome (t2, t3) was assessed and compared to t1. A concordance in the assessment of therapy response could be observed in 9/12 (75%) patients. Validation of data from patient sample analysis with Deep Sequencing is in progress.Our findings confirm the potential of ccfDNA as a biomarker for improved serum based relapse diagnosis and enables closed meshed monitoring of therapy response. A multiplex mutational analysis will be needed to translate this principle and include also other patient subpopulations (ie. KRAS2 negative). Therefore, we adapted the assay using whole genome amplification to amplify the limited amounts of ccfDNA that now allows the parallel testing of multiple mutations (work in progress). 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 4513. doi:1538-7445.AM2012-4513</jats:p

Comprehensive Analysis of miRNome Alterations in Response to Sorafenib Treatment in Colorectal Cancer Cells

MicroRNAs (miRNAs) are master regulators of drug resistance and have been previously proposed as potential biomarkers for the prediction of therapeutic response in colorectal cancer (CRC). Sorafenib, a multi-kinase inhibitor which has been approved for the treatment of liver, renal and thyroid cancer, is currently being studied as a monotherapy in selected molecular subtypes or in combination with other drugs in metastatic CRC. In this study, we explored sorafenib-induced cellular effects in Kirsten rat sarcoma viral oncogene homolog olog (KRAS) wild-type and KRAS-mutated CRC cell lines (Caco-2 and HRT-18), and finally profiled expression changes of specific miRNAs within the miRNome (&gt;1000 human miRNAs) after exposure to sorafenib. Overall, sorafenib induced a time- and dose-dependent growth-inhibitory effect through S-phase cell cycle arrest in KRAS wild-type and KRAS-mutated CRC cells. In HRT-18 cells, two human miRNAs (hsa-miR-597 and hsa-miR-720) and two small RNAs (SNORD 13 and hsa-miR-3182) were identified as specifically sorafenib-induced. In Caco-2 cells, nine human miRNAs (hsa-miR-3142, hsa-miR-20a, hsa-miR-4301, hsa-miR-1290, hsa-miR-4286, hsa-miR-3182, hsa-miR-3142, hsa-miR-1246 and hsa-miR-720) were identified to be differentially regulated post sorafenib treatment. In conclusion, we confirmed sorafenib as a potential anti-neoplastic treatment strategy for CRC cells by demonstrating a growth-inhibitory and cell cycle–arresting effect of this drug. Changes in the miRNome indicate that some specific miRNAs might be relevant as indicators for sorafenib response, drug resistance and potential targets for combinatorial miRNA-based drug strategies