Calorie restriction in humans inhibits the PI3K/AKT pathway and induces a younger transcription profile

Caloric restriction (CR) and down-regulation of the insulin/IGF pathway are the most robust interventions known to increase longevity in lower organisms. However, little is known about the molecular adaptations induced by CR in humans. Here, we report that long-term CR in humans inhibits the IGF-1/insulin pathway in skeletal muscle, a key metabolic tissue. We also demonstrate that CR induces dramatic changes of the skeletal muscle transcriptional profile that resemble those of younger individuals. Finally, in both rats and humans, CR evoked similar responses in the transcriptional profiles of skeletal muscle. This common signature consisted of three key pathways typically associated with longevity: IGF-1/insulin signaling, mitochondrial biogenesis, and inflammation. Furthermore, our data identify promising pathways for therapeutic targets to combat age-related diseases and promote health in humans.American Federation for Aging ResearchNational Center for Research Resources (U.S.) (Grant UL1 RR024992)National Institute of Diabetes and Digestive and Kidney Diseases (U.S.) (Grant P30DK056341

Integrated Proteomic Analysis of Human Cancer Cells and Plasma from Tumor Bearing Mice for Ovarian Cancer Biomarker Discovery

Background: The complexity of the human plasma proteome represents a substantial challenge for biomarker discovery. Proteomic analysis of genetically engineered mouse models of cancer and isolated cancer cells and cell lines provide alternative methods for identification of potential cancer markers that would be detectable in human blood using sensitive assays. The goal of this work is to evaluate the utility of an integrative strategy using these two approaches for biomarker discovery. Methodology/Principal Findings: We investigated a strategy that combined quantitative plasma proteomics of an ovarian cancer mouse model with analysis of proteins secreted or shed by human ovarian cancer cells. Of 106 plasma proteins identified with increased levels in tumor bearing mice, 58 were also secreted or shed from ovarian cancer cells. The remainder consisted primarily of host-response proteins. Of 25 proteins identified in the study that were assayed, 8 mostly secreted proteins common to mouse plasma and human cancer cells were significantly upregulated in a set of plasmas from ovarian cancer patients. Five of the eight proteins were confirmed to be upregulated in a second independent set of ovarian cancer plasmas, including in early stage disease. Conclusions/Significance: Integrated proteomic analysis of cancer mouse models and human cancer cell populations provides an effective approach to identify potential circulating protein biomarkers

“Topological Significance” Analysis of Gene Expression and Proteomic Profiles from Prostate Cancer Cells Reveals Key Mechanisms of Androgen Response

The problem of prostate cancer progression to androgen independence has been extensively studied. Several studies systematically analyzed gene expression profiles in the context of biological networks and pathways, uncovering novel aspects of prostate cancer. Despite significant research efforts, the mechanisms underlying tumor progression are poorly understood. We applied a novel approach to reconstruct system-wide molecular events following stimulation of LNCaP prostate cancer cells with synthetic androgen and to identify potential mechanisms of androgen-independent progression of prostate cancer.We have performed concurrent measurements of gene expression and protein levels following the treatment using microarrays and iTRAQ proteomics. Sets of up-regulated genes and proteins were analyzed using our novel concept of "topological significance". This method combines high-throughput molecular data with the global network of protein interactions to identify nodes which occupy significant network positions with respect to differentially expressed genes or proteins. Our analysis identified the network of growth factor regulation of cell cycle as the main response module for androgen treatment in LNCap cells. We show that the majority of signaling nodes in this network occupy significant positions with respect to the observed gene expression and proteomic profiles elicited by androgen stimulus. Our results further indicate that growth factor signaling probably represents a "second phase" response, not directly dependent on the initial androgen stimulus.We conclude that in prostate cancer cells the proliferative signals are likely to be transmitted from multiple growth factor receptors by a multitude of signaling pathways converging on several key regulators of cell proliferation such as c-Myc, Cyclin D and CREB1. Moreover, these pathways are not isolated but constitute an interconnected network module containing many alternative routes from inputs to outputs. If the whole network is involved, a precisely formulated combination therapy may be required to fight the tumor growth effectively

Abstract 3045: Identification of specific microRNA signatures in VHL-functional and VHL-silenced renal cell carcinoma cell lines

Abstract Objective&amp; Background: Renal cell carcinoma is the most common renal malignancy in adults. Up to 70% of clear cell renal cancers have VHL silencing either because of gene mutation or methylation. MicroRNAs are a class of small non-coding RNAs that control gene expression by targeting mRNAs and trigger either translation repression or RNA degradation. Their aberrant expression may be involved in human diseases including cancer. This study set out to investigate the influences of VHL status and the ensuing differences in miRNA profiles and biological/phenotypic effects in renal carcinoma cell lines. Methods: Normal cell line, HREC, and renal cancer cell lines that are VHL-ve cell lines, A498, A704, 786.O, and 786.O. Vec, VHL+ve cell line,786.O.VHL, VHL-WT cell line, ACHN and Caki.1 and VHL-CH3 cell line, KV6 were used for the VHL-specific genome wide miRNA profiling. miRNAs were extracted, in triplicate, from the above cell lines at 48 hrs time point by using miRVana miRNA isolation kit (Ambion). The extracted miRNAs were subjected to analysis by MEGAPlex RT primers and MEGAPlex Pre-Amp primers, followed by loading them to TaqMan Human miRNA microarrays v2.0 (Human Array A/B (Applied Biosystems). Resulting miRNAs were analysed further by using the RealTimeStatMiner v3.0 software (Integromics) followed by GeneGO pathways analysis. Results: We show that approximately 577 miRNAs were altered in human kidney cancer. 270 miRNAs and 180 miRNAs were upregulated and down-regulated in cancer cell lines when compared against normal HREC cell line, respectively. Upon analysis of differentially expressed miRNAs in RCC cell lines based on VHL status, we found that 7 miRNAs were differentially upregulated in VHL wild type RCC cells. In VHL mutant cell lines, 5 miRNAs and 6 miRNAs were differentially up and down regulated, respectively, while in RCC cell lines with wild type, but silenced (methylated) VHL, 7 and 10 miRNAs were differentially up and down regulated, respectively. Target prediction and microRNA enrichment analysis suggested that the differentially expressed miRNAs are involved in regulation of HIF, angiogenesis, invasion &amp; metastasis, extracellular matrix degradation, EMT phenomenon, metabolism and apoptosis pathways. Conclusion: This MicroRNome analysis reveals a specific spectrum of hypoxia, angiogenesis, tumor metabolism, metastasis, cell cycle and apoptotic specific miRNAs. In vitro experiments to characterize the biological significance differential miRNA expression profiles on different steps of tumorigenesis and tumor progression are underway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3045.</jats:p