Elucidation of Focal Adhesion Kinase as a Modulator of Migration and Invasion and as a Potential Therapeutic Target in Chronic Lymphocytic Leukemia.

The retention and re-migration of Chronic Lymphocytic Leukemia cells into cytoprotective and proliferative lymphoid niches is thought to contribute to the development of resistance, leading to subsequent disease relapse. The aim of this study was to elucidate the molecular processes that govern CLL cell migration to elicit a more complete inhibition of tumor cell migration. We compared the phenotypic and transcriptional changes induced in CLL cells using two distinct models designed to recapitulate the peripheral circulation, CLL cell migration across an endothelial barrier, and the lymph node interaction between CLL cells and activated T cells. Initially, CLL cells were co-cultured with CD40L-expressing fibroblasts and exhibited an activated B-cell phenotype, and their transcriptional signatures demonstrated the upregulation of pro-survival and anti-apoptotic genes and overrepresentation of the NF-κB signaling pathway. Using our dynamic circulating model, we were able to study the transcriptomics and miRNomics associated with CLL migration. More than 3000 genes were altered when CLL cells underwent transendothelial migration, with an overrepresentation of adhesion and cell migration gene sets. From this analysis, an upregulation of the FAK signaling pathway was observed. Importantly, PTK2 (FAK) gene expression was significantly upregulated in migrating CLL cells (PTK2 Fold-change = 4.9). Here we demonstrate that TLR9 agonism increased levels of p-FAK (p ≤ 0.05), which could be prevented by pharmacological inhibition of FAK with defactinib (p ≤ 0.01). Furthermore, a reduction in CLL cell migration and invasion was observed when FAK was inhibited (p ≤ 0.0001), supporting a role for FAK in both CLL migration and tissue invasion. When taken together, our data highlights the potential for combining FAK inhibition with current targeted therapies as a more effective treatment regime for CLL.

Stress hormone-mediated acceleration of breast cancer metastasis is halted by inhibition of nitric oxide synthase.

Stress hormones have been shown to be important mediators in driving malignant growth and reducing treatment efficacy in breast cancer. Glucocorticoids can induce DNA damage through an inducible nitric oxide synthase (iNOS) mediated pathway to increase levels of nitric oxide (NO). Using an immune competent mouse breast cancer model and 66CL4 breast cancer cells we identified a novel role of NOS inhibition to reduce stress-induced breast cancer metastasis. On a mechanistic level we show that the glucocorticoid cortisol induces expression of keys genes associated with angiogenesis, as well as pro-tumourigenic immunomodulation. Transcriptomics analysis confirmed that in the lungs of tumour-bearing mice, stress significantly enriched pathways associated with tumourigenesis, some of which could be regulated with NOS inhibition. These results demonstrate the detrimental involvement of NOS in stress hormone signalling, and the potential future benefits of NOS inhibition in highly stressed patients.

Disruption of Macrodomain Protein SCO6735 Increases Antibiotic Production in Streptomyces coelicolor.

ADP-ribosylation is a post-translational modification that can alter the physical and chemical properties of target proteins and that controls many important cellular processes. Macrodomains are evolutionarily conserved structural domains that bind ADP-ribose derivatives and are found in proteins with diverse cellular functions. Some proteins from the macrodomain family can hydrolyze ADP-ribosylated substrates and therefore reverse this post-translational modification. Bacteria and Streptomyces, in particular, are known to utilize protein ADP-ribosylation, yet very little is known about their enzymes that synthesize and remove this modification. We have determined the crystal structure and characterized, both biochemically and functionally, the macrodomain protein SCO6735 from Streptomyces coelicolor This protein is a member of an uncharacterized subfamily of macrodomain proteins. Its crystal structure revealed a highly conserved macrodomain fold. We showed that SCO6735 possesses the ability to hydrolyze PARP-dependent protein ADP-ribosylation. Furthermore, we showed that expression of this protein is induced upon DNA damage and that deletion of this protein in S. coelicolor increases antibiotic production. Our results provide the first insights into the molecular basis of its action and impact on Streptomyces metabolism.

Exploiting human and mouse transcriptomic data: Identification of circadian genes and pathways influencing health.

The power of the application of bioinformatics across multiple publicly available transcriptomic data sets was explored. Using 19 human and mouse circadian transcriptomic data sets, we found that NR1D1 and NR1D2 which encode heme-responsive nuclear receptors are the most rhythmic transcripts across sleep conditions and tissues suggesting that they are at the core of circadian rhythm generation. Analyzes of human transcriptomic data show that a core set of transcripts related to processes including immune function, glucocorticoid signalling, and lipid metabolism is rhythmically expressed independently of the sleep-wake cycle. We also identify key transcripts associated with transcription and translation that are disrupted by sleep manipulations, and through network analysis identify putative mechanisms underlying the adverse health outcomes associated with sleep disruption, such as diabetes and cancer. Comparative bioinformatics applied to existing and future data sets will be a powerful tool for the identification of core circadian- and sleep-dependent molecules.

The ROK family regulator Rok7B7 pleiotropically affects xylose utilization, carbon catabolite repression, and antibiotic production in streptomyces coelicolor.

Members of the ROK family of proteins are mostly transcriptional regulators and kinases that generally relate to the control of primary metabolism, whereby its member glucose kinase acts as the central control protein in carbon control in Streptomyces. Here, we show that deletion of SCO6008 (rok7B7) strongly affects carbon catabolite repression (CCR), growth, and antibiotic production in Streptomyces coelicolor. Deletion of SCO7543 also affected antibiotic production, while no major changes were observed after deletion of the rok family genes SCO0794, SCO1060, SCO2846, SCO6566, or SCO6600. Global expression profiling of the rok7B7 mutant by proteomics and microarray analysis revealed strong upregulation of the xylose transporter operon xylFGH, which lies immediately downstream of rok7B7, consistent with the improved growth and delayed development of the mutant on xylose. The enhanced CCR, which was especially obvious on rich or xylose-containing media, correlated with elevated expression of glucose kinase and of the glucose transporter GlcP. In liquid-grown cultures, expression of the biosynthetic enzymes for production of prodigionines, siderophores, and calcium-dependent antibiotic (CDA) was enhanced in the mutant, and overproduction of prodigionines was corroborated by matrix-assisted laser desorption ionization-time-of-flight analysis. These data present Rok7B7 as a pleiotropic regulator of growth, CCR, and antibiotic production in Streptomyces.

Metabolic and evolutionary insights into the closely-related species Streptomyces coelicolor and Streptomyces lividans deduced from high-resolution comparative genomic hybridization.

BACKGROUND: Whilst being closely related to the model actinomycete Streptomyces coelicolor A3(2), S. lividans 66 differs from it in several significant and phenotypically observable ways, including antibiotic production. Previous comparative gene hybridization studies investigating such differences have used low-density (one probe per gene) PCR-based spotted arrays. Here we use new experimentally optimised 104,000 × 60-mer probe arrays to characterize in detail the genomic differences between wild-type S. lividans 66, a derivative industrial strain, TK24, and S. coelicolor M145. RESULTS: The high coverage and specificity (detection of three nucleotide differences) of the new microarrays used has highlighted the macroscopic genomic differences between two S. lividans strains and S. coelicolor. In a series of case studies we have validated the microarray and have identified subtle changes in genomic structure which occur in the Asp-activating adenylation domains of CDA non-ribosomal peptide synthetase genes which provides evidence of gene shuffling between these domains. We also identify single nucleotide sequence inter-species differences which exist in the actinorhodin biosynthetic gene cluster. As the glyoxylate bypass is non-functional in both S. lividans strains due to the absence of the gene encoding isocitrate lyase it is likely that the ethylmalonyl-CoA pathway functions as the alternative mechanism for the assimilation of C2 compounds. CONCLUSIONS: This study provides evidence for widespread genetic recombination, rather than it being focussed at 'hotspots', suggesting that the previously proposed 'archipelago model' of genomic differences between S. coelicolor and S. lividans is unduly simplistic. The two S. lividans strains investigated differ considerably in genetic complement, with TK24 lacking 175 more genes than its wild-type parent when compared to S. coelicolor. Additionally, we confirm the presence of bldB in S. lividans and deduce that S. lividans 66 and TK24, both deficient in the glyoxylate bypass, possess an alternative metabolic mechanism for the assimilation of C2 compounds. Given that streptomycetes generally display high genetic instability it is envisaged that these high-density arrays will find application for rapid assessment of genome content (particularly amplifications/deletions) in mutational studies of S. coelicolor and related species.

Gene expression profiling of human cancers.

DNA microarrays allow us to visualize simultaneously the expression of potentially all genes within a cell population or tissue sample-revealing the "transcriptome." The analysis of this type of data is commonly called "gene expression profiling" (GEP) because it provides a comprehensive picture of the pattern of gene expression in a particular biological sample. For this reason microarrays are revolutionizing life sciences research and are leading to the development of novel and powerful methods for investigating cancer biology, classifying cancers, and predicting clinical outcome of cancers. Several recent high-profile reports have revealed how clustering of GEP data can clearly identify clinically (and prognostically) important subtypes of cancer among patients considered by established clinicopathological criteria to have similar tumors. Accurate "prognostic signatures" can be obtained from GEP data, which represent relatively small numbers of genes. These signatures can be valuable in directing appropriate treatment and in predicting clinical outcome, and they generally outperform other systems based on clinical and histological criteria. In this paper the basic principles of DNA microarray technology and the different types of microarray platforms available will be introduced, and the power of the technique will be illustrated by reviewing some recent GEP studies on selected cancers, including a preliminary analysis of hepatocellular carcinoma from our Palermo laboratory. GEP is likely to be adopted in the future as a key decision-making tool in the clinical arena. However, several issues relating to data analysis, reproducibility, cross-comparability, validation, and cost need to be resolved before the technology can be adopted broadly in this context.

Negative feedback regulation of dnaK, clpB and lon expression by the DnaK chaperone machine in Streptomyces coelicolor, identified by transcriptome and in vivo DnaK-depletion analysis.

The dnaK operon of Streptomyces coelicolor encodes the DnaK chaperone machine and the negative autoregulator HspR, which confers repression of the operon by binding to several inverted repeat sequences in the promoter region, dnaKp. Previous in vitro studies demonstrated that DnaK forms a specific complex with HspR bound to its operator sequences in dnaKp, and a model was proposed in which DnaK functions as a corepressor of the dnaK operon (Bucca, G., Brassington, A., Schonfeld, H.J., and Smith, C.P. (2000) Mol Microbiol 38: 1093-1103). Here we report in vivo DnaK depletion experiments which demonstrate that DnaK is a negative regulator of the dnaK operon. Cellular depletion of the DnaK chaperone leads to high-level transcription from dnaKp at the normal growth temperature. DNA microarray-based analysis of gene expression in wild-type and hspR-disruption mutant strains has identified a core cluster of genes regulated by HspR: the dnaK and clpB-SCO3660 operons and lon. These three transcription units are considered to be the direct targets of HspR. Significantly, analysis of the entire genome sequence revealed that the promoter regions of dnaK, clpB and lon are the only sequences that contain the HspR consensus binding sequence 5'-TTGAGY-N7-ACTCAA. S1 nuclease mapping confirmed that transcription of both clpB and lon is substantially enhanced at ambient temperature in strains depleted of DnaK, providing further evidence that these genes are members of the DnaK-HspR regulon. From transcriptome analysis, 17 genes were shown to be upregulated more than twofold in an hspR disruption mutant. This included the seven genes encoded by the dnaK, clpB and lon transcription units. Significantly, the other 10 genes are not heat-shock inducible in the wild type and their upregulation in the hspR mutant is considered to be an indirect consequence of enhanced synthesis of one or more components of the HspR regulon (the DnaK chaperone machine, ClpB and Lon protease).