Genome-wide analysis of the role of the antibiotic biosynthesis regulator AbsA2 in Streptomyces coelicolor A3(2)

<div><p>The AbsA1-AbsA2 two component signalling system of <i>Streptomyces coelicolor</i> has long been known to exert a powerful negative influence on the production of the antibiotics actinorhodin, undecylprodiginine and the Calcium-Dependent Antibiotic (CDA). Here we report the analysis of a <i>ΔabsA2</i> deletion strain, which exhibits the classic precocious antibiotic hyper-production phenotype, and its complementation by an N-terminal triple-FLAG-tagged version of AbsA2. The complemented and non-complemented <i>ΔabsA2</i> mutant strains were used in large-scale microarray-based time-course experiments to investigate the effect of deleting <i>absA2</i> on gene expression and to identify the <i>in vivo</i> AbsA2 DNA-binding target sites using ChIP-on chip. We show that in addition to binding to the promoter regions of <i>redZ</i> and <i>actII-orfIV</i> AbsA2 binds to several previously unidentified sites within the <i>cda</i> biosynthetic gene cluster within and/or upstream of <i>SCO3215—SCO3216</i>, <i>SCO3217</i>, <i>SCO3229—SCO3230</i>, and <i>SCO3226</i>, and we relate the pattern of AbsA2 binding to the results of the transcriptomic study and antibiotic phenotypic assays. Interestingly, dual ‘biphasic’ ChIP peaks were observed with AbsA2 binding across the regulatory genes <i>actII-orfIV</i> and <i>redZ</i> and the <i>absA2</i> gene itself, while more conventional single promoter-proximal peaks were seen at the CDA biosynthetic genes suggesting a different mechanism of regulation of the former loci. Taken together the results shed light on the complex mechanism of regulation of antibiotic biosynthesis in <i>Streptomyces coelicolor</i> and the important role of AbsA2 in controlling the expression of three antibiotic biosynthetic gene clusters.</p></div

HOX and PBX gene dysregulation as a therapeutic target in glioblastoma multiforme

Background: Glioblastoma multiforme (GBM) is the most common high-grade malignant brain tumour in adults and arises from the glial cells in the brain. The prognosis of treated GBM remains very poor with 5-year survival rates of 5%, a figure which has not improved over the last few decades. Currently, there is a modest 14-month overall median survival in patients undergoing maximum safe resection plus adjuvant chemoradiotherapy. HOX gene dysregulation is now a widely recognised feature of many malignancies. Methods: In this study we have focused on HOX gene dysregulation in GBM as a potential therapeutic target in a disease with high unmet need. Results: We show significant dysregulation of these developmentally crucial genes and specifically that HOX genes A9, A10, C4 and D9 are strong candidates for biomarkers and treatment targets for GBM and GBM cancer stem cells. We evaluated a next generation therapeutic peptide, HTL-001, capable of targeting HOX gene over-expression in GBM by disrupting the interaction between HOX proteins and their co-factor, PBX. HTL-001 induced both caspase-dependent and -independent apoptosis in GBM cell lines. Conclusion: In vivo biodistribution studies confirmed that the peptide was able to cross the blood brain barrier. Systemic delivery of HTL-001 resulted in improved control of subcutaneous murine and human xenograft tumours and improved survival in a murine orthotopic model

Exon Array Analysis of Head and Neck Cancers Identifies a Hypoxia Related Splice Variant of LAMA3 Associated with a Poor Prognosis

The identification of alternatively spliced transcript variants specific to particular biological processes in tumours should increase our understanding of cancer. Hypoxia is an important factor in cancer biology, and associated splice variants may present new markers to help with planning treatment. A method was developed to analyse alternative splicing in exon array data, using probeset multiplicity to identify genes with changes in expression across their loci, and a combination of the splicing index and a new metric based on the variation of reliability weighted fold changes to detect changes in the splicing patterns. The approach was validated on a cancer/normal sample dataset in which alternative splicing events had been confirmed using RT-PCR. We then analysed ten head and neck squamous cell carcinomas using exon arrays and identified differentially expressed splice variants in five samples with high versus five with low levels of hypoxia-associated genes. The analysis identified a splice variant of LAMA3 (Laminin α 3), LAMA3-A, known to be involved in tumour cell invasion and progression. The full-length transcript of the gene (LAMA3-B) did not appear to be hypoxia-associated. The results were confirmed using qualitative RT-PCR. In a series of 59 prospectively collected head and neck tumours, expression of LAMA3-A had prognostic significance whereas LAMA3-B did not. This work illustrates the potential for alternatively spliced transcripts to act as biomarkers of disease prognosis with improved specificity for particular tissues or conditions over assays which do not discriminate between splice variants

Identifying and validating blood mRNA biomarkers for acute and chronic insufficient sleep in humans: a machine learning approach

Acute and chronic insufficient sleep are associated with adverse health outcomes and risk of accidents. There is therefore a need for biomarkers to monitor sleep debt status. None are currently available. We applied Elastic-net and Ridge regression to entire and pre-filtered transcriptome samples collected in healthy young adults during acute total sleep deprivation and following 1 week of either chronic insufficient (< 6 h) or sufficient sleep (~8.6 h) to identify panels of mRNA biomarkers of sleep debt status. The size of identified panels ranged from 9-74 biomarkers. Panel performance, assessed by leave-one-subject-out cross-validation and independent validation, varied between sleep debt conditions. Using between-subject assessments based on one blood sample, the accuracy of classifying ‘Acute sleep loss’ was 92%, but only 57% for classifying ‘Chronic sleep insufficiency’. A reasonable accuracy for classifying ‘chronic sleep insufficiency’ could only be achieved by a within-subject comparison of blood samples. Biomarkers for sleep debt status showed little overlap with previously identified biomarkers for circadian phase. Biomarkers for acute and chronic sleep loss also showed little overlap but were associated with common functions related to the cellular stress response, such as heat shock protein activity, the unfolded protein response, protein ubiquitination and endoplasmic reticulum associated protein degradation, and apoptosis. This characteristic response of whole blood to sleep loss can further aid our understanding of how sleep insufficiencies negatively affect health. Further development of these novel biomarkers for research and clinical practice requires validation in other protocols and age groups

HOXB9 Overexpression Promotes Colorectal Cancer Progression and Is Associated with Worse Survival in Liver Resection Patients for Colorectal Liver Metastases

As is known, HOXB9 is an important factor affecting disease progression and overall survival (OS) in cancer. However, its role in colorectal cancer (CRC) remains unclear. We aimed to explore the role of HOXB9 in CRC progression and its association with OS in colorectal liver metastases (CRLM). We analysed differential HOXB9 expression in CRC using the Tissue Cancer Genome Atlas database (TCGA). We modulated HOXB9 expression in vitro to assess its impact on cell proliferation and epithelial-mesenchymal transition (EMT). Lastly, we explored the association of HOXB9 protein expression with OS, using an institutional patient cohort (n = 110) who underwent liver resection for CRLM. Furthermore, HOXB9 was upregulated in TCGA-CRC (n = 644) vs. normal tissue (n = 51) and its expression levels were elevated in KRAS mutations (p &lt; 0.0001). In vitro, HOXB9 overexpression increased cell proliferation (p &lt; 0.001) and upregulated the mRNA expression of EMT markers (VIM, CDH2, ZEB1, ZEB2, SNAI1 and SNAI2) while downregulated CDH1, (p &lt; 0.05 for all comparisons). Conversely, HOXB9 silencing disrupted cell growth (p &lt; 0.0001). High HOXB9 expression (HR = 3.82, 95% CI: 1.59&ndash;9.2, p = 0.003) was independently associated with worse OS in CRLM-HOXB9-expressing patients after liver resection. In conclusion, HOXB9 may be associated with worse OS in CRLM and may promote CRC progression, whereas HOXB9 silencing may inhibit CRC growth

Blood transcriptome based biomarkers for human circadian phase

Diagnosis and treatment of circadian rhythm sleep-wake disorders requires assessment of circadian phase of the brain’s circadian pacemaker. The gold-standard univariate method is based on collection of a 24 h time series of plasma melatonin, a suprachiasmatic nucleus driven pineal hormone. We developed and validated a multivariate whole-blood mRNA based predictor of melatonin phase which requires few samples. Transcriptome data were collected under normal, sleep-deprivation and abnormal sleep-timing conditions to assess robustness of the predictor. Partial least square regression (PLSR), applied to the transcriptome, identified a set of 100 biomarkers primarily related to glucocorticoid signaling and immune function. Validation showed that PLSR-based predictors outperform published blood-derived circadian phase predictors. When given one sample as input, the R2 of predicted vs observed phase was 0.74, whereas for two samples taken 12 h apart, R2 was 0.90. This blood transcriptome based model enables assessment of circadian phase from a few samples

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

Translational control plays an important role in the adaptive heat-shock response of Streptomyces coelicolor.

Stress-induced adaptations requiremultiple levels of regulation in all organisms to repair cellular damage. In the present study we evaluated the genome-wide transcriptional and translational changes following heat stress exposure in the soil-dwelling model actinomycete bacterium, Streptomyces coelicolor. The combined analysis revealed an unprecedented level of translational control of gene expression, deduced through polysome profiling, in addition to transcriptional changes. Our data show little correlation between the transcriptome and ‘translatome’; while an obvious downward trend in genome wide transcription was observed, polysome associated transcripts following heat-shock showed an opposite upward trend. A handful of key protein players, including the major molecular chaperones and proteases were highly induced at both the transcriptional and translational level following heat-shock, a phenomenon known as ‘potentiation’. Many other transcripts encoding cold-shock proteins, ABC-transporter systems, multiple transcription factors weremore highly polysome-associated following heat stress; interestingly, these protein families were not induced at the transcriptional level and therefore were not previously identified as part of the stress response. Thus, stress coping mechanisms at the level of gene expression in this bacterium go well beyond the induction of a relatively small number of molecular chaperones and proteases in order to ensure cellular survival at non-physiological temperatures

The prognostic significance of specific HOX gene expression patterns in ovarian cancer

OBJECTIVE: HOX genes are vital for all aspects of mammalian growth and differentiation, and their dysregulated expression is related to ovarian carcinogenesis. The aim of the current study was to establish the prognostic value of HOX dysregulation as well as its role in platinum resistance. The potential to target HOX proteins through the HOX/PBX interaction was also explored in the context of platinum resistance. METHODS: HOX gene expression was determined in ovarian cancer cell lines and primary EOCs, and compared to expression in normal ovarian epithelium and fallopian tube tissue samples. Statistical analysis included one way ANOVA and t-tests, using statistical software R and GraphPad. RESULTS: 30 of the 39 HOX genes were overexpressed in high grade serous EOC compared to normal tissue, most significant being HOXA3, A9, B13 and C10. We detected a molecular HOX gene-signature that predicted poor outcome: overexpression of HOXB4 and HOXB9 was identified in high grade serous cell lines after platinum resistance developed. Targeting the HOX/PBX dimer with the the HXR9 peptide enhanced the cytotoxicity of cisplatin in platinum-resistant ovarian cancer.CONCLUSIONS: HOX genes are highly dysregulated in ovarian cancer. High expression of HOXA13, B6, C13, D1 and D13 is predictive of poor clinical outcome. Targeting the HOX/PBX dimer in platinum–resistant cancer represents a new therapeutic option that should be further developed and tested in clinical trials