Showing papers by "Lorna W. Harries published in 2015"

The androgen receptor controls expression of the cancer-associated sTn antigen and cell adhesion through induction of ST6GalNAc1 in prostate cancer.

Abstract: Patterns of glycosylation are important in cancer, but the molecular mechanisms that drive changes are often poorly understood. The androgen receptor drives prostate cancer (PCa) development and progression to lethal metastatic castration-resistant disease. Here we used RNA-Seq coupled with bioinformatic analyses of androgen-receptor (AR) binding sites and clinical PCa expression array data to identify ST6GalNAc1 as a direct and rapidly activated target gene of the AR in PCa cells. ST6GalNAc1 encodes a sialytransferase that catalyses formation of the cancer-associated sialyl-Tn antigen (sTn), which we find is also induced by androgen exposure. Androgens induce expression of a novel splice variant of the ST6GalNAc1 protein in PCa cells. This splice variant encodes a shorter protein isoform that is still fully functional as a sialyltransferase and able to induce expression of the sTn-antigen. Surprisingly, given its high expression in tumours, stable expression of ST6GalNAc1 in PCa cells reduced formation of stable tumours in mice, reduced cell adhesion and induced a switch towards a more mesenchymal-like cell phenotype in vitro. ST6GalNAc1 has a dynamic expression pattern in clinical datasets, beingsignificantly up-regulated in primary prostate carcinoma but relatively down-regulated in established metastatic tissue. ST6GalNAc1 is frequently upregulated concurrently with another important glycosylation enzyme GCNT1 previously associated with prostate cancer progression and implicated in Sialyl Lewis X antigen synthesis. Together our data establishes an androgen-dependent mechanism for sTn antigen expression in PCa, and are consistent with a general role for the androgen receptor in driving important coordinate changes to the glycoproteome during PCa progression.

Targeted allelic expression profiling in human islets identifies cis-regulatory effects for multiple variants identified by type 2 diabetes genome-wide association studies.

Abstract: Genome-wide association studies (GWAS) have identified variation at >65 genomic loci associated with susceptibility to type 2 diabetes, but little progress has been made in elucidating the molecular mechanisms behind most of these associations. Using samples heterozygous for transcribed single nucleotide polymorphisms (SNPs), allelic expression profiling is a powerful technique for identifying cis-regulatory variants controlling gene expression. In this study, exonic SNPs, suitable for measuring mature mRNA levels and in high linkage disequilibrium with 65 lead type 2 diabetes GWAS SNPs, were identified and allelic expression determined by real-time PCR using RNA and DNA isolated from islets of 36 white nondiabetic donors. A significant allelic expression imbalance (AEI) was identified for 7/14 (50%) genes tested (ANPEP, CAMK2B, HMG20A, KCNJ11, NOTCH2, SLC30A8, and WFS1), with significant AEI confirmed for five of these genes using other linked exonic SNPs. Lastly, results of a targeted islet expression quantitative trait loci experiment support the AEI findings for ANPEP, further implicating ANPEP as the causative gene at its locus. The results of this study support the hypothesis that changes to cis-regulation of gene expression are involved in a large proportion of SNP associations with type 2 diabetes susceptibility.

The PI3K regulatory subunit gene PIK3R1 is under direct control of androgens and repressed in prostate cancer cells

Abstract: Androgen receptor (AR) signalling and the PI3K pathway mediate survival signals in prostate cancer, and have been shown to regulate each other by reciprocal negative feedback, such that inhibition of one activates the other. Understanding the reciprocal regulation of these pathways is important for disease management as tumour cells can adapt and survive when either single pathway is inhibited pharmacologically. We recently carried out genome-wide exon-specific profiling of prostate cancer cells to identify novel androgen-regulated transcriptional events. Here we interrogated this dataset for novel androgen-regulated genes associated with the PI3K pathway. We find that the PI3K regulatory subunits PIK3R1 (p85α) and PIK3R3 (p55γ) are direct targets of the AR which are rapidly repressed by androgens in LNCaP cells. Further characterisation revealed that the PIK3CA p110α catalytic subunit is also indirectly regulated by androgens at the protein level. We show that PIK3R1 mRNA is significantly under-expressed in prostate cancer (PCa) tissue, and provide data to suggest a context-dependent regulatory mechanism whereby repression of the p85α protein by the AR results in destabilisation of the PI3K p110α catalytic subunit and downstream PI3K pathway inhibition that functionally affects the properties of prostate cancer cells.

A cautionary tale: the non-causal association between type 2 diabetes risk SNP, rs7756992, and levels of non-coding RNA, CDKAL1-v1

Abstract: Aims/hypothesis Intronic single nucleotide polymorphisms (SNPs) in the CDKAL1 gene are associated with risk of developing type 2 diabetes. A strong correlation between risk alleles and lower levels of the non-coding RNA, CDKAL1-v1, has recently been reported in whole blood extracted from Japanese individuals. We sought to replicate this association in two independent cohorts: one using whole blood from white UK-resident individuals, and one using a collection of human pancreatic islets, a more relevant tissue type to study with respect to the aetiology of diabetes.

Comparison of senescence-associated miRNAs in primary skin and lung fibroblasts

Abstract: MicroRNAs are non-coding RNAs with roles in many cellular processes. Tissue-specific miRNA profiles associated with senescence have been described for several cell and tissue types. We aimed to characterise miRNAs involved in core, rather than tissue-specific, senescence pathways by assessment of common miRNA expression differences in two different cell types, with follow-up of predicted targets in human peripheral blood. MicroRNAs were profiled in early and late passage primary lung and skin fibroblasts to identify commonly-deregulated miRNAs. Expression changes of their bioinformatically-predicted mRNA targets were then assessed in both cell types and in human peripheral blood from elderly participants in the InCHIANTI study. 57/178 and 26/492 microRNAs were altered in late passage skin and lung cells respectively. Three miRNAs (miR-92a, miR-15b and miR-125a-3p) were altered in both tissues. 14 mRNA targets of the common miRNAs were expressed in lung and skin fibroblasts, of which two demonstrated up-regulation in late passage skin and lung cells (LYST; p = 0.02 [skin] and 0.02 [lung] INMT; p = 0.03 [skin] and 0.04 [lung]). ZMPSTE24 and LHFPL2 demonstrated altered expression in late passage skin cells only (p = 0.01 and 0.05 respectively). LHFPL2 was also positively correlated with age in peripheral blood (p value = 6.6 × 10−5). We find that the majority of senescence-associated miRNAs demonstrate tissue-specific effects. However, miRNAs showing common effects across tissue types may represent those associated with core, rather than tissue-specific senescence processes.

Changes in CEBPB expression in circulating leukocytes following eccentric elbow-flexion exercise

Abstract: In mouse models, CCAAT enhancer-binding protein beta (CEBPB) is necessary for M2 macrophage-mediated regeneration after muscle injury. In humans, CEBPB expression in blood was strongly associated with muscle strength. In this study we aimed to test whether CEBPB expression in blood in people is increased 2 days after exercise designed to induce muscle damage and subsequent repair. Sixteen healthy male volunteers undertook elbow flexor exercises designed to induce acute muscle micro-damage. Peripheral blood samples were collected at baseline and days 1, 2, 4 and 7 following exercise. Expression of CEBPB and related genes were analysed by qRT-PCR. Extent of muscle damage was determined by decline in maximal voluntary isometric torque and by plasma creatine kinase activity. Nine subjects had peak (day 4) creatine kinase activity exceeding 10,000 U/l. In this subgroup, CEBPB expression was elevated from baseline to 2 days post exercise (paired-samples t (1,8) = 3.72, p = 0.006). Related expression and selected cytokine changes after exercise did not reach significance. Muscle-damaging exercise in humans can be followed by induction of CEBPB transcript expression in peripheral blood. Associations between CEBPB expression in blood and muscle strength may be consistent with the CEBPB-dependent muscle repair process.

The reported healthy ageing gene expression score: lack of predictive value in two cohorts

Abstract: Sood et al. report a multi-tissue RNA signature "predictive of human health, using only peripheral blood samples". We tested this score in blood in two independent, larger cohorts and found no associations with age or related phenotypes, including strength, interleukin-6 or mortality.

The reported healthy ageing gene expression score: lack of association in two cohorts

Abstract: Sood et al. report a multi-tissue RNA signature "predictive of human health, using only peripheral blood samples". We tested this score in blood in two independent, larger cohorts and found no associations with age or related phenotypes, including muscle strength, interleukin-6 or mortality.