The role of Hsp90/Hsp70 organising protein (Hop) in the Proliferation, Survival and Migration of Breast Cancer Cells.

Hop (the Hsp90/Hsp70 organising protein) is a co-chaperone that acts as an adapter between the major molecular chaperones Hsp90 and Hsp70 during the cellular assembly of the Hsp90 complex. The Hsp90 complex regulates the stability and conformational maturation of a range of important cellular proteins, many of which are deregulated in cancer. In this study, we hypothesised that Hop knockdown inhibits proliferation and migration of cancer cells. We characterised the expression of Hop in cell models of different cancerous status, and provided evidence that Hop was upregulated in tumour cells compared to normal cell counterparts. Using an RNA interference approach, a 60-90% knockdown of Hop was achieved for up to 144 hours in the MDA-MB-231 and Hs578T breast cancer cell lines. Hop knockdown resulted in downregulation of the Hsp90 client proteins, Akt and Stat3, as well as a change in the expression of other Hsp90 co-chaperones, p23, Cdc37 and Aha1, while no change in the levels of Hsp90 or Hsp70 was observed. Silencing of Hop impaired cell proliferation in Hs578T cells but an increase in proliferation in MDA-MB-231, suggesting that the role of Hop in cancer cell proliferation was dependent on type of cancer cell. Hop knockdown in Hs578T and MDA-MB- 231 cells did not lead to any significant changes in the half maximal inhibitory concentrations (IC50) of selected small molecule inhibitors (paclitaxel, geldanamycin and novobiocin) in these cell lines after 72 hours. Hop knockdown cells were however, more sensitive than control cells to the Hsp90 inhibitors geldanamycin and novobiocin at earlier time points and in the presence of the drug transporter inhibitor, verapamil. Hop knockdown caused a decrease in cell migration as measured by the wound healing assay in both Hs578T and MDA-MB-231 cells. Hop was present in purified pseudopodia fractions of migrating cells, and immunofluorescence analysis showed that Hop colocalised with actin at the leading edges of pseudopodia, points of adhesion and at intercellular junctions of cells that have been stimulated to migrate with the chemokine stromal derived factor-1. Hop was able to bind to actin in vitro using actin cosedimentation assays, and silencing of Hop dramatically reduced the capacity of Hs578T cells to form pseudopodia. These results establish a correlation between Hop and actin dynamics, pseudopodia formation and migration in the context of Hop silencing, and collectively suggest that Hop plays a role in cancer cell migration. This study presents experimental evidence for a promising alternative to targeting Hsp90 and Hsp70 chaperones, a novel drug target in cancer therapy

The T-box transcription factor TBX3 drives proliferation by direct repression of the p21WAF1 cyclin-dependent kinase inhibitor

Background: TBX3, a member of the T-box family of transcription factors, is essential in development and has emerged as an important player in the oncogenic process. TBX3 is overexpressed in several cancers and has been shown to contribute directly to tumour formation, migration and invasion. However, little is known about the molecular basis for its role in development and oncogenesis because there is a paucity of information regarding its target genes. The cyclin-dependent kinase inhibitor p21WAF1 plays a pivotal role in a myriad of processes including cell cycle arrest, senescence and apoptosis and here we provide a detailed mechanism to show that it is a direct and biologically relevant target of TBX3. Results: Using a combination of luciferase reporter gene assays and in vitro and in vivo binding assays we show that TBX3 directly represses the p21WAF1 promoter by binding a T-element close to its initiator. Furthermore, we show that the TBX3 DNA binding domain is required for the transcriptional repression of p21WAF1 and that pseudo-phosphorylation of a serine proline motif (S190) located within this domain may play an important role in regulating this ability. Importantly, we demonstrate using knockdown and overexpression experiments that p21WAF1 repression by TBX3 is biologically significant and required for TBX3-induced cell proliferation of chondrosarcoma cells. Conclusions: Results from this study provide a detailed mechanism of how TBX3 transcriptionally represses p21WAF1 which adds to our understanding of how it may contribute to oncogenesis

DNA methylation of FKBP5 in South African women : associations with obesity and insulin resistance

CITATION: Willmer, T., et al. 2020. DNA methylation of FKBP5 in South African women : associations with obesity and insulin resistance. Clinical Epigenetics, 12:141, doi:10.1186/s13148-020-00932-3.The original publication is available at https://clinicalepigeneticsjournal.biomedcentral.comBackground: Disruption of the hypothalamic–pituitary–adrenal (HPA) axis, a neuroendocrine system associated with the stress response, has been hypothesized to contribute to obesity development. This may be mediated through epigenetic modulation of HPA axis-regulatory genes in response to metabolic stressors. The aim of this study was to investigate adipose tissue depot-specific DNA methylation differences in the glucocorticoid receptor (GR) and its co-chaperone, FK506-binding protein 51 kDa (FKBP5), both key modulators of the HPA axis. Methods: Abdominal subcutaneous adipose tissue (ASAT) and gluteal subcutaneous adipose tissue (GSAT) biopsies were obtained from a sample of 27 obese and 27 normal weight urban-dwelling South African women. DNA methylation and gene expression were measured by pyrosequencing and quantitative real-time PCR, respectively. Spearman’s correlation coefficients, orthogonal partial least-squares discriminant analysis and multivariable linear regression were performed to evaluate the associations between DNA methylation, messenger RNA (mRNA) expression and key indices of obesity and metabolic dysfunction. Results: Two CpG dinucleotides within intron 7 of FKBP5 were hypermethylated in both ASAT and GSAT in obese compared to normal weight women, while no differences in GR methylation were observed. Higher percentage methylation of the two FKBP5 CpG sites correlated with adiposity (body mass index and waist circumference), insulin resistance (homeostasis model for insulin resistance, fasting insulin and plasma adipokines) and systemic inflammation (c-reactive protein) in both adipose depots. GR and FKBP5 mRNA levels were lower in GSAT, but not ASAT, of obese compared to normal weight women. Moreover, FKBP5 mRNA levels were inversely correlated with DNA methylation and positively associated with adiposity, metabolic and inflammatory parameters. Conclusions: These findings associate dysregulated FKBP5 methylation and mRNA expression with obesity and insulin resistance in South African women. Additional studies are required to assess the longitudinal association of FKBP5 with obesity and associated co-morbidities in large population-based samples.https://clinicalepigeneticsjournal.biomedcentral.com/articles/10.1186/s13148-020-00932-3Publisher's versio

Blood-Based DNA Methylation Biomarkers for Type 2 Diabetes: Potential for Clinical Applications

Type 2 diabetes (T2D) is a leading cause of death and disability worldwide. It is a chronic metabolic disorder that develops due to an interplay of genetic, lifestyle, and environmental factors. The biological onset of the disease occurs long before clinical symptoms develop, thus the search for early diagnostic and prognostic biomarkers, which could facilitate intervention strategies to prevent or delay disease progression, has increased considerably in recent years. Epigenetic modifications represent important links between genetic, environmental and lifestyle cues and increasing evidence implicate altered epigenetic marks such as DNA methylation, the most characterized and widely studied epigenetic mechanism, in the pathogenesis of T2D. This review provides an update of the current status of DNA methylation as a biomarker for T2D. Four databases, Scopus, Pubmed, Cochrane Central, and Google Scholar were searched for studies investigating DNA methylation in blood. Thirty-seven studies were identified, and are summarized with respect to population characteristics, biological source, and method of DNA methylation quantification (global, candidate gene or genome-wide). We highlight that differential methylation of the TCF7L2, KCNQ1, ABCG1, TXNIP, PHOSPHO1, SREBF1, SLC30A8, and FTO genes in blood are reproducibly associated with T2D in different population groups. These genes should be prioritized and replicated in longitudinal studies across more populations in future studies. Finally, we discuss the limitations faced by DNA methylation studies, which include including interpatient variability, cellular heterogeneity, and lack of accounting for study confounders. These limitations and challenges must be overcome before the implementation of blood-based DNA methylation biomarkers into a clinical setting. We emphasize the need for longitudinal prospective studies to support the robustness of the current findings of this review

A pilot investigation of genetic and epigenetic variation of FKBP5 and response to exercise intervention in African women with obesity

We investigated gluteal (GSAT) and abdominal subcutaneous adipose tissue (ASAT) DNA methylation of FKBP5 in response to a 12-week intervention in African women with obesity, as well as the efect of the rs1360780 single nucleotide polymorphism (SNP) on FKBP5 methylation, gene expression and post-exercise training adaptations in obesity and metabolic related parameters. Exercise (n= 19) participants underwent 12-weeks of supervised aerobic and resistance training while controls (n= 12) continued their usual behaviours. FKBP5 methylation was measured in GSAT and ASAT using pyrosequencing. SNP and gene expression analyses were conducted using quantitative real-time PCR. Exercise training induced FKBP5 hypermethylation at two CpG dinucleotides within intron 7. When stratifed based on the rs1360780 SNP, participants with the CT genotype displayed FKBP5 hypermethylation in GSAT (p < 0.05), and ASAT displayed in both CC and CT carriers. CC allele carriers displayed improved cardiorespiratory ftness, insulin sensitivity, gynoid fat mass, and waist circumference (p < 0.05) in response to exercise training, and these parameters were attenuated in women with the CT genotype. These fndings provide a basis for future studies in larger cohorts, which should assess whether FKBP5 methylation and/or genetic variants such as the rs1360780 SNP could have a signifcant impact on responsiveness to exercise interventions.The South African Medical Research Council (SAMRC), the National Research Foundation professional development program (PDP), Tuthuka and the International Atomic Energy agency.https://www.nature.com/srepdm2022Obstetrics and Gynaecolog

Changes in subcutaneous adipose tissue microRNA expression in response to exercise training in African women with obesity

The mechanisms that underlie exercise-induced adaptations in adipose tissue have not been elucidated, yet, accumulating studies suggest an important role for microRNAs (miRNAs). This study aimed to investigate miRNA expression in gluteal subcutaneous adipose tissue (GSAT) in response to a 12-week exercise intervention in South African women with obesity, and to assess depot-specific differences in miRNA expression in GSAT and abdominal subcutaneous adipose tissue (ASAT). In addition, the association between exercise-induced changes in miRNA expression and metabolic risk was evaluated. Women underwent 12-weeks of supervised aerobic and resistance training (n = 19) or maintained their regular physical activity during this period (n = 12). Exercise-induced miRNAs were identified in GSAT using Illumina sequencing, followed by analysis of differentially expressed miRNAs in GSAT and ASAT using quantitative real-time PCR. Associations between the changes (pre- and postexercise training) in miRNA expression and metabolic parameters were evaluated using Spearman’s correlation tests. Exercise training significantly increased the expression of miR-155-5p (1.5-fold, p = 0.045), miR-329-3p (2.1-fold, p < 0.001) and miR-377-3p (1.7-fold, p = 0.013) in GSAT, but not in ASAT. In addition, a novel miRNA, MYN0617, was identified in GSAT, with low expression in ASAT. The exercise-induced differences in miRNA expression were correlated with each other and associated with changes in high-density lipoprotein concentrations. Exercise training induced adipose-depot specific miRNA expression within subcutaneous adipose tissue depots from South African women with obesity. The significance of the association between exercise-induced miRNAs and metabolic risk warrants further investigation.The South African Medical Research Council (SAMRC) and the National Research Foundation of South Africa (NRF), Competitive Programme for Rated Researchers.http://www.nature.com/scientificreportsam2023Obstetrics and Gynaecolog

Blood-Based DNA Methylation Biomarkers for Type 2 Diabetes: Potential for Clinical Applications

CITATION: Willmer, T., et al. 2018. Blood-based DNA methylation biomarkers for type 2 diabetes : potential for clinical applications. Frontiers in Endocrinology, 9:744, doi:10.3389/fendo.2018.00744.The original publication is available at https://www.frontiersin.orgENGLISH ABSTRACT: Type 2 diabetes (T2D) is a leading cause of death and disability worldwide. It is a chronic metabolic disorder that develops due to an interplay of genetic, lifestyle, and environmental factors. The biological onset of the disease occurs long before clinical symptoms develop, thus the search for early diagnostic and prognostic biomarkers, which could facilitate intervention strategies to prevent or delay disease progression, has increased considerably in recent years. Epigenetic modifications represent important links between genetic, environmental and lifestyle cues and increasing evidence implicate altered epigenetic marks such as DNA methylation, the most characterized and widely studied epigenetic mechanism, in the pathogenesis of T2D. This review provides an update of the current status of DNA methylation as a biomarker for T2D. Four databases, Scopus, Pubmed, Cochrane Central, and Google Scholar were searched for studies investigating DNA methylation in blood. Thirty-seven studies were identified, and are summarized with respect to population characteristics, biological source, and method of DNA methylation quantification (global, candidate gene or genome-wide). We highlight that differential methylation of the TCF7L2, KCNQ1, ABCG1, TXNIP, PHOSPHO1, SREBF1, SLC30A8, and FTO genes in blood are reproducibly associated with T2D in different population groups. These genes should be prioritized and replicated in longitudinal studies across more populations in future studies. Finally, we discuss the limitations faced by DNA methylation studies, which include including interpatient variability, cellular heterogeneity, and lack of accounting for study confounders. These limitations and challenges must be overcome before the implementation of blood-based DNA methylation biomarkers into a clinical setting. We emphasize the need for longitudinal prospective studies to support the robustness of the current findings of this review.https://www.frontiersin.org/articles/10.3389/fendo.2018.00744/fullPublisher's versio

Knockdown of Hop downregulates RhoC expression, and decreases pseudopodia formation and migration in cancer cell lines

The Hsp90/Hsp70 organising protein (Hop) is a co-chaperone that mediates the interaction of Hsp90 and Hsp70 molecular chaperones during assembly of Hsp90 complexes in cells. Formation of Hsp90 complexes is a key intermediate step in the maturation and homeostasis of oncoproteins and several hormone receptors. In this paper, we demonstrate that knockdown of Hop decreased migration of Hs578T and MDA-MB-231 breast cancer cells. Hop was identified in isolated pseudopodia fractions; it colocalised with actin in lamellipodia, and co-sedimented with purified actin in vitro. Knockdown of Hop caused a decrease in the level of RhoC GTPase, and significantly inhibited pseudopodia formation in Hs578T cells. Our data suggest that Hop regulates directional cell migration by multiple unknown mechanisms