Leptin is a hormone that plays a key role in controlling food intake and energy homeostasis. Skeletal muscle is an important target for leptin and recent studies have shown that leptin deficiency... Show moreLeptin is a hormone that plays a key role in controlling food intake and energy homeostasis. Skeletal muscle is an important target for leptin and recent studies have shown that leptin deficiency may lead to muscular atrophy. However, leptin deficiency-induced structural changes in muscles are poorly understood. The zebrafish has emerged as an excellent model organism for studies of vertebrate diseases and hormone response mechanisms. In this study, we explored ex-vivo magnetic resonance microimaging (μMRI) methods to non-invasively assess muscle wasting in leptin-deficient (lepb-/-) zebrafish model. The fat mapping performed by using chemical shift selective imaging shows significant fat infiltration in muscles of lepb-/- zebrafish compared to control zebrafish. T2 relaxation measurements show considerably longer T2 values in the muscle of lepb-/- zebrafish. Multiexponential T2 analysis detected a significantly higher value and magnitude of long T2 component in the muscles of lepb-/- as compared to control zebrafish. For further zooming into the microstructural changes, we applied diffusion-weighted MRI. The results show a significant decrease in the apparent diffusion coefficient indicating increased constraints of molecular movements within the muscle regions of lepb-/- zebrafish. The use of the phasor transformation for the separation of diffusion-weighted decay signals showed a bi-component diffusion system which allows us to estimate each fraction on a voxel-wise basis. A substantial difference was found between the ratio of two components in lepb-/- and control zebrafish muscles, indicating alterations in diffusion behavior associated with the tissue microstructural changes in muscles of lepb-/- zebrafish as compared to control zebrafish. Taken together, our results demonstrate that the muscles of lepb-/- zebrafish undergo significant fat infiltration and microstructural changes leading to muscle wasting. This study also demonstrates that μMRI provides excellent means to non-invasively study the microstructural changes in the muscles of the zebrafish model. Show less
Previous genome-wide association studies (GWASs) of stroke - the second leading cause of death worldwide - were conducted predominantly in populations of European ancestry(1,2). Here, in cross... Show morePrevious genome-wide association studies (GWASs) of stroke - the second leading cause of death worldwide - were conducted predominantly in populations of European ancestry(1,2). Here, in cross-ancestry GWAS meta-analyses of 110,182 patients who have had a stroke (five ancestries, 33% non-European) and 1,503,898 control individuals, we identify association signals for stroke and its subtypes at 89 (61 new) independent loci: 60 in primary inverse-variance-weighted analyses and 29 in secondary meta-regression and multitrait analyses. On the basis of internal cross-ancestry validation and an independent follow-up in 89,084 additional cases of stroke (30% non-European) and 1,013,843 control individuals, 87% of the primary stroke risk loci and 60% of the secondary stroke risk loci were replicated (P < 0.05). Effect sizes were highly correlated across ancestries. Cross-ancestry fine-mapping, in silico mutagenesis analysis(3), and transcriptome-wide and proteome-wide association analyses revealed putative causal genes (such as SH3PXD2A and FURIN) and variants (such as at GRK5 and NOS3). Using a three-pronged approach(4), we provide genetic evidence for putative drug effects, highlighting F11, KLKB1, PROC, GP1BA, LAMC2 and VCAM1 as possible targets, with drugs already under investigation for stroke for F11 and PROC. A polygenic score integrating cross-ancestry and ancestry-specific stroke GWASs with vascular-risk factor GWASs (integrative polygenic scores) strongly predicted ischaemic stroke in populations of European, East Asian and African ancestry(5). Stroke genetic risk scores were predictive of ischaemic stroke independent of clinical risk factors in 52,600 clinical-trial participants with cardiometabolic disease. Our results provide insights to inform biology, reveal potential drug targets and derive genetic risk prediction tools across ancestries. Show less
Common single-nucleotide polymorphisms (SNPs) are predicted to collectively explain 40-50% of phenotypic variation in human height, but identifying the specific variants and associated regions... Show moreCommon single-nucleotide polymorphisms (SNPs) are predicted to collectively explain 40-50% of phenotypic variation in human height, but identifying the specific variants and associated regions requires huge sample sizes1. Here, using data from a genome-wide association study of 5.4 million individuals of diverse ancestries, we show that 12,111 independent SNPs that are significantly associated with height account for nearly all of the common SNP-based heritability. These SNPs are clustered within 7,209 non-overlapping genomic segments with a mean size of around 90 kb, covering about 21% of the genome. The density of independent associations varies across the genome and the regions of increased density are enriched for biologically relevant genes. In out-of-sample estimation and prediction, the 12,111 SNPs (or all SNPs in the HapMap 3 panel2) account for 40% (45%) of phenotypic variance in populations of European ancestry but only around 10-20% (14-24%) in populations of other ancestries. Effect sizes, associated regions and gene prioritization are similar across ancestries, indicating that reduced prediction accuracy is likely to be explained by linkage disequilibrium and differences in allele frequency within associated regions. Finally, we show that the relevant biological pathways are detectable with smaller sample sizes than are needed to implicate causal genes and variants. Overall, this study provides a comprehensive map of specific genomic regions that contain the vast majority of common height-associated variants. Although this map is saturated for populations of European ancestry, further research is needed to achieve equivalent saturation in other ancestries. Show less
In this thesis, two potential therapeutic targets for diabetic nephropathy were dentified and investigated. First, we show that glomerular clusterin is upregulated in diabetic nephropathy and... Show moreIn this thesis, two potential therapeutic targets for diabetic nephropathy were dentified and investigated. First, we show that glomerular clusterin is upregulated in diabetic nephropathy and demonstrated that recombinant clusterin protein can protect the podocytes against oxidative stress in vitro. Second, we reveal that hCN1 overexpression accelerated and aggravated diabetic nephropathy in BTBR ob/ob mice. We also studied two novel zebrafish models to investigate chronic kidney disease. We showed that lepb-/- adult zebrafish have the early signs of human diabetic nephropathy, and we demonstrated that ctns mutant adult zebrafish have the kidney pathologic features of human nephropathic cystinosis. Show less
Glycemic traits are used to diagnose and monitor type 2 diabetes and cardiometabolic health. To date, most genetic studies of glycemic traits have focused on individuals of European ancestry. Here... Show moreGlycemic traits are used to diagnose and monitor type 2 diabetes and cardiometabolic health. To date, most genetic studies of glycemic traits have focused on individuals of European ancestry. Here we aggregated genome-wide association studies comprising up to 281,416 individuals without diabetes (30% non-European ancestry) for whom fasting glucose, 2-h glucose after an oral glucose challenge, glycated hemoglobin and fasting insulin data were available. Trans-ancestry and single-ancestry meta-analyses identified 242 loci (99 novel; P < 5 x 10(-8)), 80% of which had no significant evidence of between-ancestry heterogeneity. Analyses restricted to individuals of European ancestry with equivalent sample size would have led to 24 fewer new loci. Compared with single-ancestry analyses, equivalent-sized trans-ancestry fine-mapping reduced the number of estimated variants in 99% credible sets by a median of 37.5%. Genomic-feature, gene-expression and gene-set analyses revealed distinct biological signatures for each trait, highlighting different underlying biological pathways. Our results increase our understanding of diabetes pathophysiology by using trans-ancestry studies for improved power and resolution.A trans-ancestry meta-analysis of GWAS of glycemic traits in up to 281,416 individuals identifies 99 novel loci, of which one quarter was found due to the multi-ancestry approach, which also improves fine-mapping of credible variant sets. Show less
Leptin is a hormone which functions in the regulation of energy homeostasis via suppression of appetite. In zebrafish, there are two paralogues genes encoding leptin, called lepa and lepb. In a... Show moreLeptin is a hormone which functions in the regulation of energy homeostasis via suppression of appetite. In zebrafish, there are two paralogues genes encoding leptin, called lepa and lepb. In a gene expression study, we found that the lepb gene, not the lepa gene, was significantly downregulated under the state of insulin-resistant in zebrafish larvae, suggesting that the lepb plays a role in insulin homeostasis. In the current study, we characterised lepb-deficient (lepb-/-) adult zebrafish generated via a CRISPR-CAS9 gene editing approach by investigating whether the deletion of lepb gene would result in the development of type 2 diabetes mellitus (T2DM) and diabetic complications. We observed that lepb-/- adult zebrafish had an increase in body weight, length and visceral fat accumulation, compared to age-matched control zebrafish. In addition, lepb-/- zebrafish had significantly higher blood glucose levels compared to control zebrafish. These data collectively indicate that lepb-/- adult zebrafish display the features of T2DM. Furthermore, we showed that lepb-/- adult zebrafish had glomerular hypertrophy and thickening of glomerular basement membrane, compared to control zebrafish, suggesting that lepb-/- adult zebrafish develop early signs of diabetic nephropathy. In conclusion, our results demonstrate that lepb regulates glucose homeostasis and adiposity in zebrafish, and suggest that lepb-/- mutant zebrafish are a promising model to investigate the role of leptin in the development of T2DM and an attractive model to perform mechanistic and therapeutic research in T2DM and its complications. Show less
The genetically heterogeneous triple-negative breast cancer (TNBC) continues to be an intractable disease, due to lack of effective targeted therapies. Gene amplification is a major event in... Show moreThe genetically heterogeneous triple-negative breast cancer (TNBC) continues to be an intractable disease, due to lack of effective targeted therapies. Gene amplification is a major event in tumorigenesis. Genes with amplification-dependent expression are being explored as therapeutic targets for cancer treatment. In this study, we have applied Analytical Multi-scale Identification of Recurring Events analysis and transcript quantification in the TNBC genome across 222 TNBC tumors and identified 138 candidate genes with positive correlation in copy number gain (CNG) and gene expression. siRNA-based loss-of-function screen of the candidate genes has validated EGFR, MYC, ASAP1, IRF2BP2, and CCT5 genes as drivers promoting proliferation in different TNBC cells. MYC, ASAP1, IRF2BP2, and CCT5 display frequent CNG and concurrent expression over 2173 breast cancer tumors (cBioPortal dataset). More frequently are MYC and ASAP1 amplified in TNBC tumors (>30%, n = 320). In particular, high expression of ASAP1, the ADP-ribosylation factor GTPase-activating protein, is significantly related to poor metastatic relapse-free survival of TNBC patients (n = 257, bc-GenExMiner). Furthermore, we have revealed that silencing of ASAP1 modulates numerous cytokine and apoptosis signaling components, such as IL1B, TRAF1, AIFM2, and MAP3K11 that are clinically relevant to survival outcomes of TNBC patients. ASAP1 has been reported to promote invasion and metastasis in various cancer cells. Our findings that ASAP1 is an amplification-dependent TNBC driver gene promoting TNBC cell proliferation, functioning upstream apoptosis components, and correlating to clinical outcomes of TNBC patients, support ASAP1 as a potential actionable target for TNBC treatment. Show less
Roessel, S. van; Strijker, M.; Steyerberg, E.W.; Groen, J.V.; Mieog, J.S.; Groot, V.P.; ... ; Besselink, M.G. 2020
Background: The objective of this study was to validate and update the Amsterdam prediction model including tumor grade, lymph node ratio, margin status and adjuvant therapy, for prediction of... Show moreBackground: The objective of this study was to validate and update the Amsterdam prediction model including tumor grade, lymph node ratio, margin status and adjuvant therapy, for prediction of overall survival (OS) after pancreatoduodenectomy for pancreatic cancer.Methods: We included consecutive patients who underwent pancreatoduodenectomy for pancreatic cancer between 2000 and 2017 at 11 tertiary centers in 8 countries (USA, UK, Germany, Italy, Sweden, the Netherlands, Korea, Australia). Model performance for prediction of OS was evaluated by calibration statistics and Uno's C-statistic for discrimination. Validation followed the TRIPOD statement.Results: Overall, 3081 patients (53% male, median age 66 years) were included with a median OS of 24 months, of whom 38% had N2 disease and 77% received adjuvant chemotherapy. Predictions of 3-year OS were fairly similar to observed OS with a calibration slope of 0.72. Statistical updating of the model resulted in an increase of the C-statistic from 0.63 to 0.65 (95% CI 0.64-0.65), ranging from 0.62 to 0.67 across different countries. The area under the curve for the prediction of 3 -year OS was 0.71 after updating. Median OS was 36, 25 and 15 months for the low, intermediate and high risk group, respectively (P < 0.001).Conclusions: This large international study validated and updated the Amsterdam model for survival prediction after pancreatoduodenectomy for pancreatic cancer. The model incorporates readily available variables with a fairly accurate model performance and robustness across different countries, while novel markers may be added in the future. The risk groups and web-based calculator www pancreascalculaior. corn may facilitate use in daily practice and future trials. (C) 2019 Elsevier Ltd, BASO The Association for Cancer Surgery, and the European Society of Surgical Oncology. All rights reserved. Show less
Testing of ecological, biogeographic and phylogenetic hypotheses of mycorrhizal traits requires a comprehensive reference data set about plant mycorrhizal associations. Here we present a database,... Show moreTesting of ecological, biogeographic and phylogenetic hypotheses of mycorrhizal traits requires a comprehensive reference data set about plant mycorrhizal associations. Here we present a database, FungalRoot, which summarizes publicly available data about vascular plant mycorrhizal type and intensity of root colonization by mycorrhizal fungi, accompanied with rich meta-data. We compiled and digitized data about plant mycorrhizal colonization in nine wide-spread languages. The present version of the FungalRoot database contains 36,303 species-by-site observations for 14,870 plant species, tripling the previously available compiled information about plant mycorrhizal associations. Based on these data, we provide a recommended list of genus-level plant mycorrhizal associations, based on the majority of data for species and careful analysis of conflicting data. The majority of ectomycorrhizal and ericoid mycorrhizal plants are trees (92%) and shrubs (85%), respectively. The majority of arbuscular and non-mycorrhizal plant species are herbaceous (50% and 70%, respectively). Our publicly available database is a powerful resource for mycorrhizal scientists and ecologists. It features possibilities for dynamic updating and addition of data about plant mycorrhizal associations. The new database will promote research on plant and fungal biogeography and evolution, and on links between above- and belowground biodiversity and ecosystem functioning. Show less
He, J.; Wink, S.; Bont, H. de; Le Dévédec, S.; Zhang, Y.; Water, B. van de 2019
Enhanced expression and activity of protein kinases are critical in tumor cell proliferation and cancer progression. These various cancer-related kinases form intricate interdependent signaling... Show moreEnhanced expression and activity of protein kinases are critical in tumor cell proliferation and cancer progression. These various cancer-related kinases form intricate interdependent signaling networks. Evaluation of the effect of various kinase inhibitors on these networks is critical to understand kinase inhibitor efficacy in cancer therapy. The dynamic activation of some kinases can be monitored by fluorescence resonance energy transfer (FRET) biosensors with high temporal resolution. Here, we established a FRET biosensor-based high throughput imaging approach to determine ERK and AKT activity in two triple negative breast cancer (TNBC) cell lines HCC1806 and Hs578T. FRET functionality was systematically evaluated using EGF stimulation and different MEK and AKT inhibitors, respectively. Next, we assessed the effect of a kinase inhibitor library containing >350 different kinase inhibitors (KIs) on ERK and AKT kinase activity using a FRET high-throughput screening setting. Suppression of FRET-ERK activity was generally positively correlated with the proliferation phenotype against inhibitors targeting MAPK signaling in both cell lines containing FRET-ERK reporter. AKT inhibitor (AKTi) resistant HCC1806 showed decreased proliferation associated with downregulated dynamics of FRET-ERK when treated with KIs targeting protein receptor tyrosine kinase (RTK). Yet, MEK inhibitor (MEKi) resistant Hs578T showed positively correlated FRET-AKT and proliferative responses against different PI3K and AKT inhibitors. Altogether, our data demonstrate the feasibility to integrate high throughput imaging-based screening of intracellular kinase activity using FRET-based biosensors in assessing kinase specificity and possible signaling crosstalk in direct relation to therapeutic outcome. Show less
Triple-negative breast cancer (TNBC) constitutes a small subtype (~15%) of breast cancer, but causes the majority of breast cancer-related deaths. As defined by the absence of ER and PR expression... Show moreTriple-negative breast cancer (TNBC) constitutes a small subtype (~15%) of breast cancer, but causes the majority of breast cancer-related deaths. As defined by the absence of ER and PR expression and HER2 overexpression, TNBC is not curable by hormone receptor or HER2-targeted therapies. Furthermore, TNBC is highly heterogeneous and most aggressive. To date, cytotoxic chemotherapy remains the mainstay in the management of TNBC. Despite the initial response to the standard-of-care chemotherapy, TNBC often exhibits intrinsic or acquired drug resistance, and subsequently, recurs in local and distal organs. Targeted therapies have long been pursued for the treatment of TNBC, but rarely demonstrate satisfactory clinical outcomes. Therefore, improved understanding of the intricate biological basis underlying TNBC insensitivity to targeted agents and defining new therapeutic opportunities are of the upmost importance. The aim of the studies presented in this thesis was to systematically identify gene/kinase susceptibilities of refractory TNBC cells, and reveal novel potent targeted therapies for TNBC as monotherapy or in combination with approved kinase drugs. Show less
He, J.; McLaughlin, R.P.; Noord, V.E. van der; Foekens, J.A.; Martens, J.W.M.; Westen, G.J.P. van; ... ; Water, B. van de 2019
Owing to its genetic heterogeneity and acquired resistance, triple-negative breast cancer (TNBC) is not responsive to single-targeted therapy, causing disproportional cancer-related death worldwide... Show moreOwing to its genetic heterogeneity and acquired resistance, triple-negative breast cancer (TNBC) is not responsive to single-targeted therapy, causing disproportional cancer-related death worldwide. Combined targeted therapy strategies to block interactive oncogenic signaling networks are being explored for effective treatment of the refractory TNBC subtype.A broad kinase inhibitor screen was applied to profile the proliferative responses of TNBC cells, revealing resistance of TNBC cells to inhibition of the mammalian target of rapamycin (mTOR). A systematic drug combination screen was subsequently performed to identify that AEE788, an inhibitor targeting multiple receptor tyrosine kinases (RTKs) EGFR/HER2 and VEGFR, synergizes with selective mTOR inhibitor rapamycin as well as its analogs (rapalogs) temsirolimus and everolimus to inhibit TNBC cell proliferation.The combination treatment with AEE788 and rapalog effectively inhibits phosphorylation of mTOR and 4EBP1, relieves mTOR inhibition-mediated upregulation of cyclin D1, and maintains suppression of AKT and ERK signaling, thereby sensitizing TNBC cells to the rapalogs. siRNA validation of cheminformatics-based predicted AEE788 targets has further revealed the mTOR interactive RPS6K members (RPS6KA3, RPS6KA6, RPS6KB1, and RPS6KL1) as synthetic lethal targets for rapalog combination treatment.TOR signaling is highly activated in TNBC tumors. As single rapalog treatment is insufficient to block mTOR signaling in rapalog-resistant TNBC cells, our results thus provide a potential multi-kinase inhibitor combinatorial strategy to overcome mTOR-targeted therapy resistance in TNBC cells. Show less
McLaughlin, R.P.; He, J.; Noord, V.E. van der; Redel, J.; Foekens, J.A.; Martens, J.W.M.; ... ; Water, B. van de 2019
The effective treatment of triple-negative breast cancer (TNBC) remains a profound clinical challenge. Despite frequent epidermal growth factor receptor (EGFR) overexpression and reliance on... Show moreThe effective treatment of triple-negative breast cancer (TNBC) remains a profound clinical challenge. Despite frequent epidermal growth factor receptor (EGFR) overexpression and reliance on downstream signalling pathways in TNBC, resistance to EGFR-tyrosine kinase inhibitors (TKIs) remains endemic. Therefore, the identification of targeted agents, which synergise with current therapeutic options, is paramount.Compound-based, high-throughput, proliferation screening was used to profile the response of TNBC cell lines to EGFR-TKIs, western blotting and siRNA transfection being used to examine the effect of inhibitors on EGFR-mediated signal transduction and cellular dependence on such pathways, respectively. A kinase inhibitor combination screen was used to identify compounds that synergised with EGFR-TKIs in TNBC, utilising sulphorhodamine B (SRB) assay as read-out for proliferation. The impact of drug combinations on cell cycle arrest, apoptosis and signal transduction was assessed using flow cytometry, automated live-cell imaging and western blotting, respectively. RNA sequencing was employed to unravel transcriptomic changes elicited by this synergistic combination and to permit identification of the signalling networks most sensitive to co-inhibition.We demonstrate that a dual cdc7/CDK9 inhibitor, PHA-767491, synergises with multiple EGFR-TKIs (lapatinib, erlotinib and gefitinib) to overcome resistance to EGFR-targeted therapy in various TNBC cell lines. Combined inhibition of EGFR and cdc7/CDK9 resulted in reduced cell proliferation, accompanied by induction of apoptosis, G2-M cell cycle arrest, inhibition of DNA replication and abrogation of CDK9-mediated transcriptional elongation, in contrast to mono-inhibition. Moreover, high expression of cdc7 and RNA polymerase II Subunit A (POLR2A), the direct target of CDK9, is significantly correlated with poor metastasis-free survival in a cohort of breast cancer patients. RNA sequencing revealed marked downregulation of pathways governing proliferation, transcription and cell survival in TNBC cells treated with the combination of an EGFR-TKI and a dual cdc7/CDK9 inhibitor. A number of genes enriched in these downregulated pathways are associated with poor metastasis-free survival in TNBC.Our results highlight that dual inhibition of cdc7 and CDK9 by PHA-767491 is a potential strategy for targeting TNBC resistant to EGFR-TKIs. Show less
