| 1. |
- Gordon, Emma J., et al.
(författare)
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The endothelial adaptor molecule TSAd is required for VEGF-induced angiogenic sprouting through junctional c-Src activation
- 2016
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Ingår i: Science Signaling. - : American Association for the Advancement of Science (AAAS). - 1945-0877 .- 1937-9145. ; 9:437
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Tidskriftsartikel (refereegranskat)abstract
- Activation of vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) by VEGF binding is critical for vascular morphogenesis. In addition, VEGF disrupts the endothelial barrier by triggering the phosphorylation and turnover of the junctional molecule VE-cadherin, a process mediated by the VEGFR2 downstream effectors T cell-specific adaptor (TSAd) and the tyrosine kinase c-Src. We investigated whether the VEGFR2-TSAd-c-Src pathway was required for angiogenic sprouting. Indeed, Tsad-deficient embryoid bodies failed to sprout in response to VEGF. Tsad-deficient mice displayed impaired angiogenesis specifically during tracheal vessel development, but not during retinal vasculogenesis, and in VEGF-loaded Matrigel plugs, but not in those loaded with FGF. The SH2 and proline-rich domains of TSAd bridged VEGFR2 and c-Src, and this bridging was critical for the localization of activated c-Src to endothelial junctions and elongation of the growing sprout, but not for selection of the tip cell. These results revealed that vascular sprouting and permeability are both controlled through the VEGFR2-TSAd-c-Src signaling pathway in a subset of tissues, which may be useful in developing strategies to control tissue-specific pathological angiogenesis.
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| 2. |
- Kundu, Snehangshu, et al.
(författare)
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Linking FOXO3, NCOA3, and TCF7L2 to Ras pathway phenotypes through a genome-wide forward genetic screen in human colorectal cancer cells
- 2018
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Ingår i: Genome Medicine. - : Springer Science and Business Media LLC. - 1756-994X .- 1756-994X. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- Background:The Ras pathway genes KRAS, BRAF, or ERBBs have somatic mutations in similar to 60% of human colorectal carcinomas. At present, it is unknown whether the remaining cases lack mutations activating the Ras pathway or whether they have acquired mutations in genes hitherto unknown to belong to the pathway.Methods:To address the second possibility and extend the compendium of Ras pathway genes, we used genome-wide transposon mutagenesis of two human colorectal cancer cell systems deprived of their activating KRAS or BRAF allele to identify genes enabling growth in low glucose, a Ras pathway phenotype, when targeted.Results:Of the 163 recurrently targeted genes in the two different genetic backgrounds, one-third were known cancer genes and one-fifth had links to the EGFR/Ras/MAPK pathway. When compared to cancer genome sequencing datasets, nine genes also mutated in human colorectal cancers were identified. Among these, stable knockdown of FOXO3, NCOA3, and TCF7L2 restored growth in low glucose but reduced MEK/MAPK phosphorylation, reduced anchorage-independent growth, and modulated expressions of GLUT1 and Ras pathway related proteins. Knockdown of NCOA3 and FOXO3 significantly decreased the sensitivity to cetuximab of KRAS mutant but not wild-type cells.Conclusions:This work establishes a proof-of-concept that human cell-based genome-wide forward genetic screens can assign genes to pathways with clinical importance in human colorectal cancer.
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| 3. |
- Li, Xiujuan, et al.
(författare)
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Suppressed Vascular Leakage and Myocardial Edema Improve Outcome From Myocardial Infarction
- 2020
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Ingår i: Frontiers in Physiology. - : Frontiers Media SA. - 1664-042X. ; 11
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Tidskriftsartikel (refereegranskat)abstract
- Aim: The acute phase of myocardial infarction (MI) is accompanied by edema contributing to tissue damage and disease outcome. Here, we aimed to identify the mechanism whereby vascular endothelial growth factor (VEGF)-A induces myocardial edema in the acute phase of MI to eventually promote development of therapeutics to specifically suppress VEGFA-regulated vascular permeability while preserving collateral vessel formation.Methods and Results: VEGFA regulates vascular permeability and edema by activation of VEGF receptor-2 (VEGFR2), leading to induction of several signaling pathways including the cytoplasmic tyrosine kinase c-Src. The activated c-Src in turn phosphorylates vascular endothelial (VE)-cadherin, leading to dissociation of endothelial adherens junctions. A particular tyrosine at position 949 in mouse VEGFR2 has been shown to be required for activation of c-Src. Wild-type mice and mice with phenylalanine replacing tyrosine (Y) 949 in VEGFR2 (Vegfr2Y949F/Y949F) were challenged with MI through permanent ligation of the left anterior descending coronary artery. The infarct size was similar in wild-type and mutant mice, but left ventricular wall edema and fibrinogen deposition, indicative of vascular leakage, were reduced in the Vegfr2Y949F/Y949F strain. When challenged with large infarcts, the Vegfr2Y949F/Y949F mice survived significantly better than the wild-type strain. Moreover, neutrophil infiltration and levels of myeloperoxidase were low in the infarcted Vegfr2Y949F/Y949F hearts, correlating with improved survival. In vivo tyrosine phosphorylation of VE-cadherin at Y685, implicated in regulation of vascular permeability, was induced by circulating VEGFA in the wild-type but remained at baseline levels in the Vegfr2Y949F/Y949F hearts.Conclusion: Suppression of VEGFA/VEGFR2-regulated vascular permeability leads to diminished edema without affecting vascular density correlating with improved myocardial parameters and survival after MI.
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| 4. |
- Li, Xiujuan, et al.
(författare)
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VEGFR2 pY949 signalling regulates adherens junction integrity and metastatic spread
- 2016
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- The specific role of VEGFA-induced permeability and vascular leakage in physiology and pathology has remained unclear. Here we show that VEGFA-induced vascular leakage depends on signalling initiated via the VEGFR2 phosphosite Y949, regulating dynamic c-Src and VE-cadherin phosphorylation. Abolished Y949 signalling in the mouse mutant Vegfr2(Y949F/Y949F) leads to VEGFA-resistant endothelial adherens junctions and a block in molecular extravasation. Vessels in Vegfr2(Y949F/Y949F) mice remain sensitive to inflammatory cytokines, and vascular morphology, blood pressure and flow parameters are normal. Tumour-bearing Vegfr2(Y949F/Y949F) mice display reduced vascular leakage and oedema, improved response to chemotherapy and, importantly, reduced metastatic spread. The inflammatory infiltration in the tumour micro-environment is unaffected. Blocking VEGFA-induced disassembly of endothelial junctions, thereby suppressing tumour oedema and metastatic spread, may be preferable to full vascular suppression in the treatment of certain cancer forms.
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| 5. |
- Mazumder, Mohit, et al.
(författare)
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Prediction and Analysis of Canonical EF Hand Loop and Qualitative Estimation of Ca2+ Binding Affinity
- 2014
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 9:4, s. e96202-
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Tidskriftsartikel (refereegranskat)abstract
- The diversity of functions carried out by EF hand-containing calcium-binding proteins is due to various interactions made by these proteins as well as the range of affinity levels for Ca2+ displayed by them. However, accurate methods are not available for prediction of binding affinities. Here, amino acid patterns of canonical EF hand sequences obtained from available crystal structures were used to develop a classifier that distinguishes Ca2+-binding loops and non Ca2+-binding regions with 100% accuracy. To investigate further, we performed a proteome-wide prediction for E. histolytica, and classified known EF-hand proteins. We compared our results with published methods on the E. histolytica proteome scan, and demonstrated our method to be more specific and accurate for predicting potential canonical Ca2+-binding loops. Furthermore, we annotated canonical EF-hand motifs and classified them based on their Ca2+-binding affinities using support vector machines. Using a novel method generated from position-specific scoring metrics and then tested against three different experimentally derived EF-hand-motif datasets, predictions of Ca2+-binding affinities were between 87 and 90% accurate. Our results show that the tool described here is capable of predicting Ca2+-binding affinity constants of EF-hand proteins. The web server is freely available at http://202.41.10.46/calb/index.html.
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| 6. |
- Nordling, Torbjörn E. M., et al.
(författare)
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Identification of Biomarkers and Signatures in Protein Data
- 2015
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Ingår i: 2015 IEEE 11Th International Conference On E-Science. - 9781467393256 ; , s. 411-419
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Konferensbidrag (refereegranskat)abstract
- The correct diagnosis of cancer patients conventionally depends on the pathologist's experience and ability to distinguish cancer tissue from normal tissue under a microscope. Advances in technology for measuring the abundance of, e.g., proteins and mRNAs in tissue samples make it interesting to search for an optimal subset of these for classification of samples as cancer or normal. We discuss issues of identification of biomarkers that provide distinct signatures for prediction of tissues as cancer or normal, exemplified by our recent study of cancer signalling signatures in human colon cancer characterised with regards to protein abundance using high sensitivity isoelectric focusing. We show that the optimal subset for separation of cancer tissues from normal tissues does not contain any of the proteins in the top quintile in terms of significant difference between the groups according to Mann-Whitney U-test or correlation to the diagnosis. Actually, one of the proteins belongs to the tertile with the lowest significance and correlation. This highlights the weakness of the practice of only looking for significant differences in the abundance of individual proteins and raises the question of how many lifesaving discoveries that have been missed due to it. We also demonstrate how Monte Carlo simulations of the separation with random class assignment can be used to calculate p-values for observing any specific separation by chance and selection of the optimal number of proteins in the subset based on these p-values. Both selection of the optimal number of biomarkers and calculation of p-values corrected for multiple hypothesis testing are essential to obtain a subset of biomarkers that yield robust predictions for clinical use.
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| 7. |
- Padhan, Narendra, et al.
(författare)
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High sensitivity isoelectric focusing to establish a signaling biomarker for the diagnosis of human colorectal cancer
- 2016
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Ingår i: BMC Cancer. - : Springer Science and Business Media LLC. - 1471-2407 .- 1471-2407. ; 16
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Tidskriftsartikel (refereegranskat)abstract
- Background: The progression of colorectal cancer (CRC) involves recurrent amplifications/mutations in the epidermal growth factor receptor (EGFR) and downstream signal transducers of the Ras pathway, KRAS and BRAF. Whether genetic events predicted to result in increased and constitutive signaling indeed lead to enhanced biological activity is often unclear and, due to technical challenges, unexplored. Here, we investigated proliferative signaling in CRC using a highly sensitive method for protein detection. The aim of the study was to determine whether multiple changes in proliferative signaling in CRC could be combined and exploited as a "complex biomarker" for diagnostic purposes. Methods: We used robotized capillary isoelectric focusing as well as conventional immunoblotting for the comprehensive analysis of epidermal growth factor receptor signaling pathways converging on extracellular regulated kinase 1/2 (ERK1/2), AKT, phospholipase C gamma 1 (PLC gamma 1) and c-SRC in normal mucosa compared with CRC stage II and IV. Computational analyses were used to test different activity patterns for the analyzed signal transducers. Results: Signaling pathways implicated in cell proliferation were differently dysregulated in CRC and, unexpectedly, several were downregulated in disease. Thus, levels of activated ERK1 (pERK1), but not pERK2, decreased in stage II and IV while total ERK1/2 expression remained unaffected. In addition, c-SRC expression was lower in CRC compared with normal tissues and phosphorylation on the activating residue Y418 was not detected. In contrast, PLC gamma 1 and AKT expression levels were elevated in disease. Immunoblotting of the different signal transducers, run in parallel to capillary isoelectric focusing, showed higher variability and lower sensitivity and resolution. Computational analyses showed that, while individual signaling changes lacked predictive power, using the combination of changes in three signaling components to create a "complex biomarker" allowed with very high accuracy, the correct diagnosis of tissues as either normal or cancerous. Conclusions: We present techniques that allow rapid and sensitive determination of cancer signaling that can be used to differentiate colorectal cancer from normal tissue.
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| 8. |
- Padhan, Narendra
(författare)
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Highly Sensitive and Quantitative Detection of Proteins and Their Isoforms by Capillary Isoelectric Focusing Method
- 2018
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Ingår i: Journal of Visualized Experiments. - : JOURNAL OF VISUALIZED EXPERIMENTS. - 1940-087X. ; :139
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Tidskriftsartikel (refereegranskat)abstract
- Immunoblotting has become a routine technique in many laboratories for protein characterization from biological samples. The following protocol provides an alternative strategy, capillary isoelectric focusing (cIEF), with many advantages compared to conventional immunoblotting. This is an antibody-based, automated, rapid, and quantitative method in which a complete western blotting procedure takes place inside an ultrathin capillary. This technique does not require a gel to transfer to a membrane, stripping of blots, or x-ray films, which are typically required for conventional immunoblotting. Here, proteins are separated according to their charge (isoelectric point; pl), using less than a microliter (400 nL) of total protein lysate. After electrophoresis, proteins are immobilized onto the capillary walls by ultraviolet light treatment, followed by primary and secondary (horseradish peroxidase (HRP) conjugated) antibody incubation, whose binding is detected through enhanced chemiluminescence (ECL), generating a light signal that can be captured and recorded by a charge-coupled device (CCD) camera. The digital image can be analyzed and quantified (peak area) using software. This high throughput procedure can handle 96 samples at a time; is highly sensitive, with protein detection in the picogram range; and produces highly reproducible results because of automation. All of these aspects are extremely valuable when the quantity of samples (e.g., tissue samples and biopsies) is a limiting factor. The technique has wider applications as well, including screening of drugs or antibodies, biomarker discovery, and diagnostic purposes.
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| 9. |
- Padhan, Narendra, et al.
(författare)
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Highly sensitive and specific protein detection via combined capillary isoelectric focusing and proximity ligation
- 2017
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Ingår i: Scientific Reports. - : NATURE PUBLISHING GROUP. - 2045-2322. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- Detection and quantification of proteins and their post-translational modifications are crucial to decipher functions of complex protein networks in cell biology and medicine. Capillary isoelectric focusing together with antibody-based detection can resolve and identify proteins and their isoforms with modest sample input. However, insufficient sensitivity prevents detection of proteins present at low concentrations and antibody cross-reactivity results in unspecific detection that cannot be distinguished from bona fide protein isoforms. By using DNA-conjugated antibodies enhanced signals can be obtained via rolling circle amplification (RCA). Both sensitivity and specificity can be greatly improved in assays dependent on target recognition by pairs of antibodies using in situ proximity ligation assays (PLA). Here we applied these DNA-assisted RCA techniques in capillary isoelectric focusing to resolve endogenous signaling transducers and isoforms along vascular endothelial growth factor (VEGF) signaling pathways at concentrations too low to be detected in standard assays. We also demonstrate background rejection and enhanced specificity when protein detection depended on binding by pairs of antibodies using in situ PLA, compared to assays where each antibody preparation was used on its own.
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| 10. |
- Schmidt, Linnéa, et al.
(författare)
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Case-specific potentiation of glioblastoma drugs by pterostilbene
- 2016
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Ingår i: Oncotarget. - : Impact Journals, LLC. - 1949-2553. ; 7:45, s. 73200-73215
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Tidskriftsartikel (refereegranskat)abstract
- Glioblastoma multiforme (GBM, astrocytoma grade IV) is the most common malignant primary brain tumor in adults. Addressing the shortage of effective treatment options for this cancer, we explored repurposing of existing drugs into combinations with potent activity against GBM cells. We report that the phytoalexin pterostilbene is a potentiator of two drugs with previously reported anti-GBM activity, the EGFR inhibitor gefitinib and the antidepressant sertraline. Combinations of either of these two compounds with pterostilbene suppress cell growth, viability, sphere formation and inhibit migration in tumor GBM cell (GC) cultures. The potentiating effect of pterostilbene was observed to a varying degree across a panel of 41 patient-derived GCs, and correlated in a case specific manner with the presence of missense mutation of EGFR and PIK3CA and a focal deletion of the chromosomal region 1p32. We identify pterostilbene-induced cell cycle arrest, synergistic inhibition of MAPK activity and induction of Thioredoxin interacting protein (TXNIP) as possible mechanisms behind pterostilbene's effect. Our results highlight a nontoxic stilbenoid compound as a modulator of anticancer drug response, and indicate that pterostilbene might be used to modulate two anticancer compounds in well-defined sets of GBM patients.
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