Adhesion G Protein-Coupled Receptor L4 (ADGRL4/ELTD1) is an endothelial cell adhesion G protein-c... more Adhesion G Protein-Coupled Receptor L4 (ADGRL4/ELTD1) is an endothelial cell adhesion G protein-coupled receptor (aGPCR) which regulates physiological and tumour angiogenesis, providing an attractive target for anti-cancer therapeutics. To date, ADGRL4/ELTD1′s full role and mechanism of function within endothelial biology remains unknown, as do its ligand(s). In this study, ADGRL4/ELTD1 silencing, using two independent small interfering RNAs (siRNAs), was performed in human umbilical vein endothelial cells (HUVECS) followed by transcriptional profiling, target gene validation, and metabolomics using liquid chromatography-mass spectrometry in order to better characterise ADGRL4/ELTD1′s role in endothelial cell biology. We show that ADGRL4/ELTD1 silencing induced expression of the cytoplasmic metabolic regulator ATP Citrate Lyase (ACLY) and the mitochondria-to-cytoplasm citrate transporter Solute Carrier Family 25 Member 1 (SLC25A1) but had no apparent effect on pathways downstream of...
Fatty acid binding protein 4 (FABP4) is a fatty acid chaperone, which is induced during adipocyte... more Fatty acid binding protein 4 (FABP4) is a fatty acid chaperone, which is induced during adipocyte differentiation. Previously we have shown that FABP4 in endothelial cells is induced by the NOTCH1 signalling pathway, the latter of which is involved in mechanisms of resistance to antiangiogenic tumour therapy. Here, we investigated the role of FABP4 in endothelial fatty acid metabolism and tumour angiogenesis. We analysed the effect of transient FABP4 knockdown in human umbilical vein endothelial cells on fatty acid metabolism, viability and angiogenesis. Through therapeutic delivery of siRNA targeting mouse FABP4, we investigated the effect of endothelial FABP4 knockdown on tumour growth and blood vessel formation. In vitro, siRNA-mediated FABP4 knockdown in endothelial cells led to a marked increase of endothelial fatty acid oxidation, an increase of reactive oxygen species and decreased angiogenesis. In vivo, we found that increased NOTCH1 signalling in tumour xenografts led to in...
Acquired resistance to lapatinib, an inhibitor of EGFR and HER2 kinases, is common. We found that... more Acquired resistance to lapatinib, an inhibitor of EGFR and HER2 kinases, is common. We found that reactivation of EGFR, HER2 and HER3 occurred within 24 hours of lapatinib treatment after their initial dephosphorylation. This was associated with increased expression of NRG1 in cells treated with lapatinib. Exogenous NRG1 partially rescued breast cancer cells from growth inhibition by lapatinib. In addition, both parental and lapatinib-resistant breast cancer cells were sensitive to SGP1, which inhibits binding of NRG1 and other HER3 ligands. Addition of pertuzumab to lapatinib further inhibited NRG1-induced signalling, which was not fully inhibited by either drug alone. In animal model, a combination of pertuzumab to lapatinib induced a greater tumor regression than either lapatinib or pertuzumab monotherapy. This novel combination treatment may provide a promising strategy in clinical HER2-targeted therapy and may inhibit a subset of lapatinib-resistant breast cancer, although the ...
ABSTRACT Introduction Delta-like 4 (DLL4) and Jagged1 (JAG1) are two key Notch ligands that are i... more ABSTRACT Introduction Delta-like 4 (DLL4) and Jagged1 (JAG1) are two key Notch ligands that are implicated in tumour angiogenesis. DLL4-Notch signalling inhibits sprouting but promotes tumour growth through better perfused vessels. In contrast to, very little is known about JAG1- Notch signalling in tumour angiogenesis and its influence on tumour growth and progression. Objective To study the functional difference between DLL4- and JAG1- Notch signalling in vitro and in vivo. Methods Endothelial cells (ECs) were cultured on DLL4 or JAG1 coated plates and subjected to quantitative PCR to study the regulation of Notch target genes. Angiogenic assays were employed to study the functional effects of these two ligands in ECs. U87 cell lines over-expressing murine DLL4 (mDLL4) or murine JAG1 (mJAG1) were generated via lentiviral transduction. The growth of these cells in vitro or as subcutaneous tumours in mice was compared to that of control wild-type U87 cell line. Changes in vascular morphology, proliferation (MIB-1), necrosis (H&E), and markers of hypoxia (CA9) were then assessed. Results & Discussion JAG1 was less potent than DLL4 in stimulation of Notch target genes in ECs. JAG1- and DLL4- Notch signaling had different effects on vessel formation. The growth of U87 mDLL4 and U87 mJAG1 cells was slower compared to wild-type U87 cell line in vitro, however mDLL4 and mJAG1 promoted U87 tumour growth compared to wild type control cells in vivo. Both ligands did not have any effect on U87 cell proliferation but significantly reduced tumoural necrosis compared to control tumours. U87 mDLL4 tumours were less hypoxic compared to control tumours. Interestingly, tumours over- expressing mDLL4 had less but larger vessels compared to control, whereas mJAG1 produced more yet functional vessels. Conclusion JAG1- and DLL4-Notch signalling have different effects on vessel formation, which impacted on the growth of the tumours in vivo.
In this paper, we present a new experimental methodology to combine mass spectrometry (NanoSIMS) ... more In this paper, we present a new experimental methodology to combine mass spectrometry (NanoSIMS) with fluorescence microscopy to provide subcellular information on the location of small molecules in cultured cells. We demonstrate this by comparing the distribution of 5-bromo-2-deoxyuridine in the same cells given by both NanoSIMS analysis and by fluorescence immunohistochemistry. Fiducial markers in the substrates ensured that the images formed by SIMS mapping of bromine ions could be co-registered exactly with images from fluorescence microscopy. The NanoSIMS was shown to faithfully reproduce the information from fluorescence microscopy, but at a much higher spatial resolution. We then show preliminary SIMS images on the distribution of ATN-224, a therapeutic copper chelator for which there is no fluorescent marker, co-registered with conventional Lysotracker and Hoechst stains on the same cells.
The ERBB3 gene is expressed as a 6.2- and a 1.4-kb transcript. The former encodes the full-length... more The ERBB3 gene is expressed as a 6.2- and a 1.4-kb transcript. The former encodes the full-length transmembrane protein and the latter a truncated extracellular fragment consisting of 140 amino acids of the c-erbB-3 protein followed by 43 unique residues. We have examined the expression of the two ERBB3 transcripts by Northern blotting in cancer cell lines and normal human fetal and adult tissues. We expressed the truncated receptor fragment and showed that it was glycosylated, probably with a single N-linked complex sugar chain, and that the protein was a 58-kDa disulphide-linked dimer. We were able to crosslink iodinated neuregulin (NRG)-1beta to the full-length solubilised receptor but not to the truncated dimeric protein. Using Western blot analysis, the truncated protein was shown to be present in cell lysates and, using immunoelectron microscopy, in vesicular structures within cells and associated with the plasma cell membrane.
Notch signaling is an evolutionary conserved pathway that is mediated by cell-cell contact. It is... more Notch signaling is an evolutionary conserved pathway that is mediated by cell-cell contact. It is involved in a variety of developmental processes and has an essential role in vascular development and angiogenesis. Delta-like 4 (Dll4) is a Notch ligand that is up-regulated during angiogenesis. It is expressed in endothelial cells and regulates the differentiation between tip cells and stalk cells of neovasculature. Here, we present evidence that Dll4 is incorporated into endothelial exosomes. It can also be incorporated into the exosomes of tumor cells that overexpress Dll4. These exosomes can transfer the Dll4 protein to other endothelial cells and incorporate it into their cell membrane, which results in an inhibition of Notch signaling and a loss of Notch receptor. Transfer of Dll4 was also shown in vivo from tumor cells to host endothelium. Addition of Dll4 exosomes confers a tip cell phenotype on the endothelial cell, which results in a high Dll4/Notch-receptor ratio, low Notch...
Tumor endothelial markers (TEMs) that are highly expressed in human tumor vasculature compared wi... more Tumor endothelial markers (TEMs) that are highly expressed in human tumor vasculature compared with vasculature in normal tissue hold clear therapeutic potential. We report that the C-type lectin CLEC14A is a novel TEM. Immunohistochemical and immunofluorescence staining of tissue arrays has shown that CLEC14A is strongly expressed in tumor vasculature when compared with vessels in normal tissue. CLEC14A overexpression in
Adhesion G Protein-Coupled Receptor L4 (ADGRL4/ELTD1) is an endothelial cell adhesion G protein-c... more Adhesion G Protein-Coupled Receptor L4 (ADGRL4/ELTD1) is an endothelial cell adhesion G protein-coupled receptor (aGPCR) which regulates physiological and tumour angiogenesis, providing an attractive target for anti-cancer therapeutics. To date, ADGRL4/ELTD1′s full role and mechanism of function within endothelial biology remains unknown, as do its ligand(s). In this study, ADGRL4/ELTD1 silencing, using two independent small interfering RNAs (siRNAs), was performed in human umbilical vein endothelial cells (HUVECS) followed by transcriptional profiling, target gene validation, and metabolomics using liquid chromatography-mass spectrometry in order to better characterise ADGRL4/ELTD1′s role in endothelial cell biology. We show that ADGRL4/ELTD1 silencing induced expression of the cytoplasmic metabolic regulator ATP Citrate Lyase (ACLY) and the mitochondria-to-cytoplasm citrate transporter Solute Carrier Family 25 Member 1 (SLC25A1) but had no apparent effect on pathways downstream of...
Fatty acid binding protein 4 (FABP4) is a fatty acid chaperone, which is induced during adipocyte... more Fatty acid binding protein 4 (FABP4) is a fatty acid chaperone, which is induced during adipocyte differentiation. Previously we have shown that FABP4 in endothelial cells is induced by the NOTCH1 signalling pathway, the latter of which is involved in mechanisms of resistance to antiangiogenic tumour therapy. Here, we investigated the role of FABP4 in endothelial fatty acid metabolism and tumour angiogenesis. We analysed the effect of transient FABP4 knockdown in human umbilical vein endothelial cells on fatty acid metabolism, viability and angiogenesis. Through therapeutic delivery of siRNA targeting mouse FABP4, we investigated the effect of endothelial FABP4 knockdown on tumour growth and blood vessel formation. In vitro, siRNA-mediated FABP4 knockdown in endothelial cells led to a marked increase of endothelial fatty acid oxidation, an increase of reactive oxygen species and decreased angiogenesis. In vivo, we found that increased NOTCH1 signalling in tumour xenografts led to in...
Acquired resistance to lapatinib, an inhibitor of EGFR and HER2 kinases, is common. We found that... more Acquired resistance to lapatinib, an inhibitor of EGFR and HER2 kinases, is common. We found that reactivation of EGFR, HER2 and HER3 occurred within 24 hours of lapatinib treatment after their initial dephosphorylation. This was associated with increased expression of NRG1 in cells treated with lapatinib. Exogenous NRG1 partially rescued breast cancer cells from growth inhibition by lapatinib. In addition, both parental and lapatinib-resistant breast cancer cells were sensitive to SGP1, which inhibits binding of NRG1 and other HER3 ligands. Addition of pertuzumab to lapatinib further inhibited NRG1-induced signalling, which was not fully inhibited by either drug alone. In animal model, a combination of pertuzumab to lapatinib induced a greater tumor regression than either lapatinib or pertuzumab monotherapy. This novel combination treatment may provide a promising strategy in clinical HER2-targeted therapy and may inhibit a subset of lapatinib-resistant breast cancer, although the ...
ABSTRACT Introduction Delta-like 4 (DLL4) and Jagged1 (JAG1) are two key Notch ligands that are i... more ABSTRACT Introduction Delta-like 4 (DLL4) and Jagged1 (JAG1) are two key Notch ligands that are implicated in tumour angiogenesis. DLL4-Notch signalling inhibits sprouting but promotes tumour growth through better perfused vessels. In contrast to, very little is known about JAG1- Notch signalling in tumour angiogenesis and its influence on tumour growth and progression. Objective To study the functional difference between DLL4- and JAG1- Notch signalling in vitro and in vivo. Methods Endothelial cells (ECs) were cultured on DLL4 or JAG1 coated plates and subjected to quantitative PCR to study the regulation of Notch target genes. Angiogenic assays were employed to study the functional effects of these two ligands in ECs. U87 cell lines over-expressing murine DLL4 (mDLL4) or murine JAG1 (mJAG1) were generated via lentiviral transduction. The growth of these cells in vitro or as subcutaneous tumours in mice was compared to that of control wild-type U87 cell line. Changes in vascular morphology, proliferation (MIB-1), necrosis (H&E), and markers of hypoxia (CA9) were then assessed. Results & Discussion JAG1 was less potent than DLL4 in stimulation of Notch target genes in ECs. JAG1- and DLL4- Notch signaling had different effects on vessel formation. The growth of U87 mDLL4 and U87 mJAG1 cells was slower compared to wild-type U87 cell line in vitro, however mDLL4 and mJAG1 promoted U87 tumour growth compared to wild type control cells in vivo. Both ligands did not have any effect on U87 cell proliferation but significantly reduced tumoural necrosis compared to control tumours. U87 mDLL4 tumours were less hypoxic compared to control tumours. Interestingly, tumours over- expressing mDLL4 had less but larger vessels compared to control, whereas mJAG1 produced more yet functional vessels. Conclusion JAG1- and DLL4-Notch signalling have different effects on vessel formation, which impacted on the growth of the tumours in vivo.
In this paper, we present a new experimental methodology to combine mass spectrometry (NanoSIMS) ... more In this paper, we present a new experimental methodology to combine mass spectrometry (NanoSIMS) with fluorescence microscopy to provide subcellular information on the location of small molecules in cultured cells. We demonstrate this by comparing the distribution of 5-bromo-2-deoxyuridine in the same cells given by both NanoSIMS analysis and by fluorescence immunohistochemistry. Fiducial markers in the substrates ensured that the images formed by SIMS mapping of bromine ions could be co-registered exactly with images from fluorescence microscopy. The NanoSIMS was shown to faithfully reproduce the information from fluorescence microscopy, but at a much higher spatial resolution. We then show preliminary SIMS images on the distribution of ATN-224, a therapeutic copper chelator for which there is no fluorescent marker, co-registered with conventional Lysotracker and Hoechst stains on the same cells.
The ERBB3 gene is expressed as a 6.2- and a 1.4-kb transcript. The former encodes the full-length... more The ERBB3 gene is expressed as a 6.2- and a 1.4-kb transcript. The former encodes the full-length transmembrane protein and the latter a truncated extracellular fragment consisting of 140 amino acids of the c-erbB-3 protein followed by 43 unique residues. We have examined the expression of the two ERBB3 transcripts by Northern blotting in cancer cell lines and normal human fetal and adult tissues. We expressed the truncated receptor fragment and showed that it was glycosylated, probably with a single N-linked complex sugar chain, and that the protein was a 58-kDa disulphide-linked dimer. We were able to crosslink iodinated neuregulin (NRG)-1beta to the full-length solubilised receptor but not to the truncated dimeric protein. Using Western blot analysis, the truncated protein was shown to be present in cell lysates and, using immunoelectron microscopy, in vesicular structures within cells and associated with the plasma cell membrane.
Notch signaling is an evolutionary conserved pathway that is mediated by cell-cell contact. It is... more Notch signaling is an evolutionary conserved pathway that is mediated by cell-cell contact. It is involved in a variety of developmental processes and has an essential role in vascular development and angiogenesis. Delta-like 4 (Dll4) is a Notch ligand that is up-regulated during angiogenesis. It is expressed in endothelial cells and regulates the differentiation between tip cells and stalk cells of neovasculature. Here, we present evidence that Dll4 is incorporated into endothelial exosomes. It can also be incorporated into the exosomes of tumor cells that overexpress Dll4. These exosomes can transfer the Dll4 protein to other endothelial cells and incorporate it into their cell membrane, which results in an inhibition of Notch signaling and a loss of Notch receptor. Transfer of Dll4 was also shown in vivo from tumor cells to host endothelium. Addition of Dll4 exosomes confers a tip cell phenotype on the endothelial cell, which results in a high Dll4/Notch-receptor ratio, low Notch...
Tumor endothelial markers (TEMs) that are highly expressed in human tumor vasculature compared wi... more Tumor endothelial markers (TEMs) that are highly expressed in human tumor vasculature compared with vasculature in normal tissue hold clear therapeutic potential. We report that the C-type lectin CLEC14A is a novel TEM. Immunohistochemical and immunofluorescence staining of tissue arrays has shown that CLEC14A is strongly expressed in tumor vasculature when compared with vessels in normal tissue. CLEC14A overexpression in
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Papers by H. Sheldon