Browsing by Author "Lin, Salwa"

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Regulation of Human Ether À Go-Go 1 (hEAG1) Potassium Channels
(University of Leicester, 2017-08) Lin, Salwa; Mitcheson, John
Human ether à go-go (hEAG1) channel is a voltage-gated potassium channel that is only expressed in brain tissue. This channel consists of four transmembrane α helices, in which each α subunit has six domains (S1-6), S1-4 as voltage sensors and S5 and S6 are pore-forming domains. When action potential generated and subsequently the membrane potentials (Vm) depolarized, this channel repolarizes the cell back to resting potential. To date, little is known about the physiological role of EAG1 channel. In regard to diseases, ~ 70% of cancers exhibit hEAG1 overexpression. hEAG1 channel contributes to cancer proliferation indirectly through control of cytoplasmic Ca2+ (Ca2+ i). The aberrant overexpression of this channel in cancers prompted us to study its regulation by Ca2+ and hemin. To study hEAG1 regulation, currents from hEAG1-expressing Xenopus oocytes were measured by two-electrode voltage clamp (TEVC) technique. The results showed that hEAG1 currents substantially inhibited in response to increased Ca2+ i with maximal inhibition of 93.2± 2.9 %. In addition, the gating properties of hEAG1 currents in I and T was changed, in which the activation was clearly slowed without exhibition of rectification. Whereas the activity of hEAG1 channel with a deletion of PAS-cap domain in I and T substantially potentiated with 9± 1.4–fold compared to the control currents. Surprisingly, we found that upon application of extracellular hemin (5 µM) into hEAG1-expressing oocytes, the channel current significantly reduced with percentage of maximal inhibition of 24.8±5.2%. The gating of hEAG1-expressing oocytes upon persistent application of extracellular hemin interestingly resembles the gating of hEAG1 inhibited by an increase in Ca2+ i. Injection of hemin (1 µM) into hEAG1-expressing oocytes, however, showed no current inhibition. This study provides the first evidence that hemin inhibits hEAG1 channel from the extracellular side.
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The Development of the Lung Metastatic Niche in Murine Breast Cancer
(University of Oxford, 2024-05-15) Lin, Salwa; Harris, Adrian L.; Banham, Alison H.; De Val, Sarah
Cancer metastasis causes 90% of cancer deaths. Patients with secondary lung metastases often show only modest responses to chemotherapy, immunotherapy, and anti-angiogenic therapy, and can develop resistance. Remodelling of the metastatic niche and the use of alternative, non-angiogenic, vascularisation pathways such as vessel co-option are thought to contribute to resistance to therapies. However, there is only limited knowledge about the molecular mechanism underlying metastatic niche formation, and the expression profile of genes driving tumour vessel co-option is poorly understood. In part, this is due to the paucity of in vitro and in vivo models that accurately recapitulate the metastatic niche in human cancers. Here, I developed a preclinical E0771 breast cancer (BC) lung metastasis mouse model with which I investigated tumour vessel development and the metastatic niche. First, MRI imaging and histological assessments were used to study vessel growth patterns, suggesting the predominant use of vessel co-option by metastases at an early stage of development, while the induction of angiogenesis was seen during the progression to subpleural metastases. Next, I combined metastatic niche labelling with single-cell RNA sequencing (scRNA-seq) to study the transcriptome of the stroma during metastatic progression. This analysis detected a shift in endothelial cell (EC) subtypes between early and late metastatic timepoints. Interestingly, co-opted capillaries were found to enhance their OXPHOS-related genes and downregulate genes linked to tight junctions, suggesting a hyperpermeability response that may facilitate tumour cell extravasation. Further analysis of this scRNA-seq data also identified cancer-associated mesenchymal cells (CAMCs) in the late metastatic niche, which acted as the primary source of the pro-angiogenic factor VEGFA. Lineage tracing analysis using Wt1-CreERT2;tdTomato reporter mice also examined whether pleural mesothelial cells exhibited pro-metastatic capacity, but suggested that they had little capacity, migrating only underneath the subpleura and few co-expressed CAMC markers. This finding was supported by the scRNA-seq data, which suggested that pleural mesothelial cells partially contribute to the growth of the subpleural metastases but were not the primary origin of the CAMC subtypes. In conclusion, analysis of this preclinical lung metastasis model has enabled me to demonstrate the temporal and spatial heterogeneity present within the metastatic niche and distal cells, and to interrogate the gene expression patterns involved in vessel co-opting and angiogenic metastases. This data could provide important information when selecting vascular drugs to target early vs late metastasis, whilst this extensive transcriptome dataset of lung stromal cells in the metastatic niche and distal regions can provide a basis for further research into the mechanisms involved.
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