Data and Materials Availability: data for the human-cow transcriptome comparison with and without co-culture with trophoblast cells is available under GSE136299 in the Gene Expression Omnibus (GEO) database of NCBI, https://www.ncbi.nlm.nih.gov/geo/. The comparative fibroblast gene expression data is available under PRJNA564062 under SUB6229748 and SUB6264591 on the Sequence Read Archive (SRA) of NCBI, https://www.ncbi.nlm.nih.gov/sra.
Purpose: Breast cancer remains a prominent global disease affecting women worldwide despite the emergence of novel therapeutic regimens. Metastasis is responsible for most cancer-related deaths, and acquisition of a mesenchymal and migratory cancer cell phenotypes contributes to this devastating disease. The utilization of kinase targets in drug discovery have revolutionized the eld of cancer research but despite impressive advancements in kinase-targeting drugs, a large portion of the human kinome remains under-studied in cancer. NEK5, a member of the Never-in-mitosis kinase family, is an example of such an understudied kinase. Here, we characterized the function of NEK5 in breast cancer.Methods: Stably overexpressing NEK5 cell lines (MCF-7) and shRNA knockdown cell lines (MDA-MB-231, TU-BcX-4IC) were utilized. Cell morphology changes were evaluated using immuno uorescence and quanti cation of cytoskeletal components. Cell proliferation was assessed by Ki-67 staining and transwell migration assays tested cell migration capabilities. In vivo experiments with murine models were necessary to demonstrate NEK5 function in breast cancer tumor growth and metastasis.Results: NEK5 activation altered breast cancer cell morphology and promoted cell migration independent of effects on cell proliferation. NEK5 overexpression or knockdown does not alter tumor growth kinetics but promotes or suppresses metastatic potential in a cell type speci c manner, respectively. Conclusion: While NEK5 activity modulated cytoskeletal changes and cell motility, NEK5 activity affected cell seeding capabilities but not metastatic colonization or proliferation in vivo. Here we characterized NEK5 function in breast cancer systems and we implicate NEK5 in regulating speci c steps of metastatic progression.
Among mammals, the extent of placental invasion is correlated with vulnerability to malignancy. Animals with more invasive placentation (e.g. humans) are more vulnerable to malignancy, whereas animals with a non-invasive placenta (e.g. ruminants) are less likely to develop malignant cancer. To explain this correlation, we propose the hypothesis of Evolved Levels of Invasibility (ELI) positing that the permissiveness of stromal tissue to invasion is a unitary character affecting both placental and cancer invasion. We provide evidence for this hypothesis by contrasting invasion of human and bovine cancer and placental cells into a lawn of stromal cells from different species. We find that both bovine endometrial and skin fibroblasts are more resistant to invasion of placental and cancer cells than their human counterparts. Gene expression profiling identified genes with high expression in human but not bovine fibroblasts. Knocking down of a subset of them in human fibroblasts leads to significantly stronger resistance to cancer cell invasion. Comparative analysis of gene expression among mammals suggests that humans evolved higher vulnerability to malignancy than the eutherian ancestor, possibly as a correlate of more invasive placentation, and boroeutherians evolved to decrease stromal invasibility. Identifying the evolutionary determinants of stromal invasibility can provide significant insights to develop rational anti-metastatic therapeutics. ResultsCollective cell invasion behavior can be modelled in an ECM-mimetic co-culture system
For further characterization of neonatal mesenteric alpha 1-adrenoceptor populations, an extracorporeal perfusion circuit was established to control intestinal blood flow in 0-2 day old piglets. Activation of alpha 1-adrenoceptors was first documented by observing dose-dependent increases in mesenteric perfusion pressure after intra-mesenteric arterial injection of methoxamine and noradrenaline. Peripheral intravenous injections of WB 4101 (a competitive alpha 1A-adrenoceptor antagonist), but not clorethylclonidine (CEC, an alpha 1B-adrenoceptor antagonist), significantly (P < 0.05, analysis of variance) blunted mesenteric vasoconstrictor responses to those agonists. That the mesenteric vasoconstrictor response to mesenteric plexus stimulation was unaltered by CEC, but was muted by both WB 4101 and SK&F 104856 (a post-junctional alpha 1- and alpha 2-adrenoceptor antagonist) suggests that pre- and post-junctional alpha 1A-adrenoceptors are present and functional at birth.
Recent advances in immune checkpoint blockade (ICB) inhibiting programmed death-1 (PD-1) and cytotoxic T-lymphocyte-associated protein (CTLA-4) have revolutionized the standard of care for cancer treatment. However, the limited response rates to ICB across multiple cancer types suggest that new approaches and targets are clearly needed to fully elucidate the underlying biology of dysfunctional and exhausted CD8 T cells in cancer in order to achieve durable responses (cure). G protein-coupled receptors (GPCRs) are the most intensively studied drug targets, primarily due to their druggability and relevance to most physiological processes and disease conditions. However, the role of GPCRs in cancer and particularly in cancer immunology has been largely underexplored. We have applied a new computational pipeline to cross-integrate hundreds of thousands of CD8 T cells from multiple single cell RNA-seq datasets from 13 distinct cancer types. Using this approach, we identified many GPCRs that are overrepresented in exhausted CD8 T cells. A new computational strategy enabled us to predict the G protein coupling specificity of most GPCRs. This led to the discovery that Gαs-coupled GPCRs are significantly enriched in exhausted CD8 T cells. These include EP2, EP4, A2AR, β1AR, and β2AR, and we found that all of these receptors promote CD8 T cell dysfunction by inhibiting cytotoxicity and cytokine secretion. However, these receptors are also expressed in other immune cells within the tumor microenvironment. To address the direct impact of these receptors on T cell function we used a novel synthetic biology approach, involving the development of a chemogenetic CD8-restricted Gαs-DREADD (Designer Receptor Exclusively Activated by A Designer Drug) transgenic mouse model in which activation of Gαs signaling is temporally and spatially controlled. Utilizing this Gαs-DREADD model, we discovered that the Gαs-signaling axis represents a previously uncharacterized signaling axis that dampens the anti-tumor CD8 T cell activity and leads to ICB immunotherapy failure. Our central hypothesis is that secretion of GPCR ligands in the tumor microenvironment and their actions on CD8 T cells lead to the activation of the Gαs signaling cascade leading to T cell dysfunction, by decreasing cytotoxic and migratory activity that abolished the effectiveness of ICB. Our findings reveal that Gαs-coupled GPCRs may represent new targetable immune checkpoints that can be combined with ICB as part of novel multimodal precision approaches to enhance the response to immunotherapies. Citation Format: Bryan S. Yung, Victoria H. Wu, Farhoud Faraji, Robert Saddawi-Konefka, Zhiyong Wang, Alexander T. Wenzel, Miranda Song, Meghana S. Pagadala, Lauren M. Clubb, Joshua Chiou, Sanju Sinha, Marin Matic, Francesco Raimondi, Thomas Hoang, Rebecca Berdeaux, Dario A. Vignali, Ramiro Iglesias-Bartolome, Hannah Carter, Eytan Ruppin, Jill P. Mesirov, J. Silvio Gutkind. A chemogenetic approach reveals a GPCR-Gαs-PKA signaling axis promoting T cell dysfunction and cancer immunotherapy failure [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2868.
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