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Cell-based platform to test Aquaporins as targets for anticancer drug discovery
Publication . Pimpão, Catarina; Rodrigues, Maria da Graça Tavares Rebelo de Soveral; Santos, Nuno Fernando Duarte Cordeiro Correia dos
Aquaporins (AQPs) are transmembrane protein channels that facilitate the bidirectional transport of water, glycerol, H2O2, and other small solutes across cell membranes, contributing to essential physiological processes such as renal water reabsorption, skin hydration and metabolism. However, AQPs have been implicated in various pathological conditions, with their dysregulated expression being associated with metabolic disorders, inflammation and cancer. In particular, AQP1, AQP3 and AQP5 are overexpressed in multiple cancers such as hepatocellular carcinoma and pancreatic ductal adenocarcinoma (PDAC), facilitating key oncogenic processes including cell proliferation, migration and invasion. Our research group has reported the influence of AQP3 and AQP5 on the modulation of cell migration, cell biophysical and biomechanical properties in PDAC cells, highlighting the importance of these proteins in cancer development. Recent studies have proposed that AQPs may act as transceptors, exhibiting a dual transporter/receptor activity in cell membranes. Beyond their classical role as water channels, AQPs can modulate cancer-associated signaling pathways, interact with cytoskeleton elements and regulate cell-cell adhesion proteins, supporting their critical involvement in cancer biology and representing promising therapeutic targets for anticancer drug discovery. In this thesis, we aimed to investigate the individual contribution of AQP1, AQP3 and AQP5 in cancer-related cellular processes and discover novel potent and selective AQP inhibitors with anticancer activity. In addition, we studied the impact of AQP3 and AQP5 on PDAC progression using 3D cell cultures, considering their reported role in the modulation of biophysical and biomechanical properties in PDAC cells. We first sought to elucidate the individual implication of AQP1, AQP3 and AQP5 on crucial cellular processes for cancer progression. To address the challenge of cancer cells expressing multiple AQP isoforms, we developed a cell-based platform using HEK-293T cells individually overexpressing human AQP1, AQP3 or AQP5 to investigate the specific contribution of each AQP in the modulation of cell morphology, cell stiffness, cell-cell adhesion and cell biology. We found that AQP1, AQP3 and AQP5 have distinct roles in the regulation of cell stiffness and cell-cell adhesion, with AQP5 being the only AQP isoform capable of influencing cell migration and cell morphology. We hypothesized that these differences could be attributed to AQPs transceptor activity, probably interacting with the cytoskeleton or regulating the expression of cell-cell adhesion proteins. Thus, we assessed the impact of AQP1, AQP3 and AQP5 on the expression of cell-cell adhesion proteins and epithelial-mesenchymal transition (EMT) markers, as well as the role of AQP1 and AQP3 in collective cell migration. AQP1, AQP3 and AQP5 differentially regulated cell-cell adhesion proteins expression, without inducing EMT in HEK-293T cells, and AQP1 and AQP3 did not affect collective cell migration, but induced changes in the velocity of migrating cell sheets. Importantly, AQP3 triggered the expression of E-cadherin in the cell membrane of HEK-293T cells, which inherently do not express this adhesion protein. Considering the potential of AQPs as pharmacological targets, we focused on identifying efficient AQP inhibitors and evaluating their therapeutic potential. A new series of organogold compounds were assessed for their inhibitory effect on yeast cells expressing human AQP10, which pathophysiological functions are poorly characterized and with no reported inhibitors. Au(CCON)Cl2 irreversibly inhibited human AQP10, representing a valuable strategy to elucidate its physiological roles and involvement in disease mechanisms. These organogold compounds were then tested in various cellular models expressing AQP3, including red blood cells, AQP3-overexpressing HEK-293T cells and melanoma cell lines. All the organogold compounds inhibited AQP3, with Au(CNHN)Cl2 being the most potent, impairing. melanoma cell proliferation, migration and adhesion by blocking AQP3 peroxiporin activity. Moreover, we evaluated the inhibitory effect of the natural compound rottlerin on AQP1 and AQP3 activity in red blood cells and yeast cells overexpressing human AQP1 and AQP3, which revealed strong inhibition of AQP3 activity. However, when tested in HEK-293T cells overexpressing AQP1, AQP3 and AQP5, its selectivity towards AQP3 was not confirmed. Lastly, we investigated the implications of AQP3 and AQP5 on the development of spheroids derived from PDAC cell lines, using AQP3 inhibitors and individually silencing AQP3 and AQP5. Treating PDAC spheroids with the AQP3 inhibitors Auphen and DFP00173 affected spheroid morphology and growth, with similar effects observed for AQP3- and AQP5-silenced PDAC spheroids. Overall, this thesis supports the hypothesis that AQPs contribute to tumorigenesis through their transceptor activity, with AQP1, AQP3 and AQP5 playing distinct roles in cancer related cellular processes. AQP3 and AQP5 exhibited opposite actions in the modulation of cell stiffness and cell-cell adhesion, suggesting their coordinated regulatory mechanism in cancer. The newly identified AQP inhibitors could deepen our understanding of the role of AQPs in pathophysiology and also contribute to the development of therapies targeting pathologies with AQP overexpression. Finally, AQP3 and AQP5 were revealed to be critical in PDAC spheroid growth, reinforcing the important transceptor activity of these AQPs in cancer and their relevance as promising therapeutic targets.
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Fundação para a Ciência e a Tecnologia
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OE
Número da atribuição
2020.04974.BD