Temperature and WNK-SPAK/OSR1 Kinases Dynamically Regulate Antiviral Human GFP-MxA Biomolecular Condensates in Oral Cancer Cells

Phase-separated membraneless biomolecular condensates in the cytoplasm or nucleus are now recognized to play a major role in modulating diverse functions in mammalian cells, and contribute to cancer pathogenesis. Mechanisms that regulate the partitioning of components into condensed vs dispersed phases in intact cells, and those that trigger spheroid to fibril transition of condensate structure have attracted attention. We selected a common circumstance for our investigations - all of us episodically imbibe cold and warm drinks such as water, tea, or coffee subjecting our oral epithelial cells to stresses of hypotonicity and temperature. Moreover, oral cancer, in the absence of overt causes such as tobacco or alcohol, most frequently occurs in a U-shaped zone (floor of mouth, side of tongue, anterior fauces and retromolar region) reflecting the path of liquid transit through the mouth. In previous studies, we investigated the partitioning of the broad-spectrum antiviral human MxA protein (also called Mx1) between condensate (storage granule) and dispersed (antivirally active) phases in human oral cancer cells. We had observed that at 37oC, in OECM1 oral carcinoma cells, GFP-MxA condensates were exquisitely sensitive to hypotonicity – these disassembled within 1-2 min of exposure of cells to saliva-like one-third hypotonicity, and underwent spontaneous reassembly in the next 5-7 min even when continued in hypotonic medium. In the present studies we investigated whether this process was temperature sensitive representative of cold vs warm drinks. It was slowed at 5oC, and speeded up at 50oC. The involvement in this disassembly/reassembly process of WNK-SPAK/OSR1 serine-threonine kinase pathway, which regulates water and Na, K and Cl influx and efflux, was evaluated using pathway inhibitors WNK463, WNK-IN-11 and closantel. The pan-WNK inhibitor WNK463 inhibited disassembly, while the SPAK/OSR1 inhibitor closantel markedly slowed reassembly. Unexpectedly, the WNK1-selective inhibitor (WNK-IN-11), triggered dramatic and rapid (within 1 hr) spheroid to fibril transition of GFP-MxA condensates in live cells. The latter cells retained their antiviral phenotype against vesicular stomatitis virus. Thus, overall, cellular water fluxes and temperature changes dramatically but reversibly affected the GFP-MxA condensate landscape in oral epithelial cells. The WNK-SPAK/OSR1 kinases appear to be part of a mechanism for condensate recovery and restoration. The data raise a novel condensate-dysregulation hypothesis for understanding the occurrence of oral cancer along the liquid transit pathway in the mouth.