Hypoxia-Responsive HIF-1α-Dependent RhoA Signaling Promotes Motility and Aerotaxis in Polyaneuploid PC3 Cancer Cells with Increased Nuclear Rounding.

Most cancer deaths result from metastasis, yet only a subset of tumor cells can complete this process. Polyaneuploid cancer cells (PACCs), which arise via endoreplication under stressors such as hypoxia, are implicated as metastatic drivers, but how they acquire this capacity remains unclear. Here, we show that under extreme self-generated hypoxia, the polyaneuploid subset of PC3 prostate cancer cells upregulates a hypoxia-responsive, HIF-1α-dependent RhoA signaling axis that promotes aerotaxis, a behavior predictive of intravasation during metastasis. We observe an enrichment of PACCs using a membrane-based culture system in which extreme, self-generated radial oxygen gradients emerge as cellular oxygen demand exceeds supply, recapitulating key features of the tumor microenvironment. Under these conditions, PACCs exhibited elevated HIF-1α and RhoA expression relative to non-PACCs. Disruption of either HIF-1α or RhoA attenuates aerotaxis, with RhoA expression suppressed upon HIF-1α inhibition, suggesting a functional HIF-1α- dependent RhoA pathway selectively enhanced in the PACC state. Quantitative analysis of nuclear morphology further reveals that nuclear circularity and solidity, morphology metrics associated with nuclear rounding and boundary organization, increase with and saturate at high nuclear area. Notably, the hypoxic PACC population exhibits significantly greater nuclear circularity and solidity compared to non-PACCs, suggesting increased rounding and stabilization of the enlarged nuclear architecture characteristic of PACCs under extreme hypoxia. Together, these findings reveal a new functional role for HIF-1α- dependent RhoA signaling in regulating aerotaxis in polyaneuploid cancer cells, which may enable aerotactic escape from hypoxic tumor cores and entry into oxygen-rich vasculature. Additionally, the association between elevated RhoA signaling and increased nuclear circularity at larger nuclear area suggests a potential link between hypoxia-responsive migration and maintenance of enlarged nuclear architecture in the PACC state. Collectively, these characteristics highlight PACCs as a hypoxia-adapted subpopulation with relevance for anti-metastatic therapeutic strategies.