Prostate cancer is a leading cause of cancer-associated deaths in men over 60 years of age. Most patients are killed by tumor metastasis. Recent evidence has implicated a role of the tumor microenvironment and urokinase plasminogen activator (uPA) in cancer cell migration, invasion, and metastasis. Here, we examine the role of the Na⁺/H⁺ exchanger isoform 1 (NHE1) and uPA in DU 145 prostate cancer cell migration and colony formation. Knockout of NHE1 reduced cell migration. The effects of a series of novel NHE1/uPA hexamethylene-amiloride-based inhibitors with varying efficacy towards NHE1 and uPA were examined on prostate cancer cells. Inhibition of NHE1—alone, or with inhibitors combining NHE1 or uPA inhibition—generally did not prevent prostate cancer cell migration. However, uPA inhibition—but not NHE1 inhibition—prevented anchorage-dependent colony formation. Application of inhibitors at concentrations that only saturate uPA inhibition decreased tumor invasion in vivo. The results suggest that while knockout of NHE1 affects cell migration, these effects are not due to NHE1-dependent proton translocation. Additionally, while neither NHE1 nor uPA activity was critical in cell migration, only uPA activity appeared to be critical in anchorage-dependent colony formation of DU 145 prostate cancer cells and invasion in vivo.

Wnt signalling has been implicated as a driver of tumour cell metastasis, but less is known about which branches of Wnt signalling are involved and when they act in the metastatic cascade. Here, using a unique intravital imaging platform and fluorescent reporters, we visualised β-catenin/TCF-dependent and ATF2-dependent signalling activities during human cancer cell invasion, intravasation and metastatic lesion formation in the chick embryo host. We found that cancer cells readily shifted between states of low and high canonical Wnt activity. Cancer cells that displayed low Wnt canonical activity showed higher invasion and intravasation potential in primary tumours and in metastatic lesions. In contrast, cancer cells showing low ATF2-dependent activity were significantly less invasive both at the front of primary tumours and in metastatic lesions. Simultaneous visualisation of both these reporters using a double-reporter cell line confirmed their complementary activities in primary tumours and metastatic lesions. These findings might inform the development of therapies that target different branches of Wnt signalling at specific stages of metastasis.

Confined migration is essential for metastasis, yet how cells adapt their migratory and metabolic programs across stiffness-varying microenvironments remains unclear. We uncover a stiffness-dependent mechano-metabolic switch governing migration. In stiff microchannels, cells utilize the osmotic engine model (OEM), relying on NHE1 activity, front-polarization, and glycolysis. In soft microchannels, migration is OEM-independent and requires pyruvate-fueled oxidative phosphorylation (OxPHOS). This OxPHOS-driven motility depends on Arp3, β1-integrin and integrin-linked kinase, which increase membrane tension in confinement that in turn triggers TRPM7-mediated calcium influx and RhoA-/myosin-II contractility. Activating and polarizing NHE1, via overexpression, hypoxia or elevated viscosity, restore OEM- and glycolysis-dependent migration in soft microchannels, bypassing the need for actin polymerization in vitro and in chick embryos. Mitochondria addition reinstates Arp3 polarization and enhances migration in NHE1-overexpressing cells, enabling engagement of both mechanisms in vitro and in zebrafish. These findings uncover a previously unrecognized mechano-metabolic link, revealing that intracellular rewiring overrides stiffness-dependent metabolic demands.