Bioenergetic pathways regulate distinct patterns of invasion and matrix remodeling in breast cancer cells upon YAP/TAZ activation

Breast cancer invasion relies on complex interactions between intracellular signals and microenvironmental factors. Central to this process are the transcription factors YAP and TAZ, two key regulators of the Hippo signaling pathway. YAP/TAZ respond to mechanical and metabolic cues by translocating to the nucleus, where they regulate genetic programs that promote tumor progression. Within the tumor milieu, extracellular matrix (ECM) densification, alignment, and altered cell metabolism modulate the invasive phenotype of breast tumors. However, the role of YAP/TAZ activation in regulating ECM mechanics and cancer cell metabolism during invasion remains poorly understood. To address this gap, we utilized breast cancer spheroids to investigate how inhibition of either glycolysis or oxidative phosphorylation (OxPhos) influences invasion dynamics and ECM remodeling under inducible YAP/TAZ activation. MCF-10A breast cancer epithelial cells and doxycycline inducible nuclear mutants MCF-10A YAP(5SA) and TAZ(4SA) were used to generate spheroids and to conduct invasion assays in 3D collagen I. Control MCF-10A spheroids exhibited limited ECM remodeling and invaded collectively. In contrast, YAP/TAZ-activated spheroids displayed increased proliferation and invaded aggressively through a combination of collective protrusions and single cell migration. Structural and mechanical changes in collagen were monitored using 3D Magnetic Twisting Rheometry, a novel technique to assess heterogeneous stiffening of the tumor microenvironment. We found that YAP/TAZ activated spheroids aligned peritumoral collagen radially and stiffened the matrix. A Seahorse assay revealed that TAZ activation upregulates both glycolysis and OxPhos, while YAP activation primarily increases glycolysis. To explore metabolic heterogeneities during invasion, we used Fluorescence Lifetime Imaging Microscopy (FLIM) of the metabolic cofactor NAD(P)H. FLIM showed that, upon YAP/TAZ activation, the spheroid core exhibited an optical signature consistent with glycolysis, while the periphery adopted a mix of glycolysis and OxPhos. Pharmacological inhibition of metabolic pathways further elucidated the role of metabolism in spheroid invasion. In control spheroids, inhibiting either glycolysis or OxPhos hindered invasion. In contrast, in YAP/TAZ activated spheroids, inhibition of OxPhos shifted invasion from collective to single cell modality. Conversely, inhibition of glycolysis reduced single cell migration, shifting invasion towards collective migration. Taken together, our findings suggest that YAP/TAZ activation confers metabolic adaptability to invading spheroids, highlighting the crucial role of YAP/TAZ-mediated metabolic reprogramming in regulating breast cancer cell invasion modalities, as well as the associated changes in contractility and ECM remodeling.