Poster T5 - YAP/TAZ Activity Regulates a Mechano-Metabolic Crosstalk during 3D Breast Cancer Invasion

Saturday, October 26, 2024

10:00 AM - 11:00 AM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

AK

Adil Khan

PhD Candidate
The University of Texas at Dallas
Dallas, Texas, United States

Co-Author(s)

HA

Haider Ali

Undergraduate Student
The University of Texas at Dallas, United States
HR

Hrishika Rai

PhD Candidate
The University of Texas at Dallas, United States
BK

Bishant Karki

Undergraduate Student
The University of Texas at Dallas, United States

Last Author(s)

Jacopo Ferruzzi, PhD (he/him/his)

Assistant Professor
The University of Texas at Dallas
Richardson, Texas, United States

Introduction: The invasion of breast cancer cells into surrounding tissues is driven by both cell autonomous and micro-environmental factors. The interaction of cancer cells with the surrounding extra-cellular matrix (ECM) are multifaceted, and involve mechanical forces, biochemical signals and metabolic exchanges. With cancer progression, the collagen-rich ECM undergoes densification and alignment, creating fibrous pathways that facilitate cancer cell invasion [1]. This structural remodeling of the ECM is a hallmark of aggressive tumors and is closely linked to poor prognosis in breast cancer patients, likely through biophysical mechanisms [2]. In addition, metabolism is profoundly dysregulated in breast cancer, with cancer cells exhibiting heightened rates of glycolysis and oxidative phosphorylation [3]. This metabolic reprogramming supports the energetic and biosynthetic demands of cancer cells, thereby facilitating their growth and subsequent metastatic spread.
The transcriptional coactivators yes-associated protein (YAP) and the transcriptional co-activator with PDZ-binding motif (TAZ) are integral to the cellular response to mechanical signals [4] and to metabolic pathways [5]. In response to both mechanical and metabolic cues, YAP and TAZ translocate into the nucleus, where they activate the transcription of genes involved in cell proliferation, survival and migration. Despite the established roles of YAP and TAZ in mechanotransduction and metabolism, it is yet unknown how YAP/TAZ activity drives remodeling of the ECM and metabolic reprogramming of cancer cells in the context of breast cancer invasion. This study aims to fill this gap by exploring how activation of nuclear YAP/TAZ coordinates ECM mechanics with cell metabolism to facilitate invasion in complex 3D micro-environments.

Materials and

Methods: Spheroids consisting of approximately 103 cells were formed with either control MCF-10A cells or with doxycycline-inducible nuclear mutants TAZ (4SA) and YAP (5SA) [6]. 48 hours post formation, these spheroids were embedded in a 3D rat-tail collagen I network of varying concentrations (1-4 mg/ml) using established protocols [7] and cultured in MCF-10A growth media supplemented either with (+Doxy) or without (-Doxy) doxycycline. To monitor invasion over time, the spheroids were imaged using a Nikon AX confocal microscope equipped with an environment chamber (37°C, 5% CO2) and a 10× objective. Invasion dynamics were quantified using brightfield images, while confocal reflection images (using an excitation wavelength of 488nm) were collected to capture the structural remodeling of surrounding collagen. Matrix displacements due to spheroid-generated traction forces were measured from time-lapse movies to compare collective contractility [8]. The plastic micro-scale changes in ECM mechanics induced by spheroid invasion were tracked using 3D-Magnetic Twisting Rheometry (3D-MTR), a novel method developed by our laboratory. For simplicity, the collagen network was modeled as a neo-Hookean material W=c⁄2 (I_c-3) and the associated Young’s modulus E=2c(1+ν) was used to report local material properties.
To access metabolic phenotypes associated with YAP/TAZ activation, we cultured each cell type as a 2D monolayer, and performed a Seahorse Assay followed by a Click-iT EdU cell proliferation assay to compare rates of glycolysis, oxidative phosphorylation and cell proliferation. Spatio-temporal variations in cellular metabolism during 3D spheroid invasion were mapped out using Fluorescence Lifetime Imaging (FLIM) of the metabolic cofactor NAD(P)H using published methods [9].

Results, Conclusions, and Discussions: Activation of YAP/TAZ in the MCF-10A cells resulted in larger spheroids, with EdU nuclear incorporation confirming an increased rate of cell proliferation. Post embedding in low density (1-2 mg/ml) collagen, the control MCF-10A spheroids invaded collectively with modest matrix remodeling, while YAP/TAZ activated spheroids invaded more aggressively, both as contiguous multicellular strands and as single cells. Quantification of spheroid generated contractile forces shows that post 4 days of invasion, the matrix displacements are modest in control MCF-10A spheroids while the TAZ (4SA) mutants display the largest extent of matrix densification and radial alignment. These differences in cellular contractility translate into a heterogenous material stiffening of the collagen matrix with YAP/TAZ activation, as measured by 3D-MTR.
The Seahorse Assay performed on 2D monolayers of cells shows that the activation of YAP leads to an increase in glycolytic activity, while that of TAZ increases both oxidative respiratory capacity and glycolytic activity. These results are confirmed in a 3D setting, wherein FLIM measurements on invading spheroids with YAP/TAZ activation show a reduction in the ratio of free to bound NAD(P)H. With invasion, spatial heterogeneities arise in the spheroids. The core shows an optical signature consistent with glycolysis, while the collectively invading strands and the peripheral single cells adopt oxidative and glycolytic phenotypes, respectively. Confirming the metabolic patters observed with invasion, pharmacological inhibition of glycolysis (2-deoxyglucose, 5mM) and oxidative phosphorylation (rotenone/antimycin-A, 50nM each) result in the spheroids favoring collective or single-celled invasion.
In summary, tumor invasion is a complex phenomenon that depends on a structurally and mechanically remodeled ECM and on metabolic plasticity of cancer cells. Here, we show that the activation of nuclear YAP/TAZ impacts breast cancer spheroid proliferation regardless of ECM density, while migration, generation of contractile forces and collagen remodeling are especially favored in low collagen density. We also show that activation of nuclear YAP/TAZ alters the metabolic profiles of tumor spheroids in a spatially and temporally heterogeneous manner. With regards to invasion, collectively invading cells display a tendency for oxidative phosphorylation while single cell invasion appears to be fueled by a mixed oxidative and glycolytic metabolism.

Acknowledgements (Optional):