Introduction

The discipline of mechanobiology has been gaining attention over the past decade. Seminal papers have demonstrated the influence of culture substrate stiffness on cell migration¹ and stem cell differentiation².  Multiple players integrate mechanical cues from the cellular environment into the transcriptional programme of cells, such as mechanosensitive transcriptional factors like YAP/TAZ⁵. Recent work revealed that extracellular matrix (ECM) stiffness also regulates cellular metabolism^6–8 and antioxidant sensitivity of cancer cells⁹. This preprint by Torrino and colleagues focuses on the influence of the mechanical properties of the ECM on the formation of biomolecular condensates.

Biomolecular condensates are assemblies of molecules that concentrate biomolecules by liquid-liquid phase separation¹⁰. These membrane-less assemblies facilitate homeostatic responses at different scales¹¹ and play a fundamental role in the formation of solid aggregates and fibrils, which are implicated in diseases like Alzheimer’s and cancer¹². In cancer, ECM stiffening is now a well-recognized phenomenon that contributes to tumour progression and metastasis^13,14. However, how this stiffening may fine-tune metabolism to regulate condensate formation and the follow-up pathological transcriptional activity poses an open question that this preprint tries to tackle.

Key findings

Matrix stiffening enhances phase separation of biomolecular condensates by activating the polyol pathway

To investigate the influence of ECM stiffness on protein condensate formation, the authors cultured breast cancer cells on hydrogels of different stiffnesses (1 to 50 kPa) and observed the presence of YAP/TAZ aggregates which undergo phase separation. Higher ECM stiffness promoted increased number of cells bearing condensates, increased condensate number per cell and enhanced condensate intensity. Active reduction of contraction on the stiff substrate reduced condensate density similarly to growth on soft substrates.

Previous studies showed that glucose metabolism responds to mechanical cues with glycolysis being upregulated on stiffer substrates⁷. Hence the authors examined glucose metabolism in attempt to understand what mechanism may facilitate condensate precipitation. Stable tracing of [U-¹³C] glucose in cells cultured on substrates of different stiffnesses revealed increased glucose influx and higher levels of glucose-derived sorbitol, a well-known crowding agent, in response to matrix stiffening. Moreover, there was a dose-dependent relationship between stiffness and intracellular sorbitol levels.

Altogether, these results demonstrate that ECM stiffness promotes higher biological condensates formation by upregulating sorbitol production in the polyol pathway.

Mechano-dependent biomolecular condensate formation relies on intracellular sorbitol

To explain the molecular mechanism of condensate stabilization by sorbitol, the authors employed a multiscale modelling approach of molecular dynamics simulations. The approach involved simulations on two length-scales – at atomistic and residue resolution. First, atomistic potential-of-mean-force (PMF) simulations allowed to examine the interaction strength between amino acid pairs at different glucose and sorbitol concentrations. Simulations demonstrated that sorbitol changes the properties of the solvent in two ways. In presence of 100 mM sorbitol, the solvent acquires a higher affinity for π-rich residues and lower affinity for charged and spacer residues, which strengthens associative electrostatic interactions within the protein. At residue resolution, the authors used the PMF binding energies to simulate the interactions of a large set of proteins in a coarse-grained residue interaction simulation. Here, they discovered that the addition of sorbitol, but not glucose, increased the critical temperature of TAZ condensates, promoting more stable TAZ protein condensates.

To validate the prediction of their simulations, the authors next performed phase separation assays with different purified proteins known to form condensates. Consistent with their in-silico results, higher sorbitol concentrations increased solution turbidity and promoted droplet aggregation of the GFP-tagged proteins. Further, to examine if mechano-induced protein condensates depend on the mechanosensitive polyol pathway, the authors manipulated sorbitol concentrations in cells cultured on soft vs stiff substrates by silencing enzymes involved in sorbitol synthesis and conversion. Inhibiting sorbitol’s conversion into fructose or inhibiting glucose conversion into sorbitol, respectively led to increased or decreased accumulation of TAZ condensates in cells cultured on soft substrates and affected promoter occupancy by TAZ.

In summary, the authors show by simulations and experiments that upregulation of sorbitol is sufficient to promote condensate formation and that manipulating the polyol pathway modulates condensate formation.

Matrix stiffening promotes sorbitol-dependent protein condensates in breast cancer models

To highlight the context where their findings are highly relevant, the authors examined the prevalence of biological condensates in biopsies of breast cancer patients. They could show that the number of observed aggregates correlates with collagen type I depositions as an indicator of ECM stiffening and with the number of ki67-positive, highly proliferative cells.  Thus, protein condensates seem to be associated with highly proliferative and remodelled breast cancers.

Lastly, the authors tested if the stiffening of the tumour niche instructs cellular metabolism to enhance condensate formation in an orthotropic mouse model of breast cancer. Pharmacological inhibition of the lysyl-oxidase, which crosslinks fibrillar collagens and promotes ECM stiffening, allowed the authors to decrease ECM cues and to observe the metabolic consequences of perturbed ECM stiffening on condensate formation and tumour growth. Impaired ECM stiffening led to decreased intertumoral sorbitol, decreased accumulation of condensates and, most importantly, diminished tumour cell proliferation.

These results demonstrate the physiological relevance of this preprint’s findings in the context of breast cancer and a possible therapeutic application of modulating ECM stiffness in vivo to curb tumour growth.

What I like about this preprint

This preprint is fascinating to me because it reveals yet another aspect of how physical properties of the extracellular environment profoundly modulate metabolism. I am intrigued by biomolecular condensates, more so in the context of ageing and age-related diseases, and this work improves our fundamental understanding of the regulation of condensate formation. Lastly, the final experiment of the paper – in which the authors manipulated ECM stiffness in an animal and saw reduced severity of tumour progression – is a beautiful example of how mechanobiology-inspired therapeutical approaches can yield promising results and should be considered as cancer treatment strategies in future.

Questions for the authors

Since sorbitol increases macromolecular crowding, I was wondering what happens to the mechanical properties of the cells. Do their viscoelastic properties change due to increased sorbitol levels?

You showed that targeting ECM stiffness can reduce tumour growth in mouse. Can you envision a therapeutical intervention at the metabolic level, targeting the polyol pathway, instead of the ECM? If not, why would that be challenging?

What more global significance, besides in cancer, can you envision for the findings of your work? Can this mechanism be applied to understand the formation of pathological aggregates like amyloids?

References

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13. Levental, K. R. et al. Matrix Crosslinking Forces Tumor Progression by Enhancing Integrin Signaling. Cell 139, 891–906 (2009).
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Posted on: 7 September 2023 , updated on: 14 September 2023

doi: https://doi.org/10.1242/prelights.35514

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