Restoring mechanophenotype reverts malignant properties of ECM-enriched vocal fold cancer

Jasmin Kaivola, Karolina Punovuori, Megan R. Chastney, Yekaterina A. Miroshnikova, Hind Abdo, Fabien Bertillot, Fabian Krautgasser, Jasmin Di Franco, James R.W. Conway, Gautier Follain, Jaana Hagström, Antti Mäkitie, Heikki Irjala, Sami Ventelä, Hellyeh Hamidi, Giorgio Scita, Roberto Cerbino, Sara A. Wickström, Johanna Ivaska

Posted on: 19 December 2024

Preprint posted on 23 August 2024

Can restoring vocal fold mechanics combat cancer? This preprint explores how!

Selected by Teodora Piskova

Categories: biophysics, cancer biology, cell biology

Introduction

The extracellular matrix (ECM) drives cancer progression (1), but most relevant studies focus solely on solid tumours where cells are not exposed to significant forces. Unlike most cancers, vocal fold cancer (VFC) develops in a tissue that constantly experiences mechanical stress (in this case, from speaking and breathing). VFC is a rare yet aggressive cancer that grows in the squamous epithelium of the vocal cords (2). At advanced stages (T3 and T4), the vocal folds become immobile, leading to poor prognosis (3).

How cancer progresses in mechanically active tissues remains an open question. In this preprint, Kaivola and colleagues explore how ECM remodelling and mechanotransduction, the process of sensing and responding to mechanical forces, contribute to VFC progression. Importantly, they demonstrate that restoring normal tissue mechanics and motility could reverse the cancer’s aggressive features. This presents an exciting opportunity to target the mechanical environment of tumours as a novel anti-cancer therapeutic intervention.

Key Findings

Extracellular matrix remodelling alters adhesion structures and oncogenic features in vocal fold cancer

This study began by examining how ECM composition and mechanics differ between normal vocal fold tissue and VFC samples, categorized by cancer stage. In early stages (T1 and T2), the vocal folds move freely, while in advanced stages (T3 and T4), they become immobile. RNA sequencing revealed significant upregulation of ECM-related genes in cancerous tissues, including fibrillar collagens, fibronectin, and laminin-332. This ECM remodelling has consequences for the tissue mechanics and for cancer progression. Atomic force microscopy revealed that patient-derived T1 and T3 cell lines are three times stiffer compared to healthy tissue. In addition, proliferation assays on hydrogels of varying stiffness confirmed that ECM stiffening led to cancer cells exhibiting higher proliferation and migration.

Next, the authors focused on integrins – the membrane receptors that integrate the cellular cytoskeleton with the ECM. Laminin-binding integrins (α3, α6, and β4) were transcriptionally upregulated in the cancer cell lines. These integrins connect either the keratin cytoskeleton (α6β4 in hemidesmosomes), or the actin cytoskeleton (α3β1 and α6β1 in focal adhesions) with the ECM. Mass cytometry and microscopy experiments revealed that ECM-remodelling concurs with altered cell-cell and cell-ECM adhesion structures in VFC. Particularly, there were less hemidesmosomes and keratin 14 and more and smaller focal adhesions in cancer cells, while integrin α3 shifted from junctional or lamellipodial localization to endosome-like structures in advanced cancer cells.

Laminin-binding integrins lose their capacity to modulate junctional dynamics and motility in VFC

The authors then decided to explore the role of laminin-binding integrins at the cell-cell junctions in the context of VFC. The authors treated healthy and VFC cells with antibodies that block integrin α3, α6, and β1. In a 3D spheroid model, blocking integrins increased the area of healthy and T1 spheroids, suggesting reduced cell-cell compaction that allows for better spreading. T3 spheroids, however, showed no changes in spreading in response to integrin inhibition. Analysis of cell-cell junctions revealed that T3 cells had more straight and less tensile junctions, while those of healthy and T1 cells were finger-like and more tensile. These findings suggest that integrins contribute to junctional dynamics in healthy and T1 cells but lose this capacity in T3 cells. In addition, this study also found that blocking integrins (α3, α6, and β1) reduced the migratory velocity of healthy cells, but not that of T1 and T3 cells.

Phono-mimetic mechanical stimuli suppress oncogenic potential

Motivated by the observed differences in mechanical and adhesive properties, the authors sought to investigate how VFC cells respond to mechanical stimulation. The authors subjected these cells to uniaxial stretching and vibration. Stretching, which mimics the opening and closing of vocal folds, led to alignment of cells and their actin cytoskeleton in healthy and T1 cells, but not in T3 cells. Vibration caused actin stress fibre accumulation and monolayer remodelling in all cell types. Interestingly, prolonged vibration in T3 cells led to the extrusion of highly contractile cells, resembling cell extrusion in crowded epithelia—a phenomenon not observed in healthy or T1 cells.

One of the study’s most exciting findings is how these mechanical stimuli that mimic normal vocal fold movement influence oncogenic features in VFC cells. Both stretching and vibration reduced total and nuclear β-catenin levels in T1 and T3 cells. Similarly, this mechanical stimulation decreased total and nuclear levels of the oncogenic transcription factor YAP, which is known to be highly active in VFC. The ability to modulate cancer-associated transcription factors with adequate mechanical stimulation opens a new avenue to explore for potential therapy.

What I like about this preprint

To me, what stands out most about this preprint is its focus on restoring mechanical homeostasis as a potential anti-cancer intervention. This concept is both innovative and transformative, opening new avenues for cancer treatment. The study tackles the challenges of studying integrins, e.g. their functional redundancies and complex signalling pathways. This is a subject close to my heart, as I have also worked on projects examining the extracellular matrix and integrin signalling.

The authors’ approach to answer their research question is particularly exciting because it addresses cancer in a mechanically active tissue like the vocal folds. Mechanically active tissues behave differently than static ones, yet this distinction is often overlooked in cancer research. These findings may have broader implications, extending the acquired knowledge to other mechanically active systems such as cardiovascular and airway tissues.

Questions for the authors

1. On laminin isoforms:
Your study highlights the importance of laminin-332 in vocal fold cancer progression, but I noticed other laminin subunits (e.g., laminin-α1, α4, and β1) are also transcriptionally upregulated in your data. Do you have protein-level insights into which laminin isoforms are present in the healthy versus cancerous ECM? Could these have some important modulatory role for cancer progression, too?

2. On integrins at cell-cell junctions:
I find integrins at cell-cell junctions particularly intriguing. You observed less junctional integrin-α3 at advanced cancer stages without it relocating to ECM adhesions. Could you elaborate on possible functions of integrins at cell-cell junctions? Is this part of the recycling cycle, maybe acting as a reservoir, or could it involve interactions with adhesion structures?

3. On keratin network and migratory behaviour:
While T1 and T3 cells share similar focal adhesion-related characteristics, their migratory behaviours are distinct. What differs more prominently between T1 and T3 cells appears to be hemidesmosome-associated complexes and the keratin network. What role might the keratin network and its integration with the ECM play in influencing migratory behaviour in VFC?

References

1. Winkler, J., Abisoye-Ogunniyan, A., Metcalf, K. J. & Werb, Z. Concepts of extracellular matrix remodelling in tumour progression and metastasis. Nat. Commun. 11, 5120 (2020).
2. Schultz, P. Vocal fold cancer. Eur. Ann. Otorhinolaryngol., Head Neck Dis. 128, 301–308 (2011).
3. Hirano, M., Kurita, S., Matsuoka, H. & Tateishi, M. Vocal Fold Fixation in Laryngeal Carcinomas. Acta Oto-Laryngol. 111, 449–454 (1991).

Tags: cancer, ecm, mechanobiology

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