Les cavéoles sont des invaginations en forme de coupelle à la membrane plasmique. Ces organelles multifonctionnelles jouent entre autres, un rôle clé dans la mécano-protection et la signalisation cellulaire. En effet, les cavéoles ont la faculté de s’aplanir en réponse à l’augmentation de la tension membranaire, afin de protéger la cellule des contraintes mécaniques. Les cavéoles jouant un rôle clé dans la signalisation cellulaire, nous avions émis l’hypothèse que le cycle mécano-dépendent de désassemblage/réassemblage des cavéoles constitue un interrupteur mécanique de certaines voies de signalisation. Ce projet consiste à élucider le mécanisme moléculaire responsable du contrôle de la voie de signalisation JAK-STAT par la mécanique des cavéoles. Dans ces travaux, nous avons pu démontré que la cavéoline-1 (Cav1), un constitutant essentiel des cavéoles est libérée et devient hautement mobile au niveau de la membrane plasmique. Considérant les propriétés de signalisation de Cav1, Nous avons testé l’effet du désassemblage des cavéoles sur la signalisation cellulaire. Un criblage à haut débit, nous a permis identifié la voie de signalisation JAK- STAT stimulée par l’IFN-α comme voie modèle pour cette étude. En effet, la transduction du signal JAK-STAT induit par l’IFN-α est modulée par la mécanique des cavéoles. Afin de disséquer le mécanisme moléculaire responsable du contrôle de la signalisation JAK-STAT par la mécanique des cavéoles, nous avons déterminé le rôle de Cav1 dans ce contrôle. Nous avons observé que Cav1 est un régulateur négatif de la phosphorylation de STAT3 dépendante de la kinase JAK1. De plus, nous avons démontré que Cav1 interagit avec JAK1 en fonction de la tension membranaire. Nous avons également démontré que cette interaction Cav1-JAK1 fait intervenir le « scaffolding domain » de Cav1 (CSD), et que celui-ci est responsable de l’abolition de l’activité kinase de JAK1. Par conséquent, l’interaction de Cav1 avec JAK1 empêche l’activation de STAT3 par la kinase JAK1. Ces résultats démontrent que les cavéoles sont des organelles de mécano-signalisation, qui, lors d’un stress mécanique, libèrent de la Cav1 non cavéolaire capable d’inactiver la kinase JAK1, empêchant ainsi, la transduction du signal JAK-STAT. / Caveolae are small cup-shaped plasma membrane invaginations. These multifunctional organelles play a key role in cell mechanoprotection and cell signaling. Indeed our laboratory reported that caveolae have the ability to flatten out upon membrane tension increase, protecting cells from mechanical strains. Since caveolae play a key role in cell signaling we hypothesized that the mechano-dependent cycle of caveolae disassembly/reassembly may constitute a mechanical switch for signaling pathways. In this project, we elucidated the molecular mechanism underlying the control of JAK-STAT signaling by caveolae mechanics. We showed that caveolin-1 (Cav1), an essential caveolar component is released and become highly mobile at the plasma membrane under mechanical stress. Considering that caveolae are important signaling hubs at the plasma membrane, we addressed the effects of the mechanical release of Cav1 on cell signaling. Using high throughput screening, we identified the JAK-STAT signaling pathway as a candidate. To further dissect the molecular mechanism underlying the control of JAK-STAT signaling by caveolae mechanics, we addressed the role of Cav1 in the control of JAK-STAT signaling stimulated by IFN-α. We found that Cav1 was a specific negative regulator of the JAK1 dependent STAT3 phosphorylation. Furthermore, the level of Cav1 interaction with JAK1 depended on mechanical stress. We could show that Cav1-JAK1 interaction was mediated by the caveolin scaffolding domain (CSD), abolishing JAK1 kinase activity, hence, interfering with STAT3 activation upon IFN-α stimulation. Altogether our results show that caveolae are mechanosignaling organelles that disassemble under mechanical stress, releasing non-caveolar Cav1, which binds to the JAK1 kinase and inhibits its catalytic activity, preventing thereby JAK-STAT signal transduction.

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Breast cancer metastasis of a variety of vital organs is a major cause of breast cancer mortality. Autophagy has a crucial role in the metastatic breast cancer progression. As a critical mechanotransducer in the Hippo signalling pathway, YAP regulates cell proliferation and promotes autophagy. Previous publications also demonstrated extracellular matrix could regulate the nucleo-cytoplasmic transport of YAP. However, how YAP signalling connects to the interplay of autophagy and mechanotransduction in breast cancer metastasis remains entirely unknown. Through rapamycin-induced autophagy on the metastatic triple negative breast cancer (TNBC) cells, we observed upregulated YAP transcriptional activity and YAP nuclear localization in TNBC. Thus, we reported that YAP nuclear localization regulates autophagy to promote TNBC metastasis. Culturing TNBC cells on PDMS plates with various matrix stiffness demonstrated that stiff matrix promoted the migration of metastatic breast cancer cells in a YAP-dependent mechanism. Therefore, we proposed that YAP mechanotransduction promotes the migration of metastatic breast cancer cells. Then, we advance in these directions by reporting autophagy-mediated YAP nuclear localization is regulated by the response to stiff matrix when TNBC cells were cultured on different matrix stiffness upon autophagy. In conclusion, we suggest autophagy and mechanotransduction mediates YAP nuclear localization together. These findings expand the unknown gap in the convergence of YAP mechanotransduction and autophagy in metastatic breast cancer. They suggest that metastatic breast cancer cells have the potential to exhibit different YAP signalling when they colonize on a secondary location with a distinct matrix stiffness from primary location. Our study further helped to understand YAP biology and the mechanism of breast cancer metastasis that will shed light on future YAP-targeting therapeutics for metastatic breast cancer. / Thesis / Master of Applied Science (MASc)

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Breast cancer metastasis to the brain is one of the most lethal forms of metastases. Metastasis is regarded as a non-random process governed by several biomechanical factors including tissue stiffness. As brain tissue is ultrasoft and extremely heterogeneous compared to breast cancer primary sites; how are breast cancer cells able to colonize the vastly different microenvironment of the brain? As a key protein of the Hippo pathway, YAP is regarded as a mechanotransducer that is sensitive to changes in substrate stiffness. Its biochemical activity is intertwined with Piezo1, a mechanosensitive ion channel activated through plasma membrane deformation. To impact cellular function, YAP enters the nucleus and binds to the TEAD transcription domain triggering downstream expression of proteins involved in cell motility, wound healing, and metastasis. In this work, triple-negative breast cancers (TNBC) were shown to experience greater migration rates on stiff surfaces compared to soft PDMS substrates. Concurrently, cells showed YAP nuclear localization in a stiffness dependent manner. Then, mechanical characterization of human brain tissue was performed to characterize the stiffness heterogeneity in the brain associated with region specific metastasis. Five to six regions of the brain from two different patients showed similar patterns of stiffness heterogeneity with the anterior regions being generally stiffer than posterior regions. As Piezo1 is directly linked with detecting changes in biomechanical stimuli, it was used as a readout of surface stiffness to examine if cells in the brain could detect different regional stiffnesses. Comparisons of grey and white matter showed no significant difference in Piezo1 expression. As a drug screening framework, molecular dynamic simulations were performed to evaluate drug efficacy on well-characterized inflammatory mediators that are implicated in metastasis. These findings contribute to understanding the gap in knowledge surrounding the interplay between tissue stiffness and YAP mechanotransduction in the context of breast-to-brain metastasis. / Thesis / Master of Applied Science (MASc) / Breast cancer is the most common cause of cancer related deaths in women particularly when it spreads to the brain. The brain is composed of many different sub-locations comprised of different proteins that can change the tissue’s stiffness. Breast cancer can detect these changes and become more aggressive in its growth using a combination of proteins such as yes associated protein (YAP) and Piezo1. How these proteins interact in the context of breast to brain cancer metastasis however is poorly understood. This project examined the effects of surface stiffness, on YAP, and Piezo1 activity to understand how breast cancer and brain cells react to changes in surface stiffness. Results showed that on stiff surfaces YAP activity affects cancer cell migration. Also, human brain tissue was found to vary in stiffness depending on the region examined. Future investigations may shed light on therapies that could take advantage of learnings in this area to better target the spread of breast cancer.

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