京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第18838号 / 理博第4096号 / 新制||理||1589(附属図書館) / 31789 / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 阿形 清和, 教授 杤尾 豪人, 教授 森 和俊 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DFAM

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)

The Hippo tumor suppressor pathway is a highly conserved signaling pathway that regulates cell proliferation, differentiation, and organ size. Activation of the Hippo pathway leads to the phosphorylation and cytoplasmic sequestration of the pro-growth transcriptional co-activators YAP/TAZ; by contrast, impairment of the Hippo pathway enables YAP/TAZ to enter the nucleus where they bind to the TEAD transcription factors and induce the expression of genes involved in cell proliferation. Functional impairment of the Hippo pathway, and subsequent hyperactivation of YAP/TAZ, is common in many human malignancies, including melanoma. Recently, small molecule inhibitors that disrupt YAP/TAZ-TEAD binding, and thus reduce oncogenic transcriptional signaling have been discovered, but their efficacy in preventing cancer cell growth has not yet been well characterized. Moreover, no simple biomarker has been identified that can predict sensitivity to such inhibitors. We hypothesized that cells in which YAP/TAZ are enriched in the nucleus relative to the cytoplasm, indicative of an impaired Hippo pathway, would be more susceptible to TEAD inhibition. This would provide a useful biomarker to identify cancer cell lines most likely to respond to TEAD inhibition. We therefore developed and validated an automated quantification method to score nuclear:cytoplasmic YAP/TAZ localization in melanoma cell lines. This enabled us to identify “Hippo-On” and “Hippo-Off” signatures. We then treated these lines with the TEAD inhibitor MGH-CP1 and performed cell viability assays. Results from these data demonstrated that cell lines that have greater nuclear localization of YAP/TAZ are more susceptible to MGH-CP1 inhibition, suggesting that YAP/TAZ nuclear localization may be a biomarker to identify candidates for TEAD inhibitor treatment.

No description available.

Hippo signaling is a tumor suppressive signaling pathway that controls organ size by regulating cellular proliferation, apoptosis, and differentiation during development, regeneration, and homeostasis. The Hippo pathway inhibits transcriptional co-activators and Hippo pathway effectors YAP/TAZ, activation of which is often seen in cancer. Within the adult mammalian intestine, homeostasis of which requires intricate reciprocal interaction between the gut epithelium and adjacent mesenchyme, the Hippo-YAP pathway is crucial for intestinal epithelial homeostasis and regeneration. However, its role in adult mesenchymal homeostasis remains poorly understood. Here, I genetically dissect the role of mesenchymal Hippo-YAP signaling in adult intestinal homeostasis. I find that deletion of core kinases LATS1/2 or YAP activation in mesenchymal progenitor cells, but not terminally differentiated cells, disrupts signaling in the stem cell niche and mesenchymal homeostasis by inducing mesenchymal overgrowth and suppressing smooth muscle actin expression. Furthermore, inhibition of Hippo signaling in Gli1+ mesenchymal progenitors, the main source of Wnt ligands within the stem cell niche, stimulates Wnt ligand production and subsequent epithelial Wnt pathway activation, thereby driving epithelial regeneration following DSS-mediated injury as well as exacerbating APC-mediated tumorigenesis. Altogether, our data reveal a previously underappreciated requirement and the underlying mechanism for stromal Hippo-YAP signaling in adult intestinal homeostasis.

Les schwannomes sont des tumeurs bénignes se développant à partir d’une hyper-prolifération des cellules de Schwann suite à l’inactivation bi-allélique du gène NF2. Signe pathogonomique d’une pathologie rare et héréditaire, la Neurofibromatose de type 2 (NF2), ils peuvent aussi apparaître de façon sporadique. Hormis la chirurgie ou la radiothérapie, peu d’options pharmacologiques sont proposées aux patients porteurs de schwannomes, principalement à cause du peu de cibles thérapeutiques identifiées. Dans les cellules de Schwann, le phénotype cellulaire associé à la perte NF2 est une perte d’inhibition de la prolifération par le contact. Deux fonctions majeures de Merlin, produit de NF2, ont émergé au cours de ces dix dernières années. La première, démontrée par notre groupe, concerne la régulation de l’expression membranaire des récepteurs à activité tyrosine kinase (RTK) qui s’accumulent à la membrane plasmique des schwannomes humains. Le second implique Merlin dans la régulation de la voie de signalisation Hippo. Cette dernière, activée par le contact cellulaire réprime l’activité de deux co-facteurs de transcription, Yap et Taz, et régule ainsi et aussi l’inhibition de contact. Les mécanismes moléculaires par lesquels Merlin inhibe l’activité de Yap/Taz sont toutefois méconnus. Le but de nos études a été de déterminer une signature moléculaire associée à la croissance des schwannomes humains et l’importance relative de Yap/Taz. Dans une analyse protéomique à grande échelle sur des biopsies humaines, nous avons identifié à la fois l’activation spécifique de cinq RTKs que sont le PDGFRβ, Her2, Her3, Axl et Tie2 ainsi qu’une accumulation nucléaire spécifique de Yap. Nous montrons que sur la totalité des protéines étudiées, seules Yap, le PDGFRβ et P-Her3 corrèlent avec la prolifération des cellules de schwannomes humain. De plus, Yap induit la transcription des RTK activés (à l’exception de Tie2). Nous plaçons donc Yap au centre des mécanismes de régulation de la croissance des schwannomes humains et proposons que son inhibition pourrait représenter une nouvelle et prometteuse stratégie thérapeutique pour réduire la croissance de ces tumeurs. Nous apportons une nouvelle lecture des fonctions de Merlin, qui, par une potentielle interaction directe avec Yap, inhibe spécifiquement sa translocation dans le noyau indépendamment d’une régulation par la densité cellulaire ou par la voie Hippo. Par ailleurs, Merlin ne semble pas essentiel à l’activation de la voie Hippo dans les cellules de Schwann soulignant une fonction nouvelle et inattendue de Merlin dans la régulation de Yap et de la voie Hippo. Enfin nous avons étudié le rôle d’AmotL1, un puissant partenaire de Merlin et membre de la voie Hippo, dans la migration et la progression des cancers du sein. Nous mettons en évidence une fonction antagoniste de Merlin et AmotL1 dans la promotion de ces mécanismes soulignant une autre fonction nouvelle de Merlin en tant que suppresseur de la progression de cancers non associés à NF2. / Schwannomas are benign tumors arising from Schwann cell hyper-proliferation under NF2 bi-allelic inactivation. They appear in the context of a rare hereditary disease called Neurofibromatosis type 2 (NF2) or in sporadic cases. To this day, surgery and radiotherapy remain the only options to treat these patients, mainly due to the lack of therapeutical targets identified. The NF2 loss associated cellular phenotype is the loss of cell contact inhibition. Two main functions of Merlin, the NF2 product, have emerged in the last decade. The first was shown by our group and consists in the accumulation of tyrosine kinase receptors (RTK) at the plasma membrane in schwannomas. The second involves Merlin in the regulation of the Hippo signaling pathway. This pathway is activated by cell contact and inactivates a couple of transcription co-factors, Yap and Taz, then participating in cell contact inhibition of proliferation. However, the mechanisms by which Merlin inactivates Yap and Taz remain unknown. In our studies, we aimed to determine both the molecular signature of human schwannomas taking advantage of a large proteomic study, and the relative importance of Yap in the tumor suppressor function of Merlin. We could show both a specific activation of five RTKs : PDGFRβ, Her 3, Her2, Axl and Tie2 and a specific nuclear accumulation of Yap in human schwannoma. Among all the protein studied, Yap, PDGFRβ and P-Her3 are the only ones to correlate with the proliferation of human schwannoma cells. Furthermore, the activated RTK (excepted Tie2) are transcriptional targets of Yap. Hence, we found Yap as a pivotal regulator of schwannoma growth and proposed its inhibition as a new and promising therapeutical target to reduce human schwannoma growth. In addition, we show that Merlin specifically inhibits Yap nuclear translocation into the nucleus of Schwann cells by a direct interaction which is independent from the regulation by cell density and by the Hippo pathway. Moreover, Merlin expression seems not to be essential for Hippo activation in Schwann cells which brings a new and unexpected role of Merlin in Yap and Hippo regulation. In the end, we studied the role of AmotL1, a strong Hippo partner of Merlin in the migration and progression of breast cancer. We could show an antagonist function of Merlin and AmotL1 in the promotion of these mechanisms highlighting a new progression suppressor function of Merlin in cancer which are not linked to NF2 mutations.

L'intégrine β1 appartient à une large famille de récepteurs de première importance pour les interactions cellule/matrice extracellulaire. La délétion spécifique d'un régulateur négatif de l'intégrine β1, ICAP-1α, induit de sévères défauts osseux. Nous avons pu montrer que la perte d'ICAP-1α est accompagnée d'une augmentation de l'activité de l'intégrine β1, affectant le dépôt des matrices de fibronectine et de collagène de type I. De plus, nous avons pu montrer qu'ICAP-1α a une action antagoniste sur le recrutement de la kindline-2 au niveau du domaine cytoplasmique de l'intégrine β1 (Brunner et al. JCB 2011). Nous nous sommes ensuite intéressés au rôle de l'intégrine β1 elle-même dans l'ostéogenèse afin de comprendre comment les ostéoblastes intègrent les signaux du microenvironnement pour coordonner la formation et le remodelage osseux. Dans cette optique, nous avons généré un modèle de souris délétées pour l'intégrine β1 spécifiquement dans les ostéoblastes en cours de maturation. Ces souris présentent un sévère phénotype osseux caractérisé par des réductions importantes de la minéralisation et de la dynamique osseuse, ainsi que des déformations osseuses et des fractures rappelant le syndrome d'Ostéoporose Juvénile. L'analyse in vitro d'ostéoblastes n'exprimant pas l'intégrine β1 a révélé un défaut majeur de prolifération impliquant non pas la voie canonique MAPK/ERK mais plutôt un défaut d'activation du co-facteur de transcription YAP. De plus, nous avons pu montrer que les intégrines β1 régulaient le niveau d'AMP cyclique (AMPc) dans les ostéoblastes et que ceci était corrélé à l'inactivation de YAP. De même, nous avons pu relier l'inactivation de YAP à la dynamique d'endocytose des rafts. Finalement, des analyses in vivo et in vitro ont révélé un défaut fonctionnel des ostéoblastes dépourvus d'intégrine β1. Nous avons pu montrer que cette incapacité fonctionnelle était due à une réduction de la réponse au BMP-2, facteur de croissance ostéoblastique majeur, non pas au niveau de son récepteur mais probablement au niveau de l'activation des promoteurs BMP-dépendants. Nos résultats montrent ainsi que l'intégrine β1 est un régulateur clé de la prolifération ostéoblastique dépendante de YAP et de la signalisation BMP régulant la fonction ostéoblastique, la minéralisation et la formation osseuse. / Β1 integrins belong to a large family of receptors that have been shown to be of paramount importance for cell/extracellular matrix interactions. The ablation of the specific β1 integrin regulator ICAP-1α results in severe bone and mineralization defects. By combining mouse and cell biology we could demonstrate that loss of ICAP-1α was accompanied by an increase of β1 integrin activity that affects fibronectin and collagen deposition. Moreover, we could show that ICAP-1 is an important negative regulator of kindlin-2 recruitment on β1 integrin cytoplasmic domain (Brunner et al. JCB 2011). We then wanted to address the functional role of β1 integrin per se in osteogenesis and to understand how osteoblasts integrate environmental cues to coordinate bone formation and remodeling. For this we generated osteoblast specific β1 integrin deficient mice. These mice showed severe bone defects characterized by reduced bone mineralization and dynamic, as well as bending and fractures reminding human Juvenile Osteoporosis symptoms. In vitro analyses of β1 integrin deficient osteoblasts revealed proliferation defect which is not due to defective canonical MAPK/ERK pathway, but rather to defective activity of the co-transcription factor YAP. Then, we showed that β1 integrins are regulating cAMP level in osteoblasts and that the cAMP level correlates with YAP inactivation. We also linked YAP inactivation with raft endocytosis. Finally, in vivo and in vitro analyses revealed a functional incapacity of β1 integrin deficient osteoprecursors. We could show that the lazy phenotype of β1 integrin deficient osteoblasts is likely due to a reduced response to BMP signaling, a major osteoblast growth factor. Taken together, our findings demonstrate that β1 integrin is a key regulator of YAP-dependent osteoblast proliferation and BMP signaling allowing osteoblast functionality, mineralization and bone formation.

Bei verschiedenen Krebs-Typen kann die molekulare Behandlung der Krebs-Stammzellen ein effektives Ziel sein, die Therapie-Resistenz und die Metastasierung der Tumore zu hemmen. Basaler Brustkrebs enthält Zellen mit Stammzell-Eigenschaften; hingegen sind rationale Therapien gegen diese Zellen nur wenige etabliert. Ich zeige in meiner Doktorarbeit, dass Rezeptor-Tyrosinkinase-Met-Signalvermittlung die Aktivität der Hippo-Komponente YAP im basalem Brustkrebs verstärkt. Weitere Analysen zeigten erhöhte YAP-Aktivität in den Krebsstammzell-Populationen. Durch Verwendung genetischer und pharmakologischer Methoden konnte ich zeigen, dass die Interferenz mit YAP die Tumor-Bildung verzögert und die luminal-basale Transdifferenzierung verhindert sowie das Krebsstammzell-Überleben reduziert. Gen-Expressions-Analysen von YAP-Knockout-Brustdrüsen zeigten eine starke Reduzierung der Expression der β-Catenin-Zielgene, was indiziert, dass YAP für nukleare β-Catenin-Aktivität essentiell ist. Ich habe weiter gefunden, dass nukleares YAP mit β-Catenin und TEAD4 interagiert und an gemeinsamen regulatorischen Gene-Regionen überlappt. Die Analyse von proteomischen Daten von primären Brustkarzinomen des Menschen zeigte eine signifikante Hoch-Regulation der YAP-Signatur in basalem im Vergleich zu andern Brustkarzinom-Typen, was suggeriert, dass YAP-Aktivität auf den basalen Brustkrebs limitiert ist. Unsere Daten beleuchten die Wichtigkeit von YAP als essentiellen Tumor-Initiator und Krebs-Stammzell-Regulator beim basalen Brustkrebs und zeigt, dass dessen Aktivität als Prognose-Faktor wichtig ist, wenn man basalen mit luminalem Brustkrebs vergleicht. Dies suggeriert, dass Behandlung des YAP/TEAD4/β-Catenin-Komplexes mit neuen, spezifischen Pharmaka, die die Wissenschaftler in Akademie- und Industrie-Instituten im Moment entwickeln, eine potentielle therapeutische Richtung darstellen, um basalen Brustkrebs in der Zukunft zu behandeln. Dies ist der wichtigste Aspekt meiner Doktorarbeit. / In various cancer types, targeting cancer stem cells (CSCs) can serve as an effective approach for limiting therapy resistance and metastasis. While basal breast cancers encompass cells with CSC features, rational therapies remain poorly established. Here, I show that receptor tyrosine kinase (RTK) Met signalling promotes the activity of the Hippo component YAP in basal breast cancer. Further analysis revealed enhanced YAP activity within the CSC population. Utilising both genetic and pharmaceutical approaches, I show that interference of YAP activity delays tumour formation, prevents luminal to basal trans-differentiation and reduces CSC survival. Gene expression analysis of YAP knock-out mammary glands revealed a strong decrease in β-catenin target genes, suggesting that YAP is required for nuclear β-catenin activity. Mechanistically, I find that nuclear YAP interacts and overlaps with β-catenin and TEAD4 at common gene regulatory regions. Analysis of proteomic data from primary breast cancer patients identified significant upregulation of the YAP signature in basal compared to other breast cancers, suggesting that YAP activity is limited to basal breast cancers. Our data highlight the importance of YAP as a crucial tumour initiator and cancer stem cell regulator in basal breast cancer and demonstrates that its activity has prognostic value, when comparing basal with luminal breast cancers. This suggests that targeting the YAP/TEAD4/β-catenin complex through specific new drugs, which researchers in academia and industry are presently developing, is a potential therapeutic avenue for treating basal breast cancers in the future. This is the most important aspect of my thesis.

La polarité cellulaire est un déterminant essentiel dans le maintien de l’architecture tissulaire et la fonction de l’organe. Ainsi, la division cellulaire, la ciliogenèse, la prolifération, et la migration sont des évènements étroitement associés au processus de la polarisation cellulaire. L’altération de la polarité cellulaire contribue à la perte de l’intégrité des épithéliums et favorise le développement des cancers. La signalisation des lipides, telle que des phosphatidylinositols (PtdIns) joue un rôle vital dans la polarité apico-basale. Dans cette étude, nous avons développé des recherches pour mieux comprendre les mécanismes impliqués dans les effets de la phosphatase SHIP2 sur la polarité cellulaire. Nous avons pu démontrer que SHIP2 est impliquée dans la formation du site d’initiation de la formation de la lumière (AMIS) en régulant d’une part la contractilité acto-myosine induite par RhoA kinase et d’autre part YAP, un composant de la voie de signalisation Hippo. De plus, nous avons montré que l'inhibition de SHIP2 contribue à un défaut dans la formation de fuseau mitotique et dans le clivage de ce fuseau mitotique. La surexpression de SHIP2 induit une lumière large et des cils allongés attribuables à la diminution de l’expression de YAP, Aurora A et HEF1. Par contre, la diminution de l’expression de SHIP2 inhibe la formation des cils en provoquant la surexpression de YAP, Aurora A et HEF1 et ainsi l’apparition d’un phénotype multilumens. L’ensemble de nos travaux définissent un nouveau rôle de SHIP2 dans le maintien de l’intégrité et de l’homéostasie des cellules épithéliales. Nous avons aussi pu démontrer que l’expression de SHIP2 peut discriminer les différents cancers du foie (HCC, ICC et mixte) et que SHIP2 et Merlin/NF2, une protéine de la voie de signalisation Hippo, ont une forte expression dans le cholangiocarcinome (ICC) qui s’oppose à celle de YAP et de RhoA kinase. / Cell polarity is critical caracteristic for the maintenance of tissue architecture. Cell division, ciliogenesis, cell proliferation and migration are events tightly associated to cell polarization processes. Alteration in cell polarity contributes to loss of epithelium integrity and enhances cancer development. Lipids signaling, such as phosphatidylinositol (PtdIns), play a vital role in apico-basal polarity. In this study, we developed researches to better understand mechanisms implicated the role of the phosphatase SHIP2 in cell polarity. We demonstrated that SHIP2 is implicated in formation of the apical membrane initiation site (AMIS) by regulating YAP, a component of Hippo pathway, and RhoA-dependant acto-myosin contractility. Furthermore, we demonstrated that inhibition of SHIP2 contributes to defect in the formation and cleavage of the mitotic spindle. Overexpression of SHIP2 induced a large lumen with long cilia due to a decrease in YAP, Aurora A and HEF1 luminal localization. On the contrary, down regulation of SHIP2 impaired cilia outgrowth by increasing Aurora A, HEF1 and YAP luminal localization with appearance of a multilumens phenotype. Thus, our results reinforced the role of SHIP2 in maintain of integrity and homeostasis of epithelial cells. In this study, we also demonstrated that expression of SHIP2 distinguished the different types of liver cancer (HCC, ICC and mixte), and that SHIP2 and Merlin/NF2 are overexpressed in ICC which is the opposite of YAP and RhoA expression.

Breast cancer metastasis is one of the reasons why this type of cancer is destructive even after treatment as it tends to move from one organ to another increasing the risk factor for an individual. In the metastatic cascade, the tumour undergoes many different types of stress, including extracellular (ECM) stiffness. Key proteins that have been linked to the change in stiffness of the ECM are YAP and β-catenin. Both functions similarly in the manner that they need to translocate to the nucleus and bind to their respective transcription factors in order to activate their downstream genes. In parallel this seems to be on a stiffness dependent manner. Therefore, the hypothesis is that β-catenin is able to compensate for YAP function when YAP is downregulated in a stiffness dependent manner. In this work, results show a significant increase of YAP and β-catenin translocation to the nucleus of MDA-MB-231 cells when they are subject to the stiffer substrate in comparison to the softer substrate indicating increase gene expression of their respective pathways. The effect of the stiffness was then analyzed by doing single knockdown experiments with siRNA. To investigate the response of β-catenin, knocking down YAP was done, and it was shown that β-catenin translocation significantly increased on the softer matrix, while stiffer matrix showed no significant difference. Downstream gene expression also confirmed this idea with CTGF being downregulated with β-catenin knockdown and AXIN2 being downregulated with YAP knockdown. In the cell behavioural aspect, only when the double knockdown of YAP and β-catenin was done, the migration and proliferation rate had significant lowered. This echoes the idea further of the compensating effects of β-catenin to YAP. In addition, the exploration of the cytoskeleton network was investigated, as this is a key component in protein pathways, by treating the cells using LatA and Blebbistatin, affecting F-actin and myosin-II respectively. Knowing the critical role of cytoskeletal proteins in mechanotransduction, the hypothesis is that actin filaments and myosin-II mediate the YAP & β-catenin nuclear translocation activation. Findings show the direct relationship between F-actin and YAP as actin polymerization state significantly decreased when YAP was knockdown in a similar manner to when LatA was added. When myosin-II was added, both YAP and β-catenin nuclear translocation were affected, indicating its potential role in mechanotransduction. Furthermore, it was found that cell confluency and PIEZO1 activation had significant effects in YAP & β-catenin translocation. By seeding the cells with different densities, the β-catenin signalling could be visualized with IF staining, with the conclusion that at high confluency, the β-catenin translocation was alleviated. For the PIEZO1 studies, results indicate that PIEZO1 is an upstream regulator of YAP by doing single knockdown experiments and subsequently analysing YAP signalling. The findings underscore the potential significance of β-catenin as a modulator of mechanotransduction in the absence of YAP, showcasing the complexity of the protein signalling network orchestrating cellular response due to mechanical cues. Unravelling these protein interplay could offer novel insights into therapeutic targets for breast cancer mechanotransduction. Ultimately, this research adds to the understanding of the intricate protein signalling that governs mechanotransduction in breast cancer cells. The discovery of stiffness dependent YAP & β-catenin signalling, the interplay between YAP and β-catenin pathway mechanotransduction implicated by cell density, the regulation of YAP- β-catenin interplay in mechanotransduction by PIEZO1, the importance of F-actin & myosin-II in YAP & β-catenin translocation, and the YAP & β-catenin effects on cell behaviour, all help lay the groundwork for devising targeted interventions to impede cancer progression. / Thesis / Master of Applied Science (MASc) / Breast cancer is the most prominent type of cancer that exists in women and like other cancers, it can spread to other organs such as the bone, liver, and brain even though the microenvironments are different. With different proteins like yes-associated protein (YAP) regulating this microenvironmental change in the primary and secondary sites, it can flourish and become more aggressive which leads to death for the host. The interactions of these proteins and their pathways which affects the aggressiveness of the cancers are still not well understood. This project investigates the interaction between YAP and β-catenin in response to surface stiffness to understand the mechanical regulation of breast cancer metastasis. Alongside the protein signalling, cytoskeletal components, downstream gene expression, cell confluency, and membrane proteins are explored. Our results show that an increase in stiffness allow for higher nuclear translocation for YAP and β-catenin, enhancing downstream gene expression relating to migration and proliferation. Furthermore, in lower stiffness the crosstalk between YAP and β-catenin results in an inverse relationship. These findings suggest β-catenin compensates YAP function when YAP is inhibited. In terms of the cytoskeletal protein, an integral part of the cell, the intervention saw a significant alteration in the YAP & β-catenin signalling. Additionally, cell confluency played a large role in β-catenin nuclear translocation implicating the role of cell-to-cell contact in mechanotransduction. To see if mechanosensitive membrane proteins fit into the pathway, PIEZO1 studies were done and results show that it is an upstream effector of YAP, and consequently an indirect connection with β-catenin. All in all, this thesis provides insightful information in the role of stiffness matrix, cell confluency, membrane proteins and how that regulate YAP & β-catenin. This research provides the mechanism for the synergistic therapies targeting multiple proteins to prevent cancer growth and metastasis.