Mechanical Forces as Determinants of Disseminated Metastatic Cell Fate

Disseminated metastatic cancer cells represent one of the most relevant causes of disease relapse and associated death for cancer patients, and a therapeutic target of the highest priority. Still, our understanding of how disseminated cancer cells survive in the foreign metastatic environment, and eventually cause metastatic outgrowth, remains rather limited. In this review we focus on the cell microenvironment as a key regulator of cell behavior at the metastatic site, and especially on the mechanical properties of the extracellular matrix and associated integrin signaling. We discuss available evidence pointing to a pervasive role of extracellular matrix (ECM) mechanical properties in regulating cancer cell proliferation and survival after dissemination, and propose that this might represent an important bottleneck for cells invading and establishing into a novel tissue. We point to the known molecular players, how these might contribute to modulate the mechanical properties of the metastatic environment, and the response of cells to these cues. Finally, we propose that emerging knowledge on the physical interaction of disseminated metastatic cells and on the downstream mechanotransduction pathways, including YAP/TAZ (Yes-associated protein-1 and WW-domain transcription activator 1) and MRTFs (Myocardin-related transcription factors), may help to identify novel approaches for therapy

Role of YAP/TAZ in mechanotransduction

Cells perceive their microenvironment not only through soluble signals but also in term of physical and mechanical cues, such as extracellular matrix (ECM) stiffness or confined adhesiveness. By mechanotransduction systems, cells translate these stimuli into biochemical signals controlling multiple aspects of cell behavior, including growth, differentiation and cancer malignant progression; but how rigidity mechanosensing is ultimately linked to activity of nuclear transcription factors remains poorly understood. Here we report the identification of the Yorkie-homologues YAP and TAZ as nuclear relays of mechanical signals exerted by ECM rigidity and cell-shape. This regulation requires Rho activity and tension of the acto-myosin cytoskeleton but is independent from the Hippo/LATS cascade. Crucially, YAP/TAZ are functionally required for differentiation of mesenchymal stem cells induced by ECM stiffness and for survival of endothelial cells regulated by cell geometry; conversely, expression of constitutive active YAP overrules physical constraints in dictating cell behavior. These findings identify YAP/TAZ as sensors and mediators of mechanical cues instructed by the cellular microenvironment

YAP/TAZ functions and their regulation at a glance

YAP and TAZ proteins are paralog transcriptional coactivators that shuttle between the cytoplasm and the nucleus in response to multiple inputs, including the Hippo pathway; in the nucleus, they pair with DNA-binding factors of the TEAD family to regulate gene expression. Nuclear YAP/TAZ promote cell proliferation, organ overgrowth, survival to stress and dedifferentiation of post-mitotic cells into their respective tissue progenitors. YAP/TAZ are required for growth of embryonic tissues, wound healing and organ regeneration, where they are activated by cell-intrinsic and extrinsic cues. Surprisingly, this activity is dispensable in many adult self-renewing tissues, where YAP/TAZ are constantly kept in check. YAP/TAZ lay at the center of a complex regulatory network including cell-autonomous factors but also cell- and tissue-level structural features such as the mechanical properties of the cell microenvironment, the establishment of cell-cell junctions and of basolateral tissue polarity. Enhanced levels and activity of YAP/TAZ are observed in many cancers, where they sustain tumour growth, drug resistance and malignancy. In this Cell science at a glance and the accompanying poster, we review the biological functions of YAP/TAZ and their regulatory mechanisms, and highlight their position at the center of a complex signaling network

Zebrafish mutants and TEAD reporters reveal essential functions for Yap and Taz in posterior cardinal vein development

As effectors of the Hippo signaling cascade, YAP1 and TAZ are transcriptional regulators playing important roles in development, tissue homeostasis and cancer. A number of different cues, including mechanotransduction of extracellular stimuli, adhesion molecules, oncogenic signaling and metabolism modulate YAP1/TAZ nucleo-cytoplasmic shuttling. In the nucleus, YAP1/TAZ tether with the DNA binding proteins TEADs, to activate the expression of target genes that regulate proliferation, migration, cell plasticity, and cell fate. Based on responsive elements present in the human and zebrafish promoters of the YAP1/TAZ target gene CTGF, we established zebrafish fluorescent transgenic reporter lines of Yap1/Taz activity. These reporter lines provide an in vivo view of Yap1/Taz activity during development and adulthood at the whole organism level. Transgene expression was detected in many larval tissues including the otic vesicles, heart, pharyngeal arches, muscles and brain and is prominent in endothelial cells. Analysis of vascular development in yap1/taz zebrafish mutants revealed specific defects in posterior cardinal vein (PCV) formation, with altered expression of arterial/venous markers. The overactivation of Yap1/Taz in endothelial cells was sufficient to promote an aberrant vessel sprouting phenotype. Our findings confirm and extend the emerging role of Yap1/Taz in vascular development including angiogenesis

Extracellular matrix mechanical cues regulate lipid metabolism through Lipin-1 and SREBP

Extracellular matrix (ECM) mechanical cues have powerful effects on cell proliferation, differentiation and death. Here, starting from an unbiased metabolomics approach, we identify synthesis of neutral lipids as a general response to mechanical signals delivered by cell\u2013matrix adhesions. Extracellular physical cues reverberate on the mechanical properties of the Golgi apparatus and regulate the Lipin-1 phosphatidate phosphatase. Conditions of reduced actomyosin contractility lead to inhibition of Lipin-1, accumulation of SCAP/SREBP to the Golgi apparatus and activation of SREBP transcription factors, in turn driving lipid synthesis and accumulation. This occurs independently of YAP/TAZ, mTOR and AMPK, and in parallel to feedback control by sterols. Regulation of SREBP can be observed in a stiffened diseased tissue, and contributes to the pro-survival activity of ROCK inhibitors in pluripotent stem cells. We thus identify a general mechanism centered on Lipin-1 and SREBP that links the physical cell microenvironment to a key metabolic pathway

Inflammation and pancreatic cancer: molecular and functional interactions between S100A8, S100A9, NT-S100A8 and TGFβ1

BACKGROUND: In order to gain further insight on the crosstalk between pancreatic cancer (PDAC) and stromal cells, we investigated interactions occurring between TGF\u3b21 and the inflammatory proteins S100A8, S100A9 and NT-S100A8, a PDAC-associated S100A8 derived peptide, in cell signaling, intracellular calcium (Cai2+) and epithelial to mesenchymal transition (EMT). NF-\u3baB, Akt and mTOR pathways, Cai2+ and EMT were studied in well (Capan1 and BxPC3) and poorly differentiated (Panc1 and MiaPaCa2) cell lines. RESULTS: NT-S100A8, one of the low molecular weight N-terminal peptides from S100A8 to be released by PDAC-derived proteases, shared many effects on NF-\u3baB, Akt and mTOR signaling with S100A8, but mainly with TGF\u3b21. The chief effects of S100A8, S100A9 and NT-S100A8 were to inhibit NF-\u3baB and stimulate mTOR; the molecules inhibited Akt in Smad4-expressing, while stimulated Akt in Smad4 negative cells. By restoring Smad4 expression in BxPC3 and silencing it in MiaPaCa2, S100A8 and NT-S100A8 were shown to inhibit NF-\u3baB and Akt in the presence of an intact TGF\u3b21 canonical signaling pathway. TGF\u3b21 counteracted S100A8, S100A9 and NT-S100A8 effects in Smad4 expressing, not in Smad4 negative cells, while it synergized with NT-S100A8 in altering Cai2+ and stimulating PDAC cell growth. The effects of TGF\u3b21 on both EMT (increased Twist and decreased N-Cadherin expression) and Cai2+ were antagonized by S100A9, which formed heterodimers with TGF\u3b21 (MALDI-TOF/MS and co-immuno-precipitation). CONCLUSIONS: The effects of S100A8 and S100A9 on PDAC cell signaling appear to be cell-type and context dependent. NT-S100A8 mimics the effects of TGF\u3b21 on cell signaling, and the formation of complexes between TGF\u3b21 with S100A9 appears to be the molecular mechanism underlying the reciprocal antagonism of these molecules on cell signaling, Cai2+ and EMT

F-actin dynamics regulates mammalian organ growth and cell fate maintenance.

BACKGROUND & AIMS: In vitro, several data indicate that cell function can be regulated by the mechanical properties of cells and of the microenvironment. Cells measure these features by developing forces via their actomyosin cytoskeleton, and respond accordingly by transducing forces into biochemical signals that instruct cell behavior. Among these, the transcriptional coactivators YAP/TAZ recently emerged as key factors mediating multiple responses to actomyosin contractility. However, whether mechanical cues regulate adult liver tissue homeostasis, and whether this occurs through YAP/TAZ, remains largely unaddressed. METHODS & RESULTS: Here we show that the F-actin capping protein CAPZ is a critical negative regulator of actomyosin contractility and mechanotransduction. Capzb inactivation alters stress fiber and focal adhesion dynamics leading to enhanced myosin activity, increased cellular traction forces, and increased liver stiffness. In vitro, this rescues YAP from inhibition by a small geometry; in vivo, inactivation of Capzb in the adult mouse liver induces YAP activation in parallel to the Hippo pathway, causing extensive hepatocyte proliferation and leading to striking organ overgrowth. Moreover, Capzb is required for the maintenance of the differentiated hepatocyte state, for metabolic zonation, and for gluconeogenesis. In keeping with changes in tissue mechanics, inhibition of the contractility regulator ROCK, or deletion of the Yap1 mechanotransducer, reverse the phenotypes emerging in Capzb-null livers. CONCLUSIONS: These results indicate a previously unrecognized role for CAPZ in tuning the mechanical properties of cells and tissues, which is required in hepatocytes for the maintenance of the differentiated hepatocyte state and to regulate organ size. More in general, it indicates for the first time a physiological role of mechanotransduction in maintaining tissue homeostasis in mammals. LAY SUMMARY: The mechanical properties of cells and tissues (i.e. whether they are soft or stiff) are thought to be important regulators of cell behavior. A recent advancement in our understanding of these phenomena has been the identification of YAP and TAZ as key factors mediating the biological responses of cells to mechanical signals in vitro. However, whether the mechanical properties of cells and/or the mechanical regulation of YAP/TAZ are relevant for mammalian tissue physiology remains unknown. Here we challenge this issue by genetic inactivation of CAPZ, a protein that regulates the cytoskeleton, i.e. the cells' scaffold by which they sense mechanical cues. We found that inactivation of CAPZ alters cells' and liver tissue's mechanical properties, leading to YAP hyperactivation. In turn, this profoundly alters liver physiology, causing organ overgrowth, defects in liver cell differentiation and metabolism. These results reveal a previously uncharacterized role for mechanical signals for the maintenance of adult liver homeostasis.This work was supported by AIRC (Associazione Italiana per la Ricerca sul Cancro) Investigator Grant 15307, WCR (Worldwide Cancer Research) Grant 15-1192, CARIPARO Eccellenza Program 2017 and University of Padua BIRD Grant to SD, AIRC ‘Hard ROCK Café’ Fellowship to GS, Marie Sklodowska-Curie Individual Fellowship (796547) to AG, AIRC Special Program Molecular Clinical Oncology ‘5 per mille’ 10016 to SB, UK Medical Research Council and Sackler Foundation Doctoral Training Grant RG70550 to ACL, UK Medical Research Council Career Development Award G1100312/1 and an Isaac Newton Trust Research Grant 17.24(p) to KF

F-actin dynamics regulates mammalian organ growth and cell fate maintenance.

BACKGROUND & AIMS: In vitro, several data indicate that cell function can be regulated by the mechanical properties of cells and of the microenvironment. Cells measure these features by developing forces via their actomyosin cytoskeleton, and respond accordingly by transducing forces into biochemical signals that instruct cell behavior. Among these, the transcriptional coactivators YAP/TAZ recently emerged as key factors mediating multiple responses to actomyosin contractility. However, whether mechanical cues regulate adult liver tissue homeostasis, and whether this occurs through YAP/TAZ, remains largely unaddressed. METHODS & RESULTS: Here we show that the F-actin capping protein CAPZ is a critical negative regulator of actomyosin contractility and mechanotransduction. Capzb inactivation alters stress fiber and focal adhesion dynamics leading to enhanced myosin activity, increased cellular traction forces, and increased liver stiffness. In vitro, this rescues YAP from inhibition by a small geometry; in vivo, inactivation of Capzb in the adult mouse liver induces YAP activation in parallel to the Hippo pathway, causing extensive hepatocyte proliferation and leading to striking organ overgrowth. Moreover, Capzb is required for the maintenance of the differentiated hepatocyte state, for metabolic zonation, and for gluconeogenesis. In keeping with changes in tissue mechanics, inhibition of the contractility regulator ROCK, or deletion of the Yap1 mechanotransducer, reverse the phenotypes emerging in Capzb-null livers. CONCLUSIONS: These results indicate a previously unrecognized role for CAPZ in tuning the mechanical properties of cells and tissues, which is required in hepatocytes for the maintenance of the differentiated hepatocyte state and to regulate organ size. More in general, it indicates for the first time a physiological role of mechanotransduction in maintaining tissue homeostasis in mammals. LAY SUMMARY: The mechanical properties of cells and tissues (i.e. whether they are soft or stiff) are thought to be important regulators of cell behavior. A recent advancement in our understanding of these phenomena has been the identification of YAP and TAZ as key factors mediating the biological responses of cells to mechanical signals in vitro. However, whether the mechanical properties of cells and/or the mechanical regulation of YAP/TAZ are relevant for mammalian tissue physiology remains unknown. Here we challenge this issue by genetic inactivation of CAPZ, a protein that regulates the cytoskeleton, i.e. the cells' scaffold by which they sense mechanical cues. We found that inactivation of CAPZ alters cells' and liver tissue's mechanical properties, leading to YAP hyperactivation. In turn, this profoundly alters liver physiology, causing organ overgrowth, defects in liver cell differentiation and metabolism. These results reveal a previously uncharacterized role for mechanical signals for the maintenance of adult liver homeostasis.This work was supported by AIRC (Associazione Italiana per la Ricerca sul Cancro) Investigator Grant 15307, WCR (Worldwide Cancer Research) Grant 15-1192, CARIPARO Eccellenza Program 2017 and University of Padua BIRD Grant to SD, AIRC ‘Hard ROCK Café’ Fellowship to GS, Marie Sklodowska-Curie Individual Fellowship (796547) to AG, AIRC Special Program Molecular Clinical Oncology ‘5 per mille’ 10016 to SB, UK Medical Research Council and Sackler Foundation Doctoral Training Grant RG70550 to ACL, UK Medical Research Council Career Development Award G1100312/1 and an Isaac Newton Trust Research Grant 17.24(p) to KF