Methods to Study the Role of Mechanical Signals in the Induction of Cancer Stem Cells.

Microenvironmental mechanical signals are fundamental regulators of cell behavior both in physiological and in pathological context, particularly in the induction and maintenance of tumorigenic properties. It is thus of utmost importance to experimentally recreate conditions that mimic the physical attributes of real tissues to study their impact on cell behavior and in particular on the induction of cancer stem cell (CSC) properties. Here we present protocols to investigate the role of mechanical stiffness on reprogramming of primary mammary gland cells into CSCs, including the synthesis of hydrogel substrates of the desired stiffness, the isolation and culture of primary differentiated normal cells derived from the human mammary gland, and the assessment of their CSC attributes after oncogene-mediated transformation.

Aberrant MET activation impairs perinuclear actin cap organization with YAP1 cytosolic relocation.

Little is known about the signaling network responsible for the organization of the perinuclear actin cap, a recently identified structure holding unique roles in the regulation of nuclear shape and cell directionality. In cancer cells expressing a constitutively active MET, we show a rearrangement of the actin cap filaments, which crash into perinuclear patches associated with spherical nuclei, meandering cell motility and inactivation of the mechano-transducer YAP1. MET ablation is sufficient to reactivate YAP1 and restore the cap, leading to enhanced directionality and flattened nuclei. Consistently, the introduction of a hyperactive MET in normal epithelial cells, enhances nuclear height and alters the cap organization, as also confirmed by TEM analysis. Finally, the constitutively active YAP1 mutant YAP5SA is able to overcome the effects of oncogenic MET. Overall, our work describes a signaling axis empowering MET-mediated YAP1 dampening and actin cap misalignment, with implications for nuclear shape and cell motility.

YAP/TAZ as master regulators in cancer: modulation, function and therapeutic approaches.

Our understanding of the function of the transcriptional regulators YAP and TAZ (YAP/TAZ) in cancer is advancing. In this Review, we provide an update on recent progress in YAP/TAZ biology, their regulation by Hippo signaling and mechanotransduction and highlight open questions. YAP/TAZ signaling is an addiction shared by multiple tumor types and their microenvironments, providing many malignant attributes. As such, it represents an important vulnerability that may offer a broad window of therapeutic efficacy, and here we give an overview of the current treatment strategies and pioneering clinical trials.

YAP/TAZ activity in stromal cells prevents ageing by controlling cGAS-STING.

Ageing is intimately connected to the induction of cell senescence1,2, but why this is so remains poorly understood. A key challenge is the identification of pathways that normally suppress senescence, are lost during ageing and are functionally relevant to oppose ageing3. Here we connected the structural and functional decline of ageing tissues to attenuated function of the master effectors of cellular mechanosignalling YAP and TAZ. YAP/TAZ activity declines during physiological ageing in stromal cells, and mimicking such decline through genetic inactivation of YAP/TAZ in these cells leads to accelerated ageing. Conversely, sustaining YAP function rejuvenates old cells and opposes the emergence of ageing-related traits associated with either physiological ageing or accelerated ageing triggered by a mechano-defective extracellular matrix. Ageing traits induced by inactivation of YAP/TAZ are preceded by induction of tissue senescence. This occurs because YAP/TAZ mechanotransduction suppresses cGAS-STING signalling, to the extent that inhibition of STING prevents tissue senescence and premature ageing-related tissue degeneration after YAP/TAZ inactivation. Mechanistically, YAP/TAZ-mediated control of cGAS-STING signalling relies on the unexpected role of YAP/TAZ in preserving nuclear envelope integrity, at least in part through direct transcriptional regulation of lamin B1 and ACTR2, the latter of which is involved in building the peri-nuclear actin cap. The findings demonstrate that declining YAP/TAZ mechanotransduction drives ageing by unleashing cGAS-STING signalling, a pillar of innate immunity. Thus, sustaining YAP/TAZ mechanosignalling or inhibiting STING may represent promising approaches for limiting senescence-associated inflammation and improving healthy ageing.

Broadly Applicable Hydrogel Fabrication Procedures Guided by YAP/TAZ-Activity Reveal Stiffness, Adhesiveness, and Nuclear Projected Area as Checkpoints for Mechanosensing.

Mechanical signals are pivotal ingredients in how cells perceive and respond to their microenvironments, and to synthetic biomaterials that mimic them. In spite of increasing interest in mechanobiology, probing the effects of physical cues on cell behavior remains challenging for a cell biology laboratory without experience in fabrication of biocompatible materials. Hydrogels are ideal biomaterials recapitulating the physical cues that natural extracellular matrices (ECM) deliver to cells. Here, protocols are streamlined for the synthesis and functionalization of cell adhesive polyacrylamide-based (PAA-OH) and fully-defined polyethyleneglycol-based (PEG-RGD) hydrogels tuned at various rigidities for mechanobiology experiments, from 0.3 to >10 kPa.  The mechanosignaling properties of these hydrogels are investigated in distinct cell types by monitoring the activation state of YAP/TAZ. By independently modulating substrate stiffness and adhesiveness, it is found that although ECM stiffness represents an overarching mechanical signal, the density of adhesive sites does impact on cellular mechanosignaling at least at intermediate rigidity values, corresponding to normal and pathological states of living tissues. Using these tools, it is found that YAP/TAZ nuclear accumulation occurs when the projected area of the nucleus surpasses a critical threshold of approximatively 150 µm2 . This work suggests the existence of distinct checkpoints for cellular mechanosensing.

Simple yet effective methods to probe hydrogel stiffness for mechanobiology.

In spite of tremendous advances made in the comprehension of mechanotransduction, implementation of mechanobiology assays remains challenging for the broad community of cell biologists. Hydrogel substrates with tunable stiffness are essential tool in mechanobiology, allowing to investigate the effects of mechanical signals on cell behavior. A bottleneck that slows down the popularization of hydrogel formulations for mechanobiology is the assessment of their stiffness, typically requiring expensive and sophisticated methodologies in the domain of material science. Here we overcome such barriers offering the reader protocols to set-up and interpret two straightforward, low cost and high-throughput tools to measure hydrogel stiffness: static macroindentation and micropipette aspiration. We advanced on how to build up these tools and on the underlying theoretical modeling. Specifically, we validated our tools by comparing them with leading techniques used for measuring hydrogel stiffness (atomic force microscopy, uniaxial compression and rheometric analysis) with consistent results on PAA hydrogels or their modification. In so doing, we also took advantage of YAP/TAZ nuclear localization as biologically validated and sensitive readers of mechanosensing, all in all presenting a suite of biologically and theoretically proven protocols to be implemented in most biological laboratories to approach mechanobiology.

Single-cell analyses reveal YAP/TAZ as regulators of stemness and cell plasticity in Glioblastoma.

Glioblastoma (GBM) is a devastating human malignancy. GBM stem-like cells (GSCs) drive tumor initiation and progression. Yet, the molecular determinants defining GSCs in their native state in patients remain poorly understood. Here we used single cell datasets and identified GSCs at the apex of the differentiation hierarchy of GBM. By reconstructing the GSCs' regulatory network, we identified the YAP/TAZ coactivators as master regulators of this cell state, irrespectively of GBM subtypes. YAP/TAZ are required to install GSC properties in primary cells downstream of multiple oncogenic lesions, and required for tumor initiation and maintenance in vivo in different mouse and human GBM models. YAP/TAZ act as main roadblock of GSC differentiation and their inhibition irreversibly lock differentiated GBM cells into a non-tumorigenic state, preventing plasticity and regeneration of GSC-like cells. Thus, GSC identity is linked to a key molecular hub integrating genetics and microenvironmental inputs within the multifaceted biology of GBM.

ATR is essential for preservation of cell mechanics and nuclear integrity during interstitial migration.

ATR responds to mechanical stress at the nuclear envelope and mediates envelope-associated repair of aberrant topological DNA states. By combining microscopy, electron microscopic analysis, biophysical and in vivo models, we report that ATR-defective cells exhibit altered nuclear plasticity and YAP delocalization. When subjected to mechanical stress or undergoing interstitial migration, ATR-defective nuclei collapse accumulating nuclear envelope ruptures and perinuclear cGAS, which indicate loss of nuclear envelope integrity, and aberrant perinuclear chromatin status. ATR-defective cells also are defective in neuronal migration during development and in metastatic dissemination from circulating tumor cells. Our findings indicate that ATR ensures mechanical coupling of the cytoskeleton to the nuclear envelope and accompanying regulation of envelope-chromosome association. Thus the repertoire of ATR-regulated biological processes extends well beyond its canonical role in triggering biochemical implementation of the DNA damage response.

Reprogramming normal cells into tumour precursors requires ECM stiffness and oncogene-mediated changes of cell mechanical properties.

Defining the interplay between the genetic events and microenvironmental contexts necessary to initiate tumorigenesis in normal cells is a central endeavour in cancer biology. We found that receptor tyrosine kinase (RTK)-Ras oncogenes reprogram normal, freshly explanted primary mouse and human cells into tumour precursors, in a process requiring increased force transmission between oncogene-expressing cells and their surrounding extracellular matrix. Microenvironments approximating the normal softness of healthy tissues, or blunting cellular mechanotransduction, prevent oncogene-mediated cell reprogramming and tumour emergence. However, RTK-Ras oncogenes empower a disproportional cellular response to the mechanical properties of the cell's environment, such that when cells experience even subtle supra-physiological extracellular-matrix rigidity they are converted into tumour-initiating cells. These regulations rely on YAP/TAZ mechanotransduction, and YAP/TAZ target genes account for a large fraction of the transcriptional responses downstream of oncogenic signalling. This work lays the groundwork for exploiting oncogenic mechanosignalling as a vulnerability at the onset of tumorigenesis, including tumour prevention strategies.

Publisher Correction: Reprogramming normal cells into tumour precursors requires ECM stiffness and oncogene-mediated changes of cell mechanical properties.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Cell phenotypic plasticity requires autophagic flux driven by YAP/TAZ mechanotransduction.

Autophagy, besides ensuring energy metabolism and organelle renewal, is crucial for the biology of adult normal and cancer stem cells. However, it remains incompletely understood how autophagy connects to stemness factors and the nature of the microenvironmental signals that pattern autophagy in different cell types. Here we advance in these directions by reporting that YAP/TAZ transcriptionally control autophagy, being critical for autophagosomal degradation into autolysosomes. YAP/TAZ are downstream effectors of cellular mechanotransduction and indeed we found that cell mechanics, dictated by the physical property of the ECM and cytoskeletal tension, profoundly impact on autophagic flux in a YAP/TAZ-mediated manner. Functionally, by using pancreatic and mammary organoid cultures, we found that YAP/TAZ-regulated autophagy is essential in normal cells for YAP/TAZ-mediated dedifferentiation and acquisition of self-renewing properties. In tumor cells, the YAP/TAZ-autophagy connection is key to sustain transformed traits and for acquisition of a cancer stem cell state by otherwise more benign cells. Mechanistically, YAP/TAZ promote autophagic flux by directly promoting the expression of Armus, a RAB7-GAP required for autophagosome turnover and whose add-back rescues autophagy in YAP/TAZ-depleted cells. These findings expand the influence of YAP/TAZ mechanotransduction to the control of autophagy and, vice versa, the role of autophagy in YAP/TAZ biology, and suggest a mechanism to coordinate transcriptional rewiring with cytoplasmic restructuring during cell reprogramming.

The SWI/SNF complex is a mechanoregulated inhibitor of YAP and TAZ.

Inactivation of ARID1A and other components of the nuclear SWI/SNF protein complex occurs at very high frequencies in a variety of human malignancies, suggesting a widespread role for the SWI/SNF complex in tumour suppression1. However, the underlying mechanisms remain poorly understood. Here we show that ARID1A-containing SWI/SNF complex (ARID1A-SWI/SNF) operates as an inhibitor of the pro-oncogenic transcriptional coactivators YAP and TAZ2. Using a combination of gain- and loss-of-function approaches in several cellular contexts, we show that YAP/TAZ are necessary to induce the effects of the inactivation of the SWI/SNF complex, such as cell proliferation, acquisition of stem cell-like traits and liver tumorigenesis. We found that YAP/TAZ form a complex with SWI/SNF; this interaction is mediated by ARID1A and is alternative to the association of YAP/TAZ with the DNA-binding platform TEAD. Cellular mechanotransduction regulates the association between ARID1A-SWI/SNF and YAP/TAZ. The inhibitory interaction of ARID1A-SWI/SNF and YAP/TAZ is predominant in cells that experience low mechanical signalling, in which loss of ARID1A rescues the association between YAP/TAZ and TEAD. At high mechanical stress, nuclear F-actin binds to ARID1A-SWI/SNF, thereby preventing the formation of the ARID1A-SWI/SNF-YAP/TAZ complex, in favour of an association between TEAD and YAP/TAZ. We propose that a dual requirement must be met to fully enable the YAP/TAZ responses: promotion of nuclear accumulation of YAP/TAZ, for example, by loss of Hippo signalling, and inhibition of ARID1A-SWI/SNF, which can occur either through genetic inactivation or because of increased cell mechanics. This study offers a molecular framework in which mechanical signals that emerge at the tissue level together with genetic lesions activate YAP/TAZ to induce cell plasticity and tumorigenesis.

Biomaterials and engineered microenvironments to control YAP/TAZ-dependent cell behaviour.

Mechanical signals are increasingly recognized as overarching regulators of cell behaviour, controlling stemness, organoid biology, tissue development and regeneration. Moreover, aberrant mechanotransduction is a driver of disease, including cancer, fibrosis and cardiovascular defects. A central question remains how cells compute a host of biomechanical signals into meaningful biological behaviours. Biomaterials and microfabrication technologies are essential to address this issue. Here we review a large body of evidence that connects diverse biomaterial-based systems to the functions of YAP/TAZ, two highly related mechanosensitive transcriptional regulators. YAP/TAZ orchestrate the response to a suite of engineered microenviroments, emerging as a universal control system for cells in two and three dimensions, in static or dynamic fashions, over a range of elastic and viscoelastic stimuli, from solid to fluid states. This approach may guide the rational design of technological and material-based platforms with dramatically improved functionalities and inform the generation of new biomaterials for regenerative medicine applications.

YAP/TAZ upstream signals and downstream responses.

Cell behaviour is strongly influenced by physical, mechanical contacts between cells and their extracellular matrix. We review how the transcriptional regulators YAP and TAZ integrate mechanical cues with the response to soluble signals and metabolic pathways to control multiple aspects of cell behaviour, including proliferation, cell plasticity and stemness essential for tissue regeneration. Corruption of cell-environment interplay leads to aberrant YAP and TAZ activation that is instrumental for multiple diseases, including cancer.

De Novo Generation of Somatic Stem Cells by YAP/TAZ.

Here we present protocols to isolate primary differentiated cells and turn them into stem/progenitor cells (SCs) of the same lineage by transient expression of the transcription factor YAP. With this method, luminal differentiated (LD) cells of the mouse mammary gland are converted into cells that exhibit molecular and functional properties of mammary SCs. YAP also turns fully differentiated pancreatic exocrine cells into pancreatic duct-like progenitors. Similarly, to endogenous, natural SCs, YAP-induced stem-like cells ("ySCs") can be eventually expanded as organoid cultures long term in vitro, without further need of ectopic YAP/TAZ, as ySCs are endowed with a heritable self-renewing SC-like state. The reprogramming procedure presented here offers the possibility to generate and expand in vitro progenitor cells of various tissue sources starting from differentiated cells. The straightforward expansion of somatic cells ex vivo has implications for regenerative medicine, for understanding mechanisms of tumor initiation and, more in general, for cell and developmental biology studies.

Mechanobiology of YAP and TAZ in physiology and disease.

A growing body of evidence suggests that mechanical signals emanating from the cell's microenvironment are fundamental regulators of cell behaviour. Moreover, at the macroscopic scale, the influence of forces, such as the forces generated by blood flow, muscle contraction, gravity and overall tissue rigidity (for example, inside of a tumour lump), is central to our understanding of physiology and disease pathogenesis. Still, how mechanical cues are sensed and transduced at the molecular level to regulate gene expression has long remained enigmatic. The identification of the transcription factors YAP and TAZ as mechanotransducers started to fill this gap. YAP and TAZ read a broad range of mechanical cues, from shear stress to cell shape and extracellular matrix rigidity, and translate them into cell-specific transcriptional programmes. YAP and TAZ mechanotransduction is critical for driving stem cell behaviour and regeneration, and it sheds new light on the mechanisms by which aberrant cell mechanics is instrumental for the onset of multiple diseases, such as atherosclerosis, fibrosis, pulmonary hypertension, inflammation, muscular dystrophy and cancer.

Induction of Expandable Tissue-Specific Stem/Progenitor Cells through Transient Expression of YAP/TAZ.

The ability to induce autologous tissue-specific stem cells in culture could have a variety of applications in regenerative medicine and disease modeling. Here we show that transient expression of exogenous YAP or its closely related paralogue TAZ in primary differentiated mouse cells can induce conversion to a tissue-specific stem/progenitor cell state. Differentiated mammary gland, neuronal, and pancreatic exocrine cells, identified using a combination of cell sorting and lineage tracing approaches, efficiently convert to proliferating cells with properties of stem/progenitor cells of their respective tissues after YAP induction. YAP-induced mammary stem/progenitor cells show molecular and functional properties similar to endogenous MaSCs, including organoid formation and mammary gland reconstitution after transplantation. Because YAP/TAZ function is also important for self-renewal of endogenous stem cells in culture, our findings have implications for understanding the molecular determinants of the somatic stem cell state.

YAP/TAZ incorporation in the ß-catenin destruction complex orchestrates the Wnt response.

The Hippo transducers YAP/TAZ have been shown to play positive, as well as negative, roles in Wnt signaling, but the underlying mechanisms remain unclear. Here, we provide biochemical, functional, and genetic evidence that YAP and TAZ are integral components of the ß-catenin destruction complex that serves as cytoplasmic sink for YAP/TAZ. In Wnt-ON cells, YAP/TAZ are physically dislodged from the destruction complex, allowing their nuclear accumulation and activation of Wnt/YAP/TAZ-dependent biological effects. YAP/TAZ are required for intestinal crypt overgrowth induced by APC deficiency and for crypt regeneration ex vivo. In Wnt-OFF cells, YAP/TAZ are essential for ß-TrCP recruitment to the complex and ß-catenin inactivation. In Wnt-ON cells, release of YAP/TAZ from the complex is instrumental for Wnt/ß-catenin signaling. In line, the ß-catenin-dependent maintenance of ES cells in an undifferentiated state is sustained by loss of YAP/TAZ. This work reveals an unprecedented signaling framework relevant for organ size control, regeneration, and tumor suppression.

A mechanical checkpoint controls multicellular growth through YAP/TAZ regulation by actin-processing factors.

Key cellular decisions, such as proliferation or growth arrest, typically occur at spatially defined locations within tissues. Loss of this spatial control is a hallmark of many diseases, including cancer. Yet, how these patterns are established is incompletely understood. Here, we report that physical and architectural features of a multicellular sheet inform cells about their proliferative capacity through mechanical regulation of YAP and TAZ, known mediators of Hippo signaling and organ growth. YAP/TAZ activity is confined to cells exposed to mechanical stresses, such as stretching, location at edges/curvatures contouring an epithelial sheet, or stiffness of the surrounding extracellular matrix. We identify the F-actin-capping/severing proteins Cofilin, CapZ, and Gelsolin as essential gatekeepers that limit YAP/TAZ activity in cells experiencing low mechanical stresses, including contact inhibition of proliferation. We propose that mechanical forces are overarching regulators of YAP/TAZ in multicellular contexts, setting responsiveness to Hippo, WNT, and GPCR signaling.