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.

Mechanosignaling in vertebrate development.

Myriads forces are at play during morphogenesis. Their concerted activity shapes individual cells, tissues and the whole embryo, representing the most awe-inspiring marvel of developmental biology. In spite of their prevalence, the potential instructive role of cell mechanics in fate determination and patterning has remained long neglected, in part due to the difficulties in translating the physical world of cells in molecular terms. The recent discovery of the principles of mechanotransduction, of how these impact on gene expression, is however starting to change this scenario, making mechanotransduction finally amenable to experimental dissection through genetics, molecular and bioengineering approaches. Here we review this emerging field, and a series of discoveries that potently bring back cell mechanics at the centerstage of vertebrate developmental biology. We discuss the role of actomyosin contractility as integrating platform between morphogens, lateral inhibition and mechanosignaling. We also review data indicating that supracellular pulling forces, coupled with solid-to-fluid changes in the material contexture of embryonic fields, may act as overarching mechanical "organizers". The evidence also indicates that a continuum of forces is what ultimately locks "self-organizing" movements with cell fate, from the earliest pre-implantation decisions to the fine details of organogenesis. Notably, similar mechanisms are reawakened in organoids and in adult tissues during regeneration. Developmental biology has been correctly depicted, but recently often forgotten, as the "mother" of all biological disciplines. Investigations in developmental mechanics may revamp interest, and have a broad impact in the fields of regenerative medicine, stem cells and cancer biology.

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.

Epigenomic landscape of human colorectal cancer unveils an aberrant core of pan-cancer enhancers orchestrated by YAP/TAZ.

Cancer is characterized by pervasive epigenetic alterations with enhancer dysfunction orchestrating the aberrant cancer transcriptional programs and transcriptional dependencies. Here, we epigenetically characterize human colorectal cancer (CRC) using de novo chromatin state discovery on a library of different patient-derived organoids. By exploring this resource, we unveil a tumor-specific deregulated enhancerome that is cancer cell-intrinsic and independent of interpatient heterogeneity. We show that the transcriptional coactivators YAP/TAZ act as key regulators of the conserved CRC gained enhancers. The same YAP/TAZ-bound enhancers display active chromatin profiles across diverse human tumors, highlighting a pan-cancer epigenetic rewiring which at single-cell level distinguishes malignant from normal cell populations. YAP/TAZ inhibition in established tumor organoids causes extensive cell death unveiling their essential role in tumor maintenance. This work indicates a common layer of YAP/TAZ-fueled enhancer reprogramming that is key for the cancer cell state and can be exploited for the development of improved therapeutic avenues.

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.

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.

YAP and TAZ: a signalling hub of the tumour microenvironment.

YAP and TAZ are transcriptional activators pervasively induced in several human solid tumours and their functions in cancer cells are the focus of intense investigation. These studies established that YAP and TAZ are essential to trigger numerous cell-autonomous responses, such as sustained proliferation, cell plasticity, therapy resistance and metastasis. Yet tumours are complex entities, wherein cancer cells are just one of the components of a composite "tumour tissue". The other component, the tumour stroma, is composed of an extracellular matrix with aberrant mechanical properties and other cell types, including cancer-associated fibroblasts and immune cells. The stroma entertains multiple and bidirectional interactions with tumour cells, establishing dependencies essential to unleash tumorigenesis. The molecular players of such interplay remain partially understood. Here, we review the emerging role of YAP and TAZ in choreographing tumour-stromal interactions. YAP and TAZ act within tumour cells to orchestrate responses in stromal cells. Vice versa, YAP and TAZ in stromal cells trigger effects that positively feed back on the growth of tumour cells. Recognizing YAP and TAZ as a hub of the network of signals exchanged within the tumour microenvironment provides a fresh perspective on the molecular principles of tumour self-organization, promising to unveil numerous new vulnerabilities.

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.

Transcriptional addiction in cancer cells is mediated by YAP/TAZ through BRD4.

Cancer cells rely on dysregulated gene expression. This establishes specific transcriptional addictions that may be therapeutically exploited. Yet, the mechanisms that are ultimately responsible for these addictions are poorly understood. Here, we investigated the transcriptional dependencies of transformed cells to the transcription factors YAP and TAZ. YAP/TAZ physically engage the general coactivator bromodomain-containing protein 4 (BRD4), dictating the genome-wide association of BRD4 to chromatin. YAP/TAZ flag a large set of enhancers with super-enhancer-like functional properties. YAP/TAZ-bound enhancers mediate the recruitment of BRD4 and RNA polymerase II at YAP/TAZ-regulated promoters, boosting the expression of a host of growth-regulating genes. Treatment with small-molecule inhibitors of BRD4 blunts YAP/TAZ pro-tumorigenic activity in several cell or tissue contexts, causes the regression of pre-established, YAP/TAZ-addicted neoplastic lesions and reverts drug resistance. This work sheds light on essential mediators, mechanisms and genome-wide regulatory elements that are responsible for transcriptional addiction in cancer and lays the groundwork for a rational use of BET inhibitors according to YAP/TAZ biology.

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.

YAP/TAZ link cell mechanics to Notch signalling to control epidermal stem cell fate.

How the behaviour of somatic stem cells (SCs) is influenced by mechanical signals remains a black-box in cell biology. Here we show that YAP/TAZ regulation by cell shape and rigidity of the extracellular matrix (ECM) dictates a pivotal SC decision: to remain undifferentiated and grow, or to activate a terminal differentiation programme. Notably, mechano-activation of YAP/TAZ promotes epidermal stemness by inhibition of Notch signalling, a key factor for epidermal differentiation. Conversely, YAP/TAZ inhibition by low mechanical forces induces Notch signalling and loss of SC traits. As such, mechano-dependent regulation of YAP/TAZ reflects into mechano-dependent regulation of Notch signalling. Mechanistically, at least in part, this is mediated by YAP/TAZ binding to distant enhancers activating the expression of Delta-like ligands, serving as 'in cis' inhibitors of Notch. Thus YAP/TAZ mechanotransduction integrates with cell-cell communication pathways for fine-grained orchestration of SC decisions.

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 at the Roots of Cancer.

YAP and TAZ are highly related transcriptional regulators pervasively activated in human malignancies. Recent work indicates that, remarkably, YAP/TAZ are essential for cancer initiation or growth of most solid tumors. Their activation induces cancer stem cell attributes, proliferation, chemoresistance, and metastasis. YAP/TAZ are sensors of the structural and mechanical features of the cell microenvironment. A number of cancer-associated extrinsic and intrinsic cues conspire to overrule the YAP-inhibiting microenvironment of normal tissues, including changes in mechanotransduction, inflammation, oncogenic signaling, and regulation of the Hippo pathway. Addiction to YAP/TAZ thus potentially represents a central cancer vulnerability that may be exploited therapeutically.

YAP/TAZ as therapeutic targets in cancer.

The biology and regulation of YAP and TAZ, two closely related transcriptional regulators, are receiving increasing attention owing to their fundamental roles in organ growth, tissue repair and cancer. In particular, the widespread activation of YAP/TAZ in carcinomas, and the crucial role of YAP/TAZ activation for many 'hallmarks' of cancer are indicating YAP/TAZ as prime targets for designing anti-cancer drugs. Here, we start from the known modalities to regulate YAP/TAZ to highlight possible routes of therapeutic intervention.

The apical ectodermal ridge of the mouse model of ectrodactyly Dlx5;Dlx6-/- shows altered stratification and cell polarity, which are restored by exogenous Wnt5a ligand.

The congenital malformation split hand/foot (SHFM) is characterized by missing central fingers and dysmorphology or fusion of the remaining ones. Type-1 SHFM is linked to deletions/rearrangements of the DLX5-DLX6 locus and point mutations in the DLX5 gene. The ectrodactyly phenotype is reproduced in mice by the double knockout (DKO) of Dlx5 and Dlx6. During limb development, the apical ectodermal ridge (AER) is a key-signaling center responsible for early proximal-distal growth and patterning. In Dlx5;6 DKO hindlimbs, the central wedge of the AER loses multilayered organization and shows down-regulation of FGF8 and Dlx2. In search for the mechanism, we examined the non-canonical Wnt signaling, considering that Dwnt-5 is a target of distalless in Drosophila and the knockout of Wnt5, Ryk, Ror2 and Vangl2 in the mouse causes severe limb malformations. We found that in Dlx5;6 DKO limbs, the AER expresses lower levels of Wnt5a, shows scattered ß-catenin responsive cells and altered basolateral and planar cell polarity (PCP). The addition of Wnt5a to cultured embryonic limbs restored the expression of AER markers and its stratification. Conversely, the inhibition of the PCP molecule c-jun N-terminal kinase caused a loss of AER marker expression. In vitro, the addition of Wnt5a on mixed primary cultures of embryonic ectoderm and mesenchyme was able to confer re-polarization. We conclude that the Dlx-related ectrodactyly defect is associated with the loss of basoapical and PCP, due to reduced Wnt5a expression and that the restoration of the Wnt5a level is sufficient to partially reverts AER misorganization and dysmorphology.

Genome-wide association between YAP/TAZ/TEAD and AP-1 at enhancers drives oncogenic growth.

YAP/TAZ are nuclear effectors of the Hippo pathway regulating organ growth and tumorigenesis. Yet, their function as transcriptional regulators remains underinvestigated. By ChIP-seq analyses in breast cancer cells, we discovered that the YAP/TAZ transcriptional response is pervasively mediated by a dual element: TEAD factors, through which YAP/TAZ bind to DNA, co-occupying chromatin with activator protein-1 (AP-1, dimer of JUN and FOS proteins) at composite cis-regulatory elements harbouring both TEAD and AP-1 motifs. YAP/TAZ/TEAD and AP-1 form a complex that synergistically activates target genes directly involved in the control of S-phase entry and mitosis. This control occurs almost exclusively from distal enhancers that contact target promoters through chromatin looping. YAP/TAZ-induced oncogenic growth is strongly enhanced by gain of AP-1 and severely blunted by its loss. Conversely, AP-1-promoted skin tumorigenesis is prevented in YAP/TAZ conditional knockout mice. This work highlights a new layer of signalling integration, feeding on YAP/TAZ function at the chromatin level.