de-novo-generation-of-somatic-stem-cells-by-yaptaz

<em>De Novo</em> 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 ...

University of Padua School of Medicine

The assembly of the Smad complex is critical for TGFbeta signaling, yet the mechanisms that inactivate or empower nuclear Smad complexes are less understood. By means of siRNA screen we identified FAM (USP9x), a deubiquitinase acting as essential and evolutionarily conserved component in TGFbeta and bone morphogenetic protein signaling. Smad4 is monoubiquitinated in lysine 519 in vivo, a modification that inhibits Smad4 by impeding association with phospho-Smad2. FAM reverts this negative modification, re-empowering Smad4 function. FAM opposes the activity of Ectodermin/Tif1gamma (Ecto), a nuclear factor for which we now clarify a prominent role as Smad4 monoubiquitin ligase. Our study points to Smad4 monoubiquitination and deubiquitination as a way for cells to set their TGFbeta responsiveness: loss of FAM disables Smad4-dependent responses in several model systems, with Ecto being epistatic to FAM. This defines a regulative ubiquitination step controlling Smads that is parallel to those impinging on R-Smad phosphorylation.

FAM/USP9x, a deubiquitinating enzyme essential for TGFbeta signaling, controls Smad4 monoubiquitination.

TGFbeta ligands act as tumor suppressors in early stage tumors but are paradoxically diverted into potent prometastatic factors in advanced cancers. The molecular nature of this switch remains enigmatic. Here, we show that TGFbeta-dependent cell migration, invasion and metastasis are empowered by mutant-p53 and opposed by p63. Mechanistically, TGFbeta acts in concert with oncogenic Ras and mutant-p53 to induce the assembly of a mutant-p53/p63 protein complex in which Smads serve as essential platforms. Within this ternary complex, p63 functions are antagonized. Downstream of p63, we identified two candidate metastasis suppressor genes associated with metastasis risk in a large cohort of breast cancer patients. Thus, two common oncogenic lesions, mutant-p53 and Ras, selected in early neoplasms to promote growth and survival, also prefigure a cellular set-up with particular metastasis proclivity by TGFbeta-dependent inhibition of p63 function.

A Mutant-p53/Smad complex opposes p63 to empower TGFbeta-induced metastasis.

Although specific microRNAs (miRNAs) can be upregulated in cancer, global miRNA downregulation is a common trait of human malignancies. The mechanisms of this phenomenon and the advantages it affords remain poorly understood. Here we identify a microRNA family, miR-103/107, that attenuates miRNA biosynthesis by targeting Dicer, a key component of the miRNA processing machinery. In human breast cancer, high levels of miR-103/107 are associated with metastasis and poor outcome. Functionally, miR-103/107 confer migratory capacities in vitro and empower metastatic dissemination of otherwise nonaggressive cells in vivo. Inhibition of miR-103/107 opposes migration and metastasis of malignant cells. At the cellular level, a key event fostered by miR-103/107 is induction of epithelial-to-mesenchymal transition (EMT), attained by downregulating miR-200 levels. These findings suggest a new pathway by which Dicer inhibition drifts epithelial cancer toward a less-differentiated, mesenchymal fate to foster metastasis.

A MicroRNA targeting dicer for metastasis control.

Cells perceive their microenvironment not only through soluble signals but also through 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 behaviour, 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 (Yes-associated protein) and TAZ (transcriptional coactivator with PDZ-binding motif, also known as WWTR1) as nuclear relays of mechanical signals exerted by ECM rigidity and cell shape. This regulation requires Rho GTPase activity and tension of the actomyosin cytoskeleton, but is independent of 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 activated YAP overrules physical constraints in dictating cell behaviour. These findings identify YAP/TAZ as sensors and mediators of mechanical cues instructed by the cellular microenvironment.

Role of YAP/TAZ in mechanotransduction.

Cancer stem cells (CSCs) are proposed to drive tumor initiation and progression. Yet, our understanding of the cellular and molecular mechanisms that underlie CSC properties is limited. Here we show that the activity of TAZ, a transducer of the Hippo pathway, is required to sustain self-renewal and tumor-initiation capacities in breast CSCs. TAZ protein levels and activity are elevated in prospective CSCs and in poorly differentiated human tumors and have prognostic value. Gain of TAZ endows self-renewal capacity to non-CSCs. In epithelial cells, TAZ forms a complex with the cell-polarity determinant Scribble, and loss of Scribble--or induction of the epithelial-mesenchymal transition (EMT)--disrupts the inhibitory association of TAZ with the core Hippo kinases MST and LATS. This study links the CSC concept to the Hippo pathway in breast cancer and reveals a mechanistic basis of the control of Hippo kinases by cell polarity.

The Hippo transducer TAZ confers cancer stem cell-related traits on breast cancer cells.

The molecular determinants of malignant cell behaviours in breast cancer remain only partially understood. Here we show that SHARP1 (also known as BHLHE41 or DEC2) is a crucial regulator of the invasive and metastatic phenotype in triple-negative breast cancer (TNBC), one of the most aggressive types of breast cancer. SHARP1 is regulated by the p63 metastasis suppressor and inhibits TNBC aggressiveness through inhibition of hypoxia-inducible factor 1α (HIF-1α) and HIF-2α (HIFs). SHARP1 opposes HIF-dependent TNBC cell migration in vitro, and invasive or metastatic behaviours in vivo. SHARP1 is required, and sufficient, to limit expression of HIF-target genes. In primary TNBC, endogenous SHARP1 levels are inversely correlated with those of HIF targets. Mechanistically, SHARP1 binds to HIFs and promotes HIF proteasomal degradation by serving as the HIF-presenting factor to the proteasome. This process is independent of pVHL (von Hippel-Lindau tumour suppressor), hypoxia and the ubiquitination machinery. SHARP1 therefore determines the intrinsic instability of HIF proteins to act in parallel to, and cooperate with, oxygen levels. This work sheds light on the mechanisms and pathways by which TNBC acquires invasiveness and metastatic propensity.

SHARP1 suppresses breast cancer metastasis by promoting degradation of hypoxia-inducible factors.

Wnt growth factors are fundamental regulators of cell fate, but how the Wnt signal is translated into biological responses is incompletely understood. Here, we report that TAZ, a biologically potent transcriptional coactivator, serves as a downstream element of the Wnt/β-catenin cascade. This function of TAZ is independent from its well-established role as mediator of Hippo signaling. In the absence of Wnt activity, the components of the β-catenin destruction complex--APC, Axin, and GSK3--are also required to keep TAZ at low levels. TAZ degradation depends on phosphorylated β-catenin that bridges TAZ to its ubiquitin ligase β-TrCP. Upon Wnt signaling, escape of β-catenin from the destruction complex impairs TAZ degradation and leads to concomitant accumulation of β-catenin and TAZ. At the genome-wide level, a substantial portion of Wnt transcriptional responses is mediated by TAZ. TAZ activation is a general feature of Wnt signaling and is functionally relevant to mediate Wnt biological effects.

Role of TAZ as mediator of Wnt signaling.

The evolution of a solid tumor is fueled by genetic aberrations. Yet, the tumor environment often dominates over the effects of genetics: normal tissues have powerful tumor-suppressive properties that constantly tame or eliminate cells carrying transforming mutations. Critical elements of such a suppressive microenvironment are structural characteristics of normal cells and tissues, such as cell polarity, attachment to the extracellular matrix (ECM), and epithelial organization. Once these tissue-level checkpoints have been overcome, tumor growth is enhanced by recruitment of stromal cells and remodeling of the ECM. Genetic inactivation in mouse models indicates the Hippo pathway as a fundamental inhibitor of organ growth during development and as a critical tumor suppressor in epithelial tissues, such as the liver, skin, and ovaries, and soft tissues. At the centerpiece of this pathway lie two related transcriptional coactivators, YAP and TAZ, that promote tissue proliferation and the self-renewal of normal and cancer stem cells, and incite metastasis. Strikingly, YAP and TAZ are controlled by the same architectural features that first inhibit and then foster cancer growth, such as ECM elasticity, cell shape, and epithelial-to-mesenchymal transition. These findings open unexpected opportunities for the development of new cancer therapeutics targeting key YAP/TAZ regulatory inputs such as Wnt signaling, cytoskeletal contractility, G-protein-coupled receptors, or YAP/TAZ-regulated transcription.

Molecular pathways: YAP and TAZ take center stage in organ growth and tumorigenesis.

The YAP and TAZ mediators of the Hippo pathway (hereafter called YAP/TAZ) promote tissue proliferation and organ growth. However, how their biological properties intersect with cellular metabolism remains unexplained. Here, we show that YAP/TAZ activity is controlled by the SREBP/mevalonate pathway. Inhibition of the rate-limiting enzyme of this pathway (HMG-CoA reductase) by statins opposes YAP/TAZ nuclear localization and transcriptional responses. Mechanistically, the geranylgeranyl pyrophosphate produced by the mevalonate cascade is required for activation of Rho GTPases that, in turn, activate YAP/TAZ by inhibiting their phosphorylation and promoting their nuclear accumulation. The mevalonate-YAP/TAZ axis is required for proliferation and self-renewal of breast cancer cells. In Drosophila melanogaster, inhibition of mevalonate biosynthesis and geranylgeranylation blunts the eye overgrowth induced by Yorkie, the YAP/TAZ orthologue. In tumour cells, YAP/TAZ activation is promoted by increased levels of mevalonic acid produced by SREBP transcriptional activity, which is induced by its oncogenic cofactor mutant p53. These findings reveal an additional layer of YAP/TAZ regulation by metabolic cues. 

Metabolic control of YAP and TAZ by the mevalonate pathway.

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 β-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 β-TrCP recruitment to the complex and β-catenin inactivation. In Wnt-ON cells, release of YAP/TAZ from the complex is instrumental for Wnt/β-catenin signaling. In line, the β-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.

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

The transcriptional regulators YAP and TAZ are the focus of intense interest given their remarkable biological properties in development, tissue homeostasis and cancer. YAP and TAZ activity is key for the growth of whole organs, for amplification of tissue-specific progenitor cells during tissue renewal and regeneration, and for cell proliferation. In tumors, YAP/TAZ can reprogram cancer cells into cancer stem cells and incite tumor initiation, progression and metastasis. As such, YAP/TAZ are appealing therapeutic targets in cancer and regenerative medicine. Just like the function of YAP/TAZ offers a molecular entry point into the mysteries of tissue biology, their regulation by upstream cues is equally captivating. YAP/TAZ are well known for being the effectors of the Hippo signaling cascade, and mouse mutants in Hippo pathway components display remarkable phenotypes of organ overgrowth, enhanced stem cell content and reduced cellular differentiation. YAP/TAZ are primary sensors of the cell's physical nature, as defined by cell structure, shape and polarity. YAP/TAZ activation also reflects the cell "social" behavior, including cell adhesion and the mechanical signals that the cell receives from tissue architecture and surrounding extracellular matrix (ECM). At the same time, YAP/TAZ entertain relationships with morphogenetic signals, such as Wnt growth factors, and are also regulated by Rho, GPCRs and mevalonate metabolism. YAP/TAZ thus appear at the centerpiece of a signaling nexus by which cells take control of their behavior according to their own shape, spatial location and growth factor context. 

The biology of YAP/TAZ: hippo signaling and beyond.

ZO-oming on growth control by junctional proteins.

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. 

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

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.

YAP/TAZ as therapeutic targets in 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 at the Roots of Cancer.

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.

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

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.

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

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.

Article	Year
FAM/USP9x, a deubiquitinating enzyme essential for TGFbeta signaling, controls Smad4 monoubiquitination. Cell\| PubMed ID: 19135894	2009
A Mutant-p53/Smad complex opposes p63 to empower TGFbeta-induced metastasis. Cell\| PubMed ID: 19345189	2009
A MicroRNA targeting dicer for metastasis control. Cell\| PubMed ID: 20603000	2010
Role of YAP/TAZ in mechanotransduction. Nature\| PubMed ID: 21654799	2011
The Hippo transducer TAZ confers cancer stem cell-related traits on breast cancer cells. Cell\| PubMed ID: 22078877	2011
SHARP1 suppresses breast cancer metastasis by promoting degradation of hypoxia-inducible factors. Nature\| PubMed ID: 22801492	2012
Role of TAZ as mediator of Wnt signaling. Cell\| PubMed ID: 23245942	2012
Molecular pathways: YAP and TAZ take center stage in organ growth and tumorigenesis. Clinical cancer research : an official journal of the American Association for Cancer Research\| PubMed ID: 23797907	2013
Metabolic control of YAP and TAZ by the mevalonate pathway. Nature cell biology\| PubMed ID: 24658687	2014
YAP/TAZ incorporation in the β-catenin destruction complex orchestrates the Wnt response. Cell\| PubMed ID: 24976009	2014
The biology of YAP/TAZ: hippo signaling and beyond. Physiological reviews\| PubMed ID: 25287865	2014
ZO-oming on growth control by junctional proteins. Cell cycle (Georgetown, Tex.)\| PubMed ID: 25590649	2015
Genome-wide association between YAP/TAZ/TEAD and AP-1 at enhancers drives oncogenic growth. Nature cell biology\| PubMed ID: 26258633	2015
YAP/TAZ as therapeutic targets in cancer. Current opinion in pharmacology\| PubMed ID: 27262779	2016
YAP/TAZ at the Roots of Cancer. Cancer cell\| PubMed ID: 27300434	2016
Induction of Expandable Tissue-Specific Stem/Progenitor Cells through Transient Expression of YAP/TAZ. Cell stem cell\| PubMed ID: 27641305	2016
YAP/TAZ link cell mechanics to Notch signalling to control epidermal stem cell fate. Nature communications\| PubMed ID: 28513598	2017
Mechanobiology of YAP and TAZ in physiology and disease. Nature reviews. Molecular cell biology\| PubMed ID: 28951564	2017

Michelangelo Cordenonsi

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