Regulation of YAP and TAZ by Epithelial Plasticity

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Abstract

The Hippo transducers YAP and TAZ are central mediators of organ growth and tumorigenesis, regulating cell proliferation, differentiation, and epithelial stemness. In this chapter, we summarize recent findings linking the activation of YAP and TAZ to the cell’s structural and architectural features, such as cell polarity, cell shape, cell adhesion, and cytoskeletal dynamics. We examine how epithelial “plasticity” induced by epithelial-to-mesenchymal transition (EMT) promotes “Cancer Stem Cell” identity and YAP/TAZ activation, and discuss the role of TAZ as molecular determinant of self-renewal and tumor-seeding potentials in cancer cells. YAP and TAZ activation can also induce EMT, generating a self-sustaining loop. We then place special emphasis on biomechanical cues as regulators of epithelial plasticity, and as dominant regulators of YAP and TAZ nuclear localization and transcriptional activities. This regulation is mediated by physical forces, such as rigidity of the extracellular matrix, compression from neighboring cells, and tension of the actomyosin cytoskeleton. These mechanical signals hold in shape individual cells and whole tissues, and are severely disturbed in cancer. In sum, we highlight new mechanisms of YAP and TAZ regulation by cell polarity and mechanical cues. This potentially adds a new dimension to our understanding of physiology and tumorigenesis, whereby the behavior of individual cells is dictated by the integration of information about tissue architecture and mechanics mediated by YAP and TAZ.

Keywords

Epithelial plasticity Epithelial-to-mesenchymal transition Cancer stem cells Apico-basal polarity Biomechanical signals Mechanotransduction Tissue architecture Scribble Hippo Yap and TAZ 
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6.1 Overview

The Hippo pathway plays fundamental roles in the control of cell proliferation, cell survival, and cell fates in development and tissue homeostasis (Halder and Johnson 2011; Pan 2010; Ramos and Camargo 2012; Zhao et al. 2010). At the centerpiece of this signaling cascade are the transcriptional cofactors YAP and TAZ. In its most basic formulation, the pathway operates as follows: YAP and TAZ are inhibited by phosphorylation mediated by the LATS1/2 kinases, that, in turn, are activated by MST1/2 kinases, the homologues of Drosophila Hippo (Pan 2010). The mechanism of YAP/TAZ inhibition by phosphorylation is dual: degradation by the proteasome and/or sequestration in the cytoplasm by anchoring proteins (Pan 2010; Zhao et al. 2010). In recent years, however, several variations on this basic signaling module have been reported, including LATS-independent phosphorylation of YAP/TAZ, MST-independent activation of LATS, and phosphorylation-independent modalities of YAP/TAZ regulation (Dupont et al. 2011; Moleirinho et al. 2012; Schlegelmilch et al. 2011; Zhou et al. 2009). As such, the reader should be aware of a semantic issue, as the definition of what “is” the Hippo pathway has progressively blurred to include clear non-Hippo regulations and probably other pathways feeding on YAP/TAZ activity.

A main question in the Hippo field is how the activity of YAP and TAZ is regulated by extrinsic and intrinsic cellular signals. In this chapter we will summarize some emerging paradigms of YAP and TAZ regulation by epithelial-to-mesenchymal transition (EMT) and by changes in cell shape triggered by mechanical signals that the cell receives from its microenvironment. We start with the EMT phenomenon, a profound change in cell morphology that occurs during development, tissue regeneration, and tumor progression (see Sect. 1). Several lines of evidence link EMT to acquisition of phenotypic traits typical of stem cells, in both physiological and neoplastic contexts (outlined in Sect. 2) (Polyak and Weinberg 2009). EMT promotes loss of cell polarity and loss of cell–cell adhesion (Thiery et al. 2009), two events that have been long implicated in the regulation of the Hippo pathway in Drosophila and mammalian cells (Genevet and Tapon 2011).

YAP and TAZ have been also shown to be critical in controlling the amplification of stem cells and tissue progenitor cells in several tissues, playing important roles in tissue regeneration (Ramos and Camargo 2012). These functions are diverted in cancer, where YAP and TAZ serve as potent promoters of malignancy and of cancer cell “stemness,” thus recapitulating the effects of EMT (Bhat et al. 2011; Camargo et al. 2007; Cao et al. 2008; Cordenonsi et al. 2011; Lee et al. 2010; Lu et al. 2010; Moleirinho et al. 2012; Pan 2010; Schlegelmilch et al. 2011; Song et al. 2010; Zhou et al. 2011a). Despite the clear analogies between EMT and YAP/TAZ biology, only recently a direct biochemical link between EMT, cell polarity, and Hippo signaling has been revealed, along with the demonstration of a causal relationship between EMT, activation of TAZ, and induction of cancer stem cell (CSCs) traits (Cordenonsi et al. 2011) (see Sect. 3).

One of the most fascinating and recently emerged aspects of YAP and TAZ biology is their regulation by structural elements that originate at the tissue level, such as adhesion of a cell to its surrounding extracellular matrix (ECM), cell–cell junctions and the tensional forces of the cytoskeleton that keep cells, tissues, and organs in a certain shape (Halder et al. 2012). As such, these regulations can inform individual cells about properties of the tissue in which they are embedded, such as organ size and three-dimensional organization. In other words, YAP and TAZ regulations offer the unprecedented opportunity to explore one of the Holy Grails in biological research, that is, wiring signal transduction and cell biology to the overarching tissue biology (see Sect. 4).

6.2 What is EMT?

EMT is a phenotypic switch by which epithelial cells lose cell–cell adhesion and apico-basal polarity, and instead acquire motility, invasiveness, and resistance to apoptosis (Fig. 6.1) (Thiery et al. 2009). In addition, EMT includes acquisition of spread cell morphology, extended cell-ECM contacts, and capacity to degrade the basement membrane and infiltrate the underlying stroma (Fig. 6.1). Such epithelial plasticity is critical for building organs during embryogenesis and for rapid mobilization of cells for tissue repair during adult life. However, EMT is also involved in various human pathologies, most notably fibrosis and cancer (Thiery et al. 2009).
Fig. 6.1

Changes in cell architecture during EMT. See text and callouts

The most remarkable feature of EMT in the context of tumor biology is endowing cancer cells with characteristics typical of “cancer stem cells”, including the capacity to self-renew and to generate secondary tumors (Chaffer and Weinberg 2011; Mani et al. 2008; Polyak and Weinberg 2009). In addition, non-transformed epithelial cells may also undergo EMT and, in so doing, gain stemness to the extent that a single EMT-transited mammary cell is sufficient to generate an entire mammary gland when implanted into the mouse fat pad (Guo et al. 2012; Mani et al. 2008).

It is important to reflect on the fact EMT—and its reverse process mesenchymal-to-epithelial transition (MET)—also entail profound morphological changes, thus reminding us how much information can be stored in cell shape itself, irrespectively of the specific set of mutations that characterize a given cancer (Bissell and Hines 2011; Butcher et al. 2009). This provides a departure from a pure “DNA-centric” perspective in tumor biology, one whereby a specific cellular status (e.g., differentiation, capacity of tumor initiation, and chemoresistance) could be intrinsic to the shape assumed by cancer cells (i.e., epithelial vs. mesenchymal shapes). In fact, acquisition of driving mutations in potent oncogenes may not be sufficient to generate a neoplasia until tumor cells are kept in check by the three-dimensional architecture of the surrounding tissue (Bissell and Hines 2011; Podsypanina et al. 2008; Leung and Brugge 2012; Dolberg and Bissell 1984; Holst et al. 2003; Illmensee and Mintz 1976; Jonason et al. 1996; Levental et al. 2009; Michaloglou et al. 2005; Weaver et al. 1997). As such, sporadic tumor cells may remain dormant even for years before overcoming this overarching barrier, possibly by undergoing EMT. In the following section we will focus on the nature of epithelial traits targeted by the EMT, and on the intracellular and extracellular determinants of EMT.

6.2.1 The Epithelial Format: Junctional Complexes and Apico-Basal Polarity

Most of our organs contain epithelial sheets. The epithelial format emerged more than 600 million years ago to separate the inside from the outside of multicellular organisms. Epithelia have evolved specialized structures that sustain barrier, secretion and absorption functions, but also more sophisticated activities including maintenance of tissue architecture, sensing cell density, and tumor suppression (Nelson and Fuchs 2010).

The sites of cell–cell and cell-substrate contacts are the “sensorial” interfaces by which cells experience their outside world. As such, these privileged areas of the cell’s border are essential in many ways, including (a) to mediate “social cues,” such as contact inhibition of growth; (b) to orient intracellular structures coherently within the polarity and shape of the neighboring cells; and (c) to organize the cytoskeleton, adapting it to the biomechanical properties of the rest of the tissue (Fig. 6.1a) (Halder et al. 2012).

In epithelia, cells are adjoined to each other by specific junctional complexes, namely tight junctions, adherens junctions, and desmosomes (Box 6.1), and to the ECM through focal adhesions and hemidesmosomes (Nelson and Fuchs 2010). The maintenance of cellular junctions is dependent on cell polarization along the cell’s apico-basal axis; vice versa, the distinct protein complexes regulating apico-basal cell polarity (Box 6.1 and Fig. 6.2) require cell–cell adhesiveness to maintain their asymmetric localization (Martin-Belmonte and Perez-Moreno 2011). This mutually reinforcing adhesion-polarity loop provides a tumor suppressive environment to epithelial tissues. The EMT targets this system at the heart (Moreno-Bueno et al. 2008). Indeed, EMT invariably entails the loss of E-cadherin (or its cytoplasmic relocalization) causing loss of cell–cell adhesive capacity. This leads to cell depolarization that, in a vicious loop, causes further dismantling of cell–cell adhesion (Martin-Belmonte and Perez-Moreno 2011; Thiery et al. 2009). In turn, this reflects into an increased dependency on cell-ECM adhesion that licenses cell proliferation or survival (Livshits et al. 2012; Nelson et al. 2004; Paszek et al. 2005; Ruiz and Chen 2008). Furthermore, since junctional complexes have evolved tight connection to several key signal transduction pathways (Nelson and Fuchs 2010)—and to the Hippo pathway in particular (Box 6.2)—EMT entails broad effects on cell signaling.
Fig. 6.2

Protein complexes regulating epithelial cell apico-basal polarity

6.2.2 Installing EMT

How is the EMT program installed? EMT is under the control of distinct environmental cues typically acting in concert with each other and whose specific relevance may depend on the cellular context. This includes signaling from hypoxia and growth factors, including TGFβ, Wnt, Notch, Hedgehog, Interleukins, and RTK ligands (Polyak and Weinberg 2009; Thiery et al. 2009). The source of these signals is typically found in the stromal cells, but they can also be produced by epithelial cells themselves in an autocrine manner (Scheel et al. 2011). In addition, ECM stiffness is also a potent inducer of EMT-like effects (see Sect. 4 below) (Gjorevski et al. 2012). It is important to note that most of these cues have been implicated in directing differentiation or proliferation in embryonic development, or as “niche” factors for adult stem cells (Dreesen and Brivanlou 2007). This suggests that the EMT program in cancer cells may represent a hijacking of normal mechanisms of tissue formation and maintenance.

Despite the diversity of these inputs, the EMT program follows a rather stereotyped set of events. Indeed, directly or indirectly, all these inputs converge on the regulation of a group of transcription factors able to repress epithelial gene expression. This group includes Snail (SNAI1), Slug (SNAI2), ZEB1/2, and Twist (Peinado et al. 2007). Overexpression of individual members of this group can orchestrate the EMT program, instill stemness and activate the invasion-metastasis cascade (Guo et al. 2012; Wellner et al. 2009). For example, members of the TGFβ family are among the most extensively studied inducers of EMT (Heldin et al. 2009; Zavadil and Bottinger 2005). TGFβ induces EMT by inducing expression of ZEB and Snail proteins that turn off the epithelial program by repressing expression of E-Cadherin and of other junctional and polarity proteins. TGFβ can also foster EMT indirectly through activation of Rho GTPases (Bhowmick et al. 2001; Peinado et al. 2007; Wang et al. 2006; Zavadil and Bottinger 2005).

One of the mysteries of the EMT process is its duration and stability: EMT is mostly a transient phenomenon in vivo raising questions on what regulates the rate of conversion between the epithelial and EMT states (Chaffer and Weinberg 2011; Thiery et al. 2009). Work in the microRNA field has shown that mutual repression between a miRNA and its target is a very effective way to generate all-or-none responses and “one-of-the-two” cellular decisions (Inui et al. 2010). The reciprocal antagonism between miR-200 and ZEB1 or ZEB2 represents a paradigm for this feedback module. miR-200 family members are expressed in epithelial cells, where they inhibit ZEB1/2 expression preserving E-Cadherin expression; however, in mesenchymal cells, ZEB1 and ZEB2 transcriptionally repress miR-200 transcription (Gregory et al. 2008; Inui et al. 2010; Wellner et al. 2009). This mutual inhibitory loop provides robustness to the EMT phenomena, as EMT-inducing stimuli must be present for sufficient time and intensity at least to surpass the miR-200 barrier.

6.3 EMT and the Cancer Stem Cells Phenomenon

6.3.1 Cancer Stem Cells

Tumor cells are phenotypically heterogeneous, raising the question as to how this diversity is generated. In the classical view, intratumoral heterogeneity is caused by the tumor’s intrinsic genetic instability, spawning many genetically distinct subclones, sorted by Darwinian selection (Shackleton et al. 2009). Moreover, in recent years, it has become increasingly clear that a tumor is not a randomly organized collection of cells; rather, a tumor should be better envisioned as an aberrant attempt at de novo organogenesis, or as an organ “caricature” still taking advantage of the same molecular and cellular mechanisms utilized during development for epithelial self-renewal and differentiation (Egeblad et al. 2010; Pierce and Speers 1988). In fact, recapitulating the cellular hierarchies of normal tissues, tumors include a specific cell subpopulation of cancer cells—termed cancer stem cells (or tumor-initiating cells)—lodged into specific environmental niches and responsible for constant tumor regeneration (Nguyen et al. 2012; Shackleton et al. 2009; Visvader and Lindeman 2012). CSCs are operationally defined as the fraction of tumor cells specifically endowed with self-renewal, tumor-seeding, and chemoresistance potential as well as ability to generate non-CSC progeny that constitutes the rest of the tumor bulk (Visvader and Lindeman 2012).

The molecular and cellular bases of the CSC properties remain enigmatic. In fact, CSCs can be identified only retrospectively, depending on assays that measure the self-renewing potential of individual cells growing as spheres in vitro, their capacity of initiate new tumors when injected in recipient mice at limiting dilutions, or the capacity to form broad clonal descendants in vivo (Chen et al. 2012; Driessens et al. 2012; Gilbertson and Graham 2012; Gupta et al. 2009a; Nguyen et al. 2012; Schepers et al. 2012). The lack of molecular definition has contributed to a number of debates over the CSC concept, including how abundant they are, what is their relationships with normal stem cells, or whether CSCs can be generated from non-CSCs (Magee et al. 2012).

6.3.2 EMT and CSCs

What are the main evidences connecting EMT and CSCs? First, transformed human mammary epithelial cells that have undergone an EMT display an increased capacity to self-renew, to grow as soft agar colonies, and to generate tumors (Mani et al. 2008). Moreover, experimental induction of EMT promotes the formation of cells expressing cell-surface antigens that are found enriched in naturally emerging CSC populations (Mani et al. 2008). Second, EMT has been implicated in conferring metastatic potential and therapeutic resistance (Chaffer and Weinberg 2011; Gupta et al. 2009b; Moody et al. 2005; Sayan et al. 2009; Witta et al. 2006; Yang et al. 2004). The formation of secondary tumors or tumor regeneration after chemotherapy must rely on CSC-like properties. Further highlighting the link between EMT and CSCs is that fact that transcriptional inducers of EMT, such as Slug, are also instrumental for conferring stemness to normal mammary epithelia and full metastatic potential to CSC-like breast cancer cell lines (Guo et al. 2012). In line, elevated expression of EMT-inducing factors is clinically relevant, it has been detected at the tumor-stroma borders and associated to elevated incidence of metastasis, recurrence, and poor differentiation in multiple types of tumors (Peinado et al. 2007; Polyak and Weinberg 2009).

CSC representation is strongly dependent on tumor grade (with advanced tumors containing more CSCs than differentiated tumors) and microenvironmental factors (Driessens et al. 2012; Egeblad et al. 2010; Pece et al. 2010; Visvader and Lindeman 2012). Indeed, there is substantial evidence that contextual signals may expand or shrink the pool of CSCs by tuning self-renewal, differentiation or by inducing CSC-like traits in non-CSC (Chaffer et al. 2011; Quintana et al. 2010; Roesch et al. 2010). For example, TGFb and Wnt signaling have been identified as autocrine factors that maintain the stem cell state in mammary cells and collaborate to induce EMT (Scheel et al. 2011). The dynamic equilibrium between the CSCs and non-CSCs population is reminiscent of the instability and reversibility of the EMT phenotype and is indeed regulated by the same factors that control EMT.

6.3.3 Parsing EMT

The identification of EMT as a process able to endow “stemness” to epithelial cells clearly represented a critical discovery, because it anchored the operational definitions of stem cells and CSC to well-defined and well recognizable morphological features of the cell (Mani et al. 2008). That said, most of what we know about the connection between EMT and CSCs comes from studies conducted in cells cultured in vitro or isolated ex-vivo; in contrast, transition to mesenchymal cell fates has been reported only in specific tumor subsets and appears as an overall rare event (Chaffer and Weinberg 2011; Savagner 2010). This raises questions on the general relevance of EMT in human tumors. Clearly, EMT and CSCs are broad and only recently added dimensions to the cancer field, and much research is needed to attain more definitive answers. For example, EMT may not be a general feature of the whole tumor but instead it may occur only at specific locations, such as in proximity of the activated stroma at the tumor border. Moreover, epithelial cells that acquired a full EMT might be indistinguishable from fibroblasts, posing a technical challenge to their identification by routine histopathological examination. These caveats notwithstanding, it is worth noting here that part of the problem may be in our definition of EMT. Although the term EMT was originally limited to the acquisition of a fibroblast-like, spindle cell morphology, the reality of tumors may be much more variegated. “Partial-EMTs” have been described in tumors, a condition whereby cells co-express epithelial and mesenchymal markers (Klymkowsky and Savagner 2009). More critically, not all the segments of the complex EMT program may be equally necessary to confer stemness potential. loss of apicobasal polarity—a true hallmark of cancer, and primary initial step of any EMT—is more likely to be at the center stage of EMT-mediated induction of CSC.

6.4 EMT and HIPPO: EMT as Upstream Regulator of YAP and TAZ

In the above discussion, we have outlined some key aspects of EMT, its upstream inducers and the link to CSCs, but left unaddressed perhaps the most critical question: what are the molecular effectors downstream of EMT? What is executing the genetic programs of stemness, tumor progression and CSC-traits that are associated to EMT? Recent studies have highlighted, on the one hand, the fundamental role of YAP and TAZ as mediators of stemness in normal stem cells and cancer stem cells, and, on the other hand, the regulation of YAP and TAZ by cell polarity and EMT (Bhat et al. 2011; Cordenonsi et al. 2011). This indicated YAP and TAZ as ideal candidate to mediate some of the key biological effects of EMT.

6.4.1 The TAZ-CSCs Connection

TAZ has recently emerged as a primary molecular determinant of several characteristics of CSCs. TAZ is required for self-renewal and tumor-initiation capacities of breast cancer cells, as measured by the capacity of cells to grow as self-regenerating mammospheres and to form tumors once cancer cells are injected as limiting dilutions in immunocompromised mice (Cordenonsi et al. 2011). Notably, loss of TAZ impairs invasiveness, self-renewal, and tumorigenic capacity also in primary glioblastoma stem cells (GSCs), indicating that TAZ may also confer CSC-traits in tumors other than breast cancer (Bhat et al. 2011). Moreover, gain-of-TAZ endows these properties to otherwise non-CSC breast cancer cell populations (Cordenonsi et al. 2011). Importantly, if TAZ levels are experimentally induced in differentiated tumor cells, these cells generate high-grade/undifferentiated tumors (Bhat et al. 2011; Cordenonsi et al. 2011). Finally, TAZ expression is associated with expression of cell-surface antigens typical of putative CSC populations (Bhat et al. 2011; Cordenonsi et al. 2011).

There is substantial evidence that the proportion of CSCs is higher in poorly differentiated human primary tumors, namely, those routinely classified as “high-grade” malignancies by histopathological examination (Pece et al. 2010). TAZ protein levels are indeed elevated in high-grade breast cancers and glioblastomas, as assayed by immunohistochemistry in primary tumor samples (Bhat et al. 2011; Cordenonsi et al. 2011). The notion that TAZ is a CSC determinant is further supported by bioinformatic analyses of public datasets containing gene expression profiles and associated clinical history for a large collection of primary mammary tumors. First, signatures of TAZ activation (as defined by sets of TAZ target genes) identify the same tumors displaying signatures of “stemness genes” (Cordenonsi et al. 2011). Second, high TAZ is also an important clinical variable that discriminate tumors associated to poor survival and metastasis, and that are resistant to chemotherapy (Cordenonsi et al. 2011; Lai et al. 2011).

6.4.2 An EMT-Scribble-TAZ Axis in Breast Cancer

TAZ protein stabilization is induced by EMT-inducing transcription factors, such as Twist or Snail, and, crucially, TAZ is required for self-renewal induced by these EMT-promoting factors in breast cancer cells (Cordenonsi et al. 2011). Intriguingly, loss of TAZ does not revert mesenchymal cells back to an epithelial state; in other words, TAZ is downstream of EMT, and uncouples mesenchymality from EMT-induced stemness, being TAZ dispensable for the first and required for the second.

Recent evidence connects TAZ regulation by EMT to the basolateral polarity determinant Scribble (Box 6.1). The SCRIB complex plays a prominent role in human malignancies: it is downregulated or cytoplasmically mislocalized in a broad variety of tumors (including colon, breast, cervical, prostate, and lung) (Martin-Belmonte and Perez-Moreno 2011; Pearson et al. 2011; Vaira et al. 2011; Zhan et al. 2008). During the initial steps of EMT Scribble is delocalized from the membrane to the cytoplasm (Cordenonsi et al. 2011), likely as a consequence of cadherin downregulation (Navarro et al. 2005). Notably, Scribble inactivation—or its transient removal from the plasma membranes—is sufficient, per se, to increase stemness potential in mammary epithelial cells (Cordenonsi et al. 2011).

TAZ stabilization triggered by EMT is recapitulated by the sole loss-of-Scribble, indicating that loss of this polarity determinant may be sufficient to endow some of the key attributes of EMT without the need of reaching the full mesenchymal fate. Indeed, tethering Scribble to the plasma membrane can downregulate TAZ levels in cells that passed EMT (Cordenonsi et al. 2011). Mechanistically, Scribble serves as membrane-localized adaptor for TAZ and the Hippo kinases LATS and MST, leading to TAZ phosphorylation and subsequent recognition by the β-TrCP E3 ubiquitin ligase complex that causes TAZ degradation (Cordenonsi et al. 2011). Loss-of-Scribble, or its delocalization from the plasma membrane, prevents TAZ phosphorylation, leading to TAZ stabilization and nuclear activity. In addition to the Scribble-Hippo-TAZ connection, there are other mechanisms by which polarity and cell–cell junctions can control TAZ and YAP activity (see Box 6.2), although the exploitation of these mechanisms during EMT remains to be tested. We conclude that TAZ protein stabilization downstream of EMT embodies some salient characteristics so far only operationally linked to CSCs, such as tumor heterogeneity, reduced differentiation, self-renewal, tumor initiation, chemoresistance, and plasticity.

6.4.3 YAP and TAZ as Upstream Inducers of EMT

The relationship between YAP/TAZ and EMT is likely to be bidirectional, whereby EMT induces YAP and TAZ that in turn sustain the EMT program, at least in some cellular contexts. This would configure an autonomous, self-sustaining loop for enduring YAP/TAZ stabilization.

The activity of YAP as EMT inducer was noted in mammary epithelial cells since the very first report of YAP oncogenic properties (Overholtzer et al. 2006). This early observation was soon followed by others, showing that overexpression of a non-phosphorylatable form of YAP or TAZ induced EMT in MCF10A cells (Chan et al. 2008; Lei et al. 2008; Zhao et al. 2008). Thus, EMT induction in MCF10A cells is a very robust bioassay to monitor the activity of overexpressed YAP/TAZ. Little is known on how YAP and TAZ activate EMT; clearly, EMT-inducing transcription factors are likely candidates as YAP/TAZ target genes, but this hypothesis has not been tested so far.

However, EMT induction by YAP or TAZ overexpression should not be considered a general event. YAP overexpression fails to promote EMT in ovarian cancer cells or in normal bronchial epithelial cells in vitro (Zhang et al. 2011; Zhou et al. 2011b),or in hepatocytes, keratinocytes, and intestinal epithelial cells of YAP-overexpressing transgenic mice (Camargo et al. 2007; Dong et al. 2007; Schlegelmilch et al. 2011). Furthermore, endogenous YAP and TAZ activation, following genetic ablation of MST1/MST2 or Salvador (WW45), is not sufficient to trigger EMT in a variety of epithelial tissues (Lee et al. 2008, 2010; Zhou et al. 2009, 2011a). Importantly in this regard, loss of YAP or TAZ—or overexpression of a dominant-negative version of TEAD—do not typically induce gain of epithelial characteristics in mesenchymal cells (Cordenonsi et al. 2011; Dupont et al. 2011; Hong et al. 2005; Ota and Sasaki 2008). For example, as mentioned above, TAZ knockdown does not affect the mesenchymal differentiation of the metastatic or Snail-expressing breast cancer cell lines (Cordenonsi et al. 2011). The role of TAZ in high-grade glioblastoma is again a different scenario, as this is a mesenchymal type of tumor for which endogenous TAZ is pivotal for the maintenance of mesenchymal and aggressive traits (Bhat et al. 2011). How this conclusion can be generalized to other kind of mesenchymal-like tumors is unknown. Collectively these evidences indicate that YAP and TAZ can induce EMT depending on their expression levels, the experimental conditions and the cellular context.

6.5 A New Perspective on YAP/TAZ Regulation: Role of Mechanical Cues

A hallmark of cancer is the loss of tissue integrity, an event occurring even before a frank neoplasia could be even identified (Huang and Ingber 1999; Husemann et al. 2008; Podsypanina et al. 2008; Rhim et al. 2012). The identification of YAP and TAZ as downstream factors of epithelial plasticity, coupled with their potent pro-tumorigenic and pro-stemness properties, has thus clear implications in cancer research, as it sets the stage to understand molecularly the causal roles of disturbed tissue integrity for malignant transformation or dedifferentiation of tumor cells.

6.5.1 Mechanical Control of the Cancer Cell Phenotype

The above presentation has sidestepped another equally relevant issue in tumorigenesis: what is causing loss of tissue integrity in the first place? There is an increasing appreciation that mechanical inputs from the aberrant tumor microenvironment profoundly influence the tumor cell phenotype, and that may perhaps have an initiating role in tumorigenesis (Butcher et al. 2009; Egeblad et al. 2010; Huang and Ingber 2005; Jaalouk and Lammerding 2009; Provenzano and Keely 2011). Mechanical signals are pervasive elements of tissue development and homeostasis, and cells are normally subjected to different forces including stretching, compression, and pressure. These signals, typically generated by local distortions of tissue architecture, occur at the nanometer level, and thus can target individual cells with exquisite specificity; at the same time, mechanical cues can reverberate at great speed to ­distant cells though a “wave-like” propagation mediated by the semi-flexible and pre-tensed organization of the ECM network (Janmey and Miller 2011).

Mechanical signals become aberrant in cancer: tumor growth is typically accompanied by increased compression forces from the surrounding ECM and tissues, and by increased pressure of interstitial fluids caused by the tumor’s disorganized capillaries. In addition, tumors display profound changes in ECM composition and overall increase in ECM rigidity. In particular, while the normal mammary gland is soft, breast cancers are extremely stiff, due to activation of cancer-associated fibroblasts and extensive deposition of collagen (Butcher et al. 2009). Tumors also display increased expression of lysyl-oxidases (LOX), enzymes that cross-link, and thus stiffen, the collagen fibers, and tumors with the highest expression of LOX are those displaying less differentiation and poorer prognosis (Erler et al. 2006). A causal role between stiffness of the ECM and tumor progression has been recently obtained in animal models, whereby enhancing collagen cross-linking enhanced tumor progression, while targeting LOX—and thus attenuating cross-linking—reduced tumor incidence and delayed progression(Levental et al. 2009).

6.5.2 Form is Function: The Control of Cell Behavior by Cell Shape

The rigidity of the ECM is perceived by cells as an increased resisting force; this force is transmitted through integrins to the cell’s cytoskeleton (Parsons et al. 2010; Vogel and Sheetz 2006). On a stiff ECM the cell increases its inner pulling forces, namely, the tension of the actomyosin cytoskeleton, in order to balance the strong external resisting forces. As such, to an increase in ECM rigidity corresponds an increased intracellular stiffness (Ingber 2006; Mammoto et al. 2009; Parsons et al. 2010; Provenzano and Keely 2011).

Mechanical inputs can be perceived by focal adhesions, or transmitted by cell–cell junctions from neighboring cells, all impacting and depending on the organization of the cytoskeleton (Mammoto et al. 2009; Parsons et al. 2010). Thus, cells are mechanically connected to their surroundings (cells, ECM, and whole tissue) in a manner that is intimately interwoven to the cell’s own shape, and in particular cell polarity and organization of mechanosensitive and mechanoresisting elements within the cytoskeleton and junctional complexes (Butcher et al. 2009; Jaalouk and Lammerding 2009; Provenzano and Keely 2011). A cell embedded within an epithelial sheet is perfectly adapted to sustain the mechanical properties of its tissue; as such, that cell may keep its shape for as long as the mechanical features of its surroundings remain constant. However, when mechanical stresses cannot be sustained by the existing structures, the cell is forced to rewire its mechanical connections and, as such, its form. For example, mammary epithelial cells cultured on a soft ECM (i.e., reconstituted basement membrane) form spheric sheet (“acini”) of growth arrested, polarized, and differentiating cells surrounding a central lumen (Barcellos-Hoff et al. 1989). Progressive stiffening of the ECM leads to disturbed cell polarity, increased cell-ECM adhesion and contractility, and perturbed growth control (Paszek et al. 2005; Weaver et al. 1997). In other words, changes in the mechanical properties of the tumor ECM may initiate a “chain reaction” leading to changes in tissue form and architecture, with ensuing alterations in the sites of cell proliferation, asymmetric vs. symmetric cell division, and differentiation.
Fig. 6.3

Mechanical and architectural cues inform cell decisions. (a) Cell shape can be regulated by controlling the area to which cells can adhere by means of fibronecting microprinting techniques. Cell shape controls YAP and TAZ levels, nuclear vs. cytoplasmic localization and transcriptional activities, as well as a number of biological effects in distinct cellular contexts. YAP and TAZ levels mediate these different behaviors. For example, MSCs cultured in small adhesive areas differentiate as adipocytes whereas they become osteoblasts when cultured in large adhesive areas (McBeath et al. 2004). (b) Cell differentiation of pluripotent cells can be directed by ECM elasticity (see text). (Pa Pascal, is the tensile strength unit of measurement). YAP and TAZ are nuclear and active in spread cells and in cells experiencing a stiff environment (modified from Halder et al. 2012)

6.5.3 Combining Soluble and Unsoluble Cues

The events triggered by increased cellular mechanotransduction present clear analogies with processes typically described in the context of EMT induced by soluble factors, such as TGFβ. This suggests that cells experiencing high mechanical stress, such as tumor cells at the border of a stiff collagen stroma, may represent a sensitized background to the effect of EMT-inducing growth factors. Clearly, TGFβ is a very effective inducer of EMT in cells cultured on plastic, that is, a very stiff substrate (Heldin et al. 2009; Zavadil and Bottinger 2005). Intriguingly, however, when monolayers of cells grown on 2D or 3D molds of defined shapes were challenged with TGFβ, a clear spatial pattern emerged: only cells experiencing high mechanical stresses, that is, those located at edges, sharp curvatures or tips underwent EMT (Gjorevski et al. 2012; Gomez et al. 2010; Lee et al. 2011). Conversely, it is also possible that tumor cells with disturbed apico-basal polarity—by partial EMT or mutation/inactivation of polarity factors, or exposed to high levels of TGFβ—may also display increased sensitivity to mechanical gradients. Perhaps this may explain the exquisite spatial and temporal specificity of EMT induction so far observed in vivo, being transiently induced in one cell, and not in the adjacent cell, for example during wound healing and tumor progression (Gjorevski et al. 2012; Thiery et al. 2009).

6.5.4 YAP and TAZ are Downstream of Mechanical Cues

In light of the above discussion, the discovery that YAP and TAZ are activated not just by loss of polarity, but also by mechanical cues, clearly adds an entirely new dimension to our understanding of normal and pathological tissue biology. YAP and TAZ are indeed regulated by ECM stiffness, cell shape, and cytoskeletal tension (Dupont et al. 2011; Wada et al. 2011). When cells are cultured on stiff ECM, YAP, and TAZ are in the nuclei and induce target gene expression, whereas they are ­inhibited and relocalized in the cytoplasm in cells cultured on a soft ECM (Dupont et al. 2011). Cell shape can be controlled by seeding cells on microprinted fibronectin “islands” of different sizes (Chen et al. 1997) (Fig. 6.3a). YAP and TAZ are well active in cells with spread cell morphology (as cells growing at low density on plastic dishes or seeded on “big” fibronectin islands), but YAP and TAZ are inactivated in small, roundish, unspread cells seeded on small “islands” (Dupont et al. 2011; Wada et al. 2011) (Fig. 6.3a).

Crucially, YAP and TAZ are the key mediators of the biological effects of ECM stiffness and cell shape. For example, endothelial cells die when forced to remain small, while they proliferate when allowed to spread (Chen et al. 1997). The levels of YAP and TAZ dictate these opposite behaviors: if YAP and TAZ are artificially increased in small cells, these start to proliferate; in contrast, attenuation of YAP and TAZ in spread cells causes them to die (Dupont et al. 2011). Contact inhibition of growth in epithelial cells cultured at high cell density is paralleled by YAP and TAZ inhibition. Although traditionally associated to activation of the Hippo cascade by cell–cell contacts, new evidence suggests that contact inhibition may also be envisioned as consequence of reduced cell shape, due to the confinement of cell-ECM adhesion area (reviewed in (Halder et al. 2012)). A similar type of control applies to non-epithelial, fully mesenchymal cells, such as primary human mesenchymal stem cells (MSC). Analogously to the effects of morphogen gradients, these cells differentiate into distinct histotypes depending on the stiffness of the ECM in which they are cultured (Fig. 6.3b): MSCs become osteoblasts at high stiffness, muscle at intermediate stiffness and neurons or adipocytes on soft ECMs (Engler et al. 2006; McBeath et al. 2004). Again, YAP and TAZ take control of this differentiation: when the high YAP/TAZ levels of stiff MSC is lowered by siRNA-mediated knockdown, the stiff MSC behave as if they were on a soft ECM. Conversely, fates typical of elevated mechanical stimuli can be induced in soft MSC by sustaining YAP and TAZ expression (Dupont et al. 2011).

Interestingly, the regulation of YAP and TAZ by mechanical cues differs from their regulation by EMT, polarity, and the Hippo pathway. Indeed, lowering mechanical cues leads to YAP and TAZ cytoplasmic relocalization and degradation in epithelial, mesenchymal, post-EMT cells, as well as in cells depleted of LATS, or expressing LATS insensitive YAP (Dupont et al. 2011). Cell shape and mechanical cues are intimately associated to the regulation of the Rho family of small GTPase, of ROCK and MLCK, and to corresponding changes in the tensile properties and dynamics of the actomyosin cytoskeleton (Mammoto and Ingber 2009; Parsons et al. 2010; Wozniak and Chen 2009). In line with this notion, YAP and TAZ are dependent on this cytoskeletal pathway for their activity (Dupont et al. 2011; Fernandez et al. 2011; Sansores-Garcia et al. 2011; Wada et al. 2011; Zhao et al. 2012). Effective disruption of the F-actin cytoskeleton causes quantitative inactivation of YAP and TAZ in a manner largely independent from LATS(Dupont et al. 2011). This indicates that cytoskeletal inputs are simply overarching signals essential for YAP/TAZ activity, and that, probably, other regulations at the level of the Hippo cascade or other inputs may modify or cooperate with, but not completely overrule the information provided by the mechanical context (Halder et al. 2012).

6.6 Concluding Remarks and Future Perspectives

By reviewing the current status of YAP and TAZ signaling, its biological properties and regulation, we realized how many fundamental questions remain unaddressed. Here we just highlight few of them, hoping to inspire new research avenues.
  1. 1.

    What are the targets of TAZ, and possibly YAP, involved in cancer stem cells self-renewal and tumor initiation? How is YAP, and possibly TAZ, regulating the amplification of normal stem cells and progenitor cells in several normal tissues? Is there a universal “stemness” potential conferred to cells by YAP and TAZ? Answering these questions may entail investigating the connections between YAP and TAZ and regulation of symmetric vs. asymmetric cell division

     
  2. 2.

    How do YAP and TAZ control cell proliferation and survival? These biological traits are at the centerpiece of YAP and TAZ activity in cancer and organ size control. Yet, our knowledge of these processes is still limited

     
  3. 3.

    What part of the genome is controlled by YAP and TAZ? And what “package” of targets can recapitulate YAP and TAZ biological effects? These studies may reveal new avenues to tackle YAP and/or TAZ activity in basic and applied research

     
  4. 4.

    Are YAP and TAZ directly regulated by soluble growth factors? Efforts have been dedicated to study how other signaling pathways—including the TGFβ, BMP, Wnt cascade—are modified by the Hippo pathway and YAP or TAZ (Varelas and Wrana 2011). In contrast, a more direct involvement of YAP/TAZ regulation and activity in other pathways has been largely neglected. YAP and TAZ may serve as hub at the crossroad of multiple pathways

     
  5. 5.

    How are YAP and TAZ regulated by mechanical cues? This represents “the Antartica” of YAP and TAZ biology. Potential mechanisms may include the presence of unknown inhibitors unleashed in soft-cells, or of unknown activators unleashed in stiff cells

     
  6. 6.

    Are there more kinases, other than MST and LATS, that affect YAP and TAZ activity? Evidences in favor of this hypothesis have surfaced in the characterization of MST1/2 knockout livers, where YAP could be still phosphorylated by a non-LATS kinase (Zhou et al. 2009), and from the analyses of phospho-dependent but LATS-independent YAP regulation by α-catenin in keratinocytes (Schlegelmilch et al. 2011)

     
  7. 7.

    If YAP and TAZ are such powerful oncogenes, why aren’t they directly activated by mutations in human cancers? A plausible explanation may be that YAP and TAZ activity may require, in the in vivo microenvironment, the concomitant presence of several activating inputs, each one per se insufficient and not recapitulatable by missense mutations

     
  8. 8.

    Given the dominant regulation of YAP and TAZ by mechanical cues, can YAP and TAZ activity or localization be used to monitor the spatiotemporal distribution of mechanical cues in vivo?

     
  9. 9.

    Research in the Hippo field has concentrated the attention on YAP and TAZ regulation in epithelial cells. However, these factors are essential regulators of cell behaviors in fibroblasts and other non-epithelial cells, including those that infiltrate tumors and contribute to their stromal composition. Clearly, YAP and TAZ regulation in these cellular contexts must be as tight as it is in epithelia, but unlikely connected to the polarity regulatory branch. Are mechanical cues the central regulators of YAP and TAZ in these cell types? Connected to these open issues is the fact that fibroblast stiffening has been implicated in tumor progression(Goetz et al. 2011). To what extent is this dependent on cell-autonomous and non-cell-autonomous regulations of YAP or TAZ?

     
  10. 10.

    Can we identify inhibitors of TAZ activity? These molecules may be ideal candidate as anticancer stem cell therapeutics

     

Box 6.1 Molecular Composition of Junctional and Polarity Complexes

Several regulators of the Hippo pathway, and a number of YAP and TAZ binding proteins are involved in the establishment or maintenance of cell polarity and cell adhesion through adherens junctions and tight junctions, whose key features are described below. Cell-EMC adhesion will be discussed in the context of YAP/TAZ regulation by mechanical cues (see Sect. 4).

Tight junctions. All adhesion sites are composed by a basic module: transmembrane proteins recruit a number of cytoplasmic effectors, in turn linking the whole complex to cytoskeletal proteins. In the case of tight junctions (TJs) the main transmembrane components are claudins, that polymerize with each other and between adjoining cells. Other transmembrane proteins are then incorporated in such claudin mesh, including occudin and Ig-like proteins. The cytoplasmic tail of claudins interacts with ZO-1, ZO-2, ZO-3, and cingulin, that associate with F-actin (Fig. 6.1) (Nelson and Fuchs 2010).

Adherens junctions. These structures play a prominent role in connecting and transmitting forces between neighboring cells. Disruption of AJs causes loosening of cell–cell contact and disorganized tissue structure. AJs are composed of transmembrane cadherins (such as classical cadherins, e.g., E-cadherin and N-cadherin) mediating a homophilic cell–cell association, and of juxtamembrane catenins (β-, p120-, and γ-catenins) and α-catenin (Fig. 6.1). β-catenin mediates the recruitment of α-catenin, that is critical to link AJs to the actin cytoskeleton. Microtubules also associate to AJs through p120 catenin (Harris and Tepass 2010; Nelson and Fuchs 2010). The formation of TJs and of E-cadherin containing AJs is severely disturbed by EMT.

Desmosomes. These intercellular junctions provide a link between the intermediate filaments and the plasma membranes of adjoining cells, and are crucial for epithelial integrity. Intercellular adhesion is mediated by Desmoglins and Desmocollins, whose cytoplasmic domain binds to plakoglobin and plakophilins that are connected to intermediate filaments through desmoplakin. Intermediate filaments provide mechanical stability and confer resistance to mechanical stresses, due to their material properties and wiring with microtubules and microfilaments. During EMT and tumor progression, transformed epithelial cells drastically change their expression pattern of intermediate filaments, facilitating epithelial plasticity, and cell migration (Herrmann et al. 2009; Nelson and Fuchs 2010).

Apico-basal polarity complexes. There are three main cell polarity complexes in epithelial cells (see Fig. 6.2 below): the apical crumbs complex (CRB) contains the transmembrane protein CRB and the associated cytoplasmic proteins PALS1 and PATJ, and regulates the apical positioning of the TJs. The SCRIB complex—including Scribble (Scrib), Lethal Giant Larvae (LGL), and Discs Large (DLG)—organizes the basolateral plasma ­membrane domain, and is closely associated to the formation of AJs. The Par complex (composed of a-PCK, Par6, cdc42, and Par3) controls the activity and location of the CRB and SCRIB complexes promoting the formation of the border between apical and lateral domain. In addition, the Par and SCRIB complexes reciprocally inhibit each other, ­contributing to the robust spatial separation between different apical- and basal-domains (Martin-Belmonte and Perez-Moreno 2011).

Box 6.2 Cell–Cell Adhesion and Cell Polarity Complexes as Regulators of the Hippo Pathway

One of the long-standing issues in the Hippo field relates to the biochemical mechanisms by which this pathway is regulated upstream of the Hippo kinases MST1/2 and LATS1/2. Several findings point to cell–cell adhesion and cell polarity proteins as upstream regulators of YAP and TAZ.

Role of Scribble as adaptor for YAP/TAZ regulation by the Hippo kinases. See main text.

Role of Crumbs/AMOT complex in YAP/TAZ regulation. In addition to the Scribble-Hippo-TAZ connection, there are other mechanisms by which polarity and cell–cell junctions can control TAZ and YAP activity. AMOT (Angiomotin) and ZO2 are tight-junction associated proteins identified by several groups as YAP/TAZ interacting partners (Chan et al. 2011; Oka et al. 2010; Varelas et al. 2010; Wang et al. 2010; Zhao et al. 2011). In addition to AMOT, other apical proteins, including the Crumbs-associated PALS and PATJ, associate to TAZ/YAP (Varelas et al. 2010). These associations are instrumental for attenuation of YAP and TAZ activity, but the underlying mechanism is unclear. One possibility is that these proteins may simply sequester YAP and TAZ on the plasma membrane. Another possibility is that, similarly to Scribble, these proteins may serve as a supramolecular scaffold for YAP and TAZ phosphorylation. Besides binding YAP or TAZ, PALS, PATJ, and AMOT play relevant functions for junctional integrity, cell polarization, and cytoskeletal organization. Thus, a third possibility is that the YAP and TAZ regulation by these apical proteins may be secondary to loss-of-polarity and disturbed Scribble localization, or secondary to aberrant cytoskeletal organization. More studies are required to discriminate between these possibilities.

Role of E-cadherin and α-catenin as YAP regulators. The AJ components E-cadherin and α-catenin have been also implicated in YAP regulation. Expression of E-cadherin restores the density-dependent nuclear exclusion of YAP in mesenchymal cells; conversely, disturbing the E-cadherin/α-catenin complex in epithelial cells decreases phosphorylation of YAP and promotes YAP nuclear accumulation (Kim et al. 2011).

In a different study, epidermal-specific genetic depletion of α-catenin also leads to YAP dephosphorylation and nuclear accumulation (Schlegelmilch et al. 2011). The phenotype of these α-catenin mutants recapitulates the effect of YAP overexpression in transgenic mice. Interestingly, however, neither MST1/2 nor LATS1/2 are implicated in the regulation of YAP by α-catenin in keratinocytes, suggesting that other kinases may act redundantly with the canonical Hippo kinases in this and perhaps other cellular contexts.

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© Springer Science+Business Media New York 2013

Authors and Affiliations

  1. 1.Department of Biomedical SciencesUniversity of Padua School of MedicinePaduaItaly

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