Molecular Pathways: YAP and TAZ Take Center Stage in Organ Growth and Tumorigenesis

The Hippo pathway and the control of organ size.

The mammalian Hippo cascade is composed of a highly conserved kinase cassette regulating YAP and TAZ, two closely related transcriptional regulators. In its most basic formulation, the pathway is activated when the kinases MST1 and MST2, related to the Drosophila Hippo kinase, phosphorylate and activate the LATS1/2 kinases, which in turn phosphorylate and inhibit YAP and TAZ (refs. 20, 21; Fig. 1). Two cofactors, Salvador (SAV or WW45) and MOB1, are also involved as MST and/or LATS partners. NF2, encoded by the type II neurofibromatosis gene, has been implied as the first upstream regulator of hippo kinases (20, 22). The mechanism of YAP/TAZ inhibition by phosphorylation is dual: degradation by the proteasome and/or sequestration in the cytoplasm through anchoring proteins. In the nucleus, YAP and TAZ do not bind DNA directly but are coactivators that regulate gene expression by associating with specific transcription factors and in particular with TEAD factors (ref. 20; Fig. 1).

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Figure 1.

YAP and TAZ transcriptional coactivators lie at the core of a complex regulatory network that offers multiple potential therapeutic targets (exemplified by red crosses). YAP/TAZ cooperate in the nucleus with TEAD transcription factors to regulate gene transcription and promote malignant cell behaviors. Small molecules precluding TEAD interaction dampen YAP/TAZ activities (1). F-actin and small RHO GTPases, in response to cell–substrate adhesion (green dots) and to mechanical cues imposed by the ECM, are key inputs to sustain YAP/TAZ activity. Inhibitors of RHO and of the machinery that maintains tension of the F-actin cytoskeleton (such as ROCK or MLCK, not shown here) may serve to inactivate YAP/TAZ (2). WNT signaling inactivates the destruction complex containing the APC (adenomatous polyposis coli) and Axin proteins, thereby rescuing TAZ from degradation. Tankyrase inhibitors may reactivate Axin function and inhibit YAP/TAZ activity in cancers with Wnt activation or APC mutation (3). Apico-basal cell polarity (through Scribble) and NF2/Merlin regulate the activity of the LATS1/2 kinases, which phosphorylate and inhibit YAP/TAZ. GPCRs, by regulating LATS1/2 kinases and/or RHO GTPases, can either activate or repress YAP/TAZ activity. GPCR regulators might be used to dampen YAP/TAZ function (4).

The Hippo cascade constitutes an intrinsic regulator of organ size essential to stop cell growth when the organ reaches its final size; inactivation of this cascade causes excess of YAP/TAZ nuclear accumulation leading to organ overgrowth. The first indications in this direction involved conditional overexpression of YAP in the mouse liver, which caused an astonishing 4-fold increase in liver size (23). Notably, this increase was reversible, as if the organ “perceived” its size as abnormal and returned to a normal size after turning off YAP expression. After this landmark study, MST1/2- and SAV-knockout mice provided crucial genetic evidence that the Hippo cascade is required to limit tissue overgrowth. Liver-specific ablation of MST1/2 or SAV phenocopied the giant liver phenotype observed in the YAP transgenics (24, 25), and this phenotype was rescued by concomitant removal of one YAP allele. Also in the small intestine and to some extent in the skin, MST1/2 and SAV knockout, or YAP activation, all expand the progenitor cell population (26–28). The role of Hippo signaling extends to nonepithelial tissues; for example, depletion of MST1/2 or SAV in the heart causes massive cardiomegaly, due to increase in the number of cardiomyocyte (29), whereas MST1/2 inactivation in the central nervous system (CNS) promotes expansion of neural progenitors (30).

In recent years, several variations on the basic Hippo cascade have been reported, including LATS-independent phosphorylation of YAP/TAZ, MST-independent activation of LATS, and phosphorylation-independent modalities of YAP/TAZ control (25, 31–33). Thus, the reader should be aware of a potential semantic problem, as the definition of what “is” the Hippo pathway has progressively blurred to include clear non-Hippo regulations feeding on YAP/TAZ activity. For example, skin-specific depletion of MST1/2 is surprisingly inconsequential, yet YAP overexpression does cause hyperplasia and skin thickening due to amplification of undifferentiated progenitors (33). Regardless of the exact mechanism by which YAP is regulated in each tissue, results from genetic studies on YAP strongly support the remarkable conclusion that active YAP induces proliferation and self-renewal of progenitor cells (33, 34), whereas YAP inactivation leads to cell death and differentiation in multiple tissues.

How nuclear YAP/TAZ activity controls cell and organ growth remains one of the key unanswered questions. YAP and TAZ may induce, but only in specific cell types, the transcription of genes involved in cell proliferation and survival, such as Birc5, c-Myc, or cyclin D1. However, none of these growth regulators have been shown to serve as downstream mediator of YAP/TAZ effects, and, despite few exceptions (35), neither overexpression nor genetic ablation of a number of cell-cycle regulators or apoptosis mediators cause overt changes in organ size. The jury is thus still out on whether a universally valid YAP/TAZ-dependent gene expression program directing organ growth does truly exist. In another scenario, YAP/TAZ may not give precise instructions: YAP/TAZ could indeed reprogram the cellular genome to escape terminal differentiation, and in favor of an increased, or prolonged, proliferative potential. These traits could be exploited only in presence of other intrinsic (i.e., activation of proto-oncogenes) or extrinsic growth regulators, such as nutrient availability, metabolites, insulin-like growth hormones (36), or other tissue-specific growth factors (37). This remains a tempting, untested hypothesis, but perhaps consistent with the observation that, in vivo, nuclear YAP/TAZ are frequently localized in stem and immature progenitor cells (ref. 38; and M. Cordenonsi, unpublished results).

YAP/TAZ regulation by cell shape and mechanical cues.

Organ size control must involve control layers operating on a whole tissue and organ scale (39). Tissue regeneration occurring after experimental ablations or during normal homeostasis implies that cells are constantly informed on the size of the whole organ and that they are able to perceive what happens hundreds of cell diameters away. At the same time, organs are built and constantly regenerated with exquisite spatial accuracy, for example, by establishing sharp growth differentials over few cell diameters (i.e., activating one cell, but not its equally potent neighbors). It is hard to conceive these multiscale regulations as being mediated only by gradients of soluble factors or morphogens (40). An intriguing hypothesis involves regulation by mechanical signals, such as stretching, compression, and compliance of the ECM that are known to profoundly influence cell behavior (4–7). Indeed, mechanical cues can easily reverberate to distant cells, through a “wave-like” propagation mediated by the semiflexible and pretensed organization of the cell cytoskeleton and ECM network (41). Stem cells, in particular, are lodged in specific physical niches within tissues, making them primary targets of the mechanical inputs intrinsic to a given tissue architecture.

In light of this background, the discovery that YAP and TAZ are activated by mechanical cues and cytoskeletal tension added an entirely new dimension to our understanding of tissue biology (6, 31). A rigid ECM, a spread cell shape, shear stress, and a tense actin cytoskeleton are all potent inducers of YAP/TAZ nuclear localization and activity (31, 42–44). Crucially, YAP and TAZ are emerging not just as mechanotransducers but also as key functional mediators of the biologic effects of ECM stiffness and cell shape (31, 45). For example, endothelial cells die when forced to remain small, whereas they proliferate when allowed to spread (46). The levels of YAP and TAZ dictate these opposite behaviors: when YAP or TAZ are overexpressed in small cells, these start to proliferate, whereas attenuation of YAP and TAZ in spread cells causes them to die (31). Unveiling how cell mechanics and tension controls YAP/TAZ activity is a key challenge for the field, as the regulation of YAP and TAZ by mechanical cues appears independent from, and dominant over, the Hippo cascade (31). This also raises the exciting possibility, yet to be tested, that information arriving to YAP/TAZ from tissue structure and architecture through the cytoskeleton may be permissive for regulations from other cascades.

Direct targeting of YAP and TAZ.

The tumorigenic potential of YAP, and likely that of TAZ, requires association to TEAD (60). Liu-Chittenden and colleagues recently provided the proof-of-principle that disruption of the YAP/TEAD complex is indeed a valid therapeutic option. By screening a small-molecule library, they identified members of the porphyrin family as inhibitors of YAP/TEAD-dependent transcription. Intriguingly, one of these molecules, verteporfin (VP), is already in clinical use for the photodynamic therapy of macular degeneration. In animal models, administration of VP blunted liver overgrowth caused by YAP overexpression or by NF2 knockout, without overt adverse effects in other organs (60).

Rho and Rock inhibitors.

Rho and the F-actin cytoskeleton are fundamental to sustain YAP/TAZ function. For example, inhibition of Rho, or even a mild disruption of the actin cytoskeleton, leads to dramatic downregulation of YAP/TAZ transcriptional effects and inhibition of nuclear localization (31, 42, 43). Considering the fact that this occurs in a largely Hippo-independent manner (6), these discoveries opened new and exciting therapeutic scenarios, because they at least suggest that already existing Rho-inhibiting drugs may be exploited as anti-YAP/TAZ therapeutics. For example, bisphosphonates inhibit prenylation, a lipid modification required for membrane attachment of Rho and other small G-proteins. Bisphosphonates are already in use for palliative care of bone metastases. However, when used in the adjuvant setting, some compounds have provided provocative results preventing metastasis, with improved survival (61). Are these beneficial effects associated with attenuation of YAP and TAZ? While this question remains pending, another possibility to blunt YAP/TAZ function is represented by another set of enzymes regulating actin contractility, such as ROCK, MLCK, and non-muscle myosin itself. In particular, the ROCK inhibitor Y27632 and the myosin inhibitor blebbistatin have been shown to inhibit YAP/TAZ activity (31, 42). Although applications of Y27632 have been discontinued, other ROCK inhibitors are in clinical use for cardiovascular diseases (fasudil), with more potent inhibitors (AR-12286) being tested in phase III trials for glaucoma (62). Moreover, the possible use of fasudil to attenuate tissue fibrosis might be useful to interrupt the feed-forward effects between the tumor and the progressive stiffening of the stroma.

GPCR agonist and antagonists.

In keeping with the pro-YAP/TAZ role of Rho (31), recent results indicate that agonists of some G-protein–coupled receptors (GPCR), such as the bioactive lipids lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P), promote YAP/TAZ nuclear localization by sustaining Rho signaling (63). This suggests that antibodies or small-molecule agonists mimicking LPA or S1P stimulation, or activators of the enzymes that build them, may represent a therapeutic option for subsets of tumors addicted to GPCR activation.

WNT inhibitors.

In tumors characterized by mutations that inactivate APC, such as colorectal tumors, TAZ levels and activity are elevated and contribute to the malignant phenotype. It follows that, as recently shown, another route for attenuating TAZ, and possibly YAP, is the use of tankyrase inhibitors (54). These drugs restore the activity of Axin, the limiting factor of the β-catenin–and TAZ–degradation complex (64).

Finally, we speculate that the YAP/TAZ and the Hippo pathway may indeed represent a valuable target for cancer therapy and that more opportunities will emerge as we learn more on the role of YAP/TAZ in organ growth and tumorigenesis. That said, cancer selectivity and toxicity might turn into a phenomenal challenge for potent inhibitors. It is thus possible that milder yet better-tolerated inhibitors may hit an important therapeutic window: cutting “the edge” of YAP/TAZ activity required for tumor and metastasis growth while sparing normal tissues. And some of these factors may already be on our shelves.