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The Hippo signaling pathway is an emerging growth control and tumor suppressor pathway that regulates cell proliferation and stem cell functions. Defects in Hippo signaling and hyperactivation of its downstream effectors YAP and TAZ contribute to the development of cancer, suggesting that pharmacological inhibition of YAP and TAZ activity may be an effective anticancer strategy. Conversely, YAP and TAZ can also play beneficial roles in stimulating tissue repair and regeneration following injury, therefore activation of YAP and TAZ may be useful in these contexts. Recently, a complex network of intracellular and extracellular signaling pathways that modulate YAP and TAZ activities have been identified. Here we review the regulation of the Hippo signaling pathway, its functions in normal homeostasis and disease, and recent progress in the identification of small molecule pathway modulators.
The Hippo signaling pathway is an emerging growth control pathway that is conserved throughout the animal kingdom. Growing interest in the Hippo pathway is fueled by studies that demonstrate its fundamental role in organ growth control, stem cell function, regeneration, and tumor suppression 1,2,3,4,5,6. Indeed, the Hippo pathway is deregulated with high frequency in many diverse cancers suggesting that altered Hippo signaling is tightly linked to tumor initiation and/or progression 7. Hence, there is much excitement and speculation about targeting the Hippo pathway to treat a wide variety of human malignancies 8, 9.
The Hippo pathway’s main function is to negatively regulate the activity of YAP and TAZ, two homologous transcriptional co-activators that are the main downstream mediators of the Hippo pathway 10. When activated, YAP and TAZ promote cell proliferation, inhibit cell death, and are hyperactivated in many human malignancies 11,12,13, 14,7,15,16–20,21. Therapeutic intervention for cancer would thus aim at reducing or inhibiting the oncogenic function of YAP and/or TAZ. However, to date few small-molecule inhibitors have been discovered that target the Hippo pathway and a prevalent view is that most Hippo pathway signaling components are not conventional drug targets. Indeed, YAP and TAZ are transcriptional co-activators with no known catalytic activity. Moreover, no known upstream regulators that specifically promote YAP and TAZ activity have enzymatic activity 7,6. Thus, inhibiting the function of YAP and TAZ may require targeting protein-protein interactions. A further complication is that YAP and TAZ are required for tissue repair and regeneration in some contexts 22–27,28,29, raising questions as to whether systemic and chronic manipulation of Hippo signaling might have potential deleterious side effects on normal tissue function and homeostasis. However, transient activation of YAP and TAZ may help to promote tissue repair and regeneration in the context of injury 28,29. These two faces of the Hippo pathway suggest that identification and proper application of small molecular modulators of Hippo signaling may provide exciting new approaches for cancer therapy and in regenerative medicine.
The Hippo pathway relays signals from the plasma membrane into the nucleus where it regulates the expression of a battery of target genes controlling diverse cellular processes such as proliferation, survival, and differentiation 1,2,3,4,5,6. In this regard, Hippo signaling is similar to other well-known signal transduction pathways such as the EGF, TGFβ or WNT pathways. However, in contrast to these other pathways, the Hippo pathway does not appear to have dedicated extracellular signaling peptides and receptors, but rather is regulated by a network of upstream components and mechanisms, many of which are also involved in regulating cell adhesion and cell polarity 3, 6, 30. Nevertheless, the Hippo pathway bears considerable resemblance to other canonical signal transduction pathways in that many upstream regulators feed into the core of the pathway that is comprised of two serine/threonine kinases, known as the Hippo and Warts kinases in Drosophila 31–37 and the MST1/2 (Mammalian Sterile 20-like 1 and 2) and LATS1/2 (Large tumor suppressor 1/2) kinases in humans 37–40. These kinases and their essential roles in growth control were first discovered in Drosophila and function together in a novel signaling pathway, termed “the Hippo pathway” after one of its founding members 31–35. Since the initial discovery of Hippo and Warts, many additional components of the Hippo pathway have been identified and a complex signaling network that integrates multiple upstream inputs from the plasma membrane into the nucleus has emerged (Figures 1 and and2).2). In this review we will largely refer to Hippo signaling components using the mammalian nomenclature and the Drosophila components are listed in Table 1.
The core of the Hippo pathway comprises a highly conserved signaling module that functions similarly in mammals and in Drosophila and contains the MST1/2 and LATS1/2 serine/threonine kinases 31–37 and their respective scaffolding proteins SAV1 (Salvador) 41–43 and MOB1A/B 41–43, the transcriptional co-activators YAP and TAZ 44, and the TEA-domain containing sequence specific transcription factors TEAD1-4 (Figure 1) 45–49. YAP and TAZ are transcriptional co-activators that are not able to bind to DNA themselves, but they form complexes with TEADs 45–49 and other transcription factors such as SMADs 50–52, TBX5 53, 54, RUNX1/2 55 and p73 56 to regulate gene expression. The Hippo pathway is considered to be in the active state when the MST and LATS kinases are active (Figure 1A): The MST kinases in complex with SAV1 phosphorylate and activate the LATS kinases and MOB1 co-factors 34, 57–59,60, which in turn phosphorylate their downstream targets YAP and TAZ 11, 21, 44, 61–64. Phosphorylation of YAP and TAZ results in their nuclear export, cytoplasmic retention, and βTRCP dependent degradation by the proteasome 11, 21, 61, 62, 65–68. Instead of binding YAP and TAZ, the TEAD factors then form complexes with VGL4, which repress target gene expression 69, 70. Thus, when the Hippo pathway is ON, YAP and TAZ activity is inhibited and YAP/TAZ driven gene expression is suppressed. Conversely, when the Hippo pathway is OFF, YAP and TAZ accumulate in the nucleus where they drive gene expression in complex with TEAD and other transcription factors (Figure 1B) 45–49, 71–75. Thus, the Hippo pathway acts primarily by inhibiting the nuclear functions of YAP and TAZ.
A key question concerning the Hippo pathway relates to the signals and mechanisms that regulate its activity and ultimately that of YAP and TAZ. To date over 20 regulators have been identified that intersect with the core of the Hippo pathway at different levels (Figure 2) 1,2,3,4,5,6, 30. In mammals, at least four interconnected upstream branches regulate the Hippo pathway. These inputs are (1) the Crumbs complex, (2) regulators that act upstream of the MST kinases, (3) the Actin cytoskeleton, and (4) the adherens junction. Each of these inputs is described below.
The Crumbs (Crb) complex contains CRB proteins that are transmembrane proteins localizing to apical junctions which are required to specify the apical plasma membrane domain 76. CRB proteins have short intracellular domains with protein docking sites that assemble multi-protein complexes that function in cell polarity and also regulate the Hippo pathway 77–81. Most prominently for its regulation of the Hippo pathway in mammals, the CRB complex recruits members of the Angiomotin (AMOT) family of adaptor proteins that directly bind Hippo pathway components. Initial reports showed that AMOT inhibits YAP nuclear localization by directly binding to YAP and by activating LATS kinases to promote YAP phosphorylation and nuclear exclusion 77,82–87. In contrast to these studies, however, a recent report showed that AMOT is required for YAP dependent overgrowth of Nf2 mutant mouse livers, that AMOT promotes nuclear localization of YAP and that it forms a functional complex with YAP and TEADs on target gene DNA 88. AMOT may thus have growth promoting and growth-suppressing functions depending on cellular and molecular context, however, reasons for these seemingly paradoxical results is not known and requires further understanding of the function of AMOT.
A second branch of the Hippo pathway consists of kinases and other regulators that modulate the activity of the MST kinases. These include the TAO kinases and the cell polarity kinase PAR-1 that directly phosphorylate MST kinases and regulate their activity 89, 90,91. In addition, MSTs are regulated by the adaptor protein KIBRA, and in Drosophila also by the adaptor Expanded (Ex) 92–95.
A third branch of the Hippo pathway is defined by an as yet poorly understood signaling mechanism that is mediated by the actin cytoskeleton. Here, the mechanical properties of the extracellular matrix and cell-matrix attachment regulate YAP/TAZ localization and activity, in a process that requires F-actin and that is also present in Drosophila 96–100. In addition, G-protein coupled receptors (GPCRs) that relay signals from soluble extracellular cues such as lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) regulate YAP and TAZ activity through Rho GTPases that likely affect YAP/TAZ via modulating the actin cytoskeleton 101,102,103. Thus, pathways that regulate the structure of the actin cytoskeleton, for example by activating Rho signaling, affect the Hippo pathway. However, while it is generally appreciated that F-actin intersects the pathway downstream of the MST kinases, the exact mechanism is not known and may involve LATS kinase dependent and independent regulation of YAP and TAZ 96–100,104.
A fourth branch of Hippo pathway inputs emanates from the adherens junction. Engagement of E-Cadherin (E-Cad) at adherens junctions suppresses YAP nuclear localization and activity through regulating MST activity 105. Additionally, the E-cadherin associated protein α-catenin regulates YAP directly by sequestering YAP/14-3-3 complexes in the cytoplasm 106, 107. Furthermore, the junction associated Ajuba protein family members directly inhibit LATS kinase activity 108. In Drosophila, the related Zyxin protein also regulates Warts levels 109. Whether these adherens junction associated regulators of the Hippo pathway act independently of each other or whether they function coordinately to regulate Hippo signaling is not yet known. In addition, crosstalk between the different regulatory branches most likely exists. For example, CRB and adherens junctions regulate the structure of the actin cytoskeleton, and polarity proteins and junction proteins regulate each other.
In addition to these four major branches of upstream regulators of Hippo signaling, there are several other proteins that modulate the activity of the pathway, most of which have conserved functions in Drosophila (Figure 2). These include proteins that directly interact with or affect YAP and TAZ (WBP2 110,111,112, MASK1/2 113,114, ZO1/2 115, 116, HIPK2 117,118, 14-3-3, PTPN14 119–123, CK1 and βTRCP 67, 68) or that interact with the upstream kinase complexes (RASSF 124–126, PP2A 127, Salt-inducible kinases (SIKs) 128, Scribble (SCRIB), and the Scribble associated proteins Discs large (Dlg) and Lethal giant larvae (Lgl) (so far shown in Drosophila 80, 129–133)). Scrib, Dlg and Lgl form a module that regulates cell polarity and establishes the basolateral domain, indicating that the Hippo pathway is independently modulated by several junctional complexes. The Drosophila Hippo pathway is also regulated by a signaling axis from the atypical cadherin Fat that regulates the levels and activity of Warts 2,3, 30. However, whether Fat homologs are involved in vertebrate Hippo pathway regulation is not clear 134.
While the net effect of deregulated YAP/TAZ activity in many tissues is similar, the control of YAP and TAZ activity appears to be under different regulatory mechanisms in different tissues. For example, while the MST kinases are essential to inhibit YAP and TAZ in the liver 135–138, intestine 139, pancreas 140, 141, and heart 142, they appear to be dispensable in the skin 106. Presumably, in the skin, other negative regulatory mechanisms predominate, and sequestration via α-catenin and AMOT have been suggested to circumvent the need for phosphorylation by LATS kinases 106, 107. Further evidence that there is cell-type specificity in Hippo pathway regulation is the observations that the MST kinases are largely dispensable in mouse embryo fibroblasts (MEFs) 135,137, while the LATS kinases are essential to inhibit YAP and TAZ activities 143. Tissue specific requirements for upstream components is also observed in Drosophila, where Fat signaling is dominant in imaginal discs but has no significant effect in the ovarian follicle cell epithelium 144,145, 146,147,148,149,150. Likewise, Mer has minor effects in imaginal discs but is a major regulator of the Hippo pathway in follicle cells 151,145,152,153,144,154,155.
The dramatic overgrowth phenotypes caused by loss of Hippo pathway activity in Drosophila first led to the idea that the pathway may be important in the control of organ growth and act as a tumor suppressor in vertebrates. Indeed, many studies have now shown that loss of Hippo signaling or hyperactivation of YAP and TAZ promote growth and cell pluripotency depending on tissue context. Thus, loss of pathway activity in mouse models causes overgrowth of various organs such as the liver and heart and can lead to the development of cancer in the liver, skin, and intestine. Below, we highlight the phenotypes associated with loss of pathway activity in genetically engineered animal models, discuss cellular events that are controlled by YAP/TAZ, and summarize the evidence for hyperactive YAP and TAZ in human cancer.
A predominant function of the Hippo pathway is in regulating progenitor cell proliferation and organ size. In Drosophila, loss of function of the Hpo or Wts kinases, or overexpression of Yorkie (Yki), the Drosophila homolog of YAP and TAZ during development, result in severely overgrown imaginal discs leading to dramatic overgrowth of the corresponding adult structures 31–37, 44. These overgrowths arise because mutant cells have two defects. First, they have defects in regulating cell proliferation; they proliferate faster than wild-type cells and continue to proliferate when wild-type cells stop proliferating after tissues have reached their proper size. Second, mutant cells have defects in regulating apoptosis; they are resistant to apoptotic signals that are employed to eliminate extra cells. The combination of these defects then results in production of excess cells that cannot be eliminated, leading to increased organ size. Similarly in mice, YAP overexpression or loss of MST or LATS kinase activities increases liver and heart size by increasing cell number 11, 135–138, 142, 156, 157. However, the relationship between Hippo signaling and organ size is not absolute. In some tissues such as the skin and intestine, overexpression or activation of YAP causes an enlargement of the stem cell compartment in part due to a block in differentiation, but does not lead to an overall increase in organ size. 12, 106, 158. This may be due to the cellular composition of these tissues in that they continuously undergo a stem cell driven renewal program. Thus, the general conclusion from genetic studies of the Hippo pathway in mice and flies is that YAP and Yki drive cell proliferation and tissue growth. However, they may not drive growth in every tissue and they also have functions apart from regulating tissue growth, although non-growth related functions appear to be minor and related to cellular differentiation 1, 159, 160.
Several direct downstream target genes of the Hippo pathway have been identified and include genes such as cyclins, growth factors, and inhibitors of apoptosis that are involved in cell proliferation, cell survival and stem cell functions among others. In Drosophila, a combination of ChIPseq and RNAseq for Yki and the TEAD protein Scalloped (Sd) showed that Yki/Sd regulate a majority of genes expressed in imaginal discs, making them more akin to general transcriptional activators 161. At present, however, how YAP/TAZ and Yki drive cell proliferation and tissue growth is incompletely understood and likely involves the regulation of many direct target genes, some of which affect growth and survival directly while others affect these processes indirectly by global regulation of metabolism and other cellular processes 21, 161, 162,163.
The dramatic effects of YAP overexpression and hyperactivation on organ size and progenitor cell pools demonstrate a potent growth promoting activity. Indeed, there is considerable evidence that abnormal Hippo signaling is associated with a wide variety of human cancers 7. Elevated levels and nuclear localization of YAP and in some cases TAZ has been reported in a majority of solid cancers, suggesting widespread deregulation of Hippo signaling in human neoplasia 7,11,12, 15,16–20,21. Examples of abnormally elevated YAP levels and nuclear localization in human cancers include cancers in the liver, lung, breast, skin, colon and ovary 7,11,12, 15,16–20,21. In the liver, skin and colon, mouse models showed that attenuation of Hippo signaling or overexpression of YAP is sufficient to promote tumor formation 11, 12, 15, 106, 135–138. The exact mechanisms involved in transformation of normal cells to malignant tumor cells by deregulated YAP and TAZ are not known, but likely involve enhanced cell proliferation and survival coupled with acquisition of additional cancer cell phenotypes such as cancer stem cell character, epithelial to mesenchymal transition (EMT), drug resistance, and inhibition of senescence as these are all activities promoted by YAP/TAZ that are abnormally regulated in tumor cells.
The association of Hippo signaling with stem cell properties has recently been extended to cancer stem cells. In breast cancer, TAZ has been shown to be a potent stimulator of cancer stem cells in vitro 132. Overexpression of TAZ increases the ability of MFC10A cells to form mammospheres, an indication that TAZ enhances the proportion of MCF10A cells that have stem cell properties. Conversely, knockdown of endogenous TAZ inhibits mammosphere formation. TAZ is overexpressed in approximately 85% of high-grade human breast cancer with evidence for gene amplification in 15–20% of these cases 132. Taken together, these results suggest that activation of TAZ is a major event associated with breast cancer initiation and/or progression. In contrast, YAP does not appear to be effective in promoting cancer stem cell properties in the report of Cordenonsi et al., however, other studies support a role for YAP in promoting cancer-like properties in non-transformed mammary epithelial cells 15. Given that YAP and TAZ promote pluripotency in other stem cell contexts it is likely that their hyperactivation also contributes to cancer stem cell expansion, survival and self-renewal in other malignancies.
In addition to their self-renewal capacity, cancer stem cells are associated with epithelial-mesenchymal transition (EMT) a feature commonly observed in high-grade tumors and metastasis. Overexpression of YAP and/or TAZ can lead to acquisition of a mesenchymal phenotype in mammary epithelial cells suggesting Hippo signaling may play important roles in suppression of EMT 15, 61, 132. Indeed, loss of cell polarity determinants such as SCRIB leads to reduced Hippo pathway activity, activation of YAP/TAZ, EMT and cancer stem cell expansion 132. Thus, YAP/TAZ activation by loss of cell polarity may engage a positive feed-forward loop in which YAP/TAZ promotes EMT and loss of cell polarity, which then further activates YAP and TAZ. Importantly, hyperactivation of TAZ has been associated with EMT and high-grade tumors in human breast cancer and in glioma 132, 164.
Cancer stem cells are commonly resistant to chemotherapeutic agents and several recent studies indicate that hyperactivation of YAP and/or TAZ contributes to this resistance 15, 165–167. A feature of Hippo pathway inactivation and YAP/TAZ hyperactivation is increased cell survival mediated by suppression of apoptosis 11, 15, 41, 42. Several mechanisms contribute to this suppression, including transcriptional upregulation of pro-survival factors such Bcl2 family members 168. Additionally, it has been reported that the YAP/TAZ targets CTGF and Cyr61 inhibit apoptosis in liver cells 169, 170 and are responsible for taxol resistance in breast cancer cells 167. Other mechanisms likely contribute to YAP/TAZ-mediated drug resistance, including their ability to promote EMT and stem cell properties171, both of which are associated with increased resistance to chemotherapy in a variety of tumors and cell lines172.
In summary, deregulated YAP and TAZ activity promotes multiple cancer cell phenotypes beyond simply driving cell proliferation, suggesting that targeting YAP and TAZ may inhibit growth of cancer cells at many levels.
YAP/TAZ protein levels and nuclear localization are elevated in many human cancers and YAP/TAZ drive acquisition of several important cancer cell phenotypes. However, the mechanism of Hippo pathway de-regulation and YAP/TAZ activation in human cancers is not well understood. This knowledge gap is important because development of Hippo pathway based therapies would benefit from insight into molecular mechanisms that are altered and responsible for elevated YAP/TAZ activity in cancer. Few germline or somatic mutations in Hippo pathway components have been discovered in targeted and whole-genome sequencing efforts to date suggesting that these may be generally uncommon events 7. One exception to this observation is the Neurofibromatosis type 2 (NF2) locus. NF2 encodes the MERLIN tumor suppressor protein that regulates the Hippo pathway by modulating the activity of the LATS kinases by promoting their localization to the plasma membrane 92, 173,174. NF2 is mutated with high frequency in neurofibromatosis, a condition that is characterized by malignant peripheral nerve sheath tumors, and it is thus a bona fide tumor suppressor gene 175, 176. Loss of MERLIN as well as the Hippo pathway components LATS2 or SAV1 is also frequently seen in malignant mesothelioma 177.
Why NF2 and LATS2 mutations are common in neurofibromatosis and mesothelioma but uncommon in other human cancers, despite widespread deregulation of YAP/TAZ is not understood. Additionally, activating mutations in YAP or TAZ have not been reported in human cancers 7, 178, despite the ability of such mutations to drive tumor formation in mouse models and to confer tumor properties onto non-tumorigenic human cell lines 11,12, 15. One possibility is that in human cancers, mutant YAP/TAZ may not confer a growth advantage because mutant YAP/TAZ may still be kept in check by mechanisms that act independently of LATS, for example by actin mediated regulation of YAP and TAZ 104, or because they also initiate as yet unknown growth inhibitory mechanisms. In support of the latter hypothesis, YAP and TAZ are found to be genomically amplified in several human cancers including oral cancers, intracranial ependymomas, hepatocellular carcinomas, gliomas and mammary tumors 179,180,181,19,16,15. The frequency of amplification is variable, with high occurrence in oral cancers and ependymomas and relatively low frequencies observed in hepatocellular carcinomas and in breast cancer. In contrast, approximately 50% of human liver cancers display elevated YAP levels 20, and TAZ overexpression is observed in over 80% of breast cancers 18,132. These findings imply that amplification is a common mechanism in some human cancers, but other mechanisms predominate in other cancers. The exact nature of these other mechanisms is currently not known, but may include promoter methylation and epigenetic silencing of the MST and LATS kinases 182–184,185 or expression and/or upregulation of proteins that control Yap transcription 186, 187,188, 189 or stability 188.
Hence, current evidence suggests that multiple mechanisms contribute to the deregulation of YAP and TAZ in human cancers, including promoter hypermethylation, mutation, and amplification. Importantly, while specific defects that deregulate the Hippo pathway in many cancers are not known, the core components are largely unaffected by irreversible mutations or genetic aberrations 7. Hence, at least in principle, these observations provide the opportunity for pharmaceutical interventions to reactivate or restore Hippo pathway function, thereby inhibiting oncogenic YAP and TAZ activities.
Given the association of elevated and hyperactive YAP and TAZ with many cancers, direct or indirect attenuation of YAP and/or TAZ represents a rational and novel targeted approach for treatment and prevention of human malignancies. In this section, we discuss recent preclinical findings that support efforts directed towards the development of new Hippo pathway-based targeted therapies to prevent and treat malignancies.
Several lines of evidence from mouse and in vitro models show that reducing YAP activity is effective in limiting growth of tumor cells and preventing tumor formation. First, liver-specific deletion of Yap in the context of simultaneous NF2 deletion results in complete suppression of liver tumor formation induced by NF2 loss 173. Importantly, reducing, but not eliminating Yap gene dosage to 50% results in a similar suppression, indicating that partial inhibition of YAP is effective in preventing tumor formation in this model 173. In addition, expression of a dominant negative version of TEAD2 that lacks the DNA binding domain but that can still bind to YAP abolished hepatomegaly and the development of liver cancer in a NF2 mutant mouse model 190. A dosage effect for YAP was also seen in a mouse model of colon cancer where the Mst1 and Mst2 kinases were conditionally deleted in the intestinal epithelium with villin-cre 139. Villin-cre;Mst1/2 mice develop colonic adenomas by three months of age, associated with disruption of the normal colonic tissue architecture due to hyperactivation of YAP. Both of these phenotypes were effectively suppressed by removal of a single Yap allele 139. Hence these two studies show that reducing YAP dosage is effective in preventing tumor formation in situations predisposed to activation of YAP. Thus, therapeutics that target YAP may not need to fully block YAP activity for efficacy, thereby reducing the potential for negative side effects.
While these results are encouraging, whether reducing YAP dosage after tumor formation will cause a reduction in tumor burden has not yet been established. In addition, these studies activate YAP by mechanisms that are not documented in the corresponding human cancers. Hence, it will be important to test whether reducing YAP or TAZ activities in relevant genetically engineered mouse models that more closely recapitulate human cancer is effective. Also of concern is that a complete understanding of the long-term consequences of YAP and/or TAZ inhibition on normal and cancerous tissues is lacking. Potential for unexpected consequences of YAP inhibition is suggested by the finding that deletion of YAP in the mouse intestine can lead to WNT hypersensitivity with subsequent enhanced injury-induced stem cell expansion and hyperplasia 191. This effect was attributed to YAP-dependent cytoplasmic sequestration of the WNT signaling component DVL, a function of YAP that is independent of the transcriptional activity of YAP-TEAD. The growth promoting effect of YAP deletion may be due to more intestinal differentiation, resulting in gain of Paneth cells that define the stem cell niche and are a major source for WNT ligands 192. Thus, complete inhibition of YAP may have unintended consequences and result in increased colorectal tumor growth. Desirable effects may thus be best achieved by aiming to reduce the transcriptional activity of the YAP-TEAD complex, for example by disrupting the YAP-TEAD interaction or by promoting cytoplasmic localization of YAP, rather than completely removing YAP. Indeed, expression of a dominant negative TEAD2 does not cause overt liver abnormalities 22. Hence, available evidence supports the notion that selective inhibition of YAP/TEAD function may have therapeutic efficacy with minimal side effects.
Additional evidence that reducing YAP activity is effective in suppressing tumor growth comes from studies using cancer cell assays 193,186, 194–196. Results obtained with human cancer cell lines demonstrated the efficacy of reducing YAP dosage (by siRNA or shRNA depletion) in slowing or arresting the growth of tumor cells in vitro or in xenograft assays 193,186, 194–196. Other properties of cancer cells are also affected by YAP knockdown, including making cells more sensitive to chemotherapeutic reagents122, inhibition of cancer stem cell formation132, and reduction of metastatic potential 194, 197, suggesting that targeting YAP may be effective in modulating a number of cancer cell properties that are required for their expansion, survival and spread to distant tissues.
Notably, a recent study extends the potential for targeting YAP in human cancer to cancers that do not exhibit elevated levels of YAP 54. Genome-wide screens for shRNA induced synthetic lethality in a large panel of human cancer cells revealed that many tumor cell lines with activated WNT signaling are particularly sensitive to knock-down of YAP 54. In this case, YAP forms a transcriptional complex with β-catenin (the nuclear effector of canonical WNT signaling) and TBX5 (a sequence-specific DNA binding protein) that is necessary for cell survival 54. Interfering with complex assembly inhibits tumor cell survival and growth as effectively as knock down of YAP itself suggesting that the β-catenin/YAP/TBX5 complex is driving survival rather than the YAP-TEAD complex that is presumably also present in these cells. In these cancer cell lines YAP is constitutively nuclear and is not regulated by cell density indicating a non-canonical mode of YAP regulation. Indeed, LATS independent phosphorylation of YAP by the SRC family kinase YES1 is required for YAP-β-catenin-TBX5 complex formation and oncogenic properties 54. Thus, this study highlights that sensitivity to YAP inhibition may not always correlate with the levels or activity of YAP, and that TEAD-independent interactions of YAP may be important in some cancer contexts.
In summary, these studies imply that inhibiting the activity of YAP and TAZ may be effective in treating a variety of cancers, that complete inhibition is not required to show therapeutic efficacy, and that the sensitivity of a cancer cell to YAP/TAZ inhibition may not always correlate with the expression levels of YAP/TAZ.
Tissue repair and regeneration often involves stem cell activation and progenitor cell expansion and an emerging picture is that YAP and TAZ regulate the balance between stem, progenitor and differentiated cells. In general, enhanced YAP or TAZ activity is associated with stem and progenitor cell expansion coupled with inhibition of differentiation, whereas attenuation of YAP and TAZ activity tends to have the opposite effect. Examples include stem and progenitor cells of the liver, intestine, pancreas, heart, skin, and CNS 12, 106, 142, 156, 158,28, 157, 198. At present, there is little mechanistic insight into how YAP and TAZ regulate stem cell properties and inhibit differentiation. However, a recent study by Lian et al. suggests that in murine embryonic stem cells YAP directly binds to the promotors of genes that enhance pluripotency, suggesting that YAP may be a key component of the core pluripotency machinery 199. Whether this result can be generalized to tissue-specific stem cells in vivo remains to be determined.
In situations where progenitor cell expansion has been observed in vivo by YAP activation, it is not clear whether YAP functions to inhibit differentiation and expand stem cell pools or acts by reprogramming differentiated cells to more primitive and stem cell-like progenitor states. Indeed, in the case of murine embryonic stem cells and induced pluripotent cells, YAP appears to be capable of doing both 199. Enhanced YAP activity inhibits embryonic stem cell differentiation whereas inhibition of YAP leads to loss of pluripotency. Similarly, in induced pluripotent cell derivation from murine fibroblasts, YAP activity increases during the reprogramming process and negative regulators of YAP, such as the LATS kinases act as barriers to reprogramming 200. The situation in human induced pluripotent stem cells and embryonic stem cells is similar to what is observed in the mouse in that LATS kinases function as negative regulators of reprogramming 201. However, unlike in the murine situation, this effect appears to be mediated by TAZ 51. In human embryonic stem cells, TAZ is important for maintenance of the pluripotent state, while YAP is dispensable for this activity. Differences between mouse and human systems are not fully understood, but likely derive from distinct signaling pathways that regulate pluripotency and reprogramming in murine versus human cells. Taken together these results suggest that YAP and likely TAZ promote reprogramming of differentiated cells as well as expansion of stem and progenitor cell populations, both features that are important in applications to regenerative medicine.
An evolutionarily conserved essential function for YAP in tissue regeneration has been described through genetic studies of intestinal epithelial re-growth following injury. Elevated YAP expression is seen in mice treated with dextran sodium sulfate (DSS), a chemical that results in injury and inflammation of the large intestine and initiates a regenerative response 22. Mice that lack YAP in the colonic epithelium do not show overt defects in intestinal homeostasis but are unable to efficiently undergo a regenerative response upon DSS treatment 22, consistent with a role for Hippo signaling in repressing latent regenerative responses that involve stem cell activation. Another study, however, found that conditional elimination of YAP in the intestine promoted regeneration after irradiation and caused hypersensitivity to WNT ligands 191. The reasons behind the different outcomes in the Li et al. and the Cai et al. studies are unclear at the moment but may be due to differences in experimental set-up and time point of analysis. In any case, these two studies suggest that YAP has growth promoting as well as growth suppressing functions, potentially because it directly promotes progenitor cell expansion but also suppresses Paneth cell differentiation, which promotes stem cells by producing WNT ligands. The Drosophila intestine also harbors stem cells that are triggered to proliferate in response to tissue damage induced by feeding toxins or pathogens. The Drosophila YAP homolog Yki is required for this response 26,25,27,24, but unlike in mammals, Yki is activated in differentiated enterocytes which drives the expression of cytokines that then signal to stem cells and promote their proliferation 24. Thus, although Yki may not be directly involved in controlling intestinal stem cell proliferation in Drosophila, it is activated upon tissue damage. Thus, in the intestine, regulation of the Hippo pathway and activation of YAP is involved in driving regeneration of damaged tissue.
A similar situation may be operating in the liver, where loss of Hippo signaling results in expansion of oval cells 136, 202 a facultative progenitor cell population that contributes to liver repair following hepatocyte injury. In addition, YAP is required for neonatal heart regeneration in mouse 28,29. Notably, forced overexpression of an activated, S127A phospho-site mutant form of YAP in the adult mouse heart promoted heart regeneration after myocardial infarction 28. Thus, therapeutic elevation of YAP activity might prove beneficial for restoration of gut, heart, liver, and potentially other tissues following injury.
An important effect of experimental elevation of YAP activity with regard to regeneration is the promotion of cell cycle entry of non-dividing cells. This effect is seen in both the liver 11, 12 and heart 28, 142, 156,29. These are tissues that are composed of mostly quiescent cells with cellular turnover rates estimated to be on the order of one year for human hepatocytes 203 and once in a lifetime for human cardiomyocytes 204. While both cell types are normally quiescent, hepatocytes can be readily induced to re-enter the cell cycle and progress through cytokinesis following injury whereas adult cardiomyocytes do not efficiently undergo cell cycle re-entry and rarely, if at all, undergo cytokinesis. Because of this, the adult liver can regenerate while the heart cannot. Potentially relevant to these observations are the findings that, in mouse mutants of core Hippo pathway components, including Sav1, Mst1/2, there is unscheduled hepatocyte proliferation in adults 135, 202, 205, 206 and enhanced embryonic cardiomyocyte proliferation 142 Moreover, overexpression of YAP in adult murine cardiocytes induces cell cycle entry and cell division 156,28,29. Hence, transient elevation of YAP activity might prove to be useful in the repair of tissues that do not normally undergo regeneration such as the heart and to augment the proliferative capability of damaged tissues that normally regenerate, such as the liver. Altogether, while sustained activation of YAP or TAZ has clear oncogenic potential in a variety of tissues, transient up-regulation of YAP or TAZ activity by pharmacologic intervention might be useful in situations that require mobilization of stem and progenitor cell populations or even the reprogramming of adult differentiated cells.
The studies described above suggest that manipulation of the Hippo signaling pathway might be beneficial in cancer prevention and treatment as well as to expand stem cell populations for use in regenerative medicine. Targeting the Hippo pathway as an anticancer therapeutic strategy would aim to suppress YAP and TAZ activity, while targeting the Hippo pathway to facilitate regeneration and reprogramming of adult cells and tissues, would aim at elevating YAP and TAZ activity. However, although evidence is mounting that pharmacologic manipulation of Hippo signaling and YAP/TAZ activity would be beneficial, there is a need to develop effective means to manipulate Hippo signaling and YAP/TAZ activity in both a sustained and transient manner. In addition, tissue-specific control of YAP/TAZ may become important if chronic and/or long-term whole-body inhibition of YAP and/or TAZ results in deleterious side effects. This may be achieved by targeting individual branches of upstream regulators. While there is interest and potential in targeting genes and pathways that are downstream of Hippo/YAP/TAZ such as the Axl receptor kinase 207, connective tissue growth factor (CTGF, 208), EGFR signaling 209, 210, and others (reviewed in 211), below we focus on strategies to target Hippo signaling itself by manipulating pathway components.
Decades of targeted drug development efforts suggest that kinases and other proteins with enzymatic activity are attractive targets for small-molecule therapeutics 212. Within the core of the Hippo signaling pathway are two pairs of kinases, MST1/2 and LATS1/2 that restrain YAP and TAZ activity. Small-molecule inhibitors of these kinases would thus be predicted to upregulate YAP and TAZ function, which might prove beneficial in regenerative medicine applications such as ex vivo stem and progenitor cell expansion and in vivo tissue repair. A small-molecule inhibitor for MST1, 9E1, has been developed 213. This inhibitor inhibits MST activity in vitro and in cultured Hela cells as measured by Histone H2B phosphorylation in response to apoptosis, although effects on YAP/TAZ activity have not yet been reported. 9E1 shows significant although not complete selectivity for MST1 over other kinases and it provides a starting point for the development of more selective MST1 inhibitors. In addition, rational strategies to target kinases are generally effective in identifying lead compounds that can be further developed and refined by traditional medicinal chemical approaches 214, 215. On the other hand, targeting the MST or LATS kinases for anticancer therapy is more challenging since small molecule agonists would be most desirable for which there are few options for rational design. Alternatively, the tyrosine kinase YES1 or the Homeodomain interacting kinase 2 (HIPK2) may be targeted for cancer therapy in some contexts as these kinases promote YAP activity in some assays: YES1 is required for cell survival and the activation of YAP in β-catenin active cancer cell lines 54 and HIPK2 promotes YAP activity in in vitro transcriptional assays 117. While there are no reported inhibitors of HIPK2, there is evidence that YES1 inhibition can selectively target cancer cell survival and growth. The YES1 kinase phosphorylates YAP thereby promoting the formation of a YAP/β-catenin/TBX5 transcriptional complex that is essential for the proliferation of β-catenin dependent colon cancer cell lines 54. Notably, the broad range tyrosine kinase inhibitor dasatinib, which inhibits the YES1 kinase, was effective in inhibiting growth of these β-catenin dependent cell lines in vitro and to inhibit tumor formation in xenographs by interfering with YAP/β-catenin/TBX5 complex assembly independent of Hippo pathway modulation 54. Thus, Inhibition of YES1 may be effective in a subset of β-catenin and YAP dependent cancers.
The most attractive anti-cancer targets in the Hippo pathway are YAP and TAZ, as they are the key downstream mediators of the pathway. However, pharmacological inhibition of YAP and TAZ is challenging as these proteins have no known catalytic activity and function by engaging domains that facilitate protein-protein interactions with the upstream kinases LATS1/2, the 14-3-3 phosphopeptide binding proteins, the AMOT and ZO1/2 polarity proteins, and the TEAD transcription factors. In many cases, the oncogenic properties of YAP and TAZ depend on their interaction with the TEAD proteins 194,71. Genetic disruption of this interaction by mutating amino acid residues critical for YAP-TEAD or TAZ-TEAD complex formation abolishes the transforming ability of YAP and TAZ in vitro 48, and in the case of YAP, in vivo, at least in a mouse liver cancer model 190. Since the co-crystal structure of the YAP/TEAD complex has been recently determined 216–218, it is possible that this detailed structural information can be leveraged to rationally design small-molecule inhibitors of YAP/TEAD by targeting residues that line the YAP/TEAD polypeptide interaction interface.
Additional components of the Hippo pathway that could be envisioned for targeted anti-cancer therapy are proteins that are required for YAP/TAZ activity such as the MASK proteins 114,113 and WBP2 110, 111, that directly interact with YAP/TAZ, and Ajuba family proteins, which stimulate LATS activity and thus inhibit YAP/TAZ indirectly 108. However, all of these are scaffold or regulatory proteins without enzymatic activity, which would require targeting a protein-protein interface. Nevertheless, because YAP and TAZ are regulated by multiple inputs, which often show tissue specific requirements, these and other proteins may provide means to inhibit YAP/TAZ in specific tissues, thereby aiding in the development of inhibitors that show organ and/or disease type specificity.
Recently, small-molecule modulators of the EGFR-PI3K pathway have been shown to affect Hippo signaling 219. Mitogenic growth factors contained in serum as well as EGF treatment stimulated nuclear localization of YAP by inhibiting YAP phosphorylation and promoting nuclear localization 219, 220. Based on this observation, Fan et. al. screened small-molecule inhibitors of kinases and phosphatases that act downstream of EGF signaling and found that PI3K and PDK1 inhibitors, but not AKT inhibitors, were effective in blocking EGF-induced and lysophosphatidic acid (LPA)-induced YAP nuclear localization 219. Mechanistically, PDK1 physically associates with the core Hippo pathway kinase complex and this complex dissociates in response to EGF signaling thereby resulting in YAP activation 219. Thus, PI3K and PDK1 inhibitors may be effective in reducing YAP activity in cells with an intact Hippo signaling pathway that have elevated EGFR and/or reduced PTEN function.
Three recent reports link G-protein coupled receptors (GPCRs) to Hippo pathway regulation 101,102,103. LPA, sphingosine-1-phosphate (S1P), and thrombin were identified as novel signals that signal through Ga12/13 coupled GPCRs to stimulate YAP nuclear localization and activity 101,102,103. Importantly, representatives of many different types of GPCR subgroups affect YAP, demonstrating a general function of GPCRs in YAP regulation 101. GPCRs that act through Ga12/13, Gaq/11, or Gai/o stimulate YAP, while Gas coupled receptors have the opposite effect 101. GPCRs regulate YAP via Rho GTPases and the actin cytoskeleton to inhibit LATS kinases independently of MST kinases to cause dephosphorylation and nuclear accumulation of YAP 101, 102,103. Agonists of Gas-coupled receptors such as epinephrine, glucagon and the dopamine receptor agonist dihydrexidine result in enhanced YAP phosphorylation and inactivation 101. Importantly, GPCR agonists such as epinephrine also affect YAP phosphorylation in vivo causing an increase of YAP phosphorylation in the heart of injected mice 101. These results therefore indicate that YAP activity can be modulated with drugs that modulate GPCR signaling, although which agonists or antagonists will be effective in therapeutic applications remains to be determined.
The identification of F-actin as an important regulator of YAP/TAZ localization and activity opens new opportunities for small molecule modulation of Hippo signaling. The F-actin destabilizers cytochalasin D and latrunculin A and B, as well as treatment with the non-muscle myosin II inhibitor blebbistatin, the myosin light chain kinase inhibitor ML-7, the Rho inhibitor botulinum toxin C3, and the Rho kinase inhibitor Y27632 all cause nuclear export of YAP/TAZ 96,98,100,99. These results suggest that drugs that target F-actin and its modulators may also be effective in modulating YAP/TAZ activity in vivo. This approach is complicated in that the actin cytoskeleton is not a Hippo pathway specific signal transduction component but required for many basic cellular functions. Nevertheless, non-lethal levels of actin modulators may enhance the effects of other drugs that target the Hippo pathway by different mechanisms.
An attractive approach to identify small-molecule inhibitors and activators of Hippo signaling is through application of cell based high throughput screening. Indeed, a screen of approximately 3300 FDA approved drugs for inhibitors of the transcriptional activity of YAP identified 71 hits including several porphyrin compounds 190. One of them, verteporfin (VP), currently used as a photosensitizer in therapy for macular degeneration, was effective in vivo in delaying tumor progression in a NF2-depleted mouse liver model and to suppress liver overgrowth caused by overexpression of YAP using a regimen that involved repeated drug administration during the development of the cancer phenotype 190. VP was found to bind to YAP in vitro and to inhibit the interaction of YAP with TEAD 190. Although these data are exciting, future studies will be needed to determine if VP is effective in other in vitro and in vivo cancer models and also whether it is effective in the treatment of established cancers. In addition, the affinity of VP for YAP is relatively low (at the micromolar level) and higher affinity derivatives may be required.
In another cell based screen of 48 drugs for inhibitors of nuclear localization of YAP, Bao et. al. identified dobutamine, a G-protein coupled β-adrenergic receptor agonist used clinically to treat acute heart failure, as being effective in preventing nuclear accumulation of YAP and YAP-mediated transcriptional activation in osteoblastoma and HEK293 cells 221. Dobutamine treatment induced cytoplasmic translocation of YAP and phosphorylation of the main LATS phosphorylation site serine 127. Phosphorylation of this site was required for the effect of dobutamine, although it appeared to be phosphorylated by a kinase other than LATS 221. Dobutamine-mediated activation of β-adrenergic receptor likely acts through a pathway that involves Rho GTPases and F-actin, similar to other GPCRs 101,102,103.
The Hippo pathway, and in particular the YAP/TAZ-TEAD complex, is an emerging anti-cancer target and mounting evidence from mouse models as well as tissue culture assays indicates that targeting the Hippo pathway is effective in preventing disease and counteracting cellular mechanisms that promote oncogenic transformation. Although less appreciated, there is also considerable potential for application of small molecules that activate the YAP/TAZ-TEAD complex transiently for stem cell expansion and tissue repair following injury.
Work over the past decade has identified a complex network of Hippo pathway regulatory components and established robust assays by which pathway activity can be measured. These results now provide a rich landscape to identify small-molecule modulators of the Hippo pathway and a number of pharmacological modulators of Hippo signaling have already been discovered. The challenges are now to determine whether these drugs will be therapeutically useful, which combinations will be the most effective, and in what disease contexts they should be applied. Reflecting the complex regulation of the Hippo signaling pathway, these small molecules affect a variety of components and branches of the pathway. However, many of these do not specifically target Hippo pathway components and therefore another challenge is to develop novel modulators that specifically target the pathway and in particular the activity of the YAP-TEAD complex. Since many of the pathway components are adaptor proteins that may be difficult to target, it will be important to discover additional components of the Hippo pathway, especially ones that directly affect YAP/TAZ, which may lead to the identification of better drug targets. For example, the recent finding that SET7-dependent lysine monomethylation of YAP is important for cytoplasmic retention 222 as well as p300/CBP-mediated acetylation and SIRT1-mediated deacetylation of YAP 223, 224 suggests that identification and selective inhibition of YAP protein demethylases could be a novel approach to modulate YAP activity. Another challenge is to decipher how YAP/TAZ are deregulated in cancer as this will be critical in deciding which components in the pathway should be targeted.
In principle, targeting the Hippo pathway for regenerative medical applications may be more easily accomplished as the core of the pathway contains two kinases. It will thus be interesting to develop and test inhibitors of the MST and LATS kinases such as the 9E1 compound for transient stimulation of growth and regeneration of tissues following injury and in applications where stem/progenitor cell expansion is desired.
Although most attention has focused on small-molecule manipulation of YAP/TAZ activity that acts by controlling YAP/TAZ sub-cellular localization or their ability to complex with TEADs, there are other avenues for pharmacological manipulation of YAP/TAZ that warrant further investigation. For example, YAP/TAZ stability is controlled by phosphorylation-induced protein degradation 67, 68 and small molecules that enhance YAP/TAZ turnover would be expected to reduce nuclear transcriptional activities of these proteins. Finally, not all components of the Hippo pathway are required in all tissues and the pathway has tissue specific regulatory mechanisms. Targeting such tissue specific regulators provides opportunities to manipulate the pathway in specific cells, which may help in reducing toxicity and increasing therapeutic value.
In conclusion, while studies aimed at targeting the Hippo pathway for therapeutic uses are still in their infancy, promising preclinical genetic and pharmacological results have already been documented in the literature. As these studies mature, we anticipate that the full potential of harnessing the Hippo pathway in human disease prevention and treatment will be realized.