183 Andrea Perra and Marta Anna Kowalik. / International Journal of Biological & Pharmaceutical Research. 2016; 7(4): 183-191.

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YES-ASSOCIATED (YAP) – A PROMISING THERAPEUTIC TARGET FOR HEPATOCELLULAR CARCINOMA

Andrea Perra* and Marta Anna Kowalik

Oncology and Molecular Pathology Unit, Department of Biomedical Sciences, University of Cagliari, via Porcell 4, 09124 Cagliari, Italy.

ABSTRACT Hepatocellular carcinoma (HCC), the third leading cause of cancer mortality, carries a dismal prognosis and represents a major health problem. A better understanding of the molecular pathways involved in HCC development may represent an important approach for the improvement of the therapeutic strategies for this cancer. The Hippo signalling pathway, a growth- suppressive mechanism that antagonizes the transcriptional co-activator Yes-associated protein (YAP), has been recently found altered in human HCC. Moreover, using the Resistant-Hepatocyte (R-H) rat model, recently published data established that the Hippo pathway deregulation occurs already at the early stages of HCC development, making this pathway an important therapeutic target. Many strategies have been proposed to modulate the activity of the Hippo pathway, and in particular that of the final effector YAP, most of which require complex manipulations, useful to understand the functions of the pathway but difficult to apply on patients. One of the few drugs able to inhibit in vivo the pro-carcinogenic effect of YAP activation is verteporfin, which disrupts the formation of the complex between YAP and the TEAD transcription factors, causing a significant reduction of the number and size of preneoplastic foci induced in rats liver by R-H protocol. In this review, we give a short summary of Hippo pathway, providing the evidences of its deregulation in mouse, rat and human liver cancer and discuss the possibility to treat HCC with new drugs targeting the transcriptional co-activator YAP.

Key Words: Liver, Cancer, YAP, Hippo, Therapy, Verteporfin.

INTRODUCTION Liver cancer, which includes hepatocellular or cirrhosis (El-Serag et al., 2007). HCC carries a dismal carcinoma (HCC) and intrahepatic cholangiocarcinoma prognosis and only partial liver resection or liver (iCCA), represents the second cause of cancer related transplantation are potentially curative, but only a minority deaths and its incidence rate is still significantly increasing of cases is amenable to these treatments. (Bruix et al., 2015; Siegel et al., 2014). Marked variations Hepatocarcinogenesis is considered to be a very complex among geographic regions, racial and ethnic groups, and multistep process, which involves alterations in between men and women and the presence of several well- numerous signalling cascades. In particular, Wnt/β-catenin, documented environmental potentially preventable risk , Mitogen-Activated Protein Kinase, Ras, JAK/STAT, factors are reported as important epidemiologic features of Epidermal Growth Factor and Transforming HCC. This malignancy generally develops as a Growth Factor-β pathways have been extensively studied consequence of underlying liver diseases such as hepatitis through pathway-based approaches and functional experimental studies in order to identify potential Corresponding Author molecular targets (Aravalli et al., 2008). Recently, pioneering studies have reported that the Hippo signaling Andrea Perra pathway, an evolutionary highly conserved regulator of Email: [email protected] tissue growth, organ size and stem cell property, is 184 Andrea Perra and Marta Anna Kowalik. / International Journal of Biological & Pharmaceutical Research. 2016; 7(4): 183-191. involved in the pathogenesis of HCC (Dong J et al., 2007; resulting in their sequestration in the cytoplasm and, Zender L et al., 2010; Pan D et al., 2010; Forner A et al., finally, their ubiquitination-dependent proteosomal 2012). degradation. Therefore, the Hippo pathway acts by Many components of this signaling pathway, blocking the ability of YAP and TAZ to translocate in the which is composed of the upstream regulatory components, nucleus, where they interact with transcriptional factors of the Hippo core kinase components, and the downstream the TEAD family (TEAD1–4), leading to induction of transcriptional machinery, were initially discovered by target such as Axl, Birc5, CTGF, IGFBP3, ITGB2 Drosophila mosaic genetic screens (Justice RW et al., and Gli2 (Sudol M et al., 1995). 1995). Successively, genetic and biochemical studies In mammals the most conserved component of demonstrated that the Hippo pathway is highly this pathway is YAP, which is considered to be the evolutionary and functionally conserved in mammals principal and less tissue-specific nuclear effector of the (Zhao B et al., 2010). Recently, increasing number of Hippo pathway. It owes the name to its ability to interact studies confirmed that Hippo pathway perturbation can with Yes protein; however, the biological relevance of this trigger tumorigenesis in mice and altered expression of interaction remains elusive (Harvey KF et al., 2013). genes targeted by this pathway have been observed in Originally sequenced in chicken, human and mouse various human cancers (Steinhardt AA et al., 2008; Harvey homologues of YAP were identified by using cDNA to KF et al., 2013). Indeed, the Hippo pathway results probe cDNA libraries (Sudol M et al., 1995). Initially, it deregulated in a broad variety of human cancers, including was demonstrated that variations of YAP levels can hepatocellular carcinoma (Zhou D et al. 2009; Xu MZ et overcome growth arrest at confluence, in vitro, and organ al., 2009; Li H et al., 2012). In recent times, an important size control in vivo. YAP effects on liver size is goal in cancer therapy is to develop drugs able to target astonishing. Indeed, overexpression of YAP in transgenic selectively a pathway or molecule demonstrated to be mice causes an increase of the liver weight which can be crucial for tumor progression. In this regard, Hippo from 5% to 25% of body weight, largely due to increased pathway emerged as an important candidate, as critical for cell number. This effect is reversible as the enlarged liver the growth of cancer cells, but with minimal role in adult reverts to almost normal size once overexpression of YAP tissue homeostasis (Stanger BZ, 2012). In this review, we is ceased (Camargo FD et al., 2007). Moreover, sustained provide a short summary of Hippo pathway components induction of YAP expression leads to formation of and discuss the role its effector, the transcriptional co- hepatocellular adenomas and carcinomas (Dong J et al., activator YAP (Yes-associated protein), in hepato 2007). YAP activation is also associated with liver carcinogenesis and the new therapeutic strategies targeting hyperplasia, increased liver size and HCC also in non- the Hippo downstream transcriptional machinery. transgenic mice, following treatment with TCPOBOP (1,4- Bis-[2-(3,5-dichloropyridyloxy)]benzene), an agonist of The Hippo pathways and its effectors in mammals the constitutive androstane receptor (CAR) (Kowalik MA The Hippo pathway is conserved signaling et al., 2011). cascades that regulates organ size by controlling cell This intense and reversible effect on liver size proliferation and apoptosis, and participate to caused by YAP activation, places the Hippo pathway as a organogenesis and cell differentiation. These features make major mechanism inducing liver hyperplasia in mammals. the study of the alterations of this pathway extremely Furthermore, in two different animals models of YAP relevant to several human diseases, including hypertrophic activation, transgenic over-expression and drug-induced, cardiopathy, alteration of the inflammatory response, delay primary, liver tumors were observed, strengthening the in wound healing, and, recently, in primary tumors of the concept of YAP as a relevant oncogene in liver (Perra A et al., 2014). Basically, the Hippo pathway hepatocarcinogenesis, as already demonstrated for other can be divided into three parts, the upstream regulatory carcinomas (Harvey KF et al., 2013). components, the Hippo core kinase components, and the downstream transcriptional machinery, each one YAP structure and functions constituted by several (figure 1). In mammals, the The human YAP gene (also known as YAP1) is upstream components are still poorly understood, while the located at 11q13 and can be transcribed in a least 8 core kinase that controls the transcriptional machinery is isoforms, that are derived by differential splicing, that are well characterized. It contains four proteins, two of which classified into two main groups, YAP-1 (or YAP1-1) and are kinases, Mst1/2 and Lats1/2, the other two are the YAP-2 (or YAP1-2) (Gaffney CJ et al., 2012). The most adaptor/activator proteins WW45 and Mob. Upstream studied and cited isoform belongs to the YAP-2 group and regulators activate, by phosphorylation, the Mst1/2-WW45 is that containing 488 residues. The analysis of the protein complex, which, in turn, can phosphorylate and activate sequence allowed the identification of several domains, the LATS1/2-Mob complex. The major target of this common to all isoforms, that are crucial for the modulation cascade is the phosphorylation of the effectors YAP and of the Hippo pathway (figure 2). The so called Lats TAZ, that leads to their interaction with 14-3-3 proteins, recognition motifs are regions characterized by the 185 Andrea Perra and Marta Anna Kowalik. / International Journal of Biological & Pharmaceutical Research. 2016; 7(4): 183-191. sequence HxRxxS (Histidine-any-Arginine-any-any- that allow the binding with proteins containing a PPxY Serine), which can be recognized by the LATS kinase. motif (Proline-Proline-any-Tyrosine). Two consecutive This interaction is crucial for the phosphorylation of YAP WW domains are present in all isoforms, except for in these sites, with the Serine127-containing motif (S127) isoform 2, which has only one, and allow YAP to act as a being the most important for YAP inactivation. transcriptional co-activator. Indeed, these domains can S127 phosphorylation creates a binding site for interact with PPxY motifs found in several transcription 14-3-3 proteins that determine the cytoplasmic factors, such as c-Jun, AP-2, NF-E2, C/EBPalpha, sequestration of the complex. This mechanism was initially PEBP2/CBF, Runx/PEBP2, Krox-20, Krox-24, MEF2B, described by in vitro experiments where, in non-confluent Oct-4 and (Zhao B et al., 2010). The confirmation of proliferating cells, YAP was not phosphorylated and the role of YAP as a transcriptional co-activator came from localized into the nucleus, acting as a transcriptional co- the evidence that its over-expression conferred activator. Over-expression of LATS was able to induce transcription-stimulating activity on PEBP2alpha. The S127 phosphorylation of YAP, its cytoplasmic activity of YAP on some other putative interacting sequestration and cells growth arrest. Mutation of the S127 transcriptional factors was investigated and confirmed by into Ala (S127A) restored the growth activity of cells, in vitro assays, however, whether PPxY-containing regardless of LATS over-expression, and abolished growth transcriptional factors are in vivo physiological partners for arrest upon confluence (Zhao B et al., 2007). Therefore, it YAP still need confirmation (Yagi R et al., 1999). was concluded that cell to cell interactions would trigger a Another region, which exhibited strong cascade of signaling events that culminate with the Hippo transactivation property, is located in the C-terminal region pathway activation, which in turn phosphorylates YAP in of YAP (residues 276 and 472), and contains a PZD- S127, leading to enhanced interactions with 14-3-3 binding motif (TWL-COOH) that allows the interaction proteins and cytoplasmic sequestration of the complex with PDZ-motif containing proteins, such as ZO2 and (figure 1). Modulation of cell differentiation and growth NHERF2 (Huang J et al., 2005). However, the finding that rate by cell to cell and cell to extracellular matrix (ECM) N-terminal region of YAP (residue 32–121) can interact interactions, largely depends on YAP phosphorylation with the C-terminal half of TEAD family of transcriptional levels, but the results of recent studies brought to consider factor, which have neither the PPxY nor the PDZ motif that this phosphorylation could be achieved either by a (Zhao B et al., 2008, Vassilev A et al., 2001), has Hippo-dependent or -independent pathway. The questioned their importance for YAP activity. independent regulation was shown to require Rho GTPase activity and tension of the actomyosin cytoskeleton. Taken TEAD transcriptional factors as major mediators of YAP together, these data, show that YAP could be defined as the transcriptional outcome main effector of mechanical signals exerted by As previously mentioned, YAP was identified as a extracellular matrix (ECM) rigidity and cell shape, and led tight and major interacting protein for the TEAD family of to the hypothesis that actin cytoskeleton, cell morphology, transcriptional factors, that in mammals is composed by cell attachment, and mechanosensing act through a four members (TEAD1–4). The crystal structure of these common mechanism to regulate YAP activity (Wrighton proteins revealed a three-helix bundle with an KH, 2011). homeodomain fold, able to bind DNA, and a bipartite Therefore YAP function is inactivated through nuclear localization signal (NLS) located in the helix three, phosphorylation of YAP S127 that create a binding site for to promote nuclear localizzation. The N-terminal region of 14-3-3 proteins, but YAP contains other 4 HxRxxS motifs TEADs contains a conserved TEA domain involved in that could be targeted by the Hippo core kinases. Among recognizing DNA elements such as GGAATG in the these sites, the functional consequence has been promoter region of target genes, whereas the C-terminal established for the phosphorylation of serine 381 (S381), region is involved in interaction with the N-terminal region that prime the subsequent phosphorylation of S400 and of YAP. Of particular importance for this interaction is S403 by casein kinase 1 phosphorylates, leading to serine 94 (S94), as demonstrated in a recent study where recruitment of SCF E3 ubiquitin ligase and YAP mutation of this residue abolished most, if not all, the degradation (Zhao B et al., 2010). Therefore Hippo core ability of YAP to promote cellular transformation and kinase can inactivate YAP signaling, transiently regulating transcriptional activation, as assessed by microarray cytoplasmic sequestration by S127 phosphorylation, and analysis. These data suggest that interaction with TEADs is also permanently priming its proteosomal degradation by the major functional pathway through YAP mediate the S381 phosphorylation. These dual mechanism of biological outcome. Congruent with this, YAP and TEAD regulations enable Hippo pathway to tightly restrict the bind to a common set of genomic targets. In addition, functionality of YAP to prevent unwanted growth. CTGF was identified as a direct target gene of TEAD-YAP Other core sequences for YAP function are the WW complex and the promoter region of CTGF gene contains domains, that contain two conserved and consistently- several GGAATG motifs for TEAD-binding (Zhao B et positioned tryptophan (W) residues (Sudol M et al., 1995), al., 2008). 186 Andrea Perra and Marta Anna Kowalik. / International Journal of Biological & Pharmaceutical Research. 2016; 7(4): 183-191.

According to the exposed, there is a strong disease free survival was significantly longer for patients evidence to support the role of YAP as proto-oncogene in with YAP negative expression compared with patients with mammalian cells (Xu MZ et al., 2009; Li H et al., 2012; YAP positive expression (Li H et al., 2014). As reported Stanger BZ, 2012; Zender L et al., 2006; Chan SW et al., by Kim et al. (Kim GJ et al., 2013) increased YAP 2011). Genome-wide analysis using mouse model of liver expression was also more frequently found in HCCs and cancer showed amplification at 9qA1, which combined hepatocellular-cholangiocarcinoma (cHC-CCs) is syntenic to human chromosome region 11q22, which with stemness features, based on the presence of EpCAM contains YAP gene (Overholtzer M et al., 2006). Another and KRT-19 markers. With regard to TAZ, it has been study described the amplification of a smaller observed that also its up-regulation in HCC correlates with chromosomal region within 9qA1 in mouse mammary low overall survival rate after hepatic resection (Xiao H et tumors and YAP is the only gene within this narrower al., 2015). region. Amplification of 11q22 was also observed in several human cancers. YAP gene is also amplified in YAP and animal models human intracranial ependymomas, oral squamous cell A large body of evidence confirmed that Hippo carcinomas, and medulloblastomas (Baldwin C et al., pathway perturbation can trigger tumorigenesis in mice 2005; Fernandez L et al., 2009; Modena P et al., 2006; (Zhao B et al., 2010; Harvey KF et al., 2013). This Snijders AM et al., 2005), indicating a potential role for important conclusion derived from the analysis of YAP in human cancer. Moreover, YAP protein over- transgenic mice engineered to overexpress Yap under a expression has been demonstrated in approximately 45% doxycycline-inducible promoter (Dong J et al., 2007; liver cancers (Zhao B et al., 2007), with a significant Camargo FD et al., 2007). In both studies, Yap expression association with poor prognosis (Xu MZ et al., 2009; Perra led to immediate and massive hepatomegaly (a four-fold A et al., 2014). increase in liver size), associated with marked hepatocyte proliferation. Removal of doxycycline resulted in a Role of YAP in human hepatocellular carcinoma (HCC) reversion to normal size and architecture. However, Since its discovery in the past decade, much sustained expression of the Yap transgene caused the progress has been made in the Hippo/YAP signalling development of multifocal HCC. Similar phenotypes have pathway and it is increasingly clear that the deregulation of been observed in liver-specific knockout of Mst1/2 or Sav1 this cascade underlies liver tumorigenesis (Perra A et al., (Zhou D et al., 2009; Lu L et al., 2010; Song H et al., 2014; Han SX et al., 2014). Recently, YAP has been 2010; Lee KP et al., 2010). Functions of YAP in organ size reported as one of potentially interesting HCC cancer regulation and tumorigenesis have been widely confirmed genes, which have been identified and validated through in mammals, using transgenic mouse models. However, integrative functional genomics (Zender L et al., 2010). In despite recent progress, our knowledge about the role of fact, two reports identified YAP as a driving oncogene in this important growth regulatory pathway in non- human HCC 11q22 amplicons (Zender L et al., 2006; transgenic animals remained limited. Overholtzer M et al., 2006). Clinical studies further In this context, two recent studies have been confirmed the role of YAP as a relevant player implicated performed in non-genetically engineered mice and rats. In in HCC. First of all, analysis of a Chinese cohort of 177 the first (Kowalik MA et al., 2011), it was shown that pairs of tumor and adjacent non-tumor tissue from HCC HCCs develop in mice given diethylnitrosamine (DENA) patients (all surgical patients and mostly associated with and then subjected to repeated treatments with mitogen HBV infections) demonstrated that YAP was expressed in 1,4-bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP), approximately 62% of HCC cases and localized mainly in an agonist of constitutive androstane receptor (CAR). the nucleus. Importantly, YAP appeared as an independent These HCCs exhibited increased levels of YAP associated prognostic marker for overall survival and disease-free with down-regulation of microRNA-375, which is known survival (DFS) times of HCC patients and to control YAP expression. Enhanced levels of AFP and clinicopathologically associated with tumor differentiation connective tissue growth factor (CTGF), two target genes and serum α-fetoprotein (AFP) level (Xu MZ et al., 2009). of YAP were also observed in these tumors. In the second Similar results were obtained by Li et al. (Li H et al., study (Perra A et al., 2014), the authors demonstrated for 2012) on tissue arrays containing a total of 70 HCC, 21 the first time that the derangement of the Hippo pathway is normal and 17 cancer adjacent normal samples, revealing a very early event in hepatocarcinogenesis. For their high nuclear staining in more than 85% of HCC samples. experiments, they used the Resistant-Hepatocyte (R-H) rat Moreover, YAP activation in these samples was associated model of carcinogenesis (Solt DB et al., 1977), that allows with tumor aggressiveness and a higher TNM grade. Other dissecting the different steps of the carcinogenic process, studies further confirmed correlation between high YAP offering the possibility to identify phenotypically distinct expression and poor HCC prognosis (Han SX et al., 2014). lesions at well-defined timings. In this model, Next, a study of 105 patients undergoing liver preneoplastic foci and nodules (1-2 months after initiation transplantation for HCC demonstrated that HCC-specific with DENA), early HCCs (eHCC, 6-10 months) and fully 187 Andrea Perra and Marta Anna Kowalik. / International Journal of Biological & Pharmaceutical Research. 2016; 7(4): 183-191. advanced HCCs (aHCC, 14 months) develop sequentially therapeutic option (Piccolo S et al., 2013). in the liver. Remarkably, all foci of preneoplastic The pioneering proof-of-concept report which hepatocytes generated in rats as early as 4 weeks after regards the feasibility of genetic and/or pharmacological DENA treatment, displayed a marked YAP accumulation approaches to target YAP was published by Liu- and up-regulation of YAP target genes (Perra A et al., Chittenden. (Liu-Chittenden Y et al., 2012). Using a 2014). human embryonic kidney (HEK293) luciferase-based assay to screen a small molecule library for compounds Pharmacological modulation of the Hippo/YAP pathway (collection of >3300 drugs) that could inhibit YAP’s An important objective in cancer therapy is to transcriptional activity, three compounds (protoporphyrin develop drugs that selectively target a particular pathway IX, hematoporphyrin, and verteporfin), all members of the or molecule. Considering the widespread involvement of porphyrin family, were identified. Liu-Chittenden and the Hippo pathway in HCC, YAP emerged as an attractive colleagues focused their attention, in particular, on candidate, since it is critical for the growth of cancer cells, verteporfirin, already in clinical use with little side effects, but dispensable in adult tissue homeostasis (Stanger BZ, as photosensitizer used in photodynamic therapy for 2012). In this regard, it was proposed that the tumorigenic macular degeneration (Michels S et al., 2001). Recently, potential of YAP, and likely that of TAZ, requires another possibility for direct targeting of TEAD has association to TEAD. as it is reported as the major emerged and may be represented by cyclic peptides, which mediator of YAP in the Hippo pathway. TEAD is critical in both surface plasmon resonance (SPR)-based for YAP functions in gene expression, cell proliferation, competition assay and cell-based pull-down assay showed anchorage-independent growth, and epithelial- a good correlation in disrupting YAP-TEAD interaction mesenchymal transition (Zhao B et al., 2008). For this (Zhou Z et al., 2015). A detailed summary of the effects of reason, targeting the TEAD factors as selective means of genetic or pharmacological modulation of the Hippo inhibiting YAP’s oncogenic activity, appeared as a valid pathway is reported in Table 1.

Table 1. Effects of genetic or pharmacological modulation of the Hippo/YAP pathway in the liver Target Method/Drug Results Reference in vitro: YAP-mediated transcription and YAP’s ability to promote anchorage-independent growth in soft agar resulted TEAD2-DN: dominant- blocked. disruption of negative version of Liu-Chittenden et al. in vivo: in YAP overexpressing mice, expression of TEAD2- YAP-TEAD TEAD2 in which the 2012 DN completely suppressed YAP-induced liver overgrowth interaction DNA-binding domain and completely abolished YAP-induced HCC formation; has been deleted. TEAD2-DN influenced YAP’s ability to promote cell proliferation and survival. in vitro: proteolytic profiling experiments revealed that verteporfin disrupts the formation of the YAP-TEAD complex by binding to YAP and changing its conformation, disruption of thereby blocking the transcription of downstream targets. Liu-Chittenden et al. YAP-TEAD verteporfin in vivo: administration of VP blunted liver overgrowth in 2012 interaction YAP-overexpressing mice, without adverse effects in other organs; VP blocked biliary expansion, significantly reducing the number of cytocheratin-positive biliary epithelial cells in the case of NF2/Merlin deletion. in vitro: the increased luciferase activity of the Jag-1 region disruption of 1 resulted inhibited in a dose-dependent manner when either Tschaharganeh et al. YAP-TEAD verteporfin YAPS127A or TEAD4 plasmid transfection was paralleled 2013 interaction by treatment with verteporfin in vitro: verteporfin impaired the growth of HuH7 cells; as well as the viability and the colony forming ability of rat tumorigenic cells derived from HCCs generated by the disruption of Resistant-Hepatocyte (R-H) model of carcinogenesis in rat. YAP-TEAD verteporfin Perra et al. 2014 in vivo: verteporfin administration caused a significant interaction reduction in the number of GSTP-positive preneoplastic foci and of their average size in the R-H model of hepatocarcinogenesis;verteporfin exerted an inhibitory effect 188 Andrea Perra and Marta Anna Kowalik. / International Journal of Biological & Pharmaceutical Research. 2016; 7(4): 183-191.

on infiltration of liver parenchyma by oval cells in the same model. in vitro: verteporfin blocked induction of YAP target genes disruption of and hepatic stellate cells (HSC) activation. YAP-TEAD verteporfin Mannaerts et al. 2015 in vivo: in a mouse model of fibrosis (mice treated with interaction carbon tetrachloride, CCl4) verteporfin reduced fibrogenesis. dominant-negative in vitro: dominant negative mutation inhibited colony disruption of mutation (Y406H) of formation in human liver cancer cell line BEL-7404. YAP-TEAD Zhou et al. 2015 TEAD1 to abolish YAP- in vivo: a significant reduction in tumor growth rate in a interaction TEAD interaction HCC xenograft model in comparison with the control mice.

Fig 1. Schematic representation of the mammalian Hippo pathway. Although the upstream component (that are still not well characterized) activate the core kinase by phosphorylation. Activated core kinase promote the phosphorylation of YAP and the consequent cytoplasmic retention and proteasomal degradation. When Hippo signaling is inactive, YAP accumulates in the nucleus, acting as trancriptional co-activator of the TEADs.

Fig 2. Structure and principal binding domains of YAP protein. Numbers indicate the position of the most relevant residues for YAP modulation

189 Andrea Perra and Marta Anna Kowalik. / International Journal of Biological & Pharmaceutical Research. 2016; 7(4): 183-191.

CONCLUSIONS approaches are at the moment difficult to achieve. In fact, The Hippo / YAP pathway has received more and the upstream regulators, such as the GPCR transmembrane more attention in recent years, due to the discovery of its proteins, are implicated in the regulation of a large number many functions in the regulation of proliferation and of cellular functions, so that their pharmacological differentiation of a large number of cell types and tissues. modulation could lead to a high number of side effects. The demonstration of its role in regulating polarity, contact The Core kinases would be a more selective target, but inhibition and differentiation of cells, has clearly outlined they have a tumor suppressor activity, whereby their the role of this pathway in organogenesis processes and targeting would need a complex strategy of activation. For wound healing, offering a new promising approach for this reasons direct inhibition of the YAP/TEAD complex regenerative medicine. These new findings have by drugs that act as verteporfin, might be the best strategy. In fact acting on the final effector could reduce the side encouraged researchers to investigate for an involvement effects, compared to an intervention on the upstream of the Hippo in carcinogenesis, process in which is often controllers. The realization of anti-cancer drugs that have observed the loss of cell functions typically controlled by targeted YAP, however, require further efforts to define the this pathway. functionality of this transcriptional activator both in normal Now we know that the main effector of the cells than in cancer. pathway, YAP, is directly involved in liver carcinogenesis, as demonstrated by an increasing amount of data coming ACKNOWLEDGMENTS from experimental models and human hepatocellular Work in the laboratory of A.P. and M.A.K. is carcinomas. supported by the AIRC (Associazione Italiana per la This review is focused on the evidence that YAP Ricerca sul Cancro) and the FBS (Fondazione Banco di has a role in liver carcinogenesis, and on the possible use Sardegna, grant to A.P.). We apologize to colleagues of modulators of its effects in the treatment of whose work we could not cite because of space constraints. hepatocellular carcinoma, a tumor to which still lacks an effective drug therapy. CONFLICT OF INTEREST As previously discussed, it is possible to identify The authors declare that they have no conflicts of numerous drug targets in the Hippo pathway, belonging to interest. the upstream regulators or the kinase core, but both

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