IJC International Journal of Cancer

Hippo-YAP signaling pathway: A new paradigm for cancer therapy Yanlei Ma1, Yongzhi Yang1, Feng Wang1, Qing Wei2 and Huanlong Qin1 2

Department of GI Surgery, Shanghai Tenth People’s Hospital Affiliated with Tongji University, Shanghai, People’s Republic of China Department of Pathology, Shanghai Tenth People’s Hospital Affiliated with Tongji University, Shanghai, People’s Republic of China

In the past decades, the Hippo signaling pathway has been delineated and shown to play multiple roles in the control of organ size in both Drosophila and mammals. In mammals, the Hippo pathway is a kinase cascade leading from Mst1/2 to YAP and its paralog TAZ. Several studies have demonstrated that YAP/TAZ is a candidate oncogene and that other members of the Hippo pathway are tumor suppressive genes. The dysregulation of the Hippo pathway has been observed in a variety of cancers. This review chronicles the recent progress in elucidating the function of Hippo signaling in tumorigenesis and provide a rich source of potential targets for cancer therapy.

Introduction In recent decades, the basic strategy for cancer treatment has been to use “weapons of massive destruction,” an arsenal including alkylating agents, radiation and anti-metabolites, to destroy cancer cells. Although these conventional treatments may induce remission and even cure the disease, the side effects are severe and sometimes can be as lethal as the cancer itself. Due to a lack of specificity, these conventional treatments promiscuously kill rapidly dividing cells, leading to severely damaged tissues, particularly tissues with a high rate of turnover (skin, bone marrow and gut epithelium). Furthermore, some tumors were found to be intrinsically sensitive or resistant to Key words: hippo, yap, cancer, therapy Grant sponsor: National Natural Science Foundation of China; Grant number: 81372615; Grant sponsor: Shanghai Science and Technology Development Fund; Grant numbers: 12140902300 and 12410707400; Grant sponsor: Shanghai Health System Outstanding Young Talent Training Plan; Grant number: XYQ2013118; Grant sponsor: Tongji University Outstanding Youth Program; Grant number: 1501219074 DOI: 10.1002/ijc.29073 History: Received 20 Mar 2014; Accepted 2 Jul 2014; Online 10 Jul 2014 Correspondence to: Yanlei Ma, Department of GI Surgery, Shanghai Tenth People’s Hospital Affiliated to Tongji University, 301 Yanchang Road, Shanghai 200072, P. R. China, Tel.: 186-21-66300588, Fax: 186-21-66303643, E-mail: [email protected] or Huanlong Qin, Department of GI Surgery, Shanghai Tenth People’s Hospital Affiliated to Tongji University, 301 Yanchang Road, Shanghai 200072, P. R. China, Tel.: 186-21-66300588, Fax: 186-21-66303643, E-mail: [email protected] or Qing Wei, Department of Pathology, Shanghai Tenth People’s Hospital Affiliated to Tongji University, 301 Yanchang Road, Shanghai 200072, P. R. China, Tel: 186 21 66300588, Fax: 186 21 66303643. E-mail: [email protected].

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certain agents. For instance, acute promyelocytic leukemia (APL) is particularly responsive to all-trans retinoic acid (ATRA) treatment. In APL, the t(15;17) rearrangement results in the expression of the PML/RARa fusion protein, which blocks terminal myeloid differentiation. Treatment with ATRA overcomes the dominant-negative effects of PML/RARa and induces differentiation, which sensitizes APL cells to ATRA.1 With the dawn of a new age of personalized medicine, oncologists are seeking to “tailor therapy” to treat malignant diseases. The ultimate goal of such therapy is to develop target drugs that exploit the unique biological properties of a tumor that have made it selectively sensitive to the modulation of a particular pathway or molecule. ATAR in APL represents the leading example of target drugs. For example, Cetuximabis another widely known target drug used for the treatment of metastatic colorectal cancer (mCRC). However, despite their promising effectsin cancer therapy,2 targeted drugs still suffer from limited spectrum of efficacy. The pathways or molecules required for tumor growth need to be further identified for the advancement of targeted therapies. Recently, many researchers have demonstrated the involvement of the Hippo-YAP signaling pathway in the organization of size regulation, cell proliferation, apoptosis. The Hippo signaling pathway derives its name from the discovery of a set of four genes in Drosophila. These genes specify a series of kinases and adaptor proteins including the NDR family protein kinase Warts (Wts),3 the WW domain-containing protein Salvador (Sav),4 the Ste20-like protein kinase Hippo (Hpo)5 and the adaptor protein Mob-as-tumor-suppressor (Mats).6 Loss-offunction mutant clones for any of these genes results in a strong tissue overgrowth phenotype characterized by increased proliferation and decreased cell death. Subsequent researches identified the transcriptional co-activator, Yorkie (Yki) was negatively regulated by Hippo pathway. Mechanistically, Yki is phosphorylated and inactivated by Wts. Overexpression of Yki leads to tissue overgrowth, while inactivation of Yki leads to

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tissue atrophy.7 Significantly, all core factors of Hippo signalling have mammalian orthologues that functionally compensate for loss of their counterparts in flies. Hippo, Sav, Wts, Mats and Yki are homologous to mammalian Mst1/2, WW45 (also called Sav1), LATS1/2, Mob1 and YAP/TAZ respectively. One of the most interesting questions was raised whether Hippo-YAP signaling pathway is related to tumourigenesis and whether this pathway become a new paradigm for cancer therapy. Since Hippo-YAP pathway has a critical role in organ enlargement and cell contact inhibition, Hippo-YAP pathway may serve as a vulnerable target for deregulation in cancer. In fact, recent studies have implicated a direct link between dysregulation of the Hippo-YAP pathway and tumorigenesis.8,9 In this review, we will summarize the oncogenic and tumour suppressive role of the Hippo-YAP pathway components. Besides, several upstream regulators and downstream effectors will be revealed here. This review will describe a new role for the Hippo-YAP pathway in tumor development and provide a rich source of potential targets for cancer therapy.

The Hippo Kinase Cascade in Mammals and Mechanisms of YAP/TAZ Inactivation Basically, the Hippo pathway can be divided into three interlinked parts: the upstream regulatory components, the Hippo core kinase components and the downstream transcriptional machinery. The kinases Mst1/2 and Lats1/2 and two adaptors Sav1 and Mob comprised of Hippo core kinase cassette. Mst1/2 was activated by phosphorylation in response to upstream regulators. The scaffold protein Sav1 binds Mst1/2 and Lats1/2, thereby promoting the Mst1/2 phosphorylation of Lats1/2.10 Mob1 can also activate Lats1/2, and this ability is enhanced by the Mst1/2 phosphorylation of Mob1.11,12 The major target of the Hippo core kinase cascade is YAP and TAZ. YAP and TAZ interact primarily with transcriptional factors TEAD and activate expression of target genes such as IGFBP3, CTGF, ITGB2, Gli2 and Axl. However, once phosphorylated by Lats1/2, YAP/TAZ translocated to cytoplasma, sequestered by 14-3-3 and underwent ubiquitindependent degradation13,14 (Fig. 1). The oncogenic role of YAP and TAZ in cancer development Identification of YAZ, TAZ and TEAD. YAP was originally

identified in chicken as an interacting protein of Yes protein tyrosine kinase. The interaction was mediated by the SH3 domain of Yes and Pro-rich region of YAP. Due to its size of 65 kDa, the chicken protein was referred to as YAP65 (Yes-associated protein of 65 kDa).15 YAP is evolutionarily conserved; with the exception of nematodes, YAP orthologs are found in all metazoans and even in premetazoans.16 The human and mouse homologues were identified by using the YAP65 cDNA to probe human and mouse cDNA libraries.17 The human YAP gene, located at 11q13, can be transcribed into at least four isoforms. YAP has a N-terminal TEAD-interacting motif followed by WW domain. Depending on the isoform, YAP has either one or two WW domains.18 The

Hippo-YAP signaling pathway

C-terminus contains a PDZ-binding motif (TWL-COOH) for interaction with the PDZ domain of other proteins, such as ZO2 and NHERF2. The schematic depiction of YAP is shown in Figure 2. TAZ (transcriptional co-activator with PDZbinding motif) was originally identified as a 14–3–3 binding protein 7. In contrast to YAP, TAZ is only found in vertebrates.3 TAZ is homologous to YAP with a 46% amino acid sequence identity (with YAP isoform 3) and displays similar domain organization but only one WW domain.13 Due to the lack of a DNA binding domain, the strong transcriptional activation activity of YAP/TAZ remained puzzling until the interaction between transcription factors and YAP/TAZ was identified to bring YAP/TAZ to gene promoters.19 TEAD family transcription factors are major partners of YAP/TAZ in the regulation of target genes.20 The TEAD family consists of four homologous members named TEAD1–4. The domain architecture of all four TEAD genes is the same. The N-terminus contains a DNA-binding domain named TEA domain. Biochemical and functional studies have revealed that the TEA domain binds DNA elements, such as 5’-GGAATG-3’, and this element is found in the promoter regions of TEAD target genes. For example, CTGF was identified as a direct target gene of the TEAD-YAP complex, and the promoter region of the CTGF gene contains several GGAATG motifs for TEAD-binding.21,22 The C-terminal region of TEADs is involved in the interaction with YAP/TAZ.20 Importantly, S94 in YAP is important for interaction with TEADs, and the mutation of this residue abolished the formation of the YAP-TEAD complex21 (Fig. 2). Epithelial polarity controls Hippo-YAP signaling in development of cancer. Emerging evidence indicates that loss of

cell polarity contributes to tumorigenesis in epithelial tissues. Maintaining apical–basal polarity is important mechanism to control key mediators of signaling pathways involved in regulating proliferation, apoptosis and differentiation. Recent studies have linked both apical and basolateral polarity proteins to the Hippo-YAP pathway.23–27 The apical polarity protein Crumbs is found to regulate YAP/TAZ. The Crumbs complex is a well-studied tight junction-associated component that localizes to the apical domain of polarized epithelial cells. It was found that Crumbs complex including PALS1, LIN7c, AMOT interacts with TAZ/ YAP, relays cell density information by promoting TAZ/YAP phosphorylation, cytoplasmic sequestration and suppressing TGF-b pathway.26 This multifactoral interaction serves to ensure that assembly of the Hippo pathway and efficient phosphorylation of YAP/TAZ is coupled by the assembly of the Crumbs complex. Notably, Low levels of CRB3 are associated with tumor progression,28 and downregulation of YAP/TAZ inhibits EMT properties and cancer metastasis.29 Furthermore, Varelas et al. found EMT-susceptible cell lines have constitutive nuclear TAZ/YAP and defects in Crumbs complex assembly.26 Therefore, increased nuclear YAP/TAZ resulting from the loss of cell polarity may function in a forward feedback loop to further promote this process in tumerigenesis. C 2014 UICC Int. J. Cancer: 00, 00–00 (2014) V

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Figure 1. The schematic diagram of the Hippo-YAP pathway. Arrows indicate direct biochemical interactions, and blunted lines indicate activation or inhibition. The Hippo pathway core machinery consists of Mst1/2, SAV1, Lats1/2 and Mob1, and the downstream effectors are YAP, TAZ and TEADs. When the surface membrane proteins are activated, the kinase cascade is activated: the scaffold protein Sav1 binds Mst1/2 and Lats1/2, thereby promoting the Mst1/2 phosphorylation of Lats1/2. Mob1 can also activate Lats1/2, and this ability is enhanced by the Mst1/2 phosphorylation of Mob 1. The activated Lats1/2 phosphorylates YAP/TAZ and promotes its cytoplasmic sequestration by 14-3-3 and ubiquitin-dependent degradation. Non-phosphorylated YAP/TAZ transfer into the nucleus, interacts with TEADs and then drives target gene expression to promote cell proliferation and suppress apoptosis. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Basolateral polarity complexes can also regulate HippoYAP signaling. Scrib, for example, in breast cancer, acts as a scaffold to assemble Mst1/2, Lats1/2 and Taz.30 Studies in Drosophila showed loss of Scrib results in impaired Hippo-YAP pathway signaling in the epithelial tissues and sensitive to transformation by oncogenic Ras-Raf signalC 2014 UICC Int. J. Cancer: 00, 00–00 (2014) V

ing.31 Strikingly, when all cells in imaginal discs are mutant for scrib, they hyperactivate Yki, which drives growth of the discs into large neoplastic masses.32 Nevertheless, further studies should enhance our understanding of the complexity by which polarity controls Hippo-YAP signaling in cancer.

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Figure 2. Domain architecture of YAP, TAZ and TEAD. The key features of YAP and TAZ are indicated, such as the Pro-rich region, the WW domain and coiled coil region. The C-terminus contains a PZD-binding motif (TWL-COOH) for interaction with the PDZ domain of other proteins, such as ZO2 and NHERF2. The N-terminal region of TEADs contains the highly conserved TEA domain responsible for interaction with DNA elements, such as GGAATG, in the promoter region of target genes (such as CTGF and Axl). The C-terminal region of TEADs is responsible for interaction with YAP/TAZ. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

The activated YAP/TAZ exert oncogenic function via various mechanism. The Hippo pathway was initially defined as a

tumor suppressor pathway in Drosophila. As the major downstream effector of the Hippo pathway, it is not surprising that YAP functions as an oncogene. Elevated YAP/TAZ expression and nuclear localization have been observed in multiple types of human cancers, including liver cancers,33,34 colon cancers,35,36 cervical cancer,37 ovarian cancers,38–40 lung cancers,41,42 esophageal squamous cell carcinoma,43 gastric cancer44 and breast cancer45,46 (Tables 1 and 2). Among the genes induced by YAP/TAZ, CTGF is the most well-known target of the YAP-TEAD complex. Recently, the PDZ-binding motif of YAP was found to be required for CTGF transcription and oncogenic cell transforming activity.47 Several studies have reported that downstream effectors of YAP/TAZ-TEAD complex correlate with tumor development.48 Here, we will demonstrate the crosstalk between YAP/ TAZ and other signaling pathways and oncogenic function of YAP/TAZ via various mechanism. Either the aberrant Wnt or Hippo-YAP signaling pathway has been implicated in numerous cancers. It was showed majority of colorectal cancers have increased Wnt signaling49 and approximate 85% of colorectal tumors also have increased levels of nuclear YAP and increased YAP transcriptional activity.40 The mechanism of cross-regulation between these two pathway have been discovered in recent years. Interestingly, Hippo-YAP pathway can either restrict or activate Wnt pathway depending on cell or tissue type context. For example, in TAZ knockout mice, it was found b-catenin transcriptional activity was elevated in the kidneys. TAZ interacts with Dishevelled to prevent the dissociation of the b-catenin destruction complex, which results in the inhibi-

tion of Wnt response.50 In colorectal cancer cells SW480, which contain high levels of b-catenin and modest overexpression of YAP, the reporter plasmid TOPflash and YAP shRNA was co-trasfected to SW480 cells. It was found knockdown of YAP reduces TOPflash-directed luciferase expression by more than 80% without altering b-catenin overall or intranuclear abundance, strongly supporting overexpression of YAP1 enhances the transcriptional efficacy of b-catenin.51 Another study showed constitutively active form of YAP (S112A) in mice activate IGF pathway in embryonic hearts. GSK3b was inhibited by YAP during the activation of IGF pathway, which results in stabilization and nuclear translocation of b-catenin.52 The Wnt/b-catenin signaling, on the other hand, influences Hippo signaling. The Wnt/bcatenin target gene CD44 interacts with Hippo-YAP upstream regulator NF2 and therefore activates the Hippo pathway.53,54 In addition, an b-catenin/TCF-binding site was found within the first intron of the YAP gene in colon cancer cells by using ChIP-Seq and proved to be an enhancer element that functioned to mediate activation of YAP gene expression.55,56 YAP and TAZ interact with TGF-b-regulated Smads and the cytoplasmically localized TAZ/YAP prevents Smad nuclear accumulation and transcriptional activity.57 As mentioned above, components of polarity complexes, such as Crumbs, have been linked to regulation of TAZ/YAP. In dense cells, TAZ/YAP was sequestered in cytoplasma, in turn, Smads blunts transcriptional responses to TGF-b to prevent epithelial-to-mesenchymal transitions,30,58,59 indicating that translocation of YAP/TAZ from cytoplasma to nuclear may promote EMT through TGF-b pathway activation.

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Component of Hippo pathway merlin

Conserved domain FERRM

YAP/TZA activity #

RASSF1A

SARAH

#

Kibra

WW

#

Mst1/2

SARAH

#

Mob1

Mob1/phocein domain

#

Sav1

WW and SARAH domain

#

Lats1/2

NDR Ser/Thr kinase domain, PPXY motif

#

YAP/TAZ

WW, TEAD-binding domains and PZD-binding motif

TEAD

TEA and YAP/TAZ binding domains

AMOT

PPXY motif; PDZ

#

Scrib

PDZ

#

Crumbs

PPXY motif; PDZ

#

Hippo-YAP pathway was also found to cross talk with Sonic hedgehog (Shh) signaling pathway. In medulloblastoma, YAP was upregulated in human medulloblastomas with aberrant Shh signaling. Shh induces YAP1 expression and nuclear localization in cerebellar granule neuron precursors (CGNPs), which drives CGNP proliferation. Furthermore, YAP is found in cells of the perivascular niche, where proposed tumorrepopulating cells reside, suggesting that YAP is novel effector of Shh and contribute to the development of medulloblastomas.60 Recent studies showed that Hedgehog signaling in Drosophila melanogaster ovarian follicle stem cells (FSCs) induces the activity of Yki. Hedgehog signaling and Yki both regulate the rate of FSC proliferation, essential for FSC maintenance and promote increased FSC longevity and FSC duplication when in excess. Besides the authors found causal connections between Yki and Hh signaling, suggesting Coupling of Hedgehog and Hippo pathways promotes stem cell maintenance by stimulating proliferation.61 YAP-TEAD4 was found to upregulate Jag-1 to activate Notch signaling in HCC cells and mouse hepatocytes. A higher expression of YAP correlated with Jag-1 expression and Notch signaling in tumor samples and with shorter survival times of patients with HCC or colorectal cancer, indicating the utility of YAP and Notch inhibitors as therapeutics for gastrointestinal cancer.62 The roles of miRNA and lncRNA in tumorigenesis have been intensively studied in previous decades. Chaulk et al. found, at low cell densities, abundant nuclear TAZ/YAP is required for efficient pre-miRNA processing. The knockdown of TAZ/YAP in low-density cells or density-mediated sequestration of TAZ/YAP into the cytoplasm results in the defective processing of pre-miRNAs. Interestingly, Let-7is an exception to this rule and accumulates upon the loss of C 2014 UICC Int. J. Cancer: 00, 00–00 (2014) V

Table 2. The summary of YAP expression in different cancer Cancers

Cases (n)

Upregulated (%)

References

Colorectal cancer

168

72.6

36

Lung cancer

40

70

42

breast cancer

69

75.4

46

Gastric cancer

78

69.23

44

Ovarian cancer

68

94

40

Hepatocellular carcinoma

177

62

34

nuclear TAZ/YAP. Nevertheless, density-regulated TAZ/YAP localization defines a critical mechanism by which cells relay cell contact-induced cues to control miRNA biogenesis.63 The lncRNA malat1 was first identified as a critical regulator of the metastatic phenotype of lung cancer cells and later found overexpressed in many malignant diseases. In liver cancer cells, serine/arginine-rich splicing factor 1 (SRSF1) was shown to inhibit YAP activity by preventing its cooccupation with TCF/b-catenin on the Malat1 promoter. The overexpression of YAP impaired the nuclear retention of both SRSF1 and itself via an interaction with Angiomotin (AMOT), leading to the upregulation of Malat1. Thus, stabilizing the interaction of YAP and SRSF1 may be of therapeutic value in the treatment of liver cancer.64 The activated YAP/TAZ confers aggressive, stem cell like and chemoresistant properties on cancer cells. The early onset

of invasive and metastatic progression of tumors complicates treatment. YAP/TAZ and its co-factor play an important role in this process. The laboratory from the Dipartimento di Scienze Biomediche per la Salute showed that TAZ and WW domain-containing oxidoreductase (WWOX) nuclear effectors seemed important for breast cancer progression to establish bone metastatic growth. TAZ may serve as a candidate biomarker that is relevant for early therapeutic intervention against bone metastasis.65,66 Chen et al. identified the leukemia inhibitory factor receptor (LIFR) as a breast cancer metastasis suppressor. Restoring LIFR expression in highly malignant tumor cells triggers a Hippo kinase cascade and inactivates YAP.67 Collective cell migration is an essential feature in cancer progression. Cancer cells often lose contact inhibition to undergo anchorage-independent proliferation and become resistant to apoptosis. Accumulating evidence supports that tumors are highly heterogeneous and sustained by a small subpopulation of stem-like cells named cancer stem cell (CSC), a distinct celltype that is tumorigenic and capable of invasion and metastasis. These cells are considered responsible for tumor resistance to therapies. Therefore, curative therapies must eradicate CSCs. In breast cancer, the activity of TAZ is required to sustain self-renewal and tumor-initiation capacities in breast CSCs. In epithelial cells, TAZ forms a complex with the cell-polarity determinant Scribble. A loss of Scribble

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Table 1. The function and domains of mammalian Hippo pathway components

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or induction of EMT disrupts TAZ with Mst and Lats.30 Recently, a set of patient-derived breast cancer stem cell (BCSC) lines was analyzed. TAZ conferred tumorigenicity and migratory activity to differentiated, nontumorigenic breast cancer cells (dBCCs). A loss of TAZ in BCSC consistently severely impaired metastatic colonization and chemoresistance. Clinically, high expression levels of TAZ were associated with shorter disease-free survival, which identified TAZ as a novel independent negative prognostic factor or a possible therapeutic target for breast cancer.29 An interesting article just reported the functional role of YAP in quiescent colon cancer cells and dormant micrometastases during 5-FU chemotherapy; 5-fluorouracil (5-FU) resistant cells (clonal 5F31 cell) were shown to express a typical CSC-like phenotype and enter into a reversible quiescent G0 state upon reexposure to higher 5-FU concentrations. These quiescent cells downregulated nuclear YAP levels. Furthermore, YAP transcript levels were higher in the liver metastases of patients with colon cancer after 5-FU-based neoadjuvant chemotherapy and positively correlated with colon cancer relapse and shorter patient survival.68 WW domain and PDZ-binding motif regulate nuclear and function of YAP/TAZ. The WW domain of YAP/TAZ specifi-

cally binds to PPxY motif-containing proteins, such as Lats1 and the Amot family.69–71 Through binding to WW domain, the Lats kinase phophorylates YAP/TAZ and then inhibits their activity However, Zhao et al. suggests a positive role of YAP WW domains in stimulating cell proliferation and oncogenic transformation in vitro and to promote tissue overgrowth in vivo. They showed that WW domains are not required for YAP inhibition by Lats and furthermore, the PPXY motif of Lats is also dispensable for YAP inhibition.72 Moreover, Amot was found to restrict the activity of TAZ and YAP through interaction with YAP via WW domain.71 However, another study showed Amot promote nuclear translocation of YAP and act as a transcriptional cofactor of the YAP-TEAD complex to facilitate the proliferation of biliary epithelial cells and cancer development of the liver. Therefore, the function of YAP WW domains may likely vary depending on the cell and tissue context.73 The PDZ-binding domain is critical for YAP/TAZ nuclear translocation. Small molecule inhibitors that mimic the TWL peptide could be of therapeutic value. Bao et al. developed a cell-based method of screening reagents that induce the recruitment of YAP to the cytosol. They found that dobutamine could stimulate YAP translocation from the nucleus to the cytoplasm and inhibit YAP-dependent gene transcription, which suggested that dobutamine may disrupt YAP-PDZ complexes.74 Nevertheless, significant efforts are required to discover Hippo pathway stimulators or Hippo-independent YAP inhibitors. Core Hippo pathway components: Mst1/2 and Lats1/2 Mammalian Ste20-like kinases (Mst1/2). Mst1/2 proteins

have long been the subject of intense study.75 In the Hippo

Hippo-YAP signaling pathway

kinase cascade, Mst1/2 phosphorylates Lats1/2 through their SARAH coiled-coil domains.76 It also phosphorylates Mob1, which enhances Mob1’s ability to bind and activate Lats1/2.12 A single knockout of Mst1 or Mst2 neither leads to the death of mice nor obvious organ or tumor overgrowth; however, a double-knockout of Mst1 and Mst2 showed early embryonic lethality.77,78 Therefore, several studies used conditional knockout methods to investigate the role of Mst1 and Mst2 in tumorigenesis. The results showed the knockout mouse suffered from hepatomegaly and developed liver cancer.77,79 In addition, intestinal dysplasia and colonic adenomas was found.51,80 The mechanism of Mst1/2 regulation in cancer is quite complex. Accumulating evidence suggests that oxidative stress induces the activation of Mst1. A conserved antioxidant protein, Thioredoxin-1 (Trx1), physically associates with the SARAH domain of Mst1 and serves as a molecular switch to turn off the oxidative stress-induced activation of Mst1.81 Chernoff’s group has demonstrated that both Mst1 and Mst2 can interact with peroxiredoxin-1 (Prdx1) under oxidative stress conditions.82 Prdx1 is a conserved enzyme that reduces hydrogen peroxide to water and oxygen. In human hepatocarcinoma HepG2 cells, Mst1 could phosphorylate Prdx1, leading to the inactivation of Prdx1 with subsequent elevated H2O2 levels in cells. The increased level of H2O2 further activated Mst1. This forward feedback systemsuggests a tumor suppressive role of Mst1 to prevent the accumulation of mutations by DNA damage at a higher oxidant condition.82 A diverse array of Mst1/2 substrates accompanies the complex mechanisms of Mst1/2 regulation. In addition to the core Hippo pathway components Sav1, Mob1 and Lats1/2, Mst1/2 also targetsHistone H2B,83 FOXO,84 GA-binding protein (GABP)85 and Lats1/2-related kinases Ndr1/Ndr2,86 indicating its tumor suppressive effect on apoptosis and proliferation. In fact, several studies have demonstratedthe downregulation of Mst1/2 in some cancers, such as breast cancer and gastric cancer, and identified it as a predictive biomarker for cancer prognosis.87,88 Large tumor suppressor (Lats1/2). Lats (large tumor sup-

pressor) is a Ser/Thr kinase that belongs to the Ndr/LATS subfamily of AGC (protein kinase A/PKG/PKC) kinases and was originally isolated in Drosophila as a cell proliferation inhibitor.89,90 Two mammalian homologs of fly Lats, Lats1 and Lats2, were later identified and shown to be functionally conserved as tumor suppressors by regulating cell cycle progression and apoptosis.91,92 As already mentioned, the Lats1/ 2 phosphorylation of YAP/TAZ is a key event in the canonical Hippo pathway. Lats1/2 phosphorylates YAP on Ser 61/109/127/164/ 38114,93 and TAZ on Ser 66/89/117/311,94 which led to the definition of a HXRXXS/T consensus motif for Lats1/2 kinases. Lats1/2 phosphorylates YAP at Ser127 and TAZ at Ser89 to enhance cytoplasmic retention of YAP/TAZ by C 2014 UICC Int. J. Cancer: 00, 00–00 (2014) V

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Upstream regulators of Hippo pathway: RASSF1A, Kibra, Mob1 and NF2 Neurofibromatosis type II NF2/merlin. NF2/Merlin is a

cytoskeletal protein and was identified as a tumor suppressor underlying Neurofibromatosis type II.107 Merlin contains a conserved FERM domain108 and was reported to be an upstream regulator of Mst.109 Recently, Merlin was found to directly bind and recruit the Mst downstream effector kinase Lats to the plasma membrane and promote Lats phosphorylation via the Mst-Sav kinase complex, suggesting an important role of Merlin in activating the Hippo-pathway.110 The role of the Hippo pathway in the regulation of the extracellular matrix (ECM)remains unknown. Recent studies have shown that Merlin interacts with the cytoplasmic tail of CD44.111,112 CD44 is cell surface receptor for hyaluronic acid (HA), an abundant ECM component. The CD44 is frequently upregulated in malignant tumors and was a predictor of poor prognosis in several cancer types.113–115 The treatment of confluent tumor cells with HA leads to the activation of Merlin and subsequent inhibition of cell growth.54 Because Merlin is an upstream regulator of the Hippo-pathway, CD44 may exert some control over tumor growth by regulating the Hippo signaling pathway. Furthermore, CD44 plays an essential role in cancer stem cells (CSC) and has been identified as CSC biomarker in a variety of malignancies.113 Notably, in vivo studies also implicated YAP/TAZ to be crucial for the self-renewal and maintenance of stem cells. The abnormal activation of nuclear YAP/TAZ drives tissue overgrowth and bestows normal cells with cancer stem cell-like properties that may result in tumor initiation.116 Therefore, Merlin is probably connected to CD44 and Hippo the pathway, which contributes to tumorigenesis. Ras association domain family 1 isoform A (RASSF1A) acts as a strong tumor suppressor. The RASSF1A promoter is frequently hypermethylated or RASSF1A is frequently mutated in primary human cancers.117,118 Previous studies showed that RASSF1A suppresses tumors via the activation of Mst. Raf1 was found to bind to Mst2, which leads to the inhibition of the dimerization and autophosphorylation of Mst2. The Fas active receptor induces RASSF1A to compete with RAF1 in binding to Mst2 and disrupts the inhibitory complex between Raf1 and Mst2, which promotes the Mst2 phosphorylation of Lats1. The activated YAP translocates from the cytoplasm to the nucleus and forms a nuclear complex with p73, which induces the transcription of the proapoptotic puma gene.119 Another study revealed that RASSF1A can be phosphorylated by ATM on Ser131 upon DNA damage. The phosphorylated RASSF1A activated both Mst2 and Lats1, leading to the stabilization of p73. Notably, lung and ovarian tumor cell lines that retain RASSF1A expression commonly harbor polymorphisms in the region of Ser131, conveying resistance to DNA-damaging agents. This attribute may explain why the methylation of RASSF1A is observed more

Ras association domain family 1 isoform A (RASSF1A).

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increasing the interaction between yap/TAZ and 14-3-3.94 Furthermore, the phosphorylation of YAP on Ser381 by Lats1/2 leads to subsequent phosphorylation S384 and S387 by CK1delta/epsilon, which recruits the SCF(beta-TRCP) E3 ubiquitin ligase, which catalyzes YAP ubiquitination, ultimately leading to YAP degradation.95 Similar to YAP, phosphorylating Ser311 in TAZ by Lats1/2 confers a prime phosphorylation site for sequential phosphorylation Ser 314 by a CK1 and the cooperative phosphorylation recruits SCF (beta-TRCP) E3 ligase and leads to polyubiquitynation and degradation of TAZ.96 The phosphodegron-mediated degradation and phosphorylation-dependent translocation could coordinately suppress YAP/TAZ oncogenic activity. Mutation of Lats1/2 or other upstream regulators caused disruption of interaction between YAP/TAZ, 14-3-3 and b-TrCP, therefore, stabilized and nuclear localized YAP/TAZ exert oncogenic function. It was reported that mutations of Lats1/2 in cancer affect their expression and kinase activity. Also, the mutants exhibit a decreased activity in inhibiting YAP activity and tissue growth.97 Another study showed the expression and activity of Lats kinase decreased in liver cancer. Furthermore, the increased expression of YAP and its target genes Glypican-3, CTGF and Survivin was observed in liver tumor samples, suggesting a critical role of Lats1/2 induced phosphorylation-dependent translocation and degradation of YAP/TAZ in the development of liver cancer.98 Several studies have shown that Lats can be regulated by AJUBA,99 NEDD4 E3 ligase100 and protease-activated receptors (PARs)101 in cancer cells. The LIM-domain protein AJUBA is a binding partner of Lats. AJUBA was frequently downregulated in malignant mesothelioma (MM) cells. The inactivation of AJUBA showed a more dephosphorylated (activated) level of YAP. Thus, AJUBA may negatively regulate YAP activity via the Lats in MM.99 Recently, Salah et al. demonstrated NEDD4 E3 ubiquitin ligase controls Lats stability. On the protein level, NEDD4 acts as regulator of Lats via interactions between the WW domain-containing and PPxY motif-containing proteins.100 The involvement of the Hippo-pathway in the regulation of the actin cytoskeleton has recently gained much attention. Intriguingly, LATS1 can reportedly bind to actin and inhibit actin polymerization.102 Several studies have shown Lats1 interacts with Zyxin103 and LIMK1,91 two regulators of actin filament assembly. Most recently, Chan et al. and Dai et al. identified angiomotin (Amot) family members as novel substrates of Lats1/2.104,105 The N-terminal regions of Amot proteins contain a conserved HXRXXS consensus site for LATS1/2-mediated phosphorylation. Phosphorylation disrupts AMOT interaction with F-actin and correlates with reduced F-actin stress fibers and focal adhesions. Furthermore, the phosphorylation of AMOT by Lats1/2 inhibits cell migration in vitro and angiogenesis. In addition, the regulation of the actin cytoskeleton and microtubule cytoskeleton should also be considered because Zhao et al. showed that LATS1/2 activity is modulated by anti-microtubule drugs.106

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often in tumors of higher grade and correlates with a decreased responsiveness to DNA-damaging therapy.120 Interestingly, most study groups consider YAP to be an oncogenic protein; however, Matallanas et al.119 reported that nuclear YAP promotes apoptosis via inducing the transcription of the proapoptotic puma gene, indicating that YAP may exert a dual function when activated by different upstream regulators, such as RASSF1A (tumor-suppressive), and binding different partner molecules, such as p73 (tumorsuppressive) and TEAD (oncogenic). Recently, Zhang et al. found that RASSF1A regulates the oncogene Amphiregulin (AREG) via the Hippo pathway in hepatocellular carcinoma (HCC).121 AREG belongs to the EGF family and was reported to be a direct target of YAP. The overexpression of RASSF1A in HCC cell lines significantly inhibited the oncogenic functions of YAP, leading to a significant reduction in AREG secretion via the regulation of the Hippo pathway. In human samples, the expression of YAP and AREG was higher in HCC than in corresponding chronic hepatitis/cirrhosis cells, whereas RASSF1A expression was lower in HCC than in chronic hepatitis or cirrhosis cells.122 As already mentioned, the RASSF1A promoter is commonly hypermethylated in cancers. The restoration of RASSF1A by the DNA methyltransferase inhibitor Decitabinemay serves as a promising drug therapy for HCC treatment. In fact, several studies have shown that decitabine inhibits the growth of HCC cells by affecting various pathways.123–125 The RASSF1A-Hippo pathway may be a target of decitabine. Human Kibrais reportedly involved in various cellular functions via the interaction with several partners.126,127 In breast cancer, Kibra is reportedly phosphorylated by the mitotic kinases Aurora and cyclin-dependent kinase 1 during mitosis.128 The phospho-regulation of Kibra by ERK1/2 and RSK1/2 is required for proliferation and migratory activity in breast cancer cells.129 Recently, Kibra was identified as a regulator of the Hippo pathway. In Drosophila, Kibra functions together with Mer (homolog merlin in mammals) and Ex in a protein complex and regulates the Hippo kinase cascade via direct binding to Hpo (homolog Mst1/2 in mammals) and Sav.130 The loss of Kibra function causes typical loss-offunction phenotypes of Hippo components.127,130 In addition to the regulation of Mst1/2, Kibra directly regulates Lats1/2 via 2 mechanisms in mammals. First, Kibra affects Lats2 kinase activity by modulating the phosphorylation of the hydrophobic motif site (Thr1041). In addition, Kibra stabilizes Lats2 by inhibiting its ubiquitination. Furthermore, YAP overexpression induces Kibra mRNA in both murine and human cells, suggesting the evolutionary conservation of Kibra as a transcriptional target of the Hippo signaling pathway. These data indicate that a feedback loop may exist between Kibra and the Hippo pathway.131 Kibra.

As mentioned earlier, Mst play a role in the regulation of Lats kinase activities. Another highly

Mps one binder 1 (Mob1).

Hippo-YAP signaling pathway

conserved protein Mps One binder1 (MOB1) can also associate with Lats kinases. MOB1 was found essential for spindle pole body duplication and mitotic checkpoint regulation.132 However, in Hippo pathway, MOB1 was found to hyperactivate Lats kinases. MST1/2 were found to specifically phosphorylate Mob1 on Thr12 and Thr35.12 Phospho-MOB1 showed conformational changes and a higher affinity for Lats,133 which in turn phosphorylates and inactivates oncoprotein YAP/TAZ. Mutations in Mob1A have been frequently observed in human cancer cells.6 Furthermore, decreased Mob1A mRNA levels are also found in human colorectal and lung cancer samples.134,135 Moreover, the MOB1 phosphorylation of Thr12 was significantly decreased in human liver cancer samples, displaying a strong correlation with a reduction in the inactivating phosphorylation of YAP.77 An in vivo study showed that a loss of the remaining WT Mob1 allele in Mob1a(D/D) or Mob1a(D/1) mice altered the activities of the downstream Hippo mediators Lats and YAP and results in tumor development.136 Hence, researchers have identified the tumor suppressive role of Mob1 proteins in the Hippopathway and in cancer biology. The ubiquitin-proteasome system is emerging as an important control mechanism of cell biology. Praja 2 belongs to RING E3-ubiquitin ligases widely expressed in cells and several tissues.137,138 Praja2 finely tunes the stability of intracellular substrates and has a significant role in cell signaling pathway.139 For example, Praja2 degrades the regulatory subunits of PKA and therefore controls the strength and duration of PKA signaling in response to cAMP.140 Recently, Mob1 was identified as a critical praja2 substrate in Glioblastomamultiforme (GBM). Praja2 is a cancer-associated gene upregulated in GBM. MOB1 is degraded by praja2 via the ubiquitin-proteasome system, which attenuates the Hippo cascade and enhances the proliferation of glioblastoma cells. Moreover, MOB1 levels are found to be inversely correlated with praja2 levels in glioma malignancy, supporting the role of praja2 in the control of MOB1 stability in vivo.141 These findings link the ubiquitin proteasome system to the Hippo cascade and may provide a new target in the search for therapeutic interventions for cancer. Clinical perspectives

Considering the role of Hippo tumor suppresser pathway, and its effectors, the YAP/TAZ in tumerigensis, it is reasonable to indicate this pathway as potential targets for anticancer therapy. Since YAP/TAZ binds TEAD transcription factors to induce target gene expression, designing compounds that directly dissociate the interaction between YAP/TAZ and TEAD will be the most direct and effective approach to suppress YAP/TAZ-induced oncogenic events. Yi et al. identified verteporfin (VP) abrogates the interaction between YAP and TEAD. Importantly, VP significantly inhibited liver overgrowth induced by YAP overexpression or by inactivation of Nf2 in vivo,142 suggesting a promising inhibitor of YAP/TAZ. Shi et al. uncover clinical importance for VGLL4 in gastric C 2014 UICC Int. J. Cancer: 00, 00–00 (2014) V

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phatase antagonizes the function of Lats to regulate the reversible phosphorylation of TAZ. PP1A dephosphorylates TAZ at Ser89 and Ser311, promotes TAZ nuclear translocation and stabilizes TAZ by disrupting the binding to the SCF E3 ubiquitin ligase. Given the role of TAZ in human cancer, PP1A phosphatase inhibitor may severs as a promising target.145

Conclusion In summary, the past several years have witnessed an explosion of information regarding various aspects of the Hippo signaling cascade in tumorigenesis. Although the molecular aspects of the Hippo pathway and the YAP/TAZ-TEAD effector complex are clearly established, the details of the upstream regulators of the Hippo pathway and how they regulate the Hippo components during the development of the tumor require further investigation. The further understanding of how cells generate, receive and transmit Hippo signals may help us to understand the molecular paradigms for tumorigenesis. In particular, the role of the Hippo signaling pathway in tumorigenesis suggests that Hippo itself can serve as a good target for cancer treatments. Recently, several studies have developed inhibitors to inactivate YAP/TAZ. However, the efficacy in cancer treatment and the low toxicity on normal tissues of the Hippo-YAP pathway inhibition remain to be identified.

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cancer suppression. Similar to YAP, the mammalian Vestigial-like proteins VGLL4 do not contain DNA-binding domain and they also exert their transcriptional regulatory functions through pairing with TEADs via their Tondu (TDU) domain(s). Structural comparison of VGLL4-TEAD4 with YAP-TEAD4 showed that VGLL4 and YAP have partially overlapped binding sites on TEADs, thus acting as an inhibitor of each other in terms of binding TEADs. Most importantly, VGLL4 was found to suppressing YAP induced tumor growth in vitro and in vivo, suggesting disruption of YAP-TEADs interaction by a VGLL4-mimicking peptide may be a promising therapeutic strategy against YAP-driven human cancers.143 PDZ-binding motif in YAP and TAZ may serve druggable targets as well, as cytoplasma retention of YAP/TAZ can prevent them from exerting oncogenic effect in the nuclear. As mentioned earlier, polarity complex proteins are suggested to interact with the Hippo pathway and negatively regulate YAP/TAZ. For example, loss of apicobasal polarity during the EMT process changes the subcellular localization of Scribbled, which inhibits the Hippo pathway, resulting in TAZ activation.144 Thus, stabilization of cell-cell adhersion or restoration of aberrant epithelial cell polarity in cancer may be considered as therapeutic strategy. Serine/threonine protein phosphatase inhibitors is another potential target. Recently, it has been shown that PP1A phos-

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