http://informahealthcare.com/drt ISSN: 1061-186X (print), 1029-2330 (electronic) J Drug Target, Early Online: 1–9 ! 2014 Informa UK Ltd. DOI: 10.3109/1061186X.2014.983522

REVIEW ARTICLE

Hippo signaling pathway in liver and pancreas: the potential drug target for tumor therapy Delin Kong*, Yicheng Zhao*, Tong Men, and Chun-Bo Teng

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College of life science, Northeast Forestry University, Harbin, China

Abstract

Keywords

Cell behaviors, including proliferation, differentiation and apoptosis, are intricately controlled during organ development and tissue regeneration. In the past 9 years, the Hippo signaling pathway has been delineated to play critical roles in organ size control, tissue regeneration and tumorigenesis through regulating cell behaviors. In mammals, the core modules of the Hippo signaling pathway include the MST1/2-LATS1/2 kinase cascade and the transcriptional co-activators YAP/TAZ. The activity of YAP/TAZ is suppressed by cytoplasmic retention due to phosphorylation in the canonical MST1/2-LATS1/2 kinase cascade-dependent manner or the non-canonical MST1/2- and/or LATS1/2-independent manner. Hippo signaling pathway, which can be activated or inactivated by cell polarity, contact inhibition, mechanical stretch and extracellular factors, has been demonstrated to be involved in development and tumorigenesis of liver and pancreas. In addition, we have summarized several small molecules currently available that can target Hippo-YAP pathway for potential treatment of hepatic and pancreatic cancers, providing clues for other YAP initiated cancers therapy as well.

Cancer gene therapy, hepatic targeting, in vitro model, tumor targeting

Introduction Both liver and pancreas are important internal glands in mammals, and each plays an obligatory role in orchestrating the balance between lipid and glucose metabolism [1]. The liver is responsible for storage of glycogen, production of bile for lipid emulsification and secretion of a variety of serum proteins for homeostasis maintenance, whereas the pancreas is a mixed gland with both exocrine and endocrine functions: acinar is able to secrete enzymes including protease and lipase to digest food, while islet is able to secrete hormones including glucagon and insulin to control glucose levels. Liver and pancreas are both derived from the endoderm. During gastrulation, the endoderm germ layer is established and forms a primitive gut tube that is subdivided into foregut, midgut and hindgut regions [2]. Fate mapping studies indicate that the embryonic liver originates from the ventral foregut endoderm. The anterior portion of the hepatic diverticulum gives rise to the liver and intrahepatic biliary tree, while the posterior portion forms the gall bladder and extrahepatic bile ducts. Like the liver, the pancreas develops as outgrowths of the endoderm from the foregut–midgut junction at both dorsal and ventral directions. Finally, the ventral and dorsal pancreatic buds are fused to a whole pancreas. Within the pancreatic

* These authors contributed equally to this work. Address for correspondence: Chun-Bo Teng, E-mail: chunboteng@ nefu.edu.cn

History Received 5 September 2014 Revised 21 October 2014 Accepted 29 October 2014 Published online 3 December 2014

buds, the progenitor cells give rise, through a stepwise process, to endocrine, acinar and duct cells. The processes of hepatic and pancreatic regeneration in adult are well known and acknowledged [3]. In response to drug treatment or mechanical injury, mature hepatic and pancreatic cells can rescue the damage via self-proliferation. In the extensive necrosis, progenitors in organs will be induced to proliferate and differentiate to achieve tissue regeneration. However, regulatory disorders in the processes will lead to abnormal phenotypes including cancers. The Hippo signaling pathway was first defined by genetic mosaic screens in Drosophila, the core components of which included two kinases, the Ste-like kinase HIPPO and the NDR family kinase Warts (WTS), and a transcription factor Yorkie (Yki). In the year of 2005, Huang et al. [4] revealed the signaling cascade: Hippo phosphorylates and activates WTS, and then p-WTS phosphorylates but inhibits Yki via blocking its translocation into nucleus, which leads to sequestration of its target genes including CycE and Diap1. Two years later, Dong et al. [5] found that Hippo pathway was highly conserved from invertebrate to vertebrate and identified the core mammalian counterparts: MST1/2, LATS1/2 and YAP. Inactivation of the Hippo pathway induces YAP-mediated activation of various target genes that functionally result in cellular proliferation and outgrowth of organ size. Moreover, YAP has been implicated as an oncogene in solid tumors, but its exact molecular mechanism in carcinogenesis still remains unclear. It has been reported that several signaling components of the Hippo pathway are implicated as tumor

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suppressors, while the downstream effector YAP, which is negatively regulated by this signaling cascade, is proved to functionally work as an oncogene in hepatic and pancreatic cancers. Ten years of researches demonstrate that the Hippo signaling is an intriguing pathway involved in organ size control, tissue regeneration and tumorigenesis through modulating cell proliferation, differentiation and apoptosis. In this review, we will discuss the latest important findings on the Hippo signaling pathway as well as possible means by which it can be targeted in the hepatic and pancreatic development and tumorigenesis.

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The Hippo signaling pathway components Nowadays, two kinds of Hippo signaling, canonical and noncanonical, have been acknowledged. The canonical Hippo signaling pathway consists of three parts: the upstream signaling components (ligands, receptors and cytoplasmic regulatory factors), the core kinases and their adaptors, and the downstream transcriptional activators. Although the noncanonical Hippo signaling has not been fully elucidated yet, it is clear that the two ways are distinct in the manner of YAP regulation: in the canonical pathway, YAP phosphorylation is dependent on the MST1/2-LATS1/2 kinase cascade, whereas in the non-canonical one, YAP inactivation does not require MST1/2 and/or LATS1/2 [6]. In Drosophila, surface ligand Dachsous (transmembrane cadherin, DS) and its receptor FAT have been identified as a common trigger of the Hippo signaling. DS1/2 and FAT4 are the homologous mammalian proteins; however, whether they can be employed by the Hippo pathway still remains unclear [7,8]. In mammals, plasma membrane linked protein FRMD6 (Expanded in Drosophila), NF2 (MERLIN in Drosophila) and KIBRA form a complex to activate MST1/2 (Mammalian sterile 20-like kinase 1/2, HIPPO in Drosophila). With the help of adaptor proteins SAV1 (Salvador 1, SAV in Drosophila), MOBKL1A and MOBKL1B (Mps One Binder kinase activator-like 1, MATS in Drosophila), MST1/2 can phosphorylate and activate LATS1/2 (Large tumor suppressor 1/2, WTS in Drosophila) [9]. The requirement for MST1/2 to activate LATS1/2 might be cell type dependent. For instance, MST1/2 knockout in mouse livers does not significantly affect LATS1/2 phosphorylation [10]. The transcription cofactors YAP/TAZ (Yes-associated protein/Tafazzin, Yki in Drosophila) are the main downstream targets of LATS1/2. When phosphorylated by LATS1/2, p-YAP/TAZ will be bound by 14-3-3 protein and thus retained in cytoplasm [11]. In humans, the serine 127 (serine 112 in mouse) of YAP and the serine 89 (serine 87 in Mouse) of TAZ are required for interaction with 14-3-3 protein. Some other serine residues have also been shown to affect localization and function of YAP/TAZ [12]. In case of the inactivation of the MST1/2LATS1/2 kinase cascade, dephosphorylation of YAP/TAZ leads to its translocalization in nucleus, interaction with TEAD and MASK [13,14] and activation of target genes, such as Cyclin E, AXL (AXL receptor tyrosine kinase), CTGF (connective tissue growth factor) and Cyr61 (Cysteinerich angiogenic inducer 61), thereby regulation of cell proliferation, differentiation and apoptosis, as shown in Figure 1 [11,15].

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Regulation of the Hippo pathway Activated by cell polarities Both the liver and pancreas originate from the endodermal epithelia, in which cell proliferation, differentiation and apoptosis are closely related to the apical–basal polarity and contact inhibition [16]. During embryogenesis, epithelial cells also exhibit planar cell polarity. The specialized structures are orientated within the plane of the epithelial sheets [17]. In establishing planar cell polarity, the ligand and receptor, DS and FAT [18], but not the subsequent pathway of Hippo signaling [8,19], are involved. Probably, the DS-FAT downstream complex formed by FRMD6, NF2 and KIBRA is relevant to the apical polarity, given that it is located on the inner side of the apical cytoplasmic membrane. It is noteworthy that the apical–basal polarity is mainly co-regulated by the apical polarity complexes PAR3/PAR6/ aPKC (PAR) and Crumbs/PALS1/PATJ (CRB), together with the basolateral polarity complex Scribble/DLG/LGL (SCRIB) [20–22], all of which can activate Hippo signaling to regulate cell proliferation (Figure 2). CRB controls the apical polarity of the epithelium and couples Hippo signaling to sense cell density. When the breast epithelium-derived cell line Eh4 is cultured at a high density, CRB can interact with the angiostatin binding protein Agiomotin (AMOT) in the presence of PATJ. AMOT has been proved to bind to not only YAP/ TAZ on cytomembrane [9], but also to MST2 and LATS2 in cytoplasm to activate the latter, thereby promoting phosphorylation of YAP [23]. Further, p-YAP/TAZ can retain SMADs in cytoplasm so as to block the TGF-b signaling and repress the epithelial-mesenchymal transition (EMT) [24]. Another apical complex PAR can be recruited by CRB from cytoplasm to the apical membrane. It is also found to be localized at tight junctions (TJ). KIBRA, one of the Hippo regulator components, is able to bind to PAR and TJ [25]. Otherwise, the atypical protein kinase aPKC in the PAR complex can phosphorylate MST1/2, thereby regulating the activity of YAP indirectly [26]. The basolateral complex SCRIB, composed of Scribble (SCRIB), Lethal giant larvae (LGL) and Discs large (DLG), has antagonistic effect against the PAR complex, resulting in a suitable membrane proportion between apical and basolateral sides. In human breast cells, SCRIB serves as an adaptor to assemble a protein complex with MST, LATS and TAZ, which are required for MST-dependent activation of LATS and ultimate TAZ phosphorylation [27,28]. In Drosophila, loss of functional LGL in the SCRIB complex alters the localization of HIPPO [29] and leads to an overgrowth phenotype. It is likely that the LGL-HIPPO regulation is aPKC-dependent, as knockdown of aPKC in Hippo-mutant suppressed cell proliferation [30]. Activated by cell junctions It is clear that the Hippo pathway plays an important role in contact inhibition of epithelial cells, which is a wellknown phenomenon in cell proliferation regulation. When cells are in contact, they usually adhere to one another through cell–cell junctions including adherent junctions (AJ)

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Figure 1. Core kinases cascade of Hippo signaling pathway in drosophila and mammals. The core components of Hippo pathway are shown in yellow, while other components are shown in orange. The factors that promote the activity of YAP (Yes-associated protein) and TAZ (transcriptional co-activator with PDZ-binding motif) are shown in green, whereas those that inhibit YAP and TAZ activity are shown in red. Pointed arrowheads indicate activating interactions and blunt lines indicate inhibition.

and TJ. Both junctions are able to activate the Hippo pathway, since some junction components have been identified as upstream regulators of the Hippo pathway. In mammals, cell adhesion mediated by homophilic binding of E-cadherin leads to YAP inactivation through binding to MERLIN and KIBRA [31]. Besides, the homophilic binding of E-cadherin is capable of recruiting a-catenin and b-catenin. Schlegelmilch and colleagues showed that p-YAP, a-catenin and 14-3-3 can form a complex in the cytoplasm. The a-catenin–14-3-3–p-YAP complex blocks YAP from the protein phosphatase PP2A, which will dephosphorylate and activate YAP [32,33]. TJs are mainly located at the top of the basolateral sides of the adjacent epithelial cells. zonula occludens-1 (ZO-1), one of the TJ proteins, can be combined with YAP/TAZ at their WW domains with the help of the adaptor protein AMOT [34]. As described above, AMOT can interact with MST and LATS to facilitate YAP phosphorylation [23]. Interestingly, it has been recently shown that in cytoplasm the p130 splicing isoform of AMOT (AMOTp130) prevents the phosphorylation of YAP by blocking its WW domain accessible to the kinase LATS1, whereas within nucleus, AMOTp130 is associated with the transcriptional complex containing YAP and TEAD, and contributes to the regulation of a subset of YAP target genes, many of which are relevant to tumorigenesis [35]. Moreover, zonula occludens-2 (ZO-2) can be combined with YAP2 via the PDZ-binding motif,

which was then translocated into nucleus to regulate apoptosis [36]. Activated by mechanical forces Experimental data have established that cell behaviors are affected by mechanical force stimuli spreading through extracellular matrix, cell junctions and ion channels. YAP is involved in the response of cells to stresses through activation of RhoA and alteration of the filamentous actin (F-actin) cytoskeleton [6,37]. However, whether the influence of the mechanical strain on YAP is dependent on the canonical Hippo signaling still remains unclear. F-actin plays an important role in cell behaviors including adhesion, migration and proliferation [38]. Interestingly, its level is in positive correlation with YAP activity. In the F-actin-enriched monolayer cells, a high abundance of nucleus-localized YAP was observed, while cells with destructived F-actin displayed more cytoplasm-localized YAP [37,39,40]. Such association is mediated by the AMOT family proteins, such as AMOTp130, in a LATSdependent manner. F-actin can compete with YAP for the interaction with AMOTp130 thus inhibits AMOTp130mediated cytoplasmic retention of YAP, whereas LATS, in synergy with F-actin perturbations, can phosphorylate free AMOTp130 to keep it from binding to F-actin thereby retaining more YAP in the cytoplasm [41].

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Figure 2. Cell polarity and cell connection activate the Hippo pathway. The apical-basal polarity is mainly co-regulated by apical polarity-complex Par3/Par6/aPKC (PAR complexes), Crumbs/PALS1/PATJ (CRB complexes) and basolateral polarity complexes Scribble/DLG/LGL (SCRIB complexes). Cell–cell junctions including adherens junctions (AJ) and tight junctions (TJ) both formations are able to activate the Hippo signal.

In vitro, the kinase activity of LATS on YAP is shown to be modulated by small chemical molecules that interfere with microtubule (MT) assembly, indicating that MTs can regulate the Hippo pathway as well. In mammalian cells, the stability of MTs is dependent on the control of TAO-1 by PAR-1 and TAU, whereas TAO-1 is discovered to have physical interaction with the actin-regulators TESK1 and SPRED1 [42], suggesting that TAO-1 acts at the interface of F-actin and MTs [43]. Studies have shown that TAO-1 can serve as a partner of the Hippo pathway in phosphorylating HIPPO directly [44]. This discovery provides an evidence for intricate links among MTs, F-actin and Hippo signaling [44,45]. A recent study in Drosophila displayed that increasing or decreasing the physical tension of the cytoskeleton affected cell proliferation by alteration of Yki activity, which was modulated by the F-actin-binding myosin. A high concentration of myosin is usually observed at AJ, where it can bind to a-catenin with high affinity under the assistance of Ajuba LIM (JUB), a tension sensor. When cells are confronted with tension force, the binding of myosin to a-catenin, triggered by JUB, will recruit WTS to regulate YAP, thereby controlling cell proliferation [46]. A similar mechanism might act in mammalian cells given the conserved sensing apparatus and pathway molecules. It should be noted that the response of cells to the extracellular matrix hardness is distinct from that to the mechanical forces. In cells cultured on hard matrix, YAP is located in nucleus at a high abundance, while cultured on soft matrix, the YAP nucleus-localization disappears. Such

nucleus-localization shuttle of YAP according to the change of the matrix stiffness was LATS independent. Activation of YAP/TAZ is regulated not only by the abundance of F-actin but also by the tensile force produced by cytoskeleton and tonofibrils, as it is sensitive to the inhibitors of ROCK, MLCK and Myosin [47]. Despite unclear activation circuits of YAP/TAZ in response to the matrix stiffness, with its importance in the organ development and cell behaviors, future researches should focus more on the exploration of the roles of YAP/TAZ playing in this process (Figure 3). Regulation by extracellular factors Extracellular signaling molecules play important roles in cell normal physiology and pathogenesis. At present, a variety of extracellular factors have been identified to regulate cell behaviors by activating YAP/TAZ directly or cross-talking with the Hippo pathway indirectly. As reported in Drosophila, epidermal growth factor receptor (EGFR) is able to facilitate cell proliferation in an Yki-dependent way. EGFR phosphorylates the JUB protein via the RAS-RAF-MAPK kinase cascade, and then p-JUB binds to the kinase WTS and its adaptor SAV to block WTS from phosphorylating Yki, thereby releasing Yki into nucleus [48]. In mammals, there exists a similar EGFR-Hippo pathway, in which activation of EGFR or RAS promotes phosphorylation of the JUB family protein WTIP and enhances the binding of WTIP to MST and LATS to inhibit YAP phosphorylation [48]. Recently, another EGFR downstream pathway that is essential in cell growth,

Hippo signaling pathway in liver and pancreas

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Figure 3. Mechanical stretch activates the Hippo pathway. The extracellular physical tension can influence the F-actin, which can eventually alter YAP activity. GPCR, G protein-coupled receptor.

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the PI3K pathway, has been demonstrated to communicate with the Hippo pathway. When activated by EGF, PI3K phosphorylates its downstream kinase PDK1, and then pPDK1 interacts with the adaptor protein SAV to restrain LATS from phosphorylating YAP, leading to nuclear accumulation of YAP and cell proliferation [49]. In addition to growth factors, several serum factors can also regulate cell proliferation via controlling YAP activity. Lysophosphatidic acid (LPA) and sphingosine 1-phosphophate (S1P), residing in serum, act through Ga12/13-coupled receptors to inhibit the Hippo kinases LATS1/2 and then promote the nucleus localization of YAP/TAZ [50,51]. Moreover, the PI3K-PDK1 pathway mediates the inhibitory effect of LPA on the Hippo pathway as well [49]. Another serum component thrombin binds to its Ga12/13-coupled receptor PAR1 to activate RhoA and F-actin polymeration or tensile fiber formation, and then elevate YAP/TAZ activity [52]. Conversely, glucagon or epinephrine acts through their Gs-coupled receptors to activate LATS1/2 kinase activity and then inhibit YAP nuclear accumulation [50]. Extracellular matrix, including fibronectin, laminin and collagen, binds to the cell surface integrin receptors to activate integrin-linked kinase (ILK) and exerts a great effect on cell survival, proliferation and migration. Recently, ILK is found to be able to suppress the Hippo pathway via inactivation of the myosin phosphatase MYPT1-PP1, which can dephosphorylate and activate Merlin. Inhibition of ILK in breast, prostate and colon tumor cells results in activation of MST1 and LATS1 with concomitant inactivation of YAP/ TAZ and TEAD-mediated transcription [53]. Wnt family proteins, a group of well-known secreted glycoproteins, bind to a Frizzled family receptor to facilitate an accumulation of b-catenin in cytoplasm and prevent its translocation into nucleus to act as a transcriptional coactivator of the TCF/LEF family [54]. There exists an extensive crosstalk between Wnt and Hippo pathways. In the absence of Wnt, YAP/TAZ is essential for recruiting b-TrCP to the destruction complex of b-catenin and its inactivation. In the

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Figure 4. Extracellular factors activate or inhibit Hippo signaling pathway. A variety of extracellular factors have been identified activating YAP/TAZ directly or cross talking with Hippo pathway indirectly. The MAPK signal is shown in grey.

presence of Wnt, YAP/TAZ are unloaded from the destruction complex, allowing their nuclear accumulation and activation of both Wnt and YAP/TAZ dependent biological effects (Figure 4) [55].

Hippo pathway in liver and pancreas Recently, many researchers have revealed the function of Hippo pathway as a key regulator of organ growth, providing novel insight into the mechanisms that control organ size. Although much progress has been made in understanding the molecular mechanisms of signal transduction among the cascade, other aspects such as functions in hepatic and pancreatic developments have not been well explored and remain to be studied. In addition, it is considerable to

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recognize the Hippo signaling as a tumor suppressor pathway, while the YAP/TAZ-TEAD transcription factor complex represents a common target of oncogenic transformation [56]. Overactivation of the downstream effectors YAP and coactivator TAZ results in the development of cancers, which indicates that YAP or TAZ may be an effective anticancer target in tumor therapy [15,57]. Interestingly, in human cancers including medulloblastomas and esophagus tumor, none of them have been attributed to mutations or loss of the core components of the Hippo pathway [58,59]. Given that these cases have been well-summarized recently, we will discuss only hepatic and pancreatic development/cancerrelated reports in the following subsections.

Notch signaling pathway is an important co-effector in YAP-induced cell proliferation (Yimlamai et al., 2014). In HCC, YAP upregulates Jagged1 to trigger the Notch pathway and then promotes the HES1-induced survival and migration, committing high-death rate in HCC patients [75]. YAP is also found to have cross potentiation with b-catenin in hepatoblastoma. Immunohistochemical data displayed about 70% co-localization of YAP and b-catenin in the nuclei of hepatoblastoma, but not hepatocellular carcinoma and intrahepatic bile duct cancer. Additionally, acute postnatal overexpression of YAP1 and b-catenin in murine liver leads to rapid turmorigenesis, and mortality is as high as 100% within 11 weeks [76].

Regulation of liver development and tumorigenesis by the Hippo pathway

Regulation of pancreas development and tumorigenesis by the Hippo pathway

Liver development is intricately regulated by multiple signals, in which Hippo pathway plays a key role in liver size determination [60]. Conditional overexpression of YAP in transgenic mouse led to a significant augment of liver via increasing cell number. Conversely, this phenotype was reverted back to the original level when overexpression was stopped [59,61]. Similarly, loss of the Hippo signaling upstream components including MST1/2, SAV or MERLIN in hepatocytes results in enlarged liver due to excessive proliferation [62]. These data demonstrate that the Hippo signaling regulates liver growth and size in vivo in a MSTdependent manner. Interestingly, one report showed that MST1/2 inactivation in the liver resulted in deregulation of MOB1 and YAP1 but not LATS1/2, indicating that MST1/2 inactivates YAP1 in liver through an intermediary kinase distinct from LATS1/2 [10]. It has been found that liver cancer frequently harbors an amplification of the Yap gene [64]. The constitutive expression of YAP-Ser127A or liver-specific knockout of Sav1 or Mst1/2 for a long-term cause overproliferation and antiapoptotic features of the hepatic cells resembling those in liver cancer [65]. Furthermore, liver-specific knockout of the upstream regulator NF2 (MERLIN) can also lead to carcinogenesis [66,67], which has been already demonstrated to result from increased YAP in nucleus [10]. Dramatically, the out-growth of liver oval progenitors was observed in Sav1, Nf2 and Mst1/2 mutant mice [67–69]. In early views, hepatic tumorigenesis depends on a lack of differentiation of oval progenitors [70]. However, recent reports did show that activation of YAP and disorder of the Hippo pathway was capable of promoting mature hepatic cells dedifferentiation into oval cells, providing an assumption that the mature liver cells might be the origin of ductal carcinoma or other mixed types of cancer [71]. In human hepatoma cell (HCC) samples, YAP is closely related to c-Myc, and its transcription can be promoted by YAP acting on c-Abl. In turn, c-Myc is also capable of enhancing the expression of YAP [72]. In addition, YAP, activated via the non-canonical Hippo pathway, interacts with the cyclic adenosine monophosphate response element-binding protein (CREB) [73] to elevate the expression of the long non-coding RNA Malat1 (metastasis-associated lung adenocarcinoma transcript 1) to facilitate liver cancer [74].

Pancreas consists of two types of glandular tissue: exocrine acini and endocrine islets, they are responsible for producing enzymes to digest foods and hormones to sustain proper blood sugar levels, respectively [77]. During pancreas development, all exocrine and endocrine cells are derived from a same population of multipotent progenitors [78,79]. Recent reports indicate that the Hippo pathway is involved in this process. YAP1 is highly expressed in mouse pancreatic multipotent cells at E12.5 [80], then its expression disappears in endocrine progenitors and is limited in the exocrine progenitors at E16.5, which are able to form ducts and acini. During the second transition in pancreas development, overexpression of YAP1 will repress both endocrine and exocrine differentiations. Unlike in liver, loss of MST1/2 or YAP does not affect the pancreas size, whereas doxycycline-inducible overexpression of YAP in mouse significantly decreases acinar cells, increases ductal cells and causes late pancreatitis-like phenotype [80,81]. At present, it is considered that YAP promotes pancreatic progenitor cell proliferation, but inhibits their differentiation. Our study has indicated that knock-down of YAP in pancreatic progenitors leads to proliferation arrest [82]. Pancreatic ductal adenocarcinoma (PDAC) is one of the cancers with high mobility, as reported with a less than 5% survival rate in 5 years post morbidity. Using the PDAC samples, YAP1 is detected by immunostaining to be abundant in both nucleus and cytoplasm. YAP RNAi in BxPC-3 and PAN-1 cell lines significantly suppresses cell proliferation and anchorage-independent growth in soft agar, suggesting the positive regulation of YAP1 in PDAC formation [83]. Genetic detection indicates that more than 90% of PDAC patients display mutations in proto-oncogene KRAS, which constitutively activates RAF-MEK-ERK signal, resulting in abnormal cell proliferation [84,85]. In Trp53R172H mouse (KrasG12D+/+), pancreas-specific knockout of YAP prevented PDAC effectively, showing that YAP is an important downstream effectors of KRAS in PDAC [86].

Application of small molecules targeting the Hippo pathway in potential therapy of liver and pancreas cancers Lack of effective drugs leads to the high recurrence of the liver and pancreas cancers. Nowadays, doxorubicin is

Hippo signaling pathway in liver and pancreas

DOI: 10.3109/1061186X.2014.983522

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Figure 5. Some small molecules have potential application in therapy of liver and pancreas cancer. Small molecules act on Hippo pathway through inhibiting its upstream kinase or downstream effectors. SRF1, serine/arginine-rich splicing factor 1; VGLL4, vestigial-like protein 4; PPIX, protoporphyrinIX; VP, verteporfin; HP, hematoporphyrin.

commonly used in the treatment of liver cancer. However, the overexpression of YAP leads to more resistant HCCs against the doxorubicin-induced apoptosis [87]. For PDAC, there are no effective therapeutic drugs reported for KRAS so far. Since YAP is a key oncogene for liver cancer and an important cofactor for KRAS-carcinogenic PDAC, the Hippo signaling components are considered as therapeutic targets in liver and pancreas cancer. The C-terminal PDZ-binding domain of YAP is highly conserved, which is responsible for multi-regulatory effects of YAP. Loss of the PDZ-domain inhibits nucleus localization of YAP and the expression of CTGF significantly, as such the YAP-induced carcinogenesis [88]. In Drosophila, scalloped (TEAD homologue) is required in excessive cell proliferation mediated by Yki but not in normal tissue growth, which implies that suppressing the scalloped homologues may be able to selectively inhibit the YAPmediated tumor growth in mammals. It has been confirmed in mouse that dominant deactivation of TEAD in normal liver does not alter cell proliferation, but suppresses tumorigenesis induced by YAP overexpression or NF2/MERLIN inactivation [89]. Administration of verteporfin, an inhibitor of the YAP-TEAD interaction, to the YAP-overexpressed mice can weaken tumorigenesis [89] and does not significantly influence the liver development and homeostasis [66]. In a drug screen assay containing more than 3300 small molecules, besides verteporfin, more YAP-TEAD inhibitors such as porphyrin, hematoporphyrin (HP) and protoporphyrin IX (PPIX) were identified, and all of them are able to inhibit the disorders caused by the inactivation of the Hippo upstream signals [90]. In a recent report, VGLL4 and YAP were found to compete for the direct binding to TEAD. The binding domain of VGLL4 is essential for its regulatory effect, and several polypeptide analogs of VGLL4 are able to repress tumors both in vivo and in vitro [91]. Above all, disrupting the YAP–TEAD interaction by small molecules or polypeptide analogs is feasible in curing the YAP-mediated carcinogenesis.

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In addition, the chemical compound dobutamine has been shown to pharmacologically export YAP from nucleus by activating heterotrimeric G protein [63]. C19, a newly discovered small molecule inhibitor, is able to inhibit EMT and cell proliferation, so as to suppress tumorigenesis and metastasis [91]. C19 can simultaneously inhibit Hippo, Wnt and TGF-b pathways: it induces GSK3b-mediated TAZ degradation, through activation of the Hippo kinases MST/ LATS and the tumor suppressor kinase AMPK, which is an upstream regulator of the degradation complex of YAP [92]. Besides, several other small molecules have been reported to be viable in targeting the upstream components of the Hippo pathway in preliminary experiments, including adrenoceptor inhibitors (dobutamine and epinephrine), forskolin and phosphodiesterase (PDE) inhibitors, cAMP activators, LATS1/2 activators, YAP/TAZ phosphatase and inhibitors [19–21]. These molecules could be further tested as therapeutic candidates for liver, pancreas and other tissue cancers (Figure 5).

Conclusion The Hippo signaling pathway plays a significant role in the physiology and pathology of liver and pancreas. Under normal physiological conditions, the Hippo pathway is controlled precisely to guarantee cell response to the changes of polarity, density, mechanical forces and extracellular signals. Under pathological conditions, disorders of the Hippo pathway can lead to tumorigenesis of liver and pancreas. Small molecules targeting the Hippo signaling components are viable to effectively inhibit experimental tumorigenesis of liver and pancreas. Exploration of the crosstalk between Hippo signaling and other pathways in liver and pancreas will gain further insights into new strategies for modulating cell behaviors in tissue growth, organ regeneration or cancer treatment.

Acknowledgements We apologize for those primary works that are not cited in this review for space constraints. We are thankful to Dr. M.H and L.Y for their editing work on our manuscript and figures.

Declaration of interest The authors report no declarations of interest. D.K, Y.Z and T.M wrote the main manuscript, C.B.T. supervised the project and gave final approval. Research in the laboratory of C.B.T. is supported by grants from the National Natural Science Foundation of China (No. 31272520 and J1210053).

References 1. Li MD, Li CM, Wang Z. The role of circadian clocks in metabolic disease. Yale J Biol Med 2012;85:387–401. 2. Zaret KS, Grompe M. Generation and regeneration of cells of the liver and pancreas. Science 2008;322:1490–4. 3. Dor Y, Stanger BZ. Regeneration in liver and pancreas: time to cut the umbilical cord? Sci STKE 2007;2007:pe66. 4. Huang JB, Wu S, Barrera J, et al. The Hippo signaling pathway coordinately regulates cell proliferation and apoptosis by inactivating Yorkie, the Drosophila homolog of YAP. Cell 2005;122: 421–34. 5. Dong J, Feldmann G, Huang J, et al. Elucidation of a universal sizecontrol mechanism in Drosophila and mammals. Cell 2007a;130: 1120–33.

Journal of Drug Targeting Downloaded from informahealthcare.com by University of Otago on 01/02/15 For personal use only.

8

D. Kong et al.

6. Low BC, Pan CQ, Shivashankar GV, et al. YAP/TAZ as mechanosensors and mechanotransducers in regulating organ size and tumor growth. FEBS Lett 2014;588:2663–70. 7. Cappello S, Gray MJ, Badouel C, et al. Mutations in genes encoding the cadherin receptor-ligand pair DCHS1 and FAT4 disrupt cerebral cortical development. Nat Genet 2013;45:1300–8. 8. Bossuyt W, Chen CL, Chen Q, et al. An evolutionary shift in the regulation of the Hippo pathway between mice and flies. Oncogene 2014;33:1218–28. 9. Zhao B, Li L, Lu Q, et al. Angiomotin is a novel Hippo pathway component that inhibits YAP oncoprotein. Genes Dev 2011;25: 51–63. 10. Zhou D, Conrad C, Xia F, et al. Mst1 and Mst2 maintain hepatocyte quiescence and suppress hepatocellular carcinoma development through inactivation of the Yap1 oncogene. Cancer Cell 2009;16: 425–38. 11. Zhao B, Li L, Lei Q, Guan KL. The Hippo-YAP pathway in organ size control and tumorigenesis: an updated version. Genes Dev 2010;24:862–74. 12. Kanai F, Marignani PA, Sarbassova D, et al. TAZ: a novel transcriptional co-activator regulated by interactions with 14-3-3 and PDZ domain proteins. EMBO J 2000;19:6778–91. 13. Sansores-Garcia L, Atkins M, Moya IM, et al. Mask is required for the activity of the Hippo pathway effector Yki/YAP. Curr Biol 2013;23:229–35. 14. Sidor CM, Brain R, Thompson BJ. Mask proteins are cofactors of Yorkie/YAP in the Hippo pathway. Curr Biol 2013;23:223–8. 15. Zhao B, Lei QY, Guan KL. The Hippo-YAP pathway: new connections between regulation of organ size and cancer. Curr Opin Cell Biol 2008;20:638–46. 16. Ijichi H. Development of basic research of pancreatic cancer: from genetically-engineered mouse models to bedside. Nihon Shokakibyo Gakkai Zasshi 2014;111:1561–9. 17. St Johnston D, Ahringer J. Cell polarity in eggs and epithelia: parallels and diversity. Cell 2010;141:757–74. 18. Thomas C, Strutt D. The roles of the cadherins fat and dachsous in planar polarity specification in drosophila. Dev Dyn 2012;241: 27–39. 19. Irvine KD. Integration of intercellular signaling through the Hippo pathway. Semin Cell Dev Biol 2012;23:812–17. 20. Tanentzapf G, Tepass U. Interactions between the crumbs, lethal giant larvae and bazooka pathways in epithelial polarization. Nat Cell Biol 2013;5:46–52. 21. Johnson K, Wodarz A. A genetic hierarchy controlling cell polarity. Nat Cell Biol 2003;5:12–14. 22. Bilder D, Schober M, Perrimon N. Integrated activity of PDZ protein complexes regulates epithelial polarity. Nat Cell Biol 2013; 5:53–8. 23. Paramasivam M, Sarkeshik A, Yates III JR, et al. Angiomotin family proteins are novel activators of the LATS2 kinase tumor suppressor. Mol Biol Cell 2011;22:3725–33. 24. Varelas X, Samavarchi-Tehrani P, Narimatsu M, et al. The crumbs complex couples cell density sensing to Hippo-dependent control of the TGF-beta-SMAD pathway. Dev Cell 2010;19:831–44. 25. Yoshihama Y, Sasaki K, Horikoshi Y, et al. KIBRA suppresses apical exocytosis through inhibition of aPKC kinase activity in epithelial cells. Curr Biol 2011;21:705–11. 26. Buther K, Plaas C, Barnekow A, Kremerskothen, J. KIBRA is a novel substrate for protein kinase Czeta. Biochem Biophys Res Commun 2004;317:703–7. 27. Cordenonsi M, Zanconato F, Azzolin L, et al. The Hippo transducer TAZ confers cancer stem cell-related traits on breast cancer cells. Cell 2011;147:759–72. 28. Chen D, Sun Y, Wei Y, et al. LIFR is a breast cancer metastasis suppressor upstream of the Hippo-YAP pathway and a prognostic marker. Nat Med 2012;18:1511–17. 29. Grzeschik NA, Parsons LM, Allott ML, et al. Lgl, aPKC, and Crumbs regulate the Salvador/Warts/Hippo pathway through two distinct mechanisms. Curr Biol 2010;20:573–81. 30. Klezovitch O, Fernandez TE, Tapscott SJ, Vasioukhin V. Loss of cell polarity causes severe brain dysplasia in Lgl1 knockout mice. Genes Dev 2004;18:559–71. 31. Moleirinho S, Chang N, Sims AH, et al. KIBRA exhibits MSTindependent functional regulation of the Hippo signaling pathway in mammals. Oncogene 2013;32:1821–30.

J Drug Target, Early Online: 1–9

32. Silvis MR, Kreger BT, Lien WH, et al. Alpha-catenin is a tumor suppressor that controls cell accumulation by regulating the localization and activity of the transcriptional coactivator Yap1. Sci Signal 2011;4:ra33. 33. Schlegelmilch K, Mohseni M, Kirak O, et al. Yap1 acts downstream of alpha-catenin to control epidermal proliferation. Cell 2011;144: 782–95. 34. Yi C, Troutman S, Fera D, et al. A tight junction-associated Merlinangiomotin complex mediates Merlin’s regulation of mitogenic signaling and tumor suppressive functions. Cancer Cell 2011;19: 527–40. 35. Yi C, Shen Z, Stemmer-Rachamimov A, et al. The p130 isoform of angiomotin is required for Yap-mediated hepatic epithelial cell proliferation and tumorigenesis. Sci Signal 2013;6:ra77. 36. Oka T, Remue E, Meerschaert K, et al. Functional complexes between YAP2 and ZO-2 are PDZ domain-dependent, and regulate YAP2 nuclear localization and signalling. Biochem J 2010;432: 461–72. 37. Zhao B, Li L, Wang L, et al. Cell detachment activates the Hippo pathway via cytoskeleton reorganization to induce anoikis. Genes Dev 2012;26:54–68. 38. Mammoto A, Ingber DE. Cytoskeletal control of growth and cell fate switching. Curr Opin Cell Biol 2009;21:864–70. 39. Wada K, Itoga K, Okano T, et al. Hippo pathway regulation by cell morphology and stress fibers. Development 2011;138:3907–14. 40. Aragona M, Panciera T, Manfrin A, et al. A mechanical checkpoint controls multicellular growth through YAP/TAZ regulation by actin-processing factors. Cell 2013;154:1047–59. 41. Mana-capelli S, Paramasivam M, Dutta S, Mccollum D. Angiomotins link F-actin architecture to Hippo pathway signaling. Mol Biol Cell 2014;25:1676–85. 42. King I, Heberlein U. Tao kinases as coordinators of actin and microtubule dynamics in developing neurons. Commun Integr Biol 2011;4:554–6. 43. Xiao ZG, Liu H, Fu JP, et al. Cloning of common carp SOCS-3 gene and its expression during embryogenesis, GH-transgene and viral infection. Fish Shellfish Immunol 2010;28:362–71. 44. Poon CL, Lin JI, Zhang X, Harvey KF. The sterile 20-like kinase Tao-1 controls tissue growth by regulating the Salvador-WartsHippo pathway. Dev Cell 2011;21:896–906. 45. Boggiano JC, Vanderzalm PJ, Fehon RG. Tao-1 phosphorylates Hippo/MST kinases to regulate the Hippo-Salvador-Warts tumor suppressor pathway. Dev Cell 2011;21:888–95. 46. Rauskolb C, Sun S, Sun G, et al. Cytoskeletal tension inhibits Hippo signaling through an Ajuba-Warts complex. Cell 2014;158: 143–56. 47. Dupont S, Morsut L, Aragona M, et al. Role of YAP/TAZ in mechanotransduction. Nature 2011;474:179–83. 48. Reddy BV, Irvine KD. Regulation of Hippo signaling by EGFRMAPK signaling through Ajuba family proteins. Dev Cell 2013;24: 459–71. 49. Fan R, Kim NG, Gumbiner BM. Regulation of Hippo pathway by mitogenic growth factors via phosphoinositide 3-kinase and phosphoinositide-dependent kinase-1. Proc Natl Acad Sci USA 2013;110:2569–74. 50. Yu FX, Zhao B, Panupinthu N, et al. Regulation of the Hippo-YAP pathway by G-protein-coupled receptor signaling. Cell 2012;150: 780–91. 51. Miller E, Yang J, Deran M, et al. Identification of serum-derived sphingosine-1-phosphate as a small molecule regulator of YAP. Chem Biol 2012;19:955–62. 52. Regue L, Mou F, Avruch J. G protein-coupled receptors engage the mammalian Hippo pathway through F-actin: F-Actin, assembled in response to Galpha12/13 induced RhoA-GTP, promotes dephosphorylation and activation of the YAP oncogene. Bioessays 2013; 35:430–5. 53. Serrano I, Mcdonald PC, Lock F, et al. Inactivation of the Hippo tumour suppressor pathway by integrin-linked kinase. Nat Commun 2013;4:2976–88. 54. Macdonald BT, Tamai K, He X. Wnt/beta-catenin signaling: components, mechanisms, and diseases. Dev Cell 2009;17: 9–26. 55. Azzolin L, Panciera T, Soligo S, et al. YAP/TAZ incorporation in the beta-catenin destruction complex orchestrates the Wnt response. Cell 2014;158:157–70.

Hippo signaling pathway in liver and pancreas

Journal of Drug Targeting Downloaded from informahealthcare.com by University of Otago on 01/02/15 For personal use only.

DOI: 10.3109/1061186X.2014.983522

56. Johnson R, Halder G. The two faces of Hippo: targeting the Hippo pathway for regenerative medicine and cancer treatment. Nat Rev Drug Discov 2014;13:63–79. 57. Zhao Y, Khanal P, Savage P, et al. YAP-induced resistance of cancer cells to antitubulin drugs is modulated by a Hippoindependent pathway. Cancer Res 2014;74:4493–503. 58. Harvey KF, Zhang X, Thomas DM. The Hippo pathway and human cancer. Nat Rev Cancer 2013;13:246–57. 59. Gomez M, Gomez V, Hergovich A. The Hippo pathway in disease and therapy: cancer and beyond. Clin Transl Med 2014;3:22–4. 60. Dong J, Feldmann G, Huang J, et al. Elucidation of a universal sizecontrol mechanism in Drosophila and mammals. Cell 2007b;130: 1120–33. 61. Camargo FD, Gokhale S, Johnnidis J.B, et al. YAP1 increases organ size and expands undifferentiated progenitor cells. Curr Biol 2007; 17:2054–60. 62. Avruch J, Zhou D, Fitamant J, Bardeesy N. Mst1/2 signalling to Yap: gatekeeper for liver size and tumour development. Br J Cancer 2011;104:24–32. 63. Bao Y, Nakagawa K, Yang Z, et al. A cell-based assay to screen stimulators of the Hippo pathway reveals the inhibitory effect of dobutamine on the YAP-dependent gene transcription. J Biochem 2011;150:199–208. 64. Zender L, Spector MS, Xue W, et al. Identification and validation of oncogenes in liver cancer using an integrative oncogenomic approach. Cell 2006;125:1253–67. 65. Jie L, Fan W, Weiqi D, et al. The hippo-yes association protein pathway in liver cancer. Gastroenterol Res Pract 2013;2013: 187070. 66. Zhang N, Bai H, David KK, et al. The Merlin/NF2 tumor suppressor functions through the YAP oncoprotein to regulate tissue homeostasis in mammals. Dev Cell 2010;19:27–38. 67. Benhamouche S, Curto M, Saotome I, et al. Nf2/Merlin controls progenitor homeostasis and tumorigenesis in the liver. Genes Dev 2010;24:1718–30. 68. Lee KP, Lee JH, Kim TS, et al. The Hippo-Salvador pathway restrains hepatic oval cell proliferation, liver size, and liver tumorigenesis. Proc Natl Acad Sci USA 2010;107:8248–53. 69. Lu L, Li, Y, Kim SM, et al. Hippo signaling is a potent in vivo growth and tumor suppressor pathway in the mammalian liver. Proc Natl Acad Sci USA 2010;107:1437–42. 70. Alison MR. Liver stem cells: implications for hepatocarcinogenesis. Stem Cell Rev 2005;1:253–60. 71. Yimlamai D, Christodoulou C, Galli GG, et al. Hippo pathway activity influences liver cell fate. Cell 2014;157:1324–38. 72. Xiao W, Wang J, Ou C, et al. Mutual interaction between YAP and c-Myc is critical for carcinogenesis in liver cancer. Biochem Biophys Res Commun 2013;439:167–72. 73. Wang J, Ma L, Weng W, et al. Mutual interaction between YAP and CREB promotes tumorigenesis in liver cancer. Hepatology 2013; 58:1011–20. 74. Wang J, Wang H, Zhang Y, et al. Mutual inhibition between YAP and SRSF1 maintains long non-coding RNA, Malat1-induced tumourigenesis in liver cancer. Cell Signal 2014;26:1048–59. 75. Tschaharganeh DF, Chen X, Latzko P, et al. Yes-associated protein up-regulates Jagged-1 and activates the Notch pathway in

76.

77. 78.

79.

80.

81.

82.

83.

84.

85.

86.

87.

88.

89.

90.

91.

92.

9

human hepatocellular carcinoma. Gastroenterology 2013;144: 1530–42.e12. Tao J, Calvisi DF, Ranganathan S, et al. Activation of beta-catenin and Yap1 in human hepatoblastoma and induction of hepatocarcinogenesis in mice. Gastroenterology 2014;147:690–701. Gittes GK. Developmental biology of the pancreas: a comprehensive review. Dev Biol 2009;326:4–35. Gu G, Dubauskaite J, Melton DA. Direct evidence for the pancreatic lineage: NGN3+ cells are islet progenitors and are distinct from duct progenitors. Development 2002;129:2447–57. Kawaguchi Y, Cooper B, Gannon M, et al. The role of the transcriptional regulator Ptf1a in converting intestinal to pancreatic progenitors. Nat Genet 2002;32:128–34. George NM, Day, CE, Boerner BP, et al. Hippo signaling regulates pancreas development through inactivation of Yap. Mol Cell Biol 2012;32:5116–28. Gao T, Zhou D, Yang C, et al. Hippo signaling regulates differentiation and maintenance in the exocrine pancreas. Gastroenterology 2013;144:1543–53, 1553 e1. Zhang ZW, Men T, Feng RC, et al. miR-375 inhibits proliferation of mouse pancreatic progenitor cells by targeting YAP1. Cell Physiol Biochem 2013;32:1808–17. Diep CH, Zucker KM, Hostetter G, et al. Down-regulation of Yes Associated Protein 1 expression reduces cell proliferation and clonogenicity of pancreatic cancer cells. PLoS One 2012;7:e32783. Almoguera C, Shibata D, Forrester K, et al. Most human carcinomas of the exocrine pancreas contain mutant c-K-ras genes. Cell 1988;53:549–54. Kong B, Qia C, Erkan M, et al. Overview on how oncogenic Kras promotes pancreatic carcinogenesis by inducing low intracellular ROS levels. Front Physiol 2013;4:246–55. Zhang W, Nandakumar N, Shi Y, et al. Downstream of mutant KRAS, the transcription regulator YAP is essential for neoplastic progression to pancreatic ductal adenocarcinoma. Sci Signal 2014; 7:ra42. Huo X, Zhang Q, Liu AM, et al. Overexpression of Yes-associated protein confers doxorubicin resistance in hepatocellullar carcinoma. Oncol Rep 2013;29:840–6. Shimomura T, Miyamura N, Hata S, et al. The PDZ-binding motif of Yes-associated protein is required for its co-activation of TEADmediated CTGF transcription and oncogenic cell transforming activity. Biochem Biophys Res Commun 2014;443:917–23. Liu-Chittenden Y, Huang B, Shim JS, et al. Genetic and pharmacological disruption of the TEAD-YAP complex suppresses the oncogenic activity of YAP. Genes Dev 2012;26:1300–5. Li XJ, Leem SH, Park MH, Kim SM. Regulation of YAP through an Akt-dependent process by 3,30 -diindolylmethane in human colon cancer cells. Int J Oncol 2013;43:1992–8. Jiao S, Wang H, Shi Z, et al. A peptide mimicking VGLL4 function acts as a YAP antagonist therapy against gastric cancer. Cancer Cell 2014;25:166–80. Basu D, Lettan R, Damodaran K, et al. Identification, mechanism of action, and antitumor activity of a small molecule inhibitor of hippo, TGF-beta, and Wnt signaling pathways. Mol Cancer Ther 2014;13:1457–67.