Digestive and Liver Disease 47 (2015) 826–835

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Digestive and Liver Disease

journal homepage: www.elsevier.com/locate/dld

Review Article

Yes-associated protein in the liver: Regulation of hepatic

development, repair, cell fate determination and tumorigenesis

a d a,b,c a,b,c,∗

Quy Nguyen , Robert A. Anders , Gianfranco Alpini , Haibo Bai

a

Research, Central Texas Veterans Health Care System, Temple, TX, United States

b

Digestive Diseases Research Center, BaylorScott&White Healthcare, Temple, TX, United States

c

Department of Internal Medicine and Medical Physiology, Texas A&M Health Science Center, Temple, TX, United States

d

Department of Pathology, Johns Hopkins University, Baltimore, MD, United States

a r t i c l e i n f o a b s t r a c t

Article history: The liver is a vital organ that plays a major role in many bodily functions from protein production and

Received 17 March 2015

blood clotting to cholesterol, glucose and iron metabolism and nutrition storage. Maintenance of liver

Accepted 14 May 2015

homeostasis is critical for these essential bodily functions and disruption of liver homeostasis causes

Available online 22 May 2015

various kinds of liver diseases, some of which have high mortality rate. Recent research advances of

the Hippo signalling pathway have revealed its nuclear effector, Yes-associated protein, as an important

Keywords:

regulator of liver development, repair, cell fate determination and tumorigenesis. Therefore, a precise

Hippo signalling pathway

control of Yes-associated protein activity is critical for the maintenance of liver homeostasis. This review

Liver

YAP is going to summarize the discoveries on how the manipulation of Yes-associated protein activity affects

liver homeostasis and induces liver diseases and the regulatory mechanisms that determine the Yes-

associated protein activity in the liver. Finally, we will discuss the potential of targeting Yes-associated

protein as therapeutic strategies in liver diseases.

Published by Elsevier Ltd on behalf of Editrice Gastroenterologica Italiana S.r.l.

1. Introduction suppressor homolog 1/2), to the plasma membrane. Membrane

recruitment, in turn, promotes LATS1/2 phosphorylation by the

YAP (Yes-associated protein) was discovered 20 years ago in Ste-20 family protein kinase, MST1/2 (Mammalian STE20-Like Pro-

an effort to identify interacting proteins to Src family kinase tein Kinase 1/2), together with the adaptor protein SAV1 (salvador

Yes [1]. It burgeoned into a hot research objective after its homolog 1) [8]. In turn, LATS1/2, in a complex with small regulator

Drosophila ortholog, Yki, was revealed as the effector of Hippo protein MOB1 (Mps One Binder Homolog A), phosphorylates YAP.

signalling pathway in 2005 [2]. The Hippo signalling pathway was Phosphorylated YAP is sequestered in cytoplasm via interaction

recently discovered as a mechanism that controls organ size in to 14-3-3 [9]. Cytoplasmic retention of YAP renders its interac-

Drosophila [2–4]. For a detailed review about the discovery of the tion with TEA domain family transcription factors TEADs (TEA

Hippo signalling pathway in Drosophila, see reviews from Duojia domain family members) [10] to another transcription cofactor,

Pan [5,6]. This review will focus on the Hippo signalling path- Vgl4 (Vestigial like protein 4), thus stops YAP’s transcriptional out-

way in mammals. Components of the Hippo signalling pathway puts [9,11,12].

are highly conserved throughout evolution. Accordingly, loss-of- Increasing Yki activity by overexpressing Yki as well as by

function mutants flies can be rescued with their corresponding ablating Yki’s upstream negative regulators, leads to a similar over-

human orthologs [3,4,7]. The core Hippo signalling pathway is growth phenotype in Drosophila due to increased proliferation and

a kinase cascade (Fig. 1). The apical membrane-associated FERM inhibited apoptosis. These observations raised an exciting ques-

domain protein NF2 (neurofibromin 2), directly binds and recruits tion whether the Hippo pathway might play an analogous role

the Nuclear Dbf2-related family kinase, LATS1/2 (large tumour in mammals. To answer this question, mouse liver was chosen

as the model organ, since liver size is known to be tightly regu-

lated, constituting about 5% of the body weight [13]. Liver is a vital

organ that plays a major role in metabolism and has a number of

∗

Corresponding author at: Olin E. Teague Medical Center, 1901 South 1st

functions in the body, including glycogen storage, decomposition

Street, Bldg. 205, 1R58, Temple, TX 76504, United States. Tel.: +1 254 743 2502;

of red blood cells, plasma protein synthesis, hormone production,

fax: +1 254 743 0378.

E-mail address: [email protected] (H. Bai). and detoxification. It contains two kinds of epithelia, hepatocyte

http://dx.doi.org/10.1016/j.dld.2015.05.011

1590-8658/Published by Elsevier Ltd on behalf of Editrice Gastroenterologica Italiana S.r.l.

Q. Nguyen et al. / Digestive and Liver Disease 47 (2015) 826–835 827

Fig. 1. The Hippo signalling pathway in mammals. The Hippo signalling pathway negatively regulates Yes-associated protein activity. When the Hippo signalling is inactive,

Yes-associated protein accumulates in the nucleus and functions as transcriptional co-activator of the TEA domain family transcription factors. When the Hippo signalling

is active, the apical membrane-associated neurofibromin 2, directly binds and recruits the Nuclear Dbf2-related family kinase, large tumour suppressor homolog 1/2, to

the plasma membrane. Membrane recruitment, in turn, promotes large tumour suppressor homolog 1/2 phosphorylation by mammalian STE20-Like Protein Kinase 1/2,

together with the adaptor protein salvador homolog 1. In turn, large tumour suppressor homolog 1/2, in a complex with small regulator protein Mps One Binder Homolog A,

phosphorylates Yes-associated protein. Phosphorylated Yes-associated protein is sequestered in the cytoplasm via interaction to 14-3-3 proteins. Cytoplasmic retention of

Yes-associated protein renders its interaction with transcription partner TEA domain family transcription factors to another transcription cofactor, Vestigial like 4, thus stops

Yes-associated protein’s transcriptional outputs. YAP, Yes-associated protein; NF2, neurofibromin 2; LATS1/2, large tumour suppressor homolog 1/2; MST1, Mammalian

STE20-Like Protein Kinase 1/2; SAV1, salvador homolog 1; MOB1, Mps One Binder Homolog A; TEAD, TEA domain family transcription factors; Vgl4, Vestigial like 4.

and cholangiocyte, both differentiated from hepatoblast. Hepato- mature bile ducts during the first 2 weeks of life while the

cytes make up 70–85% of the liver’s cytoplasmic mass and carry out remaining unincorporated ductal plates regress [18,19]. The duc-

most functions of the liver. A specific feature of hepatocyte is that tal plates form normally but the tubulogenesis fails in Alb-Cre;

f/f

as a terminally differentiated cell, it retains ability to proliferate Yap liver, which results in bile duct paucity 2 weeks after

to replenish damaged liver. In normal conditions, hepatocytes are birth [15]. However, whether YAP affects ductal commitment

f/f

largely quiescent dividing approximately once per year. Transgenic is not clear as Yap deletion in Alb-Cre; Yap liver is not effi-

f/f

overexpression of YAP in hepatocyte immediately induces quies- cient until E18. Bile duct paucity in Alb-Cre; Yap mice induces

cent hepatocytes into cell cycle and dramatically expands the liver compensatory ductular reaction, accompanied by steatosis and

size to as much as 5 folds of the regular mouse liver weight [9]. This fibrosis in the adult liver. The hepatocytes develop normally

study provided the first direct evidence implicating the Hippo path- in these mice but with a higher turnover rate in the adult

way in mammalian organ size control. And it evoked an interesting liver. This high hepatocyte turnover may be due to reduced

question – whether YAP is normally required for liver homeosta- hepatocyte survival ability and bile duct paucity combined

sis, which might be answered using liver-specific Yap knockout together.

mice. The important role of YAP in bile duct development was further

supported by the phenotype of liver specific Nf2 knockout mice

f/f

2. Physiological role of YAP in the liver (Alb-Cre; Nf2 ) [15]. NF2 is one of the upstream tumour suppressors

of the Hippo signalling pathway and negatively regulates Yki in

2.1. Role of YAP in liver development Drosophila [20]. Consistently, liver specific Nf2 deficiency results in

a reverse phenotype to Yap deficiency on bile duct development.

Complete knockout of Yap results in early embryonic letha- As a portion of cholangiocytes on the ductal plates form primitive

lity [14]. To analyze YAP’s function at later stages of development ducts in WT livers, and no cholangiocyte on the ductal plates forms

f/f f/f

and in adult tissue, conditional Yap knockout mice were generated primitive ducts in Alb-Cre; Yap livers, in Alb-Cre; Nf2 livers all the

f/f

using loxP technique (Yap ) [15]. By crossing with Alb-Cre mice, cholangiocytes on the ductal plates become bile ducts [15]. This

f/f f/f

Yap deletion (Alb-Cre; Yap ) in both cholangiocytes and hepato- bile ducts over-tubulogenesis phenotype of Alb-Cre; Nf2 liver is

cytes starts from E13.5 and achieves efficient deletion at perinatal due to increased YAP activity and can be suppressed by partial Yap

stage (E18 and P1) [16,17], which is a critical time point during deletion [15].

intrahepatic bile duct development [18,19]. Intrahepatic bile ducts The underlying mechanism of YAP-regulated bile duct tubulo-

start developing as early as around E13.5, hepatic progenitor cells genesis remains to be identified. Several studies have suggested

adjacent to the portal veins undergo ductal commitment, forming that the Notch signalling is one of the downstream effectors of YAP

a structure known as the ductal plate. Around birth (E18 to P1), [21–24]. Notch signalling deficiency results in a same biliary phe-

tubulogenesis occurs at discrete points along the ductal plates, notype as Yap deficiency [16,25]. However, direct genetic studies

giving rise to primitive ductal structures. These primitive ductal are necessary to put Notch signalling downstream of YAP in bile

structures will incorporate into portal mesenchyme to become duct development.

828 Q. Nguyen et al. / Digestive and Liver Disease 47 (2015) 826–835

2.2. Role of YAP in repairing damaged liver human liver diseases, especially liver cancer. In this section, we will

summarize the studies revealed the links between YAP activity and

Although YAP is critical for developing liver, it is largely dis- liver diseases.

pensable in quiescent adult liver. Yap deletion in adult mouse liver

f/f

by injecting Poly dI-dC to 8 weeks old Mx1-Cre; Yap mice did not 3.1. YAP and liver cancer

affect liver histology [26]. Similarly, Nf2 deletion in adult mouse

liver with adenovirus delivered Cre recombinase (Ade-Cre) or Mx1- Role of YAP in liver cancer demonstrated by animal models. The

Cre only leads to very mild periporal hyperplasia compared to first evidence of YAP in driving liver cancer formation came from a

dramatic and widespread biliary hyperplasia in Alb-Cre mediated mouse model of liver cancer initiated from progenitor cells har-

Nf2 deletion [27]. However, YAP is required to ensure sufficient bouring p53 loss and c-myc overexpression [40]. Genome wide

regenerative proliferation in response to liver injury. Cholestatic analysis of tumours in this mouse model revealed a recurrent

injury induced by ligation of the mouse common bile duct (BDL) amplification at mouse chromosome 9qA1 that contains Yap and

triggers repair responses including cholangiocyte and hepatocyte cIAP1 (cellular inhibitor of apoptosis protein-1). Interestingly, the

proliferation, both of which peak at day 5 post-BDL and calm down syntenic region of human chromosome 11q22 is amplified in a vari-

after 14 days post-BDL [28]. YAP protein levels first increase then ety of human cancers [41–44]. Functional analysis indicated that

return to baseline correspondingly with cholangiocyte and hepa- both cIAP1 and Yap contribute to tumorigenesis and are required to

tocyte proliferation levels in isolated cholangiocytes, hepatocytes sustain rapid growth of this amplicon-containing tumour.

f/f

and whole liver [26]. Yap deletion with Mx1-Cre; Yap significantly The direct evidences of YAP in driving liver cancer came

decreases cholangiocyte proliferation, delays hepatocyte prolifer- from genetic modified mouse models with manipulations of

ation and enhances parenchymal damage after BDL [26]. It has also Hippo signalling pathway components (phenotypes summarized

been shown that YAP protein levels increase during the regener- in Tables 1 and 2). Transgenic overexpression of YAP in hepatocytes

ation response in both mouse and rat partial hepatectomy model (ApoE-rtTA; TRE Yap) leads to liver enlargement and hepatocellular

[29–31]. However, the role of YAP in the repair process after partial carcinoma (HCC) formation [9]. Similarly, increasing YAP activity by

hepatectomy remains to be evaluated. liver-specific knockout of the Hippo signalling pathway upstream

tumour suppressors also leads to HCC and/or cholangiocarcinoma

f/f

(CC) formation. Two studies of Alb-Cre; Nf2 mice revealed liver-

2.3. Role of YAP in determining liver cell fate

specific deletion of Nf2 induces HCC and biliary tumour although

with some differences in mouse survival, time course of tumour

Hepatocytes and cholangiocytes, both differentiated from hepa-

progression and the malignancy of biliary tumour, which is prob-

toblasts and/or putative liver progenitors, are the only two epithelia

ably due to different mouse genetic background [15,27]. Germline

in the liver. The relationship among hepatocytes, cholangiocytes −/−

mutants of mammalian Hippo kinase orthologs Mst1 (Mst1 ) or

and putative liver progenitors, as well as the factors that determine −/−

Mst2 (Mst2 ) are viable and fertile, without liver abnormality

their cell fate specification are long standing questions in the liver

[45], which may be due to functional redundancy of MST1 and

research field [32,33]. Recently, Yimlamai et al. provided definitive

MST2 as they share 76% identity in amino acid sequence [46].

evidence showing different YAP levels/activity could determine

However, liver specific deletion of Mst2 on Mst1 null background,

hepatic cell fates and mature hepatocytes retain the capacity of −/− f/f −/− f/f

Alb-Cre; Mst1 ;Mst2 and Ade-Cre;Mst1 ;Mst2 , develop HCC

dedifferentiation [24]. In adult mouse liver, YAP is expressed at

at 3 months of age or 10 weeks after Ade-Cre administration, respec-

high levels in bile ducts, with many cholangiocytes displaying −/− f/−

tively [47]. It was also reported that CAGGCre-ER; Mst1 ;Mst2

robust nuclear YAP expression. Conversely, YAP is expressed at

mice develop both HCC and CC by 6 months of age after postnatal

low levels in hepatocytes, where the distribution is throughout

Tamoxifen administration [48]. Similarly but less aggressively, liver

the cell [15]. Further addition of active YAP protein in cholangio-

specific knockout of Sav1 also resulted in mixed types of tumours

cytes leads to hyperplasia, but not to a ductal/progenitor-cell-like

around one year of age [49,50]. More importantly, heterozygous

appearance. The interesting observation came from overexpressing

deletion of Yap, which results in no phenotypical manifestation by

active form of YAP in mature hepatocytes, which dedifferentiates f/f

itself, significantly suppressed tumour formation in Alb-Cre; Nf2

the mature hepatocytes into ductal/progenitor cells. More impor- −/− f/f

or Alb-Cre;Mst1 ;Mst2 mice [15,34] (Table 2). These findings

tantly, this hepatocyte-dedifferentiated ductal/progenitor cell can

provided strong evidences that YAP is the driver of tumour for-

redifferentiate into hepatocyte after YAP activity levels drop [24]. In

mation in Nf2 or Mst1/2-deficient livers.

agreement, Fitamant et al. also found that increasing endogenous

Further evidences of YAP’s involvement in liver cancer came

YAP activity due to Mst1/2 deficiency results in conversion of hepa-

from the hydrodynamic injection model and the carcinogen

tocytes in periportal area to atypical ductal cells and override the

induced mouse and rat HCC models. YAP and ␤-catenin shows

zonation programme in pericentral hepatocytes [34]. Their studies

concurrent nuclear localization in human hepatoblastoma (HB)

evoked another interesting question: whether reducing YAP lev-

specimens and interacts with each other in HB cell line. Combined

els in cholangiocytes will transdifferentiate them into hepatocytes,

hydrodynamic delivery of constitutively active Yap (YapS127A) and

remains to be answered.

constitutively active ␤-catenin (N90/␤-catenin) to mouse liver

YAP also plays important role in other gastrointestinal tissues.

leads to HCC formation as early as 3 weeks after injection [51].

For example, YAP levels are critical for intestinal regeneration, pro-

Increased YAP protein levels were found in HCCs developed in mice

genitor cells maintenance and differentiation [35,36]. Increasing

given diethylnitrosamine (DENA) followed by repeated treatments

YAP levels in pancreas affects pancreatic architecture [37,38]. A

with 1,4-Bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) [52].

detailed review about the Hippo signalling in other gastrointestinal

Nuclear YAP accumulation was observed in preneoplastic hepa-

tissues can be found in reviews of Yu et al. [39]

tocytes generated in rats 4 weeks after DENA treatment, which

suggested YAP activation is an early event in liver tumorigenesis

3. YAP activity and liver diseases [53]. Taken together, all these studies provided solid evidences that

Yap is a proto-oncogene in the context of liver.

Consistent with the critical role of YAP in liver physiology, dys- Role of YAP in liver cancer demonstrated by human studies. In

regulation of YAP activity induces liver diseases in mouse models. humans, elevated YAP activity in different types of primary liver

More importantly, elevated YAP levels were constantly found in cancers was observed by many studies (summarized in Table 3).

Q. Nguyen et al. / Digestive and Liver Disease 47 (2015) 826–835 829

Table 1

Liver phenotypes of genetically modified mice with manipulations of single Hippo pathway components.

Gene Genotype Phenotype Reference

1 f/f

Amot Alb-Cre;Amot Normal [111]

f/f 13

Alb-Cre;Amot + DDC Reduced levels of ductular reactions [111]

2 −/−

Fat Fat4 Death at birth [98] f/f

Alb-Cre;Fat4 Normal [99]

3 −/−

RASSF Rassf1a Increased tumour susceptibility [100,101] −/−

Rassf5a Normal [102]

4 −/−

Nf2 Nf2 Embryonically lethal between E6.5 and E7.0 [103]

+/− 16

Nf2 Develop a variety of tumours, 9% HCC [104]

f/f

AAV-Cre; Nf2 Hepatocyte dedifferentiation [24]

f/f 17

Alb-Cre;Nf2 Over-developed bile ducts, hepatomegaly, bile duct harmatoma or CC , HCC [15,27]

f/f

Ade-Cre;Nf2 Mild periportal hyperplasia [27]

f/f 14

Ade-Cre;Nf2 + PH HCC and CC [27]

f/f 18

Mx1-Cre;Nf2 Mild OC proliferation [27]

f/f

Mx1-Cre;Nf2 +PH HCC and CC [27]

5 −/− −/−

Mst1/2 Mst1 or Mst2 Normal [45,47,48,50]

−/− +/−

Mst1 ;Mst2 Hepatomegaly, HCC by 7 months [47,48]

+/− −/−

Mst1 ;Mst2 Low incidence HCC by 15 months [47,48]

−/− −/−

Mst1 ;Mst2 Embryonically lethal at E8.5 [47,48,50,105]

f/f

Alb-Cre; Mst1/2 Hepatomegaly, HCC at 5–6 months [48,50]

−/− f/f

Alb-Cre; Mst1 ;Mst2 Hepatomegaly, HCC by 3 months [47]

−/− f/−

Alb-Cre; Mst1 ;Mst2 Hepatomegaly, HCC at 1 month [48]

−/− f/f −/− f/−

Ade-Cre; Mst1 ;Mst2 or Ade-Cre; Mst1 ;Mst2 Hepatomegaly, lethal HCC 10 weeks after Ade-Cre injection [47]

−/− f/−

CAGGCre-ER; Mst1 ;Mst2 Hepatomegaly, HCC and CC at 6 months [48]

6 −/−

Sav1 Sav1 Embryonically lethal around E17.5 to E18.5 [106]

+/−

Sav1 74% mice developed liver tumours [49]

f/f

Alb-Cre; Sav1 Hepatomegaly, HCC and CC by 13–14 months [49,50]

f/f

MMTV-Cre; Sav1 Liver tumours by 14 months [50]

f/f

Caggs-creER(T2); Sav1 Liver tumours by 14 months [50]

7 −/−

Lats1/2 Lats1 Neonatal death, infertility and growth retardation [107]

−/−

Lats2 Embryonically lethal on or before E12.5 [108]

8 −/− tr/tr

Mob1 Mob1a or Mob1b Normal [109]

−/− tr/tr

Mob1a ;Mob1b Embryonically lethal [109]

−/− tr/+

Mob1a ;Mob1b 50% liver tumours [109]

9

Yap ApoE-rtTA; TRE Yap Hepatomegaly, HCC [9]

LAP1-tTA;TetO-YapS127A Hepatomegaly [97]

Ck19-CreERT;TetO-Yap S127A Ductal hyperplasia [24]

AAV-Cre; TetO-YapS127A Hepatomegaly, Hepatocyte dedifferentiation [24]

−/−

Yap Embryonically lethal at E8.5 [14]

f/f

Alb-Cre;Yap Bile duct paucity [15] f/f

Mx1-Cre;Yap Normal [26]

f/f 15

Mx1-Cre;Yap + BDL More severe liver injury [26]

10 −/−

Taz Taz Minor skeletal defects, renal cysts [110]

11

TEAD Alb-rtTA;TRE TEAD2-DN Normal [11]

12

Vgl4 ApoE-rtTA; TRE Vgl4 Normal [12]

1 2 3 4 5

Amot, angiomotin; Fat, FAT atypical cadherin; RASSF, Ras Association (RalGDS/AF-6) Domain Family; Nf2, neurofibromin 2; Mst1/2, Mammalian STE20-Like Protein

6 7 8 9 10

Kinase 1/2; Sav1, salvador homolog 1; Lats1/2; large tumour suppressor homolog 1/2; Mob1, Mps One Binder Homolog A; Yap, Yes-associated protein; Taz, tafazzin;

11 12 13 14 15 16

TEAD, TEA domain family members; Vgl4, Vestigial like 4; DDC, 3,5-diethoxycarbonyl-1,4-dihydrocollidine; PH, partial hepatectomy; BDL, bile duct ligation; HCC,

17 18

hepatocellular carcinoma; CC, cholangiocarcinoma; OC, oval cell.

Consistently, most research groups observed more than 50% of Hippo signalling with reduced expression of MST1 [47], LATS [57]

human liver cancer tumours show elevated nuclear YAP expression and RASSF1A [59]. Several genes were proposed as YAP down-

[9,51,54–60]. Multiple mechanisms were attributed to the ele- stream targets in driving liver cancer development, including Axl

vated nuclear YAP expression, including gene amplification [40,56], (AXL receptor tyrosine kinase) [61], Survivin [56] and Amphiregulin

increased Yap mRNA levels [51,55,56] and the dysregulation of the [59]. Combining human and mouse data together, YAP is a very

Table 2

Liver phenotypes of genetically modified mice with manipulations of combined Hippo pathway components.

Gene Genotype Phenotype Reference

1 2 f/f f/f

Nf2 &Amot Alb-Cre;Nf2 ;Amot Suppressed Nf2 phenotype [111]

3 f/f f/f

Nf2&Fat4 Alb-Cre;Nf2 ; Fat4 Nf2 phenotype is not suppressed [99]

4 f/f f/f

Nf2&Yap Alb-Cre;Nf2 ; Yap Suppressed Nf2 phenotype [15]

f/f f/+

Alb-Cre;Nf2 ; Yap Suppressed Nf2 phenotype [15]

5 f/f

Nf2&TEAD Alb-Cre;Nf2 ;Alb-rtTA;TRE TEAD2-DN Suppressed Nf2 phenotype [11]

6 f/f

Nf2&Vgl4 Alb-Cre;Nf2 ;ROSA26-loxP-STOP-loxP-rtTA;TREVgl4 Completely suppressed Nf2 phenotype [12]

7 −/− f/f f/+

Mst1/2 &Yap Alb-Cre; Mst1 ;Mst2 ;Yap Suppressed Mst1/2 phenotype [34]

−/− f/f f/f

Alb-Cre; Mst1 ;Mst2 ;Yap Suppressed Mst1/2 phenotype

Yap&TEAD ApoE-rtTA;TRE Yap; TRE TEAD2-DN Suppressed YAP overexpression phenotype [11]

Yap&Vgl4 ApoE-rtTA;TRE Yap; TRE Vgl4 Suppressed YAP overexpression phenotype [12]

8 f/f

Yap&Rbpj AAV-Cre;TetO YapS127A; Rpbj Suppressed YAP overexpression phenotype [24]

1 2 3 4 5 6 7

Nf2, neurofibromin 2; Amot, angiomotin; Fat4, FAT atypical cadherin 4; Yap, Yes-associated protein; TEAD, TEA domain family members; Vgl4, Vestigial like 4; Mst1/2,

8

Mammalian STE20-Like Protein Kinase 1/2; Rbpj, recombination signal binding protein for immunoglobulin kappa J region.

830 Q. Nguyen et al. / Digestive and Liver Disease 47 (2015) 826–835

Table 3

Summary of Yes-associated protein expression survey studies in human liver cancer samples.

8

Cancer type Patients origin No. control Nuclear YAP No. tumour Nuclear YAP Paired con- Reference samples expression samples expression trol/tumour?

1 6 7 9

HCC USA N/A N/A 20 65% N [9]

HCC USA 42 5% 115 54% N [54]

10

HCC Hong Kong 177 9.0% 177 62.1% Y [55]

HCC USA 38 25% 70 85% N [57]

HCC USA 83 28% 87 71% Y [56]

HCC China 20 5% 40 42.5% N [58]

HCC China 10 Very low 70 51.4% N [60]

HCC Korea 15 20% 22 73% N [59]

HCC Korea N/A N/A 100 31% N [59]

HCC Europe N/A N/A 103 65% N [51]

2

CC USA 8 N/A 16 98% N [57]

3

ICC USA 5 None 10 90% N [56]

ICC Europe N/A N/A 62 98% N [51]

4

cHCC-CC Korea N/A N/A 58 67.2% N [59]

5

HB USA 5 N/A 22 73% Y [57]

HB Europe/USA N/A N/A 94 85% N [51]

1 2 3 4 5

HCC, hepatocellular carcinoma; CC, cholangiocarcinoma; ICC, intrahepatic cholangiocarcinoma; HCC-CC, combined hepatocellular-cholangiocarcinoma; HB, hepato-

6 7 8 9 10

blastoma; USA, United States of America; N/A, not available; YAP, Yes-associated protein; N, no; Y, yes.

promising therapeutic target for liver cancer. Further studies need Chip-qPCR assay indicated that NANOG binds to two NANOG con-

to focus on how to efficiently and specifically inhibit YAP activity. sensus sites in Yap promoter (−1294 and −323 nt). Two studies

using HCC cell lines suggested CREB regulates Yap mRNA transcrip-

3.2. YAP and other liver diseases tion but mapped CREB binding sites to different regions of Yap

promoter [58,65]. Zhang et al. mapped the CREB binding region

−

Besides liver cancer, dysregulation of YAP activity was con- on Yap promoter to be 232/+115, where is also occupied by HBx

stantly observed in biliary diseases, which is in consistence with (Hepatitis B virus X protein) to activate Yap transcription through

the role of YAP in bile duct homeostasis. Elevated nuclear YAP CREB [58]. Wang et al. mapped the CREB binding region on Yap

− −

expression was found in ductular reactions of primary sclerosing promoter to be 608/ 439 [65]. Amphiregulin and HB-EGF (epi-

cholangitis (PSC) and primary biliary cirrhosis (PBC) [26] as well dermal growth factor) treatment is able to increase Yap mRNA

as in bile duct epithelium of neonates with biliary atresia [62]. In levels through EGFR (epidermal growth factor receptor) and MEK1

cholestatic livers of young patients, hepatocytes also display robust (Dual specificity mitogen-activated protein kinase kinase 1), but it

YAP staining [63]. Whether YAP plays a role in other liver diseases is not clear how EGFR signalling increases Yap transcription [66].

is an interesting question and remains to be investigated. Yap transcription is regulated epigenetically by Menin. As a scaf-

fold protein, Menin partners with mixed-lineage leukemia (MLL)

histone methyltransferase to regulate the transcription of target

4. Regulatory mechanisms of YAP activity in the liver

genes by altering histone tail modifications [67]. In an effort to

identify the promoters occupied by Menin, Xu et al. performed

In the previous sections, we summarized accumulative evi-

ChIP-on-chip screens with Menin antibody in HepG2 cells [68].

dences showing that precise regulation of YAP activity is critical

Yap is one of the top picks with promoter occupied by Menin

for liver homeostasis and tumorigenesis. Therefore, it is impor-

coinciding with the transcriptional activation of H3 modification.

tant to completely understand the mechanisms that regulate

Interestingly, Menin epigenetically upregulates Yap transcription

YAP activity. Multiple mechanisms including mRNA transcription,

specifically in HCC cell lines as although Menin binds to the pro-

post-transcriptional modification, nucleocytoplasmic transporta-

moter of Yap in other types of cell lines including MCR-7, Wilm’s

tion and protein stability participate in regulating YAP activity. In

and A549, there are no obvious effects on H3K4me3 levels at Yap1

this section, we will review up-to-date discoveries on these regu-

foci by Menin overexpression.

latory mechanisms in the liver (Fig. 2).

4.1. Yap mRNA transcription 4.2. Post transcriptional modification to Yap mRNA

Several transcription factors including GABP (GA-binding pro- Liu et al. used bioinformatics technology to screen miRNAs that



tein), homeobox protein NANOG and CREB (cAMP response may target the 3 UTR region of Yap gene for degradation and

element-binding protein) were reported to occupy Yap promoter identified miR-375 as a candidate [69]. Luciferase reporter assay



region and upregulate Yap mRNA transcription in mouse liver or confirmed that miR-375 indeed targets the 3 UTR region of Yap

HCC cells. Wu et al. used Yap promoter region as bait to pull down gene. Ectopic expression of miR-375 decreased endogenous YAP

the binding proteins from mouse liver lysate and identified Ets protein levels as well as YAP transcriptional target – Ctgf (connec-

family transcription factor complex GABP␣/GABP␤ as candidate tive tissue growth factor) mRNA levels in PLC and 97L HCC cell lines.

[31]. Yap promoter contains 16 Ets family transcription factor bind- Consistently, elevated YAP activity correlate with down-regulation

ing site (EBS) and multiple EBS sequences were bound by GABP of miR-375 in human HCC tissues, chemical-induced mouse HCC

to regulate Yap transcription. Interestingly, GABP␤ is phosphory- model and DENA-induced rat preneoplastic hepatocytes [52,53,69].

lated and inhibited by LATS1, which indicates that Hippo signalling

regulates YAP activity through both phosphorylation and tran- 4.3. YAP protein phosphorylation/nucleocytoplasmic shuttling

scription [31]. Functional screening for oncogenes regulated by

TLR4 (Toll-like receptor 4) and NANOG using a lentiviral cDNA YAP phosphorylation mediated by the Hippo signalling rep-

library from tumour-initiating stem-like cells (TICs) in mouse and resents the major regulation strategy of YAP activity. As a

human HCC tumours revealed YAP as one of the candidates [64]. transcription coactivator, nuclear accumulation is necessary for

Q. Nguyen et al. / Digestive and Liver Disease 47 (2015) 826–835 831

Fig. 2. Mediators of Yes-associated protein activity in the liver. In the liver, Yes-associated protein activity is regulated through multiple mechanisms including transcription,

post transcriptional modification, phosphorylation and protein degradation. Transcriptional factors GA-binding protein, homeobox protein NANOG, cAMP response element-

binding protein/Hepatitis B virus X protein and scaffold protein Menin bind to Yes-associated protein promoter region to promote Yes-associated protein mRNA transcription.



miR375 interacts with 3 UTR of Yes-associated protein mRNA to promote mRNA degradation and inhibit translation. Association between Yes-associated protein and its

transcription factor partner TEA domain family members can be inhibited by competitively binding of Vestigial like protein 4 or through phosphorylation by the Hippo

pathway. Yes-associated protein Phosphorylation by the Hippo signalling pathway primes its further phosphorylation by casein kinase 1␧/␦, which leads to Yes-associated

protein degradation through proteasome. Yes-associated protein degradation can be inhibited by TEA domain family members interaction. Bile acids inhibit Yes-associated

protein phosphorylation through IQ Motif Containing GTPase Activating Protein 1. Active Hippo signalling pathway can also leads to GA-binding protein ␤ phosphorylation,

which in turn reduces Yes-associated protein transcription. YAP, Yes-associated protein; GABP, GA-binding protein; CREB, cAMP response element-binding protein; HBx,

Hepatitis B virus X protein; TEADs, TEA domain family members; Vgl4, Vestigial like 4; CK1␧/␦, casein kinase 1␧/␦; IQGAP1, IQ Motif Containing GTPase Activating Protein 1.

YAP’s function. Nuclear accumulation or cytoplasmic retention [72,73], cell density [54], cell detachment [74], F-actin cytoskele-

depends on YAP’s phosphorylation status. Dephosphorylated YAP ton [75], spectrin cytoskeleton [76,77], and cellular energy level

accumulates in nucleus while phosphorylation at Ser127 (human, [78,79] were shown to regulate YAP nuclear accumulation depend-

Ser112 in mouse YAP) by LATS1/2 kinase due to activated Hippo ent or independent of the canonical Hippo signalling. Detailed

signalling sequesters YAP in the cytoplasm via 14-3-3 interac- reviews of these upstream YAP regulators can be found elsewhere

tion [9]. In mouse liver, ablation of the Hippo signalling pathway [80,81]. Serum-derived small molecules sphingosine-1-phosphate

upstream tumour suppressors Nf2, Mst1/2 or Sav1 all resulted in (S1P) and lysophosphatidic acid (LPA) are particularly interesting

reduced levels of phosphorylated YAP, which in turn drives liver because they are the first extracellular diffusible factors regulating

tumorigenesis [15,47,49]. Using HCC cell lines, overexpression of YAP activity [82,83]. S1P and LPA act through GPCRs (G-protein-

another Hippo signalling pathway upstream tumour suppressor coupled receptors) G12/13 to inhibit the Hippo pathway kinase

RASSF1A (Ras Association (RalGDS/AF-6) Domain Family Member LATS1/2, which in turn activates YAP [83]. Whether these upstream

1) increased YAP phosphorylation [59]. inputs regulate YAP activity in the context of liver requires further

Other than the Hippo pathway, bile acid regulates YAP phos- investigation.

phorylation through IQGAP1 (IQ motif containing GTPase activating

protein 1) in the liver [63]. Phosphor-YAP levels were downreg-

4.4. YAP protein stability

−/− −/−

ulated in the presence of elevated bile acid levels in Fxr Shp

mice or WT mice fed with 1% cholic acid. IQGAP1 is required for bile

Another strategy that cell employs to regulate YAP protein levels

acid-induced YAP activation as 1% cholic acid diet failed to induce

is protein stability [11,84]. Overexpression of YAP’s transcriptional

−/−

YAP activation in Iqgap1 mice. Furthermore, adenoviral Iqgap1

partner, TEAD, significantly increased YAP protein levels but not

overexpression in WT liver was sufficient to increase YAP protein

Yap mRNA levels in mouse liver and as well as in HEK293 cells,

levels. As a key scaffolding protein that interacts with E-cadherin,

suggesting TEAD binds and stabilizes YAP, which is further sup-

IQGAP1 overexpression reduces cellular adhesion by dissociating

ported by the observation that YAP S94A (human, Ser79 in mouse

␣

-catenin from the cadherin–catenin complex [70]. These results

YAP), a mutant form defective in TEAD binding, had a shorter

indicated that elevated bile acid levels reduce hepatic cell adhesion

half-life than wild-type YAP [11]. Binding to TEAD may keep YAP

through disrupting E-cadherin junctions, which in turn activates

in active form thus inhibit YAP protein degradation initiated by

YAP. E-cadherin junction disruption has been previously reported

LATS1/2 phosphorylation. LATS1/2 can phosphorylate YAP at all the

as an evoking event of YAP activation in breast cancer cell lines [71].

five consensus HxRxxS motifs. Among these phosphorylatable ser-

In Drosophila or non-hepatic mammalian cells, multiple

ine/threonine residues, phosphorylation at S381 (human, Ser363

upstream inputs including extracellular matrix area and stiffness

in mouse YAP) primes this region for further phosphorylation by

832 Q. Nguyen et al. / Digestive and Liver Disease 47 (2015) 826–835

CK1␧/␦ (casein kinase 1␧/␦) at Ser384 (human, Ser366 in mouse alcohol-related chronic liver injury [64]. In HCC cell lines, YAP pro-

YAP) and S387 (human, Ser369 in mouse YAP) to generate a phos- motes CREB protein stability through interaction with MAPK14/p38

␤-TRCP

phorylated phosphodegron that will recruit SCF E3 ubiquitin (mitogen-activated protein kinase 14) and BTRC (beta-transducin

␤-TRCP

ligase. Ubiquitination by SCF E3 ubiquitin ligase then leads repeat containing E3 ubiquitin protein ligase) [65]. c-Myc transcrip-

YAP to proteasome degradation [84]. tion is indirectly regulated by YAP through interaction with c-Abl

Although multiple mechanisms have already been revealed, [92].

further efforts are still needed for a complete understanding of

YAP activity regulation. For example, YAP protein levels elevate 6. YAP as therapeutic target in liver cancer

in mouse livers following BDL [26]. However, the phosphorylation

status of YAP and Yap mRNA levels remain stable excluding involve- The current available data including YAP upregulation in human

ment of Hippo signalling and transcriptional regulation, which liver cancer, the tumorigenic phenotypes of mouse liver-specific

suggested other mechanisms of YAP activity regulation. Moreover, Nf2/Mst/Sav knockouts and Yap transgenic overexpression, sug-

it will be also important to define the contributions of various gests that increased YAP activity is a driving event for liver cancer

regulating mechanisms to YAP activity in different physiological formation. The fast advance of our knowledge to YAP’s regulators

contexts. and regulations made it possible to inhibit YAP activity with mul-

tiple schemes.

5. YAP transcriptional partners and targets in the liver 6.1. Reducing YAP protein levels

As a transcription coactivator, YAP does not bind to DNA directly Reducing YAP protein levels is the most direct strategy to

but, rather, partners with DNA-binding transcription factors. Stud- decrease YAP activity. Using Yap siRNA encapsulated into LNPs

ies in mammalian cells have revealed several transcription factor (siYap-LNPs), which improves the cellular uptake, stability and

partners for YAP including the p53 family p73 [85], the Runt family pharmacokinetics of the siRNA, Fitamant et al. demonstrated that

member Runx2 [86], and the TEAD/TEF family transcription factors tail vein injection of siYap-LNPs dramatically reduced liver tumour

[10]. Among these reported YAP-interacting partners, TEAD/TEF burden of Mst1/2-deficient mice [34]. Importantly, the treatment

family transcription factors are identified as the major mediators regimen was well tolerated only inducing a slight inflammation in

of YAP function in mammalian cells [87,88]. To investigate whether periportal areas that was fully resolved within a short time follow-

YAP functions through TEAD factors in intact mammalian tissues, ing discontinuation of treatment.

TEAD proteins were inactivated specifically and conditionally in the

mouse liver [11]. The mouse genome contains four highly homol- 6.2. Disrupting YAP–TEAD interaction

ogous TEAD family members, all of which are expressed in the

liver. Because different TEAD members appear to function redun- Using genetic modified mouse models, Liu-Chittenden and

dantly [89], dominant-negative scheme was employed to block colleagues demonstrated that the tumorigenic potential of YAP

the activity of all four TEAD factors with engineering a truncated in mouse liver requires association to TEAD [11], which makes

form of TEAD2 that lacks its DNA-binding domain (TEAD2-DN) [11]. YAP–TEAD interaction an ideal drug target. By screening the Johns

Co-overexpression of YAP and TEAD2-DN in mouse hepatocytes Hopkins Drug Library, they identified members of the porphyrin

potently suppressed hepatomegaly and tumorigenesis caused by family as small molecule inhibitors of YAP–TEAD interactions.

YAP overexpression or loss of NF2. Thus, TEAD is the major partner One of these inhibitors, verteporfin (VP) is used clinically as a

of YAP in the liver to induce the transcription of YAP’s downstream photosensitizer in photodynamic therapy for neovascular macu-

targets. lar degeneration. Administration of VP to YAP transgenic mice and

mRNA microarray analysis has revealed a large number of genes Nf2 knockout mice significant suppressed hepatomegaly caused by

that are induced by YAP in mouse livers [9,24,50]. Among these hepatocyte and biliary cell proliferation, respectively, in these two

candidate YAP target genes, Axl is a direct target of YAP and has mouse models, without overt adverse effects in other organs [11].

been shown to contribute to YAP-dependent oncogenic functions Additionally, VP treatment reduced cell proliferation more pro-

in HCC cell lines [61]; Ctgf [54] and Amphiregulin [90] were shown nouncedly in HuCCT1 cholangiocarcinoma cell line than it did to

to be direct targets of YAP using non-liver cell lines and found to normal cholangiocyte cell line H69, suggesting the potential of VP

be induced by YAP in mouse liver [91] and HCC cell lines [59], in treating cholangiocarcinoma [62].

respectively; in human liver cancer survey studies, expression lev- Although YAP mainly functions through TEAD, it is not the only

els of CTGF, Survivin and Glypican 3 were found to correlate with transcription cofactor of TEAD. Vgl4 competes with YAP on bind-

YAP activity levels [56,57]. Two independent studies have put YAP ing to TEAD via TDU domains [12,93,94]. Animal studies indicated

upstream of the Notch signalling pathway [23,24]. YAP activation that Vgl4 overexpression significantly suppressed transgenic YAP-

in mouse livers increases mRNA levels of several Notch pathway induced liver overgrowth and tumorigenesis [12], which suggested

components including Notch1/2, Jag1 (Jagged 1), Hes1 (hes family a potential therapeutic application of Vgl4 in cancer therapy. Jiao

bHLH transcription factor 1) and Sox9 (sex determining region Y-box and colleagues resolved the crystal structure of mouse Vgl4 tan-

9) [23,24], among which Notch2 is a direct target of YAP [24]. More dem TDU region in complex with the YBD domain of mouse TEAD4,

importantly, Notch inhibition through Rbpj (recombination signal and based on this complex structure, they designed a “Super-TDU”

binding protein for immunoglobulin kappa J region) deletion damp- peptide to inhibit YAP–TEAD interaction [94]. Administration of



ens the hepatocyte-to-ductal dedifferentiation phenotype in Yap Super-TDU peptide to Helicobacter pylori and N-methyl-N -nitro-

transgenic liver, which put the Notch signalling downstream of YAP N-nitrosoguanidine (MNNG) induced-gastric cancer mouse models

with genetic studies in intact mammalian tissue. significantly suppressed tumour numbers and tumour cell prolif-

Other than inducing target gene expression transcriptionally, eration.

YAP also exerts its influence through protein-protein interactions.

YAP interaction with SMAD7/SMAD3 in the cytoplasm results in 6.3. Targeting YAP upstream regulators

cytoplasmic retention of SMAD3, then in turn leads to inactivation

of the canonical TGF-␤ signalling, which contributes to tumour- The Hippo signalling pathway. The Hippo signalling path-

initiating stem-like cell formation in HCC caused by HCV and/or way is the major upstream negative regulator of YAP activity.

Q. Nguyen et al. / Digestive and Liver Disease 47 (2015) 826–835 833

Elevated Hippo signalling activities will efficiently inhibit YAP Conflict of interest

activity. Integrin-linked kinase (ILK) suppresses the Hippo None declared.

signalling pathway via phospho-inhibition of MYPT1-PP1 (myosin

phosphatase target subunit 1-protein phosphatase 1), leading to Funding

inactivation of NF2 [95]. Pharmaceutical inhibition of ILK with

QLT0267 in tumour cell lines and mouse xenograft models suppress Portion of this work was supported by the Dr. Nicholas C. High-

YAP-dependent tumour growth. However, this approach requires tower Centennial Chair of Gastroenterology from Scott & White,

an intact Hippo pathway signal flow in tumour cells. a VA Research Career Scientist Award, a VA Merit award and

F-actin cytoskeleton. F-actin cytoskeleton is fundamental to sus- NIH R01DK076898 grant to Dr. G. Alpini, and a grant award from

tain YAP function. A mild disruption of the actin cytoskeleton leads BaylorScott&White operational funds to H. Bai. This work is also

to dramatic inhibition of YAP nuclear localization [72,74,96]. There- supported by R01DK080736 to R. Anders.

fore, factors those contribute to the actin cytoskeleton integrity This material is the result of work supported by resources at the

may have therapeutic potential in inhibiting YAP activity. Indeed, Central Texas Veterans Health Care System. The views expressed in

the ROCK inhibitor Y27632 and the nonmuscle myosin heavy chain this article are those of the authors and do not necessarily represent

inhibitor blebbistatin have been shown to inhibit YAP activity the views of the Department of Veterans Affairs.

[72,96]. The potential of these inhibitors in treating YAP-dependent

tumours needs to be further evaluated in animal models. Acknowledgements

AMP-activated kinase (AMPK). Cellular energy stress nega-

tively regulates YAP activity through AMPK, which is a critical We apologize that we are unable to cite some important papers

energy level sensor and mediator [78,79]. Pharmacological activa- due to the scope of this review.

tion of AMPK by AMPK activators, aminoimidazole carboxamide

ribonucleotide (AICAR) or metformin, significant suppressed References

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