Mst1 and Mst2 protein kinases restrain intestinal stem
cell proliferation and colonictumorigenesis by inhibition
of Yes-associated protein (Yap) overabundance
Dawang Zhoua,b,c,d,1, Yongyou Zhange,f,1, Hongtan Wua,b,c,d, Evan Barryg,h,i, Yi Yina,b,c, Earl Lawrencej, Dawn Dawsonf,j,
Joseph E. Willisf,j, Sanford D. Markowitze,j, Fernando D. Camargog,h,i, and Joseph Avrucha,b,c,2
Departments ofaMolecular Biology and Department ofcMedicine, Harvard Medical School, Boston, MA 02115;bDiabetes Unit, Massachusetts General
Hospital, Boston, MA 02114;dState Key Laboratory of Cellular Stress Biology, School of Life Sciences, Xiamen University, Xiamen, Fujian 361005, China;
Departnents ofeMedicine andfPathology, Case Western Reserve University, Cleveland, OH 44016;jComprehensive Cancer Center, Case Medical Center,
Cleveland, OH 44016;gStem Cell Program, Children’s Hospital, Boston MA 02142;hDepartment of Stem Cell and Regenerative Biology, Harvard University,
Cambridge, MA 02138; andiHarvard Stem Cell Institute, Cambridge MA 02138
Edited by Bert Vogelstein, Johns Hopkins University, Baltimore, MD, and approved September 20, 2011 (received for review June 28, 2011)
Ablation of the kinases Mst1 and Mst2, orthologs of the Drosophila
antiproliferative kinase Hippo, from mouse intestinal epithelium
causedmarked expansion ofan undifferentiatedstemcellcompart-
ment and loss of secretory cells throughout the small and large in-
testine. Although median survival of mice lacking intestinal Mst1/
Diminished phosphorylation, enhanced abundance, and nuclear lo-
(Yap1) is evident in Mst1/Mst2-deficient intestinal epithelium, as is
strong activation of β-catenin and Notch signaling. Although bial-
lelic deletion of Yap1 from intestinal epithelium has little effect on
intestinal development, inactivation of a single Yap1 allele reduces
the continued Yap hypophosphorylation and preferential nuclear
localization, normalizes epithelial structure. Thus, supraphysiologic
Yap polypeptide levels are necessary to drive intestinal stem cell
proliferation. Yap is overexpressed in 68 of 71 human colon cancers
and in at least 30 of 36 colon cancer-derived cell lines. In colon-de-
rived cell lines where Yap is overabundant, its depletion strongly
survival. These findings demonstrate that Mst1 and Mst2 actively
suppress Yap1 abundance and action in normal intestinal epithe-
lium, an antiproliferative function that frequently is overcome in
colon cancer through Yap1 polypeptide overabundance. The dis-
pensability of Yap1 in normal intestinal homeostasis and its potent
proliferative and prosurvival actions when overexpressed in colon
cancer make it an attractive therapeutic target.
nase. Hippo is the central component of an antiproliferative
pathway that responds to signals arising from cell–cell contact to
regulate negatively the oncogenic transcriptional coactivator,
yorkie. Loss of Hippo function results in a yorkie-dependent ac-
celeratedproliferation,resistance toapoptosis,andmassive organ
overgrowth (2, 3). In mouse liver, Mst1 and Mst2 act in a re-
dundant manner to maintain hepatocyte proliferative quiescence.
Acute inactivation of both Mst1 and Mst2 in the adult liver results
in the immediate onset of hepatocyte proliferation, a doubling of
livermasswithin a weekprogressingtoa four- tofivefold increase,
followed within weeks by multifocal hepatocellular carcinoma
(HCC) (4). Albumin-Cre mediated inactivation of Mst1 and Mst2
in liver is accompanied by expansion of both the hepatocytes and
the bipotential adult liver progenitors known as “oval cells”; in
addition to HCCs and cholangiocarcinomas, these livers exhibit
many tumors with mixed cellularity, presumably reflecting an or-
igin from the Mst1/Mst2-deficient oval cells (4–6). The Mst1/
Mst2-deficient livers exhibit loss the inhibitory phosphorylation of
Yes-associated protein 1 (Yap1), the mammalian ortholog of
yorkie, and a marked increase in overall and nuclear Yap1
st1 and Mst2 are class II GC kinases (1) that are the closest
mammalian homologs of the Drosophila Hippo protein ki-
abundance. Tetracycline-induced overexpression of transgenic
Yap1 in liver also induces hepatocyte proliferation and massive
enlargement of the organ that is reversible (7, 8) but if sustained
results in the development of HCCs (8). In HCC cell lines derived
from Mst1/Mst2-null livers, depletion of Yap1 causes growth in-
hibition and extensive apoptosis, findings that support the view
that Yap1 activation is the major mechanism underlying the liver
overgrowth seen with Mst1/Mst2 inactivation (4).
These findings indicate that, as with Hippo, Mst1/Mst2 nega-
tively regulates Yap1 in mammalian liver; however, such a re-
lationship does not prevail in all mammalian tissues. Thus, in
mouse embryo fibroblasts (MEFs), cell–cell contact results in
Yap1 phosphorylation and nuclear exclusion equally well in wild-
type and Mst1/Mst2-null MEFs (4); in mouse keratinocytes, Yap
inactivation during cellular differentiation occurs independently
of Mst1 and Mst2 (9). Conversely, Mst1 negatively regulates the
proliferative response of naïve T cells to antigen receptor stimu-
lation through a Yap1-independent process (10). Thus, it appears
that the wiring upstream of Yap1 and downstream of Mst1/Mst2
has been diversified considerably in mammals compared with the
Drosophila Hippo pathway. The intestinal epithelial cell, like the
hepatocyte, is of endodermal origin; however the self-renewal
mechanisms of these two cells are radically different. Hepatocyte
self-renewal is mediated by the division of fully differentiated
adult hepatocytes that emerge from replicative quiescence ap-
proximately once per year (11). In contrast, the epithelial lining of
the small intestine turns over completely every 4–5 d through the
continuous division of intestinal stem cells located in the crypts of
Lieberkühn. These intestinal stem cells differentiate into a tran-
sient amplifying compartment and thereafter into four types of
mature cells (enterocytes, goblet cells, enteroendocrine cells, and
Paneth cells). Except for the Paneth cells, the maturing cells mi-
grate toward the tip of the villus, from which they are shed (12–
14). Previous work has shown that overexpression of Yap1 in this
epithelium results in the enhanced proliferation of the stem cell
compartment accompanied by the disappearance of all differen-
tiated cell types (7). We wished to determine the role of Mst1 and
Author contributions: D.Z., Y.Z., and J.A. designed research; D.Z., Y.Z., H.W., Y.Y., and E.L.
performed research; E.B. and F.D.C. contributed new reagents/analytic tools; D.Z., Y.Z.,
D.D., J.E.W., S.D.M., and J.A. analyzed data; and D.Z., Y.Z., S.D.M., and J.A. wrote
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1D.Z. and Y.Z. contributed equally to this work.
2To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
www.pnas.org/cgi/doi/10.1073/pnas.1110428108 PNAS Early Edition
| 1 of 9
Mst2 in intestinal epithelial homeostasis and whether these
kinases suppress Yap1 activation in this cellular milieu.
The intestine of Mst1-null and Mst2-null mice is indistinguishable
from wild-type, whereas mice deficient in both Mst1 and Mst2
exhibit embryonic lethality at embryonic day 8.5–9.5 (4, 15),
Mst1null/Mst2ff/villin-Cre mice are born in Mendelian ratios and
appear normal at birth. However, these mice exhibit a median
survival of only 13 wk (Fig. 1A) accompanied by severe wasting
(Fig. 1B). Thelengthof their smallandlargeintestine is unaltered
from wild type, but cecal size is greatly reduced, and the rectum
usually is enlarged markedly (Fig. 1C and SI Appendix, Fig. S1).
The small intestinal mucosa of Mst1null/Mst2ff/villin-Cre mice is
dysplastic: Villus structure is disorganized because of a marked
increase in undifferentiated proliferating cells (Fig. 1D) with
a dramatic decreasein cells of the secretory lineages (Fig. 1 E–G),
although the abundance of absorbtive enterocytes is better pre-
served (Fig. 1H). The colonic epithelium also is highly dysplastic,
with expansion of undifferentiated cells (Fig. 2 A and B) and se-
vere loss of goblet cells (Fig. 2B). Although cecal size is reduced
(Fig. 1C), the cecal mucosa is hyperproliferated and dysplastic (SI
Appendix, Fig. S1), and hyperproliferation of epithelia throughout
the colon is marked, especially near the rectum (Fig. 2C, Lower,
arrows in second panel from left). In the distal colon there is es-
pecially exuberant overgrowth of dysplastic epithelia and the
frequent presence of one or more adenomas extending into the
lumen (Fig. 2C, Upper). Both the small and large intestine exhibit
a marked expansion in the number of cells expressing high levels
of CD133 (SI Appendix, Fig. S2 A and B), a marker of intestinal
stem cells and transiently amplifying cells (16, 17), as well as in-
creased abundance of the well-documented stem cell markers
Leucine-rich repeat-containing G-protein coupled receptor 5
(Lgr5) and Achaete-scute complex homolog 2 (Ascl2) (Fig. 3A)
(14) and increased numbers of cells expressing CD44 and CD24,
markers associated with colon cancer stem cells (SI Appendix, Fig.
S2 A and B) (18).
In 6-wk-old wild-type mice, Mst1 and Mst2 polypeptides, visu-
alized by immunoblot, are expressed in all segments of the small
and large intestine. In the small intestine, cecum, and proximal
2D), corresponding in size to the constitutively active, caspase-
cleaved catalytic fragment that is generated in cells undergoing
apoptosis (19) but which also is predominant in normal quiescent
mouse liver (4). In the mid and distal colon, the full-length Mst1
polypeptide predominates, as is true for Mst2 throughout the in-
testine. Mst1 is absent from the small and large intestine of the
Mst1null/Mst2ff/villin-Cre mice, and the expression of Mst2 is
severely reduced in these mice (Fig. 3A). Available Mst1 and Mst2
contribution of Mst2 from nonepithelial cell types is not known;
however, the markeddecrease in Mst2observed in theintestine of
is contributed by the epithelia (Fig. 3A, Top two panels). Yap1
but is highly abundant in the colon, especially in the distal seg-
ments (Fig. 2D and Fig. 3A, Panels 3–5 from top). Yap1 immu-
nocytochemistry in the wild-type small intestine (SI Appendix, Fig.
S3, Farthest left column, top two rows) and colon (SI Appendix, Fig.
S3, Third column from the left, top two rows) shows strongest
staining in crypts that appear largely or entirely extranuclear in
stain in cells closer to the villus tip. Yap1 abundance is greatly
enhanced by elimination of Mst1 and Mst2 in all regions of the
intestine (Fig. 3 A, Panels 3 and 4 from top and B, Right and SI
Appendix, Fig. S3, Rows 1 and 2 from top). In the colon, where
from eight wild-type and 12 Mst1null/Mst2ff/villin-Cre (double-knockout, dko) mice, the latter with a average median survival of 13 wk. (B) Body weight of
Mst1null/Mst2ff/villin-Cre (dko) and wild-type mice at age 6 wk. (C) Representative examples of intestinal tract from Mst1null/Mst2ff/villin-Cre (DKO) and
wild-type mice at age 6 wk (n = 3 per group). Ce, cecum; s, stomach. (D–H) Representative samples showing absence of secretory cell lineages in the small
intestine of Mst1null/Mst2ff/villin-Cre mice (DKO) compared with wild-type mice (n = 3 per group).
Mst1null/Mst2ff/villin-Cre mice exhibit early mortality and impaired epithelial differentiation in the small intestine. (A) A Kaplan–Meier survival curve
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Yap1 abundance normally is greatest, the loss of Mst1 and Mst2
results in a decrease in Yap1(Ser127) phosphorylation (Fig. 3A,
Panel 5 from top) despite the marked up-regulation of Yap1
polypeptide. Consistent with increased Yap1 abundance and di-
minished Yap1(Ser127) phosphorylation, extensive Yap1 nuclear
localization is evident in the epithelia of the small and large in-
testine (SI Appendix, Fig. S3, Columns 2 and 4, top two rows).
Moreover, compared with the wild-type intestine, cells reactive
with anti-Ki67 are substantially more abundant in the Mst1/Mst2-
deficient intestine,both in the cryptsandextending further toward
the lumen (Fig. 3B, Left and SI Appendix, Fig. S3, Rows 3 and 4).
The hyperproliferation of undifferentiated cells and the loss of
the secretory cell populations resulting from elimination of Mst1
and Mst2 in the intestine (Figs. 1 and 2) led us to examine the
status of the Wnt and Notch pathways. Activation of the Wnt
pathway is thought to be the primary driver of proliferation in
the intestinal stem cell compartment (14, 20), acting synergisti-
cally with Notch (21). Elimination of Mst1 and Mst2 is accom-
panied by little or no change in the overall abundance of
β-catenin, nor is there a convincing difference in the abundance
of nuclear β-catenin in the Mst1/Mst2-null intestine (SI Appendix,
Fig. S3, Bottom two panels). A modest increase in the abundance
of the activated form of β-catenin (dephosphoSer37/Thr41) is
evident in the Mst1/2-deficient small and especially large in-
testine (Fig. 3A, Panel 6 from top) whereas the abundance of the
Wnt targets and stem cell markers Lgr5 (22) and Ascl2 (20) (Fig.
3A, Panels 7 and 8 from top) are increased more markedly,
suggesting enhanced β-catenin transcriptional efficacy. We
therefore inquired whether Yap1 overexpression enhances
β-catenin action. SW480 cells contain high levels of β-catenin
because of the inactivation of adenomatosis polyposis coli (APC)
(23) and show modest overexpression of Yap1. The T-cell factor
(TCF) reporter plasmid TOPflash (24) was transiently expressed
in SW480 colon cancer cells engineered to express scrambled or
Yap1-directed shRNAs in a tetracycline-stimulated manner (Fig.
3C). TOPflash-directed expression of firefly luciferase is ∼1,000
fold greater than that directed by the FOPflash vector, which
encodes a mutant inactive TCF promoter. Induction of Yap1
shRNA reduces TOPflash-directedluciferaseexpressionbymore
than 80% without altering β-catenin overall or intranuclear (Fig.
3D) abundance, strongly supporting the view that Yap1, at least
when overexpressed, enhances the transcriptional efficacy of β-
Strong activation of the Notch pathway also is evident in the
Mst1/Mst2-deficient intestines (Fig. 4). The abundance of Hairy
and enhancer of split 1 (Hes1), a transcriptional target of Notch,
is up-regulated in small and large intestines (Fig. 4A, Panel 2
from top); in contrast to its predominant localization in crypts
and transient amplifying cells in wild-type intestine, prominent
intranuclear localization of Hes1 is evident along the entire ep-
ithelial surface of the Mst1/Mst2-deficient small and, especially,
large bowel (Fig. 4B, Center column). The likelihood that the
enhanced expression of Hes1 is driven by Notch is supported by
the presence of increased amounts of intranuclear Notch in-
tracellular domain (Fig. 4A, Top) along the entire epithelial
surface of the Mst1/Mst2-deficient small and large bowel (Fig.
4B, Left). Thus, activation of the Wnt pathway, acting synergis-
tically with the strong up-regulation of Notch signaling, probably
accounts for the increased proliferation and expansion of Lgr5+
stem cells and transiently amplifying cell compartments in the
Mst1/Mst2-deficient intestinal epithelium.
Notch acts independently of Wnt to control intestinal epithelial
differentiation through Hes1 (25), which is known to inhibit
transcription of Atonal homolog1 (Atoh1/Math1) (26, 27), 2a
transcription factor that is required for the differentiation of in-
testinal secretory cell lineages (28). In the Mst1/Mst2-deficient
intestine, expression of the Notch targets Hes1 and Hairy/en-
hancer-of-split related with YRPW motif 1 (Hey1) is strongly up-
of the colonic epithelium in 6-wk-old Mst1null/Mst2ff/villin-Cre (DKO) mice. (B) Loss of goblet cells in proximal and distal colon of 6-wk-old Mst1null/Mst2ff/
villin-Cre mice. Tissues shown are representative of three mutant and three wild-type mice. Boxed areas in low-magnification (L) views are shown to the right
at higher magnification (H). Histologic views of the cecum are shown in SI Appendix, Fig. S1. (C) Formation of epithelial hyperplasia, dysplasia, and adenoma
(arrows, Upper right) in the distal colon in two Mst1null/Mst2ff/villin-Cre mice, age 15 wk (Upper) and 20 wk (Lower). (D) Expression of the Mst1, Mst2, and
Yap1 polypeptides in the mouse intestinal tract. C, colon; c, cecum; d, duodenum; i, ileum, j, jejunum; SI, small intestine.
Mst1null/Mst2ff/villin-Cre (DKO) mice exhibit loss of goblet cells, epithelial dysplasia, and adenomas in the colon. (A) Hyperproliferation and dysplasia
Zhou et al.PNAS Early Edition
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regulated (SI Appendix, Fig. S4A), and Math1 expression is
strongly reduced (Fig. 4 A, Panel 3 from top and B, Right). Thus,
overactivation of the Notch pathway, in addition to synergizing
with wingless-related MMTV integration site (Wnt) to drive stem
cell proliferation, also is responsible for the failure of differenti-
ation of the secretory lineages. Transgenic overexpression of Yap
(Ser127Ala) in the mouse intestine was shown previously to be
sufficient to cause Notch activation and a loss of cellular differ-
entiation (7). We examined the contribution of Yap1 to the acti-
vation of Notch signaling in the colon cancer-derived cell line
HCT116, which has a wild-type APC but a mutant allele of
β-catenin caused by in-frame deletion of Ser45 (29) and mild
overexpression of Yap1. Yap depletion in HCT116 cells does not
alter the abundance of β-catenin, but Ascl2 polypeptide (Fig. 4C)
isdiminishedstrongly, indicatingthat,as in theSW480cells,Yap1
directly or indirectly promotes β-catenin–stimulated gene ex-
pression. Additionally, shRNA suppression of endogenous Yap is
accompanied by a strong decrease in the abundance of the Notch
intracellular domain and in Hes1 expression, accompanied by an
increase in Math1, all consistent with down-regulation of Notch
activation (Fig. 4C). As to the mechanism of Notch activation in
mRNA encoding the Notch ligand Jagged 1 (SI Appendix, Fig.
S4A), and immunoblot ofsmallintestinal mucosal extracts verifies
the presence of increased Jagged 1 polypeptide with intense Jag-
ged 1 staining of crypts in the Mst1/Mst2-deficient small intestine
(SI Appendix, Fig. S4B). Although Jagged 1, unlike connective
tissue growth factor, is not known to be a direct transcriptional
target of Yap1 (30), Jagged 1 is a direct target of β-catenin/TCF
(31, 32). Thus, Notch activation in the Mst1/Mst2-deficient in-
testine is likely a consequence, at least in part, of Yap-stimulated
β-catenin signaling; the contributions of other Mst1/Mst2- and/or
Yap1-regulated pathways remain to be evaluated.
It was reported recently (33) that elimination of Yap1 from
the intestinal epithelium has no significant effect on epithelial
development or function in the unstressed mouse. We confirm
this finding and observe that elimination of Yap does not affect
the abundance of Ki67+cells in mouse colon (SI Appendix, Fig.
S5). Consequently, we sought to verify the importance of Yap1
to the phenotype of the Mst1/Mst2-deficient intestine. We
therefore crossed the Mst1null/Mst2ff mice with Yap1ff and
Yap1f+mice and examined the impact of elimination of one or
both Yap1 alleles on the consequences of Mst1/Mst2 deficiency
in the intestine. Removal of one or both Yap1 alleles eliminates
the premature mortality exhibited by mice lacking expression of
Mst1 and Mst2 in the intestinal epithelium (Fig.5A) and restores
epithelial differentiation (Fig. 5B). Moreover, in the intestine of
the Mst1null/Mst2ff/Yap1ff/villin-Cre mice, there are small
patches where loxP excision failed to occur (Fig. 5C). These
patches enable side-by-side comparison of epithelia that are Mst1
null but otherwise normal with epithelia lacking Mst1/Mst2 and
Yap1. These two epithelia are morphologically indistinguishable,
andthe abundance of Ki67+cells in thecrypts is unaffected by the
presence or absence of Yap1 (Fig. 5C and SI Appendix, Fig. S5).
These data demonstrate that, despite the presence of Yap1 in the
intestinal stem cells and transiently amplifying cell compartments,
Yap1 is entirely dispensable to the proliferative activity of these
cells; nevertheless, removal of the Mst1 and Mst2 kinases initiates
a Yap-dependent hyperproliferation. The ameliorative effect of
the inactivation of a single Yap1allele emphasizes the importance
of Yap1 overexpression to the phenotype incurred by Mst1/Mst2
deficiency. Thus,the levelof Yappolypeptidein the colonofwild-
type and Mst1null/Mst2ff/Yapf+/villin-Cre mice is very similar
(Fig. 5A), although in the absence of Mst1/Mst2, Yap is certainly
is intranuclear. Nevertheless, the colonic morphology of the
Mst1null Mst2ff Yapf+/villin-Cre mice is normal. Inasmuch as a
wild-type level of Yap1 in intestinal epithelium, even when pre-
dominantly intranuclear, is insufficient to promote β-catenin sig-
and loss of differentiation seen in the Mst1/Mst2-deficient in-
testine, it is clear that the ability of Mst1/Mst2 to regulate Yap1
abundance negatively is critical to their antiproliferative function.
the Mst1/Mst2-deficient intestine, Yap1 mRNA abundance is
unaltered (SI Appendix, Fig. S4A). Zhao et.al. (34) reported that
Yap polypeptide ubiquitination by β-transducin repeat-containing
protein (β-TrCP) and subsequent degradation requires phos-
at Yap1(Ser381), enabling casein kinase 1 to catalyze a processive
phosphorylation starting at Ser384. We find that elimination of
Mst1/Mst2 markedly reduces Yap1(Ser384) phosphorylation (Fig.
wt dko wt dko wt dko
Yap shRNA s 1 2 s 1 2 s 1 2 s 1 2
TOP Flash - - - - - - + + + + + +
FOP Flash + + + + + + - - - - - -
Dox - - - + + + - - - + + +
s 1 s 1
Ki67+ cells/crypt Yap+ cells/crypt
Colon Colon SI SI
Mst1null/Mst2ff/villin-Cre and wild-type mice. (A) Western blot of mucosal
epithelium from the indicated intestinal segments of 6-wk-old Mst1null/
Mst2ff/villin-Cre and wild-type mice for Mst1, Mst2 Yap, Yap(Ser127P), and
the Wnt target polypeptides as indicated. ABC, activated β-catenin. SI, small
intestine. (B) Quantitation of the number of Yap1+and Ki67+cells detected
by immunofluorescence staining of small intestine (SI) and colon of Mst1null/
Mst2ff/villin-Cre and wild-type mice. These results represent analyses of
three mice of each genotype ± SE. (n = 20 crypts per section, 3 mice of each
genotype; **P < 0.01 and ***P < 0.001 vs. WT). A representative immuno-
fluorescent image of Yap1, Ki67, and β-catenin staining is shown in SI Ap-
pendix, Fig. S3. (C) SW480 cells stably expressing lentiviral-encoded,
doxycycline-inducible scrambled (s) or either of two Yap1-directed shRNAs (1
and 2) were treated with doxycycline or carrier at time 0, transfected with
plasmids encoding either TOPflash or FOPflash and Renilla luciferase 24 h
thereafter, and were extracted 48 h after addition of doxycycline or carrier.
Luciferase activities were assayed. Firefly luciferase activity normalized for
Renilla luciferase is shown. (D) SW480 cells as in Fig. 4C expressing scrambled
(s) or Yap-directed shRNA (1) were harvested 48 h after addition of doxy-
cycline or carrier, and cytosolic and nuclear fractions were immunoblotted
for Yap, β-catenin, actin, and histone H3.
Expression of Mst1, Mst2, Yap, and Wnt targets in the intestine of
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5D), consistent with the view that diminished Yap degradation
accounts for the increase in Yap abundance, in part or in whole.
We examined the expression of Yap in human colon cancers
and in colon cancer-derived cell lines and sought to evaluate
Yap’s role in sustaining the proliferation of these lines in vitro.
Yap overexpression has been reported in a variety of cancers;
Steinhardt et.al. (35) reported that widespread Yap cytoplasmic
and nuclear staining is evident in ∼80% of 28 colonic cancers
and 44% of 16 normal colons, in which staining is localized to the
crypts (and submucosal smooth muscle cells). We performed
Yap immunohistochemistry on 71 colon carcinomas using a Yap
antibody (4), different from that used by Steinhardt et.al (35),
which in our hands exhibits high specificity (Fig. 5C) and greater
sensitivity. Two cores from each tumor were examined and
compared with normal colon. As in the mouse, normal human
colon exhibits occasional Yap+cells located at the base of the
crypt (Fig. 6A, Upper left). In comparison, 68 of 71 tumor samples
exhibited a substantial increase in overall Yap expression and in
the number of Yap+cells, although intratumoral heterogeneity
was evident, with interspersed regions of Yap−tumor cells (SI
Appendix, Fig. S7A). In the majority of these tumors, Yap was
present in both the nucleus and the cytoplasm. The distribution
of Yap staining intensity (averaged estimate from both cores) is
shown in the table in Fig. 6A.We also carried out immunoblots
for Yap1 polypeptide and Yap1(Ser127P) using extracts from 36
colonic adenoma- and cancer-derived cell lines and from epi-
thelial cells collected from the mucosa of 10 normal colons (Fig.
6B). We used blots of extracts from SW480 cells for visual nor-
malization of the blots obtained from extracts of other cells. Yap
polypeptide expression is greatly increased in the majority (at
least 30/36) of colonic cancer cell lines in comparison with nor-
mal colonic epithelial cells, which show little or no Yap immu-
noreactivity under these immunoblotting conditions (Fig. 6B).
Thus, Yap overexpression is very common in colonic cancer and
in colonic cancer-derived cell lines.
The anti-Yap(Ser127P) antibody has a greater sensitivity than
the anti-Yap polypeptide antibody: Immunoblot of the same
extracts with anti-Yap(Ser127P) demonstrates the presence of
Yap(Ser127) phosphorylation in 8/10 extracts from normal co-
lonic epithelial cells and very much stronger signals in nearly all
(∼27/30) the colon cancer cell lines that exhibit up-regulated
Yap polypeptide immunoreactivity. This result indicates that
rarely is Yap(Ser127) phosphorylation lost entirely in these co-
lonic cancer cell lines; however, whether this result reflects the
Yap(Ser127) phosphorylation state of the primary tumor is not
known, because we have observed the reappearance of Yap
(Ser127) phosphorylation on later passages of HCC-derived cell
lines obtained from HCCs in Mst1/Mst2-null livers that exhibited
no Yap(Ser127) phosphorylation in vivo or during early passages
in vitro (4).
We next sought to determine whether Yap overexpression is
functionally important to the proliferative capacity in vitro of
wt dko wt dko wt dko
- - - - - + + + + +
Yap shRNA: s 1 2 3 all s 1 2 3 all
epithelium from the indicated intestinal segments of 6-wk-old Mst1null/Mst2ff/villin-Cre and wild-type mice for Notch intracellular domain (NICD), the Notch
targets Hes1 and Math1, Stat3, and PY-Stat3. SI, small intestine. (B) Immunofluorescence staining small intestine and colon of Mst1null/Mst2ff/villin-Cre and
wild-type mice for Notch intracellular domain, Hes1, and Math1, each with DAPI. The areas in the dashed boxes are shown at higher magnification in Insets.
Results in A and B are representative of three mice of each genotype. (C) HCT116 cells were engineered to express the following tetracycline-inducible shRNAs
stably: a scrambled shRNA (s), one of three shRNAs each directed at a different segment of the Yap mRNA [Yap-sh1 (1); Yap-sh2 (2); or Yap-sh3 (3)], or all three
shRNAs together (all). Extracts were subjected to immunoblot for the polypeptides shown 24 h after addition of doxycycline (+) or carrier (−).
Notch signaling in the intestine of Mst1null/Mst2ff/villin-Cre and wild-type mice and in Yap-depleted HCT116 cells. (A) Western blot of mucosal
Zhou et al.PNAS Early Edition
| 5 of 9
these colonic cancer cell lines. We selected 11 lines reflecting
a broad range of up-regulated Yap expression, only one of which,
FET, exhibits no Yap polypeptide or Yap(Ser127P) immunore-
activity. Each line was infected with lentivirus pLKO.1 vectors
encoding two different Yap-directed shRNAs or a scrambled
shRNA control and was grown for 10 d under puromycin se-
lection. A Yap immunoblot and quantitative PCR analysis of the
SW480 line demonstrated ∼80% depletion of Yap mRNA and
polypeptide with both Yap shRNAs. All lines infected with virus
encoding Yap shRNA showed a marked inhibition in colony
formation, except for the FET line, in which the colony count
was reduced only modestly (Fig. 6C and SI Appendix, Fig. S7 B–
M). Thus, it appears that Yap, when overexpressed, contributes
strongly to the in vitro proliferative capacity of colonic cancer
Elimination of the protein kinases Mst1 and Mst2 from the in-
testinal epithelial compartment results in an expansion of stem-
like undifferentiated cells and the almost complete disappearance
of all secretory lineages. There is a marked increase in the abun-
dance of the Yap1 with a decrease in the extent of Yap1(Ser127)
phosphorylation, a site known to be regulated by Mst1/Mst2
through an intermediating protein kinase, usually Lats1 and/or
Lats2(36),aswell asa decrease in Ser384phosphorylation, a CK1
site whose phosphorylation enables β-TrCP–mediated ubiquiti-
nation. Yap1 immunoreactivity, which in the wild-type small in-
testine is evident only in the crypts and is predominantly or
entirely cytoplasmic, exhibits intense nuclear localization in the
Mst1/Mst2-deficient small intestine. In the wild-type colon, Yap is
evident largely in colonic crypt cells, and nuclear localization is
WT DKO WTDKO
o l o
l a t s i d
n i t s
n i l l am
paring the survival of wild-type, Mst1null/Mst2ff/Yapf+/villin-Cre (dkoHet), and Mst1null/Mst2ff/Yapff/villin-Cre (tko) mice. Small intestine and colonic mucosa
scrapings from these and from Mst1null/Mst2ff/villin-Cre and from Yapff/villin-Cre (Yapko) mice were extracted and immunoblotted for Yap. (B) Sections of
small intestine (SI) (ileum) and colon from wild-type (Top), Mst1null/Mst2ff/Yapf+/villin-Cre (Middle), and Mst1null/Mst2ff/Yapff/villin-Cre (Bottom) mice were
stained for lysozyme-, chromogranin-, or periodic acid Schiff (PAS)-positive material. Quantitation of the number of cells positive for these markers is shown in
the bar graphs; data are ± SE. Open bars represent WT mice; light gray bars represent Mst1/Mst2ff/villin-Cre mice; dark gray bars represent Mst1null/Mst2ff/
Yapf+/villin-Cre mice; black bars represent Mst1null/Mst2ff/Yapff/villin-Cre mice (n = 20 crypts per section, 3 mice of each genotype; ***P < 0.001 vs. WT). (C)
Section of colon from wild-type and Mst1null/Mst2ff/Yapff/villin-Cre (TKO) mice stained for Yap with or without Ki67. The TKO panels in the upper row on the
far right are from segments in which villin-Cre excision failed to occur, enabling a side-by side comparison of epithelia that are Mst1null but Mst2ff and Yap
sufficient with epithelia that lack Mst1, Mst2, and Yap1; these samples are morphologically indistinguishable. The bottom rows show that the abundance of
Ki67+cells is similar in Mst-null and in Mst1null/Mst2ff/Yapff villin-Cre epithelium. (D) Extracts of normal mouse liver and colonic mucosa, and from Mst1null/
Mst2ff/albumin-Cre mouse liver and from Mst1null/Mst2ff/villin-Cre colonic mucosa were immunoblotted for Yap and for the Yap phosphorylation sites
indicated. The intensity of the Yap polypeptide, normalized for actin intensity, was divided into the value for each of the Yap-P signals; the relative phos-
phorylation at the two sites is shown in the bar graph with values for WT liver and colon set to 1.
The effect of deleting one or both Yap alleles on the morphology of the Mst1/Mst2-deficient intestinal epithelium. (A) A Kaplan–Meier plot com-
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| www.pnas.org/cgi/doi/10.1073/pnas.1110428108Zhou et al.
detectable; in the Mst1/Mst2-deficient intestine, Yap1 is present
at much higher abundance throughout the epithelium with prom-
inent nuclear localization. Phosphorylation of Yap1 (at Ser127)
promotes Yap1 nuclear exit and also is required to initiate its
causes an increased abundance and enhanced nuclear trans-
location of Yap1. It was shown previously that overexpression
[i.e., Yap1(Ser127Ala)] is sufficient to cause an expansion of the
stem cell compartment and a loss of differentiated cell types (7)
similar to that seen with elimination of Mst1 and Mst2 in the
present study. The primacy of Yap activation and overexpression
to the phenotype of the Mst1/Mst2-null intestinal epithelium is
demonstrated by the ability of inactivation of a single Yap allele to
reverse entirely the hyperproliferation and loss of differentiation
caused by the Mst1/Mst2 deficiency.
The ability of Yap nuclear overexpression to promote the
hyperproliferation of intestinal stem cells and to inhibit intestinal
epithelial differentiation is attributable, in large part and perhaps
entirely, to an enhancement of β-catenin action and an activation
of Notch signaling. The primary effect of Yap1 overexpression
appears to be an enhancement of β-catenin transcriptional ac-
tivity rather than an increase in the overall or nuclear abundance
of β-catenin. Whether the Yap1 enhancement of β-catenin di-
rected transcription reflects a direct binding of Yap1 and its
action as a coactivator at β-catenin/TCF-regulated transcrip-
tional sites or a more indirect action of Yap1 is not known;
however, concurrent binding of Yap and β-catenin at regulatory
sites in the Sex determining region Y (SRY)-box 2 (Sox2) and
Snail homolog 2 (Snai2) genes in developing mouse heart was
reportedrecently(37). In addition to the enhancementof β-catenin
transcriptional activity, a modest increase in the abundance of the
deficient intestinal epithelium. The mechanism responsible for this
mild but reproducible β-catenin activation is not known. In Dro-
sophila the Yap1 ortholog yorkie has been shown to bind and pre-
sumably to sequester Dishevelled (Dsh) in the cytoplasm, thereby
interfering with the ability of Wnt to disrupt the β-catenin de-
struction complex (38). The loss of phosphorylation and enhanced
nuclear retention of yorkie that occurs with deletion of hippo is
proposed to decrease cytoplasmic yorkie, releasing Dsh and so fa-
cilitating Wnt’s ability to disrupt the β-catenin destruction complex
and thereby promote activation of Armadillo, the fly β-catenin
ortholog. It is not known if such a mechanism of Yap action in the
cytoplasm is operative in intestinal epithelia or, if so, whether the
loss of Yap1(Ser127) phosphorylation and enhanced nuclear re-
tention of Yap1 seen with Mst1/Mst2 deletion actually reduces
Yap1 cytoplasmic abundance sufficiently to disinhibit Dishevelled.
V400V410 V503V784 V786V451V459
N2N3 N4 N5N6N7 N8 N9
(A) Yap immunohistochemistry of colon cancer tissue microarrays. (Right) A section of normal colon shows an occasional Yap+cell in the crypt. Yap+cells also
are visualized in the lamina propria. Two cores were obtained from different regions of 71 colonic cancers; histologic sections were examined for the intensity
and subcellular localization of Yap staining and scored as in described in Materials and Methods. (Left) The averaged score of the two samples from each
tumor is shown in the table. Examples of the intratumoral heterogeneity of Yap staining are shown in SI Appendix, Fig. S5A). (B) Western blotting analysis of
YAP expression and Yap(Ser127) phosphorylation in human colon cancer cell lines and mucosal epithelium isolated from 10 normal colons. (C) A qualitative
estimate of the relative abundance of Yap polypeptide and Yap(Ser127) immunoreactivity (p-Yap1) in selected colon cancer cell lines and the extent of
inhibition of colony formation of these lines after infection with lentiviruses encoding two different Yap shRNAs. Data shown are the average of two
experiments). The primary data are shown in SI Appendix, Fig. S7.
The expression of Yap in human colon cancer tissue and cell lines and the effect of Yap shRNA on the proliferation of colon cancer-derived cell lines.
Zhou et al.PNAS Early Edition
| 7 of 9
Notch activation in the Mst1/Mst2-deficient intestine also is
likely attributable, at least in part, to loss of Yap phosphorylation
and consequent nuclear overabundance, inasmuch as transgenic
overexpression of Yap(Ser127Ala) in the mouse intestine is suf-
ficient to cause Notch activation and a loss of cellular differenti-
ation thatcanbereversedpartially by treatment witha γ-secretase
inhibitor (7). The increased expression of Jagged in the Mst1/
Mst2-deficient intestine, mediated in part through up-regulated
Wnt signaling (31, 32), probably contributes to the activation of
Notch. Although it is likely that Yap mediates the activation of
both Wnt and Notch signaling in the Mst1/Mst2-deficient in-
to-be defined outputs of Mst1/Mst2 remains to be established.
The robust proliferative response elicited by elimination of
Mst1 and Mst2 indicates that in the normal colonic epithelium
one or both of these kinases actively represses Yap1 function.
Consistent with this view is the high abundance in the small in-
testine and proximal colon of the 36-kDa Mst1 polypeptide that
is known in other settings to be constitutively active. Unfor-
tunately, immunoblot with the phospho-specific activation loop
antibody, anti-Mst1/2(Thr183P/180P) (19), has been unsuccess-
ful thus far. Direct evidence that Mst1 and/or Mst2 is active in
the unperturbed colon is the finding that the phosphorylation of
Mob1, a highly specific substrate of Mst1/Mst2 in vitro (39),
whose phosphorylation is lost in Mst1-null T cells (4) and Mst1/
Mst2-deficient liver (4), also is lost in the Mst1/Mst2-deficient
colon (SI Appendix, Fig. S6A). The mechanisms regulating Mst1/
Mst2 activation in this compartment are unknown, however, as is
true of Mst1/Mst2 regulation in most mammalian cells, apart
from T cells, where Mst1, through its association with Ras as-
sociation (RalGDS/AF-6) domain family member 5B (Rassf5B/
Nore1B/RAPL), is activated by Rap-GTP (40). Genetic and
some biochemical evidence from Drosophila indicates that Hippo
is activated by inputs arising from several varieties of cell ad-
hesion elements, e.g., the atypical cadherins Fat and Dachsous
involved in planar polarity; the apical–basal polarity assemblies
such as the Crumbs, atypical protein kinase C, and partition-
defective (Par) complexes; and the basolateral discs large-Lethal
giant larvae-scribble complexes, each of which may signal to
Hippo through the cortical actin-associated elements Kibra,
Expanded, and Merlin (2, 3, 41). The contribution of such ele-
ments to Mst1/Mst2 regulation in mammalian intestine remains
to be defined.
Indirect evidence for the loss of active Mst1/Mst1 in the wild-
Yap1 that prevails in the Mst1/Mst2-deficient intestine. In the
wild-type colon, where Yap is visualized reliably because of its
high abundance, nuclear localization of Yap1 is evident in crypt
cells. However, as one moves away from the crypt, Yap1 staining,
although persistent, weakens and is visualized primarily in the
cytosol. This pattern suggests that Mst1/Mst2 activity may be
relatively lower in the crypts and more activated as cells move
toward the lumen. Notably, despite the apparent presence of
does not diminish the abundance of Ki67+cells in this compart-
ment (Fig.5C and SI Appendix, Fig. S5A), consistent with the view
that Yap1, at the abundance found in the wild-type colon, makes
little or no contribution to epithelial proliferation. This notion is
consistent with the findings of Cai et.al. (33), who first reported
that villin-Cre–induced deletion of Yap1 did not alter colonic
development. In contrast, villin-Cre–induced deletion of Salva-
greater than 12 mo, a phenotype similar to but much milder than
that of the Mst1null/Mst2ff/villin-Cre mice. Salvador homolog 1
(Sav1) knockout in the liver (5, 42) also gives a much milder
phenotype than Mst1/Mst2 double knockout in the liver (4–6).
Thus, the phenotype of both the Mst1/Mst1 and Sav1 intestinal
knockouts indicate that Mst1 and/or Mst2 are active in the normal
intestinal epithelium and actively restrain Yap1 transcriptional
to promote proliferation. Thus, despite the evidence indicating an
active role for Yap in maintaining the phenotype of ES cells and
induced pluripotent stem cells (43), Yap1 does not contribute to
the proliferative capacity of the colonic stem cells and transient
amplifying compartments during normal mucosal turnover.
Mst1/Mst2-null intestines develop colonic adenomas within 3
mo of birth. In contrast to the polyps described in the Sav1-de-
ficient colon (33), the polypoid lesions in the Mst1/Mst2-deficient
colon do not exhibit a sawtooth/serrated architecture but appear
simply as hyperproliferative adenomas. Consistent with this con-
ventional appearance, molecular analysis indicates that an acti-
finding perhaps is not surprising in view of the highly proproli-
ferative milieu created by Mst1/Mst2 deficiency, reflected by the
increased expression of cMyc and cyclin E and diminished ex-
pression of the p21 cyclin-dependent kinase inhibitor (SI Appen-
dix, Fig. S6 B and C). In addition, IL-6 expression is up-regulated,
of Signal transducer and activator of transcription 3 (Stat3); ac-
tivated Stat3 is known to be a crucial contributant to the de-
velopment of colonic cancers, e.g., in the Dextran Sulfate sodium/
azoxymethane murine model and presumably in inflammation-
associated colon cancer (44). The early mortality of the Mst1/
Mst2ff/villin-Cre mice leaves open the question of whether these
polyps will evolve into invasive and/or metastatic cancers.
The very high prevalence of Yap1 overexpression in human
colonic cancers and derived cell lines is striking. We find a 95%
prevalence of Yap1 overexpression in colonic cancer specimens,
exceeding the 78% prevalence observed by Steinhardt et.al (35).
In addition, however, our finding that depletion of Yap1 in colon
cancer-derived cell lines that overexpress Yap1 results in a strong
inhibition of their proliferation in vitro provides strong evidence
that Yap1 is likely to be an important driver in this common
cancer. The basis for Yap1 overexpression in human colon
cancer is not known; a preliminary estimate using RNA extracted
from tissue microarrays indicates that Yap mRNA abundance is
increased approximately twofold (SI Appendix, Fig. S7N), an
increase that seems far less than the increase in Yap polypeptide.
Nevertheless, loss of Yap1(Ser127) phosphorylation appears to
be an uncommon occurrence, and additional studies will be
needed to determine the contributions of increased transcription
versus diminished degradation. In addition, the outputs of Yap1
critical to its proproliferative effect in human colon cancer re-
main to be defined. However, it is likely that the ability of Yap1
overexpression to enhance β-catenin transcriptional activity is
one important factor. The frequent overexpression and propro-
liferative impact of Yap1 in colon cancer cell lines is of particular
interest, because, if Yap1 plays little or no role in normal colonic
epithelial turnover, interference with its expression and/or out-
puts may provide attractive therapeutic targets distinct from
those of the Wnt and Notch pathways (45, 46), whose outputs are
indispensable for normal intestinal homeostasis (12–14).
Materials and Methods
Mice. Mst1−/−Mst2ff mice were generated as described previously (4). The
generation of Yap1ff mice is described in ref. 10. Villin-Cre mice were pur-
chased from the Jackson Laboratory. Mst1-null mice with Mst2 deleted in
the intestinal epithelium were generated by breeding Mst1-null/Mst2ff mice
with villin-Cre mice. Animal protocols were approved by the Institutional
Animal Care and Use Committee of Massachusetts General Hospital.
Tissue Microarray and Histological Analysis. Colonic tissues were collected
of Cleveland. Tissue microarray blocks, two cores from each tumor, were
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| www.pnas.org/cgi/doi/10.1073/pnas.1110428108Zhou et al.
retrieved by boiling the slides in Target Retrieval solution (Dakocytomation) Download full-text
for 20 min and visualized by the Envision+ System-HRP kit (Dakocytomation)
following the manufacturer’s instructions and using anti-Yap antibody
(1:200) (4). Slides were counterstained with hematoxylin to visualize the nu-
clei before microscopic analysis. Relative Yap expression and subcellular lo-
calization were classified into seven groups based on the intensity of Yap
staining and its distribution between cytoplasm and nuclei, as shown in the
table in Fig. 6.
Additional materials and methods are described in SI Appendix.
ACKNOWLEDGMENTS. We thank James K. V. Willson for the VACO colon
cancer cell lines, R. Polakiewicz (Cell Signaling Inc.) for the antiYap1(Ser384P)
antiserum, and R. T. Bronson for mouse histologic analyses. This work was
supported in part by National Institutes of Health Grants RO1 DK17776 (to
J.A.), CA136567 (to J.A.), and CA127306 (to S.D.M.), by a grant from the Stand
Up to Cancer-American Association for Cancer Research initiative (to F.D.C.),
and by institutional funds. F.D.C. is a Pew Scholar in the Biomedical Sciences.
D.Z. is supported by the Fundamental Research Funds for the Central Univer-
Planning Program of Fujian Province (Project Number 2009J1010).
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