Deficiency of the LIM-only protein FHL2 reduces intestinal tumorigenesis in Apc mutant mice.
ABSTRACT The four and a half LIM-only protein 2 (FHL2) is capable of shuttling between focal adhesion and nucleus where it signals through direct interaction with a number of proteins including beta-catenin. Although FHL2 activation has been found in various human cancers, evidence of its functional contribution to carcinogenesis has been lacking.
Here we have investigated the role of FHL2 in intestinal tumorigenesis in which activation of the Wnt pathway by mutations in the adenomatous polyposis coli gene (Apc) or in beta-catenin constitutes the primary transforming event. In this murine model, introduction of a biallelic deletion of FHL2 into mutant Apc(Delta14/+) mice substantially reduces the number of intestinal adenomas but not tumor growth, suggesting a role of FHL2 in the initial steps of tumorigenesis. In the lesions, Wnt signalling is not affected by FHL2 deficiency, remaining constitutively active. Nevertheless, loss of FHL2 activity is associated with increased epithelial cell migration in intestinal epithelium, which might allow to eliminate more efficiently deleterious cells and reduce the risk of tumorigenesis. This finding may provide a mechanistic basis for tumor suppression by FHL2 deficiency. In human colorectal carcinoma but not in low-grade dysplasia, we detected up-regulation and enhanced nuclear localization of FHL2, indicating the activation of FHL2 during the development of malignancy.
Our data demonstrate that FHL2 represents a critical factor in intestinal tumorigenesis.
Article: The LIM-only protein DRAL/FHL2 binds to the cytoplasmic domain of several alpha and beta integrin chains and is recruited to adhesion complexes.[show abstract] [hide abstract]
ABSTRACT: LIM proteins contain one or more double zinc finger structures (LIM domains) mediating specific contacts between proteins that participate in the formation of multiprotein complexes. We report that the LIM-only protein DRAL/FHL2, with four and a half LIM domains, can associate with alpha(3A), alpha(3B), alpha(7A), and several beta integrin subunits as shown in yeast two-hybrid assays as well as after overexpression in human cells. The amino acid sequence immediately following the conserved membrane-proximal region in the integrin alpha subunits or the C-terminal region with the conserved NXXY motif of the integrin beta subunits are critical for binding DRAL/FHL2. Furthermore, the DRAL/FHL2 associates with itself and with other molecules that bind to the cytoplasmic domain of integrin alpha subunits. Deletion analysis of DRAL/FHL2 revealed that particular LIM domains or LIM domain combinations bind the different proteins. These results, together with the fact that full-length DRAL/FHL2 is found in cell adhesion complexes, suggest that it is an adaptor/docking protein involved in integrin signaling pathways.Journal of Biological Chemistry 11/2000; 275(43):33669-78. · 4.77 Impact Factor
Article: Focal adhesion kinase interacts with the transcriptional coactivator FHL2 and both are overexpressed in epithelial ovarian cancer.[show abstract] [hide abstract]
ABSTRACT: Abnormal signal transduction arising from integrins and protein tyrosine kinases has been implicated in the initiation and progression of a variety of human cancers. Integrin-mediated signal transduction pathways require regulated cytoplasmic protein-protein interactions. However, little is known about integrin-associated proteins and ovarian cancer. In our study we investigated the association of pp125FAK, a cytoplasmic tyrosine kinase, involved in anchorage-independent growth of tumor cells, and the Four and a Half LIM domain (FHL) protein FHL2, which was recently shown to interact with integrins. Our data demonstrated that pp125FAK and FHL2 form a protein complex in human ovarian carcinoma. Furthermore, we showed that pp125FAK is overexpressed in epithelial ovarian cancer, but virtually absent in normal ovary. Our immunohistochemistry data showed that FHL2 protein expression is also augmented in epithelial ovarian cancer. Taken together, our results demonstrated for the first time FHL2 expression in human ovarian cancer cells, suggesting an important functional role of pp125FAK and FHL2 complex in gynecologic malignancies.Anticancer research 24(2B):921-7. · 1.73 Impact Factor
[show abstract] [hide abstract]
ABSTRACT: We have described the scaffolding protein FHL2 as a component of focal adhesion structures, to which it is recruited via binding to both alpha- or beta-integrin subunits. Using mesenchymal stem cells from wild-type and FHL2-knockout mice, we show here that inactivation of FHL2 leads to impaired assembly of extracellular matrix proteins on the cell surface and to impaired bundling of focal adhesions. Both altered properties can be restored by reexpression of recombinant FHL2 protein in FHL2-null cells. Molecular analysis of integrin-mediated signaling revealed a higher phosphorylation of FAK at tyrosine 925 in FHL2-knockout cells compared to their wild-type counterpart. Consequently, the activation of the mitogenic kinase ERK was more pronounced in knockout cells on cell adhesion. The growth factor-induced activation of ERK, however, was not altered. The perturbed organization of extracellular matrix on FHL2-null cells was improved when the increased activation of MAPK was inhibited. Our findings point to a role of FHL2 in bundling of focal adhesion structures, in integrin-mediated ERK activation, and subsequently in proper allocation of matrix proteins on the cell surface.The FASEB Journal 08/2008; 22(7):2508-20. · 5.71 Impact Factor
Deficiency of the LIM-Only Protein FHL2 Reduces
Intestinal Tumorigenesis in Apc Mutant Mice
Charlotte Labalette1,2., Yann Noue ¨t1,2., Florence Levillayer1,2, Sabine Colnot3,4, Ju Chen5, Valere
Claude6, Michel Huerre7, Christine Perret3,4, Marie-Annick Buendia1,2, Yu Wei1,2*
1De ´partement de Virologie, Institut Pasteur, Paris, France, 2Inserm U579, Paris, France, 3De ´partement d’Endocrinologie Me ´tabolisme et Cancer, Institut Cochin, Paris,
France, 4Inserm U567, Paris, France, 5Department of Medicine, University of California San Diego, La Jolla, California, United States of America, 6De ´partement
d’Anapathologie, Ho ˆpital Be ´gin, Saint Mande ´, France, 7De ´partement d’Infection et Epide ´miologie, Institut Pasteur, Paris, France
Background: The four and a half LIM-only protein 2 (FHL2) is capable of shuttling between focal adhesion and nucleus
where it signals through direct interaction with a number of proteins including b-catenin. Although FHL2 activation has
been found in various human cancers, evidence of its functional contribution to carcinogenesis has been lacking.
Methodology/Principal Findings: Here we have investigated the role of FHL2 in intestinal tumorigenesis in which
activation of the Wnt pathway by mutations in the adenomatous polyposis coli gene (Apc) or in b-catenin constitutes the
primary transforming event. In this murine model, introduction of a biallelic deletion of FHL2 into mutant ApcD14/+mice
substantially reduces the number of intestinal adenomas but not tumor growth, suggesting a role of FHL2 in the initial steps
of tumorigenesis. In the lesions, Wnt signalling is not affected by FHL2 deficiency, remaining constitutively active.
Nevertheless, loss of FHL2 activity is associated with increased epithelial cell migration in intestinal epithelium, which might
allow to eliminate more efficiently deleterious cells and reduce the risk of tumorigenesis. This finding may provide a
mechanistic basis for tumor suppression by FHL2 deficiency. In human colorectal carcinoma but not in low-grade dysplasia,
we detected up-regulation and enhanced nuclear localization of FHL2, indicating the activation of FHL2 during the
development of malignancy.
Conclusions/Significance: Our data demonstrate that FHL2 represents a critical factor in intestinal tumorigenesis.
Citation: Labalette C, Noue ¨t Y, Levillayer F, Colnot S, Chen J, et al. (2010) Deficiency of the LIM-Only Protein FHL2 Reduces Intestinal Tumorigenesis in Apc Mutant
Mice. PLoS ONE 5(4): e10371. doi:10.1371/journal.pone.0010371
Editor: Irene Oi Lin Ng, The University of Hong Kong, Hong Kong
Received October 29, 2009; Accepted March 17, 2010; Published April 28, 2010
Copyright: ? 2010 Labalette et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported in part by grants from the Association pour la Recherche sur le Cancer, the Ligue contre le Cancer (Comite de Paris) and the
French Institut National du Cancer. C.L. was supported by the Ecole Normale Superieure de Lyon and Y.N. by the Canceropele Ile-de-France. The funders had no
role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
. These authors contributed equally to this work.
The four and a half LIM-only protein 2 (FHL2) contains only
LIM domain (named after lin-11, mec-3 and Islet-1), which is a
protein-protein interaction domain. FHL2 has recently emerged as
a signalling protein that is critical in the transduction of signals
from extracellular environment and in the control of gene
expression program in response to different stimuli. In the
cytoplasm, FHL2 interacts with a- and b-integrin subunits and
focal adhesion kinase at focal adhesions where integrins bind
extracellular matrix (ECM) [1–3]. FHL2 is involved in ECM-
integrin receptor interaction, assembly of ECM proteins on the cell
surface and bundling of focal adhesions [4,5]. In response to
external stimuli, FHL2 is translocated to the nucleus where it plays
the role of transcription cofactor by interacting with numerous
transcription factors and coregulators including androgen receptor
(AR), AP1, CREB, PLZF, WT1, SKI, b-catenin, FOXO1, Runx2,
serum response factor (SRF), E4F1 and CBP/p300 [6–18]. Mice
deficient of FHL2 display defects in response to divers stimuli,
including cardiac hypertrophy under b-adrenergic stimulation,
healing defects in skin and intestinal wound and attenuated
neovascularization after corneal injury [19–22].
Several lines of evidence suggest a role of FHL2 in
carcinogenesis. FHL22/2fibroblasts are temporarily resistant to
oncogenic Ras-induced transformation . FHL2 suppression
inhibits anchorage-independent growth of cancer cell lines and
tumor formation in immuno-compromised mice . Up-
regulation of FHL2 has been found in many human cancers
[3,13,24–26]. Several studies have associated high level and
nuclear expression of FHL2 with the aggressiveness of cancer and
bad prognostic [6,27] . Moreover, numerous FHL2-interact-
ing proteins including AR, AP1, PLZF, SKI, WT1, b-catenin,
BRCA1 and E7 of human papillomavirus 16 [29,30] have primary
roles in various human cancers, implying that FHL2 may
participate in transformation process through effects on the
oncogenic activity of its partners.
We have previously demonstrated that FHL2 interacts with b-
catenin and cooperates with CBP/p300 to stimulate transcrip-
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tional activity of the b-catenin/TCF4 complex [13,18]. b-catenin
is a key effector of the Wnt signalling pathway that has central
roles not only in embryogenesis and tissue homeostasis but also in
tumorigenesis (for review, see ). In human colorectal cancer,
an overwhelming majority of cases carry mutations in Wnt
pathway components including the adenomatous polyposis coli
gene (Apc), axin and b-catenin. All the mutations lead to
constitutive activation of the Wnt signalling pathway characterized
by the formation of constitutive nuclear b-catenin/TCF complex.
Genetic modelling of activation of the Wnt signalling pathway in
the intestine has been made possible by the generation of Apc
mutant mice that recapitulate human colonic carcinogenesis.
These mice, epitomized by the ApcMin/+mouse, have been used
intensively to test genetically the ability of candidate genes to
enhance or repress adenoma formation in vivo.
In this study, we used the ApcD14/+model to address the
contribution of FHL2 activity to the intestinal transformation
process. ApcD14/+mice in which exon 14 of the Apc gene is deleted
in one allele spontaneously develop multiple polyps along their
intestinal tract . All tumors lose the Apc wild type (wt) allele,
accumulate b-catenin in the nucleus and overexpress the b-catenin
target genes cyclin D1 and c-myc . Using a genetic approach,
we demonstrate that loss of FHL2 significantly suppresses tumor
multiplicity in ApcD14/+mice. Our analysis of FHL2 expression in
murine and human intestinal lesions revealed its progressive up-
regulation and enhancement of nuclear expression during disease
development. These results provide unequivocal evidence of the
implication of FHL2 activity in the transformation process.
FHL2 deficiency reduces intestinal polyposis in ApcD14/+
To determine the in vivo effects of FHL2 in intestinal
tumorigenesis, we took advantage of FHL22/2and ApcD14/+mice
to investigate how FHL2 affects the phenotypes associated with
Apc loss [32,33]. Despite defects in bone formation [15,34],
FHL22/2mice live a normal lifespan . Histological analysis of
intestinal epithelium in FHL2-null mice revealed no major
alterations in crypt-villus architecture of the small intestine and
in the crypt structure of the colon (Fig. 1A). Moreover, FHL2-
deficient cells remained in cycle in the crypt, as demonstrated by
immunohistochemistry (IHC) for the proliferation marker Ki-67
(Fig. 1A), suggesting that FHL2 is dispensable for proliferation of
intestinal epithelial cells. No significant difference was observed in
the presence and localization of differentiated intestinal cell
lineages (enterocytes, enteroendocrine, goblet cells and Paneth
cells) in the FHL2 mutant mice (data not shown). We crossed the
ApcD14/+strain on the C57Bl/6 background  into the FHL22/2
strain on the hybrid Black Swiss-129-SV/J background .
Intercrosses from the F1 generation produced ApcD14/+mice with
three different FHL2 genotypes. Only the F2 generation was used
for further analysis in order to rule out any influence of genetic
background. In contrast to ApcD14/+mice on the C57Bl/6
background which die at 6-month-old , ApcD14/+FHL2+/+
mice on the mixed genetic background do not succumb to intestinal
adenomas until 20-month-old, which is a late stage in a mouse life
(the life span of wt animals on the same genetic background was
about 24-month-old). Because most of ApcD14/+FHL22/2mice died
at 24-month-old, the impact of FHL2 deficiency on time course of
death was not significant in ApcD14/+animals of this study (data not
Mice were first sacrificed at 3 months. Analysis of the intestine
revealed similar lengths for ApcD14/+mice of each FHL2 genotype
(data not shown). We divided the intestinal tract into small
intestine and colon segments and scored for polyp number,
location, and size. Despite few adenomas developed at this stage,
tumors occurred more frequently in ApcD14/+FHL2+/+animals
(73%, n=11) than in ApcD14/+FHL22/2littermates (40%, n=10).
We then examined the intestine at 11 months. Compared to
ApcD14/+FHL2+/+littermates, the polyp number in the small
intestine decreased by 68% in ApcD14/+FHL22/2mice and 52% in
ApcD14/+FHL2+/2mice (Figs. 2A and 2B). Precisely, while the
polyp number in ApcD14/+FHL2+/+could reach more than 40, the
great majority of ApcD14/+FHL22/2mice developed less than 9
tumors and none of the animals had more than 19 polyps (Fig. 2C).
Analysis by the chi-square test showed that the difference in the
Figure 1. Histological analysis of normal intestine in FHL22/2
mice and intestinal adenomas in ApcD14/+FHL22/2mice. A.
Normal intestinal architecture in FHL22/2mice. Intestine and colon
sections from 11 month-old wt and FHL22/2mice were stained with
H&E or Ki-67 by immunostaining. B. Intestinal adenomas in ApcD14/+
FHL2+/+and ApcD14/+FHL22/2mice. Original magnifications, X100 (A
FHL2 in Tumorigenesis
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FHL22/2mice in Fig. 2C was statistically significant (p,0.0036),
indicating that FHL2deficiency inhibits polyp formation. To test the
effect of FHL2 deficiency on tumor growth, we measured the sizes of
polyps in ApcD14/+FHL2+/+and ApcD14/+FHL22/2mice (Fig. 2D)
and carried out Wilcoxon Rank Sum test to determine if there was a
difference in tumor size between the two genotypes. The results
showed that tumor sizes in ApcD14/+FHL22/2mice were not
significantly different from those in ApcD14/+FHL2+/+mice (p.0.2,
Wilcoxon Rank Sum test), suggesting that loss of FHL2 activity does
not affect tumor growth. Histological evaluation of the adenomas
revealed no difference in gross histological characteristics or grade
between ApcD14/+FHL2+/+and ApcD14/+FHL22/2mice (see Fig. 1B).
No difference was observed in the distribution of polyps throughout
the intestine between ApcD14/+FHL2+/+and ApcD14/+FHL22/2mice
(data not shown). We next analyzed the lesions in the colon. Few
polyps developed in the colon at 11 months in ApcD14/+mice with
any of the FHL2 genotypes. However, while tumors weredetected in
FHL22/2animals (n=18) developed polyps, indicating that
the frequency of tumors in the colon is also decreased by
FHL2 deficiency. We therefore conclude that loss of FHL2
suppresses tumor formation in the intestine by probably acting on
the initiation of tumors.
b-catenin and its targets cyclin D1 and c-myc are
activated in adenomas independent of FHL2 genotype
We have previously shown that FHL2 interacts with b-catenin
and is a crucial regulator of cyclin D1 expression in fibroblasts .
To investigate the molecular mechanisms of polyp formation in
ApcD14/+FHL22/2mice, we analyzed expression of b-catenin and
its targets cyclin D1 and c-myc in tumors. Immunohistochemical
staining revealed strong nuclear expression of b-catenin in polyps
from both ApcD14/+FHL2+/+and ApcD14/+FHL22/2mice (Fig. 3A,
a and b), indicative of activation of the b-catenin/Wnt pathway.
Figure 2. Loss of FHL2 reduces intestinal polyp multiplicity. (A) Representative images of intestines from 11-month-old ApcD14/+FHL2+/+and
ApcD14/+FHL22/2mouse. Note the marked decrease in the number of polyps in ApcD14/+FHL22/2mice. (B) Total number of intestinal polyps was
counted in 11 ApcD14/+FHL2+/+, 16 ApcD14/+FHL22/+and 18 ApcD14/+FHL22/2mice at 11-month-old. Compared to ApcD14/+FHL2+/+littermates,
significant reduction in polyp number was observed in ApcD14/+FHL22/+(p,0.0046) and ApcD14/+FHL22/2mice (p,0.0001). (C) Polyp distributions
are expressed as the percentages of mice having 0–9, 10–19, 20–29, 30–39 or more than 40 polyps in the intestine. (D) FHL2 deficiency had no
effect on tumor growth. The percentages of polyps in each mouse with sizes less than 2 mm, between 2 to 5 mm and more than 5 mm are
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As direct targets of b-catenin, cyclin D1 and c-myc are closely
involved in intestinal tumorigenesis associated with activation of
Wnt signalling [35–38]. To assess the impact of FHL2 deficiency
on the targets, we examined expression of cyclin D1 and c-myc at
both RNA and protein levels in the intestine of ApcD14/+FHL22/2
mice. As shown in Fig. 3B, the mRNA levels of cyclin D1 and c-
myc were indistinguishable in nontumor intestine between ApcD14/+
increased in adenomas from mice of both genotypes. Whereas
expression of cyclin D1 in adenomas from ApcD14/+FHL22/2mice
showed significant higher level than that in tumors from ApcD14/+
FHL2+/+mice, no significant difference of c-myc expression was
observed in adenomas between ApcD14/+FHL2+/+and ApcD14/+
FHL22/2animals (Fig. 3B). In parallel, immunohistochemical
animals, and were strongly
analysis showed nuclear expression of cyclin D1 and c-myc in
the proliferative compartment of normal crypts in both ApcD14/+
FHL22/2and ApcD14/+FHL2+/+animals (Fig. 3A, e, f, g, and h).
Moreover, in keeping with the proliferative property of tumor cells,
cyclin D1 was highly expressed in all adenomas, regardless of the
FHL2 genotype (Fig. 3A, c and d). In addition, we compared
expression of other b-catenin targets such as Axin 2 and
metalloproteinase matrilysin (MMP-7) between tumors and adjacent
nontumorous tissues by real time RT-PCR [39,40]. Axin 2
and MMP-7 were uniformly activated in tumor samples, with no
significant difference among ApcD14/+FHL22/2
FHL2+/+animals (data not shown). We also performed IHC with
the intestine of ApcD14/+FHL22/2mice for c-Jun and peroxisome
proliferators-activated receptor d (PPARd), which have been shown
Figure 3. Activation of the Wnt signalling in the intestine of ApcD14/+FHL22/2mice. (A) Immunostaining for b-catenin in adenomas (a, b), for
cyclin D1 in adenomas (c, d) and normal intestine (e, f) and for c-myc in normal intestine (g, h) in ApcD14/+FHL2+/+(a, c, e, g) and ApcD14/+FHL22/2mice
(b, d, f, h). Sections were photographed at the same magnification. Original magnifications: X200. (B) Activation of the Wnt target genes cyclin D1 and
c-myc in adenomas is unaffected by FHL2 deletion. Real-time RT-PCR analysis of cyclin D1 and c-myc expression was performed with intestinal tumor
(T) and adjacent normal tissue (NT). Cyclin D1 and c-myc expression was normalized to 18S RNA and normalized values in normal intestine of ApcD14/+
The p value is calculated according to Student’s test.
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to be the Wnt targets critical for the control of intestinal
tumorigenesis [41,42]. The expression of c-Jun and PPARd was
similar between ApcD14/+FHL22/2and ApcD14/+FHL2+/+mice in
both normal and adenomatous tissues (data not shown). Taken
together, these findings indicate that Wnt signalling remains
constitutively active in the lesions of ApcD14/+FHL22/2mice and
that the inhibitive effects of FHL2 deficiency on intestinal neoplasia
are not associated with defects in the activation of c-myc and
Loss of FHL2 increases cell migration
Cell migration represents a fundamental mechanism involved in
the maintenance of epithelial homeostasis in the intestine.
Impaired cell migration correlates with tumor growth and
neoplastic progression during development of colorectal cancer
. The role of FHL2 in assembly of extracellular matrix
prompted us to examine the impact of FHL2 deficiency upon cell
migration along the crypt-villus axis. ApcD14/+FHL22/2and
ApcD14/+FHL2+/+mice were administrated with BrdU by intra-
peritoneal injection and sacrificed 2 h and 48 h later for BrdU-
positive cell scoring in longitudinal sections with the base of crypt
set as position 0. As shown in Fig. 4A, no difference was observed
between ApcD14/+FHL22/2and ApcD14/+FHL2+/+in the position
and the number of BrdU-positive cells after 2 h exposure to BrdU.
At 48 h, however, ApcD14/+FHL22/2cells moved to a higher
position than ApcD14/+FHL2+/+cells along the crypt-villus axis
(Fig. 4A). Indeed, an average of 87.4% labelled ApcD14/+FHL2+/+
cells were located at positions between 0 and 20 in longitudinal
(p,6.1026, Student’s t test), and nearly 70% of BrdU-positive
cells in ApcD14/+FHL22/2mice already migrated to positions
higher than 20 against 12.6% in ApcD14/+FHL2+/+animals
(p,1026), showing an accelerated migration rate (Figs. 4A and
4B). Moreover, the number of BrdU-positive cells in ApcD14/+
FHL22/2mice was significantly increased compared to ApcD14/+
FHL2+/+littermates (p,0.005) (Figs. 4A and 4B). As we found no
anomaly in the organization of proliferative and differentiated
compartments in ApcD14/+FHL22/2intestine (see Fig. 1A and data
not shown), the increased number of BrdU-labelled cells in these
animals might be attributed to the rapid movement of the
enterocytes from the proliferative compartment to the top of villi.
The impact of FHL2 on cell migration was independent of Apc
mutations, as the intestinal cells in FHL22/2mice moved also
faster than wt cells along the crypt-villus axis at 48 h post-BrdU
injection (Fig. 4C). These data indicate that loss of FHL2 activity
has positive effects on cell migration. As accelerated cell migration
by chemopreventive agents has been reported to be beneficial for
eliminating deleterious cells, the tumor suppression function of
FHL2 deficiency may be achieved in part through acceleration of
cell migration in ApcD14/+mice.
Up-regulation of FHL2 in human intestinal adenoma and
Next, we examined FHL2 expression in normal and tumorous
tissues from ApcD14/+mice by IHC with anti-FHL2 antibody,
which does not cross-react with any other protein, since it detects
no signal in ApcD14/+FHL22/2intestine (data not shown). In
contrast to normal epithelium where FHL2 signal was barely
detectable, strong nuclear staining of FHL2 was observed in
tumors developed in ApcD14/+mice (Fig. 5A). We then examined
FHL2 expression in the intestine of ApcD14/+animals by real time
RT-PCR. As shown in Fig. 5B, the level of FHL2 transcript was
significantly increased in tumor samples, compared to normal
adjacent intestinal tissues. To assess the expression of FHL2 in
human tumors, we analyzed five human colon adenomas with
low-grade dysplasia, five human colon adenomas with high-grade
dysplasia and five carcinomas by IHC for FHL2. Staining of
adjacent normal crypts was used as internal control. In keeping
with a previous report , FHL2 protein, undetectable in normal
tissues, was expressed in all high-grade dysplasia and carcinomas
analyzed (Fig. 5C, compare a with c and d). Interestingly, barely
detectable in low-grade dysplastic tissues, FHL2 was significantly
Figure 4. FHL2 deficiency accelerates enterocyte movement. (A)
Representative images of BrdU-positive cells along the crypt-villus axis
at 2 h and 48 h post-BrdU labelling in ApcD14/+FHL2+/+and ApcD14/+
FHL22/2mice. (B) Distribution of BrdU-positive cells at 48 h. The crypt
base was set as position 0. Three mice of each genotype were analyzed.
(C) Representative images of BrdU-positive cells along the crypt-villus
axis at 48 h post-BrdU labelling in wt and FHL22/2mice. Original
magnifications: X200 (A and C).
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increased in high-grade dysplasia and cancer cells (Fig. 5C,
compare b with c and d), showing a progressive expression pattern.
Moreover, contrasting markedly with the predominant cytoplas-
mic localization in high-grade dysplasia, intense nuclear accumu-
lation of FHL2 protein was observed in carcinomas (Fig. 5C, e).
Taken together, these data suggest that FHL2 activation in
intestinal cells may be associated with pre-cancerous and
cancerous stages and that FHL2 protein may accumulate in the
nucleus during neoplastic progression.
In this study, we provide new insights into the FHL2 function in
intestinal tumorigenesis. By employing a genetic approach in vivo,
Figure 5. Up-regulation of FHL2 in intestinal tumors from ApcD14/+mice and human colon tumors. (A) FHL2 immunostaining of normal
intestine and adenoma from ApcD14/+mouse. Original magnifications, X200. (B) Quantitative RT-PCR analysis of FHL2 expression in adenoma and
adjacent normal tissue from five ApcD14/+mice. FHL2 expression was normalized to 18S RNA. The ratio of the FHL2/18S signal in NT was arbitrarily set
at 1. The average values and standard deviations for three independent experiments from five ApcD14/+mice are shown. Average fold induction in
adenomas is 1.73 (p=0.011, Student’s t test). (C) FHL2 expression in normal human colon (a), in human low-grade (b) and high-grade colon dysplasia
(c) and in human colon carcinoma (d) by immunohistochemistry. Control staining on high-grade dysplasia and human colon carcinoma was
performed as FHL2 staining without the primary anti-FHL2 antibody. Original magnifications, X200. Part of the image of human colon carcinoma was
further enlarged to better visualize individual cells (e).
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we demonstrate that loss of FHL2 drastically suppresses
tumorigenesis in the murine intestine, which may be partly due
to the positive effects of FHL2 deficiency on cell migration in
intestinal epithelium. In the study of human colorectal tumors, we
found that FHL2 expression might be closely related to disease
stages: the level of FHL2 in high-grade dysplasia is significantly
elevated compared to normal tissues as well as low-grade dysplasia
but clearly below that in carcinomas. Moreover, cellular
localization of FHL2 switches from predominant cytoplasmic
expression in high-grade dysplasia to enhanced nuclear accumu-
lation in cancer cells.
The ApcD14/+FHL22/2mouse model underscores that animals
deficient for FHL2 are less susceptible to oncogene-induced
tumors. However, how FHL2 is involved in the proliferative and
transforming functions of the Wnt signalling in the intestine is not
clear. Previous report has shown that the D-type cyclins, in
particular cyclin D1, are dramatically down-regulated in FHL22/2
fibroblasts . The results of this study indicate that cyclin D1
expression is not dependent of FHL2 in the context of intestinal
epithelium. It appears that cyclin D1 expression was stronger
in tumors developed in ApcD14/+FHL22/2mice than those in
ApcD14/+FHL2+/+animals. As FHL2 plays an important role in
cell proliferation, it is possible that high level of cyclin D1 may
counteract FHL2 deficiency in the stimulation of tumor cell growth
in ApcD14/+FHL22/2mice. We also observed that the expression of
other b-catenin targets including c-myc, Axin2, MMP-7, c-Jun and
PPARdis not dependent of FHL2 in the intestine. Further analysis
of the involvement of Wnt and FHL2 signalling in this specific
tissue should provide insight into whether the Wnt and FHL2
signalling pathways function in an interdependent regulatory
The loss of FHL2 activity in intestinal epithelium clearly
accelerates cell migration, which constitutes a fundamental
mechanism in the control of tissue homeostasis. Apc mutation in
Apcmin/+mice is associated with decreased cell migration, resulting
in a prolonged residence time for enterocytes in the intestine .
Chemopreventive agents presumably exert their anti-tumor effects
by increasing the rates of cell migration, thus eliminating rapidly
deleterious cells [44–46]. The mechanism of the increase of cell
migration in FHL2 mutant intestine remains to be elucidated.
Because FHL2 is involved in focal adhesion, ECM-cell interaction
and assembly of the extracellular matrix [4,5], a role for FHL2 in
the regulation of cell mobility seems an attractive possibility. The
tumorigenic effects of proteins that regulate cell migration have
been previously illustrated by SPARC, a matricellular protein that
associates with the extracellular matrix and modulates cell-ECM
interaction . Deficiency of SPARC augments enterocyte
movement in Apcmin/+
mice, thereby suppressing adenoma
formation . Moreover, the positive effects of FHL2 mutation
on cell migration in ApcD14/+FHL22/2mice correlate with the
observation that FHL2 deficiency perturbs essentially adenoma
Consistent with an early report showing nuclear expression of
FHL2 in gastrointestinal cancerous tissues , our data further
indicate that high level expression and nuclear accumulation of
FHL2 in human colorectal cancer might reflect disease progres-
sion towards the malignant state. This observation is correlated
with recent discoveries that invasive breast cancers have much
stronger expression of FHL2 than premalignant ductal carcinoma
in situ samples  and that increases of nuclear FHL2 in prostate
cancer are strongly correlated with dedifferentiation of cancer cells
and with high Gleason grade . Altogether, these results support
the view that the intensity and localization of FHL2 expression in
cancer cells may serve as a biomarker in classifying tumor stage
and predicting disease outcome . Indeed, breast cancer
patients with tumors expressing low amounts of FHL2 have a
better survival compared to those with high intra-tumoral FHL2
expression  and activation of nuclear FHL2 signalling is linked
to aggressiveness and recurrence of prostate cancer . In
colorectal cancer, further examination of FHL2 expression in a
large panel of samples may permit to determine if FHL2 could be
used as a marker for stage classification of the disease.
Mechanistically, the sensor function and transcription coregulator
activity of FHL2 can provide a ready explanation for the up-
regulation and nuclear translocation of FHL2 in highly malignant
cells. In prostate cancer cells, FHL2 is strongly induced by
androgens . As a coactivator of AR, FHL2 in turn robustly
stimulates the AR activity that is critical for prostate cancer
progression [48,49]. In colorectal cancer, the molecular hallmark
is the accumulation of b-catenin in the nucleus. However,
expression of the b-catenin target genes analyzed in this study
seems not be affected by FHL2 deficiency. Nevertheless, up-
regulation of FHL2 and its nuclear accumulation may stimulate
transcription activity of not yet uncovered b-catenin targets or
other transcription complexes, which may be required for
Our data highlight FHL2 as important molecule in mediation of
the transformation process and suggest that disruption of the
FHL2 signalling may provide a viable and specific strategy for
therapeutic intervention in colorectal cancer.
Materials and Methods
Male ApcD14/+animals on the C57BL/6 background were
crossed with female FHL22/2mice on the hybrid Black Swiss-
129-SV/J background [32,33]. F1 ApcD14/+FHL2+/2mice were
intercrossed. Only the F2 ApcD14/+mice with the three FHL2
genotypes were included in the study. Mice were housed under
pathogen-free conditions. All animal procedures were carried out
in accordance with French government guidelines. All experi-
ments involving mice have been approved by Institut Pasteur.
Tumor scoring and histopathological analysis
Intestines were removed from F2 ApcD14/+mice with different
FHL2 genotypes at 3 or 11 months and fixed in 4% PFA. Polyps
were counted and measured using a Nikon dissecting microscope
at66 magnification by an observer blinded to the genotype of the
mice. Intestines were rolled in the ‘‘Swiss rolls’’ configuration and
proceeded for paraffin embedding .
Sections were cut from paraformaldehyde (PFA)-fixed paraffin-
embedded ‘Swiss rolls’. Tissues were dewaxed in xylene and
unmasked in a citric acid solution (H-3300, Vector Laboratories)
at 96uC for 45 min. Normal horse serum (2.5%; S-2000, Vector
Laboratories) was used as blocking solution. Sections were
incubated at room temperature with primary antibodies for
60 min. Endogenous peroxidase activity was blocked by incubat-
ing sections with 3% hydrogen peroxide. The sections were then
incubated with secondary antibodies (Vector Laboratories) for
30 min. The peroxidase reaction was developped using DAB
Substrate Kit (SK-4100, Vector Laboratories). Hematoxylin was
used for counterstaining. Antibodies against b-catenin and cyclin
D1 (NeoMarkers), FHL2 (MBL), c-myc (Santa Cruz) and Ki-67
(Novocastra) were used as primary antibodies. Images were
obtained on a FXA Microphot microscope equipped with a
Nikon D1 camera controlled by Nikon capture.
FHL2 in Tumorigenesis
PLoS ONE | www.plosone.org7April 2010 | Volume 5 | Issue 4 | e10371
Quantitative RT-PCR analysis
Total RNA was extracted from polyps and adjacent non
tumoral intestines removed from 11-month-old ApcD14/+mice.
Real-time PCR was performed as described previously .
Measurement of enterocyte migration
Mice at 10 weeks were administrated with Bromodeoxyuridine
(BrdU) (50 mg/kg mouse body weight), followed by immunohis-
tochemical analysis with anti-BrdU (Dako). Fields containing crypt
transverse sections were selected randomly at several locations for
BrdU positive cell counting in crypt-villus units.
Human polyp samples
Fifteen human colon specimens were obtained in Ho ˆpital Be ´gin
at Saint Mande ´ and fixed in PFA. Informed consent of patients
was obtained at the hospital and the study was performed in
accordance with European Guidelines for biomedical research.
We are grateful to Christine Neuveut and Be ´atrice Romagnolo for
insightful discussion. We thank Jennifer Dahan for technical assistance.
Conceived and designed the experiments: CL YN YW. Performed the
experiments: CL YN FL YW. Analyzed the data: CL YN FL SC VC MH
CP MAB YW. Contributed reagents/materials/analysis tools: SC JC VC
MH CP. Wrote the paper: CL MAB YW.
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