APC haploinsufficiency coupled with p53 loss sufficiently
induces mucinous cystic neoplasms and invasive pancreatic
carcinoma in mice
Tzu-Lei Kuo1, Ching-Chieh Weng1, Kung-Kai Kuo2, 7, Chiao-Yun Chen3, 7,
Deng-Chyang Wu4, 5, 7, Wen-Chun Hung6, Kuang- Hung Cheng1, 7, 8#
1
Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung, Taiwan 804.
2
Division of Hepatobiliopancreatic Surgery, Department of Surgery, 3Department of Medical
Imaging, 4Division of Gastroenterology, Department of Internal Medicine, Kaohsiung
Medical University Hospital, Kaohsiung, Taiwan 807, 5Division of Internal Medicine,
Kaohsiung Municipal Hsiao-Kang Hospital, Kaohsiung Medical University, Kaohsiung,
Taiwan 807. 6National Institute of Cancer Research, National Health Research Institutes,
Tainan, Taiwan 704. 7Center for Stem Cell Research, Kaohsiung Medical University,
Kaohsiung, Taiwan 807. 8Department of Medical Laboratory Science and Biotechnology,
Kaohsiung Medical University, Kaohsiung, Taiwan 807.
The total word count of the manuscript is: 4,416
# Correspondence should be addressed: Kuang-hung Cheng, Ph.D., Institute of
Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung Taiwan 80424, Tel:
+886 7 5252000- 5817, Fax: +886 7 5250197. E-mail: [email protected]
Keywords: APC; Haploinsufficiency; P53; Mucinous cystic neoplasms; Pancreatic
cancer
1
Abstract
Adenomatous polyposis coli (APC), a tumor suppressor gene critically involved in
familial adenomatous polyposis, is integral in Wnt/β-catenin signaling and is
implicated in the development of sporadic tumors of the distal gastrointestinal tract
including pancreatic cancer (PC). Here we report for the first time that functional APC
is required for the growth and maintenance of pancreatic islets and maturation.
Subsequently, a non-Kras mutation-induced pre-malignancy mouse model was
developed; in this model, APC haploinsufficiency coupled with p53 deletion resulted
in the development of a distinct type of pancreatic premalignant precursors, mucinous
cystic neoplasms (MCNs), exhibiting pathomechanisms identical to those observed in
human MCNs, including accumulation of cystic fluid secreted by neoplastic and
ovarian-like stromal cells, with 100% penetrance and the presence of hepatic and
gastric metastases in > 30% of the mice. The major clinical implications of this study
suggest targeting the Wnt signaling pathway as a novel strategy for managing MCN.
Introduction
Pancreatic cancer (PC) is the fourth most common cause of adult cancer mortality and
among the most lethal human cancers. The 5-year survival is only 6%(1-3). Unlike
other malignancies, no marked improvement has been achieved in PC survival. The
signature molecular alterations in PC include multiple evolutionary steps of the
2
precursor lesions of which progression involves the acquisition of muations in Kras,
Ink4a, p53, SMAD4 and APC or β-catenin(2, 4-6). Recently studies have recognized
that PC can develop from 3 distinct types of precursor lesion that affect the pancreatic
ducts: pancreatic intraepithelial neoplasms (PanINs), which are small and focal;
intraductal papillary mucinous neoplasms (IPMNs), which are moderate-sized,
papillary cystic lesions lined by mucin-producing tall columnar epithelium; and
mucinous cystic neoplasms (MCNs), comprising oligomegacysts with a single thin
layer of cuboidal and flattened epithelium and associated progesterone receptor (PR)+
as well as estrogen receptor (ER)+ ovarian-like stroma(7-9). These lesions exhibit
distinct histopathological characteristics and clinical significance but share a common
mutation profile. The basis of these biological differences is unknown but may be
associated with the cell of origin, various mutation combinations, the order of the
mutational events or other factors(5).
The APC gene was first characterized as a crucial tumor suppressor gene of the distal
gastrointestinal tract, and germ-line mutations in APC cause familial adenomatous
polyposis (FAP)(10, 11). Le Borgneeta and Farahmand et al. recently described a 14
year old girl and 29 year old man with FAP who presented with concurrent with solid
pseudopapillary tumor (SPT), a large encapsulated pancreatic mass with cystic and
3
solid components(12). Somatic mutations in APC are frequently observed in sporadic
colon and rectal tumor, however, only rare mutations (<4%) are reported in pancreatic
ductal adenocarcinomas (PDAC)(6, 13-15). It is notable that somatic mutations in
the APC gene are found more commonly in rare types of pancreatic tumors, such as
solid-pseudopapillary tumors acinar carcinomas and pancreatoblastomas(16, 17).
Alternatively, several independent studies revealed that the activation of Wnt pathway
through epigenetic downregulation of APC or secreted frizzled-related protein (SFRP)
genes or increased Wnt ligand secretion are often found associated with more
advanced human PanINs and PDAC(18-20).(21)
APC negatively regulates the Wnt/ -catenin pathway and promotes cytosolic
-catenin polyubiquitination and degradation(22, 23). During embryogenesis, the
repression of embryonic Wnt signaling is required for gastrointestinal and
hepatopancreas progenitor specifications and development(24). Wnt/β-catenin is
reactivated in PanINs, and its expression levels gradually increase during disease
progression(21, 25). In addition to targeting -catenin for degradation, APC is also
involved in microtubule dynamics, cell polarity and chromosome segregation(26-28).
Studies of genetically modified APC-deficient mouse strains demonstrated that APC
is crucial in colon, skin, thymus and nervous system development as well as neoplasia
4
as well(29-31). To understand APC-mediated tumor suppression in pancreatic
tumorigenesis, we first assessed its role in pancreatic organogenesis and whether APC
loss affects pancreatic development or homeostasis. Subsequently, a non-Kras
mutation-induced premalignancy mouse model was developed; in this model,
heterozygous loss of one APC allele coupled with p53 deletion markedly accelerated
pancreatic tumor progression in mice.
Results
Conditional APC deficiency lethally impairs fetal islet development in newborn
mice.
To assess the effects of APC deletion on pancreatic morphogenesis, APCCKO/CKO mice
were crossed with the Pdx1-Cre transgenic strain, which directs the expression of Cre
recombinase to the epithelial lineages of the pancreas during embryogenesis. The
allele was engineered to sustain Cre-mediated deletion of exon 14, resulting in loss of
the APC protein (Supplementary Figure 1). The control and heterozygous mice
exhibited
no discernible phenotypes
(Supplementary
Figure
2).
APCCKO
homozygous mice harboring Pdx1-Cre transgenic strains were documented using
allele-specific PCR genotyping and Western blot analysis to delete the locus and
5
eliminate the truncated APC protein in the pancreas, separately (Supplementary
Figure 1 and data not shown).
When
the
Pdx-1CreApcCKO/+ and
APCCKO/CKO
mice
were
interbred,
no
Pdx1-CreAPCCKO/CKO null pups were obtained after weaning (none of the 62
neonatally genotyped mice were Pdx1-CreAPCCKO/CKO positive), strongly suggesting
in utero or neonatal lethality. During embryogenesis, the various living genotypes of
the mouse embryos were recovered at a Mendelian ratio. No gross phenotypic
differences were observed between the embryos during dissection on embryonic day
(E)10, E12, E14 and E16 (Fig. 1a, i, q; data not shown). APC-positive cells were
detected in a subpopulation of pancreatic progenitor cells in developing pancreata of
Pdx1-CreAPCwt mouse embryos, but not in prenatal pancreas of Pdx1-CreAPCCKO/CKO
or Pdx1-CreAPCCKO/CKOp53L/L embryos (Fig. 1b, c, j, k, r, s). Remarkably, the
Pdx1-Cre APCCKO/CKO mice exhibited reduction in beta-cell mass and islet number
disrupting the maturation of the prenatal pancreas (Fig. 1d-f, l-n, t-v and
Supplementary Figure 3). Mechanistic analyses showed that the Pdx-1Cre-mediated
APC deficient mice exhibited altered gastrointestinal development associated with
duodenal atresia causing neonatal death (Fig. 1g, h, o, p).
6
Heterozygous loss of APC promotes MCN progression and p53 loss.
A previous study using APCMin/+ (Min, multiple intestinal neoplasia) mice reported
that the increased multiplicity and invasiveness of intestinal adenomas were
associated with p53 deficiency(32),(33). P53 is involved in the transcriptional
up-regulation of APC gene expression in response to DNA damage, and p53 is
inactivated in > 50% of PDAC, particularly late-stage tumors(34). To evaluate the
association between APC and p53 defects in PC, the phenotypes of the mice with
Pdx1-Cre-mediated deletion of the APCCKO/CKO allele on a p53lopx/loxp background
were compared. PCR genotyping of the offspring was performed as described in the
detailed Methods section (Fig. 2a). The expression levels of the altered APC and p53
alleles and β-catenin were confirmed using Western blot analysis (Fig. 2b). As
reported previously, Pdx1-Crep53L/L mice with germline wild-type APC were
generally healthy until the age of 50 weeks(35). In this setting, we still observed that
homozygous
APC
deletion
results
in
neonatal
death.
Strikingly,
all
Pdx1-CreAPCCKO/+p53L/L mice (n=51) developed a swollen abdomen with a palpable
abnormal mass between 16 to 24 weeks. Pairwise log rank tests revealed that the
average survival of Pdx1-CreAPCcko/+p53L/L mice was significantly shorter than that
of the Pdx1-Crep53L/L and Pdx1-CreAPCCKO/+ mice (p<0.01; Fig. 2c). Macroscopic
examination and histological analysis revealed that the pancreatic lesions arising from
7
the mice were MCN (Fig. 2d&e). Hyperlipidemia frequently occurs in patients with
pancreatic disease and this MCN mouse model also exhibited elevated plasma
cholesterol and triglyceride levels (Supplementary Figure 4).
For serial kinetic histopathological analysis of the pancreas, the Pdx1-CreAPCCKO/+
p53L/L mice were euthanized and autopsied at 7, 14 and 24 weeks (Fig. 2d). As shown
in Figure 2e, histopathological analysis revealed that the pancreatic tissue of the
Pdx1-CreAPCCKO/+p53L/L mice demonstrated the full spectrum of MCNs lesions,
including cystic lesions that increased in size (> 2 cm) at later time points. All of these
mice (100%) exhibited large cystic pancreata namely mucinous cystadenomas,
characterized by the presence of unilocular megacystic lesions with mucoid/watery
cyst content, and nodules or peripheral calcification on the cyst wall resembling
human MCN (Supplementary Figure 5). Alcian blue and PSA staining revealed
mucin secreting in murine MCNs (Fig. 2f). IHC assessment of the murine MCNs
stained positive for Mucin 4 (Fig. 2g). Most importantly, the MCN lesions in these
mice exhibited typical human MCN features with high levels of PR and ER
immunostaining around the stromal cells (Fig. 2h).
Immunopathological characterization of MCN in mice with conditionally
8
inactivated APC and p53.
Subsequently, the accessory signaling pathways in the murine MCN lesions were
assessed, IHC analysis of the lesions at 14 and 24 weeks implicated cellular
differentiation and early development of signaling pathways during the formation of
MCNs revealing positive staining for the epithelial ductal markers Dolichos biflorus
lectin (DBA) and E-cadherin, TGFβ1, BMP4, Wnt1, Notch1/Hes1, EGFR and
phospho-Akt, and lack of acinar (amylase) and islet (insulin) marker expression (Fig.
3 and data not shown). Tumor microenvironment analysis revealed intense collagen
type I (col-1), α-smooth muscle actin (SMA) and vimentin immunostaining
predominantly localized in the fibroblast compartment of the MCNs, indicating
abundant tumor-reactive stromal elements. Ki67 staining revealed that the
proliferative
activity
in
these
MCNs
was
similar
to
that
in
Pdx1-CreLSLKrasG12Dp53L/L mice. However, these lesions revealed more intense
TUNEL staining than did the Pdx1-CreLSLKrasG12Dp53L/L lesions (Supplementary
Figure 6).
Metastatic potential of murine MCNs in Pdx1-CreAPCCKO/+p53L/L mice.
Although most Pdx1-CreAPCCKO/+p53L/L mice had to be sacrificed at 24-26 weeks of
age, this genotype could survive for up to 32 weeks. The overall incidence of
9
pancreatic carcinoma associated with MCN in human is 15~20%(36). The
Pdx1-CreAPCCKO/+p53L/L tumors exhibited a higher incidence of metastasis and
invasion (Fig. 4a and Supplementary Table 1). The murine MCNs became
malignant PCs with nuclear anaplastic features (Fig. 4b&c), and demonstrated
stomach, duodenal or intestinal invasion or liver or lung metastasis (Fig. 4a i&ii, 4d
and Supplementary Table 1). APCCKO/+expression plus p53 knockout tumors were
substantially invasive and metastatic and, thus, consistent with aggressive human
pancreatic cystic adenocarcinoma. IHC analysis revealed persistence of high nuclear
c-myc and active-β catenin (ABC) expression levels in invasive and metastatic lesions
(Fig. 4e i-viii) as well as high levels of the metastatic protein S100A4, a target gene of
the Wnt/ -catenin pathway(37) (Fig. 4e ix-xii).
Identification of APC haploinsufficiency and p53 loss-induced cytokines by using
mouse cytokine array system.
Next, we investigated which cytokines are responsible for the rapid MCN tumor
progression in Pdx1-CreAPCCKO/+p53L/L mice, using a mouse cytokine array system to
detect 26 mouse cytokines (Fig. 5a). Potential APC haploinsufficiency and
p53-loss-induced cytokines in the cystic fluid of the pancreata included FasL,
sTNFR1, IL-1β, IL-6, macrophage inflammatory protein- 1 (MIP-1γ), keratinocyte
10
chemo- attractant (KC) and monocyte chemoattractant proteins-1 (MCP-1), of which
the expression was significantly increased compared with that in normal pancreatic
tissue. The expression of all other cytokines was similar (Fig. 5b i, ii).
To validate the cytokine array data, we investigated the protein expression levels of
FasL, TNFR1, MIP-1γ, IL-6 or MCP-1 by using Western blotting, which revealed that
IL-6, FasL, MCP-1, TNFR1, MIP-1γ and TGFβ1 proteins were highly overexpressed
in the mouse MCN tumor samples (Fig. 5c). Immunoblotting analysis also revealed
increased induction of cleaved caspase 3 in the Pdx1-Cre APC/CKO/+p53L/L pancreata
(Fig. 5c).
Concurrent heterozygous loss of APC and p53 affects the Wnt pathway to induce
MCN progression in mice.
To investigate the molecular mechanisms underlying MCN formation and progression
mediated by APC/P53 loss in the pancreas, microarray analysis was used to compare
the gene expression in early-passage PC cell lines from the Pdx1-CreAPCCKO/+p53L/L
and Pdx1-Crep53L/L models (Supplementary Figure 7). The APC/P53 tumor cells
were characterized by distinct gene expression signatures mainly annotating the
transmembrane receptor tyrosine kinase signaling pathway for regulating of cell
11
communication and adhesion. Gene Ontology (GO) revealed the upregulation of 220
genes and the downregulation of 562 genes (> 2 fold change, P< 0.05) compared with
normal Pdx1-Crep53L/L ductal cells. The analysis revealed the top 10 gene sets shown
in Figure 6a were the most significantly upregulated or downregulated in the
Pdx1-Cre APCCKO/+p53L/L cells, suggesting their potential involvement in MCN
pathogenesis (Supplementary Table 2).
To validate the microarray analysis results, APC/p53-loss-mediated regulation of the
top selected and various known Wnt target genes (TBX15, FGF7, IGFBP4, TWIST2,
TCF4 and SNAIL2) was verified using qRT-PCRs. All analyzed genes showed
significantly higher mRNA levels in the Pdx1-CreAPCCKO/+p53L/L cells than in the
Pdx-1Crep53L/L cells (p<0.01, 11-, 7.2-, 36-, 8-, 10.6-, and 8.3-fold for TBX15, FGF7,
IGFBP4, TWIST2, GCNT4, and B-CAT, respectively) (Fig. 6b). The EMT-related
genes involving increased cellular invasiveness such as Snail2, Twist2, Vimentin, and
Col-1 were up-regulated whereas the epithelial marker, including E-cadherin and
Cldn 1 exhibited significantly decreased mRNA expression levels compared with
those in the Pdx-1Crep53L/L cells (Fig. 6b). In addition, Up-regulation of several
micro-RNAs (miRNA), such as miR-19b, miR-125 and miR145 associated with the
Wnt signaling pathway, were confirmed as well(38, 39) (Fig. 6b). In conclusion, the
12
data highlight the importance of Wnt signaling pathway implicated in development of
MCNs. Moreover, IHC analysis conducted using anti-FGF7, anti-Twist2 and
anti-Tbx15 confirmed that FGF7, Tbx15 and Twist2 protein expression was
predominantly increased in premalignant MCN and carcinoma lesions (Fig. 6c).
APC haploinsufficiency and p53 loss promotes mitotic chromosome instability in
murine MCNs.
APC was found to interact with the plus ends of the microtubules and to modulate the
kinetochore-microtubule attachments. The mutated forms of APC can alter the mitotic
spindle axis orientation, resulting in chromosome missegregation(26, 40). Intriguingly,
several mitotic spindle checkpoint genes associated with chromosomal instability and
aneuploidy showed significantly higher mRNA levels in the Pdx1-CreAPCCKO/+p53L/L
cells than in the Pdx1-Crep53L/L cells (p<0.05; Fig. 6b)(41-43). To evaluate the role
of APC in mitosis and to demonstrate the underlying causes of chromosome
aneuploidy, cycling and nocodazole-arrested Pdx1-CreAPCCKO/+P53L/L cells and
Pdx1-CreP53L/L control cells were stained with propidium iodide and their DNA
content
was
analyzed
by
FACS.
A
high
percentage
of
cycling
Pdx1-CreAPCCKO/+p53L/L cells contained 4N DNA content (18% compared with 5%
in the Pdx1-CreLSLKrasG12Dp53L/L cells) (Fig. 6d). Next, cells were then arrested at
13
the G0 phase through serum starvation, and both spindle formation and cytokinesis
were analyzed during the initial mitotic phase 8 hours after release from the arrest.
Immunofluorescence co-staining for α-tubulin as well as NuMA revealed that
numerous Pdx1-CreAPCCKO/+p53L/L, but not Pdx1-CreLSLKrasp53L/L cells, with
multiple centrosomes displayed multipolar spindle formation and nucleation (Fig. 6e).
IWP-2
Wnt
pathway
inhibitor
reduces
MCN
formation
in
Pdx1-CreAPCCKO/+p53L/L mice.
To determine whether MCNs formation and progression are Wnt signaling pathway
dependent, the Wnt inhibitor IWP-2 was selected to treat primary PDAC mouse cells,
since IWP-2 can prevent Wnt/β-catenin/Tcf signaling activation following APC
loss(44).
IWP-2
strongly
inhibited
the
proliferation
of
the
primary
Pdx1-CreAPCCKO/+p53L/L cells in vitro compared with the IWP-2 treated
Pdx1-Crep53L/L, Pdx1-CreLSLKrasG12Dp53L/L and DMSO treated control cells,
indicating that active Wnt signaling is essential for the growth of PC cells derived
from the Pdx1-CreAPCCKO/+p53L/L MCN model (Fig. 7a). Western blot and
fluorogenic cleaved caspase 3 activity assays confirmed that IWP-2 treatment
effectively inhibited Wnt signaling and induced caspase 3-mediated apoptosis in the
Pdx1-CreAPCCKO/+P53L/L cells (Fig. 7b & Supplementary Figure 8). FACS analysis
14
also demonstrated that the SubG1 apoptotic population significantly increased in the
IWP-2-treated Pdx1-CreAPCCKO/+p53L/L cells after 48 hours, but not in the
IWP-2-treated Pdx1-Crep53L/L, Pdx1-Cre LSL-KrasG12Dp53L/L cells (p< 0.01, Fig. 7c).
To assess the apoptotic response to this compound in vivo, we treated
Pdx1-CreAPCCKO/+p53L/L mice (~6 wks old) with IWP-2 for 12 weeks (Fig. 7d).
Non-invasive image analysis using a 3.0 T MRI scanner (GE, Sigma HDXt,
Milwaukee, WI) with a high resolution animal coil (3.0 cm diameter) performed on
the DMSO-treated Pdx1-CreAPCCKO/+p53L/L mice clearly displayed hypertrophic
pancreas with unilocular cysts (Fig. 7e). Such lesions were significantly reduced or
absent in the pancreas of Pdx1-Cre APCCKO/+p53L/L after IWP-2 treatment for 12
weeks (p< 0.01, Fig. 7f). Gross autopsy examination and histological analysis using
H&E staining validated the anti-MCN efficacy of IWP-2 (Fig. 7e). IHC analysis
confirmed that treatment of Pdx1-Cre APCCKO/+p53L/L mice with IWP-2 resulted in
decreased total and activated protein levels of β-catenin compared with the DMSO
control groups (Fig. 7e).
Discussion
The molecular mechanisms through which APC inactivation contributes to PC
pathogenesis remain unclear, and a mouse model for studying APC dependency in PC
15
has not been developed. This is the first report stating that APC is required for islet
development (β cell development and maturation) and pancreas maturation, and loss
of APC function results in the induction of MCN formation in the context of p53 loss.
The defects in islet maturation and normal pancreatic homeostasis in the Pdx1-Cre
APCCKO/CKO mice contradict the findings reported by Strom et al., that APC loss
induces postnatal pancreatomegaly during early pancreatic development, but does not
impede pancreas neogenesis in aging mice(45). Presumably, the differences in the
flanking loxp sites for depleting the APC gene between 2 mutant mice explain the
differences in findings. Our results revealed that Pdx-1CreAPCCKO/CKO mouse
embryos exhibit a neonatally lethal duodenal stenosis, consistent with the expression
of Pdx-1 in the antral stomach and duodenum. Research findings are limited
and inconsistent regarding whether APC/ β-catenin pathway does play a critical role
in modulating Kras induced PanIN formation and progression(21, 46, 47). To
elucidate the effects of APC loss in a mutant Kras driven PC mouse model,
Pdx-1CreAPC null mice were crossed with LSL-KrasG12D mice. Our preliminary
results confirmed that the inactivation of APC impeded Kras-induced PanINs
progression (Supplementary Figure 9). Mechanistic studies dissecting how APC
loss inhibits Kras-induced PanIN formation are underway.
16
This study provided several lines of evidence demonstrating that in vivo APC
haploinsufficiency and p53 loss sufficiently promotes pancreatic cell transformation
and induces oligocystic pancreatic tumors. In contrast to our previous work
demonstrated that Kras-Smad4 mutants develop cystic tumors more predominant
resembling IPMN, but can also present IPMN mixed with multiple small cysts
(microcystic) lesions”. The observation here was supported by a recent study from the
Lewis group, who elegantly showed that activated Wnt signaling in the tumor stromal
microenvironment contributes to the development of pancreatic MCNs in
Ptf1a-Cre;LSL-Kras;elastase-tva
mice
injected
with
replication-competent-
avian-sarcoma (RCAS)-Wnt1 viruses(48). Immunohistochemical characterization of
these MCN lesions revealed the TGFβ1, BMP4, Notch1, Wnt and EGFR signaling
pathways were activated, implying involvement of multiple signaling pathways in the
development of MCN. The results obtained by applying a mouse cytokine array
system demonstrated that proinflammatory cytokines, including IL-4, IL-6, sTNFR1,
KC, MIP-1γ, MCP-1 and the neutrophil-attracting chemokines LIX (CXCL5) were
upregulated in the cystic fluid of MCNs in our mouse model(49-51). These
conclusions were confirmed using western blot analysis. MCN has been increasingly
recognized as a crucial clinical condition because of its propensity to progress to
metastatic pancreatic carcinoma. Our MCN mouse model demonstrated increased
17
nuclear staining of c-Myc, ABC, and SA100A4 is associated with increased
metastatic potential (52-56)
Gene Go pathway analysis depicted the top three molecular networks associated with
altered gene expression from primary Pdx1-CreAPCCKO/+p53L/L PDAC cell lines
versus Pdx1-Crep53L/L controls were found to be involved in cell-gap adhesion
dictating alterations in ECM cell adhesion/tight junction, the developmental Wnt
signaling pathway and the developmental regulation of the EMT (Supplementary
Figure 10). Upregulated genes included TBX15, a member of the T-box family,
which might be downstream of Wnt signaling, plays an essential role in the DV
patterning of the mouse coat. TBX15 null mice display defects in both
intramembranous bone formation and endochondral ossification(57). In addition,
Dean Tang and colleagues reported that the human prostate cancer PSA
/lo
cancer cell
population contains cancer stem cells (CSCs) that resist castration. They further
demonstrated that the PSA
/lo
cancer cell population contains cells that are relatively
quiescent and exhibit increased expression of several stem cell regulatory genes,
including NANOG, ASCL1, NKX3.1, and TBX15(58). The cross-talk of the FGF and
Wnt
pathways
may
accompany
several
biological
processes
including
tumorigenesis(59). FGF7, also known as the keratinocyte growth factor (KGF), is
18
reportedly related to cancer, including PC. For example, Niu et al., reported that FGF7
induced vascular endothelial growth factor (VEGF)-A expression in PC cells(60). The
present study provides novel information implicating FGF7 in MCN formation and
progression. In addition, IGFBP-4 is an antagonist of the Wnt/ -catenin signaling
pathway and has been associated with cell growth and metastasis. Reportedly,
IGFBP-4 expression in metastatic renal cell carcinoma (RCC) is higher than that in
primary RCC and normal human kidney tissues(61). In regard to GCNT4, a
glucosaminyl (N-Acetyl) transferase 4 Core 2, were shown to play a major role in
mucin glycan biosynthesis(62). Additional experiments are required to investigate the
precise role of these genes in APC/P53- loss-induced MCNs.
In conclusion, although the exact mechanism of the APC tumor suppressor in PC is
not entirely clear, the results of the present study clearly show that APC is a key
regulator that modulates pancreatic histopathology and the progression of PC to
metastasis. Our data suggest that molecular therapies targeting APC and the Wnt
signaling pathways may be novel effective strategies for controlling MCN
progression.
COMPETING FINANCIAL INTERESTS
19
No potential conflicts of interest were disclosed.
ACKNOWLEDGMENTS
We gratefully thank Dr. Sheau-Fang Yang,at Kaohsiung Medical University for
helping us to confirm the histologic features and pathology of MCN mice. This work
was
supported
by
grants
NSC
101-2314-B-110-001-MY2,
101-2628-
B-110-001-MY2 (to K.H. Cheng) and MOST 103-2314-B-037 -062 – (to D.C. Wu
and K.H. Cheng) from the National Science Council, Taiwan ROC, and grants
KMU-TP103G00 and KMU-TP103G01 (to D.C. Wu and K.H. Cheng) from
Kaohsiung Medical University, Kaohsiung, Taiwan.
METHODS
Genetically modified mice and mouse genotyping.
Pdx-1Cre, LSLKrasG12D, p53Loxp/Loxp, APCCKO/CKO mice, obtained from the Mouse
Models of Human Cancers Consortium (MMHCC) under material transfer
agreements, were generously made available by Drs. Andrew M. Lowy, Tyler Jacks,
Anton Berns and Raju Kucherlapati respectively(29, 63, 64). Mice were genotyped as
described by the MMHCC PCR protocols for strains 01XL5, 01XJ6, 01XC2 and
01XAA. All studies were approved by the Animal Care Committee of the University
of Kaohsiung Medical University (Animal Permit Number 10115) and all surgery and
20
euthanasia was performed using isoflurane or avertin to ensure minimal suffering.
Blood samples were obtained from the cardiac puncture of isoflurane-anesthetized
mice. The formed elements and plasma were separated through centrifugation (×3000
g, 15 min). Blood biochemistry parameters were measured with an automatic
chemistry analyzer (Hitachi 7170S, Hitachi Ltd., Tokyo, Japan). Mice embryos were
harvested at E10, E12, E14 and E16. Embryos and pancreatic tissue samples were
fixed in 10% buffered formalin overnight, washed with 1×PBS, and transferred to
70% ethanol before paraffin embedding, sectioning, and hematoxylin and eosin (H&E)
staining.
Immunohistochemistry (IHC) and immunofluorescence (IF)
H&E staining followed the standard protocol. Periodic Acid-Schiff stain (PAS) and
Alcian blue staining kits were purchased from Scy-Tek Laboratories (Logan, Utah)
and performed according to the manufacturer's protocols.Standard procedure for
IHC and IF analysis as described in detail previously, and antibodies used in these
studies are listed in Supplementary Table 3(65). Stained slides were captured
using a Carl Zeiss. Axioskop 2 plus microscope (Carl Zeiss, Thornwood, NY). IF
images were captured using a Delta Vision Personal DV Imaging System.
21
Western blot analysis
Standard procedures for immunoblotting analysis as described in detail previously(66).
The primary antibodies used in this study are listed in Supplementary Table 3
Collection of mouse pancreatic cystic fluid.
Cystic fluid was collected by placing the needle (21G) into the cystic cavity of MCNs
in Pdx1-CreAPCCKO/+p53L/L mice before autopsy. Cystic fluid was centrifuged at 2000
rpm for 10 min at 4 °C to separate the fluid from cellular components. The protein
levels of cystic fluid were determined by the BCA protein assay kit (Pierce, Rockford,
IL) and stored at – 80°C.
Mouse cytokine array analysis.
For cytokine analysis, the RayBio Mouse Inflammation Antibody Array I was
purchased from RayBiotech, Inc., Norcross, GA, USA. Sample preparation and
hybridization to the array were performed according to the manufacturer's
instructions.
TUNEL staining.
Fluorometric TUNEL staining was conducted according to the manufacturer’s
22
protocol as described for the Dead End Fluorometric TUNEL system (Promega),
which identifies apoptotic cells by fluorescein-12–dUTP labeling of fragmented DNA
staining with analysis performed as described previously(65).
RNA extraction and microarray detection.
Cells were scraped and collected by centrifugation, and total RNA was subsequently
isolated by RNeasy Mini Kit. (QIAGEN, P/N 74104). RNA quantity and purity were
assessed at 260 nm and 280nm using a Nanodrop (ND-1000; Labtech. International).
300 ng of each sample was amplified and labeled using the GeneChip WT Sense
Target Labeling and Control Reagents (900652) for Expression Analysis.
cDNA microarray analysis.
Hybridization was performed against the Affymetrix GeneChip MoGene 1.0 ST array.
The arrays were hybridized for 17 hours at 45°C and 60 rpm. Arrays were
subsequently washed (Affymetrix Fluidics Station 450) and stained with
streptavidin-phycoerythrin (GeneChip® Hybridization, Wash, and Stain Kit, 900720),
and scanned on an Affymetrix GeneChip® Scanner 3000. The resulting data was
analyzed using Expression Console software (Affymetrix) and Transcriptome
Analysis Console software (Affymetrix) with default RMA parameters. Genes
23
regulated were determined with a 2.0-fold change P value < 0.05.
GeneGo analysis.
The significant lists were uploaded from a Microsoft Excel spreadsheet onto Metacore
6.13 software (GeneGo pathways analysis) (http://www.genego.com). GeneGo
recognizes the Affymetrix identifiers and maps the tissues to the MetaCore™ data
analysis suite, generating maps to describe common pathways or molecular
connections between pancreatic tissues on the list. Graphical representations of the
molecular relationships between genes were generated using the GeneGo pathway
analysis, based upon processes showing significant (P<0.05) association.
Real-time quantitative PCR analysis (RT qPCR).
RT qPCR were carried out as described in detail previously, and the primers for RT
qPCR were listed in Supplementary Table 4(66).
Cell proliferation assay.
Standard methyl tetrazolium (MTT)-based cell growth assay as described in detail
previously(66).
Fluorescence activated cell sorting (FACS) analysis.
In vitro caspase 3 activation assays were conducted according to the manufacturer’s
24
instructions. The protocol for FACS analysis as described in detail previously(67).
Primary pancreatic cell culture.
The primary pancreatic ductal cells isolated from Pdx-1Crep53L/L mice were cultured
in HPDEC medium (DMEM/F12 serum-free medium supplemented with 0.2 ng of
EGF, 30 g/ml bovine pituitary extract and containing penicillin/streptomycin). The
mouse primary PDAC cells were cultured in RPMI-1640 medium supplemented with
10% FBS, nonessential amino acids, 100 units/mL penicillin, and 100
g/mL
streptomycin at 37°C in a 5% CO2 incubator. Primary mouse pancreatic ductal and
PDAC cells were maintained for less than 6 passages and histopathologically
characterized through SCID mice xenograft studies before performing microarray
expression profile analyses.
Xenograft SCID mice.
Specific pathogen-free, 8-week-old female C.B17/lcr-SCID mice were purchased
from BioLASCO Taiwan Co., Ltd, for the in vivo tumorigenicity study. The animals
were maintained in the animal center at the Department of Medical Research,
Kaohsiung Medical University Hospital under SPF conditions and treated according
25
to the institutional guidelines for the care and use of experimental animals. SCID mice
subcutaneously injection were conducted as previously described(66)
IWP-2 treatment.
The in vitro cytotoxicity of the Wnt inhibitor IWP-2 (Sigma-Aldrich) 20 M was
assessed by standard MTT cell proliferation assay in Pdx-1Crep53L/L normal ductal
cells, Pdx-1Cre LSLKras p53L/L and Pdx-1Cre APCL/+p53L/L PDAC cells. For in vivo
treatment, IWP-2 (5 mg) was dissolved in 100 µl of DMSO, which was then diluted
with PBS buffer solution to a final concentration of 1 mg/ml directly before use.
6-week-old Pdx-1Cre APCCKO/+p53L/L mice were administered twice weekly
intraperitoneal injections of 10 mg/kg IWP-2 or the PBS vehicle for 12 weeks (N=6
per group). At the end of the experiment, Mice were sacrificed by CO2 euthanasia after
MRI imaging, and their pancreata collected for pathohistological analysis.
Randomization was done according to genotype and blinding was applied during
histological analysis.
Magnetic Resonance Imaging (MRI).
Mice were anesthetized with 1-2 isolfurance/air, and body temperature was
maintained by air conditioning through the bore of the magnet ring. MRI scans were
26
performed using a 3T MRI scanner (GE, HDXt Sigma; GE, Milwaukee, WI) with a
high resolution animal coil (3.0 cm diameter). Mice were placed supine in the coil,
taped below the thoracic cavity on the bed to reduce respiratory motion. T2 weighted
images were acquired using a fast spin echo multi-slices sequence with TR/TE
2000/63.23 ms for coronal section and 5083/46.7 ms for axial section,, 16 echo trains,
4 averages, 2 dummy scans, field of view (FOV) = 8×4.8cm3, for coronal section and
6x6 cm2 for axial section, matrix size= 256 ×192, slice thickness= 2mm, number of
slices= 20 contiguous. Scans were captured every 10 minutes until the 90-minute
mark was reached. A glass cylinder of pure water was positioned adjacent to each
mouse as a standard reference.
Statistical analysis.
All experiments were repeated at least three times. One representative experiment is
shown. RT-qPCR and cell proliferation assays are displayed as one representative
experiment of three independent experiments, mean ± SEM. Data measured on
continuous scale was analyzed using Student’s t test and categorical data were
subjected to x2 test. p value less than 0.05 was considered significant.
Accession codes: Microarray data are available in the Gene Expression Omnibus
27
(GEO) with accession number GSE 61894.
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FIGURE LEGENDS
Figure 1. Pdx1-Cre mediated conditional deletion of APC suppresses islet and
pancreatic development and leads to fatal duodenal obstruction. Hematoxylin and
eosin (H&E) staining of sagittal sections through the abdominal cavity of E14 mouse
embryos of the indicated genotypes (a, i, q); scale bar is 2mm. H&E sections of
developing pancreas from Pdx-1CreAPCCKO/CKO (b), Pdx-1CreAPCCKO/CKOp53L/L (j)
and control Pdx-1Crep53L/L (r) embryos. Immunohistochemical staining shows an
33
APC positive subpopulation in the developing pancreata of Pdx-1Crep53L/L mouse
embryo (r, red arrow in s), but not in the Pdx-1CreAPCCKOCKO (b, c) and Pdx-1Cre
APCCKOCKOp53L/L (j, k) embryos. Immunofluorescent co-staining for insulin (red) and
amylase (green) or glucagon (green) alone were performed on sections of the pancreas
from Pdx-1CreAPCCKO/CKO (d, e, f),
Pdx-1CreAPCCKO/CKOp53L/L (l, m, n) and
control Pdx-1Crep53L/L (t, u, v) mouse embryos at E14. Nuclear DNA stained with
4’,6-diamidino -2-phenylindole (DAPI). Arrow in (e, m) points to
cell with reduced
insulin staining. Low and higher magnification sections of the duodenum mucosa
from Pdx-1CreAPCCKO/CKO (g, h), Pdx-1CreAPCCKO/CKOLp53L/L (o, p) and control
Pdx-1Crep53L/L (w, x) embryos. Arrowheads indicate the duodenal atresia (g, o).
Scale bar, 50µm
Figure 2. Concomitant APC hapoinsufficiency and p53 loss drive cystic tumors of
the pancreas in mice. (a) Specific PCR analyses to detect APC and p53 wild types
and loxp alleles from wild type, APCCKO/+, APCCOK/CKO, p53L/+, p53L/L offspring. (b)
Western blot analyses for detection of p53, β-catenin and APC protein expression in
pancreatic lysates from Pdx-1Cre control, Pdx1-Crep53L/L and Pdx1-CreAPCCKO/+
p53L/L mice. β-actin is shown as a loading control. (c) Kaplan-Meyer curve showing
significantly reduced survival time of Pdx1-CreAPCCKO/+p53L/L mice compared to
34
Pdx1-CreAPCCKO/+ wild type and Pdx1-Crep53L/L mice. (d) Gross pathology of
murine MCN lesions in Pdx1-CreAPCCKO/+p53L/L mice at different ages. (e)
Histological analysis of pancreas from wild type and Pdx1-CreAPCCKO/+p53L/L mice at
different ages by H&E staining. (f) Periodic acid-Schiff (PAS) and Alcian blue
staining revealed mucin content in cystic lesions of murine MCNs. Scale bar is 10µm
(insets), 50µm (all other images). (g) Immunohistochemistry for Mucin4 revealed
very strong expression in murine MCNs compared to normal pancreas. (h) IHC
analysis using anti-progesterone receptor (PR) and anti-estrogen receptor (ER)
antibodies show strong PR and ER expressions in the stroma of murine MCNs. IHC
staining for PR and ER confirmed the nuclear localization as well. Scale bar, 50µm.
Figure 3. Immunohistochemistry characterization of MCNs in Pdx1-CreAPCCKO/+
p53L/L mice. Premalignant MCN lesions from Pdx1-CreAPCCKO/+p53L/L mice
collected at 7 and 24 weeks and normal pancreas control were stained with Dolichos
biflorus lectin (DBA) (red), anti-E-cadherin, anti-TGFβ1, anti-BMP4, anti-Wnt1,
anti-Notch1, anti-Hes1, anti-α-smooth muscle actin (SMA), anti-collagen type1
(col-1), anti-Vimentin, anti-EGFR and anti-pAkt antibodies. Epithelial cells lining
the cysts showed positive staining for E-cadherin and DBA. Note the increasingly
intensive immunoreactivity of TGFβ1, BMP4, Wnt1, Notch1 and EGFR, and strong
nuclear expression of Hes1 and p-Akt in cystic lesions and columnar epithelium
35
following MCN progression, and also high expression of SMA, Vimentin and
collagen type 1 in surrounding stromal cells. Scale bar is 50µm.
Figure
4.
Tumor
invasion
and
metastasis
of
cystic
neoplasms
in
Pdx1-CreAPCCKO/+ p53L/L mice. (a) Gross photograph of a malignant MCN with
invasive carcinoma arising in a Pdx1-CreAPCCKO/+ p53L/L mouse. The primary
malignant MCN directly invade and compresse the proximal duodenum; also note
liver metastasis and stomach invasion. L, liver; G, gallbladder; D, duodenum; S,
stomach. Insets i, hepatic metastasis; ii, lung metastasis. (b) Histological features of
malignant MCN in Pdx1-CreAPCCKO/+p53L/L mice with high grade lesions containing
intratumoral malignant epithelium and mucin. Scale bar is 50µm. (c) High
magnification of the abnormal anaphase figures in tumor sections. Scale bar, 20µm.
(d) A 6-month old Pdx1-CreAPCCKO/+ p53L/L mouse with well differentiated hepatic
metastasis (i). PDAC invaded the duodenal (ii) and gastric wall (iii) in a 7.5-month
old Pdx1-CreAPCCKO/+ p53L/L mouse. Lung metastasis was observed in an 8-month
old Pdx1-CreAPCCKO/+ p53L/L mouse (iv). Scale bar is 50µm. (e) IHC analysis
detected intensive c-myc (i-iv), ABC (v-viii) and S100A4 (ix-xii) nuclear staining in
MCN lesions (arrows in i, v, ix), malignant MCN with associated invasive carcinoma
(iii, ix) and hepatic (iii, vii), lung (iv, viii, xii) and stomach metastasis (xi). The arrows
36
represent the areas shown at higher magnification in ii, vi and x. Scale bar is 50µm.
Figure 5. Cytokine antibody array analysis of pancreatic cyst fluid in
Pdx1-CreAPCCKO/+p53 L/L mice. (a) Template alignment of the mouse cytokines in the
array. POS, positive; NEG, negative; IL, interleukin; SDF-1, stromal cell-derived
factor 1; BLC, B-lymphocyte chemo- attractant; TAC, protachykinin; TCA-3, small
inducible cytokine A1; TIMP, tissue inhibitors of metalloproteinase; LIX, LPS
induced CXC chemokine; MCSF, macrophage colony stimulating factor; MCP-1,
monocyte chemotactic protein 1; MIG, mitogen-inducible gene; MIP-1, macrophage
inflammatory protein 1. (b) Detection of mouse cytokine expression from the cyst
fluid of two Pdx1-CreAPCCKO+p53L/L and one representative matched normal
pancreatic tissue from Pdx1-Crep53 L/L mice by the mouse cytokine array system. (c)
Western blot analysis of pancreatic lysates from Pdx1-CreAPCCKO/+p53L/L mice at
different time points for the expression of IL-6, FasL, MCP-1, MIP-γ, TGFβ1, TNFR1
and pro and cleavage caspase-3 protein compared with Pdx1-Crep53L/L control mice.
GAPDH served as a loading control.
Figure
6.
cDNA microarray
Pdx1-CreAPCCKO/+p53
L/L
analysis
and Pdx1-Crep53
37
of
L/L
primary
PDAC
cells
from
mice. (a) Heat-map presentation
of gene
profiling
of
primary
pancreatic
cells
established
from
3
Pdx1-CreAPCCKO/+p53L/L and 2 Pdx1-Crep53L/L control mice showing genes with
significantly increased (red), intermediate (black) and decreased (green) expression
levels. The numerical values give the actual values on a log 2 scale associated with
each color. The full gene name for the gene symbol is available in Supplementary
Table 2. (b) Selected target genes that showed changes in the microarray analysis
were picked for further verification by qRT-PCR. RNA pools from 3
Pdx1-CreAPCCKO/+p53L/L primary PDAC cells and 2 control Pdx1-Crep53L/L primary
ductal cells were compared and analyzed. The relative gene expression was
normalised to GAPDH expression and compared with the Pdx1-Crep53L/L control.
Data represent the means ± SD of triplicate samples. *p < 0.05, t-test. (c) IHC
analysis showed intense staining for FGF7, Tbx15 and Twist2 in premalignant MCN
lesions and PDAC compared to normal pancreas. Scale bar, 50µm. (g) FACS profiles
showing DNA content of cell populations derived from Pdx1-CreAPCCKO/+p53L/L,
Pdx1-Crep53L/L and Pdx1-CreLSLKrasp53L/L cells. Flow cytometry analysis showed
increased aneuploidy (~18%) in Pdx1-CreAPCCKO/+p53L/L PDAC cells compared to
Pdx1-Crep53L/L (~1.6%) and Pdx1-CreLSLKrasp53L/L (~3%) cells (p<0.01). (d)
Centrosome amplification in Pdx-1CreAPCCKO/+p53L/L PDAC cell lines. Primary
PDAC cells prepared from Pdx-1CreAPCCKO/+p53L/L mice exhibited greater than two
38
centrosomes as demonstrated by co-staining with the anti-NuMA (green) and
anti-α-tubulin (red) antibodies. Nuclei were stained with DAPI (blue). Scale bar is
50µm.
Figure
7.
Inhibition
of
Wnt
signaling
blocks
MCN
formation
in
Pdx1-CreAPCCKO/+p53L/L mice. (a) Cell proliferation rates were significantly reduced
in Pdx1-CreAPCCKO/+p53L/L PDAC cells treated for 3 days with 20
M of IWP-2
inhibitor, compared to Pdx1-CreLSLKrasL/+p53L/L PDAC, Pdx1-Crep53L/L cells and
untreated groups. C: vehicle treatment; IWP-2: IWP-2 treatment. (b) Immunoblotting
analysis revealed dramatically decreased c-myc, β-actenin and procaspase-3 protein
levels in Pdx1-CreAPCCKO/+P53L/L PDAC cells following IWP-2 treatment. β-actin
served
as
a
loading
control.
(c) Representative FACS
profiles
showing
Pdx1-CreAPCCKO/+p53L/L PDAC cells contained a significantly higher (p<0.01)
proportion of apoptotic cells followed by IWP-2 treatment than Pdx1-Crep53L/L and
Pdx1-Cre LSLKrasp53L/L PDAC cells after IWP-2 treatment. (d) Schematic
representation of IWP-2 treatment in Pdx1-CreAPCCKO/+p53L/L
mice. The
Pdx1-CreAPCCKO/+p53L/L mice aged 7 weeks old were given IWP-2 or vehicle
(DMSO) treatment by intraperitoneal injection (10 mg/kg twice a week) for 12 weeks.
(e) The treatment efficiency for IWP-2 was monitored by MRI at 24 weeks before
39
sacrifice. Representative MRI images of the abdomen of Pdx1-CreAPCCKO/+p53L/L
mice treated intraperitoneally with IWP-2 or vehicle (DMSO) for 12 weeks showing
the reduction of MCN formation in the IWP-2 treated Pdx1-CreAPCCKO/+p53L/L mice,
but not in the mice receiving DMSO treatment. Macroscopic appearance, H&E
histological analysis and anti-β-catenin immunostaining of murine MCNs after IWP-2
treatment or DMSO control groups. Scale bar, 50µm. (f) Quantification of cyst size
for IWP-2 treatment or DMSO control group. Mean ± SEM. * p<0.01 (n= 6 mice per
groups).
40