Polymorphic genetic control of tumor invasion in a mouse model of pancreatic neuroendocrine carcinogenesis.
ABSTRACT Cancer is a disease subject to both genetic and environmental influences. In this study, we used the RIP1-Tag2 (RT2) mouse model of islet cell carcinogenesis to identify a genetic locus that influences tumor progression to an invasive growth state. RT2 mice inbred into the C57BL/6 (B6) background develop both noninvasive pancreatic neuroendocrine tumors (PNET) and invasive carcinomas with varying degrees of aggressiveness. In contrast, RT2 mice inbred into the C3HeB/Fe (C3H) background are comparatively resistant to the development of invasive tumors, as are RT2 C3HB6(F1) hybrid mice. Using linkage analysis, we identified a 13-Mb locus on mouse chromosome 17 with significant linkage to the development of highly invasive PNETs. A gene residing in this locus, the anaplastic lymphoma kinase (Alk), was expressed at significantly lower levels in PNETs from invasion-resistant C3H mice compared with invasion-susceptible B6 mice, and pharmacological inhibition of Alk led to reduced tumor invasiveness in RT2 B6 mice. Collectively, our results demonstrate that tumor invasion is subject to polymorphic genetic control and identify Alk as a genetic modifier of invasive tumor growth.
- SourceAvailable from: ncbi.nlm.nih.gov[Show abstract] [Hide abstract]
ABSTRACT: Invasion and metastasis increase after the inhibition of VEGF signaling in some preclinical tumor models. In the present study we asked whether selective VEGF inhibition is sufficient to increase invasion and metastasis and whether selective c-Met inhibition is sufficient to block this effect. Treatment of pancreatic neuroendocrine tumors in RIP-Tag2 mice with a neutralizing anti-VEGF antibody reduced tumor burden but increased tumor hypoxia, hypoxia-inducible factor-1α, and c-Met activation and also increased invasion and metastasis. However, invasion and metastasis were reduced by concurrent inhibition of c-Met by PF-04217903 or PF-02341066 (crizotinib). A similar benefit was found in orthotopic Panc-1 pancreatic carcinomas treated with sunitinib plus PF-04217903 and in RIP-Tag2 tumors treated with XL184 (cabozantinib), which simultaneously blocks VEGF and c-Met signaling. These findings document that invasion and metastasis are promoted by selective inhibition of VEGF signaling and can be reduced by the concurrent inhibition of c-Met. SIGNIFICANCE: This report examines the mechanism of increased tumor aggressiveness after anti-VEGF therapy and presents evidence for roles of vascular pruning, hypoxia, and c-Met activation. The results show that simultaneous inhibition of c-Met and VEGF signaling not only slows tumor growth but also reduces invasion and metastasis.Cancer Discovery 03/2012; 2(3):270-87. · 15.93 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Glutamate and its receptor N-methyl-D-aspartate receptor (NMDAR) have been associated with cancer, although their functions are not fully understood. Herein, we implicate glutamate-driven NMDAR signaling in a mouse model of pancreatic neuroendocrine tumorigenesis (PNET) and in selected human cancers. NMDAR was upregulated at the periphery of PNET tumors, particularly invasive fronts. Moreover, elevated coexpression of NMDAR and glutamate exporters correlated with poor prognosis in cancer patients. Treatment of a tumor-derived cell line with NMDAR antagonists impaired cancer cell proliferation and invasion. Flow conditions mimicking interstitial fluid pressure induced autologous glutamate secretion, activating NMDAR and its downstream MEK-MAPK and CaMK effectors, thereby promoting invasiveness. Congruently, pharmacological inhibition of NMDAR in mice with PNET reduced tumor growth and invasiveness. Therefore, beyond its traditional role in neurons, NMDAR may be activated in human tumors by fluid flow consequent to higher interstitial pressure, inducing an autocrine glutamate signaling circuit with resultant stimulation of malignancy.Cell 03/2013; 153(1):86-100. · 31.96 Impact Factor
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ABSTRACT: The intracellular anaplastic lymphoma kinase (ALK) fragment shows striking homology with members of the insulin receptor family and was initially identified as an oncogenic fusion protein resulting from a translocation in lymphoma and more recently in a range of cancers. The full-length ALK transmembrane receptor of ~220 kDa was identified based on this initial work. This tyrosine kinase receptor and its ligands, the growth factors pleiotrophin (PTN) and midkine (MK) are highly expressed during development of the nervous system and other organs. Each of these genes has been implicated in malignant progression of different tumor types and shown to alter phenotypes as well as signal transduction in cultured normal and tumor cells. Beyond its role in cancer, the ALK receptor pathway is thought to contribute to nervous system development, function, and repair, as well as metabolic homeostasis and the maintenance of tissue regeneration. ALK receptor activity in cancer can be up-regulated by amplification, overexpression, ligand binding, mutations in the intracellular domain of the receptor and by activity of the receptor tyrosine phosphatase PTPRz. Here we discuss the evidence for ligand control of ALK activity as well as the potential prognostic and therapeutic implications from gene expression and functional studies. An analysis of 18 published gene expression data sets from different cancers shows that overexpression of ALK, its smaller homolog LTK (leukocyte tyrosine kinase) and the ligands PTN and MK in cancer tissues from patients correlate significantly with worse course and outcome of the disease. This observation together with preclinical functional studies suggests that this pathway could be a valid therapeutic target for which complementary targeting strategies with small molecule kinase inhibitors as well as antibodies to ligands or the receptors may be used.Frontiers in Oncology 01/2012; 2:192.
Polymorphic genetic control of tumor invasion in
a mouse model of pancreatic neuroendocrine
Matthew G. H. Chuna,b,c,d, Jian-Hua Maoc,1, Christopher W. Chiub,c, Allan Balmainc, and Douglas Hanahana,b,c,2,3
aDepartment of Biochemistry and Biophysics,bDiabetes Center, andcHelen Diller Family Comprehensive Cancer Center, University of California, San Francisco,
CA 94143; anddProgram in Biological Sciences, University of California, San Francisco, CA 94158
Contributed by Douglas Hanahan, August 31, 2010 (sent for review August 2, 2010)
Cancer is a disease subject to both genetic and environmental
influences. In this study, we used the RIP1-Tag2 (RT2) mouse
model of islet cell carcinogenesis to identify a genetic locus that
influences tumor progression to an invasive growth state. RT2
mice inbred into the C57BL/6 (B6) background develop both non-
invasive pancreatic neuroendocrine tumors (PNET) and invasive
carcinomas with varying degrees of aggressiveness. In contrast,
RT2 mice inbred into the C3HeB/Fe (C3H) background are compar-
atively resistant to the development of invasive tumors, as are RT2
C3HB6(F1) hybrid mice. Using linkage analysis, we identified a 13-
Mb locus on mouse chromosome 17 with significant linkage to the
development of highly invasive PNETs. A gene residing in this
locus, the anaplastic lymphoma kinase (Alk), was expressed at
significantly lower levels in PNETs from invasion-resistant C3H
mice compared with invasion-susceptible B6 mice, and pharmaco-
logical inhibition of Alk led to reduced tumor invasiveness in RT2
B6 mice. Collectively, our results demonstrate that tumor invasion
is subject to polymorphic genetic control and identify Alk as a ge-
netic modifier of invasive tumor growth.
anaplastic lymphoma kinase|cancer modifier genes|malignant
progression|pancreas cancer|transgenic mouse
phisms that modulate cancer susceptibility (1). Although many
investigations have focused on identifying factors that affect in-
itial tumor development (2, 3), data from both human and
mouse studies have demonstrated that genetic polymorphisms
can modulate multiple aspects of tumorigenesis, such as tumor
progression (4–6) and response to therapy (7).
In this study, we investigated the effects of genetic background
on tumor progression to an invasive growth state, motivated by
a provocative observation that mice carrying the same oncogenic
transgene but differing in genetic background developed tumors
that were markedly distinctive in their invasiveness. This model,
the RIP1-Tag2 (RT2) mouse model of islet cell carcinogenesis,
develops multiple pancreatic neuroendocrine tumors (PNET) in
a relatively synchronous and predictable multistage progression
pattern by 12–14 wk of age owing to the expression of the SV40
T antigen oncoprotein (Tag) in the pancreatic β cells (8). The
tumorigenesis pathway has predominantly been studied in RT2
mice inbred into the C57BL/6 (B6) background, and the PNETs
that arise in this genetic context display a spectrum of invasive
phenotypes and can be classified as noninvasive islet tumors (IT),
focally invasive type-1 carcinomas (IC1), and broadly invasive
type-2 carcinomas (IC2) (9). Surprisingly, we observed that when
RT2 mice were inbred into a second strain, C3HeB/Fe (C3H), the
tumors that arose were predominantly noninvasive, despite being
otherwise similar in their tumorigenesis phenotype. The impli-
cation that the invasive phenotype was influenced by genetic
background prompted our investigation, which was aimed at
mediated the susceptibility or resistance to the acquisition of the
ancer is a complex disease governed by environmental and
genetic factors, including genetic mutations and polymor-
hallmark capability for invasive growth in the RT2 mouse model
PNET Progression to Invasive Carcinoma Is Modulated by Genetic
Background. Following anecdotal observations that PNETs de-
veloping in RT2 mice inbred into the C3H background were
predominantly noninvasive, we carefully examined the distribu-
tion of the distinctive invasive phenotypes in de novo PNETs
arising in RT2 mice inbred into either the B6 or C3H genetic
backgrounds, as well as in C3HB6(F1) hybrids (F1), to determine
whether the parameter of tumor invasiveness was indeed af-
fected by genetic background (Fig. 1 A–C).
The development of invasive carcinoma lesions (IC) was
strongly suppressed in RT2 C3H mice. Whereas IC lesions con-
stitute more than half of all tumors in RT2 B6 animals at 14 wk,
less than 15% of all tumors could be classified as invasive in RT2
C3H mice (Fig. 1D). This reduction occurred in both the focally
tumor lesions (Fig. 1E). The development of IC lesions was also
lesionsin RT2F1 micewas similar to that in RT2 C3H mice(Fig. 1
D and E). These data indicate that the C3H genetic background
is resistant to the development of invasive RT2 PNETs, whereas
the F1 phenotype demonstrates that the resistant C3H back-
ground is dominant over the susceptible B6 background.
We also examined other parameters of PNET tumorigenesis
in the B6 and C3H backgrounds to determine whether additional
phenotypes were similarly affected by genetic background. The
average tumor burden per animal was significantly higher in both
RT2 C3H and RT2 F1 mice as compared with RT2 B6 mice,
whereas the average number of macroscopic tumors per animal
was higher in RT2 C3H mice as compared with RT2 B6 and RT2
F1 mice (Fig. S1). However, there were no significant differences
with regard to either the rate of tumor proliferation or tumor
apoptosis (Fig. S1). There was no indication that the driving on-
cogene was responsible for these phenotypic differences because
the levels of the Tag oncoprotein were similar in tumors isolated
from RT2 mice in the different genetic backgrounds (Fig. S2),
consistent with a previous assessment (10). Additionally, the ex-
Author contributions: M.G.H.C. and D.H. designed research; M.G.H.C. and C.W.C. per-
formed research; M.G.H.C., J.-H.M., C.W.C., A.B., and D.H. analyzed data; and M.G.H.C.
and D.H. wrote the paper.
The authors declare no conflict of interest.
Freely available online through the PNAS open access option.
1Present address: Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley,
2Present address: Swiss Institute for Experimental Cancer Research, Swiss Federal Institute
of Technology Lausanne, Lausanne CH-1015, Switzerland.
3To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| October 5, 2010
| vol. 107
| no. 40www.pnas.org/cgi/doi/10.1073/pnas.1012705107
regulator of invasion in the RT2 model as well as other cancers
(11), was not obviously different (Fig. S2).
Invasive Modifier Does Not Act in the Bone Marrow–Derived Tissue
Compartment. Because bone marrow–derived (BMD) inflam-
matory cells that supply matrix-degrading enzymes such as ca-
thepsin proteases and heparanase are functionally implicated in
the invasive phenotype in this model (12–14), we examined the
possibility that the reduced invasiveness in RT2 C3H and RT2 F1
mice was due to deficiencies in the invasion-promoting func-
tionality of BMD cells. We transferred bone marrow from B6
or F1 donor mice into RT2 F1 animals with the rationale that B6
but not F1 bone marrow would “rescue” the invasive phenotype
in recipient RT2 F1 mice if the invasive modifier operated in
this tissue compartment. RT2 F1 mice were chosen as recipients
because they develop invasive PNETs at a reduced frequency
(Fig. 1 D and E) and should also be capable of receiving bone
marrow from either B6 or F1 donors without host/donor in-
compatibility complications. In brief, we did not observe any
differences in the invasive phenotype or in any other parameter of
RT2 tumorigenesis in RT2 F1 mice whose immune systems had
been rendered B6 (Fig. S3). These results suggest that the poly-
morphic difference is operative in the cancer cells themselves or
possibly in other cellular compartments of the stroma.
In light of the evident genetic differences in the frequency of
developing invasive carcinomas in RT2 mice, we next sought to
map the putative polymorphic locus/loci associated with sus-
ceptibility vs. resistance to the invasive phenotype using standard
genetic linkage analysis.
Linkage Analysis Identifies a Region on Chromosome 17 That Is
Associated with the Development of Invasive Carcinomas in RT2 Mice.
To identify the genetic locus/loci that modify the invasive phe-
notype in RT2 mice, we performed a genome-wide linkage study.
One hundred forty-three RT2 N2 backcrossed mice, resulting
from crossing RT2 F1 male mice with B6 female mice (Fig. S4),
were scored for the incidence of IT, IC1, and IC2 tumor le-
sions in addition to the other parameters of RT2 tumorigenesis
(Dataset S1). Constitutional tail DNA was genotyped across 561
SNPs that cover the mouse genome and discriminate between
the B6 and C3H backgrounds (Dataset S1). Statistical analysis
was subsequently performed using R/qtl to determine whether
there was evidence of linkage to the development of invasive
lesions or to any of the other RT2 tumor phenotypes. Log of
odds (LOD) scores of ≥1.9 and ≥3.0 were considered suggestive
and significant linkage, respectively (15).
Using the development of IT, IC1, or IC2 PNETs as quanti-
tative traits, we observed significant linkage to four SNPs on
chromosome 17 for the development of IC2 lesions, with a peak
LOD score of 3.52 (Fig. 2A and Dataset S2). The 95% confi-
dence interval was located from 63.7 to 76.4 Mb, a 13-Mb region
that contains more than 50 annotated genes and one miRNA,
mir-1195 (Fig. 2B). Interestingly, we did not identify any locus
that was linked to the IC1 phenotype, despite the different fre-
quencies in the development of this class of tumors in RT2 B6
and RT2 C3H mice (Fig. S5 and Dataset S2).
Additionally, we observed significant linkage to the X chro-
mosome to the development of IT lesions and to the metric of
tumor number (Fig. S5 and Dataset S2). In both situations, the
linked region essentially spanned the entire chromosome, which
complicated our efforts to analyze this region in further detail.
We therefore proceeded to investigate the genes in the minimal
region of chromosome 17 that showed significant linkage to the
development of IC2 tumors.
Anaplastic Lymphoma Kinase Resides in the Chromosome 17 Minimal
Region and Is Differentially Expressed in the B6 and C3H Genetic
Backgrounds. It has previously been suggested that genetic poly-
morphisms can influence the levels of gene expression in the
context of phenotypic modifiers of complex traits (16, 17). We
therefore asked whether any of the genes located within the
minimal chromosome 17 region might be differentially expressed
staining of a noninvasive IT PNET, a focally invasive IC1 PNET, and a
broadly invasive IC2 PNET from an RT2 B6 mouse. T indicates tumor region,
and Ex indicates exocrine pancreas. Dashed lines demarcate tumor mar-
gins. (Scale bars, 200 μm.) (D) Quantification of tumor invasiveness rep-
resented as the percentage of IT tumors or total IC tumors (IC1 + IC2) in
RT2 mice on the B6, C3H, and F1 genetic backgrounds at 14 wk of age. A
minimum of 117 tumors per group was graded. *P < 0.001 by Fisher’s exact
test. (E) Same as D except IC lesions are separated into the IC1 and IC2
subclasses. *P < 0.001 by the χ2test.
PNET invasion is dependent on genetic background. (A–C) H&E
(A) LOD scores for the IC2 phenotype across the mouse genome. The IC2
phenotype shows significant linkage (LOD ≥3.0) to a region on chromosome
17. Dashed line demarcates LOD-3.0 significance cutoff. (B) Physical map of
the 95% confidence interval on chromosome 17. Map was constructed using
data from the University of California, Santa Cruz Genome Browser (http://
genome.ucsc.edu/) and the National Center for Biotechnology Information
MapViewer (http://www.ncbi.nlm.nih.gov/projects/mapview/) for the mouse
genome. Red and green arrows indicate genes that are expressed at sig-
nificantly higher and lower levels, respectively, in tumors isolated from RT2
C3H mice as compared with RT2 B6 mice.
The IC2 tumor phenotype is linked to a region on chromosome 17.
Chun et al.PNAS
| October 5, 2010
| vol. 107
| no. 40
between the parental strains and therefore contribute to the
observed differences in the invasion phenotypes.
RNA from RT2 B6 and RT2 C3H tumors were profiled by
quantitative PCR for the genes located within the minimal re-
gion on chromosome 17. This analysis revealed that a small
subset of the resident genes—Alk, Dlgap1, Emilin2, Lbh, Ltbp1,
Rab31, and Spdya—showed significant differential expression
between the B6 and C3H genetic backgrounds at the mRNA
level (Figs. 2B and 3A and Dataset S3).
We were particularly intrigued by the Alk gene, which encodes
the anaplastic lymphoma kinase. Alk mRNA levels were ∼60%
lower in RT2 C3H tumors vs. RT2 B6 tumors and ∼40% lower in
RT2 F1 tumors vs. RT2 B6 tumors, which was also reflected at
the protein level (Fig. 3 A–C). Alk expression was also reduced in
WT islets from C3H mice as compared with B6 mice, consis-
tent with Alk being expressed at higher levels in the B6 back-
ground vs. the C3H background regardless of the neoplastic state
of this tissue (Fig. 3B). Alk levels were higher in tumors com-
pared with WT islets in both genetic backgrounds, and Alk ex-
pression showed a progressive increase during the course of RT2
tumorigenesis (Fig. 3 B and D). Notably, there are no poly-
morphisms in the exonic regions of the Alk gene that differen-
tiate the B6 allele from the C3H allele (http://www.informatics.
jax.org/strains_SNPs.shtml), and therefore the Alk protein is not
intrinsically different in structure or function in these different
genetic backgrounds. Interestingly, Alk belongs to the insulin-
receptor superfamily of receptor tyrosine kinases (18), members
of which are known to influence PNET tumorigenesis in RT2
mice, including tumor invasion (9, 19). Given this association
and our observation that Alk expression levels were significantly
different between the B6 and C3H backgrounds, we sought to
explore the potential role that Alk might play in the development
of invasive RT2 tumors.
Pharmacological Inhibitor of Alk Inhibits Invasion and Other Param-
eters of PNET Tumorigenesis. We used a small molecule inhibitor
of Alk kinase activity, NVP-TAE684 (TAE684) (20), in an ex-
perimental therapeutic trial in RT2 mice, aiming to assess the
effects of reduced Alk activity on RT2 tumorigenesis, particularly
with regard to the parameter of tumor invasion.
RT2 B6 mice were treated for 4 wk with TAE684 or vehicle
using a previously defined dose regimen (20) beginning at 10 wk
of age when incipient tumors are first observed in RT2 mice (21).
RT2 B6 mice were used because they develop IC lesions at sig-
nificantly higher levels than RT2 C3H mice, and they also express
Alk in the pancreatic islets and PNETs at significantly higher
levels than RT2 C3H mice (Figs. 1 D and E and 3 A–C). This is
also the stage of RT2 tumorigenesis when there is an appreciable
increase in Alk expression levels (Fig. 3D). TAE684 was well
tolerated, and we did not observe any fluctuations in body mass
in either TAE684- or vehicle-treated mice during the course of
the trial (Fig. S6).
At the defined endpoint of the trial, TAE684-treated mice
proved to have developed ∼25% fewer macroscopic tumors than
control mice (Fig. 4B); there was a concomitant trend toward
reduced tumor burden in TAE684-treated mice, which, however,
was not statistically significant (Fig. 4A).
Notably, TAE684-treated mice developed significantly fewer
invasive lesions than control mice. There was a clear reduction
in the frequency of total IC tumors (49.7% vs. 33.3% of total
tumors in control vs. treated mice), which was accompanied by
a concomitant increase in the frequency of IT tumors (50.3% vs.
Dlgap1, Emilin2, Lbh, Ltbp1, Rab31, and Spdya obtained using a TaqMan array to profile tumors isolated from RT2 mice on the B6 and C3H backgrounds.
Seven independent tumors per genotype were analyzed in triplicate. Data shown are mean plus SE. *P < 0.05 and **P < 0.01 by the Mann–Whitney test. (B)
Real-time quantitative PCR values for Alk in a pool of islets isolated from normal WT B6 or C3H mice or in tumors (T) isolated from RT2 mice on the B6, C3H, or
F1 backgrounds. *P < 0.05 by the Mann–Whitney test. (C) Western analysis on tumor pool lysates (T) from RT2 B6 and RT2 C3H mice for Alk and β-actin. (D)
Real-time quantitative PCR values for Alk during the stages of RT2 tumorigenesis [normal WT (N), hyperplastic (H), angiogenic (A), tumor (T)] on the B6
genetic background. All real-time quantitative PCR values are shown as the percentage expression of the ribosomal protein L19 (L19).
Anaplastic lymphoma kinase (Alk) is differentially expressed in the B6 and C3H genetic backgrounds. (A) Real-time quantitative PCR values for Alk,
| www.pnas.org/cgi/doi/10.1073/pnas.1012705107Chun et al.
66.7% of total tumors in control vs. treated mice), in TAE684-
treated mice (Fig. 4C). This shift was due to a reduction in the
frequencies of both the IC1 and IC2 subclasses of invasive RT2
PNETs (Fig. 4D).
TAE684 functions by interfering with Alk kinase activity (20),
and tumors from treated RT2 mice showed reduced levels of
phosphorylated Alk (Fig. 4E). We also observed a modest but
appreciable reduction in the levels of phosphorylated Akt, one
downstream Alk target, compared with controls (Fig. 4E), con-
firming that TAE684 inhibited Alk activity in the tumors of
A considerable body of research has identified polymorphic
modifier loci scattered across the mouse genome that affect mul-
tiple aspects of cancer susceptibility and development (1, 22, 23).
Our data demonstrate that tumor progression, specifically to an
invasive growth state, is also subject to polymorphic genetic con-
trol. We identify a polymorphic locus on mouse chromosome 17
[syntenic to human chromosomes 2 (107.5–110 Mb), 5 (2.5–10
Mb), and 18 (29–34 Mb)], which influences the susceptibility of
PNETs to progress from solid adenomatous tumors to invasive
Using a prototypical mouse model of multistage tumorigenesis,
we observed that the propensity to develop an invasive phenotype
is affected by genetic background. RT2 mice inbred into the B6
background develop PNETs of varying degrees of invasiveness,
whereas RT2 mice inbred into the C3H background are largely
resistant to the development of invasive tumors. Furthermore,
RT2 F1 hybrid mice are also resistant, indicating that the C3H
genetic background is dominant-suppressive over the invasion-
prone B6 background. Linkage analysis of RT2 N2 backcross
mice, produced from backcrossing RT2 F1 mice once to the sus-
correlated with susceptibility (when the locus is homozygous B6)
documented that tumors isolated from RT2 mice undergo chro-
mosomal gains and losses at different frequencies dependent on
genetic background (10, 24). Notably, chromosome 17 is not af-
fected by copy number abnormalities in either the B6 or C3H
backgrounds, suggesting that this locus is of a class of genetic
modifiers that is not altered during tumorigenesis.
The invasion modifier locus on chromosome 17 (63.7–76.4
Mb) contains more than 50 annotated genes. Additionally, one
miRNA, mir-1195, resides in this locus, although there is no
coding change between the B6 and C3H sequences for this
miRNA (http://www.informatics.jax.org/strains_SNPs.shtml). Of
the 50 genes in the modifier locus, 7 were found to be differen-
tially expressed in the PNETs isolated from RT2 mice inbred into
the B6 and C3H backgrounds. As a first step toward auditing
candidate invasion modifier genes in this locus, we focused on
the Alk receptor tyrosine kinase, motivated in part by a series of
studies demonstrating that Alk is activated by mutation or chro-
mosomal translocation in human hematopoietic and solid can-
cers, evidently converting it into an initiating oncogene (18, 25–
27). On the basis of these and previous studies implicating Alk as
an oncogene, several small-molecule inhibitors specific to Alk
have been developed as potential therapeutics for these diseases
(20, 28). Our use of one such kinase inhibitor to probe the pos-
sible roles of Alk in PNET tumorigenesis demonstrated that Alk
promoted both tumor growth and progression; most notably,
pharmacological inhibition of Alk activity reduced tumor in-
vasiveness in RT2 B6 mice. These results are consistent with our
observation that Alk is expressed at lower levels in the tumors of
RT2 C3H mice, which are rarely invasive, as compared with the
tumors of RT2 B6 mice, which consistently develop invasive
PNETs. In comparing the B6 and C3H sequences, we did not
identify any polymorphism in either the protein-coding or un-
translated portions of the Alk mRNA that might suggest a basis
for Alk’s invasion modifier effects and/or differential expression.
However, there are four polymorphisms located within 10 kb of
the 5′-flanking region and two within 10 kb of the 3′-flanking
region, in addition to ∼300 polymorphisms residing in the large
intron 2 of the Alk gene, that distinguish the B6 and C3H alleles
(http://www.informatics.jax.org/strains_SNPs.shtml), and one or
more of these polymorphisms may account for the observed dif-
ferences in allelic expression. Our results associating Alk with
invasion are also congruent with a previous study demonstrating
that single-chain variable fragment antibodies targeting Alk can
reduce tumor cellinvasion in an in vitro setting (29).Additionally,
pharmacological inhibition of Alk hindered tumor formation in
treated with TAE684 or vehicle from 10 to 14 wk of age. Data shown are mean plus SE. Data are not statistically different. (B) Tumor number for RT2 B6 mice
treated with TAE684 or vehicle from 10 to 14 wk of age. Data shown are mean plus SE. *P < 0.05 by the Mann–Whitney test. (C) Quantification of tumor
invasiveness represented as the percentage of IT tumors or total IC tumors (IC1 + IC2) in RT2 B6 mice treated with TAE684 or vehicle from 10 to 14 wk of age. A
minimum of 83 tumors per group was graded. *P < 0.05 by Fisher’s exact test. (D) Same as C except IC tumors are separated into the IC1 and IC2 subclasses.
*P < 0.01 by the χ2test. (E) Western analysis on tumor pool lysates from TAE684- or vehicle-treated RT2 B6 mice for phospho-Alk, total Alk, phospho-Akt, total
Akt, and β-actin.
Alk inhibitor NVP-TAE684 (TAE684) reduces tumor invasiveness in RT2 mice in an experimental therapeutic trial. (A) Tumor burden for RT2 B6 mice
Chun et al.PNAS
| October 5, 2010
| vol. 107
| no. 40
RT2 mice, in accordance with earlier studies examining the on-
cogenic properties of Alk (20, 25, 26, 30). Importantly and in
contrast to the aforementioned studies in which Alk was the
driving oncogene, our results demonstrate that Alk can also act as
a tumor progression factor, being up-regulated during multistep
tumorigenesis to collaborate with an initiating oncogene (in
this case the SV40 Tag oncogene that abrogates the tumor-
suppressing activities of pRb and p53). Thus, Alk inhibition
may prove to be a useful therapy even in situations in which Alk is
not the initiating oncogene, either as a result of mutation or
Although our data implicate Alk levels as a determinant of
RT2 tumor invasion, we envision that other polymorphic invasion
modifier genes may reside in the chromosome 17 locus. The Alk
inhibitor reduced tumor invasiveness, but not to the degree seen
in the C3H background, which could reflect incomplete Alk in-
hibition or additional genetic components to the modifier effect.
Indeed, several other genes residing in this locus also showed
significant differential expression in RT2 tumors from the B6 and
C3H genetic backgrounds (Fig. 3), and one of these genes, Ltbp1,
contains a nonsynonymous coding change between the B6 and
C3H backgrounds (http://www.informatics.jax.org/strains_SNPs.
shtml). Ltbp1 encodes the latent TGF-β binding protein 1, a com-
ponent of the TGF-β pathway (31), which is known to influence
many aspects of cancer progression, including tumor invasion
and metastasis (32). Additionally, it has recently been suggested
that Emilin2, which encodes the elastin microfibril interfacer 2, is
with poorer clinical outcome, in particular relapse and poor sur-
which encodes the speedy homolog A, accelerates tumorigenesis
in a mouse model of breast cancer (34) and has also been associ-
ated with more aggressive human breast cancers (35). As such,
other genes in this locus merit future investigation.
Although bone marrow–derived inflammatory cells have been
shown to contribute to the invasiveness of RT2 PNETs (12, 13), it
does not seem that their activity is modulated by the invasion
modifier gene(s). Thus, invasive PNETs were still rare in RT2 F1
mice that received bone marrow from an invasion-permissive B6
donor (Fig. S3). Although we cannot rule out the possibility that
this modifier locus operates in other stromal cell types or in
another tissue compartment, it seems most likely that the in-
vasive modifier acts in the cancer cells.
In addition to proinvasive inflammatory cells, other factors are
known to influence progression to an invasive growth state in this
prototypical model of multistage tumorigenesis. Loss of cell–cell
adhesion complexes, including the adherens junctions mediated
by Cdh1 (11) and desmosomes (36), are associated with the de-
velopment of more-invasive tumors. Signaling through the type-1
insulin-like growth factor receptor (Igf1r) can also drive pro-
gression to an invasive state (9). The present study now establishes
a unique dimension to this multifactorial invasive growth pheno-
type, involving a polymorphic genetic modifier that can alterna-
tively override or allow these other functional effectors of invasive
growth. It remains to be determined whether the chromosome 17
invasion modifier locus identified in this study modulates any of
these functionalities or acts in a completely independent fashion.
Finally, it is pertinent to consider the translational implica-
tions of this newly identified invasion modifier. First, we suspect
that this polymorphic modifier will prove operative in other
cancer types but most likely not in all. Notably, the develop-
ment of squamous carcinoma is under distinctive polymorphic
control in mice. In this case, the B6 background is largely resis-
different oncogenic contexts—an activated Hras oncogene (37),
the HPV16 oncogenes (38), and chemical carcinogens (39). Thus,
the B6 background is permissive for invasive cancers in the pan-
creas but resistant for Hras-induced cancers in the skin. A major
determinant of skin tumor resistance is a polymorphism in the
Patched gene (Ptch1), located on mouse chromosome 13, that
introduces a nonconservative coding sequence change at the C
terminus of the protein (37). This polymorphism was not de-
tected in the present linkage analysis of invasive pancreatic tu-
mors. Therefore, both tumor types are governed by polymorphic
modifiers of invasive cancer, albeit distinctive ones. Additionally,
yet other phenotypic modifiers of metastasis are implicated in
mouse models of breast cancer (40) and in human breast cancer
(41). Given the neuroendocrine nature of the tumor type subject
to the invasion modifier reported herein, we wonder whether
similar tumor types such as small-cell lung cancer or brain cancers
might also be affected by this genetic modifier. Interestingly, Alk
has been implicated in glioblastoma (29), and as such, this tumor
type could be subject to this polymorphic modifier.
Assessing the existence of polymorphic invasion modifiers
in human cancers will be challenging. The availability of in-
creasingly cost-effective DNA sequencing of individual genomes
(both normal and cancerous) may afford inroads to identifying
polymorphisms correlating with progression to invasive carcino-
mas, particularly in organs in which both noninvasive adenomas
and invasive carcinomas are prevalent, such as the colon. Elu-
cidation of such polymorphic modifiers could well contribute to
the future of personalized medicine, whereby susceptibility vs.
resistance alleles of invasion modifiers might be factored into the
treatment for patients diagnosed with early-stage cancers.
Materials and Methods
Genetically Engineered Mice. The generation and characterization of the RT2
mouse line has been previously described (8). The RT2 line has been back-
crossed into the B6 (Charles River Laboratories) and the C3H (Jackson Lab-
oratory) genetic backgrounds more than 20 times and is effectively inbred
into these backgrounds. F1 hybrid mice and RT2 N2 mice were generated as
described (Fig. S4). Beginning at 10 wk of age, all RT2 mice received 50%
sugar food (Harlan Teklad) to relieve the effects of hypoglycemia caused by
the insulin-secreting tumors. All mice used in this study were housed and
maintained in accordance with the University of California, San Francisco
institutional guidelines governing the care of laboratory mice.
Genomic DNA Preparation, SNP Genotyping, and Linkage Analysis. Genomic
DNA was isolated from mouse tails by proteinase K (Qiagen) digestion fol-
lowed by phenol-chloroform extraction using standard methods (42). Four
micrograms of genomic DNA per animal was SNP genotyped using the
Illumina platform according to the manufacturer’s specifications, and 143
RT2 N2 mice for which tumor phenotype data were available were geno-
typed by this method. A panel of primers that discriminate between the B6
and C3H genetic backgrounds across all 19 somatic chromosomes and the X
chromosome was used (see Dataset S1 for a complete list of SNPs used in this
study). Statistical analysis was performed using R/qtl (http://www.rqtl.org/)
for the IT, IC1, IC2, tumor number, and tumor burden metrics. Because these
metrics were not normally distributed, nonparametric tests were chosen in
R/qtl. A LOD score of ≥3.0 was considered significant (15).
TAE684 Inhibitor Trial. The characterization of the Alk inhibitor NVP-TAE684
(TAE684) has been previously described (20). TAE684 was resuspended in
10% 1-methyl-2-pyrrolidinone/90% PEG 300 (vol/vol) (Sigma), and male RT2
B6 mice were administered a 10-mg/kg dose once daily or vehicle solution
alone by oral gavage from 10 to 14 wk of age. Body mass was monitored
twice weekly to adjust dosing levels and to assess any toxicity caused by
ACKNOWLEDGMENTS. We thank the Mutation Mapping and Developmen-
tal Analysis Project (Harvard Medical School, Boston, MA; National Institute
of Child Health and Human Development Grant U01-HD43430), the Partners
Healthcare Center for Genetics and Genomics (Harvard Medical School,
Boston, MA), and the University of California, San Francisco (UCSF) Helen
Diller Family Comprehensive Cancer Center Genomics Core (San Francisco,
CA) for genotyping and genomics services; the UCSF Diabetes Center
Microscopy and Islet Isolation Cores (San Francisco, CA); Nathanael Gray
(Dana-Farber Cancer Institute, Boston, MA) for the TAE684; Stephan Morris
(St. Jude Children’s Research Hospital, Memphis, TN) for advice on Alk anti-
bodies; Anguraj Sadanandam (Swiss Federal Institute of Technology Lau-
| www.pnas.org/cgi/doi/10.1073/pnas.1012705107Chun et al.
sanne, Lausanne, Switzerland) for bioinformatic analysis of the modifier
locus; Susan Cacacho, Ehud Drori, I. Celeste Rivera, Marina Vayner, and
Annie Wang for superior technical support; and Matthias Hebrok, Martin
McMahon, and members of the D.H. laboratory for advice and encourage-
ment at all stages of this project. D.H. is an American Cancer Society Re-
search Professor. This work was supported by National Cancer Institute Grant
5R01CA45234-24 (to D.H.) and by a graduate research fellowship from the
National Science Foundation (to M.G.H.C). A.B. and J.-H.M. were supported
by National Cancer Institute Grant U01-CA84244 and Department of Energy
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