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High expression of IL-13 receptor α2 in colorectal cancer is associated with invasion, liver metastasis and poor prognosis

Rodrigo Barderas+1, Rubén A. Bartolomé+1, Mª Jesús Fernandez-Aceñero2, Sofia Torres1 and J. Ignacio Casal1* ( +. Equal contribution)

1. Functional Proteomics. Department of Cellular and Molecular Medicine. Centro de

Investigaciones Biológicas (CIB-CSIC). 28040 Madrid, Spain.

2. Department of Pathology. Fundación Jiménez Díaz. Madrid. Spain

Running title: Role of IL13Rα2 in colorectal cancer metastasis

Keywords: IL13Rα2; IL13; colon cancer; metastasis; cancer survival

Grant support: R. Barderas was a recipient of a JAE-DOC Contract of the CSIC. R. Bartolome was supported by a grant to established research groups of the Asociación Española Contra el Cáncer (AECC). S. Torres was a recipient of a ProteoRed contract. This research was supported by grants BIO2009-08818 from the Spanish Ministry of Science and Innovation, established research groups (AECC) and Colomics 2 (CM) to JI Casal.

*Corresponding author: J. Ignacio Casal Functional Proteomics Laboratory Centro de Investigaciones Biológicas (CIB-CSIC) Ramiro de Maeztu, 9 28040 Madrid. Spain Phone: +34 918373112 Fax: +34 91 5360432 e-mail: icasal@cib.csic.es

Disclosures: The authors have nothing to disclose

Word counts: 4856 Figures/Tables: 6

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ABSTRACT

Autocrine secretion of cytokines by metastatic colorectal cancer cells and their role

during invasion and liver homing has been poorly characterized. In this study, we used

cytokine arrays to analyze the secretomes of poorly and highly metastatic colorectal

cancer cells. Compared with poorly metastatic cancer cells, highly metastatic cells

expressed increased levels of the immunosuppressive cytokines IL-4 and IL-13 in

addition to increased surface expression of the high affinity IL-13 receptor IL13Rα2,

suggesting that IL13Rα2 mediates IL13 effects in colorectal cancer cells. Silencing of

IL13Rα2 in highly metastatic cells led to a decrease in adhesion capacity in vitro and a

reduction in liver homing and increased survival in vivo, revealing a role for this

receptor in cell adhesion, migration, invasion and metastatic colonization. In support of

this, IL-13 signaling activated the oncogenic signaling molecules PI3K, AKT, and SRC

in highly metastatic cells.

Clinically, high expression of IL13Rα2 was associated with later stages of disease

progression and poor outcome in colorectal cancer patients. Our findings therefore

support a critical role for IL13Rα2 expression in colon cancer invasion and metastasis.

Precis:

Signaling through a cytokine decoy receptor is found to play a critical role in the

adhesion, invasion and colonization of highly metastatic colorectal cancer cells, with

potential implications for their therapeutic management

Abbreviations: IL13Rα2, interleukin 13 receptor alpha 2; RT-PCR, reverse transcriptase polymerase chain reaction; shRNA, short hairpin RNA; TMA, tissue microarray

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Introduction

Metastasis is the final step of the malignant transformation, being responsible for the

fatal outcome in cancer patients. However, metastasis is a complex process that involves

a number of different and sequential steps. Cells must detach from the primary tumor,

migrate, gain access to blood or lymphatic vessel and colonize a new target organ (1).

Therefore, metastasis cannot be considered as a single process, but as a collection of

different events, all of them exhibiting different molecular traits. Migration, invasion

and survival are fundamental aspects of the process.

Dissemination and metastasis of cancer cells depend on extensive interactions

within the tumor microenvironment (2-3). A number of these interactions are regulated

through chemokines and their receptors, which govern many different aspects of the

malignant phenotype. So, it is critical to characterize chemokines and their receptors

expression in metastatic cells. Chemokine-mediated inflammation has been shown to

play an important role in tumor biology by influencing tumor growth, invasion and

metastasis (4). Chemokines were initially described as regulators of leukocyte

trafficking and recruitment in inflammation sites. Infiltration of white blood cells,

mostly tumor-associated macrophages, and the presence of pro-inflammatory molecules

as IL-8 and IL-6 are typical features of cancer-related inflammation. Cytokines and

chemokines are produced by multiple cell types, including fibroblasts, endothelial and

epithelial cells (5), including cancer cells, which also express their G-protein coupled

receptors. These G-proteins activate a number of signaling pathways such as PLC-β,

PI3K and the MAPK cascade, which play a critical role in adhesion, migration and

survival of cancer cells (6).

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Concomitant with these effects, cytokines and chemokines also confer cancer

cells with the ability to adhere to fibronectin and collagen IV, allowing the formation of

micrometastases (7). Therefore, chemokine networks control critical steps of the

adhesion-invasion-metastasis cascade, providing the necessary signals that facilitate

cancer cell survival and growth in distant sites (3). Little is known about the role played

by the autocrine secretion of chemokines by tumor epithelial cells in cancer-related

inflammation, which might regulate the recruitment and maturation of macrophages and

other effector cells. We used the well-known KM12 cell model for the study of late

metastatic events in colorectal cancer, including liver colonization and survival of the

tumor cells in the new environment. KM12C and KM12SM epithelial cells differ only

in their metastatic properties (8). KM12SM, a highly metastatic cell line, was derived

from the poorly metastatic cell line KM12C, through successive passages in nude mice.

A previous proteomic characterization of these two colon cancer cell lines showed a

preference for expression of homing molecules in KM12SM cells (9). Moreover, a

decrease in the expression levels of enzymes from the glycolysis, pentose phosphate

pathway and amino acid transporters was observed.

To characterize the metastatic process in colorectal cancer, we analyzed the

cytokine/chemokine profiles released by colorectal metastatic cancer KM12SM cells

using antibody microarrays. These microarrays constitute a powerful tool to get a

complete overview of cytokine profiles and pathways alterations in cancer (10). We

observed higher secretion of IL-13, which was associated to a more abundant

expression of their receptor IL13Rα2 in the highly metastatic KM12 SM cells.

Silencing of IL13Rα2 revealed a major role for this receptor in cell adhesion, migration,

invasion and metastatic colonization of colorectal cancer cells. A clear increase of

expression of IL13Rα2 was observed in late stage human colon cancer tissues, showing

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a correlation between IL13Rα2 expression and poor outcome of patients with colon

cancer metastasis.

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MATERIALS AND METHODS

Cell culture

KM12C and KM12SM human colon cancer cells were obtained directly from I. Fidler´s

lab (MD Anderson Cancer Center. USA). KM12 cells were expanded in the laboratory

to prepare a large batch of working aliquots that were stored in liquid nitrogen. For each

experiment, cells were thawed and kept in culture for a maximum of 10 passages. These

two cell lines were not authenticated in our laboratory. Parental cells, and their

derivatives, were cultured in DMEM (Gibco-Life Technologies) containing 10% fetal

calf serum and antibiotics at 37ºC in a 5% CO2-humidified atmosphere. Other cell lines

were purchased directly from the American Type Culture Collection (ATCC) and

cultured as recommended. All these cell lines were authenticated by ATCC and were

passaged fewer than 6 months after purchase for all the experiments.

Human Protein Cytokine Array.

Conditioned medium from each KM12 cell type was collected after 24 and 48h in

serum-free medium and incubated with arrays containing 79 human cytokine specific

antibodies as described (10). Then, the membranes were scanned and analyzed using

Redfin, a 2D-gel image analysis software (Ludesi). Relative cytokine intensities were

normalized in comparison to control spots on the same membrane. Ratios between both

cell lines were calculated for the different cytokines at the three experiments. Individual

quantification of IL-4 and IL-13 was carried out using specific ELISA kits

(RayBiotech) (see Supplementary Methods).

Stable transformation of KM12 cells, cell adhesion and invasion assays

pLKO.1 vectors containing 5 different shRNAs for IL13Rα2 were purchased from Open

Biosystems. As shRNA control vectors, we used a scrambled shRNA and an empty pLKO.1

vector obtained from AddGene (11-12). Stable transformed cells were obtained by lentiviral

infection. Briefly, HEK293T cells were transfected with pLKO.1 vectors and the packaging

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vectors pCMV-rev, pMDLg-pRRE and pNGVL-VSVG using jetPRIME Transfection Reagent

(Polyplus). After incubating the cells for 12-15 h in serum-free medium, medium was replaced

with DMEM containing 10% FBS and antibiotics. The day after, conditioned medium

containing lentiviral particles was centrifuged, diluted 1:2-1:10 in DMEM containing 10% FBS

and antibiotics and directly added to KM12C and KM12SM cells. After three days of

incubation, infected KM12C and KM12SM CRC cells were selected using 1 μg/ml puromycin

(Sigma) during 2-3 weeks. Then, cells were cultured with medium containing 0.5 µg/ml

puromycin.

Cell adhesion and invasion using Matrigel assays were carried out according to

previously published procedures (13) (see Supplementary Methods for a full description).

Inhibitors LY294002 and PP2 were from Sigma and UO126 was from Calbiochem. For

apoptosis and proliferation assays see Supplementary Methods.

Western blot

The following antibodies were used in the study. Anti-IL4Rα, anti-IL13Rα1 and anti-α2

integrin were from Abcam, whereas anti-IL13Rα2 was from R&D Systems. AKT,

pAKT, pERK1/2, pJNK, STAT3, pSTAT3, STAT6 and pSTAT6 were from Cell

Signalling. ERK1/2, JNK and RhoGDI were from Santa Cruz Biotechnology. Tubulin

was from Sigma. Anti-β1 integrin was a gift from Dr F. Sánchez-Madrid (H. de la

Princesa. Madrid. Spain).

For western blot, KM12C and KM12SM cells were washed twice with chilled

PBS and homogenized in 0.05% SDS. Protein extracts were sonicated three times for

30s on ice, and clarified at 10000g at 4 °C. Protein extracts were run in 10% SDS-

PAGE and immunoblotting was performed following standard procedures.

Metastasis experiments in nude mice.

Swiss nude mice (Charles River) were used for intra-splenic or intra-tail inoculation.

The Ethical Committee of the Consejo Superior de Investigaciones Científicas (Madrid,

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Spain) approved the protocols used for experimental work with mice. Mice were

injected with 106 KM12 cells in 0.1 ml PBS. Mice were daily inspected for signs of

disease, such as abdominal distension, locomotive deficit, or tumor detectable by

palpation. Metastasis nodes in liver were determined by counting the number of visible

nodules in dissected livers.

Immunohistochemistry analysis

A total of 80 patients diagnosed and treated of colorectal adenocarcinoma between 2001

and 2006 in Fundación Jiménez Díaz (Madrid) were used for the study. We have

reviewed the clinical records of the patients to determine tumor stage at the time of

diagnosis and outcome (Supplementary Table S1). Hematoxylin-eosin stained sections

of the colectomy specimens were reviewed to select representative areas of the tumor to

perform immunohistochemical detection of IL13 and IL13Rα2. The working dilution

was 1/100 for IL13 and 1/500 for IL13Rα2. IL13Rα2 immunohistochemistry was

performed on a tissue microarray (TMA) using an automated system for

immunostaining (Dako Autostainer) , with antigen retrieval at high pH. The

immunostained sections were counterstained with hematoxylin and the intensity of the

membrane and cytoplasmic staining was graded as absent, weak, moderate or intense,

although for subsequent statistical analysis the cases were reclassified as positive (either

moderate or intense) or negative (either complete absence of positivity or weak staining

similar to control areas of normal colonic mucosa). In all cases, sections from normal

colonic mucosa distant from the tumor site were used as negative controls.

Statistical analyses.

Data from the patients were recorded in an Excel file. Descriptive statistics included

mean (and standard deviation) for quantitative Gaussian variables and percentages for

qualitative ones. Bivariant analysis was made with one-way ANOVA followed by

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Tukey-Kramer multiple comparison test for quantitative variables and chi-square test

for qualitative ones. In both analyses the minimum acceptable level of significance was

p < 0.05. Survival curves were plotted with Kaplan-Meier technique and compared

with the log-rank test. The level of significance for survival was also settled at a p value

< 0.05.

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RESULTS

Chemokine profiling in the secretome of KM12 cells

We investigated the production and release of 79 different chemokines, growth factors

and other immunomodulators in conditioned culture media of colorectal cancer KM12

cells by using a chemokine array. Results after 24 and 48h of culture are shown in Fig.

1A. Several chemokines, pro-angiogenic and growth factors were highly expressed in

both cell lines, among them GROα (CXCL1), IL-8 (CXCL8), Rantes (CCL5), MIP-1β

(CCL4), Eotaxin-2 (CCL24), angiogenin, MIF, osteoprotegerin or IGFBP2 (Fig. 1B).

Regarding differential expression, TIMP2, IL-4 and IL-13 were up-regulated in the

highly metastatic cells (Fig. 1A, B). IL-4 and IL-13 differences were confirmed by

using a specific ELISA, with fold-changes between 1.5-2.13, in the presence or absence

of serum, respectively (Fig. 1C).

Most of the biological effects of IL-4 and IL-13 are mediated by STAT

transcription factors (14). We analyzed KM12 cells by western blot using antibodies

specific for total and phosphorylated forms of STAT3 and STAT6 (Fig. 1D). Total and

activated STAT6 were more abundant in KM12SM cells, with no significant differences

in STAT3. High levels of STAT6 in colon cancer cells have been associated to high

invasiveness/metastasis (15). These results support a potential action of IL-4 and IL-13

on KM12 cells.

IL13Rα2 mediates IL-13 action on KM12 cells. Effects on cell adhesion

IL-4 and IL-13 usually regulate biological activity through the common type II receptor

IL4Rα/IL13Rα1 complex (16), which mediates signal transduction through the JAK-

STAT6 pathway (17). In addition, IL-13 binds a high-affinity receptor called IL13Rα2

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(18), which was initially assigned as a decoy receptor (19). We tested KM12 cells for

the presence of these receptors. IL4Rα was not detected on the surface of KM12 cells

by flow cytometry (Supplementary Fig. S1A) nor intracellular by western blot

(Supplementary Fig. S1D). IL13Rα1was not accessible on the cell surface

(Supplementary Fig. S1B), although some intracellular expression was detected using

permeabilized cells in flow cytometry (Supplementary Fig. S1C) or western blot

(Supplementary Fig. S1E). In contrast, a higher surface expression of IL13Rα2 was

observed on the surface of KM12SM than in KM12C cells by flow cytometry, 73.9%

vs. 33.9% (Fig. 2 A). Western blot analyses confirmed the increased expression of

IL13Rα2 in KM12SM cells respect to KM12C, as well as a high expression in other

metastatic colon cancer cells such as SW48 or HT29 (Fig. 2 B). Since the

IL4Rα/IL13Rα1 heterodimer for IL13 signaling was not available on the cell surface,

we hypothesized that IL13Rα2 could mediate IL-13 action in KM12 cells.

To examine the potential effect of IL13Rα2 on colorectal cancer metastasis,

IL13Rα2 was silenced on KM12C and KM12SM cells by preparing stable shRNA

transfectants. The lack of expression of IL13Rα2 was confirmed by western blot (Fig.

2C) and semi-quantitative RT-PCR (data not shown). Both analyses showed that less

than 10% of the original protein expression levels were detected after knockdown with

shRNA#23. This silencing did not affect the expression of IL13Rα1, which showed

similar intracellular levels (Supplementary Fig. S1E). To analyze the effect of

IL13Rα2-silencing on autocrine IL-13 production we quantified the levels of IL-13 by

ELISA. We observed a decrease in the expression of IL-13 in IL13Rα2-silenced cells.

The reduction was more dramatic for KM12SM cells (Supplementary Fig. S2).

Then, we analyzed the adhesive properties of the cell lines before and after

IL13Rα2-silencing using Matrigel. In basal conditions, adhesion capacity of scrambled

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KM12SM overexpressing IL13Rα2 duplicated that of KM12C cells. The decrease in

adhesion capacity after IL13Rα2 silencing was particularly significant for KM12SM

cells (>50%) and negligible for KM12C cells (Fig. 2D). Addition of IL-13 increased

significantly cell adhesion in scrambled control cells, but not in the silenced cells. The

use of anti-IL13Rα2 antibody blocked cell adhesion for IL13-treated scrambled cells,

being most relevant for KM12SM (Fig. 2D). Still, antibody blocking was not as

effective as IL13Rα2-silencing. These results suggest that IL13Rα2 also contributes to

constitutive cell adhesion in the absence of IL-13. As a control, IL13Rα1-silenced cells

(Supplementary Fig. S3A) did not show alterations in cell adhesion (Supplementary

Fig. S3B).

IL13Rα2 expression promotes cell migration and invasion in KM12 cells through

PI3K and SRC activation

To determine the effect on cell migration, we used wound healing assays and different

amounts of IL-13 for 22h. IL-13 was sufficient to promote migration in KM12 cells,

with an optimum at 10 ng/ml (Fig. 3A). KM12SM cells displayed twice more migratory

capacity than KM12C cells in presence of serum, which might contain IL-13 and other

pro-migratory factors. KM12 cells showed a much lower cell migration in wound

healing after IL13Rα2 silencing (Fig. 3B). Addition of IL-13 caused a significant

increase in the migration speed of scrambled cells (Fig. 3C). In contrast, silenced cells

were insensitive to IL-13. This inhibition was also confirmed by the use of anti-

IL13Rα2 antibodies. To study the pathways involved in the increase of cell migration

we tested different inhibitors. The increase in migration induced by IL-13 was strongly

reduced by LY294002, a PI3K inhibitor, partially reduced by PP2 (SRC inhibitor), and

not affected by UO126 (MEK1/2 inhibitor) in KM12 cells. The inhibition was more

pronounced on KM12SM cells.

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Then, we tested the ability of the cells for invasion across Matrigel (Fig. 3D). In

medium alone, the invasion was low, but twice higher for KM12SM cells. Addition of

IL-13 caused a large increase of invasion in the control cells, but not in the silenced

cells, which showed between six and ten times less invasion capacity, similar to basal

levels. As before, the use of PI3K inhibitor or anti-IL13Rα2 decreased the invasive

capacity of the scrambled cells to basal level. SRC inhibitor was also quite effective to

decrease the invasive capacity of KM12SM, but not so much on KM12C cells. No

effect was observed for MEK1/2 inhibitor. As a control, IL13Rα1 silencing in

KM12SM cells did not alter cell invasion capacity (Supplementary Fig. S3C).

To confirm the role of IL13Rα2 in cell migration and invasion, we generated

KM12 cells overexpressing this receptor, which were tested in migration and invasion

assays. Both KM12C and SM transfectants showed an increase in migration and

invasion in response to IL-13 compared to control transfectants (Supplementary Fig.

S4A-B). Collectively, these results support that IL13 action was mediated by IL13Rα2

for migration and invasion in colorectal cancer metastasis.

Pathways activation in IL13Rα2-mediated cell invasion

To study the mechanism of action of IL13Rα2 in cell invasion, we characterized the

levels of activated SRC, FAK, AKT, ERK, JNK and STAT6 in response to IL-13 or

serum (Fig. 3E). Expression of phospho-SRC and phospho-AKT was more abundant in

KM12SM cells treated with serum than with IL-13, whereas only phospho-AKT, but no

phospho-SRC, was observed in KM12C cells. These differences in phospho-SRC effect

between KM12SM and KM12C cells might explain previous differences in migration

and invasion induced by the SRC inhibitor. The presence of a double band with

different intensities in phospho-SRC between serum and IL13-treated cells suggests that

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a different member from the SRC family might be implicated in KM12SM cells, as the

antibody recognizes phosphorylation in the C-terminal tyrosine of six members of this

family. The decrease of phospho-AKT in silenced KM12SM cells treated with IL-13

confirmed the relevance of PI3K and suggests a potential effect on survival. MAPK

signaling was not affected by IL13Rα2 silencing as no differences were observed for

ERK1/2 or JNK. Activation of phospho-STAT6 was observed in KM12SM cells with

serum, but not when cells were treated only with IL13 (Fig. 3E). Therefore, IL13-

triggered activation through IL13Rα2 is STAT6-independent. Other factors must

activate STAT6 in serum-cultured KM12SM cells.

Silencing of IL13Rα2 expression in KM12 cells decreases survival, tumorigenesis

and proliferation.

The activation of the PI3K pathway has important biological effects on cell survival and

proliferation (20). To test whether IL-13 signaling via IL13α2 can modulate cell

survival, KM12 cells were subjected to apoptosis assays. In response to oxidative stress

induced by hydrogen peroxide, scrambled KM12C and KM12SM cells showed similar

levels of apoptosis (Fig. 4A). The addition of IL-13 caused a moderate effect in

promoting survival in these oxidative stress conditions. This effect was dependent on

IL13Rα2, as the addition of the antibody or the silencing of the receptor abolished the

increment in survival (Fig. 4A). The presence of LY294002, a PI3K inhibitor, inhibited

this increase in survival induced by IL-13 through IL13Rα2. These results indicate that

IL13 also affects the survival program in KM12 colorectal cancer cells through PI3K-

AKT activation mediated by the IL13Rα2 receptor. This moderate reduction in cell

apoptosis may play a role during metastasis, facilitating survival of metastatic cells.

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Then, we tested the effect of IL13Rα2 silencing on tumorigenesis through the

ability to form colonies in soft agar. KM12SM displayed a higher capacity to

proliferate in an anchorage-independent environment, when compared to KM12C (Fig.

4B). However, IL13Rα2-silenced cell lines showed a significant reduction in colony

formation, indicating a crucial role of this receptor in tumorigenesis. Regarding

proliferation, we observed only a minor effect in cells treated with IL-13 (Fig. 4C). This

IL-13 effect was blocked after incubation with anti-IL13Rα2 antibodies (Fig. 4C).

These results suggest that IL-13 signaling via IL13Rα2 does not play a major role in cell

proliferation in colorectal cancer cells.

Silencing of IL13Rα2 in KM12SM cells provokes a decrease in liver homing and an

increase in mouse survival

As a final approach to verify the role of IL13Rα2 in colon cancer invasion and

metastasis, we carried out intra-splenic injections of control and silenced KM12 cells to

analyze their ability and speed to generate metastasis in liver. IL13Rα2-silenced

KM12SM cells induced longer survival of mice than control KM12SM cells (p<0.05)

(Fig. 5A). This prolonged survival was due to the slower growth of the tumor in the

mice inoculated with IL13Rα2-silenced KM12SM cells and lower ability to colonize

liver and cause metastasis. Post-mortem analysis showed that tumors at primary

inoculation site reached similar sizes ranging between 0.7-1 cm³ in control and silenced

KM12SM cells. Poorly-metastatic KM12C cells did not cause metastasis, and only 30%

of inoculated mice developed a tumor in spleen 180 days after inoculations. Therefore,

IL13Rα2-silenced KM12SM cells lost, in a significant way, the ability to colonize the

liver, as few mice showed macroscopic metastasis after liver dissection (Fig. 5B).

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To corroborate the ability of KM12 cells for liver homing, we collected the

livers 24 h after spleen injection and carried out a PCR for amplification of human

GAPDH (Fig. 5C). Whereas human GAPDH could be detected in livers from mice

inoculated with control cells (especially KM12SM), the PCR amplification resulted in a

barely detectable band in mice inoculated with silenced cells. To examine IL13Rα2

expression in KM12SM cells as well as their invasive properties after in vivo passage,

cells were isolated from the tumors and cultured until confluence. IL13Rα2 expression

remained very low (Fig. 5D) and their invasiveness across Matrigel was not altered by

in vivo passage (Fig. 5E). Collectively, these data confirm the capacity of IL13Rα2 to

mediate homing and liver metastasis in colon cancer.

IL13Rα2 overexpression in human patients is associated to late stages and lower

overall survival

To investigate the relevance of our results in human colon cancer, we decided to study

the levels of expression of IL-13 and IL13Rα2 in tumor and adjacent normal tissue

samples. We observed IL-13 expression mainly associated to human epithelial colon

cancer cells, with weak or no IL-13 expression in the stroma of the tumors (Fig. 6A).

For statistical analysis of IL13Rα2 expression, we used a tissue microarray (TMA) with

representative sections of tumor and normal colonic mucosa from 80 patients diagnosed

and treated of colorectal adenocarcinoma and followed on the long term (more than five

years). The series was retrospectively selected. IL13Rα2 expression was not detected in

27 cases (33.7 %) and was present with either moderate or high intensity in the

remaining 53 cases (66.3%) (Fig. 6 B). Weak or no-staining was observed in all control

normal samples. We found a statistically-significant association between IL13Rα2

expression and tumor progression (T stage), with higher expression in T3 or T4 tumors

as compared with T1 or T2 (p = 0.013), lymph node involvement (higher expression in

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tumors with lymph node involvement, p = 0.013) and metastasis at the moment of

diagnosis (p = 0.038) (Fig. 6C). We found no significant association with histological

grade of the tumor, but all high grade and colloid tumors showed intense IL13Rα2

expression, as opposed to well-differentiated tumors, that were mostly negative.

Finally, we wanted to know if there was an association between IL13Rα2 expression

and survival of colon cancer patients. Survival analysis showed a clear association with

poor prognosis in terms of lower overall survival for patients with high IL13Rα2

expression (p = 0.03) (Fig. 6D).

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Discussion

Although the KM12 cell model probably does not give a complete picture of the

spontaneous metastasis in human colon cancer, actually is giving us excellent insights in

the cell adhesion, invasion and colonization of the liver in metastasis and the critical

molecules involved in these processes. Here, we have described an important role for

IL-13 and its receptor IL13Rα2 in colorectal cancer invasion and metastasis. This

conclusion was obtained from the following observations: i) IL-13 was more abundant

in the secretome of highly metastatic cells, ii) IL13Rα2 was overexpressed in highly

metastatic KM12SM cells and other metastatic cell lines compared to poorly metastatic

KM12C cells, iii) IL13Rα2-silencing decreased adhesion, invasion and clonogenicity,

iv) IL13Rα2-silencing suppressed AKT activation and promoted apoptosis, v) mice

experiments demonstrated that removal of IL13Rα2 reduced the homing capacity in

liver of KM12SM cells and increased the survival of inoculated mice and vi) human

colon cancer samples showed a high expression of IL-13 and IL13Rα2 in cancer cells.

IL13Rα2 was mainly associated to T3 or T4 tumors and to a lower overall survival.

These results confirmed that IL-13 and IL13Rα2 expression were associated to

colorectal cancer invasion and liver metastasis in cancer cells.

IL-13 has been previously associated to pathological conditions such as asthma,

autoimmune diseases and inflammatory conditions (21). It has been involved in TH2

differentiation, STAT6-dependent M2 polarization and TGF-β1 production and fibrosis

(22-23). Stimulation of mouse J774 cells with IL4 or IL-13 in combination with TNF-α

induced STAT6 phosphorylation (14) and abundant expression of IL13Rα2 on the

surface of the cells (23). IL-13 acts as an autocrine growth factor in pancreatic cancer

that promotes lymph node metastases (24) and is a major regulator of M2 macrophages

to suppress immune surveillance in metastasis (25). This counter-surveillance activity

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19

requires the expression of IL13Rα2 (26). IL13Rα2 gene expression was reported in

pancreatic and breast cancer metastasis (27-28). The almost absence of IL4Rα and

IL13Rα1 in KM12 cells (this report) and other colon cancer cells (29) indicates that IL-

13 signaling occurs through the IL13Rα2 receptor. Surprisingly, previous reports did

not detect IL13Rα2 mRNA by RT-PCR in HT-29/B6 colon cancer cells (30).

No signaling activity was initially described for IL13Rα2 due to its short

cytoplasmic domain and the lack of JAK/STAT binding sequences (19). Therefore,

IL13Rα2 was considered as a decoy receptor for IL-13 in mouse and humans (21).

However, recent studies have demonstrated that IL13Rα2 is internalized after IL-13

binding (31). It has been reported that IL13Rα2 induces MAPK signal transduction in

intestinal epithelial cells from ulcerative colitis or colorectal cancer (29) and pancreatic

cancer (28). At low concentrations, IL13Rα2 signaled through the MAPK pathway, but

at higher concentrations worked as a decoy receptor (29). In contrast, on metastatic

KM12SM cells, IL-13 action is mediated through IL13Rα2 independent from the

expression levels, as denoted by the effect on both, KM12C and KM12SM cells.

In murine macrophages, IL13Rα2 mediates AP-1 dependent, STAT-6

independent signaling, resulting in inflammation and fibrosis in vivo (23). Previous

reports in pancreatic cancer (28) and ulcerative colitis (29) showed STAT6-independent

activation in IL13Rα2 expressing cells, suggesting an inverse relationship between

IL13Rα2 expression and STAT6 activation. In colon cancer metastatic cells, signaling

through IL13Rα2 is also STAT6-independent, and is not mediated by JNK or ERK.

The lack of ERK activation could explain the minor effect of IL13Rα2 removal on cell

proliferation and might explain why IL13Rα2 overexpression does not provide any

competitive advantage in subcutaneously implanted tumors in immunodeficient mice

(32).

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In our model, IL13Rα2 signaling induced PI3K activation, as PI3K inhibitors

blocked IL-13 effects on KM12 cells. SRC activation was found exclusively on IL13-

treated KM12SM cells and might be related with the higher migration and invasion of

these cells. An alternative role for IL-13 in the increase of intestinal epithelial

permeability, mediated by the PI3K pathway without STAT6 involvement, has been

reported (33). Ulcerative colitis, a well-known risk factor for colon cancer

development, is probably driven by IL-13 (34). Also, oxazolone-induced colitis in mice

is caused by IL13-producing natural killer T cells (35). Lack of epithelial barrier

function has been reported for inflammatory bowel diseases (36-37). This increase in

permeability affected epithelial cells tight junctions, induced epithelial apoptosis and

cell restitution arrest in ulcerative colitis (30). All these processes, where IL-13 is a key

effector, contribute to severe inflammation and could constitute a connecting link

between ulcerative colitis, colon inflammation and colon cancer. In fact, we have

observed a predominant association of IL-13 expression with colon cancer epithelial

cells.

Immunohistochemical analysis of IL13Rα2 expression in a panel of metastatic

human colon cancer samples showed a clear association with late stages in human

cancer and poor outcome of patients. This worst prognosis could be attributed to the

increased invasiveness and homing capacity of cells overexpressing IL13Rα2. Liver

colonization by metastatic cells requires a different program of protein expression and

signaling activation from early metastasis. Targeting some of the cytokines or

regulatory molecules involved in metastatic invasion and homing might be a successful

approach for decreasing metastasis recurrence in colorectal cancer.

In summary, we have demonstrated a role for IL13Rα2 in adhesion, invasion,

survival and colonization of highly metastatic colon cancer cells. Our results suggest

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21

that IL13Rα2 does not function only as a decoy receptor of IL-13 in cancer cells but as

signaling mediator. Moreover, the use of IL13Rα2 expression in colon cancer patients

as a prognostic biomarker in metastatic colorectal cancer gives a pathophysiological

relevance to these findings and supports the interest of this molecule as therapeutic

target in colon cancer.

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REFERENCES

1. Nguyen DX, Massague J. Genetic determinants of cancer metastasis. Nature

reviews. 2007;8:341-52.

2. Karnoub AE, Dash AB, Vo AP, Sullivan A, Brooks MW, Bell GW, et al.

Mesenchymal stem cells within tumour stroma promote breast cancer metastasis.

Nature. 2007;449:557-63.

3. Karnoub AE, Weinberg RA. Chemokine networks and breast cancer metastasis.

Breast Dis. 2006;26:75-85.

4. Mantovani A, Allavena P, Sica A, Balkwill F. Cancer-related inflammation.

Nature. 2008;454:436-44.

5. Zlotnik A, Yoshie O. Chemokines: a new classification system and their role in

immunity. Immunity. 2000;12:121-7.

6. Balkwill F. Cancer and the chemokine network. Nat Rev Cancer. 2004;4:540-50.

7. Fernandis AZ, Prasad A, Band H, Klosel R, Ganju RK. Regulation of CXCR4-

mediated chemotaxis and chemoinvasion of breast cancer cells. Oncogene.

2004;23:157-67.

8. Morikawa K, Walker SM, Jessup JM, Fidler IJ. In vivo selection of highly

metastatic cells from surgical specimens of different primary human colon carcinomas

implanted into nude mice. Cancer Res. 1988;48:1943-8.

9. Luque-Garcia JL, Martinez-Torrecuadrada JL, Epifano C, Canamero M, Babel I,

Casal JI. Differential protein expression on the cell surface of colorectal cancer cells

associated to tumor metastasis. Proteomics. 2010;10:940-52.

10. Pena C, Garcia JM, Larriba MJ, Barderas R, Gomez I, Herrera M, et al. SNAI1

expression in colon cancer related with CDH1 and VDR downregulation in normal

adjacent tissue. Oncogene. 2009;28:4375-85.

on June 25, 2020. © 2012 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on April 13, 2012; DOI: 10.1158/0008-5472.CAN-11-4090

23

11. Sarbassov DD, Guertin DA, Ali SM, Sabatini DM. Phosphorylation and

regulation of Akt/PKB by the rictor-mTOR complex. Science. 2005;307:1098-101.

12. Stewart SA, Dykxhoorn DM, Palliser D, Mizuno H, Yu EY, An DS, et al.

Lentivirus-delivered stable gene silencing by RNAi in primary cells. RNA. 2003;9:493-

501.

13. Bartolome RA, Galvez BG, Longo N, Baleux F, Van Muijen GN, Sanchez-

Mateos P, et al. Stromal cell-derived factor-1alpha promotes melanoma cell invasion

across basement membranes involving stimulation of membrane-type 1 matrix

metalloproteinase and Rho GTPase activities. Cancer Res. 2004;64:2534-43.

14. Jiang H, Harris MB, Rothman P. IL-4/IL-13 signaling beyond JAK/STAT. J

Allergy Clin Immunol. 2000;105:1063-70.

15. Li BH, Yang XZ, Li PD, Yuan Q, Liu XH, Yuan J, et al. IL-4/Stat6 activities

correlate with apoptosis and metastasis in colon cancer cells. Biochem Biophys Res

Commun. 2008;369:554-60.

16. Aman MJ, Tayebi N, Obiri NI, Puri RK, Modi WS, Leonard WJ. cDNA cloning

and characterization of the human interleukin 13 receptor alpha chain. J Biol Chem.

1996;271:29265-70.

17. O'Shea JJ, Gadina M, Schreiber RD. Cytokine signaling in 2002: new surprises

in the Jak/Stat pathway. Cell. 2002;109 Suppl:S121-31.

18. Caput D, Laurent P, Kaghad M, Lelias JM, Lefort S, Vita N, et al. Cloning and

characterization of a specific interleukin (IL)-13 binding protein structurally related to

the IL-5 receptor alpha chain. J Biol Chem. 1996;271:16921-6.

19. Donaldson DD, Whitters MJ, Fitz LJ, Neben TY, Finnerty H, Henderson SL, et

al. The murine IL-13 receptor alpha 2: molecular cloning, characterization, and

comparison with murine IL-13 receptor alpha 1. J Immunol. 1998;161:2317-24.

on June 25, 2020. © 2012 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on April 13, 2012; DOI: 10.1158/0008-5472.CAN-11-4090

24

20. Weinberg RA. The biology of cancer. New York: Garland Science; 2007.

21. Mentink-Kane MM, Wynn TA. Opposing roles for IL-13 and IL-13 receptor

alpha 2 in health and disease. Immunol Rev. 2004;202:191-202.

22. Gordon S. Alternative activation of macrophages. Nat Rev Immunol. 2003;3:23-

35.

23. Fichtner-Feigl S, Strober W, Kawakami K, Puri RK, Kitani A. IL-13 signaling

through the IL-13alpha2 receptor is involved in induction of TGF-beta1 production and

fibrosis. Nat Med. 2006;12:99-106.

24. Formentini A, Prokopchuk O, Strater J, Kleeff J, Grochola LF, Leder G, et al.

Interleukin-13 exerts autocrine growth-promoting effects on human pancreatic cancer,

and its expression correlates with a propensity for lymph node metastases. Int J

Colorectal Dis. 2009;24:57-67.

25. Sinha P, Clements VK, Ostrand-Rosenberg S. Interleukin-13-regulated M2

macrophages in combination with myeloid suppressor cells block immune surveillance

against metastasis. Cancer Res. 2005;65:11743-51.

26. Fichtner-Feigl S, Terabe M, Kitani A, Young CA, Fuss I, Geissler EK, et al.

Restoration of tumor immunosurveillance via targeting of interleukin-13 receptor-alpha

2. Cancer Res. 2008;68:3467-75.

27. Minn AJ, Gupta GP, Siegel PM, Bos PD, Shu W, Giri DD, et al. Genes that

mediate breast cancer metastasis to lung. Nature. 2005;436:518-24.

28. Fujisawa T, Joshi B, Nakajima A, Puri RK. A novel role of interleukin-13

receptor alpha2 in pancreatic cancer invasion and metastasis. Cancer Res.

2009;69:8678-85.

on June 25, 2020. © 2012 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on April 13, 2012; DOI: 10.1158/0008-5472.CAN-11-4090

25

29. Mandal D, Levine AD. Elevated IL-13Ralpha2 in intestinal epithelial cells from

ulcerative colitis or colorectal cancer initiates MAPK pathway. Inflamm Bowel Dis.

2010;16:753-64.

30. Heller F, Florian P, Bojarski C, Richter J, Christ M, Hillenbrand B, et al.

Interleukin-13 is the key effector Th2 cytokine in ulcerative colitis that affects epithelial

tight junctions, apoptosis, and cell restitution. Gastroenterology. 2005;129:550-64.

31. Kawakami K, Taguchi J, Murata T, Puri RK. The interleukin-13 receptor alpha2

chain: an essential component for binding and internalization but not for interleukin-13-

induced signal transduction through the STAT6 pathway. Blood. 2001;97:2673-9.

32. Kawakami K, Kawakami M, Snoy PJ, Husain SR, Puri RK. In vivo

overexpression of IL-13 receptor alpha2 chain inhibits tumorigenicity of human breast

and pancreatic tumors in immunodeficient mice. J Exp Med. 2001;194:1743-54.

33. Ceponis PJ, Botelho F, Richards CD, McKay DM. Interleukins 4 and 13

increase intestinal epithelial permeability by a phosphatidylinositol 3-kinase pathway.

Lack of evidence for STAT 6 involvement. J Biol Chem. 2000;275:29132-7.

34. Bouma G, Strober W. The immunological and genetic basis of inflammatory

bowel disease. Nat Rev Immunol. 2003;3:521-33.

35. Heller F, Fuss IJ, Nieuwenhuis EE, Blumberg RS, Strober W. Oxazolone colitis,

a Th2 colitis model resembling ulcerative colitis, is mediated by IL-13-producing NK-T

cells. Immunity. 2002;17:629-38.

36. Soderholm JD, Peterson KH, Olaison G, Franzen LE, Westrom B, Magnusson

KE, et al. Epithelial permeability to proteins in the noninflamed ileum of Crohn's

disease? Gastroenterology. 1999;117:65-72.

on June 25, 2020. © 2012 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on April 13, 2012; DOI: 10.1158/0008-5472.CAN-11-4090

26

37. Schmitz H, Barmeyer C, Fromm M, Runkel N, Foss HD, Bentzel CJ, et al.

Altered tight junction structure contributes to the impaired epithelial barrier function in

ulcerative colitis. Gastroenterology. 1999;116:301-9.

on June 25, 2020. © 2012 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

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LEGENDS TO FIGURES

Figure 1. Expression of chemokines, growth factors and immunomodulators in

conditioned medium from KM12SM and KM12C cells using cytokine arrays. A)

Representative image of cytokine antibody arrays results after screening of conditioned

medium from KM12 cells. The experiment was carried out in triplicate. B) Cytokines

highly expressed in KM12 cells at 48 h. Bar graph was calculated for each cytokine

with the median value in arbitrary units of three independent assays. Inset: Signal

intensity for IL-4 and IL-13 obtained from three independent arrays. C) Expression

ratios for IL-4 and IL-13 in conditioned medium of KM12 cells in the presence or

absence of serum at 48 h after ELISA quantification. D) STAT-signaling alterations in

KM12 cells were tested by immunoblotting. Experiments were performed in triplicate

and quantified by densitometry.

Figure 2. IL13Rα2 is overexpressed on KM12SM cells and increases cell adhesion.

A) Flow cytometry analysis of KM12C and KM12SM cells. The percentage of positive

cell is shown inside each panel. B) 25 µg of protein from lysates of the indicated

colorectal carcinoma cell lines were resolved in polyacrylamide gels and subjected to

Western blot using anti-IL13Rα2 or, as a control, anti-RhoGDI antibodies C) KM12C

and KM12SM cells were infected with retroviral vectors containing different shRNAs

targeting IL13Rα2, scrambled shRNA or empty vectors, and IL13Rα2-expression was

assessed by western blot. Bands were quantified with MultiGauge software. Tubulin

was used as loading control. D) Cell adhesion to Matrigel of IL13Rα2-silenced or

control KM12 cells, pretreated for 5h with or without IL-13 plus the indicated

antibodies. Adhesion was significantly upregulated by incubation with IL-13 (**

p<0.01, *** p<0.001) and significantly decreased by incubation with anti-IL13Rα2 (ΔΔ

p<0.01, ΔΔΔ p<0.001). Data represent the mean + SD of 3 independent experiments.

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Figure 3. IL-13 increases cell migration and invasion through IL13Rα2. A) Cultures

were incubated in presence of the indicated concentrations of IL-13. Migration speed of

the cells was calculated as the distance covered in 48h. B) Wound healing assay of

KM12C and KM12SM cells incubated in medium alone or with 10% serum. Pictures

were taken 48 h after scratching. C) KM12 cells were incubated in presence or absence

of IL-13 (10 ng/ml) with the indicated antibodies and inhibitors. D) Invasion across

Matrigel of the KM12 cell lines treated as indicated. Data represent the mean + SD of 3

independent experiments. For increase in migration (* p<0.05, ** p<0.01, *** p<0.001)

and for reduction (Δ p<0.05, ΔΔ p<0.01, ΔΔΔ p<0.001). E) KM12 cells were starved in

medium, treated with IL13 (10 ng/ml) or 10% serum for 5 h and lysed. The extracts

were analyzed by western blot using the indicated antibodies. * indicates observed

changes in expression.

Figure 4. IL13Rα2 promotes cell survival and proliferation. (A) Cells were

incubated with H2O2 for 16 h in presence of medium alone or with IL-13, and in

presence or absence of LY294002 inhibitor or anti-IL13Rα2 antibodies, and subjected

to apoptosis detection assays. (B) Colony formation assay in soft agar with the indicated

cell lines. C) Proliferation and cell viability was determined by MTT assays after 24-48

h of incubation in medium with or without IL-13 (10 ng/ml) and anti-IL13Rα2

antibody. Optical density was significantly increased by addition of IL-13 (* p<0.05, **

p<0.01) and significantly inhibited by anti-IL13Rα2 (Δ p<0.05, ΔΔ p<0.01).

Figure 5. IL13Rα2 promotes liver metastasis of KM12SM cells. A) Kaplan-Meier

survival assay of nude mice inoculated with the indicated KM12 cells. Survival of mice

inoculated with IL13Rα2-silenced KM12SM cells increased significantly (*, p<0.05)

when compared to those inoculated with scrambled cells. B) Mice were examined for

macroscopic metastases in liver. Number of macroscopic metastases in liver was

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29

significantly reduced in mice inoculated with IL13Rα2-silenced KM12SM cells (**,

p<0.01). ◊ (Diamonds): number of metastases in each mouse. Average and standard

deviation are shown. C) Nude mice inoculated as in A were sacrificed 24 h after

inoculation. RNA was isolated from the liver and subjected to RT-PCR to amplify

human GAPDH. A representative experiment out of three is shown. Murine β-actin was

amplified as loading control. D) KM12 cells isolated from tumors by mechanical

disaggregation were cultured to confluence and analyzed by western blot to confirm

IL13Rα2-silencing and E) tested for invasion through Matrigel in presence or absence

of IL-13 (10ng/ml). Silenced KM12SM cells were included in the assay as a control.

Figure 6. IL13Rα2 expression is associated to human colon cancer progression and

poor survival of colorectal cancer patients. A) Immunohistochemical analysis of IL-

13 expression in human CRC tissue showed that IL-13 expression occurs preferentially

in CRC epithelial cells. B) Immunohistochemical analysis of IL13Rα2 expression in

tissue microarrays showing representative images of strong, moderate or negative

staining of different colon carcinomas and negative normal paired mucosa.

Counterstaining was made with hematoxylin. Pictures were taken at x200

magnification. C) Quantification of IL13Rα2 expression by estimation of staining

intensity was performed as described in Methods. Eighty samples were analyzed.

Positive and negative values were represented as bar graphs. p values were calculated

with the chi-square test. D) Kaplan-Meier analyses of overall survival of cancer patients

according to the expression of IL13Rα2. Significant association of IL13Rα2 expression

with lower overall survival was found by comparing differences between curves with

the log-rank test.

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20%

IL13: + - - - - - -+ + + + + - - + + + - - - + + + -LY294002: + - - + - - + - - + - - + - - + - - + - + -- -

Anti-IL13Rα2: +- - +- - +- - +- - +- - +- - +- +---

KM12C shRNA

SCRAMBLE

KM12SM shRNA

SCRAMBLE

KM12C shRNAIL13Rα2

KM12SM shRNAIL13Rα2

Anti-IL13Rα2: + + + + + + + +

CB CB

50

60

70

ngce

lls

ΔKM12C shRNA SCKM12SM shRNA SC

KM12SM shRNA IL13Rα2KM12C shRNA IL13Rα21.0

1.2

1.4

at 5

60 n

m *KM12C shRNA SCKM12SM shRNA SC

KM12SM shRNA IL13Rα2KM12C shRNA IL13Rα2

ΔΔ

10

20

30

40

of c

olon

yfo

rmi KM12SM shRNA IL13Rα2

0.2

0.4

0.6

0.8tic

al d

ensi

ty a

**

KM12SM shRNA IL13Rα2

0

% o

0.0Op

IL13 + anti-

IL13Rα2

Medium IL13 Medium + anti-

IL13Rα2

Figure 4

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N-11-4090

A

80%

100%e

mic

e

20%

40%

60%

% o

f d

isea

se-f

ree

KM12C shRNA SCRAMBLE

KM12SM shRNA SCRAMBLE

KM12C shRNA IL13Rα2

KM12SM shRNA IL13Rα2

p<0.05

0%0 20 40 60 80 100

Days after inoculation120

DB6

liver

5

4

3

sco

pic

met

asta

ses

in l

IL13Rα2

RhoGDI

Stablytransfected

cells

Tumor-isolated

celIs

50 kDa

37 kDa

E2

1

0

Nu

mb

ero

f m

acro

s

**

50

100

150

200

250

Inva

sive

cel

ls

Medium

IL13

E

C

0shRNA: SC IL13Rα2

Parental KM12SM celIs

Tumor-isolatedKM12SM celIs

SC IL13Rα2

hGAPDH

Liver Spleen

mβ-actin

Figure 5

Liver Spleen

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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on April 13, 2012; DOI: 10.1158/0008-5472.CAN-11-4090

CNormal mucosa Cancer mucosa

IL13A

Negative

Positive30

Negative

Positivep = 0.013

Negative

P iti

40

Negative

Positivep = 0.038

Negative

Positive50

Negative

Positivep = 0.013

Positive

10

15

20

25

IL13

Rα2

cou

nt

Positive

10

20

30

IL13

Rα2

cou

nt

Positive

IL13

Rα2

cou

nt

10

20

30

40

B IL13Rα2

Poorly differentiatedadenocarcinoma. Strong staining

Moderately differentiatedadenocarcinoma. Moderate staining

0

5

No Yes

Lymph Node Involvement

0

Metastasis

No Yes0

10

T1-T2 T3-T4

Tumor Grade

1.0

0.8

IL13Rα2 expressionDadenocarcinoma. Strong staining adenocarcinoma. Moderate staining

0.6

0.4

Cum

Sur

viva

l

positive

negative

Normal mucosa. Negative staining.

Moderately differentiatedadenocarcinoma. Negative staining

140120100806040200

0.2

0.0

p = 0.03

Figure 6

Overall survival

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N-11-4090

Published OnlineFirst April 13, 2012.Cancer Res   Rodrigo Barderas, Ruben A Bartolome, M Jesus Fernandez-Acenero, et al.   associated with invasion, liver metastasis and poor prognosis

2 in colorectal cancer isαHigh expression of IL-13 receptor

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