overexpression of iqgap1 in advanced colorectal cancer correlates with poor prognosis—critical...

Overexpression of IQGAP1 in advanced colorectal cancercorrelates with poor prognosis—critical role in tumor invasion

Hiroyuki Hayashi1,2, Kazuki Nabeshima1, Mikiko Aoki1, Makoto Hamasaki1, Sotaro Enatsu3, Yasushi Yamauchi2,

Yuichi Yamashita2 and Hiroshi Iwasaki1

1 Department of Pathology, Fukuoka University Hospital and School of Medicine, Fukuoka, Japan2 Department of Gastroenterological Surgery, Fukuoka University Hospital and School of Medicine, Fukuoka, Japan3 Oncology Products Medical Affairs, Lilly Research Laboratories Japan, Eli Lilly Japan, Kobe, Japan

IQGAP1 is a multifunctional protein involved in actin cytoskeleton assembly and E-cadherin-mediated cell adhesion. We

reported previously IQGAP1 overexpression in human colorectal carcinomas especially at the invasion front (IF) and that such

overexpression tended to correlate with lymph node metastasis in advanced cases. Thus, in this study, we investigated the

clinicopathological significance of IQGAP1 expression in 85 cases of pT2–3 colorectal carcinomas with special reference to its

expression pattern and prognosis, followed by analysis of the role of IQGAP1 in cancer invasion in vitro. Quantitative reverse

transcription-PCR showed significant upregulation of IQGAP1 in colorectal carcinomas compared with normal mucosa.

Immunohistochemically, IQGAP1 expression pattern was classified into diffuse (20%), IF-associated (35.3%) and focal (44.7%).

The diffuse pattern was associated with higher rates of distant metastasis. Patients with IQGAP1 overexpression and diffuse

pattern had significantly shorter survival (p < 0.0001) than others, and the diffuse pattern was an independent predictor of

poor survival by multivariate analysis. In vitro invasion assays using three human colon carcinoma cell lines showed that

IQGAP1 siRNA significantly suppressed hepatocyte growth factor (HGF)-stimulated cell invasion. HGF reduced membranous

localization of a-catenin, but did not alter localization of E-cadherin, b-catenin and IQGAP1 in membranes. Suppression of

IQGAP1 expression by siRNA did not alter membranous localization of a-catenin even in the presence of HGF. Our results

indicate that IQGAP1 plays a critical role in colon cancer cell invasion, and therefore diffuse and high expression of IQGAP1

predicts poor prognosis in patients with colorectal carcinoma.

Colorectal cancer is the third most common incidence andcauses of cancer death in both males and females in the UnitedStates,1 and it is fourth and third most common cause of can-cer death in males and females in Japan, respectively.2 The tu-mor-node-metastasis (TNM) and Dukes staging system forcolorectal cancer3 has been the most widely employed classifi-cation that provides useful prognostic information. However,the staging system, based on the extent of the tumor spread atthe time of operation, does not always account for the invasiveability and aggressiveness of the tumor itself.4 With recent pro-gress in effective adjuvant therapy, it is crucial to provide addi-tional prognostic factors within each given stage, as this is of-ten useful for clinicians to tailor the follow-up of patients toaccommodate the likelihood of more frequent metastasis thatmay require aggressive management.

Invasion is the key step in tumor progression, leading tometastasis. Based on the nature of the advancing tumor mar-gin (invasion front), colorectal carcinoma is divided into twotypes; an expanding type and an infiltrating type.5 Invasionor movement of carcinoma cells as nests, tubules or clustersof cells keeping cell–cell contact with each other, occasionallytermed collective cell migration6 or cohort migration,7 ismore or less observed in both the expanding and infiltratingtypes. In this type of epithelial cell movement, not only acti-vation of cell migration machinery is critical but also regula-tion of cell–cell contact.6,7

E-cadherin and the associated catenin complex predomi-nantly mediate the cell–cell adhesion of epithelial cells.8 Thecatenin complex includes a-catenin (102 kDa), b-catenin (92kDa) and c-catenin/plakoglobin (83 kDa). b-catenin com-bines with E-cadherin, and a-catenin links this E-cadherin-b-catenin complex to the actin cytoskeleton, which is essentialfor E-cadherin to express its full adhesive function. Remodel-ing of this adhesive sequence leads to cell detachment orloosening of cell–cell contact that enables epithelial cells tomove as clusters, and IQGAP1 is involved in the remodelingof the adhesive complexes of epithelial cells.9–11

IQGAP1 is a 189-kDa scaffolding protein that containsmultiple protein-interacting domains, such as a calponinhomology domain, a polyproline-binding domain, four cal-modulin-binding motifs and a Ras-GAP-related domain.12,13

Key words: IQGAP1, colorectal carcinoma, cell adhesion, tumor

invasion, metastasis

DOI: 10.1002/ijc.24987

History: Received 13 Jan 2009; Revised 7 Sep 2009; Accepted 6 Oct

2009; Online 23 Oct 2009

Correspondence to: Prof. K. Nabeshima, Department of Pathology,

Fukuoka University Hospital, 7-45-1 Nanakuma, Jonan-ku, 814-0180

Fukuoka, Japan, Tel.: 81-92-801-1011, E-mail: kaznabes@fukuoka-u.

ac.jp

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Int. J. Cancer: 126, 2563–2574 (2010) VC 2009 UICC

International Journal of Cancer

IJC

The interacting partners of these IQGAP1 domains includeactin, calmodulin, members of the Rho GTPase family (i.e.,Rac1 and Cdc42), Rap1, E-cadherin, b-catenin, members ofthe mitogen-activated protein kinase (MAPK) pathway andadenomatous polyposis coli.12,14 Through interaction withthese proteins, IQGAP1 regulates various basic cellular activ-ities such as cytoskeletal organization, cell–cell adhesion, cellmigration, transcription and signal transduction.

We reported previously that IQGAP1 is involved in epi-thelial type movement of human colon carcinoma cells asclusters11 and also that IQGAP1 is upregulated in humancolorectal carcinoma tissues compared with normal counter-parts.15 Moreover, IQGAP1 was strongly expressed at theinvasion front, and this invasion front-associated expressionpattern was more apparent in advanced carcinomas thanothers. Furthermore, patients with advanced carcinomaexhibiting this expression pattern showed a trend for highernodal metastasis rates, although the number of follow-upcases were small.

This study was designed to clarify the clinicopathologicalsignificance of IQGAP1 expression in advanced colorectalcarcinoma with special reference to lymph node and distantmetastases as well as its effect on the survival rate. In addi-tion, using in vitro assays, we investigated the role ofIQGAP1 in cell invasion of colorectal carcinoma with specialreference to its effect on catenin-mediated regulation of cell–cell contact.

Material and MethodsPatients and sample collection

A total of 85 advanced (pT2–3) colorectal carcinomas from48 males and 37 females [age, range, 38–92 (mean, 64.8)years] for immunohistochemical analysis were obtained fromthe colorectal carcinoma file held at the Department of Pa-thology, Fukuoka University Hospital. The 85 patients hadbeen treated at the Department of Gastroenterological Sur-gery, Fukuoka University Hospital between February 1996and December 1998. Anonymous use of redundant tissue ispart of the standard treatment agreement with patients inour hospital when no objection is expressed. The follow-upperiod was 1–70 months (mean ¼ 38.6 months). The patho-logical diagnosis included 80 cases of well to moderately dif-ferentiated adenocarcinoma, 4 cases of poorly differentiatedadenocarcinoma and 1 case of mucinous adenocarcinoma.The histological grading of carcinoma was determinedaccording to the General Rules for Clinical and PathologicalStudies on Cancer of the Colon, Rectum and Anus issued bythe Japanese Society for Cancer of the Colon and Rectum.16

Thirty cases were classified as pT2 (tumors that invade themuscularis propria) and 55 as pT3 (tumors that invadethrough the muscularis propria into the subserosa or into thenonperitonealised pericolic or perirectal tissues).17 Lymphnode metastases and distant metastases were found in 39cases (45.9%) and 22 cases (25.6%), respectively. Tumor bud-ding was defined as small clusters of carcinoma cells lying

ahead of the invasive front of the lesion as reportedpreviously.18

Real-time reverse transcriptase (RT)-PCR was conductedon frozen tissues of 35 (pT2–3) advanced colorectal carcino-mas [17 males, 18 females; age, 42–86 (mean, 65.4) years]and non-neoplastic mucosa. Tumor tissues were taken fromdeeper portions of the tumor including the invasion front,while non-neoplastic colonic mucosa was obtained from can-cer-free resection edges. All specimens were immediately fro-zen in liquid nitrogen and kept at �80�C until use. Thepathological diagnosis included 34 cases of well differentiatedto moderately differentiated adenocarcinoma and one case ofpoorly differentiated adenocarcinoma. Thirteen cases werepT2 and 22 were pT3.

Immunohistochemistry

Immunoperoxidase staining of formalin-fixed, paraffin-em-bedded tissue sections was performed using a standard bio-tin-streptavidin method. Briefly, sections were deparaffinized,rehydrated in descending alcohol dilutions. After nonspecificsites were blocked with 5% nonfat dry milk in phosphate-buffered saline (PBS) for 2 hr at 37�C, the sections wereincubated with rabbit anti-IQGAP1 monospecific antibody15

used at a 1:300 dilution overnight at 4�C. The sections werethen washed in PBS, and incubated with biotinylated anti-rabbit IgG (Dako, Carpentaria, CA) for 30 min at room tem-perature, followed by streptavidin conjugated to alkalinephosphatase (Dako) for 30 min. The reaction was identifiedwith naphthol AS-BI phosphate (Sigma Chemical, St Louis,MO) in 100 ml of 0.2 M Tris-buffered saline (TBS; pH 8.2)containing 4% hydrochloric acid and 4% nitric acid andcounterstained with Mayer’s hematoxylin or methylgreen.The immunohistochemical specificity of the antibody wasconfirmed by two types of negative controls: substituting rab-bit nonimmune IgG for the primary antibody and omittingthe primary antibody in the staining protocol. Antigen com-petition controls using anti-IQGAP1 antibody adsorbed witha 10-fold excess of IQGAP1 N-terminal peptide antigen wasalso performed.

Stained sections were evaluated semiquantitatively by twoindependent observers who were blinded to the clinical data.The whole vertical sections of carcinomas including from thetumor surface to the tumor invasion front were evenly di-vided into three compartments; the superficial, middle anddeeper one-third, and IQGAP1 staining levels were estimatedin each of them for classification of expression patterns asdescribed below. Additionally, staining levels in the whole tis-sue were also estimated. Immunostaining was considerednegative if less than 10% of the tumor cells were stained. Inspecimens considered positive, staining of the tumor wasquantitated on a scale from 1 to 4 based on the percentageof positively stained tumor cells, as described previously;15

1þ ¼ 10–25%; 2þ ¼ 25–50%; 3þ ¼ 50–75% and 4þ ¼>75%. As for the localization of reactivity, a membranous ora combined membranous and cytoplasmic reactivity was

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2564 IQGAP1 promotes tumor invasion


interpreted as positive. Moreover, IQGAP1 expression pat-terns were classified into three types: ‘‘invasion front (IF)-associated type’’ representing pronounced IQGAP1 expres-sion in the deeper one-third of carcinoma tissues includingthe invasion front, ‘‘diffuse type’’ representing expression ofIQGAP1 throughout the carcinoma tissues, and ‘‘focal type’’representing all other patterns.

RNA extraction, cDNA synthesis and real-time

quantitative RT-PCR

Messenger RNA was isolated from frozen tissue specimens,using illustraTM QuickPrep Micro mRNA purification kit (GEHealthcare Bio-Sciences, Piscataway, NJ), according to theinstructions supplied by the manufacturer. Next, the cDNAwas synthesized from 500 ng of mRNA, using Ready-To-GoT-Prime First Strand Kit (GE Healthcare Bio-Sciences). Twogene-specific oligonucleotide primer pairs for IQGAP1 andglyceraldehyde-3-phosphate dehydrogenase (GAPDH) werepurchased from Takara Bio (Otsu, Japan): (IQGAP1) For-ward, 50-GGGACCAACCAAAGTGTGTCAAC-30; Reverse,50-CTGCTCATTATTGCCTGTCTTGGA-30; (GAPDH) For-ward, 50-GCACCGTCAAGGCTGAGAAC-30; Reverese, 50-TGGTGAAGACGCCAGTGGA-30. Real-time monitoring ofPCR reactions was performed using the Light-Cycler system(Roche Applied Science, Indianapolis, IN) and SYBRVR PremixEx Taq TMII (Takara Bio), according to the instructions sup-plied by the manufacturer. Each assay was performed threetimes to verify the results. The normalized values for coloncancer and corresponding normal colonic mucosa were ana-lyzed statistically by using the Student’s t-test.

Protein extraction from frozen tissue samples

and western blotting

Proteins including IQGAP1 were extracted from tissue sam-ples using a homogenizer (polytron-aggregate; Kinematica,Luzern, Switzerland) in a lysis buffer containing 10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 2 mM ethylenediaminetetra-acetic acid (EDTA), 1% Triton X-100 and protease inhibitors(Complete Mini, Roche Applied Sciences, Penzberg, Ger-many) on ice for 30 min. The extracts were clarified by cen-trifugation at 14,000 rpm for 10 min, and subjected to so-dium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) after measurement of their protein concentra-tions using the Bio-Rad (Hercules, CA) protein assay. Afterelectrophoresis, the proteins were transferred electrophoreti-cally to Immobilon membrane (Millipore, Bedford, MA).Nonspecific sites were blocked with 2% BSA in 0.05%Tween-20/TBS, pH 7.6 (TBS-T) at 37�C for 1h and themembrane was incubated overnight at 4�C with anti-IQGAP1antibody. After washing with TBS-T, the membrane wasincubated for 1 hr with peroxidase-conjugated goat anti-rab-bit IgG. Color was developed with chemiluminescencereagents according to the instructions supplied by the manu-facturer (DuPont NEN, Boston, MA). The bands on the filmwere subjected to image analysis (NIH ImageJ version

1.3719). Statistical analysis was performed using the Student’st-test.

Cell culture

Human colon-adenocarcinoma cell line, SW837, WiDr andHT-29 cells were obtained from Dainihon Seiyaku (Osaka,Japan). These cell lines were maintained in Dulbecco’s modi-fied Eagle’s medium (DMEM; Gibco BRL, Rockville, MD),supplemented with 10% fetal calf serum (FCS), L-glutamine(746 lg/ml), sodium bicarbonate (0.2%), streptomycin(90 lg/ml) and penicillin G (90 lg/ml), pH 7.35.

In vitro invasion assays

The invasion assay based on that designed by Albin et al.19

was performed using chemotaxicells (Kurabou, Osaka, Japan)with some modifications. Briefly, colon cancer cells at latelog-phase were harvested by treatment with 0.125% trypsinand 0.5 mM EDTA treatment and 1.5 � 105cells were placedin the inner compartment of a chemotaxicell, which was sep-arated from the outer compartment by a Matrigel (12.5 lg/each surface; BD Biosciences, Bedford, MA)-coated polyvinyl-pyrrolidone-free polycarbonate filter with an 8.0-lm poresize. In both compartments, the incubation medium was se-rum-free DMEM, supplemented with 0.1% bovine serumalbumin (BSA). Hepatocyte growth factor (HGF; 100 ng/ml,Peprotech, Rocky Hill, NJ) or 10% FCS was added to thelower compartment, and serum-free DMEM added to thelower compartment served as a negative control. After incu-bation for 48 hr at 37�C in a 5% CO2 atmosphere, the filterswere fixed with 10% formalin/PBS for 30 min, and stainedwith Mayer’s hematoxylin. The cells that had migrated toareas of the lower surface of filters were manually counted in20 random fields under a microscope at a magnification of400�. Data were expressed as mean 6 SEM and differenceswere analyzed using Student’s t-test for nonpaired samples.Each assay was performed in quadruplet and repeated threetimes with similar results.

HGF-induced matrigel invasion was used as a model ofcolorectal cancer cell invasion, since expressions of HGF andits specific receptor c-Met are enhanced in colorectal cancerand related to its progression and metastases.20–23 Moreover,we previously showed expression of c-Met in SW837, WiDrand HT-29 cells and enhancement of their migration in vitroin response to HGF treatment.24

Small interfering RNA

SW837, WiDr and HT-29 cells were grown to preconfluenceand treated with small interfering RNA (siRNA) for IQGAP1(B-Bridge International, Sunnyvale, CA) or control siRNA(B-Bridge International) using Lipofectamine 2000 (Invitro-gen, Carlsbad, CA) according to the instructions provided bythe manufacturer. For IQGAP1 siRNA, RNA oligomers con-taining 21 nucleotides were synthesized in sense and anti-sense directions corresponding to human IQGAP1 at nucleo-tides 363–381 (50-UGCCAUGGAUGAGAUUGGA-30) with

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dTdT overhangs at each 30 terminus as reported previously.25

As a nonspecific siRNA control, (50-CAGUCGCGUUUGCGACUGG-30) with dTdT overhangs at 30 terminus was used.

Detection of IQGAP1 protein in tumor cells

treated with siRNA

SW837, WiDr and HT-29 cells (3.0 � 104 cells/well) wereseeded in six-well plates in serum-free DMEM. After stabili-zation of the cells over a 24-hr period, the medium was aspi-rated and replaced with serum-free DMEM, with or withoutsiRNA trasfection. After removal of the medium, the cellswere washed and scraped in PBS and collected by centrifuga-

tion at 1,000 rpm for 5min. The cells were then lysed withRIPA Lysis Buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl,1 mM EDTA, 1% NP-40; Millipore, Bedford, MA) containingprotease inhibitor cocktail tablets (Complete Mini). Lysedcells were sonicated on ice for 5 min, 3 times and centrifugedat 15,000 rpm for 20 min at 4�C. The resultant supernatantswere subjected to SDS-PAGE as described.

Cell migration on a Lab-Tek chamber slide and

immunofluorescent staining

Cell migration on a Lab-Tek chamber slide and immunofluo-rescent staining were performed as described previously.11

Figure 1. (a) IQGAP1 mRNA expression level in 35 pairs of non-neoplastic colonic mucosa and colorectal carcinoma tissues. Horizontal

lines indicate the mean values. Advanced (pT2–3) colorectal carcionoma tissues showed significantly higher IQGAP1 mRNA expression

levels compared with non-neoplastic colonic mucosa (p ¼ 0.008, by Student’s t test). (b) Expression of IQGAP1 protein in 11 pairs of

frozen non-neoplastic and tumor tissue samples analyzed by western blotting. (c) Relative expression levels of IQGAP1 protein in non-

neoplastic and colon cancer tissues (n ¼ 11). IQGAP1 expression in tumor is higher than that in normal mucosa (p < 0.001). Data are

mean 6 SEM.

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SW837 cells were seeded into compartments of an 8-well Lab-Tek tissue-culture chamber slide (Nunc, Naperville, IL; 0.8 �104 cells in 0.4 ml DMEM) and allowed to attach for 24 hr instandard culture conditions (37�C, 5% CO2 in air, 100% hu-midity). SW837 cells formed interlinked and mildly piled-upcell islands on the tissue culture glass substrate of the Lab-Tekchamber slide. The cells were treated with OPTI-MEM con-taining 0.01 pmol/ll siRNA for IQGAP1 or control siRNA for48 hr, followed by exposure to test medium [serum-freeDMEM with or without HGF (100 ng/ml)] for 24 hr. HGFtreatment alone without siRNA induces cohort type migration(cell movement in clusters keeping cell–cell contact) of SW837cells.24 After the treatments, the cells were fixed with 4% para-formaldehyde in 0.1 M phosphate buffer for 30 min at 4�C.They were then washed twice in cold PBS and treated with0.1% Triton X-100 in PBS for another 15 min at 4�C. Then thecells were incubated with 10% normal goat serum (Cedarlane,Hornby, Canada), 1% BSA (Sigma) and 0.025% thimerosal(Sigma) in PBS for 1 hr at room temperature to block nonspe-

cific binding sites. They were subsequently incubated with rab-bit anti-IQGAP1 antibody (1:500 dilution), mouse monoclonalantibodies to human E-cadherin (1:1,000 dilution, Takara Bio),human b-catenin (1:500 dilution, Transduction Laboratories,Lexington, KY) and a-catenin (1:200 dilution, TransductionLaboratories), which were diluted with the aforementionedblocking solution, for 1 hr at room temperature. The cells werethen washed in PBS, and incubated with anti-rabbit IgG goatFab0 conjugated Alexa488 or anti-mouse IgG goat Fab0 conju-gated Alexa594 (Molecular Probes, Eugene, OR) for 1 hr atroom temperature. After rinsing with PBS, the cells were im-mediately viewed under immunofluorescence microscope, BZ-8000 (Biozero, Keyence, Neu-Isenburg, Germany). For doubleimmunostaining experiments, the cells were incubated withanti-IQGAP1 rabbit antibody and anti-E-cadherin, a-cateninor b-catenin mouse monoclonal antibodies simultaneously, fol-lowed by washing in PBS and incubation with a mixture ofanti-rabbit and anti-mouse secondary antibodies describedearlier.

Figure 2. Immunohistochemical expression of IQAGAP1 in pT2–3 colorectal carcinomas. Representative cases of diffuse (a, d and g), IF-

associated (b, e and h) and focal (c, f and i) patterns. The diffuse type shows strong cytoplasmic staining with accentuation along the cell

membrane all over the tissue including the superficial (a) and deeper (d) portions as well as the invasion front (g). The IF-associated type

shows higher reactivity at the deeper portion (e) including the invasion front (h, inset), but less and focal reactivity at the superficial

portion (b, inset). The focal type shows focal and heterogeneous reactivity at both superficial (c) and deeper (f) portions including the

invasion front (i). A higher magnification view shows intense reactivity in the budding tumor cells at the invasion front (h). Original

magnifications, (a–f) �100; (g–i) �200; (inset) x400.

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Statistical analysis

Associations between categorical factors were evaluated usingthe v2 test or Fisher’s exact test. Survival curves were plottedusing the Kaplan-Meier method, and p values were calculatedusing the log rank test and ANOVA test. Multivariate analy-sis was performed by Cox’s proportional hazards regressionanalysis. A p value of < 0.05 denoted the presence of statisti-cally significant difference. All statistical evaluations wereperformed using the SPSS II software packages (SPSS Japan,Tokyo).

ResultsExpression of IQGAP1 messenger RNA and protein in

colorectal cancer tissues

In 27 of 35 pairs of samples (77%), the IQGAP1 mRNA wasexpressed at higher levels in colorectal carcinoma tissuescompared with those in non-neoplastic colonic mucosa, andthe difference was statistically significant (p ¼ 0.008, Fig. 1a).The protein expression levels, which were examined in 11frozen tissue samples by western blotting, were also higher in

carcinoma tissues by 2.5 6 0.36-fold (mean 6 SEM) com-pared with those in non-neoplastic colonic mucosa (Fig. 1b,C. p < 0.001).

Immunostaining for IQGAP1 in colorectal carcinomas

Expression of IQGAP1 protein was examined immunohisto-chemically in 85 cases of pT2–3 colorectal carcinoma (Fig. 2and Table 1). IQGAP1 expression patterns were classifiedinto 3 types; diffuse, IF-associated and focal types. In the dif-fuse type, almost all carcinoma cells stained positive in theircytoplasm and also along the membrane (Figs. 2a, 2d and2g). This type included 16 cases of well differentiated to

Table 1. IQGAP1 expression pattern and clinical characteristics

Focal IF-associated Diffuse

n 38 30 17

Age (range) 64.5 (38–83) 64.2 (41–92) 65.6 (39–80)

Gender (M:F) 28:10 12:18 8:9

Histopathology

Welldifferentiatedto moderatelydifferentiatedadenocarcinoma

34 29 17

Poorlydifferentiatedadenocarcinoma

3 1 0

Mucinousadenocarcinoma

1 0 0

Tumor budding

Positive 29 (76.3%) 28 (93.3%) 17 (100.0%)

Negative 9 (23.7%) 2 (6.7%) 0 (0.0%)

Lymph nodemetastasis1

Positive 8 (21.1%) 21 (70.0%)* 10 (58.8%)

Negative 30 (78.9%) 7 (23.3%) 6 (33.3%)

Distant metastasis2

Positive 4 (10.5%) 7 (23.3%) 11 (64.7%)*

Negative 33 (86.8%) 23 (76.7%) 6 (35.3%)

1Lymph node metastasis was not examined in 2 cases of the IF-associated type and one case of the diffuse type. 2Presence orabsence of distant metastasis was unknown in one case of the focaltype.*p < 0.0001 by ANOVA.

Figure 3. (a) Kaplan-Meier survival curves for patients of all

pathological stages (pStage I-IV) with pT2–3 colorectal cancer

according to IQGAP1 expression patterns (focal, IF-associated and

diffuse). The survival of patients with diffuse pattern was

significantly shorter than those of patients with focal or IF-

associated pattern (log rank test). (b) Kaplan-Meier survival curves

for pStage I þ II patients without lymph node metastases

according to IQGAP1 expression patterns. The survival of patients

with the diffuse pattern was significantly shorter than those of

patients with focal or IF-associated pattern (log rank test, p ¼0.004). Focal, focal type; IF-associated, invasion front-associated

type; Diffuse, diffuse type.

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moderately differentiated adenocarcinomas (18.8%). Tissuesof the IF-associated type also exhibited cytoplasmic andmembranous staining of IQGAP1 (Figs. 2b, 2e and 2h). Bud-ding tumor cells or cell clusters at the invasion front werepredominantly well stained (Fig. 2h). This type included 31well differentiated to moderately differentiated and 2 poorlydifferentiated adenocarcinomas (38.8%). Carcinomas of thefocal type showed focal and random staining, frequentlyalong the luminal border of tumor tubules (Figs. 2c, 2f and2i). This type included 33 well differentiated to moderatelydifferentiated and 2 poorly differentiated, and 1 mucinousadenocarcinomas (42.4%).

Clinicopathological analysis showed a significant correla-tion between carcinomas exhibiting the IF-associated type ofIQGAP1 expression and lymph nodes metastases, and alsobetween those showing the diffuse type of IQGAP1 expres-

sion and distant metastases (p < 0.0001, each, by ANOVAtest, Table 1). Moreover, in patients of all pathological stages(pStage I-IV), those with the diffuse type of IQGAP1 expres-sion had significantly worse prognosis in terms of survivalrate, compared to those with focal (p < 0.0001) and IF-asso-ciated (p ¼ 0.035) types of IQGAP1 expression (Fig. 3a).Even in patients of pStage I þ II (n ¼ 43, 50.6%) withoutlymph node metastases, patients with the diffuse type ofIQGAP1 expression had significantly worse prognosis com-pared to those with focal or IF-associated type of IQGAP1expression (p ¼ 0.004, Fig. 3b).

Carcinomas were also classified based on IQGAP1 expres-sion levels into high (4þ), moderate (2–3þ) and low (1þ)expression groups. Patients with high expression levels ofIQGAP1 showed significantly worse prognosis in terms of sur-vival rate, compared to those with low and moderate expres-sion levels (p < 0.0001, Fig. 4). The low expression groupincluded 2 well differentiated and 1 poorly differentiatedadenocarcinomas (3.5%), whereas moderate and high expres-sion groups included 58 well differentiated to moderately differ-entiated and 3 poorly differentiated and 1 mucinous adenocar-cinomas (76.5%) and 17 well differentiated to moderatelydifferentiated adenocarcinomas (20.0%), respectively.

Correlation between IQGAP1 expression

and clinical outcome

Univariate analysis

Within clinicopathological parameters, univariate analysisidentified a significant correlation between distant metastasis(positive) and reduced overall survival (Table 2, p < 0.001).Moreover, the diffuse pattern of IQGAP1 expression andlymph node metastasis (positive) correlated significantly withpoor patient survival (Table 2, diffuse pattern, p < 0.001;lymph node metastasis, p ¼ 0.043).

Multivariate analysis

All factors identified as significant by univariate analyses (dis-tant metastasis, lymph node metastasis and the diffuse pat-tern of IQGAP1 expression) were entered into Cox multivari-ate regression analysis. This analysis identified distantmetastasis and the diffuse type of IQGAP1 expression as sig-nificant predictors and independent factors for a shorteroverall survival (Table 3, distant metastasis, p < 0.001; diffusetype, p ¼ 0.031).

Figure 4. Kaplan-Meier survival curves for patients with low (1þ),

moderate (2–3þ) or high (4þ) levels of IQGAP1 expression. The

survival rate of patients with high IQGAP1 expression was

significantly lower than those of patients with low or moderate

levels of expression (log rank test, p < 0.0001).

Table 2. Correlation of clinicopathological parameters with overallsurvival by univariate analysis

Variable p value*

Age (<65 vs. �65) 0.07

Gender (male vs. female) 0.29

Lymph node metastasis(positive vs. negative)

0.043

Distant metastasis(positive vs. negative)

<0.001

Histology (well þ moderatelyvs. poorly differentiated)

0.93

Tumor budding (positivevs. negative)

0.92

IQGAP-1 expressionpattern (diffuse vs. focal þ IF-associated)

<0.001

*Log-rank test.

Table 3. Correlation of distant metastasis, diffuse IQGAP1expression pattern, and lymph node metastasis with overallsurvival by multivariate analysis

Variable Risk ratio 95% CI* p value

Distant metastasis 23.35 5.95–91.68 <0.001

Diffuse IQGAP1expression pattern

3.21 1.11–9.29 0.031

Lymph node metastasis 2.05 0.59–7.13 0.26

*CI, confidence interval.

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Role of IQGAP1 in colon cancer cell

invasion—In vitro studies

Effect of IQGAP1 siRNA on invasive ability

To determine the role of IQGAP1 in colon cancer invasion,we examined the effect of IQGAP1 siRNA on matrigel inva-sion using human colon cancer cell lines SW831, WiDr andHT29. The HGF-induced matrigel invasion of SW831, WiDrand HT29 was significantly suppressed by transfection ofIQGAP1 siRNA (0.01 pmol/ll) by �91, 83 and 86%, respec-tively (Fig. 5a). Treatment with IQGAP1 siRNA downregu-lated IQGAP1 expression in SW837, WiDr and HT29 cellsby about 52, 61 and 63% at 48 hr after transfection, respec-tively (Fig. 5b).

Effect of IQGAP1 siRNA on cellular expression and

localization of E-cadherin, catenins and IQGAP1

To examine a possible role of IQGAP1 during tumor cellinvasion, we analyzed its effect on E-cadherin-based cell–celladhesion. First, we examined the effect of IQGAP1 siRNA oncellular localization of E-cadherin, catenins and IQGAP1. InSW837 cells that were grown on a Lab-Tek chamber slideand transfected with unspecific control siRNA, E-cadherin,a-catenin, b-catenin and IQGAP1 were all demonstratedalong the cell membrane (Figs. 6a–6c, G-I and M-O), whichwas the same for nontreated SW837 cells (data not shown).

At 48 hr after IQGAP1 siRNA transfection, membranousexpression of IQGAP1 was markedly reduced (Figs. 6e, 6kand 6q), whereas the expression levels and localization of E-cadherin, a-catenin and b-catenin were not altered (Figs. 6d,6j and 6p).

Effect of IQGAP1 siRNA on membranous localization

of a-catenin during cell movement induced by HGF

Next, we examined the effect of IQGAP1 siRNA on membra-nous localization of E-cadherin and catenins during cellmovement induced by HGF. E-cadherin and b-catenin waslocalized to the cell membranes regardless of the presence orabsence of HGF, and both molecules were colocalized withIQGAP1 (Figs. 7a1–a3, 7a4–7a6, 7c1–7c3, 7c4–7c6). In con-trast, a-catenin was localized in cell membranes as shown ascolocalization with IQGAP1 in the absence of HGF (Figs.7b1–7b3), whereas the presence of HGF induced markedreduction of a-catenin in the membranes (Figs. 7b4–7b6).IQGAP1 was colocalized with b-catenin even in the presenceof HGF (Fig. 7c4–7c6). Transfection of IQGAP1 siRNAgreatly reduced IQGAP1 expression (Figs. 7a8–7a9, 7b8–7b9,7c8–7c9) and reversed the reduction of a-catenin localizationin membranes in the presence of HGF (Figs. 7b7 and 7b9),leading to localization of a-catenin, b-catenin and E-cadherinin the cell membranes (Figs. 7a7, 7a9, 7b7, 7b9, 7c7, 7c9).

Figure 5. Effect of IQGAP1 siRNA treatment on HGF-induced colon carcinoma cell invasion. Matrigel invasion assays were performed as

described in ‘‘Materials and Methods’’ Section. SW837, WiDr and HT29 cells were transfected with IQGAP1 siRNA or control siRNA at 0.01

pmol/ll for 48 hr before HGF stimulation. (a) HGF (100 ng/ml) significantly enhanced cancer cell invasion compared with serum-free media

(SF) control, whereas pretreatment with IQGAP1 siRNA suppressed this HGF-stimulated enhancement of invasion. Values are mean 6 SEM

(n ¼ 4). *p < 0.01 by Student’s t-test. (b) At 48 hr after transfection, the cells were lysed and analyzed by western blotting with anti-

IQGAP1 and anti-a-tubulin antibodies. NT, no treatment; C, treatment with control siRNA.

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DiscussionAlthough significant progress has been achieved in elucidat-ing the molecular roles of IQGAP1 in cellular phenomenasuch as cell migration and cell adhesion,8,26 the correlationbetween IQGAP1 expression and clinical outcomes in humanmalignant tumors have not been fully investigated. This studyis the first to show the prognostic significance of IQGAP1expression in advanced colorectal carcinoma. The main find-ing was that diffuse expression pattern of IQGAP1 signifi-cantly predict a shorter overall survival.

IQGAP1 expression in human cancers has been reportedin both in vitro and in vivo studies. In two gastric cancer celllines, IQGAP1 gene amplification correlated with correspond-ing increases in mRNA and protein expression levels.27 Com-parison of the expression profiles between primarily culturedhuman lung squamous-cell carcinoma (SCC) cells and non-neoplastic bronchial epithelial cells using suppression subtrac-tive hybridization and RT-PCR techniques identified IQGAP1as one of upregulated genes in SCC.28 Correlation betweenIQGAP1 expression and patient survival was reported inhuman glioma29 and ovarian,30 gastric31 and lung32 cancer.The expression level of IQGAP1 protein, when used in con-junction with the World Health Organization grading system,readily identified and defined a subgroup of patients withgrade III gliomas whose disease-specific survival wasshorter.29 In addition, patients with IQGAP1-negative glio-blastoma showed survival for more than 3 years.29 In ovariancancer, IQGAP1 overexpression correlated significantly withpoor prognosis by multivariate analysis.30 The diffuse expres-sion pattern at the invasion front also correlated with highhistological grade and clinicopathological stages. Examinationof IQGAP1 expression in gastric cancer revealed a trendbetween favorable prognosis and a lack of IQGAP1 expres-sion.31 In lung adenocarcinoma, patients with cytoplasmiclocalization of IQGAP1 showed a significantly better progno-sis compared to those with membranous localization orreduced expression of IQGAP1.32 Considered together, theseresult indicate that not only the presence or high levels ofIQGAP1 expression but also its expression pattern influencesprognosis. Likewise in the present study, high levels and dif-fuse pattern of IQGAP1 expression correlated with shortersurvival in patients with colorectal carcinoma. IF-associatedpattern of IQGAP1 expression correlated with higher lymphnode metastasis, whereas diffuse expression pattern was asso-ciated with distant metastasis. Furthermore, IQGAP1 expres-sion pattern may influence choice of treatment. Clinically, itis controversial whether adjuvant chemotherapy is necessaryfor patients of pStage I þ II without lymph node metastases.In this context, the diffuse pattern of IQGAP1 may aid toselect patients of pStage I þ II that may require aggressivemanagement including adjuvant chemotherapy, since the dif-fuse pattern of IQGAP1 expression significantly correlatedwith poor prognosis even in patients of pStage I þ II.

As for the relationship between IQGAP1 expression andmetastatic spread of tumor cells, Bertucci et al.33 reported the

Figure 6. Immunofluorescent localization of E-cadherin, a-catenin,

b-catenin and IQGAP1 with or without transfection of IQGAP1

siRNA in SW837 cells. After cells had been grown on a Lab-Teck

chamber slide, they were transfected with IQGAP1 or control siRNA

(0.01pmol/ll, 48 hr), followed by fixation and double

immunofluorescence staining. In cells transfected with control

siRNA, E-cadherin (a), a-catenin (g), b-catenin (m) and IQGAP1 (b,

h, n) were predominantly localized in the cell membranes.

Colocalization of IQGAP1 with E-cadherin, a-catenin and b-catenin

in cell membranes is shown in (c, i, o), respectively. IQGAP1 siRNA

transfection caused suppression of IQGAP1 expression (e, k, q),

whereas expression and localization of E-cadherin (d), a-catenin (j)

and b-catenin (p) were unchanged. a, d, g, j, m and p, visualized

with Alexa594 (red); b, e, h, k, n and q, visualized with Alexa488

(green); c, f, i, l, o and r, merge. Bars indicate 10 lm.

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Hayashi et al. 2571


IQGAP1 gene as one of the genes that were upregulated incolorectal cancer with visceral metastases, but not listed asgenes associated with lymph node metastases. Although theprecise mechanism is unknown, IQGAP1 may play a criticalrole in pathways leading to hematogenous metastasis. As astep of invasion is included twice in the metastatic cascade;once at the primary site and again at the metastatic site, weinvestigated the role of IQGAP1 in colon carcinoma invasion

using IQGAP1 siRNA and the matrigel invasion assay. Theinvasive abilities of all three human colon carcinoma celllines were greatly suppressed by transfection of IQGAP1siRNA, indicating the critical role of IQGAP1 in colon cancerinvasion. Similarly, the involvement of IQGAP1 in cellmigration and invasion was noted in human breast epithelialcells34,35 and ovarian carcinoma cells.36 Moreover, involve-ment of IQGAP1 in tumor invasiveness in vivo was also

Figure 7. Immunofluorescent localization of E-cadherin, a-catenin and IQGAP1 in the presence or absence of HGF and effect of IQGAP1

siRNA transfection. SW837 cells were grown on a Lab-Teck chamber slide, transfected with IQGAP1 or control siRNA (0.01 pmol/ll, 48 hr)

and treated with HGF (100 ng/ml), followed by fixation and double immunofluorescence staining. Without treatment, E-cadherin (a1), a-

catenin (b1), b-catenin (c1) and IQGAP1 (a2, b2, c2) were predominantly localized in the cell membranes as shown in colocalization

images (a3, b3, c3). Membranous staining of a-catenin (b4, b6) was markedly reduced in response to HGF, whereas that of E-cadherin (a4,

a6), b-catenin (c4, c6) and IQGAP1 (a5–a6, b5–b6, c5–c6) was unchanged or even stronger for IQGAP1. Transfection of cells with IQGAP1

siRNA, which suppressed IQGAP1 expression (a8, b8, c8), did not alter membranous localization of E-cadherin (a7, a9), a-catenin (b7, b9)

and b-catenin (c7, c9) even by HGF. a1, a4, a7, b1, b4, b7, c1, c4 and c7, visualized with Alexa594 (red); a2, a5, a8, b2, b5, b8, c2, c5

and c8, visualized with Alexa488 (green); a3, a6, a9, b3, b6, b9, c3, c6 and c9, merge. Bars indicate 10 lm.

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reported. Immortalized MCF-7 breast epithelial cells overex-pressing IQGAP1 formed invasive tumors when transplantedin mice, whereas tumors derived from MCF-7 cells with sta-ble knockdown of IQGAP1 were less invasive.13 In coloncancer cell line SE480, enhancement by IQGAP1 of b-cate-nin-mediated transcriptional activation, which may lead tocell invasion, was reported.37 However, in this study, translo-cation of the b-catenin from the cytoplasm to the nuclei wasnot observed during HGF-stimulated motility assays (Fig. 7).

What is the underlying mechanism(s) of IQGAP1-enhanced carcinoma cell invasion? In this study, we focusedon the role of IQGAP1 in cell–cell adhesion, since localizedrelease from cell adhesion is essential for epithelial typemovement as clusters of cells keeping cell–cell contact witheach other.7,11 In the E-cadherin-mediated cell adhesion sys-tem, a-catenin is a key molecule that links the E-cadherin–catenin complex to actin cytoskeleton. IQGAP1 is a targetmolecule of Cdc42 and Rac1 small GTPases and negativelyregulates the E-cadherin-based cell–cell adhesion by dissociat-ing a-catenin from the E-cadherin–catenin complex, resultingin loss of binding to the actin cytoskeleton.9,10 The resultantcell adhesion is weak and unstable, and probably disintegratesfairly rapidly. Recently, Noritake et al.22 proposed that E-cad-herin exists in a dynamic equilibrium between the E-cad-herin-b-catenin-a-catenin complex and the E-cadherin-b-cat-enin-IQGAP1 complex at sites of cell–cell contact. The ratiobetween these two complexes could determine the strength ofadhesion. In our in vitro study of colon cancer cells, localiza-tion of a-catenin along the cell membrane disappeared uponthe addition of HGF, whereas b-catenin and IQGAP1 wasstill noted in membranes, adding support to the aforemen-tioned hypothesis. Transfection of cells with IQGAP1 siRNA

resulted in reduction of IQGAP1 expression and consequentlocalization of a-catenin in membranes even in the presenceof HGF, possibly leading to stable cell adhesion. This mayexplain, at least in part, the reduced carcinoma cell invasioncaused by IQGAP1 siRNA transfection.

It is possible, perhaps even likely, that other IQGAP1-binding partners, such as actin cytoskeleton, calmodulin orsmall GTPases, which are implicated in cell movement usingother types of cells, contribute to IQGAP1-mediated coloncarcinoma invasion. For example, IQGAP1 serves to coupleactive Cdc42 (and Rac1) to the actin cytoskeleton, therebyenabling the Rho GTPase to induce migration of breast epi-thelial cells.35 Furthermore, Ca2þ/calmodulin signaling pro-vides an additional level to fine tune the ability of IQGAP1to increase cell migration. Moreover, using Vero cells, Wata-nabe et al.38 suggested a model in which activation of Rac1and Cdc42 in response to migration signals leads to recruit-ment of IQGAP1 in adenomatous polyposis coli (APC)which, together with CLIP-170, a microtubule-stabilizing pro-tein, form a complex that links the actin cytoskeleton andmicrotubule dynamics during cell polarization and directionalmigration. Further studies are necessary to examine thesepossibilities both in vitro and in vivo.

In conclusion, IQGAP1 is involved in colon caner cellinvasion, and may lead to vessel invasion and consequentpoor prognosis. A better understanding of the precise mecha-nisms of IQGAP1 and its interaction with other pathways isneeded before development of pharmacologic interventions.Moreover, IQGAP1, which was identified as a significantprognostic factor in the advanced stage of colon cancer, couldbe utilized to direct order-made therapy and individualize thefollow-up frequency.

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overexpression of iqgap1 in advanced colorectal cancer correlates with poor prognosis—critical...

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