Among 166 patients with colorectal cancer who underwent curative surgery between 2000 and 2007 at the Department of Gastroenterological Surgery of the Kumamoto University Hospital, Kumamoto, Japan, we selected 113 patients diagnosed with stage II and III disease, according to the 7th edition of the UICC classification (27). None of the patients had undergone preoperative chemotherapy. Tissue specimens were collected from the patients after informed consent had been obtained, in accordance with the institutional guidelines of our hospital.

The human colon carcinoma cell line, LoVo, was purchased from Riken Bioresource Center (Osaka, Japan) and was cultured in Ham’s F12 medium (Wako, Osaka, Japan). The HCT116 cell line was purchased from the ATCC (Manassas, VA, USA) and cultured in RPMI medium (Wako). All media were supplemented with 10% fetal bovine serum (Gibco, Tokyo, Japan), penicillin (100 units/ml), and streptomycin (100 μg/ml). All cells were incubated at 37°C in a humidified chamber supplemented with 5% CO₂. The anti-CD44s (clone, SFF-304) and CD44v6 (clone, VFF-18) antibodies were purchased from Bender MedSystems (Vienna, Austria). The antibodies against E-cadherin and fibronectin were purchased from BD-Biosciences (San Jose, CA, USA). The anti-vimentin antibody was purchased from Santa Cruz Biotechnology (Heidelberg, Germany). The anti-β-actin antibody was purchased from Cell Signaling Technology (Tokyo, Japan).

The immunohistochemical procedure was performed as previously described (28). In brief, after deparaffinization and rehydration of 4-μm sections, the endogenous peroxidase activity of the specimens was blocked with a methanol solution containing 3% hydrogen peroxide for 10 min at room temperature. Heat-induced antigen retrieval by microwave pretreatment in citrate buffer solution at pH 6.0 (for E-cadherin) or pH 9.0 (for CD44s, CD44v6 and vimentin) for 5–20 min was performed. Samples were incubated with the primary antibodies overnight at 4°C at the dilutions noted below. Anti-CD44s was used at a 1:100 dilution; anti-CD44v6 was used at 1:500; anti-E-cadherin was used at 1:1,200 and anti-vimentin was used at 1:50. A subsequent reaction was performed with the EnVision Plus detection system (Dako Co., Tokyo, Japan). A positive reaction was visualized with a diaminobenzidine solution, followed by counterstaining with Mayer’s hematoxylin.

We randomly selected 5 fields within the tumor invasive front under high power magnification (x400) for evaluation. Each molecule expressed on cancer cells was quantified as a percentage of the total number of stained cells. For the expression of CD44s and CD44v6, we applied a three-grade scoring system of: i) strong, ii) moderate, or iii) weak/none. Strong staining was defined as staining in >25% of the tumor cells, moderate staining was indicated when <25% of the cells were stained, and staining in <10% of the tumor cells or an absence of staining was scored as weak or none (13,15). Both CD44s and CD44v6 were considered to be highly expressed when the staining was strong. For the expression of E-cadherin and vimentin, the median value of staining was determined to be the cut-off value. The immunostaining results were independently evaluated by two investigators who were blinded with respect to the clinical and histopathologic features.

Cell invasion was assessed using the Matrigel invasion chamber (BD Biosciences, San Jose, CA, USA) as previously described (28). Cells (1×10⁵/well) transfected with either negative-control siRNA (200 nM) or CD44v6 siRNA (200 nM) were plated on Transwell chambers precoated with Matrigel in a 24-well plate. After the cells were incubated for 24 h at 37°C in a humidified incubator with 5% CO₂, the non-invading cells were removed with cotton swabs. The cells that had invaded through the membrane were fixed in 100% methanol and stained with toluidine blue. In five randomly selected fields, the number of invading cells was counted under a light microscope. Each experiment was performed in triplicate.

The migration activity was determined using the wound healing assay. A suspension of LoVo cells (2.5×10⁵/well) was poured into each well using a 12-well plate. After 24 h of incubation, the cells were transfected with siRNA and grown until subconfluence. The confluent cell layer was scratched with a pipette tip, followed by medium replacement with or without 50 ng/ml recombinant HGF (R&D Systems, Minneapolis, MN, USA). The wound distances were measured and averaged from 5 points per wound area as a baseline width. After 24 h, the width of the mean wound distance was calculated. To evaluate the ‘wound closure’, five randomly selected points along each wound were marked, and the horizontal distance the migrating cells traveled into the wound was measured. The data are reported as the means ± SD.

Total RNA was obtained from the cell lines using the RNeasy Mini kit (Qiagen, Tokyo, Japan), according to the manufacturer’s instructions. cDNA was synthesized with the SuperScript III Transcriptor First Strand cDNA Synthesis system for RT-PCR (Invitrogen, Tokyo, Japan), according to the manufacturer’s instructions. Quantitative reverse transcription PCR (qRT-PCR) was performed using a LightCycler 480 II system (Roche Diagnostics, Tokyo, Japan) as previously described (29). To perform qPCR, primers were designed using the Roche Webpage and the Universal Probe Library following the manufacturer’s recommendations. The primers used were as follows: CD44v6 forward, 5′-AACAGCTACCCA GAAGGAACAG-3′; CD44v6 reverse, 5′-CTTTGGGTGTTT GGCGATA-3′; and universal probe #145; GAPDH forward, 5′-AGCCACATCGCTCAGACAC-3′; GAPDH reverse, 5′-GC CCAATACGACCAAATCC-3′; and universal probe #60. For amplification, an initial denaturation at 95°C for 10 min was followed by 15 sec at 95°C, 15 sec at 60°C, and 13 sec at 72°C. All experiments were performed two times to confirm their reproducibility.

For isolating the proteins, cells harvested in 6-well plates were washed once in PBS and lysed in lysis buffer [Tris-HCl (pH 7.4), 25 mmol/l; NaCl, 100 mmol/l; EDTA, 2 mmol/l; Triton X-100, 1%; with 10 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mmol/l Na₃VO₄, 1 mmol/l phenylmethylsulfonyl fluoride]. Equal amounts of proteins were loaded onto 10% gels and were separated by SDS-PAGE. The resolved proteins were electrophoretically transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad Laboratories, Hercules, CA, USA). The membranes were blocked with 5% low-fat dry milk in TBS-T [25 mM Tris (pH 7.4), 125 mM NaCl, 0.4% Tween 20] for 90 min at room temperature, followed by incubation with the primary antibody at 4°C overnight. The primary antibodies for CD44v6 (1:1,000), E-cadherin (1:2,000), and vimentin (1:1,000) were the same as used for immunohistochemistry. Primary antibody for fibronectin was used at 1:1,000. The membranes were extensively washed and incubated with a 1:2,000 dilution of HRP-conjugated secondary antibody (Santa Cruz Biotechnology) for 1 h at room temperature. The membranes were washed and visualized using the chemiluminescence detection reagent kit (ECL Plus; GE Healthcare Corp., Tokyo, Japan).

Two different sequences of small interfering RNA (siRNA) targeting human CD44v6 and negative control siRNA were purchased from Qiagen (Tokyo, Japan). The sequences of CD44v6 were as follows: CD44v6#1 sense, 5′-GGCAACUCCUAGUAGUACATT-3′ and antisense, 5′-UGUACUACUAGGAGUUGCCTG-3′; CD44v6#2 sense, 5′-GAAGACUCCCAUUCGACAATT-3′ and antisense, 5′-UUGUCGAAUGGGAGUCUUCTT-3′. The cells were transfected with the CD44v6-targeting siRNA or non-silencing siRNA using Lipofectamine 2000 reagent (Qiagen) according to the manufacturer’s protocol.

Statistical analyses were performed using the StatView 5.0 software program (SAS Institute, Inc., Cary, NC, USA). The progression-free survival (PFS) and overall survival (OS) were calculated using the Kaplan-Meier method and the log-rank test was used to determine the statistical significance. A Cox proportional-hazards model was used to assess the risk ratio with simultaneous contribution from several covariates. Associations among discrete variables were assessed using the χ² test. Mean values were compared using the Student’s t-test. We analyzed the data by an ANOVA, followed by a Tukey-Kramer post-hoc test to compare multiple samples. P-values <0.05 were considered to indicate a statistically significant result.

The expression of CD44s, CD44v6 and E-cadherin was observed on the membrane of cancer cells, while vimentin expression was observed in the cytoplasm. Although CD44s expression was noted in both cancer and stromal cells, such as fibroblasts and immune cells, CD44v6 expression was localized to the cancer cells. CD44v6 expression showed a significant inverse correlation with E-cadherin expression (P=0.0007) and a positive correlation with vimentin expression (P=0.0096) (Table I), and the results from two representative patients are shown in Fig. 1A–H. In contrast, CD44s expression showed an inverse correlation with vimentin expression (P=0.031), and no correlation with E-cadherin expression (data not shown). High CD44s expression was not associated with a poor prognosis (data not shown). Based on these results, we examined the role of CD44v6 in EMT of colon cancer.

Table II shows the association between CD44v6 expression and the clinicopathological features of the 113 patients. A high level of CD44v6 expression was inversely correlated with histological differentiation of the tumor (P=0.048). To examine the prognostic value of CD44v6 expression, univariate and multivariate analyses were carried out (Table III). High CD44v6 expression was found to be an independent poor prognostic factor in disease-free survival (DFS) and overall survival (OS) (data not shown). Kaplan-Meier curves of the DFS and OS determined based on CD44v6 expression are shown in Fig. 1I and J (P=0.03 and P=0.047, respectively).

Since we hypothesized that CD44v6 has a role in the EMT phenomenon of colon cancer, we assessed whether CD44v6 knockdown impairs EMT in colon cancer cells. We transfected HCT116 and LoVo cells with two different siRNAs against CD44v6, and both reduced CD44v6 mRNA and protein expression compared with the control siRNA (Fig. 2A and B). We used siCD44v6#1 in the subsequent experiments. Knockdown of CD44v6 downregulated the protein expression of vimentin and fibronectin when compared with the control, while little change was noted in E-cadherin expression (Fig. 2B). Vimentin expression in HCT116 cells and fibronectin expression in LoVo cells were too small to compare.

The Matrigel invasion assay revealed that the knockdown of CD44v6 decreased the invasive activity of HCT116 and LoVo cells (fold-changes were 0.34 and 0.50; P=0.0003 and 0.0001) (Fig. 2C). In addition, knockdown of CD44v6 inhibited the migration of HCT116 and LoVo cells (fold-changes, 0.54 and 0.34; P=0.0085 and 0.0094, respectively) (Fig. 2D). The knockdown of CD44v6 did not cause any marked change in cell proliferation in either cell line (data not shown).

Based on previous reports indicating that CD44v6 induces cell scattering through HGF-cMet signaling in certain cancer cell cultures (24,30), we examined whether CD44v6 mediates this phenomenon. HGF stimulation of the cancer cells for 48 h led to an increase in cell migration (4.4-fold, P<0.0001) and CD44v6 knockdown decreased the HGF-mediated cell migration (0.55-fold, P<0.05) (Fig. 2E) in HCT116 cells. A similar effect was noted in LoVo cells, but the difference was not significant (data not shown). Taken together, these results indicate that CD44v6 supports HGF-induced cell scattering.