A total of 146 paired formalin-fixed paraffin-embedded CRC and adjacent normal tissue specimens were obtained from stage I-III CRC patients who received curative surgery in Ruijin hospital from May 2009 to July 2010. These patients were diagnosed by pathological analysis. According to the tumor-node-metastasis (TNM) staging system of the American Joint Committee on Cancer (AJCC, 7th edition), 63 cases (43.2%) were in disease stage I/II and 83 cases (56.8%) were in disease stage III. Written informed consent was obtained from all patients. This study was approved by the Committee of Medical Ethics at Shanghai Jiao Tong University.

Immunohistochemistry of paraffin-embedded tissue sections was performed as previously described (17). In brief, tissue sections from paraffin blocks were deparaffinised and rehydrated. Endogenous peroxidase activity was blocked with 3% H₂O₂ for 10 min. Antigen retrieval was performed in citrate buffer (10 mM, pH 6.0) for 15 min at 100°C. The sections were incubated with a primary antibody (Anti-CTTN antibody, Sigma-Aldrich, HPA057242) at 4°C overnight in a moist chamber. Finally, the visualization signal was developed with diaminobenzidine (Dako). Images of these slides stained for CTTN were scanned at ×20 magnification and captured by microscopy (DP70, Olympus, Tokyo, Japan). The final staining scores were evaluated by two pathologists without prior knowledge of the clinical data.

Human colorectal cancer cell lines (SW480 and SW620) were obtained from Shanghai Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and maintained in RPMI-1640 (Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (Invitrogen, Grand Island, NY, USA), penicillin (100 U/ml), and streptomycin (100 mg/ml) at 37°C in a humidified atmosphere of 5% CO₂.

The plasmid and lentivirus particles for CTTN overexpression or knockdown were designed and constructed by Shanghai GenePharma Co., Ltd. (Shanghai, China). SW480 cells were transduced with Lenti-GFP (vector) or Lenti-CTTN (CTTN) at 20 MOI in the presence of the 8 µg/ml polybrene (Sigma). SW620 cells were transduced with Lenti-control (control) or Lenti-shCTTN (shCTTN) at 20 MOI in the presence of the 8 µg/ml polybrene. Cells were selected stable cell lines continuously in puromycin (2 µg/ml).

CTTN-overexpressing SW480 cells were transfected with negative control (NC) or DOCK1 siRNA using Lipofectamine RNA interference MAX Reagent (Life Technologies) according to the manufacturer's instructions. The siRNAs targeting CTTN and negative control were obtained from Shanghai GenePharma Co., Ltd.

Approximately 2×10⁵ cells were harvested and resuspended in complete medium and then seeded into a 24-well plate that was precoated with matrix proteins (20 µg/ml fibronectin or 20 µg/ml laminin). After incubating for 1 h, cells were washed twice with PBS to remove the non-adherent cells. The bound cells were determined by WST-1 (Roche, Mannheim, Germany) at a wavelength of 450 nm in a microplate reader. The change in optical density was represented as fold of control.

Transwell migration assays were performed in 24-well plates with 8 µm pore polycarbonate membrane (Corning, Tewksbury, MA, USA). Cells (5×10⁵) were starved for 12 h and then added into the upper chambers of the wells in 100 µl serum-free medium. RPMI-1640 medium (600 µl) with 10% fetal bovine serum was added into the lower chamber. Cells were incubated for 36 h at 37°C to allow cell invasion into the lower chamber. The invaded cells were fixed and stained with 0.5% crystal violet. Images were taken by microscopy (Olympus) at least in 8 representative fields. Invasion assay was performed with filters pre-coated with Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) and the following protocols were the same as migration assay.

Cells were lysed with RIPA buffer (Cell Signaling Technology, Danvers, MA, USA), and the protein concentration was measured using the BCA assay (Thermo Fisher Scientific, Waltham, MA, USA). Equal amounts of protein were subjected to 10% SDS-PAGE for electrophoresis and transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). The membrane was blocked with 5% fat-free milk at room temperate, then specific antibody anti-CTTN antibody (Sigma-Aldrich), anti-DOCK1 antibody (Abcam, Cambridge, MA, USA), anti-RAC1 antibody (Sigma-Aldrich), and anti-GAPDH antibody (Cell Signaling Technology) were incubated overnight at 4°C. Immunoreactive bands were visualized with enhanced chemiluminescence (Thermo Fisher Scientific) using LAS 4000 imaging system (Fijifilm, Tokyo, Japan).

Data are presented as the mean ± standard deviation of the mean. The χ² test and the Fisher's test were used to test correlations between CTTN expression and clinicopathological parameters. Kaplan-Meier analysis with log-rank test was performed to assess patient survival. Data were analyzed using software SPSS 17.0 (SPSS Inc., Chicago, IL, USA) and a value of P<0.05 was considered to indicate a statistically significant difference.

Previous studies have shown that CTTN was increased in many types of human cancers, including colorectal cancer (11). However, the expression of CTTN in Chinese colorectal cancer was unknown. IHC was used to detect the expression of CTTN in 146 paired CRC and adjacent normal tissues. In 94 cases (64%), CTTN is expressed in the tumor tissues, mainly localized in the cell cytoplasm (Fig. 1A). In contrast, no or weak staining is seen in the cytoplasm of adjacent normal colorectal tissues (Fig. 1A). The relationship between CTTN expression and clinicopathological features is displayed in (Table I). We found CTTN expression was significantly correlated with depth of invasion (P<0.001), lymph node involvement (P<0.001), distant metastasis (P<0.031), lymph node metastasis (P<0.005), and TNM stage (P=0.012). On the contrary, no significant differences between CTTN expression and gender, age, tumor size, tumor differentiation, or preoperative CEA level were observed. To further determine if the expression of CTTN was associated with colorectal cancer patient survival, Kaplan-Meier curve analysis is performed to assess prognosis. As shown in Fig. 1B, patients with high (n=73) CTTN expression had a significantly short overall survival (OS) than those with low (n=73) CTTN expression (P=0.013). There is also a statistically significant difference in disease-free survival (DFS) between patients whose tumors had high or low CTTN expression (P<0.001) (Fig. 1C). These data strongly indicated that CTTN expression play an important role in the progression of colorectal cancer.

Since CTTN is increased in colorectal cancer patients, we knocked down the CTTN expression in SW620 cells (high invasion capability) to investigate the biological behavior. Compared with the negative control (control) and blank, the levels of CTTN protein was significantly decreased in SW620 cells transfected with shCTTN (Fig. 2A). To investigate the effect of CTTN on the migration and invasion of SW620 cells, cell adhesion was first assessed and the results indicated knockdown of CTTN caused lower adhesion capability (Fig. 2B). Furthermore, Transwell analysis showed that knockdown of CTTN decreases the migration and invasion of SW620 cells compared to control (Fig. 2C).

Next we explored whether CTTN expression impacts the invasion capability, SW480 cells (low invasion capability) were transfected with CTTN. Western blot results showed CTTN expression was significantly increased in the CTTN group compared with the control vector and blank groups (Fig. 3A). Gain of function of CTTN enhanced the adhesion capability of SW480 cells transfected with CTTN (Fig. 3B). The cell invasion assay revealed a significant increase in the average number of cells penetrating the Transwell membrane and Matrigel in the CTTN group (Fig. 3C). These findings suggested that CTTN played an important role in cell invasion.

To further determine the mechanisms that CTTN promote CRC cell invasion, we performed bioinformatic analysis to predict protein interactions. We focused on DOCK1, which has been documented as an oncogene in human cancers (Fig. 4A) (18,19). As showed in Fig. 4B, DOCK1 was significantly increased in CTTN overexpressing cells as compared with control vector and consistently decreased DOCK1 expression in SW620 cells by knockdown of CTTN. In addition, we also observed Rac1, a downstream molecule, had a similar expression (Fig. 4B). These results indicated that the expression level of DOCK1 was upregulated by CTTN in CRC cell lines and prompted us to investigate the role of DOCK1 in CTTN-mediated cell invasion.

To further investigate the role of DOCK1 in CTTN-mediated cell mobility, we knocked down DOCK1 in SW480 cells with stable overexpression of CTTN. As shown in Fig. 5A, compared to negative control (NC), transfection with the siRNA1, siRNA2, and siRNA3 results in inhibition of DOCK1 expression. siRNA2 had the most effective inhibition and was used in the remainder of the study. Then we measured the adhesion capability of SW480 cells and found DOCK1 silencing abrogated the effects of CTTN on adhesion capability (Fig. 5B). Furthermore, inhibition of DOCK1 also reduced the mean number of cells penetrating the Transwell membrane and Matrigel (Fig. 5C), suggesting that DOCK1 plays a crucial role in cell mobility of CRC cells and is involved in CTTN-mediated tumor metastasis.