Introduction

Colorectal cancer is the third most commonly diagnosed cancer in males and the second in females, with over 1.2 million new cancer cases and 608,700 deaths estimated to have occurred in 2008 [1].

The CUB and sushi multiple domains 1 (CSMD1) gene encodes a large, type I transmembrane protein located on the surfaces of neuronal and epithelial cells [2]. The human CSMD1 gene, consisting of 70 exons, spans two megabases in chromosome region 8p23.2 and encodes an 11.5-kb transcript [3]. CSMD1 expression is frequently lost in breast cancer [4], whereas CSMD1 loses allelic balance in head and neck squamous cell carcinomas (HNSCC) and lung cancers [5]. The full-length CSMD2 cDNA sequence is 12,486 bp long and is predicted to encode a protein with a molecular weight of 383 kDa [6]. Another CSMD family member is CSMD3. The expression of CSMD3 on the fetal and adult brain suggests that this gene is a good candidate for the pathogenesis of autistic spectrum disorders (ASDs) [7]. The role of CSMD2 and CSMD3 in carcinogenesis has not yet been studied so far. The great similarity between all three CSMD genes begs the question of whether CSMD2 and 3 are also likely to be tumor suppressors [6]. In a recent study of Tang et al. [8], they also found lower CSMD1 level in melanoma cells than in normal skin cells. Furthermore, they confirmed that CSMD1 exhibits antitumor activity through activation of the Smad pathway [8].

In this study, we observed significant correlations of CSMD1, CSMD2, and CSMD3 loss of function with clinical presentation of colorectal cancer patients. Therefore, our results support the idea that CSMD1, CSMD2, and CSMD3 may be used as new prognostic biomarkers for colorectal cancer.

Patients and methods

Subjects

A total of 52 patients with colorectal cancer were obtained from the Department of Intestine Surgery, Liaoning Cancer Hospital and Institute (Jan. 2008 to Dec. 2012). All patients underwent standard laboratory tests (cytology and histology). None of the patients underwent radiotherapy or chemotherapy before the operation. Informed consent was provided by all patients according to the Helsinki Declaration.

Extraction of total RNA

In this work, RNA was extracted using TRIzol solution (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the protocols recommended by the manufacturer, and RNAse-free DNase I was used to remove DNA contamination. Total RNA concentration and quantity were assessed by absorbency at 260 nm using a DNA/Protein Analyzer (DU 530, Beckman, Fullerton, CA, USA).

Real-time PCR

Real-time PCR was performed on a Rotor-Gene RG-6000A apparatus (Corbett Research, Cambridge, UK) for 40 cycles of 94 °C for 10 s, 60 °C for 10 s, and 72 °C for 15 s. Reactions (20 μl) included 2 μl of cDNA, target-specific primers, and the QuanTitect SYBR green PCR kit (QIAGEN, Valencia, CA, USA). The temperature range for analysis of melting curves was 55 to 99 °C over 30 s. The primer sequences were listed in Table 1. Relative quantitation was calculated by ΔΔCt method. Each reaction was repeated independently at three times in triplicate.

Table 1 The primers used in real-time PCR analyses
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Western blot analysis

Tissues were lysed in lysis buffer (20 mM Tris–HCl, 150 mM NaCl, 2 mM EDTA, 1 % Triton-X100) containing a protease inhibitor cocktail (Sigma-Aldrich, Saint Louis, MO, USA). Cell extract protein amounts were quantified using the BCA protein assay kit. Equivalent amounts of protein (30 μg) were separated using 12 % SDS-PAGE and transferred to a PVDF membrane (Millipore Corporation, Billerica, MA, USA). Western blot was performed using primary antibodies (Table 2). Each specific antibody binding was detected with horseradish peroxidase (HRP)-conjugated respective secondary antibodies (Amersham Biosciences, UK) and ECL solutions (Amersham Biosciences).

Table 2 The antibodies used in the Western blot analysis
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Statistics and survival analysis

Overall survival (OS) was determined using the Kaplan-Meier estimator. Kaplan-Meier survival plots were generated, and comparisons were made with log-rank statistics. Cox’s proportional hazards model was employed for multivariate analysis. For all analyses, only p < 0.05 was considered significant. All the statistical analyses and graphics were performed with GraphPad Prism 5.

Results

CSMD expression in human colorectal cancer specimens

To determine whether CSMD transcription was reduced, real-time PCR analysis for CSMD1, CSMD2, and CSMD3 expression was performed in 52 gastric cancer specimens. Results show that the levels of CSMD1, CSMD2, and CSMD3 mRNA in cancer tissue were significantly lower than in normal tissue (Fig. 1, p < 0.05). Western blot analysis was performed in order to determine the protein expression level of CSMD1, CSMD2, and CSMD3 protein. CSMD1, CSMD2, and CSMD3 protein expression in cancer tissue was significantly lower than in normal tissue (Fig. 2, p < 0.05).

Fig. 1
figure 1

The levels of CSMD1, CSMD2, and CSMD3 mRNA were measured in specimens using real-time PCR. The levels of CSMD1, CSMD2, and CSMD3 mRNA were lower in cancer tissues than in matched normal tissues (p < 0.05). GAPDH was used as an internal control. N normal, C cancer

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Fig. 2
figure 2

Representative results of four paired colorectal cancer and corresponding normal tissue by Western blot. CSMD1, CSMD2, and CSMD3 protein expressions were lower in cancer tissues than in matched normal tissues (p < 0.05). β-Actin was used as an internal control. N normal, C cancer

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CSMD expression and the clinicopathological variables

We then analyzed the potential relationship between the expression of CSMD and the clinicopathological characteristics of these patients. Unfortunately, CSMD1 expression was not associated with the clinicopathological characteristics of the patients with colorectal cancer (Table 3, p > 0.05). However, CSMD2 was associated with differentiation, lymphatic invasion, and tumor size (Table 3, p < 0.05). CSMD3 was associated with differentiation and lymphatic invasion (Table 3, p < 0.05). Cox’s proportional hazard analysis indicated that sex, age, differentiation, lymphatic invasion, tumor size, and pN category were not independent prognostic factors for colorectal cancer with CSMD protein expression (Table 4, p > 0.05). To investigate the level of CSMD with the patient survival, the survival data from 52 patients with colorectal cancer were assessed. Comparison by the Kaplan-Meier method for low versus high CSMD1 or CSMD2 expression showed a significant difference in the 5-year survival rate of the patients with colorectal cancer (Fig. 3, p < 0.05). However, CSMD3 was not associated with the patient survival (Fig. 3, p > 0.05). Furthermore, we found that the survival rates of the triple-positive patients (CSMD1+, CSMD2+, and CSMD3+) were far higher than the single-positive (CSMD1+, CSMD2+, or CSMD3+) or double-positive (CSMD1+CSMD2+, CSMD2+CSMD3+, or CSMD1+ CSMD3+) ones (Fig. 3, p < 0.05).

Table 3 Relationship between CSMD expression and clinicopathological parameters of patients with colorectal cancer
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Table 4 Multivariate analysis of clinical variables for patients with colorectal cancer
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Fig. 3
figure 3

CSMD1, CSMD2, or CSMD3 protein and prognosis of the patients with colorectal cancer. Kaplan-Meier curve survival analysis indicating that tumors without CSMD1, CSMD2, or CSMD3 expression had poorer disease-specific survival than those with these protein expressions

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Discussion

CSMD1 was cloned as a candidate suppressor of head and neck squamous cell carcinomas [3]. The great similarity between all three CSMD genes begs the question of whether CSMD2 and 3 are also likely to be tumor suppressors. In this study, we detected the first investigation of the role of all three CSMD genes in colorectal cancer. We confirmed that all three CSMD were lower in colorectal cancer tissues than in matched normal tissues. Our results agree with other studies that report reduced CSMD1 mRNA expression in prostate cancer, hepatocellular carcinoma, and non-small cell lung cancer [9–11]. Shull et al. [12] found that somatic mutations, allele loss, and DNA methylation were the main reasons for low CSMD1 expression in colorectal cancer. We will detect the cause of low CSMD1 expression in our future study. Liu et al. [13] found that CSMD3 is the second most frequently mutated gene in lung cancer. Kamal et al. [4] confirmed that loss of CSMD1 expression is associated with high tumor grade and poor survival in invasive ductal breast carcinoma. In this study, we did not find any associations of CSMD1 with the clinicopathological features of the patients with colorectal cancer. However, CSMD1 is associated with the survival rates of these patients.

CSMD2 and CSMD3 are expressed at low levels in many tissues, and that expression is highest in the central nervous system [14]. Previous studies mainly focused on the roles of CSMD2 or CSMD3 in psychosis. In the Alzheimer’s Disease Neuroimaging Initiative (ADNI) sample, genome-wide association study (GWAS) analysis of voxels of the entire brain in healthy subjects, mildly cognitively impaired patients, and Alzheimer’s patients identified the CSMD2 marker rs476463 among the most significantly associated SNPs to brain volume [15]. The expression of CSMD3 in fetal and adult brain suggests that this gene may be involved in the pathogenesis of autistic spectrum disorders (ASDs) [7]. This study offers convincing evidence for the first time that the three genes of CSMD family was downregulated in the patients with colorectal cancer and may be used as predictors of colorectal cancer.