CILP2 was overexpressed in Colorectal cancer.
Aiming at searching for potential novel prognostic markers of CRC, we firstly analyze expression data of TCGA CRC cohort from Illumina HiSeq 2000 platform, which contains 621 samples and correlating clinical and demographic information. We found that CILP2 was strongly correlated with clinical features of CRC samples in TCGA cohort, and CILP2 has not been reported in CRC before. So we focused on CILP2 and furtherly analyzed the association between CILP2 expression and CRC prognosis. To determine the role of CILP2 in colorectal cancer, we first analyzed CILP2 gene expression in 50 patients samples with paired adjacent normal tissues in TCGA cohort, and the results suggested that CILP2 gene was overexpressed significantly in tumor samples compared to paired adjacent normal tissues (Fig. 1A-1B, Fold change = 3.412, P < 0.001, Table 1). Additionally, CILP2 gene expression was upregulated in total amount of colorectal cancer samples compared with adjacent normal tissue samples in TCGA cohort (Fig. 1C, Fold change = 8.6161, P < 0.001, Table 2), indicating that CILP2 might be an potential biomarker.
To further testify upregulation of CILP2 in CRC, we detected CILP2 protein expression in a Tissue microarray (TMA) (n = 64) by immunohistochemical staining (IHC). We found that 68.75% (44/64) tumor tissues positively expressed CILP2 protein, whereas only 39.06% (25/64) of matched adjacent normal tissues positively expressed CILP2 protein. The staining result showed that CILP2 protein expression was significantly more prevalent in tumors than in matched adjacent normal tissues (Fig. 1D, P = 0.001). The representative images of CILP2 immunostaining were shown in Fig. 1E-1F.
Correlations between CILP2 expression and clinicopathological parameters in Colorectal cancer.
Furthermore, to dissect the role of CILP2 in CRC carcinogenesis, correlations between CILP2 expression and clinicopathological parameters were analyzed based on TCGA cohort (mRNA) and TMA cohort (protein), presenting in Table 3. And Table 4 showed correlation analysis results of TCGA cohort using Spearman’s test. Among 621 samples from TCGA cohort, part of clinicopathological data were missed in some cases. Median expression of CILP2 of all CRC samples was chosen as a cutoff to divide CRC samples into CILP2-high (n = 310) group and CILP2-low (n = 311) group. We observed that in TCGA cohort tumors of high CILP2 expression were positively associated with T3/4 stage (T1/2, 36.51%; T3/4, 53.35%; P = 0.001, Table 3), and with N1/2/3 stage (N0, 44.89%; N1/2/3, 74.29%; P = 0.005, Table 3). Similarly, the percentage of tumors with high CILP2 expression increased with grading of clinical stage (UICC 2010 stage) (stage I, 34.29%; stage II, 49.35%; stage III, 54.19%; stage IV, 60.00%, P < 0.001, Table 3), and distant metastasis (M0, 48.69%; M1, 60.23%, P = 0.048, Table 3). It was suggested that CILP2 gene expression was strongly correlated with T stage (P = 0.001), N stage (P = 0.005), M stage (P = 0.048), and higher clinical stage (P < 0.001), respectively in TCGA cohort (Fig. 2). However, there was no significant correlation of CILP2 expression with patients’ age or gender (P > 0.05, Table 3). In TMA cohort, tumors of positive CILP2 protein expression was significantly associated with T3/4 stage (T1/2 44.83%; T3/4, 74.29%; P = 0.022, Table 3), and higher clinical stage (UICC 2010 stage) (stage I, 35.71%; stage II, 50.00%; stage III, 70.83%; stage IV, 90.00%, P = 0.03, Table 3). However, there was no significant correlation of CILP2 protein expression with N stage (P = 0.2, Table 3), it may be due to limited set of data. Although the significant correlation was also not reached, there was a tendency that tumors with positive CILP2 protein expression were more likely to distantly metastasize compared with CILP2-negative tumors (P = 0.074, Table 3).
High CILP2 expression is associated with poor outcome of colorectal cancer patients.
Kaplan-Meier analysis was performed to investigate relationship between CILP2 expression and overall survival (OS) in TCGA cohort. There were 609 CRC samples available of prognostic information. Median expression of CILP2 of all CRC samples was chosen as a cutoff to divide CRC samples into CILP2-high (n = 305) group and CILP2-low (n = 304) group. As shown in Fig. 3, Table 5, CRC patients with high CILP2 expression exhibited a poorer OS rate compared with the lowexpression group (P = 0.003). Moreover, the univariate Cox regression analysis indicated that high CILP2 expression was strongly associated with a poor prognosis(P = 0.003). Other clinical variables, such as age (P < 0.0001), T stage (P = 0.005), N stage (P < 0.0001), M stage (P < 0.0001), and clinical stage (UICC 2010 stage) (P < 0.0001) were all associated with OS (Table 6). Moreover, the multivariate analysis revealed that high CILP2 expression (P = 0.034), age (P < 0.0001), M stage (P < 0.0001) and clinical stage (UICC 2010 stage) (P = 0.017) were independently associated with a poor prognosis (Table 6). These results suggested that CILP2 could be used as an independent prognostic predictor for colorectal cancer patients in the dataset.