Expression of CDH12 in CRC patients is correlated with tumor invasion depth and predicts poor prognosis of CRC patients
To evaluate CDH12 expression in CRC tissue, we used immunohistochemistry (IHC) staining to test tissue microarray obtained from 78 CRC patients. The pathological and statistical results were shown in Supplementary Fig. 1 and Table 1. We evaluated the IHC results and cases which demonstrate that uniformly positive staining of CDH12 was regarded as “CDH12 high expression” in the statistical analysis. Reversely, those which demonstrate heterogeneous or uniformly negative staining of CDH12 were evaluated as “CDH12 low expression.” According to these criteria, of the 78 CRC specimens, 53 tumor tissue samples exhibited a high expression of CDH12 versus 25 cases in adjacent normal tissue (P < 0.01). In addition, 15 cases expressed heterogeneously and 10 cases expressed uniformly negative which were evaluated as CDH12 low expression (Table 1). CDH12 was mainly expressed along cell membrane (Supplementary Fig. 1e, f). Then, we made a statistical analysis between staining results and clinical materials of patients which were presented in Table 1. Statistical results were shown in Tables 2 and 3; low expression of CDH12 was predominantly associated with tumor invasion depth (P = 0.02) and lymph node metastasis (P = 0.04) but has no relationship with patients’ age (P = 0.88), tumor size (P = 0.90), tumor histology (P = 0.60), and distant colonization (P = 0.36) (Table 3). Therefore, CDH12 may be involved in CRC invasion into mesenchymal tissue and lymph node metastasis.
In addition, to further evaluate the clinical prognostic significance of CDH12, Kaplan-Meier 5-year survival curve was used to evaluate the relation of CDH12 expression in CRC tissues with the survival time of CRC patients. The Kaplan-Meier survival analysis showed that patients with high CDH12 expression (IHC staining shows uniformly positive) had longer 5-year overall survival (OS) and disease free survival (DFS) than patients with low expression (IHC staining shows uniformly negative and heterogeneous intensity) (P < 0.05; Supplementary Fig. 1g, h and Fig. 1).
Expression of CDH12 in CRC cell lines
We analyzed the expression of CDH12 in seven colon cancer cell lines by using qRT-PCR. CDH12 was expressed in all these cell lines and especially high in SW1116 and SW620 but low in HCT116 (Fig. 2a). Considering the ability of tumor formation in nude mice, we have chosen SW620 and HCT116 as our experimental objects. After screening of shRNA, we used the most efficient interfering shRNA to transfect SW620 and downregulated the expression of CDH12. Interfering effect was identified by Western blot (Fig. 2b). In addition, CDH12 lentivirus was used to transfect into HCT116 to upregulate the expression of CDH12. The results showed that the expression of CDH12 was significantly increased in HCT116 compared with control groups (Fig. 2b).
High levels of CDH12 can promote cell proliferation in CRC
To verify if CDH12 is able to influence the proliferative function of CRC cell lines, we performed CCK-8 proliferation assay. Thus, we constructed CDH12 stable knockdown clone, SW620/shCDH12 cells, and the negative control clone, SW620/shNC cells. In addition, upregulated clone HCT116/CDH12 and HCT116/Vector were also constructed, respectively, with lentivirus. CCK-8 was used to test cell proliferation ability after enforcing or downregulating CDH12 expression. Proliferation abilities of cancer cells were examined at six time points (0, 24, 48, 72, 96, and 120 h). Compared with the control groups, cells expressing high levels of CDH12 (HCT116/CDH12) showed significantly high proliferational potential (P < 0.05; Fig. 3a); however, cells with lower CDH12 level (SW620/shCDH12) presented significantly decreased proliferation ability (P < 0.05; Fig. 3a).
To further elucidate the function of CDH12 on proliferation, we performed clone formation assay. Results showed that clone colony number formed in SW620/shCDH12 group is lower than control groups. At the same time, after enforcing CDH12 expression, HCT116/CDH12 cells acquired stronger ability to form clone colony (Fig. 3b). Conclusively, these data further indicated that CDH12 might be a positive regulator of proliferation in CRC cells (Fig. 4).
Enforcing/repressing CDH12 on CRC cells promotes/suppresses tumorigenicity in nude mice
To elucidate function of CDH12 in tumor development in vivo, we have chosen nude mice to perform animal experiments. The experimental results were summarized in Supplementary Fig. 3. Compared with control groups, the size and weight of tumor nodules were markedly suppressed in CDH12 downregulation group (SW620/shCDH12) (Supplementary Fig. 3a, b). Similarly, in CDH12 overexpression group (HCT116/CDH12), size and weight of tumor formed in nude mice were significantly higher than that in control groups (Supplementary Fig. 3c, d).
CDH12 promotes cell migration and invasion progression of CRC cells
CDH12 belongs to cadherin family which acts as an important regulator of cancer cell migration. To detect the influence of CDH12 on migration and invasion in CRC cells, we performed transwell assay and found that SW620 cells with downregulated CDH12 expression presented decreased invasion and migration ability compared with the control groups (P < 0.05). Consistently, ectopic expression of CDH12 in HCT116 cells promoted the migration and invasion ability of CRC cells (P < 0.05) (Fig. 5a, b, left panel). Cell numbers were counted at random five views; the average number of migratory cells was shown in Fig. 5a, b (right panel). Moreover, wound-healing assay was also used to examine migration ability of SW620/shCDH12 and HCT116/CDH12. As shown in Fig. 5c, d, after 48-h incubation with serum-free medium, the distance of the scratch wound in SW620/shCDH12 group is significantly larger compared with control groups. But in HCT116/CDH12 group, the distance of the scratch wound increased significantly compared with control group (Fig. 5c, d, left panel). Three random lines were drawn in each group, and relative length was calculated which is shown in Fig. 5c, d (right panel).
CDH12 promotes migration and invasion of CRC cells through induction of EMT
The EMT of tumor cells is widely accepted to closely correlate with cancer metastasis. During this progress, endothelial cells expressing E-cadherin switch into mesenchymal cells marking with N-cadherin expression naming “cadherin switch” . To explore whether CDH12 can promote CRC cell migration and invasion through induction of EMT, we firstly observed and compared morphology of SW620/shCDH12 and HCT116/CDH12 cells with their control group, respectively. As shown in Fig. 6b, SW620 cells expressing low CDH12 levels had a typical epithelium-like appearance not the mesenchymal phenotype which favors the metastasis of cancer cells. Whereas, HCT116/CDH12 cells with high CDH12 expression presented a fibroblastic phenotype (Fig. 7b). We then detected the change of EMT markers in SW620/shCDH12 and HCT116/CDH12; the downregulation of CDH12 led to high expression of E-cadherin in SW620/shCDH12 cells (Fig. 6a). Because we could not detect the expressions of vimentin and N-cadherin in SW620, we construct SW1116/shCDH12 cells to verify the change of EMT markers. It also showed upregulation of E-cadherin as well as downregulation of vimentin and N-cadherin in SW1116/shCDH12 cells (Fig. 6c) and the change of SW1116 cell phenotype (Fig. 6d). In addition, we found decreased expression of E-cadherin and increased expression of N-cadherin in HCT116/CDH12 cells as well as cell phenotype change (Fig. 7a, b). Immunofluence staining has also verified the downregulation of E-cadherin in SW620/shCDH12 cells and upregulation of E-cadherin in HCT116/CDH12 cells (Fig. 8).
To further characterize transcriptional factors involved in CRC EMT triggered by CDH12, we also detected the expressions of Snail and Slug in stably transfected cells versus parental cells by Western blot analysis. As shown in Fig. 6a, c, we found a significant downregulation of Snail in SW620/shCDH12 and SW1116/shCDH12 cells; in addition, high levels of Snail were found in HCT116/CDH12 cells (Fig. 7a). We did not found significant change in the expression of slug in these cells (Figs. 6a, c and 7a). This may partially indicate that Snail is required for EMT induced by CDH12. We next transfected shRNA-Snail cells into HCT116/CDH12 cells which expressed high level of Snail and examined the change of EMT markers. Expression of E-cadherin was enhanced, whereas expression of N-cadherin was downregulated in HCT116/CDH12/shRNA-Snail cells (Fig. 7c). These findings indicate that CDH12 may contribute to CRC cell metastasis by promoting EMT, and this progress is induced by targeting Snail.
Expression of CDH12 in CRC cells is modulated by MCP1
MCP1 acts as a key CC chemokine and is responsible for trafficking and activation of monocytes/macrophages through its receptor CCR2 . Recently, elevated MCP1 expression level in the tumor implicates that it is crucial for cancer growth, dissemination, and metastasis [17, 18]. MCPIP, which was originally found in human monocytes after treatment with MCP1, has been verified that it is able to combine with CDH12 gene and promote the expression of CDH12 . Therefore, we used recombinant MCP1 to stimulate CRC cells and found that MCP1 was able to induce the expression of MCPIP and consistent high expression of CDH12 in a time-dependent manner in HCT116. The premium working point was 12 h (Supplementary Fig. 5b, d). In SW620 cell line, we used recombinant MCP1 and MCPIP small interfering RNA (siRNA) to detect the expression of CDH12. Results showed that recombinant MCP1 could induce consistent high expression of MCPIP and CDH12. In addition, MCPIP siRNA was able to block the MCPIP-induced expression of CDH12 which demonstrated that CDH12 expression was MCPIP dependent (Supplementary Fig. 5a, c). That means that MCP1 can promote CDH12 expression through induction of MCPIP.