P-cadherin promotes liver metastasis and is associated with poor prognosis in colon cancer

Abstract

P-cadherin belongs to the family of classic cadherins, which is important for maintaining cellular localization and tissue integrity. Recently, it has become evident that P-cadherin contributes to the oncogenesis of many tumor types, including melanoma, prostate, breast, and colon carcinomas. Although cadherin switching is a crucial step in metastasis, the role of P-cadherin in colon cancer metastasis to the liver is unknown. In this study, we performed gene expression analysis and found that the level of P-cadherin was higher in tissue from liver metastases of colon cancer than in the corresponding primary colon cancer tissues. IHC analysis also showed that P-cadherin expression was significantly higher in liver metastases than in paired primary colorectal cancer tumors. Knockdown of P-cadherin in colon cancer cells inhibited wound healing, proliferation, and colony formation and resulted in developing fewer liver metastatic foci and reducing the tumor burden in vivo. Inhibition of P-cadherin expression also induced the up-regulation of E-cadherin and the down-regulation of β-catenin and its downstream target molecules, including survivin and c-Myc. In summary, these results uncover a novel function of P-cadherin in the regulation of colon cancer metastasis to the liver, suggesting that blocking the activity of P-cadherin or its associated signaling may be a valuable target for the treatment of hepatic metastases of colon carcinomas.

Figure 1

Expression of P-cadherin in human primary colon carcinomas and liver metastatic tissues. A: A higher level of P-cadherin mRNA was found in liver metastatic tissues (M) than in matched primary colon tumor (T) from the same patient. B: IHC analysis of P-cadherin expression in primary (N and T) and metastatic (MET) colon cancers. Results are shown as means ± SD. Statistical significance of *P < 0.05. C: Kaplan-Meier curves showing the survival of patients with colon cancer with P-cadherin staining. P-cadherin overexpression correlates with a poor prognosis in patients with colon cancer. Statistical significance of *P < 0.05, log-rank test.

Figure 2

P-cadherin regulates the migration, proliferation, and colony formation of LoVo and Ls174T cells. A: P-cadherin expression in 10 colon cancer cell lines. B: Analysis of P-cadherin expression in LoVo cells. The P-cadherin mRNA level was examined by RT-PCR in transient transfected P-cadherin RNAi-1 and RNAi-2, scramble RNAi, and parental LoVo cells. P-cadherin RNAi-1 and RNAi-2 exhibited down-regulation of P-cadherin. C: Motility of LoVo and Ls174T cells transfected with P-cadherin shRNA or vector cells was examined by wound-healing assay. Digital pictures were taken at 0, 36, and 48 hours. D: LoVo and Ls174T cells transfected with P-cadherin shRNA or vector cells were grown for 14 days in semisolid soft agar medium to monitor anchorage-independent growth. Results are shown as means ± SD. E: LoVo and Ls174T cells transfected with P-cadherin shRNA or vector cells were seeded in 96-well plates for Cell Counting Kit-8 assay. Results are shown as means ± SD. Statistical significance of *P < 0.05, sh-P-cadherin group versus vector group.

Figure 3

P-cadherin down-regulation significantly decreased tumor growth, tumor cell invasion into surrounding tissues, and liver metastasis. A: Tumor growth curves of LoVo and Ls174T and P-cadherin knockdown cells. Results are shown as means ± SD. B: H&E staining of vector groups and P-cadherin shRNA groups. The xenograft tumor showed a low invasive pattern in the P-cadherin shRNA groups. Arrows indicate area of muscular invasion. C: Down-regulation of P-cadherin in colon cancer cells dramatically inhibited the ability of the cells to generate tumor metastasis in the liver via intrasplenic injection in nude mice. Statistical plots of liver metastasis foci. Results are shown as means ± SD. Statistical significance of *P < 0.05, sh-P-cadherin group versus vector or Lovo group.

Figure 4

Association of P-cadherin expression with E-cadherin and β-catenin expression. A: Exogenous P-cadherin, E-cadherin, and N-cadherin were detected by Western blotting in two sets of LoVo and Ls174T cells transfected with P-cadherin shRNA or vector cells. B: The levels of nuclear and cytosol β-catenin were analyzed by Western blotting in two sets of LoVo and Ls174T cells transfected with P-cadherin shRNA or vector cells. C: Distribution of endogenous β-catenin in LoVo and LoVo P-cadherin shRNA cells analyzed by immunostaining using anti–P-cadherin antibody (green), β-catenin antibody (red), and DAPI (blue). In control cells, β-catenin was localized predominantly in the plasma or nuclear, but LoVo P-cadherin shRNA cells exhibited down-regulation of β-catenin from nuclear and cytoplasm. D: Distribution of endogenous β-catenin in Ls174T and Ls174T P-cadherin shRNA cells analyzed by immunostaining using anti–P-cadherin antibody (green), β-catenin antibody (red), and DAPI (blue). β-catenin was localized predominantly in the plasma or nuclear in Ls174T cells, but in Ls174T P-cadherin shRNA cells the staining of β-catenin was greatly reduced.

Figure 5

P-cadherin activated the Wnt/β-catenin pathway and inhibited the expression of E-cadherin. A: Levels of c-Myc, survivin, cyclin D1, and c-jun were analyzed by Western blotting in LoVo P-cadherin shRNA, Ls174T P-cadherin shRNA, and control cells. B: IHC study of the expression of P-cadherin, E-cadherin, and β-catenin in primary colon cancer tissues. The expression pattern of related molecules is shown in insets. C: IHC analysis of P-cadherin, E-cadherin, and β-catenin expression in experimental liver metastatic tissues. Magnification of tissues is shown in the black frames. The expression pattern of related molecules is shown in the insets. D: Immunoblot analysis of P-cadherin, E-cadherin, and EMT-related molecules in LoVo P-cadherin shRNA, Ls174T P-cadherin shRNA, and control cells.