High expression of WISP1 in colon cancer is associated with apoptosis, invasion and poor prognosis

Abstract

Colon cancer (CC) likes many epithelial-derived cancers, resulting from a complex tumorigenic process. However, the exactly mechanisms of development and progression of CC are still unknown. In this study, integrated analysis in the GSE33113 and Fudan University Shanghai Cancer Center Hospital datasets revealed that WISP1 expression was significantly increased in CC cases, positivity correlated with the advanced pathologic stage and a poor prognosis was more likely in CC patients with higher levels of WISP1. Downregulation of WISP1 inhibited cell proliferation and invasion through increasing apoptosis and blocking cell cycle at G1 phase in CC LOVO and RKO cells. Besides, Gene set enrichment analysis (GSEA) revealed that relative genes involved in the Cell adhesion molecules and Cytokine-cytokine receptor interaction pathways were enriched in WISP1-higher expression patients. Western blot analysis showed that Cell adhesion molecules pathway associated genes (ICAM- 1, VCAM-1, SDC2 and CDH2) and Cytokine-cytokine receptor interaction pathway associated genes (VEGFC, CCL18, CXCR4 and TGFBR1) were also modulated by WISP1 downregulation. Then, we found that the protein β-catenin was identified as a binding partner of WISP1 and mediated the functions of WISP1 through promoting cell proliferation and invasion in LOVO and RKO cells. Further in vivo tumor formation study in nude mice indicated that inhibition of WISP1 delayed the progress of tumor formation and inhibited PCNA expression. These results indicate that WISP1 could act as an oncogene and may serve as a promising therapeutic strategy for colon cancer.

Keywords: WISP1; apoptosis; colon cancer; invasion; poor prognosis.

Figure 1. Correlation between WISP1 expression and survival time of patients with CC

(A, B) Analysis of WISP1 expression level in CC samples by bioinformatics analysis in GSE33113 and Fudan University Shanghai Cancer Center Hospital datasets. (C) Statistical analysis on the pathological stage of CC patients in WISP1 expression from Fudan University Shanghai Cancer Center hospital. (D–F). Effect of the expression level of WISP1 on the overall survival of patients with CC in GSE33113, GSE14333 and Fudan University Shanghai Cancer Center Hospital datasets. The cut-off level was set at the median value of the WISP1 expression levels in CC patients. The WISP1-higher expression tumors have a poor prognosis compared to the WISP1-lower expression tumors. ***P < 0.001.

Figure 2. WISP1 overexpression and knockdown by shRNA in CC cell lines

(A) Real-time PCR and Western blot analysis identified significant increase in WISP1 expression in five CC cells and normal epithelial colon cells. (B, C) Real-time PCR and Western blot analysis identified significant decrease in WISP1 expression in LOVO and RKO cells treated with WISP1 shRNAs. (D) Real-time PCR and Western blot analysis identified significant increase in WISP1 expression in SW620 cells treated with pLVX-AcGFP-C1-WISP1 expressing vector (WISP1). shNC: pLVX-AcGFP-C1-scramble shRNA negative control. ***P < 0.001.

Figure 3. WISP1 shRNA inhibits cell proliferation by arresting cells at G1 phase

LOVO, RKO and SW620 cells were infected with pLVX-AcGFP-C1-shWISP1 (shWISP1) or pLVX-AcGFP-C1-WISP1 expressing vector (WISP1) after 48 h. (A–C). Cells proliferation was detected by CCK-8 assay in LOVO, RKO and SW620 cells. (D) Cell cycle profile was analyzed using flow cytometry in LOVO and RKO cells. shNC: pLVX-AcGFP-C1-scramble shRNA negative control. NC: black pLVX-AcGFP-C1 negative control. *P < 0.05, ***P < 0.001.

Figure 4. WISP1 shRNA promotes cell apoptosis

(A–C). LOVO, RKO and SW620 cells treatment with pLVX-AcGFP-C1-shWISP1 (shWISP1) or pLVX-AcGFP-C1-WISP1 expressing vector (WISP1) were stained with annexin V-fluorescein and apoptosis rates was analyzed using flow cytometry. shNC: pLVX-AcGFP-C1-scramble shRNA negative control. NC: black pLVX-AcGFP-C1 negative control. ***P < 0.001.

Figure 5. WISP1 shRNA inhibits invasion

(A–C). LOVO, RKO and SW620 cells treatment with pLVX-AcGFP-C1-shWISP1 (shWISP1) or pLVX-AcGFP-C1-WISP1 expressing vector (WISP1) and invasion was determined by transwell assays. shNC: pLVX-AcGFP-C1-scramble shRNA negative control. NC: black pLVX-AcGFP-C1 negative control. ***P < 0.001.

Figure 6. WISP1 regulates cell adhesion molecules and cytokine-cytokine receptor interaction pathways

(A, B). Genes in the Cell adhesion molecules and Cytokine-cytokine receptor interaction pathways showed significant enrichment in WISP1 high versus WISP1 low tumors in CC patients. The top portion of the figure plots the enrichment scores (ES) for each gene, whereas the bottom portion of the plot shows the value of the ranking metric moving down the list of ranked genes. (C, D). Real-time PCR and Western blot analysis identified significant decrease in ICAM-1, VCAW-1, VEGFC, CCL18 and CXCR4, while increase in SDC2, CDH2 and TGFBR1 expression in LOVO and RKO cells infected with pLVX-AcGFP-C1-shWISP1 (shWISP1). shNC: pLVX-AcGFP-C1-scramble shRNA negative control. NC: black pLVX-AcGFP-C1 negative control. ***P < 0.001.

Figure 7. WISP1 binds to β-catenin in vitro

(A) Co-immunoprecipitation showed that WISP1 interacts with β-catenin in the cell lines LOVO and RKO cells. (B) β-catenin overexpression in LOVO and RKO cells. (C, D). Cells proliferation was detected by CCK-8 assay and cell cycle profile was analyzed using flow cytometry in LOVO and RKO cells infected with pLVX-AcGFP-C1-shWISP1 (shWISP1) and pLKO.1-EGFP-β-catenin expressing vector (β-catenin). (E) Apoptosis rate was analyzed using flow cytometry and invasion was determined by transwell assays. NC: black pLKO.1-EGFP negative control. shNC: pLVX-AcGFP-C1-scramble shRNA negative control. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 8. Knockdown of WISP1 in CC cells reduces tumor growth in vivo

LOVO cells infected with pLVX-AcGFP-C1-scramble shRNA negative control (shNC) or pLVX-AcGFP-C1-shWISP1 (shWISP1) were subcutaneously injected in athymic nude mice. (A) At day 45, mice were sacrificed and tumors were weighted. (B) Tumor growth was evaluated for 45 days. (C) Histology and PCNA expression were detected by HE staining and immunohistochemistry (IHC) assays. (D) Western blot analysis identified significant decrease in WISP1, β-catenin, ICAM-1, VCAW-1, VEGFC, MMP-2 and MMP-9, while increase in E-cadherin expression in LOVO cells infected with pLVX-AcGFP-C1-shWISP1 (shWISP1). shNC: pLVX-AcGFP-C1-scramble shRNA negative control. Scale bar: 10 mm. ^#P < 0.001, ***P < 0.001.