THBS2 promotes cell migration and invasion in colorectal cancer via modulating Wnt/β-catenin signaling pathway

Abstract

Colorectal cancer (CRC) is the most common human digestive malignancy with a poor prognosis; the pathophysiology of colon cancer involves multiple linkages of regulatory networks. Recently, thrombospondin 2 (THBS2) has been extensively studied for its role in cancer progression. In this study, we evaluated the expression of THBS2 in CRC tissues and studied the possible mechanism by which THBS2 regulates CRC progression. Our results showed that the upregulation of THBS2 in CRC tissues and CRC cell lines and high expression of THBS2 was correlated with poor overall survival. The in vitro experimental data showed that THBS2 overexpression promoted CRC cell growth, invasion, and migration, while THBS2 inhibition exerted tumor-suppressive actions on CRC cells. THBS2 knockdown suppressed the activity of Wnt/β-catenin signaling. Collectively, the results implied that THBS2 exerted promotional effects on CRC cell proliferation, invasion, and migration, partly by modulating the Wnt/β-catenin signaling pathway.

Keywords: THBS2; Wnt/β-catenin; colon cancer; invasion; migration.

FIGURE 1

THBS2 is elevated in human colorectal cancer (CRC) tissues. (A) Thrombospondin 2 (THBS2) mRNA level was upregulated in CRC tissues than surrounding nontumorous tissues, which was analyzed by quantitative real‐time PCR (qRT‐PCR). **p <0.01. (B) Immunohistochemical staining analysis of THBS2 protein expression in CRC tissues and surrounding nontumorous tissues (magnification, × 200). (C). The immunohistochemistry (IHC) scores of THBS2 staining in CRC and nontumorous tissues were performed. (D) Representative western blots of THBS2 protein expression were detected in CRC and nontumorous tissues. (E). The expression of THBS2 in CRC was analyzed according to Gene Expression Profiling Interactive Analysis (GEPIA) database. *p <0.05. (F) Overall survival analysis of THBS2 in patients with CRC was performed according to GEPIA database. (G) Disease free survival analysis of THBS2 in patients with CRC was performed according to GEPIA database. (H,I) mRNA and protein expression THBS2 in normal colonic epithelial cell line, FHC and CRC cell lines (LOVO and HCT‐116) were measured using qRT‐PCR and western blotting. GAPDH was used as the control. **p <0.01 compared with FHC

FIGURE 2

Reduced thrombospondin 2 (THBS2) inhibits the aggressive phenotypes of colorectal cancer (CRC) cells. (A) LOVO and HCT116 cells were transfected with sh‐THBS2 or sh‐Ctrl. Western blot analysis was performed to detect the expression of THBS2. (B) Cell growth was measured by CCK‐8 assay after THBS2 knockdown in LOVO and HCT116 cells. (C) THBS2 knockdown inhibited the growth of LOVO and HCT116 cells which was detected by the colon formation assay. (D) The xenograft pictures of sh‐Ctrl group (n = 6) and sh‐THBS2 group (n = 6). (E) Tumor volume in the nude mice injected with sh‐THBS2 stable transfected cells was measured. (F) Wound‐healing assay was used for detecting the migration ability in THBS2 knocked down LOVO and HCT116 cells. (G) The invading cells of the transwell assay were counted under a microscope in five randomly selected fields. **p <0.01 compared with sh‐Ctrl

FIGURE 3

Upregulation of thrombospondin 2 (THBS2) promotes the aggressive phenotypes of CRC cells. (A) HCT116 cells were transfected with vector or THBS2 overexpression plasmid. Western blot analysis was performed to detect the expression of THBS2. (B) Cell growth was measured by CCK‐8 assay after THBS2 overexpression in HCT116 cells. (C) THBS2 raised the growth of LOVO cells which was detected by the colon formation assay. (D) Wound‐healing assay was used for detecting the migration ability in THBS2 overexpressing HCT116 cells. (E) The invading cells of the transwell assay were counted under a microscope in five randomly selected fields; **p <0.01 compared with vector

FIGURE 4

Thrombospondin 2 (THBS2) promotes β‐catenin nuclear translocation. (A–C) The correlating THBS2 level with Wnt/β‐catenin pathway genes (β‐catenin, MMP‐9, and Cyclin D1) were detected using TCGA. (D) Western blot of β‐catenin in the nuclear fractions of the THBS2 overexpressing CRC cells. (E) THBS2 overexpressing LOVO and HCT116 cells were transfected with the TOP/FOP‐Flash reporter plasmid, and the reporter activities were detected 48 h after transfection by a luciferase assay. (F) Western blot of MMP‐9 and cyclin D1 expression in THBS2 knocked down LOVO cells. (G) Western blot of the MMP‐9 and cyclin D1 in THBS2 overexpressing HCT116 cells. (H) Co‐IP assays revealed the association between THBS2 and β‐catenin in HCT116 and LOVO cells. Inputs were used as control. Co‐IP was performed using antibodies as indicated. (I) The representative photographs of MMP‐9 and cyclin D1 staining of subcutaneous tumor are shown (magnification, × 200). (J) The percentage of MMP‐9 positive staining and cyclin D1 positive staining in subcutaneous tumor was counted under the microscope. **p <0.01 compared with sh‐Ctrl. (K) The mRNA levels of β‐catenin in THBS2‐overexpressing LOVO and HCT116 cells were determined using qRT‐PCR assay. (L) The luciferase reporter assays were performed using the luciferase reporter plasmid linked with full‐length native promoter of β‐catenin in LOVO and HCT116 cells. (M) The β‐catenin promoter structure was constructed and luciferase activity relative to Renilla control was measured in LOVO and HCT116 cells. **p <0.01 compared with vector

FIGURE 5

Thrombospondin 2 (THBS2) promotes colorectal cancer progression via regulating Wnt/β‐catenin signaling. (A‐B) Western blot analysis of the expression of MMP‐9 and cyclin D1 in THBS2 overexpressing HCT116 and LOVO cells with the XAV939 treated. (C) THBS2 overexpressing HCT116 and LOVO cells were treated with XAV939. Growth ability was measured by colony formation assay. (D) Invasion ability was measured by transwell assay. **P <0.01 compared with vector group, ^## p <0.01 compared with vector plus 5 μM or 10 μM XAV939 group