IBSP, a potential recurrence biomarker, promotes the progression of colorectal cancer via Fyn/β-catenin signaling pathway

Abstract

Colorectal cancer (CRC) is a frequently occurring digestive system cancer and postoperative tumor metastasis and recurrence are the main reasons for the failure of CRC treatment. The aim of this study was to identifying and validating key genes associated with metastatic recurrence of CRC. RNA expression of three datasets (GSE17538, GSE32323, and GSE29623) was used for biomarker discovery. We identified integrin-binding sialoprotein (IBSP) as a candidate biomarker which was validated in three clinical cohorts (GSE41258, GSE21510, and GSE39582) and our clinical specimens. The results suggested that IBSP expression significantly increased at mRNA and protein levels among CRC cases, which was associated with metastatic recurrence, metastasis, high risk of recurrence, and poor survival in CRC. Consistent results were obtained in CRC cells. The relative level of serum IBSP evidently increased among CRC patients relative to normal controls, and downregulated after operation. As suggested by gene set enrichment analysis (GSEA), the IBSP level was associated with cell-matrix adhesion in CRC. Functional experiments in vitro showed that IBSP promoted the growth and aggressiveness of CRC, and the potential mechanism by which IBSP promoted carcinogenesis of CRC was the abnormal activation of Fyn/β-catenin signaling pathway. To sum up, findings in the present work indicate that IBSP can serve as the candidate biomarker for the diagnosis, treatment, and prognosis of CRC.

Keywords: Fyn/β-catenin signaling pathway; colorectal cancer; diagnosis and treatment; integrin-binding sialoprotein (IBSP); metastatic recurrence.

FIGURE 1

Biomarker discovery analysis in this study. (A) The significantly upregulated genes of patients with recurrence in GSE17538 dataset. (B) The significantly upregulated genes of patients with metastasis or metastatic recurrence in GSE32323 dataset. (C) The significantly upregulated genes of patients with recurrence in GSE29623 dataset. (D) Venn plots of upregulated overlapping genes

FIGURE 2

IBSP expression was upregulated in various independent datasets, clinical specimens, and CRC cell lines. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns: nonsignificant. (A) and (B) IBSP expression was specifically higher in the primary CRC and metastatic CRC, respectively, compared with normal colonic mucosa and primary CRC in GSE41258 dataset. (C) IBSP expression was specifically higher in CRC patients with metastatic recurrence, compared with CRC patients with non‐recurrence in GSE21510 dataset. (D) IBSP expression was specifically higher in the consensus molecular subtype 4 CRC patients (CMS4, with high risk of recurrence), compared with the CMS1, 2,3 CRC patients (with low risk of recurrence) in GSE39582 dataset. (E) The mRNA level of IBSP was specifically higher in the primary CRC, compared with normal colorectal mucosa in clinical specimens. (F) The mRNA level of IBSP was specifically increased in the CRC liver metastatic tumors, compared with normal liver tissues in clinical specimens. (G and H) The mRNA and protein level of IBSP were significantly increased in several CRC cell lines compared to human normal colorectal epithelial FHC cells

FIGURE 3

IBSP level in the serum act as a novel potential diagnostic biomarker for CRC. (A) IBSP level in the serum was significantly higher in CRC patients compared to healthy controls. HC: healthy controls, **** p < 0.0001. (B) The AUC was 0.8132 (95% CI, 0.7461–0.8803) of IBSP for distinguishing CRC patients from healthy controls. (C) IBSP level in the serum of CRC patients was significantly downregulated after surgical resection, suggesting that IBSP might be an indicator of prognosis monitoring.**** p < 0.0001

FIGURE 4

Biological interaction network and clinical significance of IBSP in CRC. (A) The correlations between IBSP and the differentially expressed genes in CRC. (B) According to the GSEA in GSE17538 dataset, cell‐matrix adhesion gene set was correlated with metastatic recurrence in CRC. (C) The correlation of IBSP and representative cell‐matrix adhesion‐related genes in the GSE17538 dataset. (D) IBSP expression was correlated with survival of CRC patients. Low expression of IBSP conferred a survival advantage to CRC patients

FIGURE 5

IBSP‐siRNA inhibited proliferation and induced apoptosis in CRC cells. B: blank, NC: negative control, *p < 0.05, **p < 0.01. (A) Downregulation of IBSP mRNA in CRC cells. (B) IBSP‐siRNA inhibited cell proliferation of CRC cells. (C) IBSP‐siRNA increased apoptosis of CRC cells. (D) The protein level of PCNA, Bcl2/Bax, Cyclin D1, and Cdk4 in CRC cells decreased after transfection with IBSP‐siRNA

FIGURE 6

IBSP‐siRNA inhibited invasion, migration, and epithelial–mesenchymal transition (EMT) of CRC cells. *p < 0.05, **p < 0.01, ***p < 0.001,**** p < 0.0001. (A) IBSP‐siRNA suppressed invasion and migration of CRC cells. (B) Transfection of IBSP siRNA decreased the protein level of MMP2, MMP9, N‐cadherin, and Vimentin and increased the expression of E‐cadherin

FIGURE 7

The mechanisms of IBSP function in CRC. *p < 0.05; **p < 0.01; ns: nonsignificant. (A) The LYN mRNA expression following IBSP downregulation. (B) The FYN mRNA expression following IBSP downregulation. (C) The LCK mRNA expression following IBSP downregulation. (D) Protein level of Fyn, p‐Fyn, and p‐β‐catenin decreased after transfection of IBSP‐siRNA, while β‐catenin showed no significant variation. (E) The expression of IBSP was significantly positively correlated with the expression of Fyn and β‐catenin

FIGURE 8

IBSP overexpression promoted CRC cell proliferation, invasion as well as migration via Fyn/β‐catenin pathway. *p < 0.05, **p < 0.01, ***p < 0.001,**** p < 0.0001, ns: nonsignificant. (A) Upregulation of IBSP mRNA in CRC cells. (B) IBSP overexpression promoted cell proliferation of CRC cells. (C) IBSP overexpression significantly increased the number of cell colonies. (D) IBSP overexpression promoted invasion and migration of CRC cells. (E) IBSP overexpression increased the protein level of PCNA, Bcl2, Cyclin D1, Cdk4, MMP2, MMP9, N‐cadherin, Vimentin, Fyn, p‐Fyn, and p‐β‐catenin and decreased the expression of Bax and E‐cadherin