c-Myb promotes growth and metastasis of colorectal cancer through c-fos-induced epithelial-mesenchymal transition

Abstract

c-Myb is a crucial transcription factor that participates in various biological functions; however, its role in colorectal cancer (CRC) remains poorly investigated. We first analyzed the expression and clinical significance of c-Myb in a retrospective cohort enrolling 132 CRC patients. Then, the CRISPR/Cas9 technique was used to establish c-Myb gene KO CRC cell lines. Cellular functional assays in vitro and in vivo were used to evaluate the impact of c-Myb KO in CRC cells. Finally, RNA sequencing was used to investigate the potential oncogenic mechanisms regulated by c-Myb in CRC progression and related cellular validations were accordingly carried out. As a result, c-Myb is significantly overexpressed in CRC tissues as compared with adjacent normal tissues. High expression of c-Myb is positively correlated with lymph node metastasis and poor prognosis. Univariate analysis and multivariate analysis further identify c-Myb as an independent unfavorable prognostic factor for CRC patients. c-Myb KO inhibits the proliferation, apoptosis resistance, invasion, metastasis, colony formation and in vivo tumorigenesis of CRC cells. Also, the mechanism investigation indicates that c-Myb may promote CRC progression by regulating c-fos. c-fos overexpression can rescue the inhibitory effect of c-Myb KO on the malignant characteristics of CRC cells. Finally, we find that c-Myb KO inhibits the epithelial-mesenchymal transition (EMT) molecular phenotype in CRC cells, whereas c-fos overexpression can rescue this inhibitory effect. This study suggests that c-Myb promotes the malignant progression of CRC through c-fos-induced EMT and has the potential to be a promising prognostic biomarker and therapeutic target.

Keywords: biomarker; c-Myb; c-fos; colorectal cancer; epithelial-mesenchymal transition.

Figure 1

Expression of c‐Myb in colorectal carcinoma (CRC) and matched adjacent normal tissues. A, RT‐PCR shows that the mRNA level of the c‐Myb gene was significantly higher in CRC tissues than that in matched adjacent normal tissues (n = 20, P = .005). B, Western blot shows that the protein level of c‐Myb was higher in CRC tissues than that in matched adjacent normal tissues (n = 20, P = .008). C, Representative protein bands of c‐Myb and GAPDH. D, Representative immunohistochemical staining images of c‐Myb in CRC and matched adjacent normal tissues. E, Cut‐off value of immunohistochemical staining scores is determined by receiver operating characteristic curve analysis. **P < .01

Figure 2

Prognostic significance of c‐Myb in colorectal carcinoma (CRC) patients. A, In the entire cohort, patients with high c‐Myb expression have a significantly worse overall survival (OS, upper panel, P < .001) and disease‐free survival (DFS, lower panel, P < .001) than those with low c‐Myb expression. B, In stage II CRC, patients with high c‐Myb expression have a significantly worse OS (upper panel, P = .008) and DFS (lower panel, P = .009) than those with low c‐Myb expression. C, In stage III CRC, patients with high c‐Myb expression have a significantly worse OS (upper panel, P = .002) and DFS (lower, P = .009) than those with low c‐Myb expression

Figure 3

Knockout of c‐Myb inhibits the malignant characteristics of colorectal carcinoma (CRC) cells in vitro. A, RT‐PCR shows that the mRNA level of the c‐Myb gene is higher in CRC cell lines compared with normal intestinal epithelial cells. B, Western blot shows that protein expression of c‐Myb is significantly lower in the KO group than in the negative control (NC) group. C, Optical density value of the KO group increased more slowly than that of the NC group within 5 d. D, Apoptosis rates of the KO group are higher than that of the NC group. E, Colony numbers of the KO group are lower than that of the NC group. F, Numbers of migratory cells in the KO group are lower than that in the NC group. G, Numbers of invasive cells in the KO group are lower than that in the NC group. *P < .05, **P < .01, ***P < .001, ****P < .0001. Error bar represents standard deviation, n = 3

Figure 4

Knockout of c‐Myb inhibits the growth of colorectal carcinoma (CRC) cells in vivo. A, Harvested xenografts from nude mice that were s.c. injected with HCT‐116 (upper) and SW620 cells (lower). B, Xenograft growth of the KO group is significantly inhibited as compared with that of the negative control (NC) group. C, Final xenograft weights of the KO group are lower than those of the NC group. D, Rates of proliferating cell nuclear antigen (PCNA)‐positive tumor cells in the harvested xenografts are lower in the KO group than in the NC group. E, Apoptosis rate of tumor cells in the harvested xenografts are higher in the KO group than in the NC group. *P < .05, **P < .01, ***P < .001, ****P < .0001. Error bar represents standard deviation, n = 6

Figure 5

Transcriptome sequencing and bioinformatics analysis for c‐Myb KO colorectal carcinoma (CRC) cells. A, Heat map shows the significantly expressed genes affected by c‐Myb KO in HCT116 cells. B, Gene ontology (GO) functional analysis shows the top 30 biological functions that are enriched in the significantly expressed genes. C, The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway shows the top 30 signaling pathways that are enriched in the significantly expressed genes. D, Top 10 downregulated genes are selected based on their fold change values

Figure 6

c‐Myb promotes colorectal carcinoma (CRC) progression by regulating c‐fos. A, Cytoscape software predicts that c‐Myb has interaction with c‐fos. B, RT‐PCR indicates that c‐Myb KO decreases the mRNA level of the c‐fos gene in HCT116 and SW620 cells. C, Western blot indicates that c‐Myb KO decreases the protein level of c‐fos in HCT116 and SW620 cells. D, Representative immunohistochemical staining images of c‐fos in CRC tissues. E, ChIP assay shows that c‐Myb KO effectively inhibited the binding of c‐Myb with the promoter of the c‐fos gene in HCT‐116 and SW620 cells. F, Luciferase reporter gene assay indicates that c‐Myb overexpression (OE) increases the activity of the wild type (WT) promoter of the c‐fos gene, whereas it has no impact on the activity of the mutant type (MT) promoter of the c‐fos gene, as compared with negative control (NC). **P < .01, ***P < .001. Error bar represents standard deviation, n = 3

Figure 7

Overexpression of c‐fos rescues the inhibitory effect of c‐Myb KO on the malignant characteristics of colorectal carcinoma (CRC) cells in vitro. A, RT‐PCR validates that the mRNA level of c‐fos in c‐Myb KO CRC cells is increased by c‐fos overexpression. B, Western blot validates that the protein level of c‐fos in c‐Myb KO CRC cells is increased by c‐fos overexpression. C, c‐fos overexpression accelerates the increase of the optical density value in c‐Myb KO CRC cells. D, c‐fos overexpression decreases the apoptosis rate of c‐Myb KO CRC cells. E, c‐fos overexpression increases migratory cells in c‐Myb KO CRC cells. F, c‐fos overexpression increases invasive cells in c‐Myb KO CRC cells. G, c‐fos overexpression increases colony numbers of c‐Myb KO CRC cells. NC, negative control; OE, overexpression. *P < .05, **P < .01, ***P < .001, ****P < .0001. Error bar represents standard deviation, n = 3

Figure 8

Overexpression of c‐fos rescues the inhibitory effect of c‐Myb KO in the epithelial‐mesenchymal transition (EMT) phenotype. c‐Myb KO increases E‐cadherin expression, but decreases N‐cadherin, Vimentin and snail expression in HCT‐116 (upper panel) and SW620 (lower panel) cells, whereas c‐fos overexpression could rescue this effect. NC, negative control; OE, overexpression. *P < .05, **P < .01, ***P < .001, ****P < .0001. Error bar represents standard deviation, n = 3