IGF2BP2 promotes the progression of colorectal cancer through a YAP-dependent mechanism

Abstract

To explore the effect of insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) on colorectal cancer (CRC) by recognizing the m6A modification of YAP mRNA thus activating ErbB2 expression. High expressions of IGF2BP2, YAP, and ErbB2 promoted the proliferation, migration and invasion of CRC cells and reduced their apoptosis. IGF2BP2 recognized the m6A on YAP mRNA and promoted the translation of mRNA. YAP regulated ErbB2 expression by promoting TEAD4 enrichment in ErbB2 promoter region. Therefore, IGF2BP2 promoted the expression of ErbB2 to enhance the proliferation, invasion and migration of CRC cells, to repress cell apoptosis, and to promote solid tumor formation in nude mice. IGF2BP2 activates the expression of ErbB2 by recognizing the m6A of YAP, thus affecting the cell cycle of CRC, inhibiting cell apoptosis, and promoting proliferation.

Keywords: ErbB2; IGF2BP2; YAP; apoptosis; colorectal cancer; invasion; m6A; migration; proliferation.

FIGURE 1

Silencing of insulin‐like growth factor 2 mRNA‐binding protein 2 (IGF2BP2) inhibited the proliferation, migration, and invasion of colorectal cancer (CRC) cells and promoted apoptosis. A, Expression heat map of methylation of the 6th adenosine (m6A)‐modified genes in the COAD dataset obtained from the TCGA database, *P < .05, **P < .01, ***P < .001. B, Violin chart of m6A‐modified gene expression, wherein blue represents the control group and red represents the cancer group. C, Survival curve of IGF2BP2 in COAD. D, Expression of IGF2BP2 in CRC tissues and adjacent normal tissues tested by RT‐qPCR. n = 66. *P < .05, compared with the normal group. E, Expression of IGF2BP2 in four kinds of CRC cells and normal human intestinal epithelial cells tested by RT‐qPCR. *P < .05, compared with human intestinal epithelial cells (HIEC‐6) cells. F, mRNA expression of IGF2BP2 in cells transfected with si‐IGF2BP2‐1, si‐IGF2BP2‐2, and si‐IGF2BP2‐3 tested by RT‐qPCR. G, Protein expression of IGF2BP2 in cells transfected with si‐IGF2BP2‐1, si‐IGF2BP2‐2, and si‐IGF2BP2‐3 measured by Western blot analysis. *P < .05, compared with cells transfected with si‐normal control (NC). H‐I, mRNA expression of IGF2BP2 in the oe‐NC group and the oe‐IGF2BP2 group detected by RT‐qPCR. I, Protein expression of IGF2BP2 in the oe‐NC group and the oe‐IGF2BP2 group measured by Western blot analysis. *P < .05, compared with cells transfected with oe‐NC. J, Proliferation of cells tested by CCK‐8 assay. K, Cell migration measured by the scratch test. L, Cell invasion detected by Transwell assay. M, Cell cycle distribution detected by flow cytometric analysis. N, Cell apoptosis assessed by flow cytometric analysis. *P < .05, compared with cells transfected with si‐NC. #P < .05 compared with cells transfected with oe‐NC. The results are measurement data, shown as mean ± standard deviation. Unpaired t‐test was used to compare the data between two groups, and one‐way ANOVA was used to compare the data among multiple groups. The survival rate of patients was calculated by the Kaplan‐Meier method, and univariate analysis was performed by the log‐rank test. The cell experiments were repeated three times

FIGURE 2

Insulin‐like growth factor 2 mRNA‐binding protein 2 (IGF2BP2) increased the stability of Yes‐associated protein (YAP) by binding to methylation of the 6th adenosine (m6A)‐modified YAP mRNA in colorectal cancer (CRC) cells. A, Venn diagram of COAD differential genes, IGF2BP2‐related genes in COAD^#, and the IGF2BP2‐mediated genes predicted by m6A2target showing 59 intersection genes. B, Box plot of YAP1 expression in the COAD dataset, where the left blue box is the control group and the red box on the right is the cancer group. C, Expression of YAP in CRC tissues and adjacent normal tissues detected by RT‐qPCR. n = 66. *P < .05, compared with adjacent normal tissues. D, Expression of YAP in four kinds of CRC cells and normal human intestinal epithelial cells detected by RT‐qPCR. *P < .05, compared with HIEC‐6 cells. E, Expression of YAP in cells when IGF2BP2 was silenced or overexpressed tested by RT‐qPCR. *P < .05, compared with cells transfected with si‐normal control (NC). #P < .05, compared with cells transfected with oe‐NC. F, Expression of YAP in cells when IGF2BP2 was silenced or overexpressed tested by Western blot analysis. *P < .05, compared with cells transfected with si‐NC. #P < .05 compared with cells transfected with oe‐NC. G, Expression correlation diagram of YAP1 and IGF2BP2 in the COAD^# dataset. H, Correlation analysis of IGF2BP2 and YAP in 66 cases of human CRC tissues by Pearson correlation analysis. I, m6A level of MYC mRNA detected by Me‐RIP. *P < .05, compared with cells treated by IgG. #P < .05, compared with cells transfected with si‐NC. $P < .05, compared with cells transfected with oe‐NC. J, Expression of the YAP downstream target genes CYR61 and TGF detected by RT‐qPCR in cells following IGF2BP2 silencing or overexpression. The results are measurement data, shown as mean ± standard deviation. Unpaired t‐test was used to compare the data between two groups, and one‐way ANOVA was used to compare the data among multiple groups. The cell experiments were repeated three times

FIGURE 3

Insulin‐like growth factor 2 mRNA‐binding protein 2 (IGF2BP2) promoted the proliferation, migration, and invasion of colorectal cancer (CRC) cells while attenuating cell apoptosis by stabilizing the expression of Yes‐associated protein (YAP). CRC cells were transfected with si‐IGF2BP2 or combined with oe‐YAP. A, mRNA expression of IGF2BP2 and YAP in CRC cells detected by RT‐qPCR. B, Protein expression of IGF2BP2 and YAP in CRC cells measured by Western blot analysis. C, Cell proliferation tested by CCK‐8 assay. D, Cell migration measured by scratch test. E, Invasion of cells measured by Transwell assay. F, Cell cycle distribution detected by flow cytometry. G, Cell apoptosis assessed by flow cytometry. *P < .05, compared with cells transfected with si‐normal control (NC) + oe‐NC. #P < .05, compared with cells transfected with si‐IGF2BP2 + oe‐NC. The results are measurement data, shown as mean ± standard deviation. Unpaired t‐test was used to compare the data between two groups, and one‐way ANOVA was used to compare the data among multiple groups. Data at different time points were compared by two‐way ANOVA. The cell experiments were repeated three times

FIGURE 4

Insulin‐like growth factor 2 mRNA‐binding protein 2 (IGF2BP2) upregulated the expression of ErbB2 by stabilizing the expression of Yes‐associated protein (YAP). A, Enrichment of TEAD in the ErbB2 promoter upon YAP overexpression detected by ChIP assay. *P < .05, compared with cells transfected with oe‐normal control (NC). B, Expression of ErbB2 in colorectal cancer (CRC) tissues and adjacent normal tissues detected by RT‐qPCR. n = 66. *P < .05, compared with adjacent normal tissues. C, Expression of ErbB2 in CRC cells and normal human intestinal epithelial cells detected by RT‐qPCR. *P < .05, compared with human intestinal epithelial cells (HIEC‐6) cells. D, Venn diagram of DEGs in COAD and ErbB2‐related genes in COAD^# showing 72 intersection genes. E, Bubble chart of the top 10 GO entries of 72 intersection genes obtained using KOBAS, wherein the ordinate indicates the enriched item, the horizontal indicates the number of genes enriched in the entry, and the color of the bubble indicates the significance of the enrichment‐lgP value. F, Bubble chart of the top 10 genes in the 72 intersection genes according to the KEGG analysis by KOBAS, wherein the ordinate indicates the enriched entry, the abscissa indicates the number of genes enriched in the item, and the color of the bubble indicates the significance of the enrichment‐lgP value. G, Protein expression of ErbB2 measured by Western blot analysis in cells transfected with si‐ErbB2‐1, si‐ErbB2‐2, or si‐ErbB2‐3. H, Protein expression of ErbB2 measured by Western blot analysis in cells transfected with oe‐ErbB2. *P < .05, compared with cells transfected with si‐NC or oe‐NC. I, Cell proliferation tested by CCK‐8 assay. **P < .01, compared with cells transfected with si‐NC or oe‐NC. J, Protein expression of ErbB2 detected by RT‐qPCR. K, Protein expression of ErbB2 measured by Western blot analysis. *P < .05, compared with cells transfected with si‐NC + oe‐NC. #P < .05, compared with cells transfected with si‐IGF2BP2 + oe‐NC. The results are measurement data, shown as mean ± standard deviation. Unpaired t‐test was used to compare the data between two groups and one‐way ANOVA was used to compare the data among multiple groups. The cell experiments were repeated three times

FIGURE 5

Insulin‐like growth factor 2 mRNA‐binding protein 2 (IGF2BP2) stabilized the expression of Yes‐associated protein (YAP) and upregulated the expression of ErbB2 to promote the tumorigenesis of colorectal cancer (CRC) cells in vivo. Mice were injected with cells stably transfected with oe‐IGF2BP2 + si‐normal control (NC) or oe‐IGF2BP2 + si‐YAP. A, Representative images of xenograft tumors in nude mice. B, Tumor volume in mice. C, Tumor weight in mice. D, Positive expression of ErbB2 in tumor tissues of nude mice tested by immunohistochemistry (IHC). *P < .05, compared with mice injected with cells stably transfected with oe‐NC + si‐NC. #P < .05, compared with mice injected with cells stably transfected with oe‐IGF2BP2 + si‐NC. The results are measurement data, shown as mean ± standard deviation. One‐way ANOVA was used to compare the data among multiple groups. Data of tumor volume at different time points were compared by repeated‐measures ANOVA. n = 6 for mice upon each treatment

FIGURE 6

Schematic diagram of the mechanism by which insulin‐like growth factor 2 mRNA‐binding protein 2 (IGF2BP2) affects colorectal cancer (CRC). IGF2BP2 binds to methylation of the 6th adenosine (m6A)‐modified Yes‐associated protein (YAP) mRNA and increases the stability of YAP, thus upregulating ErbB2 expression. By this mechanism, the proliferation of CRC cells is promoted, while apoptosis is inhibited, as well as the accelerated progression of CRC