A functional variant rs10409772 in FUT6 promoter regulates colorectal cancer progression through PKA/CREB signaling

Abstract

Fucosyltransferase 6 (FUT6) is overexpressed in colorectal cancer tissue according to TCGA samples and immunohistochemistry results of a tissue microarray. FUT6 effects cell migration, tumor formation and proliferation of colorectal cancer cells in different essays. FUT6 promotes cancer cell proliferation in vitro and colorectal tumorigenesis in vivo by upregulating PKA/CREB pathway activation. Moreover, FUT6 expression is regulated by rs10409772 shown in the luciferase essays, a single nucleotide polymorphism in the promoter of FUT6. Our study suggests that elevated expression of FUT6 promotes PKA/CREB signaling, which in turn augments colorectal carcinogenesis, indicating a potential therapeutic target for colorectal cancer patients with increased FUT6 expression.

Keywords: Colorectal cancer; FUT6; PKA/CREB signaling; Single nucleotide polymorphism.

Fig. 1

FUT6 is upregulated in colorectal cancer. A FUT6 expression in colon and rectal cancer tissues (red) (n = 275 and n = 92, respectively) compared with that in relative benign tissues (gray) (n = 349 and n = 318, respectively) obtained from the GEPIA online tool. B IHC staining for FUT6 in colorectal cancer tissue microarrays containing adjacent tissues and normal tissues. The complete graph of the slice is obtained under 4x zoom condition, and the graph of the square is intercepted under 20x zoom condition in Slide Viewer (0.0.2.17 version). C Analysis of FUT6 expression in colorectal cancer tissue microarrays from 34 tumor and 34 adjacent tissues. COAD, colon adenocarcinoma; READ, rectal adenocarcinoma. The data are presented as the means ± SEM. Statistical significance was analyzed by Student's t-test. ****p < <0.0001.

Fig. 2

The protein levels of FUT6 when the siRNA is transfected in colorectal cancer cells.

Fig. 3

FUT6-knokdown Impaired Cell Migration and Proliferation of Colon and Rectum Cancer In Vitro A The cell proliferation ability of FUT6-knokdown HCT116 and SW480 cells was determined by Cell Counting Kit-8 (CCK-8) assay (n = 6). B The cell migration ability of FUT6-knokdown HCT116 and SW480 cells was determined by transwell experiments (n = 3).

Fig. 4

FUT6 promote cell migration, tumor formation and proliferation of colon and rectum cancer in vitro A The cell proliferation ability of FUT6-overexpressed cells was determined by Cell Counting Kit-8 (CCK-8) assay (n = 6). B The viability of FUT6-overexpressed cells was determined by colony formation experiment. The number of colonies was record (n = 3). C The cell migration ability of FUT6-overexpressed cells was determined by transwell experiments (n = 3). The data are presented as the means ± SEM. Statistical significance was analyzed by ANOVA. **p < 0.01, ***p < 0.001.

Fig. 5

The variant rs10409772 enhance FUT6 expression. Relative reporter gene expression driven by fragments with different rs10409772 alleles in HCT116 (Fig. A) and SW480 (Fig. B) cells. Results are shown as fold changes relative to luciferase expression in cells transfected with empty vector (pGL3‐Basic). All constructs were co-transfected with pRL‐SV40 vector to standardize transfection efficiency. Results are mean ± SEM from three replicates. P values were evaluated by Student's t-test. ****p < <0.0001.

Fig. 6

The protein levels of FUT6 when pGL3-vector, rs10409772-Ref and rs10409772-Mut are transfected in colorectal cancer cells with FUT6-overexpression.

Fig. 7

FUT6 leads to increased PKA/CREB signaling. A KEGG pathway enrichment showed that FUT6 regulated the Alcoholism signaling pathway from the control and FUT6-overexpressed RNA-seq data. B Heat map analysis of different expression genes in control and FUT6-overexpressed cells. C GSEA of PKA/CREB target genes in control and FUT6-overexpressed cells. D Volcano plot of different expression genes in control and FUT6-overexpressed cells.

Fig. 8

The protein levels of the PKA/CREB pathway in FUT6-overexpressed cells and control group. Genes in control and FUT6-overexpressed cells were determined by immunoblot analysis. The data are presented as the means ± SEM. Statistical significance was analyzed by ANOVA. *p < 0.05, **p < 0.01.

Fig. 9

The protein levels of the PKA/CREB pathway in FUT6-knockdown cells and control group. Genes in control and FUT6-knockdown cells were determined by immunoblot analysis. The data are presented as the means ± SEM. Statistical significance was analyzed by ANOVA. *p < 0.05, **p < 0.01.