The eIF3a translational control axis in the Wnt/β-catenin signaling pathway and colon tumorigenesis

Abstract

Translational initiation in protein synthesis is an important regulatory step in gene expression and its dysregulation may result in diseases such as cancer. Translational control by eIF4E/4E-BP has been well studied and contributes to mTOR signaling in various biological processes. Here, we report a novel translational control axis in the Wnt/β-catenin signaling pathway in colon tumorigenesis by eIF3a, a Yin-Yang factor in tumorigenesis and prognosis. We show that eIF3a expression is upregulated in human colon cancer tissues, pre-cancerous adenoma polyps, and associates with β-catenin level and APC mutation in human samples, and that eIF3a overexpression transforms intestinal epithelial cells. We also show that eIF3a expression is regulated by the Wnt/β-catenin signaling pathway with an active TCF/LEF binding site in its promoter and that eIF3a knockdown inhibits APC mutation-induced spontaneous colon tumorigenesis in APC^min/+ mice. Together, we conclude that eIF3a upregulation in colon cancer is due to APC mutation and it participates in colon tumorigenesis by adding a translational control axis in the Wnt/β-catenin signaling pathway and that it can serve as a potential target for colon cancer intervention.

Fig. 1.. Expression of eIF3a, b, g, and i in human adenocarcinoma and adenoma colon tissues.

A. Lysates were prepared from flash frozen paired human normal (N) and adenocarcinoma (C) tissues for Western blot analysis. GAPDH was used as a loading control. B. Pearson correlation coefficient analysis between eIF3a and eIF3i fold-increase in expression in cancer over normal tissues. C. Expression of eIF3a, b, g, and i in benign colon adenoma polyps (A) in comparison with the normal (N) tissue from patient #20 as determined using Western blot.

Fig. 2.. eIF3a expression in differentiated CaCo-2 cells and tumorigenic function of eIF3a.

A. Alkaline phosphatase (AP) and sucrase (Suc) activities in CaCo-2 cells at different densities. B. Western blot analysis of eIF3a, eIF3b, and GAPDH control in CaCo-2 cells at different densities. C-E. Cell-based analysis of oncogenic function of eIF3a. Stable RIE-1 clones with eIF3a over-expression (eIF3a1 and eIF3a5) or transfected with vector control (Vec) were subjected to Western blot analysis of eIF3a and GAPDH control (C), clonogenic assay (D), and anchorage-independent growth assay in soft agar (E). F. In-vivo tumorigenic assay. The stable RIE clone with eIF3a over-expression (eIF3a1) or with empty vector control (Vec) were inoculated into female NOD/SCID mice followed by analyses of the growth of xenograft tumors. (***p < 0.001).

Fig. 3.. Regulation of eIF3a expression.

A-C. Effect of PI3K inhibitors or PTEN expression on eIF3a expression. HT29 cells were treated with Wortmannin (A) or LY294002 (B) for various times, or transiently transfected with PTEN cDNA or vector control (C) followed by Western blot analysis of eIF3a, PTEN and GAPDH control. D-E. Effect of APC expression on eIF3a expression. HT29 and CaCo-2 cells were transiently transfected with the cDNA encoding the wild-type APC or vector control followed by Western blot analysis of eIF3a, APC and GAPDH control (D) or real-time RT-PCR analysis of eIF3a mRNA in CaCo-2 cells (E). F-G. APC status and eIF3a expression in vivo. Total RNAs or lysates were prepared from whole intestinal samples of APC^min/+ and APC^+/+ mice and subjected to quantitative RT-PCR (F) and Western blot (G) analysis of eIF3a. The eIF3a mRNA level in APC^min/+ were normalized to that in the wild type control APC^+/+ mice and Western blot analysis was performed using samples from 2 individual APC^min/+ and 2 control APC^+/+ mice. (***p < 0.001).

Fig. 4.. Regulation of eIF3a expression by Wnt signaling.

A-B. Effect of TCF4 or LEF1 on eIF3a expression. CaCo-2 cells were transiently transfected with TCF4 cDNA or siRNA along with their respective vector and scrambled siRNA controls (A) or with wild-type (WT) or dominant negative mutant (DN) LEF1 cDNA along with its vector control (B) followed by Western blot analysis of eIF3a, TCF4, LEF1, or actin control. C-D. Effect of Wnt signaling pathway activation on eIF3a expression. HEK293 cells were serum-starved for 36 h followed by treatment with Wnt3a in the absence or presence of tankyrase inhibitor XAV939 (C) or transiently transfected first by vector, wild-type or dominant negative (dn) LEF1 before serum starvation and Wnt3a treatment (D), followed by Western blot analysis of LEF1, eIF3a or actin control.

Fig. 5.. Sequence and activity of eIF3a promoter.

A. Promoter sequence of human eIF3a. The putative LEF/TCF binding site GCTTTGAAA (underlined) is 279 bases upstream of the transcription start site (boxed G) and 426 bases upstream of the translation initiation codon ATG. B. Comparison between the consensus and the putative LEF/TCF binding sequences in human eIF3a gene and the schematic diagram of the luciferase reporter construct. C-D. Basal eIF3a promoter activity and its regulation by APC, β-catenin, and TCF4. Promoter construct (GL3a) shown in panel B along with a promoter-less control construct (GL) were transiently transfected into CaCo-2 cells (C) or CaCo-2 cells with over-expression of APC, TCF4 or with β-catenin knockdown (D) for determination of luciferase activity. Vector-transfected controls for APC and TCF4 overexpression or scrambled siRNA control for β-catenin knockdown were also tested, β-galactosidase was used to control transfection efficiency. a.u. = arbitrary units. E. Effect of Wnt3a and tankrase inhibitor on the promoter activity of eIF3a in HEK293 cells. F. ChIP assay of eIF3a promoter using TCF4 antibody or control normal IgG. G. Schematic drawing of deletion constructs of eIF3a promoter sequence. H. Relative promoter activity of wild-type and deletion mutant eIF3a promoters in CaCo-2 and HEK293 cells. (***p < 0.001, **p < 0.01, *p < 0.05).

Fig. 6.. eIF3a association with β-catenin expression in human colon adenocarcinoma tissues and with APC mutations.

A. Western blot analysis of β-catenin and actin control in the 20 matched pairs of human normal (N) and adenocarcinoma (C) tissues. B. Pearson correlation analysis of the fold-increase in expression in cancer over normal tissues between eIF3a and β-catenin and between eIF3i and β-catenin. C-D. Association between APC mutation and eIF3a expression in the human colon cancer TCGA dataset. WT, wild-type; FS, frameshift mutation; Stop, gaining premature stop codon; MS, missense mutation.

Fig. 7.. eIF3a knockdown inhibits APC mutation-induced colon tumor production.

A-B. Activity of three different siRNAs in knocking down eIF3a expression in NIH3T3 cells (A) and in mice (B). C-D. Effect of eIF3a knockdown on tumor formation in APCmin/+ mice. (***p < 0.001). C. IHC staining of eIF3a in intestinal tissues of APC^min/+ mice treated with scrambled control or eIF3a siRNAs. Scale bar, 50 μm.

Fig. 8.

Schematic model of the speculated translational control axis in the Wnt/β-catenin signaling and colon tumorigenesis.