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. 2013 Apr;31(3):228-36.
doi: 10.1002/cbf.2876. Epub 2012 Sep 7.

The Wnt/β-catenin/T-cell factor 4 pathway up-regulates high-mobility group A1 expression in colon cancer

Bethany M Bush  1 , Ashton T Brock, Jiayue A Deng, Ronald A Nelson, Takita Felder Sumter
Affiliations

The Wnt/β-catenin/T-cell factor 4 pathway up-regulates high-mobility group A1 expression in colon cancer

Bethany M Bush et al. Cell Biochem Funct. 2013 Apr.

Abstract

High-mobility group A1 (HMGA1) encodes proteins that act as mediators in viral integration, modification of chromatin structure, neoplastic transformation and metastatic progression. Because HMGA1 is overexpressed in most cancers and has transcriptional relationships with several Wnt-responsive genes, we explored the involvement of HMGA1 in Wnt/β-catenin/TCF-4 signalling. In adenomatous polyposis coli (APC(Min/+)) mice, we observed significant up-regulation of HMGA1 mRNA and protein in intestinal tumours when compared with normal intestinal mucosa. Conversely, restoration of Wnt signalling by the zinc induction of wild-type APC resulted in HMGA1 down-regulation in HT-29 cells. Because APC mutations are associated with mobilization of the β-catenin/TCF-4 transcriptional complex and subsequent activation of downstream oncogenic targets, we analyzed the 5'-flanking sequence of HMGA1 for putative TCF-4 binding elements. We identified two regions that specifically bind the β-catenin/TCF-4 complex in vitro and in vivo, identifying HMGA1 as an immediate target of the β-catenin/TCF-4 signalling pathway in colon cancer. Collectively, these findings strongly implicate Wnt/β-catenin/TCF-4 signalling in regulating HMGA1 to further expand the extensive regulatory network affected by Wnt/β-catenin/TCF-4 signalling.

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Figures

Figure 1
Figure 1. HMGA1 expression is upregulated in the intestinal compartment of APCMin/+ mice
(A) Semiquantitative RT-PCR shows that small intestinal mucosa and tumors isolated from mice bearing a truncated APC gene (APCMin/+) have a significant increase in HMGA1 mRNA compared to control C57B6 (B6) mice. β-actin was used as an internal control for all samples and the positive control (labeled control in this figure) was RNA isolated from the splenocytes of HMGA1 transgenic mice. (B) HMGA1 mRNA levels were confirmed in both the small and large intestinal tissues using quantitative real-time RT-PCR with primers and probes specific for murine HMGA1. ***p<0.001; **p<0.01; *p<0.05 relative to tissues from the small intestine of B6 mice (n=4). (C) Tissues from the small intestinal mucosa (M) and tumors (T) from these mice also have increased HMGA1 protein compared to analogous tissues from control (B6) mice as determined by western blot analysis.
Figure 2
Figure 2. Recovery of full length APC downregulates HMGA1 expression in HT29 cells
(A) HT29 cells bearing truncated APC were transfected so that full-length APC could be induced by the pSAR-MT vector when exposed to 100 μM zinc. β-galactosidase (control; HT29-β-Gal) and APC inducible HT29 (HT29-APC) cells were treated with 100 μM zinc for 72 hours, the protein was isolated, and HMGA1 expression determined by western blot. HT29-Apc cells treated with zinc displayed decreased expression of HMGA1 when compared to the HT29 β-galactosidase control. The western shows a representative image of three independent experiments. (B) HMGA1 mRNA levels were confirmed using quantitative real-time RT-PCR with primers and probes specific for human HMGA1. *p<0.05 relative to untreated HT29-APC. Data shown represent the mean +/− the standard deviation from three independent experiments that were conducted in triplicate.
Figure 3
Figure 3. The β-catenin/TCF-4 complex binds directly to the HMGA1 promoter region containing the TCF-4 binding consensus in HCT116 cells
(A) Identification of putative TCF-4 binding and response elements (TBEs) in the HMGA1 promoter. Sequence of the HMGA1 gene promoter is taken from the NCBI Nucleotide database (http://www.ncbi.nlm.nih.gov, reference sequence AF286367.1). (B) Chromatin immunoprecipitation experiments with sheared chromatin from HCT116 cells with endogenous overexpression of β-catenin and TCF-4 after cross-linking proteins bound to DNA with formaldehyde. The bar graph shows the percent of total input DNA immunoprecipitated with the following commercially available antibodies: rabbit Immunoglobulin G (IgG), TCF-4 (Cell Signaling), β-catenin (Santa Cruz), and histone H3 (Cell Signaling). The promoter sequence corresponding to the RPL30 promoter with TCF-4 and β-catenin antibodies was used as a negative control because there is no TCf-4 or β-catenin DNA binding site in the region amplified. Additional negative controls included rabbit IgG and no DNA (no template). The gel shows total input DNA compared to DNA immunoprecipitated with the antibodies as noted. Chromatin immunoprecipitation experiments were performed at least three times; results represent the mean +/− the standard deviation from the repeat experiments. The gel shown is taken from a representative experiment.
Figure 4
Figure 4. β-catenin/TCF-4 complexes in nuclear extracts from HCT 116 cells binds the HMGA1 promoter in vitro
Nuclear extracts were isolated from HCT116 cells and incubated with 50 fmol biotinylated DNA probe corresponding to the human HMGA1 promoter sequences beginning at −638bp (TBE1) and −1290 (TBE2) from the transcriptional initiation sequence. (A) Protein-DNA complexes were detected for biotin-labeled TBE1 and TBE2. For binding competition, a 200-fold molar excess of unlabeled DNA probe corresponding to TBE1 and TBE2 sequences was included in the reactions to serve as a specific competitor probe (sc). Unlabeled oligonucleotides bearing irrelevant sequences within the HMGA1 coding region served as non-specific competitor probe (ns). (B) Mutant (mt) biotin-labeled TBE1 and TBE2 oligonucleotides were incubated with nuclear extracts from HCT116 cells and their relative interactions compared to biotinylated wild-type (wt) oligonucleotides. Results shown are representative of three independent gel retardation assays. (C) For supershift assays, TCF-4, β-catenin, or control rabbit IgG was included. The arrows indicate the position of the specific transcription factor complexes and values below each lane reflect the relative band density compared to oligo and extracts in the absence of antibodies. Results shown are representative of four independent experiments.
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