An AXIN2 Mutant Allele Associated With Predisposition to Colorectal Neoplasia Has Context-Dependent Effects on AXIN2 Protein Function

Abstract

Heterozygous, germline nonsense mutations in AXIN2 have been reported in two families with oligodontia and colorectal cancer (CRC) predisposition, including an AXIN2 1989G>A mutation. Somatic AXIN2 mutations predicted to generate truncated AXIN2 (trAXIN2) proteins have been reported in some CRCs. Our studies of cells from an AXIN2 1989G>A mutation carrier showed that the mutant transcripts are not significantly susceptible to nonsense-mediated decay and, thus, could encode a trAXIN2 protein. In transient transfection assays, trAXIN2 was more abundant than wild-type AXIN2 protein, and in contrast to AXIN2, glycogen synthase kinase 3β inhibition did not increase trAXIN2 levels. Like AXIN2, the trAXIN2 protein interacts with β-catenin destruction complex proteins. When ectopically overexpressed, trAXIN2 inhibits β-catenin/T-cell factor-dependent reporter gene activity and SW480 CRC cell colony formation. These findings suggest the trAXIN2 protein may retain some wild-type functions when highly expressed. However, when stably expressed in rat intestinal IEC-6 cells, the trAXIN2 protein did not match AXIN2's activity in inhibiting Wnt-mediated induction of Wnt-regulated target genes, and SW480 cells with stable expression of trAXIN2 but not AXIN2 could be generated. Our data suggest the AXIN2 1989G>A mutation may not have solely a loss-of-function role in CRC. Rather, its contribution may depend on context, with potential loss-of-function when AXIN2 levels are low, such as in the absence of Wnt pathway activation. However, given its apparent increased stability in some settings, the trAXIN2 protein might have gain-of-function in cells with substantially elevated AXIN2 expression, such as Wnt pathway-defective CRC cells.

Figure 1

Analysis of AXIN2 gDNA and transcripts from a heterozygous 1989G>A allele carrier and the predicted AXIN2 truncated protein expressed. (A) The 1989G>A allele does not generate new splice elements. The sequences of the AXIN2 1989G (wild-type) and 1989A (mutant) alleles were analyzed using the SplicePort prediction algorithm. On the basis of the DNA sequence analysis, the positions of the predicted splice acceptor and donor positions in the wild-type and mutant AXIN2 alleles are indicated, with no new splice sites predicted in the mutant AXIN2 allele. The actual splice sites surrounding exon 7 are denoted with an asterisk. (B) The exonic splicing enhancer factor finder (ESEfinder) prediction identified one new predicted exon splicing enhancer factor binding site for SF1 binding in the 1989A allele. The number of each type of potential serine/arginine-rich (SR) factor binding motifs found in the AXIN2 1989G and 1989A alleles is indicated at the left. At the right, the SF1 consensus binding sequence motif is shown, with the corresponding sequences of the 1989G and A alleles, indicating the new consensus site match for the “A” allele. (C) Sequencing analysis of PCR products for the relevant region of AXIN2 exon 7 gDNA (gDNA) sequences in control DLD-1 CRC cells and (D) proband PBL samples. (E) Sequencing analysis of bulk PCR products from proband PBL cDNA identifies transcripts from both the wild-type “G” and mutant “A” AXIN2 alleles. (F) A schematic diagram of the location of the presumptive AXIN2 protein interaction domains [AXIN2 domains predicted to interact with TNKS (tankyrase), APC, GSK3, β-catenin] and DIX dimerization motif, as well as the location of the 1989G>A (W663X) nonsense mutation.

Figure 2

Analysis of AXIN2 and trAXIN2 protein abundance and regulation in transient transfection assays in HEK293T and effects of inhibitors of GSK3β, PP2A, and tankyrase on AXIN2 expression. (A) The trAXIN2 protein is expressed at increased levels in HEK293T cells relative to wild-type AXIN2. HEK293T cells were transfected with equal molar amounts of the AXIN2 or trAXIN2 expression vectors. Protein and RNA were collected for analysis. An IB from protein lysates was prepared from three separate transfection experiments for each of the two constructs, with AXIN2 and trAXIN2 proteins detected by electrochemiluminescence (ECL)-based IBs with an antibody against the FLAG-epitope. (B) Quantification of ectopically expressed AXIN2 transcripts in the transfected HEK293T cells by quantitative RT-PCR using primers in the FLAG sequence and protein levels of AXIN2 protein, based on IB analyses relative to β-actin levels, using ImageJ software. (C) A relevant portion of the AXIN1 and AXIN2 protein sequences are shown, with potential GSK3β phosphorylation sites in the proteins highlighted. (D) IB analysis of AXIN2 and trAXIN2 expression in transiently transfected HEK293T cells ectopically expressing the full-length or trAXIN2 proteins, following a 6-hour BIO and/or OA treatment, with “−” indicating no treatment and “+” indicating treatment. Shorter and longer exposures of the IBs are shown. (E) HEK293T cells were co-transfected with FLAG-tagged AXIN2 and trAXIN2 expression constructs and then treated with increasing doses of XAV939, a tankyrase inhibitor, for 24 hours after transfection, before preparing protein lysates. IB analysis to detect the ectopically expressed AXIN2 and trAXIN2 proteins was carried out with an anti-FLAG antibody.

Figure 3

The trAXIN2 protein is present in complexes with AXIN2, AXIN1, and β-catenin. (A) HEK293T cells were transfected with the indicated FLAG- or myc-tagged expression constructs. The expression of the FLAG- and myc-epitope–tagged AXIN2 and trAXIN2 proteins was assessed in IB assays to address the levels of the proteins in the input material used for IP. (B–D) HEK293T cells were transfected with the indicated plasmids. IPs were carried out with the indicated antibodies: anti–myc-epitope antibody in B and anti-FLAG-epitope antibody in C and D (for AXIN1-FLAG and β-catenin–FLAG, respectively), with the studies showing that trAXIN2 can form complexes with trAXIN2, AXIN2, AXIN1, and β-catenin. FLAG–β-catenin IPs (D) were performed following treatment with MG132 to stabilize β-catenin destruction complexes.

Figure 4

AXIN2 and trAXIN2 inhibit G418-resistant colony formation in a Wnt pathway mutant CRC cell line. G418-resistant colony formation assays were undertaken in the APC mutant SW480 CRC cell line (A and B) and in the RKO CRC cell line that lacks mutations in APC or CTNNB1 (C and D). SW480 and RKO cells were transfected with the indicated plasmids and plated in triplicate at low density under G418 drug selection for 21 days, and then the cells were fixed and stained with crystal violet, so that colonies could be visualized (A and C); experiments were undertaken three separate times to quantify colony numbers (B and D). (E) SW480 cells were transfected with the indicated expression plasmids and grown under G418 selection for 14 days and then lysed for IB analysis with the anti–FLAG-epitope antibody to assess AXIN2 and trAXIN2 protein expression.

Figure 5

AXIN2- and trAXIN2-mediated inhibition of Wnt/β-catenin/TCF transcriptional targets is context dependent. (A) Transient ectopic expression of AXIN2 and trAXIN2 suppress Wnt3a-mediated TCF reporter gene activity. HEK293T cells were transiently transfected with the pCMV-3Tag vector, AXIN2, or trAXIN2 expression constructs as well as TOPFlash reporter vector. Twenty-four hours after transfection, cells were treated with Wnt3a for 8 hours before harvesting for luciferase assays. Luciferase assays were performed in triplicate and mean and SDs are indicated. (B and C) Stable expression of AXIN2 but not trAXIN2 inhibits Wnt3a-mediated induction of endogenous Wnt/β-catenin/TCF target genes in rat intestinal IEC-6 cells. IEC-6 cells were transduced with empty retroviral expression vector construct or constructs for AXIN2 or trAXIN2, and drug selection was undertaken to create stable polyclonal cell lines. IB studies of the resultant IEC-6 cell lines show stable expression of AXIN2 or trAXIN2, as detected with anti-AXIN2 antibody (B). Stable IEC-6 transductants were treated for 16 hours with Wnt3a. The cells were then harvested, total RNA was collected, and expression of the indicated Wnt/β-catenin/TCF target genes was assessed in three separate quantitative RT-PCR experiments. The individual data points for three independent qPCR experiments with the mean of each group designated by a horizontal line are shown in C.