Elevated expression of axin2 and hnkd mRNA provides evidence that Wnt/beta -catenin signaling is activated in human colon tumors

Abstract

Genetic studies have identified mutations in key regulators of the Wnt/beta-catenin pathway in a variety of cancers, most frequently in colon cancers. However, whether the pathway is activated in clinical cancer samples is not easily determined, and therefore it is useful to find markers that could be surrogates to show activation of the Wnt/beta-catenin pathway. Gene expression profiles were analyzed in SW620, a colon cancer cell line in which beta-catenin levels are stabilized as a consequence of truncated adenomatous polyposis coli and were compared with profiles of the same cells transfected with antisense oligodeoxynucleotides. Treatment of cells with beta-catenin antisense oligodeoxynucleotides resulted in a decrease in the levels of axin2 and human naked cuticle (hnkd) mRNAs. Interestingly, the proteins encoded by both of these mRNAs are known inhibitors of the beta-catenin pathway. In 30 human cell lines derived from different origins, axin2 and hnkd were expressed only in human colon cancer cell lines that are known to have activating mutations in the Wnt/beta-catenin pathway. Further, levels of both axin2 and hnkd mRNA were also found to be elevated in about 65% of laser microdissected cells from human colon tumors compared with laser microdissected cells of normal morphology from the same patient samples. The increased expression of axin2 and hnkd correlated with truncations in adenomatous polyposis coli in the same patient samples. These results reveal that it is possible to detect activation of a carcinogenic pathway in human cancer samples with specific markers.

Figure 1

Reduction of cytosolic β-catenin levels and LEF-1 reporter activity in response to β-catenin antisense ODN transfection. (A) SW620 cell lysate was analyzed for cytosolic β-catenin levels by Western blotting 48 h posttransfection with β-catenin antisense and RC ODN in duplicated experiments. AS, antisense; RC, reverse control. (B) SW620 cells were transfected with β-catenin AS and RC ODN. After 24 h, the cells were transfected with reporter constructs as described in Methods. LEF-1 reporter activity was determined an additional 24 h later.

Figure 2

axin2 and hnkd mRNA levels are reduced in response to β-catenin antisense ODN transfection. SW620 cells were transfected with β-catenin antisense ODN. Total RNA from the cells was reverse transcribed 48 h posttransfection. axin2, hnkd, and β-catenin mRNA expression levels were determined by quantitative real-time PCR and normalized to glyceraldehyde-3-phosphate dehydrogenase. The graph represents the average of three experiments.

Figure 3

hNkd antagonizes Wnt signaling in mammalian cells and the putative promoters of axin2 and hnkd contain TCF-binding elements. (A) hNkd inhibits Wnt-1 signaling in a luciferase reporter assay in 293 cells. The data represent the average of two experiments. RLU, relative luciferase unit. (B) Schematic representation of the genomic sequence upstream of the axin2 and hnkd coding region identified as described in Methods. The putative TCF sites are shown as TBE boxes and nucleotide residues relative to the starting codon are indicated. A perfect match (CTTTGA/TA/T) is labeled with +, and an inverted perfect match is labeled as −.

Figure 4

axin2 and hnkd mRNA levels in human cell lines. Total RNA from a panel of 30 cell lines was reverse-transcribed, and axin2 and hnkd mRNA levels were determined by quantitative real-time PCR and normalized to actin. axin2 and hnkd mRNA levels are prominently elevated in human colon cancer cell lines.

Figure 5

axin2 and hnkd mRNA levels in human colon tumors. Total RNA from laser microdissected tissues was amplified and reversed transcribed as described in Methods. axin2 and hnkd mRNA levels were determined by quantitative real-time PCR and normalized to β-glucuronidase. The mutational status of APC in the patients was determined as described (Methods) and is indicated. Three patients that are wild type for APC are marked with *. The ratios of mRNA expression in tumor versus normal tissues for both axin2 and hnkd mRNA levels are shown in the table.