Adenomas in Apc1322T mutant mice are larger, more numerous and of earlier onset than those from ApcMin animals in which the mutant protein is truncated at codon 850 and lacks all β-catenin binding repeats. We have previously shown that adenomas from 1322T mice have lower nuclear β-catenin than Min adenomas. Here we found, as predicted, that lower levels of β-catenin are accompanied by generally lower expression of Wnt pathway genes, including the Wnt inhibitory factor Wif1. However, stem cell markers—including both Wnt targets such as Lgr5 and Cd44 and non-Wnt targets such as Bmi1 and Msi1—are upregulated in 1322T tumours compared with Min tumours. This suggests that the levels of Wnt signalling resulting from the homozygous 1322T mutation found in tumours are closer to the optimum for promoting expression of genes that cause the stem cell phenotype than are the Wnt levels found in Min tumours. The stem cell phenotype is thus favoured by submaximal Wnt signalling.
We observed subtle differences in the numbers of cells expressing Lgr5 in normal tissue prior to tumour initiation, suggesting that haploinsufficiency of Apc has a detectable effect on stem cell numbers. This effect may explain some of the differences in tumour initiation if, according to the cancer stem cell model of tumorigenesis, the tumour-causing ‘second hit’ first occurs in a stem cell and then spreads by clonal expansion. However, a much larger variation in stem cell number occurs in tumours of the same size and location between mouse strains than can be explained by this effect alone. Tumour progression must therefore differ as well, and cells which already have a second hit at codon 1322 favour Lgr5 expression and stem cell characteristics.
More than half of the epithelial portion of the 1322T adenomas showed Lgr5 and/or Cd44 expression which could indicate that all these cells are (cancer) stem cells. While this is possible, it is more likely that these cells have some stem cell-like characteristics such as the ability to self-replicate, or that these cells have the potential to be stem cells given the right local conditions or niche. It is notable that Bmi1 and Msi1 protein expression was detected in far fewer cells overall. While some of these differences may be explained by technical factors such as threshold levels of detection for ISH and IHC as well as differences between the levels and stability of the respective mRNAs and proteins, it may be that there are different and dynamic populations of stem-like cells that express different markers at different points in time. We must also consider that the presence of stem cell markers does not necessarily mean that all the cells they mark function as stem cells or cancer stem cells in vivo. There is currently no straightforward functional assay that identifies stem cells, but lineage tracing experiments in reporter mice—possibly in conjunction with allograft studies using tumour stem cells from 1322T and Min animals—could be used in the future to identify which marker or marker combination most accurately defines the stem cell population.
Although high levels of expression of the target gene Lgr5
indicate increased stem cells in 1322T adenomas, the functional role of the Lgr5 protein itself still remains unclear. It is therefore likely that other primary or secondary Wnt targets are important. Another Wnt pathway gene, Wif1
, was differentially expressed, with higher levels in Min tumours. While not previously known to play a major role in colorectal cancer, Wif1
expression has been shown to inhibit bladder tumour growth and osteosarcomas and is often silenced by methylation in breast carcinomas and colorectal cancer cell lines.25–28 Wif1
is unlikely to be a direct Wnt target gene, but it has been proposed that it acts as part of a negative feedback loop.29 Wif1
expression could potentially play a role in the Min adenomas, repressing tumour growth—perhaps by repressing the stem cell phenotype—despite high levels of nuclear β catenin and Wnt signalling. In support of this, Wif1
expression is generally low or absent in familial adenomatous polyposis tumours.30
Since it is counterintuitive that the feedback effect of Wif1
is stronger than the Wnt signal, it is necessary to postulate additional factors such as selective downstream effects for Wif1
to play a role in causing the phenotypic differences between the 1322T and Min animals.
Our expression arrays showed a small increase in expression of several other Wnt targets in 1322T adenomas including EphB2
. Unlike Lgr5
, these increases were not validated by Q-PCR, possibly due to the large variability between tumours, but they may still give us further insights into the mechanisms leading to tumorigenesis or increased stem cell number. The Grem1
gene is normally expressed in subepithelial myofibroblasts and found only at low levels in adenomas. Interestingly, however, it is an inhibitor of TGFβ/Bmp signalling, a pathway that is proposed to inhibit stem cell renewal,31
so higher levels of Grem1
expression, together with the lower levels of Bmp2
expression seen in 1322T tumours, could contribute to the observed stem cell phenotype. In addition, expression of the matrix metalloproteinase MMP7
correlates with poor prognosis in patients with colorectal cancer and promotes tumorigenesis in Min mice.32–34
It is also expressed highly in Paneth cells which are over-represented in 1322T adenomas. Since Paneth cells lie within the normal intestinal stem cell compartment, it is possible that MMP7 also influences stem cell numbers. Similarly, the difference in Myc
expression, although small, could play an important role in disease severity. Myc is essential for the phenotype caused by Apc
mutation in the intestine, and many of the genes upregulated after Apc loss, including the EphB receptors, are dependent on Myc expression.35
It is more difficult to explain the role of EphB in the severe 1322T phenotype. EphB signalling is important for cell compartmentalisation and migration in the intestine,36
but it is thought that high levels limit the expansion of adenomas.37
However, the expression of the EphB
genes is highest at the base of intestinal crypts in normal tissue, with EphB3
restricted exclusively to the region containing the stem cells, which could explain the high levels found in the stem cell-rich 1322T tumours.
Our use of mouse models in this study has allowed us to analyse in detail the effect of the location of specific germline and somatic Apc mutations to test the ‘just right’ hypothesis. Both our strains were kept on identical genetic backgrounds and no confounding mutations were seen at the loci. In addition, we have excluded the existence of unlinked modifiers in our mouse models by intercross experiments. While it is difficult to completely eliminate the possibility of tightly-linked modifiers in cis
, an independent targeting event resulted in the same phenotype as our 1322T model,4
suggesting that the targeted mutation alone is responsible for the severe polyposis and characteristic distribution of adenomas.
Confirmation of these results in samples from human patients has, however, proved difficult. As a result of the observations in human familial adenomatous polyposis adenomas which led to this hypothesis—namely, that a germline mutation early in APC is very rarely accompanied by an early somatic mutation—suitable samples to carry out analyses on patients with familial adenomatous polyposis are not available. In general, however, increased Lgr5 expression is seen in all familial adenomatous polyposis adenomas and the levels of nuclear β-catenin are very low (see figures 5 and 6 in online supplement), comparable to 1322T adenomas and consistent with the ‘just right’ hypothesis.
Clearly, many questions remain about how the expression of Wnt targets depends on nuclear β-catenin levels and other factors in both normal and tumour tissues. Some Wnt target genes known to be involved in intestinal tumorigenesis showed no significant difference between our two mouse models. For instance, no change was seen in Tcf1
expression which has been shown to suppress tumorigenesis in mammary tissue and also in the intestines of Min mice.38
We suggest that simple linear dependence of target levels is unlikely, and that different targets respond differently to changes in Wnt signalling. Our data confirm the operation of the ‘just right’ model of intestinal tumorigenesis1 2
and suggest that it acts through promoting the stem cell phenotype, although the underlying causes for the strong selective constraints on APC
mutations in colorectal cancer are likely to be complex.