CRC Laboratories, Department of Pathology, University Medical School, Teviot Place, Edinburgh, EH8 9AG, Scotland

Correspondence to: Alan R Clarke, CRC Laboratories, Department of Pathology, University Medical School, Teviot Place, Edinburgh, EH8 9AG, Scotland

We have analysed the pattern of -catenin expression by immunohistochemistry in mice singly or multiply mutant for Apc, p53 and Msh2. We observed increased expression of -catenin in all intestinal lesions arising on an Apc^Min+/- background. In all categories of lesion studied mosaic patterns of -catenin expression were observed, with the proportion of cells showing enhanced expression decreasing with increasing lesion size. p53 status did not alter these patterns. We also show that -catenin dysregulation marks pancreatic abnormalities occurring in Apc^Min+/- and (Apc^Min+/-, p53-/-) mice. In these mice both adenomas and adenocarcinomas of the pancreas arose and were characterized by increased expression of -catenin. We have extended these analyses to intestinal lesions arising in mice mutant for the mismatch repair gene Msh2. In these mice, increased expression of -catenin was again observed. However, in contrast with Apc^Min+/- mice, a subset of lesions retained normal expression. Taken together, these findings show that increased expression of -catenin is an efficient marker of early neoplastic change in both murine intestine and pancreas in Apc mutant mice. However, we also show that dysregulation of -catenin is not an obligate step in the development of intestinal lesions, and therefore that genetic events other than the loss of Apc function may initiate the transition from normal to neoplastic epithelium.

-catenin; Apc; p53; Msh2; intestine; pancreas

Introduction

Germline mutations in the adenomatous polyposis coli gene (APC) gene at 5q21 characterize an inherited disorder known as familial adenomatous polyposis coli (FAP) (Kinzler et al., 1991). FAP patients develop numerous adenomas throughout both the small and large intestine, some of which ultimately progress to carcinoma. However, a more general role for APC mutations in neoplasia is suggested by the fact that FAP patients have an increased predisposition to tumours of the brain, thyroid and bone and also to focal proliferative lesions (`desmoid tumours') of the connective tissue. Mutated APC has also been reported in a range of sporadic tumours, including pancreatic and gastric tumours and the majority of adenomas and carcinomas of the colorectum (Miyoshi et al., 1992; Horii et al., 1992; Nakatsuru et al., 1992). Furthermore, loss of heterozygosity at 5q21 has been observed in sporadic tumours of the breast and oesophagus (Boynton et al., 1992; Thompson et al., 1993; Kashiwaba et al., 1994).

Several different murine models of FAP have been generated by random chemical carcinogenesis (Moser et al., 1992), conventional gene targeting (Fodde et al., 1994; Oshima et al., 1995) and by the use of Cre-Lox technology (Shibata et al., 1997). All of these models are characterized by high levels of spontaneous intestinal neoplasia, confirming a role for Apc in the development of these lesions. Several observations using these models support the notion that Apc has more widespread tumour suppressor activity. First, desmoid tumours have been reported to occur spontaneously in Apc mutant mice (Shoemaker et al., 1997; Smits et al., 1998). Second, Apc^Min heterozygotes show an increased susceptibility to mammary carcinoma both spontaneously and following genotoxic stress such as carcinogen treatment or X irradiation (Moser et al., 1993, 1995; van der Houven et al., 1997). Finally, Apc heterozygosity on a p53 null background has been shown to strongly predispose to pancreatic neoplasia (Clarke et al., 1995).

How loss of function of Apc predisposes to malignancy remains unclear, however disruption of the normal function of -catenin has been implicated in this process (Rubinfeld, 1993; Su et al., 1993). Levels of -catenin are modulated by Apc through the mammalian Wnt signalling pathway, where Apc interacts with both glycogen synthase kinase 3 (GSK3) and -catenin. The central portion of Apc contains sites at which it can be phosphorylated by GSK3 and also through which it complexes with -catenin. Phosphorylation by GSK3 increases the stability of the Apc/-catenin complex and is thereby thought to increase the rate of -catenin degradation (Rubinfeld et al., 1996).

From the above it is clear that disruption of Apc function can lead to an increase in the cellular levels of -catenin. However, this is not the only potential mechanism for such an increase. Wnt-1 has been shown to regulate free pools of catenin (Papkoff et al., 1996) and both axin and the axin homologue conductin have been reported to alter -catenin activity through interaction with Apc, -catenin and GSK3 (Behrens et al., 1998; Ikeda et al., 1998; Kishida et al., 1998). The potential relevance of increased levels of -catenin becomes clear in the light of findings which show that -catenin functionally interacts with and activates members of the Tcf family of DNA binding transcription factors, including both Lef-1 and Tcf 4 (Behrens et al., 1998; Korinek et al., 1997). Activation of transcriptional signalling by -catenin-Tcf complexes has been shown to occur as a consequence of mutations in both Apc and -catenin (Morin et al., 1997; Rubinfeld et al., 1997) and mutations of -catenin have been reported in human colorectal cancers (Sparks et al., 1998). Dysregulated transcription has therefore been proposed as the basis for early neoplastic change, although the target genes through which this may be mediated remain as yet undetermined (Nusse, 1997).

-catenin also regulates E-cadherin in conjunction with -catenin, and loss of function of any of these proteins abrogates E-cadherin activities, including maintenance of the adherens junction complex. Amongst other activities this complex mediates cell-to-cell adhesion and thereby the control of cell motility (Chen et al., 1997). Modulation of cell adhesion appears to be a common mechanism in neoplastic change, and altered E-cadherin activity has been found in a number of cancers of epithelial origin including lobular breast carcinoma, colorectal carcinoma and gastric adenocarcinoma (Birchmeier and Behrens, 1994). In a transgenic murine model of pancreatic -cell carcinogenesis, loss of function of E-cadherin has been shown to be a critical step in the transition from adenoma to carcinoma (Perl et al., 1998).

Immunohistochemical analysis of both human and murine intestinal tumours has shown that both adenomas and well differentiated carcinomas are characterized by high levels of -catenin (Inomata et al., 1996; Takayama et al., 1996). However, both -catenin and E-cadherin are reported to be expressed at significantly lower levels in more aggressive malignancies, strongly suggesting that over-expression of -catenin is only crucial in early tumour development (Takayama et al., 1996).

In order to further characterize the association between tumourigenesis and dysregulation of -catenin we have analysed the pattern of -catenin expression in normal and neoplastic tissue derived from mice mutant for the tumour suppressor genes p53 and Apc. This analysis is performed on mice which carry a mutant Apc allele. Loss of the remaining wild type Apc allele results in dysregulated expression of -catenin. Because we also wished to address the possibility that dysregulation of -catenin is not an obligate step in intestinal neoplasia, we also analysed mice deficient for the DNA mismatch repair gene Msh2 (De Wind et al., 1998), a murine model of hereditary non-polyposis colorectal cancer (HNPCC). Msh2-/- mice develop lymphomas with a peak incidence at 2 - 3 months of age (De Wind et al., 1995; Reitmair et al., 1996). Of the 50% of Msh2-/- mice which survive beyond 6 months of age, 70% develop intestinal neoplasms (Reitmair et al., 1996). We report here the pattern of -catenin expression in lesions arising in mice mutant for Msh2-/- and (Msh2-/-, Apc^Min+/-).

We first investigated the pattern of expression of -catenin in intestinal lesions arising in Apc^Min and p53/Apc^Min mutants. In morphologically normal epithelium, -catenin was localized at the cell membrane. Nuclear localization was observed in some cells: these were always located at the crypt base (Figure 1a). This observation suggests a role for -catenin in the base of the crypt as -catenin is thought to mediate transcriptional regulation within the nucleus, and indeed interaction with the transcription factor Lef-1 is known to promote nuclear localization of -catenin (Huber et al., 1996).

In both Apc^Min+/- and (Apc^Min+/-, p53-/-) mice, expression of -catenin is more intense in dysplastic crypts and small adenomas. To control for staining variability between sections, changes in the intensity of expression were always scored relative to normal epithelium within the same section. The lesions were subclassified as in Clarke et al. (1995): (i) single dysplastic crypts, showing nuclear pleomorphism and stratification; (ii) complex lesions, comprising several architecturally distorted crypts in the lamina propria with virtually normal overlying surface epithelium; (iii) small adenomas, identified by the overall disturbance of architecture including the surface and distinguished from the previous category on the basis of increased size and surface involvement; (iv) large adenomas, and (v) adenocarcinoma. The pattern of -catenin staining, summarised in Figure 2, was essentially identical in Apc^Min+/- and (Apc^Min+/-, p53-/-) mice, with all features described below noted in both groups. A substantial proportion of all lesion types showed heterogeneous expression of -catenin expression, even where only single crypts were involved (type I lesions, Figure 1b,e and 2). The term `heterogeneous' is used here to describe lesions in which only a proportion of cells were characterized by increased expression. Although heterogeneous -catenin was observed in all lesions types, the proportion of cells overexpressing -catenin were at their highest in type I-III lesions (Figure 1c - d and 2). Mosaic type IV lesions showed the lowest proportion of cells staining positive for -catenin (Figure 1f). Large areas of reduced staining were observed in some late stage lesions, including those categorized both as type IV and V (Figure 1g). In all categories of lesion the predominant pattern was of increased -catenin staining within the nucleus, within the cytoplasm and also at the cell membrane. However a pattern of strong nuclear localization without concomitant cytoplasmic staining was also observed within some lesions as has been previously reported (Sheng et al., 1998).

These results show that high levels of -catenin are present in the majority of intestinal lesions, presumably as a direct consequence of perturbation of the Wnt pathway. Furthermore, areas showing high levels of -catenin included those composed of heterogeneous or single dysplastic crypts in the intestine, supporting the notion that dysregulated -catenin expression is an extremely efficient marker of early neoplastic change in the murine intestine. Lower levels of expression were seen in focal areas within some larger adenomas and adenocarcinomas, suggesting that genotypic changes which lead to elevated -catenin are only relevant to the early stages of neoplasia. This concept is supported by observations of localized areas of reduced or absent -catenin expression within some adenomas; and also by studies of human tumorigenesis, where down-regulation of both -catenin and E-cadherin has been reported in a range of carcinomas (Takayama et al., 1996).

We next investigated the pattern of expression of -catenin in intestinal lesions arising in Msh2 mutant animals and Msh2/Apc^Min mutants (summarized in Figure 2). Previous studies have shown that the Msh2 mutation predisposes to intestinal tumorigenesis and also accelerates neoplasia in Apc^Min+/- mice (De Wind et al., 1995, 1998; Reitmair et al., 1996). In Msh2-/- animals we identified type I , II and III lesions which showed normal -catenin expression (Figure 3b), a phenomenon we did not observe in Apc^Min+/-mice (Figure 3a). However, all type IV adenomas were characterized by increased levels of -catenin expression. No type V lesions were identified. In (Msh2-/-, Apc^Min+/-) mice there was a significant increase in the frequency of adenomas, as has been previously reported (Reitmair et al., 1996). The majority of these lesions stained strongly for -catenin (Figure 3c), however we again identified a small number of type I,II and III lesions (´10%) with the pattern of -catenin expression characteristic of normal cells (Figure 3d). All type IV lesions analysed showed altered -catenin expression, with an almost identical pattern to that observed in Apc^Min+/-mice. No type V lesions were identified.

We therefore successfully identified small lesions in both Msh2 and Msh2/Apc^Min mice which showed normal levels and distribution of -catenin. These findings show that dysregulated -catenin is not an obligate event in early lesion formation, and furthermore that Msh2 deficiency predisposes to such apparent -catenin-independent events. Thus, Msh-2 deficiency may predispose to dysplasia through mutations in other components of the Wnt signalling pathway which do not affect -catenin levels or indeed through mutations in other pathways. All large adenomas (type IV) were characterized by increased -catenin, showing that this degree of morphological change is absolutely associated with events which dysregulate -catenin levels.

We have previously described the phenotype of mice mutant for both p53 and Apc (Clarke et al., 1995). In addition to intestinal lesions these mice develop pancreatic neoplasia, either adenoma or acinar adenocarcinoma, with almost 100% penetrance. The cell type observed in these lesions was predominantly acinar, although some foci showed ductal transdifferentiation. The involvement of Apc in the development of pancreatic lesions was confirmed by loss of the remaining wild type allele in adenocarcinomas (Clarke et al., 1995). We therefore next wished to assess the extent of dysregulation of -catenin in these lesions.

Within morphologically normal pancreatic cells, -catenin was observed at the cell membrane, with no obvious nuclear localization. In (Apc^Min+/-p53-/-) mice all foci showing histological change were characterized by high levels of -catenin. Increased staining was also seen in foci which were virtually histologically normal. Such increased staining was never observed in pancreas samples derived from wild type mice. Foci varied in size, with some containing only single or a few cells in the plane of section (Figure 4a,b). The observation of few or single cell lesions strongly suggests that dysregulated expression occurs very early in neoplasia.

In lesions identified in formalin fixed tissues, increased -catenin expression was seen within both the nucleus and cytoplasm (Figure 4c). However, cytoplasmic staining was rarely observed in Methacarn fixed sections, suggesting that the observed cytoplasmic localisation was an artefact of tissue fixation. Using either fixation protocol we observed rare (<1%) lesions which did not show increased nuclear staining (Figure 4d). Nuclear atypia was seen in the majority of lesions, although the extent of nuclear pleomorphism varied considerably from mild to severe within each lesion (Figure 4b,e). Acinar cell lesions classified as adenoma were also characterized by increased -catenin staining. Some of these lesions contained areas of acinar-ductal transdifferentiation which were also strongly stained (Figure 4f). Adenocarcinomas contained areas in which -catenin intensity was reduced (Figure 4g), however areas of ductal differentiation within these tumours retained high levels of -catenin (Figure 4h).

These results prompted us to analyse Apc^Min mice. When these animals are maintained on a wild type background they do not develop pancreatic adenomas (Clarke et al., 1995). Surprisingly, although we confirmed absolute absence of neoplasms of the pancreas, we did find multiple foci of -catenin dysregulation identical to those observed in (Apc^Min +/-p53-/-) mice. Our previous analysis (Clarke et al., 1995) had identified focal mild dysplasia in one out of seven Apc^Min heterozygotes. Subsequent re-examination of these sections showed multiple ill-defined areas containing cells characterized by nuclear size variation. These areas overexpressed -catenin. Thus, -catenin immunohistochemistry efficiently highlighted focal areas of early histological change in the pancreas of both (Apc^Min+/-p53-/-) and Apc^Min+/-mice. We also analysed pancreatic tissue derived from Msh2-/-mice and (Msh2-/-, Apc^Min+/-) mice, neither of which develop spontaneous pancreatic neoplasms. No abnormal expression of -catenin or histological atypia was observed in Msh2-/-mice. However, in (Msh2-/-, Apc^Min+/-) mice we again identified foci of -catenin overexpression, and these did not differ in morphological appearance from those seen in Apc^Min+/-animals.

In the pancreas, dysregulated -catenin expression was seen in 100% of lesions which appeared morphologically abnormal. Previously, we had noted the presence of these lesions at high frequency only in (p53-/-, Apc^Min+/-) mice and rarely in wild type mice. The occurrence of high -catenin expression in areas of minimal histological abnormality in Apc^Min heterozygotes allows an order of genetic events to be proposed for this model of pancreatic neoplasia. In the presence of wild type p53 such dysregulated expression does not lead to neoplasia, but it is associated with nuclear size variation, raising the possibility that loss of Apc function may promote chromosomal instability. We are currently characterizing this phenomenon in greater detail. By contrast, a p53 null environment allows progression to adenoma and then adenocarcinoma. Loss of p53 is therefore essential for adenoma formation in the time frame analysed here. Notably, the requirement for genetic change differs between pancreas and intestine, as within the murine intestine p53 loss does not increase either adenoma burden or neoplastic progression (Clarke et al., 1995).

To characterize the status of the remaining Apc allele in both the intestinal and pancreatic lesions arising in Apc^Min+/- mice we performed PCR analysis on microdissected foci. Serial sections were generated and areas of increased -catenin staining identified. These areas were microdissected and DNA isolated. Loss of the remaining Apc^wt allele was assessed following PCR amplification. This approach allowed us to analyse intestinal and pancreatic lesions of, at the lowest limit, approximately 50 cells per cross section. Using this method we demonstrate loss of the remaining wild type Apc allele in both the intestinal and pancreatic lesions analysed from Apc^Min+/- and Apc^Min+/-, p53-/- mice (Figure 5). This finding is consistent with the concept that -catenin dysregulation occurs as a consequence of loss of Apc function.

Taken together, these results show that -catenin dysregulation occurs in both the intestine and pancreas, and that where present it is associated with the very first steps in the development of neoplasia. These findings demonstrate that altered expression of -catenin is a key marker of Apc dysregulation in both these tissues, and suggest that altered -catenin expression may be a useful diagnostic marker of early neoplastic change in human disease. However, we also show that -catenin dysregulation is not an obligate step in the generation of intestinal lesions in an Msh2 deficient background, and therefore that other mechanisms may underlie such early neoplastic change.

Acknowledgements

We wish to thank Hein te Riele for supply of Msh-2 deficient mice, James Going for assistance with microdissection and John Verth and his staff for animal maintenance. AR Clarke is a Royal Society University Research Fellow. This work was supported by SHERT, the Cancer Research Campaign and by a grant from the government of Thailand to R Kongkanuntn.

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Figure 1 The pattern of -catenin staining in the intestine of Apc^Min+/- and (Apc^Min+/-, p53-/-) animals. Mice mutant for Msh2 (De Wind et al., 1995), p53 (Clarke et al., 1993) and Apc^Min (Moser et al., 1992) were maintained as outbred colonies segregating for Ola/129, Balb C, SWR and C57Bl/6 genomes. Mice were monitored on a daily basis for signs of ill health and were killed when they showed signs of disease. All tissues were paraffin embedded using standard methods after overnight fixation in either buffered formalin or methacarn (four parts methanol, two parts chloroform, one part acetic acid v/v). High temperature antigen retrieval was performed (Alman et al., 1997), sections were cooled to room temperature and immersed in 1.5%H₂O₂ solution to block endogenous peroxidase for 15 min. Sections were then incubated with 1 : 50 mouse monoclonal -catenin antibody (IgG₁, clone 14, Transduction Laboratories, USA) for 60 min, and subsequently with 1 : 400 Rabbit Anti-Mouse Biotinylated secondary antibody (DAKO) for 30 min. The sections were incubated in StrepABComplex/HRP (DAKO) for 30 min. The labelled complex was developed with diaminobenzidine (DAB, 0.5 mg/ml) for 5 - 8 min. at room temperature. (a - g) Photographs demonstrating the various features observed in animals with these genotypes. All the features illustrated here were observed irrespective of p53 status. All scale bars represent 10 m (a) -catenin staining in morphologically normal crypts of the small intestine. -catenin was detected throughout the cytoplasm of epithelial cells but was strongly localized to the lateral borders. Strong nuclear localization was observed in cells at the crypt base (arrows). (b) Heterogeneous expression in a type I lesion. The majority of cells show the normal pattern of staining, with localization to the lateral borders. A subset of cells show increased cytoplasmic and nuclear staining. (c) Uniformly increased -catenin staining within a type 1 lesion. (d) Increased -catenin staining in a type II lesion. (e) Heterogeneous expression in a type II lesion. Cells showing increased -catenin showed localized to the cytoplasm and in some instances localization to the nucleus. (f) Heterogeneous expression of -catenin within a type III lesion. (g) Reduced expression within a type IV lesion. Where expression of -catenin was retained this was often localized to the nucleus

Figure 2 -catenin expression patterns within each class of intestinal lesion. (a) Percentage of each lesion type showing either upregulation of -catenin in all cells (black bars); a mosaic or heterogeneous pattern of upregulation as defined in the text (grey bars); or no upregulation (open bars). The number of lesions scored is shown over each column. Insufficient numbers of category V lesions were identified to permit scoring. (b) Histogram showing the percentage of cells expressing high levels of -catenin within lesions characterized by mosaic expression of -Catenin. Black bars, Apc^Min+/-; Grey bars (Apc^Min+/-, p53-/-); Hatched bars (Apc^Min+/-, Msh2-/-). Mean values are given for each lesion category, as defined in the text. Error bars represent SEM. Insufficient numbers of mosaic lesions were identified in Msh2-/- mice to permit analysis

Figure 3 The pattern of -catenin expression in the intestine of Msh2-/- mice (a,b) and (Msh2-/-, Apc^Min+/-) mice (c,d). Immunohistochemical analysis was performed as described in the legend to Figure 1. All scale bars represent 10 m. (a) Increased -catenin staining in a type I lesion. (b) Normal pattern of -catenin expression in a type II lesion, with -catenin strongly localized to the lateral borders. (c) Increased -catenin expression in a type II lesion. (d) Normal -catenin expression in a type II lesion, with retained localization to the lateral borders. A type I lesion showing -catenin dysregulation is indicated for comparison (arrow)

Figure 4 The pattern of -catenin staining in the pancreas of Apc^Min+/- and (Apc^Min+/-, p53-/-) mice. Immunohistochemical analysis was performed as described in the legend to Figure 1. (a - e) are representative of the patterns of -catenin staining and histological atypia observed in the pancreas of both Apc^Min+/-mice and (Apc^Min+/-, p53-/-) mice. (f - h) are representative of these patterns in pancreatic adenomas and adenocarcinomas arising in (Apc^Min+/-, p53-/-) mice. All scale bars represent 10 m. (a) Methacarn fixed. A single pancreatic acinar cell characterised by increased nuclear and cytoplasmic expression. The surrounding acinar cells are representative of the normal pattern of -catenin staining, with localization to the cell borders. (b) Methacarn fixed. Small focus of acinar cells with increased expression. These foci were often composed of cells with increased nuclear size, prominent examples of which are indicated by arrows. This focus also contains a cell (short arrow) with no increase in nuclear levels of -catenin. (c) This picture demonstrates the pattern of staining observed in formalin fixed tissues. Cells (arrowed or restricted to the upper right hand portion of this photograph) showing increased nuclear and cytoplasmic levels of -catenin staining. (d) Methacarn fixed. A dysplastic adenoma showing increased -catenin expression, but with no apparent nuclear localization. (e) Methacarn fixed. Increased -catenin staining in a pancreatic focus, showing strong nuclear localization. Again, these foci were often composed of cells with increased nuclear size. (f) Methacarn fixed. Heterogeneous -catenin expression in an adenoma containing areas of acinar-ductal transdifferentiation (arrowhead). No increase in -catenin staining was detectable in normal ducts (arrow). (g) Low levels of -catenin within an acinar adenocarcinoma. (h) Methacarn fixed. Areas of ductal differentiation within an acinar adenocarcinoma which have retained high levels of -catenin expression

Figure 5 PCR amplification of Apc alleles from microdissected lesions. PCR analysis. Histological microdissection was performed as previously described (Going and Lamb, 1996). Samples were digested in proteinase K (1 mg/ml) and 1% Tween 20. The proteinase K was subsequently heat-inactivated at 95°C, for 10 min. PCR amplification was then performed essentially as previously described (Luongo et al 1994) using the primers (5'TCTCTT CTGAGAG CAGAAGTT) and (5'ATAGCCAA AGTTATGGAA GAAGTATCA). Representative results from PCR analysis of microdissected lesions. Determination of Min status was by PCR and HindIII digest of PCR product as previously described (Luongo et al., 1994). WT, the amplification product from the wild type Apc allele. Apc^Min, the amplification product from the mutant allele. Samples were all derived form lesions arising within Apc^Min mutant mice and were as follows: lanes 1 and 2, pancreatic foci showing -catenin dysregulation; lane 3 normal pancreas; lanes 4 and 5, small intestinal lesions; lanes 6 and 7 normal intestinal epithelium. All results were obtained using microdissected areas containing a minimum of 50 cells