Human Molecular Genetics, 2002, Vol. 11, No. 13 1549-1560
© 2002 Oxford University Press
The ‘just-right’ signaling model: APC somatic mutations are selected based on a specific level of activation of the ß-catenin signaling cascade
1Centro de Investigação de Patobiologia Molecular–CIPM, Instituto Português de Oncologia Francisco Gentil, 1093 Lisbon, Portugal, 2Center for Human and Clinical Genetics, Leiden University Medical Center, PO Box 9503, 2300 RA Leiden, The Netherlands, 3Serviço de Gastrenterologia, Instituto Português de Oncologia Francisco Gentil, 1093 Lisbon, Portugal and 4Department of Surgery, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands
Received February 22, 2002; Accepted April 25, 2002
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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According to the classical interpretation of Knudson's ‘two-hit’ hypothesis for tumorigenesis, the two ‘hits’ are independent mutation events, the end result of which is loss of a tumor suppressing function. Recently, it has been shown that the APC (adenomatous polyposis coli) gene does not entirely follow this model. Both the position and type of the second hit in familial adenomatous polyposis (FAP) polyps depend on the localization of the germline mutation. This non-random distribution of somatic hits has been interpreted as the result of selection for more advantageous mutations during tumor formation. However, the APC gene encodes for a multifunctional protein, and the exact cellular function upon which this selection is based is yet unknown. In this study, we have analyzed somatic APC point mutations and loss of heterozygosity (LOH) in 133 colorectal adenomas from six FAP patients. We observed that when germline mutations result in truncated proteins without any of the seven ß-catenin downregulating 20-amino-acid repeats distributed in the central domain of APC, the majority of the corresponding somatic point mutations retain one or, less frequently, two of the same 20-amino-acid repeats. Conversely, when the germline mutation results in a truncated protein retaining one 20-amino-acid repeat, most second hits remove all 20-amino-acid repeats. The latter is frequently accomplished by allelic loss. Notably, and in contrast to previous observations, in a patient where the germline APC mutation retains two such repeats, the majority of the somatic hits are point mutations (and not LOH) located upstream and removing all of the 20-amino-acid repeats. These results indicate selection for APC genotypes that are likely to retain some activity in downregulating ß-catenin signaling. We propose that this selection process is aimed at a specific degree of ß-catenin signaling optimal for tumor formation, rather than at its constitutive activation by deletion of all of the ß-catenin downregulating motifs in APC.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Germline mutations in the APC (adenomatous polyposis coli) gene are responsible for familial adenomatous polyposis (FAP), an autosomal dominant disease characterized by the development of hundreds to thousands of colorectal adenomas (1,2). These polyps, if not removed, will ultimately progress to cancer. Mutations in APC are also detected in the majority of sporadic colorectal cancers (85%) and are implicated in the development from normal epithelium to the early adenoma stage (3–6).
APC has been shown to participate in several cellular processes, including cell cycle regulation, apoptosis, cell adhesion, cell migration, signal transduction, microtubule assembly and chromosome segregation (7–9). However, despite the fact that each of these roles is potentially linked with cancer, it appears that the tumor suppressing function of APC resides in its capacity to properly regulate intracellular ß-catenin levels (10–12). The APC protein binds to ß-catenin and, together with GSK3ß and axin/conductin, promotes its downregulation, thereby preventing signaling activity to the nucleus. In the absence of functional APC, ß-catenin accumulates in the cytoplasm and is translocated to the nucleus, where it associates with members of the TCF/LEF family of transcriptional activators, thus modulating transcription of Wnt target genes possibly involved in proliferation and apoptosis (13–15). The regulation of ß-catenin by APC is accomplished by three 15-amino-acid repeats and seven 20-amino-acid repeats that, respectively, bind and downregulate ß-catenin (16–18). In addition, three SAMP repeats that bind conductin or axin appear to be essential for optimal regulation (12,19–21). Recently, APC has been shown to contain functional nuclear export and import signals that enable it to shuttle between the cytoplasm and the nucleus (22–27). The latter appears to represent an additional Wnt signaling control mechanism by sequestering ß-catenin and transporting it out of the nucleus to the cytoplasmic proteolytic machinery.
The vast majority of APC mutations result in truncated proteins that lack all axin/conductin binding motifs and a variable number of 20-amino-acid repeats associated with the downregulation of intracellular ß-catenin levels (3,6,28–30). Whereas the germline mutations are scattered throughout the 5' half of the APC gene, the majority of somatic mutations in both sporadic and FAP-associated colorectal tumors are clustered between codons 1286 and 1513, the mutation cluster region (MCR) (3,6).
FAP represents one of the most consistent examples of genotype–phenotype correlations in hereditary cancer: a positional dependence exists between the germline mutation and the number of polyps in the gastrointestinal tract. In general, germline mutations located between codons 450 and 1600 result in stable truncated proteins and are associated with a higher polyp multiplicity (31–33). Within this group, germline mutations around codon 1300 frequently result in a profuse phenotype with patients developing over 5000 polyps (34,35). Attenuated phenotypes, characterized by a lower number of polyps and delayed age of onset, have been observed in patients with mutations located in the 5' end and 3' half of APC (36–41).
In agreement with Knudson's two-hit hypothesis, inactivation of both APC alleles can be detected in the majority of intestinal tumors already at early stages of tumor development (3–6). In the original formulation of Knudson's hypothesis, the two hits represent independent mutation events, the end-result of which is loss of a tumor suppressing function. Recently, it has been shown that APC does not entirely follow this model (42–44). The position and the type of the second hit in FAP polyps depends on the localization of the germline mutation (42). Also, in sporadic tumors, both APC hits appear to be interdependent (43,45). Apparently, somatic APC mutations are selected based on the growth advantage they provide to the tumor cell. In polyps from FAP patients with germline mutations around codon 1300, allelic loss is the most common second hit mechanism (5,42). In contrast, polyps from patients with germline mutations outside this region (5' to codon 1190 or 3' to codon 1392) showed low allelic loss frequencies and appeared to acquire second hits by point mutations within the MCR (42). It has been suggested that the explanation for the observed non-random distribution of APC somatic mutations could reside in the disturbance of a critical balance between ß-catenin binding and degradation; however, the limited point mutation data available have not allowed the authors to propose a specific selection mechanism (42). In an Apc mouse model specifically engineered to enrich for point mutations as the main second-hit mechanism, we could show that somatic Apc mutations are selected that remove all 20-amino-acid repeats, thereby completely inactivating the ß-catenin downregulating activity of Apc (44). However, in most human colorectal adenomas, truncated APC proteins are selected that retain one or two 20-amino-acid repeats. The molecular basis for this difference is yet unknown.
In this study, we have analyzed in more detail the APC ‘second hit’ in 133 polyps from six FAP patients. Apart from confirming previous observations, our results show that the dependence between germline mutation and the resulting spectrum of somatic mutations that lead successfully to tumor formation is more complex than previously suggested. Based on these new and previously published data, we propose that specific APC genotypes are selected during tumor formation based on the specific level of residual ß-catenin-downregulating activity that they can still exert, rather than on the complete inactivation of this signal transduction regulatory function of APC. According to this ‘just-right signaling’ model, the function of APC must be impaired to a specific degree to allow sufficient accumulation of nuclear ß-catenin and activation of the downstream target genes relevant for tumor formation in the intestine. Excessive ß-catenin accumulation in the nucleus has been shown to result in programmed cell death (46), and is therefore unlikely to be selected upon during tumor formation.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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We have analyzed a total of 133 colorectal adenomas from six unrelated FAP patients for somatic point mutations in exon 15 by protein truncation test (PTT) and for allelic loss. The phenotypic features, the germline mutation and the number of colorectal adenomas analyzed for each patient are described in Table 1.
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A somatic APC mutation was found in the vast majority of the adenomas investigated here (105 of 133; 79%). In these 105 adenomas, 108 mutations were detected, 79% (85 of 108) of which were point mutations and 21% (23 of 108) allelic losses. Table 2 and Figure 1 summarize these results.
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A high frequency of allelic loss and a low frequency of point mutations were observed among adenomas from the patient with the germline mutation at codon 1309. This mutation results in a truncated APC protein encompassing only one 20-amino-acid repeat. In this patient, 62% (13 of 21) of the adenomas showed allelic loss whereas 24% (5 of 21) showed a somatic point mutation. PCR amplification of a fragment encompassing the 5 bp germline deletion at codon 1309 revealed that in all LOH cases the wild-type allele was invariably lost. As depicted in Figure 1A, four out of five point mutations result in truncated proteins lacking all the 20-amino-acid repeats. Two of these point mutations were observed in adenomas also showing allelic loss.
Polyps from two patients characterized by germline mutations expected to result in short truncated proteins lacking all 20-amino-acid repeats, namely at codons 565 and 1060, were also employed for our analysis. In contrast to the codon 1309 patient, a very low frequency of allelic loss was detected in polyps from these patients. No allelic loss was observed in any of the 30 adenomas analyzed from the codon 565 patient, whereas the patient carrying the 1060 mutation showed allelic loss in only 20% (6 of 30) of the adenomas (Fig. 2). In the patient carrying the codon 1060 mutation, PCR amplification of a fragment containing the 4 bp germline deletion revealed that in all cases the wild-type allele was lost. Both allelic loss frequencies differ significantly from the codon 1309 patient (P<1x10-6 and P=0.002, respectively, Fisher's exact test). In contrast, the majority of the adenomas (70% and 63%, respectively) showed point mutations (Fig. 3), most of them clustering between codons 1286 and 1482 and resulting in truncated proteins with one or, less frequently, two 20-amino-acid repeats (Table 2). Truncating mutations leaving one 20-amino-acid repeat were observed in 15 of 21 and 13 of 19 of the point mutations respectively, whereas mutations leaving two or more 20-amino-acid repeats were found only in 6 of 21 and 4 of 19 cases respectively (Fig. 1B, C). Figure 4A shows three examples of the sequence analyses of these somatic point mutations in APC.
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T (E1322X) transversion in sample 3, an AWe have also analyzed 15 adenomas from a patient with a t(5;10)(q22;q25) translocation with one breakpoint mapped between exons 6 and 12 and the other between nucleotides 5034 and 5402 of APC (Fig. 1D). Western blot analysis using an N-terminal antibody indicated that there was no stable expression of a truncated protein, strongly suggesting that this mutation behaves as a null allele (47). As for the adenomas derived from the codon 565 patient, also in this case no allelic loss was found in any of the adenomas analyzed, which is significantly different from the allelic loss frequency found in the adenomas corresponding to the codon 1309 patient (P=1x10-4, Fisher's exact test). However, all the adenomas presented with somatic point mutations within the MCR, the vast majority of which (13 of 15) result in truncated proteins retaining one 20-amino-acid repeat (Fig. 1D, Table 2). The nature of the APC defect and the corresponding course of the disease in this patient (47) are reminiscent of attenuated polyposis (AAPC), an allelic form of FAP characterized by reduced polyp multiplicity and a delayed age of onset (36–41). For the sake of completeness, an additional attenuated patient was included (patient 5 in Table 1) characterized by a germline mutation at codon 168. This mutation is predicted to result in a very short and presumably unstable truncated protein lacking all repeats responsible for binding and downregulating ß-catenin. However, according to previous reports, alternative splicing around exons 3 and 4 occurs at low frequency, leading to the residual expression of polypeptides lacking only few functional motifs with a limited loss of the wild-type functionality (48,49). We have analyzed 10 adenomas from this patient and found no allelic loss, which is also significantly different from the allelic loss results obtained for the codon 1309 patient (P=1x10-3, Fisher's exact test). However, 8 out of 10 adenomas showed a somatic point mutation (Table 2). Similar to the patients carrying the codons 565 and 1060 germline mutations as well as the translocation patient, these mutations were localized within the MCR, the majority (6 of 8) of which left only one 20-amino-acid repeat (Fig. 1E).
Somatic mutation analysis in these five FAP patients is basically in agreement with earlier studies showing that carriers of germline mutations around codon 1300 tend to present allelic loss as the most common second hit, whereas polyps from patients with germline mutations outside this region mainly acquired second hits by point mutations within the MCR (5,42). However, patients carrying germline mutations beyond codon 1400 (i.e. retaining two or more 20-amino-acid repeats) have not been studied thoroughly for the presence of somatic point mutations, although it has been reported that allelic loss as a second hit mechanism is suppressed in the polyps of patients with comparable germline APC mutations (42). We have analyzed 27 adenomas from a FAP patient with a germline mutation at codon 1436 resulting in a truncated protein with two 20-amino-acid repeats. In agreement with previous results, allelic loss was observed in only 4 of 27 (15%) of the adenomas investigated. As in the adenomas from the previous four patients, this low frequency of allelic loss is, significantly different from the frequency of allelic loss found in the adenomas from the codon 1309 patient (P=0.0008, Fisher's exact test). Most of the adenomas of this patient (17 of 27) showed somatic point mutations, which is also significantly different from the codon 1309 patient (P=0.006, Fisher's exact test). One of these point mutations was observed in an adenoma with allelic loss. Figure 4B shows an example of a somatic point mutation sequenced from this patient.
Notably, virtually all somatic point mutations cluster upstream of the MCR, thereby resulting in truncated proteins lacking all 20-amino-acid repeats (Table 2, Fig. 1F). This localization is significantly different from that observed in polyps from patients carrying the codon 168, 565 or 1060 mutations or the translocation mutation (P<0.002, Fisher's exact test). In fact, looking at the somatic mutation spectra of all six patients as summarized in Figure 1, there appears to be a dichotomy with respect to the number of 20-amino-acid repeats encompassed by the somatic mutation. In the patients carrying the codon 168, 565 or 1060 mutations and the translocation mutation (no 20-amino-acid repeat remaining) the great majority of somatic mutations generated truncated proteins retaining one, or less frequently, two 20-amino-acid repeats. On the other hand, in the patients carrying the codon 1309 or 1436 mutations (one or two 20-amino-acid repeats left) virtually all somatic mutations completely removed all the 20-amino-acid repeats, either by allelic loss or point mutations upstream of the first 20-amino-acid repeat, respectively. This difference in the number of 20-amino-acid repeats encompassed by the somatic mutation is highly significant (P<1x10-5, Pearson chi-square test; exact). Thus, it appears that the combination of germline and somatic mutation is selected to encode a truncated protein retaining one or, less frequently, two 20-amino-acid repeats.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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It is generally accepted that different APC mutations provide a nascent colorectal tumor cell with different degrees of selective advantage to allow clonal expansion. Previously, it has been shown that the strongest selective advantage seems to be provided by somatic mutations around codon 1300. Patients presenting with germline mutations at or around codon 1300 acquire the second hit at APC by allelic loss (42) – generally believed to occur at a high spontaneous frequency (50). Polyps from patients with germline mutations elsewhere in APC acquire truncating mutations within the MCR rather than LOH, presumably to compensate for the weaker selective advantage provided by the germline mutation (42). The present study provides additional data on the interdependence between the localization of the germline mutation and the resulting spectrum of somatic events that lead successfully to tumor formation, suggesting that the selection mechanism is more complex than previously thought. All patients analyzed in this study showed a strong correlation between the position of the germline mutation and the type and localization of the second hit. According to the position of the germline mutation with respect to the 20-amino-acid repeats (ß-catenin downregulating), three different scenarios can be envisaged, as illustrated in Figure 5: (i) if the germline mutation results in a truncated protein without any of the 20-amino-acid repeats, most second hits lead to truncated proteins with one or, less frequently, two 20-amino-acid repeats; (ii) when the germline mutation results in a truncated protein with only one 20-amino-acid repeat, in the majority of the tumors the wild-type APC allele is removed by LOH; (iii) when the germline mutation leads to a truncated protein encompassing at least two 20-amino-acid repeats, the corresponding somatic mutations truncate all the 20-amino-acid repeats from the wild-type APC protein. The latter differs significantly from the localization of the somatic hits within the MCR, as was previously suggested.
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The first two scenarios are clearly supported by the results of Lamlum et al. (42). Among 21 patients with the germline mutation deleting all the 20-amino-acid repeats, only one of the 120 adenomas analyzed showed allelic loss, whereas somatic point mutations leading to the retention of one or, less frequently, two 20-amino-acid repeats comprised 19 of the 26 detected point mutations. Similar to the codon 1309 patient included in our study, the majority of adenomas from eight patients with the germline mutation encompassing one 20-amino-acid repeat showed allelic loss (39 of 49 adenomas). Also the low frequency of allelic loss (2 of 44 adenomas) observed in adenomas from six patients with the germline mutation leaving two 20-amino-acid repeats is in agreement with the third scenario proposed here. This suggests that most somatic hits in these patients will be point mutations. However, as only seven adenomas were analyzed for somatic point mutations and only two mutations were identified, it is at present unclear if most adenomas from this group of patients acquire somatic point mutations leading to truncated APC proteins lacking all 20-amino-acid repeats as here reported for the codon 1436 patient. Nevertheless, it is clear that the presence of a specific germline APC mutation defines the type and position of somatic hits that are successful in tumor formation. Several APC genotypes appear to be effective in the development of human colorectal tumors, provided that one of the mutations encodes for a truncated APC protein retaining one or two 20-amino-acid repeats. Interdependence between the somatic hits at APC is also observed in most sporadic colorectal cancers. Also, in this case, there is a clear selective advantage for one of the two hits resulting in a truncated protein retaining one or two 20-amino-acid repeats (43,45).
The underlying selection mechanism lies most likely in the ability of APC to regulate the Wnt/ß-catenin signaling pathway. Several studies have shown that truncated proteins with two or three 20-amino-acid repeats still exhibit a considerable level of residual activity, and can downregulate ß-catenin signaling almost as efficiently as full-length protein when overexpressed in vitro (18,51–53). Moreover, even when expressed at endogenous levels in mouse embryonic stem cells, a truncated Apc protein encompassing three 20-amino-acid repeats, as in Apc1572T, shows considerable more ß-catenin downregulating activity than the ApcMin protein, where all ß-catenin regulating domains are missing (M.F. Kielman et al., submitted). Also, a truncated protein with only one repeat is still capable of exporting ß-catenin out of the nucleus, and may thus contribute to reducing ß-catenin signaling activity (23,27). Thus, although virtually all of the truncated APC proteins selected for in colorectal tumorigenesis are impaired in their ß-catenin downregulating activity, they do not represent null alleles and encode for some residual ß-catenin regulating activity.
At present, the precise molecular mechanism to explain the residual downregulating activity is unclear. All the truncated APC proteins selected for in tumor formation lack the axin-binding domain and will be impaired in downregulating ß-catenin levels by the cytoplasmic APC/axin/GSK3ß destruction machinery. However, it is possible that by retaining one, two, or three 20-amino-acid repeats, increasingly more ß-catenin is displaced from the TCF/LEF transcription complexes (54), sequestered (25), and subsequently exported out of the nucleus (23,27). Truncated proteins retaining three 20-amino-acid repeats, in addition to their residual ß-catenin-binding activity, may also be more effective in exporting ß-catenin out of the nucleus owing to the nuclear export signal encompassed within the third 20-amino-acid repeat (22).
The selection for APC mutant proteins with residual activity strongly supports a model where the complete inactivation of the ß-catenin downregulating activity of APC is less advantageous for tumor formation than the retention of a specific level of residual activity. According to this ‘just-right signaling’ model, regulation of Wnt/ß-catenin signal transduction by APC must be impaired to a specific degree sufficient to allow some accumulation of nuclear ß-catenin. At the molecular level, whereas a low signaling level will mainly modulate the expression of the most accessible and most responsive genes, high doses of nuclear ß-catenin will also modulate the expression of less accessible genes, leading to a broader change in gene expression. This will increase the likelihood of conflicting downstream signals triggering the induction of an apoptotic response thereby preventing tumor formation. In accordance with this, ß-catenin overexpression has been shown to induce cell death when transfected into cell lines (46) and to promote the accumulation of transcriptionally active p53 (53), a well-known inducer of apoptosis. Also, in vivo expression of oncogenic forms of ß-catenin in the intestine of transgenic mice results in a 3–4-fold increase in the number of apoptotic cells (55). Accordingly, APC genotypes lacking all domains involved in ß-catenin regulation are expected to confer the highest level of ß-catenin signaling but are not selected for during colorectal tumor formation in humans.
As described above, the type of somatic mutations is significantly different between patients carrying a germline mutation resulting in either one or two 20-amino-acid repeats (P=0.006, Fisher's exact test), even though the end-result is the same in that most somatic hits remove all the 20-amino-acid repeats. Patients carrying germline mutations around codon 1300 (i.e. retaining one 20-amino-acid repeat) show allelic loss as the most frequent second event, whereas the codon 1436 patient (i.e. retaining two 20-amino-acid repeats) mainly acquires somatic mutations resulting in short truncated proteins lacking all the 20-amino-acid repeats. These truncated proteins, however, have retained the ability to dimerize with the germline mutant protein and may interfere with its function, thus lowering its residual activity to the optimal level for tumor formation (33,56,57).
The selection for an optimal ß-catenin signaling level for tumor formation is also supported by the spectrum of somatic mutations observed in desmoid and gastroduodenal tumors associated with FAP. However, in these tumors, the selection seems to occur for the presence of a truncated protein with two or three 20-amino-acid repeats (42,58–61). When the germline mutation results in a truncated protein that lacks all or retains at most one of the 20-amino-acid repeats, in 19 of 20 cases a selection for somatic point mutations is observed that leave two or three 20-amino-acid repeats. On the other hand, when the germline mutation results in a truncated protein retaining two or three 20-amino-acid repeats, allelic loss is the predominant type of somatic hit (6 of 8 cases). The type and localization of the somatic mutations found in these tumors are significantly different in the two groups (P=0.00005, Fisher's exact test).
Interestingly, in mouse upper-gastrointestinal-tract tumors, deletion of all the 20-amino-acid repeats seems to confer the main selective advantage, which can either be accomplished by allelic loss or a somatic point mutation upstream of all the 20-amino-acid repeats (44,62–64). Thus, our results and those of others indicate that there is an APC-controlled level of ß-catenin signaling optimal for tumor formation, which appears to differ in a tissue- and species-specific fashion.
Recently, it has been shown that the WNT regulating function of APC also depends on its capacity to export ß-catenin from the nucleus to the cytoplasm through different sequence motifs spread along its coding sequence (nuclear export signal, NES), and that loss of the NES motif located in the proximity of the third 20-amino-acid repeat around codon 1510 might confer selective advantage to the nascent tumor cell (22). However, the N-terminal NES sequence that is retained in all tumor-associated truncated APC proteins appears to play a major role in nuclear export function, since mutation of this sequence results in prominent nuclear localization (23–25,27). Selection against the NES co-localizing with the third 20-amino-acid repeat also cannot explain why truncated proteins with only one repeat are more strongly selected in colorectal tumor formation than mutant proteins lacking all repeats, whereas the ‘just-right signaling’ hypothesis can. Thus, selection for APC genotypes retaining one or, less frequently, two 20-amino-acid repeats, as observed in most FAP colorectal tumors, cannot be explained solely by selection against its nuclear export function.
In conclusion, the somatic mutation analysis of polyps from different FAP patients indicates an active selection process during tumor formation in the intestine. This selection is aimed at specific APC genotypes providing a specific level of activation of ß-catenin signaling optimal for tumorigenesis, rather than at the complete loss of the regulatory function of APC within this important signal transduction pathway. This calls for a revisitation of Knudson's ‘two-hit’ model in that, depending on the germline defect, only a specific subset of somatic mutations at the APC tumor suppressor gene will successfully lead to tumor formation in the colon and rectum.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Patients and tumor samples
Six FAP patients were included in this study, of which a total of 133 colorectal adenomas were analyzed. Table 1 shows the germline mutation presented by each patient, the corresponding phenotype and the number of colorectal adenomas analyzed.
Fresh colorectal adenomas were obtained from patients who underwent (procto)colectomy, snap-frozen in liquid nitrogen and stored at -80°C. For one patient, fresh pieces of the colon were collected immediately after surgery and stored in 70% ethanol at 4°C. Sections were cut from each adenoma for DNA isolation and for analysis of histological features. All tumors were tubular or tubulovillous adenomas with mainly low-grade dysplasia.
DNA isolation
DNA from the adenomas was isolated by proteinase K digestion (50 mM Tris–HCl pH 8.0, 100 mM NaCl, 100 mM EDTA, 1% SDS and 300 µg/ml proteinase K) at 56°C overnight, followed by phenol/chloroform extraction and ethanol precipitation.
PTT analysis
Each tumor sample was analyzed for the presence of somatic mutations in APC exon 15 using the protein truncation test (PTT). Primer sequences used to generate PTT fragments were as published elsewhere (28). APC exon 15 was divided into four overlapping fragments (codons 654–1263, 989–1700, 1595–2344 and 2101–2844, respectively). PCR was performed as described in (44). Briefly, a reaction mix was prepared containing 0.01% w/v gelatin, 200 µg/ml BSA, 10 mM Tris–HCl (pH 8.9), 50 mM KCl, 1.5 mM MgCl2, 10% (w/v) glycerol, 200 µM dNTPs, 0.25 units Taq polymerase and 6.25 pmol of each primer in a total PCR volume of 25 µl. In each PCR reaction, 200 ng of DNA was used. PTT was performed as previously described, using a TnT T7-coupled reticulocyte lysate system (Promega, Leiden, the Netherlands) with slight modifications (65).
Cloning and sequencing of mutant alleles
PCR products from tumors showing evidence of truncating mutations were sequenced directly. The position of each mutation was calculated based on the size of the resulting truncated protein. In the cases where the PTT showed a low contribution of the mutant allele, depending on the relative intensity of the truncated protein observed in the PTT, either denaturing gradient gel electrophoresis (DGGE) or cloning was used. In the cases in which the contribution of the mutant allele was considered large enough to allow visualization of the mutant homoduplex or the heteroduplex bands by DGGE, the DNA from the tumor was amplified by PCR, and DGGE was performed for the fragment containing the mutation (66). DNA was picked from the mutant homoduplex band, or alternatively from one of the heteroduplex bands, and directly used for sequencing after confirmation by DGGE that the contribution of the mutant allele was at least 50%.
When the contribution of the mutant allele did not allow visualization of the mutant homoduplex or the heteroduplex bands, the PTT fragment containing the mutation was cloned as previously described (44). DNA from colonies was isolated and submitted to PCR, followed by PTT, to discriminate between colonies containing either wild-type or mutant DNA fragments.
Based on the estimated codon, a primer was chosen to sequence the mutant clone corresponding to the truncated protein previously obtained for each tumor. Sequencing was either performed by ABI technology at the DNA Sequencing Core Unit of the Gene Technology Center at the Leiden University Medical Center or manually, using a Cycle Sequencing method (fmol sequencing kit; Promega, Leiden, the Netherlands).
Analysis of allelic loss of the APC locus
Allelic loss was analysed using two microsatellite markers on chromosome 5q close to the APC locus (D5S346 and D5S404). Each tumor and paired normal DNA were amplified by PCR in a standard PCR buffer (Gibco-BRL, Barcelona, Spain). For the patients in which the germline mutation corresponded to a 4 or 5 bp deletion (germline mutation in codons 168, 1060 and 1309), allelic loss was also analyzed by amplification of the fragment containing the germline mutation. This procedure allowed direct identification of the allelic loss whether in the somatic or in the germline allele. Products were analyzed in a denaturing polyacrylamide gel and visualized by silver staining as previously described (67). For each marker, an allelic ratio was calculated by dividing the intensity of the two alleles in the normal control DNA (DNA isolated from blood). This allelic ratio was compared with the allelic ratio obtained for the two alleles of each tumor sample. A comparative ratio greater than 1.5 was interpreted as significant, i.e. indicative of allelic loss.
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ACKNOWLEDGEMENTS |
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We thank Cátia Marques for her assistance with sequencing of the mutations, Peter de Knijff for his assistance with the statistical analyses, and Menno Kielman and Carli Tops for their helpful suggestions. This project was supported by the Portuguese Ministry of Health(1999), and by grants from the Dutch Cancer Society (98-1652) to R.S. and C.B.
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FOOTNOTES |
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* To whom correspondence should be addressed at: Center for Human and Clinical Genetics, Leiden University Medical Center, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands. Tel: +31 71 5276008; Fax: +31 71 5276075; E-mail: r.smits{at}lumc.nl
Present address: Rob van der Luijt, Department of Medical Genetics, University Medical Center Utrecht, Location WKZ, PO Box 85090, 3508 AB Utrecht, The Netherlands
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REFERENCES |
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