Colorectal Cancers Choosing Sides

Biochimica et Biophysica Acta 1816 (2011) 219–231

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Review Colorectal cancers choosing sides

Cristina Albuquerque a,1, Elvira R.M. Bakker b,2, Wendy van Veelen b,2, Ron Smits b,⁎ a Centro de Investigação de Patobiologia Molecular (CIPM), Instituto Português de Oncologia de Lisboa Francisco Gentil, Rua Prof. Lima Basto 1099-023 Lisboa, Portugal b Department of Gastroenterology and Hepatology, Erasmus MC, University Medical Center, 's Gravendijkwal 230, 3015 CE, Rotterdam, The Netherlands article info abstract

Article history: In contrast to the majority of sporadic colorectal cancer which predominantly occur in the distal colon, most Received 20 April 2011 mismatch repair deficient tumours arise at the proximal side. At present, these regional preferences have not Received in revised form 25 July 2011 been explained properly. Recently, we have screened colorectal tumours for mutations in Wnt-related genes Accepted 28 July 2011 focusing specifically on colorectal location. Combining this analysis with published data, we propose a Available online 10 August 2011 mechanism underlying the side-related preferences of colorectal cancers, based on the specific acquired genetic defects in β-catenin signalling. Keywords: Wnt/β-catenin signalling © 2011 Elsevier B.V. All rights reserved. Colorectal cancer APC Mismatch repair Tumour location Signalling dosage

Contents

1. Introduction ...... 219 2. Wnt/β-catenin signalling defects in colorectal cancer...... 220 3. Level of β-catenin signalling associated with APC and CTNNB1 mutations ...... 221 4. Tissue-speciﬁc selection for β-catenin signalling defects ...... 222 5. Possible alternative explanations for Apc-driven tumour initiation ...... 224 6. Selection for β-catenin dosage along the colorectal tract ...... 225 7. Colorectal cancers choosing sides: tumours having a functional MMR system ...... 226 8. Colorectal cancers choosing sides: mismatch repair deﬁcient tumours ...... 226 9. Concluding remarks ...... 227 Acknowledgements ...... 228 References ...... 228

1. Introduction machinery, which consists of a handful of proteins involved in the recognition and repair of DNA mismatches that occur during Epidemiological data show that 55–70% of all colorectal cancers replication [5,6]. These can be either base substitution mutations develop in the distal half of the colon, i.e. distal to the splenic flexure incorporated during DNA synthesis or, occurring more frequently, (also referred to as “left-sided colon”) [1]. However, colorectal slippage of the DNA polymerase at repetitive sequences resulting in tumours harbouring a DNA mismatch repair (MMR) deficiency form small insertions/deletions of base pairs. In case of a defective MMR an exception as they are predominantly located in the proximal part of system these mistakes are not repaired correctly, resulting in a the colon (also referred to as “right-sided colon”) [2–6] (Fig. 1). About mutator phenotype at the nucleotide level. The MMR genes most 15% of all colorectal tumours are characterised by a defect in the MMR commonly affected in colorectal cancer are MSH2, MLH1, and MSH6. About 2–3% of all colorectal cancers arise in families carrying heterozygous germline mutations in either one of these genes, leading ⁎ Corresponding author. Tel.: +31 10 7035944; fax: +31 10 7032793. to the autosomal dominant disease referred to as Lynch syndrome. E-mail address: [email protected] (R. Smits). fi 1 Tel.: +351 21 7229818; fax: +351 21 7229895. MMR de cient tumours may also arise in a sporadic context, 2 Tel.: +31 10 7033322; fax: +31 10 7032793. corresponding to 10–12% of all colorectal cancers. In contrast to

All colorectal cancers the colon differ in various aspects [7]. For example, whereas the proximal colon originates from the embryonic midgut, the distal colon is formed by the hindgut. Both sides also differ in their bile acid metabolism, water resorption, luminal content, bacterial colonisation, 30-45% 55-70% short-chain fatty acid production, and various other features. None of

proximal these aspects can however easily explain the side preferences of distal colorectal tumours. Here, we present data supporting our hypothesis that these side preferences may in part be explained by speciﬁc acquired genetic defects in β-catenin signalling along the colorectal tract. First, we will review the literature related to β-catenin signalling dosage and its importance in tumour formation.

2. Wnt/β-catenin signalling defects in colorectal cancer MMR deficient colorectal cancers The majority of colorectal tumours, including those with a mismatch repair defect, acquire mutations resulting in aberrant activation of the Wnt/β-catenin signalling pathway [8–10]. This pathway normally 70-80% 20-30% represents one of the main regulatory mechanisms to preserve tissue proximal homeostasis in the adult organism by regulating the balance between distal self-renewal, differentiation and apoptosis in several adult stem cell niches [9].AsdepictedinFig. 2, in the absence of signalling from an extracellular Wnt ligand, β-catenin is degraded by an intracellular multi- protein complex. This so-called destruction complex encompasses two kinases, GSK3 and CKI-α, the Adenomatous Polyposis Coli (APC) tumour suppressor, β-catenin (official gene name CTNNB1), and the scaffold Fig. 1. Distribution of cancers along the colorectal tract. Looking at the distribution of proteins AXIN1 and AXIN2. Formation of this complex catalyses Ser/Thr tumours along the colorectal tract, the majority is identified in the distal colon. phosphorylation of β-catenin at specific N-terminal residues, thereby However, tumours harbouring a mismatch repair (MMR) defect form an exception, as triggering its subsequent ubiquitination and proteolytic degradation. In they are predominantly located in the proximal part of the colon. The colon is divided in proximal and distal side at the splenic flexure here marked by a diagonal line. the presence of the ligand, Wnt molecules bind to the frizzled and LRP5/6 receptors and inhibit the formation of the destruction complex. β-Catenin is now free to accumulate intracellularly and translocate into most colorectal tumours, the ones with a MMR defect usually present the nucleus where it associates with members of the TCF/LEF family of with low levels of chromosomal instability and loss of heterozygosity transcription factors, thus regulating the expression of specificdown- (LOH), are more likely to be diploid, and are preferentially located in stream Wnt target genes and affecting subsequent cellular decisions. the proximal colon [5,6]. In a large number of tumour types enhanced β-catenin signalling At present it is unknown why different types of colorectal cancer strongly contributes to tumour growth, underscoring its importance occur predominantly at one side of the colorectal tract. Both sides of in maintaining tissue homeostasis throughout the organism [11,12].

Fig. 2. Schematic representation of the Wnt/β-catenin signalling pathway. (a) In the absence of an extracellular Wnt ligand, β-catenin is recruited into a destruction complex consisting of APC, Axin, GSK3 and CKI, and marked for degradation by phosphorylation at its N-terminus. The phosphorylated β-catenin binds to β-Trcp triggering ubiquitin- mediated degradation in the proteasome. As a result, no free β-catenin enters the nucleus to form transcriptional complexes with LEF/TCF and activate downstream gene expression. (b) When Wnt ligand binds to the frizzled and LRP5/6 receptors, formation of the destruction complex is inhibited. β-catenin can enter the nucleus and regulate the expression of target genes such as c-Myc. Figure adapted from reference [13] with permission from Lab. Invest., courtesy of Dr. Tong-Chuan He, University of Chicago Medical Center, Chicago, USA. C. Albuquerque et al. / Biochimica et Biophysica Acta 1816 (2011) 219–231 221

Most colorectal cancers acquire “loss of function” mutations in both majority of colorectal tumours, irrespective of the underlying form of copies of the APC gene, resulting in inefficient breakdown of intracellular genetic instability, acquire mutations resulting in aberrant activation of β-catenin and enhanced nuclear signalling. In a subset of tumours, the Wnt/β-catenin signalling pathway. oncogenic β-catenin mutations within exon 3 are observed at N-terminal phosphorylation residues, making the protein more resistant to 3. Level of β-catenin signalling associated with APC and CTNNB1 proteolytic degradation [8,9,11,13]. In both scenarios, the aberrantly mutations stabilised β-catenin constitutively activates downstream Wnt/β-catenin target genes, triggering a genetic programme that initiates tumour The APC gene consists of an open reading frame of over 8500 formation. basepairs encoding a 312 kDa protein (Fig. 3). Several motifs in the Enhanced β-catenin signalling, however, is not only involved in central domain are responsible for regulating intracellular β-catenin tumour initiation but has also been implicated in tumour progression. levels. Four 15 aa repeats bind β-catenin, whereas seven 20 aa motifs are Although the presence of APC or CTNNB1 mutations predicts constitutive involved in both binding and downregulation of β-catenin. Interspersed β-catenin signalling in each tumour cell, heterogeneous patterns of within those 20 aa repeats are three binding sites for AXIN1/AXIN2 nuclear, cytoplasmatic, and membrane-bound β-catenin are observed required for an optimal recruitment of APC into the destruction complex. within colorectal tumours [14]. Patches of cells with high levels of More recently, an additional motif, referred to as the β-catenin inhibitory nuclear β-catenin are associated with tubular branching, invasion, and domain (CID), was identified directly following the second 20 aa repeat, epithelial to mesenchymal transitions [15]. Nuclear β-catenin staining is which appears to contribute to APC's ability to regulate β-catenin also strongly correlated with tumour size and grade of dysplasia, and the signalling [31]. highest levels of nuclear β-catenin are observed at the invasion fronts The vast majority of APC mutations result in truncated proteins that of adenocarcinomas [16,17]. Based on these observations, enhanced lack all AXIN1/AXIN2 binding motifs while retaining between one and β-catenin signalling has been implicated in the induction of an invasive three 20 aa repeats associated with the down-regulation of intracellular and metastatic phenotype of colorectal tumours [18,19]. β-catenin levels. Originally, all mutations within the APC gene were Up to 85% of colorectal cancers with a functional MMR system are considered to fully impair its β-catenin downregulating activity. characterised by APC mutations, whereas CTNNB1 mutations in this However, subsequent studies have shown that most truncated proteins subset of tumours are rare [20,21]. In colorectal tumours with a MMR observed in tumours, have retained residual activity in regulating defect, APC mutations are detected in about 50% of the cases, whereas β-catenin signalling, which turns out to be of great importance for the rate of CTNNB1 mutations is 10% in sporadic MMR deficient tumours successful tumour formation. Truncated proteins with two or three 20 and reaches 30% in Lynch syndrome associated colorectal cancers aa repeats inhibit signalling almost as efficiently as full-length APC when [10,20,22–26].Inasignificant proportion of MMR deficient tumours, overexpressed in vitro [31–35]. Moreover, even when expressed at mutations have also been reported in the Wnt-related AXIN1, AXIN2,and endogenous levels, a truncated Apc protein encompassing three 20 aa TCF7L2 (also known as TCF4) genes, although the relevance of these repeats shows considerably more β-catenin downregulating activity mutations for tumour growth is unclear as they can coincide with than cells only expressing truncated proteins where all β-catenin mutations in either APC or CTNNB1 [10,27–30]. These data show that the regulating domains are lacking [36–38]. Additionally, a truncated APC

MCR of colorectal tumours MCR of desmoid and upper GI tumours 2843

146 82 65 codons

Codon Arbitrary signalling boundaries strength to nucleus

3 1513 - 1577 +

2 1431 - 1512 ++

1 1285 - 1430 +++

0 < 1285 ++++

β-catenin binding (15 aa repeat) Microtubule binding Oligomerization β-catenin downregulation (20 aa repeat) EB1/RP1 binding Armadillo repeat β-Catenin Inhibitory Domain (CID) DLG/PTP-BL binding Axin/Conductin binding

Fig. 3. APC truncations and residual β-catenin regulating activity. Compiling various studies [31–38], we have subdivided the APC truncations observed in tumours in four categories, based on the arbitrary β-catenin signalling level associated with their residual β-catenin regulating activity. The main determining factor is the number of 20 aa repeats depicted on the left. Truncated proteins with 2 repeats have to include the CID domain to acquire additional regulating activity. Codon boundaries are mentioned for each category. The subdivision shown here is used throughout the paper. Truncated proteins with 1, 2, or 3 repeats are speciﬁcally encoded by resp., 146, 82, and 65 codons. On top of the full length APC protein we have demarcated the mutation cluster regions (MCR) for APC mutations observed in colorectal, desmoid and upper GI tumours. 222 C. Albuquerque et al. / Biochimica et Biophysica Acta 1816 (2011) 219–231

811 8

1278 4 1367 1303 1338 1444 2 1417 1429

Relative luciferase activity 1555 1555

0 116

HT55 HT29

DLD1 GP5D

LS180

SW948

SW480 SW403

CACO2

LS174T HCT

COLO320 COLO205

0 0 1 1 1 1 “2” 2 3 3

Number of remaining APC repeats β-catenin mutant

Fig. 4. Using a β-catenin reporter assay, Rosin-Arbesfeld et al. showed that colorectal cancer cell lines expressing oncogenic variants of β-catenin, induce reporter activity to the same extent as cell lines expressing truncated APC proteins retaining three 20 aa repeats. For the other cell lines, the inverse correlation between the β-catenin signalling strength and the number of 20 aa repeats remaining in the truncated APC protein can be seen. Figure adapted from reference [44] with permission from EMBO J, courtesy of Dr. Mariann Bienz, MRC Laboratory of Molecular Biology, Cambridge, UK. protein with only one repeat is still capable of exporting β-catenin out of mutations, which are subsequently selected upon the growth advantage the nucleus, and may therefore reduce β-catenin signalling activity [39– they provide to the tumour cell. Mutations that do not provide the cell 41]. Thus, although nearly all of the truncated APC proteins observed in with sufficient growth advantage will not allow clonal expansion and colorectal tumorigenesis are impaired in their β-catenin downregulat- tumour formation. On the other hand, mutations which have too much ing activity, they do not represent null alleles and code for some residual impact on the cellular phenotype will not be compatible with cell β-catenin regulating activity. survival and lead to cell death. In both cases, these mutations will not Compiling the results of the above-mentioned studies, we have contribute to the somatic mutation spectrum that is finally observed in categorised the mutant APC proteins in groups based on their residual tumours. The Wnt/β-catenin signalling pathway represents a prime activity (Fig. 3). An inverse correlation is observed between the number of example for this concept, as we and others have shown that during 20 aa repeats and the resulting level of β-catenin signalling, i.e. more tumour initiation specific APC genotypes are selected on the basis of the repeats means a lower β-catenin signalling level to the nucleus. As the specificlevelofresidualβ-catenin down-regulating activity of the second 20 aa repeat by itself cannot bind β-catenin efficiently [42], resulting truncated proteins, rather than on the complete inactivation of truncated APC proteins with two repeats require the CID domain directly this signal transduction regulatory function of APC [45–51]. According following this repeat, in order to retain additional breakdown activity [31]. to this “just-right” signalling model, a specificdegreeofAPC Therefore, truncated APC proteins including two 20 aa repeats are at least impairment is required to allow sufficient accumulation of nuclear 1430 residues in length to give them a distinctive increased β-catenin β-catenin and activation of the downstream target genes relevant for regulating activity compared with truncated proteins with one repeat. The tumour formation [48].However,cellscharacterisedbyβ-catenin codon boundaries shown in Fig. 3 are used throughout the paper. signalling levels above a given threshold undergo apoptosis and hence How do oncogenic CTNNB1 mutations fit within this signalling will not contribute to tumour formation. At the molecular level, low spectrum? In most cases only one CTNNB1 allele is affected, meaning levels of β-catenin signalling mainly modulate the expression of the that only half the β-catenin produced is resistant to degradation, most accessible and responsive genes, whereas high doses of nuclear whereas the entire β-catenin signalling pool is affected in case of a full- β-catenin will also affect the expression of less responsive ones [37]. blown APC inactivation. Furthermore, Wang et al. have shown that Activation of an apoptotic response requires higher levels of β-catenin phosphorylation of β-catenin at S33/S37/T41 still occurs efficiently in signalling [52–54]. Similar results have been reported for the proto- the presence of a mutant S45 residue, suggesting that one of the oncogene c-Myc, one of the main target genes activated by the prevailing mutant forms of β-catenin in CRC is not fully resistant to Wnt/β-catenin signalling pathway [55], and required for the majority proteolytic degradation [43]. Accordingly, as depicted in Fig. 4, of Wnt/β-catenin target gene activation following Apc loss [56].A measuring the signalling activity in various colorectal cell lines tissue-specific increase in proliferation was observed in a transgenic harbouring specific APC or CTNNB1 mutations, showed that oncogenic mouse model expressing c-Myc at modest elevated levels, whereas the CTNNB1 mutations confer a low induction of a β-catenin reporter expression of pro-apoptotic genes was robustly induced when c-Myc was construct, similar to cell lines expressing truncated APC proteins expressed at high levels [57]. Thus, eliminating cells with too much retaining three 20 aa repeats [44]. As such, these observations suggest signalling of proto-oncogenes such as β-catenin or c-Myc, appears to be a that oncogenic CTNNB1 mutations result in a modest activation of the mechanism to safeguard against tumour formation. This mechanism also β-catenin signalling pathway. applies to APC-driven tumour formation. The APC gene is a large gene, subject to a variety of mutations that 4. Tissue-specific selection for β-catenin signalling defects could theoretically impair its function. Nevertheless, the mutations observed in colorectal as well as other tumour types converge to a Tumour formation is considered an evolutionary process where small region termed the mutation cluster region (MCR), coinciding clonal expansion occurs through spontaneously acquired somatic with the region in between the first 20 aa repeat and the AXIN binding C. Albuquerque et al. / Biochimica et Biophysica Acta 1816 (2011) 219–231 223 domain (Fig. 3) [11]. Different APC genotypes have been observed in carrying a germline mutation resulting in a truncated APC protein different tumour types throughout the body, suggesting that tissue- retaining one 20 aa repeat are associated with the most severe form of specific dosages of β-catenin signalling are selected to efficiently polyposis, whereas patients carrying mutations resulting in truncated trigger tumorigenesis. In about 80% of colorectal tumours with APC proteins retaining two or three 20 aa repeats are at highest risk for the mutations, at least one of the mutated alleles code for truncated APC formation of desmoid tumours and polyps in the upper GI-tract, in proteins retaining one or, less frequently, two 20 aa repeats, resulting addition to a reduced risk for severe colorectal polyposis [65,66]. in relatively high to moderate β-catenin signalling levels [31,45,47– Even more dramatic differences in tumour phenotype are observed 49]. Colorectal tumours solely expressing truncated APC proteins in Apc mutant mouse models generated by us and others [67–69] lacking all 20 aa repeats or expressing longer truncated proteins, are (Fig. 5). Mice with Apc mutations resulting in high β-catenin signalling less frequently observed. According to the “just-right” signalling levels, such as ApcMin and ApcΔ716, mainly develop intestinal tumours hypothesis, this can be explained by assuming that these truncated at high multiplicity (N100) with only a low penetrance of extra- APC proteins do not provide the optimal level of β-catenin signalling intestinal manifestations. On the other hand, age-matched animals to efficiently initiate colorectal tumour growth. carrying the hypomorphic Apc1638N mutation resulting in intermedi- Desmoid tumours (also named aggressive fibromatosis) are rare ate β-catenin signalling, are characterised by a reduced incidence of mesenchymal tumours likely selecting for low to moderate β-catenin intestinal tumours (on average 5–6) combined with a high susceptibility signalling defects. This is either accomplished by acquiring truncated for several extra-intestinal tumour types such as cutaneous cysts and APC proteins retaining two or three 20 aa repeats (~5–10% of cases), desmoid tumours [70]. More recently, the Apc1572T mouse model, or more frequently an oncogenic CTNNB1 mutation (~80%) [58–60]. characterised by a mild defect in β-catenin regulation, has been shown These truncated APC proteins with 2–3 repeats, or oncogenic CTNNB1 to develop aggressive and metastasising mammary tumours associated mutations, are also the most frequently observed alterations in polyps with nuclear accumulation of β-catenin [38], and a histological arising in the gastric and duodenal epithelium [61–64]. The spectrum appearance similar to the mammary tumours observed in transgenic of mutations in the β-catenin signalling pathway observed in these models overexpressing Wnt1 or β-catenin [71–74]. Surprisingly, tumour types, is consistent with our hypothesis that oncogenic Apc1572T mice do not develop intestinal tumours, even when followed CTNNB1 mutations provide an equivalent induction of β-catenin up to old age, suggesting that the mild induction of β-catenin signalling signalling as truncated APC proteins retaining 2–3 repeats (Fig. 4). is not sufficient to induce tumour formation in the mouse intestine [38]. The selection for specificdosagesofβ-catenin signalling is corrobo- Further support for a dominant role of β-catenin signalling dosages in rated by the phenotypes of familial adenomatous polyposis (FAP) establishing these different tumour phenotypes was provided by patients carrying specific germline mutations in the APC gene. Patients Buchert et al. [50]. By combining different Apc mutant models with a

a Structure of APC Apc mouse β-catenin signal Tumour burden Extra-intestinal gene product model following LOH in GI tract Tumours

+/ 716 +++ >100 ?

occasional +/Min +++ >100 cyst or desmoid

multiple (1-2%) +/1638N ++ 5-6 cysts and desmoids

+/1572T + 0 cysts and desmoids mammary tumours

+/1638T - 0 none

b signalling window for signalling window for signalling window for intestinal tumours cysts and desmoids mammary tumours

50 50 50 40 40 40

30 30 30

20 20 20

10 10 10

-catenin reporter activity

β 0 0 0 Wt Min Wt Min Wt Min 1638T 1572T 1638N 1638T 1572T 1638N 1638T 1572T 1638N

Fig. 5. Genotype–phenotype correlations of Apc mutant mouse models. (a) For each model the truncated Apc proteins encoded by the mutation are depicted, as well as the expected β-catenin signalling strength emerging in a nascent tumour cell following LOH of the wild type Apc allele. LOH is by far the most common spontaneous second hit in mouse models [75,76]. Please note that Apc1638N and Apc1638T truncated proteins are virtually identical as far as the position of the termination codon is concerned. However, whereas Apc1638T is present in a 1:1 ratio with wild-type Apc, in Apc1638N only ‘leaky’ amounts (1–2%) of the predicted protein are generated. Tumour multiplicity in the gastro-intestinal tract and elsewhere are indicated. Extra-intestinal tumour types have not been reported for ApcΔ716. (b) Windows of β-catenin signalling associated with specific tumour types in Apc mutant mouse models. Intestinal tumours are only observed in Apc models associated with high to moderate levels of β-catenin signalling. Desmoid tumours and cutaneous cysts prefer lower levels of signalling, while mammary tumours readily occur with the low signalling level observed in Apc1572T. The level of β-catenin signalling is derived from β-catenin reporter assays performed on embryonic stem cells homozygous for each mutation [36–38]. 224 C. Albuquerque et al. / Biochimica et Biophysica Acta 1816 (2011) 219–231 heterozygous knock-out of Ctnnb1 resulting in reduced β-catenin cell type, while simultaneously avoiding the induction of apoptosis, signalling, they observed specific β-catenin signalling thresholds for will successfully induce tumour formation. successful intestinal and liver tumour formation. How are those phenotypic differences explained in more detail? APC 5. Possible alternative explanations for Apc-driven tumour is a tumour suppressor protein, meaning that both copies have to initiation acquire a mutation before tumour formation ensues. All cells in the body of FAP patients and Apc mutant mouse models carry the same Although the concept of β-catenin signalling dosage and its impact heterozygous APC/Apc germline mutation in which the remaining wild on tumour growth is gaining acceptance, alternative explanations for type copy will be randomly hit by a number of somatic mutational APC-driven tumour formation have been presented. The APC gene mechanisms including allelic loss, insertions, deletions, and single-base encodes a large multifunctional protein implicated in various other substitutions. Of these, allelic loss by means of mitotic recombination cellular processes in addition to regulating β-catenin signalling. At its occurs at the highest spontaneous frequency, especially in the mouse C-terminus, APC binds to components of the microtubular skeleton. (more than 90%) [75–77]. Cells acquiring allelic loss through this Loss of these domains has been implicated in defects in cell migration mechanism will solely express the truncated protein originally encoded [84] and chromosomal segregation [85,86], both aspects of relevance by the germline mutation, and hence the β-catenin signalling strength for tumorigenesis. However, the Apc1638T mouse model expressing a associated with that mutation (Fig. 5). In case of the Apc1572T model, truncated Apc protein retaining normal β-catenin regulation but this implies that in the majority of emerging tumour cells, β-catenin lacking these C-terminal domains is tumour-free, showing that loss of signalling is mildly activated. According to the “just-right” signalling these domains by itself is not sufficient to induce tumour formation model this level is ideal for outgrowth of the mammary tumour, but not and requires simultaneous activation of β-catenin signalling [36]. sufficient for intestinal tumour development in the mouse. Likewise, Nevertheless, the debate is still open to what extent loss of these most nascent tumour cells in the ApcMin and ApcΔ716 models acquire microtubular functions contributes to tumour progression. high levels of β-catenin signalling, ideal for the intestine but too high for More recently, the β-catenin dogma has been challenged by Phelps mammary cells. Further support for the strong selective pressure on et al. who presented data suggesting that Apc-driven tumour formation acquiring specific Apc mutations, was provided by mutation analysis of is not initiated by nuclear accumulation of β-catenin, but instead mammary tumours developing in an Apc model associated with high depends on the transcriptional corepressor C-terminal binding protein- levels of signalling [78]. These tumours select for specificsomaticpoint 1(Ctbp1)[87]. Ctbp1 physically interacts with Apc and its levels appear mutations clustering in the codon 1521–1570 region, and are expected to increase upon Apc loss in early adenomas [87,88]. As such, changes in to result in a low level of signalling similar to the truncated protein Ctbp1 activity may represent a plausible alternative explanation for expressed by the Apc1572T model. tumour initiation. The main argument against a role for β-catenin put Importantly, oncogenic CTNNB1 mutations are also selected during forward by Phelps et al., was the lack of its nuclear detection in early tumour formation. Although the S33, S37, and S45 residues are all adenomas using immunofluorescence. As discussed by Fodde and specified by the same TCT codon within the human CTNNB1 gene, Tomlinson, while nuclear staining for β-catenin is a reliable indicator several tumour types show a strong preference for mutations in either of Wnt activation, its absence does not exclude the robust activation one of these residues. For example, whereas colorectal cancers mainly of β-catenin target genes [89]. Detection of nuclear β-catenin is more showT41andS45mutations[10,11,23,25–27,79–81] (Fig. 6), challenging than generally appreciated. For example, nuclear β-catenin tumours of the endometrium, ovarium, and pilomatricomas (of hair is not detectable in frozen colorectal tumour sections [90],isnoteasyto follicle origin) select for mutations confined to residues S33 and S37 discern using immunofluorescence on paraffin sections while this is the [11,24,58,59,82]. Overexpression experiments suggest that different case using immunoperoxidase-based methods (unpublished observa- oncogenic CTNNB1 mutations result in different levels of downstream tions), and is absent in most colorectal cancer cell lines despite strong signalling [83], likely explaining the preference for specific mutated evidence of enhanced Wnt signalling by using more sensitive approaches residues providing the optimal growth advantage for each cell type. such as β-catenin reporter assays and target gene activation. Using In summary, not all mutations in APC or CTNNB1 result in tumour immunoperoxidase-based methods, most investigators detect nuclear formation. According to the “just-right” signalling hypothesis, only accumulation of β-catenin in early adenomas [89,91]. Moreover, whereas those mutations that provide sufficient growth advantage for a certain oncogenic CTNNB1 mutations have been detected in a large number of

Fig. 6. Selection of CTNNB1 mutations in colorectal tumours. Depicted is the type and location of CTNNB1 mutations reported for colorectal tumours. N-terminal serine and threonine phosphorylation residues are indicated bold. Numbers in brackets are absolute number of tumours reported with given mutation. Although the S33, S37, and S45 residues are all speciﬁed by the same TCT codon within the human CTNNB1 gene, colorectal tumours mainly acquire mutations of S45 (33/62) or T41 (21/62), strongly suggesting that speciﬁc CTNNB1 mutations are selected during tumour formation. C. Albuquerque et al. / Biochimica et Biophysica Acta 1816 (2011) 219–231 225

Table 1 6. Selection for β-catenin dosage along the colorectal tract Distribution of APC mutations within the proximal and distal colon. APC mutations were grouped according to the number of 20 aa repeats present in the longest reported APC So far, we have for simplicity considered the colorectum as one mutation present in that tumour, as this will largely determine the residual β-catenin fi regulating activity. Divisions were made according to the codon boundaries shown in uniform organ with an overall equal selection for a speci c level of Fig. 3. Studies were only included when tumour location was properly recorded, and β-catenin signalling, favouring truncated APC proteins with one or, when the mutation analysis minimally included the entire MCR of APC up to the first less frequently, two 20 aa repeats. However, regional differences in AXIN binding domain. Furthermore, tumours with an underlying MMR defect were β-catenin signalling preference affecting tumour formation also exist excluded. A graphical representation of these data is depicted below the table. Differences in the number of repeats between both groups are highly significant along the GI-tract. Recently, we have screened a number of colorectal (pb0.001; ordinal logistic regression model fitted with SAS version 9.2, proc glimmix). tumours for mutations in Wnt-related genes with a specific focus on colorectal location, and combined these results with a meta-analysis Number of 20 aa Proximal tumours Distal tumours repeats (n=36) (n=102) of publications in which tumour location, defects in mismatch repair, and somatic mutations were reported [10]. Focussing on APC 01230123 mutations in tumours with a functional MMR machinery, we noticed Aoki et al. [93] 00100100 a different mutation spectrum between both sides of the colon (see Smith et al. [94] 0651329132 Table 1) [10,47,93–98]. Distal colonic tumours preferentially acquire Miyaki et al. [95] 02 3 1 Aust et al. [96] 01200900 mutations retaining one 20 aa repeat (63/102 tumours, i.e. 62%), Rowan et al. [47] 01132340 while long truncated APC proteins with two or three 20 aa repeats are Scholtka et al. [97] 03120832 less common in this segment of the colon (27/102 tumours, i.e. 27%). Ku et al. [98] 00010410 On the other hand, the latter mutations resulting in a moderate Albuquerque et al. [10] 00207920 signalling level are selected in tumours arising in the proximal colon 0 13 15 8 12 63 23 4 (23/36 tumours, i.e. 64%). Recently, our observation was supported by 60 data of Leedham et al. [99].AnalysingAPC mutations in proximal and Proximal distal polyps of FAP patients and a set of sporadic CRCs, the same 50 Distal prevalence for specific APC mutations along the colorectal tract was observed [99]. Proximal tumours also present with less nuclear 40 accumulation of β-catenin than their distal counterparts [100].Overall, 30 these results suggest that a gradient of β-catenin signalling dosage for tumour initiation exists along the colorectal tract with moderate levels 20

% of tumours being preferred proximally, and higher levels towards the rectum 10 (Fig. 7). This phenomenon is also observed for the location of intestinal 0 tumours in Apc mutant mouse models. Apc mutant mice to a large 0 123 Number of 20 a.a. repeats remaining extent develop tumours located in the small intestine, and select for higher signalling levels associated with short truncated proteins lacking all 20 aa domains [46,78,101]. Despite these differences between human and mouse intestinal tumour formation, clear effects on tumour location associated with β-catenin signalling levels have tumour types and expression of oncogenic β-catenin leads to the been observed. The Apc1638N model, characterised by only a development of numerous tumours in the mouse intestine [92],similar moderate increase in β-catenin signalling, develops intestinal tumours results have not yet been reported for Ctbp1. almost exclusively in the duodenum and first half of the jejunum with To date, the C-terminal microtubular functions and Ctbp1 have not a characteristic clustering at the stomach to duodenum transition been linked to the selection for specific truncated APC proteins retaining [102–104]. A proximal preference for tumour formation has also been between one and three 20 aa repeats that is observed in tumours. reported for the Apc1322T mouse model associated with a submaximal Therefore, we believe that selection for β-catenin signalling dosage level of β-catenin signalling [105]. In contrast, tumours developing in currently represents the best explanation for the tissue-specific the ApcMin and ApcΔ716 models, both resulting in a high dosage of selection of truncated APC proteins. β-catenin signalling, are preferentially selected in the distal half of the

Preferred β-catenin +++ signalling dosage ++/ +++/

APC retaining proximal APC retaining 1 Observed APC distal 2 or 3 or less often 0 or 2 mutation 20 aa repeats 20 aa repeats

Fig. 7. Selection of APC mutations and preferred β-catenin signalling strength along the colon. Focussing on tumours with a functional MMR machinery, we noticed that most tumours in the proximal colon are characterised by APC mutations resulting in a truncated protein retaining two or three 20 aa repeats associated with mild (+) to moderate (++) levels of β-catenin signalling [10,47,93–98]. This strongly suggests that the proximal colon for successful tumour initiation, prefers lower β-catenin signalling levels than the distal side. These mainly develop with APC truncated proteins retaining one or less frequently no or two repeats, resulting in a moderate (++) to high (+++) β-catenin signalling level. See Table 1 for details. 226 C. Albuquerque et al. / Biochimica et Biophysica Acta 1816 (2011) 219–231 small intestine [101,104–106]. Apc mutant models associated with with higher basal levels of β-catenin signalling [99].Basedonthis high levels of β-catenin signalling also develop some colonic tumours, observation, they postulated that this might explain the selection for especially when expression of the mutant allele is restricted to the mutations leading to a mild increase in β-catenin signalling on the distal part of the GI-tract using Cre–LoxP technology [107–109]. proximal side, as a lower increase in signalling is required to reach a Interestingly, these tumours almost exclusively develop in the distal hypothetical threshold for tumour initiation. part of the colon. Thus, both in the colon as well as the small intestine of the mouse, the proximal region seems to prefer lower levels of β-catenin 7. Colorectal cancers choosing sides: tumours having a functional signalling to initiate tumour growth than its distal counterpart. MMR system At present, the mechanisms underlying these side preferences for the optimal level of β-catenin signalling remain largely elusive. Although As mentioned previously, tumours characterised by a deficient MMR β-catenin signalling in the intestine is an important determinant in system are more frequently observed at the proximal side of the colon, establishing cell fate, it will operate in concerted action with many whereas most others occur at the distal side. We propose that these side other cellular inputs. Several differences exist between the proximal preferences may in part be explained by the specific acquired genetic and distal colon that may cooperate with the defect in β-catenin defects in β-catenin signalling along the colorectal tract. As outlined signalling to successfully initiate tumour growth [7].Forexample, below, MMR deficient tumours more frequently acquire mutations in short-chain fatty acids such as butyrate, are produced at considerably APC or CTNNB1 that are ideal for the proximal colon, whereas the ones higher levels by bacterial fermentation reactions in the proximal with a functional MMR more often acquire APC mutations optimal for colon [110]. In vitro, these fatty acids have been shown to induce an the distal colon. As depicted in Table 2 (left section), mutations leading apoptotic response when administered to colorectal cancer cell lines to truncated APC proteins retaining one 20 aa repeat are the most [111], suggesting that the higher fatty acid levels present in the commonly observed mutations in tumours with a functional MMR proximal colon reduces the apoptotic threshold, which in turn may system (181/345, i.e. 53%) [10,20,22,47,94,95,97,98,116]. In part, the lead to a preference for a moderate activation of β-catenin signalling high frequency of these mutations is likely to be explained by the fact to initiate tumour growth. A similar line of reasoning can be postulated that the region between the first and second repeat contains an for bile acids of which various structural variants also differ in their (AAAAG)2 direct repeat around codon 1309 that is prone to a deletion of concentrations along the colon [112,113]. Moreover, both sides of the 5 bps, representing one of the most common somatic APC mutations colon differ significantly in their gene expression profiles [114].For [117]. Secondly, the APC sequence between the first and second repeat example, CDX2 is expressed at higher levels in the proximal colon [115]. codes for 146 amino acid residues, which is approximately twice the Interestingly, Aoki et al. have shown that a 50% reduction in Cdx2 levels number of codons between repeats two and three and between repeat results in a strong shift from mainly small intestinal tumours towards a three and the first AXIN binding motif, 82 and 65 codons respectively large intestinal tumour phenotype in ApcΔ716 mice [106].Withrespect (Fig. 3). Thus, based on sequence length, the region in between the first to β-catenin signalling, Leedham et al. presented data suggesting that and second repeat is expected to acquire half of the mutations occurring the proximal small intestine as well as the proximal colon present within the mutation cluster region. Overall, in colorectal cells with a functional MMR system this leads to the generation of tumour cells preferentially expressing truncated APC proteins with one repeat, which Table 2 β MMR deficient tumours acquire significantly more often truncated APC proteins provides the ideal -catenin signalling level for the distal colon. retaining two or three repeats than their MMR proficient counterparts, which mainly According to the “just-right” signalling hypothesis, the same type of develop with truncated proteins retaining one such repeat. Tumours were only mutation is believed to induce an apoptotic response resulting from the included if the MMR status was clear. Tumours were included irrespective of location. high level of β-catenin signalling when occurring in the proximal colon, Differences in number of repeats between both groups are highly significant thereby preventing tumour initiation in this part of the colon. Therefore, (pb0.0001; ordinal logistic regression model fitted with SAS version 9.2, proc glimmix). we propose that the higher chance of obtaining a mutation leading to a Number of 20 aa Functional mismatch Defective mismatch truncated APC protein retaining one 20 aa repeat, shifts the balance of repeats repair (n=345) repair (n=63) tumours towards the distal colon. 0 1 2 3 012 3

Huang et al. [22] 13 24 13 5 6 4 14 2 8. Colorectal cancers choosing sides: mismatch repair deficient Konishi et al. [122] 012 1 tumours Olschwang et al. [116] 21 25 8 4 0 0 1 0 Rowan et al. [47] 2 9 7 4 013 2 In MMR deficient intestinal cells the predominant mutational Lovig et al. [20] 14 62 17 7 1 0 2 4 Smith et al. [94] 335183 mechanism consists of mutations at the nucleotide level. Overall, a Miyaki et al. [95] 02 31 defective MMR system leads to a 10–30 fold higher induction of single Domingo et al. [123] 015 3 basepair substitutions [118–120]. However, sequences most prone to Scholtka et al. [97] 0 114 4 000 1 mutations are the ones containing stretches of mono- or dinucleotide Ku et al. [98] 0 4 1 1 000 3 Albuquerque et al. [10] 7 9 4 0 006 0 repeats, of which the mutation frequency can be increased up to 100- fold, depending on the length of the repeat. As such, genes containing 60 181 75 29 7 7 33 16 these repetitive sequences in their reading frame will be most prone 60 to mutation in a MMR deficient background [5,6,121]. The APC gene contains several of these repetitive sequence features, which are 50 Functional MMR indeed more frequently mutated in MMR deficient tumours [20,22].In Defective MMR 40 Fig. 8 we have highlighted in red the sequences prone to mutation in a MMR deficient background within the region harbouring the first 30 three 20 aa repeats. As depicted in Table 3, mutations in these 20 repetitive sequences, i.e. codons 1455 (A5), 1462 (AG5) or 1554 (A6),

% of tumours are observed in about 37% (23/63) of MMR deﬁcient colorectal 10 tumours carrying APC mutations, whereas these mutations are observed in less than 5% (16/345) of tumours with a functional 0 0 123 MMR system [10,20,22,47,95,97,98,116,122,123]. Mutations at one of Number of 20 a.a. repeats remaining these hotspots lead to the generation of a truncated APC protein C. Albuquerque et al. / Biochimica et Biophysica Acta 1816 (2011) 219–231 227

Fig. 8. Expected hotspots for mutation within APC in MMR deficient tumours. The APC gene contains several mono- and dinucleotide stretches that are prone to mutation in a MMR deficient background. Here, we have highlighted in red the mononucleotide stretches of 5 or more of the same kind as well as the dinucleotide repeats with at least 4 repeats, in the region between the first 20 aa repeat and the first AXIN binding domain. Mutations at one of these hotspots lead to the generation of a truncated APC protein retaining two or three 20 aa repeats. Colour codes for structural domains are as in Fig. 3. The (AAAAG)2 direct repeat around codon 1309, often mutated in MMR proficient CRCs, is underlined. retaining two or three 20 aa repeats. The high chance of acquiring inactivating mutations in the APC gene following the second and third mutations at one of these hotspots superimposed on the normal repeat, or oncogenic CTNNB1 mutations. Both types of mutations are mutation spectrum, likely explains the high proportion of MMR associated with a low to moderate activation of β-catenin signalling. deficient tumours (49/63 tumours, i.e. 78%) that obtain truncated APC According to the “just-right” signalling model, this provides the ideal proteins retaining two or three repeats (see Table 2), which is level for successful tumour initiation in the proximal colon, whereas it considerably higher than observed in tumours with a functional MMR will not supply sufficient growth advantage in the distal colon. As a system (104/345 tumours, i.e. 30%). result, MMR deficient cells are more successful in forming a detectable In about 50% of MMR deficient tumours APC mutations are tumour on the proximal side. observed [10,20,22–26]. An additional 10% obtain oncogenic CTNNB1 mutations, resulting in a moderate signalling activity equivalent to the 9. Concluding remarks truncated APC proteins with 2–3 repeats. As such, these mutations are expected to be selected in the proximal colon, which is in accordance We propose that the side preference of colorectal tumours may in with over 80% (47/58) of tumours carrying CTNNB1 mutations being part be explained by their particular mutational mechanism, resulting detected on this side [10,25–27,80,81]. Recently, we have obtained in APC and CTNNB1 mutations leading to a specific level of β-catenin additional evidence for this observation by screening 54 MMR signalling that is ideal for either side of the colon. Obviously, our deficient tumours for CTNNB1 mutations. We confirmed that these hypothesis only applies to colorectal tumours initiated by enhanced mutations occur more frequently in proximal tumours (15/36, i.e. β-catenin signalling, which is the case for the great majority of 42%) than in their distal counterparts (1/18, i.e. 6%), irrespective of tumours with a functional MMR system. However, among the MMR sporadic or familial origin (pb0.01, Chi-square test, unpublished deficient tumours, a mutation in CTNNB1 or APC has not been observations). reported in about 40% of the tumours. There are several potential In summary, due to the underlying mutational mechanism under explanations for this apparent lower involvement of the β-catenin MMR deficient conditions, a significant proportion of cells acquire either signalling pathway in MMR deficient tumours. The first one is of a 228 C. Albuquerque et al. / Biochimica et Biophysica Acta 1816 (2011) 219–231

Table 3 In our manuscript, we have highlighted the importance of β-catenin The potential mutational hotspots, i.e. codons 1455, 1462, and 1554, marked in Fig. 8, signalling dosages for the tumorigenic process, and presented data are mutated in about 37% (23/63) of MMR deficient colorectal tumours carrying APC supporting our hypothesis that selection for specific dosages along the mutations, whereas this is only the case in less than 5% (16/345) of tumours with a functional MMR system. These differences are highly significant (Fisher exact test; colorectal tract may in part explain the side preferences of colorectal *pb0.01; **pb0.001). tumours. It shows that we should not perceive mutations in signalling pathways merely as on/off switches, but that we should appreciate the Codons Functional mismatch Defective mismatch repair (n=345) repair (n=63) nuances of signalling. The dosage-dependence as described here for β-catenin signalling will also be of relevance for other signalling 1455 1462 1554 1455 1462 1554 pathways commonly involved in cancer development. Huang et al. [22] 011212 Konishi et al. [122] 011 Olschwang et al. [116] 001000 Acknowledgements Rowan et al. [47] 004001 Lovig et al. [20] 006013 Miyaki et al. [95] 000 We are grateful to Dr. Gerard Borsboom for his help with the Domingo et al. [123] 222 statistical evaluations and to Dr. Riccardo Fodde and Dr. Peter Scholtka et al. [97] 011001 Hohenstein for their critical comments. We thank Dr. Kazuhisa Ku et al. [98] 001003 Albuquerque et al. [10] 000100 Shitoh for sharing unpublished information about tumour location of β-catenin mutant colorectal tumours. This study was financially 02145513 supported by grants from the Netherlands Organization for Scientific Research (NWO/Vidi 917.56.353) and the Dutch Cancer Society 35 Functional MMR (DDHK20053299). 30 Defective MMR Final note 25 Following acceptance of our manuscript, the DDW 2011 confer- ence abstract of Leedham et al. quoted here as reference no. 99, has 20 been submitted as a full paper (personal communication). In this paper 15 they describe the existence of a physiological Wnt signalling gradient

% of tumours 10 along the intestinal tract. Based on this observation, they indepen- dently propose a mechanism for the side preferences of colorectal 5 tumours, similar to ours. 0 14551462 1554 total hotspots Mutated codon References

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