Cited1 Deficiency Suppresses Intestinal Tumorigenesis

Conditional deletion of Apc in the murine intestine alters crypt-villus architecture and function. This process is accompanied by multiple changes in gene expression, including upregulation of Cited1, whose role in colorectal carcinogenesis is unknown. Here we explore the relevance of Cited1 to intestinal tumorigenesis. We crossed Cited1 null mice with ApcMin/+ and AhCre+Apcfl/fl mice and determined the impact of Cited1 deficiency on tumour growth/initiation including tumour multiplicity, cell proliferation, apoptosis and the transcriptome. We show that Cited1 is up-regulated in both human and murine tumours, and that constitutive deficiency of Cited1 increases survival in ApcMin/+ mice from 230.5 to 515 days. However, paradoxically, Cited1 deficiency accentuated nearly all aspects of the immediate phenotype 4 days after conditional deletion of Apc, including an increase in cell death and enhanced perturbation of differentiation, including of the stem cell compartment. Transcriptome analysis revealed multiple pathway changes, including p53, PI3K and Wnt. The activation of Wnt through Cited1 deficiency correlated with increased transcription of β-catenin and increased levels of dephosphorylated β-catenin. Hence, immediately following deletion of Apc, Cited1 normally restrains the Wnt pathway at the level of β-catenin. Thus deficiency of Cited1 leads to hyper-activation of Wnt signaling and an exaggerated Wnt phenotype including elevated cell death. Cited1 deficiency decreases intestinal tumourigenesis in ApcMin/+ mice and impacts upon a number of oncogenic signaling pathways, including Wnt. This restraint imposed by Cited1 is consistent with a requirement for Cited1 to constrain Wnt activity to a level commensurate with optimal adenoma formation and maintenance, and provides one mechanism for tumour repression in the absence of Cited1.


Introduction
Inactivation of the APC (adenomatous polyposis coli) gene marks one of the earliest events in colorectal tumourigenesis [1], an observation that has given rise to the concept of Apc as a 'cellular gatekeeper' protecting against tumourigenesis [2]. This role in suppressing tumour formation has been closely associated with its ability to regulate the level of b-catenin within cells. Thus, Apc normally forms part of the scaffold of proteins that phosphorylate b-catenin and target it for degradation. In the absence of Apc, b-catenin levels become elevated and translocates to the nucleus, where it drives increased transcription of Wnt target genes associated with cell proliferation and cell death [3].
To investigate the biological consequences of Apc loss and Wnt activation, we and others have previously used a conditional model of Apc loss. In this model, deletion of Apc is achieved through use of an inducible AhCre transgene, which is responsive to exposure to the xenobiotic b-napthoflavone. Following Cre induction and loss of function of Apc, we observe a range of rapid phenotypic changes. These include promiscuous entry of cells into S phase, loss of differentiated cell types, loss of cell polarity and disorganisation of the crypt-villus structure to the point that discrete crypts are no longer discernable. Apc deficiency also reduces the normal migration of cells along the crypt villus axis, leading to the preferential retention of Apc deficient cells. These changes may all be considered pro-tumourigenic, however we also observe a considerable stress signal within Apc deficient cells, most clearly shown by a significant elevation in apoptosis. These phenotypic changes are accompanied by the expected elevation in levels of nuclear b-catenin and marked changes in the transcriptome [3].
One of the changes we observe in the intestinal epithelial cells of AhCre + Apc fl/fl mice is a strong induction of Cited1, a bifunctional transcriptional cofactor which is able to activate or repress transcription in association with other transcription factors [4,5]. We also found this induction to be dependent upon functional c-Myc as Cited1 expression returns to basal levels in the additional absence of c-Myc, which completely rescues the phenotype of Apc deficiency [6]. These observations suggest that elevation of Cited1 may be directly associated with the preneoplastic phenotype.
Cited1 was originally identified in a mouse melanoma cell line [7]. During vertebrate development, Cited1 is expressed in progenitors of the heart, limb, axial skeleton, kidney, and placenta [8,9]. It is implicated as a key co-ordinator during renal epithelial morphogenesis [5] and is involved in mammary gland development [10]. Cited1 is required for placental development with effect on embryo growth and survival [9]. However, Cited1 null mice that survive the early postnatal period are otherwise grossly phenotypically normal [9]. Cited1 is also able to enhance TGF-b signaling and inhibit Wnt signaling depending on cellular context [5,11]. Both activation and inhibition of transcription are dependent on the CBP/p300 binding C-terminal transcription activation domain CR2, which is conserved throughout the Cited family [5,[11][12][13].
Deregulation of CITED1 has been implicated in several human cancers, including melanomas, Wilm's tumours and nephroblastomas [7,[14][15][16]. In the mouse, Cited1 is up-regulated in MMTV-Cre/FloxNeoNeuNT mammary tumours and associates with the transcription factor EGR2 to regulate the expression of the oncogene ErbB2 (HER2, Neu) [17]. Recently it has been shown that Cited1 expression, together with another transcription regulator Six2, specify self-renewing nephron progenitor cells in kidney development and it is suggested that Cited proteins may contribute to the maintenance of the self-renewing capping mesenchyme in the developing kidney [18][19][20]. Thus, although a body of studies have implicated Cited1 in both embryogenesis and carcinogenesis, its potential role in Wnt-induced intestinal tumourigenesis remains unresolved.
Given the data implicating Cited1 as a regulator of the Wnt pathway, we have tested the hypothesis that Cited1 plays a key role in intestinal tumourigenesis. We show that CITED1 is upregulated in human colorectal cancers and that Cited1 deficiency increases the survival of Apc Min/+ mice. When crossed into our acute model of Apc deficiency, we show that loss of Cited1 accentuates nearly all aspects of the Apc deficient phenotype, including the transcription of a range of oncogenic signaling pathways, including the Wnt pathway.

Cited1/CITED1 is up-regulated in the intestine of Apc deficient mouse models and human colorectal tumours
We have previously shown that deletion of Apc in the mouse intestine leads to nuclear b-catenin translocation and up-regulation of Wnt target genes, including Cited1, as scored by microarray analysis [3]. To confirm this upregulation, we analysed mouse intestinal epithelium from AhCre + Apc fl/fl and AhCre + WT (WT: wild type) mice which had been induced by intraperitoneal injection of b-napthoflavone 4 days previously [3]. Quantitative PCR analysis revealed significant upregulation of the Wnt targets c-Myc, Axin2 and Cd44 in the absence of Apc. Similarly, Cited1 showed a 15-fold increase in expression (p,0.05, Figure 1A).
To determine if CITED1 was also deregulated in human cancers, we performed a Taqman quantitative PCR on human colorectal tumour tissues. In comparison to paired normal tissues from the same patient, we observed over-expression of the human orthologues of the Wnt target genes c-MYC, AXIN2, CD44 and CITED1 (p,0.01, Figure 1B). These data demonstrate the potential transferability of our data from the acute Apc deletion mouse model to human colorectal carcinogenesis.
We next assessed Cited1 levels in adenomas developing in the Apc Min/+ mouse model of human colorectal cancer, which allows evaluation of the effects of loss of Apc function over the course of polyp development. Again, the level of Cited1 expression was significantly increased in intestinal polyps from Apc Min/+ mice compared to normal tissue from the same mouse (p,0.01, Figure 1C; Figure S1A). Detection of high levels of Cited1/ CITED1 expression in both human and murine tumours suggests that Cited1/CITED1 may play a role in intestinal tumourigenesis.
We performed in situ hybridization using a Cited1 probe designed against the deleted sequence in the Cited1 2 mouse ( Figure 1D). We observed low levels of staining (compared to expression of the housekeeping gene Polr2a) throughout the cryptvillus structure in the AhCre + WT mouse intestine (WT) with a trend to higher levels within the crypt. There was no apparent specificity for the stem cell region at the base of the crypt or for any differentiated cell type. Consistent with QPCR data, we observed an increase in the level of staining throughout the crypt-villus structure of the AhCre + Apc fl/fl mouse.

Loss of Cited1 in Apc Min/+ mice increases survival and reduces the number of intestinal adenomas
As Cited1 is upregulated in colonic tumors we next asked whether deficiency of Cited1 could inhibit intestinal adenoma formation in the Apc Min/+ mouse. To achieve this, we crossed Cited1 null (Cited1 2 ) mice onto Apc Min/+ . Given that Cited1 is on the X-chromosome, we aged male cohorts of Apc Min/+ Cited1 2 and Apc Min/+ mice until they displayed symptoms of intestinal neoplasia (rectal bleeding and paling feet). The median lifespan of Apc Min/+ mice was 230.5 days, which is increased to 515 days in the Apc Min/+ Cited1 2 mice (Log-Rank p = 0.001, Figure 2A). Cited1 2 mice had a survival rate that was not significantly different to that of WT mice (Figure 2A).
We next counted the number of adenomas in the small and large intestine ( Figure 2B). Apc Min/+ Cited1 2 mice developed significantly less tumours compared to Apc Min/+ in both the small intestine (5 versus 18.5 adenomas p,0.05) and the large intestine (1 versus 6.5 adenomas, p,0.05). The tumour distribution in the small intestine and the colon was analysed at ill health (Figure

Author Summary
Colorectal cancer is the fourth leading cause of cancer related deaths worldwide, and a key genetic change associated with this disease is mutation of the gene APC. APC encodes a protein which plays a regulatory role in the Wnt signalling pathway. To better understand the mechanisms leading to colorectal cancer after APC loss, we have used a mouse model in which we deleted Apc in the bowel and which developed several characteristics of early stage cancers. Here, we show that after Apc loss, the expression of another gene, Cited1, is increased in mice and human colorectal tumours. To study the role of Cited1 in bowel cancer after loss of Apc, we generated mice mutant for Apc (Min) or mutant for Apc and Cited1 (MinCited1). We observed that MinCited1 mice developed fewer intestinal tumours and lived longer than Min mice suggesting that Cited1 is pro-tumourigenic. However, we also observed that Cited1 deficiency actually increased many of the aspects associated with loss of Apc, including deregulation of the Wnt pathway and cell death. To explain this apparent paradox, we propose a model whereby loss of Cited1, in the context of deregulated Wnt signalling, 'over-stimulates' the Wnt pathway, the net effect of which is to inhibit tumourigenesis. S1C). There was no significant difference in the percentage of tumours found in the duodenum or jejunum of the small intestine, or in the large intestine. However, we did observe a significant increase in the percentage of tumours found in the last part of the small intestine which corresponds to the human ileum ( Figure  S1C). Total tumour burden of Apc Min/+ Cited1 2 mice was not significantly different from that of Apc Min/+ ( Figure 2C), and shared the same tubular morphology and degree of invasiveness, as assessed histologically by the frequency of invasion into the submucosa (52.8% High grade +47.16% Low grade in Apc Min/+ vs 42.8% High grade +57.4% Low grade in Apc Min/+ Cited1 2 , Chisquare x 2 = 2.1, DF = 1, p.0.05). These data suggest that mice became symptomatic of disease when they had developed an equivalent tumour burden, but that in the Cited1 mutant background this was significantly later and reflected fewer, but larger lesions at these later time points, hence implicating Cited1 in intestinal tumour initiation.
Cited1 deficiency modifies the phenotype observed immediately after Apc loss by increasing the number of Brdu positive cells and the size of the hyperplastic area To address the mechanism underlying the reduction of adenoma formation in Apc Min/+ Cited1 2 mice, we crossed Cited1 2 mice with mice conditionally mutant for Apc. We have previously demonstrated that we can achieve almost 100% recombination of the Apc fl/fl allele in the intestine using the b-napthoflavone inducible AhCre transgene to drive recombination [3]. Thus, AhCre + WT, AhCre + Apc fl/fl , AhCre + Cited1 2 , and AhCre + Apc fl/fl Cited1 2 mice were induced with b-napthoflavone and culled 4 days after the first injection to determine the role of Cited1 immediately following deletion of Apc. To confirm the level of Apc fl/fl recombination we used quantitative RT-PCR and again found that 100% of the PCR products obtained were from the recombined Apc allele ( Figure S1B). We also confirmed Cited1 deficiency in Cited1 2 mice using RT-PCR. We observed a significant 3.81 fold difference decrease in Cited1 expression in AhCre + Cited1 2 compare to AhCre + WT. The small difference observed is most likely due to the low level of expression of Cited1 in the intestine [21]. Due to the increased level of Cited1 expression after loss of Apc, Cited1 deficiency is more noticeable in the intestinal epithelial cells of AhCre + Apc fl/fl Cited1 2 mice which showed a 277.81 fold decrease compared to AhCre + Apc fl/fl mice (p,0.05 Mann-Whitney U test).
We have previously shown that the loss of Apc leads to an increase in proliferation and apoptosis and also to a loss of migration [3]. To analyse the effects of Cited1 deficiency after Apc loss, we first counted the number of cells in S phase within the crypt or hyperplastic areas (formed after Apc loss). On day 4 after b-napthoflavone induction, mice were injected with BrdU to label cells in S-phase and culled 2 hrs later ( Figure 3A). In AhCre + WT and AhCre + Cited1 2 mice the number of proliferating cells was not significantly different (AhCre + WT: 18.97 vs AhCre + Cited1 2 : 20.65 BrdU positive cells/ Crypt; p.0.05. Figure 3B). However, the number of cells in S-phase was significantly increased in the hyperplastic areas of AhCre + Apc fl/fl Cited1 2 mice compared to AhCre + Apc fl/fl mice (AhCre + Apc fl/fl : 74.13 vs AhCre + Apc fl/fl Cited1 2 : 106.4 BrdU positive cells/area, p,0.05, Figure 3B) suggesting a role for Cited1 in controlling cell proliferation in the context of active Wnt signaling.
We next analysed the histology on HE sections of the intestinal tissue from all the genotypes after b-napthoflavone induction. There were no gross changes in the crypt/villus architecture in AhCre + WT compared to AhCre + Cited1 2 mice, and induced AhCre + Apc fl/fl Cited1 2 mice had similar large aberrant crypts to those observed in AhCre + Apc fl/fl mice ( Figure 4D).
Given our findings of decreased adenoma formation in Apc Min/+ mice, we also examined the extent of the hyperplastic area within the crypts of AhCre + Apc fl/fl mice compared to AhCre + Apc fl/fl Cited1 2 mice as determined by the extent of BrdU labelling. Surprisingly, the number of cells in the hyperplastic area was greater in AhCre + Apc fl/fl Cited1 2 mice compared to AhCre + Apc fl/fl mice (Ah-Cre + Apc fl/fl : 78 cells/area vs AhCre + Apc fl/fl Cited1 2 :106 cells/area, p,0.05, Figure 4E).
We next determined migration rates by comparing the position of cells 2 hrs and 24 hrs after BrdU labelling. The difference between the 2 hrs and 24 hrs distributions for a genotype was analysed with the Kolmogorov-Smirnov test. The distribution of Brdu positive cells from 2 hrs to 24 hrs varies significantly for all genotypes (p = 0.01) indicating cell migration. Enterocytes in AhCre + WT mice and AhCre + Cited1 2 mice migrate at the same rate (20 cell positions at the 50% cumulative frequency) whereas cells in both AhCre + Apc fl/fl mice and AhCre + Apc fl/fl Cited1 2 mice show greatly reduced migration rates ( Figure 3C). Critically, although deletion of Apc in AhCre + Apc fl/fl mice results in strong suppression of migration (9 cell position migration), some movement of cells was detected in these samples ( Figure 3C). By comparison, the absence of Cited1 in AhCre + Apc fl/fl Cited1 2 mice, resulted in even less migration (2 cell position migration) than observed for enterocytes in AhCre + Apc fl/fl mice ( Figure 3C). Together, these data demonstrate that Cited1 deficiency further exacerbates both the proliferation and migration phenotypes of Apc loss in the intestine, which is surprising given that Apc Min/+ Cited1 2 mice developed significantly less intestinal tumours than Apc Min/+ mice. Consistent with these observations that proliferation is increased in AhCre + Apc fl/ fl Cited1 2 mice, we also observed a decrease in the number of differentiated entoendocrine cells and goblet cells in these mice ( Figure S5A-D).
We and others have previously demonstrated that the location and the number of paneth cells in the intestinal crypt are regulated by Wnt signalling [6,[22][23][24]. It is observed by the increased number of paneth cells after loss of Apc ( Figure S5F) and the loss of positioning at the bottom of the crypt ( Figure S5E-G). Consistent with our observations that the phenotype of Ah-Cre + Apc fl/fl mice is enhanced upon deficiency of Cited1 we also observe a change in position of the paneth cells in the hyperplastic areas of the AhCre + Apc fl/fl Cited1 2 compared to AhCre + Apc fl/fl ( Figure  S5G). This is most likely due to the increase in crypt size seen in the double mutant, which gives cells a bigger area to be distributed.

Cited1 deficiency enhances the apoptotic phenotype observed immediately after Apc loss
The increase in proliferation observed in the intestine following deletion of Apc is also associated with a dramatic increase in apoptosis [3]. We therefore examined if Cited1 was regulating apoptosis by counting apoptotic bodies in H&E sections and also scoring Caspase 3 staining. We observed no significant difference in apoptosis between AhCre + WT and AhCre + Cited1 2 mice, however, there was a significant increase in the number of apoptotic cells in AhCre + Apc fl/fl Cited1 2 mice compared to AhCre + Apc fl/fl mice, which was verified by both methods ( Figure 4A).
As mentioned above, we observed an increase in the number of cells per hyperplastic area in the AhCre + Apc fl/fl Cited1 2 samples. To verify that the increase in cell death was not an artefact of the difference in the number of cells per area, we corrected for this difference between AhCre + Apc fl/fl and AhCre + Apc fl/fl Cited1 2 mice. The normalised data confirmed increased cell death in AhCre + Apc fl/fl Cited1 2 compared to AhCre + Apc fl/fl (p,0.05) after H&E counting (AhCre + Apc fl/fl : 11.53 apoptotic cells/area vs AhCre + Apc fl/ fl Cited1 2 : 15.33 apoptotic cells/area, p,0.05) and after anti cleaved-Caspase3 staining (AhCre + Apc fl/fl : 7.32 apoptotic cells/ area vs AhCre + Apc fl/fl Cited1 2 : 9.53 apoptotic cells/area, p,0.05) ( Figure 4B-C). These data indicate that the increase in cell death is not proportional to the increase in cell proliferation. Therefore, Cited1 deficiency in a Wnt perturbed background accentuates the apoptotic response.
Scoring of Caspase3 positive cells revealed no change in the number of apoptotic cells in the intestinal tumors of Apc Min/ + Cited1 2 mice compared to Apc Min/+ mice at time of death ( Figure 4F). Therefore, the increased apoptosis we observe in the absence of Cited1 is only manifested in the context of acute Wnt activation, which underlines the role of Cited1 in restraining tumour initiation, and also implies that in those tumours that do develop in the absence of Cited1, they have developed alternate mechanisms to restrain the Wnt pathway. Cited 1 regulates several pathways including the Wnt signaling pathway We next wished to investigate the mechanism through which Cited1 may be modifying Wnt driven tumorigenesis. One possibility is a direct effect upon Wnt signaling, and in support of this, Cited1 has previously been shown to be able to bind to bcatenin and consequently inhibit Wnt induced transcription during Xenopus development [5]. Two potential TCF-4 sites were identified in the Cited1 promoter region (ctttgt and cattgaa in the 2 kb prior exon1). This implicates Cited1 in the control of the Wnt pathway, however this is not the only pathway known to be altered by Cited1. Cited1 has been shown to bind to the p300/CBP coactivators and also to Smad4, thereby enhancing their transcriptional activity [11,25]. To analyse the effects of Cited1 deficiency on various transcriptional pathways we performed a microarray analysis using the Affimetrix Chip 430 2.0 and AffylmGUI software [26]. We then submitted our microarray data to ingenuity pathway analysis software (IPA) to identify pathways significantly affected by Cited1 deficiency.

Stem cell markers are upregulated after loss of Cited1
The Wnt signalling pathway has been shown to play a critical role in intestinal homeostasis which includes stem cells maintenance. Because Cited1 loss leads to a deregulation of the Wnt pathway and due to the potential role of Cited1 in the stem cell niche in the cap mesemchyme in the developing kidney [18], we analysed the effect of Cited1 deficiency in the intestine. RT-QPCR analysis revealed a significant upregulation of several stem cell markers (Gpr49, Ascl2, Musashi and Olfm4) in AhCre + Apc fl/fl Cited1 2 tissues compared to AhCre + Apc fl/fl controls ( Figure 5C). We also performed ISH for the surrogate marker of Lgr5, Olfm4 ( Figure 5D). In AhCre + Wt and AhCre + Cited1 2 mice the location of Olfm4 expressing cells is confined to the stem cell niche at the base of the crypts. In AhCre + Apc fl/fl mice, Olfm4 expressing cells were distributed throughout the hyperplastic area. In AhCre + Apc fl/fl Cited1 2 mice Olfm4 expressing cells are also mislocalised throughout the aberrant crypts but expression is increased, consistent with our RT-QPCR data of the same tissue ( Figure 5C).
Cited1 influences the level of the active form of b-catenin in the small intestine Loss of Apc has been shown to drive an increase in total bcatenin and more importantly a re-localisation of the active form of b-catenin to the nucleus [3]. To test if Cited1 deficiency modified this phenotype, we analysed the localisation of total b-catenin in the small intestine by immuno-histochemistry ( Figure 6A). We observed a normal pattern of localisation in both AhCre + WT and AhCre + Cited1 2 mice consistent with previous findings [30]. Upon deletion of Apc, we observed nuclear translocation of b-catenin in the aberrant crypts of both AhCre + Apc fl/fl and AhCre + Apc fl/fl Cited1 2 mice ( Figure 6A) indicative of de-regulated Wnt signalling.
b-catenin regulates important cellular functions such as transcription and adhesion [31], and the cellular concentration and phosphorylation status of b-catenin has been shown to impact on these functions [31,29]. As we observe an increase in the transcription of several Wnt target genes in AhCre + Apc fl/fl Cited1 2 mice we examined the level of total b-catenin, the extent of phosphorylation at multiple sites and the ratio of transcriptionally active free b-catenin in purified intestinal epithelial cells ( Figure 6B) as previously described [32]. First, we observed a significant increase in total b-catenin accompanied by an increase in the active form of b-catenin (dephosphorylation at pS33, pS37, pT41 sites) in AhCre + Apc fl/fl and AhCre + Apc fl/fl Cited1 2 mice compared to AhCre + WT and very importantly in AhCre + Apc fl/fl mice compared to AhCre + Apc fl/fl Cited1 2 mice. These data were confirmed by western blot analysis using an antibody raised against total b-catenin ( Figure 6C) or against the active form of b-catenin (dephosphorylated sites pS33, pS37, pT41) ( Figure 6D) and were verified with a second antibody against dephosphorylated b-catenin ( Figure S2A-B). There was no significant difference in the phosphorylated (inactive) form of bcatenin (phosphorylated b-catenin at pS33, pS37, pT41 sites is degraded as a mechanism of regulating Wnt signalling) between all genotypes ( Figure 6B). b-catenin phosphorylated at pS45 (phosphorylated by casein kinase Ia as part of degradation pathway) is significantly increased in AhCre + Apc fl/fl Cited1 2 compared to Ah-Cre + WT and AhCre + Apc fl/fl .
Given that b-catenin can also be phosphorylated by Protein Kinase A (PKA) at Ser552 and Ser675 which acts to inhibit ubiquitination and therefore increase levels of active b-catenin [33], we also analysed levels of pS552 and pS675 and found phosphorylation at S675 significantly increased in AhCre + Apc fl/fl Cited1 2 tissues compared to AhCre + WT and AhCre + Apc fl/fl , demonstrating the ability of Cited1 to regulate b-catenin at multiple sites ( Figure 6B).
We also measured the intracellular free b-catenin (active bcatenin) levels by pull-down with a GST-fusion protein of the inhibitor of b-catenin and TCF-4 (ICAT). We observed a significant increase in free b-catenin in AhCre + Apc fl/fl and AhCre + Apc fl/fl Cited1 2 mice compared to AhCre + WT and noticeably a significant increase in AhCre + Apc fl/fl Cited1 2 compared to AhCre + Apc fl/fl mice ( Figure 6B). These data support our findings above which indicate that Cited1 deficiency increases the levels of active dephosphorylated b-catenin.
These data demonstrated that the active dephosphorylated form of b-catenin in purified intestinal epithelial cells is markedly increased upon Cited1 deficiency. Although the level of dephosphorylated b-catenin is increased in Cited1 2 intestinal cells compared to WT mice, it is below that observed in the AhCre + Apc fl/fl intestinal cells ( Figure 6B, 6D). As Cited1 2 mice do not develop any intestinal phenotypes such as hyperproliferation this suggests that the level of Wnt activation in Cited1 2 mice is below the critical threshold required to induce neoplasia [34]. However, when Apc is deleted in Cited1 deficient mice (AhCre + Apc fl/fl Cited1 2 ) the level of dephosphorylated b-catenin is greater than that observed with Apc loss alone, thus providing an explanation for the increased transcription of Wnt target genes observed in these mice ( Figure 5A).

Discussion
Colorectal cancer is driven by a multiplicity of different biochemical pathways, however, key amongst these is the Wnt pathway, which we and others have previously shown to activate a set of c-Myc dependent genes which are critical for the early stages of colorectal cancer [6,35]. One of these genes is Cited1, which has been found to interact at the protein level with b-catenin and thereby negatively regulate b-catenin transcription [5]. Its relevance to carcinogenesis has already been described as Cited1 up-regulation has been observed in various cancers [14,36,37]. Here, we have extended those observations and find that CITED1 is significantly up-regulated in colorectal tumours from patients and in intestinal adenomas developing in the Apc Min/+ mouse model [38]. We also previously found Cited1 to be over-expressed in intestinal epithelial cells immediately following deletion of the Wnt regulator gene Apc in AhCre + Apc fl/fl mice in a c-Myc dependent manner [6]. These data establish Cited1 as an immediate Wnt target gene in the intestine.
On the basis of these data we hypothesised that Cited1 might control b-catenin activity and thereby modulate Wnt signaling activation and its effects on colorectal tumorigenesis. To investigate this, we used microarray analysis and quantitative PCR studies to show that loss of Cited1 on an Apc deficient background does indeed impact upon a range of oncogenic signaling pathways, including Wnt. Our array data therefore show multiple effects of Cited1 deficiency including negative regulation of the Wntpathway.
To investigate the requirement of Cited1 during Wnt induced tumourigenesis, we analysed the effects of deletion of Cited1 in two well characterised mouse models of Wnt signaling activation; the Apc Min/+ mouse model of colorectal tumourigenesis and the AhCre + Apc fl/fl mouse, a conditional model of Apc loss in which the immediate phenotypic consequences of Apc deletion can be studied [3]. Surprisingly, we obtained the apparently paradoxical result that although Apc Min/+ Cited1 2 mice developed fewer intestinal tumours (associated with an increased life-span) than Apc Min/+ mice, the phenotypes induced upon conditional loss of Apc (including perturbed cell proliferation, apoptosis, differentiation and migration) were enhanced, rather than diminished, with additional loss of Cited1. Of note, we observed reduced capacity to differentiate (reflected by a reduced number of goblet cells and enteroendocrine cells), but no difference in total paneth cell numbers, although we did observe a difference in the positioning of paneth cells, which may well reflect differences in the Wnt signalling environment.
Our studies, suggest that a possible explanation for this apparent paradox is that the hyper-activated Wnt phenotype that occurs in the absence of Cited1 includes increased apoptosis. Several studies in cell culture systems already support such a model. For example, it has been reported that overexpression of b-catenin when transfected into cell lines leads to a 3-4 fold increase in cell death [39]. In addition, it has been demonstrated that high levels of c-Myc induce apoptosis in vivo [40]. This is consistent with our observations that c-Myc is overexpressed immediately following deletion of Apc in the intestine and that levels are significantly increased further with additional absence of Cited1. We interpret our data to indicate that the increase in apoptosis may counteract the increase in proliferation to the extent that the overall effect is reduced development of Wnt transformed cells and consequently inhibition of tumourigenesis.
The mechanism underlying such hyper-activation of Wnt signaling appears to be at least in part mediated through increased levels of dephosphorylated b-catenin, which we found to be upregulated in AhCre + Apc fl/fl Cited1 2 tissue compared to comparable AhCre + Apc fl/fl tissue at both the transcriptional and protein levels. Thus, we found increased levels of the dephosphorylated forms of b-catenin (T41, S33, S37). These sites when phosphorylated are involved in the degradation of b-catenin by the proteasome pathway [31]. This is accompanied by an increase in the levels of phosphorylation at serine 675 which has been shown to be phosphorylated by protein kinase A (PKA) and which has been shown to lead to inhibiting of ubiquitination of b-catenin causing its accumulation and subsequent Wnt signalling activation. We therefore show that Cited deficiency increases the pool of active bcatenin, consistent with the enhanced Wnt pathway activation we observe. It does however remain possible that Cited1 may in addition be mediating its effects downstream of b-catenin.
We cannot rule out the possibility that the other pathway changes we observe are responsible for the reduction in tumourigenesis in Apc Min/+ Cited1 2 mice, as the effects of loss of Cited1 are not exclusive to the Wnt pathway. We also cannot rule out that the effects we observe may be secondary to Cited1 deletion. For example, it is possible that some of the changes we observe may be due directly to the upregulation of c-Myc rather than a direct consequence of Cited1 loss. Functional delineation of the precise relevance of all the changes we observe requires multiple crosses onto the relevant pathways to probe such dependency. Finally, AhCre + WT, AhCre + Apc fl/fl , AhCre + Cited1 2 , and AhCre + Apc fl/fl Cited1 2 mice were maintained on an outbred background, and because the comparisons of genotypes within the same littermates was restricted due to the small number of litter size, we cannot completely rule out the effect of gene modifiers on the Cited1 loss phenotype.
Our current primary hypothesis is that Cited1 deficiency mediates its effects upon adenoma formation primarily through the apparently paradoxical derepression of the Wnt pathway. This result is however consistent with a ''just right model'' wherein a specific level of Wnt signaling activity is required for maximal tumour development and those levels of Wnt signaling above or below this level compromise tumour growth [41]. This model is further supported by recent studies on a novel mutant Apc mouse (Apc 1322T ), which has reduced Wnt signaling compared to Apc Min/+ littermates, but surprisingly develops significantly more intestinal tumours [42]. Recently it was shown by Leedham et al [43] that in normal mouse intestine, stem cell markers and Wnt target genes are expressed in a physiological gradient compatible with normal intestinal homeostasis. Pathological activation of Wnt activity using the Ctnnb1 Dex3 mouse model led to variable gradients in stem cell number and Wnt signalling activity which influenced tumour susceptibility, with regional differences in tumour predisposition throughout the length of the intestinal tract. This data, which supports the just right model hypothesis, may explain the variation we observe in the tumour distribution in the ileum between the Apc Min/+ and Apc Min/+ Cited1 2 mice models. These observations clearly show that there is not a simple linear relationship between Wnt pathway activity and tumour burden. Our data is consistent with another version of this ''just right'' concept where perturbation of Cited1 leads to increased dephosphorylated b-catenin and hyper-activation of the Wnt pathway to a level that is incompatible with maximum tumour growth (Figure 7). Notably, this relationship appears specific to the intestine as similar analysis of kidney tumorigenesis in these mice showed no effect of Cited deficiency ( Figure S3). Furthermore, to define the precise relationship between Wnt levels and tumourigenicity will require mouse modelling experiments in which Wnt activity is precisely regulated at numerous levels.
Wnt/beta-catenin signalling plays a key role in the homeostasis of the intestinal epithelium and its role in the fate and maintenance of the stem cell compartment have been clearly demonstrated [44]. Our data clearly show that Cited1 is an immediate target of Wnt signalling and is an important regulator of the Wnt pathway. The loss of Cited1 has a direct impact on stem cell status in the small intestine as we have found several stem cell markers to be upregulated including Lgr5 (Gpr49), Musashi and Olfm4. These alterations in expression could be a direct consequence of the 'hyper' activation of the Wnt pathway we observe after combined loss of Apc and Cited1. This would implicate Cited1 as an important player in Wnt dependant stem cell maintenance in the small intestine. This has been already suggested in the developing kidney where Cited1 may contribute to the maintenance of the selfrenewing capping mesenchyme [18]. By regulating the Wnt pathway, Cited1 may be an important regulator of the self-renewal compartment in the crypt of the small intestine.
Our data show that Cited1 deficiency represses tumourigenesis. The consequences of Cited1 deficiency are diverse, but in particular impact upon Wnt pathway activity. We propose a model whereby loss of Cited1, in the context of deregulated Wnt signaling, hyperactivates the Wnt pathway resulting in apoptosis of Wnt induced transformed cells and thus inhibits tumourigenesis. As Cited1 mice are fertile and viable this suggests that Cited1 represents a possible target for therapeutic intervention, where Cited1 inhibition induces cytotoxic effects due to very high Wnt signalling.

Materials and Methods
Human colorectal cancer tissue RNA samples Total RNA samples from patient colorectal tumour tissues were obtained from the Cancer Tissue Bank Research Centre (CTBRC). All colorectal cancer tissues and adjacent uninvolved colonic mucosa were obtained from surgically removed specimens with informed patient consent. Uninvolved colonic mucosa was generally 5-10 cm away from the malignant tissue.
Assaying apoptosis, number of cells per crypt, S-phase labelling in vivo and migration Apoptosis was scored from H&E or after anti cleaved-Caspase3 immuno-staining as previously described [3]. For proliferation analysis, mice were injected with 0.25 ml of BrdU (Amersham) before culling and were taken either 2 hrs (day4) or 24 hrs (day5) after BrdU injection. Staining was performed as previously described [3]. The number of cells in AhCre + Apc fl/fl and AhCre + Apc fl/fl Cited1 2 hyperplastic area was scored using the position of the last BrdU positive cells in the hyperplastic area. For each analysis, 25 full crypts or areas were scored from at least 3 mice of each genotype and time point.

In situ hybridization (ISH)
In situ hybridization of Olfm4 and Cited1 in the small intestine was performed for all genotypes using sections embedded in paraffin sectioned at 5 mm. Olfm4 hybridization was performed as described in Gregorieff et al., 2005. [48]. Cited1 hybridization was performed using a probe against the sequence deleted in the Figure 7. Schematic diagram of a ''just right'' model of Cited1 action on colorectal tumourigenesis. Loss of function of Apc is accompanied by multiple changes in gene expression, including upregulation of active b-catenin (dephospho-b-catenin), activation of Wnt and Cited1. Hence, immediately following deletion of Apc, Cited1 normally restrains the Wnt pathway at the level of b-catenin. We observe a range of rapid phenotypic changes. These include increase in proliferation and apoptosis and loss of differentiated cell types, also reduced migration, leading to the preferential retention of Apc deficient cells. These changes may all be considered pro-tumourigenic, leading to survival advantage of the tumour cell and decreased survival of the Apc Min/+ mouse compared to WT. Additional Cited1 deficiency leads to hyper-activation of Wnt signaling including upregulation of active b-catenin and an exaggerated Wnt phenotype including elevated proliferation, a further loss of cell differentiation, and most importantly increased cell death. The net effect of these changes is an increase in Apc Min/+ survival. This restraint imposed by Cited1 is consistent with a requirement for Cited1 to constrain Wnt activity to a level commensurate with optimal adenoma formation and maintenance, and provides one mechanism for tumour repression in the absence of Cited1. doi:10.1371/journal.pgen.1003638.g007 Cited1 2 allele designed by Advanced cell Diagnostics inc (ACD). RNAscope 2.0 FFPE Reagent Kit -Brown kit was used according manufacturer instructions. Negative control Probe-DapB was used together with a positive control probe Polr2a from the ACD manufacturer.

Microarray data analysis
The DNA microarray were performed from three mice of each genotype using Mouse Genome 430 2.0 Affymetrix chips at Liverpool Microarray Facility according to the manufacturer's instructions. The Microarray data were analyzed using Affylm-GUI (Affymetrix linear modeling Graphical User Interface; http://bioinf.wehi.edu.au/affylmGUI/#citation) [26]. The p values presented have been corrected for multiple testing using the BH method to control the false discovery rate. The B statistic is the log odds that the gene is differentially expressed and is adjusted for multiple testing using the assumption that 1% of genes are expected to be differentially expressed [26,[49][50][51]. Microarray data were deposited in MIAME format at www.ebi.ac.uk/ arrayexpress/ (Accession Number: E-MEXP-3202) QPCR protocols, routine methods and a description of the statistical analyses used are provided in Protocol S1. List of primers for Taqman RT-QPCR, Sybr green RT-QPCR, and Cited1 semi quantitative RT-PCR are provided in Figure S4.

Analysis of signaling pathways
Ingenuity pathway analysis (IPA) software (www.ingenuity.com) was used to determine which signaling pathways were affected by the loss of Cited1 in AhCre + WT or AhCre + Apc fl/fl mice. The comparative (AhCre + WT vs AhCre + Cited1 2 and AhCre + Apc fl/fl vs AhCre + Apc fl/fl Cited1 2 ) data from the microarray analysis were filtered for a p value of less than 0.05 and imported into the IPA software. The significance of the association between the data set and the pathway was measured in 2 ways: by the ratio and by a p value. The ratio corresponds to the number of genes from our data set that map to the ingenuity pathway divided by the total number of genes that map to the Ingenuity canonical pathway. The p value is calculated by a right tailed Fischer's exact test. The p-value associated with a pathway is a measure of the likelihood that the association between a set of focus genes in your experiment and a pathway is due to random chance.

b-catenin suspension bead array based assay
Analysis of biological function, localization, and posttranslational modification of the different forms of b-catenin were carried out as previously described [32]. Two additional assays were included in the analysis. Anti-dephospho S33/S37 and T41 (Cell Signalling Technologies) was used as an additional capture antibody to measure dephosphorylated b-catenin and GST-ICAT was employed as an additional bait protein to study free b-catenin. distribution was analysed by counting tumour burden in each of 5 cm sections along the length of the small intestine and representing this as a percentage of total tumour burden per section. Sections 1-2 (duodenum), 3-5 (jejunum) 6-8 (comparable to human Ileum). The colon was similarly divided into 1 cm sections. Section 1 corresponding to the rectum. Error bars represent standard errors. *p,0.05; statistical tests was done using Mann-Whitney U test. (TIF) ras V12 Cited1 2/+ (n = 5) (Thin solid line; p = 0.555 Log-Rank test). The median lifespan was 88 days in AhCre + Apc fl/fl K-ras V12 (n = 13), 72 days in AhCre + Apc fl/fl K-ras V12 Cited1 2 (n = 4) and 81 days in AhCre + Apc fl/fl K-ras V12 Cited1 2/+ (AhCre + Apc fl/fl K-ras V12 vs AhCre + Apc fl/ fl K-ras V12 Cited1 2 , Log Rank p = 0.732; AhCre + Apc fl/fl K-ras V12 vs AhCre + Apc fl/fl K-ras V12 Cited1 2/+ Log Rank p = 0.555). These data indicate that Cited1 deficiency does not modify the survival of mice affected by renal carcinoma and that Cited1 does not play a role in renal carcinoma induced by a loss of Apc. (TIF) Figure S4 Primers Table. Tables listing the primers D). The position and the number of paneth cells are not modified in AhCre + Cited1 2 mice compared to AhCre + WT mice (E-G). After loss of Apc, paneth cells lose their position at the bottom of the crypt and are mislocalised along the crypt-villus axis (G). We observe a change in position of the paneth cells in the hyperplastic areas of the AhCre + Apc fl/fl Cited1 2 compared to AhCre + Apc fl/fl (E-G). Statistical test was done using Kolmogorov-Smirnov test (G). Inset panels show magnifications of intestine of corresponding zone. Taken altogether, these data suggest that loss of Cited1 does not perturb the pattern of differentiation when Apc is present. However in the absence of Apc, Cited1 deficiency appears to accentuate the phenotype, with a more extreme reduction in the number of differentiated cell types. (TIF) Protocol S1 Statistical Analysis, Quantitative real-time PCR on mice samples, Cited1 status by Semi-quantitative PCR, First strand cDNA synthesis and taqman quantitative qPCR on human samples, Western Blot analysis, RNA and protein isolation from mice small intestine, Histology and Immunohistochemistry and Supplementary references are provided as supplementary informations. Primers list for Taqman RT-QPCR, Sybr green RT-QPCR and semi quantitative RT-PCR are listed in Figure S4. (DOC)