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Dual Positive and Negative Regulation of Wingless Signaling by Adenomatous Polyposis Coli

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Science  18 Jan 2008:
Vol. 319, Issue 5861, pp. 333-336
DOI: 10.1126/science.1151232

Abstract

The evolutionarily conserved Wnt/Wingless signal transduction pathway directs cell proliferation, cell fate, and cell death during development in metazoans and is inappropriately activated in several types of cancer. The majority of colorectal carcinomas contain truncating mutations in the adenomatous polyposis coli (APC) tumor suppressor, a negative regulator of Wnt/Wingless signaling. Here, we demonstrate that Drosophila Apc homologs also have an activating role in both physiological and ectopic Wingless signaling. The Apc amino terminus is important for its activating function, whereas the β-catenin binding sites are dispensable. Apc likely promotes Wingless transduction through down-regulation of Axin, a negative regulator of Wingless signaling. Given the evolutionary conservation of APC in Wnt signal transduction, an activating role may also be present in vertebrates with relevance to development and cancer.

The Wnt/Wingless (Wg) secreted proteins activate a signal transduction cascade that directs growth and differentiation in many tissues during animal development [reviewed in (1)]. Activation of target genes in response to the Wnt/Wg signal is dependent on the transcriptional activator β-catenin/Armadillo (Arm). In the absence of Wnt, four factors—APC, Axin, glycogen synthase kinase-3/Zeste white 3, and casein kinase 1—target β-catenin for phosphorylation and subsequent proteasomal degradation (28). Axin acts as a scaffold to facilitate β-catenin phosphorylation by binding β-catenin, APC, and the two kinases. Wnt-dependent down-regulation of Axin is important for β-catenin–mediated transcriptional activation (911). Mutational inactivation of negative regulatory components in the pathway and the resultant inappropriate activation of Wnt signaling is associated with the development of several types of cancer. The majority of colorectal adenomas and carcinomas contain mutations that eliminate the carboxy-terminal half of APC (1).

The Wnt/Wg signaling pathway shows considerable conservation among metazoans. Two APC homologs exist in humans, mice, and fruit flies, and the negative regulatory role of APC in Wnt signaling is conserved from flies to mammals (1215). Drosophila Apc1 and Apc2 are ubiquitously expressed, and in most cells act redundantly to negatively regulate Wg signaling (16, 17). However, in retinal photoreceptors, Apc2 activity is low enough that inactivation of Apc1 singly suffices to constitutively activate Wg signaling (13, 16). In response, all photoreceptors undergo apoptosis (13) (Fig. 1, A and B) and before their deaths some photoreceptors adopt an aberrant cell fate, as indicated by ectopic expression of homothorax and Rhodopsin 3 (18, 19) (fig. S1, A to F).

Fig. 1.

Apc2 promotes Arm-induced apoptosis in retinal photoreceptors. (A) A heterozygous Apc1Q8/+ adult eye; in each ommatidium, seven photoreceptors are observed. (B) All photoreceptors in the homozygous Apc1Q8 null mutant undergo apoptosis. (C) Apoptosis is suppressed by the heterozygous Apc233 deletion. Each image was obtained at the same magnification.

To identify genes that promote Wg signaling, we performed a genetic screen for suppressors of photoreceptor apoptosis in the Apc1Q8 null mutant (Methods). We found that apoptosis is suppressed by null and hypomorphic Apc2 alleles (Fig. 1C; fig. S2, A to C; fig. S3, A to H; and table S1). Ectopic expression of homothorax and Rhodopsin 3 is also suppressed, indicating that suppression of Wg signaling is not restricted to apoptosis (fig. S1, G to L, and fig. S4, A to F). Further, ectopic Wg signaling resulting from Arm overexpression is also partially suppressed by reduction of Apc2 (fig. S5, A to C). These data indicate that in addition to its well-established negative regulatory role, Apc2 also has an activating role in ectopic Wg signaling.

It is unknown whether this Apc-activating function in Wg signaling is also important during normal development. Previous observation of an activating function may have been obscured by the negative regulatory role of Apc1 and Apc2 and by their functional redundancy. Therefore, we developed strategies to reduce the combined activity of Apc1 and Apc2 to different extents. We eliminated all Apc1 activity using the Apc1Q8 null allele while simultaneously reducing Apc2 activity to various levels using different Apc2 alleles. We used Apc2 alleles such as Apc25-3 and Apc2G5028, which include insertions in the 5′ untranslated region and reduce Apc2 protein to low levels (Fig. 2A and table S1), and alleles that reduce Apc2 activity even further, such as the molecular null allele Apc279 and the deletion alleles Apc233 and Apc219-3 (fig. S2A and table S1).

Fig. 2.

Apc1 Apc2 double-mutant phenotypes. (A) Western blot using Apc2 carboxy-terminal antiserum on embryonic lysates. Apc2 levels are reduced in Apc25-3 and Apc2G5028 mutants. Wild-type and Apc233 mutant are shown for reference. Kinesin heavy chain is control. (B) Ventral abdomen of wild-type female. Sternites and overlying sternal bristles (St, arrow) and pleura (Pl) are indicated. (C and D) Loss of sternites and sternal bristles and expansion of pleura in homozygous panER1 mutants and homozygous Apc1Q8 Apc25-3 or Apc1Q8 Apc25-3/Apc1Q8 Apc2G5028 mutants. This phenotype is present in 65 to 70% of homozygous Apc1Q8 Apc25-3 mutant females raised at 25°C with variation in severity between individuals. (E and F) Wing-to-notum transformation (arrow) in homozygous wg1 mutants and in Apc1Q8 Apc25-3/Apc1Q8Apc219-3 mutants. Apc219-3 is a deletion allele (table S1).

Inactivation of Wg, Arm, or Arm's transcriptional co-activators dTCF/Pangolin (Pan) and Legless results in loss of sternites, which are bristle-bearing cuticular plates in the ventral abdomen (2022) (Fig. 2, B and C), and a “wingless” phenotype in which the wing blade is replaced by a duplication of the dorsal thorax or notum (2124) (Fig. 2E, and fig. S6, C to F). We observe both of these phenotypes in Apc1Q8 Apc2 double mutants that display low levels of Apc protein (Fig. 2, D and F, and fig. S6F). In contrast, abdomen and wing patterning is normal in Apc1 null mutants and in Apc2 null mutants. Therefore, we conclude that Apc not only promotes ectopic Wg signaling but is also required to promote Wg signaling during normal development. This activity is supplied redundantly by Apc1 and Apc2. Further reduction in the combined activity of Apc1 and Apc2 results in Wg hyperactivation phenotypes in the abdomen and wing, reflecting the negative regulatory role of Apc (figs. S6 and S7). Thus, Apc1 and Apc2 not only prevent signaling when Wg is absent but also promote signaling in cells responding to Wg.

To determine a mechanism by which Apc promotes Wg signaling, we examined Axin levels (Fig. 3, A to C) after simultaneous reduction of Apc1 and Apc2 (Fig. 3, D to I). Upon complete loss of Apc1 and Apc2 (in Apc1Q8 Apc279 null mutant wing clones), not only are Arm levels high (Fig. 3, M to O) but also Axin levels are increased (Fig. 3, D to F). Axin levels also increase in cells with reduced Apc activity, which retain the ability to promote some Arm degradation (in Apc1Q8 Apc233 mutant clones) (table S1 and Fig. 3, G to I and P to R). This Axin increase is present in cells regardless of their location within the wing disc and thus is not restricted to cells exposed to Wg. Furthermore, these increased Axin levels do not result from enhanced Arm-mediated transcription (fig. S8). Our results reveal that Apc1 and Apc2 negatively regulate Axin levels, suggesting a mechanism for the positive effect of Apc on Wg signaling.

Fig. 3.

Axin and Arm levels in Apc mutants. Third instar wing discs, stained with antisera on top. Genotypes at left margin. (A to C) Axins044230 homozygous mutant clones, indicated by β-gal loss [–/– in (A)], have decreased Axin signal, revealing specificity of Axin antiserum [(B) and (C)]. In some clones of cells with wild-type Axin levels, as indicated by increased β-gal signal [+/+ in (A)], there is increased Axin signal [arrowhead in (B)], as compared with Axin heterozygous (+/–) tissue. (D to I) Apc1Q8 Apc279 [–/– in (D)] and Apc1Q8 Apc233 [–/– in (G)] mutant clones display increased Axin signal. (J to L) Arm levels are high in Axin mutant clones [–/– in (J)]. (M to O) Similarly, high Arm levels are observed in Apc1Q8 Apc279 null mutant clones [–/– in (M)]. (P to R) In contrast, Apc1Q8 Apc233 mutant clones [–/– in (P)] display intermediate Arm levels. The Apc233 allele contains a deletion extending to the fifth Arm repeat (table S1), and its residual ability to down-regulate Arm likely results from production of a carboxy-terminal fragment, at levels below the threshold detectable with our antiserum.

To determine whether the Apc domains promoting Wg signaling are distinct from those promoting Arm degradation, we used three Apc2 truncation alleles that encode only amino-terminal fragments, Apc2d40, Apc2f90, and Apc2g10 (17, 25, 26) (Fig. 4A and table S1). These alleles eliminate some or all β-catenin/Arm binding sites and thus are severely compromised in promoting Arm degradation (16, 17, 26). However, in contrast with null alleles that eliminate all Apc2 coding sequences (fig. S2A and table S1), the Apc2 truncation alleles retain their positive effect on Arm-induced apoptosis and ectopic hth expression in Apc1Q8 mutant photoreceptors (Fig. 4B and fig. S9, A and B). We conclude that Apc2 amino-terminal fragments retain the ability to promote ectopic Wg signaling, whereas the β-catenin/Arm binding sites are dispensable for this activity. This conclusion is further supported by the observation that an Apc2 transgene (P[Apc2-Δ20rep]) (Fig. 4A), with an internal deletion of all five 20-amino acid repeats that are important for Arm binding and proteolysis (2), completely rescues compromised Wg transduction in wing discs resulting from reduced Apc activity (in Apc1Q8 Apc25-3/Apc1Q8 Apc219-3 mutants). Furthermore, the presence of the Apc2-Δ20rep fragment also prevents the increase in Axin (Fig. 4, C to E) observed in cells in which Apc activity is either completely lost (Apc1Q8 Apc279 mutant clones) (Fig. 3, D to F) or reduced (Apc1Q8 Apc233 mutant clones) (Fig. 3, G to I). Finally, in cells with reduced Apc activity and intermediate Arm levels (Apc1Q8 Apc233 mutant clones) (Fig. 3, P to R), the presence of the Apc2-Δ20rep fragment results in high Arm levels (Fig. 4, G to I) that are similar to those resulting from loss of Axin (fig. S10). Thus, the β-catenin/Arm binding sites are dispensable for the ability of Apc to negatively regulate Axin levels, to increase Arm levels, and to promote Wg transduction. These results support the model that Apc enhances Wg signaling by negatively regulating Axin-mediated Arm degradation.

Fig. 4.

The Apc2 amino-terminus affects Axin levels, Arm levels, and Wg signaling. (A) Human APC and Drosophila Apc2 schematic. Conserved region (gray), Arm repeats (white), β-catenin binding sites [15 amino acid repeats (light blue) and 20 amino acid repeats (dark blue)], Axin binding sites (SAMP repeats, hatched boxes), and nonsense mutations in Apc2g10, Apc2f90, and Apc2d40 are indicated. Somatic APC mutations in colorectal tumors cluster between codons 1250 and 1450 (mutation cluster region, MCR). Human APC amino-terminal fragment encoding amino acids 2-1247 (YFP-APC1247) and P[Apc2-Δ20rep] transgene are diagrammed. (B) Apoptosis is not suppressed in the Apc1Q8 Apc2g10/Apc1Q8 + retina. (C to E and G to I) Third instar wing discs stained with antisera in lower left corner. Apc1Q8 Apc233 mutant clones are indicated [–/– in (C) and (G)]. One copy of the P[Apc2-Δ20rep] transgene results in low Axin levels [(D) and (E)] and high Arm levels [(H) and (I)] in Apc1Q8 Apc233 clones. Identical results were found with two independent P[Apc2-Δ20rep] lines. (F) Model for dual Apc functions in Wg transduction. In addition to facilitating Axin-dependent Arm proteolysis, Apc also negatively regulates Axin (blue), thereby promoting Wg signaling. Our results support a proposed regulatory relationship between Axin and Apc (36) and also suggest that negative regulation of Axin by Apc maintains Arm levels within a range ensuring proper response upon Wg stimulation.

Previous experiments in Xenopus suggested that the vertebrate APC amino terminus also promotes Wnt signaling (27, 28). Indeed, we find that expression of a human APC amino-terminal fragment (29) (Fig. 4A) in a Wnt-responsive cell line (HEK293T) results in up-regulation of TCF reporter activity (pTOPFlash) (30) that is not observed with a control reporter containing mutated binding sites (pFOPFlash) (fig. S11). Therefore, human APC may also have positive and negative regulatory roles in Wnt transduction, with an amino terminus that promotes Wnt signaling.

Our results demonstrate that Apc1 and Apc2 act redundantly to regulate Wingless transduction both positively and negatively (Fig. 4F). Apc likely promotes Wingless transduction by negatively regulating Axin. For this function, the Apc amino terminus is essential, whereas the β-catenin/Arm binding sites are dispensable. We suggest that opposing activities of Apc keep Axin activity and Arm levels within a range ensuring that downstream signal transduction occurs in the presence of Wg but not in its absence. The C. elegans APC-related protein APR-1, which lacks consensus β-catenin binding sites, also promotes Wnt transduction (31, 32), further suggesting that an activating role is evolutionarily conserved. Our data also reveal that retention of an Apc amino-terminal fragment enhances ectopic Wg signaling resulting from reduction in Apc levels. This may explain why APC mutations that eliminate the carboxyl terminus, yet retain the amino terminus, are found in the majority of colonic adenomas and carcinomas (3335). By analogy to the positive effect of the Drosophila Apc amino terminus on ectopic Wg signaling, the conserved human APC amino terminus might similarly enhance ectopic Wnt signaling, accounting for its selective retention in colorectal carcinoma.

Supporting Online Material

www.sciencemag.org/cgi/content/full/319/5861/333/DC1

Materials and Methods

Table S1

Figs. S1 to S11

References

References and Notes

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