Mutations in SMAD tumor suppressor genes are involved in approximately 140,000 new cancers in the USA each year. At this time, how the absence of a functional SMAD protein leads to a tumor is unknown. However, clinical and biochemical studies suggest that all SMAD mutations are loss-of-function mutations. One prediction of this hypothesis is that all SMAD mutations cause tumors via a single mechanism. To test this hypothesis, we expressed five tumor-derived alleles of human SMAD genes and five mutant alleles of Drosophila SMAD genes in flies. We found that all of the DNA-binding domain mutations conferred gain-of-function activity, thereby falsifying the hypothesis. Furthermore, two types of gain-of-function mutation were identified – dominant negative and neomorphic. In numerous assays, the neomorphic allele SMAD4100T appears to be capable of activating the expression of WG target genes. These results imply that SMAD4100T may induce tumor formation by a fundamentally different mechanism from other SMAD mutations, perhaps via the ectopic expression of WNT target genes – an oncogenic mechanism associated with mutations in Adenomatous Polyposis Coli. Our results are likely to have clinical implications, because gain-of-function mutations may cause tumors when heterozygous, and the life expectancy of individuals with SMAD4100T is likely to be different from those with other SMAD mutations. From a larger perspective, our study shows that the genetic characterization of missense mutations, particularly in modular proteins,requires experimental verification.

Introduction

Signaling molecules of the Transforming Growth Factor-β (TGFβ)family regulate differentiation and proliferation in many species. Four TGFβ proteins have been characterized in Drosophila and each of these has a counterpart in mammals(Newfeld et al., 1999). For example, Decapentaplegic (DPP) shows significant amino acid similarity to mammalian BMP2 and BMP4. In addition, the function of these proteins appears to be conserved. In cross-species experiments, human BMP2 and BMP4 rescued dpp mutant phenotypes in Drosophila(Padgett et al., 1993), and recombinant DPP protein induced bone formation in mammalian cells(Sampath et al., 1993). The functional conservation revealed by cross-species experiments suggests that studies of TGFβ signaling in Drosophila will impact on our understanding of TGFβ signaling in mammals.

In brief, DPP signal transduction begins with a complex of transmembrane receptor serine-threonine kinases. Upon ligand binding, one of the receptors phosphorylates the cytoplasmic protein Mothers against DPP (MAD)(Newfeld et al., 1996). As a result of MAD phosphorylation, the MAD-related protein Medea (MED) is recruited to form a heteromeric complex with MAD(Wisotzkey et al., 1998). The MAD/MED complex then translocates to the nucleus and co-activates the transcription of DPP target genes (e.g. Xu et al., 1998).

All TGFβ family members use this mechanism, and MAD-related proteins(the SMAD family) are found in many species. SMAD family members contain two functionally distinct and highly conserved MAD homology (MH) domains. The amino-terminal MH1 domain appears to be responsible for transcriptional activation, whereas the carboxy-terminal MH2 domain appears necessary for forming multi-SMAD complexes (Lagna et al., 1996).

TGFβ1 was discovered through its anti-mitotic effects on fibroblast cells in culture (Barnard et al.,1990). However, TGFβ1 was unable to induce growth arrest in fibroblast-derived tumors (Fynan and Reiss, 1993). Subsequently, `loss of heterozygosity' studies have shown that human SMAD genes act as tumor suppressors in a wide variety of tissues. Homozygous mutations in SMAD2 or SMAD4 are detected in 20% of breast, 42% of colorectal, 17% of lung and 80% of pancreas tumors,as well as in the inherited cancer Autosomal Dominant Juvenile Polyposis(Eppert et al., 1996; Riggins et al., 1997; Howe et al., 1998). More recently, reporter gene assays of SMAD mutant alleles isolated from human tumors revealed a universal inability to activate transcription at wild-type levels (Dai et al., 1998; Xu and Attisano, 2000). These studies led to the hypothesis that all SMAD tumor mutations are loss-of-function mutations. One prediction of this hypothesis is that all mutations induce tumors via a single mechanism – the inability to transduce an anti-mitotic signal encoded by a TGFβ family member (e.g. Massagué et al.,2000).

However, the modular nature of SMAD proteins and our experience with mutations in Mad (e.g. Sekelsky et al., 1995) suggest that the situation is more complex. We propose the alternative hypothesis that there are multiple classes of SMAD mutation (loss of function and gain of function). A prediction of our hypothesis is that human SMAD mutations in different classes induce tumor formation via distinct mechanisms.

Here, we report a study, using the Gal4/UAS system, in which we generated phenotypes for mutant alleles of Mad and Med, as well as for mutant alleles of human SMAD2 and SMAD4 isolated from pancreatic and colon tumors. Our study establishes a set of principles for the transgenic characterization of human mutant alleles. In wild-type flies, the expression of a loss-of-function mutation does not generate a mutant phenotype, whereas the expression of a gain-of-function mutation generates a mutant phenotype. Furthermore, different classes of gain-of-function mutation generate different phenotypes. Phenotypes generated by dominant-negative alleles mimic genomic loss-of-function mutations. Phenotypes generated by neomorphic mutations are unrelated to the wild-type function of the gene.

Materials and methods

Sequencing Mad mutants

The entire locus was amplified by PCR from heterozygous animals. PCR products were sequenced using the Thermosequenase cycle sequencing kit (USB). The lesion was identified by the presence of two bases at a particular position corresponding to the mutant allele and the wild-type allele on the balancer chromosome.

Transgene constructs

The Mad1 allele contains an A615T mutation. By using PCR (Lorson et al., 1999),this mutation was copied into the Mad cDNA using a pair of complementary mutant primers. The Mad1 reverse primer 5′-CTGGACGGACGATTACTGGTCTCCCATCGC-3′ (the mutant base is shown in bold) and the M13 forward primer were used to create the 5′ Mad1 fragment. The Mad1 forward primer 5′-GCGATGGGAGACCAGTAATCGTCCGTCCAG-3′ and the M13 reverse primer were used to create the 3′ Mad1 fragment. A full-length Mad1 cDNA was produced using the M13 forward and M13 reverse primers, and the annealed 5′ and 3′ Mad1 fragments as a template. The Mad12 allele contains a C1601T mutation(Sekelsky et al., 1995). The Mad12 reverse primer 5′-GCGGAGTATCATCGCTAGGATGTGACCTCG-3′ and the M13 forward primer were used to create the 5′ Mad12 fragment. The Mad12 forward primer 5′-CGAGGTCACATCCTAGCGATGATACTCCGC-3′ and the M13 reverse primer were used to generate the 3′ Mad12 fragment. Mad mutant cDNAs were cloned into the XbaI and KpnI sites in pUAST (Brand and Perrimon,1993). SMAD4 mutant cDNAs(Schutte et al., 1996) were excised by cutting with BamHI and EcoRV, and cloned into blunted XhoI and BglII sites of pUAST. An asymmetric SacI site was used to check orientation. Additional SMAD mutant cDNAs(Riggins et al., 1996) were excised by cutting with BamHI, blunting the ends and cutting with KpnI. cDNAs were then cloned into the XbaI (blunted) and KpnI sites in pUAST. Multiple independent fly lines were generated for five mutants.

Drosophila genetics

Fly stocks were as described: In(2L)dpps6 and dpphr4 (St Johnston et al., 1990); Df(2L)C28, Mad1, Mad11and Mad12 (Sekelsky et al., 1995); Df(3R)E40 and Med7(Raftery et al., 1995); zw3M11 FRT101 and zw3sggD127 FRT101(Siegfried et al., 1992); UAS.Dpp, UAS.CA-Arm, UAS.DN-TCF, UAS.Mad, UAS.Gbb, UAS.lacZ, 24B.Gal4,32B.Gal4, 69B.Gal4, A9.Gal4, C765.Gal4, MS1096.Gal4, T80.Gal4, ap.Gal4,dll.Gal4, dpp-blink.Gal4, en.Gal4 and ptc.Gal4(Drysdale and Crosby, 2005). Dominant enhancement of dpps6/dpphr4wing phenotypes by Df(2L)C28, Mad1, Df(3R)E40 and Med7 was evaluated by examining at least 500 individuals of each genotype. Adult Gal4/UAS genotypes were generated using males from two independent lines homozygous for a UAS construct crossed to Gal4 bearing females at 25°C. Tables containing quantitative data for all Gal4/UAS genotypes are available upon request. For Gal4/UAS combinations involving SMAD2 alleles, wing size was calculated as described(Marquez et al., 2001). Clones of cells homozygous for the genetic null zw3sggD127 or the protein null zw3M11 were generated by standard methods(Siegfried et al., 1992).

Embryos and discs

Histochemical detection of β-galactosidase activity in embryos was conducted as described (Newfeld et al.,1996). Embryonic cuticles were prepared by standard methods. Vein primordia in pupal wing imaginal discs were detected with a monoclonal antibody recognizing Drosophila SRF(Marenda et al., 2004) or a riboprobe transcribed from a rhomboid cDNA(Wolff, 2000). Anterior margin bristle primordia in third instar larval wing discs were detected with a monoclonal antibody recognizing Achaete(Skeath and Carroll,1991).

Results

Mad1 and Med7 are gain-of-function alleles with dominant-negative activity

The Mad1 allele intrigued us for two reasons. First, it was the only Mad allele isolated from two screens (75,000 chromosomes in total) for modifiers of dpp adult viable phenotypes(Sekelsky et al., 1995; Su et al., 2001). By contrast,four alleles of Mad, including the Df(2L)C28 deletion, were identified in a pilot screen (1850 chromosomes) for dominant maternal enhancement of dpp embryonic lethal phenotypes(Raftery et al., 1995). Second, in an initial genetic analysis, Mad1 was a stronger enhancer of two dpp recessive embryonic lethal mutations than was Df(2L)C28 (Sekelsky et al., 1995).

In re-examining the initial data on Mad1, it became clear that certain classes of gain-of-function mutation (e.g. dominant negative) could not be detected in an assay for enhancement of lethality. The original data showed that heterozygosity for an allele with a complete loss of Mad function, such as Df(2L)C28, resulted in essentially maximum enhancement. Only 0.5-3.0% of the expected progeny survive(Sekelsky et al., 1995). In this assay, Mad mutant alleles with dominant-negative effects would appear to be loss-of-function alleles, as less than 0% of the expected progeny is not possible – 0% was seen for Mad1 enhancement of dpphr56.

We conducted a less restrictive test of the relationship between Mad1 and dpp to determine whether Mad1 was a dominant-negative allele. For the test, we exploited a dpp adult-viable mutant phenotype(Nicholls and Gelbart, 1998). The test is based upon a transheterozygous dpp mutant genotype that is adult viable with occasional wing vein defects(Fig. 1A). Truncation of Longitudinal vein 5 (L5) is observed in 11% of flies bearing the dpps6/dpphr4 genotype, while 2%display truncations of L4 and L5.

Fig. 1.

Mad1 and Med7 are dominant-negative alleles. (A) dpps6/dpphr4 wing with normal veins. Longitudinal veins 1-5, the anterior crossvein (acv) and the posterior crossvein (pcv) are shown. (B) dpps6/dpphr4Df(2L)C28 with truncated L4/L5. (C) dpps6/dpphr4 Mad1 with abnormal L2/L3/L4, no crossveins and two margin notches. (D) dpps6/dpphr4 Med7 with a truncated L5 and two margin notches.

Fig. 1.

Mad1 and Med7 are dominant-negative alleles. (A) dpps6/dpphr4 wing with normal veins. Longitudinal veins 1-5, the anterior crossvein (acv) and the posterior crossvein (pcv) are shown. (B) dpps6/dpphr4Df(2L)C28 with truncated L4/L5. (C) dpps6/dpphr4 Mad1 with abnormal L2/L3/L4, no crossveins and two margin notches. (D) dpps6/dpphr4 Med7 with a truncated L5 and two margin notches.

When we placed a single copy of Df(2L)C28 into dpps6/dpphr4 individuals, the frequency of L5 defects increased to 95%, 20% had defects in L4 and L5(Fig. 1B), and 5% had defects in L5 and L2, or L5 and the posterior crossvein. When we placed the Mad1 allele into dpps6/dpphr4 individuals, the frequency and severity of vein defects increased beyond that seen with Df(2L)C28. In flies carrying Mad1, the frequency of wings with defects in L4 and L5 increases to virtually 100%. Furthermore,10% of wings with L4 and L5 defects have missing crossveins (anterior,posterior or both) and small margin notches(Fig. 1C). We also noted that a statistically significant amount of lethality was associated with the dpps6/dpphr4 Mad1 genotype. Only 71%of the expected dpps6/dpphr4 Mad1progeny were recovered (P<0.005). No lethality was associated with either the dpps6/dpphr4 or the dpps6/dpphr4 Df(2L)C28 genotypes. Mad1 has a greater effect on dpps6/dpphr4 vein phenotypes and survival than does a deletion of Mad, indicating that Mad1 is a gain-of-function allele with dominant-negative activity.

We then identified the lesion in Mad1 and in six additional Mad alleles (Table 1). The Mad1 mutation is an amino acid substitution (Q90L) due to an A to T transversion at nucleotide 615 of the cDNA. Of thirteen sequenced Mad mutant alleles(Sekelsky et al., 1995; Chen et al., 1998) (and this report), Mad1 is the only mutation in the MH1 domain. Given that Mad1 is a dominant-negative allele with a lesion in the transcriptional activation domain, we generated the following two-part hypothesis for Mad1 dominant-negative activity. First, we propose that the MAD1 protein is unable to activate transcription, but is capable of receptor phosphorylation and of forming complexes with its partner SMAD protein Medea (MED). Second, as a result,MAD1 dominant-negative activity derives from the formation of non-functional complexes that deplete the pool of MED to the point that wild-type MAD proteins are unable to find sufficient partners for normal activity.

Table 1.

New Mad mutant sequences

AlleleLesionCodonDomainAlterationEffect*
Mad1 Missense 90 MH1 Gln to Leu Dominant negative 
Mad5 Missense 272 MH2 Arg to His Loss of function (hypomorph) 
Mad6 Missense 272 MH2 Arg to His Loss of function (hypomorph) 
MadB65.3 Missense 272 MH2 Arg to His Loss of function (hypomorph) 
Mad8 Missense 358 MH2 Ser to Leu Loss of function (hypomorph) 
Mad11 Missense 410 MH2 Gly to Asp Loss of function (hypomorph) 
Mad7 Missense 419 MH2 Val to Met Loss of function (null) 
AlleleLesionCodonDomainAlterationEffect*
Mad1 Missense 90 MH1 Gln to Leu Dominant negative 
Mad5 Missense 272 MH2 Arg to His Loss of function (hypomorph) 
Mad6 Missense 272 MH2 Arg to His Loss of function (hypomorph) 
MadB65.3 Missense 272 MH2 Arg to His Loss of function (hypomorph) 
Mad8 Missense 358 MH2 Ser to Leu Loss of function (hypomorph) 
Mad11 Missense 410 MH2 Gly to Asp Loss of function (hypomorph) 
Mad7 Missense 419 MH2 Val to Met Loss of function (null) 
*

Previously reported (Sekelsky et al.,1995), except for Mad1 and MadB65.3.

Circumstantial evidence for the first part of this hypothesis is provided by two sources. First, biochemical studies of human SMAD4 show that MH1 mutations prevent DNA binding and transcriptional activation(Dai et al., 1998; Xu and Attisano, 2000). Second, an examination of aligned SMAD MH1 sequences(Newfeld et al., 1999)revealed that the amino acid affected in the MAD1 protein, Q90, is conserved in all fly and vertebrate SMADs that participate in DNA binding(receptor-associated SMADs and co-SMADs), but is not conserved in any antagonist SMADs. Furthermore, in the structure of the SMAD3 MH1 domain bound to DNA, Q90 is one of three residues that make direct base contact(Shi et al., 1998). Nematode SMAD sequences show the same relationship between Q90 and DNA binding. Q90 is present in the unusual antagonist DAF-3. DAF-3 antagonizes TGFβ signal transduction by binding to DNA and repressing gene expression(Thatcher et al., 1999), a mechanism not used by other antagonist SMADs. In addition, Q90 is not present in the atypical receptor-associated SMADs DAF-8 and DAF-14. Both DAF-8 and DAF-14 stimulate the expression of TGFβ target genes by inhibiting DAF-3 function (Inoue and Thomas,2000). Drosophila SMAD2 is the single exception. The amino acid corresponding to Q90 in Drosophila SMAD2 falls in a run of nine amino acids unlike any other SMAD protein.

In order to experimentally test our hypothesis that the MAD1protein is transcriptionally inactive, we generated UAS.Mad1 and UAS.Mad12. The Mad12 allele has a nonsense mutation (Q417Stop) in the MH2 domain that removes the three serines phosphorylated in response to DPP signaling. The Mad12 allele behaves exactly like a deletion of Mad in both genetic and biochemical assays(Sekelsky et al., 1995; Hoodless et al., 1996). We used UAS.Mad12 as a control for overexpression of a loss-of-function mutation. We also examined UAS.Mad, as a control for overexpression of the wild-type protein.

In an initial characterization, we expressed these transgenes in wild-type wing discs and assayed their effect on vein formation in adult wings. We have shown, using Mad12 mutant clones, that vein formation is dependent on Mad activity(Marquez et al., 2001). Expression of UAS.Mad (Fig. 2B) (Marquez et al.,2001) produces excess vein tissue. Alternatively, Madgenomic loss-of-function genotypes (transheterozygous combinations of hypomorphic alleles) show reduced venation and growth defects(Fig. 2D)(Sekelsky et al., 1995). Expression of the loss-of-function allele UAS.Mad12 with 69B.Gal4 had no effect on any aspect of wing development(Fig. 2C). Expression of UAS.Mad1 with 69B.Gal4 led to vein truncations and incomplete wing outgrowth (Fig. 2E). Expression of UAS.Mad with 69B.Gal4 did not lead to defects in vein formation or wing outgrowth (data not shown). The UAS.Mad1 phenotype resembles the Madloss-of-function phenotype (compare Fig. 2D with 2E).

In addition to effects on venation, many UAS.Mad1genotypes showed significant reductions in viability. For example, 52% of the expected UAS.Mad1/T80.Gal4 flies, 55% of the UAS.Mad1/dll.Gal4 flies and 69% of the UAS.Mad1/24B.Gal4 flies were observed(P<0.005). Expression of UAS.Mad or UAS.Mad12 had no effect on viability. These studies are consistent with our genetic analysis of Mad1, supporting its identification as a dominant-negative allele.

To further test our hypothesis that the MAD1 protein is transcriptionally inactive, we examined two molecular markers for vein formation in wing imaginal discs. We analyzed Drosophila Serum response factor (SRF; Blistered – FlyBase) protein expression and rhomboid (rho) transcript accumulation. DrosophilaSRF expression is widespread in third instar wing discs but its expression is downregulated in vein primordia by rho in pupal wing discs(Fig. 3A)(Biehs et al., 1998). rho transcription in vein primordia may be directly dependent upon DPP signaling via MAD (Fig. 3E)(Yu et al., 1996). Thus, in vein development, the expansion of Drosophila SRF expression indicates a lack of rho activity and the absence of rhoactivity indicates that it is not being transcribed by MAD.

Fig. 2.

Expression of MAD1 and SMAD4130S generates phenotypes that resemble Mad loss-of-function phenotypes. (A) Wild-type wing.(B) UAS.Mad/ptc.Gal4 with extra vein tissue between L3/L4/L5. (C) UAS.Mad12/69B.Gal4 appears wild type. (D) Mad11/Mad12 with no L2, abnormal L4/L5, no crossveins and a large margin notch (not to scale). (E) UAS.Mad1/69B.Gal4 with truncated L2/L3/L4/L5, abnormal crossveins and a moderate margin notch. (F) UAS.SMAD4130S/A9.Gal4 with truncated L2/L3/L4/L5 and a small margin notch.

Fig. 2.

Expression of MAD1 and SMAD4130S generates phenotypes that resemble Mad loss-of-function phenotypes. (A) Wild-type wing.(B) UAS.Mad/ptc.Gal4 with extra vein tissue between L3/L4/L5. (C) UAS.Mad12/69B.Gal4 appears wild type. (D) Mad11/Mad12 with no L2, abnormal L4/L5, no crossveins and a large margin notch (not to scale). (E) UAS.Mad1/69B.Gal4 with truncated L2/L3/L4/L5, abnormal crossveins and a moderate margin notch. (F) UAS.SMAD4130S/A9.Gal4 with truncated L2/L3/L4/L5 and a small margin notch.

In UAS.Mad/ptc.Gal4 discs, Drosophila SRF expression is reduced in the primordia of L3, L4 and L5(Fig. 3B). This is consistent with the excess vein phenotype of UAS.Mad/ptc.Gal4 wings(Fig. 2B). In UAS.Mad12/69B.Gal4 discs, Drosophila SRF expression is essentially unaffected (Fig. 3C). This is consistent with the wild-type appearance of UAS.Mad12/69B.Gal4 wings(Fig. 2C). In UAS.Mad1/69B.Gal4 discs, Drosophila SRF expression is expanded in the primordia of L3, L4 and L5(Fig. 3D). This is consistent with the reduced venation of UAS.Mad1/69B.Gal4 wings(Fig. 2E). These results suggest that MAD1 is incapable of activating rhoand, furthermore, that MAD1 protein can block the ability of endogenous MAD to activate rho.

Examination of rho transcript accumulation in pupal wing discs expressing UAS.Mad1 confirms this interpretation. In UAS.Mad1 discs, rho expression is reduced in the primordia of L3, L4 and L5. L4 expression is the most severely affected(Fig. 3F). This is consistent with the vein phenotype of UAS.Mad1/69B.Gal4 wings(Fig. 2E). Because rhomay be a direct transcriptional target of MAD, this result supports our hypothesis that the MAD1 protein cannot activate transcription.

We then noted that of the 18 sequenced Med mutant alleles only Med7 is an MH1 domain mutation(Das et al., 1998; Hudson et al., 1998; Wisotzkey et al., 1998). Furthermore, the C99S mutation in Med7 alters one of three cysteine residues that is predicted to coordinate a zinc atom(Chai et al., 2003). We tested Med7 and the Df(3R)E40 deletion of Medin our assay for enhancement of the dpps6/dpphr4 wing phenotype. In this assay, Df(3R)E40 increased the frequency of dpps6/dpphr4 wings with L5 truncations from 11%to 28%. When we placed the Med7 allele into dpps6/dpphr4 individuals, the frequency and severity of vein defects increased beyond that seen with Df(3R)E40. In flies carrying Med7, the frequency of wings with defects in L5 and L4 was 42%. Furthermore, 15% of these wings also have defects in L2, or the posterior crossvein or margin notches(Fig. 1D). Med7 is also a gain-of-function allele with dominant-negative activity.

Fig. 3.

Expression of MAD1 results in expanded Drosophila SRF expression as a result of reduced rhomboid transcription. (A)Wild-type pupal wing disc stained for Drosophila SRF expression. Anterior is towards the top and distal to the left. L1-L5 primordia are indicated. (B) UAS.Mad/ptc.Gal4 has expanded regions without Drosophila SRF corresponding to L3/L4. (C) UAS.Mad12/69B.Gal4 appears wild type. (D) UAS.Mad1/69B.Gal4 with expanded regions of Drosophila SRF that limit the extent of L3/L4. (E) Wild-type pupal wing disc stained for rhomboid (rho) transcripts. L3/L4/L5 primordia are indicated. rho expression in L1/L2 is not yet fully developed at this stage; note the difference in the visibility of L1/L2 versus L3/L4/L5 in A. (F) UAS.Mad1/69B.Gal4 with reduced rho expression. Expression in L4 is severely reduced and distal truncations in L3/L5 are visible.

Fig. 3.

Expression of MAD1 results in expanded Drosophila SRF expression as a result of reduced rhomboid transcription. (A)Wild-type pupal wing disc stained for Drosophila SRF expression. Anterior is towards the top and distal to the left. L1-L5 primordia are indicated. (B) UAS.Mad/ptc.Gal4 has expanded regions without Drosophila SRF corresponding to L3/L4. (C) UAS.Mad12/69B.Gal4 appears wild type. (D) UAS.Mad1/69B.Gal4 with expanded regions of Drosophila SRF that limit the extent of L3/L4. (E) Wild-type pupal wing disc stained for rhomboid (rho) transcripts. L3/L4/L5 primordia are indicated. rho expression in L1/L2 is not yet fully developed at this stage; note the difference in the visibility of L1/L2 versus L3/L4/L5 in A. (F) UAS.Mad1/69B.Gal4 with reduced rho expression. Expression in L4 is severely reduced and distal truncations in L3/L5 are visible.

Perhaps dominant-negative activity explains the relative infrequency of detectable MH1 mutations in Mad and Med. Just two out of 33 sequenced mutations (6%) occur in the MH1 even though this domain covers roughly 23% of the amino acids in these proteins.

SMAD4130S and SMAD4100T are gain-of-function alleles associated with human tumors

Our identification of Mad1 and Med7as gain-of-function alleles with MH1 mutations suggested that SMAD MH1 mutations identified in human tumors might also generate gain-of-function alleles. We tested this hypothesis with a set of five SMAD mutant cDNAs derived from pancreas and colon tumors(Table 2).

Table 2.

Human SMAD tumor alleles expressed in flies

AlleleLesionCodonDomainAlterationTumorReference
SMAD4100T Missense 100 MH1 Arg to Thr Pancreas Schutte et al., 1996  
SMAD4130S Missense 130 MH1 Pro to Ser Colorectal Thiagalingam et al., 1996  
SMAD4493H Missense 493 MH2 Asp to His Pancreas Hahn et al., 1996  
SMAD4524ST Nonsense 524 MH2 Cys to Stop Colorectal Riggins et al., 1996  
SMAD2Δ344-358 Deletion 344-358 MH2 14aa deletion Colorectal Riggins et al., 1996  
AlleleLesionCodonDomainAlterationTumorReference
SMAD4100T Missense 100 MH1 Arg to Thr Pancreas Schutte et al., 1996  
SMAD4130S Missense 130 MH1 Pro to Ser Colorectal Thiagalingam et al., 1996  
SMAD4493H Missense 493 MH2 Asp to His Pancreas Hahn et al., 1996  
SMAD4524ST Nonsense 524 MH2 Cys to Stop Colorectal Riggins et al., 1996  
SMAD2Δ344-358 Deletion 344-358 MH2 14aa deletion Colorectal Riggins et al., 1996  

In studies with transgenes expressing wild-type SMAD2 or SMAD4, we showed that their phenotypes mirrored those of their fly homologs, Drosophila SMAD2 and MED, respectively(Marquez et al., 2001). This suggested to us that human SMAD proteins expressed in flies function in the same manner as their fly homologs do. By extending this idea to SMAD tumor alleles, we predict that the manner in which a mutation affects the activity of a human SMAD protein expressed in flies will inform us about the effect of that mutation on the human SMAD protein.

When SMAD alleles with mutations in the MH2 domain(UAS.SMAD4524ST,UAS.SMAD2Δ344-358 and UAS.SMAD4493H) are expressed in flies, their wings are wild type in size and appearance like those expressing the loss-of-function allele UAS.Mad12. Alternatively, wings expressing the MH1 missense mutation UAS.SMAD4130S(Fig. 2F) are similar to those expressing the dominant-negative allele UAS.Mad1(Fig. 2E). Both UAS.SMAD4130S and UAS.Mad1 engender defects in vein formation and wing outgrowth. Furthermore, UAS.SMAD4130S expression results in reduced viability like UAS.Mad1 does. For example, UAS.SMAD4130S/dll.Gal4 flies were recovered at 36% and UAS.Mad1/dll.Gal4 flies were recovered at 55% of the expected frequency. Results for SMAD4130S suggest that it too is a gain-of-function allele with dominant-negative activity.

The expression of the MH1 missense allele SMAD4100Tgenerated a wing phenotype never before reported in any study of TGFβsignaling in flies. Most wings expressing UAS.SMAD4100Tstrongly throughout the wing blade (e.g. MS1096.Gal4, C765.Gal4 or T80.Gal4) have ectopic mechanosensory bristles on the blade(Fig. 4A). In wild type, two rows of mechanosensory bristles (stout and thin) are normally found in the triple row region of the proximal anterior wing margin(Couso et al., 1994). The ectopic mechanosensory bristles seen in wings expressing UAS.SMAD4100T are largely derived from the transformation of normally `bristle-less' mechanosensory receptors called campaniform sensilla (Held, 2002).

Fig. 4.

Expression of SMAD4100T induces ectopic mechanosensory bristles on the wing blade. (A) UAS.SMAD4100T/MS1096.Gal4 wing with six ectopic mechanosensory bristles (open arrowheads). This image is a composite of three wings such that all positions for ectopic bristles are shown (the three bristles on medial L3 never appear together). (B) Dorsal L1 of wild type with two campaniform sensilla (arrowheads). (C) Dorsal L1 of UAS.SMAD4100T/MS1096.Gal4 with a stout mechanosensory bristle replacing Twin Sensillum One (open arrowhead; black arrowhead indicates Twin Sensillum Two). (D) Ventral L3 of wild type with a campaniform sensillum (arrowhead). (E) Ventral L3 of UAS.SMAD4100T/MS1096.Gal4 with a stout mechanosensory bristle replacing the sensillum (open arrowhead). (F) Dorsal L3 of wild type with three campaniform sensilla (arrowheads). (G) Dorsal L3 of UAS.SMAD4100T/MS1096.Gal4 with a thin mechanosensory bristle replacing the proximal sensillum (open arrowhead). (H) Dorsal L3 of UAS.SMAD4100T/MS1096.Gal4 with a thin mechanosensory bristle replacing the middle sensillum (open arrowhead). (I) Ectopic thin mechanosensory bristles are seen on a wing with small, unmarked clones of cells homozygous for zw3M11 (open arrowheads).

Fig. 4.

Expression of SMAD4100T induces ectopic mechanosensory bristles on the wing blade. (A) UAS.SMAD4100T/MS1096.Gal4 wing with six ectopic mechanosensory bristles (open arrowheads). This image is a composite of three wings such that all positions for ectopic bristles are shown (the three bristles on medial L3 never appear together). (B) Dorsal L1 of wild type with two campaniform sensilla (arrowheads). (C) Dorsal L1 of UAS.SMAD4100T/MS1096.Gal4 with a stout mechanosensory bristle replacing Twin Sensillum One (open arrowhead; black arrowhead indicates Twin Sensillum Two). (D) Ventral L3 of wild type with a campaniform sensillum (arrowhead). (E) Ventral L3 of UAS.SMAD4100T/MS1096.Gal4 with a stout mechanosensory bristle replacing the sensillum (open arrowhead). (F) Dorsal L3 of wild type with three campaniform sensilla (arrowheads). (G) Dorsal L3 of UAS.SMAD4100T/MS1096.Gal4 with a thin mechanosensory bristle replacing the proximal sensillum (open arrowhead). (H) Dorsal L3 of UAS.SMAD4100T/MS1096.Gal4 with a thin mechanosensory bristle replacing the middle sensillum (open arrowhead). (I) Ectopic thin mechanosensory bristles are seen on a wing with small, unmarked clones of cells homozygous for zw3M11 (open arrowheads).

Two campaniform sensilla (Twin Sensilla One and Two) are located on the dorsal surface of L1 (Fig. 4B). The most frequent transformation seen in UAS.SMAD4100T/MS1096.Gal4 wings is a stout mechanosensory bristle in place of Twin Sensillum One(Fig. 4C). A sensillum on the ventral surface of L3 has only recently been reported(Aigouy et al., 2004)(Fig. 4D). A stout mechanosensory bristle is occasionally seen in the place of this sensillum on a UAS.SMAD4100T/MS1096.Gal4 wing(Fig. 4E). Three sensilla(Sensilla Campaniformium of Dorsal Radius) are located on the dorsal surface of L3 (Fig. 4F). The second most frequent transformation seen in UAS.SMAD4100T/MS1096.Gal4 wings is a thin mechanosensory bristle replacing either the proximal (Fig. 4G), middle (Fig. 4H) or distal (not shown) sensillum at roughly equal frequencies. The most posterior ectopic bristle on a UAS.SMAD4100T/MS1096.Gal4 wing is rare, and is located on the ventral surface of L5 (Fig. 4A). We could not detect a campaniform sensillum in this location and none has been reported. Thus, five out of the six ectopic bristles on UAS.SMAD4100T/MS1096.Gal4 wings are derived from the transformation of sensilla.

Though never previously associated with TGFβ signaling, the presence of ectopic anterior margin bristles on the wing blade is not unprecedented. Wingless (WG) is a secreted signaling molecule expressed along the presumptive wing margin in imaginal discs. Numerous studies have shown that mechanosensory bristle development along the margin requires WG as well as components of the canonical WG signaling pathway (Couso et al., 1994). Alternatively, when ectopic WG signaling is activated in the presumptive wing blade, for example in zeste white 3(zw3; shaggy, sgg – FlyBase) null clones, ectopic mechanosensory bristles result (Blair,1992). Several thin mechanosensory bristles on the wing blade emanating from unmarked clones of zw3M11 cells are shown(Fig. 4I). Ectopic bristles can be generated anywhere on the wing blade by zw3M11clones.

The unexpected transformation of sensilla to bristles on wings expressing UAS.SMAD4100T suggests the hypothesis that the R100T mutation in SMAD4100T conveys a novel activity upon the encoded protein. Thus, SMAD4100T is the second gain-of-function allele associated with a human tumor. Furthermore, the similarity between the UAS.SMAD4100T wing phenotype and that of zw3M11 clones suggests a second hypothesis –that SMAD4100T has the ability to activate WG target genes.

To test our hypothesis that the R100T mutation conveys a novel activity upon SMAD4, we examined wings expressing Drosophila TGFβ family members with known roles in wing development (DPP and GBB)(Ray and Wharton, 2001). We wondered whether these genes could generate ectopic bristles on the wing blade. In these experiments, UAS.Dpp expression was absolutely lethal, and significant lethality was also associated with UAS.Gbbexpression. In the two experiments where adult flies were obtained, we found that UAS.Gbb inhibits triple row bristle formation. ap.Gal4is expressed in all dorsal cells of the wing disc(Diaz-Benjumea and Cohen,1993). We observed that the proximal 25% of the anterior margin of a UAS.Gbb/ap.Gal4 wing has no bristles(Fig. 5A). More distally, the triple row reappears but is quite irregular(Fig. 5B,C). When UAS.Gbb was driven in all cells of the wing blade with C765.Gal4 (de Celis et al.,1996), the phenotype was similar but less severe (data not shown). In summary, none of our studies of UAS.Dpp or UAS.Gbbgenerated sensilla to bristle transformation, supporting our hypothesis that UAS.SMAD4100T is a gain-of-function allele with novel activity.

Fig. 5.

Expression of GBB does not induce ectopic anterior margin bristles on the wing blade. (A) Ventral view of a UAS.Gbb/ap.Gal4 wing with all bristles missing from the proximal anterior wing margin (arrowhead). A distal portion of the anterior wing margin, indicated by the black bar, is shown at higher magnification in B,C. (B) Dorsal view: the row of stout mechanosensory bristles appears in a roughly wild-type pattern with occasional gaps. The row of widely spaced chemosensory bristles shows considerable irregularity in spacing, with some bristles displaced dorsally. (C) Ventral view: the row of alternating chemosensory and thin mechanosensory bristles is disorganized and numerous thin mechanosensory bristles are present in a region ventral to their normal location.

Fig. 5.

Expression of GBB does not induce ectopic anterior margin bristles on the wing blade. (A) Ventral view of a UAS.Gbb/ap.Gal4 wing with all bristles missing from the proximal anterior wing margin (arrowhead). A distal portion of the anterior wing margin, indicated by the black bar, is shown at higher magnification in B,C. (B) Dorsal view: the row of stout mechanosensory bristles appears in a roughly wild-type pattern with occasional gaps. The row of widely spaced chemosensory bristles shows considerable irregularity in spacing, with some bristles displaced dorsally. (C) Ventral view: the row of alternating chemosensory and thin mechanosensory bristles is disorganized and numerous thin mechanosensory bristles are present in a region ventral to their normal location.

To test the second hypothesis (that the novel activity of the SMAD4100T protein is the ability to activate WG target genes), we conducted three experiments. In the first, we tested the ability of SMAD4100T to suppress phenotypes generated by DN-TCF, a dominant-negative form of the WG pathway transcription factor Drosophila TCF (Pangolin, PAN – FlyBase). For this test, we used MS1096.Gal4, a driver strongly expressed throughout the wing blade. The wings of UAS.DN-TCF/MS1096.Gal4 flies(Fig. 6A) are very similar to those with impaired WG signaling (e.g. Couso et al., 1994). These wings are very small and lack a margin. However, if two(Fig. 6B) or three(Fig. 6C) copies of UAS.SMAD4100T are also present, then the UAS.DN-TCF/MS1096.Gal4 wing phenotype is visibly suppressed in a dosage-dependent fashion. If instead we add copies of UAS.lacZ, then the UAS.DN-TCF/MS1096.Gal4 wing phenotype is unaffected (data not shown). This result suggests that, even in the presence of a strong antagonist of WG signaling, SMAD4100T is capable of activating WG target genes involved in wing growth and margin formation.

Fig. 6.

Expression of SMAD4100T suppresses wing phenotypes generated by dominant-negative Drosophila TCF. (A) UAS.DN-TCF/MS1096.Gal4wing is only 10% of the size of a wild-type wing and has no margin. (B) UAS.DN-TCF/MS1096.Gal4 wing with two copies of UAS.SMAD4100T is roughly three-fold larger than the wing in A, and the anterior margin is restored. Transformation of Twin Sensillum One into a bristle is noted (arrowhead). (C) UAS.DN-TCF/MS1096.Gal4wing with three copies of UAS.SMAD4100T is roughly four-fold larger than the wing in A, and the anterior margin is restored. Sensillum-to-bristle transformation is noted (arrowhead). (D) UAS.SMAD4100T/MS1096.Gal4 wing has a normal margin and two sensilla-to-bristle transformations (arrowheads). (E) Wing with MS1096.Gal4 driving two copies of UAS.SMAD4100Thas a normal margin, sensilla-to-bristle transformation (arrowhead) and vein defects. (F) Wing with MS1096.Gal4 driving three copies of UAS.SMAD4100T has a reduced size, a normal margin,sensilla-to-bristle transformation (arrowhead) and vein defects.

Fig. 6.

Expression of SMAD4100T suppresses wing phenotypes generated by dominant-negative Drosophila TCF. (A) UAS.DN-TCF/MS1096.Gal4wing is only 10% of the size of a wild-type wing and has no margin. (B) UAS.DN-TCF/MS1096.Gal4 wing with two copies of UAS.SMAD4100T is roughly three-fold larger than the wing in A, and the anterior margin is restored. Transformation of Twin Sensillum One into a bristle is noted (arrowhead). (C) UAS.DN-TCF/MS1096.Gal4wing with three copies of UAS.SMAD4100T is roughly four-fold larger than the wing in A, and the anterior margin is restored. Sensillum-to-bristle transformation is noted (arrowhead). (D) UAS.SMAD4100T/MS1096.Gal4 wing has a normal margin and two sensilla-to-bristle transformations (arrowheads). (E) Wing with MS1096.Gal4 driving two copies of UAS.SMAD4100Thas a normal margin, sensilla-to-bristle transformation (arrowhead) and vein defects. (F) Wing with MS1096.Gal4 driving three copies of UAS.SMAD4100T has a reduced size, a normal margin,sensilla-to-bristle transformation (arrowhead) and vein defects.

We made two additional observations in these experiments. First, siblings to our experimental flies (those without UAS.DN-TCF) showed dosage-dependant dominant-negative phenotypes for DPP-dependent processes such as vein formation (Fig. 6D-F). Thus, above a certain threshold, UAS.SMAD4100T resembles the other MH1 mutations in our study – it has dominant-negative effects on DPP signaling. Second, we found that the UAS.DN-TCF/MS1096.Gal4genotype is male-specific lethal. In a cross of MS1096.Gal4homozygous females to UAS.DN-TCF homozygous males, we obtained 199 female progeny and no male progeny. By using the F1 females in crosses to males containing various numbers of UAS.SMAD4100Tinsertions, we obtained male progeny bearing MS1096.Gal4 and UAS.DN-TCF. The suppression of male-specific lethality suggests that SMAD4100T is able to activate WG target genes outside of wing discs.

To further support this observation, we expressed SMAD4100T in the embryonic ventral epidermis. In most abdominal segments, the ventral epidermis is composed of twelve rows of cells: six rows that secrete smooth cuticle followed by six rows that secrete protrusions called denticles(Fig. 7A). Cells choose to secrete naked cuticle or denticles according to positional information supplied, in part, by WG and Engrailed (EN)(Gritzan et al., 1999; Alexandre et al., 1999). WG signals instruct cells to secrete naked cuticle. EN is expressed just posterior to WG and an EN-expressing cell secretes the first denticle row(Fig. 7B). When a constitutively active form of the WG pathway signal transducer Armadillo(CA-ARM) is expressed via en.Gal4, the first row of denticles in all segments is transformed into naked cuticle(Fig. 7C). When SMAD4100T is expressed with en.Gal4, patches of cells within the first denticle row are transformed into naked cuticle on one or more segments of most embryos (Fig. 7D). This result is consistent with a recent report that weak global expression of CA-ARM results in a patchy loss of denticles(Hayward et al., 2005). We conclude that SMAD4100T is capable of phenocopying activated WG signaling in the ventral epidermis. These data support the idea that the ability of SMAD4100T to activate WG target genes is not context dependent.

Moving from the phenotypic to the molecular level, we looked directly at the expression of Achaete (AC) in wing discs expressing SMAD4100T. AC is expressed in the presumptive anterior wing margin(Fig. 8A) coincident with WG. Genetic analyses have shown that ac is required for the development of the margin bristles and that ac is a direct target of WG signaling(Skeath and Carroll, 1991; Blair, 1992). As a result, wing discs bearing clones of cells homozygous for a mutation in zw3 have numerous regions of ectopic AC expression in the presumptive wing blade(Fig. 8B). The same result is seen when examining AC expression in wing discs from individuals expressing UAS.SMAD4100T (Fig. 8C). In a UAS.DPP-expressing disc, the pattern of AC expression along the presumptive margin is normal, although the disc is overgrown,reflecting the ability of DPP to influence wing growth(Fig. 8D). In a UAS.GBB-expressing disc, AC expression is absent in the most proximal region of the anterior margin, reflecting the ability of GBB to inhibit bristle formation in this region (Fig. 8E). Overall, our studies of DN-TCF, CA-ARM and AC strongly support the hypothesis that the SMAD4100T protein is capable of activating the expression of WG pathway target genes.

Fig. 7.

Expression of SMAD4100T mimics the expression of constitutively active ARM. (A) Wild-type six-row denticle pattern shown with anterior to the left. The small, anteriorly pointed denticles of row one are indicated(arrowhead). (B) UAS.lacZ/en.Gal4 six-row denticle pattern; lacZ expression is coincident with row one (arrowhead). (C) UAS.CA-ARM/en.Gal4 five-row denticle pattern. The transformation of row one denticles into naked cuticle is indicated (arrowhead). (D) UAS.SMAD4100T/en.Gal4 six-row denticle pattern. The transformation of a subset of row one denticles into naked cuticle is indicated (arrowhead).

Fig. 7.

Expression of SMAD4100T mimics the expression of constitutively active ARM. (A) Wild-type six-row denticle pattern shown with anterior to the left. The small, anteriorly pointed denticles of row one are indicated(arrowhead). (B) UAS.lacZ/en.Gal4 six-row denticle pattern; lacZ expression is coincident with row one (arrowhead). (C) UAS.CA-ARM/en.Gal4 five-row denticle pattern. The transformation of row one denticles into naked cuticle is indicated (arrowhead). (D) UAS.SMAD4100T/en.Gal4 six-row denticle pattern. The transformation of a subset of row one denticles into naked cuticle is indicated (arrowhead).

Fig. 8.

Expression of SMAD4100T activates the expression of the WG target gene achaete. (A) Wild-type third instar wing disc stained for Achaete (AC). a, anterior; m, proximal margin; v, ventral surface; dis, distal margin. (B) zw3M11 clones produce ectopic AC expression on the presumptive blade (arrowheads). (C) UAS.SMAD4100T/C765.Gal4 with ectopic AC expression on the presumptive blade (arrowheads). (D) UAS.Dpp/C765.Gal4. This is a lethal genotype. The disc is overgrown (image shown at a reduced magnification) but it has wild-type AC expression. (E) UAS.Gbb/C765.Gal4 with reduced AC expression in the proximal anterior margin primordia (arrowhead).

Fig. 8.

Expression of SMAD4100T activates the expression of the WG target gene achaete. (A) Wild-type third instar wing disc stained for Achaete (AC). a, anterior; m, proximal margin; v, ventral surface; dis, distal margin. (B) zw3M11 clones produce ectopic AC expression on the presumptive blade (arrowheads). (C) UAS.SMAD4100T/C765.Gal4 with ectopic AC expression on the presumptive blade (arrowheads). (D) UAS.Dpp/C765.Gal4. This is a lethal genotype. The disc is overgrown (image shown at a reduced magnification) but it has wild-type AC expression. (E) UAS.Gbb/C765.Gal4 with reduced AC expression in the proximal anterior margin primordia (arrowhead).

Discussion

From a larger perspective, our study establishes guidelines for interpreting data from transgenic analyses of human tumor alleles. In principle, studies of this type can be applied to mutant alleles of any well-conserved tumor suppressor gene or oncogene. Furthermore, our unexpected finding that all of the tested mutations in the DNA-binding domain of SMAD genes are gain of function, whereas all of the tested mutations in the multimerization domain are loss of function, indicates that missense mutations in modular proteins should be experimentally characterized rather than defaulted to the loss-of-function category.

For SMAD tumor suppressor genes, our identification of two gain-of-function alleles of SMAD4 (dominant negative and neomorphic) falsifies the prevailing hypothesis that all SMAD tumor mutations are loss-of-function mutations. Instead, our data support an alternative hypothesis: that there are multiple classes of SMAD mutation and that each class is associated with a different mechanism of tumorigenesis.

This alternative hypothesis may also apply to other TGFβ signaling pathway components with tumor-associated mutations. For example, mutations in TGFβ receptors are found in tumors from the same tissues that exhibit SMAD mutations [e.g. pancreas (Hempen et al., 2003), colon (Peltomaki,2001), breast (Pouliot and LaBrie, 1999), lung (Zhang et al., 2004)]. However, missense mutations in TGFβ receptors conferring gain-of-function activity have not been identified in tumors,because the most common mutational assays are loss of expression and polyA tract sequencing.

An oncogenic mechanism of tumorigenesis for SMAD4100T

Our data for SMAD4100T are unprecedented in studies of TGFβsignaling in flies. This suggests that SMAD4100T may induce tumors in humans by an unexpected method. The fact that SMAD4100Texpression mimics activated WG signaling and suppresses an antagonist of WG signaling further suggests that SMAD4100T utilizes a mechanism of tumorigenesis associated with loss-of-function mutations in Adenomatous Polyposis Coli (APC). A model based on this interpretation is shown in Fig. 9.

In vertebrates and flies, APC serves as a component of the highly conserved WG/int-1 (WNT) signal transduction pathway. Like ZW3 and its homolog Glycogen Synthase Kinase-3β (GSK3β), APC functions as a WNT antagonist via participation in a cytoplasmic retention complex that prevents Armadillo (or its vertebrate homolog β-catenin) from accumulating in the nucleus in the absence of WNT signals. Studies in flies have shown that homozygous null clones bearing mutations in any member of the retention complex (ZW3, Drosophila APC and Drosophila Axin) lead to the same phenotype: ectopic anterior margin bristles on the wing blade as a result of the unregulated expression of WG target genes such as AC(Blair, 1992; Akong et al., 2002; Hamada et al., 1999). First identified in the rare inherited cancer Familial Adenomatous Polyposis,homozygous mutations in APC are now found in roughly 85% of all colon tumors(Kinzler and Vogelstein,1996). In studies of mice engineered to homozygose APC null mutations only in cells of their intestinal epithelium, the immediate consequence of APC loss was ectopic expression of WNT target genes via constitutively nuclear β-catenin(Sansom et al., 2004).

Given their roles in their respective signal transduction pathways,loss-of-function mutations in APC cause tumors by a fundamentally different mechanism than loss-of-function SMAD mutations. Specifically, inactive APC proteins cannot block a mitogenic WNT signal (an oncogenic mechanism), whereas inactive SMAD proteins cannot transduce an anti-mitotic TGFβ signal (a tumor suppressor mechanism). Interestingly, one study of SMAD4100Tin mammalian cells suggested that this allele could employ an oncogenic mechanism of tumorigenesis (Dai et al.,1999), a proposal consistent with our data.

Fig. 9.

SMAD4100T may cause tumors via an `APC-like' mechanism not previously associated with defective TGFβ signaling. Model for the mechanism of tumorigenesis used by SMAD4100T based upon its ability to activate WG target genes in flies. (Left) In the model,SMAD4100T functions in a way that mimics the repression of the ARM-destruction complex (ZW3, APC and Axin) in the WG pathway. In this illustration, we show SMAD4100T actively inhibiting the destruction complex, but SMAD4100T may interact with the WG pathway at other points, such as target promoters. SMAD4100T and proteins potentially affected by its activity are shown in red. (Right) The mechanism of tumorigenesis associated with loss-of-function mutations in APC. Loss of APC activity inhibits the β-catenin destruction complex (GSK3β, APC and Axin) leading to the overexpression of WNT target genes and Familial Adenomatous Polyposis. APC and proteins affected by the loss of APC function are shown in red.

Fig. 9.

SMAD4100T may cause tumors via an `APC-like' mechanism not previously associated with defective TGFβ signaling. Model for the mechanism of tumorigenesis used by SMAD4100T based upon its ability to activate WG target genes in flies. (Left) In the model,SMAD4100T functions in a way that mimics the repression of the ARM-destruction complex (ZW3, APC and Axin) in the WG pathway. In this illustration, we show SMAD4100T actively inhibiting the destruction complex, but SMAD4100T may interact with the WG pathway at other points, such as target promoters. SMAD4100T and proteins potentially affected by its activity are shown in red. (Right) The mechanism of tumorigenesis associated with loss-of-function mutations in APC. Loss of APC activity inhibits the β-catenin destruction complex (GSK3β, APC and Axin) leading to the overexpression of WNT target genes and Familial Adenomatous Polyposis. APC and proteins affected by the loss of APC function are shown in red.

An examination of the primary difference between the phenotypes of SMAD4100T and zw3, Apc and Axin mutant clones may shed light on SMAD4100T-associated tumor formation. The primary difference is the location of ectopic bristles on the wing blade. In wings expressing SMAD4100T, ectopic mechanosensory bristles are derived from a cell fate transformation of mechanosensory receptors(campaniform sensilla). Cells extruding sensilla and those extruding bristles are derived from Sensory Organ Precursor cells(Aigouy et al., 2004). Alternatively, zw3, Apc and Axin mutant clones generate cell fate transformations anywhere on the wing blade – regardless of cell lineage. This discrepancy suggests that SMAD4100T is not as potent an activator of WNT target genes as zw3, Apc or Axinmutations. SMAD4100T may only activate WNT target genes in cells predisposed to tumorigenesis, perhaps by pre-existing mutations.

This possibility is supported by our studies of wings with an increasing dosage of UAS.SMAD4100T. When wild-type WG signaling is present,increasing the UAS.SMAD4100T copy number did not increase the frequency of sensillum to bristle transformation. However, in wings with reduced WG signaling due to the activity of UAS.DN-TCF, increasing the copy number of UAS.SMAD4100T quantitatively increased the rescue of WG-dependent functions outside of sensilla (e.g. in wing outgrowth and anterior margin formation).

Molecular nature of the SMAD4100T-WNT pathway interaction

The transformation of campaniform sensilla to mechanosensory bristles was reported once previously. Several transheterozygous mutant genotypes of ash2, a member of the Trithorax group of transcriptional regulators,generate this phenotype (Adamson and Shearn, 1996). Although the ASH2 protein contains a zinc finger motif, its function has not yet been demonstrated biochemically. Studies of its yeast homolog suggest that ASH2 may function in chromatin remodeling and transcriptional activation as part of a complex containing histone methyltransferases (Janody et al.,2004). The similarity of SMAD4100T and ash2 mutant phenotypes, and the ability of SMAD4100T to suppress DN-TCF phenotypes, suggest that SMAD4100T contributes to the activation of WNT target genes downstream of APC, perhaps by participating in transcription factor complexes.

Three previous reports have shown physical interactions between the wild-type SMAD proteins and transcriptional effectors of WNT signaling(β-catenin and TCF). One study used Xenopus embryos to demonstrate that SMAD4/β-catenin/TCF complexes activate the transcription of the WNT target gene twin(Nishita et al., 2000). Recently, SMAD1/β-catenin/TCF complexes were detected in renal medullary dysplasia in ALK3 transgenic mice (Hu et al., 2003), and in human dysplastic renal tissue(Hu and Rosenblum, 2004). We are currently testing the possibility that SMAD4100T cooperates with ARM and/or TCF to activate the transcription of AC.

Clinical implications

At this time, therapeutic research on SMAD-associated tumors is guided by the current hypothesis that all SMAD mutations lead to tumors via a loss of a TGFβ-encoded anti-mitogenic signal. As a result, effort is focused on restoring the wild-type function in tumors by gene replacement. However, we have shown that two SMAD4 tumor alleles are gain-of-function mutations. One important feature of gain-of-function mutations is that they exert their effect even in the presence of a wild-type allele on the homologous chromosome. Thus, it seems unlikely that gene replacement will be successful in inhibiting tumorigenesis in cells with SMAD4 gain-of-function mutations

In individuals with APC mutant colon tumors (those with unregulated WG target gene expression such as Familial Adenomatous Polyposis), the transition from adenomatous polyps to carcinoma will take roughly ten years. Alternatively, for TGFβ receptor mutant colon tumors [those unable to respond to a TGFβ-encoded anti-mitogenic signal such as Hereditary Non-Polyposis Colorectal Cancer (HNPCC)], progress from adenomatous polyps to carcinoma takes less than three years(Souza, 2001). Given these data, if SMAD4100T induces tumorigenesis by an `APC-like'mechanism while SMAD4 dominant-negative and loss-of-function alleles stimulate tumorigenesis by an `HNPCC-like' mechanism, then the prognosis for cancer patients with a SMAD4100T mutation is distinctly different from that of patients with other SMAD mutations.

This raises several issues for future investigation. First, how many different mutations in SMAD4 can generate an `APC-like' gain-of-function allele? To date, three SMAD4 missense mutations near codon 100 have been identified in colon tumors [Y95N, C115R and N118K(Iacobuzio-Donahue et al.,2004)]. All of these mutations (including R100T) occur in the L2/L4 double-loop region identified in the crystal structure of the SMAD3 MH1 bound to DNA. This loop occurs at the surface of the molecule and is important for macromolecular interactions (Shi et al., 1998). Are these also gain-of-function mutations? Second,what is the relative frequency of `APC-like' gain-of-function SMAD4 alleles versus `HNPCC-like' loss-of-function alleles in tumors from various tissues?Third, can an accurate and efficient diagnostic test be developed to distinguish between `APC-like' and `HNPCC-like' alleles in tumors with a SMAD4 mutation? Answering these questions will require a continued collaboration between model organism geneticists and oncologists.

Acknowledgements

We thank G. Riggins, M. Schutte and R. Ray for cDNAs, and the Developmental Studies Hybridoma Bank for antibodies. T. Haerry, S. Hayashi, M. Hoffmann, A. Manoukian, L. Marsh, L. Raftery, E. Siegfried, K. Wharton and the Bloomington Stock Center contributed flies. A. Schmid and C. Lorson provided technical advice. A. Johnson generated the UAS.Mad1 plasmid and assisted with image analysis. B. Johnson, M. Stinchfield, B. Celaya and N. Emmert helped with fly pushing. This study was supported by the NIH (CA095875 to S.J.N.). M.B.O. is an Associate Investigator of the Howard Hughes Medical Institute.

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