1 School of Life Sciences, Arizona State University, Tempe, AZ 85287-4501,
USA
2 Howard Hughes Medical Institute, Department of Genetics, Cell Biology and
Development, University of Minnesota, Minneapolis, MN 55455, USA
Author for correspondence (e-mail:
newfeld{at}asu.edu)
Accepted 18 August 2005
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SUMMARY |
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Key words: Drosophila, SMAD genes, TGFß, Tumorigenesis, Pancreatic cancer, Colon cancer
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Introduction |
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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 TGFß 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).
TGFß1 was discovered through its anti-mitotic effects on fibroblast
cells in culture (Barnard et al.,
1990). However, TGFß1 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 TGFß 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.
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Materials and methods |
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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, Mad11
and 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/dpphr4
wing 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 ß-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
).
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Results |
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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.
|
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.
|
In order to experimentally test our hypothesis that the MAD1
protein 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, Mad
genomic 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 Mad
loss-of-function phenotype (compare Fig. 2D
with 2E).
In addition to effects on venation, many UAS.Mad1 genotypes 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. Drosophila
SRF 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 rho
activity indicates that it is not being transcribed by MAD.
|
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 rho may 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 Med
in 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.
|
SMAD4130S and SMAD4100T are gain-of-function alleles associated with human tumors
Our identification of Mad1 and Med7
as 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).
|
When SMAD alleles with mutations in the MH2 domain
(UAS.SMAD4524ST,
UAS.SMAD2344-358 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 SMAD4100T
generated a wing phenotype never before reported in any study of TGFß
signaling in flies. Most wings expressing UAS.SMAD4100T
strongly 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
).
|
Though never previously associated with TGFß 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 zw3M11
clones.
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 TGFß 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.Gbb
expression. In the two experiments where adult flies were obtained, we found
that UAS.Gbb inhibits triple row bristle formation. ap.Gal4
is 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.Gbb
generated sensilla to bristle transformation, supporting our hypothesis that
UAS.SMAD4100T is a gain-of-function allele with novel
activity.
|
|
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.
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Discussion |
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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 TGFß signaling
pathway components with tumor-associated mutations. For example, mutations in
TGFß 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 TGFß 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 TGFß
signaling in flies. This suggests that SMAD4100T may induce tumors
in humans by an unexpected method. The fact that SMAD4100T
expression 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-3ß (GSK3ß), APC functions as a WNT antagonist via
participation in a cytoplasmic retention complex that prevents Armadillo (or
its vertebrate homolog ß-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 ß-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 TGFß signal (a
tumor suppressor mechanism). Interestingly, one study of SMAD4100T
in mammalian cells suggested that this allele could employ an oncogenic
mechanism of tumorigenesis (Dai et al.,
1999), a proposal consistent with our data.
|
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
(ß-catenin and TCF). One study used Xenopus embryos to
demonstrate that SMAD4/ß-catenin/TCF complexes activate the transcription
of the WNT target gene twin
(Nishita et al., 2000).
Recently, SMAD1/ß-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
TGFß-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 TGFß receptor mutant colon tumors [those unable to
respond to a TGFß-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.
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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