1. Use tissue culture cells and transgenic mice to study the functional importance of b-catenin/TCF-4 mediated transcriptional activation in the development of colon cancer.

2. Identify target genes of the b-catenin/TCF-4 bipartite transactivator in human colonic epithelial cells by differential display and enhancer trapping.

3. Look for the functional role of dAPC in the Wingless pathway involved in the adult Drosophila compound eye development. Identify more partners of Armadillo in Wingless signaling by using a genetic screen for dominant modifiers of phenotypes in Drosophila eye development caused by constitutively active, N-terminus deletion mutant of Armadillo (deltaArm).

b. Background and Significance:

The adenomatous polyposis coli (APC) gene is a tumor suppressor gene, mutation of which is responsible for familial adenomatous polyposis (FAP). This disease is characterized by the development of multiple colorectal adenoma, of which a subset inevitably progress to malignant cancers if not surgically removed (Kinzler et al., 1996; Polakis, 1997). FAP patients have germline APC mutations, which usually results in the expression of truncated proteins, and their tumors show inactivation of the wild-type allele. Inactivation of both APC alleles also occurs frequently in sporadic colorectal adenomas.

The APC gene encodes a 300-kD protein, which has several structural domains (Fig.1B). Its N-terminus contains an oligomerization domain followed by multiple Arm repeats (protein interaction motifs also found in Drosophila Armadillo protein, the homologue of human b-catenin), while its C-terminus can bind to microtubule and the human homologue of the Drosophila discs-large (DLG) tumor suppressor. The middle part of the protein , where the majority of truncation mutations occur, contains three successive 15 aa repeats, followed by several 20-aa repeats. Both types of repeats are able to bind independently to b catenin, a cadherin-associated protein important for intercellular adhesion. Therefore, it was proposed initially that APC mutations contribute to the development of colon cancer because of their effects in intestinal cell adhesion and migration.

Recently, b-catenin has also been shown to play an essential role in Wnt/Wingless signal transduction events that alter gene expression during important developmental processes (summarized in Fig. 1A). This finding provides another, probably more important mechanism for APC to control colon cell growth. Many components of the Wnt/Wingless signaling pathway were first identified and epistatically ordered using genetic screens of Drosophila mutants that show aberrant embryonic pattern formation (Klingensmith et al., 1994; Cadigan et al., 1996). Subsequently, this pathway has been found to be highly conserved during evolution and has been implicated in cell-lineage decision in Caenorhabditis elegans, dorsal axis formation in Xenopus, and development of central nervous system as well as carcinogenesis in mammals (Cadigan et al., 1997 ). Binding of secreted Wnt/Wingless molecule to receptor Frizzled activates the cytoplasmic phosphoprotein Dishevelled. Through as yet unknown mechanisms. Dishevelled inhibits the function of the serine/threonine kinase known as glycogen synthase kinase-3 (GSK-3) in vertebrates, or zeste-white-3 (Zw3) in Drosophila. Inhibition of GSK-3 results in the accumulation of hypophosphorylated b catenin/Armadillo in the cytoplasm. Interaction of cytoplasmic b-catenin with TCF/LEF-1 transcription factors leads to relocalization of b-catenin to the nucleus and changes in target gene expression. In the absence of Wg/Wnt signal, GSK-3 acts directly or indirectly to cause the phosphorylation and degradation of b-catenin/Armadillo, possibly through the ubiquitin-proteasome pathway (Aberle et al., 1997). Cadherins and cell adhesion do not seem to be directly involved in signal transduction by b-catenin. However, sequestration of b-catenin to the cytoplasmic membrane by binding to over-expressed cadherin strongly inhibits its signaling activity in both Xenopus and Drosophila.

The upstream mechanism that regulate Wnt/Wingless signaling may vary among different species. In different occasions, activation of the Wnt receptor may or may not be the initiating event. For instance, in Xenopus it is still unclear whether Wnt and the initial receptor mediated events, which acts via the receptor Frizzled and Dishevelled, are actually involved in normal development, even though they can induce ectopic body axis formation (Vleminckx et al., 1997). In addition, unlike Drosophila, in which wg and Dtcf have similar phenotypes, pop-1, the C. elegans homologue of tcf has the opposite phenotype as Wnt, and POP-1 protein is post-transcriptionally down-regulated by the Wnt pathway (Rocheleau et al., 1997). Furthermore, upstream components of the Wnt/Wingless pathway, such as Wnt, have not been found to be involved in the carcinogenesis of colon cancer. While there may be major differences in some aspects of the Wnt/Wingless pathway among different organisms, it is clear that signaling transduction mechanisms involving b-catenin and TCF/LEF-1 transcription activators are turning out to be a common theme in developmental and cancer biology.

Currently the favored explanation for the biochemical mechanism underlying the tumor suppressor activity of the APC is that it regulates the level of b-catenin in the cytoplasm by controlling its degradation (Gumbiner et al., 1997; Munemitsu et al., 1995) In many tumor cell lines, loss of the APC function leads to b-catenin accumulation in the cytosol. Expression of wild-type APC can decrease the level of b-catenin by stimulating its degradation. Moreover, APC seems to be a very good substrate for phosphorylation by GSK-3. It has been reported that APC protein forms a complex with b-catenin and GSK-3 and is phosphorylated at the 20-aa repeats by GSK-3 in vitro, which enhances the binding affinity of these motifs to b-catenin (Rubinfeld et al., 1996), possibly leading to its degradation. In cells expressing truncated forms of APC, the reporter gene construct harboring several TCF/LEF-1 binding sites in the promoter was constitutively transcribed (Korinek et al., 1997). Importantly, when the wild-type APC was introduced into the cells, it suppressed transcriptions of the reporter gene. Together, these data are consistent with a model in which APC mediates GSK-3's function to antagonize Wnt signaling by regulating steady state level of b-catenin and, as a consequence, the ability of b-catenin to interact with TCF/LEF-1.

Many colon tumor and melanoma cell lines that have normal APC expression have been found to harbor specific mutations in b-catenin that lead to its accumulation in the cytosol and enhanced TCF/LEF-1 dependent transcriptional activity. Interestingly, these mutations occur at specific serine residues in the amino-terminal region of the b-catenin (Barth et al., 1997; Morin et al., 1997; Munemitsu et al., 1996; Rubinfeld et al., 1997), which are thought to regulate b-catenin turnover and to be phosphorylated, directly or indirectly, by GSK-3. Thus, b-catenin seems to act as an oncogene product that opposes the tumor suppressor function of APC within the same biochemical pathway.

TCF and LEF-1 proteins were found originally as enhancer binding factors for T cell-specific genes (Giese et al., 1992). Binding of TCF/LEF-1 proteins to DNA results in bending of the helix, but by themselves, these proteins are poor transcriptional activators. However, complexes between TCF/LEF-1 proteins and b-catenin act as potent transcriptional activators of reporter gene constructs containing the DNA element recognized by TCF/LEF-1(Behrens et al., 1996; Huber et al., 1996). TCF/LEF-1s are highly conserved in evolution, and are expressed in C. elegans (POP-1), Drosophila (dTCF/pangolin) and Xenopus (XTCF-3) (Brunner et al., 1997; Molenaar et al., 1996; Van de Wetering et al., 1997). Genetics epistasis analysis in Drosophila shows that dTCF/pangolin is required for Wingless signal transduction, and that it functions downstream of b-catenin/Armadillo in the Wnt/Wingless pathway. Similar result was obtained using C. elegans. The Wnt/Wingless pathway activates and/or maintains the expression of several genes, including engrailed, and ultrabithorax in Drosophila, and siamois in Xenopus(Brannon et al., 1997). TCF/LEF-1 binds to promoters of these genes. Currently a major gap of knowledge exists as to what are the target genes regulated by b-catenin/TCF complex in colon cells. Identification of these target genes will lead to a better understanding of the carcinogenesis process initiated by APC mutations.

As APC is a large, complex protein with a great diversity of domains and different phosphorylation states, it would not be surprising if it has diverse roles in signal transduction. Regulation of b-catenin signaling may be only one of the functions of the APC protein. Wnt has not yet been implicated in colon cancer, hence it is conceivable that APC participates in additional pathways that may or may not be related to Wnt/Wingless pathway. Moreover, the roles of APC homologues in the development of various model organisms are somewhat conflicting. While it is clear that APC negatively regulates b-catenin dependent transcription in colon tumor cell lines, as mentioned above, it has an activating effect on the Xenopus embryo, inducing siamois expression and body axis formation. This suggests that APC may have an important direct and positive signaling function in addition to its role in regulating b-catenin levels. Studies in Drosophila have failed to reveal a role for dAPC in the Wingless pathway that controls embryonic anterior-posterior pattern formation, even though it can down-regulate b-catenin in human cells and is expressed during many stages of Drosophila development. Apparently what we have known about APC in development and carcinogenesis is still far from complete. APC may acts as a nexus integrating different inputs and generating multiple outputs. Model organisms such as Drosophila and C. elegans are powerful tools to identify new players in both Wnt/Wingless and other new APC-related pathways, which will lead us to better understandings of the delicate network that prevents a cell from wandering into a wrong way eventually leading to cancer.

Research Design and Methods:

Specific Aim 1: Functional significance of b-catenin/TCF-4 mediated transcriptional activation in the carcinogenesis of colon cells.

It is clear that truncation of APC or oncogenic mutation of b-catenin results in the cytoplasmic accumulation of b-catenin, which leads to its nuclear localization, possibly by utilizing the nuclear localization signal of TCF/LEF-1. As a result, the level of transcription of TCF/LEF-1 reporter construct is increased in tumor cell lines. Such a change of expression pattern could leads to tumorigenic events such as inhibition of apoptosis and loss of proper cell cycle control. However given the multi-domain structure of both APC and b-catenin, and their multiple roles in cell-cell adhesion, intracellular cytoskeleton, and signal transduction, the importance of transcriptional activation by b-catenin/TCF in carcinogenesis still needs to be evaluated. It could be a minor side effect of many global changes caused by APC or b-catenin mutation, instead of being a critical tumorigenic event. Currently, no cellular gene is known to be transactivated by b-catenin/TCF/LEF-1, except that there is one report showing b-catenin/LEF-1 heterodimers binds to the murine E-cadherin promoter in vitro (Huber et al., 1996). Before setting out to search for cellular genes responsive to b-catenin/TCF regulation, it is important to assess the functional significance of this event in the carcinogenesis of colon cancer.

Dominant negative mutants of TCF/LEF-1 with deletion or mutation of the b-catenin interaction domain in the N-terminus while maintaining the HMG DNA binding domain, or vise versa, have been shown to inhibit the phenotype caused by accumulated b-catenin in both Xenopus and Drosophila. Such mutant forms of TCF/LEF-1 are useful tools in this study. A mammalian expression construct of dominant negative TCF-4 (which is the only TCF/LEF-1 family member expressed in colonic epithelium) under the control of an inducible promoter will be stably transfected into human APC -/- tumor cell lines (e.g. SW480). The phenotype of the cells expressing dominant negative TCF-4 will be compared with that of the parental tumor cell lines or the transfected cell line under noninducing culture condition. Signs of loss of tumorigenic characteristics, such as loss of the ability to grow on soft agar, or to induce tumors in nude mice, will provide evidences that the b-catenin/TCF-4 transcription activation event is critical in carcinogenesis. To show the consistency of the dominant negative effect, it will be wise to test both types of the dominant negative mutants of TCF-4 in this experiments. Since the accumulation of b-catenin is the downstream effect of APC mutation, a cell line derived from sporadic colorectal tumor patient with normal expression of full length APC protein but has oncogenic mutation of b-catenin, such as cell line HCT116, could be equally informative in this study. It should be bear in mind that a negative result could not rule out an important role for b-catenin /TCF-4 transactivation in the multi-step event of carcinogenesis. Mutations in the wild type APC allele are observed in the earliest recognizable benign tumors found in FAP individuals, suggesting that loss of wild-type APC activity plays an early role in tumorigenesis. Additional mutations at other genes, such as Ras and p53 must be accumulated before the occurrence of carcinoma and metastatic tumors. It is therefore conceivable that b-catenin/TCF-4 transcriptional activation might be dispensable to sustain the tumor cell phenotyps that we study in cell lines derived from full-fledged colorectal tumors, even though it might be critical for the early events of colorectal tumor development. One way to overcome this limitation is to develop cell line derived from the earliest observable neoplastic lesions called dysplastic aberrant crypt foci (ACF) in FAP individual, and use it in this study. It will be easier to get meaningful positive results with these cell lines. Still, given the pleiotropic effect of the APC mutation in cell-cell adhesion, cell mobility and cellular signaling , even a negative result obtained from a dysplastic cell line only with mutation in APC can not be considered as a convincing proof to neglect the role of b-catenin/TCF-4 transcription activation. However, a positive result will definitely provide a strong argument for the importance of the b-catenin/TCF transactivation in carcinogenesis.

A different approach is to study the direct effect of b-catenin/TCF-4 transactivation in a normal colonic epithelial cell line, which will avoid the difficulty to explain the negative result from the experiments described above. Accumulation of b-catenin/TCF-4 in the nucleus without dramatically altering the cytoplasmic level of b-catenin can be achieved by over-expression of TCF-4, or expression of a chimeric protein with the C-terminus of b-catenin, which is the transactivation domain, fused at the N-terminus of TCF-4, which possesses the DNA binding domain. The growth profile and tumorigenic ability of these cell lines will be compared with normal parental cell lines.

Min (multiple intestinal neoplasia) mice have a dominant truncating mutation of the murine APC gene (mAPC) at a position similar to that found in many FAP patients, and develop multiple intestinal adenomas (Shoemaker et al., 1997). A transgenic Min mouse strain carrying a dominant negative mTCF-4 under the control of the natural mTCF-4 promoter will be generated. The ability of these transgenic mice to develop intestinal adenoma, as well as other related phenotype will be compared with Min mice and mice with a control b-gal transgenic construct. Suppression of Min phenotype by dominant negative mTCF-4 will provide an additional evidence for the importance of b-catenin/TCF-4 transactivation in colon cancer development. However, additional genetic effects caused by expression of the dominant negative mTCF might complicate the interpretation of results. It will also be interesting to see if a transgenic mouse (with a wild-type genetic background) that expresses a b-catenin/mTCF-4 fusion protein under the control of an inducible promoter is more prone to developing intestinal tumor as was observed in Min mice. Meanwhile, if transgenic mice with dominant negative mTCF-4 do not show significant reduction of fitness, we can look for TCF-4 mutations in FAP individuals who do not develop tumor or develop it significantly later than other FAP individuals bearing the same APC mutation. A naturally occurring mutant TCF-4 with positive effect on reducing the tendency of FAP individuals to develop cancer will be an ultimate proof of its role in carcinogenesis.

Results obtained from various cell lines and transgenic mice, and TCF-4 mutation screen of FAP individuals will together enable us to evaluate whether transcriptional activation of b-catenin/TCF-4, and to what extent, affects the normal growth control of colorectal cells.

Specific Aim 2: Identification of target genes under the control of b-catenin/TCF-4 heterodimeric transcription activator.

Identifying genes subordinate to b-catenin/TCF-4 regulation is important not only in understanding how b catenin/TCF-4 works to control gene expression, but also in addressing the question of how changes of expression pattern leads to tumor. Does b-catenin/TCF-4 directly control genes required for cell cycle control and apoptosis? These questions cannot be answered before knowledge about the identities and functions of target genes is accumulated.

We can use PCR based differential display to detect mRNAs whose levels were affected by b-catenin/TCF 4 complex in colonic cells. In order to reduce the complexity of mRNA population to be compared and the complication caused by multiple genetic changes in tumor cell lines, I will compare the mRNAs of a normal colonic epithelial cell line with the same cell line stably transfected with constitutively active form of b-catenin (with N-terminus deletion or mutation in specific serine residues) or b-catenin/TCF-4 fusion gene construct described in the specific aim 1. We can expect to see activation of some genes by b catenin/TCF. Down-regulation of expression by b-catenin/TCF won't be surprising though, given the fact that some transcriptional activators can act as repressors under certain conditions. Differentially expressed bands will be cut of the gel and re-amplified. PCR products will be subcloned and used as probes for northern blot analysis to confirm that the corresponding mRNA is differentially transcribed in the cell lines compared. The identities of the genes encoding these mRNA and it's promoter region will be obtained by screening the cDNA and genomic library using the differentially displayed bands as probes, and sequencing.

An alternative approach will take advantage of an enhancer trap construct containing a positive/negative selection marker of a fusion gene coding for hygromycin phosphotransferase (Hy) and herpes simplex thymidine kinase(TK). This HyTK gene is driven by a promoter that has been weakened by removal of its enhancers so that the transfected trap is only expressed if it comes under the influence of an endogenous enhancer. The expression of this gene confers hygromycin resistance and gancyclovir sensitivity. Suppression results in gancyclovir resistance and hygromycin sensitivity. Cell lines expressing constitutively active form of b-catenin or b-catenin-TCF-4 fusion protein under the control of an inducible promoter (e.g. Tet-on promoter) will be used in this experiment. To isolate up-regulated enhancers, hygromycin and tetracycline (which acts to turn on the Tet-on promoter) will be added after transfection of the enhancer trap construct and 48 h of growth, and the cultures will be incubated until vigorous growing clumps of cells appear. The medium will then replaced, and the cells were grown for a day without hygromycin and tetracycline before being placed in medium containing gancyclovir. Vigorously growing clumps of cells will then be picked up for further studies. Down-regulated enhancers could also be identified by sequential selection by media containing hygromycin and gancyclovir/tetracycline. Control experiments using known natural TCF responsive promoters need to be done beforehand to determine appropriate concentrations of these selection drugs, which is critical for the success of this method.

b-catenin/TCF-4 could regulate the expression of a gene directly by acting on its promoter/enhancer, or indirectly through controlling the expression of intermediators. Identification of consensus TCF/LEF-1 DNA binding sites in the promoter region of a gene will suggest that it may be regulated directly by binding of b-catenin/TCF-4. Further experiments such as mutagenesis analysis of the promoter using a reporter construct, gel shift and footprinting analysis need to be performed to confirm it's the direct target of b catenin/TCF-4. Once a target gene is identified, it will be interesting to look if it fits into the pathways regulating cell cycle and apoptosis, as well as other aspects important in carcinogenesis. The expression pattern of the target genes, the phenotype of tissue culture cells, transgenic mice and other model organisms when these gene or their homologues are over-expressed or mutated will also provide useful information about its function.

Specific Aim 3: Functional role of dAPC in Drosophila compound eye development. A genetic screen for suppressors and enhancers of the dominant deltaArm phenotype in Drosophila eye development.

The recently identified Drosophila APC protein has 27% identity and 46% similarity overall to human APC. Furthermore, there are striking homologies to domains previously identified in human APC. dAPC is also functionally equivalent to human APC in downregulating cytoplasmic b-catenin in colon carcinoma cell lines. All these suggest that studying the function of dAPC may shed light on the function of its human homolog in carcinogenesis. However, removing zygotic dAPC expression did not alter Armadillo protein distribution during embryonic pattern formation, which is strongly influenced by the Wingless pathway. (Hayashi et al., 1997) Maternally expressed dAPC and possible gene redundance could obscure the role of dAPC in early embryo development. Wingless pathway has recently been found to be involved in the development of compound eyes of adult fly (Cadigan et al., 1996). Transgenic flies with ectopic expression of wg during eye development lack the mechanosensory bristles normally surrounding each facets of the compound eye. It is therefore reasonable to look for the role of dAPC in this aspect of Drosophila development.

First we want to know if dAPC is able to bind to Armadillo and Zeste-White 3 (Drosophila homologue of GSK-3), as what was observed with their human homologues in mammalian cells (Rubinfeld et al., 1996). This can be done by co-immunoprecipitation using Drosophila tissue culture cells. Positive result will be a good sign that dAPC may have a role in Wingless signaling. Next we want to know if overexpression of dAPC under the eye-specific promoter sevenless, which is introduced by P-element transformation, can at least partially blocks the ability of Wingless to inhibit bristle formation. We can also observe the eye phenotype caused by overexpression of dAPC alone, and see if it can be alleviated by overexpression of dTCF or Armadillo. All these results will enable us to examine if dAPC has a role in Wingless signaling in Drosophila. If we get negative or conflicting results, we may want to consider if the dAPC gene identified by Hayashi et al. is the right one, or that dAPC may play an essentially different role from that of its human homologue. The eye phenotype caused by overexpression of dAPC could also be used to search for more genes that functionally interact with dAPC by dominant modifier screening described below for deltaArm.

Since Wnt and other components of the Wnt/Wingless pathway upstream of GSK-3 hasn't been shown to involved in colon carcinogenesis, studying the signaling pathway downstream of GSK-3 and b-catenin may be more relevant to understanding colon cancer. Searching for suppressor/enhancer of the constitutively active form of b-catenin/Armadillo will identify possibly more partners of b-catenin. Theoretically, mutations in the promoter regions of Armadillo/dTCF target genes could also be identified by such a screening, which in turn may help the study of colon cancer. N-terminal deletion of Armadillo (deltaArm), the Drosophila homologue of b-catenin, is resistant to GSK-3 phosphorylation and down-regulation. This mutant is constitutively active in Wingless signaling and ubiquitous expression of deltaArm caused naked cuticle phenotype of the ventral epidermis in a larvae, similar to the phenotype caused by overexpression of Wingless. Armadillo is required for the ability of wg to inhibit eye bristles formation. Therefore it's conceivable that ectopic expression of a constitutively active form of armadillo will cause the similar phenotype in the Drosophila compound eye as ectopic expression of wg.

We can place deltaArm under the control of the sevenless enhancer/promoter which limits expression of deltaArm to a subset of cells in the development of eye. Since the Drosophila compound eye is dispensable for viability and fertility, most deleterious effects associated with a constitutively active Armadillo will be avoided. If expression of deltaArm does result in the similar phenotype as wingless, we can use the phenotype to screen for dominant suppressors and enhancers. Additional players in the Wingless pathway downstream of Armadillo as well as some target genes of Arm/TCF could be identified by such screening. In the background of sev-deltaarm, a two-fold reduction in the dosage of a downstream gene (i.e. by mutating one copy of the two present in the diploid genome) will alter the signaling efficiency and thereby visibly modify the phenotype. It is important to be able to recover modifiers as heterozygous mutations since many of the important signaling molecules in the wingless pathway may be required throughout development and would mutate to homozygous lethality. Dominant suppressors are expected to carry mutations in positively acting genes, while enhancers are expected to carry mutations in negatively acting genes.

To perform the screen, male flies will be mutagenized with either EMS or X-rays and mate to females carrying an activated sev-deltaarm construct on a balancer chromosome. F1 progeny will be scored for enhancement or suppression of the deltaarm phenotype. Mutations selected will be mapped using standard segregation analysis and balanced. Complementation testing will be carried out between all mutations on a given chromosome to determine complementation groups. After determining map positions, the mutants will complementation tested to known genes involved in Drosophila eye development. The novel mutants will then be tested against members of the Wingless pathway by examining the phenotype of double mutants to determine whether they displayed genetic interactions with other pathway members. Genetic epistasis will give us information of the order of these genes in the pathway.

At least four classes of dominant suppressors and enhancers could be identified in such a screening. (1) Mutations in bona fide signaling genes, including target genes of Armadillo/dTCF. (2) Mutations in genes that are involved in the post-translational modification of the Armadillo protein. (3) Mutations in genes that encode transcription factors for the sevenless promoter. (4) Nonspecific suppressors and enhancers. We will be interested in the first classes or mutations. To distinguish them from the other classes of mutants, a dominant negative mutant of Arm (e.g. mutation in the activation domain) will be utilized. Suppressors and enhancers that are specifically involved in signaling are expected to have the opposite effect on dominant negative Arm from that seen with constitutively active deltaArm, whereas the other three classes of mutants should have the same effect on both kinds of arm mutants.

Once a novel gene involved in signaling downstream of Armadillo is identified, we can study its function by expression pattern examination, gene disruption, overexpression, double mutant analysis, genetic epistasis analysis, suppressor and/or enhancer screening, etc. We can look for its human homologue by searching the GenBank and dbEST database, or by screening the cDNA library derived from human colon cells if it's not available from databases. Highly conserved region in cross-species sequence alignment indicates functional domains. Homology to known functional domains or supergene family will provide hints of the novel gene's function as well. We can look for mutations in these genes in colon cancer patients by PCR and SSCP. All these knowledge will enable us to understand colon cancer better and eventually leads to the cure of this disease.

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