CA2600540A1

CA2600540A1 - A novel component of the wg/wnt signaling pathway

Info

Publication number: CA2600540A1
Application number: CA002600540A
Authority: CA
Inventors: Carla Banziger; Corina Schutt; Davide Soldini; Konrad Basler
Original assignee: Individual
Current assignee: GENETICS COMPANY Inc
Priority date: 2005-03-18
Filing date: 2006-03-20
Publication date: 2006-09-21
Also published as: WO2006097336A3; WO2006097336A2; WO2006097336A9; EP1858922A2; JP2008537484A

Abstract

The present invention relates to a novel component of the Wg/Wnt signaling pathway. In particular, the invention relates to nucleic acid molecule being homologue to the sequence of the gene CG6210 (Drosophila melanogaster), of its encoded polypeptide named 3L3 or WLS, as well as derivatives, fragments and analogues thereof. The invention relates to methods for screening for a substance that inhibits or enhances the secretion of a protein of the Wnt-family as well as to antibodies binding to said (poly)peptides.

Description

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A NOVEL COMPONENT OF THE WG/WNT SIGNALING PATHWAY
BACKGROUND OF THE INVENTION

Wnt genes of vertebraten and invertebraten encode a large family of secreted, cystein rich proteins that play key roles as intercellular signaling molecules in a wide variety of biological processes (for an extensive review see (Wodarz and Nusse 1998). The first Wnt gene, mouse Wnt-1, was discovered as a proto-oncogene activated by integration of mouse mammary tumor virus in mammary tumors (Nusse and Varmus 1982). Consequently, the involvement of the Wg/Wnt pathway in cancer has been largely studied. With the identification of the Drosophila polarity gene wingless as a wnt-1 homologue (Cabrera, Alonso et al. 1987; Perrimon and Mahowald 1987; Rijsewijk, Schuermann et al.
1987), it became clear that Wnt genes are important developmental regulators. Thus, although at first glance dissimilar, biological processes like embryogenesis and carcinogenesis both rely on cell communication via identical signaling pathways.

In a current model of the canonical Wnt pathway, the secreted Wnt protein binds to Frizzle cell surface receptors and activates the cytoplasmic protein Dishevelled (Dsh). Dsh then transmits the signal to a complex of several proteins, including the protein kinase Shaggy(Sgg)/GSK3, the scaffold protein Axin and O-Catenin, the vertebrate homologue of Armadillo. In this complex (3-Catenin is targeted for degradation after being phosphorylated by Sgg. After Wnt signaling and the resulting down-regulation of Sgg activity, (3-Catenin (or its Drosophila homologue Armadillo) escape from degradation and accumulate into the cytoplasm. Free cytoplasmic R-Catenin translocates to the nucleus by a still obscure mechanism, and modulates gene transcription through binding the Tcf/Lef family of transcription factors (Grosschedl R 1999). Mutations in 0-catenin, APC, and Axin have been found in several human c cancers, suggesting that constitutive activation of canonical Wnt pathway contributes to human carcinogenesis (Uthoff SM, Eichenberger MR, McAuliffe TL, Hamilton CJ and Galandiuk S. (2001). Mol. Carcinog., 31, 56-62.
Binding of Wnt ligands to their receptors also trigger activation of noncanonical pathways, referred as Wnt signaling pathways that signal independently of 'e-catenin, which may signal through calcium flux, c-Jun NH2-terminal kinase, and G proteins. These pathways might get activated in parallel to the canonical, bCatenin dependent pathway in tumors characterized by Wnt ligand upregulation (Huguet EL, McMahon JA, McMahon AP, Bicknell R and Harris AL. (1994). Cancer Res., 54, 2615-2621.; Dale TC, Weber-Hall SJ, Smith K, Huguet EL, SUBSTITUTE SHEET (RULE 26) Jayatilake H, Gusterson BA, Shuttleworth G, O'Hare M and Harris AL. (1996).
Cancer Res., 56, 4320-4323; Vider BZ, Zimber A, Chastre E, Prevot S, Gespach C, Estlein D, Wolloch Y, Tronick SR, Gazit A and Yaniv A. (1996). Oncogene, 12, 153-158; Smith K, Bui TD, Poulsom R, Kakiamanis L, Williams G and Harris AL. (1999). Br. J. Cancer, 81, 496-502). For instance, frequent upregulation of Wnt-2 has been reported in human colorectal cancer and gastric cancers (Katoh M. (2001). Int. J. Oncol., 19, 1003-1007.). Moreover, Holcombe et al.
(Holcombe RF, Marsh JL, Waterman ML, Lin F, Milovanovic T and Truong T.
(2002). MoI.
Pathol., 55, 220-226) recently analysed the expression of specific Wnt genes in human colon cancer and malignant melanoma by in situ hybridization and their results suggest that Wnt-2 overexpression may be involved in human carcinogenesis (Pham K, Milovanovic T, Barr RJ, Truong T and Holcombe RF. (2003). Mol. Pathol., 56, 280-285.).

In addition to its role in cancerogenesis, Wnt signalling also plays a role in skeletogenesis, bone formation and fracture repair (Hartmann (2000), Holmen (2005)). For instance, upregulation of Wnt-proteins have been shown to correlate with pathobiology of rheumatoid arthritis and osteoarthritis (Sen et al. (2000), Nakamura (2005), Holmen (2005)).

Currently, there are no known therapeutic agents effectively inhibiting the Wnt pathway, either by directly inhibiting (3-Catenin transcriptional activation or by inhibiting pathway activation by Wnt ligands. This is partly due to the fact that many of the essential components required for its full activation and nuclear translocation are still unknown.
Consequently, there is an urge to understand more about this pathway in order to be able to develop effective drugs against these highly malignant diseases.

SUBSTITUTE SHEET (RULE 26) SUMMARY OF THE INVENTION

In order to identify new components required for Wg/Wnt signaling pathway activation the inventors used an approach for screening for recessive suppressors of the sev-wg phenotype (Drosophila melanogaster). In this approach a protein named 3L3 was found which is encoded by the gene CG6210. Said gene has homologues in all metazoans, and more interestingly, there is in all likelihood only one gene of this family in each species. With genetic tools it was confirmed that 3L3 plays a positive role in the Wg/Wnt signaling pathway, and in particular in the Wg/Wnt secretion pathway of all Wnt proteins. Further it has been shown that 3L3 physically interacts to Wnt-proteins. Therefore, 3L3 proteins are very promising targets for developing drugs which up- or down-regulates Wnt protein secretion and thus inhibits both the canonical and non canonical Wnt pathway.

The invention relates to a method for screening for a substance that inhibits or enhances the secretion of a protein of the Wnt-family comprising the steps of:

a) bringing a candidate substance into contact with a nucleic acid molecule with the nucleotide sequence of SEQ ID. Nos 1, 2, 3 or 7 under conditions that permit binding of said substance to said nucleic acid molecule; or b) bringing a candidate substance into contact with nucleic acid molecule coding for a (poly)peptide with the amino acid sequences of SEQ ID. Nos 4, 5, 6 or 8 under conditions that permit binding of said substance to said nucleic acid molecule; or c) bringing a candidate substance into contact with a fragment of the nucleic acid molecule according to a) or b) under conditions that permit binding of said substance to said fragment, said fragment codes for the part of the (poly)peptide with the amino acid sequences of SEQ ID. Nos 4, 5, 6 or 8 which effects Wnt-protein secretion; or d) bringing a candidate substance into contact with a derivate of the nucleic acid molecule according to a) or b) or with a derivate of the fragment according to c) under conditions that permit binding of said substance to said derivate; or e) bringing a candidate substance into contact with a nucleic acid molecule, with a fragment or with a derivate which is at least 50 % homologous to the nucleic acid molecule according to a) or b), to the fragment according to c) or to the derivate according to d), respectively;

SUBSTITUTE SHEET (RULE 26) f) detecting if the candidate substance is having inhibitory activity or enhancing activity on the secretion of a Wnt-protein.

A derivate as stated under d) is a the nucleic acid molecule according to a) or b) or a fragment according to c) with an arbitrary molecule attached to it, said candidate substance showing an affinity to said derivate which is at the most 50% increased or decreased compared to the affinity between the candidate substance and the corresponding unmodified nucleic acid molecule or fragment, respectively.

"Percent (%) homologous" with respect to the following (poly)peptide sequences is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the sequences with SEQ ID. Nos 4, 5, 6 and 8 after aligning the sequence and introducing gaps, if necessary, to achieve the maximum percentage sequence identity, and not considering any conservative amino acid substitution as part of the sequence identity. The % identity values used herein can be generated by WU-BLAST-2, which was obtained from (Tatusova TA 1999). WU-BLAST-2 uses several search parameters, most of which are set to the default values.

In a similar manner, "percent (%) homologous" with respect to the mentioned nucleic acid sequences with the SEQ ID. Nos 1, 2, 3, and 7 is defined as the percentage of nucleotide residues in a candidate sequence that are identical with the nucleotide residues in the said nucleic acid sequences. The identity values used herein can be generated using BLAST
module of WU-BLAST-2 set to the default parameters.

The (poly)peptides with the SEQ ID. Nos 4, 5, 6 and 8 encoded by nucleic acid molecules with the SEQ ID. Nos 1, 2, 3 and 7, respectively, are representatives of a novel family of proteins present in invertebrate, for example in Drosophila, leeches, slugs, snails and earthworms, and in vertebrate organisms, for example in mammals including humans, apes, monkeys, dogs, cats, rabbits, goats, pigs, hamsters, cows, horses, sheep, mice and rats. The (poly)peptides with the SEQ ID. Nos 4, 5, 6 and 8 are hereinafter referred to as 3L3 or WLS
(wntless) proteins. These proteins play an essential role in the Wg/Wnt signaling pathway and thus in the formation and maintenance of spatial arrangements and proliferation of tissues during development, and in the formation and growth of many human tumors. The (poly)peptide with the SEQ ID. No 4 is the 3L3 protein of Drosophila (3L3-PA), as well as the (poly)peptide with the SEQ ID. No 8(3L3-PB). The (poly)peptides with the SEQ
ID. Nos 5 SUBSTITUTE SHEET (RULE 26) and 6 are the 3L3 proteins of caenorhabditis (C.) elegans and human, respectively, which are structural and functional homologous of Drosophila 3L3.

In one embodiment of the invention, the nucleic acid molecule, the fragment or the derivate as stated under step e) is at least 52 % homologous to the nucleic acid molecule according to a) or b), to the fragment according to c) or to the derivate according to d), respectively, preferably at least 55 % homologous, more preferably at least 60 % homologous, even more preferably at least 65 % homologous, yet even more preferably at least 70 %
homologous.

In another preferred embodiment, the nucleic acid molecule, the fragment or the derivate as stated under step e) is at least 75 % homologous to the nucleic acid molecule according to a) or b), to the fragment according to c) or to the derivate according to d), respectively, preferably at least 80 % homologous, more preferably at least 85 % homologous, even more preferably at least 86 % homologous, yet even more preferably at least 87 %
homologous.
In a further preferred embodiment, the nucleic acid molecule, the fragment or the derivate as stated under step e) is at least 88 % homologous to the nucleic acid molecule according to a) or b), to the fragment according to c) or to the derivate according to d), respectively, preferably at least 89 % homologous, more preferably at least 90 % homologous, even more preferably at least 91 % homologous, yet even more preferably at least 92 %
homologous.
In a further preferred embodiment, the nucleic acid molecule, the fragment or the derivate as stated under step e) is at least 93 % homologous to the nucleic acid molecule according to a) or b), to the fragment according to c) or to the derivate according to d), respectively, preferably at least 94 % homologous, more preferably at least 95 % homologous, even more preferably at least 96 % homologous, yet even more preferably at least 97 %
homologous.
In a further preferred embodiment, the nucleic acid molecule, the fragment or the derivate as stated under step e) is at least 98 % homologous to the nucleic acid molecule according to a) or b), to the fragment according to c) or to the derivate according to d), respectively, preferably at least 99 % homologous.

The invention further relates to a method for screening for a substance that inhibits or enhances the secretion of a protein of the Wnt-family comprising the steps of:
SUBSTITUTE SHEET (RULE 26) a) bringing a candidate substance into contact with a (poly)peptide with the amino acid sequence of SEQ ID. Nos 4, 5, 6 or 8 under conditions that permit binding of said substance to said (poly)peptide; or b) bringing a candidate substance into contact with a (poly)peptide fragment of the (poly)peptide with the amino acid sequence of SEQ ID. Nos 4, 5, 6 or 8 under conditions that permit binding of said substance to said (poly)peptide fragment, said fragment comprises the part of the (poly)peptide with the amino acid sequences of SEQ ID. Nos 4, 5, 6 or 8 which effects Wnt-protein secretion; or c) bringing a candidate substance into contact with a derivate of the (poly)peptide according to a) or with a derivate of the (poly)peptide fragment according to b) under conditions that permit binding of said substance to said derivate; or d) bringing a candidate substance into contact with a (poly)peptide, with a (poly)peptide fragment or with a derivate which is at least 50 % homologous to the (poly)peptide according to a), to the (poly)peptide fragment according to b) or to the derivate according to c), respectively; and e) detecting if the candidate substance is having inhibitory activity or enhancing activity on the secretion of a Wnt-protein or if the candidate substance is having inhibitory activity or enhancing activity on the binding between the (poly)peptide with the amino acid sequence of SEQ ID. Nos 4, 5, 6 or 8 and a Wnt protein.

A derivate as stated under c) is a (poly)peptide with the amino acid sequence of SEQ ID. Nos 4, 5, 6 or 8, or a (poly)peptide fragment according to b) with an arbitrary molecule attached to the N- or C-terminal part or to a side chain of an amino acid, and a candidate substance showing an affinity to said derivate which is at the most 50% increased or decreased compared to the affinity between the candidate substance and the corresponding unmodified (poly)peptide or (poly)peptide fragment, respectively.
In one embodiment of the invention, the (poly)peptide, the (poly)peptide fragment or the derivate as stated under step d) is at least 52 % homologous to the (poly)peptide according to a), to the (poly)peptide fragment according to b) or to the derivate according to c), respectively, preferably at least 55 % homologous, more preferably at least 60 %
homologous, even more preferably at least 65 % homologous, yet even more preferably at least 70 % homologous.
SUBSTITUTE SHEET (RULE 26) In another preferred embodiment, the (poly)peptide, the (poly)peptide fragment or the derivate as stated under step d) is at least 75 % homologous to the (poly)peptide according to a), to the (poly)peptide fragment according to b) or to the derivate according to c), respectively, preferably at least 80 % homologous, more preferably at least 85 %
homologous, even more preferably at least 86 % homologous, yet even more preferably at least 87 % homologous.

In a further preferred embodiment, the (poly)peptide, the (poly)peptide fragment or the derivate as stated under step d) is at least 88 % homologous to the (poly)peptide according to a), to the (poly)peptide fragment according to b) or to the derivate according to c), respectively, preferably at least 89 % homologous, more preferably at least 90 %
homologous, even more preferably at least 91 % homologous, yet even more preferably at least 92 % homologous.

In a further preferred embodiment, the (poly)peptide, the (poly)peptide fragment or the derivate as stated under step d) is at least 93 % homologous to the (poly)peptide according to a), to the (poly)peptide fragment according to b) or to the derivate according to c), respectively, preferably at least 94 % homologous, more preferably at least 95 %
homologous, even more preferably at least 96 % homologous, yet even more preferably at least 97 % homologous.

In a further preferred embodiment, the (poly)peptide, the (poly)peptide fragment or the derivate as stated under step d) is at least 98 % homologous to the (poly)peptide according to a), to the (poly)peptide fragment according to b) or to the derivate according to c), respectively, preferably at least 99 % homologous.

Further, the invention relates to an antibody which specifically binds to the (poly)peptide according to step a), to the (poly)peptide fragment according step b), to the derivate according to step c) or to the (poly)peptide, the (poly)peptide fragment or the derivate according to step d), or to a (poly)peptide domain of the (poly)peptide with the amino acid sequence of SEQ ID. No 4, 5, 6 or 8, preferably to the (poly)peptide domain which is involved in Wnt-protein secretion or which binds to a Wnt-protein.

Aditionally, the invention relates to a siRNA with a target sequence being a fragment of the nucleic acid molecule coding for a (poly)peptide with the amino acid sequences of SEQ ID.
Nos 4, 5, 6 or 8, preferably with a target sequence of SEQ ID Nos 9 or 10.
SUBSTITUTE SHEET (RULE 26) In a further aspect, the invention relates to the use of said antibody or said siRNA, the nucleic acid molecule according to step a) or b) of the screening method, the fragment according to step c), the derivate according to step d) or the nucleic acid molecule, the fragment or the derivate according to step e), the (poly)peptide according to step a) of said screening method, the (poly)peptide fragment according to step b), the derivate according to step c) or the (poly)peptide, the (poly)peptide fragment or the derivate according to step d) as a drug.

The invention relates also to the use of said antibody or said siRNA, the nucleic acid molecule according to step a) or b) of the screening method, the fragment according to step c), the derivate according to step d) or the nucleic acid molecule, the fragment or the derivate according to step e), the (poly)peptide according to step a) of said screening method, the (poly)peptide fragment according to step b), the derivate according to step c) or the (poly)peptide, the (poly)peptide fragment or the derivate according to step d) for the preparation of a medicament for the treatment of Wnt-signaling related disorders, preferably cancer, bone or joint disorders or developmental disorders.

Preferred cancer types are Wnt-dependent cancer types, preferably colorectal cancer, lung cancer, nasopharyngeal carcinoma, preferably Wnt-2 dependent nasopharyngeal carcinoma, small intestinal adenocarcinoma, fundic gland polyps (gastric), gastric carcinoma, gastric (intestinal-like), gastric adenoma (without associated adenocarcinoma), gastrointestinal carcinoid tumor, esophageal adenocarcinoma, juvenile nasopharyngeal angiofibromas, melanoma, pilamatricomas, lung adenocarcinomas, ovarian carcinoma, uterine cervix, uterine endometrial, breast fibromatoses, prostate, thyroid carcinoma, hepatoblastoma, hepatocellular carcinoma, hepatocellular carcinoma associated with hepatitis C, medulloblastoma, desmoid tumor, Wilm's tumor (kidney), pancreatic (non-ductal acinar cell carcinomas), pancreatoblastoma and synovial sarcoma.

Preferred types of bone or joint disorders are osteoarthritis or rheumatoid arthritis, respectively.

Additionally, the invention relates to a pharmaceutical composition comprising said antibody or said siRNA.

In a further aspect, the invention relates to an assay for studying diseases induced by an altered Wnt secretion or for drug screening comprising the use of an organism, for example SUBSTITUTE SHEET (RULE 26) a vertebrate or an invertebrate organism, preferably selected from the group comprising Drosophila, mice, rats, rabbits, chicken, frogs, pigs, sheep, worms, for example C. elegans, and fishes, for example zebrafish, or a cell line systems, preferably human cell line systems, said organisms or cell line system, respectively, showing increased or reduced or no expression of 3L3 or express a mutated 3L3 (poly)peptide in at least one tissue or organ. "An altered Wnt secretion" means for example an increased, decreased or disrupted Wnt-protein secretion.

In a preferred embodiment of the assay said organisms express the 3L3 gene comprising at least one of the nucleic acid molecules shown in SEQ. ID Nos 1 to 3 or 7 as a transgene.

In another preferred embodiment of the assay said 3L3 gene comprises a mutation selected from the group consisting of deletions, point mutations, insertions and inversions.

Still another aspect of the invention is a method for the modification of the Wnt secretion of a cell comprising the following step:

- bringing a cell into contact with a substance that inhibts or enhances Wnt-protein secretion.
In a preferred embodiment of the method said substance is selected from said antibody or said siRNA, the nucleic acid molecule according to step a) or b), the fragment according to step c), the derivate according to step d) or the nucleic acid molecule, the fragment or the derivate according to step e), or the (poly)peptide according to step a), the (poly)peptide fragment according to step b), the derivate according to step c) or the (poly)peptide, the (poly)peptide fragment or the derivate according to step d).

The invention relates further to the use of the nucleic acid molecule according to step a) or b) of the screening method, the fragment according to step c), the derivate according to step d) or the nucleic acid molecule, the fragment or the derivate according to step e), or said antibody or said siRNA to regulate the Wnt-signaling pathway in cells, preferably to regulate cell fate determination or to control the development of stem cells in vivo and in vitro.

Additionally, the invention relates to the use of the (poly)peptide according to step a) of said screening method, the (poly)peptide fragment according to step b), the derivate according SUBSTITUTE SHEET (RULE 26) to step c) or the (poly)peptide, the (poly)peptide fragment or the derivate according to step d) to regulate the Wnt-signaling pathway in cells, preferably to regulate cell fate determination or to control the development of stem cells in vivo and in vitro.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. la-c: The CustalW protein alignment shows the high homology between the protein of Drosophila, C. elegans and human. The putative signal sequence is indicated by a dashed line above the sequence and the putative transmembrane domains are underlined (TMHMM prediction).

Fig. 2: A) Normal adult pharates as control.
B) Adult pharates homozygous mutant for 3L3 showing that aristae of the antennae are missing, and a rudimental development of all three leg pairs.
C) Adult pharates homozygous mutant for 3L3 showing that aristae of the antennae are missing, a rudimental development of all three leg pairs, and a wing-to-notum transformation (observed in 5-10% of cases) Embryo showing the germline clone phenotype.
D) and E): wild-type situation showing a normal segmentation pattern.
F) and G) embryo mutant for 3L3 showing a classical Wg loss-of-function phenotype with fusion of segments ("lawn of denticles" phenotype).

Fig. 3: A) Distalless-LacZ expression in a wild type disc.
B) Distalless-LacZ expression is reduced in 3L3 mutant tissue.
E) the same as B, but only the clone margin is shown.
D) Senseless protein expression in a wild type disc.
C) Senseless protein expression is suppressed in 3L3 mutant.
F) the same as B, but only the clone margin is shown.

Fig. 4: A) Wg protein expression in a wild type disc.
B) Wg protein expression is incresed in 3L3 mutant tissue, suggesting that in these mutant cells Wg accumulates.
C) the same as B), but only the clone margin is shown.
D) extracellular Wg protein expression is not or only weakly incresed in 3L3 mutant tissue, suggesting that the accumulation of Wg seen in B-C is mainly intracellular.

SUBSTITUTE SHEET (RULE 26) E) the same as B, but only the clone margin is shown.

Fig. 5: TOP Flash Luciferase assay. Wnt3A-V5 and siRNA_h3L3 cotransfected cells show no induction of the Wnt signaling dependent Luciferase reporter gene as the mock control, where empty vector (pcDNA3) was cotransfected with siRNA_GFP. However, Wnt3A-V5 and siRNA_GFP cotransfected cells showed a 2-fold induction of the reporter gene compared to the mock control.

Fig. 6: Co-Immunoprecipitation, IP: a-HA, blot: a-V5. h3L3-HA is interacting with Wnt3A-V5 (Lane 1). Negative controls: no interaction could be observed between CD2-HA and Wnt3A-V5 (Lane 2) and no band could be detected in EGFP transfected cells (Lane 3).

Fig. 7: siRNA against the C. elegans homologue of 3L3. A) the gonad of a wt worm.
siRNA against 3L3 leads to an abnormal arrangement of Oocytes within the gonad (B) and sometimes to a defect in distal tip cell migration (C).

Fig. 8: TOP Flash Renilla Luciferase assay. WLS RNAi showed a clear down-regulation of Wg secretion compared to GFP RNAi.
Fig. 9: Secretion of Wnt3a is impaired upon knock-down of hWLS

A) The level of Wnt3a-V5 in the supernatant of HEK-293T cells is strongly decreased by depletion of hWLS.
B) sihWLS abolishes secretion of Wntl-HA into the medium of COS-7 cells.
C) Application of sihWLS strongly decreases secretion of Wnt5a-HA compared to siGFP.

Fig. 10: B-I) Cell surface stainings of HEK-293T cells. Wnt3a-V5 cannot reach the cell surface of cells co-transfected with sihWLS (B) whereas cotransfection of siGFP leads to detectable levels of Wnt3a-V5 at the cell surface (C).
Wnt3aC77A-V5 is neither detectable at the surface of cells treated with sihWLS (D) nor siGFP (E). (F-I) HA-CD2 is detected on the cell surface (F,G) while CD2-HA is not (H,I); CD2-HA can be readily detected by conventional staining (not shown). Surface levels of HA-CD2 are not affected by sihWLS
(F).
SUBSTITUTE SHEET (RULE 26) Fig. 11: RNAi against the C. elegans WLS ortholog phenocopies the ABar spindle orientation defect of mom-2 and mom-3 mutant embryos Embryos are shown anterior to the left and ventral up. (A) Wild-type embryo, the ABar spindle (yellow) is oriented perpendicular to the spindle of ABpr (blue), i.e.
perpendicular to the optical section shown (indicated by the yellow dot). (B) In RNAi(mom-2) injected animals, the two spindles are in parallel orientation.
(C,D) In mom-3(or78) mutant embryos (C) as well as in RNAi(R06B9.6) embryos (D) the ABar and ABpr spindles are also in parallel orientation.

Fig. 12: Secretion of Wnt3a is impaired upon knock-down of hWLS
A) Lysate and culture medium of HEK-293T cells cotransfected with Shh and sihWLS show no alteration in production and secretion of Shh compared to controls (siGFP).
B) Secretion of SSHA-dTNFAN (TNF-FLAG) is not affected by depletion of WLS.

SUBSTITUTE SHEET (RULE 26) DETAILED DESCRIPTION OF THE INVENTION

The Wnt signaling cascade is essential for the development of both vertebrates and invertebrates, and has been implicated in tumorigenesis. The Drosophila Wnt genes are one of the best characterized within the Wnt-protein family, which includes more than hundred genes. In the Drosophila embryo, Wg is required for formation of parasegment boundaries and for maintenance of engrailed (en) expression in adjacent cells. The epidermis of embryo defective in Wg function shows only a rudimentary segmentation, which is reflected in an abnormal cuticle pattern. While the ventral cuticle of wild type larvae displays denticle belts alternating with naked regions, the cuticle of Wg mutant larvae is completely covered with denticles. During imaginal disc development, Wg controls dorso-ventral positional information. In the leg disc, Wg patters the future leg by the induction of ventral fate (Struhl and Basler 1993). In animals with reduced Wg activity, the ventral half of the leg develops into a mirror image of the dorsal side (Baker 1988). Accordingly, reduced Wg activity leads to the transformation of wing to notal tissue, hence the name of the gene (Sharma and Chopra 1976). In the eye disc, Wg suppresses ommatidial differentiation in favor of head cuticle development, and is involved in establishing the dorso-ventral axis across the eye field (Heberlein, Borod et al. 1998).

Additional genes have been implicated in the secretion, reception or interpretation of the Wg signaling. For instance, genetic studies in Drosophila revealed the involvement of frizzled (Dfz), Dishevelled (dsh), shaggy/zeste-white-3 (sgg/zw-3), armadillo (arm), adenomatous polyposis coli (E-apc), axin, and pangolin (pan) in Wg signaling. The genetic order of these transducers has been established in which Wg acts through Dsh to inhibit Sgg, thus relieving the repression of Arm by Sgg, resulting in the cytoplasmic accumulation of Arm and its translocation to the nucleus. In the nucleus Arm interacts with Pan to activate transcription of target genes. Vertebrate homologues have been identified for all these components (for an updated review see (Peifer and Polakis 2000), suggesting that novel identified members of the Drosophila signaling pathway may likely have vertebrate counterparts.
Mutations leading to nuclear accumulation of the mammalian homologue of Arm, R-Catenin, and consequently to constitutive activation of the Wg/Wnt pathway have been observed in many types of cancer, including colon cancer, breast cancer, melanoma, hepatocellular carcinoma, ovarian cancer, endometrial cancer, medulloblastoma pilomatricomas, and prostate cancer (Morin 1999; Polakis, Hart et al. 1999). It is now apparent that deregulation of the Wnt signaling is an important event in the genesis of these malignancies. However, SUBSTITUTE SHEET (RULE 26) there are still no known therapeutic agents effectively inhibiting the Wnt pathway, either by directly inhibiting R-Catenin transcriptional activation or by inhibiting pathway activation by Wnt ligands. This is partly due to the fact that many of the essential components required for its full activation are still unknown.
In order to identify new components required for Wingless activation the inventors used a Drosophila genetic approach to screen for recessive suppressors of the sev-wg phenotype of Drosophila melanogaster. In this approach a protein referred hereinafter to as 3L3 was found which is encoded by the gene CG6210. Said gene has homologues in all metazoans, and more interestingly, there is in all likelihood only one gene of this family in each species.
With genetic tools the inventors confirmed the role of 3L3 as a positiv component of the Wg/Wnt signaling pathway, and in particular its importance in the secretion of the Wnt-proteins. Further, it was shown by means of a binding study that 3L3 and Wnt physically interact.
In the screen for recessive suppressors of the sev-wg phenotype the inventors found 3 different alleles of the 3L3 complementation group. Two alleles have mutations in the so far uncharacterized gene CG6210. CG6210 encodes for a protein which consists of a signal sequence at the N-terminus and 7 putative transmembrane domains. One allele of the 3L3 complementation group, su20.53, contains a 47 bp deletion which leads to a frameshift in the last amino acid residue of the signal sequence and a premature stop codon.
The other suppressor su20.54 has a point mutation that leads to an amino acid residue exchange from Prolin to Serin in the first transmembrane domain. Different bioinformatics programs led to the conclusion that this protein belongs to a family of proteins of unknown function and interestingly, there is always only one protein of this family per species.
Furthermore, the protein is very well conserved in the different species, especially some of the extracellular sequences and the transmembrane-spanning domains (Fig. la-c), indicating that its function is highly specific and conserved.

Drosophila embryos are progressively subdivided into reiterated segments by the localized activities of several genes. Wingless (Wg) and Hedgehog (Hh) are the most important genes of the so-called segment polarity gene group, which eventually divides the A/P
axis into 14-15 stripes prefiguring the future segments. As a consequence, the loss of Wg or Hh function leads to a disruption of the formation of the repeating exoskeletal structures on the ventral side of the embryo, which is the most obvious phenotypic outcome of the activity of the segment polarity genes.
SUBSTITUTE SHEET (RULE 26) Genetic experiments called "germline clones" allow the inventors to generate Drosophila mutant embryos, which completely lack the protein of interest (in the present case 3L3) and then to study the segmentation pattern. In particular "germline clones" are necessary to get rid of the maternal component, which is often present in embryo for many genes.
It was observed that these 3L3 mutant embryos show a fusion of the segments, typical of a Wg or Hh loss of function (Fig. 2 D to G). This results leads to the conclusion that 3L3 is a positive component of the Wg/Wnt or Hh signaling pathway.

Wg plays a pivotal role also in the development of adult appendages of Drosophila, such as antennae, legs, and wings. Therefore inventors studied the phenotype of 3L3 mutant adult pharates, which represent flies just before hatching. They observed that all the appendages, whose development depends on Wg function, are abnormal (Fig. 2 A to C). In particular aristae of antennae are missing, legs are shorter and show segmentation defects, and in 5%
of the cases a so-called "wing-to-notum" transformations was observed, which are pathognomonic for a Wg loss of function. These findings give evidence for the conclusion that 3L3 is a positive component in the Wg/Wnt signaling pathway.

Adult appendages, e.g. wings and legs, are formed in the larva by imaginal cells, which are organized in sack-like epithelia called "imaginal discs". In particular, the wing imaginal disc comprises around 20 cells when it is formed during embryonic development and proliferates during the three larval instars to generate a disc of ca. 75'000 cells at the end of the larval life. In these imaginal discs, Wingless behaves as a morphogen, whose spatial concentration varies and to which cells respond differently at different threshold concentrations.
The inventors used in the experiments wing imaginal discs to study the effect of 3L3 mutant tissue on the expression of target genes, i.e. genes, which are activated by Wg. Two kinds of target genes are distinguished in the wing imaginal discs. On the one hand there are "long-range target genes" (such as Distalless), which are expressed also at lower Wg concentrations, and therefore are expressed at several cell-diameter distance from the Wg source. On the other hand, there are target genes, which need a high level of the morphogen for their expression (so called "short-range target genes" (such as Senseless).
After inducing 3L3 mutant clone formation in wing imaginal discs and the expression of Distalless-lacZ as a marker for the long range target genes and of the Senseless as a marker for the short range target genes (Fig. 3 A to F) was studied. In this experiment a reduction SUBSTITUTE SHEET (RULE 26) of Distalless-acZ and Senseless expression was observed, but only if the mutant tissue involved also the the Wg producing cells. These results give evidence that 3L3 in all likelihood play an important role in the production of the Wg morphogen.

To confirm the importance of 3L3 in Wg production, we tried to rescue flies were selected expressing 3L3 only in Wg producing cells. The rest of the animal was homozygous mutant for 3L3. The result was a complete rescue, what is a reference for the conclusion that the function of 3L3 is necessary only in Wg producing cells.

Further, the effect of 3L3 mutant tissue on the expression of Wg protein in wing imaginal discs was studied (Fig. 4 A to E). It was observed that in 3L3 mutant clones there is an increase in the amount of Wg expression. To better characterize this result, the inventors performed a similar experiment and looked at the wg-lacZ expression, which reveals the level of transcription of wg mRNA. The fact that no increase of the wg-lacZ
expression in 3L3 mutant tissue was present warrants the assumption that the increased staining for Wg in mutant tissue is due to a post-transcriptional process.

Finally, in order to distinguish whether the accumulation of Wg takes place intra- or extra-cellularly, the inventors examined Wg distribution using a different protocol which allows detection of extra-cellular proteins only however an accumulation of extra-cellular Wg in 3L3 mutant clones was not detectable.

These results suggest a role for 3L3 in the secretion of Wg, since its loss of function leads to an accumulation of Wg inside the Wg producing cells.
To test whether the human homologue of 3L3 (h3L3) is involved in Wnt signaling a Wnt reporter gene assay (TOP-flash) was performed. This is a luciferase based assay with 5 TCF
binding sites in front of the luciferase gene. The activity of the Wnt signaling pathway can be measured by the luminescence of the luciferase. From preceding discussed results it was known that 3L3 is important in Drosophilas Wnt producing cells. In order to mimic Wnt producing and receiving cells, one batch of human 293T cells was transfected with mWnt3A-V5 and siRNA against h3L3, another batch of cells was transfected with the TOP-flash reporter construct. 24 hours after transfection these two batches of cells were mixed and after a further incubation of 24 hours the luciferase activity was measured.
If siRNA against 3L3 was cotransfected with Wnt3A in the 'producing' cells then the level of the luciferase activity was as low as if empty vector (instead of Wnt3A) and siRNA against GFP were SUBSTITUTE SHEET (RULE 26) transfected. On the other hand if Wnt3A and siRNA against GFP were transfected a clear induction of the reporter gene was observed (Fig. 5). These results indicate that human (h) 3L3 is also a positive component of the Wnt pathway involved in the production, not reception, of the signal.
Since 3L3 is important in the Wnt producing cells it was investigated if h3L3-HA interacts with mouse (m) Wnt3A-V5. Co-immunoprecipitation experiments were performed and the results suggest that h3L3-HA interacts with mWnt3A-V5 whereas a negative control with CD2-HA, another membrane-spanning protein, did not interact with mWnt3A-V5 (Fig. 6).
In clones of wntless (WLS) mutant cells in Drosophila wing discs it was observed an accumulation of Wg in the Wg producing cells. In Kc cells we developed an assay to measure the secretion of Wg into the culture medium. The Renilla luciferase gene was fused to the N-terminus of wg and transfected into Kc cells. At the same time the cells were treated with dsRNA of WLS or GFP. WLS RNAi showed a clear down-regulation of Wg secretion compared to GFP RNAi (Fig. 8).

To determine whether this secretion defect was also the reason that in human tissue culture experiments depletion of WLS by RNAi resulted in the inability to activate the Wnt signaling cascade, we analysed the medium of Wnt secreting cells. HEK293T were cotransfected with siRNA and either Wnt3a-V5, Wntl-HA or Wnt5a-HA. The secretion of all these Wnts was significantly reduced when siRNA against WLS was cotransfected compared to siRNA against GFP. These results also indicate that in HEK293T cells the secretion of Wnts is dramatically reduced when WLS is depleted (Fig. 9; two different siRNAs (siRNAhWLS-A, siRNAhWLS-B) were validated by RT-PCR for their effectiveness to knock-down the expression of the endogenous hWLS gene in HEK-293T cells. Independent transfection of both resulted in an 85% decrease of hWLS transcripts (not shown; siRNAhWLS-B was mostly used and is henceforth referred to as sihWLS, but siRNAhWLS-A showed the same effects).
Treatment of responder cells with sihWLS, or treatment of producer cells with siRNA against GFP (siGFP) did not appreciably affect the outcome of the Wnt signalling assay.

To test whether in HEK293T cells WNT3a still can reach the extracellular surface when WLS
is downregulated, we cotransfected siRNAs and Wnt3a-V5 and then stained only the extracellular fraction of WNT3a. As a control we used CD2 with either a HA-tag at the N-terminus (extracellular) or at the C-terminus (intracellular). With the extracellular staining protocol we used we could detect only the CD2 construct with the N-terminal HA-tag. RNAi SUBSTITUTE SHEET (RULE 26) against WLS did not influence the secretion of CD2 to the extracellular surface. In contrast, the surface staining of of WNT3a-V5 was abolished in cells cotransfected with siRNA against WLS. Therefore we conclude, that WLS is necessary for the proper secretion of WNT3a (and probably other Wnt's as well) to the cell surface (Fig. 10).
In Drosophila wing discs dispatched clones showed a clear accumulation of Hh ligand but, in stark contrast, no accumulation of Hh could be observed in WLS clones. In addition WLS
mutant flies have been rescued with a wg-Ga14::UAS-WLS construct. Expression of WLS only in wg-producing cells is sufficient to rescue the lethality and supports our view that WLS is specific for Wg signaling.

In human tissue culture only the secretion of Wnt's is affected but as shown above the secretion of CD2 is unaffected by WLS RNAi. Importantly, neither the secretion of Shh nor the secretion of TNF into the medium is influenced by WLS RNAi (Fig. 12).
These results show that also the human orthologue of WLS is specific for Wnt signaling.

Our results also indicate that not only a small subset of Wnts is sensitive to WLS loss of function but that most if not all Wnt's are dependent on WLS. In C.elegans MOM-3 has been shown to be involved in many different, canonical and non-canonical Wnt pathways such as early blastomere polarization (Rocheleau, Thorpe), VPC specification (Eisenmann and Kim), Q neuroblast migration (Harris). These processes depend on three different Wnt's: MOM-2 (Rochelau, Thorpe), LIN-44 (Jiang and Sternberg) and EGL-20 (Harris).

In C. elegans two Wnt signaling pathways can be distinguished, the canonical pathway that is postembryonically active in different developmental processes and the non-canonical pathway that can only be observed in embryonic development. Non-canonical Wnt signaling controls orientation of EMS division (the EMS cell is the precursor of the future endoderm and mesoderm precursor cells) and the fate of endoderm. Loss of non-canonical Wnt signaling leads to the loss of endoderm and the development of more mesoderm.
Several components of this non-canonical Wnt signaling have been described. One of them, mom-3 (more mesoderm-3), has been genetically characterized but has not been molecularly identified so far (Eisenmann and Kim 2000; Thorpe, Schlesinger et al. 1997).
By sequencing, mutations in the homologue of 3L3 (c3L3) were found in different mom-3 alleles. Moreover, RNAi against c3L3 led to typical loss of canonical and non-canonical Wnt phenotypes such as defects in distal tip cell migration and embryonic lethality. Together, these results show that SUBSTITUTE SHEET (RULE 26) c3L3 is responsible for the mom-3 phenotype and that RNAi against c3L3 mimics a loss of mom-3 phenotype.

Wnt signaling in C.elegans is involved in many different developmental steps.
The earliest step known is the MOM-2 dependent orientation of mitotic spindles and division planes in the four to eight cell embryo. Since mom-3 worms are mutant in the wntless orthologue R06B9.6, we injected dsRNA of R06B9.6 into the gonads of wt hermaphrodites. As expected all embryos analyzed injected with dsRNA of R06B9.6, or carrying a mutant allele of mom-2 or mom-3 showed a parallel orientation of the Abar spindle to the ABpr spindle. In contrast, wt embryos had the two spindles oriented perpendicularly (Fig. 11).

In summary, the inventors found a polypeptide named 3L3 which is encoded by the gene CG6210. With genetic tools it was confirmed that 3L3 plays a role in the Wg/Wnt signaling pathway, and in particular in the Wg/Wnt secretion pathway of the Wnt proteins. Further it has been shown that 3L3 physically interacts to a Wnt-protein. Therefore 3L3 proteins are very promising targets for developing drugs which positively or negatively regulate the Wnt pathway by regulating the secretion of the Wnt ligands.

SUBSTITUTE SHEET (RULE 26) EXPERIMENTALPART
Example 1 Screen for recessive suppressors of the sev-wg phenotype:

The sev-wg transgene, which ectopically expresses Wg in the eye, leads to a rough eye phenotype that served as a phenotypic marker (Brunner et al., 1997) in the screen. Males carrying an FRT80 on the left arm and the sev-wg on the right arm of the 3rd chromosome were feeded for 12 hours with 21mM Ethyl methan sulfonate (EMS). 24 hours after the application of EMS the males were crossed to females encoding an eyeless-flp recombinase on the X chromosome and carrying an FRT80 M w+ on the 3d chromosome. Male offspring with the ey-flp and both FRT chromosomes were screened for a suppression of the rough eye phenotype. Complementation analysis between different alleles led to the complementation group 3L3 consisting of two alleles.

Example 2 Homologues in all metazoans:

By Blast search (blastp) it was found that in every sequenced metazoan organism there is only one copy of this new protein family carrying the domain of unknown function 1171 (DUF1171).
Example 3.

Membrane spanning domains:

TMHMM TOP predicted one N-terminal signal sequence and 7 putative transmembrane domains for CG6210-PB.

Example 4 Germline clones:

SUBSTITUTE SHEET (RULE 26) It is an experimental procedure used to study the phenotype of embryo of Drosophila rnelanogasterin the complete absence of the protein of interest.

Female 3'd instar larvae carrying on one chromosome the FRT and the putative mutation and on the homologue chromosome the same FRT and the ovoD mutation were heat-shocked for 1.5 hrs at 38 degrees. After hatching, virgins were crossed with males carrying the same puatative mutation and the FRT.

Female were allowed to lay eggs for one night on agar plate and embryos were then collected. They were then bleached for 3 minutes, and washed with tap water.
Then they were put into water for 24 hrs. In order to remove the vitellum they were shaken in a biphasic solution containing equal amount of Methanol and Heptane. Embryos were subsequently washed 4x with Methanol and then 4x with Triton-X100 0.1%. After 6 hours incubation at RT, they were finally mounted on a slide with Hoyer-lactate and incubated for 12-24 hours at 60 C to digest protein: Cuticles were examined under the light microscope.
Example 5 Wing imaginal discs to study the effect of 3L3 mutant tissue on the expression of target genes:

Imaginal discs are hollow sacs of cells that make adult structures during metamorphosis.
They arise as pockets in the embryonic ectoderm and grow inside the body cavity until the larva becomes a pupa, at which point they turn inside out ("evaginate") to form the body wall and appendages.

Antibody staining of imaginal discs:
Larvae were dissected in Ringer solution on ice and fixed in 200 ul tubes with PEM (200 pl)+
5% formaldehyde (10,7 p I) + 0.05% Triton X-100 (1 NI) for 20 min. After washing 4x, plus 1 hour, plus 2-3x with PBT + Na-Azid, they were incubated over night with the primary antibody (diluted in PBT + Na-Azid) at 4 degrees. Larvae were washed 5x plus 30 min with PBT + Na-Azid + 1% HINGS and incubated with the secondary antibody diluted in PBT +
Na-Azid for 2 hours, at RT. Larvae were washed again 5x plus 2 hours with PBT
+ Na-Azid and discs were mounted in PPDA. Staining in wing discs was analyzed on a confocal microscope.

SUBSTITUTE SHEET (RULE 26) Example 6 Inducing 3L3 mutant clone formation in wing imaginal discs and studying the expression of Distalless-lacZ (wg-lacZ):
Mutant clones in the wing imaginal discs represent groups of cells, which are mutant for the protein of interest. In Drosophila melanogaster this is achieved by mitotic recombination, using the yeast-specific recombinase flippase (flp) driven by the heat-shock promoter (hs-tIp).
y w hsp-flp; allele 20.53 FRT80 / TM6b flies were crossed with y w hsp-flp;
nMyc [wfJ
Minute P[y-fJ FRT80/ TM6b, and females were allowed to lay eggs for two days.
Heat-shock at 37.0 degrees for 60 minutes was made 24-72 hours AEF, and then larvae were dissected 4 days later.

In the case of Distalless-lacZ the primary antibody P-galactosidase (1:2000 rabbit, polyclonal, form Cappel ) was used.

In the case of wg-lacZ the antibody a-Wg (4D4) (1:1000 mouse, monoclonal, DSHB) was used.

Example 7 Confirmation of the importance of 3L3 in Wg production by means of the try to rescue flies expressing 30 only in Wg producing cells:

31_3 heterozygous mutant flies carrying the transgene wg-GAL4 (the transcriptional activator GAL4 is then expressed only in the cells, which express also Wg) were crossed with flies, which were also 3L3 heterozygous mutant and carried the trangene UAS-3L3 (UAS
binding sites for the GAL4 protein activate the transcription of 3L3.

Example 8 Examination of Wg distribution:

SUBSTITUTE SHEET (RULE 26) The protocol used to see extracellular Wg differs from the usual protocol in the fact that the staining with the primary antibody is performed in PBS at 4 degrees (in order to block endocytosis) for 30-60 minutes, and that fixation is done after the primary antibody in PBS, with 4% formaldehyde at 4 degrees for 20-30 minutes (Strigini and Cohen, 2000).
Example 9 The TOP Flash assay:

Cells were transfected by the Calcium-Phosphate method. 16 hours after transfection cells were washed and mixed. 24 hours after mixing, the cells were lysed with 1xPLB
from Promega and analyzed.

Example 10 Co-immunoprecipitation:
Cells were transfected by the Calcium-Phosphate method. 36 hours after transfection, cells were lysed with RIPA buffer for 1 hour 30 minutes at 4 C. The lysate was centrifuged for 30 minutes at 4 C and then incubated with Protein G Sepharose beads and rabbit a-HA
overnight at 4 C. Beads were washed 4x with TBS and then Proteins were eluted with SDS-Loading buffer from the beads at 95 C for 10 minutes. Western blotting was performed with the mouse a-V5 antibody.

Constructs:

The mWnt3A cDNA sequence was cloned into pcDNA3 followed by a V5-tag and a His-tag.
The h3L3 cDNA was cloned into pcDNA3 followed by a HA-tag. The siRNAs against 3L3 and GFP were generated by Qiagen. CD2 was cloned with a C-terminal HA-tag into pcDNA3.

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Thorpe, C.]., Schlesinger, A., et al (1997). "Wnt signaling polarizes an early C. elegans blastomere to distinguish endoderm from mesoderm." Cell;90(4): 695-705.
Thorpe, C. J., Schlesinger, A., and Bowerman, B. (2000). Wnt signalling in Caenorhabditis elegans: regulating repressors and polarizing the cytoskeleton.
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Claims

1. A method for screening for a substance that inhibits or enhances the secretion of a protein of the Wnt-family comprising the steps of:

a) bringing a candidate substance into contact with a nucleic acid molecule with the nucleotide sequence of SEQ ID. Nos 1, 2, 3 or 7 under conditions that permit binding of said substance to said nucleic acid molecule; or b) bringing a candidate substance into contact with nucleic acid molecule coding for a (poly)peptide with the amino acid sequences of SEQ ID. Nos 4, 5, 6 or 8 under conditions that permit binding of said substance to said nucleic acid molecule; or c) bringing a candidate substance into contact with a fragment of the nucleic acid molecule according to a) or b) under conditions that permit binding of said substance to said fragment, said fragment codes for the part of the (poly)peptide with the amino acid sequences of SEQ ID. Nos 4, 5, 6 or 8 which effects Wnt-protein secretion; or d) bringing a candidate substance into contact with a derivate of the nucleic acid molecule according to a) or b) or with a derivate of the fragment according to c) under conditions that permit binding of said substance to said derivate; or e) bringing a candidate substance into contact with a nucleic acid molecule, with a fragment or with a derivate which is at least 50 % homologous to the nucleic acid molecule according to a) or b), to the fragment according to c) or to the derivate according to d), respectively;
f) detecting if the candidate substance is having inhibitory activity or enhancing activity on the secretion of a Wnt-protein.

2. A method according to claim 1, characterized in that the nucleic acid molecule, the fragment or the derivate as stated under step e) is at least 88 % homologous to the nucleic acid molecule according to a) or b), to the fragment according to c) or to the derivate according to d), respectively, preferably at least 89 % homologous, more preferably at least 90 % homologous, even more preferably at least 91 %
homologous, yet even more preferably at least 92 % homologous.

3. A method for screening for a substance that inhibits or enhances the secretion of a protein of the Wnt-family comprising the steps of:
a) bringing a candidate substance into contact with a (poly)peptide with the amino acid sequence of SEQ ID. Nos 4, 5, 6 or 8 under conditions that permit binding of said substance to said (poly)peptide; or b) bringing a candidate substance into contact with a (poly)peptide fragment of the (poly)peptide with the amino acid sequence of SEQ ID. Nos 4, 5, 6 or 8 under conditions that permit binding of said substance to said (poly)peptide fragment, said fragment comprises the part of the (poly)peptide with the amino acid sequences of SEQ ID. Nos 4, 5, 6 or 8 which effects Wnt-protein secretion; or c) bringing a candidate substance into contact with a derivate of the (poly)peptide according to a) or with a derivate of the (poly)peptide fragment according to b) under conditions that permit binding of said substance to said derivate; or d) bringing a candidate substance into contact with a (poly)peptide, with a (poly)peptide fragment or with a derivate which is at least 50 % homologous to the (poly)peptide according to a), to the (poly)peptide fragment according to b) or to the derivate according to c), respectively; and e) detecting if the candidate substance is having inhibitory activity or enhancing activity on the secretion of a Wnt-protein or if the candidate substance is having inhibitory activity or enhancing activity on the binding between the (poly)peptide with the amino acid sequence of SEQ ID. Nos 4, 5, 6 or 8 and a Wnt protein.

4. A method according to claim 3, characterized in that the (poly)peptide, the (poly)peptide fragment or the derivate as stated under step d) is at least 88 %
homologous to the (poly)peptide according to a), to the (poly)peptide fragment according to b) or to the derivate according to c), respectively, preferably at least 89 % homologous, more preferably at least 90 % homologous, even more preferably at least 91 % homologous, yet even more preferably at least 92 % homologous.

5. An antibody which specifically binds to the (poly)peptide according to claim 3 step a), to the (poly)peptide fragment according to claim 3 step b), to the derivate according to claim 3 step c) or to the (poly)peptide, the (poly)peptide fragment or the derivate according to claim 3 step d), or to a (poly)peptide domain of the (poly)peptide with the amino acid sequence of SEQ ID. No 4, 5, 6 or 8, preferably to the (poly)peptide domain which is involved in Wnt-protein secretion or which binds to a Wnt-protein.

6. A siRNA with a target sequence being a fragment of the nucleic acid molecule coding for a (poly)peptide with the amino acid sequences of SEQ ID. Nos 4, 5, 6 or 8, preferably with a target sequence of SEQ ID Nos 9 or 10.

7. An antibody according to claim 5 or a siRNA according to claim 6, the nucleic acid molecule according to claim 1 step a) or b), the fragment according to claim 1 step c), the derivate according to claim 1 step d) or the nucleic acid molecule, the fragment or the derivate according to claim 1 step e), or the (poly)peptide according to claim 3 step a), the (poly)peptide fragment according to claim 3 step b), the derivate according to claim 3 step c) or the (poly)peptide, the (poly)peptide fragment or the derivate according to claim 3 step d) for the use as a drug.

8. Use of an antibody according to claim 5 or a siRNA according to claim 6, the nucleic acid molecule according to claim 1 step a) or b), the fragment according to claim 1 step c), the derivate according to claim 1 step d) or the nucleic acid molecule, the fragment or the derivate according to claim 1 step e), or the (poly)peptide according to claim 3 step a), the (poly)peptide fragment according to claim 3 step b), the derivate according to claim 3 step c) or the (poly)peptide, the (poly)peptide fragment or the derivate according to claim 3 step d) for the preparation of a medicament for the treatment of Wnt-signaling related disorders, preferably cancer, bone or joint disorders or developmental disorders.

9. A pharmaceutical composition comprising the antibody according to claim 5 or the siRNA according to claim 6, the nucleic acid molecule according to claim 1 step a) or b), the fragment according to claim 1 step c), the derivate according to claim 1 step d) or the nucleic acid molecule, the fragment or the derivate according to claim 1 step e), or the (poly)peptide according to claim 3 step a), the (poly)peptide fragment according to claim 3 step b), the derivate according to claim 3 step c) or the (poly)peptide, the (poly)peptide fragment or the derivate according to claim 3 step d).

10. An assay for studying diseases induced by a altered Wnt secretion or for drug screening comprising the use of an organism, for example vertebrates or invertebrates, preferably selected from the group comprising Drosophila, mice, rats, rabbits, chicken, frogs, pigs, sheep, worms, for example C. elegans, and fishes, for example zebrafish, or a cell line systems, preferably human cell line systems, said organisms or cell line system, respectively, showing increased or reduced or no expression of 3L3 or express a mutated 3L3 (poly)peptide in at least one tissue or organ.

11. An assay according to claim 10, characterized in that said organisms express the 3L3 gene comprising at least one of the nucleic acid molecules shown in SEQ. ID
Nos 1 to 3 or 7 as a transgene.

12. An assay according to claim 10, characterized in that said 30 gene comprises a mutation selected from the group consisting of deletions, point mutations, insertions and inversions.

13. A method for the modification of the Wnt secretion of a cell comprising the following step:

- bringing a cell into contact with a substance that inhibts or enhances Wnt-protein secretion.

14. A method according to claim 13, characterized in that said substance is the antibody according to claim 5 or the siRNA according to claim 6, the nucleic acid molecule according to claim 1 step a) or b), the fragment according to claim 1 step c), the derivate according to claim 1 step d) or the nucleic acid molecule, the fragment or the derivate according to claim 1 step e), or the (poly)peptide according to claim 3 step a), the (poly)peptide fragment according to claim 3 step b), the derivate according to claim 3 step c) or the (poly)peptide, the (poly)peptide fragment or the derivate according to claim 3 step d).

15. Use of the nucleic acid molecule according to claim 1 step a) or b), the fragment according to claim 1 step c), the derivate according to claim 1 step d) or the nucleic acid molecule, the fragment or the derivate according to claim 1 step e), or the antibody according to claim 5 or the siRNA according to claim 6 to regulate the Wnt-signaling pathway in cells, preferably to regulate cell fate determination or to control the development of stem cells.

16. Use of the (poly)peptide according to claim 3 step a), the (poly)peptide fragment according to claim 3 step b), the derivate according to claim 3 step c) or the (poly)peptide, the (poly)peptide fragment or the derivate according to claim 3 step d) to regulate the Wnt-signaling pathway in cells, preferably to regulate cell fate determination or to control the development of stem cells.