WO2012113779A1

WO2012113779A1 - Means and methods for treating a disease or disorder related to lymphangiogenesis or preventing metastasis

Info

Publication number: WO2012113779A1
Application number: PCT/EP2012/052912
Authority: WO
Inventors: Peter Petzelbauer; Heide LEB
Original assignee: Medizinische Universität Wien
Priority date: 2011-02-21
Filing date: 2012-02-21
Publication date: 2012-08-30

Abstract

The present invention relates to means and methods for use in treating a disease or disorder related to lymphangiogenesis or preventing metastasis. In particular, in context of the present invention, such means may be polynucleotides encoding Wnt-1 or functional equivalent derivatives or fragments thereof, Wnt-1 polypeptides or functional equivalent derivatives or fragments thereof, as well as pharmaceutical compositions comprising the same. The present invention also relates to means and methods for treating a disease or disorder related to lymphangiogenesis and/or for preventing metastasis by employing Wnt-1 polynucleotides and/or polypeptides. Wnt-1 polynucleotides and/or polypeptides are also to be used for treating, preventing and/or ameliorating cancer.

Description

Means and methods for treating a disease or disorder related to lymphangiogenesis or preventing metastasis

The Wnt protein family comprises 19 secreted, cysteine-rich glycoproteins that activate receptor-mediated signaling pathways which control cell differentiation, proliferation and motility (Wodarz, Ann Rev Cell Dev Biol (1998), 14: 59-88). They activate a canonical pathway, which stabilizes cytosolic β-catenin, turning it into a nuclear transcriptional regulator, where it binds to transcription factors of the T-cell factor (Tcf)/Lymphocyte- enhancer-binding factor (Lef) family of proteins. They also signal through non-canonical pathways that activate the calcineurin/NFAT (nuclear factor of activated T cells) pathway and/or transcription factors of the AP-l/Jun family (Staal, Nat Rev Immunol (2008), 8: 581- 593). Wnt-1 was initially classified as an activator of the canonical pathway, but found later to induce also NF AT -mediated gene expression (Spinsanti, J Neurochem (2008), 104: 1588- 1598; Schiavone, Biochem J (2009), 421 : 283-292). Wnt-5a was classified as a non-canonical Wnt family member but may activate β-catenin (He, Science (1997), 275: 1652-1654; Mikels, PLoS Biol (2006), 4: el 15) or even may antagonize canonical/ -catenin signaling. Thus, Wnt proteins are not intrinsically canonical or non-canonical, pathway decisions are determined by distinct sets of receptors and co-receptors (Mikels, PLoS Biol (2006), 4: el 15). In other words, pathway decisions may be cell type-specific and may depend on receptor availability.

The association of Wnt pathways with malignancies was first identified in familial adenomatous polyposis coli (Gardner, Am J Hum Genet (1962), 14: 376-390), where a mutated APC gene (Groden, Cell (1991), 66: 589-600) leads to nuclear localization of β- catenin, canonical gene transcription and to a high risk for colon cancer (Gomez Garcia, Lancet Oncol (2009), 727-735). Nuclear localization of β-catenin was subsequently used in many studies as a surrogate marker for active canonical Wnt signaling and was mostly correlated with poor outcome as, e.g., shown for breast cancer patients (Khramtsov, Am J Pathol (2010), 176: 2911-2920). Such studies also exist in melanoma, but results are contradictive: On the one hand, studies exist reporting that activation of β-catenin is a rare event in melanoma cells (Pollock, Melanoma Res (2002), 12: 183-186) and that loss of β- catenin expression plays a significant role in the progression of malignant melanoma ( ageshita, Br J Dermatol (2001), 145: 210-216). On the other hand, several studies indicate that strong β-catenin expression and stabilization is frequently found in melanoma cells and is associated with bad prognosis (Kielhorn, Int J Cancer (2003), 103: 652-656; Rimm, Am J Pathol (1999), 154: 325-329); Reifenberger, Int J Cancer (2002), 100: 549-556;; Rubinfeld, Science (1997), 275: 1790-1792; Bachmann, Clin Cancer Res (2005), 1 1 : 8606-8614; Lucero, Curr Oncol Rep (2010), 12: 314-318;). Currently, the differences are difficult to reconcile, largely because nuclear localization of β-catenin cannot necessarily be equated with its translational activity (Song, J Biol Chem (2008), 283: 25988-25999); Nusse, Trends Genet (1999), 15: 1-3). In addition, it has not yet been investigated if Wnt affects outcomes of diseases by changing the secretome of tumor cells that may target blood or lymph vessels. Moreover, no study addressed the question whether canonical or non-canonical signaling changed outcomes in melanoma and if this is a direct effect of Wnt on tumor cells or an indirect effect of Wnt or of Wnt-released secondary messengers on host stromal cells. Stroma responses are of particular importance as blood vessel angiogenesis is rate limiting for tumor nourishment and growth, and lymph vessel angiogenesis inter alia correlates with melanoma metastasis (Alitalo, Cancer Cell (2002), 1 : 219-227; Mumprecht, J Cell Mol Med (2009), 13: 1405-1416; Rinderknecht, J Cell Physiol (2008), 216: 347-354; Saaristo, Oncogene (2000), 19: 6122-6129). Finally, with regard to Wnt protein expression in melanoma, data are limited. So far, Wnt-2, Wnt-5a, Wnt-5b, Wnt- 7b, Wnt-10b were found to be expressed in both, nevi and melanoma (Pham, Mol Pathol (2003), 56: 280-285; Kashani-Sabet, Proc Natl Acad Sci U S A (2009), 106: 6268-6272; Bittner, Nature (2000), 406: 536-540; Weeraratna, Cancer Cell (2002), 1 : 279-288). Wnt-5a was found as an independent risk factor for reduced overall survival in multivariate analysis (Da Forno, Clin Cancer Res (2008), 14: 5825-5832). For Wnt-3a, it was shown that B16 melanoma overexpressing Wnt-3a exhibit decreased tumor size and decreased metastasis when implanted into mice (Chien, Proc Natl Acad Sci U S A (2009), 106: 1 193-1 198). However, this study did not investigate effects on lymph- angiogenesis. On the other hand, it was reported that Wnt-1 and Wnt-3a promote expansion of melanocytes (Dunn, Pigment Cell Res (2005), 18: 167-180). In this context, it was demonstrated that both Wnt forms act through distinct modes of function. Whereas Wnt-1 acts on melanoblast precursors to increase the number of cells that can become crest-derived melanocytes, Wnt-3a is able to bias melanoblast prompter- TV A+ cells to the crest-derived melanocyte lineage in addition to acting on melanoblast precursor cells. In another report Wnt-1 was shown to down-regulate VEGF-D (Orlandini J Biol Chem (2003), 278: 44650- 44656); yet VEGF-D is not expressed in melanoma.

A variety of angiogenic regulators are Wnt targets (e.g., Ephrins, FGF-2, FGF-18, FGF-20, endothelin-1, Cx43, uPar, MMP7 and MMP3) (Shimokowa, Cancer Res (2003), 63: 61 16- 6120; Segditsas, Hum Mol Genet (2008), 17: 3864-3875; Brabletz, Am J Pathol (1999), 155: 1033-1038; Hiendlmeyer, Cancer Res (2004), 64: 1209-1214; Kim, Oncogene (2005), 24: 597-604). Moreover, Wnt-1 itself has been shown to induce angiogenesis in vitro (Goodwin, Growth Factors (2007), 25: 25-32; Zerlin, Angiogenesis (2008), 11 : 63-69; Wright, Biochem Biophys Res Commun (1999), 263: 384-388). Finally, genetic mutations of several Wnt/Fzd genes (e.g., Wnt-2, Wnt-4, Wnt-7b, Fzd-4 and Fzd-5) resulted in abnormal vessel development (Ishikiwa, Development (2001), 128: 25-33; Monkley, Development (1996), 122: 3343-3353; Shu, Development (2002), 129: 4831-4842).

However, neo-development of vessels, particularly lymph vessels (lymphangiogenesis), is known to be associated with several diseases and disorders and metastasis (e.g., El-Chemaly, Ann N Y Acad Sci (2008), 1131 : 195-202; Patel, Seminars Ophtalmol (2009), 24: 135-138; El-Chemaly, Lymphatic Res Biol (2009), 7: 197-203; Pepper, Clin Cancer Res (2001), 7: 462-468). In melanoma, the probability of metastatic spread correlates with lymphatic vessel densities. Accordingly, there is a need for means and methods for treating, ameliorating and preventing such diseases and disorders related to lymphangiogenesis or metastasis. Thus, the technical problem underlying the present invention is the provision of novel means and methods for the medical intervention of a disease or disorder related to lymphangiogenesis or metastasis.

The technical problem underlying the present invention has been solved by the embodiments as characterized herein and particularly in the claims.

As has been surprisingly found in the present invention, Wnt-1 reduces VEGF-C expression and lymphangiogenesis and reduces and delays metastasis. This surprising finding is in clear contrast to what has been expected from the prior art, namely that Wnt-1 overexpression would result in enhanced melanoma-induced blood- and lymph-vessel neo-formation and melanoma progression.

Thus, the present invention relates to Wnt-1 polynucleotides and/or Wnt-1 polypeptides for use in

(A) treating or preventing a disease or disorder related to lymphangiogenesis, or

(B) preventing metastasis. Accordingly, the present invention relates to a polynucleotide for use in treating or preventing a disease or disorder related to lymphangiogenesis or for use in preventing metastasis, wherein said polynucleotide is selected from the group consisting of:

(a) a polynucleotide comprising or consisting of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3;

(b) polynucleotide encoding a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4;

(c) polynucleotide having a nucleotide sequence which is at least 70% identical to the sequence of SEQ ID NO: 1 or SEQ ID NO: 3 and which encodes a polypeptide exhibiting Wnt-1 activity;

(d) a polynucleotide encoding a polypeptide comprising or consisting of an amino acid sequence which is at least 70% identical to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 and which exhibits Wnt-1 activity; and

(e) a polynucleotide comprising a fragment of the nucleotide sequence of any one of (a) to (d) which is at least 24 nucleotides in length and which encodes a polypeptide having Wnt- 1 activity.

Thus the present invention relates to means and methods for the medical intervention of both, a disease or disorder related to lymphangiogenesis and metastasis.

Accordingly, the present invention relates to Wnt-1 polynucleotides and/or polypeptides for use in treating or preventing a disease or disorder related to lymphangiogenesis. The present invention further relates to Wnt-1 polynucleotides and/or polypeptides for use in preventing metastasis.

The present invention also relates to a Wnt-1 polypeptide for use in treating or preventing a disease or disorder related to lymphangiogenesis or for use in preventing metastasis, wherein said polypeptide is selected from the group consisting of:

(a) a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4;

(b) a polypeptide comprising or consisting of an amino acid sequence which is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%), 99%, 99.5% or 99.9% identical to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 and which exhibits Wnt-1 activity; and

(c) a polypeptide comprising a fragment of the amino acid sequence of (a) or (b) which is at least 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 88, 96, 104, 112, 120, 128, 136, 144, 152, 160, 168, 176, 184, 192, 200, 208, 216, 224, 232, 240, 248, 256, 264, 272, 280, 288, 296, 304, 312, 320, 328, or 336 amino acids in length and which is a polypeptide having Wnt-1 activity.

It is preferred that the polypeptide as described under item (b), supra, is a polypeptide having an amino acid sequence which is at least 70% identical to the sequence of SEQ ID NO: 2 or SEQ ID NO: 4 and which is a polypeptide exhibiting Wnt-1 activity. It is also preferred that the polypeptide as described under item (c), supra, is a fragment of at least 8 amino acids in length and which is a polypeptide having Wnt-1 activity. To assess "Wnt-1 activity" reduction of VEGF-C expression may be measured as described herein.

Accordingly, the present invention relates to a polypeptide encoded by the polynucleotide to be employed in context with the present invention for use treating or preventing a disease or disorder related to lymphangiogenesis or for use in preventing metastasis.

It is also envisaged to apply functional equivalent derivatives or fragments of Wnt-1 polypeptides and/or polynucleotides having Wnt-1 activity for the means and methods described herein. Accordingly, the present invention also relates to functional equivalent derivatives or fragments of Wnt-1 polypeptides and/or polynucleotides having Wnt-1 activity for use in treating or preventing a disease or disorder related to lymphangiogenesis. In line with this, the present invention further relates to functional equivalent derivatives or fragments of Wnt-1 polypeptides and/or polynucleotides having Wnt-1 activity for use in preventing metastasis.

The present invention further relates to a polynucleotide for use in treating or preventing a disease or disorder related to lymphangiogenesis or for use in preventing metastasis, wherein said polynucleotide is selected from the group consisting of:

(a) a polynucleotide comprising or consisting of the nucleotide sequence of SEQ ID NO:

1 (human wnt-1) or SEQ ID NO: 3 (human wnt-1 without the first 81 nucleotides, i.e. without the signal sequence);

(b) polynucleotide encoding a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 2 (human Wnt-1) or SEQ ID NO: 4 (human Wnt-1 without the first 27 amino acids, i.e. without the signal sequence);

(c) polynucleotide having a nucleotide sequence which is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to the sequence of SEQ ID NO: 1 or SEQ ID NO: 3 and which encodes a polypeptide exhibiting Wnt-1 activity;

(d) a polynucleotide encoding a polypeptide comprising or consisting of an amino acid sequence which is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 and which exhibits Wnt-1 activity; and

(e) a polynucleotide comprising a fragment of the nucleotide sequence of any one of (a) to (d) which is at least 24, 48, 72, 96, 120, 144, 168, 192, 216, 240, 264, 288, 312, 336, 360, 384, 408, 432, 456, 480, 504, 528, 552, 576, 600, 624, 648, 672, 696, 720, 744, 768, 792, 816, 840, 864, 888, 912, 936, 960, 984, 1008, 1032, 1056, 1080, or 1 104 nucleotides in length and which encodes a polypeptide having Wnt-1 activity.

It is preferred that the polynucleotide as described under item (c), supra, is a polynucleotide having a nucleotide sequence which is at least 70% identical to the sequence of SEQ ID NO: 1 or SEQ ID NO: 3 and which encodes a polypeptide exhibiting Wnt-1 activity. It is also preferred that the polynucleotide as described under item (e), supra, is a fragment of at least 24 nucleotides in length and which encodes a polypeptide having Wnt-1 activity.

The polynucleotides described under items (a) to (e), supra, and those further described herein, e.g., in the exemplified embodiments as described below, are also referred to herein as "the polynucleotide to be employed in context with the present invention". The present invention also relates to Wnt-1 polypeptides encoded by these polynucleotides for use in treating or preventing a disease or disorder related to lymphangiogenesis or for use in preventing metastasis. These polypeptides and those further described herein, e.g., in the exemplified embodiments as described below, are also referred to herein as "the polypeptide to be employed in context with the present invention".

In the prior art it was shown that when canonical Wnt proteins directly target the endothelium, they induce angiogenesis. In contrast, in context of the present invention it was demonstrated that when Wnt-1 targets melanoma cells, it changes the release of lymph-angiogenic factors and prevents lymph-angiogenesis.

The unexpected effects of Wnt-1 over-expression that severely impairs the ability of a melanoma to secrete VEGF-C, to recruit lymph vessels and to form metastasis, opens a new option; Wnt-1 could be considered as a potential anti-lymph-angiogenic factor for adjuvant treatment of melanoma and potentially also for other malignancies. For example, Wnt-1 - molecules may be used to treat cancers (e.g. melanomas) that are negative for Wnt-1 expression.

It has been found in context of the present invention and as is shown and exemplified herein, the anti-lymphangiogenic and anti-metastatic effect of Wnt-1 is calcineurin dependent and β- catenin independent. This finding is in sharp contrast to what would have been expected from Chien et al. (Chien, Proc Natl Acad Sci U S A (2009), 106: 1193-1198), namely that a decrease of metastasis would be mediated by β-catenin. In fact, the finding of the present invention that Wnt-1 reduces VEGF-C expression and lymphangiogenesis clearly contrasts data of Wnt-1 on endothelial cells in vitro: Wnt-1 and Wnt-3 have been shown to induce proliferation and migration of endothelial cells in vitro through increased Wnt/ -catenin signaling (Samarzija, Biochem Biophys Res Commun (2009), 386: 449-454; Masckauchan, Angiogenesis (2005), 8: 43-51). According to the findings of the present invention, however, Wnt-1 did not augment blood vessel formation (angiogenesis) in vivo, which one would expect based on the fact that Wnt-1 is a secreted factor and, thus, should also target stroma cells. This may be explained by the finding provided in context of the present invention that in a xenograft mouse model, Wnt-1 primarily changed gene expression patterns of melanoma cells but had only minimal effects on stroma cells (as evidenced by a concomitantly performed Affymetrix® mouse gene expression array; data not shown).

In particular, in context of the present invention, it was surprisingly found that Wnt-1 significantly reduced lymphangiogenesis and VEGF-C expression in a β-catenin independent fashion. Further, as found out in context of the present invention, Wnt-1 overexpression significantly delayed metastasis and kept the sentinel lymph node free of tumor cells for an observation period of 90 days.

The NFAT/calcineurin connection is thought to be a key regulator of VEGF-C. However, as described herein, Wnt-5a does not affect VEGF-C or lymphangiogenesis, whereas Wnt-1 clearly does. As shown herein and demonstrated in the appended examples, Wnt-1 suppresses VEGF-C even in the presence of DKK-1 (inhibitor of β-catenin dependent signaling). Together with the effects of ionomycin, which activates calcineurin and reduces VEGF-C and the inhibition of Wnt-1 -induced VEGF-C repression by CsA, these data support that Wnt-1 acts through calcineurin. As shown in the appended illustrative examples, CsA restores high VEGF-C levels in melanoma cells with a delay of 48h and the concentrations of ionomycin required to reduce VEGF-C is rather high. This delay in VEGF-C responses is also highlighted by the difference in VEGF-C inhibition in transient (50% inhibition) versus stable (90% inhibition) Wnt-1 overexpressing melanoma. This delayed response between stimulus and VEGF-C mRNA expression is in line with the concept that calcineurin does not reduce VEGF-C expression via a NFAT-dependent transcriptional regulation at the level of the VEGF-C promoter. An indication for a potential protective role of calcineurin in cancer is considered in a previous report showing that 'hyper-activated' calcineurin inhibited the formation of an effective tumor vasculature which was restored by CsA (Ryeom, Cancer Cell (2008), 13: 420-431), but lymph-angiogenesis was not evaluated. Thus, one could speculate that Wnt-1 mimics 'hyper-activated' calcineurin.

As known in the art, lymphangiogenesis is the formation of lymphatic vessels from preexisting lymphatic vessels, whereas angiogensis is the physiological process involving the growth of new blood vessels from pre-existing blood vessels. Lymphatic vessels are an important route of metastatic dissemination of tumor cells into sentinel nodes, e.g., in melanoma (Dadras, Mod Pathol (2005), 18: 1232-1242; Dadras, Am J Pathol (2003), 162: 1951-1960; He, Science (1997), 275: 1652-1654; Skobe, Am J Pathol (2001), 159: 893-903). Typical biomarkers for lymphangiogenesis are, e.g., Prox-1 and Lyve-1 , which may also be used to detect the presence of lymphangiogenesis in a given sample or tissue (Baluk, Ann N Y Acad Sci (2008), 1131 : 1-12). Of note, VEGF-C has recently been described to be an important regulator of lymphangiogenesis (Lohela, Curr Opin Cell Biol (2009), 21 : 154-165). This is also congruent with a finding of the present invention according to which VEGF-C may be used as a biomarker for Wnt-1 activity as also further described herein.

Accordingly, in context of the present invention, Wnt-1 -molceules as described herein above and below (herein referred to as, e.g., polynucleotides or polypeptides to be employed in context with the present invention; vectors comprising such polynucleotides; host cells containing such polynucleotides, vectors or polypeptides; agents as described herein and to be employed in context of the present invention; or pharmaceutical compositions comprising one or more of the preceding compounds as described herein and to be employed in context of the present invention) can be used in treating a disease or disorder related to lymphangiogenesis or in preventing metastasis. Accordingly, Wnt-1 polynucleotides and/or polypeptides as described herein may be used to treat, prevent and/or ameliorate cancer.

In context with the present invention, when reference is made to a disease or disorder related to lymphangiogenesis which can be treated as described and exemplified herein, a disease or disorder related to lymphangiogenesis is generally known in the art. It is envisaged that, in context of the present invention, the disease to be treated is cancer. This cancer may be a solid tumor. The type of cancer may be, for example, melanoma, breast cancer, lung cancer, kidney cancer, pancreatic cancer or hematopoietic tumor (lymphoma). It is also envisaged with respect to the present invention that the disease to be treated is graft rejection. This graft rejection may be, for example, lung graft rejection, kidney graft rejection, and particularly cornea graft rejection. In addition, it is also envisaged in context of the invention, that the disease to be treated is an autoimmune disorder. This autoimmune disorder may be, for example, rheumatoid arthritis or psoriasis. Furthermore, it is also envisaged in context of the invention, that the disease to be treated is oedema or impaired wound healing. In particular, in context of the present invention as described herein, a disease or disorder related to lymphangiogenesis may be selected from the group consisting of cancer, melanoma, solid tumor, breast cancer, lung cancer, kidney cancer, pancreatic cancer, hematopoietic tumor (lymphoma), graft rejection, autoimmune disorder, rheumatoid arthritis, psoriasis, oedema and impaired wound healing. In this context, examples for graft rejection are lung graft rejection, kidney graft rejection, and particularly cornea graft rejection. Accordingly, the present invention also relates to a vector comprising the polynucleotide to be employed in context with the present invention for use in treating a disease or disorder related to lymphangiogenesis or in preventing metastasis. The term "vector" as used herein particularly refers to plasmids, cosmids, viruses, bacteriophages and other vectors commonly used in genetic engineering. The polynucleotide to be employed in context with the present invention may be inserted or cloned into the vector by means and methods as well known to those skilled in the art, e.g., by using restriction enzymes and ligation protocols. In a preferred embodiment, these vectors are suitable for the transformation of cells, eukaryotic cells like fungal cells, cells of microorganisms such as yeast or prokaryotic cells. In a particularly preferred embodiment, such vectors are suitable for stable transformation of eukaryotic cells, for example to transcribe the polynucleotide to be employed in context with the present invention. Suitable vectors are known in the art. For example, in context with the present invention, the vector may be pLNCX, pcDNA3 or pBMN-I-GFP or any other vector as described and exemplified herein. Accordingly, the present invention relates to a vector such as pLNCX comprising the polynucleotide to be employed in context with the present invention. Generally, the vector may be capable of expressing said polynucleotide to be employed in context with the present invention in a eukaryotic host cell. Accordingly, in one aspect of the invention, the vector as provided is an expression vector. Generally, expression vectors have been widely described in the literature. As a rule, they may not only contain a selection marker gene and a replication-origin ensuring replication in the host selected, but also a promoter, and in most cases a termination signal for transcription. Between the promoter and the termination signal, there is preferably at least one restriction site or a polylinker which enables the insertion of a nucleic acid sequence/molecule desired to be expressed. It is to be understood that when the vector provided herein is generated by taking advantage of an expression vector known in the prior art that already comprises a promoter suitable to be employed in context of this invention, for example expression of the polynucleotide to be employed in context with the present invention, the nucleic acid molecule is inserted into that vector in a manner that the resulting vector comprises preferably only one promoter suitable to be employed in context of this invention. The promoter may generally be heterologous or homologous. The vector described herein may also encompass more than one promoter, each respective promoter may be heterologous or homologous. The skilled person knows how such insertion can be put into practice. For example, the promoter can be excised either from the nucleic acid construct or from the expression vector prior to ligation.

In an additional embodiment, the polynucleotide to employed in context with the present invention and/or the vector into which the polynucleotide to employed in context with the present invention is comprised as described herein may be transduced, transformed or transfected or otherwise introduced into a host cell. For example, the host cell is a eukaryotic or a prokaryotic cell, preferably a eukaryotic cell. As a non-limiting example, the host cell is a mammalian cell. The host cell described herein and to be employed in context with the invention described herein is intended to be particularly useful for generating the polypeptide to be employed in context with the present invention. Generally, the host cell described herein and to employed in context with the present invention may be a prokaryotic or eukaryotic cell, preferably a eukaryotic cell, comprising a polynucleotide to be employed in context with the present invention or the vector described herein and to be employed in context with the present invention or a cell derived from such a cell and containing the polynucleotide or the vector described herein and to be employed in context with the present invention. In a preferred embodiment, the host cell comprises, i.e. is genetically modified with the polynucleotide or the vector described herein and to be employed in context with the present invention in such a way that it contains the polynucleotide or the vector integrated into the genome. For example, such host cell described herein may be a human, yeast, or fungus cell. In one particular aspect, the host cell is capable to transcribe the polynucleotide to be employed in context with the present invention. An overview of examples of different corresponding expression systems to be used for generating the host cell described herein is for instance contained in Methods in Enzymology 153 (1987), 385-516, in Bitter (Methods in Enzymology 153 (1987), 516-544), in Sawers (Applied Microbiology and Biotechnology 46 (1996), 1-9), Billman-Jacobe (Current Opinion in Biotechnology 7 (1996), 500-4), Hockney (Trends in Biotechnology 12 (1994), 456-463), and in Griffiths (Methods in Molecular Biology 75 (1997), 427-440). The transformation or genetically engineering of the host cell with a polynucleotide or vector described herein and to be employed in context with the present invention can be carried out by standard methods, as for instance described in Sambrook and Russell (2001), Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, NY, USA; Methods in Yeast Genetics, A Laboratory Course Manual, Cold Spring Harbor Laboratory Press, 1990. In one aspect of the present invention, the host cell comprising the polynucleotide or the vector described herein and to be employed in context with the present invention may be a fibroblast and epithelial cell. Accordingly, the present invention relates to a fibroblast or epithelial cell comprising the polynucleotide to be employed in context with the present invention. Also, the present invention relates to a fibroblast or epithelial cell comprising the vector described herein and to be employed in context with the present invention.

The present invention also relates to a method for screening an agent capable of increasing the expression of the polynucleotide to be employed in context with the present invention and/or the polypeptide to be employed in context with the present invention, wherein said method comprises

(a) contacting the agent to be screened with a sample containing a nucleic acid molecule comprising the polynucleotide to be employed in context with the present invention and/or containing the host cell described herein and to be employed in context with the present invention;

(b) measuring

(1) the transcription level of the polynucleotide to be employed in context with the present invention,

(2) the amount of transcription product of the polynucleotide to be employed in context with the present invention,

(3) the amount of translation product of the transcription product of the polynucleotide to be employed in context with the present invention, and/or

(4) the protein level of the polypeptide to be employed in context with the present invention; and

(c) comparing the result of (b) with the corresponding result of a corresponding control sample, wherein said control sample has not been contacted with the agent to be screened.

Generally, when assessing the expression level of a polynucleotide to be employed in context with the present invention or the polypeptide to be employed in context with the present invention, methods known in the art and as also described and exemplified herein may be used. For example, for assessing the expression level (e.g., transcription level) of a polynucleotide to be employed in context with the present invention, methods such as RT- PCR or reporter gene assays such as a luciferase-assay may be employed as known in the art and as also described and exemplified herein. For example, to measure the transcription level of human Wnt-1 , cells may be transfected with Wnt-1 reporter construct in the presence of luciferase as follows. 24 hours before transfection, cells can be seeded into 12 well plates and grown to 80% to 90% confluence in serum-free medium (e.g.., OptiMeM, Gibco®). Next day, the cells may be transfected with 1 μg of reporter construct (Wnt-1 promoter cloned into a luciferase reporter vector) and 1 ng Renilla (SV40) plasmid DNA using 2 μΐ Lipofectamine 2000 (Invitrogen) (transfection mix, incubated for 20 min at RT) by methods known in the art and as also described herein. Transfection may be performed by first removing the medium from the cells and pipette 500 μΐ per well of the transfection mix onto the cells, followed by removing the transfection mix 6 h after transfection and pipette full medium on the cells. Renilla reniformis luciferase is an internal control reporter and can be used to normalize transfection efficiency in the reporter assay. 32 h after transfection, the cells may be stimulated with the agent to be tested (e.g., the agent to be tested for screening in accordance with the present invention). 48 h after transfection, cells may be lysed in 150 μΐ passive lysis buffer (1 x PLP, Promega) provided with the Dual Luciferase Assay system kit (Promega). Luciferase activity can then be measured with, e.g., Berthold Centro LB 960 luminometer and monitored with MikroWin 2000 software. Data can then be reported as normalized averages of the Luciferase/Renilla ratio.

As another non-limiting example, as mentioned, the expression level of a polynucleotide to be employed in context with the present invention may be assessed by using RT-PCR. For example, to measure the gene expression of Wnt-1 , RNA may be isolated which may then be reverse transcribed into cDNA, followed by real time PCR using specific Wnt-1 primers (optionally, for example for cells which do not endogenously express Wnt-1, before isolating RNA, cells may be transfected with Wnt-1 by methods well known in the art). Such a method for measuring gene expression of Wnt-1 may be as follows. First, RNA can be isolated using the RNeasy Mini Kit (Qiagen) according to manufacturer^'s instructions. Total RNA can be eluted with 30 μΐ of nuclease free double distilled water and stored at -80 °C. For cDNA synthesis, RNA may be reverse transcribed into first strand cDNA. 1 to 5 μg of total RNA may be mixed with 1 μΐ random hexamer primer (Roche), filled up with nuclease free H₂0 to 11 μΐ, incubated at 70 °C for 5 min, and cooled down to 4 °C. After adding 7 μΐ of the mastermix (containing 4 μΐ first strand buffer, 2 μΐ 10 mM dNTP mix and 1 μΐ RNAse inhibitor (20 U, Fermentas)), the cDNA mix may be incubated at 25 °C for 5 min. Subsequently, 1 μΐ of the Revert Aid M-MulV Reverse Transcriptase (Fermentas, Vienna, Austria) may be added and the mix can be further incubated at 25 °C for 10 min, at 42 °C for 1 h, at 70 °C for 10 min and then cooled down to 4 °C. For Real-Time Polymerase chain reaction (RT-PCR), the RT-PCR primer sets used can be purchased from Applied Biosystems (Assays-on-demand). A reaction mixture may contain 1 μg cDNA, 12.5 μΐ TaqMan Universal PCR Master Mix (Applied Biosystems), 1.25 μΐ Assay-on-demand (FAM primers, Wnt-1 : Hs00180529_ml, GAPDH: Hs99999905_ml and/or Wnt-1 primers mentioned in Table 1) and 10.25 μΐ H₂0 in a total volume of 25 μΐ. The following cycling parameters may be applied: Initial denaturation at 50 °C for 2 min and then at 95 °C for 10 min, followed by 40 cycles: 95 °C, 10 sec; 60 °C, 1 min. Each cDNA sample can be analyzed in duplicates and glyceraldehyde-3 -phosphate dehydrogenase (GAPDH) for human primers and beta-2 microglobulin for mouse primers may be used as house keeping genes. Reactions can be run on ABI Prism 7700 Sequence Detector and data analysis ma be done with the SDS 1.9.1 software package (Applied Biosystems). Changes in mRNA concentration may be determined by subtracting the CT (cycle threshold) of target gene from the CT of the house keeping gene (GAPDH) (Δ= CT gene - CT GAPDH). The mean of Δ control may be subtracted from the Δ target gene reaction (mean Δ control - Δ target gene). The difference can be calculated as

₂Λ(-ΔΔΟΤ) Furthermore, as mentioned, for assessing the expression level (e.g., the translation level or protein level) of the polypeptide to be employed in context with the present invention, methods known in the art and as also described and exemplified herein may be used. As further described herein, non-limiting examples for such assays are inter alia Western Blot, optionally paired with chromogenic dye-based protein detection techniques (such as silver or coomassie blue staining) or with fluorescence- and luminescence-based detection methods for proteins in solutions and on gels, blots and microarrays, such as immunostaining, as well as immunoprecipitation, ELISA, microarrays, and mass spectrometry as known in the art. For example, the expression of Wnt-1 on the protein level may be assessed by using a Western blot as follows. For Western blot analysis, cells may be lysed in RIPA buffer (1 ml RIPA buffer + 10 μΐ NP-40 and 10 μΐ Protease Inhibitor cocktail (Complete, Roche Diagnostics, Mannheim, Germany), incubated for 20 min on ice and then centrifuged at 15000 rpm for 15 min at 4 °C. The RIPA buffer may be composed as follows.

Ripa buffer:

amount stock final concentration

1.5 ml 1 M Tris/HCl pH 7.4 50 mM Tris/HCl pH 7,4

7.5 ml 2 M NaCl 500 mM NaCl

300 μΐ Igepal 1% Igepal

150 mg Na-DOC 0.5% Na-DOC

300 μΐ 10% SDS 0.1% SDS

1.5 ml 1 %NaN3 0.05% NaN3

18.9 ml Aqua bidest

Protein concentration of cell extracts may be determined using the Bradford method (BioRad Coomassie Reagent 5x). For SDS-polyacrylamid Gel Electrophoresis (SDS-PAGE), protein samples may be mixed with 6 x sample buffer to a final concentration of 1 x protein loading dye. Samples may be boiled for 5 min at 95 °C and centrifuged for 1 min at 15,000 rpm. 30 μg protein lysates and 5 μΐ of a prestained protein marker (Femientas) may then be loaded onto a 13% SDS polyacrylamide gel, electrophoresed (30 mA per gel for about 1 h) and blotted in a methanol containing transfer buffer for at least 1 h (100 mA). Reagents and gels used for this may be as follows. 13% SDS polyacrylamide gel: separating gel (1.5 ml 40% acrylamid, 1.75 ml 1.5 M Tris pH 8.8, 2.15 ml H₂0, 50 μΐ 10% SDS, 25 μΐ 10% APS and 2.5 μΐ TEMED) plus stacking gel (0.25 ml 40% acrylamid, 0.313 ml 1.5 M Tris pH 8.8, 1.9 ml H₂0, 25 μΐ 10% SDS and 12.5 μΐ 10% APS); 6 x sample buffer: 10 ml 10% SDS, 3 ml glycerol, 0.2 ml 2-mercaptoethanol, 7 ml stacking gel buffer and 12.5 μΐ bromphenolblue; 10 x blotting buffer: 15.15 g Tris, 72 g gylcin, ad 500 ml H₂0; 1 x blotting buffer: 50 ml 10 x blotting buffer, 75 ml methanol, ad 500 ml H₂0.

Membranes may be blocked in 1% I-block (TROPIX Bedford, MA, USA) for 1 h. For protein detection, an anti-Wnt-1 antibody may be used (e.g., goat anti -mouse Wnt-1 antibody from R&D, 1 :2500 dilution). As a secondary antibody, rabbit anti-goat IgG HRP (Zymed, 1 :50000) may be used. Bound antibodies can be visualized by chemiluminescence, (ECL plus, Amersham, Arlington Heights, IL) followed by exposure to Hyperfilm ECL (Amersham).

In context with the present invention, in particular in context with the screening method provided and described herein, when an agent to be tested in the screening method according to the present invention increases the expression of the polynucleotide to be employed in context with the present invention and or of the polypeptide to be employed in context with the present invention by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% compared to a control, it may be considered to be an agent capable of increasing the expression of the polynucleotide to be employed in context with the present invention and/or the polypeptide to be employed in context with the present invention.

The present invention also relates to a method for screening an agent capable of enhancing the activity of the polypeptide to be employed in context with the present invention, wherein said method comprises

(a) contacting the agent to be screened with a sample containing the polypeptide to be employed in context with the present invention;

(b) measuring the activity of the polypeptide to be employed in context with the present invention; and

(c) comparing the result of (b) with the corresponding result of a corresponding control sample, wherein said control sample has not been contacted with the agent to be screened

The screening methods described herein may be in vitro methods. As used herein, the term "sample" encompasses an amount of material. Said material may contain biological material, such as polynucleotides and/or polypeptides (e.g. (a) Wnt-1 polynucleotide(s) and/or (a) Wnt- 1 polypeptide(s)). For example, in context of the present invention, a sample may be an amount of buffer containing the nucleic acid molecule encoding Wnt-1 or the Wnt-1 polypeptide. It is also envisaged with respect to the present invention that a sample contains a host cell comprising the Wnt-1 polynucleotide, the Wnt-1 polypeptide and/or a vector comprising the Wnt-1 polynucleotide. A suitable "sample" in accordance with the present invention also includes (a) biological or medical sample(s), like, e.g. (a) sample(s) comprising cell(s) or tissue(s). For example, such (a) sample(s) may comprise(s) biological material of biopsies. The meaning of "biopsies" is known in the art. For instance, biopsies comprise cell(s) or tissue(s) taken, e. g. by the attending physician, from a patient/subject as described herein. Exemplarily, but not limiting, the biological or medical sample is or is derived from blood, plasma, white blood cells, urine, semen, sputum, cerebrospinal fluid, lymph or lymphatic tissues or cells, muscle cells, heart cells, cells from veins or arteries, nerve cells, cells from spinal cord, brain cells, liver cells, kidney cells, cells from the intestinal tract, cells from the testis, cells from the urogenital tract, colon cells, skin, bone, bone marrow, placenta, amniotic fluid, hair, hair and/or follicles, stem cells (embryonic, neuronal, and/or others) or primary or immortalized cell lines (lymphocytes, macrophages, or cell lines). The biological or medical sample as defined herein may also be or be derived from biopsies, for example biopsies derived from heart tissue, veins or arteries.

Generally, in context with the present invention, when assessing the activity of a polypeptide to be employed in context with the present invention, or when assessing whether a given compound has Wnt-1 activity, methods described and exemplified herein and as commonly known in the art may be used. For example, to assess Wnt-1 activity, given that Wnt-1 reduces the expression VEGF-C as described and exemplified in context with the present invention, VEGF-C expression may be measured. Accordingly, in context with the present invention, an agent capable of decreasing expression of VEGF-C may be considered to have Wnt-1 activity. In this context, expression of VEGF-C may be assessed on the transcription level, the translation level and the protein level by methods known in the art and as also described and exemplified herein. In context with the present invention, examples for measuring the transcription level of VEGF-C include reporter gene assays which commonly use reporter genes such as fluorescent proteins such as GFP, eGFP, YFP, eYFP, CFP, BFP, eBFP, luminescent proteins such as the enzymes Renilla or firefly luciferase, and β- galactosidase encoded by the lacZ gene (Inui, Nat Rev Mol Cell Biol (2010), 11 : 252-63). Other non-limiting examples include methods such as qPCR, RT-PCR, qRT-PCR, RT-qPCR, Light Cycler®, TaqMan® Platform, quantigene assay (Zhou, Anal Biochem (2000), 282: 46- 53), Northern blot, dot blot, microarrays, next generation sequencing (VanGuilder, Biotechniques (2008), 44(5): 619-26; Elvidge, Pharmacogenomics (2006), 7: 123-134; Metzker, Nat Rev Genet (2010), 11 : 31-46; Kafatos, NAR (1979), 7: 1541-1552), or the like. Furthermore, in context with the present invention, examples for measuring the translation or protein level of VEGF-C include polyacrylamide gel electrophoresis assays and related blotting techniques such as Western Blot, optionally paired with chromogenic dye-based protein detection techniques (such as silver or coomassie blue staining) or with fluorescence- and luminescence -based detection methods for proteins in solutions and on gels, blots and microarrays, such as immunostaining, as well as immunoprecipitation, ELISA, microarrays, and mass spectrometry. For measuring the transcription level of VEGF-C in context with the present invention, for example an RT-PCR or a luciferase assay may be performed. For RT- PCR, cells may be seeded in 6 well plates and allowed to reach 80% to 90% confluence. For transfection, 1 ml OptiMeM (GIBCO) may be mixed with 4 μg plasmid DNA and 6 μΐ Lipofectamine 2000 (Invitrogen) and incubated for 20 min at RT. Thereafter, the culture medium can be removed, the cells washed once with PBS and the transfection precipitate added to the cells. 5 to 6 h after incubation at 37 °C, the transfection mix may be replaced by fresh culture medium and the cells can be harvested after 72 h. RNA may be isolated using the RNeasy Mini Kit (Qiagen) according to manufacturer's instructions. Total RNA may be eluted with 30 μΐ of nuclease free double distilled water and stored at -80 °C. For cDNA synthesis, RNA may be reverse transcribed into first strand cDNA. 1 to 5 μg of total RNA may be mixed with 1 μΐ random hexamer primer (Roche), filled up with nuclease free H₂0 to 11 μΐ, incubated at 70 °C for 5 min, and cooled down to 4 °C. After adding 7 μΐ of the mastermix (containing 4 μΐ first strand buffer, 2 μΐ lOmM dNTP mix and 1 μΐ RNAse inhibitor (20 U, Fermentas)), the cDNA mix may be incubated at 25 °C for 5 min. 1 μΐ of the Revert Aid M-MulV Reverse Transcriptase (Fermentas, Vienna, Austria) may be added and the mix can be further incubated at 25 °C for 10 min, at 42 °C for 1 h, at 70 °C for 10 min and then cooled down to 4 °C. For RT-PCR, the RT-PCR primer sets used may be purchased from Applied Biosystems (Assays-on-demand, VEGF-C: Hs00153458_ml, GAPDH: Hs99999905_ml). A reaction mixture may contain 1 μg cDNA, 12.5 μΐ TaqMan Universal PCR Master Mix (Applied Biosystems), 1.25 μΐ Assay-on-demand (FAM primers) and 10.25 μΐ H₂0 in a total volume of 25 μΐ. The following cycling parameters may be applied: Initial denaturation at 50 °C for 2 min and then at 95 °C for 10 min, followed by 40 cycles: 95 °C, 10 sec; 60 °C, 1 min. Each cDNA sample may be analyzed in duplicates and glyceraldehyde- 3-phosphate dehydrogenase (GAPDH) may be used as a house keeping gene. Reactions can be run on ABI Prism 7700 Sequence Detector and data analysis may be done with the SDS 1.9.1 software package (Applied Biosystems). Changes in mRNA concentration can be determined by subtracting the CT (cycle threshold) of target gene from the CT of the house keeping gene (GAPDH) (Δ = CT gene - CT GAPDH). The mean of Δ control may be subtracted from the Δ target gene reaction (mean Δ control - Δ target gene). The difference may be calculated as 2^{a(" CT)}. Generally, in context of the present invention, for assessing whether a compound has Wnt-1 activity, a compound being capable of decreasing VEGF-C expression by at least 20%, at least 25%, at least 30%>, at least 35%, at least 40%, at least 45% or at least 50% compared to a control may be considered to be a compound having Wnt-1- activity. Furthermore, in context with the present invention, an agent that increases the activity of a polypeptide to be employed in context with the present invention (said activity being assessed based on, e.g., VEGF-C expression as described above) by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% or at least 50% compared to a control may be considered to be an agent capable of enhancing the activity of the polypeptide to be employed in context with the present invention.

The present invention further relates to agents which are capable to increase expression of the Wnt-1 polynucleotide to be employed in context with the present invention (e.g. gene expression of Wnt-1) and/or the Wnt-1 polypeptide to be employed in context with the present invention. The present invention also relates to agents which are capable of enhancing activity of the Wnt-1 polypeptide to be employed in context with the present invention. These agents may be known in the art and/or screened by using the screening methods described herein. In context with the present invention, such agents may be used for treating or preventing a disease or disorder. Accordingly, the present invention relates to a pharmaceutical composition for use in treating or preventing a disease or disorder, said pharmaceutical composition comprising a Wnt-1 polynucleotide or a Wnt-1 polypeptide, a vector comprising a Wnt-1 polynucleotide, a host cell comprising said vector or said Wnt-1 polynucleotide, and/or an agent capable of increasing the expression of said Wnt-1 polynucleotide or an agent capable of enhancing the activity of said Wnt-1 polypeptide; together with a pharmaceutically acceptable carrier or excipient. In particular, the agents as described herein may be used in treating a disease or disorder related to lymphangiogenesis or in preventing metastasis. Such a disease or disorder related to lymphangiogenesis may be, for example, cancer.

Accordingly, the present invention relates to an agent for use in treating or preventing a disease or disorder related to lymphangiogenesis or in preventing metastasis, wherein said agent is capable of increasing the expression of the polynucleotide to be employed in context with the present invention and/or the polypeptide to be employed in context with the present invention, or capable of enhancing the activity of the polypeptide to be employed in context with the present invention. This agent for use in treating or preventing a disease or disorder related to lymphangiogenesis or in preventing metastasis may be screened according to the methods for screening an agent as described herein.

Furthermore, the present invention relates to compositions comprising the polynucleotide to be employed in context with the present invention, the polypeptide to be employed in context with the present invention, the vector described in and to be employed in context with the present invention, the host cell described in and to be employed in context with the present invention, and/or the agent described in and to be employed in context with the present invention. Such a composition may further comprise a pharmaceutically acceptable carrier, excipient and/or diluent. Accordingly, the present invention relates to a pharmaceutical composition comprising the polynucleotide to be employed in context with the present invention, the polypeptide to be employed in context with the present invention, the vector described in and to be employed in context with the present invention, the host cell described in and to be employed in context with the present invention, and/or the agent described in and to be employed in context with the present invention, optionally together with a pharmaceutically acceptable carrier, excipient and/or diluent. Generally, examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc. Pharmaceutical compositions comprising such carriers can be formulated by well known conventional methods. These pharmaceutical compositions can be administered to a subject at a suitable dose, i.e. at least lmg/kg body weight, e.g. about lOmg/kg body weight to about 100 mg/kg body weight of the subject. Administration of the (pharmaceutical) composition may be effected or administered by different ways, e.g., parenterally, enterally, orally (e.g., pill, tablet, buccal, sublingual, disintegrating, capsule, thin film, liquid solution or suspension, powder, solid crystals or liquid), rectally (e.g., suppository, enema), via injection (e.g., intravenously, subcutaneously, intramuscularly, intraperitoneally, intradermally) via inhalation (e.g., intrabronchially), topically, vaginally, epicutaneously, into the eye, or intranasally. Preferably, in context with the present invention, the (pharmaceutical) composition is administered parenterally. Generally, the dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. The compositions and pharmaceutical compositions described herein and to be employed in context with the present invention may be administered locally or systemically. The compositions and pharmaceutical compositions may also be administered directly to the target site, e.g., by biolistic delivery to an internal or external target site or by catheter to a site in an artery. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like. Furthermore, also doses below or above of the exemplary ranges described herein above are envisioned, especially considering the aforementioned factors. The present invention also relates to a method for treating or preventing a disease or disorder in a subject, said method comprising administering to the subject a therapeutically effective amount of the polynucleotide to be employed in context with the present invention, the polypeptide to be employed in context with the present invention, the vector described in and to be employed in context with the present invention, the host cell described in and to be employed in context with the present invention, the agent described in and to be employed in context with the present invention, or the pharmaceutical composition described in and to be employed in context with the present invention, preferably in a suitable dose or in an effective amount. Accordingly, the present invention relates to a pharmaceutical composition for use in treating or preventing a disease or disorder, said pharmaceutical composition comprising a Wnt-1 polynucleotide or polypeptide, a vector comprising a Wnt-1 polynucleotide, a host cell comprising said vector or said Wnt-1 polynucleotide, and/or an agent capable of increasing the expression of said Wnt-1 polynucleotide or an agent capable of enhancing the activity of said Wnt-1 polypeptide; together with a pharmaceutically acceptable carrier or excipient. The present invention also relates to a method for treating or preventing a disease or disorder related to lymphangiogenesis or for preventing metastasis in a subject, said method comprising administering to the subject a therapeutically effective amount of the polynucleotide to be employed in context with the present invention, the polypeptide to be employed in context with the present invention, the vector described herein and to be employed in context with the present invention, the host cell described herein and to be employed in context with the present invention, the agent described herein and to be employed in context with the present invention, or the pharmaceutical composition described herein and to be employed in context with the present invention, preferably in a suitable dose or in an effective amount. Generally, as the skilled person readily understands, the modes of administration as described herein above for the compositions and pharmaceutical compositions to be employed in context with the present invention may also be applied to these methods. Also, as will be readily understood by the person of skill in the art, medical uses of compounds and compositions for treating or preventing diseases and disorders related to lymphangiogenesis or for preventing metastasis as provided and described herein can mutatis mutandis be construed as methods of treating or preventing the respective diseases or disorders.

Furthermore, the present invention relates to a kit suitable for performing a screening method as described herein. Such a kit may comprise one or more polynucleotides (e.g., primers or probes) and/or binding agents (e.g., antibody molecules) capable of hybridizing or binding to the polynucleotide to be employed in context of the present invention or binding the polypeptide polynucleotide to be employed in context of the present invention. Generally, in context with the present invention, when reference is made to a disease or disorder related to lymphangiogenesis which can be treated as described and exemplified herein, a disease or disorder related to lymphangiogenesis is generally known in the art. In particular, in context of the present invention as described herein, a disease or disorder related to lymphangiogenesis may be selected from the group consisting of cancer, melanoma, solid tumor, breast cancer, lung cancer, kidney cancer, pancreatic cancer, hematopoietic tumor (lymphoma), graft rejection, autoimmune disorder, rheumatoid arthritis, psoriasis, oedema and impaired wound healing. In this context, examples for graft rejection are lung graft rejection, kidney graft rejection, and particularly cornea graft rejection.

Generally, the dosages of the compounds and compositions as described and to be employed in context with the present invention and to be administered to a subject as described above may be chosen for each and every pharmaceutical embodiment and employment as specified and described herein.

Generally, the subject to be treated in context of the present invention may be mammal and is preferably human.

Generally, as used in context of the present invention, the term "Wnt-1 -molecules" encompasses the polynucleotides to be employed in the present invention, the polypeptides to be employed in context with the present invention, the vectors described herein and to be employed in context with the present invention, the host cells described herein and to be employed in context with the present invention, the agents provided herein and to be employed in context with the present invention, and compositions and pharmaceutical compositions provided herein and to be employed in context with the present invention.

As mentioned herein, "Wnt-1 -molecules" may derive from various species including of human, mouse, rat, hamster, rabbit, guinea pig, ferret, cat, dog, chicken, sheep, bovine species, horse, camel, primate or fruit fly. Accordingly, mouse "Wntl" and human "WNT1" are encompassed by the term "Wnt-1". Similarly, mouse "Wnt5a" and human WNT5a are encompassed by the term "Wnt-5a" and mouse "Wnt3a" and human "WNT3a" are encompassed by the term "Wnt-3a", respectively, and so forth. It is preferred with respect to the present invention that the Wnt-1 -molecules are of human origin. The term "Wnt-1" also relates to (a) fragment(s) of the Wnt-1 polynucleotide or polypeptide. The term "Wnt-1" further relates to (a) functional equivalent derivative(s) of the Wnt-1 polynucleotide of polypeptide. It is of note that the nucleic acid and amino acid sequences of Wnt-1 given herein are not limiting. Accordingly, the term "Wnt-1" also encompasses Wnt-1 polypeptides/nucleic acid molecules having amino acid or nucleic acid sequences being homologous to or a fragment of the amino acid or nucleotide sequences shown herein, e.g. those of the cDNA of human Wnt-1 (see SEQ ID NOs: 1 and 3) or of the amino acid sequence of human Wnt-1 (see SEQ ID NOs: 2 and 4). The term "Wnt-1" also relates to specific agonists of Wnt-1. The term "agonist", as used herein, is meant to refer to an agent that mimics or up-regulates (e.g., potentiates or supplements) the activity/functionality of a protein of interest (such as Wnt-1). An agonist can be a wild-type protein or derivative thereof having Wnt-1 activity, i.e. the ability to reduce the expression of VEGF-C as described herein. An agonist of Wnt-1 can also be a molecule that up-regulates the expression of the gene of Wnt-1 or which increases the activity of Wnt-1. An agonist of Wnt-1 can also be a protein or small molecule which increases the interaction of Wnt-1 with a target molecule. Wnt-1 polynucleotide(s) and/or polypeptide(s) may be obtained by purifying it from cells expressing/comprising Wnt-1 polynucleotide(s) and/or polypeptide(s) or (a) fragment(s) thereof or (a) functional equivalent derivative(s) thereof. These cells may overexpress Wnt-1 - molecules or may contain Wnt-1 -molecules endogenously. These cells may be prokaryotic cells such as E. coli or eukaryotic cells like, e.g., insect or mammalian cells. Wnt-1 -molecules may also be synthetically or chemically produced. Therefore solid phase peptide synthesis (SPPS) or synthetic DNA synthesis may be applied.

Generally, as used herein, the terms "polynucleotide" and "nucleic acid molecule" may be used interchangeably. The polynucleotide to be employed in context with the present invention and any other nucleic acid molecule referred to herein may be DNA molecules or RNA molecules. They may also be nucleic acid analogues, such as oligonucleotide thiophosphates, substituted ribo-oligonucleotides, LNA molecules, PNA molecules, GNA (glycol nucleic acid) molecules, TNA (threose nucleic acid) molecules, morpholino polynucleotides, or antagomir (cholesterol-conjugated) nucleic acid molecules or any modification thereof as known in the art (see, e.g., US 5,525,71 1 , US 4,71 1,955, US 5,792,608 or EP 302175 for examples of modifications). Nucleic acid molecules in context of the present invention may be naturally occurring nucleic acid residues or artificially produced nucleic acid residues. Examples for nucleic acid residues are adenine (A), guanine (G), cytosine (C), thymine (T), uracil (U), xanthine (X), and hypoxanthine (HX). In context of the present invention, thymine (T) and uracil (U) may be used interchangeably depending on the respective type of nucleic acid molecule. For example, as the skilled person is well aware of, a thymine (T) as part of a DNA corresponds to an uracil (U) as part of the corresponding transcribed mRNA. The nucleic acid molecule of the present invention may be single- or double-stranded, linear or circular, natural or synthetic, and, if not indicated otherwise, without any size limitation. The nucleic acid molecule may also comprise a promoter as further detailed herein above. The promoter may be homologous or heterologous.

In context of the present invention, "homologous" or "percent homology" means that amino acid or nucleic acid sequences have identities of at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% to the sequences shown herein, e.g. those of human Wnt-1 (e.g. SEQ ID NOs: 1 to 4), wherein the higher identity values are preferred upon the lower ones.

In accordance with the present invention, the term "identity/identities" or "percent identity/identities" in the context of two or more nucleic acid or amino acid sequences, refers to two or more sequences or subsequences that are the same, or that have a specified percentage of amino acid residues or nucleotides that are the same (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9%) identity with the nucleic acid sequences of, e.g., SEQ ID NOs: 1 and 3, or with the amino acid sequences of, e.g., SEQ ID NOs: 2 or 4, and being functional, wherein the function comprises the ability to reduce the expression of VEGF-C, when compared and aligned for maximum correspondence over a window of comparison, or over a designated region as measured using a sequence comparison algorithm as known in the art, or by manual alignment and visual inspection.

Preferably the described identity exists over a region that is at least about 50 to 100 amino acids or nucleotides in length. It is more preferred that the described identity exists over a region that is at least about 100 to 200 amino acids or nucleotides in length. It is most preferred that the described identity exists over a region that is at least about 200 to 300 amino acids or nucleotides in length. In case of nucleotide sequences, the described identity most preferably exists over a region that is at least 300, at least 400, at least 500, at least 600, at least 700, at least 800 or at least 900 nucleotides in length. Those having skill in the art will know how to determine percent identity between/among sequences using, for example, algorithms such as those based on CLUSTALW computer program ( Thompson, Nucl Acids Res (1994), 2: 4673-4680) or FASTDB ( Brutlag, Comp App Biosci (1990), 6: 237-245), as known in the art.

Although the FASTDB algorithm typically does not consider internal non-matching deletions or additions in sequences, i.e., gaps, in its calculation, this can be corrected manually to avoid an overestimation of the % identity. CLUSTALW, however, does take sequence gaps into account in its identity calculations. Also available to those having skill in this art are the BLAST and BLAST 2.0 algorithms (Altschul, Nucl Acids Res (1997), 25: 3389-3402; Altschul, J Mol Evol (1993), 36: 290-300; Altschul, J Mol Biol (1990), 215: 403-410). The BLASTN program for nucleic acid sequences uses as defaults a word length (W) of 11 , an expectation (E) of 10, M=5, N=4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length (W) of 3, and an expectation (E) of 10. The BLOSUM62 scoring matrix (Henikoff; 1989; PNAS; 89; 10915) uses alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands. In addition, the present invention relates to a polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule being degenerate as a result of the genetic code. When used in accordance with the present invention, the term "being degenerate as a result of the genetic code" means that due to the redundancy of the genetic code different nucleotide sequences code for the same amino acid.

In order to determine whether an amino acid residue or nucleotide residue in a amino acid or nucleic acid sequence corresponds to a certain position in the amino acid sequence of, e.g., SEQ ID NOs: 2 or 4, or nucleotide sequence of e.g. SEQ ID NOs: 1 or 3, the skilled person can use means and methods well-known in the art, e.g., alignments, either manually or by using computer programs.

For example, BLAST 2.0, which stands for Basic Local Alignment Search Tool BLAST (Altschul, 1997, loc. cit; Altschul, 1993, loc. cit; Altschul, 1990, loc. cit), can be used to search for local sequence alignments. BLAST, as discussed above, produces alignments of both nucleotide and amino acid sequences to determine sequence similarity. Because of the local nature of the alignments, BLAST is especially useful in determining exact matches or in identifying similar sequences. The fundamental unit of BLAST algorithm output is the High- scoring Segment Pair (HSP). An HSP consists of two sequence fragments of arbitrary but equal lengths whose alignment is locally maximal and for which the alignment score meets or exceeds a threshold or cut-off score set by the user. The BLAST approach is to look for HSPs between a query sequence and a database sequence, to evaluate the statistical significance of any matches found, and to report only those matches which satisfy the user-selected threshold of significance. The parameter E establishes the statistically significant threshold for reporting database sequence matches. E is interpreted as the upper bound of the expected frequency of chance occurrence of an HSP (or set of HSPs) within the context of the entire database search. Any database sequence whose match satisfies E is reported in the program output.

Analogous computer techniques using BLAST (Altschul, 1997, loc. cit.; Altschul, 1993, loc. cit.; Altschul, 1990, loc. cit.) are used to search for identical or related molecules in nucleotide databases such as GenBank or EMBL. This analysis is much faster than multiple membrane- based hybridizations. In addition, the sensitivity of the computer search can be modified to determine whether any particular match is categorized as exact or similar. The basis of the search is the product score which is defined as:

% sequence identity x % maximum BLAST score

100

and it takes into account both the degree of similarity between two sequences and the length of the sequence match. For example, with a product score of 40, the match will be exact within a 1-2% error; and at 70, the match will be exact. Similar molecules are usually identified by selecting those which show product scores between 15 and 40, although lower scores may identify related molecules. Another example for a program capable of generating sequence alignments is the CLUSTALW computer program ( Thompson, Nucl Acids Res (1994), 2: 4673-4680) or FASTDB ( Brutlag, Comp App Biosci (1990), 6: 237-245), as known in the art.

Generally, as used herein, if two nucleotide or amino acid sequences being compared by sequence comparisons differ in identity, then the term "identity" refers to the shorter sequence and to the part of the longer sequence that matches said shorter sequence. Therefore, when the sequences which are compared do not have the same length, the degree of identity preferably either refers to the percentage of nucleotide or amino acid residues in the shorter sequence which are identical to consecutive nucleotide or amino acid residues contained in the longer sequence or to the percentage of consecutive nucleotides or amino acids contained in the longer sequence which are identical to the nucleotide or amino acid sequence of the shorter sequence. Preferably, as described above, a gap as "part of consecutive nucleotides" or "part of consecutive amino acids" is to be counted as a mismatch. In this context, the skilled person is readily in the position to determine that part of a longer sequence that "matches" the shorter sequence. Also, these definitions for sequence comparisons (e.g., establishment of "identity" values) are to be applied for all sequences described and disclosed herein. Identity, moreover, means that there is preferably a functional and/or structural equivalence between the corresponding nucleotide or amino acid sequences. Nucleic acid or amino acid sequences having the given identity levels to the particular nucleic acid or amino acid sequences of the polynucleotides or polypeptides to be employed in context with the present invention may represent derivatives/variants of these sequences which, preferably, have the same biological function. In context of the present invention, the biological function of a polynucleotide or polypeptide to be employed in context with the present invention is Wnt-1 activity. In context with the present invention, particularly in context with the screening methods provided herein and in context with determining a polynucleotide or polypeptide having Wnt-1 activity, whether a given polynucleotide or polypeptide has Wnt-1 activity can be easily tested by methods known in the art and as also described herein.

As used herein, a "functional equivalent derivative" of a protein which displays a specific biological activity relates to derivatives of said protein having a sufficient degree of identity to display said activity. A functional equivalent derivative of Wnt-1 may be a polynucleotide or polypeptide which is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to the nucleotide or amino acid sequences of SEQ ID NOs 1 to 4. A "functional equivalent derivative" of a protein showing e.g. a specific activity may relate to a polypeptide which corresponds to a derivative of said protein which is still capable of showing said activity. For example, a "functional equivalent derivative" of Wnt-1 may be capable of showing activity of Wnt-1 , i.e. to reduce the expression of VEGF-C. Methods for determining whether a certain derivative of a protein is a functional equivalent derivative are known in the art. Preferably, a functional equivalent derivative of Wnt-1 has substantially the same biological activity as Wnt-1 itself. The explanations given herein in respect of the activity/functionality of "Wnt-1" also apply, mutatis mutandis, to a "functional equivalent derivative" of the wild-type Wnt-1. In other words, a "functional equivalent derivative" has also the activity/functionality of the wild-type Wnt-1 as defined herein. Preferably, the a "functional equivalent derivative" is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to the nucleic acid and amino acid sequences of, e.g., SEQ ID NOs: 1 to 4.

As used herein, a "fragment" of a protein which displays a specific biological activity may relate to fragments of said protein having a sufficient length to display said activity. Accordingly, a fragment of a protein showing e.g. a specific activity may relate to a polypeptide which corresponds to a fragment of said protein which is still capable of showing said activity. For example, a fragment of Wnt-1 may be capable of showing activity of Wnt-1 , i.e to reduce VEGF-C expression. A fragment of Wnt-1 as described herein may have substantially the same biological activity as Wnt-1 itself. Furthermore, a person skilled in the art will be aware that the (biological) activity/functionality as described herein often correlates with the expression level, preferably the protein or mRNA level. The term "expression" as used herein refers to the expression of a nucleic acid molecule encoding a polypeptide, whereas "activity" refers to the functionality/activity of said polypeptide, which can be determined as outlined herein. The explanations given herein in respect of the activity/functionality of "Wnt-1" also apply, mutatis mutandis, to "(a) fragment(s)" of the full length Wnt-1. In other words, a "fragment of Wnt-1" may also have the activity/functionality of the full length Wnt-1 as defined herein. Preferably, the fragment of Wnt-1 consists of at least 24, 48, 72, 96, 120, 144, 168, 192, 216, 240, 264, 288, 312, 336, 360, 384, 408, 432, 456, 480, 504, 528, 552, 576, 600, 624, 648, 672, 696, 720, 744, 768, 792, 816, 840, 864, 888, 912, 936, 960, 984, 1008, 1032, 1056, 1080, or 1 104 nucleic acids of the nucleic acid sequence of Wnt-1 (e.g. SEQ ID NO: 1 or 3) or of at least 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 88, 96, 104, 1 12, 120, 128, 136, 144, 152, 160, 168, 176, 184, 192, 200, 208, 216, 224, 232, 240, 248, 256, 264, 272, 280, 288, 296, 304, 312, 320, 328, or 336 amino acids of the amino acid sequence of Wnt-1 (e.g. SEQ ID NO: 2 or 4).

A "patient" or "subject" for the purposes of the present invention includes both humans and other animals, particularly mammals, and other organisms. Thus, the methods are applicable to both human therapy and veterinary applications. In the preferred embodiment the patient or subject is a mammal, and in the most preferred embodiment the patient or subject is a human. The terms "treatment", "treating" and the like are used herein to generally mean obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of partially or completely curing a disease and/or adverse effect attributed to the disease. The disease may be, for example, cancer. It is envisaged in context of the present invention that the disease is a disease or disorder related to lymphangiogenesis or that the disease is metastasis. The term "treatment/treating" as used herein covers any treatment of a disease in a subject and includes: (a) preventing and ameliorating an disease or disorder related to lymphangiogenesis and/or metastasis from occurring in a subject which may be predisposed to the disease; (b) inhibiting these diseases, e.g. arresting its development like the inhibition of lymphangiogenesis or metastasis; or (c) relieving the disease, e.g. causing regression of the disease, like the repression of lymphangiogenesis or metastasis. In accordance with the present invention, the term "prevention" or "preventing" of an disease means the disease per se can be hindered of developing or to develop into an even worse situation. Accordingly, it is one aspect of the present invention that Wnt-1 or a Wnt-1 agonist can be employed in avoidance of a disease or disorder related to lymphangiogenesis or of metastasis. In accordance with the present invention, Wnt-1 may also be employed before a disease or disorder related to lymphangiogenesis or metastasis develops. As disclosed and provided for herein, Wnt-1 or Wnt-1 agonists may also be employed in the amelioration and/or treatment of disorders wherein the diseased status has already developed, i.e. in the treatment of an existing disease or disorder related to lymphangiogenesis or of existing metastasis. Accordingly, the term "treatment/treating" as used herein also relates to medical intervention of an already manifested disorder, like the treatment of an already defined and manifested cancer, disease or disorder related to lymphangiogenesis or metastasis.

Generally, as used herein, particularly in context with the screening methods provided herein, the term "transcription product" of a polynucleotide, e.g., the polynucleotide to be employed in context with the present invention, is to be construed in its meaning it usually has in the art and particularly encompasses any kind of mRNA. Similarly, the term "translation product" of a transcription product as used herein is to be construed in the sense it usually has in the art and particularly encompasses proteins and polypeptides translated from an mRNA. Such proteins and polypeptides may be further processed or modified in further downstream processes within a cell. In context of the present invention, the transcription level of a given polynucleotide, e.g., the polynucleotide to be employed in context with the present invention, may be measured by means and methods well known to those of skill in the art and described herein. For example, for measuring the transcription level of a polynucleotide, reporter gene assays may be employed in which commonly used reporter genes are fluorescent proteins such as GFP, eGFP, YFP, eYFP, BFP, eBFP, luminescent proteins such as the enzymes Renilla or firefly luciferase, and β-galactosidase encoded by the lacZ gene (Inui, Nat Rev Mol Cell Biol (2010), 1 1 : 252-63). Furthermore, in context of the present invention, for measuring the amount of a transcription product, methods known in the art and as described herein may be employed. Examples for such methods are RNA amplification methods such as qPCR, RT- PCR, qRT-PCR, RT-qPCR, Light Cycler®, TaqMan® Platform, quantigene assay (Zhou, Anal Biochem (2000), 282: 46-53), Northern blot, dot blot, microarrays, next generation sequencing (VanGuilder, Biotechniques (2008), 44(5): 619-26; Elvidge, Pharmacogenomics (2006), 7: 123-134; Metzker, Nat Rev Genet (2010), 1 1 : 31-46; Kafatos, NAR (1979), 7: 1541-1552), or the like. Furthermore, in context of the present invention, for measuring the amount of the translation product of a transcription product or for measuring the protein level of a protein or polypeptide, methods known in the art and as also described herein may be used. Examples for such methods are polyacrylamide gel electrophoresis assays and related blotting techniques such as Western Blot, optionally paired with chromogenic dye-based protein detection techniques (such as silver or coomassie blue staining) or with fluorescence- and luminescence-based detection methods for proteins in solutions and on gels, blots and microarrays, such as immunostaining, as well as immunoprecipitation, ELISA, microarrays, and mass spectrometry.

In context of the present invention, to determine whether two given nucleic acid molecules are able to hybridize, e.g., whether a given polynucleotide hybridizes to a polynucleotide to be employed in context with the present invention, the hybridization may occur and be detected under physiological or artificial conditions, under stringent or non-stringent conditions. Said hybridization conditions may be established according to conventional protocols described, for example, in Sambrook, Russell "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Laboratory, N.Y. (2001); Ausubel, "Current Protocols in Molecular Biology", Green Publishing Associates and Wiley Interscience, N.Y. (1989), or Higgins and Hames (Eds.) "Nucleic acid hybridization, a practical approach" IRL Press Oxford, Washington DC, (1985). The setting of conditions is well within the skill of the artisan and can be determined according to protocols described in the art. Thus, the detection of only specifically hybridizing sequences will usually require stringent hybridization and washing conditions such as 0.1 x SSC, 0.1% SDS at 65 °C. Non-stringent hybridization conditions for the detection of homologous or not exactly complementary sequences may be set at 6 x SSC, 1% SDS at 65 °C. As is well known in the art, the length of the probe and the composition of the nucleic acid to be determined constitute further parameters of the hybridization conditions. Variations in the above conditions may be accomplished through the inclusion and/or substitution of alternate blocking reagents used to suppress background in hybridization experiments. Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. The inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility. In accordance to the invention described herein, low stringent hybridization conditions for the detection of homologous or not exactly complementary sequences may, for example, be set at 6 x SSC, 1% SDS at 65 °C. As is well known in the art, the length of the probe and the composition of the nucleic acid to be determined constitute further parameters of the hybridization conditions. Polynucleotides such as those contained in the kit provided herein which hybridize to the polynucleotide to be employed in context with the present invention also comprise fragments of the above described polynucleotides to be employed in context with the present invention. Furthermore, a hybridization complex refers to a complex between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary G and C bases and between complementary A and T (or U, respectively) bases; these hydrogen bonds may be further stabilized by base stacking interactions. A hybridization complex may be formed in solution (e.g., Cot or Rot analysis) or between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., membranes, filters, chips, pins or glass slides to which, e.g., cells have been fixed). The terms complementary or complementarity refer to the natural binding of polynucleotides under permissive salt and temperature conditions by base-pairing. For example, the sequence "A-G-T (or U, respectively)" binds to the complementary sequence "T (or U, respectively)-C-A". Complementarity between two single-stranded molecules may be "partial", in which only some of the bases of the nucleic acids bind, or it may be complete when total complementarity exists between single-stranded molecules. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. In this context, the hybridization may occur and be tested under physiological conditions or under artificial conditions as known in the art and also described herein. For example, a test to determine hybridization between a given polynucleotide and the polynucleotide to be employed in context with the present invention may be a Luciferase Assay as known in the art and as also described in technical bulletins by Promega (C8021 (psiCHEC -2 Vector), E1960 (Dual-Luciferase® Reporter Assay System)). In context of the present invention, general examples of methods suitable to determine whether a polynucleotide hybridizes to another polynucleotide are reporter gene assays in which common reporter genes are used such as fluorescent proteins (e.g., GFP, eGFP, YFP, eYFP, BFP, or eBFP), or luminescent proteins (e.g., Renilla or firefly luciferase, or β-galactosidase encoded by the lacZ gene).

Furthermore, as used herein, particularly in context of the kits provided by the present invention, e.g., a polynucleotide capable of hybridizing or binding to the polynucleotide to be employed in context of the present invention may be a polynucleotide hybridizing to the polynucleotide to be employed in context of the present invention as described herein. For example, such a polynucleotide may be a primer or a probe to be used in an assay as well known in the art and as also described and exemplified herein. Such assays may be, for example, qPC , RT-PCR, qRT-PCR, RT-qPCR, Light Cycler®, TaqMan® Platform, quantigene assay (Zhou, Anal Biochem (2000), 282: 46-53), Northern blot, dot blot, microarrays, next generation sequencing (VanGuilder, Biotechniques (2008), 44(5): 619-26; Elvidge, Pharmacogenomics (2006), 7: 123-134; Metzker, Nat Rev Genet (2010), 1 1 : 31 -46; Kafatos, NAR (1979), 7: 1541-1552), or the like. Furthermore, as used herein, particularly in context of the kits provided by the present invention, e.g., a binding molecule capable of binding to the polynucleotide or polypeptide to be employed in context with the present invention may be an antibody molecule or a fragment thereof (such as F(ab), F(ab)₂ fragments or the like) specifically binding to the polynucleotide or polypeptide to be employed in context with the present invention. Binding of an antibody or a fragment thereof to a polynucleotide can be easily detected by the skilled person using methods well known in the art such as Western Blot, ELISA, EIA or similar methods as also described and exemplified herein.

In accordance with the present invention, Wnt-1 -molecules are used to treat, prevent and/or ameliorate a disease or disorder related to lymphangiogenesis or to prevent metastasis. Accordingly, the present invention relates to a polynucleotide for use in treating or preventing a disease or disorder related to lymphangiogenesis or for use in preventing metastasis, wherein said polynucleotide is selected from the group consisting of:

1 or SEQ ID NO: 3;

(e) a polynucleotide comprising a fragment of the nucleotide sequence of any one of (a) to (d) which is at least 24 nucleotides in length and which encodes a polypeptide having Wnt-1 activity. The present invention relates to a polynucleotide for use in treating a disease or disorder related to lymphangiogenesis or in preventing metastasis, wherein said polynucleotide is selected from the group consisting of:

As mentioned, the present invention relates to the use of Wnt-1 -molecules for treating, preventing or ameliorating a disease or disorder. Thus the present invention relates to a polynucleotide for use in treating or preventing a disease or disorder, wherein said polynucleotide is selected from the group consisting of:

1 or SEQ ID NO: 3;

(d) a polynucleotide encoding a polypeptide comprising or consisting of an amino acid sequence which is at least 70% identical to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 and which exhibits Wnt-1 activity; and (e) a polynucleotide comprising a fragment of the nucleotide sequence of any one of (a) to (d) which is at least 24 nucleotides in length and which encodes a polypeptide having Wnt-1 activity.

Accordingly, the present invention relates to a polynucleotide for use in treating a disease or disorder related to lymphangiogenesis or in preventing metastasis, wherein said polynucleotide is selected from the group consisting of:

1 or SEQ ID NO: 3;

(e) a polynucleotide comprising a fragment of the nucleotide sequence of any one of (a) to (d) which is at least 24 nucleotides in length and which encodes a polypeptide having Wnt-1 activity.

In context of the present invention it is envisaged that the disease or disorder to be treated is cancer. This cancer may be a solid tumor. The type of cancer may be, for example, melanoma, breast cancer, lung cancer, kidney cancer, pancreatic cancer or hematopoietic tumor (lymphoma). It is also envisaged with respect to the present invention that the disease or disorder to be treated is graft rejection. This graft rejection may be, for example, lung graft rejection, kidney graft rejection, and particularly cornea graft rejection. In addition, it is also envisaged in context of the invention, that the disease or disorder to be treated is an autoimmune disorder. This autoimmune disorder may be, for example, rheumatoid arthritis or psoriasis. Furtheraiore, it is also envisaged in context of the invention, that the disease or disorder to be treated is oedema or impaired wound healing. The present invention relates to a polynucleotide for use in treating a disease or disorder related to lymphangiogenesis or in preventing metastasis, wherein said polynucleotide is selected from the group consisting of: (a) a polynucleotide comprising or consisting of the nucleotide sequence of SEQ ID NO:

1 (human wnt-1) or SEQ ID NO: 3 (human wnt-1 without the first 81 nucleotides, i.e. without the signal sequence); (b) polynucleotide encoding a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 2 (human Wnt-1) or SEQ ID NO: 4 (human Wnt-1 without the first 27 amino acids, i.e. without the signal sequence);

(d) a polynucleotide encoding a polypeptide comprising or consisting of an amino acid sequence which is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or.99.9% identical to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 and which exhibits Wnt-1 activity; and

(e) a polynucleotide comprising a fragment of the nucleotide sequence of any one of (a) to (d) which is at least 24, 48, 72, 96, 120, 144, 168, 192, 216, 240, 264, 288, 312, 336, 360, 384, 408, 432, 456, 480, 504, 528, 552, 576, 600, 624, 648, 672, 696, 720, 744, 768, 792, 816, 840, 864, 888, 912, 936, 960, 984, 1008, 1032, 1056, 1080, or 1 104 nucleotides in length and which encodes a polypeptide having Wnt-1 activity, wherein the disease or disorder related to lymphangiogenesis is selected from the group consisting of cancer, melanoma, solid tumor, breast cancer, lung cancer, kidney cancer, pancreatic cancer, hematopoietic tumor (lymphoma), graft rejection, lung graft rejection, kidney graft rejection, and particularly cornea graft rejection, autoimmune disorder, rheumatoid arthritis, psoriasis, oedema and impaired wound healing.

In line with this, the present invention relates to a polynucleotide for use in treating a disease or disorder related to lymphangiogenesis or in preventing metastasis, wherein said polynucleotide is selected from the group consisting of:

(d) a polynucleotide encoding a polypeptide comprising or consisting of an amino acid sequence which is at least 70% identical to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 and which exhibits Wnt-1 activity; and (e) a polynucleotide comprising a fragment of the nucleotide sequence of any one of (a) to (d) which is at least 24 nucleotides in length and which encodes a polypeptide having Wnt-1 activity,

wherein the disease or disorder related to lymphangiogenesis is selected from the group consisting of cancer, melanoma, solid tumor, breast cancer, lung cancer, kidney cancer, pancreatic cancer, hematopoietic tumor (lymphoma), graft rejection, lung graft rejection, kidney graft rejection, and particularly cornea graft rejection, autoimmune disorder, rheumatoid arthritis, psoriasis, oedema and impaired wound healing.

In one preferred aspect of the invention, the disease related to lymphangiogenesis to be treated is cancer, such as melanoma or hematopoietic tumor (lymphoma) or graft rejection, like cornea or kidney graft rejection. The present invention relates to a polynucleotide for use in treating a disease or disorder related to lymphangiogenesis or in preventing metastasis, wherein said polynucleotide is selected from the group consisting of:

(e) a polynucleotide comprising a fragment of the nucleotide sequence of any one of (a) to (d) which is at least 24, 48, 72, 96, 120, 144, 168, 192, 216, 240, 264, 288, 312, 336, 360, 384, 408, 432, 456, 480, 504, 528, 552, 576, 600, 624, 648, 672, 696, 720, 744, 768, 792, 816, 840, 864, 888, 912, 936, 960, 984, 1008, 1032, 1056, 1080, or 1104 nucleotides in length and which encodes a polypeptide having Wnt-1 activity, wherein the disease or disorder related to lymphangiogenesis is selected from the group consisting of cancer, melanoma, hematopoietic tumor (lymphoma), and cornea or kidney graft rejection. Accordingly, the present invention relates to a polynucleotide for use in treating a disease or disorder related to lymphangiogenesis or in preventing metastasis, wherein said polynucleotide is selected from the group consisting of:

(e) a polynucleotide comprising a fragment of the nucleotide sequence of any one of (a) to (d) which is at least 24 nucleotides in length and which encodes a polypeptide having Wnt-1 activity,

wherein the disease or disorder related to lymphangiogenesis is selected from the group consisting of cancer, melanoma, hematopoietic tumor (lymphoma), and cornea or kidney graft rejection.

The present invention relates to a polypeptide encoded by the polynucleotide to be employed in context with the present invention for use in treating or preventing a disease or disorder related to lymphangiogenesis or for preventing metastasis. The polypeptide to be employed in context of the present invention may comprise or consist of the amino acid of SEQ ID NO: 2 or SEQ ID NO: 4. The polypeptide to be employed in context with the present invention may comprise or consist of an amino acid sequence which is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 and exhibit Wnt-1 activity. The polypeptide to be employed in context with the present invention may comprise or consist of an amino acid sequence which is at least 70% identical to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 and exhibit Wnt-1 activity. The polypeptide to be employed in context of the present invention may be used for treating a disease or disorder related to lymphangiogenesis or in preventing metastasis. Accordingly, the present invention relates to a polypeptide encoded by the polynucleotide be employed in context of the present invention for use in treating or preventing a disease or disorder related to lymphangiogenesis or in preventing metastasis. As described herein, the polypeptide to be employed in context with the present invention may be used for treating, preventing and/or ameliorating a disease or disorder related to lymphangiogenesis. This disease or disorder related to lymphangiogenesis may be cancer. This cancer could be a solid tumor. The type of cancer may be, for example, melanoma, breast cancer, lung cancer, kidney cancer, pancreatic cancer or hematopoietic tumor (lymphoma). The disease or disorder related to lymphangiogenesis may also be graft rejection, such as lung graft rejection, kidney graft rejection, or particularly cornea graft rejection. The disease or disorder related to lymphangiogenesis may further be an autoimmune disorder, such as rheumatoid arthritis or psoriasis. Furthermore, it is also envisaged in context of the invention, that the disease or disorder to be treated is oedema or impaired wound healing. In context of the polypeptide to employed in context with the present invention, the disease or disorder related to lymphangiogenesis may be selected from the group consisting of cancer, melanoma, solid tumor, breast cancer, lung cancer, kidney cancer, pancreatic cancer, hematopoietic tumor (lymphoma), graft rejection, lung graft rejection, kidney graft rejection, and particularly cornea graft rejection, autoimmune disorder, rheumatoid arthritis, psoriasis, oedema and impaired wound healing. In one embodiment, the present invention relates to a polypeptide to employed in context of the present invention for use in treating a disease or disorder related to lymphangiogenesis selected from the group consisting of cancer, melanoma, hematopoietic tumor (lymphoma), and cornea or kidney graft rejection.

The present invention relates to a polypeptide for use in treating a disease or disorder related to lymphangiogenesis or in preventing metastasis which comprises or consists of the amino acid of SEQ ID NO: 2 or SEQ ID NO: 4. The present invention relates to a polypeptide for use in treating a disease or disorder related to lymphangiogenesis or in preventing metastasis which comprises or consists of the amino acid of SEQ ID NO: 2 or SEQ ID NO: 4, wherein the disease or disorder related to lymphangiogenesis is selected from the group consisting of cancer, melanoma, solid tumor, breast cancer, lung cancer, kidney cancer, pancreatic cancer, hematopoietic tumor (lymphoma), graft rejection, lung graft rejection, kidney graft rejection, and particularly cornea graft rejection, autoimmune disorder, rheumatoid arthritis, psoriasis, oedema and impaired wound healing. The present invention relates to a polypeptide for use in treating a disease or disorder related to lymphangiogenesis or in preventing metastasis which comprises or consists of the amino acid of SEQ ID NO: 2 or SEQ ID NO: 4, wherein the disease or disorder related to lymphangiogenesis is selected from the group consisting of cancer, melanoma, hematopoietic tumor (lymphoma), and cornea or kidney graft rejection.

The present invention relates to a polypeptide for use in treating a disease or disorder related to lymphangiogenesis or in preventing metastasis which comprises or consists of an amino acid sequence which is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 and which exhibits Wnt-1 activity. The disease or disorder related to lymphangiogenesis may be cancer. This cancer could be a solid tumor. The type of cancer may be, for example, melanoma, breast cancer, lung cancer, kidney cancer, pancreatic cancer or hematopoietic tumor (lymphoma). The disease or disorder related to lymphangiogenesis may also be graft rejection, such as lung graft rejection, kidney graft rejection, or particularly cornea graft rejection. The disease or disorder related to lymphangiogenesis may further be an autoimmune disorder, such as rheumatoid arthritis or psoriasis. Furthermore, it is also envisaged in context of the invention, that the disease or disorder to be treated is oedema or impaired wound healing. The present invention relates to a polypeptide for use in treating a disease or disorder related to lymphangiogenesis or in preventing metastasis which comprises or consists of an amino acid sequence which is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 and which exhibits Wnt-1 activity, wherein the disease or disorder related to lymphangiogenesis is selected from the group consisting of cancer, melanoma, solid tumor, breast cancer, lung cancer, kidney cancer, pancreatic cancer, hematopoietic tumor (lymphoma), graft rejection, lung graft rejection, kidney graft rejection, and particularly cornea graft rejection, autoimmune disorder, rheumatoid arthritis, psoriasis, oedema and impaired wound healing. The present invention relates to a polypeptide for use in treating a disease or disorder related to lymphangiogenesis or in preventing metastasis which comprises or consists of an amino acid sequence which is at least 50%, 55%, 60%, 65%, 70%, 75%o, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 and which exhibits Wnt-1 activity, wherein the disease or disorder related to lymphangiogenesis is selected from the group consisting of cancer, melanoma, hematopoietic tumor (lymphoma), and cornea or kidney graft rejection.

The present invention relates to a polypeptide for use in treating a disease or disorder related to lymphangiogenesis or in preventing metastasis which comprises or consists of an amino acid sequence which is at least 70% identical to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 and which exhibits Wnt-1 activity. The disease or disorder related to lymphangiogenesis may be cancer. This cancer could be a solid tumor. The type of cancer may be, for example, melanoma, breast cancer, lung cancer, kidney cancer, pancreatic cancer or hematopoietic tumor (lymphoma). The disease or disorder related to lymphangiogenesis may also be graft rejection, such as lung graft rejection, kidney graft rejection, or particularly cornea graft rejection. The disease or disorder related to lymphangiogenesis may further be an autoimmune disorder, such as rheumatoid arthritis or psoriasis. Furthermore, it is also envisaged in context of the invention, that the disease or disorder to be treated is oedema or impaired wound healing. The present invention relates to a polypeptide for use in treating a disease or disorder related to lymphangiogenesis or in preventing metastasis which comprises or consists of an amino acid sequence which is at least 70% identical to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 and which exhibits Wnt-1 activity, wherein the disease or disorder related to lymphangiogenesis is selected from the group consisting of cancer, melanoma, solid tumor, breast cancer, lung cancer, kidney cancer, pancreatic cancer, hematopoietic tumor (lymphoma), graft rejection, lung graft rejection, kidney graft rejection, and particularly cornea graft rejection, autoimmune disorder, rheumatoid arthritis, psoriasis, oedema and impaired wound healing. The present invention relates to a polypeptide for use in treating a disease or disorder related to lymphangiogenesis or in preventing metastasis which comprises or consists of an amino acid sequence which is at least 70% identical to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 and which exhibits Wnt-1 activity, wherein the disease or disorder related to lymphangiogenesis is selected from the group consisting of cancer, melanoma, hematopoietic tumor (lymphoma), and cornea or kidney graft rejection.

Further, the present invention relates to a vector comprising the polynucleotide to be employed in context with the present invention for use in treating or preventing a disease or disorder. Accordingly, the present invention relates to a pharmaceutical composition for use in treating or preventing a disease or disorder, said pharmaceutical composition comprising a vector comprising a Wnt-1 polynucleotide; together with a pharmaceutically acceptable carrier or excipient. The present invention also relates to a vector comprising the polynucleotide to be employed in context with the present invention for use in treating a disease or disorder related to lymphangiogenesis or in preventing metastasis. Accordingly, the present invention relates to a vector comprising the polynucleotide to be employed in context with the present invention for use in treating or preventing a disease or disorder related to lymphangiogenesis or in preventing metastasis. The disease or disorder related to lymphangiogenesis may be cancer. This cancer could be a solid tumor. The type of cancer may be, for example, melanoma, breast cancer, lung cancer, kidney cancer, pancreatic cancer or hematopoietic tumor (lymphoma). The disease or disorder related to lymphangiogenesis may also be graft rejection, such as lung graft rejection, kidney graft rejection, or particularly cornea graft rejection. The disease or disorder related to lymphangiogenesis may further be an autoimmune disorder, such as rheumatoid arthritis or psoriasis. Furthermore, it is also envisaged in context of the invention, that the disease or disorder to be treated is oedema or impaired wound healing. The present invention relates to a vector comprising the polynucleotide to be employed in context with the present invention for use in treating a disease or disorder related to lymphangiogenesis or in preventing metastasis, wherein the disease related to lymphangiogenesis is selected from the group consisting of cancer, melanoma, solid tumor, breast cancer, lung cancer, kidney cancer, pancreatic cancer, hematopoietic tumor (lymphoma), graft rejection, lung graft rejection, kidney graft rejection, and particularly cornea graft rejection, autoimmune disorder, rheumatoid arthritis, psoriasis, oedema and impaired wound healing. The present invention relates to a vector comprising the polynucleotide to be employed in context with the present invention for use in treating a disease or disorder related to lymphangiogenesis or in preventing metastasis, wherein the disease related to lymphangiogenesis is selected from the group consisting of cancer, melanoma, hematopoietic tumor (lymphoma), and cornea or kidney graft rejection.

The present invention relates to a host cell comprising the polynucleotide, the vector or the polypeptide to be employed in context with the present invention for use in treating or preventing a disease or disorder. Accordingly, the present invention relates to a pharmaceutical composition for use in treating or preventing a disease or disorder, said pharmaceutical composition comprising a host cell comprising a Wnt-1 polynucleotide or a vector comprising a Wnt-1 polynucleotide; together with a pharmaceutically acceptable carrier or excipient. The present invention also relates to a host cell comprising the polynucleotide, the vector or the polypeptide to be employed in context with the present invention for use in treating a disease or disorder related to lymphangiogenesis or in preventing metastasis. Accordingly, the present invention relates to a host cell comprising the polynucleotide to be employed in context with the present invention or the vector to be employed in context with the present invention for use in treating or preventing a disease or disorder related to lymphangiogenesis or in preventing metastasis. This disease or disorder related to lymphangiogenesis may be cancer. This cancer could be a solid tumor. The type of cancer may be, for example, melanoma, breast cancer, lung cancer, kidney cancer, pancreatic cancer or hematopoietic tumor (lymphoma). The disease or disorder related to lymphangiogenesis may also be graft rejection, such as lung graft rejection, kidney graft rejection, or particularly cornea graft rejection. The disease or disorder related to lymphangiogenesis may further be an autoimmune disorder, such as rheumatoid arthritis or psoriasis. Furthermore, it is also envisaged in context of the invention, that the disease or disorder to be treated is oedema or impaired wound healing. The present invention relates to a vector host cell comprising the polynucleotide, the vector or the polypeptide to be employed in context with the present invention for use in treating a disease or disorder related to lymphangiogenesis or in preventing metastasis, wherein the disease related to lymphangiogenesis is selected from the group consisting of cancer, melanoma, solid tumor, breast cancer, lung cancer, kidney cancer, pancreatic cancer, hematopoietic tumor (lymphoma), graft rejection, lung graft rejection, kidney graft rejection, and particularly cornea graft rejection, autoimmune disorder, rheumatoid arthritis, psoriasis, oedema and impaired wound healing. The present invention relates to a host cell comprising the polynucleotide, the vector or the polypeptide to be employed in context with the present invention for use in treating a disease or disorder related to lymphangiogenesis or in preventing metastasis, wherein the disease related to lymphangiogenesis is selected from the group consisting of cancer, melanoma, hematopoietic tumor (lymphoma), and cornea or kidney graft rejection. For example, the host cell may be a fibroblast or an epithelial cell.

In addition, the present invention relates to a pharmaceutical composition comprising the polynucleotide to be employed in context with the present invention, the polypeptide to be employed in context with the present invention, the vector as described herein and to be employed in context with the present invention, the host cell as described herein and to be employed in context with the present invention and/or the agent as described herein and to be employed in context with the present invention; together with a pharmaceutically acceptable carrier or excipient.

The present invention relates to a pharmaceutical composition comprising the polynucleotide, the polypeptide the vector, the host cell, and/or the agent described herein and to be employed in context with the present invention for use in treating or preventing a disease or disorder. The present invention relates to a pharmaceutical composition comprising the polynucleotide, the polypeptide the vector, the host cell, and/or the agent described herein and to be employed in context with the present invention for use in treating a disease or disorder related to lymphangiogenesis or in preventing metastasis. In line with this, the present invention relates to a pharmaceutical composition comprising the polynucleotide, the polypeptide the vector, the host cell, and/or the agent described herein and to be employed in context with the present invention for use in treating or preventing a disease or disorder related to lymphangiogenesis or in preventing metastasis. This disease or disorder related to lymphangiogenesis may be cancer. This cancer could be a solid tumor. The type of cancer may be, for example, melanoma, breast cancer, lung cancer, kidney cancer, pancreatic cancer or hematopoietic tumor (lymphoma). The disease or disorder related to lymphangiogenesis may also be graft rejection, such as lung graft rejection, kidney graft rejection, or particularly cornea graft rejection. The disease or disorder related to lymphangiogenesis may further be an autoimmune disorder, such as rheumatoid arthritis or psoriasis. Furthermore, it is also envisaged in context of the invention, that the disease or disorder to be treated is oedema or impaired wound healing. Accordingly, the present invention relates to a pharmaceutical composition comprising the polynucleotide, the polypeptide the vector, the host cell, and/or the agent described herein and to be employed in context with the present invention for use in treating a disease or disorder related to lymphangiogenesis or in preventing metastasis, wherein the disease related to lymphangiogenesis is selected from the group consisting of cancer, melanoma, solid tumor, breast cancer, lung cancer, kidney cancer, pancreatic cancer, hematopoietic tumor (lymphoma), graft rejection, lung graft rejection, kidney graft rejection, and particularly cornea graft rejection, autoimmune disorder, rheumatoid arthritis, psoriasis, oedema and impaired wound healing. The present invention relates to a pharmaceutical composition comprising the polynucleotide, the polypeptide the vector, the host cell, and/or the agent described herein and to be employed in context with the present invention for use in treating a disease or disorder related to lymphangiogenesis or in preventing metastasis, wherein the disease related to lymphangiogenesis is selected from the group consisting of cancer, melanoma, hematopoietic tumor (lymphoma), and cornea or kidney graft rejection. In context of the present invention, as described herein, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, excipient and/or diluent. In context of the present invention, as described herein, the pharmaceutical composition may be administered by different ways, e.g., parenterally, enterally, orally (e.g., pill, tablet, buccal, sublingual, disintegrating, capsule, thin film, liquid solution or suspension, powder, solid crystals or liquid), rectally (e.g., suppository, enema), via injection (e.g., intravenously, subcutaneously, intramuscularly, intraperitoneally, intraderrnally) via inhalation (e.g., intrabronchially), topically, vaginally, epicutaneously, into the eye, or intranasally. Preferably, in context with the present invention, the pharmaceutical composition is administered parenterally. In line with this, the present invention relates to the polynucleotide to be employed in context with the present invention, the polypeptide to be employed in context with the present invention, the vector as described herein and to be employed in context with the present invention, the host cell as described herein and to be employed in context with the present invention, the agent as described herein and to be employed in context with the present invention, the pharmaceutical composition as described herein and to be employed in context with the present invention, or the method for treating as described herein, wherein said polynucleotide, said polypeptide, said vector, said host cell, said agent, or said pharmaceutical composition is to be administered parenterally. In addition, the present invention relates to the polynucleotide to be employed in context with the present invention, the polypeptide to be employed in context with the present invention, the vector as described herein and to be employed in context with the present invention, the host cell as described herein and to be employed in context with the present invention, the agent as described herein and to be employed in context with the present invention, the pharmaceutical composition as described herein and to be employed in context with the present invention, or the method for treating as described herein, wherein the disease or disorder related to lymphangiogenesis is selected from the group consisting of cancer, melanoma, solid tumor, breast cancer, lung cancer, kidney cancer, pancreatic cancer, hematopoietic tumor, graft rejection, autoimmune disorder, rheumatoid arthritis, psoriasis, oedema and impaired wound healing. The polynucleotide to be employed in context with the present invention, the polypeptide to be employed in context with the present invention, the vector as described herein and to be employed in context with the present invention, the host cell as described herein and to be employed in context with the present invention, the agent as described herein and to be employed in context with the present invention, or the pharmaceutical composition as described herein and to be employed in context with the present invention can be administered to the subject at a suitable dose. The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosage for any single patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently.

The Figures show:

Figure 1 : Human melanoma expresses Wnt-1

(A) These are examples for immunohistochemistry on paraffin-embedded tissue micro-arrays containing nevi (n = 116), primary melanomas (n = 100) and melanoma metastases (n = 248) positively (top row) or negatively (lower row) staining with anti- Wnt-1 antibodies (stains red). The top right image serves as isotype control. Sections were counterstained with hematoxylin (stains blue). Wnt-1 staining was scored as strong (as exemplified by staining in melanoma, arrows), weak (as exemplified in nevus, arrow) or absent. The bar graph shows % of Wnt-1 positive lesions out of 116 nevi, 100 primary melanoma and 248 melanoma metastases. Weak expression is depicted by hatched and strong expression by black bars. ^*p<0.05.

(B) Wnt-1 , Wnt-3a and Wnt-5a mRNA expressions were analyzed in 3 mm biopsies derived from nevi (n = 7), primary melanoma (n = 10) and melanoma metastases (n = 21) by real-time PGR. Wnt mRNA levels were expressed as ACT (CT of VEGF-C minus CT of GAPDH). A ACT of 17 was taken as cut off between present (green dots) and low/absent expression (red dots). Each dot represents one patient.

(C) Examples for immunohistochemistry for VEGF-C and Wnt-1 expression in 2 melanoma metastases. The upper 2 panels show serial sections from a tissue array of a primary melanoma expressing high levels of VEGF-C and negligible Wnt-1 (corresponding areas are encircled). The lower two panels show serial sections of a metastasis with high Wnt-1 and low VEGF-C expression.

Figure 2: Delayed metastasis in Wnt-1 overexpressing human melanoma cells (A) Immunohistochemistry of control or Wnt-1 -transduced melanoma cells (M24met) injected into SCID mice, excised at a tumor volume of 400 mm³ and stained with an anti- Wnt-1 antibody (2 left images) or with isotype control antibody (right image). Orthotopically growing melanoma also colonizes the epidermis (arrow).

(B) Control and Wnt-1 overexpressing A375 melanoma cells (2x 10⁶) were injected intradermally into the right flank of SCID mice; primary tumors were removed at a mean tumor volume of 400 mm . Lymph nodes were excised after the first metastasis was palpable in each group. All lymph nodes were screened for metastasis by histological serial-sectioning. Numbers at each bar denote numbers of positive sentinel nodes/out of all animals. In Wnt-1 positive A375 tumors, no metastasis occurred within an observation period of 90 days (indicated by arrow).

Figure 3: Reduced lymphangiogenesis in Wnt-1 positive melanoma

(A) Example for H&E staining and immunohistochemistry (anti-Lyve-1 and CD 31 stains in red) of a primary M24met melanoma (control versus Wnt-1 overexpression). In control tumors, lymph vessels are dilated and filled with melanoma cells, whereas in Wnt-1 positive tumors, numbers of vessels are reduced, lumina are mostly smaller and empty (exemplified by arrows). Total lymph vessel areas were quantified as described in methods and shown in the bar graph to the right. The difference between controls and Wnt-1 ⁺ tumors was significant (mean ± SD, ^*p<0.05). n = 30 sections per group.

(B and C) Real-time PCR analysis was performed using mRNA isolated from primary M24met (B) and A375 (C) tumors using specific primers for mouse CD31, Lyve-1, Prox-1 and human VEGF-C. 2-microglobulin for mouse primers and GAPDH for human primers were used as an internal control. The difference was calculated as 2^{λ("ΔΔ€Τ)}. Values are given as mean values ± SD, *p<0.05; n = 5 mice (SCID mice) per group.

Figure 4: Wnt-1 reduces VEGF-C expression independently of the β-catenin pathway

(A) A375 melanoma cells were transfected with pLNCX control vector, Wnt-1 and Wnt-5a plasmid DNA together with luciferase reporters controlled by β- catenin/Tcf, NFAT, AP-1 or NFKB responsive elements. SV40 Renilla was used as an internal control. As positive control, stimulations with ionomycin, LiCl and TNF-oc for 16 h were used as indicated. Control cells were treated with the solvent DMSO. Luciferase activity was assessed 48 h after transfection and data are reported as normalized averages of the Luciferase/Renilla ratio. Values are given as mean values ± SD (n=3), *p<0.05 as compared to controls.

(B-C) VEGF-C mRNA expression was analyzed by real-time PGR in A375 cells stably (B) or transiently (C) overexpressing the indicated plasmids. ANTcf-4 and DKK-1 were used as inhibitors of the canonical Wnt pathway. Axin-2 mRNA expression was analyzed as a positive control for an activated canonical Wnt pathway. The inserted Western blot in (B) shows VEGF-C protein expression from serum free supematants from control and Wnt-1⁺ A375 melanoma cells.

(D) VEGF-C mRNA expression was analyzed by real-time PCR in A375 melanoma cells treated with two GSK-3 inhibitors, SB-415286 (25 μιηοΐ/ΐ and 40 μηιοΐ/ΐ) and LiCl (40 mM) for 16 h. Axin-2 mRNA expression was analyzed as positive control for an activated canonical Wnt pathway. In all experiments, GAPDH was analyzed as an internal control. The difference was calculated as 2^{a(" CT)}. Values are given as mean values ± SD, *p<0.05. Results are the mean of 3 independent experiments.

Figure 5: Wnt-1 reduces VEGF-C expression in a calcineurin-dependent manner

(A) VEGF-C mRNA expression was analyzed by real-time PCR in A375 melanoma cells, stimulated with different concentrations of ionomycin or transiently transfected with Wnt-1 or Wnt-5a and stimulated with 8 μΜ ionomycin for 16 h. IL-8 mRNA expression was determined as a positive control for an activated calcineurin/NFAT pathway.

(B) VEGF-C mRNA expression was analyzed by real-time PCR in A375 melanoma cells stably overexpressing Wnt-1 and treated with 1 μg/ml CsA for the indicated time points. As a control, Wnt-1 stable A375 cells were treated with the solvent DMSO.

(C) VEGF-C mRNA expression was analyzed by real-time PCR in A375 melanoma cells transiently expressing a pLNCX control vector, Wnt-1 or Wnt- 5a together with an empty vector or a dominant negative NFAT (dnNFAT). In all experiments, GAPDH was used as an internal control. The difference was calculated as 2^{a(" ct)}. Values are given as mean values ± SD, * p<0.05. Results are the mean of 3 independent experiments. To confirm the functionality of dnNFAT, A375 melanoma cells were transfected with a control vector or a dnNFAT plasmid DNA together with a luciferase reporter controlled by NFAT responsive elements. SV40 Renilla was used as an internal control. 32 h after transfection, A375 cells were stimulated with DMSO, ionomycin or ionomycin/PMA for 16 h. Luciferase activity was assessed 48 h after transfection and data are reported as normalized averages of the Luciferase/Renilla ratio. Values are given as mean values ± SD (n = 3), * p<0.05 as compared to controls.

Figure 6: Cyclosporine A enhances lymphangiogenesis and metastasis in Wnl-1 tumors

(A) A375 melanoma cells were injected intradermally into the right flank of SCID mice. Treatment with the calcineurin inhibitor CsA started at the time of tumor inoculation and was given daily via a gauge (50 mg/kg/day). Tumors were removed at a mean tumor volume of 400 mm^J per group, n = 8 mice per group. Animals of a treatment group were sacrificed after the first macro- metastasis was detectable in this group. Whereas Wnt-1 positive A375 tumors did not metastasize during an observation time of 60 days (0 out of 5; arrow), all CsA-treated animals carrying Wnt-1 positive A375 tumors metastasized to sentinel nodes.

(B) mRNA was prepared from primary control and Wnt-1 expressing A375 tumors, treated with or without CsA and mRNA expression for indicated genes was quantified by real-time PCR. 2-microglobulin was used as an internal control for mouse primers and GAPDH for human primers. The difference was calculated as 2^Λ<"ΔΔ0Τ). Values are given as mean values ± SD, ^* p<0.05; n = 8 mice per group.

Figure 7: Expression profiles of Wnt ligands and Frizzled receptors in M24met cells,

Mel Juso cells and A375 cells

Expression profiles of Wnt ligands and Frizzled (Fzd) receptors in M24met (1), in Mel Juso (2) and A375 (3) cells: semi-quantitative RT-PCR was prepared from M24met cells, Mel Juso cells and A375 melanoma cells to investigate the expression of Wnt and Fzd proteins. Semi-quantitative PCR amplification (45 s 96 °C, 1 min 55-63 °C, 1 min 72 °C) was performed. All PCR products were analyzed on 1.5% agarose gel containing ethidium bromide and photographed under UV radiation. The bands for Fzd-1 , Fzd-3, Fzd-10, Wnt-3a, Wnt-6, Wnt- 7, Wnt-8a, 8b, Wnt-10, Wnt-16 are unspecific primers bands. In the M24met melanoma cells (1), Wnt-5a, Wnt-6, Wnt-7b, Wnt- 10a, -10b, Fzd-6, Fzd-8 and Fzd-9 are highly expressed, whereas Wnt- 3, Wnt-9a and Fzd-2, Fzd-4, Fzd-7 are weakly expressed. In the A375 cells (3), Wnt- 7b, Wnt-9a, Wnt- 10b, Fzd-2, Fzd-6, Fzd-8 are highly expressed and Wnt-5b, Wnt-5a and Fzd-4 are weakly expressed. Figure 8: Heat-map for mRNA expression of lymphangiogenic genes

Expression profile of lymphatic genes from primary A375 and M24met melanoma cells injected into mice and excised at a tumor volume of 400 mm (n = 5/group). Mice injected with A375 melanoma cells were treated with and without CsA (50 mg/kg/day) as indicated. mRNA was prepared and subjected to Affymetrix® gene expression analysis. Heat maps show the scaled ratio of mRNA expression of each gene to the median of this gene over all arrays. Color scale indicates units of standard deviation from the mean expression of each gene (red = high expression level; blue = low expression level). VEGF-C was the only lymphatic gene where a difference in expression between control or CsA treated versus Wnt-1 was seen.

Figure 9: Lack of efficacy of Wnt-5a

VEGF-C mRNA expression was analyzed by real time PCR. GAPDH served as internal control. The difference was calculated as 2^a("aact). Values are given as mean values ± SD, *p < 0.05. Results are the mean of 3 independent experiments. A375 cells transiently over-expressing indicated plasmids.

The present invention is illustrated by the following examples. Example 1: Human samples

Human tissue arrays were produced according to routine procedures from paraffin embedded material of human nevi (n = 116), primary melanoma (n = 100) and melanoma metastases (n = 248) according to a protocol approved by the Vienna Ethics committee (085/10/2009). Briefly, 3 mm punches were taken from paraffin blocks at sites with epidermal and/or dermal melanocytes/melanoma cells and at sites excluding tumor necrosis as defined by parallel viewing of H&E-stained sections. For mRNA preparation, punch biopsies (3 mm in diameter) were taken from surgical specimen immediately after surgical removal (7 nevi, 10 primary melanoma and 21 melanoma metastases) according to a protocol approved by the Vienna Ethics committee (093/2003) and total RNA was isolated according to routine procedures (Qiagen RNeasy Mini Kit).

Example 2: Cell lines

The human melanoma cell line M24met was kindly provided by R. A. Reisfeld (Mueller, Cancer Res (1991), 51 : 2193-2198). The human melanoma cell line A375 was purchased from ATCC. M24met cells were cultured in RPMI (Roswell Park Memorial Institute standard medium, GIBCO Invitrogen), A375 cells in DMEM (Dulbecco's modified eagle medium, GIBCO Invitrogen), both supplemented with 10% FCS, 2 mM L-glutamine and 50 U/ml streptomycin/penicillin (all GIBCO Invitrogen). The amphotropic packaging cell line Phoenix (gift of H. Stockinger; Medical University of Vienna (Grignani, Cancer Res (1998), 58: 14- 19)) was cultured in DMEM supplemented with 10% FCS, 2 mM L-glutamine and 50 U/ml streptomycin/penicillin. Primary human melanoma cell lines used in Table 2 were isolated from primary human lesions and were provided by W. Berger (Medical University of Vienna).

Example 3: Antibodies, plasmids and reagents

Goat anti-mouse Wnt-1 (Order no. AF1620) and goat anti -human VEGF-C antibody (Order no. AF752) were purchased from R&D systems, rabbit anti-mouse Lyve-1 from Acris Antibodies GmbH (Order no.DP3513), rat anti-mouse PECAM-1 (CD31) (Order no. 550274) from BD Pharmingen, biotinylated secondary antibodies (anti-rat (Order no.BA-4001), anti- rabbit (Order no. BA-1000) and anti-goat IgG (Order no.BA-5000) from Vector Laboratories and HRP rabbit anti-goat IgG (Order no. 81-1620)was purchased from Zymax. CsA (Sigma- Aldrich, Order no. C3662) was solubilized in DMSO as a 10 mg/ml stock solution and diluted to final concentrations as indicated in the respective experiments. lonomycin (Sigma-Aldrich, Order no. 10634) was used in concentrations as indicated in the respective experiments. Lithium Chloride (Serva Electrophoresis, Order no. 39560) was used in a concentration of 40 mM, TNF-oc (Pepro Tech, Order no. 300-01A) and Phorbol 12-myristate 13-acetate (PMA, Sigma, Order no. P8139) in a concentration of 100 ng/ml. SB-415286, an inhibitor of GSK- 3β (Order no. 415286) was purchased from Eubio. The retroviral vectors pLNCX and Wnt- 1/pLNCX were a kind gift of A. McMahan (Harvard University, Cambridge). The Wnt- 5a/pcDNA3 plasmid was a kind gift of G. Raguenez (Institut Gustave Roussy, France). DKK- l/pcDNA3.1 was a kind gift of V.J. Hearing (National Cancer Institute, Bethesda) and ANTcf-4/pcDNA3.1 was a kind gift of R.G. Pestell (Thomas Jefferson University, Philadelphia). DKK-1 and ANTcf-4 plasmids were cloned into the retroviral vector pBMN-I- GFP, which was a kind gift of Rainer de Martin (Medical University of Vienna). The dominant negative NFAT (1-130) was a kind gift from Roger J. Davis (University of Massachusetts Medical School, Worcester). Plasmid pUBTluc containing AP-1, NFAT and NFKB luciferase reporter were a kind gift of R. Hofer-Warbinek and R. de Martin (Medical University of Vienna). The SuperTopflash reporter was a gift of R. Moon (University of Washington School Medicine, Seattle). SV40 Renilla was purchased from Promega.

Example 4: Retroviral transduction

The amphotropic packaging cell line Phoenix was transfected with retroviral pLNCX and Wnt-1 /pLNCX plasmid DNA by using the calcium phosphate precipitation to generate the retrovirus. M24met melanoma cells stably infected with Wnt-1 were treated with 600 μg ml and A375 cells with 1300 μ^πύ Geneticin (G-418, Biochrom AG), which is a selection marker. In a second step, M24met and A375 melanoma cells overexpressing Wnt-1 or the empty pLNCX vector were double infected with a second empty vector (pBMN-I-GFP), ANTcf-4- or DKK-l-pBMN-I-GFP and sorted for GFP positive cells.

Example 5: Transient transfection and luciferase reporter assay

For transient transfections, A375 melanoma cells were seeded in 6 well plates and were allowed to reach 80%-90% confluence. Next day, 1 ml OptiMeM (GIBCO) was mixed with 4 μg plasmid DNA and 6 μΐ Lipofectamine 2000 (Invitrogen) and incubated for 20 min at RT. Culture medium was removed, the cells were washed once with PBS (BioWhittaker) and the transfection precipitate was added to the cells. 5-6 hours after incubation at 37 °C, the transfection mix was replaced by fresh culture medium and the cells were harvested after 72 h.

For the luciferase assay, 24 h before transfection, A375 cells were seeded into 12 well plates and grown to 80-90% confluence. Next day, the cells were transfected with 500 ng of reporter construct (SuperTOP, NFAT, NFKB or AP-1 plasmid DNA), 500 ng plasmid DNA (empty vectors, Wnt-1, Wnt-5a or dnNFAT) and 1 ng Renilla using Lipofectamine 2000. 30 h after transfection cells were stimulated with DMSO, ionomycin, PMA, LiCl, TNF- for 16 h as indicated in the respective experiments. 48 h after transfection cells were lysed in 150 μΐ passive lysis buffer (1 x PLP, Promega) provided with the Dual Luciferase Assay system kit (Promega). Luciferase activity was measured with Berthold Centro LB 960 luminometer and monitored with MikroWin 2000 software. Data were reported as normalized averages of the luciferase/renilla ratio.

Example 6: SCID mouse model

All animal procedures were approved by the animal care and use committee of the Medical University of Vienna. This xenograft model was described previously (Loewe, Cancer Res (2006), 66: 1 1888-11896). Briefly, M24met (1 x 10⁶) or A375 (2 x 10⁶) human melanoma cells suspended in 50 μΐ PBS were injected intradermally into the right flank of 6-weak old female CB17 SCID mice (Charles River Laboratories, Germany). Tumor sizes were measured daily by using a calliper and were calculated by using the equation V = (^6)x(length)x(width)². Tumors were excised under ketamine anaesthesia after reaching a volume of 400 mm³. Defects were sutured by staples. Excised tumors were divided; the 1^st part was transferred into RNAlater (Ambion) for RNA analysis, the 2^nd fixed in 4% formaldehyde for paraffin sections, the 3^rd embedded in O.C.T. (Sakura) for cryostat sections. Subsequently, animals were monitored daily for development of palpable metastases in sentinel nodes (right axillary nodes). In the event of the first palpable macrometastasis in one group, all mice of this group were sacrificed and axillary and inguinal lymph nodes and lungs were harvested, divided and either fixed in formaldehyde or transferred into RNAlater. When indicated, animals were treated with CsA (Kwizda, 50 mg/kg/day) via a gauge starting at the day of tumor grafting and ending with removal of the lymph nodes.

Example 7: Immunohistochemistry

For histological studies paraffin embedded primary tumors and tissue arrays were stained with H&E (hematoxylin and eosin). 5 μηι sections of paraffin-embedded material were de- paraffmized, incubated in citrate buffer (Target Retrieval solution pH 6.0, Dako) and autoclaved for 10 min at 1 bar. After incubation in Real Peroxidase blocking solution (Dako), sections were incubated with indicated primary antibodies, followed by appropriate biotinylated secondary antibodies. Bound antibodies were visualized by using a horseradish- peroxidase conjugated Streptavidin kit (Novocastra) and counterstained with hematoxylin (Merck). Wnt-1 staining was quantified by two individuals blinded to the conditions based on the criteria present (unequivocal staining of >5% of melanoma or nevus cells) or absent (<5% of cells) as compared to isotype control. For CD31 staining, cryostat sections of O.C.T. embedded frozen material was used. For quantification of numbers of CD31⁺ and Lyve-1⁺ vessels, 5 random fields (0.02 mm²) were photographed using an AxioCam MRc5 digital camera (Zeiss) attached to an AH3-RFCA microscope (Olympus) and the Axio Vision Rel 4.4 Software (Zeiss). Positively stained areas were quantified by using the semi-automatic program Image Scope (Aperio Technologies) and were expressed as a percentage of the total area.

Example 8: Affymetrix chips and Real-time PCR

Human samples and SCID mouse-derived samples were homogenized with RLT lysis buffer, supplemented with Mercaptoethanol (Merck) using a mixer mill MM200 (4 min, 20 Hz, Mixer Mill MM200, Retsch). Cultured melanoma cells were lysed in RLT lysis buffer and homogenized using a QIAshredder spin column (Qiagen). Total RNA was extracted using the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. For Affymetrix® chips, 5 μg of total RNA were reverse transcribed into second strand cDNA (Affymetrix®) according to the manufacturer's instructions. Preparation of cRNA, hybridization to Human Genome U133 Plus 2.0 and scanning of the arrays were carried out according to the manufacturer's protocols (http://www.affymetrix.com). RNA signal extraction and normalization was performed as described (http://www.bioconductor.org).

To calculate differential expression between the individual sample groups, statistical comparisons are performed using the limma package implemented in the Bioconductor suite (http://www.bionconductor.org), which estimates the fold change between predefined sample groups by fitting a linear model and using an empirical Bayes method to moderate the standard errors of the estimated log fold change for each probe set. An absolute log fold change cut off of 1 was chosen and a multiple testing correction based on the false discovery rate was performed to produce adjusted p-values (http://www.jstor.org/pss/2346101).

For real-time PCR, RNA (1-3 μg) was reverse transcribed into first strand cDNA with random hexamer primers using the Revert Aid H First Strand cDNA synthesis kit (Fermentas) according to the manufacturer's instructions. The real-time PCR primer sets used were purchased from Applied Biosystems (Assays-on-demand, FAM primers: Axin-2 (Hs01063168_ml); 2-microglobulin (Mm00437762_ml); CD144 (Mm00486938_ml); CD31 (Mm01242584_ml); GAPDH (Hs99999905_ml); IL-8 (Hs00174103_ml); Lyve-1 (Mm00475056_ml); Prox-1 (Mm00435969_ml); VEGF-C (Hs00153458_ml); Wnt-1 (Hs001 80529_ml); Wnt-3a (Hs01055707_ml); Wnt-5a (Hs00180103_ml). A reaction mixture contained 1 μΐ cDNA, 12.5 μΐ TaqMan Universal PCR Master Mix (Applied Biosystems), and 1.25 μΐ Assay-on-demand in a total volume of 25 μΐ. The following cycling parameters were applied: initial denaturation at 50 °C for 2 min and then at 95 °C for 10 min followed by 40 cycles: 95 °C, 10 sec; 60 °C, 1 min. Each cDNA sample was analyzed in duplicates and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for human primers and 2-microglobulin for mouse primers were used as housekeeping genes. Reactions were run on StepOnePlus RT-PCR System (Applied Biosystem) and data analysis was done with the StepOnePlus Software v2.0 (Applied Biosystems). Changes in mRNA concentration were determined by subtracting the CT (cycle threshold) of the house keeping gene from the CT of the target gene (Δ = CT gene - CT GAPDH). The mean of Δ control was subtracted from the Δ target gene reaction (AACT = A target gene - mean Δ control). The difference was calculated as 2^{a(" CT)}.

Example 10: Westernblot

Control and Wnt-1 stable A375 cells were seeded in serum free medium for 24 h. Serum free cell supernatant was precipitated with 100% ethanol, incubated at -20 °C over night and the precipitate was harvested by centrifugation at 15000 g at 4 °C for 10 min. After washing in cold 70% ethanol, the pellet was resolubilized in sample buffer (7.5 M urea; 1.5 M thiourea; 4% CHAPS; 0.05%> SDS). 6 x SDS page loading buffer was added and the mixture was loaded onto a 10% SDS page Gel and blotted onto a PVDF membrane (Biorad). Goat anti- human VEGF-C (R&D systems) and HRP rabbit anti-goat IgG (Zymax) were used as antibodies. Example 11: Statistical analysis

All experiments were repeated at least twice and done in triplicates. Data are expressed as mean ± SD, and the statistical significance of differences was assessed by Student's two- tailed test. Differences in Wnt-1 expression in human tissues were calculated by using the Fisher's exact test (Figure 1A, bar on the lower right). p<0.05 was considered statistically significant. In Figure I B, data were analyzed by a Mann- Whitney test and p-value was corrected by a false discovery rate.

Example 12: Semi quantitative RT-PCR

2 μg of total RNA were transcribed into first strand cDNA. Semi-quantitative PCR amplification (45 s 96 °C, 1 min 55-63 °C, 1 min 72 °C) was performed with PCR reagent system (Invitrogen) using 0.5 U Taq polymerase (Invitrogen). Sequences of the primers are indicated in Table 1. Cycle numbers were additionally optimized within the linear range of individual PCR reactions. GAPDH (5'- CCATGGAGAAGGCTGGGG-3 ' , sense primer; 5'- CAAAGTTGTCATGGATGACC-3', anti-sense primer) was used as a loading control (185 bp). All PCR products were analyzed on 1.5% agarose gel containing ethidium bromide and photographed under UV radiation.

Table 1 : Primers used for RT-PCR

Protein Forward primer (5'-3') Reverse primer (5'-3')

Wnt-1 CACGACCTCGTCTACTTCGAG ACAGACACTCGTGCAGTACGC

Wnt-2 CCAGCCTTTTGGCAGGGTC GCATGTCCTGAGAGTCCATG

Wnt-2b AAGATGGTCCCAACTTCACCG CTGCCTTCTTGGGGGCTTTGC

Wnt-3 TGAACAAGCACAACAACGAG CAGTGGCATTTTTCCTTCC

Wnt-3a CAGGAACTACGTGGAGATCATG CCATCCCACCAAACTCGATGTC

Wnt-4 CCTTCTCACAGTCGTTTG CACAGCCGTCGATGGCCTT

Wnt-5a GGGAGGTTGGCTTGAACATA GAATGGCACGCAATTACCTT

Wnt-6 TGGTGCTGCGTAGTACAGTGC CCATCCTGTGGCCAGCAGTTC

Wnt-7a GCTGCCTGGGCCACCTCTTTCTCA CCCGGTGGTACAGGCCTTGCTTCT

Wnt-8b ATGTCTTTGGGGTTGGTTCCTAG TTGCTAGGAGGAAGAAGGTCAG

Wnt-1 Ob GAATGCGAATCCACAACAACAG TTGCGGTTGTGGGTATCAATGAA

Wnt-1 1 GTAAGTGCCATGGGGTGTCT GCTTCCAAGTGAAGGCAAAG

Wnt-16 TGCTCGTGCTGTTCCCCTAC ATCATGCAGTTCCATCTCTC

Fzd-1 GCCCATGAGCCCGGACTTCAC TCAGACTGTAGTCTCCCCTTG

Fzd-3 AAGGCTTCCACAGTGACACAAGG AGAGGAGAGAAAC C CCAACTAC CAC

Fzd-4 GGAAATGGTTGGGTGAAGCCTG TTTTTGATGCTGGGGTCGGG

Fzd-5 GGGCCCGTTCGTGTGCAAGTGTCG ATGCCGGCGGCCAGGAACCAG

Fzd-6 AGTCTTCAGCGGCTTGTATCTTGT GCTCCGTCCGCTTTCACCTCT Fzd-7 ACAGACTTAGCCACAGCAGCAAGG TTTCCAAATCACCCCTCGCC

Fzd-9 TTCTTCTCCACCGCCTTCAC CAGGATGACGATGGTCTTGAG

Fzd-10 TATCGGGCTCTTCTCTGTGC GACTGGGCAGGGATCTCATA

Example 13: Gene expression micro arrays

For Affymetrix® chips, 5 μg of total RNA were reverse transcribed into second strand cDNA (Affymetrix®) according to the manufacturer's instructions. Preparation of cRNA, hybridization to Human Genome U133 Plus 2.0 and scanning of the arrays were carried out according to the manufacturer's protocols (http://www.affymetrix.com). RNA signal extraction and normalization was performed as described (http://www.bioconductor.org). To calculate differential expression between the individual sample groups, statistical comparisons are performed using the limma package implemented in the Bioconductor suite (http://www.bionconductor.org), which estimates the fold change between predefined sample groups by fitting a linear model and using an empirical Bayes method to moderate the standard errors of the estimated log fold change for each probe set. An absolute log fold change cut off of 1 was chosen and a multiple testing correction based on the false discovery rate was performed to produce adjusted p-values (Benjamini, J R Statist Soc B (1995), 57: 289-300).

Example 14: Human melanoma expresses Wnt-1

In nevi, Wnt-1 expression was present in approximately 20% of cases, mostly in nests within the epidermis or papillary dermis (an example for weak Wnt-1 expression is shown in Figure 1A by arrows) and was mostly absent in the deeper dermis. In 32% of primary melanomas (p<0.05 as compared to nevi) and in 28% of melanoma metastases, Wnt-1 expression was detectable. Arrows in Figure 1A denote areas with strong Wnt-1 expression, present in less than 5% of cases (bar graph of Figure 1A). In the majority of samples, Wnt-1 expression was weak (Figure 1 A). Results of real-time PCR are shown in Figure IB. Therein, it is confirmed that Wnt-1 can be found expressed in melanoma. Of note, a direct comparison to immunohistochemistry (IHC) and mRNA data is obsolete because of the nature of samples, which for mRNA detection are tissue homogenates containing variable amounts of tumor and stroma. Wnt-3a was negligible in nevi and even more in primary melanoma as compared to nevi, and absent in metastasis. With regard to Wnt-5a, robust expression in nevi, primary melanoma and melanoma metastasis was confirmed (Bittner, Nature (2000), 406: 536-540; Da Forno, Clin Cancer Res (2008), 14: 5825-5832; Weeraratna, Cancer Cell (2002), 1 : 279- 288). Next serial sections of human melanoma were stained for Wnt-1 and VEGF-C and it was found that areas of high VEGF-C levels were mostly linked to areas with low Wnt-1 levels and vice versa (Figure 1C). Numbers of specimen with strong Wnt-1 expression as depicted in Figure 1C (bottom right) were rare (~ 5%). Example 15: Wnt-1 overexpression delays metastasis

To test biological effects of Wnt-1 in melanoma, Wnt-1 negative melanoma cell lines A375 and M24met were selected and Wnt-1 was stably overexpressed. For expression of other Wnt proteins in A375 cells, see the quantitative RT-PCR analysis in Figure 7. Following orthotopical grafting into the mouse dermis, both, A375 and M24met cells, formed tumors, colonized the mouse epidermis and expressed Wnt-1 protein (Figure 2A). Following primary tumor excision at a mean tumor volume of 400mm³, animals were monitored for sentinel lymph node metastasis. In A375 tumors, macro-metastasis occurred after 18 days in tumors expressing the control vector, whereas in Wnt-1 overexpressing A375 cells, sentinel lymph nodes were absent over an observation period of 90 days (Figure 2B). Also histological serial sections of liver, lung and brain did not reveal any metastasis.

Example 16: Wnt-1 overexpression reduces lymphangiogenesis

Histological evaluations of H&E-stained primary melanomas revealed numerous lymphatic vessels stuffed with tumor cells in control tumors (Figure 3 A, arrow), whereas in Wnt-1 positive tumors, lymphatic vessels were small spaced and mostly devoid of luminal melanoma cells (arrow, Figure 3A). Quantification of lymph vessel density by immunohistochemistry revealed a significant reduction of Lyve-1 and CD31 positive vessels in Wnt-1 expressing tumors (Figure 3A). Results were confirmed by real-time PGR. The amount of lymphatic markers Lyve-1 and Prox-1 were significantly reduced in the Wnt-1⁺ tumors compared to controls (Figure 3B for M24met and Figure 3C for A375). Also mRNA expression of the pan-endothelial marker CD31 was significantly reduced in the Wnt-1⁺ tumors compared to controls tumors (Figure 3B for M24met, Figure 3C for A375). This was largely due to the reduction in lymphatic vessels, because blood vessel markers like CD34, CXCR4 and N-cadherin were not downregulated in the Wnt-1⁺ tumor cells (data not shown). Data mining in Affymetrix® mRNA expression arrays revealed a single best correlation between VEGF-C expression and reduced lymphangiogenesis in Wnt-1 expressing melanoma (see Figure 8). Results were confirmed by real-time PCR. VEGF-C was significantly reduced in both, Wnt-1 expressing primary M24met and A375 tumors (Figure 3B for M24met and Figure 3C for A375).

Example 17: Wnt-1 reduces VEGF-C expression in a β-catenin independent manner

By Luciferase assays using SuperTop, NFAT, AP-1 and NFKB reporter constructs transfected into cultured A375 cells we investigated which pathway was activated by Wnt-1. As expected, Wnt-1 predominantly activated the canonical Wnt pathway, but also the non- canonical NFAT pathway. The AP-1 and the NFKB pathways were not triggered by Wnt-1 (Figure 4A). Similar to the in vivo tumors, effects of Wnt-1 on VEGF-C mRNA and protein expression were reproducible in cell culture: stable as well as transient overexpression of Wnt-1 significantly reduced VEGF-C expression (protein as well as mRNA) in melanoma cells (Figure 4B and 4C). This effect was not a canonical signal (i.e., not β-catenin- dependent), as concomitant overexpression of ANTcf-4 (dominant negative Tcf-4 binding DNA but lacking the β-catenin-binding site; Figure 4B) or DKK-1 (an inhibitor of the canonical Wnt pathway) did not neutralize Wnt-1 -induced reduction of VEGF-C (Figure 4B). Of note, Wnt-5a, tested for comparison as an established melanoma progression protein, did not affect VEGF-C levels (Figure 4C). To exclude further that the canonical Wnt pathway was involved in reduced VEGF-C expression, LiCl and SB-415286 (a specific GSK-3 inhibitor) which both did not lead to reduced VEGF-C expression were used, excluding further the canonical Wnt pathway for this inhibitory VEGF-C effect. For control, the β- catenin target Axin-2 was analyzed and it was found that it was up-regulated in response to GSK-3 inhibition (Figure 4D).

Example 18: Wnt-1 reduces VEGF-C expression in a calcineurin dependent manner

To test whether Wnt-1 reduces VEGF-C in a calcineurin dependent fashion, ionomycin was used, a selective calcium ionophore agent which activates the Ca /Calmodulin dependent kinase, and a dose dependent reduction of VEGF-C mRNA was found (Figure 5A). Ionomycin at a concentration of 8 μΜ reduced VEGF-C at the same level as Wnt-1 in the A375 ceils. Wnt-1 plus ionomycin did not show an additive effect. For control purposes, mRNA expression of a calcineurin/NFAT target gene - IL-8 - was analyzed and was found up-regulated after ionomycin stimulation (Figure 5A). To further confirm the role of calcineurin, the calcineurin inhibitor Cyclosporine A (CsA) was added and it restored VEGF- C expression levels in Wnt-1 expressing melanoma (Figure 5B). To analyze if Wnt-1 suppressed VEGF-C in a calcineurin/NFAT-dependent manner, a dominant negative NFAT construct (dnNFAT) was used, but this did not affect VEGF-C, indicating that Wnt-1 did not exert its effects on VEGF-C via NFAT (Figure 5C). The functionality of this dnNFAT construct was confirmed by luciferase assays using an NFAT luciferase reporter (Figure 5C). From these experiments, it can be concluded that Wnt-1 triggers the reduction of VEGF-C by activation of calcineurin but not by NFAT transcription.

Example 19: Cyclosporine A (CsA) annihilates the anti-Iymphangiogenic effects of Wnt- 1

To validate that activation of calcineurin-dependent signals were responsible for decelerated lymphangiogenesis and reduced metastasis, animals with A375 control and A375-Wnt-1 cells were fed with 50 mg/kg/day CsA via a gauge. CsA had no effect on growth of the primary tumors (Figure 6A). Importantly, in CsA fed animals, the beneficial effect of Wnt-1 was abolished. In the presence of CsA, Wnt- 1⁺ melanoma metastasized to sentinel nodes as seen in control melanoma (Figure 6A). Moreover, lymphangiogenesis as measured by Lyve-1 and Prox-1 mRNA expression as well as VEGF-C expression was increased by CsA in Wnt-1 ⁺ melanoma to the same level as seen in controls (Figure 6B). In other words, CsA abolished the anti-lymphangiogenic effect of Wnt-1.

Example 20: Effects of Wnt-1 on VEGF-C expression in primary human melanoma cell lines

Next, a set of primary human melanoma cell lines was screened for VEGF-C expression and overexpressed Wnt-1 or treated with ionomycin. In addition to A375 and M24met cells, cell lines were found exhibiting constitutive expression of high amounts of VEGF-C that responded to Wnt-1 and ionomycin by a reduced VEGF-C expression (Table 2). In particular, cell lines with CT values for VEGF-C being <24 were found to respond to Wnt-1 and ionomycin by reduced VEGF-C expression. Human melanoma lines with moderate or low VEGF-C levels did not respond to Wnt-1 or ionomycin.

Table 2: Effects of Wnt-1 on VEGF-C expression in primary human melanoma cell lines

VEGF-C mRNA expression was analyzed by real-time PCR in primary human melanoma cell lines, transiently transfected with Wnt-1 or stimulated with ionomycin for 16 h compared to strong inhibition (90%) of VEGF-C in A375 and M24met cells (75%) stably transfected with Wnt-1 ; cf. also Figure 4B). In all experiments, GAPDH was used as an internal control. The CT values of VEGF-C and GAPDH from all cell lines as well as the % of inhibition of VEGF-C mRNA after Wnt-1 transfection or after ionomycin stimulation are listed.

CT values

VEGF-C/ % of inhibition of VEGF-C mRNA BRAF status NRAS status

GAPDH

stable Wnt-1

overexpression

A375 90% BRAF^V6UOk NRAS Q61 wt

M24met 75 BRAF^wt,wt _{N RAS}061R/wt transient Wnt-1 ionomycin

overexpression stimulation

A375 20/11 40% 50% BRAF^VB0Ub NRAS Q61 wt

M24met 21/12 30% 0 _{B RAF}wt/wt NRAS^U61K/Wt

VM-54 22/14 20% 60% BRAF^veuot NRAS Q61 wt

VM-7 23/13 30% 55% BRAF ^600E,wt NRAS Q61 wt

VM-15 23/14 0 0 _{B RA}pwt/wt _{N RAS}U61K/wt

VM-47 24/13 0 0 _BRApwt/wt NRAS Q61 wt

VM-24 25/13 0 0 _{B RAF}V6U0_t NRAS Q61 wt

VM-44 26/15 0 0 g_RApV600E/wt NRAS Q61 wt

VM-30 27/12 0 0 _{B RAF}V600E

NRAS Q61 wt

VM-21 28/13 0 0 BRAF^VBUUt NRAS Q61 wt

VM-48 31/12 0 0 _BRAFv_Buu_t NRAS Q61 wt

VM-1 32/13 0 0 _BRAFV600E/wt NRAS Q61 wt VM-14 32/12 0 0 _{B RA}pV600E/wt NRAS Q61 wt

VM-10 33/13 0 0 BRAF^V6U0t NRAS Q61 wt

Also, Wnt-1 stable M24met cells were analyzed for VEGF-C expression. VEGF-C expression was more reduced in stable M24met than in Wnt-1 transient transfected M24met ; cf. results for A375.

Example 21: Wnt-5a does not reduce VEGF-C expression

Wnt-1 suppresses VEGF-C even in the presence of DKK-1 (inhibitor of beta-catenin dependent signaling). Together with the effects of ionomycin, which activates calcineurin and reduces VEGF-C and the inhibition of Wnt-1 -induced VEGF-C repression by CsA, these data suggest that Wnt-1 acts through calcineurin. The role of calcineurin in Wnt-1 -reduced VEGF- C expression raises the question, whether Wnt-5a, also an inducer of calcineurin signaling, was unable to reduce the VEGF-C expression. As shown in Figure 9, Wnt-5a does not reduce VEGF-C expression. In the experiments provided herein glycogen synthase kinase-2p (GSK3P) has been excluded to be the basis of this difference. GSK3¾ is strongly targeted by Wnt-1 and not or only weakly by Wnt-5a. GS P. activates not only β-catenin, but may also target NFAT and NFKB. However, LiCl and SB415286, inhibitors of GSK3¾ did not reduce VEGF-C expression (see Figure 4D). Moreover, Wnt-1 did not alter NFKB reporter expression levels in melanoma cells (see Figure 4A) and a dnNFAT construct did not alter VEGF-C expression in Wnt-1 positive melanoma cells (see Figure 5C). The lack of efficacy of dnNFAT in combination with the clear role of calcineurin in the Wnt-1 -induced VEGF-C inhibition deserves additional comments: In the experiments provided herein CsA restores high VEGF-C levels in melanoma cells with a delay of 48h (see Figure 5B) and the concentrations of ionomycin required to reduce VEGF-C were rather high (see Figure 5A). This delay in VEGF-C responses is also highlighted by the difference in VEGF-C inhibition in transient (50% inhibition) versus stable (90% inhibition) Wnt-1 overexpressing melanoma (see Figures 4B and C). This delayed response between stimulus and VEGF-C mRNA expression is in line with a concept that calcineurin does not reduce VEGF-C expression via a NFAT-dependent transcriptional regulation at the level of the VEGF-C promoter. An indication for a potential protective role of calcineurin in cancer is considered in a previous report showing that 'hyper-activated' calcineurin inhibited the formation of an effective tumor vasculature which was restored by CsA (Ryeom (2008) Cancer Cell, 13: 420-431), but lymph-angiogenesis was not evaluated. Thus, one could speculate that Wnt-1 mimics 'hyper- activated' calcineurin. Sequences referred to in the present application

SEQ ID NO: 1

cDNA human Wnt-1

nucleic acid sequence, homo sapiens

ATGGGGCTCTGGGCGCTGTTGCCTGGCTGGGTTTCTGCTACGCTGCTGCTGGCGCT

GGCCGCTCTGCCCGCAGCCCTGGCTGCCAACAGCAGTGGCCGATGGTGGGGTATT

GTGAACGTAGCCTCCTCCACGAACCTGCTTACAGACTCCAAGAGTCTGCAACTGG

TACTCGAGCCCAGTCTGCAGCTGTTGAGCCGCAAACAGCGGCGTCTGATACGCCA

AAATCCGGGGATCCTGCACAGCGTGAGTGGGGGGCTGCAGAGTGCCGTGCGCGA

GTGCAAGTGGCAGTTCCGGAATCGCCGCTGGAACTGTCCCACTGCTCCAGGGCCC

CACCTCTTCGGCAAGATCGTCAACCGAGGCTGTCGAGAAACGGCGTTTATCTTCG

CTATCACCTCCGCCGGGGTCACCCATTCGGTGGCGCGCTCCTGCTCAGAAGGTTC

CATCGAATCCTGCACGTGTGACTACCGGCGGCGCGGCCCCGGGGGCCCCGACTGG

CACTGGGGGGGCTGCAGCGACAACATTGACTTCGGCCGCCTCTTCGGCCGGGAGT

TCGTGGACTCCGGGGAGAAGGGGCGGGACCTGCGCTTCCTCATGAACCTTCACAA

CAACGAGGCAGGCCGTACGACCGTATTCTCCGAGATGCGCCAGGAGTGCAAGTG

CCACGGGATGTCCGGCTCATGCACGGTGCGCACGTGCTGGATGCGGCTGCCCACG

CTGCGCGCCGTGGGCGATGTGCTGCGCGACCGCTTCGACGGCGCCTCGCGCGTCC

TGTACGGCAACCGCGGCAGCAACCGCGCTTCGCGGGCGGAGCTGCTGCGCCTGG

AGCCGGAAGACCCGGCCCACAAACCGCCCTCCCCCCACGACCTCGTCTACTTCGA

GAAATCGCCCAACTTCTGCACGTACAGCGGACGCCTGGGCACAGCAGGCACGGC

AGGGCGCGCCTGTAACAGCTCGTCGCCCGCGCTGGACGGCTGCGAGCTGCTCTGC

TGCGGCAGGGGCCACCGCACGCGCACGCAGCGCGTCACCGAGCGCTGCAACTGC

ACCTTCCACTGGTGCTGCCACGTCAGCTGCCGCAACTGCACGCACACGCGCGTAC

TGCACGAGTGTCTGTGA

SEQ ID NO: 2

protein human Wnt-1

amino acid sequence, homo sapiens

MGLWALLPGWVSATLLLALAALPAALAANSSGRWWGIVNVASSTNLLTDS SLQLV

LEPSLQLLSRKQRRLIRQNPGILHSVSGGLQSAVRECKWQFRNRRWNCPTAPGPHLFG

KIVNRGCRETAFIFAITSAGVTHSVARSCSEGSIESCTCDYRRRGPGGPDWHWGGCSD

NIDFGRLFGREFVDSGE GRDLRFLMNLHNNEAGRTTVFSEMRQECKCHGMSGSCT

VRTCWMRLPTLRAVGDVLRDRFDGASRVLYGNRGSNRASRAELLRLEPEDPAHKPP

SPHDLVYFEKSPNFCT YSGRLGT AGTAGRACNS S SP ALDGCELLCCGRGHRTRTQRV

TERCNCTFHWCCHVSCRNCTHTRVLHECL

SEQ ID NO: 3

cDNA human Wnt-1 without signal peptide sequence

nucleic acid sequence, homo sapiens

GCCAACAGCAGTGGCCGATGGTGGGGTATTGTGAACGTAGCCTCCTCCACGAACC

TGCTTACAGACTCCAAGAGTCTGCAACTGGTACTCGAGCCCAGTCTGCAGCTGTT

GAGCCGCAAACAGCGGCGTCTGATACGCCAAAATCCGGGGATCCTGCACAGCGT

GAGTGGGGGGCTGCAGAGTGCCGTGCGCGAGTGCAAGTGGCAGTTCCGGAATCG

CCGCTGGAACTGTCCCACTGCTCCAGGGCCCCACCTCTTCGGCAAGATCGTCAAC CGAGGCTGTCGAGAAACGGCGTTTATCTTCGCTATCACCTCCGCCGGGGTCACCC

ATTCGGTGGCGCGCTCCTGCTCAGAAGGTTCCATCGAATCCTGCACGTGTGACTA

CCGGCGGCGCGGCCCCGGGGGCCCCGACTGGCACTGGGGGGGCTGCAGCGACAA

CATTGACTTCGGCCGCCTCTTCGGCCGGGAGTTCGTGGACTCCGGGGAGAAGGGG

CGGGACCTGCGCTTCCTCATGAACCTTCACAACAACGAGGCAGGCCGTACGACCG

TATTCTCCGAGATGCGCCAGGAGTGCAAGTGCCACGGGATGTCCGGCTCATGCAC

GGTGCGCACGTGCTGGATGCGGCTGCCCACGCTGCGCGCCGTGGGCGATGTGCTG

CGCGACCGCTTCGACGGCGCCTCGCGCGTCCTGTACGGCAACCGCGGCAGCAACC

GCGCTTCGCGGGCGGAGCTGCTGCGCCTGGAGCCGGAAGACCCGGCCCACAAAC

CGCCCTCCCCCCACGACCTCGTCTACTTCGAGAAATCGCCCAACTTCTGCACGTAC

AGCGGACGCCTGGGCACAGCAGGCACGGCAGGGCGCGCCTGTAACAGCTCGTCG

CCCGCGCTGGACGGCTGCGAGCTGCTCTGCTGCGGCAGGGGCCACCGCACGCGC

ACGCAGCGCGTCACCGAGCGCTGCAACTGCACCTTCCACTGGTGCTGCCACGTCA

GCTGCCGCAACTGCACGCACACGCGCGTACTGCACGAGTGTCTGTGA

SEQ ID NO: 4

protein human Wnt-1 without signal peptide sequence

amino acid sequence, homo sapiens

ANSSGRWWGIVNVASSTNLLTDS SLQLVLEPSLQLLSRKQRRLIRQNPGILHSVSGG

LQSAVREC WQFRNRRWNCPTAPGPHLFGKIVNRGCRETAFIFAITSAGVTHSVARS

CSEGSIESCTCDYRRRGPGGPDWHWGGCSDNIDFGRLFGREFVDSGEKGRDLRFLMN

LHNNEAGRTTVFSEMRQEC CHGMSGSCTVRTCWMRLPTLRAVGDVLRDRFDGAS

RVLYGNRGSNRASRAELLRLEPEDPAHKPPSPHDLVYFEKSPNFCTYSGRLGTAGTA

GRACNSSSPALDGCELLCCGRGHRTRTQRVTERCNCTFHWCCHVSCRNCTHTRVLH

ECL

Claims

1. Polynucleotide for use in treating or preventing a disease or disorder related to lymphangiogenesis or for use in preventing metastasis, wherein said polynucleotide is selected from the group consisting of:

2. A polypeptide encoded by the polynucleotide of claim 1 for use in treating or preventing a disease or disorder related to lymphangiogenesis or in preventing metastasis.

3. A vector comprising the polynucleotide of claim 1 for use in treating or preventing a disease or disorder related to lymphangiogenesis or in preventing metastasis.

4. A host cell comprising the polynucleotide of claim 1 or the vector of claim 3 for use in treating or preventing a disease or disorder related to lymphangiogenesis or in preventing metastasis.

5. The host cell for use according to claim 4 which is selected from the group consisting of fibroblast and epithelial cell.

6. A method for screening an agent capable of increasing the expression of the polynucleotide of claim 1 and/or the polypeptide of claim 2, wherein said method comprises

(a) contacting the agent to be screened with a sample containing a nucleic acid molecule comprising the polynucleotide of claim 1 and/or containing the host cell of claim 4;

(b) measuring

(1) the transcription level of the polynucleotide of claim 1 ,

(2) the amount of transcription product of the polynucleotide of claim 1 ,

(3) the amount of translation product of the transcription product of the polynucleotide of claim 1 , and/or

(4) the protein level of the polypeptide of claim 2; and

7. A method for screening an agent capable of enhancing the activity of the polypeptide of claim 2, wherein said method comprises

(a) contacting the agent to be screened with a sample containing the polypeptide of claim 2;

(b) measuring the activity of the polypeptide of claim 2; and

8. The method according to claim 6 or 7, wherein said method is an in vitro method.

9. An agent for use in treating or preventing a disease or disorder related to lymphangiogenesis or in preventing metastasis, wherein said agent is capable of increasing the expression of the polynucleotide of claim 1 and/or the polypeptide of claim 2, or capable of enhancing the activity of the polypeptide of claim 2.

10. The agent according to claim 9, wherein the agent is screened according to the method of claim 6 or 8, and/or screened according to claim 7 or 8.

11. A pharmaceutical composition comprising the polynucleotide of claim 1, the polypeptide of claim 2, the vector of claim 3, the host cell of claim 4 or 5, and/or the agent according to claim 9 or 10 together with a pharmaceutically acceptable carrier or excipient.

12. A pharmaceutical composition for use in treating or preventing a disease or disorder related to lymphangiogenesis or in preventing metastasis, said pharmaceutical composition comprising the polynucleotide of claim 1, the polypeptide of claim 2, the vector of claim 3, the host cell of claim 4 or 5, and/or the agent according to claim 9 or 10;

together with a pharmaceutically acceptable carrier or excipient.

13. A method for treating or preventing a disease or disorder related to lymphangiogenesis or for preventing metastasis in a subject, said method comprising administering to the subject a therapeutically effective amount of the polynucleotide of claim 1, the polypeptide of claim 2, the vector of claim 3, the host cell of claim 4 or 5, the agent according to claim 9 or 10, or the pharmaceutical composition of claim 11 or 12.

14. The polynucleotide for use according to claim 1, the polypeptide for use according to claim 2, the vector for use according to claim 3, the host cell for use according to claim 4 or 5, the agent for use according to claim 9 or 10, the pharmaceutical composition for use according to claim 1 1 or 12, or the method of claim 13, wherein said polynucleotide, said polypeptide, said vector, said host cell, said agent, or said pharmaceutical composition is to be administered parenterally.

15. The polynucleotide for use according to claim 1 or 14, the polypeptide for use according to claim 2 or 14, the vector for use according to claim 3 or 14, the host cell for use according to any one of claims 4, 5 or 14, the agent for use according to any one of claims 9, 10 or 14, the pharmaceutical composition for use according to claim 12 or 14, or the method of claim 13 or 14, wherein the disease or disorder related to lymphangiogenesis is selected from the group consisting of cancer, melanoma, solid tumor, breast cancer, lung cancer, kidney cancer, pancreatic cancer, hematopoietic tumor, graft rejection, autoimmune disorder, rheumatoid arthritis, psoriasis, oedema and impaired wound healing.

Kit suitable for performing the method according to any one of claims 6 to 8, comprising one or more polynucleotides and/or binding agents capable of hybridizing or binding to the polynucleotide of claim 1 or binding the polypeptide of claim 2.