TW201200151A - Methods and compositions related to reduced MET phosphorylation by leukocyte cell-derived chemotaxin 2 in tumor cells - Google Patents

Abstract

It was discovered that leukocyte cell-derived chemotaxin 2(LECT2) correlated with down-regulated vascular invasion in HCC patients. It was also found that LECT2 strongly reduced the growth, migration and invasiveness of HCC cells via inhibition of the phosphorylation of Met and other downstream targets in the HGF/MET pathway. The HXXXD motif of LECT2 was found to be important for its tumor inhibition mechanism. LECT2 reduced Met tyrosine phosphorylation and tumor cell invasion in other cancers, such as lung, breast, and gastric cancers, in addition to HCC. Methods and compositions for preventing, treating or diagnosing tumors, such as HCC, based on the newly discovered tumor suppression property of LECT2 are described.

Description

201200151 VI. Description of the Invention: [Technical Field of the Invention] The method and composition for reducing the carbonation of a hepatocyte growth factor receptor in a tumor cell by neutrophil leukocyte exporting factor 2 are described. [Prior Art] Hepatocellular carcinoma (HCC) is one of the most prevalent cancer types in the world, with an annual incidence of about one million (see D. Maxwell Parkin, Freddie Bray, Jacques Ferlay, Paola Pisani. 2000) Estimating the world cancer burden: Globocan. IntJ Cancer. 94: 153-6, the entire disclosure of which is incorporated herein by reference. In Taiwan, hepatocellular carcinoma continues to be the leading cause of cancer-related death in men and the second leading cause of cancer-related death in women. The survival rate of the higher-stage HCC within five years, such as Phase II to III, is about 20%, and the survival rate is less than one-third of the first period (ie, about 60%). High cancer recurrence remains the leading cause of death in HCC patients. The main adverse prognostic factors include vascular invasion (see Brian I. (2〇04) Hepatocellular carcinoma: Current management and future trends.

Gastroenterology. 127: S218-24, the entire disclosure of which is incorporated herein by reference. Vascular invasion has been found to be associated with non-vascular invasive HCC patients using high-throughput techniques of human membrane/secretory protein (TMSEC) suppression by subtractive hybridization (SSH) and oligonucleotide microarrays. There are significant differences in the performance of 20 genes in HCC patients. Specifically, neutrophil serotonin 2 (LECT2) (one of 20 existing differential genes in 201200151) has been found in vascular invasive HCC. Patients are subject to down-regulated. The LECT2 protein is isolated from the culture supernatant of phytohemagglutinin-activated human T-cell leukemia SKW-3 cells and serves as a new chemokine for neutrophils (see Yamagoe S, Yamakawa Y, Matsuo Y, Minowada J, Mizuno S, Suzuki K. (1996) Purification and primary amino acid sequence of a novel neutrophil chemotactic factor LECT2. Immunol Lett. 52: 9-13, the entire disclosure of which is hereby incorporated by reference. Since the LECT2 protein has been found to be identical to chondromodulin-II (the growth stimulating factor for chondrocytes), the LECT2 protein can be versatile. LECT2 has been shown to be associated with repair and cell growth after damage (see Toshikazu U, Hisakazu N, Kenichire G. (1999) Expression pattern of a newly recognized protein, LECT2, in hepatocellular carcinoma and its premalignant lesion. Pathol Int. 49: 147-51; and Hisakazu N, Tomomi H, Toshikazu U. (1998) Systemic expression of a newly recognized protein, LECT2, in the human body. Pathollnt. 48: 882-6, the entire contents of which are hereby incorporated by reference. e has shown that the LECT2 line is expressed in hepatocytes (see Yamagoe S, Akasaka T, Uchida T, Hachiya T, Okabe T, Yamakawa Y, Arai T, Mizuno S, Suzuki K (1997) Expression of a neutrophil chemotactic protein LECT2 In human hepatocytes 201200151 revealed by immunochemical studies using polyclonal and monoclonal antibodies to a recombinant LECT2. Biochem Biophys Res Commun. 237(1): 116-20, the entire disclosure of which is hereby incorporated by reference, and -blown) becomes substantially negative in HCC cells (see Uchida T, Nagai H, Gotoh K) , Kanagawa Η, Kouyama Η, Kawanishi Τ, Mima S, Yamagoe S. Suzuki _K, _(.l 999)^Expres.sio_n pattern^ojLa_jLe_wLy_ recognized protein, LECT2, in hepatocellular carcinoma and its premalignant lesion. Pathol Int. 49( 2): 丨 47-51, the entire disclosure of which is hereby incorporated by reference in its entirety, in its entirety, in its entirety, in the in the However, the exact role of LECT2 in cancer development (eg, migration, invasion, and metastasis) remains unreported. Identifying factors that make patients prone to death is important for tumor control. Many molecular factors have been shown to be involved in the invasion of HCC and have potential prognostic implications. These factors include changes in the number of sets of DNA chromosomes, cell proliferation, nuclear morphology, oncogenes and their receptors, such as ras, c-myc, HGF, and Met receptors (see Mann CD, Neal CP, Garcea). G, Manson MM, Dennison AR, Berry DP. (2007) Prognostic molecular markers in hepatocellular carcinoma: a systematic review. Eur J Cancer 43: 979-92, the entire disclosure of which is hereby incorporated by reference.

Met tyrosine kinase (Met or c-Met), also known as hepatocyte growth factor receptor (HGFR), is a disulfide bond that is derived from a single-stranded precursor for proteolysis and 201200151. Heterodimers. The heterodimer is formed by a single transmembrane beta bond (145 kTorr) and a fully extracellular alpha chain (5 gTorr). The extracellular fragment comprises a Sema domain (d〇main), an atypical motif consisting of more than 500 amino acids (m〇tif), which has low affinity binding activity for the ligand. The extracellular portion also includes a cysteine-rich functional domain (CyS domain) called ___Met-related sequence (MRS), and four immunoglobulin structures (IPT domain). For a typical protein-protein interaction zone. The intracellular portion of the receptor consists of a juxtamembrane section, a subsequent catalytic site, and a c-terminal regulatory tail. This membrane proximal segment is important for the regulation of receptor inhibition (see Carmen B, Walter B, Ermanno G, George F, Vande W. (2003) Met, metastasis, motility and more. Nat Rev Mol Cell Bio 4: 915-23, the entire contents of which are incorporated herein by reference. The juxtamembrane segment comprises a linear amino acid residue (eg, Ser 985 in human MET) which, once citrated, is responsible for inhibiting the activity of the receptor kinase and comprising a pathway that binds to the original oncogene product CBL. Aminic acids (eg, Tyr 1003 in human MET) are ubiquitin ligases that promote receptor polyubiquitination and cause Met degradation (see Mak HHL, Peschard P, Lin T, Naujokas MA, Zuo D, Park M, Park DM. (2007) Oncogenci activation of the Met receptor kinase fusion protein, Tpr-Met, concerning exclusion from the ecdocytic degradative pathway. Oncogene 26:7214-21, the entire disclosure of which is hereby incorporated by reference. ). The proximal membrane portion is flanked by the catalytic unit 201200151, which contains two tyrosine acids responsible for modulating the activity of the enzyme (eg, Tyr 1234 and 1235 in human MET). Finally, the C_terminal end contains two tyrosine residues (for example, Tyr 1349 and 1356 in human MET), which, when phosphorylated, create a multifunctional docking site. Call a large group of intracellular adaptors responsible for the transmission of messages via ligand-receptor interactions in cells (see Singleton PA, Salgia R, --Up-Molt Reclamation Sammani S. Mirzapoia7f> Vq) ]:, Garcia JG. (2007) CD44 regulated hepatocyte growth factor-mediated vascular integrity. Role of c-Met, Tiaml/Racl, dynamin 2, and cortactin. J Biol Chem 282: 30643-57, the full text of which is hereby incorporated herein. For reference). These two tyrosine residues have been shown to be necessary and sufficient for performing the physiological functions of Met and causing the cancer potential of Met (see John B. Rafferty, William S. Somers, Isabella).

Saint-Girons, Simon E. V. Phillips. (1989) Three-dimensional crystal structures of Escherichia coli met repressor with and without corepressor. Nature 341: 705-10 ′ This document is incorporated by reference in its entirety.

Met receptors regulate a variety of cellular events ranging from cell motility and angiogenesis to morphological differentiation and tissue regeneration. To facilitate these activities, the C-terminal region of the cytoplasm of the Met receptor serves as a docking site for many protein matrices including Grb2, Gabl, STAT3, She, SHIP-1 and Src. These matrices are characterized by multiple functional domains, including the PH, PTB, SH2, and SH3 domains, which will interact directly with Met's multi-storage C-terminal region (see Victor Martin Bolanos-Garcia, Molecular and Cellular). Biochemistry, 2〇〇5, Vol. 276: 149-157, the entire disclosure of which is incorporated herein by reference. The HGF/c-Met message delivery pathway is now considered a promising target for cancer therapy, for example, to inhibit angiogenesis, tumor growth, invasion, and metastasis (see You WK, McDonald DM. (2008) The hepatocyte growth factor/c-Met signaling pathway as a therapeutic target to inhibit angiogenesis. BMB Rep 4l (12): 833-9, the entire disclosure of which is hereby incorporated by reference. Hepatocyte growth factor (HGF, also known as dispersing factor, SF) is a multifunctional growth factor (see Maulik G, Shrikhande A, Kijima T, Ma PC, Morrison PT, Salgia R. (2002) Role of the Hepatocyte growth factor receptor, c-Met, in oncogenesis and potential for therapeutic inhibition. Cytokine Growth Factor Rev 13:41-59 and Birchmeier C, Birchmeier W, Gherardi E, Vande Woude GF. (2003) Met, metastasis, motility and more Nat Rev Mol Cell Biol 4: 915-25, the entire disclosure of which is hereby incorporated by reference. It is produced as a single chain inactive precursor protein. The mature active HGF is a heterodimer composed of an α-chain subunit and a β-chain subunit, and the heterodimerization system is bonded by a disulfide bond (refer to Nakamura T, Nishizawa). Τ, Hagiya M, Seki Τ, Shimonishi Μ, Sugimura A, Tashiro K, Shimizu S. (1989) Molecular cloning and expression of human 201200151 * hepatocyte growth factor. Nature 342:440-3, the full text of which is hereby reference). The alpha-chain subunit comprises an N-terminal hairpin functional domain and four kringle functional domains. The β-chain subunit is a serine-like protease domain lacking catalytic activity due to mutation of a necessary residue (refer to Gherardi®, Gray J, Stoker®, Perryman M, Furlong R. (1989) Purification of scatter Factor, a fibroblast-derived basic protein that modulates epith. ejlal_lnt^LacJi〇ns_and movement. Proc Natl Acad Sci USA 86:5844-48, the entire disclosure of which is hereby incorporated by reference. Binding of HGF to c-Met causes autophosphorylation of the cytoplasmic domain of Met. Depending on the development of the tumor, activation of Met can trigger different phenotypes (refer to Gao CF, Vande Woude GF. (2005) HGF/SF-Met signaling in tumor progression. Cell Res 15:49-51, the full text of which For reference here). New strategies are needed to diagnose and treat cancer. In particular, it is necessary to investigate the underlying molecular mechanisms of message transduction of cancer cells. The present invention relates to novel methods and compositions for diagnosing and/or inhibiting the invasion of cancer cells based on the signaling mechanism of cancer cell invasion (e.g., reducing MET phosphorylation via an effective amount of LECT2 in tumor cells). SUMMARY OF THE INVENTION In the present invention, it was found that LECT2 significantly reduces the proliferation of tumor cells (such as HCC cells or other tumor cells) through a message-transport pathway involving the Met receptor, for example, by reducing phosphorylation of MET in tumor cells. , 201200151 Migration and / or intrusion. Thus, in one aspect, the invention relates to a method of inhibiting at least one of proliferation, migration and invasion of tumor cells. The method comprises administering to the tumor cell a agent to increase the protein level or biological activity of the LECT2 protein or an active fragment thereof in the tumor cell, thereby reducing phosphorylation of MET in the tumor cell. In a particular embodiment, the invention relates to a method of preventing or treating hepatocellular carcinoma in a body. The method comprises administering to the individual an agent to increase the protein level or biological activity of the LECT2 protein or an active fragment thereof in the hepatocyte cancer cell of the individual, thereby reducing MET acidification in the hepatocellular carcinoma cell. Another aspect of the invention relates to a method of reducing phosphorylation of MET in tumor cells. The method comprises administering to the tumor cell an agent to increase the protein level or biological activity of the LECT2 protein or an active fragment thereof in the tumor cell. Another aspect of the invention relates to a pharmaceutical composition for preventing or treating a tumor. The composition comprises a carrier and a multi-peptide comprising an amino acid sequence of the LECT2 protein or an active fragment thereof. Alternatively, the pharmaceutical composition comprises a carrier and a polynucleotide comprising a nucleotide sequence encoding a LECT2 protein or an active fragment thereof. The multi-peptide system comprising the LECT2 protein or an active fragment thereof has at least one of the ability to reduce phosphorylation of MET in tumor cells and to bind to MET in tumor cells. 201200151 Another aspect of the invention relates to a method of determining the risk of an individual having an increased development of an invasive tumor, the method comprising: (a) obtaining a biological sample from a body; (b) measuring MET phosphate in the biological sample And (c) comparing the degree of phosphorylation measured in step (b) with the extent of the control group; when high, the individual is assessed to have an increased risk of developing an invasive tumor. In a specific embodiment, the diagnostic method comprises sequence analysis and detection of a mutation in the HXXXD motif of LECT2. A further aspect of the invention relates to a kit for diagnosing the development of an invasive tumor having an increased body, the set comprising: (a) for measuring - an individual's creature An agent for the extent of MET phosphorylation in a sample; and (1) an indication of the use of the agent to determine the risk of an individual developing an invasive tumor in a particular embodiment, the set comprising for sequence analysis and The reagent for mutating the HXXXD motif of LECT2 was extracted. The invention relates to a method for preventing or treating invasive tumors, which comprises: (a) an increased degree of acidification based on an individual's biological sample 12 201200151 and a reduced protein level of the LECT2 protein Or biological activity, determining that the individual has an increased risk of developing an invasive tumor; and (b) administering to the individual a agent to increase the protein level or biological activity of the LECT2 protein, thereby reducing the tumor cells of the individual Phosphorylation of MET. Another aspect of the invention relates to a method of identifying a compound useful for inhibiting at least one of proliferation, migration and invasion of tumor cells. The method comprises identifying a compound that enhances the performance or biological activity of LECT2 in a tumor cell, wherein the biological activity is included in the tumor cell to reduce MET acidification. Embodiments of the invention also pertain to an active fragment of an isolated LECT2 protein having at least one of the ability to reduce MET phosphorylation in tumor cells and to bind to MET in tumor cells; and a separation A polynucleotide encoding a polypeptide as described above, a vector comprising the polynucleotide, and a recombinant host cell comprising the vector. The invention also relates to the use of a multi-peptide comprising an amino acid sequence of a LECT2 protein or an active fragment thereof for the preparation of a medicament for inhibiting at least one of proliferation, migration and invasion of tumor cells, wherein The multi-peptide system has at least one of the ability to reduce phosphorylation of MET in tumor cells and to bind to MET in tumor cells. A further aspect of the invention relates to the use of a polynucleotide encoding a polypeptide comprising an amino acid sequence of a LECT2 protein or an active fragment thereof, for use in the manufacture of a medicament for inhibiting tumor cells At least one of proliferation, migration, and invasion, wherein the multi-peptide has at least one of the following: reducing MET phosphorylation in tumor cells and binding to MET in tumor cells. Other aspects, features and advantages of the present invention are disclosed in the following description (including the detailed description of the present invention and its preferred embodiments and the appended claims). The various publications, articles, and patents cited or described in the specification are hereby incorporated by reference. The discussion of documents, acts, materials, equipment, articles, and the like contained in this specification is for the purpose of providing the context of the invention. This discussion is not an admission that any or all of these matters form part of the prior art for any invention disclosed or claimed. All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art of the invention, unless otherwise defined. Unless the specific words used herein have the meaning as defined in the specification. All of the patents, published patent applications and publications cited herein are hereby incorporated by reference. It must be noted that the singular forms "a", "the", "the" and "the" are used in the <RTI ID=0.0> </ RTI> </ RTI> <RTIgt; 201200151 The term "individual" as used in this specification means any animal, particularly a mammal, especially a human, which is a compound or pharmaceutical composition that is or has been administered in accordance with embodiments of the present invention. The term "mammal" as used in this specification includes any mammal. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, etc., preferably humans. Preferably, the individual is a subject in need of treatment or prevention of a tumor, or has been the subject of observation or experimentation in the treatment or prevention of a tumor, and the tumor is characterized by hyperactivation of the Met receptor. The term "biological sample" refers to a sample obtained from an organism (e.g., a patient) or a component (e.g., a cell) derived from an organism. The sample can be any biological tissue, cell or fluid. The sample may be a "clinical sample" derived from a sample of a patient. Such samples include, but are not limited to, sputum, blood, blood cells (eg, white blood cells), amniotic fluid, blood purple, semen, bone marrow, and tissue or fine needle biopsy samples, urine, ascites, and pleural fluid, or from The cells obtained. Biological samples may also include team sections, such as frozen sections obtained for histological purposes. A biological sample can also mean a "patient sample." A "biological sample" may also comprise a substantially purified or isolated protein, membrane preparation, or cell culture. The term "operational instructions" as used in this specification, when used in the context of a package, includes a publication, recorded audio, graphic, or any other presentation medium that can be used to convey the intended use of the set. For example, the action indication can be attached or included in the container of the set. 15 201200151 In one embodiment, "treating" means ameliorating, preventing, or reversing a disease or disorder, or at least one of the apparent symptoms 'e.g., treating HCC by reducing phosphorylation of MET. In another embodiment, "treating" means modifying, preventing, or reversing at least one measurable physical parameter with respect to a disease or disorder being treated, which is not necessarily identifiable in or recognized by the mammal. . In still another embodiment, "treating" means inhibiting or slowing down ϋ or - 失 _ _ _ mil 上 "fc - c such as stable one obvious, _ _ _ shape), or physiologically (for example Stabilize a physical parameter), or both. In still another embodiment, "treating" means delaying the onset of a disease or disorder. In a particular embodiment, the compound of interest is administered as a preventative measure. As used herein, "prevention" means reducing the risk of getting a particular disease or disorder. In a preferred embodiment mode, the indicated compound is administered to a subject as a preventive measure, the individual having a risk of developing an increased tumor (eg, HCC) even if the symptoms of the tumor are not present or Report small. As used herein, "operably linked" means the function of the function between the two-nuclear phytic acid sequences. A single-stranded or double-stranded nucleic acid portion contains a two-stage nuclear (four) sequence arranged in the three portions, arranged in such a manner that the two-stage nuclear acid J to the J-system can exert physiological effects and thereby The other distinguishes. For example, the operability of the B5-(4-)-coded sequence transfer promoter sequence can be a bat paste. The operably linked nucleic acid sequence is typically as many promoter sequences; or non-contiguous, for example, 201200151 in the context of a nucleic acid sequence encoding a repressor protein. In a recombinant expression vector, "operably linked" means that the coding sequence of interest is linked to the regulatory sequence in a manner that allows expression of the coding sequence, for example, in an in vitro transcription/translation system or in an The host cell into which the vector has been introduced. As used herein, "promoter" means a portion of a gene that provides a control point for transcription of a regulated gene; the promoter may comprise a binding site for an RNA polymerase. Promoters can also contain one or more binding sites that can be used for one or more transcription factors. The promoter is usually upstream of the transcription start site ("5' end") of a gene. The promoter is typically adjacent to the transcription initiation site of the gene. However, the promoter may also be located at a distance from the origin of transcription of the gene. "Sequence" means the linear order of the monomers in a polymer, for example, the order of the amino acids in a multi-peptide, or the order of nucleotides in a polynucleotide. "Isolated protein" or "isolated multi-peptide" is substantially separated from at least one other protein present in the natural source of the protein, or when the protein is chemically synthesized, Does not contain at least one of chemical precursors or other chemicals. When preparing a protein, when other proteins or other chemicals (or referred to herein as a "contaminating protein" or "contaminating chemical") are less than about 30%, 20%, 10%, or 5% or less. And preferably less than 1% by dry weight, the protein is "substantially separated" from other proteins or other chemicals or is "substantially free" 17 201200151 Other proteins or other chemicals By. As used in this specification, an "isolated" nucleic acid molecule is substantially separated from at least one other nucleic acid molecule present in the natural source of the nucleic acid molecule, or when the nucleic acid molecule is chemically synthesized, Essentially free of at least one of chemical precursors or other chemicals. The "isolated" nucleic acid molecule can be, for example, a nucleic acid molecule that is substantially free of the genome of the organism, which is the first source of the first nuclear dragon - the molecule - At least one of the nucleotide sequences on the sides of the 5-, 1 and 3' ends. When preparing a nucleic acid molecule, when other nucleic acid molecules or other chemicals (or "contaminated nucleic acid molecules" or "contaminating chemicals" herein) are less than about 30%, 20°/❶, 10〇/ When 〇, or 5% or less, and preferably less than 1% by dry weight, the nucleic acid molecule is "substantially separated" from other nucleic acid molecules or other chemicals or "substantially not" Contains other nucleic acid molecules or other chemicals. As used herein, "recombinant" means a multi-peptide, cell, virion or organism that has been modified by a molecular biotechnology to one of the non-natural states of polynucleic acid, encoded by a polynucleic acid. The vocabulary "neutrophil leukocyte exporting factor 2", "LECT2", and "LECT2 protein" are used interchangeably in this specification, which means a multifunctional protein which can be used as a neutrophil. The factor also stimulates the growth of chondrocytes and osteoblasts, and also refers to polymorphisms and isomers encoded by transcript variants. LECT2, also known as chondromodulin II, has a high degree of sequence similarity to the chondromodulin repeat region of the myb-induced myeloid 1 201200151 protein. Examples of LECT2 include those from various mammals such as humans, monkeys, mice, rats, etc., and the amino acid sequences of such LECT2 can be found in published databases, such as NCBI PubMed. In a specific embodiment of the invention, LECT2 is a human LECT2 comprising the amino acid sequence of SEQ ID No: 2. When used in the present specification '"active fragment of LECT2" means a fragment of LECT2 which still has at least one of the following capabilities: reduction of acidification of MET in tumor cells and binding to MET in tumor cells "LECT2 An example of an active fragment can comprise an amino acid sequence having greater than about 90 or 95% amino acid sequence identity to the sequence of at least five consecutive amino acids of SEQ ID NO: 2. In a preferred embodiment, the "active fragment of LECT2" comprises from 50 to 100 amino acid residues of SEQ ID N::2. In another embodiment, the "active fragment of LECT2" comprises an HXXXD motif. It will be readily understood by those of ordinary skill in the art that based on the disclosure herein, active fragments of LECT2 can be identified using methods known in the art. For example, a LECT2 fragment capable of reducing phosphorylation of MET can be screened in an in vitro MET phosphorylation assay, or a LECT2 fragment capable of binding to MET can be screened in a binding assay. The selected molecules identified in the screening assay can be further tested in a cell based assay and/or an animal model. The words "Met tyrosine kinase", "Met receptor", "Met", "c_Met by 201200151", "c-Met", "hepatocyte growth factor receptor", or "HGFR" are used interchangeably. In the present specification, it means a proto-oncogene product which is a hepatocyte growth factor receptor and has tyrosine kinase activity, and also refers to a polytype and an isomer encoded by a transcriptional variant thereof. The primary single-stranded precursor protein is cleaved after translation to produce alpha and beta subunits, which are disulfide-bonded to form a mature receptor. Examples of METs include those from various mammals, such as human m, monkeys, mice, rats, etc., and the amino acid sequences of these METs can be found in published databases, such as NCBI PubMed. In a particular embodiment of the invention, MET is a human MET comprising an amino acid sequence such as the NCBI reference sequence: NP_000236, NP_000592. The terms "hepatocyte growth factor" and "HGF" are used interchangeably in the present specification, which means that upon binding to the c-Met receptor, it is regulated by activation of a tyrosine kinase message cascade. Cell growth, cell movement, and morphogenetic growth factors, and also refers to polymorphs and isomers encoded by transcript variants. Hepatocyte growth factor is secreted by mesenchymal cells and acts as a multifunctional cytokine that acts on cells of major epithelial origin. It plays a central role in angiogenesis, tumorigenesis, and tissue regeneration due to its ability to stimulate mitosis, cell movement, and matrix invasion. It is secreted by a single inactive polypeptide and cleaved into an alpha bond and an beta chain via a serine protease. The alpha chain and the beta chain are linked by a disulfide bond to produce an active heterodimeric molecule. This protein belongs to the original subfamily of plasmin lysin 201200151 of S1 peptide, but does not have detectable protease activity. Examples of HGF include those from various mammals, such as humans, monkeys, mice, rats, etc., and the amino acid sequences of such HGFs can be found in published databases, such as NCBI PubMed®, which is specific to one of the present inventions. In an embodiment, HGF is a human HGF comprising an amino acid sequence as in GenBank, for example, AAA64297, AAA64239 or AAA52649. As used in this specification, the term "HXXXD motif" means a motif of LECT2 that is conserved between different mammals, which is important for LECT2 to reduce the phosphorylation activity of MET. ^HXXXD motif mutations The biological activity of LECT2 is abolished or substantially reduced. Such mutations include, but are not limited to, mLECT2-l and mLECT2-l as illustrated in Figure 9. It has been found that the performance of 20 genes in vascular invasive HCC patients is significantly different compared to non-vascular invasive HCC patients. In particular, it has been found that the performance of LECT2 is associated with the inhibition of vascular invasion by HCC patients. See International Patent Application No. PCT/CN2009/074086, the entire disclosure of which is incorporated herein by reference. Human LECT2 is a basic protein of 16 thousand Daltons and is specifically expressed in the liver of adults and fetuses. Its biological functions (such as migration, invasion, and metastasis) in tumor development have not been reported. Studies of the present invention indicate that LECT2 significantly reduces tumor growth and invasion in HCC. Activation of the receptor pathway amino acid kinase (RTK) is a key pathogenic event in HCC. The present inventors have discovered that 21 201200151 LECT2 inhibits tumor invasion by inhibiting the acidification of RTKs downstream of MET and HGF/MET signaling pathways. It was further found that LECT2 inhibits HGF-induced Met activation via binding to MET, and the binding site of the binding site to HGF is noncompetitive. Mutations in the HXXXD motif of LECT2 result in the loss of tumor suppressor activity of LECT2. In addition, LECT2 has been found to reduce Met tyrosine phosphorylation and cancer cell invasion in other cancer cells such as lung, breast, and gastric cancer cells. Thus, LECT2 inhibits the invasion of cancer cells and the acidification of the Met receptor, which represents a new therapeutic modality for the treatment of tumors characterized by overactive Met receptor activity. Thus, the invention relates to a method for inhibiting at least one of proliferation, migration and invasion of tumor cells. The method comprises administering to the tumor cell a drug to increase the protein level or biological activity of the LECT2 protein or an active fragment thereof in the tumor cell, thereby reducing phosphorylation of MET in the tumor cell. Those of ordinary skill in the art will readily appreciate that the agent may also reduce phosphorylation of one or more downstream proteins in the HGF/ΜΕΤ message delivery pathway, including but not limited to Erk and Akt. Based on the disclosure herein, various agents can be used to increase the extent or biological activity of a LECT2 protein or an active fragment thereof in a tumor cell. In the embodiment of the present invention, the tumor cells can be administered - a heteropoly group comprising the LECT2 protein or an amino acid phase of the active fragment. Preferably, the polypeptide comprises a LECT2 protein, such as an amino acid sequence having SEq ID N〇: 2.

I 22 201200151 Human LECT 2. In another preferred embodiment of the season, the multi-peptide comprises an active tablet of LECT2 protein #甘上奴' wherein the active fragment has at least one of the following abilities: in tumors, sputum, Phosphorylation of MET is reduced in cells and bound to MET in tumor cells. In the case of - all &amp;# see the court, the active fragment is a fragment of SEq ID NO: 2. In another embodiment, the active fragment comprises the HXXXD motif of LECT2 or a derivative thereof. The multi-peptide can be obtained from a variety of sources. For example, it may be a native LECT2 protein or an active fragment thereof that is endogenously expressed via a mammalian cell and isolated from the cell based on the methods disclosed in the art. Preferably, the multi-success system - through the recombination of the host cells, preferably exhibits a recombination of multiple degrees of performance. Any method of recombinant performance known to those of ordinary skill in the art can be used to generate recombinant Lect2 based on the disclosure herein. The multi-peptide can have several different physical forms. It may be present as a full length nascent or unprocessed peptide, or as a partially processed multi-peptide or as a combination of processed multi-peptides. The full length of the nascent multi-peptide can be post-translationally modified to result in the formation of fragments of the entire length of the nascent peptide via a specific protein cleavage event. A fragment, or a physically ligated fragment, can have biological activity associated with the full length of the peptide, however, the extent of biological activity associated with the individual fragment can vary. The multi-peptide may also be present in a modified form including, but not limited to, a glycosylated form, a myristoylated form, a labeled acid brewing 23 201200151 (palmitoylated) form, a ribosylated form, B. Deuterated forms, ubiquitinated forms, etc. Modifications also include intramolecular cross-linking and covalent attachment to various moieties such as lipids, flavin, biotin, polyethylene glycol or derivatives thereof and the like. In addition, the modifications may also include cyclization, branching, and crosslinking. Further, an amino acid other than the conventional 20 kinds of amino acids encoded by the codon of the gene may be contained in a multi-peptide. In the present invention - the other - a m-like sample, - the protein level or biological activity of the LECT2 protein or an active fragment thereof is increased by the mu-toluene acid. The polynucleotide comprises a protein encoding the LECT2 protein or an active fragment thereof. Nucleic acid sequence. The polynucleotide comprises a regulatory sequence, e.g., a promoter operably linked to the coding sequence of the LECT2 protein or an active fragment thereof such that the LECT2 protein or an active fragment thereof is expressed in the tumor cell. Preferably, the encoded LECT2 protein comprises a LECT2 protein, such as a human LECT2 having the amino acid sequence of SEQ ID NO: 2. In another preferred embodiment, the encoded multi-peptide comprises an active fragment of a LECT2 protein, wherein the active fragment has at least one of the following abilities: reducing MET phosphorylation in tumor cells and Binding to MET in tumor cells. In one embodiment, the encoded active fragment can be a fragment of sEq IDN0:2, or a fragment comprising the HXXXD motif of LECT2 or a derivative thereof. The multi-peptide can be obtained in a variety of ways including, but not limited to, (i) in vitro amplification 'eg, via polymerase chain reaction (PCR); (ii) synthesis, 24 201200151 eg, via chemical synthesis; (iii) Recombinantly produced by colonization; (iv) purification, for example, by cleavage and electrophoresis or chromatography. The polynucleotide includes, but is not limited to, an isolated nucleic acid molecule (eg, a cDNA or genomic DNA fragment produced via PCR or restriction endonuclease treatment) independent of other sequences; and incorporation into a vector, an autologous A nucleic acid molecule that replicates a plastid, a virus (eg, a retrovirus, an adenovirus, or a vesicular virus), or a genomic DNA of a eukaryotic cell. The polynucleotide can be introduced into tumor cells using any convenient method, including, for example, electroporation, calcium phosphate precipitation, microinjection, transformation, biolistics and viral infection, liposome delivery, and the like. The polynucleotide or a portion thereof may or may not be chimeric (covalently linked) to the chromosomal DNA of the genome constituting the tumor cell. For example, the polynucleotide can be run on a free episomal element (e.g., a plastid). Alternatively, for a stably transformed or transfected cell, the polynucleotide has been inserted into the chromosome and thus can be inherited through the daughter cell through chromosomal replication. This stability is evidenced by the ability of the stably transformed or transfected cells to establish a cell line or strain that comprises a population of progeny cells containing the exogenous polynucleotide. In still another embodiment of the invention, the method comprises administering to the tumor cell a compound to enhance expression and/or biological activity of the LECT2 protein or an active fragment thereof in the tumor cell. The biological activity comprises the reduction of phosphorylation of MET in the tumor cells and/or binding to MET in the tumor cells. Such a compound 25 201200151 can be determined using the method described below. The method according to an embodiment of the present invention can be applied to various cancer cells including 4 not limited to the following groups: hepatocellular carcinoma cells, lung cancer cells, breast cancer cells, stomach: field cells, ovarian cancer cells, hypopharyngeal cancer Cells, gods, 'to glioblastoma cells, or any other tumor cell that expresses MET receptors. -------一.._ In the specific embodiment If, the present invention relates to a method for pre-preventing or treating hepatocellular carcinoma in a body, comprising administering the individual-agent The individual liver cell cancer cells enhance the protein level or biological activity of the LECT2 protein or an active fragment thereof, and the biological activity is included in the hepatocellular carcinoma cells to reduce the acidification of MET. Preferably, the LECT2 protein has the amino acid sequence of SEQ ID NO: 2. Protein therapy can be used to increase the level of protein f in LECT2 or its active fragments in a body of hepatocellular carcinoma cells. According to one embodiment of the invention, a therapeutically effective amount of LECT2 protein f or an active fragment thereof is administered to the individual to reduce phosphorylation of MET in the hepatocellular carcinoma cell and to prevent or treat the individual in the individual Hepatocellular carcinoma. Based on the disclosure herein, the Lect2 protein or active fragment thereof can be formulated and administered to the individual using methods known in the art for protein formulation and delivery. In another embodiment, 'gene therapy can introduce a nucleic acid molecule having the ability to express LECT2 protein into the hepatocellular carcinoma cell, and enhance the expression of LECT2 or an active fragment thereof in a hepatocellular carcinoma cell of a body. . The 26 201200151 method comprises administering to the individual a polynucleotide comprising a nuclear (four) sequence encoding a LECT2 egg from f or a ‘### segment thereof to reduce MET phosphorylation in hepatocellular carcinoma cells And preventing or treating hepatocellular carcinoma in the individual. A procedure for performing in vitro gene therapy is outlined in U. In general, gene therapy can involve the in vitro introduction of a functional copy of a gene (functi〇nal c〇py) into a single cell and returning the genetically engineered cells to the individual. The functional copy of the gene is under the operational control of the regulatory element, which enables the genes in the genetically engineered cells to be expressed. A number of transfection and transduction techniques and suitable performance vectors have been known to those of ordinary skill in the artefact of the art. One of the sections is described in International Patent Application No. W095/00654. In vivo gene therapy uses vectors such as adenovirus, retrovirus, vaccinia virus, bovine squamous squamous virus, herpes virus (e.g., Epstein-Barr virus). Gene transfer can also be achieved using a non-viral approach that requires in vitro infection. Such means may include calcium phosphate, DEAE-polyglucose, electroporation, and protoplast fusion. Targeted liposomes may also be used to deliver DNA to cells. As an example, a DNA molecule of a bat human LECT2 protein is first selected for colonization into retroviral vectors. The expression of the LECT2 protein from the vector can be driven by a promoter within its gene, a long terminal repeat of the retrovirus, or a promoter specific for a particular target cell. The vector can then be inserted into a cell of one of the cells to successfully express the LECT2 protein in the target cell. The gene may be in a form that allows the cell to encode enough protein to provide an effective function, and is preferably delivered to the cells. The retroviral vector is usually a preferred gene delivery vector for gene therapy, especially because of its high infectious effect and the stability of _ _ _ _ _ _ _ _ _ _ 丄 or 丄 丄 L L LECT2 DNA The gene can be transferred to cells for non-viral technology for gene therapy, wherein the techniques comprise receptor-mediated using ligand-DNA ligation or adenovirus-ligand-DNA ligation Target DNA transfer, liposome transfection membrane fusion or direct microinjection. These procedures and their variations are applicable to LECT2 gene therapy in vitro and in vivo. An operational flow of molecular methodology for gene therapy for use with the LECT2 gene has been described in Gene Therapy Protocols, edited by Paul D. Robbins, Human press, Totowa NJ, 1996. In yet another embodiment, a small molecule of compound can be used to enhance the performance or biological activity of LECT2 in hepatocellular carcinoma cells of the individual. The method comprises administering to the individual a compound to effectively enhance the expression or biological activity of LECT2 in the hepatocellular carcinoma cell, thereby reducing phosphorylation of MET in the hepatocellular carcinoma cell and preventing or Treating hepatocellular carcinoma. The effective amount of a multi-peptide, nucleic acid molecule or chemical compound of the invention administered to a respective individual during treatment may vary depending on various factors, wherein the factor 28, 201200151 contains the type, species, age, Weight, sex and medical condition; severity of treatment; route of administration; function of the kidney and liver of the patient; and selected specific peptides, nucleic acid molecules or chemical compounds. A physician or veterinarian with expertise in treatment can determine and prescribe the effective amount of the prescription required to prevent, combat or terminate the development of symptoms. Producing a non-toxic effect within the optimal range of concentrations to achieve a concentration requires a therapy based on the kinetics of the effectiveness of the active pharmaceutical ingredient on the target site. This involves considering the distribution, balance, and elimination of active pharmaceutical ingredients involved in the treatment. The methods disclosed herein can be used alone at a convenient dosage as defined by routine experimentation to achieve an optimum increase in the extent or biological activity of LECT2 while reducing any possible toxicity. In addition, co-administration or sequential administration of other agents may be desirable. When several agents are combined, the dosage administered is adjusted to achieve the desired effect. The dosage of these various agents can be independently optimized or combined to achieve a synergistic result, wherein the lesion is further reduced as compared to the use of either agent alone or with other agents. In one embodiment, the method according to an embodiment of the invention is combined with another anti-tumor treatment, including but not limited to another chemotherapy, radiation therapy, and the like. In another aspect, the present invention relates to a pharmaceutical composition for treating a tumor comprising a carrier and a multi-peptide comprising an amino acid sequence of a LECT2 protein or an active fragment thereof, wherein the multi-peptide The system has at least one of the following: reducing MET phosphorylation in tumor cells and binding to MET in tumor cells 29 201200151. In one embodiment, the pharmaceutical composition comprises a human LECT2 protein, such as the amino acid sequence of SEQ ID NO: 2 or an active fragment thereof. Another embodiment of the present invention relates to a pharmaceutical composition for treating a tumor comprising a carrier and a polynucleotide comprising a protein comprising a LECT2 protein or an active fragment thereof. The nucleotide sequence of the amino acid sequence - wherein the multi-peptide is at least one of the following: reducing MET phosphorylation in tumor cells and binding to tumor cells MET. In one embodiment, the pharmaceutical composition comprises a polynucleotide encoding a human LECT2 protein, such as a polynucleotide having the nucleotide sequence of SEQ ID NO: 1. The human LECT2 protein line, for example, has the amino acid sequence of SEQ ID NO: 2 or an active fragment thereof. In still another embodiment, the present invention relates to a pharmaceutical composition for treating a tumor comprising a carrier and a compound which enhances the expression or biological activity of the LECT2 protein or an active fragment thereof in a cell. The biological activity comprises at least one of the ability to reduce MET phosphorylation in tumor cells and to bind to MET in tumor cells. Such compounds can be determined using the methods described below. Based on the disclosure herein, any of the methods well known in the art can be used to prepare various pharmaceutical compositions in accordance with embodiments of the present invention. 201200151 Whether administered alone or in combination with an additional therapeutic agent, the therapeutically active ingredient according to embodiments of the invention may be administered by any conventional route of administration, including dosage units or containing conventional pharmaceutically acceptable Oral, topical, parenteral (including subcutaneous, intravenous, intramuscular, and intrasternal injection or infusion administration techniques) of a pharmaceutical composition of a carrier, administered by inhalation spray or rectally, and any such dose Units or pharmaceutical compositions are within the scope of the invention. Pharmaceutical compositions suitable for oral administration include solid forms such as pills, tablets, troches, and hard or soft capsules (each containing immediate release, timed release, and sustained release formulations), as well as tablets and dispersions. Powder or granules. Pharmaceutical compositions suitable for oral administration in liquid form include solutions, syrups, medicinal liquors, emulsions, and aqueous or oily suspensions. Any of these dosage forms can be prepared according to any of the methods or composite techniques known in the art for making pharmaceutical compositions. When preparing a solid oral dosage form, a pharmaceutically acceptable carrier may be optionally employed, including inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulation or disintegrating agents such as corn starch or seaweed An acid; a binding agent such as starch, gelatin, or gum arabic; and a lubricant such as magnesium stearate, stearic acid or talc. In order to provide an attractive or pharmaceutically acceptable formulation, a solid pharmaceutical composition suitable for oral administration may optionally further comprise one or more sweetening, flavoring, coloring, or preservatives. A pharmaceutical composition suitable for oral administration may also be presented as a hard or soft gelatin capsule, wherein in the case of a hard capsule, the active ingredient may be mixed with an inert solid 31 201200151 body diluent, such as calcium carbonate, calcium phosphate or Kaolin; in the case of soft capsules, the active ingredient may be mixed with water or a miscible solvent such as propylene glycol, PEG'S and ethanol' or an oily medium such as peanut oil, liquid stone, or olive oil. Aqueous suspensions may be prepared which comprise the active ingredient in admixture with excipients suitable for use in the manufacture of aqueous suspensions. Such excipients contain suspending agents such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, and sea-algae-sodium polyethene. Bismuth, gum tragacanth and gum arabic, polydextrose, polyvinylpyrrolidone or gelatin; and dispersing or wetting agents such as lecithin, polyoxyethylene stearate, Heptadecylcaoxyoxytantanol, polyoxyethylene sorbitol monooleate, and sorbitan monooleate. The aqueous suspension may also contain one or more preservatives such as ethyl or n-propyl, p-benzoic acid; one or more coloring agents, or a plurality of flavoring agents, and one or more sweeteners such as sucrose Sweetener or aspartame. An oily suspension wave is prepared by suspending the activity in a vegetable oil, such as cottonseed, olive, sesame or hazelnut oil; or suspended in a mineral oil, such as a liquid sarcophagus float V containing - a paste, For example, bee, hard, or (d) end. Sweeteners and flavoring agents as described above may be added to provide a palatable π formulation. This oily suspension can be preserved by the addition of an "antioxidant" such as ascorbic acid. 32 201200151 Dispersible powders and granules suitable for the preparation of an aqueous suspension suitable for oral administration may be admixed with all of the dispersing or wetting agents, suspending agents, and one or more preservatives discussed above. Sweeteners, flavoring agents, or coloring agents may also be present as needed. Pharmaceutical compositions suitable for oral administration can also be in the form of an oil-in-water emulsion. The oil phase may be a plant or mineral oil as described above, or a mixture thereof. Suitable emulsifiers can be naturally occurring phospholipids such as soy, lecithin, sorbitan monooleate, polyoxyethylene sorbitan monooleate. The emulsion may also contain a sweetener or flavoring agent. Syrups and medicinal liquors may also be formulated with sweeteners, for example, glycerin, propylene glycol, sorbitol or sugar. Such formulations may also contain a demulcent, preservative, and flavoring or coloring agent. The pharmaceutical composition can be further provided in a form suitable for parenteral administration (i.e., injection or infusion). The injectable aqueous or oily suspensions are optionally sterilized and may be formulated according to conventional methods using such suitable dispersing agents, wetting agents, and suspending agents. A parenterally acceptable diluent or solvent such as 1,3-butanediol, water, Ringer's solution, and isotonic sodium vapor may also be used. Cosolvents such as ethanol, propylene glycol or polyethylene glycol can also be used. In addition, sterilized, non-volatile oils have traditionally been utilized as solvents or suspension media in injectable or infusible solutions, and may contain any mild, fixed oil, such as any synthetic single or double Glyceride. Fatty acids, such as oil 33 201200151 acid, can also be used in the preparation of injectable or infusible solutions. The pharmaceutical composition may also be in the form of a suppository. The suppository can be prepared by admixing the active ingredient together with any additional additional pharmaceutical agent and a suitable non-irritating excipient which is solid at room temperature but which melts at body temperature to release the Active ingredient. Suitable materials include cocoa butter and polyethylene glycol. Creams, ointments, scent, solutions or suspensions for topical use containing the active ingredient may be prepared. Topical formulations may include cosolvents, emulsifiers, penetration enhancers, preservatives, softeners, and the like. The active ingredient according to embodiments of the present invention may also be provided as a pharmaceutical composition in the form of a liposome delivery system, such as small unilamellar vesicles, large unilamellar vesicles, and multilamellar vesicles. bubble. Liposomes can be formed from a variety of lipids, including but not limited to amphipathic lipids, such as phosphatidylcholines, sphingomyelins, phosphatidylethanolamines, and lipid-testing (phophatidylcholines), cardiolipins, phospholipidylserines, phosphatidylglycerols, phosphatidic acids, phosphatidylinositols, dimercaptos Diacyl trimethylammonium propanes, diacyl dimethylammonium propanes, and stearylamine 34 201200151 (stearylamine); neutral lipids such as triglyceride S; and combinations thereof Things. These liposomes may or may not contain cholesterol. Another aspect of the invention relates to a method of determining the risk of an individual having an increased development of an invasive tumor. The method comprises: (a) obtaining a biological sample from a body; (b) measuring the degree of phosphorylation of MET in the biological sample; and (c) comparing the degree of phosphorylation measured in step (b) with the control group Degree; wherein when the degree of phosphorylation measured in step (b) is higher than the degree of the control group, the individual is determined to have an increased risk of developing an invasive tumor. In a specific embodiment, the tumor is hepatocellular carcinoma. Based on the disclosure herein, the degree of phosphorylation of MET can be measured using any method known in the art, for example, via Western blotting. The protein level or biological activity of the LECT2 protein can be measured in a variety of ways. Based on the disclosure herein, the degree of protein of LECT2 can be measured by any of the methods known in the art for measuring multi-peptides, including but not limited to enzyme-linked immunosorbent assays using antibodies against anti-LECT2. Methods (ELISAs), Western blots, immunoprecipitation (IP), and immunohistochemistry. The protein level of LECT2 can also be measured indirectly by measuring the extent of LECT2 mRNA in the biological sample, for example, by RT-PCR. This biological activity can be measured by a binding assay of LECT2 binding to MET 35 201200151, or via acidification of MET via HGF in the presence of LECT2. In a particular embodiment, the biological activity of LECT2 is measured by detecting the presence of HXXXD mutation, which is known to reduce the biological activity. The method comprises: obtaining a biological sample from a body; obtaining a polynucleotide sequence encoding a L-EeT2 protein depleted-Η-χχ-XD base-order from the biological sample; sequencing the polynuclear choric acid sequence; Detecting mutations in the HXXXD motif; and determining the individual has an increased risk of hepatocellular carcinoma based on the mutations present in the HXXXD motif. Examples of mutations in the HXXXD motif include, but are not limited to, i) self-depleting-or a plurality of amino acids; 2) adding - one silk acid to the motif; 3) the motif - or a plurality of amino acids Replacement and so on. Based on the disclosure herein, these mutations can be measured using conventional molecular biology techniques. There are many well-known experimental techniques in the art that can be used for _mutation, such as sdective ampHfication, selective primer extension (s day pdmer extension), restriction enzyme cleavage mode, heterozygous sample and control Nucleic acid, sequence alignment, etc. Embodiments of the present application are also directed to a kit for use in stabbing today, π to determine the risk of an individual having an increased risk of developing an invasive tumor. The kit package person 36 201200151 (a) an agent for measuring the maleic acidity of MET in the biological sample of the individual; and (C) using the reagent to determine the risk of the individual having an increased risk of developing an invasive tumor Operation instructions. In a specific embodiment, the tumor is hepatocellular carcinoma. In one embodiment of the invention, the kit preferably includes a segmented carrier adapted to be secured within the enclosed compartment of the at least one trough. The carrier can include three tools for detection. For example, the kit can comprise a labeled compound or reagent having the ability to detect phosphorylated MET, a labeled compound or reagent having the ability to detect LECT2 polypeptide or mRNA encoding LECT2 polypeptide, And a means for determining the amount of the multi-peptide or mRNA in the present (for example, an antibody that binds to the multi-peptide, or an oligonucleotide probe that binds to the DNA or mRNA encoding the multi-peptide) . The kit may also include a tool for measuring biological activity, such as reagents for the MET RTK phosphorylation test. Preferably, the 'operational indication' is packaged with the reagent, for example, in a manual or package label. The label of the operational instructions explains how to use the reagent to measure the protein level and biological activity of the LECT2 protein from a biological sample, and how to assess the risk of developing an invasive tumor based on the measured extent 'eg, after the hepatocellular carcinoma, for example, If the degree of LECT2 protein or its biological activity is below a normal or control group level, it can be used to determine whether a subject has HCC or has a risk of developing HCC. According to a particular embodiment, the kit comprises: 37 201200151 a polymerase chain reaction (PCR) primer for obtaining a polynucleotide sequence encoding the HXXXD motif of the LECT2 protein from the biological sample; A sequencing primer for the polynucleotide sequence; and an indication of the operation of the individual to have an increased risk of developing an invasive tumor, such as liver cancer, based on the presence of a mutation in the HXXXD motif of LECT2. In one embodiment of the present invention, a diagnostic method and method of treatment according to embodiments of the present invention are combined to provide more effective tumor prevention and/or treatment. For example, a method according to one embodiment of the invention comprises: (a) determining, based on the degree of phosphorylation of elevated met in a biological sample of the body, the individual has an increased risk of developing an invasive tumor; And (b) administering to the individual a agent to increase the protein level or biological activity of the LEct2 protein, thereby reducing MET phosphorylation in the individual's tumor cells. In a particular embodiment, the combined method is for preventing or treating a tumor selected from the group consisting of hepatocellular carcinoma, lung cancer, breast cancer, gastric cancer, ovarian cancer, glioma, or glioblastoma, or Any other performance hall is affected by Zhao's tumor. Another aspect of the invention relates to methods for identifying compounds which inhibit at least the proliferation, migration and invasion of tumor cells. The method comprises identifying a s s substance that increases the expression or biological activity of LECT2 in a tumor cell. The biological activity is included in the tumor cell to reduce MET 38 201200151 acidification. Candidate compounds contain many chemical species. Although generally these compounds are organic compounds, preferably small organic compounds, the candidate compounds may also be biomolecules such as peptides, nucleic acids, saccharides, fatty acids, stearyl alcohol, isoprenoids. An alkene, a pyrene, a pyrimidine, a derivative or a structural analog thereof, or a combination thereof. When the compound is a mononucleotide, the compound is typically a DNA or RNA molecule, although modified nucleic acids having unnatural linkages or subunits may also be considered. The method of identifying the compound can be carried out using a conventional experimental format or in a test suitable for high throughput. The term "high throughput" means a test design that allows for easy screening of multiple samples at the same time, and may include the ability to operate mechanically. Another desirable feature of the high throughput assay is an experimental design that is optimized to reduce reagent usage or reduce the number of operations to achieve the desired assay. Examples of test formats include 96-well or 384-well plates, suspension droplets, and "laboratory wafer" microchannel wafers for liquid handling experiments. It is advantageous in the art to have a plastic mold and liquid handling apparatus such as miniaturized, or to design an improved test apparatus to operate a large number of samples using the design of the present invention. In one embodiment, the compound is identified using a reporter test to enhance its ability to enhance the expression of LECT2 in tumor cells. The method for identifying the compound comprises: (a) providing a cell expressing the indicator gene, wherein the indicator gene line is controlled by 39 201200151 LECT2 regulatory sequence from the tumor cell; (b) contacting the cell with a test compound; (c) Measuring the degree of expression of the indicator gene of the cell; and (d) identifying a test compound that increases the degree of expression of the indicator gene. By "LECT2 regulatory sequence" is meant a nucleic acid sequence that controls the expression of the open reading frame of LECT2 in the tumor cell. In one embodiment, the compound is identified by the ability to enhance the interaction between MET and LECT2 in the tumor cells. The method comprises: (a) providing a reaction mixture comprising a LECT2 protein or an active fragment thereof, Met or an active fragment thereof, and a test compound; (b) determining between the LECT2 protein or an active fragment thereof and Met or an active fragment thereof Interaction; and (c) identifying test compounds that enhance the interaction. In a preferred embodiment, the determining step comprises measuring the amount of protein phosphorylation of Met or an active fragment thereof, and the identified test compound reduces the amount of protein phosphorylation. Preferably, the reaction mixture further comprises an HGF or an active fragment thereof. The compound useful in the present invention can also be identified from a binding assay comprising: (a) contacting the Met or an active fragment thereof with a test compound and a labeled active fragment of LECT2 or LECT2; 201200151 (b) Determining the amount of the labeled LECT2 or the labeled LECT2 active fragment bound to Met or its active fragment; and (c) comparing the amount determined in step (b) with a control group for control In the group, the Met or active fragment thereof has been contacted with the labeled LECT2 or the labeled active fragment of LECT2 in the absence of the test compound. The compound useful in the present invention may also be identified as a compound that binds to Met and resembles LECT2, and comprises: (a) contacting a test compound with a test reagent comprising Met or an active fragment thereof; (b) determining Met or The biological activity of the active fragment; and (c) comparing the results determined in step (b) with the results of the control group measurement, in the control group, in the absence of the test compound, the Met or its active fragment Contact with LECT2 or an active fragment thereof. In one embodiment, the Met is in a separate membrane preparation, the assay step comprising measuring the amount of protein phosphorylation of the independent membrane preparation, and the identified test compound reducing the amount of protein phosphorylation. In another embodiment, the method further comprises testing the ability of the identified test compound to inhibit proliferation, migration or invasion of tumor cells in vivo or in vitro. Another aspect of the invention pertains to an isolated polynucleotide having a core encoding an amino acid sequence of an active fragment of LECT2 (e.g., about 51 to 100 amino acid residues of SEQ ID NO: 2) The nucleotide sequence of the active tablet 201200151 has the ability to reduce phosphorylation of Met, bind to Met, or complement the foregoing. Such a polynucleotide can be identified by screening a DNA construct containing various portions of the coding sequence of SEQ ID NO: 2 based on its ability to encode an active fragment of LECT2. Based on the disclosure herein, the screening can be performed using methods in the art, such as phage display. Another aspect of the invention pertains to an isolated multi-peptide having an active fragment of LECT2, such as from about 51 to 100 amino acid residues of SEQ ID NO: 2, which has reduced phosphorylation or binding of Met The ability to Met. The vectors and recombinant cells for the recombinant expression of the active fragment of LECT2 are also included in the embodiments of the present application. Another aspect of the invention relates to the use of a multi-peptide comprising an amino acid sequence of a LECT2 protein or an active fragment thereof for the preparation of a medicament for inhibiting at least one of proliferation, migration and invasion of tumor cells, Wherein the multi-peptide is at least one of the following: reducing MET phosphorylation in tumor cells and binding to MET in tumor cells. In a preferred embodiment, the tumor cell line is selected from the group consisting of hepatocellular carcinoma cells, lung cancer cells, breast cancer cells, gastric cancer cells, ovarian cancer cells, ingested cancer cells, glioblastoma cells, Or any other tumor cell that expresses the MET receptor. In another preferred embodiment, the multi-peptide comprises a LECT2 protein having the amino acid sequence of SEQ ID NO: 2 or an active fragment thereof. 42 201200151 A further aspect of the invention relates to the use of a polynucleotide encoding a polypeptide comprising an amino acid sequence of a LECT2 protein or an active fragment thereof for the production of a medicament for inhibiting proliferation of tumor cells, At least one of migration and invasion, wherein the multi-peptide has at least one of the following: reducing phosphorylation of MET in tumor cells and binding to MET in tumor cells. Preferably, the tumor cell line is selected from the group consisting of hepatocellular carcinoma cells, lung cancer cells, breast cancer cells, gastric cancer cells, ovarian cancer cells, ingested cancer cells, glioma cells, or any other MET-expressing MET Tumor cells of the body. In another preferred embodiment, the polynucleotide encodes a LECT2 protein having the amino acid sequence of SEQ ID NO: 2 or an active fragment thereof. The invention may be further understood by reference to the following non-limiting examples, which are to be understood by those of ordinary skill in the art. There is a more complete narrative. EXAMPLES Materials and Methods Cell Culture All cancer cell lines used were obtained from the American Culture Center (American 43 201200151).

Type Culture Collection, Rockville, Maryland, USA). Hepatocellular carcinoma cell lines were grown in DMEM medium (Life Technologies, GIBCO BRL, Rockville, Md.) at 37 ° C in a humidified atmosphere of 5% carbon dioxide - 95% air, where 'the medium has 10% Calf serum albumin (FBS) and 2 ml of L-glutamic acid (Life Technologies). Cell lines were cultured according to the supplier's recommendations. The adherent cell line was treated with trypsin-EE)TA- and detached from the culture-culture dish. A549 (human lung adenocarcinoma) is maintained and cultured in DMEM medium. CL1-5 (human lung adenocarcinoma), MB-MDA231 (human breast cancer), and N87 (human gastric cancer) were maintained and cultured in RPMI-1640 medium. All media were supplemented with 10% FBS. Transfection and establishment of a stable transgenic cell The expression vector of LECT2 was established by placing human LECT2 cDNA in the pSecTag2A eukaryotic expression vector harboring the hygromycin B gene controlled by the same promoter. The LECT2-positive expression vector (LECT2-sense) was transfected into hepatocytes using Lipofectamine 2000 reagent (Invitrogen Life tech.). A stable cell population was selected via 50 micrograms per milliliter of hydanocin B. LECT2 with significant expression in a single colony was confirmed by RT-PCR and Western blot analysis. Phosphorylation of growth factor receptor tyrosine kinases (RTKs) using the Proteome Profiler Array Kit (R&amp;D System, Minneapolis, 201200151, Minnesota, USA) by phosphotidine assay of receptor tyrosine kinase (RTKs) arrays Degree. In cold lysis buffer (1% polyethylene glycol octyl phenyl ether (Triton X-100), 1 millimolar sodium vanadate, i millimolar sodium fluoride, 0.05 millimolar concentration molybdic acid Sodium, 2 〇 microgram / ml protease inhibitor (aprotinin), 20 μg / house liter protease inhibitor (ieUpeptin), 4 μg / ml (4-肼 'this basic) A burned fluorine ((4_amidin〇phenyl) A sample of HCC cells treated with recombinant lECT2 protein was collected in methane sulfonyl fluoride, 150 mg of sodium sulphate in 5 mM milliliters of Tns-HCl, pH 7.4. The lysate was gently resuspended by pipetting up and down and shaken for 2 to 3 minutes. The lysate was then microcentrifuged at 14,000 xg for 5 minutes. The supernatant was transferred to a clean test tube and stored at _8 (rc. 500 mg of total protein from the supernatant of the HCC cells was spotted with various anti-phospho-RTK antibodies) The RTK array membrane is incubated. The acidification detection procedure is performed according to the manufacturer's protocol. The proliferation assay is tryPsinized with 9G% cluster growth (ecmfiuent gr〇Wth) cancer cells; Transfer to 24-well plate (3xl〇4 cells/well); pre-culture for 24 hours in medium - then change the medium to fresh medium' and incubate for 72 hours at 37 ° C. After the end of the culture, use The MTT assay estimated the number of viable cells. Add 200 μl of MTT solution (Sigma, St L〇uis, Missouri) (2 45 201200151 mg/ml) to each well 2 hours before the start of the subsequent experiment, followed by Reaction in the dark. The foemazan particles were then dissolved in DMSO and the absorbance at 570 nm was measured using an enzyme-linked immunosorbent assay (ELISA reader). The invasion and migration assays used 8 micron pores. The intrusion test was performed on a 24-well disk (Millipore) using Matrigel —- —* — — — — — — — — — — — _ _. _. _ A.. . ♦ _ . . (70 μg; Collaborative Biomedical, Becton Dickinson Labware' USA) coated filter for invasive testing. Place cells (lxl〇5 in 100 μl of DMEM in complete medium) on In the upper chamber, place 1 ml of the same medium in the lower chamber. After 16 hours of incubation, fix the cells with sterol for 20 minutes. Remove the cells on the upper side of the filter with cotton-tipped swabs. The filter was washed with PBS. The cells were then stained with PBS containing 0.05% crystal violet. Microscopically examined and counted cells on the underside of the filter. Transwell membranes were used in the migration assay. And fixed 2 X104 cells after 16 hours.

The FarWestern Blot is derived from the standard Western blot method to detect protein-protein interactions in vitro. Heating the cell lysate or recombinant LECT2 protein in a reduced or non-reduced Laemmli sample buffer; parsing via sodium dodecyl sulfate-polyacrylamide colloidal electrophoresis (SDS-PAGE); and electrotransfer to poly Polyvinylidene difluoride membranes (Immobilon-P 46 201200151 membran ; Millipore, Bedford, Massachusetts). This was followed by a reaction of 5% skim milk, 0.1% Tween 20 and PBS (PBST) for 1 hour, and the membrane was then reacted with recombinant Met protein overnight. The membrane was then washed in PBST and reacted with HRP-conjugated anti-Met or anti-LECT2 antibodies, developed via enhanced chemiluminescence reagent (Amersham Pharmacia Biotech, USA), and irradiated with Kodak X-Omat Blue radiographs. Take a photo. Western blotting separates total cell lysate or cell supernatant proteins by SDS-PAGE and electrotransfers to polyvinylidene fluoride membranes (Immobilon-P membrane; Millipore, Bedford' Massachusetts) . After blocking the dots in PBST, membrane binding was detected with anti-LECT2 (R&amp;D system), PY-99 (Santa Cruz Biotechnology, Santa Cruz, Calif.), p-Met and Met (cell signaling) protein. The membrane was washed and then reacted with a horseradish peroxidase-conjugated secondary antibody for 30 minutes. Antibody-bound protein bands were detected with enhanced chemiluminescent reagents (Amersham Pharmacia Biotech, USA) and photographed with Kodak X-〇mat Blue radiographs. Immunoprecipitation and Western blot analysis Cell lysates were prepared as described above and at 4. (: and with gentle rotation, the same amount of protein was reacted with specific antibodies immobilized on protein A-Sepharose beads for 2 hours. 47 201200151 Large amount of washing with lysis buffer The beads were condensed, boiled, and microcentrifuged. The protein was resolved by SDS-PAGE and the protein was transferred to a nitrocellulose membrane. After thawing in PBST, the membrane was reacted with a specific primary antibody. After washing and reacting with primary antibody Immunologically active proteins were visualized using enhanced chemiluminescence detection (Amersham, Arlington Heights 'Illinois), which specifically stated that 'these membranes were stripped and probed with other antibodies

Fc Marker Grab Test and Tube Analysis The binding of Met to Fc-tagged recombinant LECT2 (rLECT2) was analyzed by grabbing a complex bound to protein A_Sepharose via an Fc marker. rLECT2 from breast cell production was purified via Ni-NTA resin (purity greater than 95%). In combination with buffer (5 〇 millimolar sodium phosphate, pH 7 5, 5 〇〇 millimolar sodium chloride, 1% Nonnad p_4 〇 (N〇nidet P-40); final volume 150 μl The Met protein was mixed with purified rLECT2 and gently rotated at 4 C for 4 hours. The pre-equilibrated Νι-ΝΤΑ resin beads (5 μL) were added to the mixture in a binding buffer, and the mixture was gently spun at 4 C for 2 hours. The resin beads were precipitated by simple centrifugation and washing twice with the combined buffer. Proteins bound to resin beads were extracted in 5 μL of 2 χ Laemmli buffer and analyzed by Western blot analysis using anti-LECT2 antibodies. Flow Cytometry Protein Interaction Assay SK-Hepl cells grown in 90% of the clusters were trypsinized, transferred to a 48 201200151 6-well plate and cultured overnight in a medium. The medium was changed to a serum-free medium, followed by the addition of the Fc-tagged recombinant LECT2 protein for 5 minutes. The cells were resuspended in FACδ staining buffer containing anti-LECT2 antibody for 1 hour at 4 °C. The cells were washed for 5 minutes by centrifugation at 900 rpm in FACS staining buffer. Next, the secondary antibody-conjugated FITC was added to the cells and reacted in the dark for 3 minutes. The cells were kept on ice until the FACS Analyzer was ready. Dead cells were excluded by mothridine staining. Live cells were analyzed by FACS. Results Differential phosphorylation of growth factor RTKs in HCC cells with varying degrees of LECT2 protein. Phosphorylation-receptor tyrosine kinase (rtKs) arrays were used to determine phosphorylation profiles of growth factor RTKs in HCC cells with varying degrees of LECT2, for example, LECT2 overexpression or knockdown performance of stable HCC Transfectant (Fig. 1A)' or HCC cells treated with recombinant LECT2 protein or untreated (Fig. 1B). The degree of phosphorylation of growth factor RTKs in HCC cells was quantified by dot blotting. As shown in Figure 1(B), after treatment of HCC cells with recombinant LECT2 protein, the degree of phosphorylation of several growth factor RTKs was differentially affected in a dose-dependent manner (Fig. 1 (B)). In the analysis of 25 RTKs, 7 growth factor RTKs in SK-Hepl cells were highly phosphorylated. Such RTKs are EGFR, HGFR (or c-Met, Met), Tie-2, FGF-R3, 49 201200151 c-Ret, ROIU, and Dtk. Array assays were performed on samples from total cell lysate treated or untreated with recombinant LECT2 protein at a final concentration of 1 _25 or 2.5 nanomolar. After treatment with recombinant LECT2, the degree of acidification of RTKs in the following HCCs varied significantly: hgFR, Tie-2, and FGF-R3. After treatment of the following hCC with recombinant LECT2 protein, no change or little change in the degree of phosphorylation of EGFR was observed. As shown in Figure -2-, recombinant LECT2 protein inhibited phosphorylation of Met and downstream proteins in SK-Hepl cells in a dose- and time-dependent manner, for example, Erk and Akt (Fig. 2). The degree of LECT2 protein is inversely related to the acidification of Met-Litanic acid in HCC tumor tissues. In order to test the hypothesis that LECT2 can modulate the phosphorylation of Met in HCC cells, the degree of phosphorylation of LECT2 and Met was analyzed in 74 HCC tissue samples obtained from surgical resection of patients from the University of Taiwan Hospital. Using western blot analysis, it was found that a larger amount of LECT2 protein in the tissue sample was associated with less phosphorylation of Met, and less LECT2 protein and more Met phosphorylation were found in later HCC. 3)). LECT2 inhibits HGF-stimulated cell proliferation, migration and invasion in HCC cells. The HGF/Met message pathway comprising HGF and its receptor Met' has now been recognized as a promising target for cancer therapy, for example, to inhibit cell proliferation, migration and invasion. Studies have been conducted to determine whether LECT2 can regulate the performance of HCC cells through the HGF/Met message pathway. SK-Hepl cells were seeded into cell culture wells and as indicated in the MTT Cell 50 201200151 proliferation assay, the final concentration of 0, 1.25, 2.5 nanomolar concentrations bound to HGF (4G Nike/ml) The rLECT2 was processed for 72 hours. As shown in Figure 4 (A), 'HGF stimulates cell proliferation' but LECT2 inhibits HGF-stimulated cell proliferation in a dose-dependent manner. For example, the 25 nm molar concentration rLECT2 can be observed more than the 1.25 Namol concentration. Inhibition of multicellular proliferation. As also shown in Figures 4(B) and 4(C), LECT2 inhibited HGF-stimulated cell migration and invasion in a dose-dependent manner. LECT2 inhibits the dishing of Met stimulated by HGF with other RTKS in HCC cells. Western blot analysis was performed to compare the performance and phosphorylation of Met, Akt, and Erk and the cell invasive ability of cells treated or untreated with HGF, rLECT2, and SU11274 (selective small molecule inhibitors of Met). As shown in Fig. 5(A), an increase in the phosphorylation of Met and downstream proteins (e.g., Akt and Erk) was observed in HGF-treated SK-Hepl cells, which was associated with increased cell invasion. Similar to SU11274, LECT2 inhibits phosphorylation of tyrosine by MGF-induced Met, Akt, and Erk 'as indicated by a reduced message band in cells treated with rLECT2' which is associated with reduced cell invasion. Similarly, 'HGF stimulates Met phosphorylation in H C C cells of the control group, but not in stable transfectants transfected with LECT2 overexpression (Fig. 5(B)).

The downstream Met message molecule can bind to Met» during the message transfer, for example, 'Gabl can bind to the p-Tyrl349 filling site of a Met receptor' and Grb2 can bind to the p-Tyrl356 phosphorylation site of a second Met receptor. , because 51 201200151 This interaction between Gabl and Grb2 can occur on Met dimers or multimers. LECT2 reduces the acidification of Met in p-Tyrl 349, thus separating Gabl from the Met receptor (Fig. 5(C)). The biological consequences of activation by RTK are determined by the specificity of duration, intensity and message activation downstream of the receptor. In addition to internalization, transport, and degradation in lysosomes such as receptors that inhibit regulatory mechanisms, Met receptors have been identified as receptors for several PTPs. - The non-receptor proteins tyrosine acid phytase (PTPs) 1B and T cell hydratase (TCPTP) have been tolerated as negative regulators of various message pathways, including Met receptor tyrosine Kinase. It has been reported that both phosphatases interact with Met' and this interaction requires the phosphorylation of paired tyrosine (Tyr-1234/1235) in the activation loop of the Met kinase domain (i〇op) Chemical. Reference may be made to Sangwan et al., J Biol Chem. 2008; 283(49): 34374-34383, the entire disclosure of which is incorporated herein by reference. As shown in Figure 5(D), treatment with rLECT2 results in an increase in the amount of PTP1B associated with Met. These results indicate that LECT2 inhibits HGF-stimulated cell proliferation, migration and invasion in HCC cells by inhibiting Met phosphorylation, thus representing a novel mechanism for cancer therapy. LECT2 binds directly to the Met receptor. To determine if LECT2 can interact directly with Met' first test whether LECT2 and Met can form a complex on the surface of HCC cells. Co-immunoprecipitation (co_ip) is performed with lysates from HCC cells (e.g., huh-7 52 201200151 and PLC/PRF/5 cells). These cells endogenously express LECT2 and Met receptors. As shown in Figure 6 (A), the results of the co-IP study confirmed that LECT2 can interact with Met. The interaction of LECT2 with the Met receptor was further investigated using recombinant 293T cells transfected with the LECT2 and/or Met genes. Co-IP experiments confirmed the interaction between LECT2 and Met in 293T cells transfected with LECT2 and Met genes (Fig. 6(B)). The distal Western blot test showed that LECT2 was cross-linked to the extracellular domain of the Met receptor in a dose-effective manner (Fig. 6(C)). The interaction of the LECT2-Met protein and protein was further demonstrated in a test tube (Fig. 6(D)). In addition, the protein-protein interaction between LECT2 and Met was investigated by a fast and sensitive flow cytometry method and using SK-Hepl cells treated with rLECT2 protein (final concentration of 5 nanomolar concentration) for 10 minutes. Live cells were collected after rLECT2 treatment. Anti-_1^〇: D2 antibody was added to the suspended cells. After about 1 hour of reaction, FITC-fluorescent-conjugated secondary antibody was added to the cells. Flow cytometry analysis showed that SK-Hepl cells were clearly labeled with FITC, indicating the interaction of LECT2 with the cell surface (6th (E) Figure). To demonstrate the specific interaction between LECT2 and the Met receptor, the flow cytometry study was performed with SK-Hepl cells additionally treated with siRNA of Met and EGFR, where the siRNAs would each reduce Met individually. And the protein level of EGFR. As shown on the right side of Figure 6(E), to 53 201200151

Treatment of cells with Met siRNA resulted in a decrease in FITC fluorescence (the second peak from the left)&apos; but no changes were observed in cells treated with GFR siRNA or in the control group. This indicates that rLECT2 interacts with Met on the surface of SK-Hepl cells but does not interact with EGFR. The hxxxd motif of the LECT2 protein regulates tumor growth and metastasis. In order to detect the potential functional work of LECT2 involved in the regulation of tumor growth and metastasis, the results of H--%-1-5 ten-amino-acid sequence analysis showed that the HXXXD motif of LECT2 regulates tumor growth. Play an important role in the transfer. A LECT2 mutant with a mutation in the HXXXD motif was established and studied. Such a mutant comprises, for example, HQGVD (part of SEQ ID NO: 3) in place of AQGVA (SEQ ID NO: 4, shown as mLECT2-l) or AQAVA (SEQ ID NO: 5' is shown as mLECT2-2). LECT2 mutants with mutations in other amino acids were established and studied as control groups, such as mLECT2-3, mLECT2-4. See, for example, Figure 7 (A) and Figure 9. SK-Hep 1 overexpressing HXXXD mutants (eg, mLECT2-Bu mLECT2-2) compared to overexpression of wild type LECT2 or other LECT2 mutants (eg, mLECT2-3, mLECT2-4) The cells have elevated HGF-induced Met phosphorylation and cell invasion (Fig. 7(B)). Co-immunoprecipitation analysis indicated that the HXXXD mutant of LECT2 has lost all or most of its binding to Met (Fig. 7(C)). These data indicate that HXXXD is important for the interaction of LECT2 with Met and for the regulation of Met phosphorylation via 54 201200151 HGF. Recombinant LECT2 protein reduces HGF-stimulated in various cancer cells

Phosphorylation of Met. The effect of LECT2 protein on Met activation in other cancer cell lines was also investigated' including human lung cancer cells (A549), human breast cancer cells (MB-MDA-231), human gastric cancer cells (N87), and human ovarian cancer cells: ES-2. Human swallowed cancer cells: FaDu, and human glioblastoma cells: U87. Cancer cells were treated with HGF (40 ng/ml) alone or in combination with recombinant LECT2 protein (2.5 nmol concentration) for 15 minutes. Next, the activity and invasive ability of Met receptor tyrosine kinase were investigated. As shown in Fig. 8, LECT2 also inhibited HGF-induced Met acidification and HGF-induced invasion in various cancer cell tests. This indicates that LECT2 represents a new cancer treatment for tumors and cancer other than HCC. Conclusion The stimulatory role of HGF and Met receptors in tumor growth and invasion has recently been investigated. In the present study, LECT2 has been found to inhibit tumor cell proliferation, migration and invasion by inhibiting Met activation to inhibit downstream message pathways necessary for stimulating cells. Furthermore, treatment with LECT2, whether via endogenous LECT2 overexpression in HCC cells, or via administration of extrinsic LECT2 to HCC cells, reduces Met tyrosine kinase activity, thereby inhibiting HGF to HGF/Met The stimulation of the path. A specific interaction of LECT2 with the Met receptor in HCC cells has been demonstrated, for example, through the HXXXD motif of 55 201200151 LECT2 acidase LECT2. Although the tumor suppressor mechanism that is not expected to be bound by this theory is clearly associated with the association of the protein alanine _ (ΡΤΡ) with Met in visceral cells. The findings of the present invention indicate that LECT2 inhibits the lightening of the invasion of pulse cells. This _ request study is also WLEC and h% - inserted into the treatment or prevention of other new treatments that usually have the Met receptor overactivity characteristics. It will be appreciated by those skilled in the art that the above-described embodiments may be modified without departing from the broad inventive concept of the invention. Therefore, it is to be understood that the invention is not to be construed as limited The above general description, as well as the above detailed description of the present invention, will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, the present invention is shown in the preferred embodiments. However, it should be understood that the invention is not limited to the precise arrangements and tools shown. [Simplified Schematic] Figure 1 shows a photograph of a human RTK phosphorylated antibody array: a complete cell extract from human hepatocellular carcinoma cells (HCCS) is reacted on an rtK antibody array, followed by anti-峨Acidification of the tyrosine horseradish peroxidase reaction to determine the phosphorylation state; each rtk is paired in pairs with a pair of dots, with a positive control group in each corner; each pair of positive RTK dots 56 201200151 are all identified by a number to identify the corresponding RTKs listed under the array; of the 25 RTKs analyzed on the squamized RTK array, 7 or 8 growth factor RTKs are highly expressed in human HCC SK-Hepl cells Acidification of the dish: Figure 1 (A) shows that the expression of the LECT2 protein reduces the acidification of multiple receptors, alanine kinases (RTKs) in HCC: SK-Hepl/LECT2 is a stable transfectant that overexpresses LECT2; Transfectants of the SK-Hepl/Neo vector control group; Huh-7/shLECT2-2 line knocked out stable transfectants showing LECT2; and Huh-7/shLuc-based vector control group transfectants; 1 (B) shows that treatment with recombinant LECT2 protein will be fine in SK-Hepl Reduction of multiple RTKs in the cell: the array is total cells from cells treated or untreated with recombinant human LECT2-Fc protein (rLECT2) at a concentration of 1.25 nanomolar, a final concentration of 2.5 nanomolar, or a control group The lysate sample was taken; Figure 2 illustrates that LECT2 protein inhibits the phosphorylation of tyrosine in Met and downstream proteins in a dose- and time-dependent manner, as in Met, Erk and in SK-Hepl cells treated with rLECT2 Western blot analysis of phosphorylation of Akt protein indicated that 'each treatment was performed in triplicate and the assay was repeated at least three times: Figure 2 (A): treatment with rLECT2 protein (〇 to 10 nanomolar concentration) After 10 minutes, the cells were recovered 'extracted protein and the endogenous phosphorylation levels of various proteins (eg 'phosphorylation-Met, phosphorylation-Erk and phosphorylation-Akt) were detected by Western blot analysis and Met was detected. The total protein level of Erk and Akt 57 201200151 was used as a control group for loading; and SK-Hepl cells were treated with rFc protein as a marker control for recombinant cell junction Group; Figure 2(B): Tested for complete cell lysate by Western blot analysis after 0, 5, 10, 15, 30, 60, and 120 minutes of treatment with 2.5 nanomolar concentrations of rLECT2 protein Phosphorylation of _Met, phosphorylation-Erk, and phosphorylation of Akt' and detection of Me.t, E.rk, Akt-total-protein, quality-degree, as an equivalent of loading Control group; Figure 3 illustrates the inverse association between the degree of LECT2 and Met receptor activity in HCC tissue samples: anti-LECT2, anti-squaring-Met, anti-Met, and anti-beta-actin Western blotting of antibodies to determine the extent of LECT2, phosphorylation-Met, Met, and β-actin in HCC tumor tissues of various disease stages (I, II, and IV); Figure 4 illustrates LECT2 protein in HCC cells Inhibition of HGF-induced cell proliferation, migration, and invasion, each treatment was performed in three replicates, and the assay was repeated at least three times: Figure 4 (Α): exposure to rLECT2 with different concentrations (0-5 nanomolar concentration) Fresh DMEM medium (control group) or monolayer cultured H containing DGF medium containing HGF (40 ng/ml) Growth characteristics of CC SK-Hepl cells; Figure 4 (B): Sheared clusters with a blue micropipette 58 201200151 SK-Hepl monolayer' followed by exposure to rLECT2 with different concentrations (〇_5奈Mole concentration of fresh DMEM medium (control group) or DMEM medium containing HGF (40 Ng/ml) for 14 hours and observation of wound healing; Figure 4 (C): determined by Boyden chamber In vitro invasive activity of SK-Hepl cells treated with rLECT2 for 16 hours; Figure 5 shows LECT2 inhibits cell invasion and phosphorylation of Met receptor tyrosine, sequestering linker proteins from MET, but promoting in HCC cells Binding of MET to PTP1B: Figure 5 (A): rLECT2 protein inhibits phosphorylation of RTKs in HGF-induced Met and other downstream pathways in HCC cells; Figure 5 (B): LECT2 overexpresses LECT2 turnover Inhibition of HGF-induced Met phosphorylation by SK-Hepl/LECT2; however, HGF-induced is observed in the transfectants Huh-7/shLECT2-2 expressing LECT2 compared to vector-controlled cells. Increase in phosphorylation of Met; compared to control cells, The HGF-induced invasion ability of SK-Hepl/LECT2 was also reduced; Figure 5 (C): Immunoprecipitation (IP) and Western blot (WB) analysis of the effect of recombinant LECT2 on the separation of linker proteins from Met receptors; Figure 5 (D): IP and WB analysis of the effect of recombinant LECT2 on the binding of PTP1B to the Met receptor; 59 201200151 Figure 6 illustrates the IP, distal WB and flow of LECT2-Met protein interactions in HCC cells Results of cytometry analysis: Figure 6 (A): IP and WB analysis of endogenous LECT2 protein and Met from HCC cells; Figure 6 (B): Recombinant expression of LECT2 and Met in 293T cells, and recombination IP and WB analysis of recombinant LECT2 protein and Met in 293T cells; Figure 6 (C): distal western blot analysis of LECT2 and Met in a dose-dependent manner; Figure 6 (D): in a test tube Recombinant LECT2 protein binds directly to the Met receptor; and Figure 6 (E): Flow cytometry analysis of the binding of LECT2 protein to Met in SK-Hepl cells; Figure 7 illustrates the HXXXD motif of LECT2 protein Met receptor junction: Figure 7 (A): Prediction of LECT2 and Met receptor docking Stopping results; Figure 7 (B): LECT2 inhibits acidification and invasion of Met, but LECT2 with mutations in the mutated HXXXD motif (eg mLECT2-l or mLECT2-2) is not the case; 201200151 7 (C): IP and WB analysis of the binding of Met to LECT2 and the mutated LECT2 protein in HCC cells; Figure 8 shows the effect of recombinant LECT2 protein on phosphorylation of MET receptor tyrosine in other cancer cells; Cancer cells were treated with HGF (40 Ng/ml) of recombinant LECT2 protein (rLECT2) with or without binding to 2.5 nanomolar concentrations for 15 minutes; 24 hours later, the in vitro invasion activity of cancer cells was measured in a Boyden chamber; Each treatment was performed in triplicate and the experiment was repeated at least three times: Figure 8 (A): lung cancer cell line A549; Figure 8 (B): breast cancer cell line MB-MDA-231; Figure 8 (C) : Gastric cancer cell line N87; Figure 8 (D): ovarian cancer cell line ES-2; Figure 8 (E): swallowed cancer cell FaDu; and Figure 8 (F): glioma cell line U87; Figure 9 illustrates the design of a point mutation in the full length of LECT2 in the pSecTag2A vector. [Main component symbol description] (none). 201200151 117(:-028:2611^sequence table-delivery version-20110531.1\1 sequence table&lt;U0&gt;Taiwan Toyo Pharmaceutical Co., Ltd.&lt;120&gt; Neutrophil leukocyte serotonin 2 reduces liver in tumor cells Methods and compositions related to phosphorylation of cell growth factor receptors

&lt;130&gt; 688128.1WO &lt;150&gt; US 61/288,627 &lt;151&gt; 2009-12-21 &lt;160&gt; 7 &lt;170&gt; Patentln version 3.5 &lt;210&gt; 1 &lt;211&gt; 1077 &lt;212&gt; DNA &lt; 213 &gt; human &lt; 220 &gt; &lt; 223 &gt; LECT2 &lt; 400 &gt; 1 aaatcaaata gctatccatg gaatattaga acttgacttg ctccatcctc ttaaactttt 60 tgtgtctcac actaaagaaa tgagagatgc agaattctaa ggctaaatag ctaggaagta 120 ttcattcaaa cttgaatatt cttcaaagag agtgtggggg caactctaat cagaggaaga 180 aactaaagga agtaaaacca gatgttttcc accaaagccc tccttttggc tggtctgatt 240 tctaccgcac tggcagggcc atgggctaat atatgtgctg gcaagtcttc caatgagatc 300 cggacgtgtg accgccatgg ctgtggacag tactctgctc aaagaagtca gaggcctcac 360 cagggtgtgg acatcttgtg ctctgctgga tctactgtgt acgcaccatt cactggaatg 420 attgtgggcc aggagaaacc ttatcaaaac aagaatgcta tcaataatgg tgttcgaata 480 tctggaagag gtttttgtgt caaaatgttc tacattaagc caattaagta taaaggtcct 540 attaagaagg gagaaaaact tggaactcta ttgcccttgc agaaagttta tcctggcata 600 caatcgcatg tgcacattga aaactgtgac tcgagtgacc ctactgcata cctgtaaatc 660 gaa ggccaat ggtcagatct tcaaaataaa aagtcatctt aaaaacctgg atgcataccc 720 ttctcttcaa gaaatttgtg ttcacaaagg aaaaatgcat gaagggatgg ataccccatt 780 ttccatgaca tgattattac acattgcatg cctgtatcaa aacatctcac gtacctcata 840 aacatataca cctatgtacc cacaaaaatt ttttaattaa aaaaaggaaa tttgagttta 900 aatagaaaca tgataaatgc aagaaagaaa acattttgat tttaactcat tgtcactctg 960 atgttcatgt gaactggttg cttcgggctc tttgatctgt cacctatgga atctgagtgg 1020 ttttattttt tagatttctc agtcccaaag atctaagata aataaacaag agaactt 1077 &lt;210&gt; 2 &lt;211&gt; 151 &lt;212&gt; PRT &lt; 213 &gt; human &lt;220&gt;&lt;223&gt; LECT2 &lt;400&gt; 2

Met Phe Ser Thr Lys Ala Leu Leu Leu Ala Gly Leu lie Ser Thr Ala Page 1 201200151 TTYC-02826TW - Sequence Listing - Delivery Version -20110531. txt Leu Ala Gly Pro Trp Ala Asn He Cys Ala Gly Lys Ser Ser Asn Glu 20 25 30 lie Arg Thr Cys Asp Arg His Gly Cys Gly Gin Tyr Ser Ala Gin Arg 35 40 45

Ser Gin Arg Pro His Gin Gly Val Asp lie Leu Cys Ser Ala Gly Ser 50 55 60

Thr Val Tyr Ala Pro Phe Thr Gly Met lie Val Gly Gin Glu Lys Pro 65 70 75 80

Tyr Gin Asn Lys Asn Ala Me Asn Asn Gly Val Arg lie Ser Gly Arg 85 90 95

Gly Phe Cys Val Lys Met Phe Tyr lie Lys Pro lie Lys Tyr Lys Gly 100 105 110

Pro lie Lys Lys Gly Glu Lys Leu Gly Thr Leu Leu Pro Leu Gin Lys 115 120 125

Val Tyr Pro Gly lie Gin Ser His Val His lie Glu Asn Cys Asp Ser 130 135 140

Ser Asp Pro Thr Ala Tyr Leu 145 150 &lt;210&gt; 3 &lt;211&gt; 50 &lt;212&gt; PRT &lt;213&gt; Human &lt;220&gt;&lt;223&gt; LECT2 partial sequence &lt;400&gt;

Arg Pro His Gin Gly Val Asp Val Leu Cys Ser Ala Gly Ser Thr Val 15 10 15

Tyr Ala Pro Phe Thr Gly Met lie Val Gly Gin Glu Lys Pro Tyr Gin 20 25 30

Asn Lys Asn Ala lie Asn Asn Gly Val Arg lie Ser Gly Arg Gly Phe 35 40 45

Cys Val 50 &lt;210&gt; 4 &lt;211&gt; 5 &lt;212&gt; PRT &lt;213&gt; artificial sequence &lt;220&gt;&lt;223&gt; LECT2 HXXXD mutant sequence mLECT2-l &lt;400&gt;

Ala Gin Gly Val Ala 1 5 Page 2 201200151 TTYC, 02826TW - Sequence Listing - Delivery Version -20丨 10531. txt &lt;210&gt; 5 &lt;211&gt; 5 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence&lt;220&gt;&lt;223&gt; HXXXD mutation sequence of LECT2 mLECT2-2 &lt;400> 5 Ala Gin Ala Val Ala &lt;210&gt; 6 &lt;211&gt; 5 &lt;212&gt; PRT &lt;213&gt; artificial sequence &lt;220&gt;;223&gt; mutated sequence of LECT2 mLECT2-3 &lt;400&gt; 6 Ala Gin Ala Pro Ala &lt;210&gt; 7 &lt;211&gt; 5 &lt;212&gt; PRT &lt;213&gt; artificial sequence &lt;220&gt;&lt;223&gt; LECT2 Mutation sequence mLECT2-4 &lt;400&gt; 7 Ala Gly Ala Cys Ala Page 3

Claims (1)

201200151 VII. Patent application scope: 1. A method for inhibiting at least one of proliferation, migration and invasion of tumor cells, comprising administering a medicament to the tumor cells to enhance neutrophil chemotaxis in the tumor cells; The protein level or biological activity of Factor 2 (LECT2) protein or its active fragment, thereby reducing MET phosphorylation in the tumor cells. 2. The method of claim 1, which comprises administering to the tumor cell a LECT2 protein having the amino acid sequence of SEQ ID NO: 2. 3. The method of claim 1, which comprises administering to the tumor cell an active fragment of a LECT2 protein, wherein the active fragment has at least one of the following: reducing MET phosphorylation and tumor cells in tumor cells Combined with MET. 4. The method of claim 1, which comprises administering to the cell a polynucleotide comprising a nucleotide sequence encoding a LECT2 protein having the amino acid sequence of SEQ ID NO: 2. 5. The method of claim 1, which comprises administering to the cell a polynucleotide comprising a nucleotide sequence encoding an active fragment of a LECT2 protein, wherein the active fragment has at least one of the following properties: Phosphorylation of MET is reduced in tumor cells and bound to MET in tumor cells. 6. The method of claim 1, wherein the tumor cell line is selected from the group consisting of: hepatocellular carcinoma cells, lung cancer cells, breast cancer cells, gastric cancer cells, ovarian cancer cells, hypopharyngeal cancer cells, and neuroglia 62 201200151 Tumor blisters, or any other tumor cell that expresses MET. A method for preventing or treating hepatocellular carcinoma in an individual, comprising administering to the individual an agent to increase the protein level or biological activity of the LECT2 protein or an active fragment thereof in the hepatocellular carcinoma cell of the individual This reduces the acidification of MET in this hepatocellular carcinoma cell. 8. The method of claim 7, wherein the LECT2 protein line comprises the amino acid sequence of SEQ ID Ν〇: 2. A method for reducing phosphorylation of MET in a tumor cell, which comprises administering a drug to the tumor cell to increase the protein level or biological activity of the LECT2 protein or an active fragment thereof in the tumor cell. 10. The method of claim 9, wherein the LECT2 protein line comprises the amino acid sequence of SEQ ID 2. 11. A method of preventing or treating an invasive tumor comprising: (a) determining, based on the extent of elevated MET phosphorylation in a biological sample of the body, the individual has an increased risk of developing an invasive tumor; b) administering the individual one agent' to increase the protein level or biological activity of the LECT2 protein, thereby reducing squamization of MET in the individual's tumor cells. The method of claim 11, wherein the tumor is selected from the group consisting of hepatocellular carcinoma, makeup, lung cancer, breast cancer, gastric cancer, ovarian cancer, ingested carcinoma, glial maternal tumor, 忐A, etc. Express the tumor of bribes. The method, wherein the agent comprises a polypeptide comprising a LECT2 protein having the amino acid sequence of SEQ 63 201200151 ID NO: 2, or a coding comprising an amino acid sequence of SEQ ID NO: 2. Polynucleotide of the peptide of the LECT2 protein. A pharmaceutical composition for preventing or treating a tumor comprising a carrier and a polypeptide comprising an amino acid sequence of a LECT2 protein or an active fragment thereof, wherein the multi-peptide is capable of At least one of: reducing squamization of MET in tumor cells __ and binding to MET in tumor cells. 15. The pharmaceutical composition of claim 14, wherein the multipeptide peptide comprises the LECT2 protein, The LECT2 protein has the amino acid sequence of SEQ ID NO: 2. 16. The pharmaceutical composition according to claim 14, wherein the multipeptide peptide comprises an active fragment of the LECT 2. 17. a species for preventing or treating a tumor A pharmaceutical composition comprising a carrier and a polynucleotide encoding a polypeptide comprising an amino acid sequence of a LECT2 protein or an active fragment thereof, wherein the polypeptide has the following At least one of the ability to: reduce phosphorylation of MET in tumor cells and bind to MET in tumor cells. 18. The pharmaceutical composition according to claim 17, wherein the polynucleotide comprises a nucleoside encoding the LECT2 protein Acid sequence The LECT2 protein has the amino acid sequence of SEQ ID NO: 2. 19. The pharmaceutical composition of claim 17, wherein the polynucleotide comprises a nucleotide sequence encoding an active fragment of the LECT 2. 64 201200151 20 An active fragment of an isolated LECT2 protein having at least one of the following: reducing MET phosphorylation in tumor cells and binding to MET in tumor cells. Nucleotide of the active fragment of Item 20. 22. Use of a multi-peptide comprising an amino acid sequence of a LECT2 protein or an active fragment thereof for the preparation of a medicament for inhibiting proliferation, migration and invasion of tumor cells At least one of the plurality of peptides having at least one of the ability to reduce MET phosphorylation in tumor cells and to bind to MET in tumor cells. 23. The use of claim 22, wherein the tumor The cell line is selected from the group consisting of hepatocellular carcinoma cells, lung cancer cells, breast cancer cells, gastric cancer cells, ovarian cancer cells, ingested cancer cells, glioblastoma cells, or any of them. A tumor cell which exhibits a MET receptor. 25. The use of claim 22, wherein the LECT2 protein line comprises SEQ ID NO: 2. 26. a plurality of amino acid sequences encoding a LECT2 protein or an active fragment thereof The use of a polynucleotide of a peptide for the preparation of a medicament for inhibiting at least one of proliferation, migration and invasion of a tumor cell, wherein the multi-peptide has at least one of the following: reducing MET in a tumor cell Phosphorylation and binding to MET in tumor cells. 27. The use of claim 26, wherein the tumor cell line is selected from the group consisting of: hepatocellular carcinoma cells, lung cancer cells, breast cancer cells, gastric cancer cells, ovarian cancer cells, Pharyngeal cancer cells, glioblastoma cells, or any of its tumor cells that express MET receptors at 201200151. 28. The use of claim 26, wherein the LECT2 protein line comprises SEQ ID NO: 2.