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EP2515930A1 - Verfahren und zusammensetzungen im zusammenhang mit reduzierter met-phosphorylierung durch aus leukozyten gewonnenes chemotaxin 2 in tumorzellen - Google Patents

Verfahren und zusammensetzungen im zusammenhang mit reduzierter met-phosphorylierung durch aus leukozyten gewonnenes chemotaxin 2 in tumorzellen

Info

Publication number
EP2515930A1
EP2515930A1 EP10838658A EP10838658A EP2515930A1 EP 2515930 A1 EP2515930 A1 EP 2515930A1 EP 10838658 A EP10838658 A EP 10838658A EP 10838658 A EP10838658 A EP 10838658A EP 2515930 A1 EP2515930 A1 EP 2515930A1
Authority
EP
European Patent Office
Prior art keywords
met
lect2
cell
tumor cell
protein
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP10838658A
Other languages
English (en)
French (fr)
Other versions
EP2515930A4 (de
Inventor
Min-Liang Kuo
Yu-Ling Wu
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
TTY Biopharm Co Ltd
Original Assignee
TTY Biopharm Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by TTY Biopharm Co Ltd filed Critical TTY Biopharm Co Ltd
Publication of EP2515930A1 publication Critical patent/EP2515930A1/de
Publication of EP2515930A4 publication Critical patent/EP2515930A4/de
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/195—Chemokines, e.g. RANTES
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • Hepatocellular carcinoma is one of the most common types of cancer in the world, with annual incidence of approximately 1 million cases (1). In Taiwan, it continues to be the leading cause of cancer-related death among men and the second among woman.
  • the five year survival rate of HCC with higher stages, e.g., stages ⁇ , is about 20%, which is less than one third of that with stage I, i.e., about 60%.
  • High cancer recurrence is still the major cause of death of HCC patients.
  • the major poor prognostic factors included vascular invasion (2).
  • LECT2 was isolated as a new chemotactic factor for neutrophils from the culture supematants of phytohemagglutinin-activated human T cell leukemia SKW-3 cell, see Yamagoe et al (3).
  • LECT2 protein may be multifunctional, since it was found to be identical to chondromodulin- ⁇ , a growth stimulator of chondrocytes.
  • LECT2 has been shown to be related to the repair and cell growth after damage (4, 5). It has been shown that LECT2 is expressed in hepatocytes (17), and that LECT2 becomes essentially negative in full-blown HCC cells (18), suggesting an important role of LECT2 in the HCC cells in addition to the activation of neutrophils.
  • the precise roles of LECT2 in cancer progression such as migration, invasion and metastasis, have not been reported.
  • Met tyrosine kinase (Met or c-Met), also known as hepatocyte growth factor receptor (HGFR)
  • HGFR hepatocyte growth factor receptor
  • the heterodimer is formed by a single-pass transmembrane beta chain (145 kDa) and a completely extracellular alpha chain (50 kDa).
  • the extracellular segment contains a Sema domain, an atypical motif made of over 500 amino acids, which has a low affinity binding activity for the ligand.
  • the extracellular portion also comprises a cysteine-rich domain (Cys domain) known as Met-related sequence (MRS), and four immunoglobulin-like structures (IPT domains), a typical protein-protein interaction region.
  • Cys domain cysteine-rich domain
  • MCS Met-related sequence
  • IPT domains immunoglobulin-like structures
  • the intracellular portion of the receptor is made of a juxtamembrane section followed by a catalytic site and a C-terminal regulatory tail.
  • the juxtamembrane segment is vital for receptor downregulation (7).
  • a serine residue e.g., Ser 985 in human MET
  • a tyrosine e.g., Tyr 1003 in human MET
  • the juxtamembrane portion is flanked by the catalytic site, which contains two tyrosines (e.g., Tyr 1234 and 1235 in human MET) responsible for regulating the enzyme activity.
  • the C-terminal tail contains two tyrosine residues (e.g., Tyr 1349 and 1356 in human MET) that when phosphorylated create a multifunctional docking site capable of recruiting a vast cohort of intracellular adaptors in charge of signal transduction inside the cell triggered by the ligand-receptor interaction (9). These two tyrosine residues have been demonstrated to be both essential and sufficient to execute Met physiological functions and to elicit Met oncogenic potential (10).
  • tyrosine residues e.g., Tyr 1349 and 1356 in human MET
  • Met receptor regulates multiple cellular events, ranging from cell motility and angiogenesis to morphological differentiation and tissue regeneration.
  • the cytoplasmic C-terminal region of Met receptor acts as a docking site for multiple protein substrates, including Grb2, Gabl , STAT3, She, SHIP-1 and Src. These substrates are characterized by the presence of multiple domains, including the PH, PTB, SH2 and SH3 domains, which directly interact with the multisubstrate C-terminal region of Met. See Victor Martin Bolanos-Garcia, Molecular and Cellular Biochemistry, 2005, Vol. 276:149-157.
  • HGF Hepatocyte growth factor
  • SF scatter factor
  • Mature active HGF is a heterodimer composed of an alpha-chain subunit and a beta-chain subunit that are linked by a disulfide bond (14).
  • the alpha-chain subunit contains an N-terminal hairpin domain and four kringle domains.
  • the beta- chain subunit is a serine-protease-like domain lacking catalytic activity due to mutations in essential residues (15).
  • the binding of HGF to c-Met triggers autophosphorylation of the cytoplasmic domain of Met. Met activation may induce different phenotypes depending on tumor progression (16).
  • the present invention relates to new methods and compositions to diagnose and/or suppress the invasiveness of cancer cells based on signal-transduction molecular mechanisms of the invasiveness of cancer cells, e.g., by reducing MET phosphorylation with effective amount of LECT2 in tumor cells.
  • LECT2 strongly reduced proliferation, migration and/or invasiveness of a tumor cell, such as HCC cells or other tumor cells, via a signal transduction pathway involving Met receptor, e.g., by reducing MET phosphorylation in the tumor cell.
  • the present invention relates to a method of suppressing at least one selected from the group consisting of the proliferation, migration and invasiveness of a tumor cell.
  • the method comprises administering to the tumor cell an agent to increase protein level or biological activity of a LECT2 protein or an active fragment thereof in the tumor cell to thereby reduce phosphorylation of MET in the tumor cell.
  • the present invention relates to a method of preventing or treating hepatocellular carcinoma in a subject.
  • the method comprises administering to the subject an agent to increase protein level or biological activity of a LECT2 protein or an active fragment thereof in a hepatocellular carcinoma cell of the subject to thereby reduce phosphorylation of MET in the hepatocellular carcinoma cell.
  • Another general aspect of the present invention relates to a method of reducing phosphorylation of MET in a tumor cell.
  • the method comprises administering to the tumor cell an agent to increase protein level or biological activity of a LECT2 protein or an active fragment thereof in the tumor cell.
  • Another general aspect of the present invention relates to a pharmaceutical composition for preventing or treating a tumor.
  • the composition comprises a carrier and a polypeptide comprising the amino acid sequence a LECT2 protein or an active fragment thereof.
  • the pharmaceutical composition comprises a carrier and a polynucleotide comprising a nucleotide sequence encoding a LECT2 protein or an active fragment thereof.
  • the polypeptide comprising the LECT2 protein or the active fragment thereof is capable of at least one of reducing phosphorylation of MET in a tumor cell and binding to the MET in the tumor cell.
  • Another general aspect of the present invention relates to a method of identifying a subject having an increased risk of developing an invasive tumor.
  • the method comprises:
  • step (b) wherein when the level measured from step (b) is higher than that of the control, the subject is identified as having the increased risk of developing an invasive tumor.
  • the diagnostic method comprises sequence analysis and detection of a mutation in the HXXXD motif of LECT2.
  • kits for identifying a subject having an increased risk of developing an invasive tumor comprises:
  • the kit comprises reagents for sequence analysis and detection of a mutation in the HXXXD motif of LECT2.
  • Yet another general aspect of the invention relates to a method of preventing or treating an invasive tumor, comprising:
  • Another general aspect of the present invention relates to a method of identifying a compound useful for suppressing at least one selected from the group consisting of the proliferation, migration and invasiveness of a tumor cell.
  • the method comprises identifying a compound increasing expression or biological activity of LECT2 in the tumor cell, wherein the biological activity comprises reducing MET phosphorylation in the tumor cell.
  • Embodiments of the present invention also relates to an isolated active fragment of a LECT2 protein capable of at least one of reducing phosphorylation of MET in a tumor cell and binding to the MET in the tumor cell, as well as an isolated polynucleotide encoding such a polypeptide, a vector comprising the polynucleotide, and a recombinant host cell comprising the vector.
  • the present invention also relates to use of a polypeptide comprising the amino acid sequence of a LECT2 protein or an active fragment thereof for the preparation of a medicament for suppressing at least one of the proliferation, migration and invasiveness of a tumor cell, wherein the polypeptide is capable of at least one of reducing phosphorylation of MET in a tumor cell and binding to the MET in the tumor cell.
  • a further general aspect of the present invention relates to use of a polynucleotide encoding a polypeptide comprising the amino acid sequence of a LECT2 protein or an active fragment thereof for the preparation of a medicament for suppressing at least one of the proliferation, migration and invasiveness of a tumor cell, wherein the polypeptide is capable of at least one of reducing phosphorylation of MET in a tumor cell and binding to the MET in the tumor cell.
  • Figure 1 include pictures of human RTK phosphorylation antibody arrays: the whole cell extracts from the human hepatocellular carcinoma cells (HCCs) were incubated on the RTK antibody array, phosphorylation status was determined by subsequent incubation with anti- phosphotyrosine horseradish peroxidase; each RTK was spotted in duplicate with the pairs of dots in each corner spotted with positive controls; each pair of positive RTK dots is denoted by a number, with the identity of the corresponding RTKs listed below the arrays; out of 25 the RTKs analyzed in the phosphorylation RTK array, 7 or 8 growth factor RTKs were highly phosphorylated in human HCC SK-Hepl cells;
  • FIG. 1 (A) shows that expression of LECT2 protein decreased phosphorylation of multiple receptor tyrosine kinases (RTKs) in HCC: SK-Hepl/LECT2 is a stable transfectant overexpressing LECT2; SK-Hepl/Neo is the vector control transfectant; Huh-7/shLECT2-2 is a stable transfectant with knockdown expression of LECT2; and Huh-7/shLuc is a vector control transfectant;
  • RTKs multiple receptor tyrosine kinases
  • Figure 1 (B) shows that treatment with recombinant LECT2 protein decreased multiple RTKs in SK-Hepl cells: the arrays were done with samples from total cell lysate of cells treated with or without the recombinant human LECT2-Fc protein (rLECT2) at a final concentration of 1.25 nM, 2.5 nM, or the controls;
  • FIG. 2 illustrate that LECT2 protein inhibited tyrosine phosphorylation of Met and downstream proteins in a dose-dependent and time-dependent manner as shown by Western blot analysis of the phosphorylation of Met, Erk and Akt proteins in SK-Hepl cells treated with rLECT2, each treatment was performed in triplicate, and the assays were repeated at least three times:
  • Figure 3 illustrate an inverse association of the LECT2 level and Met receptor activity in HCC tissue samples: Western blot was performed using anti-LECT2, anti-p-Met, anti-Met and anti- ⁇ - Actin antibodies to determine the levels of LECT2, p-Met, Met and ⁇ -Actin in HCC tumor tissues at various disease stages I, II and TV;
  • Figure 4 illustrate that LECT2 protein suppressed HGF-induced cell proliferation, migration, and invasion in HCC cells, each treatment was performed in triplicate, and the assays were repeated at least three times:
  • Figure 4(A) the growth properties of monolayer cultured HCC SK-Hepl cells exposed to fresh DMEM medium (control) or DMEM medium containing HGF (40 ng/mL) with different concentrations of rLECT2 (0-5 nM);
  • FIG. 4(B) a confluent SK-Hepl monolayer was sheared with a blue pipette tip, then exposed to fresh DMEM medium (control) or DMEM medium containing HGF (40 ng/mL) with different concentrations of rLECT2 (0-5 nM) for 14 hours, closure of the wound was observed;
  • Figure 4(C) in vitro invasion activity of SK-Hepl cells treated with rLECT2 for 16 hours as measured by the Boyden chamber;
  • Figure 5 show that LECT2 inhibited cell invasion and Met receptor tyrosine phosphorylation, dissociated the adaptor protein from MET, but increased the association of MET with PTP1B in HCC cells;
  • FIG. 5(B) LECT2 suppressed HGF-induced Met phosphorylation in LECT2-overexpressing transfectant SK-Hepl /LECT2, but increased HGF-induced Met phosphorylation was observed in LECT2-knockdown transfectants Huh-7/shLECT2-2, as compared with the vector control cells, the HGF-induced invasion ability of SK-Hepl /LECT2 was also reduced as compared to the control cells;
  • FIG. 5(C) Immunoprecipitation (IP) and Western Blot (WB) analyses of the effect of recombinant LECT2 on the dissociation of adaptor proteins from Met receptor;
  • FIG. 6 illustrate results from the IP, Far WB and flow cytometry analyses of LECT2-Met protein interaction in HCC cells:
  • Figure 6(A) ⁇ and WB analyses of endogenous LECT2 protein and Met from HCC cells
  • Figure 6(B) Recombinant expression of LECT2 and Met in 293T cells, and IP and WB analyses of the recombinant LECT2 protein and Met in the recombinant 293T cells;
  • Figure 6(D) Direct binding of recombinant LECT2 protein to Met receptor in a tube assay
  • Figure 6(E) Flow cytometry analyses of the LECT2 protein and Met binding assay in SK- Hepl cell
  • Figure 7 illustrate that the HXXXD motif of LECT2 protein was associated with Met receptor:
  • Figure 7(A) The docking results of the prediction models of LECT2 and Met receptor docking;
  • FIG. 7(B) LECT2 inhibited Met phosphorylation and invasion, but not mutant LECT2 having a mutated HXXXD motif, e.g., mLECT2-l or mLECT2-2;
  • FIG. 7(C) IP and WB analyses of the association of Met with LECT2 and mutant LECT2 proteins in HCC cells
  • Figure 8 show the effect of recombinant LECT2 protein on Met receptor tyrosine phosphorylation in other cancer cells; the cancer cells were treated with HGF 40 ng/mL combined with or without 2.5 nM recombinant LECT2 protein (rLECT2) for 15 minutes; in vitro invasion activity of the cancer cells was measured with the Boyden chamber after 24 hours; each treatment was performed in triplicate, and the assays were repeated at least three times:
  • FIG. 8(A) Lung cancer cell lines A549;
  • Figure 8(B) Breast cancer cell lines MB-MDA-231 ;
  • FIG. 8(C) Gastric cancer cell lines N87;
  • Figure 9 illustrates the design of point mutations of LECT2-full length in pSecTag2A vector.
  • the term "subject” means any animal, preferably a mammal, most preferably a human, to whom will be or has been administered compounds or pharmaceutical compositions according to embodiments of the invention.
  • the term "mammal” as used herein, encompasses 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., more preferably, a human.
  • a subject is in need of, or has been the object of observation or experiment of, treatment or prevention of a tumor characterized by Met receptor excessive activity.
  • biological sample refers to a sample obtained from an organism (e.g., patient) or from components (e.g., cells) of an organism.
  • the sample may be of any biological tissue, cell(s) or fluid.
  • the sample may be a "clinical sample” which is a sample derived from a patient. Such samples include, but are not limited to, sputum, blood, blood cells (e.g., white cells), amniotic fluid, plasma, semen, bone marrow, and tissue or fine needle biopsy samples, urine, peritoneal fluid, and pleural fluid, or cells therefrom.
  • Biological samples may also include sections of tissues such as frozen sections taken for histological purposes.
  • a biological sample may also be referred to as a "patient sample.”
  • a "biological sample” may also include a substantially purified or isolated protein, membrane preparation, or cell culture.
  • the term "instructions" when used in the context of a kit includes a publication, a recording, a diagram or any other medium of expression which can be used to communicate the usefulness of the kit for its designated use.
  • the instructions can, for example, be affixed to or included within a container for the kit.
  • treatment refers to an amelioration, prophylaxis, or reversal of a disease or disorder, or at least one discernible symptom thereof, for example, treating a HCC by reducing the phosphorylation of MET.
  • treatment refers to an amelioration, prophylaxis, or reversal of at least one measurable physical parameter related to the disease or disorder being treated, not necessarily discernible in or by the mammal.
  • treatment refers to inhibiting or slowing the progression of a disease or disorder, either physically, e.g., stabilization of a discernible symptom, physiologically, e.g., stabilization of a physical parameter, or both.
  • treatment or “treating” refers to delaying the onset of a disease or disorder.
  • compounds of interest are administered as a preventative measure.
  • prevention or “preventing” refers to a reduction of the risk of acquiring a given disease or disorder.
  • the specified compounds are administered as a preventative measure to a subject having an increased risk of developing a tumor, such as HCC, even though symptoms of the tumor are absent or minimal.
  • operably linked refers to a functional relationship between two nucleotide sequences.
  • a single-stranded or double-stranded nucleic acid moiety comprises the two nucleotide sequences arranged within the nucleic acid moiety in such a manner that at least one of the two nucleotide sequences is able to exert a physiological effect by which it is characterized upon the other.
  • a promoter sequence that controls transcription of a coding sequence is operably linked to that coding sequence.
  • Operably linked nucleic acid sequences can be contiguous, typical of many promoter sequences, or non-contiguous, in the case of, for example, nucleic acid sequences that encode repressor proteins.
  • operably linked is intended to mean that the coding sequence of interest is linked to the regulatory sequence(s) in a manner that allows for expression of the coding sequence, e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell.
  • a "promoter” refers to a portion of a gene that provides a control point for regulated gene transcription.
  • a promoter can include a binding site for R A polymerase.
  • a promoter can also include one or more binding sites for one or more transcription factors.
  • a promoter is often upstream of ("5' to") the transcription initiation site of a gene.
  • a promoter is typically adjacent to the transcriptional start site of the gene. However, a promoter can also be located at a distance from the transcriptional start site of the gene.
  • Sequence means the linear order in which monomers occur in a polymer, for example, the order of amino acids in a polypeptide or the order of nucleotides in a polynucleotide.
  • isolated protein or “isolated polypeptide” is one that is substantially separated from at least one of the other proteins present in the natural source of the protein, or is substantially free of at least one of the chemical precursors or other chemicals when the protein is chemically synthesized.
  • a protein is "substantially separated from” or “substantially free of other protein(s) or other chemical(s) in preparations of the protein when there is less than about 30%, 20%, 10%, or 5% or less, and preferably less than 1% (by dry weight) of the other protein(s) or the other chemical(s) (also referred to herein as a "contaminating protein” or a "contaminating chemical”).
  • an "isolated" nucleic acid molecule is one that is substantially separated from at least one of the other nucleic acid molecules present in the natural source of the nucleic acid, or is substantially free of at least one of the chemical precursors or other chemicals when the nucleic acid molecule is chemically synthesized.
  • An "isolated" nucleic acid molecule can also be, for example, a nucleic acid molecule that is substantially free of at least one of the nucleotide sequences that naturally flank the nucleic acid molecule at its 5' and 3' ends in the genomic DNA of the organism from which the nucleic acid is derived.
  • a nucleic acid molecule is "substantially separated from” or “substantially free of other nucleic acid molecule(s) or other chemical(s) in preparations of the nucleic acid molecule when there is less than about 30%, 20%, 10%, or 5% or less, and preferably less than 1%, (by dry weight) of the other nucleic acid molecule(s) or the other chemical(s) (also referred to herein as a "contaminating nucleic acid molecule” or a "contaminating chemical”).
  • recombinant refers to a polynucleotide, a polypeptide encoded by a polynucleotide, a cell, a viral particle or an organism that has been modified using molecular biology techniques to something other than its natural state.
  • leukocyte cell-derived chemotoxin 2 LECT2
  • LECT2 protein LECT2 protein
  • LECT2 chondromodulin-II
  • LECT2 has high sequence similarity to the chondromodulin repeat regions of the chicken myb-induced myeloid 1 protein.
  • Examples of LECT2 include that from various mammals, such as human, monkey, murine, rat, etc., the amino acid sequences of which can be found from published databases, such as the NCBI PubMed.
  • LECT2 is a human LECT2 comprising the amino acid sequence of SEQ ID No:2.
  • an “active fragment of LECT2” refers to a fragment of LECT2 that is still capable of at least one of reducing MET phosphorylation in a tumor cell and binding to MET in the tumor cell.
  • Examples of “active fragment of LECT2” can comprise an amino acid sequence that has greater than about 90 or 95% amino acid sequence identity, to the sequence of at least five consecutive amino acids of SEQ K ) NO:2.
  • the "active fragment of LECT2” comprises 50-100 amino acid residues of SEQ ID NO:2.
  • the "active fragment of LECT2" comprises the HXXXD motif.
  • the active fragment of LECT2 can be identified using methods known in the art. For example, fragments of LECT2 can be screened for their ability to reduce MET phosphorylation in an in vitro MET phosphorylation assay, or for their ability to bind MET in a binding assay. The hit molecules identified from the screening assay can be further tested in a cell based assay and/or an animal model.
  • Metal tyrosine kinase all refer to proto- oncogene product that is the hepatocyte growth factor receptor and has tyrosine-kinase activity, as well as its polymorphisms and isoforms encoded by transcript variants.
  • the primary single chain precursor protein is post-translationally cleaved to produce the alpha and beta subunits, which are disulfide linked to form the mature receptor.
  • Examples of MET include that from various mammals, such as human, monkey, murine, rat, etc., the amino acid sequences of which can be found from published databases, such as that from the NCBI PubMed.
  • MET is a human MET comprising the amino acid sequence as that in NCBI Reference Sequence: NP 000236, NP 000592, etc.
  • Hepatocyte growth factor and "HGF,” as used herein interchangeably, all refer to a growth factor that regulates cell growth, cell motility, and morphogenesis by activating a tyrosine kinase signaling cascade after binding to the c-Met receptor, as well as its polymorphisms and isoforms encoded by transcript variants.
  • Hepatocyte growth factor is secreted by mesenchymal cells and acts as a multi-functional cytokine on cells of mainly epithelial origin. Its ability to stimulate mitogenesis, cell motility, and matrix invasion gives it a central role in angiogenesis, tumorogenesis, and tissue regeneration.
  • HGF human growth factor-1
  • HGF is a human HGF comprising the amino acid sequence as that in GenBank, e.g., AAA64297, AAA64239 or AAA52649.
  • HXXXD motif refers to a motif of the LECT2 conserved among different mammals, which is important for LECT2's activity to reduce the phosphorylation of MET. Mutations in the HXXXD motif resulted in abolished or greatly reduced biological activity of LECT2 Such mutations include, but are not limited to mLECT2-l and mLECT2-l illustrated in Fig. 9.
  • LECT2 is a 16-kDa basic protein and is specifically expressed in the adult and fetal livers. Its biological functions in tumor progression, such as migration, invasion and metastasis, have not been reported. Studies from the present invention indicated that LECT2 strongly reduced tumor growth and invasiveness in HCC. Activation of the receptor tyrosine kinases (TK) are critical pathogenetic event in HCC. It was discovered from the present invention that LECT2 inhibited tumor invasion via inhibition of phosphorylation of MET and downstream RTKs in the HGF/MET signal transduction pathway.
  • TK receptor tyrosine kinases
  • LECT2 inhibited HGF- induced Met activation by binding to MET at a site noncompetitive with the binding site of HGF Mutations in the HXXXD motif of LECT2 resulted in the loss of the tumor suppression activity of LECT2.
  • LECT2 also reduced Met tyrosine phosphorylation and cancer cells invasion in other cancer cells, such as the lung, breast, and gastric cancer cells. Accordingly, LECT2 suppresses the invasiveness and Met receptor phosphorylation of tumor cells, which represents a novel therapeutic approach for treating tumors characterized by Met receptor excessive activity.
  • the present invention relates to a method of suppressing at least one selected from the group consisting of the proliferation, migration and invasiveness of a tumor cell.
  • the method comprises administering to the tumor cell an agent to increase protein level or biological activity of a LECT2 protein or an active fragment thereof in the tumor cell to thereby reduce MET phosphorylation in the tumor cell.
  • the agent can also reduce phosphorylation of one or more downstream proteins in the HGF/MET signal transduction pathway, including but not limited to, Erk and Akt.
  • a polypeptide comprising the amino acid sequence of a LECT2 protein or an active fragment thereof can be administered to the tumor cell.
  • the polypeptide comprises a LECT2 protein, such as a human LECT 2 having the amino acid sequence of SEQ ID NO: 2.
  • the polypeptide comprises an active fragment of LECT2 protein, which is capable of at least one of reducing phosphorylation of MET and binding to MET in a tumor cell.
  • the active fragment is a fragment of SEQ ID NO: 2.
  • the active fragment comprises the HXXXD motif of LECT2 or a derivative thereof.
  • the polypeptide can be obtained from various sources.
  • it can be a native LECT2 protein or an active fragment thereof expressed by a mammalian cell endogenously and isolated from the cell using methods known in the art in view of the present disclosure.
  • the polypeptide is a recombinant polypeptide expressed by a recombinant host cell, preferably at an increased expression level. Any of the methods of recombinant expression known to those skilled in the art can be used to produce the recombinant LECT2 in view of the present disclosure.
  • the polypeptide can have several different physical forms. It can exist as a full-length nascent or unprocessed polypeptide, or as a partially processed polypeptide or as a combination of processed polypeptides
  • the full-length nascent polypeptide can be postranslationally modified by specific proteolytic cleavage events that result in the formation of fragments of the full-length nascent polypeptide.
  • a fragment, or physical association of fragments can have the biological activity associated with the full-length polypeptide; however, the degree of biological activity associated with individual fragments can vary.
  • the polypeptide can also exist as a modified form, including but not limited to, glycosylated forms, myristoylated forms, palmitoylated forms, ribosylated forms, acetylated forms, ubiquitinated forms, etc. Modifications also include intra-molecular crosslinking and covalent attachment to various moieties such as lipids, flavin, biotin, polyethylene glycol or derivatives thereof, etc. In addition, modifications may also include cyclization, branching and cross-linking. Further, amino acids other than the conventional twenty amino acids encoded by the codons of genes may also be included in a polypeptide.
  • the protein level or biological activity of a LECT2 protein or an active fragment thereof is increased by administering to the tumor cell a polynucleotide comprising a nucleotide sequence encoding the LECT2 protein or the active fragment thereof.
  • the polynucleotide comprises a regulatory sequence, such as a promoter, operably linked to the coding sequence of the LECT2 protein or the active fragment thereof, which directs the expression of the LECT2 protein or the active fragment thereof in the tumor cell.
  • the encoded LECT2 protein comprises a LECT2 protein, such as a human LECT 2 having the amino acid sequence of SEQ ID NO: 2.
  • the encoded polypeptide comprises an active fragment of LECT2 protein, which is capable of at least one of reducing phosphorylation of MET and binding to MET in a tumor cell.
  • the encoded active fragment can be a fragment of SEQ ID NO:2, or a fragment comprises the HXXXD motif of LECT2 or a derivative thereof.
  • the polynucleotide can be obtained by various means, including, but not limited to: (i) amplification in vitro by, for example, polymerase chain reaction (PCR); (ii) synthesis by, for example, chemical synthesis; (iii) recombinantly produced by cloning; or (iv) purified, as by cleavage and electrophoretic or chromatographic separation.
  • PCR polymerase chain reaction
  • the polynucleotide includes, without limitation, separate nucleic acid molecules (e.g., cDNA or genomic DNA fragments produced by PCR or restriction endonuclease treatment) independent of other sequences, as well as nucleic acid molecules that are incorporated into a vector, an autonomously replicating plasmid, a virus (e.g., a retrovirus, adenovirus, or herpes virus), or into the genomic DNA of an eukaryote.
  • nucleic acid molecules e.g., cDNA or genomic DNA fragments produced by PCR or restriction endonuclease treatment
  • nucleic acid molecules that are incorporated into a vector, an autonomously replicating plasmid, a virus (e.g., a retrovirus, adenovirus, or herpes virus), or into the genomic DNA of an eukaryote.
  • the polynucleotide can be introduced into the tumor cell using any suitable method including, for example, electroporation, calcium phosphate precipitation, microinjection, transformation, biolistics and viral infection, liposome delivery, etc.
  • the polynucleotide can be maintained on an episomal element, such as a plasmid.
  • the polynucleotide has become integrated into the chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the stably transformed or transfected cell to establish cell lines or clones comprised of a population of daughter cells containing the exogenous polynucleotide.
  • the method comprises administering to the tumor cell a compound that increases expression and/or the biological activity of the LECT2 protein or the active fragment thereof in the tumor cell.
  • the biological activity comprises reducing MET phosphorylation in the tumor cell and/or binding to MET in the tumor cell.
  • Such compound can be identified using methods described below.
  • the method according to embodiments of the present invention can be used with various tumor cells, including, but not limited to that selected from the group consisting of a hepatocellular carcinoma cell, a lung cancer cell, a breast cancer cell, a gastric cancer cell, an ovarian carcinoma cell, a hypopharyngeal carcinoma cells, a Glioblastoma cells, or any other tumor cell that expresses a Met receptor.
  • a hepatocellular carcinoma cell including, but not limited to that selected from the group consisting of a hepatocellular carcinoma cell, a lung cancer cell, a breast cancer cell, a gastric cancer cell, an ovarian carcinoma cell, a hypopharyngeal carcinoma cells, a Glioblastoma cells, or any other tumor cell that expresses a Met receptor.
  • the present invention relates to a method of preventing or treating hepatocellular carcinoma in a subject, comprising administering to the subject an agent to increase protein level or biological activity of a LECT2 protein or an active fragment thereof in a hepatocellular carcinoma cell of the subject, wherein the biological activity comprises reducing MET phosphorylation in the hepatocellular carcinoma cell.
  • the LECT2 protein has the amino acid sequence of SEQ ID NO: 2.
  • Protein therapy can be used to increase the protein level of LECT2 or an active fragment thereof in a hepatocellular carcinoma cell of the subject.
  • a therapeutically effective amount of LECT2 protein or an active fragment thereof is administered to the subject to thereby reduce MET phosphorylation in the hepatocellular carcinoma cell and prevent or treat hepatocellular carcinoma in the subject.
  • the LECT2 protein or an active fragment thereof can be formulated and administered to the subject using methods in the art for protein formulation and delivery, in view of the present disclosure.
  • gene therapy can be used to increase the expression of LECT2 or an active fragment thereof in a hepatocellular carcinoma cell of the subject by introducing a nucleic acid molecule capable of expressing LECT2 protein in the hepatocellular carcinoma cell.
  • the method comprises administering to the subject a polynucleotide comprising a nucleotide sequence encoding the LECT2 protein or the active fragment thereof to thereby reduce MET phosphorylation in the hepatocellular carcinoma cell and prevent or treat hepatocellular carcinoma in the subject.
  • gene therapy can involve introduction in vitro of a functional copy of a gene into a cell(s) of a subject, and returning the genetically engineered cell(s) to the subject.
  • the functional copy of the gene is under operable control of regulatory elements, which permit expression of the gene in the genetically engineered cell(s).
  • Numerous transfection and transduction techniques as well as appropriate expression vectors are well known to those of ordinary skill in the art, some of which are described in PCT application WO95/00654.
  • In vivo gene therapy uses vectors such as adenovirus, retroviruses, vaccinia virus, bovine papilloma virus, and herpes virus such as Epstein- Barr virus. Gene transfer can also be achieved using non-viral means requiring infection in vitro. Such means can include calcium phosphate, DEAE dextran, electroporation, and protoplast fusion. Targeted liposomes can also be potentially beneficial for delivery of DNA into a cell.
  • a DNA molecule encoding a human LECT2 protein can be first cloned into a retroviral vector.
  • the expression of LECT2 protein from the vector can be driven from its endogenous promoter or from the retroviral long terminal repeat or from a promoter specific for certain target cells.
  • the vector can then be introduced into a cell of a subject to successfully express LECT2 proteins in the target cells.
  • the gene can be preferably delivered to those cells in a form which can be used by the cell to encode sufficient protein to provide effective function.
  • Retroviral vectors are often a preferred gene delivery vector for gene therapy especially because of their high efficiency of infection and stable integration and expression.
  • LECT2 encoding DNA can be transferred into cells for gene therapy by non-viral techniques including receptor-mediated targeted DNA transfer using ligand-DNA conjugates or adenovirus-ligand-DNA conjugates, lipofection membrane fusion or direct microinjection. These procedures and variations thereof are suitable for ex vivo as well as in vivo LECT2 gene therapy. Protocols for molecular methodology of gene therapy suitable for use with the LECT2 gene are described in Gene Therapy Protocols, edited by Paul D. Robbins, Human press, Totowa NJ, 1996.
  • a small molecule compound can be used to increase the expression or biological activity of LECT2 in the hepatocellular carcinoma cell of the subject.
  • the method comprises administering to the subject a compound effective to increase the expression or biological activity of LECT2 in the hepatocellular carcinoma cell to thereby reduce MET phosphorylation in the hepatocellular carcinoma cell and prevent or treat hepatocellular carcinoma in the subject.
  • the effective amount of polypeptides, nucleic acid molecules or chemical compounds of the invention administered to individuals can vary according to a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular polypeptide, nucleic acid molecule or chemical compound employed.
  • a physician or veterinarian of specialized skill in the treatment can determine and prescribe the effective amount required to prevent, counter or arrest the progress of the condition.
  • Optimal precision in achieving concentrations within the range that yields efficacy without toxicity requires a regimen based on the kinetics of the active pharmaceutical ingredient's availability to target sites. This involves a consideration of the distribution, equilibrium and elimination of the active pharmaceutical ingredient involved in the therapy.
  • the method disclosed herein can be used alone at appropriate dosages defined by routine testing in order to obtain optimal increase of the LECT2 level or biological activity while minimizing any potential toxicity.
  • co-administration or sequential administration of other agents may be desirable.
  • the dosages of administration are adjusted when several agents are combined to achieve desired effects. Dosages of these various agents can be independently optimized and combined to achieve a synergistic result wherein the pathology is reduced more than it would be if either agent were used alone.
  • the method according to embodiments of the present invention is combined with another anti-tumor therapy, including, but are not limited to another chemotherapy, radiotherapy, etc.
  • the present invention relates to a pharmaceutical composition for treating a tumor, comprising a carrier and a polypeptide comprising the amino acid sequence a LECT2 protein or an active fragment thereof, wherein the polypeptide is capable of at least one of reducing phosphorylation of MET in a tumor cell and binding to the MET in the tumor cell.
  • the pharmaceutical composition comprises a human LECT2 protein, e.g., that having 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, which comprises a carrier and a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence a LECT2 protein or an active fragment thereof, wherein the polypeptide is capable of at least one of reducing phosphorylation of MET in a tumor cell and binding to the MET in the tumor cell.
  • the pharmaceutical composition comprises a polynucleotide that encodes a human LECT2 protein, e.g., that having the amino acid sequence of SEQ ID NO: 2, or an active fragment thereof.
  • the present invention relates to a pharmaceutical composition for treating a tumor, comprising a carrier and a compound that increases expression or biological activity of a LECT2 protein or an active fragment thereof in the cell.
  • the biological activity comprises at least one of reducing phosphorylation of MET in a tumor cell and binding to the MET in the tumor cell.
  • Such compound can be identified using methods described below.
  • the therapeutic active ingredient according to embodiments of the present invention can be administered by any known route of administration, including, orally, topically, parenterally (including subcutaneous, intravenous, intramuscular, and intrasternal injection or infusion administration techniques), by inhalation spray or rectally in dosage units or pharmaceutical compositions containing conventional pharmaceutically acceptable carriers and any such dosage units or pharmaceutical compositions are within the scope of the present invention.
  • compositions adapted for oral administration include solid forms such as pills, tablets, caplets, and hard or soft capsules (each including immediate release, timed release, and sustained release formulations) as well as lozenges and dispersible powders or granules.
  • Liquid forms of pharmaceutical compositions adapted for oral administration include solutions, syrups, elixirs, emulsions, and aqueous or oily suspensions. Any of these dosage forms may be prepared according to any method or compounding technique known in the art for the manufacture of pharmaceutical compositions.
  • compositions adapted for oral administration may further include one or more sweetening agents, flavoring agents, coloring agents, or preserving agents in order to provide attractive or palatable preparations.
  • compositions adapted for oral administration may also be presented as hard or soft gelatin capsules, wherein the active ingredient may be mixed with an inert solid diluent, such as calcium carbonate, calcium phosphate or kaolin in the case of the former or with water or miscible solvents such as propylene glycol, PEG's and ethanol, or an oil medium such as peanut oil, liquid paraffin, or olive oil in the case of the latter.
  • an inert solid diluent such as calcium carbonate, calcium phosphate or kaolin in the case of the former or with water or miscible solvents such as propylene glycol, PEG's and ethanol, or an oil medium such as peanut oil, liquid paraffin, or olive oil in the case of the latter.
  • Aqueous suspensions can be prepared that contain the active ingredient(s) in admixture with excipients suitable for the manufacture of aqueous suspensions.
  • excipients include suspending agents, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia, dextran, polyvinylpyrrolidone or gelatin; and dispersing or wetting agents such as lecithin, polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate, and polyethylene sorbitan monooleate.
  • Aqueous suspensions may also contain one or more preservatives, such as ethyl or n- propyl, p-hydroxybenzoate; one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose, saccharine or aspartame.
  • Oily suspensions may be formulated by suspending the active lngredient(s) in a vegetable oil, such as cottonseed, olive, sesame or coconut oil, or in a mineral oil, such as liquid paraffin.
  • the oily suspensions may contain a thickening agent, such as beeswax, hard paraffin, or cetyl alcohol. Sweetening agents such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation.
  • Such oily suspensions may be preserved by the inclusion of an antioxidant such as ascorbic acid.
  • Dispersible powders and granules suitable for the preparation of an aqueous suspension suitable for oral administration can provide the active ingredient(s) in admixture with a dispersing or wetting agent, suspending agent, and one or more preservatives, all of which have been discussed above.
  • a dispersing or wetting agent suspending agent
  • preservatives all of which have been discussed above.
  • Sweetening, flavoring, or coloring agents may also be present, if desired.
  • compositions suitable for oral administration may also be presented in the form of an oil-in-water emulsion.
  • the oily phase may be a vegetable or mineral oil, such as those described above, or mixtures of these.
  • Suitable emulsifying agents may be naturally-occurring phosphatides, such as soy bean, lecithin, sorbitan monooleate, or polyoxyethylene sorbitan monooleate.
  • the emulsions may also contain sweetening or flavoring agents.
  • Syrups and elixirs may be formulated with sweetening agents, for example, glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative and flavoring or coloring agents.
  • sweetening agents for example, glycerol, propylene glycol, sorbitol or sucrose.
  • Such formulations may also contain a demulcent, a preservative and flavoring or coloring agents.
  • compositions may be further provided in a form adapted for parenteral administration, i.e., by injection or infusion.
  • injectable aqueous or oleaginous suspensions are desirably sterile and may be formulated according to known methods using suitable dispersing, wetting and suspending agents as mentioned above.
  • a parenterally-acceptable diluent or solvent may also be utilized, such as 1 ,3-butanediol, water, Ringer's solution, and isotonic sodium chloride.
  • Cosolvents such as ethanol, propylene glycol or polyethylene glycols may also be used.
  • sterile, fixed oils are conventionally employed as solvents or suspending mediums in injectable or infusible solutions, and these may include any bland fixed oil, such as any of the synthetic mono- or diglycerides.
  • Fatty acids such as oleic acid also may be utilized in the preparation of injectable or infusible solutions.
  • the pharmaceutical composition may also be presented in the form of a suppository.
  • Suppositories can be formulated by mixing the active ingredient(s) and any additional desired therapeutic agent(s) with a suitable non-irritating excipient that is solid at room temperature but molten at body temperature, thereby releasing the active ingredient(s).
  • suitable materials include cocoa butter and polyethylene glycols.
  • Topical formulations may include cosolvents, emulsifiers, penetration enhancers, preservatives, emollients, and the like.
  • the active ingredients according to embodiments of the present invention can also be provided in a pharmaceutical composition in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles, and multilamellar vesicles.
  • Liposomes can be formed from a variety of lipids, including but not limited to amphipathic lipids such as phosphatidylcholines, sphingomyelins, phosphatidylethanolamines, phophatidylcholines, cardiolipins, phosphatidylserines, phosphatidylglycerols, phosphatidic acids, phosphatidylinositols, diacyl trimethylammonium propanes, diacyl dimethylammonium propanes, and stearylamine, neutral lipids such as triglycerides, and combinations thereof. They may either contain cholesterol or may be cholesterol-free.
  • Another general aspect of the present invention relates to a method of identifying a subject having an increased risk of developing an invasive tumor. The method comprises:
  • step (b) when the level measured from step (b) is higher than that of the control, the subject is identified as having the increased risk of developing the invasive tumor
  • the tumor is hepatocellular carcinoma.
  • the phosphorylation level of MET can be measured using any method known in the art in view of the present disclosure, for example, by Western blot.
  • the protein level or biological activity of LECT2 protein can be measured by various means.
  • the protein level of LECT2 can be measured by any method for measuring a polypeptide known to those skilled in the art, including, but not limited to, enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, and immunohistochemistry, using an anti-LECT2 antibody.
  • the protein level of LECT2 can also be measured indirectly by measuring the level of LECT2 mRNA in the biological sample, e.g., via RT- PCR.
  • the biological activity can be measured by binding assay of the ability of LECT2 to bind to MET, or by phopshorylation of MET by HGF in the presence of LECT2.
  • the biological activity of LECT2 is measured by detecting the present of an HXXXD mutation, which is known to reduce the biological activity.
  • the method comprises:
  • mutations in the HXXXD motif include, but are not limited to: 1) a deletion of one or more amino acids from the motif; 2) an addition of one or more amino acids to the motif; 3) a substitution of one or more amino acids of the motif; etc.
  • mutations can be detected using known molecular biology techniques in view of the present disclosure.
  • assay techniques known in the art which can be used for detecting mutations, such as selective amplification, and selective primer extension, restriction enzyme cleavage patterns, hybridizing a sample and control nucleic acids, sequence comparison, etc.
  • Embodiments of the present application also relates to a kit for identifying a subject having an increased risk of developing an invasive tumor.
  • the kit comprises:
  • the tumor is hepatocellular carcinoma.
  • such a kit preferably comprises a compartmentalized carrier suitable to hold in close confinement at least one container.
  • the carrier can contain a means for detection.
  • the kit can comprise a labeled compound or agent capable of detecting the phosphorylated MET, a labeled compound or agent capable of detecting the LECT2 polypeptide or mRNA encoding the LECT2 polypeptide and means for determining the amount of the polypeptide or mRNA in a sample (e.g., an antibody which binds the polypeptide or an oligonucleotide probe which binds to DNA or mRNA encoding the polypeptide).
  • the kit can also comprise a means for the biological activity measurement, such as reagents for MET RTK phosphorylation assay.
  • a means for the biological activity measurement such as reagents for MET RTK phosphorylation assay.
  • instructions are packaged with the reagents, for example, in a pamphlet or package label.
  • the labeling instructions explain how to use the reagents to measure protein level or biological activity of LECT2 protein from a biological sample and how to assess the risk of developing invasive tumor, such as advanced stage of hepatocellular carcinoma, based on the measured level, e.g., for determining whether a test subject is suffering from or is at risk of developing HCC if the level of LECT2 protein or its biological activity is below a normal or control level.
  • the kit comprises:
  • PCR polymerase chain reaction
  • a combined method according to an embodiment of the present invention comprises:
  • the combined method is used to prevent or treat a tumor selected from the group consisting of a hepatocellular carcinoma, a lung cancer, a breast cancer, a gastric cancer, an ovarian carcinoma, a hypopharyngeal carcinoma, a glioblastoma, or any other tumor that expresses the MET.
  • Another general aspect of the present invention relates to a method of identifying a compound useful for suppressing at least one selected from the group consisting of the proliferation, migration and invasiveness of a tumor cell.
  • the method comprises identifying a compound increasing expression or biological activity of LECT2 in the tumor cell, wherein the biological activity comprises reducing MET phosphorylation in the tumor cell.
  • candidate compounds encompass numerous chemical classes. Although typically they are organic compounds, preferably, small organic compounds, candidate compounds also can be biomolecules such as peptides, nucleic acid, saccharides, fatty acids, sterols, isoprenoids, purines, pyrimidines, derivatives or structural analogs of the above, or combinations thereof and the like. Where the compound is a nucleic acid, the compound typically is a DNA or RNA molecule, although modified nucleic acids having non-natural bonds or subunits are also contemplated.
  • the compound identification methods can be performed using conventional laboratory formats or in assays adapted for high throughput.
  • high throughput refers to an assay design that allows easy screening of multiple samples simultaneously, and can include the capacity for robotic manipulation.
  • Another desired feature of high throughput assays is an assay design that is optimized to reduce reagent usage, or minimize the number of manipulations in order to achieve the analysis desired.
  • assay formats include 96-well or 384-well plates, levitating droplets, and "lab on a chip" microchannel chips used for liquid handling experiments. It is well known by those in the art that as miniaturization of plastic molds and liquid handling devices are advanced, or as improved assay devices are designed, that greater numbers of samples can be performed using the design of the present invention.
  • the compound is identified by its ability to increase the expression of LECT2 in a tumor cell using a reporter assay.
  • the compound identification method comprises: (a) providing a cell expressing a reporter gene under the control of a LECT2 regulatory sequence from the tumor cell;
  • LECT2 regulatory sequence refers to a nucleic acid sequence that can control the expression of the LECT2 open reading frame in the tumor cell.
  • the compound is identified by its ability to increase the interaction between MET and LECT2 in the tumor cell.
  • the method comprises:
  • the determining step comprises measuring the amount of protein phosphorylation of the Met or the active fragment thereof, and the identified test compound decreases the amount of protein phosphorylation.
  • the reaction mixture further comprises an HGF or an active fragment thereof.
  • the compound useful for the present invention can also be identified from a binding assay, comprising:
  • step (c) comparing the amount determined in step (b) with a control measurement wherein the Met or active fragment thereof has been contacted with the labeled LECT2 or the labeled active fragment of LECT2 in the absence of test compound.
  • the compound useful for the present invention can also be identified as a compound that binds a Met and mimics LECT2, comprising:
  • step (b) determining a biological activity of the Met or active fragment thereof; and (c) comparing the result determined in step (b) with that of a control measurement wherein the Met or the active fragment thereof was contacted with LECT2 or an active fragment thereof in the absence of the test compound.
  • the Met is in an isolated membrane preparation
  • the determining step comprises measuring the amount of protein phosphorylation of the isolated membrane preparation, and the identified test compound decreases the amount of protein phosphorylation.
  • the method further comprises testing the identified test compound for its ability to suppress proliferation, migration or invasiveness of the tumor cell in vivo or in vitro.
  • Another general aspect of the invention relates to an isolated polynucleotide having a nucleotide sequence encoding an amino acid sequence of an active fragment of LECT2, such as about 51-100 amino acid residues of SEQ ID NO:2, that is capable of reducing the phosphorylation of Met or binding to Met, or a complement thereof.
  • Such polynucleotide can be identified by screening DNA constructs containing various portions of the coding sequences of SEQ ID NO:2 for their ability to encode an active fragment of LECT2.
  • the screening can be conducted using methods in the art, such as phage display.
  • Another general aspect of the present invention relates to an isolated polypeptide having an active fragment of LECT2, such as about 51-100 amino acid residues of SEQ ID NO:2, that is capable of reducing the phosphorylation of Met or binding to Met.
  • the vector and recombinant cells for recombinant expression of the active fragment of LECT2 are also encompassed by embodiments of the present application.
  • Another general aspect of the present invention relates to use of a polypeptide comprising the amino acid sequence of a LECT2 protein or an active fragment thereof for the preparation of a medicament for suppressing at least one of the proliferation, migration and invasiveness of a tumor cell, wherein the polypeptide is capable of at least one of reducing phosphorylation of MET in a tumor cell and binding to the MET in the tumor cell.
  • the tumor cell is selected from the group consisting of a hepatocellular carcinoma cell, a lung cancer cell, a breast cancer cell, a gastric cancer cell, an ovarian carcinoma cell, a hypopharyngeal carcinoma cells, a Glioblastoma cells, or any other tumor cell that expresses a MET receptor.
  • the polypeptide comprises a LECT2 protein having the amino acid sequence of SEQ ID NO:2 or an active fragment thereof.
  • Yet another general aspect of the present invention relates to use of a polynucleotide encoding a polypeptide comprising the amino acid sequence of a LECT2 protein or an active fragment thereof for the preparation of a medicament for suppressing at least one of the proliferation, migration and invasiveness of a tumor cell, wherein the polypeptide is capable of at least one of reducing phosphorylation of MET in a tumor cell and binding to the MET in the tumor cell.
  • the tumor cell is selected from the group consisting of a hepatocellular carcinoma cell, a lung cancer cell, a breast cancer cell, a gastric cancer cell, an ovarian carcinoma cell, a hypopharyngeal carcinoma cells, a Glioblastoma cells, or any other tumor cell that expresses a MET receptor.
  • the polynucleotide encodes a LECT2 protein having the amino acid sequence of SEQ ID NO:2 or an active fragment thereof.
  • Hepatocellualr carcinoma cells were grown in DMEM medium (Life Technologies Inc., GIBCO BRL, Rockville, Md.) with 10% fetal bovine serum (FBS) and 2 mM L-glutamine (Life Technologies Inc.) at 37 °C in a humidified atmosphere of 5% CC>2-95% air. Cells were cultured according to the supplier's recommendations. Adherent cells were detached from the culture dishes by treatment with trypsin-EDTA.
  • A549 human lung adenocarcinoma was maintained and cultured in DMEM medium.
  • CLl -5 human lung adenocarcinoma
  • MB-MDA231 human breast cancer
  • N87 human gastric cancer
  • the expression vector LECT2 was constructed by placing the human LECT2 cDNA in the pSecTag2A eukaryotic expression vector containing the hygromycin B gene under the control of the same promoter.
  • the LECT2 -sense expression constructs were transfected into hepatoma cells using Lipofectamine 2000 reagent (Invitrogen Life tech.). Stable cell populations were selected by 50 /z g/mL hygromycin B. Single clones were confirmed to have prominent expression of LECT2 by RT-PCR and Western Blot analysis.
  • RTKs Receptor Tyrosine Kinases
  • RTKs growth factor receptor tyrosine kinases
  • the lysates were gently resuspended by pipetting up and down and rocked at 2-8 °C for 30 minutes. The lysates were then microcentrifuged at 14,000 x g for 5 minutes. The supernatant was transferred into a clean test tube and stored at -80°C. Five-hundred micrograms of total proteins from the HCC cell supernatant were incubated with RTK array membranes spotted with various anti-phospho-RTK antibodies. The phosphorylation detection procedures were performed according to the manufacturer's protocol.
  • Cancer cells at 90% confluent growth were trypsinized, transferred to 24-well plates (3 x 10 4 cells/well), and preincubated in a culture medium for 24 hours. The culture medium was then changed to fresh medium and incubated at 37°C for 72 hours. At the end of this period, numbers of viable cells were estimated using the MTT assay.
  • a solution of MTT (Sigma, St Louis, MO) (2 mg/mL), 200 piL, was added into each well 2 hours before experiments turn over, then incubated in the darkness.
  • the foemazan grain was then dissolved in DMSO, and the absorbance at 570 nm was read using an ELISA reader.
  • Invasion assays were performed using transwell inserts for 24-well plate containing 8- ⁇ m pores (Millipore). Matrigel (70 g; Collaborative Biomedical, Becton Dickinson Labware, USA) coated filters were used for invasion assay. Cells (1 ⁇ 10 5 in 100 /zL of DMEM complete medium) were placed in the upper chamber, and 1 mL of the same medium was placed in the lower chamber. After 16 hours in the culture, cells were fixed with methanol for 20 min. Cells on the upper side of the filters were removed with cotton-tipped swabs, and the filters were washed with PBS. Cells were then stained with 0.05 % crystal violet in PBS. Cells on the underside of the filters were viewed and counted under a microscope. Transwell membranes were used for migration assay and 2 x 10 4 cells were fixed after 16 hours
  • the membranes were then washed in PBST and incubated with the HRP- conjugated anti-Met or anti-LECT2 antibody, and revealed by enhanced chemiluminescence reagents (Amersham Pharmacia Biotech, USA) and photographed with Kodak X-Omat Blue autoradiography film.
  • Proteins in the total cell lysates or cell supernatant were separated by SDS-PAGE and electrotransferred to a polyvinylidene difluoride membrane (Immobilon-P membrane; Millipore Corp., Bedford, MA). After blot blocking in PBST, membrane-bound proteins were probed with primary antibodies against LECT2 (R&D system), PY-99 (Santa Cruz Biotechnology, Santa Cruz, CA), p-Met and Met (cell signaling). The membrane was washed and then incubated with horseradish peroxidase-conjugated secondary antibodies for 30 min. Antibody-bound protein bands were detected with enhanced chemiluminescence reagents (Amersham Pharmacia Biotech, USA) and photographed with Kodak X-Omat Blue autoradiography film.
  • the cellular lysates were prepared as described previously, and equal amounts of protein were incubated with specific antibody immobilized onto protein A-Sepharose beads for 2 h at 4 °C with gentle rotation. The beads were washed extensively with lysis buffer, boiled, and microcentrifuged. Proteins were resolved by SDS-PAGE and transferred to nitrocellulose membrane. After blocking in PBST, the membrane was incubated with specific primary antibodies. After washing and incubating with secondary antibodies, immunoreactive proteins were visualized using enhanced chemiluminescence detection (Amersham, Arlington Heights, IL). Where indicated, the membranes were stripped and reprobed with another antibody.
  • the binding of Met to the recombinant Fc-tagged LECT2 was analyzed by Fc tag pull-down of bound complexes with protein A-Sepharose.
  • the rLECT2 was purified from mammary cells by Ni-NTA resin (more than 95% purity). Met protein was mixed with the purified rLECT2 in a binding buffer (50 mM sodium phosphate, pH 7.5, 500 mM Nacl, 1% Nonidet P-40, final volume 150 ⁇ 1), and incubated at 4°C for 4 h, with gentle rotation.
  • Ni-NTA resin beads 50 ⁇ 1 pre- equilibrated in binding buffer were added to the mixture and incubated at 4°C for 2 h with gentle rotation. The resin beads were sedimented by brief centrifugation and washed three times with the binding buffer. The proteins associated with the resin beads were extracted with 50 ⁇ 1 of 2X Laemmli buffer and analyzed by Western blot analysis using anti-LECT2 antibody.
  • SK-Hepl cells at 90% confluent growth were trypsinized, transferred to a 6-well plate and incubated in a culture medium overnight.
  • the culture medium was changed to a serum-free medium, then added with recombinant Fc-tagged LECT2 protein for 5 minutes.
  • the cells were resuspended in FACS staining buffer containing anti-LECT2 antibody for 1 hr at 4°C .
  • the cells were washed with FACS staining buffer by centrifugation at 900rpm for 5minutes.
  • a secondary antibody- conjugated FITC was added to the cells and incubated in the dark for 30 minutes.
  • the cells were kept on ice until the FACS analyzer was ready. Dead cells were gated out by propidium iodide staining. Live cells were analyzed by FACS.
  • Phosphorylation-receptor tyrosine kinases (RTKs) array was used to determine the phosphorylation profile of growth factor RTKs in HCC cells having different levels of LECT2, e.g., stable HCC transfectants with overexpression or knockdown expression of LECT2 ( Figure 1 A), or HCC cells treated with or without recombinant LECT2 protein ( Figure IB).
  • the phosphorylation level of growth factor RTKs in HCC cells was quantified by dot blotting.
  • FIG. IB the phosphorylation levels of several growth factor RTKs were differentially affected after the HCC cells were treated with recombinant LECT2 protein in a dose- dependent manner (Figure IB).
  • 7 growth factor RTKs were highly phosphorylated in SK-Hepl cells.
  • These RTKs are EGFR, HGFR (or c-Met, Met), Tie-2, FGF-R3, c-Ret, ROR1 , and Dtk.
  • the array was done with samples from total cell lysate treated with or without recombinant LECT2 protein at 1.25 or 2.5 nM final concentration.
  • the phosphorylation level of the following RTKs in HCC was significantly altered after the treatment with recombinant LECT2: HGFR, Tie-2, and FGF-R3 No or little change of the phosphorylation level of EGFR was observed after the HCC was treated with the recombinant LECT2 protein.
  • the LECT2 protein level was inversely associated with Met tyrosine phosphorylation in HCC tumor tissues.
  • LECT2 may regulate Met phosphorylation in HCC cells
  • the levels of LECT2 and Met phosphorylation were analyzed in 74 surgically resected HCC tissue samples from patients in the National Taiwan University Hospital. Using Western Blot analysis, it was found that stronger LECT2 protein expression correlated with lower Met phosphorylation in the tissue samples, and that less LECT2 protein and more Met phosphorylation were found in the higher stage HCC (Figure 3).
  • HGF Met signaling pathway which includes HGF and its receptor Met, is now recognized as a promising target for cancer therapy, e.g., for the inhibition of cell proliferation, migration and invasion. Studies were conducted to determine whether LECT2 regulates HCC cell phenotypes via the HGF Met signaling pathway.
  • SK-Hepl cells were seeded onto the cell culture wells and treated with HGF (40 ng/mL) in combination with rLECT2 at 0, 1.25, 2.5 nM final concentration as indicated for 72 hr in MTT cell proliferation assay.
  • HGF serum-derived neurotrophic factor
  • LECT2 inhibited HGF stimulated cell proliferation in a dosage dependent fashion, e.g., more inhibition of cell proliferation was observed with 2.5 nM rLECT2 than that with 1.25 nM.
  • LECT2 inhibited HGF stimulated cell migration and invasion in a dosage dependent fashion.
  • the signaling molecules downstream of Met can bind to Met during signal transduction.
  • Gabl can bind to the phosphorylation site p-Tyrl349 on one Met receptor
  • Grb2 can bind to the phosphorylation site p-Tyrl 356 on a second Met receptor, so that an interaction between
  • Gabl and Grb2 can occur on Met dimmers or multimers.
  • LECT2 decreased Met phosphorylation at p-Tyrl349, thus dissociated Gabl from the Met receptor ( Figure 5C).
  • Met receptor has been identified as a substrate for several PTPs.
  • the non-receptor protein-tyrosine phosphatases (PTPs) IB and T-cell phosphatase (TCPTP) have been implicated as negative regulators of multiple signaling pathways, including the Met receptor-tyrosine kinases. It has been reported that both phosphatases interact with Met and that these interactions require phosphorylation of twin tyrosines (Tyr- 1234/1235) in the activation loop of the Met kinase domain.
  • LECT2 binds directly to Met receptor. To determine whether LECT2 could interact directly with Met, it was first tested whether LECT2 and Met formed a complex on the surface of HCC cells.
  • Co-immunoprecipitation was conducted with lysates form HCC cells, e.g., huh-7 and
  • PLC PPvF/ 5 cells which endogenously express LECT2 and Met receptor.
  • results from the co-IP study demonstrated that LECT2 interacts with Met.
  • HXXXD motif of LECT2 protein regulates tumor growth and metastasis.
  • LECT2 mutants having mutations in the HXXXD motif were constructed and studied. Such mutants include, for example, that harboring an AQGVA (SEQ ID NO:4, shown as mLECT2-l) or AQAVA (SEQ ID NO:5, shown as mLECT2-2) instead of a HQGVD (part of SEQ ID NO:3).
  • LECT2 mutants having mutations in other amino acids were also constructed and studied as a control, such as mLECT2-3, mLECT2-4. See, e.g., Figure 7A and Figure 9.
  • HXXXD mutants e.g., mLECT2-l, mLECT2-2
  • HXXXD mutants had increased HGF-induced Met phosphorylation and cell invasion, as compared with that overexpressing the wild type LECT2 or other LECT2 mutants (e.g., mLECT2-3, mLECT2-4)
  • Co- immunoprecipitation analysis indicated that the HXXXD mutant of LECT2 had lost all or most of its binding activity to Met (Figure 7C).
  • LECT2 protein reduced HGF-stimulated Met phosphoryaltion in various cancer cells.
  • LECT2 represents a novel cancer therapy for other tumors and cancers in addition to HCC.
  • LECT2 inhibits tumor cell proliferation, migration and invasion by inhibiting Met activation, thereby suppressing the downstream signaling pathway necessary to stimulate the cells.
  • the treatment with LECT2 either by endogenous overexpression of LECT2 in HCC cells or by administering external LECT2 to HCC cells, has reduced Met tyrosine kinase activity, thus inhibited the HGF stimulation to the HGF Met pathway.
  • a direct specific interaction between LECT2 and Met receptor in HCC cells was demonstrated, e.g., via the HXXXD motif of LECT2. While not wishing to be bound by the theory, the tumor inhibition mechanism of LECT2 appears to be related to protein tyrosine phosphatases (FTP) IB associated with Met in HCC cells.
  • FTP protein tyrosine phosphatases
  • LECT2 suppresses the invasiveness and Met receptor phosphorylation of HCC cells.
  • Studies of the present application also indicated that LECT2 represents a novel therapeutic approach for treating or preventing other tumors generally characterized by Met receptor excessive activity.

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EP10838658.2A 2009-12-21 2010-12-20 Verfahren und zusammensetzungen im zusammenhang mit reduzierter met-phosphorylierung durch aus leukozyten gewonnenes chemotaxin 2 in tumorzellen Withdrawn EP2515930A4 (de)

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UCHIDA T ET AL: "Expression pattern of a newly recognized protein, LECT2, in hepatocellular carcinoma and its premalignant lesion", PATHOLOGY INTERNATIONAL, CARLTON SOUTH, AU, vol. 49, no. 2, 1 February 1999 (1999-02-01), pages 147-151, XP009159532, ISSN: 1320-5463, DOI: 10.1046/J.1440-1827.1999.00836.X [retrieved on 2002-03-01] *
YANG ET AL: "Leukocyte cell-derived chemotaxin 2 (LECT2) gene expression can influence intravasation and metastatic ability of hepatoma cells through in vivo study", AMERICAN ASSOCIATION FOR CANCER RESEARCH. PROCEEDINGS OF THE ANNUAL MEETING, AMERICAN ASSOCIATION FOR CANCER RESEARCH, US, vol. 50, 1 January 2009 (2009-01-01), XP009167645, ISSN: 0197-016X *

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