WO2007132967A1 - An analyzing method for measuring the changes of glycosylation in various glycoprotein - Google Patents

Abstract

The present invention relates to an analyzing method of measuring the changes of glycosylation in various glycoproteins which participate in carcinogenesis and metastasis.

Description

[DESCRIPTION] [Invention Title]

AN ANALYZING METHOD FOR MEASURING THE CHANGES OF GLYCOSYLATION IN VARIOUS GLYCOPROTEIN [Technical Field]

<i> The present invention is concerned with a measurement and analyzing method of changes in glyco-structures associated with tumorigenesis and metastasis. In detail, changes in N-glycan of glycoproteins are measured and onset and metastasis of cancer are estimated based on the measurement. [Background Art]

<2> Properties of proteins have been analyzed predominantly by 2- dimensional (2-D) electrophoresis. Today, highly sophisticated mass spectrometers like MALDI-TOF and amino acid sequencers have been developed, and the development has led to "proteomics era", a kind of post-genomics. However, proteomic approaches have a limitation in that it shows a fixed state of proteome rather than a dynamic pattern. In fact, the complicated signal transduction in cells generally exhibits a dynamism in the expression level of proteins and post-translational modification.

<3> Moreover, it is difficult to detect the proteins related to signal transduction by displaying 2-D gels since the proteins exist in a minimal level in cells. Protein glycosylation allows us to overcome these limitations and to observe cells in a more dynamic point of view. Changes in protein that can not be distinguished by simple staining are easily monitored when investigating alterations in glycosylation using a specific lectin.

<4> Recently, this approach enables us to overcome the limitations of conventional proteomics and is differentiated with proteomics, termed "glycomics". Glycomics is mainly based on pursuit of alterations in protein glycosylation, a kind of post-translational modification.

<5> One of the biological disturbances is an aberrant glycosylation of proteins, which is induced by a signal of a certain oncogene and causes, in turn, a dysfunction of cell adhesion, cell-cell recognition and eventually tumorigenesis and cancer malignancy (Hakomori and Kannagi , 1983, J. Natl. Cancer Inst., 71: 231-251; Feizi, 1985, Nature, 314: 53-57). mRNA is translated on an endoplasmic reticulum and, in case of a glycoprotein, core glycan moiety is produced there. After that, the glycoproteins are translocated to Golgi body where supplementary glycans are attached by such glycosyltransferase as illustrated in Fig. 1. The glycosyltransferases are activated by a signal transduction elicited by the specific oncogenes, such as ras, raf, ets, and so on. One of the interesting glycosyltransferases is FUT8, which catalyzes an addition of fucose to the core moiety of N-glycan. Alpha-fetoprotein(AFP) and antitrypsin are widely known to well exemplify an importance of fucosylation in cancer malignancy (Miyoshi , E., Ko, J.H. et al., 1999, Biochim. Biophys. Acta 1473: 9-20). Recently, FUT8 responsible for the fucosylation was purified and the cDNA was cloned by Taniguchi et al . When fut8-transfected hepatoma Hep3B (Hep3B-FT) was injected to the spleen of athymic nude mice, tumor formation in the liver was dramatically suppressed in the transfectants (Miyoshi, E., Ko, J.H. et al., 1999, Cancer Research 59: 2237-2243). Lectin Lens culinaris agglutinin (LCA) recognizes the fucose attached to the innermost GIcNAc of N-glycan but is used to detect core N- glycan for the low specificity.

<6> The glycosyltransferase GnT-V is an enzyme catalyzing such reaction that N-acetylglucosamine is attached onto the J31.6 site of the basic sugar chain of a glycoprotein and is known to be directly associated with cancer invasion and metastasis(Dennis, et al . , 1987, Science, 236: 582-585). As for glycoproteins, basic sugar chain is formed in endoplasmic reticulum after protein is synthesized, which moves to Golgi apparatus. Then, sugars are added thereto by various glycotransferases resulted from various vital phenomena of cells. Primary sugar chains are formed by catalyzing of six types of N-acetylglucosaminyltransferases (I—VI) as shown in Fig. land especially GnT-V forming β 1,6-N-acetylglucosamine sugar chain has been believed to be deeply associated with tumorigenesis and metastasis. GnT-V is located in Golgi apparatus. This enzyme makes target proteins be secreted to or out of the cell surface by causing the changes of sugar chains. At this time, glycoproteins recognize surface proteins of target cells and then adhere thereto, causing a cancer.

<7> GnT-V was first noticed by the report of Dennis et al. (1987) that the βl,6 branches were remarkably represented as cancer tissues were growing or during metastasis(Dennis, et al . , 1987, Science, 236: 582-585). A cell surface protein gpl30 is one or major target proteins of GnT-V and shows high metastasis activity when βl,6 N-acetylglucosamine is added. GnT-V knockout mice were established in which GnT-V was deficient in their embryonic stem (ES) cells and to which polyomavirus middle T antigen (reffered "PyMT hereinafter) viral oncogene was introduced in order to induce a cancer. Resultingly, the growth of cancer and metastasis induced by PyMT were remarkably inhibited in GnT-V knockout mice comparing with another mormal mice proup in which only PyMT was over-expressed (Granovsky et al . , 2000, Nature Med., 6: 306-312), and the growth of βl,6 branch caused high metastasis especially in mice with breast cancer. Recent studies support that the GnT-V activity to 33 types of hepatocellular carcinoma (HCC) tissues is fifty times as high as it's activity to normal tissues and four times as high as to cancer surrounding tissues (Yao et al . , 1998, J Cancer Res. Clin. Oncol., 124: 27-30). As shown above, GnT-V relates to cancer metastasis and represents high matastasis activity in various tissues.

<8> GnT-V was purified in the human lung cancer cell and the mouse kidney, the cDNA was cloned, and the promoter and genomic structure was resolved (Gu et al., 1993, J Biochem, 113:614-619; Soreibah et al., 1993, J Biol. Chem. , 268:15381-15385; Kang et al., 1996, J. Biol. Chem., 271:26706-26712). The inventor of the present invention has reported that the transcription factor ets-1 is involved in the expression of GnT-V (Ko, et al., 1999, J. Biol. Chem., 274(33): 22941-22948). As the dietary style becomes Westernized, colon cancer shows a higher incidence rate in the Orient. However, diagnosis of colon cancer relies on the complicated and expensive methods like colonic endoscope, demanding a more convenient and simple diagnostic methods, for instance, using urine or blood. [Disclosure] [Technical Problem]

<9> Thus, the present inventors detected βl,6 N-acetylglucosamine in which sugars were attached by GnT-V in cancer-induced cells and found out novel glycoproteins showing the changes of sugar chains by analyzing amino acid sequences with a mass spectrometer. The present invention was completed with the result that the aberrant glycosylation of the novel proteins plays a critical role in cancer invasion and metastasis.

<io> It is an object of the present invention to provide a diagnostic kit for cancer to detect changes in glycan structures associated with tumorigenesis and metastasis. [Technical Solution]

<ii> To achieve the above object, the inventors found out and selected glycoproteins that are assumed to be useful in a diagnostic kit for cancer.

<i2> The present invention is not restricted to a specific cancer type and can be applied to various cancer type including colon, gastric, lung, liver, uterine, breast and pancreatic cancer.

<13> The present invention is to provide method and kit for detecting βl,6- GIcNAc moiety in N-I inked glycoproteins derived from tumor or metastatic cancer cells. GnT-V responsible for attaching βl,6-GlcNAc is involved in tumorigenesis and cancer malignancy irrespective of cancer type and βl,6- GIcNAc moiety can be recognized by phytohaemagglutinin-L4 (L₄-PHA).

[Advantageous Effects]

<14> As stated above, the present invention provide a clue that one can diagnose cancer more efficiently and precisely when glycosyl alteration is detected simultaneously with the protein expression levels. Expression level and glycosyl alteration are detected with the specific antibody and lectin respectively as in Fig. 34. Cumulative studies indicate that many of the proteins, including TIMP-I, in Table 1 are expressed in brain, kidney, liver, intestine, gastric, rectal, and cervical tissues. Therefore, the proteins could be used for diagnosis of other cancer than colon cancer.

[Description of Drawings] <i5> Fig.l illustrates the structures of N-I inked glycan linkage catalyzed by various glycosyltransferases and the structures of N-I inked glycan linkage that recognized by L-PHA. <i6> Fig. 2 illustrates the reaction in which GnT-V catalyzes the addition of βl,6-GIcNAc to the core of N-glycan. <i7> Fig. 3 shows that an expression level of GnT-V is quite low in the mock, and the stable transfectant expresses GnT-V highly as accessed by

Northern blot. <18> Fig. 4 shows 2-D gels and L-PHA blots of secreted proteins in the mock and GnT-V transfectants. <19> Fig. 5 exemplifies an identification of proteins of interest using

ESI/Q-TOF by showing the chromatogram of a peptide of TIMP-I. <20> Fig. 6 shows the co-crystallized structure of TIMP-I and MMP-3, as published previously, by which the inhibitory mechanism of TIMP-I toward MMP-

3 is elucidated. <2i> Fig. 7 gives a brief explanation to the properties of TIMP family members in terms of expression and glycosylation. <22> Fig. 8 describes the scheme for action mechanisms and structures of

MMPs that have been known to date. <23> Fig. 9 shows the positions of N-glycans on TIMP-1 and the primary structures of TIMP-I and the glycosylation mutants. <24> Fig. 10 indicates an expression level of TIMP-I in the mock and the glycosylation mutants of the transfectant WiDr cells. <25> Fig. 11 shows the glycan structures of TIMP-I, which was isolated in

WiDr÷mock and WiDr:GnT-V, and subjected to 2-D electrophoresis and lectin blotting using L-PHA and DSA, recognizing Jβl,6-GIcNAc moiety and lactosamine, respectively.

<26> Fig. 12 elucidates the path through which N-glycan becomes aberrant. <27> Fig. 13 shows a scheme for measuring migration of cells in a Transwell kit and the results of migration assay. <28> Fig. 14 shows a scheme for measuring invasion of cells in a Matrigel kit where the pore is coated with matrix compounds and the results of invasion assay. <29> Fig. 15 indicates no difference in an expression of MMP-2 and MMP-9 between mock and GnT-V transfectant of WiDr, as accessed by zymography. <30> Fig. 16 illustrates the mechanism by which non-fluorogenic substrate becomes fluorogenic by MMP attack. <3i> Fig. 17 shows an inhibition of fluorogenic substrate-hydrolysis activity of MMP-2 by TIMP-I and the TIMP-I mutant proteins. <32> Fig. 18 shows an inhibition of fluorogenic substrate-hydrolysis activity of MMP-9 by TIMP-I and the TIMP-I mutant proteins. <33> Fig. 19 shows zymographic data demonstrating a difference in gelatinases (MMP-2 and MMP-9) inhibition of TIMP-I and the TIMP-I mutants. <34> Fig. 20 is a time-course trace of MMP-2 activity in the presence of

TIMP-I purified from TIMP-I: mock and kinetically represents the MMP-2 inhibition by TIMP-I. <35> Fig. 21 is a time-course trace of MMP-2 activity in the presence of

TIMP-I purified from TIMP-I: GnT-V and kinetically represents the MMP-2 inhibition by TIMP-I. <36> Fig. 22 shows a difference in time-course trace of MMP-2 inhibition between TIMP-I: mock and TIMP-I: GnT-V. <37> Fig. 23 is a time-course trace of MMP-9 activity in the presence of

TIMP-I purified from TIMP-I: mock and kinetically represents the MMP-9 inhibition by TIMP-I. <38> Fig. 24 is a time-course trace of MMP-9 activity in the presence of

TIMP-I purified from TIMP-I: GnT-V and kinetically represents the MMP-9 inhibition by TIMP-I. <39> Fig. 25 shows a difference in MMP-9 inhibition between TIMP-I: mock and

TIMP-I: GnT-V. <40> Fig. 26 shows a tumor that formed in nude mice subcutaneousIy injected with TIMP-1-expressing WiDr cells. <4i> Fig. 27 shows a growth pattern of tumor in nude mice injected with the mutant transfectants of GnT-V: TIMP-I in a time-dependent manner (n=4). <42> Fig. 28 shows a growth pattern of tumor in nude mice injected with the mutant transfectants of mock: TIMP-1 in a time-dependent manner (n=4). <43> Fig. 29 is the immuno-pathological data that show WiDr cells invading into the muscle tissues of a nude mouse. <44> Fig. 30 shows the size of tumors that formed in nude mice injected with

T-WT:GnT-V, T-N30/78Q:GnT-V, and wild type WiDr eel ls(total n=30). <45> Fig. 31 shows the expression level of GnT-V using RT-PCR and TIMP-I expression using immunoblotting in the tissues of colon cancer patients with various stages. <46> Fig. 32 shows data where the normal tissues and cancer tissues from 10 representative colon cancer cases of each stage were compared with respect to

TIMP-I expression levels, aberrant glycosylation, and transcription levels of

GnT-V gene. <47> Fig. 33 illustrates the mechanism for enhanced cancer metastasis that occurs through an aberrant glycosylation of TIMP-1, which has a weaker inhibitory activity of MMPs. <48> Fig. 34 shows a scheme for detecting a candidate glycoprotein bilaterally by using antibody and lectin.

[Best Mode] <49> The present invention relates to a method of immunochemical detection of glycoproteins by treating antibody in control and experimental cells and a detection kit using the method. <50> The present invention relates to the method of immunochemical detection of glycoproteins by treating antibody in the media of control and experimental cells and a detection kit using the method. <5i> More specifically, the present invention relates to a detection method of glycoproteins through lectin blot using L₄-PHA which recognizes βl,6-

NAcGIc sugar branch and LCA which recognizes αl,6 fucose sugar branch. <52> The present invention relates to a detection method for changes of glyco-structure of one or more proteins associated with tumorigenesis and cancer metastasis containing following steps;

<53> ( i ) A step in which antibodies for at least one selected from a group that are assumed to be involved in tumorigenesis and cancer metastasis consisting of α-1-antitrypsin, angiotensinogen, βhexosaminidase β chain precursor, cathepsin D preproprotein, cathepsin X precursor, chain H of IgGl, dipeptidyl aminopeptidase II, discoidin receptor tyrosine kinase isoform b, dystroglycan 1 precursor, granulins precursor, heat shock 70 kDa protein, heat shock protein 1(9OkDa) β , heparan sulfate proteoglycan perlecan, hexosaminidase A preproprotein, Ig K chain V-III, IgG Fc binding protein, laminin receptor-like protein 5, legumain precursor, Met proto-oncogene precursor, N-acetylgalactosamine-6-sulfatase, N-acetyl-glucosamine-6- sulfatase, neogenin homolog 1, prolyl 4-hydroxylase, prosaposin, protective protein for b-galactosidase, protein tyrosine kinase 6, protein tyrosine kinase 7, ribonuclease T2 precursor, serine protease 22, tumor-associated calcium signal transducer 1 precursor, tumor rejection antigen-l(gp96) and Zn- α-2-glycoprotein are covalently bound to matrix.

<54> (ii) A step in which protein samples are added to the antibody-bound matrix and induced to interact each other, followed by washing out.

<55> (iii) A step in which interactions are induced by treating monoclonal antibody for the proteins mentioned above in control groups and by treating lectins that recognize an altered N-glycan in experimental groups respectively, followed by washing out.

<56> (iv) A step in which secondary antibody labeled with chromogenic enzyme is treated in the monoclonal antibody-bound control groups or chromogenic enzyme-labeled receptor for ligand bound on lectin is treated in the lectin- treated experimental groups, followed by washing out.

<57> (v) A step in which a chromo-substrate is treated to emit lights, a light intensity is measured.

<58> The present invention relates to a cancer-diagnostic kit comprising matrix covalently bound with antibody for at least one selected from the a group consisting of α-l-antitrypsin, angiotensinogen, βhexosaminidase β chain precursor, cathepsin D preproprotein, cathepsin X precursor, chain H of IgGl, dipeptidyl aminopeptidase II, discoidin receptor tyrosine kinase isoform b, dystroglycan 1 precursor, granulins precursor, heat shock 70 kDa protein, heat shock protein 1(9OkDa) β , heparan sulfate proteoglycan perlecan, hexosaminidase A preproprotein, Ig K chain V-HI, IgG Fc binding protein, laminin receptor-like protein 5, legumain precursor, Met proto- oncogene precursor, N-acetylgalactosamine-6-sulfatase, N-acetyl-glucosamine- 6-sulfatase, neogenin homolog 1, prolyl 4-hydroxylase, prosaposin, protective protein for b-galactosidase, protein tyrosine kinase 6, protein tyrosine kinase 7, ribonuclease T2 precursor, serine protease 22, tumor-associated calcium signal transducer 1 precursor, tumor rejection antigen-l(gp96) and Zn-α-2-glycoprotein, the antibody for the glycoprotein, ligand-labeled lectin, chromogenic enzyme-labeled secondary antibody and chromogenic enzyme- labeled receptor for the ligand.

<59> The matrix mentioned above can be selected among a group consisting of nitrocellulose membrane, polyvinyl-based well plates, polystyrene-based well plates and glass-based slide glass.

<60> The chromogenic enzymes mentioned above can be a peroxidase, an alkaline phosphatase and so on.

<6i> The ligand and receptor can be exemplified by biotin and avidin, respectively.

<62> The ELISA kit mention above can contain chromogenic substrates such as ABTS [2,2' azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)], OPD (o- phenylenediamine), or TMB (tetramehtyl benzidine).

<63> The present invention relates to a detection method for changes of glyco-structure of one or more proteins associated with tumorigenesis and cancer metastasis containing following steps;

<64> ( i ) A step in which a sample preparation is covalently bound onto the matrix in control group and experimental group! <65> (ii) A step in which antibody for at least one selected from a group consisting of the following proteins that associated with tumorigenesis and cancer metastasis is bound in control group; α-l-antitrypsin, angiotensinogen, βhexosaminidase β chain precursor, cathepsin D preproprotein, cathepsin X precursor, chain H of IgGl, dipeptidyl aminopeptidase II, discoidin receptor tyrosine kinase isoform b, dystroglycan 1 precursor, granulins precursor, heat shock 70 kDa protein, heat shock protein 1(9OkDa) β , heparan sulfate proteoglycan perlecan, hexosaminidase A preproprotein, Ig K chain V-UI, IgG Fc binding protein, laminin receptor- like protein 5, legumain precursor, Met proto-oncogene precursor, N- acetylgalactosamine-6-sulfatase, N-acetyl-glucosamine-6-sulfatase, neogenin homolog 1, prolyl 4-hydroxylase, prosaposin, protective protein for b- galactosidase, protein tyrosine kinase 6, protein tyrosine kinase 7, ribonuclease T2 precursor, serine protease 22, tumor-associated calcium signal transducer 1 precursor, tumor rejection antigen-l(gp96) and Zn- α-2- glycoprotein, and ligand-labeled lectins are added to the matrix for detecting alteration in glycan structures in experimental group, followed by washing out .

<66> (iii) A step in which secondary antibody labeled with chromogenic enzyme is treated in the antibody-bound control groups and chromogenic enzyme- labeled receptor for ligand bound on lectin is treated in the lectin-treated experimental group rexpectiveIy, followed by washing out.

<67> (iv) A step in which a chromo-substrate is treated to emit lights, a light intensity is measured.

<68>

<69> Changes in expression level and N-glycan structures of the glycoproteins mentioned above can be useful for a kit for cancer diagnosis, in that the glycoproteins involved in tumorigenesis and cancer metastasis reside on the cell surface or secretes out and thus they can be analyzed from urine or blood.

<70> The diagnostic method comprises two major steps; a step for collecting samples from patients and for analyzing expression level of proteins that are assumed to be involved in tumorigenesis and cancer metastasis and changes in

N-glycan structures from the collected samples. <7i> In the first step, samples are collected from blood or urine preferentially by routine isolation method of serum. In the second step, any method based on antigen-antibody interaction such as enzyme-linked immunosorbent assay and immunoblot can be used. <72> The inventors provide a detecting method for the protein expression level and changes in N-glycan structures using antibodies for the glycoproteins involved in tumorigenesis and cancer metastasis as a preferable examp1e. <73> A kind of analysis method for analyzing the protein expression level and changes in N-glycan structures using enzyme-linked immunosorbant assay comprises the following steps: <74> 1) A step in which antibodies to the glycoproteins involved in tumorigenesis and cancer metastasis are covalently bound onto the matrix; <75> 2) A step in which sample such as serum, is admixed to interact, followed by washing out; <76> 3) A step in which labeled antibodies or L₄-PHA is treated;

<77> 4) A step in which chromogenic enzyme or fluorogen-labeled secondary antibodies are treated following washing out;

<78> 5) A step in which light intensities are measured following treatment of chromogenic or fluorogenic substrates.

<79> The matrix can be selected among the nitrocellulose membrane, polyvinyl-based 96-well plates, polystyrene-based 96-well plates and glass- based slide glass.

<80> In the step 3, biotin can be labeled, the biotin-labeled antibodies can be used for quantitative and qualitative analysis, and the biotin-labeld L₄-

PHA can be used for detecting the change of βl,6-GlcNAc moiety. <8i> The enzymes mentioned above can be a peroxidase or an alkaline phosphatase and fluorogens can be FITC or RITC, etc.

<82> ABTS [2,2' azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)], OPD (o- phenylenediamine), or TMB (tetramehtyl benzidine) can be used as a chromogenic substrates, and especially, OPD that reacts with a peroxidase can be used in the present invention.

<83> A biological microchip or microarray system can be used for high through-put analysis in the diagnostic method.

<84> The present invention is to provide method and kit for detecting the changes of βl,6-GIcNAc moiety in N-linked glycoproteins derived from tumor or metastatic cancer cells and is not restricted to a specific cancer type and can instead be applied to every cancer type including colon, gastric, lung, liver, uterine, breast and pancreatic cancer.

<85> Proteins involved in tumorigenesis and cancer metastasis are selected from the following glycoproteins that show changes in N-glycan structures; α -1-antitrypsin, angiotensinogen, βhexosaminidase β chain precursor, cathepsin D preproprotein, cathepsin X precursor, chain H of IgGl, dipeptidyl aminopeptidase II, discoidin receptor tyrosine kinase isoform b, dystroglycan 1 precursor, granulins precursor, heat shock 70 kDa protein, heat shock protein 1(9OkDa) β , heparan sulfate proteoglycan perlecan, hexosaminidase A preproprotein, Ig K chain V-HI, IgG Fc binding protein, laminin receptor- like protein 5, legumain precursor, Met proto-oncogene precursor, N- acetylgalactosamine-6-sulfatase, N-acetyl-glucosamine-6-sulfatase, neogenin homolog 1, prolyl 4-hydroxylase, prosaposin, protective protein for b- galactosidase, protein tyrosine kinase 6, protein tyrosine kinase 7, ribonuclease T2 precursor, serine protease 22, tumor-associated calcium signal transducer 1 precursor, tumor rejection antigen-l(gp96) and Zn- α-2- glycoprotein.

<86> a-l-antitrypsin(AAT) is a member of a serine protease family. The effects of polymorphonuclear neutrophil (PMN) conditioned medium alone, and supplemented with serine proteinase inhibitor alpha-1 antitrypsin (AAT) and its C-terminal fragment (C-36 peptide), on cultured lung cancer cells were measured. As a result, PMN-conditioned medium loses its effects on cell proliferation, invasiveness and IL-8 release in the presence of AAT, whereas VEGF is up-regulated by 3.7-fold (pO.001) compared to controls. These indicate that the proliferation and invasion of cancer cells are modulated by exogenously present alpha-1 antitrypsin (Zelvyte I et al, 2004, Cancer Cell Int., 4, 7)

<87> Angiotensin II is a vasoconstrictor which is directly produced from angiotensinogen at acidic pH by active trypsin. When angiotensin II concentration and angiotensin converting enzyme (ACE) activity were measured in tissues from normal pancreas, pancreatic ductal cancers, colon cancers, and hepatocellular carcinomas, tissue angiotensin II levels in pancreatic ductal cancer were significantly higher than those of normal pancreas, colon cancers, or hepatocellular carcinomas. However, there was no significant difference in the ACE activity in tissue between them. This indicates an evidence of an ACE-independent, angiotensin II-generating system in pancreatic ductal cancer tissues (Ohta T et al , 2003, Int. J. Oncol. 23, 593- 598).

<88> Aberrant crypt foci (ACF), consisting of morphologically irregular crypts, are thought to be precancerous lesions for colon cancers. When a relationship between the length, rim diameter, and width of hexosaminidase("Hex")-positive crypts was investigated, transcription levels of Hex alpha and Hex beta subunits were significantly lowered (PO.002). This decrease could be a molecular marker for precancerous enzyme-altered ACF (Tsukamoto T et al , 2001, Jpn. J. Cancer Res, 92, 109-118).

<89> When the mRNA levels of cathepsins B, D, H, and L were investigated in colorectal carcinoma patients with different clinical stages using real-time quantitative reverse transcriptase polymerase chain reaction, cathepsins B, D and L were significantly higher in metastatic lesions than in primary tumors, suggesting that they may be potential molecular targets in colorectal carcinoma progression and metastasis (Tsukamoto T et al , 2006, Cancer 106, 1489-1497) . <90> The expression pattern of cathepsin X, a serine protease, was assessed in malignant and non-malignant prostatic tissue samples of 56 cases after prostatectomy. Cathepsin X was quantified at both protein and mRNA levels using several detection methods: Western blotting, immunohistochemistry, quantitative RT-PCR, and in situ hybridization. As a result, prostatic intraepithelial neoplasias (PINs) and prostate carcinomas stained highly positive for cathepsin X, showing a significant difference to the staining of normal prostate glands. In contrast, relatively weak and heterogeneous staining was observed for cathepsins F, B, and L. The high expression levels of cathepsin X both in PIN and invasive adenocarcinomas of the prostate suggest that cathepsin X may play a role in the early tumorigenesis of prostate cancer (Nagler DK et al, 2004, Prostate 60, 109-119).

<9i> When a protein composed of a soluble single-chain TCR was constructed that is genetically linked to the constant domain of an IgGl H chain, the Ag recognition portion of the protein binds to an unmutated peptide derived from human p53 (aa 264-272) presented in the context of HLA-A2.1, whereas the IgGl H chain provides effector functions (Mosquera LA et al, 2005, J. Immunol. YlA, 4381-4388).

<92> To investigate the possibility of its being a marker enzyme for tumor cells, the activity of dipeptidyl aminopeptidase (DPP) II , a membrane-bound enzyme, in cultured human carcinoma cells such as HeLa, KB and K-44, was examined. The DPP II activity was 6- to 24-fold higher in carcinoma cell lines than in human fibroblast. These clear enzymatic differences between carcinoma cells and normal human fibroblast may be useful as a marker of malignancy (Komatsu M et al , 1987, J. Natl. Cancer Inst. 78, 863-868).

<93> The discoidin receptor tyrosine kinase isoform b, a transmembrane protein, is activated by various types of collagen and consists of three isoforms-DDR Ia, b, and c-as a result of alternative splicing in the cytoplasmic region (Alves F et al, 2001 FASEB J. 15, 1321-1323).

<94> Dystroglycan (DG), a non-integrin adhesion molecule, is a pivotal component of the dystrophin-glycoprotein complex, that is expressed in skeletal muscle, and exists in a wide variety of tissues at the interface between the basement membrane (BM) and the cell membrane. There is growing evidence indicating the abnormalities of DG found in human cancers, and the implications of these findings with respect to our understanding of cancer pathogenesis and to the development of novel strategies for a better management of cancer patients (Sgambato A 2005, J. Cell Physiol. 205, 163- 169).

<95> Granulin(-epithelin) precursor (GEP) is a novel growth factor. cDNA microarray study indicated that GEP was over-expressed in hepatocellular carcinoma (HCC). The clinical significance of GEP expression and its potential as a therapeutic target in HCC was investigated. A total of 110 pairs of HCCs and adjacent non-tumor liver tissues, and 22 normal liver tissues were examined. The GEP RNA level was examined by quantitative reverse transcription-PCR, and protein localization by immunohistochemistry. The GEP function was examined by transfection experiments. The RNA levels of the HCCs were significantly higher than those of the non-tumor liver tissues and normal livers (P O.001). GEP protein staining was observed in tumor cytoplasm, and the GEP protein levels of the HCCs were also significantly higher than those of the non-tumor liver tissues and normal livers (P O.001). GEP is an important factor for HCC growth, invasion, and metastasis. GEP has the potential to serve as a tumor marker and therapeutic target (Cheung ST et al , 2004, Clin. Cancer Res. 10, 7629-7636).

<96> When the constitution of each isoform of progesterone receptor(PR) was studied by using different monoclonal antibodies raised against PR-A/B (JZB39 and KD68), against PR-B (PR6 and KC 146), and against hsp90 and hsp70 heat shock proteins (9D2 and Ab72, respectively), hsp90 was present in isoforms 1 (680 kDa), 2 (600 kDa) and 3 (361 kDa) exclusively, whereas hsp70 remained strongly bound to isoforms 4 (224 kDa) and 5 (193 kDa) (Lemoisson E, 1994, Ann. Biol. Clin. 52, 433-443).

<97> Genetic studies associated the CAPB(Carcinoma Prostate Brain) locus with familial risk of brain and prostate cancers. It has been identified heparin sulfate proteoglycan perlecan as a candidate gene for CAPB(Datta MW et al, 2006, MoI. Cancer 5, 9).

<98> The presence of the variant form of beta-hexosaminidase was investigated from a large and heterogeneous group of cancer patients (with different primary sites and differing degrees of metastatic involvement), from normal controls and pathological controls with nonmalignant diseases. Comparison of total serum beta-hexosaminidase activity levels and the percentage of the total activity comprised of beta-hexosaminidase B (Hex B) revealed no significant differences (P greater than 0.01) between the three groups. However, analytical isoelectric focusing indicated that the variant beta-hexosaminidase was present in 80% of 108 cancer patients, 37% of 27 pathological controls and 11% of 18 normal controls (Plucinsky MC 1986, Cancer 58, 1484-1487). This may arise from the heterogeneity of glycans attached beta-hexosaminidase.

<99> A 72-year-old woman with dural plasmacytoma was revealed to have an immunoglobulin (Ig) G-kappa multiple myeloma (MM) (Haegelen C et al , 2006, J. Neurosurg. 104, 608-610).

<ioo> Targeting receptor activator of nuclear factor kappaB ligand (RANKL) with osteoprotegerin (OPG) prevents the osteolytic activity of CaP and its ability to establish tumor in bone. sRANK-Fc is an effective inhibitor of RANKL that diminishes progression of CaP growth in bone through inhibition of bone remodeling (Zhang J et al , 2003, Cancer Res.63, 7883-7890).

<ioi> Human MCF-7 breast carcinoma cells possess a receptor-like moiety on their surface that has a high binding affinity (Kd = 2nM) for laminin, a glycoprotein localized in basement membranes. Laminin preferentially stimulates (8-fold) MCF-7 cells to attach to type IV (basement membrane) collagen, whereas fibronectin stimulates attachment only 2-fold for these cells on type I collagen. The attachment properties of two other human breast carcinoma cell lines to type IV collagen were also studied. The attachment of ZR-75-1 cells was stimulated 4-fold by laminin and 5-fold by fibronectin, whereas T47-D cell attachment was stimulated 2-fold by laminin and 7-fold by fibronectin. The receptor for laminin on the surface of these tumor cells may be involved in the initial interaction of tumor cells via laminin with the vascular basement membrane to facilitate invasion and subsequent promotion of metastasis (Terranova ,VP et al, 1983, Proc. Natl. Acad. ScL 80, 444-448).

<iO2> Asparagine endopeptidase(AEP) or lrgumain in higher animal is lysosomal cystein protease. Progesterone and glucocorticoids such as dexamethasone mediate distinct biological functions, yet they bind to receptors that recognize the same consensus DNA response element. In breast cancer, progestins are associated with the onset and progression of tumors, whereas glucocorticoids are growth-suppressive in mammary cancer cells. PR- and GR- positive Ishikawa H endometrial cancer cells were treated with vehicle, dexamethasone (10OnM) or progesterone (10OnM) for 2h, 6h, 12h and 24h, and RNA was isolated. Affymetrix microarrays were performed using the human HG- U133A chip, querying the expression of 22,000 genes. The transcript for cysteine 1 (legumain), a gene associated with metastasis, was strongly down- regulated by progesterone (Davies S et al, 2006, Gynecol. Oncol. 101, 62-70).

<i03> The proto-oncogene c-Met has been suggested to be associated with progression of squamous cell carcinoma of the head and neck. In a retrospective study, a cohort of 84 0SCC(0ral squamous cell carcinoma) patients was investigated for c-Met expression and its cellular localization by immunohistochemistry. Sixty-nine cases (82.2%) of OSCC showed immunopositivity, with a mainly membranous expression and scattered areas also showing a cytoplasmic localization, whereas 15 cases (17.8%) did not show c-Met. No statistical association was found between c-Met expression and any variables considered at baseline, apart from the higher number of c-Met positivity in females (p = 0.026). Among positive tumors, well-differentiated areas showed low or absent cytoplasmic expression, while low-differentiated areas showed both membranous and cytoplasmic posit ivity. In terms of prognostic significance, c-Met expression was found to have an independent association with a poorer overall survival rate (p = 0.036). On the basis of these results, it is possible to suggest c-Met as an early marker of poor prognosis, a hallmark of aggressive biological behavior in OSCC, suggested to be useful in identifying cases of OSCC before the relapse (Lo Muzio L et al , 2006, Tumor Biol.27, 115-121).

<i04> Mucopolysaccharidosis type IV A (Morquio A) is caused by a deficiency of N-acetylgalactosamine-6-sulfatase (GALNS), an enzyme capable of cleaving the sulfate group from both N-acetylgalactosamine-6-sulfate and galactose-6- sulfate. Galactose-6-sulfatase (GALS) activity in leukocytes and fibroblasts of the affected family members was clearly deficient. Molecular genetic analysis of the GALNS gene revealed that two different point mutations segregate in the family, which correlated well with the clinical phenotype (Tylki-Szymanska A et al , 1998, Clin. Genet.53, 369-374).

<iO5> Mucopolysaccharidosis type III D is the least common of the four subtypes of Sanfilippo syndrome. It is caused by a deficiency of N- acetylglucosamine-6-sulphatase, which is one of the enzymes involved in the catabolism of heparan sulphate. The patient was found to be homozygous for a single base pair deletion (cll69delA), which will cause a frame-shift and premature termination of the protein (Beesley CE et al , 2003, J. Met. Genet. 40, 192-194).

<iO6> Neogenin is expressed in cap cells that have been suggested to be mammary stem or precursor cells. The nontumorigenie M13SV1 cells and normal tissues showed stronger expression of neogenin than the M13SV1R2N1 cells and the paired cancer tissues. In the tissue array, all (8/8) of the normal breast tissues showed strong neogenin expression, while 93.5% (43/46) of breast cancer tissues had either no expression or only moderate levels of neogenin expression (Lee JE et al , 2005, BMC Cancer 5, 154).

<iO7> Mammalian cells respond to changes in oxygen through a conserved pathway that is regulated by the hypoxia-inducible factor (HIF). The alpha subunit of HIF is targeted for degradation under normoxic conditions by a ubiquitin-ligase complex that recognizes a hydroxylated proline residue in HIF. A conserved family of HIF prolyl-4-hydoxylase (HPH) enzymes was identified that appear to be responsible for this posttranslational modification. HPH is an essential component of the pathway through which cells sense oxygen (Bruick RK et al , 2001, Science 294, 1337-1340).

<iO8> It was revealed that prosaposin (PSAP) is a secreted protein expressed in androgen-independent (AI) prostate cancer cells. Southern hybridization, quantitative real-time polymerase chain reaction, and/or single nucleotide polymorphism (SNP) array analysis also revealed the genomic amplification of PSAP in the metastatic AI prostate cancer cell lines, PC-3, DU-145, MDA-PCa 2b, M-12, and NCI-H660 (Koochekpour S et al 2005, Genes Chromosomes Cancer 44, 351-364).

<i09> Galactosialidosis is an autosomal recessive lysosomal storage disease caused by a combined deficiency of lysosomal beta-galactosidase and neuraminidase as a result of a primary defect in the protective protein/cathepsin A (PPCA). Two Dutch cases of early infantile galactosialidosis represented with neonatal ascites. The activity of protective protein for beta-galactosidase was decreased in urine, leukocytes, and fibroblasts(Groener J et al, 2003, MoI. Genet. Metab.78, 22-228).

<πo> Receptor tyrosine kinase 6 has an unusual ectodomain. The 150 amino acids in the amino terminus of the receptor is homologous to a putative phospholipid-binding sequence that is found also in other cell adhesion molecules such as the neuronal A5 antigen and coagulation factors V and VIII (Perez JL et al , 1994, Oncogene 9, 211-219).

<iii> The recently cloned protein tyrosine kinase 7(=colon carcinoma kinase 4) oncogene contains an evolutionarily conserved GxxxG motif in its single transmembrane domain (TMD). It has been suggested that this pairwise glycine motif may provide a strong driving force for transmembrane helix-helix interactions (Kobus FJ et al, 2005, Biochemistry 44, 1464-1470).

<ii2> The region 6q27 from human chromosome 6 has been reported to contain one or more tumor suppressor genes on the basis of cytogenetic, molecular and functional studies. RNASET2, a precursor for ribonuclease T2, is a secreted glycoprotein and has an effect on the metastatic behavior of the highly- invasive ovarian cancer cell line HEY3MET2. RNASET2-mediated suppression of tumorigenesis and metastasis was not affected by a double point mutation targeted to the putative ribonuclease catalytic sites, suggesting that tumor suppression by RNASET2 is not mediated by its ribonuclease activity (Acquati F et al, 2005, Int. J. oncol.26, 1159-1168).

<H3> A cDNA that encodes a novel serine protease 22, prosemin, from human brain was isolated. The amino acid sequence of prosemin shows significant homology to prostasin, gamma-tryptase, and testisin. prosemin is expressed and secreted from various kinds of cancer cells, such as glioma, pancreas, prostate, and ovarian cell lines. It may be used as a candidate tumor marker (Mitsui S et al, 2005, FEBS J.272, 4911-4923).

<ii4> Tumor-associated calcium signal transducer 1-precursor, a cell surface antigen, an epithelial glycoprotein, defined by the monoclonal antibody HEA 125, is expressed on virtually all epithelial cell membranes but not on mesodermal or neural cell membranes. The cDNA encoding Tumor-associated calcium signal transducer 1-precursor was isolated by HEA 125 antibody enrichment of colon tumor cDNA expressed transiently in COS cells. Tumor- associated calcium signal transducer 1-precursor is a cell surface molecule involved in cell-cell or cell-matrix interaction (Simon B et al, 1990, Proc. Natl. Acad. ScL 87, 2755-2759).

<ii5> Tumor rejection antigen-l(gp96) is a 96-kDa glycoprotein of the endoplasmic reticulum that is believed to be an antigen involved in peptide transport by major histocompatibility complex (MHC) class I molecules. This function implies that gp96 carries a large array of different peptides that represent the antigenicity of the cell and can serve all MHC class I molecules(Demine R et al , 2005, J. Biol. Chem.280, 17573-17578).

<ii6> Zn-alpha-2-glycoprotein is one of the proteins present in breast cyst fluids, being found at levels 30-50 times its plasma concentration. Using an immunoperoxidase technique the distribution of this glycoprotein has been studied in a range of non-mammary tissues and carcinomas, as well as in normal, benign and malignant breast specimens. Zn-alpha-2-glycoprotein was demonstrated in 16 of 33 invasive carcinomas, 15 of which were eosinophilic on haematoxylin and eosin(HE) staining, and in one of three non-invasive carcinomas. No staining was apparent in other non-mammary tissues and carcinomas apart from weak reactivity of serous cells of the parotid gland. Zn- alpha-2-glycoprotein is, therefore, a reliable immunohistochemical marker of apocrine cell differentiation (Bundred NJ et al , 1987, Histopathology 11, 603-610).

<ii7> As can be seen in reference mentioned above, most proteins were reported to show alterations in expression level, but in a DNA or mRNA level. However, there is little evidence that points to changes in terms of glycosylation. Thus, the present invention has significance in that pursuit of glycan changes in some glycoproteins is useful for cancer diagnosis.

<ii8> The diagnostic kit of the present invention can be used to quantify the expression level of proteins and to measure an altered glycan structure by employing ELISA, in which antibodies raised against proteins of interest are immobilized onto a 96-well microtiter plate.

<ii9> The diagnostic kit of the present invention includes antibodies raised against the candidate proteins, matrix, an appropriate buffer, a secondary antibody labeled with chromogenic enzyme or fluorescents, and L4-PHA or LCA for recognizing altered glycan structure.

<i20> The matrix can be chosen among the nitrocellulose membrane, polyvinyl or polystyrene 96-well plate, or slide glass. The labeled enzyme can be a peroxidase or an alkaline phosphatase, and FITC or RITC can be used for a fluorescent material. The substrate can be 2,2' azino^~bis(3- ethylbenzothiazoline-6-sulfonic acid), o-phenylenediamine, or tetramethyl benzidine.

<i2i> The present invention can utilize the automated analytical equipments with biological chips to diagnose cancer. For instance, protein chips are designed to chase the altered glycan structures by immobilizing the candidate proteins of interest onto a glass plate. This kit contains the proteins of interest, an appropriate buffer, and L₄-PHA. <122> Cancer metastasis highly depends on cell recognition and adhesion, and proteins involved in those functions reside mainly on plasma membrane or secrete out and thus could be found in blood or urine. As mentioned previously, the inventors selected WiDr in that the cells show low expression level of GnT-V and they adopted WiDr^GnT-V as a model cell line for glycomic study. Protein samples were retrieved from the conditioned media of WiDr cells, run on 2-D gels, and subjected to the comparative lectin blot using L₄-

PHA. Candidate proteins that show glycosyl change and are assumed to be involved in cancer malignancy were screened. <123> Proteins of interest were spotted from the 2-D gels, digested, and the digested peptide was sequenced using ESI/Q-TOF mass analyzer. Proteins were identified against a database and compared in terms of molecular mass, isoelectric point and sequences. <i24> [Table 1]

<125> a; Sequence coverage refers to the percentage of peptides that was identified by mass spectrometer.

<126> b; Total score is a sum of the score values obtained from each of an individual peptide. Score is -10 x Log (P), where P is the probability that the observed match is a random event; it is based on NCBInr database using the MASCOT searching program as MS/MS data.

<127> c; Molecular weight (Mr) and isoelectric point (pi) are theoretical values where glycan residues were not considered for the calculations. The theoretical values are prone to be changed by an attachment of glycans to peptides and thus to be different from the experimental values estimated on 2-D gels.

<128> <129> Tissue inhibitor of metal loproteinase-1 (TIMP-I) is an inhibitor protein for metal loproteinases (MMPs) involved in the degradation of extracellular matrix. That is, it keeps normal conditions by inhibiting MMPs responsible for cell migration and invasion. TIMP-1 loses the inhibitory activity upon an aberrant glycosylation of attachment of βl,6-GlcNAc. To demonstrate this, decisive evidence was provided in the present invention through the following examples.

<130>

[Mode for Invention]

<i3i> The present invention will be explained in more detail with reference to the following Examples. However, the present invention are not restricted to the following examples. And, the other proteins in Table 1 involved in cancer can be used as markers of the present invention.

<132> <Example 1> Enrichment of glycoprotein and 2-D electrophoresis <i33> As mentioned previously, the inventors selected WiDr in that the cells show low expression level of GnT-V (Fig. 3) and adopted WiDr:GnT-V as a model cell line for glycomic study. WiDr^GnT-V showed a high angiogenesis in a CAM assay (Ro JH et al, unpublished data). Cancer metastasis highly depends on cell recognition and adhesion, and proteins involved in cell recognition and adhesion exist mainly on plasma membrane or secrete out and thus could be found in blood or urine. Control cell line (WiDr :mock) and experimental cell line (WiDr :GnT-V) were cultured in RPMI1640(gibco BRL, USA) containing 10% FCS. GnT-V overexpressing cell line was established from WiDr that was purchased from American Type Tissue Culture (ATCC), into which an expression vector cloned with GnT-V gene was transfected. Colony was allowed to form in a media containing 350mg/ml G418, and the stable transfectant was selected by Northern blot analysis. When the cells were confluent in CO2 incubator, the cells were washed two times with PBS. For protein preparations, cells were cultured in serum-free RPMI 1640 media for 2 days, from which the total secretome was isolated and subjected to concentration in 80% acetone. Washed three times with acetone, the dry pellet was solubilized in lysis buffer(8M urea, 4% w/v CHAPS, ImM DTT, 0.2% v/v carrier ampholyte, Bromophenol Blue) and allowed to stand for Ih at room temperature. Immobline dry strips (Pharmacia, pH 3-10, 13cm) were loaded with 0.5mg of proteins and allowed to rehydrate for 18-22hr. Isoelectric focusing was performed at 20^°C using a Multiphor II electrophoresis unit(Pharmacia) according to manufacturer's instructions. Equilibrated in buffer containing SDS and mercaltoethanol , the strips were inserted into 12% SDS-PAGE gels. After running, the gels were either stained with colloidal G-250 Coomassie Brilliant BlueCBiosafe; Bio- Rad) or Western-blotted onto PVDF membrane (Milliphore, USA). The membrane blots were treated with biotin-labeled Lens culinaris agglutinin (LCA) or lectin phytohaemagglutinin-L₄ (L₄-PHA) (Sigma) for Ih, followed by treatment with avidin coupled to horseradish peroxidase(Vecstatin) and visualized by enhanced chemiluminescence(Amersham). To compare with control cell line(WiDr:Mock), the results of Coomasie brilliant blue staining and L₄-PHA lectin blotting following the isoelectric focusing are shown in Fig.4.

<134> <Example 2> Protein identification using ESI/Q-TOF mass analyzer

<135> Spots that showed a differential display were excised from the coomassie-stained gels, destained in 30% methanol and 100% acetonitrile, subsequently, and tryptic-digested overnight using modified porcine trypsin (Promega). Mass spectrometric analyses were performed using a Q-TOF MS (Micromass) equipped with a nano-ESKElectrospray Ionization) source, in which each peptide is forced to be resolved and thus peptide sequencing is possible. Fig. 5 shows a peptide sequence of TIMP-I as an example. Proteins were identified by blasting the determined sequence against a database. Molecular weight, pi, and peptides matched were compiled in Table 1. To minimize the possibility of systematic errors, the proteins that were exactly matched to at least two peptides with significant score values and no miss were screened.

<i36> <Exaraple 3> TIMP-I and cancer metastasis

<137> TIMP-I was select to validate our strategy for discovery of marker proteins. Fig. 6 shows the cocrystal structure of TIMP-1 and MMP-3 as published in Nature, 1999, in which the glycosylation sites of TIMP-I is adjacent to the active site of MMP-3. As shown in Fig. 7, TIMP-I is a sole glycoprotein among the TIMP family members. In addition, TIMP-I has been arguably reported on the effects on cancer malignancy. MMPs is responsible for degrading ECM molecules and more than 30 members have been known to date as described in Fig. 8.

<138> <Example 4> Establishment of transfectants of TIMP-I and the glycosylation mutants

<139> In order to examine the effects of GnT-V-initiated alterations of N- 1 inked glycans on TIMP-I on cancer cell behavior, site-directed mutageneses were performed in which either or both of Asn30 and Asn78 were replaced with GIn (Fig. 9). The Gln30 mutant is called T-N30Q, the Gln78 mutant is called T-N78Q and Gln30/Gln78 mutant is called T-N30/78Q. TIMP-I mutant genes were generated using the standard Megaprimer methods, where either or both of Asn

78 and Asn were changed to GIn. Wild-type timp-1 and the mutant genes were cloned into pcDNA 3.1 hygro(+) plasmid vector (Invitrogen). The repetitive transfections and selections were conducted so as to meet two requirements'- The first is that the amounts of TIMP-I recombinant protein (rTIMP-1) secreted are as much as possible so as to minimize the effects of cognate TIMP-I. The other is that the amounts of rTIMP-1 proteins secreted are equal among the transfectants in order to exclude the differences in cancer cell behavior arising from difference in the levels of secreted TIMP-I. Stable transfectants satisfying both requirements were selected based on Western blot analysis (Fig. 10). The secretion level of cognate TIMP-I was 18.40.8% of that for rTIMP-1 in WiDr:GnT-V cells and negligible in case of WiDr:mock cells. The molecular mass of the mature form of TIMP-I is ca. 28.5 kDa, of which N-I inked glycans account for about 8 kDa (Caterina NC et al , 1998, Biochim Biophys Acta 14, 21-34). The eradication of either of the two N- 1inked glycosylations produced rTIMP-1 mutant proteins whose molecular masses were reduced by 4kDa, confirming that the intended transfectant cells were produced.

<i40> <Example 5>Estimation of glycan structure of TIMP-I altered by GnT-V <i4i> To deduce the structure of aberrant glycan of TIMP-I, the cognate TIMP- 1 was purified both from WiDr:mock and WiDr:GnT-V. Anti-TIMP-1 monoclonal antibody(Santa Cruz Biotechnology) was conjugated with CNBr-activated Sepharose 4B column. TIMP-I was purified by the IgG_anti-τiMP-i conjugated

Sepharose 4B column. The concentration of TIMP-I protein was calculated using molar constant 26,50OM cm . The purified TIMP-I was subjected to 2-DE followed by immunoblot using anti-TIMP-I antibody, and lectin blot analysis using L₄-PHA and Datura stramonium agglutinin (DSA) (Fig. 11). As shown in

Fig. 12, L₄-PHA recognizes b 1,6 N-acetylglucosamine branch, a product of GnT-

V, and lectin DSA recognizes lactosamine. TIMP-I from WiDr÷mock was divided into one main spot in the basic region and two minor ones in the acidic region on 2-DE gels, indicating that only small fraction of TIMP-I possibly carry an acidic glycosyl residue such as sialic acid. None of the subdivisions carried βl,6-GIcNAc or lactosamine linkages. However, the majority of the aberrant TIMP-I showed βl,6-GIcNAc linkages, and extended polylactosamine glycan moieties. Moreover, TIMP-I was divided into multi- spots on 2-DE gels, showing an increment of heterogeneity. The aberrantly appended glycans on TIMP-I from WiDr:GnT-V were estimated to be ca. 2kDa, which corresponds to around 10 or more monomeric saccharides.

<i42> <Example 6> Effects of the aberrant glycosylation of TIMP-I on in vitro cell migration and invasion

<143> Cumulative studies indicating that an increase in GnT-V activity correlates with the high invasive/metastatic potential of cancer cells and the fact that TIMP-I is associated with the potential prompted us to examine the role of N-I inked glycosylation of TIMP-I in the metastatic potential of colon cancer cell. For the purpose, the migration and invasion properties of each TIMP-I transfectant were investigated in vitro. As shown in Fig. 13, cell migration assays were performed using 12-well Transwell chambers (Corning Inc.) with δμπrpore size polycarbonate inserts (Guo H-B et al, 2002, Cancer Res. 62, 6837-6845). Cells migrating or invading to the lower surface of the filters were fixed in methanol, stained with Toluidine blue, and counted with a microscope at X400. Concerning cell migration, GnT-V affected cell migration, but in a TIMP-1-independent manner. Little difference in the motility was observed among the TIMP-1 transfectants of WiDr:mock cells as well as WiDr÷GnT-V cells. However, GnT-V conferred a higher motility on WiDr cells irrespective of the mutational status of TIMP-I, indicating that the increment of cell motility is actually aided by GnT-V, but possibly mediated by other mediator proteins or through a signal transduction pathway independent of TIMP-I as reported previously.

<i44> Meanwhile, glycan moieties on TIMP-I affected the cell invasion significantly (Fig. 14). T-N30/78Q:GnT-V cells showed a dramatically slow cell invasion compared with T-WT:GnT-V, and T-N30Q and T-N78Q were intermediate between them. However, little difference was found among the TIMP-I transfectants of WiDr:mock cells. Collectively, these data strongly suggest that aberrant TIMP-I glycosylation, but not glycosylation itself, affects in vitro cell invasion.

<145> In vitro cell invasion involves two distinct processes: the hydrolysis of basement membranes coated throughout the 8μm-pores and migration through the pores. Since no difference in cell migration itself was observed (Fig. 13), we reasoned that the different cell invasion originated from differences in the hydrolysis rate of basement membrane. Treatment of gelatinase inhibitors significantly nullified the invasiveness of WiDr cells, indicating the involvement of hydrolysis of the basement membrane catalyzed by MMP-2 and/or MMP-9 (Garbett EA et al , 1999, MoI. Pathol. 52, 140-145; Ogiwara K et al, 2005, Proc. Natl. Acad. ScL 102, 8442-8447). In view of the result that the cell invasion rate was nearly the same among mock cells of TIMP-I transfectant , that the differences in cell invasion could arise from the changes in gelatinases-inhibitory ability of TIMP-I induced by depletion of N-I inked glycosylation of TIMP-I could be eliminated. Removal of the carbohydrates from human TIMP-I by treatment with N-glycosidase F has been shown to have no measurable effect on the inhibitory activity (Caterina NC et al, 1998, Biochim. Biophys. Acta 14, 21-34). A difference in the migration and invasion rate between WiDr:mock and WiDr: GnT-V did not arise from a different expression of MMP-2 and MMP-9 (Fig. 3c), and moreover, the transfection of TIMP-I or the mutant gene did not alter the expression pattern of gelatinases (data not shown). Taken together, the results suggest that the aberration in TIMP-I affects its inhibitory ability toward gelatinases and thus the rate in the hydrolysis of Matrigel coating materials. An increase in the hydrolysis rate through the aberration of TIMP- 1 would eventually lead to enhanced invasiveness of WiDr cells.

<146> <Exaraple 7> Effects of the aberrant glycosylation of TIMP-I on gelatinases inhibition: Fluorogenic substrate-based approach

<147> To confirm that the TIMP-I aberration results in mitigated inhibition on gelatinases, the proteolytic activities of gelatinases were monitored in the presence of various gelatinases inhibitors. ProMMP-2 and proMMP-9 (Calbiochem), were activated by incubation with an equal molar ratio of active MMP-3 (Sigma) at 37°C for 4h and with ImM p-aminophenylmercuric acetate (APMA) at 37°C for 2h, respectively. The active gelatinases were purified on a gelatin-Sepharose (Sigma) column according to the previous procedures (Fridman, R. et al, 1992, /. Biol. Chem. 267, 15398-15405). Following the gelatinases (50ng) were incubated with equal molar ratio of TIMP-I or MMP inhibitors and 8 mM substrates, DABCYL-GABA-PQGL-E(EDANS)-AK-NH₂

(Calbiochem) in 5OmM Tris-HCl buffer (pH 7.5) containing 15OmM NaCl, 5mM CaCl₂, O.lmM ZnCl₂, 0.02% Brij-35 at 4°C for Ih. As shown in Fig. 16, non- fluorogenic substrate DABCYL-GABA-PQGL-E(EDANS)-AK-NH₂ was hydrolyzed by gelatinases to be fluorogenic DABCYL-GABA-PQG, which was kinetically measured in an LS 45 Luminescence Spectrometer(PerkinElmer) at an excitation and emission wavelengths of 338 and 495nm, respectively. When gelatinases were pre-incubated with equal molar ratios of TIMP-I, the steady state of the hydrolysis reaction reached directly after initiation with the pattern of nearly zero-order kinetics (Inset of Fig. 17). The slope of formation of fluorescent products was used to determine the relative activity of gelatinases. Consistent with previous reports(Caterina NC et al, 1998, Biochim. Biophys. Acta 14, 21-34; Stricklin GP, 1986, Coll relat Res 6, 219- 228), our results showed that the depletion of the cognate N-I inked glycans on TIMP-I has little effect on gelatinase inhibition (Figs. 17, 18). Rather, the aberrant glycans were responsible for the mitigated inhibition on gelatinases. rTIMP-1 and the mutant proteins purified from WiDr÷mock retained wild-type levels of inhibitory activity, whereas T-WT from WiDr:GnT-V showed a significant loss of gelatinase inhibition.

<148> <Example 8> Effects of the aberrant glycosylation of TIMP-I on gelatinases inhibition". Zymographic approach

<149> Inhibition of gelatinases by TIMP-I was confirmed by gelatin zymography (Fig. 19), where the gelatinolytic ability of the latent and active forms of MMP-2 and MMP-9 were measured in the presence of various inhibitors. Latent and active forms of gelatinases were run on 12% SDS-PAGE gel copolymerized with 0.5% (w/v) gelatin and 5//g/m# rTIMP-1 or the mutant proteins.

Gelatinases and recombinant proteins in the gel were allowed to bind at 4⁰C

°c overnight, and the embedded gelatin was allowed to be hydrolyzed at 37 for

12h in 5OmM Tris-HCl (pH 7.5) containing 15OmM NaCl, 5mM CaCl₂, O.lmM ZnCl₂, and 0.02% (w/v) Brij-35. We reasoned that if TIMP-I is covalently incorporated in gelatin-copolymerized gels, it could tether gelatinases in the vicinity and, thereby, prevent from the gelatinolytic activity. The results show that rTIMP-1 isolated from WiDr÷mock cells retained an inhibitory effect on both the latent and active forms of MMP-9. However, TIMP-I from T-WT:GnT-V failed to tightly inhibit the gelatinolytic activity of MMP-9. The pattern of inhibition of MMP-2 by TIMP-I was not identical to the invasion assays and the fluorogenic assays, which might be due to restricted interactions of TIMP-I and MMP-2 in gels. Nevertheless, TIMP-I from T-WTiGnT-V showed mitigated inhibition on MMP-2 compared with TIMP-1 from T-Q30/78G:mock. These results suggest that the aberrantly-attached glycans play a role as interferences in the TIMP-I inhibition on the gelatinases.

<i50> <Example 9> Effects of the aberrant glycosylation of TIMP-I on gelatinases inhibition: Kinetic approach

<i5i> To address the question of why the aberrant glycosylation of TIMP-I results in a significant loss of gelatinases inhibition, we investigated the binding properties of TIMP-I and gelatinases. Kinetic parameters (Ji_0n, k_ou, Ky) for TIMP-1/gelatinase interaction were determined according to the previous procedure (Olson et al, 1997, J. Biol. Chetn. 272, 29975-29983) with some modifications. Briefly, the first-order binding constants (k) were determined under the following conditions. InM of active gelatinases were added into the reaction mixture containing 8mM fluorescent substrate and TIMP-I. TIMP-l:mock and TIMP-I:GnT-V were varied from 0 to 6nM and 0 to 25nM, respectively. Progress curves were recorded at 37°C in an LS 45 Spectrometer (PerkinElmer). The curves were fitted to Math Figure 1: <i52> [Math Figure 1]

IP]= v_a, + {vo-v_s){λ-Q^k')lk

<153> in which [P] is the product concentration, Vo and v_s are the initial and steady-state velocities, respectively, and k is pseudo first-order rate constant of inhibition: v_s, and k were calculated with regression method using

SigmaPlot (SPSS Science, Inc.). The second-order rate constant (k_oπ) was calculated by the linear regression of k as a function of TIMP-I concentration. The k_ott values were estimated from the time-course for the dissociation of the gelatinase-TIMP-1 complex. The complexes were prepared by incubation of equimolar amounts of gelatinases and TIMP-I (ImM) at 37^°C for Ih. Complex dissociation was achieved by diluting the complexes 1,000-fold in a cuvette containing the substrate. After equilibrium was reached, the recorded time-response curves were fitted to Math Fig.l. The negative of the obtained values were used as an approximation of £_Off- The inhibition constantsCK,) were calculated by K₁ - Ic₀U I -fen-

<154> The binding shows slow, tight-binding competitive inhibition and exhibits time-dependent inhibition and, as a result, gelatinases activities over time showed a curvilinear function on the progress curves. Fig. 20 and

Fig.21 show the time-courses for MMP-2 activity in the presence of various concentrations of TIMP-I:mock and TIMP-I:GnT-V, respectively. The kinetic pattern for MMP-9 was similar to that for MMP-2(Fig. 23-25). Kinetic parametersC^ k_o//, K_;) for gelatinases inhibition by TIMP-I were calculated were compiled as follows. As expected, the aberrantly glycosylated TIMP-I was found to loosely bind to active gelatinases with a lower k_on and to dissociate more efficiently as assessed by the higher k_off. K, for aberrant TIMP-

1/gelatinases interactions was found to be 7.2-fold higher than that for wild-type TIMP-l/MMP-2. The increase in inhibition constant was more dramatic for the TIMP-l/MMP-9 interaction, with the K, being increased by 11.4-fold.

These results indicate that the TIMP-1 aberration leads to a shift in equilibria for the TIMP-1/gelatinases interactions toward the dissociation process of the complex molecules. As a result, the tight binding and control of active gelatinases by TIMP-I is loosened, resulting in a higher bioavailability of uncomplexed, uninhibited, free gelatinases.

<155>

<i56> <Example 10> In vivo experiment for assessment of effect of aberrant glycosylation of TIMP-I

<i57> Cancer cell proliferation was reported to be decreased in MMP-9- deficient mice compared with wild-type mice (Coussens LM et al, 2000, Cell 103, 481-490) and reduced metastasis and angiogenesis of melanoma have been observed in mice that were genetically modified to lack MMP-2 expression (Itoh T et al, 1998, Cancer Res. 58, 1048-1051), indicating the critical role of gelatinases activity in tumor progression. A rationale in which an altered TIMP-I inhibition on gelatinases might affect cancer cell proliferation and/or invasive/metastatic potential in vivo in some manner, prompted us to test this possibility in a nude mouse system. Six-week-old C.By.Cg-Foxnf"

(nude) mice were housed and maintained in the animal facility under specific pathogen-free conditions with continuous microbiological monitoring. Sterilized commercial diet (Harlan, Indiana) and water were given ad libitum.

6

Cells (2 x 10 cells in 100m£ of PBS) were inoculated subcutaneousIy into the femur of nude mice. The nude mice (n=4 or 10/group) were inspected daily and tumor size was measured three times weekly in 2 dimensions with calipers. The

2 tumor volumes were calculated using the formula: length x (width) /2. For histopathological examination, subcutaneous tumor masses were sectioned from the sacrificed mice and fixed in 10% neutral-buffered formalin. After fixation, the specimens were routinely processed, embedded in paraffin and stained with hematoxylin and eosin (H&E).

<158> Each TIMP-I transfectant of WiDr:mock or WiDr:GnT-V cells were inoculated subcutaneousIy into the femurs of nude mice. The tumor began to form 2 weeks after the inoculations, independent of TIMP-I variations(Fig. 26). When equal number of the cells were inoculated, the tumor growth was remarkably faster in the mice inoculated with T-WT:GnT-V(n=4) than those with T-N30/78Q:GnT-V cells (Fig. 27). Little difference in tumorigenesis could, however, be found among TIMP-I transfectants of WiDr:mock cells (Fig. 28). A histopathological analysis of the tumor formed indicates that the tumor cells, bordered with dashed lines, exhibited a growth property in which tumor cells proliferate by infiltrating into skeletal muscle fibers(solid arrow) and the surrounding endomysiurn(open arrow), the major component of which is wisps of collagen (Fig. 29). Since relatively large deviations, originating from the small population of mice tested, were seen, we performed the mouse experiment on a larger scale(n=30). A representative group of cells comprising T-WT:GnT-V, T-N30/78Q:GnT-V, T-WT:mock and parental WiDr were each tested in 30 mice with a higher confidence level (Fig. 30). As expected, only minute amounts of tumor mass were formed in the T-N30/78Q:GnT-V-inoculated mice (p<0.01, compared with T-WT=GnT-V), in great contrast to the tumor mass of T-WTGnT-V. The tumor mass of parental WiDr cells (p<0.05, compared with T-WT:GnT-V) was somewhat larger than that of T-WT:mock (pO.Ol, compared with T-WT--GnT-V), which appears to be due to lower level of TIMP-I expression in parental WiDr cells than in T-WT:mock cells

<159>

<16O> <Example 11> Relations of TIMP-1 aberration with colon cancer invasion and metastasis in colon cancer tissues

<i6i> The relationship between TIMP-I aberration and cancer progressions has not been defined yet, but both in vitro experiments and in vivo data provided sufficient circumstantial evidence for an association of the aberration in cancer progression of colon cancer patients. To deduce the involvement of aberrant glycosylation of TIMP-I in colon cancer, both normal and tumor tissues of colon cancer cases were analyzed in terms of expression level and TIMP-I glycosylation.

<i62> As shown in Fig. 31, the expression level of TIMP-1 did not reflect the progression of colon cancer. TIMP-1 expression was elevated in almost all colon cancers compared to its paired normal tissues consistent with previous report (Egeblad M et al, 2002 Nature Rev. 2, 161-174; Heppner KJ et al, 1996, Am. J. Pathol. 149, 273-282; Baker EA et al , 2000, Br. J. Surg. 87, 1215- 1221; Yoshikawa T et al , 2001, Cancer 91, 1739-1744; Huang LW et al , 2000, Gynecol. Oncol. 11, 369-376), but the extent of the elevation was independent of Astler-Coller colon cancer stages, an index of colon cancer progression and commonly a metastatic parameter. Rather, at the stages where cell invasion and spreading to near tissues is prosperous, i.e. Astler-Coller grade C and D, an aberrantly glycosylated TIMP-1 was found in more than 70% of colon cancer tissues tested, which is quite different from the case of group I. Interestingly, the transcription level of GnT-V was estimated by RT- PCR to be increased in a cancer stage-dependent manner, and concomitantly with the promotion of TIMP-1 aberration. When GnT-V overflows by a signal under cancerous conditions, the transferase is likely to promote TIMP-1 aberration and thus cancer malignancy. Fig. 32 shows the representative results of 10 cases. Cases 1, 4 and 5 showed a marked increase in bl,6- GlcNAc-attached aberration in TIMP-I glycosylation and, in agreement with Fig. 11, a slight increase in molecular mass. Those cases showed that an elevated transcription of GnT-V (Fig. 33) revealed a relatively high cancer stage and a clinically high tumor invasion to remote sites, especially metastasizing to regional lymph nodes (data not shown). To our knowledge, the aberrant glycosylation of TIMP-I has not been reported to correlate with the cancer invasion and metastasis in vivo or in vitro. Our data strongly suggest that the aberrant glycosylation of TIMP-I induced by GnT-V is closely associated with the elevated invasion/metastasis potential in colon cancer cells. One of the intriguing features is that TIMP-I has seemingly discordant, dual functions; TIMP-1 not only inhibits cancer progression by abrogating MMPs, but also has effects on cancer cell growth and survival in an MMP- dependent or -independent manner. Actually, overexpression of TIMP-1 inhibits tumor growth and metastasis of melanoma (Khokha R et al , 1994, J. Natl. Cancer Inst. 86, 299-304) and suppresses the metastatic potential of human gastric cells (Watanabe M et al, 1996, Cancer 77, 1676-1680) and oral squamous cell carcinoma (Nii M et al, 2000, Int. J. Oncol. 16, 11-124). However, this is quite contradictory to the tendency that TIMP-1 is upregulated in many cancer types (Egeblad M et al , 2002 Nature Rev. 2, 161- 174; Heppner KJ et al , 1996, Am. J. Pathol. 149, 273-282; Baker EA et al , 2000, Br. J. Surg. 87, 1215-1221; Yoshikawa T et al , 2001, Cancer 91, 1739- 1744; Huang LW et al, 2000, Gynecol. Oncol. 77, 369-376) and the reports that a high level of TIMP-1 correlates with a poor prognosis (Coussens LM et al, 1996, Chem. Biol. 3, 895-904; Reed JC 2001, Curr. Opin. Oncol. 11, 68-75). Moreover, high preoperative plasma TIMP-1 levels are associated with a short survival of patients with colorectal cancer (HoIten-Andersen MN et al, 2000, Clin. Cancer Res. 6, 4292-4299), lung cancer (Ylisirnio S et al, 2000 Anticancer Res. 20, 1311-1316), and gastric cancer (Yoshikawa T et al, 2000, Cancer Lett. 151, 81-86). If the effects of TIMP-I on cancer development and progression are taken into account only in terms of "quantity" without consideration of "quality" incessant debates over the genuine role of TIMP-I in biological systems would occur and, although both aspects are relevant, it would fail to explain clearly whether TIMP-1 is pro-oncogenic or not.

[Industrial Applicability] As described, the present invention provides a clinically useful kit for more efficacious diagnosis of cancer via concomitant assay of protein level and changes in glycosylation.

Claims

[CLAIMS] [Claim 1] a detection method for change of glyco-structure of one or more proteins associated with tumorigenesis and cancer metastasis containing following steps;

( i ) A step in which antibodies for at least one selected from a group that are assumed to be involved in tumorigenesis and cancer metastasis consisting of α-l-antitrypsin, angiotensinogen, βhexosaminidase β chain precursor, cathepsin D preproprotein, cathepsin X precursor, chain H of IgGl, dipeptidyl aminopeptidase II, discoidin receptor tyrosine kinase isoform b, dystroglycan 1 precursor, granulins precursor, heat shock 70 kDa protein, heat shock protein 1(9OkDa) β , heparan sulfate proteoglycan perlecan, hexosaminidase A preproprotein, Ig K chain V-III, IgG Fc binding protein, laminin receptor-like protein 5, legumain precursor, Met proto-oncogene precursor, N-acetylgalactosamine-6-sulfatase, N-acetyl-glucosamine-6- sulfatase, neogenin homolog 1, prolyl 4-hydroxylase, prosaposin, protective protein for b-galactosidase, protein tyrosine kinase 6, protein tyrosine kinase 7, ribonuclease T2 precursor, serine protease 22, tumor-associated calcium signal transducer 1 precursor, tumor rejection antigen-l(gp96) and Zn- α.-2-glycoprotein are covalently bound to matrix.

(ii) A step in which protein sample is added to the antibody-bound matrix and induced to interact each other, followed by washing out.

(iii) A step in which interactions are induced by treating monoclonal antibody for the proteins mentioned above in control groups and by treating lectins that recognize an altered N-glycan in experimental groups respectively, followed by washing out.

(iv) A step in which secondary antibody labeled with chromogenic enzyme is treated in the monoclonal antibody-bound control groups or chromogenic enzyme-labeled receptor for ligand bound on lectin is treated in the lectin- treated experimental groups, followed by washing out.

(v) A step in which a chromo-substrate is treated to emit lights, a light intensity is measured. [Claim 2] a detection method for changes of glyco-structure of one or more proteins associated with tumorigenesis and cancer metastasis containing following steps;

( i ) A step in which a sample preparation is covalently bound onto the matrix in control group and experimental group;

(ii) A step in which antibody for at least one selected from a group consisting of the following proteins that associated with tumorigenesis and cancer metastasis is bound in control group; α-1-antitrypsin, angiotensinogen, βhexosaminidase β chain precursor, cathepsin D preproprotein, cathepsin X precursor, chain H of IgGl, dipeptidyl aminopeptidase II, discoidin receptor tyrosine kinase isoform b, dystroglycan 1 precursor, granulins precursor, heat shock 70 kDa protein, heat shock protein 1(9OkDa) β, heparan sulfate proteoglycan perlecan, hexosaminidase A preproprotein, Ig K chain V-EI, IgG Fc binding protein, laminin receptor- like protein 5, legumain precursor, Met proto-oncogene precursor, N- acetylgalactosamine-6-sulfatase, N-acetyl-glucosamine-6-sulfatase, neogenin homolog 1, prolyl 4-hydroxylase, prosaposin, protective protein for b- galactosidase, protein tyrosine kinase 6, protein tyrosine kinase 7, ribonuclease T2 precursor, serine protease 22, tumor-associated calcium signal transducer 1 precursor, tumor rejection antigen-l(gp96) and Zn- α-2- glycoprotein, and ligand-labeled lectins are added to the matrix for detecting alteration in glycan structures in experimental group, followed by washing out .

(iii) A step in which secondary antibody labeled with chromogenic enzyme is treated in the antibody-bound control groups and chromogenic enzyme- labeled receptor for ligand bound on lectin is treated in the lect in-treated experimental group rexpectively, followed by washing out.

(iv) A step in which a chromo-substrate is treated to emit lights, a light intensity is measured. [Claim 3]

The method according to claim 1, wherein said protein sample is acquired from blood or urine. [Claim 4]

The method according to claim 1, wherein said glyco-structure is sugar chain branches of N-linked βl,6 N-acetylglucosamine. [Claim 5]

The method according to claim 1, wherein said glyco-structure is sugar chain branches of N-linked αl,6 fucose. [Claim 6]

The method according to claim 1, wherein said lectin is at least one selected from a group consisting lectin phytohaemagglutinine 4(L₄-PHA) and lens culinaris agglutinin(LCA). [Claim 7] cancer-diagnostic kit comprising matrix covalently bound with antibody for at least one selected from the a group consisting of α-l-antitrypsin, angiotensinogen, βhexosaminidase β chain precursor, cathepsin D preproprotein, cathepsin X precursor, chain H of IgGl, dipeptidyl aminopeptidase II, discoidin receptor tyrosine kinase isoform b, dystroglycan 1 precursor, granulins precursor, heat shock 70 kDa protein, heat shock protein 1(9OkDa) β , heparan sulfate proteoglycan perlecan, hexosaminidase A preproprotein, Ig K chain V-HI, IgG Fc binding protein, laminin receptor- like protein 5, legumain precursor, Met proto-oncogene precursor, N- acetylgalactosamine-6-sulfatase, N-acetyl-glucosamine-6-sulfatase, neogenin homolog 1, prolyl 4-hydroxylase, prosaposin, protective protein for b- galactosidase, protein tyrosine kinase 6, protein tyrosine kinase 7, ribonuclease T2 precursor, serine protease 22, tumor-associated calcium signal transducer 1 precursor, tumor rejection antigen-l(gp96) and Zn- α-2- glycoprotein, antibody for the glycoprotein, ligand-labeled lectin, chromogenic enzyme-labeled secondary antibody and chromogenic enzyme-labeled receptor for the ligand. [Claim 8]

The kit according to claim 7, wherein said cancer is colon, gastric, lung, liver, uterine, breast or pancreatic cancer. [Claim 9]

The kit according to claim 7, wherein said matrix is selected from a group consisting of nitrocellulose membrane, polyvinyl-based well plates, polystyrene-based well plates and glass-based slide glass. [Claim 10]

The kit according to claim 7, wherein said chromogenic enzyme is selected from a group consisting of peroxidase and alkaline phosphatase. [Claim 11]

The kit according to claim 7, wherein said ligand and receptor are biotin and avidin. [Claim 12]

The kit according to claim 7, wherein chromogenic substrates is comprised supplementarily. [Claim 13]

The kit according to claim 7, wherein said lectin is at least one selected from a group consisting lectin phytohaemagglutinine 4(L₄-PHA) and lens culinaris agglutinin(LCA).