HK1190170A - Biomarker dact1 for gastric cancer - Google Patents

Abstract

The present invention provides a method for diagnosing and determining prognosis of gastric cancer in a subject by detecting suppressed expression of the dact1 gene, which in some cases is due to elevated methylation level in the genomic sequence of this gene. A kit and device useful for such a method are also provided. In addition, the present invention provides a method for treating gastric cancer by increasing dact1 gene expression or activity.

Description

DACT1 as biomarker of gastric cancer

Background

Gastric cancer (gastric cancer), also known as gastric cancer (stomach cancer), is the fourth most common cancer worldwide, with about 1,000,000 cases diagnosed each year. Gastric cancer is a disease with a high mortality rate (about 800,000 deaths per year), which makes it the second most common cause of cancer death worldwide after lung cancer. The incidence of gastric cancer is significantly higher in men and in developing countries, including many asian countries.

In the early stage of gastric cancer, there are usually no clinical symptoms or only ambiguous symptoms, and thus many cases cannot be diagnosed until the disease reaches the late stage. This often leads to a poorer prognosis: metastasis occurs in 80-90% of individuals diagnosed with gastric cancer, with 65% of individuals diagnosed early having a 6 month survival rate and less than 15% of individuals diagnosed late having a 6 month survival rate.

Because of the prevalence of gastric cancer and its severe impact on the life expectancy of patients, new methods of diagnosing, monitoring and treating gastric cancer are needed. The present invention fulfills this need and other related needs.

Summary of The Invention

In a first aspect, the present invention provides a method of detecting gastric cancer in an individual. The method comprises the following steps: (a) measuring the expression level of DACT1 in a sample taken from the subject; and (b) comparing the expression level obtained in step (a) with a standard control. Detection of a decrease in the expression level of DACT1 when compared to a standard control indicates that the individual may have gastric cancer. Typically, the sample used in the method is a gastric mucosal sample, for example a gastric mucosal sample comprising gastric epithelial cells.

In some embodiments, the expression level of DACT1 is the DACT1 protein level. In other embodiments, the expression level of DACT1 is the DACT1mRNA level. When measuring the level of DACT1 protein, step (a) may comprise performing an immunoassay with an antibody that specifically binds to DACT1 protein. For example, Western blot analysis can be used. In other cases, step (a) may comprise mass spectrometry or hybridization-based assays, such as hybridization to microarrays, fluorescent probes, or molecular beacons.

When measuring DACT1mRNA levels, step (a) may, in some cases, comprise an amplification reaction, such as a Polymerase Chain Reaction (PCR), in particular a reverse transcriptase-PCR (RT-PCR). In other cases, the detection step may comprise a polynucleotide hybridization assay, such as a Southern blot analysis or Northern blot analysis or an in situ hybridization assay. For example, polynucleotide probes can be used in polynucleotide hybridization assays that hybridize to the complement of SEQ ID NO 1, 4, or 6 or above. In some cases, a polynucleotide probe may include a detectable moiety.

In some embodiments, when the patient is indicated as having gastric cancer after performing the first round of the above method steps, the method may further comprise repeating step (a) with the same type of sample from the individual after a period of time. An increase in the expression level of DACT1 after the period of time, compared to the amount of expression level of DACT1 of the initial step (a), is indicative of an improvement in gastric cancer, while a decrease is indicative of a worsening of gastric cancer.

In a second aspect, the present invention provides a method of detecting gastric cancer in an individual. The method comprises the following steps: (a) treating a sample taken from said individual with an agent capable of differentially modifying methylated DNA and unmethylated DNA; and (b) determining whether each CpG in the CpG-containing genomic sequence is methylated or unmethylated, the CpG-containing genomic sequence being at least a segment of SEQ ID NO 1 or 6 and comprising at least one CpG. When the presence of a methylated CpG is detected in the CpG-containing genomic sequence, this is an indication that the individual may have gastric cancer.

In some embodiments, the CpG-containing genomic sequence contains two or more CpG, and when at least 50% of all CpG are methylated, the individual is indicated as having gastric cancer. In some cases, the CpG-containing genomic sequence is a segment of at least 15, 20, 50 or more contiguous nucleotides of SEQ ID No. 1 or 6. In other cases, the CpG-containing genomic sequence is SEQ ID NO 1 or 6. In one embodiment of the claimed method, the CpG-containing genomic sequence is SEQ ID NO 6 and when at least 5 of all CpG in the CpG-containing genomic sequence are methylated, indicating that the individual is likely to have gastric cancer.

In some examples, the sample used in the claimed method is a gastric mucosal sample. In other examples, when the individual is indicated to have gastric cancer after the first round of the method, the method further comprises repeating steps (a) and (b) with the same type of sample from the individual after a period of time. An increase in the number of methylated CpG detected after a period of time compared to the number of methylated CpG determined in the initial step (b) is indicative of a worsening of gastric cancer, whereas a decrease is indicative of an improvement of gastric cancer.

In some embodiments, the agent used in the claimed method that is capable of differentially modifying methylated DNA and unmethylated DNA is an enzyme that preferentially cleaves methylated DNA, an enzyme that preferentially cleaves unmethylated DNA, or bisulfite. In other embodiments, step (b) of the method comprises an amplification reaction; or step (b) may comprise sequencing the DNA molecule.

In a third aspect, the present invention provides a kit for detecting gastric cancer in an individual, the kit comprising (1) a standard control that provides an average amount of DACT1 protein or DACT1 mRNA; and (2) reagents capable of specifically and quantitatively identifying DACT1 protein or DACT1 mRNA. In some cases, the agent may be an antibody that specifically binds to DACT1 protein; alternatively, the agent may be a polynucleotide probe that hybridizes to DACT1 mRNA. For example, the polynucleotide probe has a nucleotide sequence shown by the complement of SEQ ID NO. 1, 4 or 6 or more. The reagent may include a detectable moiety. In other cases, the kit may further comprise two oligonucleotide primers for specifically amplifying at least a segment of SEQ ID NO 2 or 3 or the complement thereof in an amplification reaction. Typically, the kit may also include an instruction manual.

In a fourth aspect, the present invention provides a method of inhibiting gastric cancer cell growth. The claimed method comprises the steps of: gastric cancer cells are contacted with an effective amount of (1) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO. 5, or (2) a nucleic acid comprising a polynucleotide sequence encoding SEQ ID NO. 5. In some embodiments, the nucleic acid is an expression cassette comprising a promoter operably linked to a polynucleotide sequence encoding SEQ ID No. 5. A variety of promoters may be used in the method, for example, the promoter may be an epithelial cell-specific promoter. In other embodiments, the nucleic acid comprises the polynucleotide sequence set forth in SEQ ID NO. 2 or 3. In other embodiments, the gastric cancer cells are located in the patient.

In a fifth aspect, the invention provides an isolated nucleic acid having a nucleotide sequence at least 95% identical to a stretch of about 20-100 contiguous nucleotides of the complement of SEQ ID NO 1, 2, 3, 4 or 6 or more. In some embodiments, the nucleic acid has a nucleotide sequence identical to a stretch of about 20-100 consecutive nucleotides of the complement of SEQ ID NO 1, 2, 3, 4, or 6 or more. In other embodiments, the nucleic acid is conjugated to a detectable moiety.

In addition, the present invention provides a kit for detecting gastric cancer. The kit comprises: (1) an agent that differentially modifies methylated DNA and unmethylated DNA, and (2) an indicator that determines whether each CpG in the CpG-containing genomic sequence is methylated or unmethylated after treatment of a sample from an individual undergoing gastric cancer detection with the agent. The CpG-containing genomic sequence is at least one segment of SEQ ID NO 1 or 6 and comprises at least one CpG. The present invention also provides a composition for inhibiting the growth of gastric cancer cells. The composition comprises an effective amount of (1) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO. 5 (e.g., a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO. 5) or (2) a nucleic acid comprising or consisting of a polynucleotide sequence encoding SEQ ID NO. 5 (e.g., a nucleic acid sequence comprising the polynucleotide sequence of SEQ ID NO. 2 or 3) and a pharmaceutically acceptable carrier. Thus, the present invention also provides the use of a polypeptide comprising the amino acid sequence set forth in SEQ ID NO. 5 (e.g., a polypeptide consisting of the amino acid sequence of SEQ ID NO. 5) or a nucleic acid comprising a polynucleotide sequence encoding SEQ ID NO. 2 or 3 (e.g., a nucleic acid sequence comprising the polynucleotide sequence of SEQ ID NO. 2 or 3 or a nucleic acid sequence consisting of the polynucleotide sequence of SEQ ID NO. 2 or 3) for the preparation of a medicament for inhibiting gastric cancer cell growth. Furthermore, the present invention provides the use of a polynucleotide sequence comprising or consisting of a segment of SEQ ID NO 1, 2, 3, 4 or 6 or a segment of the complement thereof for the preparation of a kit for the detection of gastric cancer. The segment is typically about 20-100 contiguous nucleotides of SEQ ID NO 1, 2, 3, 4 or 6 or the complement thereof.

Drawings

FIG. 1 shows DACT1mRNA expression in normal tissues and gastric cell lines in one embodiment.

FIG. 2 shows the results of bisulfite genomic sequencing of the DACT1 promoter (SEQ ID NOS:15-18) in a gastric cancer cell line according to one embodiment.

FIG. 3 shows the effect of demethylating agents on DACT1 expression in one embodiment.

FIG. 4 shows the effect of DACT1 α expression on transfected cells in one embodiment of a colony formation assay.

FIG. 5 shows the effect of DACT1 α expression on transfected cells in one embodiment of a cell growth curve.

Figure 6 shows the inhibitory effect of DACT 1a on cell spreading, F-actin formation, cell migration and invasive ability in one embodiment.

FIG. 7 shows the growth inhibitory effect of DACT1 α expression in one embodiment of nude mice.

FIG. 8 shows the methylation status of DACT1 (SEQ ID NOS16 and 18) and Kaplan-Meier analysis of gastric cancer surviving patients in primary gastric cancer and normal stomach tissue samples in one embodiment.

Definition of

The term "DACT 1 gene" or "DACT 1 protein" as used herein refers to any naturally occurring variant or mutant, interspecies homolog or ortholog, or artificial variant of the human DACT1 gene or DACT1 protein. The human DACT1 gene is located on chromosome 14q 23.1. The cDNA sequence of the human wild-type DACT1 gene is shown in GenBank accession No. NM-016651 (provided herein as SEQ ID NO: 3), which encodes the 798 amino acid DACT1 protein (provided herein as SEQ ID NO: 5). The DACT1 protein within the meaning of the present application typically has at least 80% or 90% or 95% or more sequence identity with the human wild-type DACT1 protein.

Herein, the terms "gastric cancer" and "gastric cancer" have the same meaning, and refer to cancer of the stomach or gastric cells. Such cancers may be adenocarcinomas that occur in the gastric lining (mucosa or gastric epithelium) and may also occur in the pyloric, somatic or cardiac portions (inferior, somatic and superior) of the stomach. "gastric cancer cells" are gastric epithelial cells that are characteristic of gastric cancer, and include precancerous cells, which are in the early stage of transition to cancer cells or are prone to transition to cancer cells. Such cells may exhibit one or more phenotypic traits characteristic of cancer cells.

As used herein, the term "or" is generally intended to include "and/or" unless the context clearly indicates otherwise.

The term "gene expression" as used herein is used to refer to the transcription of DNA to form an RNA molecule encoding a particular protein (e.g., the human DACT1 protein), or to the translation of a protein encoded by a polynucleotide sequence. In other words, the term "gene expression" herein includes both mRNA levels and protein levels encoded by a gene of interest (e.g., the human DACT1 gene).

As used herein, the term "biological sample" or "sample" includes tissue sections such as biopsy and autopsy samples, as well as frozen sections taken for histological purposes, or processed versions of any of these samples. Biological samples include blood or blood components or products (e.g., serum, plasma, platelets, red blood cells, etc.), sputum or saliva, lymphoid and tongue tissue, cultured cells (e.g., primary cultures, explanted tissues, and transformed cells), stool, urine, gastric biopsies, and the like. Biological samples are typically obtained from eukaryotes, which may be mammals, which may be primates, and which may be human individuals.

As used herein, the term "biopsy" refers to the process of removing a tissue sample for diagnostic or prognostic evaluation as well as to the tissue sample itself. Any biopsy technique known in the art may be applied to the diagnostic and prognostic methods of the present invention. The biopsy technique applied will depend on the type of tissue to be evaluated (e.g., tongue, colon, prostate, kidney, bladder, lymph nodes, liver, bone marrow, blood cells, stomach tissue, etc.), among other factors. Representative biopsy techniques include, but are not limited to, excisional biopsy, incisional biopsy, needle biopsy, surgical biopsy, and bone marrow biopsy, and may include colonoscopy. Those skilled in the art are well known for a variety of biopsy techniques and can select between them and perform them with minimal experimentation.

As used herein, the term "isolated" nucleic acid molecule refers to a nucleic acid molecule that is separated from other nucleic acid molecules with which the isolated nucleic acid molecule is normally associated. Thus, an "isolated" nucleic acid molecule includes, but is not limited to, a nucleic acid molecule that does not contain a nucleotide sequence that is naturally present in the genome of the organism from which the isolated nucleic acid is derived at one or both ends of the nucleic acid (e.g., a cDNA or genomic DNA fragment produced by PCR or restriction endonuclease digestion). Such isolated nucleic acid molecules are typically introduced into a vector (e.g., a cloning vector or an expression vector) for ease of manipulation or for the production of fused nucleic acid molecules. In addition, an isolated nucleic acid molecule can include an engineered nucleic acid molecule, such as a recombinant or synthetic nucleic acid molecule. For example, nucleic acid molecules present within hundreds to millions of other nucleic acid molecules in a nucleic acid library (e.g., a cDNA or genomic library) or a gel containing restriction digested genomic DNA (e.g., agarose or polyacrylamide) are not "isolated" nucleic acids.

The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in either single-or double-stranded form, and polymers thereof. Unless specifically limited, the term includes nucleic acids containing known analogs of natural nucleotides that have similar binding properties to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly includes conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, Single Nucleotide Polymorphisms (SNPs) and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed base and/or deoxyinosine residues (Batzer et al, Nucleic Acid Res.19:5081(1991); Ohtsuka et al, J.biol.chem.260: 2605-membered-rings 2608(1985); and Rossolini et al, mol.cell.Probes 8:91-98 (1994)). The term nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene.

The term "gene" means a segment of DNA involved in the production of polypeptide chains, which comprises regions (leader and trailer) in the context of coding regions involved in transcription/translation and regulation of transcription/translation of the gene product, as well as intervening sequences (introns) between individual coding segments (exons).

In the present application, the terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. These terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. These terms, as used herein, include amino acid chains of any length, including full-length proteins (i.e., antigens), in which the amino acid residues are linked by covalent peptide bonds.

The term "amino acid" refers to naturally occurring and synthetic amino acids as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code and those amino acids which are subsequently modified, for example, hydroxyproline, γ -carboxyglutamic acid and O-phosphoserine. For the purposes of this application, an amino acid analog refers to a compound that has the same basic chemical structure as a naturally occurring amino acid, i.e., the carbon to which the hydrogen, carboxyl, amino, and R groups are attached, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. These analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. For the purposes of this application, an amino acid mimetic refers to a compound that has a structure that is different from the usual chemical structure of an amino acid, but functions in a similar manner to a naturally occurring amino acid.

Amino acids can include amino acids with non-naturally occurring D-chirality (as disclosed in WO 01/12654), which can improve the stability (e.g., half-life), bioavailability, and other properties of polypeptides comprising one or more such D-amino acids. In some cases, one or more amino acids of the therapeutic polypeptide have D-chirality, and possibly all amino acids have D-chirality.

Amino acids may be referred to herein by the well-known three-letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical nomenclature Commission. Nucleotides, likewise, may be referred to by their accepted single-letter codes.

The term "identical" or percent "identity" as used herein when describing two or more polynucleotide or amino acid sequences refers to: two or more sequences or subsequences that are the same, or two or more sequences or subsequences that have a specified percentage of amino acid residues or nucleotides that are the same (e.g., a variant DACT1 protein used in the methods of the invention (e.g., for treating gastric cancer) have at least 80% sequence identity, preferably 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a reference sequence (e.g., a wild-type human DACT1 protein), when compared and aligned for maximum correspondence over a comparison window or designated region, as determined using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Such sequences are referred to as "substantially identical". With respect to polynucleotide sequences, this definition also refers to the complement of the test sequence. Preferably, identity exists over a region that is at least about 50 amino acids or nucleotides in length, or more preferably, over a region that is 75-100 amino acids or nucleotides in length.

For sequence comparison, one sequence is typically used as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters may be used, or optional parameters may be specified. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the program parameters. For sequence comparisons of nucleic acids and proteins, the BLAST and BLAST 2.0 algorithms and default parameters discussed below are used.

As used herein, a "comparison window" includes a segment having any one of a number of contiguous positions selected from the group consisting of 20 to 600, typically about 50 to about 200, more typically about 100 to about 150, wherein a sequence can be compared after optimal alignment with a reference sequence having the same number of contiguous positions. Methods of sequence alignment for comparison are well known in the art. Optimal alignment of sequences for comparison can be performed by: such as the local homology algorithm of Smith & Waterman, adv.Appl.Math.2:482(1981), the homology alignment algorithm of Needleman & Wunsch, J.Mol.biol.48:443(1970), the similarity search method of Pearson & Lipman, Proc.Nat' l.Acad.Sci.USA 85:2444(1988), which can be implemented by computer (GAP, BESTFIT, FASTA and TFASTA of Wisconsin Genetics Software Package (Wisconsin Genetics Software Package), Genetics computer Group (Genetics computer Group), Science 575 Dr., Madison, Wis), or by manual alignment and visual inspection (see, for example, Current Protocols in Molecular Biology (Aumbel.1995)).

Examples of algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms described in Altschul et a1., (1990) J.Mol.biol.215: 403-. Software for running BLAST analysis is publicly available through the National Center for biotechnology Information website ncbi. The algorithm first involves identifying high scoring sequence pairs (HSPs) by identifying short characters of length W in the query sequence that match or meet some positive threshold score T when aligned with a character of the same length in a database sequence. T is referred to as the neighbor score threshold (Altschul et al, supra). These initial adjacent character hits serve as the basis for initiating searches to find longer HSPs containing them. Character hits are then extended in both directions along each sequence, provided that the cumulative alignment score can be increased. For nucleotide sequences, the parameters M (reward score for pairs of matching residues; always greater than 0) and N (penalty score for mismatching residues; always less than 0) can be used to calculate a cumulative score. For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Stopping the character hit extension in each direction when: when the accumulated comparison score decreases by X amount from the maximum implementation value; (ii) when the cumulative score reaches zero or below zero due to accumulation of one or more negative-scoring residue alignments; or at the end of either sequence. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) used a word length (W) of 28, an expectation (E) of 10, M =1, N = -2, and double-strand comparisons as default parameters. For amino acid sequences, the BLASTP program uses a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix as default parameters (see, Henikoff and Henikoff, proc. natl. acad. sci. usa89:10915 (1989)).

The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Nat' l.Acad. Sci. USA 90: 5873. 5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P (N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a test nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

As described below, an indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with an antibody raised against the polypeptide encoded by the second nucleic acid. Thus, for example, two peptides are generally substantially identical if one polypeptide differs from the other polypeptide only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules, or their complements, are capable of hybridizing to each other under stringent conditions, as described below. Another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequences.

As used herein, the terms "stringent hybridization conditions" and "high stringency" refer to conditions under which a probe hybridizes to its target subsequence (typically in a complex mixture of nucleic acids) and not to other sequences. Stringent conditions are sequence dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. Extensive guidance to Nucleic acid Hybridization is found in Tijssen, technique Biochemistry and Molecular Biology- -Hybridization with Nucleic acid Probes, "Overview of protocols for Hybridization and the strategy of Nucleic acid assays (Overview of Hybridization principles and Nucleic acid assay strategies)," 1993, and is readily understood by those skilled in the art. Typically, stringent conditions are selected to be about 5-10 ℃ below the thermal melting point (Tm) for the specific sequence at a defined ionic strength, pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (at Tm, 50% of the probes are occupied at equilibrium due to the presence of excess target sequence). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, the positive signal is at least 2 times background, preferably 10 times background hybridization. Exemplary stringent hybridization conditions can be as follows: 50% formamide, 5 XSSC and 1% SDS, incubated at 42 ℃, or 5 XSSC, 1% SDS, incubated at 65 ℃, and washed in 0.2 XSSC and 0.1% SDS at 65 ℃.

For nucleic acids that do not hybridize to each other under stringent conditions, if the polypeptides they encode are substantially identical, the nucleic acids are still substantially identical. This can occur, for example, when a copy of a nucleic acid is produced using the maximum codon degeneracy permitted by the genetic code. In these cases, the nucleic acid typically hybridizes under moderately stringent hybridization conditions. Exemplary "moderately stringent hybridization conditions" include: hybridization was performed in a buffer of 40% formamide, 1M NaCl, 1% SDS at 37 ℃ and washing in 1 XSSC at 45 ℃. Positive hybridization was at least twice background. One skilled in the art will readily recognize that other hybridization and wash conditions may be used to provide conditions of similar stringency. Other guidance for determining hybridization parameters is provided in many references, for example, Current Protocols in Molecular Biology, ed.

An "expression cassette" is a nucleic acid construct, produced recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular polynucleotide in a host cell. The expression cassette may be part of a plasmid, viral genome or nucleic acid fragment. Typically, an expression cassette includes a polynucleotide to be transcribed operably linked to a promoter. In this context, "operably linked" means that two or more genetic elements (such as a polynucleotide coding sequence and a promoter) are placed in relative positions that allow the elements to perform an appropriate biological function (such as the promoter directing transcription of the coding sequence). Other elements that may be present in an expression cassette include elements that enhance transcription (e.g., enhancers) and terminate transcription (e.g., terminators), as well as elements that confer some binding affinity or antigenicity to the recombinant protein produced by the expression cassette.

The term "bisulfite" as used herein includes all types of bisulfite salts, such as sodium bisulfite, which can chemically convert cytosine (C) to uracil (U) without chemically modifying methylated cytosines, and thus can be used to modify DNA sequences based on differences in the methylation state of DNA.

As used herein, an agent that "differentially modifies" methylated DNA or unmethylated DNA includes any agent that reacts differentially with methylated and unmethylated DNA in a process by which a distinguishable product or quantitatively distinguishable result (e.g., degree of binding or precipitation) is produced from methylated and unmethylated DNA, thereby allowing the methylation state of the DNA to be identified. Such processes may include, but are not limited to, chemical reactions (such as conversion of unmethylated C → U by bisulfite), enzymatic treatment (such as cleavage by methylation dependent endonucleases), binding, and precipitation. Thus, an enzyme that preferentially cleaves methylated DNA is an enzyme that cleaves DNA molecules with significantly higher efficiency when the DNA is methylated, whereas an enzyme that preferentially cleaves unmethylated DNA exhibits significantly higher efficiency when the DNA is unmethylated. In the context of the present invention, an agent that "differentially modifies" methylated or unmethylated DNA also refers to any agent that, in its binding to DNA sequences or in precipitation of DNA sequences, is capable of exhibiting differential capability depending on the methylation state of those DNA sequences. One class of such agents consists of methylated DNA binding proteins.

As used herein, a "CpG-containing genomic sequence" refers to a segment of DNA sequence located at a defined position in the genome of an individual. Typically, a "CpG-containing genomic sequence" is at least 15 contiguous nucleotides in length and contains at least one CpG pair. In some cases, it may be at least 18, 20, 25, 30, 50, 80, 100, 150, 200, 250, or 300 contiguous nucleotides in length and contain at least 2, 3, 4,5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 CpG pairs. For any "CpG-containing genomic sequence" at a given location (e.g., within a region of the human DACT1 genomic sequence, such as a region containing a promoter and exon 1), nucleotide sequence variations may exist between individuals, and even between alleles of the same individual. In addition, a "CpG-containing genomic sequence" may include a nucleotide sequence that is transcribed or not transcribed into a protein product, and the nucleotide sequence may be a protein coding sequence, a non-protein coding sequence (e.g., a transcription promoter), or a combination thereof.

The term "immunoglobulin" or "antibody" (used interchangeably herein) refers to an antigen binding protein having a substantially four-polypeptide chain structure consisting of two heavy chains and two light chains, the chains being stabilized, for example, by interchain disulfide bonds, the antigen binding protein having the ability to specifically bind an antigen. Both heavy and light chains fold into domains.

The term "antibody" also refers to antigen-binding and epitope-binding fragments of antibodies, e.g., Fab fragments, that can be used in immunoaffinity assays. There are many well characterized antibody fragments. Thus, for example, pepsin digests the antibody C-terminal of the disulfide bond in the hinge region, producing the Fab dimer F (ab)'₂The Fab is itself linked to V via a disulfide bond_H-C_H1 linked light chain. F (ab)'₂Can be reduced under mild conditions to disrupt the disulfide bond in the hinge region and thus (Fab')₂The dimer is converted to Fab' monomer. The Fab' monomer is essentially a Fab with a portion of the hinge region (see, e.g., Fundamental Immunology, Paul, ed., Raven Press, n.y. (1993) for a more detailed description of other antibody fragments). Although different antibodies are defined based on the digestion of intact antibodiesBody fragments, it will be understood by those skilled in the art that fragments may be synthesized chemically, de novo or by using recombinant DNA techniques. Thus, the term antibody also includes antibody fragments produced by modification of whole antibodies or antibody fragments synthesized using recombinant DNA techniques.

The phrase "specifically binds," when used in the context of describing the binding relationship of a particular molecule to a protein or peptide, refers to a binding reaction that is capable of determining the presence of a protein in a heterogeneous population of proteins and other biological agents. Thus, under the specified binding assay conditions, a specified binding agent (e.g., an antibody) binds to a particular protein at least twice background and does not substantially bind in significant amounts to other proteins present in the sample. Under such conditions, specific binding of an antibody may require selection of an antibody specific for a particular protein, or specific for a protein but not specific for a similar "sister" protein of that protein. A variety of immunoassay formats can be used to select antibodies specifically immunoreactive with a particular protein or in a particular format. For example, solid phase ELISA immunoassays are commonly used to select Antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual (1988) for a description of the immunization patterns and conditions that can be used to determine specific immunoreactivity). Typically, the specific or selective binding reaction is at least twice background signal or noise, more typically 10 to 100 times more background. On the other hand, the term "specifically binds" when it relates to the formation of a double-stranded complex of one polynucleotide sequence with another polynucleotide sequence describes "polynucleotide hybridization" based on Watson-Crick base pairing, as described in the definition of the term "polynucleotide hybridization method".

As used herein, "increase" or "decrease" refers to a detectable positive or negative change in amount as compared to a comparative control, e.g., an established standard control (such as an average expression level of DACT1mRNA or protein in non-cancerous stomach tissue). The increase is a positive change, typically at least 10% or at least 20% or 50% or 100% of the control value, and may be up to at least 2-fold, at least 5-fold or even 10-fold of the control value. Likewise, a decrease is a negative change of typically at least 10% or at least 20%, 30% or 50% or even up to at least 80% or 90% of the control value. Other terms indicating a change or difference in amount from a comparative reference are used in the same manner as described above in this application, such as "more", "less", "higher" and "lower". In contrast, the term "substantially the same" or "substantially no change" indicates little or no change in amount from the standard control value, typically within ± 10%, or ± 5%, or ± 2% of the standard control, or even a smaller change from the standard control.

As used herein, "polynucleotide hybridization method" refers to a method for detecting the presence and/or amount of a predetermined polynucleotide sequence based on its ability to form Watson-Crick base pairs with polynucleotide probes of known sequence under suitable hybridization conditions. Examples of such hybridization methods include Southern blotting, Northern blotting and in situ blotting.

As used herein, "primer" refers to an oligonucleotide that can be used in an amplification method, such as Polymerase Chain Reaction (PCR), to amplify a nucleotide sequence based on a polynucleotide sequence corresponding to a gene of interest (e.g., the cDNA or genomic sequence of human DACT1, or a portion thereof). Typically, at least one PCR primer used to amplify a polynucleotide sequence is sequence specific for the polynucleotide sequence. The exact length of the primer depends on a variety of factors, including temperature, source of primer, and method used. For example, for diagnostic and prognostic applications, oligonucleotide primers typically contain at least 10, or 15, or 20, or 25 or more nucleotides, but they may contain fewer nucleotides or more, depending on the complexity of the target sequence. Factors involved in determining the appropriate length of a primer are well known to those skilled in the art. Specific embodiments of the primers used are shown in Table 1 herein, wherein their specific applications are indicated. In this context, the term "primer pair" denotes a primer pair which hybridizes to the opposite strand of a target DNA molecule or to a region of the target DNA flanking the nucleotide sequence to be amplified. As used herein, the term "primer site" refers to a region of a target DNA or other nucleic acid to which a primer hybridizes.

A "label", "detectable label" or "detectable moiety" is a moiety that can be detected spectrophotometrically, photochemically, biochemically, immunochemically, chemically, or by other physical means. For example, useful markers include³²P, fluorescent dyes, electron density reagents, enzymes (e.g., enzymes commonly used in ELISA), biotin, digoxigenin, or haptens and proteins that can be made detectable (e.g., by incorporating a radioactive component into the peptide) or used to detect antibodies specifically reacting with the peptide. Typically, a detectable label is attached to a probe or molecule having defined binding characteristics (e.g., a polypeptide or polynucleotide having a known binding specificity) to allow for easy detection of the presence of the probe (and its bound target).

As used herein, a "standard control" refers to a predetermined amount or concentration of a polynucleotide sequence or polypeptide (e.g., DACT1mRNA or protein) present in an established normal disease-free tissue sample (e.g., a normal gastric epithelial tissue sample). Standard control values are suitable for use in the methods of the invention as a basis for comparing the amount of DACT1mRNA or protein present in a test sample. The established samples used as standard controls provide the average amount of DACT1mRNA or protein in general in gastric epithelial tissue samples (e.g., gastric mucosa) for normal healthy persons who do not have any gastric disorders as defined conventionally, particularly gastric cancer. The standard control value may vary depending on the nature of the sample and other factors such as the sex, age, race of the individual on which the control value is established.

When describing a healthy person who does not suffer from any gastric disorder (particularly gastric cancer) as conventionally defined, the term "average" refers to the amount of certain properties, particularly human DACT1mRNA or DACT1 protein, present in the stomach tissue (e.g., epithelial tissue or gastric mucosa) of a randomly selected human of healthy persons who do not suffer from any gastric disorder (particularly gastric cancer). The selected population should include a sufficient number of persons such that the average amount of DACT1mRNA or protein in the gastric mucosa of these individuals reflects with reasonable accuracy the corresponding amount of DACT1 mRNA/protein in the healthy population. Furthermore, the selected population is typically of similar age to the individual whose stomach tissue sample was used to test for indications of gastric cancer. Also, other factors such as gender, race, medical history should be considered, and it is preferred that the characteristics between the test individual and the selected group of individuals establish an "average" value are closely matched.

The term "amount" as used herein refers to the amount of a polynucleotide or polypeptide of interest (e.g., human DACT1mRNA or protein) present in a sample. The amount may be expressed in absolute terms, i.e. the total amount of polynucleotide or polypeptide in the sample, or in relative terms, i.e. the concentration of polynucleotide or polypeptide in the sample.

The term "treating" or "treatment" as used herein describes an act that results in the elimination, alleviation, reversal or prevention or delay of the onset or recurrence of any symptoms of the associated disorder. In other words, "treating" a disorder includes both therapeutic and prophylactic intervention in the disorder.

The term "effective amount" as used herein refers to an amount of a given substance that is sufficient in amount to produce a desired effect. For example, an effective amount of a polynucleotide encoding DACT1mRNA is the amount of the polynucleotide that achieves an increase in the expression level or biological activity of DACT1 protein such that gastric cancer symptoms are reduced, reversed, eliminated, prevented, or delayed in onset in a patient to whom the polynucleotide is administered for therapeutic purposes. An amount sufficient to achieve this is defined as a "therapeutically effective dose". The dosage range will vary with the nature of the therapeutic agent being administered and other factors such as the route of administration and the severity of the patient's condition.

The term "subject" or "subject in need of treatment" as used herein includes subjects seeking medical assistance due to having a risk of, or actually having, gastric cancer. Individuals also include those currently undergoing treatment for whom improvements in treatment regimens are sought. Individuals in need of treatment include individuals exhibiting symptoms of gastric cancer or at risk of having gastric cancer or symptoms thereof. For example, individuals in need of treatment include individuals with a genetic predisposition or family history of gastric cancer, individuals who have previously suffered from the associated symptoms, individuals who have been exposed to a triggering substance or event, and individuals who suffer from chronic or acute symptoms of the disorder. The "individual in need of treatment" may be at any age of life.

"inhibitors," "activators," and "modulators" of DACT1 protein refer to inhibitory, activating, or regulatory molecules, such as ligands, agonists, antagonists, and homologs and mimetics thereof, identified using in vitro and in vivo assays of DACT1 protein binding or signal transduction, respectively. The term "modulator" includes inhibitors and activators. For example, an inhibitor is an agent that partially or completely blocks carbohydrate binding, reduces activity of the DACT1 protein, prevents activity of the DACT1 protein, delays activation of the DACT1 protein, inactivates the DACT1 protein, desensitizes activity of the DACT1 protein, or down-regulates activity of the DACT1 protein. In some cases, the inhibitor binds, directly or indirectly, to the DACT1 protein, e.g., a neutralizing antibody. Inhibitors as used herein are synonymous with inactivators and antagonists. For example, an activator is an agent that stimulates the activity of the DACT1 protein, increases the activity of the DACT1 protein, promotes the activity of the DACT1 protein, enhances the activation of the DACT1 protein, sensitizes the activity of the DACT1 protein, or up-regulates the activity of the DACT1 protein. Modulators include DACT1 protein ligands or binding partners, including modified naturally occurring ligands and synthetically designed ligands, antibodies and antibody fragments, antagonists, agonists, small molecules including carbohydrate-containing molecules, sirnas, RNA aptamers, and the like.

Detailed Description

I. Introduction to the design reside in

Gastric cancer patients often face a severe prognosis due to the lack of well-defined symptomatic properties in their early stages of progression. Thus, early detection of gastric cancer is critical to improving patient survival.

The inventor firstly finds that the expression of DACT1 protein in gastric cancer cells is inhibited. This suppressed expression of DACT1 protein was due to increased methylation within the DACT1 genomic sequence, particularly the promoter region of the gene, resulting in decreased transcription of DACT1 mRNA. This discovery provides an important means for detecting, monitoring and treating gastric cancer.

General procedure

The practice of the present invention utilizes conventional techniques in the field of molecular biology. Basic textbooks disclosing the general methods used in the present invention include Sambrook and Russell, molecular cloning, Laboratory Manual (3 rd edition.2001); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al, eds., 1994)).

For nucleic acids, the size is given in kilobases (kb) or in basepairs (bp). Nucleic acid size is an estimate obtained from agarose or acrylamide gel electrophoresis, from sequenced nucleic acids, or from published DNA sequences. For proteins, the size is given in kilodaltons (kDa) or number of amino acid residues. Protein size is estimated from gel electrophoresis, from sequenced proteins, from deduced amino acid sequences, or from published protein sequences.

For example, commercially unavailable oligonucleotides can be synthesized chemically using an automated synthesizer according to the solid phase phosphoramidite triester method first described by Beaucage and Caruthers, tetrahedron Lett.22:1859-1862(1981), as described by Van Devanter et al, Nucleic Acids Res.12:6159-6168 (1984). Purification of the oligonucleotides is carried out using any art-recognized strategy, such as native acrylamide gel electrophoresis or anion exchange High Performance Liquid Chromatography (HPLC) as described by Pearson and Reanier, J.Chrom.255:137-149 (1983).

Sequences of interest used in the present invention, e.g., the polynucleotide sequence of the human DACT1 Gene and synthetic oligonucleotides (e.g., primers), can be verified using a chain termination method for sequencing double-stranded templates, e.g., as described in Wallace et al, Gene16:21-26 (1981).

Obtaining of tissue samples and analysis of DACT1mRNA or DNA

The present invention relates to measuring the amount of DACT1mRNA or analyzing the methylation pattern of DACT1 genomic DNA in human gastric tissue (especially gastric epithelial samples) as a means to detect the presence of gastric cancer, assess the risk of developing gastric cancer, and/or monitor the progression or treatment efficacy of gastric cancer. Thus, the first step in practicing the present invention is to obtain a gastric epithelial tissue sample from a test individual and extract mRNA or DNA from the sample.

A.Acquisition and preparation of gastric tissue samples

Gastric tissue samples are obtained from a person to be detected or monitored for gastric cancer using the methods of the invention. Collection of a gastric epithelial tissue sample from an individual is performed following standard procedures typically followed by a hospital or clinic, such as during an endoscopic examination. Appropriate amounts of gastric epithelium are collected and may be stored according to standard procedures prior to further preparation.

Analysis of DACT1mRNA or DNA present in a patient's gastric epithelial sample of the invention may be performed using, for example, the gastric mucosa. Methods for preparing tissue samples for nucleic acid extraction are well known to those skilled in the art. For example, a sample of the stomach mucosa of an individual should first be treated to disrupt the cell membrane in order to release the nucleic acids contained within the cell.

B.Extraction and quantification of RNA

There are many methods for extracting mRNA from biological samples. The general method of mRNA preparation can be followed (e.g., as described in Sambrook and Russell, Molecular Cloning: A laboratory Manual 3d ed., 2001). mRNA can also be obtained from biological samples of test individuals using various commercially available reagents or kits, such as Trizol reagent (Invitrogen, Carlsbad, Calif.), Oligotex Direct mRNA kit (Qiagen, Valencia, Calif.), RNeasy Mini kit (Qiagen, Hilden, Germany), andseries 9600 (Promega, Madison, Wis.). Combinations of multiple of these approaches may also be used.

It is necessary to eliminate all DNA contamination from RNA preparations. Therefore, the sample should be handled carefully, treated thoroughly with DNase, and appropriate negative controls used in the amplification and quantification steps.

1. PCR-based quantitative determination of mRNA levels

After extraction of mRNA from the sample, the amount of human DACT1mRNA can be quantified. Preferred methods for determining mRNA levels are amplification-based methods, e.g. by Polymerase Chain Reaction (PCR), especially reverse transcription-polymerase chain reaction (RT-PCR).

Prior to the amplification step, a DNA copy (cDNA) of human DACT1mRNA must be synthesized. This is achieved by reverse transcription, which can be performed as a separate step or in a homogeneous (heterologous) reverse transcription-polymerase chain reaction (RT-PCR), which is a modified polymerase chain reaction for amplification of RNA. Suitable methods for ribonucleic acid PCR amplification are described below: romero and Rotbart, Diagnostic molecular biology: Principles and Applications pp.401-406; Perssing et al, eds., MayoFoundation, Rochester, MN,1993; Egger et al, J.Clin.Microbiol.33: 1442-; and U.S. patent No. 5,075,212.

General methods of PCR are well known in the art and are not described in detail herein. For a review of the principles of PCR Methods, protocols and design of primers, see, e.g., Innis, et al, PCRProtocols: A Guide to Methods and Applications, Academic Press, Inc.N.Y., 1990. PCR reagents and protocols are also available from commercial suppliers, such as Roche molecular systems.

Most commonly, PCR is performed in an automated process using thermostable enzymes. In this process, the temperature of the reaction mixture is automatically cycled through the denaturation zone, the primer annealing zone, and the extension reaction zone. Instruments particularly suitable for this purpose are commercially available.

Although PCR amplification of target mRNA species is commonly used in the practice of the present invention, it will be appreciated by those skilled in the art that amplification of these mRNA species in maternal blood samples can be achieved by any known method, such as Ligase Chain Reaction (LCR), transcription mediated amplification and semi-conserved sequence replication or Nucleic Acid Sequence Based Amplification (NASBA), each of which provides sufficient amplification. Recently developed branched-chain DNA techniques can also be used to quantitatively determine the amount of mRNA markers in maternal blood. For a review of branched-chain DNA signal amplification for direct quantification of nucleic acid sequences in clinical samples, see Nolte, adv.Clin.chem.33:201-235, 1998.

2. Other methods of quantification

DACT1mRNA can also be detected using other standard techniques known to those skilled in the art. Although an amplification step is typically performed prior to the detection step, the method of the invention does not necessarily require amplification. For example, whether or not an amplification step is performed in advance, mRNA can be identified by size fractionation (e.g., gel electrophoresis). After running in agarose or polyacrylamide gels and labeling with ethidium bromide according to well-known techniques (see, e.g., Sambrook and Russell, supra), the presence of a band of the same size as compared to a standard control indicates the presence of the target mRNA, and then the amount of the target mRNA is compared to the control based on the intensity of the band. Alternatively, oligonucleotide probes specific for DACT1mRNA can be used to detect the presence of the mRNA and indicate the amount of mRNA by comparison to a standard control based on the intensity of the signal provided by the probe.

Sequence-specific probe hybridization is a well-known method for detecting specific nucleic acids, including other types of nucleic acids. Under sufficiently stringent hybridization conditions, the probe will hybridize specifically only to substantially complementary sequences. The stringency of the hybridization conditions can be relaxed to allow different amounts of sequence mismatches.

Many hybridization patterns are known in the art, including but not limited to liquid phase, solid phase, or mixed phase hybridization assays. The following documents provide an overview of various hybridization assay formats: singer et al, Biotechniques 4:230,1986, Haase et al, Methods In Virology, pp.189-226,1984, Wilkinson, In situ Hybridization, Wilkinson ed., IRL Press, Oxford university Press, Oxford, and Hames and Higgins eds, Nucleic acid Hybridization, A Practical Approach, IRL Press, 1987.

The hybridization complex is detected according to known techniques. Nucleic acid probes that are capable of specifically hybridizing to a target nucleic acid (i.e., mRNA or amplified DNA) can be labeled by any of several methods commonly used to detect the presence of hybridized nucleic acids. A commonly used detection method is to utilize³H、¹²⁵I、³⁵S、¹⁴C or³²Autoradiography of P-labeled probes. The choice of radioisotope depends on research preferences in view of ease of synthesis, stability and half-life of the selected isotope. Other labels include compounds (e.g., biotin and digoxigenin) that bind to anti-ligands or antibodies labeled with fluorophores, chemiluminescent reagents, and enzymes. Alternatively, the probes may be directly conjugated to labels such as fluorophores, chemiluminescent reagents and enzymes. The choice of label depends on the sensitivity desired, the ease of conjugation to the probe, stability requirements and the instrumentation available.

Probes and primers necessary for the practice of the present invention can be synthesized and labeled using well-known techniques. For example, oligonucleotides useful as probes and primers can be chemically synthesized using an automated synthesizer according to the solid phase phosphoramidite triester method first described by Beaucage and Caruthers, Tetrahedron Lett.22:1859-1862,1981, as described by Van Devanter et al, Nucleic Acids Res.12:6159-6168, 1984. For example, purification of the oligonucleotide can be carried out by native acrylamide gel electrophoresis or anion exchange HPLC as described by Pearson and Reanier, J.Chrom.255:137-149, 1983.

C.Detection of methylation in DACT1 genomic sequences

The methylation status of segments of the DACT1 genomic sequence containing one or more CpG (cytosine-guanine dinucleotide) pairs was investigated to provide an indication as to whether the test individual has gastric cancer, whether the individual is at risk of developing gastric cancer, or whether the gastric cancer in the individual is worsening or improving.

Typically, the segment of DACT1 genomic sequence analyzed for methylation patterns includes a 5' untranslated region (such as a promoter region) and includes one or more CpG nucleotide pairs. For example, SEQ ID NO 1 or 6 or a portion thereof can be used to determine how many CpG pairs are methylated and how many CpG pairs are unmethylated within a sequence. The sequence being analyzed should be long enough to contain at least 1 CpG dinucleotide pair, and detection of methylation at this CpG site is generally sufficient to indicate the presence of gastric cancer cells. The sequence being analyzed is typically at least 15 or 20 contiguous nucleotides in length, and may be longer, having at least 25, 30, 50, 100, 200, 300, 400 or more contiguous nucleotides. There are at least 1, usually 2 or more, and often 3, 4,5, 6, 7, 8, 9 or more CpG nucleotide pairs within a sequence. In the case of analyzing the methylation status of a plurality (2 or more) of CpG sites, the tested individual is considered to have gastric cancer or an increased risk of developing gastric cancer when at least 50% of CpG pairs within the analyzed genomic sequence are confirmed to be methylated. For example, SEQ ID NO 1 (i.e., DACT1 genomic sequence segment (-556 to-383 relative to the transcription start site)) and SEQ ID NO 6 (i.e., DACT1 genomic sequence segment (-18 to +102 relative to the transcription start site)) contain several CpG pairs. In established gastric cancer cell lines and samples obtained from gastric cancer, some or most of the CpG pairs within this region are found to be methylated, while non-cancerous gastric epithelial cells show very few, if any, methylated CpG sites. To determine the methylation pattern of DACT1 genomic sequences, it is very useful to perform bisulfite treatment followed by DNA sequencing, since bisulfite converts unmethylated cytosines (C) to uracils (U) while leaving methylated cytosines unchanged, allowing direct identification by the DNA sequencing process. Optionally, an amplification process such as PCR is included after bisulfite conversion and prior to DNA sequencing.

DNA extraction and treatment

Methods for extracting DNA from biological samples are well known and routinely practiced in the field of molecular biology, see, e.g., Sambrook and Russell, supra. RNA contamination should be eliminated to avoid interference with DNA analysis. The DNA is then treated with an agent that modifies the DNA in a manner that is differentially methylated, i.e., that produces a distinct and distinguishable chemical structure from methylated cytosine (C) residues and unmethylated C residues after treatment. Typically, such agents are capable of reacting with unmethylated C residues within a DNA molecule to convert each unmethylated C residue to a uracil (U) residue, while the methylated C residues remain unchanged. This conversion of unmethylated C → U allows detection and comparison of methylation status based on changes in the primary sequence of the nucleic acid. An exemplary reagent suitable for this purpose is a bisulfite salt, such as sodium bisulfite. Methods for chemical modification of DNA using bisulfite are well known in the art (see, e.g., Herman et al, Proc. Natl. Acad. Sci. USA93: 9821-.

As will be appreciated by those skilled in the art, any other agent not mentioned herein but having the same property of differentially modifying methylated and unmethylated DNA chemically (or by any other mechanism) may be used in the practice of the present invention. Methylation-specific modification of DNA can also be achieved, for example, by methylation-sensitive restriction enzymes, some of which typically cleave unmethylated DNA fragments but not methylated DNA fragments, while others (e.g., methylation-dependent endonuclease McrBC) cleave methylated cytosine-containing DNA but not unmethylated DNA. Furthermore, a combination of chemical modification and restriction enzyme treatment may be used to carry out the invention, for example, in conjunction with bisulfite restriction enzyme analysis (COBRA) (Xiong et al 1997 Nucleic acids sRs.25 (12): 2532-2534). Other available methods for detecting DNA methylation include, for example, analysis of Methylation Sensitive Restriction Endonucleases (MSRE) by Southern blot or PCR analysis, methylation specific or methylation sensitive PCR (MS-PCR), methylation sensitive single nucleotide primer extension (Ms-SnuPE), High Resolution Melting (HRM) analysis, bisulfite sequencing, pyrophosphate sequencing, methylation specific single strand conformation analysis (MS-SSCA), methylation specific denaturing gradient gel electrophoresis (MS-DGGE), methylation specific melting curve analysis (MS-MCA), methylation specific denaturing high performance liquid chromatography (MS-DHPLC), methylation specific Microarrays (MSO). These assays may be PCR analysis, quantitative analysis using fluorescent labels, or Southern blot analysis. Exemplary methylation-sensitive DNA cleaving agents include, for example, restriction enzymes, AatII, AciI, AclI, AgeI, AscI, Asp718, AvaI, BbrP1, BceAI, BmgBI, BsaAI, BsaHI, BseEI, BsiWI, BsmBI, BspDI, BsrFI, BssHII, BstBI, BstUI, ClaI, EagI-HFTM, FauI, FseI, FspI, HaeII, HgaI, HhaI, HinP1I, HpaII, Hpy99I, HpyCH4IV, KasI, MluI, NarI, NgoMIV, NotI-HFTM, NruI, Nbt.BsmAI, PaeR7I, PspXI, PvuI, RsII, SacgII, SagIII, SalaI, SfoBI, or TspI.

2. Optional amplification and sequence analysis

After modifying the DNA in a methylation-differential manner, sequence-based analysis was performed on the treated DNA so that the methylation state of the DACT1 genomic sequence could be determined. Optionally, an amplification reaction is performed after the methylation specific modification prior to sequence analysis. Various polynucleotide amplification methods are well established and are frequently used in research. For example, general methods of Polymerase Chain Reaction (PCR) for amplification of polynucleotide sequences are known in the art and are not described in detail herein. For a review of the principles of PCR Methods, Protocols and design of primers, see, e.g., Innis, et al, PCR Protocols: A guides to Methods and Applications, Academic Press, Inc.N.Y., 1990. PCR reagents and protocols are also available from commercial suppliers, such as Roche molecular Systems.

Although PCR amplification is commonly used in the practice of the present invention, one skilled in the art will recognize that amplification of the relevant genomic sequences can be achieved by any known method, such as Ligase Chain Reaction (LCR), transcription-mediated amplification and semi-conserved sequence replication or nucleic acid sequence-based amplification (NASBA), each of which can provide sufficient amplification.

Techniques for determining polynucleotide sequences are also well established and widely practiced in related research fields. For example, the rationale and general techniques for polynucleotide sequencing are described in various research reports and papers on molecular biology and recombinant genetics, e.g., Wallace et al, supra, Sambrook and Russell, supra, and Ausubel et al, supra. Either manual or automated DNA sequencing methods routinely practiced in research laboratories may be used in the practice of the present invention. Other means for detecting changes in polynucleotide sequence (e.g., C → U) suitable for practicing the methods of the invention include, but are not limited to, mass spectrometry, primer extension, polynucleotide hybridization, real-time PCR, melting curve analysis, high resolution melting analysis, heteroduplex analysis, pyrosequencing, and electrophoresis.

Quantification of the polypeptide

A.Obtaining a sample

The first step in practicing the present invention is to obtain a sample of gastric epithelium from an individual to be tested, assessed or monitored for gastric cancer, risk of developing gastric cancer or severity/progression of the condition. The same type of sample should be taken from the control group (normal individuals who do not suffer from any gastric diseases, especially tumors) and the test group (for example, individuals to be tested for the possible presence of gastric cancer). As stated in the above section, standard procedures routinely used in hospitals and clinics are generally followed for this purpose.

To detect the presence of gastric cancer in a test individual or to assess the risk of developing gastric cancer, a gastric mucosal sample of the individual patient may be taken and the level of human DACT1 protein measured and then compared to a standard control. A test subject is considered to have, or be at increased risk of developing, gastric cancer if a decrease in human DACT1 protein levels is observed when compared to control levels. To monitor disease progression or assess efficacy in gastric cancer patients, samples of the gastric epithelium of individual patients may be taken at different time points, so that the levels of human DACT1 protein may be measured to provide information indicative of the disease state. For example, a patient is considered to have an improved severity of gastric cancer or is considered to be effective in receiving treatment when the patient's DACT1 protein level shows a trend of generally increasing over time. The tendency of the patient to have unchanged or even continuously reduced levels of DACT1 protein indicates worsening of the disease and ineffectiveness of the treatment given to the patient. Generally, lower levels of DACT1 protein observed in a patient are indicative of a more severe form of gastric cancer and a poorer prognosis of the disease, e.g. manifested by a shorter life expectancy, a higher metastasis rate, resistance to treatment, etc.

B.Preparation of samples for DACT1 protein detection

Stomach tissue samples from individuals are suitable for use in the present invention and may be obtained by well known methods and methods set forth in the preceding section. In certain applications of the present invention, gastric mucosa may be the preferred sample type.

C.Determination of the level of human DACT1 protein

Any particular class of protein, such as the DACT1 protein, can be detected using a variety of immunological assays. In some embodiments, a sandwich assay may be performed by capturing a polypeptide from a test sample with an antibody having specific binding affinity for the polypeptide. It can then be detected with a labeled antibody having specific binding affinity for the polypeptide. The immunological assay may be carried out using a microfluidic device, such as a microarray protein chip. Proteins of interest (e.g., human DACT1 protein) can also be detected by gel electrophoresis (such as two-dimensional gel electrophoresis) and Western blot analysis using specific antibodies. Alternatively, standard immunohistological techniques using appropriate antibodies can be used to detect a given protein (e.g., human DACT1 protein). Monoclonal and polyclonal antibodies (including antibody fragments having the desired binding specificity) can be used for specific detection of polypeptides. Such antibodies and their binding fragments with specific binding affinity for a particular protein (e.g., human DACT1 protein) can be generated by known techniques.

Other methods may also be used to perform the determination of DACT1 protein levels in the present invention. For example, a variety of techniques have been developed to rapidly and accurately quantify target proteins (even in large numbers of samples) based on mass spectrometry techniques. These methods involve highly accurate instruments such as triple quadrupole (triple Q) instruments using Multiple Reaction Monitoring (MRM) techniques, matrix assisted laser desorption ionization/ionization time of flight tandem mass spectrometers (MALDI TOF/TOF), ion trap instruments using selective ion detection (SIM) mode, and electrospray ionization (ESI) based QTOP mass spectrometers. See, e.g., Pan et al, JProteome Res.2009 February;8(2): 787-.

V. establishing a Standard control

To establish a standard control for carrying out the method of the invention, a group of healthy persons is first selected that do not suffer from any gastric disorder as defined conventionally, in particular any form of tumor, such as gastric cancer. In order to screen and/or monitor gastric cancer using the methods of the present invention, these individuals are eligible for appropriate parameters, if available. Optionally, the individuals have the same gender, similar age, or similar ethnic background.

The health status of selected individuals is confirmed by well-established, routinely used methods, including but not limited to routine physical examination of individuals and a general review of their medical history.

Furthermore, the selected group of healthy individuals must have a reasonable sample size, so that it can be reasonably assumed that the average amount/concentration of human DACT1mRNA or DACT1 protein of the gastric tissue samples obtained from the group represents the normal or average level of the total healthy population. Preferably, the selected group comprises at least 10 persons.

Once the mean value of DACT1mRNA or DACT1 protein was established based on the individual values in each individual of the selected healthy control group, the mean or median or representative value or characteristic was considered as a standard control. The standard deviation was also determined in the same procedure. In some cases, separate standard controls may be established for separately defined groups having different characteristics (such as age, gender, or ethnic background).

Treatment of gastric cancer

By proving the correlation between the inhibition expression of the DACT1 protein and gastric cancer, the invention also provides a means for treating gastric cancer patients: by increasing the expression or biological activity of DACT1 protein. Gastric cancer treatment, as used herein, includes reducing, reversing, alleviating, or eliminating one or more symptoms of gastric cancer, and preventing or delaying the onset of one or more associated symptoms.

A.Increasing DACT1 expression or activity

1. Nucleic acid encoding DACT1 protein

Enhancement of the expression of the DACT1 gene can be achieved by using a nucleic acid encoding a functional DACT1 protein. The nucleic acid may be a single-stranded nucleic acid (such as mRNA) or a double-stranded nucleic acid (such as DNA) that can be translated into an active form of DACT1 protein under favorable conditions.

In one embodiment, the DACT1 encoding nucleic acid is provided in the form of an expression cassette, typically produced recombinantly, having a promoter operably linked to a polynucleotide sequence encoding a DACT1 protein. In some cases, the promoter is a universal promoter that directs gene expression in all or most tissue types. In other cases, the promoter is one that directs gene expression specifically in epithelial cells (particularly gastric epithelial cells). Administration of the nucleic acid increases DACT1 protein expression in a target tissue (e.g., gastric epithelium). Since the GenBank accession NM-016651 of the cDNA sequence of the human DACT1 gene is known and is provided herein as SEQ ID NO. 3, suitable nucleic acids encoding DACT1 can be obtained from such sequences, species homologs, and variants of these sequences.

DACT1 protein

The disease can also be effectively treated by administering an effective amount of active DACT1 protein directly to a patient suffering from gastric cancer and exhibiting inhibited expression or activity of DACT1 protein. This can be achieved, for example, by administering a recombinantly produced biologically active DACT1 protein to a gastric cancer patient. Formulations and methods for delivering protein or polypeptide based therapeutics are well known in the art.

Activators of DACT1 protein

An increase in the activity of the DACT1 protein may be achieved with an agent that activates the expression of the DACT1 protein or enhances the activity of the DACT1 protein. For example, a demethylating agent (e.g., 5-Aza) may be able to activate DACT1 gene expression by removing the inhibition of DACT1 gene expression caused by methylation of the promoter region of the DACT1 gene. Other activators may include transcriptional activators specific for the DACT1 promoter and/or enhancer. Such activators may be screened and identified using the DACT1 expression assay described in the examples herein.

An agonist of DACT1 protein (such as an activating antibody) is another activator of DACT1 protein. Such activators act by enhancing the biological activity of the DACT1 protein, typically (but not necessarily) by direct binding to the DACT1 protein and/or its interacting proteins. Initial screening of such agonists may begin with a binding assay that identifies molecules that physically interact with the DACT1 protein.

B.Pharmaceutical composition

1. Preparation

The compounds of the invention are useful in the preparation of pharmaceutical compositions or medicaments. The pharmaceutical composition or medicament may be administered to an individual for the treatment of gastric cancer.

The compounds used in the present invention (e.g., DACT1 protein, nucleic acid encoding DACT1 protein, activator of DACT1 gene expression) can be used for the preparation of pharmaceutical compositions or medicaments comprising an effective amount of the compound in combination or admixture with excipients or carriers suitable for the application.

An exemplary pharmaceutical composition for enhancing expression of DACT1 comprises (i) an expression cassette comprising a polynucleotide sequence encoding a human DACT1 protein as described herein, and (ii) a pharmaceutically acceptable excipient or carrier. The terms "pharmaceutically acceptable" and "physiologically acceptable" are used synonymously herein. For use in the therapeutic methods described herein, a therapeutically effective dose of the expression cassette can be provided.

The DACT1 protein or nucleic acid encoding the DACT1 protein can be administered via liposomes, which function to target the conjugate to a particular tissue and increase the half-life of the composition. Liposomes include emulsions, foaming agents, micelles, insoluble monolayers, liquid crystals, phospholipid dispersants, lamellar layers, and the like. In these formulations, the inhibitor to be delivered, alone or in combination with molecules or other therapeutic or immunogenic compositions that are capable of binding to receptors widely present in, for example, the targeted cells (e.g., skin cells), is incorporated as part of the liposome. Thus, liposomes filled with a protein or nucleic acid of the invention can be directed to a treatment site where the liposomes then deliver a selected protein or nucleic acid composition. Liposomes for use in the present invention are formed from standard vesicle-forming lipids, which typically include neutral and negatively charged phospholipids and sterols (e.g., cholesterol). The choice of lipid is generally guided by considerations such as liposome size, acid instability and stability of the liposomes in the blood stream. Various methods are available for preparing liposomes, as described, for example, in Szoka et al (1980) ann.rev.biophyls.bioeng.9: 467, U.S. Pat. nos. 4,235,871, 4,501,728, and 4,837,028.

The pharmaceutical compositions or medicaments for use in the present invention may be prepared by standard techniques using one or more physiologically acceptable carriers or excipients. Suitable Pharmaceutical carriers are described herein and in "Remington's Pharmaceutical Sciences" of e.w. martin. The compounds and agents of the present invention and their physiologically acceptable salts and solvates may be formulated for administration by any suitable route, including inhalation, topical, nasal, oral, parenteral or rectal administration.

Typical dosage forms for topical administration include creams, ointments, sprays, lotions and plasters. However, the pharmaceutical composition may be formulated for any type of administration, for example, intradermal injection, subcutaneous injection, intravenous injection, intramuscular injection, intranasal injection, intracranial injection, intratracheal injection, intraarterial injection, intraperitoneal injection, intravesical injection, intrapleural injection, intracoronary injection, or intratumoral injection using a syringe or other device. Dosage forms for administration by inhalation (e.g., aerosol) or oral, rectal or vaginal administration are also contemplated.

2. Route of administration

Suitable dosage forms for topical application to, for example, the skin and eyes are preferably aqueous solutions, ointments, creams or gels well known in the art. Such dosage forms may contain solubilizers, stabilizers, tonicity enhancing agents, buffers and preservatives.

Suitable dosage forms for transdermal administration include an effective amount of a compound or agent of the invention and a carrier. Preferred carriers include absorbable pharmaceutically acceptable solvents for aiding passage through the skin of the host. For example, a transdermal device takes the form of a bandage comprising a support member, a reservoir containing the compound and optionally a carrier, an optional rate controlling barrier that delivers the compound to the skin of the host at a controlled and predetermined rate over an extended period of time, and means to secure the device to the skin. Matrix transdermal dosage forms may also be used.

For oral administration, the pharmaceutical composition or medicament may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients. Preferred are tablets and gelatin capsules comprising: active ingredient (i.e. DACT1 protein or nucleic acid encoding DACT1 protein), and (a) diluents or fillers such as lactose, glucose, sucrose, mannitol, sorbitol, cellulose (e.g. ethylcellulose, microcrystalline cellulose), sugar gums, pectin, polyacrylates and/or dibasic calcium phosphate, calcium sulfate, (b) lubricants such as silica, talc, stearic acid, magnesium or calcium stearate, metal stearates, colloidal silicon dioxide, hydrogenated vegetable oils, corn starch, sodium benzoate, sodium acetate and/or polyethylene glycol; for tablets, it may also contain (c) binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone and/or hydroxypropylcellulose; if desired, (d) a disintegrating agent such as starch (e.g., potato starch or sodium starch), glycolate, agar, alginic acid or a sodium salt thereof, or a foaming mixture; (e) wetting agents, such as sodium lauryl sulfate and/or (f) absorbents, colorants, fragrances and sweeteners.

The tablets may also be coated with a film or enteric coated according to methods known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may be prepared in a conventional manner with pharmaceutically acceptable additives such as suspending agents, for example, sorbitol syrup, cellulose derivatives or hydrogenated edible fats; emulsifiers, such as lecithin or gum arabic; non-aqueous media such as almond oil, oily esters, ethyl alcohol or fractionated vegetable oils; and preservatives, such as methyl or propyl p-hydroxybenzoate or sorbic acid. Optionally, the formulation may also contain buffer salts, flavoring agents, coloring agents and/or sweetening agents. Formulations for oral administration may be suitably formulated to achieve controlled release of the active compound, if desired.

The compounds and agents of the invention may be formulated for parenteral administration by injection, for example by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. Injectable compositions are preferably aqueous isotonic solutions or suspensions, and suppositories are preferably prepared from fatty emulsions or suspensions. The compositions may be sterile and/or contain adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, dissolution promoters, salts for regulating the osmotic pressure and/or buffers. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle (e.g., sterile, pyrogen-free water) before use. In addition, they may contain other therapeutically valuable substances. The compositions are prepared according to conventional mixing, granulating or coating methods, respectively, and contain about 0.1 to 75%, preferably about 1 to 50%, of the active ingredient.

For administration by inhalation, the active ingredient (e.g., DACT1 protein or a nucleic acid encoding DACT1 protein) may be conveniently delivered as an aerosol spray from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a given amount. Capsules or cartridges of, for example, gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

The compounds and agents of the present invention may also be formulated in rectal compositions, for example, suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.

In addition, the active ingredient may be formulated as a depot preparation. Such long acting dosage forms may be administered by implantation (e.g., subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the active ingredient may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or may be formulated as sparingly soluble derivatives, for example, as a sparingly soluble salt.

The pharmaceutical composition or medicament of the invention comprises (i) an effective amount of a compound described herein that increases the level or activity of DACT1 protein, and (ii) another therapeutic agent. When used with a compound of the invention, the therapeutic agents can be used alone, sequentially or in combination with one or more other such therapeutic agents (e.g., a first therapeutic agent, a second therapeutic agent, and a compound of the invention). Administration may be by the same or different routes of administration or may be carried out together in the same pharmaceutical formulation.

3. Dosage form

The pharmaceutical composition or medicament may be administered to an individual in a therapeutically effective dose to prevent, treat or manage gastric cancer as described herein. The pharmaceutical composition or medicament is administered in an amount sufficient to elicit an effective therapeutic response in the subject.

The dose of active agent administered depends on the individual's weight, age, individual condition, surface area or volume of the area to be treated, and the form of administration. The size of the dose is also determined by the presence, nature and extent of any adverse reactions that accompany the administration of a particular compound in a particular individual. For example, each DACT1 protein or nucleic acid encoding a DACT1 protein is likely to have a unique dose. A unit dose for oral administration to a mammal of about 50 to 70kg may contain about 5 to 500mg of the active ingredient. In general, the dosage of the active compounds of the present invention is that which is sufficient to achieve the desired effect. The optimal dosage regimen may be calculated from measurements of the accumulation of the agent in the individual. Typically, the dose may be administered once or more times daily, weekly, or monthly. The optimal dosage, dosing method and repetition rate can be readily determined by one skilled in the art.

To achieve the desired therapeutic effect, the compound or agent may be administered in a therapeutically effective daily dose divided into multiple days. Thus, therapeutically effective administration of a compound for treating a related condition or disease described herein in a subject requires periodic (e.g., daily) administration for a period of 3 days to two weeks or more. Typically, the agent is administered for at least 3 consecutive days, typically at least 5 consecutive days, more typically at least 10 consecutive days, and sometimes 20, 30, 40 or more consecutive days. Although continuous daily administration is the preferred route to achieve a therapeutically effective dose, a therapeutically beneficial effect can be achieved even without daily administration of the agent, provided that the administration is repeated frequently enough to maintain a therapeutically effective concentration of the agent in the individual. For example, the agent may be administered every other day, every third day, or once a week if a higher dosage range is used and tolerated by the individual.

The optimal dose, toxicity or therapeutic efficacy of a compound or agent can vary with the relative potency of the individual compound or agent, and can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the LD₅₀(dose leading to 50% of population deaths) and ED₅₀(a therapeutically effective dose in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD₅₀/ED₅₀. Agents that exhibit large therapeutic indices are preferred. Although agents exhibiting toxic side effects may be used, consideration should be given to designing a delivery system that targets such agents to the site of the affected tissue, thereby minimizing potential damage to normal cells and reducing side effects.

For example, data obtained from cell culture assays and animal studies can be used to formulate a range of dosage for use in humans. The dosage of such compounds is preferably such that ED is included₅₀But within a range of circulating concentrations with little or no toxicity. The dosage may vary within this range depending upon the dosage form and route of administration utilized. For any agent used in the methods of the invention, a therapeutically effective dose can first be estimated from cell culture assays. Can be dosed in animal models to achieve IC including cell culture assays₅₀(concentration of agent that achieves half the maximum inhibition of symptoms). This information can be used to more accurately determine the dose available to humans. For example, levels in plasma can be measured by High Performance Liquid Chromatography (HPLC). Typically, for a typical individual, the dose equivalent of the agent is about 1ng/kg to 100 mg/kg.

Exemplary doses of the DACT1 protein or nucleic acid encoding the DACT1 protein described herein are provided. When administered intravitreally, the dosage of the nucleic acid encoding DACT1 (e.g., expression cassette) can be 0.1-0.5 mg/eye (e.g., 5-30 mg/kg). Small organic compound activators may be administered orally at 5-1000mg, or by intravenous infusion at 10-500 mg/ml. The monoclonal antibody activator may be administered by intravenous injection or infusion in the following amounts: 50-500mg/ml (over a period of 120 minutes); 1-500mg/kg (over a period of 60 minutes); or 1-100mg/kg (bolus) 5 times per week. The DACT1 protein or peptide activator may be administered subcutaneously at 10-500 mg; administered intravenously at 0.1-500mg/kg twice daily or about 50mg once weekly or 25mg twice weekly.

The pharmaceutical compositions of the present invention may be administered alone or in combination with at least one other therapeutic compound. Exemplary advantageous therapeutic compounds include systemic and topical anti-inflammatory agents, analgesic agents, antihistamines, anesthetic compounds, and the like. The other therapeutic compound may be administered simultaneously with the primary active ingredient (e.g., the DACT1 protein or a nucleic acid encoding the protein), even in the same composition. Other therapeutic compounds may also be administered alone in a composition or dosage form different from the main active ingredient. Some doses of the principal component (such as DACT1 protein or a nucleic acid encoding DACT1 protein) may be administered concurrently with other therapeutic compounds, while other doses may be administered separately, depending on the particular condition and characteristics of the individual.

The dosage of the pharmaceutical composition of the present invention can be adjusted during the course of treatment, depending on the severity of the symptoms, the frequency of relapse, and the physiological response to the treatment regimen. The person skilled in the art is usually involved in such an adjustment of the treatment regimen.

Kit and device

The present invention provides compositions and kits for performing the methods described herein to assess levels of DACT1mRNA or DACT1 protein in an individual that can be used for a variety of purposes, such as detecting or diagnosing the presence of gastric cancer, determining the risk of developing gastric cancer, and monitoring the progression of gastric cancer in a patient.

Kits for performing the determination of DACT1mRNA levels typically include at least one oligonucleotide useful for specifically hybridizing to at least a segment of the coding sequence of DACT1 or its complement. Optionally, the oligonucleotide is labeled with a detectable moiety. In some cases, the kit may comprise at least two oligonucleotide primers that can be used in the amplification of at least one segment of DACT1 DNA or mRNA by PCR, in particular by RT-PCR.

Kits for performing the determination of DACT1 protein levels typically include at least one antibody useful for specifically binding to the amino acid sequence of DACT1 protein. Optionally, the antibody is labeled with a detectable moiety. The antibody may be a monoclonal antibody or a polyclonal antibody. In some cases, the kit may include at least two different antibodies, one for specific binding to DACT1 protein (i.e., a primary antibody) and the other for detection of the primary antibody (i.e., a secondary antibody), which is typically linked to a detectable moiety.

Typically, the kit further comprises a suitable standard control. The standard controls indicate the mean value of the DACT1 protein or DACT1mRNA of gastric epithelium in healthy individuals without gastric cancer. In some cases, the standard control may be provided as a set value. In addition, the kit of the present invention may provide an operation manual for instructing the user to analyze the test sample and to evaluate the presence, risk or status of gastric cancer in the test subject.

In another aspect, the invention may also be embodied in an apparatus or system comprising one or more such apparatuses, which apparatus or system is capable of carrying out all or part of the method steps described herein. For example, in some cases, after receiving a gastric tissue sample (e.g., a gastric mucosa sample) taken from a subject for detecting gastric cancer, assessing the risk of developing gastric cancer, or monitoring the progression of a condition, the device or system performs the following steps: (a) determining the amount or concentration of DACT1mRNA or DACT1 protein in the sample; (b) comparing the amount or concentration to a standard control value; and (c) providing a result indicative of whether gastric cancer is present in the individual or whether the individual is at risk of developing gastric cancer, or whether there is a change (i.e., worsening or improvement) in the gastric cancer disease state of the individual. In other cases, the device or system of the present invention performs the tasks of steps (b) and (c) after step (a) is performed and the quantity or concentration from (a) is input to the device. Preferably, the device or system is partially or fully automated.

Examples

The following examples are offered by way of illustration only and not by way of limitation. Those skilled in the art will recognize that various noncritical parameters may be changed or modified to produce substantially the same or similar results.

Materials and methods

Human stomach sample

Tissue sample

Paraffin-embedded tumor tissue samples were obtained from 205 gastric cancer patients diagnosed at the first subsidiary hospital of the university of zhongshan, guangzhou, china, between 1 month 1999 and 12 months 2006. In addition, 20 age-matched upper gastroscopy normal individuals were recruited as controls. The study protocol was approved by the ethical committee for clinical research at the medical college of zhongshan university.

Tumor cell lines

Ten gastric cancer cell lines (AGS, Kato III, MKN28, MKN45, N87, SNU1, SNU16, SNU719, BGC823 and MGC803) and one normal gastric epithelial cell line (GES1) were used in this study. The cell lines were maintained in RPMI-1640 or DMEM medium (Gibco BRL, Rockville, Md.) containing 10% fetal bovine serum.

Analysis of Gene expression

RNA isolation

Total RNA was isolated using Qiazol reagent (Qiagen, Valencia, CA, USA). First, about 5 to 10X 10⁶Cells or 30mg tissue were homogenized in 1mL Qiazol reagent and incubated for 10 minutes at room temperature. To each sample was added 0.2mL of chloroform. The mixture was shaken vigorously for 15 seconds and allowed to stand at room temperature for an additional 3 minutes. The sample was centrifuged at 12,000g for 20 minutes at 4 ℃ and the sample was separated into two layers. Transfer the upper aqueous phase containing RNA to a new tubeMixed with 0.7ml isopropanol, incubated at room temperature for 10 minutes and then centrifuged at 12,000g for 10 minutes at 4 ℃. After discarding the supernatant, washing the RNA precipitate twice with 1mL of 75% ethanol; air-dry for 5 minutes and re-dissolve the RNA with RNase-free water. DNA contamination was removed by digestion with RNase-free DNase I (GE Healthcare, Buckinghamshire, England). The quality and yield of total RNA were determined by measuring the absorbance at 260nm/280nm using a NanoDrop ND-1000(NanoDrop Technologies, Wilmington, DE, USA). Purified RNA was stored at-80 ℃ prior to use.

cDNA Synthesis

cDNA was synthesized using the MultiScriptbe reverse transcriptase kit (Applied Biosystems, Foster City, Calif., USA). The reaction mixture contained 1 Xreverse transcriptase buffer, 1 XdNTP, 1 Xrandom primer (kit provided), 2.5U/. mu.L reverse transcriptase, 1U/. mu.L RNase inhibitor and 2. mu.g total RNA. The mixture was incubated at 25 ℃ for 10 minutes, then 37 ℃ for 120 minutes, then 85 ℃ for 5 minutes to inactivate the enzyme. The cDNA was stored at-80 ℃ prior to other applications.

Semi-quantitative reverse transcription PCR (RT-PCR)

Semi-quantitative RT-PCR was performed in a total volume of 25. mu.L for reactions containing GeneAmp1 XPCR buffer II (applied biosystems), 2.5mM MgCl₂200. mu.M of each dNTP, 200nM of each primer, 0.5U AmpliTaq Gold DNA polymerase (Applied Biosystems) and 30-50ng cDNA. The PCR program started with an initial denaturation at 95 ℃ for 10 min, followed by 32-35 amplification cycles (94 ℃ for 30 sec, annealing temperature for 30 sec, and 72 ℃ for 30 sec), with a final extension at 72 ℃ for 10 min. The PCR bands were observed under UV light and photographed. Expression of the target gene was normalized with the expression of the housekeeping gene β -actin as an internal control. All primers used to amplify the transcripts are listed in table 1.

DNA methylation analysis

Genomic DNA extraction

Genomic DNA was isolated from GC cell lines and tissue samples using a DNA mini kit (Qiagen) following the kit protocol. Approximately 25mg of sample were lysed in 180. mu.L of QIAamp ATL buffer and 20. mu.L of proteinase K in a 1.5mL microfuge tube at 56 ℃ for 1 hour. Add 4. mu.L of RNase A (100mg/ml, QIAGEN) and mix with vortex pulses for 15 seconds, then incubate at room temperature for 2 minutes. Then, 200. mu.L of AL buffer was added to the lysate and the sample was incubated at 70 ℃ for 10 minutes. After addition of 200. mu.L of absolute ethanol, the solution was mixed for 15 seconds with a vortex pulse. Lysates were then purified using a QIAamp column according to the manufacturer's instructions. Genomic DNA was diluted in 200. mu.L of DNase-free water. The quality and the obtained amount of DNA were determined by measuring the absorbance at 260nm/280nm using NanoDropND-1000 (NanoDrop).

Sodium bisulfite conversion

Sodium metabisulfite modified genomic DNA as described in Tao et al, hum. mol. Gene, 11(18): 2091-2101. Briefly, 5. mu.g of genomic DNA dissolved in 30. mu.L of TE buffer (Sigma-Aldrich) was mixed with 3.3. mu.L of 3mM NaOH to a final concentration of 0.3mM and incubated at 37 ℃ for 15 minutes. The denatured DNA was mixed with 333. mu.L of bisulfite solution and treated at 55 ℃ for 4 hours with exclusion of light. The bisulfite solution was prepared as 2.4M sodium metabisulfite (pH 5.0-5.2) (Sigma-Aldrich) and 0.5mM hydroquinone (Sigma-Aldrich). The treated DNA was desalted and purified using the Qiaex II kit (Qiagen) following the protocol provided with the kit. Then, the DNA was treated with 0.3M NaOH at 37 ℃ for 15 minutes and precipitated with 3M ammonium acetate and 3 volumes of ethanol. The recovered DNA was dissolved in 100. mu.LTE buffer (pH 8.0) and stored at-20 ℃.

Demethylation treatment with 5-Aza-2' -deoxycytidine ("5-Aza

At 1X 10 per 100mm dish⁵The cells were seeded at a density of (2) and allowed to grow for 24 hours. Then, cells were treated with 2 μ M5-Aza-2' -deoxycytidine ("5-Aza") (Sigma-Aldrich) for 5 days. Supplement with 5-Aza daily. Gene expression of DACT1 was assessed using semi-quantitative RT-PCR.

Methylation Specific PCR (MSP)

Methylation-specific and non-methylation-specific primers were designed to assess methylation status in GC cell lines. The PCR mixture contained 1 XPCR buffer II (applied biosystems), 2mM MgCl₂200 μ M of each dNTP, 600nM of each primer, 0.5U AmpliTaq GoldDNA polymerase (Applied Biosystems) and 20ng bisulfite treated DNA. The PCR program was 95 ℃ for 10 min, followed by 38 amplification cycles (94 ℃ for 30 sec, 60 ℃ for 30 sec, and 72 ℃ for 30 sec), with a final extension at 72 ℃ for 5 min. MSP bands were observed under UV light and photographed.

Direct Bisulfite Genomic Sequencing (BGS)

The bisulfite treated DNA was amplified using the primers listed in Table 1. PCR amplification of DNA treated with 2. mu.L bisulfite produced approximately 170bp PCR products containing 9 CpG dinucleotides in the DACT1 promoter region. The amplified BGS product was sequenced. Sequencing analysis was performed by SeqScape software (Applied Biosystems, Foster City, Calif.).

Array comparative genomic hybridization (Array CGH)

The DNA from the five gastric cancer cell lines (MKN45, MKN28, katoii, N87, SUN1) and the normal stomach tissue reference sample was differentially labeled using different fluorophores and hybridized to Array-CGH (Agilent Technologies, Santa Clara, CA). The results were analyzed by Agilent G4175AA CGH Analytics 3.4(Agilent Technologies). Then, the fluorescence intensity ratio of the gastric cancer cell line to the normal reference DNA was calculated, thereby evaluating copy number variation at a specific position of the genome. Two probes were used to detect copy number changes of the DACT1 gene: locus I is located 58177255 to 58177309 of chr14 and locus II is located 58179290 to 58179349 of chr 14.

Biological function analysis

Cloning of DACT1 alpha and construction of expression vector

Full-length cDNA of the DACT1 α gene expression vector was generated by PCR cloning. Total RNA from human stomach (Ambion, Austin, TX, USA) was reverse transcribed into cDNA. The sequence corresponding to the Open Reading Frame (ORF) of DACT 1a was amplified by PCR. The PCR product was cloned into pCDNA3.1 expression vector and pBABE-puro vector.

DACT1 alpha Gene transfection

The cells were cultured at approximately 6X 10⁵Cells/well were seeded in 6-well plates without antibiotics and incubated for about 24 hours until the cell density reached about 90% confluence. Then, the cells were transfected with 2. mu.g of DACT 1. alpha. and a control vector (pCDNA3.1), respectively, using Lipofectamine 2000 (Invitrogen). Lipofectamine 2000 (6.0. mu.L) diluted in 125. mu.L Opti-MEM (Invitrogen) was incubated for 5 minutes at room temperature. Then, the plasmid DNA diluted in 125. mu.L of Opti-MEM was mixed with the Lipofectamine mixture. At 5% CO₂After incubation in an incubator at 37 ℃ for 24-48 hours, cells were harvested for testing for transgene expression. For stable cell lines, cells were passaged at a ratio of 1: 10 into fresh growth medium containing the appropriate concentration of neomycin (G418) (Invitrogen). Stably transfected cells were harvested after 14-21 days of selection for functional assays.

Colony formation assay

Two days after transfection, cells were subsequently dispensed in 6-well plates containing RPMI1640 containing 10% FBS and 500. mu.g/mL neomycin (G418) at a ratio of 1: 20. After 14-18 days of selection, cells were fixed with 70% ethanol for 10 minutes and stained with 0.5% crystal violet solution for 10 minutes. Colonies of greater than 50 cells per colony were counted. Experiments were performed in triplicate samples of three independent samples.

Annexin V apoptosis assay

Annexin V is a protein that can bind to a cell membrane that has undergone apoptosis but has not lost membrane integrity. The proportion of apoptotic cells was assessed using annexin V and 7-amino-actinomycin (7-AAD) double staining. Briefly, wash with 1 × PBSThe washed cells were resuspended in 100. mu.L of ice-cold annexin binding buffer (10mM HEPES, 140mM NaCl and 2.5mM CaCl)₂pH7.4), containing 5. mu.L of Alexa Fluor 488-conjugated annexin V (Invitrogen) and 2. mu.L of 7-AAD staining solution (50. mu.g/mL). After incubation for 15 minutes at room temperature, the cells were mixed with an additional 400 μ L of ice-cold annexin binding buffer and analyzed using a flow cytometer.

Cell spreading assay and F-actin staining

Stably transfected BGC823 and MGC803 cells with pcDNA3.1-DACT1 or pcDNA3.1 were collected by trypsinization, washed twice with DMEM medium and resuspended on coverslips and plates. Cells were allowed to spread in DMEM for 6 hours and then photographed. Coverslips with spread cells were fixed with 3% paraformaldehyde for F-actin staining. Cells on the coverslips were permeabilized with TritonX-100 and then blocked. For F-actin staining, cells were stained with rhodamine-phalloidin (Invitrogen). Finally, the cells were washed and mounted with DAPI-containing mounting medium (Vector Laboratories, Burlingame, CA). Images were captured by fluorescence microscopy.

Cell migration and invasion assay

Wound healing assays were performed to analyze cell migration. AGS and MGC803 stably transfected with pcDNA3.1-DACT1 or pcDNA3.1 control vectors were inoculated into 6-well plates. After 24 hours, a monolayer of cells was manually scraped off with the tip of a plastic pipette and then washed with PBS. The cells were photographed at 0 and 24 hours of incubation (phase contrast microscopy). The distance traveled by the cells was measured between the two boundaries of the acellular region and the results were expressed as the ratio of DACT1 transfected group to pcDNA3.1 transfected cells. For measuring cell invasion activity, BD BioCoat was used^TMGrowth Factor Reduced MATRIGEL^TMInvasion Chamber (BD Biosciences) was subjected to a perforation (Transwell) assay.

In vivo tumorigenesis

For the production of retroviruses, use is made ofpBABE-puro-DACT1 alpha or pBABE-puro empty vector, two packaging plasmids pUMVC (Addgene) and pCMV-VSV-G (Addgene) were co-transfected into 293FT cells (Invitrogen) at a ratio of 1:0.9: 0.1. At 24 hours post transfection, cells were provided with fresh medium. At 48 hours post-transfection, supernatants containing retrovirus pBABE-puro-DACT1 α or pBABE-puro control were collected and stored at-80 ℃. To generate stable cell lines expressing DACT1 α and control cell lines, supernatants containing retroviral pBABE-puro-DACT1 α or pBABE-puro controls were added to BGC823 cells. After 24 hours of transduction, the retroviral-containing medium was removed and replaced with fresh medium containing the antibiotic puromycin (Invitrogen) at 0.5. mu.g/ml for selection. Cells stably expressing DACT1 or control BGC823 cells (5X 10 in 0.2mL PBS⁵Cells) were injected subcutaneously into the left or right dorsal side of 4-week-old male Balb/c nude mice (4/group), respectively. Tumor volumes were measured every 3 days until week 3. Tumor volume (mm) was estimated by measuring the longest and shortest diameters of tumors³) And calculated as follows: volume = (shortest diameter) 2 × (longest diameter) × 0.5. Animal care and all experimental protocols were approved by the animal ethics committee of chinese university in hong kong. After 3 weeks, mice were sacrificed, tumors were weighed and fixed in formalin for histological analysis.

Statistical analysis

Data are presented as mean ± Standard Deviation (SD). Differences between the 2 pre-selected groups were compared using independent student t-test. Differences in mouse cell growth curves or tumor growth rates were determined by variable repeat assay (ANOVA). The correlation between DACT1 methylation and clinical physiological characteristics of gastric cancer patients was compared using the Pearson's chi-square test. Kaplan-Meier survival curves and log rank test were used to evaluate overall survival data corresponding to the methylation status of DACT 1. When P is less than 0.05, the data is considered statistically significant; when P is less than 0.01, the data is considered statistically significant.

Results

Silencing or Down-Regulation of DACT1 in gastric cancer

DACT1 is expressed in most human tissues

DACT1 was expressed in all normal adult and fetal tissues tested as well as in the normal human gastric epithelial cell line (GES1) (fig. 1A).

DACT1 is epigenetically inhibited in cancer cell lines

The mRNA expression of DACT1 was silenced or reduced in 9 of 10 lung cancer cell lines. To illustrate the role of promoter methylation in the downregulation of DACT1, DACT1 methylation status was examined by MSP. Methylation was observed in 5 silent cell lines (KatoIII, AGS, MKN45, SNU719, MGC803), but not detected in normal gastric epithelial cells GES1 (fig. 1B). The methylation status of DACT1 was confirmed by BGS. BGS results are consistent with those of MSP, with dense methylation found in methylated cell lines (MKN45 and KatoIII), but not in unmethylated MKN28 and normal stomach tissue (fig. 2).

After demethylation, DACT1 expression can be restored

To test whether methylation could directly mediate DACT1 silencing, three silenced cell lines (MKN28, AGS, KatoIII) were treated with demethylating agent 5-Aza. This treatment restored DACT1 expression in AGS and KatoIII cell lines (fig. 3), suggesting that transcriptional silencing of DACT1 in gastric cancer cells is mediated by promoter methylation.

Functional assay

Inhibition of cell proliferation by DACT1 alpha

The in vitro biological effect of DACT1 α on cell growth was detected in cell lines that do not express DACT1 α (AGS, BGC823 and MGC803) by colony formation assay and cell growth curves. The colonies formed in the three cells transfected with DACT1 α were significantly fewer in number and significantly smaller in size than the cells transfected with the control vector (fig. 4). With this finding, abnormal expression of DACT1 α in AGS cells significantly reduced the cell growth curve (fig. 5). Both colony formation assay and cell growth curve assay strongly demonstrated that DACT1 α is able to inhibit cell growth of GC cells in vitro.

Induction of apoptosis by DACT1 alpha

To examine the contribution of apoptosis to the observed growth inhibition of cells transfected with DACT1 α, apoptosis was determined by flow cytometry using double staining of annexin V-APC and 7-AAD. The results showed an increase in the number of early apoptotic cells in cells transfected with DACT1 α compared to the number of early apoptotic cells in cells transfected with the control vector (BGC 823: 2.73% + -0.05% vs. 3.60% + -0.45%, P = 0.03; MGC 803: 2.36% + -0.25% vs. 3.63% + -0.25%).

DACT1 alpha for inhibiting spreading, migration and invasion of gastric cancer cells

Re-expression of DACT1 α showed a significant reduction in cell spreading (fig. 6A). The extent of spreading of individual cells was substantially inhibited in BGC823(P <0.01) and MGC803 cells (P =0.001) transfected with DACT1 α compared to control cells (fig. 6A). Meanwhile, DACT1 α inhibited actin microfilament formation in BGC823 and MGC803 cells by rhodamine-labeled phalloidin staining (fig. 6B). DACT1 α re-expression slowed "wounds" that showed cell migration scraping at the AGS and MGC803 cell borders (fig. 6C). Quantitative analysis at 24 hours confirmed a significant reduction in wound closure in AGS (P <0.01) and MGC803(P <0.05) cells transfected with DACT1 α compared to control cells transfected with vector (fig. 6C). In addition, DACT1 α significantly reduced cell invasiveness as measured by puncture assay in AGS (P <0.05) and MGC803(P <0.01) cells, respectively (fig. 6D).

In vivo tumor suppression

BGC823 cells were transfected with retrovirus pBABE-puro-DACT1 α or pBABE-puro control and selected with puromycin to generate cell lines stably expressing DACT1 α and control cell lines. BGC823 cells stably expressing DACT1 α or control BGC823 cells were injected subcutaneously into the left or right dorsal side of 4-week-old male Balb/c nude mice to compare the tumor growth pattern in vivo. The in vivo tumor growth curve for BGC823 stably transfected with DACT1 α or empty vector is shown in FIG. 7A. Tumor volume in DACT1 α transfected nude mice was significantly smaller than vehicle control mice (P < 0.001). At the end of the experiment, tumors were isolated and weighed. The average tumor weight in DACT1 α transfected nude mice was significantly lower than that of vehicle control mice (P <0.01) (fig. 7B), suggesting that DACT1 α acts as a tumor suppressor in gastric carcinogenesis.

Methylation status in gastric cancer patients

Methylation status in gastric cancer tissue and Normal stomach tissue

Clinical use of DACT1 methylation was assessed in 205 primary gastric cancer samples and 20 healthy stomach tissue samples. Of 205 gastric cancer cases, 29.3% (60/205) detected partial and dense promoter methylation of DACT1, but not in 20 healthy stomach tissue samples (FIG. 8A).

Association between DACT1 methylation and clinical features

DACT1 methylation was associated with advanced tumor size (P <0.05), lymph node metastasis (P <0.05) and distant metastasis (P =0.05) (table 2). DACT1 methylation was more frequent in TNM stage III/IV than in stage I/II cases (P <0.0005) (Table 2). Moreover, as shown in the Kaplan-Meier survival curve, gastric cancer patients with DACT1 methylation had significantly shorter survival (P =0.007, log rank test) compared to gastric cancer patients without DACT1 methylation (fig. 8B).

All patents, patent applications, and other publications (including GenBank accession numbers) cited in this application are incorporated by reference herein in their entirety and for all purposes.

TABLE 1 DNA sequences of primers used in this study

TABLE 2 clinicopathological characteristics of lung cancer patients determined by methylation status of DACT1 promoter

Claims (43)

1. A method of detecting gastric cancer in an individual comprising the steps of:

(a) measuring the expression level of DACT1 in a sample taken from the subject; and

(b) comparing the expression level obtained in step (a) with a standard control,

wherein a decrease in the expression level of DACT1 when compared to a standard control indicates that the individual has gastric cancer.

2. The method of claim 1, wherein the sample is a gastric mucosal sample.

3. The method of claim 1, wherein the expression level of DACT1 is the level of DACT1 protein.

4. The method of claim 1, wherein the expression level of DACT1 is a DACT1mRNA level.

5. The method of claim 3, wherein step (a) comprises performing an immunoassay with an antibody that specifically binds DACT1 protein.

6. The method of claim 5, wherein step (a) comprises Western blot analysis.

7. The method of claim 1, wherein step (a) comprises mass spectrometry or hybridization to a microarray, fluorescent probe, or molecular beacon.

8. The method of claim 4, wherein step (a) comprises an amplification reaction.

9. The method of claim 8, wherein the amplification reaction is a Polymerase Chain Reaction (PCR).

10. The method of claim 9, wherein the PCR is reverse transcriptase-PCR (RT-PCR).

11. The method of claim 4, wherein the detecting step comprises a polynucleotide hybridization assay.

12. The method of claim 11, wherein the polynucleotide hybridization assay is Southern blot analysis or Northern blot analysis.

13. The method of claim 11, wherein the polynucleotide hybridization assay is an in situ hybridization assay.

14. The method of claim 11, wherein a polynucleotide probe is used in the polynucleotide hybridization assay to hybridize to a complement of SEQ ID NOs1, 4, or 6 or more.

15. The method of claim 14, wherein the polynucleotide probe comprises a detectable moiety.

16. The method of claim 1, further comprising repeating step (a) with the same type of sample from the individual after a period of time when the individual is indicated as having gastric cancer, wherein an increase in the level of DACT1 expression after the period of time as compared to the amount of initial step (a) is indicative of an improvement in gastric cancer and a decrease is indicative of a deterioration in gastric cancer.

17. A method of detecting gastric cancer in an individual comprising the steps of:

(a) treating a sample taken from the individual with an agent that differentially modifies methylated DNA and unmethylated DNA; and

(b) determining whether each CpG in the CpG-containing genomic sequence is methylated or unmethylated, wherein the CpG-containing genomic sequence is at least a segment of SEQ ID NO 1 or 6 and comprises at least one CpG,

wherein the presence of a methylated CpG in the CpG-containing genomic sequence is indicative of the individual having gastric cancer.

18. The method of claim 17, wherein the CpG-containing genomic sequence comprises two or more cpgs, and wherein at least 50% of all cpgs are methylated is indicative that the individual has gastric cancer.

19. The method of claim 17 or 18, wherein the CpG-containing genomic sequence is a segment of at least 15 contiguous nucleotides of SEQ ID NO 1 or 6.

20. The method of claim 17 or 18, wherein the CpG-containing genomic sequence is a segment of at least 20 contiguous nucleotides of SEQ ID NO 1 or 6.

21. The method of claim 17 or 18, wherein the CpG-containing genomic sequence is a segment of at least 50 contiguous nucleotides of SEQ ID NO 1 or 6.

22. The method of claim 17 or 18, wherein the CpG-containing genomic sequence is SEQ ID NO 1 or 6.

23. The method of claim 17, wherein the CpG-containing genomic sequence is SEQ id No. 6, and wherein at least 5 of all CpG are methylated is indicative that the individual has gastric cancer.

24. The method of claim 17, wherein the sample is a gastric mucosal sample.

25. The method of claim 17, further comprising repeating steps (a) and (b) with the same type of sample from the individual after a period of time when the individual is indicated as having gastric cancer, wherein an increase in the number of methylated CpG after the period of time as compared to the number of methylated CpG determined in the initial step (b) is indicative of a worsening of gastric cancer and a decrease is indicative of an improvement of gastric cancer.

26. The method of claim 17, wherein the agent capable of differentially modifying methylated DNA and unmethylated DNA is an enzyme that preferentially cleaves methylated DNA, an enzyme that preferentially cleaves unmethylated DNA, or bisulfite.

27. The method of claim 17, wherein step (b) comprises an amplification reaction.

28. The method of claim 17, wherein step (b) comprises sequencing of the DNA molecule.

29. A kit for detecting gastric cancer in an individual comprising (1) a standard control providing an average amount of DACT1 protein or DACT1 mRNA; and (2) reagents capable of specifically and quantitatively identifying DACT1 protein or DACT1 mRNA.

30. The kit of claim 29, wherein the reagent is an antibody that specifically binds to DACT1 protein.

31. The kit of claim 29, wherein said reagent is a polynucleotide probe that is capable of hybridizing to said DACT1 mRNA.

32. The kit of claim 31, wherein the polynucleotide probe has a nucleotide sequence set forth as the complement of SEQ id No. 1, 4 or 6 or more.

33. The kit of claim 29, wherein the reagent comprises a detectable moiety.

34. The kit of claim 29, further comprising two oligonucleotide primers capable of specifically amplifying at least a segment of SEQ ID No. 2 or 3 or the complement thereof in an amplification reaction.

35. The kit of claim 29, further comprising an instruction manual.

36. A method of inhibiting the growth of a gastric cancer cell comprising contacting the gastric cancer cell with an effective amount of a polypeptide comprising the amino acid sequence set forth in SEQ ID NO. 5 or a nucleic acid comprising a polynucleotide sequence encoding SEQ ID NO. 5.

37. The method of claim 36, wherein the nucleic acid is an expression cassette comprising a promoter operably linked to the polynucleotide sequence encoding SEQ id No. 5.

38. The method of claim 37, wherein the promoter is an epithelial cell-specific promoter.

39. The method of claim 36 or 37, wherein the nucleic acid comprises the nucleotide sequence set forth in SEQ ID NO 2 or 3.

40. The method of claim 36, wherein the gastric cancer cells are in a patient.

41. An isolated nucleic acid having a nucleotide sequence at least 95% identical to a segment of about 20-100 consecutive nucleotides of the complement of SEQ ID NO 1, 2, 3, 4 or 6 or more.

42. The nucleic acid of claim 41, having a nucleotide sequence identical to a stretch of about 20-100 consecutive nucleotides of the complement of SEQ ID NO 1, 2, 3, 4 or more.

43. The nucleic acid of claim 41 or 42, conjugated to a detectable moiety.