Methods and compositions for detecting cancers

Abstract

In certain aspects, the invention provides isolated SLC5A8 (also referred to as Hui1) nucleic acid molecules, which encode novel sodium solute symporter members. The invention also provides antisense nucleic acid molecules, recombinant expression vectors containing SLC5A8 nucleic acid molecules, host cells into which the expression vectors have been introduced, and nonhuman transgenic animals in which a SLC5A8 gene has been introduced or disrupted. The invention still further provides isolated SLC5A8 proteins, fusion proteins, antigenic peptides, and anti-SLC5A8 antibodies. Diagnostic methods utilizing compositions of the invention are also provided. In other aspects, the invention provides methods and compositions for detecting and treating SLC5A8-associated cancer. Differential methylation of the SLC5A8 nucleotide sequences has been observed in SLC5A8-associated cancer, such as colon cancer, breast cancer, thyroid cancer, or stomach cancer.

We claim:  
     
         1 . An isolated polypeptide comprising an amino acid sequence selected from the group consisting of: 
 a) an amino acid sequence at least 95% identical to SEQ ID NO: 1; and    b) an amino acid sequence encoded by a nucleic acid that hybridizes under high stringency conditions to a nucleic acid of any one of SEQ ID NOS: 3 or 4, wherein said polypeptide is a cell surface protein.    
     
     
         2 . The isolated polypeptide of  claim 1 , wherein the polypeptide comprises a transmembrane domain as set forth in any one of SEQ ID NOS: 19-31.  
     
     
         3  An isolated antibody, or fragment thereof, which is specifically immunoreactive with an epitope of an amino acid sequence as set forth in SEQ ID NO: 1.  
     
     
         4 . The antibody of  claim 3 , wherein said antibody is selected from the group consisting of: a polyclonal antibody, a monoclonal antibody, an Fab fragment and a single chain antibody.  
     
     
         5 . The antibody of  claim 3 , wherein said antibody is labeled with a detectable label.  
     
     
         6 . An isolated nucleic acid selected from the group consisting of: 
 a) a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 2, or a complement thereof,    b) a nucleic acid molecule that encodes a polypeptide comprising the amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 7; and    c) a nucleic acid molecule that hybridizes under stringent conditions to SEQ ID NO: 2.    
     
     
         7 . The nucleic acid of  claim 6 , further comprising a vector nucleic acid sequence.  
     
     
         8 . A host cell which contains the nucleic acid of  claim 6 .  
     
     
         9 . A method for producing the polypeptide of  claim 1 , comprising culturing the host cell of  claim 5  under conditions in which the nucleic acid molecule is expressed.  
     
     
         10 . A method for detecting the presence of the polypeptide of  claim 1  in a sample, comprising: a) contacting the sample with an antibody which selectively binds to the polypeptide of  claim 1;  and b) determining whether the antibody binds to the polypeptide in the sample.  
     
     
         11 . A kit for detecting a human SCL5A8 polypeptide comprising: (i) an antibody of  claim 3;  and (ii) a detectable label for detecting said antibody.  
     
     
         12 . A method for detecting the presence of the nucleic acid of  claim 6  in a sample, comprising: 
 a) contacting the sample with the probe or primer of  claim 6;  and  
 b) determining whether the probe or primer binds to a nucleic acid in the sample.  
 
     
     
         13 . A kit comprising the probe or primer of  claim 6  and instructions for use.  
     
     
         14 . A method for identifying a compound which binds to the polypeptide of  claim 1 , comprising: 
 a) contacting the polypeptide, or a cell expressing the polypeptide of  claim 1 , with a test compound; and    b) determining whether the polypeptide binds to the test compound.    
     
     
         15 . A method for modulating the activity of the polypeptide of  claim 1 , comprising contacting the polypeptide or a cell expressing the polypeptide of  claim 1  with a compound which binds to the polypeptide in a sufficient concentration to modulate the activity of the polypeptide.  
     
     
         16 . A method of inhibiting aberrant activity of a SLC5A8-expressing cell, comprising contacting the cell with a compound that modulates the activity or expression of the polypeptide of  claim 1 , in an amount which is effective to reduce or inhibit the aberrant activity of the cell.  
     
     
         17 . The method of any of claims  14 - 16 , wherein the compound is selected from the group consisting of a peptide, a phosphopeptide, a small organic molecule, an antibody, and a peptidomimetic.  
     
     
         18 . The method of any of claims  14 - 17 , wherein the cell is found in the colon, kidney, lung, esophagus, small bowel, stomach, thyroid, uterus, and breast.  
     
     
         19 . A method of treating or preventing a disorder characterized by aberrant activity of a SLC5A8-expressing cell, in a subject, comprising administering to the subject an effective amount of a compound that modulates the activity or expression of the polypeptide of  claim 1 , such that the aberrant activity of the SLC5A8-expressing cell is reduced or inhibited.  
     
     
         20 . A transgenic mouse having germline and somatic cells comprising a chromosomally incorporated transgene that disrupts the genomic SLC5A8 gene and inhibits expression of said gene, wherein said disruption comprises insertion of a selectable marker sequence resulting in said transgenic mouse exhibiting increased susceptibility to the formation of tumors as compared to the wildtype mouse.  
     
     
         21 . The transgenic mouse of  claim 20 , wherein said mouse is homozygous for said disruption.  
     
     
         22 . The transgenic mouse of  claim 20 , wherein said mouse is heterozygous for said disruption.  
     
     
         23 . A transgenic mouse having germline and somatic cells in which at least one allele of a genomic SLC5A8 gene is disrupted by a chromosomally incorporated transgene, which transgene inhibits the expression of said genomic SLC5A8 gene, wherein (i) said genomic SLC5A8 gene encodes a SLC5A8 protein; and (ii) said disruption comprises insertion of a selectable marker sequence, which replaces all or a portion of the genomic SLC5A8 gene or is inserted into the coding sequence of said genomic SLC5A8 gene; and (iii) said transgenic mouse has increased susceptibility to the development of neoplasms.  
     
     
         24 . Isolated mammalian cells comprising a diploid genome including a chromosomally incorporated transgene, which transgene disrupts the genomic SLC5A8 gene and inhibits expression of said gene.  
     
     
         25 . The cells of  claim 24 , which cells are mouse cells.  
     
     
         26 . A method for generating a mouse and mouse embryonic stem cells having a functionally disrupted endogenous SLC5A8 gene, comprising the steps of: 
 (i) constructing a transgene construct including (a) a recombination region having all or a portion of the endogenous SLC5A8 gene, which recombination region directs recombination of the transgene with the endogenous SLC5A8 gene; and (b) a marker sequence which provides a detectable signal for identifying the presence of the transgene in a cell;    (ii) transferring the transgene into embryonic stem cells of a mouse;    (iii) selecting embryonic stem cells having a correctly targeted homologous recombination between the transgene and the SLC5A8 gene;    (iv) transferring said cells identified in step (iii) into a mouse blastocyst and implanting the resulting chimeric blastocyst into a female mouse; and    (v) selecting offspring harboring an endogenous SLC5A8 gene allele comprising the correctly targeted recombination.    
     
     
         27 . A method of evaluating the carcinogenic potential of an agent comprising: (i) contacting the transgenic mouse of  claim 20  with a test agent; and (ii) comparing the number of transformed cells in a sample from the treated mouse with the number of transformed cells in a sample from an untreated transgenic mouse or transgenic mouse treated with a control agent, wherein the difference in the number of transformed cells in the treated mouse, relative to the number of transformed cells in the absence of treatment or treatment with a control agent, indicates the carcinogenic potential of the test compound.  
     
     
         28 . A method of evaluating an anti-proliferative activity of a test compound, comprising: 
 (i) providing a transgenic mouse of  claim 20  having germline and somatic cells in which the expression of the SLC5A8 gene is inhibited by said chromosomally incorporated transgene, or a sample of cells derived therefrom;    (ii) contacting the transgenic mouse or the sample of cells with a test agent; and    (iii) determining the number of transformed cells in a specimen from the transgenic mouse or in the sample of cells,    wherein a statistically significant decrease in the number of transformed cells, relative to the number of transformed cells in the absence of the test agent, indicates the test compound is a potential anti-proliferative agent.    
     
     
         29 . A method for detecting differential methylation patterns in a SLC5A8 nucleotide sequence, comprising: 
 a) obtaining a sample from a patient;    b) assaying said sample for the presence of methylation within a nucleotide sequence as set forth in any one of SEQ ID NOS: 12-13 or fragments thereof;    c) obtaining a sample from a healthy subject;    d) assaying for the presence of methylation in a nucleotide sequence as set forth in any one of SEQ ID NOS: 12-13 or fragments thereof; and    e) comparing the methylation patterns in the sample from the patient to the methylation patterns in the normal sample.    
     
     
         30 . A method for detecting a SLC5A8-associated cancer, comprising: 
 a) obtaining a sample from a patient; and    b) assaying said sample for the presence of methylation within a nucleotide sequence as set forth in any one of SEQ ID NOS: 12-13 or fragments thereof;    wherein methylation of said nucleotide sequence is indicative of a SLC5A8-associated cancer.    
     
     
         31 . The method of any one of claims  29  and  30 , wherein the sample is a bodily fluid selected from the group consisting of blood, serum, plasma, a blood-derived fraction, stool, urine, and a colonic effluent.  
     
     
         32 . The method of  claim 31 , wherein the bodily fluid is obtained from a subject suspected of having or is known to have a SLC5A8-associated cancer.  
     
     
         33 . The method of  claim 32 , wherein said SLC5A8-associated cancer is selected from the group consisting of: colon cancer, breast cancer, thyroid cancer, and stomach cancer.  
     
     
         34 . The method of any one of claims  29  and  30 , comprising assaying for the presence of methylation within the SLC5A8 sequence as set forth in SEQ ID NO: 14.  
     
     
         35 . The method of any of claims  29 - 34 , wherein the assay is methylation-specific PCR.  
     
     
         36 . The method of  claim 35 , comprising: 
 a) treating DNA from the sample with a compound that converts non-methylated cytosine bases in the DNA to a different base;    b) amplifying a region of the compound converted SLC5A8 nucleotide sequence with a forward primer and a reverse primer; and    c) analyzing the methylation patterns of said SLC5A8 nucleotide sequences.    
     
     
         37 . The method of  claim 35 , comprising: 
 a) treating DNA from the sample with a compound that converts non-methylated cytosine bases in the DNA to a different base;    b) amplifying a region of the compound converted SLC5A8 nucleotide sequence with a forward primer and a reverse primer; and    c) detecting the presence and/or amount of the amplified product.    
     
     
         38 . The method of  claim 35 , wherein the forward primers are selected from SEQ ID NOS: 8 and 10.  
     
     
         39 . The method of  claim 35 , wherein the reverse primers are selected from SEQ ID NOS: 9 and 11.  
     
     
         40 . The method of  claim 35 , wherein the compound used to treat DNA is a bisulfite compound.  
     
     
         41 . The method of any of claims  29 - 34 , wherein the assay comprises using a methylation-specific restriction enzyme.  
     
     
         42 . The method of  claim 41 , wherein said methylation-specific restriction enzyme is selected from HpaII, SmaI, SacII, EagI, MspI, BstUI, and BssHII.  
     
     
         43 . The method of  claim 41 , further comprising a pair of primers selected from SEQ ID NOS: 5-7.  
     
     
         44 . A method for detecting a SLC5A8-associated cancer in a subject, comprising detecting SLC5A8 protein or nucleic acid expression in a sample from the subject.  
     
     
         45 . The method of  claim 44 , wherein the sample is a bodily fluid selected from the group consisting of blood, serum, plasma, a blood-derived fraction, stool, urine, and a colonic effluent.  
     
     
         46 . The method of  claim 45 , wherein the bodily fluid is from a subject suspected of having or known to have a SLC5A8-associated cancer.  
     
     
         47 . The method of  claim 46 , wherein the SLC5A8-associated cancer is selected from the group consisting of: colon cancer, breast cancer, thyroid cancer, and stomach cancer.  
     
     
         48 . The method of  claim 44 , wherein the SLC5A8 protein is detected by immunoassays.  
     
     
         49 . A method for identifying an agent which enhances SLC5A8 protein or nucleic acid expression in a diseased cell associated with SLC5AS gene silencing, comprising: 
 a) contacting the cell with a sufficient amount of the agent under suitable conditions;    b) quantitatively determining the amount of SLC5A8 protein or nucleic acid; and    c) comparing the amount of SLC5A8 protein or nucleic acid with the amount of SLC5A8 protein or nucleic acid in the absence of the agent,    wherein a greater amount of SLC5A8 protein or nucleic acid in the presence of the agent than i n t he absence o f t he a gent i ndicates t hat t he a gent e nhances S LC5A8 p rotein o r nucleic acid expression.    
     
     
         50 . The method of  claim 49 , wherein said SLC5A8 gene silencing is due to differential methylation of a SLC5A8 nucleotide sequence.  
     
     
         51 . The method of  claim 50 , wherein differential methylation occurs within a SLC5A8 nucleotide sequence set forth in any one of SEQ ID NOS: 12-13 or fragments thereof.  
     
     
         52 . The method of  claim 49 , wherein the diseased cell is from a subject having colon neoplasia.  
     
     
         53 . A method for monitoring over time a SLC5A8-associated cancer comprising: 
 a) detecting the methylation status of a SLC5A8 nucleotide sequence in a sample from the subject for a first time; and    
       b) detecting the methylation status of the SLC5A8 nucleotide sequence in a sample from the same subject at a later time;  
       wherein absence of methylation in the SLC5A8 nucleotide sequence taken at a later time and the presence of methylation in the SLC5A8 nucleotide sequence taken at the first time is indicative of cancer regression;  
       wherein presence of methylation in the SLC5A8 nucleotide sequence taken at a later time and the absence of methylation in the SLC5A8 nucleotide sequence taken at the first time is indicative of cancer progression.  
     
     
         54 . The method of  claim 53 , wherein the sample is a bodily fluid selected from the group consisting of blood, serum, plasma, a blood-derived fraction, stool, urine, and a colonic effluent.  
     
     
         55 . The method of  claim 53 , wherein the SLC5A8-associated cancer is selected from the group consisting of: colon cancer, breast cancer, thyroid cancer, and stomach cancer.  
     
     
         56 . A method for treating a SLC5A8-associated proliferative disease in a subject, comprising administering to the subject a sufficient amount of a compound, wherein the compound modulates the SLC5A8 protein or nucleic acid expression.  
     
     
         57 . The method of  claim 56 , wherein the disease is associated with methylation of a SLC5A8 nucleic acid sequence, and the compound induces SLC5A8 expression.  
     
     
         58 . The method of  claim 57 , the compound is a demethylation agent selected from 5azacytidine and 5-deoxy-azacytidine.  
     
     
         59 . The method of  claim 56 , wherein the SLC5A8-associated proliferative disease is selected from the group consisting of: thyroid nodular hyperplasia, thyroid adenoma, thyroid cancer, colon neoplasia, breast cancer, and stomach cancer.  
     
     
         60 . A method for treating a SLC5A8-associated cancer in a subject, comprising administering to the subject a vector containing a SLC5A8 nucleic acid which is operably linked to a heterologous promoter.  
     
     
         61 . The method of  claim 60 , wherein the SLC5A8 nucleic acid encodes a polypeptide at least 90% identical to SEQ ID NO: 1.  
     
     
         62 . The method of  claim 60 , wherein the cancer is a colon neoplasia.  
     
     
         63 . A bisulfite-converted methylated SLC5A8 nucleotide sequence selected from the group consisting of: 
 a) a nucleotide sequence of any one of SEQ ID NOS: 15-18 or a fragment thereof;    b) a complement of any one of SEQ ID NOS: 15-18; and    c) a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of any one of SEQ ID NOS: 15-18.    
     
     
         64 . Oligonucleotide primers for detecting methylation of a SLC5A8 nucleotide sequence, selected from SEQ ID NOS: 5-11.  
     
     
         65 . A kit for detecting a SLC5A8-associated cancer in a subject, comprising at least two primers of  claim 64 .  
     
     
         66 . The kit of  claim 65 , further comprising a compound to convert a template DNA.  
     
     
         67 . The kit of  claim 66 , wherein the compound is bisulfite.  
     
     
         68 . The kit of  claim 67 , wherein each primer comprises at least a CpG dinucleotide.  
     
     
         69 . A method of converting a nucleic acid sequence at least 95% identical to any one of SEQ ID NOs: 12-13 or fragments thereof, to a bisulfite converted sequence comprising: 
 a) providing a nucleotide acid having a nucleotide sequence as set forth in any one of SEQ ID NOS: 12-13 or fragments thereof; and    b) adding a bisulfite compound, whereby the unmethylated cytosine bases of the CpG islands are converted to a different base.    
     
     
         70 . The method of  claim 69 , wherein the unmethylated cytosine is converted to a uracil.  
     
     
         71 . A nucleic acid sequence as prepared by the method of  claim 69 .  
     
     
         72 . An isolated or recombinant methylated SLC5A8 nucleic acid, comprising a nucleotide sequence as set forth in any one of SEQ ID NOS: 12-13 or fragments thereof, wherein the cytosine of the CpG island is methylated.  
     
     
         73 . An isolated or recombinant SLC5A8 nucleic acid, selected from the group consisting of: 
 a) a nucleotide sequence as set forth in any one of SEQ ID NOS: 12-13 or a fragment thereof;    b) a complement of any one of SEQ ID NOS: 12-13;    c) a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of any one of SEQ ID NOS: 12-13;    d) a nucleotide sequence that is at least 98% identical to the nucleotide sequence of any one of SEQ ID NOS: 12-13; and    e) a nucleotide sequence comprising at least 50 consecutive base pairs of any one of SEQ ID NOS: 12-13,    wherein the SLC5A8 nucleotide sequence is differentially methylated in a SLC5A8associated disease cell.    
     
     
         74 . A method for detecting colon cancer, comprising: 
 a) obtaining a sample from a patient; and    b) assaying said sample for the presence of methylation of nucleotide sequences within at least two genes selected from the group consisting of: SLC5A8, HLTF, p16, and hMLH1;    wherein methylation of nucleotide sequences within the two genes is indicative of colon cancer.    
     
     
         75 . The method of  claim 74 , wherein the sample is a bodily fluid selected from the group consisting of blood, serum, plasma, a blood-derived fraction, stool, urine, and a colonic effluent.  
     
     
         76 . The method of  claim 74 , wherein the bodily fluid is obtained from a subject suspected of having or is known to have colon cancer.  
     
     
         77 . A kit for detecting colon cancer in a subject, comprising primers for detecting methylation of nucleotide sequence within at least two genes selected from the group consisting of: SLC5A8, HLTF, p16, and hMLH1, 
 wherein the primers for detecting methylation of SLC5A8 nucleotide sequence are selected from SEQ ID NOS: 5-11;    wherein the primers for detecting methylation of HLTF nucleotide sequence are selected from 5′-TGGGGTTTCGTGGTTTTTTCGCGC-3′, 5′-CCGCGAATCCAATCAAACGTCGACG-3′, 5′-ATTTTTGGGGTTTTGTGGTTTTTTTGTGT-3′, 5′-ATCACCACAAATCCAATCAAACATCAACA-3′, 5′-GCACGACTAAAAAATAAATCGCCGCG-3′, 5′-AAACACACAACTAAAAAATAAATCACCACA-3′, 5′-TAAAACCTCGTAACTTTCCCGCGCG-3′, 5′-GTCGCGAGTTTAGTTAGACGTCGAC-3′, 5′-TCCTAAAACCTCATAACTTTCCCACACA-3′, 5′-AGTTGTTGTGAGTTTAGTTAGATGTTGAT-3′   wherein the primers for detecting methylation of hMLH 1 nucleotide sequence are selected from 5′AACGAATTAATAGGAAGAGCGGATAGCG-3′, 5′-CGTCCCTCCCTAAAACGACTACTACCC-3′, 5′-CGTTTTTTTTTGAAGCGGTTATTGTTTGT-3′, and 5′-AACGAACCAATAAAAAAAACAAACAACG-3′   
     
     
         78 . The kit of  claim 77 , further comprising a compound to convert a template DNA.  
     
     
         79 . The kit of  claim 78 , wherein the compound is bisulfite.