Genomic Screen for Epigenetically Silenced Genes Associated With Cancer

Abstract

A method of identifying epigenetically silenced genes, e.g., methylation silenced genes, in cancer cells is provided. In addition, methods of identifying a cancer by detecting epigenetic silencing of gene expression are provided, as are methods of treating a subject having such a cancer, for example, a colorectal cancer and/or gastric cancer. Reagents for practicing such methods also are provided.

1 . A method for identifying a cell that exhibits or is predisposed to exhibiting unregulated growth, comprising detecting, in a test cell, hypermethylation of at least one gene comprising SFRP1, SFRP2, SFRP4, SFRP5, or a combination thereof, thereby identifying the test cell as a cell that exhibits or is predisposed to exhibiting unregulated growth. 
     
     
         2 . The method of  claim 1 , wherein at least one gene comprises SFRP1, SFRP2, or a combination thereof. 
     
     
         3 . The method of  claim 1 , wherein the cell exhibiting, or predisposed to exhibiting unregulated growth, is a neoplastic cell. 
     
     
         4 . The method of  claim 3 , wherein the neoplastic cell is a premalignant cell. 
     
     
         5 . The method of  claim 3 , wherein the neoplastic cell is a cancer cell. 
     
     
         6 . The method of  claim 5 , wherein the cancer cell is a carcinoma or a sarcoma. 
     
     
         7 . The method of  claim 5 , wherein the cancer cell is a colorectal cancer cell or a gastric cancer cell. 
     
     
         8 . The method of  claim 1 , wherein detecting hypermethylation comprises contacting a region comprising a regulatory region of the nucleic acid molecule comprising the gene with a methylation sensitive restriction endonuclease, which cleaves a recognition site in the regulatory region comprising a methylated cytosine residue of a CpG dinucleotide, whereby cleavage of the nucleic acid molecule is indicative of the hypermethylation status of the gene of the test cell. 
     
     
         9 . The method of  claim 8 , wherein the methylation sensitive restriction endonuclease is Acc III, Ban I, BstN I, Msp I, or Xma I. 
     
     
         10 . The method of  claim 1 , wherein detecting hypermethylation comprises contacting a regulatory region of the nucleic acid molecule comprising the gene of the test cell with a chemical reagent that selectively modifies either an unmethylated cytosine residue or a methylated cytosine residue, and detecting a product generated due to said contacting, wherein the product is indicative of methylation of a cytosine residue in a CpG dinucleotide of the gene, thereby detecting the methylation status of the gene of the test cell. 
     
     
         11 . The method of  claim 10 , wherein detecting the product comprises an electrophoresis method, a chromatography method, a spectrometry method, a mass spectrometry method, or a combination thereof. 
     
     
         12 . The method of  claim 10 , wherein the chemical reagent is hydrazine, thereby producing a hydrazine treated regulatory region of the gene,
 the method further comprising contacting the hydrazine treated regulatory region with a reagent that cleaves hydrazine modified cytosine residues to generate a product comprising fragments of the nucleic acid molecule comprising the gene,   separating the fragments according to molecular weight, and   detecting a gap at a position known to contain a cytosine residue in the regulatory region of the gene, wherein the gap is indicative of methylation of a cytosine residue in the CpG dinucleotide in the gene, thereby detecting the methylation status of the gene of the test cell.   
     
     
         13 . The method of  claim 12 , wherein the reagent that cleaves the hydrazine modified cytosine residue is piperidine. 
     
     
         14 . The method of  claim 10  wherein the chemical reagent comprises bisulfite ions, whereby unmethylated cytosine residues in the regulatory region of the gene are converted to bisulfite modified cytosine residues,
 the method further comprising exposing the bisulfite ion treated gene to alkaline conditions, whereby bisulfite modified cytosine residues are converted to uracil residues, and   detecting an amount or distribution of uracil residues in the regulatory region of the bisulfite ion treated gene of the test cell,   wherein a decrease in the amount or distribution of uracil residues in the regulatory region of gene from the test cell, as compared to the amount or distribution of uracil residues in a corresponding bisulfite ion treated unmethylated gene following exposure to alkaline conditions, is indicative of methylation of cytosine residues in CpG dinucleotides in the regulatory region of the gene, thereby detecting methylation status of the gene of the test cell.   
     
     
         15 . The method of  claim 14 , wherein detecting the amount or distribution of uracil residues comprises determining the nucleotide sequence of the bisulfite modified regulatory region of the gene following exposure to alkaline conditions. 
     
     
         16 . The method of  claim 14 , wherein detecting the amount or distribution of uracil residues comprises contacting the bisulfite ion treated gene sequence, following exposure to alkaline conditions, with an oligonucleotide that selectively hybridizes to the regulatory region of the gene containing uracil residues, and
 detecting selective hybridization of the oligonucleotide.   
     
     
         17 . The method of  claim 16 , wherein the oligonucleotide has a nucleotide sequence as set forth in SEQ ID NO:23, 24, 111, 112, 115, 116, 119, 120, 125, 126, 129, 130, 133, 134, 139, 140, 143, or 144. 
     
     
         18 . The method of  claim 16 , wherein the oligonucleotide comprises a detectable label, and wherein detecting selective hybridization comprises detecting the label. 
     
     
         19 . The method of  claim 18 , wherein the detectable label is a radioisotope, a paramagnetic isotope, a luminescent compound, a chemiluminescent compound, a fluorescent compound, a metal chelate, an enzyme, a substrate for an enzyme, a receptor, or a ligand for a receptor. 
     
     
         20 . The method of  claim 16  wherein the oligonucleotide is a substrate for a primer extension reaction, and wherein detecting selective hybridization comprises detecting a product of the primer extension reaction. 
     
     
         21 . The method of  claim 20 , wherein the oligonucleotide has a nucleotide sequence as set forth in SEQ ID NO:23, 24, 111, 112, 115, 116, 119, 120, 125, 126, 129, 130, 133, 134, 139, 140, 143, or 144. 
     
     
         22 . The method of  claim 16 , wherein detecting the amount or distribution of uracil residues comprises
 contacting the regulatory region of the gene with an amplification primer pair comprising a forward primer and a reverse primer under conditions suitable for amplification, wherein at least one primer of the primer pair comprises an oligonucleotide that selectively hybridizes to a nucleotide sequence of the regulatory region containing uracil residues,   whereby generation of an amplification product is indicative of methylation of cytosine residues in CpG dinucleotides in the regulatory region of the gene, thereby detecting the methylation status of the gene of the test cell.   
     
     
         23 . The method of  claim 22 , wherein the amplification primer pair comprises a primer pair as set forth in SEQ ID NO:23 and 24, SEQ ID NOS:111 and 112, SEQ ID NOS:115 and 116, SEQ ID NOS:119 and 120, SEQ ID NOS:125 and 126, SEQ ID NOS:129 and 130, SEQ ID NOS:133 and 134, SEQ ID NOS:139 and 140, or SEQ ID NOS:143 and 144. 
     
     
         24 . The method of  claim 14 , wherein detecting the amount or distribution of uracil residues comprises
 contacting in the regulatory region of the gene with a first amplification primer pair and a second amplification primer pair under conditions suitable for amplification,
 wherein the first amplification primer pair comprises a forward primer and a reverse primer, wherein at least one primer of the first primer pair comprises an oligonucleotide that selectively hybridizes to a nucleotide sequence of the regulatory region of the gene containing uracil residues, and 
 wherein the second amplification primer pair comprises a forward primer and a reverse primer, wherein both primers of the second primer pair selectively hybridize to a nucleotide sequence of the regulatory region of the gene containing cytosine residues, but not to a corresponding nucleotide sequence of the regulatory region of the gene containing uracil residues, and 
 wherein an amplification product, if any, generated by the first primer pair has a first length, and wherein an amplification product, if any, generated by the second primer pair has a second length, which is different from the first length, 
   whereby the length of the amplification products is indicative of uracil residues and, therefore, methylation of cytosine residues in CpG dinucleotides in the regulatory region of the gene, thereby detecting the methylation status of the gene of the test cell.   
     
     
         25 . The method of  claim 1 , wherein detecting hypermethylation comprises
 a) contacting the test cell with a demethylating agent, and   b) detecting increased expression of an RNA encoded by the gene as compared to a level of expression of the RNA in a test cell not contacted with a demethylating agent.   
     
     
         26 . The method of  claim 25 , wherein the demethylating agent comprises a methyltransferase inhibitor. 
     
     
         27 . The method of  claim 26 , wherein the methyltransferase inhibitor comprises 5-aza-2′-deoxycytidine. 
     
     
         28 . The method of  claim 1 , which is performed in a high throughput format, wherein the test cell, or extract of the test cell, comprises one of a plurality of test cells, or extracts of the test cells, or a combination thereof. 
     
     
         29 . The method of  claim 28 , wherein each of the test cells, or extracts of the test cells, of the plurality is the same or different, or a combination thereof. 
     
     
         30 . The method of  claim 28 , further comprising detecting methylation, if any, of cytosine residues in a CpG dinucleotide in a CpG island of the regulatory region of the gene in a corresponding cell exhibiting regulated growth, or an extract of the corresponding cell. 
     
     
         31 . The method of  claim 28 , wherein the test cells, or extracts of the test cell, are arranged in an array. 
     
     
         32 . The method of  claim 31 , wherein the array is an addressable array. 
     
     
         33 . The method of  claim 28 , wherein the test cells, or extracts of the test cells, are on a microchip, a glass slide, or a bead. 
     
     
         34 . The method of  claim 1 , wherein the test cell comprises a sample obtained from a subject. 
     
     
         35 . The method of  claim 34 , wherein the subject is a human subject. 
     
     
         36 . The method of  claim 34 , wherein the sample comprises an organ sample, a tissue sample, or a cell sample. 
     
     
         37 . The method of  claim 36 , wherein the sample comprises a gastrointestinal tract sample, a liver sample, a skin sample, a lymph node sample, a kidney sample, a lung sample, a muscle sample, a bone sample, or a brain sample. 
     
     
         38 . The method of  claim 37 , wherein the gastrointestinal tract sample comprises a stomach sample, a small intestine sample, a colon sample, or a rectal sample. 
     
     
         39 . The method of  claim 34 , wherein the sample comprises a biological fluid. 
     
     
         40 . The method of  claim 39 , wherein the biological fluid comprises bone marrow, blood, serum, lymph, cerebrospinal fluid, saliva, sputum, stool, urine, or ejaculate.