Urothelial cancer and methods of detection and targeted therapy

Abstract

The invention provides methods for detecting a cellular proliferative disorder (e.g., urothelial cancer) in a subject by assessing the methylation status of the GULP1 promoter in a nucleic acid sample. The methods of the invention are useful for diagnostic, prognostic as well as therapeutic regimen predictions.

What is claimed is: 
     
         1 . A method for detecting a urothelial cell proliferative disorder associated with engulfment adaptor PTB domain containing 1 (GULP1) in a subject comprising:
 a) contacting a target nucleic acid in a sample of urothelial tissue or biological fluid from the subject containing urothelial cells with a reagent which detects GULP1, wherein the reagent detects methylation of the promoter region of GULP1 when the target nucleic acid is DNA, and wherein the reagent detects the level of GULP1 RNA when the target nucleic acid is RNA; and   b) detecting GULP1 target nucleic acid, wherein hypermethylation of the promoter of GULP1 DNA, or decreased levels of GULP1 RNA, as compared with the level of GULP1 RNA in a normal cell, is indicative of a urothelial cell proliferative disorder.   
     
     
         2 . The method of  claim 1 , wherein the subject is a mammalian subject. 
     
     
         3 . The method of  claim 1 , wherein the biological fluid is selected from the group consisting of a biopsy specimen, a tissue specimen, ejaculate, urine and blood. 
     
     
         4 . The method of  claim 1 , wherein the reagent which detects methylation of the promoter region of GULP1 is a restriction endonuclease. 
     
     
         5 . The method of  claim 4 , wherein the restriction endonuclease is methylation sensitive. 
     
     
         6 . The method of  claim 5 , wherein the restriction endonuclease is selected from the group consisting of MspI, HpaII, BssHII, BstUI and NotI. 
     
     
         7 . The method of  claim 1 , wherein the reagent is a nucleic acid probe. 
     
     
         8 . The method of  claim 7 , wherein the nucleic acid probe comprises a nucleic acid sequence as recited in SEQ ID NO: 7 or SEQ ID NO: 13. 
     
     
         9 . The method of  claim 7 , wherein the probe is detectably labeled. 
     
     
         10 . The method of  claim 8 , wherein the label is selected from the group consisting of a radioisotope, a bioluminescent compound, a chemiluminescent compound, a fluorescent compound, a metal chelate, and an enzyme. 
     
     
         11 . The method of  claim 1 , wherein the cell is an epithelial cell. 
     
     
         12 . The method of  claim 1 , wherein the cell proliferative disorder is carcinoma of the bladder, ureters, kidney, or renal pelvis. 
     
     
         13 . The method of  claim 1 , wherein the methylation of the promoter region is detected by contacting the target nucleic acid with GULP1 oligonucleotide primers. 
     
     
         14 . The method of  claim 12 , wherein the primers comprise at least one selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 11 and SEQ ID NO: 12. 
     
     
         15 . The method of  claim 1 , wherein the detecting comprises contacting the nucleic acids with an agent that modifies nonmethylated cytosine residues, amplifying CpG-containing nucleic acids by means of CpG-specific oligonucleotide primers, wherein the oligonucleotide primers distinguish between modified methylated and nonmethylated nucleic acid, and detecting the methylated CpG-containing promoter region based on the presence or absence of amplification products produced in said amplifying step. 
     
     
         16 . The method of  claim 14 , wherein the primers comprise at least one selected from SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 11 and SEQ ID NO: 12. 
     
     
         17 . The method of  claim 14 , wherein the amplifying step comprises a polymerase chain reaction. 
     
     
         18 . The method of  claim 14 , wherein the modifying agent is bisulfite. 
     
     
         19 . The method of  claim 14 , wherein cytosine is modified to uracil. 
     
     
         20 . A method for monitoring the effectiveness of a therapeutic regimen in a subject having a urothelial cell proliferative disorder associated GULP1 comprising:
 a) contacting a target nucleic acid in a sample of urothelial tissue or biological fluid from the subject containing urothelial cells with a reagent which detects GULP1, wherein the reagent detects methylation of the promoter region of GULP1 when the target nucleic acid is DNA, and wherein the reagent detects the level of GULP1 RNA when the target nucleic acid is RNA, wherein the contacting of the sample occurs prior to, simultaneously with and/or following a course of treatment; and   b) detecting GULP1 target nucleic acid, wherein a reduction of hypermethylation of the promoter of GULP1 DNA, or increased levels of GULP1 RNA, as compared with the level of GULP1 RNA prior to treatment, is indicative of effectiveness of a therapeutic regimen for treatment of urothelial cell proliferative disorder in the subject.   
     
     
         21 . The method of  claim 19 , wherein the therapeutic regimen is chemotherapy. 
     
     
         22 . The method of  claim 20 , wherein the chemotherapy is paclitaxel or cisplatin. 
     
     
         23 . A method of treating a urothelial cell proliferative disorder associated with GULP1 in a subject comprising:
 contacting a GULP1 containing nucleic acid sequence in the subject with an agent that reduces methylation of or demethylates the promoter region of GULP1, wherein the promoter region is hypermethylated as compared with a subject not having a urothelial cell proliferative disorder, thereby increasing expression of the GULP1 gene and ameliorating the symptoms associated with the disorder.   
     
     
         24 . The method of  claim 22 , wherein the subject is treated with a therapeutic regimen comprising administration of one or more chemotherapeutic agents. 
     
     
         25 . The method of  claim 23 , wherein the chemotherapeutic agent is administered in combination with a demethylating agent. 
     
     
         26 . The method of  claim 22 , wherein the demethylating agent is 5-azacytidine, 5-aza-2-deoxycytidine or zebularine. 
     
     
         27 . A method for providing a prognosis to a subject having a urothelial cell proliferative disorder associated GULP1 comprising:
 a) contacting a target nucleic acid in a sample of urothelial tissue or biological fluid from the subject containing urothelial cells with a reagent which detects GULP1, wherein the reagent detects methylation of the promoter region of GULP1 when the target nucleic acid is DNA, and wherein the reagent detects the level of GULP1 RNA when the target nucleic acid is RNA, wherein the contacting of the sample occurs prior to, simultaneously with and/or following a course of treatment;   b) detecting GULP1 target nucleic acid; and   c) providing a prognosis to the subject based on (b), wherein a reduction of hypermethylation of the promoter of GULP1 DNA, or increased levels of GULP1 RNA, as compared with the level of hypermethylation or GULP1 RNA as compared to a reference sample of the subject, is indicative of an increase likelihood of cancer-free survival in the subject.   
     
     
         28 . The method of  claim 26 , wherein the subject is undergoing a therapeutic regimen. 
     
     
         29 . The method of  claim 27 , wherein the therapeutic regimen is chemotherapy. 
     
     
         30 . The method of  claim 28 , wherein the chemotherapy is paclitaxel or cisplatin. 
     
     
         31 . A method of treating a urothelial cell proliferative disorder associated with GULP1 in a subject comprising:
 administering to a subject having or at risk of having the urothelial cell proliferative disorder with an agent that inhibits activation of Nrf2 pathway, thereby treating the urothelial cell proliferative disorder associated with GULP1 in the subject.   
     
     
         32 . The method of  claim 30 , wherein the agent is administered with a chemotherapeutic agent. 
     
     
         33 . The method of  claim 31 , wherein the chemotherapeutic agent is paclitaxel or cisplatin.