Methods for Diagnosis and Treatment of Endometrial Cancer

Abstract

The present invention discloses methods of using endothelial membrane protein 2 (EMP2) as a biomarker for stratification of endometrial premalignancy, diagnosing, staging and imaging of endometrial neoplasia. Further, methods for identifying pharmaceutical/therapeutic modalities are described, including compositions which modulate glycolipid-enriched lipid raft microdomains (GEMs).

1 . A method for determining the likelihood of an endometrial cell becoming cancerous, comprising:
 determining the level of endothelial membrane protein 2 (EMP2) polypeptide or polynucleotide in a test sample, wherein increased levels of EMP2 polypeptide or polynucleotide in the test sample relative to a control sample correlates with the endometrial cells having an increased likelihood of becoming cancerous.   
     
     
         2 . The method of  claim 1 , wherein the determining comprises performing Northern blotting, Western blotting, protein gel electrophoresis, immunoprecipitation, ELISA, PCR, RT-PCR, differential display, serial analysis of gene expression, array analysis, and/or immunohistochemistry. 
     
     
         3 . The method of  claim 2 , comprising performing immunohistochemistry on a group of endometrial cells using an anti-endothelial membrane protein 2 (EMP2) antibody or antigen binding fragment thereof; and
 determining the binding of the antibody or antigen binding fragment thereof to the endometrial cells, wherein an increased amount of antibody or antigen-binding fragment thereof bound to the endometrial cells relative to a control group correlates with the endometrial cells having an increased likelihood of becoming cancerous.   
     
     
         4 . The method of  claim 3 , wherein the antibody binds to an amino acid sequence as set forth in SEQ ID NO: 1. 
     
     
         5 . The method of  claim 1 , further comprising determining the frequency of detecting EMP2 in a sample and comparing the frequency of detection with multiple variables to generate multivariate models for the identification of variables demonstrating statistical significance for patient survival. 
     
     
         6 . The method of  claim 5 , wherein the multiple variables are ER, PR, vascular, stage, diagnosis, disease status, and survival status. 
     
     
         7 . The method of  claim 5 , wherein the multivariate model is disease free survival, which model demonstrates significance for stage, diagnosis, and the presence of EMP2. 
     
     
         8 . A method for monitoring the progression of endometrial premalignancy in a subject, comprising:
 determining the level of endothelial membrane protein 2 (EMP2) polypeptide or polynucleotide in an endometrial cell from an endometrial tissue sample obtained at a first time;   determining the level of EMP2 polypeptide or polynucleotide in an endometrial cell from an endometrial tissue sample obtained at a second time; and   comparing the levels of EMP2 polypeptide or polynucleotide in the endometrial cell at the first and second times, wherein increased levels of EMP2 polypeptide or polynucleotide at the second time relative to the first time correlates with progression of endometrial premalignancy to a cancerous stage.   
     
     
         9 . The method of  claim 8 , wherein the determining comprises performing Northern blotting, Western blotting, protein gel electrophoresis, immunoprecipitation, ELISA, PCR, RT-PCR, differential display, serial analysis of gene expression, array analysis, and/or immunohistochemistry. 
     
     
         10 . The method of  claim 9 , comprising determining the expression of EMP2 in endometrial cells by immunohistochemistry. 
     
     
         11 . The method of  claim 10 , wherein an anti-EMP2 antibody binds an amino acid sequence as set forth in SEQ ID NO: 1. 
     
     
         12 . The method of  claim 8 , wherein the subject is undergoing endometrial drug therapy at the premalignant stage or malignant stage. 
     
     
         13 . A method of monitoring the stage of endometrial cancer in a subject comprising:
 identifying a subject presenting endometrial cancer;   determining epithelial membrane protein 2 (EMP2) polypeptide or polynucleotide level in a sample of endometrial tissue from the subject to establish a baseline EMP2 level for the subject;   measuring EMP2 polypeptide or polynucleotide level in an endometrial tissue sample obtained from the same subject at subsequent time points; and   comparing the measured EMP2 polypeptide or polynucleotide level with the baseline EMP2 polypeptide or polynucleotide level, wherein an increase in measured EMP2 polypeptide or polynucleotide level in the subject versus baseline EMP2 polypeptide or polynucleotide level is associated with a cancer which is progressing, and wherein a decrease in measured EMP2 polypeptide or polynucleotide level versus baseline EMP2 polypeptide or polynucleotide level is associated with a cancer which is regressing or in remission.   
     
     
         14 . A method for screening for a candidate compound that affects endometrial premalignant phenotype comprising:
 culturing endometrial tissue or cells;   determining the level of endothelial membrane protein 2 (EMP2) polypeptide or polynucleotide in the cultured tissue or cells at a first time point;   contacting the cultured tissue or cells with a candidate compound;   determining the level EMP2 polypeptide or polynucleotide in the cultured tissue or cells subsequent to compound contact; and   comparing the polypeptide or polynucleotide levels of EMP2 before and after compound contact, wherein a change in the level of EMP2 polypeptide or polynucleotide after compound contact correlates with a compound induced alteration in the level of EMP2.   
     
     
         15 . The method of  claim 14 , wherein an increase in the level of EMP2 correlates with the onset of or progression of an endometrial premalignant cell phenotype. 
     
     
         16 . The method of  claim 14 , wherein a decrease in the level of EMP2 correlates with the regression of an endometrial premalignant phenotype. 
     
     
         17 . The method of  claim 16 , wherein the determining comprises performing Northern blotting, Western blotting, protein gel electrophoresis, immunoprecipitation, ELISA, PCR, RT-PCR, differential display, serial analysis of gene expression, array analysis, and/or immunohistochemistry. 
     
     
         18 . The method of  claim 17 , comprising determining the expression of EMP2 in endometrial tissue or cells by immunohistochemistry. 
     
     
         19 . The method of  claim 18 , comprising contacting the cultured endometrial tissue or cells with an anti-EMP2 antibody or antigen-binding fragment thereof;
 quantitating the binding of the antibody or fragment thereof to the cultured tissue or cells at a first time point;   contacting the cultured tissue or cells with a candidate compound;   quantitating the binding of the antibody or fragment thereof to the cultured tissue or cells subsequent to compound contact; and   comparing the amount of antibody binding before and after compound contact.   
     
     
         20 . The method of  claim 19 , wherein the anti-EMP2 antibody binds to a protein comprising an amino acid as set forth in SEQ ID NO: 1. 
     
     
         21 . The method of  claim 14 , wherein the endometrial tissue or cells are not suspected of having endometrial cancer. 
     
     
         22 . The method of  claim 14 , wherein the candidate compound is a modulator of a progesterone receptor DNA binding domain, NF-κB, a serum response element, or PPAR. 
     
     
         23 . The method of  claim 22 , wherein the candidate compound is an NF-κB inhibitor. 
     
     
         24 . The method of  claim 23 , wherein the compound is a heterocyclic compound represented by formula (I) or a pharmaceutically acceptable salt thereof: 
       
         
           
           
               
               
           
         
       
       wherein R 1  is a cycloalkyl group, a cycloalkyl group having a substituent(s), wherein when the cycloalkyl group is a cyclopropyl group said cyclopropyl group has a substituent(s), a cycloalkenyl group or a cycloalkenyl group having a substituent(s); each R 2  and R 3  is a hydrogen atom or an alkyl group; R 4  is an alkyl group, an alkyl group having a substituent(s), an alkenyl group, an alkenyl group having a substituent(s), a cycloalkyl group, a cycloalkyl group having a substituent(s), a cycloalkenyl group, a cycloalkenyl group having a substituent(s), an aryl group, an aryl group having a substituent(s), an aromatic heterocyclic group having at least one hetero-atom within a ring or an aromatic heterocyclic group having a substituent(s) and at least one hetero-atom within a ring; A is a heterocyclic ring or a heterocyclic ring having a substituent(s);
 B is an aromatic ring, an aromatic ring having a substituent(s), a heterocyclic ring or a heterocyclic ring having a substituent(s); n is an integer selected from 0 to 6; —Y— is an interatomic bond, —CO—, —CO—O—, —CO—NR 5 —, —CS—NR 6 , —SO—, —SO 2 —, wherein each of R 5  and R 6  respectively is a hydrogen atom or an alkyl group; wherein —X— is an interatomic bond, —O—, —O—CHR 7 —, —CHR 8 —O—, —O—CO—, —CO—O—, —O—CS—, —CS—O—, —S—, —SO—, —SO 2 —S—CHR 9 —, —CHR 10 —S—, —S—CO—, —CO—S—, —S—CS—, —CS—S—, —SO 2 —NR 11 —, —NR 12 —SO 2 —, —NR 13 —, —NR 14 —, —CHR 15 —, —CHR 16 , —NR 17 —, —CO—, —C(═NOR 18 )—, —C(═.CHR 19 )—, —CO—CHR 20 —, —CHR 21 , —CO—, —CO—NR 22 —, —NR 23 , —CO—, —CR 24 , R 25 —, —CHR 26 , —CHR 27 —, —CR 28 , ═CR 29 —, —O—CHR 30 , —CHR 31 —, wherein each of R 7 , R 8 , R 9 , R 10 , R 15 , R 16 , R 20 , R 21 , R 24 , R 28 , R 29 , R 30  and R 31  respectively is either of a hydrogen atom or an alkyl group; each of R 11 , R 12 , R 13 , R 14 , R 17 , R 18 , R 19 , R 22  and R 23  respectively is either of a hydrogen atom, an alkyl group or an acyl group; each of R 26  and R 27  respectively is either of a hydrogen atom, a hydroxy group or an alkyl group; and R 25  is a hydrogen atom, a hydroxy group, an alkyl group, an alkyl group having a substituent(s), a mercapto group, an alkoxy group, an alkylthio group, an acyloxy group, an amino group, an alkylamino group, an amino group substituted with an amino protective group, a carboxyl group, an alkoxycarbonyl group, an aminocarbonyl group, or a cyano group. 
 
     
     
         25 . The method of  claim 22 , wherein the candidate compound indirectly inhibits the serum response element. 
     
     
         26 . The method of  claim 25 , wherein the compound is: 
       
         
           
           
               
               
           
         
       
     
     
         27 . The method of  claim 22 , wherein the candidate compound is a PPAR antagonist of Formula (I) or (II), or pharmaceutically acceptable salts or solvates thereof, 
       
         
           
           
               
               
           
         
       
       where in Formula (I) X is O, S, or NH;
 and R is methyl, ethyl, n-propyl, i-propyl, cyclopropyl, n-butyl, phenyl, or —CH 2 OCH 3 , 
 
       
         
           
           
               
               
           
         
       
       and where in Formula (II) X is C or N;
 and R is methyl, ethyl, n-propyl, i-propyl, —CH 2 OCH 3 , or —CO 2 CH 3 . 
 
     
     
         28 . A method for molecular analysis of endometrial samples comprising:
 providing one or more endometrial samples, which samples include at least one control;   exposing the one or more samples to different biological reagents that react with one or more biological markers, wherein at least one of the markers is epithelial membrane protein 2 (EMP2);   performing one or more assays to detect one or more of the biological markers in the samples;   analyzing the assays to determine whether a reaction with a biological marker has occurred in the one or more samples,   wherein an increase in the amount EMP2 in a sample relative to the control correlates with endometrial cancer (EC).   
     
     
         29 . The method of  claim 28 , wherein the biological reagents are selected from the group consisting of antibodies or EMP2-binding fragments thereof, oligonucleotide probes, oligonucleotide primers, aptamers, polypeptides, peptides, ligands, hormones, lipids, carbohydrates, lectins, and small molecules. 
     
     
         30 . The method of  claim 28 , wherein assays comprise performing Northern blotting, Western blotting, protein gel electrophoresis, immunoprecipitation, ELISA, PCR, RT-PCR, differential display, serial analysis of gene expression, array analysis, and/or immunohistochemistry. 
     
     
         31 . The method of  claim 30 , wherein the assay is an array analysis comprising providing an array having a plurality of sections, each section comprising a plurality of endometrial samples, which samples include at least one control;
 exposing different sections to different biological reagents that react with one or more biological markers, wherein at least one of the markers is EMP2;   performing one or more assays to detect one or more of the biological markers in the samples;   obtaining images of the different sections after performing the one or more assays; and   analyzing the images to determine whether a reaction with a biological marker has occurred in the different samples.   
     
     
         32 . The method of  claim 28 , wherein the molecular analysis is an analysis of tissue, cellular, or subcellular distribution of the biological marker. 
     
     
         33 . The method of  claim 28 , wherein the plurality of endometrial samples are obtained from different subjects. 
     
     
         34 . The method of  claim 33 , further comprising annotating information about the subjects and associating that information with the results of the image analysis, thereby obtaining correlations between the information and observed reactions. 
     
     
         35 . The method of  claim 34 , wherein obtaining images comprises capturing digital images and storing the digital images. 
     
     
         36 . The method of  claim 35 , wherein analyzing the images further comprises quantifying the reaction between the reagent and the biological marker. 
     
     
         37 . The method of  claim 30 , wherein the assay is immunohistochemistry. 
     
     
         38 . The method of  claim 37 , further comprising exposing the samples to an antibody directed against SEQ ID NO: 1. 
     
     
         39 . A method of treating endometrial cancer in a subject in need thereof comprising administering a therapeutically effect amount of a pharmaceutical composition comprising a compound identified by the method of  claim 15 , wherein the composition inhibits EMP2 expression, thereby modulating the regulation of GPI-lipid rafts and/or caveolae. 
     
     
         40 . The method of  claim 39 , further comprising combining the administration of the pharmaceutical composition with a surgical modality. 
     
     
         41 . The method of  claim 39 , further comprising administering a nonestrogenic progesterone derivative. 
     
     
         42 . The method of  claim 41 , wherein the derivative is hydroxyprogesterone caproate, medroxyprogesterone acetate, or megestrol acetate. 
     
     
         43 . The method of  claim 39 , further comprising administering a cytotoxic chemotherapeutic modality. 
     
     
         44 . The method of  claim 43 , where the chemotherapeutic modality is cyclophosphamide, doxorubicin, cisplatin, or combinations thereof. 
     
     
         45 . The method of  claim 39 , wherein the modulation decreases surface expression of integrins. 
     
     
         46 . The method of  claim 39 , wherein the modulation decreases fibronectin binding. 
     
     
         47 . The method of  claim 39 , further comprises the administering of a ribozyme directed against EMP2 mRNA. 
     
     
         48 . A kit comprising:
 an agent which detects the level of epithelial membrane protein 2 (EMP2);   a container comprising an agent for detecting the level of EMP2 in a sample;   a control; and   instructions to provide guidance for carrying out an assay embodied by the kit and for making a determination of the level of EMP2 based upon that assay.   
     
     
         49 . The kit of  claim 48 , wherein the agent is an antibody or EMP2-binding fragment thereof, oligonucleotide probe, oligonucleotide primer, aptamer, polypeptide, peptide, ligand, hormone, lipid, carbohydrate, lectin, or small molecule. 
     
     
         50 . The method of  claim 49 , wherein the agent is an anti-EMP2 antibody or an antigen-binding fragment thereof. 
     
     
         51 . The kit of  claim 48 , wherein the antibody or antigen-binding fragment thereof is attached to a substrate, which substrate is applied to a sample from a patient or to a surface that may contain EMP2 and the surface of the substrate is then processed to assess whether specific binding occurs between the antibody and EMP2 or other component of the sample. 
     
     
         52 . The kit of  claim 51 , wherein the substrate is a dipstick. 
     
     
         53 . A method of treating endometrial cancer in a subject in need thereof comprising monitoring the level of EMP2 in combination with one or more therapeutic regimens. 
     
     
         54 . The method of  claim 53 , wherein the therapeutic regimen comprises a surgical modality. 
     
     
         55 . The method of  claim 53 , wherein the therapeutic regimen comprises administering a nonestrogenic progesterone derivative. 
     
     
         56 . The method of  claim 55 , wherein the derivative is hydroxyprogesterone caproate, medroxyprogesterone acetate, or megestrol acetate. 
     
     
         57 . The method of  claim 53 , further comprising administering a cytotoxic chemotherapeutic modality. 
     
     
         58 . The method of  claim 57 , where the chemotherapeutic modality is cyclophosphamide, doxorubicin, cisplatin, or combinations thereof. 
     
     
         59 . The method of  claim 57 , further comprising administering an antibody directed against EMP2.