Screening For Anti-Cancer Compounds Using Netrin-1 Activity

Abstract

The subject matter of the present invention relates to an in vitro method for the screening of anti-cancer compounds based on the capacity for these compound to interact with netrin-1 receptor and/or to inhibit the dimerization of the intracellular domain of the netrin-1 receptor expressed in tumor cells. The invention also relates to a method for predicting the presence of metastatic or aggressive cancer, or for determining the efficiency of an anti-cancer treatment based on the measuring of the expression level of netrin-1. The invention further comprises kits and compounds as a medicament for the treatment of cancer such as metastatic breast cancer, related to the overexpression of netrin-1 by the tumor cells.

1 . Method for selecting a compound for the prevention or the treatment of cancer, wherein said method comprises the following steps of:
 a) obtaining a medium containing netrin-1, or a fragment thereof, and a netrin-1 receptor, or a fragment thereof, wherein:
 said netrin-1, or a fragment thereof, and said netrin-1 receptor, or a fragment thereof, is able to specifically interact together to form a binding pair, and/or 
 said netrin-1, or a fragment thereof, is able to induce the dimerization or multimerization of said netrin-1 receptor, or a fragment thereof, particularly the intracellular domain of said netrin-1 receptor; 
   b) contacting said medium with the compound to be tested;   c) measuring the inhibition of the interaction between netrin-1, or a fragment thereof, and said netrin-1 receptor, or a fragment thereof, and/or
 determining whether said compound inhibit inhibits the dimerization or multimerization of said netrin-1 receptor, or a fragment thereof, particularly the dimerization of the intracellular domain of said netrin-1 receptor; and 
   d) selecting said compound if:
 the measuring in step c) demonstrates a significantly inhibition of the interaction between netrin-1, or a fragment thereof, and netrin-1 receptor, or a fragment thereof, in presence of said compound, and/or 
 the determination in step c) demonstrates a significantly inhibition of the dimerization or multimerization of said netrin-1 receptor, or a fragment thereof, in presence of said compound, particularly the dimerization of the intracellular domain of said netrin-1 receptor. 
   
     
     
         2 . The method according to  claim 1 , wherein said cancer to be prevented or treated is a cancer wherein tumoral cells express or overexpress netrin-1. 
     
     
         3 . The method according to  claim 1  or  2 , wherein said cancer to be prevented or treated is selected from the group consisting of breast cancer, colorectal cancer, lung cancer, neuroblastoma, glioma, acute myeloid leukemia, sarcoma, melanoma, ovanan adenocarcinoma, renal adenocarcinoma pancreatic adenocarcinoma, uterus adenocarcinoma, stomach adenocarcinoma, kidney adenocarcinoma and rectal adenocarcinoma. 
     
     
         4 . The method according to  claim 1 , wherein said cancer to be prevented or treated is a metastatic cancer or an aggressive cancer. 
     
     
         5 . The method according to  claim 1 , wherein at step a) said netrin-1 receptor is selected from the group consisting of DCC, UNC5H, neogenin and the adenosine A2b. 
     
     
         6 . The method according to  claim 5 , wherein at step a) said netrin-1 receptor is selected from the group of DCC, UNC5H1, UNC5H2 and UNC5H3. 
     
     
         7 . The method according to  claim 1 , wherein at step a):
 said netrin-1 receptor fragment comprises or is the intracellular domain of the netrin-1 receptor, or part thereof able to interact with netrin-1; and/or   said netrin-1 receptor fragment comprises or is the intracellular domain of the netrin-1 receptor, or part thereof able to dimerize or multimerize in presence of netrin-1.   
     
     
         8 . The method according to  claim 1 , wherein at step a) said netrin-1 or/and said netrin 1 receptor are from mammal, particularly from mouse, rat or human. 
     
     
         9 . The method according to  claim 1 , wherein at step a) said netrin-1 is from chicken. 
     
     
         10 . The method according to  claim 1 , wherein said netrin-1 or/and said netrin-1 receptor and/or the compound to be tested is labelled by a marker able to be directly or indirectly measured. 
     
     
         11 . The method according to  claim 1 , wherein at step c):
 the measuring of the inhibition of the interaction between netrin-1, or a fragment thereof, and said netrin-1 receptor, or a fragment thereof, is carried out by immunoassay   
       (particularly by ELISA or by Immunoradiometric Assay (IRMA)), by Scintillation Proximity Assay (SPA) or by Fluorescence Resonance Energy Transfer (FRET); and/or
 the dimerization or multimerization, or its inhibition, of said netrin-1 receptor, or fragment thereof, particularly the intracellular domain, is carried out by immunoprecipitation or FRET. 
 
     
     
         12 . The method according to  claim 1 , wherein at step a) said medium comprises cells which express at their surface membrane an endogenous netrin-1 receptor or a recombinant netrin-1 receptor, particularly at least the extracellular domain of a recombinant netrin-1 receptor. 
     
     
         13 . The method according to  claim 12 , wherein at step a) said medium comprises cells which express recombinant netrin-1 receptor. 
     
     
         14 . The method according to  claim 12 , wherein at step a) said medium comprises tumoral cells which express endogenously said netrin-1 receptor at their membrane surface and which express or overexpress netrin-1, and wherein at step c) the inhibition of the interaction between netrin-1 and its netrin-1 receptor in presence of the compound to be tested, is measured by the apoptosis or cells death induced by the presence of the compound to be tested. 
     
     
         15 . The method according to  claim 14 , wherein at step a) said medium comprises metastatic tumoral cells, particularly cells selected from the group consisting of 4T1 cells, CAL51 cells, T47D cells, SKBR7 cells, IMR32 cells, GL26 cells and H358 cells. 
     
     
         16 . A method according to  claim 1  for selecting a compound for the prevention or the treatment of cancer, wherein said method comprises the following steps of:
 a) obtaining a medium comprising a mammal cell expressing an endogenous or a recombinant netrin-1 receptor, or a fragment thereof comprising at least its intracellular domain, preferably a tumor cell, more preferably a cell presenting dimerization or multimerization of its netrin-1 receptor intracellular domain or a cell wherein its netrin-1 receptor intracellular domain is able to dimerize or multimerize in presence of netrin-1;   b) contacting said medium with the compound to be tested, optionally the medium further containing netrin-1, or a fragment thereof able to interact with the extracellular domain of the netrin-1 receptor;   c) determining whether the dimerization or multimerization of said netrin-1 receptor intracellular domain is inhibited in the presence of said compound to be tested;   d) optionally, determining whether the presence of the compound to be tested induces the cell death of said mammal cell; and   e) selecting said compound if the determination in step c) demonstrates a significant inhibition of the dimerization or multimerization of the intracellular domain of said netrin-1 receptor and/or if the determination in step d) demonstrates the cell death of said mammal cell.   
     
     
         17 . An in vitro method for predicting the presence of a metastatic or an aggressive cancer in a patient having a primary tumor from a biopsy of said patient containing primary tumors cells, said method comprising the following step of:
 (a) measuring the netrin-1 expression level in said biopsy.   
     
     
         18 . The method according to  claim 17 , wherein an increase of the netrin-1 expression level in said biopsy, compared with expression of netrin-1 in a non-metastatic primary tumor biopsies or in a non-aggressive cancer biopsies is significant of the presence of a metastatic cancer or an aggressive cancer. 
     
     
         19 . The method according to  claim 17 , wherein a ratio superior to 2 between netrin-1 expression in the biopsy to be tested and in the non-metastatic reference biopsy is significant of the presence of a metastatic cancer. 
     
     
         20 . A method for determining in vitro the efficiency of an anti-cancer treatment for a patient or for in vitro selecting patients who respond to a specific anti-cancer treatment, said method comprising the following steps of:
 (a) obtaining a primary tumor biopsy of said treated patient; and   (b) measuring of the netrin-1 expression level in said biopsy, wherein the efficiency of said anti-cancer treatment is correlated with the decrease of the amount of the netrin-1 expression level measured in said biopsy, or wherein the selected patients who respond to a specific anti-cancer treatment are patients where the amount of the netrin-1 expression level measured in their biopsy has been decreased after said specific treatment.   
     
     
         21 . A method according to  claim 20 , wherein said cancer induced an overexpression of netrin-1 and/or is a metastatic or an aggressive cancer. 
     
     
         22 . The method according to  claim 17 , wherein the measured netrin-1 expression product is the RNA encoding netrin-1, particularly measured by a quantitative real time reverse PCR method. 
     
     
         23 . The method according to  claim 17 , wherein the expression level of netrin-1 which is measured is the measuring of the netrin-1 protein level, particularly by a method using specific antibodies able to specifically recognize said netrin-1 protein. 
     
     
         24 . The method according to  claim 17 , wherein the primary tumor is a primary tumor of a cancer selected from the group consisting of breast cancer, colorectal cancer, lung cancer, neuroblastoma, glioma, acute myeloid leukemia, sarcoma, melanoma, ovarian adenocarcinoma, renal adenocarcinoma pancreatic adenocarcinoma, uterus 5 adenocarcinoma, stomach adenocarcinoma, kidney adenocarcinoma and rectal adenocarcinoma. 
     
     
         25 . Kit for the selection of a compound for the prevention or the treatment of cancer, wherein said kit comprises:
 a netrin-1 receptor protein, or a fragment thereof able to specifically interact with the netrin-1 protein to form a binding pair, preferably recombinant protein; and   netrin-1 protein, or a fragment thereof able to specifically interact with said netrin-1 receptor protein to form a binding pair, preferably recombinant protein.   
     
     
         26 . Kit for the selection of a compound for the prevention or the treatment of cancer, wherein said kit comprises:
 tumoral cells which express netrin-1 receptor and which express or overexpress netrin-1, particularly cells from tumoral cell line, preferably selected from the group consisting of 4T1 cells, CAL51 cells, T47D cells, SKBR7 cells, IMR32 cells, GL26 cells and H358 cells; and, optionally,   netrin-1 protein, or a fragment thereof able to specifically interact with said netrin-1 receptor protein to form a binding pair, preferably a recombinant netrin-1 protein.   
     
     
         27 . A compound selected from the group consisting of:
 a compound selected by the method of  claim 1 ;   a compound comprising an extracellular domain of netrin-1 receptor or fragment thereof able to specifically inhibit the interaction between the netrin-1 and said netrin-1 receptor, and/or able to inhibit the dimerization or multimerization of said netrin-1 receptor, or a fragment thereof, particularly the intracellular domain of said netrin-1 receptor; and   a monoclonal or polyclonal antibody directed specifically against netrin-1 or netrin-1 receptor, particularly directed to the extracellular domain of said netrin-1 receptor or to the netrin-1 fragment able to interact with the extracellular domain of said netrin-1 receptor, as a medicament.   
     
     
         28 . A compound according to  claim 27 , wherein said extracellular domain of netrin-1 receptor or fragment thereof is selected from the group of DCC, UNC5H, neogenin and the adenosine A2b. 
     
     
         29 . A compound according to  claim 27 , wherein said compound comprising an extracellular domain of netrin-1 receptor is selected from the group consisting of DCC, DCC-EC-Fc and DCC-5Fbn. 
     
     
         30 . Method of treatment for inducing the apoptosis or the cell death of tumor cells which have acquired the selective advantage to escape netrin-1 dependence receptors induced apoptosis, preferably by elevated netrin-1 level, in a patient comprising administering a compound able to inhibit this selective advantage in a tumor cell in said patient in need thereof. 
     
     
         31 . Method for the prevention or for the treatment of cancer in a patient comprising administering a compound according to  claim 27  in said patient in need thereof. 
     
     
         32 . (canceled) 
     
     
         33 . The method according to  claim 31 , wherein said cancer is a metastatic or an aggressive cancer. 
     
     
         34 . The method according to  claim 30 , wherein said cancer is selected from the group consisting of breast cancer, colorectal cancer, lung cancer, neuroblastoma, glioma, acute myeloid leukemia, sarcoma, melanoma, ovarian adenocarcinoma, renal adenocarcinoma pancreatic adenocarcinoma, uterus adenocarcinoma, stomach adenocarcinoma, kidney adenocarcinoma and rectal adenocarcinoma. 
     
     
         35 . The method or the use according to  claim 29 , wherein the primary tumor cells of said cancer express or overexpress netrin-1. 
     
     
         36 . A method, comprising the step of determining the level of netrin-1 expression as a marker for the identification of a metastatic or an aggressive cancer in a patient. 
     
     
         37 . The method according to  claim 36 , wherein said metastatic or aggressive cancer is selected from the group consisting of breast cancer, colorectal cancer, lung cancer, neuroblastoma, glioma, acute myeloid leukemia, sarcoma, melanoma, ovarian adenocarcinoma, renal adenocarcinoma pancreatic adenocarcinoma, uterus adenocarcinoma, stomach adenocarcinoma, kidney adenocarcinoma and rectal adenocarcinoma. 
     
     
         38 . The method according to  claim 36 , wherein said cancer is selected from breast or colorectal cancer.