Crk-a novel target for cancer therapy

Abstract

This disclosure identifies a novel apoptotic compensatory proliferation signaling complex (ASCS) involving ExoT's cellular target, Crk1. This disclosure demonstrates that in cells induced to undergo apoptosis, Crk1 forms globular complexes which associate with plasma membrane in the apoptotic cell and are released from the dying cell to its healthy neighbor, inducing it to proliferate. ExoT prevents the ACSC assembly while inducing apoptosis, indicating that apoptotic program cell death and compensatory proliferation are distinct and can be uncoupled from each other. Without being bound to any particular theory, it is believed that Crk1 functions as a scaffold for the ACSC assembly and that its ADP-ribosylation by ExoT interferes with this process while resulting in anoikis apoptotic cell death.

1 . A composition for identifying an anti-cancer therapeutic agent, wherein said therapeutic agent inhibits the assembly of an apoptotic compensatory signaling complex (ACSC), said composition comprising:
 a cell that expresses a protein selected from the group consisting of Crk protein, CrkI protein, CrkI/SH2(DN), CrkI/SH3(DN) and a combination thereof; and   an agent for detecting the assembly of ACSC.   
     
     
         2 . The composition of  claim 1  wherein the protein is Crk or CrkI. 
     
     
         3 . A method for inducing apoptosis in cancerous cells, said cancerous cells comprising a protein selected from the group consisting of Crk, CrkI, CrkI/SH2(DN), CrkI/SH3(DN) and a combination thereof as a scaffold for an apoptotic compensatory signaling complex (ACSC),
 said method comprising contacting said cancerous cells with Exotoxin T (ExoT).   
     
     
         4 . The method of  claim 3  wherein the protein is Crk or CrkI. 
     
     
         5 . (canceled) 
     
     
         6 . A method of inhibiting apoptotic compensatory signaling complex (ACSC) assembly while inducing apoptosis, the method comprising:
 exposing a cell population to exotoxin T (ExoT).   
     
     
         7 . The method of  claim 6  wherein the cell population comprises at least one protein selected from the group consisting of Crk, CrkI, CrkI/SH2(DN), CrkI/SH3(DN) and a combination thereof. 
     
     
         8 . The method of  claim 6  wherein the protein is Crk or CrkI. 
     
     
         9 . An assay for identifying a therapeutically active compound that inhibits apoptotic compensatory signaling complex (ACSC)-induced cell proliferation, the assay comprising:
 a cell population comprising a scaffold for the ACSC;   an apoptosis-inducing agent; and   a readout system for monitoring the cell population for a proliferation capacity index, or for apoptosis and a proliferation capacity index,   wherein a proliferation capacity index of the cell population exposed to a test compound that is less than a proliferation capacity index of a control is indicative of a therapeutically active compound.   
     
     
         10 . The assay of  claim 9 , wherein the scaffold comprises a protein selected from the group consisting of Crk, CrkI, CrkI/SH2(DN), CrkI/SH3(DN), and a combination thereof. 
     
     
         11 . The assay of  claim 10 , wherein the protein is Crk or CrkI. 
     
     
         12 . The assay of  claim 10 , wherein the protein is a recombinant expressed protein. 
     
     
         13 . The assay of  claim 10 , wherein the cells of said cell population comprise an expression vector comprising a nucleotide sequence that encodes the protein. 
     
     
         14 . The assay of  claim 9 , wherein the test compound induces apoptosis. 
     
     
         15 . The assay of  claim 9 , wherein readout system monitors the proliferation capacity index of the test compound relative to a proliferation capacity index of Exotoxin T to identify the therapeutically active compound. 
     
     
         16 . The assay of  claim 9 , wherein the readout system comprises a microscopic system. 
     
     
         17 . The assay of  claim 16 , wherein the microscopy system comprises video microscopy or fluorescence microscopy. 
     
     
         18 . The assay of  claim 9 , wherein the cell population comprise cancer cells. 
     
     
         19 . A method of identifying a therapeutically active compound that inhibits apoptotic compensatory signaling complex (ACSC)-induced cell proliferation, the method comprising:
 providing an in vitro cell population comprising a scaffold for the ACSC;   adding an apoptosis-inducing agent to the cell population;   culturing the cell population in the presence of the apoptosis-inducing agent;   measuring a proliferation capacity index of a portion of the cultured cell population exposed to a test compound;   measuring a proliferation capacity index of a portion the cultured cell population exposed to a control; and   wherein a decrease in the proliferation capacity index of the cell population exposed to the test compound, as compared to the proliferation capacity index of the cell population exposed to the control, is indicative of a therapeutically active compound.   
     
     
         20 . The method of  claim 19 , wherein the scaffold comprises a protein selected from the group consisting of Crk, CrkI, CrkI/SH2(DN), CrkI/SH3(DN), and a combination thereof. 
     
     
         21 . The method of  claim 20 , wherein the cells of said cell population comprise an expression vector comprising a nucleotide sequence that encodes the protein. 
     
     
         22 . The method of  claim 19 , wherein the test compound induces apoptosis. 
     
     
         23 . The method of  claim 19 , comprising monitoring the proliferation capacity index using microscopy.