Tyrosine-phosphorylated wbp2, a novel cancer target and biomarker

Abstract

WW-binding protein 2 (WBP2) has been demonstrated in different studies to be a tyrosine kinase substrate, to activate ERα/PR transcription and to play a role in breast cancer. However, the role of WBP2 tyrosine phosphorylation in regulating ER function and breast cancer biology is unknown. Here, we established WBP2 as a tyrosine phosphorylation target of estrogen signaling via EGFR crosstalk. Using dominant negative, constitutively active mutants, RNAi and pharmacological studies, we demonstrated that phosphorylation of WBP2 at Tyr192 and Tyr231 could be regulated by c-Src and c-Yes kinases. We further showed that abrogating WBP2 phosphorylation impaired >60% of ERα reporter activity putatively by blocking nuclear entry of WBP2 and its interaction with ERα. Compared to vector control, overexpression of WBP2 and its phospho-mimic mutant in MCF7 resulted in larger tumors in mice, induced loss of cell-cell adhesion, enhanced cell proliferation, anchorage-independent growth, migration and invasion in both estrogen-dependent and -independent manner, events of which could be substantially abolished by overexpression of phosphorylation-defective mutant. Wnt/3-catenin inhibitor FH535 blocked phospho-WBP2-mediated cancer cell growth more pronouncedly than tamoxifen and fulvestrant, in part by reducing the expression of ERα.

What is claimed is: 
     
         1 . A method of treating a patient afflicted with cancer, comprising administering to the patient an agent that interferes with phosphorylation of tyrosine Y192 and/or Y231 in the polypeptide of SEQ ID NO: 1. 
     
     
         2 . The method of  claim 1 , wherein the agent comprises an isolated phosphorylation site-specific antibody that specifically binds to a WW-domain binding protein of SEQ ID: NO. 1 only when the polypeptide is phosphorylated at tyrosine Y192, at tyrosine Y231, or at tyrosine Y192 and tyrosine Y231, wherein the antibody does not bind the polypeptide of SEQ ID No: 1 when it is not phosphorylated at said tyrosine. 
     
     
         3 . The method of  claim 2 , wherein the isolated phosphorylation site-specific antibody is an immunoglobulin comprising an immunoglobulin heavy chain and/or an immunoglobulin light chain. 
     
     
         4 . The method of  claim 3 , wherein the immunoglobulin is an IgG1 kappa immunoglobulin. 
     
     
         5 . The method of  claim 4 , wherein the IgG1 kappa immunoglobulin comprises a human IgG1 constant region within a heavy chain of the immunoglobulin and a human constant region within a light chain of the immunoglobulin. 
     
     
         6 . The method of  claim 3 , wherein the immunoglobulin comprises fully or partially human framework regions within the heavy chain and within the light chain. 
     
     
         7 . The method of  claim 3 , wherein the immunoglobulin comprises murine framework regions within the heavy chain and within the light chain. 
     
     
         8 . The method of  claim 1 , wherein the agent comprises a peptide capable of binding to (a) a portion of SEQ ID NO: 1 that includes phosphorylated tyrosine Y192, phosphorylated tyrosine Y231, or phosphorylated tyrosine Y192 and phosphorylated tyrosine Y231 and (b) amino acids flanking either side of phosphorylated tyrosine Y192, phosphorylated tyrosine Y231, or phosphorylated tyrosine Y192 and phosphorylated tyrosine Y231. 
     
     
         9 . The method of  claim 8 , wherein the peptide comprises (a) SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6 and (b) a nuclear localization signal. 
     
     
         10 . The method of  claim 1 , wherein the agent comprises an isolated phosphorylation site-specific aptamer that specifically binds to a WW-domain binding protein of SEQ ID NO: 1 only when the polypeptide is phosphorylated at tyrosine Y192, or phosphorylated at tyrosine Y192 and phosphorylated at tyrosine Y231, wherein the aptamers does not bind the polypeptide of SEQ ID NO: 1 when it is not respectively phosphorylated at tyrosine Y192, or phosphorylated at tyrosine Y192 and phosphorylated at tyrosine Y231. 
     
     
         11 . The method of  claim 1 , wherein the agent comprises a small interfering RNA. 
     
     
         12 . The method of  claim 11 , wherein the small interfering RNA comprises SEQ ID NO: 2. 
     
     
         13 . The method of  claim 1 , wherein the agent comprises an anti-WNT pathway compound, an anti-EGFR compound, an anti-SRC compound, an anti-YES compound, an anti-EF2 compound, an anti-WBP2 compound or an anti-ER compound. 
     
     
         14 . The method of  claim 13 , wherein the agent comprises FH535 of formula 1 
       
         
           
           
               
               
           
         
       
     
     
         15 . The method of  claim 13 , wherein the agent comprises fulvestrant of formula 2 
       
         
           
           
               
               
           
         
       
     
     
         16 . A method of treating a patient afflicted with cancer, comprising administering to the patient a composition comprising an agent that interferes with phosphorylation of tyrosine Y192 and/or Y231 in the polypeptide of SEQ ID NO: 1 and an anti-estrogen receptor compound. 
     
     
         17 . The method of  claim 16 , wherein the anti-estrogen receptor compound is an FH535 of formula 1 
       
         
           
           
               
               
           
         
       
     
     
         18 . The method of  claim 16 , wherein the anti-estrogen receptor compound is fulvestrant of formula 2