Mig-6 Knockout Mice and Elucidation of Association of Mig-6 With Early Onset Degenerative Joint Disease and Role As A Tumor Suppressor

Abstract

The molecular mechanism underlying degenerative joint disease, also known as osteoarthritis (OA), is not fully understood. Disruption of mitogen inducible gene 6 (Mig-6) in mice by homologous recombination (KO mice) led to early onset OA as revealed by simultaneous enlargement and deformity of multiple joints, degradation of articular cartilage and the development of bony outgrowths or osteophytes within the joint space. The latter appeared to be derived from proliferation of mesenchymal progenitor cells followed by differentiation into chondrocytes. Because of the striking similarity to human OA, Mig-6 KO mice arc a useful animal model for studying the mechanism of this disease and for testing new drugs or therapies for treating OA. These KO mice also developed epithelial hyperplasia, adenoma, and adenocarcinoma in organs such as lung, gallbladder, and bile duct. Mig-6 is therefore a tumor suppressor gene and is a candidate gene for the frequent Ip36 genetic alterations found in lung cancer. It can be used as a tumor biomarker as well as a target for cancer therapy. Mig-6 is located in human chromosome Ip36, a locus frequently associated with human lung cancer. Mig-6 is a negative regulator of EGF signaling, and like EGF, was induced by HGF/SF in human lung cancer cell lines. Frequently the receptors EGFR and Met were co-expressed, and Mig-6 was induced by both EGF and HGF/SF in a MAPK-dependent fashion. Not all tumor lines express Mig-6 in response to either EGF or HGF/SF. In these cases, missense and nonsense mutations in the Mig-6 coding region were found, as was evidence for Mig-6 transcriptional silencing.

1 - 27 . (canceled) 
     
     
         28 . A method for detecting a structurally or functionally abnormal mig-6 gene in a subject, the method comprising detecting in a sample of cells, tissue or nucleic acid from said subject
 (a) the presence of a mutation in the coding sequence of the mig-6 gene;   (b) a decrease or absence of expression of the mig-6 gene;   (c) increased expression of the mig-6 gene secondary to downstream blockade in a signalling pathway in which Mig-6 is a participant;   (d) the presence of a mutation or decreased activity in a promoter of the mig-6 gene; or   (e) abnormal methylation of at least a part of the mig-6 gene thereby detecting a structurally or functionally abnormal mig-6 gene.   
     
     
         29 . The method of  claim 28  wherein the presence of an abnormal mig-6 gene indicates that the subject has increased susceptibility to the development of any disease or condition associated with decreased or absent mig-6 function, compared to a subject with a structurally or functional normal mig-6 gene. 
     
     
         30 . The method of  claim 29  wherein the presence of an abnormal mig-6 gene indicates that the subject has increased susceptibility to the development of osteoarthritis. 
     
     
         31 - 33 . (canceled) 
     
     
         34 . The method of claim  33  wherein the nucleic acid of said sample is subject to RT-PCR prior to said detecting. 
     
     
         35 . The method of  claim 34  wherein the RT-PCR is performed using the following primers: 
       
         
           
                 
                 
               
                     
                   [SEQ ID NO: 8] 
                 
                     
                   (a) forward prime 5′-TCTTCCACCGTTGCCAATC-S′; 
                 
             
                
                
               
            
           
         
         
           
                 
               
                   [SEQ ID NO: 9] 
                 
                   (b) reverse primer 5′TTCCACCTCACAGTCTGTGTCAT-S′; 
                 
                   and 
                 
                     
                 
                   [SEQ H) NO: 10] 
                 
                   (c) TaqMan Probe 5′-CTGAAGCCCTCTCTCT-3′. 
                 
             
                
                
                
                
                
                
               
            
           
         
       
     
     
         36 . The method of  claim 28 , wherein the subject is heterozygous for the mutant mig-6 gene. 
     
     
         37 . The method of  claim 28 , wherein the subject is homozygous for the mutant mig-6 gene. 
     
     
         38 . The method of  claim 28 , wherein expression of the mig-6 gene is detected using hybridization to a nucleic acid microarray. 
     
     
         39 . The method of  claim 28 , wherein expression of the mig-6 gene is detected by measuring the presence or quantity of the Mig-6 protein in the sample. 
     
     
         40 - 41 . (canceled) 
     
     
         42 . The method of  claim 28  wherein the subject is a human. 
     
     
         43 . A method for predicting increased susceptibility in a subject to the development of osteoarthritis comprising;
 sampling cells, tissue or nucleic acid from a subject;   analyzing said sample of cells, tissue or nucleic acids; and   detecting in said sample cells, tissue or nucleic acids the presence of a structurally or functionally abnormal mig-6 gene;   wherein the detection allows for the prediction of development of osteoarthritis in the subject.   
     
     
         44 . The method of  claim 43  wherein the detection of a mutation in the mig-6 gene from said sample indicates a structurally or functionally abnormal mig-6 gene. 
     
     
         45 . The method of  claim 43  wherein the detection of a decrease or absence of expression of the mig-6 gene from said sample indicates a structurally or functionally abnormal mig-6 gene. 
     
     
         46 . The method of  claim 43  wherein the detection of abnormal methylation of at least a part of the mig-6 gene from said sample indicates a structurally or functionally abnormal mig-6 gene. 
     
     
         47 . The method of  claim 43  wherein the detection of one or more of any combination of:
 a) a mutation in the mig-6 gene; 
 b) a decrease or absence of expression of the mig-6 gene; 
 c) abnormal methylation of at least a part of the mig-6 gene; in said sample is an indication of increased susceptibility of the patient to the development of osteoarthritis. 
 
     
     
         48 . The method of  claim 47  wherein said subject is a human. 
     
     
         49 . The method of  claim 43  wherein the nucleic acid of said sample is subject to RT-PCR during said analysis. 
     
     
         50 . The method of  claim 49  wherein the RT-PCR is performed using the following 
       
         
           
                 
                 
               
                     
                   [SEQ ID NO: 8] 
                 
                     
                   (a) forward prime 5′-TCTTCCACCGTTGCCAATC-S′; 
                 
             
                
                
               
            
           
         
         
           
                 
               
                   [SEQ ID NO: 9] 
                 
                   (b) reverse primer 5′-TTCCACCTCACAGTCTGTGTCAT-S′; 
                 
                   and 
                 
                     
                 
                   [SEQ H) NO: 10] 
                 
                   (c) TaqMan Probe 5′-CTGAAGCCCTCTCTCT-3′. 
                 
             
                
                
                
                
                
                
               
            
           
         
       
     
     
         51 . The method of  claim 44 , wherein the subject is heterozygous for the mutant mig-6 gene. 
     
     
         52 . The method of  claim 44 , wherein the subject is homozygous for the mutant mig-6 gene. 
     
     
         53 . The method of  claim 45 , wherein expression of the mig-6 gene is detected using hybridization to a nucleic acid microarray. 
     
     
         54 . The method of  claim 45 , wherein expression of the mig-6 gene is detected by measuring the presence or quantity of the Mig-6 protein in the sample.