New markers for the epithelial and proliferative or mesenchymal invasive phenotype of human neoplasias

Abstract

The present invention relates to a new Ena/VASP protein isoform, uses thereof, diagnostic methods and kits comprising the same.

1 . A method for predicting proliferative or invasive behaviour of a pre neoplastic lesion or of a neoplastic lesion comprising:
 detecting in vitro or in vivo expression of hMena11a and hMenaΔv6 splicing variants of hMena in a biological sample comprising cells of said lesion;   wherein detecting expression of hMena11a and not of hMenaΔv6 indicates a proliferative behaviour, whereas detecting expression of hMenaΔv6 and not of hMena11a indicates an invasive behaviour.   
     
     
         2 . The method according to  claim 1 , wherein said lesion is selected from the group consisting of: pancreatic, breast, colorectal, gastric, ovarian, lung, prostate, urothelial, head and neck, esophageal, and skin tumours including melanoma. 
     
     
         3 . The method according to  claim 1 , wherein said biological sample comprises at least: a specimen of a pre neoplastic or neoplastic lesion tissue, pre neoplastic or neoplastic lesion cells, or blood-free circulating neoplastic lesion cells. 
     
     
         4 . The method according to  claim 1 , wherein said detection of one or both said splicing variants is carried out by detection of a corresponding protein by immunohistochemistry. 
     
     
         5 . The method according to  claim 4 , wherein a polyclonal or a monoclonal antibody or an immunologically active fragment thereof specific for isoform 11a of the hMena protein is used for hMena11a detection, and/or a polyclonal or a monoclonal antibody or an immunologically active fragment thereof specific for isoform Δv6 of the hMena protein is used for hMenaΔv6 detection. 
     
     
         6 . The method according to  claim 5 , wherein said antibody or fragment thereof specific for the hMena11a isoform is specific for an epitope comprised in SEQ ID NO: 9. 
     
     
         7 . The method according to  claim 5 , wherein said antibody or fragment thereof specific for the hMenaΔv6 isoform is specific for an epitope comprised in SEQ ID NO: 2 or SEQ ID NO: 3. 
     
     
         8 . The method according to  claim 1 , wherein detection of one or both said splicing variants is carried out by detecting transcription products comprising SEQ ID NO: 8 or fragments thereof and transcription products comprising the junction between exon 5 and 7 of the hMena gene. 
     
     
         9 . The method according to  claim 8 , wherein said detection is carried out by hybridising a mRNA or cDNA obtained from said biological sample with an oligonucleotide comprising SEQ ID NO: 8 and/or its complementary sequence or a fragment thereof and with another oligonucleotide comprising from 797 to 807 of SEQ ID NO: 1 or and/or of their complementary nucleotides in the sequence complementary to SEQ ID NO: 1. 
     
     
         10 . The method according to  claim 8 , wherein said detection is carried out by PCR by amplifying a mRNA or cDNA obtained from said biological sample with a primer pair amplifying SEQ ID NO: 8 or a fragment thereof and with another primer pair amplifying a fragment of SEQ ID NO: 1 comprising nucleotides from 797 to 807 of SEQ ID NO: 1. 
     
     
         11 . A method for predicting proliferative or invasive behaviour of a pre neoplastic lesion or of a neoplastic lesion comprising:
 detecting in vitro or in vivo presence of antibodies specific for hMena11a and antibodies specific for hMenaΔv6 isoforms of hMena in a body fluid sample;   wherein detecting presence of antibodies specific for hMena11a and not of hMenaΔv6 indicates a proliferative behaviour, whereas detecting presence of antibodies specific for hMenaΔv6 and not of hMena11a indicates an invasive behaviour.   
     
     
         12 . The method according to  claim 11 , wherein said lesion is selected from the group consisting of: pancreatic, breast, colorectal, gastric, ovarian, lung, prostate, urothelial, head and neck, esophageal, and skin tumours including melanoma. 
     
     
         13 . The method according to  claim 11 , wherein said body fluid samples comprises at least: blood, serum, breast milk, peritoneal fluid, pleural fluid, pericardial fluid, or lymph. 
     
     
         14 . A kit for predicting proliferative or invasive behaviour of a pre neoplastic or neoplastic lesion comprising reagents for detecting expression of hMena11a and hMenaΔv6 splicing variants in a biological sample of said pre neoplastic or neoplastic lesion wherein said reagents are selected from the group consisting of: a) a polyclonal or a monoclonal antibody or an immunologically active fragment thereof specific for isoform 11a of the hMena protein and a polyclonal or a monoclonal antibody or an immunologically active fragment thereof specific for isoform Δv6 of the hMena protein; b) a labelled oligonucleotide comprising SEQ ID NO: 8 and/or its complementary sequence or a fragment thereof and another labelled oligonucleotide comprising from 797 to 807 of SEQ ID NO: 1 or and/or their complementary nucleotides in the sequence complementary to SEQ ID NO: 1; c) a primer pair amplifying SEQ ID NO: 8 or a fragment thereof and another primer pair amplifying a fragment of SEQ ID NO: 1 comprising from 797 to 807 of SEQ ID NO: 1; and d) peptides for detection of antibodies specific for hMena11a and antibodies specific for hMenaΔv6 isoforms of hMena. 
     
     
         15 . The kit according to  claim 14 , wherein said antibody or fragment thereof specific for the hMena11a isoform is specific for an epitope comprised in SEQ ID NO: 9 or an epitope specific for hMena11a isoform, and wherein said antibody or fragment thereof specifically bind a protein having SEQ ID NO: 2 or an epitope specific for hMenaΔv6 isoform or a peptide having SEQ ID NO: 3. 
     
     
         16 . The kit according to  claim 14 , further comprising at least one solid support for detection of expression of hMena11a and hMenaΔv6 splicing variants. 
     
     
         17 - 28 . (canceled) 
     
     
         29 . A molecule selected from the group consisting of:
 (i) a nucleic acid comprising a sequence set forth in SEQ ID NO: 1 coding for hMenaΔv6, a fragment thereof comprising from 797 to 807 of SEQ ID NO: 1, the complement of said sequence set forth in SEQ ID NO: 1 coding for hMenaΔv6, or the complement of said fragment;   (ii) a protein comprising a sequence set forth in SEQ ID NO: 2 coding for hMenaΔv6 or a fragment thereof comprising from 267 to 270 of SEQ ID NO: 2;   (iii) a polyclonal or monoclonal antibody or an immunologically active fragment thereof specifically binding a protein comprising SEQ ID NO: 2, an epitope specific for hMenaΔv6, or a peptide comprising SEQ ID NO: 3; and   (iv) an siRNA specific for SEQ ID NO: 1 or a fragment thereof comprising from 797 to 807 of SEQ ID NO: 1;   (v) an inhibitor of at least one or more of hMenaΔv6 expression, functionality, or oligomerisation; and   (vi) a candidate compound that inhibits invasive behaviour of a pre neoplastic or neoplastic lesion or cells thereof.   
     
     
         30 . A pharmaceutical composition for inhibiting invasiveness of a pre neoplastic or neoplastic lesion or cells thereof comprising a molecule of  claim 29  and at least a pharmaceutically acceptable carrier and/or excipient. 
     
     
         31 . A method for inhibiting invasive behaviour of a pre neoplastic or neoplastic lesion or cells thereof, said method comprising inhibiting at least expression, functionality, or oligomerisation of hMenaΔv6 with an inhibitor of  claim 29 . 
     
     
         32 . A method for screening of a candidate compound of  claim 29 , said method comprising contacting said compound with a cell or tissue expressing hMenaΔv6; wherein reduction in at least one or more of hMenaΔv6 expression, functionality, or oligomerisation is indicative that said compound might inhibit invasive behaviour of a pre neoplastic or neoplastic lesion or cells thereof.