Use of nucleolin as a biomarker for lymphangiogenesis in cancer prognosis and therapy

Abstract

The present invention relates to a method of identifying cancer subjects, in particular human patients, who are suitable for anti-lymphangiogenesis therapy to prevent tumor growth and tumor metastasis. The present invention also relates to a new approach, which uses nucleolin as a bait to search and screen for lymphangiogenesis inhibitors or cancer suppressors, which function in a manner that is analogous to endostatin. The invention is based upon the discovery that nucleolin is specifically expressed on lymphangiogenic vessels and functions as a specific receptor for endostatin, and thus is involved in the signal transduction pathway of endostatin as an anti-lymphangiogenesis inhibitor. The present invention also discloses that cell surface nucleolin on lymphatic endothelial cells is a biomarker for lymphangiogenic vessels, which could be used for the prediction of tumor metastasis.

1 . A method of determining the likelihood of lymphatic metastasis in a subject with cancer, comprising the step of determining the presence of cell surface nucleolin on lymphatic vessels in a cancer tissue sample or a tissue sample from lymph node adjacent to the cancer tissue from said subject, wherein the presence of cell surface nucleolin on lymphatic vessels in the sample is indicative of higher likelihood of lymphatic metastasis in said subject. 
     
     
         2 . A method of determining the prognosis of a subject with cancer, comprising the step of determining the presence of cell surface nucleolin on lymphatic vessels in a cancer tissue sample or a tissue sample from lymph node adjacent to the cancer tissue from said subject, wherein the presence of cell surface nucleolin on lymphatic vessels in the sample is indicative of an unfavorable prognosis of said subject. 
     
     
         3 . A method of determining the susceptibility of a cancer in a subject to endostatin therapy, comprising the step of determining the presence of cell surface nucleolin on lymphatic vessels in a cancer tissue sample or a tissue sample from lymph node adjacent to the cancer tissue from said subject, wherein the presence of cell surface nucleolin on lymphatic vessels in the sample is indicative of a higher susceptibility of the cancer to endostatin therapy. 
     
     
         4 . A method of screening a nucleolin-specific lymphangiogenesis inhibitor, comprising the steps of applying an appropriate binding assay to a pool of candidate molecules, thereby obtaining a plurality of nucleolin-specific molecules that specifically interact with cell surface nucleolin; testing each of the plurality of nucleolin-specific molecules for its effectiveness of inhibiting lymphangiogenesis by carrying out an anti-lymphangiogenesis assay; and selecting a nucleolin-specific molecule which is effective in inhibiting lymphangiogenesis as demonstrated by the anti-lymphangiogenesis assay. 
     
     
         5 . A method of treating a cancer subject having or suspected to have lymphatic metastasis, comprising the steps of determining the presence of cell surface nucleolin on lymphatic vessels in a cancer tissue sample or a tissue sample from lymph node adjacent to the cancer tissue from said subject, and administering a nucleolin-specific anti-lymphangiogenic agent to a subject who is determined as positive for the presence of cell surface nucleolin on lymphatic vessels in the sample. 
     
     
         6 . A method of preventing lymphatic metastasis in a cancer subject, comprising the steps of determining the presence of cell surface nucleolin on lymphatic vessels in a cancer tissue sample or a tissue sample from lymph node adjacent to the cancer tissue from said subject, and administering a nucleolin-specific anti-lymphangiogenic agent to a subject who is determined as positive for the presence of cell surface nucleolin on lymphatic vessels in the sample. 
     
     
         7 . A method of preventing or inhibiting the proliferation and/or migration of lymphatic endothelial cells in a cancer subject, comprising the steps of determining the presence of cell surface nucleolin on lymphatic vessels in a cancer tissue sample or a tissue sample from lymph node adjacent to the cancer tissue from said subject, and administering a nucleolin-specific anti-lymphangiogenic agent to a subject who is determined as positive for the presence of cell surface nucleolin on lymphatic vessels in the sample. 
     
     
         8 . The method of  claim 5 , wherein the nucleolin-specific anti-lymphangiogenic agent is endostatin or a conjugate comprising endostatin. 
     
     
         9 . The method of  claim 8 , wherein the conjugate is endostatin covalently linked to a modifier capable of enhancing its half-life and/or biological activity. 
     
     
         10 . The method of  claim 9 , wherein the modifier is PEG molecule. 
     
     
         11 . The method of  claim 10 , wherein the PEG molecule is covalently linked to the N-terminal a amino group of endostatin. 
     
     
         12 . The method of  claim 11 , wherein the PEG molecule has a molecular weight of 20-40 kD. 
     
     
         13 . The method of  claim 8 , wherein the conjugate is endostatin linked to a cytotoxic agent. 
     
     
         14 . The method of  claim 5 , wherein the nucleolin-specific anti-lymphangiogenic agent comprises a nucleolin-specific molecule selected by the method of  claim 4 . 
     
     
         15 . The method of  claim 5 , wherein the nucleolin-specific anti-lymphangiogenic agent comprises an anti-nucleolin antibody. 
     
     
         16 . The method of  claim 15 , wherein the anti-nucleolin antibody is linked to a cytotoxic agent. 
     
     
         17 . The method of  claim 1 , wherein the subject is a mammal. 
     
     
         18 . The method of  claim 17 , wherein the subject is a human. 
     
     
         19 . The method of  claim 1 , wherein the step of determining the presence of cell surface nucleolin on lymphatic vessels is performed by carrying out an immunoassay. 
     
     
         20 . The method of  claim 19 , wherein the immunoassay is performed with an antibody that specifically binds to nucleolin. 
     
     
         21 . The method of  claim 20 , wherein the antibody is a monoclonal antibody or antigen binding fragment thereof. 
     
     
         22 . The method of  claim 20 , wherein the antibody is polyclonal antibody. 
     
     
         23 . A method of selecting an lymphangiogenesis inhibitor having the ability to inhibit lymphatic endothelial proliferation and/or migration when added to proliferating lymphatic endothelial cells in vitro, comprising the steps of performing a suitable assay to identify and obtain a nucleolin-specific molecule that specifically interact with surface nucleolin on lymphatic endothelial cells; testing the nucleolin-specific molecule thus obtained for its effectiveness in inhibiting lymphatic endothelial cell proliferation and/or migration; and selecting a nucleolin-specific molecule which is effective in inhibition of lymphatic endothelial cell proliferation and/or migration.