Assays for screening for or identifying an agent or molecule that can block or inhibit AVB3 integrin from forming a complex with KRAS
In alternative embodiments, the invention provides compositions and methods for overcoming or diminishing or preventing Growth Factor Inhibitor resistance in a cell, or, a method for increasing the growth-inhibiting effectiveness of a Growth Factor inhibitor on a cell, or, a method for re-sensitizing a cell to a Growth Factor Inhibitor, comprising for example, administration of a combination of a TBK1 inhibitor and an RTK inhibitor. In alternative embodiments, the cell is a tumor cell, a cancer cell or a dysfunctional cell. In alternative embodiments, the invention provides compositions and methods for determining: whether an individual or a patient would benefit from or respond to administration of a Growth Factor Inhibitor, or, which individuals or patients would benefit from a combinatorial approach comprising administration of a combination of: at least one growth factor and at least one compound, composition or formulation used to practice a method of the invention, such as an NFKB inhibitor, such as a lenalidomide or a REVLIMID™, or IKK inhibitor; or an inhibitor of Galectin-3.
1. A method or assay for screening for and/or identifying an agent or molecule that can block or inhibit integrin αvβ3 protein from forming a complex with a KRAS polypeptide on a cell or a tumorsphere surface, or for
screening for an agent or molecule that can block or inhibit integrin αvβ3 protein/KRAS polypeptide complex formation by its ability to block or inhibit the binding of Galectin-3 protein to integrin αvβ3 protein,
wherein the method or assay consists or
(a) providing a test agent or a test molecule;
(b) providing a Galectin-3 protein and a cell or a tumorsphere expressing an integrin αvβ3 protein and the KRAS polypeptide ;
(c) mixing or combining the test agent or molecule with the Galectin-3 protein and the cell under conditions wherein the Galectin-3 protein and the integrin αvβ3 protein are capable of binding to each other;
(d) determining or measuring if the test agent or molecule inhibits or blocks the binding of the Galectin-3 protein to the integrin αvβ3 protein, and
(e) determining or measuring if the test agent or molecule inhibits or blocks the integrin αvβ3 protein from forming a complex with the KRAS polypeptide on the cell or tumorsphere surface.
2. The method or assay of claim 1 , wherein the screening is in vitro or in vivo.
3. The method or assay of claim 1 , wherein the determining or measuring comprises use of a positive and a negative controls .
4. The method or assay of claim 1 , wherein the Galectin-3 protein is a synthetic protein, a recombinant protein and/or an isolated protein.
5. The method or assay of claim 1 , wherein for determining or measuring if the test agent or molecule inhibits or blocks the binding of the Galectin-3 protein to the integrin αvβ3 protein, the Galectin-3 protein or the integrin αvβ3 protein is immobilized on a surface.
6. The method or assay of claim 5 , wherein the amount of Galectin-3 protein bound to the immobilized integrin αvβ3 protein is measured or quantified using a fluorescent anti-Galectin-3 antibody or Galectin-3 protein binding fragment thereof.
7. The method or assay of claim 1 , wherein the test agent or the test molecule comprises a small molecule, a protein, a peptide, an antibody, a nucleic acid, a sugar or a polysaccharide, a fatty acid or a fat.
8. The method or assay of claim 3 , wherein use of a positive and a negative control comprises comparing the amount of inhibiting or blocking of the Galectin-3 protein to the integrin αvβ3 protein in the presence of the test agent or molecule to the amount of inhibiting or blocking of the Galectin-3 protein to the integrin αvβ3 protein in the absence of the test agent or molecule as negative control, and/or in the presence of known inhibitor or blocker of the binding of the Galectin-3 protein to the integrin αvβ3 protein as positive control,
and optionally the positive and/or negative control assays are done under conditions identical to the test assay with the test agent or molecule.
9. The method or assay of claim 5 , wherein the surface is a gel, a glass, a membrane, or a plastic.
10. The method or assay of claim 5 , wherein the Galectin-3 protein or the integrin αvβ3 protein is immobilized on a plate surface.
11. The method or assay of claim 5 , wherein the Galectin-3 protein or the integrin αvβ3 protein is immobilized on a well of a surface.
12. The method or assay of claim 6 , wherein the amount of Galectin-3 protein bound to the immobilized integrin αvβ3 protein is measured or quantified as fluorescent anti-Galectin-3 protein immunoreactivity.
13. The method of claim 1 , wherein the KRAS polypeptide is an oncogenic or an active wild-type KRAS polypeptide.
14. The method of claim 1 , wherein the cell is a cell line.
15. The method of claim 14 , wherein the cell line is a pancreatic or a lung cell line.
16. The method of claim 14 , wherein the cell line is a tumor cell line.
17. The method of claim 1 , wherein the cell is a tumor cell.
18. The method of claim 17 , wherein the tumor cell is an epithelial tumor cell, a lung tumor cell or a pancreatic tumor cell.
19. The method of claim 1 , wherein determining or measuring if the test agent or molecule inhibits or blocks the integrin αvβ3 protein from forming a complex with the KRAS polypeptide on the cell or tumorsphere surface comprises the determining or measuring if the integrin αvβ3 protein co-precipitates in a complex with the KRAS polypeptide.
20. The method of claim 1 , wherein determining or measuring if the test agent or molecule inhibits or blocks the integrin αvβ3 protein from forming a complex with the KRAS polypeptide on the cell or tumorsphere surface comprises the determining or measuring if the integrin αvβ3 protein and the KRAS polypeptide co-localize to a membrane cluster in the cell or tumorsphere surface.
21. The method of claim 17 , wherein determining or measuring if the test agent or molecule inhibits or blocks the integrin αvβ3 protein from forming a complex with the KRAS polypeptide on the tumor cell surface comprises the determining or measuring if tumorsphere formation by the tumor cell is inhibited.
22. The method of claim 1 , wherein the determining or measuring if the test agent or molecule inhibits or blocks the integrin αvβ3 protein from forming a complex with the KRAS polypeptide on the cell or tumorsphere surface comprises culturing or growing the cell under anchorage-independent growth conditions.