Compositions and methods for identifying anti-cancer, anti-metastatic and anti-stress agents

Abstract

In alternative embodiments, provided are products of manufacture, such as assays, chimeric nucleic acids and nucleic acid constructs, recombinant cells, and methods, comprising use of beta3-integrin (ITGB3) promoters operatively linked to a reporter, for drug screening, and in particular, screening for agents that inhibit cancer cell survival and metastasis. In alternative embodiments, compositions and methods as provided herein also can be used to identifying novel pathways that lead to acquired resistance, stemness, and anchorage independent growth; and characterizing distinct populations of cancer cells within a tumor microenvironment.

What is claimed is: 
     
         1 . A nucleic acid construct, or a chimeric or recombinant nucleic acid, comprising:
 (a) a beta3-integrin (ITGB3) promoter, or a functional subsequence thereof; and   (b) a reporter nucleic acid, or a nucleic acid encoding a reporter or a marker protein or compound,   wherein optionally the reporter or marker protein or compound is a bioluminescent or a fluorescent protein or compound, or a luciferase or a green fluorescent protein (GFP),   and optionally the nucleic acid construct or a chimeric or recombinant nucleic acid further comprises an ITGB3 distal promoter and/or enhancer, or SEQ ID NO:2, or a subsequence of SEQ ID NO:2 ( FIG. 5 ) that can function as an ITGB3 distal promoter and/or enhancer.   
     
     
         2 . The nucleic acid construct, or a chimeric or a recombinant nucleic acid of  claim 1 , wherein the beta3-integrin (ITGB3) promoter, or a functional subsequence thereof comprises all, substantially all or a portion of a human beta3-integrin (ITGB3) promoter, or a functional subsequence thereof. 
     
     
         3 . The nucleic acid construct, or a chimeric or a recombinant nucleic acid of  claim 1 , further comprising, or being contained in, an expression cassette, a vector, a plasmid, a phagemid or an artificial chromosome. 
     
     
         4 . An expression cassette, a vector, a plasmid, a phagemid or an artificial chromosome comprising or having contained within a nucleic acid construct, or a chimeric or a recombinant nucleic acid of  claim 1 . 
     
     
         5 . A recombinant or an engineered cell or cell line comprising a nucleic acid construct, or a chimeric or a recombinant nucleic acid of  claim 1 ,
 wherein optionally the cell is a beta3-integrin negative cell, or a cell unable to express a functional amount of a homologous beta3-integrin, and optionally the beta3-integrin negative cell is a beta3-integrin knockout cell,   and optionally the nucleic acid construct or the chimeric or recombinant nucleic acid, or the expression cassette, vector, plasmid, phagemid or artificial chromosome are stably integrated into the cell's chromosome, or are stably episomally expressed,   and optionally the cell is a cancer cell or a cancer cell line, or a carcinoma cell line or an immortalized cell line.   
     
     
         6 . A non-human transgenic organism comprising the recombinant or the engineered cell of  claim 5 . 
     
     
         7 . A method for identifying or screening for an agent or a compound:
 which inhibits beta3-integrin (ITGB3) promoter-driven integrin beta-3 (ITGB3) expression and phenotypes resulting from ITGB3 upregulation,   that inhibits, negatively affects or decreases cancer cell survival;   that inhibits or decreases the amount of metastasis or circulating tumor cells, or patient derived xenografts (PDX);   that reverses cancer cell acquired resistance to a drug;   that inhibits Slug activation, tumorsphere formation and tumor initiation in human breast cancer cells;   that reverses stemness; and/or   that inhibits, negatively affects or decreases anchorage independent growth of a cancer cell,   comprising:   (a) providing a test compound,   wherein optionally the test compound comprises or is a biologic, a drug, a small molecule or a small molecule drug, a bio-molecule, a protein, a lipid, a polysaccharide, or a nucleic acid;   (b) providing a recombinant or an engineered cell, or a cell free expression system, comprising a nucleic acid construct, or a chimeric or recombinant nucleic acid of  claim 1 ,   wherein the beta3-integrin (ITGB3) promoter or the functional subsequence thereof expresses the reporter nucleic acid, or the reporter or marker protein,   and optionally the beta3-integrin (ITGB3) promoter or the functional subsequence thereof is constitutively active and the reporter nucleic acid, or the reporter or marker protein is constitutively expressed,   and optionally the beta3-integrin (ITGB3) promoter or the functional subsequence thereof is inducibly expressed resulting in inducible expression of the reporter nucleic acid, or the reporter or marker protein,   and optionally the beta3-integrin (ITGB3) promoter or the functional subsequence thereof is inducibly expressed by addition of a chemical or a drug, such as an erlotinib, or a conditioned media, such as a conditioned media from serum deprived cells or cancer cells;   (c) administering to or contacting the test compound with the recombinant or engineered cell, or to the cell free expression system, and measuring or determining the level of the reporter nucleic acid, or the reporter or marker protein,   wherein measuring or determining an increase in the level of the reporter nucleic acid, or the reporter or marker protein indicates that the test compound is an beta3-integrin (ITGB3) promoter inducer,   and wherein measuring or determining a decrease in the level of the reporter nucleic acid, or the reporter or marker protein indicates that the test compound is a beta3-integrin (ITGB3) promoter inhibitor, and optionally also is a compound that can:
 inhibit, negatively affect or decrease cancer cell survival; 
 inhibit or decrease the amount of metastasis; 
 reverse cancer cell acquired resistance to a drug; 
 reverse stemness; and/or 
 inhibit, negatively affect or decrease anchorage independent growth of a cancer cell. 
   
     
     
         8 . The method of  claim 7 , further comprising administering a negative control compound known to not affect beta3-integrin (ITGB3) promoter activity. 
     
     
         9 . A recombinant or an engineered cell or cell line comprising an expression cassette, a vector, a plasmid, a phagemid or an artificial chromosome of  claim 4 ,
 wherein optionally the cell is a beta3-integrin negative cell, or a cell unable to express a functional amount of a homologous beta3-integrin, and optionally the beta3-integrin negative cell is a beta3-integrin knockout cell,   and optionally the nucleic acid construct or the chimeric or recombinant nucleic acid, or the expression cassette, vector, plasmid, phagemid or artificial chromosome are stably integrated into the cell's chromosome, or are stably episomally expressed,   and optionally the cell is a cancer cell or a cancer cell line, or a carcinoma cell line or an immortalized cell line.   
     
     
         10 . A non-human transgenic organism comprising the recombinant or the engineered cell of  claim 9 . 
     
     
         11 . The method of  claim 7 , wherein the nucleic acid construct, or a chimeric or recombinant nucleic acid comprises or is contained in an expression cassette, a vector, a plasmid, a phagemid or an artificial chromosome.