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

Patent metadata

Field	Value
Publication number	US-11021755-B2
Application number	US-201815926494-A
Country	US
Kind code	B2
Filing date	Mar 20, 2018
Priority date	Mar 18, 2015
Publication date	Jun 1, 2021
Grant date	Jun 1, 2021

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.

First claim

What is claimed is:

1. A method for identifying or screening for an agent or a compound:
that inhibits or decreases the amount of metastasis or circulating tumor cells;
that reverses cancer cell acquired resistance to a drug; and/or
that inhibits, negatively affects or decreases anchorage independent growth of a cancer cell,
comprising:
(a) providing a test compound;
(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 comprising
a beta3-integrin (ITGB3) promoter operably linked to a nucleic acid encoding a reporter or a marker protein or marker compound,
wherein inhibiting activity of the beta3-integrin (ITGB3) promoter results in decreased expression and measurable levels of the nucleic acid encoding the reporter or marker protein or marker compound;
(c) measuring or determining the level of the nucleic acid encoding the reporter or marker protein or marker compound before adding the test compound, and
(d) 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 nucleic acid encoding the reporter or marker protein or marker compound,
wherein measuring or determining an increase in the level of the nucleic acid encoding the reporter or marker protein or marker compound, as compared to the level measured in step (c), indicates that the test compound is an beta3-integrin (ITGB3) promoter inducer,
and wherein measuring or determining a decrease in the level of the nucleic acid encoding the reporter or marker protein or marker compound, as compared to the level measured in step (c), indicates that the test compound is a beta3-integrin (ITGB3) promoter inhibitor,
and by measuring or determining a decrease in the level of the nucleic acid encoding the reporter or marker protein or marker compound, as compared to the level measured in step (c), indicates that the test compound can:
inhibit or decrease the amount of metastasis or circulating tumor cells; reverse cancer cell acquired resistance to a drug; and/or inhibit, negatively affect or decrease anchorage independent growth of a cancer cell.

2. The method of claim 1 , further comprising administering a negative control compound known to not affect beta3-integrin (ITGB3) promoter activity.

3. The method of claim 1 , 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.

4. The method of claim 1 , wherein 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.

5. The method of claim 1 , wherein the reporter or marker protein or compound comprises a bioluminescent or a fluorescent protein or compound, or a luciferase or a green fluorescent protein (GFP).

6. The method of claim 1 , wherein the nucleic acid construct or a chimeric or recombinant nucleic acid further comprises an ITGB3 enhancer.

7. The method of claim 1 , wherein the ITGB3 promoter is an ITGB3 distal promoter.

8. The method of claim 7 , wherein the ITGB3 distal promoter comprises a sequence as set forth in SEQ ID NO:2.

9. The method of claim 1 , wherein the beta3-integrin (ITGB3) promoter is constitutively active and the reporter nucleic acid, or the reporter or marker protein is constitutively expressed.

10. The method of claim 1 , wherein the beta3-integrin (ITGB3) promoter is inducibly expressed before addition of the test compound, resulting in inducible expression of the reporter nucleic acid, or the reporter or marker protein.

11. The method of claim 10 , wherein the beta3-integrin (ITGB3) promoter is inducibly expressed by addition of a chemical or a drug or a conditioned media.

12. The method of claim 11 , wherein the conditioned media is obtained from serum deprived cells or cancer cells.

13. The method of claim 1 , wherein the beta3-integrin (ITGB3) promoter is inducibly expressed by addition of erlotinib.

Assignees

• Univ California

Inventors

• Cheresh David
• Gozo Maricel
• Yebra Mayra
• Seguin Laetitia

Classifications

• G01N33/57557

  of other specific parts of the body, e.g. brain · CPC title

• G01N33/57555

  of the prostate · CPC title

• C07K14/70557

  Integrin beta3-subunit-containing molecules, e.g. CD41, CD51, CD61 · CPC title

• C12N15/00

  Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor (mutants or genetically engineered microorganisms, per se C12N1/00, C12N5/00, C12N7/00; new plants per se A01H; plant reproduction by tissue culture techniques A01H4/00; new animals per se A01K67/00; use of medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases, gene therapy A61K48/00) · CPC title

• C12Q2600/158

  Expression markers · CPC title