# US 8,465,912 B2 · Methods for the identification of agents that inhibit mesenchymal-like tumor cells or their formation

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- Number: US 8,465,912 B2
- Document ID: US8465912B2
- Status: Granted
- Kind: B2
- Application number: 12/660,444
- Filed: 2010-02-26
- Published: 2010-09-02
- Granted: 2013-06-18
- Earliest claimed: 2009-02-27
- Assignees: OSI Pharmaceuticals, Inc.
- Applicants: Julie Kan
- Inventors: Julie Kan
- Web page: https://patentgrep.com/patent/US8465912B2

## CPC

- C12N 5/0693: {Tumour cells; Cancer cells}
- A61P 35/00: Antineoplastic agents
- G01N 33/5011: {for testing antineoplastic activity}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

The present invention provides tumor cell preparations for use as models of the EMT process for use in the identification of anti-cancer agents, wherein said tumor cell preparations comprise cells of the epithelial tumor cell line H1650, which are stimulated by receptor ligands to induce EMT, or which have been engineered to inducibly express a protein that stimulates EMT. The present invention also provides methods of identifying potential anti-cancer agents by using such tumor cell preparations to identify agents that inhibit EMT, stimulate MET, or inhibit the growth of mesenchymal-like cells. Such agents should be particularly useful when used in conjunction with other anti-cancer drugs such as EGFR and IGF-1R kinase inhibitors, which appear to be less effective at inhibiting tumor cells that have undergone an EMT.

## Claims

### Claim 1

A method of identifying an agent that inhibits tumor cells from undergoing an epithelial to mesenchymal transition, comprising:
contacting a sample of cells of the epithelial tumor cell line H1650 with a test agent to be screened,
contacting the sample with a single or dual protein ligand preparation that induces an epithelial-to-mesenchymal transition in H1650 cells,
determining whether the test agent inhibits the tumor cells in the sample from undergoing an epithelial to mesenchymal transition, by comparing the level of a biomarker whose level is indicative of the EMT status of the sample tumor cells to the level of the same biomarker in an identical sample of identically treated H1650 cells not contacted with the test agent,
and thus determining whether the test agent is an agent that inhibits tumor cells from undergoing an epithelial to mesenchymal transition.

### Claim 2 (depends on 1)

The method of claim 1, wherein the single protein ligand that induces an epithelial-to-mesenchymal transition in H1650 cells is selected from any of the protein ligands that bind to and activate the EGF receptor or the HGF receptor.

### Claim 3 (depends on 2)

The method of claim 2, wherein the single protein ligand that induces an epithelial-to-mesenchymal transition in H1650 cells is selected from, EGF, TGF-α, HB-EGF, amphiregulin, betacellulin, epiregulin, epigen, and HGF.

### Claim 4 (depends on 3)

The method of claim 3, wherein the single protein ligand that induces an epithelial-to-mesenchymal transition in H1650 cells is selected from EGF and HGF.

### Claim 5 (depends on 4)

The method of claim 4, wherein the single protein ligand that induces an epithelial-to-mesenchymal transition in H1650 cells is EGF, optionally supplemented with TNF-α.

### Claim 6 (depends on 4)

The method of claim 4, wherein the single protein ligand that induces an epithelial-to-mesenchymal transition in H1650 cells is HGF, optionally supplemented with EGF, TGF-beta, TNF-α, or OSM.

### Claim 7 (depends on 1)

The method of claim 1, wherein the dual protein ligands that induce an epithelial-to-mesenchymal transition in H1650 cells are a protein ligand that binds to a receptor that activates the signal transduction pathways activated by the binding of oncostatin M to its receptor or a protein ligand that binds to a receptor that activates the signal transduction pathways activated by the binding of TGF-beta to its receptor, plus one ligand that binds to the TNFα receptor and activates the same signal transduction pathways that are activated by the binding of TNFα to its receptor.

### Claim 8 (depends on 7)

The method of claim 7, wherein the dual protein ligands that induce an epithelial-to-mesenchymal transition in H1650 cells are oncostatin-M plus TNFα, or TGF-beta plus TNFα.

### Claim 9 (depends on 7)

The method of claim 7, wherein TGF-beta is TGFbeta-1, TGFbeta-2, TGFbeta-3, or heterodimers thereof.

### Claim 10 (depends on 1)

The method of claim 1, wherein the biomarker whose level is indicative of the EMT status of the sample tumor cells is an epithelial cell biomarker.

### Claim 11 (depends on 10)

The method of claim 10, wherein the epithelial cell biomarker is E-cadherin, CDH1 promoter activity, cytokeratin 8, cytokeratin 18, P-cadherin or erbB3.

### Claim 12 (depends on 1)

The method of claim 1, wherein the biomarker whose level is indicative of the EMT status of the sample tumor cells is a mesenchymal cell biomarker.

### Claim 13 (depends on 12)

The method of claim 12, wherein the mesenchymal cell biomarker is vimentin, fibronectin, N-cadherin, CDH1 methylation, zeb1, twist, FOXC2 or snail.

### Claim 14

A method of identifying an agent that inhibits tumor cells that have undergone an epithelial to mesenchymal transition, comprising
contacting a sample of cells of the epithelial tumor cell line H1650 with a single or dual protein ligand preparation to induce an epithelial-to-mesenchymal transition in the H1650 cells,
contacting the sample of cells with a test agent to be screened,
determining whether the test agent inhibits mesenchymal-like H1650 cell growth,
and thus determining whether it is an agent that inhibits the growth of tumor cells that have undergone an epithelial to mesenchymal transition.

### Claim 15 (depends on 14)

The method of claim 14, wherein the single protein ligand that induces an epithelial-to-mesenchymal transition in H1650 cells is selected from any of the protein ligands that bind to and activate the EGF receptor or the HGF receptor.

### Claim 16 (depends on 15)

The method of claim 15, wherein the single protein ligand that induces an epithelial-to-mesenchymal transition in H1650 cells is selected from, EGF, TGF-α, HB-EGF, amphiregulin, betacellulin, epiregulin, epigen, and HGF.

### Claim 17 (depends on 16)

The method of claim 16, wherein the single protein ligand that induces an epithelial-to-mesenchymal transition in H1650 cells is selected from EGF and HGF.

### Claim 18 (depends on 17)

The method of claim 17, wherein the single protein ligand that induces an epithelial-to-mesenchymal transition in H1650 cells is EGF, optionally supplemented with TNF-α.

### Claim 19 (depends on 17)

The method of claim 17, wherein the single protein ligand that induces an epithelial-to-mesenchymal transition in H1650 cells is HGF, optionally supplemented with EGF, TGF-beta, TNF-α, or OSM.

### Claim 20 (depends on 14)

The method of claim 14, wherein the dual protein ligands that induce an epithelial-to-mesenchymal transition in H1650 cells are a protein ligand that binds to a receptor that activates the signal transduction pathways activated by the binding of oncostatin M to its receptor or a protein ligand that binds to a receptor that activates the signal transduction pathways activated by the binding of TGF-beta to its receptor, plus one ligand that binds to the TNFα receptor and activates the same signal transduction pathways that are activated by the binding of TNFα to its receptor.

### Claim 21 (depends on 20)

The method of claim 20, wherein the dual protein ligands that induce an epithelial-to-mesenchymal transition in H1650 cells are oncostatin-M plus TNFα, or TGF-beta plus TNFα.

### Claim 22 (depends on 19)

The method of claim 19, wherein TGF-beta is TGFbeta-1, TGFbeta-2, TGFbeta-3, or heterodimers thereof.

### Claim 23 (depends on 14)

The method of claim 14, comprising, after the step of
determining whether the test agent inhibits the growth of tumor cells that have undergone an epithelial to mesenchymal transition, the additional steps of
determining whether an agent that inhibits mesenchymal-like H1650 tumor cell growth, also inhibits epithelial H1650 tumor cell growth,
and thus determining whether it is an agent that specifically inhibits the growth of tumor cells that have undergone an epithelial to mesenchymal transition.

### Claim 24 (depends on 23)

The method of claim 23, wherein in the step of
determining whether the test agent inhibits mesenchymal-like H1650 tumor cell growth, it is determined that the test agent does so by stimulating apoptosis of said tumor cells.

### Claim 25 (depends on 23)

The method of claim 23, wherein in the step of
determining whether the test agent inhibits mesenchymal-like H1650 tumor cell growth, it is determined that the test agent does so by inhibiting proliferation of said tumor cells.

### Claim 26

A method of identifying an agent that stimulates mesenchymal-like tumor cells to undergo a mesenchymal to epithelial transition, comprising
contacting a sample of cells of the epithelial tumor cell line H1650 with a single or dual protein ligand preparation to induce an epithelial-to-mesenchymal transition in the H1650 cells,
contacting the sample of cells with a test agent to be screened,
determining whether the test agent stimulates the mesenchymal-like H1650 cells in the sample to undergo a mesenchymal to epithelial transition, by comparing the level of a biomarker whose level is indicative of the EMT status of the sample tumor cells to the level of the same biomarker in an identical sample of mesenchymal-like H1650 cells not contacted with the test agent,
and thus determining whether the test agent is an agent that stimulates mesenchymal-like tumor cells to undergo a mesenchymal to epithelial transition.

### Claim 27 (depends on 26)

The method of claim 26, wherein the single protein ligand that induces an epithelial-to-mesenchymal transition in H1650 cells is selected from any of the protein ligands that bind to and activate the EGF receptor or the HGF receptor.

### Claim 28 (depends on 27)

The method of claim 27, wherein the single protein ligand that induces an epithelial-to-mesenchymal transition in H1650 cells is selected from, EGF, TGF-α, HB-EGF, amphiregulin, betacellulin, epiregulin, epigen, and HGF.

### Claim 29 (depends on 28)

The method of claim 28, wherein the single protein ligand that induces an epithelial-to-mesenchymal transition in H1650 cells is selected from EGF and HGF.

### Claim 30 (depends on 29)

The method of claim 29, wherein the single protein ligand that induces an epithelial-to-mesenchymal transition in H1650 cells is EGF, optionally supplemented with TNF-α.

### Claim 31 (depends on 29)

The method of claim 29, wherein the single protein ligand that induces an epithelial-to-mesenchymal transition in H1650 cells is HGF, optionally supplemented with EGF, TGF-beta, TNF-α, or OSM.

### Claim 32 (depends on 26)

The method of claim 26, wherein the dual protein ligands that induce an epithelial-to-mesenchymal transition in H1650 cells are a protein ligand that binds to a receptor that activates the signal transduction pathways activated by the binding of oncostatin M to its receptor or a protein ligand that binds to a receptor that activates the signal transduction pathways activated by the binding of TGF-beta to its receptor, plus one ligand that binds to the TNFα receptor and activates the same signal transduction pathways that are activated by the binding of TNFα to its receptor.

### Claim 33 (depends on 32)

The method of claim 32, wherein the dual protein ligands that induce an epithelial-to-mesenchymal transition in H1650 cells are oncostatin-M plus TNFα, or TGF-beta plus TNFα.

### Claim 34 (depends on 32)

The method of claim 32, wherein TGF-beta is TGFbeta-1, TGFbeta-2, TGFbeta-3, or heterodimers thereof.

### Claim 35 (depends on 26)

The method of claim 26, wherein the biomarker whose level is indicative of the EMT status of the sample tumor cells is an epithelial cell biomarker.

### Claim 36 (depends on 35)

The method of claim 35, wherein the epithelial cell biomarker is E-cadherin, CDH1 promoter activity, cytokeratin 8, cytokeratin 18, P-cadherin or erbB3.

### Claim 37 (depends on 26)

The method of claim 26, wherein the biomarker whose level is indicative of the EMT status of the sample tumor cells is a mesenchymal cell biomarker.

### Claim 38 (depends on 37)

The method of claim 37, wherein the mesenchymal cell biomarker is vimentin, fibronectin, N-cadherin, CDH1 methylation, zeb1, twist, FOXC2 or snail.

### Claim 39

A method of preparing a composition comprising a chemical compound which inhibits the growth of tumor cells that have undergone an epithelial to mesenchymal transition, which comprises
contacting a sample of cells of the epithelial tumor cell line H1650 with a test agent to be screened,
contacting the sample with a single or dual protein ligand preparation that induces an epithelial-to-mesenchymal transition in H1650 cells,
determining whether the test agent inhibits the tumor cells in the sample from undergoing an epithelial to mesenchymal transition, by comparing the level of a biomarker whose level is indicative of the EMT status of the sample tumor cells to the level of the same biomarker in an identical sample of identically treated H1650 cells not contacted with the test agent,
and thus determining whether the test agent is an agent that inhibits tumor cells from undergoing an epithelial to mesenchymal transition,
and admixing the test agent so identified with a carrier, thereby preparing said composition.

### Claim 40

A method of preparing a composition comprising a chemical compound which inhibits the growth of tumor cells that have undergone an epithelial to mesenchymal transition, which comprises
contacting a sample of cells of the epithelial tumor cell line H1650 with a single or dual protein ligand preparation to induce an epithelial-to-mesenchymal transition in the H1650 cells,
contacting the sample of cells with a test agent to be screened,
determining whether the test agent inhibits mesenchymal-like H1650 cell growth,
and thus determining whether it is an agent that inhibits the growth of tumor cells that have undergone an epithelial to mesenchymal transition,
and admixing the test agent so identified with a carrier, thereby preparing said composition.

### Claim 41

A method of preparing a composition comprising a chemical compound which inhibits the growth of tumor cells that have undergone an epithelial to mesenchymal transition, which comprises
contacting a sample of cells of the epithelial tumor cell line H1650 with a single or dual protein ligand preparation to induce an epithelial-to-mesenchymal transition in the H1650 cells,
contacting the sample of cells with a test agent to be screened,
determining whether the test agent stimulates the mesenchymal-like H1650 cells in the sample to undergo a mesenchymal to epithelial transition, by comparing the level of a biomarker whose level is indicative of the EMT status of the sample tumor cells to the level of the same biomarker in an identical sample of mesenchymal-like H1650 cells not contacted with the test agent,
and thus determining whether the test agent is an agent that stimulates mesenchymal-like tumor cells to undergo a mesenchymal to epithelial transition,
and admixing the test agent so identified with a carrier, thereby preparing said composition.