Method and compositions for inhibiting tumorigenesis

• US 20050112707A1
• Filed: 08/31/2004
• Published: 05/26/2005
• Est. Priority Date: 06/20/1997
• Status: Active Grant

• Current Assignee: New York University Medical Center (New York University)
• Original Assignee: New York University Medical Center (New York University)
• Inventors: Altaba, Ariel Ruiz i, Rom, William, Wong, Kam-Meng Tchou, Sanchez, Pilar
• Granted Patent: US 7,741,298 B2
• Time in Patent Office: Days
• US Class Current: 435/7.230
• CPC Class Codes: A61K 35/12 Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells vaccines or medicinal preparations containing antigens or antibodies A61K39/00
• CPC Class Codes: C12N 2501/11 Epidermal growth factor [EGF]
• CPC Class Codes: C12N 2501/41 Hedgehog proteins; Cyclopamine (inhibitor)
• CPC Class Codes: C12N 2506/08 from cells of the nervous system
• CPC Class Codes: C12N 5/0619 Neurons
• CPC Class Codes: G01N 2800/52 Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
• CPC Class Codes: G01N 33/5082 Supracellular entities, e.g. tissue, organisms
• CPC Class Codes: G01N 33/5743 of skin, e.g. melanoma

Abstract

The present invention relates to compounds, small interfering RNAs and compositions and methods of inhibiting tumorigenesis using agents that inhibit the sonic hedgehog and GLI signaling pathway, including agents that inhibit GLI expression, synthesis and/or function. The present invention also relates to particular biomarkers that can be used in the diagnosis and prognosis of cancer. Methods of treating cancer, including glioblastomas, medulloblastomas, basal cell carcinomas, prostate cancer and small cell and non-small cell lung cancer are also provided using small organic compounds, siRNAs and blocking antibodies that inhibit or block the SHH/GLI pathway.

Claims

1. A method of inducing a tumor cell to undergo apoptosis comprising administering an antagonist to the SHH/GLI signaling pathway in the cell, wherein said administering results in tumor cell death and prevention from metastasis.

2. The method of claim 1, wherein said tumor cell is selected from the group consisting of gliomas, medulloblastomas, primitive neuroectodermal tumors (PNETS), basal cell carcinoma, small cell lung cancers, large cell lung cancers, tumors of the gastrointestinal tract, rhabdomyosarcomas, soft tissue sarcomas, pancreatic tumors and prostate tumors.

14. The method of either of claims 1 or 4, wherein said antagonist comprises an agent that increases negative, or decreases positive acting elements affecting SHH-GLI signaling, said agents selected from the group consisting of small molecules that alter PKA kinase activity, small molecules that alter GSK3 kinase activity, small molecules that alter CK 1 kinase activity and small molecules that alter the activity of DYRK1.

18. The method of any one of claims 1-17, wherein said GLI molecule may be selected from the group consisting of GLI1, GLI2 and GLI3.

44. An antibody to the GLI protein of either one of claims 18 or 43.

45. The antibody of claim 44, which is a polyclonal antibody.

46. The antibody of claim 44, which is a monoclonal antibody.

47. An immortal cell line that produces a monoclonal antibody according to claim 46.

48. The antibody of either of claims 45 or 46, wherein said antibody is a blocking antibody and is effective as a therapeutic agent to prevent the proliferation of tumor cells in vivo.

49. A pharmaceutical composition comprising the antibody of either of claims 45 or 46 and a pharmaceutically acceptable carrier.

50. The antibody of claim 48, wherein said antibody is coupled to an anti-tumor drug or radioisotope for use in treating aggressive and highly invasive tumor cells in situ.

51. The antibody of either of claims 45 or 46, wherein said antibody is effective as a diagnostic agent to aid in identification of aggressive and highly invasive tumor cells in situ or in vivo.

52. The antibody of claim 51, wherein said antibody is coupled to an imaging reagent and used for in situ or in vivo imaging of aggressive and highly invasive tumor cells in situ or in vivo.

3. A method of screening for apoptotic resistant tumor cells, comprising measuring the level of expression of a GLI protein or a nucleic acid encoding said GLI protein in said tumor cells, wherein enhanced expression of said GLI protein or nucleic acid encoding said GLI protein correlates with apoptosis resistance in said tumor cells.

8. The method of either one of claims 3 or 7, wherein said GLI protein or nucleic acid encoding said GLI protein is selected from the group consisting of GLI1, GLI2 and GLI3.

4. A method of treating apoptotic resistant tumor cells in vivo or in vitro comprising administering an antagonist of the SHH/GLI signaling pathway to said tumor cells.

6. The method of either one of claims 4 or 5, wherein said administering results in making the apoptotic resistant tumor cells apoptotic sensitive.

10. The method of any one of claims 4-9, wherein said tumor cells are selected from the group consisting of gliomas, medulloblastomas, primitive neuroectodermal tumors (PNETS), basal cell carcinoma, small cell lung cancers, large cell lung cancers, tumors of the gastrointestinal tract, rhabdomyosarcomas, soft tissue sarcomas, pancreatic tumors and prostate tumors.

11. The method of either of claims 4 or 9, wherein said antagonist is selected from the group consisting of an antibody to GLI1, GLI2, GLI3, an siRNA molecule, an antisense nucleic acid, and a small organic molecule that antagonizes or inhibits the expression and/or activity of at least one of the GLI proteins and/or nucleic acid molecules encoding said at least one GLI protein.

12. The method of claim 11, wherein said siRNA molecule is selected from the group consisting of SEQ ID NOs:

29, 31 and 32.

13. The method of either of claims 4 or 9, wherein said antagonist is cyclopamine and derivatives and analogs thereof.

5. A method of treating a subject suffering from a cancerous condition, wherein said condition is characterized by the presence of apoptotic resistant tumor cells, comprising administering an antagonist of the SHH/GLI signaling pathway to said subject.

7. A method of predicting resistance of a tumor cell to chemotherapeutic agents, said method comprising measuring the level of expression of at least one GLI protein or nucleic acid encoding said at least one GLI protein in said tumor cell, whereby an increased level of expression of at least one GLI protein or nucleic acid encoding said at least one GLI protein in said tumor cell is predictive of resistance of said tumor cell to chemotherapeutic agents.

9. A method of overcoming resistance to chemotherapeutic agents in tumor cells, comprising administering an antagonist to the SHH/GLI signaling pathway to the cell, wherein said administering results in increased sensitivity of the tumor cell to said chemotherapeutic agent and results in subsequent tumor cell death and prevention from metastasis.

15. A method of treating a tumor in an animal subject, said method comprising administering an antagonist of the SHH/GLI signaling pathway, wherein said administering results in induction of apoptosis in said tumor, in increased sensitivity to chemotherapeutic agents, and in prevention of metastasis of said tumor.

16. The method of claim 15, wherein said tumor is selected from the group consisting of gliomas, medulloblastomas, primitive neuroectodermal tumors (PNETS), basal cell carcinoma, small cell lung cancers, large cell lung cancers, tumors of the gastrointestinal tract, rhabdomyosarcomas, soft tissue sarcomas, pancreatic tumors and prostate tumors.

17. The method of claim 15, wherein said antagonist is selected from the group consisting of an antibody to a GLI molecule, an siRNA molecule specific for a GLI molecule, an antisense nucleic acid specific for a GLI molecule, a small organic molecule that antagonizes the expression and/or activity of at least one GLI molecule, cyclopamine or analogs thereof, and any agent that inhibits the SHH/GLI signaling pathway.

19. The antagonist of claim 17, wherein said siRNA molecule is selected from the group consisting of SEQ ID NOs:

29, 31 and 32.

20. A method of identifying a subject that is likely to have an aggressive and highly invasive form of cancer comprising:

determining the level of GLI in a tissue specimen from said subject;

wherein when the level of GLI determined is significantly elevated in tumor cells relative to that determined in benign cells of the same specimen, the animal subject is identified as being likely to have an aggressive form of cancer.

21. The method of claim 20, wherein said cancer is selected from the group consisting of gliomas, medulloblastomas, primitive neuroectodermal tumors (PNETS), basal cell carcinoma, small cell lung cancers, large cell lung cancers, tumors of the gastrointestinal tract, rhabdomyosarcomas, soft tissue sarcomas, pancreatic tumors and prostate tumors.

22. The method of claim 20, wherein the determining is performed in situ or in vitro.

23. The method of claim 22, wherein said tissue specimen is selected from the group consisting of tumor tissue, normal benign tissue collected from the same site as the tumor tissue, whole blood, serum, plasma, urine and cerebrospinal fluid.

24. The method of claim 22, wherein the determining is performed by a method that uses a probe selected from the group consisting of an antibody specific for GLI;

a set of primers specific for an mRNA encoding GLI; and

a nucleotide probe specific for an mRNA encoding GLI.

25. A method of identifying an animal subject that is likely to have a slow growing form of cancer comprising detecting low GLI levels in a tumor obtained from a tumor tissue sample from the subject;

wherein when the GLI is barely detectable in the tumor tissue sample from the subject, and is comparable to the levels detected in the benign cells of the same specimen, the subject is identified as being likely to have a slow growing and non-invasive cancer.

28. The method of claim 25, wherein said determining is performed in situ or in vitro.

29. The method of claim 28, wherein said determining is performed by a method that uses a probe selected from the group consisting of an antibody specific for GLI;

a set of primers specific for an mRNA encoding GLI; and

a nucleotide probe specific for an mRNA encoding GLI.

30. The method of claim 25, further comprising determining the level of GLI in a tissue sample from the animal subject;

wherein when the level of GLI determined is up-regulated by not greater than 50% in tumor cells relative to that determined in benign cells of the same specimen, the animal subject is identified as being likely to have a slow growing cancer.

26. A method of identifying an animal subject that is likely to have a slow growing form of lung cancer comprising detecting low GLI levels in a sample of tumorous lung tissue obtained from the subject;

wherein when the GLI is barely detectable in the sample of tumorous lung tissue from the subject and is comparable to the levels detected in a sample obtained from non-cancerous lung tissue from a normal subject known to be free of lung cancer, the subject is identified as being likely to have a slow growing and non-invasive lung cancer.

27. The method of claim 26, wherein said sample of tumorous lung tissue is selected from the group consisting of lung tissue obtained from a subject suspected of having small cell lung cancer and a subject suspected of having non-small cell lung cancer.

31. An isolated siRNA molecule selected from the group consisting of SEQ ID NOs:

29, 31 and 32, wherein said siRNA molecule inhibits the proliferation of tumor cells.

32. A pharmaceutical composition comprising the isolated siRNA molecule of claim 31, wherein said composition is effective in treating tumor cells that are highly invasive and aggressive lung tumor cells.

33. An isolated nucleic acid probe specific for GLI;

wherein said probe is used to identify GLI in a tumor sample, and wherein said probe can differentiate between highly invasive and aggressive tumors and benign tumors in a patient sample.

34. The isolated nucleic acid probe of claim 33, wherein the GLI is a mammalian GLI.

35. The GLI of claim 34, wherein said mammalian GLI is human GLI.

36. A recombinant DNA molecule that is operatively linked to an expression control sequence;

wherein the recombinant DNA molecule comprises a nucleic acid that encodes human GLI.

37. An expression vector containing the recombinant DNA molecule of claim 36.

38. A cell that has been transformed with the expression vector of claim 37, wherein said GLI protein is expressed by the cell.

39. The cell of claim 38, which is a mammalian cell.

40. A method of expressing a recombinant GLI protein in a cell containing the expression vector of claim 37 comprising culturing the cell in an appropriate cell culture medium under conditions that provide for expression of recombinant GLI by the cell.

41. The method of claim 40, further comprising the step of purifying the recombinant GLI.

42. The purified form of the recombinant GLI of claim 41.

43. An antigenic or proteolytic fragment of the GLI of claim 42.