Figure 1.
Kinase array analysis demonstrates high levels of phosphorylated EGFR, ERBB2, CREB and p53.
020913 and 060919 GBM oncospheres demonstrated activation of multiple receptor tyrosine kinases (A and C), and non-receptor tyrosine kinases (B and D). The RTKs that were phosphorylated (A and C) are numbered as follows: 1. p-EGFR 2. p-MER 3. p-TIE2 4. p-ERBB2 5. p-ERBB3 6. p-MSP R 7. p-ERBB4 8. p-FGFR3 9. p-FGFR4 10. p-M-CSF R 11. p-Insulin R 12. p-c-RET 13. p-EPHA1 14. p-IGF-IR 15. p-EPHA2 16. p-ROR2 17. p-EPHA3 18. p-DLK 19.p-TIE1 20. p-EPHA4 21. p-ROR1. Similarly, non receptor tyrosine kinases (B and D) that were phosphorylated are numbered as follows: 1. p-TOR (S2448) 2. p-SRC (Y419) 3. p-p38α (T180/Y182) 4. p-MEK1/2 (S218/S222, S222/S226) 5. p-CREB (S133) 6. p- LYN (Y397) 7. p-YES (Y426) 8. p-CHK-2 (T68) 9. p-ERK1/2 (T202/Y204, T185/Y187) 10. p-MSK1/2 (S376/S360) 11. p-HSP27 (S78/S82) 12. p-FGR (Y412) 13. p-JNK pan (T183/Y185, T221/Y223) 14. p-AMPKα1 (T174) 15. p-AMPKα2 (T172) 16. p-STAT2 (Y689) 17. p-STAT3 (Y705) 18. p-STAT6 (Y641) 19. p-GSK-3α/β (S21/S9) 20. p-AKT (Y473) 21. p-β-CATENIN 22. p- STAT5b (Y699) 23. p-STAT5a/b (Y699) 24. p- Akt (T308) 25. p-p70 S6 kinase (T421/S424) 26. p-p70 S6 kinase (T229) 27. p-STAT1 (Y701) 28. p-p53 (S392) 29. p-p53 (S46) 30. p-p53 (S15) 31. p-RSK1/2/3 (S380) 32. p-RSK1/2 (S221) 33. p-STAT4 (Y693) 34. p-p27 (T157) 35. p-c-JUN (S63) 36. p-eNOS (S1177) 37. p-PAXILLIN (Y118) 38. p-PLCγ (Y783) 39. p-PYK2 (Y402). Figure 1E: Schematic representation of activation of multiple tyrosine kinases in GBM oncosphere lines. Only the kinases with a known role in cell proliferation and/or transcription have been depicted.
Figure 2.
Drug combinations consisting of multi tyrosine kinase inhibitor sunitinib works best in reducing growth and inducing apoptosis in GBM oncospheres.
Fold change in proliferation is shown for 020913 (A) and 060919 (B) cells when treated with FDA-approved RTK inhibitors at 25% of their IC50 concentration. Combination of gefitinib (5 µM) and sunitinib (10 µM) demonstrate synergism in inhibiting cell growth. Other combinations of gefitinib and imatinib (15 µM), sunitinib and sorafenib (1 µM), and imatinib and sunitinib also showed increased growth inhibition of GBM cells. C and D: Caspase 3/7 assay demonstrating that sunitinib alone induces caspase 3/7 expression in 020913 cells (C) and 060919 cells (D), whereas treatment with Gefitinib, Imatinib and Sorafenib did not show caspase 3/7 release. Also, combinations containing sunitinib did not demonstrate an increased caspase release.
Figure 3.
Oncospheres treated with sunitinib and gefitinib show no regrowth after treatment. 020913 cells (A) and 060919 cells (B) were treated for 24 hours with RTK inhibitors as single agents or in combination at 25% of their IC50 concentration.
The drug was withdrawn after 24 hours and the cells were allowed to grow for 2 weeks. Treated cells were analyzed for their growth kinetics using alamar blue assay. GBM cells treated with the RTK combination consisting of gefitinib and sunitinib were unable to re-grow, whereas GBM cells treated with other drugs or combinations survived and re-grew. Analysis of neurosphere formation ability demonstrated that 020913 cells treated with gefitinib (C) or sunitinib (D) or other combinations like sunitinib and sorafenib (F) could form neurospheres after withdrawal of the drug, whereas cells treated with gefitinib and sunitinib (E) could not form any neurospheres.
Figure 4.
Kinase inhibition after treatment with RTK inhibitors.
A. Combining receptor tyrosine kinase inhibitors suppress downstream effectors of growth factor signaling. p-STAT3 was blocked only by the combination treatment, whereas p-AKT was blocked by all the drugs either as single agents or in combinations. The combinations of gefitinib and sunitinib as well as sunitinib and sorafenib were able to inhibit p-AKT, p-MAPK and p-STAT3 in both the GBM oncosphere lines.
Figure 5.
A: Mice implanted intracranially with 020913 GBM oncosphere cells were treated with gefitinib (75 mg/kg), sunitinib (15 mg/kg) and a combination of gefitinib (75 mg/kg) and sunitinib (15 mg/kg
). Gefitinib alone could significantly (p = 0.0001) improve survival in the animals compared to control animals. Sunitinib did not show any efficacy either when used alone (p = 0.13, compared to control) or when combined with gefitinib (p = 0.18, compared to gefitinib alone). Figure 5B: Rats were implanted intracranially with 9L tumor 1 mm3 tumor pieces. Rats were then treated with gefitinib (50 mg/kg), sunitinib (8 mg/kg) and a combination of gefitinib (50 mg/kg) and sunitinib (8 mg/kg). None of the drugs including the combination of gefitinib and sunitinib showed any efficacy (p = 0.9).