OBJECTIVE: Glioblastoma Multiforme (GBM), one of the most malignant forms of primary brain tumors, is characterized by its highly heterogenous genetic composition, aggressive infiltration of surrounding tissue, and resistance to current treatments. Gene expression analysis has characterized GBM into four main types, with a significant portion belonging to the Classical subtype, typified by overexpression and/or mutation of the epidermal growth factor receptor (EGFR). Also common to this subtype of GBM is the loss of crucial tumor suppressor genes Ink4A/ARF and PTEN, which contribute to the invasive nature and unregulated proliferation that underlie the GBM pathology. The high rate of tumor recurrence post treatment with surgical resection, chemotherapy, and radiation has driven the pursuit of more effective molecularly targeted therapies. This study was undertaken to determine the effects of two types of small molecule tyrosine kinase inhibitors on cells overexpressing wild-type EGFR in the context of their respective complements of tumor suppressor genes.
METHODS: Several cell lines were established from mouse models of EGFR wild-type (EGFRWT) driven gliomagenesis and treated with 10 μM of type I tyrosine kinase inhibitors Gefitinib (Iressa®, Astra Zeneca), CI-1033 (Canertinib, Pfizer), or Dimethyl Sulfoxide vehicle. Cells were exposed to each drug treatment as part of a time course ranging from 0 to 24 hours and then evaluated by trypan blue exclusion and Western blot analysis for cell viability and molecular and biochemical effects respectively.
RESULTS: Evaluation of cell viability indicated that CI-1033 caused a greater increase in cell death than gefitinib when compared to control treated cells regardless of the tumor suppressors lost. Gefitinib was found to cause cell death only in cells expressing the PTEN tumor suppressor whereas CI-1033 showed similar levels of cell death for cells deficient in Ink4A/ARF or both Ink4A/ARF and PTEN tumor suppressors. Western blot analysis revealed that CI-1033 more effectively inhibited EGFR compared to gefitinib. Treatment with both gefitinib and CI-1033 effectively blocked phosphorylation of EGFR, but this effect was less pronounced with gefitinib treatment. Further analysis of downstream signaling molecules showed a greater presence of cleaved caspase 3, a hallmark of apoptosis, in gefitinib treated cells expressing PTEN than in those cells treated with CI-1033. Cells deficient in both Ink4A/ARF and PTEN did not demonstrate any induction of cleaved caspase 3 following either treatment.
CONCLUSIONS: Based on the significant differences in cell viability between treatments, CI-1033 is an overall more effective inhibitor of EGFRWT expressing cells lacking PTEN, while gefitinib and CI-1033 were found to be similarly effective in cells expressing PTEN. The results of western blot analysis indicate that total and irreversible EGFR inhibition may be necessary to induce cell death in a manner that effectively terminates downstream cell signaling. It is likely that CI-1033, unlike gefitinib, induces apoptosis in a caspase-independent manner, which may be one of the many differences in downstream effects produced by these two drugs. Further research is necessary to determine the extent to which each inhibitor shuts down proliferative cell signaling pathways such as PI3K-AKT and MEK-ERK signaling pathways downstream of EGFR. Overall, these data indicate that genotype plays an important role in the determination of therapeutic response and may aid in the evaluation of clinical prognoses.
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/16128 |
| Date | 08 April 2016 |
| Creators | Blazar, Ilyse Natasha |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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