Kinases found in signalling pathways leading to growth and proliferation can become mutated leading to aberrant cell cycle progression and tumourigenesis. Inhibitors of these oncogenic kinases have already shown clinical effect, with imatinib, an inhibitor of the BCRlAbl fusion kinase found in chronic myeloid leukaemia producing excellent clinical responses. The molecular basis behind this effect has been shown to be due to induction of intrinsic apoptosis, through upregulation of the pro-apoptotic BH3-only proteins of the Bcl-2 family. Our aim is to investigate the effect of kinase inhibition on cells with other oncogenic kinases, including two novel kinases. Malignancies that express oncogenic kinases include mutant epidermal growth factor receptor (EGFR) in non-small cell lung cancer (NSCLC) and B-Raf in colon cancer and melanoma. Currently it is not well understood how signalling pathways link the induction of apoptosis through the upregulation of BH3-only genes. To address this, the key signalling pathways regulated after kinase inhibition were studied, focussing on the ERK, AKT, p38 an JNK pathways and a comprehensive study of the levels of all the BH3-only proteins was carried out. Inhibition of MEK1 alone kills B-Raf mutant melanoma and colon cancer cells, whereas B- Raf wildtype cells were more resistant, with the induction of Bim and Bmf correlating with mutant status. Inhibition of mutant EGFR in NSCLC with Iressa resulted in cell death, with Bim and Puma becoming induced. The combination of MEK and AKT inhibition of EGFR mutant NSCLC cells did not result in appreciable cell death, although Bim was induced, indicating that a decrease of pro-survival members of the Bcl-2 family by another pathway is also needed to induce cell death. In addition, the signalling pathways evoked by two novel fusion kinases CD? 4-ROS and SLC34A2-ROS were also investigated as additional potential oncogenic kinases. By utilising a variety of techniques, including flow cytometry with various anti-CD? 4 antibodies, immunofluoresence using antibodies against c-ROS and cell surface biotinylation assays, a lack of cell surface expression was revealed, contrary to earlier predictions. Furthermore, neither c-ROS fusion protein appeared to confer robust pro-survival signalling, although they were able to stimulate ERK activation and ROS kinase inhibition did not induce apoptosis. Finally, CD?4-ROS was unable to transform Ba/F3 or NIH-3T3 cells, suggesting these two fusion kinases are not a good candidate to target in therapeutic strategies. Together, these studies further our knowledge of oncogenic kinases and indicate that signals downstream of these kinases are complex, being both cell and context dependent. Not only induction of the pro-apoptotic BH3-only proteins is required to induce cell death but also a decrease in the pro-survival members, plus release from any sequestration. We anticipate that the knowledge gained will help to develop future therapies, suggesting combination therapies will be the most effective.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:580534 |
| Date | January 2012 |
| Creators | Harris, Claire Louise |
| Publisher | University of Southampton |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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