- Title
- Regulation of apoptosis induced by targeting the RAF/MEK/ERK pathway in human melanoma
- Creator
- Lai, Fritz Shien Choong
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2014
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- Melanoma remains a major health issue in Australia due to its high morbidity and mortality rates. Although targeting survival signaling pathways, particularly BRAFv600E has achieved an unprecedented clinical response, tumour recurrences often occur due to development of resistance mechanisms. The mechanisms of melanoma drug resistance are often associated with reactivation of MEK/ERK signaling and crosstalk with the PI3K/Akt pathway. It has been reported that targeting oncogenic BRAF can inhibit cell proliferation, however recent evidence suggests that induction of programmed cell death is a major determinant of BRAFv600E melanoma responses to selective BRAF inhibitors. Therefore, multiple therapeutic approaches have incorporated targeting cell death pathways into current melanoma treatment regimens in hope of a better clinical outcome. The overall aim of this thesis is to investigate and identify potential strategies of overcoming BRAF inhibitor resistance in human melanoma through induction of cell death. Chapter Three demonstrates that exposure of BRAFv600E melanoma cell lines to the BRAF inhibitor PLX4720 for prolonged periods allows a proportion of resistant cells to grow, albeit with reduced proliferative rate. The continual growth of PLX-resistant cell lines is largely due to reactivation of ERK which is independent of MEK and CRAF but may be mediated by the PI3K/Akt pathway. LY294002, a PI3K inhibitor, blocked the rebound activation of ERK and subsequently induced apoptosis in the presence of PLX4720. Similarly, siRNA knockdown of Akt3 inhibited reactivation of ERK suggesting that cotargeting mutant BRAF and the PI3K/Akt pathway may overcome acquired BRAF inhibitor resistance of human melanomas by induction of apoptosis. To further understand the mechanisms of BRAF inhibitor resistance, cultures from paired pre- and post-treatment primary melanoma tissue biopsies were established. Studies in Chapter Four showed an increase in growth rates in the post-treatment samples despite the presence of apoptotic activity as evidenced by activation of caspase-3 and PARP cleavage. This was also associated with upregulation of Bims and SRp55 and a decrease in Mcl-1 and Bcl-2. Changes in activation of MEK, ERK and Akt between the paired pre- and post-treatment samples may contribute to acquired BRAF inhibitor resistance. Results in Chapter Four indicated that this phenomenon was mainly due to insufficient inhibition of ERK activation, leading to resistance against PLX4720-induced apoptosis. The findings in Chapter Five demonstrated that epigenetic alterations such as upregulation of histone deacetylases (HDACs), may act as mediators of melanoma resistance against therapy. This observation was confirmed upon targeting HDACs with the FDA-approved HDAC inhibitor SAHA in the presence of PLX4720 to induce synergistic killing of BRAFv600E melanomas. The combination of SAHA and PLX4720 also sensitised PLX-resistant melanoma cell lines and post-treatment patient biopsy cultures to induction of apoptosis suggesting that co-targeting HDACs and oncogenic BRAF may improve therapeutic outcome of patients receiving BRAF inhibitor monotherapy. Although activation of the caspase cascade typically mediates apoptotic signaling, induction of necrosis has been reported to be capable of executing death in a caspaseindependent manner. Chapter Six demonstrated that cell death induced by SAHA and PLX4720 was indeed due to necrosis as evidenced by early uptake of Annexin V and propidium iodide, release of HMGB1, plasma membrane rupture and loss of nuclear and cytoplasmic contents. However, induction of necrosis was independent of RIPK1 and RIPK3 suggesting that the combination of SAHA and PLX4720 can bypass the canonical necrotic pathways in order to kill BRAFv600E melanomas. Nevertheless, administration of SAHA and the clinically available BRAF inhibitor vemurafenib cooperatively inhibits melanoma xenograft growth in vivo suggesting that this combination therapy can potentially overcome BRAF inhibitor resistance in human melanoma.
- Subject
- melanoma; BRAF; HDAC; cell death; resistance; thesis by publication
- Identifier
- http://hdl.handle.net/1959.13/1051145
- Identifier
- uon:15254
- Rights
- Copyright 2014 Fritz Shien Choong Lai
- Language
- eng
- Full Text
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| Thumbnail | File | Description | Size | Format | |||
|---|---|---|---|---|---|---|---|
| View Details Download | ATTACHMENT01 | Abstract | 12 MB | Adobe Acrobat PDF | View Details Download | ||
| View Details Download | ATTACHMENT02 | Thesis | 22 MB | Adobe Acrobat PDF | View Details Download |