Research Doctorate - Doctor of Philosophy (PhD) / Melanoma is a skin cancer that remains a major public health problem in Australia because of its high incidence and the high morbidity and mortality associated with the disease. Melanoma has proven largely resistant to many chemotherapeutic and biological agents. Hope for a new approach in treatment of melanoma comes from the identification of the mechanisms employed in induction of apoptosis by ER stress and the possible resistance mechanisms in melanoma cells against ER stress-induced apoptosis. At the beginning of this study, little was known about the effects of ER stress on melanoma. The aim of this thesis was to elucidate the mechanisms of ER stress-induced apoptosis, the interaction between ER stress pathways and other signalling pathways in melanoma, thus to provide more information in identification of treatment approaches that will increase the sensitivity of melanoma to apoptosis induced by ER stress. Studies in Chapter 3 show that most melanoma cells are relatively resistant to ER stress-induced apoptosis except one cell line Me1007. However, inhibition of the MEK/ERK sensitizes melanoma cells to ER stress-induced apoptosis. This is mediated, at least in part, by caspase-4 activation and is associated with inhibition of the ER chaperone GRP78 expression. Moreover, inhibition of the MEK/ERK pathway reduces the level of GRP78 expression as well as its up-regulation by ER stress. Therefore, when the MEK/ERK is inhibited, caspase-4 is released from its complex with GRP78 and activated to mediated apoptosis. Chapter 4 demonstrates that up-regulation of the anti-apoptotic Bcl-2 family member Mcl-1 is one of the mechanisms critical for protection of melanoma cells against ER stress-induced apoptosis. Inhibition of Mcl-1 by siRNA renders melanoma cells sensitive to apoptosis induced by the ER stress inducers Thapsigargin (TG) or Tunicamycin (TM) mediated by PUMA and Noxa. ER stress up-regulates the BH3-only proteins PUMA and Noxa, but not Bim and BIK in melanoma cells, through transcriptional mechanisms, but the increase of Noxa but not PUMA is dependent on p53. Up-regulation of Mcl-1 is also due to increased transcription that involved the IRE1α and ATF6 signaling pathways of the unfolded protein response. In addition, activation of the MEK/ERK signaling pathway appears to be necessary for optimal up-regulation of Mcl-1. Melanoma cells are largely unresponsive to chemotherapy-induced apoptosis. Activation of the Unfolded Protein Response (UPR) by ER stress has profound effects on the sensitivity of melanoma cells to clinically relevant chemotherapeutic drugs and those in development for clinical use. In Chapter 5, the DNA-damaging drugs Cisplatin and Adriamycin, and the histone deacetylase inhibitors Suberic Bishydroxamate (SBHA) and Sodium Butyrate (NaB) further activate the UPR, indicative of induction of ER stress. The MEK inhibitors U0126 and AZD6244 reduce GRP78 expression levels; however, microtubule-targeting drugs Vincristine and Docetaxel do not change the GRP78 level. Knockdown of the IREα and ATF6 pathway of the UPR, and GRP78 by siRNA results in increased sensitivity of melanoma cells to these compounds. Studies in Chapter 6 show that treatment with either Tunicamycin (TM) or Thapsigargin (TG) selectively up-regulates TRAIL-R2 expression and enhances TRAIL-induced apoptosis in melanoma cells. This appears to be cooperatively mediated by the ATF6 and IRE1α signaling pathways and GADD153/CHOP. However, although siRNA knockdown of ATF6 or IRE1α inhibits up-regulation of TRAIL-R2, it sensitizes melanoma cells to TRAIL-induced apoptosis. Thus, it appears that regulation of TRAIL-R2 expression is not the only means by which the UPR regulates TRAIL-induced apoptosis in melanoma. The UPR may also antagonize TRAIL-induced apoptotic signaling by an intracellular mechanism(s). Study of a melanoma cell line Me1007 in Chapter 7 is the only cell line sensitive to ER stress-induced apoptosis, shows that apoptosis in this cell line is induced by ER stress via a caspase-8-mediated pathway. The high sensitivity of Me1007 to ER stress-induced apoptosis is associated with low expression levels of the apoptosis repressor with caspase recruitment domain (ARC) protein. In resistant cell lines, ARC is expressed at relatively high levels, which may effectively inhibit activation of caspase 8. Therefore, ARC appears to be critical in blocking activation of casapse-8 in melanoma cells subjected to ER stress.
| Identifer | oai:union.ndltd.org:ADTP/280740 |
| Date | January 2008 |
| Creators | Jiang, Chen Chen |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | Copyright 2008 Chen Chen Jiang |
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