Medical Biology - Theses
Permanent URI for this collection
Search Results
1 - 3 of 3
-
Item
-
ItemTumour suppression by p53 and therapeutic targeting of the p53-MDM2 interaction( 2014)The transcription factor p53 plays an essential role in tumour suppression and in the way that cancer cells respond to DNA damage-inducing anti-cancer therapies. Activated in response to a diverse range of cellular stresses p53 can coordinate the expression of ~600 targets genes. Through this p53 is able to regulate activity in a broad range of downstream effector pathways including; cell cycle arrest, cellular senescence, apoptotic cell death, coordination of DNA repair and coordination of metabolism. Many conventional anti-cancer strategies act through induction of extensive DNA damage in tumour cells. Such therapeutics limit cancer cell growth and promote tumour regression, in a large part, through DNA damage-induced activation of p53 and its downstream effector pathways e.g. apoptosis (via PUMA and NOXA), cell cycle arrest and cellular senescence (via p21). Concerns that genotoxic strategies for anti-cancer treatment may lead to further mutations within cancer cells (leading to treatment resistance) or in non-cancerous tissues (leading to secondary cancers) has led to the development of novel anti-cancer therapeutics that can activate p53 via non-genotoxic mechanisms. Small molecule inhibitors of MDM2, such as Nutlin3a, are the first of such compounds to enter clinical trials. Utilising gene-targeted mice lacking p21, PUMA and/or NOXA, I have shown that PUMA- (and to a lesser extent NOXA-) mediated apoptosis and not p21-mediated cell cycle arrest and/or senescence is essential to drive treatment responses to Nutlin3a in both non-transformed lymphoid and malignant Eμ-Myc lymphoma cells. In addition to its fundamental role in cellular responses to DNA damage, p53 has been shown to play an essential role in the suppression of cancer development, with its loss frequently observed in human cancers (e.g. Burkitt’s lymphoma) and in animal models of MYC-driven lymphomagenesis (e.g. Eμ-Myc mice). Apoptosis induced downstream of p53 was originally thought to be the sole effector process required to limit MYC-driven lymphomagenesis. However loss of PUMA fails to completely remove the requirement for mutation/deletion of p53 in Eμ-Myc lymphoma or accelerate lymphoma onset in mice to the same extent as loss of a single allele of p53. Together these data suggest that processes in addition to PUMA- (and NOXA-) mediated apoptosis are required for suppression of MYC-driven cancers. The CDK inhibitor, p21, has been suggested to be critical effector of tumour suppression downstream of p53. To determine if p21 and PUMA cooperate in p53-mediated suppression of MYC-driven lymphomagenesis I generated Eμ-Myc mice lacking p21 (or both p21 and PUMA). Surprisingly, lymphoma onset in Eμ-Myc;p21-/- mice was profoundly delayed. This suggests that within this context p21 acts to promote, rather than suppress, lymphomagenesis. The delay in lymphoma onset observed in Eμ-Myc;p21-/- mice was largely overcome with the concomitant loss of PUMA, suggesting that induction apoptosis is required for the survival advantage observed in these mice. Cell cycle arrest mediated by p21 may promote lymphoma development by allowing nascent cancer cells to arrest and repair damage to DNA. This process if impaired, e.g. by deletion of p21, may allow continued replication of cells bearing aberrant changes to their DNA, and this may in turn trigger clearance of these cells by PUMA-mediated apoptosis. Loss of p53 protein activity, either directly, e.g. through deletion or mutation of p53, or indirectly, e.g. through over-expression of MDM2, is observed in almost all human cancers. Interestingly, the majority of mutations to p53 observed in human cancer occur within the p53’s DNA binding domain and thus impair its transcriptional activity. This suggests that p53’s ability to block early “cell-transforming” events is critically linked to its ability to modulate transcription of its ~600 target genes. Given p53-mediated transcriptional induction of PUMA, NOXA and p21 (and hence induction of apoptosis, cell cycle arrest and/or cellular senescence) is critical for p53-mediated responses to DNA damage (and enforced oncogene expression), it was widely believed that these same processes would also be essential for p53’s ability to suppress spontaneously forming cancers. In order to gain concrete insight into whether apoptosis (via PUMA and NOXA) and/or cell cycle arrest and/or senescence (via p21) are required for p53-mediated suppression of spontaneous tumour development, I generated mice completely lacking all three of these critical effectors. In stark contrast to p53-deficient mice, which all succumb to spontaneous thymic lymphoma and/or sarcoma development by 250 days of age, p21-/-Puma-/-Noxa-/- mice remain tumour free for up to 500 days. These results demonstrate beyond doubt that suppression of spontaneous tumorigenesis by p53 does not require PUMA/NOXA-mediated apoptosis or p21-mediated cell cycle arrest and cellular senescence. The discovery that processes are not essential for p53-mediated tumour suppression (despite their critical importance for cellular responses to acute DNA damage and enforced oncogene expression) re-launches the search for the p53 target genes and effector processes that are critical to prevent cancer development. Gaining insight into the mechanisms by which p53 suppresses early “cell-transforming” events will have important ramifications for the development of novel strategies for cancer therapy and possibly even cancer prevention.
-
ItemBIM is critical for DNA damage-induced apoptosis and enforces tumour suppression( 2013)Apoptosis is a highly regulated, ordered form of cell death that is critical for a wide variety of physiological processes. Loss of key apoptotic regulators or other defects in apoptosis can result in either excessive or insufficient removal of cells leading to severe consequences, such as embryonic lethality, autoimmunity or cancer. The BCL-2 family of proteins are key regulators of apoptosis that control the ‘point of no return’ integrating diverse upstream signalling pathways to determine whether a cell will live or die under conditions of stress. Due to their ability to regulate cell survival, abnormalities in the expression of the BCL-2 family members are frequently observed in human cancer. Furthermore due to their ability to initiate apoptosis signalling, the BH3-only subfamily of the BCL-2 protein family are required for mediating tumour cell killing following treatment with many chemotherapeutic agents. Accordingly, mutation or loss of the BH3-only proteins, or their upstream regulators, is associated with chemoresistance and poor treatment response. The tumour suppressor p53 is a critical direct transcriptional activator of the genes encoding the BH3-only proteins PUMA and NOXA. Mutations in p53, with consequent loss of its transcriptional activity, constitute the most frequent abnormality in human cancer and are associated with poor response to anti-cancer therapeutics, particularly those that cause DNA damage. Since p53 functions upstream of the BH3-only proteins in apoptosis signalling, therapies that act independently of p53 to induce the expression of BH3-only proteins are likely to be more efficacious for the treatment of patients that harbour p53-deficient tumours. In this thesis I describe the characterisation of the mechanisms by which DNA damage can induce apoptosis in the absence of p53 function, focussing on those that are relevant to the treatment of p53-deficient tumours. To do this I generated a panel of p53-deficient thymic lymphoma-derived cell lines and determined their ability to undergo apoptosis in response to DNA damaging agents that induce different types of DNA lesions, such as γ-irradiation, etoposide and cisplatin. I have examined changes in protein expression of members of the BCL-2 family following treatment with these DNA damaging agents and identified those members of the BH3-only sub-group that are up-regulated. By generating lymphoma-derived cell lines from mice that lack both p53 and select members of the BH3-only proteins, I have been able to determine which are essential for the induction of DNA damage-induced apoptosis in the absence of p53. Through this approach I have identified novel mediators of DNA damage induced-apoptosis both in lymphoma cell lines and also in primary non-transformed cells deficient for p53. I then sought to determine whether this newly identified pathway plays a critical role in the elimination of pre-leukaemic cells that have sustained physiological DNA damage in vivo (e.g. due to replication induced stress or the stress elicited by oncogene activation). To address this cohorts of mice deficient for p53 plus candidate transducers of this pathway were aged and the lymphoma incidence was compared to those lacking p53 alone. The additional loss of this novel DNA damage induced pathway provoked a pronounced acceleration in lymphoma onset in the p53-deficient mice and also resulted in a more aggressive tumour phenotype. The importance of this tumour suppressor pathway was further characterised by ageing p53-heterozygous mice deficient for pathway members and comparing their tumour incidence to that of mice lacking only a single allele of p53; revealing a profound acceleration of tumour development in this context. BIM has been shown to be a critical tumour suppressor in the Eμ-Myc mouse model of lymphoma and recent evidence has shown that the pro-survival BCL-2 family member BCL-XL is critical to promote survival of cells undergoing neoplastic transformation in this model. To gain further insight into the processes that govern cell survival during neoplastic transformation, I performed experiments to determine whether concomitant loss of BIM would be sufficient to abrogate the delay in lymphomagenesis observed in the absence of BCL-XL. Loss of a single allele of Bim was sufficient to revert this delay and loss of both alleles of Bim provoked a further acceleration in lymphoma onset. Thus through my investigations in vitro into the manner in which apoptosis can be induced in cells lacking p53 and my studies utilising various mouse models of lymphoma development I have identified a novel apoptotic pathway important for the elimination of in p53-deficient pre-leukaemic cells and malignant lymphoma cells. These data provide new mechanistic insights into DNA damage induced apoptosis in vitro and for tumour suppression in vivo.