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This book is about the p53 gene, one of the most frequently mutated or deleted genes in human cancers. The frequent occurrence of inactivated p53 implicates this gene product in the genesis of many human cancers. The p53 gene can suppress the growth of cancer cells and the transformation process by oncogenes. The p53 protein is a transcription factor that can repress or activate promoters containing one of three p53 DNA-binding motifs. The activity of p53 is regulated by phosphorylation and other transcription factors. Replacement of the p53 function or restoration of the p53 biochemical pathway is a focus of gene therapy.
The current year (2004) marks the Silver Anniversary of the discovery of the p53 tumor suppressor. The emerging ?eld ?rst considered p53 as a viral antigen and then as an oncogene that cooperates with activated ras in transforming primary cells in culture. Fueling the concept of p53 acting as a transforming factor, p53 expression was markedly elevated in various transformed and tumorigenic cell lines when compared to normal cells. In a simple twist of fate, most of the studies conducted in those early years inadvertently relied on a point mutant of p53 that had been cloned from a normal mouse genomic library. A bona ?de wild-type p53 cDNA was subsequently isolated, ironically, from a mouse teratocarcinoma cell line. A decade after its discovery, p53 was shown to be a tumor suppressor that protects against cancer. It is now recognized that approximately half of all human tumors arise due to mutations within the p53 gene. As remarkable as this number may seem, it signi?cantly underrepresents how often the p53 pathway is targeted during tumorigenesis. It is my personal view, as well as many in the p53 ?eld, that the p53-signaling pathway is corrupted in nearly 100% of tumors. If you are interested in understanding cancer and how it develops, you must begin by studying p53 and its pathway. After demonstrating that p53 functions as a tumor suppressor the ?eld exploded and p53 became a major focus of scientists around the world.
Among several genetic alterations in human cancer, mutations in the TP53 tumour suppressor gene represent the most common, occurring in approximately 50% of all human cancers. The majority of these mutations in p53 are missense mutations, resulting in cancer cells expressing stable, full-length mutated p53 proteins. Missense mutant p53's exhibit loss of tumour suppressive property of wild-type p53, dominant negative effects that can inactivate any wild-type p53 protein, and gain-of-function (GOF) properties that promote tumour progression and metastasis. Evidence suggests that cancer cells depend on the sustained expression of mutant p53 GOF. Thus, identifying the common downstream factor that drive mutant p53 GOF can provide an attractive approach to therapeutically target mutant p53 expressing tumours. This thesis presents the study of the characterization and functional analysis of mutant p53 secreted factors called "the mutant p53 secretome". In particular, the thesis aims at identifying the critical secreted effector of mutant p53 GOF that can serve as a potential therapeutic target for treatment of mutant p53 expressing tumours. Furthermore, the thesis investigates the association of the identified factor within the secretome with clinical parameters such as patient's survival. This thesis makes several original contributions to the field of cancer research, which are briefed below. Firstly, the mutant p53 induced secretome was characterized using quantitative proteomics of conditioned medium from mutant p53 expressing inducible H1299 human lung cancer cells. The majority of the identified secreted proteins were the transcriptional targets influenced by mutant p53. Alpha-1 antitrypsin (A1AT) was selected for further investigation, as it was the protein showing the highest expression in the mutant p53 secretome. The role of A1AT in driving the oncogenic activity of mutant p53 in human lung cancer cells was explored. A1AT was shown to drive mutant p53 induced invasion in lung cancer cell lines. Ablation of A1AT using antibodies and gene knockdown approaches inhibited the mutant p53 driven invasion, providing a rational to investigate the development of antibody-based cancer therapies that target A1AT. The clinical association of A1AT was further investigated in tissue microarray (TMA) samples of lung adenocarcinoma (ADC) patients. Mutant p53 expression was shown to correlate with A1AT, which validates in vivo that A1AT is a bonafide target of mutant p53. Furthermore, elevated expression of A1AT was demonstrated to correlate with increased local invasion and poor prognosis of lung ADC patients. Mutant p53 is reported to function as an aberrant transcription factor that can interact with other transcription factors to reprogram the cellular transcriptome of cancer cells. The mechanism of regulation of A1AT by mutant p53 was confirmed to involve p63. The role of A1AT in driving the mutant p53 induced invasive behavior of breast cancer cells was also explored, and a relationship of A1AT with p53 status and with different subtypes of breast cancer was established. In p53 mutant basal-like subtypes, A1AT expression was shown to drive invasion and treatment with anti-A1AT antibodies inhibited invasion. This suggests that the A1AT-targeted are potential therapies in various cancer types and its regulation in breast cancer may also extend beyond p53. Collectively, these studies provide new insights into the invasive behavior of mutant p53 that are manifested through aberrant secretion of extracellular proteins. The identification of A1AT as a critical and indispensable effector of mutant p53 gain-of-function offers a new therapeutic options for treatment of p53 mutant tumours. The findings in this thesis involve significant elements of novelty describing how mutant p53 influences the cellular secretome.
Since the discovery of p53 as a tumor suppressor, numerous methods have evolved to reveal the unique structural features and biochemical functions of this protein. Several unique properties of p53 posed a challenge to understa- ing its normal function in the initial phase of its research. The low levels of p53 in normal cells, its stabilization under situations of genotoxic stress, induction of growth arrest, and apoptosis with stabilization of the protein, obstructed the visibility of its normal, unmutated function. The property of p53 that can sense a promoter and transactivate or inhibit is still not well understood. It is still not known whether it is the absence of the protein that causes tumorigenesis, or if its mutants have a dominant role in inducing cancer. p53 Protocols comprises eighteen chapters for the study of the diverse properties of p53 and related proteins. The methods included are invaluable for delineating the function of other proteins that may function as tumor suppr- sors or growth suppressors. The chapters are not presented in any schematic order, for the importance and diversity of the functions of p53 make it imp- sible to organize them suitably. We have made a sincere effort to collect the methods most useful to those investigators working on tumor suppressors or growth suppressors. The purpose of p53 Protocols is not only to provide investigators with methods to analyze similar biochemical functions, but also to familiarize them with the associated problems that arose during the course of investigations.
This thesis presents the first report of the comprehensive and quantitative analysis of the effects of tumor-derived mutations on the tetrameric structure of tumor suppressor protein p53, which plays a central role in maintaining genomic integrity. Inactivation of p53 via mutation of its gene is a key step in tumorigenesis. Biophysical analyses revealed that the stability of the mutant peptides varied widely. Formation of a tetrameric structure is to be critical for protein–protein interactions, DNA binding, and the post-translational modification of p53. A small destabilization of the tetrameric structure therefore could result in dysfunction of tumor suppressor activity. This work suggests that the threshold for loss of tumor suppressor activity, in terms of the disruption of p53’s tetrameric structure, could be extremely low. Furthermore, functional control of p53 via tetramer formation was demonstrated, based on the structure–function analysis of mutant p53. The results disclosed that relatively small changes in tetramer formation, induced by the stabilization or inhibition of homo-tetramerization, could control p53 function.
P53 is the most commonly mutated tumor suppressor gene in human cancers. Activation of p53 maintains the genomic integrity and protects the organism against the propagation of cells that carry damaged DNA with potentially oncogenic mutations. While p53 protein is normally short-lived and kept at low levels in a relatively inactive form, upon genotoxic and cellular stresses p53 is transiently stabilized and activated as a transcription factor. The increase in protein level and transcriptional activity is attributed largely to the various posttranslational modifications of p53, including the N-terminal phosphorylation, and the multiple C-terminal modifications such as ubiquitination, acetylation, sumoylation, neddylation and methylation. Specifically, the six Lysine residues at p53 C-terminus can be posttranslationally modified by diverse mechanisms, among which the ubiquitination of those residues has been thought to be required for Mdm2-mediated ubiquitin-directed proteasomal degradation, and the acetylation is suggested to activate p53 transcriptional activity and contribute to its stabilization. To investigate the physiological functional outcome of the C-terminal modifications in regulating p53 stability and activity, we introduced Lysine to Arginine missense mutations at the six Lysine residues (K6R) into the endogenous p53 locus in mouse embryonic stem cells (ESCs). Unexpectedly, analysis of mouse ESCs, mouse embryonic fibroblast (MEFs) and thymocytes has concluded that ubiquitination of C-terminal Lysine residues is not required for efficient p53 degradation either before or after DNA damage. The outcome of loss of all potential posttranslational modifications is a modestly impaired p53 activity after DNA damage in a promoter-specific manner. Phosphorylation of Ser46 of human p53 is suggested to play an important role in activating p53-dependent apoptosis. To address the physiological role of Ser46 phosphorylation, we introduced Ser46 to Alanine mutation (S46A) into the human p53 knock-in (HUPKI) allele in mice. Consistent with the previous cell line studies, transactivation of p53 targeting apoptotic genes is preferentially affected by the mutation and p53-dependent apoptosis after DNA damage is partially impaired in mutant thymocytes and in E1A/Ras-overexpressed MEFs. In addition, Ser46 phosphorylation may contribute to preventing spontaneous immortalization of cultured MEFs and to oncogene Ras-induced premature senescence of MEFs.
The three sections of this volume present currently available cancer gene therapy techniques. Part I describes the various aspects of gene delivery. In Part II, the contributors discuss strategies and targets for the treatment of cancer. Finally, in Part III, experts discuss the difficulties inherent in bringing gene therapy treatment for cancer to the clinic. This book will prove valuable as the volume of preclinical and clinical data continues to increase.
This book describes the Notch signaling pathway with a focus on molecular mechanisms. The Notch signaling pathway is a seemingly simple pathway that does not involve any second messenger. Upon ligand binding two consecutive proteolytic cleavages of the NOTCH receptor release the Notch intracellular domain from the membrane. The Notch intracellular domain migrates into the nucleus and activates gene expression. Recently, new technologies allowed us to better understand this pivotal signaling cascade and revealed new regulatory mechanisms. The different chapters cover many aspects of the Notch signaling focusing on the mechanisms governing the receptor/ligand interaction as well as on the downstream intracellular signaling events. Aspects of both canonical and non-canonical signaling are discussed and the function of Notch signaling in physiological and pathological contexts are elucidated. This book is not only intended for experts but it should also be a useful resource for young, sprouting scientists or interested scientists from other research areas, who may use this book as a stimulating starting point for further discoveries and developments.