Dissecting Mechanisms of Transformation Following Loss of p53 and RB

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2023

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AbstractFor over 30 years the cancer biology field has scrutinized the mechanisms behind p53 and RB mediated tumor suppression (Fields and Jang 1990; Kern et al. 1991; Raycroft, Wu, and Lozano 1990; Dyson 1998; Classon and Harlow 2002; Knudsen and Knudsen 2008) Together, these genes regulate complex interconnecting pathways responsible for the regulation of cell growth, cell death, and genomic integrity (Sherr and McCormick 2002). Not surprisingly, the pathways regulated by these two tumor suppressors are almost universally disrupted during the development of cancer (Hanahan and Weinberg 2000). By nature, tumor suppressors play a key role in preserving the integrity of the genome. Whether in response to injury or in maintaining stable gene expression patterns, p53 and RB have been well established as two of the most important factors preventing genomic instability, epigenetic deregulation, transformation, and tumorigenesis. However, the precise mechanism by which p53 or RB mediate tumor suppression remains unclear (Tiwari, Jones, and Abrams 2018). Over the last decade, several studies in genetically engineered mouse models have demonstrated that the canonical functions of these two tumor suppressors fail to fully explain their tumor suppressive capabilities (Mello and Attardi 2018; Janic et al. 2018; T. Li et al. 2012). Moreover, expanding literature continues to highlight a strong correlation between transposable element de-repression and several types of cancer, however, no definitive link between transposable elements and tumorigenesis has been established (Tiwari, Jones, and Abrams 2018; Wylie et al. 2016; Tiwari et al. 2020; Rodriguez-Martin et al. 2020). At present, the role that genomic repetitive elements play in tumorigenesis remains an open question. Here, to elucidate a possible mechanism of transformation we sought to investigate the link between the concurrent loss of p53 and RB and the de-repression of transposons. While individually, p53 and RB have been implicated in separate transposable element defense mechanisms, each responsible for preventing the expression of transposons (Ishak et al. 2016; Wylie et al. 2016; Tiwari et al. 2020; Dick et al. 2018; Tiwari, Jones, and Abrams 2018), my thesis work seeks to understand the role these repetitive elements play in transformation. Specifically, I investigate how the combined loss of p53 and RB affect the expression of transposons and how this relates to the development of cancer. Using genetically engineered mouse models we derived several lines of mouse embryonic fibroblasts (MEFs) which, through cre-lox technology, allowed for the deletion of floxed tumor suppressors (H. Kim et al. 2018). We derived MEFs from mice containing floxed p53, RB, both p53 and RB as well as several p53 and RB mutants. Generating primary MEFs allowed us to interrogate the loss of p53 and RB in a non-cancerous context, free of additional mutations (Todaro and Green 1963; Xu 2005). Moreover, by choosing MEFs as a model system, we could harness and observe the transformation process in a minimally manipulated system. Accordingly, we transformed normal Wild Type (WT) MEF cells and induced transformation by recombining loxP sites flanking p53 and RB. In dissecting whether loss of p53 and RB affected transposable element expression we showed that loss of p53 or RB alone each partially derepress long interspersed nuclear element 1 (LINE1) transposable elements, but remarkably co-deletion of p53 and RB together not only transform cells but also simultaneously induce massive expression of LINE1. Additionally, through the use of p53 transactivation domain mutants, we showed that the ability of p53 to repress transposable elements is a non-canonical function linked to tumor suppression. Interrogation of short interspersed nuclear elements (SINEs) produced similar findings. Furthermore, the derepression of both LINEs and SINEs appears to be regulated by the modification of the H3K9me3 histone mark. These results further correlate the expression of transposable elements to transformation and tumorigenesis. Complementary work revealed that loss of both p53 and RB in MEFs not only derepressed transposable elements and transformed cells, but in the process, massively rearranged the three-dimensional (3-D) chromatin landscape. This change in 3-D chromatin architecture resulted in the loss of intrachromosomal loops and the subsequent overexpression of several oncogenes. To assess the scope of the rearrangement of chromatin architecture, we developed a UCSC genome browser based atlas, mapping thousands DNA loops stemming from promoters, enhancers and silencers. We find that while the general structure of topologically associated domains are largely stable (Dixon et al. 2012), following deletion of p53 or RB, local chromatin contacts are vastly reorganized. Moreover, the reorganization of DNA loops was found to affect gene expression in a context dependent manner. These results support a model of p53 and RB as guardians of the genome that cooperate to maintain the chromatin architecture required for normal cellular function and prevent changes in DNA topology that promote uncontrolled growth associated with cellular transformation. More than half of all cancer patients have mutations in p53 or RB and many harbor evidence of deregulated transposable elements amidst a disordered chromatin landscape. Here, we explore the distinct and overlapping genomic regulatory processes that p53 and RB cooperatively maintain to preserve genomic integrity and prevent cancer. Overall, my work implicates transposable element derepression as a mechanism that can promote the development of cancer and moreover, provides a genome wide atlas of how p53 and RB can affect the chromatin landscape to regulate gene expression in a context dependent manner. Moreover, my work provides mechanistic insight into the tissue specific functions of p53 and RB. Taken together, this thesis provides new insights into the mechanisms by which p53 and RB prevent cancer.

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Lopez, Omar Magaña (2023). Dissecting Mechanisms of Transformation Following Loss of p53 and RB. Dissertation, Duke University. Retrieved from https://hdl.handle.net/10161/27637.

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