Date of Award
8-12-2024
Degree Name
Doctor of Philosophy (PhD)
Degree Type
Dissertation
Abstract
The p14ARF tumor suppressor is the second most commonly mutated gene in Non-Small Cell Lung Cancer (NSCLC). p14ARF (p19ARF in mice) is encoded by the cyclin-dependent kinase inhibitor 2A (CDKN2A) gene. The p14ARF tumor suppressor is known to play a fundamental role in cancer progression by regulating senescence, apoptosis, and proliferation. The primary function of p14ARF is to respond to oncogenic signals and activate the p53 tumor suppressor by inhibiting and sequestering the HDM2 (MDM2) oncogene. However, recent studies have uncovered multiple p53-independent functions of p14ARF, opening the door to a myriad of possible vulnerabilities in p14ARF-mutant tumors. Many current therapeutic efforts in cancer biology focus on inhibiting the function of aberrant oncogenes whose activities are known to increase proliferation and cell growth. Unfortunately, these efforts have had limited success in lung cancer, where the five-year survival rate remains unchanged. To address this significant unmet medical need, I took a novel approach; rather than inhibiting oncogenes, I reestablished the function of the p14ARF tumor suppressor in NSCLC to block tumor growth and proliferation. The long-term goal of this project is to determine the genetic requirements and clinical significance of p14ARF tumor suppression in humans, leading to the development of p14ARF-targeted cancer therapeutics. To achieve this, I first established the origin and minimal domain responsible for cell sensitivity to p14ARF. In silico methods were used to identify conserved amino acid residues within p14ARF and the corresponding minimal domain(s) were investigated. I utilized the lentiviral delivery system to exogenously express a library of contiguous small p14ARF deletion mutants to establish the p14ARF domain that is critical for its function in arresting NSCLC cells in vitro. Following this initial set of experiments, I wished to identify small molecules capable of mimicking p14ARF. Years of research has been dedicated to the recovery of lost tumor suppressor genes and their functions, but a major obstacle has been finding efficient and non-toxic ways of delivering these molecules into the cell. Recent advances in chemistry peptide systems have led to the consistent and efficient uptake of arginine-rich cell-penetrating peptides (CPPs). Based on this peptide delivery technology, I synthesized peptides containing p14ARF residues 1-14, 1-10, 5-14, and 16-28 followed by a polyarginine tail; I employed this cell-penetrating peptide technology to deliver these domains into multiple NSCLC cell lines. My work has established that cell-penetrating peptides can deliver p14ARF domains into NSCLC cells and that these p14ARF peptides exhibit a sequence specific effect in inhibiting cell proliferation and promoting cell death. As in vitro studies provided promising results, this project could easily be transferred to in vivo studies given the established use of cell-penetrating peptides in mouse models. I next sought to determine the nucleolar impact of p14ARF and p14ARF CPPs based on their interaction with NPM1. The nucleolus has multiple non-membrane bound sub-compartments; these can act as quality control mechanisms and prevent the formation of potentially toxic protein aggregates. The NPM1 protein participates in the oncogenic stress response through its interaction with p14ARF, and it requires p14ARF’s terminal amino acid sequence for binding. The interaction of these proteins alters the phase separation within the nucleolus and immobilizes both proteins, leading to a decrease in both proteins’ functions. This assessment is supported by research finding a decrease in various NPM1 functions following p14ARF overexpression. Therefore, investigating the ability of p14ARF CPPs to alter functional outputs of NPM1 (ribosome biogenesis, protein synthesis, etc.) will further determine the ability of p14ARF CPPs to function as wildtype p14ARF. CPPs containing p14ARF residues 1-14, 1-10, 5-14, and 16-28 followed by a poly-arginine tail were tagged with biotin. Immunoprecipitation reactions evaluated the binding partners of the corresponding minimal domains. After treatment of these p14ARF CPPs, ribosome biogenesis, total protein per cell, cell death, and cell cycle arrest were measured. A decrease in ribosome biogenesis and total protein per cell were observed post-treatment with CPPs containing amino acids 1-14. We saw an increase in cell death when cells were treated with CPPs containing amino acids 1-14, 1-10, and 5-14, but not when cells were treated with amino acids 16-28. We did not see any significant change in cell cycle arrest post-CPP treatment. A NPM1 rescue experiment was conducted in hopes of better understanding how significant the CPP-NPM1 interaction is for the phenotypic response of cell death. We saw a slight decrease in efficacy of the CPP treatment in cells with an NPM1 mutant incapable of binding p14ARF. This partial rescue suggests that the CPPs are interacting with NPM1 as well as other molecular partners to induce the cell death response. These pathways could be further elucidated by a pull-down experiment with the CPP’s in the cells. Performing mass spectrometry on these samples would showcase the multiple pathways these CPPs are likely acting on, providing new avenues to potentially exploit in further therapeutic research. Additionally, fluorescence recovery after photobleaching (FRAP) could evaluate the physical effect of these CPPs on the nucleolus, further assessing their ability to function as wildtype p14ARF. This dissertation will aid the development of precision medicine-based therapy in the treatment of lung cancer. Additionally, it will provide an understanding of which amino acid residues within the p14ARF tumor suppressor are required for its cellular function in preventing cancer. These findings will provide a more refined explanation of p14ARF’s basic p53-dependent and -independent functions in cancer. This increased knowledge will provide a valuable resource to the wider molecular oncology field by laying the foundation for more precise therapeutic cancer treatments, ones that could be used to either restore p14ARF function or inhibit downstream targets of p14ARF.
Language
English (en)
Chair and Committee
Jason Weber
Committee Members
Abby Green; Jessica Silva Fisher; Jieya Shao; Joshua Rubin
Recommended Citation
Grobelny Guthrie, Anna Louise, "The clinical significance and genetic origin of p14ARF" (2024). Arts & Sciences Electronic Theses and Dissertations. 3295.
https://openscholarship.wustl.edu/art_sci_etds/3295