Studies have shown that gain-of- function mutant p53, AKT, and NFκB promote invasion and metastasis in tumor cells. Signals transduced by AKT and p53 are integrated via negative feedback between the two pathways. Tumor derived p53 was also indicated to induce NFκB gene expression. Due to the close relationship between p53/AKT and p53/NFκB, we hypothesized that AKT and NFκB can enhance motility in cells expressing mutant p53. Effects on cell motility were determined by scratch assays. CXCL5- chemokine is also known to induce cell motility. We hypothesized that enhanced cell motility by AKT and NFκB is mediated, in part, by CXCL5. CXCL5 expression levels in the presence and absence of inhibitors were determined by qRT-PCR. We also hypothesized that gain-of-function mutant p53 contributes to the activation of AKT. The effect of mutant p53 on AKT phosphorylation was investigated with a Ponasterone A- inducible mutant cell line (H1299/R175H) and vector control. These results indicated that AKT and NFκB enhance motility in cells expressing mutant p53 and this enhanced motility is, in part, mediated by CXCL5. However, AKT phosphorylation was independent of mutant p53.

Breast cancer is the most frequently diagnosed form of cancer in women and the second leading cause of cancer-related deaths. Breast cancer is a heterogeneous disease consisting of many types of tissue neoplasia, and there appears to be no model of how a particular lesion develops into an aggressive, malignant, invasive carcinoma. Genetic mutation and aberrant epigenetic regulation are among the most common events that lead to neoplasia. In breast cancer, p53 mutation is the most common genetic defect related to a single gene. Therefore, this dissertation focuses on the mechanisms and consequences of p53 mutation during breast tumorigenesis. Genome-wide analysis of gene expression and epigenetic modifications in a panel of breast cancer cell lines suggested that p53 mutation and aberrant epigenetic silencing were cooperating mechanisms in the silencing of wild-type p53 target genes during cancer progression. Therefore, models of p53 inactivation were created in non-malignant human mammary epithelial cells to determine the role of p53 mutation on the epigenetic status of its target genes and the acquisition of malignant phenotypes. Comparisons of each model demonstrated that differing modes of p53 inactivation produced different functional consequences. Loss of wild-type p53 function alone ablated the normal cellular response to external stress stimuli, but had no affect on the expression of genes or epigenetic status in untreated cells. Introduction of missense mutant p53 protein caused very few changes when the protein was expressed at low levels. However, accumulation of mutant p53 caused a variety of gene expression changes and interfered with endogenous wild-type p53. The accumulation of mutant p53 also caused an increase in migration and invasion of the cells that expressed it. Interestingly, epigenetic aberrations were not detected in response to any of the p53 manipulations. These data suggest that accumulation of missense mutation is particularly dangerous to normal cells. They also suggest that p53 mutation and epigenetic aberration are two distinct mechanisms, which overlap and cooperate during tumorigenesis. These data suggest that treatment strategies for human breast cancer should include modalities to target both defects for increased efficacy.

Cutaneous squamous cell carcinoma (cSCC) is a type of non-melanoma skin cancer that is the 4th most common cancer registration in Scotland after BCC, lung and breast cancer. Over 30,000 cSCC incidences are reported each year in the United Kingdom. In addition, around 1 in 4 skin cancer deaths in the UK are due to cSCCs. Amongst those highly prone to developing cSCCs include organ transplant recipient, immunosuppressed, recessive dystrophic epidermolysis bullosa (RDEB) and Xeroderma Pigmentosum (XP) patients. cSCC patients that display regional metastasis have a 5-year survival rate of 25-50%, whilst this rate is close to 0% in RDEB patients with multiple cSCCs. Wild-type p53 (wt-p53) has been shown to prevent cSCC development and induce tanning and sunburn responses in skin cells. However, TP53 mutations are found in over half of all human cancers and cSCC is no exception as TP53 mutational frequency in cSCCs is around 64-87.5% (Durinck et al, 2011; South et al, 2014). The majority of TP53 mutations in cSCCs are UV-signature missense mutations, highlighting UV-radiation as one of the main risk factors for cSCC development. Mutant p53 proteins can lose wt-p53 functions, have dominant-negative effects against wt-p53 and acquire gain of function (GOF) activities. Mutant p53 GOF activity is induced by the accumulation of mutant p53 in tumour cells. Mutant p53 accumulation is not due to intrinsic properties of the mutants but requires other cellular events, possibly those known to stabilise wt-p53 under cellular stress. It is known that the TP53 mutations and mutant p53 accumulation are early steps in cSCC development. This makes skin an excellent system to investigate the early changes to p53. We have investigated the potential of targeting mutant p53 for cSCC therapy and mechanisms that promote mutant p53 accumulation in cSCCs. We selected low-passage cSCC cell lines that express hotspot mutant p53 proteins, in cSCCs and in general, by analysing TP53 mutational data from the IARC database and next generation sequencing studies performed on cSCC primary tumours by Dr South at Ninewells Hospital, Dundee. cSCC cell lines were generated from immunocompetent, transplant and RDEB patients by Dr South’s group at Ninewells Hospital, Dundee. We found that: 1. PRIMA-1MET, a small molecule reported to restore wt-p53 activity, lacked tumour selectivity as it is able to reduce cell viability in both normal skin and cSCC cells with similar potency. cSCC cell lines are relatively resistant to PRIMA-1MET compared to cell lines derived from other tumour types. 2. Mutant p53 knockdown studies performed on cSCC cell lines suggest that some p53 mutants play a pro-proliferative role. However, there is no evidence for a pro-migratory role of mutant p53 in cSCC. 3. There are no clear alterations in DNA-damage response pathways or the general ubiquitin proteasome system that could contribute to mutant p53 stabilisation in cSCC. 4. Heat shock factor 1 (HSF-1) is upregulated in cSCC compared to normal human keratinocytes (NHK). HSP90 inhibitors, 17-AAG and 17-DMAG, reduce mutant p53 protein levels suggesting that HSP90 plays a role in stabilising mutant p53 in cSCCs. 5. PR-619, a broad range deubiquitinating enzyme (DUB) inhibitor, reduces mutant p53 protein levels in a range of cSCC cell lines. This is rescued by the addition of bortezomib suggesting that DUBs can play a role in protecting mutant p53 from proteasomal degradation. Expression of HAUSP and USP10, which have been shown to stabilise wild-type p53, is generally elevated in cSCC compared to NHK. However, knockdown of these DUBs does not reduce protein levels of mutant p53 in cSCC cell lines. 6. A potential isoform of MDMX (51 kDa) is strongly upregulated in all cSCC cell lines examined. There is an association between the ability of MDMX siRNAs to deplete the 51 kDa protein and reduce mutant p53 protein levels and stability. Furthermore we show that the protein can form complexes with MDM2 in vitro and in cSCC cells. We propose that the MDMX isoform is able to stabilise mutant p53 in cSCC cells through this interaction with MDM2.

Lung cancer is the most common cause of cancer-related mortality worldwide with current treatments providing limited therapeutic benefit in most cases. TP53 (Trp53, p53) mutations occur in approximately 50% of lung adenocarcinoma cases and are associated with poor prognosis and so novel therapies that target these p53 mutant lung tumours are urgently needed. Despite the high frequency of p53 mutations in lung tumours, the impact these mutations have on response to therapy remains unclear in this cancer type. The aim of my project is to characterise the gain-of-function and dominant-negative effects of p53 mutations in lung tumours and to identify ways of therapeutically targeting these p53 mutant tumours based on dependencies and susceptibilities that our analysis uncovers. To characterise the gain-of-function and dominant-negative effects of p53 mutations I compared p53 mutant murine lung tumour cells that endogenously express either a contact (R270H, equivalent to R273H in humans) or conformational (R172H, equivalent to R175H in humans) p53 mutant protein and p53 null lung tumour cell lines; both in the presence and absence of wild-type p53. Interestingly, transcriptional and functional analysis uncovered metabolic gain-of-functions that are specific to the type of p53 mutation. Upregulation of mevalonate pathway expression was observed only in R270H lung tumours and consequently R172H and R270H lung tumours displayed distinct sensitivities to simvastatin, a mevalonate pathway inhibitor widely used in the clinic. Furthermore, the transcriptional signature underlying this sensitivity to simvastatin was also present in human lung tumours with contact p53 mutations, indicating that these findings may be clinically relevant. On the other hand, our analysis of the potential dominant-negative effects of the p53 mutants on wild-type p53 demonstrated that wild-type p53 was able to induce typical p53 target genes to a similar level in p53 null and mutant cells. Furthermore, wild-type p53 restoration resulted in comparable tumour suppressive responses in p53 mutant and null tumours and thus, p53-restoration therapy will likely be of benefit to patients with p53 mutations in lung cancer. Hence, I have demonstrated that lung tumours harbouring contact and conformational p53 mutations display common and distinct therapeutic susceptibilities.

Indiana University-Purdue University Indianapolis (IUPUI) / Myelodysplastic syndrome (MDS) is a clonal disease arising from mutated hematopoietic stem cells (HSCs). MDS stem cells originate from pre-leukemic HSCs, which have enhanced competitive advantage over wild-type (WT) HSCs but normal differentiation capacity. Recently, acquired somatic gain-of-function (GOF) TP53 mutations were identified in the blood of aged healthy individuals as well as in patients with MDS. However, the role of GOF TP53 mutations in clonal hematopoiesis and the pathogenesis of MDS is largely unknown. Based upon our previous studies and clinical findings, I hypothesized that GOF mutant p53 drives the development of pre-leukemic HSCs with enhanced competitive advantage, leading to clonal expansion and the pathogenesis of MDS. To test my hypothesis, I examined HSC behaviors in young p53+/+ and p53R248W/+ mice. I discovered that p53R248W enhances the repopulating potential of HSCs without affecting terminal differentiation. I also found that GOF mutant p53 protects HSCs from genotoxic stress and promotes their expansion. To investigate the role of mutant p53 in the pathogenesis of hematological malignancies, I monitored disease development in p53+/+ and p53R248W/+ mice and observed that some mutant p53 mice develop MDS during aging. Therefore, I demonstrated that GOF mutant p53 enhances the repopulating potential of HSCs and drives the development of pre-leukemic HSCs, predisposing aged mutant p53 mice to MDS development. Mechanistically, I found that mutant p53 increases the chromatin accessibility to genes important for HSC maintenance, including pluripotent gene Sox2 and chemokine gene Cxcl9. By performing biochemical experiments, I discovered that GOF mutant p53, but not WT p53, interacts with histone methyltransferase EZH2 and enhances histone H3 lysine 27 trimethylation (H3K27me3) at genes, including Mef/Elf4 and Gadd45g, that negatively regulate HSC self-renewal. Collectively, these findings demonstrated that GOF mutant p53 drives pre-leukemic HSC development through modulating epigenetic pathways. Thus, our studies have uncovered novel mechanistic and functional links between GOF mutant p53 and epigenetic regulators in pre-leukemic HSCs. This research may identify epigenetic regulator EZH2 as a novel target for the prevention and treatment of MDS patients with TP53 mutations.

No description available.

Head and neck squamous cell carcinoma (HNSCC) is the 6th most common type of cancer in the western hemisphere with a five-year survival rate of only 50% for patients with a localized tumor, which decreases significantly to as low as 5% for those patients with tumors that have metastasized to distant sites of the body. It has been found that both mutant p53 and epidermal growth factor receptor (EGFR) signaling pathways function to increase the expression of CXCL5, which has been identified as a key mediator in the process of tumor metastasis. Previous data from our lab suggested that the p53 homolog, p63, may function as a negative regulator of CXCL5 and that mutant p53 may inhibit this molecule to elevate CXCL5 expression levels. In the current study we utilized an model system in which the H179L p53 mutant was expressed in HN4 cells to investigate the hypothesis that mutant p53 enhances expression of CXCL5 by both interfering with p63 function and cooperating with EGFR/EPS8 signaling, leading to increased cell proliferation and motility. The results of the current study indicate a role for mutant p53 in head and neck squamous cell carcinoma proliferation, migration and tumorigenicity, possibly through enhancement of CXCL5 expression. We were able to show that mutant p53 expression caused an increase in the expression of this chemokine in addition to increasing proliferation and migration of the cells compared to the vector control. Additionally, we showed that p63 protein is a negative regulator of CXCL5 that is downregulated in the cells expressing mutant p53, which suggests that through direct interaction, mutant p53 may function to inhibit p63 function as well as target it for degradation. These results support the hypothesis that GOF mutant p53 enhances expression of CXCL5 by interfering with p63 function in cancer cells. The results of the current study results also showed that upon treatment with EGF, HN4 cells expressing mutant p53 express elevated levels of CXCL5; and that the mutant p53-expressing HN4 cells cooperate with EGFR/EPS8 signaling to further deregulate chemokine expression. These data taken together suggest there are complex interactions taking place between mutant p53, p63, EGFR signaling, and CXCL5 to regulate the biological processes that promote tumor progression that could lead to metastasis. Additional studies are needed to further elucidate the molecules involved in the mutant p53 mechanism that promotes tumorigenesis.

The tumour suppressor p53 is often disrupted by missense mutations that can result in p53 protein accumulation and acquisition of novel oncogenic activities. Various studies have demonstrated that DNA-damaging drugs currently used in the clinic aimed at activating wild type p53, can also stabilise and activate mutant p53 oncogenic functions and thereby paradoxically enhance tumour progression, resulting in poor response to the treatment. In this study we aimed to investigate whether, like in wt p53, post-translational modifications (PTMs) drive such drug-induced mutant p53 accumulation and activation. For this purpose, we generated plasmids expressing non-phosphorylatable and phospho-mimic versions of R175H mutant p53 and tested them in different cell line models. We demonstrated that in response to DNA damage mutant p53 is accumulated and phosphorylated and these phenomena appeared to be mediated by ATM and ATR kinases. DNA-damage induced acetylation was also observed and occurred in a S15 phosphorylation-dependent manner. This suggested a role of the HAT p300, which is recruited by phosphorylated S15. Of note, other works have shown that p300 is required to trigger some oncogenic functions of mutant p53. We then aimed at developing systems to explore mutant p53 functions and their dependence on PTMs. Although we showed that cell growth is compromised upon endogenous mutant p53 depletion, exogenous expression of mutant p53 or its phosphorylation-site forms did not result in a successful rescue in our experimental conditions, thus we were unable to use this strategy to test the effect of PTMs. Ectopic expression of R175H mutant p53 or its phosphorylayion-site versions did not interfere with the growth rate and response to chemotherapy of the p53-null cell line H1299. We also found that mutant p53 phosphorylation does not affect subcellular localisation of mutant p53 and mutant p53-mediated inhibition of p63. Interestingly, ectopically expressed mutant p53 enhanced cell migration in H1299 cells. Notably, our results suggested an apparent threshold effect of mutant p53 levels required to induce migration. Due to the difficulty of obtaining cell lines expressing similar levels of the different phosphorylation-site mutants, the determination of the role of phosphorylation in mutant p53-induced migration was not conclusive. Remarkably, we found that, while S15 and S20 phosphorylation decreased MDM2-dependent degradation, only phosphorylated S20 interfered with CHIP-induced turnover in H1299 cells. Overall our data suggest that, despite exhibiting opposite biological effects, mutant and wt p53 can share upstream regulatory mechanisms and thus present phosphorylation as a promising target to prevent mutant p53 stabilisation and activation and improve response to therapy. Our results also highlight the challenge of developing a good system for determining the effects of the mutant p53 protein and its regulation by PTMs.

Protein p53 je jednou z nejdůležitějších molekul v lidském těle. P53 reguluje celou řadu procesů v buňce, jako je například oprava DNA, buněčný cyklus nebo indukce apoptózy. Protein p53 je známý i jako „strážce genomu“. DNA vazebné schopnosti proteinu p53 jsou důležité pro normální vývoj a růst buňky. Mutace genu pro p53 mohou vést ke ztrátě jeho DNA vazebných vlastností a funkce nádorového supresoru, což muže způsobit rozvoj rakoviny. Teoretická část této diplomové práce je zaměřena na popis vlastností, funkce a mechanismus aktivace proteinu p53 a popis lokálních sekundárních struktur DNA. Hlavním cílem experimentální části byla produkce čtyř mutantních forem proteinů p53 a wild-type p53 proteinu a studium jejich vazebných vlastnosti s různými lokálními sekundárními strukturami DNA. Pomoci Gateway klonovacího systému byly připraveny čtyři expresní vektory, které byly použity pro produkci proteinů v bakteriálním expresním systému. Celkem byly úspěšně připraveny čtyři mutantní formy a wild-type p53 protein. Jejich vazebné vlastnosti byly studovány gelovou retardační analýzu. Výsledky naznačují různé DNA-vazebné vlastnosti wild-type p53 a studovaných mutantních forem tohoto proteinu. Všechny mutantní proteiny ztratily schopnost sekvenčně specificky vázat DNA, zatímco nespecifická interakce s DNA byla pozorována u tří ze čtyř mutantních forem. Jeden ze studovaných mutantních proteinů se vázal jenom na superhelikální formu DNA.

New drug discoveries and new approaches towards diagnosis and treatment have improved cancer therapeutics remarkably. One of the most influential and effective discoveries in the field of cancer therapeutics was antimetabolites, such as the antifolates. The interest in antifolates increased as some of the antifolates showed responses in cancers, such as mesothelioma, leukemia, and breast cancers. When pemetrexed (PTX) was discovered, our laboratory had established that the primary mechanism of action of pemetrexed is to inhibit thymidylate 22 synthase (TS) (E. Taylor et al., 1992). Preclinical studies have shown that PTX has a broad range of antitumor activity in human and murine models of cancer (Adjei, 2000; Adjei, 2004; S. Chattopadhyay, Moran, & Goldman, 2007; Miller et al., 2000). Accordingly, in February 2004, the FDA issued first-line treatment approval for pemetrexed in malignant pleural mesothelioma and in 2008 for first line treatment for locally advanced or metastatic NSCLC (reviewed in (Rollins & Lindley, 2005). As an antifolate this level of therapeutic activity of PTX against lung cancers was surprising and atypical (Hazarika, White, Johnson, & Pazdur, 2004). This led us to the question whether the effects of pemetrexed on other folate-dependent targets could explain the clinical activity of the drug. Our lab showed that, in addition to inhibiting thymidylate synthase, PTX also inhibits aminoimidazolecarboxamide ribonucleotide formyltransferase (AICART), the second folate-dependent enzyme of de novo purine synthesis. Inhibition of AICART leads to massive accumulation of its substrate 5-amino-4-imidazolecarboxamide ribonucleotide (ZMP), causing activation of AMP-dependent kinase (AMPK), which ultimately leads to suppression of mTORC1 signaling, a central regulator of cell growth and proliferation. This secondary mechanism could explain the unusual activity of PTX against mesothelioma and lung cancers. The large proportion of lung cancers are either null or mutant for p53 function. Therefore, this thesis focused on defining what the role of p53 is in the PTX-mediated AMPK activation and mTORC1 inhibition and how the loss of p53 affects mTORC1 signaling. These two questions proved to be interlinked. Chapter 2 investigates this relationship in detail. We found that, upon loss of p53, mTORC1 signaling is enhanced to a significant degree in colon carcinoma and lung cancer cell lines. Clearly, this observation required explanation. We found that the major factors responsible for these differences in mTORC1 activity upon loss of p53 23 were lower levels of two p53 target genes Tuberin (TSC2) and sestrin2. Immunoprecipitation studies of mTORC1 complexes from p53 wt and p53 null cells revealed quite interesting differences in the components of the mTORC1 complex. Immunoprecipitates from p53 null cells had higher levels of mTOR and lower levels of TSC2 and PRAS40 bound to raptor. This suggested that, in comparision to p53 competent cells, p53 null cells have more mTORC1 complex with enhanced activity due to decreased interaction of TSC2 and PRAS40, both of which are inhibitors of mTORC1. These observations explained the higher mTORC1 in p53 null cells and laid the foundation for determining the role of p53 in PTX-activated AMPK and mTORC1 inhibition. In the experiments described in Chapter 3, we found that PTX-mediated AMPK activation inhibited mTORC1 regardless of the p53 status in colon carcinoma cells. This suggested that mTORC1 inhibition by PTX was either independent of p53 mediated negative regulation of mTORC1 or was somewhere bypassing it. Therefore, we compared the effects of PTX with the classic AMPK activator aminoimidazolecarboxamide ribonucleoside (AICAR). In spite of a common mechanism of AMPK activation, namely, expansion of cellular ZMP levels, signaling from AMPK activated by PTX or AICAR were quite different. PTX-activated AMPK phosphorylated the mTORC1 component Raptor but not tuberin (TSC2), whereas AICARactivated AMPK phosphorylated both the targets. This differential behavior of two AMPK activators was due to differential behavior of p53 under these two treatments. Both, AICAR and PTX treatment led to increase in p53 levels but the p53 that accumulated after AICAR treatment was transcriptionally active while the p53 that accumulated after PTX treatment was not. Transcription of p53 targets, including TSC2 and sestrin2, was activated in AICAR- but not in PTX-treated cells. In the absence of p53 function, TSC2 was deficient and mTORC1 activity 24 enhanced, but Raptor phosphorylation by AMPK following PTX was robust and independent of both p53 and TSC2. Therefore we concluded that p53 deficiency suppresses TSC2 and upregulates mTORC1, but AMPK-phosphorylation of Raptor after pemetrexed treatment was sufficient to suppress mTORC1, even in TSC2 deficiency. This suggested pemetrexed as a drug for treatment of Tuberous Sclerosis, a genetic disease caused by functional inactivity of TSC1 or TSC2 due to point mutations in these genes. Mutation of p53 is one of the most common genetic alterations in human cancers and tumors. Cancers that express mutant p53 tend to be more aggressive, resistant to chemotherapy and show worse prognosis then p53-null tumors (Elledge et al., 1993; Olivier et al., 2006). This tumor-promoting activity of mutant p53 has been correlated with acquired and novel transcriptional activities of mutant p53. It has been shown that mutp53 can activate the transcription of cell growth promoting genes, such as, NFκB2, PCNA, MDR1, Axl, EGFR, hTERT, and HSP70, which are not usually transcriptional targets of wt p53. Interestingly, we found that whereas DNA damaging drugs enhance the acquired oncogenic transcriptional activities of mutp53, PTX interferes with this transcription activation. We also found in Chapter 4 that PTX can limit or block the DNA damaging drug-mediated increment of transcriptional activation of mutp53. This suggests that blockade of transcriptional activation of mutp53 by pemetrexed may provide an additional therapeutic benefit in mutp53 bearing cancers. As discussed in Chapter Three, although pemetrexed (with TdR) increases the levels of p53 and its binding to the promoter of its target gene, p21, this p53 is transcriptionally inactive. In order to understand the mechanism of the pemetrexed-mediated transcriptional defect of wt p53, we studied the PTX-mediated signaling towards ATM and ATR and their effects on their substrates Chk2 and Chk1, respectively. These studies suggested that the difference between 25 signaling under AICAR treatment and PTX treatment was that, unlike PTX, AICAR treatment was leading to DNA damage, followed by Chk2 phosphorylation at Thr68. We found there were three major differences between AICAR and pemetrexed (+ TdR) mediated signaling: AICAR caused DNA damage, followed by ATM mediated phosphorylation of Chk2 at Thr68 and phosphorylation of p53 at Ser15 all of which lead to activation of p53 transcriptional activity, events which do not take place under PTX treatment. Studies aimed at understanding the effects of PTX on wt and mutp53 transcriptional activities are discussed in detail in Chapters Three and Four of this dissertation. Overall, we concluded that PTX interferes with the transcription activity of wild type as well as gain-of-function mutant p53. The blockade of DNA damaging agent-mediated enhancement of mutp53 transcription activity by PTX, suggests the clinical relevance of PTX in carcinomas with mutp53. We suggest that this could be one of the contributing factors in the effects of PTX against human lung cancers.