Dissertations / Theses on the topic 'Mutant p53 gain of function'

p53 is mutated in 50% of all human cancers, and up to 70% of lung cancer. Mutant p53 is usually expressed at elevated levels in cancer cells and has been correlated with a poor prognosis. Cancer cells that express mutant p53 show an increase in oncogenic phenotypes including an increase in growth rate, resistance to chemotherapeutic drugs, and an increase in motility and tumorigenicity to name a few. We have identified several genes involved in cell growth and survival that are upregulated by expression of common p53 mutants: NFκB2, Axl, and epidermal growth factor receptor (EGFR). The aim of this study was to determine the role NFκB2, Axl, and EGFR play in mutant p53’s gain of function (GOF) phenotype and to determine a mechanism for upregulation of mutant p53 target gene upregulation. Inhibition of mutant p53 in various cancer cell lines using RNAi in the form of transient siRNA transfection or stable shRNA cell line generation caused a decrease in the gain of function ability of those cells in the form of reduced chemoresistance, reduced cell growth and motility, and a reduction in tumor formation. Additionally, inhibition of NFκB2, Axl, and EGFR also showed similar effects. Promoter deletion analysis of the NFκB2 promoter did not show a specific mutant p53 response element needed for mutant p53 mediated transactivation. Similarly, deletion of the p53/p63 binding site on the Axl promoter did not inhibit mutant p53 transactivation. Sequence analysis of the NFκB2, Axl, and EGFR promoters revealed several transcription factor binding sites located throughout the promoters. ChIP analysis of mutant p53 and the promoter-specific transcription factor binding revealed that in the presence of mutant p53, individual transcription factor binding is increased to the NFκB2, Axl, and EGFR promoters as well as an increase in acetylated histone binding. This data suggests that mutant p53 promotes an increase in transcription by inducing acetylation of histones via recruitment of transcription factors to the promoters of mutant p53 target genes.

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.

p53 is a transcription factor with tumour suppressive attributes which is known to be mutated in over half of human cancers. As well as compromising the ability of p53 to function as a transcription factor, mutations in p53 often result in a gain-of-function phenotype which is characterised by increased ability of cancer cells to migrate and invade. This is mediated by the ability of mutant p53 to increase recycling of α5β1 integrin and receptor tyrosine kinases (RTK) from endosomes to the plasma membrane; a process which is dependent on the Rab11 effector, Rab Coupling Protein (RCP) and the phosphatidic acid generating enzyme, diacylglycerol kinase-α (DGKα). Despite accumulating evidence linking RCP/DGKα-dependent receptor recycling to invasive migration, the mechanisms by which mutant p53 controls endosomal trafficking were still unclear when the current study was instigated. Initial experiments indicated that the mutant p53 gain-of-function phenotype was not cell autonomous, and could be passed to p53 null cells by incubating them with conditioned medium from mutant p53 (R273H)-expressing cells. Furthermore, fractionation approaches indicated that the mutant p53 phenotype was transmitted between cells by a microvesicle vector. Upon treatment with microvesicles collected from mutant p53 expressing cells, p53 null cells displayed increased α5β1 integrin and RTK recycling and the consequent invasive/migratory behaviour that was dependent on these RCP and DGKα-regulated trafficking events. Despite a requirement for RCP in the response of p53 null cells to microvesicles, this Rab11 effector was not required for the production of pro-invasive microvesicles. Rather, mutant p53-expressing cells relied on Rab35 (but not Rab27a or Rab27b) for the production and/or release of microvesicles that were capable of transferring mutant p53’s gain-of-function phenotype. An in-depth RNA sequencing analysis indicated that microvesicles from mutant p53 cells influenced the endocytic trafficking and migratory characteristics of p53 null cells without detectably altering mRNA expression in these recipient cells. This indicated the possibility that microvesicles from mutant p53-expressing cells may act directly on the endomembrane system of recipient cells. Immunoprecipitation experiments indicated that there was a physical interaction between Rab35 and podocalyxin (PODXL), a highly-charged sialomucin which is known to directly influence membrane organisation. Additionally, PODXL was detectable in microvesicular preparations by mass spectrometry. Microvesicles purified from mutant p53-expressing cells in which PODXL had been knocked down using siRNA, had significantly reduced capacity to promote integrin/RTK recycling and mutant p53-like migratory behaviour in p53 null cells, indicating that PODXL, as well as Rab35, is a key factor responsible for transmitting mutant p53’s gain-of-function phenotype between cells. In addition to being incapable of influencing the migration of other cells, Rab35 knockdown cells themselves migrated with the characteristics of p53 null cells. Interestingly, microvesicles from mutant p53-expressing cells restored mutant p53-like migratory behaviour in these Rab35 knockdown cells. These data indicate that Rab35 and PODXL-dependent production of phenotype altering microvesicles not only influences the migration of neighbouring cells in a paracrine fashion, but may constitute an autocrine link between mutant p53 and integrin trafficking in the mutant p53 cells themselves. Finally, I have found that p53 null cells may be educated by microvesicles from mutant p53-expressing cells to themselves release cell migration-altering microvesicles, providing further evidence supporting the existence of microvesicle-based autocrine/paracrine mechanisms that may act to propagate mutant p53’s invasive gain-of-function within both homogeneous and heterogeneous populations of tumour cells.

It was hypothesized that the C-terminal sequences for mutant p53 would be required for oligomerization. and oligomerization may be critical for gain-of-function. An N-terminal deletion mutant of p53 that deletes amino acids 1-293 was used as a tool to perform hetero-oligomerization studies. This mutant retains the entire oligomerization domain but dispenses off the transactivation domain and a large portion of the sequence- specific DNA-binding domain. Co-transfection experiments show that p53 del. 1-293 forms hetero-oligomeric complexes with p53-D281G. Also. co-expression of p53 del. 1- 293 with p53-D281G inhibited p53-D28lG-mediated transactivation of the EGFR and MDRl promoters suggesting that hetero-oligomerization inactivates transcriptional functions of mutant p53. The interaction of p53 deli 1-293 and p53-D281G reduced transactivation potential of p53-D281G in stably transfected 10(3) murine cells. Therefore, the data presented supports the idea that proper oligomeric forms of mutant p53 are required for its transactivation function. Expression of mutant p53-D2810 also resulted in increased growth rate (H1299 cells), decreased chemosensitivity (H1299 and 21PT cells) and increased plating efficiency (Saos-2 cells). Expression of a transactivation deficient mutant p53 did not induce gain-of-function properties (increased growth rate and decreased chemosensitivity). Unlike the other gain-of-function properties tested, soft agar plating efficiency in Saos-2 cells was not significantly affected by the expression of a transactivation deficient mutant p53, suggesting that transactivation may not be the only factor affecting this gain-of-function property In order to identify the genes responsible for the observed phenotypes, global gene expression analyses were carried out using p53-null H1299 cell stably transfected to express mutant p53 (-Rl75H, -R273H and -D281G). A thorough and stringent analysis revealed 150 genes up-regulated by the expression of mutant p53. Up-regulation of a number of these genes was confirmed by QPCR and transient transcriptional promoter analyses; expression of the transactivation deficient mutant p53-D2810 (L22Q/W23S) did not result in up-regulation of the tested genes further supporting the idea that transactivation of genes is directly related to gain-of-function phenotypes. Using the ASNS gene as a model, this transactivation by mutant p53 was concentration dependent and that the increased transcription did indeed result in increased protein levels.

2009/2010
Mutations in the TP53 gene are among the most frequent genetic alterations in human cancers. As a consequence of these mutations p53 loses its tumour suppressor functions and may acquire novel oncogenic activities (gain of function) sustaining tumour formation and progression. Many in vivo studies highlighted that mutant p53 gain of function is associated with elevated protein levels, supporting the notion that in tumour cells altered signalling could stabilize and activate mutant p53, with mechanisms similar to those required to stimulate wild-type p53. The aim of my PhD work was to investigate the mechanisms underlying mutant p53 gain of function, focusing on factors that might link cancer-related signalling with mutant p53 activity. An intriguing candidate for this role is the phosphorylationdependent prolyl isomerase Pin1, that transduces phosphorylation signalling into conformational changes affecting the functions of its substrates, as ours and other laboratories have reported for wild-type p53. Despite Pin1 supports wild-type p53 functions, Pin1 is frequently overexpressed in human tumours and has been shown to promote both Her2/Neu/Ras and Notch1 dependent transformation. So we reasoned that the physiological role of Pin1 as a component of checkpoint mechanisms might be subverted during tumourigenesis, thereby turning it into an essential partner of mutant p53 and a critical amplifier of its oncogenic functions. Indeed, we now demonstrate that Pin1 enhances tumourigenesis in a Li-Fraumeni mouse model and cooperates with mutant p53 in Ras-dependent cell transformation. In human breast cancer cells, Pin1 promotes both mutant p53 dependent inhibition of the anti-metastatic factor p63 and the induction of a mutant p53 transcriptional program to increase tumor aggressiveness. Accordingly, we have identified a transcriptional signature (the Pin1/mutant p53 signature) that is associated with poor prognosis in breast cancer and, in a cohort of patients, Pin1 over-expression influences the prognostic value of p53 mutation. Considering that TP53 mutation is more frequent in tumors with higher risk of recurrence such as triple-negative cases and that some of the Pin1/mutant p53 signature genes are over-expressed in triple negative breast cancers, our findings carry therapeutic implications for this kind of cancers and possibly also for other tumours bearing mutant p53 and high levels of Pin1.
XXII Ciclo
1983

Head and neck squamous cell carcinoma (HNSCC) is one of the ten most common cancers worldwide, with a survival rate of less than 50%. A class of mutant p53, known as gain of function (GOF) mutant p53, has been found to be expressed in tumors in these patients. GOF mutant p53 not only loses the wild type tumor suppressor functions, but also gains aberrant functions that have been linked to tumorigenesis. In this current study, we utilized a model system consisting of cells derived from HNSCC tumors in order to investigate our hypothesis that GOF mutant p53 enhances, and p63 inhibits, EPS8 and CXCL5 expression and promoter activity. We found decreased EPS8 expression, CXCL5 expression, and cellular migration associated with the loss of GOF mutant p53. This indicates an enhancing role of GOF mutant p53 in cellular migration and expression of these target genes. The loss of GOF mutant p53 was also associated with decreased EPS8 and CXCL5 promoter activity, indicating upregulation of these target gene promoters by GOF mutant p53. We found increased EPS8 expression,CXCL5 expression, and cellular migration with the loss of p63 in cell expressing high levels of p63. This indicates an ixinhibitory role of p63 on the expression of these target genes and cellular migration. Loss of p63 was also associated with increased EPS8 and CXCL5 promoter activity, indicating p63 may be downregulating these target gene promoters. Based on our knowledge of EPS8 and CXCL5 in tumorigenesis, our findings suggest that GOF mutant p53 and p63 play opposing rolesin HNSCC tumorigenesis. Additional studies are needed to further elucidate the mechanism by which GOF mutant p53 and p63 regulate EPS8 and CXCL5 expression and promoter activity.

Recent advances in understanding lung biology have shown evidence for the existence of resident lung stem cells. Independent studies in identifying and characterizing these somatic lung stem cells have shown the potential role of these cells in lung repair and regeneration. Understanding the functional characteristics of these tissue resident stem/progenitor cells has gained much importance with increasing evidence of cancer stem cells, cells in a tumor tissue with stem cell characteristics. Lung cancer is most commonly characterized by loss of p53 function which results in uncontrolled cell divisions. Incidence of p53 point mutations is highest in lung cancer, with a high percentage of missense mutations as a result of tobacco smoking. Certain point mutations in p53 gene results in its oncogenic gain of functions (GOF), with enhanced tumorigenic characteristics beyond the loss of p53 function. However, there are no available data on characterization of lung stem cells carrying GOF mutations and correlating them with those of normal stem cells, in this study, for the first time we show that percentage of Sca-1 expressing subpopulation is significantly higher in the lungs of mice carrying p53 GOF mutations than those in lungs isolated from p53+/+ wild type mice. Further, we successfully established lung cells differentially expressing two cell surface markers, Sca-1 and PDGFR-α, with results demonstrating existence of different subpopulations of cells in the lung. Results from our project demonstrate the importance of p53 GOF mutations as correlated with specific lung cell subpopulations.

Le système immunitaire joue un rôle important dans le contrôle et l'éradication du cancer. Des acteurs majeurs de la réponse immune antitumorale sont les cellules tueuses naturelles (ou cellules NK) et les lymphocytes T cytotoxiques (ou CTL), capable de reconnaitre et détruire des cellules tumorales par l’exocytose de perforine et de granzymes contenus dans leur granule cytotoxique. Il a été montré au sein du laboratoire l’implication de la protéine suppresseur de tumeur p53 dans cette voie apoptotique. Or, plus de 50% des tumeurs humaines présentent des mutations inactivatrices de p53 ce qui favorise le développement tumoral. De ce fait, l’inactivation fréquente de p53 dans les tumeurs humaines pourrait leur permettre d’échapper à la destruction par les CTL et les cellules NK.Dans ce contexte, mes travaux de thèse ont montré que la réactivation pharmacologique de la fonction de p53 sauvage dans des cellules tumorales exprimant une p53 mutée augmente leur susceptibilité à la lyse induite par les cellules NK grâce à l’induction d’un processus d’autophagie. De plus, j’ai cherché à déterminer le lien entre les mutations de p53 et l’expression à la surface des cellules tumorales de PD-L1 qui empêche l’activation optimale des cellules cytotoxiques et conduit à leur épuisement. Mes travaux actuels suggèrent que l’expression de p53 mutantes induits une surexpression de PD-L1 à la surface des cellules cancéreuses. Les mécanismes expliquant ce phénomène sont en cours d’études
Immune system plays an important role in the control and destruction of cancer cells. The major effectors of antitumor immune response are Natural Killer (NK) cells and the cytotoxic T lymphocytes, which recognize et destroy tumor cells by exocytosis of perforin and granzymes contained in cytotoxic granules. It has been previously shown in the laboratory that the tumor suppressor p53 plays an important role in this apoptotic pathway. However more than 50% of human tumors have p53 inactivating mutations which favor tumor development. Consequently, frequent p53 inactivation in human tumor could enable them to escape from destruction by cytotoxic immune cells. In this context, my thesis work has shown that the pharmacological reactivation of wild type p53 function in cancer cells expressing a mutated p53 increased their susceptibility to NK cell-mediated apoptosis cells through the induction of an autophagic process. Moreover, I tried to determine the link between p53 mutations and the expression of the immune checkpoint ligand PD-L1 which prevent efficient activation of cytotoxic cells and promote immune cells exhaustion. My work suggests that the expression of p53 mutants promotes an the expression of PD-L1 at the cancer cell surface. The study of the underlying mechanisms is still in progress

A number of recent studies suggest that expression of mutant p53 with mutations in certain codons show a gain of function and some of the characteristics of an oncoprotein. In order to study gain of function mutation and eliminate the potential of a dominant negative interaction with endogenous wild type p53 protein, p53 knockout mouse astrocytes were used. A retrovirus system was used to introduce mutant p53 genes into these p53-null astrocytes. Immunohistochemical staining and western blot experiments showed the expression of mutant p53 protein in these cells after infection with the mutant p53 retroviruses. Cell growth experiment did not suggest growth advantages for mutant p53 expressing astrocytes over vector control cells. Data from clonogenic survival assays following exposure to etoposide or cisplatin suggested that mutant p53 expressing cells with a point mutation at codon 273 may be resistant to apoptosis induced by etoposide. In contrast, p53 with a point mutation at codon 248 may sensitize cells to the apoptotic effects of etoposide and cisplatin.

p53 is an important tumor suppressor gene which is mutated in ~50% of all human cancers. Some of the p53 mutants appear to have acquired novel functions beyond merely losing wild-type functions. To investigate these gain-of-function effects in vivo, we interbred MMTV-v-Ha-ras transgenic mice to either p53-/- knock-out mice or p53R172H/+ knock-in mice to generate mice of three different genotypes: MMTV-ras, MMTV-ras/p53-/-, and MMTV-ras/p53R172H/R172H. Male mice of each of these genotypes were characterized with regard to age of salivary tumor onset and the tumors were characterized with regard to mean growth rates, proliferation fraction, apoptotic levels, and tumor histopathology, as well as responses to doxorubicin treatment. Microarray analysis was also performed to profile gene expression.The MMTV-ras/p53-/- and MMTV-ras/p53R172H/R172H mice display similar properties in age of tumor onset, tumor growth rates, and tumor histopathology, as well as response to doxorubicin. However, a subset of genes show differential expression between the two groups of tumor , and do not appear to be regulated by wild-type p53. At the same time, the MMTV-ras/p53R172H/R172H and MMTV-ras/p53+/+ tumors share similar expression levels of a group of genes that are differentially expressed in the MMTV-ras/p53-/- tumors. Thus, the gain-of-function effects may be caused in part by perturbed regulation of genes not normally regulated by wild-type p53, in addition to imbalances in the regulation of normal p53 target genes.

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease characterized by degeneration of motorneurons leading to progressive paralysis and eventually death due to respiratory failure. So far, no effective treatments are available for this devastating disease. Interestingly, genetic forms of ALS show the same pathological alterations observed in the most frequent sporadic cases. For this reason, studying the role of single disease causing mutations is important to gain insight into the pathophysiological events that lead to disease development. Ten years ago a mutation in the gene encoding for VAPB was associated with a dominantly inherited form of ALS (termed ALS8). Moreover, sporadic ALS patients have reduced levels of the endogenous protein. VAPB belongs, along with its homologue VAPA, to the VAP protein family: ER resident tail anchored proteins that, thanks to their binding partners, are involved in several cellular functions like lipid transport, ER stress and membrane contact site formation. The ALS-associated mutation P56S dramatically alters VAPB structure, thus preventing the binding to its physiological interactors and causing its aggregation. The cellular and molecular mechanisms underlying pathogenicity of mutant VAPB are still poorly understood. It is still unclear whether P56S-VAPB-generated inclusions exert a direct toxic function or whether reduced levels of the wt protein are sufficient to trigger the disease. An intermediate situation, where both mechanisms contribute to ALS development, could also be possible. The aim of my thesis is to analyze the contribution of mutant VAPB gain or loss of function in the development of ALS. To reach this goal, I used two complementary approaches: I analyzed cellular models that either express moderate levels of P56S-VAPB or have reduced levels of the endogenous protein. Our findings would be relevant not only to understand the pathogenesis of ALS8, but also for forms of sporadic ALS, in which VAPB levels are reduced.

Basal-like breast cancers (BLBC) are an aggressive sub-type of breast cancer which associates with high rates of proliferation and metastasis. However, the molecular mechanisms underlying these tumours are still largely unknown as most BLBC are also triple-receptor negative, which is defined by the lack of expression of oestrogen (ER) and progesterone (PR) receptors, with normal HER2 status. Consequently, there are currently no targeted therapies available for these tumours, which display the worst prognosis of all breast cancer subtypes. TP53 mutations occur within 20% of all breast tumours, however, this rate is observed to increase dramatically to approximately 85% within the basal-like subtype. Moreover, TP53 missense mutations can lead to the production of mutant proteins which possess novel oncogenic capabilities, known as Gain-of-Function (GOF) p53 mutants. These GOF mutants may display wild-type protein conformation (contact mutants) or exhibit conformational alterations to the tertiary protein structure (structural mutants). We have shown that siRNA-mediated knockdown of endogenous mutant p53, particularly contact GOF mutants significantly reduces survival of BLBC cells. We also demonstrate that contact, but not structural, p53 mutant proteins can directly bind to the transcription factor Etsl, and are also involved in upregulation of its expression. We show that mutant p53 interaction with Etsl is a critical event for both survival of these cells and co-regulation of downstream target genes. We have identified several novel mutant p53-specific transcriptional targets, some of which play important roles in the proliferation and survival of BLBC cells. Additionally, we have putatively detected an alternatively-spliced p53 protein isoform following over-expression of a structural GOF p53 mutant in non-tumourigenic and tumour-derived cell lines.

Loss of p53 function is a critical event in tumour development. It has been well documented that p53 mutation can lead to an oncogenic gain of function (GOF) associated with poorer prognosis and therapeutic response. Such mutants exert their gain-of-function properties by modifying cells through altered transcriptional activity as well as novel protein-protein interactions, but the molecular details of the mechanisms involved are still not entirely understood. Recent reports indicate that mutant p53 increases TGFβ-dependent metastatic potential of cancer cells via inhibition of p63 function and also promotes invasion through increased integrin (aSpl) and EGFR recycling, which is also linked with suppression of transcriptionally active p63 in normal epithelial cells, lung cancer cells and invasive breast cancer cells. The aim of this study was to investigate the mechanism of mutant p53 GOF in squamous cell carcinoma of the head and neck (SCCHN) cells. To avoid the potentially confounding impact of studying cells from biologically distinct anatomical sub-sites, the studies described here focused upon the most common sub-type of SCCHN -laryngeal squamous cell carcinoma (LSCC). Two hot-spot p53 mutants representing two distinct mutational classes have been chosen for these studies: R17SH (structural mutation) and R273H (contact mutation), both of which have been previously demonstrated to have GOF properties and have been documented to occur in laryngeal cancer. The mechanism of mutant p53 GOF was investigated by identifying protein interactions of mutant p53 and by studying gene expression changes in LSCC cells expressing these p53 mutants. In addition, the functional consequences of mutant p53 expression in LSCC cells were investigated in terms of response to radiation (a primary treatment modality for LSCC) and motility/invasiveness (key determinants of metastatic potential). A number of proteins have been identified in the mutant p53 containing protein complexes in LSCC cells and one of these, HSP70, has been confirmed by immunoprecipitation to selectively interact with the p53-175H mutant. p53-273H-expressing cells and to a smaller extent p53-175H-expressing cells display increased motility. A stable knock-down of the mutant p53 has been shown to reduce the motility of cells suggesting that p53-273H contributes to increased

Senescence stimuli activate multiple tumor suppressor pathways to initiate cycle arrest and a differentiation program characteristic of senescent cells. We performed a two-stage, gain-of function screen to select for the genes whose enhanced expression can bypass replicative senescence. We uncovered multiple genes known to be involved in p53 and Rb regulation, ATM regulation and two components of the CST complex involved in preventing telomere erosion and additional genes such as REST and FOXO4 that have been implicated in aging. Among the new genes now implicated in senescence we identified DLX2, a Homeobox transcription factor that has been shown to be required for tumor growth, metastasis and associates with poor cancer prognosis. Growth analysis showed that DLX2 expression led to increased cellular replicative lifespan. We found that DLX2 expression inhibited p53 activation, and DLX2 reduced the protein level of upstream activator kinases ATM and DNA-PK. Our data suggest that DLX2 expression reduces the protein components of the TTI1/TTI2/TEL2 complex, a key complex required for the proper folding and stabilization of ATM, DNA-PK and other members of the PIKK family. Over-expression of DLX2 exhibit mutual exclusivity with p53 alteration in cancer patients, suggesting DLX2 may attenuate the p53 pathway during tumor formation. Our functional screen identified novel players that may promote tumorigenesis by regulating the ATM-p53 pathway and senescence.
Medical Sciences

The tumor suppressor gene TP53 sits at the crux of response to cellular stresses. This is the most frequently inactivated gene in human tumors, being the target of somatic mutations. The protein product of TP53 is p53, and plays a crucial role in anti-proliferative signals through the induction of apoptosis, senescence, and cell-cycle arrest when activated by stresses such as genotoxic chemotherapeutic drugs. Therefore, the status of TP53 mutation in a tumor has profound implications for the tumorigenic potential as well as the response to anti-cancer therapies. Indeed, numerous studies have shown a predictive and prognostic value of TP53 mutations to the response to chemotherapy, but just as many studies show no significant contribution of TP53 mutations to chemotherapy response. This controversy is partly due to the lack of standard methods of TP53 mutation detection, but more importantly, it is due to the categorization of all TP53 mutations into one group. Certain mutations in TP53 can confer a mutant p53 with new, gained activities, not normally present in the WT p53 protein. These have been commonly called "gain of function" (GOF) p53 proteins, and some GOF p53 proteins can even confer oncogenic properties. However, not all gained functions are necessarily implicated in oncogenicity. Using stringent criteria, we have defined a select group of GOF TP53 mutations that do function as oncogenic proteins as oncomorphic TP53 mutations. In this work, we utilize data available from a large patient population through The Cancer Genome Atlas (TCGA) as well as data available from the University of Iowa Gynecologic Oncology Tumor Bank to examine the association of oncomorphic TP53 mutations with patient outcome using advanced serous ovarian cancer as a model. We demonstrate that oncomorphic TP53 mutations are associated with worse progression-free survival, chemoresistance, and higher rates of recurrence than other mutations in TP53 that have no evidence of oncomorphic abilities. We identify molecular alterations in patients with oncomorphic TP53 mutations, particularly the increased expression of β-catenin. We also observe that oncomorphic p53 proteins lose the normal protein:protein interactions with the microRNA microprocessing complex, implicating the role of dysregulated miRNAs in pathways associated with chemoresistance. The cumulative results from our studies provide human evidence for the consideration of different classes of TP53 mutations. Patients with oncomorphic TP53 mutations deserve careful follow-up therapy and may require novel treatment regimens to improve outcomes. We propose that stratification of patients should be considered based upon the individual TP53 mutation identified from their tumors.

Le mutazioni del gene TP53, che compromettono la funzione trascrizionale di p53, si verificano in più del 50% dei tumori umani, tra cui l’adenocarcinoma pancreatico, e rendono le cellule tumorali più resistenti alle terapie convenzionali. Negli ultimi anni, molti sforzi sono stati indirizzati per identificare molecole-riattivanti la proteina p53 mutata ri-conferendole una conformazione wild-type e attiva trascrizionalmente. Nella presente tesi di Dottorato, dimostriamo che due di queste molecole, CP-31398 e RITA, possono indurre inibizione della crescita di cellule di adenocarcinoma pancreatico umano, apoptosi e autofagia attivando il legame p53/DNA e la fosforilazione di p53 (ser15), senza modificare la quantità della proteina totale. Questi effetti si verificano sia nelle linee cellulari di adenocarcinoma pancreatico p53 wild-type che nelle p53 mutate (mutp53), mentre sono molto meno pronunciati nelle normali linee cellulari primarie di fibroblasti umani. Inoltre, CP-31398 e RITA regolano l'asse SESN1- 2/AMPK/mTOR inducendo la fosforilazione di AMPK in Thr172, che ha un ruolo cruciale nella risposta autofagica. Il ruolo protettivo dell'autofagia con inibizione della crescita delle cellule da CP-31398 e RITA è supportato dall’ osservazione che l'inibitore dell’AMPK compound C o gli inibitori dell’autofagia clorochina e 3-metiladenina sensibilizzano le linee cellulari di adenocarcinoma pancreatico alla risposta apoptotica indotta dalle molecole riattivanti p53. I nostri risultati dimostrano per la prima volta il ruolo di sopravvivenza dell'autofagia indotta dalle molecole riattivanti p53 in cellule tumorali p53 mutate. Le proteine p53 mutate perdono non solo la loro funzione di soppressore tumorale, ma anche acquistano nuove proprietà oncogene note come (GOF- “gain of function”) "guadagno di funzione". Generalmente, le proteine p53 mutate sono sovra-espresse nelle cellule tumorali e promuovono le attività GOF aumentando la proliferazione delle cellule tumorali e la loro resistenza ad una varietà di farmaci chemioterapici comunemente utilizzati in pratica clinica. Con il guadagno di funzione la proteina mutp53 si comporta come fattore di trascrizione oncogenico interagendo con altri regolatori trascrizionali, come E2F1, NF-Y e VDR. Inoltre, le proteine p53 mutate possono anche interagire con proteine oncosoppressorie inibendo le loro funzioni. Con questo lavoro dimostriamo che la gemcitabina (GEM), chemioterapico standard, attiva fortemente mutp53 stimolando la sua fosforilazione (Ser15) e la traslocazione nucleare. Questi eventi provocano l’attivazione della GOF in cellule tumorali p53 mutate, come evidenziato dalla stimolazione del ciclo cellulare promossa dai geni, come Cdk1 e CCNB1, dopo il trattamento con GEM. Inoltre, abbiamo dimostrato che il silenziamento di mutp53 aumenta la sensibilità delle cellule tumorali a GEM e che l'aggiunta di CP-31398 o RITA al trattamento con GEM può sinergicamente indurre apoptosi in linee cellulari di adenocarcinoma pancreatico sia p53 wt che p53 mutate, e che questi effetti non sono presenti in cellule tumorali che non esprimono il gene per p53. Questa combinazione di composti induce fortemente la fosforilazione di p53 in Ser15 (senza modificare la concentrazione totale di p53), l'apoptosi, e la formazione dell'autofagosoma. Inoltre, abbiamo dimostrato che l'autofagia provocata da GEM/CP-31398 ha un ruolo protettivo per le cellule tumorali. Infatti, l'aggiunta del inibitori dell’autofagia, clorochina o 3-metiladenina, aumenta l'apoptosi indotta dal trattamento GEM/CP-31398. I nostri risultati supportano lo sviluppo di una strategia antitumorale basata sull'inibizione autofagia associata al trattamento combinato di molecole riattivanti p53 con la chemioterapia tradizionale, nell’adenocarcinoma pancreatico sia con p53wt che con p53 mutato.
TP53 gene mutations compromising p53 transcriptional function occur in more than 50% of human cancers, including pancreatic adenocarcinoma, and render cancer cells more resistant to conventional therapy. In the last few years, many efforts have been addressed to identify p53-reactivating molecules able to restore the wild-type transcriptionally competent conformation of the mutated proteins. In the present thesis, we show that two of these compounds, CP-31398 and RITA, can induce cell growth inhibition, apoptosis, and autophagy by activating p53/DNA binding and p53 phosphorylation (Ser15), without affecting the total amount of p53. These effects occur in both wild-type and mutant p53 (mutp53) pancreatic adenocarcinoma cell lines, whereas they are much less pronounced in normal human primary fibroblasts. Furthermore, CP-31398 and RITA regulate the axis SESN1- 2/AMPK/mTOR by inducing AMPK phosphorylation in Thr172, which has a crucial role in the autophagic response. The protective role of autophagy in cell growth inhibition by CP-31398 and RITA is supported by the finding that the AMPK inhibitor compound C or the autophagy inhibitors chloroquine or 3-methyladenine sensitize pancreatic adenocarcinoma cell lines to the apoptotic response induced by p53-reactivating molecules. Our results demonstrate for the first time a survival role for autophagy induced by p53 reactivating molecules in p53 mutant cancer cells. Mutp53 proteins not only lose their tumor suppressive function but also gain new oncogenic properties known as “gain-of-function” (GOF). Generally, mutp53 proteins are over-expressed in cancer cells and promote GOF activities enhancing the proliferation of cells and their resistance to a variety of chemotherapeutic drugs commonly used in the clinical practice. GOF activities are carried out because mutp53 proteins behave as oncogenic transcription factors by interacting with other transcriptional regulators, such as E2F1, NF-Y and VDR. Moreover, these p53 mutant proteins can also interact with oncosuppressor proteins inhibiting their function. Here, we show that the standard drug gemcitabine (GEM) strongly activates mutp53 by stimulating both its phosphorylation (Ser15) and nuclear translocation. These events result in the stimulation of mutp53 GOF in cancer cells bearing mutant p53, as revealed by the stimulation of cell cycle promoting genes, as Cdk1 and CCNB1, after GEM treatment. Furthermore, we demonstrate that silencing of mutp53 strongly increases sensitivity of cancer cells to GEM and that the addition of CP-31398 or RITA to GEM treatment can synergistically induce apoptotic cell death in both wt and mutant p53 pancreatic adenocarcinoma cell lines, whereas these effects are missing in p53-null cancer cells. This drug combination strongly induces p53 phosphorylation in Ser15 (without affecting the total amount of p53), apoptosis, and autophagosome formation. Furthermore, we demonstrate that autophagy stimulation by GEM/CP-31398 has a protective role for cancer cells. In fact, the addition of the autophagy inhibitors, chloroquine or 3-methyladenine, increases apoptosis induced by GEM/CP-31398 treatment. Our results support the development of an anti-tumoral strategy based on autophagy inhibition associated to the combined treatment of p53-reactivating molecules with standard chemotherapy, for both wild-type and mutant p53 pancreatic adenocarcinoma cell types.

p53 is the most frequent target for mutation in human tumors and mutation at this locus is a common and early event in breast carcinogenesis. Breast tumors with mutated p53 often contain abundant levels of this mutant protein, which has been postulated to actively contribute to tumorigenesis by acquiring pro-oncogenic ("gain-of-function") properties. To elucidate how mutant p53 might contribute to mammary carcinogenesis, we employed a three-dimensional (3D) culture model of breast cancer. When placed in a laminin-rich extracellular matrix, non-malignant mammary epithelial cells form structures highly reminiscent for many aspects of acinar structures found in vivo. On the other hand, breast cancer cells, when placed in the same environment, form highly disorganized and sometimes invasive structures. Modulation of critical oncogenic signaling pathways has been shown to phenotypically revert breast cancer cells to a more acinar-like morphology. We examined the role of mutant p53 in this context by generating stable, regulatable p53 shRNA derivatives of mammary carcinoma cell lines to deplete endogenous mutant p53. We demonstrated that, depending on the cellular context, mutant p53 depletion is sufficient to significantly reduce invasion or in some cases actually induce a phenotypic reversion to more acinar-like structures in breast cancer cells grown in 3D culture. Additionally, using stable overexpression of a panel of tumor-derived p53 mutants in non-malignant mammary epithelial cells, we found that mutant p53 is sufficient to disrupt normal acinar morphogenesis. Genome-wide expression analysis identified the mevalonate pathway as significantly upregulated by mutant p53. Statins and sterol biosynthesis intermediates revealed that this pathway is both necessary and sufficient for the phenotypic effects of mutant p53 on mammary tissue architecture. We then showed that mutant p53 associates with sterol gene promoters at least partly via SREBP transcription factors. Finally, p53 mutation correlates with highly expressed mevalonate pathway genes in human breast tumors and elevated expression of the mevalonate pathway correlates with a poor prognosis in breast cancer. We also queried a number of pathways/proteins that had previously been implicated in breast cancer and shown to be sufficient to bring about a phenotypic reversion in 3D culture to search for additional mechanisms by which mutant p53 might contribute to mammary carcinogenesis. Using this approach, we identified integrin β4 as a novel target of mutant p53 in breast cancer cells and demonstrated that stable knockdown of integrin β4 is sufficient to dramatically reduce invasive processes in breast cancer cells grown in 3D culture. We also show that mutant p53 associates with the promoter of ITGB4, the gene encoding integrin β4. Finally, we demonstrated that inhibition of NF-κB, a downstream mediator of integrin β 4 signaling, can mimic the phenotypic effects of mutant p53 depletion. These findings contribute to our understanding of breast carcinogenesis and may offer novel prognostic indicators and therapeutic targets for tumors bearing mutations in p53.

The TP53 tumour suppressor gene is mutated in approximately 50% of all human cancers. The majority of these mutations are missense mutations resulting in the expression of a mutated form of the full-length p53 protein. This mutant protein exhibits a loss of tumour suppressive activity, dominant-negative activity to inactivate functional p53 and gain-of-function properties to drive tumour progression and metastasis. Investigation into mutant p53-mediated oncogenic pathways and the mechanisms through which they are controlled plays an integral role in identifying new therapeutic targets for a range of mutant p53-expressing tumours. To model the initial events that occur in cancer following sporadic p53 mutation, an isogenic panel of cell lines was established in the p53 null, H1299 lung cancer cell line, expressing wild-type or various p53 hotspot mutants under the control of an inducible promoter. These cell lines were harnessed to investigate a range of wildtype and mutant p53 functions. The induced wild-type p53 protein is demonstrated to be transcriptionally and biologically active, and its function can be further mediated by DNA damaging agents or expression of regulatory proteins. Conversely, induced mutant p53 exhibits a loss of the majority of the normal wild-type transcriptional activity while mediating gain-of-function, oncogenic phenotypes in H1299 cells. This system is demonstrated to provide an important platform with which to investigate both wild-type and mutant p53 function. Mutant p53 is reported to function as an aberrant transcription factor, re-programming the cellular transcriptome to enhance oncogenic pathways. The mechanisms underlying this were specifically examined through expression microarray analysis, which identified a number of mutant p53-regulated targets. Surprisingly, these targets were predominately also direct targets of wild-type p53. A novel mechanism for mutant p53 activity is subsequently suggested, whereby mutant p53 is recruited to the DNA through its interaction with p63. A key function of mutant p53 is its ability to drive tumourigenesis through the initiation of a range of oncogenic pathways. Through utilising the inducible system, mutant p53 is demonstrated to influence mitotic pathways, resulting in multinucleation, and enhance the invasive and migratory properties of cancer cells. Importantly, an endogenous protein, ANKRD11, is identified with the capacity to suppress the oncogenic properties of mutant p53 and provide a potential target for the development of new cancer therapeutics. The role of mutant p53 in driving the invasive and metastatic potential of breast cancer cells was further explored and a relationship between mutant p53 and a micro-RNA (miR-155) established. Mutant p53 expression is shown to correlate with miR-155 expression, with miR-155 target genes involved in invasive pathways. ZNF652 is specifically identified as a target of miR-155 and loss of ZNF-652 is correlated with increased invasion and poor prognosis in breast cancer. Collectively, these studies identify key mechanisms through which mutant p53 functions to enhance tumourigenesis and importantly identify novel targets, ANKRD11, miR-155 and ZNF652, for the development of cancer therapies.
Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 2011

碩士
中國醫藥大學
基礎醫學研究所碩士班
99
CXCR7 was identified as a novel, alternate receptor for CXCL12 that promotes tumor growth and metastasis in breast cancer and other malignancies. However, the expression and function of CXCR7 in tumor progression is controversial. This issue may be due to complex genetic alterations. Here, we show that two human breast tumor cell lines (MCF-7 and MDA-MB-231) have different expression levels of CXCR7. The poorly invasive MCF-7 cell line was up-regulation of CXCR7, whereas the highly invasive MDA-MB-231 cell line was down-regulation of CXCR7. The p53 levels are associated with CXCR7 expression. The p53 gain-of-function mutation in MDA-MB-231 cells suppressed CXCR7 expression via up-regulation of hypermethylated in cancer 1 (HIC1), a transcriptional repressor of CXCR7. Knockdown of p53 in MDA-MB-231 cells decreased HIC1 expression and further increased CXCR7 expression. However, overexpresion of p53 in MCF-7 increased HIC1 expression and further decreased CXCR7 expression. To investigate the function role of CXCR7 in breast cancer progression, Lentiviral expression and knockdown systems were used to overexpress CXCR7 in MDA-MB-231 cells and knockdown CXCR7 in MCF-7 cells, respectively. We observed that overexpression of CXCR7 in MDA-MB-231 cells promoted cell proliferation and migration whereas knockdown of CXCR7 in MCF-7 decreased the chemotaxis ability. Interestingly, the invasion ability was increased by knockdown of CXCR7 in MCF-7. Taken together, these results suggest that p53 mutant mediates CXCR7 expression and CXCR7 has multiple roles in breast cancer progression.

The relatively small number of MAPKKs encoded in the Arabidopsis genome suggests that this particular class of kinases acts as a point of convergence within the plant's 'integration of external stimuli and their transduction to elicit biological responses. In an effort to gain information about the function of the MAPKK, AtMKK9 , in Arabidopsis, I have characterized several aspects of the phenotype of DEX:CA-MKK9-FLAG transgenic plants, which express an inducible constitutively active version of AtMKK9, CA-MKK9. I have found that CA-MKK9 expression can control the production of ethylene by activating a downstream MAPK, AtMPK6, which is known to promote the stabilization of ethylene biosynthesis enzymes. CA-MKK9 induction was correlated with an increase in AtMPK6 activity in planta, and was rapidly followed by production of a burst of ethylene in the induced plant tissues. I hypothesized that CA-MKK9 directly activates AtMPK6, and demonstrated that a recombinant version of CA-MKK9 was capable of phosphorylating AtMPK6 in vitro. In addition, the production of the hormone was abolished when C A - MKK9was expressed in a mpk6 knock-out background, thus proving that AtMPK6 is required for CA-MKK9 -mediated ethylene biosynthesis. I have also confirmed preliminary data from our laboratory suggesting that CA-MKK9 plays a role in oxidative programmed cell death. The necrotic lesions induced by CA-MKK9 were still observed in the mpk6 background, indicating that programmed cell death was triggered by CA-MKK9 activity independently of AtMPK6 activity and of ethylene overproduction. In addition, in order to investigate short-term transcriptional events triggered by CA-MKK9, I attempted to capture the transcriptional profile of DEX:CA-MKK9-FLAG plants using two-channel oligonucleotide microarrays. The CA-MKK9 - affected genes included a number of genes involved in the octadecanoid pathway, and their promoters were enriched in ABRE-like elements. However, my attempts to validate the microarray results using additional biological replicates and quantitative real-time PCR revealed that the majority of these early-response microarray results were apparently false positives, indicating that the microarray experiment was probably inappropriately constructed for capturing early transcriptional responses to CA-MKK9 using the dexamethasone-inducible system.
Science, Faculty of
Botany, Department of
Graduate

博士
國立臺灣大學
病理學研究所
84
p53 is one of the most frequently mutated genes in human cancer. Naturally occurring mutations of p53 are mainly located within a region containing residues 100 to 300, and are predominantly of missense type, resulting in loss of the protein''s DNA-binding activity. Here we show that some p53 mutations also represses the p53 N-terminal activation domain. The repression activity is conformational dependent. Interestingly, the central region of a temperature-sensitive mutant p53N247I possesses a movable and regulable inacti- vation function. It represses other activities present on the same polypeptide chain without strict regard to the configuration of that polypeptide at the non-permissive temperature (37℃)butnot at the permissive one (30℃). Furthermore, this mutant p53 region exhibits no other activity and its function is independent of endogenous p53 status. Among those of human temperature-sensitive p53 mutants, the central region of p53N247I is optimal in demonstrating such an autonomous inactivation function. By deletion studies, the minimal region containing the activity is mapped to residues 101-298 of p53N247I. The central region of p53N247I appears to function in a manner that is intramolecular distance-dependent and does not alter the subcellular compartmentalization of the GAL4 fusion protein. Furthermore, de novo protein synthesis is not required for the activity of the central region of p53N247I. These results strongly suggest that the central region can be used as a movable regulatory cassette, a powerful tool for thermal regulation of chimeric proteins.

Dissertação de mestrado em Genética Molecular
Machado-Joseph Disease (MJD), also known as spinocerebellar ataxia type 3 (SCA3), is an autosomal dominant neurodegenerative disorder which involves the cerebellar, pyramidal, extrapyramidal, motor neuron and oculomotor systems. MJD is caused by an expanded CAG repeat in the coding region of the ataxin-3 gene; for this reason it belongs to the group of polyglutamine (polyQ) associated diseases. The normal function of the ataxin-3 protein (ATXN3) remains mostly unknown, although there are some data suggesting a role for this protein in the modulation of protein degradation (either by the UPS or through autophagy) in association with its deubiquitylase (DUB) activity. Other evidence suggests a role in transcription regulation, in the cellular response(s) to stress and in the cytoskeleton organization as well as in cellular adhesion. In this work we studied the normal function of ATXN3 in neurons, using a novel neuroblastoma SH-SY5Y cell-based approach. We characterized all the different cell lines, assessing cell differentiation and survival, morphology, adhesion and cytoskeleton features, in cells overexpressing wild-type (ATXN3_28Q) and expanded (ATXN3_83Q) ATXN3 as well as cells expressing a catalytic mutant version of this protein (ATXN3_C14A). We also evaluated the transcriptomic profile and protein expression of the cell lines and further investigated the pathways underlying the cellular changes and cytoskeletal regulators. Moreover, in cells expressing both mutant and expanded ATXN3, we found a decreased expression of α-5 integrin (ITGA5) and inhibition of its downstream partners’ activity. The findings described in this study led us to hypothesize that the DUB activity of ATXN3 underlie the neuronal phenotype regulation and also that the expansion of the polyQ tract causes partial loss of function.
A doença de Machado-Joseph (DMJ), também conhecida como ataxia espinocerebelar 3, é uma desordem autossómica dominante que envolve os sistemas cerebelar, piramidal, extrapiramidal, motor neuronal e oculomotor. A DMJ é causada por uma expansão no codão CAG na região codificante do gene da ataxina-3; por esta razão, pertence ao grupo das doenças associadas às poliglutaminas (poliQ). A função normal da proteína ataxina-3 permanece desconhecida, embora alguns dados sugiram um papel da modulação da degradação proteica (quer pelo UPS ou por autofagia) em associação com a sua actividade como deubiquitilase (DUB). Outras evidências apontam para funções relacionadas com regulação da transcrição, como resposta celular ao stress, no citoesqueleto e adesão celular. Neste trabalho estudámos a função normal da ATXN3 em neurónios, usando uma nova abordagem com uma linha neuronal SY5Y de neuroblastoma. Caracterizámos as diferentes linhas celulares, nomeadamente diferenciação celular e sobrevida, morfologia, propriedades de adesão e citoesqueleto em células sobreexpressando a estirpe selvagem da ATXN3 (ATXN3_28Q), a forma expandida (ATXN3_83Q) assim como células sobre-expressando uma versão catalítica mutante (ATXN3_C14A) desta proteína. Avaliámos as diferenças na expressão transcripcional e proteica entre as linhas celulares e investigámos quais as vias de sinalização envolvidas nas alterações celulares observadas e reguladores do citoesqueleto. As células sobre-expressando ambas as formas mutante e expandida, da proteína apresentaram uma expressão diminuída da α5 integrina (ITGA5) bem como uma inibição da actividade desta proteína ao longo da via. As evidências resultantes deste estudo levaram-nos a colocar a hipótese que a actividade DUB da ATXN3 está na base da regulação do fenótipo neuronal e também que a expansão do tracto de poliglutaminas causa uma perda parcial da função da ataxina-3.