Thèses sur le sujet « Metabolism, Cell Cycle, Cancer, Kras »

Mammalian cells proliferate, differentiate or die in response to extracellular signals as growth factors and nutrients. Cancer is essentially a disease in which cells have lost responsiveness to many of these signals and to normal checks on cell proliferation. Therefore, it may not be surprising that tumor cells, in order to meet the increased requirements of proliferation, often display fundamental changes in pathways of energy metabolism and nutrient uptake (Garber, 2006). In particular, several studies shown that the process of tumorigenesis is often associated with altered metabolism of two major nutrients, glucose and glutamine (Mazurek et al, 1998; Chiaradonna et al., 2006; Gaglio et al., 2009). Moreover, this metabolic changes and cellular sensitivity to these nutrients can be induced or influenced by oncogenic transformation as i.e. Ras mutation (Chiaradonna et al., 2006; Gaglio et al., 2009) that has been found in 25% of human cancers. Glucose and glutamine are involved in multiple pathways required for cell proliferation and survival both in normal and transformed cells. The role of these pathways in the survival of transformed cells is mostly based either from the fact that the pathways in question can be regulated by oncogenes, or that cell death following nutrients shortage is associated with changes in the activation state of these pathways. In particular, in this work has been studied the response of K-Ras transformed cells to glucose and glutamine availability. Transformed cells have been shown to have a particular dependence on aerobic glycolysis compared to normal cells. Indeed, proliferation analysis of asynchronous K-Ras transformed fibroblasts as compared to normal cells, grown both in high and low glucose (25mM and 1mM), indicated that transformed cells showed an higher proliferation potential in 25mM glucose and lost completely this proliferative advantage in low glucose (1mM). Moreover, the strong reduction of glucose availability, observed at 72hrs in cells grown in 1mM glucose, induced an enhanced apoptosis only in transformed cells. Indeed, the apoptotic process activated in normal cells was glucose independent and probably correlated to prolonged contact inhibition. This effect has been validated by both Annexin V/PI staining and caspases-3 activation. Since transformed cells were characterized by strong reduction of proliferation as well as apoptosis at low glucose concentration, it has been analyzed the effects of Ras activation and glucose shortage on the cell cycle machinery, in particular during G1/S transition in synchronized normal and transformed cells. These results indicated that the timing of G1/S transition execution was glucose independent in both cell lines. Therefore, oncogenic Ras expression is able to induce a greater sensitivity to glucose shortage as compared to normal cells (decreased proliferation and enhanced apoptosis) only if the glucose shortage is persistent. Another metabolic adaptation of cancer cells, that has been studied, is their propensity to exhibit increased glutamine consumption. The results indicated that in asynchronous normal cells, contact inhibition, regardless of glutamine availability, brings to down-regulation of Akt that together with AMPK up-regulation, observed at low glutamine, concurs to TOR pathway inactivation. As a result, the expression of cyclin D, E and A is down regulated, pRb phosphorylation is strongly reduced, p27kip1 level is increased and its localization becomes preferentially nuclear, establishing therefore a condition that bring to a G1- cell cycle arrest. In synchronous normal cells, glutamine shortage slows the G2/M transition, indicating a possible role of glutamine in such cell cycle phase. In K-ras transformed cells, in which the level of activated Ras-GTP is very high (Nagase et al., 1995) and the contact inhibition is less efficient (Nagase et al., 1995), the deprivation of glutamine affects Akt and AMPK in a way opposite to that observed in normal cells, leaving the TOR pathway at least partially activated. This event allows sizable expression of cyclin D (at least until 72 hrs), E and A, sustained pRb phosphorylation, decreased p27kip1 and its preferential cytoplasmic localization, conditions that, taken together, promote entrance into S phase. Surprisingly, in condition of glutamine shortage, transformed asynchronous cells accumulate in S phase. In synchronous transformed cells, glutamine shortage slows both the G1 to S and the G2/M transitions. Since glutamine is an important intermediate in purine and pyrimidine biosynthesis, glutamine exhaustion could deplete intracellular nucleotide pools, bringing in turn to a failure in the execution of a normal cell cycle (Christofk et al., 2008; Martinez-Diez et al., 2006). This hypothesis has been confirmed by the fact that the proliferation defect of transformed cells is rescued by adding the four deoxyribonucleotides (precursors of DNA polymerization) to low glutamine medium. Moreover, experiments performed in synchronized transformed cells have been shown that low-glutamine medium causes a 2 hrs delay in entering into S phase after serum re-addition. This effect on cell cycle timing is worsened by complete absence of glutamine, in which 4 hrs delay in entering into S phase was observed. These data strongly indicated that the effect of glutamine limitation in transformed cells was first to slow down the S phase traverse, then, when a more severe limitation was established, to stuck a large fraction of the cells population in S-phase. Indeed, addition of a mix of 10µM deoxyribonucleotides reverted completely S phase reaching. Therefore in cells exhibiting high metabolic rates, such as rapidly dividing cancer cells grown in vitro, glutamine, being the most readily available amino acid used as energy source, may became the major source to sustain protein and nucleic acid synthesis (Ziegler et al., 2001), especially when glucose levels are low and energy demand is high. However, analysis of the levels of mRNA, proteins and above all of ATP in normal and transformed cells grown in high and low glutamine availability, did not show particular differences, suggesting an important role of glutamine for nucleotides synthesis in K-ras transformed cells. In conclusion, glutamine shortage in K-ras transformed cells limits proliferation by inducing abortive S phase entrance, while glucose shortage in the same system enhanced cell death (Lopez-Rios et al., 2007; Mankoff et al., 2007). The differential effects of glutamine and glucose on cell viability are not a property of the transformed phenotype per se, but rather depend on the specific pathway being activated in transformation. It has been previously shown that nutrient shortage influence cell proliferation and G1/S transition of K-Ras transformed fibroblasts. To understand how intracellular and extracellular signals are transmitted to the cell-cycle machinery and how the latter adjusts its frequencies accordingly is one of the major challenges in molecular biomedicine. To this aim, a computational model of the cell cycle based on experimental data has been developed. Indeed biochemical and genetic studies can be combined with bioinformatics and biosystems approaches in order to sketch a plan of the regulatory circuits governing cell cycle progression in normal cells, firstly, and then in transformed cells. Taking in consideration that the trespassing of the Restriction Point influences the timing of the cell cycle execution and that such timing is influenced both by growth factors and nutrients availability, it has been initially identified the restriction point in normal mouse fibroblasts, synchronized in G0 by serum starvation and stimulated to re-enter into S-phase by readdition of serum. During the time course of re-entering into cell cycle, from G0 to G1/S phase, and in agree with restriction point reaching, it has been observed a gradual increase of cyclin D and cyclin E, a constant expression of Cdk4 and Cdk2 and an abrupt decrease of p27Kip1. Moreover, in quiescent cells, has been observed a completely nuclear localization of p27Kip1 and more cytoplasmic localization of Cdk4 and Cdk2. These data agreed with other results since, in most cases, the concentration of the kinase subunit is relatively constant, whereas the concentration of the cyclin subunit oscillates. This detailed study of G1/S transition in normal fibroblasts allowed a novel mathematical model develop. Because tumor cells often display a reduced dependence on growth factors or an increased dependence on some nutrients, an understanding of the cell cycle and a dynamical computational model that include regulatory aspect might help explain the changes leading to tumor formation.

The cell cycle is an essential process in all living organisms that must be carefully regulated to ensure successful cell growth and division. Disregulation of the cell cycle is a key contributing factor towards the formation of cancerous cells. Understanding events at a cellular level is the first step towards comprehending how cancer manifests at an organismal level. Mathematical modelling can be used as a means of formalising and predicting the behaviour of the biological systems involved in cancer. In response, cell cycle models have been constructed to simulate and predict what happens to the mammalian cell over a time course in response to variable parameters.Current cell cycle models rarely account for certain precursors of cell growth such as energy usage and the need for non-essential amino acids as fundamental building blocks of macromolecules. Normal and cancer cell metabolism differ in the way they derive energy from glucose. In addition, normal and cancer cells also demonstrate different levels of gene expression. Two versions of a mammalian cell cycle and metabolism model, based on ordinary differential equations (ODEs) that respond to fluctuations in glucose concentration levels, have been developed here for the normal and cancer cell scenarios. Sensitivity analysis is performed for both normal and cancer cells using these cell cycle and metabolism models to investigate which kinetic reaction steps have a greater effect over the cell cycle period. Detailed analysis of the models and quantitatively assessing metabolite levels at various stages of the cell cycle may offer novel insights into how the glycolytic rate varies during the cell cycle for both normal and cancer cells.The results of the sensitivity analysis are used to identify potential drug targets in cancer therapy. Combinations of these individual targets are also investigated to compare the different effects of single and multiple drug compounds on the time it takes to complete a cell division cycle.

L’objectif de cette thèse est d’étudier comment la cellule mammifère adapte son métabolisme aux étapes du cycle cellulaire. Le cycle cellulaire est l’ensemble des étapes menant une cellule à se diviser. Le rôle du métabolisme est de fournir à la cellule les éléments et l’énergie dont elle a besoin pour fonctionner. En particulier, à chaque étape du cycle cellulaire, la cellule a besoin de différents éléments pour pouvoir, à terme, se diviser correctement. Il est donc crucial pour la cellule de coordonner le métabolisme et le cycle cellulaire et en particulier de contrôler ce que le métabolisme produit au cours du cycle cellulaire. Pour mieux comprendre ce lien entre ces deux processus, nous avons étudié comment un modèle mathématique du métabolisme répondait à différentes variations imposées par le cycle cellulaire et nous avons comparé ces réponses à la littérature. Satisfaits des résultats obtenus, nous avons alors construit un modèle hybride représentant l’évolution du métabolisme au cours du cycle cellulaire. Nous retrouvons dans ce modèle hybride les grandes variations connues des voies métaboliques au cours des phases du cycle cellulaire ainsi que des variations expérimentales des métabolites énergétiques et redox. Encouragés par ces résultats, nous avons finalement perturbé notre modèle hybride pour retrouver des tendances du métabolisme dues au cancer, un ensemble de maladies touchant à la fois le cycle cellulaire et le métabolisme
The goal of this thesis is to study how the mammal cell adjusts its metabolism to the steps of the cell cycle. The cell cycle is the series of events leading a cell to divide itself. The purpose of the metabolism is to supply the cell with all the elements and the energy it needs to work. In particular, at every step of the cell cycle, the cell needs different elements to properly divide itself. So, it is crucial for the cell to coordinate the metabolism and the cell cycle and in particular to control what the metabolism produces through the cell cycle. To have a better understanding of the links between these two processes, we studied how a mathematical model representing the metabolism answered to different variations imposed by the cell cycle and we compared those answers to the literature. Satisfied by the results, we therefore built a hybrid model representing the evolution of the metabolism through the cell cycle. We recover in this hybrid model the main known variations of the metabolism through the cycle’s phases as well as experimental variations of the energetic and redox metabolites. Encouraged by these results, we finally disturbed our hybrid model to recover metabolic tendencies due to cancer, a set of diseases affecting both the metabolism and the cell cycle

O câncer de ovário (OvCa) se destaca dentre as neoplasias ginecológicas por ser um dos mais letais e de difícil diagnóstico. O OvCa ocorre devido ao acúmulo de alterações celulares progressivas promovidas por mutações no genoma de uma célula que, consequentemente, alteram as complexas vias de regulação celular que respondem a fatores internos, como reprogramação genética, ou externos, como a resposta a fatores de crescimento, que juntamente com outras alterações moleculares favorecem a progressão e a metástase. Uma importante etapa da cascata metastática é a transição epitélio-mesenquimal (EMT), um processo bem orquestrado que resulta na perda do fenótipo epitelial e aquisição do fenótipo mesenquimal pelas células tumorais, que adquirem um caráter mais invasivo e migratório, além de se tornarem mais resistentes às drogas. A desregulação de fatores de transcrição como ZEB1, TWIST e SNAI1, vias de sinalização, microRNAs e fatores de crescimento incluindo EGF, TGF? e HGF podem desencadear a EMT. Após a eficiente indução da EMT com EGF na linhagem epitelial de adenocarcinoma de ovário humano Caov-3, foi realizada a análise proteômica quantitativa detalhada, baseada na análise de frações subcelulares enriquecidas em proteínas de membrana, citosol e núcleo, obtidas por centrifugação diferencial e subsequente fracionamento de proteínas por SDS-PAGE, a fim de compreender mais profundamente os mecanismos moleculares modulados pela EMT no OvCa. A partir da análise dos dados coletados em um sistema de espectrometria de massas de alta resolução acoplados a cromatografia líquida (LCMS/MS) e com o auxílio da bioinformática foram identificadas redes de interação proteína-proteína diferencialmente expressas, relacionadas principalmente com a regulação do ciclo celular e do metabolismo. A indução da EMT por EGF resultou na ativação de importantes vias de sinalização, tais como PI3K/Akt/mTOR e Ras/MAPK Erk, além da parada do ciclo celular na fase G1 regulada pelo aumento dos níveis de p21Waf1/Cip1, independentemente de p53, e diminuição de proteínas checkpoint. Através da proteômica dirigida, o monitoramento de reações múltiplas (MRM) revelou que, após a indução da EMT por EGF, o metabolismo das células Caov-3 foi alterado de uma maneira bastante peculiar. O estudo proteômico descrito permitiu a correlação entre processo da EMT induzido por EGF com o controle translacional, a regulação do ciclo celular e a alteração do metabolismo energético.
Ovarian cancer (OvCa) stands out among gynecological malignancies for being one of the most lethal and difficult to diagnose. OvCa occurs due to the accumulation of progressive cell changes promoted by mutations in the cell genome which, consequently, alter the complex cellular regulation pathways that respond to internal factors, such as genetic reprogramming, or external, such as response to growth factors, which together with other molecular changes favor the progression and metastasis. An important step of the metastatic cascade is the epithelial-mesenchymal transition (EMT), a well-orchestrated process that results in the loss of epithelial phenotype and acquisition of mesenchymal phenotype by tumor cells that acquire a more invasive and migratory character, and become more resistant to drugs. Deregulation of transcription factors such as ZEB1, TWIST and SNAI1, signaling pathways, microRNAs and growth factors including EGF, TGF? and HGF can trigger EMT. After an efficient EMT induction by EGF in the epithelial cell line of human adenocarcinoma ovarian Caov-3, detailed quantitative proteomic analysis was performed based on analysis of subcellular fractions enriched in proteins from membrane, cytosol and nucleus, obtained by differential centrifugation and subsequent fractionation of proteins by SDS-PAGE, in order to understand deeply the molecular mechanisms modulated by EMT in OvCa. From the analysis of data collected in a highresolution mass spectrometry system coupled to liquid chromatography (LC-MS/MS) and with the aid of bioinformatics were identified protein-protein interaction networks differentially expressed, mainly related to regulation cell cycle and metabolism. EGF induced-EMT resulted in the activation of major signaling pathways such as PI3K/Akt/mTOR and Ras/MAPK Erk, in addition to G1 phase cell cycle arrest regulated by increased levels of p21Waf1/Cip1, regardless of p53, and reduction of checkpoint proteins. Through the targeted proteomics, multiple reaction monitoring (MRM) showed that after EGF induced-EMT, Caov-3 cells metabolism was changed in a very particular way. The proteomic study described allowed the correlation between EMT process induced by EGF with translational control, regulation of cell cycle and the change in the energy metabolism.

KRAS est parmi les gènes les plus fréquemment mutés dans les cancers humains, tel que ~ 45% des cancers colorectaux (CCR). Malgré les efforts déployés pour réduire son potentiel oncogénique, KRAS muté est fréquemment associé à la résistance aux médicaments et est extrêmement difficile à cibler sur le plan thérapeutique. Les protéines à la surface cellulaire sont souvent dérégulées dans les cancers et sont des cibles thérapeutiques attrayantes en raison de leur accessibilité aux anticorps. Nous avons séquençé les ARNm de cellules épithéliales intestinales exprimant KRAS muté et observé que ces dernières présentaient des changements importants dans les gènes codant pour des protéines de surface cellulaire. Par conséquent, notre objectif était d'identifier de nouvelles cibles thérapeutiques exprimées à la surface de cellules transformées par l’oncogène KRAS. En utilisant une approche de pointe en protéomique de surface cellulaire, nous avons identifié plusieurs protéines différentiellement exprimées dans les cellules avec KRAS muté par rapport à leurs homologues de type sauvage. Nous avons ensuite effectué un crible CRISPR/Cas9 basé sur les protéines de surface cellulaire, qui a révélé que la perte de la protéine Atp7a affectait de manière différentielle les cellules épithéliales intestinales, en fonction de leur statut KRAS. De façon intéressante, nous avons constaté que ATP7A était régulé à la hausse dans les cellules avec KRAS muté par rapport à leurs homologues de type sauvage. ATP7A a un double rôle dans les cellules; alors qu'il est essentiel pour la maturation des enzymes dépendantes du cuivre (Cu), ATP7A protège les cellules d'une toxicité excessive induite par le Cu (cuproptose). Chez l'homme, les mutations dans ATP7A entraînent des troubles caractérisés par des déficiences systémiques dans le transport et les niveaux de Cu. Chez les animaux et dans les modèles de culture cellulaire, tel que les cellules épithéliales intestinales, les niveaux intracellulaires de Cu sont directement corrélés avec l'abondance post-transcriptionnelle d'ATP7A. Dans le même ordre d'idées, nous avons observé que les cellules de CCR avec KRAS muté avaient relativement plus de Cu intracellulaire, et la surexpression d'ATP7A protégeait les cellules KRAS muté de la cuproptose, par rapport à leurs homologues de type sauvage. Nous avons également observé que la croissance in vivo des xénogreffes KRAS mutées était réduite lorsque les souris étaient nourries avec un régime pauvre en Cu. Le Cu est utilisé par plusieurs enzymes qui régulent des fonctions cellulaires critiques, notamment la respiration mitochondriale, la motilité cellulaire et la prolifération. Nous montrons que les cellules mutantes KRAS étaient plus sensibles au chélateur de Cu, ammonium tetrathiomolybdate (TTM), par rapport aux cellules de type sauvage. De plus, les cellules avec KRAS muté traitées avec le TTM ont présenté des activités réduites de MEK1/2 dépendant du Cu et de l'enzyme de la chaîne de transport d'électrons mitochondriale, cytochrome c oxidase (CCO). Nous avons été surpris de constater que le transporteur de Cu de haute affinité, CTR1, est régulé à la baisse dans les cellules avec KRAS muté, et avons donc émis l'hypothèse que les cellules KRAS mutées doivent absorber le Cu par d'autres moyens. Ainsi, nous avons constaté que la macropinocytose agit comme une voie non canonique d'approvisionnement en Cu dans les cellules avec KRAS muté. Le traitement de cellules in vivo avec l'inhibiteur de la macropinocytose, EIPA, a inhibé l'expression d'ATP7A et diminué le Cu biodisponible dans les xénogreffes KRAS mutées. En conclusion, nos résultats montrent que les cellules avec KRAS muté augmentent les niveaux de Cu et d'ATP7A pour soutenir la tumorigenèse en augmentant l'activité cuproenzymatique et diminuant la cuproptose. Cette étude est pertinente pour le cancer, car les tissus tumoraux contiennent fréquemment des niveaux de Cu plus élevés que les tissus normaux. Des études récentes ont mis en évidence un potentiel de repositionnement du chélateur de Cu TTM, qui est disponible en clinique et utilisé pour traiter les troubles du Cu. Nos résultats démontrent que la biodisponibilité du Cu pourrait être exploitée pour traiter le CCR avec KRAS muté avec de tels inhibiteurs. Les travaux futurs comprennent l'identification de stratégies combinatoires qui peuvent être améliorer les effets anti-cancéreux de la chélation du Cu.
KRAS is amongst the most frequently mutated genes driving human cancers, including ~ 45% of colorectal cancers (CRC). Despite intense efforts to curb its oncogenic potential, mutant KRAS is frequently associated with drug resistance and is extremely challenging to target therapeutically. Cell-surface proteins are often spatially dysregulated in cancers and are attractive therapeutic targets due to their easy accessibility. We performed RNA sequencing of mutant KRAS-expressing intestinal epithelial cells and observed that cells undergoing transformation exhibited dramatic changes in cell surface-coding genes. Therefore, our goal was to identify novel druggable targets expressed at the cell surface of mutant KRAS-transformed cells. Using a cutting-edge cell surface proteomics approach, we identified several differentially expressed proteins at the surface of KRAS-mutant cells compared to wild-type counterparts. We then performed a cell surface based CRISPR/Cas9 screen, which revealed that loss of the copper exporter Atp7a differentially affected the fitness of intestinal epithelial cells, depending on their KRAS status. Interestingly, we found that ATP7A was upregulated in KRAS-mutant cells compared to wild-type counterparts. ATP7A has a dual role in cells; while it is essential for maturation of copper (Cu)-dependent enzymes, ATP7A protects cells from excess Cu-induced toxicity (cuproptosis). In humans, ATP7A mutations result in disorders characterized by systemic deficiencies in Cu transport and levels. In animals and in tissue culture models, including intestinal epithelial cells, intracellular Cu levels are directly correlated with the post-transcriptional abundance of ATP7A. In line with this, we observed that KRAS-mutant CRC cells and tissues had relatively more intracellular Cu, and ATP7A-overexpression protected KRAS-mutant cells from cuproptosis, compared to wild-type counterparts. We also observed that in vivo growth of KRAS-mutant xenografts was reduced when mice were fed a Cu-deficient diet. Cu is utilized by several enzymes that regulate critical cellular functions including mitochondrial respiration, cell motility and proliferation. We show that KRAS-mutant cells were more sensitive to the Cu chelating drug ammonium tetrathiomolybdate (TTM), compared to wild-type cells. Moreover, TTM-treated KRAS-mutant cells displayed reduced activities of Cu-dependent MEK1/2 and mitochondrial electron transport chain enzyme, cytochrome c oxidase (CCO). We were surprised to find that the high-affinity CTR1 importer is downregulated in KRAS-mutant cells, and so we hypothesized that KRAS cells must uptake Cu through alternate means. In accordance with this, we found that macropinocytosis acts as a non-canonical Cu-supply route in KRAS-mutant cells. In vivo, treating cells with the macropinocytosis inhibitor EIPA, inhibited the expression of ATP7A and decreased bioavailable Cu in KRAS xenografts. In conclusion, our results show that KRAS-mutant cells increase Cu and ATP7A levels, likely to support tumorigenesis by elevating cuproenzymatic activity and parallelly dealing with cuproptosis. This study is relevant to cancer as tumor tissues and patients contain higher Cu levels than normal controls. Recent studies have highlighted a potential for repurposing the clinically available copper chelator TTM, which is used to treat Cu disorders. Our results demonstrate that copper bioavailability could be exploited to treat KRAS-mutated CRC with such inhibitors. Future work includes identification of combinatorial strategies that may be synthetic lethal to copper chelation.

Chemotherapy is a treatment that uses cytotoxic drugs to kill rapidly dividing cancer cells. There are many anti-cancer chemotherapeutic drugs used alone or in combination with others to kill cancerous cells, and some of these, are of plant origin. Naturally occurring compounds, such as Taxol, are used in chemotherapy and have very specific, unique, molecular targets. However, according to the World Health Organization (Ekor, 2014), approximately eighty percent of the world’s population depends on natural compounds from traditional medicine and these compounds are widely used in complementary medicine as anti-cancer drugs (Foster et al., 2000). Traditional Chinese medicine (TCM) uses treatments that contain multiple natural compounds, a number of which have been claimed to be of therapeutic benefit to cancer sufferers (Chung et al., 2015). Some TCM preparations have shown anti-cancer, anti-migratory and anti-metastatic properties in laboratory settings (Wang et al., 2009;Pan et al., 2011;Qu et al., 2016). Research suggests that TCM natural compound mixtures might synergistically trigger therapeutic benefits through the action of multiple components affecting multiple regulatory signaling targets (Wang et al., 2008). Compound Kushen injection (CKI) is a TCM anticancer agent which has been approved by the Chinese State Food and Drug Administration to treat solid tumors in combination with chemotherapy drugs in clinics for pain relief, cancer metastasis and enhancement of the immune system since 1995, and is used to treat approximately 30,000 patients daily. Although a large body of evidence has suggested CKI has anti-cancer properties (Xu et al., 2011;Gao et al., 2018) the anti-cancer mechanisms attributable to specific compounds within the mixture remain unknown. CKI contains multiple alkaloid and flavonoid compounds and the main bioactive compounds such as matrine and oxymatrine have shown to affect cancer cells in the lab. However, other medicinal herbs containing these two compounds as main components have demonstrated patient toxicity. It is therefore important to better understand the effects of CKI, particularly with respect the contributions of individual compounds within the mixture. In this thesis, I describe a multi-disciplinary approach including analytical chemistry, cellular assays and transcriptome analysis to explore the effects of several major compounds present in CKI. Through the application of a subtractive fractionation method that removed individual compounds one, two or three at a time, I have been able to map these compounds and their interactions to specific pathways based on altered gene expression profiles. This has illuminated the roles of several major compounds of CKI, that on their own, have no, or minimal, activity in our bioassay. This approach has enabled us to identify the interactions between compounds in a mixture as shown by the response of cancer cell cultures. Using a systems biology approach along with cellular migration and invasion assays, I have mapped the activity of related proteins and pathways which may contribute to the migrastatic activity of CKI. Altogether, this thesis presents an initial characterization of the underlying mechanistic changes induced by CKI. First, by comparing differentially expressed genes across treatment combinations generated using our subtractive fractionation approach, I identified specific candidate pathways that were altered by the removal of compounds from the mixture. Second, by using transcriptome data of a breast cancer cell line, the effects of CKI on candidate anti-migratory pathways for six different cancer cell lines were assessed. These experiments identified specific candidate target pathways through which CKI might act. These approaches can be used to understand the roles and interactions of individual compounds from any complex natural compound mixture whose biological activity cannot be associated with purified compounds.
Thesis (Ph.D.) -- University of Adelaide, School of Biological Sciences, 2019

Dichloroacetate (DCA) it a metabolic reprogramming agent that is used to target the unique metabolism of cancer cells, but is not always effective in colorectal cancer cells. In HCT116 cells, DCA was unable to induce apoptosis, but did decrease proliferation when compared to untreated cells. A decrease in full length Mcl-1 protein expression 7 hours following DCA treatment did not correspond with changes in mRNA production or changes in expression of inhibitory binding partners, but may be due to altered proteasomal degradation. Similar reduction in levels of a lower molecular weight Mcl-1 band occurred, which did not result from alternative splicing or from caspase-mediated cleavage. Mcl-1 showed primarily nuclear localization within the cell, and expression changes in full-length Mcl-1 were seen in nuclear lysate but not cytoplasmic lysate after 7 hours of DCA treatment. Changes in nuclear Mcl-1 expression did not correspond with cell cycle arrest or progression. These results suggest that proteasomal degradation of Mcl-1 may be altered following treatment with DCA, and this change may be associated with decreased proliferation, independent of cell cycle arrest. This may indicate a novel role of nuclear Mcl-1 in response of colorectal cancer to DCA exposure.
Final thesis for Leanne Delaney in partial fulfillment of requirements for the degree of Master of Science in Biomedical Sciences
NSERC

Research Doctorate - Doctor of Philosophy (PhD)
Endometrial cancer is one of the most common female cancers in industrialized countries. Traditional risk factors associated with endometrial cancer are well understood and include excessive exposure to estrogen or estrogen unopposed by progesterone. However, variations in the genes that influence these hormones and their association with endometrial cancer have not been well investigated. By studying genetic variation in endometrial cancer, novel markers of risk may be discovered that can be used to identify women at high risk and for the implementation of specialised treatments. Polymorphisms in the genes involved in the following pathways; hormone biosynthesis, hormone receptors, estrogen metabolism, DNA repair and cell cycle control, have been suggested to be involved in the initiation and development of endometrial cancer. The focus of this study was to examine genetic variants in these pathways to assess the existence of an association with the risk of endometrial cancer. In the first part of this study, the COMT V158M polymorphism was examined in a hereditary non-polyposis colorectal cancer (HNPCC) cohort to determine its association with disease expression. The heterozygous genotype was over-represented in women with endometrial/ovarian cancer that did not harbour mismatch repair (MMR) gene mutations. This result suggested that the COMT V158M polymorphism may alter the risk of developing HNPCC related endometrial/ovarian cancer in MMR mutation negative women. Since COMT is involved in the metabolism of estrogen and that estrogen is the main risk factor for endometrial cancer development, closer examination was warranted to determine the association of genetic variation involved in hormone-related pathways and endometrial cancer risk, outside of the context of an inherited predisposition to disease. In the second part of this study, a cohort of 191 women with endometrial cancer and 291 healthy control women were genotyped for polymorphisms in genes involved in hormone biosynthesis, hormone receptors, estrogen metabolism, DNA repair and cell cycle control. The results revealed that variations in estrogen receptor alpha (ESR1) and beta (ESR2), and the androgen receptor (AR), were associated with an increase and decrease in endometrial cancer risk, respectively. Additionally, polymorphisms in CYP1A1, CYP1B1, GSTM1 and GSTP1 were related to a decrease in endometrial cancer risk. A trend was observed for the cyclin D1 870 G>A polymorphism and an increase in endometrial cancer risk, however, this result did not reach significance. Taken together, these results revealed that perturbations in the hormone receptors and estrogen metabolism genes, may aid in the identification of women at high risk of developing endometrial cancer. Interestingly, stratification of the women with endometrial cancer revealed that combinations of polymorphisms in TP53 and MDM2 were associated with higher grades of cancer. This finding may possibly have significant implications as women with reduced apoptotic ability, due to combinations of polymorphisms in these genes, have an increased risk of presenting with higher grades of endometrial cancer, that are associated with lower survival rates. In summary, the results of this thesis showed that variation in the estrogen and androgen receptors, and estrogen metabolism genes, may alter the risk of developing endometrial cancer. Moreover, polymorphisms in the cell cycle control genes, TP53 and MDM2, appear to be associated with higher grades of endometrial cancer. This study of polymorphisms may help explain genetic differences in individual susceptibility to endometrial cancer and are markers of risk that aid in the development of effective and personalised strategies to prevent disease development. This study has improved the understanding of genetic variation associated with endometrial cancer risk. It has the potential to enhance our ability to treat women with endometrial cancer through improved identification and treatment strategies, by virtue of the genetic variation identified, that appears to predispose to disease.

Research Doctorate - Doctor of Philosophy (PhD)
Endometrial cancer is one of the most common female cancers in industrialized countries. Traditional risk factors associated with endometrial cancer are well understood and include excessive exposure to estrogen or estrogen unopposed by progesterone. However, variations in the genes that influence these hormones and their association with endometrial cancer have not been well investigated. By studying genetic variation in endometrial cancer, novel markers of risk may be discovered that can be used to identify women at high risk and for the implementation of specialised treatments. Polymorphisms in the genes involved in the following pathways; hormone biosynthesis, hormone receptors, estrogen metabolism, DNA repair and cell cycle control, have been suggested to be involved in the initiation and development of endometrial cancer. The focus of this study was to examine genetic variants in these pathways to assess the existence of an association with the risk of endometrial cancer. In the first part of this study, the COMT V158M polymorphism was examined in a hereditary non-polyposis colorectal cancer (HNPCC) cohort to determine its association with disease expression. The heterozygous genotype was over-represented in women with endometrial/ovarian cancer that did not harbour mismatch repair (MMR) gene mutations. This result suggested that the COMT V158M polymorphism may alter the risk of developing HNPCC related endometrial/ovarian cancer in MMR mutation negative women. Since COMT is involved in the metabolism of estrogen and that estrogen is the main risk factor for endometrial cancer development, closer examination was warranted to determine the association of genetic variation involved in hormone-related pathways and endometrial cancer risk, outside of the context of an inherited predisposition to disease. In the second part of this study, a cohort of 191 women with endometrial cancer and 291 healthy control women were genotyped for polymorphisms in genes involved in hormone biosynthesis, hormone receptors, estrogen metabolism, DNA repair and cell cycle control. The results revealed that variations in estrogen receptor alpha (ESR1) and beta (ESR2), and the androgen receptor (AR), were associated with an increase and decrease in endometrial cancer risk, respectively. Additionally, polymorphisms in CYP1A1, CYP1B1, GSTM1 and GSTP1 were related to a decrease in endometrial cancer risk. A trend was observed for the cyclin D1 870 G>A polymorphism and an increase in endometrial cancer risk, however, this result did not reach significance. Taken together, these results revealed that perturbations in the hormone receptors and estrogen metabolism genes, may aid in the identification of women at high risk of developing endometrial cancer. Interestingly, stratification of the women with endometrial cancer revealed that combinations of polymorphisms in TP53 and MDM2 were associated with higher grades of cancer. This finding may possibly have significant implications as women with reduced apoptotic ability, due to combinations of polymorphisms in these genes, have an increased risk of presenting with higher grades of endometrial cancer, that are associated with lower survival rates. In summary, the results of this thesis showed that variation in the estrogen and androgen receptors, and estrogen metabolism genes, may alter the risk of developing endometrial cancer. Moreover, polymorphisms in the cell cycle control genes, TP53 and MDM2, appear to be associated with higher grades of endometrial cancer. This study of polymorphisms may help explain genetic differences in individual susceptibility to endometrial cancer and are markers of risk that aid in the development of effective and personalised strategies to prevent disease development. This study has improved the understanding of genetic variation associated with endometrial cancer risk. It has the potential to enhance our ability to treat women with endometrial cancer through improved identification and treatment strategies, by virtue of the genetic variation identified, that appears to predispose to disease.

Radiotherapy is a major treatment modality used to treat muscle-invasive bladder cancer, with patient outcomes similar to surgery. However, radioresistance is a significant factor in treatment failure. Cell-free extracts of muscle-invasive bladder tumors are defective in nonhomologous end-joining (NHEJ), and this phenotype may be used clinically by combining radiotherapy with a radiosensitizing drug that targets homologous recombination, thereby sparing normal tissues with intact NHEJ. The response of the homologous recombination protein RAD51 to radiation is inhibited by the small-molecule tyrosine kinase inhibitor imatinib. Stable RT112 bladder cancer Ku knockdown (Ku80KD) cells were generated using short hairpin RNA technology to mimic the invasive tumor phenotype and also RAD51 knockdown (RAD51KD) cells to show imatinib's pathway selectivity. Ku80KD, RAD51KD, nonsilencing vector control, and parental RT112 cells were treated with radiation in combination with either imatinib or lapatinib, which inhibits NHEJ and cell survival assessed by clonogenic assay. Drug doses were chosen at approximately IC40 and IC10 (nontoxic) levels. Imatinib radiosensitized Ku80KD cells to a greater extent than RAD51KD or RT112 cells. In contrast, lapatinib radiosensitized RAD51KD and RT112 cells but not Ku80KD cells. Taken together, our findings suggest a new application for imatinib in concurrent use with radiotherapy to treat muscle-invasive bladder cancer. Cancer Res; 73(5); 1611-20. (c)2012 AACR.