Dissertations / Theses on the topic 'STK10'

La maladie rénale chronique (MRC) représente un lourd fardeau pour la santé publique, et pourtant, les mécanismes moléculaires qui régissent la progression de la maladie restent mal compris. Parmi les voies de signalisation potentielles, la voie du récepteur du facteur de croissance épidermique (EGFR) joue un rôle crucial dans la progression de la MRC. Cependant, une inhibition prolongée de l'EGFR n'est pas une option envisageable pour les patients atteints de MRC en raison du risque d'effets indésirables importants. Une compréhension approfondie de la signalisation de l'EGFR est essentielle pour développer des stratégies thérapeutiques plus ciblées et plus sûres. Dans nos travaux antérieurs, nous avons identifié la kinase sérine-thréonine 10 (STK10) comme un partenaire clé de l'EGFR lors de l'activation médiée par un ligand. Ce projet vise à élucider les mécanismes moléculaires sous-jacents à la progression de la MRC, avec pour objectif d'identifier de nouvelles cibles thérapeutiques. Notre recherche se concentre sur la compréhension de la voie de signalisation EGFR-STK10 dans les cellules rénales. Nous avons employé une approche intégrée combinant la phosphoprotéomique impartiale et la biotinylation dépendante de la proximité (TurboID), suivie par la spectrométrie de masse, afin d'explorer l'activation de STK10, ses partenaires d'interaction et ses voies de signalisation lors de l'activation médiée par un ligand de l'EGFR. Grâce à l'analyse phosphoprotéomique, nous avons caractérisé les phosphorylations de STK10 suite à l'activation de l'EGFR par EGF et TGFa, Nous avons identifié deux sites de phosphorylation, S191 et T195, dans la boucle d'activation de STK10, qui sont spécifiquement régulés à la hausse suite à l'activation de l'EGFR par TGFa, mais pas par EGF. De plus, nous avons observé que la phosphorylation en aval d'ERM, une cible connue de STK10, est significativement augmentée uniquement lors de la stimulation par TGFa. L'inhibition pharmacologique ou la réduction génétique de STK10 a empêché l'augmentation de ces phosphosites et l'activation d'ERM, suggérant une activation spécifique au ligand de STK10 dans les cellules épithéliales tubulaires rénales. Mécaniquement, nos analyses ont identifié un enrichissement de protéines impliquées dans le trafic des récepteurs, suggérant que STK10 joue un rôle dans la régulation du tri de l'EGFR. Notamment, nous avons démontré que l'inhibition pharmacologique et la réduction génétique de STK10 n'affectent pas l'activation précoce de l'EGFR, mais entravent le trafic à long terme du récepteur suite à la stimulation par TGFa. Dans les cellules shSTK10, l'EGFR est significativement plus dégradé que dans les cellules contrôles, ce qui suggère que STK10 pourrait favoriser le recyclage du récepteur, conduisant à son activation soutenue. L'activation soutenue de l'EGFR a été impliquée dans plusieurs modèles de MRC, contribuant à des processus pathologiques. L'analyse ontologique des gènes a révélé que STK10 est impliqué dans différents processus pathologiques associés à la progression de la MRC et qui sont médiés par une activation constante de l'EGFR. L'inhibition de STK10 in vitro a effectivement bloqué certains de ces processus délétères tels que la migration cellulaire, la croissance et la prolifération dans les cellules épithéliales rénales. Ainsi, l'inhibition de STK10 pourrait bloquer ces processus et potentiellement ralentir la progression de la MRC. En conclusion, cette étude mène à de nouvelles perspectives sur la voie STK10 dans le contexte de la signalisation de l'EGFR dans les cellules rénales. Elle met en lumière un nouveau rôle potentiel pour cette kinase dans la régulation du trafic des récepteurs suite à la stimulation par TGFa, probablement en favorisant son recyclage et son activation soutenue, conduisant à des processus pathogéniques. L'inhibition de STK10 devrait être davantage testée pour évaluer son potentiel en tant que stratégie thérapeutique pour la MRC
Chronic Kidney Disease (CKD) is a major public health burden, yet the molecular mechanisms driving its progression remain poorly understood. Among the potential pathways, the Epidermal Growth Factor Receptor (EGFR) signaling axis plays a critical role in CKD progression. However, prolonged inhibition of EGFR is not a feasible option for CKD patients due to the risk of significant adverse effects. Therefore, a deeper understanding of EGFR signaling is essential for developing more targeted and safer therapeutic strategies. In our previous work, we identified Serine-Threonine Kinase 10 (STK10) as a key partner of EGFR during ligand-mediated activation. The project aims to unravel the molecular mechanisms underlying CKD progression, with the goal of identifying new therapeutic targets. Our research focuses on understanding the EGFR-STK10 signaling pathway in kidney cells. To investigate this, we employed an integrated approach combining unbiased phosphoproteomics and proximity-dependent biotinylation (TurboID), followed by mass spectrometry, to explore STK10 activation, its interacting partners, and its signaling pathways, upon EGFR mediated ligand activation. Through phosphoproteomic analysis, we characterized STK10 phosphorylations following EGFR activation by EGF and TGFa. We identified two phosphorylation sites, S191 and T195, in the STK10 activation loop, which are specifically upregulated following EGFR activation by TGFa, but not by EGF. Additionally, we observed that downstream phosphorylation of ERM, a known STK10 target, is significantly increased only upon TGFa treatment. Pharmacological inhibition or genetic knockdown of STK10 using shRNA successfully prevented the upregulation of these phosphosites and the activation of ERM, suggesting a ligand-specific activation of STK10 in kidney epithelial tubular cells. Mechanistically, our analyses identified an enrichment of proteins involved in receptor trafficking, suggesting that STK10 plays a role in regulating EGFR sorting. Notably, we demonstrated that pharmacological inhibition or genetic downregulation of STK10 does not affect early EGFR activation but does impair the long-term trafficking of the receptor following TGFa treatment. In shSTK10 cells, EGFR is significantly more degraded than in control cells, suggesting that STK10 might favor the recycling of the receptor, leading to its sustain activation. Sustained EGFR activation has been implicated in several CKD models, contributing to pathological processes. Importantly, gene ontology analysis revealed that STK10 is involved in different pathological processes associated with CKD progression mediated by constant EGFR activation. Inhibition of STK10 in vitro effectively blocked some of these deleterious processes such as cell migration, growth, and proliferation in kidney epithelial cells. Thus, inhibition of STK10 could block these processes and potentially slow down CKD progression. In conclusion, this study provides new insights into the STK10 pathway in the context of EGFR signaling in kidney cells. It highlights a potential novel role for this kinase in regulating receptor trafficking upon TGFa treatment, likely promoting its recycling and sustained activation, leading to pathogenic processes. STK10 inhibition should be further tested to evaluate its potential as a therapeutic strategy for CKD

LKB1 is the second most commonly altered tumour suppressor gene in lung adenocarcinoma, the most prevalent form of lung cancer. LKB1 is a "master kinase" that has been shown to phosphorylate up to 13 downstream targets. We hypothesised that LKB1 loss is associated with an increased dependency on alternative, targetable pathways. The overall aims of this project were to better understand the role of LKB1 loss in lung cancer and to identify novel approaches to selectively target LKB1 mutated cells. We generated isogenic cells with or without LKB1 and used these to study the effect of LKB1 on cell proliferation. Importantly, we used a range of models including 2D culture, 3D spheroids and, sub-cutaneous and orthotopic xenograft models. To understand the role of LKB1 loss in lung cancer, the effect of LKB1 on mRNA expression was analysed using whole genome RNA Sequencing. To identify novel approaches to selectively target LKB1 mutated cells, we used biological screening methods and also investigated the effect of several metabolic inhibitors. We found that loss of LKB1 expression had no effect on cell proliferation in 2D culture, but was associated with increased growth in 3D spheroids, sub-cutaneous and orthotopic xenografts, as well as greater metastasis in a lung orthotopic model. Gene ontology analysis of the transcriptome identified that genes associated with cAMP signalling and cytoskeletal organisation were differentially expressed between LKB1 deficient and proficient cells. We confirmed that cAMP signalling was increased in LKB1 deficient cells, though there was no difference in sensitivity between LKB1 deficient and proficient cells to cAMP signalling modulators. The bioactive small molecule screen showed that LKB1 deficient cells underwent apoptosis more slowly and therefore, were less sensitive to many compounds, compared with LKB1 proficient cells. Screening in 3D spheroids was a novel approach that we used to identify microtubule inhibitors as potentially selective compounds acting in LKB1 deficient cells. Our RNASeq data suggests that there was a metabolic shift from oxidative phosphorylation to aerobic glycolysis in LKB1 deficient cells, although this did not affect sensitivity to complex I inhibitors. Importantly, LKB1 deficient cells were more sensitive to glucose and glutamine deprivation which suggests that targeting these metabolic pathways may hold the greatest promise to selectively inhibit proliferation in LKB1 mutated cells.

Les carcinomes hépatocellulaires (CHC) mutés CTNNB1 ont des caractéristiques phénotypiques propres en termes de polarité et de métabolisme (absence de stéatose). Nous avons émis l’hypothèse que ce phénotype pouvait être secondaire à l’activation du gène suppresseur de tumeurs LKB1 qui code une Ser/Thr kinase multitâches.Nous avons tout d’abord montré qu’il existait effectivement un dialogue complexe entre les voies Wnt/β-Caténine et LKB1 dans le foie. Les mutations de CTNNB1 sont en effet capables d’induire l’expression protéique de LKB1 dans des lignées hépatomateuses humaines, et les CHC mutés CTNNB1 présentent une expression protéique accrue de LKB1 et une signature transcriptionnelle d’activation de LKB1. De plus, dans deux modèles murins d’invalidation hépatospécifique de Lkb1, LKB1 est apparu comme requis pour l’activation complète du programme transcriptionnel de β-Caténine mais de façon dépendante du stade de développement et du contexte nutritionnel. Enfin, la signalisation LKB1 est apparue comme nécessaire à la survie des hépatocytes activés pour β-Caténine dans deux modèles murins différents.Nous avons aussi caractérisé les rôles métaboliques de LKB1 dans le foie. L’invalidation hépatospécifique de Lkb1 induisait une augmentation progressive de la masse grasse corporelle avec utilisation préférentielle des glucides comme substrat énergétique. Il existait une activation de la néoglucogenèse hépatique avec hyperglycémie et une lipogenèse accrue avec accumulation hépatocytaire de lipides. Enfin, nous avons mis en évidence une activation paradoxale de la signalisation AKT dans les hépatocytes, même à jeun, et une dépendance énergétique aux acides aminés. Enfin, nous avons identifié une nouvelle isoforme protéique de LKB1 délétée de son domaine N-Terminal et d’une partie de son domaine kinase. D’expression tissulaire préférentiellement musculaire et myocardique, cette isoforme catalytiquement inactive se comportait comme dominant positif sur l’activation de l’AMPK par la forme conventionnelle mais comme dominant négatif dans l’activité polarisation induite par LKB1. Enfin, elle était capable d’induire, en l’absence de la forme conventionnelle, la prolifération cellulaire et la tumorigenèse chez la souris nude. Elle pourrait exercer des rôles métaboliques particuliers dans les tissus fortement oxydatifs et des rôles oncogéniques dans certains contextes
CTNNB1-Mutated hepatocellular carcinomas (HCC) share a specific polarity and metabolic phenotype without steatosis. We hypothesized that such phenotype could imply the tumor suppressor gene LKB1 that encodes for a multi-Task Ser/Thr kinase.We first demonstrated that a complex crosstalk indeed exists in the liver between LKB1 and the Wnt/β-Catenin pathway. LKB1 proteic expression was controlled by mutant β-Catenin in hepatomatous cell line and CTNNB1-Mutated HCCs had an enhanced LKB1 proteic expression as well a transcriptomic signature of LKB1 activation. In two mouse model of liver-Specific invalidation of Lkb1, we showed that LKB1 was required for full activation of the β-Catenin transcriptomic program, but it depended on the developmental stage and nutritional context. At least, LKB1 appeared to be required for the survival of β-Catenin activated liver cells in two other mouse models.Then, we wanted to caracterize the metabolic roles of LKB1 in the liver. Liver-Specific invalidation of Lkb1 progressively raised the body fat mass and we observed that carbohydrates were preferred as whole-Body energetic fuel. In the liver, gluconeogenesis and lipogenesis were enhanced, resulting in mild hyperglycemia and lipid accumulation in the hepatocytes. At least, we identified an aberrant activation of the AKT signaling in the liver, even during fasting, and an energetic dependence towards amino acids.At least, we identified a novel LKB1 proteic isoform that is deleted of its N-Terminal domain and part of its kinase domain. Highly expressed in the muscle and in the heart, this catalytically inactive isoform however acted as a positive dominant towards AMPK activation by full length LKB1 but as a negative dominant towards LKB1-Induced cell polarization. This isoform is also able to enhance cell proliferation and to induce tumors in a xenograft model, even when expressed alone. It could play specific metabolic roles in oxidative tissues and could be oncogenic in some contexts

La phosphorylation de protéines correspond à l’addition covalente d’un groupement phosphate (PO4 3-) par une protéine kinase sur un substrat. Cette réaction est réversible : la déphosphorylation est catalysée par des protéines phosphatases. Chez S. aureus, la sérine/thréonine kinase Stk1 phosphoryle des acides aminés sérines et thréonines et a été montrée impliquée dans la régulation de la virulence du pathogène : nous avons approfondi les connaissances sur ce mécanisme en identifiant trois nouveaux substrats et en étudiant les effets de la phosphorylation sur leur activité : - l’enzyme LuxS, responsable de la synthèse de l’AI-2 impliqué dans la communication intra bactérienne, voit son activité enzymatique drastiquement diminuée en étant phosphorylée par Stk1 sur un site thréonine unique (T14) ; - le régulateur CcpA, dont la fixation sur l’ADN module l’expression de nombreux gènes de virulence, est phosphorylée par Stk1 sur deux sites (T18 et T33) et cette phosphorylation diminue l’affinité de la protéine régulatrice CcpA pour l’ADN de ses gènes cibles ; - l’élément réponse du système à deux composants SaeR est phosphorylé sur deux sites thréonines (T87 et T192) de la région impliquée dans l’affinité de SaeR pour l’ADN. Les rôles de la kinase Stk1 sont donc multiples et liés à la régulation de la virulence de S. aureus. Les autres voies mettant en jeu la phosphorylation de protéines bactériennes, comme les systèmes à deux composants ou le système CcpA/HPr, sont couplées à cette phosphorylation par la sérine/thréonine kinase : ces résultats soulignent à la fois la diversité et la complexité de la régulation des mécanismes responsables de la virulence de S. aureus
Protein phosphorylation consists in the catalyzed addition of a phosphate group on a substrate. This reversible reaction is ensured by both kinase and phosphatase proteins. S. aureus is a human prokaryote pathogen and a part of its virulence is known to be regulated by the serine/threonine kinase Stk1, which phosphorylates serine or threonine residues of its substrates. We investigated the mechanisms of this virulence regulation and newly identified three substrates of Stk1: the quorumsensing LuxS protein, the catabolite carbon protein CcpA and the two components system response element SaeR. LuxS is phosphorylated on a unique threonine residue in position 14 and phosphorylation dramatically influences its enzymatic activity on AI-2 production. CcpA phosphorylation on two threonine residues in the DNA-binding region of the protein (T18 and T33) decreases the affinity of the protein for its targeted DNA sequences. Besides, Stk1 also phosphorylates the response element SaeR on two threonine residues (T87 and T192) in the DNAbinding region. Therefore Stk1 kinase plays numerous roles in S. aureus virulence regulation and the complexity of this regulation pattern increases when considering that three of the phosphorylation pathways in prokaryotes are crossed over: the two components system phosphorylation, the HPr/HPrK system and the serine/threonine kinase proteins phosphorylation. These results highlight the need to focus on Stk1 as a key element in the complexity of virulence regulation in S. aureus

Skeletal muscle contraction results in the phosphorylation and activation of the AMP-activated protein kinase (AMPK) by an upstream kinase, AMPKK. The LKB1-STRAD-MO25 complex is the major AMPKK in skeletal muscle; however, LKB1-STRAD-MO25 activity is not increased by muscle contraction. This relationship suggests that phosphorylation of AMPK by LKB1-STRAD-MO25 during skeletal muscle contraction may be regulated by allosteric mechanisms. In this study we tested an array of metabolites including glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), fructose 1,6-bisphosphate (F1,6-P2), 3-phosphoglycerate (3PG), glucose-1-phosphate (G1P), glucose-1,6-bisphosphate (G1,6-P2), adenosine diphosphate (ADP), carnitine (Carn), acetyl-carnitine (Acarn), inosine monophosphate (IMP), inosine, and ammonia for allosteric regulation. We found that 3PG stimulated LKB1-STRAD-MO25 activity and allowed for increased AMPK phosphorylation. 3PG did not stimulate LKB1-STRAD-MO25 activity toward the peptide substrate LKB1tide. These results have identified 3PG as an AMPK-specific regulator of AMPK phosphorylation and activation by LKB1-STRAD-MO25.

This dissertation is devoted to the study of the molecular biology of major tumor suppressors, defined as those that prevent the cellular processes identified as the hallmarks of cancer. Specifically, the major tumor suppressors pRb and STK11 are explored in the context of osteosarcoma and lung cancer, respectively. RB1 was the first tumor suppressor gene discovered. Over four decades of work have revealed that the Rb protein (pRb) is a master regulator of biological pathways influencing virtually every aspect of intrinsic cell fate including cell growth, cell-cycle checkpoints, differentiation, senescence, self-renewal, replication, genomic stability and apoptosis. While these many processes may account for a significant portion of RB1's potency as a tumor suppressor, a small, but growing stream of evidence suggests that RB1 also significantly influences how a cell interacts with its environment, including cell-to-cell and cell-to-extracellular matrix interactions. Chapter 2 highlights pRb's role in the control of cell adhesion and how alterations in the adhesive properties of tumor cells may drive the deadly process of metastasis. Chapter 3 defines a role for pRb as a suppressor of the progression to metastasis by upregulating integrin α10. Transcription of this integrin subunit is herein found to be pRb-dependent in mouse osteoblasts. Classic pRb partners in cell cycle control, E2F1 and E2F3, do not repress transcription of integrin α10 and phosphorylation of pRb is not necessary for activation of the integrin α10 promoter. Promoter deletion revealed a pRb responsive region between -108bp to -55bp upstream of the start of the site of transcription. pRb activation of transcription also leads to increased levels of integrin α10 protein and a greater concentration of the integrin α10 protein at the cell membrane of mouse osteoblasts. These higher levels of integrin α10 correspond to increased binding to collagen substrate. Consistent with our findings in mouse osteoblasts, we found that integrin α10 is significantly underexpressed in multiple solid tumors that have frequent inactivation of the pRb pathway. Bioinformatically, we identified data consistent with an 'integrin switch' that occurs in multiple solid tumors consisting of underexpression of integrins α7, α8, and α10 with concurrent overexpression of integrin β4. pRb promotes cell adhesion by inducing expression of integrins necessary for cell adhesion to a substrate. We propose that pRb loss in solid tumors exacerbates aggressiveness by debilitating cellular adhesion, which in turn facilitates tumor cell detachment and metastasis. Lung cancer is the leading cause of cancer-related death in the U.S. and additional targeted therapies are desperately needed to treat these patients. STK11 is the third most frequently mutated gene in lung adenocarcinoma following only KRAS and TP53, yet its mutational status is not currently clinically evaluated and no therapies have been approved to specifically target its pathway. A deep understanding of the complex pathways controlled by STK11 and their alterations in cancer are required to develop effective therapies for patients with loss-of-function mutations. In Chapter 4 we present the current understanding of STK11, focusing on its molecular biology and therapeutic implications, including a compilation of studies evaluating STK11 somatic mutations in human lung cancer tissue and how the frequency of these mutations varies across histological subtypes and patient populations. Finally, we review the strategies being used to target STK11-deficient cancers at the clinical trial, pre-clinical, and basic science levels as well as proposing potential new therapies that might benefit this patient population. STK11 is a tumor-suppressor commonly mutated in lung adenocarcinoma (LuAd). There are a number of agents that may selectively target the deregulated pathways in STK11 mutated tumors, and thus, identifying the subset of adenocarcinomas that harbor these mutations could have significant clinical benefit. In Chapter 5, we characterized a cohort of 442 adenocarcinoma patients with respect to STK11 mutation status and subset of this cohort using immunochemistry, gene expression, and western blotting. We found that measuring STK11 mutation status is complicated by the fact that many STK11 mutations lead to expression of a stable protein that is indistinguishable from wild type (WT) via immunohistochemistry. To circumvent this, we used published cell line mutation and gene expression data to derive a signature correlating with STK11 mutation status. This signature was validated in the cohort of 442 lung adenocarcinomas and strongly correlates with mutation status (ROC curve AUC = 85.29). These data suggest that STK11; mutation status may be best assessed by measuring the downstream targets included in our signature.

O câncer de mama é uma doença multifatorial causada pela combinação de fatores de risco genéticos e ambientais. Polimorfismos genéticos de baixa penetrância têm sido associados a esta doença, porém existem poucos estudos publicados sobre a prevalência destes na população brasileira. No presente estudo, determinamos a freqüência alélica e genotípica dos polimorfismos ER)-397 PvuII C/T, ER)-351 XbaI A/G, PR PROGINS e STK15 F31I e investigamos a relação destes com fatores de risco estabelecidos para câncer de mama. Foram estudadas 750 mulheres sem câncer de mama, envolvidas em um programa de rastreamento mamográfico no Sul do Brasil, uma região com altos índices de incidência da doença. As freqüências genotípicas do polimorfismo PROGINS não foram significativamente diferentes das encontradas em populações brasileiras e nãobrasileiras. A distribuição dos genótipos de ER)-397 PvuII C/T, ER)-351 XbaI A/G e STK15 F31I, no entanto, foram significativamente diferentes da maioria dos estudos previamente publicados, enfatizando a necessidade de análises de populações específicas para avaliação do impacto de polimorfismos de baixa penetrância no risco para câncer de mama. Adicionalmente, a distribuição dos haplótipos de ER) (ER)-397 PvuII - ER)-351 XbaI) foram significativamente diferentes das descritas em outras populações. A média estimada do risco de desenvolver câncer de mama ao longo da vida na amostra estudada foi de 7.8%, a maioria (97.5%) das mulheres apresentavam achados normais ao exame de mamografia (BIRADS 1 e 2), e nenhum fator de ri sco reprodutivo para câncer de mama foi identificado na amostra. No entanto, a média do índice de massa corporal foi 29.6 e 41.1% das mulheres apresentavam índice de massa corporal a 30. Ao analisamos a relação entre os três polimorfismos e fatores de risco já estabelecidos, as seguintes associações estatisticamente significativas foram encontradas: (a) genótipo GG de ER)-351 e menarca a 14 anos, (b) genótipos A2A2 e A1A2 de PR PROGINS e maior estimativa de risco de desenvolver câncer de mama em 5-anos em mulheres pósmenopáusicas; (c) genótipos A2A2 e A1A2 PR PROGINS e maior índice de massa corporal em mulheres pós-menopáusicas; (d) genótipo AT e AA de STK15 F31I e densidade mamográfica moderadamente densa (tecido fibroglandular em 50-75% do tecido mamário) em mulheres pré-menopáusicas; (e) genótipo TT de STK15 F31I e mamas menos densas (tecido fibroglandular em 0-50% do tecido mamário) em mulheres pré-menopáusicas e (f) genótipo TT de STK15 F31I e idade da menarca a 12 anos. Estudos de caso-controle adicionais são necessários para melhor compreensão da relação entre estes e outros polimorfismos e risco para câncer de mama na população brasileira.
Breast cancer is a multifactorial disease caused by a combination of genetic and environmental risk factors. Highly prevalent low-penetrance genetic polymorphisms have been associated with an increased risk of developing the disease. Only a few studies have been published about the prevalence of such polymorphisms in the highly heterogeneous Brazilian population. In the present study, we have determined the allelic and genotypic frequencies of the ER)-397 PvuII C/T, ER)-351 XbaI A/G, PR PROGINS and STK15 F31I polymorphisms and investigated their relationship with established breast cancer risk factors. We studied 750 breast cancer-unaffected women enrolled in a mammographic screening program in Southern Brazil, a region with particularly high breast cancer incidence rates. Genotypic frequencies for PROGINS were not significantly different from previous studies in Brazilian and non-Brazilian individuals. The distribution of ER)- 397 PvuII C/T, ER)-351 XbaI A/G and STK15 F31I genotypes, however, was significantly different from most of the previously published reports, emphasizing the need for population-specific analyses in evaluating the impact of low-penetrance gene polymorphisms on breast cancer risk. Furthermore, the distribution of ER) haplotypes using the ER)-397 PvuII C/T and ER)-351 XbaI A/G polymorphic markers was also distinct from that described in other populations. The mean estimated lifetime risk of developing breast cancer in the sample was 7.8%, most of the women (97.5%) had normal mammographic image results (BIRADS 1 and 2) and no significant reproductive risk factors for breast cancer were identified in the sample. However, mean body mass index was 29.6 and 41.1% of the women had a body mass index a 30. When analyzing the relationship between the three polymorphisms and other established breast cancer risk factors the following significant associations were found between: (a) ER)-351 GG genotype and menarche a 14 years; (b) PROGINS A2 allele and higher mean estimated 5-year risk of developing breast cancer in postmenopausal women; (c) A1A2 and A2A2 genotypes and higher mean body mass index in postmenopausal women; (d) STK15 F31I AT and AA genotypes and moderately dense (50-75% of the breast with fibroglandular tissue) in premenopausal women; (e) STK15 F31I TT genotype and less dense (0-50% of the breast with fibroglandular tissue) in premenopausal women and (f) STK15 F31I TT genotype and later age at menarche (a 12 years). Additional case-control studies are necessary to clarify the relationship between this and other polymorphisms and breast cancer risk in the Brazilian population.

Both the prenatal and postnatal environment have a profound impact on the risk for adult diseases. A model of catch-up growth following fetal growth restriction was set in Wistar rats to study changes in the gene expression of energetic metabolism–related genes. Excessive adipose tissue accumulation and hypertrophy, decreased circulating adiponectin and triglycerides levels, higher HOMA-IR values and reduced expression of STK11, DLK1 and SIRT1 genes in retroperitoneal adipose, hepatic and muscular tissues were observed in pups with catch-up growth at postnatal day 42, that is related to visceral fat accumulation and insulin resistance. In conclusion, catch-up growth following prenatal growth restriction leads to changes in STK11, DLK1 and SIRT1 gene expression. These genes could be new therapeutic targets for early prevention on fetal programming of metabolic disorders in adulthood.
Els ambients pre i postnatal poden augmentar el risc de desenvolupar malalties en l’edat adulta. S’ha dissenyat un model animal de creixement recuperador postnatal secundari a restricció de pes prenatal en rates Wistar per estudiar canvis en l’expressió de gens de regulació metabòlica. Les cries amb creixement recuperador postnatal presenten acumulació excessiva i hipertròfia del teixit adipós visceral, disminució dels nivells l’adiponectina i triglicèrids circulants, augment del valor de HOMA-IR i baixa expressió dels gens STK11, DLK1 i SIRT1 en els teixits adipós, hepàtic i muscular en el dia postnatal 42 que es relaciona amb acumulació de greix visceral i resistència a la insulina. En conclusió, el creixement recuperador postnatal secundari a restricció de pes prenatal provoca canvis en l’expressió dels gens STK11, DLK1 i SIRT1, els quals podrien ser noves dianes terapèutiques per a la prevenció precoç de la programació fetal dels desordres metabòlics en l’edat adulta.

Les réactions de phosphorylation / déphosphorylation chez les bactéries régulent plusieurs fonctions cellulaires telles que croissance, différenciation, pathogénie, résistance aux antibiotiques, réponse au stress, formation des biofilms ainsi que plusieurs processus impliqués dans le métabolisme secondaire. Cependant, les signaux qui déclenchent la cascade de signalisation par phosphorylation/déphosphorylation intracellulaire restent encore peu connus. Staphylococcus aureus est une bactérie à Gram-positif pathogène pour l‟homme. Elle est l‟une des principales causes des infections nosocomiales et ce pathogène opportuniste est capable de provoquer de multiples infections allant du furoncle à la septicémie. Nos études se sont basées sur la caractérisation aux niveaux structural et fonctionnel de deux protéines de cette bactérie : une sérine/thréonine kinase nommée Stk1 ainsi que l‟un de ses substrats, la triose phosphate isomérase. Stk1 a déjà été identifiée comme responsable de la phosphorylation de plusieurs enzymes impliquées dans le métabolisme central de la bactérie ainsi que dans les phénomènes de virulence et de résistance à l‟antibiotique phosphomycine. Cependant, à ce jour, aucune caractérisation structurale n‟a été conduite sur cette kinase. Nous avons ainsi mené une étude cristallographique de plusieurs domaines de cette protéine et nous présentons, plus particulièrement, la structure de trois domaines extracellulaires dits « PASTA », ainsi qu‟un modèle tridimensionnel de la protéine entière. Les domaines PASTA sont spécifiques des Ser/Thr kinases et des Penicillin-Binding Proteins et sont impliqués dans la synthèse du peptidoglycane. Par conséquent, la connaissance de la structure de ces domaines chez Stk1 pourrait servir de base à la conception rationnelle de nouveaux inhibiteurs à visée thérapeutique. Enfin, nous avons démontré que l‟activité de l‟un des substrats de Stk1, la triose phosphate isomérase, était régulée par phosphorylation / déphosphorylation, et nous avons décrit le mécanisme qui contrôle son activation/inactivation réversible
The phosphorylation /dephosphorylation reactions in bacteria regulate various cellular functions such as growth, differentiation, pathogenicity, antibiotic resistance, stress response, biofilm formation as well as several processes involved in secondary metabolism. However, detailed understanding of their complete signaling pathways induced by phosphorylation/dephosphorylation is still unclear. Staphylococcus aureus is a Gram-positive bacterium and a human pathogen. It is one of the primary causes of nosocomial infections and this opportunist pathogen is able to cause multiple infections ranging from furuncle to septicemia. This study is focused on the structural and functional characterizations of two proteins from this bacterium: the serine/threonine kinase Stk1 and one of its substrates, the triose phosphate isomerase. Stk1 has been previously identified as responsible for the phosphorylation of several enzymes involved in the central metabolism of this bacterium as well as virulence and resistance to the antibiotic phosphomycin. However, no structural characterization has been done to date. We have performed a crystallographic study of several domains of this protein. We now present the structure of three extracellular domains designated “PASTA” in addition to the 3D molecular model of the entire protein. PASTA domains are specific to bacterial Ser/Thr kinases and to Penicillin-Binding Proteins which are involved in the peptidoglycan synthesis. Thus, the structural knowledge of PASTA domains from Stk1 could be of particular interest in the rational drug-design of new inhibitors with therapeutic aims. Finally, we have demonstrated that triose phosphate isomerase activity is regulated by phosphorylation/dephosphorylation and we have described the reversible activation/inactivation mechanism

Le carcinome pulmonaire est une affection grave et fréquente dont la prise en charge a été bouleversée ces dernières années, tant sur le plan diagnostique que pronostique ou « théranostique » avec l’avènement des « thérapies ciblées ». Ces dernières permettent une nette amélioration de la survie et du confort des patients éligibles, mais ne sont pas sans compliquer le travail médical, depuis le diagnostic de la maladie jusqu’au suivi régulier du patient, sans oublier le choix des traitements ou les problèmes techniques posés par la multiplication arborescente des altérations moléculaires à rechercher à partir d’un tissu tumoral souvent peu abondant dans ce contexte particulier de l’oncologie thoracique. Ce travail de thèse collige 5 travaux de recherche selon deux angles d’approche : les marqueurs moléculaires pronostiques et « théranostiques » du cancer pulmonaire, et les procédures de diagnostic in vivo de cette pathologie. Le premier axe comporte 4 articles. Les deux premiers concernent l’évaluation d’une nouvelle technique moléculaire, la LD-RT-PCR, dans la détection des translocation géniques du cancer pulmonaire : la première étude est une étude de faisabilité, la deuxième est un travail de validation. Le troisième article explore l’association entre la présence d’une mutation STK11 dans les carcinomes pulmonaires et l’expression de PDL1. Enfin, le quatrième article est une étude de cas illustrant l’importance de l’approche morphologique du cancer pulmonaire. Le second axe est représenté par un travail comparant une technique d’imagerie in vivo par voie endoscopique utilisant la micro-endoscopie confocale par laser avec l’approche microscopique conventionnelle
Lung cancer is a serious and frequent condition for which the management strategies have been dramatically modified in recent years, from a diagnostic, prognostic and “theranostic” perspective, most notably with the introduction of “targeted therapies”. The latter have demonstrated dramatic improvement in both quality of life and survival rates of eligible patients, yet consequently highlight new complications in diagnosis, treatment options or technical considerations which can be attributed to the growing number of molecular alterations to be detected from limited tissue samples frequently encountered in thoracic oncology. This work combines 5 different research papers from 2 different angles: prognostic and “theranostic” molecular markers of lung cancer, as well as in vivo diagnostic procedures of lung cancer. The first angle encompasses 4 articles. The first two evaluate a new molecular technique, LD-RT-PCR, to detect gene translocation in lung cancer. The third article explores the association between STK11 mutations in lung cancer and the expression of PDL1. Finally, the fourth article is a case report illustrating the importance of a morphological approach to lung cancer. The second angle compares in vivo imaging techniques by endoscopy using confocal laser microendoscopy alongside a conventional microscopic approach

碩士
國立成功大學
環境醫學研究所
97
Objective: We hereby propose a study to evaluate whether arsenic exposure and functional STK15 (Aurora A) Phe31Ile and p53 Pro72Arg polymorphisms are associated with the risk of bladder cancer in southwestern Taiwan. Design: We conducted a hospital-based case-control study and recruit participants through teaching hospitals in southern Taiwan with bladder cancer patients as cases and non-cancer patients as controls. All the participants were interviewed using a standard questionnaire to collect data on demographic characteristics, smoking habits, drinking water history, and other relevant risk factors. The genotypes were determined using polymerase chain reaction-restricted fragment length polymorphism (PCR-RFLP). Result: We collected 59 bladder cancer patients (46 men and 13 women) as cases and 201 non-cancer patients (with 110 men and 91 women) as controls. Among demographic characteristics, we found that the male gender, lower educational level, and smoking were associated with a higher risk of bladder cancers. In addition, we found that drinking well over fifteen years and in southwestern Taiwan with high arsenic concentration were more frequent in bladder cancer group than in the non-cancer group. In gene polymorphism analysis, we found that both STK15 Phe31Ile (T>A) mutant type (AA) and mutant allele (A) associated with a higher risk of bladder cancers OR=2.1 (95% CI=0.8-5.5, p=0.39) and OR= 1.7 (95% CI=0.7-4.4, p=0.17). Furthermore, mutant type (AA) was more frequent in bladder cancer patients than in non-cancer controls 50.8% vs. 39.3%. p53 Pro72Arg (C>G) with one mutant allele (GC) was associated with a high risk of bladder cancers OR=1.4 (95% CI=0.7-2.7, p=0.54). An increase in mutant allele (A) of STK15 Phe31Ile (T>A) was associated with higher risk of bladder cancers in non-arsenic exposure area, but an increase in mutant allele (A) of STK15 Phe31Ile (T>A) was associated with a lower risk of bladder cancers in high-arsenic exposure area. An increase in mutant allele (C) of p53 Pro72Arg (C>G) was associated with a lower risk of bladder cancers in non-arsenic exposure area, but an increase in mutant allele (C) of p53 Pro72Arg (C>G) was associated with a higher risk of bladder cancers in high-arsenic exposure area. Regardless of geno-types, an increase in arsenic concentration was associated with a higher risk of bladder cancers. When both STK15 Phe31Ile and p53 Pro72Arg carried mutant alleles, we would find a higher risk in bladder cancers OR=1.1 (95% CI=0.3-4.7). Gene polymorphisms act as a modifier of bladder cancers in high CAE concentration group, OR=4.5 (95% CI=2.1-9.6, p<0.001). Conclusion: STK15 play an important role in G2/M of cell cycle control: STK15 Phe31Ile (T>A) with mutant type (AA) and mutant allele (A) were related to higher risk of bladder cancers. When both STK15 Phe31Ile and p53 Pro72Arg carried mutant alleles, we would find a higher risk in bladder cancers.

碩士
國立陽明大學
口腔生物研究所
96
Oral squamous cell carcinoma (OSCC) is one of commonly diagnosed cancers all over the world. It has been one of the 10 leading causes of death from cancer in Taiwan since 1991. In the past decades, incidence and mortality rate of OSCC increased remarkably. Although the technologies of diagnosis and therapy have been remarkably developed, the 5-year survival rate of OSCC has not been improved significantly. Our laboratory has found that chromosome 20q was a hotspot for gene amplification in OSCC. STK15 (Aurora A) is a serine/threonine kinase which has been identified as a regulator of the mitosis and it localizes on 20q13.2-13.3. The amplification and overexpression of STK15 was found in many cancers; however, the alterations of STK15 was rarely reported in OSCC. In this study, we attempted to identify the role of STK15 in OSCC, which may provide us the insight to develop a potential therapeutic regimen for increasing survival rate of human. We used real-time PCR, immunohistochemistry and Western blot to analyze OSCC cell lines, buccal brushes from high risk individuals, primary tumors, recurrent tumors, and nodal metastasis. We found that the copy number of STK15 increased following the disease progression from high risk non-tumor tissue to primary tumors, then to recurrent tumors, although the incidence of copy number amplification was only 11.6%. Cytosolic STK15 was detected in most tissues examined and some cases had both cytosol and nuclear STK15 expression. Interestingly, the percentage of nuclear STK15 expression increased following the progression from non-cancerous tissue to primary tumor, then to metastatic tumor. Furthermore, higher nuclear STK15 expression was significantly associated with the worse survival rate of OSCC patients. We further elucidated the functions of STK15 in OSCC by using shRNA to knockdown STK15 in OECM-1 cells. The results showed that the proliferation, migration and anchorage-independent growth were decreased and autophagy was induced after STK15 knockdown in OECM-1 cells. This study provides clinicopathological and functional evidences suggesting that STK15 is oncogenic for OSCC. Preclinical therapeutic trials is underway to clarify if the knockdown of STK15 bestows potential therapeutic efficacy for controlling OSCC.

Colorectal cancer (CRC) is one of the most common forms of cancer in Western nations, it is however uncommon in sub-Saharan Africa. In South Africa there is an approximate ten-fold lower incidence of CRC in black patients compared to Caucasian patients. This could be due to differences in lifestyles and environment that exist between the various population groups. Underlying molecular events could also account for the difference in susceptibility to colorectal cancer. Mutations in the Peutz-Jeghers syndrome (PJS) gene, STK11, predispose to amongst others colorectal cancer. To examine the role of this gene in South African patients with CRC, DNA from 208 tumours (104 black patients, 104 Caucasian patients) was screened for STK11 mutations via PCR-SSCP analysis. In total 8 novel missense mutations, one of which was germline, were identified in seven tumours (~3.4% 7/208) from 5 black and two Caucasian patients. One tumour from a Caucasian patient was found to be a compound heterozygote. Peutz-Jeghers syndrome was thus diagnosed in 0.96% (1/104) of black patients via a germ line mutation. Thus 4.8% (5/104) of tumours from black patients and 1.9% (2/104) of tumours from Caucasian patients harbour STK11 missense mutations. In addition, 3 synonymous and 5 intronic mutations were detected in a further 73 tumours from black patients, whereas only 3 synonymous and 5 intronic mutations were detected in 25 tumours from Caucasian patients. The present study is the sixth to suggest that somatic mutation of the STK11 gene in sporadic colorectal cancer of Caucasians is an infrequent event. However, this is only the second study of a non-Western population to show somatic mutations in sporadic cases of CRC. Furthermore with regard to the anatomic site of tumours with somatic missense mutations, the present study found that for black patients 7.69% (2/26) of the left-sided tumours, 2% (1/50) of rectal tumours and 4.54% (1/22) of right-sided tumours harboured mutations. Thus the frequency of missense mutations of left-sided CRC tumours compared to right-sided tumours was not significantly elevated (÷2-test, 1df, p = 0.881) in the black population. This study represents the first investigation into the role of the STK11 gene in putative sporadic cases of CRC from both black and Caucasian South African patients. The observed mutations clearly show that mutations of the STK11 gene are infrequent in the CRCs of the South African Caucasian population, and more frequent in the South African black population. This may be a reflection of the differences in lifestyle and incidence of CRC in the different populations.
Dissertation (MSc (Human Genetics))--University of Pretoria, 2006.
Genetics
unrestricted

碩士
國立臺灣大學
藥理學研究所
105
Cell migration is important for embryonic development, tissue regeneration and cancer metastasis. Recent research identified cell migration-related genes, but how they crosstalk with each other remain unclear. To resolve this mystery, we conducted a “two-hit” cell migration screen using short hairpin RNA (shRNA) and small molecules inhibitors. Among our candidates, we are particularly interested in STK40, a putative serine/threonine kinase. Previous literature reports showed that STK40 knockdown increased cell motility. However, our two-hit screen revealed STK40 knockdown plus PD98059 (MEK inhibitor) resulted in a dramatic suppression of cell motility. We therefore hypothesize that STK40 interacts with MAPK signaling to regulate cell migration. 　　To validate this hypothesis, we first conducted various cell migration assays by analyzing different parameters to investigate how STK40 regulates cell migration in epithelial and endothelial cells. We discovered that STK40 improved speed in both cell lines and improved coordination in SAS. Our immunofluorescent assays also showed that altering STK40 level altered E-cadherin and phalloidin, which may explain STK40 improvement in coordination. Further exploration will aim at how STK40 and MAPK regulate cell migration, hoping to disclose the mechanism of the regulation of STK40 in cell migration.