abstract: LKB1/STK11 is a serine/threonine kinase first identified in C.elegans as a gene important for cell polarity and proliferation. Mutations in LKB1 are the primary cause of Peutz-Jegher’s cancer syndrome, an autosomal dominantly inherited disease, in which patients are predisposed to benign and malignant tumors. Past studies have focused on defining LKB1 functions in various tissue types, for example LKB1 regulates axonal polarization and dendritic arborization by activating downstream substrates in excitatory neurons of the developing neocortex. However, the implications of LKB1, specifically in the developing cortical inhibitory GABAergic interneurons is unknown. LKB1 deletion was achieved by using Cre-lox technology to induce LKB1 loss in cells localized in the medial ganglionic eminence (MGE) that express Nkx2.1 and generate cortical GABAergic neurons. In this research study it is suggested that LKB1 plays a role in GABAergic interneuron specification by specifically regulating the expression of parvalbumin during the development of fast-spiking interneurons. Preliminary evidence suggest LKB1 also modulates the number of Nkx2.1-derived oligodendrocytes in the cortex. By utilizing a GABAergic neuron specific LKB1 deletion mutant, we confirmed that the loss of parvalbumin expression was due to a GABAergic neuron autonomous function for LKB1. These data provide new insight into the cell specific functions of LKB1 in the developing brain. / Dissertation/Thesis / Masters Thesis Biology 2019

Ventilatory drive is mediated by respiratory central pattern generators that are located in the brainstem, which are continuously modulated by specialised peripheral and central chemoreceptors to adjust ventilatory patterns according to changes in arterial PO2. These specialised oxygen-sensing chemoreceptors are activated in response to acute reductions in arterial PO2 and ultimately trigger a respiratory response that acts to restore oxygen-levels. However, the molecular mechanism by which mammals are able to regulate their breathing pattern in such a manner during hypoxia remains controversial. Therefore, the studies performed in this thesis aimed to investigate the possibility that this process may be mediated by the liver kinase B 1 (LKB1)/ AMP-activated protein kinase (AMPK) signalling pathway, which is central to cellular adaptations to metabolic stress. This first involved the development of transgenic mice in which Lkb1 or AMPK were deleted. Global knockout of Lkb1 (Sakamoto, 2006) or AMPK activity (Viollet et al., 2009) are embryonic lethal. Thus, the Cre/loxP system was used to develop transgenic mice that had either Lkb1 or both isoforms of the AMPK catalytic α- subunit (α1 and α2) conditionally knocked out in catecholaminergic cells (including therein hypoxia-activated cells of the brainstem and carotid body) by driving Cre expression through a tyrosine-hydroxylase-specific promoter region. The consequent effects on the ventilatory response to hypoxia were then examined using unrestrained whole-body plethysmography. This demonstrated that, in contrast to the hyperventilation evoked in controls, increased ventilation was virtually abolished in the Lkb1 and AMPK α1 and α2 double knockouts during hypoxia. Both knockout mice also exhibited periods of hypoventilation with frequent apnoeas during hypoxia. Additionally, studies on single AMPK α1 and AMPK α2 knockouts identified that the ventilatory dysfunction in AMPK α1 and α2 double knockouts was primarily caused by AMPK α1 deletion. In contrast, the severe ventilatory abnormalities exhibited during hypoxia following the deletion of Lkb1 and AMPK in catecholaminergic cells were mostly reversed upon exposure of mice to hypoxia with hypercapnia. Also, the ventilatory response to hypercapnia alone was without any major effect as a result of Lkb1 deletion or the dual-deletion of AMPK α1 and α2 catalytic subunits in catecholaminergic cells. This thesis therefore demonstrates, for the first time, that the LKB1-AMPK signalling pathway is key to respiratory adaptations during hypoxia, by regulating catecholaminergic oxygen-sensing cells, thus protecting against hypoventilation and apnoeas. The LKB1-AMPK signaling pathway can thereby determine oxygen and energy supply at both a cellular and whole-body level.

Cell signalling determines physiological responses to many cellular stimuli and environmental changes. The transforming growth factor-beta (TGFβ)/bone morphogenetic protein (BMP) signalling pathways begin by binding of ligand to the heterodimeric receptor complex, followed by activation of Smads that translocate to the nucleus to regulate transcription of genes that further mediate cellular physiology. The TGFβ/BMP pathways are very important for proper tissue development and homeostasis, thus precise spatial and temporal regulation of the signalling pathway is required and achieved by many positive and negative signalling regulators. This thesis work identified the liver kinase B1 (LKB1) pathway as a negative regulator of TGFβ/BMP signalling pathways. In the first paper, we established LKB1 as a negative regulator of TGFβ signalling and TGFβ-induced epithelial to mesenchymal transition (EMT). LKB1 impairs Smad4 binding capacity to DNA leading to suppressed TGFβ-activated gene transcription. The second paper describes further the mechanism of LKB1 negative regulation on BMP signalling, by mediating BMP type I receptor degradation resulting in inhibition of BMP-induced cell differentiation. Downstream of LKB1, salt inducible kinase 1 (SIK1) is a TGFβ target gene and its expression is up-regulated by Smad2/3/4-mediated gene transcription. The third paper elucidates the mechanism of SIK1 transcriptional induction via an enhancer element located 3’ of the gene and SIK1-mediated type I TGFβ receptor degradation, which requires the activity of Smad7 and of the Smurf2 ubiquitin ligase. The fourth manuscript finds sucrose non-fermenting (SNF) 1-like kinase 2 (NUAK2) as another TGFβ target gene and its up-regulation results in modification of the mammalian target of rapamycin (mTOR) pathway that controls protein synthesis. NUAK2 cooperates with LKB1 leading to Raptor phosphorylation and inhibition of mTOR-mediated protein synthesis. Collectively, this thesis work has provided a functional link between two important signalling pathways, the metabolic LKB1 pathway and TGFβ/BMP pathway.

STK11/LKB1, a serine/threonine protein kinase, is a key upstream kinase of adenine monophosphate-activated protein kinase (AMPK), a necessary kinase in the control of metabolism for maintaining energy homeostasis. Although it has become clear that LKB1 is mutated in a significant number of Peutz¡VJeghers syndrome (PJS) and sporadic cancers, most frequently in adenocarcinoma of the lung, little is known about how the LKB1 signaling regulates the metabolic process and energy production underlying hypoxia and increased radiosensitivity of lung tumor. Here, we employed lung cancer cells as a model system to dissect the functional roles of LKB1 signaling in human lung adenocarcinoma. We found that LKB1 inhibits lung cancer cell migration, transformation and chemo-resistance in vitro after we restored LKB1 expression in LKB1 null A549 and H460 lung cancer cells. We also found that LKB1 prevents UV-induced DNA damage in human lung cancer cell lines by comet assay and activated UV-induced apopotsis by MTT assays. Furthermore, we designed a systems biology approach to provide a comprehensive protein-protein interaction analysis in order to elucidate the LKB1 tumor suppressor network in vivo. We employed Immunoprecipitation-HPLC- Mass Spectrometry (IP-LC-MS) to identify the novel proteins interacting with LKB1 under different cellular stress conditions. We have identified that LKB1 is involved in CFTR synthesis pathway underlying normoxia condition and participates in the glycolysis and gluconeogenesis pathways underlying hypoxia condition. Together, our findings indicated that LKB1 is involved in the regulation of cell migration, energy metabolism and DNA repair in lung cancer cells, and should provides insights to further exploit the concept of deranged cancer bioenergetics and aberrant growth signals to achieve more effective and selective strategies for lung cancer patients.

Lung cancer is the second leading cause of cancer-related death in Taiwan in 2003. However, lung cancer has become the first in 2004. Discovering the new molecular targets may provide new methods for the treatments of this devastating disease. LKB1, a serine/threonine kinase, is a new tumor suppressor gene with spontaneous mutations and/or deletions found in human cancers. Reports have recently demonstrated that LKB1 inactivating mutations in lung adenocarcinomas of sporadic origin, including primary tumors and lung cancer cell lines. In this study, we investigated LKB1 gene inactivation frequencies in 110 Taiwan patients with non-small cell lung carcinomas (NSCLC) and 7 lung cancer cell lines. LKB1 inactivation was screened by polymerase chain reaction (PCR), sequencing, co-amplification and loss of heterozygosity (LOH) analysis. In addition, LKB1 expressions were determined in clinical samples by immunohistochemistry (IHC) and in cell lines by reverse-transcriptase PCR and western blot analysis. The results showed five out of 110 (4.5%) patients with LKB1 gene exon 8 deletions. Two out these 5 patients were also found with exon 7 deletions. An identical mutation at codon 354 (Phe to Leu) were found in 4 out of 65 (6.2%) patients. The nature of this mutation was found to be a new LKB1 polymorphism by single strand conformation polymorphism (SSCP) assay and sequencing analysis after compared to normal controls. Various point mutations were also found in 3 out of 7 cell lines. In addition, 11 out of 81 (13.6%) patients were found with LOH. Finally, reduced expressions of LKB1 were observed in lung cancer clinical samples. These data suggest that LKB1 may be a tumor suppressor gene that involved in the carcinogenesis of NSCLC.

<p>Obesity is the latest epidemic of the 21^st century. Indeed, numerous studies have associated obesity with an increased risk of developing several health conditions, including cancer. Moreover, modest increases in body mass index increase the risk of developing cancer, especially cancer of the kidney. Although the mechanism mediating this increased risk is unknown, the plasma level of adiponectin is known to be inversely correlated with body weight and risk of developing kidney cancer.</p> <p>Tumour suppression via adiponectin is believed to be mediated through adiponectin receptor-1, which activates AMPK by LKB1 and suppresses pathways upregulated in cancer by inhibiting mTOR. Consistent with the anti-tumourigenic properties of this pathway, several cancers display reduced AdipoR1 and LKB1 expression and/or increased mTOR activity. In this study we identified reduced AdipoR1 and LKB1 protein expression in patients’ renal cell carcinomas and quantified the reduction in LKB1, on tissue microarrays containing 201 RCC patients, to be significant.</p> <p>Targeted knockdown of LKB1 in CRL-1932 cells (shLKB1) was accompanied by a reduction in AdipoR1, and recapitulated our observations in RCC tumours. These shLKB1 cells were unable to execute established events of adiponectin-AMPK signalling and, presented increased proliferation and invasion abilities <em>in vitro</em> and tumour growth <em>in vivo</em>. Collectively, these results suggest that a reduced plasma level of adiponectin coupled with a downregulation of AdipoR1 and LKB1 expression, disrupts the tumour-suppressive adiponectin-AMPK signalling pathway, and rationalizes the association of obesity with the development of RCC.</p> / Master of Science in Medical Sciences (MSMS)

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.

Des mutations du gène STK11 furent initialement décrites comme responsable du syndrome Peutz-Jeghers, dont la gravité est lliée à une incidence accrue d’apparition de tumeurs. Le produit de ce gène, la sérine/thréonine kinase LKB1, a une expression ou une activité catalytique réduite, voir perdue, consécutivement à des mutations somatiques dans plusieurs types de cancer mais principalement du poumon (30% des NSCLC). Cette kinase est considérée de ce fait comme un suppresseur de tumeur d’importance. Les mécanismes moléculaires responsables de sa propriété suppresseur de tumeur restent à identifier. En effet, alors que sa fonction dans le métabolisme cellulaire, au travers de l’activation de la kinase AMPK, fut longtemps privilégiée, elle est actuellement remise en cause au profit de sa fonction de régulatrice de la signalisation Wnt canonique. Mes travaux de thèse confortent cette éventualité dans le cas des tumeurs pulmonaires (NSCLC). En effet, parmi les deux complexes fonctionnels que forme LKB1 avec les pseudokinases STRADα ou β, mes résultats démontrent que seul celui impliquant STRADβ intervient dans la régulation de la voie Wnt. Pour cela, le complexe LKB1/STRADβ se localise au niveau du cil primaire et participe à l’activation de la kinase MARK3. Ces résultats, étayés par un modèle murin invalidé pour STRADβ ainsi que l’analyse, a posteriori, de bases de données transcriptomiques adossées aux données cliniques de patients atteints de NSCLC, suggèrent que l’activité suppresseur de tumeur de LKB1 est associée à sa localisation et à sa fonction au niveau du cil primaire en participant à l’activation de MARK3 et à la régulation de la signalisation Wnt canonique. / STK11 gene mutations were originally identified as responsible for the Peutz-Jeghers syndrome of which severity is mainly related to an increase incidence of tumor development. The product of this gene the serine/threonine kinase LKB1 gets its activity or its expression reduced, sometimes even lost, following somatic mutations in several types of cancer such as pancreas, liver but mainly from lung. Indeed, almost 30% of non-small cell lung carcinoma (NSCLC) does not express anymore or only an inactive form, has led to consider this kinase as tumor suppressor of importance. While there is no doubt of the involvement of its catalytic activity molecular mechanisms responsible for its tumor suppressor properties remain to be identified. Indeed, whereas its function as regulator of cellular metabolism through AMPK has been favor for a while, it is currently re-assess to benefit to its regulator function on canonical Wnt signaling. My thesis work, reinforce this eventuality in NSCLC. Indeed, among the two functional complexes formed by LKB1 through its association with STRADα or β pseudokinases, my results show that only the complex related to STRADβ is involved in the canonical Wnt pathway regulation. For that, LKB1/STRADβ complex localizes at primary cilia and participates to MARK3 kinase activation. These results strengthened by a STRADβ knockout mouse model and an a posteriori transcriptomic analysis of lung adenocarcinoma patient datasets related to their clinical records, suggest that LKB1 tumour suppressor activity is associated with its localization and its function at primary cilia participating in the activation of MARK3 and thus regulation of canonical Wnt signaling.

Le gène suppresseur de tumeur LKB1 code une sérine/thréonine kinase qui régule le métabolisme énergétique et la polarité cellulaire. Son action biologique s'exerce en partie via la protéine kinase activée par l'AMP (AMPK), substrat de LKB1 dont la phosphorylation stimule l'activité catalytique. Nous avons récemment mis en évidence une interaction entre LKB1 et la déacétylase HDAC6. HDAC6 régule principalement l'état d'acétylation de protéines localisées dans le cytoplasme telles que la molécule chaperon HSP90, la tubuline α, et la cortactine. HDAC6 contrôle la stabilité des protéines liées à HSP90 mais agit aussi sur la polarité et l'adhérence des cellules. De plus, HDAC6 répond à différentes situations de stress cellulaire en favorisant le transport des protéines polyubiquitinées vers les aggrésomes, où celles ci sont dégradées, et en promouvant la formation des granules de stress, complexes ribonucléoprotéiques participant au stockage des ARNm et au blocage de la traduction. Mon projet de recherche a porté sur les conséquences fonctionnelles de l'interaction entre LKB1 et HDAC6. J'ai ainsi pu montrer que la formation de ce complexe est renforcée en condition de stress oxydatif et thermique. Dans cette situation biologique, LKB1 interfère avec la capacité de HDAC6 à fixer les protéines ubiquitinylées, et par conséquent prévient la formation des aggrésomes et des granules de stress. A l'inverse, LKB1 stimule l'activité déacétylase de HDAC6, et cette action de LKB1 est requise pour la migration orientée des cellules ainsi que pour la polarisation apico-basale dans un modèle de culture d'entérocytes. Ce travail nous a ainsi permis d'identifier un nouvel effecteur de LKB1 qui intervient dans la réponse au stress et dans la polarisation cellulaire. Il s'agit aussi de la première mise en évidence d'une régulation de l'activité de liaison à l'ubiquitine de HDAC6. Ces données suggèrent que LKB1, via son effet sur HDAC6, pourrait limiter la réponse adaptative des cellules soumises à des stress exogènes et endogènes, comme ceux que les cellules en voie de transformation rencontrent dans leur microenvironnement, une propriété qui pourrait s'avérer essentielle pour son activité de suppresseur de tumeur / The tumor suppressor LKB1 is a serine-threonine kinase that acts as a critical regulator of energy homeostasis and cell polarity 1,2. LKB1 relays its intracellular signal through the AMP-activated protein kinase (AMPK) as well as twelve additional members of the AMPK sub-family 3-5. However, despite the identification of these LKB1 effectors, the mechanisms that underlie LKB1-mediated biological effects remain incompletely understood. We now report that LKB1 interacts with and phosphorylates HDAC6, a deacetylase that protects cells against extrinsic insults through its ability to ligate polyubiquinated misfolded proteins and to dynamically associate with both the microtubule and the actin cytoskeleton networks 6. We further found that the formation of the LKB1-HDAC6 complex was promoted in response to diverse stressful stimuli. As a consequence, HDAC6 ubiquitin-binding activity was inhibited, thus impeding the formation of aggresomes and stress granules, two transient cellular structures that, respectively, prevent the accumulation of aggregated proteins 7 and remodel messenger ribonucleoprotein complexes following stresses that block translation 8. Collectively, these data identify HDAC6 as a key downstream component of the LKB1 signalling pathway. Our findings further suggest that LKB1, via its inhibitory effect on HDAC6 ubiquitin-binding activity, limits the cellular adaptive response to a protracted stress, a distinctive biological property that is likely to contribute to its tumor-suppressive function

The pathogenesis of heart failure (HF) involves compensatory left ventricular hypertrophy. Reactive oxygen species (ROS) are elevated in HF and mediate myocardial hypertrophy. ROS also mediate formation of lipid peroxidation byproducts, yet little is known about their role in promoting hypertrophy. One lipid peroxidation byproduct, 4-hydroxy-trans-2-nonenal (HNE) is a reactive aldehyde that forms covalent adducts on proteins. HNE levels are also elevated in HF and may mediate hypertrophy via HNE-adduct formation. LKB1 - a tumor suppressor protein - regulates cellular growth through activation of the downstream kinase AMPK. Activation of AMPK suppresses functions that consume ATP and simultaneously activates processes to generate energy. The LKB1 protein is inhibited by oxidants, but whether this results in myocardial hypertrophy is unclear. I hypothesized that HNE can directly promote cardiac hypertrophy via the modification of LKB1. In HEK293T cells I observed that HNE adducts inhibit activity of LKB1 through direct oxidative modification. Mutation of LKB1 Lys-96 or Lys-97 resulted in less HNE-LKB1 adduct formation. Mutation of LKB1 Lys-97 prevented the inhibitory effect of HNE, suggesting that HNE-adduction at this residue is sufficient to inhibit LKB1. In cardiomyocytes HNE inhibited both LKB1 and AMPK, increased phosphorylation of mTOR, p70S6K, and S6K, and increased protein synthesis. HNE also activated Erk1/2, which contributed to S6K activation but was not required for cellular growth. Hypertrophic S6K activation was dependent on mTOR. Mice fed a high-fat high-sucrose (HFHS) diet have myocardial hypertrophy that can be prevented by antioxidants. Hearts of HFHS mice have HNE-LKB1 adducts, inhibited LKB1 activity, yet no change in AMPK activation. Mice lacking aldehyde dehydrogenase 2 (ALDH2), an enzyme involved in HNE detoxification, have increased myocardial hypertrophy when fed HFHS diet yet have increased LKB1 activity. In summary HNE directly causes hypertrophy in cardiomyocytes. This occurs through inhibition of LKB1 and in part through Erk1/2 activation. In HFHS-fed mice HNE-LKB1 adduct formation is associated with decreased LKB1 activity. Impairing detoxification of reactive aldehydes in the ALDH2-KO mice is sufficient to increase myocardial hypertrophy, but this appears to be independent of LKB1. This study demonstrates a novel mechanism of cardiac hypertrophy caused by reactive aldehydes.