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.

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.

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.

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.

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