LKB1 Deficiency Research Articles

Abstract 1839: LKB1 deficiency enhances sensitivity to energetic stress induced by erlotinib treatment in non-small cell lung cancer (NSCLC) cells

Abstract The tumor suppressor LKB1 is inactivated in 20-30% of non-small cell lung cancer (NSCLC) patient tumors. Because LKB1 is the master kinase phosphorylating the energy-sensing kinase AMPK, dysregulation of the LKB1-AMPK signaling axis causes loss of cellular homeostasis in response to metabolic inhibition or external stress-induced mitochondrial insults. Erlotinib is used clinically in NSCLC patients to target mutationally activated EGFR and is known to induce mitochondrial-mediated apoptosis. We investigated the metabolic response and mitochondrial function of LKB1-deficient NSCLC cells treated with erlotinib. LKB1-deficient cells exhibited enhanced sensitivity to erlotinib treatment in vitro and in vivo despite having wild-type EGFR. We found that this enhanced response was due to altered energetic metabolism and mitochondrial dysfunction in LKB1-deficient cells, which prevents homestatic maintenance of ATP and reactive oxygen species (ROS) levels in response to erlotinib treatment. Despite negative regulation of mTOR activity by LKB1-AMPK signaling, erlotinib effectively blocked mTOR signaling in LKB1-deficient cells, resulting in inhibition of cell growth, as well as activation of apoptosis. These findings will be important for designing targeted treatments for LKB1-deficient NSCLC patient tumors harboring wild-type EGFR, which disrupt metabolic and signaling pathways known to regulate energy metabolism in these cells. Citation Format: Young Mi Whang, Serk In Park, Irina A. Trenary, Changki Lee, Jacob M. Kaufman, David P. Carbone, Jamey D. Young. LKB1 deficiency enhances sensitivity to energetic stress induced by erlotinib treatment in non-small cell lung cancer (NSCLC) cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1839. doi:10.1158/1538-7445.AM2014-1839

An AMPK-Independent Signaling Pathway Downstream of the LKB1 Tumor Suppressor Controls Snail1 and Metastatic Potential

The serine/threonine kinase LKB1 is a tumor suppressor whose loss is associated with increased metastatic potential. In an effort to define biochemical signatures of metastasis associated with LKB1 loss, we discovered that the epithelial-to-mesenchymal transition transcription factor Snail1 was uniquely upregulated upon LKB1 deficiency across cell types. The ability of LKB1 to suppress Snail1 levels was independent of AMPK but required the related kinases MARK1 and MARK4. In a screen for substrates of these kinases involved in Snail regulation, we identified the scaffolding protein DIXDC1. Similar to loss of LKB1, DIXDC1 depletion results in upregulation of Snail1 in a FAK-dependent manner, leading to increased cell invasion. MARK1 phosphorylation of DIXDC1 is required for its localization to focal adhesions and ability to suppress metastasis in mice. DIXDC1 is frequently downregulated in human cancers, which correlates with poor survival. This study defines an AMPK-independent phosphorylation cascade essential for LKB1-dependent control of metastatic behavior.

Neuron-derived Neurotrophic Factor Functions as a Novel Modulator That Enhances Endothelial Cell Function and Revascularization Processes

Strategies to stimulate revascularization are valuable for cardiovascular diseases. Here we identify neuron-derived neurotrophic factor (NDNF)/epidermacan as a secreted molecule that is up-regulated in endothelial cells in ischemic limbs of mice. NDNF was secreted from cultured human endothelial cells, and its secretion was stimulated by hypoxia. NDNF promoted endothelial cell network formation and survival in vitro through activation of Akt/endothelial NOS (eNOS) signaling involving integrin αvβ3. Conversely, siRNA-mediated knockdown of NDNF in endothelial cells led to reduction of cellular responses and basal Akt signaling. Intramuscular overexpression of NDNF led to enhanced blood flow recovery and capillary density in ischemic limbs of mice, which was accompanied by enhanced phosphorylation of Akt and eNOS. The stimulatory actions of NDNF on perfusion recovery in ischemic muscles of mice were abolished by eNOS deficiency or NOS inhibition. Furthermore, siRNA-mediated reduction of NDNF in muscles of mice resulted in reduction of perfusion recovery and phosphorylation of Akt and eNOS in response to ischemia. Our data indicate that NDNF acts as an endogenous modulator that promotes endothelial cell function and ischemia-induced revascularization through eNOS-dependent mechanisms. Thus, NDNF can represent a therapeutic target for the manipulation of ischemic vascular disorders.

Transdifferentiation of lung adenocarcinoma in mice with Lkb1 deficiency to squamous cell carcinoma

Lineage transition in adenocarcinoma (ADC) and squamous cell carcinoma (SCC) of non-small cell lung cancer, as implicated by clinical observation of mixed ADC and SCC pathologies in adenosquamous cell carcinoma, remains a fundamental yet unsolved question. Here we provide in vivo evidence showing the transdifferentiation of lung cancer from ADC to SCC in mice: Lkb1-deficient lung ADC progressively transdifferentiates into SCC, via a pathologically mixed mAd-SCC intermediate. We find that reduction of lysyl oxidase (Lox) in Lkb1-deficient lung ADC decreases collagen disposition and triggers extracellular matrix remodelling and upregulates p63 expression, a SCC lineage survival oncogene. Pharmacological Lox inhibition promotes the transdifferentiation, whereas ectopic Lox expression significantly inhibits this process. Notably, ADC and SCC show differential responses to Lox inhibition. Collectively, our findings demonstrate the de novo transdifferentiation of lung ADC to SCC in mice and provide mechanistic insight that may have important implications for lung cancer treatment.

Abstract B07: Loss of LKB1 promotes CXCL8 production via NF-κB and WNT signaling in human bronchial epithelial cells.

Abstract Loss of LKB1/STK11, a tumor suppressor gene, characterizes 5-39% of primary non-small cell lung cancers (NSCLC). In a murine model of lung carcinogenesis with inducible KRAS activation, concurrent mutation of LKB1 (KrasG12D, Lkb1-/-) yields a higher frequency of NSCLC tumors and metastasis compared to KRAS mutation only. In vitro studies have shown that following LKB1 loss, tumor cells acquire increased migration and invasion through activation of the SRC kinase family. Although these previous findings offer a possible explanation for LKB1-dependent tumor progression, few mechanisms have been proposed to delineate how LKB1 deficiency contributes to pulmonary carcinogenesis. In this study, we utilize human bronchial epithelial cells (HBECs) immortalized in the absence of viral onco-proteins that represent a useful model to study the early events of lung carcinogenesis. Knockdown of LKB1 in HBECs (LKB1-KD HBECs) leads to numerous cancer-associated phenotypes, including increased proliferation (when combined with KRAS mutation), preference for glycolytic metabolism and induction of epithelial-to-mesenchymal transition. More importantly, LKB1-KD HBECs secrete higher levels of multiple inflammatory factors, most prominent of which is the pro-angiogenic chemokine CXCL8. By binding to its membrane receptors CXCR1/2 which are universally expressed by neutrophils, macrophages, endothelial cells and pulmonary epithelial cells, CXCL8 is associated with chemotaxis, angiogenesis, tumorigenicity and metastasis. Our data indicate that knockdown of LKB1 in HBECs leads to transcriptional upregulation and elevated secretion of CXCL8. Moreover, the NF-κB and WNT pathways are activated and mediate the increased release of CXCL8 following LKB1 loss in HBECs. Conversely, re-introduction of wildtype LKB1 in LKB1-null NSCLC tumor cells decreases CXCL8 production and reduces NF-κB and WNT signaling activity. These findings suggest that LKB1 deficiency drives augmentation of CXCL8 in the developing tumor microenvironment via LKB1-dependent NF-κB and WNT signaling in HBECs. In future functional studies, we will determine if the LKB1-CXCL8 axis yields LKB1-dependent angiogenesis and tumorigenesis. Delineating the contribution of LKB1 to these malignant phenotypes may yield a more thorough understanding of the pathogenesis of NSCLC, which is needed to create a pathway for the application of novel early detection and chemoprevention strategies. NIH/NCI #U01CA152751 (SMD, TCW), NCI #U01CA152751-S1 (SMD, TCW), NCI #U01CA152751-AS (SMD, KK), NCI SPORE #P50CA70907 (JDM, JEL) Citation Format: RUI LI, Tonya C. Walser, Kostyantyn Krysan, David Shackelford, Jill E. Larsen, John D. Minna, Steven M. Dubinett. Loss of LKB1 promotes CXCL8 production via NF-κB and WNT signaling in human bronchial epithelial cells. [abstract]. In: Proceedings of the AACR-IASLC Joint Conference on Molecular Origins of Lung Cancer; 2014 Jan 6-9; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2014;20(2Suppl):Abstract nr B07.

Abstract C55: Investigating mechanisms of gastrointestinal polyposis and osteoblastic tumor formation induced by mesenchymal Lkb1 deficiency

Abstract Lkb1 has emerged as an important tumor suppressor gene in both sporadic and hereditary cancer. Inactivating germline mutations in the LKB1 gene underlie familial Peutz-Jeghers Syndrome (PJS), a cancer-prone disorder characterized by predisposition to gastrointestinal polyposis. Targeted disruption of Lkb1 in mesenchymal smooth muscle cells was sufficient to recapitulate PJS-type polyposis in mice (Katajisto et al, 2008) and Lkb1 heterozygous mice develop osteoblastic tumors (Robinson et al, 2008). To investigate the mechanism(s) by which Lkb1 loss in mesenchymal tissues drives tumorigenesis, we deleted Lkb1 using two mesenchymal deletors (Fsp1-Cre and Twist2-Cre) expressed in mesenchymal progenitors. Heterozygous deletion of Lkb1 with either Fsp1-Cre (Lkb1flox/+;Fsp1-Cre) or Twist2-Cre (Lkb1flox/+;Twist2-Cre) results in both polyposis and osteoblastic tumor. Homozygous Lkb1 deletion using Fsp1-Cre (Lkb1flox/flox;Fsp1-Cre) leads to rapid polyposis and osteoblastic tumors with complete penetrance, indicating that complete loss of Lkb1 is advantageous for tumorigenesis. AMPK, a key substrate of Lkb1 connected to several other tumor suppressors, has been proposed to mediate Lkb1 tumor suppressive function. However, homozygous deletion of both AMPKα1 and AMPKα2 using Fsp1-Cre does not lead to tumorigenesis. These results implicate other Lkb1 substrate kinases in tumor suppression and suggest Lkb1 loss in a common progenitor underlies mesenchymal tumorigenesis. Katajisto P, et al., (2008) LKB1 signaling in mesenchymal cells required for suppression of gastrointestinal polyposis. Nat Genet, 40(4): p. 455-9 Robinson J, et al., (2008) Osteogenic tumours in Lkb1-deficient mice. Exp Mol Pathol, 85(3): p. 223-6 Citation Format: Kaisa Laajanen, Saara Ollila, Iris PL Wong, Kari Vaahtomeri, Gustavo Leone, Benoit Viollet, Makela P. Tomi. Investigating mechanisms of gastrointestinal polyposis and osteoblastic tumor formation induced by mesenchymal Lkb1 deficiency. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C55.

The LKB1 Tumor Suppressor as a Biomarker in Mouse and Human Tissues

Germline mutations in the LKB1 gene (also known as STK11) cause the Peutz-Jeghers Syndrome, and somatic loss of LKB1 has emerged as causal event in a wide range of human malignancies, including melanoma, lung cancer, and cervical cancer. The LKB1 protein is a serine-threonine kinase that phosphorylates AMP-activated protein kinase (AMPK) and other downstream targets. Conditional knockout studies in mouse models have consistently shown that LKB1 loss promotes a highly-metastatic phenotype in diverse tissues, and human studies have demonstrated a strong association between LKB1 inactivation and tumor recurrence. Furthermore, LKB1 deficiency confers sensitivity to distinct classes of anticancer drugs. The ability to reliably identify LKB1-deficient tumors is thus likely to have important prognostic and predictive implications. Previous research studies have employed polyclonal antibodies with limited success, and there is no widely-employed immunohistochemical assay for LKB1. Here we report an assay based on a rabbit monoclonal antibody that can reliably detect endogenous LKB1 protein (and its absence) in mouse and human formalin-fixed, paraffin-embedded tissues. LKB1 protein levels determined through this assay correlated strongly with AMPK phosphorylation both in mouse and human tumors, and with mRNA levels in human tumors. Our studies fully validate this immunohistochemical assay for LKB1 in paraffin-embedded formalin tissue sections. This assay should be broadly useful for research studies employing mouse models and also for the development of human tissue-based assays for LKB1 in diverse clinical settings.

Mitochondrial and performance adaptations to exercise training in mice lacking skeletal muscle LKB1

LKB1 and its downstream targets of the AMP-activated protein kinase family are important regulators of many aspects of skeletal muscle cell function, including control of mitochondrial content and capillarity. LKB1 deficiency in skeletal and cardiac muscle (mLKB1-KO) greatly impairs exercise capacity. However, cardiac dysfunction in that genetic model prevents a clear assessment of the role of skeletal muscle LKB1 in the observed effects. Our purposes here were to determine whether skeletal muscle-specific knockout of LKB1 (skmLKB1-KO) decreases exercise capacity and mitochondrial protein content, impairs accretion of mitochondrial proteins after exercise training, and attenuates improvement in running performance after exercise training. We found that treadmill and voluntary wheel running capacity was reduced in skmLKB1-KO vs. control (CON) mice. Citrate synthase activity, succinate dehydrogenase activity, and pyruvate dehydrogenase kinase content were lower in KO vs. CON muscles. Three weeks of treadmill training resulted in significantly increased treadmill running performance in both CON and skmLKB1-KO mice. Citrate synthase activity increased significantly with training in both genotypes, but protein content and activity for components of the mitochondrial electron transport chain increased only in CON mice. Capillarity and VEGF protein was lower in skmLKB1-KO vs. CON muscles, but VEGF increased with training only in skmLKB1-KO. Three hours after an acute bout of muscle contractions, PGC-1α, cytochrome c, and VEGF gene expression all increased in CON but not skmLKB1-KO muscles. Our findings indicate that skeletal muscle LKB1 is required for accretion of some mitochondrial proteins but not for early exercise capacity improvements with exercise training.

Metabolic and Functional Genomic Studies Identify Deoxythymidylate Kinase as a Target in LKB1-Mutant Lung Cancer

The LKB1/STK11 tumor suppressor encodes a serine/threonine kinase, which coordinates cell growth, polarity, motility, and metabolism. In non-small cell lung carcinoma, LKB1 is somatically inactivated in 25% to 30% of cases, often concurrently with activating KRAS mutations. Here, we used an integrative approach to define novel therapeutic targets in KRAS-driven LKB1-mutant lung cancers. High-throughput RNA interference screens in lung cancer cell lines from genetically engineered mouse models driven by activated KRAS with or without coincident Lkb1 deletion led to the identification of Dtymk, encoding deoxythymidylate kinase (DTYMK), which catalyzes dTTP biosynthesis, as synthetically lethal with Lkb1 deficiency in mouse and human lung cancer lines. Global metabolite profiling showed that Lkb1-null cells had a striking decrease in multiple nucleotide metabolites as compared with the Lkb1-wild-type cells. Thus, LKB1-mutant lung cancers have deficits in nucleotide metabolism that confer hypersensitivity to DTYMK inhibition, suggesting that DTYMK is a potential therapeutic target in this aggressive subset of tumors.

Marked phenotypic differences of endurance performance and exercise-induced oxygen consumption between AMPK and LKB1 deficiency in mouse skeletal muscle: changes occurring in the diaphragm

LKB1 phosphorylates members of the AMP-activated protein kinase (AMPK) family. LKB1 and AMPK in the skeletal muscle are believed to regulate not only fuel oxidation during exercise but also exercise capacity. LKB1 was also required to prevent diaphragm fatigue, which was shown to affect exercise performance. Using mice expressing dominant negative (DN) mutants of LKB1 and AMPK, specifically in the skeletal muscle but not in the heart, we investigated the roles of LKB1 and AMPK activity in exercise performance and the effects of these kinases on the characteristics of respiratory and locomotive muscles. In the diaphragm and gastrocnemius, both AMPK-DN and LKB1-DN mice showed complete loss of AMPKα2 activity, and LKB1-DN mice showed a reduction in LKB1 activity. Exercise capacity was significantly reduced in LKB1-DN mice, with a marked reduction in oxygen consumption and carbon dioxide production during exercise. The diaphragm from LKB1-DN mice showed an increase in myosin heavy chain IIB and glycolytic enzyme expression. Normal respiratory chain function and CPT I activity were shown in the isolated mitochondria from LKB1-DN locomotive muscle, and the expression of genes related to fiber type, mitochondria function, glucose and lipid metabolism, and capillarization in locomotive muscle was not different between LKB1-DN and AMPK-DN mice. We concluded that LKB1 in the skeletal muscle contributes significantly to exercise capacity and oxygen uptake during exercise. LKB1 mediated the change of fiber-type distribution in the diaphragm independently of AMPK and might be responsible for the phenotypes we observed.

Abstract 1975: AICAR induces cell death in LKB1-deficient NSCLC.

Abstract AICAR is a well known AMPK activator, and AICAR-induced AMPK activation has been shown to inhibit cell proliferation or induce apoptosis through various mechanisms, such as the inactivation of metabolic enzyme involved ATP consumption, the inhibition of the of fatty acid synthesisPI3K/AKT pathway, the activation of cell cycle regulators, or the activation of the JNK pathway. LKB1 is an upstream AMPK kinase, and energetic stress-induced AMPK activation usually requires LKB1. Interestingly, AICAR has also been shown to induce caspase-3 cleavage in LKB1- null MEF and ovarian cancer cells, indicating AICAR can induce cell killing through an AMPK- independent mechanism. Because LKB1 is most frequently inactivated in non-small cell lung cancer, we evaluated the effect of AICAR in lung cancer cell lines. AICAR preferentially induced caspase-3 cleavage in LKB1- mutant NSCLC cell lines H157, A549, H460 and H1944, but not in LKB1- wild type cell lines H1299, H358, H1650 and H1792. Transient depletion of LKB1 by RNAi in H1299 and H1792 cells promoted AICAR-induced caaspase-3 cleavage in the absence of AMPK activation, supporting the notion that AICAR induces cell killing in LKB1-depleted cells through an AMPK- independent mechanism. In LKB1-null H460 cells, AICAR treatment led to a significant increase in S phase cell populations, and G1 cell cycle arrest by serum starvation is was sufficient to prevent AICAR-induced cell death. These data indicated that AICAR-induced cell killing in LKB1- deficient cells is cell cycle dependent. In summary, the ability of AICAR to preferentially induce cell death in LKB1- deficient NSCLC suggestsed that LKB1 deficiency may be a therapeutic target for the treatment of NSCLC. Citation Format: Fakeng Liu, Wei Zhou, Xiuju Liu, Shi-yong Sun, Fadlo Khuri, Yu-long He, Diansheng Zhong. AICAR induces cell death in LKB1-deficient NSCLC. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1975. doi:10.1158/1538-7445.AM2013-1975

Abstract 348: LKB1 deficiency confers lung adenocarcinoma phenotypic plasticity with squamous transdifferentiation potential.

Abstract The existence and related mechanisms of phenotypic plasticity and dynamic lineage conversion of lung cancers, as implicated by clinical observation of mixed adenocarcinoma (ADC) and squamous cell carcinoma (SCC) pathologies in adenosquamous cell carcinoma (Ad-SCC), remain as fundamental yet unsolved questions. Here we find that about 13% human lung Ad-SCC harbor genetic alterations of tumor suppressor LKB1. Through lineage tracing experiments and pathological analyses in mouse models of human lung cancer, we further show that LKB1 deficiency confers lung adenocarcinoma phenotypic plasticity to progressively transdifferentiate into squamous cell carcinoma (SCC), via pathologically mixed Ad-SCC as intermediate. Down-regulation of lysyl oxidase (LOX) following reduced hypoxia level in LKB1-deficient lung adenocarcinoma decreases collagen disposition and triggers extracellular matrix remodeling and eventually up-regulates p63 expression, the SCC lineage survival oncogene. Moreover, pharmacological inhibition of Lox enzymatic activity promotes the transdifferentiation, whereas ectopic Lox expression conversely increases collagen deposition and significantly inhibits this process. ADC and SCC show differential response to pharmacological Lox inhibition. Notably, human lung Ad-SCC with LKB1 genetic alterations show pathology-specific patterns of LOX expression and collagen deposition in adenomatous and squamous portions as seen from animal models. These data uncover an essential role and related molecular mechanism of LKB1 deficiency in determining lung adenocarcinoma phenotypic plasticity. Citation Format: Xiangkun Han, Fuming Li, Zhaoyuan Fang, Yijun Gao, Rong Fang, Fei Li, Yihua Sun, Jufeng Xia, Shun Yao, Li Li, Haiquan Chen, Yingyong Hou, Xinyuan Liu, Hongbin Ji. LKB1 deficiency confers lung adenocarcinoma phenotypic plasticity with squamous transdifferentiation potential. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 348. doi:10.1158/1538-7445.AM2013-348

The CRTC1-NEDD9 Signaling Axis Mediates Lung Cancer Progression Caused by LKB1 Loss

Somatic mutation of the tumor suppressor gene LKB1 occurs frequently in lung cancer where it causes tumor progression and metastasis, but the underlying mechanisms remain mainly unknown. Here, we show that the oncogene NEDD9 is an important downstream mediator of lung cancer progression evoked by LKB1 loss. In de novo mouse models, RNAi-mediated silencing of Nedd9 inhibited lung tumor progression, whereas ectopic NEDD9 expression accelerated this process. Mechanistically, LKB1 negatively regulated NEDD9 transcription by promoting cytosolic translocation of CRTC1 from the nucleus. Notably, ectopic expression of either NEDD9 or CRTC1 partially reversed the inhibitory function of LKB1 on metastasis of lung cancer cells. In clinical specimens, elevated expression of NEDD9 was associated with malignant progression and metastasis. Collectively, our results decipher the mechanism through which LKB1 deficiency promotes lung cancer progression and metastasis, and provide a mechanistic rationale for therapeutic attack of these processes.

LKB1 suppresses melanoma metastasis: the answer is YES

<scp>LKB</scp>1 suppresses melanoma metastasis: the answer is <scp>YES</scp>

LKB1/STK11 Inactivation Leads to Expansion of a Prometastatic Tumor Subpopulation in Melanoma

Germline mutations in LKB1 (STK11) are associated with the Peutz-Jeghers syndrome (PJS), which includes aberrant mucocutaneous pigmentation, and somatic LKB1 mutations occur in 10% of cutaneous melanoma. By somatically inactivating Lkb1 with K-Ras activation (±p53 loss) in murine melanocytes, we observed variably pigmented and highly metastatic melanoma with 100% penetrance. LKB1 deficiency resulted in increased phosphorylation of the SRC family kinase (SFK) YES, increased expression of WNT target genes,and expansion of a CD24(+) cell population, which showed increased metastatic behavior invitro and invivo relative to isogenic CD24(-) cells. These results suggest that LKB1 inactivation in the context of RAS activation facilitates metastasis by inducing an SFK-dependent expansion of a prometastatic, CD24(+) tumor subpopulation.

Abstract LB-260: Identification of DTYMK and CHEK1 as therapeutic targets in LKB1 mutant non-small cell lung cancer

Abstract The LKB1 tumor suppressor encodes a key metabolic sensor that integrates cell growth and metabolism. LKB1 is mutationally inactivated in multiple adult malignancies, including &amp;gt;20% of lung cancers, often simultaneously with activating KRAS mutations. LKB1 mutations are an important predictor of poor outcome and resistance to current therapeutic approaches. We employed an integrative approach to define novel therapeutic targets in Lkb1 mutant lung cancers. Matched cell lines from genetically engineered mouse models of cancer driven by activated Kras alone or in combination with Lkb1 deletion, were employed in high-throughput RNAi, kinase inhibitor, and metabolite screens. These screens identified knockdown of either Dtymk (deoxythymidylate kinase) or Chek1 (checkpoint kinase 1) as synthetically lethal with Lkb1 deficiency in both mouse and human lung cancer cell lines, and revealed that Lkb1 inactivation conferred marked sensitivity to treatment with CHEK1 inhibitors. Lkb1 deficient cells had a distinct metabolic profile, characterized by striking decreases in multiple nucleotide metabolites. Knockdown of DTYMK inhibited dTTP biosynthesis and, consequently, DNA synthesis, and knockdown of CHEK1 caused accumulation of DNA damage. We hypothesize that Lkb1 loss enhances dependence on these enzymes due to broad defects in nucleotide metabolism. Our studies support the development of therapies target DTYMK and CHEK1 in LKB1 mutant non-small cell lung cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-260. doi:1538-7445.AM2012-LB-260

Tumor suppressor function of Liver kinase B1 (Lkb1) is linked to regulation of epithelial integrity

Although loss of epithelial integrity is a hallmark of advanced cancer, it remains poorly understood whether genetic alterations corrupting this integrity causally facilitate tumorigenesis. We show that conditional deletion of tumor suppressor gene Lkb1 (Par-4) in the mammary gland compromises epithelial integrity manifested by mislocalization of cell polarity markers, lateralization of tight junctions, deterioration of desmosomes and basement membrane (BM), and hyperbranching of the mammary ductal tree. We identify the desmosomal BM remodelling serine protease Hepsin as a key factor mediating Lkb1 loss-induced structural alterations in mammary epithelium and BM fragmentation. Although loss of Lkb1 alone does not promote mammary tumorigenesis, combination of Lkb1 deficiency with oncogenic c-Myc leads to dramatic acceleration in tumor formation. The results coupling Lkb1 loss-mediated epithelial integrity defects to mislocalization of serine protease Hepsin and to oncogenic synergy with c-Myc imply that Lkb1 loss facilitates oncogenic proliferation by releasing epithelial cells from structural BM boundaries.

Intestinal renin–angiotensin system is stimulated after deletion of Lkb1

Background and aimsLKB1 is a serine-threonine kinase, mutation of which can lead to the development of multiple benign intestinal hamartomas (Peutz–Jeghers syndrome). In this study, the authors investigate the mechanisms...

LKB1 Haploinsufficiency Cooperates With Kras to Promote Pancreatic Cancer Through Suppression of p21-Dependent Growth Arrest

Background & AimsPatients carrying germline mutations of LKB1 have an increased risk of pancreatic cancer; however, it is unclear whether down-regulation of LKB1 is an important event in sporadic pancreatic cancer. In this study, we aimed to investigate the impact of LKB1 down-regulation for pancreatic cancer in mouse and human and to elucidate the mechanism by which Lkb1 deregulation contributes to this disease.MethodsWe first investigated the consequences of Lkb1 deficiency in a genetically modified mouse model of pancreatic cancer, both in terms of disease progression and at the molecular level. To test the relevance of our findings to human pancreatic cancer, we investigated levels of LKB1 and its potential targets in human pancreatic cancer.ResultsWe definitively show that Lkb1 haploinsufficiency can cooperate with oncogenic KrasG12D to cause pancreatic ductal adenocarcinoma (PDAC) in the mouse. Mechanistically, this was associated with decreased p53/p21-dependent growth arrest. Haploinsufficiency for p21 (Cdkn1a) also synergizes with KrasG12D to drive PDAC in the mouse. We also found that levels of LKB1 expression were decreased in around 20% of human PDAC and significantly correlated with low levels of p21 and a poor prognosis. Remarkably, all tumors that had low levels of LKB1 had low levels of p21, and these tumors did not express mutant p53.ConclusionsWe have identified a novel LKB1-p21 axis that suppresses PDAC following Kras mutation in vivo. Down-regulation of LKB1 may therefore serve as an alternative to p53 mutation to drive pancreatic cancer in vivo.

Abstract 5109: LKB1 in Vascular Endothelial Cells is Essential for Revascularization in a Mouse Ischemic Hind-limb Model

Recent reports have indicated that LKB1 is a regulator of AMP-activated protein kinase (AMPK) signaling in many tissues including liver, skeletal muscle and cardiac myocytes. In vascular endothelial cells, AMPK signaling is essential for cell function and angiogenic responses to tissue ischemia. Homozygous LKB1 deficiency in mice leads to embryonic lethality associated with abnormal vascular development. Here, we investigated whether heterozygous LKB1 deficiency in endothelial cells has a role in regulating revascularization to tissue ischemia in hind limb ischemic mouse model. We also explored the role of AMPK signaling downstream of LKB1 in vascular endothelial cells. Homozygous vascular endothelial cell deletion of LKB1, using the conditional loxP gene regulation system crossed with Tie2-Cre transgenic mice, led to an embryonic lethal phenotype. Thus, we produced endothelial specific reduced LKB1 model, Lkb1 flox/ + ;Tie2 Tg/ + (heterozygous-KO) mice. LKB1 expression level was decreased to half in the isolated endothelial cells from heterozygous-KO mice compared with those from Lkb1 flox/ + ;Tie2 +/+ (control) mice, but mice were normal in viability, body weight and vascular density. Revascularization of hind limb following ischemic surgery was significantly impaired in heterozygous-KO mice compared with control mice as evaluated by laser Doppler Flow method and capillary density analyses at day 14 and 28 after surgery. In cultured endothelial cells, reduction of LKB1, using both siRNA and adenovirus expressing non-functional kinase dead LKB1 protein, attenuated differentiation into network structures that was accompanied by diminished phosphorylation levels of AMPK (Thr-172) and acetyl-CoA carboxylase (ACC) (Ser-79). Conversely, LKB1 over-expression by adenovirus (Ad-LKB1) augmented differentiation into network structure with elevated phosphorylation levels of AMPK, ACC and eNOS (Ser-1177). The augmented network structure induced by Ad-LKB1 was abrogated by the co-transduction of a dominant negative mutant of AMPK. Taken together, our observations suggest that LKB1-AMPK axis in endothelial cells plays a pivotal role in the revascularization response to tissue ischemia.