LKB1 Levels Research Articles

Evidence that Adiponectin Fuels LKB1/ERα Interaction in Breast Cancer Cells

BACKGROUNDObesity is characterized by a chronic and excessive growth of adipose tissue and it is increasingly recognized as an oncogenic factor. Due to its endocrine function, adipose tissue secretes several bioactive molecules known as adipocytokines. Among these, adiponectin plays a key role in the pathogenesis of different obesity‐associated diseases, including malignancies, such as breast cancer.The main pathway through which adiponectin exerts its effects is LKB1/AMPK/mTOR signaling. It has been reported that LKB1 plays an essential role in mammary carcinogenesis, regulating apoptosis, proliferation, angiogenesis, energy metabolism and cell polarity. Moreover, low levels of LKB1 have been associated with a more aggressive tumor phenotype and correlate with a poor prognosis. In contrast, LKB1 overexpression induces inhibition of tumor growth, angiogenesis and metastases. In the recent years, our research group has identified ERα as an important regulator of adiponectin action in breast cancer, clarifying that the role of this cytokine seems to be dependent on cell phenotypes.Here, we provided novel insights about the effects of adiponectin on the functional interaction between ERα and LKB1 in ERα‐positive (MCF‐7 and ZR75) and negative (MDA‐MB‐231) breast cancer cells.METHODSRNA levels were assessed by RT‐PCR. Protein expressions were evaluated by immunoblotting in the presence or absence of ERα siRNA and antiestrogen ICI 182, 780. To study promoter activity, transient transfection assays were done. The recruitment of ERα on LKB1 promoter was evaluated by ChIP and DAPA assays. Immunofluorescence assays were performed to evaluate LKB1 localization and function.RESULTSWe demonstrated that adiponectin increased mRNA and protein levels of LKB1 in ERα‐positive breast cancer cells MCF‐7 and ZR75, whereas no significant changes were observed in ERα‐negative MDA‐MB‐231 cells. This up‐regulatory effect was due to the direct action of adiponectin on the promoter region of LKB1 gene. For instance, in MCF‐7 cells, transiently transfected with plasmids containing the full length or a series of 5′ deleted segments of human LKB1 promoter, we identified a sequence (−1626 to −1598) containing Estrogen Responsive Elements (ERE). ChIP and DAPA assays evidenced an increased association of ERα on ERE‐containing sequence proving the recruitment of ERα on LKB1 promoter in adiponectin‐treated cells. Noteworthy, in the presence of ERα siRNA and the pure antiestrogen ICI 182, 780 the upregulation of LKB1 expression was no longer noticeable. It is extremely intriguing to remark how the increasing intracellular pool of LKB1 upon adiponectin was mostly sequestered in the nuclear compartment as coactivator of ERα, which in turn uncoupled it from AMPK interaction, generally leading to the inhibition of cellular energy expenditure.CONCLUSIONSOur results demonstrated how ERα may impact LKB1/AMPK functional interaction upon adiponectin exposure and in such way may negatively interfere in switching cellular energy balance.Support or Funding InformationAssociazione Italiana Ricerca sul Cancro (AIRC) Grants IG‐18602; Progetti di Ricerca di Interesse Nazionale‐Ministero Istruzione Università e Ricerca Grant 2015B7M39T_001This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Prognostic Impact of DNA Repair Protein Expression in Non-Small Cell Lung Cancers Treated with Platinum-Based Chemotherapy and Subsequent Curative Lung Resection

Objective: Multimodal treatments that include preoperative platinum-based chemotherapy are fundamental to the treatment of advanced non-small cell lung cancer (NSCLC). This study aimed to investigate the predictive value of DNA repair protein expression in surgically resected NSCLCs in terms of prognosis and responses to platinum-containing chemotherapy. Methods: This retrospective study included 136 patients with NSCLC who were treated with preoperative platinum-based chemotherapy, followed by curative lung resection. ATM, RAD51, LKB1, H2AX, and SIRT1 expression levels were analyzed in resected tumor specimens via immunostaining and were used to classify patients and compare survival and responses to chemotherapy. Results: SIRT1 expression correlated significantly with improved responses to platinum-based chemotherapy (odds ratio, 2.28; p = 0.024), progression-free survival (hazard ratio [HR], 0.74; p = 0.036), overall survival (HR, 0.63; p = 0.006), and tumor-bearing survival (HR, 0.62; p = 0.014). After adjusting for clinical variables, the HR of SIRT1 expression remained significant for overall survival (HR, 0.59; p = 0.039) but not for progression-free survival (HR, 0.74; p = 0.183). No prognostic stratification was observed for the other 4 markers. Conclusion: Patients with SIRT1-expressing NSCLC had superior responses to chemotherapy and longer survival durations than those with SIRT1-negative cancer.

Molecular cloning, polymorphism, and expression analysis of the LKB1/STK11 gene and its association with non-specific digestive disorder in rabbits.

Liver kinase B1 (LKB1, also called STK11) encodes a serine/threonine kinase mutated in Peutz-Jeghers cancer syndrome characterized by gastrointestinal polyposis. Although LKB1 plays an important role in regulating energy homeostasis, cell growth, and metabolism via activation of adenosine monophosphate (AMP)-activated protein kinase (AMPK), nothing is known about its molecular characteristics and possible involvement in non-specific digestive disorder (NSDD) of rabbits. In the present study, we first cloned the coding sequence (CDS) of rabbit LKB1, which consisted of 1317bp encoding 438 amino acids (AAs) and contained a highly conserved S_TKc kinase domain. Its deduced AA sequence showed 87.93-91.10% similarities with that of other species. In order to determine its involvement in NSDD, a NSDD rabbit model was built by a dietary fiber deficiency. The polymorphic site of LKB1 was then investigated in both healthy and NSDD groups using directing sequencing. Our results suggested that a synonymous variant site (840 c. G > C, CCC→CCG) existed in its S_TKc domain, which was associated with susceptibility to NSDD. Furthermore, qPCR was utilized to examine the mRNA levels of LKB1 and its downstream targets (i.e., PRKAA2, mTOR and NF-kβ) in several intestinal-related tissues from both healthy and NSDD groups. Significant changes in their expression levels between two groups indicated that impaired LKB1 signaling contributed to the intestinal abnormality in NSDD rabbits. Taken together, it could be concluded that LKB1 might be a potential candidate gene affecting the occurrence of rabbit NSDD. This information may serve as a basis for further investigations on rabbit digestive diseases.

In This Issue

HomeCirculation ResearchVol. 121, No. 9In This Issue Free AccessIn BriefPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessIn BriefPDF/EPUBIn This Issue Ruth Williams Ruth WilliamsRuth Williams Search for more papers by this author Originally published13 Oct 2017https://doi.org/10.1161/RES.0000000000000178Circulation Research. 2017;121:1021LKB1 and Atherosclerosis (p 1047)Download figureDownload PowerPointThe kinase LKB1 slows foam cell formation and atherosclerosis progression in mice, report Liu et al.Atherosclerosis begins with the gradual accumulation of fatty deposits on blood vessel walls. Monocytes are recruited to the lesions where they convert to macrophages to phagocytose lipids in the lesions. Continued lipid uptake by the macrophages transforms them into foam cells, which is a critical step in plaque development. However, mechanisms underlying this transformation remain unclear. Liu and colleagues now show that the kinase LKB1, previously identified as having cardioprotective and anti-inflammatory effects, is a negative regulator of foam cell development. They found that, in mice prone to atherosclerosis, the levels of LKB1 in plaque macrophages decreases with disease progression. This decrease prevented LKB1-directed phosphorylation of scavenger receptor A (SRA)—a principle macrophage receptor responsible for lipid uptake and foam cell conversion. The team went on to show that LKB1-directed phosphorylation of SRA promotes the receptor’s degradation. They also found that atherogenesis was accelerated in atherosclerosis-prone mice with LKB1-lacking macrophages. As in mice, levels of LKB1 are also low in humans with atherosclerosis, suggesting the kinase may have a similar role in both mice and humans. Thus, boosting LKB1 levels in macrophages might be a new way to slow or prevent the progression of atherosclerosis.Endothelial TRPM2 in PMN Transmigration (p 1081)Download figureDownload PowerPointMittal et al examine the molecular details of neutrophil transmigration across the vascular endothelium.Neutrophils must exit the blood to get to sites of injury or infection, and they do this by squeezing through or between the endothelial cells—a process known as transmigration. Neutrophils generated reactive oxygen species (ROS) for killing bacteria, but recent evidence suggests ROS generation may also help neutrophils exit the blood. Indeed, exposure of endothelial cells in culture to the ROS hydrogen peroxide induces the opening of interendothelial junctions. And this process was shown to require calcium entry into endothelial cells via the cation channel TRPM2. Mittal and colleagues have now investigated this TRPM2-dependent mechanism in mice. The team engineered mice with endothelium-specific conditional deletion of TRPM2 and found that, when the mice were injected with bacterial toxin, the animals exhibited reduced inflammation (fewer neutrophils exiting the blood) compared with wild-type controls. Upon examining the molecular details of TRPM2-dependent transmigration, they found that neutrophil released ROS-induced production of ADPR, a TRPM2 activator, which was essential for the channel’s action. Together, these results suggest that, in cases of excessive inflammation, inhibition of TRPM2 might be an effective anti-inflammatory approach.CV Event Prediction by Machine Learning in MESA (p 1092)Download figureDownload PowerPointMachine learning strategy identifies clinical outcome predictors in large epidemiological study, report Ambale-Venkatesh et al.Epidemiological studies have led to the identification of several risk factors for cardiovascular disease, such as smoking, high blood pressure, and high cholesterol. But large epidemiological studies can provide many millions of data points from hundreds of variables, making the identification of other risk factors and outcome predictors a complex task. Ambale-Venkatesh and colleagues reasoned that the pattern recognition power of machine learning might make it an effective tool for mining such datasets. The team applied a machine learning technique to data from the Multi-Ethnic Study of Atherosclerosis (MESA), which includes 735 clinical variables (such as imaging, biomarkers, and questionnaires) from 6814 middle-aged to elderly individuals assessed over 12 years. They found that, compared with traditional data-mining approaches, machine learning predicted clinical outcomes with greater accuracy. It also identified nontraditional risk predictors, such as imaging scores, electrocardiography, and serum biomarkers for certain clinical outcomes, as opposed to more traditional predictors, such as ethnicity, gender, and medication use. The authors conclude that machine learning methods may be valuable for linking subclinical disease markers with outcomes in large cohort studies. Previous Back to top Next FiguresReferencesRelatedDetails October 13, 2017Vol 121, Issue 9 Advertisement Article InformationMetrics © 2017 American Heart Association, Inc.https://doi.org/10.1161/RES.0000000000000178 Originally publishedOctober 13, 2017 PDF download Advertisement

Abstract 361: Loss of cholangiocyte primary cilia induces LKB1 downregulation and defective AMPK signaling

Abstract Cholangiocarcinoma (CCA) is a malignancy arising from cholangiocytes, the epithelial cells lining the biliary tree. CCA is an uncommon, but devastating cancer that is increasing in incidence. Over the past 3 decades, 5-years survival rates have remained at 10%. Although surgical resection and liver transplantation are potentially curative therapies, most patients are diagnosed at late stages and are not eligible for these options. Therefore, it is imperative to identify novel targets leading to new therapeutic strategies for this devastating disease. Cholangiocytes express primary cilia that function as chemo, mechano, and osmosensors controlling several molecular pathways. We showed cilia are absent in CCA cells, and experimental deciliation of normal cholangiocytes induced a malignant like phenotype, with significant invasion and proliferation, suggesting the loss of cilia could be associated with CCA development. LKB1 is a tumor suppressor described to be expressed in primary cilia in MDCK cells, and is involved in AMPK activation through a ciliary dependent mechanism. AMPK functions as metabolic and stress check points. Interestingly, patients with intrahepatic CCA and low expression of LKB1 have poor prognosis. Therefore, we hypothesized that primary cilia function as tumor suppressor organelles through a LKB1-AMPK-p53 pathway. To test this hypothesis, first LKB1 subcellular localization was evaluated by confocal microscopy. We show for the first time that LKB1 is enriched in cholangiocyte primary cilia in a normal human cholangiocyte cell line (NHC). However, in experimental deciliated NHC and the CCA cell line KMCH, LKB1 is found dispersed in the cell cytoplasm. Next, we analyzed cellular expression levels of LKB1, AMPK (T172), p53 (s15), p53 and p21 by western blot. We found experimentally deciliated cholangiocytes and CCA cells exhibit defective AMPK signaling characterized by lower levels of LKB1 (-70% and -85%), AMPK (T172) (-72% and -71%), p53 (-37% and -83%), p53 (S15) (-38% and -100%) and p21 (-59% and -87%) relative to NHC. These molecular characteristics correlated with increased cell proliferation in deciliated cells (28%) and CCA cells (27%). Finally, we attempted to rescue the phenotype by using the AMPK activator, ampkinone. This treatment induced 20% inhibition of proliferation on experimental deciliated cholangiocytes and 18% in CCA cells, while not affecting NHC cells and the inhibition was associated with phosphorylation of AMPK and p21 up-regulation. In summary, our data suggest cholangiocyte primary cilia may normally function as tumor suppressors via a mechanism involving LKB1 and AMPK. The loss of cilia in CCA impairs LKB1-AMPK-p53-p21 signaling inducing a proliferative phenotype that may be rescued by specific activation of AMPK, warranting further studies to assess its use as a potential therapeutic approach. Citation Format: Adrian P. Mansini, Kristen M. Thelen, Sergio A. Gradilone. Loss of cholangiocyte primary cilia induces LKB1 downregulation and defective AMPK signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 361. doi:10.1158/1538-7445.AM2017-361

AMPK and SIRT1 activation contribute to inhibition of neuroinflammation by thymoquinone in BV2 microglia

Thymoquinone is a known inhibitor of neuroinflammation. However, the mechanism(s) involved in its action remain largely unknown. In this study, we investigated the roles of cellular reactive oxygen species (ROS), 5′ AMP-activated protein kinase (AMPK) and sirtuin 1 (SIRT1) in the anti-neuroinflammatory activity of thymoquinone. We investigated effects of the compound on ROS generation in LPS-activated microglia using the fluorescent 2′,7′-dichlorofluorescin diacetate (DCFDA)-cellular ROS detection. Immunoblotting was used to detect protein levels of p40phox, gp91phox, AMPK, LKB1 and SIRT1. Additionally, ELISA and immunofluorescence were used to detect nuclear accumulation of SIRT1. NAD+/NADH assay was also performed. The roles of AMPK and SIRT1 in anti-inflammatory activity of thymoquinone were investigated using RNAi and pharmacological inhibition. Our results show that thymoquinone reduced cellular ROS generation, possibly through inhibition of p40phox and gp91phox protein. Treatment of BV2 microglia with thymoquinone also resulted in elevation in the levels of LKB1 and phospho-AMPK proteins. We further observed that thymoquinone reduced cytoplasmic levels and increased nuclear accumulation of SIRT1 protein and increased levels of NAD+. Results also show that the anti-inflammatory activity of thymoquinone was abolished when the expressions of AMPK and SIRT1 were suppressed by RNAi or pharmacological antagonists. Pharmacological antagonism of AMPK reversed thymoquinone-induced increase in SIRT1. Taken together, we propose that thymoquinone inhibits cellular ROS generation in LPS-activated BV2 microglia. It is also suggested that activation of both AMPK and NAD+/SIRT1 may contribute to the anti-inflammatory, but not antioxidant activity of the compound in BV2 microglia.

Identification of the Energy Stress Sensor AMPK As Therapeutic Target in Acute Lymphoblastic Leukemia

Background and Hypothesis: While often transformed by the same oncogenes, biological and clinical characteristics of B-cell lineage and myeloid leukemias markedly differ. For instance, BCR-ABL1 tyrosine kinase drives both chronic myeloid leukemia (CML) and B cell lineage Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL). While the majority of CML patients achieve long-term disease-free survival under treatment, Ph+ ALL patients invariably relapse within months after initial remission. Here, we investigated whether the distinct characteristics of Ph+ ALL and CML have a metabolic basis and identified the metabolic sensor LKB1 and its substrate AMPK as novel therapeutic targets in pre-B ALL.Results: Metabolic measurements revealed strikingly higher AMP:ATP ratios with concomitant decreases in ATP production in patient-derived Ph+ ALL cells when compared to CML cells. These findings indicate a state of chronic energy depletion in pre-B ALL cells. Energy deficit activates the LKB1-AMPK energy sensor pathway to stimulate glucose uptake to restore ATP levels. Notably, LKB1 levels and activity of its substrate AMPKα were higher in patient-derived Ph+ ALL cells compared to CML cells. To study the consequences of inducible deletion of Lkb1, murine BCR-ABL1-driven myeloid lineage (CML) and B cell precursor (Ph+ ALL) Lkb1fl/fl leukemia cells were generated. We found that Lkb1-deletion increased glycolysis, ATP levels and proliferation in myeloid leukemia, consistent with the common notion that LKB1 is an established tumor suppressor. On the contrary, loss of Lkb1 function resulted in diminished glycolytic activity, impaired mitochondrial functions and cell death in pre-B ALL cells. C/EBPa-mediated reprogramming of B-cell into myeloid identity reversed the detrimental effects of Lkb1-deletion, restoring glycolysis, energy levels and survival of B→ myeloid reprogrammed cells. To study Lkb1 early in B cell lineage in vivo, Lkb1fl/fl mice were crossed with Mb1-Cretg/+ mice. Loss of Lkb1 function strikingly eradicated early B cell progenitor cells in vivo. Reduced survival fitness upon Lkb1 deletion in pre-B ALL cells was largely rescued by metabolites that can enter the TCA cycle and thus provide ATP. Importantly, we found that inducible deletion of Lkb1 delayed onset of pre-B ALL and prolonged survival of transplant recipient mice in vivo. Similar to observations made with deletion of Lkb1 in murine BCR-ABL1 pre-B ALL cells, both loss of Ampka function and small molecule inhibition of AMPK (BML-275) resulted in cell death as well as reduced glycolytic activity and mitochondrial function in BCR-ABL1 pre-B ALL cells. Moreover, prolonged overall survival was observed in transplant recipient mice injected with Ampka-deficient pre-B ALL cells. Finally, BML-275 synergized with glucocorticoids, a central component of all main therapy regimen in pre-B ALL, in eradicating patient-derived pre-B ALL cells.Conclusions: Taken together, our findings showed that B-cell lineage leukemia, unlike myeloid leukemia, critically depend on LKB1/AMPK signaling for survival. Our findings also showed that LKB1/AMPK can be leveraged to provide a novel therapeutic avenue in pre-B ALL. DisclosuresHochhaus:BMS: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; ARIAD: Honoraria, Research Funding.

Anti-neuroinflammatory Effect of Emodin in LPS-Stimulated Microglia: Involvement of AMPK/Nrf2 Activation.

AMPK/Nrf2 signaling regulates multiple antioxidative factors and exerts neuroprotective effects. Emodin is one of the main bioactive components extracted from Polygonum multiflorum, a plant possessing important activities for human health and for treating a variety of diseases. This study examined whether emodin can activate AMPK/Nrf2 signaling and induce the expression of genes targeted by this pathway. In addition, the anti-neuroinflammatory properties of emodin in lipopolysaccharide (LPS)-stimulated microglia were examined. In microglia, the emodin treatment increased the levels of LKB1, CaMKII, and AMPK phosphorylation. Emodin increased the translocation and transactivity of Nrf2 and enhanced the levels of HO-1 and NQO1. In addition, the emodin-mediated expression of HO-1 and NQO1 was attenuated completely by an AMPK inhibitor (compound C). Moreover, emodin decreased dramatically the LPS-induced production of NO and PGE2 as well as the protein expression and promoter activity of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). In addition, emodin effectively inhibited the production of pro-inflammatory cytokines, TNF-α and IL-6, and reduced the level of IκBα phosphorylation, leading to the suppression of the nuclear translocation, phosphorylation, and transactivity of NF-κB. Emodin also suppressed the LPS-stimulated activation of STATs, JNK, and p38 MAPK. The anti-inflammatory effects of emodin were reversed by transfection with Nrf-2 and HO-1 siRNA and by a co-treatment with an AMPK inhibitor. These results suggest that emodin isolated from P. multiflorum can be used as a natural anti-neuroinflammatory agent that exerts its effects by inducing HO-1 and NQO1 via AMPK/Nrf2 signaling in microglia.

The miR-17∼92 microRNA Cluster Is a Global Regulator of Tumor Metabolism

A central hallmark of cancer cells is the reprogramming of cellular metabolism to meet the bioenergetic and biosynthetic demands of malignant growth. Here, we report that the miR-17∼92 microRNA (miRNA) cluster is an oncogenic driver of tumor metabolic reprogramming. Loss of miR-17∼92 in Myc(+) tumor cells leads to a global decrease in tumor cell metabolism, affecting both glycolytic and mitochondrial metabolism, whereas increased miR-17∼92 expression is sufficient to drive increased nutrient usage by tumor cells. We mapped the metabolic control element of miR-17∼92 to the miR-17 seed family, which influences cellular metabolism and mammalian target of rapamycin complex 1 (mTORC1) signaling through negative regulation of the LKB1 tumor suppressor. miR-17-dependent tuning of LKB1 levels regulates both the metabolic potential of Myc(+) lymphomas and tumor growth invivo. Our results establish metabolic reprogramming as a central function of the oncogenic miR-17∼92 miRNA cluster that drives the progression of MYC-dependent tumors.

Antiaging Gene Klotho Deficiency Promoted High-Fat Diet-Induced Arterial Stiffening via Inactivation of AMP-Activated Protein Kinase.

Klotho was originally discovered as an aging-suppressor gene. The objective of this study is to investigate whether klotho gene deficiency affects high-fat diet (HFD)-induced arterial stiffening. Heterozygous Klotho-deficient (KL(+/-)) mice and WT littermates were fed on HFD or normal diet. HFD increased pulse wave velocity within 5 weeks in KL(+/-) mice but not in wild-type mice, indicating that klotho deficiency accelerates and exacerbates HFD-induced arterial stiffening. A greater increase in blood pressure was found in KL(+/-) mice fed on HFD. Protein expressions of phosphorylated AMP-activated protein kinase-α (AMPKα), phosphorylated endothelial nitric oxide synthase (eNOS), and manganese-dependent superoxide dismutase (Mn-SOD) were decreased, whereas protein expressions of collagen I, transforming growth factor-β1, and Runx2 were increased in aortas of KL(+/-) mice fed on HFD. Interestingly, daily injections of an AMPKα activator, 5-aminoimidazole-4-carboxamide-3-ribonucleoside, abolished the increases in pulse wave velocity, blood pressure, and blood glucose in KL(+/-) mice fed on HFD. Treatment with 5-aminoimidazole-4-carboxamide-3-ribonucleoside for 2 weeks not only abolished the downregulation of phosphorylated AMPKα, phosphorylated eNOS, and Mn-SOD levels but also attenuated the increased levels of collagen I, transforming growth factor-β1, Runx2, superoxide, elastic lamellae breaks, and calcification in aortas of KL(+/-) mice fed on HFD. In cultured mouse aortic smooth muscle cells, cholesterol plus KL-deficient serum decreased phosphorylation levels of AMPKα and LKB1 (an important upstream regulator of AMPKα activity) but increased collagen I synthesis, which can be eliminated by activation of AMPKα by 5-aminoimidazole-4-carboxamide-3-ribonucleoside. In conclusions, Klotho deficiency promoted HFD-induced arterial stiffening and hypertension via downregulation of AMPKα activity.

Isoflurane suppresses the self-renewal of normal mouse neural stem cells in a p53-dependent manner by activating the Lkb1-p53-p21 signalling pathway.

Isoflurane is widely used in anaesthesia for surgical operations. However, whether it elicits unwanted side effects, particularly in neuronal cells, remains to be fully elucidated. The Lkb1-p53-p21 signalling pathway is able to modulate neuronal self-renewal and proliferation. Furthermore, the suppression of Lkb1-dependent p21 induction leads to apoptosis. In the present study, whether Lkb1-p53-p21 signalling is involved in the response to isoflurane was investigated. A comparison of mouse primary, wild-type neural stem cells (WT NSCs) with the p53−/− NSC cell line, NE-4C, revealed that isoflurane inhibited proliferation in a dose-, a time- and a p53-dependent manner. However, flow cytometric analysis revealed that the concentration of isoflurane which caused 50% inhibition (the IC50 value) induced cell cycle arrest in WT NSCs. Furthermore, the protein expression levels of LKB1, p53 and p21 were increased, although those of nestin and survivin decreased, following treatment of WT NSCs with isoflurane. On the other hand, isoflurane induced the phosphorylation of Ser15 in p53 in WT NSCs, which was associated with p53 binding to the p21 promoter, and consequentially, the transcriptional activation of p21. All these events were abrogated in NE-4C cells. Taken together, the present study has demonstrated that isoflurane suppresses the self-renewal of normal mouse NSCs by activating the Lkb1-p53-p21 signalling pathway.

Abstract 1124: Transcriptional control of B cell identity restricts metabolic fitness in human leukemia

Abstract Oncogenic lesions in multi-potent progenitor cells often give rise to either B-cell or myeloid lineage leukemia. While transformed by the same oncogenes (e.g. BCR-ABL1, RAS), B-lineage and myeloid leukemias are distinct diseases. Given that oncogenic tyrosine kinase signaling (e.g. BCR-ABL1) imposes significant metabolic requirements on energy supply, biogenesis and metabolic fitness, we studied whether the divergent characteristics of myeloid and B-lineage leukemias have a metabolic basis. Metabolic analyses revealed that B-lineage acute lymphoblastic leukemia (Ph+ ALL) cells proliferate at maximum capacity of their glycolytic machinery. In contrast to myeloid leukemia (CML), B-lineage ALL cells lack metabolic adaptive fitness in response to metabolic fluctuations. C/EBPα-mediated reprogramming of B-lineage cells into the myeloid lineage induced glycolytic gene expression (Insr, Slc2a1, G6pdx, G6pd2, and Hk3). Frequent genetic lesions of transcription factors that determine B cell identity (IKZF1, PAX5, EBF1) partially mitigate B cell-instrinsic metabolic liability. Reconstitution of PAX5 expression in patient-derived B-lineage ALL cells reduced metabolic fitness by impacting glucose metabolism. Using genetic and metabolic experiments, we identified the metabolic liability observed in B-lineage ALL is in part dependent on the serine/threonine kinase LKB1. In agreement with previous studies, Cre-mediated deletion of Lkb1 induced proliferation in myeloid leukemia. Surprisingly, Lkb1 deletion led to apoptosis and decreased leukemogenic capacity in B-lineage leukemia. Consistent with the above observations, Arf, p53 and p27 levels were reduced in Lkb1-deficient myeloid leukemia cells, while Lkb1 deletion in B-lineage ALL cells up-regulated Arf, p53 and p27 levels. Enhanced glucose consumption and lactate production were observed in Lkb1-deficient myeloid leukemia cells. In contrast, loss of Lkb1 led to defective glycolytic and mitochondrial activity in B-lineage ALL. Lkb1 deletion in B-lineage ALL caused global accumulation of metabolites, suggesting that LKB1 is required for maintaining metabolic homeostasis. Moreover, loss of Lkb1 decreased protein levels of mitochondrial, anti-apoptotic BCL-2 family proteins, BCL-xL and MCL1, in B-lineage ALL. Reverse Phase Protein Array analyses revealed that LKB1 levels positively correlated with BCL-xL and MCL1 in patient-derived Ph+ ALL samples (n = 51) as well as other subtypes of B-lineage ALL (n = 183; MDACC, 1983-2007). Importantly, C/EBPα-mediated reprogramming of B-lineage ALL cells to the myeloid linage relieved dependency on LKB1. Taken together, we showed that transcriptional control of B cell identity causes unique metabolic liability. B-lineage ALL cells exhibit unique reliance on LKB1 for metabolic homeostasis and survival. Our findings revealed LKB1 as a potential therapeutic target in B-lineage ALL. Note: This abstract was not presented at the meeting. Citation Format: Lai N. Chan, Daniel Braas, Christian Hurtz, Seyedmehdi Shojaee, Huimin Geng, Valeria Cazzaniga, Carina Ng, Behzad Kharabi Masouleh, Yi Hua Qiu, Nianxiang Zhang, Kevin R. Coombes, Thomas Ernst, Giovanni Cazzaniga, Andreas Hochhaus, Steven Kornblau, Thomas Graeber. Transcriptional control of B cell identity restricts metabolic fitness in human leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1124. doi:10.1158/1538-7445.AM2015-1124

Deoxypodophyllotoxin suppresses tumor vasculature in HUVECs by promoting cytoskeleton remodeling through LKB1-AMPK dependent Rho A activatio.

Angiogenesis plays a critical role in the growth and metastasis of tumors, which makes it an attractive target for anti-tumor drug development. Deoxypodophyllotoxin (DPT), a natural product isolated from Anthriscus sylvestris, inhibits cell proliferation and migration in various cancer cell types. Our previous studies indicate that DPT possesses both anti-angiogenic and vascular-disrupting activities. Although the RhoA/ RhoA kinase (ROCK) signaling pathway is implicated in DPT-stimulated cytoskeleton remodeling and tumor vasculature suppressing, the detailed mechanisms by which DPT mediates these effects are poorly understood. In the current study, we found that DPT promotes cytoskeleton remodeling in human umbilical vein endothelial cells (HUVECs) via stimulation of AMP-activated protein kinase (AMPK) and that this effect is abolished by either treatment with a selective AMPK inhibitor or knockdown. Moreover, the cellular levels of LKB1, a kinase upstream of AMPK, were enhanced following DPT exposure. DPT-induced activation of AMPK in tumor vasculature effect was also verified by transgenic zebrafish (VEGFR2:GFP), Matrigel plug assay, and xenograft model in nude mice. The present findings may lay the groundwork for a novel therapeutic approach in treating cancer.

Abstract A14: LKB1 loss is associated with Akt1 phosphorylation in head and neck cancer

Abstract Head and neck cancer is a multifactorial disease including a group of biologically similar cancers and representing 3% of all malignant tumors. Its incidence has been rising in the last years with more than half a million cases and leading to 300 000 deaths yearly. The most common tumor type is squamous cell carcinoma but recent studies have shown that the tumors may display a very heterogenous nature. Although the survival rates have improved over the past decades due to the use of concurrent therapeutic modalities the molecular mechanism of the disease is still obscure. Besides tobacco and alcohol consumption several other factors including viral infection, diet, inflammation and occupational or genetic factors have been implicated with the disease. Recent findings suggest an important role for genetic factors in head and neck carcinogenesis. Alterations in the signaling pathways leading to the activation of oncogenes and inhibition of tumor suppressor genes are being increasingly implicated with the development of the disease. Head and neck cancer is associated with the enhanced expression and activity of different signaling pathways which lead to phosphorylation of several kinases and activation of a number of transcription factors. In order to better understand the molecular mechanisms of head and neck carcinogenesis it is essential to characterize the molecular changes and their involvement in the alterations of the cellular control. One of the key signaling pathways in head and neck cancer is the PI3K-Akt-MTOR pathway which has a crucial role in cell growth and survival. Dysregulation of this pathway generates a favourable oncogenic environment, and has been documented in a variety of transformed cells and human tumors. PIK3CA and Akt are critical genes in the PI3K signaling pathway. It has been shown that exons 9 and 20 of the PIK3CA gene are frequently mutated in various cancers. On the other hand, elevated expression of phosphorylated Akt reflects PI3K pathway activation. It has also been known that the master kinase LKB1 is a tumor suppressor and exerts its effect on multiple signaling pathways. Therefore, in this study we investigated the effect of LKB1on the mRNA expression levels and PIK3CA mutations in relation to the Akt phosphorylation rate in HNSCC. For this purpose, we analyzed LKB1, PIK3CA and Akt1 expression levels by qRT-PCR in association with PIK3CA exon 9 and 20 mutations and p-Akt levels in 100 HNSCC tumors and corresponding non-cancerous tissue samples. Down-regulation of the LKB1 mRNA expression level was observed in 63.3% of the cases and this down-regulation was associated with Akt1 phosphorylation. Our results indicate that activation of PI3K pathway is significantly associated with the down-regulation of LKB1. Citation Format: Nejat Dalay, Seda Ekizoglu, Soydan Dogan, Emin Karaman, Damla Ulker, Nur Buyru. LKB1 loss is associated with Akt1 phosphorylation in head and neck cancer. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr A14.

MicroRNA-17 promotes normal ovarian cancer cells to cancer stem cells development via suppression of the LKB1-p53-p21/WAF1 pathway.

The mechanism underlying the development of human ovarian cancer is poorly understood. The liver kinase protein, LKB1, is hypothesized to play a pivotal role in tumor cell proliferation and invasion capacity through regulation of p53 and p21/WAF1 expression. Previous studies suggest LKB1 may, in turn, be regulated by microRNA-17. Here, we examined the role of miR-17 in the expression of LKB1 and the downstream effects on proliferation and invasion capacity of normal ovarian cancer cells (OCCs) and ovarian stem cells. In this study, both the mRNA and protein expression levels of LKB1, p53, and p21 decreased in OCCs following transfection with a miR-17 expression plasmid. MiR-17 expression affected cell cycle regulation and stimulated the proliferation and invasion capacity of OCCs in vitro. ChIP assays indicated that the binding efficiency of p53 to the p21/WAF1 gene promoter was much lower in miR-17 transfected OCCs than in OCCs transfected with a mutated miR-17. Co-immunoprecipitation and western blotting showed significantly lower levels of p53 and p53 Ser15-pho in the miR-17 transfected OCCs as compared to the mutant miR-17 transfected OCCs. Xenograft experiments confirmed that suppression of tumor growth in vivo occurred in the absence of functional miR-17. These findings suggest that mature miR-17 expression may have an important role in the pathogenesis of human ovarian tumors through its interference with the LKB1-p53-p21/WAF1 pathway expression by epigenetic modification. These findings are of potential importance in the identification of novel therapeutic targets in human ovarian cancer.

LKB1 and AMPK regulate synaptic remodeling in old age.

Age-related decreases in neural function result in part from alterations in synapses. To identify molecular defects that lead to such changes, we focused on the outer retina, in which synapses are markedly altered in old rodents and humans. We found that the serine/threonine kinase LKB1 and one of its substrates, AMPK, regulate this process. In old mice, synaptic remodeling was accompanied by specific decreases in the levels of total LKB1 and active (phosphorylated) AMPK. In the absence of either kinase, young adult mice developed retinal defects similar to those that occurred in old wild-type animals. LKB1 and AMPK function in rod photoreceptors where their loss leads to aberrant axonal retraction, the extension of postsynaptic dendrites and the formation of ectopic synapses. Conversely, increasing AMPK activity genetically or pharmacologically attenuates and may reverse age-related synaptic alterations. Together, these results identify molecular determinants of age-related synaptic remodeling and suggest strategies for attenuating these changes.

Loss of LKB1 and PTEN tumor suppressor genes in the ovarian surface epithelium induces papillary serous ovarian cancer

Epithelial ovarian cancer presents mostly with serous, endometrioid or mucinous histology but is treated as a single disease. The development of histotype-specific therapy has been challenging because of the relative lack of studies attributing disrupted pathways to a distinct histotype differentiation. mTOR activation is frequently associated with poor prognosis in serous ovarian cancer, which is the most common and most deadly histotype. However, the mechanisms dysregulating mTOR in the pathogenesis of ovarian cancer are unknown. We detected copy number loss and correlated lower expression levels of LKB1, TSC1, TSC2 and PTEN tumor suppressor genes for upstream regulators of mTOR activity in up to 80% in primary ovarian serous tumor databases, with LKB1 allelic loss-predominant. Reduced LKB1 protein was usually associated with increased mTOR activity in both serous ovarian cancer cell lines and primary tumors. Conditional deletion of Lkb1 in murine ovarian surface epithelial (OSE) cells caused papillary hyperplasia and shedding but not tumors. Simultaneous deletion of Lkb1 and Pten, however, led to development of high-grade ovarian serous histotype tumors with 100% penetrance that expressed WT1, ERα, PAX8, TP53 and cytokeratin 8, typical markers used in the differential diagnosis of serous ovarian cancer. Neither hysterectomy nor salpingectomy interfered with progression of ovarian tumorigenesis, suggesting that neither uterine nor Fallopian tube epithelial cells were contributing to tumorigenesis. These results implicate LKB1 loss in the OSE in the pathogenesis of serous ovarian cancer and provide a compelling rationale for investigating the therapeutic potential of targeting LKB1 signaling in patients with this deadly disease.

LKB1 is a central regulator of tumor initiation and pro-growth metabolism in ErbB2-mediated breast cancer

BackgroundGermline and somatic mutations in STK11, the gene encoding the serine/threonine kinase LKB1, are strongly associated with tumorigenesis. While loss of LKB1 expression has been linked to breast cancer, the mechanistic role of LKB1 in regulating breast cancer development, metastasis, and tumor metabolism has remained unclear.MethodsWe have generated and analyzed transgenic mice expressing ErbB2 in the mammary epithelium of LKB1 wild-type or LKB1-deficient mice. We have also utilized ErbB2-expressing breast cancer cells in which LKB1 levels have been reduced using shRNA approaches. These transgenic and xenograft models were characterized for the effects of LKB1 loss on tumor initiation, growth, metastasis and tumor cell metabolism.ResultsWe demonstrate that loss of LKB1 promotes tumor initiation and induces a characteristic shift to aerobic glycolysis (‘Warburg effect’) in a model of ErbB2-mediated breast cancer. LKB1-deficient breast cancer cells display enhanced early tumor growth coupled with increased cell migratory and invasive properties in vitro. We show that ErbB2-positive tumors deficient for LKB1 display a pro-growth molecular and phenotypic signature characterized by elevated Akt/mTOR signaling, increased glycolytic metabolism, as well as increased bioenergetic markers both in vitro and in vivo. We also demonstrate that mTOR contributes to the metabolic reprogramming of LKB1-deficient breast cancer, and is required to drive glycolytic metabolism in these tumors; however, LKB1-deficient breast cancer cells display reduced metabolic flexibility and increased apoptosis in response to metabolic perturbations.ConclusionsTogether, our data suggest that LKB1 functions as a tumor suppressor in breast cancer. Loss of LKB1 collaborates with activated ErbB2 signaling to drive breast tumorigenesis and pro-growth metabolism in the resulting tumors.

LKB1 Inhibits Breast Cancer Partially through Repressing the Hedgehog Signaling Pathway

Constitutive activation of the Hedgehog (Hh) signaling pathway has been implicated in the development of many human malignancies. Hh targets, such as Patched (PTCH), smoothened (SMO), Sonic hedgehog (SHH) and glioma-associated oncogene homologue 1 (GLI1), are markers of Hh signaling activation and expressed in most Hh-associated tumors. The protein kinase LKB1 has been shown to slow proliferation and induce cell-cycle arrest in many cell lines. In this study, we observed that activated LKB1 decreased the expression of factors related to Hh reporter activity in MDA-MB-231 breast cancer cells, including of SMO, SHH and GLI1. In contrast, LKB1 siRNA increased the expression of these target genes. The same results were shown to inhibit the Hh factors Sufu and Hip. Furthermore, we also observed negative correlation between LKB1 and glioma-associated oncogene homologue 1 (GLI1) in three breast cancer cell lines. Meanwhile, LKB1 siRNA rescued the inhibition of cell growth by 3-Keto-N-(aminoethyl-N'-aminocaproyldihydrocinnamoyl) cyclopamine (KAAD-cyclopamine), an antagonist of the Hh element SMO, which suggests that LKB1 acts as the downstream of SMO. In vivo, LKB1 siRNA increased tumor growth in the mammary fat pad, and the expression levels of Hh displayed similar results in vitro. Overexpression of the LKB1 protein in human breast cancers is associated with the expression of Hh. We found that breast carcinomas with detectable Hh had weak or undetectable expression of LKB1, whereas tumors that expressed high levels of LKB1 had undetectable Hh signaling. In this study, we find that LKB1 are negatively correlated with the expression of Hh related transcription factors. These findings suggest that LKB1 may inhibit tumorigenesis by regulating Hh signaling in certain cancers.

Could a Possible Crosstalk between AMPK and TGF-β Signaling Pathways Be a Key Player in Benign and Malignant Salivary Gland Tumors?

Background: Salivary gland tumors (SGTs) are known for their specific heterogeneity and ambiguous outcome for the affected patients. The LKB1 and SMAD4 genes are pivotal components of important signaling pathways, including AMPK and TGF-β. To our knowledge, no study has reported an association between the expression levels of these genes in SGTs. The expression levels of LKB1 and SMAD4 were evaluated to clarify their possible crosstalk in SGTs. Materials and Methods: A total of 50 fresh tissue specimens were obtained from patients with SGTs, including pleomorphic adenoma (PA), Warthin’s tumor (WT), intermediate grade mucoepidermoid carcinoma (MEC), salivary duct carcinoma (SDC), and carcinoma ex pleomorphic adenoma (CexPA), as well as 8 normal samples. Quantitative real-time polymerase chain reaction was performed for all samples with specific primers. Results: Data were analyzed using statistical methods. PA, WT, MEC, and SDC showed a significant decrease in LKB1 levels, but the gene was up-regulated in CexPA. SMAD4 was overexpressed in all samples. Conclusion: The results suggest a possible link between downregulation of LKB1 and overexpression of SMAD4 in SGTs. LKB1 depletion leads to upregulation of SMAD4, promoting epithelial-mesenchymal transition in tumor cells. Therefore, LKB1 and SMAD4 could be key players in inducing tumor heterogeneity in SGTs.