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Aims/hypothesis Although targeted in extrapancreatic tissues by several drugs used to treat type 2 diabetes, the role of AMP-activated protein kinase (AMPK) in the control of insulin secretion is still debatable. Previous studies have used pharmacological activators of limited selectivity and specificity, and none has examined in primary pancreatic beta cells the actions of the latest generation of highly potent and specific activators that act via the allosteric drug and metabolite (ADaM) site. Methods AMPK was activated acutely in islets isolated from C57BL6/J mice, and in an EndoC-βH3 cell line, using three structurally distinct ADaM site activators (991, PF-06409577 and RA089), with varying selectivity for β1- vs β2-containing complexes. Mouse lines expressing a gain-of-function mutation in the γ1 AMPK subunit (D316a) were generated to examine the effects of chronic AMPK stimulation in the whole body, or selectively in the beta cell. Results Acute (1.5 h) treatment of wild-type mouse islets with 991, PF-06409577 or RA089 robustly stimulated insulin secretion at high glucose concentrations (ppp2+ dynamics in response to 991 (AUC, ppppp Conclusions/interpretation AMPK activation exerts complex, time-dependent effects on insulin secretion. These observations should inform the design and future clinical use of AMPK modulators. Graphical abstract

Diabetic cardiomyopathy (DCM) is associated with oxidative stress and augmented inflammation in the heart. Neuraminidases (NEU) 1 has initially been described as a lysosomal protein which plays a role in the catabolism of glycosylated proteins. We investigated the role of NEU1 in the myocardium in diabetic heart. Streptozotocin (STZ) was injected intraperitoneally to induce diabetes in mice. Neonatal rat ventricular myocytes (NRVMs) were used to verify the effect of shNEU1 in vitro. NEU1 is up-regulated in cardiomyocytes under diabetic conditions. NEU1 inhibition alleviated oxidative stress, inflammation and apoptosis, and improved cardiac function in STZ-induced diabetic mice. Furthermore, NEU1 inhibition also attenuated the high glucose-induced increased reactive oxygen species generation, inflammation and, cell death in vitro. ShNEU1 activated Sirtuin 3 (SIRT3) signaling pathway, and SIRT3 deficiency blocked shNEU1-mediated cardioprotective effects in vitro. More importantly, we found AMPKα was responsible for the elevation of SIRT3 expression via AMPKα-deficiency studies in vitro and in vivo. Knockdown of LKB1 reversed the effect elicited by shNEU1 in vitro. In conclusion, NEU1 inhibition activates AMPKα via LKB1, and subsequently activates sirt3, thereby regulating fibrosis, inflammation, apoptosis and oxidative stress in diabetic myocardial tissue.

Metformin is an anti-diabetic drug and has anticancer effects on various cancers. Several studies have suggested that metformin reduces cell proliferation and stimulates cell-cycle arrest and apoptosis. However, the definitive molecular mechanism of metformin in the pathophysiological signaling in endometrial tumorigenesis and metastasis is not clearly understood. In this study, we examined the effects of metformin on the cell viability and apoptosis of human cervical HeLa and endometrial HEC-1-A and KLE cancer cells. Metformin suppressed cell growth in a dose-dependent manner and dramatically evoked apoptosis in HeLa cervical cancer cells, while apoptotic cell death and growth inhibition were not observed in endometrial (HEC-1-A, KLE) cell lines. Accordingly, the p27 and p21 promoter activities were enhanced while Bcl-2 and IL-6 activities were significantly reduced by metformin treatment. Metformin diminished the phosphorylation of mTOR, p70S6K and 4E-BP1 by accelerating adenosine monophosphate-activated kinase (AMPK) in HeLa cancer cells, but it did not affect other cell lines. To determine why the anti-proliferative effects are observed only in HeLa cells, we examined the expression level of liver kinase B1 (LKB1) since metformin and LKB1 share the same signalling system, and we found that the LKB1 gene is not expressed only in HeLa cancer cells. Consistently, the overexpression of LKB1 in HeLa cancer cells prevented metformin-triggered apoptosis while LKB1 knockdown significantly increased apoptosis in HEC-1-A and KLE cancer cells. Taken together, these findings indicate an underlying biological/physiological molecular function specifically for metformin-triggered apoptosis dependent on the presence of the LKB1 gene in tumorigenesis.

Erythropoiesis is a tightly regulated multistage process in which hematopoietic stem cells (HSCs) proliferate progressively and differentiate to form mature red blood cells (RBCs). This process is metabolically demanding, requiring the coordinated activity of multiple organs to utilize iron and establish homeostasis. Central to erythropoiesis is the production of erythropoietin (Epo), driven by tissue oxygen status. Liver kinase B1 (LKB1) acts as a central regulator to control cellular metabolism, cell polarity and proliferation during development and stress response. As all of these fundamental cellular processes regulated by LKB1 are present in erythropoiesis, we hypothesize that LKB1 may be involved in this coordination. LKB1 exerts its control of metabolism, polarity, and proliferation through AMP-activated protein kinase (AMPK) and 12 other AMPK-related effector kinases. LKB1 regulates quiescence and metabolic homeostasis of HSCs through AMPK-dependent and AMPK-independent mechanisms. Whole-body knockout studies in mice have revealed that loss of AMPK results in shortened erythrocyte life span, increased osmotic fragility, hemolytic destruction of RBCs, splenomegaly, and anemia. Several questions still remain: Does LKB1 affect steady-state erythropoiesis? Does LKB1 play a role in the erythroid iron deprivation response? How does LKB1 affect the maturation of erythroblasts to become mature RBCs? To determine the role of LKB1 in erythropoiesis, we crossed mice carrying loxP-flanked STK11/LKB1 alleles with EpoR-GFP-Cre mice, which will lead to gene excision just prior to erythroid commitment. These animals were then studied in steady-state and stress conditions. Mice with erythroid loss of LKB1 were found to have elevated serum Epo levels but unexpectedly had normal RBC counts with no evidence of anemia. An increase in Epo is normally accompanied by an increase in RBCs. These findings suggest a poor response to Epo – which could be due to shortened lifespan of the RBCs, ineffective erythropoiesis, or resistance to Epo. Mice with erythroid loss of LKB1 had RBC lifespans similar to those of control animals. Erythroid LKB1 was found to be dispensable for an appropriate response to anemic challenge in mice. Also, LKB1 was found to have a very limited role in a mouse model of iron deprivation. Ex vivo analysis revealed that erythroid loss of LKB1 results in accelerated maturation of erythroid progenitors as evidenced by an accumulation of CD71/Ter119 double positive cells when compared to control. Inhibition of the LKB1 downstream kinase AMPK recapitulates this accelerated maturation. Conversely, agonist stimulation of AMPK delays maturation. These findings reveal a previously unreported role for LKB1. Therefore, LKB1 is identified as a regulator of Epo homeostasis and the Epo-EpoR axis.

Liver Kinase B1 (LKB1) is a serine/threonine kinase that can regulate energy metabolism and skeletal muscle growth. In the present study, LKB1 cDNA of triploid crucian carp (Carassius auratus) was cloned. The cDNA contains a complete open reading frame (ORF), with a length of 1326 bp, encoding 442 amino acids. Phylogenetic tree analysis showed that the LKB1 amino acid sequence of the triploid crucian carp had a high sequence similarity and identity with carp (Cyprinus carpio). Tissue expression analysis revealed that LKB1 was widely expressed in various tissues. LKB1 expressions in the brain were highest, followed by kidney and muscle. In the short-term LKB1 activator and inhibitor injection experiment, when LKB1 was activated for 72 h, expressions of myogenic differentiation (MyoD), muscle regulatory factor (MRF4), myogenic factor (MyoG) and myostatin 1 (MSTN1) were markedly elevated and the content of inosine monophosphate (IMP) in muscle was significantly increased. When LKB1 was inhibited for 72 h, expressions of MyoD, MyoG, MRF4 and MSTN1 were markedly decreased. The long-term injection experiment of the LKB1 activator revealed that, when LKB1 was activated for 15 days, its muscle fibers were significantly larger and tighter than the control group. In texture profile analysis, it showed smaller hardness and adhesion, greater elasticity and chewiness. Contrastingly, when LKB1 was inhibited for 9 days, its muscle fibers were significantly smaller, while the gap between muscle fibers was significantly larger. Texture profile analysis showed that adhesion was significantly higher than the control group. A feeding trial on triploid crucian carp showed that with dietary lysine-glutamate dipeptide concentration increasing, the expression of the LKB1 gene gradually increased and was highest when dipeptide concentration was 1.6%. These findings may provide new insights into the effects of LKB1 on fish skeletal muscle growth and muscle quality, and will provide a potential application value in improvement of aquaculture feed formula.

Liver kinase B1 (LKB1) is a serine/threonine protein kinase that acts as a key tumor suppressor protein by activating its downstream kinases, such as AMP-activated protein kinase (AMPK). However, the regulatory actions of LKB1 and AMPK on DNA damage response (DDR) remain to be explored. In this study, we investigated the function of LKB1 in DDR induced by cisplatin, a representative DNA-damaging agent, and found that LKB1 stabilizes and activates p53 through the c-Jun N-terminal kinase (JNK) pathway, which promotes cisplatin-induced apoptosis in human fibrosarcoma cell line HT1080. On the other hand, we found that AMPKα1 and α2 double knockout (DKO) cells showed enhanced stabilization of p53 and increased susceptibility to apoptosis induced by cisplatin, suggesting that AMPK negatively regulates cisplatin-induced apoptosis. Moreover, the additional stabilization of p53 and subsequent apoptosis in AMPK DKO cells were clearly canceled by the treatment with the antioxidants, raising the possibility that AMPK suppresses the p53 activation mediated by oxidative stress. Thus, our findings unexpectedly demonstrate the reciprocal regulation of p53 by LKB1 and AMPK in DDR, which provides insights into the molecular mechanisms of DDR.

Background and Aims Butyric acid is an intestinal microbiota-produced short-chain fatty acid, which exerts salutary effects on alleviating nonalcoholic fatty liver disease (NAFLD). However, the underlying mechanism of butyrate on regulating hepatic lipid metabolism is largely unexplored. Methods A mouse model of NAFLD was induced with high-fat diet feeding, and sodium butyrate (NaB) intervention was initiated at the eighth week and lasted for 8 weeks. Hepatic steatosis was evaluated and metabolic pathways concerning lipid homeostasis were analyzed. Results Here, we report that administration of NaB by gavage once daily for 8 weeks causes an augmentation of insulin-induced gene (Insig) activity and inhibition of lipogenic gene in mice fed with high-fat diet. Mechanistically, NaB is sufficient to enhance the interaction between Insig and its upstream kinase AMP-activated protein kinase (AMPK). The stimulatory effects of NaB on Insig-1 activity are abolished in AMPKα1/α2 double knockout (AMPK−/−) mouse primary hepatocytes. Moreover, AMPK activation by NaB is mediated by LKB1, as evidenced by the observations showing NaB-mediated induction of phosphorylation of AMPK, and its downstream target acetyl-CoA carboxylase is diminished in LKB1–/– mouse embryonic fibroblasts. Conclusions These studies indicate that NaB serves as a negative regulator of hepatic lipogenesis in NAFLD and that NaB attenuates hepatic steatosis and improves lipid profile and liver function largely through the activation of LKB1-AMPK-Insig signaling pathway. Therefore, NaB has therapeutic potential for treating NAFLD and related metabolic diseases., Graphical abstract

Lipopolysaccharide (LPS) is an endotoxin involved in a number of acute and chronic inflammatory syndromes. Although LPS‐induced signalling has been extensively studied, there are still mysteries remaining to be revealed. In the current study, we used high‐throughput phosphoproteomics to profile LPS‐initiated signalling and aimed to find novel mediators. A total of 448 phosphoproteins with 765 phosphorylation sites were identified, and we further validated that the phosphorylation of MARK2 on T208 was important for the regulation on LPS‐induced CXCL15 (human IL‐8 homolog), IL‐1β, IL‐6 and TNF‐α release, in which LKB1 had a significant contribution. In summary, induction of cytokines by LPS in mouse macrophage is regulated by LKB1‐MARK2 signals. Our study provides new clues for further exploring the underlying mechanisms of LPS‐induced diseases, and new therapeutic approaches concerning bacterial infection may be derived from these findings.

Inactivating mutations in the liver kinase B1 (LKB1) tumor suppressor gene underlie Peutz-Jeghers syndrome (PJS) and occur frequently in various human cancers. We previously showed that LKB1 regulates centrosome duplication via PLK1. Here, we report that LKB1 further helps to maintain genomic stability through negative regulation of survivin, a member of the chromosomal passenger complex (CPC) that mediates CPC targeting to the centromere. We found that loss of LKB1 led to accumulation of misaligned and lagging chromosomes at metaphase and anaphase and increased the appearance of multi- and micro-nucleated cells. Ectopic LKB1 expression reduced these features and improved mitotic fidelity in LKB1-deficient cells. Through pharmacological and genetic manipulations, we showed that LKB1-mediated repression of survivin is independent of AMPK, but requires p53. Consistent with the key influence of LKB1 on survivin expression, immunohistochemical analysis indicated that survivin is highly expressed in intestinal polyps from a PJS patient. Lastly, we reaffirm a potential therapeutic avenue to treat LKB1-mutated tumors by demonstrating the increased sensitivity to survivin inhibitors of LKB1-deficient cells.

The spectrum of kinase alterations displays distinct functional characteristics and requires kinase mutation‐oriented strategies for therapeutic interference. Besides phosphotransferase activity, protein abundance, and intermolecular interactions, particular patient‐mutations promote pathological kinase conformations. Despite major advances in identifying lead molecules targeting clinically relevant oncokinase functions, still many kinases are neglected and not part of drug discovery efforts. One explanation is attributed to challenges in tracking kinase activities. Chemical probes are needed to functionally annotate kinase functions, whose activities may not always depend on catalyzing phospho‐transfer. Such non‐catalytic kinase functions are related to transitions of full‐length kinase conformations. Recent findings underline that cell‐based reporter systems can be adapted to record conformation changes of kinases. Here, we discuss the possible applications of an extendable kinase conformation (KinCon) reporter toolbox for live‐cell recording of kinase states. KinCon is a genetically encoded bioluminescence‐based biosensor platform, which can be subjected for measurements of conformation dynamics of mutated kinases upon small molecule inhibitor exposure. We hypothesize that such biosensors can be utilized to delineate the molecular modus operandi for kinase and pseudokinase regulation. This should pave the path for full‐length kinase‐targeted drug discovery efforts aiming to identify single and combinatory kinase inhibitor therapies with increased specificity and efficacy.

Liver kinase B1 (LKB1) is a tumor suppressor gene, the inactivation of which occurs frequently in different tumor types. However, whether LKB1 is associated with the clinical features of gastric cancer (GC) and regulating tumor immunity is unknown. In this study, we showed that LKB1 is highly expressed in the serum of healthy individuals (n = 176) compared to GC patients (n = 416) and is also associated with clinical outcomes and good survival rates in GC patients. Furthermore, genes associated with immune checkpoints and T cell activation, such as PD−1, PD−L1, CD8A, CD8B, CD28, and GZMM, were shown to be highly expressed in GC subgroups with high LKB1 expression. Compared with fresh gastric cancerous tissues, LKB1 was highly expressed in CD3+CD8+ and CD3+CD8+CD28+ T cells in fresh adjacent non-cancerous tissues. CD3+CD8+ T cells produced an IFN−γ anti−cancer immune response. Furthermore, the proportion of CD3+CD8+ T cells that expressed LKB had a positive correlation with IFN−γ expression. Moreover, GC patients with low LKB1 expression had a poor objective response rate, and worse progression-free survival and overall survival when treated with pembrolizumab. In conclusion, LKB1 may be a potential immune checkpoint in GC patients.

The natural bioactive compound capsaicin has been reported to have anticancer activity, although the underlying mechanism of action has not been completely clarified. Herein, we investigated the mechanism whereby capsaicin exerts antitumor effects on prostate cancer cells. We found that capsaicin activated AMP-activated kinase (AMPK) and promoted cell death in the LKB1-expressing prostate cancer cell lines LNCaP and PC3, but not in the liver kinase B1 (LKB1)-null cell line DU-145. Capsaicin treatment stimulated LKB1 phosphorylation and activated AMPK in LKB1-expressing cells. In addition, LKB1 silencing in LNCaP and PC3 cells abrogated capsaicin-induced AMPK activation, while the overexpression of LKB1 by lentiviral infection in DU-145 cells induced capsaicin-triggered AMPK phosphorylation. Moreover, the calcium/calmodulin-dependent kinase kinase 2 (CaMKK2) inhibitor STO-609 did not modify the activation of AMPK induced by capsaicin, suggesting a CaMKK2-independent mechanism. Capsaicin-induced LKB1 phosphorylation was dependent on the transient receptor potential cation channel subfamily V member 1 (TRPV1), since TRPV1 knocked down by shRNA abolished LKB1 and AMPK phosphorylation in LKB1-expressing cells. Altogether, our results showed that capsaicin affected AMPK activity in an LKB1- and TRPV1-dependent fashion, linking TRPV1 with cell fate. These data also suggest that capsaicin may be a rational chemotherapeutic option for prostate tumors.

Lung homeostasis and regeneration depend on lung epithelial progenitor cells. Lkb1 (Liver Kinase B1) has known roles in the differentiation of airway epithelial cells during embryonic development. However, the effects of Lkb1 in adult lung epithelial progenitor cell regeneration and its mechanisms of action have not been determined. In this study, we investigated the mechanism by which Lkb1 regulates lung epithelial progenitor cell regeneration. Organoid culture showed that loss of Lkb1 significantly reduced the proliferation of club cells and alveolar type 2 (AT2) cells in vitro. In the absence of Lkb1, there is a slower recovery rate of the damaged airway epithelium in naphthalene-induced airway epithelial injury and impaired expression of surfactant protein C during bleomycin-induced alveolar epithelial damage. Moreover, the expression of autophagy-related genes was reduced in club cells and increased in AT2 cells, but the expression of Claudin-18 was obviously reduced in AT2 cells after Lkb1 knockdown. On the whole, our findings indicated that Lkb1 may promote the proliferation of lung epithelial progenitor cells via a niche-dependent pathway and is required for the repair of the damaged lung epithelium.

The energy sensor AMP kinase (AMPK) and the master scaffolding protein, AXIN, are two major regulators of biological processes in metazoans. AXIN-dependent regulation of AMPK activation plays a crucial role in maintaining metabolic homeostasis during glucose-deprived and energy-stressed conditions. The two proteins are also required for muscle function. While studies have refined our knowledge of various cellular events that promote the formation of AXIN-AMPK complexes and the involvement of effector proteins, more work is needed to understand precisely how the pathway is regulated in response to various forms of stress. In this review, we discuss recent data on AXIN and AMPK interaction and its role in physiological changes leading to improved muscle health and an extension of lifespan. We argue that AXIN-AMPK signaling plays an essential role in maintaining muscle function and manipulating the pathway in a tissue-specific manner could delay muscle aging. Therefore, research on understanding the factors that regulate AXIN-AMPK signaling holds the potential for developing novel therapeutics to slow down or revert the age-associated decline in muscle function, thereby extending the healthspan of animals.

Intramuscular fat (IMF) deposition is one of the most important factors to affect meat quality in livestock and induce insulin resistance and adverse metabolic phenotypes for humans. However, the key regulators involved in this process remain largely unknown. Although liver kinase B1 (LKB1) was reported to participate in the development of skeletal muscles and classical adipose tissues. Due to the specific autonomic location of intramuscular adipocytes, deposited between or within muscle bundles, the exact roles of LKB1 in IMF deposition need further verified. Here, we cloned the goat LKB1 coding sequence with 1,317 bp, encoding a 438 amino acid peptide. LKB1 was extensively expressed in detected tissues and displayed a trend from decline to rise during intramuscular adipogenesis. Functionally, knockdown of LKB1 by two individual siRNAs enhanced the intramuscular preadipocytes differentiation, accompanied by promoting lipid accumulation and inducing adipogenic transcriptional factors and triglyceride synthesis-related genes expression. Conversely, overexpression of LKB1 restrained these biological signatures. To further explore the mechanisms, the RNA-seq technique was performed to compare the difference between siLKB1 and the control group. There were 1,043 differential expression genes (DEGs) were screened, i.e., 425 upregulated genes and 618 downregulated genes in the siLKB1 group. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis predicted that the DEGs were mainly enriched in the focal adhesion pathway and its classical downstream signal, the PI3K-Akt signaling pathway. Specifically, knockdown of LKB1 increased the mRNA level of focal adhesion kinase (FAK) and vice versa in LKB1-overexpressed cells, a key component of the activated focal adhesion pathway. Convincingly, blocking this pathway by a specific FAK inhibitor (PF573228) rescued the observed phenotypes in LKB1 knockdown adipocytes. In conclusion, LKB1 inhibited goat intramuscular adipogenesis through the focal adhesion pathway. This work expanded the genetic regulator networks of IMF deposition and provided theoretical support for improving human health and meat quality from the aspect of IMF deposition.

Targeting the first protein complex of the mitochondrial electron transport chain (MC1) in cancer has become an attractive therapeutic approach in the recent years, given the metabolic vulnerabilities of cancer cells. The anticancer effect exerted by the pleiotropic drug metformin and the associated reduction in hypoxia‐inducible factor 1α (HIF‐1α) levels putatively mediated by MC1 inhibition led to the development of HIF‐1α inhibitors, such as BAY87‐2243, with a more specific MC1 targeting. However, the development of BAY87‐2243 was stopped early in phase 1 due to dose‐independent emesis and thus there is still no clinical proof of concept for the approach. Given the importance of mitochondrial metabolism during cancer progression, there is still a strong therapeutic need to develop specific and safe MC1 inhibitors. We recently reported the synthesis of compounds with a novel chemotype and potent action on HIF‐1α degradation and MC1 inhibition. We describe here the selectivity, safety profile and anti‐cancer activity in solid tumors of lead compound EVT‐701. In addition, using murine models of lung cancer and of Non‐Hodgkin's B cell lymphoma we demonstrated that EVT‐701 reduced tumor growth and lymph node invasion when used as a single agent therapy. LKB1 deficiency in lung cancer was identified as a potential indicator of accrued sensitivity to EVT‐701, allowing stratification and selection of patients in clinical trials. Altogether these results support further evaluation of EVT‐701 alone or in combination in preclinical models and eventually in patients., The present study elucidates the mechanism of action and supports further preclinical and clinical investigation of EVT‐701, either as a single agent or in combination, for the treatment of solid tumors. LKB1 deficiency in lung cancer was identified as potential indicator of accrued sensitivity to EVT‐701, allowing stratification and selection of patients in clinical trials

Conditional genetically engineered mouse models (GEMMs) of non-small cell lung cancer (NSCLC) harbor common oncogenic driver mutations of the disease, but in contrast to human NSCLC these models possess low tumor mutational burden (TMB). As a result, these models often lack tumor antigens that can elicit host adaptive immune responses, which limits their utility in immunotherapy studies. Here, we establish Kras-mutant murine models of NSCLC bearing the common driver mutations associated with the disease and increased TMB, by in vitro exposure of cell lines derived from GEMMs of NSCLC [KrasG12D (K), KrasG12DTp53−/−(KP), KrasG12DTp53+/−Lkb1−/− (KPL)] to the alkylating agent N-methyl-N-nitrosourea (MNU). Increasing the TMB enhanced host anti-tumor T cell responses and improved anti-PD-1 efficacy in syngeneic models across all genetic backgrounds. However, limited anti-PD-1 efficacy was observed in the KPL cell lines with increased TMB, which possessed a distinct immunosuppressed tumor microenvironment (TME) primarily composed of granulocytic myeloid-derived suppressor cells (G-MDSCs). This KPL phenotype is consistent with findings in human KRAS-mutant NSCLC where LKB1 loss is a driver of primary resistance to PD-1 blockade. In summary, these novel Kras-mutant NSCLC murine models with known driver mutations and increased TMB have distinct TMEs and recapitulate the therapeutic vulnerabilities of human NSCLC. We anticipate that these immunogenic models will facilitate the development of innovative immunotherapies in NSCLC.

Lung cancer with loss-of-function of the LKB1 tumor suppressor is a common aggressive subgroup with no effective therapies. LKB1-deficiency induces constitutive activation of cAMP/CREB-mediated transcription by a family of three CREB-regulated transcription coactivators (CRTC1-3). However, the significance and mechanism of CRTC activation in promoting the aggressive phenotype of LKB1-null cancer remain poorly characterized. Here, we observed overlapping CRTC expression patterns and mild growth phenotypes of individual CRTC-knockouts in lung cancer, suggesting functional redundancy of CRTC1-3. We consequently designed a dominant-negative mutant (dnCRTC) to block all three CRTCs to bind and co-activate CREB. Expression of dnCRTC efficiently inhibited the aberrantly activated cAMP/CREB-mediated oncogenic transcriptional program induced by LKB1-deficiency, and specifically blocked the growth of human and murine LKB1-inactivated lung cancer. Collectively, this study provides direct proof for an essential role of the CRTC-CREB activation in promoting the malignant phenotypes of LKB1-null lung cancer and proposes the CRTC-CREB interaction interface as a novel therapeutic target.

Background: In the past few decades, many lines of evidence implicate the importance of liver kinase B1 (LKB1) as a tumor suppressor gene in the development and progression of solid tumours. However, the prognostic and clinicopathological value of LKB1 in patients with lung cancer are controversial. This article aimed to investigate the latest evidence on this question. Methods: A systematic literature searched in the PubMed, Web of Science, Embase, Cochrane library, Scopus until September 20, 2020. The association between overall survival (OS), relapse-free survival (RFS), progression-free survival (PFS), clinicopathological features and LKB1 were analysed by meta-analysis. Results: Eleven studies including 1507 patients were included in this meta-analysis. The pooled results revealed that low LKB1 expression was significantly associated with poor overall survival (OS) (HR = 1.67, 95% CI: 1.07–2.60, P = .024) in lung cancer. However, no association was found between LKB1 expression and DFS/PFS (HR = 1.29, 95% CI: 0.70–2.39, P = .410). Pooled results showed that low LKB1 expression was associated with histological differentiation (poor vs moderate or well, OR = 4.135, 95% CI:2.524–6.774, P

Accumulating evidence suggests that a reduction in the number of Foxp3+ regulatory T cells (Tregs) contributes to the pathogenesis of acute graft-versus-host disease (aGVHD), which is a major adverse complication that can occur after allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, the precise features and mechanism underlying the defects in Tregs remain largely unknown. In this study, we demonstrated that Tregs were more dramatically decreased in bone marrow compared with those in peripheral blood from aGVHD patients and that bone marrow Treg defects were negatively associated with hematopoietic reconstitution. Tregs from aGVHD patients exhibited multiple defects, including the instability of Foxp3 expression, especially in response to IL-12, impaired suppressor function, decreased migratory capacity, and increased apoptosis. Transcriptional profiling revealed the downregulation of Lkb1, a previously identified critical regulator of murine Treg identity and metabolism, and murine Lkb1-regulated genes in Tregs from aGVHD patients. Foxp3 expression in human Tregs could be decreased and increased by the knockdown and overexpression of the Lkb1 gene, respectively. Furthermore, a loss-of-function assay in an aGVHD murine model confirmed that Lkb1 deficiency could impair Tregs and aggravate disease severity. These findings reveal that Lkb1 downregulation contributes to multiple defects in Tregs in human aGVHD and highlight the Lkb1-related pathways that could serve as therapeutic targets that may potentially be manipulated to mitigate aGVHD.

Purpose The purpose of this study was to investigate the prognostic significance of liver kinase b1 (LKB1) loss in patients with operable colon cancer (CC). Materials and Methods Two hundred sixty-two specimens from consecutive patients with stage III or high-risk stage II CC, who underwent surgical resection with curative intent and received adjuvant chemotherapy with fluoropyrimidine and oxaliplatin, were analyzed for LKB1 protein expression loss, by immunohistochemistry as well as for KRAS exon 2 and BRAFV600E mutations by Sanger sequencing and TS, ERCC1, MYC, and NEDD9 mRNA expression by real-time quantitative reverse transcription polymerase chain reaction. Results LKB1 expression loss was observed in 117 patients (44.7%) patients and correlated with right-sided located primaries (p=0.032), and pericolic lymph nodes involvement (p=0.003), BRAFV600E mutations (p=0.024), and TS mRNA expression (p=0.041). Patients with LKB1 expression loss experienced significantly lower disease-free survival (DFS) (hazard ratio [HR], 1.287; 95% confidence interval [CI], 1.093 to 1.654; p=0.021) and overall survival (OS) (HR, 1.541; 95% CI, 1.197 to 1.932; p=0.002), compared to patients with LKB1 expressing expressing tumors. Multivariate analysis revealed LKB1 expression loss as independent prognostic factor for both decreased DFS (HR, 1.217; 95% CI, 1.074 to 1.812; p=0.034) and decreased OS (HR, 1.467; 95% CI, 1.226 to 2.122; p=0.019). Conclusion Loss of tumoral LKB1 protein expression, constitutes an adverse prognostic factor in patients with operable CC.

Polycystic ovary syndrome (PCOS) is a major cause of anovulatory sterility in women, and most PCOS patients exhibit hyperandrogenism (HA). Liver kinase b1 (LKB1) is a tumor suppressor that has recently been reported to be involved in PCOS. However, the mechanism by which LKB1 affects HA has not previously been elucidated. We report here that ovarian LKB1 levels are significantly decreased in a female mouse model of HA. Moreover, we report that LKB1 expression is inhibited by elevated androgens via activation of androgen receptors. In addition, LKB1 treatment was observed to suppress androgen synthesis in theca cells and promote estrogen production in granulosa cells by regulating steroidogenic enzyme expression. As expected, LKB1 knockdown inhibited estrogen levels and enhanced androgen levels, and LKB1‐transgenic mice were protected against HA. The effect of LKB1 appears to be mediated via IGF‐1 signaling. In summary, we describe here a key role for LKB1 in controlling sex hormone levels.

Sirtuin 1 (SIRT1) is known to play a role in a variety of tumorigenesis processes by deacetylating histone and non‐histone proteins; however, antitumour effects by suppressing SIRT1 activity in non‐small cell lung cancer (NSCLC) remain unclear. This study was designed to scrutinize clinicopathological significance of SIRT1 in NSCLC and investigate effects of metformin on SIRT1 inhibition. This study also evaluated new possibilities of drug combination using a SIRT1 inhibitor, tenovin‐6, in NSCLC cell lines. It was found that SIRT1 was overexpressed in 300 (62%) of 485 formalin‐fixed paraffin‐embedded NSCLC tissues. Its overexpression was significantly associated with reduced overall survival and poor recurrence‐free survival after adjusted for histology and pathologic stage. Thus, suppression of SIRT1 expression may be a reasonable therapeutic strategy for NSCLC. Metformin in combination with tenovin‐6 was found to be more effective in inhibiting cell growth than either agent alone in NSCLC cell lines with different liver kinase B1 (LKB1) status. In addition, metformin and tenovin‐6 synergistically suppressed SIRT1 expression in NSCLC cells regardless of LKB1 status. The marked reduction in SIRT1 expression by combination of metformin and tenovin‐6 increased acetylation of p53 at lysine 382 and enhanced p53 stability in LKB1‐deficient A549 cells. The combination suppressed SIRT1 promoter activity more effectively than either agent alone by up‐regulating hypermethylation in cancer 1 (HIC1) binding at SIRT1 promoter. Also, suppressed SIRT1 expression by the combination synergistically induced caspase‐3‐dependent apoptosis. The study concluded that metformin with tenovin‐6 may enhance antitumour effects through LKB1‐independent SIRT1 down‐regulation in NSCLC cells.

For many decades, kinases have predominantly been characterized as oncogenes and drivers of tumorigenesis, because activating mutations in kinases occur in cancer with high frequency. The oncogenic functions of kinases relate to their roles as growth factor receptors and as critical mediators of mitogen‐activated pathways. Indeed, some of the most promising cancer therapeutic agents are kinase inhibitors. However, cancer genomics studies, especially screens that utilize high‐throughput identification of loss‐of‐function somatic mutations, are beginning to shed light on a widespread role for kinases as tumor suppressors. The initial characterization of tumor‐suppressing kinases— in particular members of the protein kinase C (PKC) family, MKK4 of the mitogen‐activated protein kinase kinase family, and DAPK3 of the death‐associated protein kinase family— laid the foundation for bioinformatic approaches that enable the identification of other tumor‐suppressing kinases. In this review, we discuss the important role that kinases play as tumor suppressors, using several examples to illustrate the history of their discovery and highlight the modern approaches that presently aid in the identification of tumor‐suppressing kinases. © 2018 IUBMB Life, 71(6):738–748, 2019

The discovery of liver kinase B1 (LKB1) as an upstream kinase for AMP-activated protein kinase (AMPK) led to the identification of several related kinases that also rely on LKB1 for their catalytic activity. Among these, the salt-inducible kinases (SIKs) have emerged as key regulators of metabolism. Unlike AMPK, SIKs do not respond to nucleotides, but their function is regulated by extracellular signals, such as hormones, through complex LKB1-independent mechanisms. While AMPK acts on multiple targets, including metabolic enzymes, to maintain cellular ATP levels, SIKs primarily regulate gene expression, by acting on transcriptional regulators, such as the cAMP response element-binding protein-regulated transcription coactivators and class IIa histone deacetylases. This review describes the development of research on SIKs, from their discovery to the most recent findings on metabolic regulation.

Uterine carcinoma (UC) is the most common gynecologic malignancy in the United States. TP53 mutant UCs cause a disproportionate number of deaths due to limited therapies for these tumors and the lack of mechanistic understanding of their fundamental vulnerabilities. Here we sought to understand the functional and therapeutic relevance of TP53 mutations in UC. We functionally profiled targetable TP53 dependent DNA damage repair and cell cycle control pathways in a panel of TP53 mutant UC cell lines and patient-derived organoids. There were no consistent defects in DNA damage repair pathways. Rather, most models demonstrated dependence on defective G2/M cell cycle checkpoints and subsequent upregulation of Aurora kinase-LKB1-p53-AKT signaling in the setting of baseline mitotic defects. This combination makes them sensitive to Aurora kinase inhibition. Resistant lines demonstrated an intact G2/M checkpoint, and combining Aurora kinase and WEE1 inhibitors, which then push these cells through mitosis with Aurora kinase inhibitor-induced spindle defects, led to apoptosis in these cases. Overall, this work presents Aurora kinase inhibitors alone or in combination with WEE1 inhibitors as relevant mechanism driven therapies for TP53 mutant UCs. Context specific functional assessment of the G2/M checkpoint may serve as a biomarker in identifying Aurora kinase inhibitor sensitive tumors.

Simple Summary Cancers arising from the lining of the uterus, endometrial cancers, are the most common gynecologic malignancy in the United States. Once endometrial cancer escapes the uterus and grows in distant locations, there are limited therapeutic options. The most aggressive and lethal endometrial cancers carry alterations in the protein p53, which is a critical guardian of many cellular functions. The role of these p53 alterations in endometrial cancer is not well understood. The goal of this work was to use p53 altered models of endometrial cancer to understand which, if any, therapeutically targetable vulnerabilities these p53 alterations may confer in endometrial cancer. Here we show that many of these p53 altered cells have problems with cell division which can be targeted with novel single and combination therapies. These discoveries may lead to relevant new therapies for difficult to treat advanced stage endometrial cancers. Abstract Uterine carcinoma (UC) is the most common gynecologic malignancy in the United States. TP53 mutant UCs cause a disproportionate number of deaths due to limited therapies for these tumors and the lack of mechanistic understanding of their fundamental vulnerabilities. Here we sought to understand the functional and therapeutic relevance of TP53 mutations in UC. We functionally profiled targetable TP53 dependent DNA damage repair and cell cycle control pathways in a panel of TP53 mutant UC cell lines and patient-derived organoids. There were no consistent defects in DNA damage repair pathways. Rather, most models demonstrated dependence on defective G2/M cell cycle checkpoints and subsequent upregulation of Aurora kinase-LKB1-p53-AKT signaling in the setting of baseline mitotic defects. This combination makes them sensitive to Aurora kinase inhibition. Resistant lines demonstrated an intact G2/M checkpoint, and combining Aurora kinase and WEE1 inhibitors, which then push these cells through mitosis with Aurora kinase inhibitor-induced spindle defects, led to apoptosis in these cases. Overall, this work presents Aurora kinase inhibitors alone or in combination with WEE1 inhibitors as relevant mechanism driven therapies for TP53 mutant UCs. Context specific functional assessment of the G2/M checkpoint may serve as a biomarker in identifying Aurora kinase inhibitor sensitive tumors.

Background The oncogenic PIM kinases and the tumor-suppressive LKB1 kinase have both been implicated in the regulation of cell growth and metabolism, albeit in opposite directions. Here we investigated whether these kinases interact with each other to influence AMPK activation and tumorigenic growth of prostate and breast cancer cells. Methods We first determined how PIM and LKB1 kinases affect AMPK phosphorylation levels. We then used in vitro kinase assays to demonstrate that LKB1 is phosphorylated by PIM kinases, and site-directed mutagenesis to identify the PIM target sites in LKB1. The cellular functions of PIM and LKB1 kinases were evaluated using either pan-PIM inhibitors or CRISPR/Cas9 genomic editing, with which all three PIM family members and/or LKB1 were knocked out from PC3 prostate and MCF7 breast cancer cell lines. In addition to cell proliferation assays, we examined the effects of PIM and/or LKB1 loss on tumor growth using the chick embryo chorioallantoic membrane (CAM) xenograft model. Results We provide both genetic and pharmacological evidence to demonstrate that inhibition of PIM expression or activity increases phosphorylation of AMPK at Thr172 in both PC3 and MCF7 cells, but not in their derivatives lacking LKB1. This is explained by our observation that all three PIM family kinases can phosphorylate LKB1 at Ser334. Wild-type LKB1, but not its phosphodeficient derivative, can restore PIM inhibitor-induced AMPK phosphorylation in LKB1 knock-out cells. In the CAM model, loss of LKB1 enhances tumorigenicity of PC3 xenografts, while cells lacking both LKB1 and PIMs exhibit slower proliferation rates and form smaller tumors. Conclusion PIM kinases are novel negative regulators of LKB1 that affect AMPK activity in an LKB1-dependent fashion. The impairment of cell proliferation and tumor growth in cells lacking both LKB1 and PIMs indicates that these kinases possess a shared signaling role in the context of cancer. These data also suggest that PIM inhibitors may be a rational therapeutic option for LKB1-deficient tumors.

The NAD+-dependent deacetylase Sirt1 has been implicated in the prevention of many age-related diseases, including cancer, type 2 diabetes, and cardiovascular disease. Resveratrol, a plant polyphenol, exhibits antiaging, antitumor, and vascular protection effects by activating Sirt1. However, the molecular mechanism of Sirt1 activation as induced by resveratrol remains unclear. By knockdown/rescue experiments, fluorometric Sirt1 activity assay, immunoprecipitation, and pull-down assays, we identify here that the tumor suppressor LKB1 (liver kinase B1) as a direct activator of Sirt1 elicited by resveratrol. Resveratrol promotes the binding between LKB1 and Sirt1, which we first reported, and this binding leads to LKB1-mediated phosphorylation of Sirt1 at three different serine residues in the C terminus of Sirt1. Mechanistically, LKB1-mediated phosphorylation increases intramolecular interactions in Sirt1, such as the binding of the C terminus to the deacetylase core domain, thereby eliminating DBC1 (Deleted in Breast Cancer 1, Sirt1 endogenous inhibitor) inhibition and promoting Sirt1–substrate interaction. Functionally, LKB1-dependent Sirt1 activation increases mitochondrial biogenesis and respiration through deacetylation and activation of the transcriptional coactivator PGC-1α. These results identify Sirt1 as a context-dependent target of LKB1 and suggest that a resveratrol-stimulated LKB1-Sirt1 pathway plays a vital role in mitochondrial metabolism, a key physiological process that contributes to numerous age-related diseases.

© 2021 by the authors., The dysregulation of autophagy is important in the development of many cancers, including thyroid cancer, where V600EBRAF is a main oncogene. Here, we analyse the effect of V600EBRAF inhibition on autophagy, the mechanisms involved in this regulation and the role of autophagy in cell survival of thyroid cancer cells. We reveal that the inhibition of V600EBRAF activity with its specific inhibitor PLX4720 or the depletion of its expression by siRNA induces autophagy in thyroid tumour cells. We show that V600EBRAF downregulation increases LKB1-AMPK signalling and decreases mTOR activity through a MEK/ERK-dependent mechanism. Moreover, we demonstrate that PLX4720 activates ULK1 and increases autophagy through the activation of the AMPK-ULK1 pathway, but not by the inhibition of mTOR. In addition, we find that autophagy blockade decreases cell viability and sensitize thyroid cancer cells to V600EBRAF inhibition by PLX4720 treatment. Finally, we generate a thyroid xenograft model to demonstrate that autophagy inhibition synergistically enhances the anti-proliferative and pro-apoptotic effects of V600EBRAF inhibition in vivo. Collectively, we uncover a new role of AMPK in mediating the induction of cytoprotective autophagy by V600EBRAF inhibition. In addition, these data establish a rationale for designing an integrated therapy targeting V600EBRAF and the LKB1-AMPK-ULK1-autophagy axis for the treatment of V600EBRAF-positive thyroid tumours., This research was supported by grants from the Ministerio de Ciencia, Innovación y Universidades of Spain (SAF 2014-53639-R) and from UAH-Comunidad de Madrid (CAM) (CM/JIN/2019-019).

LKB1-signaling has prominent roles in cancer development and metastasis. This report evaluates LKB1-signaling pathway gene expression associations with patient survival in overall breast cancer, specific subtypes, as well as pre- and post-chemotherapy. Subtypes analyzed were based on intrinsic molecular subtyping and traditional biomarker classifications. Intrinsic molecular subtypes included were Luminal-A, Luminal-B, HER2-enriched, and Basal-like. The biomarker subtypes assessed were Estrogen-Receptor Positive (ER+) and Negative (ER-), Wild-Type TP53 (WT-TP53) & Mutant-TP53, and Triple-Negative Breast Cancer (TNBC). Additionally, comparisons were made between these subtypes and breast cancer overall, and analyses between LKB1 signaling to patient survival before and after chemotherapy were made. We used the Kaplan-Meier Online Tool (KM Plotter) to correlate the relationship between mRNA expression of known LKB1 scaffolding proteins (CAB39 and LYK5), and downstream signaling targets (AMPK, MARK1, MARK2, MARK3, MARK4, NUAK1, NUAK2, PAK1, SIK1, SIK2, BRSK1, BRSK2, SNRK, and QSK), and patient survival across each subtype and treatment group. Our findings provide evidence that LKB1-signaling is associated with improved survival in overall breast cancer. Stratification into breast cancer subtypes show a more complicated relationship; NUAK2, for example, is correlated with improved survival in ER- but is worse in ER+ breast cancer. In evaluating the association of LKB1-signaling pathway expression with relapse free survival of varying breast cancer tumors exposed to chemotherapy or treatment-naive tumors, our data provides baseline knowledge for understanding the pathway dynamics that affect survival and therefore are linked to pathology. This establishes a foundation for studying LKB1 targets with the goal of identifying druggable targets.

Over the last 20 years, it has been shown that complex signaling cascades are involved in zinc excitotoxicity. Free zinc rapidly induces PKC activation, which causes reactive oxygen species (ROS) production at least in part through NADPH oxidase. It also promotes neuronal nitric oxide synthase, thereby increasing nitric oxide (NO) production. Extracellular signal-regulated kinase activation and Egr-1 transcription factor activity were quickly induced by zinc, too. These concurrent actions of kinases consequently produce oxygen free radical, ROS, and NO, which may cause severe DNA damage. Following the excessive activity of poly(ADP-ribose) polymerase-1 depletes NAD+/ATP in the cells. Zinc excitotoxicity exhibits distinct characteristics of apoptosis, too. Activation of caspase-3 is induced by liver kinase B1 (LKB1)-AMP-activated kinase (AMPK)-Bim cascade signaling and induction of p75NTR receptors and p75NTR-associated Death Executor. Thus, zinc excitotoxicity is a mechanism of neuronal cell death showing various cell death patterns. In addition to the above signaling cascades, individual intracellular organelles also play a crucial role in zinc excitotoxicity. Mitochondria and lysosomes function as zinc reservoirs, and as such, are capable of regulating zinc concentration in the cytoplasm. However, when loaded with too much zinc, they may undergo mitochondrial permeability transition pore (mPTP) opening, and lysosomal membrane permeabilization (LMP), both of which are well-established mechanisms of cell death. Since zinc excitotoxicity has been reported to be associated with acute brain injuries, including stroke, trauma, and epilepsy, we performed to find the novel AMPK inhibitors as therapeutic agents for these diseases. Since we thought acute brain injury has complicated neuronal death pathways, we tried to see the neuroprotection against zinc excitotoxicity, calcium-overload excitotoxicity, oxidative damage, and apoptosis. We found that two chemicals showed significant neuroprotection against all cellular neurotoxic models we tested. Finally, we observed the reduction of infarct volume in a rat model of brain injury after middle cerebral artery occlusion (MCAO). In this review, we introduced the AMPK-mediated cell death mechanism and novel strategy for the development of stroke therapeutics. The hope is that this understanding would provide a rationale for acute brain injury and eventually find new therapeutics.

Liver kinase B1 (LKB1), also known as serine/threonine kinase 11 (STK11) is the major energy sensor for cells to respond to metabolic stress. Autophagy degrades and recycles proteins, macromolecules, and organelles for cells to survive starvation. To assess the role and cross-talk between autophagy and Lkb1 in normal tissue homeostasis, we generated genetically engineered mouse models where we can conditionally delete Stk11 and autophagy essential gene, Atg7, respectively or simultaneously, throughout the adult mice. We found that Lkb1 was essential for the survival of adult mice, and autophagy activation could temporarily compensate for the acute loss of Lkb1 and extend mouse life span. We further found that acute deletion of Lkb1 in adult mice led to impaired intestinal barrier function, hypoglycemia, and abnormal serum metabolism, which was partly rescued by the Lkb1 loss-induced autophagy upregulation via inhibiting p53 induction. Taken together, we demonstrated that autophagy and Lkb1 work synergistically to maintain adult mouse homeostasis and survival.

Esophageal cancer is a type of gastrointestinal carcinoma and is among the 10 most common causes of cancer death worldwide. However, the specific mechanism and the biomarkers in the proliferation and metastasis of esophageal tumors are still unclear. Therefore, the development of several natural products which could inhibit esophageal tumors deserve attention. In the present study, different sources of cancer cells were used to select the sensitive cell line (esophageal cancer cell KYSE450) and the proper dose of angustoline, which were utilized in the following cell viability, migration and invasion assays. Then the lipidomic detection of clinical samples (tissue and blood plasma) from esophageal cancer patients was performed, to screen out the specific phospholipid metabolites [PC (16:0/18:1) and LPC (16:0)]. Considering lysophosphatidylcholine acyltransferase 2 (LPCAT2) was tightly relative with phospholipids conversion, serine/threonine-protein kinase 11 (LKB1), 5'-monophosphate (AMP)-activated protein kinase (AMPK) and embryonic lethal, and abnormal vision, drosophila-like 1 (ELAVL1) were investigated, to evaluate their expression levels in esophageal tumor tissue and KYSE450 cells. Additionally, KYSE450 tumor bearing mouse model was constructed, the role of angustoline in inhibiting esophageal tumors through regulating LKB1/AMPK/ELAVL1/LPCAT2 pathway was validated, and found that the conversion from LPC (16:0) to PC (16:0/18:1) was blocked by angustoline in some degree. The above results for the first time proved that angustoline suppressed esophageal tumors through activating LKB1/AMPK and inhibiting ELAVL1/LPCAT2, which consequently blocked phospholipid remodeling from LPC (16:0) to PC (16:0/18:1).

In quiescent cells, primary cilia function as a mechanosensor that converts mechanic signals into chemical activities. This unique organelle plays a critical role in restricting mechanistic target of rapamycin complex 1 (mTORC1) signaling, which is essential for quiescent cells to maintain their quiescence. Multiple mechanisms have been identified that mediate the inhibitory effect of primary cilia on mTORC1 signaling. These mechanisms depend on several tumor suppressor proteins localized within the ciliary compartment, including liver kinase B1 (LKB1), AMP-activated protein kinase (AMPK), polycystin-1, and polycystin-2. Conversely, changes in mTORC1 activity are able to affect ciliogenesis and stability indirectly through autophagy. In this review, we summarize recent advances in our understanding of the reciprocal regulation of mTORC1 and primary cilia.

Epithelial ovarian cancer (EOC) has a unique mode of metastasis, where cells shed from the primary tumour, form aggregates called spheroids to evade anoikis, spread through the peritoneal cavity, and adhere to secondary sites. We previously showed that the master kinase Liver kinase B1 (LKB1) is required for EOC spheroid viability and metastasis. We have identified novel (nua) kinase 1 (NUAK1) as a top candidate LKB1 substrate in EOC cells and spheroids using a multiplex inhibitor beads-mass spectrometry approach. We confirmed that LKB1 maintains NUAK1 phosphorylation and promotes its stabilization. We next investigated NUAK1 function in EOC cells. Ectopic NUAK1-overexpressing EOC cell lines had increased adhesion, whereas the reverse was seen in OVCAR8-NUAK1KO cells. In fact, cells with NUAK1 loss generate spheroids with reduced integrity, leading to increased cell death after long-term culture. Following transcriptome analysis, we identified reduced enrichment for cell interaction gene expression pathways in OVCAR8-NUAK1KO spheroids. In fact, the FN1 gene, encoding fibronectin, exhibited a 745-fold decreased expression in NUAK1KO spheroids. Fibronectin expression was induced during native spheroid formation, yet this was completely lost in NUAK1KO spheroids. Co-incubation with soluble fibronectin restored the compact spheroid phenotype to OVCAR8-NUAK1KO cells. In a xenograft model of intraperitoneal metastasis, NUAK1 loss extended survival and reduced fibronectin expression in tumours. Thus, we have identified a new mechanism controlling EOC metastasis, through which LKB1-NUAK1 activity promotes spheroid formation and secondary tumours via fibronectin production.

Previous reports have shown that excess calorie intake promotes p53 dependent senescence in mouse adipose tissues. The objective of the current study was to address the mechanism underlying this observation, i.e. adipocyte aging. Using cultured 3T3-L1 cells, we investigated the involvement of energy regulators Sirt1, AMPK, and LKB1 in senescence. Fifteen days post differentiation, Sirt1 knock-down increased senescence-associated beta-galactosidase (SA-β-Gal) staining by 20-40% (p

Intratumoral recruitment of immune cells following innate immune activation is critical for anti-tumor immunity and involves cytosolic dsDNA sensing by the cGAS/STING pathway. We have previously shown that KRAS-LKB1 (KL) mutant lung cancer, which is resistant to PD-1 blockade, exhibits silencing of STING, impaired tumor cell production of immune chemoattractants, and T cell exclusion. Since the vasculature is also a critical gatekeeper of immune cell infiltration into tumors, we developed a novel microfluidic model to study KL tumor-vascular interactions. Notably, dsDNA priming of LKB1-reconstituted tumor cells activates the microvasculature, even when tumor cell STING is deleted. cGAS-driven extracellular export of 2'3' cGAMP by cancer cells activates STING signaling in endothelial cells and cooperates with type 1 interferon to increase vascular permeability and expression of E selectin, VCAM-1, and ICAM-1 and T cell adhesion to the endothelium. Thus, tumor cell cGAS-STING signaling not only produces T cell chemoattractants, but also primes tumor vasculature for immune cell escape.

Deleted in Breast Cancer 1 (DBC1) is an important metabolic sensor. Previous studies have implicated DBC1 in various cellular functions, notably cell proliferation, apoptosis, histone modification, and adipogenesis. However, current reports about the role of DBC1 in tumorigenesis are controversial and designate DBC1 alternatively as a tumor suppressor or a tumor promoter. In the present study, we report that polyoma small T antigen (PyST) associates with DBC1 in mammalian cells, and this interaction leads to the posttranslational downregulation of DBC1 protein levels. When coexpressed, DBC1 overcomes PyST-induced mitotic arrest and promotes the exit of cells from mitosis. Using both transient and stable modes of PyST expression, we also show that cellular DBC1 is subjected to degradation by LKB1, a tumor suppressor and cellular energy sensor kinase, in an AMP kinase-independent manner. Moreover, LKB1 negatively regulates the phosphorylation as well as activity of the prosurvival kinase AKT1 through DBC1 and its downstream pseudokinase substrate, Tribbles 3 (TRB3). Using both transient transfection and stable cell line approaches as well as soft agar assay, we demonstrate that DBC1 has oncogenic potential. In conclusion, our study provides insight into a novel signaling axis that connects LKB1, DBC1, TRB3, and AKT1. We propose that the LKB1–DBC1–AKT1 signaling paradigm may have an important role in the regulation of cell cycle and apoptosis and consequently tumorigenesis.

AMPK is a crucial regulator of energy homeostasis that acts downstream of its upstream kinase liver kinase B1 (LKB1) and calcium/calmodulin-dependent protein kinase 2 (CaMKK2). LKB1 primarily phosphorylates AMPK after energy stress, whereas calcium-mediated activation of AMPK requires CaMKK2, although the regulatory mechanisms of calcium-mediated AMPK activation remain unclear. Using biochemical, microscopic, and genetic approaches, we demonstrate that the stromal interaction molecule (STIM)2, a calcium sensor, acts as a novel regulator of CaMKK2-AMPK signaling. We reveal that STIM2 interacts with AMPK and CaMKK2 and that the increase in intracellular calcium levels promotes AMPK colocalization and interaction with STIM2. We further show that STIM2 deficiency attenuates calcium-induced but not energy stress-induced AMPK activation, possibly by regulating the CaMKK2-AMPK interaction. Together, our results identify a previously unappreciated mechanism that modulates calcium-mediated AMPK activation.-Chauhan, A. S., Liu, X., Jing, J., Lee, H., Yadav, R. K., Liu, J., Zhou, Y., Gan B. STIM2 interacts with AMPK and regulates calcium-induced AMPK activation.

Summary Cancer cells display metabolic plasticity to survive stresses in the tumor microenvironment. Cellular adaptation to energetic stress is coordinated in part by signaling through the liver kinase B1 (LKB1)-AMP-activated protein kinase (AMPK) pathway. Here, we demonstrate that miRNA-mediated silencing of LKB1 confers sensitivity of lymphoma cells to mitochondrial inhibition by biguanides. Using both classic (phenformin) and newly developed (IM156) biguanides, we demonstrate that elevated miR-17∼92 expression in Myc+ lymphoma cells promotes increased apoptosis to biguanide treatment in vitro and in vivo. This effect is driven by the miR-17-dependent silencing of LKB1, which reduces AMPK activation in response to complex I inhibition. Mechanistically, biguanide treatment induces metabolic stress in Myc+ lymphoma cells by inhibiting TCA cycle metabolism and mitochondrial respiration, exposing metabolic vulnerability. Finally, we demonstrate a direct correlation between miR-17∼92 expression and biguanide sensitivity in human cancer cells. Our results identify miR-17∼92 expression as a potential biomarker for biguanide sensitivity in malignancies., Graphical Abstract, Highlights IM156 is a newly developed biguanide with more potency than phenformin IM156 inhibits mitochondrial respiration to cause bioenergetic stress in lymphoma miR-17-dependent silencing of LKB1 reduces adaptation to bioenergetic stress miR-17∼92 expression correlates with biguanide sensitivity in human cancer cells, Izreig et al. show that miR-17∼92 expression in cancer cells sensitizes them to biguanide treatment by disrupting bioenergetic stability. These data implicate miR-17∼92 expression as a potential biomarker for biguanide sensitivity in hematological malignancies and possibly solid tumors.

The serine/threonine kinase LKB1, act as a tumor suppressor, has been reported in several sporadic cancers. However, how the loss of LKB1 promotes melanoma invasion and metastasis remains incompletely understood. In this study, we inactivated LKB1expression by RNA interference in BRAF mutation and wild type melanoma cells respectively. We found LKB1 inactivation cooperate with BRAF V600E lead to melanoma cells more aggressive by a series of experiments including wound scratch test, Transwell assay. While single alteration, either LKB1 loss or BRAF V600E, fails to enhance melanoma cells invasion ability. Mechanistically, LKB1 loss synergism with BRAF V600E resulted in the activation of the PI3K/Akt/mTOR signaling pathway and significant up-regulation expression of MMP-2. In addition, LKB1 expression in human melanoma tissues was negatively associated with MMP-2 expression in the presence of BRAF V600E. Thus, our findings indicate a probable explanation on LKB1 function as a tumor suppressor in melanoma and a new therapeutic strategy for melanoma by targeting on BRAF and LKB1 together.

// Tsung-Ming Chang 1, * , Yan-Shen Shan 2, 3, * , Pei-Yi Chu 4, 1, 5, * , Shih Sheng Jiang 1 , Wen-Chun Hung 1 , Yu-Lin Chen 1 , Hsiu-Chi Tu 1 , Hui-You Lin 1 , Hui-Jen Tsai 1, 6, 7 and Li-Tzong Chen 1, 6, 7, 8 1 National Institute of Cancer Research, National Health Research Institutes, Tainan, Taiwan 2 Department of Surgery, National Cheng Kung University Hospital, Tainan, Taiwan 3 Institute of Clinical Medicine, National Cheng Kung University, Tainan, Taiwan 4 Department of Pathology, Show Chwan Memorial Hospital, Changhua, Taiwan 5 School of Medicine, College of Medicine, Fu Jen Catholic University, New Taipei City, Taiwan 6 Department of Internal Medicine, National Cheng Kung University Hospital, Tainan, Taiwan 7 Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan 8 Institute of Molecular Medicine, National Cheng Kung University, Tainan, Taiwan * These authors contributed equally to this work Correspondence to: Hui-Jen Tsai, email: hjtsai@nhri.org.tw Keywords: pancreatic neuroendocrine tumor, PTEN, LKB1, mTOR, c-Myc Received: June 15, 2017 Accepted: September 03, 2017 Published: September 16, 2017 ABSTRACT Pancreatic neuroendocrine tumor (pNET) is an uncommon type of pancreatic neoplasm. Low Phosphatase and Tensin Homologue (PTEN) expression and activation of the mechanistic target of rapamycin (mTOR) pathway have been noted in pNETs, and the former is associated with poor survival in pNET patients. Based on the results of the RADIANT-3 study, everolimus, an oral mTOR inhibitor, has been approved to treat advanced pNETs. However, the exact regulatory mechanism for the mTOR pathway in pNETs remains largely unknown. PTEN and liver kinase B1 (LKB1) are well-known for their regulatory role in the mTOR pathway. We evaluated the expression of PTEN and LKB1 in 21 pNET patients, and low PTEN and LKB1 expression levels were noted in 48% and 24% of the patients, respectively. Loss of PTEN and LKB1 synergistically promoted cell proliferation of pNET, attenuated the sensitivity of cells to mTOR inhibitors and enhanced c-Myc expression, which back-regulated PTEN, AKT, mTOR and its downstream effectors. For pNET cells with low expression levels of PTEN and LKB1, silencing the expression of c-Myc by shRNA reduced their proliferative rate, while adding either c-Myc inhibitor or AMP-activated protein kinase activator reversed their resistance to mTOR inhibitors in vitro and in vivo . Furthermore, high c-Myc expression was subsequently identified in 81% of pNETs, suggesting that up-regulation of c-Myc expression in pNETs may occur through PTEN/LKB1-dependent and PTEN/LKB1-independent regulation. The results delineated the regulation of PTEN and LKB1 on the AKT/mTOR/c-Myc axis and suggested that both c-Myc and mTOR are potential therapeutic targets for pNET.

Background: The most prevalent form of bone cancer is osteosarcoma (OS), which is associated with poor prognosis in case of metastases formation. Mice harbouring liver kinase B1 (LKB1+/−) develop osteoblastoma-like tumours. Therefore, we asked whether loss of LKB1 gene has a role in the pathogenesis of human OS. Methods: Osteosarcomas (n=259) were screened for LKB1 and sirtuin 1 (SIRT1) protein expression using immunohistochemistry and western blot. Those cases were also screened for LKB1 genetic alterations by next-generation sequencing, Sanger sequencing, restriction fragment length polymorphism and fluorescence in situ hybridisation approaches. We studied LKB1 protein degradation through SIRT1 expression. MicroRNA expression investigations were also conducted to identify the microRNAs involved in the SIRT1/LKB1 pathway. Results: Forty-one per cent (106 out of 259) OS had lost LKB1 protein expression with no evident genetic anomalies. We obtained evidence that SIRT1 impairs LKB1 protein stability, and that SIRT1 depletion leads to accumulation of LKB1 in OS cell lines resulting in growth arrest. Further investigations revealed the role of miR-204 in the regulation of SIRT1 expression, which impairs LKB1 stability. Conclusions: We demonstrated the involvement of sequential regulation of miR-204/SIRT1/LKB1 in OS cases and showed a mechanism for the loss of expression of LKB1 tumour suppressor in this malignancy.

Liver kinase B1 (LKB1) is a serine/threonine protein kinase ubiquitously expressed in mammalian cells. It was first identified in Peutz-Jeghers syndrome as a tumor suppressor gene. Whether endothelial LKB1 regulates angiogenesis and tumor growth is unknown. In this study, we generated endothelial cell-specific LKB1-knockout (LKB1endo-/-) mice by crossbreeding vascular endothelial-cadherin-Cre mice with LKB1flox/flox mice. Vascular endothelial growth factor (VEGF) level was highly co-stained in endothelial cells but not in macrophages in LKB1endo-/- mice. Consistently, LKB1endo-/- mouse tissues including the lung, skin, kidney and liver showed increased vascular permeability. Tumors implanted in LKB1endo-/- mice but not macrophage-specific LKB1-knockout mice grew faster and showed enhanced vascular permeability and increased angiogenesis as compared with those implanted in wild-type mice. Injection of VEGF-neutralizing antibody but not the isotype-matched control antibody decreased endothelial-cell angiogenesis and tumor growth in vivo. Furthermore, LKB1 deletion enhanced mouse retinal and cell angiogenesis, and knockdown of VEGF by small-interfering RNA decreased endothelial cell proliferation and migration. Re-expression of LKB1 or knockdown of VEGF receptor 2 decreased the overproliferation and -migration observed in LKB1endo-/- cells. Mechanistically, LKB1 could bind to the VEGF transcription factor, specificity protein 1 (Sp1), which then inhibited the binding of Sp1 to the VEGF promoter to reduce VEGF expression. Endothelial LKB1 may regulate endothelial angiogenesis and tumor growth by modulating Sp1-mediated VEGF expression.

The AMP-activated protein kinase (AMPK) acts as a cellular energy sensor. Once switched on by increases in cellular AMP : ATP ratios, it acts to restore energy homeostasis by switching on catabolic pathways while switching off cell growth and proliferation. The canonical AMP-dependent mechanism of activation requires the upstream kinase LKB1, which was identified genetically to be a tumour suppressor. AMPK can also be switched on by increases in intracellular Ca 2+ , by glucose starvation and by DNA damage via non-canonical, AMP-independent pathways. Genetic studies of the role of AMPK in mouse cancer suggest that, before disease arises, AMPK acts as a tumour suppressor that protects against cancer, with this protection being further enhanced by AMPK activators such as the biguanide phenformin. However, once cancer has occurred, AMPK switches to being a tumour promoter instead, enhancing cancer cell survival by protecting against metabolic, oxidative and genotoxic stresses. Studies of genetic changes in human cancer also suggest diverging roles for genes encoding subunit isoforms, with some being frequently amplified, while others are mutated.

Entender los mecanismos moleculares relacionados con la melanomagénesis es clave a la hora de encontrar nuevas estrategias preventivas y terapéuticas contra esta letal enfermedad. La epidemiología indica que hay factores genéticos y medioambientales asociados con el desarrollo y la progresión del melanoma. En este estudio, hemos generado el modelo animal B/L/UV que nos permite estudiar la cooperación entre el oncogén, BrafV600E, el factor medioambiental, de la radiación ultravioleta (UVR) y la pérdida del supresor tumoral, Lkb1. En resumen, el modelo animal B/L/UV presentado aquí es un potente sistema para estudiar la patogénesis del melanoma. Este modelo animal demuestra el importante rol de la UVR en la prevención de la senescencia inducida por BrafV600E, un requisito imprescindible en la progresión del melanoma en este específico contexto genético. Además, otro hecho evidente en la importancia de Lkb1 en la inducción in vivo de esta enfermedad, que tiene un efecto relevante en los tumores, afectando su iniciación, inestabilidad genética y su heterogeneidad histológica. El análisis genético de los tumores destaca el papel de las vías de señalización Rho, Netrin1 y SAPK/JNK en la melanomagénesis. Understanding the molecular mechanisms related with melanomagenesis is key in order to find new preventive and therapeutical approaches against this lethal disease. Epidemiology indicates that there are genetic- and environmental-factors associated with melanoma development and progression. In this study, we have generated the B/L/UV animal model that allows us to study the cooperation between the oncogene, BrafV600E, the environmental insult, ultraviolet radiation (UVR), and the loss of a tumor suppressor, Lkb1. In summary, the B/L/UV animal model presented here is a strong system to study the melanoma pathogenesis. This animal model demonstrates the important role of UVR in preventing the BrafV600E-induced senescence, a requisite in melanoma progression in this specific mutational context. Moreover, a patent fact is the importance of Lkb1 in in vivo induction of this disease, which has a relevant effect in the tumors, affecting its onset, its genetic instability, and its histological heterogeneity. The genetic analysis of tumors highlights the important role of Rho-, Netrin1- and SAPK/JNK-signaling pathway in melanomagenesis.

Vascular calcification is a common pathological feature of atherosclerosis, chronic kidney disease, vascular injury, and aging. Liver kinase B1 (LKB1) plays pivotal roles in cellular processes such as apoptosis, metabolism, and cell cycle regulation. In addition, growing evidence has indicated that LKB1 functions as a tumor suppressor gene. However, its role in vascular calcification has not been reported. LKB1flox/flox mice were hybridized with SM22-CreERT2 transgenic mice and adult mice received tamoxifen to obtain smooth muscle-specific LKB1-knockout (LKB1SMKO) mice. LKB1 expression was decreased under calcifying conditions, and LKB1 overexpression had a protective effect on vascular calcification. However, high mobility group box 1 (HMGB1) overexpression partially counteracted the promotion of vascular calcification induced by LKB1 overexpression. Mechanically, LKB1 could bind to HMGB1 to promote HMGB1 degradation. Furthermore, LKB1SMKO mice showed intensified vascular calcification, which was alleviated by treatment with the HMGB1 inhibitor glycyrrhizic acid. Based on our results, LKB1 may inhibit vascular calcification via inhibiting HMGB1 expression., Graphical abstract Image 1, Highlights • LKB1 expression was reduced under calcifying conditions. • LKB1 overexpression had a protective effect on vascular calcification. • Binding of LKB1 to HMGB1 promoted HMGB1 degradation. • LKB1 may inhibit vascular calcification by inhibiting HMGB1 expression.

AMP-activated protein kinase (AMPK) is a key regulator of cellular energy balance. In response to metabolic stress, it acts to redress energy imbalance through promotion of ATP-generating catabolic processes and inhibition of ATP-consuming processes, including cell growth and proliferation. While findings that AMPK was a downstream effector of the tumour suppressor LKB1 indicated that it might act to repress tumourigenesis, more recent evidence suggests that AMPK can either suppress or promote cancer, depending on the context. Prior to tumourigenesis AMPK may indeed restrain aberrant growth, but once a cancer has arisen, AMPK may instead support survival of the cancer cells by adjusting their rate of growth to match their energy supply, as well as promoting genome stability. The two isoforms of the AMPK catalytic subunit may have distinct functions in human cancers, with the AMPK-α1 gene often being amplified, while the AMPK-α2 gene is more often mutated. The prevalence of metabolic disorders, such as obesity and Type 2 diabetes, has led to the development of a wide range of AMPK-activating drugs. While these might be useful as preventative therapeutics in individuals predisposed to cancer, it seems more likely that AMPK inhibitors, whose development has lagged behind that of activators, would be efficacious for the treatment of pre-existing cancers.

Liver kinase B1 (LKB1) functions as a tumor suppressor encoded by STK11, a gene that mutated in Peutz-Jeghers syndrome and in sporadic cancers. Previous studies showed that LKB1 participates in IR- and ROS-induced DNA damage response (DDR). However, the impact of LKB1 mutations on targeted cancer therapy remains unknown. Herein, we demonstrated that LKB1 formed DNA damage-induced nuclear foci and co-localized with ataxia telangiectasia mutated kinase (ATM), γ-H2AX, and breast cancer susceptibility 1 (BRCA1). ATM mediated LKB1 phosphorylation at Thr 363 following the exposure of cells to ionizing radiation (IR). LKB1 interacted with BRCA1, a downstream effector in DDR that is recruited to sites of DNA damage and functions directly in homologous recombination (HR) DNA repair. LKB1 deficient cells exhibited delayed DNA repair due to insufficient HR. Notably, LKB1 deficiency sensitized cells to poly (ADP-ribose) polymerase (PARP) inhibitors. Thus, we have demonstrated a novel function of LKB1 in DNA damage response. Cancer cells lacking LKB1 are more susceptible to DNA damage-based therapy and, in particular, to drugs that further impair DNA repair, such as PARP inhibitors.