Kinase LKB1 Research Articles

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.

Halting Mitochondria at Axonal Branch Points

A pathway involving the kinases LKB1 and NUAK1 signals mitochondria to stop at the sites of axon branching.

LKB1 Controls Human Bronchial Epithelial Morphogenesis through p114RhoGEF-Dependent RhoA Activation

LKB1 is a Ser/Thr kinase that plays an important role in controlling both energy metabolism and cell polarity in metazoan organisms. LKB1 is also a tumor suppressor, and homozygous, inactivating mutations are found in a wide range of human cancers. In lung cancer, inactivating mutations are found in 10 to 50% of cases, but the consequences of functional loss in this context are poorly understood. We report here that LKB1 is required for the maturation of apical junctions in the human bronchial epithelial cell line 16HBE14o- (16HBE). This activity is dependent on an interaction with the Rho guanine nucleotide exchange factor p114RhoGEF but is independent of LKB1 kinase activity. Together, LKB1 and p114RhoGEF control RhoA activity in these cells to promote apical junction assembly.

PAS Kinase as a Nutrient Sensor in Neuroblastoma and Hypothalamic Cells Required for the Normal Expression and Activity of Other Cellular Nutrient and Energy Sensors

PAS kinase (PASK) is a nutrient sensor that is highly conserved throughout evolution. PASK-deficient mice reveal a metabolic phenotype similar to that described in S6 kinase-1 S6K1-deficient mice that are protected against obesity. Hypothalamic metabolic sensors, such as AMP-activated protein kinase (AMPK) and the mammalian target of rapamycin (mTOR), play an important role in feeding behavior, the homeostasis of body weight, and energy balance. These sensors respond to changes in nutrient levels in the hypothalamic areas involved in feeding behavior and in neuroblastoma N2A cells, and we have recently reported that those effects are modulated by the anorexigenic peptide glucagon-like peptide-1 (GLP-1). Here, we identified PASK in both N2A cells and rat VMH and LH areas and found that its expression is regulated by glucose and GLP-1. High levels of glucose decreased Pask gene expression. Furthermore, PASK-silenced N2A cells record an impaired response by the AMPK and mTOR/S6K1 pathways to changes in glucose levels. Likewise, GLP-1 effect on the activity of AMPK, S6K1, and other intermediaries of both pathways and the regulatory role at the level of gene expression were also blocked in PASK-silenced cells. The absence of response to low glucose concentrations in PASK-silenced cells correlates with increased ATP content, low expression of mRNA coding for AMPK upstream kinase LKB1, and enhanced activation of S6K1. Our findings indicate that, at least in N2A cells, PASK is a key kinase in GLP-1 actions and exerts a coordinated response with the other metabolic sensors, suggesting that PASK might play an important role in feeding behavior.

Carving Axon Arbors to Fit: Master Directs One Kinase at a Time

Pyramidal neurons in the cortex require the master kinase LKB1 for early axon specification. Courchet etal. now uncover a later role for LKB1 and its tango with the downstream effector kinase NUAK1 in controlling terminal axonal branching through influencing mitochondrial motility in axons.

Interplay between the intracellular energy sensor AMP-activated protein kinase (AMPK) and the estrogen receptor activities in regulating rat pituitary tumor cell (GH3) growth in vitro

Estrogen receptor α has a role in regulating rat somatolactotroph tumor cell growth (GH3 cells). AMP-activated protein kinase (AMPK) is a metabolic checkpoint which is able to negatively regulate intracellular signaling downstream of growth factors receptors in conditions increasing cellular AMP levels. We have recently reported on the role of AMPK activation in affecting viability and proliferation of GH3 cells. In the present study, we investigated the interplay between ER- and AMPK-pathways. Results can be regarded as relevant to the development of novel multi-targeted pharmacological therapies against pituitary tumors. We confirmed that estradiol (E2) and the ER antagonist fulvestrant exert stimulatory and inhibitory effects, respectively on GH3 cell growth in a competitive manner. The upstream kinase LKB1 is known to phosphorylate and activate AMPK. Here we showed that neither E2 nor fulvestrant caused a downregulation of LKB1 expression and phospho-AMPK levels in GH3 cells. Actually, fulvestrant strongly reduced the phosphorylation of ACC, which is a direct target of AMPK and a known index of AMPK activity. 2-deoxyglucose, a compound reducing glucose utilization, caused an increase in AMPK activity vs baseline and was able to hinder the stimulatory effect of E2 on cell viability, confirming that the exposure of GH3 cells to estrogens does not prevent them from being responsive to the inhibitory activity of compounds activating AMPK. Finally, the AMPK activator AICAR (AMP analog) did not cause further decrease in cell viability in the course of co-treatments with fulvestrant versus fulvestrant alone, in agreement with impaired phospho-AMPK activity in the presence of the anti-estrogen.

Abstract 2157: Targeting the Maternal Embryonic Leucin zipper Kinase (MELK) in gastric cancers.

Abstract Gastric cancer (GC) is the fourth most common cancer, and the second leading cause of cancer-related death in the world. The high mortality rate of gastric cancer is due to late-stage diagnosis and unpredictable outcome with empiric treatments. There are no tools to stratify therapies for ERBB2-negative GC (90% of patients). Even in ERBB2-positive patients, much better therapies are desirable. To identify novel GC drivers and therapeutic targets, we conducted a whole-transcriptome RNA-seq analysis in Caucasian cancers and paired normal gastric tissues. We identified 879 differentially expressed genes with FDR<0.01 & fold change (FC) >2. We next compared our results with those of Cui J. et al. (Nucl. Acids Res. 2011) which identified differential expressions in 80 tumor-normal pairs of Asian GC. With an FDR <0.01 & FC>2, we found 308 genes overlapping, including 30 kinases. Among the kinases, we found consistent high expression of the Maternal Embryonic Leucine zipper Kinase (MELK), the only member of the member of the Snf1/AMPK serine/threonine kinase family not regulated by LKB1 kinase, but rather through autophosphorylation. MELK is putatively involved in mitotic progression, inhibition of mRNA splicing, cytokinesis, and apoptosis. MELK overexpression has been found in other cancers, including human glioblastoma and breast cancers where it promotes tumorigenesis and portends to worse outcome. The precise in vivo regulation, function and crystal/NMR structures of MELK are still unknown; however the evidence that three MELK substrates are critical regulators of the mitotic entry, underscores the hypothesis that MELK inhibition may offer important novel therapeutic strategy. We investigated MELK protein expression in a panel of 30 gastric cell lines, and found high levels of MELK protein in 14/30. Similar results were observed in a panel of 22 colon carcinoma lines. MELK blockade using siRNA affected cytokinesis with the formation of polynucleated cells and aberrant mitotic spindle. Ongoing in vitro and in vivo investigations include cells with MELK knockdown by shRNA as well as MELK -/-, MELK +/+ cells, and MELK point mutation cells generated by Zinc Finger Nuclease Technology, and cells carrying MELK kinase dead; and identification of novel small compounds inhibiting MELK activity. Because of the increased expression in a broad spectrum of cancer sites, and the putative role in orchestrating mitotic entry, we believe that MELK offers important targeted therapeutic windows: 1) it will enhance the efficacy of antineoplastic drugs targeting mitosis; 2) by hindering the ability of tumor initiating cells to proliferate, it will impact the repopulation phase following chemo-radiation therapy, and therefore could be incorporated into molecular targeted strategies circumventing resistance to conventional therapy in MELK positive cancers. Citation Format: Julie G. Izzo, Kevin N. Dalby, Han Liang, Hui Yao, Brian P. James, Saira Ahmed, Xiuping Liu, Chang-Gong Liu, Yon Hui Kim, Garth Powis. Targeting the Maternal Embryonic Leucin zipper Kinase (MELK) in gastric cancers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2157. doi:10.1158/1538-7445.AM2013-2157 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Abstract 5534: Metformin increases AMPKα activity by inhibition of AMPKα and cell cycle proliferation in Asian gastric cancer.

Abstract The LKB1/AMPK signaling pathway has been well elucidated and recent evidence suggests its involvement in cancer cell biology, demonstrating that the reinforcement of the tumor suppressive functions of LKB1/AMPK is a valuable therapeutic strategy for cancers. Interest in metformin as a novel anticancer agent for breast cancer and other solid tumors continue to grow, currently being investigated in several cancer types in both neoadjuvant and metastatic settings. The biological effect of metformin on cancer cells is driven by its ability to activate AMPK through upstream kinase LKB1, tumor suppressor gene in epithelial tissues. Metformin increases intracellular AMP level, which allosterically activates AMPK. We have previously identified the AMPKα as a modulator in gastric cancer (GC) and through experimental evidence. We show the impact of LKB1/AMPK modulation of HNF4α, a dramatic suppression of cancer cell growth. GC samples were collected and sequenced on SOLiD v 3.0 for both WT-seq and small RNA-seq. Computational analysis showed that 356 out of 18,890 genes were identified as GC related differentially expressed genes in the five-group comparison (normal, tumor stage I, II, III or IV). 28 genes were identified as stage-specific differentially expressed genes, and 13 out of the 28 genes were within the network between HNF4α and HNF1α. In order to test the anti-proliferation activity of metformin in GC cell lines associated with activation of PRKAA1, PRKAA2 and LKB1, and by HNF4α suppression, 4 GC cell lines (NCI-N87, AGS, HS 746T and MKN 45) were treated with metformin. Both PRKAA1/2 showed increased gene expression level when the cells were treated with 10mM of metformin. As for STK11 (LKB1 gene) and HNF4A gene expression level, LKB1 increased and HNF4A decreased with metformin treatment on all four cell lines. Metformin treated NCI-N87 and AGS show that it is involved in cell cycle arrest. Western-blot analysis shows, decreased protein expression of Cyclin A/B and D1 on metformin treated. Lastly, NCI-N87 xenograft study show metformin treated suppression of tumor progression compared to non-treated mouse. During the 28 day treatment of metformin, PRKAA1 and PRKAA2 expression level increased compared to the untreated with. Consistent with in vitro assay, LKB1 level was elevated in the metformin treated tumor compared to the non-treated, and HNF4α level decreased over time in the metformin treated tumor. Study shows that AMPK is a strong therapeutic tumor suppressor target and that metformin is a potential drug for Asian early gastric cancer patient. In our research in progress, we observe potential relationships between the Wnt pathway and AMPKα in light of WNT druggability with metformin. In conclusion LKB1/AMPK by HNF4α inhibition suggests metformin could be a candidate for gastric cancer treatment, probably in combination with conventional chemotherapy and/or as a maintenance therapy. Citation Format: Hae Rim Jung, Hae Ryung Chang, Hye-Hyun Seo, Robert Lemos, Hee Seo Park, Han Liang, Garth Powis, Yon Hui Kim. Metformin increases AMPKα activity by inhibition of AMPKα and cell cycle proliferation in Asian gastric cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5534. doi:10.1158/1538-7445.AM2013-5534

Abstract 1277: Utilizing uterine cancer models to study malignant transformation driven by Lkb1 loss.

Abstract The serine-threonine kinase LKB1 is an upstream component of the AMPK-mTOR pathway and regulates cell survival and growth. Inherited germline mutations in LKB1 predispose women to endocervical malignancies whereas somatic mutations in LKB1 promote cervical cancer progression. Our lab has shown that tissue specific knockout of Lkb1 in the endometrial epithelium of mice facilitates growth of highly invasive, lethal tumors that are well differentiated and retain structural polarity. Additionally, stable shRNA mediated knockdown of LKB1 in human immortalized endocervical cells drives anchorage-independent growth. To investigate potential pathways involved in LKB1 mediated oncogenic transformation, we have employed a microarray based approach comparing hits from human immortalized endocervical cell lines and mouse tissue to identify specific genes that are differentially regulated when LKB1 function is absent. Preliminary data suggests that pro-angiogenic and inflammatory mediators are upregulated in LKB1 deficient cells. We are currently assessing the overall contribution of these factors to growth and invasiveness in our model systems and the mechanisms by which Lkb1 loss facilitates their activation. These pathways can be potential therapeutic targets in other cancers characterized by Lkb1 loss of function mutations. Citation Format: Christopher G. Peña, Yuji Nakada, Diego Castrillon. Utilizing uterine cancer models to study malignant transformation driven by Lkb1 loss. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1277. doi:10.1158/1538-7445.AM2013-1277

LKB1 Mediates the Development of Conventional and Innate T Cells via AMP-Dependent Kinase Autonomous Pathways

The present study has examined the role of the serine/threonine kinase LKB1 in the survival and differentiation of CD4/8 double positive thymocytes. LKB1-null DPs can respond to signals from the mature α/β T-cell-antigen receptor and initiate positive selection. However, in the absence of LKB1, thymocytes fail to mature to conventional single positive cells causing severe lymphopenia in the peripheral lymphoid tissues. LKB1 thus appears to be dispensable for positive selection but important for the maturation of positively selected thymocytes. LKB1 also strikingly prevented the development of invariant Vα14 NKT cells and innate TCR αβ gut lymphocytes. Previous studies with gain of function mutants have suggested that the role of LKB1 in T cell development is mediated by its substrate the AMP-activated protein kinase (AMPK). The present study now analyses the impact of AMPK deletion in DP thymocytes and shows that the role of LKB1 during the development of both conventional and innate T cells is mediated by AMPK-independent pathways.

α-SNAP inhibits AMPK signaling to reduce mitochondrial biogenesis and dephosphorylates Thr172 in AMPKα in vitro

The AMP-activated protein kinase (AMPK) regulates metabolism in normal and pathological conditions and responds to nutrients, hormones, anti-diabetic drugs and physical exercise. AMPK is activated by kinase LKB1 and inactivated by phosphatases whose identities remain uncertain. Here we show that AMPK associates with α-SNAP, an adapter that enables disassembly of cis-SNARE complexes formed during membrane fusion. Knockdown of α-SNAP activates AMPK to phosphorylate its endogenous substrates ACC and Raptor and provokes mitochondrial biogenesis. AMPK phosphorylation is rescued from α-SNAP RNAi by LKB1 knockdown or expression of wild type but not mutated α-SNAP. Recombinant wild type but not mutated α-SNAP dephosphorylates pThr172 in AMPKα in vitro. Over-expression of wild-type but not mutated α-SNAP prevents AMPK activation in cells treated with agents to elevate AMP concentration. The mouse α-SNAP mutant hyh (hydrocephalus with hop gait) shows enhanced binding and inhibition of AMPK. By negatively controlling AMPK, α-SNAP therefore potentially coordinates membrane trafficking and metabolism.

Caenorhabditis elegans PIG-1/MELK Acts in a Conserved PAR-4/LKB1 Polarity Pathway to Promote Asymmetric Neuroblast Divisions

Asymmetric cell divisions produce daughter cells with distinct sizes and fates, a process important for generating cell diversity during development. Many Caenorhabditis elegans neuroblasts, including the posterior daughter of the Q cell (Q.p), divide to produce a larger neuron or neuronal precursor and a smaller cell that dies. These size and fate asymmetries require the gene pig-1, which encodes a protein orthologous to vertebrate MELK and belongs to the AMPK-related family of kinases. Members of this family can be phosphorylated and activated by the tumor suppressor kinase LKB1, a conserved polarity regulator of epithelial cells and neurons. In this study, we present evidence that the C. elegans orthologs of LKB1 (PAR-4) and its partners STRAD (STRD-1) and MO25 (MOP-25.2) regulate the asymmetry of the Q.p neuroblast division. We show that PAR-4 and STRD-1 act in the Q lineage and function genetically in the same pathway as PIG-1. A conserved threonine residue (T169) in the PIG-1 activation loop is essential for PIG-1 activity, consistent with the model that PAR-4 (or another PAR-4-regulated kinase) phosphorylates and activates PIG-1. We also demonstrate that PIG-1 localizes to centrosomes during cell divisions of the Q lineage, but this localization does not depend on T169 or PAR-4. We propose that a PAR-4-STRD-1 complex stimulates PIG-1 kinase activity to promote asymmetric neuroblast divisions and the generation of daughter cells with distinct fates. Changes in cell fate may underlie many of the abnormal behaviors exhibited by cells after loss of PAR-4 or LKB1.

The Tumor Suppressor Kinase LKB1 Activates the Downstream Kinases SIK2 and SIK3 to Stimulate Nuclear Export of Class IIa Histone Deacetylases

Histone deacetylases 4 (HDAC4), -5, -7, and -9 form class IIa within the HDAC superfamily and regulate diverse physiological and pathological cellular programs. With conserved motifs for phosphorylation-dependent 14-3-3 binding, these deacetylases serve as novel signal transducers that are able to modulate histone acetylation and gene expression in response to extracellular cues. Here, we report that in a PKA-sensitive manner the tumor suppressor kinase LKB1 acts through salt-inducible kinase 2 (SIK2) and SIK3 to promote nucleocytoplasmic trafficking of class IIa HDACs. Both SIK2 and SIK3 phosphorylate the deacetylases at the conserved motifs and stimulate 14-3-3 binding. SIK2 activates MEF2-dependent transcription and relieves repression of myogenesis by the deacetylases. Distinct from SIK2, SIK3 induces nuclear export of the deacetylases independent of kinase activity and 14-3-3 binding. These findings highlight the difference among members of the SIK family and indicate that LKB1-dependent SIK activation constitutes an important signaling module upstream from class IIa deacetylases for regulating cellular programs controlled by MEF2 and other transcription factors.

Loss of lkb1 Expression Reduces the Latency of ErbB2-Mediated Mammary Gland Tumorigenesis, Promoting Changes in Metabolic Pathways

The tumor suppressor kinase LKB1 is mutated in a broad range of cancers however, the role of LKB1 mammary gland tumorigenesis is not fully understood. Evaluation of human breast cancer tissue microarrays, indicate that 31% of HER2 positive samples lacked LKB1 expression. To expand on these observations, we crossed STK11fl/fl mice with mice genetically engineered to express activated Neu/HER2-MMTV-Cre (NIC) under the endogenous Erbb2 promoter, to generate STK11−/−/NIC mice. In these mice, the loss of lkb1 expression reduced the latency of ErbB2-mediated tumorigenesis compared to the latency of tumorigenesis in NIC mice alone. Analysis of STK11−/−/NIC mammary tumors revealed hyperactivation of mammalian target of rapamycin (mTOR) through both mTORC1 and mTORC2 pathways as determined by the phosphorylation status of ribosomal protein S6 and AKT. Furthermore, STK11−/−/NIC mammary tumors had elevated ATP levels along with changes in metabolic enzymes and metabolites. The treatment of primary mammary tumor cells with specific mTOR inhibitors AZD8055 and Torin1, that target both mTOR complexes, attenuated mTOR activity and decreased expression of glycolytic enzymes. Our findings underscore the existence of a molecular interplay between LKB1-AMPK-mTORC1 and ErbB2-AKT-mTORC2 pathways with mTOR at its epicenter, suggestive that loss of LKB1 expression may serve as a marker for hyperactivated mTOR in HER2 positive breast cancer and warranting further investigation into therapeutics that target LKB1-AMPK-mTOR and glycolytic pathways.

Abstract A59: Dixdc1 links the LKB1 tumor suppressor to Snail, noncanonical Wnt signaling, and metastasis

Abstract The kinase LKB1 is a tumor suppressor mutated in familial and sporadic human cancers, whose loss is uniquely coupled with metastatic progression. Although LKB1 controls growth and metabolism through phosphorylation of AMPK, it also phosphorylates the related MARK/ Par1 kinases, which play conserved roles in cell polarity and cytoskeletal dynamics. In a screen for novel targets of LKB1-dependent signals, we identified the scaffold protein DIXDC1 as a substrate of MARK1 which controls cell migration and invasion. DIXDC1 localizes to focal adhesions, and like LKB1, its depletion results in hyperactivation of focal adhesion kinase and subsequent increases in the EMT-transcription factors Snail and Slug. Depletion of LKB1 or DIXDC1 results in Snail-dependent induction of the noncanonical Wnt5a and Wnt5b, which are required for the increased invasive behavior of these cells. Studies on a panel of murine lung adenocarcinoma cell lines from defined stages of metastatic progression reveals that DIXDC1 depletion was sufficient to promote metastasis, while Wnt5a and Wnt5b are required for metastatic behavior in these cells. DIXDC1 is frequently downregulated in human cancers, and inversely correlates with Snail and Wnt5 family members expression. This study reveals the first downstream effector of LKB1 in tumor suppression that is independent of AMPK and identifies a novel signaling pathway that provides a molecular explanation for how LKB1 controls EMT and metastasis. Citation Format: Jonathan M. Goodwin, Robert U. Svensson, David B. Shackelford, Monte M. Winslow, Reuben J. Shaw. Dixdc1 links the LKB1 tumor suppressor to Snail, noncanonical Wnt signaling, and metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A59.

Lkb1 regulates organogenesis and early oncogenesis along AMPK-dependent and -independent pathways

The tumor suppressor Lkb1/STK11/Par-4 is a key regulator of cellular energy, proliferation, and polarity, yet its mechanisms of action remain poorly defined. We generated mice harboring a mutant Lkb1 knockin allele that allows for rapid inhibition of Lkb1 kinase. Culturing embryonic tissues, we show that acute loss of kinase activity perturbs epithelial morphogenesis without affecting cell polarity. In pancreas, cystic structures developed rapidly after Lkb1 inhibition. In lung, inhibition resulted in cell-autonomous branching defects. Although the lung phenotype was rescued by an activator of the Lkb1 target adenosine monophosphate-activated kinase (AMPK), pancreatic cyst development was independent of AMPK signaling. Remarkably, the pancreatic phenotype evolved to resemble precancerous lesions, demonstrating that loss of Lkb1 was sufficient to drive the initial steps of carcinogenesis ex vivo. A similar phenotype was induced by expression of mutant K-Ras with p16/p19 deletion. Combining culture of embryonic tissues with genetic manipulation and chemical genetics thus provides a powerful approach to unraveling developmental programs and understanding cancer initiation.

Protective effect of ganodermanondiol isolated from the Lingzhi mushroom against tert-butyl hydroperoxide-induced hepatotoxicity through Nrf2-mediated antioxidant enzymes

Ganodermanondiol, a biologically active compound, was isolated from the Lingzhi mushroom (Ganoderma lucidum). The present study examined the protective effects of ganodermanondiol against tert-butyl hydroperoxide (t-BHP)-induced hepatotoxicity. Ganodermanondiol protected human liver-derived HepG2 cells through nuclear factor-E2-related factor 2 (Nrf2) pathway-dependent heme oxygenase-1 expressions. Moreover, ganodermanondiol increased cellular glutathione levels and the expression of the glutamine-cysteine ligase gene in a dose-dependent manner. Furthermore, ganodermanondiol exposure enhanced the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) and its upstream kinase activators, LKB1 and Ca(2+)/calmodulin-dependent protein kinase-II (CaMKII). This study indicates that ganodermanondiol exhibits potent cytoprotective effects on t-BHP-induced hepatotoxicity in human liver-derived HepG2 cells, presumably through Nrf2-mediated antioxidant enzymes and AMPK.

LKB1-regulated adaptive mechanisms are essential for neuronal survival following mitochondrial dysfunction

Mitochondrial dysfunction plays an important role in the etiology of neurodegenerative diseases. However, the progressive nature of neuronal loss in genetic models of mitochondrial dysfunction suggests the presence of compensatory mechanisms promoting neuronal survival under these conditions. Here, we identified the energy metabolism kinase LKB1 as a key regulator of the compensatory mechanisms activated in neurons, following mitochondrial dysfunction. To accomplish this, we have created an in vivo neurodegenerative model based on the deletion of the mitochondrial protein apoptosis-inducing factor (AIF) in postmitotic neurons. Loss of mitochondrial function caused by AIF deletion induced several adaptive mechanisms, including increased glycolysis and mitochondrial biogenesis. Importantly, the activation of these adaptive mechanisms was abrogated by the deletion of one allele of LKB1, resulting in impaired neuronal survival. Because loss of mitochondrial function is a central mechanism implicated in neurodegenerative diseases, modulation of LKB1-dependent pathways may represent an important strategy to preserve neuronal survival and function.

Abstract PL03-03: The LKB1-AMPK pathway coordinates metabolic reprogramming with growth control

Abstract The serine/threonine kinase LKB1 is a tumor suppressor gene mutated in the familial cancer condition Peutz-Jeghers syndrome (PJS), as well as in 30% of sporadic non-small cell lung cancer (NSCLC). One of the critical substrates of LKB1 is the AMP-activated protein kinase (AMPK). AMPK is a highly conserved sensor of cellular energy status found in all eukaryotic cells that restores metabolic homeostasis following stress. Thus LKB1 is a unique energy-state sensitive regulator of growth and metabolic reprogramming via its effects on AMPK. Our laboratory has performed a three-pronged screen to identify novel substrates of AMPK that may mediate its effects on metabolism and growth control. These studies have led to the identification of components of the mTOR signaling pathway (raptor, TSC2), the autophagy pathway (ULK1), and transcriptional regulators of metabolism (Srebp1, HDAC4/5/7) all as direct substrates of AMPK. Collectively, these studies uncovered novel conserved effectors of LKB1 and AMPK that mediate their role as a metabolic checkpoint coordinating cell growth with energy status. The connection of AMPK to tumor suppression has also led many to examine whether compounds that normally serve to activate AMPK, may in fact exhibit anti-cancer activities. Notably, the most widely used type 2 diabetes therapeutic in the US and worldwide, metformin, is a mitochondrial OXPHOS inhibitor which activates AMPK. We have therefore examined the potential anti-cancer effects of metformin and its more potent analog phenformin, in the context of genetically engineered mouse models of lung cancer. Whereas cells containing an intact LKB1-AMPK pathway respond to this metabolic stress by undergoing growth arrest, cells lacking LKB1 are unable to sense the damage and continue to divide ultimately undergoing apoptosis. Thus we examined whether this class of compounds may show selective therapeutic efficacy in preclinical trials in Kras-dependent lung cancer mouse models with loss of LKB1 as compared to those bearing loss of p53. Citation Format: Reuben J. Shaw. The LKB1-AMPK pathway coordinates metabolic reprogramming with growth control. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr PL03-03.

Post-translational modification of serine/threonine kinase LKB1 via Adduction of the Reactive Lipid Species 4-Hydroxy-trans-2-nonenal (HNE) at lysine residue 97 directly inhibits kinase activity.

Oxidative stress is pathogenic in a variety of diseases, but the mechanism by which cellular signaling is affected by oxidative species has yet to be fully characterized. Lipid peroxidation, a secondary process that occurs during instances of free radical production, may play an important role in modulating cellular signaling under conditions of oxidative stress. 4-Hydroxy-trans-2-nonenal (HNE) is an electrophilic aldehyde produced during lipid peroxidation that forms covalent adducts on proteins, altering their activity and function. One such target, LKB1, has been reported to be inhibited by HNE adduction. We tested the hypothesis that HNE inhibits LKB1 activity through adduct formation on a specific reactive residue of the protein. To elucidate the mechanism of the inhibitory effect, HEK293T cells expressing LKB1 were treated with HNE (10 μm for 1 h) and assayed for HNE-LKB1 adduct formation and changes in LKB1 kinase activity. HNE treatment resulted in the formation of HNE-LKB1 adducts and decreased LKB1 kinase activity by 31 ± 9% (S.E.) but had no effect on the association of LKB1 with its adaptor proteins sterile-20-related adaptor and mouse protein 25. Mutation of LKB1 lysine residue 97 reduced HNE adduct formation and attenuated the effect of HNE on LKB1 activity. Taken together, our results suggest that adduction of LKB1 Lys-97 mediates the inhibitory effect of HNE.