Tumor Suppressor Liver Kinase B1 Research Articles

LKB1 prevents ILC2 exhaustion to enhance antitumor immunity

Group 2 innate lymphoid cells (ILC2s) play crucial roles in mediating allergic inflammation. Recent studies also indicate their involvement in regulating tumor immunity. The tumor suppressor liver kinase B1 (LKB1) inactivating mutations are associated with a variety of human cancers; however, the role of LKB1 in ILC2 function and ILC2-mediated tumor immunity remains unknown. Here, we show that ablation of LKB1 in ILC2s results in an exhausted-like phenotype, which promotes the development of lung melanoma metastasis. Mechanistically, LKB1 deficiency leads to a marked increase in the expression of programmed cell death protein-1 (PD-1) in ILC2s through the activation of the nuclear factor of activated Tcell pathway. Blockade of PD-1 can restore the effector functions of LKB1-deficient ILC2s, leading to enhanced antitumor immune responses invivo. Together, our results reveal that LKB1 acts to restrain the exhausted state of ILC2 to maintain immune homeostasis and antitumor immunity.

Identification of a novel mitochondria-localized LKB1 variant required for the regulation of the oxidative stress response

The tumor suppressor Liver Kinase B1 (LKB1) is a multifunctional serine/threonine protein kinase that regulates cell metabolism, polarity, and growth and is associated with Peutz-Jeghers Syndrome and cancer predisposition. The LKB1 gene comprises 10 exons and 9 introns. Three spliced LKB1 variants have been documented, and they reside mainly in the cytoplasm, although two possess a nuclear-localization sequence (NLS) and are able to shuttle into the nucleus. Here, we report the identification of a fourth and novel LKB1 isoform that is, interestingly, targeted to the mitochondria. We show that this mitochondria-localized LKB1 (mLKB1) is generated from alternative splicing in the 5' region of the transcript and translated from an alternative initiation codon encoded by a previously unknown exon 1b (131bp) hidden within the long intron 1 of LKB1 gene. We found by replacing the N-terminal NLS of the canonical LKB1 isoform, the N-terminus of the alternatively spliced mLKB1 variant encodes a mitochondrial transit peptide that allows it to localize to the mitochondria. We further demonstrate that mLKB1 colocalizes histologically with mitochondria-resident ATP Synthase and NAD-dependent deacetylase sirtuin-3, mitochondrial (SIRT3) and that its expression is rapidly and transiently upregulated by oxidative stress. We conclude that this novel LKB1 isoform, mLKB1, plays a critical role in regulating mitochondrial metabolic activity and oxidative stress response.

PKCq modulates LKB1 isoforms downstream of IL-6 signaling to mediate iTreg-Th17 cell plasticity

Abstract Following activation, CD4 T cells undergo metabolic and transcriptional changes responding to external cues and differentiate into T helper (Th) cells. This differentiation process was perceived to be terminal. However, it has been shown that T cells exhibit plasticity between subsets in highly inflammatory environments. This phenomenon is prevalent in autoimmune conditions, such as colitis, in which high levels of IL-6 promote plasticity between regulatory T (Treg) cells and Th17 cells. Protein Kinase C theta (PKCq) is a serine/threonine kinase that promotes Th17 differentiation while negatively regulating Treg polarization. The tumor suppressor liver kinase B1 (LKB1) encoded by Stk11, a serine/threonine kinase, enforces energy-preserving mechanisms such as fatty acid oxidation, a major energy source for Tregs. Recent studies demonstrate loss of LKB1 disrupts Treg survival and function. Stk11 can be alternatively spliced into a short variant by the inclusion of a cryptic exon. However, specificity of LKB1 isoforms influencing Th plasticity has not been elucidated. Our study shows that the expression of the Stk11 short splice variant (Stk11S) is upregulated in Th17 compared to induced (i)Tregs. We demonstrate that, in Th17 cells heterogeneous ribonucleoprotein L-like (hnRNPLL), a splicing factor promoting cryptic exon inclusion, binds to transcripts of Stk11 and correlates with increased expression of Stk11S. We further show in iTregs exposed to IL-6, Prkcq, Stk11S, and Rorc are upregulated, suggesting iTreg-Th17 plasticity is induced in response to IL-6 resulting in Stk11 splicing downstream of PKCq. Thus, PKCq may destabilize iTreg programming through its regulation of Stk11 splicing under pro-inflammatory conditions. Supported by grants from the department of defense.

Endothelial cell-specific expression of serine/threonine kinase 11 modulates dendritic cell differentiation

In the bone marrow, classical and plasmacytoid dendritic cells (DC) develop from the macrophage-DC precursor (MDP) through a common DC precursor (CDP) step. This developmental process receives essential input from the niche in which it takes place, containing endothelial cells (EC) among other cell types. Here we show that targeted deletion of serine/threonine kinase 11 (Stk11) encoding tumor suppressor liver kinase b1 (Lkb1) in mouse ECs but not DCs, results in disrupted differentiation of MDPs to CDPs, severe reduction in mature DC numbers and spontaneous tumorigenesis. In wild type ECs, Lkb1 phosphorylates polypyrimidine tract binding protein 1 (Ptbp1) at threonine 138, which regulates stem cell factor (Scf) pre-mRNA splicing. In the absence of Lkb1, exon 6 of Scf is spliced out, leading to the loss of Scf secretion. Adeno-associated-virus-mediated delivery of genes encoding either soluble Scf or the phosphomimetic mutant Ptbp1T138E proteins rescued the defects of MDP to CDP differentiation and DC shortage in the endothelium specific Stk11 knockout mice. In summary, endothelial Stk11 expression regulates DC differentiation via modulation of Scf splicing, marking the Stk11-soluble-Scf axis as a potential cause of DC deficiency syndromes.

LKB1 deficiency upregulates RELM-\u03b1 to drive airway goblet cell metaplasia

Targeting airway goblet cell metaplasia is a novel strategy that can potentially reduce the chronic obstructive pulmonary disease (COPD) symptoms. Tumor suppressor liver kinase B1 (LKB1) is an important regulator of the proliferation and differentiation of stem/progenitor cells. In this study, we report that LKB1 expression was downregulated in the lungs of patients with COPD and in those of cigarette smoke-exposed mice. Nkx2.1Cre; Lkb1f/f mice with conditional loss of Lkb1 in mouse lung epithelium displayed airway mucus hypersecretion and pulmonary macrophage infiltration. Single-cell transcriptomic analysis of the lung tissues from Nkx2.1Cre; Lkb1f/f mice further revealed that airway goblet cell differentiation was altered in the absence of LKB1. An organoid culture study demonstrated that Lkb1 deficiency in mouse airway (club) progenitor cells promoted the expression of FIZZ1/RELM-α, which drove airway goblet cell differentiation and pulmonary macrophage recruitment. Additionally, monocyte-derived macrophages in the lungs of Nkx2.1Cre; Lkb1f/f mice exhibited an alternatively activated M2 phenotype, while expressing RELM-α, which subsequently aggravated airway goblet cell metaplasia. Our findings suggest that the LKB1-mediated crosstalk between airway progenitor cells and macrophages regulates airway goblet cell metaplasia. Moreover, our data suggest that LKB1 agonists might serve as a potential therapeutic option to treat respiratory disorders associated with goblet cell metaplasia.

Resveratrol-induced Sirt1 phosphorylation by LKB1 mediates mitochondrial metabolism

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.

Autophagy Inhibition Sensitizes Liver Kinase B1 (LKB1)‐Deficient Kras‐Driven Lung Tumors to MEK Inhibitor Trametinib

Tumor suppressor Liver Kinase B1 (LKB1) activates 5’-adenosine monophosphate protein kinase (AMPK) and maintains energy homeostasis in response to energy crises. LKB1 and KRAS are the third most frequent co-mutations detected in non-small cell lung cancer (NSCLC), causing aggressive tumor growth, metastases and resistance to standard chemotherapy as well as immunotherapy. Thus, identifying a novel treatment for patients harboring co-mutations in LKB1 and KRAS is urgently needed. Autophagy degrades and recycles the building blocks for cancer cells to survive metabolic challenges. Using genetically engineered mouse models (GEMMs), we have previously demonstrated that autophagy compensates for Lkb1 loss for Kras-driven lung tumorigenesis; loss of an autophagy-essential gene Atg7 dramatically impaired tumor initiation and tumor growth in KrasG12D/+;Lkb1-/- (KL) lung tumors. This is in sharp contrast to Lkb1 wild-type (WT) (KrasG12D/+;p53-/- (KP)) tumors that are less sensitive to autophagy gene ablation. To further value our discoveries in clinical translational ability, we treated mouse lung tumor-derived cell lines (TDCLs) with FDA-approved autophagy inhibitor hydroxychloroquine (HCQ) and found that KL TDCLs were much sensitive to HCQ-induced cell death compared with KP TDCLs. Furthermore, a combination treatment of HCQ with mitogen-activated protein kinase kinase (MAPKK/MEK) inhibitor Trametinib showed synergistic anti-proliferative effects in KL TDCLs, but not in KP TDCLs. To elucidate the underlying mechanism of increased sensitivity of KL TDCLs to Trametinib by autophagy ablation, we performed metabolomic profiling of KL TDCLs with Trametinib, HCQ, or combination treatment. We found that several glycolytic and TCA cycle intermediates, amino acids, and ATP levels were significantly upregulated upon treatment with Trametinib, which were significantly reduced by the combination treatment. In addition, we found that the combination treatment significantly reduced mitochondrial membrane potential, basal respiration as well as ATP production in KL TDCLs compared with the single agents. However, these effects were not observed in KP TDCLs. Similarly, we found that LKB1-mutant human lung cancer cell lines were much more sensitive to the combination treatment than LKB1 WT cells. Finally, we performed in vivo tumor assay using allograft mouse models and GEMMs to validate our in vitro observations. We found anti-tumor synergistic effects of the combination treatment in KL tumor growth, with no such effect in KP tumor growth. Taken together, our observations suggest that autophagy upregulation in Lkb1-deficient tumors cause resistance to Trametinib treatment by maintaining energy homeostasis for cell survival. Therefore, a combination of autophagy and MEK inhibition could be a novel therapeutic strategy to specifically treat LKB1-deficient NSCLC.

Optimization of WZ4003 as NUAK inhibitors against human colorectal cancer.

NUAK, the member of AMPK (AMP-activated protein kinase) family of protein kinases, is phosphorylated and activated by the LKB1 (liver kinase B1) tumor suppressor protein kinase. Recent work has indicated that NUAK1 is a key component of the antioxidant stress response pathway, and the inhibition of NUAK1 will suppress the growth and survival of colorectal tumors. As a promising target for anticancer drugs, few inhibitors of NUAK were developed. With this goal in mind, based on NUAK inhibitor WZ4003, a series of derivatives has been synthesized and evaluated for anticancer activity. Compound 9q, a derivative of WZ4003 by removing a methoxy group, was found to be the most potential one with stronger inhibitory against NUAK1/2 enzyme activity, tumor cell proliferation and inducing apoptosis of tumor cells. By invivo efficacy evaluations of colorectal SW480 xenografts, 9q suppresses tumor growth more effectively with an excellent safety profile invivo and is therefore seen as a suitable candidate for further investigation.

Autophagy Inhibition Sensitizes Liver Kinase B1 (LKB1)‐Deficient Kras‐Driven Lung Tumors to MEK Inhibitor Trametinib

Tumor suppressor liver kinase B1 (LKB1) activates 5’‐adenosine monophosphate protein kinase (AMPK) and plays an essential role in maintaining energy homeostasis. LKB1 mutations are the third most frequent mutation detected in non‐small cell lung cancer (NSCLC). Compared to p53 mutation, co‐mutations in LKB1 with KRAS causes aggressive tumor growth and resistance to chemotherapy as well as immunotherapy. Thus, identifying a novel target to improve LKB1‐deficient Kras‐driven NSCLC treatment is urgently needed. Our previous work demonstrates that autophagy inhibition is synthetically lethal in KrasG12D/+;Lkb1−/− (KL) mediated tumorigenesis; in contrast to intact autophagy KL lung tumors, loss of an autophagy‐essential gene Atg7 dramatically impaired both tumor initiation and tumor growth. This is in sharp contrast to wild‐type (WT) LKB1 (KrasG12D/+;p53−/−(KP)) tumors that are less sensitive to autophagy gene ablation. These observations indicate that LKB1 mutations predispose KRAS NSCLC to autophagy inhibition and that LKB1 mutations could be explored as a predictive biomarker for precision lung cancer therapy. To further value our discoveries in clinical translational ability, we treated mouse lung tumor derived cell lines (TDCLs) with FDA approved autophagy inhibitor hydroxychloroquine (HCQ) and found that the KL TDCLs were much more sensitive to HCQ‐induced cell death compared with Lkb1‐WT KP TDCLs. Furthermore, a combination treatment of HCQ with mitogen‐activated protein kinase kinase (MAPKK or MEK) inhibitor Trametinib showed synergistic anti‐proliferative effects in KL TDCLs, but not in KP TDCLs. To elucidate the underlying mechanism of increased sensitivity of KL TDCLs to Trametinib by autophagy ablation, we performed metabolomics profiling of KL TDCLs with Trametinib, HCQ alone or in combination treatment. We found that the levels of several glycolytic and TCA cycle intermediates, as well as amino acids were significantly upregulated upon treatment with Trametinib, which were significantly reduced by the combined treatment with HCQ and trametinib. Moreover, Trametinib treatment led to an increased level of ATP, which was decreased by the combination of HCQ and Trametinib. Similarly, we observed anti‐tumor synergistic effects of the combination treatment in KL allograft tumor growth. Our observations suggest that KL TDCLs resist to Trametinib treatment by upregulating autophagy to maintain energy homeostasis for cancer cell survival. Therefore, a combination therapy of autophagy and MEK inhibition could be a novel therapeutic strategy to specifically treat LKB1‐deficient NSCLC.Support or Funding InformationThis research has been supported by the research funds from NIH K22 CA190521 grant, American Cancer Society Early Investigator Pilot Award, Lung Cancer Research Foundation grant and startup funding from Rutgers Cancer Institute of New Jersey.

LKB1-AMPK Axis Negatively Regulates Ferroptosis by Inhibiting Fatty Acid Synthesis

Ferroptosis is a form of regulated necrotic cell death characterized by an iron-dependent lipid peroxidation. Although mounting evidence has demonstrated that diverse cellular metabolic processes contribute to ferroptosis, the detail mechanism by which cellular metabolism regulates ferroptosis is largely unknown. Here, we demonstrated that AMP-activated protein kinase (AMPK), a highly conversed master regulator of cellular energy homostasis is a crucial negative regulator of ferroptosis. Activation of AMPK by glucose starvation or a pharmaceutical activator suppresses ferroptotic cell death, and genetic knocking out of AMPK sensitizes cells to ferroptosis. Mechanistically, ferroptosis inducers activate AMPK which in turn phosphorylates acetyl-CoA carboxylase 1 (ACC1), leading to inhibition of fatty acid synthesis, lipid peroxide accumulation and ferroptosis. Importantly, loss of function of tumor suppressor liver kinase B1 (LKB1) sensitizes mouse embryonic fibroblasts (MEFs) and human non-small cell lung carcinoma cell lines to ferroptosis. Collectively, our results demonstrate the vital role of LKB1-AMPK-ACC1-FAS axis in dictating ferroptotic cell death, and suggest that malignant mutations in LKB1-AMPK signalling could predict the responsiveness of cancer cells to future ferroptosis-inducing therapies.

4-hydroxyphenylpyruvate dioxygenase promotes lung cancer growth via pentose phosphate pathway (PPP) flux mediated by LKB1-AMPK/HDAC10/G6PD axis

4-hydroxyphenylpyruvate dioxygenase (HPD) is an important modifier of tyrosine metabolism. However, the precise contribution of HPD to cancer metabolism and tumorigenesis remains unclear. In this study, we found that HPD was highly expressed in lung cancer and its higher expression correlated with poor prognosis in lung cancer patients. Suppressed HPD expression was sufficient to decrease oxidative pentose phosphate pathway (PPP) flux, leading to reduced RNA biosynthesis and enhanced reactive oxygen species (ROS) level, attenuated cancer cell proliferation, and tumor growth. Mechanistically, HPD not only promotes tyrosine catabolism leading to increased acetyl-CoA levels, the source of histone acetylation, but also stimulates histone deacetylase 10 (HDAC10) translocation from the nucleus into the cytoplasm mediated by tumor suppressor liver kinase B1 (LKB1)–AMP-activated protein kinase (AMPK) signaling. Both controlled histone acetylation modification, which enhanced transcription of the important PPP enzyme Glucose-6-Phosphate Dehydrogenase (G6PD). Thus, this study reveals HPD as a novel regulator of LKB1–AMPK signaling-mediated HDAC10 nuclear location, which contributes to G6PD expression in promoting tumor growth, which is a promising target for lung cancer treatment.

Autophagy modulates lipid metabolism in Liver Kinase B1 (LKB1)‐deficient Kras‐driven lung tumorigenesis

Tumor suppressor liver kinase B1 (LKB1) activates 5′‐adenosine monophosphate protein kinase (AMPK) to maintain energy homeostasis. LKB1 mutations are the third most frequent mutation detected in non‐small cell lung cancer (NSCLC). Compared to p53 mutation, mutations in LKB1 causes more aggressive tumor growth and resistance to chemotherapy as well as immunotherapy. Thus, identifying a novel target to improve LKB1‐deficient NSCLC treatment is urgently needed. Autophagy degrades and recycles macromolecules for cells to survive starvation. Using genetically engineered mouse models for NSCLC with Kras activation and LKB1 loss (KL), we found that autophagy (Atg7) deficiency prevents tumor initiation, reduced tumor growth and extended the mouse lifespan compared to mice bearing Atg7 wild‐type (WT) tumors. Most importantly, tumor growth inhibition by Atg7 deficiency was much more dramatic in KL than Kras/p53 (KP) lung tumors. Furthermore, we generated mouse tumor‐derived cell lines (TDCLs) and found that Atg7 null TDCLs were more sensitive to starvation‐induced cell death than Atg7 WT TDCLs, which can be rescued by palmitate supplementation, indicating that autophagy is required to maintain free fatty acid (FA) level for cells to survive starvation. We further performed 13C glucose tracing study and found that Atg7 deletion increased de novo FA synthesis and reduced FAs elongation. Fasnall, a selective FA synthase inhibitor, inhibited Atg7 null cell proliferation. Treatment with Orlistat, a lipase inhibitor, did not impact cell proliferation in nutrient‐rich conditions but accelerated starvation‐induced Atg7 null cell death, indicating that lipase‐medicated lipid degradation compensates for autophagy loss to provide free FAs in starvation. A carnitine palmitoyltransferase I inhibitor, Etomoxir treatment, accelerated the starvation‐induced Atg7 null cell death and reduced oxygen consumption rate (OCR) of Atg7 null cells in starvation, compared with Atg7 WT cells, indicating that autophagy‐deficient cells rely more on FA oxidation for energy production in nutrient deprivation. To probe the significance of autophagy in human lung cancer cell lines with LKB1 mutation, we knocked down ATG7 using siRNA. ATG7 knockdown (KD) inhibited clonogenic survival of LKB1 mutant cells in starvation but had no effect in LKB1 WT cells. Supplementation of glutamine or palmitate rescued the survival of LKB1 mutant cells with ATG7 KD in starvation and maintained OCR, demonstrating that KRAS‐driven human lung cancer cells with mutant LKB1 rely on autophagy to maintain mitochondrial metabolism to survive energy crises. Taken together, our results demonstrate that autophagy modulates lipid metabolism for KL cells to survive metabolic stress. Thus, a combination of autophagy inhibition with interruption of lipid metabolism could be a novel therapeutic strategy to specifically treat LKB1‐deficient NSCLC.Support or Funding InformationThis research has been supported by the research funds from K22 CA190521 (NIH) grant, American Cancer Society Early Investigator Pilot Award, Lung Cancer Research Foundation grant and startup funding from Rutgers Cancer Institute of New Jersey.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Autophagy modulates lipid metabolism to maintain metabolic flexibility for Lkb1-deficient Kras-driven lung tumorigenesis.

Loss of tumor suppressor liver kinase B1 (LKB1) promotes cancer cell proliferation but also leads to decreased metabolic plasticity in dealing with energy crises. Autophagy is a protective process involving self-cannibalization to maintain cellular energy homeostasis during nutrient deprivation. We developed a mouse model for Lkb1-deficient lung cancer with conditional deletion of essential autophagy gene Atg7 to test whether autophagy compensates for LKB1 loss for tumor cells to survive energy crises. We found that autophagy ablation was synthetically lethal during Lkb1-deficient lung tumorigenesis in both tumor initiation and tumor growth. We further found that autophagy deficiency causes defective intracellular recycling, which limits amino acids to support mitochondrial energy production in starved cancer cells and causes autophagy-deficient cells to be more dependent on fatty acid oxidation (FAO) for energy production, leading to reduced lipid reserve and energy crisis. Our findings strongly suggest that autophagy inhibition could be a strategy for treating LKB1-deficient lung tumors.

Tumor Suppressor LKB1 inhibits both the mRNA Expression and the Amplification of hTERC by the Phosphorylation of YAP in Lung Cancer Cells.

Liver kinase B1 (LKB1) is a critical tumor suppressor that is frequently mutated in human cancers. LKB1 has serine/threonine protein kinase activity, which regulates gene expression by phosphorylation of Yes-Associated protein (YAP). The phosphorylation-dependent YAP shuttling is critically important intracellular mechanism in the Hippo pathway. In our previous study, we found that the amplification of hTERC was significant higher in the bronchial brushing cells of patients with lung cancer, however, the underlying molecular mechanism is not clear. In this study, we showed that LKB1 overexpression could phosphorylate YAP and promoted its nuclear rejection. Silencing LKB1 could dephosphorylate YAP and promoted its entry into the nucleus. Here, we found that LKB1 inhibited the mRNA expression and the amplification of hTERC. YAP further up-regulated hTERC at mRNA and gene amplification levels. Therefore, we suggest that LKB1 may inhibit the expression and amplification of hTERC through the axis of LKB1-pYAP(YAP)-hTERC.

Oncogenic miR-744 promotes prostate cancer growth through direct targeting of LKB1.

Prostate cancer (PCa) is one of the most common malignancies worldwide, and with a limited number of treatments for this type of cancer, its incidence is rapidly increasing. Patients presenting with PCa are likely to experience disease recurrence, which represents a considerable clinical challenge. MicroRNAs (miRNAs) have been widely characterized as a critical regulator in a number of types of cancer, including PCa. miRNA-744 (miR-744) has been reported to be involved in cancer regulation; however, its role in PCa remained poorly understood. In a recent study, it was demonstrated that miR-744 was overexpressed in prostate tissue from PCa patients when compared with the surrounding tissues, and knockdown of miR-744 resulted in reduced cell growth. In addition, an increased population of apoptotic cells was detected upon miR-744 knockdown, together with a decrease in cell proliferation. Cell cycle analysis demonstrated a higher number of cells in the G1 phase and lower numbers in the S phase following miR-744 silencing. The levels of key proteins involved in cell cycle progression (cyclin D1, cyclin-dependent kinase 4, and proliferating cell nuclear antigen) were increased, whereas those proteins responsible for cell cycle inhibition (cyclin-dependent kinase inhibitor p21) were decreased. The tumor suppressor liver kinase B1 (LKB1) was revealed to be a potential target of miR-744, suggesting its potential mechanism of action. LKB1 levels were negatively correlated with miR-744, and LKB1 was indicated to be a direct target of miR-744. Furthermore, it was revealed that by targeting LKB1, miR-744 may regulate adenosine monophosphate-activated protein kinase (AMPK); the AMPK signaling pathway was activated by miR-744 knockdown, with subsequent inhibition of the mammalian target of rapamycin (mTOR) signaling pathway. Taken together, these results demonstrated that miR-744 promoted cell growth through the AMPK signaling pathway, by targeting LKB1. The present study revealed a novel insight into the biological function of miR-744 in PCa, and that miR-744 may be a potential therapeutic target.

Implication and Regulation of AMPK during Physiological and Pathological Myeloid Differentiation.

AMP-activated protein kinase (AMPK) is a heterotrimeric serine/threonine kinase consisting of the arrangement of various α β, and γ isoforms that are expressed differently depending on the tissue or the cell lineage. AMPK is one of the major sensors of energy status in mammalian cells and as such plays essential roles in the regulation of cellular homeostasis, metabolism, cell growth, differentiation, apoptosis, and autophagy. AMPK is activated by two upstream kinases, the tumor suppressor liver kinase B1 (LKB1) and the calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) through phosphorylation of the kinase on Thr172, leading to its activation. In addition, AMPK inhibits the mTOR pathway through phosphorylation and activation of tuberous sclerosis protein 2 (TSC2) and causes direct activation of unc-51-like autophagy activating kinase 1 (ULK1) via phosphorylation of Ser555, thus promoting initiation of autophagy. Although it is well established that AMPK can control the differentiation of different cell lineages, including hematopoietic stem cells (HSCs), progenitors, and mature hematopoietic cells, the role of AMPK regarding myeloid cell differentiation is less documented. The differentiation of monocytes into macrophages triggered by colony stimulating factor 1 (CSF-1), a process during which both caspase activation (independently of apoptosis induction) and AMPK-dependent stimulation of autophagy are necessary, is one noticeable example of the involvement of AMPK in the physiological differentiation of myeloid cells. The present review focuses on the role of AMPK in the regulation of the physiological and pathological differentiation of myeloid cells. The mechanisms of autophagy induction by AMPK will also be addressed, as autophagy has been shown to be important for differentiation of hematopoietic cells. In addition, myeloid malignancies (myeloid leukemia or dysplasia) are characterized by profound defects in the establishment of proper differentiation programs. Reinduction of a normal differentiation process in myeloid malignancies has thus emerged as a valuable and promising therapeutic strategy. As AMPK seems to exert a key role in the differentiation of myeloid cells, notably through induction of autophagy, we will also discuss the potential to target this pathway as a pro-differentiating and anti-leukemic strategy in myeloid malignancies.

Transcription factor-7–like 2 (TCF7L2) gene acts downstream of the Lkb1/Stk11 kinase to control mTOR signaling, β cell growth, and insulin secretion

Variants in the transcription factor-7–like 2 (TCF7L2/TCF4) gene, involved in Wnt signaling, are associated with type 2 diabetes. Loss of Tcf7l2 selectively from the β cell in mice has previously been shown to cause glucose intolerance and to lower β cell mass. Deletion of the tumor suppressor liver kinase B1 (LKB1/STK11) leads to β cell hyperplasia and enhanced glucose-stimulated insulin secretion, providing a convenient genetic model for increased β cell growth and function. The aim of this study was to explore the possibility that Tcf7l2 may be required for the effects of Lkb1 deletion on insulin secretion in the mouse β cell. Mice bearing floxed Lkb1 and/or Tcf7l2 alleles were bred with knockin mice bearing Cre recombinase inserted at the Ins1 locus (Ins1Cre), allowing highly β cell–selective deletion of either or both genes. Oral glucose tolerance was unchanged by the further deletion of a single Tcf7l2 allele in these cells. By contrast, mice lacking both Tcf7l2 alleles on this background showed improved oral glucose tolerance and insulin secretion in vivo and in vitro compared with mice lacking a single Tcf7l2 allele. Biallelic Tcf7l2 deletion also enhanced β cell proliferation, increased β cell mass, and caused changes in polarity as revealed by the “rosette-like” arrangement of β cells. Tcf7l2 deletion also increased signaling by mammalian target of rapamycin (mTOR), augmenting phospho-ribosomal S6 levels. We identified a novel signaling mechanism through which a modifier gene, Tcf7l2, lies on a pathway through which LKB1 acts in the β cell to restrict insulin secretion.

Tumor suppressor LKB1 inhibits the progression of gallbladder carcinoma and predicts the prognosis of patients with this malignancy.

Gallbladder carcinoma(GBC) represents the most common fatal tumors of the biliary tract. The 3-year or 5-year survival rate for patients with this disease are 30 and 5%, respectively. Liver kinase B1 (LKB1), a primary upstream kinase of adenosine monophosphate-activated protein kinase (AMPK) necessary for maintaining cell metabolism and energy homeostasis, has been found to be an important tumor suppressor gene in recent years, and its inactivation has also found to be closely associated with tumor growth, metastasis and cancer stem cell (CSC) proliferation. Nevertheless, the function of LKB1 in GBC remains unclear. In this study, we found that the expression of LKB1 in GBC tissues was decreased compared with that in non-cancerous tissues. LKB1 overexpression suppressed the proliferation, metastasis and expansion of GBC CSCs. Mechanically, LKB1 suppressed GBC cell progression via the JAK/signal transducer and activator of transcription 3 (STAT3) pathway. The use of the JAK2 inhibitor, AZD‑1480, attenuated the suppressive effects of LKB1 overexpression on the growth, metastasis and self-renewal ability of the GBC cells, which further demonstrated that JAK/STAT3 was involved in the LKB1-induced suppression of GBC cell growth, metastasis and self-renewal ability. More importantly, the decreased expression of LKB1 was a predictor of a poor prognosis of patients with GBC. On the whole, our data indicate that LKB1 inhibits GBC cell growth, metastasis and self-renewal ability by disrupting JAK/STAT3 signaling, and may thus prove to be a novel prognostic biomarker for patients with GBC.

Abstract 4969: Autophagy modulates lipid metabolism to support Liver Kinase B1 (LKB1) - deficient lung tumor growth

Abstract Autophagy degrades and recycles macromolecules for cells to survive starvation. In genetically engineered mouse models (GEMMs) for human non-small cell lung cancer (NSCLC), autophagy supports Kras-driven lung tumor growth with or without Trp53. Tumor suppressor liver kinase B1 (LKB1) activates 5'-adenosine monophosphate protein kinase (AMPK) to maintain energy homeostasis. LKB1 mutations are detected in 20-30% of NSCLC, causing aggressive tumor growth and resistance to chemotherapy. Identifying novel target to improve LKB1-deficient tumor treatment is urgently needed. Using GEMMs for NSCLC with oncogenic Kras and LKB1 loss (KL), we found that autophagy deficiency increased the survival of the mice bearing Atg7-/- tumors compared to mice bearing wild-type (WT) tumors. To determine the mechanism of autophagy in supporting LKB1-deficient Kras-driven lung tumor growth, tumor derived cell lines (TDCLs) were generated from the lung tumors of these mice. We found that Atg7 null TDCLs were more sensitive to glucose and glutamine deprivation-induced cell death compared to Atg7 WT TDCLs. Atg7 null TDCLs were also more sensitive to starvation-induced cell death than Atg7 WT TDCLs, which can be rescued by glucose, glutamine, pyruvate, lactate and nucleotide supplementation. Thus, autophagy is required for KL TDCLs to tolerate metabolic stress. Furthermore, we observed that palmitate supplementation successfully rescued starvation-induced Atg7 null TDCLs death, indicating that autophagy is required to maintain free fatty acid level for cells to survive starvation. We further performed metabolomics in TDCLs in normal and starvation conditions and found that level of amino acids and intermediates for glycolysis and TCA cycle metabolism were significantly lower in Atg7 null TDCLs compared to Atg7 WT TDCLs during HBSS starvation. Surprisingly, we observed a significant increase in the levels of biotin, a precursor for fatty acid synthesis, in Atg7 null TDCLs than that in WT TDCLs, indicating that accumulation of biotin in autophagy deficient cells might be due to defective fatty acid synthesis. In support of this, we found that the lipid droplets accumulation is significantly lower in Atg7 null KL TDCLs than that in Atg7 WT cells. To further evaluate the functional consequences of autophagy-mediated lipid metabolism in supporting KL tumor growth, we treated Atg7 null and WT cells with etomoxir, an irreversible inhibitor of carnitine palmitoyltransferase-1 (CPT-1) to inhibit fatty acid oxidation, in normal and starvation conditions. We found that Atg7 null TDCLs are much more sensitive to etomoxir treatment than WT cells. Taken together, autophagy plays a critical role in supporting lipid metabolism for cells to survive metabolic stress. Thus, a combination of autophagy inhibition with interruption of lipid metabolism could be a novel therapeutic strategy to treat LKB1-deficient lung tumor. Citation Format: Vrushank D. Bhatt, Zhixian Hu, Xiaoyang Su, Jessie Yanxiang Guo. Autophagy modulates lipid metabolism to support Liver Kinase B1 (LKB1) - deficient lung tumor growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4969.

Autophagy modulates lipid metabolism to support Liver Kinase B1 (LKB1)‐deficient lung tumor growth

Autophagy degrades and recycles macromolecules for cells to survive starvation. In genetically engineered mouse models (GEMMs) for human non‐small cell lung cancer (NSCLC), autophagy supports Kras‐driven lung tumor growth with or without Trp53. Tumor suppressor liver kinase B1 (LKB1) activates 5′‐adenosine monophosphate protein kinase (AMPK) to maintain energy homeostasis. LKB1 mutations are detected in 20–30% of NSCLC, causing aggressive tumor growth and resistance to chemotherapy. Identifying novel target to improve LKB1‐deficient tumor treatment is urgently needed. Using GEMMs for NSCLC with oncogenic Kras and LKB1 loss (KL), we found that autophagy deficiency increased the survival of the mice bearing Atg7−/− tumors compared to mice bearing wild‐type (WT) tumors. To determine the mechanism of autophagy in supporting LKB1‐deficient Kras‐driven lung tumor growth, tumor‐derived cell lines (TDCLs) were generated from the lung tumors of these mice. We found that Atg7 null TDCLs were more sensitive to glucose and glutamine deprivation‐induced cell death compared to Atg7 WT TDCLs. Atg7 null TDCLs were also more sensitive to starvation‐induced cell death than Atg7 WT TDCLs, which can be rescued by glucose, glutamine, pyruvate, lactate and nucleotide supplementation. Thus, autophagy is required for KL TDCLs to tolerate metabolic stress. Furthermore, we observed that palmitate supplementation successfully rescued starvation‐induced Atg7 null TDCLs death, indicating that autophagy is required to maintain the free fatty acid level for cells to survive starvation. We further performed metabolomics in TDCLs in normal and starvation conditions and found that level of amino acids and intermediates for glycolysis and TCA cycle metabolism were significantly lower in Atg7 null TDCLs compared to Atg7 WT TDCLs during HBSS starvation. Surprisingly, we observed a significant increase in the levels of biotin, a precursor for fatty acid synthesis, in Atg7 null TDCLs than that in WT TDCLs, indicating that accumulation of biotin in autophagy deficient cells might be due to defective fatty acid synthesis. In support of this, we found that the lipid droplets accumulation is significantly lower in Atg7 null KL TDCLs than that in Atg7 WT cells. To further evaluate the functional consequences of autophagy‐mediated lipid metabolism in supporting KL tumor growth, we treated Atg7 null and WT cells with etomoxir, an irreversible inhibitor of carnitine palmitoyltransferase‐1 (CPT‐1) to inhibit fatty acid oxidation, in normal and starvation conditions. We found that Atg7 null TDCLs are much more sensitive to etomoxir treatment than WT cells. Taken together, autophagy plays a critical role in supporting lipid metabolism for cells to survive metabolic stress. Thus, a combination of autophagy inhibition with interruption of lipid metabolism could be a novel therapeutic strategy to treat LKB1‐deficient lung tumor.Support or Funding InformationThis research has been supported by the research funds from K22 CA190521 (NIH) grant, American Cancer Society Early Investigator Pilot Award and start‐up funding from Rutgers Cancer Institute of New Jersey.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.