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.     

3D view to tumor suppression: lkb1, polarity and the arrest of oncogenic c-myc

Machiavelli wrote, in his famous political treatise Il Principe, about disrupting organization by planting seeds of dissension or by eliminating necessary support elements. Tumor cells do exactly that by disrupting the organized architecture of epithelial cell layers during progression from contained benign tumor to full-blown invasive cancer. However, it is still unclear whether tumor cells primarily break free by activating oncogenes powerful enough to cause chaos or by eliminating tumor suppressor genes guarding the order of the epithelial organization. Studies in Drosophila have exposed genes that encode key regulators of the epithelial apicobasal polarity and which, upon inactivation, cause disorganization of the epithelial layers and promote unscheduled cell proliferation. These polarity regulator/tumor suppressor proteins, which include products of neoplastic tumor suppressor genes (nTSGs), are carefully positioned in polarized epithelial cells to maintain the order of epithelial structures and to impose a restraint on cell proliferation. In this review, we have explored the presence and prevalence of somatic mutations in the human counterparts of Drosophila polarity regulator/tumor suppressor genes across the human cancers. The screen points out LKB1, which is a causal genetic lesion in Peutz-Jeghers cancer syndrome, a gene mutated in certain sporadic cancers and a human homologue of the fly polarity gene par-4. We review the evidence linking Lkb1 to polarity regulation in the scope of our recent results suggesting a coupled role for Lkb1 as an architect of organized acinar structures and a suppressor of oncogenic c-Myc. We finally present models to explain how Lkb1-dependent formation of epithelial architecture is coupled to suppression of normal and oncogene-induced proliferation.     

Loss of Lkb1 in Adult β Cells Increases β Cell Mass and Enhances Glucose Tolerance in Mice

The Lkb1 tumor suppressor exerts its biological effects through phosphorylation and consequent activation of the AMP kinase (AMPK) family. Extensive genetic and biochemical evidence supports a role for Lkb1 in cell cycle arrest, establishment of cell polarity, and cellular energy metabolism. However, the role of Lkb1 and the AMPK family in β cell function in vivo has not been established. We generated conditional knockout mice with a deletion of the Lkb1 gene in the β cell compartment of pancreatic islets; these mice display improved glucose tolerance and protection against diet-induced hyperglycemia. Lkb1^−/− β cells are hypertrophic because of elevated mTOR activity; they also proliferate more and secrete more insulin in response to glucose. These data indicate that inhibiting Lkb1 activity in β cells may facilitate β cell expansion and glucose tolerance in vivo.     

The LKB1/AMPK polarity pathway

The LKB1 tumor suppressor kinase is an activator of the AMP-activated protein kinase (AMPK), a metabolic gauge that responds to variations of cellular energetic levels by favoring catabolic versus anabolic processes. Recent studies have provided substantial evidence that LKB1 and AMPK control cell polarity from invertebrates to mammals. This review examines how the LKB1–AMPK pathway, in conjunction with other positional signals, converts energy-sensing information into the activation of Myosin II to maintain epithelial-cell architecture but also to complete cell division. This molecular link between polarity and metabolism may constitute an ancient stress-response protective mechanism that was co-opted for tumor suppression during evolution.     

LKB1 and AMPK maintain epithelial cell polarity under energetic stress

LKB1 is mutated in both familial and spontaneous tumors, and acts as a master kinase that activates the PAR-1 polarity kinase and the adenosine 5'monophosphate-activated kinase (AMPK). This has led to the hypothesis that LKB1 acts as a tumor suppressor because it is required to maintain cell polarity and growth control through PAR-1 and AMPK, respectively. However, the genetic analysis of LKB1-AMPK signaling in vertebrates has been complicated by the existence of multiple redundant AMPK subunits. We describe the identification of mutations in the single Drosophila melanogaster AMPK catalytic subunit AMPKalpha. Surprisingly, ampkalpha mutant epithelial cells lose their polarity and overproliferate under energetic stress. LKB1 is required in vivo for AMPK activation, and lkb1 mutations cause similar energetic stress-dependent phenotypes to ampkalpha mutations. Furthermore, lkb1 phenotypes are rescued by a phosphomimetic version of AMPKalpha. Thus, LKB1 signals through AMPK to coordinate epithelial polarity and proliferation with cellular energy status, and this might underlie the tumor suppressor function of LKB1.     

LKB1 and AMPK in cell polarity and division

LKB1 and AMP-activated protein kinase (AMPK) are serine-threonine kinases implicated in key cellular pathways, including polarity establishment and energy sensing, respectively. Recent in vivo analyses in Drosophila have demonstrated vital roles for both AMPK and LKB1--in part through the myosin regulatory light chain--in cell polarity and cell division. Evidence from mammalian experiments also supports non-metabolic functions for LKB1 and AMPK. This review examines unanticipated AMPK functions for initiating and maintaining cell polarity and completing normal cell division. The ability of AMPK to sense energy status might be coupled with fundamental cell biological functions.     

肝激酶B1和造血干细胞内稳态

肝激酶B1(liver kinase B1,LKB1),又名丝氨酸/苏氨酸蛋白激酶11(STK11),是一种蛋白激酶,可磷酸化AMP激活的蛋白激酶和12种其他AMPK相关激酶。LKB1还是一种肿瘤抑制蛋白,生殖细胞LKB1基因突变可引发家族性黑斑息肉综合征,而体细胞突变可造成多种肿瘤发生。小鼠Lkb1的失活可导致造血干细胞(HSC)静息的丧失、快速的HSC消耗、严重的全血细胞减少和最终的致死。Lkb1缺陷的HSC细胞显示出线粒体缺陷、膜电位减少和细胞ATP耗竭。这些结果说明LKB1是一种HSC内稳态和造血过程中的新调节因子。     

Liver kinase B1 (LKB1) in the pathogenesis of UVB-induced murine basal cell carcinoma

LKB1, a known tumor suppressor, is mutated in Peutz–Jeghers Syndrome (PJS). It is responsible for the enhanced cancer risk in patients with PJS. Dysregulation of LKB1-dependent signaling also occurs in various epithelial cancers. UVB alters the expression of LKB1, though its role in the pathogenesis of skin cancer is unknown. Here we describe upregulation of LKB1 expression in UVB-induced murine basal cell carcinoma (BCC) and in human skin tumor keratinocytes. AMP-kinase and acetyl Co-A carboxylase, the downstream LKB1 targets, are also enhanced in this neoplasm. In addition, p-Akt, a kinase which inactivates GSK3β by its phosphorylation, is enhanced in BCCs. Consistently, an accumulation of p-GSK3β and an increase in activated nuclear β-catenin are found. mTOR signaling, which is also inhibited by LKB1, remains upregulated in BCCs. However, a marked decrease in the expression of sestrins, which function as potent negative regulators of mTOR is observed. Metformin, a known chemical inducer of this pathway, was found effective in immortalized HaCaT keratinocytes, but failed to activate the LKB1-dependent signaling in human carcinoma A431 cells. Thus, our data show that the LKB1/AMPK axis fails to regulate mTOR pathway, and a complex regulatory mechanism exists for the persistent mTOR activation in murine BCCs.     

LKB1 and Notch Pathways Interact and Control Biliary Morphogenesis

Background

LKB1 is an evolutionary conserved kinase implicated in a wide range of cellular functions including inhibition of cell proliferation, regulation of cell polarity and metabolism. When Lkb1 is inactivated in the liver, glucose homeostasis is perturbed, cellular polarity is affected and cholestasis develops. Cholestasis occurs as a result from deficient bile duct development, yet how LKB1 impacts on biliary morphogenesis is unknown.

Methodology/Principal Findings

We characterized the phenotype of mice in which deletion of the Lkb1 gene has been specifically targeted to the hepatoblasts. Our results confirmed that lack of LKB1 in the liver results in bile duct paucity leading to cholestasis. Immunostaining analysis at a prenatal stage showed that LKB1 is not required for differentiation of hepatoblasts to cholangiocyte precursors but promotes maturation of the primitive ductal structures to mature bile ducts. This phenotype is similar to that obtained upon inactivation of Notch signaling in the liver. We tested the hypothesis of a functional overlap between the LKB1 and Notch pathways by gene expression profiling of livers deficient in Lkb1 or in the Notch mediator RbpJκ and identified a mutual cross-talk between LKB1 and Notch signaling. In vitro experiments confirmed that Notch activity was deficient upon LKB1 loss.

Conclusion

LKB1 and Notch share a common genetic program in the liver, and regulate bile duct morphogenesis.     

Stromal Liver Kinase B1 [STK11] Signaling Loss Induces Oviductal Adenomas and Endometrial Cancer by Activating Mammalian Target of Rapamycin Complex 1

Germline mutations of the Liver Kinase b1 (LKB1/STK11) tumor suppressor gene have been linked to Peutz-Jeghers Syndrome (PJS), an autosomal-dominant, cancer-prone disorder in which patients develop neoplasms in several organs, including the oviduct, ovary, and cervix. We have conditionally deleted Lkb1 in Müllerian duct mesenchyme-derived cells of the female reproductive tract and observed expansion of the stromal compartment and hyperplasia and/or neoplasia of adjacent epithelial cells throughout the reproductive tract with paratubal cysts and adenomyomas in oviducts and, eventually, endometrial cancer. Examination of the proliferation marker phospho-histone H3 and mammalian Target Of Rapamycin Complex 1 (mTORC1) pathway members revealed increased proliferation and mTORC1 activation in stromal cells of both the oviduct and uterus. Treatment with rapamycin, an inhibitor of mTORC1 activity, decreased tumor burden in adult Lkb1 mutant mice. Deletion of the genes for Tuberous Sclerosis 1 (Tsc1) or Tsc2, regulators of mTORC1 that are downstream of LKB1 signaling, in the oviductal and uterine stroma phenocopies some of the defects observed in Lkb1 mutant mice, confirming that dysregulated mTORC1 activation in the Lkb1-deleted stroma contributes to the phenotype. Loss of PTEN, an upstream regulator of mTORC1 signaling, along with Lkb1 deletion significantly increased tumor burden in uteri and induced tumorigenesis in the cervix and vagina. These studies show that LKB1/TSC1/TSC2/mTORC1 signaling in mesenchymal cells is important for the maintenance of epithelial integrity and suppression of carcinogenesis in adjacent epithelial cells. Because similar changes in the stromal population are also observed in human oviductal/ovarian adenoma and endometrial adenocarcinoma patients, we predict that dysregulated mTORC1 activity by upstream mechanisms similar to those described in these model systems contributes to the pathogenesis of these human diseases.     

LKB1 Suppresses p21-activated Kinase-1 (PAK1) by Phosphorylation of Thr109 in the p21-binding Domain

The serine/threonine protein kinase LKB1 is a tumor suppressor gene mutated in Peutz-Jeghers syndrome patients. The mutations are found also in several types of sporadic cancer. Although LKB1 is implicated in suppression of cell growth and metastasis, the detailed mechanisms have not yet been elucidated. In this study, we investigated the effect of LKB1 on cell motility, whose acquisition occurs in early metastasis. The knockdown of LKB1 enhanced cell migration and PAK1 activity in human colon cancer HCT116 cells, whereas forced expression of LKB1 in Lkb1-null mouse embryonic fibroblasts suppressed PAK1 activity and PAK1-mediated cell migration simultaneously. Notably, LKB1 directly phosphorylated PAK1 at Thr¹⁰⁹ in the p21-binding domain in vitro. The phosphomimetic T109E mutant showed significantly lower protein kinase activity than wild-type PAK1, suggesting that the phosphorylation at Thr¹⁰⁹ by LKB1 was responsible for suppression of PAK1. Consistently, the nonphosphorylatable T109A mutant was resistant to suppression by LKB1. Furthermore, we found that PAK1 was activated in the hepatocellular carcinomas and the precancerous liver lesions of Lkb1(+/−) mice. Taken together, these results suggest that PAK1 is a direct downstream target of LKB1 and plays an essential role in LKB1-induced suppression of cell migration.     

The LKB1 tumor suppressor controls spindle orientation and localization of activated AMPK in mitotic epithelial cells

Orientation of mitotic spindles plays an integral role in determining the relative positions of daughter cells in a tissue. LKB1 is a tumor suppressor that controls cell polarity, metabolism, and microtubule stability. Here, we show that germline LKB1 mutation in mice impairs spindle orientation in cells of the upper gastrointestinal tract and causes dramatic mislocalization of the LKB1 substrate AMPK in mitotic cells. RNAi of LKB1 causes spindle misorientation in three-dimensional MDCK cell cysts. Maintaining proper spindle orientation, possibly mediated by effects on the downstream kinase AMPK, could be an important tumor suppressor function of LKB1.     

Cryptotanshinone induces G1 cell cycle arrest and autophagic cell death by activating the AMP-activated protein kinase signal pathway in HepG2 hepatoma

AMP-activated protein kinase (AMPK) performs a pivotal function in energy homeostasis via the monitoring of intracellular energy status. Once activated under the various metabolic stress conditions, AMPK regulates a multitude of metabolic pathways to balance cellular energy. In addition, AMPK also induces cell cycle arrest or apoptosis through several tumor suppressors including LKB1, TSC2, and p53. LKB1 is a direct upstream kinase of AMPK, while TSC2 and p53 are direct substrates of AMPK. Therefore, it is expected that activators of AMPK signal pathway might be useful for treatment or prevention of cancer. In the present study, we report that cryptotanshinone, a natural compound isolated from Salvia miltiorrhiza, robustly activated AMPK signaling pathway, including LKB1, p53, TSC2, thereby leading to suppression of mTORC1 in a number of LKB1-expressing cancer cells including HepG2 human hepatoma, but not in LKB1-deficient cancer cells. Cryptotanshinone induced HepG2 cell cycle arrest at the G1 phase in an AMPK-dependent manner, and a portion of cells underwent apoptosis as a result of long-term treatment. It also induced autophagic HepG2 cell death in an AMPK-dependent manner. Cryptotanshinone significantly attenuated tumor growth in an HCT116 cancer xenograft in vivo model, with a substantial activation of AMPK signal pathways. Collectively, we demonstrate for the first time that cryptotanshinone harbors the therapeutic potential for the treatment of cancer through AMPK activation.     

AMPK/LKB1 Signaling in Epithelial Cell Polarity and Cell Division

Cells must coordinate diverse processes including cell division, cell migration, and cell polarity with the cell’s metabolic status. How single molecules coordinate these seemingly distinct cell biological events remains relatively unexplored. AMP-activated protein kinase (AMPK) sits at a unique position as a proposed energy sensor that can interface with diverse signaling molecules ranging from LKB1 to mammalian target of rapamycin (mTOR), affecting processes from ribosomal biogenesis to actin regulation. Determining biologically relevant direct kinase targets remains challenging. Alternatively, one can genetically inactivate a kinase and subsequently characterize cellular and whole animal phenotypes without the kinase’s activity. Recent genetic studies inactivating AMPK activity in Drosophila indicate unanticipated roles for AMPK as a regulator of epithelial polarity, consistent with known roles of an upstream activator, LKB1 as a PAR (partioning defective) mutant in Caenorhabditis elegans and polarity regulator. Additional genetic analyses demonstrate that both AMPK and LKB1 function are required for faithful chromosomal segregation during mitosis. At least some of these apparently divergent phenotypes may be mediated through myosin regulatory light chain, and presumably the acto-myosin complex, which can affect both polarity and cell division. Chromosomal integrity defects could also be consistent with LKB1’s role as a known human tumor suppressor gene. Elucidating the molecular players that interface with AMPK and their potential energy dependent regulation remains an important challenge to fully understand AMPK signaling.     

Targeting LKB1 signaling in cancer

The serine/threonine kinase LKB1 is a master kinase involved in cellular responses such as energy metabolism, cell polarity and cell growth. LKB1 regulates these crucial cellular responses mainly via AMPK/mTOR signaling. Germ-line mutations in LKB1 are associated with the predisposition of the Peutz–Jeghers syndrome in which patients develop gastrointestinal hamartomas and have an enormously increased risk for developing gastrointestinal, breast and gynecological cancers. In addition, somatic inactivation of LKB1 has been associated with sporadic cancers such as lung cancer. The exact mechanisms of LKB1-mediated tumor suppression remain so far unidentified; however, the inability to activate AMPK and the resulting mTOR hyperactivation has been detected in PJS-associated lesions. Therefore, targeting LKB1 in cancer is now mainly focusing on the activation of AMPK and inactivation of mTOR. Preclinical in vitro and in vivo studies show encouraging results regarding these approaches, which have even progressed to the initiation of a few clinical trials. In this review, we describe the functions, regulation and downstream signaling of LKB1, and its role in hereditary and sporadic cancers. In addition, we provide an overview of several AMPK activators, mTOR inhibitors and additional mechanisms to target LKB1 signaling, and describe the effect of these compounds on cancer cells. Overall, we will explain the current strategies attempting to find a way of treating LKB1-associated cancer.     

LKB1 is required for hepatic bile acid transport and canalicular membrane integrity in mice

LKB1 is a 'master' protein kinase implicated in the regulation of metabolism, cell proliferation, cell polarity and tumorigenesis. However, the long-term role of LKB1 in hepatic function is unknown. In the present study, it is shown that hepatic LKB1 plays a key role in liver cellular architecture and metabolism. We report that liver-specific deletion of LKB1 in mice leads to defective canaliculi and bile duct formation, causing impaired bile acid clearance and subsequent accumulation of bile acids in serum and liver. Concomitant with this, it was found that the majority of BSEP (bile salt export pump) was retained in intracellular pools rather than localized to the canalicular membrane in hepatocytes from LLKB1KO (liver-specific Lkb1-knockout) mice. Together, these changes resulted in toxic accumulation of bile salts, reduced liver function and failure to thrive. Additionally, circulating LDL (low-density lipoprotein)-cholesterol and non-esterified cholesterol levels were increased in LLKB1KO mice with an associated alteration in red blood cell morphology and development of hyperbilirubinaemia. These results indicate that LKB1 plays a critical role in bile acid homoeostasis and that lack of LKB1 in the liver results in cholestasis. These findings indicate a novel key role for LKB1 in the development of hepatic morphology and membrane targeting of canalicular proteins.     

LKB1 Deficiency in Tie2-Cre-expressing Cells Impairs Ischemia-induced Angiogenesis

LKB1 is a tumor suppressor protein whose loss leads to HIF1α-mediated activation of a proangiogenic program in intestinal polyps. LKB1 is also protein kinase regulator of AMP-activated protein kinase (AMPK) signaling, which is essential for endothelial cell responses to tissue ischemia. To discern whether LKB1 signaling is either pro- or antiangiogenic, we investigated ischemia-induced revascularization in mice that were deficient for LKB1 in Tie2-Cre-expressing cells. Whereas homozygous deletion of LKB1 led to embryonic lethality, heterozygous LKB1-knock-out (KO) (Lkb1^flox/+;Tie2^Tg/+) mice were viable. Unchallenged heterozygous LKB1-KO mice displayed normal capillary density, but the revascularization of hind limb following ischemic surgery was significantly impaired as evaluated by laser Doppler flow and capillary density measurements. Reduction of LKB1 in cultured endothelial cells, using either small interfering RNA or an adenovirus expressing nonfunctional kinase-dead LKB1 protein, attenuated endothelial proliferation, migration, and differentiation into network structures on Matrigel that was accompanied by diminished AMPK phosphorylation at Thr-172. Conversely, adenovirus-mediated LKB1 overexpression (Ad-LKB1) augmented network structure formation, and this was associated with elevated AMPK phosphorylation. The augmented differentiation of endothelial cells into network structures induced by Ad-LKB1 was abrogated by the co-transduction of a dominant negative mutant of AMPK. These observations suggest that the LKB1-AMPK signaling axis in endothelial cells is a positive regulator of the revascularization response to tissue ischemia.     

Opinion: alternative views of AMP-activated protein kinase

Genes most closely related to adenosine monophosphate (AMP)-activated protein kinase, including SAD kinases and Par-1 regulate cell polarity, although AMP-activated protein kinase (AMPK) modulates cellular energy status. LKB1 (Par-4) is required for normal activation of AMPK in the liver and also regulates cell polarity. AMPK is proposed to inhibit energy consuming activity while initiating energy producing activity during energy limitation. Demonstration that metformin, a common drug for Type 2 diabetes, requires LKB1 for full therapeutic benefit has increased interest in AMPK signaling. Despite the potential importance of AMPK signaling for diabetes, metabolic syndrome and even cancer, the developmental processes regulated by AMPK in genetically mutant animals require further elucidation. Mouse conditional null mutants for AMPK activity will allow genetic elucidation of AMPK function in vivo. This perspective focuses on sequence and structural moieties of AMPK and genetic analysis of AMPK mutations. Interestingly, the predicted protein structure of the carboxy-terminus of AMPKα resembles the carboxy-terminal KA-1 domain of MARK3, a Par-1 orthologue.     

AMPK links energy status to cell structure and mitosis

AMP-activated protein kinase (AMPK) is known as an important cellular energy sensor, but its in vivo role has not been fully understood. Recent studies provided surprising results that AMPK regulates cell polarity and mitosis under the control of tumour suppressor LKB1. Moreover, these newly found in vivo functions of AMPK are regulated by energy status in a cell autonomous manner. These findings provide novel insights into the physiological function of AMPK and the treatment of AMPK-related diseases such as cancer and diabetes.