Molecular mechanisms of tumor suppression by LKB1

The LKB1 tumor suppressor gene is frequently mutated in sporadic lung adenocarcinomas and cervical cancers and germline mutations are causative for Peutz-Jeghers syndrome characterized by gastrointestinal polyposis. The intracellular LKB1 kinase is implicated in regulating polarity, metabolism, cell differentiation, and proliferation – all functions potentially contributing to tumor suppression. LKB1 acts as an activating kinase of at least 14 kinases mediating LKB1 functions in a complex signaling network with partial overlaps. Regulation of the LKB1 signaling network is highly context dependent, and spatially organized in various cellular compartments. Also the mechanisms by which LKB1 activity suppresses tumorigenesis is context dependent, where recent observations are providing hints on the molecular mechanisms involved.     

Regulation of PCTAIRE1 protein stability by AKT1, LKB1 and BRCA1

PCTAIRE1, also known as CDK16, is a cyclin-dependent kinase that is regulated by cyclin Y. It is a member of the serine-threonine family of kinases and its functions have primarily been implicated in cellular processes like vesicular transport, neuronal growth and development, myogenesis, spermatogenesis and cell proliferation. However, as extensive studies on PCTAIRE1 have not yet been conducted, the signaling pathways for this kinase involved in governing many cellular processes are yet to be elucidated in detail. Here, we report the association of PCTAIRE1 with important cellular proteins involved in major cell signaling pathways, especially cell proliferation. In particular, here we show that PCTAIRE1 interacts with AKT1, a key player of the PI3K signaling pathway that is responsible for promoting cell survival and proliferation. Our studies show that PCTAIRE1 is a substrate of AKT1 that gets stabilized by it. Further, we show that PCTAIRE1 also interacts with and is degraded by LKB1, a kinase that is known to suppress cellular proliferation and also regulate cellular energy metabolism. Moreover, our results show that PCTAIRE1 is also degraded by BRCA1, a well-known tumor suppressor. Together, our studies highlight the regulation of PCTAIRE1 by key players of the major cell signaling pathways involved in regulating cell proliferation, and therefore, provide crucial links that could be explored further to elucidate the mechanistic role of PCTAIRE1 in cell proliferation and tumorigenesis.     

LKB1 in lung cancerigenesis: a serine/threonine kinase as tumor suppressor

Lung cancer is featured with high mortality, with a 15% five-year survival rate worldwide. Genetic alterations, such as loss of function of tumor suppressor genes, frequently contribute to lung cancer initiation, progression and metastasis. Liver kinase B1 (LKB1), as a serine/threonine kinase and tumor suppressor, is frequently mutated and inactivated in non-small cell lung cancer (NSCLC). Recent studies have provided strong evidences that LKB1 loss promotes lung cancerigenesis process, especially lung cancer progression and metastasis. This review will summarize recent progress on how LKB1 modulates the process of lung cancerigenesis, emphasizing on LKB1 downstream signaling pathways and biological functions. We will further discuss the potential development of prognostic biomarkers or therapeutic targets in lung cancer clinic based on the molecular alteration associated with deregulated LKB1 signaling.     

LKB1及其生物学功能

LKB1基因是一种保守的抑癌基因,其编码产物LKB1即丝氨酸-苏氨酸激酶11(serine/threonine kinase,STK11)。LKB1与细胞极性调节、男性精子形成、肿瘤及细胞代谢等方面有关。本文阐述了近年来LKB1的最新研究进展。     

LKB1 tumor suppressor protein: PARtaker in cell polarity

The LKB1 (also called serine/threonine kinase 11) tumor suppressor gene was cloned in 1998 by linkage analysis of Peutz-Jeghers cancer syndrome patients. Mammalian LKB1 has been implicated as a regulator of multiple biological processes and signaling pathways, including the control of cell-cycle arrest, p53-mediated apoptosis, Wnt signaling, transforming growth factor (TGF)-beta signaling, ras-induced cell transformation, and energy metabolism. The Caenorhabditis elegans and Drosophila melanogaster LKB1 homologs, termed PAR4 and dLKB1, respectively, regulate cell polarity. Recently, mammalian LKB1 was found to be active only in a complex with two other proteins--STRAD and MO25--and to induce complete polarization of intestinal epithelial cells in a cell-autonomous fashion. In this article, we summarize the findings regarding LKB1 over the past six years. In addition, we discuss LKB1 in polarity in the context of both the other PAR proteins and its tumor suppressive activities.     

High-level expression of functional tumor suppressor LKB1 in Escherichia coli

The human LKB 1 tumor suppressor has been implicated as an important regulator of many cellular processes and signaling pathways, indicating that it could be a good candidate for anticancer drugs. The failure of its obtain high-level expression has been a major obstacle to study its protein structure and function in vitro. Here, we describe the high-level expression of human LKB 1 in Escherichia coli and show its kinase activity and anticancer effects on a tumor cell line. The gene encoding LKB 1 was optimized by replacing rare codons with codons frequently used in E. coli and synthesized with overlapping primers. The recombinant His-LKB 1 was expressed in hosts BL21（DE3） （BL） and Rosetta-gami（DE3）pLysS （RG）. His- LKB 1 from BL was present mainly as inclusion body. The soluble His-LKB 1 from RG accounted for 34. 1% of total proteins and the yield of purified His-LKB 1 was approximately 92 μg/ml. Purified His-LKB 1 protein from both hosts was functionally active, as shown by reversible autophosphorylation and kinase activity in the absence of any other associated kinase. The growth inhibitory ratio of the purified BL-derived and RG-derived His-LKB 1 on hepatic carcinoma SMMC-7721 cells was 24.97% and 45.68%, respectively, and both could produce significant cell-cycle arrest.     

Insulin-Like Growth Factor-1 Signaling Regulates miRNA Expression in MCF-7 Breast Cancer Cell Line

In breast carcinomas, increased levels of insulin-like growth factor 1 (IGF-1) can act as a mitogen to augment tumorigenesis through the regulation of MAPK and AKT signaling pathways. Signaling through these two pathways allows IGF-1 to employ mechanisms that favor proliferation and cellular survival. Here we demonstrate a subset of previously described tumor suppressor and oncogenic microRNAs (miRNAs) that are under the direct regulation of IGF-1 signaling. Additionally, we show that the selective inhibition of either the MAPK or AKT pathways prior to IGF-1 stimulation prevents the expression of previously described tumor suppressor miRNAs that are family and cluster specific. Here we have defined, for the first time, specific miRNAs under the direct regulation of IGF-1 signaling in the estrogen receptor positive MCF-7 breast cancer cell line and demonstrate kinase signaling as a modulator of expression for a small subset of microRNAs. Taken together, these data give new insights into mechanisms governing IGF-1 signaling in breast cancer.     

Loss of TRB3 Alters Dynamics of MLK3-JNK Signaling and Inhibits Cytokine-activated Pancreatic Beta Cell Death

Disabling cellular defense mechanisms is essential for induction of apoptosis. We have previously shown that cytokine-mediated activation of the MAP3K MLK3 stabilizes TRB3 protein levels to inhibit AKT and compromise beta cell survival. Here, we show that genetic deletion of TRB3 results in basal activation of AKT, preserves mitochondrial integrity, and confers resistance against cytokine-induced pancreatic beta cell death. Mechanistically, we find that TRB3 stabilizes MLK3, most likely by suppressing AKT-directed phosphorylation, ubiquitination, and proteasomal degradation of MLK3. Accordingly, TRB3^−/− islets show a decrease in both the amplitude and duration of cytokine-stimulated MLK3 induction and JNK activation. It is well known that JNK signaling is facilitated by a feed forward loop of sequential kinase phosphorylation and is reinforced by a mutual stabilization of the module components. The failure of TRB3^−/− islets to mount an optimal JNK activation response, coupled with the ability of TRB3 to engage and maintain steady state levels of MLK3, recasts TRB3 as an integral functional component of the JNK module in pancreatic beta cells.     

Skeletal muscle-selective knockout of LKB1 increases insulin sensitivity, improves glucose homeostasis, and decreases TRB3

LKB1 is a tumor suppressor that may also be fundamental to cell metabolism, since LKB1 phosphorylates and activates the energy sensing enzyme AMPK. We generated muscle-specific LKB1 knockout (MLKB1KO) mice, and surprisingly, found that a lack of LKB1 in skeletal muscle enhanced insulin sensitivity, as evidenced by decreased fasting glucose and insulin concentrations, improved glucose tolerance, increased muscle glucose uptake in vivo, and increased glucose utilization during a hyperinsulinemic-euglycemic clamp. MLKB1KO mice had increased insulin-stimulated Akt phosphorylation and a > 80% decrease in muscle expression of TRB3, a recently identified Akt inhibitor. Akt/TRB3 binding was present in skeletal muscle, and overexpression of TRB3 in C2C12 myoblasts significantly reduced Akt phosphorylation. These results demonstrate that skeletal muscle LKB1 is a negative regulator of insulin sensitivity and glucose homeostasis. LKB1-mediated TRB3 expression provides a novel link between LKB1 and Akt, critical kinases involved in both tumor genesis and cell metabolism.     

LKB1 promotes metabolic flexibility in response to energy stress

The Liver Kinase B1 (LKB1) tumor suppressor acts as a metabolic energy sensor to regulate AMP-activated protein kinase (AMPK) signaling and is commonly mutated in various cancers, including non-small cell lung cancer (NSCLC). Tumor cells deficient in LKB1 may be uniquely sensitized to metabolic stresses, which may offer a therapeutic window in oncology. To address this question we have explored how functional LKB1 impacts the metabolism of NSCLC cells using ¹³C metabolic flux analysis. Isogenic NSCLC cells expressing functional LKB1 exhibited higher flux through oxidative mitochondrial pathways compared to those deficient in LKB1. Re-expression of LKB1 also increased the capacity of cells to oxidize major mitochondrial substrates, including pyruvate, fatty acids, and glutamine. Furthermore, LKB1 expression promoted an adaptive response to energy stress induced by anchorage-independent growth. Finally, this diminished adaptability sensitized LKB1-deficient cells to combinatorial inhibition of mitochondrial complex I and glutaminase. Together, our data implicate LKB1 as a major regulator of adaptive metabolic reprogramming and suggest synergistic pharmacological strategies for mitigating LKB1-deficient NSCLC tumor growth.     

The Tumor Suppressor Gene TUSC2 (FUS1) Sensitizes NSCLC to the AKT Inhibitor MK2206 in LKB1-dependent Manner

TUSC2-defective gene expression is detected in the majority of lung cancers and is associated with worse overall survival. We analyzed the effects of TUSC2 re-expression on tumor cell sensitivity to the AKT inhibitor, MK2206, and explored their mutual signaling connections, in vitro and in vivo. TUSC2 transient expression in three LKB1-defective non-small cell lung cancer (NSCLC) cell lines combined with MK2206 treatment resulted in increased repression of cell viability and colony formation, and increased apoptotic activity. In contrast, TUSC2 did not affect the response to MK2206 treatment for two LKB1-wild type NSCLC cell lines. In vivo, TUSC2 systemic delivery, by nanoparticle gene transfer, combined with MK2206 treatment markedly inhibited growth of tumors in a human LKB1-defective H322 lung cancer xenograft mouse model. Biochemical analysis showed that TUSC2 transient expression in LKB1-defective NSCLC cells significantly stimulated AMP-activated protein kinase (AMPK) phosphorylation and enzymatic activity. More importantly, AMPK gene knockdown abrogated TUSC2-MK2206 cooperation, as evidenced by reduced sensitivity to the combined treatment. Together, TUSC2 re-expression and MK2206 treatment was more effective in inhibiting the phosphorylation and kinase activities of AKT and mTOR proteins than either single agent alone. In conclusion, these findings support the hypothesis that TUSC2 expression status is a biological variable that potentiates MK2206 sensitivity in LKB1-defective NSCLC cells, and identifies the AMPK/AKT/mTOR signaling axis as an important regulator of this activity.     

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.     

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.     

A Mouse Model Uncovers LKB1 as an UVB-Induced DNA Damage Sensor Mediating CDKN1A (p21WAF1/CIP1) Degradation

Exposure to ultraviolet (UV) radiation from sunlight accounts for 90% of the symptoms of premature skin aging and skin cancer. The tumor suppressor serine-threonine kinase LKB1 is mutated in Peutz-Jeghers syndrome and in a spectrum of epithelial cancers whose etiology suggests a cooperation with environmental insults. Here we analyzed the role of LKB1 in a UV-dependent mouse skin cancer model and show that LKB1 haploinsufficiency is enough to impede UVB-induced DNA damage repair, contributing to tumor development driven by aberrant growth factor signaling. We demonstrate that LKB1 and its downstream kinase NUAK1 bind to CDKN1A. In response to UVB irradiation, LKB1 together with NUAK1 phosphorylates CDKN1A regulating the DNA damage response. Upon UVB treatment, LKB1 or NUAK1 deficiency results in CDKN1A accumulation, impaired DNA repair and resistance to apoptosis. Importantly, analysis of human tumor samples suggests that LKB1 mutational status could be a prognostic risk factor for UV-induced skin cancer. Altogether, our results identify LKB1 as a DNA damage sensor protein regulating skin UV-induced DNA damage response.     

The LKB1 tumor suppressor kinase in human disease

Inactivating germline mutations in the LKB1 gene underlie Peutz-Jeghers syndrome characterized by hamartomatous polyps and an elevated risk for cancer. Recent studies suggest the involvement of LKB1 also in more common human disorders including diabetes and in a significant fraction of lung adenocarcinomas. These observations have increased the interest towards signaling pathways of this tumor suppressor kinase. The recent breakthroughs in understanding the molecular functions of the LKB1 indicate its contribution as a regulator of cell polarity, energy metabolism and cell proliferation. Here we review how the substrates and cellular functions of LKB1 may be linked to Peutz-Jeghers syndrome and other diseases, and discuss how some of the molecular changes associated with altered LKB1 signaling might be used in therapeutic approaches.     

Important role of the LKB1-AMPK pathway in suppressing tumorigenesis in PTEN-deficient mice

The LKB1 tumour suppressor phosphorylates and activates AMPK (AMP-activated protein kinase) when cellular energy levels are low, thereby suppressing growth through multiple pathways, including inhibiting the mTORC1 (mammalian target of rapamycin complex 1) kinase that is activated in the majority of human cancers. Blood glucose-lowering Type 2 diabetes drugs also induce LKB1 to activate AMPK, indicating that these compounds could be used to suppress growth of tumour cells. In the present study, we investigated the importance of the LKB1-AMPK pathway in regulating tumorigenesis in mice resulting from deficiency of the PTEN (phosphatase and tensin homologue deleted on chromosome 10) tumour suppressor, which drives cell growth through overactivation of the Akt and mTOR (mammalian target of rapamycin) kinases. We demonstrate that inhibition of AMPK resulting from a hypomorphic mutation that decreases LKB1 expression does not lead to tumorigenesis on its own, but markedly accelerates tumour development in PTEN(+/-) mice. In contrast, activating the AMPK pathway by administration of metformin, phenformin or A-769662 to PTEN(+/-) mice significantly delayed tumour onset. We demonstrate that LKB1 is required for activators of AMPK to inhibit mTORC1 signalling as well as cell growth in PTEN-deficient cells. Our findings highlight, using an animal model relevant to understanding human cancer, the vital role that the LKB1-AMPK pathway plays in suppressing tumorigenesis resulting from loss of the PTEN tumour suppressor. They also suggest that pharmacological inhibition of LKB1 and/or AMPK would be undesirable, at least for the treatment of cancers in which the mTORC1 pathway is activated. Most importantly, our results demonstrate the potential of AMPK activators, such as clinically approved metformin, as anticancer agents, which will suppress tumour development by triggering a physiological signalling pathway that potently inhibits cell growth.     

Modulator of Apoptosis 1 (MOAP-1) Is a Tumor Suppressor Protein Linked to the RASSF1A Protein

Modulator of apoptosis 1 (MOAP-1) is a BH3-like protein that plays key roles in cell death or apoptosis. It is an integral partner to the tumor suppressor protein, Ras association domain family 1A (RASSF1A), and functions to activate the Bcl-2 family pro-apoptotic protein Bax. Although RASSF1A is now considered a bona fide tumor suppressor protein, the role of MOAP-1 as a tumor suppressor protein has yet to be determined. In this study, we present several lines of evidence from cancer databases, immunoblotting of cancer cells, proliferation, and xenograft assays as well as DNA microarray analysis to demonstrate the role of MOAP-1 as a tumor suppressor protein. Frequent loss of MOAP-1 expression, in at least some cancers, appears to be attributed to mRNA down-regulation and the rapid proteasomal degradation of MOAP-1 that could be reversed utilizing the proteasome inhibitor MG132. Overexpression of MOAP-1 in several cancer cell lines resulted in reduced tumorigenesis and up-regulation of genes involved in cancer regulatory pathways that include apoptosis (p53, Fas, and MST1), DNA damage control (poly(ADP)-ribose polymerase and ataxia telangiectasia mutated), those within the cell metabolism (IR-α, IR-β, and AMP-activated protein kinase), and a stabilizing effect on microtubules. The loss of RASSF1A (an upstream regulator of MOAP-1) is one of the earliest detectable epigenetically silenced tumor suppressor proteins in cancer, and we speculate that the additional loss of function of MOAP-1 may be a second hit to functionally compromise the RASSF1A/MOAP-1 death receptor-dependent pathway and drive tumorigenesis.     

Liver kinase B1 regulates the centrosome via PLK1

Liver kinase B1 (LKB1) is a tumor suppressor mutationally inactivated in Peutz–Jeghers syndrome (PJS) and various sporadic cancers. Although LKB1 encodes a kinase that possesses multiple functions, no individual hypothesis posed to date has convincingly explained how loss of LKB1 contributes to carcinogenesis. In this report we demonstrated that LKB1 maintains genomic stability through the regulation of centrosome duplication. We found that LKB1 colocalized with centrosomal proteins and was situated in the mitotic spindle pole. LKB1 deficiency-induced centrosome amplification was independent of AMP-activated protein kinase (AMPK), a well-defined substrate of LKB1. Cells lacking LKB1 exhibited an increase in phosphorylated and total Polo-like kinase 1 (PLK-1), NIMA-related kinase 2 (NEK2), and ninein-like protein (NLP). Overexpression of active PLK1 (T210D) reversed the inhibition of LKB1 on centrosome amplification. In contrast, depletion of PLK1 with siRNA or suppression of PLK1 kinase activity with BTO-1 (5-Cyano-7-nitro-2-benzothiazolecarboxamide-3-oxide) abrogated LKB1 deficiency-induced centrosome amplification. We further characterized that LKB1 phosphorylated and activated AMPK-related kinase 5 (NUAK1 or ARK5) that in turn increased the phosphorylation of MYPT1, enhanced the binding between MYPT1–PP1 and PLK1, and conferred an effective dephosphorylation of PLK1. More importantly, we noted that LKB1-deficient cells exhibited multiple nuclear abnormalities, such as mitotic delay, binuclear, polylobed, grape, large, and micronuclear. Immediate depletion of LKB1 resulted in the accumulation of multiploidy cells. Expression of LKB1 is reversely correlated with the levels of PLK1 in human cancer tissues. Thus, we have uncovered a novel function of LKB1 in the maintenance of genomic stability through the regulation of centrosome mediated by PLK1.