Breaking the epithelial polarity barrier in cancer: the strange case of LKB1/PAR-4

The PAR clan of polarity regulating genes was initially discovered in a genetic screen searching for genes involved in asymmetric cell divisions in the Caenorhabditis elegans embryo. Today, investigations in worms, flies and mammals have established PAR proteins as conserved and fundamental regulators of animal cell polarization in a broad range of biological phenomena requiring cellular asymmetries. The human homologue of invertebrate PAR-4, a serine–threonine kinase LKB1/STK11, has caught attention as a gene behind Peutz–Jeghers polyposis syndrome and as a bona fide tumour suppressor gene commonly mutated in sporadic cancer. LKB1 functions as a master regulator of AMP-activated protein kinase (AMPK) and 12 other kinases referred to as the AMPK-related kinases, including four human homologues of PAR-1. The role of LKB1 as part of the energy sensing LKB1-AMPK module has been intensively studied, whereas the polarity function of LKB1, in the context of homoeostasis or cancer, has gained less attention. Here, we focus on the PAR-4 identity of LKB1, discussing the weight of evidence indicating a role for LKB1 in regulation of cell polarity and epithelial integrity across species and highlight recent investigations providing new insight into the old question: does the PAR-4 identity of LKB1 matter in cancer?     

A Highlights from MBoC Selection: LKB1 kinase-dependent and -independent defects disrupt polarity and adhesion signaling to drive collagen remodeling during invasion

LKB1 is a serine/threonine kinase and a commonly mutated gene in lung adenocarcinoma. The majority of LKB1 mutations are truncations that disrupt its kinase activity and remove its C-terminal domain (CTD). Because LKB1 inactivation drives cancer metastasis in mice and leads to aberrant cell invasion in vitro, we sought to determine how compromised LKB1 function affects lung cancer cell polarity and invasion. Using three-dimensional models, we show that LKB1 kinase activity is essential for focal adhesion kinase–mediated cell adhesion and subsequent collagen remodeling but not cell polarity. Instead, cell polarity is overseen by the kinase-independent function of its CTD and more specifically its farnesylation. This occurs through a mesenchymal-amoeboid morphological switch that signals through the Rho-GTPase RhoA. These data suggest that a combination of kinase-dependent and -independent defects by LKB1 inactivation creates a uniquely invasive cell with aberrant polarity and adhesion signaling that drives invasion into the microenvironment.     

MARK/Par1 Kinase Is Activated Downstream of NMDA Receptors through a PKA-Dependent Mechanism

The Par1 kinases, also known as microtubule affinity-regulating kinases (MARKs), are important for the establishment of cell polarity from worms to mammals. Dysregulation of these kinases has been implicated in autism, Alzheimer’s disease and cancer. Despite their important function in health and disease, it has been unclear how the activity of MARK/Par1 is regulated by signals from cell surface receptors. Here we show that MARK/Par1 is activated downstream of NMDA receptors in primary hippocampal neurons. Further, we show that this activation is dependent on protein kinase A (PKA), through the phosphorylation of Ser431 of Par4/LKB1, the major upstream kinase of MARK/Par1. Together, our data reveal a novel mechanism by which MARK/Par1 is activated at the neuronal synapse.     

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.     

LKB1 Represses Focal Adhesion Kinase (FAK) Signaling via a FAK-LKB1 Complex to Regulate FAK Site Maturation and Directional Persistence

Liver kinase β1 (LKB1, also known as STK11) is a serine/threonine kinase that has multiple cellular functions including the regulation of cell polarity and motility. Murine proteomic studies show that LKB1 loss causes aberrant adhesion signaling; however, the mechanistic underpinnings of this relationship are unknown. We show that cells stably depleted of LKB1 or its co-activator STRADα have increased phosphorylation of focal adhesion kinase (FAK) at Tyr³⁹⁷/Tyr⁸⁶¹ and enhanced adhesion to fibronectin. LKB1 associates in a complex with FAK and LKB1 accumulation at the cellular leading edge is mutually excluded from regions of activated Tyr³⁹⁷-FAK. LKB1-compromised cells lack directional persistence compared with wild-type cells, but this is restored through subsequent pharmacological FAK inhibition or depletion, showing that cell directionality is mediated through LKB1-FAK signaling. Live cell confocal imaging reveals that LKB1-compromised cells lack normal FAK site maturation and turnover, suggesting that defects in adhesion and directional persistence are caused by aberrant adhesion dynamics. Furthermore, re-expression of full-length wild-type or the LKB1 N-terminal domain repressed FAK activity, whereas the kinase domain or C-terminal domain alone did not, indicating that FAK suppression is potentially regulated through the LKB1 N-terminal domain. Based upon these results, we conclude that LKB1 serves as a FAK repressor to stabilize focal adhesion sites, and when LKB1 function is compromised, aberrant FAK signaling ensues, resulting in rapid FAK site maturation and poor directional persistence.     

LKB1 is a master kinase that activates 13 kinases of the AMPK subfamily, including MARK/PAR-1

We recently demonstrated that the LKB1 tumour suppressor kinase, in complex with the pseudokinase STRAD and the scaffolding protein MO25, phosphorylates and activates AMP-activated protein kinase (AMPK). A total of 12 human kinases (NUAK1, NUAK2, BRSK1, BRSK2, QIK, QSK, SIK, MARK1, MARK2, MARK3, MARK4 and MELK) are related to AMPK. Here we demonstrate that LKB1 can phosphorylate the T-loop of all the members of this subfamily, apart from MELK, increasing their activity >50-fold. LKB1 catalytic activity and the presence of MO25 and STRAD are required for activation. Mutation of the T-loop Thr phosphorylated by LKB1 to Ala prevented activation, while mutation to glutamate produced active forms of many of the AMPK-related kinases. Activities of endogenous NUAK2, QIK, QSK, SIK, MARK1, MARK2/3 and MARK4 were markedly reduced in LKB1-deficient cells. Neither LKB1 activity nor that of AMPK-related kinases was stimulated by phenformin or AICAR, which activate AMPK. Our results show that LKB1 functions as a master upstream protein kinase, regulating AMPK-related kinases as well as AMPK. Between them, these kinases may mediate the physiological effects of LKB1, including its tumour suppressor function.     

Protein Kinase A Rescues Microtubule Affinity-regulating Kinase 2-induced Microtubule Instability and Neurite Disruption by Phosphorylating Serine 409

Microtubule affinity-regulating kinase 2 (MARK2)/PAR-1b and protein kinase A (PKA) are both involved in the regulation of microtubule stability and neurite outgrowth, but whether a direct cross-talk exists between them remains unclear. Here, we found the disruption of microtubule and neurite outgrowth induced by MARK2 overexpression was blocked by active PKA. The interaction between PKA and MARK2 was confirmed by coimmunoprecipitation and immunocytochemistry both in vitro and in vivo. PKA was found to inhibit MARK2 kinase activity by phosphorylating a novel site, serine 409. PKA could not reverse the microtubule disruption effect induced by a serine 409 to alanine (Ala) mutant of MARK2 (MARK2 S409A). In contrast, mutation of MARK2 serine 409 to glutamic acid (Glu) (MARK2 S409E) did not affect microtubule stability and neurite outgrowth. We propose that PKA functions as an upstream inhibitor of MARK2 in regulating microtubule stability and neurite outgrowth by directly interacting and phosphorylating MARK2.     

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 STK11^fl/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.     

Linked decreases in liver kinase B1 and AMP-activated protein kinase activity modulate matrix catabolic responses to biomechanical injury in chondrocytes

Introduction

AMP-activated protein kinase (AMPK) maintains cultured chondrocyte matrix homeostasis in response to inflammatory cytokines. AMPK activity is decreased in human knee osteoarthritis (OA) chondrocytes. Liver kinase B1 (LKB1) is one of the upstream activators of AMPK. Hence, we examined the relationship between LKB1 and AMPK activity in OA and aging cartilages, and in chondrocytes subjected to inflammatory cytokine treatment and biomechanical compression injury, and performed translational studies of AMPK pharmacologic activation.

Methods

We assessed activity (phosphorylation) of LKB1 and AMPKα in mouse knee OA cartilage, in aging mouse cartilage (6 to 24 months), and in chondrocytes after mechanical injury by dynamic compression, via immunohistochemistry or western blot. We knocked down LKB1 by siRNA transfection. Nitric oxide, matrix metalloproteinase (MMP)-3, and MMP-13 release were measured by Griess reaction and ELISA, respectively.

Results

Knockdown of LKB1 attenuated chondrocyte AMPK activity, and increased nitric oxide, MMP-3 and MMP-13 release (P <0.05) in response to IL-1β and TNFα. Both LKB1 and AMPK activity were decreased in mouse knee OA and aged knee cartilage, and in bovine chondrocytes after biomechanical injury. Pretreatment of bovine chondrocytes with AMPK activators AICAR and A-769662 inhibited both AMPKα dephosphorylation and catabolic responses after biomechanical injury.

Conclusion

LKB1 is required for chondrocyte AMPK activity, thereby inhibiting matrix catabolic responses to inflammatory cytokines. Concurrent loss of LKB1 and AMPK activity in articular chondrocytes is associated with OA, aging and biomechanical injury. Conversely, pharmacologic AMPK activation attenuates catabolic responses to biomechanical injury, suggesting a potentially novel approach to inhibit OA development and progression.     

SRC kinase activator CDCP1 promotes hepatocyte growth factor–induced cell migration/invasion of a subset of breast cancer cells

Cancer invasion and metastasis are the major causes of cancer patient mortality. Various growth factors, including hepatocyte growth factor (HGF), are known to promote cancer invasion and metastasis, but the regulatory mechanisms involved are not fully understood. Here, we show that HGF-promoted migration and invasion of breast cancer cells are regulated by CUB domain–containing protein 1 (CDCP1), a transmembrane activator of SRC kinase. In metastatic human breast cancer cell line MDA-MB-231, which highly expresses the HGF receptor MET and CDCP1, we show that CDCP1 knockdown attenuated HGF-induced MET activation, followed by suppression of lamellipodia formation and cell migration/invasion. In contrast, in the low invasive/nonmetastatic breast cancer cell line T47D, which had no detectable MET and CDCP1 expression, ectopic MET expression stimulated the HGF-dependent activation of invasive activity, and concomitant CDCP1 expression activated SRC and further promoted invasive activity. In these cells, CDCP1 expression dramatically activated HGF-induced membrane remodeling, which was accompanied by activation of the small GTPase Rac1. Analysis of guanine nucleotide exchange factors revealed that ARHGEF7 was specifically required for CDCP1-dependent induction of HGF-induced invasive ability. Furthermore, immunofluorescence staining demonstrated that CDCP1 coaccumulated with ARHGEF7. Finally, we confirmed that the CDCP1-SRC axis was also crucial for HGF and ARHGEF7-RAC1 signaling in MDA-MB-231 cells. Altogether, these results demonstrate that the CDCP1-SRC-ARHGEF7-RAC1 pathway plays an important role in the HGF-induced invasion of a subset of breast cancer cells.     

Microtubule affinity–regulating kinase 4 with an Alzheimer's disease-related mutation promotes tau accumulation and exacerbates neurodegeneration

Accumulation of the microtubule-associated protein tau is associated with Alzheimer''s disease (AD). In AD brain, tau is abnormally phosphorylated at many sites, and phosphorylation at Ser-262 and Ser-356 plays critical roles in tau accumulation and toxicity. Microtubule affinity–regulating kinase 4 (MARK4) phosphorylates tau at those sites, and a double de novo mutation in the linker region of MARK4, ΔG316E317D, is associated with an elevated risk of AD. However, it remains unclear how this mutation affects phosphorylation, aggregation, and accumulation of tau and tau-induced neurodegeneration. Here, we report that MARK4^ΔG316E317D increases the abundance of highly phosphorylated, insoluble tau species and exacerbates neurodegeneration via Ser-262/356–dependent and –independent mechanisms. Using transgenic Drosophila expressing human MARK4 (MARK4^wt) or a mutant version of MARK4 (MARK4^ΔG316E317D), we found that coexpression of MARK4^wt and MARK4^ΔG316E317D increased total tau levels and enhanced tau-induced neurodegeneration and that MARK4^ΔG316E317D had more potent effects than MARK4^wt. Interestingly, the in vitro kinase activities of MARK4^wt and MARK4^ΔG316E317D were similar. When tau phosphorylation at Ser-262 and Ser-356 was blocked by alanine substitutions, MARK4^wt did not promote tau accumulation or exacerbate neurodegeneration, whereas coexpression of MARK4^ΔG316E317D did. Both MARK4^wt and MARK4^ΔG316E317D increased the levels of oligomeric forms of tau; however, only MARK4^ΔG316E317D further increased the detergent insolubility of tau in vivo. Together, these findings suggest that MARK4^ΔG316E317D increases tau levels and exacerbates tau toxicity via a novel gain-of-function mechanism and that modification in this region of MARK4 may affect disease pathogenesis.     

Regulation of Focal Adhesion Kinase Activation,Breast Cancer Cell Motility,and Amoeboid Invasion by the RhoA Guanine Nucleotide Exchange Factor Net1

Net1 is a RhoA guanine nucleotide exchange factor (GEF) that is overexpressed in a subset of human cancers and contributes to cancer cell motility and invasion in vitro. However, the molecular mechanism accounting for its role in cell motility and invasion has not been described. In the present work, we show that expression of both Net1 isoforms in breast cancer cells is required for efficient cell motility. Although loss of Net1 isoform expression only partially blocks RhoA activation, it inhibits lysophosphatidic acid (LPA)-stimulated migration as efficiently as knockdown of RhoA itself. However, we demonstrate that the Net1A isoform predominantly controls myosin light-chain phosphorylation and is required for trailing edge retraction during migration. Net1A interacts with focal adhesion kinase (FAK), localizes to focal adhesions, and is necessary for FAK activation and focal adhesion maturation during cell spreading. Net1A expression is also required for efficient invasion through a Matrigel matrix. Analysis of invading cells demonstrates that Net1A is required for amoeboid invasion, and loss of Net1A expression causes cells to shift to a mesenchymal phenotype characterized by high β₁-integrin activity and membrane type 1 matrix metalloproteinase (MT1-MMP) expression. These results demonstrate a previously unrecognized role for the Net1A isoform in controlling FAK activation during planar cell movement and amoeboid motility during extracellular matrix (ECM) invasion.     

FAK competes for Src to promote migration against invasion in melanoma cells

Melanoma is one of the most deadly cancers because of its high propensity to metastasis, a process that requires migration and invasion of tumor cells driven by the regulated formation of adhesives structures like focal adhesions (FAs) and invasive structures like invadopodia. FAK, the major kinase of FAs, has been implicated in many cellular processes, including migration and invasion. In this study, we investigated the role of FAK in the regulation of invasion. We report that suppression of FAK in B16F10 melanoma cells led to increased invadopodia formation and invasion through Matrigel, but impaired migration. These effects are rescued by FAK WT but not by FAK^Y397F reexpression. Invadopodia formation requires local Src activation downstream of FAK and in a FAK phosphorylation-dependant manner. FAK deletion correlates with increased phosphorylation of Tks-5 (tyrosine kinase substrate with five SH3 domain) and reactive oxygen species production. In conclusion, our data show that FAK is able to mediate opposite effects on cell migration and invasion. Accordingly, beneficial effects of FAK inhibition are context dependent and may depend on the cell response to environmental cues and/or on the primary or secondary changes that melanoma experienced through the invasion cycle.Patients with spreading melanoma diseases have a very poor prognosis with a 5-year survival rate <5%. The metastatic spread of melanoma is a complex process involving several genetic alterations. In melanoma,¹ as in many highly invasive cancer cell types like head and neck squamous cell carcinoma² or breast carcinoma,³ specialized matrix-degrading organelles termed invadopodia have been identified. Invadopodia consist of dynamic actin-based protrusions of 0, 1 to 2 μm in diameter emanating from the ventral edge of tumor cells.⁴ Besides their actin scaffold, these structures are enriched in proteolytic enzymes such as matrix metalloproteinases (MMPs), which mediate extracellular matrix (ECM) degradation. Indeed, MMP are upregulated in invasive melanoma and there is extensive evidence that they have a role in promoting the dissemination of melanoma.^{5, 6, 7} Several proteins like integrins, Src and paxillin, found at sites of cell adhesion to the matrix, are also present in invadopodia.^{8, 9} On the other hand, other proteins like the Src substrate proteins cortactin¹⁰ and the tyrosine kinase substrate with five SH3 domain (Tks-5)¹¹ are specifically localized at invadopodia and not found at focal adhesion (FA). In addition, reactive oxygen species (ROS)¹² have been localized at invadopodia and are supposed to have a prominent role in inducing invadopodia function.^{13, 14} Although significant efforts have been made to characterize components of invadopodia, the precise mechanisms of their regulation, especially in a melanoma context, remain poorly understood.Tumor invasion is a multistep process that requires cell adhesion to the environing substratum, migration and invasion. In many cell types, migration requires fine control of FA turn-over. FAs are formed by the cluster of up to 200 proteins¹⁵ ensuring cell anchorage to the ECM. The cyclic process of FA formation and disruption is crucial for cell migration. Because both anchorage and migration involve cellular interactions with ECM components, FAs are endowed with transmembrane ECM receptor proteins such as integrins that interact with ECM molecules and intra-cellular proteins composed of scaffold proteins, as well as signal-transducing molecules. Among those, focal adhesion kinase (FAK) is a crucial signaling protein that integrates signals from integrins to the actin filaments during cell migration.¹⁶ Structurally, FAK is a 125-kDa protein that contains an N-terminal 4.1-ezrin–radixin–moesin domain, a central kinase domain and a C-terminal domain that contains the focal adhesion targeting site.¹⁷ The phosphorylation of FAK at Y397 creates a binding site for Src, which can phosphorylate other tyrosines on the FAK sequence, thus creating new binding sites for SH2 domain-containing proteins.FAK is involved in many aspects of the metastatic process and thus, overexpression, hyperphosphorylation and/or elevated activity of FAK have been reported in a variety of human cancers, including sarcomas and carcinomas of the breast, colon, thyroid, prostate, oral cavity, liver, stomach and ovary.¹⁸ In human melanoma cell lines, early studies reported high FAK expression and requirement of FAK for cell substrate adhesion.¹⁹ Later, it was reported that FAK promotes the aggressive melanoma phenotype.²⁰ Indeed, immunohistochemical analyses revealed high levels of FAK phosphorylation at Tyr397 and Tyr576, a marker of FAK kinase activity, in late-stage cutaneous and uveal melanoma, which correlated with their increased invasion and migration properties.²¹ Furthermore, melanoma differentiation-associated gene-9 (mda-9)/syntenin was also reported to mediate adhesion-dependant activation of protein kinase Cα (PKCα) and FAK in melanoma cells. Thus, inhibiting either mda-9/syntenin or PKCα suppressed fibronectin-induced formation of integrin-β1/FAK/c-Src signaling complexes and reduced migration and invasion toward fibronectin.²² Therefore, FAK appears to be a major player of melanoma invasion, but how this kinase controls the formation and proteolytic activity of invadopodia in melanoma cells was never investigated.In this study, we uncovered a surprising negative regulation of invadopodia activity in B16F10 cells by FAK. The depletion of FAK was associated with increased ROS production and Tks-5 phosphorylation. Using mutation of FAK at Tyr397, a binding site for Src, we found that these sites are implicated in FAK-mediated inhibition of invadopodia activity. In addition, we report that this mutation induced decreased migration speed but increased invasive properties. Taken together, our data suggest a competition between FA and invadopodia substrates for Src phosphorylation that might depend on environmental cues, thus leading to the engagement of either migration or degradation pathways.     

Lumican – Derived Peptides Inhibit Melanoma Cell Growth and Migration

Lumican, a small leucine-rich proteoglycan of the extracellular matrix, presents potent anti-tumor properties. Previous works from our group showed that lumican inhibited melanoma cell migration and tumor growth in vitro and in vivo. Melanoma cells adhered to lumican, resulting in a remodeling of their actin cytoskeleton and preventing their migration. In addition, we identified a sequence of 17 amino acids within the lumican core protein, named lumcorin, which was able to inhibit cell chemotaxis and reproduce anti-migratory effect of lumican in vitro. The aim of the present study was to characterize the anti-tumor mechanism of action of lumcorin. Lumcorin significantly decreased the growth in monolayer and in soft agar of two melanoma cell lines – mice B16F1 and human SK-MEL-28 cells – in comparison to controls. Addition of lumcorin to serum free medium significantly inhibited spontaneous motility of these two melanoma cell lines. To characterize the mechanisms involved in the inhibition of cell migration by lumcorin, the status of the phosphorylation/dephosphorylation of proteins was examined. Inhibition of focal adhesion kinase phosphorylation was observed in presence of lumcorin. Since cancer cells have been shown to migrate and to invade by mechanisms that involve matrix metalloproteinases (MMPs), the expression and activity of MMPs were analyzed. Lumcorin induced an accumulation of an intermediate form of MMP-14 (~59kDa), and inhibited MMP-14 activity. Additionally, we identified a short, 10 amino acids peptide within lumcorin sequence, which was able to reproduce its anti-tumor effect on melanoma cells. This peptide may have potential pharmacological applications.     

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.     

Fisetin Inhibits Human Melanoma Cell Invasion through Promotion of Mesenchymal to Epithelial Transition and by Targeting MAPK and NFκB Signaling Pathways

Malignant melanoma is responsible for approximately 75% of skin cancer-related deaths. BRAF plays an important role in regulating the mitogen-activated protein kinase (MAPK) signaling cascade in melanoma with activating mutations in the serine/threonine kinase BRAF occurring in 60–70% of malignant melanomas. The BRAF-MEK-ERK (MAPK) pathway is a key regulator of melanoma cell invasion. In addition, activation of NFκB via the MAPK pathway is regulated through MEK-induced activation of IKK. These pathways are potential targets for prevention and treatment of melanoma. In this study, we investigated the effect of fisetin, a phytochemical present in fruits and vegetables, on melanoma cell invasion and epithelial-mesenchymal transition, and delineated the underlying molecular mechanism. Treatment of multiple human malignant melanoma cell lines with fisetin (5–20 µM) resulted in inhibition of cell invasion. BRAF mutated melanoma cells were more sensitive to fisetin treatment, and this was associated with a decrease in the phosphorylation of MEK1/2 and ERK1/2. In addition, fisetin inhibited the activation of IKK leading to a reduction in the activation of the NFκB signaling pathway. Treatment of cells with an inhibitor of MEK1/2 (PD98059) or of NFκB (caffeic acid phenethyl ester) also reduced melanoma cell invasion. Furthermore, treatment of fisetin promoted mesenchymal to epithelial transition in melanoma cells, which was associated with a decrease in mesenchymal markers (N-cadherin, vimentin, snail and fibronectin) and an increase in epithelial markers (E-cadherin and desmoglein). Employing three dimensional skin equivalents consisting of A375 cells admixed with normal human keratinocytes embedded onto a collagen-constricted fibroblast matrix, we found that treatment of fisetin reduced the invasive potential of melanoma cells into the dermis and increased the expression of E-cadherin with a concomitant decrease in vimentin. These results indicate that fisetin inhibits melanoma cell invasion through promotion of mesenchymal to epithelial transition and by targeting MAPK and NFκB signaling pathways.     

DAPK activates MARK1/2 to regulate microtubule assembly, neuronal differentiation, and tau toxicity

Death-associated protein kinase (DAPK) is a key player in several modes of neuronal death/injury and has been implicated in the late-onset Alzheimer's disease (AD). DAPK promotes cell death partly through its effect on regulating actin cytoskeletons. In this study, we report that DAPK inhibits microtubule (MT) assembly by activating MARK/PAR-1 family kinases MARK1/2, which destabilize MT by phosphorylating tau and related MAP2/4. DAPK death domain, but not catalytic activity, is responsible for this activation by binding to MARK1/2 spacer region, thereby disrupting an intramolecular interaction that inhibits MARK1/2. Accordingly, DAPK(-/-) mice brain displays a reduction of tau phosphorylation and DAPK enhances the effect of MARK2 on regulating polarized neurite outgrowth. Using a well-characterized Drosophila model of tauopathy, we show that DAPK exerts an effect in part through MARK Drosophila ortholog PAR-1 to induce rough eye and loss of photoreceptor neurons. Furthermore, DAPK enhances tau toxicity through a PAR-1 phosphorylation-dependent mechanism. Together, our study reveals a novel mechanism of MARK activation, uncovers DAPK functions in modulating MT assembly and neuronal differentiation, and provides a molecular link of DAPK to tau phosphorylation, an event associated with AD pathology.     

Integrin-binding Protein Nischarin Interacts with Tumor Suppressor Liver Kinase B1 (LKB1) to Regulate Cell Migration of Breast Epithelial Cells

Biallelic inactivation of LKB1, a serine/threonine kinase, has been detected in 30% of lung adenocarcinomas, and inhibition of breast tumor growth has been demonstrated. We have identified the tumor suppressor, Nischarin, as a novel binding partner of LKB1. Our mapping analysis shows that the N terminus of Nischarin interacts with amino acids 44–436 of LKB1. Time lapse microscopy and Transwell migration data show that the absence of both Nischarin and LKB1 from an invasive breast cancer cell line (MDA-MB-231) enhances migration as measured by increased distance and speed of migrating cells. Our data suggest that this is a result of elevated PAK1 and LIMK1 phosphorylation. Moreover, the absence of Nischarin and LKB1 increased tumor growth in vivo. Consistent with this, the percentage of S phase cells was increased, as demonstrated by flow cytometry and enhanced cyclin D1. The absence of Nischarin and LKB1 also led to a dramatic increase in the formation of lung metastases. Our studies, for the first time, demonstrate functional interaction between LKB1 and Nischarin to inhibit cell migration and breast tumor progression. Mechanistically, we show that these two proteins together regulate PAK-LIMK-Cofilin and cyclin D1/CDK4 pathways.