Activation of tumor suppressor LKB1 by honokiol abrogates cancer stem-like phenotype in breast cancer via inhibition of oncogenic Stat3.

Tumor suppressor and upstream master kinase Liver kinase B1 (LKB1) plays a significant role in suppressing cancer growth and metastatic progression. We show that low-LKB1 expression significantly correlates with poor survival outcome in breast cancer. In line with this observation, loss-of-LKB1 rendered breast cancer cells highly migratory and invasive, attaining cancer stem cell-like phenotype. Accordingly, LKB1-null breast cancer cells exhibited an increased ability to form mammospheres and elevated expression of pluripotency-factors (Oct4, Nanog and Sox2), properties also observed in spontaneous tumors in Lkb1-/- mice. Conversely, LKB1-overexpression in LKB1-null cells abrogated invasion, migration and mammosphere-formation. Honokiol (HNK), a bioactive molecule from Magnolia grandiflora increased LKB1 expression, inhibited individual cell-motility and abrogated the stem-like phenotype of breast cancer cells by reducing the formation of mammosphere, expression of pluripotency-factors and aldehyde dehydrogenase activity. LKB1, and its substrate, AMP-dependent protein kinase (AMPK) are important for HNK-mediated inhibition of pluripotency factors since LKB1-silencing and AMPK-inhibition abrogated, while LKB1-overexpression and AMPK-activation potentiated HNK's effects. Mechanistic studies showed that HNK inhibited Stat3-phosphorylation/activation in an LKB1-dependent manner, preventing its recruitment to canonical binding-sites in the promoters of Nanog, Oct4 and Sox2. Thus, inhibition of the coactivation-function of Stat3 resulted in suppression of expression of pluripotency factors. Further, we showed that HNK inhibited breast tumorigenesis in mice in an LKB1-dependent manner. Molecular analyses of HNK-treated xenografts corroborated our in vitro mechanistic findings. Collectively, these results present the first in vitro and in vivo evidence to support crosstalk between LKB1, Stat3 and pluripotency factors in breast cancer and effective anticancer modulation of this axis with HNK treatment.

Collagen phagocytosis is regulated by the guanine nucleotide exchange factor Vav2.

Collagen phagocytosis is a crucial alpha2beta1-integrin-dependent process that mediates extracellular matrix remodeling by fibroblasts. We showed previously that after initial contact with collagen, activated Rac1 accelerates collagen phagocytosis but the Rac guanine nucleotide exchange factors (GEFs) that regulate Rac are not defined. We examined here the GEFs that regulate collagen phagocytosis in mouse fibroblasts. Collagen binding enhanced Rac1 activity (5-20 min) but not Cdc42 or RhoA activity. Analysis of collagen bead-associated proteins showed enrichment with Vav2, which correlated temporally with increased Rac1 activity. Knockdown of Vav2 prevented Rac activation, recruitment of Rac1 to collagen bead binding sites, and collagen bead binding, but knockdown of Sos-1 or beta-Pix had no effect on Rac activation or collagen binding. Vav2 was associated with the nucleotide-free Rac1 mutant (G15ARac1) after collagen binding. Collagen bead binding promoted phosphorylation of Vav2, which temporally correlated with Rac1 activation and which required Src kinase activity. Blockage of Src activity prevented collagen bead-induced Rac activation and collagen bead binding. Collectively these data indicate that Vav2 regulates the Rac1 activity associated with the binding step of collagen phagocytosis.

Novel splice isoforms of STRADalpha differentially affect LKB1 activity, complex assembly and subcellular localization.

STRADalpha is a pseudokinase that forms a heterotrimeric complex with the scaffolding protein MO25 and the tumor suppressor serine threonine protein kinase LKB1. Mutations in the LKB1 gene are responsible for the Peutz-Jeghers Syndrome (PJS) characterized by a predisposition to hamartomatous polyps and hyperpigmentation of the buccal mucosa. Mutations in LKB1 have also been observed in some sporadic tumours unrelated to PJS. The LKB1/STRAD/MO25 complex is involved in the regulation of numerous signaling pathways including metabolism, proliferation and cellular polarity of human intestinal epithelial cells. Cell polarization, together with tissue-restricted transcription, represents the main feature of enterocyte differentiation. Since a full-length STRADalpha transcript has not been identified thus far in these cells, we evaluated the expression of endogenous STRADalpha in five colorectal cancer cell lines characterized by their diverse ability to differentiate in vitro. We report herein the discovery of several novel splice isoforms of STRADalpha that differentially affect the kinase activity, complex assembly, subcellular localization of LKB1 and the activation of the LKB1-dependent AMPK pathway.

LKB1, the multitasking tumour suppressor kinase.

Mutations in the lkb1 gene are found in Peutz-Jeghers syndrome (PJS), with loss of heterozygosity or somatic mutations at the lkb1 locus, suggesting the gene product, the serine/threonine kinase LKB1, may function as a tumour suppressor. Patients with PJS are at a greater risk of developing cancers of epithelial tissue origin. It is widely accepted that the presence of hamartomatous polyps in PJS does not in itself lead to the development of malignancy. The signalling mechanisms that lead to these PJS related malignancies are not well understood. However, it is evident from the recent literature that LKB1 is a multitasking kinase, with unlimited potential in orchestrating cell activity. Thus far, LKB1 has been found to play a role in chromatin remodelling, cell cycle arrest, Wnt signalling, cell polarity, and energy metabolism, all of which may require the tumour suppressor function of this kinase and/or its catalytic activity.

LKB1 associates with Brg1 and is necessary for Brg1-induced growth arrest.

Inactivating mutations in the serine-threonine kinase LKB1 (STK11) are found in most patients with Peutz-Jeghers syndrome; however the function of LKB1 is unknown. We found that LKB1 binds to and regulates brahma-related gene 1 (Brg1), an essential component of chromatin remodeling complexes. The association requires the N terminus of LKB1 and the helicase domain of Brg1 and LKB1 stimulates the ATPase activity of Brg1. Brg1 expression in SW13 cells induces the formation of flat cells indicative of cell cycle arrest and senescence. Expression of a kinase-dead mutant of LKB1, SL26, in SW13 cells blocks the formation of Brg1-induced flat cells, indicating that LKB1 is required for Brg1-dependent growth arrest. The inability of mutants of LKB1 to mediate Brg1-dependent growth arrest may explain the manifestations of Peutz-Jeghers syndrome.

Vav2 is required for cell spreading.

Vav2 is a widely expressed Rho family guanine nucleotide exchange factor highly homologous to Vav1 and Vav3. Activated versions of Vav2 are transforming, but the normal function of Vav2 and how it is regulated are not known. We investigated the pathways that regulate Vav2 exchange activity in vivo and characterized its function. Overexpression of Vav2 activates Rac as assessed by both direct measurement of Rac-GTP and cell morphology. Vav2 also catalyzes exchange for RhoA, but does not cause morphologic changes indicative of RhoA activation. Vav2 nucleotide exchange is Src-dependent in vivo, since the coexpression of Vav2 and dominant negative Src, or treatment with the Src inhibitor PP2, blocks both Vav2-dependent Rac activation and lamellipodia formation. A mutation in the pleckstrin homology (PH) domain eliminates exchange activity and this construct does not induce lamellipodia, indicating the PH domain is necessary to catalyze nucleotide exchange. To further investigate the function of Vav2, we mutated the dbl homology (DH) domain and asked whether this mutant would function as a dominant negative to block Rac-dependent events. Studies using this mutant indicate that Vav2 is not necessary for platelet-derived growth factor- or epidermal growth factor-dependent activation of Rac. The Vav2 DH mutant did act as a dominant negative to inhibit spreading of NIH3T3 cells on fibronectin, specifically by blocking lamellipodia formation. These findings indicate that in fibroblasts Vav2 is necessary for integrin, but not growth factor-dependent activation of Rac leading to lamellipodia.

Target gene therapy for alpha-fetoprotein-producing hepatocellular carcinoma by E1B55k-attenuated adenovirus.

Gene therapy using replication-competent adenovirus that selectively propagates in tumor cells may be an effective treatment for cancer. We developed an adenovirus that would be replication specific for hepatocellular carcinoma (HCC). Based on our finding that the E1B55k-deficient adenovirus was able to replicate in human primary hepatocytes, we therefore designed an adenovirus carrying E1A and attenuated E1B gene driven by the alpha-fetoprotein promoter (Adv-AFP-E1AdB), thus restricting the replication specificity in AFP-producing HCC. Replication of Adv-AFP-E1AdB in primary hepatocytes was practically negligible 4 days after infection. Although Adv-AFP-E1AdB replicated slowly in AFP-producing HCC, it efficiently destroyed HCC cells independent of their p53 status. Experiments were conducted in vivo using systemic administration of Adv-AFP-E1AdB and we observed tumor size reduction in nude mice having liver cancer. The use of replication-competent adenovirus deficient of the E1B gene coupled to an AFP-targeting strategy may be a safe and efficacious treatment for HCC.

TAZ: a novel transcriptional co-activator regulated by interactions with 14-3-3 and PDZ domain proteins.

The highly conserved and ubiquitously expressed 14-3-3 proteins regulate differentiation, cell cycle progression and apoptosis by binding intracellular phosphoproteins involved in signal transduction. By screening in vitro translated cDNA pools for the ability to bind 14-3-3, we identified a novel transcriptional co-activator, TAZ (transcriptional co-activator with PDZ-binding motif) as a 14-3-3-binding molecule. TAZ shares homology with Yes-associated protein (YAP), contains a WW domain and functions as a transcriptional co-activator by binding to the PPXY motif present on transcription factors. 14-3-3 binding requires TAZ phosphorylation on a single serine residue, resulting in the inhibition of TAZ transcriptional co-activation through 14-3-3-mediated nuclear export. The C-terminus of TAZ contains a highly conserved PDZ-binding motif that localizes TAZ into discrete nuclear foci and is essential for TAZ-stimulated gene transcription. TAZ uses this same motif to bind the PDZ domain-containing protein NHERF-2, a molecule that tethers plasma membrane ion channels and receptors to cytoskeletal actin. TAZ may link events at the plasma membrane and cytoskeleton to nuclear transcription in a manner that can be regulated by 14-3-3.

Adenovirus-mediated drug-sensitivity gene therapy for hepatocellular carcinoma.

Selective gene therapy represents a potent approach in cancer treatment that utilizes a cell's own nontoxic suicide genes. Currently, the suicide genes under investigation mediate sensitivity by encoding viral or bacterial enzymes that convert inactive prodrug into toxic antimetabolites that inhibit nucleic acid synthesis (1,2).

Association of protein kinase Cmu with type II phosphatidylinositol 4-kinase and type I phosphatidylinositol-4-phosphate 5-kinase.

Protein kinase Cmu (PKCmu), also named protein kinase D, is an unusual member of the PKC family that has a putative transmembrane domain and pleckstrin homology domain. This enzyme has a substrate specificity distinct from other PKC isoforms (Nishikawa, K., Toker, A., Johannes, F. J., Songyang, Z., and Cantley, L. C. (1997) J. Biol. Chem. 272, 952-960), and its mechanism of regulation is not yet clear. Here we show that PKCmu forms a complex in vivo with a phosphatidylinositol 4-kinase and a phosphatidylinositol-4-phosphate 5-kinase. A region of PKCmu between the amino-terminal transmembrane domain and the pleckstrin homology domain is shown to be involved in the association with the lipid kinases. Interestingly, a kinase-dead point mutant of PKCmu failed to associate with either lipid kinase activity, indicating that autophosphorylation may be required to expose the lipid kinase interaction domain. Furthermore, the subcellular distribution of the PKCmu-associated lipid kinases to the particulate fraction depends on the presence of the amino-terminal region of PKCmu including the predicted transmembrane region. These results suggest a novel model in which the non-catalytic region of PKCmu acts as a scaffold for assembly of enzymes involved in phosphoinositide synthesis at specific membrane locations.

The formation of diradylglycerol molecular species in murine peritoneal macrophages varies dose-dependently with dietary purified eicosapentaenoic and docosahexaenoic ethyl esters.

Substantial effects of dietary fish oil-derived fatty acid ethyl esters on the metabolism of diradylglycerol (DG) have recently been described. We undertook to isolate the separate effects of (n-3) eicosapentaenoic acid (EPA) and (n-3) docosahexaenoic acid (DHA) on DG metabolism. For 3 wk, male C57BL/6 mice were fed one of six purified diets in which the lipid component was either 3 g/100 g corn oil ethyl ester (COEE) (control diet) or 1 g/100 g COEE plus 2 g/100 g of EPA ethyl ester (EPEE), DHA ethyl ester (DHEE) or an EPEE:DHEE mixture. Peritoneal macrophages were analyzed for DG content and for molecular species distributions of DG and phospholipid classes. We found that the degree of incorporation of EPA and DHA into DG in macrophages was dependent on the dietary concentration of EPEE and DHEE, under basal conditions and after stimulation with platelet-activating factor, phorbol myristate acetate and ionomycin. Incorporation of EPA and DHA into phospholipids was also significant and dose dependent in each phospholipid class. For both DG and phospholipid molecular species, the incorporation of EPA in the sn-2 position was considerably greater than that of species with DHA under conditions of equimolar dietary content. These results demonstrate that 1) incorporation of EPA and DHA into DG are independent and dependent on dietary content, 2) EPA is incorporated with greater affinity than DHA and 3) these effects on DG metabolism seem to result from corresponding effects on parent membrane phospholipids. Physiologically and therapeutically relevant differences may exist between EPA and DHA.

Acyl chain dependence of diacylglycerol activation of protein kinase C activity in vitro.

Stimulation of protein kinase C (PKC) activity in lipid vesicles in vitro was achieved by pure molecular species of diacylglycerol (DAG), specifically 1-stearoyl-2-acyl-sn-glycerol substituted with 2-arachidonoyl,2-eicosapentaenoyl or 2-docosahexaenoyl (SAG, SEG, and SDG, respectively). PKC activity was measured in lipid vesicles containing 30 mol% 1-palmitoyl-2-oleoyl-sn-glycerol-3-phospho-L-serine (POPS), 68-70 mol% 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC), and 0-2 mol% DAG in the presence of 20 microM calcium. Our results demonstrate that amplification of PKC activity differs significantly among these molecular species of DAG. In particular, SDG at 0.5 mol% is more potent in increasing PKC activity than is dioleoylglycerol (DOG), SEG, or SAG, and SAG and SDG at 1.0 and 2.0 mol% have similar potencies which are greater than those of DOG or SEG. These findings demonstrate that sn-2 substitutions in DAG by specific n-3 and n-6 polyunsaturated fatty acids increase the potency of DAG to stimulate PKC activity in vitro.

n-3 polyunsaturated fatty acid-induced changes in the molecular species composition of diradylglycerol in murine peritoneal macrophages remain stable during incubation ex vivo.

Three-to four-week-old C57BL/6 mice were maintained for four weeks on diets in which the 10% lipid component was the ethyl esters of either corn oil or n-3 polyunsaturated fatty acids (n-3 PUFA). Incubation of macrophages ex vivo for 14 h, a normal incubation time used for macrophage studies, in the absence of fetal calf serum, did not diminish the extent of incorporation of 20:5n-3 (eicosapentaenoic acid) and 22:6n-3 (docosahexaenoic acid) moieties into membrane phospholipids and into diradylglycerol (DG) relative to that after a very abbreviated incubation time. We conclude that studies examining the effects of dietary n-3 PUFA on DG formation and related physiological effects in macrophages can be performed after a normal ex vivo incubation of at least 14 h without experiencing a significant loss of incorporated n 3 PUFA.

Formation of second messenger diradylglycerol in murine peritoneal macrophages is altered after in vivo (n-3) polyunsaturated fatty acid supplementation.

The consequences of macrophage membrane incorporation of (n-3) polyunsaturated fatty acids (PUFA) on diradylglycerol (DG) formation and phospholipase-mediated signal transduction in vivo remain largely unknown. Three-to four-week-old C57BL/6 mice were randomly assigned to diets in which the 10% lipid component of the purified diet was either corn oil ethyl ester (control) or (n-3) ethyl ester [(n-3) PUFA-enriched]. After 4 wk, thioglycollate-elicited peritoneal macrophages were harvested and assayed for (1) total DG mass, (2) DG molecular species fraction analyses by argentation TLC and (3) separation of phospholipid classes and analyses of their molecular species fractions. We found that basal DG mass and the increase in DG mass after stimulation with platelet-activating factor or phorbol myristate acetate were similar in cells from control and (n-3) PUFA-enriched diet groups, whereas ionomycin-stimulated DG formation was less in the cells from the (n-3) PUFA-enriched diet group. Analyses of DG molecular species fractions showed that the proportions of species with five and six double bonds were markedly greater in the (n-3) PUFA-enriched diet group macrophages compared with those of the control diet group. The molecular species fractions of all phospholipid classes including phosphatidylinositol showed substantial incorporation of (n-3) PUFA. These results demonstrate that dietary enrichment with (n-3) PUFA induces marked changes in DG metabolism in murine peritoneal macrophages. These changes may contribute to the mechanisms of antiinflammatory, antithrombotic and antineoplastic actions associated with dietary (n-3) PUFA supplementation.