BACH1 Stabilization by Antioxidants Stimulates Lung Cancer Metastasis.

For tumors to progress efficiently, cancer cells must overcome barriers of oxidative stress. Although dietary antioxidant supplementation or activation of endogenous antioxidants by NRF2 reduces oxidative stress and promotes early lung tumor progression, little is known about its effect on lung cancer metastasis. Here, we show that long-term supplementation with the antioxidants N-acetylcysteine and vitamin E promotes KRAS-driven lung cancer metastasis. The antioxidants stimulate metastasis by reducing levels of free heme and stabilizing the transcription factor BACH1. BACH1 activates transcription of Hexokinase 2 and Gapdh and increases glucose uptake, glycolysis rates, and lactate secretion, thereby stimulating glycolysis-dependent metastasis of mouse and human lung cancer cells. Targeting BACH1 normalized glycolysis and prevented antioxidant-induced metastasis, while increasing endogenous BACH1 expression stimulated glycolysis and promoted metastasis, also in the absence of antioxidants. We conclude that BACH1 stimulates glycolysis-dependent lung cancer metastasis and that BACH1 is activated under conditions of reduced oxidative stress.

Control of the innate immune response by the mevalonate pathway.

Deficiency in mevalonate kinase (MVK) causes systemic inflammation. However, the molecular mechanisms linking the mevalonate pathway to inflammation remain obscure. Geranylgeranyl pyrophosphate, a non-sterol intermediate of the mevalonate pathway, is the substrate for protein geranylgeranylation, a protein post-translational modification that is catalyzed by protein geranylgeranyl transferase I (GGTase I). Pyrin is an innate immune sensor that forms an active inflammasome in response to bacterial toxins. Mutations in MEFV (encoding human PYRIN) result in autoinflammatory familial Mediterranean fever syndrome. We found that protein geranylgeranylation enabled Toll-like receptor (TLR)-induced activation of phosphatidylinositol-3-OH kinase (PI(3)K) by promoting the interaction between the small GTPase Kras and the PI(3)K catalytic subunit p110δ. Macrophages that were deficient in GGTase I or p110δ exhibited constitutive release of interleukin 1ß that was dependent on MEFV but independent of the NLRP3, AIM2 and NLRC4 inflammasomes. In the absence of protein geranylgeranylation, compromised PI(3)K activity allows an unchecked TLR-induced inflammatory responses and constitutive activation of the Pyrin inflammasome.

Podocyte Geranylgeranyl Transferase Type-I Is Essential for Maintenance of the Glomerular Filtration Barrier.

SIGNIFICANCE STATEMENT: A tightly regulated actin cytoskeleton attained through balanced activity of RhoGTPases is crucial to maintaining podocyte function. However, how RhoGTPases are regulated by geranylgeranylation, a post-translational modification, has been unexplored. The authors found that loss of the geranylgeranylation enzyme geranylgeranyl transferase type-I (GGTase-I) in podocytes led to progressive albuminuria and foot process effacement in podocyte-specific GGTase-I knockout mice. In cultured podocytes, the absence of geranylgeranylation resulted in altered activity of its downstream substrates Rac1, RhoA, Cdc42, and Rap1, leading to alterations of ß1-integrins and actin cytoskeleton structural changes. These findings highlight the importance of geranylgeranylation in the dynamic management of RhoGTPases and Rap1 to control podocyte function, providing new knowledge about podocyte biology and glomerular filtration barrier function. BACKGROUND: Impairment of the glomerular filtration barrier is in part attributed to podocyte foot process effacement (FPE), entailing disruption of the actin cytoskeleton and the slit diaphragm. Maintenance of the actin cytoskeleton, which contains a complex signaling network through its connections to slit diaphragm and focal adhesion proteins, is thus considered crucial to preserving podocyte structure and function. A dynamic yet tightly regulated cytoskeleton is attained through balanced activity of RhoGTPases. Most RhoGTPases are post-translationally modified by the enzyme geranylgeranyl transferase type-I (GGTase-I). Although geranylgeranylation has been shown to regulate activities of RhoGTPases and RasGTPase Rap1, its significance in podocytes is unknown. METHODS: We used immunofluorescence to localize GGTase-I, which was expressed mainly by podocytes in the glomeruli. To define geranylgeranylation's role in podocytes, we generated podocyte-specific GGTase-I knockout mice. We used transmission electron microscopy to evaluate FPE and measurements of urinary albumin excretion to analyze filtration barrier function. Geranylgeranylation's effects on RhoGTPases and Rap1 function were studied in vitro by knockdown or inhibition of GGTase-I. We used immunocytochemistry to study structural modifications of the actin cytoskeleton and ß1 integrins. RESULTS: Depletion of GGTase-I in podocytes in vivo resulted in FPE and concomitant early-onset progressive albuminuria. A reduction of GGTase-I activity in cultured podocytes disrupted RhoGTPase balance by markedly increasing activity of RhoA, Rac1, and Cdc42 together with Rap1, resulting in dysregulation of the actin cytoskeleton and altered distribution of ß1 integrins. CONCLUSIONS: These findings indicate that geranylgeranylation is of crucial importance for the maintenance of the delicate equilibrium of RhoGTPases and Rap1 in podocytes and consequently for the maintenance of glomerular integrity and function.

Epithelial RAC1-dependent cytoskeleton dynamics controls cell mechanics, cell shedding and barrier integrity in intestinal inflammation.

OBJECTIVE: Increased apoptotic shedding has been linked to intestinal barrier dysfunction and development of inflammatory bowel diseases (IBD). In contrast, physiological cell shedding allows the renewal of the epithelial monolayer without compromising the barrier function. Here, we investigated the role of live cell extrusion in epithelial barrier alterations in IBD. DESIGN: Taking advantage of conditional GGTase and RAC1 knockout mice in intestinal epithelial cells (Pggt1b iΔIEC and Rac1 iΔIEC mice), intravital microscopy, immunostaining, mechanobiology, organoid techniques and RNA sequencing, we analysed cell shedding alterations within the intestinal epithelium. Moreover, we examined human gut tissue and intestinal organoids from patients with IBD for cell shedding alterations and RAC1 function. RESULTS: Epithelial Pggt1b deletion led to cytoskeleton rearrangement and tight junction redistribution, causing cell overcrowding due to arresting of cell shedding that finally resulted in epithelial leakage and spontaneous mucosal inflammation in the small and to a lesser extent in the large intestine. Both in vivo and in vitro studies (knockout mice, organoids) identified RAC1 as a GGTase target critically involved in prenylation-dependent cytoskeleton dynamics, cell mechanics and epithelial cell shedding. Moreover, inflamed areas of gut tissue from patients with IBD exhibited funnel-like structures, signs of arrested cell shedding and impaired RAC1 function. RAC1 inhibition in human intestinal organoids caused actin alterations compatible with arresting of cell shedding. CONCLUSION: Impaired epithelial RAC1 function causes cell overcrowding and epithelial leakage thus inducing chronic intestinal inflammation. Epithelial RAC1 emerges as key regulator of cytoskeletal dynamics, cell mechanics and intestinal cell shedding. Modulation of RAC1 might be exploited for restoration of epithelial integrity in the gut of patients with IBD.

Intussusceptive Angiogenesis in Human Metastatic Malignant Melanoma.

Angiogenesis supplies oxygen and nutrients to growing tumors. Inhibiting angiogenesis may stop tumor growth, but vascular endothelial growth factor inhibitors have limited effect in most tumors. This limited effect may be explained by an additional, less vascular endothelial growth factor-driven form of angiogenesis known as intussusceptive angiogenesis. The importance of intussusceptive angiogenesis in human tumors is not known. Epifluorescence and confocal microscopy was used to visualize intravascular pillars, the hallmark structure of intussusceptive angiogenesis, in tumors. Human malignant melanoma metastases, patient-derived melanoma xenografts in mice (PDX), and genetically engineered v-raf murine sarcoma viral oncogene homolog B1 (BRAF)-induced, phosphatase and TENsin homolog deleted on chromosome 10 (PTEN)-deficient (BPT) mice (BrafCA/+Ptenf/fTyr-Cre+/0-mice) were analyzed for pillars. Gene expression in human melanoma metastases and PDXs was analyzed by RNA sequencing. Matrix metalloproteinase 9 (MMP9) protein expression and T-cell and macrophage infiltration in tumor sections were determined with multiplex immunostaining. Intravascular pillars were detected in human metastases but rarely in PDXs and not in BPT mice. The expression of MMP9 mRNA was higher in human metastases compared with PDXs. High expression of MMP9 protein as well as infiltration of macrophages and T-cells were detected in proximity to intravascular pillars. MMP inhibition blocked formation of pillars, but not tubes or tip cells, in vitro. In conclusion, intussusceptive angiogenesis may contribute to the growth of human melanoma metastases. MMP inhibition blocked pillar formation in vitro and should be further investigated as a potential anti-angiogenic drug target in metastatic melanoma.

Rho-GTPase dependent leukocyte interaction generates pro-inflammatory thymic Tregs and causes arthritis.

Conditional mutation of protein geranylgeranyltransferase type I (GGTase-I) in macrophages (GLC) activates Rho-GTPases and causes arthritis in mice. Knocking out Rag1 in GLC mice alleviates arthritis which indicates that lymphocytes are required for arthritis development in those mice. To study GLC dependent changes in the adaptive immunity, we isolated CD4+ T cells from GLC mice (CD4+GLCs). Spleen and joint draining lymph nodes (dLN) CD4+GLCs exhibited high expression of Cdc42 and Rac1, which repressed the caudal HOXA proteins and activated the mechanosensory complex to facilitate migration. These CDC42/RAC1 rich CD4+GLCs presented a complete signature of GARP+NRP1+IKZF2+FOXP3+ regulatory T cells (Tregs) of thymic origin. Activation of the ß-catenin/Lef1 axis promoted a pro-inflammatory Th1 phenotype of Tregs, which was strongly associated with arthritis severity. Knockout of Cdc42 in macrophages of GLC mice affected CD4+ cell biology and triggered development of non-thymic Tregs. Knockout of Rac1 and RhoA had no such effects on CD4+ cells although it alleviated arthritis in GLC mice. Disrupting macrophage and T cell interaction with CTLA4 fusion protein reduced the Th1-driven inflammation and enrichment of thymic Tregs into dLNs. Antigen challenge reinforced the CD4+GLC phenotype in non-arthritic heterozygote GLC mice and increased accumulation of Rho-GTPase expressing thymic Tregs in dLNs. Our study demonstrates an unexpected role of macrophages in stimulating the development of pro-inflammatory thymic Tregs and reveal activation of Rho-GTPases behind their arthritogenic phenotype.

Glucosylceramide synthase deficiency in the heart compromises ß1-adrenergic receptor trafficking.

AIMS: Cardiac injury and remodelling are associated with the rearrangement of cardiac lipids. Glycosphingolipids are membrane lipids that are important for cellular structure and function, and cardiac dysfunction is a characteristic of rare monogenic diseases with defects in glycosphingolipid synthesis and turnover. However, it is not known how cardiac glycosphingolipids regulate cellular processes in the heart. The aim of this study is to determine the role of cardiac glycosphingolipids in heart function. METHODS AND RESULTS: Using human myocardial biopsies, we showed that the glycosphingolipids glucosylceramide and lactosylceramide are present at very low levels in non-ischaemic human heart with normal function and are elevated during remodelling. Similar results were observed in mouse models of cardiac remodelling. We also generated mice with cardiomyocyte-specific deficiency in Ugcg, the gene encoding glucosylceramide synthase (hUgcg-/- mice). In 9- to 10-week-old hUgcg-/- mice, contractile capacity in response to dobutamine stress was reduced. Older hUgcg-/- mice developed severe heart failure and left ventricular dilatation even under baseline conditions and died prematurely. Using RNA-seq and cell culture models, we showed defective endolysosomal retrograde trafficking and autophagy in Ugcg-deficient cardiomyocytes. We also showed that responsiveness to ß-adrenergic stimulation was reduced in cardiomyocytes from hUgcg-/- mice and that Ugcg knockdown suppressed the internalization and trafficking of ß1-adrenergic receptors. CONCLUSIONS: Our findings suggest that cardiac glycosphingolipids are required to maintain ß-adrenergic signalling and contractile capacity in cardiomyocytes and to preserve normal heart function.

Targeting Filamin A Reduces Macrophage Activity and Atherosclerosis.

BACKGROUND: The actin-binding protein FLNA (filamin A) regulates signal transduction important for cell locomotion, but the role of macrophage-specific FLNA during atherogenesis has not been explored. METHODS: We analyzed FLNA expression in human carotid atherosclerotic plaques by immunofluorescence. We also produced mice with Flna-deficient macrophages by breeding conditional Flna-knockout mice ( Flna o/fl) with mice expressing Cre from the macrophage-specific lysosome M promoter ( LC). Atherosclerosis in vivo was studied by transplanting bone marrow from male Flna o/fl/ LC mice to atherogenic low-density lipoprotein receptor-deficient ( Ldlr-/-) mice; and by infecting Flna o/fl and Flna o/fl/ LC mice with AdPCSK9 (adenoviral vector overexpressing proprotein convertase subtilisin/kexin type 9). Furthermore, C57BL/6 mice were infected with AdPCSK9 and then treated with the calpain inhibitor calpeptin to inhibit FLNA cleavage. RESULTS: We found that macrophage FLNA expression was higher in advanced than in intermediate human atherosclerotic plaques. Flna o/fl/ LC macrophages proliferated and migrated less than controls; expressed lower levels of phosphorylated AKT and ERK1/2; exhibited reduced foam cell formation and lipid uptake; and excreted more lipids. The deficiency of Flna in macrophages markedly reduced the size of aortic atherosclerotic plaques in both Ldlr-/-BMT: Flnao/fl/LC and AdPCSK9-infected Flna o/fl/ LC mice. Intima/media ratios and numbers of CD68-positive macrophages in atherosclerotic plaques were lower in Flna-deficient mice than in control mice. Moreover, we found that STAT3 interacts with a calpain-cleaved carboxyl-terminal fragment of FLNA. Inhibiting calpain-mediated FLNA cleavage with calpeptin in macrophages reduced nuclear levels of phosphorylated STAT3, interleukin 6 secretion, foam cell formation, and lipid uptake. Finally, calpeptin treatment reduced the size of atherosclerotic plaques in C57BL/6 mice infected with AdPCSK9. CONCLUSIONS: Genetic inactivation of Flna and chemical inhibition of calpain-dependent cleavage of FLNA impaired macrophage signaling and function, and reduced atherosclerosis in mice, suggesting that drugs targeting FLNA may be useful in the treatment of atherosclerosis.

Inhibiting PGGT1B Disrupts Function of RHOA, Resulting in T-cell Expression of Integrin α4ß7 and Development of Colitis in Mice.

BACKGROUND & AIMS: It is not clear how regulation of T-cell function is altered during development of inflammatory bowel diseases (IBD). We studied the mechanisms by which geranylgeranyltransferase-mediated prenylation controls T-cell localization to the intestine and chronic inflammation. METHODS: We generated mice with T-cell-specific disruption of the geranylgeranyltransferase type I, beta subunit gene (Pggt1b), called Pggt1bΔCD4 mice, or the ras homolog family member A gene (Rhoa), called RhoaΔCD4 mice. We also studied mice with knockout of CDC42 or RAC1 and wild-type mice (controls). Intestinal tissues were analyzed by histology, multiphoton and confocal microscopy, and real-time polymerase chain reaction. Activation of CDC42, RAC1, and RHOA were measured with G-LISA, cell fractionation, and immunoblots. T cells and lamina propria mononuclear cells from mice were analyzed by flow cytometry or transferred to Rag1-/- mice. Mice were given injections of antibodies against integrin alpha4beta7 or gavaged with the RORC antagonist GSK805. We obtained peripheral blood and intestinal tissue samples from patients with and without IBD and analyzed them by flow cytometry. RESULTS: Pggt1bΔCD4 mice developed spontaneous colitis, characterized by thickening of the intestinal wall, edema, fibrosis, accumulation of T cells in the colon, and increased expression of inflammatory cytokines. Compared with control CD4+ T cells, PGGT1B-deficient CD4+ T cells expressed significantly higher levels of integrin alpha4beta7, which regulates their localization to the intestine. Inflammation induced by transfer of PGGT1B-deficient CD4+ T cells to Rag1-/- mice was blocked by injection of an antibody against integrin alpha4beta7. Lamina propria of Pggt1bΔCD4 mice had increased numbers of CD4+ T cells that expressed RORC and higher levels of cytokines produced by T-helper 17 cells (granulocyte-macrophage colony-stimulating factor, interleukin [IL]17A, IL17F, IL22, and tumor necrosis factor [TNF]). The RORC inverse agonist GSK805, but not antibodies against IL17A or IL17F, prevented colitis in Pggt1bΔCD4 mice. PGGT1B-deficient CD4+ T cells had decreased activation of RHOA. RhoAΔCD4 mice had a similar phenotype to Pggt1bΔCD4 mice, including development of colitis, increased numbers of CD4+ T cells in colon, increased expression of integrin alpha4beta7 by CD4+ T cells, and increased levels of IL17A and other inflammatory cytokines in lamina propria. T cells isolated from intestinal tissues from patients with IBD had significantly lower levels of PGGT1B than tissues from individuals without IBD. CONCLUSION: Loss of PGGT1B from T cells in mice impairs RHOA function, increasing CD4+ T-cell expression of integrin alpha4beta7 and localization to colon, resulting in increased expression of inflammatory cytokines and colitis. T cells isolated from gut tissues from patients with IBD have lower levels of PGGT1B than tissues from patients without IBD.

Deficiency of Isoprenylcysteine Carboxyl Methyltransferase (ICMT) Leads to Progressive Loss of Photoreceptor Function.

UNLABELLED: Retinal neurons use multiple strategies to fine-tune visual signal transduction, including post-translational modifications of proteins, such as addition of an isoprenyl lipid to a carboxyl-terminal cysteine in proteins that terminate with a "CAAX motif." We previously showed that RAS converting enzyme 1 (RCE1)-mediated processing of isoprenylated proteins is required for photoreceptor maintenance and function. However, it is not yet known whether the requirement for the RCE1-mediated protein processing is related to the absence of the endoproteolytic processing step, the absence of the subsequent methylation step by isoprenylcysteine methyltransferase (ICMT), or both. To approach this issue and to understand the significance of protein methylation, we generated mice lacking Icmt expression in the retina. In the absence of Icmt expression, rod and cone light-mediated responses diminished progressively. Lack of ICMT-mediated methylation led to defective association of isoprenylated transducin and cone phosphodiesterase 6 (PDE6α') with photoreceptor membranes and resulted in decreased levels of transducin, PDE6α', and cone G-protein coupled receptor kinase-1 (GRK1). In contrast to our earlier findings with retina-specific Rce1 knock-out mice, rod PDE6 in Icmt-deficient mice trafficked normally to the photoreceptor outer segment, suggesting that the failure to remove the -AAX is responsible for blocking the movement of PDE6 to the outer segment. Our findings demonstrate that carboxyl methylation of isoprenylated proteins is crucial for maintenance of photoreceptor function. SIGNIFICANCE STATEMENT: In this report, we show that an absence of isoprenylcysteine methyltransferase-mediated protein methylation leads to progressive loss of vision. Photoreceptors also degenerate, although at a slower pace than the rate of visual loss. The reduction in photoresponses is due to defective association of crucial players in phototransduction cascade. Unlike the situation with RCE1 deficiency, where both methylation and removal of -AAX were affected, the transport of isoprenylated proteins in isoprenylcysteine methyltransferase-deficient retinas was not dependent on methylation. This finding implies that the retention of the -AAX in PDE6 catalytic subunits in Rce1(-/-) mice is responsible for impeding their transport to the rod photoreceptor outer segment. In conclusion, lack of methylation of isoprenylcysteines leads to age-dependent photoreceptor dysfunction.

Role of the C-terminal basic amino acids and the lipid anchor of the Gγ2 protein in membrane interactions and cell localization.

Heterotrimeric G proteins are peripheral membrane proteins that frequently localize to the plasma membrane where their presence in molar excess over G protein coupled receptors permits signal amplification. Their distribution is regulated by protein-lipid interactions, which has a clear influence on their activity. Gßγ dimer drives the interaction between G protein heterotrimers with cell membranes. We focused our study on the role of the C-terminal region of the Gγ2 protein in G protein interactions with cell membranes. The Gγ2 subunit is modified at cysteine (Cys) 68 by the addition of an isoprenyl lipid, which is followed by the proteolytic removal of the last three residues that leaves an isoprenylated and carboxyl methylated Cys-68 as the terminal amino acid. The role of Cys isoprenylation of the CAAX box has been defined for other proteins, yet the importance of proteolysis and carboxyl methylation of isoprenylated proteins is less clear. Here, we showed that not only geranylgeranylation but also proteolysis and carboxyl methylation are essential for the correct localization of Gγ2 in the plasma membrane. Moreover, we showed the importance of electrostatic interactions between the inner leaflet of the plasma membrane and the positively charged C-terminal domain of the Gγ2 subunit (amino acids Arg-62, Lys-64 and Lys-65) as a second signal to reach the plasma membrane. Indeed, single or multiple point mutations at Gγ2 C-terminal amino acids have a significant effect on Gγ2 protein-plasma membrane interactions and its localization to charged Ld (liquid disordered) membrane microdomains. This article is part of a Special Issue entitled: Membrane Lipid Therapy: Drugs Targeting Biomembranes edited by Pablo V. Escribá.

Loss of one copy of Zfp148 reduces lesional macrophage proliferation and atherosclerosis in mice by activating p53.

RATIONALE: Cell proliferation and cell cycle control mechanisms are thought to play central roles in the pathogenesis of atherosclerosis. The transcription factor Zinc finger protein 148 (Zfp148) was shown recently to maintain cell proliferation under oxidative conditions by suppressing p53, a checkpoint protein that arrests proliferation in response to various stressors. It is established that inactivation of p53 accelerates atherosclerosis, but whether increased p53 activation confers protection against the disease remains to be determined. OBJECTIVE: We aimed to test the hypothesis that Zfp148 deficiency reduces atherosclerosis by unleashing p53 activity. METHODS AND RESULTS: Mice harboring a gene-trap mutation in the Zfp148 locus (Zfp148(gt/+)) were bred onto the apolipoprotein E (Apoe)(-/-) genetic background and fed a high-fat or chow diet. Loss of 1 copy of Zfp148 markedly reduced atherosclerosis without affecting lipid metabolism. Bone marrow transplantation experiments revealed that the effector cell is of hematopoietic origin. Peritoneal macrophages and atherosclerotic lesions from Zfp148(gt/+)Apoe(-/-) mice showed increased levels of phosphorylated p53 compared with controls, and atherosclerotic lesions contained fewer proliferating macrophages. Zfp148(gt/+)Apoe(-/-) mice were further crossed with p53-null mice (Trp53(-/-) [the gene encoding p53]). There was no difference in atherosclerosis between Zfp148(gt/+)Apoe(-/-) mice and controls on a Trp53(+/-) genetic background, and there was no difference in levels of phosphorylated p53 or cell proliferation. CONCLUSIONS: Zfp148 deficiency increases p53 activity and protects against atherosclerosis by causing proliferation arrest of lesional macrophages, suggesting that drugs targeting macrophage proliferation may be useful in the treatment of atherosclerosis.

Farnesyltransferase haplodeficiency reduces neuropathology and rescues cognitive function in a mouse model of Alzheimer disease.

Isoprenoids and prenylated proteins have been implicated in the pathophysiology of Alzheimer disease (AD), including amyloid-ß precursor protein metabolism, Tau phosphorylation, synaptic plasticity, and neuroinflammation. However, little is known about the relative importance of the two protein prenyltransferases, farnesyltransferase (FT) and geranylgeranyltransferase-1 (GGT), in the pathogenesis of AD. In this study, we defined the impact of deleting one copy of FT or GGT on the development of amyloid-ß (Aß)-associated neuropathology and learning/memory impairments in APPPS1 double transgenic mice, a well established model of AD. Heterozygous deletion of FT reduced Aß deposition and neuroinflammation and rescued spatial learning and memory function in APPPS1 mice. Heterozygous deletion of GGT reduced the levels of Aß and neuroinflammation but had no impact on learning and memory. These results document that farnesylation and geranylgeranylation play differential roles in AD pathogenesis and suggest that specific inhibition of protein farnesylation could be a potential strategy for effectively treating AD.

Targeting GGTase-I activates RHOA, increases macrophage reverse cholesterol transport, and reduces atherosclerosis in mice.

BACKGROUND: Statins have antiinflammatory and antiatherogenic effects that have been attributed to inhibition of RHO protein geranylgeranylation in inflammatory cells. The activity of protein geranylgeranyltransferase type I (GGTase-I) is widely believed to promote membrane association and activation of RHO family proteins. However, we recently showed that knockout of GGTase-I in macrophages activates RHO proteins and proinflammatory signaling pathways, leading to increased cytokine production and rheumatoid arthritis. In this study, we asked whether the increased inflammatory signaling of GGTase-I-deficient macrophages would influence the development of atherosclerosis in low-density lipoprotein receptor-deficient mice. METHODS AND RESULTS: Aortic lesions in mice lacking GGTase-I in macrophages (Pggt1b▵/▵) contained significantly more T lymphocytes than the lesions in controls. Surprisingly, however, mean atherosclerotic lesion area in Pggt1b▵/▵ mice was reduced by ≈60%. GGTase-I deficiency reduced the accumulation of cholesterol esters and phospholipids in macrophages incubated with minimally modified and acetylated low-density lipoprotein. Analyses of GGTase-I-deficient macrophages revealed upregulation of the cyclooxygenase 2-peroxisome proliferator-activated-γ pathway and increased scavenger receptor class B type I- and CD36-mediated basal and high-density lipoprotein-stimulated cholesterol efflux. Lentivirus-mediated knockdown of RHOA, but not RAC1 or CDC42, normalized cholesterol efflux. The increased cholesterol efflux in cultured cells was accompanied by high levels of macrophage reverse cholesterol transport and slightly reduced plasma lipid levels in vivo. CONCLUSIONS: Targeting GGTase-I activates RHOA and leads to increased macrophage reverse cholesterol transport and reduced atherosclerosis development despite a significant increase in inflammation.

Silencing RhoA inhibits migration and invasion through Wnt/ß-catenin pathway and growth through cell cycle regulation in human tongue cancer.

Ras homolog gene family member A (RhoA) has been identified as a critical regulator of tumor aggressive behavior. In this study, we assessed the role of RhoA in the mechanisms underlying growth, migration, and invasion of squamous cell carcinoma of tongue (TSCC). Stable RhoA knockdown of TSCC cell lines SCC-4 and CAL27 were achieved using Lentiviral transfection. The effects of RhoA depletion on cell migration, invasion, and cell proliferation were determined. The possible underlying mechanism of RhoA depletion on TSCC cell line was also evaluated by determining the expression of Galectin-3 (Gal-3), ß-catenin, and matrix metalloproteinase-9 (MMP-9) in vivo. Meanwhile, the underlying mechanism of TSCC growth was studied by analysis of cyclin D1/2, p21CIP1/WAF1, and p27Kip1 protein levels. Immunohistochemical assessments were performed to further prove the alteration of Gal-3 and ß-catenin expression. We found that, in mice injected with human TSCC cells in the tongue, RhoA levels were higher in primary tumors and metastasized lymph nodes compared with those in the normal tissues. Silencing of RhoA significantly reduced the tumor growth, decreased the levels of Gal-3, ß-catenin, MMP-9, and cyclin D1/2, and increased the levels of p21CIP1/WAF1 and p27Kip1. In vitro, RhoA knockdown also led to inhibition of cell migration, invasion, and proliferation. Our data suggest that RhoA plays a significant role in TSCC progression by regulating cell migration and invasion through Wnt/ß-catenin signaling pathway and cell proliferation through cell cycle regulation, respectively. RhoA might be a novel therapeutic target of TSCC.

RAS-converting enzyme 1-mediated endoproteolysis is required for trafficking of rod phosphodiesterase 6 to photoreceptor outer segments.

Prenylation is the posttranslational modification of a carboxyl-terminal cysteine residue of proteins that terminate with a CAAX motif. Following prenylation, the last three amino acids are cleaved off by the endoprotease, RAS-converting enzyme 1 (RCE1), and the prenylcysteine residue is methylated. Although it is clear that prenylation increases membrane affinity of CAAX proteins, less is known about the importance of the postprenylation processing steps. RCE1 function has been studied in a variety of tissues but not in neuronal cells. To approach this issue, we generated mice lacking Rce1 in the retina. Retinal development proceeded normally in the absence of Rce1, but photoreceptor cells failed to respond to light and subsequently degenerated in a rapid fashion. In contrast, the inner nuclear and ganglion cell layers were unaffected. We found that the multimeric rod phosphodiesterase 6 (PDE6), a prenylated protein and RCE1 substrate, was unable to be transported to the outer segments in Rce1-deficient photoreceptor cells. PDE6 present in the inner segment of Rce1-deficient photoreceptor cells was assembled and functional. Synthesis and transport of transducin, and rhodopsin kinase 1 (GRK1), also prenylated substrates of RCE1, was unaffected by Rce1 deficiency. We conclude that RCE1 is essential for the intracellular trafficking of PDE6 and survival of photoreceptor cells.

Targeting the protein prenyltransferases efficiently reduces tumor development in mice with K-RAS-induced lung cancer.

RAS and RHO proteins, which contribute to tumorigenesis and metastasis, undergo posttranslational modification with an isoprenyl lipid by protein farnesyltransferase (FTase) or protein geranylgeranyltransferase-I (GGTase-I). Inhibitors of FTase and GGTase-I were developed to block RAS-induced malignancies, but their utility has been difficult to evaluate because of off-target effects, drug resistance, and toxicity. Moreover, the impact of FTase deficiency and combined FTase/GGTase-I deficiency has not been evaluated with genetic approaches. We found that inactivation of FTase eliminated farnesylation of HDJ2 and H-RAS, prevented H-RAS targeting to the plasma membrane, and blocked proliferation of primary and K-RAS(G12D)-expressing fibroblasts. FTase inactivation in mice with K-RAS-induced lung cancer reduced tumor growth and improved survival, similar to results obtained previously with inactivation of GGTase-I. Simultaneous inactivation of FTase and GGTase-I markedly reduced lung tumors and improved survival without apparent pulmonary toxicity. These data shed light on the biochemical and therapeutic importance of FTase and suggest that simultaneous inhibition of FTase and GGTase-I could be useful in cancer therapeutics.

ROS-lowering doses of vitamins C and A accelerate malignant melanoma metastasis.

Oxidative stress is a barrier of migration and metastasis for malignant melanoma cells. Consequently, reducing oxidative stress with the antioxidant N-acetylcysteine (NAC) stimulates melanoma cell migration in vitro and metastasis in vivo. However, it is not yet known whether the NAC effect is shared with other antioxidants. Here, we screened 104 redox-active compounds and identify 27 that increase migration of human malignant melanoma cells in two doses. Validation experiments in four cell lines and four drug doses resulted in a list of 18 compounds which were ranked based on their ability to increase migration and reduce ROS levels; vitamin C (VitC) ranked as number one, followed by the vitamin E analogue Trolox and several carotenoids and Vitamin A-related compounds. Four diet-relevant compounds from this list-VitC, ß-carotene, retinyl palmitate, and canthaxanthin-were selected and found to accelerate metastasis in mice with BRAFV600E-driven malignant melanoma. Genomics analyses revealed that the transcription factor BACH1 is activated following antioxidant administration and knockout of Bach1 in mouse melanoma cells reduced lymph node and liver metastasis in xenograft mouse models. We conclude that a broad range of antioxidants accelerate melanoma migration and metastasis and that BACH1 is functionally linked to melanoma metastasis in vivo.

A MYC-controlled redox switch protects B lymphoma cells from EGR1-dependent apoptosis.

Refractory and relapsed B cell lymphomas are often driven by the difficult-to-target oncogene MYC. Here, we report that high MYC expression stimulates proliferation and protects B lymphoma cells from apoptosis under normal oxidative stress levels and that compounds including N-acetylcysteine (NAC) and vitamin C (VitC) induce apoptosis by reducing oxidative stress. NAC and VitC injections effectively reduce tumor growth in lymphoma cells with high MYC expression but not in those with low MYC expression. MYC knockdown confers tumor resistance to NAC and VitC, while MYC activation renders B cells sensitive to these compounds. Mechanistically, NAC and VitC stimulate MYC binding to EGR1 through Cys117 of MYC, shifting its transcriptional output from cell cycle to apoptosis gene expression. These results identify a redox-controlled mechanism for MYC's role in maintaining proliferation and preventing apoptosis, offering a potential therapeutic rationale for evaluating NAC or VitC in patients with MYC-driven B cell lymphoma.