guanosine-triphosphate and Melanoma

Signal transduction pathways post-translationally regulating nucleotide metabolism remain largely unknown. Guanosine monophosphate reductase (GMPR) is a nucleotide metabolism enzyme that decreases GTP pools by converting GMP to IMP. We observed that phosphorylation of GMPR at Tyr267 is critical for its activity and found that this phosphorylation by ephrin receptor tyrosine kinase EPHA4 decreases GTP pools in cell protrusions and levels of GTP-bound RAC1. EPHs possess oncogenic and tumor-suppressor activities, although the mechanisms underlying switches between these two modes are poorly understood. We demonstrated that GMPR plays a key role in EPHA4-mediated RAC1 suppression. This supersedes GMPR-independent activation of RAC1 by EPHA4, resulting in a negative overall effect on melanoma cell invasion and tumorigenicity. Accordingly, EPHA4 levels increase during melanoma progression and inversely correlate with GMPR levels in individual melanoma tumors. Therefore, phosphorylation of GMPR at Tyr267 is a metabolic signal transduction switch controlling GTP biosynthesis and transformed phenotypes.

Topics: GMP Reductase; Guanosine Triphosphate; Humans; Melanoma; Nucleotides; Phosphorylation; Receptor, EphA4

While several high-penetrance melanoma risk genes are known, variation in these genes fail to explain melanoma susceptibility in a large proportion of high-risk families. As part of a melanoma family sequencing study, including 435 families from Mediterranean populations we identified a novel NRAS variant (c.170A > C, p.D57A) in an Italian melanoma-prone family. This variant is absent in exomes in gnomAD, ESP, UKBiobank, and the 1000 Genomes Project, as well as in 11,273 Mediterranean individuals and 109 melanoma-prone families from the US and Australia. This variant occurs in the GTP-binding pocket of NRAS. Differently from other RAS activating alterations, NRAS D57A expression is unable to activate MAPK-pathway both constitutively and after stimulation but enhances EGF-induced PI3K-pathway signaling in serum starved conditions in vitro. Consistent with in vitro data demonstrating that NRAS D57A does not enrich GTP binding, molecular modeling suggests that the D57A substitution would be expected to impair Mg2 + binding and decrease nucleotide-binding and GTPase activity of NRAS. While we cannot firmly establish NRAS c.170A > C (p.D57A) as a melanoma susceptibility variant, further investigation of NRAS as a familial melanoma gene is warranted.

Topics: Cell Line, Tumor; Germ-Line Mutation; GTP Phosphohydrolases; Guanosine Triphosphate; Humans; Melanoma; Membrane Proteins; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-akt; Skin Neoplasms

The C-terminal guanine nucleotide exchange factor (GEF) module of Trio (TrioC) transfers signals from the Gα

Topics: Binding Sites; Carcinogenesis; Cell Line, Tumor; Crystallography, X-Ray; GTP-Binding Protein alpha Subunits, Gq-G11; Guanosine Triphosphate; HEK293 Cells; Humans; Melanoma; Models, Molecular; Mutation; Protein Binding; Protein Domains; Rho Guanine Nucleotide Exchange Factors; rhoA GTP-Binding Protein; Signal Transduction; Uveal Neoplasms

Melanoma progression is associated with increased invasion and, often, decreased levels of microphthalmia-associated transcription factor (MITF). Accordingly, downregulation of MITF induces invasion in melanoma cells; however, little is known about the underlying mechanisms. Here, we report for the first time that depletion of MITF results in elevation of intracellular GTP levels and increased amounts of active (GTP-bound) RAC1, RHO-A and RHO-C. Concomitantly, MITF-depleted cells display larger number of invadopodia and increased invasion. We further demonstrate that the gene for guanosine monophosphate reductase (GMPR) is a direct MITF target, and that the partial repression of GMPR accounts mostly for the above phenotypes in MITF-depleted cells. Reciprocally, transactivation of GMPR is required for MITF-dependent suppression of melanoma cell invasion, tumorigenicity and lung colonization. Moreover, loss of GMPR accompanies downregulation of MITF in vemurafenib-resistant BRAF

Topics: Animals; Cell Line, Tumor; Disease Models, Animal; Disease Progression; Ectopic Gene Expression; Extracellular Matrix; Female; Gene Expression Regulation, Neoplastic; GMP Reductase; Guanosine Triphosphate; Heterografts; Humans; Intracellular Space; Melanocytes; Melanoma; Melanoma, Experimental; Mice; Microphthalmia-Associated Transcription Factor; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasms; rho GTP-Binding Proteins

Ras guanosine triphosphatases (GTPases) regulate signaling pathways only when associated with cellular membranes through their C-terminal prenylated regions. Ras proteins move between membrane compartments in part via diffusion-limited, fluid phase transfer through the cytosol, suggesting that chaperones sequester the polyisoprene lipid from the aqueous environment. In this study, we analyze the nature of the pool of endogenous Ras proteins found in the cytosol. The majority of the pool consists of farnesylated, but not palmitoylated, N-Ras that is associated with a high molecular weight (HMW) complex. Affinity purification and mass spectrographic identification revealed that among the proteins found in the HMW fraction is VPS35, a latent cytosolic component of the retromer coat. VPS35 bound to N-Ras in a farnesyl-dependent, but neither palmitoyl- nor guanosine triphosphate (GTP)-dependent, fashion. Silencing VPS35 increased N-Ras's association with cytoplasmic vesicles, diminished GTP loading of Ras, and inhibited mitogen-activated protein kinase signaling and growth of N-Ras-dependent melanoma cells.

Topics: Animals; Cell Line, Tumor; Cytosol; Dogs; Gene Silencing; Green Fluorescent Proteins; GTP Phosphohydrolases; Guanosine Triphosphate; HEK293 Cells; Humans; Jurkat Cells; Lipoylation; Madin Darby Canine Kidney Cells; Melanoma; Membrane Proteins; Molecular Weight; Mutant Proteins; Protein Binding; Protein Prenylation; Protein Transport; Subcellular Fractions; Vesicular Transport Proteins

NRAS mutation at codons 12, 13, or 61 is associated with transformation; yet, in melanoma, such alterations are nearly exclusive to codon 61. Here, we compared the melanoma susceptibility of an NrasQ61R knock-in allele to similarly designed KrasG12D and NrasG12D alleles. With concomitant p16INK4a inactivation, KrasG12D or NrasQ61R expression efficiently promoted melanoma in vivo, whereas NrasG12D did not. In addition, NrasQ61R mutation potently cooperated with Lkb1/Stk11 loss to induce highly metastatic disease. Functional comparisons of NrasQ61R and NrasG12D revealed little difference in the ability of these proteins to engage PI3K or RAF. Instead, NrasQ61R showed enhanced nucleotide binding, decreased intrinsic GTPase activity, and increased stability when compared with NrasG12D. This work identifies a faithful model of human NRAS-mutant melanoma, and suggests that the increased melanomagenecity of NrasQ61R over NrasG12D is due to heightened abundance of the active, GTP-bound form rather than differences in the engagement of downstream effector pathways.. This work explains the curious predominance in human melanoma of mutations of codon 61 of NRAS over other oncogenic NRAS mutations. Using conditional "knock-in" mouse models, we show that physiologic expression of NRASQ61R, but not NRASG12D, drives melanoma formation.

Topics: Alleles; AMP-Activated Protein Kinase Kinases; Animals; Cell Line, Tumor; Cell Transformation, Neoplastic; Codon; Extracellular Signal-Regulated MAP Kinases; Gene Deletion; Gene Order; Genes, ras; Genetic Loci; Genotype; Guanosine Triphosphate; Humans; Melanoma; Mice; Mitogen-Activated Protein Kinases; Mutation; Neoplasm Metastasis; Oncogene Proteins, Fusion; Phosphatidylinositol 3-Kinases; Protein Binding; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins B-raf; Tumor Burden

Melanoma is one of the most aggressive types of human cancers, and the mechanisms underlying melanoma invasive phenotype are not completely understood. Here, we report that expression of guanosine monophosphate reductase (GMPR), an enzyme involved in de novo biosynthesis of purine nucleotides, was downregulated in the invasive stages of human melanoma. Loss- and gain-of-function experiments revealed that GMPR downregulates the amounts of several GTP-bound (active) Rho-GTPases and suppresses the ability of melanoma cells to form invadopodia, degrade extracellular matrix, invade in vitro, and grow as tumor xenografts in vivo. Mechanistically, we demonstrated that GMPR partially depletes intracellular GTP pools. Pharmacological inhibition of de novo GTP biosynthesis suppressed whereas addition of exogenous guanosine increased invasion of melanoma cells as well as cells from other cancer types. Our data identify GMPR as a melanoma invasion suppressor and establish a link between guanosine metabolism and Rho-GTPase-dependent melanoma cell invasion.

Topics: Animals; Cell Line, Tumor; Cell Movement; Extracellular Matrix; GMP Reductase; Guanosine Triphosphate; HCT116 Cells; Humans; IMP Dehydrogenase; Melanoma; Mice; Phenotype; Purine Nucleosides; rac1 GTP-Binding Protein; rho GTP-Binding Proteins; RNA Interference; RNA, Small Interfering; Transplantation, Heterologous

RAC1 is a small, Ras-related GTPase that was recently reported to harbor a recurrent UV-induced signature mutation in melanoma, resulting in substitution of P29 to serine (RAC1(P29S)), ranking this the third most frequently occurring gain-of-function mutation in melanoma. Although the Ras family GTPases are mutated in about 30% of all cancers, mutations in the Rho family GTPases have rarely been observed. In this study, we demonstrate that unlike oncogenic Ras proteins, which are primarily activated by mutations that eliminate GTPase activity, the activated melanoma RAC1(P29S) protein maintains intrinsic GTP hydrolysis and is spontaneously activated by substantially increased inherent GDP/GTP nucleotide exchange. Determination and comparison of crystal structures for activated RAC1 GTPases suggest that RAC1(F28L)--a known spontaneously activated RAC1 mutant--and RAC1(P29S) are self-activated in distinct fashions. Moreover, the mechanism of RAC1(P29S) and RAC1(F28L) activation differs from the common oncogenic mutations found in Ras-like GTPases that abrogate GTP hydrolysis. The melanoma RAC1(P29S) gain-of-function point mutation therefore represents a previously undescribed class of cancer-related GTPase activity.

Topics: Amino Acid Substitution; Animals; Cell Surface Extensions; Chlorocebus aethiops; COS Cells; Crystallography, X-Ray; Enzyme Activation; Genetic Association Studies; Guanosine Triphosphate; Humans; Hydrolysis; Kinetics; Melanoma; Mice; Microscopy, Fluorescence; Models, Molecular; Mutation, Missense; NIH 3T3 Cells; Oncogenes; rac1 GTP-Binding Protein; Recombinant Fusion Proteins; Signal Transduction; Static Electricity

Recently we reported that activation of Epac1, an exchange protein activated by cAMP, increases melanoma cell migration via Ca 2+ release from the endoplasmic reticulum (ER). G-protein βγ subunits (Gβγ) are known to act as an independent signaling molecule upon activation of G-protein coupled receptor. However, the role of Gβγ in cell migration and Ca 2+ signaling in melanoma has not been well studied. Here we report that there is crosstalk of Ca 2+ signaling between Gβγ and Epac in melanoma, which plays a role in regulation of cell migration.. SK-Mel-2 cells, a human metastatic melanoma cell line, were mainly used in this study. Intracellular Ca 2+ was measured with Fluo-4AM fluorescent dyes. Cell migration was examined using the Boyden chambers.. The effect of Gβγ on Epac-induced cell migration was first examined. Epac-induced cell migration was inhibited by mSIRK, a Gβγ -activating peptide, but not its inactive analog, L9A, in SK-Mel-2 cells. Guanosine 5', α-β-methylene triphosphate (Gp(CH2)pp), a constitutively active GTP analogue that activates Gβγ, also inhibited Epac-induced cell migration. In addition, co-overexpression of β1 and γ2, which is the major combination of Gβγ, inhibited Epac1-induced cell migration. By contrast, when the C-terminus of β adrenergic receptor kinase (βARK-CT), an endogenous inhibitor for Gβγ, was overexpressed, mSIRK's inhibitory effect on Epac-induced cell migration was negated, suggesting the specificity of mSIRK for Gβγ. We next examined the effect of mSIRK on Epac-induced Ca 2+ response. When cells were pretreated with mSIRK, but not with L9A, 8-(4-Methoxyphenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (8-pMeOPT), an Epac-specific agonist, failed to increase Ca 2+ signal. Co-overexpression of β1 and γ2 subunits inhibited 8-pMeOPT-induced Ca 2+ elevation. Inhibition of Gβγ with βARK-CT or guanosine 5'-O-(2-thiodiphosphate) (GDPβS), a GDP analogue that inactivates Gβγ, restored 8-pMeOPT-induced Ca 2+ elevation even in the presence of mSIRK. These data suggested that Gβγ inhibits Epac-induced Ca 2+ elevation. Subsequently, the mechanism by which Gβγ inhibits Epac-induced Ca 2+ elevation was explored. mSIRK activates Ca 2+ influx from the extracellular space. In addition, W-5, an inhibitor of calmodulin, abolished mSIRK's inhibitory effects on Epac-induced Ca 2+ elevation, and cell migration. These data suggest that, the mSIRK-induced Ca 2+ from the extracellular space inhibits the Epac-induced Ca 2+ release from the ER, resulting suppression of cell migration.. We found the cross talk of Ca 2+ signaling between Gβγ and Epac, which plays a major role in melanoma cell migration.

Topics: Amino Acid Sequence; beta-Adrenergic Receptor Kinases; Calcium Channel Blockers; Calcium Signaling; Calmodulin; Cell Line, Tumor; Cell Movement; GTP-Binding Protein beta Subunits; GTP-Binding Proteins; Guanine Nucleotide Exchange Factors; Guanosine Diphosphate; Guanosine Triphosphate; Humans; Melanoma; Molecular Sequence Data; Neoplasm Proteins; Peptide Fragments; Peptides; Recombinant Fusion Proteins; Recombinant Proteins; Thionucleotides

This study shows that the small GTP-binding protein ADP-ribosylation factor 6 (ARF6) is an important regulator of tumor growth and metastasis. Using spontaneous melanoma tumor growth assays and experimental metastasis assays in nude mice, we show that sustained activation of ARF6 reduces tumor mass growth but significantly enhances the invasive capacity of tumor cells. In contrast, mice injected with tumor cells expressing a dominantly inhibitory ARF6 mutant exhibited a lower incidence and degree of invasion and lung metastasis compared with control animals. Effects on tumor growth correlate with reduced cell proliferation capacity and are linked at least in part to alterations in mitotic progression induced by defective ARF6 cycling. Furthermore, phospho-ERK levels in subcultured cells from ARF6(GTP) and ARF6(GDP) tumor explants correlate with invasive capacity. ARF6-induced extracellular signal-regulated kinase (ERK) signaling leads to Rac1 activation to promote invadopodia formation and cell invasion. These findings document an intricate role for ARF6 and the regulation of ERK activation in orchestrating mechanisms underlying melanoma growth, invasion, and metastases.

Topics: ADP-Ribosylation Factor 6; ADP-Ribosylation Factors; Animals; Cell Growth Processes; Cell Line, Tumor; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Guanosine Diphosphate; Guanosine Triphosphate; Humans; Lung Neoplasms; Male; Melanoma; Mice; Phospholipase D

Tumor cell invasion through the extracellular matrix is accompanied by the formation of invadopodia, which are actin-rich protrusions at the adherent surface of cells at sites of extracellular matrix degradation. Using the invasive human melanoma cell line LOX as a model system, we demonstrate that the ADP-ribosylation factor 6 (ARF6) GTPase is an important regulator of invadopodia formation and cell invasion. We show that ARF6 localizes to invadopodia of LOX cells. Sustained activation of ARF6 significantly enhances the invasive capacity of melanoma as well as breast tumor cell lines, whereas dominant negative ARF6 abolishes basal cell invasive capacity as well as invasion induced by growth factors. Furthermore, using biochemical assays, we show that enhanced invasive capacity is accompanied by the activation of endogenous ARF6. Finally, we provide evidence that ARF6-enhanced melanoma cell invasion depends on the activation of the extracellular signal-regulated kinase (ERK), and that the ARF6 GTPase cycle regulates ERK activation. This study describes a vital role for ARF6 in melanoma cell invasion and documents a link between ARF6-mediated signaling and ERK activation.

Topics: ADP-Ribosylation Factor 6; ADP-Ribosylation Factors; Cell Line, Tumor; Enzyme Activation; Growth Substances; Guanosine Triphosphate; Humans; Hydrolysis; MAP Kinase Signaling System; Melanoma; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Neoplasm Invasiveness

Phospholipase D (PLD) is a highly regulated enzyme involved in lipid-mediated signal transduction processes affecting vesicular trafficking and cytoskeletal reorganization. It is regulated by protein kinase C, adenosine diphosphate (ADP)-ribosylation factors and Rho family proteins, and both protein kinase C and Rho family proteins have been implicated in the metastatic potential of melanoma. We analysed PLD in four human melanoma cell lines and in primary human melanocytes. Melanoma cell lines showed phosphatidylcholine-hydrolysing, phosphatidylinositol 4,5-bisphosphate-dependent PLD activity, which was activated by phorbol ester and a non-hydrolysable guanosine triphosphate (GTP) analogue in a dose-dependent and synergistic manner, whereas primary melanocytes exhibited only low PLD activity compared with the melanoma cell lines. As determined by reverse transcription polymerase chain reaction, both splicing variants of PLD1, PLD1a and PLD1b, and the isoenzyme PLD2, are expressed in melanoma cells and melanocytes. Western blot analysis showed that PLD1 expression was low in primary melanocytes in contrast to melanoma cells, which is in agreement with our finding of low activity. Interestingly, Rho protein mRNA was elevated in all melanoma cell lines. We conclude that in human melanoma cells, the PLD activity that is stimulated by phorbol ester requires ADP-ribosylation factor, protein kinase C and Rho proteins for full activity, and most probably represents the isoenzyme PLD1.

Topics: Adenosine Diphosphate; ADP-Ribosylation Factors; Alternative Splicing; Blotting, Western; Cell Division; Cell Line, Tumor; Cells, Cultured; DNA, Complementary; Dose-Response Relationship, Drug; Enzyme Activation; Gene Expression Regulation, Enzymologic; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Humans; Immunoblotting; Melanocytes; Melanoma; Neoplasm Metastasis; Oleic Acid; Phorbol Esters; Phospholipase D; Protein Isoforms; Protein Kinase C; Reverse Transcriptase Polymerase Chain Reaction; rho GTP-Binding Proteins; RNA; RNA, Messenger

Topics: Adenosine Triphosphate; Cell Membrane; Cytoskeleton; Guanosine Triphosphate; Melanoma; Melanosomes; Microscopy, Electron; Models, Biological; Myosins; Protein Binding; rab GTP-Binding Proteins; Tumor Cells, Cultured

The Ser122 --> Pro mutation in human nucleoside diphosphate kinase (NDK)-B/Nm23-H2 was recently found in melanoma cells. In comparison to the wild-type enzyme, steady state activity of NDKS122P with ATP and TDP as substrates was slowed down 5-fold. We have utilized transient kinetic techniques to analyze phosphoryl transfer between the mutant enzyme and various pairs of nucleoside triphosphates and nucleoside diphosphates. The two half-reactions of phosphorylation and dephosphorylation of the active site histidine residue (His118) were studied separately by making use of the intrinsic fluorescence changes which occur during these reactions. All apparent second order rate constants are drastically reduced, falling 5-fold for phosphorylation and 40-200-fold for dephosphorylation. Also, the reactivity of the mutant with pyrimidine nucleotides and deoxy nucleotides is more than 100-fold reduced compared with the wild-type. Thus, the rate-limiting step of the NDK-BS122P-catalyzed reaction is phosphoryl transfer from the phospho-enzyme intermediate to the nucleoside diphosphate and not phosphoryl transfer from the nucleoside triphosphate to the enzyme as was found for the wild-type protein. This results in a pronounced shift of the equilibrium between unphosphorylated and phosphorylated enzyme. Moreover, like the Killer-of-prune mutation in Drosophila NDK and the neuroblastoma Ser120 --> Gly mutation in human NDK-A/Nm23-H1, the Ser122 --> Pro substitution in NDK-B affects the stability of the protein toward heat and urea. These significantly altered properties may be relevant to the role of the mutant enzyme in various intracellular processes.

Topics: Adenosine Triphosphate; Base Sequence; DNA Primers; Guanosine Triphosphate; Humans; Melanoma; Monomeric GTP-Binding Proteins; Mutation; NM23 Nucleoside Diphosphate Kinases; Nucleoside-Diphosphate Kinase; Phosphorylation; Transcription Factors; Tumor Cells, Cultured

The effects of the alpha-melanocyte-stimulating hormone (alpha-MSH) (10(-7)-10(-5) M) and the beta-adrenoceptor agonist isoprenaline (10(-9)-10(-4) M) on adenylate cyclase (AC) activity were investigated in homogenates of the human IGR 1 melanoma cells with or without additional GTP. Basal AC activity was increased by the administration of 10 microM GTP. Alpha-MSH had no effect on cyclic AMP (cAMP) accumulation, while isoprenaline stimulated AC activity in a dose-dependent manner.

Topics: Adenylyl Cyclases; Cells, Cultured; Cyclic AMP; Guanosine Triphosphate; Humans; Isoproterenol; Melanocyte-Stimulating Hormones; Melanoma

The liver was the organ most frequently involved in 50 patients with primary melanomas of the choroid or ciliary body, who were treated with enucleation or cobalt plaque radiotherapy and who subsequently developed systemic metastasis. Forty-seven (94%) of the patients had clinical or laboratory evidence of liver metastasis. The median survival time following treatment of the primary choroidal melanoma was 28 months (range, 7.7 to 123.1 months). Results of pretreatment studies, including serum liver enzyme levels, liver scans, or both, were abnormal in six (12%) of the patients studied. As a group, the eight patients who died within one year following ocular surgery had a significantly higher prevalence of pretreatment laboratory abnormalities. Extraocular extension of the choroidal melanoma was also present in four (50%) of these eight patients. Conversely, no patient who survived longer than the median survival period (28 months) had abnormal pretreatment laboratory findings.

Topics: Adult; Aged; Alkaline Phosphatase; Aspartate Aminotransferases; Female; Guanosine Triphosphate; Humans; L-Lactate Dehydrogenase; Liver Neoplasms; Male; Melanoma; Middle Aged; Radiography; Uveal Neoplasms