Histone deacetylase inhibitors induce growth arrest and differentiation in uveal melanoma.

PURPOSE: Metastasis is responsible for the death of most cancer patients, yet few therapeutic agents are available which specifically target the molecular events that lead to metastasis. We recently showed that inactivating mutations in the tumor suppressor gene BAP1 are closely associated with loss of melanocytic differentiation in uveal melanoma (UM) and metastasis. The purpose of this study was to identify therapeutic agents that reverse the phenotypic effects of BAP1 loss in UM. EXPERIMENTAL DESIGN: In silico screens were done to identify therapeutic compounds predicted to differentiate UM cells using Gene Set Enrichment Analysis and Connectivity Map databases. Valproic acid (VPA), trichostatin A, LBH-589, and suberoylanilide hydroxamic acid were evaluated for their effects on UM cells using morphologic evaluation, MTS viability assays, bromodeoxyuridine incorporation, flow cytometry, clonogenic assays, gene expression profiling, histone acetylation and ubiquitination assays, and a murine xenograft tumorigenicity model. RESULTS: Histone deacetylase (HDAC) inhibitors induced morphologic differentiation, cell-cycle exit, and a shift to a differentiated, melanocytic gene expression profile in cultured UM cells. VPA inhibited the growth of UM tumors in vivo. CONCLUSIONS: These findings suggest that HDAC inhibitors may have therapeutic potential for inducing differentiation and prolonged dormancy of micrometastatic disease in UM.

ABCB1 identifies a subpopulation of uveal melanoma cells with high metastatic propensity.

Metastasis of tumor cells to distant organs is the leading cause of death in melanoma. Yet, the mechanisms of metastasis remain poorly understood. One key question is whether all cells in a primary tumor are equally likely to metastasize or whether subpopulations of cells preferentially give rise to metastases. Here, we identified a subpopulation of uveal melanoma cells expressing the multidrug resistance transporter ABCB1 that are highly metastatic compared to ABCB1(-) bulk tumor cells. ABCB1(+) cells also exhibited enhanced clonogenicity, anchorage-independent growth, tumorigenicity and mitochondrial activity compared to ABCB1(-) cells. A375 cutaneous melanoma cells contained a similar subpopulation of highly metastatic ABCB1(+) cells. These findings suggest that some uveal melanoma cells have greater potential for metastasis than others and that a better understanding of such cells may be necessary for more successful therapies for metastatic melanoma.

Protein O-GlcNAc modulates motility-associated signaling intermediates in neutrophils.

The modification of serine/threonine residues on cytoplasmic and nuclear proteins by N-acetylglucosamine (O-GlcNAc) is suggested to play a role in the regulation of a variety of signal transduction pathways. We have previously shown that glucosamine (GlcNH(2)), a metabolic precursor of O-GlcNAcylation, increases (2)O-GlcNAc and enhances motility in neutrophils. Here, we extend this correlation by showing that a mechanistically distinct means of increasing O-GlcNAc, achieved by inhibition of O-GlcNAc removal with O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc), increases basal cellular motility and directional migration induced by the chemoattractant formyl-methionine-leucine-phenylalanine (fMLP). Furthermore, we demonstrate that O-GlcNAc modulates the activities of signaling intermediates known to regulate neutrophil movement. GlcNH(2) and PUGNAc increase both the basal and fMLP-induced activity of a central mediator of cellular motility, the small GTPase Rac. Phosphoinositide 3-kinase, an important regulator of Rac activity and neutrophil motility, is shown to regulate the signaling pathway on which GlcNH(2) and PUGNAc act. Rac is an important upstream regulatory element in p38 and p44/42 mitogen-activated protein kinase (MAPK) signaling in neutrophils, and these MAPKs are implicated in chemotactic signal transduction. We show that GlcNH(2) and PUGNAc treatment increases p42/44 and p38 MAPK activities and that these increases are associated with activation of upstream MAPK kinases. These data indicate that O-GlcNAcylation is an important signaling element in neutrophils that modulates the activities of several critical signaling intermediates involved in the regulation of cellular movement.

Neutrophils exhibit rapid agonist-induced increases in protein-associated O-GlcNAc.

A variety of cytoplasmic and nuclear proteins can be modified on serine and threonine residues by O-linked beta-N-acetylglucosamine (O-GlcNAc), although the effects of this modification on protein and cellular functions are not completely defined. The sugar donor for the O-GlcNAc transferase that catalyzes this post-translational modification is UDP-N-acetylglucosamine (UDP-GlcNAc), a product of the hexosamine biosynthesis pathway (HBP). Here, the dynamics of the O-GlcNAc modification are examined in the physiological context of agonist-induced signal transduction using neutrophils. Formylated Met-Leu-Phe (fMLF) is shown to stimulate a rapid and transient increase in protein O-GlcNAcylation in both immunoblot and immunofluorescence imaging assays using O-GlcNAc-specific antibodies. In high performance liquid chromatography analyses of HBP metabolic activity, short term exposure to an exogenous substrate of the HBP, glucosamine (GlcNH(2)), leads to increased GlcNH(2) 6-phosphate and then UDP-GlcNAc levels. The GlcNH(2) treatments also increase O-GlcNAcylation and augment the aforementioned fMLF-associated increase. In functional assays, GlcNH(2) pre-treatment selectively augments fMLF-induced chemotaxis but has little effect on respiratory burst activity. Furthermore, augmenting levels of O-GlcNAc in the absence of agonist is sufficient to stimulate chemotaxis. These data demonstrate that neutrophils possess a functionally significant O-GlcNAcylation pathway that is robustly induced by stimulation with agonist. We propose that O-GlcNAcylation plays an important role in rapid and dynamic neutrophil signal transduction, especially with respect to chemotaxis.