Tumor angiogenesis is a key event in cancer progression. Here, we report that tumors can stimulate tumor angiogenesis by secretion of galectin-1. Tumor growth and tumor angiogenesis of different tumor models are hampered in galectin-1-null (gal-1 −/− ) mice. However, tumor angiogenesis is less affected when tumor cells express and secrete high levels of galectin-1. Furthermore, tumor endothelial cells in gal-1 −/− mice take up galectin-1 that is secreted by tumor cells. Uptake of galectin-1 by cultured endothelial cells specifically promotes H-Ras signaling to the Raf/mitogen-activated protein kinase/extracellular signal-regulated kinase (Erk) kinase (Mek)/Erk cascade and stimulates endothelial cell proliferation and migration. Moreover, the activation can be blocked by galectin-1 inhibition as evidenced by hampered membrane translocation of H-Ras.GTP and impaired Raf/Mek/Erk phosphorylation after treatment with the galectin-1-targeting angiogenesis inhibitor anginex. Altogether, these data identify galectin-1 as a proangiogenic factor. These findings have direct implications for current efforts on galectin-1-targeted cancer therapies. Cancer Res; 70(15); 6216-24. ©2010 AACR.

1. Ras signaling and oncogenesis depend on the dynamic interplay of Ras with distinctive plasma membrane (PM) microdomains and various intracellular compartments. Such interaction is dictated by individual elements in the carboxy-terminal domain of the Ras proteins, including a farnesyl isoprenoid group, sequences in the hypervariable region (hvr)-linker, and palmitoyl groups in H/N-Ras isoforms. 2. The farnesyl group acts as a specific recognition unit that interacts with prenyl-binding pockets in galectin-1 (Gal-1), galectin-3 (Gal-3), and cGMP phosphodiesterase delta. This interaction appears to contribute to the prolongation of Ras signals in the PM, the determination of Ras effector usage, and perhaps also the transport of cytoplasmic Ras. Gal-1 promotes H-Ras signaling to Raf at the expense of phosphoinositide 3-kinase (PI3-K) and Ral guanine nucleotide exchange factor (RalGEF), while galectin-3 promotes K-Ras signaling to both Raf and PI3-K. 3. The hvr-linker and the palmitates of H-Ras and N-Ras determine the micro- and macro-localizations of these proteins in the PM and in the Golgi, as well as in 'rasosomes', randomly moving nanoparticles that carry palmitoylated Ras proteins and their signal through the cytoplasm.4. The dynamic compartmentalization of Ras proteins contributes to the spatial organization of Ras signaling, promotes redistribution of Ras, and provides an additional level of selectivity to the signal output of this regulatory GTPase.

Purpose: Farnesylthiosalicylic acid (FTS) is a Ras inhibitor that dislodges all active Ras isoforms from the membrane.We assessed the ability of FTS to reverse the transformed phenotype of neurofibromatosis type 1 (NF1)^associated tumor cell lines of malignant peripheral nerve sheath tumor (MPNST). Experimental Design: nf1 mutations were genotyped, allelic losses were analyzed, and neurofibromin expression levels were determined in MPNST cell lines ST88-14, S265P21, and 90-8. The effects of FTS on GTP-bound Ras (Ras-GTP) and its prominent downstream targets, as well as on cell morphology, anchorage-dependent and anchorage-independent growth, and tumor growth in mice, were assessed. Results: The MPNSTcell lines were biallelic, NF1inactive, and neurofibromin deficient. We show that FTS treatment shortened the relatively long duration of Ras activation and signaling to extracellular signal-regulated kinase, Akt, and RalA in all NF1-deficient MPNST cell lines (NF1 cells) to that observed in a non-NF1, normally expressing neurofibromin MPNSTcell line. These effects of FTS led to lower steady-state levels of Ras-GTP and its activated targets. Both anchoragedependent and anchorage-independent growth of NF1cells were dose dependently inhibited by FTS, and the inhibition correlated positively with Ras-GTP levels. NF1cells were found to possess strong actin stress fibers, and this phenotype was also corrected by FTS. NF1tumor growth in a nude mouse model was inhibited by oral FTS. Conclusions: FTS treatment of NF1cells normalized Ras-GTP levels, resulting in reversal of the transformed phenotype and inhibition of tumor growth. FTS may therefore be considered as a potential drug for the treatment of NF1.

The Arf1-directed GTPase-activating protein ArfGAP1 is a Golgi-localized protein that controls the dynamics of the COPI coat of carriers that mediate transport in the endoplasmic reticulumGolgi shuttle. Previously the interaction of ArfGAP1 with the Golgi was allocated to a portion of the non-catalytic, carboxyl part of the protein, but the mechanism of this interaction has not been established. In this study we identify a short stretch in the non-catalytic part of ArfGAP1 (residues 204 -214) in which several hydrophobic residues contribute to Golgi localization. Even single alanine replacement of two of these residues (Leu-207 and Trp-211) strongly diminished Golgi localization. Mutations in the hydrophobic residues also diminished the in vitro activity of ArfGAP1 on Arf1 bound to Golgi membranes. The stretch containing the hydrophobic residues was recently shown to mediate the binding of ArfGAP1 to loosely packed lipids of highly curved liposomes (Bigay, J., Casella, J. F., Drin, G., Mesmin, B., and Antonny, B. (2005) EMBO J. 24, 2244 -2253). Whereas short fragments containing the hydrophobic stretch were not Golgi-localized, a proximal 10-residue inframe insertion that is present in new ArfGAP1 isoforms that we identified in brain and heart tissues could confer Golgi localization on these fragments. This localization was abrogated by alanine replacement of residues Phe-240 or Trp-241 of the insertion sequence but not by their replacement with leucines. Our findings indicate that ArfGAP1 interacts with the Golgi through multiple hydrophobic motifs and that alternative modes of interaction may exist in tissue-specific ArfGAP1 isoforms.Membrane traffic in the endoplasmic reticulum-Golgi shuttle is mediated by the COPI 3 and COPII trafficking systems. The COPII system mediates the initial exit from the endoplasmic reticulum, whereas subsequent transport to the Golgi apparatus and retrograde Golgi to endoplasmic reticulum traffic involves COPI carriers. The COPI and COPII coats are composed of evolutionarily distinct sets of proteins yet follow similar pathways of coat assembly and disassembly (for recent reviews see Refs. 1-3). In both systems a small GTPase (Arf1 and Sar1 for COPI and COPII, respectively) plays a key regulatory role. Following activation by a guanine nucleotide exchange protein, the GTPase translocates from cytosol to the organelle membrane, where it initiates the process of coat formation by the direct binding of coat subunits (4 -6). The coat in turn recruits cargo and polymerizes causing membrane deformation to form a bud.In the second phase of the GTPase cycle of Arf1 and Sar1, bound GTP is hydrolyzed with the aid of a GTPase-activating protein (GAP). The hydrolysis of GTP is a prerequisite for coat dissociation, and its inhibition leads to the accumulation of coated vesicles that are prevented from fusing with the target membrane (4, 7). The GAP for Sar1 is a subunit of the first layer of the COPII coat, the Sec23/24 complex (8). ArfGAPs constitute a large family of proteins containing a hi...