Galit Elad-Sfadia

Galectin-1 binds oncogenic H-Ras to mediate Ras membrane anchorage and cell transformation

Ras genes, frequently mutated in human tumors, promote malignant transformation. Ras transformation requires membrane anchorage, which is promoted by Ras farnesylcysteine carboxymethylester and by a second signal. Previously we showed that the farnesylcysteine mimetic, farnesylthiosalicylic acid (FTS) disrupts Ras membrane anchorage. To understand how this disruption contributes to inhibition of cell transformation we searched for new Ras-interacting proteins and identified galectin-1, a lectin implicated in human tumors, as a selective binding partner of oncogenic H-Ras(12V). The observed size of H-Ras(12V)-galectin-1 complex, which is equal to the sum of the molecular weights of Ras and galectin-1 indicates a direct binding interaction between the two proteins. FTS disrupted H-Ras(12V)-galectin-1 interactions. Overexpression of galectin-1 increased membrane-associated Ras, Ras-GTP, and active ERK resulting in cell transformation, which was blocked by dominant negative Ras. Galectin-1 antisense RNA inhibited transformation by H-Ras(12V) and abolished membrane anchorage of green fluorescent protein (GFP)-H-Ras(12V) but not of GFP-H-Ras wild-type (wt), GFP-K-Ras(12V), or GFP-N-Ras(13V). H-Ras(12V)-galectin-1 interactions establish an essential link between two proteins associated with cell transformation and human malignancies that can be exploited to selectively target oncogenic Ras proteins.

Spatiotemporal Organization of Ras Signaling: Rasosomes and the Galectin Switch

Summary1. 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 δ. 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.

Galectin‐3/MAC‐2, Ras and PI3K activate complement receptor‐3 and scavenger receptor‐AI/II mediated myelin phagocytosis in microglia

The removal of degenerated myelin is essential for repair in Wallerian degeneration that follows traumatic injury to axons and in autoimmune demyelinating diseases (e.g., multiple sclerosis). Microglia can remove degenerated myelin through phosphatidylinositol‐3‐kinase (PI3K)‐dependent phagocytosis mediated by complement receptor‐3 (CR3/MAC‐1) and scavenger receptor‐AI/II (SRAI/II). Paradoxically, these receptors are expressed in microglia after injury but myelin is not phagocytosed. Additionally, Galectin‐3/MAC‐2 is expressed in microglia that phagocytose but not in microglia that do not phagocytose, suggesting that Galectin‐3/MAC‐2 is instrumental in activating phagocytosis. S‐trans, trans‐farnesylthiosalicylic (FTS), which inhibits Galectin‐3/MAC‐2 dependent activation of PI3K through Ras, inhibited phagocytosis. K‐Ras‐GTP levels and PI3K activity increased during normal phagocytosis and decreased during FTS‐inhibited phagocytosis. Galectin‐3/MAC‐2, which binds and stabilizes active Ras, coimmunoprecipitated with Ras and levels of the coimmunoprecipitate increased during normal phagocytosis. A role for Galectin‐3/MAC‐2 dependent activation of PI3K through Ras, mostly K‐Ras, is thus suggested. An explanation may thus be offered for deficient phagocytosis by microglia that express CR3/MAC‐1 and SRAI/II without Galectin‐3/MAC‐2 and efficient phagocytosis when CR3/MAC‐1 and SRAI/II are co‐expressed with Galectin‐3/MAC‐2.

Oncogenic synergism between ErbB1, nucleolin and mutant Ras

Alterations in the ErbB family of growth factor receptors, their signaling components, and mutational activation of Ras proteins are major contributors to malignant transformation. Recently, mutant Ras was shown to be capable of activating ErbB receptors in a ligand-independent manner. Furthermore, it was observed that nucleolin, a transcriptional regulator and ribosome biogenesis factor, can bind both K-Ras and the cytoplasmic tail of ErbB receptors to enhance ErbB receptor activation. However, the functional significance of these interactions to cancer pathogenesis has not been probed. Here, we show that endogenous nucleolin interacts simultaneously in vivo with endogenous Ras and ErbB1 (EGFR) in cancer cells. The C-terminal 212 amino acids of nucleolin were determined to be sufficient to interact with ErbB1 and all Ras protein isoforms (H-, N-, and K-Ras). Nucleolin partially colocalizes with Ras at the plasma membrane. Moreover, activated but not wild-type Ras facilitates nucleolin interaction with ErbB1 and stabilizes ErbB1 receptor levels. Most importantly, these three oncogenes synergistically facilitate anchorage-independent cell growth in vitro and tumor growth in vivo. Our findings suggest strategies to target nucleolin as a general approach to inhibiting ErbB- and Ras-driven cancers.

Suppression of lung cancer tumor growth in a nude mouse model by the Ras inhibitor salirasib (farnesylthiosalicylic acid)

Aberrant Ras pathway functions contribute to the malignant phenotype of lung cancers. Inhibitors of Ras might therefore be considered as potential drugs for lung cancer therapy. Here, we show that the Ras inhibitor farnesylthiosalicylic acid (salirasib) inhibits proliferation of human lung cancer cells harboring a mutated K-ras gene (A549, H23, or HTB54) or overexpressing a growth factor receptor (H1299 or HTB58) and enhances the cytotoxic effect of the chemotherapeutic drug gemcitabine. Salirasib inhibited active K-Ras in A549 cells, reversed their transformed morphology, and inhibited their anchorage-independent growth in vitro. Tumor growth in A549 and HTB58 cell nude mouse models was inhibited by i.p. administration of salirasib. P.o. formulated salirasib also inhibited A549 cell tumor growth. Our results suggest that p.o. salirasib may be considered as a potential treatment for lung cancer therapy. [Mol Cancer Ther 2007;6(6):1765–1773]

Inhibition of Intimal Thickening in the Rat Carotid Artery Injury Model by a Nontoxic Ras Inhibitor

Background—Neointimal formation with and without previous vascular injury is common after balloon dilation and in transplant arteriosclerosis. It involves proliferation and migration of medial smooth muscle cells and inflammation, processes that are regulated by Ras proteins and their down-stream effectors. Farnesylthiosalicylate (FTS) is a Ras inhibitor that interferes with Ras membrane anchorage and affects Ras proteins in their active state. In the present study, we tested the hypothesis that systemic administration of FTS will suppress intimal thickening in the rat carotid injury model. Methods and Results—The effects of FTS on rat vascular smooth muscle cells (VSMC) and splenocytes proliferation were evaluated in vitro. The in vivo effects of FTS on the neointima of balloon-injured male Wistar rats, treated daily for 2 weeks with FTS (5 mg/kg weight, intraperitoneally) were evaluated by determination of Ras, Ras-GTP, and active ERK levels (3 days after injury), and by quantitative determination of the extent of intimal thickening and immunohistochemistry for Ras, iNOS, NFkB, and Ki-67 (2 weeks after injury). FTS inhibited VSMC and splenocyte proliferation as well as interferon-&ggr; secretion by splenocytes in a dose-dependent manner. Compared with controls, FTS treatment resulted in a strong decrease in Ras-GTP and active ERK, and it significantly reduced intimal thickening after the injury. Ras expression appeared predominantly at areas of neointima regardless of the treatment group. NFkB and iNOS-positive cell numbers were reduced in sections of FTS treated rats. Conclusion—FTS appears to act as a potent inhibitor of intimal thickening in a model of experimental arterial injury.

The Ras Inhibitor S-trans, trans-Farnesylthiosalicylic Acid Exerts Long-Lasting Neuroprotection in a Mouse Closed Head Injury Model

Traumatic brain injury activates N-methyl-D-aspartate receptors (NMDAR) inducing activation of the Ras protein (a key regulator of cell growth, survival, and death) and its effectors. Thus, trauma-induced increase in active Ras-GTP might contribute to traumatic brain injury pathology. Based on this hypothesis, a new concept of neuroprotection is proposed, examined here by investigating the effect of the Ras inhibitor S-trans, trans-farnesylthiosalicylic acid (FTS) in a mouse model of closed head injury (CHI). Mice subjected to CHI were treated systemically 1 h later with FTS (5 mg/kg) or vehicle. After 1 h, Ras-GTP in the contused hemisphere showed a significant (3.8-fold) increase, which was strongly inhibited by FTS (82% inhibition) or by the NMDA-receptor antagonist MK-801 (53%). Both drugs also decreased active (phosphorylated) extracellular signal-regulated kinase. FTS prevented the CHI-induced reduction in NMDAR binding in cortical, striatal, and hippocampal regions, measured by [3H]-MK-801 autoradiography, and decreased lesion size by 50%. It also reduced CHI-induced neurologic deficits, indicated by the highly significant (P < 0.0001) 60% increase in extent of recovery. Thus, FTS provided long-term neuroprotection after CHI, rescuing NMDAR binding in the contused hemisphere and profoundly reducing neurologic deficits. These findings suggest that nontoxic Ras inhibitors such as FTS may qualify as neuroprotective drugs.

The Ras Antagonist, Farnesylthiosalicylic Acid (FTS), Decreases Fibrosis and Improves Muscle Strength in dy2J/dy2J Mouse Model of Muscular Dystrophy

The Ras superfamily of guanosine-triphosphate (GTP)-binding proteins regulates a diverse spectrum of intracellular processes involved in inflammation and fibrosis. Farnesythiosalicylic acid (FTS) is a unique and potent Ras inhibitor which decreased inflammation and fibrosis in experimentally induced liver cirrhosis and ameliorated inflammatory processes in systemic lupus erythematosus, neuritis and nephritis animal models. FTS effect on Ras expression and activity, muscle strength and fibrosis was evaluated in the dy2J/dy2J mouse model of merosin deficient congenital muscular dystrophy. The dy2J/dy2J mice had significantly increased RAS expression and activity compared with the wild type mice. FTS treatment significantly decreased RAS expression and activity. In addition, phosphorylation of ERK, a Ras downstream protein, was significantly decreased following FTS treatment in the dy2J/dy2J mice. Clinically, FTS treated mice showed significant improvement in hind limb muscle strength measured by electronic grip strength meter. Significant reduction of fibrosis was demonstrated in the treated group by quantitative Sirius Red staining and lower muscle collagen content. FTS effect was associated with significantly inhibition of both MMP-2 and MMP-9 activities. We conclude that active RAS inhibition by FTS was associated with attenuated fibrosis and improved muscle strength in the dy2J/dy2J mouse model of congenital muscular dystrophy.

The association between let-7, RAS and HIF-1α in Ewing Sarcoma tumor growth.

Ewing Sarcoma (ES) is the second most common primary malignant bone tumor in children and adolescents. microRNAs (miRNAs) are involved in cancer as tumor suppressors or oncogenes. We studied the involvement of miRNAs located on chromosomes 11q and 22q that participate in the most common translocation in ES. Of these, we focused on 3 that belong to the let-7 family. We studied the expression levels of let-7a, and let-7b and detected a significant correlation between low expression of let-7b and increased risk of relapse. let-7 is known to be a negative regulator of the RAS oncogene. Indeed, we detected an inverse association between the expression of let-7 and RAS protein levels and its downstream target p-ERK, following transfection of let-7 mimics and inhibitors. Furthermore, we identified let-7 as a negative regulator of HIF-1α and EWS-FLI-1. Moreover, we were able to show that HIF-1α directly binds to the EWS-FLI-1 promoter. Salirasib treatment in-vitro resulted in the reduction of cell viability, migration ability, and in the decrease of cells in S-phase. A significant reduction in tumor burden and in the expression levels of both HIF-1α and EWS-FLI-1 proteins were observed in mice after treatment. Our results support the hypothesis that let-7 is a tumor suppressor that negatively regulates RAS, also in ES, and that HIF-1α may contribute to the aggressive metastatic behavior of ES. Moreover, the reduction in the tumor burden in a mouse model of ES following Salirasib treatment, suggests therapeutic potential for this RAS inhibitor in ES.

Ras Chaperones: New Targets for Cancer and Immunotherapy

The Ras inhibitor S-trans,trans-farnesylthiosalicylic acid (FTS, Salirasib®) interferes with Ras membrane interactions that are crucial for Ras-dependent signaling and cellular transformation. FTS had been successfully evaluated in clinical trials of cancer patients. Interestingly, its effect is mediated by targeting Ras chaperones that serve as key coordinators for Ras proper folding and delivery, thus offering a novel target for cancer therapy. The development of new FTS analogs has revealed that the specific modifications to the FTS carboxyl group by esterification and amidation yielded compounds with improved growth inhibitory activity. When FTS was combined with additional therapeutic agents its activity toward Ras was significantly augmented. FTS should be tested not only in cancer but also for genetic diseases associated with abnormal Ras signaling, as well as for various inflammatory and autoimmune disturbances, where Ras plays a major role. We conclude that FTS has a great potential both as a safe anticancer drug and as a promising immune modulator agent.