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Supplementary Methods, Figure Legends 1-4 from Phenotypic Reversion of Invasive Neurofibromin-Deficient Schwannoma by FTS: Ras Inhibition Reduces BMP4/Erk/Smad Signaling

Neurofibromin-deficient (Nf1−/−) malignant peripheral nerve sheath tumors (MPNST) are highly invasive, refractory to chemotherapy, and characterized by overactivated Ras. Ras activates mitogenic pathways and regulates morphogenic programs—such as those induced by bone morphogenetic proteins (BMP) and TGF-β. The role of such a cross-talk in determining the phenotype and transformation potential of MPNSTs is unknown. Here, we used MPNST cell lines and selective Ras inhibition with S-trans,trans-farnesylthiosalicylic-acid (FTS; salirasib) in conjunction with specific inhibitors of TGF-β and BMP signaling. FTS perturbed signaling of BMP4 and TGF-β1 to Smad-dependent and Erk-dependent pathways. Furthermore, FTS inhibited motility and spreading, reduced the gelatinase secretion, eliminated the expression and activation of regulators of cell–matrix interaction, and altered gene expression. These phenomena are indicative of a phenotypic reversion of NF1-defficient cells by FTS. Inhibition of BMP4 and TGF-β by noggin and SB-431542, respectively, mimicked the FTS-mediated effects on adhesion, spreading, and cell morphology. This strongly suggests that a cross-talk among TGF-β superfamily ligands and Ras plays a significant role in the transformation of NF1−/− MPNSTs. Our results support the therapeutic potential of FTS, in conjuncture with BMP and TGF-β pathway inhibitors, toward the inhibition of mitogenic and morphogenic signaling pathways and the alleviation of NF1 symptoms. Mol Cancer Ther; 10(8); 1317–26. ©2011 AACR.

Supplementary Figure 4 from Phenotypic Reversion of Invasive Neurofibromin-Deficient Schwannoma by FTS: Ras Inhibition Reduces BMP4/Erk/Smad Signaling

Supplementary Figure 3 from Phenotypic Reversion of Invasive Neurofibromin-Deficient Schwannoma by FTS: Ras Inhibition Reduces BMP4/Erk/Smad Signaling

Supplementary Figure 1 from Phenotypic Reversion of Invasive Neurofibromin-Deficient Schwannoma by FTS: Ras Inhibition Reduces BMP4/Erk/Smad Signaling

Supplementary Figure 2 from Phenotypic Reversion of Invasive Neurofibromin-Deficient Schwannoma by FTS: Ras Inhibition Reduces BMP4/Erk/Smad Signaling

Supplementary Figures 1-5 from p53 Regulates the Ras Circuit to Inhibit the Expression of a Cancer-Related Gene Signature by Various Molecular Pathways

Supplementary Movie S4 from Ras and Its Signals Diffuse through the Cell on Randomly Moving Nanoparticles

Supplementary Materials and Methods, Table 1 from p53 Regulates the Ras Circuit to Inhibit the Expression of a Cancer-Related Gene Signature by Various Molecular Pathways

Supplementary Figure Legends 1-13 from p53 Regulates the Ras Circuit to Inhibit the Expression of a Cancer-Related Gene Signature by Various Molecular Pathways

Supplementary Table 5 from Gene Expression Signature of Human Cancer Cell Lines Treated with the Ras Inhibitor Salirasib (S-Farnesylthiosalicylic Acid)

Supplementary Table 4 from Gene Expression Signature of Human Cancer Cell Lines Treated with the Ras Inhibitor Salirasib (S-Farnesylthiosalicylic Acid)

Deregulation of Ras pathways results in complex abnormalities of multiple signaling cascades that contribute to human malignancies. Ras is therefore considered an appropriate target for cancer therapy. In light of the complexity of the deregulated Ras pathway, it is important to decipher at the molecular level the response of cancer cells to Ras inhibitors that would reregulate it. In the present study, we used gene expression profiling as a robust method for the global dissection of gene expression alterations that resulted from treatment with the Ras inhibitor S-farnesylthiosalicylic acid (FTS; salirasib). Use of a ranking-based procedure, combined with functional analysis and promoter sequence analysis, enabled us to decipher the common and most prominent patterns of the transcriptional response of five different human cancer cell lines to FTS. Remarkably, the analysis identified a distinctive core transcriptional response to FTS that was common to all cancer cell lines tested. This signature fits well to a recently described deregulated Ras pathway signature that predicted sensitivity to FTS. Taken together, these studies provide strong support for the conclusion that FTS specifically reregulates defective Ras pathways in human tumor cells. Ras pathway reregulation by FTS was manifested by repression of E2F-regulated and NF-Y–regulated genes and of the transcription factor FOS (all of which control cell proliferation), repression of survivin expression (which blocks apoptosis), and induction of activating transcription factor–regulated and Bach2-regulated genes (which participate in translation and stress responses). Our results suggest that cancer patients with deregulated Ras pathway tumors might benefit from FTS treatment. [Cancer Res 2007;67(7):3320–8]

Supplementary Figures 1-5 from Tumor Cells Secrete Galectin-1 to Enhance Endothelial Cell Activity

Supplementary Table 2 from Gene Expression Signature of Human Cancer Cell Lines Treated with the Ras Inhibitor Salirasib (S-Farnesylthiosalicylic Acid)

Spatiotemporal modulation of Ras signaling from different intracellular compartments requires mechanisms allowing Ras and its signals to navigate across cells. Here, we describe one mechanism by which clusters of palmitoylated H-Ras and N-Ras isoforms but not nonpalmitoylated K-Ras diffuse through the cytoplasm, independently of ATP, on fast, randomly moving, small cytosolic nanoparticles (“rasosomes”). Rasosomes forced to diffuse out of live cells and trapped by Ras antibody beads appear as round structures of 80- to 100-nm diameter. Association of H-Ras with rasosomes requires Ras palmitoylation and the hypervariable sequence (hvr) upstream of the palmitoylated cysteines. H-Ras hvr mutants that fail to interact with rasosomes are biologically inactive. Epidermal growth factor stimulation rapidly increases active H-Ras-GTP and phosphorylated extracellular signal-regulated kinase (ERK) on rasosomes. Similarly, rasosomes carrying H-Ras(G12V) but not H-Ras are loaded with active ERK. Thus, the rasosome represents a hitherto unknown particle that enables Ras signal information to spread rapidly across cells. (Cancer Res 2006; 66(4): 1974-81)

Supplementary Table 1 from Gene Expression Signature of Human Cancer Cell Lines Treated with the Ras Inhibitor Salirasib (S-Farnesylthiosalicylic Acid)

Supplementary Figure S3 from Ras and Its Signals Diffuse through the Cell on Randomly Moving Nanoparticles

Supplementary Figures 6-13 from p53 Regulates the Ras Circuit to Inhibit the Expression of a Cancer-Related Gene Signature by Various Molecular Pathways

Glia cells are involved in upper motor neuron degeneration in amyotrophic lateral sclerosis (ALS). Protease activated receptor 1 (PAR1) pathway is related to brain pathologies. Brain PAR1 is located on peri-synaptic astrocytes, adjacent to pyramidal motor neurons, suggesting possible involvement in ALS. Brain thrombin activity in superoxide dismutase 1 (SOD1) mice was measured using a fluorometric assay, and PAR1 levels by western blot. PAR1 was localized using immunohistochemistry staining. Treatment targeted PAR1 pathway on three levels, thrombin inhibitor TLCK (N-Tosyl-Lys-chloromethylketone), PAR1 antagonist SCH-79797 and the Ras intracellular inhibitor FTS (S-trans-trans-farnesylthiosalicylic acid). Mice were weighed and assessed for motor function and survival. SOD1 brain thrombin activity was increased (p &lt, 0.001) particularly in the posterior frontal lobe (p = 0.027) and hindbrain (p &lt, 0.01). PAR1 levels were decreased (p &lt, 0.001, brain, spinal cord, p &lt, 0.05). PAR1 and glial fibrillary acidic protein (GFAP) staining decreased in the cerebellum and cortex. SOD1 mice lost weight (&ge, 17 weeks, p = 0.047), and showed shorter rotarod time (&ge, 14 weeks, p &lt, 0.01). FTS 40mg/kg significantly improved rotarod scores (p &lt, 0.001). Survival improved with all treatments (p &lt, 0.01 for all treatments). PAR1 antagonism was the most efficient, with a median survival improvement of 10 days (p &lt, 0.0001). Our results support PAR1 pathway involvement in ALS.

Summary The transition from naive to primed state of pluripotent stem cells is hallmarked by epithelial-mesenchymal transition, metabolic switch from oxidative phosphorylation to aerobic glycolysis, and changes in the epigenetic landscape. Since these changes are also seen as putative hallmarks of neoplastic cell transformation, we hypothesized that oncogenic pathways may be involved in this process. We report that the activity of RAS is repressed in the naive state of mouse embryonic stem cells (ESCs) and that all three RAS isoforms are significantly activated upon early differentiation induced by LIF withdrawal, embryoid body formation, or transition to the primed state. Forced expression of active RAS and RAS inhibition have shown that RAS regulates glycolysis, CADHERIN expression, and the expression of repressive epigenetic marks in pluripotent stem cells. Altogether, this study indicates that RAS is located at a key junction of early ESC differentiation controlling key processes in priming of naive cells., Graphical Abstract, Highlights • RAS is activated upon early differentiation of mouse embryonic stem cells (mESCs) • RAS repression by LIF prevents early differentiation • RAS regulates the transition from naive to primed state of mESCs • RAS regulates glycolysis, chromatin remodeling, and CADHERIN expression, Altshuler et al. report that RAS activation positively regulated key processes of naive-primed transition of mouse embryonic stem cells, including changes in metabolism, chromatin remodeling, and the switch in CADHERIN expression. Pharmacological inhibition of RAS attenuated cellular priming, suggesting that RAS inhibition may be potentially useful for converting human cells into ground state and for efficient somatic cellular reprogramming.

// Nir Rainy 1, 2, 3 , Morad Zayoud 1, 2 , Yoel Kloog 3 , Oded Rechavi 3 , Itamar Goldstein 1, 2 1 Sheba Cancer Research Center, Chaim Sheba Academic Medical Center, Tel Hashomer, Ramat Gan 5262100, Israel 2 Sackler School of Medicine, Tel-Aviv University, Tel-Aviv 6997801, Israel 3 Department of Neurobiology, The George S. Wise Faculty of Life Sciences & Sagol School of Neuroscience, Tel-Aviv University, Tel-Aviv 6997801, Israel Correspondence to: Itamar Goldstein, email: Itamar.Goldstein@sheba.health.gov.il Keywords: microRNAs, herpesviruses, lymphoma, oncomiRs, viral carcinogenesis Received: November 08, 2015 Accepted: May 08, 2016 Published: May 26, 2016 ABSTRACT The two human lymphotrophic γ-herpesviruses, Kaposi's sarcoma herpesvirus (KSHV) and Epstein-Barr virus (EBV), are a recognized cause of human cancer, encoding multiple miRs that are major players in carcinogenesis. Previously, we discovered that EBV-encoded miRs transfer between infected B and T lymphocytes. To further explore the biological significance of the spreading of γ-herpesvirus-encoded miRs on carcinogenesis, we focused on KSHV-miR-K12-11 (miR-K12-11) that is unique in having an identical seed sequence with the oncomiR hsa -miR-155, implicated in B cell lymphomas development. Here, we show for the first time that miR-K12-11 transfers in vitro from KSHV-infected BCBL-1 and BC-1 lymphoma lines to T cells. The transferred miR-K12-11 is active in the adopting T cells and binds its canonical target, the 3’-UTR of BACH1. Importantly, we show that the transfer of miR-K12-11 from BCBL-1 to Jurkat cells correlates with inhibition of the innate type-I interferons response to viral dsRNAs downstream of IKKe, a validated miR-K12-11 target. Finally, we show that miR-K12-11 spreading is not reduced by blocking the classical ceramide-dependent exosome secretion pathway. In summary, we report for the first time that intercellular viral oncomiR spreading is an additional mechanism employed by KSHV to inhibit host anti-viral immunity and consequently promote oncogenesis.

Brain metastases are resistant to chemotherapy and carry a poor prognosis. Studies have shown that tumor cells are surrounded by activated astrocytes, whose cytoprotective properties they exploit for protection from chemotherapy-induced apoptosis. The mechanism of such astrocytic protection is poorly understood. A non-mutational mechanism of resistance to chemotherapy that is receiving increased attention is the regulation of gene translation mediated by small noncoding RNAs (sRNAs), and particularly microRNAs (miRNAs). With the aim of examining the role of astrocytic sRNAs in promoting resistance of human lung tumor PC14 cells to chemotherapy-induced apoptosis, here we used a miRNA microarray to compare sRNA profiles of human lung tumor cells cultured with and without astrocytes. We found that sRNAs are transferred from astrocytes to PC14 cells in a contact-dependent manner. Transfer was rapid, reaching a plateau after only 6 hours in culture. The sRNA transfer was inhibited by the broad-spectrum gap-junction antagonist carbenoxolone, indicating that transfer occurs via gap junctions. Among the transferred sRNAs were several that are implicated in survival pathways. Enforced expression of these sRNAs in PC14 cells increased their resistance to the chemotherapeutic agent paclitaxel. These novel findings might be of clinical relevance for the treatment of patients with brain metastases.

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.

The Ras oncogene transmits signals, which regulate various cellular processes including cell motility, differentiation, growth and death. Since Ras signalling is abnormally activated in more than 30% of human cancers, Ras and its downstream signalling pathways are considered good targets for therapeutic interference. Ras is post-translationally modified by the addition of a farnesyl group, which permits its attachment to the plasma membrane. Exploiting this knowledge, a synthetic Ras inhibitor, S-trans, trans-farnesylthiosalicylic acid (FTS; Salirasib), was developed. FTS resembles the farnesylcysteine group of Ras, and acts as an effective Ras antagonist. In the present review, the effect of FTS in combination with various other drugs, as tested in vitro and in vivo, and its therapeutic potential are discussed. As reviewed, FTS cooperates with diverse therapeutic agents, which significantly improves treatment outcome. Therefore, combinations of FTS with other agents have a potential to serve as anti-cancer or anti-inflammatory therapies.

Background Ras-GTPases are vital for normal T cell activation, and downstream effectors of Ras include the MEK/ERK, PI3-kinase/AKT, mTOR/p70S6-kinase, and NF-kB pathways. Somatic mutations in NRAS cause an autoimmune lymphoproliferative disorder and T cells from Rheumatoid Arthritis (RA) patients exhibit perturbation of the Ras/MEK/ERK pathway. The small molecule Farnesylthiosalicylic acid (FTS) inhibits the interaction between Ras-GTPases and prenyl-binding chaperones vital for proper plasma membrane localization and downstream signaling [1]. Previous pre-clinical studies suggest that FTS has an immunomodulatory effect in various animal models of autoimmunity [2]. Objectives To test in the Lewis rat adjuvant induced arthritis (AIA) and in the DBA/1 mouse collagen type-II induced arthritis (CIA) models the therapeutic immunomodulatory effect of FTS alone or combined with methotrexate (MTX). Methods Arthritis was induced in 8–12 week old male Lewis rats by complete Freund9s adjuvant (CFA) injection and in male DBA/1 mice by collagen type-II (CII) immunization. Animals were treated prophylactically with once daily oral FTS (100 mg/kg); weekly i.p injection of MTX (0.5 mg/kg), oral FTS combined with MTX, or daily oral vehicle solution (0.5% carboxy methyl cellulose; CMC). Arthritis severity was scored daily from disease onset until study termination. In addition, we measured multiple disease- and drug-related immunological/molecular biomarkers. Results AIA severity was significantly reduced by FTS treatment compared to CMC controls (Figure 1A, P P P Conclusions FTS, a first-in-class oral selective Ras-GTPases inhibitor, exhibits a potent immunomodulatory effect in two classical murine model of arthritis, coupled with the inhibition of the TH17 response to relevant arthritogenic-antigens. Thus, Ras-signaling-blockade is a promising novel therapeutic approach for RA. References Kloog Y, Cox AD. Prenyl-binding domains: potential targets for Ras inhibitors and anti-cancer drugs. Semin Cancer Biol. 2004 Aug; 14(4):253–261. Mor A, Aizman E, Chapman J, Kloog Y. Immunomodulatory properties of farnesoids: the new steroids? Curr Med Chem. 2013; 20(10):1218–1224. Disclosure of Interest None declared

High percentage of human cancers involves alteration or mutation in Ras proteins, including the most aggressive malignancies, such as lung, colon and pancreatic cancers. FTS (Salirasib) is a farnesylcysteine mimetic, which acts as a functional Ras inhibitor, and was shown to exert anti-tumorigenic effects in vitro and in vivo. Previously, we have demonstrated that short-term treatment with FTS also induces protective autophagy in several cancer cell lines. Drug resistance is frequently observed in cancer cells exposed to prolonged treatment, and is considered a major cause for therapy inefficiency. Therefore, in the present study, we examined the effect of a prolonged treatment with FTS on drug resistance of HCT-116 human colon cancer cells, and the involvement of autophagy in this process. We found that cells grown in the presence of FTS for 6 months have become resistant to FTS-induced cell growth inhibition and cell death. Furthermore, we discovered that the resistant cells exhibit altered autophagy, reduced apoptosis and changes in Ras-related signaling pathways following treatment with FTS. Moreover we found that while FTS induces an apoptosis-related cleavage of p62, the FTS-resistant cells were more resistant to apoptosis and p62 cleavage.

Hepatic stellate cells (HSCs) have a key role in the formation of hepatic fibrosis. The active form of vitamin D, 1,25(OH)2D3, has been found to have antiproliferative and antifibrotic effects in various tissues including liver. Farnesylthiosalicylic acid (FTS), a novel Ras antagonist, was also found to inhibit hepatic fibrosis. The purpose of this study was to examine the antiproliferative and antifibrotic effects of the combined treatment of 1,25(OH)2D3 and FTS on primary cultured HSCs. Primary HSCs, isolated from rat’s livers, were treated with 1,25(OH)2D3, FTS or a combination of both. Proliferation was assessed by bromodeoxyuridine. Expression of p-ERK, ERK, Ras-GTP, total-Ras, CyclinD1 and fibrotic markers was measured by western blotting analysis and real-time PCR. Cytotoxicity was assessed by lactate dehydrogenase method. The combined treatment inhibited HSCs proliferation by threefold. The effect was synergistic and non-cytotoxic. In concordance, the combined treatment suppressed CyclinD1 expression by ~2-fold, whereas 1,25(OH)2D3 or FTS alone showed a significantly lower inhibitory effect. The effect of the combined treatment on CyclinD1 expression was mediated via Ras-GTP and p-ERK signal transduction pathway. The effect on fibrotic markers showed that 1,25(OH)2D3 decreased collagen Iα1 expression by ~40 %, FTS by ~50 % and the combined treatment by ~60 %. 1,25(OH)2D3 inhibited tissue inhibitor of metalloproteinases-1 (TIMP-1) expression by 20 %. FTS alone or 1,25(OH)2D3 + FTS inhibited TIMP-1 expression by 60 %. FTS inhibited transforming growth factor-β (TGF-β) expression by 25 %, while 1,25(OH)2D3 had no effect. Although the combination of 1,25(OH)2D3 and FTS did not demonstrate an additive antifibrotic effect, it showed a synergistic antiproliferative effect on primary HSCs. Therefore, the combined treatment may have a potential therapeutic value in the initiation of fibrotic process.

Many cases of heritable environmental responses have been documented but the underlying mechanisms are largely unknown. Recently, inherited RNA interference has been shown to act as a multigenerational genome surveillance apparatus. We suggest that inheritance of regulatory RNAs is at the root of many other epigenetic phenomena, the trigger that induces other epigenetic mechanisms, such as the depositing of histone modifications and DNA methylation. In addition, we explore the possibility that interacting organisms influence each other's transcriptomes by exchanging heterologous non-coding RNAs.

// Eran Schmukler 1 , Yoel Kloog 1 and Ronit Pinkas-Kramarski 1 1 Department of Neurobiology. Tel-Aviv University, Ramat-Aviv, Israel. Correspondence: Pinkas-Kramarski Ronit, email: // Keywords : Autophagy, Ras, cancer, oncogene Received : January 6, 2014 Accepted : February 13, 2014 Published : February 13, 2014 Abstract Autophagy, a process of self-degradation and turnover of cellular components, plays a complex role in cancer. Evidence exists to show that autophagy may support tumor growth and cell survival, whereas it can also contribute to tumor suppression and have anti-survival characteristics in different cellular systems. Numerous studies have described the effects of various oncogenes and tumor suppressors on autophagy. The small GTPase Ras is an oncogene involved in the regulation of various cell-signaling pathways, and is mutated in 33% of human cancers. In the present review, we discuss the interplay between Ras and autophagy in relation to oncogenesis. It appears that Ras can upregulate or downregulate autophagy through several signaling pathways. In turn, autophagy can affect the tumorigenicity driven by Ras, resulting in either tumor progression or repression, depending on the cellular context. Furthermore, Ras inhibitors were shown to induce autophagy in several cancer cell lines.

Summary Rheumatoid arthritis (RA) is an autoimmune disease characterized by pronounced inflammation and leucocyte infiltration in affected joints. Despite significant therapeutic advances, a new targeted approach is needed. Our objective in this work was to investigate the anti-inflammatory effects of the Ras inhibitor farnesylthiosalicylic acid (FTS) on adjuvant-induced arthritis (AIA) in rats, an experimental model for RA. Following AIA induction in Lewis rats by intradermal injection of heat-killed Mycobacterium tuberculosis, rats were treated with either FTS or dexamethasone and assessed daily for paw swelling. Joints were imaged by magnetic resonance imaging and computerized tomography and analysed histologically. The anti-inflammatory effect of FTS was assessed by serum assay of multiple cytokines. After adjuvant injection rats demonstrated paw swelling, leucocyte infiltration, cytokine secretion and activation of Ras-effector pathways. Upon FTS treatment these changes reverted almost to normal. Histopathological analysis revealed that the synovial hyperplasia and leucocyte infiltration observed in the arthritic rats were alleviated by FTS. Periarticular bony erosions were averted. Efficacy of FTS treatment was also demonstrated by inhibition of CD4+ and CD8+ T cell proliferation and of interferon (IFN)-γ, tumour necrosis factor (TNF)-α, interleukin (IL)-6 and IL-17 release. The Ras effectors PI3K, protein kinase B (AKT), p38, and extracellular-regulated kinase (ERK) were significantly attenuated and forkhead box protein 3 (FoxP3) transcription factor, a marker of regulatory T cells, was significantly increased. Thus, FTS possesses significant anti-inflammatory and anti-arthritic properties and accordingly shows promise as a potential therapeutic agent for RA. Its effects are apparently mediated, at least in part, by a decrease in proinflammatory cytokines.

LIM kinases (LIMKs) are important cell cytoskeleton regulators that play a prominent role in cancer manifestation and neuronal diseases. The LIMK family consists of two homologues, LIMK1 and LIMK2, which differ from one another in expression profile, intercellular localization, and function. The main substrate of LIMK is cofilin, a member of the actin-depolymerizing factor (ADF) protein family. When phosphorylated by LIMK, cofilin is inactive. LIMKs play a contributory role in several neurodevelopmental disorders and in cancer growth and metastasis. We recently reported the development and validation of a novel LIMK inhibitor, referred to here as T56-LIMKi, using a combination of computational methods and classical biochemistry techniques. Here we report that T56-LIMKi inhibits LIMK2 with high specificity, and shows little or no cross-reactivity with LIMK1. We found that T56-LIMKi decreases phosphorylated cofilin (p-cofilin) levels and thus inhibits growth of several cancerous cell lines, including those of pancreatic cancer, glioma and schwannoma. Because the most promising in-vitro effect of T56-LIMKi was observed in the pancreatic cancer cell line Panc-1, we tested the inhibitor on a nude mouse Panc-1 xenograft model. T56-LIMKi reduced tumor size and p-cofilin levels in the Panc-1 tumors, leading us to propose T56-LIMKi as a candidate drug for cancer therapy.

High glucose uptake and increase blood flow is a characteristic of most metastatic tumors. Activation of Ras signaling increases glycolytic flux into lactate, de novo nucleic acid synthesis and uncoupling of ATP synthase from the proton gradient. Met tyrosine kinase receptor signaling upon activation by its ligand, hepatocyte growth factor/scatter factor (HGF/SF), increases glycolysis, oxidative phosporylation, oxygen consumption, and tumor blood volume. Ras is a key factor in Met signaling. Using the Ras inhibitor S-trans,trans-farnesylthiosalicylic acid (FTS), we investigated interplay between HGF/SF-Met–Ras signaling, metabolism, and tumor blood-flow regulation. In vitro, HGF/SF-activated Met increased Ras activity, Erk phosphorylation, cell motility and glucose uptake, but did not affect ATP. FTS inhibited basal and HGF/SF-induced signaling and cell motility, while further increasing glucose uptake and inhibiting ATP production. In vivo, HGF/SF rapidly increased tumor blood volume. FTS did not affect basal blood-flow but abolished the HGF/SF effect. Our results further demonstrate the complex interplay between growth-factor-receptor signaling and cellular and tumor metabolism, as reflected in blood flow. Inhibition of Ras signaling does not affect glucose consumption or basal tumor blood flow but dramatically decreases ATP synthesis and the HGF/SF induced increase in tumor blood volume. These findings demonstrate that the HGF/SF-Met–Ras pathway critically influences tumor-cell metabolism and tumor blood-flow regulation. This pathway could potentially be used to individualize tumor therapy based on functional molecular imaging, and for combined signaling/anti-metabolic targeted therapy.

// Eran Schmukler 1 , Eya Wolfson 1 , Roni Haklai 1 , Galit Elad-Sfadia 1 , Yoel Kloog 1 and Ronit Pinkas-Kramarski 1 1 Department of Neurobiology. Tel-Aviv University, Ramat-Aviv, Israel. Correspondence: Ronit Pinkas-Kramarski, email: // Keywords : Autophagy, Ras, Transformation, signal transduction Received : October 14, 2013 Accepted : November 18, 2013 Published : November 20, 2013 Abstract The Ras family of small GTPases transmits extracellular signals that regulate cell growth, differentiation, motility and death. Ras signaling is constitutively active in a large number of human cancers. Ras can also regulate autophagy by affecting several signaling pathways including the mTOR pathway. Autophagy is a process that regulates the balance between protein synthesis and protein degradation. It is important for normal growth control, but may be defective in diseases. Previously, we have shown that Ras inhibition by FTS induces autophagy, which partially protects cancer cells and may limit the use of FTS as an anti-cancer drug. Since FTS is a non toxic drug we hypothesized that FTS and chloroquine (an autophagy inhibitor) will synergize in cell growth inhibition and cell death. Thus, in the present study, we explored the mechanism of each individual drug and their combined action. Our results demonstrate that in HCT-116 and in Panc-1 cells, FTS induces autophagy, which can be inhibited by chloroquine. Furthermore, the combined treatment synergistically decreased the number of viable cells. Interestingly, the combined treatment enhanced apoptotic cell death as indicated by increased sub-G1 cell population, increased Hoechst staining, activation of caspase 3, decrease in survivin expression and release of cytochrome c. Thus, chloroquine treatment may promote FTS-mediated inhibition of tumor cell growth and may stimulate apoptotic cell death.

Farnesylthiosalisylic acid (FTS) is a potent non-toxic anticancer drug that targets oncogenic and pathologically activated Ras. The mechanism of action of FTS is well understood. It interferes with the binding of activated Ras proteins to their escort chaperons and with Ras tethering to the plasma membrane. This agent has been evaluated successfully in phase II clinical trials of pancreatic and lung cancer patients. It is generally agreed that Ras proteins play an important role in cancer, but they also drive activation of the immune system. Therefore we hypothesized that inhibiting Ras might be beneficial in autoimmune and inflammatory conditions. Over the past decade we have extensively studied the effects of FTS in multiple animal models of such diseases. We were able to show potent anti-inflammatory properties of FTS in autoimmune disease models such as systemic lupus erythematous, antiphospholipd syndrome, Guillain-Barré syndrome, multiple sclerosis, and inflammatory bowel diseases. Its potential was also shown in type I and type II diabetes. Animal models of contact dermatitis, allergic inflammation, and proliferative nephritis were studied as well. We have also investigated the molecular mechanisms, signaling pathways, and inflammatory mediators underlying these conditions. In this review we summarize our (and others) published data, and conclude that FTS has great potential as a safe anti-inflammatory drug.

// Raya Faigenbaum 1 , Roni Haklai 1 , Gilad Ben-Baruch 2 and Yoel Kloog 1 1 Department of Neurobiology, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel 2 Department of Obstetrics and Gynecology, Sheba Medical Center, Tel Hashomer, Sackler School of Medicine,Tel Aviv University, Tel Aviv, Israel Correspondence: Yoel Kloog, email: // Keywords : Endometrial cancer, medroxyprogesterone acetate, FTS, Ras, ERα, ERβ Received : February 11, 2013 Accepted : February 26, 2013 Published : February 27, 2013 Abstract Type 2 endometrial carcinoma (EC) is a poorly differentiated EC. Unlike type 1 EC, which responds to hormonal treatment (progestins), type 2 EC is refractory to hormonal treatment because of its low expression of active estrogen and progesterone receptors (ER, PR). The aim of this study was to develop a novel drug combination designed to treat these aggressive type 2 EC tumors without surgery and with fertility potential preserved. We examined the effects of combined treatment with the progestin medroxyprogesterone acetate (MPA) and the Ras inhibitor S-farnesylthiosalicylic acid (FTS; Salirasib). Because FTS can induce cell differentiation in tumor cells, we examined whether FTS could induce re-differentiation of type 2 EC cells, thereby sensitizing them to MPA. We found that FTS reduced Ras-GTP, phospho-Akt, and phospho-ERK, and that these reductions all correlated with a decrease in ERα phosphorylation. Combined treatment with FTS and MPA induced stronger reduction in USPC1 type 2 EC cell numbers than the reduction induced by either drug alone. MPA caused ERα degradation. Death of the cells was caused by MPA but not by FTS. The phosphorylated ERα induces gene transcription manifested by enhanced cell proliferation and survival. The combination of FTS and MPA, by reducing the mRNA expression of ERα-mediated genes (i.e. PR , c-fos and ps2 / TFF1 ), inhibited tumor growth and enhanced the death of type 2 EC cells. These promising results might herald a novel treatment for the highly aggressive, incurable type 2 endometrial carcinoma.

Restenosis (renarrowing of the blood vessel wall) and cancer are two different pathologies that have drawn extensive research attention over the years. Antiproliferative drugs such as paclitaxel inhibit cell proliferation and are therefore effective in the treatment of cancer as well as neointimal hyperplasia, which is known to be the main cause of restenosis. Antiproliferative drugs are highly hydrophobic and their release from porous biodegradable structures is therefore advantageous. The release profiles of four antiproliferative drugs from highly porous polymeric structures were studied in this study in light of the physical properties of both the host polymers and the drug molecules, and a qualitative model was developed. The chemical structure of the polymer chain directly affects the drug release profile through water uptake in the early stages or degradation and erosion in later stages. It also affects the release profile indirectly, through the polymer's 3D porous structure. However, this effect is minor. The drug volume and molecular area dominantly affect its diffusion rate from the 3D porous structure and the drug's solubility parameter compared with that of the host polymer has some effect on the drug release profile. This model can also be used to describe release mechanisms of other hydrophobic drugs from porous structures. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Efrat Mashiach-Farkash *,1 , Roni Rak *,3 , Galit Elad-Sfadia 3 , Roni Haklai 3 , Shmuel Carmeli 2 , Yoel Kloog 3 and Haim J. Wolfson 1 1 The Blavatnik School of Computer Science, Raymond and Beverly Sackler Faculty of Exact Sciences, Tel-Aviv University, Tel Aviv, Israel 2 School of Chemistry, Raymond and Beverly Sackler Faculty of Exact Sciences, Tel-Aviv University, Tel Aviv, Israel 3 Department of Neurobiology, The George S. Wise Faculty of Life Sciences, Tel-Aviv University, Tel Aviv, Israel * denotes equal contributors Received: June 21, 2012; Accepted: July 06, 2012; Published: July 07, 2012; Keywords: Cofilin, Ras, Rac, Rho, LIMK. Abbreviations: ADF, actin-depolymerizing factor; CSRD, cysteine/serine-rich domain; CTD, C-terminal domain; FCS, fetal calf serum; FTS, S-trans, trans-farnesyl thiosalicyclic acid; GAP, GTPase activating protein; GRD, GAP-related domain; LIMK, LIM kinase; MEF, Mouse embryonic fibroblast; NF1, neurofibromin; NF1-/-, neurofibromin deficient ;Pak1, p21-activated kinase 1; PI3K, phospatidylinositol 3-kinase. Correspondence: Yoel Kloog, email: // // Abstract Neurofibromin regulates cell motility via three distinct GTPase pathways acting through two different domains, the Ras GTPase-activating protein-related domain (GRD) and the pre-GRD domain. First, the GRD domain inhibits Ras-dependent changes in cell motility through the mitogen activated protein cascade. Second, it also regulates Rho-dependent (Ras-independent) changes by activating LIM kinase 2 (LIMK2), an enzyme that phosphorylates and inactivates cofilin (an actin-depolymerizing factor). Third, the pre-GRD domain acts through the Rac1 GTPase, that activate the P21 activated kinase 1 (PAK1)-LIMK1-cofilin pathway. We employed molecular modeling to identify a novel inhibitor of LIMK1/2. The active sites of an ephrin-A receptor (EphA3) and LIMK2 showed marked similarity (60%). On testing a known inhibitor of EphA3, we found that it fits to the LIMK1/2-ATP binding site and to the latter’s substrate-binding pockets. We identified a similar compound, T56-LIMKi, and found that it inhibits LIMK1/2 kinase activities. It blocked the phosphorylation of cofilin which led to actin severance and inhibition of tumor cell migration, tumor cell growth, and anchorage-independent colony formation in soft agar. Because modulation of LIMK by neurofibromin is not affected by the Ras inhibitor Salirasib, we examined the combined effect of Salirasib and T56-LIMKi each of which can affect cell motility by a distinct pathway. We found that their combined action on cell proliferation and stress-fiber formation in neurofibromin-deficient cells was synergistic. We suggest that this drug combination may be developed for treatment of neurofibromatosis and cancer.