A major concern in targeted drug therapy is that the inhibition of receptors and signaling molecules in tumor cells may also affect similar components in the tumor microenvironment or in the immune system, with undefined consequences for inhibition of tumor growth. One example is given by the Ras inhibitor salirasib (Farnesythiosalycilic acid, FTS), which in addition to its antitumor activity in mice and humans also exhibits anti-inflammatory activity. Here we show three major effects through which Ras inhibition by FTS provides a favorable antitumor environment in immune-competent mice with subcutaneous or intracranial tumors. First, FTS exhibited antitumor activity in intracranial immune-competent tumor-bearing mice and increased their survival relative to tumor-bearing immune-compromised mice. Second, FTS induced an increase in regulatory T cells in mouse splenocytes, in which Foxp3+ T cells did not interfere with the tumor growth inhibitory effects of FTS. Third, FTS induced an increase in antitumor cytotoxic T-cell reactivity in glioma cells by downregulating their own expression of Foxp3. This downregulation induced a TGF-β-associated mechanism in glioma cells altering the tumor microenvironment and causing reduced resistance of the tumor to the immune system. These results are important as they might explain some of the major beneficial effects of Ras inhibitors. They may provide an experimental framework for examination of the impact of other anticancer drugs on cancer and the immune system.

The three oncogenes, ErbB receptors, Ras proteins and nucleolin may contribute to malignant transformation. Previously, we demonstrated that nucleolin could bind both Ras protein and ErbB receptors. We also showed that the crosstalk between the three proteins facilitates anchorage independent growth and tumor growth in nude mice, and that inhibition of this interaction in prostate and colon cancer cells reduces tumorigenicity. In the present study, we show that treatment with Ras and nucleolin inhibitors reduces the oncogenic effect induced by ErbB1 receptor in U87-MG cells. This combined treatment enhances cell death, reduces cell proliferation and cell migration. Moreover, we demonstrate a pivotal role of nucleolin in ErbB1 activation by its ligand. Nucleolin inhibitor prevents EGF-induced receptor activation and its downstream signaling followed by reduced proliferation. Furthermore, inhibition of Ras by Salirasib (FTS), mainly reduces cell viability and motility. The combined treatment, which targets both Ras and nucleolin, additively reduces tumorigenicity both in vitro and in vivo. These results suggest that targeting both nucleolin and Ras may represent an additional opportunity for inhibiting cancers, including glioblastoma, that are driven by these oncogenes.

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.

Autophagy, a process of regulated turnover of cellular constituents, is essential for normal growth control but may be defective under pathological conditions. The Ras/PI3K/mTOR signaling pathway negatively regulates autophagy. Ras signaling has been documented in a large number of human cancers. In this in-vitro study we examined the effect of the Ras inhibitor Salirasib (S-trans, trans-farnesylthiosalicylic acid; FTS) on autophagy induction and cell viability. We show that Ras inhibition by FTS induced autophagy in several cell lines, including mouse embryonic fibroblasts and the human cancer cell lines HeLa, HCT-116 and DLD-1. The autophagy induced by FTS seems to inhibit the cell death induced by FTS, since in the absence of autophagy the death of FTS-treated cells was enhanced. Therefore, inhibition of autophagy may promote the inhibition of tumor cell growth and the cell death mediated by FTS.

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.

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.

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 IKKε, 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.

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.