The small GTPase proteins, Ras and Rheb, serve as molecular switches regulating cell proliferation, differentiation and apoptosis. Ras also regulates Rheb by inactivating the tuberous sclerosis complex (TSC), which includes products of the TSC1 and TSC2 genes encoding hamartin (TSC1) and tuberin (TSC2), respectively, and acts as a Rheb-specific GTPase-activating protein. Loss of function of TSC1 or TSC2 results in an increase in active Rheb.GTP with the consequent translational abnormalities and excessive cell proliferation characteristic of the genetic disorders, tuberous sclerosis and lymphangioleiomyomatosis (LAM). To determine whether inactivation of Rheb, Ras or both might be a potential treatment for LAM, we used TSC2-null ELT3 cells as a LAM model. The cells were treated with the Ras inhibitor S-trans,transfarnesylthiosalicylic acid (FTS; salirasib), which mimics the C-terminal S-farnesyl cysteine common to Ras and Rheb. This C-terminus is critical for their attachment to cellular membranes and for their biological activities. Untreated, the ELT3 cells expressed significant amounts of Rheb but little Ras.GTP, and this phenotype was reversed by TSC2 reexpression. Treatment with FTS decreased Ras.GTP only slightly in the TSC2-null cells, but reduced their overactive Rheb as well as their proliferation, migration and tumor growth. Notably, TSC2 reexpression in these ELT3 cells rescued them from the inhibitory effect of FTS. Evidently, therefore, FTS blocks active Rheb in TSC2-null ELT3 cells and may have therapeutic potential for LAM.Lymphangioleiomyomatosis (LAM) is a rare lung disorder affecting mainly women of childbearing age. It is characterized by abnormal migration of smooth muscle cells into the lung and their proliferation in the lung and pulmonary lymphatics, leading to obstruction of tissue, loss of pulmonary function and eventually death.1-4 LAM can be sporadic or linked to tuberous sclerosis complex (TSC), an autosomal dominant inherited disorder characterized by benign tumors (hamartomas) in various organs. 2 The origin of LAM cells remains unknown. 2LAM cells in both sporadic and TSC-linked disorders carry inactivating mutations in both alleles of the TSC1 or TSC2 genes.

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.

Galectin-3 (Gal-3) is a pleiotropic-galactoside-binding protein expressed at relatively high levels in human neoplasms. Its carbohydrate recognition domain (CRD) contains a hydrophobic pocket that can accommodate the farnesyl moiety of K-Ras. Binding of K-Ras to Gal-3 stabilizes K-Ras in its active (GTP-bound) state. Gal-3, which does not interact with N-Ras, was nevertheless shown to reduce N-Ras-GTP in BT-549 cells by an unknown mechanism that we explored here. First, comparative analysis of various cancer cell lines (glioblastomas, breast cancer cells and ovarian carcinomas) showed a positive correlation between low N-Ras-GTP/high K-Ras-GTP phenotype and Gal-3 expression levels. Next we found that epidermal growth factor-stimulated GTP loading of N-Ras, but not of K-Ras, is blocked in cells expressing high levels of Gal-3. Activation of Ras guanine nucleotide releasing proteins (RasGRPs) by phorbol 12-myristate 13acetate (PMA) or downregulation of Gal-3 by Gal-3 shRNA increased the levels of N-Ras-GTP in Gal-3 expressing cells. We further show that the N-terminal domain of Gal-3 interacts with and inhibits RasGRP4-mediated GTP loading on N-Ras and H-Ras proteins. Growth of BT-549 cells stably expressing the Gal-3 N-terminal domain was strongly attenuated. Overall, these experiments demonstrate a new control mechanism of Ras activation in cancer cells whereby the Gal-3 N-terminal domain inhibits activation of N-Ras and H-Ras proteins.

Mutations in tuberous sclerosis (TSC) genes cause the genetic disorder TSC, as well as other neoplasms, including lymphangioleiomyomatosis (LAM) and angiomyolipomas (AMLs). AMLs are benign renal tumors occur both in sporadic LAM and in TSC. As they carry the same mutations, AML cell lines serve as a model for TSC and LAM. Rheb/mammalian target of rapamycin complex 1 (mTORC1) pathway is chronically activated in TSC-deficient cells, and this activation can be diminished using the appropriate inhibitors. Rapamycin (sirolimus) is a known specific inhibitor of mTORC1, whereas S-trans,trans-farnesylthiosalicylic acid (FTS; salirasib) has been shown to inhibit Rheb. To examine the effect of the Rheb/mTOR inhibition pathway, we used human TSC2-deficient AML cells, derived from a LAM patient. FTS indeed inhibited Rheb in these cells and attenuated their proliferation. After comparative treatments with FTS or rapamycin or by re-expression of TSC2, we carried out a gene array analysis. This yielded a substantial number of commonly altered genes, many of which we identified as downstream targets of the interferon (IFN) regulatory factor 7 (IRF7) transcription factor, a central activator of the IFN type 1 immune response. Furthermore, nuclear localization of IRF7 was impaired by each of the three treatments. Interestingly, the phenomena seen on FTS or rapamycin treatment were selective for TSC2-deficient cells. Moreover, knockdown of IRF7 by siRNA mimicked the decrease in number of the abovementioned genes and also inhibited AML cell proliferation. Altogether, these findings support FTS as a potential treatment for TSC and its related pathologies and IRF7 as a novel target for treatment.