Ras inhibition enhances autophagy,which partially protects cells from death

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.     

Addition of a histone deacetylase inhibitor redirects tamoxifen-treated breast cancer cells into apoptosis,which is opposed by the induction of autophagy

Modulation of estrogen signaling is one of the most successful modalities for the treatment of estrogen receptor (ER)-positive breast cancer, yet de novo and acquired resistance are frequent. Recent data suggests that the induction of autophagy may play a considerable role in promoting tumor cell survival and resistance to anti-estrogen therapy. Hence, bypassing autophagy may offer a novel strategy to enhance the anti-tumor efficacy of anti-estrogens. Histone deacetylases (HDAC) are involved in the regulation of steroid hormone receptor mediated cell signaling and their inhibition potentiates the anti-tumor effects of anti-estrogens. However, the mechanism underlying this anti-tumor activity is poorly understood. In this report, we show that the addition of an HDAC inhibitor redirects the response of ER-positive breast cancer cells when treated with tamoxifen from growth arrest to apoptotic cell death. This redirection requires functional ER signaling and is mediated by a depletion of Bcl-2 and an induction of Bax and Bak, manifesting in cytochrome C release and PARP cleavage. With combined treatment, a subpopulation of cells is refractory to apoptosis and exhibit a strong induction of autophagy. Inhibition of autophagy in these cells, using siRNA directed against Beclin-1 or treatment with chloroquine, further promotes the induction of apoptosis. Thus, supporting prior reports that autophagy acts as a survival mechanism, our findings demonstrate that HDAC and autophagy inhibition directs autophagy-protected cells into apoptotic cell death, which may impair development of tamoxifen resistance.     

Hypoxia-induced autophagy promotes tumor cell survival and adaptation to antiangiogenic treatment in glioblastoma

Antiangiogenic therapy leads to devascularization that limits tumor growth. However, the benefits of angiogenesis inhibitors are typically transient and resistance often develops. In this study, we explored the hypothesis that hypoxia caused by antiangiogenic therapy induces tumor cell autophagy as a cytoprotective adaptive response, thereby promoting treatment resistance. Hypoxia-induced autophagy was dependent on signaling through the hypoxia-inducible factor-1α (HIF-1α)/AMPK pathway, and treatment of hypoxic cells with autophagy inhibitors caused a shift from autophagic to apoptotic cell death in vitro. In glioblastomas, clinically resistant to the VEGF-neutralizing antibody bevacizumab, increased regions of hypoxia and higher levels of autophagy-mediating BNIP3 were found when compared with pretreatment specimens from the same patients. When treated with bevacizumab alone, human glioblastoma xenografts showed increased BNIP3 expression and hypoxia-associated growth, which could be prevented by addition of the autophagy inhibitor chloroquine. In vivo targeting of the essential autophagy gene ATG7 also disrupted tumor growth when combined with bevacizumab treatment. Together, our findings elucidate a novel mechanism of resistance to antiangiogenic therapy in which hypoxia-mediated autophagy promotes tumor cell survival. One strong implication of our findings is that autophagy inhibitors may help prevent resistance to antiangiogenic therapy used in the clinic.     

Interfering with the interaction between ErbB1, nucleolin and Ras as a potential treatment for glioblastoma

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.     

Farnesyl thiosalicylic acid inhibits the growth of melanoma cells through a combination of cytostatic and pro-apoptotic effects

Novel classes of drug that interfere with the signalling of the small G-protein Ras, the so-called Ras antagonists, are showing much promise as novel anti-cancer agents. In this study, we demonstrate that the novel Ras antagonist farnesylthiosalicylic acid (FTS) inhibits the growth of Colo 853 melanoma cells through a combination of cytostatic and pro-apoptotic effects. Furthermore, these phenomena are seen under conditions of cell attachment and in the presence of serum. Treatment of Colo 853 cells with FTS led to time-dependent inhibition of constitutive Akt, retinoblastoma protein (pRB) and ERK activity, with a concurrent loss of Akt expression. Inhibition of Akt and ERK activity induces apoptosis in other human cancer cell lines. Here it is demonstrated that inhibition of Akt, or ERK and Akt in combination, leads to cell cycle arrest but not apoptosis in melanoma cells. FTS treatment was also found to upregulate activity of the stress-activated p38 MAP kinase. Inhibition of p38 MAP kinase, using the selective inhibitor SB 203580, followed by FTS treatment, significantly increased the proportion of apoptotic cells after 72 hr, possibly suggesting a modulatory role for p38 MAP kinase in FTS-induced melanoma cell apoptosis.     

The Ras inhibitor farnesylthiosalicylic acid as a potential therapy for neurofibromatosis type 1.

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 MPNST cell lines were biallelic, NF1 inactive, 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 MPNST cell line. These effects of FTS led to lower steady-state levels of Ras-GTP and its activated targets. Both anchorage-dependent and anchorage-independent growth of NF1 cells were dose dependently inhibited by FTS, and the inhibition correlated positively with Ras-GTP levels. NF1 cells were found to possess strong actin stress fibers, and this phenotype was also corrected by FTS. NF1 tumor growth in a nude mouse model was inhibited by oral FTS. CONCLUSIONS: FTS treatment of NF1 cells 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 Ras inhibitor S-trans,trans-farnesylthiosalicylic acid chemosensitizes human tumor cells without causing resistance.

Ras transformation requires Ras membrane anchorage, which is promoted by a farnesylcysteine carboxymethyl ester and by additional sequences specific to each Ras isoform. We showed previously that S-trans,trans-farnesylthiosalicylic acid (FTS) disrupts Ras membrane anchorage and that this disturbance contributes to inhibition of cell transformation and tumor growth. Most tumor cells develop resistance to anticancer agents. Here we examined whether tumor cells develop resistance to FTS and evaluated the therapeutic potential of FTS combined with cytotoxic drugs, because oncogenic Ras promotes antiapoptotic signals in tumors of epithelial origin. We showed that Panc-1 pancreatic cancer cells, SW480 colon cancer cells, and H-ras (EJ)-transformed Rat-1 fibroblasts exposed to FTS for prolonged periods (>6 months) do not escape FTS-induced growth inhibition and do not develop drug resistance. These cells continued to express reduced amounts of Ras, exhibit a reversed phenotype, and show an altered response to the cytotoxic drugs doxorubicin and gemcitabine. FTS-treated Panc-1 or SW480 cells acquired sensitivity to the cytotoxic drugs, whereas FTS-treated EJ cells lost sensitivity to doxorubicin, reflecting the opposite effects of oncogenic Ras on the survival of epithelial cells and fibroblasts. Treatment with FTS led to a marked increase in sensitivity to gemcitabine of the formerly resistant SW480 cells and a 100-fold increase in sensitivity to gemcitabine of Panc-1 cells. Such treatment in mice with preexisting Panc-1 tumors provided a synergistic effect of FTS and gemcitabine, leading to enhanced inhibition of tumor growth and a 65% increase in survival rate.     

Differential sensitivity of melanoma cell lines with differing B-Raf mutational status to the new oncogenic B-Raf kinase inhibitor UI-152

Activating mutations in B-Raf kinase are common in malignant melanoma, an aggressive tumor of neuroectodermal origin. In the present study, the antiproliferative effect of the new oncogenic B-Raf targeting drug UI-152 on two types of melanoma cell lines with differing B-Raf mutational status was examined, and the underlying mechanisms were investigated. In cellular assays, UI-152 displayed high selectivity for tumor cells bearing B-Raf(V600E), showing more than 1000-fold higher inhibition of their proliferation than wild-type B-Raf-bearing cells. As expected, UI-152 completely abolished MEK-ERK phosphorylation in A375P cells harboring B-Raf(V600E). In SK-MEL-2 cells expressing B-Raf(WT), UI-152 caused the paradoxical activation of the MAPK pathway but to a much lesser extent than that observed of other oncogenic B-Raf inhibitors. These data suggest that UI-152 may be a more ideal B-Raf inhibitor capable of preserving potency against oncogenic B-Raf while minimizing the paradoxical activation of MAPK signaling. In addition, we showed that UI-152 treatment of A375P cells simultaneously induced cellular autophagy and apoptosis. However, autophagy inhibition with 3-methyladenine and inhibition of apoptosis by overexpression of the X-linked inhibitor of apoptosis failed to rescue melanoma cells from UI-152-induced cell death, implying that apoptosis and autophagy may cooperate in the induction of cell death in UI-152-treated cells. Collectively, our data suggest that UI-152 may be an effective B-Raf inhibitor and a potential therapeutic strategy for B-Raf(WT) and Ras mutant melanoma.     

Ras inhibition by FTS attenuates brain tumor growth in mice by direct antitumor activity and enhanced reactivity of cytotoxic lymphocytes

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.     

TW01001, a novel piperazinedione compound,induces mitotic arrest and autophagy in non-small cell lung cancer A549 cells

Here, we report that TW01001, a novel piperazinedione compound, could be a new mitotic inhibitor for the treatment of non-small cell lung cancer by the following observations in A549 cells: (1) induction of cells to accumulate at G2/M phase, which ultimately led to cell apoptotic death, (2) accumulation of p53 and inhibition of survival signalings, and (3) induction of p53-independent autophagy. Taken together, our data suggested that TW01001 induces autophagy-p53-signaling pathway to cause mitotic arrest and cell growth inhibition in A549 cells and provides the framework for further development as a novel therapeutic agent for lung cancer treatment.     

Autophagy and autophagic cell death are next targets for elimination of the resistance to tyrosine kinase inhibitors

Autophagy, a cellular degradation system has been demonstrated in some hematopoietic malignant cell lines, but there is much still remaining to be known about its role and the mechanisms. We observed the excessive autophagy in chronic myelogenous leukemia (CML) cell line, K562, associated with treatment of 12-O-tetradecanoyl-phorbol-13-acetate (TPA), which can induce K562 cells to differentiate into megakaryocytic lineage. Confocal microscopic analysis demonstrated that autophagic cells did not express a megakaryocyte marker, the CD41 molecule, indicating that the autophagy was independent of megakaryocytic differentiation. After remarkable autophagic degradation, the cells finally underwent autophagic cell death (APCD). On the other hand, a block of TPA-induced autophagy by chloroquine rapidly promoted cell death that was not APCD. This result suggested that autophagy regulated two mechanisms in K562 cells: both the cell survival system and APCD. To confirm that autophagy regulates the cell survival system in K562 cells, imatinib was used to induce cell death in K562 cells. Autophagy has not been considered during imatinib treatment; nonetheless, co-treatment with imatinib and chloroquine markedly enhanced imatinib-induced cell death, compared to K562 cells treated only with imatinib. Furthermore, imatinib-resistant cell lines, BaF3/T315I and BaF3/E255K, also underwent cell death by co-treatment with imatinib and chloroquine. From these data, we concluded that autophagy is deeply related to the cell survival system and that inhibition of autophagy accelerates TPA- or imatinib-induced cell death. The block of autophagy could be a new strategy in the treatment of CML.     

Antagonistic effects of chloroquine on autophagy occurrence potentiate the anticancer effects of everolimus on renal cancer cells

Renal cell carcinoma is an aggressive disease often asymptomatic and weakly chemo-radiosensitive. Currently, new biologic drugs are used among which everolimus, an mTOR inhibitor, that has been approved for second-line therapy. Since mTOR is involved in the control of autophagy, its antitumor capacity is often limited. In this view, chloroquine, a 4-alkylamino substituted quinoline family member, is an autophagy inhibitor that blocks the fusion of autophagosomes and lysosomes. In the present study, we evaluated the effects of everolimus alone or in combination with chloroquine on renal cancer cell viability and verified possible synergism. Our results demonstrate that renal cancer cells are differently sensitive to everolimus and chloroquine and the pharmacological combination everolimus/chloroquine was strongly synergistic inducing cell viability inhibition. In details, the pharmacological synergism occurs when chloroquine is administered before everolimus. In addition, we found a flow autophagic block and shift of death mechanisms to apoptosis. This event was associated with decrease of Beclin-1/Bcl^-2 complex and parallel reduction of anti-apoptotic protein Bcl^-2 in combined treatment. At last, we found that the enhancement of apoptosis induced by drug combination occurs through the intrinsic mitochondrial apoptotic pathway activation, while the extrinsic pathway is involved only partly following its activation by chloroquine. These results provide the basis for new therapeutic strategies for the treatment of renal cell carcinoma after appropriate clinical trial.     

A Ras inhibitor tilts the balance between Rac and Rho and blocks phosphatidylinositol 3-kinase-dependent glioblastoma cell migration

Glioblastoma multiforme are highly aggressive tumors for which no adequate treatment has yet been developed. Glioblastoma multiforme show large amounts of active Ras, considered an appropriate target for directed therapy. Here, we show that the Ras inhibitor S-trans, trans-farnesyl thiosalicylic acid (FTS) can avert the transformation of human glioblastoma multiforme cells by inhibiting both their migration and their anchorage-independent proliferation. FTS, by down-regulating Ras activity in glioblastoma multiforme cells, inhibited phosphatidylinositol 3-kinase signaling, resulting in decreased activity of Rac-1. At the same time, activation of RhoA was increased. These two small GTPases are known to control the arrangement of the actin cytoskeleton. By tilting the balance between Rac-1 and RhoA activities, FTS caused the glioblastoma multiforme cells to undergo profound changes in morphology, including rearrangement of actin into stress fibers and assembly of focal adhesions, both of which are governed by RhoA signaling. These morphologic changes allowed strong attachment of the cells to the matrix, rendering them immobile. The results show that FTS should be considered as a candidate drug for glioblastoma multiforme therapy because it targets not only cell proliferation but also cell migration and invasion, which together constitute the most problematic aspect of these malignancies.     

Farnesylthiosalicylic acid blocks mammalian target of rapamycin signaling in breast cancer cells

Estradiol (E2) stimulates proliferation of hormone-dependent breast cancer and exerts downstream effects on growth factors and their receptors. Key among the pathways' mediating growth factor action is the MAP kinase signaling cascade and the PI-3 kinase pathway with its downstream effector mTOR. We postulated that farnesylthiosalicylic acid (FTS), a novel anti-Ras drug, could effectively inhibit hormone-dependent breast cancer because Ras activates both the MAP kinase and the PI3 kinase pathways. Wild-type MCF-7 cells and a long-term estrogen-deprived subline (LTED) were used to examine the effect of FTS on cell growth and on several biochemical parameters. FTS inhibited growth of both cell lines by reducing proliferation and inducing apoptosis. These effects correlated best with blockade of phosphorylation of PHAS-I and p70 S6 kinase, 2 downstream effectors of mTOR. We observed only minimal inhibition of Akt, an effector upstream of mTOR. Taken together, these findings demonstrate a novel effect of FTS to inhibit mTOR signaling and also suggest that mTOR has a key role in breast cancer cell proliferation. Unexpectedly, only minimal inhibition of MAP kinase occurred in response to FTS at concentrations that markedly reduced cell growth. These later data provide support for the concept that FTS exerts its effects predominantly by blocking mTOR and to a lesser effect by inhibition of MAP kinase in breast cancer cells.     

Chronic mTOR activation promotes cell survival in Merkel cell carcinoma

Merkel cell carcinoma (MCC) is an aggressive skin cancer with rising incidence. In this study, we demonstrate that mTOR activation and suppressed autophagy is common in MCCs. mTOR inhibition in two primary human MCC cell lines induces autophagy and cell death that is independent of caspase activation but can be attenuated by autophagy inhibition. This is the first study to evaluate mTOR and autophagy in MCC. Our data suggests a potential role of autophagic cell death upon mTOR inhibition and thus uncovers a previously underappreciated role of mTOR signaling and cell survival, and merits further studies for potential therapeutic targets.