Ras-induced cellular events

Ras is a crucial regulator of cell growth in eukaryotic cells. Activated Ras can stimulate signal transduction cascades, leading to cell proliferation, differentiation or apoptosis. It is also one of the most commonly mutated genes in both solid tumours and haematologic neoplasias. In leukaemia and tumours, aberrant Ras signalling can be induced directly by Ras mutation or indirectly by altering genes that associate with Ras or its signalling pathways. A requisite for Ras function is localization to the plasma membrane, which is induced by the post-translational modification farnesylation. Molecules that interfere with this Ras modification have been used as antitumour agents. Ras is emerging as a dual regulator of cell functions, playing either positive or negative roles in the control of proliferation or apoptosis. The diversity of Ras-mediated effects may be related in part to the differential involvement of Ras homologues in distinct cellular processes or to the expanding array of Ras effectors.     

Ras-mediated cell proliferation and cell death: some clues from the interleukin-2 receptor system

Oncoproteins of the Ras family have been extensively studied because of their implication in human cancer. Their roles have been primarily assigned to the commandment of cell proliferation and suppression of apoptosis, which has also been demonstrated by the involvement of Ras activation in the signal transduction pathways triggered by most cytokine receptors. Nevertheless, the functions of Ras proteins have been extended in the last years by the findings showing that they can also act as promoters or enhancers of apoptosis in various systems and conditions. These considerations have raised the issue as to how the signals delivered by Ras are regulated and translated in terms of cellular responses, suggesting that signal complementation may direct the final fate of cells. As an example, the interleukin-2 receptor system may represent a useful model in which the meaning of Ras signals may be evaluated in terms of interactions with other simultaneous signalling events, since knowledge of the biochemical events triggered by the interaction of interleukin-2 with its cell surface receptor in lymphocytes has allowed the proposal of a complete signalling model arranged in three independent channels, one of which is mediated by Ras.This work was supported by grants from CICYT and Pharmacia-Upjohn.     

Novel aspects of Ras proteins biology: regulation and implications.

The importance of Ras proteins as crucial crossroads in cellular signaling pathways has been well established. In spite of the elucidation of the mechanism of RAS activation by growth factors and the delineation of MAP kinase cascades, the overall framework of Ras interactions is far from being complete. Novel regulators of Ras GDP/GTP exchange have been identified that may mediate the activation of Ras in response to changes in intracellular calcium and diacylglycerol. The direct activation of Ras by free radicals such as nitric oxide also suggests potential regulation of Ras function by the cellular redox state. In addition, the array of Ras effectors continues to expand, uncovering links between Ras and other cellular signaling pathways. Ras is emerging as a dual regulator of cellular functions, playing either positive or negative roles in the regulation of proliferation and apoptosis. The signals transmitted by Ras may be modulated by other pathways triggered in parallel, resulting in the final order for proliferation or apoptosis. The diversity of ras-mediated effects may be related in part to differential involvement of Ras homologues in distinct cellular processes. The study of Ras posttranslational modifications has yielded a broad battery of inhibitors that have been envisaged as anti-cancer agents. Although an irreversible modification, Ras isoprenylation appears to be modulated by growth factors and by the activity of the isoprenoid biosynthetic pathway, which may lead to changes in Ras activity.     

α‐Mangostin suppresses the de novo lipogenesis and enhances the chemotherapeutic response to gemcitabine in gallbladder carcinoma cells via targeting the AMPK/SREBP1 cascades

High rates of de novo lipid synthesis have been discovered in certain kinds of tumours, including gallbladder cancer (GBC). Unlike several other tumours, GBC is highly insensitive to standard adjuvant therapy, which makes its treatment even more challenging. Although several potential targets and signalling pathways underlying GBC chemoresistance have been revealed, the precise mechanisms are still elusive. In this study, we found that α‐Mangostin, as a dietary xanthone, repressed the proliferation and clone formation ability, induced cell cycle arrest and the apoptosis, and suppressed de novo lipogenesis of gallbladder cancer cells. The underlying mechanisms might involve the activation of AMPK and, therefore, the suppression of SREBP1 nuclear translocation to blunt de novo lipogenesis. Furthermore, SREBP1 silencing by siRNA or α‐mangostin enhanced the sensitivity of gemcitabine in gallbladder cancer cells. In vivo studies also displayed that MA or gemcitabine administration to nude mice harbouring NOZ tumours can reduce tumour growth, and moreover, MA administration can significantly potentiate gemcitabine‐induced inhibition of tumour growth. Corroborating in vitro findings, tumours from mice treated with MA or gemcitabine alone showed decreased levels of proliferation with reduced Ki‐67 expression and elevated apoptosis confirmed by TUNEL staining, furthermore, the proliferation inhibition and apoptosis up‐regulation were obviously observed in MA combined with gemcitabine treatment group. Therefore, inhibiting de novo lipogenesis via targeting the AMPK/SREBP1 signalling by MA might provide insights into a potential strategy for sensitizing GBC cells to chemotherapy.     

The tuberous sclerosis complex: balancing proliferation and survival

Mutations in genes encoding either hamartin [TSC1 (tuberous sclerosis complex 1)] or tuberin (TSC2) result in a multisystem disorder characterized by the development of benign tumours and hamartomas in several organs. The TSC1 and TSC2 proteins form a complex that lies at the crossroad of many signalling pathways integrating the energy status of the cell with signals induced by nutrients and growth factors. The TSC1/2 complex is a critical negative regulator of mTORC1 [mTOR (mammalian target of rapamycin) complex 1], and by that controls anabolic processes to promote cell growth, proliferation and survival. In the present paper, we review recent evidence highlighting the notion that the TSC1/2 complex simultaneously controls mTOR-dependent and mTOR-independent signals critical for the balancing of cell proliferation and cell death.     

EGF signalling pathway regulates colon cancer stem cell proliferation and apoptosis

Objectives

Cancer stem cells (CSCs) compose a subpopulation of cells within a tumour that can self‐renew and proliferate. Growth factors such as epidermal growth factor (EGF) and basic fibroblast growth factor (b‐FGF) promote cancer stem cell proliferation in many solid tumours. This study assesses whether EGF, bFGF and IGF signalling pathways are essential for colon CSC proliferation and self‐renewal.

Material and methods

Colon CSCs were cultured in serum‐free medium (SFM) with one of the following growth factors: EGF, bFGF or IGF. Characteristics of CSC gene expression were evaluated by real time PCR. Tumourigenicity of CSCs was determined using a xenograft model in vivo. Effects of EGF receptor inhibitors, Gefitinib and PD153035, on CSC proliferation, apoptosis and signalling were evaluated using fluorescence‐activated cell sorting and western blotting.

Results

Colon cancer cell HCT116 transformed to CSCs in SFM. Compared to other growth factors, EGF was essential to support proliferation of CSCs that expressed higher levels of progenitor genes (Musashi‐1, LGR5) and lower levels of differential genes (CK20). CSCs promoted more rapid tumour growth than regular cancer cells in xenografts. EGFR inhibitors suppressed proliferation and induced apoptosis of CSCs by inhibiting autophosphorylation of EGFR and downstream signalling proteins, such as Akt kinase, extracellular signal‐regulated kinase 1/2 (ERK 1/2).

Conclusions

This study indicates that EGF signalling was essential for formation and maintenance of colon CSCs. Inhibition of the EGF signalling pathway may provide a useful strategy for treatment of colon cancer.     

Modifications and intracellular trafficking of FADD/MORT1 and caspase-8 after stimulation of T lymphocytes

The adaptor protein FADD/MORT1 is essential for apoptosis induced by 'death receptors', such as Fas (APO-1/CD95), mediating aggregation and autocatalytic activation of caspase-8. Perhaps surprisingly, FADD and caspase-8 are also critical for mitogen-induced proliferation of T lymphocytes. We generated novel monoclonal antibodies specific for mouse FADD and caspase-8 to investigate whether cellular responses, apoptosis or proliferation, might be explained by differences in post-translational modification and subcellular localisation of these proteins. During both apoptosis signalling and mitogenic activation, FADD and caspase-8 aggregated in multiprotein complexes and formed caps at the plasma membrane but they did not colocalise with lipid rafts. Interestingly, mitogenic stimulation, but not Fas ligation, induced a unique post-translational modification of FADD. These different modifications may determine whether FADD and caspase-8 induce cell death or proliferation.     

Suppression of apoptotic death in hematopoietic cells by signalling through the IL-3/GM-CSF receptors.

Interleukin 3 (IL-3) and granulocyte-macrophage colony stimulating factor (GM-CSF) exert their biological functions through acting on a specific receptor which consists of a ligand-specific alpha subunit and the shared common beta subunit. Inhibition by genistein of a subset of IL-3/GM-CSF-mediated signals, including c-myc induction, resulted in the abrogation of DNA synthesis, however, IL-3 still protected cells from apoptotic cell death. Conversely, a C-terminal truncated form of the GM-CSF receptor, which is missing a critical cytoplasmic region required for activation of the Ras/Raf-1/MAP kinase pathway, induced DNA synthesis, but failed to prevent cell death in response to GM-CSF. Consequently, cells died by apoptosis in the presence of GM-CSF, despite displaying a transient mitogenic response. However, expression of activated Ras protein complemented defective signalling through the mutant receptor and supported long-term proliferation in concert with GM-CSF. These results indicate that IL-3 and GM-CSF prevent apoptosis of hematopoietic cells by activating a signalling pathway distinct from the induction of DNA synthesis and that long-term cell proliferation requires the activation of both pathways.     

The Ras/Raf/ERK signalling pathway drives Schwann cell dedifferentiation

Schwann cells are a regenerative cell type. Following nerve injury, a differentiated myelinating Schwann cell can dedifferentiate and regain the potential to proliferate. These cells then redifferentiate during the repair process. This behaviour is important for successful axonal repair, but the signalling pathways mediating the switch between the two differentiation states remain unclear. Sustained activation of the Ras/Raf/ERK cascade in primary cells results in a cell cycle arrest and has been implicated in the differentiation of certain cell types, in many cases acting to promote differentiation. We therefore investigated its effects on the differentiation state of Schwann cells. Surprisingly, we found that Ras/Raf/ERK signalling drives the dedifferentiation of Schwann cells even in the presence of normal axonal signalling. Furthermore, nerve wounding in vivo results in sustained ERK signalling in associated Schwann cells. Elevated Ras signalling is thought to be important in the development of Schwann cell-derived tumours in neurofibromatosis type 1 patients. Our results suggest that the effects of Ras signalling on the differentiation state of Schwann cells may be important in the pathogenesis of these tumours.     

Death Receptor-Induced Apoptosis Signalling Regulation by Ezrin Is Cell Type Dependent and Occurs in a DISC-Independent Manner in Colon Cancer Cells

Ezrin belongs to the ERM (ezrin-radixin-moesin) protein family and has been demonstrated to regulate early steps of Fas receptor signalling in lymphoid cells, but its contribution to TRAIL-induced cell death regulation in adherent cancer cells remains unknown. In this study we report that regulation of FasL and TRAIL-induced cell death by ezrin is cell type dependant. Ezrin is a positive regulator of apoptosis in T-lymphoma cell line Jurkat, but a negative regulator in colon cancer cells. Using ezrin phosphorylation or actin-binding mutants, we provide evidence that negative regulation of death receptor-induced apoptosis by ezrin occurs in a cytoskeleton- and DISC-independent manner, in colon cancer cells. Remarkably, inhibition of apoptosis induced by these ligands was found to be tightly associated with regulation of ezrin phosphorylation on serine 66, the tumor suppressor gene WWOX and activation of PKA. Deficiency in WWOX expression in the liver cancer SK-HEP1 or the pancreatic Mia PaCa-2 cell lines as well as WWOX silencing or modulation of PKA activation by pharmacological regulators, in the colon cancer cell line SW480, abrogated regulation of TRAIL signalling by ezrin. Altogether our results show that death receptor pro-apoptotic signalling regulation by ezrin can occur downstream of the DISC in colon cancer cells.     

Ribosome Synthesis and MAPK Activity Modulate Ionizing Radiation-Induced Germ Cell Apoptosis in Caenorhabditis elegans

Synthesis of ribosomal RNA by RNA polymerase I (RNA pol I) is an elemental biological process and is key for cellular homeostasis. In a forward genetic screen in C. elegans designed to identify DNA damage-response factors, we isolated a point mutation of RNA pol I, rpoa-2(op259), that leads to altered rRNA synthesis and a concomitant resistance to ionizing radiation (IR)-induced germ cell apoptosis. This weak apoptotic IR response could be phenocopied when interfering with other factors of ribosome synthesis. Surprisingly, despite their resistance to DNA damage, rpoa-2(op259) mutants present a normal CEP-1/p53 response to IR and increased basal CEP-1 activity under normal growth conditions. In parallel, rpoa-2(op259) leads to reduced Ras/MAPK pathway activity, which is required for germ cell progression and physiological germ cell death. Ras/MAPK gain-of-function conditions could rescue the IR response defect in rpoa-2(op259), pointing to a function for Ras/MAPK in modulating DNA damage-induced apoptosis downstream of CEP-1. Our data demonstrate that a single point mutation in an RNA pol I subunit can interfere with multiple key signalling pathways. Ribosome synthesis and growth-factor signalling are perturbed in many cancer cells; such an interplay between basic cellular processes and signalling might be critical for how tumours evolve or respond to treatment.     

Oncogenic Ras in tumour progression and metastasis

The ras genes give rise to a family of related GTP-binding proteins that exhibit potent transforming potential. Mutational activation of Ras proteins promotes oncogenesis by disturbing a multitude of cellular processes, such as gene expression, cell cycle progression and cell proliferation, as well as cell survival, and cell migration. Ras signalling pathways are well known for their involvement in tumour initiation, but less is known about their contribution to invasion and metastasis. This review summarises the role and mechanisms of Ras signalling, especially the role of the Ras effector cascade Raf/MEK/ERK, as well as the phosphatidylinositol 3-kinase/Akt pathway in Ras-mediated transformation and tumour progression. In addition, it discusses the impact of Rho GTPases on Ras-mediated transformation and metastasis.     

Cell cycle arrest of hematopoietic cell lines after treatment with ceramide is commonly associated with retinoblastoma activation

BACKGROUND: Leukaemia cells differ from their normal counterparts in that their ability to properly regulate survival, proliferation, differentiation, and apoptosis is aberrant. Understanding the molecular mechanisms controlling cell proliferation and developing therapeutic strategies to correct nonfunctional regulatory mechanisms are emerging areas of medical research. Ceramide, a metabolite of membrane sphingomyelin hydrolysis, has recently emerged as a key regulator of cellular proliferation, differentiation, and apoptosis in leukaemia cells. METHODS: Leukaemia cell lines were treated with a biologically active analogue of ceramide, C(2)-ceramide. Cell cycle status was assessed flow cytometrically using propidium iodide. Induction of apoptosis was confirmed by annexin V staining of externalised phosphatidylserine and retinoblastoma activation was determined by Western blotting. RESULTS: C(2)-ceramide induced activation of retinoblastoma tumour suppressor protein, G(0)/G(1) cell cycle arrest, or apoptosis in leukaemia cell lines. In addition, these effects differed depending upon cell type, thus confirming the pleiotropic nature of the ceramide signalling pathway. Most cells studied responded to exogenous C(2)-ceramide by entering growth arrest, evidently resulting from activation of retinoblastoma protein, and by displaying some degree of apoptosis. CONCLUSIONS: Taken together, these findings suggest that signalling via ceramide has novel therapeutic applications for treatment of leukaemia.     

Oncogenic Ras promotes butyrate-induced apoptosis through inhibition of gelsolin expression

Activation of Ras promotes oncogenesis by altering a multiple of cellular processes, such as cell cycle progression, differentiation, and apoptosis. Oncogenic Ras can either promote or inhibit apoptosis, depending on the cell type and the nature of the apoptotic stimuli. The response of normal and transformed colonic epithelial cells to the short chain fatty acid butyrate, a physiological regulator of epithelial cell maturation, is also divergent: normal epithelial cells proliferate, and transformed cells undergo apoptosis in response to butyrate. To investigate the role of k-ras mutations in butyrate-induced apoptosis, we utilized HCT116 cells, which harbor an oncogenic k-ras mutation and two isogenic clones with targeted inactivation of the mutant k-ras allele, Hkh2, and Hke-3. We demonstrated that the targeted deletion of the mutant k-ras allele is sufficient to protect epithelial cells from butyrate-induced apoptosis. Consistent with this, we showed that apigenin, a dietary flavonoid that has been shown to inhibit Ras signaling and to reverse transformation of cancer cell lines, prevented butyrate-induced apoptosis in HCT116 cells. To investigate the mechanism whereby activated k-ras sensitizes colonic cells to butyrate, we performed a genome-wide analysis of Ras target genes in the isogenic cell lines HCT116, Hkh2, and Hke-3. The gene exhibiting the greatest down-regulation by the activating k-ras mutation was gelsolin, an actin-binding protein whose expression is frequently reduced or absent in colorectal cancer cell lines and primary tumors. We demonstrated that silencing of gelsolin expression by small interfering RNA sensitized cells to butyrate-induced apoptosis through amplification of the activation of caspase-9 and caspase-7. These data therefore demonstrate that gelsolin protects cells from butyrate-induced apoptosis and suggest that Ras promotes apoptosis, at least in part, through its ability to down-regulate the expression of gelsolin.     

Dominant inhibitory Ras delays Sindbis virus-induced apoptosis in neuronal cells.

Mature neurons are more resistant than dividing cells or differentiating neurons to Sindbis virus-induced apoptotic death. Therefore, we hypothesized that mitogenic signal transduction pathways may influence susceptibility to Sindbis virus-induced apoptosis. Since Ras, a 21-kDa GTP-binding protein, plays an important role in cellular proliferation and neuronal differentiation, we investigated the effect of an inducible dominant inhibitory Ras on Sindbis virus-induced death of a rat pheochromocytoma cell line, PC12 cells. Dexamethasone induction of dominant inhibitory Ras (Ha Ras(Asn17)) expression in transfected PC12 cell lines (MMTV-M17-21 and GSrasDN6 cells) resulted in a marked delay in Sindbis virus-induced apoptosis, compared with infected, uninduced cells. The delay in death after Sindbis virus infection in induced versus uninduced PC12 cells was not associated with differences in viral titers or viral infectivity. No delay in Sindbis virus-induced apoptosis was observed in Ha Ras(Asn17)-transfected PC12 cells if dexamethasone induction was initiated less than 12 h before Sindbis virus infection or in wild-type PC12 cells infected with a chimeric Sindbis virus construct that expresses Ha Ras(Asn17). The delay in Sindbis virus-induced apoptosis in induced Ha Ras(Asn17)-transfected PC12 cells was associated with a decrease in cellular DNA synthesis as measured by 5'-bromo-2'-deoxyuridine incorporation. Thus, in PC12 cells, inducible dominant inhibitory Ras inhibits cellular proliferation and delays Sindbis virus-induced apoptosis. These findings suggest that a Ras-dependent signaling pathway is a determinant of neuronal susceptibility to Sindbis virus-induced apoptosis.     

Modulation of the Ras/Raf/MEK/ERK pathway by Ca(2+), and calmodulin

Ras activation induces a variety of cellular responses that depend on the specific activated effector, the intensity and amplitude of its activation, and the cellular type. Transient activation followed by a sustained but low signal of the Ras/Raf/MEK/ERK pathway is a common feature of cell proliferation in many systems. On the contrary, sustained, high activation is linked with either senescence or apoptosis in fibroblasts and to differentiation in neurones and PC12 cells. The temporal regulation of the pathway is relevant and not only depends on the specific receptor activated but also on the presence of diverse modulators of the pathway. We review here evidence showing that calcium (Ca(2+)) and calmodulin (CaM) are able to regulate the Ras/Raf/MEK/ERK pathway. CaM-binding proteins (CaMBPs) as Ras-GRF and CaM-dependent protein kinase IV (CaMKIV) positively modulate ERK1/2 activation induced by either NGF or membrane depolarisation in neurones. In fibroblasts, CaM binding to EGF receptor and K-Ras(B) may be involved in the downregulation of the pathway after its activation, allowing a proliferative signalling.     

DRacGAP, a novel Drosophila gene, inhibits EGFR/Ras signalling in the developing imaginal wing disc

We have identified a novel Drosophila gene, DRacGAP, which behaves as a negative regulator of &Rgr;-family GTPases DRac1 and DCdc42. Reduced function of DRacGAP or increased expression of DRac1 in the wing imaginal disc cause similar effects on vein and sensory organ development and cell proliferation. These effects result from enhanced activity of the EGFR/Ras signalling pathway. We find that in the wing disc, DRac1 enhances EGFR/Ras-dependent activation of MAP Kinase in the prospective veins. Interestingly, DRacGAP expression is negatively regulated by the EGFR/Ras pathway in these regions. During vein formation, local DRacGAP repression would ensure maximal activity of Rac and, in turn, of Ras pathways in vein territories. Additionally, maximal expression of DRacGAP at the vein/intervein boundaries would help to refine the width of the veins. Hence, control of DRacGAP expression by the EGFR/Ras pathway is a previously undescribed feedback mechanism modulating the intensity and/or duration of its signalling during Drosophila development.