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.     

Oncogenic Ras-induced proliferation requires autocrine fibroblast growth factor 2 signaling in skeletal muscle cells

Constitutively activated Ras proteins are associated with a large number of human cancers, including those originating from skeletal muscle tissue. In this study, we show that ectopic expression of oncogenic Ras stimulates proliferation of the MM14 skeletal muscle satellite cell line in the absence of exogenously added fibroblast growth factors (FGFs). MM14 cells express FGF-1, -2, -6, and -7 and produce FGF protein, yet they are dependent on exogenously supplied FGFs to both maintain proliferation and repress terminal differentiation. Thus, the FGFs produced by these cells are either inaccessible or inactive, since the endogenous FGFs elicit no detectable biological response. Oncogenic Ras-induced proliferation is abolished by addition of an anti-FGF-2 blocking antibody, suramin, or treatment with either sodium chlorate or heparitinase, demonstrating an autocrine requirement for FGF-2. Oncogenic Ras does not appear to alter cellular export rates of FGF-2, which does not possess an NH(2)-terminal or internal signal peptide. However, oncogenic Ras does appear to be involved in releasing or activating inactive, extracellularly sequestered FGF-2. Surprisingly, inhibiting the autocrine FGF-2 required for proliferation has no effect on oncogenic Ras-mediated repression of muscle-specific gene expression. We conclude that oncogenic Ras-induced proliferation of skeletal muscle cells is mediated via a unique and novel mechanism that is distinct from Ras-induced repression of terminal differentiation and involves activation of extracellularly localized, inactive FGF-2.     

Requirement of regulated activation of Ras for response of MDCK cells to hepatocyte growth factor/scatter factor

Hepatocyte growth factor/scatter factor (HGF/SF) induces cell scattering, migration, and branching tubule formation of MDCK cells. To examine the role of the Ras protein in the HGF/SF-induced responses, we constructed MDCK cell clones expressing either inducible dominant-negative Ras or constitutively activated Ras and analyzed their effects on responses of cells to HGF/SF. Induced expression of dominant-negative Ras prevented cell dissociation required for cell scattering, migration, and cystic formation as well as branching morphology required for branching tubule formation. Constitutively activated Ras induced cell dissociation, but not a scattered fibroblastic morphology even in the presence of HGF/SF. MDCK cells expressing constitutively activated Ras migrated at a level similar to that of wild-type MDCK cells stimulated by HGF/SF. MDCK cells expressing constitutively activated Ras showed disorganized growth in three-dimensional culture and did not form the branching tubule structures. These results indicate that activation of the Ras protein is essential for the cell scattering, migration, and branching tubule formation of MDCK cells induced by HGF/SF, and a properly regulated activation is required for some stages of the HGF/SF-induced responses of MDCK cells.     

Staurosporines Disrupt Phosphatidylserine Trafficking and Mislocalize Ras Proteins

Oncogenic mutant Ras is frequently expressed in human cancers, but no anti-Ras drugs have been developed. Since membrane association is essential for Ras biological activity, we developed a high content assay for inhibitors of Ras plasma membrane localization. We discovered that staurosporine and analogs potently inhibit Ras plasma membrane binding by blocking endosomal recycling of phosphatidylserine, resulting in redistribution of phosphatidylserine from plasma membrane to endomembrane. Staurosporines are more active against K-Ras than H-Ras. K-Ras is displaced to endosomes and undergoes proteasomal-independent degradation, whereas H-Ras redistributes to the Golgi and is not degraded. K-Ras nanoclustering on the plasma membrane is also inhibited. Ras mislocalization does not correlate with protein kinase C inhibition or induction of apoptosis. Staurosporines selectively abrogate K-Ras signaling and proliferation of K-Ras-transformed cells. These results identify staurosporines as novel inhibitors of phosphatidylserine trafficking, yield new insights into the role of phosphatidylserine and electrostatics in Ras plasma membrane targeting, and validate a new target for anti-Ras therapeutics.     

Geranylgeranylated, but not farnesylated, RhoB suppresses Ras transformation of NIH-3T3 cells

RhoB is a low molecular weight GTPase that is both farnesylated (RhoB-F) and geranylgeranylated (RhoB-GG) in cells. Based on data from rodent cell models, it has been suggested that RhoB displays differential effects on cell transformation, according to the nature of its prenylation. To test directly this hypothesis, we generated GTPase-deficient RhoB mutants that are exclusively either farnesylated or geranylgeranylated. We show that in Ras-transformed murine NIH-3T3 cells, RhoB-F enhances, whereas RhoB-GG and RhoB (F/GG) suppresses anchorage-dependent and -independent cell growth as well as tumor growth in nude mice. We then demonstrate that Ras constitutive activation of the tumor survival pathways Akt and NF-kappa B are blocked by RhoB-GG, but not by RhoB-F, providing further support for the opposing role of RhoB-F and RhoB-GG in Ras malignant transformation in NIH-3T3 cells. In addition, both RhoB (F/GG) and RhoB-GG induce apoptosis in Ras-transformed NIH-3T3 cells whereas RhoB-F has no effect. Our data demonstrate that RhoB-F and RhoB-GG which differ only by a 5-carbon isoprene behave differently in rodent cells highlighting the important role of prenyl groups in protein function and emphasize the potency of RhoB to regulate negatively the oncogenic signal.     

Therapeutic levels of the hydroxmethylglutaryl-coenzyme A reductase inhibitor lovastatin activate ras signaling via phospholipase D2

Hydroxmethylglutaryl (HMG)-coenzyme A (CoA) reductase inhibitors (statins) lower serum cholesterol but exhibit pleiotropic biological effects that are difficult to ascribe solely to cholesterol depletion. Here, we investigated the effect of lovastatin on protein prenylation and cell signaling. We show that high concentrations (50 μM) of lovastatin inhibit Ras, Rho, and Rap prenylation but that therapeutic levels of lovastatin (50 nM to 500 nM) do not. In contrast, depletion of cellular cholesterol by therapeutic levels of lovastatin increased Ras GTP loading and mitogen-activated protein kinase (MAPK) activation in human umbilical vein endothelial cells and rodent fibroblasts. Elevated Ras signaling was not seen in statin-treated cells if cholesterol levels were maintained by supplementation. Activation of Ras-MAPK signaling was a consequence of, and dependent on, activation of phospholipase D2 (PLD2). Expression of dominant interfering PLD2 or biochemical inhibition of PLD2 abrogated Ras and MAPK activation induced by lovastatin. In contrast, ectopic expression of wild-type PLD2 enhanced Ras and MAPK activation in response to therapeutic levels of lovastatin. Statin-induced cholesterol depletion also modestly activated the epidermal growth factor receptor (EGFR), resulting in downregulation of EGFR expression. These results suggest that statins modulate key cell signaling pathways as a direct consequence of cholesterol depletion and identify the EGFR-PLD2-Ras-MAPK axis as an important statin target.     

Ras signaling is essential for lens cell proliferation and lens growth during development

The vertebrate ocular lens is a simple and continuously growing tissue. Growth factor-mediated receptor tyrosine kinases (RTKs) are believed to be required for lens cell proliferation, differentiation and survival. The signaling pathways downstream of the RTKs remain to be elucidated. Here, we demonstrate the important role of Ras in lens development by expressing a dominant-negative form of Ras (dn-Ras) in the lens of transgenic mice. We show that lens in the transgenic mice was smaller and lens growth was severely inhibited as compared to the wild-type lens. However, the lens shape, polarity and transparency appeared normal in the transgenic mice. Further analysis showed that cell proliferation is inhibited in the dn-Ras lens. For example, the percentage of 5-bromo-2'-deoxyuridine (BrdU)-labeled cells in epithelial layer was about 2- to 3-fold lower in the transgenic lens than in the wild-type lens, implying that Ras activity is required for normal cell proliferation during lens development. We also found a small number of apoptotic cells in both epithelial and fiber compartment of the transgenic lens, suggesting that Ras also plays a role in cell survival. Interestingly, although there was a delay in primary fiber cell differentiation, secondary fiber cell differentiation was not significantly affected in the transgenic mice. For example, the expression of beta- and gamma-crystallins, the marker proteins for fiber differentiation, was not changed in the transgenic mice. Biochemical analysis indicated that ERK activity, but not Akt activity, was significantly reduced in the dn-Ras transgenic lenses. Overall, our data imply that the RTK-Ras-ERK signaling pathway is essential for cell proliferation and, to a lesser extent, for cell survival, but not for crystallin gene expression during fiber differentiation. Thus, some of the fiber differentiation processes are likely mediated by RTK-dependent but Ras-independent pathways.     

Prevention of farnesylation of c-Ha-Ras protein enhances synergistically the cytotoxic action of doxorubicin in cycling but not in quiescent cells

Ras, the product of a proto-oncogene, is a GTP-hydrolyzing enzyme found mutated in approximately 50% of human cancers. "Gain of function" mutations of Ras lead to an escape of transformed cells from cell-cycle control, rendering them independent to stimulation by growth factors, giving them almost unlimited proliferation capacity. The cytosolic precursor isoform of Ras is biologically inactive. After several post-translational modifications, Ras is anchored to the plasma membrane and, thereby, the protein becomes activated. The finding that lipid modifications of Ras protein, particularly farnesylation, are essential for its signal transduction activity, gave rise to the concept that blocking farnesyl protein transferase (FPTase), the enzyme catalyzing the first step in the Ras modification cascade, would prevent proper membrane anchoring and provide an improved approach in the cure of tumors harboring Ras mutations. In the present study we used transformed rat cells overexpressing a temperature-sensitive p53 protein, adopting wt conformation at 32 degrees C and mutant conformation at 37 degrees C. We treated the cells growing at 32 or 37 degrees C with doxorubicin alone, or in combination with inhibitors of FPTase. Combined treatment was more efficient and the same inhibition of cell proliferation was reached at lower DOX concentrations. The treatment strongly affected the growth rate of tumor cells but only negligibly of normal cells. However, the inhibitors of FPTase prevented the membrane anchoring in both situations. These results show two striking advantages of the combined treatment: the desired cytostatic effect on tumor cells at lower drug concentrations and clearly reduced adverse effects on quiescent cells.     

Activated H-Ras regulates hematopoietic cell survival by modulating Survivin

Survivin expression and Ras activation are regulated by hematopoietic growth factors. We investigated whether activated Ras could circumvent growth factor-regulated Survivin expression and if a Ras/Survivin axis mediates growth factor independent survival and proliferation in hematopoietic cells. Survivin expression is up-regulated by IL-3 in Ba/F3 and CD34+ cells and inhibited by the Ras inhibitor, farnesylthiosalicylic acid. Over-expression of constitutively activated H-Ras (CA-Ras) in Ba/F3 cells blocked down-modulation of Survivin expression, G0/G1 arrest, and apoptosis induced by IL-3 withdrawal, while dominant-negative (DN) H-Ras down-regulated Survivin. Survivin disruption by DN T34A Survivin blocked CA-Ras-induced IL-3-independent cell survival and proliferation; however, it did not affect CA-Ras-mediated enhancement of S-phase, indicating that the anti-apoptotic activity of CA-Ras is Survivin dependent while its S-phase enhancing effect is not. These results indicate that CA-Ras modulates Survivin expression independent of hematopoietic growth factors and that a CA-Ras/Survivin axis regulates survival and proliferation of transformed hematopoietic cells.     

Increased p21(ras) activity in human fibroblasts transduced with survivin enhances cell proliferation

Survivin is critically involved in mitosis and when overexpressed enhances the activity of the Aurora B kinase, a serine-threonine kinase belonging to the family of oncogenic Aurora/IpI1p-related kinases. Both proteins interact with Ras GTPase-activating protein suggesting an impact on the Ras pathway. This study aimed at defining the role of survivin in proliferation and potential transformation of cells. When survivin was overexpressed in normal human lung fibroblasts, the characteristic track lanes of fibroblasts were disturbed and the rate of cell proliferation was increased. An enhanced level of p21(ras) mRNA and protein expression and concomitant rise in levels of activated p21(ras) were observed. Despite increased proliferation cell survival remained dependent on serum and cells were not able to form colonies in soft agar assays. These data suggest that overexpression of survivin increases cell growth but, despite the increase in active p21(ras), is not sufficient to transform primary cells. Yet, in addition to its anti-apoptotic function it might contribute to the accelerated growth of tumour cells by increasing p21(ras) activity.     

Ras-mutant Cancer Cells Display B-Raf Binding to Ras That Activates Extracellular Signal-regulated Kinase and Is Inhibited by Protein Kinase A Phosphorylation

The small G protein Ras regulates proliferation through activation of the mitogen-activated protein (MAP) kinase (ERK) cascade. The first step of Ras-dependent activation of ERK signaling is Ras binding to members of the Raf family of MAP kinase kinase kinases, C-Raf and B-Raf. Recently, it has been reported that in melanoma cells harboring oncogenic Ras mutations, B-Raf does not bind to Ras and does not contribute to basal ERK activation. For other types of Ras-mutant tumors, the relative contributions of C-Raf and B-Raf are not known. We examined non-melanoma cancer cell lines containing oncogenic Ras mutations and express both C-Raf and B-Raf isoforms, including the lung cancer cell line H1299 cells. Both B-Raf and C-Raf were constitutively bound to oncogenic Ras and contributed to Ras-dependent ERK activation. Ras binding to B-Raf and C-Raf were both subject to inhibition by the cAMP-dependent protein kinase PKA. cAMP inhibited the growth of H1299 cells and Ras-dependent ERK activation via PKA. PKA inhibited the binding of Ras to both C-Raf and B-Raf through phosphorylations of C-Raf at Ser-259 and B-Raf at Ser-365, respectively. These studies demonstrate that in non-melanocytic Ras-mutant cancer cells, Ras signaling to B-Raf is a significant contributor to ERK activation and that the B-Raf pathway, like that of C-Raf, is a target for inhibition by PKA. We suggest that cAMP and hormones coupled to cAMP may prove useful in dampening the effects of oncogenic Ras in non-melanocytic cancer cells through PKA-dependent actions on B-Raf as well as C-Raf.     

Vascular endothelial growth factor induction of the angiogenic phenotype requires Ras activation.

We investigated the role of Ras in vascular endothelial growth factor (VEGF)-mediated signal transduction and the promotion of angiogenic changes primary endothelial cells. We find that VEGF potently induces Ras activation and that this step is essential for the stimulation by VEGF of several cellular changes associated with angiogenesis, including proliferation, migration, and branching morphogenesis in three-dimensional culture. Inhibition of Ras signaling induced subtle changes in the actin architecture but had no effect on the phosphatidylinositol 3-kinase (PI3K) or p38 signaling pathways. In contrast, activation of ERK was largely dependent on Ras. Although inhibiting ERK activity completely suppressed cell proliferation and partially blocked in vitro differentiation, neither ERK nor PI3K activity was required for VEGF-induced migration. These data provide the first direct demonstration that inhibition of Ras signal transduction is anti-angiogenic. Interestingly, VEGF signal transduction bifurcates both upstream and downstream of Ras, with different Ras-dependent signals controlling endothelial cell proliferation and migration, essential components of the angiogenic response.     

The farnesyltransferase inhibitor,FTI-2153, inhibits bipolar spindle formation during mitosis independently of transformation and Ras and p53 mutation status

Recently, we have shown that the farnesyltransferase inhibitor FTI-2153 induces accumulation of two human lung cancer cell lines in mitosis by inhibiting bipolar spindle formation during prometaphase. Here we investigate whether this mitotic arrest depends on transformation, Ras and/or p53 mutation status. Using DAPI staining (DNA) and immunocytochemistry (microtubules), we demonstrate that in normal primary foreskin fibroblasts (HFF), as well as in several cancer cell lines of different origins including human ovarian (OVCAR3), lung (A-549 and Calu-1) and fibrosarcoma (HT1080), FTI-2153 inhibits bipolar spindle formation and induces a rosette morphology with a monopolar spindle surrounded by chromosomes. In both malignant cancer cell lines and normal primary fibroblasts, the percentage of prometaphase cells with bipolar spindles decreases from 67-92% in control cells to 2-28% in FTI-2153 treated cells. This inhibition of bipolar spindle formation correlates with an accumulation of cells in prometaphase. The ability of FTI-2153 to inhibit bipolar spindle formation is not dependent on p53 mutation status since both wild-type (HFF, HT1080 and A-549) and mutant (Calu-1 and OVCAR3) p53 cells were equally affected. Similarly, both wild-type (HFF and OVCAR3) and mutant (HT1080, Calu-1 and A-549) Ras cells accumulate monopolar spindles following treatment with FTI-2153. However, two cell lines, NIH3T3 (WT Ras and WT p53) and the human bladder cancer cell line, T-24 (mutant H-Ras and mutant p53) are highly resistant to FTI-2153 inhibition of bipolar spindle formation. Finally, the ability of FTI-2153 to inhibit tumor cell proliferation does not correlate with inhibition of bipolar spindle formation. Taken together these results demonstrate that the ability of FTI-2153 to inhibit bipolar spindle formation and accumulate cells in mitosis is not dependent on transformation, Ras or p53 mutation status. Furthermore, in some cell lines, FTIs inhibit growth by mechanisms other than interfering with the prophase/metaphase traverse.     

SmgGDS-558 regulates the cell cycle in pancreatic,non-small cell lung,and breast cancers

Oncogenic mutation or misregulation of small GTPases in the Ras and Rho families can promote unregulated cell cycle progression in cancer. Post-translational modification by prenylation of these GTPases allows them to signal at the cell membrane. Splice variants of SmgGDS, named SmgGDS-607 and SmgGDS-558, promote the prenylation and membrane trafficking of multiple Ras and Rho family members, which makes SmgGDS a potentially important regulator of the cell cycle. Surprisingly little is known about how SmgGDS-607 and SmgGDS-558 affect cell cycle-regulatory proteins in cancer, even though SmgGDS is overexpressed in multiple types of cancer. To examine the roles of SmgGDS splice variants in the cell cycle, we compared the effects of the RNAi-mediated depletion of SmgGDS-558 vs. SmgGDS-607 on cell cycle progression and the expression of cyclin D1, p27, and p21 in pancreatic, lung, and breast cancer cell lines. We show for the first time that SmgGDS promotes proliferation of pancreatic cancer cells, and we demonstrate that SmgGDS-558 plays a greater role than SmgGDS-607 in cell cycle progression as well as promoting cyclin D1 and suppressing p27 expression in multiple types of cancer. Silencing both splice variants of SmgGDS in the cancer cell lines produces an alternative signaling profile compared with silencing SmgGDS-558 alone. We also show that loss of both SmgGDS-607 and SmgGDS-558 simultaneously decreases tumorigenesis of NCI-H1703 non-small cell lung carcinoma (NSCLC) xenografts in mice. These findings indicate that SmgGDS promotes cell cycle progression in multiple types of cancer, making SmgGDS a valuable target for cancer therapeutics.     

Long glucocorticoid-induced leucine zipper (L-GILZ) protein interacts with ras protein pathway and contributes to spermatogenesis control

Correct function of spermatogonia is critical for the maintenance of spermatogenesis throughout life, but the cellular pathways regulating undifferentiated spermatogonia proliferation, differentiation, and survival are only partially known. We show here that long glucocorticoid-induced leucine zipper (L-GILZ) is highly expressed in spermatogonia and primary spermatocytes and controls spermatogenesis. Gilz deficiency in knock-out (gilz KO) mice leads to a complete loss of germ cell lineage within first cycles of spermatogenesis, resulting in male sterility. Spermatogenesis failure is intrinsic to germ cells and is associated with increased proliferation and aberrant differentiation of undifferentiated spermatogonia and with hyperactivity of Ras signaling pathway as indicated by an increase of ERK and Akt phosphorylation. Spermatogonia differentiation does not proceed beyond the prophase of the first meiotic division due to massive apoptosis associated with accumulation of unrepaired chromosomal damage. These results identify L-GILZ as a novel important factor for undifferentiated spermatogonia function and spermatogenesis.     

甲羟戊酸通路对细胞生长调控作用的研究进展

甲羟戊酸(MVA)通路对细胞生长具有重要的调节作用,MVA及其衍生物通过对蛋白质异戊烯化和N糖基化修饰而影响Ras蛋白、生长因子及受体的功能、细胞内信号转导和细胞的生长。MVA通路参与血管活性物质生成的调节是其调节细胞生长的另一机制。MVA生成的限速酶羟甲基戊二酸单酰辅酶A(HMGCoA)则受MVA通路衍生物的反馈抑制。HMGCoA还原酶抑制剂通过抑制MVA及其衍生物的生成而抑制细胞的生长和增殖。     

Low proliferation and high apoptosis of osteoblastic cells on hydrophobic surface are associated with defective Ras signaling

The hydrophobic (HPB) nature of most polymeric biomaterials has been a major obstacle in using those materials in vivo due to low compatibility with cells. However, there is little knowledge of the molecular detail to explain how surface hydrophobicity affects cell responses. In this study, we compared the proliferation and apoptosis of human osteoblastic MG63 cells adhered to hydrophilic (HPL) and hydrophobic surfaces. On the hydrophobic surface, less formation of focal contacts and actin stress fibers, a delay in cell cycle progression, and an increase in apoptosis were observed. By using fibroblast growth factor 1 (FGF1) as a model growth factor, we also investigated intracellular signaling pathways on hydrophilic and hydrophobic surfaces. The activation of Ras, Akt, and ERK by FGF1 was impaired in MG63 cells on the hydrophobic surface. The overexpression of constitutively active form of Ras and Akt rescued those cells from apoptosis and recovered cell cycle progression. Furthermore, their overexpression also restored the actin cytoskeletal organization on the hydrophobic surface. Finally, the proliferative, antiapoptotic, and cytoskeletal effects of constitutively active Ras in MG63 cells on the hydrophobic surface were blocked by wortmannin and PD98059 that inhibit Akt and ERK activation, respectively. Therefore, our results suggest that the activation of Ras and its downstream molecules Akt and ERK to an appropriate level is one of crucial elements in the determination of osteoblast cell responses. The Ras pathway may represent a cell biological target that should be considered for successful surface modification of biomaterials to induce adequate cell responses in the bone tissue.