VEGF activates protein kinase C-dependent, but Ras-independent Raf-MEK-MAP kinase pathway for DNA synthesis in primary endothelial cells.

KDR/FIk-1 tyrosine kinase, one of the two VEGF receptors induces mitogenesis and differentiation of vascular endothelial cells. We have previously reported that a major target molecule of KDR/Flk-1 kinase is PLC-gamma, and that VEGF induces activation of MAP kinase, mainly mediated by protein kinase C (PKC) in the NIH3T3 cells overexpressing KDR/FIk-1 (Takahashi and Shibuya, 1997). However, the signal transduction initiated from VEGF in endothelial cells remains to be elucidated. In primary sinusoidal endothelial cells which showed strictly VEGF-dependent growth, we found that VEGF stimulated the activation of Raf-1-MEK-MAP kinase cascade. To our surprise, an important regulator, Ras was not efficiently activated to a significant level in response to VEGF. Consistent with this, dominant-negative Ras did not block the VEGF-induced phosphorylation of MAP kinase. On the other hand, PKC-specific inhibitors severely reduced VEGF-dependent phosphorylation of MEK, activation of MAP kinase and subsequent DNA synthesis. A potent PI3 kinase inhibitor, Wortmannin, could not inhibit either of them. These results suggest that in primary endothelial cells, VEGF-induced activation of Raf-MEK-MAP kinase and DNA synthesis are mainly mediated by PKC-dependent pathway, much more than by Ras-dependent or PI3 kinase-dependent pathway.     

Retroviral gene transfer of dominant negative raf-1 mutants suppresses ha-ras-induced transformation and delays tumor formation

Activating mutants of ras are among the most frequently found genetic alterations in human cancers. Therefore, Ras appears to be an attractive target for therapeutic intervention using gene transfer. The protein kinase Raf-1 acts as a direct downstream effector of Ras and is involved in Ras-induced cellular transformation. Using the NIH3T3 fibroblast-derived tumor cell line PEJ, which expresses oncogenic Ha-rasG12V, we analyzed whether dominant negative mutants of Raf-1 can inhibit Ras-mediated transformation. Retroviral gene transfer was used to stably transduce PEJ cells with three different dominant negative mutants of Raf-1. This resulted in reversion of the transformed phenotype in vitro as evidenced by an increase in contact inhibition and reduced anchorage-independent growth. However, tumor formation in nude mice was significantly delayed only by one of these mutants. Therefore, dominant negative mutants of the oncoprotein Myc, which is known to synergize with Raf-1 in tumor formation, were transduced into PEJ cells expressing a dominant negative Raf mutant. This leads to killing of the cells. These results indicate that although interference with Ras-induced transformation using dominant negative mutants of Raf is feasible and effective in vitro using retroviral vectors, an additional block (e.g., that of Myc) is necessary to kill PEJ cells. These results also indicate that interference with Ras-dependent signaling is not sufficient for inhibition of tumor formation of PEJ cells in vivo.     

Role of Ras in signal transduction from the nerve growth factor receptor: relationship to protein kinase C, calcium and cyclic AMP.

A dominant inhibitory ras mutant (Ha-ras Asn-17) has been used to investigate the role of Ras in nerve growth factor (NGF)-mediated signal transduction in PC12 cells. Expression of Ha-Ras Asn-17 blocks neuronal differentiation of these cells in response to NGF treatment. The Ha-Ras Asn-17 block was bypassed by treatment with NGF plus dibutyryl cAMP or NGF plus the Ca2+ ionophore ionomycin, but not by NGF plus 12-O-tetradecanoyl phorbol acetate (TPA). Direct stimulation of the cAMP or Ca2+ pathways thus appeared to act synergistically with a Ras-independent NGF signaling pathway. This Ras-independent pathway was also distinct from protein kinase C, since its activity was not affected by protein kinase C down-regulation. It thus appears that NGF stimulation generates a Ras-independent intracellular signal that contributes to neuronal differentiation independently of the cAMP, Ca2+ or protein kinase C second messenger systems. Since TPA did not bypass the Ha-Ras Asn-17 block to differentiation, protein kinase C also did not appear to be sufficient for Ras-dependent pathways mediating NGF-induced differentiation. Down-regulation experiments further indicated that protein kinase C was not required for NGF induction of early response genes via either Ras-dependent or Ras-independent pathways. Moreover, the formation of inositol phosphates and mobilization of intracellular calcium in response to NGF was not inhibited in PC12 cells expressing the Ha-Ras Asn-17 protein. Therefore, although calcium was able to bypass the Ha-Ras Asn-17 block to PC12 differentiation, Ras activity was not required for activation of phospholipase C in response to NGF. It thus appears that both Ras-dependent and Ras-independent signaling pathways contribute to NGF-induced PC12 cell differentiation independently of the cAMP, calcium and protein kinase C second messenger systems.     

Ras mediates radioresistance through both phosphatidylinositol 3-kinase-dependent and Raf-dependent but mitogen-activated protein kinase/extracellular signal-regulated kinase kinase-independent signaling pathways

Cells transformed by the oncogenic small GTPase, Ras, display a radioresistant phenotype in response to ionizing radiation (IR). To determine the mechanisms by which Ras mediates radioresistance in epithelial cells, we assessed the importance of three major survival pathways that can be activated by Ras [phosphatidylinositol 3-kinase (PI3-K)>Akt, nuclear factor kappaB (NF-kappaB), and Raf>mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK)>extracellular signal-regulated kinase] as necessary or sufficient for Ras-mediated radioresistance in matched pairs of RIE-1 rat intestinal epithelial cells expressing oncogenic Ras or empty vector (RIE-Ras and RIE-vector). Inhibiting PI3-K with LY294002 sensitized RIE-1 cells to IR in a dose-dependent manner, indicating that PI3-K is necessary for radioresistance, whereas inhibition of NF-kappaB with the super-repressor IkappaBalpha had little effect on survival. Expression of either the constitutively active catalytic subunit of PI3-K, p110alpha-CAAX, or the Ras effector domain mutant 12V/40C, which retains binding to PI3-K but is impaired in binding to other Ras effectors, was sufficient to confer partial radioresistance. Expression of either a constitutively active form of the serine/threonine kinase Raf-1 or the Ras effector domain mutant 12V/35S, which retains binding to Raf but is impaired in binding to other Ras effectors, was also sufficient to confer partial radioresistance. Surprisingly, however, even complete inhibition of MEK activity by using U0126 resulted in no change in post-IR survival whatsoever. Thus, whereas Raf contributes to Ras-mediated radioresistance, this is accomplished through a MEK-independent pathway. Taken together, these results indicate that multiple pathways, including both PI3-K-dependent and Raf-dependent but MEK-independent signaling, are required for Ras-mediated radioresistance in epithelial cells. Finally, we demonstrate that Ras-mediated radioresistance can be uncoupled from Ras-mediated transformation, in that PI3-K is required for radioresistance but not transformation, whereas MEK and NF-kappaB are required for transformation but not radioresistance in RIE-1 epithelial cells.     

Transfection of constitutively active mitogen-activated protein/extracellular signal-regulated kinase kinase confers tumorigenic and metastatic potentials to NIH3T3 cells

Cellular growth and differentiation are controlled by multiple extracellular signals, many of which activate extracellular signal-regulated kinase (ERK)/mitogen-activated protein (MAP) kinases. Components of the MAP kinase pathways also cause oncogenic transformation in their constitutively active forms. Moreover, expression of activated ras can confer metastatic potential upon some cells. Activation of MAP kinases requires phosphorylation of both Thr and Tyr in the catalytic domain by a family of dual-specificity kinases, called MEKs (MAP kinase/ERK kinase). MEK1 is activated by phosphorylation at Ser218 and Ser222 by Raf. Mutation of these two sites to acidic residues, specifically [Asp218], [Asp218, Asp222], and [Glu218, Glu222], results in constitutively active MEK1. Using these mutant variants of MEK1, we showed previously that transfection of NIH/3T3 or Swiss 3T3 cells causes morphological transformation and increases growth on soft agar, independent of ERK activity. The transformed cell lines show increased expression of matrix metalloproteinases 2 and 9 and cathepsin L, proteinases that have been implicated in the metastatic process. We tested NIH3T3 cells transfected with the [Asp218] or [Asp218, Asp222] for metastatic potential after i.v. injection into athymic mice. Parental 3T3 cells formed no tumors grossly or histologically. However, all MEK1 mutant transformants formed macroscopic metastases. Thus, like activated Ras, MEK1 can confer both tumorigenic and metastatic potential upon NIH3T3 cells. These results refine the mechanism through which ras could confer tumorigenic and metastatic potential (ie., the critical determinants of tumorigenic and metastatic potential are downstream of MEK1).     

Signal transduction pathways through TRK-A and TRK-B receptors in human neuroblastoma cells.

Little is known about the signal transduction pathways of TRK family receptors in neuroblastoma (NB) cells. In this study, an NB cell line, designated MP-N-TS, was established from an adrenal tumor taken from a 2-year-old boy. This cell line expressed both TRK-A and TRK-B receptors, which is rare in a single NB cell line. Therefore, the MP-N-TS cell line was used to determine whether the signal transduction through these constitutive receptors is functional. Three neurotrophins, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-4 / 5 (NT-4 / 5), induced tyrosine phosphorylation of panTRK, and BDNF and NT-4 / 5 induced tyrosine phosphorylation of TRK-B. Tyrosine phosphorylation of panTRK and / or TRK-B by the neurotrophins was inhibited in the presence of a tyrosine kinase inhibitor K252a. Tyrosine phosphorylation of Src homologous and collagen (Shc), extracellular signal-regulated kinase (ERK)-1 and ERK-2, and phospholipase C-gamma1 (PLC-gamma1) was increased by the three neurotrophins and the increase was inhibited in the presence of K252a. Activation of Ras, detected as the GTP-bound form of Ras, was induced by the three neurotrophins. The neurotrophins did not modulate the expressions of TRK-A or TRK-B mRNA, but they did induce the expression of c-fos mRNA. Exogenous NGF induced weak neurite outgrowth, whereas exogenous BDNF and NT-4 / 5 induced distinct neurite outgrowth. Exogenous BDNF and NT-4 / 5 increased the number of viable cells, while NGF did not. Our results demonstrate that the signal transduction pathways through TRK-A and TRK-B in MP-N-TS cells are functional and similar, and the main downstream signaling pathways from the three neurotrophins are mitogen-activated protein kinase (MAPK) cascades through Shc, activated Ras, ERK-1 and ERK-2, and the transduction pathway through PLC-gamma1. Further, BDNF and NT-4 / 5 increased cell viability. The MP-N-TS cell line should be useful for clarifying the TRK-A and TRK-B signaling pathways responsible for the different prognoses in patients with NB.     

Role of mitogen-activated protein kinases in the induction of parathyroid hormone-related peptide

Tumor production of parathyroid hormone-related protein (PTHRP) is responsible for most cases of hypercalcemia of malignancy. The transplantable rat Leydig tumor H-500 is known to cause hypercalcemia in rats by the release of abundant PTHRP and to closely reproduce the human syndrome. We have demonstrated recently that Ras oncogene can stimulate PTHRP gene expression in Fr3T3 fibroblasts in vitro and cause hypercalcemia in vivo. Using rat Leydig tumor H-500 cells, we have investigated the role of effector pathways downstream of Ras in serum-induced PTHRP expression. The Ras inhibitors B-1086 and Lovastatin decreased PTHRP mRNA expression. i.p. administration of B-1086 (50-100 mg/kg/day) into H-500 tumor-bearing male Fischer rats resulted in a dose-dependent reduction in tumor volume, serum calcium, plasma PTHRP, and tumoral PTHRP mRNA expression. Transient transfection of dominant-negative Ras (Ras N17) and Raf (Raf C4B) reduced, whereas activated Raf-1 (Raf BXB) increased, basal expression of PTHRP in H-500 cells. A similar decrease in PTHRP production was seen with a mitogen-activated protein kinase kinase (MEK) inhibitor (PD 098059), implicating the involvement of Ras/Raf/MEK/extracellular signal-regulated kinase (ERK) pathway. In addition, stimulation with UV light, which can activate c-Jun NH2-terminal kinase (JNK), or expression of an activated form of Rac (Rac V12) was sufficient to increase PTHRP mRNA. Moreover, a dominant-negative Rac (Rac N17) blocked serum-induced PTHRP gene expression. Collectively, these results demonstrate that PTHRP is induced via both Raf-ERK and Rac-JNK mediated pathways, effects which can be blocked by chemical inhibitors and dominant-negative mutants of these pathways in vitro and in vivo. Availability of selective inhibitors of Ras signaling molecules may therefore add to our existing armamentarium to control hypercalcemia of malignancy.     

Demonstration of a genetic therapeutic index for tumors expressing oncogenic BRAF by the kinase inhibitor SB-590885

Oncogenic BRAF alleles are both necessary and sufficient for cellular transformation, suggesting that chemical inhibition of the activated mutant protein kinase may reverse the tumor phenotype. Here, we report the characterization of SB-590885, a novel triarylimidazole that selectively inhibits Raf kinases with more potency towards B-Raf than c-Raf. Crystallographic analysis revealed that SB-590885 stabilizes the oncogenic B-Raf kinase domain in an active configuration, which is distinct from the previously reported mechanism of action of the multi-kinase inhibitor, BAY43-9006. Malignant cells expressing oncogenic B-Raf show selective inhibition of mitogen-activated protein kinase activation, proliferation, transformation, and tumorigenicity when exposed to SB-590885, whereas other cancer cell lines and normal cells display variable sensitivities or resistance to similar treatment. These studies support the validation of oncogenic B-Raf as a target for cancer therapy and provide the first evidence of a correlation between the expression of oncogenic BRAF alleles and a positive response to a selective B-Raf inhibitor.     

Functional analysis of the regulatory requirements of B-Raf and the B-Raf(V600E) oncoprotein

The BRAF(V600E) mutation is found in approximately 6% of human cancers and mimics the phosphorylation of the kinase domain activation segment. In wild-type B-Raf (B-Raf(wt)), activation segment phosphorylation is thought to cooperate with negative charges within the N-region for full activation. In contrast to Raf-1, the N-region of B-Raf is constitutively negatively charged owing to the presence of residues D447/D448 and the phosphorylation of S446. Therefore, it has been suggested that this hallmark predisposes B-Raf for oncogenic activation. In this study, we demonstrate that neutralizing mutations of these residues (in particular S446 and S447), or uncoupling of B-Raf from Ras-guanine 5'-triphosphate (GTP), strongly reduce the biological activity of B-Raf in a PC12 cell differentiation assay. We also confirm that S365 is a 14-3-3 binding site, and determine that mutation of this residue rescues the impaired biological activity of B-Raf proteins with a neutralized N-region, suggesting that the N-region opposes a 14-3-3-mediated transition into an inactive conformation. However, in the case of B-Raf(V600E), although complete N-region neutralization resulted in a 2.5-fold reduction in kinase activity in vitro, this oncoprotein strongly induced PC12 differentiation or transformation and epithelial-mesenchymal transition of MCF-10A cells regardless of its N-region charge. Furthermore, the biological activity of B-Raf(V600E) was independent of its ability to bind Ras-GTP. Our analysis identifies important regulatory differences between B-Raf(wt) and B-Raf(V600E) and suggests that B-Raf(V600E) cannot be inhibited by strategies aimed at blocking S446 phosphorylation or Ras activation.     

ERK signalling and oncogene transformation are not impaired in cells lacking A-Raf

Previous studies have indicated an important role for the Raf family of protein kinases in controlling cellular responses to extracellular stimuli and activated oncogenes, through their ability to activate the MEK/ERKs. To investigate the specific role of A-Raf in this process we generated A-Raf deficient mouse embryonic fibroblasts (MEFs) and embryonic stem (ES) cells by gene targeting and characterized their ability to undergo proliferation, differentiation, apoptosis, ERK activation, and transformation by oncogenic Ras and Src. The A-Raf deficient cells are not disrupted for any of these processes, despite the fact that this protein is normally expressed at high levels in both cell types. This implies either that A-Raf plays no role in MEK/ERK activation, that its function is fully compensated by other Raf proteins or MEK kinases or that its role in MEK/ERK activation is highly tissue-specific. Interestingly, B-Raf and Raf-1 activity towards MEK as measured by the immunoprecipitation kinase cascade assay are both significantly increased in the A-Raf deficient MEFs.     

Inhibition of PLC-gamma1 activity converts nerve growth factor from an anti-mitogenic to a mitogenic signal in CHO cells.

Nerve growth factor (NGF) treatment of Chinese hamster ovary fibroblast (CHO) cells exogenously expressing 2.5x105 TrkA receptors (CHO/TrkA) results in inhibition of serum and insulin-like growth factor-I (IGF-I) stimulated cell proliferation in a dose-dependent manner. Furthermore, NGF does not stimulate [3H]thymidine incorporation and inhibits IGF-I mediated DNA synthesis in CHO/TrkA cells. NGF and IGF-I induce extracellular-signal regulated kinase 1 (ERK1) and ERK2 activation, but NGF is able to stimulate a higher and more sustained activation of these enzymes compared with IGF-I. Cotreatment with NGF and IGF-I yields an ERK1/2 activity profile similar to that of NGF treatment alone. While pretreatment with mitogen activated protein kinase kinase (MKK) inhibitor PD98059 (30 microM) results in 100% inhibition of IGF-I stimulated MAPK phosphorylation (IC50<1 microM), NGF mediated MAPK phosphorylation is only decreased by 50% (IC50=3 microM). NGF, but not IGF-I, stimulates tyrosine phosphorylation and activation of PLC-gamma1 which can be inhibited in a dose-dependent manner by phosphoinositide-specific phospholipase C (PI-PLC) inhibitor U73122 (IC50=4 microM). Pretreatment with U73122 (IC50=7 microM) results in an 87% inhibition of NGF mediated MAPK phosphorylation, while cotreatment with PD98059 and U73122 results in 97% inhibition. U73122 pretreatment has no effect on NGF stimulated Akt activation. NGF, but not IGF-I, stimulates the tyrosine phosphorylation of Suc1-associated neurotrophic factor-induced tyrosine phosphorylation target (SNT-1)/fibroblast growth factor receptor substrate 2 (FRS2) which can be completely prevented by pretreatment with 10 microM U73122. Finally, inhibition of PI-PLC results in NGF's ability to stimulate DNA synthesis in the absence and presence of IGF-I.     

C-Raf is required for the initiation of lung cancer by K-Ras(G12D)

The Ras/Raf/MEK/ERK (extracellular signal-regulated kinase) pathway is primarily responsible for mitogenesis in metazoans, and mutational activation of this pathway is common in cancer. A variety of selective chemical inhibitors directed against the mitogen-activated protein kinase pathway are now available for clinical investigation and thus the determination of the importance of each of the kinases in oncogenesis is paramount. We investigated the role of two Raf kinases, B-Raf and C-Raf, in Ras oncogenesis, and found that although B-Raf and C-Raf have overlapping functions in primary mesenchymal cells, C-Raf but not B-Raf is required for the proliferative effects of K-Ras(G12D) in primary epithelial cells. Furthermore, in a lung cancer mouse model, C-Raf is essential for tumor initiation by oncogenic K-Ras(G12D), whereas B-Raf is dispensable for this process. Our findings reveal that K-Ras(G12D) elicits its oncogenic effects primarily through C-Raf and suggest that selective C-Raf inhibition could be explored as a therapeutic strategy for K-Ras-dependent cancers.     

K252a is a selective inhibitor of the tyrosine protein kinase activity of the trk family of oncogenes and neurotrophin receptors.

K252a, an efficient serine/threonine protein kinase inhibitor (IC50s of 10 to 30 nM), has been shown to block the neuronal differentiation of rat pheochromocytoma PC12 cells induced by nerve growth factor (NGF). In this report, we demonstrate that K252a is a potent inhibitor (IC50 of 3 nM) of the tyrosine protein kinase activity of the NGF receptor gp140trk, the product of the trk protooncogene. K252a also inhibits the kinase activity of its transforming alleles, the trk oncogenes, and of the related neurotrophin receptors gp145trkB and gp145trkC, the products of the other known members of the trk gene family, trkB and trkC. In contrast, K252a has no effect (even at micromolar concentrations) on other tyrosine protein kinases such as the receptors for EGF and PDGF and the products of the v-src and v-fms oncogenes. In addition, K252a rapidly reverts the transformed phenotype of NIH3T3 cells transformed by either autocrine stimulation of the trk family of receptors by their cognate ligands or by expression of trk oncogenes isolated from human tumors. The selectivity of K252a for the catalytic activity of the trk family of kinases should help to establish the structural basis for the rational design of highly specific tyrosine protein kinase inhibitors.     

v-Src suppresses SHPS-1 expression via the Ras-MAP kinase pathway to promote the oncogenic growth of cells

We investigated the effect of cell transformation by v-src on the expression and tyrosine phosphorylation of SHPS-1, a putative docking protein for SHP-1 and SHP-2. We found that transformation by v-src virtually inhibited the SHPS-1 expression at mRNA level. While nontransforming Src kinases including c-Src, nonmyristoylated forms of v-Src had no inhibitory effect on SHPS-1 expression, transforming Src kinases including wild-type v-Src and chimeric mutant of c-Src bearing v-Src SH3 substantially suppressed the SHPS-1 expression. In cells expressing temperature sensitive mutant of v-Src, suppression of the SHPS-1 expression was temperature-dependent. In contrast, tyrosine phosphorylation of SHPS-1 was rather activated in cells expressing c-Src or nonmyristoylated forms of v-Src. SHPS-1 expression in SR3Y1 was restored by treatment with herbimycin A, a potent inhibitor of tyrosine kinase, or by the expression of dominant negative form of Ras. Contrary, active form of Mekl markedly suppressed SHPS-1 expression. Finally, overexpression of SHPS-1 in SR3Y1 led to the drastic reduction of anchorage independent growth of the cells. Taken together, our results suggest that the suppression of SHPS-1 expression is a pivotal event for cell transformation by v-src, and the Ras-MAP kinase cascade plays a critical role in the suppression.     

Is B-Raf a good therapeutic target for melanoma and other malignancies?

The RAF family members, A-Raf, B-Raf, and C-Raf (or Raf-1), are intermediate molecules in the mitogen-activated protein (MAP) kinase [Ras/Raf/MAP kinase/extracellular signal-regulated kinase (Erk) kinase (MEK)/Erk] pathway, which relays extracellular signals from the cell membrane to the nucleus via a cascade of phosphorylation events ultimately promoting cancer development. This pathway is activated by mutation in approximately 7% of all human cancers. B-Raf is one of the proteins frequently mutated to an active form during tumor development. Therefore, B-Raf is an attractive cancer target but lack of clinical efficacy using agents targeting this protein has raised serious doubts about its therapeutic utility. Design of more effective B-Raf inhibitory agents, targeting other members of the signaling cascade for greater clinical efficacy or inhibiting B-Raf in combination with other targets, is being evaluated to resolve these perplexing issues. Here, we discuss recent progress, using preclinical models and clinical studies, to resolve the controversy of whether B-Raf would be a good therapeutic target for melanoma and other malignancies.     

TC21 and Ras share indistinguishable transforming and differentiating activities.

Constitutively activated mutants of the Ras-related protein TC21/R-Ras2 cause tumorigenic transformation of NIH3T3 cells. However, unlike Ras, TC21 fails to bind to and activate the Raf-1 serine-threonine kinase. Thus, whereas Ras transformation is critically dependent on Raf-1 TC21 activity is promoted by activation of Raf-independent signaling pathways. In the present study, we have further compared the functions of Ras and TC21. First we determined the basis for the inability of TC21 to activate Raf-1. Whereas Ras can interact with the two distinct Ras-binding sequences in NH2-terminus of Raf-1, designated RBS1 and Raf-Cys, TC21 could only bind Raf-Cys. Thus, the inability of TC21 to bind to RBS1 may prevent it from promoting the translocation of Raf-1 to the plasma membrane. Second, we found that TC21 is an activator of the JNK and p38, but not ERK, mitogen-activated protein kinase cascades and that TC21 transforming activity was dependent on Rac function. Thus, like Ras, TC21 may activate a Rac/JNK pathway. Third, we determined if TC21 could cause the same biological consequences as Ras in three distinct cell types. Like Ras, activated TC21 caused transformation of RIE-1 rat intestinal epithelial cells and terminal differentiation of PC12 pheochromocytoma cells. Finally, activated TC21 blocked serum starvation-induced differentiation of C2 myoblasts, whereas dominant negative TC21 greatly accelerated this differentiation process. Therefore, TC21 and Ras share indistinguishable biological activities in all cell types that we have evaluated. These results support the importance of Raf-independent pathways in mediating the actions of Ras and TC21.