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Biomedical subjects

Richard Marais

Publications and source records attributed to Richard Marais.

35 records · Page 2Linked to original sources
PubMed2004

B-RAF is a therapeutic target in melanoma.

B-RAF is a serine/threonine-specific protein kinase that is mutated in approximately 70% of human melanomas. However, the role of this signalling molecule in cancer is unclear. Here, we show that ERK is constitutively activated in melanoma cells expressing oncogenic B-RAF and that this activity is required for proliferation. B-RAF depletion by siRNA blocks ERK activity, whereas A-RAF and C-RAF depletion do not affect ERK signalling. B-RAF depletion inhibits DNA synthesis and induces apoptosis in three melanoma cell lines and we show that the RAF inhibitor BAY43-9006 also blocks ERK activity, inhibits DNA synthesis and induces cell death in these cells. BAY43-9006 targets B-RAF signalling in vivo and induces a substantial growth delay in melanoma tumour xenografts. Our data demonstrate that oncogenic B-RAF activates ERK signalling, induces proliferation and protects cells from apoptosis, demonstrating that it is an important therapeutic target and thus provides novel strategies for clinical management of melanoma and other cancers.

Benzenesulfonates↗
PubMed2004

V599EB-RAF is an oncogene in melanocytes.

The oncogenic version of B-RAF, (V599E)B-RAF, is found in approximately 70% of human melanomas. However, the role that this oncogene plays in melanoma is unclear because (V559E)B-RAF is also found in approximately 80% of benign nevi. We have examined the role of oncogenic B-RAF in the early stages of melanoma by expressing (V599E)B-RAF in cultured melanocytes. In these cells, (V599E)B-RAF induced constitutive mitogen activated ERK-activating kinase (MEK) and extracellular signal-regulated kinase (ERK) signaling, 12-O-tetradecanoylphorbol-13-acetate-independent growth, and tumorigenicity in nude mice. Intriguingly, in RAS-transformed melanocytes, B-RAF depletion did not block MEK-ERK signaling or cell cycle progression. Similarly, B-RAF depletion blocked MEK-ERK signaling in human melanoma cells harboring oncogenic B-RAF, but not in melanoma cells harboring oncogenic RAS. Thus, although B-RAF can act as a potent oncogene in the early stages of melanoma by signaling through MEK and ERK, it is not required for this signaling in RAS-transformed melanocytes due to innate redundancy within the pathway. These findings have important implications for future therapeutic strategies.

Animals↗
PubMed2004

Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF.

Over 30 mutations of the B-RAF gene associated with human cancers have been identified, the majority of which are located within the kinase domain. Here we show that of 22 B-RAF mutants analyzed, 18 have elevated kinase activity and signal to ERK in vivo. Surprisingly, three mutants have reduced kinase activity towards MEK in vitro but, by activating C-RAF in vivo, signal to ERK in cells. The structures of wild type and oncogenic V599EB-RAF kinase domains in complex with the RAF inhibitor BAY43-9006 show that the activation segment is held in an inactive conformation by association with the P loop. The clustering of most mutations to these two regions suggests that disruption of this interaction converts B-RAF into its active conformation. The high activity mutants signal to ERK by directly phosphorylating MEK, whereas the impaired activity mutants stimulate MEK by activating endogenous C-RAF, possibly via an allosteric or transphosphorylation mechanism.

Allosteric Regulation↗
PubMed2004

Guilty as charged: B-RAF is a human oncogene.

RAF proteins are components of a conserved signaling pathway that regulates cellular responses to extracellular signals. Despite over 20 years of research into the regulation and function of the RAF proteins, it was only realized recently that the B-RAF isoform is mutated at a high frequency in human cancer. Here we review the rapid progress made in our understanding of B-RAF as an oncogene and of its role in cancer.

Amino Acid Sequence↗
PubMed2004

The RAF proteins take centre stage.

Since their discovery over 20 years ago, the RAF proteins have been intensely studied. For most of that time, the focus of the field has been the C-RAF isoform and its role as an effector of the RAS proteins. However, a report that implicates B-RAF in human cancer has highlighted the importance of all members of this protein kinase family and recent studies have uncovered intriguing new data relating to their complex regulation and biological functions.

Amino Acid Sequence↗
PubMed2004

The Brn-2 transcription factor links activated BRAF to melanoma proliferation.

Malignant melanoma, an aggressive and increasingly common cancer, is characterized by a strikingly high rate (70%) of mutations in BRAF, a key component of the mitogen-activated protein (MAP) kinase signaling pathway. How signaling events downstream from BRAF affect the underlying program of gene expression is poorly understood. We show that the Brn-2 POU domain transcription factor is highly expressed in melanoma cell lines but not in melanocytes or melanoblasts and that overexpression of Brn-2 in melanocytes results in increased proliferation. Expression of Brn-2 is strongly upregulated by Ras and MAP kinase signaling. Importantly, the Brn-2 promoter is stimulated by kinase-activating BRAF mutants and endogenous Brn-2 expression is inhibited by RNA interference-mediated downregulation of BRAF. Moreover, silent interfering RNA-mediated depletion of Brn-2 in melanoma cells expressing activated BRAF leads to decreased proliferation. The results suggest that the high levels of Brn-2 expression observed in melanomas link BRAF signaling to increased proliferation.

Animals↗
PubMed2003

Protein kinase A blocks Raf-1 activity by stimulating 14-3-3 binding and blocking Raf-1 interaction with Ras.

Cyclic AMP (cAMP) blocks Raf-1 activation by stimulating its phosphorylation on serine 43 (Ser43), serine 233 (Ser233), and serine 259 (Ser259). We show here that phosphorylation of all three sites blocks Raf-1 binding to Ras.GTP in vivo and that cAMP stimulates binding of 14-3-3 proteins to Ser233 and Ser259. We also show that Raf-1 and protein kinase A (PKA) form a complex in vivo that is disrupted by cAMP and that ablation of PKA by use of small interfering RNA blocks phosphorylation by cAMP. The ability of PKA to block Raf-1 activation is ablated by the PKA inhibitor H89. These studies suggest that Raf-1 and cAMP form a signaling complex in cells. Upon activation of PKA, Raf-1 is phosphorylated and 14-3-3 binds, blocking Raf-1 recruitment to the plasma membrane and preventing its activation.

14-3-3 Proteins↗
PubMed2003

Self-immolative nitrogen mustards prodrugs cleavable by carboxypeptidase G2 (CPG2) showing large cytotoxicity differentials in GDEPT.

Nineteen novel potential self-immolative prodrugs and their corresponding drugs have been synthesized for gene-directed enzyme prodrug therapy (GDEPT) with carboxypeptidase G2 (CPG2) as the activating enzyme. The compounds are derived from o- and p-amino and p-methylamino aniline nitrogen mustards. Their aqueous stability, kinetics of drug release by CPG2, and cytotoxicity in the colon carcinoma cell line WiDr, expressing either surface-tethered CPG2 (stCPG2(Q)3) or control beta-galactosidase, are assessed. The effect of various structural features on stability, kinetics of activation, and biological activity is discussed. The p-methylamino prodrugs are the most stable compounds from this series, with the largest cytotoxicity differentials between CPG2-expressing and nonexpressing cells. The most potent compounds in all series are prodrugs of bis-iodo nitrogen mustards. 4-[N-[4'-Bis(2' '-iodoethyl)aminophenyl]-N'-methylcarbamoyloxymethyl]phenylcarbamoyl-l-glutamic acid, compound 39b, is 124-fold more cytotoxic to WiDr cells expressing CPG2 than to cells expressing beta-galactosidase. An additional six compounds show better cytotoxicity differential than the published N-[4-[(2-chloroethyl)(2-mesyloxyethyl)amino]benzoyl]-l-glutamic acid (CMDA) prodrug.

Aniline Mustard↗
PubMed2003

Gene regulation in cancer gene therapy strategies.

Regulation of expression in gene therapy is considered to be a very desirable goal, preventing toxic effects and improving biological efficacy. A variety of systems have been reported in an ever widening range of applications, this paper describes these systems with specific reference to cancer gene therapy.

Animals↗
PubMed2002

Calmodulin prevents activation of Ras by PKC in 3T3 fibroblasts.

We have shown previously (Villalonga, P., López- Alcalá, C., Bosch, M., Chiloeches, A., Rocamora, N., Gil, J., Marais, R., Marshall, C. J., Bachs, O., and Agell, N. (2001) Mol. Cell. Biol. 21, 7345-7354) that calmodulin negatively regulates Ras activation in fibroblasts. Hence, anti-calmodulin drugs (such as W13, trifluoroperazine, or W7) are able to induce Ras/ERK pathway activation under low levels of growth factors. We show here that cell treatment with protein kinase C (PKC) inhibitors abolishes W13-induced activation of Ras, Raf-1, and ERK. Consequently, PKC activity is essential for achieving the synergism between calmodulin inhibition and growth factors to activate Ras. Furthermore, whereas the activation of PKC by 12-O-tetradecanoylphorbol-13-acetate (TPA) does not induce Ras activation in 3T3 cells, activation is observed if calmodulin is simultaneously inhibited. This indicates that calmodulin is preventing Ras activation by PKC. Treatment of cells with epidermal growth factor receptor or platelet-derived growth factor receptor tyrosine kinase inhibitors does not abrogate the activation of Ras by calmodulin inhibition. This implies that epidermal growth factor receptor and platelet-derived growth factor receptor tyrosine kinase activities are dispensable for the activation of Ras by TPA plus W13, and, therefore, Ras activation is not a consequence of the transactivation of those receptors by the combination of the anti-calmodulin drug plus TPA. Furthermore, K-Ras, the isoform previously shown to bind to calmodulin, is the only one activated by TPA when calmodulin is inhibited. These data suggest that direct interaction between K-Ras and calmodulin may account for the inability of PKC to activate Ras in 3T3 fibroblasts. In vitro experiments showed that the phosphorylation of K-Ras by PKC was inhibited by calmodulin, suggesting that calmodulin-dependent modulation of K-Ras phosphorylation by PKC could be the mechanism underlying K-Ras activation in fibroblasts treated with TPA plus W13.

3T3 Cells↗
PubMed2002

Mutations of the BRAF gene in human cancer.

Cancers arise owing to the accumulation of mutations in critical genes that alter normal programmes of cell proliferation, differentiation and death. As the first stage of a systematic genome-wide screen for these genes, we have prioritized for analysis signalling pathways in which at least one gene is mutated in human cancer. The RAS RAF MEK ERK MAP kinase pathway mediates cellular responses to growth signals. RAS is mutated to an oncogenic form in about 15% of human cancer. The three RAF genes code for cytoplasmic serine/threonine kinases that are regulated by binding RAS. Here we report BRAF somatic missense mutations in 66% of malignant melanomas and at lower frequency in a wide range of human cancers. All mutations are within the kinase domain, with a single substitution (V599E) accounting for 80%. Mutated BRAF proteins have elevated kinase activity and are transforming in NIH3T3 cells. Furthermore, RAS function is not required for the growth of cancer cell lines with the V599E mutation. As BRAF is a serine/threonine kinase that is commonly activated by somatic point mutation in human cancer, it may provide new therapeutic opportunities in malignant melanoma.

3T3 Cells↗
PubMed2002

Three new prodrugs for suicide gene therapy using carboxypeptidase G2 elicit bystander efficacy in two xenograft models.

Three new prodrugs, [prodrug 1: 4-[bis(2-iodoethyl)amino]-phenyloxycarbonyl-L-glutamic acid; prodrug 2: 3-fluoro-4-[bis(2-chlorethyl)amino]benzoyl-L-glutamic acid; and prodrug 3: 3,5-difluoro-4-[bis(2-iodoethyl)amino]benzoyl-L-glutamic acid] have been assessed for use with a mutant of carboxypeptidase G2 (CPG2, glutamate carboxypeptidase, EC 3.4.17.11,) engineered to be tethered to the outer tumor cell surface (stCPG2(Q)3) as the activating enzyme in suicide gene therapy systems. All three of the prodrugs produce much greater cytotoxicity differentials between stCPG2(Q)3- and control beta-galactosidase (beta-gal)-expressing breast carcinoma MDA MB 361 and colon carcinoma WiDr cells (70- to 450-fold) than was previously observed (19- to 27-fold) with 4-[(2-chloroethyl)(2-mesyloxyethyl)amino]benzoyl-L-glutamic acid (CMDA). Prodrug 1 is the most effective antitumor agent in xenografts in mice inoculated with 100% stCPG2(Q)3-expressing MDA MB 361 cells, whereas prodrugs 2 and 3 are most effective when the percentage of stCPG2(Q)3-expressing cells is 50% or 10%. In nude mice bearing xenografts arising from inocula of 100% stCPG2(Q)3-expressing WiDr cells, prodrug 2 is the most effective antitumor agent. All three of the prodrugs produced histological evidence of substantial bystander cell killing in WiDr xenografts in which only 10% or 50% of the cells inoculated were expressing stCPG2(Q)3. We conclude that all three of the prodrugs are more effective therapeutically with stCPG2(Q)3 than is the previously described prodrug CMDA and, also, that the optimal choice of prodrug varies among different tumor types and that prodrugs, optimized for their bystander effect, are effective when only low percentages of cells in a tumor express CPG2.

Animals↗
PubMed2002

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.

Animals↗
PubMed2002

Cyclic AMP blocks cell growth through Raf-1-dependent and Raf-1-independent mechanisms.

It is widely accepted that cyclic AMP (cAMP) can block cell growth by phosphorylating Raf-1 on serine 43 and inhibiting signaling to extracellular signal-regulated protein kinase. We show that the suppression of Raf-1 by cAMP is considerably more complex than previously reported. When cellular cAMP is elevated, Raf-1 is phosphorylated on three residues (S43, S233, and S259), which work independently to block Raf-1. Both Ras-dependent and Ras-independent processes are disrupted. However, when cAMP-insensitive versions of Raf-1 are expressed in NIH 3T3 cells, their growth is still strongly suppressed when cAMP is elevated. Thus, although Raf-1 appears to be an important cAMP target, other pathways are also targeted by cAMP, providing alternative mechanisms that lead to suppression of cell growth.

1-Methyl-3-isobutylxanthine↗
PubMed2002

14-3-3 antagonizes Ras-mediated Raf-1 recruitment to the plasma membrane to maintain signaling fidelity.

We have investigated the role that S259 phosphorylation, S621 phosphorylation, and 14-3-3 binding play in regulating Raf-1 activity. We show that 14-3-3 binding, rather than Raf-1 phosphorylation, is required for the correct regulation of kinase activity. Phosphorylation of S621 is not required for activity, but 14-3-3 binding is essential. When 14-3-3 binding to conserved region 2 (CR2) was disrupted, Raf-1 basal kinase activity was elevated and it could be further activated by (V12,G37)Ras, (V23)TC21, and (V38)R-Ras. Disruption of 14-3-3 binding at CR2 did not recover binding of Raf-1 to (V12,G37)Ras but allowed more efficient recruitment of Raf-1 to the plasma membrane and stimulated its phosphorylation on S338. Finally, (V12)Ras, but not (V12,G37)Ras, displaced 14-3-3 from full-length Raf-1 and the Raf-1 bound to Ras. GTP was still phosphorylated on S259. Our data suggest that stable association of Raf-1 with the plasma membrane requires Ras-mediated displacement of 14-3-3 from CR2. Small G proteins that cannot displace 14-3-3 fail to recruit Raf-1 to the membrane efficiently and so fail to stimulate kinase activity.

14-3-3 Proteins↗
PubMed2002

Expression of kinase suppressor of Ras in the normal adult and embryonic mouse.

Recent studies indicate that kinase suppressor of Ras (KSR)is a scaffold protein for the Ras/Raf/MEK/ERK signaling cascade in mammals. To help determine the in vivo function of KSR, we have examined the tissue-specific distribution of this protein in the embryonic and adult mouse using a rat monoclonal antibody raised against the mouse protein. Western blot analysis indicates that the protein is expressed at highest levels in the adult brain. It is also expressed at low levels in bladder, ovary, testis, and lung, but the protein is not detectable in any other adult tissue. However, reverse transcription-PCR analysis shows that Ksr transcripts are detected in all adult tissues except the liver. A variant containing a differentially spliced exon in the CA4 domain is observed in brain, cerebellum, ovary, and intestine. The protein is also expressed throughout the E6.5 embryo and at high levels in the neuroepithelium of the E10.5 embryo. At this embryonic stage, expression is also detected at lower levels in the limb and tail buds as well as in the myocardium.

Animals↗