Classical RAS proteins are not essential for paradoxical ERK activation induced by RAF inhibitors.
Ontology highlight
ABSTRACT:
SUBMITTER: Lai LP
PROVIDER: S-EPMC8812530 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:B-Raf inhibitors have been used for the treatment of some B-Raf-mutated cancers. They effectively inhibit B-Raf/MEK/ERK signaling in cancers harboring mutant B-Raf, but paradoxically activates MEK/ERK in Ras-mutated cancers. Death receptor 5 (DR5), a cell surface pro-apoptotic protein, triggers apoptosis upon ligation with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or aggregation. This study focused on determining the effects of B-Raf inhibition on DR5 expression and DR5 activation-induced apoptosis in Ras-mutant cancer cells. Using chemical and genetic approaches, we have demonstrated that the B-Raf inhibitor PLX4032 induces DR5 upregulation exclusively in Ras-mutant cancer cells; this effect is dependent on Ras/c-Raf/MEK/ERK signaling activation. PLX4032 induces DR5 expression at transcriptional levels, largely due to enhancing CHOP/Elk1-mediated DR5 transcription. Pre-exposure of Ras-mutated cancer cells to PLX4032 sensitizes them to TRAIL-induced apoptosis; this is also a c-Raf/MEK/ERK-dependent event. Collectively, our findings highlight a previously undiscovered effect of B-Raf inhibition on the induction of DR5 expression and the enhancement of DR5 activation-induced apoptosis in Ras-mutant cancer cells and hence may suggest a novel therapeutic strategy against Ras-mutated cancer cells by driving their death due to DR5-dependent apoptosis through B-Raf inhibition.
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Project description:Small molecules targeting aberrant RAF activity, like vemurafenib (PLX4032), are highly effective against cancers harboring the V600E BRAF mutation and are now approved for clinical use against metastatic melanoma. However, in tissues showing elevated RAS activity and in RAS mutant tumors, these inhibitors stimulate RAF dimerization, resulting in inhibitor resistance and downstream "paradoxical" ERK activation. To understand the global signaling response of cancer cells to RAF inhibitors, we profiled the temporal changes of the phosphoproteome of two colon cancer cell lines (Colo205 and HCT116) that respond differently to vemurafenib. Comprehensive data mining and filtering identified a total of 37,910 phosphorylation sites, 660 of which were dynamically modulated upon treatment with vemurafenib. We established that 83% of these dynamic phosphorylation sites were modulated in accordance with the phospho-ERK profile of the two cell lines. Accordingly, kinase substrate prediction algorithms linked most of these dynamic sites to direct ERK1/2-mediated phosphorylation, supporting a low off-target rate for vemurafenib. Functional classification of target proteins indicated the enrichment of known (nuclear pore, transcription factors, and RAS-RTK signaling) and novel (Rho GTPases signaling and actin cytoskeleton) ERK-controlled functions. Our phosphoproteomic data combined with experimental validation established novel dynamic connections between ERK signaling and the transcriptional regulators TEAD3 (Hippo pathway), MKL1, and MKL2 (Rho serum-response elements pathway). We also confirm that an ERK-docking site found in MKL1 is directly antagonized by overlapping actin binding, defining a novel mechanism of actin-modulated phosphorylation. Altogether, time-resolved phosphoproteomics further documented vemurafenib selectivity and identified novel ERK downstream substrates.
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Project description:The ASPP2 (also known as 53BP2L) tumor suppressor is a proapoptotic member of a family of p53 binding proteins that functions in part by enhancing p53-dependent apoptosis via its C-terminal p53-binding domain. Mounting evidence also suggests that ASPP2 harbors important nonapoptotic p53-independent functions. Structural studies identify a small G protein Ras-association domain in the ASPP2 N terminus. Because Ras-induced senescence is a barrier to tumor formation in normal cells, we investigated whether ASPP2 could bind Ras and stimulate the protein kinase Raf/MEK/ERK signaling cascade. We now show that ASPP2 binds to Ras-GTP at the plasma membrane and stimulates Ras-induced signaling and pERK1/2 levels via promoting Ras-GTP loading, B-Raf/C-Raf dimerization, and C-Raf phosphorylation. These functions require the ASPP2 N terminus because BBP (also known as 53BP2S), an alternatively spliced ASPP2 isoform lacking the N terminus, was defective in binding Ras-GTP and stimulating Raf/MEK/ERK signaling. Decreased ASPP2 levels attenuated H-RasV12-induced senescence in normal human fibroblasts and neonatal human epidermal keratinocytes. Together, our results reveal a mechanism for ASPP2 tumor suppressor function via direct interaction with Ras-GTP to stimulate Ras-induced senescence in nontransformed human cells.
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Project description:RAF inhibitors selectively block ERK signaling in BRAF-mutant melanomas and have defined a genotype-guided approach to care for this disease. RAF inhibitors have the opposite effect in BRAF wild-type tumor cells, where they cause hyperactivation of ERK signaling. Here, we predict that RAF inhibitors can enhance T cell activation, based upon the observation that these agents paradoxically activate ERK signaling in BRAF wild-type cells. To test this hypothesis, we have evaluated the effects of the RAF inhibitor BMS908662 on T cell activation and signaling in vitro and in vivo. We observe that T cell activation is enhanced in a concentration-dependent manner and that this effect corresponds with increased ERK signaling, consistent with paradoxical activation of the pathway. Furthermore, we find that the combination of BMS908662 with CTLA-4 blockade in vivo potentiates T cell expansion, corresponding with hyperactivation of ERK signaling in T cells detectable ex vivo. Lastly, this combination demonstrates superior anti-tumor activity, compared to either agent alone, in two transplantable tumor models. This study provides clear evidence that RAF inhibitors can modulate T cell function by potentiating T cell activation in vitro and in vivo. Paradoxical activation of ERK signaling in T cells offers one mechanism to explain the enhanced antitumor activity seen when RAF inhibitors are combined with CTLA-4 blockade in preclinical models.
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Project description:Connector enhancer of Ksr (CNK) is a conserved multidomain protein essential for Ras signaling in Drosophila melanogaster and thought to be involved in Raf kinase activation. However, the precise role of CNK in Ras signaling is not known, and mammalian CNKs are proposed to have distinct functions. Caenorhabditis elegans has a single CNK homologue, cnk-1. Here, we describe the role of cnk-1 in C. elegans Ras signaling and its requirements for LIN-45 Raf activation. We find that cnk-1 positively regulates multiple Ras signaling events during development, but, unlike Drosophila CNK, cnk-1 does not appear to be essential for signaling. cnk-1 mutants appear to be normal but show cell-type-specific genetic interactions with mutations in two other Ras pathway scaffolds/adaptors ksr-1 and sur-8. Genetic epistasis using various activated LIN-45 Raf transgenes shows that CNK-1 promotes LIN-45 Raf activation at a step between the dephosphorylation of inhibitory sites in the regulatory domain and activating phosphorylation in the kinase domain. Our data are consistent with a model in which CNK promotes Raf phosphorylation/activation through membrane localization, oligomerization, or association with an activating kinase.
| S-EPMC1187957 | biostudies-literature
Project description:The kinases of the Raf family have been intensively studied as activators of the mitogen-activated protein kinase kinase/extra-cellular signal-regulated kinase (ERK) module in regulated and deregulated proliferation. Genetic evidence that Raf is required for ERK activation in vivo has been obtained in lower organisms, which express only one Raf kinase, but was hitherto lacking in mammals, which express more than one Raf kinase. Ablation of the two best studied Raf kinases, B-Raf and Raf-1, is lethal at midgestation in mice, hampering the detailed study of the essential functions of these proteins. Here, we have combined conventional and conditional gene ablation to show that B-Raf is essential for ERK activation and for vascular development in the placenta. B-Raf-deficient placentae show complete absence of phosphorylated ERK and strongly reduced HIF-1alpha and VEGF levels, whereas all these parameters are normal in Raf-1-deficient placentae. In addition, neither ERK phosphorylation nor development are affected in B-raf-deficient embryos that are born alive obtained by epiblast-restricted gene inactivation. The data demonstrate that B-Raf plays a nonredundant role in ERK activation during extraembyronic mammalian development in vivo.
| S-EPMC1360532 | biostudies-literature
Project description:Oncogenic mutations of ras and B-raf frequently occur in many cancer types and are critical for cell transformation and tumorigenesis. Death receptor 5 (DR5) is a cell surface pro-apoptotic death receptor for tumor necrosis factor-related apoptosis-inducing ligand and has been targeted in cancer therapy. The current study has demonstrated induction of DR5 expression by the oncogenic proteins Ras and B-Raf and revealed the underlying mechanisms. We demonstrated that both Ras and B-Raf induce DR5 expression by enforced expression of oncogenic Ras (e.g. H-Ras12V or K-Ras12V) or B-Raf (i.e. V600E) in cells and by analyzing gene expression array data generated from cancer cell lines and from human cancer tissues. This finding is further supported by our results that knockdown of endogenous K-Ras or B-Raf (V600E) reduced the expression of DR5. Importantly, we have elucidated that Ras induces DR5 expression through co-activation of ERK/RSK and JNK signaling pathways and subsequent cooperative effects among the transcriptional factors CHOP, Elk1, and c-Jun to enhance DR5 gene transcription. Moreover, we found that the majority of cancer cell lines highly sensitive to the DR5 agonistic antibody AMG655 have either Ras or B-Raf mutations. Our findings warrant further study on the biology of DR5 regulation by Ras and B-Raf, which may provide new insight into the biology of Ras and B-Raf, and on the potential impact of Ras or B-Raf mutations on the outcome of DR5-targeted cancer therapy.
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Project description:Epidermal growth factor (EGF) and Ras mitogenic signal transduction pathways are frequently activated in breast carcinoma and inhibit mammary differentiation and apoptosis. HC11 mouse mammary epithelial cells, which differentiate and synthesize beta-casein following growth to confluency and stimulation with lactogenic hormones, were used to study EGF-dependent signaling during differentiation. Blocking Mek-Erk or phosphotidylinositol-3-kinase (PI-3 kinase) signaling with specific chemical inhibitors enhanced beta-casein promotor-driven luciferase activity. Because EGF stimulation of HC11 cells resulted in the activation of Ras, the effect of activated Ras (RasV12) or dominant negative (DNRasN17) on lactogen induced differentiation was examined. HC11 cell lines expressing RasV12 or DNRasN17 under the control of a tetracycline (tet)-responsive promotor were constructed. Activated RasV12 expression resulted in reduced tyrosine phosphorylation of Stat5 and a delay in beta-casein expression in response to prolactin. However, the expression of tet-regulated DNRasN17 and adenovirus-encoded DNRasN17 enhanced Stat5 tyrosine phosphorylation, Stat5 DNA binding, and beta-casein transcription. The expression of DNRasN17 blocked the activation of the Mek-Erk pathway by EGF but did not prevent the phosphorylation of AKT, a measure of activation of the PI-3-kinase pathway. Moreover, the expression of DNRasN17 prevented the block to lactogenic differentiation induced by EGF. Stimulation of HC11 cells with prolactin resulted in the association of the SHP2 phosphatase with Stat5, and this association was prevented by DNRasN17 expression. These results demonstrate that in HC11 cells DNRas inhibits the Mek-Erk pathway and enhances lactogenic hormone-induced differentiation. This occurs, in part, by inhibiting the association of the SHP2 phosphatase with Stat5.
| S-EPMC1586098 | biostudies-literature
Project description:First-generation RAF inhibitors paradoxically induce ERK signaling in normal and tumor cells exhibiting RAS activity. Compound-induced RAF dimerization through stabilization of the RAF ON/active state by inhibitors has emerged as a critical contributing factor. RAF inhibitors also enhance RAS-RAF association. Although this event is thought to play a key role in priming RAF activation, the underlying mechanism is not known. Here we report that RAF inhibitors induce the disruption of intramolecular interactions between the kinase domain and its N-terminal regulatory region independently of RAS activity. This provides a molecular basis to explain the induction of RAS-RAF association by RAF inhibitors, as well as the co-operativity observed between RAS activity and RAF kinase inhibitors in driving RAF activation. Profiling of second-generation RAF inhibitors confirmed their improved mode of action, but also revealed liabilities that allowed us to discern two properties of an ideal RAF inhibitor: high-binding affinity to all RAF paralogs and maintenance of the OFF/autoinhibited state of the enzyme.
| S-EPMC5662619 | biostudies-literature
Project description:Multiple myeloma (MM) is a plasma cell malignancy that is still considered to be incurable in most cases. A dominant mutation cluster has been identified in RAS/RAF genes, emphasizing the potential significance of RAS/RAF/MEK/ERK signaling as a therapeutic target. As yet, however, the clinical relevance of this finding is unclear as clinical responses to MEK inhibition in RAS-mutant MM have been mixed. We therefore assessed RAS/RAF mutation status and MEK/ERK pathway activation by both targeted sequencing and phospho-ERK immunohistochemistry in 180 tissue biopsies from 103 patients with newly diagnosed MM (NDMM) and 77 patients with relapsed/refractory MM (rrMM). We found a significant enrichment of RAS/BRAF mutations in rrMM compared to NDMM (P=0.011), which was mainly due to an increase of NRAS mutations (P=0.010). As expected, BRAF mutations were significantly associated with activated downstream signaling. However, only KRAS and not NRAS mutations were associated with pathway activation compared to RAS/BRAFwt (P=0.030). More specifically, only KRASG12D and BRAFV600E were consistently associated with ERK activation (P<0.001 and P=0.006, respectively). Taken together, these results suggest the need for a more specific stratification strategy consisting of both confirmation of protein-level pathway activation as well as detailed RAS/RAF mutation status to allow for a more precise and more effective application of targeted therapies, for example, with BRAF/MEK inhibitors in MM.
| S-EPMC5523069 | biostudies-literature