A two-dimensional ERK-AKT signaling code for an NGF-triggered cell-fate decision

Department of Chemical and Systems Biology, Stanford University, Stanford, CA 94305, USA.
Molecular cell (Impact Factor: 14.02). 12/2011; 45(2):196-209. DOI: 10.1016/j.molcel.2011.11.023
Source: PubMed


Growth factors activate Ras, PI3K, and other signaling pathways. It is not well understood how these signals are translated by individual cells into a decision to proliferate or differentiate. Here, using single-cell image analysis of nerve growth factor (NGF)-stimulated PC12 cells, we identified a two-dimensional phospho-ERK (pERK)-phospho-AKT (pAKT) response map with a curved boundary that separates differentiating from proliferating cells. The boundary position remained invariant when different stimuli were used or upstream signaling components perturbed. We further identified Rasa2 as a negative feedback regulator that links PI3K to Ras, placing the stochastically distributed pERK-pAKT signals close to the decision boundary. This allows for uniform NGF stimuli to create a subpopulation of cells that differentiates with each cycle of proliferation. Thus, by linking a complex signaling system to a simpler intermediate response map, cells gain unique integration and control capabilities to balance cell number expansion with differentiation.

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    • "Although the MAPK signaling and PI3K/AKT signaling pathways feature multiple interconnections, they are commonly considered as two distinct pathways [53]. Sharing EGFR as an activating upstream growth factor receptor, the MAPK and PI3K/AKT axes mediate different cellular outcomes by complex temporal phosphorylation patterns, rather than by exclusive activation of a single cascade [54]. The parental RKO cells harbor prominent mutations in both axes of this signaling network, namely B-RafV600E and p110αH1047R. "
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    ABSTRACT: Background Colorectal cancers carrying the B-Raf V600E-mutation are associated with a poor prognosis. The purpose of this study was to identify B-RafV600E-mediated traits of cancer cells in a genetic in vitro model and to assess the selective sensitization of B-RafV600E-mutant cancer cells towards therapeutic agents. Methods Somatic cell gene targeting was used to generate subclones of the colorectal cancer cell line RKO containing either wild-type or V600E-mutant B-Raf kinase. Cell-biologic analyses were performed in order to link cancer cell traits to the BRAF-mutant genotype. Subsequently, the corresponding tumor cell clones were characterized pharmacogenetically to identify therapeutic agents exhibiting selective sensitivity in B-RafV600E-mutant cells. Results Genetic targeting of mutant BRAF resulted in restoration of sensitivity to serum starvation-induced apoptosis and efficiently inhibited cell proliferation in the absence of growth factors. Among tested agents, the B-Raf inhibitor dabrafenib was found to induce a strong V600E-dependent shift in cell viability. In contrast, no differential sensitizing effect was observed for conventional chemotherapeutic agents (mitomycin C, oxaliplatin, paclitaxel, etoposide, 5-fluorouracil), nor for the targeted agents cetuximab, sorafenib, vemurafenib, RAF265, or for inhibition of PI3 kinase. Treatment with dabrafenib efficiently inhibited phosphorylation of the B-Raf downstream targets Mek 1/2 and Erk 1/2. Conclusion Mutant BRAF alleles mediate self-sufficiency of growth signals and serum starvation-induced resistance to apoptosis. Targeting of the BRAF mutation leads to a loss of these hallmarks of cancer. Dabrafenib selectively inhibits cell viability in B-RafV600E mutant cancer cells.
    Molecular Cancer 05/2014; 13(1):122. DOI:10.1186/1476-4598-13-122 · 4.26 Impact Factor
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    • "This also applied to mTORC2-mediated growth factor signaling pathways, as suggested by the periodic Akt activation pattern upon stimulation by insulin or growth factors (Purvis and Lahav, 2013). To this end, mTORC1-mediated inhibition of mTORC2 through Sin1 phosphorylation might be one of such mechanisms, in addition to de-phosphorylation of Akt and multiple other negative feedback mechanism, to ensure that mTORC2 is only activated in a “pulse” manner (Chen et al., 2012). Therefore, between these two mTOR-containing complexes, it is plausible that mTORC1 exhibits constant basal activity whereas the mTORC2 complex is only transiently activated following external stimuli. "
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    ABSTRACT: Mammalian target of rapamycin (mTOR) plays essential roles in cell proliferation, survival and metabolism by forming at least two functional distinct multi-protein complexes, mTORC1 and mTORC2. External growth signals can be received and interpreted by mTORC2 and further transduced to mTORC1. On the other hand, mTORC1 can sense inner-cellular physiological cues such as amino acids and energy states and can indirectly suppress mTORC2 activity in part through phosphorylation of its upstream adaptors, IRS-1 or Grb10, under insulin or IGF-1 stimulation conditions. To date, upstream signaling pathways governing mTORC1 activation have been studied extensively, while the mechanisms modulating mTORC2 activity remain largely elusive. We recently reported that Sin1, an essential mTORC2 subunit, was phosphorylated by either Akt or S6K in a cellular context-dependent manner. More importantly, phosphorylation of Sin1 at T86 and T398 led to a dissociation of Sin1 from the functional mTORC2 holo-enzyme, resulting in reduced Akt activity and sensitizing cells to various apoptotic challenges. Notably, an ovarian cancer patient-derived Sin1-R81T mutation abolished Sin1-T86 phosphorylation by disrupting the canonical S6K-phoshorylation motif, thereby bypassing Sin1-phosphorylation-mediated suppression of mTORC2 and leading to sustained Akt signaling to promote tumorigenesis. Our work therefore provided physiological and pathological evidence to reveal the biological significance of Sin1 phosphorylation-mediated suppression of the mTOR/Akt oncogenic signaling, and further suggested that misregulation of this process might contribute to Akt hyper-activation that is frequently observed in human cancers.
    Protein & Cell 01/2014; 5(3). DOI:10.1007/s13238-014-0021-8 · 3.25 Impact Factor
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    • "These results were consistent with some of the earlier reports exploring neurite outgrowth [43-45] but not others [15-18,39-42]. A recent systems-based study revealed a two-dimensional Erk-Akt signaling code that was critical in governing PC12 cells proliferation and differentiation [46]. Thus, the controversy surrounding the involvement of P38 and Akt would be more adequately addressed using systems-based approaches in the future. "
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    ABSTRACT: Synergistic multi-ligand treatments that can induce neuronal differentiation offer valuable strategies to regulate and modulate neurite outgrowth. Whereas the signaling pathways mediating single ligand-induced neurite outgrowth, such as Akt, extracellular signal-regulated kinase (Erk), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (P38), have been extensively studied, the mechanisms underlying multi-ligand synergistic neurite outgrowth are poorly understood. In an attempt to gain insight into synergistic neurite outgrowth, PC12 cells were treated with one of three combinations: pituitary adenylate cyclase-activating peptide (PACAP) with epidermal growth factor (EP), basic fibroblast growth factor (FP), or nerve growth factor (NP) and then challenged with the appropriate kinase inhibitors to assess the signaling pathways involved in the process. Response surface analyses indicated that synergistic neurite outgrowth was regulated by distinct pathways in these systems. Synergistic increases in the phosphorylation of Erk and JNK, but not Akt or P38, were observed with the three growth factor-PACAP combinations. Unexpectedly, we identified a synergistic increase in JNK phosphorylation, which was involved in neurite outgrowth in the NP and FP, but not EP, systems. Inhibition of JNK using the SP600125 inhibitor reduced phosphorylation of 90 kDa ribosomal S6 kinase (P90RSK) in the NP and FP, but not EP, systems. This suggested the involvement of P90RSK in mediating the differential effects of JNK in synergistic neurite outgrowth. Taken together, these findings reveal the involvement of distinct signaling pathways in regulating neurite outgrowth in response to different synergistic growth factor-PACAP treatments. Our findings demonstrate a hitherto unrecognized mechanism of JNK-P90RSK in mediating synergistic neurite outgrowth induced by the co-treatment of growth factors and PACAP.
    BMC Neuroscience 12/2013; 14(1):153. DOI:10.1186/1471-2202-14-153 · 2.67 Impact Factor
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