Computational Modeling of PI3K/AKT and MAPK Signaling Pathways in Melanoma Cancer

Background

Malignant melanoma is an aggressive tumor of the skin and seems to be resistant to current therapeutic approaches. Melanocytic transformation is thought to occur by sequential accumulation of genetic and molecular alterations able to activate the Ras/Raf/MEK/ERK (MAPK) and/or the PI3K/AKT (AKT) signalling pathways. Specifically, mutations of B-RAF activate MAPK pathway resulting in cell cycle progression and apoptosis prevention. According to these findings, MAPK and AKT pathways may represent promising therapeutic targets for an otherwise devastating disease.

Result

Here we show a computational model able to simulate the main biochemical and metabolic interactions in the PI3K/AKT and MAPK pathways potentially involved in melanoma development. Overall, this computational approach may accelerate the drug discovery process and encourages the identification of novel pathway activators with consequent development of novel antioncogenic compounds to overcome tumor cell resistance to conventional therapeutic agents. The source code of the various versions of the model are available as S1 Archive.     

Plasticity of the MAPK Signaling Network in Response to Mechanical Stress

Cells display versatile responses to mechanical inputs and recent studies have identified the mitogen-activated protein kinase (MAPK) cascades mediating the biological effects observed upon mechanical stimulation. Although, MAPK pathways can act insulated from each other, several mechanisms facilitate the crosstalk between the components of these cascades. Yet, the combinatorial complexity of potential molecular interactions between these elements have prevented the understanding of their concerted functions. To analyze the plasticity of the MAPK signaling network in response to mechanical stress we performed a non-saturating epistatic screen in resting and stretched conditions employing as readout a JNK responsive dJun-FRET biosensor. By knocking down MAPKs, and JNK pathway regulators, singly or in pairs in Drosophila S2R+ cells, we have uncovered unexpected regulatory links between JNK cascade kinases, Rho GTPases, MAPKs and the JNK phosphatase Puc. These relationships have been integrated in a system network model at equilibrium accounting for all experimentally validated interactions. This model allows predicting the global reaction of the network to its modulation in response to mechanical stress. It also highlights its context-dependent sensitivity.     

Canonical and Alternative MAPK Signaling

The archetype of MAPK cascade activation is somewhat challenged by the most recent discovery of unexpected phosphorylation patterns, alternative activation mechanisms and sub-cellular localization, in various members of this protein kinase family. In particular, activation by autophosphorylation pathways has now been described for the three best understood MAPK subgroups: ERK1/2; JNK1/2 and p38α/β. Also, a form of dosage compensation between homologues has been shown to occur in the case of ERK1/2 and JNK1/2. In this paper we summarize the MAPK activation pathway, with an emphasis on non-canonical examples. We use this information to propose a model for MAPK signal transduction that considers a cross-talk between MAPKs with different activation loop sequence motifs and unique C-terminal extensions. We highlight the occurrence of non-canonical substrate specificity during MAPK auto-activation, in strong connection with MAPK homo- and hetero-dimerization events.     

SEC23A Inhibit Melanoma Metastatic through Secretory PF4 Cooperation with SPARC to Inhibit MAPK Signaling Pathway

Metastasis of melanoma to the distant organs is a multistep process in which the tumor microenvironment (TME) may play an important role. However, the relationship between metastatic progression and TME is intricate. In the present study, using melanoma derivative cell lines OL (oligometastatic) and POL (polymetastatic) that differ in their metastatic colonization capability, we have elucidated a new mechanism involving “SEC23A-PF4-MAPK/ERK axis” in which PF4 transported by COPII hinders metastasis through inhibition of MAPK/ERK signaling pathway. Furthermore, SPARC can act cooperatively to enhance the inhibition of Pf4 on ERK phosphorylation and melanoma cell metastasis. Our findings show the possibility of targeting cancer cell secretome for therapeutic development.     

SPARC Controls Melanoma Cell Plasticity through Rac1

Cell transition to a more aggressive mesenchymal-like phenotype is a hallmark of cancer progression that involves different steps and requires tightly regulated cell plasticity. SPARC (Secreted Protein Acidic and Rich in Cysteine) is a matricellular protein that promotes this transition in various malignant cell types, including melanoma cells. We found that suppression of SPARC expression in human melanoma cells compromised cell migration, adhesion, cytoskeleton structure, and cell size. These changes involved the Akt/mTOR pathway. Re-expression of SPARC or protein addition restored all the cell features. Suppression of SPARC expression was associated with increased Rac1-GTP levels and its membrane localization. Expression of the dominant negative mutant of Rac1 counteracted almost all the changes observed in SPARC-deficient cells. Overall, these data suggest that most of the SPARC-mediated effects occurred mainly through the blockade of Rac1 activity.     

MAPK信号通路与脂肪细胞分化

促分裂原活化的蛋白激酶(MAPK)通路是真核细胞重要的信号转导通路,主要有ERK、p38和JNK三条途径,参与调控多种细胞应答和生理病理过程。该文重点讨论了MAPK对脂肪细胞分化的调控。其中ERK对脂肪细胞分化的调节具有多样性,随分化进程不同表现为不同的调控功能,p38和JNK也通过不同的机制对脂肪细胞分化发挥相异的调节作用。MAPK信号转导与脂肪分化的紧密联系,使其可能成为调控与脂分化密切相关的代谢疾病如肥胖、糖尿病等的一条关键通路。     

Mechanisms of Activity-dependent Plasticity in Cellular Nitric Oxide-cGMP Signaling

Cellular responsiveness to nitric oxide (NO) is shaped by past history of NO exposure. The mechanisms behind this plasticity were explored using rat platelets in vitro, specifically to determine the relative contributions made by desensitization of NO receptors, which couple to cGMP formation, and by phosphodiesterase-5 (PDE5), which is activated by cGMP and also hydrolyzes it. Repeated delivery of brief NO pulses (50 nm peak) at 1-min intervals resulted in a progressive loss of the associated cGMP responses, which was the combined consequence of receptor desensitization and PDE5 activation, with the former dominating. Delivery of pulses of differing amplitude showed that NO stimulated and desensitized receptors with similar potency (EC₅₀ = 10–20 nm). PDE5 activation was highly sensitive to NO, with a single pulse peaking at 2 nm being sufficient to evoke a 50% loss of response to a subsequent near-maximal NO pulse. However, the activated state of the PDE subsided quickly after removal of NO, the half-time for recovery being 25 s. In contrast, receptor desensitization reverted much more slowly, the half-time being 16 min. Accordingly, with long (20-min) exposures, NO concentrations as low as 600 pm provoked significant desensitization. The results indicate that PDE5 activation and receptor desensitization subserve distinct short term and longer term roles as mediators of plasticity in NO-cGMP signaling. A kinetic model explicitly describing the complex interplay between NO concentration, cGMP synthesis, PDE5 activation, and the resulting cGMP accumulation successfully simulated the present and previous data.Nitric oxide (NO) is an intercellular messenger molecule in most tissues of the body and exerts physiological effects by binding to receptors possessing intrinsic guanylyl cyclase (GC)⁴ activity. The receptor proteins are known by various names, including the homogenate-based one, soluble guanylyl cyclase, but here we simply call them NO receptors because this terminology is conceptually more informative in a cellular context. Synthesis of cGMP from GTP that follows receptor activation can engage a number of downstream targets, including cGMP-dependent protein kinase, to bring about alterations in cell function, such as smooth muscle relaxation and neural transmission (1–3).In common with other hormone or transmitter signaling pathways, the sensitivity of the NO-cGMP pathway is subject to short term and long term regulation. Enduring exposure of cells to NO (hours or more) leads to a loss of NO responsiveness that in the cardiovascular system contributes to the clinical problem of tolerance to nitrovasodilator therapy (4–6). One mechanism here is a gradual loss of the NO receptor mRNA and protein (7, 8). Conversely, a chronic lack of NO leads to supersensitivity that has been attributed to increased NO receptor activity (9, 10).Short term regulatory mechanisms serve to shape acute cellular cGMP responses to NO and may involve NO receptor desensitization, reducing the rate of cGMP formation (11), activation of phosphodiesterases (PDEs) that consume cGMP (12), or combinations of the two (13, 14). These short term mechanisms may also be quite sustained. For example, relatively brief (5 min) exposures of cells to NO can reduce responsiveness to NO an hour later, a phenomenon that correlated with increased activity of PDE5 (15). This PDE isoform, which is inhibited by drugs like sildenafil (Viagra) used to treat erectile dysfunction, contains a non-catalytic cGMP binding site whose occupation stimulates catalytic activity (16, 17). Reciprocally, shortly after removing the endothelial source of NO, blood vessels develop a supersensitivity to NO-induced relaxation, an effect that was speculated to reflect increased NO receptor responsiveness (18).Addressing directly the relative contributions of NO receptors and PDEs to the regulation of NO-cGMP signaling is problematic in complex tissues because it is very difficult to measure their activities accurately. Rat platelets maintained in vitro, on the other hand, have merit as an experimental model for these purposes because they are physiological NO targets, homogenous, and are also extremely small (about 1 μm in diameter), minimizing problems of compartmentation of the signaling proteins and of diffusion delays in access of applied agents to the cell interior. In addition, they exist naturally in suspension, which is ideal for kinetic studies, they have an abundance of NO receptors of just one type (α1β1), and the only detectable PDE that hydrolyzes NO-evoked cGMP signals is PDE5 (13). Exposure of these cells to persistent NO generates only very transient cGMP responses, mostly terminating within 10 s of NO application; this profile has been explained by a combination of receptor desensitization and PDE5 enhancement (13). The questions addressed here are whether or not these adaptations persist beyond the period of NO application to influence subsequent cellular responsiveness and, if so, for how long, and what are their relative contributions to the plasticity? Central to the feasibility of obtaining answers was our recent development of a method for delivering repeated NO pulses of known amplitude and duration (14), allowing controlled conditioning NO exposures to be followed by test exposures after selected time intervals in NO-free solution.     

Signaling for Vesicle Mobilization and Synaptic Plasticity

The hypothesis that release of classical neurotransmitters and neuropeptides is facilitated by increasing the mobility of small synaptic vesicles (SSVs) and dense core vesicles (DCVs) could not be tested until the advent of methods for visualizing these secretory vesicles in living nerve terminals. In fact, fluorescence imaging studies have only since 2005 established that activity increases secretory vesicle mobility in motoneuron terminals and chromaffin cells. Mobilization of DCVs and SSVs appears to be due to liberation of hindered vesicles to promote quicker diffusion. However, F-actin and synapsin, which have been featured in mobilization models, are not required for activity-dependent increases in the mobility of DCVs or SSVs. Most recently, the signaling required for sustained mobilization has been identified for Drosophila motoneuron DCVs and shown to increase synaptic transmission. Specifically, presynaptic endoplasmic reticulum ryanodine receptor-mediated Ca2+ release activates Ca2+/calmodulin-dependent kinase II to mobilize DCVs and induce post-tetanic potentiation (PTP) of neuropeptide release in the Drosophila neuromuscular junction. The shared signaling for increasing vesicle mobility and PTP links vesicle mobilization and synaptic plasticity.     

RhoA-p38MAPK信号转导通路及其进展

RhoA属于小G蛋白家族成员,p38MAPK属于丝/苏氨酸蛋白激酶家族成员.本文从巨噬细胞、肌肉细胞、成纤维细胞、神经元和肿瘤几个方面阐述RhoA-p38MAPK信号通路的研究进展,此信号通路在细胞骨架的改变和肿瘤的发生发展方面有望成为新的研究热点.     

RhoA—p38MAPK信号转导通路及其进展

RhoA属于小G蛋白家族成员,p38MAPK属于丝/苏氨酸蛋白激酶家族成员。本文从巨噬细胞、肌肉细胞、成纤维细胞、神经元和肿瘤几个方面阐述RhoA-p38MAPK信号通路的研究进展,此信号通路在细胞骨架的改变和肿瘤的发生发展方面有望成为新的研究热点。     

巨噬细胞免疫调变信号——PKA与PKC对MAPK信号通路的调节

以前的研究工作表明,细菌脂多糖（ＬＰＳ）可以调变抑制性巨噬细胞为增强Ｔ、Ｂ淋巴细胞及ＮＫ细胞活性,同时又能保持或增强其抗肿瘤效应。忆报道了在这一复杂的免疫调变过程中伴随有蛋白激酶Ｃ（ＰＫＣ）和促分裂原活化蛋白激酶（ＭＡＰＫ）信号转导通路的激活。为了探索免疫调变过程中其他信号对ＭＡＰＫ通路的影响,以ＬＰＳ调变小鼠腹腔抑制性巨噬细胞为模型,研究了ｃＡＭＰ／ＰＫＡ和佛波酯（ＰＭＡ）／ＰＫＣ信号对ＭＡＰＫ     

Mechanisms and Constraints on Yeast MAPK Signaling Specificity

The survival of cells relies on their ability to respond specifically to diverse environmental signals. Surprisingly, intracellular signaling pathways often share the same or homologous protein components, yet undesirable crosstalk is, in general, suppressed. This signaling specificity has been well studied in the yeast model system Saccharomyces cerevisiae, where the mitogen-activated protein kinase (MAPK) cascades are repeatedly employed in mediating distinct biological processes including pheromone-induced mating and filamentous growth under starvation. Although various mechanisms have been proposed to interpret the yeast MAPK signaling specificity, a consistent theory is still lacking. Here, we present a mathematical model that shows signaling specificity can arise through asymmetric hierarchical inhibition. The parameters of our model are, where possible, based on experimental data that allow us to determine the constraints imposed by signaling specificity on these parameters. Our model is in broad agreement with experimental observations to date and generates testable predictions that may stimulate further research.     

Case Studies of Categorical Data-Derived Adjustment Factors

Investigations were performed on representative compounds from five different therapeutic classes to evaluate the use of categorical data-derived adjustment factors to account for interindividual variability. The five classes included antidepressants, angiotensin converting enzyme (ACE) inhibitors, nonsteroidal anti-inflammatory drugs (NSAIDS), cholesterol lowering agents, and antibiotics. Each of the case studies summarized the mode of action of the class responsible for both the therapeutic and adverse effects and the key pharmacodynamic (PD) and pharmacokinetic (PK) parameters that determine the likelihood of these responses for individual compounds in the class. For each class, an attempt was made to identify the key factors that determine interindividual variability and whether there was a common basis to establish a categorical default adjustment factor that could be applied across the class (or at least across specific subclasses within the class). Linking the PK and PD parameters to the critical endpoint used to establish a safe level of exposure was an important underlying theme throughout the investigations. Despite the wealth of PK and PD information in the published literature on the surrogate compounds representing these classes, it was difficult to derive a categorical adjustment factor that could be applied broadly within each class. The amount of information available may have hindered rather than helped the evaluations. Derivation of categorical defaults for different classes of “common” chemicals may be more straightforward if sufficient data are available. In a few cases (e.g., tricyclic antibiotics, ACE inhibitors and selected antiinflammatory agents) categorical defaults could be proposed, although it is unclear whether the reduction in uncertainty resulting from their application would be offset by the additional uncertainties that may have resulted from their application. Residual uncertainties may remain depending on the level of confidence in the underlying assumptions used to support the categorical defaults. Regardless of the conclusions on the utility of categorical defaults, these investigations provided further support for the use of data-derived adjustment factors on a compound-specific basis.