Long noncoding RNAs in the mTOR signaling network: biomarkers and therapeutic targets

As an evolutionarily conserved serine/threonine kinase of the phosphoinositide 3-kinase (PI3K) related kinase family, the mechanistic/mammalian target of rapamycin (mTOR) plays vital roles in the PI3K/AKT/mTOR pathway, participating in different cellular processes including cell survival, metabolism and proliferation. Aberrant activity of this signaling pathway may lead to oncogenesis. Over the last two decades, great progress has been made in the understanding of mTOR activation and how its response is counteracted for maintaining tissue homeostasis. Besides regulatory proteins and microRNAs, long noncoding RNA (lncRNA) is another emerging critical layer of the intricate modulatory architecture for the control of the mTOR signaling circuit. Also, the production of numerous lncRNAs is induced by mTOR treatment. These findings offer new perspectives for designing novel diagnostic and therapeutic strategies. In this review, we summarize the interactions between the mTOR signaling pathway and lncRNAs in the development and progression of various types of tumors, focusing on the mechanisms of these interactions, and also discuss the potential use of lncRNAs as biomarkers and therapeutic targets for malignancies.     

Insulin-like growth factor I-stimulated melanoma cell migration requires phosphoinositide 3-kinase but not extracellular-regulated kinase activation

Dysregulated signaling contributes to altered cellular growth, motility, and survival during cancer progression. We have evaluated the ability of several factors to stimulate migration in WM1341D, a cell line derived from an invasive human vertical growth phase melanoma. Basic fibroblast growth factor, hepatocyte growth factor, interleukin-8, and CCL27 each slightly increased migration. Insulin-like growth factor I (IGF-I), however, stimulated a 15-fold increase in migration. This response required the IGF-I receptor, which activates phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathways. Both pathways have been implicated in migration in a variety of cell types, but the signaling required for IGF-I-induced melanoma cell migration is not well defined. IGF-I-stimulated activation of MAPK/ERK signaling in WM1341D cells was inhibited by U0126, but a 33-fold higher dose of U0126 was needed to inhibit IGF-I-stimulated cellular migration. In contrast, similar concentrations of either wortmannin or LY294002 were required to inhibit both IGF-I-induced PI3K activation and migration. These results indicate that IGF-I-stimulated migration of WM1341D cells requires PI3K activation but is independent of MAPK/ERK signaling. Determining the contributions of IGF-I signaling pathways to migration will help us to understand melanoma progression and may lead to new therapeutic targets of this highly metastatic cancer.     

PTEN/PI3K and MAPK signaling in protection and pathology following CNS injuries

Brain and spinal cord injuries initiate widespread temporal and spatial neurodegeneration, through both necrotic and programmed cell death mechanisms. Inflammation, reactive oxidation, excitotoxicity and cell-specific dysregulation of metabolic processes are instigated by traumatic insult and are main contributors to this cumulative damage. Successful treatments rely on prevention or reduction of the magnitude of disruption, and interfering with injurious cellular responses through modulation of signaling cascades is an effective approach. Two intracellular signaling pathways, the phosphatase and tensin homolog (PTEN)/phosphatidylinositol 3-kinase (PI3K) and mitogenactivated protein kinase (MAPK) signaling cascades play various cellular roles under normal and pathological conditions. Activation of both pathways can influence anatomical and functional outcomes in multiple CNS disorders. However, some mechanisms involve inhibiting or enhancing one pathway or the other, or both, in propagating specific downstream effects. Though many intracellular mechanisms contribute to cell responses to insult, this review examines the evidence exploring PTEN/PI3K and MAPK signaling influence on pathology, neuroprotection, and repair and how these pathways may be targeted for advancing knowledge and improving neurological outcome after injury to the brain and spinal cord.     

Induction of Thyroid Gene Expression and Radioiodine Uptake in Melanoma Cells: Novel Therapeutic Implications

Both the MAP kinase and PI3K/Akt pathways play an important role in the pathogenesis of melanoma. We conducted the present study to test the hypothesis that targeting the two pathways to potently induce cell inhibition accompanied with thyroid iodide-handling gene expression for adjunct radioiodine ablation could be a novel effective therapeutic strategy for melanoma. We used specific shRNA approaches and inhibitors to individually or dually suppress the MAP kinase and PI3K/Akt pathways and examined the effects on a variety of molecular and cellular responses of melanoma cells that harbored activating genetic alterations in the two pathways. Suppression of the MAP kinase and PI3K/Akt pathways showed potent anti-melanoma cell effects, including the inhibition of cell proliferation, transformation and invasion, induction of G₀/G₁ cell cycle arrest and, when the two pathways were dually suppressed, cell apoptosis. Remarkably, suppression of the two pathways, particularly simultaneous suppression of them, also induced expression of genes that are normally expressed in the thyroid gland, such as the genes for sodium/iodide symporter and thyroid-stimulating hormone receptor. Melanoma cells were consequently conferred the ability to take up radioiodide. We conclude that dually targeting the MAP kinase and PI3K/Akt pathways for potent cell inhibition coupled with induction of thyroid gene expression for adjunct radioiodine ablation therapy may prove to be a novel and effective therapeutic strategy for melanoma.     

Synergistic targeted inhibition of MEK and dual PI3K/mTOR diminishes viability and inhibits tumor growth of canine melanoma underscoring its utility as a preclinical model for human mucosal melanoma

Human mucosal melanoma (MM), an uncommon, aggressive and diverse subtype, shares characteristics with spontaneous MM in dogs. Although BRAF and N‐RAS mutations are uncommon in MM in both species, the majority of human and canine MM evaluated exhibited RAS/ERK and/or PI3K/mTOR signaling pathway activation. Canine MM cell lines, with varying ERK and AKT/mTOR activation levels reflective of naturally occurring differences in dogs, were sensitive to the MEK inhibitor GSK1120212 and dual PI3K/mTOR inhibitor NVP‐BEZ235. The two‐drug combination synergistically decreased cell survival in association with caspase 3/7 activation, as well as altered expression of cell cycle regulatory proteins and Bcl‐2 family proteins. In combination, the two drugs targeted their respective signaling pathways, potentiating reduction of pathway mediators p‐ERK, p‐AKT, p‐S6, and 4E‐BP1 in vitro, and in association with significantly inhibited solid tumor growth in MM xenografts in mice. These findings provide evidence of synergistic therapeutic efficacy when simultaneously targeting multiple mediators in melanoma with Ras/ERK and PI3K/mTOR pathway activation.     

PI3K: a potential therapeutic target for cancer

Phosphatidylinositol 3-kinase (PI3K), one member of lipid kinase family, has been demonstrated to play a key role in regulating cell proliferation, adhesion, survival, and motility. Recent studies indicate that PI3K related signaling pathway is one of the most commonly activated pathways in human cancers. Accordingly, pharmacological inhibition of key nodes in this signaling cascade has been a focus in developmental therapeutics. To date, Inhibitors targeting PI3K or nodes in this pathway, AKT and mTOR, are best studied and have reached clinical trials. In this review, we will focus on recent progress on understanding of PI3Ks signaling pathway and the development of PI3K inhibitors.     

Epac1‐induced cellular proliferation in prostate cancer cells is mediated by B‐Raf/ERK and mTOR signaling cascades

cAMP‐dependent, PKA‐independent effects on cell proliferation are mediated by cAMP binding to EPAC and activation of Rap signaling. In this report, we employed the analogue 8‐CPT‐2‐O‐Me‐cAMP to study binding to EPAC and subsequent activation of B‐Raf/ERK and mTOR signaling in human cancer cells. This compound significantly stimulated DNA synthesis, protein synthesis, and cellular proliferation of human 1‐LN prostate cancer cells. By study of phosphorylation‐dependent activation, we demonstrate that EPAC‐mediated cellular effects require activation of the B‐Raf/ERK and mTOR signaling cascades. RNAi directed against EPAC gene expression as well as inhibitors of ERK, PI 3‐kinase, and mTOR were employed to further demonstrate the role of these pathways in regulating prostate cancer cell proliferation. These studies were then extended to several other human prostate cancer cell lines and melanoma cells with comparable results. We conclude that B‐Raf/ERK and mTOR signaling play an essential role in cAMP‐dependent, but PKA‐independent, proliferation of cancer cells. J. Cell. Biochem. 108: 998–1011, 2009. © 2009 Wiley‐Liss, Inc.     

Mitogen-activated protein kinases as key players in osmotic stress signaling

BackgroundOsmotic stress arises from the difference between intracellular and extracellular osmolality. It induces cell swelling or shrinkage as a consequence of water influx or efflux, which threatens cellular activities. Mitogen-activated protein kinases (MAPKs) play central roles in signaling pathways in osmotic stress responses, including the regulation of intracellular levels of inorganic ions and organic osmolytes.Scope of reviewThe present review summarizes the cellular osmotic stress response and the function and regulation of the vertebrate MAPK signaling pathways involved. We also describe recent findings regarding apoptosis signal-regulating kinase 3 (ASK3), a MAP3K member, to demonstrate its regulatory effects on signaling molecules beyond MAPKs.Major conclusionsMAPKs are rapidly activated by osmotic stress and have diverse roles, such as cell volume regulation, gene expression, and cell survival/death. There is significant cell type specificity in the function and regulation of MAPKs. Based on its activity change during osmotic stress and its regulation of the WNK1-SPAK/OSR1 pathway, ASK3 is expected to play important roles in osmosensing mechanisms and cellular functions related to osmoregulation.General significanceMAPKs are essential for various cellular responses to osmotic stress; thus, the identification of the upstream regulators of MAPK pathways will provide valuable clues regarding the cellular osmosensing mechanism, which remains elusive in mammals. The elucidation of in vivo MAPK functions is also important because osmotic stress in physiological and pathophysiological conditions often results from changes in the intracellular osmolality. These studies potentially contribute to the establishment of therapeutic strategies against diseases that accompany osmotic perturbation.     

Mitochondrial reactive zones in antiviral innate immunity

Mitochondria are multi-functioning organelles that participate in a wide range of biologic processes from energy metabolism to cellular suicide. Mitochondria are also involved in the cellular innate immune response against microorganisms or environmental irritants, particularly in mammals. Mitochondrial-mediated innate immunity is achieved by the activation of two discrete signaling pathways, the NLR family pyrin domain-containing 3 inflammasomes and the retinoic acid-inducible gene I-like receptor pathway. In both pathways, a mitochondrial outer membrane adaptor protein, called mitochondrial antiviral signaling MAVS, and mitochondria-derived components play a key role in signal transduction. In this review, we discuss current insights regarding the fundamental phenomena of mitochondrial-related innate immune responses, and review the specific roles of various mitochondrial subcompartments in fine-tuning innate immune signaling events. We propose that specific targeting of mitochondrial functions is a potential therapeutic approach for the management of infectious diseases and autoinflammatory disorders with an excessive immune response.     

Akt regulates TPP1 homodimerization and telomere protection

Telomeres are specialized structures at the ends of eukaryotic chromosomes that are important for maintaining genome stability and integrity. Telomere dysfunction has been linked to aging and cancer development. In mammalian cells, extensive studies have been carried out to illustrate how core telomeric proteins assemble on telomeres to recruit the telomerase and additional factors for telomere maintenance and protection. In comparison, how changes in growth signaling pathways impact telomeres and telomere‐binding proteins remains largely unexplored. The phosphatidylinositol 3‐kinase (PI3‐K)/Akt (also known as PKB) pathway, one of the best characterized growth signaling cascades, regulates a variety of cellular function including cell proliferation, survival, metabolism, and DNA repair, and dysregulation of PI3‐K/Akt signaling has been linked to aging and diseases such as cancer and diabetes. In this study, we provide evidence that the Akt signaling pathway plays an important role in telomere protection. Akt inhibition either by chemical inhibitors or small interfering RNAs induced telomere dysfunction. Furthermore, we found that TPP1 could homodimerize through its OB‐fold, a process that was dependent on the Akt kinase. Telomere damage and reduced TPP1 dimerization as a result of Akt inhibition was also accompanied by diminished recruitment of TPP1 and POT1 to the telomeres. Our findings highlight a previously unknown link between Akt signaling and telomere protection.     

PI3K is involved in L‐selectin‐ and PSGL‐1‐mediated neutrophil rolling on E‐selectin via F‐actin redistribution and assembly

L‐selectin and P‐selectin glycoprotein ligand‐1 (PSGL‐1) are adhesion molecules that play critical roles in neutrophil rolling during inflammation and lymphocyte homing. On the other hand they also function as signaling receptors to induce cytoskeleton changes. The present study is to investigate the signaling kinases responsible for the F‐actin changes mediated by L‐selectin and PSGL‐1 during neutrophil rolling on E‐selectin. Western blot analysis demonstrated that PI3K activation, peaking within 5 min, was induced by ligation of L‐selectin and PSGL‐1 with E‐selectin, and that Vav1 (the pivotal downstream effector of PI3K signaling pathway involved in cytoskeleton regulation) was recruited to the membrane and tyrosine‐phosphorylated, depending on PI3K. Furthermore, the F‐actin redistribution and assembly mediated by ligation with E‐selectin were blocked by LY294002, a PI3K specific inhibitor. Additional experiments showed that PI3K activity was involved in neutrophil rolling on E‐selectin. However, Syk/Zap70, the well‐known upstream kinase of PI3K, was not involved in this event. These data suggest that PI3K is required for the F‐actin‐based cytoskeleton changes during neutrophil rolling on E‐selectin, which may consequently regulate the rolling event. J. Cell. Biochem. 110: 910–919, 2010. © 2010 Wiley‐Liss, Inc.     

Data-Derived Modeling Characterizes Plasticity of MAPK Signaling in Melanoma

The majority of melanomas have been shown to harbor somatic mutations in the RAS-RAF-MEK-MAPK and PI3K-AKT pathways, which play a major role in regulation of proliferation and survival. The prevalence of these mutations makes these kinase signal transduction pathways an attractive target for cancer therapy. However, tumors have generally shown adaptive resistance to treatment. This adaptation is achieved in melanoma through its ability to undergo neovascularization, migration and rearrangement of signaling pathways. To understand the dynamic, nonlinear behavior of signaling pathways in cancer, several computational modeling approaches have been suggested. Most of those models require that the pathway topology remains constant over the entire observation period. However, changes in topology might underlie adaptive behavior to drug treatment. To study signaling rearrangements, here we present a new approach based on Fuzzy Logic (FL) that predicts changes in network architecture over time. This adaptive modeling approach was used to investigate pathway dynamics in a newly acquired experimental dataset describing total and phosphorylated protein signaling over four days in A375 melanoma cell line exposed to different kinase inhibitors. First, a generalized strategy was established to implement a parameter-reduced FL model encoding non-linear activity of a signaling network in response to perturbation. Next, a literature-based topology was generated and parameters of the FL model were derived from the full experimental dataset. Subsequently, the temporal evolution of model performance was evaluated by leaving time-defined data points out of training. Emerging discrepancies between model predictions and experimental data at specific time points allowed the characterization of potential network rearrangement. We demonstrate that this adaptive FL modeling approach helps to enhance our mechanistic understanding of the molecular plasticity of melanoma.     

Pathological roles of MAPK signaling pathways in human diseases

The mammalian family of mitogen-activated protein kinases (MAPKs) includes extracellular signal-regulated kinase (ERK), p38, and c-Jun NH₂-terminal kinase (JNK), with each MAPK signaling pathway consisting of at least three components, a MAPK kinase kinase (MAP3K), a MAPK kinase (MAP2K), and a MAPK. The MAPK pathways are activated by diverse extracellular and intracellular stimuli including peptide growth factors, cytokines, hormones, and various cellular stressors such as oxidative stress and endoplasmic reticulum stress. These signaling pathways regulate a variety of cellular activities including proliferation, differentiation, survival, and death. Deviation from the strict control of MAPK signaling pathways has been implicated in the development of many human diseases including Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS) and various types of cancers. Persistent activation of the JNK or p38 signaling pathways has been suggested to mediate neuronal apoptosis in AD, PD, and ALS, whereas the ERK signaling pathway plays a key role in several steps of tumorigenesis including cancer cell proliferation, migration, and invasion. In this review, we summarize recent findings on the roles of MAPK signaling pathways in human disorders, focusing on cancer and neurodegenerative diseases including AD, PD, and ALS.     

The role of mitogen- and stress-activated protein kinase pathways in melanoma

Recent discoveries have increased our comprehension of the molecular signaling events critical for melanoma development and progression. Many oncogenes driving melanoma have been identified, and most of them exert their oncogenic effects through the activation of the RAF/MEK/ERK mitogen-activated protein kinase (MAPK) pathway. The c-Jun N-terminal kinase (JNK) and p38 MAPK pathways are also important in melanoma, but their precise role is not clear yet. This review summarizes our current knowledge on the role of the three main MAPK pathways, extracellular regulated kinase (ERK), JNK, and p38, and their impact on melanoma biology. Although the results obtained with BRAF inhibitors in melanoma patients are impressive, several mechanisms of acquired resistance have emerged. To overcome this obstacle constitutes the new challenge in melanoma therapy. Given the major role that MAPKs play in melanoma, understanding their functions and the interconnection among them and with other signaling pathways represents a step forward toward this goal.     

Tamoxifen inhibits tumor cell invasion and metastasis in mouse melanoma through suppression of PKC/MEK/ERK and PKC/PI3K/Akt pathways

In melanoma, several signaling pathways are constitutively activated. Among these, the protein kinase C (PKC) signaling pathways are activated through multiple signal transduction molecules and appear to play major roles in melanoma progression. Recently, it has been reported that tamoxifen, an anti-estrogen reagent, inhibits PKC signaling in estrogen-negative and estrogen-independent cancer cell lines. Thus, we investigated whether tamoxifen inhibited tumor cell invasion and metastasis in mouse melanoma cell line B16BL6. Tamoxifen significantly inhibited lung metastasis, cell migration, and invasion at concentrations that did not show anti-proliferative effects on B16BL6 cells. Tamoxifen also inhibited the mRNA expressions and protein activities of matrix metalloproteinases (MMPs). Furthermore, tamoxifen suppressed phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt through the inhibition of PKCα and PKCδ phosphorylation. However, other signal transduction factor, such as p38 mitogen-activated protein kinase (p38MAPK) was unaffected. The results indicate that tamoxifen suppresses the PKC/mitogen-activated protein kinase kinase (MEK)/ERK and PKC/phosphatidylinositol-3 kinase (PI3K)/Akt pathways, thereby inhibiting B16BL6 cell migration, invasion, and metastasis. Moreover, tamoxifen markedly inhibited not only developing but also clinically evident metastasis. These findings suggest that tamoxifen has potential clinical applications for the treatment of tumor cell metastasis.