u-0126 and Rhabdomyosarcoma

The human rhabdomyosarcoma cell line TE671 has been used extensively to study different aspects of muscle biology. However, its ability to differentiate and form myotubes has not been explored. Here, we examined muscle differentiation when we specifically stopped proliferation of human TE671 (WT-TE671) cells by using 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene (U0126), an MAPK inhibitor. Our data show that treated cells initiated fusion, and myotube formation and that expression levels of dysferlin and myogenin were increased, whereas those of pax7 were decreased. Treatment of WT-TE671 cells with vitamin D3 alone and cotreatment with U0126 also promoted dysferlin expression. In addition, we knocked out the DYSF gene, which is involved in muscle differentiation, using CRISPR/Cas9 technology in WT-TE671 cells (Dysf-KO TE671). No dysferlin expression was observed before and after U0126 treatment. Although myogenin expression was absent in vehicle-treated Dysf-KO TE671 cells, after addition of U0126, myogenin reached levels similar to WT-TE671. This widely available source of human cells appropriately treated with U0126 may represent a useful model to study human muscle physiology in vitro. This dysferlin-deficient cell line should allow the study of pathophysiological pathways involved in dysferlin-deficient muscle and constitute a tool for high-throughput screening of therapeutic compounds for patients with dysferlinopathy and other muscle diseases.

Topics: Butadienes; Cell Differentiation; Cell Line, Tumor; CRISPR-Associated Protein 9; CRISPR-Cas Systems; Enzyme Inhibitors; Humans; Mitogen-Activated Protein Kinase Kinases; Muscular Diseases; Nitriles; Rhabdomyosarcoma

In a previous study the ERK1/2 pathway was found to be crucially involved in positive regulation of the enterovirus A 71(EV-A71) IRES (vIRES), thereby contributing to the efficient replication of an important human enterovirus causing death in young children (<5yrs) worldwide. This study focuses on unraveling more about the detailed mechanism of ERK's involvement in this regulation of vIRES. Through the use of siRNAs and specifically pharmacological inhibitor U0126, the ERK cascade was shown to positively regulate EV-A71-mediated cleavage of eIF4GI that established the cellular conditions which favour vIRES-dependent translation. Site-directed mutagenesis of the viral 2A protease (2A

Topics: Antiviral Agents; Butadienes; Cell Line, Tumor; Enterovirus A, Human; Enterovirus Infections; Enzyme Activation; HEK293 Cells; Humans; Internal Ribosome Entry Sites; Mutagenesis, Site-Directed; Nitriles; Polyproteins; Rhabdomyosarcoma; RNA, Small Interfering; Signal Transduction; Viral Proteins; Virus Replication

Rhabdomyosarcoma (RMS) frequently exhibits concomitant activation of the PI3K/Akt/mTOR and the Ras/MEK/ERK pathways. Therefore, we investigated whether pharmacological cotargeting of these two key survival pathways suppresses RMS growth. Here, we identify a synthetic lethal interaction between PI3K/Akt/mTOR and Ras/MEK/ERK pathway inhibition in RMS. The dual PI3K/mTOR inhibitor PI103 and the MEK inhibitor UO126 synergize to trigger apoptosis in several RMS cell lines in a highly synergistic manner (combination index <0.1), whereas either agent alone induces minimal cell death. Similarly, genetic knockdown of p110α and MEK1/2 cooperates to induce apoptosis. Molecular studies reveal that cotreatment with PI103/UO126 cooperates to suppress PI3K/Akt/mTOR and Ras/MEK/ERK signaling, whereas either compound alone is not only less effective to inhibit signaling, but even cross-activates the other pathway. Accordingly, PI103 alone increases ERK phosphorylation, while UO126 enhances Akt phosphorylation, consistent with negative crosstalks between these two signaling pathways. Furthermore, PI103/UO126 cotreatment causes downregulation of several antiapoptotic proteins such as XIAP, Bcl-xL and Mcl-1 as well as increased expression and decreased phosphorylation of the proapoptotic protein BimEL, thus shifting the balance towards apoptosis. Consistently, PI103/UO126 cotreatment cooperates to trigger Bax activation, loss of mitochondrial membrane potential, caspase activation and caspase-dependent apoptosis. This identification of a synthetic lethal interaction between PI3K/mTOR and MEK inhibitors has important implications for the development of novel treatment strategies in RMS.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Apoptosis Regulatory Proteins; Butadienes; Cell Line, Tumor; Class I Phosphatidylinositol 3-Kinases; Dose-Response Relationship, Drug; Drug Synergism; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Furans; Humans; MAP Kinase Kinase 1; MAP Kinase Kinase 2; Membrane Potential, Mitochondrial; Molecular Targeted Therapy; Nitriles; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Pyridines; Pyrimidines; ras Proteins; Rhabdomyosarcoma; RNA Interference; Signal Transduction; Time Factors; TOR Serine-Threonine Kinases; Transfection

Multimodal treatment has improved the outcome of many solid tumors, and in some cases the use of radiosensitizers has significantly contributed to this gain. Activation of the extracellular signaling kinase pathway (MEK/ERK) generally results in stimulation of cell growth and confers a survival advantage playing the major role in human cancer. The potential involvement of this pathway in cellular radiosensitivity remains unclear. We previously reported that the disruption of c-Myc through MEK/ERK inhibition blocks the expression of the transformed phenotype; affects in vitro and in vivo growth and angiogenic signaling; and induces myogenic differentiation in the embryonal rhabdomyosarcoma (ERMS) cell lines (RD). This study was designed to examine whether the ERK pathway affects intrinsic radiosensitivity of rhabdomyosarcoma cancer cells. Exponentially growing human ERMS, RD, xenograft-derived RD-M1, and TE671 cell lines were used. The specific MEK/ERK inhibitor, U0126, reduced the clonogenic potential of the three cell lines, and was affected by radiation. U0126 inhibited phospho-active ERK1/2 and reduced DNA protein kinase catalytic subunit (DNA-PKcs) suggesting that ERKs and DNA-PKcs cooperate in radioprotection of rhabdomyosarcoma cells. The TE671 cell line xenotransplanted in mice showed a reduction in tumor mass and increase in the time of tumor progression with U0126 treatment associated with reduced DNA-PKcs, an effect enhanced by radiotherapy. Thus, our results show that MEK/ERK inhibition enhances radiosensitivity of rhabdomyosarcoma cells suggesting a rational approach in combination with radiotherapy.

Topics: Animals; Butadienes; Cell Line, Tumor; Combined Modality Therapy; DNA Repair; Down-Regulation; Enzyme Inhibitors; Female; Humans; Mice; Mice, Nude; Mitogen-Activated Protein Kinase Kinases; Nitriles; Radiation-Sensitizing Agents; Rhabdomyosarcoma; Signal Transduction; Xenograft Model Antitumor Assays

p21WAF1, implicated in the cell cycle control of both normal and malignant cells, can be induced by p53-dependent and independent mechanisms. In some cells, MEKs/ERKs regulate p21WAF1 transcriptionally, while in others they also affect the post-transcriptional processes. In myogenic differentiation, p21WAF1 expression is also controlled by the myogenic transcription factor MyoD. We have previously demonstrated that the embryonal rhabdomyosarcoma cell line undergoes growth arrest and myogenic differentiation following treatments with TPA and the MEK inhibitor U0126, which respectively activate and inhibit the ERK pathway. In this paper we attempt to clarify the mechanism of ERK-mediated and ERK-independent growth arrest and myogenic differentiation of embryonal and alveolar rhabdomyosarcoma cell lines, particularly as regards the expression of the cell cycle inhibitor p21WAF1.. p21WAF1 expression and growth arrest are induced in both embryonal (RD) and alveolar (RH30) rhabdomyosarcoma cell lines following TPA or MEK/ERK inhibitor (U0126) treatments, whereas myogenic differentiation is induced in RD cells alone. Furthermore, the TPA-mediated post-transcriptional mechanism of p21WAF1-enhanced expression in RD cells is due to activation of the MEK/ERK pathway, as shown by transfections with constitutively active MEK1 or MEK2, which induces p21WAF1 expression, and with ERK1 and ERK2 siRNA, which prevents p21WAF1 expression. By contrast, U0126-mediated p21WAF1 expression is controlled transcriptionally by the p38 pathway. Similarly, myogenin and MyoD expression is induced both by U0126 and TPA and is prevented by p38 inhibition. Although MyoD and myogenin depletion by siRNA prevents U0126-mediated p21WAF1 expression, the over-expression of these two transcription factors is insufficient to induce p21WAF1. These data suggest that the transcriptional mechanism of p21WAF1 expression in RD cells is rescued when MEK/ERK inhibition relieves the functions of myogenic transcription factors. Notably, the forced expression of p21WAF1 in RD cells causes growth arrest and the reversion of anchorage-independent growth.. Our data provide evidence of the key role played by the MEK/ERK pathway in the growth arrest of Rhabdomyosarcoma cells. The results of this study suggest that the targeting of MEK/ERKs to rescue p21WAF1 expression and myogenic transcription factor functions leads to the reversal of the Rhabdomyosarcoma phenotype.

Topics: Biomarkers; Butadienes; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p21; Down-Regulation; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation, Neoplastic; Humans; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase Kinases; MyoD Protein; Myogenin; Nitriles; p38 Mitogen-Activated Protein Kinases; Phenotype; Rhabdomyosarcoma; Tetradecanoylphorbol Acetate; Transcription, Genetic