Carmela Ciccarelli

PKCα-mediated ERK, JNK and p38 activation regulates the myogenic program in human rhabdomyosarcoma cells

We have previously suggested that PKCα has a role in 12-O-Tetradecanoylphorbol-13-acetate (TPA)-mediated growth arrest and myogenic differentiation in human embryonal rhabdomyosarcoma cells (RD). Here, by monitoring the signalling pathways triggered by TPA, we demonstrate that PKCα mediates these effects by inducing transient activation of c-Jun N-terminal protein kinases (JNKs) and sustained activation of both p38 kinase and extracellular signal-regulated kinases (ERKs) (all referred to as MAPKs). Activation of MAPKs following ectopic expression of constitutively active PKCα, but not its dominant-negative form, is also demonstrated. We investigated the selective contribution of MAPKs to growth arrest and myogenic differentiation by monitoring the activation of MAPK pathways, as well as by dissecting MAPK pathways using MEK1/2 inhibitor (UO126), p38 inhibitor (SB203580) and JNK and p38 agonist (anisomycin) treatments. Growth-arresting signals are triggered either by transient and sustained JNK activation (by TPA and anisomycin, respectively) or by preventing both ERK and JNK activation (UO126) and are maintained, rather than induced, by p38. We therefore suggest a key role for JNK in controlling ERK-mediated mitogenic activity. Notably, sarcomeric myosin expression is induced by both TPA and UO126 but is abrogated by the p38 inhibitor. This finding indicates a pivotal role for p38 in controlling the myogenic program. Anisomycin persistently activates p38 and JNKs but prevents myosin expression induced by TPA. In accordance with this negative role, reactivation of JNKs by anisomycin, in UO126-pre-treated cells, also prevents myosin expression. This indicates that, unlike the transient JNK activation that occurs in the TPA-mediated myogenic process, long-lasting JNK activation supports the growth-arrest state but antagonises p38-mediated myosin expression. Lastly, our results with the MEK inhibitor suggest a key role of the ERK pathway in regulating myogenic-related morphology in differentiated RD cells.

Down-regulation of c-Myc following MEK/ERK inhibition halts the expression of malignant phenotype in rhabdomyosarcoma and in non muscle-derived human tumors

BackgroundExpression of c-myc proto-oncogene is inappropriate in a wide range of human tumors, and is a downstream target of Ras/Raf/ERK pathway, which promotes c-Myc stability by enhancing c-Myc expression and activity.The aim of this study was to investigate whether the oncogenic phenotype in the human muscle-derived Rhabdomyosarcoma (RD) cell line and in non muscle-derived human tumor cell lines (SW403, IGR39 and PC3) can be blocked by disrupting the c-Myc pathway either by means of pharmacological MEK/ERK inhibition or by direct inactivation of the c-Myc protein.ResultsWe demonstrate that, in all the tumor cell lines used, the MEK/ERK inhibitor U0126 rapidly induces c-Myc de-phosphorylation, which is followed by a marked reduction in its expression level, by inhibition of proliferation and by reversion of anchorage-independent growth. These data suggest that the targeting of pathways controlling c-Myc expression or stability reverses deregulated growth of different tumor-derived cell lines. Indeed, in RD cells, we found a marked down-regulation of cyclins E2, A and B and CDK2, all of which are known to be targets of c-Myc. Moreover, ectopic MadMyc chimera, a c-Myc function antagonist, causes dramatic growth arrest, CDK and cyclin modulation as well as inhibition of anchorage-independent growth in RD cells, as occurs in U0126-treated cells. In particular, we found that the mere inhibition of c-Myc by MadMyc chimera rescues the myogenic program, MHC expression and the acquisition of the myogenic-like phenotype in RD cells.ConclusionOur data provide evidence of the key role played by the MEK/ERK pathway in the growth arrest and transformation phenotype of Rhabdomyosarcoma and of non muscle-derived tumor cell lines. In fact, MEK/ERK inhibitor, U0126, induces growth arrest, anchorage-dependent growth of these cell lines. In addition, the results of this study demonstrate that the direct inactivation of c-Myc by Mad/Myc chimera rescues myogenic program and leads to the reversal of the Rhabdomyosarcoma phenotype. In conclusion these data strongly suggest that the targeting of c-Myc by means of the MEK inhibitor can be tested as a promising strategy in anti-cancer therapy.

MEK/ERK inhibitor U0126 affects in vitro and in vivo growth of embryonal rhabdomyosarcoma

We reported previously that the disruption of c-Myc through mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) inhibition blocks the expression of the transformed phenotype in the embryonal rhabdomyosarcoma (ERMS) cell line (RD), thereby inducing myogenic differentiation in vitro. In this article, we investigate whether MEK/ERK inhibition, by the MEK/ERK inhibitor U0126, affects c-Myc protein level and growth of RMS tumor in an in vivo xenograft model. U0126 significantly reduced RMS tumor growth in RD cell line-xenotransplanted mice. Immunobiochemical and immunohistochemical analysis showed (a) phospho-active ERK levels were reduced by U0126 therapy and unaltered in normal tissues, (b) phospho-Myc and c-Myc was reduced commensurate with phospho-ERK inhibition, and (c) reduction in Ki-67 and endothelial (CD31) marker expression. These results indicate that MEK/ERK inhibition affects growth and angiogenic signals in tumor. The RD-M1 cultured xenograft tumor-derived cell line and the ERMS cell line TE671 responded to U0126 by arresting growth, down-regulating c-Myc, and initiating myogenesis. All these results suggest a tight correlation of MEK/ERK inhibition with c-Myc down-regulation and arrest of tumor growth. Thus, MEK inhibitors may be investigated for a signal transduction-based targeting of the c-Myc as a therapeutic strategy in ERMS. [Mol Cancer Ther 2009;8(3):543–51]

p21WAF1 expression induced by MEK/ERK pathway activation or inhibition correlates with growth arrest, myogenic differentiation and onco-phenotype reversal in rhabdomyosarcoma cells

Backgroundp21WAF1, 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.Resultsp21WAF1 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.ConclusionOur 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.

Vitamin D Protects Human Endothelial Cells from H2O2 Oxidant Injury Through the Mek/Erk-Sirt1 Axis Activation

Endothelium homeostasis alterations govern the pathogenesis of cardiovascular diseases. Several studies show that vitamins anti-oxidant proprieties rescue the endothelial functions adversely affected by oxidative stress in several diseases. We investigated the vitamin D anti-oxidant potential in human endothelial cells exposed to H2O2 oxidative stress. Vitamin D protected endothelial cells against H2O2 oxidative stress counteracting the superoxide anion generation, the apoptosis and blocking the extrinsic caspase cascade by positively controlling phospho-active ERKs level. MEKs/ERKs inhibitor U0126 reverted the vitamin D anti-oxidant effects. Characterizing the vitamin D downstream effector, we found that vitamin D up-regulated SirT-1 and reverted the SirT-1 down-regulation induced by H2O2. ERKs activation by vitamin D strictly correlated with SirT-1 protein accumulation since both MEKs/ERKs inhibition and ERK1/2 silencing decreased SIRT-1. SirT-1 inhibition by Sirtinol reverted the vitamin D anti-oxidant effects. Thus, vitamin D significantly reduced the endothelial malfunction and damage caused by oxidative stress, through the activation of MEKs/ERKs/SirT-1 axis.

MEK/ERK Inhibitor U0126 Increases the Radiosensitivity of Rhabdomyosarcoma Cells In vitro and In vivo by Downregulating Growth and DNA Repair Signals

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. Mol Cancer Ther; 10(1); 159–68. ©2011 AACR.

Nerve Growth factor regulation of cyclin D1 in PC12 cells through a p21RAS extracellular signal-regulated kinase pathway requires cooperative interactions between Sp1 and nuclear factor-kappaB.

The PC12 pheochromocytoma cell line responds to nerve growth factor (NGF) by exiting from the cell cycle and differentiating to induce extending neurites. Cyclin D1 is an important regulator of G1/S phase cell cycle progression, and it is known to play a role in myocyte differentiation in cultured cells. Herein, NGF induced cyclin D1 promoter, mRNA, and protein expression via the p21(RAS) pathway. Antisense- or small interfering RNA to cyclin D1 abolished NGF-mediated neurite outgrowth, demonstrating the essential role of cyclin D1 in NGF-mediated differentiation. Expression vectors encoding mutants of the Ras/mitogen-activated protein kinase pathway, and chemical inhibitors, demonstrated NGF induction of cyclin D1 involved cooperative interactions of extracellular signal-regulated kinase, p38, and phosphatidylinositol 3-kinase pathways downstream of p21(RAS). NGF induced the cyclin D1 promoter via Sp1, nuclear factor-kappaB, and cAMP-response element/activated transcription factor sites. NGF induction via Sp1 involved the formation of a Sp1/p50/p107 complex. Cyclin D1 induction by NGF governs differentiation and neurite outgrowth in PC12 cells.

Angiotensin-converting-enzyme inhibition counteracts angiotensin II-mediated endothelial cell dysfunction by modulating the p38/SirT1 axis.

Objective: Oxidative stress has been linked to endothelial dysfunction and angiotensin II stimulates the reactive oxygen species production contributing to several cardiovascular diseases. We have studied the chain of events induced by angiotensin-converting-enzyme (ACE) activation in vascular umbilical vein endothelial cells (HUVECs) by using an ACE inhibitor such as zofenoprilat. Methods: We used specific assay to measure the superoxide anion production, tetrazolium bromide (MTT) assay for cell viability, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay for cell apoptosis, and western blot for protein analysis in the study. Results: Zofenoprilat counteracts the superoxide anion production and cell apoptosis induced by angiotensin I treatment by blocking the extrinsic caspase cascade, NF-kB and p38 activation. p38 inhibitor SB203580 reverted the angiotensin II oxidant effects while the p38 constitutively activation, by MKK6 transfection, abrogated the zofenoprilat effects. Characterizing the zofenoprilat downstream effector we found that zofenoprilat reverted the SirT-1 downregulation induced by angiotensin II. p38 activation by angiotensin II was strictly correlated with SirT1 protein downregulation; SB203580 significantly prevented SirT1 downregulation induced by angiotensin II while the p38 constitutive activation abolished SIRT1 protein basal levels. p38 directly bound SirT1 sequestering it in the cytoplasm. SirT1 inhibition by sirtinol annulled zofenoprilat action while SirT1 overexpression reverted the cytotoxic effects of angiotensin II. Finally, zofenoprilat negatively controlled angiotensin I receptor protein expression through SirT1. Conclusion: The p38-SirT1 axis is found markedly relevant in modulating the cardiovascular benefit deriving from ACE-inhibitors and might represent a novel target for innovative drugs in cardiovascular prevention.

Hypoxia sustains glioblastoma radioresistance through ERKs/DNA-PKcs/HIF-1α functional interplay

The molecular mechanisms by which glioblastoma multiforme (GBM) refracts and becomes resistant to radiotherapy treatment remains largely unknown. This radioresistance is partly due to the presence of hypoxic regions, which are frequently found in GBM tumors. We investigated the radiosensitizing effects of MEK/ERK inhibition on GBM cell lines under hypoxic conditions. Four human GBM cell lines, T98G, U87MG, U138MG and U251MG were treated with the MEK/ERK inhibitor U0126, the HIF-1α inhibitor FM19G11 or γ-irradiation either alone or in combination under hypoxic conditions. Immunoblot analysis of specific proteins was performed in order to define their anti‑oncogenic or radiosensitizing roles in the different experimental conditions. MEK/ERK inhibition by U0126 reverted the transformed phenotype and significantly enhanced the radiosensitivity of T98G, U87MG, U138MG cells but not of the U251MG cell line under hypoxic conditions. U0126 and ERK silencing by siRNA reduced the levels of DNA protein kinase catalytic subunit (DNA-PKcs), Ku70 and K80 proteins and clearly reduced HIF-1α activity and protein expression. Furthermore, DNA-PKcs siRNA-mediated silencing counteracted HIF-1α activity and downregulated protein expression suggesting that ERKs, DNA-PKcs and HIF-1α cooperate in radioprotection of GBM cells. Of note, HIF-1α inhibition under hypoxic conditions drastically radiosensitized all cell lines used. MEK/ERK signal transduction pathway, through the sustained expression of DNA-PKcs, positively regulates HIF-1α protein expression and activity, preserving GBM radioresistance in hypoxic condition.

Factor-V expression in platelets from human megakaryocytic culture.

The origin of platelet‐factor‐V has long been discussed. To elucidate whether and when human platelet‐factor‐V is synthesized by megakaryocytes, we utilized in vitro‐generated megakaryocytes capable of producing platelets. Factor‐V gene was silent in purified progenitors and megakaryocytic precursors but was expressed in late culture phase and maintained also in platelets. Similarly, factor‐V protein was expressed in mature proplatelet‐bearing megakaryocytes (immunofluorescence analysis); it was also detectable in cultured megakaryocytes and platelets (Western blotting) and within permeabilized cultured platelets (flow cytometry). The absence of other cells in our culture system indicates conclusively that human megakaryocytes synthesize factor‐V.