Background: Expression 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. Results: We 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. Conclusion: Our 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.

Epigenetic modifications play a key role in the patho-physiology of many tumors and the current use of agents targeting epigenetic changes has become a topic of intense interest in cancer research. DNA methyltransferase (DNMT) inhibitors represent a promising class of epigenetic modulators. Research performed yielded promising anti-tumorigenic activity for these agents in vitro and in vivo against a variety of hematologic and solid tumors. These epigenetic modulators cause cell cycle and growth arrest, differentiation and apoptosis. Rationale for combining these agents with cytotoxic therapy or radiation is straightforward since the use of DNMT inhibitor offers greatly improved access for cytotoxic agents or radiation for targeting DNA-protein complex. The positive results obtained with these combined approaches in preclinical cancer models demonstrate the potential impact DNMT inhibitors may have in treatments of different cancer types. Therefore, as the emerging interest in use of DNMT inhibitors as a potential chemo- or radiation sensitizers is constantly increasing, further clinical investigations are inevitable in order to finalize and confirm the consistency of current observations.

We have examined acetylcholine (ACh)-elicited potentials or currents in current- or voltage-clamped cultured myotubes exposed to 12-O-tetradecanoyl-phorbol-13-acetate (TPA), a potent tumor promoter that activates protein kinase C. Although this agent had little action on either membrane resting potential or electrical resistance, a reversible decrease in ACh sensitivity was induced on 3-4-d-old chick myotubes. Depression of transmitter action by TPA was extended to 7-8-d mouse myotubes only when they were treated with phosphatidylserine. Glyceryl dioleate had effects on myotubes similar to those of TPA but with a reduced efficacy. We conclude that the activation of protein kinase C might be involved with the capacity of ACh receptors to respond to transmitter stimulation.

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]

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.