Down-regulation of c-Myc following MEK/ERK inhibition halts the expression of malignant phenotype in rhabdomyosarcoma and in non muscle-derived human tumors
© Marampon et al; licensee BioMed Central Ltd. 2006
Received: 20 April 2006
Accepted: 09 August 2006
Published: 09 August 2006
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.
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.
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.
The Myc protein, which has been shown to play an essential role in the control of cell proliferation, growth, differentiation and apoptosis [1, 2], is a member of the basic region/helix-loop-helix/leucine zipper (b/HLH/Zip) family of transcriptional regulators that is capable of both transactivation and transrepression [1, 3] of a large number of target genes [4, 5] through heterodimerization with its biological partner Max . Members of the Myc family are activated in many, if not most, human tumors  and the strong selection for c-Myc over-expression in tumors appears to reflect the ability of c-Myc to provide constitutive signals that promote cellular transformation . It has recently been reported that Ras controls c-Myc protein accumulation resulting from ERK-mediated stabilization of c-Myc by Ser62 phosphorylation, whereas subsequent Thr58 phosphorylation by glycogen-synthase kinase-3 (GSK-3) is required for c-Myc degradation . Thus, Ras activates AKT, which in turn inactivates GSK3, leading to the block of c-Myc degradation pathway. Consequently, the frequent Ras mutations in human cancer  and concomitant deregulation of c-Myc suggest a possible synergistic relationship of c-Myc and Ras in the disruption of normal cell growth regulation . Indeed, inhibition of the MEK/ERK pathway in v-Ki-ras rat fibroblasts, MDA-MB231 and HBC4 breast cancer cell lines, and c-Myc depletion by siRNA in MCF7 and over-expression of a c-Myc antagonist, Mxi1, in prostate carcinoma DU145, all induce reversion of the malignant phenotype [9–12].
Both the c-Myc and Ras/MEK/ERK pathways play an important role in the progression of the G1-cell cycle phase by enhancing cyclins expression [13, 14] and CDK/cyclin complex activities [15, 16]. In addition, c-Myc constitutive expression suppresses expression of the cell cycle inhibitors p21WAF1 and p27KIP1 .
Interestingly, osteogenic sarcoma, harbouring conditional alleles of c-Myc, differentiate into mature bone under brief c-Myc inactivation ; likewise, transgenic mice that conditionally express c-Myc in liver develop hepatocarcinoma that is reversed following c-Myc inactivation . Accordingly, the down-regulation of c-Myc results in the attenuation of both cell division and cell growth as well as in the protection against some apoptotic processes [20, 21].
Given the synergistic relationship between MEK/ERK and c-Myc in cell growth and malignant transformation, the blocking of the MEK/ERK pathway  might conceivably be used against cancer.
The embryonal rhabdomyosarcoma cell line (RD) consists of muscle-derived precursors that fail to complete the differentiation program , probably owing to the action of mutated N-Ras proto-oncogene , mutated tumor suppressor p53  and over-expressed c- or N-Myc .
Since we found that U0126, a MEK/ERK pathway inhibitor, induces p21WAF1 expression  and promotes G1 cell cycle arrest and myogenic differentiation in RD cells , we decided to investigate whether the MEK/ERK pathway and c-Myc might cooperate in cell growth and transformation control in RD cells. Furthermore, in order to investigate the effect of MEK/ERK inhibition on non-muscle-derived cell lines we used colon adenocarcinoma- (SW403), melanoma- (IGR39), prostate-derived cell lines (PC3), all bearing mutated Ras and deregulated c-Myc [29–31].
We found that the disruption of the MEK/ERK pathway, by means of the MEK inhibitor U0126, dramatically decreased c-Myc expression level, inducing growth inhibition and reversion of anchorage-independent growth in all the cell lines used. Moreover, we show that direct inactivation of c-Myc by the MadMyc chimera protein, a repressor of c-Myc activity, causes growth arrest, reversion of anchorage-independent growth and myogenic differentiation in RD cells.
MEK/ERK inhibitor drastically reduces c-Myc expression
While the expression level of Max isoforms (21 and 22 Kda), which heterodimerize with c-Myc , was unaffected (Fig 1A), the amount of c-Myc associated with Max was dramatically reduced in U0126-treated cells, as shown by immunoprecipitation experiments (Fig 1C). Equal amounts of Max were detected in the immunocomplex (Fig 1C). Taken together, these results indicate that c-Myc is a down stream target of ERKs and MEK/ERK inhibition mediates loss of c-Myc and of the c-Myc/Max heterodimer, providing one possible molecular mechanism of growth arrest i.e. that induced by the MEK inhibitor U0126.
Effects of U0126 on G0/G1 arrest and cell cycle regulator expression in RD cell lines
Blockade of functional c-Myc induces growth arrest
Anchorage-independent growth of RD cells is inhibited by U0126-mediated c-Myc down regulation and rescued by c-Myc over-expression
Molecular and morphological myogenic-like phenotype is induced by MadMyc chimera and is attenuated by forced c-Myc expression
Taken together, these results demonstrate that the mere inhibition of c-Myc can rescue the myogenic program in RD cells by myogenic transcription factor activation, MHC expression and myogenic-like phenotype acquisition.
U0126 down-regulates c-Myc and counteracts the oncophenotype of non-muscle-derived tumor cell lines
Moreover, the colony-forming assay in soft agar showed that the colony formation of the IGR39, SW403 and PC3 tumor cell lines was abolished by U0126, whereas numerous, large colonies were present in the untreated cells (Fig 11B).
These data show that cell transformation of different tumor-derived cell lines is halted by inhibition of MEK/ERK pathway followed by c-Myc down-regulation.
The pharmacological inhibitors of Ras/MEK/ERK signalling are arousing considerable interest on account of their potential therapeutic uses [22, 37]. In this paper, we addressed the issue of whether MEK/ERK inhibition, by targeting c-Myc, prevents the transformed phenotype expression in RD cells as well as in a number of tumor cell lines that express a mutated version of ras and over-express c-Myc. The efficient growth inhibition induced by the MEK inhibitor U0126 in RD, colon carcinoma, prostate and melanoma cell lines clearly demonstrates that the MEK/ERK pathway is a pre-requisite for the aberrant growth of these cells. Indeed, U0126 permanently inhibits phospho-ERKs in all tumor cell lines used. It is noteworthy that both c-Myc phosphorylation and c-Myc expression itself decreased in RD cells as well as in all the non muscle tumor cell lines examined following MEK/ERK inhibition. Conversely, in muscle and non-muscle untransformed cell lines, U0126, while transiently inhibiting phospho-ERKs, only slightly inhibits growth and does not down-regulate c-Myc. This result is consistent with no major effects of MEK/ERK inhibition on proliferation status of muscle and non-muscle untransformed cell lines. All together these data are in line with the notion that c-Myc is a downstream target of MEK/ERK pathway and suggest that aberrant growth of different tumor cell lines can be halted by targeting c-Myc following MEK/ERK inhibition. Although c-Myc has previously been reported to be a downstream target of MEKs/ERKs  the correlation between ERK-mediated c-Myc stability and aberrant growth, though inferable from recent studies in the literature [37, 38], has so far received little attention.
Besides inducing growth arrest, U0126 also abolished, in the cell lines used here, anchorage-independent growth, as demonstrated by the lack of clones in the soft agar assay. In addition, in RD cells the comparison of growth in soft agar in the presence of U0126 or TPA demonstrates that while TPA only reduces the growth potential of RD cells, U0126 is also able to abolish anchorage-independent growth. The failure of TPA to abolish anchorage-independent growth can be explained by its inability to induce p21WAF1 and its positive effects on c-Myc and cyclin D1 expression in non-adherent RD cultures. Conversely, the U0126-mediated arrest of growth in non-adherent cultures can be due to the drastic c-Myc down-regulation and cyclin D1, known to be involved in cell transformation [12, 16, 39]. In addition, the experiment in suspension cultures suggest that MEK/ERK inhibitor, U0126, may have cytostatic effects . These results demonstrate that the mere inhibition of growth potential is not sufficient to prevent the transformed phenotype expression.
Recent studies in the literature report, on the one hand, that MAPKs and c-Myc cooperate in promoting invasive growth  and, on the other, that targeted disruption of c-Myc suppresses cell transformation and tumor formation . The Ras-MAPK pathways are, however, currently receiving attention owing to the therapy potential they offer , while a number of papers reporting that c-Myc inactivation results in tumor inhibition and regression [11, 12, 18]. Our data attempt to demonstrate a possible link between these two major targets in a cascade in which MEK/ERK kinases lie upstream of the oncogenic molecule c-Myc which, in turn, induces neoplastic transformation. In fact, we here show that ERKs and particularly ERK2, are upstream kinases of c-Myc in RD cells as demonstrated by siRNA results. These results are in line with data reported by others that c-Myc stability and accumulation is regulated by ERK-mediated phosphorylation of ser62 . Moreover, it is evident the relationship between MEK/ERK inhibition, c-Myc down regulation and blockade of cell transformation in the cell lines here used. This functional correlation is highly relevant in the field of possible new therapeutic approaches for some human tumor, including rhabdomyosarcoma.
In an attempt to determine the specific role of c-Myc in sustaining aberrant growth as well as cell transformation and inhibition of differentiation, we used RD cells on account of their ability to undergo growth arrest and myogenic differentiation upon MEK/ERK inhibition [27, 28]. Our data show that MEK/ERK inhibition down-regulates cyclin E2, A and B and CDK2, all of which are known to be transcriptional targets of c-Myc [13, 15, 44]. It can, consequently, be hypothesized that the disruption of the c-Myc network by ERK depletion is responsible for the failed expression of the relevant cell-cycle proteins.
Hypothesising that c-Myc expression alone sustains the program for deregulated growth as well as transformation and inhibition of differentiation, we stably over-expressed MadMyc chimera in RD cells to specifically block c-Myc activity . We found that growth of MadMyc-over-expressing RD cells is arrested, as demonstrated by p21WAF1 enhanced expression and cyclin D1, A and B and CDK2 down-regulation, as also observed in U0126-treated cells. Furthermore, myogenic differentiation is induced in MadMyc-expressing RD cells, as shown in this study by the restored transcriptional function of myogenic transcription factors and MHC expression. It is noteworthy that induction of myogenic differentiation in MadMyc chimera expressing cells does not imply a myogenin or MyoD increased expression level neither down-regulation of pospho-ERKs which are instead enhanced. This is in agreement with the role of ERKs in fusion and late differentiation processes during myogenic differentiation . Importantly, MadMyc-stably-expressing cells do not exhibit anchorage-independent growth, which is instead enhanced in c-Myc-over-expressing cells. On the other hand, forced expression of c-Myc attenuated the U0126-mediated anchorage-independent growth inhibition and differentiative effects in RD cells. These experiments demonstrate that c-Myc over-expression rescues oncogenic phenotype repressed by MEK inhibitor U0126. Worthy of note is also the fact that the role of mutated Ras in aberrant growth of RD cells is compromised by the selective disruption of c-Myc in MadMyc-expressing cells demonstrating that c-Myc is indispensable to the maintaining of Ras/MEK/ERK-mediated oncogenic phenotype.
Our data provide evidence that the cooperation between MEK/ERK and c-Myc pathways play a major role in the expression of transformed phenotype in muscle and non muscle-derived transformed cell lines. Importantly, our results show for the first time that the disruption of c-Myc pathway either directly or indirectly drammatically impairs the expression of transformed phenotype inducing myogenic differentiation in RD cells. In conclusion these data strongly suggest that the targeting of c-Myc by means of the MEK/ERK inhibitor can be tested as a promising strategy in anti-cancer therapy.
Cell cultures and treatments
The embryonal Rhabdomyosarcoma (RD), the prostate carcinoma PC3 (ATCC, Rockville MD), the melanoma IGR39 and colon adenocarcinoma SW403 (DSMZ, Braunschweig, Germany) human cancer cell lines were cultured in Dulbecco modified Eagle medium (DMEM), supplemented with glutamine, gentamycin (GIBCO-BRL Gaithersburg, MD) and 10% (RD, PC3 and SW403) or 15% (IGR39) heat-inactivated foetal bovine serum (Hyclone, Logan UT). C2C12 and NIH3T3 (ATCC) were grown in DMEM supplemented with glutamine, gentamycin and 10% heat-inactivated foetal bovine serum. One day after plating, cells were treated with 10 μM U0126 kinase inhibitors (Promega, Madison, WI) or 10-7 M TPA (Sigma, St Louis, MO) for the times shown in the figures.
Cells were harvested in phosphate buffered saline, sedimented and lysed in 10 mM Tris pH 7, 50 mM NaCl, 1% NP40, 1 mM ZnCl2, additioned with protease and phosphatase inhibitors. Protein extracts were clarified by centrifugation. Supernatant, normalized as equal amounts of proteins, were incubated with Max antibody (H-2) (Santa Cruz Biotechnology, Santa Cruz CA) at 4°C for 3 hrs. 30 μl of protein-G Plus (Santa Cruz Biotechnology) were added to collect immunocomplexes. Protein G-bound immunocomplexes were washed 6 times with extraction buffer and processed for SDS-PAGE and immunoblotting.
Cells were lysed in 2% SDS containing phosphatase and protease inhibitors sonicated for 30 sec. Proteins of whole cell lysates were assessed using the Lowry method , and equal amounts were separated on SDS-PAGE. The proteins were transferred to a nitrocellulose membrane (Schleicher & Schuell, BioScience GmbH, Germany) by electroblotting. Immunoblottings were performed with the following antibodies: anti-c-Myc polyclonal (N-262) or monoclonal (9E10), anti-phospho c-Myc (Thr 58/Ser 62-R), anti-Max (H-2), anti-phospho ERK1/2 (E-4), anti-ERK2 (C-14 positive also for ERK1), anti-p21WAF1 (C-19), anti-p27 (F-8), anti-Cyclin-E (HE12), -A (H-432), -D1 (M 20) and -B (H-20), CDK2 (M2) and 4 (H-22), -pRb (C-15), anti-myogenin (F-D5), a-tubulin (B-7), MyoD (C-20) (all from Santa Cruz Biotechnology) and anti-MHC (MF20, gift from Fichman D). Peroxidase-conjugate anti-mouse or anti-rabbit IgG (Amersham-Pharmacia Biotech, UK or Santa Cruz) were used for enhanced chemiluminescence (ECL) detection.
Plasmids and transfection
One day after plating, RD cells were transfected with plasmids using Lipofectamine Plus reagent (Invitrogen, Italy) according to the manufacturer's instructions. For the luciferase assay, the CMV or the c-Myc (kindly provided by Dr. L.G. Larsson) or MadMyc chimera plasmid (kindly provided by Dr. R. Bernards) were co-tranfected in RD cells together with pMyo84-luc (kindly provided by B.M. Scicchitano described in ). Total lysates were processed for luciferase activity according to the manufacturer's instructions (Promega Italia).
RD stably transfected cells were obtained transfecting cells with a plasmid encoding c-Myc, MadMyc chimera or empty vector CMV, all carrying G418-neomycin resistance. Polyclonal populations of CMV, c-Myc and MadMyc chimera expressing cells were selected using 0.4 mg/ml of G418-neomycin (Sigma) for three weeks. RNA interference experiments were performed with siRNA for ERK1 and ERK2 (Sancta Cruz Biotechnology) using Lipofectamine 2000 reagent (Invitrogen, Italy), according to the manufacturer's instructions. Briefly, cells were plated at 40–50% confluence and transfected after 24 hr with 100 nM siRNA, which we ascertained was sufficient to detect maximum fluorescence using fluorescein-conjugated control siRNA.
Cells were fixed in 4% paraformaldehyde and washed; non-specific binding sites were blocked with 3% BSA in PBS for 20 min at room temperature. Cells were then incubated for 1 hr at RT with a 1:100 dilution of the anti-MHC (MF20), specific mouse monoclonal antibody. After rinsing with PBS, the cells were incubated with anti-mouse IgG-Cy3 and DAPI (Zymed, Invitrogen, Italia).
Suspension cell cultures and colony-forming assays in semisolid agar
RD cells were initiated as adherent cultures, detached and seeded in 50 ml Falcon tube at 5 × 104 cells/ml in a total volume of 12 ml of same medium as adherent cultures and after 1 day additioned with TPA or U0126. All tubes were placed in an orbital shaker (~120 rpm) in a 37°C humidified incubator with 5% CO2.
Colony-forming assays were based on standard methods. Briefly, 2 × 104 cells were resuspended in 4 ml of 0.33% special Noble agar (Difco, Detroit, MI) and plated (6 cm plate) in growth medium-containing 0.5% soft agar. Colonies were photographed 14 days after plating.
Cell proliferation assay and FACS analysis
Cells from adherent and suspension culture were counted using hemocytometer, and tested for exclusion of trypan blue. Data are expressed as average of triplicate + standard error.
For FACS analysis cells were harvested by trypsin-EDTA and washed; pellets were resuspended in 0,3 ml 50% FCS in PBS, additioned with 0,9 ml 70% ethanol and left O/N in the dark at 4°C before FACS analysis (Coulter Epics XL Flow Cytometer, Beckman Coulter CA, USA).
rhabdomyosarcoma cell line
Mitogen-activated protein Extracellular Kinase
Extracellular signal-Regulated protein Kinase.
We are very grateful to Dr A. Floridi for his generous help and support throughout this work. We are also grateful B. Di Iulio for her useful help and F. Padula for FACS analysis. We thank Lewis Baker for reviewing the English in the manuscript. We are most grateful to "Cassa Edile di Roma e Provincia" for supporting in part our work. This work was supported by grants from MIUR to BMZ and from University of L'Aquila.
- Grandori C, Cowley SM, James LP, Eisenman RN: The Myc/Max/Mad network and the transcriptional control of cell behavior. Annu Rev Cell Dev Biol. 2000, 16: 653-699. 10.1146/annurev.cellbio.16.1.653View ArticlePubMedGoogle Scholar
- Henriksson M, Luscher B: Proteins of the Myc network: essential regulators of cell growth and differentiation. Adv Cancer Res. 1996, 68: 109-182.View ArticlePubMedGoogle Scholar
- Oster SK, Ho CS, Soucie EL, Penn LZ: The myc oncogene: MarvelouslY Complex. Adv Cancer Res. 2002, 84: 81-154.View ArticlePubMedGoogle Scholar
- Watson JD, Oster SK, Shago M, Khosravi F, Penn LZ: Identifying genes regulated in a Myc-dependent manner. J Biol Chem. 2002, 277: 36921-36930. 10.1074/jbc.M201493200View ArticlePubMedGoogle Scholar
- Menssen A, Hermeking H: Characterization of the c-MYC-regulated transcriptome by SAGE: identification and analysis of c-MYC target genes. Proc Natl Acad Sci USA. 2002, 99: 6274-6279. 10.1073/pnas.082005599PubMed CentralView ArticlePubMedGoogle Scholar
- Blackwood EM, Eisenman RN: Max: a helix-loop-helix zipper protein that forms a sequence-specific DNA-binding complex with Myc. Science. 1991, 251: 1211-1217.View ArticlePubMedGoogle Scholar
- Sears R, Nuckolls F, Haura E, Taya Y, Tamai K, Nevins JR: Multiple Ras-dependent phosphorylation pathways regulate Myc protein stability. Genes Dev. 2000, 14: 2501-2514. 10.1101/gad.836800PubMed CentralView ArticlePubMedGoogle Scholar
- Bos JL: ras oncogenes in human cancer: a review. Cancer Res. 1989, 49: 4682-4689.PubMedGoogle Scholar
- Fukazawa H, Uehara Y: U0126 reverses Ki-ras-mediated transformation by blocking both mitogen-activated protein kinase and p70 S6 kinase pathways. Cancer Res. 2000, 60: 2104-2107.PubMedGoogle Scholar
- Fukazawa H, Noguchi K, Murakami Y, Uehara Y: Mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) inhibitors restore anoikis sensitivity in human breast cancer cell lines with a constitutively activated extracellular-regulated kinase (ERK) pathway. Mol Cancer Ther. 2002, 1: 303-309.PubMedGoogle Scholar
- Taj MM, Tawil RJ, Engstrom LD, Zeng Z, Hwang C, Sanda MG, Wechsler DS: Mxi1, a Myc antagonist, suppresses proliferation of DU145 human prostate cells. Prostate. 2001, 47: 194-204. 10.1002/pros.1063View ArticlePubMedGoogle Scholar
- Wang YH, Liu S, Zhang G, Zhou CQ, Zhu HX, Zhou XB, Quan LP, Bai JF, Xu NZ: Knockdown of c-Myc expression by RNAi inhibits MCF-7 breast tumor cells growth in vitro and in vivo. Breast Cancer Res. 2005, 7: R220-R228. 10.1186/bcr975PubMed CentralView ArticlePubMedGoogle Scholar
- Jansen-Durr P, Meichle A, Steiner P, Pagano M, Finke K, Botz J, Wessbecher J, Draetta G, Eilers M: Differential modulation of cyclin gene expression by MYC. Proc Natl Acad Sci USA. 1993, 90: 3685-3689. 10.1073/pnas.90.8.3685PubMed CentralView ArticlePubMedGoogle Scholar
- Ussar S, Voss T: MEK1 and MEK2, different regulators of the G1/S transition. J Biol Chem. 2004, 279: 43861-43869. 10.1074/jbc.M406240200View ArticlePubMedGoogle Scholar
- Berns K, Hijmans EM, Bernards R: Repression of c-Myc responsive genes in cycling cells causes G1 arrest through reduction of cyclin E/CDK2 kinase activity. Oncogene. 1997, 15: 1347-1356. 10.1038/sj.onc.1201280View ArticlePubMedGoogle Scholar
- Liu JJ, Chao JR, Jiang MC, Ng SY, Yen JJ, Yang-Yen HF: Ras transformation results in an elevated level of cyclin D1 and acceleration of G1 progression in NIH 3T3 cells. Mol Cell Biol. 1995, 15: 3654-3663.PubMed CentralPubMedGoogle Scholar
- Gartel AL, Shchors K: Mechanisms of c-myc-mediated transcriptional repression of growth arrest genes. Exp Cell Res. 2003, 283: 17-21. 10.1016/S0014-4827(02)00020-4View ArticlePubMedGoogle Scholar
- Jain M, Arvanitis C, Chu K, Dewey W, Leonhardt E, Trinh M, Sundberg CD, Bishop JM, Felsher DW: Sustained loss of a neoplastic phenotype by brief inactivation of MYC. Science. 2002, 297: 102-104. 10.1126/science.1071489View ArticlePubMedGoogle Scholar
- Shachaf CM, Kopelman AM, Arvanitis C, Karlsson A, Beer S, Mandl S, Bachmann MH, Borowsky AD, Ruebner B, Cardiff RD, Yang Q, Bishop JM, Contag CH, Felsher DW: MYC inactivation uncovers pluripotent differentiation and tumour dormancy in hepatocellular cancer. Nature. 2004, 431: 1112-1117. 10.1038/nature03043View ArticlePubMedGoogle Scholar
- Johnston LA, Prober DA, Edgar BA, Eisenman RN, Gallant P: Drosophila myc regulates cellular growth during development. Cell. 1999, 98: 779-790. 10.1016/S0092-8674(00)81512-3View ArticlePubMedGoogle Scholar
- Mateyak MK, Obaya AJ, Sedivy JM: c-Myc regulates cyclin D-Cdk4 and -Cdk6 activity but affects cell cycle progression at multiple independent points. Mol Cell Biol. 1999, 19: 4672-4683.PubMed CentralPubMedGoogle Scholar
- Kohno M, Pouyssegur J: Pharmacological inhibitors of the ERK signaling pathway: application as anticancer drugs. Prog Cell Cycle Res. 2003, 5: 219-224.PubMedGoogle Scholar
- Merlino G, Helman LJ: Rhabdomyosarcoma – working out the pathways. Oncogene. 1999, 18: 5340-5348. 10.1038/sj.onc.1203038View ArticlePubMedGoogle Scholar
- Chardin P, Yeramian P, Madaule P, Tavitian A: N-ras gene activation in the RD human rhabdomyosarcoma cell line. Int J Cancer. 1985, 35: 647-652.View ArticlePubMedGoogle Scholar
- Germani A, Fusco C, Martinotti S, Musaro A, Molinaro M, Zani BM: TPA-induced differentiation of human rhabdomyosarcoma cells involves dephosphorylation and nuclear accumulation of mutant P53. Biochem Biophys Res Commun. 1994, 202: 17-24. 10.1006/bbrc.1994.1887View ArticlePubMedGoogle Scholar
- Dias P, Kumar P, Marsden HB, Gattamaneni HR, Kumar S: N- and c-myc oncogenes in childhood rhabdomyosarcoma. J Natl Cancer Inst. 1990, 82: 151-View ArticlePubMedGoogle Scholar
- Ciccarelli C, Marampon F, Scoglio A, Mauro A, Giacinti C, De Cesaris P, Zani BM: p21WAF1 expression induced by MEK/ERK pathway activation or inhibition correlates with growth arrest, myogenic differentiation and onco-phenotype reversal in rhabdomyosarcoma cells. Mol Cancer. 2005, 4: 41- 10.1186/1476-4598-4-41PubMed CentralView ArticlePubMedGoogle Scholar
- Mauro A, Ciccarelli C, De Cesaris P, Scoglio A, Bouche M, Molinaro M, Aquino A, Zani BM: PKCalpha-mediated ERK, JNK and p38 activation regulates the myogenic program in human rhabdomyosarcoma cells. J Cell Sci. 2002, 115: 3587-3599. 10.1242/jcs.00037View ArticlePubMedGoogle Scholar
- Field JK, Spandidos DA: The role of ras and myc oncogenes in human solid tumours and their relevance in diagnosis and prognosis (review). Anticancer Res. 1990, 10: 1-22.PubMedGoogle Scholar
- Peehl DM: Oncogenes in prostate cancer. An update. Cancer. 1993, 71: 1159-1164. 10.1002/1097-0142(19930201)71:3+<1159::AID-CNCR2820711439>3.0.CO;2-UView ArticlePubMedGoogle Scholar
- Polsky D, Cordon-Cardo C: Oncogenes in melanoma. Oncogene. 2003, 22: 3087-3091. 10.1038/sj.onc.1206449View ArticlePubMedGoogle Scholar
- Dean M, Levine RA, Ran W, Kindy MS, Sonenshein GE, Campisi J: Regulation of c-myc transcription and mRNA abundance by serum growth factors and cell contact. J Biol Chem. 1986, 261: 9161-9166.PubMedGoogle Scholar
- Waters CM, Littlewood TD, Hancock DC, Moore JP, Evan GI: c-myc protein expression in untransformed fibroblasts. Oncogene. 1991, 6: 797-805.PubMedGoogle Scholar
- Rivard N, Boucher MJ, Asselin C, L'Allemain G: MAP kinase cascade is required for p27 downregulation and S phase entry in fibroblasts and epithelial cells. Am J Physiol. 1999, 277: C652-C664.PubMedGoogle Scholar
- Miner JH, Wold BJ: c-myc inhibition of MyoD and myogenin-initiated myogenic differentiation. Mol Cell Biol. 1991, 11: 2842-2851.PubMed CentralPubMedGoogle Scholar
- Scicchitano BM, Spath L, Musaro A, Molinaro M, Rosenthal N, Nervi C, Adamo S: Vasopressin-dependent myogenic cell differentiation is mediated by both Ca2+/calmodulin-dependent kinase and calcineurin pathways. Mol Biol Cell. 2005, 16: 3632-3641. 10.1091/mbc.E05-01-0055PubMed CentralView ArticlePubMedGoogle Scholar
- Downward J: Targeting RAS signalling pathways in cancer therapy. Nat Rev Cancer. 2003, 3: 11-22. 10.1038/nrc969View ArticlePubMedGoogle Scholar
- Ponzielli R, Katz S, Barsyte-Lovejoy D, Penn LZ: Cancer therapeutics: targeting the dark side of Myc. Eur J Cancer. 2005, 41: 2485-2501. 10.1016/j.ejca.2005.08.017View ArticlePubMedGoogle Scholar
- Li YJ, Song R, Korkola JE, Archer MC, Ben David Y: Cyclin D1 is necessary but not sufficient for anchorage-independent growth of rat mammary tumor cells and is associated with resistance of the Copenhagen rat to mammary carcinogenesis. Oncogene. 2003, 22: 3452-3462. 10.1038/sj.onc.1206411View ArticlePubMedGoogle Scholar
- Gysin S, Lee SH, Dean NM, McMahon M: Pharmacologic inhibition of RAF-->MEK-->ERK signaling elicits pancreatic cancer cell cycle arrest through induced expression of p27Kip1. Cancer Res. 2005, 65: 4870-4880. 10.1158/0008-5472.CAN-04-2848View ArticlePubMedGoogle Scholar
- Gao CF, Xie Q, Su YL, Koeman J, Khoo SK, Gustafson M, Knudsen BS, Hay R, Shinomiya N, Vande Woude GF: Proliferation and invasion: plasticity in tumor cells. Proc Natl Acad Sci USA. 2005, 102: 10528-10533. 10.1073/pnas.0504367102PubMed CentralView ArticlePubMedGoogle Scholar
- Hatakeyama S, Watanabe M, Fujii Y, Nakayama KI: Targeted destruction of c-Myc by an engineered ubiquitin ligase suppresses cell transformation and tumor formation. Cancer Res. 2005, 65: 7874-7879.PubMedGoogle Scholar
- Yeh E, Cunningham M, Arnold H, Chasse D, Monteith T, Ivaldi G, Hahn WC, Stukenberg PT, Shenolikar S, Uchida T, Counter CM, Nevins JR, Means AR, Sears R: A signalling pathway controlling c-Myc degradation that impacts oncogenic transformation of human cells. Nat Cell Biol. 2004, 6: 308-318. 10.1038/ncb1110View ArticlePubMedGoogle Scholar
- Yin XY, Grove L, Datta NS, Katula K, Long MW, Prochownik EV: Inverse regulation of cyclin B1 by c-Myc and p53 and induction of tetraploidy by cyclin B1 overexpression. Cancer Res. 2001, 61: 6487-6493.PubMedGoogle Scholar
- Bennett AM, Tonks NK: Regulation of distinct stages of skeletal muscle differentiation by mitogen-activated protein kinases. Science. 1997, 278: 1288-1291. 10.1126/science.278.5341.1288View ArticlePubMedGoogle Scholar
- Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein measurement with the Folin phenol reagent. J Biol Chem. 1951, 193: 265-275.PubMedGoogle Scholar
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