Panel Of Rhabdomyosarcoma Cell Lines Research Articles

Next-generation sequencing reveals a novel role of lysine-specific demethylase 1 in adhesion of rhabdomyosarcoma cells.

Lysine-specific demethylase 1 (LSD1), a histone lysine demethylase with the main specificity for H3K4me2, has been shown to be overexpressed in rhabdomyosarcoma (RMS) tumor samples. However, its role in RMS biology is not yet well understood. Here, we identified a new role of LSD1 in regulating adhesion of RMS cells. Genetic knockdown of LSD1 profoundly suppressed clonogenic growth in a panel of RMS cell lines, whereas LSD1 proved to be largely dispensable for regulating cell death and short-term survival. Combined RNA and ChIP-sequencing performed to analyze RNA expression and histone methylation at promoter regions revealed a gene set enrichment for adhesion-associated terms upon LSD1 knockdown. Consistently, LSD1 knockdown significantly reduced adhesion to untreated surfaces. Importantly, precoating of the plates with the adhesives collagen I or fibronectin rescued this reduced adhesion of LSD1 knockdown cells back to levels of control cells. Using KEGG pathway analysis, we identified 17 differentially expressed genes (DEGs) in LSD1 knockdown cells related to adhesion processes, which were validated by qRT-PCR. Combining RNA and ChIP-sequencing results revealed that, within this set of genes, SPP1, C3AR1, ITGA10 and SERPINE1 also exhibited increased H3K4me2 levels at their promoter regions in LSD1 knockdown compared to control cells. Indeed, LSD1 ChIP experiments confirmed enrichment of LSD1 at their promoter regions, suggesting a direct transcriptional regulation by LSD1. By identifying a new role of LSD1 in the modulation of cell adhesion and clonogenic growth of RMS cells, these findings highlight the importance of LSD1 in RMS.

Anaplastic lymphoma kinase aberrations correlate with metastatic features in pediatric rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most frequent soft tissue tumor in childhood and arises from immature mesenchymal cells committed to skeletal muscle differentiation. Anaplastic Lymphoma Kinase (ALK) is a receptor tyrosine kinase aberrantly expressed in several cancers. Moreover, ALK full-length receptor protein has been observed in RMS, although its clinical and functional significance is yet controversial. The role of ALK and its clinical relevance were investigated in a selected cohort of 74 FFPE pediatric RMS and a panel of RMS cell lines, evaluating its gene and protein status, utilizing Fluorescent In Situ Hybridization (FISH), immunohistochemistry (IHC) and Western blot approaches. Moreover, to get insight into its possible therapeutic relevance, effects of ALK silencing on cell proliferation, invasion and apoptosis were studied in RMS cells. ALK IHC positivity was significantly correlated with gene copy number gain, the alveolar subtype, PAX3/7-FOXO1 rearrangements, the presence of metastasis at diagnosis and a worse overall outcome. Furthermore, EML4-ALK fusion gene associated with higher protein expression was identified in an embryonal RMS. ALK silencing in RH30 ALK positive cells strongly inhibited invasion capability. Overall, our data suggest a potential role of ALK in pediatric RMS.

Abstract PR16: Targeting the chromatin architecture established by PAX3-FOXO1 in rhabdomyosarcoma

Abstract Master transcription factors establish enhancers to regulate cell identity genes by recruiting epigenetic machinery, and are sequentially exchanged during changes in cell identity (ie, differentiation). Commonly, the fusion of transcription factors profoundly alters proper progression of cell identity, serving as the signature oncogenic event in many malignancies. The most common soft tissue cancer of childhood, rhabdomyosarcoma (RMS), is characterized by an inability to exit the proliferative myoblast-like state, presumably by blocking myogenic transcription factors from advancing the active enhancer landscape. This is achieved by either chromosomal translocation resulting in the oncogenic fusion transcription factor PAX3/7-FOXO1 (Fusion-Positive alveolar subtype, FP-RMS) or mutations in the tyrosine kinase/RAS/PIK3C axis (Fusion-Negative embryonal subtype, FN-RMS). Patients who harbor a PAX-fusion typically relapse despite aggressive therapy and have very poor survival. Here we hypothesized that the PAX3-FOXO1 fusion gene causes epigenetic reprogramming resulting in increased proliferation and a failure to terminally differentiate. Furthermore we hypothesized that disrupting the epigenetic machinery recruited by this fusion gene would provide a tractable target for therapy. We mapped the landscape of epigenetic alterations caused by the PAX3-FOXO1 fusion gene using a combination of RNA-seq, DNase hypersensitivity, and ChIP-seq against histone marks and transcription factors in cell lines and models of FP-RMS. We found high expression of several master transcription factors (including MYOD1, MYOG, MYCN, and SOX8) in FP-RMS primary tumors and cell lines, resembling human skeletal muscle myoblasts. ChIP-seq revealed that PAX3-FOXO1 is exclusively bound to distal, active enhancers and the histone modification most enriched surrounding PAX3-FOXO1 was acetylated H3K27. Furthermore we found that the introduction of the fusion gene into fibroblast cells opened up the chromatin at these same sites, completely rewiring the active enhancer landscape, recapitulating a transcriptome locked in a myoblast-like state. Genome-wide profiling of MYOD1, MYOG and MYCN reveals that all three master regulators collaborative bind at nearly every PAX3-FOXO1 driven super enhancer (SE), while typical enhancers (TEs) rarely have more than two of these four. PAX3-FOXO1 has a 7-fold preference for SEs over TEs. We also find that PAX3-FOXO1 bound, myogenic enhancers are decommissioned throughout normal skeletal muscle differentiation. To identify small molecules that would inhibit the PAX3-FOXO1 induced epigenetic machinery we treated a panel of FP-RMS cell lines with 1912 targeted agents and chemical probes at multiple concentrations and measured cell viability. Classes of molecules selectively potent for PAX3-FOXO1 driven cells (as compared to normal fibroblasts) hit connected biologically relevant targets including SE controlled receptor tyrosine kinases (including FGFR4, IGF1R, ALK), and transcriptional cofactors involved in SE complexes (including HDACs and BRD). In an expanded panel of RMS cell lines we confirmed that FP-RMS is selectively sensitive to the BET bromodomain inhibitors with the most potent being JQ1. These inhibitors selectively suppress PAX3-FOXO1 dependent transcription as measured by reporter assays and RNA-seq analysis. Indeed, coactivators of looped chromatin p300, MED1 and BRD4 excessively co-localize with PAX3-FOXO1 genome wide. In vivo, JQ1 selectively ablated PAX3-FOXO1 dependent SE driven transcription, and significantly delayed tumor progression in xenografts of PAX3-FOXO1 driven cell lines. In conclusion we found that PAX3-FOXO1 establishes myogenic super enhancers that are sensitive to BET bromodomain inhibition which constitutes a novel therapeutic strategy for children with PAX-fusion driven rhabdomyosarcoma. This abstract is also presented as Poster A16. Citation Format: Berkley E. Gryder, Marielle E. Yohe, Jack Shern, Hsien-Chao Chou, Young Song, Rajesh Patidar, Sam Li, Sivasish Sindiri, Abigail Cleveland, Hongling Liao, Xinyu Wen, Xiaohu Zhang, Lesley Mathews-Griner, Rajarshi Guha, Paul Shinn, Marc Ferrer, Scott Martin, Madhu Lal, Craig Thomas, Javed Khan. Targeting the chromatin architecture established by PAX3-FOXO1 in rhabdomyosarcoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr PR16.

Abstract A25: Reprogramming RAS-driven rhabdomyosarcoma via MEK inhibition

Abstract PAX-fusion negative rhabdomyosarcoma (RMS) arises from skeletal muscle precursors that have failed to differentiate normally despite the expression of the myogenic master transcription factor, MYOD1. The cure rate for relapsed or refractory fusion negative RMS is poor despite aggressive multi-modality treatment. Novel treatment approaches such as the use of targeted therapies including those that induce skeletal muscle differentiation might improve overall survival for patients with fusion negative RMS. Genetic studies have shown that the most common single nucleotide variant in fusion negative RMS is an oncogenic change in one of the RAS isoforms, namely NRAS, HRAS or KRAS. In this study, we hypothesized that targeting aberrant RAS activity releases the differentiation block in fusion negative RMS and sought to unravel the underlying epigenetic mechanisms through which RAS signaling drives oncogenic transcription in RMS. To achieve this goal, we combined high-throughput drug screening with biochemical, RNAseq and ChIPseq assays across a panel of RMS cell lines driven by oncogenic RAS mutations. Critically, we demonstrated that expression of oncogenic RAS was necessary for survival of these RAS-mutated RMS cells. In addition, overexpression of mutant RAS isoforms in C2C12 myoblasts inhibited myogenic differentiation induced by low-serum conditions. This differentiation block was mediated primarily by engagement of the RAF-MEK-ERK MAP kinase pathway. In corroboration with these observations, an unbiased screen of the ability of small molecules to impact cell viability demonstrated that inhibitors of the MAP kinase pathway were the most potently selective class of molecules for RAS-mutated RMS. In particular, trametinib, an allosteric, non-ATP competitive inhibitor of MEK1/2, was the most consistently potent MEK inhibitor in RAS-mutated RMS cell lines. Trametinib treatment induced G1 arrest and skeletal muscle differentiation in RAS-mutated RMS cell lines. Trametinib also slowed tumor growth and prolonged survival in xenograft models of RAS-mutated RMS. To determine the mechanism by which MEK inhibition induced skeletal muscle differentiation in RAS-mutated RMS, we analyzed changes in gene expression, transcription factor deposition and histone modification in RMS cells treated with trametinib. Trametinib treatment increased expression of myogenic transcription factors, such as MYOG and MEF2C, and decreased expression of transcription factors important for proliferation, such as MYC and ID3, in RAS-mutated RMS cells. ChIPseq experiments demonstrated that this transcriptional reprogramming was driven in part by changes in the active enhancer landscape, since H3K27ac deposition at MYH3, TTNT2 and other muscle-specific loci increased with trametinib treatment. Both MYC and MYOD1 bound the active enhancers induced by trametinib treatment in RAS-mutated RMS, despite an overall decrease in MYC expression. Finally, we found significant ERK2 deposition on the MYOG promoter in the untreated cells. ERK2 is known to recruit the Polycomb repressive machinery at developmental loci in embryonic stem cells and therefore aberrant ERK2 activity may facilitate repression of MYOG expression in RAS-mutated RMS. In summary, our data support a model of RAS-driven RMS in which aberrant ERK activity drives tumor cell proliferation, in part through increased expression and stability of MYC, and prevents myogenic differentiation, in this case through alterations in the enhancer landscape and interactions with the Polycomb repressive machinery. Future work is aimed at identifying rational combinations of trametinib and direct epigenetic modulators that synergistically drive RAS-mutated RMS differentiation with the goal of providing measurable clinical benefit in relapsed or refractory RAS-mutated RMS. Citation Format: Marielle E. Yohe, Berkley E. Gryder, Jack F. Shern, Young K. Song, Hongling Liao, Hsein-Chao Chou, Sivasish Sindiri, Arnulfo Mendoza, Xiaohu Zhang, Rajarashi Guha, Diana C. Haines, James P. Madigan, Jun S. Wei, Marc Ferrer, Craig J. Thomas, Javed Khan. Reprogramming RAS-driven rhabdomyosarcoma via MEK inhibition. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr A25.

Abstract A44: Proprotein convertase furin in rhabdomyosarcoma progression: A potential target for pediatric sarcomas therapy?

Abstract To improve therapies for embryonal pediatric tumors such as rhabdomyosarcoma (RMS), the most common soft tissue sarcoma in children, we have selected, by in vivo phage display, for peptides that bind to RMS with high specificity. We have shown that coupling of doxorubicin to RMS-P3, our lead RMS-targeting peptide, increases its therapeutic impact in a xenograft mouse model for RMS. We have identified furin as potential target for RMS binding peptide RMS-P3. Furin is a member of the proprotein convertases (PC), a family of ten serine proteases, which convert inactive proproteins into their active form. We have found consistent expression of furin in a panel of RMS cell lines and in RMS biopsies at the mRNA level. To elucidate the contribution of furin to RMS growth we have generated a panel of RMS cell lines either overexpressing furin, or a specific intrinsic PC inhibitor called α1-PDX, as well as RMS cell lines were expression of furin has been stably reduced by shRNA. These cells have been tested for their growth, migration and invasion potential in vitro as well as for their growth in vivo as subcutaneous xenografts. The results suggest a direct correlation between furin activity and RMS cells migration and invasive properties in vitro as well as RMS tumor growth in vivo. Interestingly, alveolar RMS cells display a higher furin activity compared to embryonal RMS cells. Alveolar RMS cells growth in vivo cannot be further enhanced by furin overexpression, in contrast to embryonal RMS cells, suggesting a causal link between high furin activity and alveolar RMS malignant phenotype. Among the known furin substrates, which are processed and activated by furin, and involved in tumor growth, migration and invasion, we found a direct correlation between furin activity and IGFR1 activation. Our results suggest that furin plays a relevant role in RMS progression through enhanced activation of the IGF1R pathway, among others, and that it might represent a viable target for therapeutic intervention in RMS. This warrants further studies in the development of novel therapeutic approaches for RMS based both on specific inhibition of its activity and on targeted drug delivery by furin targeting peptides. Citation Format: Patricia Jaaks, Valentina D'Alessandro, Beat W. Schäfer, Michele Bernasconi.{Authors}. Proprotein convertase furin in rhabdomyosarcoma progression: A potential target for pediatric sarcomas therapy? [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A44.

Abstract 3109: ALK as a valid therapeutic target for the treatment of rhabdomyosarcoma

Abstract Anaplastic Lymphoma Kinase (ALK) is a transmembrane receptor tyrosine kinase, a member of the insulin receptor family, with a tightly controlled expression pattern. Deregulated ALK expression has been linked to the development of several types of cancers. Typically, chromosomal translocations involving ALK generate constitutively expressed chimeric oncoproteins, such as NPM-ALK in anaplastic large cell lymphoma and EML4-ALK in non-small cell lung carcinoma. However, activating mutations in ALK, and increased copy number that result in aberrant gene expression, have also been observed in neuroblastoma and colorectal cancers. Based on the above observations, several small molecule ALK inhibitors have been developed and are in clinical trials for a variety of tumors expressing this kinase. This has resulted in the use of crizotinib for the treatment of EML4-ALK + lung cancers. Recently several large scale immunohistochemistry and genome sequencing studies of pediatric rhabdomyosarcoma (RMS) revealed aberrant ALK expression. It was found that ALK mRNA expression was significantly different in alveolar (ARMS) and embryonic RMS (ERMS), the two major histological subtypes in children. Thus, ALK expression might play a role in tumorigenesis for this disease, and this kinase may represent a valid therapeutic target for the treatment of RMS. To answer this question we analyzed ALK expression in a large panel of RMS cell lines, and pediatric tumor samples by quantitative PCR and immunoblotting. We also compared the sensitivity of these cells to a series of ALK inhibitors. Our results indicate that the majority of ARMS cell lines demonstrated significantly higher ALK mRNA expression as compared to ERMS, and to normal human myoblast control cells. This was confirmed by western analysis. Growth inhibition assays confirmed that all RMS lines were more sensitive to crizotinib and LDK-378 than control lines, consistent with the hypothesis that ALK is a driver of cell growth in these tumors. Based on our in vitro analysis, we suggest that ALK inhibition may represent an effective therapeutic modality for the treatment of ALK+ RMS. Preclinical studies to confirm or disprove this hypothesis are currently underway. Supported by St. Jude Children's Research Hospital and by the American Lebanese Syrian Associated Charities Citation Format: Monika Wierdl, Lyudmila Tsurkan, Viktor Tollemar, Lying Chi, Elizabeth Stewart, Michael A. Dyer, Philip M. Potter. ALK as a valid therapeutic target for the treatment of rhabdomyosarcoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3109. doi:10.1158/1538-7445.AM2014-3109

Abstract 827: Identification of the insulin-like growth factor 2 mRNA binding protein (IGF2BP1) as an important regulator of cIAP1 translation and apoptosis in rhabdomyosarcomas.

Abstract Rhabdomyosarcoma, a neoplasm characterized by undifferentiated myoblats-like cells, is the most common soft tissue sarcoma of childhood and represents 3-4% of all childhood cancers. IGF2BP1 is an oncofetal protein that was first identified in the rhabdomyosarcoma RD cell line and was shown to be overexpressed in a variety of cancers. Our group has previously identified IGF2BP1 as a potential translation modulator of the cellular inhibitor of apoptosis protein 1 (cIAP1), a key regulator of the NFκB signaling pathway and of caspase-8 mediated cell death in mammalian cells. In this study, we report that IGF2BP1 and cIAP1 expression is upregulated in a panel of rhabdomyosarcoma cell lines. We also show that IGF2BP1 is a positive regulator of cIAP1 translation, specifically through an internal ribosome entry site (IRES) mechanism. Finally, we report that altering the levels of cIAP1 in two rhabdomyosarcoma cell lines, RH36 and RH41, either by IGF2BP1 knock-down or by a Smac mimetic coumpound, sensitizes these cells to TNFα-mediated cell death. Our results identify IGF2BP1 and cIAP1 as important regulators of apoptosis in rhabdomyosarcomas. Citation Format: Mame Daro Faye, Tyson Graber, Stephanie Langlois, Kyle Cowan, Martin Holcik. Identification of the insulin-like growth factor 2 mRNA binding protein (IGF2BP1) as an important regulator of cIAP1 translation and apoptosis in rhabdomyosarcomas. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 827. doi:10.1158/1538-7445.AM2013-827

Abstract 4332: Proprotein convertases as target for therapy of pediatric sarcomas

Abstract One of the biggest challenges of treating children with cancer is achieving complete remission, while ensuring that they will be able to lead normal, productive life in adulthood. To improve therapies for embryonal pediatric tumors such as rhabdomyosarcoma (RMS) the most common soft tissue sarcoma in children, we have selected by in vivo phage display for peptides that bind to RMS with high specificity. We have shown that coupling of Doxorubicin to RMS-P3, a RMS targeting peptide, considerably increases its therapeutic impact in a mouse model for RMS. We have identified proprotein convertases (PC), a family of ten serine proteases which convert inactive proprotein into their active form, as targets RMS binding peptides. Now we aimed at elucidating the contribution to RMS growth in vivo of the two proprotein convertases, furin and PC7, which are highly expressed in RMS tumors. To this end, we have generated a panel of RMS cell lines overexpressing furin, overexpressing a specific intrinsic PC inhibitor called α1-PDX, as well as cell lines were expression of furin or PC7 has been stably blocked by shRNA. These cells have been tested for their growth, migration and invasion potential in vitro as well as for their growth in vivo as subcutaneous xenografts. Histological analysis of tumor sections, in particular of microvessel density and angiogenesis has been performed to reveal if PC activity correlates with RMS progression. Our results confirm a strong correlation between PCs activity and RMS growth in vivo and suggest furin and PC7 as viable targets for therapeutic intervention and warrant further studies in the development of novel therapeutic approaches based both on specific inhibition of PCs protease activity and on targeted drug delivery by PCs targeting peptides. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4332. doi:1538-7445.AM2012-4332

Bone marrow derived mesenchymal stem/stromal cells transduced with full length human TRAIL repress the growth of rhabdomyosarcoma cells in vitro

Rhabdomyosarcoma (RMS) is the most frequent juve nal cancer originating from skeletal muscle and patient survival is poor in case of metastatic disease. New target ed therapeutics are critically needed. In a recent Hematologica paper Kuci et al. 1 reported that in vitro cul tured cytokine-induced killer (CIK) cells effectively lysed the targeted rhabdomyosarcoma (RMS) cell lines and proposed that CIK cells may be used as a novel adoptive immunotherapy for the treatment of patients with RMS after allogeneic stem cell transplantation. The authors showed that TRAIL expression con tributes to the antitumor effect of CIK cells by inducing caspase activation in rhabdomyosarcoma cell lines. Furthermore, they provided evidence that not every RMS cell line is equally sensitive for TRAIL-mediated CIKinduced cytotoxicity. We have previously shown for the first time on a panel of RMS cell lines that around half of them were highly sensitive for the hr-TRAIL-induced caspase-mediated apoptosis. 2 RMS cell line type did not determine their response to TRAIL as several alveolar RMS cell lines were sensitive to TRAIL while an embrional RMS cell line was resistant. 2 Further research revealed that resistance of RMS cells to TRAIL can be the result of dysregulation of various molecular components such as FLIP overexpres sion, 2 casein kinase II (CK2) overactivity 3 or Bcl-2 overex pression. 3,4

Pharmacologic inhibition of cyclin-dependent kinase 4/6 activity arrests proliferation in myoblasts and rhabdomyosarcoma-derived cells

Myoblast cell cycle exit and differentiation are mediated in part by down-regulation of cyclin D1 and associated cyclin-dependent kinase (Cdk) activity. Because rhabdomyosarcoma may represent a malignant tumor composed of myoblast-like cells failing to exit the cell cycle and differentiate, we considered whether excess Cdk activity might contribute to this biology. Cyclin D-dependent Cdk4 and Cdk6 were expressed in most of a panel of six human rhabdomyosarcoma-derived cell lines. Cdk4 was expressed in 73% of alveolar and embryonal rhabdomyosarcoma tumors evaluated using a human tissue microarray. When challenged to differentiate by mitogen deprivation in vitro, mouse C2C12 myoblasts arrested in G(1) phase of the cell cycle, whereas four in the panel of rhabdomyosarcoma cell lines failed to do so. C2C12 myoblasts maintained in mitogen-rich media and exposed to a Cdk4/Cdk6 inhibitor PD 0332991 accumulated in G(1) cell cycle phase. Similar treatment of rhabdomyosarcoma cell lines caused G(1) arrest and prevented cell accumulation in vitro, and it delayed growth of rhabdomyosarcoma xenografts in vivo. Consistent with a role for Cdk4/Cdk6 activity as a regulator of myogenic differentiation, we observed that PD 0332991 exposure promoted morphologic changes and enhanced the expression of muscle-specific proteins in cultured myoblasts and in the Rh30 cell line. Our findings support the concept that pharmacologic inhibition of Cdk4/Cdk6 may represent a useful therapeutic strategy to control cell proliferation and possibly promote myogenic differentiation in rhabdomyosarcoma.

An anti-apoptotic role for NGF receptors in human rhabdomyosarcoma

The expression and biological function of Nerve Growth Factor (NGF) receptors was studied in a panel of rhabdomyosarcoma cell lines derived from embryonal and alveolar histotype. All the cell lines expressed both the high affinity receptor TrkA and the low affinity receptor p75NTR. Treatment with exogenous NGF did not considerably alter rhabdomyosarcoma cell growth or differentiation, but significantly inhibited spontaneous apoptosis as well as apoptosis, and induced by serum starvation or apoptosis induced by treatment with cycloheximide (CHX). Rhabdomyosarcoma cell lines expressed NGF and other neurotrophins and trace amounts of NGF protein were found in the supernatants of rhabdomyosarcoma cell cultures. Blocking the putative autocrine loop with an anti-NGF antibody resulted in an increase in apoptosis compared with control cultures. These data suggest that the simultaneous presence of both high and low affinity NGF receptors engaged by endogenous or exogenous NGF might contribute to the escape from apoptosis exhibited by the rhabdomyosarcoma cells.