transforming-growth-factor-beta and Rhabdomyosarcoma

Myostatin is a negative regulator of muscle mass. Important advances in our understanding of the complex biology of this factor have revealed the therapeutic potential of antagonizing the myostatin pathway. Here we present the rationale for evaluating anti-myostatin therapies in human muscle-wasting disorders.

Topics: Adipose Tissue; Animals; Humans; Muscles; Muscular Dystrophies; Myostatin; Rhabdomyosarcoma; Signal Transduction; Transforming Growth Factor beta

Rhabdomyosarcoma (RMS) is the most common soft tissue cancer in children. Treatment outcomes, particularly for relapsed/refractory or metastatic disease, have not improved in decades. The current lack of novel therapies and low tumor mutational burden suggest that chimeric antigen receptor (CAR) T-cell therapy could be a promising approach to treating RMS. Previous work identified FGF receptor 4 (FGFR4, CD334) as being specifically upregulated in RMS, making it a candidate target for CAR T cells. We tested the feasibility of an FGFR4-targeted CAR for treating RMS using an NSG mouse with RH30 orthotopic (intramuscular) tumors. The first barrier we noted was that RMS tumors produce a collagen-rich stroma, replete with immunosuppressive myeloid cells, when T-cell therapy is initiated. This stromal response is not seen in tumor-only xenografts. When scFV-based binders were selected from phage display, CARs targeting FGFR4 were not effective until our screening approach was refined to identify binders to the membrane-proximal domain of FGFR4. Having improved the CAR, we devised a pharmacologic strategy to augment CAR T-cell activity by inhibiting the myeloid component of the T-cell-induced tumor stroma. The combined treatment of mice with anti-myeloid polypharmacy (targeting CSF1R, IDO1, iNOS, TGFbeta, PDL1, MIF, and myeloid misdifferentiation) allowed FGFR4 CAR T cells to successfully clear orthotopic RMS tumors, demonstrating that RMS tumors, even with very low copy-number targets, can be targeted by CAR T cells upon reversal of an immunosuppressive microenvironment.

Topics: Animals; Cell Line, Tumor; Humans; Immunotherapy, Adoptive; Mice; Polypharmacy; Receptor, Fibroblast Growth Factor, Type 4; Receptors, Antigen, T-Cell; Receptors, Chimeric Antigen; Rhabdomyosarcoma; Transforming Growth Factor beta; Tumor Microenvironment

PAX3-FOXO1 (PAX3-FKHR) is the fusion protein produced by the genomic translocation that characterizes the alveolar subtype of Rhabdomyosarcoma, a pediatric sarcoma with myogenic phenotype. PAX3-FOXO1 is an aberrant but functional transcription factor. It retains PAX3-DNA-binding activity and functionally overlaps PAX3 function while also disturbing it, in particular its role in myogenic differentiation. We herein show that PAX3-FOXO1 interferes with normal FOXO function. PAX3-FOXO1 affects FOXO-family member trans-activation capability and the FOXO-dependent TGF-β response. PAX3-FOXO1 may contribute to tumor formation by inhibiting the tumor suppressor activities which are characteristic of both FOXO family members and TGF-β pathways. The recognition of this mechanism raises new questions about how FOXO family members function.

Topics: Cell Differentiation; Cell Line; Cell Line, Tumor; Forkhead Box Protein O1; Forkhead Transcription Factors; HEK293 Cells; Humans; Oncogene Proteins, Fusion; Paired Box Transcription Factors; PAX3 Transcription Factor; Rhabdomyosarcoma; Transforming Growth Factor beta; Translocation, Genetic

We describe a patient with insufficient bone regeneration of the tibia after bone transport over an intramedullary nail, in whom union was ultimately achieved after exchange nailing and intramedullary application of rh-bone morphogenetic protein-7 at the site of distraction.

Topics: Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Bone Neoplasms; Child; Female; Fractures, Stress; Humans; Ilizarov Technique; Leg Length Inequality; Osteogenesis, Distraction; Recombinant Proteins; Rhabdomyosarcoma; Tibia; Tibial Fractures; Transforming Growth Factor beta

Myostatin, also called GDF-8, a secreted growth and differentiating factor that belongs to the transforming growth factor-beta superfamily, is a known negative regulator of myogenesis in vivo. Overexpression of GDF-8 contributes to the lack of differentiation in human rhabdomyosarcoma (RMS) cells. We investigated whether a retrovirus-based RNA interference (RNAi) system against GDF-8 expression in human RMS cells would enhance myogenic differentiation.. A retrovirus-based RNAi system was developed that utilized the U6-RNA polymerase III promoter to drive efficient expression and deliver the GDF8-specific short hairpin RNAs (shRNAs) in human RMS cell A204. In this system, the retrovirus vector was integrated into the host cell genome and allowed stable expression of shRNAs. GDF-8 expression was determined by real-time polymerase chain reaction and western blotting analysis. An 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed to determine the cell proliferation. Myogenic differentiation markers were monitored by western blotting analysis. Cell cycle and apoptosis was determined by propidium iodide staining and analysed in a flow cytometer.. In the siGDF8 A204 cell pools, the levels of both GDF-8 mRNA and protein were dramatically reduced by this RNAi system. In differentiation conditions, inhibition of myostatin synthesis led to enhanced cell cycle withdrawal, consequently stimulated myogenic differentiation and increased the rate of tumor cell apoptosis.. The results demonstrate that deactivation of myostatin by using retrovirus-based RNAi thus may be useful for therapy in rhabdomyosarcomas.

Topics: Apoptosis; Cell Cycle; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Gene Expression Regulation, Neoplastic; Genetic Vectors; Humans; Myostatin; Promoter Regions, Genetic; Retroviridae; Rhabdomyosarcoma; RNA Interference; RNA, Messenger; RNA, Small Interfering; Transduction, Genetic; Transforming Growth Factor beta; Tumor Cells, Cultured

Transforming growth factor-beta (TGF-beta) signals through membrane-bound heteromeric serine/threonine kinase receptors. Upon ligand binding, TGF-beta activates intracellular Smad proteins and regulates proliferation and apoptosis in various cell types. To demonstrate the effects of TGF-beta/Smad signal on growth and apoptosis of human embryonal rhabdomyosarcoma (RMS) cells, a strategy of RNAi-mediated 'gene silencing' of Smad4 was used to interrupt endogenous TGF-beta/Smad signaling in an RMS cell line, RD, and the regulation of exogenous TGF-beta1 to growth and apoptosis of the cells was also determined. Physiologically, TGF-beta/Smad signaling was essential for the normal growth of RD. The interruption of endogenous TGF-beta/Smad signaling by RNAi significantly suppressed the growth of RD cells and dramatically induced apoptosis of RD cells. Exogenous TGF-beta1 also inhibited the growth of RD cells, but had no effect on apoptosis. It also partially counteracted the growth inhibition and apoptosis induced by Smad4 silencing in RD cells. These findings provide a new insight into how TGF-beta/Smad signaling regulates the growth and apoptosis of cancer cells. Moreover, as a powerful tool, shRNA interference suppresses endogenous Smad4 gene expression and subsequently modulates cell growth and apoptosis, which may provide a novel basis for the development of rational intervention strategies in RMS therapy.

Topics: Apoptosis; Cell Nucleus; Cell Proliferation; Gene Expression; Gene Silencing; Humans; Rhabdomyosarcoma; RNA Interference; RNA, Messenger; RNA, Small Interfering; Signal Transduction; Smad4 Protein; Transforming Growth Factor beta; Tumor Cells, Cultured

Transforming growth factor-beta (TGF-beta) can inhibit the growth of most epithelial and endothelial cells. The growth regulative role of TGF-beta on soft tissue sarcoma was seldom reported. Here we examined TGF-beta1 effects on the growth of human rhabdomyosarcoma cell RD and searched the relative molecular mechanism.. The viability of RD was examined by [(3)H]-thymidine incorporation and [3-(4,5-dimethylthiazol-z-yl)-2,5-diphenyl tetrazolium bromide] (MTT) assay. RD cell cycle was analysed by flow cytometry. The protein and mRNA of cell cycle regulative factors in RD were detected by Western blot and reverse transcription-polymerase chain reaction (RT-PCR), respectively. The kinase activity of cdk2 or cdk4 was examined by immunoprecipitation and kinase assay. Immunofluorescent staining was used to detect the location of cell cycle regulative factors in RD by laser scanning confocal microscope.. TGF-beta1 inhibits RD proliferation by G1-arrest in cell cycle progression. TGF-beta1 can prominently up-regulate P27 of RD, then augment P27 to bind cyclinE-cdk2 complexes, which effectively suppress cdk2 kinase activity. P21 increased and c-myc decreased in RD due to TGF-beta1. Both P15 and cdk4 have not been involved in the growth inhibitory event. TGF-beta1 treatment induced P27 to congregate around nucleus. P21 pervaded from nucleus to both nucleus and cytoplasm by TGF-beta1 treatment.. TGF-beta1 inhibits the proliferation of human rhabdomyosarcoma cell line RD and induces RD G1-arrest. This course is accomplished by TGF-beta1 up-regulating P27 to suppress cdk2 kinase activity. The induction of P21 and down-regulation of C-myc might participate in the growth-arrest event.

Topics: Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p27; G1 Phase; Genes, myc; Humans; Rhabdomyosarcoma; RNA, Messenger; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Suppressor Proteins

Rhabdomyosarcoma (RMS) tumors are the most common soft-tissue sarcomas in childhood. In this investigation, we show that myostatin, a skeletal muscle-specific inhibitor of growth and differentiation is expressed and translated in the cultured RMS cell line, RD. The addition of exogenous recombinant myostatin inhibits the proliferation of RD cells cultured in growth media, consistent with the role of myostatin in normal myoblast proliferation inhibition. However, unlike normal myoblasts, upregulation of p21 was not observed. Rather, myostatin signalling resulted in the specific downregulation of both Cdk2 and its cognate partner, cyclin-E. The analysis of Rb reveals that there was no change in its phosphorylation status with myostatin treatment, consistent with D-type-cyclin-Cdk4/6 complexes being active in the absence of p21. Moreover, the activity of Rb appeared to be unchanged between treated and nontreated RD cells, as determined by the ability of Rb to bind E2F1. The examination of NPAT, a substrate of cyclin-E-Cdk2 involved in the transcriptional activation of replication-dependent histone gene expression, revealed that it undergoes a loss of phosphorylation with myostatin treatment. Supporting this, a downregulation in H4-histone gene expression was observed. These results suggest that myostatin could potentially be used as an inhibitor of RMS proliferation and define a previously uncharacterized, Rb-independent mechanism for the inhibition of muscle precursor cell proliferation by myostatin.

Topics: CDC2-CDC28 Kinases; Cell Cycle; Cell Cycle Proteins; Cell Division; Cell Line, Tumor; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinases; Gene Expression Regulation, Neoplastic; Histones; Humans; Myostatin; Nuclear Proteins; Phosphorylation; Proto-Oncogene Proteins; Retinoblastoma Protein; Rhabdomyosarcoma; Transforming Growth Factor beta

Transforming growth factor-beta (TGF-beta) is a multifunctional regulator of cell growth and differentiation, whose actions are highly cell type specific. To study the role of the TGF-beta1 autocrine loop in regulating growth and myogenic differentiation in the human rhabdomyosarcoma cell line, RD, an attempt was made to establish a framework for the expression of several components of TGF-beta1/Smad signalling pathway at the mRNA and protein levels by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis in RD cells compared with the normal myoblasts. Higher exogenous concentration of TGF-beta1 was necessary to reach a growth-inhibition effect, whereas TGF-beta1 downregulated the expression of myosin heavy-chain mRNA at lower concentrations than that was required for growth inhibition. Treatment with TGF-beta1 significantly decreased the number of sarcomeric actin and myosin-expressing cells. In this study, we have shown that RD cells displayed higher expression of TbetaRI, TbetaRII, Smad2 and Smad4 at both the mRNA and protein levels than myoblasts. Smad3 and Smad7 mRNA were expressed at higher level in RD cells than in myoblasts. The staining patterns of TbetaR and Smads suggest that they may transduce different TGF-beta1 signalling in RD cells than in myoblasts. TGF-beta1 signalling induced a rapid relocation of Smad2 to the nucleus; in contrast, Smad4 remained localized to the cytoplasm unless it was coexpressed with Smad2. These studies suggest that signalling from the cell surface to the nucleus through Smad proteins is a required component of TGF-beta1-induced cell response in RD cells. The RD cell line is a suitable model to study the TGF-beta autocrine loop involved in growth and differentiation of RMS.

Topics: Autocrine Communication; Blotting, Western; Cell Differentiation; Cell Division; Cell Survival; Depression, Chemical; DNA-Binding Proteins; Dose-Response Relationship, Drug; Fluorescent Antibody Technique; Humans; Microscopy, Confocal; Myosin Heavy Chains; Receptors, Transforming Growth Factor beta; Reverse Transcriptase Polymerase Chain Reaction; Rhabdomyosarcoma; RNA, Messenger; Smad Proteins; Smad2 Protein; Smad4 Protein; Soft Tissue Neoplasms; Statistics, Nonparametric; Trans-Activators; Transforming Growth Factor beta; Translocation, Genetic; Tumor Cells, Cultured

Rhabdomyosarcomas (RMSs) are one of the most common solid tumor of childhood. Rhabdomyosarcoma (RMS) cells fail to both complete the skeletal muscle differentiation program and irreversibly exit the cell cycle as a consequence of an active repression exerted on the muscle-promoting factor MyoD. Myostatin is a negative regulator of normal muscle growth, we have thus studied its possible role in RMS cells. Here, we present evidence that overexpression of myostatin is a common feature of RMS since both subtypes of RMS (embryonal RD and alveolar Rh30 cells) express high levels of myostatin when compared to nontumoral skeletal muscle cells. Interestingly, we found that inactivation of myostatin through overexpression of antisense myostatin or of follistatin (a myostatin antagonist) constructs enhanced differentiation of RD cells. In addition, RD and Rh30 cells treated with blocking antimyostatin antibodies progress into the myogenic terminal differentiation program. Finally, our results suggest that high levels of myostatin could impair MyoD function in RMS cells. These results show that an autocrine myostatin loop contributes to maintain RMS cells in an undifferentiating stage and suggest that new therapeutic approaches could be exploited for the treatment of RMS based on inactivation of myostatin protein.

Topics: Antibodies; Cell Differentiation; Cell Line, Tumor; Fluorescent Antibody Technique; Humans; Myostatin; Rhabdomyosarcoma; Transforming Growth Factor beta

The aim was to investigate the influence of pulmonary metastases of the rhabdomyosarcoma R1H on the radiation response of the lung of the WAG/Rij rat.. Three groups of animals were investigated: metastases-free animals treated with fractionated irradiation of the lungs; metastases-bearing animals receiving no irradiation; and metastases-bearing animals treated with fractionated irradiation initiated 14, 21 or 28 days after induction of pulmonary metastases of the R1H-tumour by i.v. injection of viable tumour cells. Metastases were thus treated at various well-defined sizes. Total doses of 20-60Gy were applied in fractions of 2 Gy within 11 days. Complication rate and survival time were used as endpoints.. About 2 months after onset of irradiation treatment, animals had to be sacrificed because of severe respiratory distress either caused by irradiation-induced lung damage (median 57 days, range 36-77 days), or because of development of lung metastases (65, 20-160 days). A decrease of the ED(50) (dose required to induce lethal lung damage in 50% of irradiated animals) was determined for metastases-bearing animals. This effect increased with metastatic volume.. The results suggest that the presence of tumours in the lung decreased the lung tolerance to radiation. This effect can hardly be explained by a reduction in functional lung volume by metastatic volume.

Topics: Animals; Female; Lung; Lung Neoplasms; Male; Radiation Tolerance; Rats; Rhabdomyosarcoma; Transforming Growth Factor beta

Transforming growth factor beta (TGF) is a well-known inhibitor of myogenic differentiation as well as an autocrine product of rhabdomyosarcoma cells. We studied the role of the TGF-beta autocrine loop in regulating growth and myogenic differentiation in the human rhabdomyosarcoma cell line, RD. We previously reported that the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) induces growth arrest and myogenic differentiation in these cells, which constitutively express muscle regulatory factors. We show that TPA inhibits the activation of secreted latent TGF-beta, thus decreasing the concentration of active TGF-beta to which the cells are exposed. This event is mediated by the TPA-induced alteration of the uPA/PAI serine-protease system. Complete removal of TGF-beta, mediated by the ectopic expression of a soluble type II TGF-beta receptor dominant negative cDNA, induces growth arrest, but does not trigger differentiation. In contrast, a reduction in the TGF-beta concentration, to a range of 0.14-0.20 x 10(-2) ng/ml (which is similar to that measured in TPA-treated cells), mimics TPA-induced differentiation. Taken together, these data demonstrate that cell growth and suppression of differentiation in rhabdomyosarcoma cells require overproduction of active TGF-beta; furthermore, they show that a 'critical' concentration of TGF-beta is necessary for myogenic differentiation to occur, whereas myogenesis is abolished below and above this concentration. By impairing the TGF-beta autocrine loop, TPA stabilizes the factor concentration within the range compatible for differentiation to occur. In contrast, in human primary muscle cells a much higher concentration of exogenous TGF-beta is required for the differentiation inhibitory effect and TPA inhibits differentiation in these cells probably through a TGF-beta independent mechanism. These data thus clarify the mechanism underlying the multiple roles of TGF-beta in the regulation of both the transformed and differentiated phenotype.

Topics: Animals; Aprotinin; Autocrine Communication; Cell Differentiation; Cell Division; Cells, Cultured; Dose-Response Relationship, Drug; Humans; Muscle, Skeletal; Mutation; Myosin Heavy Chains; Pepstatins; Plasminogen; Plasminogen Inactivators; Protein Precursors; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Rhabdomyosarcoma; RNA, Messenger; Tetradecanoylphorbol Acetate; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator

The transforming growth factor (TGF)-betas are a highly conserved group of potent multifunctional cell regulatory proteins with variable effects on cell growth and differentiation. Most of the small round cell group of childhood tumors are thought to arise from either primitive mesenchyme or neuroectoderm and show evidence of skeletal muscle or neural differentiation, and rarely both. To investigate the possibility that the TGF-betas have a role in the growth or differentiation of these neoplasms, we used antibodies specific for peptide sequences of the three known mammalian TGF-beta isoforms (TGF-betas 1, 2, and 3) to probe for TGF-beta protein expression in a total of 49 cases. TGF-beta 1 immunoreactivity was present in 16/17 (94%) of rhabdomyosarcomas, and the staining intensity was usually strong. TGF-beta 1 was also present in three of three ectomesenchymomas. In contrast, TGF-beta 1 was absent in all but one out of nine poorly differentiated neuroblastomas. Differentiating neuronal cells of ganglioneuroblastomas, however, were strongly positive for TGF-beta 1. Ewing's sarcomas and peripheral primitive neuroectodermal tumors had a less consistent, but usually positive, staining pattern. TGF-beta 3 staining patterns were very similar to those of TGF-beta 1. TGF-beta 2 immunoreactivity was only rarely detected in this group of tumors. The results suggest different roles for TGF-betas 1 and 3 in neuroblastoma and rhabdomyosarcoma. Expression of TGF-betas 1 and 3 is associated with neuronal differentiation of neuroblastoma. In contrast, these proteins may promote the growth of rhabdomyosarcoma by suppressing differentiation.

Topics: Adolescent; Adult; Antibodies, Monoclonal; Carcinoma, Small Cell; Child; Child, Preschool; Ganglioneuroma; Humans; Infant; Mesenchymoma; Molecular Probes; Neuroblastoma; Neurosecretory Systems; Rhabdomyosarcoma; Sarcoma, Ewing; Transforming Growth Factor beta