Insulin-like growth factor-I has different effects on myogenin induction and cell cycle progression in human alveolar and embryonal rhabdomyosarcoma cells

Alveolar rhabdomyosarcoma (RMS) has a much poorer outcome than embryonal RMS. In this study, we found that IGF-I affected the induction of myogenin and cell cycle progression in alveolar RMS cells, but not in embryonal RMS cells. IGF-I enhanced the induction of myogenin protein in alveolar RMS SJ-Rh30 and KP-RMS-MS cells as it did in myoblast C2C12 cells, but not in embryonal RMS RD or KP-RMS-KH cells. IGF-I induction of myogenin protein was blocked by anti-IGF-IR monoclonal antibody alphaIR-3 and the mTOR-specific inhibitor rapamycin. In Rh30mTOR-rr cells, which stably express a rapamycin-resistant mutant mTOR, rapamycin did not inhibit IGF-I induction of myogenin protein. These data suggest that IGF-I induces myogenin in alveolar RMS cells through the IGF-IR/mTOR pathway. In C2C12 cells, IGF-I induces myogenin protein followed by cell cycle arrest leading to myogenic differentiation. IGF-I promoted G1-S cell cycle progression without any signs of terminal differentiation in alveolar RMS cells. On the other hand, IGF-I promoted neither cell cycle arrest nor G1-S cell cycle progression in embryonal RMS cells. In alveolar RMS SJ-Rh30 cells, 4E-BP1, one of two effectors downstream of mTOR, was continuously hyperphosphorylated by IGF-I, whereas in embryonal RMS RD cells, 4E-BP1 was only transiently hyperphosphorylated. These findings suggest that the different effects of IGF-I on myogenin induction and cell cycle progression in alveolar and embryonal RMS cells are due to a difference of phosphorylation status of 4E-BP1. These different responses to IGF-I help to explain immunohistochemical and clinical behavioral differences between alveolar and embryonal RMS.     

FGFR4 signaling couples to Bim and not Bmf to discriminate subsets of alveolar rhabdomyosarcoma cells

Biological heterogeneity represents a major obstacle for cancer treatment. Therefore, characterization of treatment‐relevant tumor heterogeneity is necessary to develop more effective therapies in the future. Here, we uncovered population heterogeneity among PAX/FOXO1‐positive alveolar rhabdomyosarcoma by characterizing prosurvival networks initiated by FGFR4 signaling. We found that FGFR4 signaling rescues only subgroups of alveolar rhabdomyosarcoma cells from apoptosis induced by compounds targeting the IGF1R‐PI3K‐mTOR pathway. Differences in both proapoptotic machinery and FGFR4‐activated signaling are involved in the different behavior of the phenotypes. Proapoptotic stress induced by the kinase inhibitors is sensed by Bim/Bad in rescue cells and by Bmf in nonrescue cells. Anti‐apoptotic ERK1/2 signaling downstream of FGFR4 is long‐lasting in rescue and short‐termed in most non‐rescue cells. Gene expression analysis detected signatures specific for these two groups also in biopsy samples. The different cell phenotypes are present in different ratios in alveolar rhabdomyosarcoma tumors and can be identified by AP2β expression levels. Hence, inhibiting FGFR signaling might represent an important strategy to enhance efficacy of current RMS treatments.     

Heparanase activity in alveolar and embryonal rhabdomyosarcoma: implications for tumor invasion

Background  

Rhabdomyosarcoma (RMS) is a malignant soft tissue sarcoma of childhood including two major histological subtypes, alveolar (ARMS) and embryonal (ERMS) RMS. Like other human malignancies RMS possesses high metastatic potential, more pronounced in ARMS than in ERMS. This feature is influenced by several biological molecules, including soluble factors secreted by tumor cells, such as heparanase (HPSE). HPSE is an endo-β-D-glucuronidase that cleaves heparan sulphate proteoglycans.     

Strategies to inhibit FGFR4 V550L-driven rhabdomyosarcoma

Background Rhabdomyosarcoma (RMS) is a paediatric cancer driven either by fusion proteins (e.g., PAX3-FOXO1) or by mutations in key signalling molecules (e.g., RAS or FGFR4). Despite the latter providing opportunities for precision medicine approaches in RMS, there are currently no such treatments implemented in the clinic.Methods We evaluated biologic properties and targeting strategies for the FGFR4 V550L activating mutation in RMS559 cells, which have a high allelic fraction of this mutation and are oncogenically dependent on FGFR4 signalling. Signalling and trafficking of FGFR4 V550L were characterised by confocal microscopy and proteomics. Drug effects were determined by live-cell imaging, MTS assay, and in a mouse model.Results Among recently developed FGFR4-specific inhibitors, FGF401 inhibited FGFR4 V550L-dependent signalling and cell proliferation at low nanomolar concentrations. Two other FGFR4 inhibitors, BLU9931 and H3B6527, lacked potent activity against FGFR4 V550L. Alternate targeting strategies were identified by RMS559 phosphoproteomic analyses, demonstrating that RAS/MAPK and PI3K/AKT are essential druggable pathways downstream of FGFR4 V550L. Furthermore, we found that FGFR4 V550L is HSP90-dependent, and HSP90 inhibitors efficiently impeded RMS559 proliferation. In a RMS559 mouse xenograft model, the pan-FGFR inhibitor, LY2874455, did not efficiently inhibit growth, whereas FGF401 potently abrogated growth.Conclusions Our results pave the way for precision medicine approaches against FGFR4 V550L-driven RMS.Subject terms: Growth factor signalling, Paediatric cancer, Oncogenes, Sarcoma     

Isolation of genes differentially expressed in human primary myoblasts and embryonal rhabdomyosarcoma

Using a subtractive hybridization method, we have cloned 48 cDNAs which are expressed in human primary myoblasts but down-regulated in the embryonal-rhabdomyosarcoma (RMS) cell line RD. Twenty-nine sequences could be identified as coding for previously known gene products, while 19 encode unknown proteins. Twelve clones coding for known proteins that were highly down-regulated in the RD cells were chosen for further analysis on Northern blots containing additional normal and RMS cells. The expression pattern of TGF-β-induced gene product-3 (βigH3), inhibitory G-protein alpha sub-unit (Gα_i2), osteoblast-specific factor-2 (OSF-2), 22-kDa smooth-muscle protein (SM22), clone A3351 (homologous to mouse talin), testican, thrombospondin-1 and thrombospondin-2 suggests involvement of these proteins in the genesis of the neoplastic phenotype. Among the clones with unknown sequence, several are identical or homologous to expressed sequence tags or known cDNAs, such as integrins or laminin. These results suggest that several isolated clones might have an important role in the determination or maintenance of the normal phenotype, and thus their loss is possibly involved in the progression of malignancy. © 1996 Wiley-Liss, Inc.     

Expression and activity of vascular endothelial growth factor and metalloproteinases in alveolar and embryonal rhabdomyosarcoma cell lines

Rhabdomyosarcoma (RMS) is a malignant tumour of skeletal muscle origin which includes two major histological subtypes: alveolar rhabdomyosarcoma (ARMS), the more aggressive, and embryonal rhabdomyosarcoma (ERMS). In order to establish whether the higher metastatic potential of ARMS cells may depend on differential expression of specific matrix metalloproteinases (MMPs) and angiogenesis-related factors, we studied the expression of MMP-2, MT1-MMP, TIMP-2, VEGF and VEGF receptors in four ARMS (RH30, RH4, RH18, RH28), three ERMS (RD, RH36, SMS-CTR) and one undifferentiated sarcoma cell line (A204). Semi-quantitative analysis of MMP-2 revealed high levels of expression in 3 out of 4 ARMS cell lines whereas, among ERMS, only RH36 showed comparable levels of the protease. TIMP-2 and MT1-MMP showed no significant differences among cell lines. in vitro invasiveness was also evaluated. The MMP-2-overexpressing RH30 cells were more invasive than RD cells, which expressed low levels of MMP-2. Exogenous expression of the ARMS specific PAX3-FKHR chimeric protein in RD cells increased MMP-2 activity and invasiveness. Of the three main VEGF isoforms only VEGF165 and VEGF121 were detected in RMS lines: ARMS expressed both isoforms, whereas the ERMS cell line SMS-CTR and the undifferentiated sarcoma cell line A204 showed the VEGF121 isoform only. All RMS cell lines expressed VEGFR-1 at mRNA as well as at protein level. The VEGFR-2, on the contrary, was undetectable with the sole exception of the RH28 cell line. Overall, our data suggest that a high level of MMP-2 protein and VEGF/VEGFR expression may contribute to the metastatic phenotype of ARMS cells and that exogenously induced PAX3-FKHR expression increases MMP-2 secretion and invasive capability of RMS cells.     

GLI inhibitor GANT-61 diminishes embryonal and alveolar rhabdomyosarcoma growth by inhibiting Shh/AKT-mTOR axis

Rhabdomyosarcoma (RMS) typically arises from skeletal muscle. Currently, RMS in patients with recurrent and metastatic disease have no successful treatment. The molecular pathogenesis of RMS varies based on cancer sub-types. Some embryonal RMS but not other sub-types are driven by sonic hedgehog (Shh) signaling pathway. However, Shh pathway inhibitors particularly smoothened inhibitors are not highly effective in animals. Here, we show that Shh pathway effectors GLI1 and/or GLI2 are over-expressed in the majority of RMS cells and that GANT-61, a specific GLI1/2 inhibitor dampens the proliferation of both embryonal and alveolar RMS cells-derived xenograft tumors thereby blocking their growth. As compared to vehicle-treated control, about 50% tumor growth inhibition occurs in mice receiving GANT-61 treatment. The proliferation inhibition was associated with slowing of cell cycle progression which was mediated by the reduced expression of cyclins D1/2/3 & E and the concomitant induction of p21. GANT-61 not only reduced expression of GLI1/2 in these RMS but also significantly diminished AKT/mTOR signaling. The therapeutic action of GANT-61 was significantly augmented when combined with chemotherapeutic agents employed for RMS therapy such as temsirolimus or vincristine. Finally, reduced expression of proteins driving epithelial mesenchymal transition (EMT) characterized the residual tumors.     

Cytogenetic and cellular characteristics of a human embryonal rhabdomyosarcoma cell line, RMS-YM

A human tumour cell line, designated RMS-YM, was established from a childhood rhabdomyosarcoma. The monolayer cells were polygonal, round or spindle-shaped. The cells became multilayered and formed many focal piles when confluent. RMS-YM became stable with a doubling time of about 30 h and has been maintained for 104 passages to date. Tumourigenicity of the cells was confirmed by heterotransplantation into nude mice. Morphological features were similar to those of the primary tumour, and myofibrils were found by electron microscopy. The expression of desmin and human myoglobin, and high levels of striated muscle system specific enzymes were recognised. Chromosomal analysis revealed possible gene amplification in the form of homogeneously staining regions. Oncogene analysis was performed on the primary tumour and the cell line, but neither N-myc nor N-ras genes were amplified, nor were Ki-ras, Ha-ras or N-ras genes mutated at the 12th, 13th and 61st codons. The RMS-YM cell line may provide a system to identify novel genes which are amplified in rhabdomyosarcoma.     

High intrinsic radiosensitivity of a newly established and characterised human embryonal rhabdomyosarcoma cell line

A new human rhabdomyosarcoma cell line (HX170c) has been established from a paratesticular embryonal tumour in a 5-year-old male. The cells grew as an adherent monolayer with a doubling time of 32 h and showed pleomorphic features. Intermediate filament analysis revealed the line to be mesenchymal in origin (reactivity to vimentin and desmin antibodies). The line was tumorigenic in nude mice, possessed elevated levels of creatine phosphokinase (mainly of the MM isoenzyme form) and had a near diploid mean chromosome number of 50. In vitro cell cloning determinations gave colony forming efficiencies of 0.01% in soft agar and 24% in a monolayer anchorage-dependent assay. Radiosensitivity determinations using a monolayer clonogenic assay with feeder layer support showed the cells to be among the more radiosensitive human tumour cell types (surviving fraction at 2 Gy of 0.26) that have been investigated. Furthermore, experiments utilising continuous low dose rate radiation at 3.2 cGy min-1, showed that, under these experimental conditions, the cells possessed only a very low capacity to recover from radiation-induced damage (dose reduction factor at 1% cell survival of 1.07 for 150 versus 3.2 cGy min-1). As other human tumour cells of an embryonal cell origin (e.g. neuroblastoma and germ cell tumours of the testis) have also been shown to be radiosensitive it appears that sensitivity to radiation may be a common property of this group of tumours.     

Simultaneous targeting of insulin-like growth factor-1 receptor and anaplastic lymphoma kinase in embryonal and alveolar rhabdomyosarcoma: A rational choice

BackgroundRhabdomyosarcoma (RMS) is an aggressive soft tissue tumour mainly affecting children and adolescents. Since survival of high-risk patients remains poor, new treatment options are awaited. The aim of this study is to investigate anaplastic lymphoma kinase (ALK) and insulin-like growth factor-1 receptor (IGF-1R) as potential therapeutic targets in RMS.Patients and methodsOne-hundred-and-twelve primary tumours (embryonal RMS (eRMS)86; alveolar RMS (aRMS)26) were collected. Expression of IGF-1R, ALK and downstream pathway proteins was evaluated by immunohistochemistry. The effect of ALK inhibitor NVP-TAE684 (Novartis), IGF-1R antibody R1507 (Roche) and combined treatment was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays in cell lines (aRMS Rh30, Rh41; eRMS Rh18, RD).ResultsIGF-1R and ALK expression was observed in 72% and 92% of aRMS and 61% and 39% of eRMS, respectively. Co-expression was observed in 68% of aRMS and 32% of eRMS. Nuclear IGF-1R expression was an adverse prognostic factor in eRMS (5-year survival 46.9 ± 18.7% versus 84.4 ± 5.9%, p = 0.006). In vitro, R1507 showed diminished viability predominantly in Rh41. NVP-TAE684 showed diminished viability in Rh41 and Rh30, and to a lesser extent in Rh18 and RD. Simultaneous treatment revealed synergistic activity against Rh41 and Rh30.ConclusionCo-expression of IGF-1R and ALK is detected in eRMS and particularly in aRMS. As combined inhibition reveals synergistic cytotoxic effects, this combination seems promising and needs further investigation.     

Prostatic embryonal rhabdomyosarcoma in adults. A clinicopathologic review.

Embryonal rhabdomyosarcoma of the prostate is a rare, highly malignant tumor that occurs predominantly in male infants and children, in whom it is the most common prostatic sarcoma. Six cases occurring in adults have been published, and the authors report three additional cases. The natural history is characterized by rapid growth, with the typical formation of large pelvic or abdominal masses, often leading renal failure due to bilateral ureteric obstruction. The tumor eventually disseminates widely, mainly to the lungs, bone, liver, and serosal surfaces, and unlike most other sarcomas, regional lymph node metastases are common. Combined modality therapy has resulted in marked improvement in survival rates and reduced surgical morbidity for children with these tumors. However, in adults the prognosis remains poor, with all patients dying of disseminated disease within 16 months of histologic diagnosis (mean survival, 8 months).     

Two distinct tumor cell growth-inhibiting factors from a human rhabdomyosarcoma cell line

Tumor cell growth-inhibiting factors (TIFs) have been shown to inhibit the growth of tumor cell lines in culture. TIF-1, the first TIF to be described, is a low-molecular-weight, acid- and heat-stable polypeptide with no antiviral activity. A second class of TIFs (TIF-2) has now been isolated from the conditioned media of a human rhabdomyosarcoma cell line and partially purified by polyacrylamide gel filtration, cation exchange, and reverse-phase high-pressure liquid chromatography. Partially purified preparations of TIF-2 inhibit the growth of a variety of human tumor cells in soft agar and monolayer cultures and are mitogenic for normal human and mouse cells. TIF-2 has no antiviral activity. The growth-inhibitory effects of TIF-2 are reversible when the affected cells are no longer exposed to the factor. Although both TIF-1 and TIF-2 are obtained from the same source, they can be distinguished by their molecular weight, heat lability, elution pattern from reverse-phase high-pressure liquid chromatography, and their effect on the growth of mink lung epithelial cells. The growth of a human tumor cell variant, selected for resistance to growth inhibition by TIF-1, is inhibited by TIF-2. TIFs may therefore be a family of related polypeptides which selectively inhibit the growth of tumor cells.     

2-deoxyglucose induces Noxa-dependent apoptosis in alveolar rhabdomyosarcoma

Alveolar and embryonal rhabdomyosarcomas are childhood tumors that do not respond well to current chemotherapies. Here, we report that the glycolytic inhibitor 2-deoxyglucose (2-DG) can efficiently promote cell death in alveolar, but not embryonal, rhabdomyosarcoma cell lines. Notably, 2-DG also induced cell differentiation accompanied by downregulation of PAX3/FOXO1a, the chromosome translocation-encoded fusion protein that is a central oncogenic driver in this disease. Cell death triggered by 2-DG was associated with its ability to activate Bax and Bak. Overexpression of the antiapoptotic Bcl-2 homologues Bcl-x(L) and Mcl-1 prevented apoptosis, indicating that cell death proceeds through the mitochondrial pathway. Mechanistic investigations indicated that Mcl-1 downregulation and Noxa upregulation were critical for 2-DG-induced apoptosis. In addition, 2-DG promoted eIF2α phosphorylation and inactivation of the mTOR pathway. Mcl-1 loss and cell death were prevented by downregulation of the endoplasmic reticulum (ER) stress-induced protein ATF4 and by incubating cells in the presence of mannose, which reverted 2-DG-induced ER stress but not ATP depletion. Thus, energetic stresses created by 2-DG were not the primary cause of cell death. Together, our findings suggest that glycolysis inhibitors such as 2-DG may be highly effective in treating alveolar rhabdomyosarcoma and that Noxa could offer a prognostic marker to monitor the efficacy of such agents.