Embryonal Rhabdomyosarcoma Cell Research Articles

Impairment of Lysosomal Activity as a Therapeutic Modality Targeting Cancer Stem Cells of Embryonal Rhabdomyosarcoma Cell Line RD

Rhabdomyosarcoma is the most frequent soft tissue sarcoma in children and adolescents, with a high rate of relapse that dramatically affects the clinical outcome. Multiagent chemotherapy, in combination with surgery and/or radiation therapy, is the treatment of choice. However, the relapse rate is disappointingly high and identification of new therapeutic tools is urgently needed. Under this respect, the selective block of key features of cancer stem cells (CSC) appears particularly promising. In this study, we isolated rhabdomyosarcoma CSC with stem-like features (high expression of NANOG and OCT3/4, self-renewal ability, multipotency). Rhabdomyosarcoma CSC showed higher invasive ability and a reduced cytotoxicity to doxorubicin in comparison to native cells, through a mechanism unrelated to the classical multidrug resistance process. This was dependent on a high level of lysosome acidity mediated by a high expression of vacuolar ATPase (V-ATPase). Since it was not associated with other paediatric cancers, like Ewing’s sarcoma and neuroblastoma, V-ATPase higher expression in CSC was rhabdomyosarcoma specific. Inhibition of lysosomal acidification by the V-ATPase inhibitor omeprazole, or by specific siRNA silencing, significantly enhanced doxorubicin cytoxicity. Unexpectedly, lysosomal targeting also blocked cell growth and reduced the invasive potential of rhabdomyosarcoma CSC, even at very low doses of omeprazole (10 and 50 µM, respectively). Based on these observations, we propose lysosome acidity as a valuable target to enhance chemosensitivity of rhabdomyosarcoma CSC, and suggest the use of anti-V-ATPase agents in combination with standard regimens as a promising tool for the eradication of minimal residual disease or the prevention of metastatic disease.

Abstract A47: Self-renewing cellular compartments in embryonal rhabdomyosarcoma are modulated by hedgehog signaling

Abstract Rhabdomyosarcoma (RMS) is a heterogeneous group of malignancies with features of impaired skeletal muscle differentiation and represents the majority of soft tissue cancers in the pediatric age group. Current treatment regimens show clinical benefit but this has reached a plateau. Since there is significant risk of adverse side effects and secondary tumors later in life, identifying and targeting sub-populations with higher tumorigenic potential and self renewal capacity would not only decrease tumor burden but also lower the probability of relapse. Using in vitro self renewal assays we could identify self-renewing cellular compartments within embryonal RMS (eRMS) cell lines. These compartments showed upregulation of several components of the sonic hedgehog pathway. Here, we now show using both genetic loss- and gain-of-function experiments and clinically relevant small molecule modulators that Hedgehog signalling is important for determining the self-renewal, tumor initiation capability both in vitro as well as in vivo. In addition, the hedgehog pathway can also influence chemoresistance of eRMS cells and altered the differentiation status of the cells by directly modulating the expression of various stem cell associated transcription factors. The pathway was found to be heterogeneously activated within eRMS patient samples in a comprehensive tissue microarray analysis. Importantly, the presence of Hedgehog-active cells identified using effector transcription factors could negatively predict patient survival in a retrospective analysis of this patient cohort. This work elucidates both a novel and central role played by the Hedgehog pathway in eRMS and underscores the importance of studying tumor heterogeneity in pediatric cancer. Citation Format: Sampoorna Satheesha, Amandine Bovay, Gabriele Manzella, Peter Bode, Simone Feuchtgruber, Ewa Koscielniak, Reka Belle, Elisa Casanova, Nurhak Dogan, Beat W. Schäfer. Self-renewing cellular compartments in embryonal rhabdomyosarcoma are modulated by hedgehog signaling. [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 A47.

Abstract A66: NF-kappa B is a potential therapeutic target in a subset of rhabdomyosarcomas

Abstract Rhabdomyosarcoma (RMS) is a tumor that arises from skeletal muscle progenitor cells and is the most common soft tissue sarcoma of childhood. RMS cells are characterized by impaired myogenesis and do not undergo terminal differentiation. Nuclear factor-kappa B (NF-κB) is a family of transcription factors found in virtually all mammalian cells that is commonly associated with inflammation and has been implicated in numerous malignancies. NF-κB p65 containing dimers are the most prevalent active species of NF-κB. Increased NF-κB activity has been shown to disrupt myogenesis by suppressing the differentiation of myoblasts through a regulatory circuit involving microRNA miR-29. However, the role of NF-κB in RMS is largely unexplored. The purpose of this study is to investigate the role of NF-κB in RMS tumorigenesis and evaluate its potential as a therapeutic target in this disease. NF-κB activity was surveyed in a panel of embryonal RMS (ERMS) and alveolar RMS (ARMS) cell lines. Four out of 8 RMS cell lines (2 ERMS and 2 ARMS) demonstrated increased NF-κB activation at baseline as assessed by increased levels of p65 and phosphorylated p65 on western blotting, nuclear localization of p65 on EMSA super shift, and increased NF-κB transcriptional activity on a luciferase reporter assay. RMS cell lines, including TTC442 (ERMS), also demonstrated dose-dependent cytotoxicity after treatment with Bortezomib, a proteasome inhibitor. BAY 16-1942, a specific inhibitor of IκB kinase-β (IKK-β), was ineffective as a single agent; however, doxorubicin-induced cytotoxicity was enhanced by up to 39% in TTC442 cells treated concurrently with this agent. RH30 (ARMS), TTC442 (ERMS), and RD (ERMS) cells were transfected with IκB-SR, a doxycycline-inducible super-repressor form of IκBα, and effects on proliferation and clonogenic activity were assessed. WST-1 assay of cellular proliferation revealed growth impairment of TTC442 cells expressing the IκB-SR mutant, with average values of only 63% and 60% of control cells after 72 and 96 hours in culture. In addition, TTC442 and RH30 cells expressing the IκB-SR mutant formed 35% and 29% fewer colonies, respectively, compared to control cells when plated in low concentration. No differences in proliferation or clonogenic capacity were observed in RD cells, a line that did not show consistent NF-κB activation in our initial survey. These data indicate that NF-κB is activated in a subset of RMS cells and suggest that modulation of NF-κB activity may affect baseline tumor growth, sensitivity to chemotherapy, and clonogenic potential of these cells. These findings support the hypothesis that NF-κB is a promising new therapeutic target for RMS. Citation Format: Catherine M. Albert, David M. Loeb, Ido Paz-Priel. NF-kappa B is a potential therapeutic target in a subset of rhabdomyosarcomas. [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 A66.

Abstract A39: The novel atypical retinoid ST1926 is active against rhabdomyosarcoma and induces cell cycle arrest in S and G2/M phases

Abstract Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children with two subtypes: embryonal rhabdomyosarcoma (ERMS) and alveolar rhabdomyosarcoma (ARMS). With current treatment strategies, most children with recurrent or high-risk disease fare poorly, identifying a need for novel therapeutic approaches. The novel retinoid, ST1926, is an orally available compound belonging to the class of synthetic atypical retinoids and is currently in clinical trials. ST1926 is showing promise because of its increased specificity and reduced toxicity and its unique antitumor mechanism of action that appears different from that of classical all-trans retinoic acid (ATRA). We are investigating the therapeutic potential of this novel synthetic retinoid ST1926, in treatment of RMS. Cell viability assessed by MTT assay showed that ST1926 was a much more potent inhibitor of cell proliferation than ATRA, in both ERMS and ARMS cell lines in vitro. In fact, ST1926 effectively inhibited cell proliferation in all tested cell lines, at concentrations as low as 0.5 micromolar, which is easily achievable in vivo. Unlike ATRA, ST1926 did not induce morphological differentiation in RMS cells. Further analysis showed that ST1926 induced accumulation of cells in S and/or in G2/M phases of the cell cycle, as well as induction of apoptosis in some cell lines, in a p53-independent manner. We are currently investigating the mechanistic details of the cell cycle inhibitory activities of ST1926 in RMS cells. This data identifies ST1926 as an interesting possible therapeutic agent in RMS, and in vivo preclinical studies utilizing this agent are currently underway. Citation Format: Hussein Basma, Loai Dweik, Nadine Darwiche, Claudio Pisano, Raya Saab. The novel atypical retinoid ST1926 is active against rhabdomyosarcoma and induces cell cycle arrest in S and G2/M phases. [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 A39.

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 A68: Posttranslational modifications of the oncogenic fusion protein EWS/FLI1

Abstract Ewing sarcoma is a pediatric tumor that affects bone and soft tissues. It is characterized in the majority of cases by the presence of a chromosomal translocation that leads to the expression of the oncogenic transcription factor, EWS/FLI1. Continuing expression of EWS/FLI1 is essential for tumor growth, cellular transformation and tumor cell survival, suggesting it being an important target for therapy. However, very little is known about the regulation of its oncogenic activity that might be modulated by posttranslational modifications (PTMs). PTMs can modulate protein function in several different ways such as by altering its conformation, stability, activity, protein-protein and protein-DNA interactions, cellular localization etc. Here, we aimed to identify and characterize PTMs of EWS/FLI1 to understand their relevance for its oncogenic potential in order to identify novel therapeutic approaches. We purified flag-EWS/FLI1 from A673 cells (Ewing cell line) and analyzed it by mass spectrometry to detect PTMs. For the first time, we were able to identify novel Ser/Thr residues which are phosphorylated in EWS/FLI1 apart from the phospho-Thr79 residue which was previously described. Except Thr79, all phosphorylation sites are present in the FLI1 part of the fusion protein. Furthermore, EWS/FLI1 was found to be methylated at a single arginine residue. Non evidence of acetylation of EWS/FLI1 was detected. Individual mutations of the phospho-Ser/Thr to alanine or the methylated arginine to lysine did not affect the cellular localization of EWS/FLI1. In order to assess whether phosphorylation of EWS/FLI1 has an impact on its transcriptional activity, different cellular systems were used to overexpress EWS/FLI1 wild type (wt) and mutants and to measure EWS/FLI1 target genes level by qRT-PCR. Transient overexpression of EWS/FLI1 wt and mutants in Ewing cell lines (A673 and RD ES) did not lead to a sufficient upregulation of EWS/FLI1 target genes. However, overexpression of EWS/FLI1 by retroviral transduction of RD (embryonal rhabdomyosarcoma cell line) and hMSC (human messenchymal stem cells) induced significant upregulation of EWS/FLI1 target genes when comparing EWS/FLI1 wt with EWS/FLI1 R386N, a DNA binding site mutant completely lacking transcriptional activity. With these systems, we are now in the process to elucidate the importance of individual PTMs of EWS/FLI1. Preliminary results indicate that phosphorylation of the fusion protein may be necessary for full transcriptional activity by increasing its stability. Characterization of PTMs in EWS/FLI1 is therefore expected to lead to the discovery of new target options for the treatment of Ewing sarcoma. Citation Format: Laura A. Lopez Garcia, Maria E. Gierisch, Beat W. Schäfer. Posttranslational modifications of the oncogenic fusion protein EWS/FLI1. [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 A68.

Abstract 764: A role for GLI1 in the development of multidrug resistance in rhabdomyosarcoma (RMS) cells

Abstract RMS is the most common sarcoma of childhood. About 30% of patients with localized tumors will recur following treatment. The outcome for patients with recurrent RMS remains poor. Therefore, development of chemotherapy resistance during RMS therapy represents an important problem and novel approaches to prevent or reverse drug resistance are essential. Activation of multidrug transporter genes, including MDR1, MRP1, LRP and TAP1 represents an important mechanism for drug resistance in RMS. However, the mechanism of expression of multidrug resistance genes in RMS is incompletely understood. Recent reports have suggested a role for the Hedgehog (HH) signaling pathway and its downstream mediator GLI1 in acquisition of a multidrug resistance phenotype in esophageal adenocarcinoma, glioblastoma, and myeloid leukemia. Since approximately 60% of embryonal RMS (ERMS) and more rarely alveolar RMS (ARMS) express HH pathway components, we hypothesized that HH pathway activation may up-regulate the expression of multidrug resistance genes in RMS. We demonstrated expression of HH pathway components, including GLI1, in ERMS (RD, Rh18 and Ruch-2) and ARMS (Rh30 and Rh41) cell lines by qRT PCR and/or Western blot. We found variable expression of MDR1, MRP1, LRP and TAP1 by qRT PCR in these cell lines. Treatment of RMS cells with GLI1 siRNA (RD, Rh18 and Rh41) or the GLI1 antagonist GANT61 (Rh18 and Rh30) caused down-regulation LRP and TAP1 expression. In addition, GLI1, MDR1, LRP and TAP1 expression was increased in vincristine resistant UKF Rhb-1 RMS cells compared with parental cells (obtained from Dr. Cinatl) by qRT PCR. Treatment of vincristine resistant UKF Rhb-1 cells with vincristine together with either GANT61 or Cpd#33 (GLI1 inhibitors), each at a dose, which did not reduce cell viability individually, significantly decreased cell viability by MTT assay. Our results suggest that GLI1 up-regulates a subset of multidrug resistance genes in RMS cells and that GLI1-inhibitors may reduce multidrug resistance. Citation Format: Joon Won Yoon, Marilyn Lamm, King-Fu Leong, Stephen Iannaccone, Philip Iannaccone, David Walterhouse. A role for GLI1 in the development of multidrug resistance in rhabdomyosarcoma (RMS) cells. [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 764. doi:10.1158/1538-7445.AM2014-764

Defective RAGE activity in embryonal rhabdomyosarcoma cells results in high PAX7 levels that sustain migration and invasiveness.

Rhabdomyosarcoma is a muscle-derived malignant tumor mainly affecting children. The most frequent variant, embryonal rhabdomyosarcoma (ERMS) is characterized by overexpression of the transcription factor, PAX7 which prevents ERMS cells from exiting the cell cycle and terminally differentiating. However, a role for PAX7 in the invasive properties of ERMS cells has not been investigated in detail thus far. Here we show that ectopic expression of receptor for advanced glycation end-products (RAGE) in human ERMS cells results in the activation of a RAGE/myogenin axis which downregulates PAX7 by transcriptional and post-translational mechanisms, as in normal myoblasts, and reduces metastasis formation. High PAX7 sustains migration and invasiveness in ERMS cells by upregulating EPHA3 and EFNA1 and downregulating NCAM1 thus decreasing the neural cell adhesion molecule (NCAM)/polysialylated-NCAM ratio. Microarray gene expression analysis shows that compared with the RAGE(-ve) TE671/WT cells and similarly to primary human myoblasts, TE671/RAGE cells show upregulation of genes involved in muscle differentiation and cell adhesion, and downregulation of cell migration related and major histocompatibility complex class I genes. Our data reveal a link between PAX7 and metastasis occurrence in ERMSs, and support a role for the RAGE/myogenin axis in metastasis suppression. Thus, low RAGE expression in ERMS primary tumors may be predictive of metastatic behavior.

PET/MR imaging and optical imaging of metastatic rhabdomyosarcoma in mice.

The combination of PET and MR imaging synergizes molecular and morphologic information, allowing better diagnosis in cancer patients. The diagnosis of tumor recurrence in rhabdomyosarcoma is extremely challenging and could be improved with PET/MR imaging. The aim of this study was to validate PET/MR imaging in a disseminated rhabdomyosarcoma mouse model. One million alveolar (Rh30) and embryonal (RD) rhabdomyosarcoma cells with stably transfected mCherry and Gaussia luciferase were injected intraperitoneally into NOD/LtSz-scid-IL2Rγnull mice. Nine animals were treated with vincristine (0.75 μg/g/d). Tumor growth was monitored on the basis of serum luciferase activity, optical imaging (OI) of the fluorescent protein mCherry, and sequential PET/MR imaging with 3'-deoxy-3'-(18)F-fluorothymidine ((18)F-FLT) and (18)F-FDG. Immunohistochemical Ki-67 and glucose transporter 1 analysis was used to evaluate tumor cell density and proliferative and metabolic activity. The injection of rhabdomyosarcoma cells led to intraperitoneal tumor growth in 34 of 37 mice (Rh30) and 4 of 9 animals (RD). OI revealed inconsistent results for tumors located near the liver. The detection of tumors in the peritoneal cavity was exclusively possible with sequential PET/MR imaging. PET studies with (18)F-FLT MR imaging were more reliable than (18)F-FDG comparing the tracer uptake and correlation with tumor weight. Treatment with vincristine led to reduced tumor growth, which was efficiently detected with (18)F-FDG PET and MR imaging. Total tumor burden as estimated by PET/MR imaging correlated with the serum luciferase activity. We established a unique model of metastatic rhabdomyosarcoma with a high frequency of tumor occurrence and easy monitoring of the tumor growth based on reporter gene expression. The accurate detection of rhabdomyosarcoma requires high soft-tissue contrast provided by the MR imaging and high tracer uptake for PET, which was achieved with (18)F-FLT as the tracer before and (18)F-FDG after treatment with vincristine. PET/MR imaging allows improved diagnosis of experimental rhabdomyosarcoma and therefore might influence clinical therapeutic decisions in the future.

Chemosensitization of rhabdomyosarcoma cells by the histone deacetylase inhibitor SAHA

Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma in childhood with a dismal prognosis, highlighting the need for novel treatment strategies. Here, we identify a novel synthetic lethal interaction between the histone deacetylase inhibitor (HDACI) SAHA and anticancer drugs in RMS cells. Importantly, SAHA significantly increases chemotherapeutic drug-induced apoptosis in both embryonal and alveolar RMS cell lines, including several anticancer agents that are used in the clinic for the treatment of RMS such as Doxorubicin, Etoposide, Vincristine and Cyclophosphamide. Calculation of combination index (CI) reveals that the interaction of SAHA and Doxorubicin or Etoposide is synergistic. Mechanistically, SAHA causes acetylation of histone H3 protein in RMS cells, indicating that SAHA alters the chromatin context. Also, cotreatment with SAHA and Doxorubicin changes the ratio of pro- and antiapoptotic Bcl-2 proteins with downregulation of Mcl-1 and Bcl-xL, dephosphorylation of Bcl-2 and upregulation of BimEL, thus shifting the balance towards apoptosis. Consistently, SAHA and Doxorubicin cooperate to stimulate activation of Bax and Bak, caspase activation and caspase-dependent apoptosis. Overexpression of Bcl-2 significantly rescues SAHA/Doxorubicin-mediated apoptosis, underscoring the requirement of the mitochondrial apoptotic pathway for the synergistic induction of apoptosis by SAHA and Doxorubicin. Caspase-dependent apoptotic cell death is confirmed by the use of the broad-range caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk), which significantly decreases SAHA/Doxorubicin-triggered apoptosis. In conclusion, these findings demonstrate that the HDACI SAHA represents a promising strategy to prime RMS cells for chemotherapy-induced apoptosis and warrants further investigation in combination regimens.

Hyper-Activation of Notch3 Amplifies the Proliferative Potential of Rhabdomyosarcoma Cells

Rhabdomyosarcoma (RMS) is a pediatric myogenic-derived soft tissue sarcoma that includes two major histopathological subtypes: embryonal and alveolar. The majority of alveolar RMS expresses PAX3-FOXO1 fusion oncoprotein, associated with the worst prognosis. RMS cells show myogenic markers expression but are unable to terminally differentiate. The Notch signaling pathway is a master player during myogenesis, with Notch1 activation sustaining myoblast expansion and Notch3 activation inhibiting myoblast fusion and differentiation. Accordingly, Notch1 signaling is up-regulated and activated in embryonal RMS samples and supports the proliferation of tumor cells. However, it is unable to control their differentiation properties. We previously reported that Notch3 is activated in RMS cell lines, of both alveolar and embryonal subtype, and acts by inhibiting differentiation. Moreover, Notch3 depletion reduces PAX3-FOXO1 alveolar RMS tumor growth in vivo. However, whether Notch3 activation also sustains the proliferation of RMS cells remained unclear. To address this question, we forced the expression of the activated form of Notch3, Notch3IC, in the RH30 and RH41 PAX3-FOXO1-positive alveolar and in the RD embryonal RMS cell lines and studied the proliferation of these cells. We show that, in all three cell lines tested, Notch3IC over-expression stimulates in vitro cell proliferation and prevents the effects of pharmacological Notch inhibition. Furthermore, Notch3IC further increases RH30 cell growth in vivo. Interestingly, knockdown of Notch canonical ligands JAG1 or DLL1 in RMS cell lines decreases Notch3 activity and reduces cell proliferation. Finally, the expression of Notch3IC and its target gene HES1 correlates with that of the proliferative marker Ki67 in a small cohort of primary PAX-FOXO1 alveolar RMS samples. These results strongly suggest that high levels of Notch3 activation increase the proliferative potential of RMS cells.

MiR-214 and N-ras regulatory loop suppresses rhabdomyosarcoma cell growth and xenograft tumorigenesis.

Rhabdomyosarcoma (RMS) is a childhood malignant soft tissue cancer that is derived from myogenic progenitors trapped in a permanent mode of growth. Here, we report that miR-214 is markedly down-regulated in human RMS cell lines. Although not required for embryogenesis in mice, miR-214 suppresses mouse embryonic fibroblast (MEF) proliferation. When re-introduced into RD cells, a line of human embryonal RMS cells, miR-214 showed inhibition of tumor cell growth, induction of myogenic differentiation and apoptosis, as well as suppression of colony formation and xenograft tumorigenesis. We show that in the absence of miR-214, expression of proto-oncogene N-ras is markedly elevated in miR-214(-/-) MEFs, and manipulations of miR-214 levels using microRNA mimics or inhibitor in RD cells reciprocally altered N-ras expression. We further demonstrate that forced expression of N-ras from a cDNA that lacks its 3'-untranslated region neutralized the pro-myogenic and anti-proliferative activities of miR-214. Finally, we show that N-ras is a conserved target of miR-214 in its suppression of xenograft tumor growth, and N-ras expression is up-regulated in xenograft tumor models as well as actual human RMS tissue sections. Taken together, these data indicate that miR-214 is a bona fide suppressor of human RMS tumorigensis.

Pharmacological inhibition of EZH2 as a promising differentiation therapy in embryonal RMS

BackgroundEmbryonal Rhabdomyosarcoma (RMS) is a pediatric soft-tissue sarcoma derived from myogenic precursors that is characterized by a good prognosis in patients with localized disease. Conversely, metastatic tumors often relapse, leading to a dismal outcome. The histone methyltransferase EZH2 epigenetically suppresses skeletal muscle differentiation by repressing the transcription of myogenic genes. Moreover, de-regulated EZH2 expression has been extensively implied in human cancers. We have previously shown that EZH2 is aberrantly over-expressed in RMS primary tumors and cell lines. Moreover, it has been recently reported that EZH2 silencing in RD cells, a recurrence-derived embryonal RMS cell line, favors myofiber-like structures formation in a pro-differentiation context. Here we evaluate whether similar effects can be obtained also in the presence of growth factor-supplemented medium (GM), that mimics a pro-proliferative microenvironment, and by pharmacological targeting of EZH2 in RD cells and in RD tumor xenografts.MethodsEmbryonal RMS RD cells were cultured in GM and silenced for EZH2 or treated with either the S-adenosylhomocysteine hydrolase inhibitor 3-deazaneplanocin A (DZNep) that induces EZH2 degradation, or with a new class of catalytic EZH2 inhibitors, MC1948 and MC1945, which block the catalytic activity of EZH2. RD cell proliferation and myogenic differentiation were evaluated both in vitro and in vivo.ResultsHere we show that EZH2 protein was abnormally expressed in 19 out of 19 (100%) embryonal RMS primary tumors and cell lines compared to their normal counterparts. Genetic down-regulation of EZH2 by silencing in GM condition reduced RD cell proliferation up-regulating p21Cip1. It also resulted in myogenic-like differentiation testified by the up-regulation of myogenic markers Myogenin, MCK and MHC. These effects were reverted by enforced over-expression of a murine Ezh2, highlighting an EZH2-specific effect. Pharmacological inhibition of EZH2 using either DZNep or MC inhibitors phenocopied the genetic knockdown of EZH2 preventing cell proliferation and restoring myogenic differentiation both in vitro and in vivo.ConclusionsThese results provide evidence that EZH2 function can be counteracted by pharmacological inhibition in embryonal RMS blocking proliferation even in a pro-proliferative context. They also suggest that this approach could be exploited as a differentiation therapy in adjuvant therapeutic intervention for embryonal RMS.

Bioenergetic properties of human sarcoma cells help define sensitivity to metabolic inhibitors

Sarcomas represent a diverse group of malignancies with distinct molecular and pathological features. A better understanding of the alterations associated with specific sarcoma subtypes is critically important to improve sarcoma treatment. Renewed interest in the metabolic properties of cancer cells has led to an exploration of targeting metabolic dependencies as a therapeutic strategy. In this study, we have characterized key bioenergetic properties of human sarcoma cells in order to identify metabolic vulnerabilities between sarcoma subtypes. We have also investigated the effects of compounds that inhibit glycolysis or mitochondrial respiration, either alone or in combination, and examined relationships between bioenergetic parameters and sensitivity to metabolic inhibitors. Using 2-deoxy-D-glucose (2-DG), a competitive inhibitor of glycolysis, oligomycin, an inhibitor of mitochondrial ATP synthase, and metformin, a widely used anti-diabetes drug and inhibitor of complex I of the mitochondrial respiratory chain, we evaluated the effects of metabolic inhibition on sarcoma cell growth and bioenergetic function. Inhibition of glycolysis by 2-DG effectively reduced the viability of alveolar rhabdomyosarcoma cells vs. embryonal rhabdomyosarcoma, osteosarcoma, and normal cells. Interestingly, inhibitors of mitochondrial respiration did not significantly affect viability, but were able to increase sensitivity of sarcomas to inhibition of glycolysis. Additionally, inhibition of glycolysis significantly reduced intracellular ATP levels, and sensitivity to 2-DG-induced growth inhibition was related to respiratory rates and glycolytic dependency. Our findings demonstrate novel relationships between sarcoma bioenergetics and sensitivity to metabolic inhibitors, and suggest that inhibition of metabolic pathways in sarcomas should be further investigated as a potential therapeutic strategy.

Pan-Mammalian Target of Rapamycin (mTOR) Inhibitor AZD8055 Primes Rhabdomyosarcoma Cells for ABT-737-induced Apoptosis by Down-regulating Mcl-1 Protein

The PI3K/mammalian Target of Rapamycin (mTOR) pathway is often aberrantly activated in rhabdomyosarcoma (RMS) and represents a promising therapeutic target. Recent evaluation of AZD8055, an ATP-competitive mTOR inhibitor, by the Preclinical Pediatric Testing Program showed in vivo antitumor activity against childhood solid tumors, including RMS. Therefore, in the present study, we searched for AZD8055-based combination therapies. Here, we identify a new synergistic lethality of AZD8055 together with ABT-737, a BH3 mimetic that antagonizes Bcl-2, Bcl-xL, and Bcl-w but not Mcl-1. AZD8055 and ABT-737 cooperate to induce apoptosis in alveolar and embryonal RMS cells in a highly synergistic fashion (combination index < 0.2). Synergistic induction of apoptosis by AZD8055 and ABT-737 is confirmed on the molecular level, as AZD8055 and ABT-737 cooperate to trigger loss of mitochondrial membrane potential, activation of caspases, and caspase-dependent apoptosis that is blocked by the pan-caspase inhibitor Z-VAD-fmk. Similar to AZD8055, the PI3K/mTOR inhibitor NVP-BEZ235, the PI3K inhibitor NVP-BKM120 and Akt inhibitor synergize with ABT-737 to trigger apoptosis, whereas no cooperativity is found for the mTOR complex 1 inhibitor RAD001. Interestingly, molecular studies reveal a correlation between the ability of different PI3K/mTOR inhibitors to potentiate ABT-737-induced apoptosis and to suppress Mcl-1 protein levels. Importantly, knockdown of Mcl-1 increases ABT-737-induced apoptosis similar to AZD8055/ABT-737 cotreatment. This indicates that AZD8055-mediated suppression of Mcl-1 protein plays an important role in the synergistic drug interaction. By identifying a novel synergistic interaction of AZD8055 and ABT-737, our findings have important implications for the development of molecular targeted therapies for RMS.

Abstract C152: Characterizing rhabdomyosarcoma and osteosarcoma cellular metabolism.

Abstract Sarcomas represent a diverse group of malignancies with distinct molecular and pathological features. A better understanding of the alterations associated with specific sarcoma subtypes is critically important to improve sarcoma treatment. A renewed interest in the metabolic properties of cancer cells, as well as the recognition of altered cellular metabolism as a critical hallmark of cancer, has led to an exploration of targeting metabolic dependencies of cancer cells as a therapeutic strategy. In this study, we have characterized key bioenergetic properties of human rhabdomyosarcoma and osteosarcoma cell lines in order to identify potential differences in sarcoma subtypes. We have also investigated the effects of compounds that inhibit glycolysis or mitochondrial respiration, either alone or in combination, and examined relationships between bioenergetic parameters and sensitivity to metabolic inhibitors. Glycolytic and mitochondrial bioenergetic profiles were generated using Seahorse extracellular flux analysis to measure extracellular acidification rates and oxygen consumption rates, and significant differences in glycolytic and respiratory bioenergetics between rhabdomyosarcoma and osteosarcoma cell lines were identified. Using 2-deoxy-D-glucose (2-DG), a competitive inhibitor of glycolysis, oligomycin, an inhibitor of mitochondrial ATP synthase, and metformin, a widely-used diabetes drug and inhibitor of complex I of the mitochondrial respiratory chain, we evaluated the effects of metabolic inhibition on sarcoma cell growth and bioenergetic function. Inhibition of glycolysis by 2-DG effectively reduced the viability of alveolar rhabdomyosarcoma cells versus embryonal rhabdomyosarcoma, osteosarcoma, and normal cells, whereas inhibition of mitochondrial respiration by oligomycin or metformin did not significantly affect viability. Combined inhibition of glycolysis and respiration decreased cell growth more effectively than either treatment alone in all sarcoma lines tested. Additionally, inhibition of glycolysis significantly reduced intracellular ATP levels in all sarcoma lines tested, and sensitivity to 2-DG-induced growth inhibition was related to respiratory rates and glycolytic dependency. Our findings suggest that inhibition of metabolic pathways in sarcomas should be further investigated as a potential therapeutic strategy. Funded by NCI Contract No. HHSN261200800001E. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C152. Citation Format: Sameer Issaq, Beverly Teicher, Anne Monks. Characterizing rhabdomyosarcoma and osteosarcoma cellular metabolism. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C152.

TDP1 and PARP1 Deficiency Are Cytotoxic to Rhabdomyosarcoma Cells

Rhabdomyosarcoma is the most common soft tissue sarcoma in children. Metastatic rhabdomyosarcoma in children has a 5-year event-free survival rate of <30%, and a recent clinical trial with irinotecan, a topoisomerase I inhibitor, failed to improve outcome. Therefore, it was surmised that failure of irinotecan may be the result of overexpression of the DNA repair enzyme tyrosyl-DNA phosphodiesterase (TDP1), which processes topoisomerase I-DNA complexes resulting from topoisomerase I inhibitor treatment. Using human tissue microarrays and gene expression arrays, a marked overexpression of TDP1 protein and mRNA in RMS tumors was observed. Critically, knockdown of TDP1 or inhibition of poly(ADP-ribose) polymerase-1 (PARP-1), an enzyme in the same complex as TDP1, sensitized rhabdomyosarcoma cell lines to analogues of irinotecan. Interestingly, BRCA1/2 mutations or altered expression was not detectable in rhabdomyosarcoma cells; however, TDP1 knockdown and PARP-1 inhibition alone were cytotoxic to a subset of rhabdomyosarcoma cells, suggesting that they harbor genetic lesions in DNA repair components that have synthetic lethal interactions with loss of TDP1 or PARP1 function. Furthermore, culturing embryonal rhabdomyosarcoma cells in serum/nutrient-restricted medium increased cellular cytotoxicity upon PARP-1 inhibition and was intrinsically cytotoxic to alveolar, though not embryonal rhabdomyosarcoma cells. The results of these studies suggest a compensatory role for TDP1 in rhabdomyosarcoma after topoisomerase-I based therapy and further demonstrate that TDP1 knockdown, PARP-1 inhibition, and dietary restriction have therapeutic validity. Selective targeting of TDP1 and/or PARP-1 in rhabdomyosarcoma induces cytotoxicity and sensitizes to DNA damaging agents.

Identification of target genes of PAX3-FOXO1 in alveolar rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is a soft tissue sarcoma categorized into two major subtypes: alveolar RMS (ARMS) and embryonal RMS (ERMS). Most ARMS express the PAX3-FOXO1 (P3F) fusion oncoprotein generated by the 2;13 chromosomal translocation. In the present study, the downstream target genes of P3F were identified by analyzing two independent sets of gene expression profiles: primary RMS tumors and RD ERMS cells transduced with inducible P3F constructs. We found 34 potential target genes (27 upregulated and 7 downregulated) that were significantly and differentially expressed between P3F-positive and P3F-negative categories, both in primary RMS tumors and in the inducible P3F cell culture system. Gene ontology analysis of microarray data of the inducible P3F cell culture system employed indicated apoptosis, cell death, development, and signal transduction as overrepresented significant functional categories found in both upregulated and downregulated genes. Therefore, among the 34 potential target genes, the expression of cell death-related [Gremlin1, cysteine knot superfamily 1, BMP antagonist 1 (GREM1) and death-associated protein kinase 1 (DAPK1)] and development-related [myogenic differentiation 1 (MYOD1) and hairy/enhancer-of-split related with YRPW motif 1 (HEY1)] genes were further investigated. The differential expression of GREM1, DAPK1, MYOD1 and HEY1 was confirmed in independent tumors and inducible cell culture systems. The expression of GREM1, DAPK1 and MYOD1 were significantly upregulated; HEY1 was significantly downregulated in independent P3F-positive ARMS tumors and transcriptionally active P3F cells, compared to those in ERMS tumors and transcriptionally inactive P3F cells. This study identified target genes of P3F and suggested that four downstream targets (GREM1, DAPK1, MYOD1 and HEY1) can contribute to the biological activities of P3F involved in growth suppression or cell death and myogenic differentiation.

Integrated analysis of RNAi screens in pediatric rhabdomyosarcoma.

10040 Background: Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma. Embryonal RMS (ERMS) are characterized by 11p15 LOH, while alveolar RMS (ARMS) harbor a translocation between PAX3 or PAX7 and FOXO1. Relapsed or metastatic RMS has a poor prognosis with a 5-year survival rate of 25%. We hypothesized that a reduction in proliferation of ARMS cell lines as result of the loss of function (LOF) of a specific protein via RNAi likely point to novel targets that exhibit “synthetic lethality” with the PAX3/7-FOXO1 translocation, and can be used to reveal unique ARMS vulnerabilities. Methods: We are using two complementary screening strategies to identify genes critical for RMS cell growth and survival. One RNAi screening strategy used an inducible LOF shRNA screen of 15,000 shRNA -5,000 genes- introduced into RH30 (ARMS) or RD (ERMS) cells. Data analysis after subtraction of targets related to common survival pathways revealed 40 genes with close association with RMS cell growth. Follow up analysis included further comparison of the effects of gene specific LOF in ARMS and ERMS cell lines and assessment in orthotopic xenografts. To increase the strength of our findings, we have also conducted parallel siRNA screens of the human kinome targeting 704 kinase genes (3 siRNAs per gene) in RH30 or RD cells. Results: Of the 40 genes identified by shRNA screening, 15 genes were identified as selectively needed for ARMS cell growth. Follow up analysis of one of these genes, TNK-2 (tyrosine kinase, non-receptor 2) in orthotopic xenografts, confirmed that LOF of TNK2 markedly decreases ARMS cell growth. The siRNA screen has identified a further putative 26 genes that selectively reduce ARMS cell growth. These screens have led us to focus on members of the ERK, PI3K and IGF1R pathways as potential targets for the treatment of ARMS. Conclusions: RNAi screening affords an unbiased method for the discovery and elucidation of gene function. Ongoing efforts are focused on the integration of results obtained to gain further insight into the signaling networks influencing alveolar rhabdomyosarcoma biology and thus identify a small group of druggable genes that could represent alveolar rhabdomyosarcoma specific vulnerabilities.

Abstract 2648: Self-renewing cellular compartments in embryonal rhabdomyosarcoma are modulated by Hedgehog signaling.

Abstract Rhabdomyosarcoma (RMS) is a heterogeneous group of malignancies with features of impaired skeletal muscle differentiation and represents the majority of the soft tissue cancers in the pediatric age group. Current treatment regimens offer some clinical respite but at a significant risk of averse side effects and secondary tumors later in life. Identifying and targeting sub-populations with higher tumorigenic potential and self renewal capacity not only decreases tumor burden but also lowers the probability of relapse. Using the sphere formation assay we could select for self-renewing cells from the heterogeneous monolayer cultures of embryonal RMS (eRMS) cell lines, RD and Rh36, which had multipotent differentiation ability in vitro and greater tumorigenic potential in vivo. These cells also showed increased expression of embryonic stem cell genes. Interestingly, quantitative gene expression analysis revealed increased expression of various components of the Hedgehog pathway, a developmental pathway important in eRMS biology. Inhibition or activation of the pathway in vitro by small molecule agents led to increased or decreased self renewing ability respectively. Furthermore we observed that eRMS adherent monolayer cultures contained a sub-population of GLI1-positive cells which could be modulated by pathway manipulation. Importantly, inhibition of the hedgehog pathway in vitro led to significantly delayed tumor initiation in vivo. Ongoing experiments aim at confirming these results on the genetic level. We postulate that the Hedgehog pathway sustains a primitive cellular compartment necessary for tumor initiation within embryonal RMS cell lines. Citation Format: Sampoorna Satheesha, Nurhak Dogan, Elisa A. Casanova, Dagmar Walter, Beat W. Schäfer. Self-renewing cellular compartments in embryonal rhabdomyosarcoma are modulated by Hedgehog signaling. [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 2648. doi:10.1158/1538-7445.AM2013-2648