Rhabdomyosarcoma Tumorigenesis Research Articles

Abstract 5830: A novel Notch-YAP circuit drives stemness and tumorigenesis in embryonal rhabdomyosarcoma

Abstract Introduction: Rhabdomyosarcoma (RMS), a tumor characterized by skeletal muscle features, is the most common soft tissue sarcoma in children and adolescents. The standard of care treatment has not changed in over 40 years, resulting in a stagnant survival rate of less than 30% for high risk groups. Previous studies have identified the developmental pathways Notch and YAP as potent oncogenic signals in the embryonal subtype of RMS (eRMS). Understanding the signaling between Notch and YAP in cancers driven by these pathways, such as eRMS, will be crucial to designing rational combination therapies. Experimental Procedures: For in vitro assays, we utilized 3D sphere cell culture methods that permit the study of stem cell behavior. To analyze signaling, we performed genetic gain and loss-of-function (dox-inducible shRNAs and constitutively active constructs), and pharmacologic inhibition. Target validation was assessed by qPCR and immunoblot, and mechanism was determined by ChIP and limiting dilution assays. For in vivo experiments, we performed subcutaneous xenografts in immunocompromised mice. Immunohistochemistry was used to analyze protein expression in human and murine tumors. Statistical analysis was performed to assess significant differences. Results: We have discovered a novel Notch-YAP circuit that drives cancer cell stemness and tumorigenesis. In eRMS 3D spheres, Notch transcriptionally upregulates YAP and increases its activity, while YAP transcriptionally upregulates the Notch ligands JAG1 and DLL1 and the core Notch transcription factor RBPJ to provide positive feedback. In vivo, YAP suppression decreases Notch signaling, stemness, and proliferation. This circuit boosts expression of the stem cell gene SOX2, which is functionally required for eRMS cell stemness. Inhibiting one node of the circuit, for example through Notch pharmacologic blockade, is not sufficient to shut down the circuit, as stemness and cell growth can be rescued by ectopic YAP. Rather, dual inhibition of Notch and YAP is necessary to silence the circuit’s signaling and sphere growth. Importantly, nuclear Notch and YAP protein expression correlate in eRMS patient tumor samples, suggesting this circuit is intact in human eRMS tumors. Conclusions: While unidirectional signaling between these two pathways has been reported in metazoan development and rarely in human malignancy, this is the first finding of an intact bidirectional circuit between Notch and YAP that supports cancer cell stemness and tumorigenesis. This study provides mechanistic insight into the long-standing question of how eRMS cancer stem cells maintain plasticity. Additionally, while this signaling is identified here in RMS, it could be applicable to many cancer types that are Notch and YAP-dependent. Lastly, this work provides evidence and rationale for research into combination therapies targeting this Notch-YAP signaling circuit. Citation Format: Katherine K. Slemmons, Lisa E.S. Crose, Stefan Riedel, Manuela Sushnitha, Brian Belyea, Corinne M. Linardic. A novel Notch-YAP circuit drives stemness and tumorigenesis in embryonal rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5830. doi:10.1158/1538-7445.AM2017-5830

Abstract 3873: Novel non-genetic mechanisms drive rhabdomyosarcoma tumorigenesis

Abstract Most pediatric solid tumors originate from perturbations during development, however the mechanisms that drive these disruptions and promote tumor progression are still poorly understood. We believe that epigenetic deregulation of cancer genes and developmental pathways is a common driver in pediatric cancers, especially those with a low mutation rate. To test this novel idea, we are focusing on rhabdomyosarcoma (RMS), a developing muscle tumor with striking genetic differences between two of the major subtypes. We hypothesize that differential epigenetic deregulation of differentiation programs and cancer genes between the major subtypes are essential for tumorigenesis. Indeed, our preliminary integrative analysis reveal distinct epigenetic landscapes between the two subtypes particularly at the loci of HOX cluster genes A-C, including an oncogenic HOXC long non-coding RNA (lncRNA) called HOTAIR. Knockdown of HOTAIR led to derepression of HOXD10, a candidate tumor suppressor, and a decrease in RMS proliferation, migration, and invasion. Thus, we believe HOX gene deregulation is required for tumorigenesis, and their implication in several adult cancers suggests this currently unappreciated mechanism is likely to be more broadly relevant. In addition, we identified a group of RMS specific novel lncRNAs, of which one demonstrates an essential role in cell survival. Our innovative approach to integrate epigenetic data sets from tumor types with varying genetic differences shed light on the mechanisms that drive tumorigenesis, which may lead to identifying previously unknown potential therapeutic targets. Since current therapeutic options have not changed over the last twenty-five years, these data will especially provide a strong translational impact for children with RMS, whose survival outcomes are less than 20% in the case of metastatic disease. Note: This abstract was not presented at the meeting. Citation Format: Justina McEvoy, Michael Dyer, Xiang Chen. Novel non-genetic mechanisms drive rhabdomyosarcoma tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3873. doi:10.1158/1538-7445.AM2017-3873

Novel Roles for Staufen1 in Embryonal and Alveolar Rhabdomyosarcoma via c-myc-dependent and -independent events

Rhabdomyosarcoma is the most common soft tissue sarcoma in children and young adults. Rhabdomyosarcomas are skeletal muscle-like tumours that typically arise in muscle beds, and express key myogenic regulatory factors. However, their developmental program remains blocked in the proliferative phase with cells unable to exit the cell cycle to fuse into myotubes. Recently, we uncovered a key role for the RNA-binding protein Staufen1 during myogenic differentiation through the regulation of c-myc translation. Given the known implication of c-myc in rhabdomyosarcoma, we hypothesized in the current work that Staufen1 controls rhabdomyosarcoma tumorigenesis. Here, we report for the first time the novel role of Staufen1 in cancer, specifically in rhabdomyosarcoma. We demonstrate that Staufen1 is markedly upregulated in human rhabdomyosarcoma tumours and cell lines as compared to normal skeletal muscle. Moreover, we show that Staufen1 promotes the tumorigenesis of embryonal and alveolar rhabdomyosarcoma subtypes both in cell culture and in animal models. Finally, our data demonstrate that Staufen1 has differential roles in embryonal versus alveolar rhabdomyosarcoma through the control of proliferative and apoptotic pathways, respectively. Together, these results provide the first evidence for Staufen1’s direct implication in cancer biology. Accordingly, Staufen1 thus represents a novel target for the development of future therapeutic strategies for rhabdomyosarcoma.

Identification of tumor-initiating cells derived from two canine rhabdomyosarcoma cell lines.

Cancer stem cells or tumor-initiating cells (TICs) are a small subpopulation of cells that have the capacity to self-renew, differentiate and initiate tumors. These cells may function in tumor initiation, aggression and recurrence. Whether spheres derived from canine rhabdomyosarcoma cells have stem cell-like properties is unclear. We induced sphere formation in the canine rhabdomyosarcoma cell lines, CMS-C and CMS-J, and characterized the spheres in vitro and in vivo. Sphere-forming cells were more resistant to vincristine, mitoxantrone and doxorubicin than adherent cells. Xenograft transplantation demonstrated that 1 × 103 sphere-forming cells derived from CMS-C were sufficient for tumor formation. The sphere assay showed that the sphere-forming cells were present in these tumors. These results suggest that the spheres derived from canine rhabdomyosarcoma cells may possess characteristics of TICs. This study provides the foundation for elucidating the contribution of TICs to rhabdomyosarcoma tumorigenesis.

Interfering with Hedgehog Pathway: New Avenues for Targeted Therapy in Rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most frequent pediatric soft-tissue tumor accounting for about 7% of childhood malignancies. Multimodal therapy is the standard treatment for individuals with RMS but generally fails to cure high-risk group patients and can result in long-term side effects. Therefore, understanding the mechanisms driving RMS might help to find new candidate targets for more specific and effective therapeutic modalities. One of the molecular machineries which is often deregulated in cancer and specifically involved in tumorigenesis of RMS, is Hedgehog (Hh) signaling. There is increasing evidence that targeting this developmental pathway may hold promise in future treatment strategies for RMS. In this review, we discuss the contribution of the Hh pathway in RMS, the challenges of inhibiting this embryonic signaling in children with an update on recent preclinical data and ongoing clinical trials.

Wnt Signaling in Rhabdomyosarcoma - A Potential Targeted Therapy Option.

Rhabdomyosarcoma (RMS) is the most common soft tissue malignancy in the pediatric population and recapitulates many of the phenotypic and biological features of embryonic skeletal muscle. RMS is pathologically characterized by myogenic differentiation arrest. Dysregulation of pathways essential for myogenesis like contribute to the tumorigenesis of RMS. The Wnt signaling pathway is one of the major pathways required for normal myogenesis. Aberrant Wnt signaling has also been demonstrated in many types of cancer. The role of Wnt pathway in the pathogenesis of RMS is beginning to be appreciated. This review will go over current knowledge on biological roles of Wnt pathway in RMS and potential translational application in therapeutics.

FoxF1 and FoxF2 transcription factors synergistically promote rhabdomyosarcoma carcinogenesis by repressing transcription of p21Cip1 CDK inhibitor.

The role of Forkhead Box F1 (FoxF1) transcription factor in carcinogenesis is not well characterized. Depending on tissue and histological type of cancer, FoxF1 was shown to be either oncogene or tumor suppressor. Alveolar rhabdomyosarcoma (RMS) is the most aggressive pediatric soft tissue sarcoma. While FoxF1 is highly expressed in alveolar RMS, the functional role of FoxF1 in RMS is unknown. The present study demonstrates that expression of FoxF1 and its closely related transcription factor FoxF2 are essential for rhabdomyosarcoma tumor growth. Depletion of FoxF1 or FoxF2 in rhabdomyosarcoma cells decreased tumor growth in orthotopic mouse models of RMS. The decreased tumorigenesis was associated with the reduced tumor cell proliferation. Cell cycle regulatory proteins Cdk2, Cdk4/6, Cyclin D1 and Cyclin E2 were decreased in FoxF1- and FoxF2-deficient RMS tumors. Depletion of either FoxF1 or FoxF2 delayed G1-S cell cycle progression, decreased levels of phosphorylated Rb and increased protein levels of the CDK inhibitors, p21Cip1 and p27Kip1. Depletion of both FoxF1 and FoxF2 in tumor cells completely abrogated RMS tumor growth in mice. Overexpression of either FoxF1 or FoxF2 in tumor cells was sufficient to increase carcinogenesis in orthotopic RMS mouse model. FoxF1 and FoxF2 directly bound to and repressed transcriptional activity of p21Cip1 promoter through −556/−545 bp region, but did not affect p27Kip1 transcription. Knockdown of p21Cip1 restored cell cycle progression in the FoxF1- or FoxF2-deficient tumor cells. Altogether, FoxF1 and FoxF2 promoted RMS tumorigenesis by inducing tumor cell proliferation via transcriptional repression of p21Cip1 gene promoter. Due to robust oncogenic activity in RMS tumors, FoxF1 and FoxF2 may represent promising targets for anti-tumor therapy.

Abstract 2440: FEM1A regulation of the NF-κB/YY1/miR-29 circuitry in rhabdomyosarcoma

Abstract Introduction: Rhabdomyosarcoma (RMS), a malignant tumor related to defective skeletal muscle differentiation, is the most common soft tissue neoplasm of children, and metastatic RMS continues to have a poor prognosis. The canonical form of transcription factor NF-κB, a p50/p65 heterodimer, regulates normal myogenic differentiation, and is dysregulated in RMS tumorigenesis. NF-κB is active in myoblasts and blocks myogenic differentiation; for myogenic differentiation to occur, NF-κB must be down-regulated, which leads to down-regulation of YY1 and de-repression of miR-29, a microRNA that promotes myogenic differentiation. Conversely, NF-κB is up-regulated in RMS, leading to activation of YY1, down-regulation of miR-29, and suppression of myogenic differentiation. The mechanisms of NF-κB regulation in these contexts are incompletely understood. FEM1A is a gene most highly expressed in cardiac and skeletal muscle. We have shown that FEM1A is up-regulated early during skeletal muscle differentiation, and is consistently down-regulated in RMS, both in human RMS and in mouse RMS genetic models. However, whether down-regulation of FEM1A plays a role in RMS tumorigenesis, or is just a marker of defective myogenic differentiation, is unknown. In macrophages, FEM1A has been shown to inhibit canonical NF-κB, both by directly binding p50, and by binding and preventing the degradation of p105, the precursor of p50 that also acts as a cytoplasmic inhibitor of NF-κB activation. Therefore, we tested the hypothesis that over-expression of FEM1A in RMS cells decreases NF-κB activation, which in turn results in decreased expression of YY1, leading to increased expression of miR-29, promoting RMS myogenic differentiation. Methods: Cultured RMS13 rhabdomyosarcoma cells were used. To determine if FEM1A regulates NF-κB/YY1/miR-29 circuitry, resulting in RMS13 differentiation, we transfected RMS13 cells with HA-FEM1A using X-treme Gene HP (Roche). Results: Using immunoblotting, we observed that FEM1A overexpression decreases p50 and p65 translocation to the nucleus, decreases YY1 protein levels, and increases myosin heavy chain expression, a marker of skeletal muscle differentiation, in RMS13 cells. Using qPCR, we observed that FEM1A overexpression increases miR-29 gene expression in RMS13 cells. Conclusion: These data demonstrate that FEM1A negatively modulates NF-κB and YY1, and promotes miR-29 expression and myogenic differentiation, in RMS cells. This indicates that FEM1A down-regulation may play a role in RMS tumorigenesis, rather than just being a marker of defective differentiation. Results from our study have the potential to improve understanding of RMS tumor biology, and could allow the FEM1A/NF-κB/YY1/miR-29 circuitry to become a target for molecular-based therapy of RMS. Citation Format: Samira C. Grifoni, Joseph F. Maher. FEM1A regulation of the NF-κB/YY1/miR-29 circuitry in rhabdomyosarcoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2440.

Abstract 2461: Rhabdomyosarcoma tumors recur by a PAX3-FOXO1-independent mechanism in a human myoblast xenograft model

Abstract The PAX3-FOXO1 fusion gene is generated by a 2;13 chromosomal translocation, and is a characteristic feature of a subset of rhabdomyosarcoma (RMS) with aggressive behavior and poor prognosis. This study utilizes an inducible expression system in human myoblasts to dissect the molecular mechanism of PAX3-FOXO1 action during RMS tumorigenesis and progression.Constitutive and doxycycline-inducible expression constructs were used to generate a human myoblast cell line that constitutively expresses MYCN and reversibly expresses PAX3-FOXO1, respectively. PAX3-FOXO1 expression was assessed by Western blotting, qRT-PCR and immunohistochemistry. Oncogenicity in these engineered myoblasts was studied in vitro by focus formation and in vivo by intramuscular injection of NOD-SCID mice. Cell proliferation, apoptosis and myogenic differentiation were assessed by western blot or immunohistochemical assays.In focus formation assays, doxycycline-treated myoblasts expressing constitutive MYCN and doxycycline inducible PAX3-FOXO1 showed a high level of oncogenic transformation. When doxycycline was removed during the course of this assay, smaller foci formed with prominent myogenic differentiation and cell death. Intramuscular injection of these engineered myoblasts resulted in RMS tumor formation when fusion protein expression was induced by feeding mice a doxycycline-supplemented diet. After small palpable tumors formed, doxycycline withdrawal resulted in decreased PAX3-FOXO1 expression and tumor regression. Microscopic examination of regressing tumors revealed widespread myogenic differentiation and cell death. In most cases, the tumors recurred several weeks later despite the absence of inducing agent. Analysis of recurrent tumor samples revealed that a subset emerged in the absence of PAX3-FOXO1 expression. A cell line generated from a PAX3-FOXO1-independent recurrence demonstrated transformation in vitro in the absence of doxycycline. Though cell lines derived from primary tumors were dependent on PAX3-FOXO1 and differentiated when doxycycline was removed, the recurrent tumor-derived cells did not differentiate under these conditions and instead proliferated continuously. Furthermore, reinjection of these recurrent tumor-derived cells resulted in tumor formation in mice in the absence of doxycycline, and even more rapid tumor formation in the presence of doxycycline.Our study provides evidence that the PAX3-FOXO1 fusion protein is necessary to develop primary tumors but recurrent tumors can develop by a PAX3-FOXO1-independent mechanism. The recurrent tumors are postulated to have acquired secondary oncogenic events that were activated or selected by initial PAX3-FOXO1 expression. These secondary events have an additive oncogenic effect with PAX3-FOXO1 expression, and then contribute to tumor recurrence in the absence of PAX3-FOXO1 expression. Citation Format: Puspa R. Pandey, Bishwanath Chatterjee, Javed Khan, Stephen M. Hewitt, Markku Martti Miettinen, Frederic G. Barr. Rhabdomyosarcoma tumors recur by a PAX3-FOXO1-independent mechanism in a human myoblast xenograft model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2461.

Targeting Focal Adhesion Kinase Suppresses the Malignant Phenotype in Rhabdomyosarcoma Cells

Despite the tremendous advances in the treatment of childhood solid tumors, rhabdomyosarcoma (RMS) continues to provide a therapeutic challenge. Children with metastatic or relapsed disease have a disease-free survival rate under 30%. Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase that is important in many facets of tumorigenesis. Signaling pathways both upstream and downstream to FAK have been found to be important in sarcoma tumorigenesis, leading us to hypothesize that FAK would be present in RMS and would impact cellular survival. In the current study, we showed that FAK was present and phosphorylated in pediatric alveolar and embryonal RMS tumor specimens and cell lines. We also examined the effects of FAK inhibition upon two RMS cell lines utilizing parallel approaches including RNAi and small molecule inhibitors. FAK inhibition resulted in decreased cellular survival, invasion, and migration and increased apoptosis. Furthermore, small molecule inhibition of FAK led to decreased tumor growth in a nude mouse RMS xenograft model. The findings from this study will help to further our understanding of the regulation of tumorigenesis in RMS and may provide desperately needed novel therapeutic strategies for these difficult-to-treat tumors.

Role of the YAP Oncoprotein in Priming Ras-Driven Rhabdomyosarcoma.

Rhabdomyosarcoma (RMS), a cancer characterized by features of skeletal muscle histogenesis, is the most common soft tissue sarcoma of childhood and adolescence. Survival for high-risk groups is less than 30% at 5 years. RMS also occurs during adulthood, with a lower incidence but higher mortality. Recently, mutational profiling has revealed a correlation between activating Ras mutations in the embryonal (eRMS) and pleomorphic (pRMS) histologic variants of RMS, and a poorer outcome for those patients. Independently, the YAP transcriptional coactivator, an oncoprotein kept in check by the Hippo tumor suppressor pathway, is upregulated in eRMS. Here we show that YAP promotes cell proliferation and antagonizes apoptosis and myogenic differentiation of human RMS cells bearing oncogenic Ras mutations in cell culture studies in vitro and in murine xenografts in vivo. Pharmacologic inhibition of YAP by the benzoporphyrin derivative verteporfin decreased cell proliferation and tumor growth in vivo. To interrogate the temporal contribution of YAP in eRMS tumorigenesis, we used a primary human cell-based genetic model of Ras-driven RMS. Constitutively active YAP functioned as an early genetic lesion, permitting bypass of senescence and priming myoblasts to tolerate subsequent expression of hTERT and oncogenic Ras, which were necessary and sufficient to generate murine xenograft tumors mimicking RMS in vivo. This work provides evidence for cooperation between YAP and oncogenic Ras in RMS tumorigenesis, laying the foundation for preclinical co-targeting of these pathways.

Abstract 3273: PAX3-FOXO1 is essential for initiation but not for recurrence during rhabdomyosarcoma tumorigenesis

Abstract The PAX3-FOXO1 fusion gene, which is generated by a 2; 13 chromosomal translocation, is a characteristic feature of fusion-positive rhabdomyosarcoma (RMS), a major RMS subtype associated with aggressive behavior and poor prognosis. This study utilizes a novel inducible expression system in human myoblasts to dissect the molecular mechanism and contribution of PAX3-FOXO1 in RMS tumorigenesis.A human myoblast cell line (immortalized by BMI1 and TERT expression) was transduced with a retroviral construct that constitutively expresses MYCN and a lentiviral based- doxycycline inducible construct that variably and reversibly expresses PAX3-FOXO1. Focus formation and animal xenograft experiments were performed to study transformation in vitro and tumorigenesis in vivo, respectively. Myogenic differentiation was assessed by light microscopy and by western blot or immunohistochemical assays of muscle-specific protein expression.PAX3-FOXO1-transduced myoblasts treated with doxycycline demonstrated a time- and dose-dependent increase in expression of PAX3-FOXO1 mRNA and protein and its downstream targets genes; doxycycline withdrawal led to cessation of fusion protein expression. Though myoblasts expressing PAX3-FOXO1 or MYCN alone did not show any evidence of transformation in culture, combined PAX3-FOXO1 and MYCN expression resulted in myoblast transformation. Under differentiation-promoting culture conditions, combined PAX3-FOXO1 and MYCN expression inhibited myogenic differentiation. Intramuscular injection of myoblasts with MYCN and PAX3-FOXO1 resulted in rapid RMS tumor formation in NOD-SCID mice when fusion protein expression was induced by feeding mice a doxycycline-containing diet. Myoblasts with MYCN expression alone did not form any tumors while PAX3-FOXO1 induction without MYCN expression resulted in RMS tumors only after a much longer latency period. After tumors formed from myoblasts expressing PAX3-FOXO1 with or without MYCN, down-regulation of PAX3-FOXO1 expression by doxycycline withdrawal resulted in tumor regression associated with widespread myogenic differentiation. However, the regressed tumors slowly grew back in the absence of doxycycline induction demonstrating a PAX3-FOXO1-independent oncogenic mechanism for recurrence.The PAX3-FOXO1 fusion protein collaborated with MYCN in the initial stage of RMS tumorigenesis to promote dysregulated cell proliferation and inhibit myogenic differentiation. Though most cells in the initial tumor were dependent on the fusion protein, recurrent tumors formed in which the fusion protein was not required to maintain the tumorigenic phenotype. Citation Format: Puspa R. Pandey, Stephen M. Hewitt, Markku M. Miettinen, Frederic G. Barr. PAX3-FOXO1 is essential for initiation but not for recurrence during rhabdomyosarcoma tumorigenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3273. doi:10.1158/1538-7445.AM2015-3273

Abstract 2102: TBX2 promotes rhabdomyosarcoma (RMS) tumorigenesis by repression of PTEN expression

Abstract RMS is most common soft tissue sarcoma in children. We previously identified TBX2 as an oncogene and showed that TBX2 is required for tumor formation. Here, we identified the tumor suppressor PTEN as a novel target of TBX2 in RMS and normal muscle. PTEN, which regulates the PI3K-AKT pathway, is repressed in RMS. Our work shows that TBX2 functions to regulate PTEN expression and thus, PI3K-AKT signaling, to enhance the malignancy of RMS tumors. Both quantitative real time PCR and western blot assays were used to assay PTEN expression at the level of mRNA and protein respectively. Immunofluorescence staining was performed to assay the protein expression in human RMS tumor samples. Muscle tissue slides of proliferating myoblasts were assayed by co-immunofluorescence staining, and the proliferating myoblasts were induced by activating satellite cells with cardiotoxin induced muscle damage. Reporter constructs for luciferase assay were based on PTEN promoter. Chromatin immunoprecipitation (ChIP) assays were used to detect protein binding on gene promoters. Xenograft assays were conducted to assay TBX2 function in tumor malignancy. We have found that PTEN was up-regulated upon muscle differentiation, but little expression of PTEN was observed in RMS cells. Although TBX2 was expressed in proliferating myoblasts, it was highly down-regulated upon myogenic differentiation. Human RMS tumor samples showed a down-regulation of PTEN and an up-regulation of TBX2 compared with normal tissues. Over-expression of TBX2 repressed PTEN expression in both C2C12 myoblasts and 10T1/2 fibroblasts. Either depletion of TBX2 or a dominant negative TBX2 increased PTEN expression in RMS cells. Luciferase assays confirmed the regulation of PTEN by TBX2 and ChIP assays demonstrated that TBX2 recruited HDAC1 to the PTEN promoter for repression. The role of HDAC1 in repression of PTEN was confirmed by treatment with HDAC inhibitors, which abrogated the repression of PTEN by TBX2. This work demonstrates that the high expression of TBX2 in RMS is involved in the activation of PI3K-AKT signaling pathway by repressing PTEN, underlying the oncogenic function of TBX2 in tumor cells. Citation Format: Bo K. Zhu, Judith K. Davie. TBX2 promotes rhabdomyosarcoma (RMS) tumorigenesis by repression of PTEN expression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2102. doi:10.1158/1538-7445.AM2015-2102

Abstract 3278: Distinct methylation profiles characterize fusion-positive and fusion-negative rhabdomyosarcoma

Abstract BACKGROUND: Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of childhood. Based on the presence or absence of a characteristic fusion gene (either PAX3-FOXO1 or PAX7-FOXO1), RMS comprises two major subtypes: fusion-positive (FP) and fusion-negative (FN). The goal of this study is to analyze DNA methylation differences between FP and FN tumors and to determine how these methylation differences contribute to biological differences between FP and FN RMS. METHODS: DNA methylation profiles of 37 RMS tumors (20 FP and 17 FN) and 10 RMS cell lines (5 FP and 5 FN) as well as 8 normal tissues (4 muscles and 4 bone marrows) were assessed using DNA methylation arrays. Expression array analysis was performed on 58 RMS tumors (33 FP and 25 FN, including 16 with matched methylation data). Bioinformatic methods including classification, differential methylation, differential expression, methylation-expression correlation and gene set analysis were carried out. RESULTS: Unsupervised clustering clearly distinguished the FP and FN subsets in 37 RMS primary tumors and in 10 RMS cell lines. The FP tumors showed substantially lower overall levels of methylation compared to FN tumors. Integrative methylation and gene expression analysis revealed that methylation differences between FP and FN tumors could either be positively or negatively associated with mRNA expression. Though there was no significant difference in the distribution of PAX3-FOXO1 binding sites between genes with and without differential methylation, PAX3-FOXO1 binding sites were enriched among genes that were both differentially methylated and differentially expressed. An 11-gene DNA methylation signature, classifying the RMS tumors into FP and FN subsets, was established in 37 RMS primary tumors and validated in 10 RMS cell lines. Pyrosequencing studies of 6 of the methylation signature genes independently confirmed differential methylation between FP and FN cases for the CpG site queried on the methylation array as well as other nearby CpG sites. To further investigate the relationship between DNA methylation and expression, we focused on the EMILIN1 gene, which shows higher methylation and lower mRNA expression in FP tumors compared to FN tumors. After treating RMS cell lines with 5-Aza-2′-deoxycytidine, the EMILIN1 gene demonstrated decreased DNA methylation and increased mRNA expression in multiple FP cell lines. CONCLUSION: FP and FN RMS tumors possess characteristic methylation profiles that contribute to the expression differences between these fusion subtypes. These findings indicate an important role of epigenetics in RMS tumorigenesis, suggest novel approaches for identifying fusion status, and may find new therapeutic targets in RMS. Citation Format: Wenyue Sun, Bishwanath Chatterjee, Yonghong Wang, Holly S. Stevenson, Daniel C. Edelman, Paul S. Meltzer, Frederic G. Barr. Distinct methylation profiles characterize fusion-positive and fusion-negative rhabdomyosarcoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3278. doi:10.1158/1538-7445.AM2015-3278

Clonality and evolutionary history of rhabdomyosarcoma.

To infer the subclonality of rhabdomyosarcoma (RMS) and predict the temporal order of genetic events for the tumorigenic process, and to identify novel drivers, we applied a systematic method that takes into account germline and somatic alterations in 44 tumor-normal RMS pairs using deep whole-genome sequencing. Intriguingly, we find that loss of heterozygosity of 11p15.5 and mutations in RAS pathway genes occur early in the evolutionary history of the PAX-fusion-negative-RMS (PFN-RMS) subtype. We discover several early mutations in non-RAS mutated samples and predict them to be drivers in PFN-RMS including recurrent mutation of PKN1. In contrast, we find that PAX-fusion-positive (PFP) subtype tumors have undergone whole-genome duplication in the late stage of cancer evolutionary history and have acquired fewer mutations and subclones than PFN-RMS. Moreover we predict that the PAX3-FOXO1 fusion event occurs earlier than the whole genome duplication. Our findings provide information critical to the understanding of tumorigenesis of RMS.

Abstract A57: The secreted Wnt inhibitor SFRP3 is required for PAX3-FOXO1-positive alveolar rhabdomyosarcoma tumorigenesis

Abstract Introduction: This study aims to understand the contribution of the Wnt pathway inhibitor secreted frizzled related protein 3 (SFRP3) to alveolar rhabdomyosarcoma tumorigenesis. Rhabdomyosarcoma is the most common pediatric soft tissue sarcoma and demonstrates features of skeletal muscle. Of the two predominant (pediatric) subtypes, embryonal (eRMS) and alveolar (aRMS), aRMS has the poorer prognosis, with a 5-year survival rate of <30%. The majority of aRMS tumors express the fusion protein PAX3/7-FOXO1. As PAX3/7-FOXO1 is not currently druggable, our goal is to identify proteins that are downstream from or cooperate with PAX3-FOXO1 (PF) to enable tumorigenesis with the hope that these proteins may be more amenable to pharmacological inhibition. Experimental procedures: Microarray analysis using the Affymetrix U133A platform was performed to compare the transcriptomes of human skeletal muscle (HSMM) cells stably expressing either an empty vector or PF. Only genes that were differentially regulated at least three-fold were considered for further study. The microarray was validated and SFRP3 mRNA levels were assessed using RT-PCR. Lentiviral technology was used to stably express shRNAs in human PF-positive aRMS cell lines Rh28 and Rh30. Cell growth, cell proliferation, apoptosis, and cell cycle analysis were measured by MTT assay, BrdU assay, immunoblot for cleaved caspase 3, and by PI staining followed by analysis by flow cytometry, respectively. For the xenograft tumors in mice, doxycycline-inducible SFRP3 shRNAs were stably expressed in aRMS cells. These cells were injected into the flanks of SCID/beige mice to generate the tumors. Induction of the shRNAs by doxycycline administration began once the tumors were palpable. All animal work was performed under institution approved IACUC protocols. Results: In a microarray analysis of the transcriptomes of human skeletal muscle myoblasts (HSMM) with an empty vector or PF, it was noted that the Wnt pathway was preferentially represented in HSMMs with PF. Of these, SFRP3 was selected for further investigation as its mRNA levels were elevated in PF-positive aRMS cells, but not eRMS or HSMM cells. shRNAs against SFRP3 were generated and stably expressed in Rh28 and Rh30 cells, validated to suppress SFRP3, and shown to profoundly decrease cell growth (p<0.001). Associated with this decreased cell growth was a decrease in cell proliferation (p<0.001), an increase in apoptosis, and an increase in the percentage of cells at G1, suggesting a cell cycle arrest. Consistent with a G1 cell cycle arrest, shRNAs against SFRP3 also increased the levels of p21, an inhibitor of cell cycle progression. To assess the effects of SFRP3 shRNAs on tumor growth in a xenograft model of aRMS, a doxycycline-inducible system was used, allowing the shRNAs to be expressed only after the tumors formed. In this model, suppression of SFRP3 reduced tumor growth and tumor weight by more than three-fold. Further, in a pilot study, the combination of SFRP3 suppression with vincristine, a standard chemotherapeutic agent used in the treatment of aRMS, was more effective at reducing cell growth in vitro than with either treatment alone and ablated tumor growth in vivo. Conclusions: In conclusion, SFRP3 is necessary for the growth of aRMS cells both in vitro and in vivo. Inhibition of SFRP3 combined with standard of care chemotherapy should be explored further as SFRP3 is a promising new target in the treatment of PF-positive aRMS. Citation Format: Julie Kephart, Rosanne Jones, Lisa Crose, Jen-Tsan Ashley Chi, Corinne Linardic. The secreted Wnt inhibitor SFRP3 is required for PAX3-FOXO1-positive alveolar rhabdomyosarcoma tumorigenesis. [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 A57.

PAX3-NCOA2 fusion gene has a dual role in promoting the proliferation and inhibiting the myogenic differentiation of rhabdomyosarcoma cells

We analyzed a complex chromosomal translocation in a case of embryonal rhabdomyosarcoma (RMS) and showed that it generates the fusion gene PAX3 (paired box 3)-NCOA2 (nuclear receptor coactivator 2). To understand the role of this translocation in RMS tumorigenesis, we established two types of stable mouse myoblast C2C12 cell lines expressing PAX3-NCOA2 and PAX3-FOXO1A (forkhead box O1A), respectively. Compared with control cells, PAX3-NCOA2 cells grew faster, were more motile, were less anchorage dependent, progressed more quickly through the G1/S phase of cell cycle and showed greater transcriptional activation of the PAX3 consensus-binding site. However, PAX3-NCOA2 cells proliferated more slowly and differentiated more weakly than did PAX3-FOXO1A cells. Both PAX3-NCOA2 cells and PAX3-FOXO1A cells formed tumors in nude mice, although the PAX3-NCOA2-induced tumors grew more slowly. Our results may explain why NCOA2 rearrangement is mainly found in embryonal rhabdomyosarcoma, which has a better prognosis than alveolar rhabdomyosarcoma, which expresses the PAX3-FOXO1A fusion gene. These results indicate that the PAX3-NCOA2 fusion gene has a dual role in the tumorigenesis of RMS: promotion of the proliferation and inhibition of the myogenic differentiation of RMS cells.

Abstract 2630: Tumor suppressor CASZ1 inhibits normal myoblast and rhabdomyosarcoma (RMS) migration.

Abstract Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of childhood, and is thought to arise from primitive mesenchymal cells committed to skeletal muscle differentiation. Less than 30% of patients with metastatic disease survive even with the most intensive treatment regimens. Approximately 50% of patients with RMS have intermediate risk and the event-free survival of the intermediate risk patients is approximately 70%. However, even within this group, the survival of patients with metastatic embryonal tumors is much poorer (∼50%). Thus understanding the molecular mechanism of RMS metastasis and the development of therapeutic strategies to inhibit RMS metastasis is critical for these patients. We have reported that zinc finger transcription factor CASZ1 functions as a tumor suppressor gene in neuroblastoma. In these studies we found that CASZ1 regulates genes involved in cell movement and many pertaining to skeletal/muscle system development. To understand whether the induction of muscle developmental genes by CASZ1 was an anomaly associated with pathology, we evaluated the expression and regulation of CASZ1 in normal myoblasts. CASZ1 expression significantly increased when the C2C12 murine myoblast cell line differentiated upon serum withdrawal. Knockdown of CASZ1 in myoblasts causes a 30% increase in cell migration (p<0.02) as determined using an Essen IncuCyte scratch-wound healing assay. Furthermore knock-down of CASZ1 in C2C12 cells leads to a 50% decrease in expression of the skeletal muscle development gene myogenin while over-expression of CASZ1 leads to 2-fold increase in myogenin mRNA. Since CASZ1 regulates aspects of normal muscle development, we hypothesized that its dysregulation in RMS may impact tumor progression. To test this hypothesis, we first evaluated the expression of CASZ1 in primary RMS tumors. We found that CASZ1 mRNA level in embryonal RMS patients’ samples is ∼1.5 lower than in normal muscle or alveolar RMS (p<0.001). To investigate the role of CASZ1 in embryonal RMS cell growth and migration, we developed a Tet-On CASZ1 expression system using the RD RMS cell line. Overexpression of CASZ1 does not affect RD cell proliferation based on MTS and IncuCyte cell confluence assays. Over-expression of CASZ reduces cell motility by 20% (p<0.01) using an Essen IncuCyte scratch-wound healing assay. Knockdown of CASZ1 in RD cells increases cell migration by 40% (p<0.01) but does not affect cell proliferation. However when CASZ1 is expressed in the presence of a muscle inducing differentiation cue, 12-O-tetradecanoylphorbol-13-acetate, TPA, the proliferation of RD cells is suppressed an additional 40% (p<0.01). Taken together, our results suggest that regulation of myogenin expression and cell migration by CASZ1 in C2C12 maybe integral to proper myogenic differentiation, and dysregulation of CASZ1 during early myogenesis may contribute to embryonal RMS tumorigenesis and metastasis. Citation Format: Zhihui Liu, Wilson Chan, Carol J. Thiele. Tumor suppressor CASZ1 inhibits normal myoblast and rhabdomyosarcoma (RMS) migration. [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 2630. doi:10.1158/1538-7445.AM2013-2630 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Abstract 2535: Regulation of Chk1 by the mTOR pathway is essential for cancer cells to complete DNA replication in response to replication stress

Abstract Oncogenic signaling leads to DNA replication stress, DNA damage and consequently DNA damage response (DDR), a barrier for tumorigenesis. For survival and proliferation, cancer cells must overcome this oncogene-induced DDR, but the molecular mechanism by which this is achieved is largely unknown. Here we report that the mTOR pathway prevents either spontaneous or drug induced DNA damage by sustaining ATR-Chk1 checkpoint in pediatric rhabdomyosarcoma (RMS) both in vivo and in vitro. Pharmacological inhibition of mTOR signaling by the selective TOR kinase inhibitor AZD8055 lead to spontaneous DNA damage and apoptosis as demonstrated by the induction of H2AX phosphorylation and PARP1 cleavage both in cell culture and tumor xenografts of RMS. Treatment RMS Rh30 cells with AZD8055 enhanced hydroxyurea induced H2AX phosphorylation and attenuated ATR-Chk1 checkpoint activation, as shown by decreased levels of phospho-Chk1(S345). We further found that mTOR signaling controls CHK1 expression at the transcriptional level via enhancing the activity of cyclin dependent kinases (CDKs). Consistent with its essential role for cells to survive DNA replication stress, siRNA-mediated knockdown of Chk1, or inhibition of Chk1 function by a Chk1 kinase inhibitor resulted in H2AX phosphorylation, while ectopic increase of Chk1 attenuated the DNA damage induced by mTOR kinase inhibition. Most importantly, the mTOR kinase inhibitor prevented the slow S-phase progression following DNA replication stress, which was accompanied with a decrease of Chk1 protein levels and phospho-Chk1(S345). Our data suggest that the TORC1/TORC2 pathway promotes RMS tumorigenesis by enhancing the cell survival functions of the ATR-Chk1 checkpoint in response to DNA replication stress. Our findings discovered an important unexpected functional linkage between TOR signaling and the ATR-Chk1 checkpoint signaling cascade and may impact targeting these two prominent cancer cell signaling pathways. Supported by CA77776. 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 2535. doi:1538-7445.AM2012-2535

Embryonic signaling pathways and rhabdomyosarcoma: contributions to cancer development and opportunities for therapeutic targeting.

Rhabdomyosarcoma is the most common soft tissue sarcoma of childhood and adolescence, accounting for approximately 7% of childhood cancers. Current therapies include nonspecific cytotoxic chemotherapy regimens, radiation therapy, and surgery; however, these multimodality strategies are unsuccessful in the majority of patients with high-risk disease. It is generally believed that these tumors represent arrested or aberrant skeletal muscle development, and, accordingly, developmental signaling pathways critical to myogenesis such as Notch, WNT, and Hedgehog may represent new therapeutic targets. In this paper, we summarize the current preclinical studies linking these embryonic pathways to rhabdomyosarcoma tumorigenesis and provide support for the investigation of targeted therapies in this embryonic cancer.