Proliferation Of Rhabdomyosarcoma Cells Research Articles

Phospholipase Cδ-4 (PLCδ4) Acts as a Nuclear Player to Influence Cyclin B Expression in the Embryonal Rhabdomyosarcoma Cell Lines RD and A204.

Rhabdomyosarcoma (RMS), the most common form of sarcoma typical of pediatric age, arises from the malignant transformation of the mesenchymal precursors that fail to differentiate into skeletal muscle cells. Here, we investigated whether the protein phospholipase C δ4 (PLCδ4), a member of the PLC family involved in proliferation and senescence mechanisms of mesenchymal stromal stem cells, may play a role in RMS. Our molecular and morpho-functional data reveal that PLCδ4 is highly expressed in the fusion-negative, p53-positive, SMARCB1 heterozygous mutated embryonal RMS (ERMS) cell line A204, while it is poorly expressed in the ERMS cell lines RD (fusion-negative, MYC amplification, N-RAS (Q61H), homozygous mutated p53) and Hs729 (homozygous mutated p53) and the alveolar rhabdosarcoma (ARMS) cell line SJCRH30 (RH30; fusion positive, heterozygous mutated RARA, polyheterozygous mutated p53). To characterize the role of PLCδ4, the RD cell line was stably transfected with wild-type PLCδ4 (RD/PLCδ4). Overexpressed PLCδ4 mainly localized to the nucleus in RD cells and contributed to the phosphorylation of PRAS40 (T246), Chk2(T68), WNK1(T60), and Akt 1/273 (S473), as revealed by proteome profiler array analysis. Overexpression of PLCδ4 in RD cells enhanced cyclin B1 expression and resulted in G2/M-phase cell cycle arrest. In contrast, siRNA-mediated knockdown of PLCδ4 in A204 cells resulted in reduced cyclin B1 expression. Our study identifies a novel role for nuclear PLCδ4 as a regulator of cyclin B1 via Akt-dependent phosphorylation. The modulation of PLCδ4 expression and its downstream targets could represent a crucial signaling pathway to block embryonal RMS cell proliferation.

Improving anti-tumor efficacy of low-dose Vincristine in rhabdomyosarcoma via the combination therapy with FOXM1 inhibitor RCM1.

Rhabdomyosarcoma (RMS) is a highly metastatic soft-tissue sarcoma that often develops resistance to current therapies, including vincristine. Since the existing treatments have not significantly improved survival, there is a critical need for new therapeutic approaches for RMS patients. FOXM1, a known oncogene, is highly expressed in RMS, and is associated with the worst prognosis in RMS patients. In the present study, we found that the combination treatment with specific FOXM1 inhibitor RCM1 and low doses of vincristine is more effective in increasing apoptosis and decreasing RMS cell proliferation in vitro compared to single drugs alone. Since RCM1 is highly hydrophobic, we developed innovative nanoparticle delivery system containing poly-beta-amino-esters and folic acid (NPFA), which efficiently delivers RCM1 to mouse RMS tumors in vivo. The combination of low doses of vincristine together with intravenous administration of NPFA nanoparticles containing RCM1 effectively reduced RMS tumor volumes, increased tumor cell death and decreased tumor cell proliferation in RMS tumors compared to RCM1 or vincristine alone. The combination therapy was non-toxic as demonstrated by liver metabolic panels using peripheral blood serum. Using RNA-seq of dissected RMS tumors, we identified Chac1 as a uniquely downregulated gene after the combination treatment. Knockdown of Chac1 in RMS cells in vitro recapitulated the effects of the combination therapy. Altogether, combination treatment with low doses of vincristine and nanoparticle delivery of FOXM1 inhibitor RCM1 in a pre-clinical model of RMS has superior anti-tumor effects and decreases CHAC1 while reducing vincristine toxicity.

PROX1 transcription factor controls rhabdomyosarcoma growth, stemness, myogenic properties and therapeutic targets

Rhabdomyosarcoma (RMS) is an aggressive pediatric soft-tissue cancer with features of skeletal muscle. Because of poor survival of RMS patients and severe long-term side effects of RMS therapies, alternative RMS therapies are urgently needed. Here we show that the prospero-related homeobox 1 (PROX1) transcription factor is highly expressed in RMS tumors regardless of their cell type of origin. We demonstrate that PROX1 is needed for RMS cell clonogenicity, growth and tumor formation. PROX1 gene silencing repressed several myogenic and tumorigenic transcripts and transformed the RD cell transcriptome to resemble that of benign mesenchymal stem cells. Importantly, we found that fibroblast growth factor receptors (FGFR) mediated the growth effects of PROX1 in RMS. Because of receptor cross-compensation, paralog-specific FGFR inhibition did not mimic the effects of PROX1 silencing, whereas a pan-FGFR inhibitor ablated RMS cell proliferation and induced apoptosis. Our findings uncover the critical role of PROX1 in RMS and offer insights into the mechanisms that regulate RMS development and growth. As FGFR inhibitors have already been tested in clinical phase I/II trials in other cancer types, our findings provide an alternative option for RMS treatment.

A GRIP-1-EZH2 switch binding to GATA-4 is linked to the genesis of rhabdomyosarcoma through miR-29a.

Terminal differentiation failure is an important cause of rhabdomyosarcoma genesis, however, little is known about the epigenetic regulation of aberrant myogenic differentiation. Here, we show that GATA-4 recruits polycomb group proteins such as EZH2 to negatively regulate miR-29a in undifferentiated C2C12 myoblast cells, whereas recruitment of GRIP-1 to GATA-4 proteins displaces EZH2, resulting in the activation of miR-29a during myogenic differentiation of C2C12 cells. Moreover, in poorly differentiated rhabdomyosarcoma cells, EZH2 still binds to the miR-29a promoter with GATA-4 to mediate transcriptional repression of miR-29a. Interestingly, once re-differentiation of rhabdomyosarcoma cells toward skeletal muscle, EZH2 was dispelled from miR-29a promoter which is similar to that in myogenic differentiation of C2C12 cells. Eventually, this expression of miR-29a results in limited rhabdomyosarcoma cell proliferation and promotes myogenic differentiation. We thus establish that GATA-4 can function as a molecular switch in the up- and downregulation of miR-29a expression. We also demonstrate that GATA-4 acts as a tumor suppressor in rhabdomyosarcoma partly via miR-29a, which thus provides a potential therapeutic target for rhabdomyosarcoma.

Abstract 2305: Centriolin depletion inhibits cellular proliferation of rhabdomyosarcoma cells

Abstract Rhabdomyosarcoma (RMS), the most common pediatric soft tissue sarcoma, accounts for approximately five percent of all childhood cancers. It develops from rhabdomyoblasts and can arise in a variety of anatomic locations. Recent findings have assisted the prediction of disease prognosis and treatment regimens based on genetic and cellular differences among the major types of RMS. One potential target is a cellular appendage called the primary cilium. Primary cilia are thought to play a critical role in the regulation of myogenesis, a process deregulated in RMS. During myogenesis, Gli protein accumulates at the primary cilia in myoblasts and induces proliferation. The centrosome protein, centriolin (CNTRL), is known to be a key structural component of primary cilia. Therefore, we hypothesized that disruption of CNTRL, using CRISPR/Cas9, may affect Gli protein function and, therefore, the proliferation of these cells. Two cell lines were used: A204, a rhabdoid cell line, and CCL-136, an embryonal RMS cell line. When CNTRL was depleted and analyzed 2 days post-transfection, a significant reduction in cell viability was observed in CCL-136 compared to control, whereas no significant increase in cell death in A204 was observed. Gli1 expression decreased in both cells lines when CNTRL was eliminated, while cell cycle progression was also reduced, as confirmed by immunofluorescence staining of Ki67. Unexpectedly, primary cilia formation, indicated by Arl13B expression, increased in A204 with CNTRL loss, with no perceptible change in CCL-136 cells. To assess differentiation potential, A204 and CCL-136 were cultured in differentiation media using 2% horse serum. Separately, cells were cultured in 0% FBS (serum) to test differentiation potential under starvation conditions. As expected, both cell lines expressed robust levels of MyoG, a muscle differentiation marker, with 2% horse serum. However, loss of CNTRL leads to a perceptible decrease in MyoG in the CCL-136, and a slight decrease in A204. Additionally, wound healing assays were conducted and showed CCL-136 had inherently greater ability to migrate 24 hours post-wound compared to A204., However, CCL-136 showed diminished migration under stressed conditions. Interestingly, cell migration was significantly diminished in A204 under starvation conditions when CNTRL was depleted (p<0.05). We show that CNTRL loss inhibits the cell cycle in two different forms of RMS. This arrest occurs with a corresponding reduction in Gli1 expression. This confirms previous studies that suggest Gli1 is a critical factor in maintaining the proliferative state. In addition, our results show that in cells normally producing primary cilia, like A204, CNTRL loss increases the number of ciliated cells. Taken together, our data support the possibility of inhibiting cellular proliferation of RMS cells through manipulation of CNTRL and suggest that CNTRL may be a viable future treatment target. Citation Format: Yoshinori Takeda, Ariel Cole, Caitlin Boyle, Adam Gromley. Centriolin depletion inhibits cellular proliferation of rhabdomyosarcoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2305.

Heparanase-1 is downregulated in chemoradiotherapy orbital rhabdomyosarcoma and relates with tumor growth as well as angiogenesis.

To determine the role of heparanase-1 (HPSE-1) in orbital rhabdomyosarcoma (RMS), and to investigate the feasibility of HPSE-1 targeted therapy for RMS. Immunohistochemistry was performed to analyze HPSE-1 expression in 51 cases of orbital RMS patients (including 28 cases of embryonal RMS and 23 cases of alveolar RMS), among whom there were 27 treated and 24 untreated with preoperative chemoradiotherapy. In vitro, studies were conducted to examine the effect of HPSE-1 silencing on RMS cell proliferation and tube formation of human umbilical vein endothelial cells (HUVECs). RD cells (an RMS cell line) and HUVECs were infected with HPSE-1 shRNA lentivirus at a multiplicity of infection (MOI) of 10 and 30 separately. Real-time PCR and Western blot were applied to detect the mRNA and protein expression levels of HPSE-1. Cell viability of treated or control RD cells was evaluated by cell counting kit-8 (CCK-8) assay. Matrigel tube formation assay was used to evaluate the effect of HPSE-1 RNAi on the tube formation of HUVECs. Immunohistochemistry showed that the expression rate of HPSE-1 protein was 92.9% in orbital embryonal RMS and 91.3% in orbital alveolar RMS. Tissue from alveolar orbital RMS did not show relatively stronger staining than that from the embryonal orbital RMS. However, despite the types of RMS, comparing the cases treated chemoradiotherapy with those untreated, we have observed that chemoradiotherapy resulted in weaker staining in patients' tissues. The expression levels of HPSE-1 declined significantly in both the mRNA and protein levels in HPSE-1 shRNA transfected RD cells. The CCK-8 assay showed that lentivirus-mediated HPSE-1 silencing resulted in significantly reduced RD cells viability in vitro. Silencing HPSE-1 expression also inhibited VEGF-induced tube formation of HUVECs in Matrigel. HPSE-1 silencing may be a promising therapy for the inhibition of orbital RMS progression.

Gallium(III) Complex with Cloxyquin Ligands Induces Ferroptosis in Cancer Cells and Is a Potent Agent against Both Differentiated and Tumorigenic Cancer Stem Rhabdomyosarcoma Cells.

In this work, gallium(III) complex with cloxyquin (5-chloro-8-quinolinol, HClQ) ligands is shown to effectively inhibit proliferation of rhabdomyosarcoma cells, the frequent, aggressive, and poorly treatable cancer of children. It offers striking selectivity to cancer cells compared to noncancerous human fibroblasts. The data reveal that the complex induces ferroptosis in rhabdomyosarcoma cells, likely due to interfering with iron metabolism. Importantly, it can kill both bulk and stem rhabdomyosarcoma cells. To the best of our knowledge, this is the first compound based on metal other than Fe capable of inducing ferroptosis in cancer cells.

BET inhibition therapy counteracts cancer cell survival, clonogenic potential and radioresistance mechanisms in rhabdomyosarcoma cells.

The antitumour effects of OTX015, a first-in-class BET inhibitor (BETi), were investigated as a single agent or in combination with ionizing radiation (IR) in preclinical in vitro models of rhabdomyosarcoma (RMS), the most common childhood soft tissue sarcoma. Herein, we demonstrated the upregulation of BET Bromodomain gene expression in RMS tumour biopsies and cell lines compared to normal skeletal muscle. In vitro experiments showed that OTX015 significantly reduced RMS cell proliferation by altering cell cycle modulators and apoptotic related proteins due to the accumulation of DNA breaks that cells are unable to repair. Interestingly, OTX015 also impaired migration capacity and tumour-sphere architecture by downregulating pro-stemness genes and was able to potentiate ionizing radiation effects by reducing the expression of different drivers of tumour dissemination and resistance mechanisms, including the GNL3 gene, that we correlated for the first time with the RMS phenotype. In conclusion, our research sheds further light on the molecular events of OTX015 action against RMS cells and indicates this novel BETi as an effective option to improve therapeutic strategies and overcome the development of resistant cancer cells in patients with RMS.

“Verteporfin exhibits anti-proliferative activity in embryonal and alveolar rhabdomyosarcoma cell lines”

Rhabdomyosarcoma (RMS) is a pediatric tumor, which arises from muscle precursor cells. Recently, it has been demonstrated that Hippo Pathway (Hpo), a pathway that regulates several physiological and biological features, is involved in RMS tumorigenesis. For instance, an upregulation of the Hpo downstream effector Yes-Associated Protein 1 (YAP) leads to the development of embryonal rhabdomyosarcoma (eRMS) in murine activated muscle satellite cells. On the other hand, the YAP paralog transcriptional co-activator with PDZ-binding motif (TAZ) is overexpressed in alveolar rhabdomyosarcoma (aRMS) patients with poor survival.YAP and TAZ exhibit both cytoplasmic and nuclear functions. In the nucleus, YAP binds TEADs (TEA domain family members) factors and together they constitute a complex that is able either to activate the transcription of several genes such as MYC, Tbx5 and PAX8 or to maintain the stability of others like p73. Due to the key role of YAP and TAZ in cancer, the identification and/or development of new compounds able to block their activity might be an effective antineoplastic strategy. Verteporfin (VP) is a molecule able to stop the formation of YAP/TEAD complex in the nucleus.The aim of this study is to evaluate the action of VP on RMS cell lines.This work shows that VP has an anti-proliferative activity on all RMS cell lines analyzed. Depending on RMS cell lines, VP affects cell cycle differently. Moreover, VP is able to decrease YAP protein levels, and to induce the activation of apoptosis mechanism through the cleavage of PARP-1. In addition, Annexin V assay showed the activation of apoptosis and necrosis after VP treatment.In summary, the ability of VP to disrupt RMS cell proliferation could be a novel and valuable strategy to improve the therapeutic approaches in treating rhabdomyosarcoma.

Trichostatin A Inhibits Rhabdomyosarcoma Proliferation and Induces Differentiation through MyomiR Reactivation

Rhabdomyosarcoma (RMS) is a malignant tumour of soft tissues, occurring mainly in children and young adults. RMS cells derive from muscle cells, which due to mutations and epigenetic modifications have lost their ability to differentiate. Epigenetic modifications regulate expression of genes responsible for cell proliferation, maturation, differentiation and apoptosis. HDAC inhibitors suppress histone acetylation; therefore, they are a promising tool used in cancer therapy. Trichostatin A (TsA) is a pan-inhibitor of HDAC. In our study, we investigated the effect of TsA on RMS cell biology. Our findings strongly suggest that TsA inhibits RMS cell proliferation, induces cell apoptosis, and reactivates tumour cell differentiation. TsA up-regulates miR-27b expression, which is involved in the process of myogenesis. Moreover, TsA increases susceptibility of RMS cells to routinely used chemotherapeutics. In conclusion, TsA exhibits anti-cancer properties, triggers differentiation, and thereby can complement an existing spectrum of chemotherapeutics used in RMS therapy.

Targeting ALK in pediatric RMS does not induce antitumor activity in vivo.

The anaplastic lymphoma kinase (ALK) has been demonstrated to be a valid clinical target in diseases such as anaplastic large cell lymphoma and non-small cell lung cancer. Recent studies have indicated that ALK is overexpressed in pediatric rhabdomyosarcoma (RMS) and hence we hypothesized that this kinase may be a suitable candidate for therapeutic intervention in this tumor. We evaluated the expression of ALK in a panel of pediatric RMS cell lines and patient-derived xenografts (PDX), and sensitivity to ALK inhibitors was assessed both in vitro and in vivo. Essentially, all RMS lines were sensitive to crizotinib, NVP-TAE684 or LDK-378 in vitro, and molecular analyses demonstrated inhibition of RMS cell proliferation following siRNA-mediated reduction of ALK expression. However, in vivo PDX studies using ALK kinase inhibitors demonstrated no antitumor activity when used as single agents or when combined with standard of care therapy (vincristine, actinomycin D and cyclophosphamide). More alarmingly, however, crizotinib actually accelerated the growth of these tumors in vivo. While ALK appears to be a relevant target in RMS in vitro, targeting this kinase in vivo yields no therapeutic efficacy, warranting extreme caution when considering the use of these agents in pediatric RMS patients.

C-type natriuretic peptide in combination with sildenafil attenuates proliferation of rhabdomyosarcoma cells.

Rhabdomyosarcoma (RMS) is a malignant mesenchymal tumor and the most common soft tissue sarcoma in children. Because of several complications associated with intensive multimodal therapies, including growth disturbance and secondary cancer, novel therapies with less toxicity are urgently needed. C‐type natriuretic peptide (CNP), an endogenous peptide secreted by endothelial cells, exerts antiproliferative effects in multiple types of mesenchymal cells. Therefore, we investigated whether CNP attenuates proliferation of RMS cells. We examined RMS patient samples and RMS cell lines. All RMS clinical samples expressed higher levels of guanylyl cyclase B (GC‐B), the specific receptor for CNP, than RMS cell lines. GC‐B expression in RMS cells decreased with the number of passages in vitro. Therefore, GC‐B stable expression lines were established to mimic clinical samples. CNP increased cyclic guanosine monophosphate (cGMP) levels in RMS cells in a dose‐dependent manner, demonstrating the biological activity of CNP. However, because cGMP is quickly degraded by phosphodiesterases (PDEs), the selective PDE5 inhibitor sildenafil was added to inhibit its degradation. In vitro, CNP, and sildenafil synergistically inhibited proliferation of RMS cells stably expressing GC‐B and decreased Raf‐1, Mitogen‐activated protein kinase kinase (MEK), and extracellular signal‐regulated kinase (ERK) phosphorylation. These results suggested that CNP in combination with sildenafil exerts antiproliferative effects on RMS cells by inhibiting the Raf/MEK/ERK pathway. This regimen exerted synergistic effects on tumor growth inhibition without severe adverse effects in vivo such as body weight loss. Thus, CNP in combination with sildenafil represents a promising new therapeutic approach against RMS.

Crizotinib-induced antitumour activity in human alveolar rhabdomyosarcoma cells is not solely dependent on ALK and MET inhibition.

BackgroundRhabdomyosarcoma (RMS) is the most commonly diagnosed malignant soft tissue tumour in children and adolescents. Aberrant expression of Anaplastic Lymphoma Kinase (ALK) and MET gene has been implicated in the malignant progression of RMS, especially in the alveolar subtype. This observation suggests that crizotinib (PF-02341066), a kinase inhibitor against ALK and MET, may have a therapeutic role in RMS, although its antitumour activity in this malignancy has not yet been studied.MethodsRH4 and RH30 alveolar RMS (ARMS) cell lines were treated with crizotinib and then assessed by using proliferation, viability, migration and colony formation assays. Multiple approaches, including flow cytometry, immunofluorescence, western blotting and siRNA-based knock-down, were used in order to investigate possible molecular mechanisms linked to crizotinib activity.ResultsIn vitro treatment with crizotinib inhibited ALK and MET proteins, as well as Insulin-like Growth Factor 1 Receptor (IGF1R), with a concomitant robust dephosphorylation of AKT and ERK, two downstream kinases involved in RMS cell proliferation and survival. Exposure to crizotinib impaired cell growth, and accumulation at G2/M phase was attributed to an altered expression and activation of checkpoint regulators, such as Cyclin B1 and Cdc2. Crizotinib was able to induce apoptosis and autophagy in a dose-dependent manner, as shown by caspase-3 activation/PARP proteolytic cleavage down-regulation and by LC3 activation/p62 down-regulation, respectively. The accumulation of reactive oxygen species (ROS) seemed to contribute to crizotinib effects in RH4 and RH30 cells. Moreover, crizotinib-treated RH4 and RH30 cells exhibited a decreased migratory/invasive capacity and clonogenic potential.ConclusionsThese results provide a further insight into the molecular mechanisms affected by crizotinib in ARMS cells inferring that it could be a useful therapeutic tool in ARMS cancer treatment.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-015-0228-4) contains supplementary material, which is available to authorized users.

Autoregulatory loop between TGF-β1/miR-411-5p/SPRY4 and MAPK pathway in rhabdomyosarcoma modulates proliferation and differentiation.

The origin of rhabdomyosarcoma (RMS) remains controversial. However, specific microRNAs (miRNAs) are downregulated in RMS and it is possible that re-expression of these miRNAs may lead to differentiation. Transforming growth factor-β1 (TGF-β1) is known to block differentiation of RMS. We therefore analyzed miRNA microarrays of RMS cell lines with or without TGF-β1 knockdown and identified a novel anti-oncogene miR-411-5p. Re-expression of miR-411-5p inhibited RMS cell proliferation in vitro and tumorigenicity in vivo. Using a luciferase reporting system and sequence analysis, the potential target of miR-411-5p was identified as sprouty homolog 4 (SPRY4), which inhibits protein kinase Cα-mediated activation of mitogen-activated protein kinases (MAPKs), especially p38MAPK phosphorylation. These results revealed an inverse correlation between TGF-β1/SPRY4 and miR-411-5p levels. SPRY4 small interfering RNA and miR-411-5p both activated p38MAPK phosphorylation and also promoted apoptosis and myogenic differentiation, indicated by increased caspase-3, myosin heavy chain, and myosin expression. SPRY4 and miR-411 mRNA levels correlated with TGF-β1 expression levels in RMS tissues, which was confirmed by immunohistochemical staining for TGF-β1, SPRY4, and phosphorylated p38MAPK proteins. Overall, these results indicate that miR-411-5p acts as an RMS differentiation-inducing miRNA prompting p38MAPK activation via directly downregulating SPRY4. These results establish an autoregulatory loop between TGF-β1/miR-411-5p/SPRY4 and MAPK in RMS, which governs the switch between proliferation and differentiation.

MicroRNA-27a Contributes to Rhabdomyosarcoma Cell Proliferation by Suppressing RARA and RXRA.

BackgroundRhabdomyosarcomas (RMS) are rare but very aggressive childhood tumors that arise as a consequence of a regulatory disruption in the growth and differentiation pathways of myogenic precursor cells. According to morphological criteria, there are two major RMS subtypes: embryonal RMS (ERMS) and alveolar RMS (ARMS) with the latter showing greater aggressiveness and metastatic potential with respect to the former. Efforts to unravel the complex molecular mechanisms underlying RMS pathogenesis and progression have revealed that microRNAs (miRNAs) play a key role in tumorigenesis.Methodology/Principal FindingsThe expression profiles of 8 different RMS cell lines were analyzed to investigate the involvement of miRNAs in RMS. The miRNA population from each cell line was compared to a reference sample consisting of a balanced pool of total RNA extracted from those 8 cell lines. Sixteen miRNAs whose expression discriminates between translocation-positive ARMS and negative RMS were identified. Attention was focused on the role of miR-27a that is up-regulated in the more aggressive RMS cell lines (translocation-positive ARMS) in which it probably acts as an oncogene. MiR-27a overexpressing cells showed a significant increase in their proliferation rate that was paralleled by a decrease in the number of cells in the G1 phase of the cell cycle. It was possible to demonstrate that miR-27a is implicated in cell cycle control by targeting the retinoic acid alpha receptor (RARA) and retinoic X receptor alpha (RXRA).ConclusionsStudy results have demonstrated that miRNA expression signature profiling can be used to classify different RMS subtypes and suggest that miR-27a may have a therapeutic potential in RMS by modulating the expression of retinoic acid receptors.

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.

RBPJ Is a Novel Target for Rhabdomyosarcoma Therapy

The Notch pathway regulates a broad spectrum of cell fate decisions and differentiation processes during fetal and postnatal development. In addition, the Notch pathway plays an important role in controlling tumorigenesis. However, the role of RBPJ, a transcription factor in the Notch pathway, in the development of tumors is largely unknown. In this study, we focused on the role of RBPJ in the pathogenesis of rhabdomyosarcoma (RMS). Our data showed that Notch pathway genes were upregulated and activated in human RMS cell lines and patient samples. Inhibition of the Notch pathway by a γ-secretase inhibitor (GSI) decreased the in vitro proliferation of RMS cells. Knockdown of RBPJ expression by RNAi inhibited the anchorage-independent growth of RMS cells and the growth of xenografts in vivo. Additionally, overexpression of RBPJ promoted the anchorage-independent growth of RMS cells. Further, we revealed that RBPJ regulated the cell cycle in RMS xenograft tumors and decreased proliferation. Our findings suggest that RBPJ regulates the RMS growth, and that the inhibition of RBPJ may be an effective therapeutic approach for patients with RMS.

Cytostatic and cytotoxic effects of tyrphostin AG1296 on RMS cells

Aim of the studyThe aim of the work was to determine the influence of tyrphostin AG1296, an inhibitor of platelet-derived growth factor receptor (PDGFR) tyrosine kinase, on autocrine growth of rhabdomyosarcoma (RMS) cells.Materials and methodsRMS cells were cultured in serum-free DMEM/F12 medium. Modified crystal violet (CV) and MTT methods were used to determine the RMS cells’ proliferation and viability. Influence of the investigated inhibitor on cell apoptosis or necrosis was determined by differential staining with Hoechst No. 33258 and propidium iodide.ResultsThe AG1296 inhibitor affects RMS cell proliferation in a dose-dependent way at the concentration range 1–100 µM. At concentrations above 25 µM there was 100% inhibition of growth of these cells and a cytotoxic effect was noticed. 50% inhibition of RMS cells proliferation (IC50) was observed at concentration 6.65 ±0.44 µM (determined by CV method) and 7.30 ±0.26 µM (determined by MTT method). The differential staining method shows that this inhibitor causes a cytotoxic effect.ConclusionThe results of these experiments indicate that autocrine growth of RMS cells is regulated by at least one autocrine loop, involving platelet-derived growth factor (PDGF) and its receptor (PDGFR).The fact that tyrphostin AG1296 is able to complete inhibition of RMS cell growth in vitro gives a chance for providing a new group of antitumor drugs, which may be more effective than the medicines used so far.

A set of imprinted genes required for normal body growth also promotes growth of rhabdomyosarcoma cells

In many normal tissues, proliferation rates decline postnatally, causing somatic growth to slow. Previous evidence suggests that this decline is due, in part, to decline in the expression of growth-promoting imprinted genes including Mest, Plagl1, Peg3, Dlk1, and Igf2. Embryonal cancers are composed of cells that maintain embryonic characteristics and proliferate rapidly in childhood. We hypothesized that the abnormal persistent rapid proliferation in embryonal cancers occurs in part because of abnormal persistent high expression of growth-promoting imprinted genes. Analysis of microarray data showed elevated expression of MEST, PLAGL1, PEG3, DLK1, and IGF2 in various embryonal cancers, especially rhabdomyosarcoma, as compared to nonembryonal cancers and normal tissues. Similarly, mRNA expression, assessed by real-time PCR, of MEST, PEG3, and IGF2 in rhabdomyosarcoma cell lines was increased as compared to nonembryonal cancer cell lines. Furthermore, siRNA-mediated knockdown of MEST, PLAGL1, PEG3, and IGF2 expression inhibited proliferation in Rh30 rhabdomyosarcoma cells. These findings suggest that the normal postnatal downregulation of growth-promoting imprinted genes fails to occur in some embryonal cancers, particularly rhabdomyosarcoma, and contributes to the persistent rapid proliferation of rhabdomyosarcoma cells and, more generally, that failure of the mechanisms responsible for normal somatic growth deceleration can promote tumorigenesis.

Retinoic acid fails to induce cell cycle arrest with myogenic differentiation in rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Current treatment strategies do not cure most children with recurrent or high-risk disease, underlying the need for novel therapeutic approaches. Retinoic acid has been shown to induce differentiation in a variety of cells including skeletal myoblasts and neuroblasts. In the setting of minimal residual disease, retinoic acid improves survival in neuroblastoma, another poorly differentiated childhood tumor. Whether such an approach is useful for rhabdomyosarcoma has not yet been investigated. Several in vitro studies have demonstrated an appreciable effect of retinoic acid on human RMS cellular proliferation and differentiation. We assessed the efficacy of ATRA on rhabdomyosarcoma, in vitro and in vivo, using cell lines and xenografts. ATRA slowed RMS cell proliferation, and promoted a more differentiated myogenic phenotype in both alveolar and embryonal RMS cell lines. Treatment of cultured murine myoblasts with retinoids increased Myogenin expression, but did not induce cell cycle arrest. Despite the favorable in vitro effects, ATRA failed to delay relapse of minimal residual disease using human RMS xenografts in immuno-suppressed NOD-SCID (NSG) mice. Interestingly, tumors that recurred after ATRA treatment showed evidence of enhanced muscle differentiation. Our results indicate that ATRA could increase the expression of some genes associated with muscle differentiation in rhabdomyosarcoma cells, but there was no benefit of single-agent therapy in an MRD model, likely because cell cycle arrest was uncoupled from the pro-differentiation effects of retinoids.