EPICYCLE: A confirmatory preclinical study of the anti-rhabdomyosarcoma efficacy of BET bromodomain and cyclin-dependent kinase 9 inhibitors.

Facioscapulohumeral muscular dystrophy (FSHD) is linked to the deletion of the D4Z4 arrays at chromosome 4q35. Recent studies suggested that aberrant expression of double homeobox 4 (DUX4) from the last D4Z4 repeat causes FSHD. The aim of this study is to determine transcriptomic responses to ectopically expressed DUX4 in human and mouse cells of muscle lineage. We expression profiled human rhabdomyosarcoma (RD) cells and mouse C2C12 cells transfected with expression vectors of DUX4 using the Affymetrix Human Genome U133 Plus 2.0 Arrays and Mouse Genome 430 2.0 Arrays, respectively. A total of 2267 and 150 transcripts were identified to be differentially expressed in the RD and C2C12 cells, respectively. Amongst the transcripts differentially expressed in the RD cells, MYOD and MYOG (2 fold, p<0.05), and six MYOD downstream targets were up-regulated in RD but not C2C12 cells. Furthermore, 13 transcripts involved in germline function were dramatically induced only in the RD cells expressing DUX4. The top 3 IPA canonical pathways affected by DUX4 were different between the RD (inflammation, BMP signaling and NRF-2 mediated oxidative stress) and the C2C12 cells (p53 signaling, cell cycle regulation and cellular energy metabolism). Amongst the 40 transcripts shared by the RD and C2C12 cells, UTS2 was significantly induced by 76 fold and 224 fold in the RD and C2C12 cells, respectively. The differential expression of MYOD, MYOG and UTS2 were validated using real-time quantitative RT-PCR. We further validated the differentially expressed genes in immortalized FSHD myoblasts and showed up-regulation of MYOD, MYOG, ZSCAN4 and UTS2. The results suggest that DUX4 regulates overlapped and distinct groups of genes and pathways in human and mouse cells as evident by the selective up-regulation of genes involved in myogenesis and gametogenesis in human RD and immortalized cells as well as the different molecular pathways identified in the cells.

Novel Pleiotropic Effects of Thrombin in Regulation of Metastatic Potential of Human Rhabdomyosarcoma (RMS) Cells – Identification of Negative PAR1 and PAR3 Receptor Crosstalk.

The erythropoietin receptor (EpoR) is expressed by cells from the erythroid lineage; however, evidence has accumulated that it is also expressed by some solid tumors. This is an important observation, because recombinant erythropoietin (EPO) is employed in cancer patients to treat anemia related to chemo/radiotherapy. In our studies we employed eight rhabdomyosarcoma (RMS) cell lines (three alveolar-type RMS cell lines and five embrional-type RMS cell lines), and mRNA samples obtained from positive, PAX7-FOXO1-positive, and fusion-negative RMS patient samples. Expression of EpoR was evaluated by RT-PCR, gene array and FACS. The functionality of EpoR in RMS cell lines was evaluated by chemotaxis, adhesion, and direct cell proliferation assays. In some of the experiments, RMS cells were exposed to vincristine (VCR) in the presence or absence of EPO to test whether EPO may impair the therapeutic effect of VCR. We report for a first time that functional EpoR is expressed in human RMS cell lines as well as by primary tumors from RMS patients. Furthermore, EpoR is detectably expressed in both embryonal and alveolar RMS subtypes. At the functional level, several human RMS cell lines responded to EPO stimulation by enhanced proliferation, chemotaxis, cell adhesion, and phosphorylation of MAPKp42/44 and AKT. Moreover, RMS cells became more resistant to VCR treatment in the presence of EPO. Our findings have important potential clinical implications, indicating that EPO supplementation in RMS patients may have the unwanted side effect of tumor progression.

Introduction: The molecular basis for loss of cell differentiation in the process of carcinogenesis is not well understood but differentiation status of the given tumor strongly influences its progression and metastatic ability and therefore the overall patient survival. Rhabdomyosarcoma (RMS) is one of most frequent soft tissue tumor occurring in children and adolescent. The precise molecular mechanism responsible for the disruption of myogenesis, characteristic for RMS tumors, is not fully understood. Activation of MET receptor has been shown to influence proliferation, survival and migration of RMS cells and MET is rapidly downregulated at the onset of myogenic differentiation. However, the influence of MET receptor on differentiation status of RMS has not been studied at all. Objectives: We have focused on the induction of differentiation as a novel approach for RMS treatment and looked at the role of MET receptor in this process. Materials and Methods: The expression of muscles specific genes (MyoD, Myogenin) was evaluated using RT-PCR and western blot in differentiated RMS cells and RMS cells with downregulated MET receptor. Number of apoptotic cells was studied by Annexin V binding assay. In order to study the differentiation potential of MET negative cells, RMS animal model in NOD-SCID mice was developed. We studied tumor morphology (H&amp;E staining), maturity (desmin staining) and proliferation rate (Ki67 staining). Results: We found that in control cells (poorly maturated RH30 cell line), the expression of MyoD (present in undifferentiated muscle cells) was high whereas during differentiation it decreased. On the other hand, Myogenin expression (present in more matured muscle cells) markedly increased at the same time. We noticed increased number of apoptotic cells in differentiation medium with 2 % of horse serum. We also obtained RMS cell lines with stable downregulation of MET receptor. The expression of differentiation related genes in MET negative cells was similar to serum differentiated RMS cells. MyoD expression was almost completely reduced in these cells. Analysis of tumor lesions formed in NOD-SCID mice showed that tumors with downregulation of MET were much more differentiated and their proliferation rate was much lower. MET negative tumors were also less metastatic in comparison to wild type controls. These data are in concordance with decreased expression of MyoD in vitro and suggest higher differentiation level of MET negative tumors in vivo. Summary: In this study, for the first time, we have shown that differentiation of RMS cells is connected to the decrease of expression and signaling of MET receptor. These findings might have the significant clinical implication for the treatment of RMS cells because they suggest that induction of differentiation of RMS cells by e.g. blocking MET receptor might have influence on the aggressiveness/metastatic potential of these tumors. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A112.

Hamster BHK21 tumor cells and human rhabdomyosarcoma (RD) cells responded to cytochalasin B (CB) by becoming highly multinucleated. Nearly 80% of the BHK cells contained four or more nuclei after 7 days of exposure to CB, and 35% of these had at least seven nuclei. Similar frequencies of multinucleation were obtained with RD cells. Administration of 10(-3) M theophylline and dibutyryl adenosine 3',5'-cyclic monophosphate (Bu2cAMP) at 5X10(-4) M or 10(-3) M to CB-treated RD or BHK cells greatly reduced the frequency of cells with five or more nuclei. The frequency of cells with seven or more nuclei was reduced to less than 5%. Along with this reduction in highly multinucleated cells was an increase in the incidence of binucleated cells. Bu2cAMP was toxic and caused many CB-treated BHK cells to detach from the culture surface, but not all cells were killed. Bu2cAMP had little toxic effect on CB-treated RD cells. These observations indicated that the inhibition of high degrees of multinucleation were not the result of nonspecific toxic effects of Bu2cAMP but that nuclear division was limited by it. The effect of Bu2cAMP on density-dependent inhibition of growth was also studied. Addition of only theophylline and Bu2cAMP to either BHK or RD cells resulted in growth to significantly lower saturation densities. The toxicity of Bu2cAMP on cells in crowded cultures apparently caused the limited propagation. Bu2cAMP resulted in significant cell killing or detachment but, once the lower saturation densities were reached, cell death was minimized. Thus Bu2cAMP did not restore contact inhibition per se. It was also found that untreated RD cells grew to lower concentration densities than expected from the microscopic inspection of cells in situ. Microscopic inspection revealed high concentration densities and numerous mitoses. This apparent contradiction was due to the ability of RD cells to fuse upon the attainment of confluence and to produce multinucleated cells without the aid of CB.

Novel Pleiotropic Effects of Thrombin in Regulation of the Metastatic Potential of Human Rhabdomyosarcoma (RMS) Cells.

Differential gene expressions of the MAPK signaling pathway in enterovirus 71-infected rhabdomyosarcoma cells

Our study demonstrated the biological changes produced in the Rhabdomyosarcoma (RD) cell line triggered by the photochemical reaction between PHOTOGEM® (photosensitizing agent) and He-Ne laser light (wavelength = 632.8 nm of red light). The basic parameters of photodynamic therapy were optimized to study photosensitizer localization/uptake, PHOTOGEM® absorption spectra, cytotoxic effects, phototoxic effects, and morphological changes in the RD cell line. The experiment included three steps. First, spectrometric measurements were obtained to optimize the absorbance and optimal density of PHOTOGEM® in the experimental biological model (RD cell line). A neutral red assay was used to estimate the loss of cellular viability. Second, the RD cell line containing PHOTOGEM® was irradiated with laser light (dose up to 100 J/cm2). In addition, a non-fluorescent compound was used to determine the intracellular production of reactive oxygen species. The treated cells were examined and photographed. The optical density of the PHOTOGEM®-exposed RD cell line was insignificant after 0–2 h but increased significantly after 20 and 24 h. A photosensitizer concentration of 120 μg/ml and a red He-Ne laser dose of 100 J/cm2 having wavelength 632.8 nm produced the maximum phototoxic effect in the RD cell line. The cell viability of the PHOTOGEM®-labeled RD cells decreased to 65% in the absence of the laser dose, but there was significant cell viability loss under suitable laser exposure (100 J/cm2).

Comparison of human rhabdomyosarcoma, HEp-2, and human foreskin fibroblast cells for the isolation of herpes simplex virus from clinical specimens

The protective effect of the human serum albumin, purified from globulins, (HSAGF) and antibodies to hFcRn was studied in RD cells infected with several strains and clones of species B enteroviruses possessing different receptor specificity (echoviruses 3, 9, 11, 30 and coxsackieviruses A9, B4, B5). It was shown that HSA-GF at concentrations of 4% or less protected RD cells from infection with echoviruses 3, 9, 11 and coxsackievirus A9. The antibodies to hFcRn at concentrations of 2.5 ug/mL or less demonstrated the similar spectrum of protective activity in RD cells against infection with echoviruses 3, 9, 11, 30 and coxsackievirus A9. The protective effect of HSA-GF or the antibodies to hFcRn was not observed in RD cells infected with coxsackieviruses B4 and B5 that need coxsackievirus-adenovirus receptor for uncoating. The usage of the previously characterized echovirus 11 clonal variants with different receptor specificity allowed us to define the function of hFcRn as a canyon-binding uncoating receptor in RD cells. The kinetics and magnitude of the observed protective effects correlated with receptor specificity of the enteroviruses used in this work supporting the two-step interaction of DAF-dependent echoviruses with the cellular receptors. In this study, the function of hFcRn was defined in RD cells as a canyon-binding and uncoating receptor for echoviruses and coxsackievirus A9. The two-step interaction of DAF-dependent echoviruses during entry into the cells was confirmed: initially with the binding receptor DAF and subsequently with the uncoating receptor hFcRn.

Background: Rhabdomyosarcoma (RMS) is the most common soft tissue of pediatric sarcoma, and studies demonstrate that RMS arises from skeletal muscle precursor cells. RMS, genetically and histologically, is divided into two subtypes: PAX-FOXO1 fusion positive (alveolar) RMS (aRMS), which is driven by chromosomal translocation involving PAX3 or PAX7 genes with FOXO1 and PAX-FOXO1 fusion negative (embryonal) RMS (eRMS), which is marked by mutations in RAS isoforms and some genes such as TP53, PIK3CA, CTNNB1 and FGFR4. Chromatin was one of the earliest identified targets for cancer therapy. Several chromatin remodeling proteins are associated with cancer progression processes such as proliferation, differentiation, apoptosis, and tumorigenesis. Components of chromatin remodeling complexes, such as the imitation switch (ISWI) complex, have been classified as both oncogenes and tumor suppressors. The ISWI family protein, SMARCA1 (also known as SNF2L), has been implicated in tumorigenesis for several cancer types. ISWI complexes regulate cell differentiation and proliferation in other cell systems, but their impact in myogenesis is not well understood. In this study, we will characterize the function of SMARCA1 in RMS cells and skeletal muscle. We hypothesize that SMARCA1 acts to modulate chromatin accessibility, and drive RMS tumorigenic growth. Methods: We will use RNA-seq, ATAC-seq, and CUT&amp;RUN to study the impact of SMARCA1 deletion in RMS cells. Furthermore, phenotypic experiments will be performed to determine the influence of SMARCA1 on differentiation. Results: SMARCA1 is expressed at a high level in RMS tissues but not in muscle tissues. Furthermore, our findings show that SMARCA1 is modestly upregulated during the differentiation in normal myogenesis. Curiously, eRMS cell lines show no change in the protein level of SMARCA1 when treated with a MEK inhibitor, trametinib. Analysis of gene clusters in RMS transcriptomic data of indicated that SMARCA1 co-expresses with a RAS effector, RALA. SMARCA1 also clustered with HDAC2. In addition, SMARCA1 interacted with HDAC2 in RMS cells as determined by co-immunoprecipitation experiments. Conclusions: This study will deepen our understanding of how SMARCA1 impacts RMS differentiation and proliferation and may credential SMARCA1 as a novel therapeutic target in rhabdomyosarcoma. Citation Format: Ashwaq K Aljabri, Katie E Hebron, Yuliya Kriga, Juan Manuel Caravaca, Aiysha Althobaiti, Alex Emmons, Stacey Stauffer, Angela Kim, Lauren Stoak, Morgan Porter, Jyoti Shetty, Peter Fitzgerald, Bao Tran, Matthew Geisler, Judith K Davie, Marielle E Yohe. The role of SMARCA1 in Rhabdomyosarcoma and skeletal muscle differentiation [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_A23.

BackgroundMicroRNAs act as post-transcriptional regulators that repress translation or degrade mRNA transcripts. Each microRNA has many mRNA targets and each mRNA may be targeted by several microRNAs. Skeletal muscles express a plethora of microRNA genes that regulate muscle development and function by controlling the expression of protein-coding target genes. To expand our understanding of the role of microRNA, specifically bta-miR-365-3p, in muscle biology, we investigated its functions in regulating primary bovine myoblast proliferation and differentiation.ResultsFirstly, we found that bta-miR-365-3p was predominantly expressed in skeletal muscle and heart tissue in Chinese Qinchuan beef cattle. Quantitative PCR and western blotting results showed that overexpression of bta-miR-365-3p significantly reduced the expression levels of cyclin D1 (CCND1), cyclin dependent kinase 2 (CDK2) and proliferating cell nuclear antigen (PCNA) but stimulated the expression levels of muscle differentiation markers, i.e., MYOD1, MYOG at both mRNA and protein level. Moreover, downregulation of bta-miR-365-3p increased the expression of CCND1, CDK2 and PCNA but decreased the expression of MYOD1 and MYOG at both mRNA and protein levels. Furthermore, flow cytometry, EdU proliferation assays and immunostaining results showed that increased levels of bta-miR-365-3p suppressed cell proliferation but promoted myotube formation, whereas decreased levels of bta-miR-365-3p resulted in the opposite consequences. Finally, we identified that activin A receptor type I (ACVR1) could be a direct target of bta-miR-365-3p. It was demonstrated that bta-miR-365-3p can bind to the 3’UTR of ACVR1 gene to regulate its expression based on dual luciferase gene reporter assays. Consistently, knock-down of ACVR1 was associated with decreased expressions of CDK2, CCND1 and PCNA but increased expression of MYOG and MYOD1 both at mRNA and protein level.ConclusionCollectively, these data suggested that bta-miR-365-3p represses proliferation but promotes differentiation of bovine myoblasts through several biological mechanisms involving downregulation of ACVR1.

p16INK4A (p16) tumour suppressor induces growth arrest by inhibiting function of cyclin-dependent kinase (CDK)4 and CDK6. Homozygous p16 gene deletion is frequent in primary rhabdomyosarcoma (RMS) cells as well as derived cell lines. To confirm the significance of p16 gene deletion in tumour biology of RMS, a temperature-sensitive p16 mutant (E119G) gene was retrovirally transfected into the human RMS cell line RD, which has homozygous gene deletion of p16 gene. Decrease from 40°C (restrictive) to 34°C (permissive) culture temperature reduced CDK6-associated kinase activity and induced G1 growth arrest. Moreover, RD-p16 cells cultured under permissive condition demonstrated differentiated morphology coupled with expressions of myogenin and myosin light chain. These suggest that deletion of p16 gene may not only facilitate growth but also inhibit the myogenic differentiation of RD RMS cells. © 1999 Cancer Research Campaign

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

Simple SummaryCDK9, in combination with Cyclin T1, is one of the major regulators of RNA Polymerase II mediated productive transcription of critical genes in any cell. The activity of CDK9 is significantly up-regulated in a wide variety of cancer entities, to aid in the overexpression of genes responsible for the regulation of functions, which are beneficial to the cancer cells, like proliferation, survival, cell cycle regulation, DNA damage repair and metastasis. Enhanced CDK9 activity, therefore, leads to poorer prognosis in many cancer types, offering the rationale to target it using small-molecule inhibitors. Several, increasingly specific inhibitors, have been developed, some of which are presently in clinical trials. Other approaches being tested involve combining inhibitors against CDK9 activity with those against CDK9’s upstream regulators like BRD4, SEC and HSP90; or downstream effectors like cMYC and MCL-1. The inhibition of CDK9’s activity holds the potential to be a highly effective anti-cancer therapeutic.Cyclin Dependent Kinase 9 (CDK9) is one of the most important transcription regulatory members of the CDK family. In conjunction with its main cyclin partner—Cyclin T1, it forms the Positive Transcription Elongation Factor b (P-TEFb) whose primary function in eukaryotic cells is to mediate the positive transcription elongation of nascent mRNA strands, by phosphorylating the S2 residues of the YSPTSPS tandem repeats at the C-terminus domain (CTD) of RNA Polymerase II (RNAP II). To aid in this process, P-TEFb also simultaneously phosphorylates and inactivates a number of negative transcription regulators like 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) Sensitivity-Inducing Factor (DSIF) and Negative Elongation Factor (NELF). Significantly enhanced activity of CDK9 is observed in multiple cancer types, which is universally associated with significantly shortened Overall Survival (OS) of the patients. In these cancer types, CDK9 regulates a plethora of cellular functions including proliferation, survival, cell cycle regulation, DNA damage repair and metastasis. Due to the extremely critical role of CDK9 in cancer cells, inhibiting its functions has been the subject of intense research, resulting the development of multiple, increasingly specific small-molecule inhibitors, some of which are presently in clinical trials. The search for newer generation CDK9 inhibitors with higher specificity and lower potential toxicities and suitable combination therapies continues. In fact, the Phase I clinical trials of the latest, highly specific CDK9 inhibitor BAY1251152, against different solid tumors have shown good anti-tumor and on-target activities and pharmacokinetics, combined with manageable safety profile while the phase I and II clinical trials of another inhibitor AT-7519 have been undertaken or are undergoing. To enhance the effectiveness and target diversity and reduce potential drug-resistance, the future of CDK9 inhibition would likely involve combining CDK9 inhibitors with inhibitors like those against BRD4, SEC, MYC, MCL-1 and HSP90.