Differentiation Of Rhabdomyosarcoma Cells Research Articles

The Wnt-pathway corepressor TLE3 interacts with the histone methyltransferase KMT1A to inhibit differentiation in Rhabdomyosarcoma.

Rhabdomyosarcoma tumor cells resemble differentiating skeletal muscle cells, which unlike normal muscle cells, fail to undergo terminal differentiation, underlying their proliferative and metastatic properties. We identify the corepressor TLE3 as a key regulator of rhabdomyosarcoma tumorigenesis by inhibiting the Wnt-pathway. Loss of TLE3 function leads to Wnt-pathway activation, reduced proliferation, decreased migration, and enhanced differentiation in rhabdomyosarcoma cells. Muscle-specific TLE3-knockout results in enhanced expression of terminal myogenic differentiation markers during normal mouse development. TLE3-knockout rhabdomyosarcoma cell xenografts result in significantly smaller tumors characterized by reduced proliferation, increased apoptosis and enhanced differentiation. We demonstrate that TLE3 interacts with and recruits the histone methyltransferase KMT1A, leading to repression of target gene activation and inhibition of differentiation in rhabdomyosarcoma. A combination drug therapy regime to promote Wnt-pathway activation by the small molecule BIO and inhibit KMT1A by the drug chaetocin led to significantly reduced tumor volume, decreased proliferation, increased expression of differentiation markers and increased survival in rhabdomyosarcoma tumor-bearing mice. Thus, TLE3, the Wnt-pathway and KMT1A are excellent drug targets which can be exploited for treating rhabdomyosarcoma tumors.

Genomic and Epigenetic Changes Drive Aberrant Skeletal Muscle Differentiation in Rhabdomyosarcoma

Rhabdomyosarcoma (RMS), the most common soft-tissue sarcoma in children and adolescents, represents an aberrant form of skeletal muscle differentiation. Both skeletal muscle development, as well as regeneration of adult skeletal muscle are governed by members of the myogenic family of regulatory transcription factors (MRFs), which are deployed in a highly controlled, multi-step, bidirectional process. Many aspects of this complex process are deregulated in RMS and contribute to tumorigenesis. Interconnected loops of super-enhancers, called core regulatory circuitries (CRCs), define aberrant muscle differentiation in RMS cells. The transcriptional regulation of MRF expression/activity takes a central role in the CRCs active in skeletal muscle and RMS. In PAX3::FOXO1 fusion-positive (PF+) RMS, CRCs maintain expression of the disease-driving fusion oncogene. Recent single-cell studies have revealed hierarchically organized subsets of cells within the RMS cell pool, which recapitulate developmental myogenesis and appear to drive malignancy. There is a large interest in exploiting the causes of aberrant muscle development in RMS to allow for terminal differentiation as a therapeutic strategy, for example, by interrupting MEK/ERK signaling or by interfering with the epigenetic machinery controlling CRCs. In this review, we provide an overview of the genetic and epigenetic framework of abnormal muscle differentiation in RMS, as it provides insights into fundamental mechanisms of RMS malignancy, its remarkable phenotypic diversity and, ultimately, opportunities for therapeutic intervention.

The Different Impact of ERK Inhibition on Neuroblastoma, Astrocytoma, and Rhabdomyosarcoma Cell Differentiation.

Aberrant ERK activity can lead to uncontrolled cell proliferation, immortalization, and impaired cell differentiation. Impairment of normal cell differentiation is one of the critical stages in malignant cell transformation. In this study, we investigated a relationship between ERK tyrosine kinase activity and the main differentiation features (changes in cell morphology and expression of genes encoding differentiation markers and growth factor receptors) in SH-SY5Y neuroblastoma, U-251 astrocytoma, and TE-671 rhabdomyosarcoma cells. ERK activity was assessed using a reporter system that enabled live measurements of ERK activity in single cells. We demonstrated that suppression of ERK activity by selective ERK inhibitors, in contrast to a commonly used differentiation inducer, retinoic acid, leads to significant changes in TE-671 cell morphology and expression of the myogenic differentiation marker genes PROM1, MYOG, and PAX7. There was a relationship between ERK activity and morphological changes at an individual cell level. In this case, SH-SY5Y cell differentiation induced by retinoic acid was ERK-independent. We showed that ERK inhibition increases the sensitivity of TE-671 cells to the EGF, IGF-1, and NGF growth factors, presumably by reducing basal ERK activity, and to the BDNF growth factor, by increasing expression of the TrkB receptor.

EGR1 Interacts with TBX2 and Functions as a Tumor Suppressor in Rhabdomyosarcoma

BackgroundEGR1, one of the immediate‐early response genes, can function as a tumor suppressor gene or as an oncogene in cancer. The function of EGR1 has not been fully characterized in rhabdomyosarcoma (RMS), a pediatric cancer derived from the muscle linage.MethodsEGR1 expression was characterized by RNA and protein analysis in RMS cells. EGR1 was ectopically expressed in RH30 cells by transfection of an EGR1 expression plasmid and selection of stable cell lines. A combination of techniques such as proliferation assays, scratch assays and soft agar assays were performed with RH30 cells expressing EGR1 to demonstrate the loss of oncogenic growth properties. Differentiation of RMS cells was detected by immunohistochemistry and RNA and protein analysis. Co‐immunoprecipitation studies were used to identify the interaction between EGR1 and TBX2 and gene expression changes were detected by RNA and protein analysis. TUNEL assays and RNA and protein analysis were used to characterize apoptosis in EGR1 expressing RH30 cells.ResultsWe found that EGR1 is downregulated in the alveolar RMS (ARMS) subtype but expressed at levels comparable to normal skeletal muscle in embryonal RMS (ERMS). We found that over expression of EGR1 in ARMS significantly decreased cell proliferation, mobility, and anchorage‐independent growth. EGR1 also promoted differentiation in RMS cells by inducing the expression of several genes involved in muscle differentiation including myosin heavy chain (MHC), MYOD1 and MYOG. We also found that EGR1 interacts with TBX2, which we have shown functions as an oncogene in RMS. The interaction inhibits EGR1 dependent gene expression, which includes the cell cycle regulators CDKN1A and PTEN as well as other important cell growth drivers such as NDRG1 and CST6. We also found that EGR1 induces apoptosis by triggering the intrinsic apoptosis pathway. EGR1 also sensitized RMS cells to chemotherapeutic agents.ConclusionsWe show that EGR1 is a potent tumor suppressor in RMS and that EGR1 and TBX2 antagonize each other's function. Our data suggest that activation of EGR1 may improve the therapeutic targeting of RMS.Support or Funding InformationHigher Committee for Education Development in Iraq (HCED)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A novel retinoid X receptor agonist, UAB30, inhibits rhabdomyosarcoma cells in vitro

BackgroundWhile patients with early-stage rhabdomyosarcoma (RMS) have seen steady improvement in prognosis over the last 50 y, those with advanced-stage or high-grade disease continue to have a dismal prognosis. Retinoids have been shown to cause growth suppression and terminal differentiation in RMS cells, but the toxicities associated with retinoic acid limit its use. Rexinoids provide an alternative treatment approach to retinoic acid. Rexinoids primarily bind the retinoid X receptor with minimal retinoic acid receptor binding, the entity responsible for many of the toxicities of retinoid therapies. UAB30 is a novel rexinoid with limited toxicities. We hypothesized that UAB30 would lead to decreased cell survival in RMS. Materials and methodsTwo RMS cell lines, one embryonal (RD) subtype and one alveolar (St. Jude Cancer Research Hospital 30) subtype, were used. Cells were treated with UAB30, and cytotoxicity, proliferation, mobility, and apoptosis were evaluated. ResultsUAB30 significantly decreased RMS tumor cell viability and proliferation. Invasion, migration, and attachment-independent growth were reduced following UAB30 treatment. UAB30 also resulted in apoptosis and G1 cell cycle arrest. UAB30 affected both the alveolar and embryonal RMS cell lines in a similar fashion. ConclusionsThe results of these studies suggest a potential therapeutic role for the low-toxicity synthetic retinoid X receptor selective agonist, UAB30, in RMS treatment.

SRC family kinase (SFK) inhibition reduces rhabdomyosarcoma cell growth in vitro and in vivo and triggers p38 MAP kinase-mediated differentiation.

Recent data suggest that SRC family kinases (SFKs) could represent potential therapeutic targets for rhabdomyosarcoma (RMS), the most common soft-tissue sarcoma in children. Here, we assessed the effect of a recently developed selective SFK inhibitor (a pyrazolo[3,4-d]pyrimidine derivative, called SI221) on RMS cell lines. SI221, which showed to be mainly effective against the SFK member YES, significantly reduced cell viability and induced apoptosis, without affecting non-tumor cells, such as primary human skin fibroblasts and differentiated C2C12 cells. Moreover, SI221 decreased in vitro cell migration and invasion and reduced tumor growth in a RMS xenograft model. SFK inhibition also induced muscle differentiation in RMS cells by affecting the NOTCH3 receptor-p38 mitogen-activated protein kinase (MAPK) axis, which regulates the balance between proliferation and differentiation. Overall, our findings suggest that SFK inhibition, besides reducing RMS cell growth and invasive potential, could also represent a differentiation therapeutic strategy for RMS.

Alternative Splicing of MEF2C pre-mRNA Controls Its Activity in Normal Myogenesis and Promotes Tumorigenicity in Rhabdomyosarcoma Cells

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Many cellular disruptions contribute to the progression of this pediatric cancer, including aberrant alternative splicing. The MEF2 family of transcription factors regulates many developmental programs, including myogenesis. MEF2 gene transcripts are subject to alternate splicing to generate protein isoforms with divergent functions. We found that MEF2Cα1 was the ubiquitously expressed isoform that exhibited no myogenic activity and that MEF2Cα2, the muscle-specific MEF2C isoform, was required for efficient differentiation. We showed that exon α in MEF2C was aberrantly alternatively spliced in RMS cells, with the ratio of α2/α1 highly down-regulated in RMS cells compared with normal myoblasts. Compared with MEF2Cα2, MEF2Cα1 interacted more strongly with and recruited HDAC5 to myogenic gene promoters to repress muscle-specific genes. Overexpression of the MEF2Cα2 isoform in RMS cells increased myogenic activity and promoted differentiation in RMS cells. We also identified a serine protein kinase, SRPK3, that was down-regulated in RMS cells and found that expression of SRPK3 promoted the splicing of the MEF2Cα2 isoform and induced differentiation. Restoration of either MEF2Cα2 or SPRK3 inhibited both proliferation and anchorage-independent growth of RMS cells. Together, our findings indicate that the alternative splicing of MEF2C plays an important role in normal myogenesis and RMS development. An improved understanding of alternative splicing events in RMS cells will potentially reveal novel therapeutic targets for RMS treatment.

Abstract 3558: The role of miRNA in PAX3-FKHR positive rhabdomyosarcoma

Abstract Rhabdomyosarcoma is the most commonly diagnosed pediatric soft tissue cancer. Tumors may develop in several locations, but all tumors express markers of undifferentiated skeletal muscle tissue. Of the two main rhabdomyosarcoma subtypes, the alveolar subtype leads to a much poorer prognosis than the embryonal subtype. Alveolar rhabdomyosarcoma is characterized by a chromosomal translocation between either chromosomes 1 and 13, or more commonly 2 and 13, which creates the fusion genes PAX7-FKHR or PAX3-FKHR, respectively. The fusion genes encode a protein that contains the PAX DNA binding domain and the FKHR transactivation domain, but the mechanism of disease of the fusion protein has not yet been fully elucidated. Here we use gain of function and loss of function techniques to study the effect of PAX3-FKHR on miRNA expression. Using small RNA-seq, we show that PAX3-FKHR expression leads to an upregulation of microRNAs mir-495 and miR-543. Examination of the mir-495 and mir-543 genes revealed conserved PAX3 binding sites. We further investigate the effect of the microRNAs on rhabdomyosarcoma cell differentiation and tumorigenicity. Citation Format: Shannon Muir, Jason Nathanson, Melissa Wilbert, Gene Yeo, Frank Furnari, Karen Arden, Webster Cavenee. The role of miRNA in PAX3-FKHR positive rhabdomyosarcoma. [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 3558. doi:10.1158/1538-7445.AM2014-3558

Overexpression of the skNAC gene in human rhabdomyosarcoma cells enhances their differentiation potential and inhibits tumor cell growth and spreading.

Skeletal and heart muscle-specific variant of the alpha subunit of nascent polypeptide complex (skNAC) is exclusively present in striated muscle cells. During skeletal muscle cell differentiation, skNAC expression is strongly induced, suggesting that the protein might be a regulator of the differentiation process. Rhabdomyosarcoma is a tumor of skeletal muscle origin. Since there is a strong inverse correlation between rhabdomyosarcoma cell differentiation status and metastatic potential, we analyzed skNAC expression patterns in a set of rhabdomyosarcoma cell lines: Whereas RD/12 and RD/18 cells showed a marked induction of skNAC gene expression upon the induction of differentiation-similarly as the one seen in nontransformed myoblasts-skNAC was not induced in CCA or Rh30 cells. Overexpressing skNAC in CCA and Rh30 cells led to a reduction in cell cycle progression and cell proliferation accompanied by an upregulation of specific myogenic differentiation markers, such as Myogenin or Myosin Heavy Chain. Furthermore, in contrast to vector-transfected controls, a high percentage of the cells formed long, Myosin Heavy Chain-positive, multinucleate myotubes. Consistently, soft agar assays revealed a drop in the metastatic potential of skNAC-overexpressing cells. Taken together, these data indicate that reconstitution of skNAC expression can enhance the differentiation potential of rhabdomyosarcoma cells and reduces their metastatic potential, a finding which might have important therapeutic implications.

MiR-206 integrates multiple components of differentiation pathways to control the transition from growth to differentiation in rhabdomyosarcoma cells

BackgroundSimilar to replicating myoblasts, many rhabdomyosarcoma cells express the myogenic determination gene MyoD. In contrast to myoblasts, rhabdomyosarcoma cells do not make the transition from a regulative growth phase to terminal differentiation. Previously we demonstrated that the forced expression of MyoD with its E-protein dimerization partner was sufficient to induce differentiation and suppress multiple growth-promoting genes, suggesting that the dimer was targeting a switch that regulated the transition from growth to differentiation. Our data also suggested that a balance between various inhibitory transcription factors and MyoD activity kept rhabdomyosarcomas trapped in a proliferative state.MethodsPotential myogenic co-factors were tested for their ability to drive differentiation in rhabdomyosarcoma cell culture models, and their relation to MyoD activity determined through molecular biological experiments.ResultsModulation of the transcription factors RUNX1 and ZNF238 can induce differentiation in rhabdomyosarcoma cells and their activity is integrated, at least in part, through the activation of miR-206, which acts as a genetic switch to transition the cell from a proliferative growth phase to differentiation. The inhibitory transcription factor MSC also plays a role in controlling miR-206, appearing to function by occluding a binding site for MyoD in the miR-206 promoter.ConclusionsThese findings support a network model composed of coupled regulatory circuits with miR-206 functioning as a switch regulating the transition from one stable state (growth) to another (differentiation).

The effect of the NF-kappa B inhibitors curcumin and lactacystin on myogenic differentiation of rhabdomyosarcoma cells

Rhabdomyosarcoma is a soft tissue sarcoma mainly seen in children. Despite considerable progress within the last few years, therapeutic approaches for this type of tumor are still limited. The respective tumor cells originate from myogenic precursor cells and are characterized by a blockade in their differentiation program. Interestingly, there is a direct inverse correlation between the differentiation status of a specific rhabdomyosarcoma cell and its metastatic potential. Thus, here, we tested whether the ubiquitous transcription factor NF-κB, which regulates myogenic differentiation and is also a promising therapeutic target in the treatment of other types of tumors, might be an interesting candidate for the development of novel rhabdomyosarcoma treatment strategies. For this purpose, we analyzed NF-κB activity (classical pathway) in myoblasts with different differentiation potential, specifically in three different rhabdomyosarcoma cell lines. In addition, we inhibited NF-κB activity in these cells and analyzed the effects on myogenic differentiation. We show that after the induction of differentiation, NF-κB activity declines rapidly in normal myoblasts, but only slightly in rhabdomyosarcoma cells. However, after treatment of the cells with two different small-molecule NF-κB-inhibiting compounds, the IKK inhibitor curcumin and the proteasome inhibitor lactacystin, we found that neither curcumin nor lactacystin promoted myogenic differentiation in either normal myoblasts or rhabdomyosarcoma cells. Taken together, our data suggest that treatment with curcumin or lactacystin might not be a suitable approach in the treatment of rhabdomyosarcoma.

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.

Abstract 1985: The activation of GSK3 by HGF-MET axis is responsible for differentiation of rhabdomyosarcoma cells

Abstract Introduction: Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma among children. At the late stages (metastatic) the outcome for the patients is very poor. The molecular basis for loss of cell differentiation in the process of carcinogenesis is not well understood. Differentiation status of rhabdomyosarcoma cells strongly influences its progression and metastatic ability and therefore the overall patient survival. At the onset of myogenic differentiation MET receptor is rapidly downregulated and on the other hand the activation of MET receptor in rhabdomyosarcoma has been shown to influence proliferation, survival and migration. Aim: Dissecting the role of HGF-MET receptor axis and GSK3 activation in the process of RMS differentiation. Materials and methods: Cell lines used in experiments: RH30 (ARMS), SMS-CTR (ERMS). Real time RT-PCR and western blotting were used to evaluate gene expression and the activation of various intracellular signaling pathways, respectively. The evaluation of protein activation in RMS cell lines was performed after stimulation with HGF. Results: We found that, during differentiation process the expression of MyoD, characteristic for poorly differentiated rhabdomyosarcoma cells decreased and the expression of myogenin, protein characteristic for differentiated/ matured muscle cells increased. That was paralleled by decreased expression of MET receptor. In RMS cell lines with stable downregulation of MET receptor the expression of differentiation related genes was similar to differentiated RMS cells. When we study the activation of GSK3 we observed the phosphorylation of GSK3 beta on serine 9 after HGF stimulation. The intracellular pathway responsible for GSK3 beta phosphorylation turn out to be PI3K-AKT dependent. We observed also the accumulation of beta-catenin in the nucleus of RMS cells stimulated with HGF. This effect was father augmented when HGF was used together with BIO, small molecular inhibitor of GSK3 beta. Conclusion and future directions: In this study we have shown that the expression and signaling of MET receptor is responsible for differentiation of RMS cells. Moreover we postulate that MET receptor exerts its influence on RMS cells differentiation through GSK3 beta activation. 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 blocking MET receptor could inhibit growth of RMS tumors. Finally, we think that also GSK3 beta could be used as the new therapeutic target to induce differentiation of RMS. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1985. doi:10.1158/1538-7445.AM2011-1985

Distinct roles for miR‐1 and miR‐133a in the proliferation and differentiation of rhabdomyosarcoma cells

Rhabdomyosarcoma is the most common soft tissue sarcoma in the pediatric population. As this tumor has an undifferentiated myogenic phenotype, agents that promote differentiation hold particular promise as part of a novel therapeutic approach to combat this type of cancer. In this report, we focus on the contribution of two microRNAs (miRNAs) in rhabdomyosarcomas. Levels of miR-1 and miR-133a are drastically reduced in representative cell lines from each major rhabdomyosarcoma subtype (embryonal and alveolar). Introduction of miR-1 and miR-133a into an embryonal rhabdomyosarcoma-derived cell line is cytostatic, thereby suggesting a tumor suppressor-like role for these myogenic miRNAs. Transcriptional profiling of cells after miR-1 and miR-133a expression reveals that miR-1 (but not miR-133a) exerts a strong promyogenic influence on these poorly differentiated tumor cells. We identify mRNAs that are down-regulated by these miRNAs and propose roles for miR-1 and miR-133a in repressing isoforms of genes that are normally not expressed in muscle. Finally, we show that mRNA targets of miR-1 and miR-133a are up-regulated in rhabdomyosarcomas, suggesting a causative role for these miRNAs in the development of rhabdomyosarcomas. More important, these results point to the promise of enhancing rhabdomyosarcoma therapy using miRNAs as agents that mediate cytostasis and promote muscle differentiation.

Abstract A112: Cell differentiation and downregulation of MET receptor as novel approaches for rhabdomyosarcoma treatment

Abstract 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&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.

MyoD and E-protein heterodimers switch rhabdomyosarcoma cells from an arrested myoblast phase to a differentiated state

Rhabdomyosarcomas are characterized by expression of myogenic specification genes, such as MyoD and/or Myf5, and some muscle structural genes in a population of cells that continues to replicate. Because MyoD is sufficient to induce terminal differentiation in a variety of cell types, we have sought to determine the molecular mechanisms that prevent MyoD activity in human embryonal rhabdomyosarcoma cells. In this study, we show that a combination of inhibitory Musculin:E-protein complexes and a novel splice form of E2A compete with MyoD for the generation of active full-length E-protein:MyoD heterodimers. A forced heterodimer between MyoD and the full-length E12 robustly restores differentiation in rhabdomyosarcoma cells and broadly suppresses multiple inhibitory pathways. Our studies indicate that rhabdomyosarcomas represent an arrested progress through a normal transitional state that is regulated by the relative abundance of heterodimers between MyoD and the full-length E2A proteins. The demonstration that multiple inhibitory mechanisms can be suppressed and myogenic differentiation can be induced in the RD rhabdomyosarcomas by increasing the abundance of MyoD:E-protein heterodimers suggests a central integrating function that can be targeted to force differentiation in muscle cancer cells.

Synthesis and evaluation of new 5-fluorouracil antitumor cell differentiating derivatives

Three new antitumour drugs containing two 5-fluorouracil moieties at both ends of the structure and a two amide bond linker were synthesized. Appropriated bis-acetal were reacted with two equivalents of 5-FU to afford the desired compounds. These drugs were evaluated for their ability to induce myogenic maturation in vitro on human rhabdomyosarcoma cells in an experimental model. Compounds 5 and 6 induced morphological and phenotypical differentiation in rhabdomyosarcoma cells at 4.5 and 3.5 μM, respectively. These new cell differentiating agents could be used as an alternative to selective destruction of undifferentiated cells. A potential role of the differentiation therapy as an alternative approach to the treatment of rhabdomyosarcomas is suggested.

Induction of Terminal Differentiation by the c-Jun Dimerization Protein JDP2 in C2 Myoblasts and Rhabdomyosarcoma Cells

Muscle cell differentiation is a result of a complex interplay between transcription factors and cell signaling proteins. Proliferating myoblasts must exit from the cell cycle prior to their differentiation. The muscle regulatory factor and myocyte enhancer factor-2 protein families play a major role in promoting muscle cell differentiation. Conversely, members of the AP-1 family of transcription factors that promote cell proliferation antagonize muscle cell differentiation. Here we tested the role of the c-Jun dimerization protein JDP2 in muscle cell differentiation. Endogenous expression of JDP2 was induced in both C2C12 myoblast and rhabdomyosarcoma (RD) cells programmed to differentiate. Ectopic expression of JDP2 in C2C12 myoblast cells inhibited cell cycle progression and induced spontaneous muscle cell differentiation. Likewise, constitutive expression of JDP2 in RD cells reduced their tumorigenic characteristics and restored their ability to differentiate into myotubes. JDP2 potentiated and synergized with 12-O-tetradecanoylphorbol-13-acetate to induce muscle cell differentiation of RD cells. In addition, JDP2 induced p38 activity in both C2 and RD cells programmed to differentiate. This is the first demonstration of a single transcription factor that rescues the myogenic program in an otherwise non-differentiating cancer cell line. Our results indicate that the JDP2 protein plays a major role in promoting skeletal muscle differentiation via its involvement in cell cycle arrest and activation of the myogenic program.

From a classic approach in cancer chemotherapy towards differentiation therapy: acyclic and cyclic seven-membered 5-fluorouracil O,N-acetals.

Novel derivatives of 5-fluorouracil (5-FU) possessing a broader spectrum of antitumor activity and fewer toxic side effects than 5-FU have been sought. Herein, we report three different types of 5-FU O,N-acetals: a) a novel class of 5-fluorouracil-containing acyclonucleosides. The antitumor activities of such compounds were assessed against HEp human cells showing that (RS)-1-¿[3-(2-hydroxyethoxy)-1-cyclopentoxy]propyl¿-5-fluorouracil 3c is 4-fold more active than 5-FU. (RS)-1-¿[3-(2-hydroxyethoxy)-1-isopropoxy]propyl¿-5-fluorouracil 3b has important potential advantages over 5-FU because of its lower toxicity and its ability to induce myogenic differentiation in rhabdomyosarcoma cells. Our results suggest that this drug may be useful for differentiation therapy in this type of tumor; b) within the cyclic prodrugs of 5-FU, a series of new ring-expanded isosteres (1,4-oxaheteroepanes) of Ftorafur were synthesized. The level of diastereoselectivity in the preparation of cis and trans 1-(3-chloromethyl)-1,4-dioxepan-5-yl)-5-fluorouracil, although modest, suggests a potentially general approach for controlling the stereochemistry of this unexplored class of reactions involving the preparation of 5-FU seven-membered O,N-acetals; c) new 5-FU acyclic analogs containing two 5-FU moieties at both ends of the molecules with a linker having two amide bonds have been designed and synthesized. These bis(5-FU-O,N-acetals) show interesting antineoplastic activities against the HT-29 cell line.

TGF-beta autocrine loop regulates cell growth and myogenic differentiation in human rhabdomyosarcoma cells.

Transforming growth factor beta (TGF) is a well-known inhibitor of myogenic differentiation as well as an autocrine product of rhabdomyosarcoma cells. We studied the role of the TGF-beta autocrine loop in regulating growth and myogenic differentiation in the human rhabdomyosarcoma cell line, RD. We previously reported that the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) induces growth arrest and myogenic differentiation in these cells, which constitutively express muscle regulatory factors. We show that TPA inhibits the activation of secreted latent TGF-beta, thus decreasing the concentration of active TGF-beta to which the cells are exposed. This event is mediated by the TPA-induced alteration of the uPA/PAI serine-protease system. Complete removal of TGF-beta, mediated by the ectopic expression of a soluble type II TGF-beta receptor dominant negative cDNA, induces growth arrest, but does not trigger differentiation. In contrast, a reduction in the TGF-beta concentration, to a range of 0.14-0.20 x 10(-2) ng/ml (which is similar to that measured in TPA-treated cells), mimics TPA-induced differentiation. Taken together, these data demonstrate that cell growth and suppression of differentiation in rhabdomyosarcoma cells require overproduction of active TGF-beta; furthermore, they show that a 'critical' concentration of TGF-beta is necessary for myogenic differentiation to occur, whereas myogenesis is abolished below and above this concentration. By impairing the TGF-beta autocrine loop, TPA stabilizes the factor concentration within the range compatible for differentiation to occur. In contrast, in human primary muscle cells a much higher concentration of exogenous TGF-beta is required for the differentiation inhibitory effect and TPA inhibits differentiation in these cells probably through a TGF-beta independent mechanism. These data thus clarify the mechanism underlying the multiple roles of TGF-beta in the regulation of both the transformed and differentiated phenotype.