A bioengineering method for modeling alveolar Rhabdomyosarcoma and assessing chemotherapy responses.
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ABSTRACT:
SUBMITTER: Stefanek E
PROVIDER: S-EPMC8374652 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Alveolar rhabdomyosarcoma (aRMS) is an aggressive myogenic childhood malignancy, not infrequently presenting as incurable metastatic disease. To identify therapeutic targets, we performed an unbiased tyrosine kinome RNA interference screen in primary cell cultures from a genetically engineered, conditional mouse model of aRMS. We identified ephrin receptor B4 (EphB4) as a target that is widely expressed in human aRMS and that portends a poor clinical outcome in an expression level-dependent manner. We also uncovered cross-talk of this ephrin receptor with another receptor tyrosine kinase, PDGFRβ, which facilitates PDGF ligand-dependent, ephrin ligand-independent activation of EphB4 converging on the Akt and Erk1/2 pathways. Conversely, EphB4 activation by its cognate ligand, EphrinB2, did not stimulate PDGFRβ; instead, apoptosis was paradoxically induced. Finally, we showed that small-molecule inhibition of both PDGFRβ and EphB4 by dasatinib resulted in a significant decrease in tumor cell viability in vitro, as well as decreased tumor growth rate and significantly prolonged survival in vivo. To our knowledge, these results are the first to identify EphB4 and its cross-talk with PDGFRβ as unexpected vital determinants of tumor cell survival in aRMS, with EphB4 at the crux of a bivalent signaling node that is either mitogenic or proapoptotic.
| S-EPMC4035936 | biostudies-literature
Project description:Despite advances in multi-modal treatment approaches, clinical outcomes of patients suffering from PAX3-FOXO1 fusion oncogene-expressing alveolar rhabdomyosarcoma (ARMS) remain dismal. Here we show that PAX3-FOXO1-expressing ARMS cells are sensitive to pharmacological ataxia telangiectasia and Rad3 related protein (ATR) inhibition. Expression of PAX3-FOXO1 in muscle progenitor cells is not only sufficient to increase sensitivity to ATR inhibition, but PAX3-FOXO1-expressing rhabdomyosarcoma cells also exhibit increased sensitivity to structurally diverse inhibitors of ATR. Mechanistically, ATR inhibition leads to replication stress exacerbation, decreased BRCA1 phosphorylation and reduced homologous recombination-mediated DNA repair pathway activity. Consequently, ATR inhibitor treatment increases sensitivity of ARMS cells to PARP1 inhibition in vitro, and combined treatment with ATR and PARP1 inhibitors induces complete regression of primary patient-derived ARMS xenografts in vivo. Lastly, a genome-wide CRISPR activation screen (CRISPRa) in combination with transcriptional analyses of ATR inhibitor resistant ARMS cells identifies the RAS-MAPK pathway and its targets, the FOS gene family, as inducers of resistance to ATR inhibition. Our findings provide a rationale for upcoming biomarker-driven clinical trials of ATR inhibitors in patients suffering from ARMS.
| S-EPMC9314382 | biostudies-literature
Project description:Many recurrent chromosome translocations in cancer result in the generation of fusion genes that are directly implicated in the tumorigenic process. Precise modeling of the effects of cancer fusion genes in mice has been inaccurate, as constructs of fusion genes often completely or partially lack the correct regulatory sequences. The reciprocal t(2;13)(q36.1;q14.1) in human alveolar rhabdomyosarcoma (A-RMS) creates a pathognomonic PAX3-FOXO1 fusion gene. In vivo mimicking of this translocation in mice is complicated by the fact that Pax3 and Foxo1 are in opposite orientation on their respective chromosomes, precluding formation of a functional Pax3-Foxo1 fusion via a simple translocation. To circumvent this problem, we irreversibly inverted the orientation of a 4.9 Mb syntenic fragment on chromosome 3, encompassing Foxo1, by using Cre-mediated recombination of two pairs of unrelated oppositely oriented LoxP sites situated at the borders of the syntenic region. We tested if spatial proximity of the Pax3 and Foxo1 loci in myoblasts of mice homozygous for the inversion facilitated Pax3-Foxo1 fusion gene formation upon induction of targeted CRISPR-Cas9 nuclease-induced DNA double strand breaks in Pax3 and Foxo1. Fluorescent in situ hybridization indicated that fore limb myoblasts show a higher frequency of Pax3/Foxo1 co-localization than hind limb myoblasts. Indeed, more fusion genes were generated in fore limb myoblasts via a reciprocal t(1;3), which expressed correctly spliced Pax3-Foxo1 mRNA encoding Pax3-Foxo1 fusion protein. We conclude that locus proximity facilitates chromosome translocation upon induction of DNA double strand breaks. Given that the Pax3-Foxo1 fusion gene will contain all the regulatory sequences necessary for precise regulation of its expression, we propose that CRISPR-Cas9 provides a novel means to faithfully model human diseases caused by chromosome translocation in mice.
| S-EPMC4319822 | biostudies-literature
Project description:The highly aggressive muscle cancer alveolar rhabdomyosarcoma (ARMS) is one of the most common soft tissue sarcoma of childhood, yet the outcome for the unresectable and metastatic disease is dismal and unchanged for nearly three decades. To better understand the pathogenesis of this disease and to facilitate novel preclinical approaches, we previously developed a conditional mouse model of ARMS by faithfully recapitulating the genetic mutations observed in the human disease, i.e., activation of Pax3:Fkhr fusion gene with either p53 or Cdkn2a inactivation. In this report, we show that this model recapitulates the immunohistochemical profile and the rapid progression of the human disease. We show that Pax3:Fkhr expression increases during late preneoplasia but tumor cells undergoing metastasis are under apparent selection for Pax3:Fkhr expression. At a whole-genome level, a cross-species gene set enrichment analysis and metagene projection study showed that our mouse model is most similar to human ARMS when compared with other pediatric cancers. We have defined an expression profile conserved between mouse and human ARMS, as well as a Pax3:Fkhr signature, including the target gene, SKP2. We further identified 7 "druggable" kinases overexpressed across species. The data affirm the accuracy of this genetically engineered mouse model.
| S-EPMC2789740 | biostudies-literature
Project description:Gene fusions and fusion products were thought to be unique features of neoplasia. However, more and more studies have identified fusion RNAs in normal physiology. Through RNA sequencing of 27 human noncancer tissues, a large number of fusion RNAs were found. By analyzing fusion transcriptome, we observed close clusterings between samples of same or similar tissues, supporting the feasibility of using fusion RNA profiling to reveal connections between biological samples. To put the concept into use, we selected alveolar rhabdomyosarcoma (ARMS), a myogenic pediatric cancer whose exact cell of origin is not clear. PAX3-FOXO1 (paired box gene 3 fused with forkhead box O1) fusion RNA, which is considered a hallmark of ARMS, was recently found during normal muscle cell differentiation. We performed and analyzed RNA sequencing from various time points during myogenesis and uncovered many chimeric fusion RNAs. Interestingly, we found that the fusion RNA profile of RH30, an ARMS cell line, is most similar to the myogenesis time point when PAX3-FOXO1 is expressed. In contrast, full transcriptome clustering analysis failed to uncover this connection. Strikingly, all of the 18 chimeric RNAs in RH30 cells could be detected at the same myogenic time point(s). In addition, the seven chimeric RNAs that follow the exact transient expression pattern as PAX3-FOXO1 are specific to rhabdomyosarcoma cells. Further testing with clinical samples also confirmed their specificity to rhabdomyosarcoma. These results provide further support for the link between at least some ARMSs and the PAX3-FOXO1-expressing myogenic cells and demonstrate that fusion RNA profiling can be used to investigate the etiology of fusion-gene-associated cancers.
| S-EPMC5135356 | biostudies-literature
Project description:Aqueous mistletoe extracts from the European mistletoe (Viscum album) contain mainly mistletoe lectins and viscotoxins as cytotoxic compounds. Lipophilic triterpene acids, which do not occur in conventional mistletoe preparations, were solubilised with β-cyclodextrins. The combination of an aqueous extract (viscum) and a triterpene-containing extract (TT) recreated a whole mistletoe extract (viscumTT). These extracts were tested on rhabdomyosarcoma in vitro, ex vivo, and in vivo with regard to anticancer effects. Viscum and viscumTT inhibited cell proliferation and induced apoptosis effectively in a dose-dependent manner in vitro and ex vivo, whereas TT showed only moderate inhibitory effects. viscumTT proved to be more effective than the single extracts and displayed a synergistic effect in vitro and a stronger effect in vivo. viscumTT induced apoptosis via the extrinsic and intrinsic pathways, evidenced by the loss of mitochondrial membrane potential and activation of CASP8 and CASP9. CASP10 inhibitor inhibited apoptosis effectively, emphasising the importance of CASP10 in viscumTT-induced apoptosis. Additionally, viscumTT changed the ratio of apoptosis-associated proteins by downregulation of antiapoptotic proteins such as XIAP and BIRC5, thus shifting the balance towards apoptosis. viscumTT effectively reduced tumour volume in patient-derived xenografts in vivo and may be considered a promising substance for rhabdomyosarcoma therapy.
| S-EPMC5534308 | biostudies-other
Project description:Genomic and clinical analyses of major amplification events in alveolar rhabdomyosarcoma Affymetrix SNP 50K XbaI chips were used to anaylyze 57 alveolar rhabdomyosarcoma samples and selected cases were further analyzed using Affymetrix SNP 250K StyI chips
2010-10-16 | E-GEOD-24715 | biostudies-arrayexpress
Project description:The development of three-dimensional cell culture techniques has allowed cancer researchers to study the stemness properties of cancer cells in in vitro culture. However, a method to grow PAX3-FOXO1 fusion-positive rhabdomyosarcoma (FP-RMS), an aggressive soft tissue sarcoma of childhood, has to date not been reported, hampering efforts to identify the dysregulated signaling pathways that underlie FP-RMS stemness. Here, we first examine the expression of canonical stem cell markers in human RMS tumors and cell lines. We then describe a method to grow FP-RMS cell lines as rhabdospheres and demonstrate that these spheres are enriched in expression of canonical stemness factors as well as Notch signaling components. Specifically, FP-RMS rhabdospheres have increased expression of SOX2, POU5F1 (OCT4), and NANOG, and several receptors and transcriptional regulators in the Notch signaling pathway. FP-RMS rhabdospheres also exhibit functional stemness characteristics including multipotency, increased tumorigenicity in vivo, and chemoresistance. This method provides a novel practical tool to support research into FP-RMS stemness and chemoresistance signaling mechanisms.
| S-EPMC7888706 | biostudies-literature
Project description:Alveolar rhabdomyosarcoma is an aggressive skeletal muscle cancer of childhood. Our initial studies of rhabdomyosarcoma gene expression for patients enrolled in a national clinical trial suggested that platelet-derived growth factor receptor A (PDGFR-A) may be a mediator of disease progression and metastasis. Using our conditional mouse tumor models that authentically recapitulate the primary mutations and metastatic progression of alveolar rhabdomyosarcomas in humans, we found by immunoblotting and immunokinase assays that PDGFR-A and its downstream effectors, mitogen-activated protein kinase and Akt, were highly activated in both primary and metastatic tumors. Inhibition of PDGFR-A by RNA interference, small molecule inhibitor or neutralizing antibody had a dramatic effect on tumor cell growth both in vitro and in vivo, although resistance evolved in one-third of tumors. These results establish proof-of-principal for PDGFR-A as a therapeutic target in alveolar rhabdomyosarcoma.
| S-EPMC2813858 | biostudies-literature
Project description:Alveolar rhabdomyosarcoma (aRMS) is a myogenic childhood sarcoma frequently associated with a translocation-mediated fusion gene, Pax3:Foxo1a.We investigated the complementary role of Rb1 loss in aRMS tumor initiation and progression using conditional mouse models.Rb1 loss was not a necessary and sufficient mutational event for rhabdomyosarcomagenesis, nor a strong cooperative initiating mutation. Instead, Rb1 loss was a modifier of progression and increased anaplasia and pleomorphism. Whereas Pax3:Foxo1a expression was unaltered, biomarkers of aRMS versus embryonal rhabdomyosarcoma were both increased, questioning whether these diagnostic markers are reliable in the context of Rb1 loss. Genome-wide gene expression in Pax3:Foxo1a,Rb1 tumors more closely approximated aRMS than embryonal rhabdomyosarcoma. Intrinsic loss of pRb function in aRMS was evidenced by insensitivity to a Cdk4/6 inhibitor regardless of whether Rb1 was intact or null. This loss of function could be attributed to low baseline Rb1, pRb and phospho-pRb expression in aRMS tumors for which the Rb1 locus was intact. Pax3:Foxo1a RNA interference did not increase pRb or improve Cdk inhibitor sensitivity. Human aRMS shared the feature of low and/or heterogeneous tumor cell pRb expression.Rb1 loss from an already low pRb baseline is a significant disease modifier, raising the possibility that some cases of pleomorphic rhabdomyosarcoma may in fact be Pax3:Foxo1a-expressing aRMS with Rb1 or pRb loss of function.
| S-EPMC4177545 | biostudies-literature