Differentiation Of Rhabdomyosarcoma Research Articles

Novel small molecule DMAMCL induces differentiation in rhabdomyosarcoma by downregulating of DLL1

Rhabdomyosarcoma (RMS), a mesenchymal tumor occurring in the soft tissue of children, is associated with a defect in differentiation. This study unveils a novel anti-tumor mechanism of dimethylaminomicheliolide (DMAMCL), which is a water-soluble derivative of Micheliolide. First, we demonstrate that DMAMCL inhibits RMS cell growth without obvious cell death, leading to morphological alterations, enhanced expression of muscle differentiation markers, and a shift from a malignant to a more benign metabolic phenotype. Second, we detected decreased expression of DLL1 in RMS cells after DMAMCL treatment, known as a pivotal ligand in the Notch signaling pathway. Downregulation of DLL1 inhibits RMS cell growth and induces morphological changes similar to the effects of DMAMCL. Furthermore, DMAMCL treatment or loss of DLL1 expression also inhibits RMS xenograft tumor growth and augmented the expression of differentiation markers. Surprisingly, in C2C12 cells DMAMCL treatment or DLL1 downregulation also induces cell growth inhibition and an elevation in muscle differentiation marker expression. These data indicated that DMAMCL induced RMS differentiation and DLL1 is an important factor for RMS differentiation, opening a new window for the clinical use of DMAMCL as an agent for differentiation-inducing therapy for RMS treatment.

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.

Genomic and epigenetic changes drive aberrant skeletal muscle differentiation in rhabdomyosarcoma.

Genomic and epigenetic changes drive aberrant skeletal muscle differentiation in rhabdomyosarcoma.

Abstract A003: ASAP1 regulates myogenic differentiation in rhabdomyosarcoma by modulating YAP localization

Abstract Despite aggressive, multimodal therapies, the prognosis of patients with refractory or recurrent rhabdomyosarcoma (RMS) has not improved in four decades. RMS is thought to arise due to defective differentiation in skeletal muscle precursor cells. Differentiation-inducing therapy may improve outcomes for RMS patients with advanced disease. In RAS-mutant PAX fusion-negative RMS (FN-FMS), targeting ERK1/2 activation through MEK1/2 inhibition (MEKi) induces differentiation, slows tumor growth, and extends survival in preclinical studies. However, the duration of therapeutic response is short lived. Identifying additional targets for differentiation therapy is necessary. We propose that ASAP1, an Arf GTPase-activating protein (Arf GAP) highly expressed in FN-RMS and implicated in breast and colorectal cancer progression as well as osteogenic and retinal endothelium differentiation, contributes to FN-RMS differentiation. We find that knockdown (KD) of ASAP1 inhibits differentiation in myoblasts and FN-RMS cells, and its overexpression enhances differentiation. Moreover, myogenic differentiation-associated genes are not enriched upon loss of ASAP1. We discover that KD of ASAP1 homologs, ASAP2 and ASAP3, also blocks differentiation. However, loss of a paralogous Arf GAP, ARAP1, does not, indicating that ASAP regulates differentiation through a mechanism not explained by GAP activity alone. Interestingly, KD of Arf1 or Arf5, small GTPases inactivated by ASAP, also blocks differentiation of FN-RMS, suggesting a novel relationship between Arf and ASAP. Using RNAseq, qPCR, and immunoblotting techniques, we show that loss of ASAP blocks myogenic transcription factor expression. To determine the mechanism by which ASAP1 regulates myogenic transcription factor expression, we investigate the PI3K/AKT, MAPK, and Hippo pathways, which are known to be regulated by ASAP1. While the PI3K/AKT and MAPK pathways are unaffected, downstream components of the Hippo pathway are modulated by ASAP1 KD in FN-RMS cells treated with MEKi. YAP and TAZ are transcriptional coactivators that promote proliferation. Upon Hippo pathway activation, YAP/TAZ activity is blocked by phosphorylation at a nuclear exclusion site. Using cell fractionation and immunoblotting techniques, we find that induction of differentiation by MEKi increases TAZ phosphorylation, excluding it from the nucleus and blocking pro-proliferative transcription. Further, upon ASAP1 KD, TAZ phosphorylation is blocked, restoring nuclear localization, and inhibiting MEKi-induced differentiation. In conclusion, we discover that ASAP1 regulates MEKi-induced differentiation of FN-RMS cells by modulating TAZ localization and supports targeting the YAP pathway as a strategy for FN-RMS differentiation therapy. Our work also identifies ASAP1 as a potential effector of Arf1 activity, a novel interaction of these two proteins. The results described herein provide a deeper understanding of differentiation in FN-RMS and establish the groundwork for advancing differentiation therapy in FN-RMS. Citation Format: Katie E. Hebron, Olivia Feehan-Nelson, Angela Kim, Xiaoying Jian, Sofia A Girald, Paul Randazzo, Marielle E. Yohe. ASAP1 regulates myogenic differentiation in rhabdomyosarcoma by modulating YAP localization [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr A003.

Abstract 6193: TWIST2 promotes tumor progression and represses myogenic differentiation in rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS) is an aggressive pediatric soft tissue sarcoma of myoblast origin. RMS tumors are unable to terminally differentiate into syncytial muscle despite elevated expression of the muscle master regulators, MyoD and Myf5. Fusion-positive RMS (FP-RMS) and fusion-negative RMS (FN-RMS) have emerged as the two major RMS subtypes. The pathogenesis of FP-RMS is commonly defined by the balanced translocation of chromosomes 2 and 3 resulting in the misexpression of a chimeric PAX3/7-FOXO1 oncoprotein that derails normal differentiation pathways. However, the molecular basis of FN-RMS is poorly understood as single nucleotide variations in genes like KRAS, NRAS, and TP53 have limited recurrence (less than 10% of all patient tumors). In contrast, DNA copy number variations are highly recurrent and the genes they encode represent vulnerabilities in FN-RMS. Strikingly, we discovered that 72% of FN-RMS patient tumors harbor copy number amplifications at loci of TWIST1, TWIST2, or both, resulting in the upregulation of TWIST gene expression. We demonstrated that depletion of TWIST2 using a doxycycline inducible shRNA system caused cell cycle arrest and increased myogenic differentiation in xenograft models of FN-RMS. To interrogate whether TWIST2 can directly regulate the expression of these genes, we performed RNA-seq and ChIP-seq for TWIST2, H3K27Ac, and MYOD in TWIST2 knockdown and control FN-RMS cells. Utilizing this genome-wide data of chromatin binding, we comprehensively demonstrated that TWIST2 competes with MYOD preferentially at a subset of enhancers required for myogenic differentiation, causing repression of these loci. Interestingly, while MYOD binding is gained at myogenic loci in the absence of TWIST2, binding of MYOD is not lost at growth-promoting, oncogenic enhancers despite a decrease in their associated gene expression. This suggests that TWIST2 interacts with a different slew of transcription factors or chromatin regulators to activate gene expression. To interrogate this possibility, we are characterizing the TWIST2 protein interactome in FN-RMS cells using BioID as well as motifs enriched at TWIST2-regulated peaks. By studying TWIST2-associated pathways that affect RMS growth and differentiation, we see an opportunity to unlock novel therapeutic strategies for RMS. Citation Format: Akansha Shah, Lei Guo, Priscilla Jaichander, Kenian Chen, Karla Cano Hernandez, Lin Xu, Rhonda Bassel-Duby, Eric Olson, Ning Liu. TWIST2 promotes tumor progression and represses myogenic differentiation in rhabdomyosarcoma [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 6193.

PAX7 Is a Sensitive Marker of Skeletal Muscle Differentiation in Rhabdomyosarcoma and Tumors With Rhabdomyosarcomatous Differentiation in the Female Genital Tract.

In the female genital tract, rhabdomyosarcoma may occur in "pure" form or as a heterologous constituent of a biphasic neoplasm such as carcinosarcoma or adenosarcoma. Discriminating rhabdomyosarcoma from its histologic mimics relies on confirmation of skeletal muscle differentiation by morphology or immunohistochemistry (IHC), which can be challenging to interpret in some cases owing to limited expression. PAX7, a transcription factor expressed in mammalian muscle progenitor cells, has been reported in up to 86% of soft tissue rhabdomyosarcomas by IHC. To determine whether PAX7 IHC could augment current approaches to identify rhabdomyosarcoma in gynecologic malignancies, we assessed PAX7, myogenin, and MyoD1 IHC on whole tissue sections from 100 gynecologic tumors: 50 with rhabdomyosarcomatous differentiation and 50 with features mimicking rhabdomyosarcoma. PAX7 expression was present in 96% (48/50) of gynecologic tumors with rhabdomyosarcomatous differentiation and was absent in all rhabdomyosarcoma mimics; it was more diffusely expressed than myogenin in 16 cases and was positive in a greater percentage of tumor cells in 28 cases. PAX7 and myogenin were typically coexpressed, and no rhabdomyosarcoma exhibited complete absence of both markers; however, 2 myogenin-negative tumors were PAX7-postive. Morphologically, PAX7 localized to the nuclei of primitive-appearing cells, whereas myogenin was observed in maturing rhabdomyoblasts including strap cells. Our findings highlight the utility of PAX7 as a complementary diagnostic marker of rhabdomyosarcomatous differentiation in gynecologic tumors. PAX7 should be used in combination with other markers of skeletal muscle differentiation, namely myogenin, and may be particularly helpful in cases where myogenin and/or MyoD1 expression is limited.

Abstract B42: Mutant RAS represses CASZ1, a novel regulator of MYOD and MYOG, to inhibit embryonal rhabdomyosarcoma differentiation

Abstract Embryonal rhabdomyosarcoma (ERMS) is a childhood cancer that expresses myogenic master regulatory factor MYOD but fails to differentiate. Zinc finger transcription factor CASZ1 is a tumor suppressor and orchestrates cell-fate specification, commitment, and differentiation in many cell types during development. CASZ1 is expressed during skeletal myogenesis; however, whether CASZ1 plays a role in skeletal muscle cell differentiation and RMS pathogenesis is unknown. Here we uncover that CASZ1 crosstalks with myogenic regulatory factors Myf5, MyoD, and Myog to regulate myogenesis in C2C12 cells. Public transcriptome data show that CASZ1 mRNA levels are lower in ERMS compared to normal muscle (p<0.001). CASZ1 levels decrease upon oncogenic activation of RAS-MEK pathway and increase when ERMS undergoes terminal differentiation after MEK inhibitor trametinib (MEKi) treatment. Analysis of super-enhancer landscape in MEKi-induced differentiated ERMS cells reveals a core transcriptional regulatory circuitry (CRC) including CASZ1, MYOD, and MYOG. Loss of CASZ1 attenuates MEKi-induced myogenic programming in ERMS. Restoration of CASZ1 in ERMS cells induces muscle differentiation. ChIP-seq, ATAC-seq, and RNA-seq results show that CASZ1 directly upregulates expression of skeletal muscle differentiation genes and represses non-muscle genes through affecting chromatin accessibility, enhancer maintenance, and super-enhancer activity. Next-generation sequencing of primary RMS tumors identified a nonsynonymous single-nucleotide variant causing an arginine-to-cysteine switch (R25C). When evaluated in 293T and ERMS cells, R25C impairs CASZ1 nuclear localization and fails to activate target genes’ transcription. Tumor xenograft studies show that CASZ1 suppresses RMS tumor growth (p<0.005), while the CASZ1-R25C mutant has no effect. Taken together, loss of CASZ1 activity, due to RAS-MEK signaling or genetic alteration, impairs ERMS differentiation, contributing to RMS tumorigenesis. Citation Format: Zhihui Liu, Xiyuan Zhang, Haiyan Lei, Norris Lam, Oliver Yockey, Max Xu, Arnulfo Mendoza, Jun S Wei, Javed Khan, Marielle E. Yohe, Jack F. Shern, Carol J. Thiele. Mutant RAS represses CASZ1, a novel regulator of MYOD and MYOG, to inhibit embryonal rhabdomyosarcoma differentiation [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B42.

CASZ1 induces skeletal muscle and rhabdomyosarcoma differentiation through a feed-forward loop with MYOD and MYOG

Embryonal rhabdomyosarcoma (ERMS) is a childhood cancer that expresses myogenic master regulatory factor MYOD but fails to differentiate. Here, we show that the zinc finger transcription factor CASZ1 up-regulates MYOD signature genes and induces skeletal muscle differentiation in normal myoblasts and ERMS. The oncogenic activation of the RAS-MEK pathway suppresses CASZ1 expression in ERMS. ChIP-seq, ATAC-seq and RNA-seq experiments reveal that CASZ1 directly up-regulates skeletal muscle genes and represses non-muscle genes through affecting regional epigenetic modifications, chromatin accessibility and super-enhancer establishment. Next generation sequencing of primary RMS tumors identified a single nucleotide variant in the CASZ1 coding region that potentially contributes to ERMS tumorigenesis. Taken together, loss of CASZ1 activity, due to RAS-MEK signaling or genetic alteration, impairs ERMS differentiation, contributing to RMS tumorigenesis.

A rare malignant thyroid carcinosarcoma with aggressive behavior and DICER1 gene mutation: a case report with literature review

BackgroundMalignant biphasic tumor also known as carcinosarcoma is an uncommon neoplasm that is composed of both malignant epithelial and mesenchymal components. Most reported cases of carcinosarcoma affect the female genital tract; however, other sites including head and neck, lung, and breast have been described. Carcinosarcoma of the thyroid is an extremely rare and aggressive malignancy with an ominous clinical course similar to anaplastic carcinoma.Case presentationWe report a case of a 45-year-old female who was found to have a biphasic thyroid carcinosarcoma. Her clinical course declined significantly shortly after she underwent a total thyroidectomy and she developed distant metastases to the lungs. Histopathological features of the primary and metastatic tumor were identical. The tumor is composed of an intimately intermixed epithelial component of poorly differentiated follicular thyroid carcinoma and a spindle cell sarcoma with rhabdomyosarcoma differentiation. Molecular analysis using a next-generation sequencing based assay revealed a DICER1 (E1705K) point mutation in neoplastic cells.ConclusionTo our knowledge, the E1705K point mutation within the DICER1 gene is the first reported mutation in carcinosarcoma of the thyroid. A comprehensive review of the relevant literature is also included for discussion.

SPRY2 is a novel MET interactor that regulates metastatic potential and differentiation in rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is a predominantly pediatric soft-tissue cancer where the tumor cells exhibit characteristics of the developing skeletal muscle, and the two most common sub-types are embryonal and alveolar RMS. Elevated activation of the receptor tyrosine kinase (RTK) MET is frequent in RMS and is thought to cause increased tumor metastasis and lack of differentiation. However, the reasons underlying dysregulated MET expression and activation in RMS are not well understood. Therefore, we explored the role of Sprouty 2 (SPRY2), a modulator of RTK signaling, in regulating MET. We identify SPRY2 as a novel MET interactor that colocalizes with and binds MET in both embryonal and alveolar RMS. We find that depletion of SPRY2 leads to MET degradation, resulting in reduced migratory and clonogenic potential, and induction of differentiation in both embryonal and alveolar RMS, outcomes that are identical to depletion of MET. Activation of the ERK/MAPK pathway, known to be crucial for regulating cell migration and whose inhibition is required for myogenic differentiation, was downregulated upon depletion of MET or SPRY2. This provides a direct connection to the decreased migration and induction of differentiation upon depletion of MET or SPRY2. Thus, these data indicate that SPRY2 interacts with MET and stabilizes it in order to maintain signaling downstream of MET, which keeps the ERK/MAPK pathway active, resulting in metastatic potential and inhibition of differentiation in RMS. Our results identify a novel mechanism by which MET signaling is stabilized in RMS, and is a potential target for therapeutic intervention in RMS.

EWSR1 fusion proteins mediate PAX7 expression in Ewing sarcoma.

PAX7 is a paired-box transcription factor that is required for the developmental specification of adult skeletal muscle progenitors in mice. We previously demonstrated PAX7 expression as a marker of skeletal muscle differentiation in rhabdomyosarcoma. Here, using analyses of published whole-genome gene expression microarray data, we identify PAX7 as a gene with significantly increased expression in Ewing sarcoma in comparison to CIC-DUX4 round cell sarcoma. Analysis of PAX7 in a large cohort of 103 Ewing sarcoma cases by immunohistochemistry revealed expression in 99.0% of cases (102/103). PAX7 expression was noted in cases demonstrating three distinct Ewing sarcoma EWSR1 translocations involving FLI1, ERG, and NFATc2. No PAX7 expression was observed in any of 27 cases of CIC-DUX4 sarcoma by immunohistochemistry (0%; 0/27). Exploring the mechanism of PAX7 expression in Ewing sarcoma using curated RNA- and ChIP-sequencing data, we demonstrate that the EWSR1 fusion protein is required for PAX7 expression in Ewing sarcoma and identify a candidate EWSR1-FLI1-bound PAX7 enhancer that coincides with both a consensus GGAA repeat-containing binding site and a peak of regulatory H3K27 acetylation. Taken together, our findings provide mechanistic support for the utility of PAX7 immunohistochemistry in the diagnosis of Ewing sarcoma, while linking this sarcoma of uncertain histogenesis to a key transcriptional regulator of mammalian muscle progenitor cells.

CRISPR screen identifies the NCOR/HDAC3 complex as a major suppressor of differentiation in rhabdomyosarcoma

Dysregulated gene expression resulting from abnormal epigenetic alterations including histone acetylation and deacetylation has been demonstrated to play an important role in driving tumor growth and progression. However, the mechanisms by which specific histone deacetylases (HDACs) regulate differentiation in solid tumors remains unclear. Using pediatric rhabdomyosarcoma (RMS) as a paradigm to elucidate the mechanism blocking differentiation in solid tumors, we identified HDAC3 as a major suppressor of myogenic differentiation from a high-efficiency Clustered regularly interspaced short palindromic repeats (CRISPR)-based phenotypic screen of class I and II HDAC genes. Detailed characterization of the HDAC3-knockout phenotype in vitro and in vivo using a tamoxifen-inducible CRISPR targeting strategy demonstrated that HDAC3 deacetylase activity and the formation of a functional complex with nuclear receptor corepressors (NCORs) were critical in restricting differentiation in RMS. The NCOR/HDAC3 complex specifically functions by blocking myoblast determination protein 1 (MYOD1)-mediated activation of myogenic differentiation. Interestingly, there was also a transient up-regulation of growth-promoting genes upon initial HDAC3 targeting, revealing a unique cancer-specific response to the forced transition from a neoplastic state to terminal differentiation. Our study applied modifications of CRISPR/CRISPR-associated endonuclease 9 (Cas9) technology to interrogate the function of essential cancer genes and pathways and has provided insights into cancer cell adaptation in response to altered differentiation status. Because current pan-HDAC inhibitors have shown disappointing results in clinical trials of solid tumors, therapeutic targets specific to HDAC3 function represent a promising option for differentiation therapy in malignant tumors with dysregulated HDAC3 activity.

Abstract B05: MicroRNA-206 drives rhabdomyosarcoma differentiation through downregulation of PAX7

Abstract The goal of this work is to understand the mechanism of microRNA-206 (miR-206) induced differentiation in rhabdomyosarcoma (RMS). RMS is the most common soft tissue sarcoma of childhood with both histology and gene expression suggestive that RMS tumor cells resemble a developmental arrest of skeletal muscle. miR-206 is a skeletal muscle specific miRNA expression increases through normal muscle development and has decreased levels in RMS compared to normal skeletal muscle. Exogneous miR-206 replacement drives differentiation and cell cycle exit of RMS. However, the target genes responsible for the relief of the development arrest are largely unknown. Using a combinatorial approach with mRNA microarrays and tandem mass tag (TMT) proteome profiling we identified 165 mRNAs and 272 proteins downregulated in miR-206 mimic transfected RMS cells. Potential targets were validated with 3'UTR reporter assays as well as real-time PCR and immunoblot from miR-206 mimic transfected cells. Further analysis with genetic rescue identified the key targets responsible for the RMS differentiation block as PAX7, PAX3, CCND2, and NOTCH3. However, siRNA knockdown of these targets alone and in a pooled combination failed to induce differentiation to the same extent as miR-206 despite knockdown levels comparable to miR-206. Thus, target identification collectively is key to understanding the mechanism of miR-206 function in RMS, suggesting the one miRNA-one target model to be insufficient for the complex phenotype of miR-206 induced differentiation. Genetic deletion of miR-206 in a mouse model of RMS accelerated and exacerbated tumor development indicating that both in vitro and in vivo miR-206 possesses tumor suppressive properties. In addition, Notch3 and Pax7 levels were increased in knockout tumors compared to wild type, providing further evidence for these genes as key targets of miR-206 in RMS. These results illustrate that miR-206 relieves the differentiation arrest in RMS and suggests that miR-206 replacement therapy could be a potential therapeutic strategy. Citation Format: Jason A. Hanna, Matthew R. Garcia, Jonathan C. Go, David Finkelstein, Kiran Kodali, Vishwajeeth Pagala, Eric N. Olson, Junmin Peng, Mark E. Hatley. MicroRNA-206 drives rhabdomyosarcoma differentiation through downregulation of PAX7. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr B05.

Constitutive activation of MET signaling impairs myogenic differentiation of rhabdomyosarcoma and promotes its development and progression.

Rhabdomyosarcoma (RMS) is a soft tissue sarcoma, which may originate from impaired differentiation of mesenchymal stem cells (MSC). Expression of MET receptor is elevated in alveolar RMS subtype (ARMS) which is associated with worse prognosis, compared to embryonal RMS (ERMS). Forced differentiation of ARMS cells diminishes MET level and, as shown previously, MET silencing induces differentiation of ARMS. In ERMS cells introduction of TPR-MET oncogene leads to an uncontrolled overstimulation of the MET receptor downstream signaling pathways. In vivo, tumors formed by those cells in NOD-SCID mice display inhibited differentiation, enhanced proliferation, diminished apoptosis and increased infiltration of neutrophils. Consequently, tumors grow significantly faster and they display enhanced ability to metastasize to lungs and to vascularize due to elevated VEGF, MMP9 and miR-378 expression. In vitro, TPR-MET ERMS cells display enhanced migration, chemotaxis and invasion toward HGF and SDF-1. Introduction of TPR-MET into MSC increases survival and may induce expression of early myogenic factors depending on the genetic background, and it blocks terminal differentiation of skeletal myoblasts. To conclude, our results suggest that activation of MET signaling may cause defects in myogenic differentiation leading to rhabdomyosarcoma development and progression.

Treatment Outcomes in Completely Resected Stage I to Stage IV Uterine Carcinosarcoma With Rhabdomyosarcoma Differentiation

To evaluate overall survival (OS) and progression-free survival (PFS) after adjuvant therapy in stage I to stage IV uterine carcinosarcoma with rhabdomyosarcoma differentiation. Memorial Sloan-Kettering Cancer Center medical records from 1990 to 2012 were reviewed. Patients who received chemotherapy with or without radiation therapy (RT), or RT alone, for completely resected stage I to stage IV uterine carcinosarcoma with rhabdomyosarcoma differentiation were included. Of 53 patients, International Federation of Gynecology and Obstetrics stage distribution was as follows: I, 13 (24.5%); II, 8 (15.1%); III, 13 (24.5%); and IV, 19 (35.9%). Forty-one (77.4%) of 53 patients received adjuvant chemotherapy, and 34% of the patients who received chemotherapy also received pelvic RT or intravaginal brachytherapy (IVRT). Twelve (22.6%) of the 53 patients received only pelvic RT with/without IVRT. Paclitaxel-carboplatin was the most commonly used adjuvant chemotherapy treatment. The median PFS for the entire cohort was 13.4 months (95% confidence interval [CI], 10.5-17.0). The median OS for the entire cohort was 23.0 months (95% CI, 16.9-34.3). The median PFS periods by stage were 15.9 months for stages I/II versus 11.2 months for stages III/IV (P = 0.012). Median OS was not reached in the early-stage cohort. The median OS for the late-stage cohort was 20.9 months (P = 0.004). The median PFS periods by treatment were 10.4 months for pelvic RT with/without IVRT group versus 13.1 months for chemotherapy with/without pelvic RT with/without IVRT group (P = 0.498). The median OS periods by treatment were 23.6 months for chemotherapy with/without pelvic RT with/without IVRT group versus 16.9 months for pelvic RT with/without IVRT group (P = 0.501). The results suggest that chemotherapy alone or in combination with RT is associated with longer PFS and OS compared to RT alone. Only the stage of disease significantly affected PFS and OS.

Mechanisms of impaired differentiation in rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood, with presumed skeletal muscle origins, because of its myogenic phenotype. RMS is composed of two main subtypes, embryonal RMS (eRMS) and alveolar RMS (aRMS). Whereas eRMS histologically resembles embryonic skeletal muscle, the aRMS subtype is more aggressive and has a poorer prognosis. In addition, whereas the genetic profile of eRMS is not well established, aRMS is commonly associated with distinct chromosome translocations that fuse domains of the transcription factors Pax3 and Pax7 to the forkhead family member FOXO1A. Both eRMS and aRMS tumor cells express myogenic markers such as MyoD, but their ability to complete differentiation is impaired. How this impairment occurs is the subject of this review, which will focus on several themes, including signaling pathways that converge on Pax-forkhead gene targets, alterations in MyoD function, epigenetic modifications of myogenic promoters, and microRNAs whose expression patterns in RMS alter key regulatory circuits to help maintain tumor cells in an opportunistically less differentiated state.

TGF-β1 suppression of microRNA-450b-5p expression: a novel mechanism for blocking myogenic differentiation of rhabdomyosarcoma

Transforming growth factor beta 1 (TGF-β1) is the most potent inhibitor of myogenic differentiation (MyoD) of rhabdomyosarcoma (RMS); however, the underlying mechanisms of this inhibition remain unclear. In this study, we identified novel TGF-β1-related microRNAs (miRNAs); among these, miR-450b-5p is significantly regulated by TGF-β1. We provide evidence that TGF-β1 exerts it function by suppressing miR-450b-5p. Both in cultured cells and tumor implants, miR-450b-5p significantly arrested the growth of RMS and promoted its MyoD. Utilizing a bioinformatics approach, we identified miR-450b-5p target mRNAs. Among these candidates, only the expression of ecto-NOX disulfide-thiol exchanger 2 (ENOX2) and paired box 9 (PAX9) was augmented by miR-450b-5p knockdown examined by western blot; the engineered inhibition antagonized TGF-β1-mediated differentiation inhibition. Furthermore, we found that the Smad3 and Smad4 pathways, but not Smad2, are the principal mediator of TGF-β1 suppression of miR-450b-5p. Taken together, these results suggest that disrupting the TGF-β1 suppression of miR-450b-5p, or knockdown of ENOX2 and PAX9, are effective approaches in inducing RMS MyoD.

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.

Effect of Notch3 to regulate rhabdomyosarcoma growth in vitro and in vivo.

10068 Background: Rhabdomyosarcoma is a pediatric soft tissue sarcoma that originates from skeletal muscle progenitors that proliferate indefinitely. Notch signaling inhibits myogenesis and has been reported to be abnormally activated in rhabdomyosarcomas. However, the mechanism underlying the deregulation of Notch signaling in rhabdomyosarcoma remains unclear. Methods: The activation of Notch signaling was investigated in primary rhabdomyosarcomas and cell lines. The role of Notch was evaluated through gene silencing in tumor cell lines in vitro and on a xenografts model in vivo. Results: Notch3 and Notch1 were over-expressed and activated in rhabdomyosarcoma cell lines when compared to cultured myoblasts. Moreover, they were over-expressed, along with their target gene HES1, in a cohort of 5 fusion-positive and 24 fusion-negative rhabdomyosarcoma primary samples as compared to skeletal muscle tissues. Notch3 expression was significantly higher in rhabdomyosarcoma specimens with respect to Notch1, irrespective to the fusion-gene status (P<0.05). Furthermore, in the whole cohort, patients with HES1 expression levels over the median value showed poor overall survival and shorter recurrence free interval (P=0.0001 for both). Notch3 targeted down-regulation severely impaired proliferation of both alveolar and embryonal rhabdomyosarcoma cell lines hampering their tumorigenic properties in vitro and tumor xenografts growth in vivo. Conclusions: Altogether, these results support a role for Notch3 as an inhibitor of differentiation in rhabdomyosarcoma and identify the Notch3 signaling pathway as a new potential target for therapy in this soft tissue sarcoma.