RESUMO
Rhabdomyosarcoma (RMS) is a solid tumor whose metastatic progression can be accelerated through interleukin-4 receptor alpha (Il4ra) mediated interaction with normal muscle stem cells (satellite cells). To understand the function of Il4ra in this tumor initiation phase of RMS, we conditionally deleted Il4ra in genetically-engineered RMS mouse models. Nullizygosity of Il4ra altered the latency, site and/or stage distribution of RMS tumors compared to IL4RA intact models. Primary tumor cell cultures taken from the genetically-engineered models then used in orthotopic allografts further defined the interaction of satellite cells and RMS tumor cells in the context of tumor initiation: in alveolar rhabdomyosarcoma (ARMS), satellite cell co-injection was necessary for Il4ra null tumor cells engraftment, whereas in embryonal rhabdomyosarcoma (ERMS), satellite cell co-injection decreased latency of engraftment of Il4ra wildtype tumor cells but not Il4ra null tumor cells. When refocusing on Il4ra wildtype tumors by single cell sequencing and cytokine studies, we have uncovered a putative signaling interplay of Il4 from T-lymphocytes being received by Il4ra + rhabdomyosarcoma tumor cells, which in turn express Ccl2, the ligand for Ccr2 and Ccr5. Taken together, these results suggest that mutations imposed during tumor initiation have different effects than genetic or therapeutic intervention imposed once tumors are already formed. We also propose that CCL2 and its cognate receptors CCR2 and/or CCR5 are potential therapeutic targets in Il4ra mediated RMS progression.
Assuntos
Subunidade alfa de Receptor de Interleucina-4 , Animais , Camundongos , Subunidade alfa de Receptor de Interleucina-4/genética , Subunidade alfa de Receptor de Interleucina-4/metabolismo , Rabdomiossarcoma/genética , Rabdomiossarcoma/patologia , Rabdomiossarcoma/metabolismo , Humanos , Células Satélites de Músculo Esquelético/metabolismo , Rabdomiossarcoma Alveolar/genética , Rabdomiossarcoma Alveolar/patologia , Rabdomiossarcoma Alveolar/metabolismo , Modelos Animais de Doenças , Rabdomiossarcoma Embrionário/genética , Rabdomiossarcoma Embrionário/patologia , Rabdomiossarcoma Embrionário/metabolismo , Transdução de Sinais , Receptores de Superfície CelularRESUMO
Alveolar rhabdomyosarcoma (ARMS), an invasive subtype of rhabdomyosarcoma (RMS), is associated with chromosomal translocation events resulting in one of two oncogenic fusion genes, PAX3-FOXO1 or PAX7-FOXO1. ARMS patients exhibit an overexpression of the pleiotropic cytokine transforming growth factor beta (TGF-ß). This overexpression of TGF-ß1 causes an increased expression of a downstream transcription factor called SNAIL, which promotes epithelial to mesenchymal transition (EMT). Overexpression of TGF-ß also inhibits myogenic differentiation, making ARMS patients highly resistant to chemotherapy. In this review, we first describe different types of RMS and then focus on ARMS and the impact of TGF-ß in this tumor type. We next highlight current chemotherapy strategies, including a combination of the FDA-approved drugs vincristine, actinomycin D, and cyclophosphamide (VAC); cabozantinib; bortezomib; vinorelbine; AZD 1775; and cisplatin. Lastly, we discuss chemotherapy agents that target the differentiation of tumor cells in ARMS, which include all-trans retinoic acid (ATRA) and 5-Azacytidine. Improving our understanding of the role of signaling pathways, such as TGF-ß1, in the development of ARMS tumor cells differentiation will help inform more tailored drug administration in the future.
Assuntos
Rabdomiossarcoma Alveolar , Rabdomiossarcoma , Humanos , Rabdomiossarcoma Alveolar/genética , Rabdomiossarcoma Alveolar/metabolismo , Rabdomiossarcoma Alveolar/patologia , Fator de Crescimento Transformador beta , Fator de Crescimento Transformador beta1 , Fatores de Transcrição Box Pareados/genética , Transição Epitelial-Mesenquimal , Rabdomiossarcoma/genética , Proteínas de Fusão Oncogênica/genéticaRESUMO
Neopetrotaurines A-C (1-3), unusual alkaloids possessing two isoquinoline-derived moieties that are linked via a unique taurine bridge, were isolated from a Neopetrosia sp. marine sponge. These new compounds have proton-deficient structural scaffolds that are difficult to unambiguously assign using only conventional 2- and 3-bond 1H-13C and 1H-15N heteronuclear correlation data. Thus, the application of LR-HSQMBC and HMBC NMR experiments optimized to detect 4- and 5-bond long-range 1H-13C heteronuclear correlations facilitated the structure elucidation of these unusual taurine-bridged marine metabolites. Neopetrotaurines A-C (1-3) showed significant inhibition of transcription driven by the oncogenic fusion protein PAX3-FOXO1, which is associated with alveolar rhabdomyosarcoma, and cytotoxic activity against PAX3-FOXO1-positive cell lines.
Assuntos
Alcaloides , Poríferos , Rabdomiossarcoma Alveolar , Animais , Rabdomiossarcoma Alveolar/metabolismo , Linhagem Celular , Alcaloides/farmacologia , Isoquinolinas/farmacologiaRESUMO
Although a rare disease, rhabdomyosarcoma (RMS) is one of the most common cancers in children the more aggressive and metastatic subtype is the alveolar RMS (ARMS). Survival outcomes with metastatic disease remain dismal and the need for new models that recapitulate key pathological features, including cell-extracellular matrix (ECM) interactions, is warranted. Here, we report an organotypic model that captures cellular and molecular determinants of invasive ARMS. We cultured the ARMS cell line RH30 on a collagen sponge in a perfusion-based bioreactor (U-CUP), obtaining after 7 days a 3D construct with homogeneous cell distribution. Compared to static culture, perfusion flow induced higher cell proliferation rates (20% vs. 5%), enhanced secretion of active MMP-2, and upregulation of the Rho pathway, associated with cancer cell dissemination. Consistently, the ECM genes LAMA1 and LAMA2, the antiapoptotic gene HSP90, identified in patient databases as hallmarks of invasive ARMS, were higher under perfusion flow at mRNA and protein level. Our advanced ARMS organotypic model mimics (1) the interactions cells-ECM, (2) the cell growth maintenance, and (3) the expression of proteins that characterize tumor expansion and aggressiveness. In the future, the perfusion-based model could be used with primary patient-derived cell subtypes to create a personalized ARMS chemotherapy screening system.
Assuntos
Rabdomiossarcoma Alveolar , Rabdomiossarcoma , Criança , Humanos , Rabdomiossarcoma Alveolar/metabolismo , Linhagem Celular , Perfusão , Técnicas de Cultura de Células em Três Dimensões , Linhagem Celular TumoralRESUMO
BACKGROUND: Anomalies of the FOXO1 gene in alveolar rhabdomyosarcoma are associated with a worse clinical prognosis, which determines the high value of studying the status of this gene when choosing a therapy strategy. The «gold standard¼ for determining FOXO1 gene rearrangements is currently the fluorescent in situ hybridization (FISH) technique. OBJECTIVE: Study of the relationship between canonical FOXO1 translocation and immunohistochemical expression of new surrogate markers in alveolar rhabdomyosarcoma to determine their predictive value. MATERIAL AND METHODS: 139 cases of rhabdomyosarcoma were retrospectively studied. The study used tissue matrix technology (TMA). On sections obtained from TMA blocks, the FISH technique was implemented using the locus-specific probe MetaSystems XL FOXO1 Break Apart (Metasystems, Germany). Immunohistochemical studies were performed on similar sections from TMA blocks with OLIG2 (Cell Marque Antibodies, clone 211F1.1) and MUC4 (Cell Marque Antibodies, clone 8G7) antibodies. RESULTS: The final expression analysis and statistical processing using a 2x2 contingency table and Fisher's exact test passed 111 cases (76 without FOXO1 rearrangement and 35 with rearrangement). The specificity of OLIG2 and MUC4 expression for FOXO1-rearranged alveolar rhabdomyosarcoma was 85.53% and 80.26%, respectively (p<0.01). CONCLUSION: The present study confirms the high predictive value of the expression of surrogate markers OLIG2 and MUC4 in determining the genetic status of alveolar rhabdomyosarcoma, which makes it possible to predict with high specificity the detection of the FOXO1 gene rearrangement.
Assuntos
Rabdomiossarcoma Alveolar , Humanos , Rabdomiossarcoma Alveolar/diagnóstico , Rabdomiossarcoma Alveolar/genética , Rabdomiossarcoma Alveolar/metabolismo , Fatores de Transcrição Box Pareados/genética , Fatores de Transcrição Box Pareados/metabolismo , Hibridização in Situ Fluorescente/métodos , Proteína Forkhead Box O1/genética , Estudos Retrospectivos , Biomarcadores , Translocação Genética/genética , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismoRESUMO
Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of childhood characterized by the inability to exit the proliferative myoblast-like stage. The alveolar fusion positive subtype (FP-RMS) is the most aggressive and is mainly caused by the expression of PAX3/7-FOXO1 oncoproteins, which are challenging pharmacological targets. Here, we show that the DEAD box RNA helicase 5 (DDX5) is overexpressed in alveolar RMS cells and that its depletion and pharmacological inhibition decrease FP-RMS viability and slow tumor growth in xenograft models. Mechanistically, we provide evidence that DDX5 functions upstream of the EHMT2/AKT survival signaling pathway, by directly interacting with EHMT2 mRNA, modulating its stability and consequent protein expression. We show that EHMT2 in turns regulates PAX3-FOXO1 activity in a methylation-dependent manner, thus sustaining FP-RMS myoblastic state. Together, our findings identify another survival-promoting loop in FP-RMS and highlight DDX5 as a potential therapeutic target to arrest RMS growth.
Assuntos
RNA Helicases DEAD-box , Rabdomiossarcoma Alveolar , Rabdomiossarcoma Embrionário , Rabdomiossarcoma , Linhagem Celular Tumoral , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Regulação Neoplásica da Expressão Gênica , Antígenos de Histocompatibilidade , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Proteínas de Fusão Oncogênica/metabolismo , Fatores de Transcrição Box Pareados/genética , RNA Helicases/metabolismo , Rabdomiossarcoma/metabolismo , Rabdomiossarcoma Alveolar/genética , Rabdomiossarcoma Alveolar/metabolismo , Rabdomiossarcoma Alveolar/patologiaRESUMO
Rhabdomyosarcoma (RMS) is an aggressive pediatric soft tissue sarcoma characterized by a very poor prognosis when relapses occur after front-line therapy. Therefore, a major challenge for patients' management remains the identification of markers associated with refractory and progressive disease. In this context, cancer autoantibodies are natural markers of disease onset and progression, useful to unveil novel therapeutic targets. Herein, we matched autoantibody profiling of alveolar RMS (ARMS) patients with genes under regulatory control of PAX3-FOXO1 transcription factor and revealed fibroblast growth factor 8 (FGF8) as a novel ARMS tumor antigen of diagnostic, prognostic, and therapeutic potential. We demonstrated that high levels of FGF8 autoantibodies distinguished ARMS patients from healthy subjects and represented an independent prognostic factor of better event-free survival. FGF8 was overexpressed in ARMS tumors compared to other types of pediatric soft tissue sarcomas, acting as a positive regulator of cell signaling. Indeed, FGF8 was capable of stimulating ARMS cells migration and expression of pro-angiogenic and metastasis-related factors, throughout MAPK signaling activation. Of note, FGF8 was found to increase in recurrent tumors, independently of PAX3-FOXO1 expression dynamics. Risk of recurrence correlated positively with FGF8 expression levels at diagnosis and reduced FGF8 autoantibodies titer, almost as if to suggest a failure of the immune response to control tumor growth in recurring patients. This study provides evidence about the crucial role of FGF8 in ARMS and the protective function of natural autoantibodies, giving new insights into ARMS biology and laying the foundations for the development of new therapeutic strategies.
Assuntos
Rabdomiossarcoma Alveolar , Rabdomiossarcoma Embrionário , Autoanticorpos/uso terapêutico , Fator 8 de Crescimento de Fibroblasto , Humanos , Imunidade , Recidiva Local de Neoplasia , Fator de Transcrição PAX3 , Fatores de Transcrição Box Pareados/genética , Fatores de Transcrição Box Pareados/metabolismo , Fatores de Transcrição Box Pareados/uso terapêutico , Rabdomiossarcoma Alveolar/genética , Rabdomiossarcoma Alveolar/metabolismo , Rabdomiossarcoma Alveolar/patologia , Rabdomiossarcoma Embrionário/genética , Rabdomiossarcoma Embrionário/metabolismoRESUMO
Chimeric transcription factors drive lineage-specific oncogenesis but are notoriously difficult to target. Alveolar rhabdomyosarcoma (RMS) is an aggressive childhood soft tissue sarcoma transformed by the pathognomonic Paired Box 3-Forkhead Box O1 (PAX3-FOXO1) fusion protein, which governs a core regulatory circuitry transcription factor network. Here, we show that the histone lysine demethylase 4B (KDM4B) is a therapeutic vulnerability for PAX3-FOXO1+ RMS. Genetic and pharmacologic inhibition of KDM4B substantially delayed tumor growth. Suppression of KDM4 proteins inhibited the expression of core oncogenic transcription factors and caused epigenetic alterations of PAX3-FOXO1-governed superenhancers. Combining KDM4 inhibition with cytotoxic chemotherapy led to tumor regression in preclinical PAX3-FOXO1+ RMS subcutaneous xenograft models. In summary, we identified a targetable mechanism required for maintenance of the PAX3-FOXO1-related transcription factor network, which may translate to a therapeutic approach for fusion-positive RMS.
Assuntos
Rabdomiossarcoma Alveolar , Rabdomiossarcoma , Carcinogênese/genética , Linhagem Celular Tumoral , Criança , Proteína Forkhead Box O1/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Histona Desmetilases com o Domínio Jumonji/genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Fator de Transcrição PAX3/genética , Fator de Transcrição PAX3/metabolismo , Fatores de Transcrição Box Pareados/genética , Fatores de Transcrição Box Pareados/metabolismo , Fatores de Transcrição Box Pareados/uso terapêutico , Rabdomiossarcoma/genética , Rabdomiossarcoma Alveolar/genética , Rabdomiossarcoma Alveolar/metabolismo , Rabdomiossarcoma Alveolar/patologiaRESUMO
B7-H3 (also known as CD276) is associated with aggressive characteristics in various cancers. Meanwhile, in alveolar rhabdomyosarcoma (ARMS), PAX3-FOXO1 fusion protein is associated with increased aggressiveness and poor prognosis. In the present study, we explored the relationship between PAX3-FOXO1 and B7-H3 and the biological roles of B7-H3 in ARMS. Quantitative real time PCR and flow cytometry revealed that PAX3-FOXO1 knockdown downregulated B7-H3 expression in all the selected cell lines (Rh-30, Rh-41, and Rh-28), suggesting that PAX3-FOXO1 positively regulates B7-H3 expression. Gene expression analysis revealed that various genes and pathways involved in chemotaxis, INF-γ production, and myogenic differentiation were commonly affected by the knockdown of PAX3-FOXO1 and B7-H3. Wound healing and transwell migration assays revealed that both PAX3-FOXO1 and B7-H3 were associated with cell migration. Furthermore, knockdown of PAX3-FOXO1 or B7-H3 induced myogenin expression in all cell lines, although myosin heavy chain induction varied depending on the cellular context. Our results indicate that PAX3-FOXO1 regulates B7-H3 expression and that PAX3-FOXO1 and B7-H3 are commonly associated with multiple pathways related to an aggressive phenotype in ARMS, such as cell migration and myogenic differentiation block.
Assuntos
Antígenos B7/metabolismo , Diferenciação Celular , Movimento Celular , Proteína Forkhead Box O1/metabolismo , Desenvolvimento Muscular , Fator de Transcrição PAX3/metabolismo , Rabdomiossarcoma Alveolar/metabolismo , Western Blotting , Linhagem Celular Tumoral , Regulação para Baixo , Citometria de Fluxo , Técnicas de Silenciamento de Genes , Humanos , Miogenina/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Rabdomiossarcoma Alveolar/patologia , TranscriptomaRESUMO
Rhabdomyosarcoma (RMS), is the most frequent soft tissue tumor in children that originates from disturbances in differentiation process. Mechanisms leading to the development of RMS are still poorly understood. Therefore, by analysis of two RMS RH30 cell line subclones, one subclone PAX7 negative, while the second one PAX7 positive, and comparison with other RMS cell lines we aimed at identifying new mechanisms crucial for RMS progression. RH30 subclones were characterized by the same STR profile, but different morphology, rate of proliferation, migration activity and chemotactic abilities in vitro, as well as differences in tumor morphology and growth in vivo. Our analysis indicated a different level of expression of adhesion molecules (e.g., from VLA and ICAM families), myogenic microRNAs, such as miR-206 and transcription factors, such as MYOD, MYOG, SIX1, and ID. Silencing of PAX7 transcription factor with siRNA confirmed the crucial role of PAX7 transcription factor in proliferation, differentiation and migration of RMS cells. To conclude, our results suggest that tumor cell lines with the same STR profile can produce subclones that differ in many features and indicate crucial roles of PAX7 and ID proteins in the development of RMS.
Assuntos
Diferenciação Celular , Fator de Transcrição PAX7/metabolismo , Rabdomiossarcoma Alveolar/metabolismo , Animais , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas Inibidoras de Diferenciação/genética , Proteínas Inibidoras de Diferenciação/metabolismo , Camundongos Endogâmicos NOD , Camundongos SCID , MicroRNAs/genética , MicroRNAs/metabolismo , Invasividade Neoplásica , Fator de Transcrição PAX7/genética , Rabdomiossarcoma Alveolar/genética , Rabdomiossarcoma Alveolar/patologia , Transdução de Sinais , Carga TumoralRESUMO
Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma. The two predominant histologic variants of RMS, embryonal and alveolar rhabdomyosarcoma (eRMS and aRMS, respectively), carry very different prognoses. While eRMS is associated with an intermediate prognosis, the 5-year survival rate of aRMS is less than 30%. The RMS subtypes are also different at the molecular level-eRMS frequently has multiple genetic alterations, including mutations in RAS and TP53, whereas aRMS often has chromosomal translocations resulting in PAX3-FOXO1 or PAX7-FOXO1 fusions, but otherwise has a "quiet" genome. Interestingly, mutations in RAS are rarely found in aRMS. In this study, we explored the role of oncogenic RAS in aRMS. We found that while ectopic oncogenic HRAS expression was tolerated in the human RAS-driven eRMS cell line RD, it was detrimental to cell growth and proliferation in the human aRMS cell line Rh28. Growth inhibition was mediated by oncogene-induced senescence and associated with increased RB pathway activity and expression of the cyclin-dependent kinase inhibitors p16 and p21. Unexpectedly, the human eRMS cell line RMS-YM, a RAS wild-type eRMS cell line, also exhibited growth inhibition in response to oncogenic HRAS in a manner similar to aRMS Rh28 cells. This work suggests that oncogenic RAS is expressed in a context-dependent manner in RMS and may provide insight into the differential origins and therapeutic opportunities for RMS subtypes.
Assuntos
Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Rabdomiossarcoma Alveolar/metabolismo , Rabdomiossarcoma Embrionário/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Senescência Celular , Regulação Neoplásica da Expressão Gênica , Humanos , ImmunoblottingRESUMO
PURPOSE: Recent work has highlighted the therapeutic potential of targeting autophagy to modulate cell survival in a variety of diseases including cancer. Recently, we found that the RNA-binding protein Staufen1 (STAU1) is highly expressed in alveolar rhabdomyosarcoma (ARMS) and that this abnormal expression promotes tumorigenesis. Here, we asked whether STAU1 is involved in the regulation of autophagy in ARMS cells. METHODS: We assessed the impact of STAU1 expression modulation in ARMS cell lines (RH30 and RH41), non-transformed skeletal muscle cells (C2C12) and STAU1-transgenic mice using complementary techniques. RESULTS: We found that STAU1 silencing reduces autophagy in the ARMS cell lines RH30 and RH41, while increasing their apoptosis. Mechanistically, this inhibitory effect was found to be caused by a direct negative impact of STAU1 depletion on the stability of Beclin-1 (BECN1) and ATG16L1 mRNAs, as well as by an indirect inhibition of JNK signaling via increased expression of Dual specificity phosphatase 8 (DUSP8). Pharmacological activation of JNK or expression silencing of DUSP8 was sufficient to restore autophagy in STAU1-depleted cells. By contrast, we found that STAU1 downregulation in non-transformed skeletal muscle cells activates autophagy in a mTOR-dependent manner, without promoting apoptosis. A similar effect was observed in skeletal muscles obtained from STAU1-overexpressing transgenic mice. CONCLUSIONS: Together, our data indicate an effect of STAU1 on autophagy regulation in ARMS cells and its differential role in non-transformed skeletal muscle cells. Our findings suggest a cancer-specific potential of targeting STAU1 for the treatment of ARMS.
Assuntos
Autofagia/genética , Proteínas do Citoesqueleto/genética , Perfilação da Expressão Gênica/métodos , Músculo Esquelético/metabolismo , Proteínas de Ligação a RNA/genética , Rabdomiossarcoma Alveolar/genética , Animais , Apoptose/genética , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Proteína Beclina-1/genética , Proteína Beclina-1/metabolismo , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , Células Cultivadas , Proteínas do Citoesqueleto/metabolismo , Regulação para Baixo/genética , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Músculo Esquelético/citologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Interferência de RNA , Proteínas de Ligação a RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rabdomiossarcoma Alveolar/metabolismo , Rabdomiossarcoma Alveolar/patologia , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismoAssuntos
Neoplasias Encefálicas/patologia , Glândula Pineal/patologia , Rabdomiossarcoma Alveolar/patologia , Biomarcadores Tumorais/líquido cefalorraquidiano , Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Sistema Nervoso Central/patologia , Criança , Diagnóstico Diferencial , Regulação Neoplásica da Expressão Gênica/genética , Glioma/diagnóstico , Glioma/patologia , Humanos , Hipertensão Intracraniana , Masculino , Coativador 2 de Receptor Nuclear/genética , Coativador 2 de Receptor Nuclear/metabolismo , Fator de Transcrição 2 de Oligodendrócitos/metabolismo , Proteínas de Fusão Oncogênica/genética , Fator de Transcrição PAX3/genética , Fator de Transcrição PAX3/metabolismo , Rabdomiossarcoma Alveolar/diagnóstico , Rabdomiossarcoma Alveolar/genética , Rabdomiossarcoma Alveolar/metabolismoRESUMO
AIMS: Mucin 4 (MUC4) is a transmembrane glycoprotein normally expressed by several human epithelial surfaces, including those of the colon, vagina, and respiratory tract. Although MUC4 overexpression is seen in various carcinomas, its expression among mesenchymal neoplasms is fairly specific to low-grade fibromyxoid sarcoma and sclerosing epithelioid fibrosarcoma. Having observed unanticipated anti-MUC4 immunoreactivity in rhabdomyosarcoma, we aimed to further characterise its expression. METHODS AND RESULTS: Expression of MUC4 was assessed by immunohistochemistry in a total of 97 rhabdomyosarcomas using formalin-fixed paraffin-embedded tissue sections. MUC4 was expressed by 21 of 26 PAX3/7-FOXO1 fusion-positive cases, wherein immunoreactivity, varying from weak to strong, was present in 20-100% of neoplastic cells. With the exception of one sclerosing rhabdomyosarcoma showing immunoreactivity in 20% of cells, MUC4 was not expressed by embryonal (n = 28), sclerosing (n = 20), or pleomorphic (n = 23) rhabdomyosarcomas. Analysing published gene expression microarray data from a separate cohort of 33 fusion-positive and 25 fusion-negative rhabdomyosarcomas, we found on average a 11.4-fold increased expression in fusion-positive tumours (P = 0.0004). CONCLUSIONS: MUC4 is expressed to a variable extent in the majority of PAX3/7-FOXO1 fusion-positive (alveolar) rhabdomyosarcomas, while expression in other rhabdomyosarcoma subtypes is rare.
Assuntos
Neoplasias de Cabeça e Pescoço/metabolismo , Mucina-4/metabolismo , Rabdomiossarcoma Alveolar/metabolismo , Neoplasias de Tecidos Moles/metabolismo , Adulto , Feminino , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Rabdomiossarcoma Alveolar/patologia , Neoplasias de Tecidos Moles/patologiaRESUMO
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.
Assuntos
Células-Tronco Neoplásicas/metabolismo , Receptores Notch/metabolismo , Rabdomiossarcoma Alveolar/metabolismo , Rabdomiossarcoma Alveolar/patologia , Transdução de Sinais , Biomarcadores , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Células-Tronco Neoplásicas/patologiaRESUMO
Numerous cell lines for human alveolar rhabdomyosarcoma (ARMS) have been developed and are widely used to study biological processes of this myogenic cancer. The present study investigated the resemblance of commonly used ARMS cell lines to primary tumors in regards to gene expression. RNA-sequencing data was retrieved from published datasets for 4 commonly used ARMS cell lines and 35 ARMS primary tumors. The genes with most variable expression across primary tumors were used to calculate rank-based Spearman's correlation. The observed median correlations ranged from 0.36 to 0.61. RH-41 showed the highest median correlation while KYM-1 was the least correlated cell line. A significant number of genes dysregulated between tumors and non-tumors also exhibited similar expression patterns between tumors and cell lines, including The findings suggest that ARMS cell lines exhibit changes in gene expression compared to primary tumors and may not be completely representative of the disease process.
Assuntos
RNA Mensageiro/genética , Rabdomiossarcoma Alveolar/genética , Transcriptoma/genética , Linhagem Celular Tumoral , Bases de Dados Genéticas , Expressão Gênica/genética , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Modelos Biológicos , Rabdomiossarcoma Alveolar/metabolismo , Rabdomiossarcoma Alveolar/patologia , Rabdomiossarcoma Embrionário/genética , Rabdomiossarcoma Embrionário/metabolismo , Rabdomiossarcoma Embrionário/patologia , Análise de Sequência de RNA/métodos , Estatísticas não ParamétricasRESUMO
In this case report we evaluate the genetics of and scientific basis of therapeutic options for a 14-yr-old male patient diagnosed with metastatic PAX3-FOXO1 fusion positive alveolar rhabdomyosarcoma. A distinguishing genetic feature of this patient was a germline RET C634F mutation, which is a known driver of multiple endocrine neoplasia type 2A (MEN2A) cancer. Through sequential DNA and RNA sequencing analyses over the patient's clinical course, a set of gene mutations, amplifications, and overexpressed genes were identified and biological hypotheses generated to explore the biology of RET and coexisting signaling pathways in rhabdomyosarcoma. Somatic genetic abnormalities identified include CDK4 amplification and FGFR4 G388R polymorphism. Because of the initial lack of patient-derived primary cell cultures, these hypotheses were evaluated using several approaches including western blot analysis and pharmacological evaluation with molecularly similar alveolar rhabdomyosarcoma cell lines. Once a primary cell culture became available, the RET inhibitor cabozantinib was tested but showed no appreciable efficacy in vitro, affirming with the western blot negative for RET protein expression that RET germline mutation could be only incidental. In parallel, the patient was treated with cabozantinib without definitive clinical benefit. Parallel chemical screens identified PI3K and HSP90 as potential tumor-specific biological features. Inhibitors of PI3K and HSP90 were further validated in drug combination synergy experiments and shown to be synergistic in the patient-derived culture. We also evaluated the use of JAK/STAT pathway inhibitors in the context of rhabdomyosarcomas bearing the FGFR4 G388R coding variant. Although the patient succumbed to his disease, study of the patient's tumor has generated insights into the biology of RET and other targets in rhabdomyosarcoma.
Assuntos
Estudos de Associação Genética , Predisposição Genética para Doença , Mutação em Linhagem Germinativa , Proteínas Proto-Oncogênicas c-ret/genética , Rabdomiossarcoma Alveolar/diagnóstico , Rabdomiossarcoma Alveolar/genética , Adolescente , Alelos , Substituição de Aminoácidos , Biópsia , Análise Mutacional de DNA , Genótipo , Humanos , Imuno-Histoquímica , Masculino , Fenótipo , Tomografia por Emissão de Pósitrons , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-ret/metabolismo , Radiografia Torácica , Rabdomiossarcoma Alveolar/tratamento farmacológico , Rabdomiossarcoma Alveolar/metabolismoRESUMO
Fibroblast growth factor receptor 4 (FGFR4) aberrant expression and activity have been linked to the pathogenesis of a variety of cancers including rhabdomyosarcomas (RMS). We found that treatment of alveolar rhabdomyosarcoma (aRMS) cells with Guadecitabine (SGI-110), a next-generation DNA methyltransferase inhibitor (DNMTi), resulted in a significant reduction of FGFR4 protein levels, 5â¯days post treatment. Chromatin immunoprecipitation-sequencing (ChIP-seq) in aRMS cells revealed attenuation of the H3K4 mono-methylation across the FGFR4 super enhancer without changes in tri-methylation of either H3K4 or H3K27. These changes were associated with a significant reduction in FGFR4 transcript levels in treated cells. These decreases in H3K4me1 in the FGFR4 super enhancer were also associated with a 240-fold increase in KDM5B (JARID1B) mRNA levels. Immunoblot and immunofluorescent studies also revealed a significant increase in the KDM5B protein levels after treatment in these cells. KDM5B is the only member of KDM5 (JARID1) family of histone lysine demethylases that catalyzes demethylation of H3K4me1. These data together suggest a pleiotropic effect of DNMTi therapy in aRMS cells, converging to significantly lower FGFR4 protein levels in these cells.
Assuntos
Antineoplásicos/farmacologia , Azacitidina/análogos & derivados , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos/metabolismo , Rabdomiossarcoma Alveolar/tratamento farmacológico , Azacitidina/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sequenciamento de Cromatina por Imunoprecipitação , Regulação para Baixo/efeitos dos fármacos , Elementos Facilitadores Genéticos , Histonas/metabolismo , Humanos , Histona Desmetilases com o Domínio Jumonji/metabolismo , Lisina/metabolismo , Proteínas Nucleares/metabolismo , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos/genética , Proteínas Repressoras/metabolismo , Rabdomiossarcoma Alveolar/genética , Rabdomiossarcoma Alveolar/metabolismo , Rabdomiossarcoma Alveolar/patologiaRESUMO
Rhabdomyosarcoma (RMS) is the most common soft tissue malignancy in childhood and adolescence. Patients with the most aggressive histological variant have an unfavorable prognosis due to a high metastasis incidence. Lysyl oxidase-like 2 (LOXL2) is a lysyl oxidase, member of a family of extracellular matrix (ECM) crosslinking enzymes that recently have emerged as important regulators of tumor progression and metastasis. We report that LOXL2 is overexpressed in RMS, suggesting a potential role for LOXL2 in RMS oncogenic progression. Consistently, transient and stable LOXL2 knockdown decreased cell migratory and invasive capabilities in two ARMS cell lines. Furthermore, introduction of LOXL2 in RMS non-expressing cells using wild type or mutated (catalytically inactive) constructs resulted in increased cell migration, cell invasion and number and incidence of spontaneous lung metastasis in vivo, independently of its catalytic activity. To further study the molecular mechanism associated with LOXL2 expression, a pull-down assay on LOXL2-transfected cells was performed and analyzed by mass spectrometry. The intermediated filament protein vimentin was validated as a LOXL2-interactor. Thus, our results suggest an oncogenic role of LOXL2 in RMS by regulating cytoskeleton dynamics and cell motility capabilities.
Assuntos
Aminoácido Oxirredutases/metabolismo , Biomarcadores Tumorais/metabolismo , Carcinogênese/patologia , Neoplasias Pulmonares/secundário , Rabdomiossarcoma Alveolar/patologia , Aminoácido Oxirredutases/genética , Animais , Apoptose , Biocatálise , Biomarcadores Tumorais/genética , Carcinogênese/genética , Carcinogênese/metabolismo , Movimento Celular , Proliferação de Células , Progressão da Doença , Transição Epitelial-Mesenquimal , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Camundongos , Camundongos Nus , Rabdomiossarcoma Alveolar/genética , Rabdomiossarcoma Alveolar/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Pseudoglandular schwannoma (PGS) is a rare morphological variant of benign schwannoma. PGS is quite distinct from the somewhat better characterized glandular schwannoma variant. PGS is characterized by the presence of gland-like structures lined with pseudocolumnar or cuboidal-like neoplastic Schwann cells lining variably sized cystic space cells. Herein, we describe a rare case of PGS in a 17-year-old adolescent boy with a neck mass 8.5 cm in diameter present for 2 years with a recent increase in size. Fine-needle aspiration (FNA) demonstrated abundant rhabdoid-like cuboidal cells, plasmacytoid cells, binucleation, clusters of round to oval cells with scant cytoplasm, and a lack of stromal cells with spindle or oval nuclei. These findings were challenging and were reminiscent of alveolar rhabdomyosarcoma on FNA. Magnetic resonance imaging was also suggestive of malignancy. However, the histologic picture and the immunohistochemical analysis of the resected mass were consisted with PGS. The numerous rhabdoid-like cuboidal neoplastic Schwann cells with a plasmacytoid appearance, paving cystic spaces, and the lack of blunt-ended proliferating stromal cells on smears mimicked alveolar rhabdomyosarcoma on FNA. To our knowledge, cytological features of PGS have not been previously reported. This is the first published report of an unusual PGS case located in the neck region causing a diagnostic dilemma on cytology.