RESUMEN
Dedifferentiated liposarcoma (DDLPS) is an aggressive mesenchymal cancer marked by amplification of MDM2, an inhibitor of the tumor suppressor TP53. DDLPS patients with higher MDM2 amplification have lower chemotherapy sensitivity and worse outcome than patients with lower MDM2 amplification. We hypothesized that MDM2 amplification levels may be associated with changes in DDLPS metabolism. Six patient-derived DDLPS cell line models were subject to comprehensive metabolomic (Metabolon) and lipidomic (SCIEX 5600 TripleTOF-MS) profiling to assess associations with MDM2 amplification and their responses to metabolic perturbations. Comparing metabolomic profiles between MDM2 higher and lower amplification cells yielded a total of 17 differentially abundant metabolites across both panels (FDR < 0.05, log2 fold change < 0.75), including ceramides, glycosylated ceramides, and sphingomyelins. Disruption of lipid metabolism through statin administration resulted in a chemo-sensitive phenotype in MDM2 lower cell lines only, suggesting that lipid metabolism may be a large contributor to the more aggressive nature of MDM2 higher DDLPS tumors. This study is the first to provide comprehensive metabolomic and lipidomic characterization of DDLPS cell lines and provides evidence for MDM2-dependent differential molecular mechanisms that are critical factors in chemoresistance and could thus affect patient outcome.
RESUMEN
Liposarcomas are tumors arising in white adipose tissue (WAT) with avidity for local recurrence. Aggressive dedifferentiated liposarcomas (DDLS) may arise from well-differentiated subtypes (WDLS) upon disease progression, however, this key issue is unresolved due in large part to knowledge gaps about liposarcoma cellular composition. Here, we wished to improve insights into liposarcoma cellular hierarchy. Tumor section analysis indicated that the populations, distinguishable based on the expression of CD34 (a marker of adipocyte progenitors) and CD36 (a marker of adipocyte differentiation), occupy distinct intra-tumoral locations in both WDLS and DDLS. Taking advantage of these markers, we separated cells from a panel of fresh human surgical specimens by fluorescence-activated cell sorting (FACS). Based on chromosome analysis and the culture phenotypes of the composing populations, we demonstrate that malignant cells comprise four mesenchymal populations distinguished by the expression of CD34 and CD36, while vascular (CD31+) and hematopoietic (CD45+) components are non-neoplastic. Finally, we show that mouse xenografts are derivable from both CD36-negative and CD36-positive DDLS cells, and that each population recreates the heterogeneity of CD36 expression in vivo. Combined, our results show that malignant cells in WDLS and DDLS can be classified according to distinct stages of adipogenesis and indicate immunophenotypic plasticity of malignant liposarcoma cells.
Asunto(s)
Adipocitos/patología , Liposarcoma/patología , Adipocitos/citología , Adipocitos/metabolismo , Animales , Diferenciación Celular , Modelos Animales de Enfermedad , Citometría de Flujo , Xenoinjertos , Humanos , Inmunofenotipificación , Liposarcoma/genética , Liposarcoma/inmunología , Ratones , Ratones Endogámicos NOD , Ratones SCID , FenotipoRESUMEN
Liposarcoma can be an aggressive, debilitating, and fatal malignancy. In this study, we identifed miRNAs associated with the differentiation status of liposarcoma to gain insight into the basis for its progression. miRNA expression profiles determined in human tumors and normal fat specimens identified a dedifferentiated tumor expression signature consisting of 35 miRNAs. Deregulated miRNA expression was confirmed in a second independent sample cohort. The miR-155 was the most overexpressed miRNA and functional investigations assigned an important role in the growth of dedifferentiated liposarcoma cell lines. Transient or stable knockdown of miR-155 retarded tumor cell growth, decreased colony formation, and induced G(1)-S cell-cycle arrest in vitro and blocked tumor growth in murine xenografts in vivo. We identified casein kinase 1α (CK1α) as a direct target of miR-155 control which enhanced ß-catenin signaling and cyclin D1 expression, promoting tumor cell growth. In summary, our results point to important functions for miR-155 and ß-catenin signaling in progression of liposarcoma, revealing mechanistic vulnerabilities that might be exploited for both prognostic and therapeutic purposes.
Asunto(s)
Quinasa de la Caseína I/genética , Liposarcoma/genética , MicroARNs/fisiología , Oncogenes , Transducción de Señal/fisiología , beta Catenina/fisiología , Animales , Secuencia de Bases , Línea Celular Tumoral , Proliferación Celular , Ciclina D1/fisiología , Regulación Neoplásica de la Expresión Génica , Humanos , Liposarcoma/patología , Ratones , Datos de Secuencia MolecularRESUMEN
Well-differentiated liposarcoma (WDLS) is one of the most common malignant mesenchymal tumors and dedifferentiated liposarcoma (DDLS) is a malignant tumor consisting of both WDLS and a transformed nonlipogenic sarcomatous component. Cytogenetically, WDLS is characterized by the presence of ring or giant rod chromosomes containing several amplified genes, including MDM2, TSPAN31, CDK4, and others mainly derived from chromosome bands 12q13-15. However, the 12q13-15 amplicon is large and discontinuous. The focus of this study was to identify novel critical genes that are consistently amplified in primary (nonrecurrent) WDLS and with potential relevance for future targeted therapy. Using a high-resolution (5.0 kb) "single nucleotide polymorphism"/copy number variation microarray to screen the whole genome in a series of primary WDLS, two consistently amplified areas were found on chromosome 12: one region containing the MDM2 and CPM genes, and another region containing the FRS2 gene. Based on these findings, we further validated FRS2 amplification in both WDLS and DDLS. Fluorescence in situ hybridization confirmed FRS2 amplification in all WDLS and DDLS tested (n = 57). Real time PCR showed FRS2 mRNA transcriptional upregulation in WDLS (n = 19) and DDLS (n = 13) but not in lipoma (n = 5) and normal fat (n = 9). Immunoblotting revealed high expression levels of phospho-FRS2 at Y436 and slightly overexpression of total FRS2 protein in liposarcoma but not in normal fat or preadipocytes. Considering the critical role of FRS2 in mediating fibroblast growth factor receptor signaling, our findings indicate that FRS2 signaling should be further investigated as a potential therapeutic target for liposarcoma.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Liposarcoma/genética , Liposarcoma/patología , Proteínas de la Membrana/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Western Blotting , Estudios de Casos y Controles , Diferenciación Celular/genética , Mapeo Cromosómico , Cromosomas Humanos Par 12 , Variaciones en el Número de Copia de ADN , Proteínas Ligadas a GPI/genética , Amplificación de Genes , Humanos , Hibridación Fluorescente in Situ , Liposarcoma/metabolismo , Proteínas de la Membrana/metabolismo , Metaloendopeptidasas/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosforilación , Polimorfismo de Nucleótido Simple , Proteínas Proto-Oncogénicas c-mdm2/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Factores de Crecimiento de Fibroblastos/genética , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Reproducibilidad de los Resultados , Estudios Retrospectivos , Transducción de SeñalRESUMEN
PURPOSE: MET signaling has been suggested a potential role in malignant peripheral nerve sheath tumors (MPNST). Here, MET function and blockade were preclinically assessed. EXPERIMENTAL DESIGN: Expression levels of MET, its ligand hepatocyte growth factor (HGF), and phosphorylated MET (pMET) were examined in a clinically annotated MPNST tissue microarray (TMA) incorporating univariable and multivariable statistical analyses. Human MPNST cells were studied in vitro and in vivo; Western blot (WB) and ELISA were used to evaluate MET and HGF expression, activation, and downstream signaling. Cell culture assays tested the impact of HGF-induced MET activation and anti-MET-specific siRNA inhibition on cell proliferation, migration, and invasion; in vivo gel-foam assays were used to evaluate angiogenesis. Cells stably transduced with anti-MET short hairpin RNA (shRNA) constructs were tested for growth and metastasis in severe combined immunodeficient (SCID) mice. The effect of the tyrosine kinase inhibitor XL184 (Exelixis) targeting MET/VEGFR2 (vascular endothelial growth factor receptor 2) on local and metastatic MPNST growth was examined in vivo. RESULTS: All three markers were expressed in MPNST human samples; pMET expression was an independent prognosticator of poor patient outcome. Human MPNST cell lines expressed MET, HGF, and pMET. MET activation increased MPNST cell motility, invasion, angiogenesis, and induced matrix metalloproteinase-2 (MMP2) and VEGF expression; MET knockdown had inverse effects in vitro and markedly decreased local and metastatic growth in vivo. XL184 abrogated human MPNST xenograft growth and metastasis in SCID mice. CONCLUSIONS: Informative prognosticators and novel therapies are crucially needed to improve MPNST management and outcomes. We show an important role for MET in MPNST, supporting continued investigation of novel anti-MET therapies in this clinical context.