Dataset Information

MRNA and protein levels of FUS, EWSR1, and TAF15 are upregulated in liposarcoma.

ABSTRACT: Translocations or mutations of FUS, EWSR1, and TAF15 (FET) result in distinct genetic diseases. N-terminal translocations of any FET protein to a series of transcription factors yields chimeric proteins that contribute to sarcomagenesis, whereas mutations in the conserved COOH-terminal domain of wild-type FUS were recently shown to cause familial amyotrophic lateral sclerosis. We thus investigated whether the loss of one FUS allele by translocation in liposarcoma may be followed by mutations in either the remaining FUS allele or the paralogous EWSR1. Furthermore, we investigated the strength of the FET promoters and their contributions to sarcomagenesis given the proteins' frequent involvement in oncogenic translocations. We sequenced the respective genomic regions of both FUS and EWSR1 in 96 liposarcoma samples. Additionally, we determined FET transcript and protein levels in several liposarcoma cell lines. We did not observe sequence variations in either FUS or EWSR1. However, protein copy numbers reached an impressive 0.9 and 5.5 Mio of FUS and EWSR1 per tumor cell, respectively. Compared with adipose-derived stem cells, FUS and EWSR1 protein expression levels were elevated on average 28.6-fold and 7.3-fold, respectively. TAF15 mRNA levels were elevated on average 3.9-fold, although with a larger variation between samples. Interestingly, elevated TAF15 mRNA levels did not translate to strongly elevated protein levels, consistent with its infrequent occurrence as translocation partner in tumors. These results suggest that the powerful promoters of FET genes are predominantly responsible for the oncogenic effect of transcription factor translocations in sarcomas.

SUBMITTER: Spitzer JI

PROVIDER: S-EPMC3056538 | biostudies-literature | 2011 May

REPOSITORIES: biostudies-literature

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mRNA and protein levels of FUS, EWSR1, and TAF15 are upregulated in liposarcoma.

Spitzer Jessica I JI Ugras Stacy S Runge Simon S Decarolis Penelope P Antonescu Christina C Tuschl Tom T Singer Samuel S

Genes, chromosomes & cancer 20110222 5

Translocations or mutations of FUS, EWSR1, and TAF15 (FET) result in distinct genetic diseases. N-terminal translocations of any FET protein to a series of transcription factors yields chimeric proteins that contribute to sarcomagenesis, whereas mutations in the conserved COOH-terminal domain of wild-type FUS were recently shown to cause familial amyotrophic lateral sclerosis. We thus investigated whether the loss of one FUS allele by translocation in liposarcoma may be followed by mutations in ...[more]

PMID: 21344536

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Project description:Accumulation of the DNA/RNA binding protein fused in sarcoma as cytoplasmic inclusions in neurons and glial cells is the pathological hallmark of all patients with amyotrophic lateral sclerosis with mutations in FUS as well as in several subtypes of frontotemporal lobar degeneration, which are not associated with FUS mutations. The mechanisms leading to inclusion formation and fused in sarcoma-associated neurodegeneration are only poorly understood. Because fused in sarcoma belongs to a family of proteins known as FET, which also includes Ewing's sarcoma and TATA-binding protein-associated factor 15, we investigated the potential involvement of these other FET protein family members in the pathogenesis of fused in sarcoma proteinopathies. Immunohistochemical analysis of FET proteins revealed a striking difference among the various conditions, with pathology in amyotrophic lateral sclerosis with FUS mutations being labelled exclusively for fused in sarcoma, whereas fused in sarcoma-positive inclusions in subtypes of frontotemporal lobar degeneration also consistently immunostained for TATA-binding protein-associated factor 15 and variably for Ewing's sarcoma. Immunoblot analysis of proteins extracted from post-mortem tissue of frontotemporal lobar degeneration with fused in sarcoma pathology demonstrated a relative shift of all FET proteins towards insoluble protein fractions, while genetic analysis of the TATA-binding protein-associated factor 15 and Ewing's sarcoma gene did not identify any pathogenic variants. Cell culture experiments replicated the findings of amyotrophic lateral sclerosis with FUS mutations by confirming the absence of TATA-binding protein-associated factor 15 and Ewing's sarcoma alterations upon expression of mutant fused in sarcoma. In contrast, all endogenous FET proteins were recruited into cytoplasmic stress granules upon general inhibition of Transportin-mediated nuclear import, mimicking the findings in frontotemporal lobar degeneration with fused in sarcoma pathology. These results allow a separation of fused in sarcoma proteinopathies caused by FUS mutations from those without a known genetic cause based on neuropathological features. More importantly, our data imply different pathological processes underlying inclusion formation and cell death between both conditions; the pathogenesis in amyotrophic lateral sclerosis with FUS mutations appears to be more restricted to dysfunction of fused in sarcoma, while a more global and complex dysregulation of all FET proteins is involved in the subtypes of frontotemporal lobar degeneration with fused in sarcoma pathology.

Project description:BackgroundLiposarcomas are the most common class of soft tissue sarcomas, and myxoid liposarcoma is the second most common liposarcoma. EWSR1-DDIT3 is a chimeric fusion protein generated by the myxoid liposarcoma-specific chromosomal translocation t(12;22)(q13;q12). Current studies indicate that multipotent mesenchymal cells are the origin of sarcomas. The mechanism whereby EWSR1-DDIT3 contributes to the phenotypic selection of target cells during oncogenic transformation remains to be elucidated.Methodology/principal findingsReporter assays showed that the EWSR1-DDIT3 myxoid liposarcoma fusion protein, but not its wild-type counterparts EWSR1 and DDIT3, selectively repressed the transcriptional activity of cell lineage-specific marker genes in multipotent mesenchymal C3H10T1/2 cells. Specifically, the osteoblastic marker Opn promoter and chondrocytic marker Col11a2 promoter were repressed, while the adipocytic marker Ppar-?2 promoter was not affected. Mutation analyses, transient ChIP assays, and treatment of cells with trichostatin A (a potent inhibitor of histone deacetylases) or 5-Aza-2'-deoxycytidine (a methylation-resistant cytosine homolog) revealed the possible molecular mechanisms underlying the above-mentioned selective transcriptional repression. The first is a genetic action of the EWSR1-DDIT3 fusion protein, which results in binding to the functional C/EBP site within Opn and Col11a2 promoters through interaction of its DNA-binding domain and subsequent interference with endogenous C/EBP? function. Another possible mechanism is an epigenetic action of EWSR1-DDIT3, which enhances histone deacetylation, DNA methylation, and histone H3K9 trimethylation at the transcriptional repression site. We hypothesize that EWSR1-DDIT3-mediated transcriptional regulation may modulate the target cell lineage through target gene-specific genetic and epigenetic conversions.Conclusions/significanceThis study elucidates the molecular mechanisms underlying EWSR1-DDIT3 fusion protein-mediated phenotypic selection of putative target multipotent mesenchymal cells during myxoid liposarcoma development. A better understanding of this process is fundamental to the elucidation of possible direct lineage reprogramming in oncogenic sarcoma transformation mediated by fusion proteins.

Project description:An increasing number of epithelioid vascular lesions, in particular tumors from the benign and low-grade end of the spectrum, have been characterized by recurrent gene fusions. As a result, the detection of these molecular markers have improved the classification of diagnostically challenging cases. However, despite the significant progress, there are occasional lesions that do not fit in known histologic or molecular groups. Herein, we present five such unclassified epithelioid vascular lesions, which occurred in the bone and showed a distinct morphology composed of alternating vasoformative and solid growth and mild to moderate nuclear pleomorphism. The variegated morphologic appearance resembled that of composite hemangioendothelioma, being distinct from both epithelioid hemangioma and epithelioid hemangioendothelioma, and consistently showed cytologic atypia. Due to their unusual morphologic appearance and negative molecular work-up, targeted transcriptome sequencing was performed in two cases showing the presence of NFATC2 fusions with either EWSR1 or FUS genes. Three additional bone tumors with EWSR1 gene rearrangements were identified by FISH screening of a large cohort of 45 fusion-negative epithelioid vascular neoplasms, one fused to NFATC2 while two others to NFATC1. There were three females and two males, with a wide age range at presentation, mean of 44 years. The lesions occurred in the pelvis, maxillary sinus, and humerus. Two patients presented with polyostotic disease, both located in the pelvic bones. Two patients had available follow-up, one developed two local recurrences in the humerus over a 15-year period, while the other showed no recurrence 4 years subsequent to an en-bloc resection. Tumors were positive for CD31 and ERG, while negative for EMA, CK, synaptophysin, and chromogranin. FISH confirmed this abnormality in all cases, none of them being associated with gene amplifications. Further studies are needed to establish the pathogenetic relationship of this rare molecular subset with other epithelioid vascular tumors and to determine its clinical behavior.

Dataset Information

MRNA and protein levels of FUS, EWSR1, and TAF15 are upregulated in liposarcoma.

Publications

mRNA and protein levels of FUS, EWSR1, and TAF15 are upregulated in liposarcoma.

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