Human Clear Cell Sarcoma Research Articles

EWSR1::ATF1 Orchestrates the Clear Cell Sarcoma Transcriptome in Human Tumors and a Mouse Genetic Model.

Clear cell sarcoma (CCS) is a rare, aggressive malignancy that most frequently arises in the soft tissues of the extremities. It is defined and driven by expression of one member of a family of related translocation-generated fusion oncogenes, the most common of which is EWSR1::ATF1. The EWSR1::ATF1 fusion oncoprotein reprograms transcription. However, the binding distribution of EWSR1::ATF1 across the genome and its target genes remain unclear. Here, we interrogated the genomic distribution of V5-tagged EWSR1::ATF1 in tumors it had induced upon expression in mice that also recapitulated the transcriptome of human CCS. ChIP-sequencing of V5-EWSR1::ATF1 identified previously unreported motifs including the AP1 motif and motif comprised of TGA repeats that resemble GGAA-repeating microsatellites bound by EWSR1::FLI1 in Ewing sarcoma. ChIP-sequencing of H3K27ac identified super enhancers in the mouse model and human contexts of CCS, which showed a shared super enhancer structure that associates with activated genes.

Functional genomics of human clear cell sarcoma: genomic, transcriptomic and chemical biology landscape for clear cell sarcoma.

Systemic therapy for metastatic clear cell sarcoma (CCS) bearing EWSR1-CREB1/ATF1 fusions remains an unmet clinical need in children, adolescents, and young adults. To identify key signaling pathway vulnerabilities in CCS, a multi-pronged approach was taken: (i) genomic and transcriptomic landscape analysis, (ii) integrated chemical biology interrogations, (iii) development of CREB1/ATF1 inhibitors, and (iv) antibody-drug conjugate testing (ADC). The first approach encompassed DNA exome and RNA deep sequencing of the largest human CCS cohort yet reported consisting of 47 patient tumor samples and 8 cell lines. Sequencing revealed recurrent mutations in cell cycle checkpoint, DNA double-strand break repair or DNA mismatch repair genes, with a correspondingly low to intermediate tumor mutational burden. DNA multi-copy gains with corresponding high RNA expression were observed in CCS tumor subsets. CCS cell lines responded to the HER3 ADC patritumab deruxtecan in a dose-dependent manner in vitro, with impaired long term cell viability. These studies of the genomic, transcriptomic and chemical biology landscape represent a resource 'atlas' for the field of CCS investigation and drug development. CHK inhibitors are identified as having potential relevance, CREB1 inhibitors non-dependence of CCS on CREB1 activity was established, and the potential utility of HER3 ADC being used in CCSis found.

Establishment and Characterisation of Human Clear Cell Sarcoma and Malignant Melanoma Cell Lines.

Clear cell sarcoma of soft tissue (CCSST) and conventional malignant melanoma (MM) are rare and aggressive tumours with similarities in morphology and the expression of melanocytic markers. We established two CCSST cell lines (FU-CCSST-1 and FU-CCSST-2) from soft tissues of the patella and supraclavicular. A MM cell line (FU-MM-1) was established from lymph node metastases of subungual malignant melanoma. FU-CCSST-2 cells were transplantable to immunodeficient mice. Immunohistochemical studies demonstrated tumour cells were negative for cytokeratin AE1/AE3 and positive for S100 protein, HMB45, Melan-A, CD146 and SOX10 in all specimens. FU-CCSST-1 and FU-CCSST-2 harboured t(12;22)(q13;q12) translocations with expression of the EWSR1/ATF1 fusion gene. FU-MM-1 demonstrated loss of the short arm of chromosome 9 and harboured wild-type BRAF (codon 469 and 600) and NRAS (codon 12, 13 and 61). We report the establishment and characterisation of CCSST and MM cell lines that may have utility in the study of pathogenic mechanisms and development of novel therapeutic reagents.

CD151 confers metastatic potential to clear cell sarcoma of the soft tissue in animal model.

Cluster of differentiation 151 (CD151) is a potent therapeutic target for regulating tumor metastasis. In the present study, the role of CD151 in clear cell sarcoma of soft tissue was examined using a xenoplanted tumor model, which had high rates of metastasis. A clear cell sarcoma cell line, HS-MM, which was transplanted to the aponeuroses of the thighs, the most affected sites of human clear cell sarcoma, exhibited robust lymphatic invasion and nodal metastasis in SCID-beige mice. Serial in vivo passaging of peritoneally disseminated tumor cells accelerated the metastatic activity, which was accompanied by increased CD151 expression, and were designated as HS-MMhigh. Notably, inoculation of anti-CD151 antibody significantly suppressed the lymphatic invasion, peritoneal dissemination and distant metastasis of the present clear cell sarcoma model without affecting local tumor growth at the transplantation site. Small interfering RNA (siRNA)-mediated downregulation of CD151 did not alter cell proliferation, but significantly inhibited Matrigel invasion activity of HS-MMhigh cells. Downregulation of CD151 impaired matrix metalloproteinase-9 activity and phosphorylation of SMAD3 protein in HS-MMhigh cells. The present results suggest that CD151 may contribute to invasion and metastasis of clear cell sarcoma of soft tissue. Therefore, CD151 may serve as a potent target to regulate metastasis of clear cell sarcoma.

Establishment and Characterization of a Novel Human Clear-cell Sarcoma of Soft-tissue Cell Line, RSAR001, Derived from Pleural Effusion of a Patient with Pleural Dissemination.

Clear cell sarcoma (CCS) of soft tissue is exceedingly rare and frequently exhibits aggressive behavior. Toward the goals of improving the aggressive course and poor prognosis of CCS, and establish new therapeutic methods, molecular genetic and biological characterizations of CCS are required. A new human CCS cell line (designated RSAR001) was established from the pleural effusion of a 44-year-old man with multiple lung metastases and pleural dissemination. The cell line and its xenograft were characterized including their morphology, immunohistochemistry, cytogenetic analysis, reverse transcription-polymerase chain reaction, direct sequencing analysis, and fluorescence in situ hybridization analysis. The cell line has been maintained for over 12 months with more than 50 passages. RSAR001 cells exhibited a fascicular or diffuse growth pattern of short spindle- or oval-shaped cells with clear cytoplasm in heterotransplanted tumor, that was similar to the primary tumor. Immunophenotypically, RSAR001 cells in vitro and in vivo exhibited almost the same characteristics as the primary tumor. Cytogenetic analyses revealed a translocation, t(12;22)(q13;q12). Reverse transcription-polymerase chain reaction and direct sequencing analysis detected transcripts of the Ewing sarcoma breakpoint region 1-activating transcription factor 1 (EWSR1-ATF1) type 1 fusion gene. Fluorescence in situ hybridization using a break-apart probe for the EWSR1 gene on 22q12 showed a rearrangement. These findings indicate that the RSAR001 cell line harbors EWSR1-ATF1 type 1 chimeric fusion gene, which is specific to CCS. RSAR001 cells might be useful for investigating biological behaviors and developing new treatments such as molecular-targeting antitumor drugs or immunological drugs for CCS.

Trabectedin is a promising antitumor agent potentially inducing melanocytic differentiation for clear cell sarcoma.

Clear cell sarcoma is an aggressive soft tissue sarcoma and highly resistant to conventional chemotherapy and radiation therapy. This devastating disease is defined by EWSR1‐ATF1 fusion gene resulting from chromosomal translocation t(12;22)(q13;q12) and characterized by melanocytic differentiation. A marine‐derived antineoplastic agent, trabectedin, inhibits the growth of myxoid liposarcoma and Ewing sarcoma by causing adipogenic differentiation and neural differentiation, respectively. In this study, we examined the antitumor effects and mechanism of action of trabectedin on human clear cell sarcoma cell lines. We showed that trabectedin decreased the cell proliferation of five clear cell sarcoma cell lines in a dose‐dependent manner in vitro and reduced tumor growth of two mouse xenograft models. Flow cytometry and immunoblot analyses in vitro and immunohistochemical analysis in vivo revealed that trabectedin‐induced G2/M cell cycle arrest and apoptosis. Furthermore, trabectedin increased the expression of melanocytic differentiation markers along with downregulation of ERK activity in vitro and the rate of melanin‐positive cells in vivo. These results suggest that trabectedin has potent antitumor activity against clear cell sarcoma cells by inducing cell cycle arrest, apoptosis, and, in part, by promoting melanocytic differentiation through inactivation of ERK signaling. Our present study indicates that trabectedin is a promising differentiation‐inducing agent for clear cell sarcoma.

Abstract 2141: TAS-115, a novel and highly potent VEGFR/MET inhibitor, shows prominent antitumor efficacy by restoring HGF-mediated hypoxia-resistant phenotype in clear cell sarcoma

Abstract <Background> Hepatocyte growth factor (HGF) / HGF receptor (MET) signaling is considered to be involved in chemoresistance to cancer treatment. Notably, many reports have described reduced sensitivity to vascular endothelial growth factor receptor (VEGFR)-targeted inhibitors in patients with high serum HGF levels compared to those with low serum levels. This study investigated whether HGF directly influenced sensitivity to VEGFR-targeted inhibitors in human clear cell sarcoma (CCS) xenograft models using human HGF knock-in (hHGF KI) mice, and evaluated the potency of TAS-115. <Material and methods> MET signaling pathway in SU-CCS-1, a human CCS cell line, were analyzed using Western blotting analysis. To study the effects of hypoxia in SU-CCS-1 cells, culture plates were placed in airtight jars under anaerobic conditions and cell viability was assessed by ATP-based assay. In the in vivo study, SU-CCS-1 cells were subcutaneously implanted into hHGF KI mice, and compounds were orally administered once daily for 14 consecutive days. Tumor vessel density (TVD) was determined by immunohistochemistry using anti-CD31 antibody. <Results> SU-CCS-1 cells expressed MET, and exogenous HGF phosphorylated MET and its downstream factors in SU-CCS-1 cells. Exogenous HGF also significantly enhanced SU-CCS-1 cell proliferation. TAS-115 inhibited HGF-induced MET phosphorylation and SU-CCS-1 cell proliferation in a dose-dependent manner at concentrations higher than 10 nM. In contrast, pazopanib, a VEGFR-targeted multi-kinase inhibitor, could not block HGF-induced phenotypes in SU-CCS-1 cells, even at 1 μM. To evaluate the effects of HGF on the antitumor activities of TAS-115 and pazopanib against SU-CCS-1 cells, these agents were administered in hHGF KI and wild-type (WT) mice bearing subcutaneous SU-CCS-1 tumors. TAS-115 completely suppressed tumor growth and reduced TVD in SU-CCS-1 tumor tissue in both models at a dose of 200 mg/kg/d. In contrast, pazopanib suppressed tumor growth at a dose of 100 mg/kg/d in WT mice but not in hHGF KI mice. However, pazopanib significantly reduced TVD in SU-CCS-1 tumor tissue in both models. HGF is therefore suggested to impact SU-CCS-1 cells rather than mouse endothelial cells. We considered that HGF modulated cell proliferation or survival in SU-CCS-1 cells under hypoxia. Actually, hypoxic conditions induced apoptosis in SU-CCS-1 cells, and endogenous HGF dose-dependently attenuated hypoxia-related apoptosis. TAS-115 clearly restored HGF-mediated cytoprotection under hypoxia but pazopanib did not. <Conclusion> These results suggest that effects of VEGFR-targeted inhibitors are attenuated by HGF-mediated hypoxia resistance, and simultaneous inhibitors of the MET and VEGFR axes, such as TAS-115, are more effective in sarcoma patients with high serum HGF than inhibition of the VEGFR axis alone. Citation Format: Hidenori Fujita, Yukari Yamada, Miki Terasaka, Yayoi Fujioka, Naomoto Harada, Akihiro Hashimoto, Kazutaka Miyadera, Kenichi Matsuo, Kazuhiko Yonekura. TAS-115, a novel and highly potent VEGFR/MET inhibitor, shows prominent antitumor efficacy by restoring HGF-mediated hypoxia-resistant phenotype in clear cell sarcoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2141.

Boron neutron capture therapy as new treatment for clear cell sarcoma: Trial on different animal model

Clear cell sarcoma (CCS) is a rare malignant tumor with a poor prognosis. In our previous study, the tumor disappeared under boron neutron capture therapy (BNCT) on subcutaneously-transplanted CCS-bearing animals. In the present study, the tumor disappeared under this therapy on model mice intramuscularly implanted with three different human CCS cells. BNCT led to the suppression of tumor-growth in each of the different model mice, suggesting its potentiality as an alternative to, or integrative option for, the treatment of CCS.

Modeling Clear Cell Sarcomagenesis in the Mouse: Cell of Origin Differentiation State Impacts Tumor Characteristics

Clear cell sarcoma (CCS) of tendons and aponeuroses is a deadly soft-tissue malignancy resembling melanoma, with a predilection for young adults. EWS-ATF1, the fusion product of a balanced chromosomal translocation between chromosomes 22 and 12, is considered the definitional feature of the tumor. Conditional expression of the EWS-ATF1 human cDNA in the mouse generates CCS-like tumors with 100% penetrance. Tumors, developed through varied means of initiating expression of the fusion oncogene, model human CCS morphologically, immunohistochemically, and by genome-wide expression profiling. We also demonstrate that although fusion oncogene expression in later stages of differentiation can transform mesenchymal progenitor cells and generate tumors resembling CCS generally, expression in cells retaining stem cell markers permits the full melanoma-related phenotype.

EWS/ATF1 expression induces sarcomas from neural crest–derived cells in mice

Clear cell sarcoma (CCS) is an aggressive soft tissue malignant tumor characterized by a unique t(12;22) translocation that leads to the expression of a chimeric EWS/ATF1 fusion gene. However, little is known about the mechanisms underlying the involvement of EWS/ATF1 in CCS development. In addition, the cellular origins of CCS have not been determined. Here, we generated EWS/ATF1-inducible mice and examined the effects of EWS/ATF1 expression in adult somatic cells. We found that forced expression of EWS/ATF1 resulted in the development of EWS/ATF1-dependent sarcomas in mice. The histology of EWS/ATF1-induced sarcomas resembled that of CCS, and EWS/ATF1-induced tumor cells expressed CCS markers, including S100, SOX10, and MITF. Lineage-tracing experiments indicated that neural crest-derived cells were subject to EWS/ATF1-driven transformation. EWS/ATF1 directly induced Fos in an ERK-independent manner. Treatment of human and EWS/ATF1-induced CCS tumor cells with FOS-targeted siRNA attenuated proliferation. These findings demonstrated that FOS mediates the growth of EWS/ATF1-associated sarcomas and suggest that FOS is a potential therapeutic target in human CCS.

Boron neutron capture therapy (BNCT) selectively destroys human clear cell sarcoma in mouse model

Clear cell sarcoma of tendons and aponeuroses (CCS) is a rare malignant tumor with no effective treatment. This study demonstrates the efficacy of BNCT with the use of human CCS-bearing nude mice. Groups A and C were administered saline, and groups B and D were injected with p-borono-l-phenylalanine-fructose complex. Groups C and D were then irradiated with thermal neutrons. The tumors in only group D disappeared, demonstrating that BNCT is a potentially new option for the treatment of human CCS.

The melanocyte inducing factor MITF is stably expressed in cell lines from human clear cell sarcoma.

Clear cell sarcoma (CCS) is associated with the EWS/ATF1 oncogene that is created by chromosomal fusion of the Ewings Sarcoma oncogene (EWS) and the cellular transcription factor ATF1. The melanocytic character of CCS suggests that the microphthalmia-associated transcription factor (Mitf), a major inducer of melanocytic differentiation, may be miss-expressed in CCS. Accordingly, we show that the mRNA and protein of the melanocyte-specific isoform of Mitf (Mitf-M) are present in several cultured CCS cell lines (Su-ccs-1, DTC1, Kao, MST-1, MST-2 and MST-3). The above cell lines thus provide a valuable experimental resource for examining the role of Mitf-M in both CCS and melanocyte differentiation. Melanocyte-specific expression of Mitf-M is achieved via an ATF-dependent melanocyte-specific cAMP-response element in the Mitf-M promoter, and expression of Mitf-M in CCS cells suggests that EWS/ATF1 (a potent and promiscuous activator of cAMP-inducible promoters) may activate the Mitf-M promoter. Surprisingly, however, the Mitf-M promoter is not activated by EWS/ATF1 in transient assays employing CCS cells, melanocytes or nonmelanocytic cells. Thus, our results indicate that Mitf-M promoter activation may require an appropriate chromosomal context in CCS cells or alternatively that the Mitf-M promoter is not directly activated by EWS/ATF1.

Establishment and characterisation of a human clear cell sarcoma model in nude mice.

We have established a new experimental model of human clear cell sarcoma, UM-CCS1, using serial subcutaneous transplantation of intact tumour tissue in nude mice. The heterotransplanted nude mouse tumours retained characteristic morphological features of the primary clear cell sarcoma. Immunohistochemical analysis showed the retained expression patterns of S-100 protein, melanoma-associated antigen HMB-45 and vimentin in the xenografts as compared to the primary tumour. DNA index showed low variations both between the xenografts in the same passage and between the serial passages. Cytogenetic analysis of the primary tumour and the xenografts showed the unbalanced translocation der(6)t(6;12)(p23;q13). Based on the combined genetic data a reasonable interpretation of our findings is that there was a complex chromosomal rearrangement resulting in a cytogenetically cryptic EWS-ATF1 fusion gene. Analysis of cell kinetics using in vivo incorporation of iododeoxyuridine and flow cytometry showed generally short potential doubling time (T(pot)) of the xenografts. Volume doubling time showed low variations without correlation with T(pot). The retained phenotypic and genotypic characteristics of the primary tumour and the morphological and structural stability over time makes the model suitable for studies on the tumour biology and treatment of clear cell sarcoma.

Histamine enhanced antitumor effect of docetaxel and dacarbazine in human clear cell sarcoma xenografts in nude mice

Histamine enhanced antitumor effect of docetaxel and dacarbazine in human clear cell sarcoma xenografts in nude mice

Microfilament-undercoated plasmalemmal infoldings in a human clear-cell sarcoma

Unusual structures often found in the cytoplasm of tumor cells in a clear-cell sarcoma appeared as multilayered, concentric, oval, spiral, parallel arrays of cisternae in various planes of section. It was demonstrated that the cisternal membrane and cavity were continuous with plasmalemmas of tumor cells and the extracellular space, respectively, suggesting that the structures were formed by the intracytoplasmic infoldings of plasmalemmas. Another characteristic found in the structures was orderly microfilaments with an average diameter of 6.5 nm which were placed between the confronting plasmalemmas in the infoldings. The filaments which underlay the infolded plasmalemmas ran parallel to each other along the cytoplasmic surfaces of plasmalemmas approximately 15 nm apart. The regularly arranged filaments were found in the infolded plasmalemmas, but not beneath any other area of plasmalemmas. The short ends of long filaments appeared to bend toward the inner surfaces of plasmalemma and to be directly connected with the surface proper. These results show that the filaments may be closely associated with the plasmalemmal infoldings and included as the same category of plasmalemmal undercoat. Additionally, the biological significance of the structures is discussed.