Epithelioid Sarcoma Cell Line Research Articles

Expression of Forkhead Box M1 and Anticancer Effects of FOXM1 Inhibition in Epithelioid Sarcoma

Epithelioid sarcoma (ES) is a rare aggressive sarcoma that, unlike most soft-tissue sarcomas, shows a tendency toward local recurrence and lymph node metastasis. Novel antitumor agents are needed for ES patients. Forkhead box transcription factor 1 (FOXM1) is a member of the Forkhead transcription factor family and is associated with multiple oncogenic functions; FOXM1 is known to be overexpressed and correlated with pathogenesis in various malignancies. In this study, we immunohistochemically analyzed FOXM1 expression levels and their clinical, clinicopathologic, and prognostic significance in 38 ES specimens. In addition, to investigate potential correlations between FOXM1 downregulation and oncologic characteristics, we treated ES cell lines with thiostrepton, a naturally occurring antibiotic that inhibits both small interfering RNA (siRNA) and FOXM1. In the analyses using ES samples, all 38 specimens were diagnosed as positive for FOXM1 by immunohistochemistry. We separated specimens into high (n = 19) and low (n = 19) FOXM1–protein expression groups by staining index score, and into large (n = 12), small (n = 25), and unknown (n = 1) tumor-size groups using a cutoff of 5 cm maximum diameter. Although there were significantly more samples with high FOXM1 expression in the large tumor group (P = .013), there were no significant differences with respect to age (P = 1.00), sex (P = .51), primary site of origin (P = .74), histologic subtypes (P = 1.00), depth (P = .74), or survival rate (P = .288) between the high and low FOXM1–protein expression groups. In the in vitro experiments using ES cell lines, FOXM1 siRNA and thiostrepton successfully downregulated FOXM1 mRNA and protein expression. Furthermore, downregulation of FOXM1 inhibited cell proliferation, drug resistance against chemotherapeutic agents, migration, and invasion and caused cell cycle arrest in the ES cell lines. Finally, cDNA microarray analysis data showed that FOXM1 regulated cIAP2, which is one of the apoptosis inhibitors activated by the TNFα-mediated NF-κB pathway. In conclusion, the FOXM1 gene may be a promising therapeutic target for ES.

Photoimmunotheranostics of epithelioid sarcoma by targeting CD44 or EGFR

Epithelioid sarcoma (ES) is a rare soft tissue neoplasm with high recurrence rates. Wide surgical resection remains the only potential curative treatment. ES presents most commonly on the fingers, hands and forearm, making light-based cancer cell-targeted therapies such as near-infrared photoimmunotherapy (NIR-PIT) that is target-specific, but with limited penetration depth, suitable for ES treatment. We established that CD44 and EGFR were overexpressed in ES patient samples and in the VA-ES-BJ human ES cell line. NIR-PIT of VA-ES-BJ cells using antibody photosensitizer conjugates, prepared by conjugating a CD44 or EGFR monoclonal antibody to the photosensitizer IR700, confirmed that NIR-PIT with both conjugates resulted in cell death. Neither treatment with NIR light alone nor treatment with the conjugates but without NIR light were effective. CD44-IR700-PIT resulted in greater cell death than EGFR-IR700-PIT, consistent with the increased expression of CD44 by VA-ES-BJ cells. In tumors, EGFR-IR700 exhibited a higher tumor-to-normal ratio, as determined by in vivo fluorescence imaging, and a higher anti-tumor growth effect, compared to CD44-IR700. No antitumor effect of the EGFR antibody or the photosensitizer conjugate alone was observed in vivo. Our data support evaluating the use of EGFR-IR700-PIT in the management of ES for detecting and eliminating ES cells in surgical margins, and in the treatment of superficial recurrent tumors.

A Modular Composite Device of Poly(Ethylene Oxide)/Poly(Butylene Terephthalate) (PEOT/PBT) Nanofibers and Gelatin as a Dual Drug Delivery System for Local Therapy of Soft Tissue Tumors.

In the clinical management of solid tumors, the possibility to successfully couple the regeneration of injured tissues with the elimination of residual tumor cells left after surgery could open doors to new therapeutic strategies. In this work, we present a composite hydrogel–electrospun nanofiber scaffold, showing a modular architecture for the delivery of two pharmaceutics with distinct release profiles, that is potentially suitable for local therapy and post-surgical treatment of solid soft tumors. The composite was obtained by coupling gelatin hydrogels to poly(ethylene oxide)/poly(butylene terephthalate) block copolymer nanofibers. Results of the scaffolds’ characterization, together with the analysis of gelatin and drug release kinetics, displayed the possibility to modulate the device architecture to control the release kinetics of the drugs, also providing evidence of their activity. In vitro analyses were also performed using a human epithelioid sarcoma cell line. Furthermore, publicly available expression datasets were interrogated. Confocal imaging showcased the nontoxicity of these devices in vitro. ELISA assays confirmed a modulation of IL-10 inflammation-related cytokine supporting the role of this device in tissue repair. In silico analysis confirmed the role of IL-10 in solid tumors including 262 patients affected by sarcoma as a negative prognostic marker for overall survival. In conclusion, the developed modular composite device may provide a key-enabling technology for the treatment of soft tissue sarcoma.

CHIR99021, trough GSK-3β Targeting, Reduces Epithelioid Sarcoma Cell Proliferation by Activating Mitotic Catastrophe and Autophagy.

Epithelioid sarcoma (ES) is a rare disease representing <1% of soft tissue sarcomas. Current therapies are based on anthracycline alone or in combination with ifosfamide or other cytotoxic drugs. ES is still characterized by a poor prognosis with high rates of recurrence. Indeed, for years, ES survival rates have remained stagnant, suggesting that conventional treatments should be revised and improved. New therapeutic approaches are focused to target the key regulators of signaling pathways, the causative markers of tumor pathophysiology. To this end, we selected, among the drugs to which an ES cell line is highly sensitive, those that target signaling pathways known to be dysregulated in ES. In particular, we found a key role for GSK-3β, which results in up-regulation in tumor versus normal tissue samples and associated to poor prognosis in sarcoma patients. Following this evidence, we evaluated CHIR99021, a GSK-3 inhibitor, as a potential drug for use in ES therapy. Our data highlight that, in ES cells, CHIR99021 induces cell cycle arrest, mitotic catastrophe (MC) and autophagic response, resulting in reduced cell proliferation. Our results support the potential efficacy of CHIR99021 in ES treatment and encourage further preclinical and clinical studies.

Correction: SMARCB1/INI1 Genetic Inactivation Is Responsible for Tumorigenic Properties of Epithelioid Sarcoma Cell Line VAESBJ.

In the original version of [this article][1] ([1][2]), Fig. 5D contained two erroneously swapped blots and one accidentally duplicated blot. The error has been corrected in the latest online HTML and PDF versions of the article. The authors regret this error. 1. 1.[↵][3]1. Brenca M, 2.

Histone deacetylase inhibitor panobinostat induces antitumor activity in epithelioid sarcoma and rhabdoid tumor by growth factor receptor modulation

BackgroundEpithelioid sarcomas and rhabdoid tumors are rare, aggressive malignancies with poor prognosis. Both are characterized by INI1 alterations and deregulation of growth factor receptors albeit their interaction has not been elucidated.MethodsIn this study, we investigated the activity of a panel of epigenetic modulators and receptor tyrosine kinase inhibitors in vitro on respective cell lines as well as on primary patient-derived epithelioid sarcoma cells, and in vivo on xenografted mice. Focusing on histone deacetylase (HDAC) inhibitors, we studied the mechanism of action of this class of agents, its effect on growth factor receptor regulation, and changes in epithelial-to-mesenchymal transition by using cell- and RT-qPCR-based assays.ResultsPan-HDAC inhibitor panobinostat exhibited potent anti-proliferative activity at low nanomolar concentrations in A204 rhabdoid tumor, and VAESBJ/GRU1 epithelioid sarcoma cell lines, strongly induced apoptosis, and resulted in significant tumor growth inhibition in VAESBJ xenografts. It differentially regulated EGFR, FGFR1 and FGFR2, leading to downregulation of EGFR in epithelioid sarcoma and to mesenchymal-to-epithelial transition whereas in rhabdoid tumor cells, EGFR was strongly upregulated and reinforced the mesenchymal phenotype. All three cell lines were rendered more susceptible towards combination with EGFF inhibitor erlotinib, further enhancing apoptosis.ConclusionsHDAC inhibitors exhibit significant anticancer activity due to their multifaceted actions on cytotoxicity, differentiation and drug sensitization. Our data suggest that the tailored, tissue-specific combination of HDAC inhibitors with therapeutics which target cellular salvage mechanisms might increase their therapeutic relevance.

CD73 complexes with emmprin to regulate MMP-2 production from co-cultured sarcoma cells and fibroblasts

BackgroundInteraction between cancer cells and fibroblasts mediated by extracellular matrix metalloproteinase inducer (emmprin, CD147) is important in the invasion and proliferation of cancer cells. However, the exact mechanism of emmprin mediated stimulation of matrix metalloprotease-2 (MMP-2) production from fibroblasts has not been elucidated. Our previous studies using an inhibitory peptide against emmprin suggested the presence of a molecule on the cell membrane which forms a complex with emmprin. Here we show that CD73 expressed on fibroblasts interacts with emmprin and is a required factor for MMP-2 production in co-cultures of sarcoma cells with fibroblasts.MethodsCD73 along with CD99 was identified by mass spectrometry analysis as an emmprin interacting molecule from a co-culture of cancer cells (epithelioid sarcoma cell line FU-EPS-1) and fibroblasts (immortalized fibroblasts cell line ST353i). MMP-2 production was measured by immunoblot and ELISA. The formation of complexes of CD73 with emmprin was confirmed by immunoprecipitation, and their co-localization in tumor cells and fibroblasts was shown by fluorescent immunostaining and proximity ligation assays.ResultsStimulated MMP-2 production in co-culture of cancer cells and fibroblasts was completely suppressed by siRNA knockdown of CD73, but not by CD99 knockdown. MMP-2 production was not suppressed by CD73-specific enzyme inhibitor (APCP). However, MMP-2 production was decreased by CD73 neutralizing antibodies, suggesting that CD73-mediated suppression of MMP-2 production is non-enzymatic. In human epithelioid sarcoma tissues, emmprin was immunohistochemically detected to be mainly expressed in tumor cells, and CD73 was expressed in fibroblasts and tumor cells: emmprin and CD73 were co-localized predominantly on tumor cells.ConclusionThis study provides a novel insight into the role of CD73 in emmprin-mediated regulation of MMP-2 production.

Emmprin, released as a microvesicle in epithelioid sarcoma, interacts with fibroblasts.

Emmprin (extracellular matrix metalloproteinase inducer, CD147) is a glycosylated transmembrane protein, consisting of two immunoglobulin domains, that stimulates the production of matrix metalloproteinases (MMPs) by tumor-associated fibroblasts. These effects play important roles in tumor invasion and metastasis. However, the precise mechanisms by which emmprin acts on fibroblasts have not been fully elucidated, especially in sarcoma cells. Previously, we demonstrated that emmprin, expressed in conditioned medium collected from the epithelioid sarcoma cell line (FU-EPS-1), stimulates MMP-2 production via interactions with fibroblasts. In this study, we used microvesicles derived from sarcoma cells, and determined whether emmprin exists in the microvesicles, which enhance the production of MMP-2 via fibroblasts. Microvesicles released from FU-EPS-1 cells were shown to contain full-length emmprin, identified as a 45-kDa protein characterized by polylactosamine glycosylation. Microvesicles collected from FU-EPS-1 cells transfected with emmprin-specific siRNA or transduced with shRNA displayed significantly reduced MMP-2 production by fibroblasts compared with those from control-transfected cells. Our findings show that emmprin is released through microvesicle shedding in sarcoma cells, and emmprin in microvesicles regulates MMP-2 production by influencing the activity of fibroblasts located at sites distant from the tumor cells.

The genomic landscape of epithelioid sarcoma cell lines and tumours.

We carried out whole genome and transcriptome sequencing on four tumour/normal pairs of epithelioid sarcoma. These index cases were supplemented with whole transcriptome sequencing of three additional tumours and three cell lines. Unlike rhabdoid tumour (the other major group of SMARCB1-negative cancers), epithelioid sarcoma shows a complex genome with a higher mutational rate, comparable to that of ovarian carcinoma. Despite this mutational burden, SMARCB1 mutations remain the most frequently recurring event and are probably critical drivers of tumour formation. Several cases show heterozygous SMARCB1 mutations without inactivation of the second allele, and we explore this further in vitro. Finding CDKN2A deletions in our discovery cohort, we evaluated CDKN2A protein expression in a tissue microarray. Six out of 16 cases had lost CDKN2A in greater than or equal to 90% of cells, while the remaining cases had retained the protein. Expression analysis of epithelioid sarcoma cell lines by transcriptome sequencing shows a unique profile that does not cluster with any particular tissue type or with other SWI/SNF-aberrant lines. Evaluation of the levels of members of the SWI/SNF complex other than SMARCB1 revealed that these proteins are expressed as part of a residual complex, similarly to previously studied rhabdoid tumour lines. This residual SWI/SNF is susceptible to synthetic lethality and may therefore indicate a therapeutic opportunity.

Abstract LB-251: Histone deacetylase inhibition for the treatment of epithelioid sarcoma; novel cross talk between epigenetic components

Abstract Objective Enhanced knowledge of epithelioid sarcoma (ES) molecular determinants will hopefully aid the development of urgently needed targeted therapies. The hallmark loss of INI1 causes a disruption in epigenetics, potentially contributing to the development of the disease. Recently, we have demonstrated that HDAC inhibitors (HDACi) to have efficacious value for the treatment various genetically complex sarcomas. Here, we aim to evaluate the potential therapeutic efficacy of HDACi in ES. Methods Three ES cell lines were used for this study: Epi-544, HS-ES, VAESBJ. MTS and clonogenic assays were utilized to evaluate the effect of HDACi on cell line growth. Cell cycle FACS and annexin V PI/FACS analysis with WB for cleaved caspase 3 were used to determine the effects of HDACi on cell cycle and apoptosis, respectively. In vivo growth effects of HDACi were evaluated using SCID mouse ES xenograft models. An Illumina gene array was used to evaluate HDACi-induced gene change. Results HDACi inhibited ES cell growth and colony formation. HDACi induced G2 cell cycle arrest and enhanced apoptosis in all ES cell lines tested. Similarly, HDACi abrogated ES xenograft growth and increased apoptosis in vivo. Due to the HDACi mechanisms of function, one of the major consequences of this therapy is gene expression modulation. Using an Illumina gene array, we sought to identify genes responsible for the in vitro and in vivo anti-ES effects observed. Array analysis showed Survivin be significantly down-regulated in response to HDACi treatment. HDACi-induced decrease of Survivin was confirmed via WB. For enrichment analysis of our genes set with published annotated gene signatures, the Molecular Signatures Database (MSigDB) was used. MSigDB analysis identified a curated gene set of regulated genes after EZH2 knockdown (KD) with significant commonality of our gene array. We demonstrated HDACi-induced decrease of EZH2. EZH2 KD resulted in reduced cell growth and increased apoptosis in VAESBJ cells. Conclusion HDACs play a role in the progression of ES, where HDACi abrogate ES cell growth and enhance apoptosis in vitro and in vivo. Gene array analysis revealed various genes modulated by HDACi that may contribute to the progression of the disease. Anti-ES effects may be in part through the HDACi-induced repression of EZH2. Citation Format: Gonzalo Lopez, Yechun Song, Dennis Ruder, Chad Creighton Creighton, Svetlana Bolshakov, Xiaoli Zhang, Dina Lev, Raphael Pollock. Histone deacetylase inhibition for the treatment of epithelioid sarcoma; novel cross talk between epigenetic components. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-251. doi:10.1158/1538-7445.AM2015-LB-251

High Expression of CD109 Antigen Regulates the Phenotype of Cancer Stem-Like Cells/Cancer-Initiating Cells in the Novel Epithelioid Sarcoma Cell Line ESX and Is Related to Poor Prognosis of Soft Tissue Sarcoma

Epithelioid sarcoma (ES) is a relatively rare, highly malignant soft tissue sarcoma. The mainstay of treatment is resection or amputation. Currently other therapeutic options available for this disease are limited. Therefore, a novel therapeutic option needs to be developed. In the present study, we established a new human ES cell line (ESX) and analyzed the characteristics of its cancer stem-like cells/cancer-initiating cells (CSCs/CICs) based on ALDH1 activity. We demonstrated that a subpopulation of ESX cells with high ALDH1 activity (ALDHhigh cells) correlated with enhanced clonogenic ability, sphere-formation ability, and invasiveness in vitro and showed higher tumorigenicity in vivo. Next, using gene expression profiling, we identified CD109, a GPI-anchored protein upregulated in the ALDHhigh cells. CD109 mRNA was highly expressed in various sarcoma cell lines, but weakly expressed in normal adult tissues. CD109-positive cells in ESX predominantly formed spheres in culture, whereas siCD109 reduced ALDH1 expression and inhibited the cell proliferation in vitro. Subsequently, we evaluated the expression of CD109 protein in 80 clinical specimens of soft tissue sarcoma. We found a strong correlation between CD109 protein expression and the prognosis (P = 0.009). In conclusion, CD109 might be a CSC/CIC marker in epithelioid sarcoma. Moreover, CD109 is a promising prognostic biomarker and a molecular target of cancer therapy for sarcomas including ES.

A novel angiomatoid epithelioid sarcoma cell line, Asra-EPS, forming tumors with large cysts containing hemorrhagic fluid in vivo

BackgroundWhereas we can use several human epithelioid sarcoma (ES) cell lines for basic and preclinical research, an angiomatoid ES cell line has not been reported to date. We have treated a case of an angiomatoid ES developing in the right upper extremity of a 67-year-old man.MethodsAn angiomatoid ES cell line, Asra-EPS was newly established and characterized for its morphology, growth rate and chromosomal analysis. Tumorigenicity of Asra-EPS cells was also analyzed in athymic nude mice.ResultsAsra-EPS cells were round, polygonal or spindle-shaped with an abundant cytoplasm and have been maintained continuously in vitro for over 150 passages during more than 15 months. These cells secreted cancer antigen 125 (CA 125), interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) into the culture medium. Asra-EPS cells were tumorigenic when implanted in nude mice with tumors reaching a volume of 1000 mm3 at around 50 days. Histological features of tumors formed in mice were essentially the same as those of the original tumor, exhibiting a multinodular proliferation of eosinophilic epithelioid and spindle-shaped cells with prominent areas of hemorrhage and blood-filled cystic spaces strikingly corresponding to the potential of hemorrhagic cyst formation in the original tumor. They showed immunopositive staining for cytokeratins (AE1/AE3 and CAM5.2), epithelial membrane antigen (EMA), vimentin, CD31, CD34 and CA 125, but negative for integrase interactor 1 (INI-1) and factor VIII-related antigen.ConclusionsThe established cell line represents a biologically relevant new tool to investigate the molecular pathology of human angiomatoid ES and to evaluate the efficacy of novel therapeutics both in vitro and in vivo.

SMARCB1/INI1 Genetic Inactivation Is Responsible for Tumorigenic Properties of Epithelioid Sarcoma Cell Line VAESBJ

Epithelioid sarcoma is a rare soft tissue neoplasm that usually arises in the distal extremities of young adults. Epithelioid sarcoma presents a high rate of recurrences and metastases and frequently poses diagnostic dilemmas. We previously reported loss of tumor suppressor SMARCB1 protein expression and SMARCB1 gene deletion in the majority of epithelioid sarcoma cases. Unfortunately, no appropriate preclinical models of such genetic alteration in epithelioid sarcoma are available. In the present report, we identified lack of SMARCB1 protein due to a homozygous deletion of exon 1 and upstream regulatory region in epithelioid sarcoma cell line VAESBJ. Restoration of SMARCB1 expression significantly affected VAESBJ cell proliferation, anchorage-independent growth, and cell migration properties, thus supporting the causative role of SMARCB1 loss in epithelioid sarcoma pathogenesis. We investigated the translational relevance of this genetic background in epithelioid sarcoma and showed that SMARCB1 ectopic expression significantly augmented VAESBJ sensitivity to gamma irradiation and acted synergistically with flavopiridol treatment. In VAESBJ, both activated ERBB1/EGFR and HGFR/MET impinged on AKT and ERK phosphorylation. We showed a synergistic effect of combined inhibition of these 2 receptor tyrosine kinases using selective small-molecule inhibitors on cell proliferation. These observations provide definitive support to the role of SMARCB1 inactivation in the pathogenesis of epithelioid sarcoma and disclose novel clues to therapeutic approaches tailored to SMARCB1-negative epithelioid sarcoma.

Effects of TRAIL and taurolidine on apoptosis and proliferation in human rhabdomyosarcoma, leiomyosarcoma and epithelioid cell sarcoma

Soft tissue sarcomas (STS) are a heterogeneous group of malignant tumours representing 1% of all malignancies in adults. Therapy for STS should be individualised and multimodal, but complete surgical resection with clear margins remains the mainstay of therapy. Disseminated soft tissue sarcoma still represents a therapeutic dilemma. Commonly used chemotherapeutic agents such as doxorubicin and ifosfamide have proven to be effective in fewer than 30% in these cases. Therefore, we tested the apoptotic and anti-proliferative in vitro effects of TNF-related apoptosis-inducing ligand (TRAIL) and taurolidine (TRD) on rhabdomyosarcoma (A-204), leiomyosarcoma (SK-LMS-1) and epithelioid cell sarcoma (VA-ES-BJ) cell lines. Viability, apoptosis and necrosis were quantified by FACS analysis (propidium iodide/Annexin V staining). Gene expression was analysed by DNA microarrays and the results validated for selected genes by rtPCR. Protein level changes were documented by western blot analysis. Cell proliferation was analysed by BrdU ELISA assay. The single substances TRAIL and TRD significantly induced apoptotic cell death and decreased proliferation in rhabdomyosarcoma and epithelioid cell sarcoma cells. The combined use of TRAIL and TRD resulted in a synergistic apoptotic effect in all three cell lines, especially in rhabdomyosarcoma cells leaving 18% viable cells after 48 h of incubation (p<0.05). Analysis of the differentially regulated genes revealed that TRD and TRAIL influence apoptotic pathways, including the TNF-receptor associated and the mitochondrial pathway. Microarray analysis revealed remarkable expression changes in a variety of genes, which are involved in different apoptotic pathways and cross talk to other pathways at multiple levels. This in vitro study demonstrates that TRAIL and TRD synergise in inducing apoptosis and inhibiting proliferation in different human STS cell lines. Effects on gene expression differ relevantly in the sarcoma entities. These results provide experimental support for in vivo trials assessing the effect of TRAIL and TRD in STS and sustain the approach of individualized therapy.

Combining EGFR and mTOR blockade for the treatment of epithelioid sarcoma.

Molecular deregulations underlying epithelioid sarcoma (ES) progression are poorly understood yet critically needed to develop new therapies. Epidermal growth factor receptor (EGFR) is overexpressed in ES; using preclinical models, we examined the ES EGFR role and assessed anti-ES EGFR blockade effects, alone and with mTOR inhibition. EGFR and mTOR expression/activation was examined via tissue microarray (n = 27 human ES specimens; immunohistochemistry) and in human ES cell lines (Western blot and quantitative reverse transcriptase PCR). Cell proliferation, survival, migration, and invasion effects of EGFR and mTOR activation treated with erlotinib (anti-EGFR small-molecule inhibitor) alone and combined with rapamycin were assessed in cell culture assays. In vivo growth effects of erlotinib alone or with rapamycin were evaluated using severe combined immunodeficient mouse ES xenograft models. EGFR was expressed and activated in ES specimens and cell lines. EGFR activation increased ES cell proliferation, motility, and invasion and induced cyclin D1, matrix metalloproteinase (MMP) 2, and MMP9 expression. EGFR blockade inhibited these processes and caused significant cytostatic ES growth inhibition in vivo. mTOR pathway activation at varying levels was identified in all tissue microarray-evaluable ES tissues; 88% of samples had no or reduced PTEN expression. Similarly, both ES cell lines showed enhanced mTOR activity; VAESBJ cells exhibited constitutive mTOR activation uncoupled from EGFR signaling. Most importantly, combined erlotinib/rapamycin resulted in synergistic anti-ES effects in vitro and induced superior tumor growth inhibition in vivo versus single agent administration. EGFR and mTOR signaling pathways are deregulated in ES. Preclinical ES model-derived insights suggest that combined inhibition of these targets might be beneficial, supporting evaluations in clinical trials.

Epithelioid Sarcoma and Unclassified Sarcoma with Epithelioid Features: Clinicopathological Variables, Molecular Markers, and a New Experimental Model

Epithelioid sarcoma (ES) and unclassified sarcoma with epithelioid features (USEF) are clinically and therapeutically unresolved. We compared ES and USEF patients' clinical behavior, treatment, outcome, and molecular marker expression. Furthermore, preclinical ES study models were developed to enable comprehensive benchside investigations. A database of ES and USEF patients (n = 116) treated since 1992 was created. A clinically annotated ES-USEF tissue microarray (TMA) was assayed for tumor-related markers. Newly established human and commercially available ES cell lines were characterized and tested in vivo. ES and USEF patients presenting with localized disease exhibited 22% and 25% local recurrence rates, 35% and 19% nodal metastasis rates, and 41% and 53% distant metastasis rates (median follow-up, 54 months and 39 months, respectively). The 5- and 10-year disease-specific survival rates were 88% and 43% and 52% and 42% (ES and USEF, respectively). TMA immunohistochemistry identified integrase interactor (INI)-1 loss, cancer antigen 125, and p53 nuclear expression as significantly more common in ES than USEF cases. Both cell lines preserved ES morphological and biochemical characteristics in vitro and in vivo; loss of INI-1 was shown to occur in both lines. Enhanced knowledge of ES and USEF clinical behavior, marker expression, and molecular determinants, extended via experimental models, will hopefully accelerate development of urgently needed effective targeted therapies for ES and USEF.

Serum CA 125 expression as a tumor marker for diagnosis and monitoring the clinical course of epithelioid sarcoma

We report here, our experience of seven patients with epithelioid sarcomas and their serum CA 125 levels, as well as the results of an in vitro and in vivo study of CA 125 expression in epithelioid sarcoma cells and xenografts using three epithelioid sarcoma cell lines. In the clinical study, the serum CA 125 levels of seven epithelioid sarcoma patients were examined at multiple time points. Expression of the MUC16 gene that encodes the CA 125 sequence was examined using RT-PCR methods in three epithelioid sarcoma cell lines, FU-EPS-1, SFT-8606 and NEPS, and the CA 125 protein in each cell lysate was examined by Western blot using anti-CA 125 clone OC125 antibody. The concentration of CA 125 in the conditioned medium of each cell line was also measured. In five of the seven epithelioid sarcoma patients, CA 125 levels reflected regression and progression of their disease. The CA 125 concentrations in the conditioned medium of FU-EPS-1, SFT-8606 and NEPS cells were 259, 252, and 6 U/ml, respectively. Strong expression of MUC16 mRNA was shown in FU-EPS-1 and SFT-8606 cells: correspondingly, a thick band was observed by Western blot analysis in only FU-EPS-1 and SFT-8606 cells. We concluded that epithelioid sarcoma cells produce and secrete CA 125 into the blood serum and that the elevation of serum CA 125 correlates with disease progression. Therefore, measuring the serum CA 125 level should provide an useful index for diagnosing and monitoring the course of epithelioid sarcoma.

Paclitaxel (Taxol®)-induced apoptosis in human epithelioid sarcoma cell lines is enhanced by upregulation of CD95 ligand (FasL/Apo-1L)

Metastasizing epithelioid sarcoma (ES) is an extremely aggressive tumor, because conventional chemotherapy and irradiation are largely ineffective. Here, we analyzed the impact of the CD95-mediated drug-induced apoptosis in ES cell lines. The effects of paclitaxel (Taxol) and 5-FU were determined by MTT assay. The extent of apoptosis was analyzed by light microscopy and Annexin V staining (flow cytometry). The expression of death receptors and ligands was defined by RT-PCR, Western blotting and flow cytometry. All cell lines expressed CD95, but not the CD95 ligand. The CD95 activation resulted in apoptosis and cell death in all cell lines. Both paclitaxel and 5-FU are able to trigger apoptosis, and furthermore, to upregulate CD95, whereas only paclitaxel increases CD95 ligand expression. Neutralizing antibodies directed against CD95 ligand effectively inhibited paclitaxel-induced cell death, thereby providing evidence for a direct involvement of the CD95 system in paclitaxel-induced apoptosis. Concomitant upregulation of CD95 receptor and ligand may significantly enhance the response of ES to anticancer drugs. As evident from the differential response of our clonal ES subpopulations to paclitaxel and 5-FU, effective activation of the CD95 system depends on intrinsic properties of both the chemotherapeutic agent and target cell population.

Emmprin in epithelioid sarcoma: Expression in tumor cell membrane and stimulation of MMP‐2 production in tumor‐associated fibroblasts

Emmprin is a transmembrane glycoprotein on tumor cells that stimulates peritumoral fibroblasts to produce matrix metalloproteinases (MMPs). Emmprin and the induced MMPs play a crucial role in tumor progression, invasion and metastasis of human carcinomas (epithelial malignancies). However, only a few reports have addressed its role in soft tissue sarcomas. This study investigated the expression and role of emmprin in epithelioid sarcoma (ES). Immunoblot studies of 2 ES cell lines showed that they express emmprin, and co-culture of these ES cells with dermal fibroblasts resulted in upregulation of gelatinase A (MMP-2) in fibroblasts, as shown by zymography, immunoblotting and enzyme immunoassay. This stimulation was inhibited by an activity-blocking peptide against emmprin and by antiemmprin antibody. In addition, in vivo, immunohistochemical analysis of 5 ES patient cases demonstrated diffuse emmprin expression in ES cells and MMP-2 expression in both ES cells and peritumoral fibroblasts. The histopathological findings that peritumoral fibroblasts that were not in direct contact with emmprin-expressing ES cells exhibit upregulated MMP-2 prompted us to look for a soluble form of emmprin. Soluble full-length emmprin released from ES cells was detected in conditioned medium and shown to stimulate MMP-2 production by fibroblasts. In conclusion, emmprin is expressed in ES in both membrane and soluble forms and stimulates MMP-2 production via interactions with fibroblasts, which could play a role in ES cell stromal invasion and vascular involvement.

Prognostic significance of dysadherin expression in epithelioid sarcoma and its diagnostic utility in distinguishing epithelioid sarcoma from malignant rhabdoid tumor

Dysadherin is a cancer-associated cell membrane glycoprotein, which downregulates E-cadherin and promotes metastasis. We studied the clinicopathological features in 72 cases of epithelioid sarcoma and in six cases of malignant rhabdoid tumor, and also assessed the immunohistochemical expression of dysadherin, E-cadherin and MIB-1 in epithelioid sarcoma and malignant rhabdoid tumor cases. In addition, we compared dysadherin mRNA expression between epithelioid sarcoma and malignant rhabdoid tumor cell lines, using RT-PCR and real-time quantitative RT-PCR analysis. Immunohistochemical dysadherin expression was more frequently observed in proximal-type epithelioid sarcoma (71%) in comparison with distal-type epithelioid sarcoma (36%) (P=0.037). Furthermore, seven proximal-type epithelioid sarcoma cases mimicking malignant rhabdoid tumor (histologically classified as the large cell type, accompanied by frequent rhabdoid cells and located in deep soft tissue) were all positive for dysadherin (100%), whereas dysadherin expression was not detected at all in any of the true six malignant rhabdoid tumors (0%). Cell lines established from proximal-type epithelioid sarcoma revealed significantly higher levels of dysadherin mRNA expression, compared with the levels seen in malignant rhabdoid tumor cell lines by real-time quantitative RT-PCR (P=0.0433). Epithelioid sarcoma patients with dysadherin expression survived for a significantly shorter time than those without dysadherin expression (P=0.001). In multivariate analysis, dysadherin immunopositivity (P=0.0004) was one of the two independent adverse prognostic factors. We conclude that dysadherin expression in epithelioid sarcoma is a significant poor prognostic factor and that it is a powerful diagnostic marker for distinguishing epithelioid sarcoma, including the proximal-type epithelioid sarcoma, from malignant rhabdoid tumor. In epithelioid sarcoma, especially in proximal-type epithelioid sarcoma, increased cell disadhesion and motility by dysadherin plays an important role to acquire aggressive biological behavior. However, in malignant rhabdoid tumor, cell growth cycle that is regulated by hSNF5/INI1 gene seems to be critical to lethal biological behavior rather than dysadherin.