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Ewing’s Sarcoma Ewing’s sarcoma, an aggressive cancer of bone and soft tissue, primarily affects children and young adults. A t(11;22) translocation is noted in 85 to 90% of cases. Management inclu...

To assess the role of the canonical Wnt pathway via activation of β-catenin in tumor progression of conjunctival melanoma. β-Catenin localization was assessed by immunohistochemistry in 43 conjunctival nevi, 48 primary acquired melanoses (PAM, conjunctival melanocytic intraepithelial neoplasia), and 44 conjunctival melanomas. Activation of the canonical or the noncanonical Wnt pathway was tested in vitro in 4 conjunctival melanoma cell lines with stimulation of either Wnt5a or Wnt3a. Wound healing assays were performed with Wnt5a. Nuclear β-catenin expression was found in 16% of the nevi, in 15% of the melanomas, and in 4% of the PAM. Membranous β-catenin expression was identified in all the nevi and PAM and in 97.7% of the melanomas. In vitro, Wnt5a stimulation in the 4 conjunctival melanomas and in 1 skin melanoma cell line did not induce nuclear translocation of β-catenin, nor did it increase cell motility in the wound healing assays. Wnt3a stimulation did not induce nuclear localization of β-catenin in any of the cell lines tested. In conjunctival melanoma, nuclear localization and activation of β-catenin appear to be limited, suggesting that inhibition of ARF6, responsible for β-catenin activation, in subsets of skin melanoma may not represent a treatment option for this tumor. In vitro, Wnt3a or Wnt5a did not induce nuclear β-catenin localization.

Aims: Conjunctival and cutaneous melanoma partially share similar clinical and molecular backgrounds. As 5-hydroxymethylcytosine (5-hmC) loss has been demonstrated in cutaneous melanoma, we decided to assess if similar changes were occurring in conjunctival melanoma. Methods: 5-methylcytosine (5-mC), 5-hmC and TET2 were respectively identified by immunohistochemistry and RNA ISH in 40 conjunctival nevi and 37 conjunctival melanomas. Clinicopathological correlations were established. Results: 5-mC, TET2 and 5-hmC were respectively identified in 67.5%, 95% and 100% of conjunctival nevi and in 81.1%, 35.1% and 54% of conjunctival melanomas. A significant 5-hmC and TET2 loss was identified in conjunctival melanoma comparing to nevus, as well as a significant correlation between TET2 and 5-hmC expression. In the melanomas, 5-hmC expression was only significantly associated with local lymphatic invasion, but not with other clinicopathological parameters. There was a correlation between TET2 expression and the localization of the tumors. 5-mC expression was not associated with any clinicopathological parameters. Conclusions: We identified a significant 5-hmC loss in conjunctival melanoma similar to cutaneous melanoma. This loss may possibly be attributed to TET2 loss or IDH1 mutations. 5-hmC loss in conjunctival melanoma may help in the differential diagnosis between atypical conjunctival nevus and conjunctival melanoma.

The embryonic transcription factors TWIST1/2 are frequently overexpressed in cancer, acting as multifunctional oncogenes. Here we investigate their role in neuroblastoma (NB), a heterogeneous childhood malignancy ranging from spontaneous regression to dismal outcomes despite multimodal therapy. We first reveal the association of TWIST1 expression with poor survival and metastasis in primary NB, while TWIST2 correlates with good prognosis. Secondly, suppression of TWIST1 by CRISPR/Cas9 results in a reduction of tumor growth and metastasis colonization in immunocompromised mice. Moreover, TWIST1 knockout tumors display a less aggressive cellular morphology and a reduced disruption of the extracellular matrix (ECM) reticulin network. Additionally, we identify a TWIST1-mediated transcriptional program associated with dismal outcome in NB and involved in the control of pathways mainly linked to the signaling, migration, adhesion, the organization of the ECM, and the tumor cells versus tumor stroma crosstalk. Taken together, our findings suggest TWIST1 as novel therapeutic target in NB.

Targeting of the most aggressive tumor cell subpopulations is key for effective management of most solid malignancies. However, the metastable nature of tumor heterogeneity, which allows cells to transition between strong and weak tumorigenic phenotypes, and the lack of reliable markers of tumor-promoting properties hamper identification of the most relevant cells. To overcome these obstacles, we designed a functional microRNA (miR)-based live-cell reporter assay to identify highly tumorigenic cells in xenotransplants of primary Ewing sarcoma (EwS) 3D cultures. Leveraging the inverse relationship between cell pluripotency and miR-145 expression, we successfully separated highly tumorigenic, metastasis-prone (miR-145 low ) cells from poorly tumorigenic, nonmetastatic (miR-145 high ) counterparts. Gene expression and functional studies of the two cell populations identified the EPHB2 receptor as a prognostic biomarker in patients with EwS and a major promoter of metastasis. Our study provides a simple and powerful means to identify and isolate tumor cells that display aggressive behavior.

Cancer evolution determines molecular and morphologic intratumor heterogeneity and challenges the design of effective treatments. In lung adenocarcinoma, disease progression and prognosis are associated with the appearance of morphologically diverse tumor regions, termed histologic patterns. However, the link between molecular and histologic features remains elusive. Here, we generated multiomics and spatially resolved molecular profiles of histologic patterns from primary lung adenocarcinoma, which we integrated with molecular data from >2,000 patients. The transition from indolent to aggressive patterns was not driven by genetic alterations but by epigenetic and transcriptional reprogramming reshaping cancer cell identity. A signature quantifying this transition was an independent predictor of patient prognosis in multiple human cohorts. Within individual tumors, highly multiplexed protein spatial profiling revealed coexistence of immune desert, inflamed, and excluded regions, which matched histologic pattern composition. Our results provide a detailed molecular map of lung adenocarcinoma intratumor spatial heterogeneity, tracing nongenetic routes of cancer evolution., SIGNIFICANCE: Lung adenocarcinomas are classified based on histologic pattern prevalence. However, individual tumors exhibit multiple patterns with unknown molecular features. We characterized nongenetic mechanisms underlying intratumor patterns and molecular markers predicting patient prognosis. Intratumor patterns determined diverse immune microenvironments, warranting their study in the context of current immunotherapies.

PurposeEndosialin/tumor endothelial marker-1 (TEM1) is an attractive theranostic target expressed by the microenvironment of a wide range of tumors, as well as by sarcoma and neuroblastoma cells. We report on the radiolabeling and preclinical evaluation of the scFv78-Fc, a fully human TEM1-targeting antibody fragment cross-reactive with mouse TEM1.ProceduresThe scFv78-Fc was conjugated with the chelatorp-SCN-Bn-CHX-A”-DTPA, followed by labeling with indium-111. The number of chelators per molecule was estimated by mass spectrometry. A conventional saturation assay, extrapolated to infinite antigen concentration, was used to determine the immunoreactive fraction of the radioimmunoconjugate. The radiopharmaceutical biodistribution was assessed in immunodeficient mice grafted with Ewing’s sarcoma RD-ES and neuroblastoma SK-N-AS human TEM1-positive tumors. The full biodistribution studies were preceded by a dose-escalation experiment based on the simultaneous administration of the radiopharmaceutical with increasing amounts of unlabeled scFv78-Fc. Radiation dosimetry extrapolations to human adults were obtained from mouse biodistribution data according to established methodologies and additional assumptions concerning the impact of the tumor antigenic sink in the cross-species translation.Results[111In]CHX-DTPA-scFv78-Fc was obtained with a radiochemical purity > 98 % after 1 h incubation at 42 °C and ultrafiltration. It showed good stability in human serum and > 70 % immunoreactive fraction. Biodistribution data acquired in tumor-bearing mice confirmed fast blood clearance and specific tumor targeting in both xenograft models. The radiopharmaceutical off-target uptake was predominantly abdominal. After a theoretical injection of [111In]CHX-DTPA-scFv78-Fc to the reference person, the organs receiving the highest absorbed dose would be the spleen (0.876 mGy/MBq), the liver (0.570 mGy/MBq) and the kidneys (0.298 mGy/MBq). The total body dose and the effective dose would be 0.058 mGy/MBq and 0.116 mSv/MBq, respectively.Conclusions[111In]CHX-DTPA-scFv78-Fc binds specifically to endosialin/TEM1in vitroandin vivo. Dosimetry estimates are in the range of other monoclonal antibodies radiolabeled with indium-111. [111In]CHX-DTPA-scFv78-Fc could be potentially translated into clinic.

Ewing sarcoma (EwS) is associated with poor prognosis despite current multimodal therapy. Targeting of EWS-FLI1, the fusion protein responsible for its pathogenesis, and its principal downstream targets has not yet produced satisfactory therapeutic options, fueling the search for alternative approaches. Here, we show that the oncofetal RNA-binding protein LIN28B regulates the stability of EWS-FLI1 mRNA in ~10% of EwSs. LIN28B depletion in these tumors leads to a decrease in the expression of EWS-FLI1 and its direct transcriptional network, abrogating EwS cell self-renewal and tumorigenicity. Moreover, pharmacological inhibition of LIN28B mimics the effect of LIN28B depletion, suggesting that LIN28B sustains the emergence of a subset of EwS in which it also serves as an effective therapeutic target.

Patients with melanoma resistant to RAF/MEK inhibitors (RMi) are frequently resistant to other therapies, such as immune checkpoint inhibitors (ICI), and individuals succumb to their disease. New drugs that control tumor growth and favorably modulate the immune environment are therefore needed. We report that the small-molecule CX-6258 has potent activity against both RMi-sensitive (RMS) and -resistant (RMR) melanoma cell lines. Haspin kinase (HASPIN) was identified as a target of CX-6258. HASPIN inhibition resulted in reduced proliferation, frequent formation of micronuclei, recruitment of cGAS, and activation of the cyclic GMP-AMP synthase (cGAS)–stimulator of interferon genes (STING) pathway. In murine models, CX-6258 induced a potent cGAS-dependent type-I IFN response in tumor cells, increased IFNγ-producing CD8+ T cells, and reduced Treg frequency in vivo. HASPIN was more strongly expressed in malignant compared with healthy tissue and its inhibition by CX-6258 had minimal toxicity in ex vivo–expanded human tumor-infiltrating lymphocytes (TIL), proliferating TILs, and in vitro differentiated neurons, suggesting a potential therapeutic index for anticancer therapy. Furthermore, the activity of CX-6258 was validated in several Ewing sarcoma and multiple myeloma cell lines. Thus, HASPIN inhibition may overcome drug resistance in melanoma, modulate the immune environment, and target a vulnerability in different cancer lineages. Significance: HASPIN inhibition by CX-6258 is a novel and potent strategy for RAF/MEK inhibitor–resistant melanoma and potentially other tumor types. HASPIN inhibition has direct antitumor activity and induces a favorable immune microenvironment.

Metastases are responsible for the vast majority of cancer-related deaths, but, despite intense efforts to understand their underlying mechanisms with the goal of uncovering effective therapeutic targets, treatment of metastatic cancer has progressed minimally. In this review, we examine the biological programs currently proposed to be key drivers of metastasis. On the basis of evidence from a growing body of research, we discuss to what extent the cellular and molecular mechanisms that are suggested to underlie cancer cell dissemination are specific to the metastatic process, as opposed to representing natural primary tumor progression. Our review highlights the contrast between the abundance of insight gained into the events that constitute the metastatic cascade and the paucity of therapeutic options.

Synovial sarcoma (SyS) is an aggressive neoplasm driven by the SS18–SSX fusion, and is characterized by low T cell infiltration. Here, we studied the cancer–immune interplay in SyS using an integrative approach that combines single-cell RNA sequencing (scRNA-seq), spatial profiling and genetic and pharmacological perturbations. scRNA-seq of 16,872 cells from 12 human SyS tumors uncovered a malignant subpopulation that marks immune-deprived niches in situ and is predictive of poor clinical outcomes in two independent cohorts. Functional analyses revealed that this malignant cell state is controlled by the SS18–SSX fusion, is repressed by cytokines secreted by macrophages and T cells, and can be synergistically targeted with a combination of HDAC and CDK4/CDK6 inhibitors. This drug combination enhanced malignant-cell immunogenicity in SyS models, leading to induced T cell reactivity and T cell–mediated killing. Our study provides a blueprint for investigating heterogeneity in fusion-driven malignancies and demonstrates an interplay between immune evasion and oncogenic processes that can be co-targeted in SyS and potentially in other malignancies. Single-cell transcriptional profiling of primary human synovial sarcoma tumors suggests that combinatorial treatment with HDAC and CDK4/CDK6 inhibitors could enhance tumor immunogenicity.

Synovial sarcoma is an aggressive mesenchymal neoplasm, driven by the SS18-SSX fusion, and characterized by immunogenic antigens expression and exceptionally low T cell infiltration levels. To study the cancer-immune interplay in this disease, we profiled 16,872 cells from 12 human synovial sarcoma tumors using single-cell RNA-sequencing (scRNA-Seq). Synovial sarcoma manifests antitumor immunity, high cellular plasticity and a core oncogenic program, which is predictive of low immune levels and poor clinical outcomes. Using genetic and pharmacological perturbations, we demonstrate that the program is controlled by the SS18-SSX driver and repressed by cytokines secreted by macrophages and T cells in the tumor microenvironment. Network modeling predicted that SS18-SSX promotes the program through HDAC1 and CDK6. Indeed, the combination of HDAC and CDK4/6 inhibitors represses the program, induces immunogenic cell states, and selectively targets synovial sarcoma cells. Our study demonstrates that immune evasion, cellular plasticity, and cell cycle are co-regulated and can be co-targeted in synovial sarcoma and potentially in other malignancies.

Uveal melanoma (UM) is a rare intraocular tumor that, similar to cutaneous melanoma, originates from melanocytes. To gain insights into its genetics, we performed whole-genome sequencing at very deep coverage of tumor-control pairs in 33 samples (24 primary and 9 metastases). Genome-wide, the number of coding mutations was rather low (only 17 variants per tumor on average; range 7–28), thus radically different from cutaneous melanoma, where hundreds of exonic DNA insults are usually detected. Furthermore, no UV light-induced mutational signature was identified. Recurrent coding mutations were found in the known UM drivers GNAQ, GNA11, BAP1, EIF1AX, and SF3B1. Other genes, i.e., TP53BP1, CSMD1, TTC28, DLK2, and KTN1, were also found to harbor somatic mutations in more than one individual, possibly indicating a previously undescribed association with UM pathogenesis. De novo assembly of unmatched reads from non-coding DNA revealed peculiar copy-number variations defining specific UM subtypes, which in turn could be associated with metastatic transformation. Mutational-driven comparison with other tumor types showed that UM is very similar to pediatric tumors, characterized by very few somatic insults and, possibly, important epigenetic changes. Through the analysis of whole-genome sequencing data, our findings shed new light on the molecular genetics of uveal melanoma, delineating it as an atypical tumor of the adult for which somatic events other than mutations in exonic DNA shape its genetic landscape and define its metastatic potential.

Summary Ewing sarcoma (EwS) is associated with poor prognosis despite current multimodal therapy. Targeting of EWS-FLI1, the fusion protein responsible for its pathogenesis, and its principal downstream targets has not yet produced satisfactory therapeutic options, fueling the search for alternative approaches. Here, we show that the oncofetal RNA-binding protein LIN28B regulates the stability of EWS-FLI1 mRNA in ~10% of EwSs. LIN28B depletion in these tumors leads to a decrease in the expression of EWS-FLI1 and its direct transcriptional network, abrogating EwS cell self-renewal and tumorigenicity. Moreover, pharmacological inhibition of LIN28B mimics the effect of LIN28B depletion, suggesting that LIN28B sustains the emergence of a subset of EwS in which it also serves as an effective therapeutic target.

Synovial sarcoma is a highly aggressive soft tissue malignancy that often affects adolescents and young adults. It is associated with a unique chromosomal translocation that results in the formation and expression of the fusion gene SS18-SSX, which underlies its pathogenesis. Although SS18-SSX provides a potentially unique therapeutic target, all attempts to neutralise it have been unsuccessful thus far. When complete surgical removal of the tumour fails, therapy is limited to largely ineffective cytotoxic drug regimens. Nevertheless, recent discoveries about the mechanisms of SS18-SSX protein function have provided insight into potential alternative therapeutic strategies. SS18-SSX displays oncogenic activity through protein-protein interactions and participation in chromatin remodelling complexes. This review summarises our current understanding of the function of SS18-SSX and the mechanisms by which it alters the epigenetic landscape of permissive cells to induce transformation and the subsequent development of synovial sarcoma.

Metastasis is a multi-step process in which direct crosstalk between cancer cells and their microenvironment plays a key role. Here, we assessed the effect of paired tumor-associated and normal lung tissue mesenchymal stem cells (MSCs) on the growth and dissemination of primary human lung carcinoma cells isolated from the same patients. We show that the tumor microenvironment modulates MSC gene expression and identify a four-gene MSC signature that is functionally implicated in promoting metastasis. We also demonstrate that tumor-associated MSCs induce the expression of genes associated with an aggressive phenotype in primary lung cancer cells and selectively promote their dissemination rather than local growth. Our observations provide insight into mechanisms by which the stroma promotes lung cancer metastasis., Highlights • Distinct gene expression profiles distinguish normal lung and tumor-associated MSCs. • MSCs induce EMT- and hypoxia-related genes in primary tumor cells and promote their metastatic potential. • A 4-gene T-MSC signature is involved in MSC-induced metastasis promotion. The tumor microenvironment, which includes mesenchymal stem cells (MSCs) among many other stromal cell types, plays a fundamental role in cancer metastasis. Although MSCs are suggested to participate in tumor progression, most studies thus far have been performed on bone marrow-derived MSCs and cancer cell lines. Using primary human pulmonary MSCs and paired lung cancer cells, we show that tumor cells modulate MSCs to acquire properties, including a four-gene signature, which allow them to promote tumor dissemination. Our results provide insight into the mutual cancer cell-stromal cell modulation that drives tumor dissemination.

Medulloblastoma is the most frequent malignant pediatric brain tumor and is divided into at least four subgroups known as WNT, SHH, Group 3, and Group 4. Here, we characterized gene regulation mechanisms in the most aggressive subtype, Group 3 tumors, through genome-wide chromatin and expression profiling. Our results show that most active distal sites in these tumors are occupied by the transcription factor OTX2. Highly active OTX2-bound enhancers are often arranged as clusters of adjacent peaks and are also bound by the transcription factor NEUROD1. These sites are responsive to OTX2 and NEUROD1 knockdown and could also be generated de novo upon ectopic OTX2 expression in primary cells, showing that OTX2 cooperates with NEUROD1 and plays a major role in maintaining and possibly establishing regulatory elements as a pioneer factor. Among OTX2 target genes, we identified the kinase NEK2, whose knockdown and pharmacologic inhibition decreased cell viability. Our studies thus show that OTX2 controls the regulatory landscape of Group 3 medulloblastoma through cooperative activity at enhancer elements and contributes to the expression of critical target genes.Significance: The gene regulation mechanisms that drive medulloblastoma are not well understood. Using chromatin profiling, we find that the transcription factor OTX2 acts as a pioneer factor and, in cooperation with NEUROD1, controls the Group 3 medulloblastoma active enhancer landscape. OTX2 itself or its target genes, including the mitotic kinase NEK2, represent attractive targets for future therapies. Cancer Discov; 7(3); 288–301. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 235

To analyze the activity of mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinases/mechanistic target of rapamycin (PI3K/mTOR) pathways in benign and malignant conjunctival melanocytic proliferations and explore whether specific inhibitors can suppress growth of conjunctival melanoma (CJM) cells.; The presence of a BRAF V600E mutation and activation of ERK, MEK, S6, and AKT were assessed with immunohistochemistry in 35 conjunctival nevi and 31 melanomas. Three CJM cell lines were used: CRMM1, carrying the BRAF V600E mutation; CRMM2, harboring the NRAS Q61L mutation; and T1527A, with a BRAF G466E mutation. WST-1 assays were performed with a BRAF inhibitor (vemurafenib), two MEK inhibitors (trametinib, selumetinib), a PI3K inhibitor (pictilisib), and a dual PI3K/mTOR inhibitor (dactolisib). The phosphorylation of ERK, MEK, and S6 were tested with western blots and apoptosis with cleaved caspase-3 immunostaining.; A BRAF V600E mutation was detected in 42.6% of nevi and in 35.5% of CJM. MEK and ERK activation were higher in CJM, occurring in 62.9% and 45.7% of the nevi and 90.3% and 96.8% of the CJM, respectively. There was also a significant increase in S6 activation in CJM (90.3%) compared with the nevi (20%). CRMM1 was sensitive to trametinib and the PI3K inhibitors but only marginally to vemurafenib. CRMM2 was moderately sensitive to pictilisib, whereas T1527A was resistant to all drugs tested.; The MAPK pathway activity in CJM is increased, not only as a consequence of the BRAF V600E mutation. Targeted therapy may be useful for patients with CJM, especially those with activating BRAF mutations, whereas NRAS-mutated melanomas are relatively resistant.

Although human tumours are shaped by the genetic evolution of cancer cells, evidence also suggests that they display hierarchies related to developmental pathways and epigenetic programs in which cancer stem cells (CSCs) can drive tumour growth and give rise to differentiated progeny1. Yet, unbiased evidence for CSCs in solid human malignancies remains elusive. Here we profile 4,347 single cells from six IDH1 or IDH2 mutant human oligodendrogliomas by RNA sequencing (RNA-seq) and reconstruct their developmental programs from genome-wide expression signatures. We infer that most cancer cells are differentiated along two specialized glial programs, whereas a rare subpopulation of cells is undifferentiated and associated with a neural stem cell expression program. Cells with expression signatures for proliferation are highly enriched in this rare subpopulation, consistent with a model in which CSCs are primarily responsible for fuelling the growth of oligodendroglioma in humans. Analysis of copy number variation (CNV) shows that distinct CNV sub-clones within tumours display similar cellular hierarchies, suggesting that the architecture of oligodendroglioma is primarily dictated by developmental programs. Subclonal point mutation analysis supports a similar model, although a full phylogenetic tree would be required to definitively determine the effect of genetic evolution on the inferred hierarchies. Our single-cell analyses provide insight into the cellular architecture of oligodendrogliomas at single-cell resolution and support the cancer stem cell model, with substantial implications for disease management.

Background: Blocking the mechanistic target of rapamycin complex-1 (mTORC1) with chemical inhibitors such as rapamycin has shown limited clinical efficacy in cancer. The tumor microenvironment is characterized by an acidic pH which interferes with cancer therapies. The consequences of acidity on the anti-cancer efficacy of mTORC1 inhibitors have not been characterized and are thus the focus of our study. Methods: Cancer cell lines were treated with rapamycin in acidic or physiological conditions and cell proliferation was investigated. The effect of acidity on mTORC1 activity was determined by Western blot. The anticancer efficacy of rapamycin in combination with sodium bicarbonate to increase the intratumoral pH was tested in two different mouse models and compared to rapamycin treatment alone. Histological analysis was performed on tumor samples to evaluate proliferation, apoptosis and necrosis. Results: Exposing cancer cells to acidic pH in vitro significantly reduced the anti-proliferative effect of rapamycin. At the molecular level, acidity significantly decreased mTORC1 activity, suggesting that cancer cell proliferation is independent of mTORC1 in acidic conditions. In contrast, the activation of mitogen-activated protein kinase (MAPK) or AKT were not affected by acidity, and blocking MAPK or AKT with a chemical inhibitor maintained an anti-proliferative effect at low pH. In tumor mouse models, the use of sodium bicarbonate increased mTORC1 activity in cancer cells and potentiated the anti-cancer efficacy of rapamycin. Combining sodium bicarbonate with rapamycin resulted in increased tumor necrosis, increased cancer cell apoptosis and decreased cancer cell proliferation as compared to single treatment. Conclusions: Taken together, these results emphasize the inefficacy of mTORC1 inhibitors in acidic conditions. They further highlight the potential of combining sodium bicarbonate with mTORC1 inhibitors to improve their anti-tumoral efficacy.

SummaryWe have recently demonstrated that human pediatric mesenchymal stem cells can be reprogrammed toward a Ewing sarcoma family tumor (ESFT) cancer stem cell (CSC) phenotype by mechanisms that implicate microRNAs (miRNAs). Here, we show that the miRNA profile of ESFT CSCs is shared by embryonic stem cells and CSCs from divergent tumor types. We also provide evidence that the miRNA profile of ESFT CSCs is the result of reversible disruption of TARBP2-dependent miRNA maturation. Restoration of TARBP2 activity and systemic delivery of synthetic forms of either of two of its targets, miRNA-143 or miRNA-145, inhibited ESFT CSC clonogenicity and tumor growth in vivo. Our observations suggest that CSC self-renewal and tumor maintenance may depend on deregulation of TARBP2-dependent miRNA expression.

Cancer stem cells that display tumor-initiating properties have recently been identified in several distinct types of malignancies, holding promise for more effective therapeutic strategies. However, evidence of such cells in sarcomas, which include some of the most aggressive and therapy-resistant tumors, has not been shown to date. Here, we identify and characterize cancer stem cells in Ewing's sarcoma family tumors (ESFT), a highly aggressive pediatric malignancy believed to be of mesenchymal stem cell (MSC) origin. Using magnetic bead cell separation of primary ESFT, we have isolated a subpopulation of CD133+ tumor cells that display the capacity to initiate and sustain tumor growth through serial transplantation in nonobese diabetic/severe combined immunodeficiency mice, re-establishing at each in vivo passage the parental tumor phenotype and hierarchical cell organization. Consistent with the plasticity of MSCs, in vitro differentiation assays showed that the CD133+ cell population retained the ability to differentiate along adipogenic, osteogenic, and chondrogenic lineages. Quantitative real-time PCR analysis of genes implicated in stem cell maintenance revealed that CD133+ ESFT cells express significantly higher levels of OCT4 and NANOG than their CD133− counterparts. Taken together, our observations provide the first identification of ESFT cancer stem cells and demonstration of their MSC properties, a critical step towards a better biological understanding and rational therapeutic targeting of these tumors. [Cancer Res 2009;69(5):1776–81]

Sarcomas account for less than 10% of all human malignancies that are believed to originate from as yet poorly defined mesenchymal progenitor cells. They constitute some of the most aggressive adult and childhood cancers in that they have a high metastatic proclivity and are typically refractory to conventional chemo- and radiation therapy. Ewing's sarcoma is a member of Ewing's family tumors (ESFT) and the second most common solid bone and soft tissue malignancy of children and young adults. It is associated in 85% of cases with the t(11;22)(q24:q12) chromosomal translocation that generates fusion of the 5' segment of the EWS gene with the 3' segment of the ETS family gene FLI-1. The resulting EWS-FLI-1 fusion protein is believed to behave as an aberrant transcriptional activator that contributes to ESFT development by altering the expression of its target genes in a permissive cellular environment. Although ESFTs are among the best studied sarcomas, the mechanisms involved in EWS-FLI-1-induced transformation require further elucidation and the primary cells from which ESFTs originate need to be identified. This review will highlight some of the most recent discoveries in the field of Ewing sarcoma biology and origins.

Sarcomas comprise some of the most aggressive solid tumours that, for the most part, respond poorly to chemo- and radiation therapy and are associated with a sombre prognosis when surgical removal cannot be performed or is incomplete. Partly because of their lower frequency, sarcomas have not been studied as intensively as carcinomas and haematopoietic malignancies, and the molecular mechanisms that underlie their pathogenesis are only beginning to be understood. Even more enigmatic is the identity of the primary cells from which these tumours originate. Over the past 25 years, however, several non-random chromosomal translocations have been found to be associated with defined sarcomas. Each of these translocations generates a fusion gene believed to be directly related to the pathogenesis of the sarcoma in which it is expressed. The corresponding fusion proteins provide a unique tool not only to study the process of sarcoma development, but also to identify cells that are permissive for their putative oncogenic properties. This is the first of two reviews that cover the mechanisms whereby specific fusion/mutant gene products participate in sarcoma development and the cellular context that may provide the necessary permissiveness for their expression and oncogenicity. Part 1 of the review focuses on sarcomas that express fusion genes containing TET gene family products, including EWSR1, TLS/FUS, and TAFII68. Part 2 (J Pathol 2007; DOI: 10.1002/path.2008) summarizes our current understanding of the genetic and cellular origins of sarcomas expressing fusion genes exclusive of TET family members; it also covers soft tissue malignancies harbouring specific mutations in RTK-encoding genes, the prototype of which are gastrointestinal stromal tumours (GIST).

A subset of sarcomas is associated with specific chromosomal translocations that give rise to fusion genes believed to participate in transformation and oncogenesis. Identification of the primary cell environment that provides permissiveness for the oncogenic potential of these fusion genes is essential to understand sarcoma pathogenesis. We have recently shown that expression of the EWS-FLI-1 fusion protein in primary mesenchymal progenitor cells (MPCs) suffices to develop Ewing's sarcoma-like tumors in mice. Because most sarcomas bearing unique chromosomal translocations are believed to originate from common progenitor cells, and because MPCs populate most organs, we expressed the sarcoma-associated fusion proteins FUS/TLS-CHOP, EWS-ATF1, and SYT-SSX1 in MPCs and tested the tumorigenic potential of these cells in vivo. Whereas expression of EWS-ATF1 and SYT-SSX1 failed to transform MPCs, FUS-CHOP–expressing cells formed tumors resembling human myxoid liposarcoma. Transcription profile analysis of these tumors revealed induction of transcripts known to be associated with myxoid liposarcoma and novel candidate genes, including PDGFA, whose expression was confirmed in human tumor samples. MPCFUS-CHOP and the previously described MPCEWS-FLI-1 tumors displayed distinct transcription profiles, consistent with the different target gene repertoires of their respective fusion proteins. Unexpectedly, a set of genes implicated in cell survival and adhesion displayed similar behavior in the two tumors, suggesting events that may be common to primary MPC transformation. Taken together, our observations suggest that expression of FUS-CHOP may be the initiating event in myxoid liposarcoma pathogenesis, and that MPCs may constitute one cell type from which these tumors originate. (Cancer Res 2006; 66(14): 7016-23)

Ewing's sarcoma is a member of Ewing's family tumors (EFTs) and the second most common solid bone and soft tissue malignancy of children and young adults. It is associated in 85% of cases with the t(11;22)(q24:q12) chromosomal translocation that generates fusion of the 5′ segment of the EWS gene with the 3′ segment of the ETS family gene FLI-1. The EWS-FLI-1 fusion protein behaves as an aberrant transcriptional activator and is believed to contribute to EFT development. However, EWS-FLI-1 induces growth arrest and apoptosis in normal fibroblasts, and primary cells that are permissive for its putative oncogenic properties have not been discovered, hampering basic understanding of EFT biology. Here, we show that EWS-FLI-1 alone can transform primary bone marrow–derived mesenchymal progenitor cells and generate tumors that display hallmarks of Ewing's sarcoma, including a small round cell phenotype, expression of EFT-associated markers, insulin like growth factor-I dependence, and induction or repression of numerous EWS-FLI-1 target genes. These observations provide the first identification of candidate primary cells from which EFTs originate and suggest that EWS-FLI-1 expression may constitute the initiating event in EFT pathogenesis. (Cancer Res 2005; 65(24): 11459-68)

The cellular heterogeneity of solid tumors has intrigued researchers for over a century. Whereas much of the heterogeneity can be attributed to reactive host tissue stromal cells that comprise a gr...

Plasticity in cancer stem–like cells (CSC) may provide a key basis for cancer heterogeneity and therapeutic response. In this study, we assessed the effect of combining a drug that abrogates CSC properties with standard-of-care therapy in a Ewing sarcoma family tumor (ESFT). Emergence of CSC in this setting has been shown to arise from a defect in TARBP2-dependent microRNA maturation, which can be corrected by exposure to the fluoroquinolone enoxacin. In the present work, primary ESFT from four patients containing CD133+ CSC subpopulations ranging from 3% to 17% of total tumor cells were subjected to treatment with enoxacin, doxorubicin, or both drugs. Primary ESFT CSC and bulk tumor cells displayed divergent responses to standard-of-care chemotherapy and enoxacin. Doxorubicin, which targets the tumor bulk, displayed toxicity toward primary adherent ESFT cells in culture but not to CSC-enriched ESFT spheres. Conversely, enoxacin, which enhances miRNA maturation by stimulating TARBP2 function, induced apoptosis but only in ESFT spheres. In combination, the two drugs markedly depleted CSCs and strongly reduced primary ESFTs in xenograft assays. Our results identify a potentially attractive therapeutic strategy for ESFT that combines mechanism-based targeting of CSC using a low-toxicity antibiotic with a standard-of-care cytotoxic drug, offering immediate applications for clinical evaluation. Cancer Res; 74(22); 6610–22. ©2014 AACR.

Ewing's sarcoma family tumors (ESFT) express the EWS-FLI-1 fusion gene generated by the chromosomal translocation t(11;22)(q24;q12). Expression of the EWS-FLI-1 fusion protein in a permissive cellular environment is believed to play a key role in ESFT pathogenesis. However, EWS-FLI-1 induces growth arrest or apoptosis in differentiated primary cells, and the identity of permissive primary human cells that can support its expression and function has until now remained elusive. Here we show that expression of EWS-FLI-1 in human mesenchymal stem cells (hMSC) is not only stably maintained without inhibiting proliferation but also induces a gene expression profile bearing striking similarity to that of ESFT, including genes that are among the highest ESFT discriminators. Expression of EWS-FLI-1 in hMSCs may recapitulate the initial steps of Ewing's sarcoma development, allowing identification of genes that play an important role early in its pathogenesis. Among relevant candidate transcripts induced by EWS-FLI-1 in hMSCs, we found the polycomb group gene EZH2, which we show to play a critical role in Ewing's sarcoma growth. These observations are consistent with our recent findings using mouse mesenchymal progenitor cells and provide compelling evidence that hMSCs are candidate cells of origin of ESFT. [Cancer Res 2008;68(7):2176–85]

We have recently shown that microRNAs (miRNAs) play essential roles is the generation of the cancer stem cell (CSC) phenotype in Ewing sarcoma familly tumors (ESFT). Here we compared the miRNA expression profile of ESFT CSCs to that of embryonic stem cells and CSCs from different tumor, which seem to be shared by these different cell types. Our data suggests that the mechanisms implicated in the miRNAs CSC signature rely on a reversible TARBP-2 dependent miRNA maturation defect. Restoration of TARBP2 activity either through chemical compounds or systemic delivery of synthetic forms of two of its targets, miRNA-143 or miRNA-145, inhibited ESFT CSC growth in vitro and in vivo. Taken together our observations provide new therapeutic strategies through restoration of normal miRNA expression profile, inhibiting CSC self-renewal and tumor maintenance. Citation Format: Claudio De Vito, Nicolo Riggi, Sandrine Cornaz, Ivan Stamenkovic. Disregulation of TARBP-2 dependent miRNA maturation : A potential new therapeutic target in Ewing sarcoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1958. doi:10.1158/1538-7445.AM2013-1958

In Response: Our study was directed toward identifying a primary cell that is permissive for EWS-FLI-1-mediated transformation. Following the observation that EWS-FLI-1 transforms primary mouse mesenchymal progenitor cells (MPC) to form Ewing's sarcoma–like tumors ( [1][1]), we tested these