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Ewing sarcoma (EWS) is an aggressive bone and soft tissue tumor with high susceptibility to metastasize. The underlying molecular mechanisms leading to EWS metastases remain poorly understood. Epigenetic changes have been implicated in EWS tumor growth and progression. Linking epigenetics and metastases may provide insight into novel molecular targets in EWS and improve its treatment. Here, we evaluated the effects of a selective G9a histone methyltransferase inhibitor (BIX01294) on EWS metastatic process. Our results showed that overexpression of G9a in tumors from EWS patients correlates with poor prognosis. Moreover, we observe a significantly higher expression of G9a in metastatic EWS tumor as compared to either primary or recurrent tumor. Using functional assays, we demonstrate that pharmacological G9a inhibition using BIX01294 disrupts several metastatic steps in vitro, such as migration, invasion, adhesion, colony formation and vasculogenic mimicry. Moreover, BIX01294 reduces tumor growth and metastases in two spontaneous metastases mouse models. We further identified the sialidase NEU1 as a direct target and effector of G9a in the metastatic process in EWS. NEU1 overexpression impairs migration, invasion and clonogenic capacity of EWS cell lines. Overall, G9a inhibition impairs metastases in vitro and in vivo through the overexpression of NEU1. G9a has strong potential as a prognostic marker and may be a promising therapeutic target for EWS patients., Research in the EDA and OMT labs are supported by “Asociación Española Contra el Cáncer” (AECC). EDA lab is also supported by the Ministry of Economy and Competitiveness of Spain-FEDER (CIBERONC, PI2000003, RD06/0020/0059). DGD and LHP are supported by CIBERONC (CB16/12/00361)S. LHP is funded by the Consejería de Salud de la Junta de Andalucía (PI-0013-2018). SRV is supported by “Asociación Alba Pérez lucha contra el cáncer infantil”. The OMT lab is supported by the Catalan Agency for the Management of University and Research Grants (AGAUR 2017 SGR 332).

Secreted protein acidic and rich in cysteine (SPARC) is a matricellular glycoprotein overexpressed by several cancers. Because SPARC shows high binding affinity to albumin, we reasoned that pediatric sarcoma xenografts expressing SPARC would show enhanced uptake and accumulation of nanoparticle albumin-bound (nab)-paclitaxel, a potent anticancer drug formulation. We first evaluated the expression of SPARC in patient-derived xenografts (PDXs) of Ewing sarcoma, rhabdomyosarcoma and osteosarcoma, finding variable SPARC gene expression that correlated well with SPARC protein measured by immunoblotting. We revealed that the activity of the fusion gene chimera EWSR1-FLI1, the genetic driver of Ewing sarcoma, leads to lower expression of the gene SPARC in these tumors, likely due to enriched acetylation marks of the histone H3 lysine 27 at regions including the SPARC promoter and potential enhancers. Then, we used SPARC-edited Ewing sarcoma cells (A673 line) to demonstrate that SPARC knocked down (KD) cells accumulated significantly less amount of nab-paclitaxel in vitro than SPARC wild type (WT) cells. In vivo, SPARC KD and SPARC WT subcutaneous xenografts in mice achieved similar maximum intratumoral concentrations of nab-paclitaxel, though drug clearance from SPARC WT tumors was significantly slower. We confirmed such SPARC-mediated long-term intratumoral accumulation of nab-paclitaxel in Ewing sarcoma PDX with high expression of SPARC, which accumulated significantly more nab-paclitaxel than SPARC-low PDX. SPARC-high PDX responded better to nab-paclitaxel than SPARC-low tumors, although these results should be taken cautiously, given that the PDXs were established from different patients that could have specific determinants predisposing response to paclitaxel. In addition, SPARC KD Ewing sarcoma xenografts responded better to soluble docetaxel and paclitaxel than to nab-paclitaxel, while SPARC WT ones showed similar response to soluble and albumin-carried drugs. Overall, our results show that pediatric sarcomas expressing SPARC accumulate nab-paclitaxel for longer periods of time, which could have clinical implications for chemotherapy efficacy., AMC acknowledges funding from AECC Scientific Foundation, MINECO (SAF2011-22660), European Union Seventh Framework Programme (FP7/2007-2013) under Marie Curie International Reintegration Grant (PIRG-08-GA-2010-276998) and ISCIII-FEDER (CP13/00189 and CPII18/00009). EDA, OMT and JM acknowledge support of AECC Scientific Foundation (GCB13131578). NU acknowledges funding by the Basque Government, Research Groups of the Basque University System (Project No. IT 1186-19). We thank support of the Central Service of Analysis in Alava, SGIker (UPV/EHU/ERDF, EU) and Xarxa de Bancs de Tumors de Catalunya (XBTC) sponsored by Pla Director d'Oncologia de Catalunya.

Purpose:Endoglin (ENG; CD105) is a coreceptor of the TGFβ family that is highly expressed in proliferating endothelial cells. Often coopted by cancer cells, ENG can lead to neo-angiogenesis and vasculogenic mimicry in aggressive malignancies. It exists both as a transmembrane cell surface protein, where it primarily interacts with TGFβ, and as a soluble matricellular protein (sENG) when cleaved by matrix metalloproteinase 14 (MMP14). High ENG expression has been associated with poor prognosis in Ewing sarcoma, an aggressive bone cancer that primarily occurs in adolescents and young adults. However, the therapeutic value of ENG targeting has not been fully explored in this disease.Experimental Design:We characterized the expression pattern of transmembrane ENG, sENG, and MMP14 in preclinical and clinical samples. Subsequently, the antineoplastic potential of two novel ENG-targeting monoclonal antibody–drug conjugates (ADC), OMTX503 and OMTX703, which differed only by their drug payload (nigrin-b A chain and cytolysin, respectively), was assessed in cell lines and preclinical animal models of Ewing sarcoma.Results:Both ADCs suppressed cell proliferation in proportion to the endogenous levels of ENG observed in vitro. Moreover, the ADCs significantly delayed tumor growth in Ewing sarcoma cell line–derived xenografts and patient-derived xenografts in a dose-dependent manner.Conclusions:Taken together, these studies demonstrate potent preclinical activity of first-in-class anti-ENG ADCs as a nascent strategy to eradicate Ewing sarcoma.

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PURPOSE The aim of this study was to assess the cost-effectiveness of using next-generation sequencing (NGS) versus single-gene testing (SgT) for the detection of genetic molecular subtypes and oncogenic markers in patients with advanced non–small-cell lung cancer (NSCLC) in the setting of Spanish reference centers. METHODS A joint model combining decision tree with partitioned survival models was developed. A two-round consensus panel was performed to describe clinical practice of Spanish reference centers, providing data on testing rate, prevalence of alterations, turnaround times, and treatment pathways. Treatment efficacy data and utility values were obtained from the literature. Only direct costs (euros, 2022), obtained from Spanish databases, were included. A lifetime horizon was considered, so a 3% discount rate for future costs and outcomes was considered. Both deterministic and probabilistic sensitivity analyses were performed to assess uncertainty. RESULTS A target population of 9,734 patients with advanced NSCLC was estimated. If NGS was used instead of SgT, 1,873 more alterations would be detected and 82 more patients could potentially be enrolled in clinical trials. In the long term, using NGS would provide 1,188 additional quality-adjusted life-years (QALYs) in the target population compared with SgT. On the other hand, the incremental cost of NGS versus SgT in the target population was €21,048,580 euros for a lifetime horizon (€1,333,288 for diagnosis phase only). The obtained incremental cost-utility ratios were €25,895 per QALY gained, below the standard cost-effectiveness thresholds. CONCLUSION Using NGS in Spanish reference centers for the molecular diagnosis of patients with metastatic NSCLC would be a cost-effective strategy over SgT.

Endoglin (ENG) is a mesenchymal stem cell (MSC) marker typically expressed by active endothelium. This transmembrane glycoprotein is shed by matrix metalloproteinase 14 (MMP14). Our previous work demonstrated potent preclinical activity of first-in-class anti-ENG antibody-drug conjugates as a nascent strategy to eradicate Ewing sarcoma (ES), a devastating rare bone/soft tissue cancer with a putative MSC origin. We also defined a correlation between ENG and MMP14 expression in ES. Herein, we show that ENG expression is significantly associated with a dismal prognosis in a large cohort of ES patients. Moreover, both ENG/MMP14 are frequently expressed in primary ES tumors and metastasis. To deepen in their functional relevance in ES, we conducted transcriptomic and proteomic profiling of in vitro ES models that unveiled a key role of ENG and MMP14 in cell mechano-transduction. Migration and adhesion assays confirmed that loss of ENG disrupts actin filament assembly and filopodia formation, with a concomitant effect on cell spreading. Furthermore, we observed that ENG regulates cell-matrix interaction through activation of focal adhesion signaling and protein kinase C expression. In turn, loss of MMP14 contributed to a more adhesive phenotype of ES cells by modulating the transcriptional extracellular matrix dynamics. Overall, these results suggest that ENG and MMP14 exert a significant role in mediating correct spreading machinery of ES cells, impacting the aggressiveness of the disease., E.A.’s laboratory is supported by ISCIII-FEDER (PI20/00003), CIBERONC (CB16/12/00361), PAIDI-Junta de Andalucía (P18-RT-735), Fundación CRIS Contra el Cáncer, Asociación Candela Riera and Asociación Pablo Ugarte. A.T.A. is supported by Juan de la Cierva Incorporación fellowship (IJC-2018-036767-I); P.P.-C. is sponsored by the Fundación María García Estrada. J.O.-P is supported by Ph.D. Grant Plan Propio from the University of Seville. J.D.-M is supported by CIBERONC (CB16/12/00361). C.S.-A. is supported by the European Social Fund and the Junta de Andalucía (Talento Doctores 2020, DOC_01473). This work was supported by grants from the Consejería de Salud (Junta de Andalucía, grants No PI-0036-2017, PI-0040-2017, and PI-0061-2020) awarded to J.D.-M, A.T.A. and C. S.-A., respectively. This work was also supported by the GEIS-Fundación Mari Paz Jiménez Casado (IV beca trienal) granted to J.D.-M, the 13ª GEIS-Beca Buesa granted to A.T.A. and CRIS (Cancer Research Innovation Spain) granted to J.D.-M and E.A. The laboratory of T.G.P.G. is supported by the Barbara and Wilfried Mohr Foundation. The lab of J.A. is supported by the Instituto de Salud Carlos III (ISCIII), grant number PI20CIII/00020; Asociación Pablo Ugarte, grant numbers TRPV205/18, TPI-M 1149/13; Asociación Candela Riera; Asociación Todos Somos Iván & Fundación Sonrisa de Alex, grant reference: TVP333-19.

Tumor growth depends on the vascular system, either through the expansion of blood vessels or novel adaptation by tumor cells. One of these novel pathways is vasculogenic mimicry (VM), which is defined as a tumor-provided vascular system apart from endothelial cell-lined vessels, and its origin is partly unknown. It involves highly aggressive tumor cells expressing endothelial cell markers that line the tumor irrigation. VM has been correlated with high tumor grade, cancer cell invasion, cancer cell metastasis, and reduced survival of cancer patients. In this review, we summarize the most relevant studies in the field of angiogenesis and cover the various aspects and functionality of aberrant angiogenesis by tumor cells. We also discuss the intracellular signaling mechanisms involved in the abnormal presence of VE-cadherin (CDH5) and its role in VM formation. Finally, we present the implications for the paradigm of tumor angiogenesis and how targeted therapy and individualized studies can be applied in scientific analysis and clinical settings.

The majority of current cancer therapies are aimed at reducing tumour growth, but there is lack of viable pharmacological options to reduce the formation of metastasis. This is a paradox, since more than 90% of cancer deaths are attributable to metastatic progression. Integrin alpha9 (ITGA9) has been previously described as playing an essential role in metastasis; however, little is known about the mechanism that links this protein to this process, being one of the less studied integrins. We have now deciphered the importance of ITGA9 in metastasis and provide evidence demonstrating its essentiality for metastatic dissemination in rhabdomyosarcoma and neuroblastoma. However, the most translational advance of this study is to reveal, for the first time, the possibility of reducing metastasis by pharmacological inhibition of ITGA9 with a synthetic peptide simulating a key interaction domain of ADAM proteins, in experimental metastasis models, not only in childhood cancers but also in a breast cancer model., This research was supported by grants from: Institut Català d’Oncologia (ICO); Instituto de Salud Carlos III (PI18/00398 and FI18/00178); ACCIÓ (COMRDI15-1-0014); Fundació la Marató de TV3; Fundació A. BOSCH; Rotary Clubs Barcelona Eixample, Barcelona Diagonal, Santa Coloma de Gramanet, München-Blutenburg, Deutschland Gemeindienst e.V. and others from Barcelona and province; Eric Abidal Foundation; Del Hospital a la catedral Initiative by Xavi Vallès; and Mi compañero de viaje Association.

Vasculogenic Mimicry (VM) refers to the capacity to form a blood network from aggressive cancer cells in an independent way of endothelial cells, to provide nutrients and oxygen leading to enhanced microenvironment complexity and treatment failure. In a previous study, we demonstrated that VE-Cadherin and its phosphorylation at Y658 modulated kaiso-dependent gene expression (CCND1 and Wnt 11) through a pathway involving Focal Adhesion kinase (FAK). In the present research, using a proteomic approach, we have found that β-catenin/TCF-4 is associated with nuclear VE-cadherin and enhances the capacity of malignant melanoma cells to undergo VM in cooperation with VE-Cadherin; in addition, preventing the phosphorylation of Y658 of VE-cadherin upon FAK disabling resulted in VE-Cadherin/β-catenin complex dissociation, increased β-catenin degradation while reducing TCF-4-dependent genes transcription (C-Myc and Twist-1). Uveal melanoma cells knockout for VE-Cadherin loses β-catenin expression while the rescue of VE-Cadherin (but not of the phosphorylation defective VE-Cadherin Y658F mutant) permits stabilization of β-catenin and tumor growth reduction in vivo experiments. In vivo, the concomitant treatment with the FAK inhibitor PF-271 and the anti-angiogenic agent bevacizumab leads to a strong reduction in tumor growth concerning the single treatment. In conclusion, the anomalous expression of VE-Cadherin in metastatic melanoma cells (from both uveal and cutaneous origins), together with its permanent phosphorylation at Y658, favors the induction of the aggressive VM phenotype through the cooperation of β-catenin with VE-Cadherin and by enhancing TCF-4 genes-dependent transcription.

The Hippo pathway is an evolutionarily conserved modulator of developmental biology with a key role in tissue and organ size regulation under homeostatic conditions. Like other signaling pathways with a significant role in embryonic development, the deregulation of Hippo signaling contributes to oncogenesis. Central to the Hippo pathway is a conserved cascade of adaptor proteins and inhibitory kinases that converge and regulate the activity of the oncoproteins YAP and TAZ, the final transducers of the pathway. Elevated levels and aberrant activation of YAP and TAZ have been described in many cancers. Though most of the studies describe their pervasive activation in epithelial neoplasms, there is increasing evidence pointing out its relevance in mesenchymal malignancies as well. Interestingly, somatic or germline mutations in genes of the Hippo pathway are scarce compared to other signaling pathways that are frequently disrupted in cancer. However, in the case of sarcomas, several examples of genetic alteration of Hippo members, including gene fusions, have been described during the last few years. Here, we review the current knowledge of Hippo pathway implication in sarcoma, describing mechanistic hints recently reported in specific histological entities and how these alterations represent an opportunity for targeted therapy in this heterogeneous group of neoplasm., E.d.Á.’s laboratory is supported by ISCIIIFEDER (PI20/00003) to E.d.Á., Consejería de Salud y Familias, Junta de Andalucía (PE-0186-2018) to E.d.Á. and (PI-0061-2020) to C.S.-A. and E.d.Á., GEIS (Beca José María Buesa) to A.T.A., GEIS-Fundación Mari Paz Jiménez Casado (Beca Trienal a la Investigación en Sarcomas) to J.D.-M., Fundación CRIS Contra el Cáncer, Asociación Pablo Ugarte, Fundación María García Estrada and CIBERONC. C.S.-A. is supported by the European Social Fund and the Junta de Andalucía (Talento Doctores 2020, DOC_01473), J.O.-P. is granted by a pre-doctoral fellowship from the VI Plan Propio from the Universidad de Sevilla; A.T.A. is supported Juan de la Cierva Incorporación fellowship (IJC-2018-036767-I), and J.D.-M. is supported by CIBERONC (CB16/12/00361).