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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.

Chemotherapy is one of the most established and effective treatments for almost all types of cancer. However, the elevated toxicity due to the non-tumor-associated effects, development of secondary malignancies, infertility, radiation-induced fibrosis and resistance to treatment limit the effectiveness and safety of treatment. In addition, these multiple factors significantly impact quality of life. Over the last decades, our increased understanding of cancer epigenetics has led to new therapeutic approaches and the promise of improved patient outcomes. Epigenetic alterations are commonly found in cancer, especially the increased expression and activity of histone deacetylases (HDACs). Dysregulation of HDACs are critical to the development and progression of the majority of tumors. Hence, HDACs inhibitors (HDACis) were developed and now represent a very promising treatment strategy. The use of HDACis as monotherapy has shown very positive pre-clinical results, but clinical trials have had only limited success. However, combinatorial regimens with other cancer drugs have shown synergistic effects both in pre-clinical and clinical studies. At the same time, these combinations have enhanced the efficacy, reduced the toxicity and tumor resistance to therapy. In this review, we will examine examples of HDACis used in combination with other cancer drugs and highlight the synergistic effects observed in recent preclinical and clinical studies. This work was supported by Instituto de Salud Carlos III_FEDER (PI17/00464 and CB16/12/00361), by the Consejería de Salud, Junta de Andalucía (PI-0013-2018), and by Fundación María García Estrada.

Ewing sarcoma (ES) is the second most common bone malignancy affecting children and young adults with poor prognosis due to high metastasis incidence. Our group previously described that EphA2, a tyrosine kinase receptor, promotes angiogenesis in Ewing sarcoma (ES) cells via ligand-dependent signaling. Now we wanted to explore EphA2 ligand-independent activity, controlled upon phosphorylation at S897 (p-EphA2S897 ), as it has been linked to metastasis in several malignancies. By reverse genetic engineering we explored the phenotypic changes after EphA2 removal or reintroduction. Gene expression microarray was used to identify key players in EphA2 signaling. Mice were employed to reproduce metastatic processes from orthotopically implanted engineered cells. We established a correlation between ES cells aggressiveness and p-EphA2S897 . Moreover, stable overexpression of EphA2 in low EphA2 expression ES cells enhanced proliferation and migration, but not a non-phosphorylable mutant (S987A). Consistently, silencing of EphA2 reduced tumorigenicity, migration and invasion in vitro, and lung metastasis incidence in experimental and spontaneous metastasis assays in vivo. A gene expression microarray revealed the implication of EphA2 in cell signaling, cellular movement and survival. ADAM19 knockdown by siRNA technology strongly reproduced the negative effects on cell migration observed after EphA2 silencing. Altogether, our results suggest that p-EphA2S897 correlates with aggressiveness in ES, so blocking its function may be a promising treatment.

Wilms tumour (WT) is the most common renal tumour in children. Most WT patients respond to chemotherapy, but subsets of tumours develop resistance to chemotherapeutic agents, which is a major obstacle in their successful treatment. Multidrug resistance transporters play a crucial role in the development of resistance in cancer due to the efflux of anticancer agents out of cells. The aim of this study was to explore several human multidrug resistance transporters in 46 WT and 40 nonneoplastic control tissues (normal kidney) from patients selected after chemotherapy treatment SIOP 93–01, SIOP 2001. Our data showed that the majority of the studied multidrug resistance transporters were downregulated or unchanged between tumours and control tissues. However, BCRP1, MDR3 and MRP1 were upregulated in tumours versus control tissues. MDR3 and MRP1 overexpression correlated with highrisk tumours (SIOP classification) (p = 0.0022 and p < 0.0001, respectively) and the time of disease-free survival was significantly shorter in patients with high transcript levels of MDR3 (p = 0.0359). MDR3 and MRP1 play a role in drug resistance in WT treatment, probably by alteration of an unspecific drug excretion system. Besides, within the blastemal subtype, we observed patients with low MDR3 expression were significantly associated with a better outcome than patients with high MDR3 expression. We could define two types of blastemal WT associated with different disease outcomes, enabling the stratification of blastemal WT patients based on the expression levels of the multidrug resistance transporter MDR3. Ministerio de Economía y Competitividad PI1401466, RD06/0020/0059, PI1100018, CD06/00001 Red Tematica de Investigacion Cooperativa en Cancer RD12/0036/0017 Unión Europea FP7-HEALTH- 2011-two-stage, Project ID 278742 EUROSARC Instituto de Salud Carlos III FIS PI13/02282

Resumen del trabajo presentado a la EACR-OECI Conference: "Precision Medicine for Cancer", celebrada en Neumünster Abbey (Luxemburgo) del 1 al 4 de marzo de 2015.-- et al., An essential aspect of breast cancer is its different evolution among patients with the same histopathological disease. Moreover, cancer is a tissue growing in the context of a complex organism, thus it can be identified two main sources of variability responsible for the disease behavior: intrinsic and extrinsic factors which act, respectively, mainly inside the tumor cells and outside them at local or systemic levels. Our aim is to identify intrinsic factors to the tumor cells responsible for the different responses of breast cancer to chemotherapy with Doxorubicin and Docetaxel. For this purpose, we collected tumors developed in a cohort of genetically heterogeneous mice from a backcross between a resistant strain to breast cáncer (C57BL/6) and a susceptible one (FVB) which overexpress the cNeu/ErbB2 protooncogene controlled by the MMTV prometer. The backcross mice were genotyped by SNP analysis. To identify tumor intrinsic factors controlling the response to chemotherapy, we transplanted 125 tumors collected from the backcross mice into singenic F1-C57/FVB mice to remove variability coming from the host compartments. Each tumor was transplanted into two F1 reciplent mice; each one was treated with Doxorubicin or Docetaxel, and we studied tumor response to treatment. Linkage analysis permits us to identify QTL (Quantitative Trait Loci) controlling susceptibility to mammary cancer and evolution of the disease in the backcross population, and the specific intrinsic QTL associated with different chemotherapy responses in the F1 mice. Moreover, we are studying molecular and signalling pathways that control chemotherapy responses and the QTL associated with them. The identification of breast cancer susceptibility genes and their pathways associated with different response to chemotherapy will be important for the prediction of human breast cancer evolution during therapy, and to learn about the mechanisms involved in resistance to chemotherapy, thus it would help to develop new preventivo and therapeutic strategies.

Resumen del póster presentado al VII Simposium Bases Biológicas del Cáncer y Terapias Personalizadas, celebrado en el Centro de Investigación del Cáncer (CIC-IBMCC) del 21 al 22 de mayo de 2015.-- et al., Este póster ha ganado el 1er premio en el Concurso de Pósters de Oncología Básica y Traslacional en Oncología para Jóvenes Investigadores, celebrado durante el VII Simposium Bases Biológicas del Cáncer y Terapias Personalizadas., An essential aspect of breast cancer is its different evolution among patients with the same histopathological disease. Moreover, cancer is a tissue growing in the context of a complex organism, thus it can be identified two main sources of variability responsible for the disease behavior: intrinsic and extrinsic factors which act, respectively, mainly inside the tumor cells and outside them at local or systemic levels. Our aim is to identify intrinsic factors to the tumor cells responsible for the different responses of breast cancer to chemotherapy with Doxorubicin and Docetaxel. For this purpose, we collected tumors developed in a cohort of genetically heterogeneous mice from a backcross between a resistant strain to breast cancer (C57BL/6) and a susceptible one (FVB) which overexpress the cNeu/ErbB2 protooncogene controlled by the MMTV promoter. The backcross mice were genotyped by SNP analysis. To identify tumor intrinsic factors controlling the response to chemotherapy, we transplanted 125 tumors collected from the backcross mice into singenic F1-C57/FVB mice to remove variability coming from the host compartments. Each tumor was transplanted into two F1 recipient mice; each one was treated with Doxorubicin or Docetaxel, and we studied tumor response to treatment. Linkage analysis permits us to identify QTL (Quantitative Trait Loci) controlling susceptibility to mammary cancer and evolution of the disease in the backcross population, and the specific intrinsic QTL associated with different chemotherapy responses in the F1 mice. Moreover, we are studying molecular and signalling pathways that control chemotherapy responses and the QTL associated with them. The identification of breast cancer susceptibility genes and their pathways associated with different response to chemotherapy will be important for the prediction of human breast cancer evolution during therapy, and to learn about the mechanisms involved in resistance to chemotherapy, thus it would help to develop new preventive and therapeutic strategies.

[ES] Los tumores de mama y de pulmón son la causa principal de muerte por cáncer en el Mundo Occidental. La muerte viene, sobre todo, determinada por la diseminación a distancia o metástasis; aunque en el cáncer de pulmón también tienen relevancia las complicaciones debidas a la progresión local. Por ello, estamos interesados en comprender mejor los mecanismos moleculares y celulares que determinan la mala evolución de estos tumores. Con el fin de predecir la mala evolución de distintos tipos de cáncer, diversos laboratorios han descrito una serie de patrones de expresión génica con un fin pronóstico. Un problema no resuelto es identificar cuáles de esos genes, además de un valor pronóstico, están particularmente implicados en la patogenia tumoral y tendrían un interés terapéutico, por lo que serían dianas ideales para evitar la evolución de la enfermedad. Uno de esos genes, cuya sobreexpresión se ha implicado en el mal pronóstico y la progresión de diversos tipos de cáncer, incluidos mama y pulmón, es el gen SNAI2/SLUG. La proteína SNAI2/SLUG regula fundamentalmente procesos de transición epitelio-mesenquimal y de inhibición de la apoptosis. Por lo primero, la célula tumoral adquiere características mesenquimales con pérdida de adhesión y capacidad de movimiento, fenómenos que definen la metástasis. Por lo segundo, la célula tumoral no responde a las señales de muerte celular y, ello favorecería la iniciación tumoral, el crecimiento local, y la supervivencia de la célula tumoral lejos de su microambiente. Ello sugiere que el gen SNAI2/SLUG podría ser un candidato ideal para ser utilizado como diana terapeútica, cuya inhibición podría interferir con varias fases del desarrollo tumoral. Nuestro objetivo global fue evaluar in vivo la importancia del gen SNAI2/SLUG en el desarrollo y evolución de los cánceres de mama y pulmón, como evaluación indirecta de su interés como diana terapéutica. Para ello, se determinó el papel del gen Snai2/Slug en el inicio y progresión del cáncer de pulmón in vivo, además de en la susceptibilidad, desarrollo y diseminación del cáncer de mama in vivo. Por otro lado, se estudiaron los fenómenos celulares y moleculares por los que el gen Snai2/Slug contribuye a la patogenia de los cánceres de mama y de pulmón. Con este proyecto se pretendió mejorar nuestros conocimientos de los mecanismos moleculares y celulares que determinan el inicio, la progresión y diseminación tumoral en diferentes tipos de cáncer con alta mortalidad en el mundo, como son los cánceres de mama y pulmón. Y, en concreto, pretendimos evaluar la importancia del gen Snai2/Slug en la patogenia de la susceptibilidad y evolución de estos tipos de cáncer. Ello será un indicador indirecto de la pertinencia y utilidad de la inhibición farmacológica del gen Snai2/Slug o sus productos sobre las diferentes fases del desarrollo tumoral. Ello permitiría mejorar el manejo clínico de estas enfermedades., [EN] Breast tumors and lung cancers are the leading cause of cancer death in the Western World. Death comes mainly determined by distant spread or metastasis, although lung cancer also have relevance complications due to local progression. Therefore, we are interested in better understanding the molecular mechanisms and cellular determinants of poor prognosis of these tumors.

Wilms tumor (WT) is an embryonal malignant neoplasm of the kidney that accounts for 6–7% of all childhood cancers. WT seems to derive from multipotent embryonic renal stem cells that have failed to differentiate properly. Since mechanisms underlying WT tumorigenesis remain largely unknown, the aim of this study was to explore the expression of embryonic stem cell (ESC) markers in samples of WT patients after chemotherapy treatment SIOP protocol, as the gene expression patterns of ESC are like those of most cancer cells. We found that expression of ESC markers is heterogeneous, and depends on histological WT components. Interestingly, among ESC markers, HMGA2 was expressed significantly stronger in the blastemal component than in the stromal and the normal kidney. Moreover, two subsets of patients of WT blastemal type were identified, depending on the expression levels of HMGA2. High HMGA2 expression levels were significantly associated with a higher proliferation rate (p=0.0345) and worse patient prognosis (p=0.0289). The expression of HMGA2 was a stage-independent factor of clinical outcome in blastemal WT patients. Our multivariate analyses demonstrated the association between LIN28B–LET7A–HMGA2 expression, and the positive correlation between HMGA2 and SLUG expression (p=0.0358) in blastemal WT components. In addition, patients with a poor prognosis and high HMGA2 expression presented high levels of MDR3 (multidrug resistance transporter). Our findings suggest that HMGA2 plays a prominent role in the pathogenesis of a subset of blastemal WT, strongly associated with relapse and resistance to chemotherapy., LHP and DJGD are supported by Red Temática de Investigación Cooperativa en Cáncer (RD12/0036/0017). CS is supported by a contract from Nicolás Monardes Program, Consejería de Salud, Junta de Andalucía. Research in Enrique de Alava’s lab is supported by the Ministry of Economy and Competitiveness of Spain-FEDER (CIBERONC, PI1700464, RD06/0020/0059, FMGE) and the European Commission (FP7-HEALTH-2011-two-stage, Project ID 278742 EUROSARC). DJGD is a PhD researcher funded by the Consejería de Salud, Junta de Andalucía (PI-0197-2016). Research in Carmen Sáez’s lab is also supported by research grants from the Instituto de Salud Carlos III, FIS PI13/02282.

Ewing sarcoma (ES) is an aggressive tumor defined by EWSR1 gene fusions that behave as an oncogene. Here we demonstrate that RING1B is highly expressed in primary ES tumors, and its expression is independent of the fusion oncogene. RING1B-depleted ES cells display an expression profile enriched in genes functionally involved in hematological development but RING1B depletion does not induce cellular differentiation. In ES cells, RING1B directly binds the SCN8A sodium channel promoter and its depletion results in enhanced Nav1.6 expression and function. The signaling pathway most significantly modulated by RING1B is NF-κB. RING1B depletion results in enhanced p105/p50 expression, which sensitizes ES cells to apoptosis by FGFR/SHP2/STAT3 blockade. Reduced NaV1.6 function protects ES cells from apoptotic cell death by maintaining low NF-κB levels. Our findings identify RING1B as a trait of the cell-of-origin and provide a potential targetable vulnerability., IHM is a Miguel Servet investigator (Instituto de Salud Carlos III, ISCIII). This work was supported by the Xarxa de Bancs de Tumors de Catalunya sponsored by Pla Director d’Oncologia de Catalunya and by grants from ISCIII-FEDER (PS09/00973; RIC RD12/0042/0014, Red HERACLES), Ministerio de Economía y Competitividad (MEC) (SAF2012-31089 and SAF2015-69762-R), FEDER Funds and by the Asociación Española Contra el Cáncer (AECC). E.Á.’s lab is supported by the AECC, MEC-FEDER (RD12/0036/0017, PT13/0010/0056, RTC-2014-2102-1, ISCIII Sara Borrell CD06/00001, PI12/03102, PI14/01466), the European FP7 Projects EuroSARC (FP7-HEALTH-2011-two-stage, Project 278742 EUROSARC), Euroewing (FP7-HEALTH.2013.2.4.1-1, Project 602856), Fundación Memoria de DM Solorzano Barruso, Fundación Cris contra el Cáncer, Pablo Ugarte Foundation, and Fundación M. García Estrada. O.M.T. is funded by ISCIII-FEDER (CES12/021) and the AECC. AMC is funded by the European FP7 projects (FP7/2007-2013) under a Marie Curie International Reintegration Grant (PIRG-08- GA-2010-276998) and ISCIII-FEDER (CP13/00189). The Ewing group at the Developmental Tumor Biology Laboratory, Hospital Sant Joan de Déu, is supported by the AECC and the generous donations from Pablo Ugarte Foundation.