Selective histone methyltransferase G9a inhibition reduces metastatic development of Ewing sarcoma through the epigenetic regulation of NEU1.

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.

Clonogenic Assays to Detect Cell Fate in Mitotic Catastrophe.

Mitotic catastrophe (MC) is a cell death modality induced by DNA damage that involves the activation of cell cycle checkpoints such as the "DNA structure checkpoint" and "spindle assembly checkpoint" (SAC) leading to aberrant mitosis. Depending on the signal, MC can drive the cell to death or to senescence. The suppression of MC favors aneuploidy. Several cancer therapies, included microtubular poisons and radiations, trigger MC. The clonogenic assay has been used to study the capacity of single cells to proliferate and to generate macroscopic colonies and to evaluate the efficacy of anticancer drugs. Nevertheless, this method cannot analyze MC events. Here, we report an improved technique based on the use of human colon cancer HCT116 stable expressing histone H2B-GFP and DsRed-centrin proteins, allowing to determine the capacity of cells to proliferate, and to determine changes in the nucleus and centrosomes.

LOXL2 promotes oncogenic progression in alveolar rhabdomyosarcoma independently of its catalytic activity.

Rhabdomyosarcoma (RMS) is the most common soft tissue malignancy in childhood and adolescence. Patients with the most aggressive histological variant have an unfavorable prognosis due to a high metastasis incidence. Lysyl oxidase-like 2 (LOXL2) is a lysyl oxidase, member of a family of extracellular matrix (ECM) crosslinking enzymes that recently have emerged as important regulators of tumor progression and metastasis. We report that LOXL2 is overexpressed in RMS, suggesting a potential role for LOXL2 in RMS oncogenic progression. Consistently, transient and stable LOXL2 knockdown decreased cell migratory and invasive capabilities in two ARMS cell lines. Furthermore, introduction of LOXL2 in RMS non-expressing cells using wild type or mutated (catalytically inactive) constructs resulted in increased cell migration, cell invasion and number and incidence of spontaneous lung metastasis in vivo, independently of its catalytic activity. To further study the molecular mechanism associated with LOXL2 expression, a pull-down assay on LOXL2-transfected cells was performed and analyzed by mass spectrometry. The intermediated filament protein vimentin was validated as a LOXL2-interactor. Thus, our results suggest an oncogenic role of LOXL2 in RMS by regulating cytoskeleton dynamics and cell motility capabilities.

DNA methylation profiling identifies PTRF/Cavin-1 as a novel tumor suppressor in Ewing sarcoma when co-expressed with caveolin-1.

Epigenetic modifications have been shown to be important in developmental tumors as Ewing sarcoma. We profiled the DNA methylation status of 15 primary tumors, 7 cell lines, 10 healthy tissues and 4 human mesenchymal stem cells lines samples using the Infinium Human Methylation 450K. Differential methylation analysis between Ewing sarcoma and reference samples revealed 1166 hypermethylated and 864 hypomethylated CpG sites (Bonferroni p < 0.05, δ-ß-value with absolute difference of >0.20) corresponding to 392 and 470 genes respectively. Gene Ontology analysis of genes differentially methylated in Ewing sarcoma samples showed a significant enrichment of developmental genes. Membrane and cell signal genes were also enriched, among those, 11 were related to caveola formation. We identified differential hypermethylation of CpGs located in the body and S-Shore of the PTRF gene in Ewing sarcoma that correlated with its repressed transcriptional state. Reintroduction of PTRF/Cavin-1 in Ewing sarcoma cells revealed a role of this protein as a tumor suppressor. Restoration of caveolae in the membrane of Ewing sarcoma cells, by exogenously reintroducing PTRF, disrupts the MDM2/p53 complex, which consequently results in the activation of p53 and the induction of apoptosis.

Caveolin-1 promotes Ewing sarcoma metastasis regulating MMP-9 expression through MAPK/ERK pathway.

Ewing sarcoma (ES) is a bone and soft tissue sarcoma affecting mostly children and young adults. Caveolin-1 (CAV1) is a well-known target of EWS/FLI1, the main driver of ES, with an oncogenic role in ES. We have previously described how CAV1 is able to induce metastasis in ES via matrix metalloproteinase-9 (MMP-9). In the present study we showed how CAV1 silencing in ES reduced MEK1/2 and ERK1/2 phosphorylation. Accordingly, chemical inhibition of MEK1/2 resulted in reduction in MMP-9 expression and activity that correlated with reduced migration and invasion. IQ Motif Containing GTPase Activating Protein 1 (IQGAP1) silencing reduced MEK1/2 and ERK1/2 phosphorylation and MMP-9 expression. Furthermore, IQGAP1 silenced cells showed a marked decrease in their migratory and invasive capacity. We demonstrated that CAV1 and IQGAP1 localize in close proximity at the cellular edge, thus IQGAP1 could be the connecting node between CAV1 and MEK/ERK in ES metastatic phenotype. Analysis of the phosphorylation profile of CAV1-silenced cells showed a decrease of p-ribosomal protein S6 (RPS6). RPS6 can be phosphorylated by p90 ribosomal S6 kinases (RSK) proteins. CAV1-silenced cells showed reduced levels of p-RSK1 and treatment with U0126 provoked the same effect. Despite not affecting ERK1/2 and RPS6 phosphorylation status neither MMP-9 expression nor activity, RSK1 silencing resulted in a reduced migratory and invasive capacity in vitro and reduced incidence of metastases in vivo in a novel orthotopic model. The present work provides new insights into CAV1-driven metastatic process in ES unveiling novel key nodes.

The importance of being dead: cell death mechanisms assessment in anti-sarcoma therapy.

Cell death can occur through different mechanisms, defined by their nature and physiological implications. Correct assessment of cell death is crucial for cancer therapy success. Sarcomas are a large and diverse group of neoplasias from mesenchymal origin. Among cell death types, apoptosis is by far the most studied in sarcomas. Albeit very promising in other fields, regulated necrosis and other cell death circumstances (as so-called "autophagic cell death" or "mitotic catastrophe") have not been yet properly addressed in sarcomas. Cell death is usually quantified in sarcomas by unspecific assays and in most cases the precise sequence of events remains poorly characterized. In this review, our main objective is to put into context the most recent sarcoma cell death findings in the more general landscape of different cell death modalities.

"(Not) all (dead) things share the same breath": identification of cell death mechanisms in anticancer therapy.

During the last decades, the knowledge of cell death mechanisms involved in anticancer therapy has grown exponentially. However, in many studies, cell death is still described in an incomplete manner. The frequent use of indirect proliferation assays, unspecific probes, or bulk analyses leads too often to misunderstandings regarding cell death events. There is a trend to focus on molecular or genetic regulations of cell demise without a proper characterization of the phenotype that is the object of this study. Sometimes, cancer researchers can feel overwhelmed or confused when faced with such a corpus of detailed insights, nomenclature rules, and debates about the accuracy of a particular probe or assay. On the basis of the information available, we propose a simple guide to distinguish forms of cell death in experimental settings using cancer cell lines.

Caveolin-1 is down-regulated in alveolar rhabdomyosarcomas and negatively regulates tumor growth.

Rhabdomyosarcoma is the most common soft tissue sarcoma of childhood and adolescence. Despite advances in therapy, patients with histological variant of rhabdomyosarcoma known as alveolar rhabdomyosarcoma (ARMS) have a 5-year survival of less than 30%. Caveolin-1 (CAV1), encoding the structural component of cellular caveolae, is a suggested tumor suppressor gene involved in cell signaling. In the present study we report that compared to other forms of rhabdomyosarcoma (RMS) CAV1 expression is either undetectable or very low in ARMS cell lines and tumor samples. DNA methylation analysis of the promoter region and azacytidine-induced re-expression suggest the involvement of epigenetic mechanisms in the silencing of CAV1. Reintroduction of CAV1 in three of these cell lines impairs their clonogenic capacity and promotes features of muscular differentiation. In vitro, CAV1-expressing cells show high expression of Caveolin-3 (CAV3), a muscular differentiation marker. Blockade of MAPK signaling is also observed. In vivo, CAV1-expressing xenografts show growth delay, features of muscular differentiation and increased cell death. In summary, our results suggest that CAV1 could function as a potent tumor suppressor in ARMS tumors. Inhibition of CAV1 function therefore, could contribute to aberrant cell proliferation, leading to ARMS development.

An approach to the histomorphological and histochemical variations of the humerus cortical bone through human ontogeny.

For many years, clinical and non-clinical investigations have investigated cortical bone structure in an attempt to address questions related to normal bone development, mineralisation, pathologies and even evolutionary trends in our lineage (adaptations). Research in the fields of medicine, materials science, physical anthropology, palaeontology, and even archaeobiology has contributed interesting data. However, many questions remain regarding the histomorphological and histochemical variations in human cortical bone during different stages of life. In the present work, we describe a study of long bone cortex transformations during ontogeny. We analysed cross-sections of 15 human humeri histomorphologically and histochemically from perinatal to adult age, marking and quantifying the spatial distribution of bone tissue types using GIS software and analysing the mineral composition and crystallinity of the mineralised cortex using Raman spectroscopy and X-ray diffraction. Our results allowed us to propose that human cortical bone undergoes three main 'events' through ontogeny that critically change the proportions and structure of the cortex. In early development, bone is not well mineralised and proportionally presents a wide cortex that narrows through the end of childhood. Before reaching complete maturity, the bone mineral area increases, allowing the bone to nearly reach the adult size. The medullary cavity is reduced, and the mineral areas have a highly ordered crystalline structure. The last event occurs in adulthood, when the 'oldest' individuals present a reduced mineralised area, with increasing non-mineralised cavities (including the medullary cavity) and reduced crystalline organisation.

Resveratrol and aspirin eliminate tetraploid cells for anticancer chemoprevention.

Tetraploidy constitutes a genomically metastable state that can lead to aneuploidy and genomic instability. Tetraploid cells are frequently found in preneoplastic lesions, including intestinal cancers arising due to the inactivation of the tumor suppressor adenomatous polyposis coli (APC). Using a phenotypic screen, we identified resveratrol as an agent that selectively reduces the fitness of tetraploid cells by slowing down their cell cycle progression and by stimulating the intrinsic pathway of apoptosis. Selective killing of tetraploid cells was observed for a series of additional agents that indirectly or directly stimulate AMP-activated protein kinase (AMPK) including salicylate, whose chemopreventive action has been established by epidemiological studies and clinical trials. Both resveratrol and salicylate reduced the formation of tetraploid or higher-order polyploid cells resulting from the culture of human colon carcinoma cell lines or primary mouse epithelial cells lacking tumor protein p53 (TP53, best known as p53) in the presence of antimitotic agents, as determined by cytofluorometric and videomicroscopic assays. Moreover, oral treatment with either resveratrol or aspirin, the prodrug of salicylate, repressed the accumulation of tetraploid intestinal epithelial cells in the Apc(Min/+) mouse model of colon cancer. Collectively, our results suggest that the chemopreventive action of resveratrol and aspirin involves the elimination of tetraploid cancer cell precursors.

Antiapoptotic activity of argon and xenon.

Although chemically non-reactive, inert noble gases may influence multiple physiological and pathological processes via hitherto uncharacterized physical effects. Here we report a cell-based detection system for assessing the effects of pre-defined gas mixtures on the induction of apoptotic cell death. In this setting, the conventional atmosphere for cell culture was substituted with gas combinations, including the same amount of oxygen (20%) and carbon dioxide (5%) but 75% helium, neon, argon, krypton, or xenon instead of nitrogen. The replacement of nitrogen with noble gases per se had no effects on the viability of cultured human osteosarcoma cells in vitro. Conversely, argon and xenon (but not helium, neon, and krypton) significantly limited cell loss induced by the broad-spectrum tyrosine kinase inhibitor staurosporine, the DNA-damaging agent mitoxantrone and several mitochondrial toxins. Such cytoprotective effects were coupled to the maintenance of mitochondrial integrity, as demonstrated by means of a mitochondrial transmembrane potential-sensitive dye and by assessing the release of cytochrome c into the cytosol. In line with this notion, argon and xenon inhibited the apoptotic activation of caspase-3, as determined by immunofluorescence microscopy coupled to automated image analysis. The antiapoptotic activity of argon and xenon may explain their clinically relevant cytoprotective effects.

Transgenerational cell fate profiling: a method for the graphical presentation of complex cell cycle alterations.

The illicit generation of tetraploid cells constitutes a prominent driver of oncogenesis, as it often precedes the development of aneuploidy and genomic instability. In addition, tetraploid (pre-)malignant cells display an elevated resistance against radio- and chemotherapy. Here, we report a strategy to preferentially kill tetraploid tumor cells based on the broad-spectrum kinase inhibitor SP600125. Live videomicroscopy revealed that SP600125 affects the execution of mitosis, impedes proper cell division and/or activates apoptosis in near-to-tetraploid, though less so in parental, cancer cells. We propose a novel graphical model to quantify the differential response of diploid and tetraploid cells to mitotic perturbators, including SP600125, which we baptized "transgenerational cell fate profiling." We speculate that this representation constitutes a valid alternative to classical "single-cell fate" and "genealogical" profiling and, hence, may facilitate the analysis of cell fate within a heterogeneous population as well as the visual examination of cell cycle alterations.

An immunosurveillance mechanism controls cancer cell ploidy.

Cancer cells accommodate multiple genetic and epigenetic alterations that initially activate intrinsic (cell-autonomous) and extrinsic (immune-mediated) oncosuppressive mechanisms. Only once these barriers to oncogenesis have been overcome can malignant growth proceed unrestrained. Tetraploidization can contribute to oncogenesis because hyperploid cells are genomically unstable. We report that hyperploid cancer cells become immunogenic because of a constitutive endoplasmic reticulum stress response resulting in the aberrant cell surface exposure of calreticulin. Hyperploid, calreticulin-exposing cancer cells readily proliferated in immunodeficient mice and conserved their increased DNA content. In contrast, hyperploid cells injected into immunocompetent mice generated tumors only after a delay, and such tumors exhibited reduced DNA content, endoplasmic reticulum stress, and calreticulin exposure. Our results unveil an immunosurveillance system that imposes immunoselection against hyperploidy in carcinogen- and oncogene-induced cancers.

Mapping human long bone compartmentalisation during ontogeny: a new methodological approach.

Throughout ontogeny, human bones undergo differentiation in terms of shape, size and tissue type; this is a complex scenario in which the variations in the tissue compartmentalisation of the cortical bone are still poorly understood. Currently, compartmentalisation is studied using methodologies that oversimplify the bone tissue complexity. Here, we present a new methodological approach that integrates a histological description and a mineral content analysis to study the compartmentalisation of the whole mineralised and non-mineralised tissues (i.e., spatial distribution in long bone sections). This new methodology, based on Geographical Information System (GIS) software, allows us to draw areas of interest (i.e., tracing vectorial shapes which are quantifiable) in raw images that are extracted from microscope and compared them spatially in a semi-automatic and quantitative fashion. As an example of our methodology, we have studied the tibiae from individuals with different age at death (infant, juvenile and adult). The tibia's cortical bone presents a well-formed fibrolamellar bone, in which remodelling is clearly evidenced from early ontogeny, and we discuss the existence of "lines of arrested growth". Concurrent with the histological variation, Raman and FT-IR spectroscopy analyses corroborate that the mineral content in the cortical bone changes differentially. The anterior portion of the tibia remains highly pierced and is less crystalline than the rest of the cortex during growth, which is evidence of more active and continuous remodelling. Finally, while porosity and other "non-mineralised cavities" are largely modified, the mineralised portion and the marrow cavity size persist proportionally during ontogeny.

Selective killing of p53-deficient cancer cells by SP600125.

The genetic or functional inactivation of p53 is highly prevalent in human cancers. Using high-content videomicroscopy based on fluorescent TP53(+/+) and TP53(-/-) human colon carcinoma cells, we discovered that SP600125, a broad-spectrum serine/threonine kinase inhibitor, kills p53-deficient cells more efficiently than their p53-proficient counterparts, in vitro. Similar observations were obtained in vivo, in mice carrying p53-deficient and -proficient human xenografts. Such a preferential cytotoxicity could be attributed to the failure of p53-deficient cells to undergo cell cycle arrest in response to SP600125. TP53(-/-) (but not TP53(+/+) ) cells treated with SP600125 became polyploid upon mitotic abortion and progressively succumbed to mitochondrial apoptosis. The expression of an SP600125-resistant variant of the mitotic kinase MPS1 in TP53(-/-) cells reduced SP600125-induced polyploidization. Thus, by targeting MPS1, SP600125 triggers a polyploidization program that cannot be sustained by TP53(-/-) cells, resulting in the activation of mitotic catastrophe, an oncosuppressive mechanism for the eradication of mitosis-incompetent cells.

Autophagic removal of micronuclei.

Macroautophagy is known to participate in the quality control and turnover of cytoplasmic organelles, yet there is little evidence that macroautophagy targets nuclei in mammalian cells. Here, we investigated whether autophagy may target micronuclei, which arise as a result of deficient bipolar chromosome segregation in cells exposed to cell cycle perturbations. After removal of several distinct cell cycle blockers (nocodazole, cytochalasin D, hydroxyurea or SP600125), cells manifested an increase in the frequency of micronuclei (positive for histone H2B-RFP) as well as an increase in autophagic puncta (positive for GFP-LC3) over several days. A small but significant percentage of micronuclei co-localized with GFP-LC3 in autophagy-competent cells and this co-localization was lost after knockdown of ATG5 or ATG7. Electron microscopy analyses confirmed autophagic sequestration of micronuclei. "Autophagic micronuclei" (GFP-LC3⁺) were also decorated with p62/SQSTM1, while non-autophagic (GFP-LC3⁻) micronuclei where p62/SQSTM1 negative. In addition, GFP-LC3⁺ micronuclei exhibited signs of envelope degradation and γH2AX⁺ DNA damage foci, yet stained less intensively for chromatin markers, whereas GFP-LC3⁻ micronuclei were surrounded by an intact envelope and rarely exhibited markers or DNA damage. These results indicate that micronuclei can be subjected to autophagic degradation. Moreover, it can be speculated that removal of micronuclei may contribute to the genome-stabilizing effects of autophagy.

Autophagy-dependent anticancer immune responses induced by chemotherapeutic agents in mice.

Antineoplastic chemotherapies are particularly efficient when they elicit immunogenic cell death, thus provoking an anticancer immune response. Here we demonstrate that autophagy, which is often disabled in cancer, is dispensable for chemotherapy-induced cell death but required for its immunogenicity. In response to chemotherapy, autophagy-competent, but not autophagy-deficient, cancers attracted dendritic cells and T lymphocytes into the tumor bed. Suppression of autophagy inhibited the release of adenosine triphosphate (ATP) from dying tumor cells. Conversely, inhibition of extracellular ATP-degrading enzymes increased pericellular ATP in autophagy-deficient tumors, reestablished the recruitment of immune cells, and restored chemotherapeutic responses but only in immunocompetent hosts. Thus, autophagy is essential for the immunogenic release of ATP from dying cells, and increased extracellular ATP concentrations improve the efficacy of antineoplastic chemotherapies when autophagy is disabled.

Cytofluorometric purification of diploid and tetraploid cancer cells.

During malignant transformation, cells can increase their ploidy and hence become polyploid (mostly tetraploid). Frequently, however, tetraploid cells undergo asymmetric divisions, in turn entailing a reduction in ploidy and the acquisition of a pseudo-diploid, aneuploid state. To investigate such a stepwise aneuploidization process, we developed a cytofluorometric method (based on the heterogeneity in cell size and/or chromatin content) that allows for the cloning and subsequent functional analysis of cells with distinct ploidies. Here, we detail this methodology, which has been instrumental for investigating the functional link between ploidy status and oncogenesis.

Involvement of p38alpha in the mitotic progression of p53(-/-) tetraploid cells.

The tumor suppressor protein p53 plays a major role in preserving genomic stability. p53 suppresses a pathway leading from normal diploidy to neoplastic aneuploidy (via an intermediate metastable stage of tetraploidy) at two levels: first by preventing the generation/survival of tetraploid cells, and second by repressing their aberrant multipolar division. Here, we report the characterization of p53(-/-) tetraploid cells, which-at difference with both their p53(-/-) diploid and their p53(+/+) tetraploid counterparts-manifest a marked hyperphosporylation of the mitogen-activated protein kinase MAPK14 (best known as p38alpha) that is particularly strong during mitosis. In p53(-/-) tetraploid cells, phosphorylated p38alpha accumulated at centrosomes during the metaphase and at midbodies during the telophase. Selective knockdown or pharmacological inhibition of p38alpha had a dramatic effect on p53(-/-) (but not p53(+/+)) tetraploids, causing the activation of the spindle assembly checkpoint, an arrest during the metaphase, a major increase in abnormal bipolar and monopolar mitoses, as well as an increment in the generation of multinuclear cells. We conclude that the mitotic progression of p53(-/-) (but not p53(+/+)) tetraploids heavily relies on p38alpha, revealing a novel function for this protein in the context of aneuploidizing cell divisions.