Endothelial Secreted Factors Suppress Mitogen Deprivation-Induced Autophagy and Apoptosis in Glioblastoma Stem-Like Cells

International audienceRapidly growing and highly vascularized tumors, such as glioblastoma multiforme, contain heterogeneous areas within the tumor mass, some of which are inefficiently supplied with nutrients and oxygen. While the cell death rate is elevated in such zones, tumor cells are still suspected to grow and survive independently of extracellular growth factors. In line with this, glioblastoma stem-like cells (GSCs) are found closely associated with brain vasculature in situ, and as such are most likely in a protected microenvironment. However, the behavior of GSCs under deprived conditions has not been explored in detail. Using a panel of 14 patient-derived GSCs, we report that ex vivo mitogen deprivation impaired self-renewal capability, abolished constitutive activation of the mTor pathway, and impinged on GSC survival via the engagement of autophagic and apoptotic cascades. Moreover, pharmacological inhibition of the mTor pathway recapitulated the mitogen deprivation scenario. In contrast, blocking either apoptosis or autophagy, or culturing GSCs with endothelial-secreted factors partly restored mTor pathway activation and rescued GSC survival. Overall, our data suggest that GSCs are addicted to mTor, as their survival and self-renewal are profoundly dependent on this signaling axis. Thus, as mTor governs the fate of GSCs under both deprivation conditions and in the presence of endothelial factors, it could be a key target for therapeutic purposes

Mapping Bromodomains in breast cancer and association with clinical outcome

A specific family of proteins that participate in epigenetic regulation is the bromodomain (BRD) family of proteins. In this work, we aimed to explore the expression of the BRD family at a transcriptomic level in breast cancer, and its association with patient survival. mRNA level data from normal breast and tumor tissues were extracted from public datasets. Gene set enrichment analysis (GSEA) was performed to identify relevant biological functions. The KM Plotter Online tool was used to evaluate the relationship between the presence of different genes and patient clinical outcome. mRNA level data from HER2+ breast cancer patients sensible and resistant to trastuzumab were also evaluated. The BRD family was an enriched function. In HER2 positive tumors the combined analyses of BRD2, BAZ1A, TRIM33 and ZMYND8 showed a detrimental relapse free survival (RFS). Similarly, the combined analysis of BRD2, BAZ1A, PHIP, TRIM33, KMT2A, ASH1L, PBRM1, correlated with an extremely poor overall survival (OS). The prognosis was confirmed using an independent dataset from TCGA. Finally, no relation between expression of BRD genes and response to trastuzumab was observed in the HER2 population. Upregulation of some BRD genes is associated with detrimental outcome in HER2 positive tumors, regardless trastuzumab treatment

Evaluation of transcriptionally regulated genes identifies NCOR1 in hormone receptor negative breast tumors and lung adenocarcinomas as a potential tumor suppressor gene

Regulation of transcription is a key process in cellular homeostasis. It depends on regulators that either repress or stimulate the transcription of genes, therefore controlling different biological functions. The Nuclear Receptor Corepressor 1 (NCOR1) is one of those co-repressors that regulate the transcription by facilitating the recruitment of HDAC1, 2, 3, 4, 5 and 7. In our article, by using an in silico approach, we evaluate the mutational status of NCOR1 in breast and lung tumors. We identified that NORC1 is mutated in more than 3% of breast tumors and lung adenocarcinomas and linked this fact with detrimental outcome in some subtypes, particularly in those that are hormone receptor negative. In addition to these findings, as mutations in this gene are deleterious, we confirmed that high levels of this gene were linked with good prognosis in the same tumor subtypes. Findings in the same direction were identified in lung adenocarcinomas, with mutations associated with detrimental prognosis and high expression with better outcome. In conclusion, hereby we describe the presence and prognostic role of mutations in the NCOR1 gene in hormone receptor negative breast and lung adenocarcinomas, and we also confirm that NCOR1 is a tumor suppressor gene. Further studies should be performed to explore therapeutic mechanisms to restore its function

Cost Effective Use of a Thiosulfinate-Enriched Allium sativum Extract in Combination with Chemotherapy in Colon Cancer

In this work, we sought to investigate the effects of a thiosulfinate-enriched garlic extract, co-administered with 5-fluorouracil (5-FU) or oxaliplatin chemotherapy, on the viability of colon cancer cells (Caco-2 and HT-29). We also addressed the economic feasibility of a new combined treatment of this thiosulfinate-enriched garlic extract, with oxaliplatin that could reduce the dosage and costs of a monotherapy. The thiosulfinate-enriched garlic extract not only enhanced the impact of 5-FU and oxaliplatin (500 µM) in decreasing Caco-2 and HT-29 viability, but also showed a higher effect than standard 5-FU and oxaliplatin chemotherapy as anti-cancer agents. These results provided evidences for the combination of lyophilized garlic extract and 5-FU or oxaliplatin as a novel chemotherapy regimen in colon cancer cells that may also reduce the clinical therapy costs.En este trabajo, buscamos investigar los efectos de un extracto de ajo enriquecido con tiosulfinato, coadministrado con quimioterapia con 5-fluorouracilo (5-FU) u oxaliplatino, sobre la viabilidad de las células de cáncer de colon (Caco-2 y HT-29). . También abordamos la viabilidad económica de un nuevo tratamiento combinado de este extracto de ajo enriquecido con tiosulfinato, con oxaliplatino que podría reducir la dosis y los costes de una monoterapia. El extracto de ajo enriquecido con tiosulfinato no solo mejoró el impacto del 5-FU y el oxaliplatino (500 µM) en la disminución de la viabilidad de Caco-2 y HT-29, sino que también mostró un efecto mayor que el 5-FU estándar y la quimioterapia con oxaliplatino como anticancerígeno agentes

Pathogenic Microenvironment from Diabetic–Obese Visceral and Subcutaneous Adipocytes Activating Differentiation of Human Healthy Preadipocytes Increases Intracellular Fat, Effect of the Apocarotenoid Crocetin

In diabetes mellitus type 2 (DM2), developed obesity is referred to as diabesity. Implementation of a healthy diet, such as the Mediterranean, prevents diabesity. Saffron is frequently used in this diet because of its bioactive components, such as crocetin (CCT), exhibit healthful properties. It is well known that obesity, defined as an excessive accumulation of fat, leads to cardiometabolic pathology through adiposopathy or hypertrophic growth of adipose tissue (AT).This is related to an impaired adipogenic process or death of adipocytes by obesogenic signals. We aimed to evaluate the effect of the pathogenic microenvironment and CCT, activating differentiation of healthy preadipocytes (PA). For this, we used human cryopreserved PA from visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) depots obtained from healthy and obese-DM2 donors. We studied the effect of a metabolically detrimental (diabesogenic) environment, generated by obese-DM2 adipocytes from VAT (VdDM) or SAT (SdDM), on the viability and accumulation of intracellular fat of adipocytes differentiated from healthy PA, in the presence or absence of CCT (1 or 10 µM). Intracellular fat was quantified by Oil Red O staining. Cytotoxicity was measured using the MTT assay. Our results showed that diabesogenic conditions induce cytotoxicity and provide a proadipogenic environment only for visceral PA. CCT at 10 µM acted as an antiadipogenic and cytoprotective compound

Mitogen deprivation reduced glioblastoma stem-like cell survival.

<p>a-h) GSCs were deprived from mitogens for 1 to 3 days. GSCs growing in mitogen-supplemented medium were used as a control (C). a) The number of secondary neurospheres per field of view (NS/FOV) was counted after 3 days of deprivation in GSCs #1-4. b) Sox2 levels (green) were analyzed by confocal in GSCs #1-4. Nuclei were counterstained with DAPI (blue). Scale bar: 15 μm. c-d) Expression of Sox2 and Nestin as well as GADPH as a control, was evaluated by RT-PCR, while GFAP expression was analyzed by confocal analysis. Differentiation of GSC#4 was induced as described in methods. e) PI incorporation was measured by flow cytometry (10.000 events) in GSCs #1–14 and the percentage of cell death was estimated. Graph represents mean+s.d. of three independent experiments. Student’s t-test: ^***<i>P</i><0.001, ^**<i>P</i><0.01. f) Flow cytometry analysis (10.000 events) of Annexin V-FITC/Propidium Iodide (PI) staining was used to measure cell death in GSC#1. Number of cells either viable (Annexin V/PI –/–) or in early (Annexin V/PI +/–), late apoptosis (Annexin V/PI +/+) and necrotic (Annexin V/PI –/+) phases was expressed as percentage of total population. g) DNA profile was analyzed by flow cytometry with PI staining in GSC#1. Percentage of apoptotic cells was calculated based on sub-G₁ peak. Student’s t-test: ^***<i>P</i><0.001. h) Western blot analysis was conducted using the indicated antibodies in GSC#1. Each panel is representative of three independent experiments.</p

Mitogen deprivation triggers both autophagy and apoptosis.

<p>a-e) GSCs were deprived from mitogens for 1 to 3 days. GSCs growing in mitogen-supplemented medium were use as a control (C). a) Electron microscopy analysis of mitogen-starved GSC#1. <i>Left panels</i>. Cells presented morphological signs of apoptosis, including cytoplasmic blebbing, nuclear fragmentation and chromatin condensation. Scale bars: 2 μm. <i>Right panels</i>. Different stages of autophagy can be seen, such as pre-autophagosome and early autophagosome (upper panels) and late autophagosomes (autolysosomes/amphisomes, lower panels). Scale bars: 50 nm. b) Western blot analysis was conducted using the indicated antibodies in GSC#1. ** non-processed form; * processed form. c-e) LC3B puncta, Atg12 aggregates and mitochondrial cytochrome c release were analyzed by confocal microscopy. Scale bars: 5 μm (c,d) and 2.5 μm (e). f) GSC#1 were pre-treated with vehicle (DMSO) or QVD (10 μM) for 45 min and mitogen-deprived for 3 days in the absence or presence of the drug. Flow cytometry analysis (10.000 events) of Annexin V-FITC/PI staining was used to measure cell death in GSC#1. Number of cells either viable (Annexin V/PI –/–) or in early (Annexin V/PI +/–), late apoptosis (Annexin V/PI +/+) and necrotic (Annexin V/PI –/+) phases was expressed as percentage of total population. g) GSC#1 were transfected with siRNA against Beclin-1 or a control siRNA (siC) for 48 hours. Cells were mitogen-deprived as indicated and collected 5 days post-transfection. Beclin-1 protein levels were analyzed by western-blot and cell death was examined as in f). h) GSC#1 were treated with vehicle (DMSO) or Chloroquine (CQ) (25 μM, XX min) in mitogen-supplemented medium (C) and in mitogen-deprived conditions for 3 days (3d). Flow cytometry analysis (10.000 events) of Annexin V-FITC/PI staining was used to measure cell death in GSC#1 as in f). Graph represents mean+s.d. of three independent experiments. Each panel is representative of three independent experiments.</p

mTOR inhibition provokes autophagy and apoptosis in GSC.

<p>a-c) GSC#1 were treated with DMSO (–), LY294002 (LY, 10 μM), Rapamycin (RP, 50nM), PP242 (PP, 1 μM) and PI103 (PI, 10 μM). a) Protein lysates were analyzed after 1 day by western blot for the indicated antibodies. b) LC3B puncta were examined under confocal microscope after 3 days. Scale bars: 5 μm. c) Flow cytometry analysis of Annexin V-FITC/PI staining was performed as described in 2f). d-g) GSC#1 were pre-treated with vehicle (DMSO) or QVD (10 μM) for 45 min and mitogen-deprived for 3 days in the absence or presence of the drug (d-e). Alternatively, GSC#1 were transfected with siRNA against Beclin-1 or a control siRNA (siC) for 48 hours. Cells were mitogen-deprived as indicated and collected 5 days post-transfection (f-g). Protein extracts were analyzed by western blot for the indicated antibodies (d, f). ** non-processed form; * processed form. LC3B staining was examined by confocal microscopy (e, g). Scale bars: 5 μm. Graph represents mean+s.d. of three independent experiments. Each panel is representative of three independent experiments.</p

Brain endothelial cells protect GSC against mitogen deprivation-induced autophagy and apoptosis.

<p>a-d) GSC#1 were cultured for 3 days with control medium (C), deprivation medium (3d) or endothelial-derived conditioned medium (EC-CM). a) Electron microscopy analysis revealed a protective effect of EC-CM against apoptosis when compared to 3 day-starved cells. Scale bars: 1 μm. b) Protein extracts were analyzed by western blot for the indicated antibodies. ** non-processed form; * processed form. c) GSCs #1–14 were cultured for 3 days with deprivation medium or EC-CM and PI incorporation was measured by flow cytometry. Percent of cell death was represented as the mean+s.d. of three independent experiments. Student’s t-test: ^***<i>P</i><0.001, ^**<i>P</i><0.01, ^*<i>P</i><0.05. d) LC3B puncta was examined by confocal microscopy. Scale bars: 5 μm. e-f) Annexin V-FITC/PI staining (e), as well as PI staining alone (f), were used to evaluate apoptosis as described in 1f-g. Each panel is representative of three independent experiments. g) Schematic representation of the central role of mTor in regulating mitogen deprivation-induced autophagy and apoptosis in GSCs (left panel). The positive effect of endothelial factors on this pathway is illustrated (right panel).</p

Genetic mutational status of genes regulating epigenetics: Role of the histone methyltransferase KMT2D in triple negative breast tumors.

PurposeEpigenetic regulating proteins like histone methyltransferases produce variations in several functions, some of them associated with the generation of oncogenic processes. Mutations of genes involved in these functions have been recently associated with cancer, and strategies to modulate their activity are currently in clinical development.MethodsBy using data extracted from the METABRIC study, we searched for mutated genes linked with detrimental outcome in invasive breast carcinoma (n = 772). Then, we used downstream signatures for each mutated gene to associate that signature with clinical prognosis using the online tool "Genotype-2-Outcome" (http://www.g-2-o.com). Next, we performed functional annotation analyses to classify genes by functions, and focused on those associated with the epigenetic machinery.ResultsWe identified KMT2D, SETD1A and SETD2, included in the lysine methyltransferase activity function, as linked with poor prognosis in invasive breast cancer. KMT2D which codes for a histone methyltransferase that acts as a transcriptional regulator was mutated in 6% of triple negative breast tumors and found to be linked to poor survival. Genes regulated by KMT2D included RAC3, KRT23, or KRT14, among others, which are involved in cell communication and signal transduction. Finally, low expression of KMT2D at the transcriptomic level, which mirror what happens when KMT2D is mutated and functionally inactive, confirmed its prognostic value.ConclusionIn the present work, we describe epigenetic modulating genes which are found to be mutated in breast cancer. We identify the histone methyltransferase KMT2D, which is mutated in 6% of triple negative tumors and linked with poor survival