Sphingolipid distribution at mitochondria-associated membranes (MAMs) upon induction of apoptosis.

The levels and composition of sphingolipids and related metabolites are altered in aging and in common disorders such as diabetes and cancers, as well as in neurodegenerative, cardiovascular, and respiratory diseases. Changes in sphingolipids have been implicated as being an essential step in mitochondria-driven cell death. However, little is known about the precise sphingolipid composition and modulation in mitochondria or related organelles. Here, we used LC-MS/MS to analyze the presence of key components of the ceramide metabolic pathway in vivo and in vitro in purified ER, mitochondria-associated membranes (MAMs), and mitochondria. Specifically, we analyzed the sphingolipids in the three pathways that generate ceramide: sphinganine in the de novo ceramide pathway, SM in the breakdown pathway, and sphingosine in the salvage pathway. We observed sphingolipid profiles in mouse liver, mouse brain, and a human glioma cell line (U251). We analyzed the quantitative and qualitative changes of these sphingolipids during staurosporine-induced apoptosis in U251 cells. Ceramide (especially C16-ceramide) levels increased during early apoptosis possibly through a conversion from mitochondrial sphinganine and SM, but sphingosine and lactosyl- and glycosyl-ceramide levels were unaffected. We also found that ceramide generation is enhanced in mitochondria when SM levels are decreased in the MAM. This decrease was associated with an increase in acid sphingomyelinase activity in MAM. We conclude that meaningful sphingolipid modifications occur in MAM, the mitochondria, and the ER during the early steps of apoptosis.

Cytosine methylation of mature microRNAs inhibits their functions and is associated with poor prognosis in glioblastoma multiforme.

BACKGROUND: Literature reports that mature microRNA (miRNA) can be methylated at adenosine, guanosine and cytosine. However, the molecular mechanisms involved in cytosine methylation of miRNAs have not yet been fully elucidated. Here we investigated the biological role and underlying mechanism of cytosine methylation in miRNAs in glioblastoma multiforme (GBM). METHODS: RNA immunoprecipitation with the anti-5methylcytosine (5mC) antibody followed by Array, ELISA, dot blot, incorporation of a radio-labelled methyl group in miRNA, and miRNA bisulfite sequencing were perfomred to detect the cytosine methylation in mature miRNA. Cross-Linking immunoprecipiation qPCR, transfection with methylation/unmethylated mimic miRNA, luciferase promoter reporter plasmid, Biotin-tagged 3'UTR/mRNA or miRNA experiments and in vivo assays were used to investigate the role of methylated miRNAs. Finally, the prognostic value of methylated miRNAs was analyzed in a cohorte of GBM pateints. RESULTS: Our study reveals that a significant fraction of miRNAs contains 5mC. Cellular experiments show that DNMT3A/AGO4 methylated miRNAs at cytosine residues inhibit the formation of miRNA/mRNA duplex and leading to the loss of their repressive function towards gene expression. In vivo experiments show that cytosine-methylation of miRNA abolishes the tumor suppressor function of miRNA-181a-5p miRNA for example. Our study also reveals that cytosine-methylation of miRNA-181a-5p results is associated a poor prognosis in GBM patients. CONCLUSION: Together, our results indicate that the DNMT3A/AGO4-mediated cytosine methylation of miRNA negatively.

Mitochondria transfer from tumor-activated stromal cells (TASC) to primary Glioblastoma cells.

The tumor microenvironment (TME) controls many aspects of cancer development but little is known about its effect in Glioblastoma (GBM), the main brain tumor in adults. Tumor-activated stromal cell (TASC) population, a component of TME in GBM, was induced in vitro by incubation of MSCs with culture media conditioned by primary cultures of GBM under 3D/organoid conditions. We observed mitochondrial transfer by Tunneling Nanotubes (TNT), extracellular vesicles (EV) and cannibalism from the TASC to GBM and analyzed its effect on both proliferation and survival. We created primary cultures of GBM or TASC in which we have eliminated mitochondrial DNA [Rho 0 (ρ0) cells]. We found that TASC, as described in other cancers, increased GBM proliferation and resistance to standard treatments (radiotherapy and chemotherapy). We analyzed the incorporation of purified mitochondria by ρ0 and ρ+ cells and a derived mathematical model taught us that ρ+ cells incorporate more rapidly pure mitochondria than ρ0 cells.

Low-Dose Pesticide Mixture Induces Accelerated Mesenchymal Stem Cell Aging In Vitro.

The general population is chronically exposed to multiple environmental contaminants such as pesticides. We have previously demonstrated that human mesenchymal stem cells (MSCs) exposed in vitro to low doses of a mixture of seven common pesticides showed a permanent phenotype modification with a specific induction of an oxidative stress-related senescence. Pesticide mixture also induced a shift in MSC differentiation toward adipogenesis. Thus, we hypothesized that common combination of pesticides may induce a premature cellular aging of adult MSCs. Our goal was to evaluate if the prolonged exposure to pesticide mixture could accelerate aging-related markers and in particular deteriorate the immunosuppressive properties of MSCs. MSCs exposed to pesticide mixture, under long-term culture and obtained from aging donor, were compared by bulk RNA sequencing analysis. Aging, senescence, and immunomodulatory markers were compared. The protein expression of cellular aging-associated metabolic markers and immune function of MSCs were analyzed. Functional analysis of the secretome impacts on immunomodulatory properties of MSCs was realized after 21 days' exposure to pesticide mixture. The RNA sequencing analysis of MSCs exposed to pesticide showed some similarities with cells from prolonged culture, but also with the MSCs of an aged donor. Changes in the metabolic markers MDH1, GOT and SIRT3, as well as an alteration in the modulation of active T cells and modifications in cytokine production are all associated with cellular aging. A modified functional profile was found with similarities to aging process. Stem Cells 2019;37:1083-1094.

Low-Dose Pesticide Mixture Induces Senescence in Normal Mesenchymal Stem Cells (MSC) and Promotes Tumorigenic Phenotype in Premalignant MSC.

Humans are chronically exposed to multiple environmental pollutants such as pesticides with no significant evidence about the safety of such poly-exposures. We exposed mesenchymal stem cells (MSC) to very low doses of mixture of seven pesticides frequently detected in food samples for 21 days in vitro. We observed a permanent phenotype modification with a specific induction of an oxidative stress-related senescence. Pesticide mixture also induced a shift in MSC differentiation towards adipogenesis but did not initiate a tumorigenic transformation. In modified MSC in which a premalignant phenotype was induced, the exposure to pesticide mixture promoted tumorigenic phenotype both in vitro and in vivo after cell implantation, in all nude mice. Our results suggest that a common combination of pesticides can induce a premature ageing of adult MSC, and as such could accelerate age-related diseases. Exposure to pesticide mixture may also promote the tumorigenic transformation in a predisposed stromal environment. Abstract Video Link: https://youtu.be/mfSVPTol-Gk Stem Cells 2017;35:800-811.

Pharmacological targeting of apelin impairs glioblastoma growth.

Glioblastoma are highly aggressive brain tumours that are associated with an extremely poor prognosis. Within these tumours exists a subpopulation of highly plastic self-renewing cancer cells that retain the ability to expand ex vivo as tumourspheres, induce tumour growth in mice, and have been implicated in radio- and chemo-resistance. Although their identity and fate are regulated by external cues emanating from endothelial cells, the nature of such signals remains unknown. Here, we used a mass spectrometry proteomic approach to characterize the factors released by brain endothelial cells. We report the identification of the vasoactive peptide apelin as a central regulator for endothelial-mediated maintenance of glioblastoma patient-derived cells with stem-like properties. Genetic and pharmacological targeting of apelin cognate receptor abrogates apelin- and endothelial-mediated expansion of glioblastoma patient-derived cells with stem-like properties in vitro and suppresses tumour growth in vivo. Functionally, selective competitive antagonists of apelin receptor were shown to be safe and effective in reducing tumour expansion and lengthening the survival of intracranially xenografted mice. Therefore, the apelin/apelin receptor signalling nexus may operate as a paracrine signal that sustains tumour cell expansion and progression, suggesting that apelin is a druggable factor in glioblastoma.

HB-EGF is associated with DNA damage and Mcl-1 turnover in human glioma cell lines treated by Temozolomide.

Temozolomide (TMZ) is the main chemotherapeutic agent used for treating newly diagnosed Glioblastoma Multiforme (GBM), the most frequent malignant brain tumors in adults. This alkylating agent induces DNA double strand breaks (DSBs) which in turn lead to apoptosis by activating the Bcl-2 controlled mitochondrial pathway. However, GBM invariably recur as tumors become resistant to TMZ. We investigated the implication of EGFR ligands in this resistance and we found that the pro Heparin Binding Epidermal Growth Factor (proHB-EGF) expression is linked to the early response to TMZ in human glioma cell lines. However, HB-EGF does not affect apoptosis per se although its expression is associated with the degradation of Mcl-1. HB-EGF is implicated in DSBs repair as silencing of HB-EGF increased γH2AX foci half-life as well as USP9X expression, two features that could be linked to the observed effect on Mcl-1. Our data demonstrate a new role for HB-EGF in TMZ treated cell lines.

Differentiation-related response to DNA breaks in human mesenchymal stem cells.

We have recently shown that the in vitro differentiation of human mesenchymal stem cells (hMSCs) was accompanied by an increased sensitivity toward apoptosis; however, the mechanism responsible for this shift is not known. Here, we show that the repair of DNA double-strand breaks (DSBs) was more rapid in undifferentiated hMSCs than in differentiated osteoblasts by quantification of the disappearance of γ-H2AX foci in the nuclei after γ-irradiation-induced DNA damage. In addition, there was a marked and prolonged increase in the level of nuclear Ku70 and an increased phosphorylation of DNA-PKcs. This was accompanied by an augmentation in the phosphorylation of ATM in hMSCs post-irradiation suggesting the nonhomologous end joining repair mechanism. However, when hMSCs were induced to differentiate along the osteogenic or adipogenic pathways; irradiation of these cells caused an expeditious and robust cell death, which was primarily apoptotic. This was in sharp contrast to undifferentiated hMSCs, which were highly resistant to irradiation and/or temozolomide-induced DSBs. In addition, we observed a 95% recovery from DSB in these cells. Our results suggest that apoptosis and DNA repair are major safeguard mechanisms in the control of hMSCs differentiation after DNA damage.

Comparison of spheroids formed by rat glioma stem cells and neural stem cells reveals differences in glucose metabolism and promising therapeutic applications.

Cancer stem cells (CSCs) are thought to be partially responsible for cancer resistance to current therapies and tumor recurrence. Dichloroacetate (DCA), a compound capable of shifting metabolism from glycolysis to glucose oxidation, via an inhibition of pyruvate dehydrogenase kinase was used. We show that DCA is able to shift the pyruvate metabolism in rat glioma CSCs but has no effect in rat neural stem cells. DCA forces CSCs into oxidative phosphorylation but does not trigger the production of reactive oxygen species and consecutive anti-cancer apoptosis. However, DCA, associated with etoposide or irradiation, induced a Bax-dependent apoptosis in CSCs in vitro and decreased their proliferation in vivo. The former phenomenon is related to DCA-induced Foxo3 and p53 expression, resulting in the overexpression of BH3-only proteins (Bad, Noxa, and Puma), which in turn facilitates Bax-dependent apoptosis. Our results demonstrate that a small drug available for clinical studies potentiates the induction of apoptosis in glioma CSCs.

Spatial Distribution of Immune Cells in Primary and Recurrent Glioblastoma: A Small Case Study.

Only a minority of patients with glioblastoma (GBM) respond to immunotherapy, and always only partially. There is a lack of knowledge on immune distribution in GBM and in its tumor microenvironment (TME). To address the question, we used paired primary and recurrent tumors from GBM patients to study the composition and the evolution of the immune landscape upon treatment. We studied the expression of a handful of immune markers (CD3, CD8, CD68, PD-L1 and PD-1) in GBM tissues in 15 paired primary and recurrent GBM. In five selected patients, we used Nanostring Digital Spatial Profiling (DSP) to obtain simultaneous assessments of multiple biomarkers both within the tumor and the microenvironment in paired primary and recurrent GBM. Our results suggest that the evolution of the immune landscape between paired primary and recurrent GBM tumors is highly heterogeneous. However, our study identifies B3-H7 and HLA-DR as potential targets in primary and recurrent GBM. Spatial profiling of immune markers from matched primary and recurrent GBM shows a nonlinear complex evolution during the progression of cancer. Nonetheless, our study demonstrated a global increase in macrophages, and revealed differential localization of some markers, such as B7-H3 and HLA-DR, between GBM and its TME.

A Simple 3D Cell Culture Method for Studying the Interactions between Human Mesenchymal Stromal/Stem Cells and Patients Derived Glioblastoma.

We have developed a 3D biosphere model using patient-derived cells (PDCs) from glioblastoma (GBM), the major form of primary brain tumors in adult, plus cancer-activated fibroblasts (CAFs), obtained by culturing mesenchymal stem cells with GBM conditioned media. The effect of MSC/CAFs on the proliferation, cell-cell interactions, and response to treatment of PDCs was evaluated. Proliferation in the presence of CAFs was statistically lower but the spheroids formed within the 3D-biosphere were larger. A treatment for 5 days with Temozolomide (TMZ) and irradiation, the standard therapy for GBM, had a marked effect on cell number in monocultures compared to co-cultures and influenced cancer stem cells composition, similar to that observed in GBM patients. Mathematical analyses of spheroids growth and morphology confirm the similarity with GBM patients. We, thus, provide a simple and reproducible method to obtain 3D cultures from patient-derived biopsies and co-cultures with MSC with a near 100% success. This method provides the basis for relevant in vitro functional models for a better comprehension of the role of tumor microenvironment and, for precision and/or personalized medicine, potentially to predict the response to treatments for each GBM patient.

Challenges in glioblastoma research: focus on the tumor microenvironment.

Glioblastoma (GBM) is the most deadly type of malignant brain tumor, despite extensive molecular analyses of GBM cells. In recent years, the tumor microenvironment (TME) has been recognized as an important player and therapeutic target in GBM. However, there is a need for a full and integrated understanding of the different cellular and molecular components involved in the GBM TME and their interactions for the development of more efficient therapies. In this review, we provide a comprehensive report of the GBM TME, which assembles the contributions of physicians and translational researchers working on brain tumor pathology and therapy in France. We propose a holistic view of the subject by delineating the specific features of the GBM TME at the cellular, molecular, and therapeutic levels.

The Activation of Mesenchymal Stem Cells by Glioblastoma Microvesicles Alters Their Exosomal Secretion of miR-100-5p, miR-9-5p and let-7d-5p.

Glioblastoma (GBM) is the most aggressive brain tumor, and despite initial response to chemo- and radio-therapy, the persistence of glioblastoma stem cells (GSCs) unfortunately always results in tumor recurrence. It is now largely admitted that tumor cells recruit normal cells, including mesenchymal stem cells (MSCs), and components of their environment, to participate in tumor progression, building up what is called the tumor microenvironment (TME). While growth factors and cytokines constitute essential messengers to pass on signals between tumor and TME, recent uncovering of extracellular vesicles (EVs), composed of microvesicles (MVs) and exosomes, opened new perspectives to define the modalities of this communication. In the GBM context particularly, we investigated what could be the nature of the EV exchange between GSCs and MSCs. We show that GSCs MVs can activate MSCs into cancer-associated fibroblasts (CAFs)-like cells, that subsequently increase their secretion of exosomes. Moreover, a significant decrease in anti-tumoral miR-100-5p, miR-9-5p and let-7d-5p was observed in these exosomes. This clearly suggests a miRNA-mediated GBM tumor promotion by MSCs exosomes, after their activation by GBM MVs.

Three-dimensional in vitro culture models in oncology research.

Cancer is a multifactorial disease that is responsible for 10 million deaths per year. The intra- and inter-heterogeneity of malignant tumors make it difficult to develop single targeted approaches. Similarly, their diversity requires various models to investigate the mechanisms involved in cancer initiation, progression, drug resistance and recurrence. Of the in vitro cell-based models, monolayer adherent (also known as 2D culture) cell cultures have been used for the longest time. However, it appears that they are often less appropriate than the three-dimensional (3D) cell culture approach for mimicking the biological behavior of tumor cells, in particular the mechanisms leading to therapeutic escape and drug resistance. Multicellular tumor spheroids are widely used to study cancers in 3D, and can be generated by a multiplicity of techniques, such as liquid-based and scaffold-based 3D cultures, microfluidics and bioprinting. Organoids are more complex 3D models than multicellular tumor spheroids because they are generated from stem cells isolated from patients and are considered as powerful tools to reproduce the disease development in vitro. The present review provides an overview of the various 3D culture models that have been set up to study cancer development and drug response. The advantages of 3D models compared to 2D cell cultures, the limitations, and the fields of application of these models and their techniques of production are also discussed.

Cellular Heterogeneity and Cooperativity in Glioma Persister Cells Under Temozolomide Treatment.

We have observed a drug-tolerant/persister state in a human glioblastoma (GBM) cell line after exposure to temozolomide, the standard-of-care chemotherapeutic agent for GBM. We used a multicolor lentiviral genetic barcode labeling to follow cell population evolution during temozolomide treatment. We observed no change in the distribution of the different colored populations of cells in persister or resistant cells suggesting that pre-existing minor subpopulations, which would be expected to be restricted to a single color, were not amplified/selected during the response to the drug. We have previously identified four genes (CHI3L1, FAT2, KLK5, and HB-EGF) that were over-expressed during the persister stage. Single-cell analysis of these four genes indicated that they were expressed in different individual cells ruling out the existence of a single persister-specific clone but suggesting rather a global answer. Even so, the transitory silencing of CHI3L1, FAT2, or KLK5 influenced the expression of the other three genes and the survival of U251 cells in absence of temozolomide. Since proteins encoded by the four genes are all localized in the extracellular matrix or interact within the extracellular compartment, we propose that cellular interactions and communications are important during the persister stage before the acquisition of chemo-resistance. Thus, persisters might be a new therapeutically relevant target in GBM.

Proximity ligation in situ assay for monitoring the global DNA methylation in cells.

BACKGROUND: DNA methylation has a central role in the epigenetic control of mammalian gene expression, and is required for X inactivation, genomics imprinting and silencing of retrotransposons and repetitive sequences. Thus, several technologies have been developed to measure the degree of DNA methylation. RESULTS: We here present the development of the detection of protein-protein interactions via the adaptation of the proximity ligation in situ technology to evaluate the DNA methylation status in cells since the quantification of Dnmt1/PCNA interaction in cells reflects the degree of DNA methylation. CONCLUSION: This method being directly realizable on cells, it appears that it could suggest a wide range of applications in basic research and drug development. More particularly, this method is specially adapted for the investigations realized from a weak quantity of biologic materiel such as stem cells or primary cultured tumor cells for examples.

Increase in intracellular PGE2 induces apoptosis in Bax-expressing colon cancer cell.

BACKGROUND: NSAIDs exhibit protective properties towards some cancers, especially colon cancer. Yet, it is not clear how they play their protective role. PGE2 is generally shown as the only target of the NSAIDs anticancerous activity. However, PGE2 known targets become more and more manifold, considering both the molecular pathways involved and the target cells in the tumour. The role of PGE2 in tumour progression thus appears complex and multipurpose. METHODS: To gain understanding into the role of PGE2 in colon cancer, we focused on the activity of PGE2 in apoptosis in colon cancer cell lines. RESULTS: We observed that an increase in intracellular PGE2 induced an apoptotic cell death, which was dependent on the expression of the proapoptotic protein Bax. This increase was induced by increasing PGE2 intracellular concentration, either by PGE2 microinjection or by the pharmacological inhibition of PGE2 exportation and enzymatic degradation. CONCLUSIONS: We present here a new sight onto PGE2 in colon cancer cells opening the way to a new prospective therapeutic strategy in cancer, alternative to NSAIDs.

TOM20-mediated transfer of Bcl2 from ER to MAM and mitochondria upon induction of apoptosis.

In this work, we have explored the subcellular localization of Bcl2, a major antiapoptotic protein. In U251 glioma cells, we found that Bcl2 is localized mainly in the ER and is translocated to MAM and mitochondria upon induction of apoptosis; this mitochondrial transfer was not restricted to the demonstrator cell line, even if cell-specific modulations exist. We found that the Bcl2/mitochondria interaction is controlled by TOM20, a protein that belongs to the protein import machinery of the mitochondrial outer membrane. The expression of a small domain of interaction of TOM20 with Bcl2 potentiates its anti-apoptotic properties, which suggests that the Bcl2-TOM20 interaction is proapoptotic. The role of MAM and TOM20 in Bcl2 apoptotic mitochondrial localization and function has been confirmed in a yeast model in which the ER-mitochondria encounter structure (ERMES) complex (required for MAM stability in yeast) has been disrupted. Bcl2-TOM20 interaction is thus an additional player in the control of apoptosis.

C-terminal residues regulate localization and function of the antiapoptotic protein Bfl-1.

Unlike other antiapoptotic members of the Bcl-2 family, Bfl-1 does not contain a well defined C-terminal transmembrane domain, and whether the C-terminal tail of Bfl-1 functions as a membrane anchor is not yet clearly established. The molecular modeling study of the full-length Bfl-1 performed within this work suggests that Bfl-1 may co-exist in two distinct conformational states: one in which its C-terminal helix alpha9 is inserted in the hydrophobic groove formed by the BH1-3 domains of Bfl-1 and one with its C terminus. Parallel analysis of the subcellular localization of Bfl-1 indicates that even if Bfl-1 may co-exist in two distinct conformational states, most of the endogenous protein is tightly associated with the mitochondria by its C terminus in both healthy and apoptotic peripheral blood lymphocytes as well as in malignant B cell lines. However, the helix alpha9 of Bfl-1, and therefore the binding of Bfl-1 to mitochondria, is not absolutely required for the antiapoptotic activity of Bfl-1. A particular feature of Bfl-1 is the amphipathic character of its C-terminal helix alpha9. Our data clearly indicate that this property of helix alpha9 is required for the anchorage of Bfl-1 to the mitochondria but also regulates the antiapoptotic function Bfl-1.

Folate supplementation limits the aggressiveness of glioma via the remethylation of DNA repeats element and genes governing apoptosis and proliferation.

PURPOSE: We have investigated whether the folate supplementation could be used to limit the aggressiveness of glioma through the DNA remethylation because (a) the cancer genome is characterized by a low level of DNA methylation (or 5-methylcytosine, 5 mC); and (b) folate is the main generator of S-adenosyl-methionine, the methyl donor molecule in the DNA methylation reaction catalyzed by the DNA methyltranferases. EXPERIMENTAL DESIGN: The effects of folate supplementations were analyzed on the global DNA methylation status, the methylation status of DNA repeat element, the sensitivity of temozolomide-induced apoptosis, and the proliferation index of glioma cells. Finally, we analyzed whether the DNA methylation level could be used as a prognostic factor and/or a biomarker in an antiglioma therapy using folate supplementation as an adjuvant. RESULTS: Our data show that gliomagenesis is accompanied by a reduction in 5 mC levels and that this low level of 5 mC is a poor prognostic factor in Glioblastoma Multiforme patients. We also show that folate supplementation enhanced the DNA remethylation through the Sp1/Sp3-mediated transcriptional up-regulation of genes coding for Dnmt3a and Dnmt3b proteins, two de novo methyltranferases. Finally, we show that the folate-induced DNA methylation limits proliferation and increases the sensitivity to temozolomide-induced apoptosis in glioma cells through methylation of the genes implicated in these processes (PDGF-B, MGMT, survivin, and bcl-w). CONCLUSION: This study suggests that folate supplementation could be a promising adjuvant for the future design of antiglioma therapies in preclinical and/or clinical studies.