Epigenetic Mechanisms Histone Deacetylase-Dependent Regulate the Glioblastoma Angiogenic Matrisome and Disrupt Endothelial Cell Behavior In Vitro.

Glioblastoma (GBM) is the most aggressive brain tumor and different efforts have been employed in the search for new drugs and therapeutic protocols for GBM. Epitranscriptomics has shed light on new druggable Epigenetic therapies specifically designed to modulate GBM biology and behavior such as Histone Deacetylase inhibitors (iHDAC). Although the effects of iHDAC on GBM have been largely explored, there is a lack of information on the underlaying mechanisms HDAC-dependent that modulate the repertoire of GBM secreted molecules focusing on the set of Extracellular Matrix (ECM) associated proteins, the Matrisome, that may impact the surrounding tumor microenvironment. To acquire a better comprehension of the impacts of HDAC activity on the GBM Matrisome, we studied the alterations on the Matrisome-associated ECM regulators, Core Matrisome ECM glycoproteins, ECM-affiliated proteins and Proteoglycans upon HDAC inhibition in vitro as well as their relationship with glioma pathophysiological/clinical features and angiogenesis. For this, U87MG GBM cells were treated for with iHDAC or vehicle (control) and the whole secretome was processed by Mass Spectrometry NANOLC-MS/MS. In silico analyses revealed that proteins associated to the Angiogenic Matrisome (AngioMatrix), including Decorin, ADAM10, ADAM12 and ADAM15 were differentially regulated in iHDAC versus control secretome. Interestingly, genes coding for the Matrisome proteins differentially regulated were found mutated in patients and were correlated to glioma pathophysiological/clinical features. In vitro functional assays, using HBMEC endothelial cells exposed to the secretome of control or iHDAC treated GBM cells, coupled to 2D and 3D GBM cell culture system, showed impaired migratory capacity of endothelial cells and disrupted tubulogenesis in a Fibronectin and VEGF independent fashion. Collectively, our study provides understanding of epigenetic mechanisms HDAC-dependent to key Matrisomal proteins that may contribute to identify new druggable Epigenetic therapies or gliomagenesis biomarkers with relevant implications to improve therapeutic protocols for this malignancy.

O-GlcNAcylation protein disruption by Thiamet G promotes changes on the GBM U87-MG cells secretome molecular signature.

Glioblastoma (GBM) is a grade IV glioma highly aggressive and refractory to the therapeutic approaches currently in use. O-GlcNAcylation plays a key role for tumor aggressiveness and progression in different types of cancer; however, experimental evidence of its involvement in GBM are still lacking. Here, we show that O-GlcNAcylation plays a critical role in maintaining the composition of the GBM secretome, whereas inhibition of OGA activity disrupts the intercellular signaling via microvesicles. Using a label-free quantitative proteomics methodology, we identified 51 proteins in the GBM secretome whose abundance was significantly altered by activity inhibition of O-GlcNAcase (iOGA). Among these proteins, we observed that proteins related to proteasome activity and to regulation of immune response in the tumor microenvironment were consistently downregulated in GBM cells upon iOGA. While the proteins IGFBP3, IL-6 and HSPA5 were downregulated in GBM iOGA cells, the protein SQSTM1/p62 was exclusively found in GBM cells under iOGA. These findings were in line with literature evidence on the role of p62/IL-6 signaling axis in suppressing tumor aggressiveness and our experimental evidence showing a decrease in radioresistance potential of these cells. Taken together, our findings provide evidence that OGA activity may regulate the p62 and IL-6 abundance in the GBM secretome. We propose that the assessment of tumor status from the main proteins present in its secretome may contribute to the advancement of diagnostic, prognostic and even therapeutic tools to approach this relevant malignancy.

Fourier transform infrared spectroscopy of developing bone mineral: from amorphous precursor to mature crystal.

Bone mineral development has been described to proceed through an amorphous precursor prior to apatite crystallization. However, further analytical approaches are necessary to identify specific markers of amorphous mineral components in bone. Here, we establish an original Fourier transform infrared (FTIR) spectroscopy approach to allow the specific identification of the amorphous and/or crystalline nature of bone mineral. Using a series of standards, our results demonstrate that obtaining the second derivative of the FTIR spectra could reveal a peak specifically corresponding to amorphous calcium phosphate (ACP) at ∼992 cm-1. The intensity of this peak was strongly correlated to ACP content in standard mixtures. The analysis of a variety of bones showed that a clear ACP peak could be identified as a specific marker of the existence of an amorphous mineral component in developing bones. In contrast, the ACP peak was not detected in the mature bones. Moreover, subjecting developing bones to ex vivo crystallization conditions led to a clear reduction of the ACP peak, further substantiating the conversion of amorphous mineral precursor into mature apatite crystals. Analysis of mineralization in osteogenic cell cultures corroborated our observations, showing the presence of ACP as a major transient component in early mineralization, but not in the mature matrix. Additionally, FTIR imaging revealed that ACP was present in areas of matrix development, distributed around the edges of mineralizing nodules. Using an original analytical approach, this work provides strong evidence to support that bone mineral development is initiated by an amorphous precursor prior to apatite crystallization.

Emerging role of glycosylation in the polarization of tumor-associated macrophages.

Tumors are formed by several cell types interacting in a complex environment of soluble and matrix molecules. The crosstalk between the cells and extracellular components control tumor fate. Macrophages are highly plastic and diverse immune cells that are known to be key regulators of this complex network, which is mostly because they can adjust their metabolism and reprogram their phenotype and effector function. Here, we review the studies that disclose the central role of metabolism and tumor microenvironment in shaping the phenotype and function of macrophages, highlighting the importance of the hexosamine biosynthetic pathway. We further discuss growing evidence of nutrient-sensitive protein modifications such as O-GlcNAcylation and extracellular glycosylation in the function and polarization of tumor-associated macrophages.

The availability of the embryonic TGF-ß protein Nodal is dynamically regulated during glioblastoma multiforme tumorigenesis.

BACKGROUND: Glioblastoma (GBM) is the most common primary brain tumor presenting self-renewing cancer stem cells. The role of these cells on the development of the tumors has been proposed to recapitulate programs from embryogenesis. Recently, the embryonic transforming growth factor-ß (TGF-ß) protein Nodal has been shown to be reactivated upon tumor development; however, its availability in GBM cells has not been addressed so far. In this study, we investigated by an original approach the mechanisms that dynamically control both intra and extracellular Nodal availability during GBM tumorigenesis. METHODS: We characterized the dynamics of Nodal availability in both stem and more differentiated GBM cells through morphological analysis, immunofluorescence of Nodal protein and of early (EEA1 and Rab5) and late (Rab7 and Rab11) endocytic markers and Western Blot. Tukey's test was used to analyze the prevalent correlation of Nodal with different endocytic markers inside specific differentiation states, and Sidak's multiple comparisons test was used to compare the prevalence of Nodal/endocytic markers co-localization between two differentiation states of GBM cells. Paired t test was used to analyze the abundance of Nodal protein, in extra and intracellular media. RESULTS: The cytoplasmic distribution of Nodal was dynamically regulated and strongly correlated with the differentiation status of GBM cells. While Nodal-positive vesicle-like particles were symmetrically distributed in GBM stem cells (GBMsc), they presented asymmetric perinuclear localization in more differentiated GBM cells (mdGBM). Strikingly, when subjected to dedifferentiation, the distribution of Nodal in mdGBM shifted to a symmetric pattern. Moreover, the availability of both intracellular and secreted Nodal were downregulated upon GBMsc differentiation, with cells becoming elongated, negative for Nodal and positive for Nestin. Interestingly, the co-localization of Nodal with endosomal vesicles also depended on the differentiation status of the cells, with Nodal seen more packed in EEA1/Rab5 + vesicles in GBMsc and more in Rab7/11 + vesicles in mdGBM. CONCLUSIONS: Our results show for the first time that Nodal availability relates to GBM cell differentiation status and that it is dynamically regulated by an endocytic pathway during GBM tumorigenesis, shedding new light on molecular pathways that might emerge as putative targets for Nodal signaling in GBM therapy.

Type I interferon response in astrocytes promotes brain metastasis by enhancing monocytic myeloid cell recruitment.

Cancer metastasis to the brain is a significant clinical problem. Metastasis is the consequence of favorable interactions between invaded cancer cells and the microenvironment. Here, we demonstrate that cancer-activated astrocytes create a sustained low-level activated type I interferon (IFN) microenvironment in brain metastatic lesions. We further confirm that the IFN response in astrocytes facilitates brain metastasis. Mechanistically, IFN signaling in astrocytes activates C-C Motif Chemokine Ligand 2 (CCL2) production, which further increases the recruitment of monocytic myeloid cells. The correlation between CCL2 and monocytic myeloid cells is confirmed in clinical brain metastasis samples. Lastly, genetically or pharmacologically inhibiting C-C Motif Chemokine Receptor 2 (CCR2) reduces brain metastases. Our study clarifies a pro-metastatic effect of type I IFN in the brain even though IFN response has been considered to have anti-tumor effects. Moreover, this work expands our understandings on the interactions between cancer-activated astrocytes and immune cells in brain metastasis.

Live Cell Imaging Supports a Key Role for Histone Deacetylase as a Molecular Target during Glioblastoma Malignancy Downgrade through Tumor Competence Modulation.

Glioblastoma (GBM) is the most aggressive tumor of the central nervous system, and the identification of the mechanisms underlying the biological basis of GBM aggressiveness is essential to develop new therapies. Due to the low prognosis of GBM treatment, different clinical studies are in course to test the use of histone deacetylase inhibitors (iHDACs) in anticancer cocktails. Here, we seek to investigate the impact of HDAC activity on GBM cell behavior and plasticity by live cell imaging. We pharmacologically knock down HDAC activity using two different inhibitors (TSA and SAHA) in two different tumor cell types: a commercial GBM cell line (U87-MG) and primary tumor (GBM011). Upon 72 hours of in vitro iHDAC treatment, GBM cells presented a very unusual elongated cell shape due to tunneling tube formation and independent on TGF-ß signaling epithelial to mesenchymal transition. Live cell imaging revealed that voltage-sensitive Ca++ signaling was disrupted upon HDAC activity blockade. This behavior was coupled to vimentin and connexin 43 gene expression downregulation, suggesting that HDAC activity blockade downgrades GBM aggressiveness mostly due to tumor cell competence and plasticity modulation in vitro. To test this hypothesis and access whether iHDACs would modulate tumor cell behavior and plasticity to properly respond to environmental cues in vivo, we xenografted GBM oncospheres in the chick developing the neural tube. Remarkably, upon 5 days in the developing neural tube, iHDAC-treated GBM cells ectopically expressed HNK-1, a tumor-suppressor marker tightly correlated to increased survivor of patients. These results describe, for the first time in the literature, the relevance of iHDACs for in vivo tumor cell morphology and competence to properly respond to environmental cues. Ultimately, our results highlight the relevance of chromatin remodeling for tumor cell plasticity and shed light on clinical perspectives aiming the epigenome as a relevant therapeutic target for GBM therapy.

Epigenetic Control of Macrophage Shape Transition towards an Atypical Elongated Phenotype by Histone Deacetylase Activity.

Inflammatory chronic pathologies are complex processes characterized by an imbalance between the resolution of the inflammatory phase and the establishment of tissue repair. The main players in these inflammatory pathologies are bone marrow derived monocytes (BMDMs). However, how monocyte differentiation is modulated to give rise to specific macrophage subpopulations (M1 or M2) that may either maintain the chronic inflammatory process or lead to wound healing is still unclear. Considering that inhibitors of Histone Deacetylase (HDAC) have an anti-inflammatory activity, we asked whether this enzyme would play a role on monocyte differentiation into M1 or M2 phenotype and in the cell shape transition that follows. We then induced murine bone marrow progenitors into monocyte/macrophage differentiation pathway using media containing GM-CSF and the HDAC blocker, Trichostatin A (TSA). We found that the pharmacological inhibition of HDAC activity led to a shape transition from the typical macrophage pancake-like shape into an elongated morphology, which was correlated to a mixed M1/M2 profile of cytokine and chemokine secretion. Our results present, for the first time, that HDAC activity acts as a regulator of macrophage differentiation in the absence of lymphocyte stimuli. We propose that HDAC activity down regulates macrophage plasticity favoring the pro-inflammatory phenotype.