Dataset Information

O-GlcNAcase activity is necessary for the molecular signature of the Glioblastoma secretome

ABSTRACT: Glioblastoma (GBM) is a grade IV glioma highly aggressive and refractory to the therapeutic approaches currently in use nowadays. Although sugar metabolism through the hexosamine biosynthetic pathway (HBP) plays a key role in tumor aggressiveness and progression in different types of cancer, whether the HBP might be a targetable strategy for GBM is still lacking experimental evidence. Here we show that the HBP enzyme O-GlcNAcase (OGA) plays a critical role in GBM secretome signature. Using a label-free quantitative proteomics methodology, we identified 51 proteins in the GBM secretome whose abundance was significantly altered. Among these proteins, we observed that proteins related to proteasome activity and autophagy were consistently down-regulated in GBM cells upon OGA activity inhibition (iOGA). While the proteins IGFBP3 and HSPA5 were down regulated in iOGA GBM cells, the protein SQSTM1/p62 was a only identified in iOGA GBM cells. These in silico findings were in line with experimental evidence showing a decrease in autophagy in iOGA cells followed by a decrease in radio-resistance. Taken together our findings consistently bridge the protein profile in the secretome with functional evidence of autophagy regulation in GBM by the OGA activity. 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.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

DISEASE(S): Glioblastoma

SUBMITTER: Joseph Evaristo

LAB HEAD: Fábio César Sousa Nogueira

PROVIDER: PXD019496 | Pride | 2021-09-09

REPOSITORIES: Pride

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Publications

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

Oliveira-Nunes Maria Cecilia MC Julião Glaucia G Menezes Aline A Mariath Fernanda F Hanover John A JA Evaristo Joseph Albert Medeiros JAM Nogueira Fábio César Sousa FCS Dias Wagner Barbosa WB de Abreu Pereira Denise D Carneiro Katia K

Clinical proteomics 20210426 1

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 ...[more]

PMID: 33902430

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Project description:Glioblastoma (GBM), the most malignant primary brain tumor, is characterized by widespread heterogeneity, leading to poor and unpredictable clinical outcomes. Recent studies have provided evidences that the tumor microenvironment has a critical role in regulating tumor growth by establishing a complex network of interactions with tumor cells. Here we investigate how GBM cells modulate glial cells, and how this modulation can influence back on the malignant phenotype of GBM cells. Primary mouse glial cultures were established and cultured in serum-free media (unprimed) or exposed to secretome derived from GL261 GBM cells (primed). Conditioned media (CM) from each glial culture were collected and a proteomic analysis was conducted. Glial cells CM (unprimed and primed) were also used in GL261 GBM cells to evaluate its impact in critical hallmarks of GBM cells, including viability, migration, and activation of tumor-related intracellular signaling pathways. The proteomic analysis revealed that the pre-exposure of glial cells to CM from GBM cells led to the upregulation of several proteins related to inflammatory response, cell adhesion and extracellular structure organization within the secretome of primed glial cells, consistent with a pattern of reactive astrogliosis. At the functional levels, CM derived from unprimed glial cells favored an increase in cell migration capacity, while CM from primed glial cells was more efficient in promoting GBM cells viability. These effects on GBM cells were accompanied by activation of particular intracellular cancer-related pathways, mainly the MAPK/ERK pathway, which is a known regulator of cell proliferation. Together, our results demonstrate that glial cells can have a different impact on the progression of GBM tumors, suggesting that the secretome of GBM cells is able to modulate the secretome of neighboring glial cells, in a way that regulates the “go-or-grow” phenotypic switch of GBM cells. Together, our results suggest that glial cells can impact on the pathophysiology of GBM tumors, and that the secretome of GBM cells is able to modulate the secretome of neighboring glial cells, in a way that regulates the “go-or-grow” phenotypic switch of GBM cells.

Project description:Glioblastoma (GBM) carries a dismal prognosis largely due to acquired resistance to the standard treatment, which incorporates the chemotherapy temozolomide (TMZ). Inhibiting the proteasomal pathway is an emerging strategy, where combination treatments are under clinical investigation. We hypothesized that pre-treatment of GBM with bortezomib (BTZ) might sensitize glioblastoma to TMZ by abolishing autophagy survival signals to augment DNA damage and apoptosis. P3 patient-derived GBM cells as well as the tumor cell lines U87, HF66, A172 and T98G were investigated for clonogenic survival after single or combined treatment with TMZ and BTZ in vitro. Change in autophagic flux was examined after experimental treatments in conjunction with inhibitors of autophagy or downregulation of autophagy-related genes -5 and -7 (ATG5 and ATG7, respectively). Autophagic flux was increased in TMZ-resistant P3 and T98G cells as indicated by diminished levels of the autophagy markers LC3A/B-II and increased STX17, higher protein degradation and no formation of p62 bodies nor induction of apoptosis. In contrast, BTZ treatment attenuated ULK1 mRNA, total and phosphorylated protein, and accumulated LC3A/B-II, p62 and autophagosomes analogously to Baf1 and chloroquine autophagy inhibitors. These autophagosomes did not fuse with lysosomes, indicated by attenuated STX17 expression and reduced degradation of long-lived proteins, which culminated in enhanced caspase-3/8 dependent apoptosis. BTZ synergistically enhanced TMZ efficacy, attenuated tumor cell proliferation, triggered ATM/Chk2 DNA damage signalling to further augment caspase-3/8 mediated apoptosis in the TMZ resistant P3 and T98G GBM cells. Genetic or chemical inhibition of autophagy (with CRISPR-CAs9 ATG5, ATG7 shRNA, MRT68921 or VPS34-IN1) abrogated BTZ efficacy and rescued BTZ+ TMZ treated GBM cells from death. We conclude that Bortezomib ameliorates temozolomide resistance through ATG5/7-dependent abrogated autophagic flux and may be amenable in combination treatment regimens for TMZ refractory GBM patients.

Dataset Information

O-GlcNAcase activity is necessary for the molecular signature of the Glioblastoma secretome

Publications

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

Similar Datasets

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