RESUMO
GRP94 is an ATP-dependent chaperone able to regulate pro-oncogenic signaling pathways. Previous studies have shown a critical role of GRP94 in brain metastasis (BrM) pathogenesis and progression. In this work, an untargeted lipidomic analysis revealed that some lipid species were altered in GRP94-deficient cells, specially GM2 and GM3 gangliosides. The catalytic pathway of GM2 is affected by the low enzymatic activity of ß-Hexosaminidase (HexA), responsible for the hydrolysis of GM2 to GM3. Moreover, a deficiency of the GM2-activator protein (GM2-AP), the cofactor of HexA, is observed without alteration of gene expression, indicating a post-transcriptional alteration of GM2-AP in the GRP94-ablated cells. One plausible explanation of these observations is that GM2-AP is a client of GRP94, resulting in defective GM2 catabolic processing and lysosomal accumulation of GM2 in GRP94-ablated cells. Overall, given the role of gangliosides in cell surface dynamics and signaling, their imbalance might be linked to modifications of cell behaviour acquired in BrM progression. This work indicates that GM2-AP could be an important factor in ganglioside balance maintenance. These findings highlight the relevance of GM3 and GM2 gangliosides in BrM and reveal GM2-AP as a promising diagnosis and therapeutic target in BrM research.
Assuntos
Neoplasias Encefálicas/secundário , Carcinoma/secundário , Proteína Ativadora de G(M2)/biossíntese , Gangliosídeo G(M2)/análise , Gangliosídeo G(M3)/análise , Glicoproteínas de Membrana/fisiologia , Proteínas de Neoplasias/fisiologia , Animais , Neoplasias Encefálicas/metabolismo , Carcinoma/metabolismo , Linhagem Celular Tumoral , Meios de Cultivo Condicionados/química , Regulação para Baixo , Feminino , Proteína Ativadora de G(M2)/genética , Regulação Neoplásica da Expressão Gênica , Genes Reporter , Humanos , Lipidômica , Lisossomos/metabolismo , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/genética , Camundongos , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Neoplasias de Mama Triplo Negativas/patologia , Cadeia alfa da beta-Hexosaminidase/biossíntese , Cadeia alfa da beta-Hexosaminidase/genéticaRESUMO
The formation of neurotoxic beta-amyloid fibrils in Alzheimer's disease (AD) is suggested to involve membrane rafts and to be promoted, in vitro, by enriched concentrations of gangliosides, particularly GM1, and the cholesterol therein. In our study, the presence of rafts and their content of the major membrane lipids and gangliosides in the temporal cortex, reflecting late stages of AD pathology, and the frontal cortex, presenting earlier stages, has been investigated. Whole tissue and isolated detergent-resistant membrane fractions (DRMs) were analysed from 10 AD and 10 age-matched control autopsy brains. DRMs from the frontal cortex of AD brains contained a significantly higher concentration (micromol/micromol glycerophospholipids), of ganglioside GM1 (22.3 +/- 4.6 compared to 10.3 +/- 6.4, p <0.001) and GM2 (2.5 +/- 1.0 compared to 0.55 +/- 0.3, p <0.001). Similar increases of these gangliosides were also seen in DRMs from the temporal cortex of AD brains, which, in addition, comprised significantly lower proportions of DRMs. Moreover, these remaining rafts were depleted in cholesterol (from 1.5 +/- 0.2 to 0.6 +/- 0.3 micromol/micromol glycerophospholipids, p <0.001). In summary, we found an increased proportion of GM1 and GM2 in DRMs, and accelerating plaque formation at an early stage, which may gradually lead to membrane raft disruptions and thereby affect cellular functions associated with the presence of such membrane domains.