RESUMO
Expression of ATP-binding cassette B5 (ABCB5) has been demonstrated to confer chemoresistance, enhance cancer stem cell properties and associate with poor prognosis in hepatocellular carcinoma (HCC). The aim of this study was to evaluate the genetic variations of ABCB5 in HCC patients with reference to healthy individuals and the clinicopathological significance. A pilot study has examined 20 out of 300 pairs HCC and paralleled blood samples using conventional sequencing method to cover all exons and exon/intron regions to investigate whether there will be novel variant sequence and mutation event. A total of 300 HCC and 300 healthy blood DNA samples were then examined by Sequenom MassARRAY genotyping and pyrosequencing for 38 SNP and 1 INDEL in ABCB5. Five novel SNPs were identified in ABCB5. Comparison of DNA from blood samples of HCC and healthy demonstrated that ABCB5 SNPs rs75494098, rs4721940 and rs10254317 were associated with HCC risk. Specific ABCB5 variants were associated with aggressive HCC features. SNP rs17143212 was significantly associated with ABCB5 expression level. Nonetheless, the paralleled blood and tumour DNA sequences from HCC patients indicated that ABCB5 mutation in tumours was not common and corroborated the TCGA data sets. In conclusion, ABCB5 genetic variants had significant association with HCC risk and aggressive tumour properties.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Animais , Povo Asiático/genética , Carcinoma Hepatocelular/etnologia , DNA de Neoplasias/genética , Intervalo Livre de Doença , Éxons/genética , Predisposição Genética para Doença , Genótipo , Humanos , Mutação INDEL , Íntrons/genética , Estimativa de Kaplan-Meier , Neoplasias Hepáticas/etnologia , Mutação , Neoplasias/genética , Projetos Piloto , Polimorfismo de Nucleotídeo Único , Modelos de Riscos Proporcionais , RNA Mensageiro/biossíntese , RNA Neoplásico/biossíntese , Risco , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Especificidade da Espécie , Vertebrados/genéticaRESUMO
BACKGROUND: Granulin-epithelin precursor (GEP) is a secretory growth factor, which has been demonstrated to control cancer growth, invasion, drug resistance and immune escape. Our previous studies and others also demonstrated its potential in targeted therapy. Comprehensive characterization of GEP partner on cancer cells are warranted. We have previously shown that GEP interacted with heparan sulfate on the surface of liver cancer cells and the interaction is crucial for GEP-mediated signaling transduction. This study aims to characterize GEP protein partner at the cell membrane with the co-immunoprecipitation and mass spectrometry approach. METHODS: The membrane fraction from liver cancer model Hep3B was used for capturing binding partner with the specific monoclonal antibody against GEP. The precipitated proteins were analyzed by mass spectrometry. After identifying the GEP binding partner, this specific interaction was validated in additional liver cancer cell line HepG2 by co-immunoprecipitation using GRP78 and GEP antibodies, respectively, as the bait. GRP78 transcript levels in hepatocellular carcinoma (HCC) clinical samples (n = 77 pairs) were examined by real-time quantitative RT-PCR. GEP and GRP78 protein expressions were investigated by immunohistochemistry on paraffin sections. RESULTS: We identified the GEP-binding protein as 78-kDa glucose-regulated protein (GRP78, also named heat shock 70-kDa protein 5, HSPA5). This interaction was validated in independent HCC cell lines. Increased GRP78 mRNA levels were demonstrated in liver cancer tissues compared with the paralleled liver tissues (t-test, P = 0.002). GRP78 and GEP transcript levels were significantly correlated (Spearman's correlation, P = 0.001), and the proteins were also detectable in the cytoplasm of liver cancer cells by immunohistochemical staining. CONCLUSIONS: GRP78 and GEP are interacting protein partners in liver cancer cells and may play a role in GEP-mediated cancer progression in HCC.
Assuntos
Carcinoma Hepatocelular/metabolismo , Proteínas de Choque Térmico/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Neoplasias Hepáticas/metabolismo , Transdução de Sinais , Linhagem Celular Tumoral , Chaperona BiP do Retículo Endoplasmático , Feminino , Regulação Neoplásica da Expressão Gênica , Proteínas de Choque Térmico/genética , Células Hep G2 , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Masculino , Pessoa de Meia-Idade , Progranulinas , Ligação ProteicaRESUMO
Granulin-epithelin precursor (GEP) is a pluripotent secretory growth factor which promotes cancer progression in a number of human cancers. However, how cancer cells interact with GEP remains unknown. In this study, we aimed to identify the cell surface-binding partner of GEP on liver cancer cells. Human recombinant GEP (rGEP) was expressed and purified to homogeneity. The rGEP was shown to trigger phosphorylation of AKT and ERK1/2 in liver cancer cells. We demonstrated cell surface attachment of rGEP, which was blocked by prebinding of platelet-derived growth factor-AA, platelet-derived growth factor-BB and fibroblast growth factor-2. Therefore, heparan sulfate (HS) had been reasoned as the binding partner of rGEP. Heparinase digestion validated the role of HS on supporting the attachment. The heparin-binding domain of GEP was mapped to RRH(555-557) in the C-terminal region. Suppression of the HS polymerase exostosin-1 reduced the rGEP binding and rGEP-mediated signaling transduction. Suppression of a specific HS proteoglycan, glypican-3, also showed a partial reduction of rGEP binding and an inhibition on rGEP-mediated activation of AKT. Furthermore, glypican-3 was shown to correlate with the expressions of GEP in clinical samples (Spearman's ρ = 0.363, P = 0.001). This study identified HS, partly through glypican-3, as a novel binding partner of GEP on the surface of liver cancer cells.
