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NFkB signaling and ISGylation associated with BRCA1-mutated fallopian tube epithelium

ABSTRACT: Germline BRCA1 or BRCA2 mutations (mtBRCA1 and mtBRCA2) dramatically increase risk for high-grade serous ovarian cancer (HGSOC), the most commonly diagnosed histotype. Other risk factors for this cancer, which originates primarily in the distal fallopian tube epithelium (FTE), implicate ovulation. To test whether mtBRCA1 or mtBRCA2 FTE cells respond differently to peri-ovulatory follicular fluid (FF) exposure than control patient FTE, gene expression profiles from primary FTE cultures were compared at baseline, 24h after FF exposure, and 24h after FF replacement with culture medium. Hierarchical clustering revealed both FF exposure and BRCA mutation status affect gene expression, with BRCA1 mutation having the greatest impact. Analysis revealed increased NFκB and EGFR signaling at baseline, with increased interferon signaling after recovery from FF exposure in mtBRCA1 samples. Inhibition of EGFR signaling and ISGylation by increased BRCA1 expression was verified in an immortalized FTE cell line, OE-E6/E7, stably transfected with BRCA1. Suppression of ISG15 and ISGylated protein levels by BRCA1 expression was found to be mediated by decreased NFκB signaling and was transiently suppressed by FF exposure. This study demonstrates increased NFκB signaling associated with decreased BRCA1 expression resulting in increased ISG15 and ISGylation following FF exposure, which could represent potential targets for chemoprevention.

ORGANISM(S): Homo sapiens

PROVIDER: GSE98699 | GEO | 2018/05/31

REPOSITORIES: GEO

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Project description:Filamentous hemagglutinin (FHA), Bordetella pertussis' key adhesin, is both present at the cell surface and released in the extracellular milieu. To comprehend the role free FHA plays in the pathogenic process, we analyzed the global transcriptional changes in human peripheral blood mononuclear cells (PBMC) after exposure to that purified virulence factor. This analysis was undertaken with four different FHA preparations (FHA-1, FHA-2, FHA-3, and FHA-4), purified from four different B. pertussis strains. Cultures comprising 3x106 PBMC were stimulated for 0.5, 2, 4, and 6 hours with 5 ug/ml of each purified B. pertussis FHA. Control PBMC cultures were simultaneously treated with similar volumes of elution buffers (Mock-1 and Mock-2), and sampled at the same time points. The array experiment was composed of 27 samples. Most of the transcripts known to be part of the "common host-transcriptional response" that mediates inflammation were among the 1,235 array elements (representing 683 known unique genes) showing a greater than 3-fold change in transcript abundance between FHA-treated and untreated cells. We also identified an FHA-specific signature not observed in cells stimulated with B. pertussis LPS or heat-killed B. pertussis. This response, mounted by 13 genes, largely (69%, 9/13 genes) interferon (IFN)-regulated genes. Further analysis revealed that 18.3% (125 out of 683) of the FHA-responsive genes were in fact regulated by IFN. These included several major players of the antiviral IFN type I response, as well as three key components of the ISGylation pathway. Induction of the ubiquitin-like protein ISG15 and its specific protease USP18 was confirmed at the mRNA level by relative real-time polymerase chain reaction (RT-PCR). Western-blot analysis demonstrated the presence of both free ISG15 and several ISGylated conjugates in FHA-stimulated PBMC cell lysate. Intracellular FACS analysis provided further evidence that monocytes and a natural-killer (NK)-enriched cell population are the primary producers of ISG15 after FHA stimulation. Our results revealed new activities of free B. pertussis FHA: activation of the host IFN and ISGylation pathways. The consequences of such activation are yet to be resolved, but we hypothesize that FHA utilizes the ISGylation pathway or USP18 to regulate the IFN response, which in turn sensitizes the host to the infection and induces cell death. Groups of assays that are related as part of a time series. Infection: treatment with purified B. pertussis filamentous hemagglutinin (FHA) at 0.005 mg/mL Time: Samples were collected after various treatment time (hours) Keywords: time_series_design Computed

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NFkB signaling and ISGylation associated with BRCA1-mutated fallopian tube epithelium

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