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Mitochondrial DNA in the tumor microenvironment activates neutrophils and is associated with worsened outcomes in patients with advanced epithelial ovarian cancer
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Advanced cancer causes necrosis and releases damage-associated molecular patterns (DAMPs). Mitochondrial DAMPs activate neutrophils, including generation of neutrophil extracellular traps (NETs), which are injurious, thrombogenic, and implicated in metastasis. We hypothesized that extracellular mitochondrial DNA (mtDNA) in ascites from patients with epithelial ovarian cancer (EOC) would correlate with worsened outcomes.Banked ascites supernatants from patients with newly diagnosed advanced EOC were analyzed for mtDNA, neutrophil elastase, and activation of healthy donor neutrophils and platelets. TCGA was mined for expression of SELPand ELANE. The highest quartile of ascites mtDNA correlated with reduced progression-free survival (PFS) and a higher likelihood of disease progression within 12-months following primary surgery (n=68, log-rank, p=0.0178). NETs were detected in resected tumors. Ascites supernatants chemoattracted neutrophils, induced NETs, and activated platelets. Ascites exposure rendered neutrophils suppressive, based on abrogation of ex vivostimulated T cell proliferation. Increased SELPmRNA expression correlated with worsened overall survival (n=302, Cox model, p=0.02).In this single-center retrospective analysis, ascites mtDNA correlated with worsened PFS in advanced EOC. Our results support mitochondrial and other DAMPs activating neutrophil and platelet responses that facilitate metastasis and obstruct anti-tumor immunity. These pathways are potential prognostic markers and therapeutic targets.

Keywords [en]
Gynecological cancers, ovarian cancer, innate immunity, inflammation, tumor microenvironment, neutrophils, platelets, thrombosis, metastasis
National Category
Cell and Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-147420OAI: oai:DiVA.org:umu-147420DiVA, id: diva2:1203615
Available from: 2018-05-03 Created: 2018-05-03 Last updated: 2018-06-09
In thesis
1. The release of histone proteins from cells via extracellular vesicles
Open this publication in new window or tab >>The release of histone proteins from cells via extracellular vesicles
2018 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

Histones are chromatin-associated proteins localized to the nucleus. However, extracellular histones are present in biofluids from healthy individuals and become elevated under disease conditions, such as neurodegeneration and cancer. Hence, extracellular histones may have important biological functions in healthy and diseased states, which are not understood. Histones have been reported in the proteomes of extracellular vesicles (EVs), including microvesicles and exosomes. The main aim of this thesis was to determine whether or not extracellular histones are secreted via EVs/exosomes.

In an initial study (Paper I), I optimized methods for human embryonic kidney (HEK293) cell culture, transfection and protein detection using western blotting.

In the main study (Paper II), I used oligodendrocyte cell lines (rat OLN-93 and mouse Oli-neu) to investigate the localization of histones to EVs. Western blotting of EVs purified from OLN-93 cell-conditioned media confirmed the presence of linker and core histones in them. Immunolocalization and transmission electron microscopy confirmed that histones are localized to EVs, as well as intraluminal vesicles (ILVs) within multivesicular bodies (MVBs). This suggests that histones are secreted via the MVB/exosome pathway.

Localization of histones in EVs was investigated by biochemical/proteolytic degradation and purification followed by western blotting. Surprisingly, histones were associated with the membrane but not the luminal fraction. Overexpression of tagged histones in HEK293 cells confirmed their conserved, membrane localization. OLN-93 cell EVs contained both double stranded and single stranded DNA but nuclease and protease digestion showed that the association of histones and DNA with EVs was not interdependent.

The abundance of histones in EVs was not affected by differentiation in Oli-neu cells. However, histone release was upregulated as an early response to cellular stress in OLN-93 cells and occurred before the release of markers of stress including heat shock proteins. Interestingly, a notable upregulation in secretion of small diameter (50-100 nm) EVs was observed following heat stress, suggesting that a sub-population of vesicles may be involved specifically in histone secretion in response to stress. Proteomic analyses identified the downregulation of endosomal sorting complex required for transport (ESCRT) as a possible mechanism underlying increased histone secretion.

In Paper III, I developed methods to quantify extracellular histone proteins in human ascites samples from ovarian cancer patients.

 

In summary, we show for the first time that membrane-associated histones are secreted via the MVB/exosome pathway. We demonstrate a novel pathway for extracellular histone release that may have a role in both health and disease. 

Place, publisher, year, edition, pages
Umeå: Umeå University, 2018. p. 74
Keywords
Histone, extracellular vesicles, exosomes, extracellular histones extracellular DNA, cellular stress, proteomics, ESCRT complex, Lrrn1, mid-hindbrain organizer, ovarian cancer, ascites
National Category
Cell and Molecular Biology Cell Biology
Identifiers
urn:nbn:se:umu:diva-147409 (URN)978-91-7601-888-0 (ISBN)
Presentation
2018-05-28, E04_ R-1, Umeå, 13:00 (English)
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Supervisors
Available from: 2018-05-04 Created: 2018-05-03 Last updated: 2018-06-09Bibliographically approved

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Muthukrishnan, UmaGilthorpe, Jonathan D.Urban, Constantin F.

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