The heterogeneity of exosomal populations has hindered our understanding
of their biogenesis, molecular composition, biodistribution, and functions. By
employing asymmetric-flow field-flow fractionation (AF4), we identified two
exosome subpopulations (large exosome vesicles, Exo-L, 90-120 nm; small exosome
vesicles, Exo-S, 60-80 nm) and discovered an abundant population of
non-membranous nanoparticles termed “exomeres” (~35 nm).
Exomere proteomic profiling revealed an enrichment in metabolic enzymes and
hypoxia, microtubule and coagulation proteins and specific pathways, such as
glycolysis and mTOR signaling. Exo-S and Exo-L contained proteins involved in
endosomal function and secretion pathways, and mitotic spindle and IL-2/STAT5
signaling pathways, respectively. Exo-S, Exo-L, and exomeres each had unique
N-glycosylation, protein, lipid, and DNA and RNA profiles
and biophysical properties. These three nanoparticle subsets demonstrated
diverse organ biodistribution patterns, suggesting distinct biological
functions. This study demonstrates that AF4 can serve as an improved analytical
tool for isolating and addressing the complexities of heterogeneous nanoparticle
subpopulations.
A switch from oxidative phosphorylation to glycolysis is frequently observed in cancer cells and is linked to tumor growth and invasion, but the underpinning molecular mechanisms controlling the switch are poorly understood. In this report we show that Notch signaling is a key regulator of cellular metabolism. Both hyper- and hypoactivated Notch induce a glycolytic phenotype in breast tumor cells, although by distinct mechanisms: hyperactivated Notch signaling leads to increased glycolysis through activation of the phosphatidylinositol 3-kinase/AKT serine/threonine kinase pathway, whereas hypoactivated Notch signaling attenuates mitochondrial activity and induces glycolysis in a p53-dependent manner. Despite the fact that cells with both hyper- and hypoactivated Notch signaling showed enhanced glycolysis, only cells with hyperactivated Notch promoted aggressive tumor growth in a xenograft mouse model. This phenomenon may be explained by that only Notch-hyperactivated, but not -hypoactivated, cells retained the capacity to switch back to oxidative phosphorylation. In conclusion, our data reveal a role for Notch in cellular energy homeostasis, and show that Notch signaling is required for metabolic flexibility.
Notch signaling is frequently hyperactivated in breast cancer, but how the enhanced signaling contributes to the tumor process is less well understood. In this report, we identify the proinflammatory cytokine interleukin-6 (IL-6) as a novel Notch target in breast tumor cells. Enhanced Notch signaling upregulated IL-6 expression, leading to activation of autocrine and paracrine Janus kinase/signal transducers and activators of transcription signaling. IL-6 upregulation was mediated by non-canonical Notch signaling, as it could be effectuated by a cytoplasmically localized Notch intracellular domain and was independent of the DNA-binding protein CSL. Instead, Notch-mediated IL-6 upregulation was controlled by two proteins in the nuclear factor (NF)-κB signaling cascade, IKKα and IKKβ (inhibitor of nuclear factor kappa-B kinase subunit alpha and beta, respectively), as well as by p53. Activation of IL-6 by Notch required IKKα/IKKβ function, but interestingly, did not engage canonical NF-κB signaling, in contrast to IL-6 activation by inflammatory agents such as lipopolysaccharide. With regard to p53 status, IL-6 expression was upregulated by Notch when p53 was mutated or lost, and restoring wild-type p53 into p53-mutated or -deficient cells abrogated the IL-6 upregulation. Furthermore, Notch-induced transcriptomes from p53 wild-type and -mutated breast tumor cell lines differed extensively, and for a subset of genes upregulated by Notch in a p53-mutant cell line, this upregulation was reduced by wild-type p53. In conclusion, we identify IL-6 as a novel non-canonical Notch target gene, and reveal roles for p53 and IKKα/IKKβ in non-canonical Notch signaling in breast cancer and in the generation of cell context-dependent diversity in the Notch signaling output.
Highlights d Metastasis inducers lead to a decline in CAF-1 suppressing canonical H3 incorporation d EMT and metastatic colonization occur as a function of CAF-1 levels d Histone H3.3 variant is essential for tumor progression and aggressive phenotypes d HIRA-mediated H3.3 gap filling induces a pro-metastatic transcriptional reprogramming
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