NFκB activation by hypoxic small extracellular vesicles drives oncogenic reprogramming in a breast cancer microenvironment

Abstract

Small extracellular vesicles (sEV) contribute to the crosstalk between tumor cells and stroma, but the underlying signals are elusive. Here, we show that sEV generated by breast cancer cells in hypoxic (sEV^HYP), but not normoxic (sEV^NORM) conditions activate NFκB in recipient normal mammary epithelial cells. This increases the production and release of inflammatory cytokines, promotes mitochondrial dynamics leading to heightened cell motility and disrupts 3D mammary acini architecture with aberrant cell proliferation, reduced apoptosis and EMT. Mechanistically, Integrin-Linked Kinase packaged in sEV^HYP via HIF1α is sufficient to activate NFκB in the normal mammary epithelium, in vivo. Therefore, sEV^HYP activation of NFκB drives multiple oncogenic steps of inflammation, mitochondrial dynamics, and mammary gland morphogenesis in a breast cancer microenvironment.

Fig. 1:. Breast cancer-derived sEV^HYP activate NFκB in MCF10A recipient cells.

A Bioinformatics analysis of NFκB target genes upregulated by sEV^HYP in recipient MCF10A cells by RNA-Seq. B MCF10A cells treated with sEV^NORM or sEV^HYP for 24 h were analyzed for nuclear accumulation of p50 NFκB by fluorescence microscopy. Representative images. Scale bar, 25 μm. Ctrl, control. Cyan, nuclei; yellow, p50 NFκB. C The conditions are as in B and nuclear accumulation of p50 NFκB was quantified at the indicated increasing concentrations of sEV^NORM or sEV^HYP. Mean±SD (two independent experiments). D MCF10A cells treated with sEV^NORM or sEV^HYP were incubated with pXSC or transfected with p65 NFκB -directed siRNA (si-p65) and analyzed for expression of the indicated NFκB target genes, by qRT-PCR. Mean±SD (N=3). E Conditioned media from MCF10A cells treated as in D were analyzed for cytokine release by LEGENDplex. Mean±SD (N=4). F sEV-induced nuclear accumulation of p50 NFκB was quantified in the presence or absence of pXSC. For panels C-F, numbers correspond to p values by 1-way Anova with Tukey’s posttest.

Fig. 2:. NFκB regulation of sEV^HYP-dependent mitochondrial dynamics and cell motility.

A, B MCF10A cells treated with sEV^HYP were incubated with or without pXSC and analyzed for changes in mitochondrial volume indicative of organelle fusion (>1.3-fold, positive y-scale) or fission (<0.7-fold, negative y-scale) over 40-sec intervals (A) and the number of mitochondrial fusion and fission events was quantified (B). Each line corresponds to a single cell. Mean±SD (N=3). Numbers correspond to p values by unpaired t-test. C MCF10A cells treated with sEV^NORM or sEV^HYP were imaged after 24 h by confocal microscopy and mitochondrial accumulation at the cortical cytoskeleton was quantified. Mean±SD (N=3). D MCF10A cells as in C were analyzed for single cell motility in 2D contour plots with quantification of speed of cell movements (Velocity, Vel, μm/min) and distance traveled by individual cells (Dis, μm). The cutoff velocities for slow (black, <0.53 μm/min)- or fast (red, >0.53 μm/min)-moving cells are indicated. E MCF10A cells transfected with siCtrl or si-p65 NFκB were treated with sEV^NORM or sEV^HYP and analyzed for directional cell migration in a wound closure assay at the indicated time intervals. Mean±SD (N=3). F MCF10A cells incubated with sEV^NORM or sEV^HYP were treated with pXSC or transfected with si-p65 NFκB and analyzed for migration on PET inserts.Mean±SD (N=3). For panels C and F, numbers correspond to p values by 1-way Anova with Tukey’s posttest.

Fig. 3:. NFκB regulation of 3D mammary acini morphogenesis.

A MCF10A acini in 3D culture seeded in the presence of Ctrl, sEV^NORM or sEV^HYP with or without pXSC (added once at t=0) were analyzed by fluorescence microscopy 21 d after seeding (representative images; scale bar, 15 μm). B The conditions are as in A and the expression of p50 NFκB, ILK, Ki-67, cleaved (Cl) caspase 3, survivin, SNAIL and vimentin was quantified. Magenta, nuclei; yellow, individual protein of interest. MFI, mean fluorescence intensity. Mean±SD (N=3). Numbers correspond to p values by 1-way Anova with Tukey’s posttest.

Fig. 4:. sEV-induced EMT in the mammary gland, in vivo.

A, B AT3-derived sEV^HYP were injected in the abdominal mammary gland of C57BL/6 mice and tissue samples harvested after 6 weeks were analyzed by immunohistochemistry (A) and reactivity with antibodies to ILK or vimentin was quantified (B). Ctrl, control. Representative images. Scale bar, 100 μm. C, D The conditions are as in A and mammary gland tissues harvested after 6 weeks were analyzed by immunofluorescence (C, representative images) and changes in expression of p50 NFκB, ILK, E-cadherin and SNAIL were quantified (D). Scale bar, 50 μm. MFI, mean fluorescence intensity. Mean±SD. Numbers correspond to p values by unpaired t-test.