Targeting USP8 inhibits Cancer Progression and Prevents EV-TβRII-Induced Exhaustion of CD8+ T cells (2022)
Figure 6

Figure 6. Pharmacological inhibition of USP8 represses TGF-β signalling and prevents the exosomal secretion of TβRII
a, Structure of the USP8 inhibitor,9-ethyloxyimino-9H-indeno [1,2-b] pyrazine-2,3-dicarbonitrile.

b, Left: screening procedure for cellular components critical for the secretion of EV-TβRII; Right: nanosight analysis of GFP positive EVs derived from MDA-MB-231 cells expressing TβRII-GFP and pre-treated with anti-cancer compounds. n = 3 technical replicates.

c, Immunoblot analysis of total cell lysates derived from MDA-MB-231 breast cancer cell treated with USP8 inhibitor and TGF-β (2.5 ng/ml) as indicated.

d, Effect of USP8 inhibitor (5 μM) on SMAD3/SMAD4-dependent CAGA12-Luc transcriptional response induced by TGF-β (2.5 ng/ml) for 16 h in HEK293T cells transfected with control vector (Co.vec) or USP8 wt/cs. n = 3 biological replicates.

e, Heatmap shows differentially expressed genes in control DMSO-treated or USP8 inhibitor (5 μM)-treated MDA-MB-231 cells for 24 h (left panel). Volcano plot of transcriptome proﬁles between control and USP8 inhibitor treated MDA-MB-231 cells (right panel). Red and green dots represent genes significantly up-regulated and down-regulated in control MDA-MB-231 cells. (p < 0.05, false discovery rate [FDR] < 0.1).

f, qRT-PCR analysis of control (DMSO-treated) or USP8 inhibitor (5 μM for 24 h) treated MDA-MB-231 cells. Relative mRNA levels are shown as a heatmap.

g, Gene signatures indicating the up-regulated epithelial-mesenchymal-transition (EMT) (upper panel) and breast cancer progenitors-related gene set (bottom panel) are significantly enriched in control cells; shown by pre-ranked gene-set enrichment analysis (GSEA).

h, The down-regulated TGF-β signaling (upper panel) and metastasis-related gene sets (bottom panel) are significantly enriched in USP8 inhibitor-treated MDA-MB-231 cells; shown by pre-ranked gene-set enrichment analysis (GSEA).

i, Go enrichment analysis of significant up- and down-regulated gene sets in control DMSO versus USP8 inhibitor treated MDA-MB-231 cells.

j, The total concentration of EVs by nanosight analysis (left) and the percentage of TβRII+ EVs by FACS (right) from 4T1 and MDA-MB-231 cells treated with USP8 inhibitor (5 μM) for 24 h. n = 3 technical replicates.

k-n, Experimental procedure in vivo: MDA-MB-231-Luc cells (1 × 105 per mouse) were intracardially injected into nude mice (n = 7 for each group). USP8 inhibitor (1 mg/kg) was injected at day 1 via intraperitoneal injection every the other day for three weeks. Normalized BLI signals (left panel) and BLI imaging of three representive nude mice from each group at week 6 (right panel) were shown (k). Percentage of metastasis-free survival in each experimental group followed in time (l). Number of bone metastasis nodules (m). Percentage of TβRII+ crEVs in plasma samples from mice (left panel) and immunoblot analysis of metastatic tumor nodule in mice and circulating EVs from plasma (right panel) (n).
Data information: *p < 0.05 (two-tailed Student's t test (d, j, m, n (left)) or two-way ANOVA (k, l)). Data are shown as mean + SD (b, d, j) or as means ± SD (k, m, n (left)).