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. 2021 Oct 12;10:e71596. doi: 10.7554/eLife.71596

Figure 6. Pharmacological inhibition of ubiquitin-specific protease 28 (USP28) prevents human lung squamous cell carcinoma (LSCC) tumour progression and reduces c-MYC protein levels in xenograft models.

(A) Small interfering RNA (siRNA)-mediated knockdown of USP28 decreases c-MYC, c-JUN, and Δp63 protein levels in human LUDLU-1 LSCC cells. (B) USP28 inhibition using FT206 (0.2 and 0.4 μM) reduces c-MYC, c-JUN, and Δp63 protein levels in human LUDLU-1 LSCC cells. (C) USP28 inhibition using FT206 decreases cell proliferation in human LSCC (NCI-H520, CALU-1, and LUDLU-1) cell lines (n = 8). Graphs indicate mean ± SEM. (D, E, F) In vivo tumour graft growth curves of human LSCC (NCI-H520, CALU-1, and LUDLU-1) cell lines subcutaneously injected in flanks of immunocompromised mice. Animals with palpable tumours were treated with vehicle or FT206 (75 mg/kg) via oral gavage. Plots indicate mean ± SD of the tumour volumes. p Values calculated from two-way analysis of variance (ANOVA) with Bonferroni’s multiple comparisons test (NCI-H520 n = 4 vehicle and 4 FT206; CALU-1 n = 3 vehicle and 3 FT206; LUDLU-1 n = 3 vehicle and 3 FT206). (G, H, I) Mice treated as in D, E, and F, respectively. Plots showing the weight of xenograft tumours at the end point. Student’s two-tailed t test was used to calculate p values (NCI-H520 n = 4 vehicle and 4 FT206; CALU-1 n = 3 vehicle and 3 FT206; LUDLU-1 n = 3 vehicle and 3 FT206). (J, K, L) c-MYC immunohistochemistry stainings of NCI-H520, CALU-1, and LUDLU-1 xenografts in mice treated as in D, E, and F, respectively. Scale bars, 50 μm. Source data for C, D, E, F, G, H, and I.

Figure 6—source data 1. USP28 inhibition impairs tumour growth in human LSCC xenografts.

Figure 6.

Figure 6—figure supplement 1. USP25 deletion does not affect c-MYC,c-JUN and Δp63 protein levels.

Figure 6—figure supplement 1.

(A) Immunoblot of endogenous ubiquitin-specific protease 25 (USP25), c-JUN, c-MYC, and Δp63 in USP25-depleted lung squamous cell carcinoma (LSCC) cells. VINCULIN served as loading control. (B) Immunoblot of endogenous c-MYC and c-JUN in LSCC cells upon FT206 treatment (IC50 doses display in panel D). VINCULIN served as loading control. (C) Immunoblot of endogenous c-MYC and c-JUN in lung adenocarcinoma (LADC) cells upon FT206 treatment (IC50 doses display in panel D). VINCULIN served as loading control. (D) IC50 values (doses that inhibits 50% of the cell viability) were calculated after exposure of human LADC and LSCC cells to different concentrations of FT206 compound. (E) Immunoblot of endogenous c-MYC and c-JUN in USP28 mutant LSCC cells upon FT206 treatment. The LSCC cell line SKMES contains a nonsense mutation in Usp28 (c.193G > T). VINCULIN served as loading control.