Fig 2: Knockdown of RPN2 decreased luminal B BC cell dissemination in vivo. Luminal B ER+ T47D cells, transfected with negative control siRNA (siRNA-C) or siRNA targeting RPN2 (siRNA-RPN2) were injected + estradiol (E2) ± neutrophils (Neu) into zebrafish transgenic embryos with green fluorescent blood vessels and analyzed as described in materials and methods. (A) Migration in vitro (n = 6). (B) In vivo dissemination of transfected T47D in presence of E2 ± Neu (n = 23–26). Scale bar = 100 µm. (n = 23–26). (C) Western blot analysis for confirmation of siRNA-RPN2 transfection and VCAM-1, ICAM-1, and MUC-1 expression. (D) Focal adhesions area (n = 5–6). Scale bar = 10 µm. (E) Proliferation (n = 6). Representative images of zebrafish embryos with disseminated luminal B T47D BC cells and immunocytochemistry analysis of vinculin expression are shown. Arrows show disseminated T47D and arrowheads show focal adhesions. BV = blood vessels. Data are presented as mean ± SEM. Two-tailed Student’s t-test *P < 0.05, ***P < 0.001, ns, not significant. Data are represented of at least two independent experiments.

Fig 3: 14-3-3zeta-mediated RPN2 upregulation is required for the mtp53 stability.(a) Detected peptides that differed between RPN2 knockdown cell line (MM231-LN shRPN2) and control (MM231-LN shNC). MS peak of 14-3-3Zeta (1433Z) in MM231-LN shNC (Right) and MM231-LN shRPN2 (Left) (indicated by arrows). (b) Gel-like views of MS peaks with retention time (RT) along the vertical axes (top) and distribution of the mean peak intensity of duplicates (bottom) across 6 samples. (c) Spot intensity of 14-3-3Z in MM231-LN shNC and MM231-LN shRPN2. (d) 14-3-3zeta reduced wtp53 expression. Lysates from MCF7 cells, transfected as indicated, were immunoblotted as shown. (e) and (f) RPN2 stabilized mtp53 in MM231-LN cells. Lysates from MM231-LN cells, transfected as indicated, were immunoblotted as shown. (g) and (h) 14-3-3zeta-dependent feedback regulation of mtp53 protein stability. Full-length gels and blots are shown in supplementary figure 17 and 18.

Fig 4: RPN2 is essential for the maintenance of the CSC fraction.(a) The CSC fraction derived from MCF7-ADR and MM231-LN cells. (b) MCF7-ADR and MM231-LN cells were segregated by fluorescence-activated cell sorting (FACS) into CD44high/CD24low and CD44high/CD24high subsets; sorted subsets were then compared for RPN2 expression by quantitative real-time PCR (qRT-PCR). Each data point is the average of three experiments. (c) Immunohistochemistry (IHC) for RPN2 (Top panel) and E-cadherin (Bottom panel) in MM231-LN tumors. MM231-LN xenografts were grown for 5–6 weeks after fat pad injection. Sections are representative of at least four mice analyzed per group. Scale bar, 200 μm. (d) RPN2 knockdown suppressed Snail expression via GSK3β activation. In MM231-LN cells, RPN2 and Snail mRNA expression was monitored by qRT-PCR after 72 h of treatment with a GSK3β inhibitor (CHIR99021, 3 μM). (n = 3, *P < 0.05, **P < 0.01,***P < 0.001).

Fig 5: RPN2 knockdown suppressed the heat shock proteins via GSK3ß activation.(a) RPN2 knockdown suppressed the expression of HSP27 and HSP70 and reduced HSP90 protein stability. MM231-LN shNC and MM231-LN shRPN2 cells were treated with cycloheximide (50 µg/ml) for 0–8 h. Cell lysates were subjected to western blotting with anti-HSP27, anti-HSP70, anti-HSP90 and anti-actin antibodies. (b) HSF1 restored the HSP27 expression in MM231-LN shRPN2 cells. After transfection of pFlag-HSF1 into MM231-LN shRPN2 cells, Cell lysates were subjected to western blotting with anti-Flag (top), anti-HSP27 (middle), anti-actin (bottom) antibodies. (c) HSF1 transcriptional activity in RPN2 knockdown cell line. HSP70 promoter-driven cypridina luciferase activity (HSP70-CLuc) was normalized to the thymidine kinase promoter-driven gaussia luciferase activity (TK-GLuc). MM231-LN shNC or MM231-LN shRPN2 cells were transfected with 200 ng of pHSP70-Cluc in combination with 200 ng of pTK-Gluc. Transfection and luciferase assays were performed as described in experimental procedure. (d) HSF1 restored the HSP70 expression in MM231-LN shRPN2 cells. MM231-LN shRPN2 cells were transfected with pFlag-HSF1 with 200 ng of pHSP70-Cluc, or pSV40-Cluc in combination with 100 ng of pTK-Gluc, and luciferase activity was measured at 48 h after transfection. (e) GSK3ß inhibitor restored HSF1 transcriptional activity. Six hours after transfection, cells were exposed to CHIR99021 (3, 10 µM) or an equivalent volume of dimethyl sulfoxide as a control added to the growth medium (CHIR99021, 0 µM). Treatments lasted for 72 h, after which the luciferase activity was examined. (f) Rescue experiment using MM231-LN shRPN2-UTR cells. RPN2-MF restored the HSP70 activity in MM231-LN shRPN2-UTR cells. pHSP70-Cluc was transfected into MM231-LN shRPN2-UTR cells with 300 ng of pCMV-RPN2-MF, or pEGFP-N1 in combination with 100 ng of pTK-Gluc, and luciferase activity was measured at 48 h after transfection. Full-length gels and blots are shown in supplementary figure 15.