Targeting proteasome-associated deubiquitinases as a novel strategy for the treatment of estrogen receptor-positive breast cancer

Abstract

Estrogen receptor α (ERα) is expressed in ~67% of breast cancers and is critical to their proliferation and progression. The expression of ERα is regarded as a major prognostic marker, making it a meaningful target to treat breast cancer (BCa). However, hormone receptor-positive BCa was sometimes irresponsive or even resistant to classic anti-hormonal therapies (e.g., fulvestrant and tamoxifen). Hence, novel anti-endocrine therapies are urgent for ERα⁺ BCa. A phase II study suggested that bortezomib, an inhibitor blocking the activity of 20 S proteasomes, intervenes in cancer progression for anti-endocrine therapy in BCa. Here we report that proteasome-associated deubiquitinases (USP14 and UCHL5) inhibitors b-AP15 and platinum pyrithione (PtPT) induce growth inhibition in ERα⁺ BCa cells. Further studies show that these inhibitors induce cell cycle arrest and apoptosis associated with caspase activation, endoplasmic reticulum (ER) stress and the downregulation of ERα. Moreover, we suggest that b-AP15 and PtPT block ERα signaling via enhancing the ubiquitin-mediated degradation of ERα and inhibiting the transcription of ERα. Collectively, these findings demonstrate that proteasome-associated deubiquitinases inhibitors b-AP15 and PtPT may have the potential to treat BCa resistant to anti-hormonal therapy.

Fig. 1. b-AP15 and PtPT suppress the growth of BCa cells.

a, b BCa cells were treated with or without b-AP15. c, d BCa cells were treated with or without PtPT. Cell viability was tested using MTS assays. Mean ± SD of three independent experiments. *p < 0.05, ^#p < 0.01, the two-sided t-test. BCa cells were treated with b-AP15 (e) and PtPT (f) for 24 h, then were transplanted in 30% agarose for 10 days. The shown images were from one of three independent experiments

Fig. 2. Inhibition of USP14 and UCHL5 induces cell death in BCa cells.

a BCa cells were treated with the indicated concentrations of b-AP15 for 24 h. Cells were harvested and stained with Annexin V-FITC/PI followed by flow cytometry. Representative images and b quantification of cell death were shown. Mean ± SD of three independent experiments. *p < 0.05. c BCa cells were exposed to the indicated concentrations of PtPT for 24 h. Cells were collected and stained with Annexin V-FITC/PI followed by flow cytometry. Representative images and (d) quantification of cell death were shown. Mean ± SD of three independent experiments. *p < 0.05. e BCa cells were treated with indicated concentrations of b-AP15, or b-AP15 (1 μM) for the indicated time. Total proteins were extracted. The expression of PARP, Bcl-2 and cleaved caspase-3 were tested by western blot. GAPDH was used as a loading control (f) BCa cells were treated with indicated concentrations of PtPT, or PtPT (5 μM) for indicated time. Total proteins were extracted. The expression of PARP, Bcl-2 and cleaved caspase-3 were tested by western blot. GAPDH was shown as a loading control

Fig. 3. b-AP15 or PtPT trigger ER stress in BCa cells.

a, b BCa cells were treated with b-AP15 (0, 0.5, 1, 2 μM) or PtPT (0, 2.5, 5, 7.5 μM) for 24 h. Proteins lysates were subjected to western blot analysis for total ubiquitinated proteins (Ub-prs), K48-linked ubiquitin, P-eIF2α, eIF2α, HSP70,CHOP. GAPDH was shown as a loading control. c, d BCa cells were treated with b-AP15 (1 μM) or PtPT (5 μM) for 0, 6, 12, 24 h. The expression of proteins as mentioned above was detected using western blot analysis

Fig. 4. b-AP15 or PtPT induce cell cycle arrest in BCa cells.

a BCa cells were exposed to b-AP15 for 24 h. Cells were fixed and stained with PI followed by flow cytometry. Representative images and (b) distribution of cell cycle stages is shown. c BCa cells were exposed to PtPT for 24 h. Cells were fixed and stained with PI followed by flow cytometry. Representative images and (d) distribution of cell death are shown. e BCa cells were treated with indicated concentrations of b-AP15, or b-AP15 (1 μM) for indicated time. Total proteins were extracted. The expression of CDK4, cyclinD1 and P21 were tested by western blot. GAPDH was shown as a loading control. f BCa cells were treated with indicated concentrations of PtPT, or PtPT (5 μM) for the indicated time. Total proteins were extracted. The expression of CDK4, cyclinD1 and P21 were tested by western blot. GAPDH was probed as a loading control

Fig. 5. Two inhibitors of USP14 and UCHL5 decrease ERα and IGF1R levels.

a, c BCa cells were exposed to b-AP15 or PtPT for 24 h, or b-AP15 (1 μM) / PtPT (5 μM) for multiple length of time. ERα and IGF1R levels were detected using western blot analysis. The shown images is from one of three independent experiments. GAPDH was probed as a loading control. b, d Relative quantifications of ERα and IGF1R expression were shown. *p < 0.05, ^#p < 0.01

Fig. 6. USP14 and UCHL5 inhibitors affect ERα and IGF1R protein levels but not their localization.

a, b BCa cells (a: MCF-7; b: T47D) post b-AP15 (1 μM) or PtPT (5 μM) treatment for 12 h were fixed and incubated with ERα antibody or IGF1R antibody overnight at 4 °C. Then cells were washed and incubated with secondary Cy3-conjugated antibodies. Nucleus was stained by DAPI. Immunofluorescence microscopy shows endogenous ER α and IGF1R (orange) and nucleus (blue). Three independent experiments were performed

Fig. 7. USP14 and UCHL5 inhibitors increase the ubiquitination of ERα and decrease ERα-mediated transcription activity.

a Total RNAs were collected from T47D cells exposed to b-AP15 or PtPT for 12 h and subjected to RT-qPCR analysis for ERα and ERα-targeted gene, PS2 mRNA expression. Mean ± SD (n = 3). *p < 0.05, ^#p < 0.01. b T47D cells were treated with cycloheximide (CHX) for 0, 6, 12, 24 h and co-treatment of CHX and b-AP15 or PtPT at the same time. Protein lysates were collected and subjected to western blot assay for ERα protein level. Representative images were shown. c Relative quantifications of ERα band density were calculated. *p < 0.05. d The total proteins were collected from T47D cells treated with b-AP15 (1 μM) or PtPT (5 μM) for 24 h and MG132 (10 μM) for 6 h before it was collected, immunoprecipitated with ERα antibody beads and immunoblotted for ERα and ubiquitin (Ub). e T47D cells were transfected with luciferase reporter plasmid containing estrogen receptor elements (EREs). After transfection for 24 h, cells were exposed to b-AP15 and PtPT for 24 h. Protein lysates were collected and dual-luciferase assay was used to measure relative luciferase activity. Three independent experiments were performed.*p < 0.05