USP36 promotes tumorigenesis and tamoxifen resistance in breast cancer by deubiquitinating and stabilizing ERα

Abstract

Background: Breast cancer is the most prevalent cancer in women globally. Over-activated estrogen receptor (ER) α signaling is considered the main factor in luminal breast cancers, which can be effectively managed with selective estrogen receptor modulators (SERMs) like tamoxifen. However, approximately 30-40% of ER + breast cancer cases are recurrent after tamoxifen therapy. This implies that the treatment of breast cancer is still hindered by resistance to tamoxifen. Recent studies have suggested that post-translational modifications of ERα play a significant role in endocrine resistance. The stability of both ERα protein and its transcriptome is regulated by a balance between E3 ubiquitin ligases and deubiquitinases. According to the current knowledge, approximately 100 deubiquitinases are encoded in the human genome, but it remains unclear which deubiquitinases play a critical role in estrogen signaling and endocrine resistance. Thus, decoding the key deubiquitinases that significantly impact estrogen signaling, including the control of ERα expression and stability, is critical for the improvement of breast cancer therapeutics.

Methods: We used several ER positive breast cancer cell lines, DUB siRNA library screening, xenograft models, endocrine-resistant (ERα-Y537S) model and performed immunoblotting, real time PCR, RNA sequencing, immunofluorescence, and luciferase activity assay to investigate the function of USP36 in breast cancer progression and tamoxifen resistance.

Results: In this study, we identify Ubiquitin-specific peptidase 36 (USP36) as a key deubiquitinase involved in ERα signaling and the advancement of breast cancer by deubiquitinases siRNA library screening. In vitro and in vivo studies showed that USP36, but not its catalytically inactive mutant (C131A), could promote breast cancer progression through ERα signaling. Conversely, silencing USP36 inhibited tumorigenesis. In models resistant to endocrine therapy, silencing USP36 destabilized the resistant form of ERα (Y537S) and restored sensitivity to tamoxifen. Molecular studies indicated that USP36 inhibited K48-linked polyubiquitination of ERα and enhanced the ERα transcriptome. It is interesting to note that our results suggest USP36 as a novel biomarker for treatment of breast cancer.

Conclusion: Our study revealed the possibility that inhibiting USP36 combined with tamoxifen could provide a potential therapy for breast cancer.

Keywords: Breast cancer; ERα; Stability; Tamoxifen resistance; USP36; Ubiquitin.

Fig. 1

Identification of USP36 as a novel mediator of ER signaling in ER positive breast cancer. A The flowchart shows the siRNA screening procedure for identifying novel deubiquitinases involved in modulating ER signaling. Each of the 76 human DUB genes was knocked down in MCF-7 cells with 20 μM pooled siRNAs. After 48 h, the quantitative gene expression analysis was detected by real-time PCR. B The relative expression level of TFF1(PS2) in MCF-7 cells transfected with DUBs in the screening library. The real-time PCR results of siControl is normalized to 1. C The expression of USP36 in breast cancer tissues (n = 1097) and normal tissues (n = 114) from TCGA database (https://www.genome.gov/). D The expression of USP36 in ER positive breast cancer tissues (n = 566) and normal tissues (n = 114) from TCGA database (https://www.genome.gov/). E–F Kaplan − Meier analysis showing relapse-free survival depending on USP36 expression levels in ER positive and ER negative breast cancer from public meta-analysis data (https://kmplot.com). G-H Immunohistochemistry (IHC) detecting USP36 expression in 65 breast cancer samples and 55 normal breast tissue (G). Statistical analysis of USP36 expression in G (H). I Volcano map of RNA-seq data from MCF-7 cell lines treated with siControl or siUSP36. |log2Fold change|> 1 and P value < 0.05 are set as screening criteria. J KEGG analysis of downregulated genes in RNA-seq data from MCF-7 cell lines treated with siControl or siUSP36 with threshold criteria of P < 0.05 and fold change > 1.5. K Gene set enrichment analysis (GSEA) shows enrichment of estrogen response genes in RNA-seq data from MCF-7 cell lines treated with siControl or siUSP36. L Heatmap of relationship of USP36 and differentially ER pathway related genes in RNA-seq data with threshold criteria of P < 0.05 and fold change > 1.5. M Gene set enrichment analysis (GSEA) shows enrichment of estrogen response genes in breast cancer tissue from TCGA data with threshold criteria of P < 0.05 and fold change > 1.5. All P values were calculated by unpaired two-tailed Student’s t tests. *P < 0.05; **P < 0.01; ***P < 0.001. (C-L)

Fig. 2

USP36 depletion inhibits ERα positive breast cancer progression in vivo and in vitro. A-B Real-time PCR was performed to determine USP36 mRNA levels in MCF-7 and T47D cells following USP36 siRNA treatment 48 h. C-D MCF-7 and T47D cells transfected with siControl or siUSP36 for 48 h were tested for viability using the CCK-8 assay at the indicated time points. E–H Colony formation (left panel) of MCF-7 and T47D cells transfected with scrambled siRNA or two independent USP36 siRNAs for 48 h. F and H show the quantitative analysis of the colony formation assay results. I-L MCF-7 and T47D cells were tested for their migration ability using Transwell assays. J and L show the quantitative analysis of the Transwell assays results. M-P MCF-7 and T47D cells were tested for their migration ability using wound healing assays. N and P show the quantitative analysis. Q-T The percentage of apoptotic cells was determined by FACS analysis after MCF-7 and T47D cells were treated with USP36 siRNA for 48 h. PI and Annexin V staining were performed on the cells. U A representative image of a tumor derived from a nude mouse injected with stably transfected shControl or shUSP36 MCF-7 cells is shown. V-W The tumor volume (V) and weight (W) in nude mice subcutaneously inoculated with stably transfected shControl or shUSP36 MCF-7 cells.Three independent experiments were conducted to obtain the results shown in Panels A-W. All the data are presented as the means ± SDs. *P < 0.05; **P < 0.01; ***P < 0.001 for comparisons (Student’s t test)

Fig. 3

USP36 instead of USP36 C131A overexpression promotes ERα positive breast cancer progression in vivo and in vitro. A-B Immunoblot analysis showing the expression level of USP36 in MCF-7 and T47D cells transfected with Flag or Flag-USP36 WT or Flag-USP36 C131A plasmid. β-Actin was used as the internal control. C-D MCF-7 and T47D cells transfected with Flag or Flag-USP36 WT or Flag-USP36 C131A plasmid for 48 h were tested for viability using the CCK-8 assay at the indicated time points. E–H Colony formation (left panel) of MCF-7 and T47D cells transfected with indicated plasmid for 48 h. F and H show the quantitative analysis of the colony formation assay results. I-L MCF-7 and T47D cells transfected with indicated plasmid for 48 h, were tested for their migration ability using Transwell assays. J and L show the quantitative analysis of the Transwell assays results. M-P MCF-7 and T47D cells transfected with indicated plasmid for 48 h, were tested for their migration ability using wound healing assays. N and P show the quantitative analysis. Q A representative image of a tumor derived from a nude mouse injected with stably MCF-7 cells as indicated is shown. R-S The tumor volume (R) and weight (S) in nude mice subcutaneously inoculated with stably transfected MCF-7 cells as indicated. Three independent experiments were conducted to get the results shown in Panels C-S. All the data are presented as the means ± SDs. *P < 0.05; **P < 0.01; ***P < 0.001 for comparisons (Student’s t test)

Fig. 4

USP36 instead of USP36 C131A is required for ERα signaling in breast cancer. A-B USP36 and ERα protein levels were determined by western blotting. MCF-7 and T47D cells in charcoal-stripped FBS and phenol red-free DMEM were transiently transfected with 20 nM siControl or 20 nM siUSP36 and then treated with 10 nM estradiol or vehicle for 6 h. Cell lysates were immunoblotted with the indicated antibodies. β-Actin was used as internal control. C-D USP36 and ERα protein levels were determined by western blotting. MCF-7 and T47D cells in charcoal-stripped FBS and phenol red-free DMEM were transiently transfected with Flag or Flag-USP36 for 48 h, and then treated with 10 nM estradiol or vehicle for 6 h. Cell lysates were immunoblotted with the indicated antibodies. β-Actin was used as internal control. E–F; I-J; O GREB1, PKIB, PS2 and PDZK1 mRNA levels were determined by real-time PCR after treatment with indicated method in MCF-7 and T47D cells. G-H; K-L; P ERE-luciferase activity was detected by luciferase assays in MCF-7 and T47D cells. M–N USP36 and ERα protein levels were determined by western blotting. MCF-7 cells were transiently transfected with Flag or Flag-USP36 WT or Flag-USP36 C131A plasmid for 48 h. β-Actin was used as the internal control. Q-R Immunohistochemical (IHC) staining to evaluate USP36 and ERα expression in HCC tissues. In Panels E-R, the results are representative of three independent experiments. All the data are presented as the means ± SDs. *P < 0.05; **P < 0.01; ***P < 0.001 (Student’s t test)

Fig. 5

USP36 promotes breast cancer progression via ERα. A Immunoblot analysis showing the expression level of ERα and USP36 in MCF-7 cells stably transduced with shUSP36, transfected with Flag or Flag-ERα plasmid. β-Actin was used as the internal control. B GREB1, PKIB, PS2 and PDZK1 mRNA levels were determined by real time PCR after treatment with Flag or Flag-ERα plasmid for 48 h in MCF-7 cells stably transduced with shUSP36. C MCF-7 cells stably transduced with shUSP36 were transfected with Flag or Flag-ERα plasmid for 48 h. The CCK-8 assays were used to detect the cell viability at the indicated time points. D-E MCF-7 cells stably transduced with shUSP36 were transfected with Flag or Flag-ERα plasmid for 48 h. Transwell assays were used to detect migration ability. E shows the quantitative analysis of the Transwell assays results. F-G MCF-7 cells stably transduced with shUSP36 were transfected with Flag or Flag-ERα plasmid for 48 h. Wound healing assays were used to detect migration ability. G shows the quantitative analysis. H-I MCF-7 cells stably transduced with shUSP36 were transfected with Flag or Flag-ERα plasmid for 48 h. The percentage of apoptotic cells was determined by FACS analysis. PI and Annexin V staining were performed on the cells. I shows the quantitative analysis. Three independent experiments were conducted to get the results shown in Panels A-I. All the data are presented as the means ± SDs. **P < 0.01; ***P < 0.001 for comparisons (Student’s t test)

Fig. 6

USP36 associates with ERα and modulates ERα stability in breast cancer cell. A Immunofluorescence staining assay showing the localization patterns of USP36 and ERα in MCF-7 cells. Intracellular localization of USP36 (green) and ERα (red) is shown. Nucleus (blue) were stained with DAPI. Scale bar, 20 µM. B-C Immunoprecipitation assay showing the endogenous interaction between USP36 and ERα. For examining the endogenous interaction between USP36 and ERα, lysates of MCF-7 cells were precipitated with anti-ERα or anti-USP36 antibodies, and the precipitates were examined by immunoblotting with 2% input sample. D Schematic of the ERα protein, along with the ERα deletion mutants (residues 1–180, 1–300, 180–595 and 300–595) used in the Co-IP assays. Schematic of the USP36 protein, along with the USP36 deletion mutants (residues 1–420; 421–800 and 801–1121) used in the Co-IP assays. E–F Immunoprecipitation assay showing AF1 domain is required for ERα to interact with USP36. HEK293T cells were co-transfected with 2 µg USP36 plasmid and full-length HA-ERα or mutant ERα (1–180, 1–300, 180–595 and 300–595). After 24 h, the cells were treated with 10 μM MG132 for 6 h. Then, the cells were harvested with NP-40 lysis buffer. Co-IP was performed using an anti-Flag antibody, and the possible interacting ERα domains were detected with anti-HA antibody. G Immunoprecipitation assay showing USP36 interacts with ERα through its USP domain (1–420). Co-IP was performed using an anti-HA antibody, and the possible interacting USP36 domains were detected with anti-Flag antibody. H; K; N USP36 and ERα protein levels were determined by western blotting. Cells by treat with 10 μM proteasome inhibitor MG132 for 6 h. I-J; L-M; O-P USP36 and ERα protein levels were determined by western blotting. MCF-7 cells were treated with 100 μM cycloheximide (CHX) for the indicated times. The expression of ERα protein was estimated by ImageJ software and is represented graphically in the right panel (J, M, P). In Panels A-C, E-I, K-L, N–O, the results are representative of three independent experiments. All the data are presented as the means ± SDs. *P < 0.05; **P < 0.01 (Student’s t test)

Fig. 7

USP36 regulates ERα protein stability by inhibiting K48-linked polyubiquitination of ERα. A Polyubiquitinated ERα was detected via western blotting. HEK293T cells were co-transfected with 1 µg Flag-ERα plasmid, 0.5 µg HA-Ub plasmid and 0.5 µg Myc-tag or Myc-USP36 plasmids, plasmids in HEK293T cells upon MG132 treatment and then immunoblotted with the indicated antibodies. B Polyubiquitinated ERα was detected via western blotting. MCF-7 cells were transfected with 1 µg Flag-ERα plasmid, 0.5 µg HA-Ub plasmid and 20 μM USP36 siRNA upon MG132 treatment for 6 h and then immunoblotted with the indicated. C-D K48-specific polyubiquitinated ERα was detected via western blotting in HEK293T and MCF-7 cells with indicated antibodies. E–F K48R-specific polyubiquitinated ERα was detected via western blotting in HEK293T and MCF-7 cells with indicated antibodies. G Total polyubiquitinated ERα was detected via western blotting in HEK293T cells with indicated antibodies. H K48-specific polyubiquitinated ERα was detected via western blotting in HEK293T cells with indicated antibodies. I K48R-specific polyubiquitinated ERα was detected via western blotting in HEK293T cells with indicated antibodies. All the results are representative of three independent experiments

Fig. 8

USP36 inhibition could restore tamoxifen sensitivity in endocrine resistant breast cancer model. A The Kaplan–Meier analysis conducted on data sourced from the meta-analysis available at (https://kmplot.com) revealed a significant correlation between elevated levels of USP36 expression and decreased survival rates among breast cancer patients undergoing tamoxifen treatment. B USP36 depletion reduces the protein level of ERα and ERα Y537S in MCF-7 Y537S cells. Immunoblotting of cell lysates will be performed using specific antibodies with β-actin as the loading control. C Depleting USP36 restores tamoxifen’s inhibitory effect on MCF-7 Y537S cells. Cells will be transfected with siControl or siUSP36 for 24 h, treated with 1 μM tamoxifen for 12 h, and cell metabolic activity will be measured using the CCK-8 assay at specified time points. D Luciferase assay demonstrates that USP36 depletion can restore the inhibitory effect of tamoxifen on luciferase activity in MCF-7 Y537S cells. E Depleting USP36 restores tamoxifen’s inhibitory effect on ERα target genes in MCF-7 Y537S cells. Cells will be transfected with siControl or siUSP36 for 24 h, then treated with 1 μM tamoxifen for 12 h. RNA will be extracted for gene expression analysis, with each group analyzed in triplicate. F-G Transwell assay demonstrates that USP36 depletion reduces the migratory ability of MCF-7 Y537S cells. H-I Wound healing assay demonstrates that USP36 depletion reduces the migratory ability of MCF-7 Y537S cells. J-L In a xenograft model, deletion of USP36 restored the inhibitory effect of tamoxifen on MCF-7 Y537S. Tumor growth was monitored in vivo in different groups of mice by measuring tumor growth (J), tumor volume (K) and weight (L). Three independent experiments were conducted to get the results shown in Panels C-L. All the data are presented as the means ± SDs. *P < 0.05; **P < 0.01; ***P < 0.001 for comparisons (Student’s t test)

Fig. 9

A hypothetical model of the mechanism of USP36 regulation of ERα signaling. The inhibition of USP36 decreases breast cancer progression via modulating ERα K48-linked deubiquitinating, which subsequently suppresses ERα signaling activity and tamoxifen resistance (draw by biorender)