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Targeting the USP7-CDK1 axis suppresses estrogen receptor-positive breast cancer progression

Cancer Cell International volume 25, Article number: 60 (2025) Cite this article

Abstract

Estrogen receptor-positive breast cancer (ERPBC) accounts for approximately 70% of breast cancers in women worldwide. The therapeutic strategy process for ERPBC is well-established and significantly reduces the mortality rate. The discovery of new therapeutic targets remains essential for ERPBC patients with metastasis or endocrine resistance. This study indicated that USP7 is highly expressed in ERBPC and promotes tumor progression and metastasis. Inhibition of USP7 activity repressed proliferation, induced apoptosis, suppressed migration and invasive activities, and reversed the epithelial-mesenchymal transition of ERPBC. Mass spectrometry analysis indicated that USP7 regulates CDK1 expression, which is highly expressed and correlates with a poor overall survival rate in ERPBC. USP7 directly interacts with CDK1 and regulates its stability. The combined inhibition of USP7 and CDK1 by GNE-6776 and Ro-3306 synergistically represses the malignant process and metastasis of ERPBC. These findings proved that targeting USP7 and CDK1 is a potential strategy for overcoming endocrine resistance in patients with advanced ERPBC.

Introduction

Breast cancer is a heterogeneous disease with subtypes identified according to molecular and prognostic classifications [1], and estrogen receptor-positive breast cancer (ERPBC) accounts for approximately 70% of all breast cancers diagnosed [2]. The prognosis of early-stage ERPBC is outstanding, and mortality rates for the advanced stage have consequently decreased over time because of early detection and advancements in treatment. The most significant developments have been a consequence of the privation of estrogen signaling, which has evolved from the use of selective estrogen receptor modulators to aromatase inhibitors and selective estrogen receptor degraders [3]. Treatment for ERPBC in the metastatic setting comprises endocrine therapy with targeted CDK 4/6 inhibitors, which are effective for a median of approximately 2 years. Recently, new treatments have been explored to improve outcomes for patients with advanced or metastatic ERPBC, including novel anti-ERPBC genes and combination therapies that target multiple pathways involved in the growth and survival of ERPBC. These therapies may improve outcomes in patients with advanced or metastatic disease.

Ubiquitin-specific-processing protease 7 (USP7) is a deubiquitinating enzyme stabilizing several oncogenic proteins, such as MDM2 and c-Myc, promoting tumor growth and survival [4, 5]. USP7 has been identified as a critical regulator of immune checkpoint proteins in cancer cells, including PD-L1 and CTLA-4 [6]. Inhibition of USP7 enhances antitumor immune responses and sensitizes cancer cells to immunotherapy. USP7 is highly expressed in several types of cancer, including ovarian, pancreatic, and lung cancer, and USP7 dysregulation has been associated with poor prognosis and resistance to therapy. Therefore, USP7 is a potential biomarker for cancer diagnosis and prognosis [7], and USP7 inhibitors are currently being developed and tested in preclinical and clinical studies [8].

Cell cycle dysregulation is a significant hallmark of malignancies that causes uncontrolled cell proliferation and subsequent tumor progression. Cyclin-dependent kinase 1 (CDK1) is a critical regulator of cell cycle progression that triggers mitotic entry and controls the initial phase of mitosis [9]. Moreover, CDK1 regulates DUB3 phosphorylation and BMAL1-UHRF1 pathway to promoter tumor metastasis [10, 11]. The dysregulation of CDK1 in malignancy can therefore have greater significance than that of other interphase CDKs that are involved in cell cycle checkpoints. CDK1 overexpression has been observed in many cancers, including breast cancer, and is a biomarker associated with poor prognosis and tumor progression [12, 13]. An earlier study showed that the use of CDK1 inhibitors was associated with favorable treatment outcomes in BC, indicating a potential role of targeting CDK1 in BC treatment [14]. Nonetheless, the upstream regulators of CDK1 remain unknown.

This study investigated the additional characteristics of USP7 in ERPBC by exploring the relationship between the expression of USP7 and CDK1. We investigated the expression and effects of USP7 in ERPBC and demonstrated that CDK1 levels are regulated by USP7, which promotes tumor progression in ERPBC.

Materials and methods

Chemicals and cell culture

Diethyl sulfoxide (DMSO) was obtained from VWR Chemicals, LLC. (Mastsonford Rd., USA). GNE-6776 (purity ≥ 95%) was obtained from MedChemExpress (NJ, USA). Ro-3306 (purity ≥ 95%) was purchased from Cayman (MI, USA). GNE 6776 and Ro-3306 were solubilized in DMSO and stored at − 20℃. MCF7, T47D, MDA-MB-231, and MDA-MB-468 cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). All cells were incubated in Dulbecco’s modified minimal essential medium containing high glucose concentration and 10% fetal bovine serum.

Protein extraction and western blot analysis

The protocols used for protein extraction and western blotting have been described previously [15]. RIPA buffer containing protease inhibitors was used for protein extraction. Cell lysates were harvested by centrifugation at 13,000 rpm at 4 °C for 10 min, and protein content was determined using the Bradford method (Bio-Rad Laboratories). For western blot analysis, 15–50 µg of cell lysates from each cell line with/without GNE-6776 or Ro-3306 treatment were loaded onto 8–12% SDS-PAGE gels and transferred onto nitrocellulose membranes (GE Healthcare Life Science, Bucks, UK). Membranes were incubated with antibodies overnight at 4 °C and probed with antibodies (listed in Supplementary Table 1). Signals were detected using an ECL chemiluminescence kit (GE Healthcare Life Science). Membranes were photographed using the Invitrogen iBright FL1500 Imaging System (Thermo Fisher Scientific, MA, USA).

Lentivirus infection experiment

A lentivirus containing short hairpin RNAs (shRNAs) was generated using a lentiviral vector (pLKO.1-puro) in 293T cells. The sequences and clonal designations of the pLKO-scrambled plasmid and other pLKO plasmids that caused the knockdown of USP7 have been detailed before [16].

Mass spectrometry analysis

The endogenous USP7 expression was suppressed in HepG2 cells by a lentivirus infection system carrying USP7 shRNA and followed that USP7 wild-type or inactive mutant (K443R) was transiently expressed in HepG1-USP7-silencing cells. The cell was lysed following the protocol for protein extraction. The iTRAQ-based mass spectrometry analysis was performed to investigate the downregulated expression of targets in USP7-silencing cells with transient expression of USP7 mutants versus it with transient expression of USP7 wild-type (Tools Biotechnology, R.O.C).

Plasmids

The HA-CDK1 plasmids were obtained from Addgene (plasmid #1888) The various plasmids are shown in Supplementary Table 3.

MTT assay

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to detect cell viability. To detect the effect of USP7 suppression on ERPBC cell lines, Cells were plated in 96-well plates for 0, 24, 48, and 72 h. To determine the cytotoxicity of drugs on ERPBC cell lines, MCF7 and T47D cells were plated in 96-well plates for 24 h and then treated with various concentrations of GNE-6776 or Ro-3306 (10 µM) for 72 h. Plates were then washed with 1× PBS and 30 µL of MTT added per well, followed by incubation for 3–4 h at 37℃. The resulting blue formazan crystals were dissolved in DMSO, and absorption was detected at 595 nm using an ELISA plate reader. The relative value was compared with the initial value at 0 h.

BrdU proliferation assay

BrdU proliferation was performed using the BrdU Cell Proliferation Assay Kit (BioVision, CA, USA). Cells were plated in 96-well plates overnight, fresh culture medium was added, and incubation was continued for 96 h. BrdU was then added to wells and incubated at 37 °C for 2–4 h. Cell proliferation was detected according to the manufacturer’s instructions. Absorption was analyzed at 450 nm using an ELISA plate reader. The relative value was compared with the control.

Colony formation assay

Cells (8 × 103) were plated in a 12-well plate, and the culture medium was changed every 3 days. After two weeks, the cells were washed with PBS and fixed with methanol at 25oC for 30 min. Fixed cells were stained with 0.5% crystal violet (Sigma, St. Louis, MO, USA) for 1 h, washed with water, and air-dried. Cells were photographed, and crystal violet was dissolved in 33% acetic acid, and absorption was measured at 550 nm using an ELISA plate reader.

Apoptosis analysis

Cells (3 × 104) were plated in 12-well plates after 24 h and treated with 10 µM of GNE-6776 or 10 µM of Ro-3306 for 96 h. Cells were harvested and incubated with Muse™ Annexin V & Dead Cell kit reagent (Merck Millipore, Billerica, MA, USA) at room temperature for 20 min in the dark. Apoptotic cells were analyzed using a MuseTM Cell Analyzer (Merck Millipore), and apoptosis levels were measured using MUSE 1.7 Analysis software (Merck Millipore).

Mitochondrial membrane potential analysis

Cells (3 × 104) were plated in 12-well plates after 24 h and treated with 10 µM of GNE-6776 or 10 µM of Ro-3306 for 72 h. Harvested cells were resuspended in Muse™ MitoPotential Kit reagent (Merck Millipore) and incubated at 37℃ for 20 min. The mitochondrial membrane potential (MMP) of cells was measured using the Muse™ Cell Analyzer (Merck Millipore), and the total depolarized percentage was analyzed using MUSE 1.7 Analysis software (Merck Millipore).

RNA extraction and real-time quantitative PCR

RNA purification, cDNA synthesis, and real-time quantitative PCR (qPCR) were performed as described previously [16]. Total RNA was isolated from cultured cells using Trizol (Invitrogen Life Technologies, Carlsbad, CA, USA). Quantitative real-time PCR was performed on a PRISM ABI7700 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) with standard program settings. Each reaction contained 0.1 µg of cDNA and 80 µmol/L of primers in a 1X SYBR Green Mixture (ABI), with a total volume of 50 µL. The qPCR primers are shown in Supplementary Table 2.

Coimmunoprecipitation and GST pulldown assays

CoImmunoprecipitation (CoIP) experiments were performed by incubating different antibodies (Supplementary Table 1) as described previously [16]. GST pulldown assays were performed by coincubating HA-CDK1 with GST-USP7 1–209 or GST-USP7 210–500 protein and glutathione–agarose (Sigma). The purification of GST proteins was performed as previously described [15]. The pulled-down HA-CDK1 was detected by western blotting.

In vitro migration and invasion assay

Falcon® Cell Culture Inserts (CORNING) and BD BioCoat Matrigel Invasion Chambers (Becton Dickinson) were used for migration (2 × 10⁴ cells/well) and invasion (2 × 10⁵ cells/well containing 12 µL Matrigel with growth factor, BD) assays, conducted in 24-well plates for 20 and 30 h, respectively. Control-, USP7-silencing, and reconstitution of vector control or CDK1 in USP7-silencing breast cancer cells were used in migration and invasion assays. In the combination treatment experiments, breast cancer cells were treated with GNE-6776 (50 mg/mL stock solution) with or without Ro-3366 for 10 h, after which the medium was replaced with fresh media. The scale bar represents 200 μm.

In vitro deubiquitination assays

Wild-type Flag-USP7 and dominant-negative mutants (K443R) were expressed in 293T cells, which were lysed in RIPA buffer. Immunoprecipitation was performed with anti-Flag-conjugated agarose (Sigma) and eluted using Flag peptides (100 mg/mL) in DUB buffer (50 mM Tris-HCl pH 8.0, 50 mM NaCl, 1 mM EDTA, 10 mM DTT, 5% glycerol). Myc-ubiquitinated HA-CDK1 was expressed in 293T cells with MG132 treatment, precipitated with anti-HA-conjugated protein A beads (Sigma), and eluted with 100 mM of HA peptide (Sigma) in DUB buffer. The detailed process has been described previously [16].

In vivo xenograft studies

All animal experiments were performed in accordance with the AAALAC international guidelines for animal care. Eight-week-old female SCID mice (NLAC, Taiwan) were housed under specific pathogen-free conditions and provided food and water ad libitum. Due to the high cost of GNE-6776 and the requirement for compliance with animal ethics guidelines violation, the ERPBC cell lines with suppression of USP7 were used to evaluate the effect of the USP7-CDK1 axis on ERPBC proliferation. On day 0, orthotopic mammary tumors were inoculated with MCF7 and T47D-USP7i silencing cells (5 × 107 cells in Matrigel). Tumors were measured once a week using digital microcalipers, and volumes were calculated as volume (width2 × length) / 2). The average tumor volume reached 50–100 mm3 on day 20, and mice were then injected with Ro-3306 at 3 mg/kg per mouse (n = 6) intraperitoneally, and six mice were administered with solvent treatment as a control (n = 6). Tumors were measured twice weekly, and once the maximal tumor volume was reached (400–600 mm3), mice were euthanized. Tumor volumes are represented as mean volume ± s. d.

Primary cell models

Primary cells were isolated from the tumor tissue of breast patients with the estrogen receptor subtype using the tumor dissociation kit (Miltenyi Biotec., #130-095-929) and the gentlMACS dissociation kit (Miltenyi Biotec., #130-093-235). The isolated primary ERPBC cells were treated with GNE-6776 and Ro-3306, followed by MTT assay and Annexin V apoptosis analysis.

Statistical analysis

Data are presented as means ± standard deviation (mean ± SD) and at least three independent experiments. Statistical analysis was conducted using either a Student’s t-test or ANOVA to assess significance among groups across three independent experiments. A p-value of < 0.05 was set as the threshold for statistical significance, which is indicated in each figure. * Significant differences are represented as: *,#p < 0.05, **,##p < 0.01, ***,###p < 0.001.

Results

USP7 plays an essential role in the malignant progression and metastasis of estrogen receptor-positive breast cancer

To investigate the role of USP7 in ERPBC, bioinformatic analysis was conducted using the UALCAN database (https://ualcan.path.uab.edu/analysis.html), which indicated that higher expression of USP7 was observed in primary breast tumors (Fig. 1A), stages 1 to 3 of breast cancer compared with normal breast (Fig. 1B), and ERPBC (luminal type) compared with normal and triple-negative subtypes (Fig. 1C). Moreover, a high expression of USP7 resulted in poor overall survival in patients with ERPBC, according to the Kaplan–Meier analysis (https://kmplot.com/analysis/) (Fig. 1D). Western blotting was performed to confirm that USP7 was highly expressed in ERPBC cell lines (MCF7 and T47D) compared with that in TNBC cell lines (MDA-MB-231 and MDA-MB-468) (Fig. 1E and F). Therefore, we hypothesized that USP7 upregulation was highly correlated with the malignant progression of ERPBC.

Fig. 1
figure 1

USP7 promotes the malignant progression of ERPBC. (A–C). USP7 levels in normal tissues and breast cancer. Data shown were obtained from UALCAN (https://ualcan.path.uab.edu/). Data set: Gene: USP7; TCGA dataset: breast cancer; USP7 expression in (A): comparison between sample types; (B): comparison between normal tissues and stages of breast cancer; (C): comparison between normal types and subclasses of breast cancer. (D) The overall survival rate correlated with the levels of USP7 in ERPBC using the Kaplan–Meier Plotter (https://kmplot.com/analysis/). Data set: Gene: USP7; Sample type: ERPBC. (E) USP7 levels in two subtypes of breast cancer cell lines assessed via Western blot assay. (F) Quantitative analysis of USP7 expression in ERPBC and TNBC cell lines

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To further investigate the role of USP7 in the malignant progression of ERPBC, USP7 silencing was performed in two ERPBC cell lines (MCF7 and T47D) using a lentivirus infection system carrying shRNA (Fig. 2A and B). MCF7 and T47D cells with USP7 silencing exhibited repressed cell viability compared with control cells. (Fig. 2C). BrdU assay analysis indicated that USP7 silencing decreased the synthesis of new DNA in proliferating MCF7 and T47D cells (Fig. 2D). The colony formation assay indicated that USP7 repression caused lower growth ability of MCF7 and T47D cells than that of control cells (Fig. 2E and F).

Fig. 2
figure 2

USP7 induces the proliferation of ERPBC cells. (A) USP7 expression in MCF7 (upper) and T47D (lower)-USP7-silenced cells. (B) Quantitative analysis of USP7 protein levels in ERPBC cell lines with USP7 suppression. (C) The proliferation ability of MCF7 (left) and T47D (right) cells with USP7 knockdown assessed via MTT assays in a time-dependent manner. (D) Proliferation of MCF7- (left) and T47D- (right) USP7–silenced cells assessed via BrdU incorporation assay. (E) Growth of MCF7- (left) and T47D- (right) USP7-silenced cells assessed via tumor formation assay. (F) Quantitative analysis of MCF7 (left) and T47D (right) cell growth with USP7 silencing using a tumor formation assay

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We then explored the role of USP7 in ERPBC metastasis. In vitro migration assays indicated that the migration of MCF7 and T47D cells with USP7 silencing was repressed compared with that of the control (Fig. 3A and B). The decreased invasiveness of USP7-silenced MCF7 and T47D cells was also observed using in vitro invasion assays (Fig. 3C and D). The expressions of epithelial-mesenchymal transition (EMT) markers and EMT-related transcriptional factors were detected in MCF7 and T47D cells with suppression of USP7. The expressions of epithelial-mesenchymal markers (E-cadherin, γ-catenin, Vimentin, and N-cadherin) and EMT-related transcription factors (Twis1t, Snail, Slug, and ZEB1) in MCF7 and T47D cells with suppression of USP7. The results indicated USP7 silencing causes the upregulation of epithelial markers, E-cadherin and γ-catenin, and downregulation of mesenchymal markers, Vimentin and N-cadherin, Twist1, Snail, Slug, and ZEB1 in ERPBC cell lines (Supplementary Fig. 1A and C).

Fig. 3
figure 3

USP7 enhances the metastasis of ERPBC cells. (A) Migration of MCF7 (upper) and T47D (lower) cells with silencing assessed via an in vitro migration assay. (B) Quantitative analysis of migration of MCF7- (left) and T47D- (right) USP7-silenced cells. (C) Invasiveness of MCF7 (upper) and T47D (lower) cells with silencing assessed via an in vitro invasion assay. (D) Quantitative analysis of MCF7- (left) and T47D- (right) USP7-silenced cell invasiveness

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Suppression of USP7 activity induces apoptosis and suppresses metastasis in estrogen receptor-positive breast cancer cells

To clarify the function of USP7 in ERPBC, an inhibitor for USP7, GNE-6776, was used. IC50 values for MCF7 and T47D cells at 72 and 96 h were 27.2 and 31.4 µM and 31.8 and 37.4 µM, respectively (Fig. 4A) and were higher than that in the literature due to the different cultural conditions and experimental process [17]. GNE-6676 treatment dose-dependently induced apoptosis of MCF7 and T47D cells (Fig. 4B and C). MMP, which is correlated with apoptosis, was evaluated to assess the effect of GNE-6776 on ERPBC. The MMP in MCF7 and T47D cells under a serial dose of GNE-6776 treatment was depolarized in a dose-dependent manner (Fig. 4D and E). GNE-6776 treatment was used to investigate its effect on ERPBC metastasis. GNE-6776 treatment dose-dependently downregulated the migration activity of MCF7 and T47D cells (Fig. 5A and B). GNE-6776 treatment also suppressed the invasive activity of MCF7 and T47D cells in a dose-dependent manner (Fig. 5C and D). Inhibition of USP7 activity also caused the increase in the expressions of epithelial markers, E-cadherin and γ-catenin, and the decrease in expressions of mesenchymal markers, Vimentin and N-cadherin, and EMT-related transcription factors, Twist1, Snail, Slug, and ZEB1 (Supplementary Fig. 1B and D).

Fig. 4
figure 4

Inhibition of USP7 induces apoptosis of ERPBC cells. (A) IC50 values of USP7 inhibitor (GNE-6776) for MCF7 (left) and T47D (right) cells at 72 and 96 h. (B) Apoptotic levels of MCF7 (upper) and T47D (lower) cells treated with GNE-6776 assessed via an Annexin V-dependent flow cytometry assay. (C) Quantitative analysis of GNE-6776-induced apoptosis of MCF7 (upper) and T47D (lower) cells. (D) Depolarization of the mitochondrial membrane potential (MMP) of MCF7 (upper) and T47D (lower) cells treated with GNE-6776 as assessed by flow cytometry. (E) Quantitative analysis of GNE-6776-induced dysfunction of MMP in MCF7 (upper) and T47D (lower) cells

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Fig. 5
figure 5

Suppression of metastasis ability by USP7 inhibitor in ERPBC cells. (A) Image of GNE-6776-treated MCF7 (upper) and T47D (lower) cells in the in vitro migration assay. (B) Quantitative analysis of the migration of GNE-6776-treated MCF7 (left) and T47D (right) cells. (C) Invasiveness of GNE-6776-treated MCF7 (upper) and T47D (lower) cells assessed via an in vitro invasion assay. (D) Quantitative analysis of the invasiveness of MCF7 (left) and T47D (right) cells after GNE-6776 treatment

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USP7 directly regulates CDK1 stabilization in estrogen receptor-positive breast cancer

USP7 is a deubiquitinating enzyme. The novel downstream target regulated by USP7 was investigated via mass spectrometry analysis of HepG2-USP7-silenced cells with overexpression of wild-type USP7 and an inactive mutant (K443R) (Fig. 6A). The 350 proteins were downregulated in USP7-suppressed cells with re-expression of the K443R mutant, most of them were highly correlated with cell cycle. Interestingly, one of the downregulated proteins was cyclin-dependent kinase 1, CDK1 (also called CDC2) (the value shown in Supplementary Table 4) exhibited a higher expression in breast cancer cells than in normal types (Fig. 6B). CDK1 was also highly expressed in the luminal subtype of breast cancer cells than in normal breast cells (Fig. 6C). Patients with breast cancer exhibiting elevated CDK1 expression had a lower overall survival rate, as indicated by the Kaplan–Meier analysis (Fig. 6D). The bioinformatic evidence about USP7 and CDK1 raised a possibility that USP7 regulates CDK1 expression to promote ERPBC progression.

Fig. 6
figure 6

USP7 regulates CDK1 expression in ERPBC cells. (A) GO analysis of mass spectrometry for comparison between the re-expression of negative-dominant mutant (K443R) and wild-type (WT) USP7-suppressing cells. (B–C) mRNA levels in normal tissues and breast cancer. Data shown were obtained from UALCAN (https://ualcan.path.uab.edu/). Data set: Gene: USP7; TCGA dataset: breast cancer. mRNA expression levels in (B): comparison between sample types; (C): comparison between normal types and subclasses of breast cancer. (D) The overall survival rate correlated with mRNA levels in ER-positive breast cancer using Kaplan–Meier Plotter (https://kmplot.com/analysis/). Data set: Gene: CDK1; Sample type: ERPBC. (E) Protein levels of CDK family members in GNE-6776-treated MCF7 (left) and T47D (right) cells assessed via western blotting. (F) Quantitative analysis of CDK1, CDK2, and CDK4 protein levels in GNE-6776-treated MCF7 (upper) and T47D (lower) cells. (G) Expression of USP7 and CDK1 in MCF7 (left) and T47D (right) cells with USP7 suppression assessed via western blotting. (H) Quantitative analysis of CDK1 and USP7 protein levels in MCF7 (left) and T47D (left)-suppressing cells. (I) CDK1 and USP7 levels in 293T cells with transient overexpression of CDK1 and a serial dose of USP7 assessed via western blotting. (J) Quantitative analysis of CDK1 and USP7 protein levels in 293T cells. with transient overexpression of CDK1 and a serially diluted dose of USP7. (K) mRNA levels in MCF7- (left) and T47D- (right) USP7-suppressed cells assessed via qPCR

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Moreover, only the expression of CDK1 was downregulated in MCF7 and T47D cells treated with a serial dose of GNE-6776 compared with that of other CDK family members, CDK2 and CDK4 (Fig. 6E and F). The expression of CDK1 was also downregulated in MCF7-USP7- and T47D-USP7-silenced cells compared with that in control cells, as shown by western blotting (Fig. 6G and H). The expression of Flag-USP7 in 293T cells was upregulated in a dose-dependent manner more than that of HA-CDK1 (Fig. 6I and J). The mRNA level of CDK1 was unchanged in MCF7 and T47D cells with the suppression of USP7 (Fig. 6K). Our results indicate that USP7 regulates the protein expression of CDK1 in ERPBC. To evaluate the function of USP7 on the CDK1-regulated cell cycle, the cell cycle alternations were assessed in USP7 inhibitor-treated MCF7 and T47D cells by flow cytometry analysis. The results indicated that G2/M cell cycle arrest was observed in the ERPBC cell line under GNE-6776 treatment since CDK1 plays a key role in cell cycle progression through the G2/M phase transition (Supplementary Fig. 2A and B). This evidence strengthens the effect of the relationship between USP7 and CDK1 on ERPBC proliferation.

To further understand that the USP7-CDK1 axis regulates ERPBC progression. The activities of viability, migration, and invasion of MCF7-USP7-silenced and T47D-USP7-silenced cells with expression of CDK1 were assessed by MTT and in vitro migration and invasion assays. The results showed that the repressed viability and downregulated migration and invasion activities by suppressing USP7 expression were rescued in ERPBC-USP7-silenced cells with transient expression of CDK1 lines (supplementary Fig. 3A to F). To prove that USP7 is a specific deubiquitinating enzyme for CDK1, a CoIP experiment was performed to detect the interaction between USP7 and CDK1 by using ERPBC-ctrl cell as a positive control and ERPBC-USP7-silenced cell as a negative control. Immunoprecipitating USP7 with anti-USP7 antibodies detected endogenous CDK1 in MCF7 and T47D cells, but this was not observed in MCF7 and T47D cells with USP7 silencing (Fig. 7A). The transient overexpression of USP7 and CDK1 in 293T cells was performed to detect the interaction between Flag-USP7 and HA-CDK1 using CoIP. HA-CDK1 was detected in 293T cells by immunoprecipitating Flag-USP7 with anti-Flag antibodies (Fig. 7B). A similar result was observed in the reverse CoIP experiment (Fig. 7C). We then investigated the direct interaction between USP7 and CDK1, and a transient expression system was first performed in 293T cells with HA-CDK1 overexpression and Flag-USP7 truncated domains. The CoIP experiment indicated that HA-CDK1 interacted with the N-terminal domain (residues: 1–500) of Flag-USP7 (Fig. 7D, left), which also interacted with HA-CDK1 in the reverse CoIP experiment (Fig. 7D, right). The N-terminal domain of USP7 contains the catalytic domain and is a protein-protein region [16]. The GST pulldown assay was performed to explore the direct interaction between USP7 and CDK1 and showed that residues 210–500 of USP7 contained the region that directly interacted with CDK1 (Fig. 7E). The in vitro deubiquitination assay showed that purified K48-linked polyubiquitinated CDK1 was downregulated significantly (0.53 fold) by incubation with wild-type USP7, and this was changed slightly (0.83 fold) when incubated with a domain-negative mutant of USP7 (USP7 K443R) (Fig. 7F). Our findings showed that USP7 promotes tumor progression and metastasis of ERPBC via direct regulation of CDK1 stability.

Fig. 7
figure 7

USP7 directly regulates CDK1 stability in ERPBC cells. (A) Interaction between USP7 and CDK1 in MCF7- (left) and T47D- (right) USP7-suppressed cells assessed via immunoprecipitation of USP7 with anti-USP7 antibodies and western blotting. (B) Interaction between CDK1 and USP7 in 293T cells with transient expression of HA-tag CDK1 and Flag-tagged USP7 assessed via immunoprecipitation of CDK1 with anti-Flag antibodies and western blotting. (C) Interaction between CDK1 and USP7 in 293T cells with transient expression of HA-tag CDK1 and Flag-tag USP7 assessed via immunoprecipitation of CDK1 with anti-HA antibodies and western blotting assay. (D) Interaction between the truncated domain of USP7 and CDK1 in 293T cells with the transient expression of HA-tag CDK1 and either the 1–500 or 500–1100 amino acid mutants of Flag-tag USP7 assessed via immunoprecipitation with anti-Flag (left) or anti-HA (right) antibodies and western blotting. (E) Direct interaction between purified truncated mutant of GST-USP7 and purified HA-CDK1 via GST pulldown assay. (F) Levels of K48-linked polyubiquitinated CDK1 incubated with wild-type (WT) or domain-negative mutant (K443R) of purified USP7 assessed via an in vitro deubiquitination assay with anti-Ub K48 antibodies. TRAF: Tumor necrosis factor Receptor–Associated Factor; CD: Catalytic domain; Ubl: ubiquitin-like

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Co-inhibition for USP7 and CDK1 synergistically induces apoptosis and suppresses metastatic activities in estrogen receptor-positive breast cancer

To prove the relationship between USP7 and CDK1 in the ERPBC progression, MCF7 and T47D cells were treated with the USP7 inhibitor GNE-6776 and the CDK1 inhibitor Ro-3306. The IC50 values of Ro-3306 for MCF7 and T47D cells were first detected via the MTT assay. IC50 values for MCF7 and T47D cells were 27.8 and 15.1 µM at 48 h and decreased to 18.5 and 9.5 µM at 72 h. (Fig. 8A). The viability of MCF7 and T47D cells treated with 10 µM GNE-6776 and Ro-3306 was synergistically decreased compared with that following single treatments with GNE-6776 and Ro-3306 (Fig. 8B). The combination treatment with GNE-6776 and Ro-3306 also synergistically induced apoptosis in MCF7 and T47D cells compared with single treatments with GNE-6776 and Ro-3306 (Fig. 8C and D). Increasing depolarization of MMP in MCF7 and T47D cells treated with GNE-6776 and Ro-3306 was also observed (Fig. 8E-F). The combination treatment with GNE-6776 and Ro-3306 synergistically suppressed migration and invasive activity in MCF7 and T47D cells (Fig. 9A–D).

Fig. 8
figure 8

Dual inhibition of USP7 and CDK1 synergistically causes apoptosis in ERPBC cells. (A) IC50 value of CDK inhibitor (Ro-3306) for MCF7 (left) and T47D (right) cells at 48 and 72 h. (B) Viability of MCF7 (left) and T47D (right) cells following combined GNE-6776 and Ro-3306 treatment assessed via MTT assay. (C) Apoptosis levels of MCF7 (upper) and T47D (lower) cells following combined GNE-6776 and Ro-3306 treatment assessed via Annexin V-based flow cytometry analysis. (D) Quantitative analysis for apoptotic levels of MCF7 (left) and T47D (right) cells with GNE-6776 and Ro-3306 treatment. (E) Depolarization of mitochondria membrane potential (MMP) following combined treatment of MCF7 (upper) and T47D (lower) cells with GNE-6776 and Ro-3306 assessed via flow cytometry analysis. (F) Quantitative analysis of depolarized levels of MMP in combined GNE-6776- and Ro-3306-treated MCF7 (left) and T47D (right) cells

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Fig. 9
figure 9

Synergistic suppression of combination treatment targeting USP7 and CDK1 on metastasis of ERPBC cells. (A) Image of combined GNE-6776- and Ro-3306-treated migration of MCF7 (upper) and T47D (lower) cells using in vitro migration assay. (B) Quantitative analysis of the migration of MCF7 (left) and T47D (right) cells under GNE-6776 and Ro-3306 treatment. (C) Image of the invasiveness of MCF7 (upper) and T47D (lower) cells treated with GNE-6676 and Ro-3306 assessed via an in vitro invasion assay. (D) Quantitative analysis of the invasiveness of MCF7 (left) and T47D (right) cells after combined treatment with GNE-6776 and Ro-3306

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Targeting USP7-CDK1 axis exhibits significant therapeutic activity in a xenograft mouse model and primary cell models of estrogen receptor-positive breast cancer

The control and the USP7-silenced of MCF7 and T47D cells (approximately 5 × 107 cells per mouse) were subcutaneously injected into NOD SCID female mice. These mice were intraperitoneally injected with Ro-3306 (3 mg/kg) thrice a week after tumors grew to the appropriate size (the diameter is about 0.5 to 0.7 cm). The tumor volumes in 3 mg/kg of the Ro-3306 treatment group were significantly decreased at 87.5% and 90.2% at day 50, respectively, compared with those in untreated mice in the MCF7-USP7-si and T47D-USP7-si xenograft models. (Fig. 10A and B). Since repression of USP7 downregulated the growth of MCF7 and T47D cells, the tumor size in the untreated control group at day 25 is comparable to that in the USP7-si group without treatment at day 50 (Fig. 10B and supplementary Fig. 4B). The tumor volumes in the 3 mg/kg of Ro-3306 group were slightly decreased at 30.2% and 50.4% at day 25, respectively, and lowered at 56.9% and 45.8% at day 35, compared with those in untreated mice (supplementary Fig. 4A and B). Moreover, the administration of Ro-3306 at 3 mg/kg did not present observable toxic effects on mice, and body weights remained stable in the USP7-silenced group (Fig. 10C and supplementary Fig. 4C). Primary breast cancer cells from patients with ER-positive subtype were isolated and cotreated with GNE-6776 and Ro-3306 for MTT and Annexin V apoptosis assays. Synergistic repression of viability and apoptotic induction in primary ERPBC cells with combined treatment with GNE-6776 and Ro-3306 were consistent with the results of cell line experiments (Fig. 10D to F). Overall, these results suggest that inhibition by targeting the USP7–CDK1 axis has a valid therapeutic effect on ERPBC.

Fig. 10
figure 10

Effect of inhibition of USP7 and CDK1 on ERPBC in ex vivo primary cell and in vivo xenograft models. (A) Eight-week-old female SCID mice were subcutaneously injected with MCF7 and T47D breast cancer cells. Tumors were excised (six mice per group) on day 50 after euthanizing mice. (B & C) Tumor sizes and body weights were measured twice per week and calculated using the formula V = (L × W2) / 2. Statistics are represented as mean volume ± SD (D) Primary ERPBC cells were cotreated with GNE-6776 and Ro-3306 for the MTT assay. (E) Apoptosis levels of primary ERPBC cells treated with GNE-6776 and Ro-3306 were analyzed via an Annexin V apoptosis assay. (F) Quantitative analysis of apoptosis induced by co-treatment of GNE-6776 and Ro-3306 in primary cells (G) A graphic model for the findings in this report

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Discussion

The current study investigated the function and molecular pathways of USP7 in ERPBC and demonstrated that USP7 plays a critical role by targeting CDK1. Herein, functional assays showed that the inhibition of USP7 decreased cell proliferation migration and invasion abilities but increased apoptosis in ERPBC. The online bioinformatics data from the UCLAN database and the Kaplan-Meier Method showed that higher expressions of USP7 and CDK1 were observed in 563 cases of breast cancer with luminal type compared with normal breast, and both elevated USP7 and CDK1 expressions present a poorer survival rate in breast cancer patients with luminal type. Moreover, our study showed that USP7 promotes breast cancer progression via the regulation of CDK1 stability. The USP7-CDK1 axis exhibits high potential co-biomarkers to evaluate estrogen-receptor-positive breast cancer malignancy in clinical therapy. Their correlation should be clarified further in the future.

We discovered that USP7 is a novel deubiquitinating enzyme for CDK1 activity and highly expressed in ERPBC. The cell cycle is a highly regulated and tightly controlled cell process organized by checkpoints involving CDKs and cyclins, among which CDK1 is central to cell cycle progression [9, 18]. Alternations in CDK1 activity often cause unrestricted cell proliferation, a hallmark of malignant breast tumors [19,20,21]. Therefore, identifying mechanisms that may interfere with the stability of CDK1 is relevant for understanding malignant progression and metastasis in BC. The USP7/CDK1 axis may regulate the cell cycle and play a crucial role in regulating ERPBC malignancy. On the other hand, the literature found that short-term inhibition of USP7 activity causes uncontrolled activation of CDK1 to promote cell death [22]. However, our finding indicated that long-term inhibition of USP7 activity or USP7 silencing downregulates CDK1 expression to induce apoptosis. The literature indicated that long-term stress exposure triggers different molecular mechanisms in cells to exhibit different cellular physiology compared with short-term stress exposure [23, 24]. Moreover, our previous finding indicated that USP7 could be K48-linked polyubiquitinated, and this translational modification causes USP7 to function as a protein scaffold to regulate downstream target gene expression transcriptionally, except acting as a deubiquitinating enzyme [16, 25]. The possible molecular mechanism of the discrepancy in CDK1 between the two studies was that USP7 was still K63-linked polyubiquitinated in cancer cells under short-term USP7 inhibitor treatment and controlled active status of remaining CDK1 via transcriptional regulation of CDK1 phosphorylation/dephosphorylation. However, CDK1 stabilization was downregulated and the remaining CDK1 proceeded into the protein degradation stage in cancer cells treated with USP7 inhibitor for a long-term time or suppression of USP7 expression, thus causing G2/M cell cycle arrest and induction of apoptosis.

Breast cancer is a heterogeneous disease, classified into several subtypes, such as ERPBC, based on the molecular features of the tumor. USP7 is a well-studied deubiquitinating enzyme that removes ubiquitin from a substrate molecule to prevent it from proteosome-dependent degradation, and previous studies have shown that USP7 plays an essential role in breast cancer progression [26, 27]. Earlier studies have also demonstrated that USP7 is essential for completing DNA replication and stabilizing proteins involved in numerous oncogenic pathways [28,29,30]. Consistent with these assessments, the analysis from The Cancer Genome Atlas and UALCAN datasets showed the upregulation of USP7 transcript in breast cancer with a greater significant expression in ERPBC. Moreover, a higher USP7 mRNA level is associated with worse outcomes, which is consistent with an earlier study that showed the continued risk of recurrence in patients with ERPBC after their initial diagnosis [31].

The role of USP7 in the malignant process of ERPBC is not well understood, although USP7 inhibitors for cancer therapy are under development in a clinical trial [8]. USP7 has been indicated to regulate the tumorigenesis of ERPBC by stabilizing estrogen receptor α [27]. Therapeutic strategies for ERPBC in clinical medicine have been well established. However, overcoming endocrinal resistance to ERPBC is critical for prolonging the lifetime of patients with advanced or metastatic ERPBC. Insensitivity to anti-ER drugs, such as fulvestrant and tamoxifen, was observed in the endocrine resistance of ERPBC. This study indicated that USP7 directly stabilizes CDK1 expression to regulate the cell growth and metastatic activity of ERPBC. Both USP7 and CDK1 were highly expressed in ERPBC and were related to its poor survival curves. Single-drug treatment exhibits stronger toxicity on mammalian cells and causes the cancer patient stronger side effects. The combinational treatment of USP7 inhibitor and CDK1 inhibitor with lower doses synergistically repressed the ERPBC proliferation and metastasis and further provided a potential therapeutic strategy in clinical breast therapy.

Targeting CDKs may have unintended consequences because they play an important role in normal cell processes. Therefore, understanding the pathways by which CDKs contribute to cancer and normal cell functions is critical to balance the possible benefits of CDK inhibitors in terms of risks and toxicities. The current study not only investigated the function and regulation of the USP7/CDK1 axis at the cellular level in vitro but also characterized the in vivo functions of USP7 in ERPBC. However, ERPBC carcinogenesis and development can be complex. The current study confirmed the interaction between USP7 and CDK1, but further studies are necessary to determine other downstream pathways.

Data availability

No datasets were generated or analysed during the current study.

Abbreviations

USP7:

Ubiquitin-Specific-processing Protease 7

CDK1:

Cyclin-Dependent Kinase 1

ERPBC:

Estrogen receptor-positive breast cancer

CDK4/6:

Cyclin-Dependent Kinase 4 and 6

MDM2:

Murine Double Minute 2

PD-L1:

Programmed Cell Death Ligand 1

CTLA-4:

Cytotoxic T-lymphocyte Associated protein 4

BC:

Breast Cancer

UALCAN:

The University of ALabama at Birmingham CANcer

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Acknowledgements

We thank H.T.L. at Changhua Christian Hospital for experimental assistance in this study.

Funding

This work was supported to H.T.W. by the Ministry of Science and Technology Summit grant (MOST-110-2314-B-371-007-MY3) and Changhua Christian Hospital (112-CCH-IRP-090; 112-CCH-IRP-100); Y.T.L. by Chang Gung Memorial Hospital (CMRPG3M1841); K.W.H. by the Ministry of Science and Technology Summit and Frontier grants (MOST 111-2628-B-039-007-MY3), by China Medical University (CMU112-MF-06), and the “Drug Development Center, China Medical University” from The Featured Areas Research Center Program within the framework of the Higher Education Sprout Project (Ministry of Education, Taiwan).

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Author notes

  1. Joseph Lin, Yueh-Te Lin and Kai-Wen Hsu contributed equally to this work.

Authors and Affiliations

  1. Cancer Research Center, Changhua Christian Hospital, Changhua, 500, Taiwan, R.O.C.

    Joseph Lin, Yi-En Liu, Yun-Cen Chen, Dar-Ren Chen & Han-Tsang Wu

  2. Cancer Genome Research Center, Department of Medical Research and Development, Chang Gung Memorial Hospital at Linkou, Taoyuan, 333, Taiwan, R.O.C.

    Yueh-Te Lin

  3. Research Center for Cancer Biology, Institute of New Drug Development, China Medical University, Taichung, 404, Taiwan, R.O.C.

    Kai-Wen Hsu

  4. Drug Development Center, Program for Cancer Biology and Drug Discovery, China Medical University, Taichung, 404, Taiwan, R.O.C.

    Kai-Wen Hsu

  5. Institute of Translational Medicine and New Drug Development, China Medical University, Taichung, 404, Taiwan, R.O.C.

    Kai-Wen Hsu

  6. Comprehensive Breast Cancer Center, Changhua Christian Hospital, Changhua, 500, Taiwan, R.O.C.

    Joseph Lin, Yung-Liang Yeh, Hsin-Ya Huang & Dar-Ren Chen

  7. Department of Animal Science and Biotechnology, Tunghai University, Taichung, 407, Taiwan, R.O.C.

    Chang-Chi Hsieh

  8. School of Medicine, Chung Shan Medical University, Taichung, 402, Taiwan, R.O.C.

    Dar-Ren Chen

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Contributions

Joseph Lin: Study conception and design, primary tumor collection. Yueh-Te Lin: Study conception and design, Data acquisition and analysis. Kai-Wen Hsu: Study conception and design, Data acquisition and analysis. Yi-En Liu: Data acquisition and analysis. Yun-Cen Chen: Data acquisition and analysis. Yung-Liang Yeh: Primary tumor collection. Hsin-Ya Huang: Primary tumor collection. Chang-Chi Hsieh: Study conception and design. Dar-Ren Chen: primary tumor collection, Data interpretation, final approval and overall responsibility for the published work. Han-Tsang Wu: Study conception and design, Data acquisition and analysis, final approval and overall responsibility for the published work.

Corresponding authors

Correspondence to Dar-Ren Chen or Han-Tsang Wu.

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Ethics approval and consent to partipate

The animal experiment in this study followed the 3Rs guideline (Replacement, Reduction, Refinement) and was approved by the IACUC of Changhua Christian Hospital. The approval number is CCH-AE-107-010, and the approval date is 21 December 2018. The primary tumor was obtained from estrogen-receptor-positive breast cancer patients who underwent surgical resection at Changhua Christian Hospital. The approval number is 230724, and the approval date is 24 November 2023.

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Lin, J., Lin, YT., Hsu, KW. et al. Targeting the USP7-CDK1 axis suppresses estrogen receptor-positive breast cancer progression. Cancer Cell Int 25, 60 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12935-025-03693-2

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Cancer Cell International

ISSN: 1475-2867

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