USP7 Induces Chemoresistance in Triple-Negative Breast Cancer via Deubiquitination and Stabilization of ABCB1

Triple-negative breast cancer (TNBC) accounts for 15–20% of all breast cancer. TNBC does not express the estrogen receptor, progesterone receptor, or human epidermal growth factor receptor 2. Cytotoxic chemotherapy and surgery are the current therapeutic strategies for TNBC patients, but the chemoresistance of TNBC limits the efficiency of this strategy and shortens the lifespan of patients. The exploration of targeted therapy is ongoing in TNBC research. The aim of the present study was to identify the mechanism underlying acquired resistance in TNBC through the exploration of the relationship between the expression of USP7 and of ABCB1. We found that ubiquitin specific protease 7 (USP7) is a potential therapeutic target for overcoming the chemoresistance of TNBC. USP7 overexpression increased the chemoresistance of TNBC, while the knockdown of USP7 effectively increased the chemosensitivity of chemoresistant TNBC. A USP7 inhibitor effectively induced apoptosis and suppressed metastasis in chemoresistant TNBC. We further clarified that USP7 is a specific deubiquitinating enzyme for ABCB1 that plays an essential role in drug resistance. USP7 directly interacted with ABCB1 and regulated its stability. We concluded that USP7 promotes the chemoresistance of TNBC by stabilizing the ABCB1 protein.


Introduction
Breast cancer (BC) is the most commonly diagnosed malignancy globally. In 2020, an estimated 2.26 million cases were diagnosed, with 685,000 deaths resulting from the disease, according to the World Health Organization [1]. BC is a heterogeneous disease, and is currently divided into three major subtypes based on gene expression profiles: estrogen receptor (ER) positive (luminal A and B), human epidermal growth factor receptor (HER2) positive, and triple-negative BC (TNBC) [2,3]. Different biological subtypes are further associated with discrepancies in treatment responses and disease-specific outcomes [4,5]. The outcome for patients diagnosed with HER2 enriched or ER-positive BCs has been improved by the development of targeted therapies which significantly reduce the risk of death and relapse. However, in patients with TNBC, treatment options remained limited, and the prognosis is relatively poor. TNBC tumors are resistant to endocrine therapy and HER2-targeting treatments, making cytotoxic chemotherapy the only approved and validated treatment in adjuvant and neoadjuvant settings [6].
Although optimal protocols are yet to be determined, anthracycline and taxane-based regimens are presently the backbone of systemic therapeutics for TNBC. A platinumbased regimen has produced favorable results in neoadjuvant and metastatic settings [7]. However, the development of chemoresistance often limits the efficacy of current standard therapeutic regimens, and the establishment of an optimal treatment for TNBC remains the biggest challenge in increasing the overall survival rate in this group of patients. Many mechanisms which may contribute to the development of anticancer drug resistance have been studied [8]. Of these, ATP-binding cassette (ABC) transporter-mediated drug efflux has received considerable attention [9,10]. The overexpression of ABC transporters is related to both anthracycline and taxane resistance in breast cancer, since doxorubicin and paclitaxel are substrates of p-glycoprotein (Pgp) encoded by the ABCB1 gene [11][12][13][14]. Thus, a rational approach to overcoming chemoresistance is to target the ABCB1 transporter using activity inhibitors. A wide range of ABCB1 transporter inhibitors have been studied, but they are either too toxic to be beneficial, lack specificity, or have little impact on drug accumulation [15][16][17]. Regardless of these poor initial results, research into ABCB1 transporter inhibitors is ongoing. These findings suggest that the development of TNBC chemoresistance may be related to multiple pathways, and further exploration of the mechanisms that directly induce ABCB1 overexpression is warranted.
Ubiquitination is a reversible, post-translational modification that covalently conjugates a ubiquitin (Ub) moiety onto a substrate molecule, a process which may further regulate the activity, interactions, and stability of substrate proteins [18]. Ubiquitination of protein is constantly reversed by deubiquitinating enzymes (DUBs) that remove Ub from substrates and therefore keep them from proteosome-dependent degradation [19]. Ubiquitin specific protease (USP) 7 is one of the most widely studied DUBs functioning in proteosome-dependent degradation [20]. A large number of studies have demonstrated an association between USP7 expression and the development of drug resistance in multiple malignancies, in response to treatment with chemotherapeutic agents [21]. Raised USP7 expression may suggest a poor tumor prognosis; therefore, it has been considered by some researchers to be a marker for prognosis and a potential drug target for anticancer therapy [22].
Dysfunction of the ubiquitination and proteasomal system, especially the degradation of ABCB1, plays a crucial role in anticancer drug resistance [23,24]. These results led us to investigate the regulation of the stability of ABCB1. Ubiquitination modification functions are not limited to protein degradation, but also include the regulation of other biological processes, including DNA replication and repair, receptor response, protein trafficking, gene transcription, and protein-protein interactions [25]. In the present study, we thought to identify the mechanism underlying acquired resistance in TNBC through the exploration of the relationship between the expression of USP7 and of ABCB1. We evaluated the expression and effects of USP7 in chemoresistant TNBC cells, and showed that ABCB1 expression is directly regulated by USP7, a member of the DUBs, which in turn regulates the chemoresistance of TNBC through the USP7-ABCB1 pathway.

Chemicals
Doxorubicin (98% of purity) and paclitaxel (98% of purity) were obtained from Cayman Chemical (East Ellsworth Rd., MI, USA). GNE-6776 (95% of purity) was obtained from Carbosynth (Old Station Business Park, Compton, UK). The stock solution of GNE 6776 was dissolved in dimethyl sulfoxide (DMSO) (Sigma, St. Louis, MO, USA) and stored at −20 • C. The less than 0.1% of DMSO in culture medium was used in all treatments.

Cell Culture
MDA-MB-231 cells was purchased from the ATCC (American Type Culture Collection). MDA-MB-231 cells was treated with doxorubicin or paclitaxel, and selected resistance cells, MDA-MB-231-DoxR and MDA-MB-231-PtxR, respectively. MDA-MB-231-DoxR cells were incubated in DMEM (Dulbecco's modified Minimal Essential Medium) medium with high glucose, and 10% of FBS containing 5 nM of doxorubicin and MDA-MB-231-PtxR cells was incubated in DMEM (Dulbecco's modified Minimal Essential Medium) medium with high glucose and 10% of FBS containing 2 nM of paclitaxel. The cell lines tested negative for mycoplasma contamination.

Plasmids
The HA-ABCB1 plasmids were obtained from Addgene (plasmid #10957). The various plasmids were shown in Supplementary Table S3.

Colony Formation Assay
The cells were seeded in 12-well plates for 2-3 weeks later, fixed in methanol at room temperature for 20 min, and then stained with 0.5% crystal violet (Sigma, St. Louis, MO, USA) for 35 min. Images were obtained using a digital camera. The crystal violet was dissolved in 33% acetic acid. Absorption was measured at 550 nm using an ELISA plate reader.

Migration and Invasion Assays
Falcon ® Cell Culture Inserts (CORNING) and BD BioCoat Matrigel Invasion Chambers (Becton Dickson) were used for migration (2 × 10 4 cells/well) and invasion (2 × 10 5 cells/well containing 12 µL matrigel with growth factor, BD) assays, which were performed using 24-well plates for 18 and 24 h, respectively, as described previously. GNE-6776 (50 mg/mL in stock) was used to treat cells for 12 h, and then plates were replaced with fresh medium. The scale bar represents 200 µm.

In-Vivo Xenograft Studies
All animal work was carried out in accordance with The AAALAC international for animal care. Animal experiment in this study was followed by 3Rs guideline and approved by Changhua Christian Hospital. The approval number: CCH-AE-107-010 and the data of approval is 21st of December, 2018.
Seven-to eight-week-old female SCID mice (NLAC, Taiwan) were housed under specific pathogen-free conditions and provided with food and water ad libitum. As described previously [20,22], we numbered mammary ducts 1-10 in the female mice. On day 0, orthotopic mammary tumors were inoculated with the breast tumor cell line MDA-MB-231-DoxR and MDA-MB-231-PaxR (10 × 10 7 cells in saline) at the mammary duct. Tumors were measured twice a week with digital microcalipers, and volumes were calculated (volume = (width 2 × length)/2). Tumors were measured twice weekly, and once the maximal tumor volume was reached (600 to 800 mm 3 ), mice were sacrificed. Tumor volumes are represented as mean volume ± s.d. The number of observed nodules in the lungs was calculated and represented as mean volume ± s.d. The scale bar respresents 1 cm.

Protein Extraction, Western Blot Analysis, RNA Extraction, and Quantitative Real-Time PCR
The extraction of proteins from cells and Western blot analysis was performed as described [27]. The characteristics of the antibodies used were listed (Supplementary Table S1). RNA purification, cDNA synthesis, and quantitative real-time PCR analysis were performed as described before [6]. The sequences of primers used in the real-time PCR experiment were shown (Supplementary Table S2).

Co-Immunoprecipitation and GST Pull-Down Assays
Co-immunoprecipitation experiments were performed by incubating different antibodies (Supplementary Table S1) and were described before [23]. GST pull-down assays were performed by incubating HA-CDK1 with GST-USP7 1-209 or GST-USP7 210-500 protein and Glutathione-Agarose (Sigma, St. Louis, MO, USA). Purification of GST proteins was described [22]. The pulled-down HA-CDK1 protein was detected by Western blot analysis.

Deubiquitination Assays
For the in vivo deubiquitination assay, knockdown of USP7 silencing in MDA-MB-231 cells was performed by lentivirus infection system with shRNA. MDA-MB-231-USP7silencing cells were treated with 50 mM of MG132 (Sigma, St. Louis, MO, USA) to prevent CDK1 protein degradation for 12-16 h and lysed by RIPA buffer. For the in vitro deubiquitination assay, Flag-USP7 wild type and its mutants were expressed in 293T cells, lysed by RIPA buffer, immunoprecipitated by anti-Flag agarose (Sigma, St. Louis, MO, USA), and eluted by Flag peptides (100 mg/mL) in the DUB buffer (50 mM Tris-HCl pH 8.0, 50 mM NaCl, 1 mM EDTA, 10 mM DTT, 5% glycerol). The Myc-ubiquitinated HA-CDK1 were expressed in 293T cells with MG132 treatment, precipitated by protein A beads conjugating with anti-HA antibodies (Sigma, St. Louis, MO, USA), and eluted by 100 mM of HA peptide (Sigma, St. Louis, MO, USA) in the DUB buffer. The process in detail was described before [23].

Statistical Analysis
Data are represented as the mean ± SEM for biological triplicate experiments. All error bar analysis was by STDEV. One-way ANOVA was used to compare different groups at the same concentration; a p value of <0.05 was considered statistically significant.

USP7 Regulates Drug Resistant Activity in Chemoresistant TNBC
To  and PtxR cells at serial doses of Dox and Ptx in a dose-dependent manner ( Figure 3H,I). The MMP-related proteins Bcl2 and BclxL were markedly decreased; BAX and Bim were highly increased in MDA-MB-231-DoxR and PtxR-USP7-silencing cells under 10 nM of Dox and 8 nM of Ptx treatment, respectively ( Figure 3J). The similar results were also observed in MDA-MB-468-DoxR and MDA-MB-468-PtxR cells with USP7 suppression (Supplementary Figure S2B-E).

USP7 Enhances the Metastatic Activity in Chemoresistant TNBC
Since drug resistance causes cancer cells to exhibit increased proliferation and aggression, we further assessed the metastatic ability of chemoresistant TNBC with USP7 suppressed. The migration ability of MDA-MB-231-DoxR-USP7-silencing and MDA-MB-231-PtxR-USP7-silencing cells was repressed compared with control, according to in vitro migration assays ( Figure 4A

USP7 Enhances the Metastatic Activity in Chemoresistant TNBC
Since drug resistance causes cancer cells to exhibit increased proliferation and aggression, we further assessed the metastatic ability of chemoresistant TNBC with USP7 suppressed. The migration ability of MDA-MB-231-DoxR-USP7-silencing and MDA-MB-231-PtxR-USP7-silencing cells was repressed compared with control, according to in vitro migration assays ( Figure 4A,B). The decreased invasive activity of MDA-MB-231-DoxR-USP7-silencing and MDA-MB-231-PtxR-USP7-silencing cells was assessed using in vitro invasion assays ( Figure 4C,D). The markers for epithelial-mesenchymal transition (EMT) were evaluated in MDA-MB-231-DoxR-USP7-silencing and MDA-MB-231-PtxR-USP7-silencing cells using western blotting analysis. Increased levels of E-cadherin and plakoglobin, and decreased levels of vimentin and N-cadherin were observed compared with control ( Figure 4E)

USP7 Regulates ABCB1 Expression in TNBC
We further explored downstream candidates regulated by USP7 involved in the chemoresistance of TNBC. The ABC family members ABCB1, ABCC1, and ABCG2, which are related to drug resistance in several types of cancer, were first evaluated in MDA-MB-231 cells. Only ABCB1 expression was significantly downregulated in MDA-MB-231-USP7-silencing cells compared with control ( Figure 8A). The mRNA level of ABCB1 was not changed in MDA-MB-231-USP7-silencing cells compared with control ( Figure 8B). Reexpression of USP7 rescues the protein level of ABCB1 in MDA-MB-231-USP7-silencing cells ( Figure 8C). The expression of ABCB1 in different TNBC cell lines (MDA-MB-231, BT20, and BT549) was higher than its levels in ER-positive (ER+) BC cell lines ( Figure 8D). Higher expression of ABCB1 was observed in MDA-MB-231-DoxR and MDA-MB-231-PtxR cells than in parental cells ( Figure 8E). The expression of ABCB1 was repressed in MDA-MB-231-DoxR-USP7-silencing and MDA-MB-231-PtxR-USP7-silencing cells (Figure 8F). USP7 upregulated ABCB1 expression in a dose-dependent manner in 293T cells by transient transfection assay ( Figure 8G). Re-expression of ABCB1 in MDA-MB-231-DoxR/PtxR cells promoted chemoresistance activity in response to Dox and Ptx, respectively ( Figure 8H,I).

USP7 Regulates ABCB1 Expression in TNBC
We further explored downstream candidates regulated by USP7 involved in the chemoresistance of TNBC. The ABC family members ABCB1, ABCC1, and ABCG2, which are related to drug resistance in several types of cancer, were first evaluated in MDA-MB-231 cells. Only ABCB1 expression was significantly downregulated in MDA-MB-231-USP7silencing cells compared with control ( Figure 8A). The mRNA level of ABCB1 was not changed in MDA-MB-231-USP7-silencing cells compared with control ( Figure 8B). Reexpression of USP7 rescues the protein level of ABCB1 in MDA-MB-231-USP7-silencing cells ( Figure 8C). The expression of ABCB1 in different TNBC cell lines (MDA-MB-231, BT20, and BT549) was higher than its levels in ER-positive (ER+) BC cell lines ( Figure 8D). Higher expression of ABCB1 was observed in MDA-MB-231-DoxR and MDA-MB-231-PtxR cells than in parental cells ( Figure 8E). The expression of ABCB1 was repressed in MDA-MB-231-DoxR-USP7-silencing and MDA-MB-231-PtxR-USP7-silencing cells ( Figure 8F). USP7 upregulated ABCB1 expression in a dose-dependent manner in 293T cells by transient transfection assay (Figure 8G). Re-expression of ABCB1 in MDA-MB-231-DoxR/PtxR cells promoted chemoresistance activity in response to Dox and Ptx, respectively ( Figure 8H,I).

USP7 Is a Specific Deubiquitinating Enzyme for ABCB1
USP7 is a deubiquitinating enzyme that removes the subtract from the K48-linked polyubiquitin chain and thus stabilizes its protein expression. We first performed coimmunoprecipitation (CoIP) assay experiments to detect interactions between USP7 and ABCB1. ABCB1 was observed in MDA-MB-231-USP7-silencing cells by pulling down the USP7 with ant-USP7 antibodies ( Figure 9A). Flag-tag USP7 interacts with ABCB1 in 293T cells with transient expression of Flag-tag USP7, according to immunoprecipitation experiments using anti-Flag antibodies ( Figure 9B). The N-terminal domain (1-500 aa) of USP7 is the domain which interacted with ABCB1 ( Figure 9C). The reverse experiment also showed the same result ( Figure 9D). USP7 directly interacts with ABCB1 according to GST-pull down assays, and the USP7 domain directly interacting with ABCB1 is 1-209 aa ( Figure 9E). We previously identified that 1-209 aa of USP7 is the protein-protein interaction region [27]. In vivo deubiquitination assays were performed to detect whether ABCB1 is a substrate for USP7 by treating MDA-MB-231-USP7-silencing cells with MG132 and then immunoprecipitating with endogenous ABCB1. Knockdown of USP7 increased the K48-linked polyubiquitin chain of ABCB1 compared with control ( Figure 9F). We further evaluated whether USP7 is a deubiquitinating enzyme for ABCB1 by performing in vitro deubiquitination assays. Purified flag-tag USP7 wild-type (WT) removed K48-linked polyubiquitin chains from purified polyUb-HA-ABCB1. The level of K48-linked polyubiquitin chains of ABCB1 incubated with a loss of function mutant (K443R) of USP7 [27] was similar to that of ABCB1 without USP7 ( Figure 9G).

USP7 Is a Specific Deubiquitinating Enzyme for ABCB1
USP7 is a deubiquitinating enzyme that removes the subtract from the K48-linked polyubiquitin chain and thus stabilizes its protein expression. We first performed co-immunoprecipitation (CoIP) assay experiments to detect interactions between USP7 and ABCB1. ABCB1 was observed in MDA-MB-231-USP7-silencing cells by pulling down the USP7 with ant-USP7 antibodies ( Figure 9A). Flag-tag USP7 interacts with ABCB1 in 293T cells with transient expression of Flag-tag USP7, according to immunoprecipitation experiments using anti-Flag antibodies ( Figure 9B). The N-terminal domain (1-500 aa) of USP7 is the domain which interacted with ABCB1 ( Figure 9C). The reverse experiment also showed the same result ( Figure 9D). USP7 directly interacts with ABCB1 according to GST-pull down assays, and the USP7 domain directly interacting with ABCB1 is 1-209 aa ( Figure 9E). We previously identified that 1-209 aa of USP7 is the protein-protein interaction region [27]. In vivo deubiquitination assays were performed to detect whether ABCB1 is a substrate for USP7 by treating MDA-MB-231-USP7-silencing cells with MG132 and then immunoprecipitating with endogenous ABCB1. Knockdown of USP7 increased the K48-linked polyubiquitin chain of ABCB1 compared with control ( Figure 9F). We further evaluated whether USP7 is a deubiquitinating enzyme for ABCB1 by performing in vitro deubiquitination assays. Purified flag-tag USP7 wild-type (WT) removed K48-linked polyubiquitin chains from purified polyUb-HA-ABCB1. The level of K48-linked polyubiquitin chains of ABCB1 incubated with a loss of function mutant (K443R) of USP7 [27] was similar to that of ABCB1 without USP7 ( Figure 9G).

USP7-Silencing Chemoresistant TNBC Exhibits Significantly Reduced Tumorigenesis and Lung Metastasis in Orthotopic BC Mouse Models
We orthotopically injected the MDA-MB-231-DoxR and MDA-MB-231-PtxR cell lines into female NOD SCID mice. On Day 16, the tumor volumes of mice orthotopically injected with USP7-silencing cell lines mice began to decline in comparison with control mice ( Figure 10A,B), even though the tumors of MDA-MB-231-DoxR and MDA-MB-231-PtxR xenograft models had different growth rates. The tumor disappeared in one MDA-MB-231-PtxR USP7-silencing xenograft model. USP7-silencing cell lines produced significantly decreased numbers of nodules in mice lungs compared to the control group ( Figure 10C,D). Taken together, these results suggested that USP7 plays an important role in tumorigenesis and metastasis in BC xenograft mouse models.

USP7-Silencing Chemoresistant TNBC Exhibits Significantly Reduced Tumorigenesis and Lung Metastasis in Orthotopic BC Mouse Models
We orthotopically injected the MDA-MB-231-DoxR and MDA-MB-231-PtxR cell lines into female NOD SCID mice. On Day 16, the tumor volumes of mice orthotopically injected with USP7-silencing cell lines mice began to decline in comparison with control mice ( Figure 10A,B), even though the tumors of MDA-MB-231-DoxR and MDA-MB-231-PtxR xenograft models had different growth rates. The tumor disappeared in one MDA-MB-231-PtxR USP7-silencing xenograft model. USP7-silencing cell lines produced significantly decreased numbers of nodules in mice lungs compared to the control group (Figure 10C,D). Taken together, these results suggested that USP7 plays an important role in tumorigenesis and metastasis in BC xenograft mouse models.

Discussion
TNBC accounts for 10-20% of annually diagnosed BC, cases and often occurs in younger females [28]. The absence of ER, PR expression, and HER2 amplification currently makes chemotherapy the most effective medical treatment [6]. Although optimal protocols are yet to be determined, taxane and anthracycline-based regimens are presently the backbone of systemic therapeutic options for TNBC. Platinum-based regimens have produced favorable results in neoadjuvant and metastatic settings [7]. Despite having a worse prognosis, patients with TNBC tumors have better pathological complete response (pCR) rates than non-TNBC patients after neoadjuvant chemotherapy [29,30]. However, Liedtke et al. demonstrated that TNBC patients with residual disease are more likely to suffer from early recurrence and die from metastatic disease [29]. The discrepancies between high sensitivity to neoadjuvant therapy and poor clinical prognosis driven by higher relapse rates among patients with residual disease suggests that a considerable proportion of TNBC tumors become drug resistant during treatment, or are inherently resistant.
USP7 was initially believed to function as a p53 deubiquitinating enzyme, resulting in the stabilization of p53 [31,32]. Consistent with this assessment, one study also showed a correlation between low USP7 expression levels and poor patient prognosis in non-small cell lung cancer [33]. However, USP7 was demonstrated to have an oncogenic function in BC, acting by regulating the stability of geminin, an inhibitor of the DNA replication licensing factor, whose levels were shown to be prognostic for poor BC survival in another study [34]. USP7 was shown to deubiquitinate and stabilize the ERα subunit, promoting cell proliferation and tumor growth in ER-positive BC by inhibiting cell cycle arrest and apoptosis [35]. These findings suggest that overexpression of USP7 is an indicator of poor prognosis for BC patients. Upregulation of USP7 is frequently observed in various types of cancer, and the apparent contribution of this upregulation to carcinogenesis has led to speculation that USP7 could be an effective target in anticancer therapies.
In the current study, we utilized established cell lines in which MDA-MB-231 BC cells were selected for survival in increasing concentration of doxorubicin (MDA-MB-231-DoxR) or paclitaxel (MDA-MB-231-PtxR), to better understand the mechanism underlying acquired resistance in TNBC. Cell lines selected under identical conditions with USP7 knockdown were used for comparison. We found that USP7 expression promoted and regulated the chemoresistance of TNBC cells. Silencing of USP7 expression decreased the proliferative and metastatic ability of chemoresistant TNBC cells. A USP7 inhibitor exerted anticancer activity by inducing apoptosis and repressing metastasis of resistant TNBC. We further found that TNBC chemoresistance is regulated by the USP7-ABCB1 pathway. A number of scientific reports have proposed that USP7 is a key effector protein in the emergence of resistance to doxorubicin and paclitaxel chemotherapies in various cancers [20,36,37]. We discovered for the first time that USP7 is a novel regulator of ABCB1 activity and expression. Therefore, USP7 may contribute to resistance via its role in stabilizing ABCB1, which is responsible for the apical transport of these two agents. We revealed that modulation of USP7 expression levels and treatment with USP7 inhibitor led to the downregulation of ABCB1 through the USP7-ABCB1 pathway in anthracycline and taxane-resistant TNBC cells. Besides, PIK3CA (p110α), markers for cell cycle (CDK6, cyclin B1) and proliferation (Ki67, PCNA) were downregulated in chemoresistant TNBC cells by USP7 inhibitor (Supplementary Figure S7) Other mechanisms which may contribute to the development of anticancer drug resistance have also been studied [8]. Among them, ABC transporter-mediated drug efflux has received considerable attention [9,10]. Forty-nine different ABC transporters have been identified in humans, classified into seven subfamilies, A-G [38]. ABCB1 is the most well-studied member of the family, and a large number of studies have demonstrated that ABCB1 expression increases after chemotherapy [39,40]. ABCB1 expression was shown to be higher and more frequently observed in TNBC patients than in ER-positive subtypes, even before the administration of cytotoxic agents [41]. This observation suggests that this transporter may also provide an intrinsic growth advantage to cancer cells and promote their aggressive nature.
Our results support those of many previous studies which have found an ABCB1mediated resistance mechanism in doxorubicin-and paclitaxel-resistant BC cell lines [11,12]. This mechanism is of considerable importance in TNBC, as doxorubicin (anthracycline) and paclitaxel (taxane), which are used routinely in first-line chemotherapy protocols, are well-studied substrates of the ABCB1 transporter [11,12]. Doxorubicin is a cell cycle agent that acts directly on DNA to interfere with DNA transcription, therefore preventing mRNA synthesis and causing cell death. Paclitaxel belongs to a class of cytotoxic drugs, taxanes, that act on the G2 and M phases of the cell cycle to promote the irreversible accumulation of tubulin, resulting in abnormal chromosomal partitioning during the formation of the mitotic spindle. This phenomenon prevents proper mitotic cell division and eventually leads to cell death [18]. Because cancer cells with drug transporters are responsible for resistance to chemotherapy agents, earlier trials have attempted to combine chemotherapeutic drugs with newer target agents to understand the mechanisms underlying tumor invasion, that might reduce TNBC-associated mortality rates [42]. However, the addition of the ABCB1 inhibitor has not succeeded in restoring sensitivity to chemotherapy or improving clinical outcomes in BC patients [15][16][17]. Therefore, most trials were terminated due to severe side effects, and no health authority-approved inhibitors of ABC transporter currently exist [43]. These data also suggested the development of TNBC chemoresistance may be related to the number and diversity of pathways involved, and further exploration of mechanisms that directly induce ABCB1 overexpression is warranted. In future directions, another prominent approach to targeting these transporters to overcome chemoresistance is by inhibiting their expression, rather than using an ABCB1 inhibitor to suppress their activity. We found that modification of USP7 expression might be a valuable tactic to overcome the limitations of anthracycline and taxane resistance. Our results demonstrated pharmacological inhibition of USP7, which further downregulated ABCB1 expression, suppressed tumor growth, and considerably restored the effectiveness of doxorubicin and paclitaxel. We further used the mouse xenograft model to identify the regulatory role of USP7 in TNBC chemoresistance in vivo. The results clearly demonstrated that xenograft tumors from USP7-silencing cell lines were significantly smaller than those of controlled mice. Moreover, silencing of USP7 also markedly inhibited lung tumor metastases in mouse models. Another future focus is how these USP7 inhibitors should be implemented, whether as a single agent or combined treatment. Due to the extensive heterogeneity of TNBC and existence of crosstalk between signaling pathways, it will be interesting to learn whether and how different mechanisms regulate the TNBC chemoresistance independently or coordinately in the future study. Going forward, the requirement of incorporating and validating biomarkers in the future clinical studies of TNBC is also evident.

Conclusions
Collectively, our data produced previously unreported evidence that both doxorubicinand paclitaxel-induced chemoresistance could be reversible following combination treatment with a USP7 inhibitor. In addition, we report for the first time that ABCB1 is a novel substrate of USP7, and USP7 maintains the protein stability of ABCB1. We revealed that modulation of USP7 expression levels and treatment with USP7 inhibitor led to the downregulation of ABCB1 via the USP7-ABCB1 pathway in anthracycline and taxane-resistant TNBC cells. Our findings provide preclinical evidence that USP7 is an attractive target for treatment of drug resistant TNBC, acting via regulation of the USP7-ABCB1 pathway, and offer a rationale for further assessment of USP7 inhibitor for the treatment of TNBC.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/cells11203294/s1, Figure S1: The role of USP family members in MDA-MB-468 cells under chemo-drug treatment; Figure S2: The role of USP7 in chemoresistant activity of MDA-MB-468 cells with chemo-drug resistance. Figure S3: The role of USP7 in migration and invasive activity of chemo-drug-resistant MDA-MB-468 cells. Figure S4: The effect of USP7 inhibitor on chemo-drug-resistant MDA-MB-468 cells. Figure S5: The effect of USP7 inhibitor on migration and invasive activities of chemo-drug-resistant MDA-MB-468 cells. Figure S6: Correlation between USP7 and ABCB1 in vivo.; Table S1: List of proteins tested by and characteristics of the corresponding antibodies. Table S2: Sequence of the oligonucleotides for Quantitative real-time PCR assays. Table S3: List of plasmid constructs used.