Ubiquitin-Specific Peptidase 8 Modulates Cell Proliferation and Induces Cell Cycle Arrest and Apoptosis in Breast Cancer by Stabilizing Estrogen Receptor Alpha

Breast cancer (BC) is the most common neoplastic and lethal malignancy in women. Although antiendocrine therapy is the main treatment for estrogen receptor alpha (ERα)-positive BC, the development of resistance is a major clinical complication. In this study, we aimed to explore the role of ubiquitin-specific peptidase 8 (USP8) in ERα signaling and identify potential targets for endocrine resistance. Public databases were used to analyze USP8 expression, prognosis, clinical characteristics, and immune cell infiltration. Immunohistochemistry and western blot assays were used to detect protein levels and ERα signaling. Quantitative reverse transcription-PCR was used to measure ERα target gene expression. The cell counting kit-8, wound-healing, clone formation, and Transwell assays were used to investigate the effects of USP8 depletion or inhibition on cell proliferation, migration, and invasion. An immunofluorescence assay was used for localizing USP8 and ERα, and a protein stability assay was performed for detecting the degradation of ERα protein. The cell cycle and apoptosis were assessed using flow cytometry. USP8 was highly expressed in the luminal subtype of BC and was associated with poor prognosis. The infiltration levels of many immune cells were positively correlated with USP8 expression. Depletion of USP8 dramatically decreased the ERα signaling activity and weakened the proliferation, migration, and invasion capabilities of BC cells. USP8 knockdown markedly induced apoptosis and cell cycle arrest (G0/G1). Colocalization analysis and protein stability assays indicated a probable mechanism by which USP8 regulates ERα. Our study demonstrates that USP8 might be crucial in BC development and may be considered a potential target for treating ER-positive BC malignancies in vitro.


Introduction
Breast cancer (BC) is the most prevalent and lethal malignancy in women worldwide [1]. BC is classifed into diverse clinical subsets, according to estrogen receptor alpha (ERα), progesterone receptor (PR), and human epidermal growth factor 2 (HER2) expression [2]. Among these types, ERpositive tumors, also referred to as luminal BC, are the predominant subtype, accounting for nearly 80% of cases [3]. Previous studies have indicated that most ER-positive BC patients can beneft from anti-ERα therapy; nevertheless, approximately half of them may develop drug resistance. ERα plays a crucial role in the development and progression of BC, as it contributes to the expression of oncogenic proteins and induces cell cycle progression [4]. Posttranslational modifcations are dynamic biological processes involved in ERα stability, which may lead to the amplifcation of ERα signaling and tamoxifen resistance [5]. E3 ligases [6][7][8] appear to facilitate ERα signaling by stabilizing the ERα protein. Because ERα plays a signifcant role in the development of drug resistance in luminal BC [9], it is important to explore this mechanism in detail for better management of endocrine therapy.
Several studies have demonstrated that tumorinfltrating immune cells (TICs) are strongly associated with BC progression [17]. Immune components are likely to provide evidence of an immunotherapy response in BC [18]. Studies have shown a correlation between cancer prognosis and degree of immune cell infltration [19][20][21]. However, the biological infuence of TICs in ER-positive BC still requires further investigation.
In our study, we aimed to explore the role of USP8 in ERα signaling and identify potential targets for endocrine resistance.

Public Data Retrieval and
Bioinformatics. USP8 expression was analyzed using UALCAN (https://ualcan.path. uab.edu). Survival time and status were obtained from TCGA dataset (https://portal.gdc.com) for estimating BC prognosis. For Kaplan-Meier curves, p-values and hazard ratios with 95% confdence intervals were obtained using log-rank tests and univariate Cox proportional hazards regression. Te characteristics of patients with BC from the UALCAN and cBioPortal (https://www.cbioportal.org/) online tools were listed, and the mRNA expression matrix of the cancer cells was acquired from the CCLE dataset (https:// portals.broadinstitute.org/ccle) [22]. Te analysis was performed using the R software package ggplot2 (v4. 1.3). Correlations between USP8 and ERα targets, apoptosisrelated, and cell cycle-regulated genes are presented as scatter plots of TCGA data using Pearson's correlation analysis. (GSEA) and Analysis of Immune Infltration. Te c2.cp.keggv7.2 gene and hallmark collections were obtained from the molecular signatures database (MSigDB) and analyzed using the GSEA via homonymous software. Te signifcant gene sets conformed to the standards of "nominal (NOM) p value <0.05" and "false discovery rate (FDR) q-value <0.25."

Gene Set Enrichment Analysis
Te proportion of TIC profles across BC cases was evaluated using CIBERSORT [23]. Only cases with a p value <0.05 were selected for the follow-up analysis. Te correlation between USP8 expression and tumor purity and immune cell infltration levels in BC and luminal subtypes was investigated using the Tumor Immune Estimation Resource (TIMER) 2.0 platform (https://timer.compgenomics.org/).

Cell Lines and Cell
Culture. Cells were acquired from the American Type Culture Collection (ATCC), and human BC cell lines MCF7 and T47D were cultured in minimum essential medium and Roswell Park Memorial Institute-1640 medium (Biosharp, China) supplemented with 10% fetal bovine serum (FBS). HEK293T cells were grown in Dulbecco's Modifed Eagle Medium supplemented with 10% FBS. All cells were grown at 37°C in a humidifed 5% CO 2 incubator. 17β-Estradiol (E2; Sigma-Aldrich) was dissolved in ethanol when required for the assays. (IHC). BC specimens were obtained from the remaining tissues of patients who underwent surgery from September 2021 to March 2022 at the Zhongnan Hospital of Wuhan University. All diagnoses were confrmed by two experienced pathologists. ER, PR, HER2, and triple-negative BC (TNBC) subtypes were included. Specifc primary antibodies against USP8 were used for IHC. Immunohistochemical scores were assessed using the ImageJ software.

RNA Extraction and Quantitative
Reverse Transcription-PCR (qRT-PCR) Analysis. Total RNA was extracted from cancer cells using a HiPure Total RNA Mini Kit (Magen), according to the manufacturer's protocol. cDNA was synthesized using a cDNA reverse transcription kit (Abclonal). We used a total volume of 10 μL, including 2 × SYBR Master Mix (Abclonal), template cDNA, and a mixture of each forward and reverse primer. Nuclease-free water was used to dilute components. Te qRT-PCR was conducted in triplicate. Relative expression was normalized to that of ubiquitously expressed 36B4 and calculated using the ΔΔCT method. Te primer sequences used are listed in Supplementary Table S1.

Western Blot (WB)
Analysis. MCF7 and T47D cells were lysed using the RIPA extraction reagent (Servicebio) supplemented with protease and phosphatase inhibitors. Total protein was separated using 10% or 12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a 0.45 μm polyvinylidene fuoride membrane (Millipore). Te membrane was then successively blocked in Trisbufered saline with 0.05% Tween-20 containing 5% skim milk for one hour, treated with primary antibodies overnight and secondary antibodies for an hour (Supplementary  Tables S2 and S3). An enhanced chemiluminescence WB substrate was used to develop the blots.

Cell Counting Kit-8 (CCK-8) Assay for Cell Viability.
BC cells were plated in clear-bottom 96-well plates and processed according to the manufacturer's instructions. CCK-8 solution was added to each well, and the absorbance at 450 nm was measured every 24 h.

2.9.
Wound-Healing (WH) Assay. Monolayer-confuent human breast cells were wounded with a single pass of a 200 μL pipette tip, and cell medium was replaced with 1% FBS. Te ImageJ software was used to assess the WH rate with the following equation: WH rate(%) � (initial wound area − nonhealed area) initial wound area . (1)

Colony Formation Assay.
After incubation for 2 weeks, colonies were fxed with paraformaldehyde and stained with 0.1% crystal violet. Only colonies with more than 50 cells were counted. Te colony formation rate was estimated as the number of colonies/number of seeded cells × 100.
2.11. Transwell Assay. Transwell assays were used to assess cell migration and invasion capacity. For the invasion assay, the upper chambers were coated with Matrigel (BD BioCoat, USA). Indicated cells resuspended in serum-defcient medium (200 μL) were seeded into the upper chambers, whereas the bottom wells were flled with complete medium (600 μL). After 24 h, the cells migrating through the membrane were fxed with 4% paraformaldehyde and stained with 0.1% crystal violet and then counted under a microscope.

Flow Cytometry
Analysis. MCF7 and T47D cells were adjusted to a density of 1 × 10 6 cells/mL, inoculated in 6-well plates, and incubated at 37°C and 5% CO 2 . Cells were harvested, washed with phosphate-bufered saline, and centrifuged at 300 × g. Following the manufacturers' protocol, we resuspended the cell pellets in 1 mL DNA staining solution and incubated them at 37°C for 30 min without light. For apoptosis analysis, the pellets were resuspended in a mixture of 5 μL fuorescein isothiocyanate (FITC)/ Annexin V and 10 μL PI staining solution combined with 500 μL 1 × binding bufer and incubated for 10 min. Cell cycle distribution and apoptosis were monitored using fow cytometry with a FACScan fow cytometer (Beckman, cat. #FC500, USA).

USP8 Is Associated with Poor Outcome of BC and with ERα and PR Protein Levels in Human BC Specimens.
USP8 is highly expressed in BC according to the CPTAC database ( Figure 1(a)). Te clinicopathological characteristics correlating with USP8 expression are shown in Table 1. USP8 expression showed signifcant diferences according to race and the ER, PR, and PAM50 status. Te survival Kaplan-Meier analysis of TCGA database revealed that USP8 is associated with poor prognosis in BC patients (Figures 1(b)-1(d)). In the BC cohort, USP8 expression was signifcantly associated with the ER and PR status, N stage, and PAM50 subtype but had no signifcant association with the HER2 status (Figures 1(e)-1(h)). Consistently, the level of USP8 protein was high in BC tissues, especially in ERpositive BC patients (Figure 1(i)). Based on the CCLE dataset, USP8 was also highly expressed in MCF7 and T47D BC cell lines (Figure 1(j)), consistent with our own fnding (Figures 1(k) and 1(l)). As USP8 is highly expressed in the luminal BC subtype and is related to ERα protein levels, the correlation between USP8 and ERα target gene expression suggests that USP8 is positively associated with PS2, PDZK1, GREB1, and CCND1 (Figure 1(m)). Moreover, USP8 was also positively correlated with BCL2 and negatively correlated with BAX ( Figure 1(m)). Furthermore, USP8 was positively associated with CDK2, CDK4, and CDK6 ( Figure 1(m)).

Relationship between USP8 and Proportion of TIC
Subtypes. Using the CIBERSORT method, we constructed the immune cell profles of 22 BC cases and analyzed the proportion of TIC subtypes (Figures 2(a) and 2(b)). Four TIC subtypes, found to have a strong link with USP8 Journal of Oncology  (Figure 3(a)). Overall, DUB-IN-3 reduced the proliferation rate of MCF-7 cells (Figure 3(b))-the higher the concentration, the more obvious the inhibition of cell proliferation, which was more apparent with the addition of tamoxifen (Figure 3(c)). Next, we used shRNA in BC cell lines. Te knockdown efciency of USP8 was verifed at both the protein and transcriptional levels ( Figure S1). USP8 knockdown also increased the sensitivity of MCF7 and T47D cells to ta-

USP8 as a Putative
Marker for Stabilizing ERα. GSEA was carried out individually for the high-and low-USP8-expression groups. For the c2 gene set in MSigDB, genes in the high-USP8-expression group were mainly enriched in metabolism-related biological processes (Figure 4(f )). Simultaneously, genes in the low-USP8-expression group were enriched in oxidative phosphorylation, protease, and ribosome-related processes (Figure 4(g)). Similarly, several immune activities and metabolic functions were enriched in the estrogen response in both early and late phases of hallmark gene sets (Figures 4(h) and 4(i)).

USP8 May Interact with ERα in the Cytoplasm and Modulate ERα Protein
Stability. An immunofuorescence assay showed that USP8 was localized to both the nucleus and the cytoplasm, whereas ERα was mainly localized to the nucleus, in BC cells ( Figure 5(i)). USP8 knockdown signifcantly reduced the level of ERα protein, which could be reverted by the proteasome inhibitor MG132 (Figure 5(g)).
As CHX inhibits protein synthesis, the protein half-life assay indicated that USP8 knockout in MCF-7 cells evidently impaired the endogenous stability of ERα ( Figure 5(h)).

Discussion
Te present results indicate that USP8 is upregulated in BC tissues and is associated with poor prognosis and immune cell infltration. USP8 was localized to both the cytoplasm and nucleus, and its knockdown attenuated ERα signaling activity, cell proliferation, migration, and invasion, probably via ubiquitination of ERα. USP8 depletion stimulated cell cycle arrest and apoptosis. ER-positive BC is characterized by slow disease progression and relatively good prognosis, while 30% of patients sufer from metastases or endocrine resistance due to clinical heterogeneity [24]. Various mechanisms have been proposed to elucidate endocrine resistance, including changes in ER regulators and diferent signaling pathways [25][26][27][28][29]. Posttranslational modifcations have been studied for their role in ERα signaling, among which ubiquitination of ERα is a crucial factor in endocrine insensitivity [6][7][8]30]. When ERα is stimulated with estrogen, it can be transferred to the nucleus and bind to cis-regulatory DNA region of target genes, promoting gene expression [31]. Tamoxifen, an estrogen receptor modulator, has tissue-specifc agonistic and antagonistic efects on ER [32].
USPs are the most common DUBs; they can eliminate ubiquitin chains from the target proteins and may be involved in regulating the protein ubiquitination process [33]. USP8 has a catalytic domain located on its C-terminus and is upregulated in various malignancies [12]. We sought to elucidate the association between USP8 and ER signaling, as this mechanism remains unclear.
Our study shows that USP8 is highly expressed in BC samples in public databases and is associated with poor overall survival. USP8 was found to be upregulated in ERpositive BC compared with that in other subtypes, which lays the foundation for subsequent research.
Although anti-ER treatment is the primary therapy for ER-positive BC, immunotherapy cannot be neglected. Interestingly, while meaningful responses to immune modulation appear to be limited to TNBC [34], our fndings on the   caused by its disrupting efect on regulatory T-cell functions [35]. A recent study showed that USP8 might be an immunomodulatory target that enhances the efcacy of anti-PD-1/PD-L1 in treating human carcinomas [36]. Compared with the existing work related to immune therapy in ER-      Journal of Oncology positive BC patients [37], our study also shows that high USP8 expression may indicate better outcomes and responses to immune therapy due to the high proportions of TICs. USP8 plays a vital role in potentiating cell proliferation in lung and cervical cancers [13,15], leading to cell cycle dysregulation and accelerated apoptosis [16,38,39]. Similarly, our study confrms that depletion of both DUB-IN-3 and USP8 inhibited cell viability, which was inhibited more severely at higher concentrations of tamoxifen [40]. And tamoxifen sensitivity was further increased after USP8 knockdown. USP8 knockdown also suppressed the colony formation and migration capacity of BC cells. Furthermore, shUSP8 increased the apoptosis rate, with BAX (proapoptotic) upregulation and BCL2 (antiapoptotic) downregulation. USP8 depletion also caused cell cycle arrest at the G0/G1 phase in two diferent BC cell lines, which is in line with previous studies [39]. We further hypothesized that the cell cycle-related proteins CDK2/4/6 and cyclin D1 were reduced in the shUSP8 group. We next observed that the depletion of USP8 decreased ERα protein levels. qRT-PCR and bioinformatics analyses showed that shUSP8 downregulated PS2, PDZK1, GREB1, and CCND1. WB assays demonstrated that USP8 stabilized ERα. When cells were treated with 10 nM E2 for 6 h, both USP8 and ERα protein levels were rescued (Figures 5(e)and 5(f )). Inhibition of the proteasomal degradation pathway with MG132 had a remarkable efect on stabilizing ERα protein levels even when USP8 was depleted. Based on the inhibitory efects of CHX on protein translation, we found   that USP8 knockdown in MCF7 and T47D cells decreased the stability of endogenous ERα, which presented a shortened half-life in the shUSP8 group. Immunofuorescence analysis revealed that USP8 is localized to both the nucleus and cytoplasm, whereas ERα is mainly localized to the nucleus, indicating the location of interaction of both the proteins. In addition, we performed GSEA and found that high USP8 expression correlated with the estrogen response.

Conclusions
Overall, we verifed the role of USP8 in ER-positive BC cells as an ER αstabilization-mediating deubiquitinase. USP8 expression is higher in ER-positive BC, and upregulation of USP8 mediates cell proliferation and apoptosis and facilitates the cell cycle of BC cells. We provide in vitro evidence that USP8 is a crucial mediator of endocrine resistance in ER-positive BC and could be a novel therapeutic target for treating endocrine-resistant cancers. Further research into the direct interaction between USP8 and ERα is warranted to determine the exact interaction between the two proteins.

Data Availability
All data supporting the results of this study are shown in this published article and supplementary documents.

Conflicts of Interest
Te authors declare that there are no conficts of interest. Table S1: primer sequence used for qRT-PCR.