Background
Invasive breast carcinoma (BC) is the leading cause of all new cancer diagnoses in women [
1]. Tumor protein p53 pathway inactivation plays an important role in the process of BC tumor genesis. Wild-type p53 is present in approximately 70% of BC cases [
2], and the p53 pathway is partially abrogated through inactivation of various signals or effector elements [
3]. Accordingly, the role of p53 signaling in BC tumorigenesis has attracted considerable attention [
4]. In addition to point mutations and gene deletions, post-translational protein modification abnormalities have been confirmed to be an important mechanism underlying inactivation of p53. Among these abnormalities, ubiquitination is more complex than phosphorylation and acetylation [
5]. Most research focuses on the regulatory effect of ubiquitin ligase on the ubiquitination of p53 [
6]; such research includes the well-known ubiquitination enzyme murine double minute 2 (Mdm2) [
7,
8]. Recently, the N-terminal p53 TAD and Mdm2 pBD regions were studied to discover anticancer drug molecules [
4]. However, limited success was achieved due to tumor recurrence [
9] or
TP53 gene mutations [
10]. The intriguing nature of the regulation of p53 signaling and its role in tumorigenesis are certainly perplexing due to the complexity involved [
4]. Therefore, identifying more strategies to stabilize p53 is particularly important.
The ubiquitination of many proteins has been well documented to be reversed by deubiquitinating enzymes (DUBs), which belong to a superfamily of cysteine proteases and metalloproteases that cleave ubiquitin-protein bonds. The human genome encodes approximately 100 DUBs [
11] that can be classified into the following six families: ubiquitin-specific proteases (USPs), ubiquitin car boxy-terminal hydrolases (UCHs), ovarian tumor (OTUs) proteases, Machado-Joseph disease protein domain proteases (MJDs), JAMM/MPN domain-associated metallopeptidases (JAMMs), and monocyte chemotactic protein-induced proteins (MCPIPs).
In BC, numerous DUBs [
11], including breast cancer-promoting DUBs and cancer-suppressing DUBs, are aberrantly expressed. However, only two deubiquitination enzymes can deubiquitinate and stabilize p53 [
11],and USP7 (HAUSP) might represent the first example [
12]. However, TSPYL5 can bind USP7 and suppress its ability to deubiquitinate and stabilize p53 [
13]. In addition, an interesting feedback loop exists in p53 regulation because USP7 also binds, deubiquitinates and stabilizes Mdm2 more potently under physiologic conditions [
14,
15] and stabilizes p53 under genotoxic stress conditions [
16,
17]. USP10 can deubiquitinate cytoplasmic p53 and inhibit MDM2-mediated p53 nuclear export and degradation. USP10 can also shuttle into the nucleus and stabilize p53 when DNA damage occurs [
18]. However, USP10 may stabilize both wild-type p53 and mutant p53 [
19] and is more highly expressed in breast cancer tissue than in adjacent normal tissue [
20]. Unsurprisingly, such an important tumor suppressor is controlled by multiple DUBs. However, few DUBs have been found in breast cancer, and the mechanisms regulating p53 deubiquitination remain enigmatic.
Our previous study found that OTU deubiquitinase 3 (OTUD3) can deubiquitinate and stabilizes PTEN [
21]. In the current study, we found that the expression of OTUD3 was decreased in BC and proved for the first time that OTUD3 is an enzyme related to the deubiquitination of p53. Compared with PTEN, high expression levels of OTUD3 and p53 are more indicative of a better prognosis in BC. This study further elucidated the influence of OTUD3 on BC cell biological function and its molecular mechanism and suggests that OTUD3 should be explored as a therapeutic target in breast cancer.
Methods
Kaplan-Meier plotter
Correlations between the mRNA expression levels of
OTUD3, TP53 and
PTEN and the prognosis of BC were assessed with the Kaplan-Meier Plotter tool [
22,
23] (
http://kmplot.com/analysis/index). BC patients were divided into two groups according to median expression levels (high expression vs. low expression). A Kaplan-Meier survival chart was used in the analysis to evaluate the relapse-free survival (RFS) of the patients, and the risk ratio (HR) and its 95% confidence interval (CI) and the log- rank test were used to calculate the
p-value.
Cells and tumor tissues
Two human breast cancer cell lines, MCF-7 and DU4475, were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The MCF7 cells were cultured at 37 °C in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS (HyClone, USA) under a 5% CO2 atmosphere. The DU4475 cells were cultured in RPMI-1640 medium supplemented with 10% FBS. This study was approved by the Human Ethics Review Committee of Qilu Hospital (Qingdao) of Shandong University. The use of eighty paired breast cancer tissues and matched adjacent normal tissues was approved by the Department of Pathology of Qilu Hospital (Qingdao) of Shandong University. All patients underwent surgical resection at Qilu Hospital (Qingdao) of Shandong University. Informed consent was obtained from all subjects or their relatives.
Antibodies and reagents
An anti-OTUD3 antibody (HPA028544) for immunohistochemistry (IHC) and the proteasome inhibitor MG132 were purchased from Sigma-Aldrich, USA. An anti-OTUD3 antibody (ab107646), wild-type anti-p53 antibody (ab131442), and anti-p21 antibody (ab109520) for western blotting were purchased from Abcam, United Kingdom. An anti-glyceraldehyde 3-phosphate dehydrogenase antibody (anti-GAPDH) and secondary antibodies were purchased from Santa Cruz Biotechnology, Inc., USA. Anti-Myc and anti-Flag antibodies were obtained from MBL, BEIJING B&M BIO TECH CO.,LTD, Beijing, China.
Immunohistochemistry
IHC was performed by using the avidin-biotin complex method, including heat-induced antigen-retrieval procedures. Incubation with an antibody against OTUD3 (1:100 dilution; HPA028544) was carried out at 4 °C for 18 h. All staining was assessed by a quantitative imaging method (inForm, PerkinElmer) utilizing continuous measurement and pathologists blinded to the sample origins and subject outcomes. The widely accepted German semi-quantitative scoring system based on the staining intensity and area was used. Each specimen was assigned a score according to the intensity of nuclear, cytoplasmic, and/or membrane staining (no staining = 0; weak staining = 1, moderate staining = 2, and strong staining = 3) and the extent of stained cells (0% = 0, 1–24% = 1, 25 = 49% = 2, 50–74% = 3, and 75–100% = 4). The final immunoreactive score was determined by multiplying the intensity score by the extent score and ranged from 0 (minimum) to 12 (maximum).
Lentivirus infection
Lentiviruses carrying shRNA targeting human OTUD3 lentiviral vectors (GV112) were obtained from GeneChem. We constructed lentiviruses carrying overexpression lentiviral vectors. The viruses were used to infect cells in the presence of polybrene. After forty-eight hours, MCF7 or DU4475 cells were cultured in medium containing puromycin for the selection of stable clones. The clones with stable OTUD3 knockdown were identified and verified by western blotting. The shRNA sequences were as follows: OTUD3 no. 1: 5′-TGGAAATCAGGGCTTAAAT-3′; no. 2, 5′-GAGTTACACATCGCATATC-3′; no. 3, 5′-CGTCTGCCATCGCATATTA-3′; and non-targeting control, 5′-TTCTCCGAACGTGTCACGT-3′.
Western blot (WB) analysis
The cells and tissue specimens were lysed using RIPA buffer (Sigma-Aldrich, St. Louis, MO, USA). The protein samples were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes (Millipore). The membranes were blocked with 5% non-fat milk at room temperature for 2 h and incubated overnight with primary antibodies at 4 °C. After washing with TBST three times for 15 min each, the membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies for 2 h at room temperature with slight shaking. GAPDH was used as the loading control. The immunoreactive bands were visualized using SuperSignal West Pico Chemiluminescent Substrates (Thermo Fisher Scientific, USA).
Protein half-life assay
For the p53 half-life assay, the MCF7 and DU4475 cells were grown in 2-cm plates to approximately 60% confluence, and then the cells were transfected with OTUD3 shRNAs. After twenty-four hours, the cells were treated with the protein synthesis inhibitor cycloheximide (CHX, Sigma, 10 μg ml-1) for the indicated durations before collection.
Immunoprecipitation
The cultured cells were lysed with HEPES lysis buffer (20 mM HEPES, pH 7.2, 50 mM NaCl, 0.5% Triton X-100, 1 mM NaF and 1 mM dithiothreitol) supplemented with Protease Inhibitor Cocktail Tablets (Roche). The immunoprecipitations were performed using the indicated primary antibody and protein A/G agarose beads (Santa Cruz) at 4 °C. Then, the immunocomplexes were washed with HEPES lysis buffer four times. Both the lysates and immunoprecipitates were examined using the indicated primary antibodies, followed by incubation with the appropriate secondary antibody and SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific).
GST pulldown assays
Bacterial-expressed GST and GST-p53 bound to glutathione-Sepharose 4B beads (from GE) were incubated with Myc-OTUD3-expressing MCF7 cells for 2 h at 4 °C. Then, the beads were washed with GST binding buffer (100 mM NaCl, 10 mM Tris,50 mM NaF, 2 mM EDTA, 0.5 mM Na3VO4 and 1% Nonidet P40) four times, and the proteins were eluted and subjected to western blotting.
Ubiquitylation assay
The cells were treated with 20 mM MG132 proteasome inhibitor for 8 h. Then, the cells were washed with PBS and lysed in 0.5 ml of HEPES buffer (20 mM HEPES, pH 7.2, 50 mM NaCl, 1 mM NaF, and 0.5% Triton X 100) supplemented with 0.1% SDS and a protease inhibitor cocktail (Roche, Germany). The lysates were centrifuged to obtain the cytosolic proteins. Briefly, the individual samples were incubated with primary antibodies for 3 h, followed by incubation with protein A/G agarose beads (Santa Cruz) for another 8 h at 4 °C. The beads were washed three times with HEPES buffer. The proteins were released from the beads by boiling in 40 ml of 26SDS-PAGE sample buffer for 10 min. The samples were subjected to a WB analysis.
Proliferation assay
The cells were plated in 96-well plates (100-μl cell suspensions, 1*104 cells ml− 1) and assayed for MTS (3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium, inner salt, Sigma) reduction. Twenty-four hours after plating, 0.05 mg ml-1 MTS reagent (Promega) was added to each well, and the cells were incubated at 37 °C for 4 h, followed by absorbance measurement at 490 nm. The sample values were standardized to those of wells containing medium alone.
Apoptosis assays
First,cells were treated with cisplatin (10 mM, 24 h, Sigma-Aldrich, USA). After incubation, the cells were washed with PBS and stained with fluorescein isothiocyanate-Annexin V and propidium iodide according to the manufacturer’s protocol (Beijing Biosea Biotechnology Annexin V Kit). Then, the apoptotic cells (Annexin V-positive, propidium iodide-negative) were determined by flow cytometry.
Cells were resuspended in DMEM containing 0.35% low-melting agarose (Sigma) and 10% FBS and seeded onto a coating of 0.7% low-melting agarose in DMEM containing 10% FBS. The plates were incubated at 37 °C and 5% CO2, and the colonies were scored 3 weeks after preparation. Colonies larger than 0.1 mm in diameter were scored as positive.
Statistical analysis
Differences between two independent groups were evaluated using an unpaired Student’s t-test. Chi-square tests and one-way ANOVA were used to compare the groups. Correlation analysis was performed using Spearman’s rank correlation coefficient. All IHC and WB statistical analyses were performed with GraphPad Prism 7.00 and SPSS 19.0(IBM Corp, USA). All other results are expressed as the mean ± standard deviation (SD) of three independent experiments unless stated otherwise. All statistical tests were two-sided, and p-values< 0.05 (*) or < 0.01 (**) were considered statistically significant.
Discussion
Our experiment proved for the first time that OTUD3 is a tumor-suppressing DUB in BC. The online database analysis showed that BC patients with high OTUD3 and p53 expression have better RFS, thus revealing a potential prognostic biomarker of BC. In addition, the mRNA expression of OTUD3 was lower in BC tissue than in normal adjacent tissue and was unrelated to the staging or molecular type. The clinical sample study of the Qilu cohort further proved that the protein expression of OTUD3 in BC tissues was lower than that in adjacent tissues. OTUD3 expression in cancer tissues was independent of the molecular type and histological classification. Therefore, the absence of OTUD3 is associated with the occurrence of BC. As a tumor suppressor gene, OTUD3 may serve as a new biomarker of the occurrence and development of BC. Targeting the OTUD3 upstream and downstream pathways may be a useful therapeutic strategy because BC cells may have lost the expression of such tumor-suppressing DUBs.
Functional p53 prevents the progression of cancer by increasing growth inhibition in the form of apoptosis, senescence and/or autophagy [
40]. Deubiquitination is a major mechanism that stabilizes p53 and induces apoptosis. Regulation of p53 ubiquitination and deubiquitination in BC is of great interest but remains poorly understood. Our study proves for the first time that downregulation of OTUD3 in clinical BC samples highly coincides with downregulation of p53. OTUD3 can directly interact with and stabilize p53 through deubiquitination in BC cells. The N-terminal of OTUD3 contains an OTU domain that directly participates in the binding of the T2 and T3 sequences of p53. Decreased OTUD3 expression may be an important mechanism underlying the loss of TP53 function in breast cancer cells carrying WT TP53 alleles. Both breast cancer cell lines used in this study were p53 wild-type BC cells—the luminal BC cell line MCF-7 and the TNBC cell line DU4475. The functional experiments using BC cells further confirmed OTUD3 anticancer function. OTUD3 supplements enzymes that can regulate p53 by deubiquitination and participates in protein-protein interactions in BC. Thus, OTUD3 is of great significance.
The major causes of death from breast cancer are relapse, drug resistance and metastasis, which are highly related to dysregulation of the MDM family [
41‐
43]. The MDM family comprises the E3 ligase MDM2 and its close homologue MDM4 (alternatively termed MDMX). MDM2 is a vital regulator of tumor suppressor p53 activity in the breast [
7,
8,
44] and has been identified as an independent prognostic biomarker in BC [
45]. The complex between MDM2 and p53 is largely formed by the interaction between the N-terminal domain of MDM2 and the N-terminal transactivation (TA) domain of p53 (residues 15–29) [
46,
47]. The N-terminal domain of p53 contains the main Mdm2 binding site. The finding that OTUD3 potentially binds the N-terminus of p53 may suggest that both Mdm2 and OTUD3 compete for the same binding site in p53, possibly explaining the observed effects of OTUD3 overexpression and knockdown on p53 ubiquitination and p53 levels in cells. This spatially separates MDM2 from p53, resulting in the stabilization of the p53 protein and allowing p53 to regulate gene transcription, leading to p21 and BAX expression, cell cycle arrest, and/or cell death.
We found that OTUD3 deletion is generally associated with the obliteration of WT p53 in BC, suggesting that OTUD3 loss may be selected by tumors to disrupt the p53 pathway. Although our findings reveal an important mechanism by which p53 can be stabilized by direct deubiquitination and imply that OTUD3 might function as a tumor suppressor in vivo through the stabilization of p53, many questions remain unanswered. Our study found that the OTUD3 mRNA level was not related to DNA methylation through an online database; thus, the reason for the decrease in OTUD3 expression in BC remains to be further explained. In addition, the effect of OTUD3 on other key regulators in the p53 pathway must also be examined.
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