The Ubiquitin Peptidase UCHL1 Induces G0/G1 Cell Cycle Arrest and Apoptosis Through Stabilizing p53 and Is Frequently Silenced in Breast Cancer

Background Breast cancer (BrCa) is a complex disease driven by aberrant gene alterations and environmental factors. Recent studies reveal that abnormal epigenetic gene regulation also plays an important role in its pathogenesis. Ubiquitin carboxyl- terminal esterase L1 (UCHL1) is a tumor suppressor silenced by promoter methylation in multiple cancers, but its role and alterations in breast tumorigenesis remain unclear. Methodology/Principal Findings We found that UCHL1 was frequently downregulated or silenced in breast cancer cell lines and tumor tissues, but readily expressed in normal breast tissues and mammary epithelial cells. Promoter methylation of UCHL1 was detected in 9 of 10 breast cancer cell lines (90%) and 53 of 66 (80%) primary tumors, but rarely in normal breast tissues, which was statistically correlated with advanced clinical stage and progesterone receptor status. Pharmacologic demethylation reactivated UCHL1 expression along with concomitant promoter demethylation. Ectopic expression of UCHL1 significantly suppressed the colony formation and proliferation of breast tumor cells, through inducing G0/G1 cell cycle arrest and apoptosis. Subcellular localization study showed that UCHL1 increased cytoplasmic abundance of p53. We further found that UCHL1 induced p53 accumulation and reduced MDM2 protein level, and subsequently upregulated the expression of p21, as well as cleavage of caspase3 and PARP, but not in catalytic mutant UCHL1 C90S-expressed cells. Conclusions/Significance UCHL1 exerts its tumor suppressive functions by inducing G0/G1cell cycle arrest and apoptosis in breast tumorigenesis, requiring its deubiquitinase activity. Its frequent silencing by promoter CpG methylation may serve as a potential tumor marker for breast cancer.


Introduction
Breast cancer has been the most common malignancy and the major cause of cancer-related mortality of women worldwide [1]. Although there have been major improvement in the diagnosis and treatment of breast cancer in recent years, early detection methods for breast cancer are still limited. Abnormal promoter methylation is the major mechanism for the inactivation of tumor suppressor genes (TSGs) in tumorigenesis [2,3]. Increased evidences have demonstrated that aberrant promoter methylation is a promising tumor marker for the early detection of multiple malignancies including breast cancer [4,5,6]. Thus, identification of more epigenetically-disrupted TSGs in breast cancer is needed.
Here, we report that UCHL1 is frequently methylated in breast cancer cell lines and primary tumors, but rarely in normal breast tissues and mammary epithelial cells, well correlated with its downregulation or silencing. Promoter methylation of UCHL1 is significantly correlated with pathologic stage of breast cancer and progesterone receptor status. Ectopic UCHL1 expression in breast tumor cells suppresses cell growth, induces G0/G1 arrest and apoptosis through disrupting p53 signaling, depending on its deubiquitinase (DUB) activity, suggesting that UCHL1 is a functional tumor suppressor and potential tumor marker for this cancer.

Reduced expression of UCHL1 in breast cancer
As both UCHL1 and USP10 increase p53 stability by deubiquitination, we first examined the expression of UCHL1 and USP10 in normal human tissues including breast tissues, as well as mammary epithelial and breast cancer cell lines, using RT-PCR. UCHL1 expression was frequently downregulated or silenced in breast cancer cell lines, but broadly expressed in all the normal adult tissues and mammary epithelial cell lines ( Fig. 1A and B), while USP10 is widely expressed in both normal tissues and breast cancer cell lines. Western blot confirmed UCHL1 expression in protein level in breast cancer cell lines, consistent with mRNA level (Fig. 1C).
We further analyzed UCHL1 expression using a tissue microarray carrying 30 breast cancer tissues and paired adjacent non-cancerous tissues by immunohistochemistry. The immunostaining quantification of UCHL1 was analyzed by using Image Pro-Plus (IPP) including 3 parameters: density mean, area sum, and integrated optical density (IOD). Result showed that UCHL1 expression was significantly reduced in breast cancer tissues compared to the adjacent noncancerous tissues (p,0.05) (Fig. 1D). These results suggest that UCHL1 is frequently downregulated in breast cancer.
Promoter methylation of UCHL1 and restoration of UCHL1 expression by demethylation We next investigated whether promoter CpG methylation was responsible for the silencing of UCHL1 in breast cancer. MSP analysis showed that UCHL1 was frequently methylated in 9/10 (90%) breast cancer cell lines, well correlated with expression levels, while no UCHL1 methylation was found in two normal mammary epithelial cell lines ( Fig. 2A).
We then treated methylated and silenced breast cell lines, MB231, MB435 and T47D, with demethylation reagent 5-aza-29- deoxycytidine (Aza) and trichostatin A (TSA). RT-PCR showed that UCHL1 expression was dramatically restored after treatment in these cell lines, together with increase in unmethylated alleles of the UCHL1 promoter (Fig. 2B). These results demonstrate that promoter methylation of UCHL1 mediates its silencing in breast cancer.

UCHL1 methylation and its clinical correlation in breast tumors
To address whether methylation occurs in primary tumors, we analyzed the promoter methylation of UCHL1 in 66 breast tumor samples, 20 breast tumor adjacent tissues, and 28 normal breast tissues using MSP. UCHL1 promoter methylation was observed in 53 of 66 (80%) primary tumors, 3 of 20 (15%) adjacent normal tissues and 1 of 28 (3.5%) normal breast tissues (Fig. 3, Table 1). Detailed BGS analysis of representative samples further confirmed the MSP data (Fig. 3D).
We further analyzed the correlation of UCHL1 methylation with clinicopathological features ( Table 2). UCHL1 methylation was statistically correlated with clinical stage and progesterone receptor (PR) status. However, there was no association of UCHL1 methylation with other clinicopathological characteristics of patients, including age, histological type, tumor size, lymph node metastasis, oestrogen receptor (ER) and Hormone Receptor (HR) status. These results indicate that promoter methylation of UCHL1 plays an important role in breast tumorigenesis and might be a potential tumor marker for this cancer.

Ectopic UCHL1 expression inhibited colony formation and proliferation of breast cancer cells
Silencing of UCHL1 by promoter methylation in breast cancer indicated that UCHL1 might be a functional tumor suppressor in breast tumorigenesis. We thus performed colony formation assay of MB231 and MCF-7 cells which had complete methylation and  silencing of UCHL1. Ectopic expression of UCHL1 markedly suppressed the colony formation abilities of breast tumor cells, compared with vector-transfected cells (down to ,20% and 70%, respectively; Fig. 4). The effects of UCHL1 expression on cell growth at 24, 48 and 72 hrs were also assessed by CCK-8 assay. In UCHL1-expressing MB231 cells, cell growth was significantly decreased at 48 h and 72 h (p,0.05) (Fig. 4D), but increased in vector-expressing cells in a time-dependent manner. These data suggest that UCHL1 suppresses cell proliferation of breast cancer.

UCHL1 induced G0/G1cell cycle arrest and apoptosis of breast tumor cells
We further investigated the effects of UCHL1 on cell cycle and apoptosis of breast cancer cells. Representative results of cell-cycle  Figure 5A. Flow cytometry analysis revealed a statistically significant increase in the number of UCHL1expressing cells with G0/G1phase (21% increase, p,0.05; Fig. 5A) accompanied by the decrease of S and G2-M cells, compared to control-transfected cells (Fig. 5B). Next, we evaluated the apoptotic effect of UCHL1 in breast cancer cells using TUNEL and annexin V-FITC/PI staining assays. TUNEL assay showed that apoptosis was obviously induced in UCHL1-transfected MB231 cells (Fig. 6A). Representative Annexin V-FITC staining was shown in Figure 6A. Similar to that measured by the TUNEL assay, the percentage of Annexin V-PI-positive cells was increased in UCHL1-transfected MB231 cells and reached 71.8%, compared with controls (Fig. 6B). These results indicate that the inhibitory effect of cell proliferation by UCHL1 is most likely mediated by G0/G1cell cycle arrest and apoptosis.
Effect of UCHL1 on cell cycle and apoptosis is related to p53 accumulation, requiring its deubiquitinase activity As both growth arrest and apoptosis are associated with the induction of p53, and UCHL1 has been identified increased p53 accumulation by deubiquitination, we firstly examined the possible correlation between UCHL1 expression and p53 cytoplasmic localization. Immunostaining showed that UCHL1 is primarily located in cytoplasm as expected, and increased the abundance of cytoplasmic p53 in UCHL1-expressing MB231 cells (Fig. 7A), but not in the controls (data not shown), suggesting that UCHL1 contributes to cytoplasmic retention of p53.
We then assessed the expression of p53 in UCHL1-expressed MB231 cells, using its catalytic mutant UCHL1 C90S as a control in addition to the vector only [10]. Western blot showed that UCHL1 promoted p53 accumulation in breast tumor cells, along with the reduction of MDM2, while UCHL1 C90S did not increase p53 accumulation, but partly decreased the expression of MDM2 (Fig. 7B). In MB231-expressing UCHL1 cells, p53 protein level was showed 9-fold or greater changes compared to the controls, but no any change was observed in UCHL1 C90Sexpressing cells (Fig. 7C). We further found the expression of p21 was dramatically increased in UCHL1-expressing cells, which are the key G1/S cell cycle regulators and p53 downstream target genes, as well as cleaved-caspase 3 and PARP (Fig. 7B), while little or no upregulation of these markers was observed in UCHL1 C90Sexpressing cells. Thus, upregulated p53-signaling by UCHL1 through its DUB activity was involved in G0/G1 cell cycle arrest and apoptosis in breast cancer cells.

Discussion
In the present study, we analyzed the epigenetic alteration of UCHL1, its tumor suppressive functions and related-mechanisms in breast cancer. We found that UCHL1 was abundantly expressed in normal breast tissues and normal mammary epithelial cell lines, but frequently downregulated or silenced in breast cancer cell lines and primary tumors due to its promoter methylation, indicating that aberrant promoter methylation is a major cause for UCHL1 disruption in breast cancer. In primary tumors, UCHL1 methylation was associated with clinical stage and progesterone receptor status, indicating its potential as tumor marker for this cancer. Functionally, ectopic expression of UCHL1 suppressed the colony formation and cell proliferation of breast tumor cells through inducing G0/G1cell cycle arrest and apoptosis. Furthermore, we found that the p53 accumulation is mainly due to its cytoplasmic retention by UCHL1, depending on its DUB activity, which is responsible for the tumor suppressive function of UCHL1. Thus, UCHL1, acts as a functional tumor suppressor by inhibiting cell proliferation and inducing apoptosis, but is epigenetically-silenced in breast cancer.
Abnormal of ubiquitin-proteasome signaling pathway is closely associated with multiple tumorigenesis, including breast cancer [8]. Malfunctions in the ubiquitin-proteasome system enhance the effects of oncoproteins, reduce the protein levels of tumor suppressor proteins, further leading to the inhibition of apoptosis of tumor cells and promotion of cell proliferation [18].
UCHL1, located at 4p14, was first reported as a member of the ubiquitin proteasome pathway [19], and plays an important role in controlling intracellular ubiquitin levels in cells undergoing ubiquitin-dependent protein degradation [9,20]. Promoter methylation has been identified to be the major cause for UCHL1 downregulation or silencing in multiple malignancies. Although high expression of UCHL1 was reported to predict early recurrence in patients with invasive breast cancer [21], other evidences indicated the potential of UCHL1 as tumor suppressor in breast cancer. Overexpression of UCHL1 has been found to induce apoptosis in MCF-7 cells [22]. Using genomic screening upregulated genes by demethylation agent treatment in breast cancer cells, Fujikane et al found that UCHL1 was methylated in primary breast tumors [23], consistent with our studies.
Our previous work demonstrated that UCHL1 could activate the p14ARF-p53 signaling pathway by deubiquitinating p53 and p14ARF as well as ubiquitinating MDM2, which might be through its two opposing enzyme activities, hydrolase and ligase, further resulting in its tumor suppressive role in NPC tumorigenesis [11,15]. Although USP10 has been reported to regulate p53 localization and stability by deubiquitinating p53 directly [24], our data showed that USP10, unlike UCHL1, is widely expressed in both normal tissues and breast cancer cell lines, and no any expression correlation in mRNA level between USP10 and UCHL1 was found in breast cancer, indicating UCHL1 is mainly responsible for p53 stability in breast cancer. UCHL1 is primarily located in cytoplasm exerting its ubiquitinase fucntion, and p53 is also reported to be localized in cytoplasm in quiescent mammary gland without hormone treatment, which is not responsible for p21 transcription [25]. Another report suggested that cytoplasmic p53 was usually wide type and detected in normal breast tissue while mutated p53 is located in nucleus in breast cancer tissues [26]. In this study, we found that UCHL1 retained p53 in the cytoplasm substantially. Furthermore, using catalytic mutant UCHL1 C90S as a control, the accumulation of p53 mediated by UCHL1 was observed, subsequently, p21, as key p53 downstream target genes and regulators of cell cycle G1/S checkpoint, as well as cleaved-caspase 3 and PARP, were obviously upregulated, accompanied by UCHL1-mediated p53 activation, but not in UCHL1 C90Sexpressing breast cancer cells. We also found a dramatic reduction of MDM2 in UCHL1-expressing cells, but minor change was observed in UCHL1 C90S-transfected cells, which is well correlated with the function of UCHL1 C90S, lacking hydrolase activity but maintaining binding affinity for ubiquitin. However, the study on the mechanism of the negative correlation between UCHL1 and MDM2 level needs to further investigated. Thus, ensuring proper p53 signaling is tightly related to UCHL1-induced tumor suppression in breast pathogenesis.
Recent studies have shown that UCHL1 methylation is correlated with tumor cell differentiation, lymph node metastasis and poor prognosis, thus as a tumor marker [12,13,23,27,28]. UCHL1 promoter methylation is an independent prognostic factor for ESCC survival and thus a valuable tumor marker for ESCC progression [13]. We also identified that UCHL1 methylation as a biomarker for HCC and other digestive tumors previously [15]. In breast cancer, we found that the promoter methylation of UCHL1 was a promising marker indicative of breast cancer progression.
In summary, we found that UCHL1 possesses tumor-suppressive functions in breast tumor cells requiring its DUB activity, and is frequently silenced by promoter methylation, thus as a potential tumor marker for breast cancer. Our study further extends the current understanding of the role of epigenetically-disrupted tumor suppressor gene in breast tumorigenesis.  [29,30]. Human normal mammary epithelial cell lines HMEpC and HMEC (CA-830-05a, Applied Biosystems, Foster City, CA) were used as controls. All cell lines were maintained in RPMI 1640 (Gibco-BRL, Karlsruhe, Germany) supplemented with 10% fetal bovine serum (FBS) (PAA Laboratories, Linz, Austria), 100 U/ml penicillin and 100 mg/ml streptomycin, at 37uC in a humidified atmosphere containing 5% CO 2 [29,30,31]. RNA samples of human normal adult tissues were purchased commercially (Stratagene, La Jolla, CA; Millipore Chemicon, Billerica, MA and BioChain Institute, Hayward, CA). DNA samples of breast tumor, adjacent tissues and normal breast tissues have been described previously [32,33].

Cell lines and tumor samples
Some fresh breast tumors, adjacent non-cancerous tissues and normal breast tissues were obtained from patients treated by primary surgery at the First Affiliated Hospital Surgery Department of Chongqing Medical University (Chongqing, China). All samples were evaluated and subjected to histological diagnosis by expert pathologists. Grading of tumors was achieved by staining with hematoxylin and eosin (H&E). The clinical data, including race, age, site of primary tumor, stage, receptor status and tumor differentiation, were also obtained. All patients provided informed consent for the study to retain and analyze their tissues for research purposes. The samples were immediately snap-frozen following resection and stored in liquid nitrogen until processing. The study was approved by Institutional Review Board of the Chongqing Medical University. The written informed consent was obtained.

DNA and RNA preparation
DNA and RNA were isolated as previously described [34,35]. Briefly, RNA was extracted using TRIzol reagent (Life Technologies Inc., Carlsbad, CA). DNA was extracted using Qiagen DNeasy Tissue kit (Qiagen, Inc., Valencia, CA). Extracted DNA and RNA were quantified using spectrophotometry analysis. Samples were stored at 220uC or 280uC until use.

Tissue microarray and immunohistochemistry
To evaluate the expression levels of UCHL1 protein in breast cancer tissues, tissue microarray (TMA) was constructed using paraffin-embedded, formalin-fixed tissues from 31 patients, including primary tumor and adjacent tissues, and one pair as positive controls (Biochip Co., Ltd., Shanghai, China). Immunohistochemistry was performed using UCHL1 polyclonal antibody (ab10404, Abcam). Briefly, sections were deparaffinized, subjected to microwave antigen retrieval for 15 min in sodium citrate solution (pH 6.0) and then incubated with 3% hydrogen peroxide to block endogenous peroxidase activity. The sections were incubated with primary antibody (1:1000 dilution) overnight at 4uC, followed by second antibody (1:2000 dilution) at 37uC for one hour. Finally, the slides were counterstained with hematoxylin. Negative control was performed by replacing the primary antibody with PBS.

Flow cytometry analysis of cell cycle and apoptosis
Flow cytometry analyses of cell cycle and apoptosis were described previously [29,39]. For cell cycle analysis, cells were fixed in ice-cold 70% ethanol and stained with propidium iodide (PI). The cell-cycle profiles were assayed using the Elite ESP flow cytometry at 488 nm, and the data were analyzed using the CELL Quest software (BD Biosciences, San Jose, CA). For apoptosis analysis, Annexin V-FITC/PI staining was also performed using flow cytometry according to the manufacturer's guidelines. Briefly, cells were incubated with PI and Annexin V-fluorescein isothiocynate in the darkness at room temperature. Flow cytometric analysis was immediately performed.

Colony formation assay
For monolayer culture, freshly seeded tumor cells (2610 5 /well) were plated in a 6-well plate, cultured overnight, and transfected with pcDNA3.1-UCHL1 or pcDNA3.1 plasmid using Lipofectamine 2000 (Invitrogen, CA). Cells were plated in a new 6-well plate 48 h post-transfection, and selected for 1-2 weeks using G418 (1200 mg/ml), while untransfected cells would not survive G418 selection. Surviving colonies ($50 cells per colony) were counted after staining with Giemsa14 days post-transfection, counted and photographed. Each experiment was run in triplicate, and performed for three times.

Cell proliferation assay
For the analysis of cell growth rate, MB231 cells transfected as above and plated in 96-well plate at a density of 5610 3 cells/well, and cell proliferation assay was measured at 24, 48 and 72 h using the Cell Counting Kit-8 (CCK-8) (Dojindo Molecular Technologies, Japan) according to the company's instruction. Spectrometric absorbance at 490 nm was measured using a microplate reader. The data were obtained from three independent cultures and each experiment was repeated four times.

TUNEL assay
Analysis of apoptotic cells was performed using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining kit following the manufacturer's instruction (Roche, CA). Briefly, MB231 cells were grown on glass coverslips and transfected with pcDNA3.1-UCHL1 or pcDNA3.1 plasmid and cultured for 48 h. Transfected cells were then washed with PBS, fixed with 4% paraformaldehyde in PBS, and incubated with primary antibodies. The nuclei were counterstained with DAPI (Roche, CA). TUNEL-positive cells had a pyknotic nucleus with dark green fluorescent staining, indicative of apoptosis. The TUNEL reaction was also visualized by chromogenic staining with DAB (002114, Invitrogen, CA). Images of the sections were taken using a fluorescence microscope (Leica DM IRB).

Indirect Immunofluorescence
Cells grown on coverslips were stained by indirect immunofluorescence as described previously [39]. Briefly, cells were incubated with primary antibodies against UCHL1 and p53, and then incubated with Alexa Fluor 555-(Invitrogen Molecular Probes, Carlsbad, CA) or FITC-conjugated (Dako, Denmark) secondary antibody against mouse or rabbit Ig G. Cells were then counterstained with DAPI and imaged with a fluorescence microscope (Leica DM IRB).

Statistical analysis
Statistical analyses were performed using SPSS version 16 software. The expression analysis of UCHL1 between BrCa tissues and the corresponding adjacent tissues were assessed using the Student's t-test. Comparisons of categorical variables were performed using the x 2 test or 2-tailed t-test. Fisher's exact test was used when appropriate. Differences were considered statistically significant if a p value was less than 0.05.