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 
. 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 
, located at 4p14, was first reported as a member of the ubiquitin proteasome pathway 
, and plays an important role in controlling intracellular ubiquitin levels in cells undergoing ubiquitin-dependent protein degradation 
. 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 
, 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 
. 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 
, 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 
. Although USP10 has been reported to regulate p53 localization and stability by deubiquitinating p53 directly 
, 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
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 
. 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 
. 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 C90S
-expressing 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 
promoter methylation is an independent prognostic factor for ESCC survival and thus a valuable tumor marker for ESCC progression 
. We also identified that UCHL1
methylation as a biomarker for HCC and other digestive tumors previously 
. 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.