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 C90Sexpressed cells

Effects of UCHL1 on the colony formation and cell proliferation of breast cancer cells.

<p>(A) Representative colony formation assay. (B) Quantitative analysis of colony formation. The numbers of G418-resistant colonies in vector-transfected controls were set to 100%. Values are expressed as mean±SD from three experiments, and the asterisks indicate statistical significance compared to controls (<i>p</i><0.01). (C) Expression of UCHL1 by RT-PCR in vector- and UCHL1-transfected MB231 and MCF-7 cells. (D) CCK-8 cell proliferation assay for vector- and UCHL1-transfecetd MB231 cells. Asterisks indicate a significant level of proliferation compared to controls (<i>p</i><0.05).</p

Downregulation of <i>UCHL1</i> in breast cancer.

<p>(A and B) <i>UCHL1</i> and <i>USP10</i> expression in normal adult tissues (A), mammary epithelial cell lines and breast cancer cell lines (B), were evaluated using semi-quantitative RT-PCR, with <i>GAPDH</i> as a control. (C) UCHL1 protein expression was detected breast cancer cell lines by western blot. (D) Immunohistochemical analysis of UCHL1 in paired adjacent non-cancerous breast tissues and primary breast tumors.</p

Clinicopathological features and <i>UCHL1</i> methylation of breast tumors.

<p>Clinicopathological features and <i>UCHL1</i> methylation of breast tumors.</p

<i>UCHL1</i> methylation in breast cancer cell lines and reactivation of <i>UCHL1</i> by demethylation.

<p>(A) <i>UCHL1</i> is frequently methylated in breast cancer cell lines. (B) Pharmacological demethylation restores <i>UCHL1</i> expression. A+T: Aza and TSA treatment. M, methylated; U, unmethylated.</p

Detection of apoptosis induced by UCHL1 using TUNEL assay and Annexin V-FITC/PI staining in MB231 cells.

<p>(A) Apoptotic cells detected by TUNEL staining. (B) Induction of apoptosis detected by flow cytometric analysis with Annexin V-FITC and PI-staining.</p

UCHL1 promotes the activation of p53 signaling depending on its deubiquitinase activity.

<p>(A) The subcellular localization of UCHL1 and p53 was analyzed in <i>UCHL1</i>-transfected MB231 cells. (B) Western blot analysis of cell cycle- and apoptosis-related proteins in vector-, <i>UCHL1</i> and <i>UCHL1 C90S</i>-transfected MB231 cells. β-actin was used as a control. (C) Quantification of p53 protein expression by Western blot using scanning and ImageJ software. Each sample was normalized to β-actin content.</p

Methylation status of the <i>UCHL1</i> promoter in primary breast tumors.

<p>Note: BrCa, breast cancer; BA, breast cancer adjacent tissues; BNP, breast normal tissues.</p

Effect of UCHL1 on cell cycle distribution of MB231 cells.

<p>(A) Representative cell cycle analysis. (B) Summarized flow cytometry data. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029783#s2" target="_blank">Results</a> are represented as average±S.D and are based on three independent experiments. Statistical significance was determined using Student's t-test (p<0.001).</p

Promoter methylation of <i>UCHL1</i> in primary breast tumors.

<p>(A, B, C) Representative analysis of <i>UCHL1</i> methylation by MSP in normal breast tissues, breast tumor adjacent tissues and primary tumors. M, methylated; U, unmethylated. (D) Detailed BGS analysis of <i>UCHL1</i> promoter in primary tumor and normal breast tissue. Circles, single CpG sites analyzed; row of circles, individual promoter allele cloned, randomly selected and sequenced; Filled circle, methylated; empty circle, unmethylated.</p