The authors have declared that no competing interests exist.
Conceived and designed the experiments: QT. Performed the experiments: YX TX XL QL CL SL ZW WP. Analyzed the data: TX LL. Contributed reagents/materials/analysis tools: GR LT. Wrote the paper: YX TX LL QT.
Krüppel-associated box-containing zinc finger proteins (KRAP-ZFPs) are well recognized as key regulators of transcription, which play a crucial role in the regulation of cell proliferation, differentiation, apoptosis and tumorigenesis. We previously identified a KRAP-ZFP protein ZNF545 acting as a tumor suppressor involved in tumor pathogenesis. However, its expression and biological function in breast cancer remain elusive. In this study, we found that ZNF545 was frequently downregulated in estrogen receptor-positive (ER+), progesterone receptor-positive (PR+) and human epidermal growth factor receptor 2-negative (HER2−) breast tumor tissues compared with paired adjacent non-tumor tissues. We further examined its expression and methylation in breast cancer cell lines by semi-quantitative RT-PCR and methylation-specific PCR. We found that ZNF545 was silenced by promoter methylation in MCF7 cell line, and its expression could be restored by demethylation, concomitant with increased unmethylated alleles. ZNF545 methylation was detected in 29% of breast tumor tissues, but not in normal breast tissues, suggesting tumor-specific methylation of ZNF545 in breast cancer. Ectopic expression of ZNF545 in MCF7 cells inhibited cell proliferation through inducing cell cycle G0/G1 arrest and apoptosis, thus as a tumor suppressor. Moreover, ZNF545 upregulated mRNA and protein levels of c-Jun/AP1, BAX, p53 and Caspase 3. Taken together, these results demonstrate that ZNF545 inhibits breast tumor cell proliferation through inducing apoptosis and is disrupted by promoter methylation in breast cancer.
Breast cancer is one of the most common malignancies and the leading cause of cancer death in females worldwide, accounting for 23% (1,380,000) of all cancer cases and 14% (458,400) of cancer deaths in 2008
Zinc finger proteins (ZFPs), the largest transcription factor family, are only present in tetrapod vertebrate genomes
We previously demonstrated that Zinc-finger protein 545 (ZNF545) is a novel KRAB-ZFP member frequently methylated in multiple common tumors. Here, we examined expression and methylation of ZNF545 in breast cancer, and further assessed its tumor suppressive functions in breast cancer.
A series of breast tumor cell lines (BT549, MDA-MB-231, MDA-MB468, MCF-7, T47D, SK-BR-3) were used. These cells were from ATCC (American type culture collection). Cell lines were maintained in RPMI 1640 (Gibco BRL, Karlsruhe, Germany) supplemented with 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA), and 100 U/ml of penicillin and streptomycin at 37°C in moist air including 5% CO2
Clinicopathological features | Number (n = 128) | |||
methylated | unmethylated | |||
0.232 | ||||
≤40 | 14 | 2 (14%) | 12 (86%) | |
>40 | 102 | 33 (32%) | 69 (68%) | |
unknown | 12 | 2 (17%) | 10 (83%) | |
0.658 | ||||
I | 7 | 2 (29%) | 5 (71%) | |
II | 81 | 26 (32%) | 55 (68%) | |
III | 6 | 2 (33%) | 4 (67%) | |
unknown | 34 | 7 (21%) | 27 (79%) | |
0.302 | ||||
<2.0 cm | 42 | 10 (24%) | 32 (76%) | |
≥2.0 cm≤5.0 cm | 63 | 21 (33%) | 42 (67%) | |
>5.0 cm | 9 | 4 (44%) | 5 (56%) | |
unknown | 14 | 2 (14%) | 12 (86%) | |
0.371 | ||||
Positive | 54 | 18 (33%) | 36 (67%) | |
Negative | 60 | 17 (28%) | 43 (72%) | |
unknown | 14 | 2 (14%) | 12 (86%) | |
0.501 | ||||
Positive | 54 | 19 (35%) | 35 (65%) | |
Negative | 43 | 12 (28%) | 31 (72%) | |
unknown | 31 | 5 (16%) | 6 (84%) | |
0.403 | ||||
Positive | 44 | 14 (32%) | 30 (68%) | |
Negative | 53 | 17 (32%) | 36 (68%) | |
unknown | 31 | 6 (19%) | 25 (81%) | |
0.068 | ||||
>+++ | 6 | 2 (33%) | 4 (67%) | |
++ | 50 | 21 (42%) | 29 (58%) | |
<+ | 39 | 8 (21%) | 31 (79%) | |
unknown | 32 | 6 (19%) | 26 (81%) | |
0.540 | ||||
Positive | 37 | 13 (35%) | 24 (65%) | |
Negative | 49 | 14 (29%) | 35 (71%) | |
unknown | 42 | 10 (24%) | 32 (76%) | |
0.762 | ||||
1 | 35 | 10 (29%) | 25 (71%) | |
2 | 54 | 17 (31%) | 37 (69%) | |
3 | 25 | 8 (32%) | 17 (68%) | |
4 | 1 | 0 (0%) | 1 (100%) | |
unknown | 13 | 2 (15%) | 11 (85%) |
Genomic DNA was extracted from cell lines and tissues using DNAzol Reagent (Invitrogen, Rockville, MD, USA) and the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocols. Total RNA was isolated from cell lines and tissues using TRI Reagent (Molecular Research Center, Cincinnati, OH). The integrity of DNA and RNA were detected by gel electrophoresis. Samples were stored at −80°Cuntil use.
As previously described
The reverse transcriptase–polymerase chain reaction (RT-PCR) was performed as previously described
Bisulfite modification of DNA and methylation-specific PCR (MSP) were performed as previously described
Monolayer culture was performed. MCF7 cells (2×105 per well) were plated in six-well plates and transfected with pcDNA3.1(+)-Flag-ZNF545 plasmid or control vector (4 µg each) using Lipofectamine-2000 (Invitrogen, Carlsbad, CA). Forty-eight hours later, transfectants were collected, re-plated, and selected for two weeks in the presence of 100 µg/ml G418 in MCF7 cells. Surviving colonies were stained with Gentian Violet and visible colonies (≥50 cells) were counted. All the experiments were performed in triplicate wells three times.
MCF7 cells were cultured in six-well plates at a density of 2×105 cells/well and grown overnight. Cultures were then transiently transfected with pcDNA3.1(+)-Flag-ZNF545 plasmid or the control vector using Lipofectamine-2000 (Invitrogen, Carlsbad, CA). Forty-eight hours later, 2×103 cells were replated in 96-well plates. After 24 h, 48 h, and 72 h, proliferation was measured using the Cell Counting Kit-8 (CCK-8, Beyotime, Shanghai, China)
MCF7 cells were cultured in 6-well plates at a density of 2×105 cells/well and grown overnight. Cultures were then transiently transfected with 4 µg pcDNA3.1(+)-Flag- ZNF545 plasmid or the control vector using Lipofectamine-2000 (Invitrogen, Carlsbad, CA) following the manufacturer's protocol. After 48 h, cells were collected and centrifuged at 800 rpm for 5 min. Cells were then washed with PBS twice and fixed in ice-cold 70% ethanol for 1 h, and treated with 100 µl of 50 mg/l propidium iodide for 30 min at 4°C in the dark. Data were analyzed by CELL Quest software (BD Biosciences, San Jose, CA, USA). Apoptosis was examined using acridine orange/ethidium bromide (AO/EB) fluorescence staining. Transfectants (1×105 cells) were replated in six-well plates. After 24 hours, cells were washed three times with phosphate-buffered saline (PBS) then stained with AO/EB for 5 min and visualized immediately under a fluorescence microscope (LEICA CTR4000B). The percentage of apoptotic cells was then calculated by the formula: percentage of apoptotic cell (%) = (amount of apoptotic cell/total cell examined) ×100%.
Forty-eight hours after transfection, cells were harvested and lysed in M-PER Mammalian Protein Extraction Reagent (Pierce, Thermo Scientific, Cramlington, UK) containing a protease inhibitor cocktail (Sigma Aldrich, St. Louis, MO). A total of 50 µg of protein lysates were separated by sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto a PVDF membrane (Bio-Rad, Hercules, CA, USA). The primary antibodies were used: c-Jun (#9165, cell signaling Technology, Danvers, MA), p-c-Jun (#2361, cell signaling),cleaved caspase-3 (#9661, Cell Signaling), p53 (sc-126, Santa Cruz, CA), BAX (#9942, cell signaling), and GAPDH (Southern Biotech,Birmingham, USA) was used as a control. Proteins were visualized using an enhanced chemiluminescence kit (Amersham Pharmacia Biotech, Piscataway, NJ, USA).
Statistical analyses were performed with SPSS software (version 16). Student's
We firstly examined ZNF545 expression in paired breast tumor tissues with different ER/PR/HER2 status by quantitative real-time PCR (qRT-PCR). We found that expression of ZNF545 was downregulated in 89.5% (17/19) of Luminal A (ER+/PR+/HER2−) subtypes, but increased in 69.2% (9/13) of triple negative (ER−/PR−/HER2−) and 87.5%(7/8) of Luminal B (ER+/PR+/HER2+) breast cancer tissues, compared with their adjacent non-tumor tissues (
(A) ZNF545 expression in primary breast tumor tissues and paired surgical margin tissues were evaluated using quantitative RT-PCR analysis. (B) Reduced expression of ZNF545 in breast cancer. Data extracted from cancer microdatabase Oncomine:
Variable | No.paired cases | Expression rate | p-value |
0.0147 | |||
Downregulation | 1 | 12.5% | |
Upregulation | 7 | 87.5% | |
0.0419 | |||
Downregulation | 17 | 89.5% | |
Upregulation | 2 | 10.5% | |
0.0252 | |||
Downregulation | 4 | 30.8% | |
Upregulation | 9 | 69.2% |
Dataset | Tissue type | Sample number | Median of expression intensity (log 2) | Fold change | |
TCGA Breast | Breast | 61 | 0.463 | ||
ICBC | 3 | −0.368 | −1.835 | 0.002 | |
MBC | 3 | 0.064 | −1.259 | 0.007 | |
IC | 76 | 0.150 | −1.264 | 1.14E-05 | |
IDC | 389 | 0.167 | −1.329 | 2.87E-09 | |
ILC | 36 | 0.275 | −1.230 | 0.014 | |
Zhao breast | Breast | 3 | 0.480 | ||
LC | 19 | −0.180 | −1.482 | 2.30E-04 | |
IDC | 38 | 0.145 | −1.196 | 3.49E-04 | |
Karnoub breast | Breast | 15 | −0.385 | ||
ILC | 7 | −0.797 | −1.475 | 0.006 | |
Finak breast | Breast | 6 | −0.105 | ||
IC | 53 | −2.768 | −5.909 | 6.26E-13 |
Data extracted from cancer microdatabase Oncomine:
Abnormal promoter methylation is an important mechanism for TSG silencing in carcinogenesis
To investigate ZNF545 promoter methylation in breast tumor tissues, MSP was used to examine 128 primary breast carcinomas tissues and seven normal breast tissues. ZNF545 methylation was detected in 37 out of 128 (29%) breast cancer tissue, but not in normal breast tissues (
(A) Methylation of
Tissue | Samples number | Frequency of methylation | ||
methylated | unmethylated | |||
Breast tumor | 128 | 37 | 91 | 29% |
Normal breast | 7 | 0 | 7 | 0 |
We further analyzed the relationship of ZNF545 methylation with clinicopathological features of breast cancer patients, including age, tumor grade, tumor size, lymph node metastasis, ER status, PR status and HER2 status, but no significant correlation was observed (
To further determine whether ZNF545 is a functional TSG in breast cancer, the effect of ZNF545 on MCF7 cell proliferation was examined by colony formation assay and CCK8 assay. Results showed that ZNF545 markedly reduced the efficiency of MCF7 colony formation to ∼10% compared to controls (***
(A) Representative colony formation assay and Quantitative analysis of colony formation. The numbers of G418-resistant colonies in vector-transfected controls were set to 100%, Values are expressed as the mean±SD from three experiments, and the asterisk indicates the statistical significance compared to the controls (***, p<0.001). (B) Expression of ZNF545 by RT-PCR in vector- and ZNF545-transfected MCF-7 cells. (C) CCK-8 assay for cell proliferation on vector- and ZNF545-transfecetd MCF7 cells. Asterisks indicate a significant level of proliferation compared with controls (**, p<0.01).
Flow cytometric analysis of cell cycle and AO/EB staining were used to assess the mechanism of ZNF545 in inhibiting cell proliferation. ZNF545 obviously increased the number of MCF7 cells in the G0-G1 phase from 47.42% to 52.36%(*
(A) Anlysis of cell cycle distribution of vector-, ZNF545-transfected MCF7 cells. The distribution and percentage of cells in G1, S and G2/M phase of the cell cycle are indicated. B) Representative flow cytometry histograms of cell cycle alterations. The assay was performed in triplicate (*,
AO/EB staining is used to detect early apoptotic cells (stained green with yellowish dots as well as nuclear fragmentation and blebbing of cytoplasm) and late apoptotic cells (stained orange with condensed and often fragmented nuclei). AO/EB staining showed uniformly green control cells with normal morphology, whereas green early apoptotic cells with chromatin condensation occurred in ZNF545-expressing MCF7 cells, with orange later apoptotic cells with fragmented chromatin and apoptotic bodies also appearing (
We also examined its potential downstream target genes in ZNF545-expressing MCF7 cells by qRT-PCR, and found that ZNF545 could upregulate expression of c-Jun/AP1, BAX, p53 and Caspase 3 in mRNA level and protein level (
ZNF545 is reportedly a functional tumor suppressor in multiple cancers and is silenced by promoter methylation by our group
DNA methylation is a pivotal regulatory mechanism in gene transcription
ZNF545 is a novel member of the KRAB-containing zinc finger protein (KRAB-ZFP) family identified
In summary, this report is the first to show that ZNF545 is a functional TSG in breast cancer, through inhibiting cell growth and inducing apoptosis, and its tumor-specific methylation may serve as a potential tumor marker for breast cancer.