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Carcinogenesis Advance Access originally published online on August 13, 2008
Carcinogenesis 2008 29(11):2195-2202; doi:10.1093/carcin/bgn194
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© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Adiponectin stimulates Wnt inhibitory factor-1 expression through epigenetic regulations involving the transcription factor specificity protein 1

Jing Liu1,2,3, Janice B.B. Lam1,2,3,5,{dagger}, Kim H.M. Chow1,2,3, Aimin Xu1,2,3, Karen S.L. Lam2,3, Randall T. Moon4 and Yu Wang1,3,*

1 Department of Pharmacology
2 Department of Medicine
3 Research Center of Heart, Brain, Hormone and Healthy Aging, University of Hong Kong, Hong Kong, China
4 Howard Hughes Medical Institute, University of Washington School of Medicine, Seattle, WA 98195, USA
5 School of Biological Sciences, University of Auckland, Auckland 1003, New Zealand

* To whom correspondence should be addressed. Department of Pharmacology, University of Hong Kong, Faculty of Medicine Building, 21 Sassoon Road, Pokfulam, Hong Kong, China. Tel: +852 28192864; Fax: +852 28170859; Email: yuwanghk{at}hku.hk


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
 References
 
Adiponectin (ADN) is an adipokine possessing growth inhibitory activities against various types of cancer cells. Our previous results demonstrated that ADN could impede Wnt/beta-catenin-signaling pathways in MDA-MB-231 human breast carcinoma cells [Wang,Y. et al. (2006) Adiponectin modulates the glycogen synthase kinase-3 beta/beta-catenin signaling pathway and attenuates mammary tumorigenesis of MDA-MB-231 cells in nude mice. Cancer Res., 66, 11462–11470]. Here, we extended our studies to elucidate the effects of ADN on regulating the expressions of Wnt inhibitory factor-1 (WIF1), a Wnt antagonist frequently silenced in human breast tumors. Our results showed that ADN time dependently stimulated WIF1 gene and protein expressions in MDA-MB-231 cells. Overexpression of WIF1 exerted similar inhibitory effects to those of ADN on cell proliferations, nuclear beta-catenin activities, cyclin D1 expressions and serum-induced phosphorylations of Akt and glycogen synthase kinase-3 beta. Blockage of WIF1 activities significantly attenuated the suppressive effects of ADN on MDA-MB-231 cell growth. Furthermore, our in vivo studies showed that both supplementation of recombinant ADN and adenovirus-mediated overexpression of this adipokine substantially enhanced WIF1 expressions in MDA-MB-231 tumors implanted in nude mice. More interestingly, we found that ADN could alleviate methylation of CpG islands located within the proximal promoter region of WIF1, possibly involving the specificity protein 1 (Sp1) transcription factor and its downstream target DNA methyltransferase 1 (DNMT1). Upon ADN treatment, the protein levels of both Sp1 and DNMT1 were significantly decreased. Using silencing RNA approaches, we confirmed that downregulation of Sp1 resulted in an increased expression of WIF1 and decreased methylation of WIF1 promoter. Taken together, these data suggest that ADN might elicit its antitumor activities at least partially through promoting WIF1 expressions.

Abbreviations: ADN, adiponectin; DMEM, Dulbecco’s modified Eagle’s medium; DNMT1, DNA methyltransferase 1; FBS, fetal bovine serum; GSK-3β, glycogen synthase kinase-3 beta; mRNA, messenger RNA; MSP, methylation-specific polymerase chain reaction; PCR, polymerase chain reaction; siRNA, small interfering RNA; Sp1, specificity protein 1; WIF1, Wnt inhibitory factor-1


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
 References
 
Recent epidemiological studies suggest that obesity increases the risks of cancer development, especially those of lifestyle-related cancers, such as breast, prostate, endometrium, colon and gallbladder cancers (1). Breast cancer is the most frequent cancer in women and represents the second leading cause of cancer death among women. Obesity is a well-known risk factor for breast cancer after menopause and associated with late-stage disease and poor prognosis (2–6). Although the detailed mechanisms remain exclusive, many clinical and experimental evidences suggest that the dysregulated production of adipokines might play causative roles in obesity-related mammary carcinogenesis (3,7,8). Notably, adipocyte is one of the predominant stromal cell types in the microenvironment of mammary tissue and the proximity suggests that adipokines produced from adipocytes could be critically involved in the development of mammary tumors (3,9).

Adiponectin (ADN) is an adipokine possessing diversified beneficial functions against obesity-related diseases (10). Unlike most of adipokines that are increased in obesity, the circulating levels of ADN are inversely correlated with obesity and insulin resistance, two risk factors of breast cancer (3,8). Obese women having reduced serum ADN levels are at an increased risk for developing breast cancer (11–15). Moreover, tumors in women with low serum ADN levels are more likely to show a biologically aggressive phenotype. We and others have demonstrated that ADN could inhibit the proliferation of several different types of human breast cancer cells, including MDA-MB-231, T47D and MCF7 cells (11,16–20). In MDA-MB-231 cells, ADN treatment blocks serum-stimulated phosphorylation of Akt and glycogen synthase kinase-3 beta (GSK-3β) and suppresses the intracellular accumulation of beta-catenin and its nuclear activities (19). These clinical and experimental evidences suggest that ADN could act as a negative regulator in the development of breast cancer, and replenishment of this protein might represent a novel therapeutic strategy in obesity-related breast cancer diseases (15).

Aberrant activations of Wnt/beta-catenin-signaling pathway play key roles in many types of carcinogenesis (21). Wnt1 was the first oncogene identified in naturally occurring mammary tumors in mice (22). Transgenic mice overexpressing beta-catenin driven by mouse mammary tumor virus showed increased predisposition to mammary tumors and lobuloalveolar hyperplasia (23). In human, an elevated level of nuclear and/or cytoplasmic beta-catenin can be detected in >50% of breast carcinomas, which correlates with the expression of the beta-catenin target genes and poor prognosis (24). Overexpressions of WNT2, WNT7B, WNT10B, WNT2B and WNT9A have been shown in human breast cancer tissues compared with normal tissue (25,26). On the other hand, mutations of the key regulatory molecules in Wnt-signaling cascades are rare in human breast cancer patients (27). Recent evidences suggest that the gene expressions of several Wnt antagonists, such as Wnt inhibitory factor-1 (WIF1) and secreted frizzled-related protein 1, are inactivated due to promoter hypermethylation and epigenetic silencing, which may contribute to the upregulated Wnt/beta-catenin-signaling in breast cancer (28–33). In this study, we show that ADN treatment augments the gene and protein expressions of WIF1 in MDA-MB-231 cells in culture and tumors in nude mice, which might at least in part contribute to its tumor-suppressive activities. Our results also suggest that epigenetic regulations are involved in the stimulatory effects of ADN on WIF1 expressions.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Supplementary material
 Funding
 References
 
Materials
Antibodies against WIF1, specificity protein 1 (Sp1), DNMT1 and beta-actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against total and phosphorylated Akt (Ser473), phosphorylated GSK-3β (Ser9) and β-catenin were from Cell Signaling (Beverly, MA). Anti-cyclin D1 rabbit monoclonal antibody was from Upstate (Lake Placid, NY). SYBR® GreenERTM qPCR Supermix, DAB substrate kit, Lipofectamine transfection reagent, TRIzol® and pcDNA3.1+ vector were from Invitrogen (Carlsbad, CA). Crystal violet, sodium bisulfite, hydroquinone, 5-azacytidine and hematoxylin were from Sigma (St Louis, MO). pGEM®-T Easy Vector Kit, ImProm-IITM Reverse Transcription System and Bright-GloTM luciferase assay system were from Promega (Madison, WI). TOP/FOPflash (T-cell factor–lymphoid enhancer factor-1 reporter plasmid) and Trichostatin A were from Upstate. The human breast carcinoma MDA-MB-231, T47D and BT474 cells and human colon carcinoma HT29 and SW480 were obtained from American Type Culture Collection (Manassas, VA) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin, streptomycin and fungisome in 5% CO2 at 37°C. Recombinant full-length ADN was produced as we described previously (19).

Construction of the mammalian expression vector for WIF1 and CTNNB1 gene
RNA was extracted from the lung tissues of C57 mice using TRIzol® reagent according to the manufacturer’s instructions. One microgram of total RNA was used for the synthesis of first strand complementary DNA using ImProm-IITM Reverse Transcription System. The primers for cloning the full-length murine WIF1 gene were 5'-CTGCAAGCTTAGGTCCTGAGCAGCATG-3' (forward) and 5'-CGTATCTAGATCA-CTTATCGTCGTCATCCTTGTAATC-CCAGATGTAATTGGATTCAGG-3' (reverse). The mammalian expression vector pcDNA-WIF1 was generated by cloning the HindIII and XbaI restriction enzyme-digested polymerase chain reaction (PCR) fragment into pcDNA3.1+ vector. This vector (pcDNA-WIF1) was used for transfection of MDA-MB-231 cells for expressing the full-length WIF1 with a FLAG-tag at the C-terminus. Note that mouse and human WIF1 proteins share 94% sequence homology. The full-length human beta-catenin gene was PCR amplified from a prokaryotic expression vector M57 pPET28a-TEV-full-length human beta-catenin (http://www.addgene.org/pgvec1?f=c&cmd=findpl&identifier=17198) using the forward primer 5'-CTGGGATCCGACAATGGCTACTCAAGCTG-3' and reverse primer 5'-CTGTCTAGATTA-CTTATCGTCGTCATCCTTGTAATC-CAGGTCAGTATCAAACCAGG-3'. The mammalian expression vector pcDNA-beta-catenin was generated by subcloning the BamHI and XbaI restriction enzyme-digested PCR fragment into the mammalian expression vector pcDNA3.1+.

Quantitative reverse transcription–PCR assay
Total RNA was isolated from MDA-MB-231 cells or tumor tissue samples and used for the synthesis of complementary DNA. Quantitative reverse transcription–PCR was performed using SYBR® GreenERTM qPCR Supermix. The reactions were carried out on a 7000 Sequence Detection System (Applied Biosystems, Foster City, CA). Quantification was achieved using Ct values that were normalized with beta-actin as internal control. The primers were 5'-GTGTGAAATCAGCAAATGCC-3' (forward) and 5'-GTCTTCCATGCCAACCTTCT-3' (reverse) for human WIF1 and 5'-TGACCCAGATCATGTTTGAGA-3' (forward) and 5'-AGTCCATCACGATGCCAGT-3' (reverse) for human beta-actin.

Western blotting
Total cell lysates were extracted from MDA-MB-231 cells and tumor tissues and quantified by BCA Protein Reagent Kit (Pierce, Rockford, IL). Fifty micrograms of protein were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride membranes. Following blocking, membranes were probed with various primary antibodies to determine the levels of WIF1, Akt, GSK3β, beta-catenin, cyclin D1, Sp1, DNMT1 and actin. Immunoreactive antibody–antigen complexes were visualized with the enhanced chemiluminescence reagents from GE Healthcare (Uppsala, Sweden).

Bisulfite genomic sequencing and methylation-specific polymerase chain reaction
Genomic DNA was extracted by standard phenol–chloroform procedures. Two micrograms of genomic DNA was treated with 3 M sodium bisulfite and 10 mM hydroquinone at 50°C overnight. After desalting, PCR was performed for amplifying human WIF1 and WNT5A promoter. The primers for bisulfite-treated DNA templates are listed in Table I. For bisulfite genomic sequencing, the PCR products were cloned into pGEM®-T Easy Vector and transformed into competent E. coli. DH5{alpha}. White clones were selected for sequencing using T7 promoter primer.


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  		Table I. List of bisulfite sequencing primers for WIF1 and WNT5A promoter

 
For methylation-specific polymerase chain reaction (MSP), bisulfite-treated genomic DNA was amplified using either a methylation-specific or an unmethylation-specific primer set corresponding to the human WIF1 promoter region from –488 to –290. The methylation-specific primers were 5'-GGGCGTTTTATTGGGCGTAT-3' (forward) and 5'-AAACCAACAATCAACGAAAC-3' (reverse). The unmethylation-specific primers were 5'-GGGTGTTTTATTGGGTGTAT-3' (forward) and 5'-AAACCAACAATCAACAAAAC-3' (reverse). Agarose gel electrophoresis was performed for visualization of the MSP products.

Pyrosequencing
After bisulfite modification, PCR amplification was performed using bisulfite genomic sequencing primer as the forward and both the methylated and unmethylated MSP primers as the reverse primers (the reverse primer is biotinylated). The biotinylated PCR product was purified by using streptavidin-Sepharose beads. The pyrosequencing reaction was done automatically by using a PSQ 96MA system along with a SNP Reagent Kit. The unbiased sequencing primer for the Sp1 site of the WIF1 promoter was 5'-GTATTGTGAATGTAGTTT-3'. All the reactions were performed as recommended by the manufacturer’s instructions (Pyrosequencing, Westborough, MA).

Small interfering RNA knocking down of Sp1 and WIF1 expressions
Small interfering RNA (siRNA) was used for suppressing the expression of Sp1 in MDA-MB-231 cells. The siRNA sequences for Sp1 were 5'-GAGGAAAUGACUCGUAGUCtt-3' (sense strand) and 5'-GACUACGAGUCAUUUCCUCtt-3' (antisense strand). Ten microliters of 20 µM siRNA or scramble siRNA control were introduced into the cultured cells using Lipofectamine 2000 reagent according to the manufacturer’s instructions. The downregulation of Sp1 or WIF1 protein levels was confirmed by western blotting analysis. WIF1 siRNA was obtained from Santa Cruz Biotechnology for suppressing WIF1 messenger RNA (mRNA) expression in T47D and BT474 cells.

Crystal violet staining and cell proliferation measurement
Crystal violet staining is a rapid and sensitive method for cell number measurement in monolayer cultures (34,35). In this method, cell nuclei are stained with the crystal violet dye and the excess dye is washed out before optical density measurements at 570 nm. The readings reflect the number of cells in the sample. For proliferation measurement, MDA-MB-231 cells were seeded at a density of 4 x 103 in 96-well plates. Twenty-four hours later, cells were transfected with or without 0.15 µg of pcDNA3.1+ control or pcDNA-WIF1 plasmids using Lipofectamine reagents. After starvation in 0.5% FBS DMEM, cells were then incubated with 0.5 or 10% FBS DMEM in the presence or absence of ADN for 24, 48 and 72 h as we described previously (19). At the end of treatment, the media was removed and cells were washed with phosphate-buffered saline, fixed with 100 µl of 100% methanol followed by staining with 0.2% crystal violet dye solution in 20% methanol for 15 min. Excess dye was removed by gentle washing with double-distilled water followed by air drying. Stained cells were solubilized with 1% sodium dodecyl sulfate and quantified using µQuant MQX200 microplate reader (Biotek Instruments Inc., Highland Park, VT). For neutralization experiment, 2 µg/ml of anti-WIF1 antibody (WIF1 IgG) was added to the starvation and treatment media for complete blockade of endogenous WIF1 activities. For knocking down endogenous WIF1 expression, T47D and BT474 cells were transfected with WIF1 siRNA or scramble siRNA using Lipofectamine 2000 reagents. Insulin-stimulated cell proliferation was evaluated in the presence or absence of ADN as we described previously (19).

Beta-catenin/T-cell factor–lymphoid enhancer factor-1 transcription reporter assay
The nuclear activity of endogenous beta-catenin was analyzed using the TOPflash/FOPflash reporter system as described previously (19). Briefly, cells were seeded at a density of 2 x 104 in 24-well plates and treated as indicated. Cells were cotransfected with 0.15 µg TOPflash reporter plasmid with pcDNA3.1+ or pcDNA-WIF1. Twenty-four and 48 h after transfection, cells were washed with phosphate-buffered saline and lysed with compatible lysis buffer. Cell lysates were collected and luciferase activity was measured using Bright-GloTM Luciferase Assay System on Lumat LB9507 and expressed in relative luciferase units. Luciferase activity was normalized against control Renilla luciferase signal.

Statistics
All results were derived from at least three independent experiments. All animal experiments were performed with six to eight samples per group. Data are shown as mean values ± standard deviations. Comparison between groups was done using Student’s unpaired t-test. In all statistical comparisons, P < 0.05 was used to indicate a significant difference.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Supplementary material
 Funding
 References
 
ADN treatment augments the expressions of WIF1 in MDA-MB-231 cells
WIF1 can act as an antagonist for Wnt signaling by directly interacting with Wnt proteins (29,36). Diminished expression of WIF1 has been found in both human breast tumor tissues and human breast carcinoma cell lines, which might contribute to the aberrant activation of Wnt/beta-catenin signaling (28,33). We reported previously that ADN could inhibit MDA-MB-231 human breast carcinoma cell growth and attenuate tumor development in nude mice, partly through modulating the Wnt/beta-catenin-signaling pathways (19). In this study, we found that ADN treatment could enhance the expressions of WIF1 in MDA-MB-231 cells. Real-time PCR analysis demonstrated that after 24 h treatment with ADN, the mRNA levels of WIF1 were significantly increased by 1.6- and 2.5-fold in 0.5% FBS and 10% FBS DMEM culture conditions, respectively (Figure 1A). The elevated WIF1 expression lasted until 72 h after treatment. In addition, a much lower level of WIF1 mRNA was observed in cells treated with 10% FBS at the time points of 12, 24, 48 and 72 h, comparing with those of the 0.5% FBS group. Moreover, it seems that WIF1 expression gradually decreased after prolonged culturing, suggesting that its gene expression might also be related to cell cycle progression or cellular confluence status. However, due to the limitation of the cell culture system, we could not extend the experiment for longer period for investigating these possibilities. The stimulatory effects of ADN on WIF1 protein expression were confirmed by western blotting (Figure 1B) and immunocytochemistry analysis (supplementary Figure 6A is available at Carcinogenesis Online).


Figure 1
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  		Fig. 1. ADN treatment increases the mRNA and protein levels of WIF1 in MDA-MB-231 cells (A and B) and the cell-derived tumors in nude mice (C and D). MDA-MB-231 cells were treated with or without ADN (15 µg/ml) in the presence of 0.5 or 10% FBS for different periods and collected for quantitative reverse transcription–PCR (A) and western blotting analysis (B) for monitoring the expression levels of WIF1. Three approaches were employed for evaluating the in vivo effects of ADN (19). Firstly, MDA-MB-231 cells pretreated without (cell alone) or with (ADN protein) ADN were implanted into the mammary fat pad of nude mice. Alternatively, recombinant adenovirus expressing luciferase (Ad-Luc) or ADN (Ad-ADN) (108 p.f.u. per mouse) was either locally injected into tumor at day 14 or tail vein injected into nude mice at day 7 after the initial inoculation of the cells. Tumor tissues were collected at 5 weeks after cell implantation for quantitative reverse transcription–PCR (C) and western blotting analysis (D) of WIF1 expressions. *P < 0.05 and **P < 0.01 versus control groups without ADN treatment, n = 6. Results were derived from at least three independent experiments.

 
In our previous study, we used different strategies for evaluating ADN’s antitumor activities in nude mice, including pretreatment of the MDA-MB-231 cells with recombinant ADN protein before implantation and intratumor or tail vein injection of the recombinant adenovirus encoding ADN in nude mice (19). To investigate whether ADN could enhance WIF1 expressions in mammary tumor tissues, we then analyzed its gene and protein levels in MDA-MB-231 tumors derived from nude mice treated with ADN using these three approaches, which showed suppressive effects on tumor development (19). Our results from real-time PCR (Figure 1C), western blotting (Figure 1D) as well as immunohistochemistry analysis (supplementary Figure 6B is available at Carcinogenesis Online) revealed that ADN treatment could significantly augment both the gene and protein expressions of WIF1 in mammary tumor tissues. WIF1 expression in the xenograft that had been incubated with ADN protein before transplantation was elevated to similar levels as those treated with single injection of recombinant adenovirus, indicating that the effects elicited by this hormone were not transient but could be kept even when the proteins were removed, possibly through permanent modifications on the gene or at the transcriptional levels. Taken together, these data demonstrated that ADN could stimulate the expressions of WIF1 in MDA-MB-231 cells in vitro and in vivo.

Increased expression of WIF1 at least partially mediates the inhibitory effects of ADN on MDA-MB-231 cell growth
ADN treatment blocks serum-induced phosphorylations of Akt and GSK3β, suppresses intracellular accumulation of beta-catenin and its nuclear activities and reduces the expression of cyclin D1 in MDA-MB-231 cells (19). To investigate whether increased WIF1 expression could imitate ADN’s effect, we performed transient transfection experiments in MDA-MB-231 cells using the expression vector (pcDNA-WIF1) encoding full-length WIF1 protein. Comparing with pcDNA-transfected cells, WIF1 overexpression significantly decreased beta-catenin and cyclin D1 levels (Figure 2A) and attenuated serum-stimulated phosphorylations of Akt and GSK-3β (Figure 2B). In addition, the nuclear activities of beta-catenin were also significantly decreased by 20–30% in cells transiently transfected with pcDNA-WIF1 and cultured under either reduced serum (0.5% FBS) or high serum (10% FBS) conditions (Figure 2C and D). These results suggested that elevation of WIF1 levels could exert signaling and biological effects similar to those of ADN in MDA-MB-231 cells. Indeed, overexpression of WIF1 significantly inhibited MDA-MB-231 cell proliferation at 24, 48 and 72 h after transfection (Figure 3A and B). On the other hand, neutralization of WIF1 activities by adding its specific antibody to the culture medium completely reversed its suppressive effects on cell proliferation in 0.5% FBS-treated group. Importantly, the inhibitory effects of ADN on MDA-MB-231 cell growth were largely attenuated by coincubating with WIF1 antibody, at all time points evaluated under both 0.5 and 10% FBS culture conditions (Figure 3C and D). These results suggest that elevated WIF1 expressions might at least partially contribute to the inhibitory effects of ADN on MDA-MB-231 cell proliferation and Wnt/beta-catenin signaling.


Figure 2
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  		Fig. 2. Overexpression of WIF1 suppresses nuclear beta-catenin activities and attenuates serum-stimulated phosphorylations of Akt and GSK3β in MDA-MB-231 cells. (A) MDA-MB-231 cells transiently transfected with the expression vectors pcDNA3.1+ (control) and pcDNA-WIF1 (WIF1) were collected at 48 h after transfection for western blotting analysis, using antibodies recognizing the FLAG epitope (for detecting FLAG-tagged WIF1), beta-catenin or cyclin D1. (B) Transiently transfected MDA-MB-231 cells cultured under serum-free conditions were stimulated with 10% FBS for 60, 90 and 120 min and then harvested for detecting phosphorylated Akt (P-Akt) and phosphorylated GSK3β (P-GSK3β) by western blotting analysis. (C) Nuclear beta-catenin activities were evaluated in 0.5 and 10% FBS DMEM culture conditions using TOPflash/FOPflash reporter assays as described in Materials and Methods. *P < 0.05 and **P < 0.01 versus controls, n = 6–8, representative results from three independent studies were shown.

 


Figure 3
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  		Fig. 3. Overexpression of WIF1 inhibits MDA-MB-231 cell growth and mediates the antiproliferative effects of ADN. Upper panel: the expression vectors pcDNA3.1+ (control) and pcDNA-WIF1 (WIF1) were transfected into MDA-MB-231 cells as described in Figure 2. Cell proliferation was evaluated in both 0.5% FBS DMEM (A) and 10% FBS DMEM culture conditions (B) in the presence or absence of WIF1 neutralization antibody (WIF1 IgG). Bottom panel: MDA-MB-231 cells cultured in DMEM containing 0.5% FBS (C) or 10% FBS (D) were treated without (vehicle) or with 15 µg/ml ADN in the presence or absence of WIF1 IgG for 24 h. At the end of treatment, cell proliferation was evaluated as described in Materials and Methods. Note that neutralization of WIF1 attenuated the inhibitory effects of ADN on MDA-MB-231 cell proliferation. *P < 0.05 and **P < 0.01, n = 6–8. Results are derived from three independent studies.

 
ADN modulates the methylations of CpG islands located within the proximal region of WIF1 promoter
Several studies have shown that WIF1 promoter is aberrantly hypermethylated in both human breast tumor cell lines and mammary tumors, which correlates to the absence or low levels of WIF1 expression (28,33). To elucidate the potential mechanisms underlying the stimulatory effects of ADN on WIF1 expression, we then examined the promoter methylation status of WIF1 in MDA-MB-231 cells. Bisulfite sequencing of human WIF1 promoter (–467 to +1) revealed that ADN treatment resulted in a decreased average percentage (27.86%) of the methylated CpG islands comparing with vehicle-treated cells (37.35%). The increased levels of unmethylated CpG islands in ADN-treated samples were further confirmed by MSP analysis (Figure 4A). Especially, we found that the methylations of several CpG islands located within the WIF1 promoter region from –467 to –382, which includes a Sp1-binding site (5'-GGCGGG-3'), were profoundly attenuated in cells treated with ADN (Figure 4B and supplementary Figure 7 is available at Carcinogenesis Online). Pyrosequencing results demonstrated that in untreated MDA-MB-231 cells, the average percentage of methylated CpG1-4 was ~76.76%, whereas the methylation percentage was reduced to ~24.24% by ADN treatment (Figure 4C). We also analyzed the promoter methylation status of WIF1 in MDA-MB-231 tumors collected from nude mice. Bisulfite genomic sequencing analysis revealed that in tumors treated with ADN through protein pretreatment, intratumor or tail vein injections, the average percentage of methylated CpG islands within the WIF1 promoter region from –467 to –382 was ~25, 29.15 and 34.18%, which were much lower than their respective control groups, 66.68, 58.33 and 62.5%. The methylation changes induced by ADN treatment were further confirmed by MSP analysis (Figure 4D). Consistently, the percentage of methylated CpG dinucleotides within the Sp1-binding region was also significantly decreased (Figure 4E). These results demonstrated that ADN could facilitate demethylation of CpG islands in WIF1 promoter, which might contribute to its stimulatory activities on WIF1 expressions. Note that the reduced CpG methylation of WIF1 promoter in MDA-MB-231 cells could only be observed at 24 h after ADN treatment, but not during the early stages (data not shown), suggesting that it might not elicit its effects through de novo demethylation.


Figure 4
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  		Fig. 4. ADN promotes demethylation of the CpG islands located within the proximal region of WIF1 promoter. The methylated status of human WIF1 promoter (from –467 to –382) in MDA-MB-231 cells treated with or without ADN for 24 h was evaluated by MSP (A), bisulfite genomic sequencing (B) and pyrosequencing (C) analysis. The in vivo effects of ADN treatment on WIF1 promoter methylation were also assessed by MSP (D) and bisulfite genomic sequencing (E) analysis using tumor tissues collected from nude mice treated with or without ADN as described in Figure 1. Note that the methylations of CpG dinucleotides of Sp1 transcription factor-binding region could be largely reduced by ADN treatment.

 
ADN downregulates Sp1, a transcription factor negatively involved in regulating WIF1 expressions
It has been reported previously that the 85 bp region within WIF1 promoter (from –382 to –467) containing the Sp1-binding site displays variable degrees of CpG island methylation in human breast cancer cells (28). 5-Azacytidine treatment also caused significant demethylation of CpG islands within this region, which was accompanied by increased protein and mRNA expressions of WIF1 (supplementary Figure 8 is available at Carcinogenesis Online). Note that coincubation with Trichostatin A could not further enhance the demethylation effects of 5-azacytidine on WIF1 promoter, but dramatically augmented the mRNA levels to ~32-fold higher, suggesting that in addition to DNA methylation, other mechanisms such as histone acetylation might be involved in regulating WIF1 expressions. Sp1 belongs to a family of transcription factors that can bind GC/GT-rich promoter elements (37). DNA methyltransferase 1 (DNMT1), an enzyme involved in WIF1 gene silencing through methylation of cytosine residues, can be upregulated by Sp1 (28,38). During our studies on the methylation status of WIF1 promoter in different cancer cell lines, including MDA-MB-231, BT474, T47D, HT29 and SW480 cells, we noticed that the increased numbers of CpG methylation sites within this region were negatively correlated with the gene and protein levels of WIF1 but positively related to Sp1, DNMT1 and intracellular β-catenin accumulations and its nuclear activities (supplementary Figure 9 is available at Carcinogenesis Online). For example, in T47D cells, the WIF1 expression was about five times higher than that of MDA-MB-231 cells, and the eight CpG dinucleotides in Sp1-binding region were barely methylated. These informations indicated that the methylation/demethylation of the Sp1-binding site might be critically involved in regulating WIF1 expression levels.

We subsequently investigated whether ADN could affect Sp1 and DNMT1 protein expression levels. Our results demonstrated that while ADN could increase WIF1 expressions, it dramatically reduced the levels of both Sp1 and DNMT1 in MDA-MB-231 cells (Figure 5A). Downregulation of Sp1 by specific siRNA silencing also decreased DNMT1 levels but increased WIF1 expressions, resembling the effects of ADN (Figure 5B). Note that coincubation with ADN had no additive effects on these changes (data not shown), suggesting that the increased WIF1 expressions in ADN-treated MDA-MB-231 cells might be attributed to its effects on Sp1 and the downstream target DNMT1. In addition, real-time PCR results showed that the mRNA levels of WIF1 gene were significantly increased in cells transfected with Sp1 siRNA (Figure 5C). MSP analysis revealed that knocking down of Sp1 enhanced the demethylation of WIF1 promoter (Figure 5D), which might be related to the decreased DNMT1 levels (28). In summary, these results suggested that ADN might elicit its stimulatory effects on WIF1 gene expressions and promoter demethylations through inhibiting Sp1 and DNMT1 expressions in MDA-MB-231 cells.


Figure 5
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  		Fig. 5. ADN downregulates the expression levels of Sp1 and DNMT1, two critical factors involved in regulating WIF1 expression and promoter hypermethylation. Protein levels of Sp1 and DNMT1 in MDA-MB-231 cells treated with or without ADN were assessed by western blotting analysis (A). MDA-MB-231 cells transfected with or without siRNA for Sp1 were cultured in the presence or absence of ADN for 24 h. The protein expression levels of Sp1, DNMT1 and WIF1 were measured by western blotting analysis (B) and WIF1 mRNA levels measured by real-time PCR analysis (C). The methylation status of WIF1 promoter was evaluated by MSP (D) analysis. **P < 0.01, n = 6. Results were derived from three independent studies.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Supplementary material
 Funding
 References
 
Lower ADN levels are believed to be causatively associated with increased risks of breast cancer diseases (11–15). The inhibitory effects of ADN on human breast carcinoma cell growth and tumor development have been confirmed by many cellular and animal studies (11,16–19). The antiproliferative activities of ADN have also been demonstrated in other types of cancer cells, including the endometrial cancer, gastric cancer and prostate cancer cells (15). Despite the detailed molecular mechanisms remain elusive, it has been found that ADN can elicit distinctive signaling pathways in a cell type-dependent manner. We have reported that ADN could negatively regulate the Wnt/beta-catenin pathways in MDA-MB-231 human breast carcinoma cells (19). Here, we presented both in vitro and in vivo evidences demonstrating that ADN treatment enhanced the mRNA and protein expressions of a Wnt antagonist, WIF1, which might at least partially mediate the inhibitory effects of ADN on Wnt/beta-catenin signaling (Figures 1, 2 and 3). In MDA-MB-231 cells, overexpression of WIF1 could suppress intracellular accumulation of beta-catenin and its nuclear activities, decrease cyclin D1 expression levels and inhibit cell proliferation (Figure 2). The inhibitory effects of WIF1 on nuclear beta-catenin activities could also be observed in T47D cells transiently overexpressing human beta-catenin protein (supplementary Figure 10 is available at Carcinogenesis Online). Importantly, blockage of WIF1 activities stimulated the cell growth and significantly attenuated its own or the growth inhibitory effects of ADN (Figure 3). Similar blockage effects could also be observed in BT474 and T47D cells, two human breast carcinoma cell lines having relative high levels of WIF1 expressions (supplementary Figure 11 is available at Carcinogenesis Online). The stimulatory effects of ADN on WIF1 expressions could be observed after 24 h of chronic treatment (Figure 1). This is timely consistent with our observations that serum-induced phosphorylations of Akt and GSK3β could only be blocked in MDA-MB-231 cells pretreated with ADN for 24 h but not in those cells without pretreatment (19), suggesting that the increased WIF1 levels might participate in the signaling and biological effects induced by this hormone. Indeed, the phosphorylations of Akt and GSK3β could be attenuated by increasing WIF1 expressions (Figure 2B) (39). These results collectively suggest that the WIF1 might play an important role in mediating the antitumor activities of ADN in MDA-MB-231 cells.

Downregulation of WIF1 is found in breast, prostate, lung, bladder, colorectal, gastrointestinal, nasopharyngeal and esophageal carcinomas and may be an early event in tumourigenesis in these tissues (28,33,40–42). Promoter hypermethylation is the major culprit contributing to WIF1 gene silencing. Interestingly, our results showed that ADN treatment significantly decreased the numbers of methylated CpG islands within WIF1 promoter in MDA-MB-231 cells and in tumor tissues derived from nude mice (Figure 4). As the effects are observed after prolonged treatment with ADN, we could not exclude the possibilities of selection for pre-existing subpopulations. However, we believe that these effects are related to the downregulation of the transcription factor Sp1 based on the following evidences: firstly, the methylations of several CpG islands within or close to the Sp1-binding region were dramatically decreased in ADN-treated MDA-MB-231 cells and tumors; secondly, ADN considerably downregulated the protein levels of Sp1 and its target DNMT1, which might be directly involved in hypermethylation of WIF1 promoter (28); thirdly, knocking down of Sp1 expressions increased both the gene and protein expressions of WIF1 and decreased its promoter methylation. To further confirm our findings, we have tested whether ADN could enhance the promoter demethylation of another gene, WNT5A, which contain multiple Sp1-binding sites (43). Our results revealed that ADN significantly decreased the methylations of CpG dinucleotides located within four GC box/Sp1 DNA-binding sequences and increased the gene expression of WNT5A by ~13-folds at 48 h after treatment (supplementary Figure 12 is available at Carcinogenesis Online). These results suggest that ADN could promote the demethylation of WIF1 and WNT5A promoter, possibly through a Sp1-related mechanism.

Sp1 is involved in many biological processes, including cell proliferation, apoptosis, differentiation and transformation, and also plays an important role in tumor progression, invasion and metastasis (37). Although the expression patterns of Sp1 in various tumors are different, increased Sp1 expressions have been consistently observed in specimens of breast cancer comparing with adjacent breast tissue (44). Moreover, Sp1 staining in cancer tissue is positively correlated to TNM classification of malignant tumors stage, tumor invasion and lymph node metastasis (45). There is growing evidence suggesting that Sp proteins play critical roles in the growth and metastasis of mammary tumor cells. Depending on the gene context, Sp1 is able to enhance or repress promoter activity (46). Elevated Sp1 has been directly associated with increased expression of genes that are critically involved in tumorigenesis and metastatic progression, such as vascular endothelial growth factor, urokinase plasminogen activator and its receptor, cyclin D1 and breast cancer 1, possibly through regulations at the transcriptional level (37,47). On the other hand, genes involved in antioxidative processes, including methionine sulfoxide reductase B1 and glutathione S-transferase that are downregulated in cancer cells, have been shown to contain heavily methylated CpG dinucleotides within Sp1-binding region of their promoters (48,49). It has been reported that Sp1 can bind to both unmethylated and methylated GC-rich promoters for recruiting factors involved in DNA methylation, such as DNMT1 and methyl CpG-binding proteins (49). In addition, Sp1 can stimulate DNMT1 expressions, which might also contribute to the increased promoter methylation of its target genes. Despite these information, the roles of Sp1 and the detailed epigenetic mechanisms underlying ADN-mediated WIF1 expression are not clear and currently under investigation in our laboratory.

Hypermethylation within CpG islands represent the most common mechanism contributing to the inactivation of tumor suppressor genes in cancers. Methylation changes also occur during aging, chronic inflammation, infections and diet interventions. It has been noticed that age- or inflammation-related methylation applies to different regions within a promoter from those associated with tumor development. These pretumor methylation sites might form the seeds for methylation in core regions that are responsible for gene silencing. Indeed, studies have revealed that methylations at Sp1 sites occur at the early stage of tumor development and could promote dense methylations and gene silencing observed at the late stage of tumor development (48). It will be interesting to investigate whether the methylation of the CpG islands within Sp1-binding region in WIF1 promoter occurs at the early stage of breast cancer development and facilitates subsequent WIF1 gene silencing as well as dense methylation of WIF1 promoter.

In summary, we have demonstrated that ADN could regulate the expressions of a tumor suppressor gene, WIF1, through promoting epigenetic activations. The increased WIF1 might be critically involved in the antitumor effects of ADN by mediating its inhibitory activities on Wnt/beta-catenin signaling. As WIF1 gene is normally highly expressed in normal breast epithelial cells, our results, together with previously reported studies, suggest that decreased ADN levels might be etiologically associated with WIF1 gene silencing observed in clinical breast cancer patients.


   Supplementary material
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
Funding
 References
 
Supplementary Figures 6–12 can be found at http://carcin.oxfordjournals.org/


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
References
 
University of Hong Kong (200511159013 to Y.W.); Hong Kong Research Grant Council (HKU 778007 to Y.W., HKU 7645/06M to A.X.).


   Footnotes
 
{dagger} Co-first author. Back


   Acknowledgments
 
We thank Dr Patrick Y.Wang for helping with the bioinformatics analysis and Ms Carol C.F.Lau for performing the pyrosequencing analysis.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
 References
 

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Received April 13, 2008; revised July 21, 2008; accepted August 8, 2008.


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K. Kawakami, H. Hirata, S. Yamamura, N. Kikuno, S. Saini, S. Majid, Y. Tanaka, K. Kawamoto, H. Enokida, M. Nakagawa, et al.
Functional Significance of Wnt Inhibitory Factor-1 Gene in Kidney Cancer
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