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Secreted frizzled-related protein-5 (sFRP-5) has been identified as one of the secreted antagonists that bind Wnt protein. However, the functional significance of sFRP-5 in renal cell cancer (RCC) has not been reported. We hypothesized that sFRP-5 may be epigenetically downregulated through DNA methylation and histone modification and function as a tumor suppressor gene in RCC. Using tissue microarray and real-time RT-PCR, we found that sFRP-5 was significantly downregulated in kidney cancer tissues and cell lines, respectively. DNA bisulfite sequencing of the sFRP-5 promoter region in RCC cell lines showed it to be densely methylated whereas there was few promoter methylation in normal kidney. The sFRP-5 expression was restored and the acetylation of H3 and H4 histones associated with the sFRP-5 promoter region were significantly increased after treatment with demethylation agent (5-Aza-dc) and histone deacetylase inhibitor (TSA). When RCC cells were transfected with the sFRP-5 gene, significant inhibition of anchorage independent colony formation and cell invasion were observed compared to controls. The sFRP-5 transfection also significantly induced apoptosis in RCC cells. In conclusion, this is the first report documenting that the sFRP-5 is downregulated by promoter methylation and histone acetylation and functions as a tumor suppressor gene by inducing apoptosis in RCC cells.
The Wnt signaling pathway plays an important role during animal development.1,2 The main feature of the Wnt signaling pathway is the regulation of the transcription factor β-catenin, which together with members of the T cell factor lymphoid enhance factor (TCF/LEF) family, promotes the transcription of several genes that result in proliferation and reduced apoptosis.3,4 Recently, the Wnt/ β-catenin pathway has been shown to be involved in tumorigenesis involving several cell types. Aberrant expression of Wnt signaling has been reported in various human cancers, including breast cancer, colorectal cancer, non-small cell lung cancer (NSCLC) and bladder cancer.5-8
Secreted frizzled-related proteins (sFRPs), a family of five secreted glycoproteins, have been identified as possible negative modulators of the Wnt signal transduction pathway.9 sFRPs block Wnt signalling either by interacting with Wnt proteins to prevent them from binding to Fz (Frizzled) proteins those are receptors for Wnt and positive mediators of the Wnt signaling pathway, or by forming non-functional complexes with Fz proteins.10,11
Recently, it has become clear that aberrant epigenetic silencing of tumor suppressor genes by promoter DNA hypermethylation and histone deacetylation plays an important role in the pathogenesis of human cancer.12 Many candidate tumor suppressor genes silenced by DNA methylation and/or histone modification also have been reported in RCC.13-15 In addition, members of the sFRP family such as sFRP-1 and sFRP-2 have been reported to be epigenetically silenced in RCC.16-19
Secreted frizzled-related protein-5 (sFRP-5) is also a member of the sFRP family and downregulation by promoter hypermethylation has been reported recently in various human malignancies including gastric, cervical, hepatocellular, pancreatic, oral squamous cell, breast, colon and bladder cancer.5,20-28 With regard to RCC, a previous report from our laboratory showed that the methylation frequency of Wnt antagonists in clinical samples could serve as biomarkers for RCC,29 however, there have been no other previous reports investigating the function of sFRP-5 in RCC and the involvement of histone modifications in sFRP-5 repression. Therefore, in the present study, we hypothesized that sFRP-5 is epigenetically downregulated and functions as a tumor suppressor gene in RCC. To address this issue, we examined sFRP-5 mRNA and protein expression levels in RCC cell lines and human RCC tissues. We also looked at the methylation status of the sFRP-5 promoter region by bisulfite sequencing and the histone acetylation by ChIP assay. In addition, we overexpressed sFRP-5 by transfection in a RCC cell line (A498) and studied its effect on colony formation, invasion and apoptosis.
A tissue microarray which consisted of twelve ccRCCs and matched adjacent tissue 1.5 cm away from the carcinoma was obtained from US Biomax, Inc. (KD481; Rockville, MD). Detailed information on the tumor samples can be found at http://www.biomax.us/index.php. Immunostaining was done on the tissue microarray using the UltraVision Detection System (Thermo Scientific, Seattle, WA) following the manufacture's protocol. Antigen retrieval was carried out by microwaving the slide in 10 mmol/L sodium citrate buffer. The slide was incubated overnight with a 1:100 dilution of anti-sFRP-5 antibody (cat#; LS-B307, LifeSpan Biosciences, Inc., Minneapolis, MN). A pathologist not involved in the present study evaluated the immunostaining under blind conditions. Immunohistochemical staining was graded on an arbitrary scale from 0 to 2; 0 representing negative expression (0–20% positive cells), 1 representing weakly positive expression (20–50% positive cells) and 2 representing strongly positive expression (50–100% positive cells). The renal tubular cells in normal tissues and tumorous cells (not stromal cells) in cancer tissues were scored and the scale was determined according to the average number of positive cells in 10 random fields of the slide.19
We used four human kidney cancer cell lines, Caki1, Caki2, ACHN and A498 obtained from the American Type Culture Collection (ATCC, Manassas, VA). The Caki1, Caki2 and A498 cell lines were incubated in RPMI 1640 medium supplemented with 10% fetal bovine serum. ACHN cells were maintained in Eagle's minimal essential medium supplemented with 10% fetal bovine serum. These cells were maintained in a humidified incubator (5% CO2) at 37 °C. For 5-aza-2’-deoxycytidine (5-Aza-dc; Sigma-Aldrich, St. Louis, MO) treatment, cells were treated with 5-Aza-dc (5μM) for 4 days. Culture medium and 5-Aza-dc were replaced daily. For the combined treatment, 300 nM of trichostatin A (TSA; Millipore, Bedford, MA) was added during the last 24 hours. Total RNA and genomic DNA were isolated using the RNeasy Mini kit (Qiagen, Valencia, CA) and QIAamp DNA mini kit (Qiagen), respectively, following the manufacturer's directions.
Bisulfite modification of genomic DNA was done using the EpiTect Bisulfite kit (Qiagen) following the manufacturer's protocol. The Genomic DNA from adult human normal kidney tissue (cat#; D1234142-50, BioChain, Hayward, CA) was also modified and used as a control. Primers for bisulfite genomic sequencing PCR were designed by using the online program MethPrimer30 or by referring to a previous report.24 The primer sequences and PCR conditions are shown in Table 1. The fragment amplified corresponded to the sFRP-5 promoter region −404 to −128 (the ATG start codon of sFRP-5 was defined as +1). All reactions were done as described previously from our laboratory.19 The amplification products were confirmed by electrophoresis on a 2% agarose gel and sequenced directly by an outside vendor (McLab, South San Francisco, CA).
Reverse transcription reactions were carried out with 2 μg of total RNA using a Reverse Transcription System Kit (Promega, Madison, WI). Semi-quantitative RT-PCR was done using REDTaq (Sigma-Aldrich). The primer sequences and PCR conditions are shown in Table 1. Quantitative real-time RT-PCR analysis was performed in triplicate with an Applied Biosystems Prism 7500 Fast Sequence Detection System using TaqMan universal PCR master mix according to the manufacturer's specifications (Applied Biosystems Inc., Foster City, CA). The TaqMan primer IDs purchased from Applied Biosystems are as follows, sFRP-5; Hs00169366_ml and GAPDH; Hs99999905_m1. The total RNA from adult human normal kidney tissue (cat#; R1234142-50, BioChain) was used as a control. The thermal cycler conditions were as follows: hold for 20 sec at 95 °C, followed by two-step PCR for 40 cycles of 95 °C for 3 sec, followed by 60°C for 30 sec. Levels of RNA expression were determined using the 7500 Fast System SDS software version 1.3.1 (Applied Biosystems).
ChIP assays were performed on cell line DNA using the EZ-ChIP Kit (Millipore, Billerica, MA) and followed the manufacturer's protocols. Chromatin was prepared from confluent cells and sonicated by using a Bioruptor (Diagenode, Sparta, NJ), then immunoprecipitated with each antibody. Antibodies for acetylated histone H3 (AcH3, 06-599, Millipore) and acetylated histone H4 (AcH4, 06-866, Millipore) were used in the immunoprecipitations. The immunoprecipitated DNA was eluted in a total volume of 200μl, and 5 μl of each DNA sample were analyzed by semi-quantitative PCR and quantitative real time PCR using SYBR® Green PCR Master Mix (Applied Biosystems). Semi-quantitative PCR was performed as described previously.19 The thermal cycler conditions for quantitative real time PCR were as follows: hold for 10 min at 95 °C, followed by three-step PCR for 40 cycles of 95 °C for 30 sec, 60°C for 30 sec and 72°C for 30 sec. Cycle threshold (CT) value was used for relative quantification of sFRP-5 expression. Fold changes relative to input ΔCT were calculated as 2−ΔΔCT.31 PCR primers were designed to amplify the corresponding promoter region of sFRP-5 (−180 to −81; the ATG start codon of sFRP-5 was defined as +1). The primer pairs used for ChIP assays and PCR conditions are shown in Table 1.
Plasmids containing the human full-length cDNA fragment of sFRP-5 (sFRP-5; cat#RC208391) and empty vector (EV; cat#PS100001) were purchased from Origene (Rockville, MD). A498 cells (1 × 105 cells per well of 6-well plate) were transfected with either sFRP-5 or EV using FuGENE HD (Roche Applied science, Indianapolis, IN) according to the manufacturer's protocol. The transfected A498 cells were selected with 400μg/ml of G418 (Invitrogen, Carlsbad, CA). Cell lines stably expressing sFRP-5 (A498-sFRP-5) and transfected with empty vector (A498-EV) were maintained with 200μg/ml of G418.
Soft agar colony formation was done with EV-transfected stable A498 cells (A498-EV) and sFRP-5-transfected stable A498 cells (A498-sFRP-5) using a CytoSelect Cell Transformation Assay kit (Cell Biolabs, San Diego, CA) according to the manufacturer's protocol. Cells were incubated 7 days in semisolid agar media before being solubilized and detected by using the provided MTT solution in a microplate reader (Spectra MAX 190; Molecular Devices Co., Sunnyvale, CA) at OD570nm. Data are the mean ± S.D. of 6 independent experiments. Invasion assay was conducted with A498-EV and A498-sFRP-5 using the BioCoat Matrigel Invasion Chambers (BD Biosciences, Bedford, MA). Cells were incubated for 22 h at 37°C in a 5% CO2 atmosphere. Those cells that migrated through the membrane to the lower surface were stained with Hema-3 (Fisher Scientific, Middletown, VA) and counted with a microscope. Four random fields were chosen for each membrane, and the results were shown as migrated cells per field.
A498 cells transiently transfected with EV or sFRP-5 were harvested 72 hours after transfection by trypsinization and stained using an ANNEXIN V-FITC /7-AAD KIT (Beckman Caulter, Inc. Fullerton, CA) according to the manufacturer's protocol and immediately analyzed by flow cytometry (Cell Lab Quanta SC; Beckman Coulter, Inc.). Experiments were done in triplicate.
Data are shown as mean values ± standard deviation (SD). The Student's t-test was used to compare the two different groups. P values of less than 0.05 were regarded as statistically significant. All statistical analyses were performed using StatView version 5.0 for Windows.
To examine the expression levels of sFRP-5 in malignant and normal kidney tissues, immunostaining was performed on a tissue microarray using antibody to sFRP-5. The macroscopic appearance of the tissue microarray and typical immunostaining of sFRP-5 in normal and ccRCC specimens (x40 and x200) are shown in Fig.1A. The staining was much stronger in normal specimens than in ccRCC. The mean expression score (1.864±0.351) in normal specimens (N=22) was significantly higher compared to that (1.000±0.590) in ccRCC specimens (N=24) (p<0.0001; Fig.1b). This result suggests that sFRP-5 protein expression is downregulated in human ccRCC.
The methylation status at 17 CpG sites of the sFRP-5 CpG island was characterized in four RCC cell lines and normal kidney DNA by bisulfite genomic sequencing (Fig. 2a, b and c). These analyses indicated that the CpG islands in four RCC cell lines showed essentially the same methylation status. The promoter regions checked in these RCC cell lines were densely methylated whereas the same region in normal kidney was mostly unmethylated.
We checked sFRP-5 mRNA expression in RCC cell lines and in human normal kidney RNA by real time RT-PCR. As shown in Figure 3a, sFRP-5 mRNA expression was significantly downregulated in RCC cell lines compared to human normal kidney RNA (p<0.01). We also examined the changes in sFRP-5 expression after treatment of cells with 5-Aza-dc, an inhibitor of DNA methyltransferases (DNMTs) and TSA, a histone deacetylase (HDAC) inhibitor. After the combined treatment with 5-Aza-dc and TSA, sFRP-5 expression was restored in all RCC cell lines tested (Fig. 3b). Treatment using only 5-Aza-dc also restored sFRP-5 mRNA expression indicating that downregulation of sFRP-5 expression in RCC cell lines correlates with CpG methylation of the sFRP-5 promoter.
To further investigate whether the transcriptional upregulation of sFRP-5 was associated with alternations in histone acetylation status at the promoter region, we examined local histone acetylation in the chromatin associated with the sFRP-5 promoter region using AcH3 and AcH4 antibodies by ChIP assay. We found that 5-Aza-dc and TSA treatment significantly increased the state of acetylation of H3 and H4 histones associated with an open, transcriptionally active regulatory region in the sFRP-5 gene in all cell lines (Fig.4a and b). There was no PCR amplification in control immunoprecipitations without antibody (data not shown).
Next, we examined the effect of sFRP-5 overexpression in A498 cells using soft agar colony formation and cell invasion assays. As shown in Figure 5a(1) and (2) left, sFRP-5 transcription was significantly increased at 72 hrs after transient transfection with sFRP-5 but not in untreated (UT) cells or those treated with empty vector (EV) (p<0.01). Stable cell line A498-sFRP5 also had significantly higher levels of sFRP-5 transcription compared to A498-EV (p<0.01, Fig. 5a (2) right) and we used these stable cell lines for soft agar colony formation and invasion assays. In soft agar colony formation assay, we found a significant reduction of anchorage independent colony formation in A498-sFRP-5 compared to A498-EV (p<0.05, Fig. 5b). As shown in Figure 5c, the number of invasive cells of A498-sFRP-5 (89.67±4.33) was also significantly decreased compared to that of A498-EV (62.33±3.06; p<0.05). These results suggest that sFRP-5 expression reduced anchorage independent colony formation efficacy and the invasive ability of A498 cells.
We also evaluated apoptosis by using flow cytometry with A498 cells which were transiently transfected with EV or sFRP-5. As shown in Figure 6a, the apoptotic cell fraction was 6.17% in EV-transfected A498 cells and 16.45% in those transfected with sFRP-5 at 72 hours after transfection. Thus sFRP-5 transient transfection significantly induced apoptosis in A498 cells (p<0.05; Fig. 6b).
RCC is the third most common genitourinary cancer, with 54,390 predominantly male cases and 13,010 deaths in the United States in 2008.32 Recently, the Wnt signaling pathway has been shown to be involved in tumorigenesis involving several cell types.5-8 The sFRPs have been identified as possible antagosists of the Wnt signaling pathway9 and sFRP-5, a member of the sFRP family, has been reported to be downregulated by promoter hypermethylation in various human cancers.5, 20, 22-27
In this study we found that sFRP-5 was significantly downregulated at the protein level in human ccRCC tissue microarrays and at the mRNA level in human RCC cell lines. Bisulfite sequencing of the sFRP-5 promoter region showed it to be densely methylated in RCC cell lines whereas there was few methylation in normal human kidney DNA. In addition, treatment with demethylation agent (5-Aza-dc) and histone deacetylase inhibitor (TSA) restored sFRP-5 mRNA expression in RCC cell lines. Our results indicate that downregulation of sFRP-5 expression in RCC cell lines and human cancer tissues correlate with CpG methylation of the sFRP-5 promoter. These results are consistent with studies in other types of cancer mentioned above. However, these previous reports did not examined histone modifications in the sFRP-5 gene. Several tumor suppressor genes are known to be repressed even though they have hypomethylated promoters.33, 34 This indicates that although DNA methylation is a major epigenetic mechanism for gene silencing, there are other epigenetic silencing pathways independent of DNA methylation.35 It is generally known that aberrant DNA methylation and histone modifications work together to silence many tumor suppressor genes in human cancer.36,37
Therefore, we examined the effect of a DNA methyltransferase inhibitor (5-Aza-dc) and a histone deacetylase inhibitor (TSA) on histone acetylation (AcH3, AcH4) associated with the sFRP-5 promoter region. We found that 5-Aza-dc and TSA treatment altered the state of chromatin modification by increasing the acetylation of histones associated with sFRP-5 genes. These data suggest that the induction of sFRP-5 gene is at least partially mediated by changes in histone acetylation. Taken together, the sFRP-5 gene may be downregulated by epigenetic events including DNA methylation and histone modification.
Using A498 cells transfected with a sFRP-5 expression plasmid, we also demonstrated that re-expression of sFRP-5 significantly inhibited anchorage independent colony formation and cell invasion. These results indicate that sFRP-5 functions as a tumor suppressor in RCC cells. Recent studies have revealed that sFRPs are tumor suppressor candidates.5,14,17,38 The expression of sFRPs is frequently silenced by promoter hypermethylation in a variety of cancers. Restored expression of sFRPs has been shown to inhibit cell growth in vitro and tumor growth in vivo.38 For instance, Gumz et al. reported that stable reexpression of sFRP-1 in RCC cells resulted in decreased expression of Wnt target genes, decreased growth in cell culture, inhibition of anchorage-independent growth, and decreased tumor growth in athymic nude mice.17 As for sFRP-5, Zhao et al. have shown that the expression of sFRP-5 was inversely correlated with the expression of MMP-7 and MT1-MMP, well known as positive regulators of invasion and metastasis, in gastric cancer cells.20 Also, Lin et al. have found that the restoration of sFRP-5 suppressed colony formation, invasive ability and inhibited expression of Wnt/ β-catenin downstream genes such as c-myc and cyclin D1 in cervical cancer cells.21 Thus our current studies are in agreement with these previous reports.
In the present study we have also shown that restoration of sFRP-5 expression significantly induces apoptosis in RCC cells. Targeted inhibition of the Wnt signaling pathway by sFRP-5 transfection has been shown in other cancers.23,27 Positive Wnt signaling has been reported to have anti-apoptotic activity in several cell types39,40 and our results and others suggest that sFRP-5 restoration leads to increased apoptosis in several types of cancer cells.
Recently, there is increasing evidence showing that some sFRPs have oncogenic functions. Under certain circumstances, some sFRPs are overexpressed in cancer41 and have growth promoting42 or anti-apoptotic effects.43 The apparently contradictory roles of sFRPs in these studies may be due to the different Wnt ligands present in different cells, biphasic responses to different concentrations of sFRPs, and the binding affinities and specificities of different sFRPs for Wnts.10 Further work will be needed to have a better understanding of the specific relationship between sFRPs including sFRP-5 and Wnt signaling.
In conclusion, we have shown for the first time that sFRP-5 is frequently downregulated in RCC cell lines and clinical ccRCC tissues. Our results suggest that the downregulation is caused by a combination of DNA methylation and histone modification. In addition, sFRP-5 overexpression inhibited colony formation and cell invasion and induced apoptosis in RCC cells. These results suggest that sFRP-5 functions as a tumor suppressor gene in RCC and that restoration of sFRP-5 may offer a new therapeutic approach for the treatment of RCC patients.
We thank Dr. Roger Erickson for his support and assistance with the preparation of the manuscript. This study was supported by Grants RO1CA130860, and T32DK007790 from the NIH, VA REAP award, and Merit Review grants (PI: R.D.).