Secreted Frizzled-Related Protein 4 Is Silenced by Hypermethylation and Induces Apoptosis in B-Catenin – Deficient Human Mesothelioma Cells
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Research Article Secreted Frizzled-Related Protein 4 Is Silenced by Hypermethylation and Induces Apoptosis in B-Catenin – Deficient Human Mesothelioma Cells Biao He,1 Amie Y. Lee,1 Sina Dadfarmay,1 Liang You,1 Zhidong Xu,1 Noemi Reguart,1,2 Julien Mazieres,1,3 Iwao Mikami,1 Frank McCormick,1 and David M. Jablons1 1Thoracic Oncology Laboratory, Department of Surgery, Comprehensive Cancer Center, University of California, San Francisco, California; 2Medical Oncology Service, Institut Catala` d’Oncologia, Hospital Germans Trias i Pujol, Barcelona, Spain; and 3Department Innovation Therapeutique et Oncologie Moleculaire, Institut National de la Sante et de la Recherche Medicale U563, Institut Claudius Regaud, Toulouse, France Abstract acute lymphoblastic leukemia (7), and renal clear cell carcinoma The secreted frizzled-related proteins (SFRPs) function as (8). Forced expression of Dkk-1 in brain tumor (9) and cervical negative regulators of Wnt signaling and have important cancer (10), or Dkk-3 in non–small cell lung cancer (4) and implications in tumorigenesis. Frequent promoter hyper- osteosarcoma (11), inhibits cell growth. Down-regulation of SFRPs methylation of SFRPs has been identified in human cancer. was found in several cancers, including mesothelioma (12), breast Restoration of SFRP function attenuates Wnt signaling and cancer (13), cervical cancer (14), and gastric cancer (15, 16). Loss induces apoptosis in a variety of cancer types. Wnt signaling of SFRP family expression was also found to be associated with is known to inhibit apoptosis through activation of BB-catenin/ promoter hypermethylation in mesothelioma and colorectal Tcf–mediated transcription. Recently, we identified aberrant cancer tissue samples (12, 16–18). Furthermore, restoration of Wnt activation as a result of Dishevelled overexpression in SFRPs in colon cancer cell lines carrying downstream mutations h malignant mesothelioma. Here, we report that silencing suppressed Wnt/ -catenin-dependent transcription and induced of SFRP4 is correlated with promoter hypermethylation in apoptosis, suggesting that Wnt signaling may be regulated in a BB-catenin–deficient mesothelioma cell lines. Reexpression of quantitative manner at different levels (18). However, there are few h SFRP4 in these BB-catenin–deficient mesothelioma cell lines reports that have linked -catenin-independent pathway(s) to the blocks Wnt signaling, induces apoptosis, and suppresses Wnt-dependent apoptotic inhibition (19). Here, we report that h growth. Conversely, knocking down SFRP4 by small interfer- SFRP4 is methylation silenced in -catenin-deficient mesothelio- ing RNA in cell lines expressing both SFRP4 and BB-catenin ma cell lines. Furthermore, we show that reexpressing SFRP4 in stimulates Wnt signaling, promotes cell growth, and inhibits these cell lines down-regulates Dishevelled (Dvl), induces apopto- h chemodrug-induced apoptosis. Our results suggest that sis, and suppresses cell growth, suggesting that -catenin- methylation silencing of SFRP4 may play an important role independent pathway(s) may be important for the apoptotic in aberrant Wnt activation in mesothelioma even in the inhibition caused by Wnt activation. absence of BB-catenin. Our data also suggest that BB-catenin– independent noncanonical pathway(s) may be involved in the Materials and Methods apoptotic inhibition caused by activation of Wnt signaling. Cell Lines. The human mesothelioma cell lines H28 and H2052 were (Cancer Res 2005; 65(3): 743-8) obtained from American Type Culture Collection (Manassas, VA). The human mesothelioma cell line MS-1 was obtained from NIH (Bethesda, Introduction MD). These cell lines were cultured in RPMI 1640 supplemented with 10% fetal bovine serum, penicillin (100 IU/mL), and streptomycin (100 Ag/mL). The activation of the canonical Wnt pathway is mediated by Normal human small airway epithelial cells (primary culture) were h -catenin (1, 2). However, noncanonical pathways act indepen- obtained from Clonetics (Walkersville, MD) and cultured in Clonetics h dently of -catenin (1, 2). At least two classes of Wnt antagonists SAGM Bullet kit. All cells were cultured at 37jC in a humid incubator with have been reported (1). The first class, including secreted Frizzled- 5% CO2. related protein (SFRP) family and Wnt inhibitory factor 1, binds Semiquantitative Reverse Transcription-PCR. Total RNA from cell directly to Wnt ligands. The second class, including the Dickkopf lines was isolated using Qiagen RNeasy Mini Kit (Valencia, CA). Reverse (Dkk) family, binds to low-density lipoprotein receptor–related transcription-PCR (RT-PCR) was done in GeneAmp PCR system 2700 protein 5/6. Recently, both SFRP and Dkk families have been (Applied Biosystems, Foster City, CA) using One-Step RT-PCR kit from implicated in oncogenesis. For example, the production of Dkk-1 Invitrogen Life Technologies (Carlsbad, CA). Primers for RT-PCR were by myeloma cells is associated with the presence of lytic bone obtained from Operon Technologies, Inc. (Alameda, CA). Primer sequences lesions in patients with multiple myeloma (3). Dkk-3 has been for amplifying the human SFRP4 were described previously (17). Glyceraldehyde-3-phosphate dehydrogenase was used as internal found silenced by methylation in non–small cell lung cancer (4–6), control. Methylation and Sequencing Analysis. Genomic DNA from cell lines was extracted using DNA STAT-60 reagent (TEL-TEST, Inc., Friendswood, TX). Bisulfite modification of genomic DNA was done by using EZ DNA Requests for reprints: David M. Jablons, Thoracic Oncology Laboratory, methylation kit (Zymo Research, Orange, CA). Bisulfite-treated genomic Department of Surgery, Comprehensive Cancer Center, University of California, DNA was amplified using primers described previously for human SFRP4 1600 Divisadero Street, C322C, Campus Box 1674, San Francisco, CA 94115. Phone: 415-353-7502; Fax: 415-502-3179; E-mail: [email protected]. (17). The amplified 230-bp product corresponds to À384 to À154 in the #2005 American Association for Cancer Research. SFRP4 promoter region (the start codon ATG of SFRP4 is defined as 0). www.aacrjournals.org 743 Cancer Res 2005; 65: (3). February 1, 2005 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2005 American Association for Cancer Research. Cancer Research 5-Aza-2V-deoxycytidine (Sigma, St. Louis, MO) treatment was done as Cruz, CA). Anti-caspase-3 antibody was purchased from Oncogene (Cam- described previously (17). For amplification of SFRP4 genomic region, bridge, MA). Anti-c-Myc antibody was purchased from Cell Signaling primers were designed according to the SFRP4 genomic sequence in Technology (Beverly, MA). Anti-h-actin antibody was purchased from chromosome 7. Untreated genomic DNA isolated from the cell lines were Sigma-Aldrich Corp. (St. Louis, MO). Anti-h-catenin antibody was used as template. The primer sequences and the length of fragments purchased from Transduction Laboratories (Lexington, KY). For detecting h amplified are listed in Table 1. The PCR products were extracted from alteration of -catenin, cytosolic extracts were prepared and examined as the agarose gel using Qiagen QIAquick Gel Extraction Kit and were described previously (20). RNA Interference. RNA interference experiments were done as subsequently sequenced at the DNA Sequencing Core Facility, Compre- described previously (19). The ion-exchange high-performance liquid hensive Cancer Center, University of California (San Francisco, CA). chromatography–purified small interfering RNA (siRNA; SFRP4 siRNA Western Blotting. Standard protocol was used. Anti-Dvl-3 and anti- and nonsilencing siRNA control, >97% pure) were purchased from Qiagen- Survivin antibodies were obtained from Santa Cruz Biotechnology (Santa Xeragon (Germantown, MD). The targeted sequence of SFRP4 siRNA is 5V- AAGTCCCGCTCATTACAAATT-3V, corresponding to +701 to +721 of the human SFRP4 cDNA sequence (the start codon ATG is defined as +1). To Table 1. Primers used for amplifying SFRP4 analyze proliferation, 3 Â 104 cells were transfected with siRNA in a 24-well genomic region plate. After transfection, viable cells (trypan blue exclusion) were collected by trypsinization and counted at various time points. Experiments were Primer Sequence Size (bp) done in triplicate. Apoptosis Analysis. Cells were harvested by trypsinization and stained 1F:5V-ATGTTCCTCTCCATCCTAGT-3V 350 using an Annexin V FITC Apoptosis Detection Kit (Oncogene) according to R: 5V-TGGTTGTACATCTTCATGAG-3V the manufacturer’s protocol. Then, stained cells were immediately analyzed 2F:5V-CTCATGAAGATGTACAACCA-3V 420 by flow cytometry (FACScan, Becton Dickinson, Franklin Lakes, NJ). R: 5V-TAGCAACACCTGTTTACTGC-3V Alimta (LY231514, multitargeted antifolate, pemetrexed) was supplied by 3F:5V-GGGAGCCTTCGATGATGTTT-3V 470 Eli Lilly (Indianapolis, IN). Alimta was diluted in sterile physiologic solution R: 5V-GGTAACCTGGGCCAGATAAA-3V at a concentration of 10 mg/mL. The solution was divided into aliquots, 4F:5V-TTATCCAGGCTTTATCTGGC-3V 430 stored at À80jC, and diluted in culture medium before each experiment. R: 5V-CCAAGCACAAATCGATGTGA-3V Transient Transfection and Colony Formation Assay. For transient 5F:5V-TCACATCGATTTGTGCTTGG-3V 400 transfection experiments, cells (2 Â 105) were plated in six-well plates 24 V V R: 5-TAATCCCAGCAGTTTGGGAA-3 hours before transfection. LipofectAMINE 2000 (Invitrogen Life Technolo- V V 6F:5-TTCCCAAACTGCTGGGATTA-3 390 gies) was used to mediate transfection using 5.0 Ag SFRP4 cDNA construct R: 5V-CGTTGCCAAAGTTGGCTTCA-3V