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[Cancer Research 65, 743-748, February 1, 2005]
© 2005 American Association for Cancer Research

Molecular Biology, Pathobiology and Genetics

Secreted Frizzled-Related Protein 4 Is Silenced by Hypermethylation and Induces Apoptosis in ß-Catenin–Deficient Human Mesothelioma Cells

Biao He1, Amie Y. Lee1, Sina Dadfarmay1, Liang You1, Zhidong Xu1, Noemi Reguart1,2, Julien Mazieres1,3, Iwao Mikami1, Frank McCormick1 and David M. Jablons1

1 Thoracic Oncology Laboratory, Department of Surgery, Comprehensive Cancer Center, University of California, San Francisco, California; 2 Medical Oncology Service, Institut Catalàd'Oncologia, Hospital Germans Trias i Pujol, Barcelona, Spain; and 3 Department Innovation Therapeutique et Oncologie Moleculaire, Institut National de la Sante et de la Recherche Medicale U563, Institut Claudius Regaud, Toulouse, France

Requests for reprints: David M. Jablons, Thoracic Oncology Laboratory, Department of Surgery, Comprehensive Cancer Center, University of California, 1600 Divisadero Street, C322C, Campus Box 1674, San Francisco, CA 94115. Phone: 415-353-7502; Fax: 415-502-3179; E-mail: jablonsd{at}surgery.ucsf.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
The secreted frizzled-related proteins (SFRPs) function as negative regulators of Wnt signaling and have important implications in tumorigenesis. Frequent promoter hypermethylation of SFRPs has been identified in human cancer. Restoration of SFRP function attenuates Wnt signaling and induces apoptosis in a variety of cancer types. Wnt signaling is known to inhibit apoptosis through activation of ß-catenin/Tcf–mediated transcription. Recently, we identified aberrant Wnt activation as a result of Dishevelled overexpression in malignant mesothelioma. Here, we report that silencing of SFRP4 is correlated with promoter hypermethylation in ß-catenin–deficient mesothelioma cell lines. Reexpression of SFRP4 in these ß-catenin–deficient mesothelioma cell lines blocks Wnt signaling, induces apoptosis, and suppresses growth. Conversely, knocking down SFRP4 by small interfering RNA in cell lines expressing both SFRP4 and ß-catenin stimulates Wnt signaling, promotes cell growth, and inhibits chemodrug-induced apoptosis. Our results suggest that methylation silencing of SFRP4 may play an important role in aberrant Wnt activation in mesothelioma even in the absence of ß-catenin. Our data also suggest that ß-catenin–independent noncanonical pathway(s) may be involved in the apoptotic inhibition caused by activation of Wnt signaling.

Key Words: human • mesothelioma • methylation • apoptosis • Wnt signaling


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
The activation of the canonical Wnt pathway is mediated by ß-catenin (1, 2). However, noncanonical pathways act independently of ß-catenin (1, 2). At least two classes of Wnt antagonists have been reported (1). The first class, including secreted Frizzled-related protein (SFRP) family and Wnt inhibitory factor 1, binds directly to Wnt ligands. The second class, including the Dickkopf (Dkk) family, binds to low-density lipoprotein receptor–related protein 5/6. Recently, both SFRP and Dkk families have been implicated in oncogenesis. For example, the production of Dkk-1 by myeloma cells is associated with the presence of lytic bone lesions in patients with multiple myeloma (3). Dkk-3 has been found silenced by methylation in non–small cell lung cancer (4–6), acute lymphoblastic leukemia (7), and renal clear cell carcinoma (8). Forced expression of Dkk-1 in brain tumor (9) and cervical cancer (10), or Dkk-3 in non–small cell lung cancer (4) and osteosarcoma (11), inhibits cell growth. Down-regulation of SFRPs was found in several cancers, including mesothelioma (12), breast cancer (13), cervical cancer (14), and gastric cancer (15, 16). Loss of SFRP family expression was also found to be associated with promoter hypermethylation in mesothelioma and colorectal cancer tissue samples (12, 16–18). Furthermore, restoration of SFRPs in colon cancer cell lines carrying downstream mutations suppressed Wnt/ß-catenin-dependent transcription and induced apoptosis, suggesting that Wnt signaling may be regulated in a quantitative manner at different levels (18). However, there are few reports that have linked ß-catenin-independent pathway(s) to the Wnt-dependent apoptotic inhibition (19). Here, we report that SFRP4 is methylation silenced in ß-catenin-deficient mesothelioma cell lines. Furthermore, we show that reexpressing SFRP4 in these cell lines down-regulates Dishevelled (Dvl), induces apoptosis, and suppresses cell growth, suggesting that ß-catenin-independent pathway(s) may be important for the apoptotic inhibition caused by Wnt activation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Cell Lines. The human mesothelioma cell lines H28 and H2052 were obtained from American Type Culture Collection (Manassas, VA). The human mesothelioma cell line MS-1 was obtained from NIH (Bethesda, MD). These cell lines were cultured in RPMI 1640 supplemented with 10% fetal bovine serum, penicillin (100 IU/mL), and streptomycin (100 µg/mL). Normal human small airway epithelial cells (primary culture) were obtained from Clonetics (Walkersville, MD) and cultured in Clonetics SAGM Bullet kit. All cells were cultured at 37°C in a humid incubator with 5% CO2.

Semiquantitative Reverse Transcription-PCR. Total RNA from cell lines was isolated using Qiagen RNeasy Mini Kit (Valencia, CA). Reverse transcription-PCR (RT-PCR) was done in GeneAmp PCR system 2700 (Applied Biosystems, Foster City, CA) using One-Step RT-PCR kit from Invitrogen Life Technologies (Carlsbad, CA). Primers for RT-PCR were obtained from Operon Technologies, Inc. (Alameda, CA). Primer sequences for amplifying the human SFRP4 were described previously (17). Glyceraldehyde-3-phosphate dehydrogenase was used as internal 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 methylation kit (Zymo Research, Orange, CA). Bisulfite-treated genomic DNA was amplified using primers described previously for human SFRP4 (17). The amplified 230-bp product corresponds to –384 to –154 in the SFRP4 promoter region (the start codon ATG of SFRP4 is defined as 0). 5-Aza-2'-deoxycytidine (Sigma, St. Louis, MO) treatment was done as described previously (17). For amplification of SFRP4 genomic region, primers were designed according to the SFRP4 genomic sequence in chromosome 7. Untreated genomic DNA isolated from the cell lines were used as template. The primer sequences and the length of fragments amplified are listed in Table 1. The PCR products were extracted from the agarose gel using Qiagen QIAquick Gel Extraction Kit and were subsequently sequenced at the DNA Sequencing Core Facility, Comprehensive Cancer Center, University of California (San Francisco, CA).


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  		Table 1. Primers used for amplifying SFRP4 genomic region

 
Western Blotting. Standard protocol was used. Anti-Dvl-3 and anti-Survivin antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-caspase-3 antibody was purchased from Oncogene (Cambridge, MA). Anti-c-Myc antibody was purchased from Cell Signaling Technology (Beverly, MA). Anti-ß-actin antibody was purchased from Sigma-Aldrich Corp. (St. Louis, MO). Anti-ß-catenin antibody was purchased from Transduction Laboratories (Lexington, KY). For detecting alteration of ß-catenin, cytosolic extracts were prepared and examined as described previously (20).

RNA Interference. RNA interference experiments were done as described previously (19). The ion-exchange high-performance liquid chromatography–purified small interfering RNA (siRNA; SFRP4 siRNA and nonsilencing siRNA control, >97% pure) were purchased from Qiagen-Xeragon (Germantown, MD). The targeted sequence of SFRP4 siRNA is 5'-AAGTCCCGCTCATTACAAATT-3', corresponding to +701 to +721 of the human SFRP4 cDNA sequence (the start codon ATG is defined as +1). To analyze proliferation, 3 x 104 cells were transfected with siRNA in a 24-well plate. After transfection, viable cells (trypan blue exclusion) were collected by trypsinization and counted at various time points. Experiments were done in triplicate.

Apoptosis Analysis. Cells were harvested by trypsinization and stained using an Annexin V FITC Apoptosis Detection Kit (Oncogene) according to the manufacturer's protocol. Then, stained cells were immediately analyzed by flow cytometry (FACScan, Becton Dickinson, Franklin Lakes, NJ).

Alimta (LY231514, multitargeted antifolate, pemetrexed) was supplied by Eli Lilly (Indianapolis, IN). Alimta was diluted in sterile physiologic solution at a concentration of 10 mg/mL. The solution was divided into aliquots, stored at –80°C, and diluted in culture medium before each experiment.

Transient Transfection and Colony Formation Assay. For transient transfection experiments, cells (2 x 105) were plated in six-well plates 24 hours before transfection. LipofectAMINE 2000 (Invitrogen Life Technologies) was used to mediate transfection using 5.0 µg SFRP4 cDNA construct in pCDNA3 vector (kindly provided by Dr. Amir Rattner) or 5.0 µg empty pCDNA3 vector as control according to the manufacturer's protocol. Transfected cells were striped and plated on 10 cm cell culture dishes at 48 hours after transfection. The cells were then selected by G418 (400 µg/mL). Colonies were stained with 0.5% methylene blue and were counted 3 weeks after the transfection.

Statistical Analysis. Data are mean ± SD. The Student's t test was used for comparing activities of different constructs and treatments.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
SFRP4 Is Silenced by Promoter Hypermethylation in ß-Catenin–Deficient Human Mesothelioma Cell Lines. Previously, we showed that human mesothelioma cell lines H28 and MS-1 lack cytosolic ß-catenin protein and have no Tcf/Lef transcriptional activity (19). To further assess the role of upstream Wnt signaling in these cells, we examined SFRP4 expression (Fig 1A). We found that the SFRP4 transcript was missing in both H28 and MS-1 cell lines. As a control, we found that SFRP4 was expressed in a normal primary cell culture small airway epithelial cell and a mesothelioma cell line H2052 (Fig. 1A). To investigate cause of the missing SFRP4 transcript in these two ß-catenin-deficient cell lines, we first examined the possibility of deletion or rearrangement of the gene by genomic sequencing (Fig. 1B). We were able to amplify fragments covering the whole genomic region of SFRP4 gene (including both exons and introns) from both cell lines H28 and MS-1 (data not shown). Sequencing of these fragments showed no molecular abnormalities, such as deletions, mutations, or rearrangements, when compared with the wild-type genomic sequence (chromosome 7; Fig. 1B).



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  		Figure 1. Correlation of promoter methylation with silencing of SFRP4 in ß-catenin-deficient mesothelioma cell lines H28 and MS-1. A, semiquantitative RT-PCR results for SFRP4 expression. B, sequencing of genomic region of the SFRP4 gene (both exons and introns). Part of the intron between exons 2 and 3 of the SFRP4 gene from cell line H28. C, bisulfite-sequencing analysis of the SFRP4 promoter region. Unmethylated ({square}) and methylated ({blacksquare}) CpG islands in the promoter region. We sequenced three individual clones of PCR products amplified from bisulfite-treated genomic DNA for each sample. D, reactivation of SFRP4 expression by 5-aza-2'-deoxycytidine treatment. Semiquantitative RT-PCR was done after 5-aza-2'-deoxycytidine treatment (1.0 µmol/L for 96 hours).

 
Aberrant promoter hypermethylation has been found to be an important mechanism in the inactivation of tumor suppressor genes in cancer (21). Therefore, we next analyzed the methylation status of the CpG islands in the SFRP4 promoter region in these cell lines. Both cell lines, H28 and MS-1, lacking SFRP4 expression were found to be hypermethylated (Fig. 1C). In contrast, no hypermethylation was seen in the control cells, small airway epithelial cell and H2052, that express SFRP4. In addition, we confirmed that the SFRP4 expression was restored after the demethylating agent, 5-aza-2'-deoxycytidine, treatment in MS-1 and H28 cells (Fig. 1D). Therefore, our results indicate that the SFRP4 expression is silenced in ß-catenin-deficient mesothelioma cell lines and the silencing correlates with dense CpG methylation of the SFRP4 promoter.

To further investigate the relationship between loss of SFRP4 expression and ß-catenin level, we compared the expression and methylation status of SFRP4 with ß-catenin levels in a variety of cell lines Table 2). In all the cancer cell lines expressing ß-catenin that we examined, 9 of 18 (50%) showed methylation silencing of SFRP4, 3 of 18 (16.7%) showed SFRP4 down-regulation and partial methylation, and 6 of 18 (33.3%) expressed SFRP4 and showed no methylation. This result indicates that silencing of SFRP4 also correlates with the promoter methylation in cancer cells expressing ß-catenin. Taken together, our data suggest that loss of SFRP4 expression in ß-catenin-deficient mesothelioma cells is not likely a result of loss of ß-catenin.


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  		Table 2. SFRP4 methylation silencing and cytosolic ß-catenin status in human cell lines

 
Restoration of SFRP4 in ß-Catenin–Deficient Cell Lines Induces Apoptosis and Suppresses Cell Growth. Our finding of methylation silencing of SFRP4 in H28 and MS-1 cells raises the possibility that upstream Wnt signaling is still functional in apoptotic inhibition in cells lacking ß-catenin. To answer this question, we restored the SFRP4 expression in these ß-catenin-deficient cells. Interestingly, restoration of SFRP4 was able to kill these cells; this cell killing was largely due to apoptosis induction (Fig. 2A). In contrast, when we transfected the SFRP4 construct into the mesothelioma cell line H2052, where SFRP4 is not silenced and the promoter is not methylated, no apoptosis induction was observed (Fig. 2B). The blockade of Wnt signaling by SFRP4 restoration in H28 cells was confirmed by analyzing the expression of Wnt signaling effectors (Fig. 2C). Dvl-3, a Wnt intracellular mediator, as well as Survivin, an apoptosis inhibitor and also a Wnt downstream target gene (22), were both down-regulated after restoration of SFRP4 in H28 cells. The cell line MS-1 also showed similar apoptotic and downstream effects after restoration of SFRP4 (data not shown). As a control, in H2052 cells, no change in cytosolic ß-catenin, Dvl-3, and Survivin was observed after transfection of the SFRP4 construct, consistent with the presence of endogenous SFRP4 in these H2052 cells (Fig. 2C). Furthermore, after selection of drug-resistant colonies for 3 weeks, we found that the colony numbers of SFRP4-transfected cells significantly decreased compared with that of empty vector-transfected cells (P < 0.001 for both MS-1 and H28; Fig. 3A and B). However, in H2052 cells, no significant difference was seen in colony numbers between SFRP4 and empty vector-transfected cells after drug selection (P = 0.45; Fig. 3C). Taken together, these data suggest that the silencing of SFRP4 in mesothelioma H28 and MS-1 cells may be critical for their survival and that ß-catenin-independent mechanism(s) might be responsible for the apoptosis and growth suppression induced by blocking Wnt signaling in these cells. ß-catenin/Tcf transcription was thought to be required for Wnt-mediated cell survival and it inhibits apoptosis by preventing cytochrome c release and subsequent caspase activation (23, 24). To our knowledge, our report is the first demonstration that ß-catenin is not necessary for SFRP-induced apoptosis in cancer.



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  		Figure 2. Restoration of SFRP4 induces apoptosis in ß-catenin-deficient mesothelioma cells. A and B, Annexin V analysis of apoptosis after restoration of SFRP4 in H28 and H2052 cells, respectively. FL1-H (X axis), Annexin V-FITC staining; FL3-H (Y axis), propidium iodide staining. C, analysis of downstream effectors after SFRP4 restoration in H28 and H2052 cells. Semiquantitative RT-PCR was used to examine the SFRP4 expression. Cytosolic proteins were prepared and used in the Western blots. ß-Actin served as loading control.

 


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  		Figure 3. Restoration of SFRP4 suppresses the growth of ß-catenin-deficient mesothelioma cells. Colony formation assay using cell lines MS-1 (A), H28 (B), and H2052 (C). Columns, average colony numbers in triplicate experiments; bars, SD.

 
Knockdown of SFRP4 by siRNA in SFRP4-Expressing Cells Promotes Cell Growth and Inhibits Chemodrug-Induced Apoptosis. To further explore the function of SFPR4 in canonical and noncanonical Wnt signaling pathways during tumorigenesis, we used siRNA to silence SFRP4 expression in H2052 cells that express both SFRP4 and ß-catenin (Fig. 4). Interestingly, in H2052 cells treated with SFRP4 siRNA, we found increased expression of cytosolic ß-catenin, Wnt target genes: c-Myc and Survivin (Fig. 4A). Our explanation is that siRNA knockdown of SFPR4 reduces the Wnt-inhibiting effect of SFRP4 and allows more Wnt molecules to bind to its receptor complex, which in turn enhances the canonical pathway. Enhanced activation of the canonical pathway results in stabilization of more free ß-catenin, which accumulates in the cytoplasm. This result is also supported by a recent observation that the ß-catenin level correlated inversely with SFRP4 level in endometrial sarcomas (25). Both Survivin and c-Myc have been known to promote cell proliferation and inhibit apoptosis. Consistently, we observed increased growth rate in SFRP4 siRNA-treated H2052 cells (Fig. 4B). To examine whether increased Survivin level by SFRP4 siRNA treatment inhibits apoptosis, we treated H2052 cells with a chemodrug, Alimta, 1 day after transfection with control and SFRP4 siRNA. Alimta is a novel multifunctional antifolate antimetabolite (26) and is now considered a standard in the treatment of mesothelioma. After 3 days of Alimta treatment (at a concentration of 1 µg/mL), we observed significant apoptosis induction (>30%; P < 0.03) in H2052 cells transfected with control siRNA but not with SFRP4 siRNA (Fig. 4C). We also observed up-regulation of the cleaved (active) form of caspase-3 (apoptosis activator) and down-regulation of Survivin (apoptosis inhibitor) after Alimta treatment in control siRNA-transfected H2052 cells (Fig. 4D). In contrast, Survivin level was significantly up-regulated in SFRP4 siRNA-transfected cells. Although Alimta down-regulated the Survivin level, it was still higher than that in control siRNA-transfected cells before Alimta treatment (Fig. 4D), consistent with no apoptosis induction and caspase-3 activation by Alimta in these SFRP4 siRNA-transfected cells. Taken together, our data show that knockdown of SFRP4 in SFRP4-expressing mesothelioma cells further enhances canonical Wnt signaling, inhibits apoptosis, and promotes cell growth, suggesting a critical role of SFRP4 during the development of mesothelioma.



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  		Figure 4. Silencing SFRP4 by siRNA in SFRP4-expressing H2052 cells. A, analysis of canonical Wnt pathway downstream effectors after SFRP4 siRNA treatment. Semiquantitative RT-PCR was used to confirm siRNA-directed down-regulation of the SFRP4 expression. B, analysis of cell proliferation after SFRP4 siRNA treatment. Numbers of viable cells transfected with SFRP4 siRNA (100 nmol/L; {square}) and nonsilencing control siRNA (100 nmol/L; {circ}). Points, means; bars, SD. C, graph of apoptosis induction by Alimta in siRNA-treated H2052 cells analyzed by Annexin V staining. Columns, mean percentage of apoptotic cells; bars, SD. Experiments were done in triplicate, and a total of 2 x 104 cells were analyzed in each individual experiment. D, Western blot analysis of apoptosis pathway effectors after Alimta and siRNA treatment. In all Western blots, cytosolic proteins were prepared and ß-actin served as loading control.

 
Previous studies have shown that SFRPs can sensitize cells to proapoptotic stimuli through ß-catenin down-regulation (27). However, involvement of ß-catenin-independent mechanism(s) in Wnt-induced apoptotic inhibition has rarely been reported. Recently, we found that blockade of Wnt-1 signaling induces apoptosis partially through the Dvl/c-Jun-NH2-kinase–dependent pathway (19, 28), the role of which in Wnt signaling is not well understood (29). In this study, we show that the restoration of SFRP4 in ß-catenin-deficient mesothelioma cell lines induces apoptosis, suppresses cell growth, and down-regulates Wnt signaling. Moreover, down-regulation of SFRP4 in ß-catenin-intact mesothelioma cells inhibits drug-induced apoptosis and stimulates cell growth through further enhancing canonical Wnt signaling pathway. Therefore, our data suggest that SFRP4 may induce apoptosis not only through the canonical pathway but also through ß-catenin-independent noncanonical pathways, such as c-Jun-NH2-kinase-dependent pathways.

Recently, Suzuki et al. (17) reported that SFRPs were preferentially hypermethylated in human colorectal cancers carrying downstream mutations and that those colorectal cancer cells retained sensitivity to upstream Wnt signaling (18), suggesting that upstream regulation of Wnt signaling may be actively and importantly involved in tumorigenesis even in colorectal cancer carrying downstream activating mutations. Our current finding of SFRP4 silencing by promoter hypermethylation in ß-catenin-deficient mesothelioma cells, together with our recent report of Dvl (an upstream mediator of Wnt signaling) overexpression in mesothelioma (20), provides more evidence that upstream Wnt signaling may be important during the development of cancer, especially mesothelioma.


   Acknowledgments
 
Grant support: Larry Hall Memorial Trust and Kazan, McClain, Edises, Abrams, Fernandez, Lyons & Farrise Foundation.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Drs. Amir Rattner (Department of Molecular Biology and Genetics, School of Medicine, Johns Hopkins University, Baltimore, MD) for kindly providing the SFRP4 cDNA construct and Hiromu Suzuki (Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD) for kindly providing the primer sequences of methylation analysis and RT-PCR for SFRP4.

Received 5/25/04. Revised 11/19/04. Accepted 11/29/04.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

  1. Giles RH, van Es JH, Clevers H. Caught up in a Wnt storm: Wnt signaling in cancer. Biochim Biophys Acta 2003;1653:1–24.[Medline]
  2. Taketo MM. Shutting down Wnt signal-activated cancer. Nat Genet 2004;36:320–2.[CrossRef][Medline]
  3. Tian E, Zhan F, Walker R, et al. The role of the Wnt-signaling antagonist DKK1 in the development of osteolytic lesions in multiple myeloma. N Engl J Med 2003;349:2483–94.[Abstract/Free Full Text]
  4. Nozaki I, Tsuji T, Iijima O, et al. Reduced expression of REIC/Dkk-3 gene in non-small cell lung cancer. Int J Oncol 2001;19:117–21.[Medline]
  5. Tsuji T, Nozaki I, Miyazaki M, et al. Antiproliferative activity of REIC/Dkk-3 and its significant down-regulation in non-small-cell lung carcinomas. Biochem Biophys Res Commun 2001;289:257–63.[CrossRef][Medline]
  6. Kobayashi K, Ouchida M, Tsuji T, et al. Reduced expression of the REIC/Dkk-3 gene by promoter-hypermethylation in human tumor cells. Gene 2002;282:151–8.[CrossRef][Medline]
  7. Roman-Gomez J, Jimenez-Velasco A, Agirre X, et al. Transcriptional silencing of the Dickkopfs-3 (Dkk-3) gene by CpG hypermethylation in acute lymphoblastic leukaemia. Br J Cancer 2004;91:707–13.[Medline]
  8. Kurose K, Sakaguchi M, Nasu Y, et al. Decreased expression of REIC/Dkk-3 in human renal clear cell carcinoma. J Urol 2004;171:1314–8.[CrossRef][Medline]
  9. Shou J, Ali-Osman F, Multani AS, Pathak S, Fedi P, Srivenugopal KS. Human Dkk-1, a gene encoding a Wnt antagonist, responds to DNA damage and its overexpression sensitizes brain tumor cells to apoptosis following alkylation damage of DNA. Oncogene 2002;21:878–89.[CrossRef][Medline]
  10. Mikheev AM, Mikheeva SA, Liu B, Cohen P, Zarbl H. A functional genomics approach for the identification of putative tumor suppressor genes: Dickkopf-1 as suppressor of HeLa cell transformation. Carcinogenesis 2004;25:47–59.[Abstract/Free Full Text]
  11. Hoang BH, Kubo T, Healey JH, et al. Dickkopf 3 inhibits invasion and motility of Saos-2 osteosarcoma cells by modulating the Wnt-ß-catenin pathway. Cancer Res 2004;64:2734–9.[Abstract/Free Full Text]
  12. Lee A, He B, You L, et al. Expression of the secreted frizzled-related protein gene family is downregulated in human mesothelioma. Oncogene 2004;23:6672–6.[CrossRef][Medline]
  13. Ugolini F, Charafe-Jauffret E, Bardou VJ, et al. WNT pathway and mammary carcinogenesis: loss of expression of candidate tumor suppressor gene SFRP1 in most invasive carcinomas except of the medullary type. Oncogene 2001;20:5810–7.[CrossRef][Medline]
  14. Ko J, Ryu KS, Lee YH, et al. Human secreted frizzled-related protein is down-regulated and induces apoptosis in human cervical cancer. Exp Cell Res 2002;280:280–7.[CrossRef][Medline]
  15. To KF, Chan MW, Leung WK, et al. Alterations of frizzled (FzE3) and secreted frizzled related protein (hsFRP) expression in gastric cancer. Life Sci 2001;70:483–9.[CrossRef][Medline]
  16. Caldwell GM, Jones C, Gensberg K, et al. The Wnt antagonist sFRP1 in colorectal tumorigenesis. Cancer Res 2004;64:883–8.[Abstract/Free Full Text]
  17. Suzuki H, Gabrielson E, Chen W, et al. A genomic screen for genes upregulated by demethylation and histone deacetylase inhibition in human colorectal cancer. Nat Genet 2002;31:141–9.[CrossRef][Medline]
  18. Suzuki H, Watkins DN, Jair KW, et al. Epigenetic inactivation of SFRP genes allows constitutive WNT signaling in colorectal cancer. Nat Genet 2004;36:417–22.[CrossRef][Medline]
  19. You L, He B, Uematsu K, et al. Inhibition of Wnt-1 signaling induces apoptosis in ß-catenin-deficient mesothelioma cells. Cancer Res 2004;64:3474–8.[Abstract/Free Full Text]
  20. Uematsu K, Kanazawa S, You L, et al. Wnt pathway activation in mesothelioma: evidence of Dishevelled overexpression and transcriptional activity of ß-catenin. Cancer Res 2003;63:4547–51.[Abstract/Free Full Text]
  21. Baylin SB. Mechanisms underlying epigenetically mediated gene silencing in cancer. Semin Cancer Biol 2002;12:331–7.
  22. Kim PJ, Plescia J, Clevers H, Fearon ER, Altieri DC.Survivin and molecular pathogenesis of colorectal cancer. Lancet 2003;362:205–9.[CrossRef][Medline]
  23. Chen S, Guttridge DC, You Z, et al. Wnt-1 signaling inhibits apoptosis by activating ß-catenin/T cell factor mediated transcription. J Cell Biol 2001;152:87–96.[Abstract/Free Full Text]
  24. You Z, Saims D, Chen S, et al. Wnt signaling promotes oncogenic transformation by inhibiting c-Myc-induced apoptosis. J Cell Biol 2002;157:429–40.[Abstract/Free Full Text]
  25. Hrzenjak A, Tippl M, Kremser ML, et al. Inverse correlation of secreted frizzled-related protein 4 and ß-catenin expression in endometrial stromal sarcomas. J Pathol 2004;204:19–27.[CrossRef][Medline]
  26. Hanauske AR. The role of Alimta in the treatment of malignant pleural mesothelioma: an overview of preclinical and clinical trials. Lung Cancer 2004;45 Suppl 1:S121–4.[Medline]
  27. Kawano Y, Kypta R. Secreted antagonists of the Wnt signalling pathway. J Cell Sci 2003;116:2627–34.[Abstract/Free Full Text]
  28. Lee AY, He B, You L, et al. Dickkopf-1 antagonizes Wnt signaling independent of ß-catenin in human mesothelioma. Biochem Biophys Res Commun 2004;323:1246–50.[CrossRef][Medline]
  29. Veeman MT, Axelrod JD, Moon RT. A second canon. Functions and mechanisms of ß-catenin independent Wnt signaling. Dev Cell 2003;5:367–77.[CrossRef][Medline]



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Clin. Cancer Res., January 15, 2006; 12(2): 383 - 391.
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C.-l. Huang, D. Liu, J. Nakano, S. Ishikawa, K. Kontani, H. Yokomise, and M. Ueno
Wnt5a Expression Is Associated With the Tumor Proliferation and the Stromal Vascular Endothelial Growth Factor--An Expression in Non-Small-Cell Lung Cancer
J. Clin. Oncol., December 1, 2005; 23(34): 8765 - 8773.
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