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Molecular Biology and Genetics |
Thoracic Oncology Laboratory [K. U., L. Y., B. H., Z. X., K. L., F. M., D. M. J.], Department of Medicine [S. K., B. M. P.], University of California, San Francisco Cancer Center, San Francisco, California 94115, and The Fourth Department of Internal Medicine, Nippon Medical School, Tokyo 113-8602, Japan [K. U.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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PDZ-Dvl) was created and stably transfected into LRK1A, REN, and H513. The effect of PDZ-Dvl on mesothelioma growth was assayed in vitro (colony formation assay in soft agar) and in vivo (s.c. implantation in athymic mice NCRNU-M). In mesothelioma cells tested, PDZ-Dvl-mediated inhibition of Dvl decreased cytosolic ß-catenin levels, diminished Tcf-mediated transcription, and suppressed tumorigenesis of LRK1A and REN in vitro and in vivo. PDZ-Dvl also down-regulated expression of c-myc in REN and COX-2 in H513. Our data suggest that in malignant pleural mesothelioma, Wnt signaling is activated through Dvl overexpression and downstream signaling through ß-catenin. Inhibition of this signaling leads to significant antitumor effects. These results demonstrate Dvl overexpression in human cancer and, specifically, that Wnt signaling plays a role in mesothelioma pathogenesis. These data offer possible new avenues for therapeutic intervention. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Previously, we have analyzed, by cDNA array, differential gene expression in early-stage, resected human mesothelioma. We found consistent evidence of Wnt pathway activation; a pathway not previously known to be active in mesothelioma and, typically, only active in normal cells during development. Wnt signaling results in ß-catenin accumulation and transcriptional activation of specific target genes during development. Recently, Matsuzawa and Reed (4) and Liu et al. (5) demonstrated that Siah-1 links the p53 pathway to ß-catenin and promotes its degradation (4 , 5) . Most malignant pleural mesothelioma (>80%) have wild-type p53 but homozygous deletion of p14, resulting in functional inactivation of the p53 pathway. Inactivation of the p53 pathway may promote accumulation of ß-catenin. Dysregulation of ß-catenin signaling because of primary ß-catenin mutation is an important event in the genesis of several human malignancies (6) but is not found in malignant pleural mesothelioma.
The Dvl3 family proteins (Dsh in Drosophila, Dvl in mammals) are membrane-proximal signaling intermediates in the Wnt pathway (7, 8, 9, 10, 11) . Downstream of Dvl is the enzyme GSK3ß, a serine-threonine kinase that is a negative regulator of insulin and Wnt signaling (12) . In the absence of a Wnt signal, GSK3ß phosphorylates ß-catenin and induces its ubiquitination and proteolytic degradation (13 , 14) . The presence of a Wnt signal inhibits GSK3ß, which stabilizes ß-catenin, allowing it to translocate to the nucleus, where it acts as an essential cofactor for Tcf/Lef-dependent transcription (15 , 16) . ß-Catenin-Tcf/Lef induces transcription of important downstream target genes, c-myc(17) , cyclinD1 (18) , among others, many of which have been implicated in cancer.
To confirm the activity of Wnt signaling in mesotheliomas, we performed Western blotting of Dvl and cytosolic ß-catenin in fresh and cultured mesothelioma cells. In addition, we evaluated the transcriptional activity of ß-catenin using a Tcf-dependent luciferase reporter gene. In this study, we demonstrate that mesothelioma cells overexpress Dvl. Furthermore, we show that overexpression of Dvl causes cytoplasmic accumulation of ß-catenin, transcriptional activity, and tumor growth. Lastly, we demonstrate that activation of the Dvl-ß-catenin signaling pathway contributes to tumorigenesis and that targeted inhibition of Dvl inhibits tumor growth.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cellular Fractionation, Western Blot, and Immunohistochemistry.
Whole cell lysates were obtained with M-Per mammalian protein extraction reagent (Pierce, Rockford, IL). Cytoplasmic fractions were prepared according to a protocol described previously (21)
. Briefly, the cell pellets were resuspended in ice-cold hypotonic buffer [20 mM Tris (pH 7.5), 25 mM sodium fluoride, and 1 mM EDTA] containing a protease inhibitor mixture tablet (Roche, Indianapolis, IN). Cells were lysed by incubation on ice for 20 min, then subjected to ultracentrifugation at 100,000 x g for 30 min at 4°C. The supernatant aliquots (20 µg) containing the cytoplasmic fraction or the whole cell lysate aliquots (20 µg) were separated on 4–15% gradient SDS-polyacrylamide gels and transferred to Immobilon-P (Millipore, Bedford, MA) membranes for Western blotting. Antigen-antibody complexes were detected by enhanced chemiluminescence blotting analysis system (Amersham Pharmacia Biotech, Piscataway, NJ). To compare the expression level, a gel densitometry (UN-SCAN-IT, Silk Scientific, Orem, UT) was used. The following primary antibodies were used: Dvl-1 (3F12) and Dvl-3 (4D3; all Santa Cruz Biotechnology, Santa Cruz, CA); ß-actin (Sigma Chemical Co., St. Louis, MO); ß-catenin and GSK-3ß (Transduction Laboratories, Lexington, KY); APC (Oncogene, Boston, MA); COX-2 (Cayman, Ann Arbor, MI); and Tcf-4 (Exalpha Biologicals, Inc., Boston, MA). Immunohistochemistry was done using snap-frozen, ornithine carbamyl transferase-embedded tumors that had been cut into 6-µm sections. The primary antibody, ß-catenin monoclonal antibody (Transduction Laboratories), was applied at 1:100 dilution, and tissues were incubated for 1 h at room temperature. Universal Labelled Streptavidin-Biotin+ system (Dako, Carpinteria, CA) and the secondary antibody were then applied. 3,3'-Diaminobenzidine was used as the chromogen, and the slides were counterstained with hematoxylin.
Sequencing Analysis.
Genomic DNA from microdissected tissues was isolated using Puregene kit (Gentra Systems, Inc., Minneapolis, MN). Exon 3 of ß-catenin was amplified using PCR. The primers design was based on the exon 3 sequence in GenBank, and the primer sequences are: forward, 5'-tcgtatttatagctgatt-3' and reverse, 5'-taatactcttaccagcta-3'. The PCR products were applied for sequencing using Applied Biosystems 377 DNA sequencer (Applied Biosystems, Foster City, CA). RNA was extracted from cell lines using an RNeasy mini kit (Qiagen, Inc., Valencia, CA) and reverse transcribed using Superscript II (Life Technologies, Inc., Rockville, MD). Whole coding region of ß-catenin was amplified using LA TaqDNA polymerase (Panvera Corporation, Madison, WI). The primer sequences are forward, 5'-ataatagtcgacgtgattataaagatgatgatgataaaatggctactcaagctgatttg-3' and reverse, 5'-ataatagcggccgcttattacaggtcagtatcaaacca-5'. The PCR products were cloned into SalI/NotI site of pSV-SPORT1 (Life Technologies, Inc.) vector to be applied for sequencing.
Transfection and Luciferase Assays.
Transfection was performed using Lipofectamine Plus reagent (Life Technologies, Inc.) according to the manufacturer’s instructions. Cells were seeded into 24-well plates and then transiently transfected with 0.25 µg of the TOPFLASH or FOPFLASH (kindly provided by Dr. Hans Clevers) reporter plasmids (22)
or plus activator plasmid. The cells were incubated for 24 h at 37°C, washed once with PBS, and then lysed to measure luciferase reporter gene expression by dual-luciferase reporter assay system (Promega, Madison, WI). The luciferase activity was normalized to Renilla luciferase activity from cotransfected internal control plasmid pRL-TK. Tcf-mediated gene transcription was determined by the ratio of pTOPFLASH:pFOPFLASH luciferase activity, each corrected for luciferase activities of the pRL-TK reporter. All experiments were performed in duplicate, a minimum of three times. To analyze the effect of Dvl on ß-catenin signaling, a pCS-mouse Dvl-1 and a PDZ-Dvl construct (kindly provided by Dr. Patricia C. Salinas) were cotransfected with TOPFLASH or FOPFLASH for luciferase assays. Empty pCS2+ vector was added to control for the amount of plasmid DNA.
Creation of Cell Lines.
To obtain cell lines expressing wild-type PDZ-Dvl, the XhoI/EcoRI mouse PDZ-Dvl-1 fragment of the pCS-mouse PDZ-Dvl-1 was cloned into the XhoI/EcoRI site of pLXN. In the same manner, pLXN-mouse Dvl-1 was constructed from pCS-mouse Dvl-1. To prepare retroviral stocks, Phoenix A cells were transfected with pLXN-neo-Dvl-1, PDZ-dvl-1, or pLXN-neo (empty vector) using Lipofectamine Plus reagent. Retroviral constructs were introduced into LRK1A, REN, and H513 and selected after culture in neomycin (1600 µg/ml; 7 days).
cDNA Expression Array.
For analyzing different gene expression in empty vector and PDZ-dvl-1-introduced cells, Atlas human cancer 1.2 array (Clontech Laboratories, Inc., Palo Alto, CA) was used. Total RNA was extracted from the cell lines using an RNeasy mini kit (Qiagen, Inc.) according to the manufacturer’s instructions. The materials provided with the kit were used, and the recommended protocol was followed in all steps. Five µg of total RNA was converted into 33P-labeled cDNAs for hybridization. The hybridized Atlas membranes were exposed to X-ray film for 3 days.
Soft Agar Assays.
Cells (1 x 106) were placed in RPMI 1640 plus 15% FCS in 0.35% (w/v) low melting temperature agar between layers of 0.7% low melting temperature agar. After 4 weeks, colonies were stained with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma Chemical Co.), and colonies containing >100 cells were scored. Colony assays were performed a minimum of six times, and results are reported as a mean of six experiments.
Tumorigenicity Assays.
All animal experiments were performed in accordance with institutional guidelines. Athymic mice NCRNU-M (4–5 weeks old; Taconic, Germantown, NY) were maintained in pathogen-free conditions. For s.c. implantation, 4 x 106 cells were injected s.c. into anesthetized mice. Tumors were measured every 7 days, and tumor volumes were calculated using width (a) and length (b) measurements (a2 x b/2, where a < b).
Statistical Methods.
Results are expressed as means ± SD. All statistical comparisons were made with a two-sided Student’s t test. P of <0.05 was considered to be statistically significant.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Furthermore, five additional malignant mesothelioma cells tested (two primary malignant pleural mesothelioma cultured cells and three cell lines, LRK1A, REN, and H513) had high levels of Dvl-3 and cytosolic ß-catenin, compared with normal pleural controls. Immunohistochemical analysis of several of the tumor cells, demonstrated cytoplasmic, nuclear, and membrane bound ß-catenin (Fig. 1B)
. We found no mutation in exon 3 of ß-catenin in 13 mesothelioma tissues, including the cases tested by the Western blot analysis and two malignant effusions. Exon 3 was selected for mutational analysis because it encodes the NH2-terminal regulatory domain of ß-catenin previously found to contain activating mutations (Fig. 1C
; Ref. 23
). Furthermore, we detected no mutation in the whole coding region of ß-catenin in three mesothelioma cell lines (LRK1A, REN, and H513).
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. Transcriptional activity mediated by Tcf-ß-catenin protein complexes was assayed as a ratio to reporter gene activity in mesothelioma cell lines with significant overexpression of Dvl and cytosolic ß-catenin (Fig. 3)
. Mesothelioma cells with high levels of cytosolic ß-catenin, including cells from malignant pleural mesothelioma effusions, LRK1A, REN, and H513 cell lines showed a significant fold increase (1.5–2.4-fold, P < 0.01) in Tcf-mediated gene transcriptional activity of ß-catenin (pTOPFLASH/pFOPFLASH). In contrast, normal mesothelial cells, which have minimal expression of cytosolic ß-catenin, showed no difference.
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PDZ-Dvl Inhibits Endogenous Dvl and Stabilizes Cytosolic ß-Catenin in Mesothelioma and Down-Regulated the Downstream Target Genes.
; Refs. 27
, 28
). This activity was inhibited by a pCS-mouse Dvl-1 construct by cotransfection of pCS-cDNA-encoding PDZ-Dvl-1. Furthermore, Tcf-dependent transcriptional activity of ß-catenin in LRK1A was reduced by transfection of pCS-PDZ-Dvl-1 (from 2.1- to 1.3-fold, P < 0.05), whereas transfection of pCS-Dvl-1 enhanced Tcf-dependent transcriptional activity of ß-catenin (from 2.1- to 3.8-fold, P < 0.05), indicating that ß-catenin Tcf-mediated transcription in these cells is regulated significantly by Dvl.
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PDZ-Dvl-1 and wild-type Dvl-1 into LRK1A, REN, and H513 cell lines, respectively (Fig. 4B)
. Retrovirus transfection of pLXN-PDZ-Dvl-1 induced expression of PDZ-Dvl-1 protein, which significantly reduced the expression of cytosolic ß-catenin in all cells tested compared with controls (P < 0.05; Fig. 4C
). These results demonstrate that Dvl regulates cytosolic ß-catenin in mesothelioma cells.
Using Atlas human cancer 1.2 array, c-myc expression in REN was shown to be down-regulated by PDZ-Dvl-1 transfection (Fig. 5A)
. On the other hand, COX-2, which has been confirmed to be one of target genes of Wnt/ß-catenin pathway, was down-regulated by PDZ-Dvl-1 transfection using Western blot analysis (Fig. 5B)
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PDZ-Dvl Inhibits Tumorigenicity of Mesothelioma Cell Lines in Soft Agar and in Athymic Mice.PDZ-Dvl-1 in LRK1A, REN, and H513 through retroviral transfection using empty vector as control. After selection, cells were plated in 0.35% soft agar and colonies scored after 28 days. Colony formations of LRK1A and REN transfected with PDZ-Dvl-1 decreased substantially compared with control (P < 0.01; Fig. 6, A and B
). H513 was unable to grow in soft agar. In addition, the in vivo growth of both LRK1A and REN s.c. tumors in athymic mice was inhibited significantly by transfection with a PDZ-Dvl-1 mutant compared with control (P < 0.05 and P < 0.005, respectively; Fig. 7
).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We sought to determine whether wild-type Dvl or PDZ-Dvl could control the expression of cytosolic ß-catenin and Tcf transcription in malignant pleural mesothelioma. Previously, it was shown that Dvl can activate Tcf-dependent transcriptional activity in mammalian cells (27
, 28)
and that the PDZ domain of Dvl appears critical for this activity (27)
. Our studies confirm that in malignant pleural mesothelioma, Dvl can enhance Tcf-dependent transcriptional activity of ß-catenin and that, conversely, PDZ-Dvl inhibits significantly this activity. Furthermore, the stable expression of PDZ-Dvl reduces the expression of cytosolic ß-catenin in malignant pleural mesothelioma with accompanied reduction of c-myc expression in REN and COX-2 in H513. C-Myc was the first target gene of the ß-catenin signaling pathway to be demonstrated in humans, and overexpression of c-myc has been detected in malignant mesotheliomas (17
, 29) . COX-2 has been showed to be a downstream target of the Wnt signaling pathway, and high expression of COX-2 has been proposed to be correlated with poor survival in malignant mesothelioma (30, 31, 32)
. Finally, multiple malignant pleural mesothelioma analyzed have high expression of Dvl and cytosolic ß-catenin suggesting activation of Wnt signaling.
We wondered whether these transcriptional activities had biological relevance and whether they affected cell growth or tumorigenicity in mesothelioma cells. Growth characteristics of malignant pleural mesothelioma cells transfected stably with either empty vector or PDZ-Dvl correlated with the transcriptional activity of ß-catenin and tumorigenesis. Colony formation and growth in athymic mice of malignant pleural mesothelioma cell lines, LRK1A and REN (which have high ß-catenin potential transcriptional activity), were reduced significantly after transfection with PDZ-Dvl. These results suggest that malignant pleural mesothelioma cells may require this critical Dvl-ß-catenin pathway for tumor formation.
In conclusion, our data suggest that in malignant pleural mesothelioma, the Wnt pathway is activated through Dvl overexpression and downstream signaling through ß-catenin. These malignant pleural mesothelioma tumors appear to be driven, in part, by the transcriptional regulation of translocated ß-catenin. Additional investigation of Wnt signaling and Dvl-ß-catenin activation in mesothelioma may offer new avenues for intervention and development of effective novel therapies.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by the Larry Hall Memorial Trust and the Kazan, McClain, Edises, Abrams, Fernandez, Lyons & Farrise Foundation. 
2 To whom requests for reprints should be addressed, at Section of General Thoracic Surgery, University of California, San Francisco, Campus Box 1674, 1600 Divisadero Street, Suite 322C, San Francisco, CA 94115. Phone: (415) 353-7502; Fax: (415) 353-9525; E-mail: JablonsD{at}surgery.ucsf.edu
3 The abbreviations used are: Dvl, Dishevelled; GSK3ß, glycogen synthase kinase-3ß; Tcf, T-cell factor; Lef, lymphocyte enhancer binding factor; PDZ-Dvl, a PDZ domain deletion mutant; COX-2, cyclooxygenase-2.
Received 1/24/03. Accepted 5/13/03.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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