Purpose: Resistance to apoptosis is a hallmark of cancer and correlates with aggressiveness of tumors and poor prognosis. The Wnt/β-catenin pathway plays a pivotal role in the genesis of colorectal cancer by mechanisms not fully elucidated yet. Previous studies have linked regulation of osteoprotegerin (OPG) in bone to Wnt/β-catenin signaling. As OPG also serves as a decoy receptor for tumor necrosis factor–related apoptosis-inducing ligand (TRAIL), we hypothesized that OPG might play a role in mediating resistance to apoptosis in colorectal cancer cells.

Experimental Design: Expression analysis and functional studies in human colorectal cancer cell lines and determination of expression in primary tumors and sera from patients with colorectal cancer.

Results: We found production of OPG in colorectal cancer cells to be regulated by β-catenin/Tcf-4. Addition of exogenous OPG to colorectal cancer cells caused resistance to TRAIL. Similarly, accumulation of OPG in medium of cultivated cells caused resistance to TRAIL, and this could be reverted by removal of OPG. Furthermore, OPG levels were significantly increased in serum of patients with advanced disease.

Conclusions: We conclude that the Wnt/β-catenin pathway contributes to carcinogenesis and cancer cell survival by driving expression of OPG. Expression of the survival factor OPG might provide colorectal cancer cells with an essential growth advantage and contribute to cell invasion and metastasis. Inhibition of OPG expression might offer a new therapeutic approach for the treatment of patients with colorectal tumors overexpressing OPG and make these tumors sensitive to TRAIL-induced apoptosis.

Loss of sensitivity to apoptosis is a hallmark of cancer. The mechanism of how cells lose the ability to undergo apoptosis is thought to occur through several mechanisms, which include loss of membrane-bound death receptors and impairment of components of the extrinsic and intrinsic apoptotic pathways. This is supported by in vitro studies from cell lines as well as clinical studies showing a positive correlation between loss of proapoptotic membrane receptors and dedifferentiation, tumor size, poor prognosis, and tumor recurrence (1). Sensitization of cancer cells to proapoptotic stimuli including administration of apoptosis-inducing ligands such as TRAIL has therefore become a new approach in cancer therapy and proved safe in first clinical trials (2). Understanding the specific mechanisms leading to resistance to apoptosis will therefore allow for the development of new targeted therapeutic options. In particular, well-characterized signaling pathways known to play important roles in carcinogenesis should be considered concerning their contribution to cell immortalization by causing resistance to apoptosis. The Wnt/β-catenin signaling pathway is tightly regulated and has important functions in development, tissue homeostasis, and regeneration. Deregulation of the Wnt/β-catenin pathway is frequently found in various human cancers, and its activation is involved in 80% of colorectal cancers (3). However, the multiplicity of the mechanisms by which deranged Wnt/β-catenin signaling participates in carcinogenesis is not fully understood yet.

Osteoprotegerin (OPG; TNFRSF11b) was initially found to contribute to homeostasis of bone turnover due to its capability of binding to receptor activator of nuclear factor κB. Subsequent investigations revealed that OPG also acts as a decoy receptor and binds to TRAIL, neutralizing its function. Several reports suggest a role of OPG mediating resistance to apoptosis in cancer (reviewed by Holen et al.; ref. 4). OPG is thought to exert a protective antiapoptotic action in OPG-expressing tumors by overcoming the physiologic mechanism of tumor surveillance exerted by TRAIL. Here, we report on the expression, regulation, and function of OPG in colorectal cancer and on OPG serum levels in patients with various stages of colorectal cancer. We show that the Wnt/β-catenin pathway regulates OPG in colorectal cancer cells, that OPG mediates resistance to TRAIL-induced apoptosis, and that its serum levels are highly elevated in patients with advanced colorectal cancers.

Analysis of OPG in medium and serum. OPG concentrations in medium and serum of 40 healthy individuals, 22 patients with colonic adenomas, and 127 patients with colorectal cancers were assayed by ELISA (Raybiotech) according to the manufacturer's instructions. The patients with colorectal cancers included 10 patients with International Union Against Cancer (UICC) I, 34 patients with UICC II, 43 patients with UICC III, and 40 patients with UICC IV stage disease. Of the patients with stage IV disease, only one had overt bone metastases.

Cell culture. Cell lines were from American Type Culture Collection and cultured under standard conditions as recommended. To assess OPG production, cells were seeded in 12-well plates and grown for 3 d in the same medium. Medium from three different wells for assessment of triplicate values were harvested every 24 h.

Short interfering RNAs were purchased from Dharmacon as on-target validated siRNAs (Standard siGENOME siRNATM). Transfection of siRNAs was done with Lipofectamine 2000 (Invitrogen); controls were done with noncoding sequences for β-galactosidase (sense, UUAUGCCGAUCGCGUCACAUU; antisense, PUGUGACGCGAUCGGCAUAAUU) purchased from Dharmacon. Cells (1.5 × 105) were seeded in 12-well plates and, after overnight incubation, transfected with Oligofectamine (Invitrogen) according to the protocol of the manufacturer. Human recombinant TRAIL was purchased from Sigma. Recombinant OPG and OPG antibody was from R&D. DNA fragmentation was quantified as the fraction of cells with subdiploid DNA content using flow cytometry as described (5). For preconditioned medium, 3 × 106 cells were seeded in a 10-cm dish and let in culture for 5 d. OPG-depleted cell culture medium were obtained essentially as described (6). Briefly, preconditioned sera were centrifuged and filtered (0.2 μm; Sartorius) to remove cells from the supernatant, and incubated with antibodies against OPG (R&D). OPG immunocomplexes were removed by centrifugation after overnight incubation with Protein G Agarose (Upstate).

For reporter gene assays in HeLa cells, cells were transfected with 200 ng of a luciferase reporter gene construct for OPG (kindly provided by Rudolf Grosschedl, Max-Planck Institute for Immunobiology, Freiburg, Germany), 200 ng of constitutively active β-galactosidase reporter plasmid pCH110 (Amersham Pharmacia Biosciences), and the pcDNA3-based plasmids S33Y-β-catenin, Tcf4, and VP16Tcf (Kolligs Genes Dev, 2000). Transfections were done with Nanofectin I (PAA Laboratories) as recommended by the manufacturer, and luciferase activity was measured 48 h later (Promega) according to the manufacturer's instructions using a luminescence reader (Turner). To normalize luciferase activity, β-galactosidase activity was determined using standard methods.

Western blot analysis. Cell lysates were prepared with radioimmunoprecipitation assay lysis buffer (TBS, 0.5% deoxycholate, 0.1% SDS, and 1% Nonidet P-40), and equal amounts of protein were separated by electrophoresis in discontinuous SDS-polyacrylamide gels. The antibody incubation steps were done in TBS containing 0.05% Tween 20 and 5% nonfat dry milk. The anti–β-catenin antibody (Transduction Labs) and the anti–β-actin antibody (Sigma-Aldrich) were used at 1:2,500 and 1:10,000 dilutions, respectively. The secondary horseradish peroxidase–conjugated antibody goat antimouse (Amersham Biosciences) was used at 1:10,000. The blots were subjected to enhanced chemiluminescence (Amersham) and exposed to hyperfilm enhanced chemiluminescence (Amersham).

Quantitative reverse transcription-PCR. RNA was extracted from cells and tissues using Trizol reagent (Invitrogen). RNA was reverse transcribed after DNase treatment with random hexamer primers (final concentration, 2.5 ng/μL) using SuperScript II reverse transcriptase as described by the manufacturer (Invitrogen). The oligonucleotide primers used will be made available on request. PCR reactions were run on a Realplex4 Mastercycler Epgradient S (Eppendorf). Resulting PCR products were monitored by melting point analysis and agarose gel electrophoresis. PCR efficiency was determined by analyzing serial dilutions of cDNA.

Overexpression of OPG in colorectal cancer cells. OPG, a secreted member of the tumor necrosis factor receptor superfamily of receptors, has previously been reported to act as a soluble decoy receptor for receptor activator of nuclear factor κB ligand (RANKL; for review, see Theoleyre et al.; ref. 7). OPG prevents binding of RANKL to its receptor, receptor activator of nuclear factor κB, and inhibits osteoclast differentiation and, hereby, contributes to bone homeostasis. Subsequent studies showed that OPG also functions as a soluble receptor for TRAIL and exerts antiapoptotic functions in prostate and breast cancer cells (6, 8). As only very little is known about expression and function of OPG in colorectal cancer (9), we initially studied the expression of OPG in colorectal cancer cells. For this, we assessed OPG expression by reverse transcription-PCR in microdissected colorectal cancers and matched normal epithelium of the same patients (Fig. 1A) and in several colorectal cancer cell lines (Fig. 1B). OPG was expressed at higher levels in the four primary colorectal cancers than in matched normal colon epithelium and expressed in all colorectal cancer cell lines tested with the exception of RKO cells. Interestingly, in contrast to the other cell lines, RKO cells do not contain a mutation of downstream components of the Wnt-pathway. Correspondingly, OPG, secreted by colorectal cancer cells lines carrying mutations of the adenomatous polyposis coli gene, SW480, or β-catenin gene, HCT116, accumulated in cell culture medium (Fig. 1C).

Fig. 1.

OPG is overexpressed in colorectal cancer cells. A, OPG mRNA expression as measured by quantitative reverse transcription-PCR in microdissected colorectal cancers and matched normal colon epithelium from the same patients. B, OPG mRNA expression in colorectal cancer cell lines. C, time course of OPG concentration in medium of SW480 and HCT116 colorectal cancer cell lines. Values are expressed as fold increase of OPG concentration in cell culture supernatants at day 2 and 3 versus concentrations measured after overnight incubation.

Fig. 1.

OPG is overexpressed in colorectal cancer cells. A, OPG mRNA expression as measured by quantitative reverse transcription-PCR in microdissected colorectal cancers and matched normal colon epithelium from the same patients. B, OPG mRNA expression in colorectal cancer cell lines. C, time course of OPG concentration in medium of SW480 and HCT116 colorectal cancer cell lines. Values are expressed as fold increase of OPG concentration in cell culture supernatants at day 2 and 3 versus concentrations measured after overnight incubation.

Close modal

OPG expression is regulated by β-catenin in colon cancer cells. As the Wnt/β-catenin pathway is deregulated in the majority of colorectal cancers and it has previously been reported that expression of OPG is regulated by β-catenin/Tcf in osteoblasts (10), we assessed whether Wnt/β-catenin contributes to the regulation of OPG in colorectal cancers. For this purpose, SW480 cells were transiently transfected with siRNA directed against β-catenin. This resulted in effective silencing of β-catenin expression on both protein and mRNA levels (Fig. 2A and B). Reduction of cellular β-catenin levels coincided with a strong reduction of OPG mRNA expression and expression of well-known target genes of β-catenin, including c-Myc and Dickkopf-1 (Fig. 2B; ref. 3). Correspondingly, only very little OPG accumulated in the supernatants of SW480 cells transfected with siRNA directed against β-catenin compared with cells transfected with control siRNA (Fig. 2C). Luciferase reporter assays confirmed that transcription from the human OPG promoter in epithelial cells is dependent on the presence of β-catenin and Tcf-4 (Fig. 2D). Strongest induction of reporter activity was observed upon transfection of a construct expressing a fusion of Tcf4 to the transactivation domain of VP16, further supporting the role of Tcf-4 in the regulation of OPG.

Fig. 2.

OPG production is regulated by β-catenin. A, Western blot analysis of β-catenin in SW480 cells after either incubation with vehicle, a β-catenin-siRNA targeting sequence (si-βcat), or a control siRNA (si-βgal). B, mRNA expression analysis of β-catenin (β-cat), OPG, c-Myc, and Dickkopf-1 (DKK1) as determined by quantitative reverse transcription-PCR in cells 3 d after transfection of β-catenin–targeting siRNA (β) or control siRNA (c). C, time course of OPG concentration in the supernatant of SW480 cells after transfection with siRNA targeting β-catenin as determined by ELISA. Twenty-four, 48, and 72 h after transfection of siRNAs, medium was collected and assayed for OPG. D, OPG reporter assay after transient transfection of HeLa cells with the firefly luciferase reporter gene construct for OPG, expression constructs pcDNA3/S33Y-β (S33Y-β-catenin), pcDNA3/Tcf4 (Tcf-4), and pcDNA3/VP16Tcf4 (expressing a chimeric fusion protein consisting of the VP16 transactivation and the Tcf-4 DNA binding domains) or empty vector pcDNA3, as indicated. Transfection efficiency was controlled for by cotransfection of a β-galactosidase expressing plasmid. Columns, mean of a representative experiment done in triplicate; bars, SD.

Fig. 2.

OPG production is regulated by β-catenin. A, Western blot analysis of β-catenin in SW480 cells after either incubation with vehicle, a β-catenin-siRNA targeting sequence (si-βcat), or a control siRNA (si-βgal). B, mRNA expression analysis of β-catenin (β-cat), OPG, c-Myc, and Dickkopf-1 (DKK1) as determined by quantitative reverse transcription-PCR in cells 3 d after transfection of β-catenin–targeting siRNA (β) or control siRNA (c). C, time course of OPG concentration in the supernatant of SW480 cells after transfection with siRNA targeting β-catenin as determined by ELISA. Twenty-four, 48, and 72 h after transfection of siRNAs, medium was collected and assayed for OPG. D, OPG reporter assay after transient transfection of HeLa cells with the firefly luciferase reporter gene construct for OPG, expression constructs pcDNA3/S33Y-β (S33Y-β-catenin), pcDNA3/Tcf4 (Tcf-4), and pcDNA3/VP16Tcf4 (expressing a chimeric fusion protein consisting of the VP16 transactivation and the Tcf-4 DNA binding domains) or empty vector pcDNA3, as indicated. Transfection efficiency was controlled for by cotransfection of a β-galactosidase expressing plasmid. Columns, mean of a representative experiment done in triplicate; bars, SD.

Close modal

OPG protects colorectal cancer cells from TRAIL-induced apoptosis. TRAIL triggers apoptosis by binding to the death receptors DR4 and DR5 (11). Although TRAIL is present in tumors where it is produced by monocytes and is known as the principal mediator of acquired antitumor activity (12), many colorectal cancers show resistance to TRAIL due to various mechanisms including expression of antagonistic decoy receptors (for review, see van Geelen et al.; ref. 13). Thus far, three antagonistic TRAIL receptors, DcR1, DcR2, and OPG have been described (14). It has been reported that OPG serves as a survival factor for human prostate and mammary cancer cells (6, 8). We therefore assessed the role of OPG in mediating resistance of colorectal cancer cells to TRAIL-induced apoptosis. Four colorectal cancer cell lines were treated with increasing amounts of TRAIL, and a dose-dependent increase in apoptosis was observed (data not shown; Fig. 3A-C). Concomitant addition of OPG decreased TRAIL-induced apoptosis dependent on the dose of recombinant OPG added (data not shown; Fig. 3C). OPG was able to inhibit TRAIL-induced apoptosis in cells showing high (i.e., HCT116), intermediate (i.e., LoVo and SW480), and low (i.e., DLD1) sensitivity to TRAIL-induced apoptosis. In agreement with OPG specifically antagonizing TRAIL-induced apoptosis, apoptosis induced by treatment of HCT116 cells with increasing concentrations of etoposid could not be inhibited by OPG (data not shown). Moreover, removal of endogenous OPG secreted into the cell culture medium from HCT116 and DLD1 cells led to an increase in sensitivity to TRAIL-induced apoptosis by 18% to 19% (Fig. 3D). Finally, to link β-catenin expression to sensitivity to TRAIL-induced apoptosis in colorectal cancer cells, the expression of β-catenin was silenced by siRNA. This resulted in an increase in sensitivity to TRAIL-induced apoptosis (Fig. 3E). This supports the role of β-catenin as a regulator of OPG and inhibitor of TRAIL-induced apoptosis.

Fig. 3.

OPG inhibits TRAIL-induced apoptosis in colorectal cancer cells and endogenous OPG production acts as a survival factor in cancer cells by preventing TRAIL-mediated apoptosis. A, typical fluorescence-activated cell sorting patterns of HCT116 and SW480 cells: unstimulated cells (left), cells stimulated with 100ng/mL TRAIL (middle) or with 100 ng/mL TRAIL, and addition of 1 μg/mL of recombinant human OPG (right). B and C, apoptosis rates as determined by the fraction of sub-G1 events by fluorescence-activated cell sorting after stimulation with increasing amounts of TRAIL for 24 h as indicated, with or without addition of recombinant OPG. Columns, mean of three experiments; bar, SD. D, sensitization of cells to TRAIL-induced apoptosis after depletion of OPG in medium compared with control medium (conditioned). Cells were exposed to 5 ng TRAIL for 24 h. Columns, mean of representative experiments done in triplicates; bars, SD. E, siRNA-based silencing of β-catenin expression results in sensitization for TRAIL-induced apoptosis. Columns, mean of representative experiments done in triplicates are shown; bars, SD.

Fig. 3.

OPG inhibits TRAIL-induced apoptosis in colorectal cancer cells and endogenous OPG production acts as a survival factor in cancer cells by preventing TRAIL-mediated apoptosis. A, typical fluorescence-activated cell sorting patterns of HCT116 and SW480 cells: unstimulated cells (left), cells stimulated with 100ng/mL TRAIL (middle) or with 100 ng/mL TRAIL, and addition of 1 μg/mL of recombinant human OPG (right). B and C, apoptosis rates as determined by the fraction of sub-G1 events by fluorescence-activated cell sorting after stimulation with increasing amounts of TRAIL for 24 h as indicated, with or without addition of recombinant OPG. Columns, mean of three experiments; bar, SD. D, sensitization of cells to TRAIL-induced apoptosis after depletion of OPG in medium compared with control medium (conditioned). Cells were exposed to 5 ng TRAIL for 24 h. Columns, mean of representative experiments done in triplicates; bars, SD. E, siRNA-based silencing of β-catenin expression results in sensitization for TRAIL-induced apoptosis. Columns, mean of representative experiments done in triplicates are shown; bars, SD.

Close modal

Serum OPG concentrations are increased in patients with metastasized colorectal cancers. Serum OPG concentrations have been reported to be increased in patients with prostate cancer, which progressed after androgen ablation therapy (15), and in patients with colorectal and pancreatic cancers compared with healthy individuals (16). To better understand the expression of OPG in patients with colorectal cancers, we assessed OPG concentration in sera from healthy individuals, patients with colonic adenomas, and patients with various stages of colorectal cancers. There was no significant difference in age between healthy individuals and cancer patients. The mean serum concentration in healthy individuals was 512 pg/mL; in patients with colorectal cancer, serum OPG levels were found to be in the range between 700 and 1,453 pg/mL (Fig. 4). We observed a significant increase in serum OPG concentrations in patients with UICC stage III and IV cancer compared with healthy individuals (P = 0.0359 and P < 0.0001, respectively). Moreover, a significant increase in OPG serum levels was observed in patients with metastatic disease compared with patients with locally advanced disease (UICC stage IV versus stage III; P < 0,00001). The majority of the stage IV patients had liver metastases, only one patient had overt bone metastases, ruling out that bone metastasis might be the underlying cause for elevated OPG serum levels.

Fig. 4.

Serum OPG concentration is increased in patients with late-stage colorectal cancer. OPG serum levels in 40 healthy individuals, 22 patients with colorectal adenomas, and patients with different stages of colorectal cancers (10 UICC I, 34 UICC II, 43 UICC III, and 40 UICC IV) and mean serum levels.

Fig. 4.

Serum OPG concentration is increased in patients with late-stage colorectal cancer. OPG serum levels in 40 healthy individuals, 22 patients with colorectal adenomas, and patients with different stages of colorectal cancers (10 UICC I, 34 UICC II, 43 UICC III, and 40 UICC IV) and mean serum levels.

Close modal

The fact that OPG is regulated by β-catenin/Tcf-4 in colorectal cancer underlines the importance of this pathway in providing cancer cells with growth as well as survival signals allowing for proliferation and evasion from apoptosis (17, 18). Among the factors regulated by Wnt/β-catenin that function in the regulation of apoptosis, e.g., Fas antigen, Caspase 3, Clusterin, Survivin, and Cyclooxygenase-2 (for review, see Ilyas; ref. 18), OPG as an extracellular factor has the potential to become a therapeutic target. Targeting of deregulated β-catenin/Tcf signaling in cancer has thus far been hampered by the fact that the Wnt-pathway physiologically functions in tissue homeostasis and regeneration of several tissues including intestinal epithelium, skin, and hematopoetic cells (3, 17, 19). Therefore, an approach targeting central signaling components such as β-catenin and Tcf-4 might not be feasible. A strategy interfering with target genes of the Wnt-pathway, which have critical functions in cancer, might prove more promising. Exogenous administration of TRAIL has been shown to be a rational and promising tumor therapy that overcomes resistance to apoptosis and has proven to be safe in first clinical trials (2). Although several cancers are sensitive to TRAIL-induced apoptosis, others are resistant to TRAIL or acquire resistance during therapy (14). Antagonizing antiapoptotic factors such as OPG by neutralizing antibodies might therefore extend the number of tumors sensitive to TRAIL. Moreover, determination of OPG serum concentrations in patients could serve as a tool to screen for OPG-producing tumors and might be useful as a predictive marker for TRAIL resistance.

Our results indicate that production and release of OPG into the serum of patients affected by late stage metastatic tumors is likely to exert a protective antiapoptotic effect to tumor cells when these invade and metastasize. This systemic increase of OPG in patients affected by late-stage colorectal cancers is in accordance with data showing that loss of receptor-mediated apoptosis is a hallmark of cancer correlating with recurrence rates and poor prognosis (20). This notion is further supported by the fact that TRAIL receptor knockout mice or mice in which TRAIL receptors were blocked exhibited enhanced tumor formation and formation of metastases (21). Moreover, expression of OPG correlated with depth of tumor invasion, nodal metastases, and advanced tumor stage in patients with gastric carcinoma (22). In conclusion, we provide first evidence that Wnt/β-catenin signaling contributes to resistance to apoptosis by regulating OPG secretion in colorectal cancer. Our data suggest that inhibition of OPG expression might sensitize colorectal cancers to endogenous TRAIL produced by monocytes as well as exogenous administration of TRAIL. Determination of OPG serum levels might prove a useful biomarker that helps identify patients with TRAIL-resistance due to OPG overexpression.

No potential conflicts of interest were disclosed.

Grant support: Deutsche Forschungsgemeinschaft (KFO128) KO 1826/3-1 (F.T. Kolligs) and GO 417/4-1 and EI 480/2-1 (B. Göke).

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 Rudolf Grosschedl for providing the OPG reporter construct.

1
Eichhorst ST. Modulation of apoptosis as a target for liver disease.
Expert Opin Ther Targets
2005
;
9
:
83
–99.
2
Duiker EW, Mom CH, de Jong S, et al. The clinical trail of TRAIL.
Eur J Cancer
2006
;
42
:
2233
–40.
3
Herbst A, Kolligs FT. Wnt signaling as a therapeutic target for cancer.
Methods Mol Biol
2007
;
361
:
63
–91.
4
Holen I, Shipman CM. Role of osteoprotegerin (OPG) in cancer.
Clin Sci Lond
2006
;
110
:
279
–91.
5
Nicoletti I, Migliorati G, Pagliacci MC, Grignani F, Riccardi C. A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry.
J Immunol Methods
1991
;
139
:
271
–9.
6
Holen I, Croucher PI, Hamdy FC, Eaton CL. Osteoprotegerin (OPG) is a survival factor for human prostate cancer cells.
Cancer Res
2002
;
62
:
1619
–23.
7
Theoleyre S, Wittrant Y, Tat SK, Fortun Y, Redini F, Heymann D. The molecular triad OPG/RANK/RANKL: involvement in the orchestration of pathophysiological bone remodeling.
Cytokine Growth Factor Rev
2004
;
15
:
457
–75.
8
Holen I, Cross SS, Neville-Webbe HL, et al. Osteoprotegerin (OPG) expression by breast cancer cells in vitro and breast tumours in vivo-a role in tumour cell survival?
Breast Cancer Res Treat
2005
;
92
:
207
–15.
9
Pettersen I, Bakkelund W, Smedsrod B, Sveinbjornsson B. Osteoprotegerin is expressed in colon carcinoma cells.
Anticancer Res
2005
;
25
:
3809
–16.
10
Glass DA, Bialek P, Ahn JD, et al. Canonical Wnt signaling in differentiated osteoblasts controls osteoclast differentiation.
Dev Cell
2005
;
8
:
751
–64.
11
Pitti RM, Marsters SA, Ruppert S, Donahue CJ, Moore A, Ashkenazi A. Induction of apoptosis by Apo-2 ligand, a new member of the tumor necrosis factor cytokine family.
J Biol Chem
1996
;
271
:
12687
–90.
12
Takeda K, Smyth MJ, Cretney E, et al. Critical role for tumor necrosis factor-related apoptosis-inducing ligand in immune surveillance against tumor development.
J Exp Med
2002
;
195
:
161
–9.
13
Van Geelen CM, de Vries EG, de Jong S. Lessons from TRAIL-resistance mechanisms in colorectal cancer cells: paving the road to patient-tailored therapy.
Drug Resist Updat
2004
;
7
:
345
–58.
14
Zhang L, Fang B. Mechanisms of resistance to TRAIL-induced apoptosis in cancer.
Cancer Gene Ther
2005
;
12
:
228
–37.
15
Eaton CL, Wells JM, Holen I, Croucher PI, Hamdy FC. Serum osteoprotegerin (OPG) levels are associated with disease progression and response to androgen ablation in patients with prostate cancer.
Prostate
2004
;
59
:
304
–10.
16
Lipton A, Ali SM, Leitzel K, et al. Serum osteoprotegerin levels in healthy controls and cancer patients.
Clin Cancer Res
2002
;
8
:
2306
–10.
17
Clevers H. Wnt/β-catenin signaling in development and disease.
Cell
2006
;
127
:
469
–80.
18
Ilyas M. Wnt signalling and the mechanistic basis of tumour development.
J Pathol
2005
;
205
:
130
–44.
19
Barker N, Clevers H. Mining the Wnt pathway for cancer therapeutics.
Nat Rev Drug Discov
2006
;
5
:
997
–1014.
20
Bachmann MF, Wong BR, Josien R, Steinman RM, Oxenius A, Choi Y. TRANCE, a tumor necrosis factor family member critical for CD40 ligand-independent T helper cell activation.
J Exp Med
1999
;
189
:
1025
–31.
21
Cretney E, Takeda K, Yagita H, Glaccum M, Peschon JJ, Smyth MJ. Increased susceptibility to tumor initiation and metastasis in TNF-related apoptosis-inducing ligand-deficient mice.
J Immunol
2002
;
168
:
1356
–61.
22
Ito R, Nakayama H, Yoshida K, et al. Expression of osteoprotegerin correlates with aggressiveness and poor prognosis of gastric carcinoma.
Virchows Arch
2003
;
443
:
146
–51.