Cancer Research Meeting Calendar Sign up for Cancer Research eTOC's
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
Cancer Research 67, 10642, November 15, 2007. doi: 10.1158/0008-5472.CAN-07-2015
© 2007 American Association for Cancer Research
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Thiyagarajan, S.
Right arrow Articles by Spiegelman, V. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Thiyagarajan, S.
Right arrow Articles by Spiegelman, V. S.

Priority Reports

Role of GLI2 Transcription Factor in Growth and Tumorigenicity of Prostate Cells

Saravanan Thiyagarajan1, Neehar Bhatia1, Shannon Reagan-Shaw1, Diana Cozma2, Andrei Thomas-Tikhonenko2, Nihal Ahmad1 and Vladimir S. Spiegelman1

1 Department of Dermatology and Paul P. Carbone Comprehensive Cancer Center, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin and 2 Department of Pathobiology, University of Pennsylvania, Philadelphia, Pennsylvania

Requests for reprints: Vladimir S. Spiegelman, Department of Dermatology, University of Wisconsin School of Medicine and Public Health, 445 Henry Mall, Room 301, Madison, WI 53706. Phone: 608-265-8197; Fax: 608-263-2919/608-263-5223; E-mail: spiegelman{at}dermatology.wisc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Aberrant activation of the Hedgehog (Hh) signaling pathway has been reported in various cancer types including prostate cancer. The GLI2 transcription factor is a primary mediator of Hh signaling. However, its relative contribution to development of prostate tumors is poorly understood. To establish the role of GLI2 in maintaining the tumorigenic properties of prostate cancer cells, we developed GLI2-specific small hairpin RNA. Knockdown of GLI2 in these cells resulted in significant down-regulation of the Hh signaling pathway, followed by inhibition of colony formation, anchorage-independent growth, and growth of xenografts in vivo. Conversely, ectopic expression of Gli2 in nontumorigenic prostate epithelial cells resulted in accelerated cell cycle progression, especially transition through G2-M, and augmented proliferation. Altogether, our findings suggest that GLI2 plays a critical role in the malignant phenotype of prostate cancer cells, and GLI2 may potentially become an attractive therapeutic target for the treatment of prostate cancer. [Cancer Res 2007;67(22):10642–6]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
The role of the Hedgehog (Hh) signaling pathway has been well documented in vertebrate embryonic development, stem cell maintenance, and tumorigenesis (reviewed in refs. 1, 2). Each of the three Hh genes (Sonic hedgehog, Desert hedgehog, and Indian hedgehog) encodes a secreted signaling peptide that binds to a membrane-bound 12-span transmembrane receptor called Patched (Ptc). Binding of Hh ligand to the Ptc receptor on the target cell alleviates Ptc-mediated suppression of a seven-span transmembrane protein called Smoothened (Smo). Smo activation initiates an intracellular signal transduction cascade that ultimately activates expression of Hh target genes.

Transcriptional activation of Hh target genes in mammals occurs through the actions of three related proteins, GLI1, GLI2, and GLI3 (3, 4). GLI3 acts primarily as a transcriptional repressor (5), whereas GLI2 is reported to be the primary activator of Hh signaling and GLI1 is a secondary target, downstream of GLI2, which also acts as a transcriptional activator (6). GLI1 is a transcriptional target of GLI2, and its up-regulation in response to the activation of the Hh signaling pathway depends on GLI2 protein stabilization. Therefore, GLI1 amplifies Hh-induced, GLI2-mediated transcription of GLI target genes.

The Gli2 transcription factor is indispensable for mouse development, as Gli2 knockout mice die prenatally and exhibit neural tube defects including a complete loss of the floor plate and a reduction in V3 interneurons (7, 8). Gli2 was also shown to be essential for normal prostate development (9). On the other hand, mice lacking Gli1 seem to be normal and fertile, suggesting that the GLI1 protein is not required for Hh signaling in mouse (10).

Recent studies have shown the importance of Hh signaling in human prostate cancer (11–14). Compelling data imply that elevated pathway activity is associated with high-grade and metastatic prostate cancer and show that pathway manipulation can modulate invasiveness and metastasis (12), but the role of ligand-dependent pathway activation in prostate cancer cells and the role of GLI1 in tumor cell behavior have been controversial (15). Although down-regulation of GLI1 has been shown to inhibit prostate tumorigenesis (13), the major mode of GLI1 regulation is the induction of its transcription by GLI2. Therefore, GLI1 amplifies GLI-dependent transcriptional outcome initially induced by GLI2. We have recently shown that stabilization of GLI2 protein could contribute to the overexpression of GLI2 protein in prostate cancer cells and primary tumors (16). In the present study, we have investigated the significance of GLI2 expression in prostate cell growth and tumorigenesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
DNA constructs. pcDNA3.1-Flag-Gli2 was a kind gift of Drs. M. Grachtchouk and A. Dlugosz (University of Michigan, Ann Arbor, MI). pGL3-Bcl2promo luciferase was generously provided by Dr. F. Aberger (University of Salzburg, Salzburg, Austria; ref. 17), K17 luciferase was from Dr. P. Coulombe (Johns Hopkins University, Baltimore, MD), 8x3'Gli BS-LucII (8 directly repeated copies of 3'Gli binding site from HNF3ß floor plate enhancer cloned into p{delta}51LucII; ref. 18) was kindly provided by Dr. H. Sasaki (Osaka University, Osaka, Japan), and pSV40 ß-gal was from Promega. The packaging plasmids pCMV{Delta}R8.2 and pCMV-VSV-G were obtained from Dr. Didier Trono (University of Geneva, Geneva, Switzerland) and Dr. Robert Weinberg (MIT, Cambridge, MA), respectively (Addgene plasmids 12263 and 8454). Construction of GLI2 small hairpin RNA (shRNA) was done according to Ambion recommendations. Briefly, siRNA sequences were selected using the siRNA Target Finder and Design Tool.3 The annealed shRNA insert was cloned into the modified pLVTHM vector (19). The following GLI2 target sequence was used: 5'-GATCTGGACAGGGATGACT-3'.

Tissue culture and transfections. 293T human embryo kidney cells and prostate cancer cell lines 22R{nu}1, PC3, LnCaP, DU145, and RWPE1 were purchased from American Type Culture Collection. Prostate cancer cells and 293T were grown in DMEM-RPMI in the presence of 10% fetal bovine serum (FBS) and antibiotics at 37°C and 5% CO2. RWPE1 cells were cultured in keratinocyte serum-free medium supplemented with bovine pituitary extract and epidermal growth factor from Invitrogen. RWPE1 stable clones were selected in complete medium containing G418 (Sigma-Aldrich) over 2 weeks. Transfections were done using the calcium phosphate procedure or lipofection with LipofectAMINE 2000 (Invitrogen).

Lentiviral-mediated shRNA delivery. For producing lentiviruses, 293T cells grown at 60% to 70% confluence were transfected with 10 µg of pLVTHM-Gli2 shRNA, 10 µg of pCMV{Delta}R8.2, and 7.5 µg of pCMV-VSV-G plasmids by calcium phosphate method (20). The next day (day 1), complete medium without antibiotics was replaced to the cells. The virus was collected as media supernatant on days 2 and 3, passed through 0.45-µm filter, and stored at –80°C. At the time of transduction, 22R{nu}1 cells at 50% to 60% confluence were infected by adding 10 mL of media containing lentiviruses twice at 12-h interval, and then green fluorescent protein expression was analyzed 24 h after second infection.

Antibodies and Western blotting. Antibodies against Gli2 (G20), ß-actin, and horseradish peroxidase–conjugated secondary antibodies were purchased from Santa Cruz Biotechnology. Immunoblotting procedures were done as described elsewhere (16).

Luciferase reporter assays. Prostate cells were transfected with 8x3'Gli BS-LucII reporter, K17 luciferase reporter plasmid, or pGL3-Bcl2promo luciferase reporter plasmid, pSV40 ß-gal (Promega), and respective shGLI2 expression plasmids. Luciferase and ß-galactosidase activities were done using Luciferase Reporter Assay Reagent (Promega) and ß-gal assay reagent (Pierce Biotechnology), respectively, according to the manufacturers' recommendations.

Colony formation assay. Scrambled and GLI2 shRNAs were cotransfected with pTk-Puro in the indicated cell lines at a ratio of 19:1. The transfected cells were subcultured and selected with puromycin (Sigma; the final concentration of puromycin in the media varied for the cell lines from 1 to 3 µg/mL). Colonies that formed in 3 to 5 weeks were fixed with 10% formalin for 2 h, washed with PBS, stained with 2% Gentian violet (Ricca Chemical Company) for 45 min, and air-dried.

Growth in soft agar. 22R{nu}1 prostate cancer cells were infected with lentiviruses encoding GLI2 shRNA or scrambled shRNA. GLI2 shRNA– and scrambled shRNA–expressing 22R{nu}1 cells were grown in six-well plates in complete media, trypsinized, washed with PBS, counted, and mixed at a concentration of 10,000 cells/2 mL of RPMI, 0.33% agar. The cells in 0.33% agar were plated over 2 mL of RPMI, 0.5% agar that had been allowed to harden in a six-well dish. Cells were fed 0.5 mL of RPMI twice a week and allowed to grow for 3 weeks. The cells were fixed with 10% formalin and stained with Gentian violet. Colony formation was determined under low magnification (x10) in an inverted microscope.

Xenograft growth. For the tumor growth studies, transduced 22R{nu}1 cells infected with lentiviruses encoding GLI2 shRNA or scrambled shRNA and BALB/c athymic (nude) male (NxGen Biosciences, Inc.) mice, 5 weeks old, were used. Cells (1 x 106) in 0.1 mL of culture medium were mixed with 0.1 mL of Matrigel (BD Biosciences) and were injected s.c. into one flank of the mice. Tumors were measured and volume was calculated using the following formula: volume = 1/2 (4{pi}/3) (L1/2) (L2/2) (H) = 0.5238 L1L2H, where L1 is the long diameter, L2 is the short diameter, and H is the height. The time to a target mean tumor volume of 500 mm3 is defined as the elapsed time from the date of cell implantation to the date when a 500-mm3 target is reached or when the mouse was sacrificed. Of the 15 mice in the study, 10 reached a target tumor volume of 500 mm3 by day 72, at which point experiments were concluded. A Kaplan-Meier survival analysis with the corresponding log-rank analysis was done using S-plus Software (Insightful). A linear regression analysis was used to measure the rate of mean tumor volume growth as a function of time using S-plus Software (Insightful). P < 0.05 was considered to be statistically significant.

Proliferation assays. For measurement of proliferation, RWPE1 cells stably transfected with pcDNA3.1 or pcDNA3.1-Flag-Gli2 cells were plated at 2 x 103 per well into 96-well flat-bottomed microtiter plates (Falcon) in triplicate. Twenty microliters of CellTiter 96 AQueous One Solution Reagent (Promega) were added to each well of the assay plate containing the cells in 100 µL of culture medium. The plate was incubated for 2 h at 37°C in a humidified 5% CO2 chamber. The absorbance was recorded at 490 nm using an ELISA reader. The proliferation was assessed 24, 48, and 72 h after the initial reading.

Cell cycle analysis. Cell cycle analysis was carried out in stable RWPE1 clones of pcDNA3.1+ (control) and cells expressing Gli2 by propidium iodide staining. Cells were synchronized by starving for growth factors and medium supplements for 24 h and harvested at different time points, 0, 6, 12, and 24 h, after adding the complete media to the cells. For fixing, 95% ethanol was added to cell suspension, incubated for 45 min at 4°C, and then stored at –20°C overnight. Fixed cells were pelleted at 300 x g for 10 min, washed twice with PBS and once with staining buffer (PBS, 2% FBS, and 0.01% NaN3), and treated with RNase A (100 µg/mL) for 30 min at 37°C. Propidium iodide (25 µg/mL; Molecular Probes) staining was carried out at 37°C for ≥30 min and stored at 4°C until flow analysis.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Different GLI2-targeting shRNA constructs were designed and cloned in the vector that can be used for both transfection and lentiviral-mediated delivery. The construct most efficient in inhibiting GLI2 expression was chosen for our experiments. It down-regulates endogenous GLI2 expression in both transient transfection of 293T cells (Fig. 1A ) and lentiviral delivery in 22R{nu}1 prostate cancer cells (Fig. 2C , inset). Prostate cancer cells exhibited high constitutive levels of Hh pathway activity as compared with nontumorigenic immortalized prostate epithelial cells (RWPE1), measured by three different Gli-dependent luciferase reporter constructs (8x3'Gli BS-LucII, pGL3-Bcl2promo, and K17-driven luciferase). These data are in line with previous observations that the Hh signaling pathway is constitutively activated in prostate cancer cells (11, 12, 16). GLI2 knockdown repressed all three reporter activities in LnCaP, DU145, and 22R{nu}1 cells, averaging 3.2-, 3.0-, and 4.1-fold inhibitions, respectively (Fig. 1B–D). These data show that GLI2 plays a significant role in the pathway activation, and down-regulation of GLI2 is efficient in inhibiting transcriptional outcome of the Hh signaling pathway in prostate cancer cells.


Figure 1
View larger version (13K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 1. shRNA-mediated inhibition of GLI2 inhibits GLI-dependent transcription in prostate cancer cells. A, immunoblot analysis of GLI2 protein isolated from 293T cells transfected with shRNA plasmids as indicated. Representative of two independent experiments. Prostate cancer cells were transfected with pGL3-Bcl2promo luciferase (B), K17-luciferase (C), 8x3'Gli BS-LucII (D), pSV40 ß-gal (Promega), and shRNA expression plasmids as indicated. Luciferase activity was estimated using luciferase reporter assay reagent (Promega). ß-Galactosidase was used for normalization and estimated using ß-galactosidase assay reagent (Pierce). *, P < 0.01, compared with cells transfected with scrambled shRNA (Student's t test).

 

Figure 2
View larger version (16K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 2. Knockdown of GLI2 suppresses proliferation and anchorage-independent growth of prostate cancer cells. A, the colony formation assay was carried out in RWPE1 and prostate cancer cell lines transfected with scrambled and GLI2 shRNAs. The transfected cells were selected with puromycin (Sigma), allowed to form colonies, fixed with 10% formalin, stained with 2% Gentian violet (Ricca Chemical Company), and analyzed statistically. Anchorage-independent growth of 22R{nu}1 cells was studied. Cells infected with GLI2 shRNA– or scrambled shRNA–encoding lentiviruses were grown in six-well plates at a concentration of 10,000 per well as described. Colony formation was determined under low magnification (x10) in an inverted microscope (B) and counted (C). Inset, levels of GLI2 expression analyzed by immunoblotting with Gli2 antibody (G20). *, P < 0.01, compared with cells infected with scrambled shRNA (Student's t test).

 
Colony formation assay revealed that down-regulation of GLI2 leads to substantial inhibition of growth of all four human prostate cancer cell lines (DU145, PC3, 22R{nu}1, and LnCaP) examined as compared with scrambled shRNA control, whereas GLI2 knockdown in RWPE1 cells did not significantly affect the number of colonies (Fig. 2A). Taken together with our previous observation that GLI2 is overexpressed in primary human prostate tumors and prostate cancer cell lines (16). These data suggest that GLI2 is important for the proliferation and survival of prostate cancer cells.

To investigate the role of GLI2 in maintaining tumorigenic properties of prostate cancer cells, we used lentiviral-mediated delivery of GLI2 shRNA. 22R{nu}1 cells infected with lentiviruses encoding GLI2-specific shRNA exhibited 5-fold decrease in the number of colonies formed in soft agar as compared with cells infected with lentiviruses encoding scrambled shRNA (Fig. 2B and C). These data show that knockdown of GLI2 dramatically inhibits anchorage-independent growth of 22R{nu}1 prostate cancer cells.

We next investigated whether inhibition of GLI2 function affects the growth of prostate cancer cells in vivo. Inhibition of GLI2 expression by lentiviral-mediated introduction of GLI2 shRNA significantly inhibited the growth of 22R{nu}1 xenograft in nude mice (Fig. 3 ). The linear regression analysis showed that tumors expressing GLI2-specific shRNA grew much slower than xenografts expressing scrambled shRNA (average, 3.1 and 20.7 mm3/d, respectively; Fig. 3A). The average time to a target tumor volume of 500 mm3 was also significantly longer in GLI2-shRNA–expressing than in scrambled shRNA–expressing xenografts (64 and 44 days, respectively; Fig. 3B). The observed differences were statistically significant, with P < 0.05 according to a log-rank analysis (Fig. 3C). Together, these data suggest that inhibition of GLI2 is efficient in suppressing tumorigenic properties of prostate cancer cells both in vitro and in vivo.


Figure 3
View larger version (15K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 3. GLI2 down-regulation inhibits growth of 22R{nu}1 xenografts. Tumor growth studies were done in nude mice injected with 22R{nu}1 cells expressing GLI2 shRNA or scrambled shRNA. The time to a target mean tumor volume of 500 mm3 is defined as the elapsed time from the date of treatment to the date when the 500 mm3 target is reached or when the mouse is sacrificed. A, a linear regression analysis was used to measure both the rate of mean tumor or carcinoma area growth and tumor multiplicity as a function of time using S-plus Software. B, a Kaplan-Meier survival analysis with the corresponding log-rank analysis of Kaplan-Meier data (C) was done using S-plus Software (Insightful), * P < 0.05.

 
As discussed earlier, GLI2 protein is expressed at very low levels in RWPE1 cells (Fig. 4A , inset) and normal prostate epithelial cells (16). To assess the GLI2 function in these cells, the RWPE1 cells were stably transfected with pcDNA3.1 (as control) or pcDNA3.1-Flag-Gli2 plasmids. RWPE1 cells that stably express Gli2 proliferated at a much faster rate than control transfected cells (Fig. 4A). Interestingly, ectopic expression of Gli2 in RWPE1 cells resulted in both faster growth and higher saturation density (data not shown). Cell cycle analysis revealed accumulation of these cells in S phase (~50% cells transfected with Gli2, as compared with ~20% cells transfected with empty vector) and almost complete disappearance of cells in G2-M (Fig. 4B). These data suggest that ectopic expression of Gli2 accelerates cell cycle, especially transition through G2-M, and stimulates proliferation of nontumorigenic prostate epithelial cells.


Figure 4
View larger version (27K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 4. Overexpression of Gli2 accelerates growth of RWPE1 cells. A, cell proliferation assay was measured in RWPE1 cells stably expressing pcDNA3.1 or pcDNA3.1-Flag-Gli2 as indicated. The proliferation was estimated with CellTiter 96 AQueous One Solution Reagent (Promega) 24, 48, and 72 h after the initial reading at 490 nm using an ELISA reader. The levels of GLI2 in these RWPE1 clones were analyzed by immunoblotting with Gli2 antibody (G20, Santa Cruz biotechnology). B, cell cycle analysis was carried in stable RWPE1 clones of pcDNA3.1+ (control) and cells expressing Gli2 by propidium iodide staining. Cells were synchronized by starving on serum-free medium without growth factors and medium supplements for 24 h and then harvested 24 h after addition of serum. Cells fixed with 95% ethanol were treated with RNase A (100 µg/mL) for 30 min at 37°C and with propidium iodide (25 µg/mL; Molecular Probes) for ≥30 min at 37°C for flow analysis.

 
We provided evidence that GLI2 transcription factor plays a critical role in the growth and tumorigenicity of prostate cells. Previous studies concentrated on the role of GLI1 oncogene in prostate tumorigenesis. Because GLI1 itself is a transcriptional target of the Hh pathway, GLI1 mRNA expression serves as a reliable indicator of activated Hh signaling, and elevated GLI1 expression was linked with cancer development and progression (12, 13). Our data showed that down-regulation of GLI2 alone is very efficient in inhibiting the transcriptional output of Hh signaling in prostate cancer cells. GLI2 transcription factor significantly contributes to malignant transformation of prostate cancer cells. GLI2 may become an attractive target for therapeutic intervention, especially for patients with high-grade and/or metastatic prostate cancer. The first small-molecule inhibitors of GLI transcription factors efficient in inhibiting the growth of prostate cancer cells have recently been identified (21). These, as well as potential Gli2-specific inhibitors, may become powerful therapeutics for the management of prostate cancer and other malignancies characterized by constitutive activation of the Hh signaling pathway.

Future studies are needed to analyze the mechanisms responsible for overexpression of GLI2 in prostate cancer and the role of Gli2 activation in response to paracrine and autorcrine Hh signaling in prostate cancer cells.


   Acknowledgments
 
Grant support: NCI grant 1R01CA121851 (V.S. Spiegelman), American Cancer Society Award RSG-02-140-01-CNE (V.S. Spiegelman), the Wisconsin Partnership Fund for a Healthy Future (V.S. Spiegelman), and NCI grants 1R01CA122334 and 1R01CA102709CA 92900 (A. Thomas-Tikhonenko).

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. F. Aberger, P. Coulombe, D. Trono, R. Weinberg M. Grachtchouk, Dlugosz, and H. Sasaki for their generous gifts of reagents; Dr. I. Siddiqui for the help with xenograft experiments; and Dr. S.Y. Fuchs for critical reading of the manuscript.


   Footnotes
 
3 http://www.ambion.com/techlib/misc/siRNA_finder.html Back

Received 5/30/07. Revised 8/17/07. Accepted 10/ 2/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

  1. Riobo NA, Manning DR. Pathways of signal transduction employed by vertebrate Hedgehogs. Biochem J 2007;403:369–79.[CrossRef][Medline]
  2. Rubin LL, de Sauvage FJ. Targeting the Hedgehog pathway in cancer. Nat Rev Drug Discov 2006;5:1026–33.[CrossRef][Medline]
  3. Lum L, Beachy PA. The Hedgehog response network: sensors, switches, and routers. Science 2004;304:1755–9.[Abstract/Free Full Text]
  4. Bijlsma MF, Spek CA, Peppelenbosch MP. Hedgehog: an unusual signal transducer. Bioessays 2004;26:387–94.[CrossRef][Medline]
  5. Dai P, Akimaru H, Tanaka Y, Maekawa T, Nakafuku M, Ishii S. Sonic Hedgehog-induced activation of the Gli1 promoter is mediated by GLI3. J Biol Chem 1999;274:8143–52.[Abstract/Free Full Text]
  6. Matise MP, Joyner AL. Gli genes in development and cancer. Oncogene 1999;18:7852–9.[CrossRef][Medline]
  7. Ding Q, Motoyama J, Gasca S, et al. Diminished Sonic hedgehog signaling and lack of floor plate differentiation in Gli2 mutant mice. Development 1998;125:2533–43.[Abstract]
  8. Matise MP, Epstein DJ, Park HL, Platt KA, Joyner AL. Gli2 is required for induction of floor plate and adjacent cells, but not most ventral neurons in the mouse central nervous system. Development 1998;125:2759–70.[Abstract]
  9. Doles J, Cook C, Shi X, Valosky J, Lipinski R, Bushman W. Functional compensation in Hedgehog signaling during mouse prostate development. Dev Biol 2006;295:13–25.[CrossRef][Medline]
  10. Park HL, Bai C, Platt KA, et al. Mouse Gli1 mutants are viable but have defects in SHH signaling in combination with a Gli2 mutation. Development 2000;127:1593–605.[Abstract]
  11. Fan L, Pepicelli CV, Dibble CC, et al. Hedgehog signaling promotes prostate xenograft tumor growth. Endocrinology 2004;145:3961–70.[Abstract/Free Full Text]
  12. Karhadkar SS, Steven Bova G, Abdallah N, et al. Hedgehog signalling in prostate regeneration, neoplasia and metastasis. Nature 2004;431:707–12.[CrossRef][Medline]
  13. Sanchez P, Hernandez AM, Stecca B, et al. Inhibition of prostate cancer proliferation by interference with SONIC HEDGEHOG-GLI1 signaling. Proc Natl Acad Sci U S A 2004;101:12561–6.[Abstract/Free Full Text]
  14. Sheng T, Li C, Zhang X, et al. Activation of the hedgehog pathway in advanced prostate cancer. Mol Cancer 2004;3:29.[CrossRef][Medline]
  15. Zhang J, Lipinski R, Shaw A, Gipp J, Bushman W. Lack of demonstrable autocrine hedgehog signaling in human prostate cancer cell lines. J Urol 2007;177:1179–85.[CrossRef][Medline]
  16. Bhatia N, Thiyagarajan S, Elcheva I, et al. Gli2 is targeted for ubiquitination and degradation by ß-TrCP ubiquitin ligase. J Biol Chem 2006;281:19320–6.[Abstract/Free Full Text]
  17. Regl G, Kasper M, Schnidar H, et al. Activation of the BCL2 promoter in response to Hedgehog/GLI signal transduction is predominantly mediated by GLI2. Cancer Res 2004;64:7724–31.[Abstract/Free Full Text]
  18. Sasaki H, Hui C, Nakafuku M, Kondoh H. A binding site for Gli proteins is essential for HNF-3ß floor plate enhancer activity in transgenics and can respond to Shh in vitro. Development 1997;124:1313–22.[Abstract]
  19. Wiznerowicz M, Trono D. Conditional suppression of cellular genes: lentivirus vector-mediated drug-inducible RNA interference. J Virol 2003;77:8957–61.[Abstract/Free Full Text]
  20. Graham FL, van der Eb AJ. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 1973;52:456–67.[CrossRef][Medline]
  21. Lauth M, Bergstrom A, Shimokawa T, Toftgard R. Inhibition of GLI-mediated transcription and tumor cell growth by small-molecule antagonists. Proc Natl Acad Sci U S A 2007;104:8455–60.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
S. Dennler, J. Andre, F. Verrecchia, and A. Mauviel
Cloning of the Human GLI2 Promoter: TRANSCRIPTIONAL ACTIVATION BY TRANSFORMING GROWTH FACTOR-{beta} VIA SMAD3/{beta}-CATENIN COOPERATION
J. Biol. Chem., November 13, 2009; 284(46): 31523 - 31531.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Thiyagarajan, S.
Right arrow Articles by Spiegelman, V. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Thiyagarajan, S.
Right arrow Articles by Spiegelman, V. S.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online