help button home button Endocrine Society Molecular Endocrinology ENDO 08 Sessions Library
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Molecular Endocrinology, doi:10.1210/me.2006-0048
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
Right arrow Citation Map
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 NURSA Molecule Pages Link
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Dong, Z.
Right arrow Articles by Lu, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Dong, Z.
Right arrow Articles by Lu, S.
Molecular Endocrinology 20 (10): 2315-2325
Copyright © 2006 by The Endocrine Society

Vav3 Oncogene Is Overexpressed and Regulates Cell Growth and Androgen Receptor Activity in Human Prostate Cancer

Zhongyun Dong, Yin Liu, Shan Lu, Amy Wang, Kiwon Lee, Lu-Hai Wang, Monica Revelo and Shan Lu

Departments of Pathology (Y.L., K.L., M.R., S.L.) and Medicine (Z.D., S.L., A.W.,), University of Cincinnati College of Medicine, Cincinnati, Ohio 45267; and Department of Microbiology (L.-H.W.), Mount Sinai School of Medicine, New York, New York 10029

Address all correspondence and requests for reprints to: Shan Lu, Department of Pathology, University of Cincinnati College of Medicine, 231 Albert Sabin Way, Cincinnati, Ohio 45267. E-mail: shan.lu{at}uc.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The purpose of this research was to investigate the role of Vav3 oncogene in human prostate cancer. We found that expression of Vav3 was significantly elevated in androgen-independent LNCaP-AI cells in comparison with that in their androgen-dependent counterparts, LNCaP cells. Vav3 expression was also detected in other human prostate cancer cell lines (PC-3, DU145, and 22Rv1) and, by immunohistochemistry analysis, was detected in 32% (26 of 82) of surgical specimens of human prostate cancer. Knockdown expression of Vav3 by small interfering RNA inhibited growth of both androgen-dependent LNCaP and androgen-independent LNCaP-AI cells. In contrast, overexpression of Vav3 promoted androgen-independent growth of LNCaP cells induced by epidermal growth factor. Overexpression of Vav3 enhanced androgen receptor (AR) activity regardless of the presence or absence of androgen and stimulated the promoters of AR target genes. These effects of Vav3 could be attenuated by either phosphatidylinositol 3-kinase (PI3K) inhibitors or dominant-negative Akt and were enhanced by cotransfection of PI3K. Moreover, phosphorylation of Akt was elevated in LNCaP cells overexpressing Vav3, which could be blocked by PI3K inhibitors. Finally, we ascertained that the DH domain of Vav3 was responsible for activation of AR. Taken together, our data show that overexpression of Vav3, through the PI3K-Akt pathway, inappropriately activates AR signaling axis and stimulates cell growth in prostate cancer cells. These findings suggest that Vav3 overexpression may be involved in prostate cancer development and progression.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
COMPELLING DATA SHOW that the elevated androgen receptor (AR) activity and phosphatidylinositol 3-kinase (PI3K)-Akt signaling play an important role in prostate cancer development and progression (1, 2, 3, 4). The PI3K-Akt pathway is a cell growth and survival pathway for human cancers. Elevated PI3K-Akt signaling, such as mediated by phosphatase and tensin analog (PTEN) deletion and mutation, contributes to prostate cancer development and progression. Prostate-specific deletion of murine PTEN, a tumor suppressor gene that shuts off PI3K-Akt signaling, induces the prostatic intraepithelial neoplasia lesions, which later progress to invasive prostate cancer in mice (5). Multiple growth factors and cytokines, including epidermal growth factor (EGF), signal through the PI3K-Akt pathway and inappropriately activate AR in prostate cancer cells, which can be accomplished by direct AR phosphorylation by active Akt (6, 7, 8). PI3K-Akt signaling can also induce phosphorylation and inactivation of glycogen synthase kinase 3ß, resulting in an increased nuclear level of ß-catenin. Consequently, coactivator ß-catenin enhances AR activity to stimulate prostate cancer cell growth and survival (9, 10). Studies from our laboratory as well as others demonstrate that elevated PI3K-Akt signaling is critical for androgen-independent growth of human prostate cancer cells (11). It is noteworthy, however, that an elevation of Akt phosphorylation is observed in 45.8% of prostate cancer specimens (12) and a loss of or reduced PTEN protein expression occurs in approximately 30% of human prostate cancers (13, 14), suggesting the presence of other mechanisms that activate PI3K-Akt signaling in prostate cancer.

Vav gene is an oncogene identified in cell transformation experiments (15). Vav family proteins include three members (Vav1, Vav2, and Vav3) in mammalian cells (16, 17, 18). Vav1 is primarily expressed in hematopoietic lineages, whereas Vav2 and Vav3 are more ubiquitously expressed (19). Vav proteins share a common structure, including an N-terminal calponin homology (CH) domain involved in Ca+2 mobilization and transforming activity, an acidic region containing three regulatory tyrosines, a Dbl homology (DH) domain with a conserved region that promotes the exchange of GDP for GTP on Rac/Rho GTPases, a pleckstrin homology (PH) domain binding to phosphatidylinositol triphosphate (PIP3) that enables its movement to the inner face of the plasma membrane, two Src-homology (SH)3 domains interacting with proteins containing proline-rich sequences, and a SH2 domain interacting with the proteins containing phosphorylated tyrosines (20, 21). Vav proteins function as guanosine nucleotide exchange factors (GEFs) for the Rho/Rac family proteins. Independent of their GEF activity, Vav proteins also work as adapter molecules. Tyrosine phosphorylation by receptor or cytoplasmic protein tyrosine kinase is required for Vav protein activation. In the nonphosphorylation state, the DH domain is masked by the N-terminal extension containing the tyrosine residue Y174. Phosphorylation of Y174 exposes the DH domain, which in turn interacts with substrate proteins (22). Upon ligand stimulation of EGF receptor, Ros, insulin receptor, and IGF receptor, Vav3 is activated and physically associated with a variety of signaling molecules, including Rac1, Cdc42, PI3K, shc, Grb2, and phospholipase C{gamma}, leading to alteration in cell morphology and cell transformation (23). Similarly, overexpression of Vav3 leads to PI3K activation and focus formation in NIH3T3 cells. Vav3-induced cell transformation can be blocked by overexpression of PTEN or PI3K inhibitor LY294002 (24). However, the role of Vav3 in human cancer has not been described.

The purpose of this study was to determine the potential roles of Vav3 in human prostate cancer. We found that Vav3 was overexpressed in human prostate cancer and in androgen-independent human prostate cancer cells. Overexpression of Vav3 promoted prostate cancer cell growth and activated AR, regardless of the absence or presence of androgen. The effects of Vav3 on cell growth and AR activity were mediated by activation of the PI3K-Akt pathway. These findings suggest that overexpression of Vav3 inappropriately activates AR and stimulates cell growth, which may play important roles in human prostate cancer development and/or progression.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Vav3 Gene Expression in Prostate Cancer Cells and Human Prostate Cancer Specimens
Previously, we established an androgen-independent cell line LNCaP-AI from its parental androgen-dependent LNCaP cells (25). Taking advantage of the same genetic background between LNCaP and LNCaP-AI cells, we performed oligonucleotide DNA microarray analysis to identify genes that may contribute to androgen-independent growth. One of the most striking findings in this study is overexpression of Vav3 oncogene in LNCaP-AI cells. To evaluate Vav3 protein expression, we generated a rabbit anti-Vav3 antibody against a peptide located at the C terminus of Vav3, which is specific for Vav3 of both human and mouse origin and does not cross with Vav1 and Vav2. As shown in Fig. 1AGo, the 98-kDa Vav3 was overexpressed in LNCaP-AI cells as compared with that in LNCaP cells. Similarly, Vav3 protein was detected in three other androgen-independent human prostate cancer cell lines (PC3, DU145, and 22RV1) (Fig. 1AGo).


Figure 1
View larger version (62K):
[in this window]
[in a new window]
  		Fig. 1. Expression Analysis of Vav3 in Prostate Cancer Cells and in Human Prostate Cancer Specimens

A, Vav3 expression in prostate cancer cell lines. Cell extracts (100 µg) were used for Western blot analysis of Vav3 expression in LNCaP and LNCaP-AI cells, as well as PC3, DU145, and 22RV1 cells. ß-Actin is served as loading control. B, IHC analysis of Vav3 expression in human prostate cancer specimens. I, The normal prostate tissues are shown an area of unremarkable prostate glands and stroma with negative immunoreactivity for Vav3. II, An area of prostatic adenocarcinoma Gleason score 6 (3+3) with moderate to intense diffuse immunoreactivity for Vav3. III, An area of prostatic adenocarcinoma Gleason score 8 (4+4) with intense diffuse immunoreactivity for Vav3. The microphotographs of immunohistochemical staining are shown at original magnification of x400.

 
To determine the potential role of Vav3 in prostate cancer, we evaluated Vav3 expression in surgical specimens of human prostate cancer by immunohistochemistry (IHC). Eighty two tumor tissues and 10 normal tissues from patients of 38- to 87-yr-old men were analyzed (Table 1Go). Vav3 protein was detected in 26 tumor tissues (32%). All normal prostate tissues showed negative immunoreactivity for Vav3. As shown in Fig. 1BGo, Vav3 protein was located in the tumor epithelium but not stroma or normal prostate tissues. A significant difference was found for Vav3 gene positive (n = 26) and negative (n = 56) prostate cancer specimens (P < 0.001) using binomial method and log link function. A retrospective power analysis was carried out to determine the association of Vav3 expression and Gleason score and showed that the study had approximately 52% power to detect a significant trend given the observed data, assuming a significance level of {alpha} = 5%. Therefore, our sample size is not large enough to demonstrate the association between Vav3 expression and Gleason score in prostate cancer.


View this table:
[in this window]
[in a new window]
  		Table 1. Summary of ICH Analysis of Vav3 Expression in Prostate Cancer Specimens Based on Gleason Score

 
Vav3 Is Involved in Growth of Prostate Cancer Cells
To determine the role of Vav3 in growth of prostate cancer cells, we used small interfering RNA (siRNA) technology to reduce Vav3 gene expression. We designed a small interfering Vav (siVav)3–247 oligonucleotide specific for Vav3 gene and control-247 oligonucleotide. After transfection with different concentrations of siVav3–247 or control-247, LNCaP cells were cultured in stripped medium supplemented with 10–8 M dihydrotestosterone (DHT), and LNCaP-AI cells were cultured in stripped medium in a 96-well plate. As shown in Fig. 2AGo, transfection of siVav3–247, but not control-247, inhibited both androgen-dependent growth in LNCaP cells and androgen-independent growth in LNCaP-AI cells in a dose-dependent manner. At 1.0 pM/well of siRNA concentration, transfection of siVav3–247 inhibited growth of LNCaP and LNCaP-AI cells by 53% and 65%, respectively. In contrast, transfection of the same amount of control-247 resulted in 15% inhibition in LNCaP cells and 18% in LNCaP-AI cells. Knockdown expression of Vav3 by siVav3–247 was confirmed at the protein level (Fig. 2BGo). Densitometry analysis of Vav3 relative to ß-actin showed that the Vav3 levels decreased by 80% and 45% on d 3 and d 5, respectively, in LNCaP cells transfected with siVav3–247. In contrast, transfection of control-247 reduced Vav3 by only 22% and 1% in LNCaP cells on d 3 and d 5, respectively.


Figure 2
View larger version (29K):
[in this window]
[in a new window]
  		Fig. 2. Knockdown Expression of Vav3 Inhibits Cell Growth

A, LNCaP and LNCaP-AI cells (5000/well in 96-well plate) were transiently transfected with siVav3–247 or control-247 at the concentrations of 0, 0.25, 0.5, and 1.0 pM/well using Lipofectamine 2000 for overnight. Then, LNCaP cells were cultured in stripped medium with 10–8 M DHT, and LNCaP-AI cells were cultured in stripped medium for 5 d, followed by MTT assay. The data were presented as percent inhibition. B, Expression analysis of Vav3 upon Vav3 siRNA treatment. LNCaP cells were transfected with 5 pM/well of siVav3–247 or control-247 in six-well plates for 3 and 5 d. The cell extracts were prepared and subjected to Western blot analysis for Vav3. ß-Actin served as loading control.

 
We then determined whether overexpression of Vav3 in LNCaP cells could stimulate cell growth. LNCaP cells were stably transfected with wild-type Vav3, constitutive active Vav3*, or empty vector pHEF to derive LNCaP-Vav3, LNCaP-Vav3*, and LNCaP-HEF cell lines, respectively. Overexpression of 98-kDa Vav3 and 75-kDa Vav3* was confirmed at the protein level (Fig. 3AGo). Overexpression of Vav3 or Vav3* did not alter AR expression in these cells (Fig. 3BGo). We found that overexpression of constitutive active Vav3* significantly stimulated cell growth in the absence of androgen. On d 6, the OD reading, which reflects the cell density, in wells containing LNCaP-Vav3* cells, was 1.6-fold higher than those containing LNCaP-HEF cells (Fig. 3CGo). Overexpression of wild-type Vav3 potentiated growth-stimulatory effects of EGF (Fig. 3DGo). In the cultures containing 1.6 ng/ml of EGF, the density of LNCaP-Vav3 cells was 3-fold higher than that of LNCaP-HEF cells. Overexpression of Vav3 or Vav3* did not significantly stimulate LNCaP cell growth in the presence of DHT in 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, which may be because the robust DHT effect overshadowed Vav3-mediated cell growth in the short term of cell growth experiment. Therefore, our data suggest that overexpression of Vav3 promoted androgen-independent growth in prostate cancer cells.


Figure 3
View larger version (35K):
[in this window]
[in a new window]
  		Fig. 3. The Role of Vav3 in Prostate Cancer Cell Growth

A and B, Vav3 expression in LNCaP-Vav3, LNCaP-Vav3*, and LNCaP-pHEF cell lines. Cell extracts (100 µg) from each cell line were used for Western blot analysis of Vav3 and AR expression. ß-Actin served as loading control. C, LNCaP-HEF or LNCaP-Vav3* cells (5000 cells per well) were seeded into 96-well plates in stripped medium. MTT staining for 1 h was performed on d 0, d 1, d 3, and d 6. D, LNCaP-HEF and LNCaP-Vav3 cells were cultured in stripped medium in the presence of various concentrations of EGF for 5 d, followed by MTT assay. The error bar represents the average of triplicate samples of each experiment.

 
Vav3 Activates AR via the PI3K-Akt Pathway
To determine the role of Vav3 in activation of AR, we investigated whether overexpression of Vav3 could regulate AR-dependent luciferase reporter activities. Hela cells were transiently cotransfected with an ARE-driven luciferase reporter without or with an AR expression vector, as well as Vav3 or Vav3*. As shown in Fig. 4AGo, minimal luciferase activity was detected in cells without cotransfection of AR, regardless of stimulation by DHT or transfection of Vav3 or Vav3*. Expression of AR moderately increased the basal level of the androgen response element (ARE)-driven luciferase activity, which was elevated in cells treated with DHT. Similarly, this AR-dependent luciferase activity was enhanced by approximately 1.9- and 3.5-fold in cells overexpressed with Vav3 and Vav3*, respectively (Fig. 4AGo). These data indicate that the elevated ARE promoter activity in Hela cells was mediated by AR. Furthermore, Vav3 dramatically enhanced AR activity at the below physiological level of androgen (Fig. 4BGo). The effects of Vav3 on AR activation were verified in LNCaP-AI cells, which had higher and more consistent transfection efficiency in transient transfection experiments. Data in Fig. 4CGo show that overexpression of Vav3 enhanced AR activity in both the presence and absence of its ligand DHT in LNCaP-AI cells. In agreement with its effects on androgen-independent growth of cells overexpressing Vav3 (Fig. 3DGo), overexpression of Vav3 potentiated EGF-stimulated AR activity in LNCaP-AI cells (Fig. 4DGo). On the other hand, we determined whether blocking AR activity compromised Vav3-mediated AR activation. We found that the AR antagonist Flutamide inhibited AR activation mediated by both DHT and Vav3 (Fig. 4EGo).


Figure 4
View larger version (30K):
[in this window]
[in a new window]
  		Fig. 4. Vav3 Is Involved in AR Activation

A and B, Hela cells (105 cells per well in 12-well plate) were cotransfected with pGL3-ARE-E4-Luc (0.25 µg), expression vector (100 ng) for Vav3, Vav3*, or empty vector pHEF, as well as expression vector (25 ng) for AR, or empty vector pCMV, respectively. Then, the cells were treated with DHT (10–8 M) (panel A) or 10–12 M and 10–11 M DHT (panel B). C and D, LNCaP-AI cells (2 x 105 cells per well in 12-well plates) were cotransfected with pGL3-ARE-E4-Luc (1 µg), expression vector (0.5 µg) for Vav3, or empty vector pHEF, respectively. Then, the cells were treated with DHT (10–8 M) (panel C) or EGF (10 ng/ml) (panel D). E, LNCaP-AI cells were cotransfected with pGL3-ARE-E4-Luc (1 µg) and expression vector for Vav3* (0.5 µg). Then, the cells were treated with Flutamide (10 µM) without or with DHT (10–10 M). All transfection and drug treatments are in stripped medium for 24 h, followed by luciferase assay. Renilla luciferase as an internal control was used to normalize the data. Data are presented as the mean (±SD) of duplicate values of a representative experiment that was independently repeated five times.

 
PI3K-Akt signaling enhances AR activity and is thought to be critical for prostate cancer development and progression (6, 11). Because Vav3 is a potential upstream regulator of the PI3K-Akt pathway (23, 24), we investigated whether overexpression of Vav3 could affect PI3K-Akt signaling-mediated AR activation. Hela cells were transiently cotransfected with ARE-Luc reporter, AR expression vector, Vav3*, and p85 (the PI3K-regulatory subunit) and/or p110 (the catalytic subunit). As shown in Fig. 5AGo, transfection of Vav3* with either subunit or both subunits of PI3K stimulated AR activity. AR activity was further enhanced in cells cotransfected with both Vav3* and either subunits of PI3K. To determine the role of endogenous PI3K in Vav3-induced AR activation, we investigated the effects of PI3K inhibitors LY294002 and Wortmannin on AR activation. After transfection of LNCaP-AI cells with expression vector for Vav3 or Vav3* and luciferase reporter driven by ARE or the promoters of PSA and p21WAF/CIP genes, both are AR-responsive genes (26, 27), the cells were treated for 24 h with 5 µM of LY294002 or 0.5 µM of Wortmannin. PI3K inhibitors blocked both basal and Vav3- or Vav3*-stimulated activation of ARE promoter in the absence (Fig. 5BGo) and presence (Fig. 5CGo) of DHT. The PI3K inhibitors also blocked Vav3-mediated activation of PSA (Fig. 5DGo) and p21WAF/CIP (Fig. 5EGo) promoters. Consistently, the basal and Vav3*-stimulated p21WAF/CIP promoter activity was inhibited by cotransfection of dominant-negative Akt (Fig. 5FGo). These data suggest that Vav3 activates AR, which is mediated by PI3K-Akt signaling.


Figure 5
View larger version (39K):
[in this window]
[in a new window]
  		Fig. 5. Vav3 Activates AR via the PI3K-Akt Pathway

A, Hela cells were cotransfected with pGL3-ARE-E4-Luc (0.25 µg), expression vector (50 ng) for Vav3, Vav3*, or empty vector pHEF, and expression vector (50 ng) for p85, p110, p85+p110, or empty vector pCMV, as well as expression vector (25 ng) for AR, or empty vector pCMV, respectively. B and C, LNCaP-AI cells were cotransfected with pGL3-ARE-E4-Luc (1 µg) and expression vector for Vav3* (0.5 µg). Then, the cells were treated with LY294002 (10 µM) or Wortmannin (0.5 µM) without (panel B) or with DHT (panel C). D and E, LNCaP-AI cells were cotransfected with PSA(–4.3)-Luc (1 µg) (panel D) or p21(–215)-Luc (1 µg) (panel E), as well as expression vector (0.5 µg) for Vav3, Vav3*, or empty vector pHEF, respectively. Then, the cells were treated with LY294002 or Wortmannin. F, LNCaP-AI cells were cotransfected with p21(–215)-Luc reporter (1 µg/well in 12-well plates), dominant-negative Akt (0.25 µg) or empty vector pUS, as well as Vav3* expression vector (0.25 µg) or empty vector pHEF, respectively, in stripped medium. All transfection and drug treatments are in stripped medium for 24 h, followed by luciferase assay. Renilla luciferase as an internal control was used to normalize the data. Data are presented as the mean (±SD) of duplicate values of a representative experiment that was independently repeated for five times.

 
We directly investigated effects of Vav3 and PI3K-Akt signaling on expression of the androgen-responsive p21WAF/CIP gene at the protein level by Western blot analysis. LNCaP cells stably transfected with Vav3 expressed higher level of Vav3 (Fig. 3AGo) and p21WAF/CIP (Fig. 6Go). Treatment with DHT (10–8 M) enhanced p21WAF/CIP expression in control (LNCaP-HEF) and Vav3-engineered LNCaP-Vav3 cells (Fig. 6AGo). Moreover, although there was no discernable difference in Akt expression levels, phosphorylated Akt was significantly elevated in LNCaP-Vav3 cells relative to that in LNCaP-HEF cells (Fig. 6BGo). Treatment with LY294002 reduced the levels of phosphorylated Akt in both LNCaP-HEF and LNCaP-Vav3 cells. These data indicated that overexpression of Vav3 enhanced AR-target gene expression, which was mediated by activation of PI3K-Akt signaling.


Figure 6
View larger version (46K):
[in this window]
[in a new window]
  		Fig. 6. Effects of Vav3 on p21WAF/CIP Expression and Akt Phosphorylation

A, LNCaP-HEF and LNCaP-Vav3 cells were treated with DHT (10–8 M) in stripped medium for 24 h, followed by Western blot analysis for p21WAF/CIP. B, LNCaP-HEF and LNCaP-Vav3 cells were treated with LY294002 in stripped medium for 24 h, followed by Western blot analysis for p21WAF/CIP, Akt, and Phospho-Akt. ß-Actin served as loading control.

 
The DH Domain of Vav3 Is Responsible for Activation of AR
To dissect the functional domain of Vav3 involved in AR activation, we generated truncated constructs of Vav3* with either deletion of the DH domain (Vav3*-{Delta}DH) that has GEF activity or the SH domain that has adaptor activity (Vav3*-{Delta}SH) (Fig. 7AGo). We found that the ARE promoter activity was moderately enhanced in cells cotransfected with Vav3 and was significantly elevated in cells cotransfected with Vav3* in Hela cells (Fig. 7BGo). Deletion of the DH domain of Vav3* abolished its activity in activation of ARE promoter. In sharp contrast, the removal of the SH domain did not alter the effects of Vav3* on ARE promoter activation (Fig. 7BGo). This experiment was also confirmed in LNCaP cells without cotranfection of AR expression vector. Because the Vav3*-{Delta}DH construct still contains the PH domain with PIP3 binding activity, these data also suggest that binding of PIP3 does not play an essential role for AR activation by Vav3*. However, our data did not exclude the regulatory role of the PH domain for Vav3 activity in AR activation. This finding indicates that the DH domain was responsible for activation of AR.


Figure 7
View larger version (26K):
[in this window]
[in a new window]
  		Fig. 7. Determination of the Functional Domain of Vav3 Involved in AR Activation

A, Deletion constructs of Vav3 gene. B, Hela cells (105 cells per well in 12-well plates) were cotransfected pGL3-ARE-E4-Luc (0.25 µg) with expression vector (100 ng) for Vav3, Vav3*, Vav3*-{Delta}DH, Vav3*-{Delta}SH, or empty vector pHEF, as well as expression vector (25 ng) AR for 24 h, respectively, followed by luciferase assay. Renilla luciferase as an internal control was used to normalize the data. Data are presented as the mean (±SD) of duplicate values of a representative experiment that was independently repeated five times. CH, Calponin homology; AD, Acidic domain.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
We have investigated the potential roles of Vav3 in human prostate cancer. We found that Vav3 protein was detected in all human prostate cancer cell lines examined. The expression of Vav3 was further elevated in androgen-independent human prostate cancer cells in comparison with their androgen-dependent counterpart. Moreover, Vav3 protein was detected in approximately 30% of human prostate cancer tissues. Down-regulation of Vav3 inhibited both androgen-dependent and androgen-independent growth of human prostate cancer cells. In sharp contrast, overexpression of Vav3 significantly enhanced EGF-stimulated androgen-independent growth. These data suggest that overexpression of Vav3 is involved in prostate cancer cell growth and that Vav3 may play an important role in human prostate cancer development and progression to androgen-independent growth.

AR is expressed in most prostate cancers, and elevated AR activity is thought to be involved in prostate cancer development and progression (28, 29). AR mutation and gene amplification are common alterations in advanced human prostate cancer and are associated with the disease progression (30, 31). Mutations in the ligand-binding domain generate promiscuous AR, which accepts a broad ligand spectrum for activation, such as by unfavorable adrenal androgen and AR antagonists (32, 33). Overexpression of steroid hormone receptor coactivators, such as SRC1, TIFII, SRC3/RAC3, and ARA55, dramatically enhances AR activity (34, 35, 36). However, probably more importantly, AR can be activated by phosphorylation (37, 38). For instance, activation of PI3K-Akt signaling up-regulates AR activity (3, 6, 11). Data presented in this paper reveal that overexpression of Vav3 could be a novel mechanism that inappropriately activates AR in human prostate cancer cells. This effects is mediated by PI3K-Akt signaling. Four lines of evidence support this conclusion. First, overexpression of Vav3 activates both liganded and unliganded AR in prostate cancer cells, which can be inhibited by Flutamide. Vav3 dramatically enhances AR activity by androgen at concentrations of below the physiological level. Second, overexpression of Vav3 enhances promoter activities of AR-target genes PSA and p21WAF/CIP, which can be attenuated by PI3K inhibitors or dominant-negative Akt. On the other hand, AR activity is enhanced by cotransfection of Vav3 and PI3K. Third, overexpression of Vav3 leads to up-regulation of endogenous p21WAF/CIP expression, which is further enhanced by DHT. Constitutive phosphorylation of Akt is elevated in LNCaP cells overexpressing Vav3, which can be blocked by PI3K inhibitors. Finally, the DH domain of Vav3 is identified to be responsible for regulation of AR activity. Moreover, we found that overexpression of Vav3 potentiates EGF-stimulated AR activation and prostate cancer cell growth. Previous studies have demonstrated that EGF stimulates prostate cancer cell growth, which is inhibited by AR antagonists Casodex and ICI 182780, suggesting an AR-mediated mechanism (39, 40).

During preparation of this manuscript, one most recent publication by Lyons and Burnstein (41) also showed that Vav3 up-regulates AR activity and supports androgen-independent growth in prostate cancer cells. However, in contrast to our finding that AR activation by Vav3 is mediated by PI3K-Akt signaling in LNCaP cells, they found that effects of Vav3 in PC3 cells were not inhibited by PI3K inhibitor. Because two different cell lines were used in these studies, the discrepancy could be due to the heterogeneity of prostate cancer cell lines. We identified that the DH domain of Vav3 is responsible for AR activation by deletion mutation analysis, which is an approach by taking advantage of multiple distinct function domains of Vav3. We found that deletion of the DH domain, but not the PH domain, abolished the Vav3-mediated AR activation. Our data did not exclude the regulatory effect of the PH domain, a PIP3-binding site, for Vav3 activity, which may be what was observed by the study of Lyons and Burstein.

Recently, we reported that the signaling level of the PI3K-Akt pathway is elevated in LNCaP-AI cells relative to that in LNCaP cells and is critical for their androgen-independent growth (11). This elevated PI3K-Akt signaling in LNCaP-AI cells is not due to PTEN, because PTEN is mutated in LNCaP cells (4). Data presented in this paper show that overexpression of Vav3 is at least partially responsible for the further elevation of PI3K-Akt signaling in LNCaP-AI cells. Our finding suggests that Vav3 overexpression is an alternative mechanism that leads to elevated PI3K-Akt signaling and AR activity in human prostate cancer, which may be of great importance in prostate cancer development and progression. Further studies are necessary to determine whether Vav3 activates AR by direct binding or enhances nuclear localization and/or AR chromosome occupancy. We will also address whether Vav3 overexpression and loss of or reduced PTEN protein expression occur additively or mutually exclusively in human prostate cancer and whether overexpression of Vav3 in prostate cancer and further elevation in androgen-independent cells, e.g. LNCaP-AI vs. LNCaP cells, are due to Vav3 gene amplification or an up-regulation of Vav3 gene transcription.

In summary, this study revealed that Vav3 is overexpressed in human prostate cancer specimens and is further elevated in androgen-independent prostate cancer cells. Vav3 overexpression, independent of PTEN, is involved in aberrant cell growth, AR activation, and induction of AR target genes in human prostate cancer cells. These effects were mediated by PI3K-Akt signaling. Therefore, overexpression of Vav3 could be a new marker for human prostate cancer. Targeting Vav3 could be an efficacious therapeutic modality for prostate cancers, especially those androgen-independent diseases.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Reagents
RPMI 1640 medium was purchased from Invitrogen (Carlsbad, CA). Fetal bovine serum (FBS) and charcoal/dextran-treated FBS were purchased from HyClone Laboratories, Inc. (Logan, UT). Antibodies against Akt and phospho-Akt were purchased from Cell Signaling Technology, Inc. (Beverly, MA). Anti-p21 antibody was obtained from Oncogene Research Products (La Jolla, CA). Human Vav3-specific Stealth Select RNA interference (siVav3–247: 5'-CCCAGTTTCTCTGTTTGAAGAACAT-3') and its control (control-247: 5'-CCCTTCTCTGTTTGTAAAGAGACAT-3') were designed by a software in Invitrogen website, and we purchased both oligos from Invitrogen. The transfection reagent Lipofectamine 2000 was purchased from Invitrogen.

Anti-Vav3 antibody from Upstate Biotechnology, Inc. (Charlottesville, VA) was used for IHC analysis. We also generated our custom anti-Vav3 antibody from the C terminus of Vav3 peptide sequence (CMELVEYYKHHSLKEGFRT). We determined Vav3 expression from normal mouse prostate tissues and LNCaP cells, and no nonspecific band was detected by this antibody. Wild-type Vav3 showed a 98-kDa band, and Vav3* showed a 75-kDa band. Our custom Vav3 antibody was used for Western blot analysis in this study.

Cell Culture
The human prostate adenocarcinoma cell lines (LNCaP, PC-3, DU145, and 22Rv1) and cervical carcinoma cell line Hela were obtained from ATCC (Manassas, VA) and maintained in RPMI 1640 medium supplemented with 10% FBS (complete medium) at 37 C in 5% CO2. The androgen-independent LNCaP-AI cells were derived from LNCaP cells by long-term culture of the latter in RPMI 1640 medium supplemented with 10% charcoal/dextran-treated FBS (stripped medium) (25). LNCaP-AI cells were maintained in stripped medium.

Plasmids
The construction and structure of pBEF-Vav3, pHEF-Vav3*, and control vector pHEF were detailed in our previous study (24). Vav3* is a constitutive active form of Vav3 due to the CH domain and acidic region deletion at the N terminus (Fig. 7AGo). PSA(–4.3)-Luc and pGL3-ARE-E4-Luc, in which luciferase reporter gene is driven by a 4.3-kb PSA promoter and four copies of ARE fused with adenovirus E4 minimal promoter at the upstream, respectively, were generously provided by Dr. Zhengxin Wang. The p21(–215)-Luc reporter containing 215 bp of p21WAF/CIP promoter sequence including ARE was detailed in our previous studies (26, 27). The PI3K expression vectors were gifts from Dr. Anke Klippel (atugen AG, Berlin, Germany).

Vav3*-{Delta}SH was generated by deletion SH2 and SH3 fragments at the C terminus of Vav3* using BamHI site, and subsequently a linker (5'-GATCCATAATGATGC-3') containing stop codon was inserted in frame. Vav3*-{Delta}DH was generated by PCR amplification of PH domain with an upstream primer (5'-CGAGAATTCGCATGGAGCCGTGGAAGCAG-3') containing a translation start codon ATG in frame and downstream primer (5'-CCTGGATCCACCTGTTTAGGA-3'') containing a BamHI site, and this PCR fragment was inserted at the BamHI site of Vav3* sequence with the DH and PH domain deletion using the BamHI site.

Cell Growth Assay
Tumor cell growth was estimated by the MTT assay as previously described (25). Briefly, LNCaP and LNCaP-AI cells were seeded into 96-well cell culture plates at a density of 5 x 103 cells per well in stripped medium. After incubation in 5% CO2 at 37 C overnight, the cells were treated with various concentrations of drugs in stripped medium for 5 d. At the end of incubation, 20 µl of MTT (2.5 mg/ml in PBS) was added to each well, and the cells were further incubated for 1 h at 37 C to allow complete reaction between the dye and the enzyme mitochondrial dehydrogenase in the viable cells. After removal of the residual dye and medium, 100 µl of dimethylsulfoxide was added to each well, and the absorbance at 570 nm was measured using BMG Microplate Reader (BMG Labtech, Inc., Durham, NC).

Western Blot Analysis
Western blot analysis was performed as previously described (25). Briefly, aliquots of samples with the same amount of protein, determined using the Bradford assay (Bio-Rad Laboratories, Inc., Hercules, CA), were mixed with loading buffer (final concentrations of 62.5 mM Tris-HCl, pH 6.8, 2.3% SDS, 100 mM dithiothreitol, and 0.005% bromophenol blue), boiled, fractionated in a SDS-PAGE, and transferred onto a 0.45-µm nitrocellulose membrane (Bio-Rad). The filters were blocked with 2% fat-free milk in PBS and probed with first antibody in PBS containing 0.1% Tween 20 (PBST) and 1% fat-free milk. The membranes were then washed four times in PBST and incubated with horseradish peroxidase-conjugated secondary antibody (Bio-Rad) in PBST containing 1% fat-free milk. After washing four times in PBST, the membranes were visualized using the enhanced chemiluminescence Western blotting detection system (Amersham Pharmacia Biotech, Arlington Height, IL). For Western blot analysis of Vav3 expression, milk-free Tris-buffered saline buffer (pH 8.0) was used for the first antibody overnight at 4 C.

Transient Transfection Assay
Cells (105/well) were seeded in 12-well tissue culture plates. Next day, Optifect-mediated transfection was used for the transient transfection assay according to the protocol provided by Invitrogen/Life Technologies, Inc. The cells were then treated with hormone or drugs in stripped medium for 24 h. Subsequently, the cell extracts were prepared and luciferase activity was assessed in a Berthold Detection System (Pforzheim, Germany) using a kit (Promega Corp., Madison, WI) following the manufacturer’s instruction. For each assay, cell extract (20 µl) was used, and the reaction was started by injection of 50 µl of luciferase substrate. Each reaction was measured for 10 sec in the Luminometer. Luciferase activity was defined as light units/mg protein.

IHC Analysis of Vav3 Expression
The paraffin-embedded tissue sections of human prostate cancer specimens for IHC analysis are from our prostate cancer tissue bank (Department of Pathology, University of Cincinnati College of Medicine) and prostate cancer tissue array obtained from US Biomax, Inc. (Rockville, MD).

The paraffin-embedded tissue sections or tissue arrays were deparaffinized in xylene, rehydrated in graded alcohol, and transferred to PBS. The slides were treated in a microwave oven (1500 W) at 80% power output for 2 x 5 min in 0.1 M citric acid to retrieve antigens, followed by a treatment with 3% H2O2 in methanol (vol/vol) to block endogenous peroxidase. The treated slides were incubated in blocking buffer (5% BSA) at room temperature for 1 h and then with the blocking buffer containing approximately 1–2 µg/ml of rabbit anti-Vav3 antibody (Upstate Biotechnology, Inc., Lake Placid, NY) or rabbit IgG (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) overnight at 4 C. After washing, the slides were incubated with a biotinylated secondary antibody (from BioGenex Laboratories, San Ramon, CA), followed by washing and incubation with the streptavidin-conjugated peroxidase (BioGenex). A positive reaction was visualized by incubating the slides with stable diaminobenzidine and counterstaining with Gill’s hematoxylin (BioGenex). After washing three times with PBS, the slides were mounted with Universal Mount mounting medium (Fisher Scientific, Pittsburgh, PA). The intensity and extent of cytoplasm-positive labeling for Vav3 antibody were assessed semiquantitatively and scored as 0 (no staining), 1+ (weak and focal staining in <25% of tissue), 2+ (moderate intensity in ~25–50% of tissue), and 3+ (strong and diffused staining in >50% of tissue).


   ACKNOWLEDGMENTS
 
We thank Dr. Zengxin Wang (University of Texas M. D. Anderson Cancer Center, Houston, TX) and Dr. Anke Klippel (atugen AG, Berlin, Germany) for providing us with plasmids.


   FOOTNOTES
 
This work was supported by Cancer Center Foundation, University of Cincinnati College of Medicine.

Author disclosure: Z.D., Y.L., S.L., A.W., K.L., L.H.W., M.R., and S.L. have nothing to declare.

First Published Online June 8, 2006.

Abbreviations: AR, Androgen receptor; ARE, androgen response element; DH, Dbl homology; DHT, dihydrotestosteroe; EGF, epidermal growth factor; FBS, fetal bovine serum; GEF, guanosine nucleotide exchange factor; IHC, immunohistochemistry; MTT assay, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; PBST, PBS containing 0.1% Tween 20; PH, pleckstrin homology; PI3K, phosphatidylinositol 3-kinase; PIP3, phosphatidylinositol triphosphate; PSA, prostate-specific antigen; PTEN, phosphatase and tensin analog; SH, Src-homology; siRNA, small interfering RNA; siVav, small interfering Vav.

Received for publication January 27, 2006. Accepted for publication June 1, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Whang YE, Wu X, Suzuki H, Reiter RE, Tran C, Vessella RL, Said JW, Isaacs WB, Sawyers CL 1998 Inactivation of the tumor suppressor PTEN/MMAC1 in advanced human prostate cancer through loss of expression. Proc Natl Acad Sci USA 95:5246–5250[Abstract/Free Full Text]
  2. Cairns P, Okami K, Halachmi S, Halachmi N, Esteller M, Herman JG, Jen J, Isaacs WB, Bova GS, Sidransky D 1997 Frequent inactivation of PTEN/MMAC1 in primary prostate cancer. Cancer Res 57:4997–5000[Abstract/Free Full Text]
  3. Murillo H, Huang H, Schmidt LJ, Smith DI, Tindall DJ 2001 Role of PI3K signaling in survival and progression of LNCaP prostate cancer cells to the androgen refractory state. Endocrinology 142:4795–4805[Abstract/Free Full Text]
  4. Vlietstra RJ, van Alewijk DC, Hermans KG, van Steenbrugge GJ, Trapman J 1998 Frequent inactivation of PTEN in prostate cancer cell lines and xenografts. Cancer Res 58:2720–2723[Abstract/Free Full Text]
  5. Wang S, Gao J, Lei Q, Rozengurt N, Pritchard C, Jiao J, Thomas GV, Li G, Roy-Burman P, Nelson PS, Liu X, Wu H 2003 Prostate-specific deletion of the murine Pten tumor suppressor gene leads to metastatic prostate cancer. Cancer Cell 4:209–221[CrossRef][Medline]
  6. Wen Y, Hu MC, Makino K, Spohn B, Bartholomeusz G, Yan DH, Hung MC 2000 HER-2/neu promotes androgen-independent survival and growth of prostate cancer cells through the Akt pathway. Cancer Res 60:6841–6845[Abstract/Free Full Text]
  7. Craft N, Shostak Y, Carey M, Sawyers CL 1999 A mechanism for hormone-independent prostate cancer through modulation of androgen receptor signaling by the HER-2/neu tyrosine kinase. Nat Med 5:280–285[CrossRef][Medline]
  8. Yeh S, Lin HK, Kang HY, Thin TH, Lin MF, Chang C 1999 From HER2/Neu signal cascade to androgen receptor and its coactivators: a novel pathway by induction of androgen target genes through MAP kinase in prostate cancer cells. Proc Natl Acad Sci USA 96:5458–5463[Abstract/Free Full Text]
  9. Yang F, Li X, Sharma M, Sasaki CY, Longo DL, Lim B, Sun Z 2002.Linking ß-catenin to androgen-signaling pathway. J Biol Chem 277:11336–11344
  10. Sharma M, Chuang WW, Sun Z 2002 Phosphatidylinositol 3-kinase/Akt stimulates androgen pathway through GSK3ß inhibition and nuclear ß-catenin accumulation. J Biol Chem 277:30935–30941[Abstract/Free Full Text]
  11. Lu S, Ren CX, Liu Y, Epner DE 2006 The PI3K-Akt signaling is involved in regulation of p21WAF/CIP expression and androgen-independent growth in prostate cancer cells. Int J Oncol 28:245–251[Medline]
  12. Ayala G, Thompson T, Yang G, Frolov A, Li R, Scardino P, Ohori M, Wheeler T, Harper W 2004 High levels of phosphorylated form of Akt-1 in prostate cancer and non-neoplastic prostate tissues are strong predictors of biochemical recurrence. Clin Cancer Res 10:6572–6578[Abstract/Free Full Text]
  13. Thomas GV, Horvath S, Smith BL, Crosby K, Lebel LA, Schrage M, Said J, De Kernion J, Reiter RE, Sawyers CL 2004 Antibody-based profiling of the phosphoinositide 3-kinase pathway in clinical prostate cancer. Clin Cancer Res 10:8351–8356[Abstract/Free Full Text]
  14. Halvorsen OJ, Haukaas SA, Akslen LA 2003 Combined loss of PTEN and p27 expression is associated with tumor cell proliferation by Ki-67 and increased risk of recurrent disease in localized prostate cancer. Clin Cancer Res 9:1474–1479[Abstract/Free Full Text]
  15. Katzav S, Martin-Zanca D, Barbacid M 1989 vav, a novel human oncogene derived from a locus ubiquitously expressed in hematopoietic cells. EMBO J 8:2283–2290[Medline]
  16. Bustelo XR 2000 Regulatory and signaling properties of the Vav family. Mol Cell Biol 20:1461–1477[Free Full Text]
  17. Bustelo XR 2001 Vav proteins, adaptors and cell signaling. Oncogene 20:6372–6381[CrossRef][Medline]
  18. Trenkle T, McClelland M, Adlkofer K, Welsh J 2000 Major transcript variants of VAV3, a new member of the VAV family of guanine nucleotide exchange factors. Gene 245:139–149[CrossRef][Medline]
  19. Swat W, Fujikawa K 2005 The vav family: at the crossroads of signaling pathways. Immunol Res 32:259–266[CrossRef][Medline]
  20. Zugaza JL, Lopez-Lago MA, Caloca MJ, Dosil M, Movilla N, Bustelo XR 2002 Structural determinants for the biological activity of Vav proteins. J Biol Chem 277:45377–45392[Abstract/Free Full Text]
  21. Moores SL, Selfors LM, Fredericks J, Breit T, Fujikawa K, Alt FW, Brugge JS, Swat W 2000 Vav family proteins couple to diverse cell surface receptors. Mol Cell Biol 20:6364–6373[Abstract/Free Full Text]
  22. Aghazadeh B, Lowry WE, Huang XY, Rosen MK 2000 Structural basis for relief of autoinhibition of the Dbl homology domain of proto-oncogene Vav by tyrosine phosphorylation. Cell 102:625–633[CrossRef][Medline]
  23. Zeng L, Sachdev P, Yan L, Chan JL, Trenkle T, McClelland M, Welsh J, Wang LH 2000 Vav3 mediates receptor protein tyrosine kinase signaling, regulates GTPase activity, modulates cell morphology, and induces cell transformation. Mol Cell Biol 20:9212–9224[Abstract/Free Full Text]
  24. Sachdev P, Zeng L, Wang LH 2002 Distinct role of phosphatidylinositol 3-kinase and Rho family GTPases in Vav3-induced cell transformation, cell motility, and morphological changes. J Biol Chem 277:17638–17648[Abstract/Free Full Text]
  25. Lu S, Tsai SY, Tsai MJ 1999 Molecular mechanisms of androgen-independent growth of human prostate cancer LNCaP-AI cells. Endocrinology 140:5054–5059[Abstract/Free Full Text]
  26. Lu S, Liu M, Epner DE, Tsai SY, Tsai MJ 1999 Androgen regulation of the cyclin-dependent kinase inhibitor p21 gene through an androgen response element in the proximal promoter. Mol Endocrinol 13:376–384[Abstract/Free Full Text]
  27. Lu s, Jenster G, Epner DE 2000 Androgen induction of cylin-dependent kinase inhibitor p21 gene: role of androgen receptor and transcription factor Sp1 complex. Mol Endocrinol 14:753–760[Abstract/Free Full Text]
  28. Linja MJ, Savinainen KJ, Saramaki OR, Tammela TL, Vessella RL, Visakorpi T 2001 Amplification and overexpression of androgen receptor gene in hormone-refractory prostate cancer. Cancer Res 61:3550–3555[Abstract/Free Full Text]
  29. Ruizeveld de Winter JA, Janssen PJ, Sleddens HM, Verleun-Mooijman MC, Trapman J, Brinkmann AO, Santerse AB, Schroder FH, van der Kwast TH 1994 Androgen receptor status in localized and locally progressive hormone refractory human prostate cancer. Am J Pathol 144:735–746[Abstract]
  30. Taplin ME, Bubley GJ, Shuster TD, Frantz ME, Spooner AE, Ogata GK, Keer HN, Balk SP 1995 Mutation of the androgen-receptor gene in metastatic androgen-independent prostate cancer. N Engl J Med 332:1393–1398[Abstract/Free Full Text]
  31. Visakorpi T, Hyytinen E, Koivisto P, Tanner M, Keinanen R, Palmberg C, Palotie A, Tammela T, Isola J, Kallioniemi OP 1995 In vivo amplification of the androgen receptor gene and progression of human prostate cancer. Nat Genet 9:401–406[CrossRef][Medline]
  32. Veldscholte J, Ris-Stalpers C, Kuiper GG, Jenster G, Berrevoets C, Claassen E, van Rooij HC, Trapman J, Brinkmann AO, Mulder E 1990 A mutation in the ligand binding domain of the androgen receptor of human LNCaP cells affects steroid binding characteristics and response to anti-androgens. Biochem Biophys Res Commun 173:534–540[CrossRef][Medline]
  33. Kokontis J, Ito K, Hiipakka RA, Liao S 1991 Expression and function of normal and LNCaP androgen receptors in androgen-insensitive human prostatic cancer cells. Altered hormone and antihormone specificity in gene transactivation. Receptor 1:271–279[Medline]
  34. Gregory CW, He B, Johnson RT, Ford OH, Mohler JL, French FS, Wilson EM 2001 A mechanism for androgen receptor-mediated prostate cancer recurrence after androgen deprivation therapy. Cancer Res 61:4315–4319[Abstract/Free Full Text]
  35. Fujimoto N, Mizokami A, Harada S, Matsumoto T 2001 Different expression of androgen receptor coactivators in human prostate. Urology 58:289–294[CrossRef][Medline]
  36. Culig Z, Comuzzi B, Steiner H, Bartsch G, Hobisch A 2004 Expression and function of androgen receptor coactivators in prostate cancer. J Steroid Biochem Mol Biol 92:265–271[CrossRef][Medline]
  37. Nazareth LV, Weigel NL 1996 Activation of the human androgen receptor through a protein kinase A signaling pathway. J Biol Chem 271:19900–19907[Abstract/Free Full Text]
  38. Sadar MD 1999 Androgen-independent induction of prostate-specific antigen gene expression via cross-talk between the androgen receptor and protein kinase A signal transduction pathways. J Biol Chem 274:7777–7783[Abstract/Free Full Text]
  39. Migliaccio A, Di Domenico M, Castoria G, Nanayakkara M, Lombardi M, de Falco A, Bilancio A, Varricchio L, Ciociola A, Auricchio F 2005 Steroid receptor regulation of epidermal growth factor signaling through Src in breast and prostate cancer cells: steroid antagonist action. Cancer Res 65:10585–10593[Abstract/Free Full Text]
  40. Culig Z, Hobisch A, Cronauer MV, Radmayr C, Trapman J, Hittmair A, Bartsch G, Klocker H 1994 Androgen receptor activation in prostatic tumor cell lines by insulin-like growth factor-I, keratinocyte growth factor, and epidermal growth factor. Cancer Res 54:5474–5478[Abstract/Free Full Text]
  41. Lyons LS, Burnstein KL 2006 Vav3, a Rho GTPase guanine nucleotide exchange factor, increases during progression to androgen independence in prostate cancer cells and potentiates androgen receptor transcriptional activity. Mol Endocrinol 20:1061–1072[CrossRef]

NURSA Molecule Pages Link:

Nuclear Receptors:   AR
Ligands:   Dihydrotestosterone



This article has been cited by other articles:


Home page
 Cancer Res.Home page
Y. Liu, J. Q. Mo, Q. Hu, G. Boivin, L. Levin, S. Lu, D. Yang, Z. Dong, and S. Lu
Targeted Overexpression of Vav3 Oncogene in Prostatic Epithelium Induces Nonbacterial Prostatitis and Prostate Cancer
Cancer Res., August 1, 2008; 68(15): 6396 - 6406.
[Abstract] [Full Text] [PDF]

Home page
 Hum Mol GenetHome page
J. Yan, S. Chen, Y. Zhang, X. Li, Y. Li, X. Wu, J. Yuan, A. G. Robling, R. Karpur, R. J. Chan, et al.
Rac1 mediates the osteoclast gains-in-function induced by haploinsufficiency of Nf1
Hum. Mol. Genet., April 1, 2008; 17(7): 936 - 948.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
L. S. Lyons, S. Rao, W. Balkan, J. Faysal, C. A. Maiorino, and K. L. Burnstein
Ligand-Independent Activation of Androgen Receptors by Rho GTPase Signaling in Prostate Cancer
Mol. Endocrinol., March 1, 2008; 22(3): 597 - 608.
[Abstract] [Full Text] [PDF]

Home page
 Endocr. Rev.Home page
H. V. Heemers and D. J. Tindall
Androgen Receptor (AR) Coregulators: A Diversity of Functions Converging on and Regulating the AR Transcriptional Complex
Endocr. Rev., December 1, 2007; 28(7): 778 - 808.
[Abstract] [Full Text] [PDF]

Home page
 Molecular Cancer TherapeuticsHome page
S. Lu, A. Wang, S. Lu, and Z. Dong
A novel synthetic compound that interrupts androgen receptor signaling in human prostate cancer cells
Mol. Cancer Ther., July 1, 2007; 6(7): 2057 - 2064.
[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
Right arrow Citation Map
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 NURSA Molecule Pages Link
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Dong, Z.
Right arrow Articles by Lu, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Dong, Z.
Right arrow Articles by Lu, S.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals