Vav3, a Rho GTPase Guanine Nucleotide Exchange Factor, Increases ...

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Dec 29, 2005 - The AR antagonist, bicalutamide, significantly diminished androgen-mediated induction of PSA-Luc confirming participation by AR (Fig. 4).

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Molecular Endocrinology 20(5):1061–1072 Copyright © 2006 by The Endocrine Society doi: 10.1210/me.2005-0346

Vav3, a Rho GTPase Guanine Nucleotide Exchange Factor, Increases during Progression to Androgen Independence in Prostate Cancer Cells and Potentiates Androgen Receptor Transcriptional Activity Leah S. Lyons and Kerry L. Burnstein Department of Molecular and Cellular Pharmacology, University of Miami Miller School of Medicine, Miami, Florida 33136 The progression of prostate cancer from androgen dependence to androgen independence is often accompanied by enhanced androgen receptor (AR) transcriptional activity. We observed a marked increase in the expression of Vav3, a Rho GTPase guanine nucleotide exchange factor (GEF), during the progression of human prostate cancer LNCaP cells to the androgen-independent derivative, LNCaP-R1. GEFs activate Rho family GTPases by promoting the exchange of GDP for GTP. Reporter gene assays showed that Vav3 potentiated AR transcriptional activity, and knock down of Vav3 resulted in decreased AR transactivation. Vav3 also increased androgen-induced levels of prostate-specific antigen mRNA. Furthermore, Vav3 enhanced AR activity at subnanomolar concentra-

tions of androgen. This finding is particularly relevant because low androgen levels may be present in prostate tissue of patients undergoing androgen deprivation therapy. Enhancement of AR activity by Vav3 required amino terminal activation function 1 (AF1) of AR; however, Vav3 did not interact with AR or increase AR levels. Neither GEF function nor the C-terminal domains of Vav3 were required for Vav3-mediated enhancement of AR activity; however, the pleckstrin homology domain was obligatory. These data show that Vav3 levels rise during progression to androgen independence and support continued AR signaling (even under conditions of low androgen) by a novel GEF-independent cross-talk mechanism. (Molecular Endocrinology 20: 1061–1072, 2006)

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tion of AR transcriptional activity by growth factors [especially IGF, epidermal growth factor (EGF), and keratinocyte growth factor], AR gene amplification, somatic AR mutations that result in broader ligand specificity, and changes in expression of AR coactivators or corepressors, which favor AR transcriptional activity (2, 6). A limited number of cell lines reproduce the transition from androgen dependence to independence. Prolonged culture of the androgen-dependent human prostate cancer cell line, LNCaP, in androgen-depleted media results in the emergence of an androgenindependent derivative, LNCaP-R1 (3). LNCaP-R1 cells retain many of the seminal features of clinical androgen-independent prostate cancer including enhanced AR activity, which drives prostate-specific antigen (PSA) expression even under conditions of very low androgen concentrations, as well as robust growth in androgen-depleted media in culture and in vivo (3, 8, 9). Gene expression profiling of LNCaP and its androgen-independent derivative LNCaP-R1 revealed a striking increase in the expression of Vav3, a Rho family guanine nucleotide exchange factor (GEF), in the androgen-independent cells. Vav3 is a broadly expressed, multidomain protein and the most recently identified member of the Vav subfamily of diffuse B-

IGNALING BY THE androgen receptor (AR), a member of the nuclear receptor superfamily, is required for normal prostate development and promotes growth of prostate tumors. Androgen deprivation therapy, a common treatment for non-organconfined prostate cancer, results in decreased tumor burden and symptomatic relief. Unfortunately, the majority of patients undergoing this treatment eventually relapse with tumors that progress under conditions of low androgen (1, 2). Androgen-independent cancers generally maintain functional AR (reviewed in Refs. 1–5). Several mechanisms have been proposed to explain continued AR signaling in androgen-independent prostate cancer. These include inappropriate stimulaFirst Published Online December 29, 2005 Abbreviations: AF, Activation function; AR, androgen receptor; CSS, charcoal-stripped FBS; DBL, diffuse B-cell lymphoma; DH, DBL homology; EGF, epidermal growth factor; FBS, fetal bovine serum; GEF, guanine nucleotide exchange factor; PH, pleckstrin homology; PI3K, phosphatidylinositol 3-kinase; PSA, prostate-specific antigen; PSA-Luc, PSAluciferase reporter plasmid; RNAi, RNA interference; siRNA, small interfering RNA. Molecular Endocrinology is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving the endocrine community.

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cell lymphoma (DBL) proteins (10). Interestingly, Vav3 is recruited to and activated by the EGF receptor, IGF receptor, and phosphatidylinositol 3-kinase (PI3K) pathways, which have been strongly implicated in prostate cancer progression (11–15). Like other DBL family GEFs, Vav proteins contain the canonical DBL homology (DH) and pleckstrin homology (PH) domains arranged in tandem (16). Vav3 supports the exchange of GDP for GTP on Rho A, Rho G, and Rac1 (17). Although the DH/PH tandem domain organization is critical for GEF activity of most DBL family members (18), Vav3 does not require the PH domain for GEF activity, suggesting distinct biological roles for the Vav3 PH domain (10, 19, 20). Because enhanced androgen responsiveness is observed in LNCaP-R1 cells as well as in other models of progressed prostate cancer (3, 21), we investigated the potential effects of Vav3 on AR transcriptional activity. Vav3 significantly enhanced AR transcriptional activity but did not cause up-regulation of AR levels. Additionally, Vav3 enhanced AR transcriptional activity at subnanomolar doses of androgen, suggesting a role for Vav3 in the maintenance of AR signaling after androgen deprivation therapy. Mutational analysis of Vav3 demonstrated that the action of Vav3 on AR does not require GEF activity or the Vav3 C-terminal domains but is dependent on the Vav3 PH domain.

RESULTS Vav3 Is Up-Regulated during Progression of LNCaP to Androgen Independence Gene expression profiling initially revealed that Vav3 mRNA was elevated in androgen-independent LNCaP-R1 cells compared with the parental androgen-dependent LNCaP cell line. This finding was confirmed by semiquantitative RT-PCR (data not shown)

and ribonuclease protection assays (Fig. 1A). We found that Vav3 mRNA is expressed in even higher amounts in another androgen-independent, human prostate cancer cell line, ALVA31 (Fig. 1A). Immunoprecipitation of Vav3 protein from LNCaP, LNCaP-R1, and ALVA31 cells showed that the rank order of Vav3 protein expression was the same as for Vav3 mRNA (Fig. 1B). To determine whether Vav3 levels are increased in other models of prostate cancer progression, we examined LAPC4, a human androgen-dependent prostate cancer cell line (22) after androgen withdrawal. RT-PCR analysis was performed in a semiquantitative manner (as described in Materials and Methods) by removing samples during the linear range of amplification. We observed a 3-fold elevation of Vav3 mRNA after 3 months of androgen withdrawal but not at earlier time points (Fig. 1C and data not shown). Vav3 Enhances the Transcriptional Activity of Wild-Type and Mutant AR Because LNCaP-R1 cells (3, 8, 9, 23) similar to other models of progressed prostate cancer (1, 21, 24, 25) exhibit increased androgen responsiveness, we examined the effects of Vav3 on AR transcriptional activity. We used a PSA-luciferase reporter plasmid (PSA-Luc) consisting of the PSA promoter and 5⬘ flanking region, which contains both the distal (⫺5325 to ⫺4023) and proximal (⫺542 to ⫹12) ARE-containing enhancer regions but lacks the intervening sequences. Androgen induction of PSA-Luc was significantly greater in LNCaP cells transfected with Vav3 compared with empty vector (Fig. 2A). Direct analysis of PSA mRNA by semiquantitative RT-PCR in LNCaP cells showed that introduction of Vav3 compared with empty vector resulted in a 3.8-fold enhancement of androgen induction of PSA mRNA (Fig. 2B). To substantiate the role of Vav3 in enhancing AR activity, we used RNA interference to specifically down-regulate Vav3 in ALVA31 and LNCaP-R1 cells.

Fig. 1. Vav3 Levels Increase during Progression of LNCaP Cells to Androgen Independence A, Vav3 mRNA was examined by ribonuclease protection assays. Forty micrograms of total RNA from the indicated cell lines were hybridized to Vav3- and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)- (loading control) specific probes. Protected RNAs were visualized by autoradiography. Jurkat (T-cell line) RNA served as a positive control. B, Vav3 protein was immunoprecipitated from the indicated cell lines. Vav3-transfected LNCaP cells served as a positive control. After immunoprecipitation, complexes were immunoblotted for Vav3. Actin from total cell lysates is shown as a loading control. C, LAPC-4 cells were cultured in either 10% FBS or 10% CSS. Vav3 and actin mRNA were examined by RT-PCR during the linear range of amplification using Vav3-and actin-specific primers.

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one of the most common somatic AR mutations identified in advanced prostate cancer (26, 27). Thus, Vav3 stimulated the transcriptional activity of both mutated and wild-type AR. Vav3 Increases AR Transcriptional Activity at Low Androgen Levels Recent data suggest that low levels of androgen persist in prostate tumors of patients who have undergone androgen deprivation therapy and have relapsed to androgen independence (28, 29). We evaluated Vav3 effects on AR transcriptional activity at nonsaturating levels of androgen. Vav3 stimulated AR regulation of PSA-Luc at doses as low as 0.01 nM R1881, whereas control cells showed little activation of PSALuc at this same dose. Fold induction at 0.01 nM R1881 was 1.5-fold in the absence of Vav3 and 3.4fold in cells expressing Vav3; at 0.1 nM, induction was 3.4 for control and 10.8 in Vav3-transfected cells (Fig. 5). These experiments show that Vav3 increases AR transcriptional activity at androgen concentrations found in the prostate glands of men who relapse from androgen deprivation therapy (28, 29).

Fig. 2. Vav3 Enhances AR Transcriptional Activity A, LNCaP cells were transfected with Vav3 or equivalent amounts of the empty vector and the reporter plasmid, PSALuc. Cells were treated with either vehicle or 5 nM R1881. Luciferase activity was determined 48 h after transfection. Data are plotted as relative luciferase units (RLU ⫾ SEM) and are representative of three experiments performed in triplicate. Significance refers to differences between R1881treated vector and Vav3 transfected samples. B, LNCaP cells were transfected with Vav3 or empty vector. Cells were treated with vehicle or 5 nM R1881 for 48 h. RT-PCR was conducted during the linear range of amplification using PSA and actin-specific primers. Data are representative of four independent experiments.

Transfection of Vav3-specific small interfering RNAs (siRNA) [designated Vav3 RNA interference (RNAi)] significantly decreased levels of Vav3 protein and mRNA but did not reduce mRNA levels of the closely related Vav2 (Fig. 3, A and B). This knockdown of Vav3 resulted in significantly decreased AR transcriptional activity (Fig. 3C). PSA-Luc assays were also conducted in the PC3 prostate cancer cell line because these cells express low levels of Vav3 and no detectable AR. Thus, PC3 cells permitted us to determine the effects of Vav3 on expressed, wild-type AR. Introduction of Vav3 into PC3 cells resulted in a 268 (⫾21)% increase in fold induction compared with vector-transfected control cells (not shown). The AR antagonist, bicalutamide, significantly diminished androgen-mediated induction of PSA-Luc confirming participation by AR (Fig. 4). These findings are consistent with results obtained in LNCaP cells, which contain an AR T877A mutation,

Vav3 Enhancement of AR Activity Does Not Occur through Up-Regulation of AR Levels or through Direct Interaction with AR We examined the possibility that Vav3 enhances AR activity by elevating AR levels. Although expression of AR is elevated after androgen treatment in LNCaP cells due to increased AR stability (30), introduction of Vav3 did not result in a further increase in AR (Fig. 6, A and B). AR levels were also comparable in the absence of hormone in Vav3- and vector-transfected controls (Fig. 6, A and B). Furthermore, Vav3 did not elevate levels of transiently transfected AR in the PC3 cell line (data not shown). Because a variety of proteins enhance AR transcriptional activity by direct interaction with AR (31), we tested whether Vav3 and AR physically interact. Coimmunoprecipitation experiments in PC3 cells transfected with AR and Vav3 failed to show interaction between AR and Vav3 (Fig. 7). However, interaction between AR and the steroid hormone coactivator protein E6-AP was observed after androgen treatment (Fig. 7). E6-AP interaction with both progesterone and ARs is strongly ligand-dependent (Ref. 32 and personal communication Dr. Z. Nawaz, University of Miami, Miami, FL). The lack of a demonstrable interaction between Vav3 and AR suggests that Vav3 exerts effects on AR through mechanisms distinct from those described for AR coactivator proteins. Effects of Vav3 on AR Require the AR Activation Function 1 (AF-1) Region The human AR contains two well-characterized activating functions: AF1, the major transcriptional activa-

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Fig. 3. Specific Knockdown of Vav3 Decreases AR Transcriptional Activity A, ALVA31 cells were transfected with a Vav3 cDNA (5 ␮g) and no siRNA, Vav3-specific siRNA (Vav3 RNAi) or the scrambled (SCR) control. Forty-eight hours after transfection, lysates were immunoblotted for Vav3 and actin. B, ALVA31 and LNCaP-R1 cells were transfected with either the Vav3 siRNA or the scrambled siRNA. Forty-eight hours after transfection, total RNA was isolated. RT-PCR was conducted during the linear range of amplification using Vav3- (left panels), Vav2- (right panel), and actin-specific primers. C, ALVA31 and LNCaP-R1 cells were transfected with the PSA-Luc reporter plasmid, a ␤-galactosidase plasmid, AR and either Vav3-specific siRNA or a scrambled control. Cells were treated with either vehicle or 5 nM R1881. Luciferase activity was determined 48 h after transfection as described. Data from three triplicate experiments are plotted as fold induction (ligand/no ligand ⫾ SEM) percent of control (scrambled RNAi transfected) samples. Significance was determined using a two-tailed Student’s t test and refers to differences in fold induction between Vav3 RNAi-transfected and scrambled control RNAi transfected samples.

tion region, is located in the N-terminal domain, and AF2 is present in the C-terminal ligand binding domain. To determine whether either of these regions is required for the effects of Vav3 on AR, PSA-Luc assays were conducted in PC3 cells transfected with wild-type AR mutants lacking the AF1 or AF2 regions (33, 34). Deletion of the AF1 region of AR abolished AR transcriptional activity, and Vav3 could not rescue transactivation by the ⌬ AF1 AR mutant (Fig. 8A). As expected, AF2 deletion resulted in constitutive AR activity (35, 36). ⌬ AF2 AR activity was increased by Vav3 (Fig. 8B). Thus, AF1 but not AF2 is required for the effects of Vav3 on AR. Enhancement of AR Transcriptional Activity Does Not Involve Signaling through the PI3K Pathway Vav3 activates the PI3K pathway (18), and several reports suggest that activation of PI3K can modulate AR transcriptional activity (37–41). To determine

whether the enhancement of AR transcriptional activity by Vav3 involves signaling through the PI3K pathway, the effects of the specific PI3K inhibitor, LY294002 were determined. LY294002 did not affect the ability of Vav3 to enhance AR transcriptional activity (Fig. 9A) but did, as expected, reduce levels of Akt phosphorylation (data not shown). To investigate the involvement of this pathway in Vav3-mediated effects on AR without the use of pharmacologic inhibitors, the effects of Vav3 on the transcriptional activity of two putative AR Akt phosphorylation site mutants (S790A and S213A) was examined. These AR mutations eradicate the ability of Akt to phosphorylate AR on these residues in vitro (41). Although mutation of these residues decreased androgen-inducible PSA-Luc activity, neither abolished the capacity of Vav3 to enhance AR transcriptional activity (Fig. 9B), suggesting that signaling through the PI3K pathway is not involved in the effects of Vav3 on AR.

Lyons and Burnstein • Vav3 Potentiates AR Transcriptional Activity

Fig. 4. Effects of Vav3 on AR Transcriptional Activity Are Diminished by the AR Antagonist Bicalutamide PC3 cells were transfected with Vav3 or empty vector, PSA-Luc reporter and AR. Twenty-four hours after transfection, cells were treated with either vehicle or R1881 and either vehicle or 20 ␮M bicalutamide. Luciferase activity was determined 24 h after treatment. Data from three triplicate experiments are plotted as fold induction relative to control (vector transfected samples) in the absence of bicalutamide (% control RLU/␤-gal ⫾ SEM).

Enhancement of AR Transcriptional Activity Does Not Require the Vav3 C-Terminal Domains and Is GEF Independent The C-terminal region of Vav3 consists of two SH3 domains flanking a single SH2 domain. These domains are potential sites of protein/protein interactions and are used in recruitment of Vav proteins to growth factor receptors (16, 42). To examine the contribution of the Vav3 C-terminal domains to the effects of Vav3 on AR, we created a Vav3 deletion mutant lacking the entire C-terminal cassette (Vav3 ⌬C) (Fig. 10A). Vav3 ⌬C enhanced the transcriptional activity of AR to the same extent as wild-type Vav3 (Fig. 10B).

Fig. 5. Vav3 Enhances AR Transcriptional Activity at Low Doses of Androgen PC3 cells were transfected with Vav3 or empty vector, PSA-Luc reporter and AR. Cells were treated with either vehicle or the indicated amount of R1881. Luciferase activity was determined. Means of triplicate samples are plotted as relative luciferase units (RLU ⫾ SEM) and are representative of three experiments.

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Fig. 6. Vav3 Potentiation of AR Transcriptional Activity Is Not Due to Up-Regulation of AR A, LNCaP cells were transfected with Vav3 or empty vector. Cells were treated with control vehicle or 5 nM R1881 for 48 h. AR levels were determined by Western blotting. Androgen stabilization of AR protein is evident. B, Bands from three independent experiments were quantified by densitometry. Data are plotted as relative AR levels per actin ⫾ SEM.

Because certain Rho family proteins can influence steroid hormone receptor transcriptional activity (43, 44), we examined whether Vav3 GEF activity was required for enhancement of AR transcriptional activity. We generated two distinct Vav3 mutant proteins containing alterations in the DBL GEF domain. One GEF mutant was generated by changing three conserved

Fig. 7. Vav3 Is Not Detected in AR Immuno-Complexes PC3 cells transfected with AR or empty vector and either Vav3 or E6-AP were treated with vehicle or 5 nM R1881 for 24 h. Cell lysates were immunoprecipitated (IP) for AR. Immune complexes were subjected to SDS-PAGE and immunoblotted (IB) for Vav3, AR, and E6-AP. Right panel, 10% of lysate input was immunoblotted with the indicated antibodies. For IgG controls, R1881-treated lysates were combined and equal volumes were immunoprecipitated with AR antibodies or IgG.

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DBL GEFs, the PH domain of Vav3 appears to be dispensable for GEF activity (10). Because recent studies suggest that the Vav PH domain may have distinct biological roles (19), we examined the contribution of the Vav3 PH domain to Vav3-mediated AR effects. We generated Vav3 mutants lacking either the entire PH domain (Vav3⌬PH) or containing a single point mutation in an invariant tryptophan (W493L) that is predicted to drastically alter PH domain structure and function (10, 19, 20) (Fig. 11A). Neither PH mutant potentiated AR transcriptional activity despite expression levels similar to the wild-type protein (Fig. 11, B and C). Thus, although several domains of Vav3 are not required for enhancement of AR transcriptional activity, the PH domain is indispensable.

DISCUSSION

Fig. 8. The AF1 Region of AR Is Required for Vav3-Mediated Enhancement of AR Transcriptional Activity PC3 cells were transfected with wild-type AR or AR containing a deletion of the AF1 region (⌬ AF1) (A) or AF-2 (B), the PSA-Luc reporter, Vav3, or empty vector and ␤-galactosidase. Cells were treated with either vehicle or 5 nM R1881. Samples were assayed for luciferase activity as described. Means of triplicate samples are plotted as relative luciferase activity/␤-galactosidase ⫾ SEM. Significance was determined using a two-tailed Student’s t test and represents differences between R1881-treated vector and Vav3-transfected samples.

leucines (336–338) to glutamine, isoleucine, and phenylalanine (Vav3 QIF). Mutation of these conserved amino acids eradicates GEF activity of the closely related Vav2 protein (45). We constructed another GEF mutant termed VAV3 ISOIII in which residues 336–342 were changed from LLLQELV to IIIQDAA. This mutation eliminates Vav3 GEF activity (46). Mutations within the DBL homology domain of Vav3 had no significant effect on the ability of Vav3 to enhance AR transcriptional activity (Fig. 10B), showing that the actions of Vav3 on AR are GEF independent. Mutants were expressed at levels similar to the wild-type protein (Fig. 10C). The Vav3 PH Domain Is Essential for Vav3 Effects on AR Transcriptional Activity The DH/PH tandem organization is invariant in all DBL GEFs including Vav proteins. However, unlike other

The mechanisms involved in the progression of prostate cancer to androgen independence are unresolved. However, it is increasingly apparent that AR signaling is intact in androgen-independent disease, and more importantly may be required for progression to this state. There is also evidence that in men with recurrent androgen-independent prostate cancer, androgen levels may reach the nanomolar range in regions of the prostate that surround tumors (28, 29). These levels of androgens are sufficient to activate a sensitized or overexpressed AR (24). In fact, heightened androgen sensitivity may permit survival and growth of prostate cancer cells in patients undergoing androgen deprivation therapy. Gregory et al. (24) found that AR protein was stabilized and exhibited increased sensitivity to androgen in androgen-independent derivatives of both LNCaP and CWR22 cells. These androgen-independent cells required four orders of magnitude lower (femtomolar range) levels of dihydrotestosterone for growth than that required for androgen-dependent cells (47). Our findings support a role for Vav3 in this regard because introduction of Vav3 resulted in enhanced AR transactivation at subnanomolar concentrations of androgen. We show that expression of the Rho family GEF, Vav3, was increased after long-term androgen deprivation of LNCaP and LAPC-4 cells. The mechanisms responsible for up-regulation of Vav3 levels under conditions of androgen withdrawal are unknown. However, it is unlikely that the AR directly downregulates levels of Vav3 mRNA because these changes appear to require prolonged androgen deprivation as shown in LNCaP and LAPC-4 cells. In addition, we did not detect androgen-mediated downregulation of Vav3 mRNA in LNCaP-R1 cells, nor did we observe increases in Vav3 mRNA after androgen deprivation of LNCaP cells over a period of 0–5 d (data not shown). Vav3 potentiation of AR transcriptional activity does not occur through up-regulation of AR levels. Further-

Lyons and Burnstein • Vav3 Potentiates AR Transcriptional Activity

Fig. 9. Enhancement of AR Transcriptional Activity by Vav3 Does Not Involve Signaling through the PI3 Kinase Pathway A, PC3 cells were transfected with Vav3 or empty vector, the PSA-Luc reporter, AR, and ␤-galactosidase. Cells were treated with either vehicle or LY294002 (LY) and vehicle or 5 nM R1881. PSA-Luc activity was determined. Data from three triplicate experiments are plotted as fold induction ⫾ SEM. B, PC3 cells were transfected with Vav3 or empty vector, the AR AKT phosphorylation site mutants AR S790A (top panel), or AR S213A (bottom panel), the PSA-Luc reporter, and ␤galactosidase. Cells were treated with either vehicle or 5 nM R1881. Samples were assayed for luciferase activity. Means of triplicate samples are plotted as relative luciferase activity/ B-galactosidase ⫾ SEM. Significance was determined using a two-tailed Student’s t test and represents differences between R1881-treated vector and Vav3 transfected samples.

more, we did not detect interaction of Vav3, a cytoplasmic protein (10, 48), with AR suggesting that the mechanism by which Vav3 enhances AR transcriptional activity is distinct from the well-characterized AR coactivators such as the p160 (Src1) and ARA families of coactivators. The action of Vav3 on AR likely involves cross talk with other signaling pathways. Although not well understood, many mechanisms of AR

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cross talk have been proposed to result in continued AR signaling under conditions of androgen deprivation. Numerous studies have highlighted the role of growth factor receptor pathways such as EGF, IGF, and keratinocyte growth factor and the cytokine IL-6 in the activation of AR under conditions of low androgen concentrations (11, 49, 50). Vav proteins are recognized primarily for their GEF activity, especially in hematopoietic cells, mediating B and T cell signaling cascades. Our results indicate that the effects of Vav3 on AR do not require GEF activity of Vav3. Mutations within the DBL homology domain did not affect Vav3 potentiation of AR activity. Furthermore, a Vav3 mutant (Vav3⌬C) lacking the entire Cterminal cassette (SH3, SH2, SH3) retains the ability to enhance AR transcriptional activity to the same degree as wild-type Vav3. This mutant is not expected to possess GEF activity because the absence of the SH2 domain should not allow recruitment to and activation of Vav3 by membrane-associated growth factor receptors. Although GEF-independent activities of Vav1 and Vav2 have been described (45, 51), GEF-independent actions of Vav3 have not been reported. Our findings showing a GEF-independent role for Vav3 in the modulation of steroid hormone responsiveness are novel. Rubino et al. (44) found that Brx, a Dbl family GEF, coactivates estrogen receptors; however, these effects of Brx require GEF activity. Several studies implicate the PI3K/Akt pathway in the activation of AR through mechanisms that involve the phosphorylation of AR by the downstream PI3K target Akt (38, 41, 52). These reports prompted us to examine the possibility that Vav3 signaling through PI3K may underlie Vav3-mediated enhancement of AR activity. However, we found no diminution in the ability of Vav3 to enhance AR transcriptional activity in the presence of the PI3K inhibitor LY294002. Similarly, transcriptional activity of both AR Akt phosphorylation site mutants (AR S790A and AR S213A) was enhanced by Vav3. However, Vav3 stimulation of AR S790A transcriptional activity was somewhat compromised compared with wild-type AR. In addition, Vav3 activation of PI3 kinase requires Rho GTPase signaling (18), yet we show that the Vav3 effects on AR are GEF independent. Together, these data suggest that the PI3 kinase pathway is not involved in the enhancement of AR activity by Vav3. Our studies using Vav3 ⌬PH and the Vav3 PH domain site mutant (Vav3 W493L) show that the PH domain of Vav3 is necessary for the actions of Vav3 on AR. PH domains classically have two roles in DBL family proteins: membrane targeting and modulation of GEF activity (19). Although the PH domain of many DBL family GEFs is essential for GEF activity, the role of this domain for Vav proteins is unclear because an intact PH domain was found to be dispensable for the catalytic activity of the DH domains of Vav2 and Vav3 (10, 19). Nevertheless, the PH domain is important for biological actions of Vav2 such as transformation and membrane association. Thus, the Vav2 PH domain

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Fig. 10. Vav3 Enhancement of AR Transcriptional Activity Is GEF Independent and Does Not Require the C-Terminal Domains of Vav3 A, Schematic illustrating region of Vav3 C-terminal deletion and DBL domain (GEF) mutations. CH, Calponin homology; AD, acidic domain. B, PC3 cells were transfected with the Vav3, Vav3 GEF mutant (Vav3 QIF, or ISOII), or Vav3 C-terminal deletion mutant (Vav3 ⌬ C) plasmids, the PSA-Luc reporter, AR, and ␤-galactosidase. Cells were treated with either vehicle or 5 nM R1881. Samples were assayed for luciferase activity (summary of three to six experiments, each performed in triplicate). Data are plotted as fold induction relative to control (% control RLU/␤-gal ⫾ SEM). Solid horizontal line denotes control values and is set to 100%. C, Lysates from experiments in B were immunoblotted for Vav3 and actin (loading control). No significant differences were found using a one-way ANOVA.

may mediate cellular events that are distinct from those associated with GEF activity. Studies examining the PH domain of Vav1 have yielded contradictory results in which the Vav1 PH domain is either required, not required, or negatively regulates GEF activity (20, 53–55). Vav3 enhances ligand-dependent AR transcriptional activity, possibly through the interaction of the Vav3 PH domain with the plasma membrane or with membrane-associated proteins. The PH domain of Vav3 may mediate interactions between Vav3 and membrane-associated proteins that represent signaling modules that ultimately influence AR transcriptional activity. In support of this, Vav3 is localized to membranes in NIH3T3 cells (10), T cells (56), and PC12 cells (57). Furthermore, the PH domain of Vav2 is important for membrane localization (19). This study demonstrates that Vav3 levels increase during progression of prostate cancer cells to androgen independence. Furthermore, Vav3 participates in a novel mechanism of AR potentiation even at subnanomolar concentrations of androgen. Vav3 upregulation therefore provides a plausible basis for con-

tinued AR action in androgen-independent prostate cancer.

MATERIALS AND METHODS Cell Culture and Prostate Cancer Cell Lines The human prostate cancer cell lines LNCaP.FGC (ATCC catalog no. CRL 1740; batch F-11701) and PC-3 (ATCC catalog no. CRL 1435; batch F-11154) were obtained from American Type Culture Collection (Manassas, VA). The human prostate carcinoma cell line ALVA31 (7) was generously provided by Drs. Stephen Loop and Richard Ostensen (Department of Veteran Affairs Medical Center, Tacoma, WA). The human prostate cancer cell line LNCaP 104-R1 (3, 9) was generously provided by Drs. John Kokontis and Shutsung Liao (University of Chicago). LAPC-4 cells were generously provided by Dr. Charles Sawyers (University of California, Los Angeles, CA). Cell culture media (RPMI-1640 and DMEM) were obtained from Invitrogen Life Technologies (Gaithersburg, MD). Fetal bovine serum (FBS) was obtained from Hyclone (Logan, UT). LNCaP-R1 cells were grown in DMEM supplemented with 50 IU/ml penicillin, 50 ␮g/ml streptomycin (Invitrogen Life Technologies), and 10% charcoal-stripped

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Fig. 11. Vav3 Enhancement of AR Transcriptional Activity Requires the Vav3 PH Domain A, Schematic showing region of Vav3 PH domain mutations or deletion. B, PC3 cells were transfected with the Vav3 or Vav3 PH domain mutants (⌬ PH or W493L), the PSA-Luc reporter, AR, and ␤-galactosidase. Cells were treated with vehicle or 5 nM R1881. Samples were assayed for luciferase activity as described. Data (three to six experiments performed in triplicate) are plotted as fold induction relative to control ⫾ SEM. Significance was determined using a one way ANOVA and refers to differences in fold induction compared with wild-type Vav3-transfected samples. Solid horizontal line is set to 100% and denotes control values. C, Lysates from experiments in B were immunoblotted for Vav3 and actin (loading control).

FBS (CSS). LNCaP, ALVA31, Jurkat, and PC-3 cell lines were cultured in RPMI supplemented with 100 IU/ml penicillin, 100 ␮g/ml streptomycin, 2 mM L-glutamine (Invitrogen Life Technologies) and 10% FBS. The LAPC-4 cell line was cultured in Isocove’s media (Invitrogen Life Technologies) supplemented with 15% FBS, 10 nM dihydrotestosterone, 100 IU/ml penicillin, 100 ␮g/ml streptomycin, and 2 mM L-glutamine. Androgen deprivation of the LAPC-4 cell line was achieved by culturing the cells in Isocove’s media supplemented with 10% CSS for 3 months. Plasmids and Their Construction The human AR cDNA (pCMVhAR) was provided by Dr. Michael McPhaul (University of Texas, Southwestern Medical Center). Wild-type AR and the AR ⌬AF1, ⌬AF2 mutants and the AR AKT phosphorylation site mutants were the generous gifts of Dr. Zafar Nawaz. Vav3 pIRES2 enhanced green fluorescent protein and Vav3 pBluescript were kindly provided by Dr. Michael McClelland (Sidney Kimmell Cancer Center, San Diego, CA). The PSA Luciferase plasmid was kindly provided by Dr. Carlos Perez-Stable. All PCR-based approaches were performed using the Expand Hi Fidelity PCR system (Roche Applied Bioscience, Indianapolis, IN). For subcloning purposes, the Vav3 cDNA was inserted into pCMV HA (CLONTECH, division of BD Biosciences, Mountain View, CA) by digestion of pIRES2 EGFP Vav3 with EcoR1 and Sal1 and ligation of the Vav3 cDNA fragment into the same sites of pCMV HA to generate pCMV HA Vav3. The Vav3 QIF and ISOIII GEF mutants and all subsequent cloning using sitedirected mutagenesis was achieved using the QuikChange

site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer’s instructions. Mutagenic primers for Vav3 QIF were as follows: forward, 5⬘-GTG TTT TAA AGT ACC ACC AGA TCT TCC AGG AAC TGG TCA AAC ATA CC-3⬘; reverse, 5⬘-GGT ATG TTT GAC CAG TTC CTG GAA AAT CTG GTG GTA CTT TAA AAC AC-3⬘ and for Vav3 ISO III; forward, 5⬘-CGT GTT TTA AAG TAC CAC ATC ATC ATC CAG GAC GCG GCC AAA CAT ACC ACT GAT CCG-3⬘; reverse, 5⬘-CGG ATC AGT GGT ATG TTT GGC CGC ATC CTG GAT GAT GAT GTG GTA CTT TAA AAC ACG-3⬘. Vav3 ⌬PH was generated by site-directed mutagenesis by insertion of an Nsi 1 site at position 1209. This site was inserted immediately 5⬘ of the PH domain. The Vav3 cDNA contains a natural Nsi site at position 1598, which is at the 3⬘ end of the PH domain. The PH domain was released by digestion with Nsi1, and the vector was religated to generate Vav3 ⌬ PH and reformation of one Nsi1 site. Mutagenic primers were as follows: forward, 5⬘-CCC TTC GTG AAA TTA AAC AGT TTC AGC TAT CTA TGC ATA ATT TGA ACC AAC CAG TTT TGC TTT TTG G-3⬘; reverse, 5⬘-CCA AAA AGC AAA ACT GGT TGG TTC AAA TTA TGC ATA GAT AGC TGA AAC TGT TTA ATT TCA CGA AGG G-3⬘. The Vav3 W493L mutant was created by site-directed mutagenesis using the following mutagenic primers: forward, 5⬘-GCA AAA CAA AAG ATT TAA AGA AGA AAT TGC TAG AAC AGT TTG AAA TGG CTT TGT C-3⬘; reverse, 5⬘ GAC AAA GCC ATT TCA AAC TGT TCT AGC AAT TTC TTC TTT AAA TCT TTT GTT TTG C-3⬘. Antisense and sense Vav3 constructs were generated by PCR amplification of Vav3 nucleotides 452-1872 using the following primers: forward, 5⬘-TTCCTGATTTAATAGATGAAACCC-3⬘; reverse, 5⬘-TGTTTAGGAGTTCTTCGCAGTCC-3⬘. The amplified Vav3 product

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Lyons and Burnstein • Vav3 Potentiates AR Transcriptional Activity

was then cloned into the pAMP cloning vector (Promega Biosciences, San Luis, CA) to generate pAMP Vav3 452 ⫺1872. pAMP Vav3 was digested with HindIII and EcoR1 for the sense orientation and Kpn1 and Not1 for the antisense orientation and cloned into the same sites of pCDNA3. The Vav3 C-terminal deletion mutant lacking the entire SH3-SH2SH3 cassette (Vav3 ⌬C) was constructed with a standard PCR-based approach using the following primer pair: forward primer, 5⬘-CTT CCT GAT TTA ATA GAT GAA ACC C-3⬘; and reverse primer, GGT AAA CCT GGA TCC ACC TGT TTA GG-3⬘. The amplified product was inserted into the PGEM TA cloning vector (Promega Biosciences) and then digested with Nsi1 and Sal1. The resulting fragment was subcloned into the same sites of pCMVHA Vav3 to generate pCMV HA Vav3 ⌬ C. pCMV HA Vav3⌬C deletion construct was then digested with EcoR1 and Sal1 and the Vav3 fragment was inserted into the same sites of pIRES2 EGFP to generate pIRES 2 EGFP Vav3⌬C. All mutant cDNAs were sequenced and protein expression was analyzed by Western blotting. RNA Interference Vav3-specific siRNAs were constructed using the Silencer siRNA construction kit (Ambion Corp., Austin, TX) according to the manufacturer’s protocol. Sense and antisense strands of Vav3-specific siRNAs were generated using the following oligonucleotides. Sense, 5⬘-AAC GTG AGA AAT GTC CTT ATG CCT GTC TC-3⬘; antisense, 5⬘-AAC ATA AGG ACA TTT CTC ACG CCT GTC TC-3⬘. Scrambled controls were as follows: sense, 5⬘-AAT TTT GCC GGA TTA CGG AAA CCT GTC TC-3⬘; antisense, 5⬘-AAT TTC CGT AAT CCG GCA AAA CCT GTC TC-3⬘.

the Promega luciferase assay kit. ␤-galactosidase activity was measured as an internal control for transfection efficiency. For luciferase assays using RNAi, cells were transfected with 4 nM control or Vav3-specific RNAi. Immunoprecipitations and Western Blotting For coimmunoprecipitation, PC-3 cells were seeded in 100 mM plates as described and transfected the next day using Lipofectamine with 2 ␮g CMVhAR and 10 ␮g either E6-AP or Vav3 constructs. After transfection, cells were refed with RPMI supplemented with 3% CS-FBS and incubated overnight. Cells were then treated with either vehicle or 5 nM R1881 for 24 h. Forty-eight hours after transfection, cells were lysed in ice-cold Nonidet P-40 lysis buffer [20 mM TrisHCl (pH 7.4) 120 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, 10% glycerol, 1 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride, 10 ␮l/ml protease inhibitor cocktail]. Lysates were passed through a 26-gauge needle five times, incubated with a 50% slurry of Protein A prebound to the appropriate antibody (AR C-19 or control IgG; both from Santa Cruz Biotechnologies, Santa Cruz, CA) and then rotated for 2 h at 4 C. Beads were collected by centrifugation and washed three times with ice-cold lysis buffer. Immune complexes were collected by the addition of 30 ␮l 2⫻ sodium dodecyl sulfate sample buffer. Bound fractions were subjected to SDS-PAGE followed by Western blotting for AR (AR-C-19; Santa Cruz), E6-AP (E6-AP H-182; Santa Cruz), or Vav3 (antiVav3; Upstate, Waltham, MA), and visualized by enhanced chemiluminescence (ECL) (Amersham Pharmacia Biotech, Piscataway, NJ). RT-PCR

Cell Transfections and Reporter Gene Assays All transfections except those for RNAi experiments were carried out using the cationic lipid reagent Lipofectamine (Invitrogen Life Technologies) according to the manufacturer’s instructions. Transfections for RNAi experiments were conducted using Lipofectamine plus (Invitrogen Life Technologies) according to the manufacturer’s protocol. For immunoprecipitations, approximately 8 ⫻ 106 cells were seeded in 100-mm dishes 16–20 h before transfections. Immediately before transfection, cells were washed and refed with unsupplemented DMEM. Cells were incubated with DNA/Lipofectamine complexes for 5 h at 37 C. After incubation, plates were refed with fresh media and incubated for 48 h before experiments to allow maximal gene expression. For luciferase assays, RNAi experiments and analysis of Vav3 mutant expression, 16–20 h before transfection, cells were plated at a density of approximately 2 ⫻ 106 cells in 60-mm dishes. Immediately before transfection, media were replaced with unsupplemented DMEM. For luciferase assays, and Vav3 mutant expression studies, cells were transfected with 5 ␮g reporter PSA luc, 250 ng CMVhAR or AR mutants, 250 ng of either vector or Vav3 cDNA, and 0.5 ␮g CMV-␤-galactosidase. After a 4- to 5-h incubation with DNA/lipid complexes, plates were refed with RPM1 containing 3% CS-FBS and treated with either ethanol (vehicle) or the indicated concentrations of synthetic androgen, methyltrienolone (R1881) from DuPont-New England Nuclear (Boston, MA). Studies using the AR antagonist bicalutamide were carried out as described above with the exception that after transfection, cells were allowed to recover for 18 h. After recovery, cells were treated with bicalutamide (20 ␮M) for 15 min before the addition of 1–5 nM R1881. For experiments using the PI3 kinase inhibitor LY294002 (Calbiochem-Novabiochem, San Diego, CA), cells were treated with LY294002 or vehicle 16 h after transfection. R1881 or vehicle was added to samples 15 min after treatment with LY294002. Forty-eight hours after transfection, cells were harvested, lysed, and assessed for protein expression by Western blotting and luciferase activity using

Total RNA was harvested using the Trizol method according to the manufacturer’s protocol (Invitrogen Life Technologies). Ten micrograms of total RNA were reverse transcribed using Superscript (Invitrogen Life Technologies) reverse transcriptase. For amplification of PSA mRNA, 8 ␮l of the RT reaction were subjected to PCR using the following forward and reverse primers: forward, 5⬘-GAT GAC TCC AGC CAC GAC CT-3; reverse, 5⬘-CAC AGA CAC CCC ATC CTA TC-3⬘. These primers result in the amplification of an 800-bp fragment. To control for cDNA input in PCRs, a 420-bp fragment of ␤-actin was amplified using the following primers: forward, 5⬘-GTG GGG CGC CCC AGG CAC CA-3⬘; reverse, 5⬘-CTC CTT AAT GTC ACG CAC GAT TTC-3⬘. For experiments using semiquantitiative RT-PCR, the linear range of amplification for PSA, actin, and Vav3 mRNA was determined by removing aliquots at various cycle intervals during the PCR. Once this range was determined, experiments were performed by removing aliquots at two- to three-cycle intervals during the linear range. For experiments requiring quantification, bands were analyzed using the National Institutes of Health Image J image analysis program. Ribonuclease Protection Assays Ribonuclease protection assays were performed using the RPAIII kit according to the manufacturer’s protocol (Ambion). Vav3 radiolabeled probes for ribonuclease protection assays were generated by in vitro transcription of Vav3 cDNA cloned into pBluescript (Stratagene). For antisense probe generation, the pBluescript vector was digested with Xho1 to generate a probe of approximately 250 bp. Digestion with ribonucleases resulted in the production of a Vav3-specific protected fragment of approximately 190 bp. Glyceraldehyde-3-phosphate dehydrogenase was used as an internal loading control and unprotected and protected fragments corresponded to 200 and 110 bp, respectively. Ribonuclease protection assays were analyzed on denaturing 15% polyacrylamide gels.

Lyons and Burnstein • Vav3 Potentiates AR Transcriptional Activity

Acknowledgments We are grateful to Drs. Michael McClelland (Sidney Kimmel Cancer Center), Charles Sawyers (UCLA), Zafar Nawaz (University of Miami), and Carlos Perez-Stable (University of Miami) for generously providing reagents. We thank Dr. Daniel Gioeli for insightful comments on the manuscript, and Dr. Zafar Nawaz for helpful discussions. We also appreciate the assistance of Carol Maiorino, Yassin Flores, Zhengying Wang, Shuyun Rao, and Joanne Faysal (all from the University of Miami).

14.

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16. Received August 29, 2005. Accepted December 22, 2005. Address all correspondence and requests for reprints to: Dr. Kerry L. Burnstein, 1600 Northwest 10th Avenue, Miami, Florida 33136. E-mail: [email protected] This work was supported by National Institutes of Health Grants DK065281 and DK45478 (to K.L.B.). L.L. was supported by a predoctoral fellowship from the PhRMA Foundation. Author disclosure: K.B. and L.L. have nothing to declare.

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