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[Cancer Research 65, 9906-9913, November 1, 2005]
© 2005 American Association for Cancer Research

Cell and Tumor Biology

The Role of DOC-2/DAB2 in Modulating Androgen Receptor–Mediated Cell Growth via the Nongenomic c-Src–Mediated Pathway in Normal Prostatic Epithelium and Cancer

Jian Zhoul1, Gina Hernandez1, Szu-Wei Tu1, Chien-Ling Huang2, Ching-Ping Tseng2 and Jer-Tsong Hsieh1

1 Department of Urology, University of Texas Southwestern Medical Center, Dallas, Texas and 2 School of Medical Technology, Chang Gung University, Tao-Yuan, Taiwan, Republic of China

Requests for reprints: Jer-Tsong Hsieh, Department of Urology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9110. Phone: 214-648-3988; Fax: 214-648-8786; E-mail: JT.Hsieh{at}UTSouthwestern.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Prostate cancer is initially responsive to androgen ablation, but prostate cancer tumors invariably progress to an androgen-independent state that is ultimately lethal. The onset of the androgen-independent prostate cancer is often associated with up-regulation of the androgen receptor that can cause antagonists to exhibit agonistic activity, which could lead to the failure of androgen ablation therapy. We describe a unique protein—DOC-2/DAB2 (differentially expressed in ovarian cancer-2/disabled 2)—that antagonizes androgen receptor–mediated cell growth in prostate cancer cells via interaction with c-Src protein. This interaction causes inactivation of Erk and Akt proteins critical for proliferation and survival of prostate cancer cells. However, DOC-2/DAB2 does not change the capacity of androgen receptor to regulate the transcription of androgen-responsive reporter genes, indicating that DOC-2/DAB2 selectively inhibits androgen receptor–mediated cell growth in androgen-independent prostate cancer by disrupting the androgen receptor/c-Src complex. In normal prostatic epithelia, DOC-2/DAB2 protein levels are more abundant than androgen receptor protein levels and reduced endogenous DOC-2/DAB2 protein levels in these cells by DOC-2/DAB2 RNA interference result in enhancing androgen receptor–mediated cell growth. We conclude that DOC-2/DAB2 can modulate androgen receptor–mediated cell growth in both normal and malignant prostatic epithelial cells and the outcome of this study could evolve into a new therapeutic strategy of prostate cancer.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Primary prostate cancer is an androgen-dependent disease. Since 1990, prostate cancer has been the most common nonskin cancer in American men, surpassing even lung cancer. Because the mean age of the American population continues to increase whereas mortality from other causes continues to decrease, the incidence and mortality of prostate cancer are likely to stay at a relatively high level because prostate cancer is a typical aging-related disease. Although surgery can effectively control primary prostate cancer, androgen-independent prostate cancer can only delay by androgen ablation therapy. Overwhelming evidence indicates that androgen receptor mutation and/or aberrant expression underlie recurrent androgen-independent prostate cancer (1, 2). A recent study (3) indicates that overexpression of androgen receptor can cause antagonists to exhibit agonistic activity and also hypersensitizes androgen receptor activity in androgen-independent prostate cancer, which provides these cells a growth advantage even in an androgen-deprived milieu. Therefore, it is critical to develop a new strategy to suppress aberrant androgen receptor–mediated proliferation in androgen-independent prostate cancer cells.

We have recently reported that DOC-2/DAB2 can suppress the activity of c-Src kinase (4) that has been shown to be involved in androgen-induced cell proliferation of LNCaP cells (5–8). DOC-2/DAB2 (differentially expressed in ovarian cancer-2/disabled 2) belongs to the disabled gene and is first identified in ovarian cancers as a candidate tumor suppressor gene (9). The loss of DOC-2/DAB2 expression has also been associated with several cancers, such as breast, colon, choriocarcinoma, and prostate (10–13). Increased expression of DOC-2/DAB2 can suppress cell proliferation in ovarian cancer, choriocarcinoma, and prostate cancer (12–14). We have shown that DOC-2/DAB2 can antagonize the protein kinase C activity in prostate cancer cells by recruiting a novel Ras GTPase activation protein DAB2IP (or DIP1/2), resulting in the suppression of protein kinase C–elicited Ras activation (15, 16). The COOH-terminal domain of DOC-2/DAB2 contains three proline-rich domains known to interact with several proteins containing SH3 domains (4, 15, 17, 18). For example, DOC-2/DAB2 can prevent growth factor receptor binding protein 2 (Grb2) from binding to son of sevenless by interacting with Grb2 and inhibit c-Src activation. This interaction results in the suppression of both nerve growth factor– and epidermal growth factor (EGF)–mediated signal transduction (18). Thus, DOC-2/DAB2 seems to be a key homeostatic factor in modulating the mitogen-induced signal cascade in prostate cancer cells.

To examine the role of DOC-2/DAB2 in androgen receptor–mediated signal transduction, we observed an inverse relationship between androgen receptor and DOC-2/DAB2 expression in normal prostatic epithelia and prostate cancer. The elevated androgen receptor levels were observed in androgen-independent prostate cancer cells where the DOC-2/DAB2 levels were relatively low. Increased DOC-2/DAB2 in androgen-independent prostate cancer cells can inhibit androgen receptor–mediated cell growth in these cells. In contrast, androgen can further enhance cell proliferation if endogenous DOC-2/DAB2 protein levels are knocked down by RNA interference in normal prostatic epithelia. DOC-2/DAB2 is able to disrupt the binding of androgen receptor to c-Src protein and such interaction is critical for androgen receptor–mediated cell growth in prostatic epithelium as well as in prostate cancer cells. Taken together, DOC-2/DAB2 can modulate androgen receptor–elicited cell growth via nongenomic pathway (5, 19, 20) and down-regulation of DOC-2/DAB2 gene expression can lead to the increased androgen receptor activity in androgen-independent prostate cancer cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents and cell culture. The DOC-2/DAB2 expression vector pCI-neo-T7-p82 (T7-p82), c-Src expression vector, and hemagglutinin-tagged Erk2 expression vector have been previously described (4). The PSA gene promoter-luciferase and Probasin gene promoter-luciferase vectors were obtained from Drs. Trapman (Department of Pathology, Erasmus Medical Center, Rotterdam, the Netherlands) and Matusik (Department of Cell and Developmental Biology, Vanderbilt University, Nashville, TN), respectively (21, 22). Both the RNA interference expression vectors for DOC-2/DAB2 (2112) and its control vector (U6) were described in a previous study (23, 24). LNCaP, C4-2, p59-23 clone (13), and COS cells were maintained in T medium containing 5% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA). PrEC1, PrEC2, PrEC3, SWNPC2, SWPC1, SWPC3, and PZ-HPV7 cells (25) were cultured in PrEGM medium (Cambrex BioScience, Walkersville, Inc.). LAPC4, MDAPCa2a, and MDAPCa2b cells were maintained in different media as described in original publications (26). Antibodies used in this study are as follows: DOC-2/DAB2 ({alpha}DOC-2; Transduction Laboratories, Lexington, KY); androgen receptor ({alpha}AR; BD Bioscience PharMingen, San Diego, CA); phospho-Erk1/2 ({alpha}pErk; Cell Signaling, Beverley, MA); Erk2 ({alpha}Erk2; Santa Cruz, Santa Cruz, CA); phopho-Src Tyr416 ({alpha}pSrc416; Upstate, Charlottesville, VA); Src ({alpha}Src; Oncogene, Cambridge, MA); phospho-AKT Ser473 ({alpha}pAKT473; Cell Signaling); AKT ({alpha}AKT; Cell Signaling); and actin ({alpha}Actin; Sigma, St. Louis, MO).

Cell growth assay. Cells were plated in 96-well plates (103 per well) with T medium containing either 0.5% charcoal-stripped FBS (for C4-2 or p59-23) or PrEGM (for PZ-HPV7). For C4-2 or p59-23 cells, dihydrotestosterone and/or PP2 were added into cells 48 hours after plating and medium was changed every 48 hours. For PZ-HPV7 cells, RNA interference expression vector (2112 or U6) was transfected into cells using LipofectAMINE 2000 (Invitrogen) according to the instructions of the manufacturer, with a total amount of DNA of 1 µg/well. Twenty-four hours after transfection, cells were trypsinized and plated for another 24 hours; then, dihydrotestosterone was added and medium was changed every 48 hours. At the indicated time, total cell number was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Roche, Indianapolis, IN).

Transfection and luciferase assay. Cells (5 x 105 cells/well) were plated in a six-well plate (Costar, Acton, MA) with 70% to 80% confluence before transfection. The transfection was carried out using LipofectAMINE PLUS (Invitrogen) according to the instructions of the manufacturer with total amount of DNA of 1 µg/well. The luciferase assay was carried out as previously described (18). The fold of induction was calculated as follows: {treatment [luciferase activity (treatment – background)] / [ß-gal activity (treatment – background)]} / {ethanol control [luciferase activity (control – background)] / [ß-gal activity (control – background)]}.

Coimmunoprecipitation, pull-down assay, and Western blot analysis. LNCaP cells were cotransfected with both hemagglutinin-tagged Erk2 expression plasmid and DOC-2/DAB2 expression plasmid (or the control plasmid) for 24 hours, then treated with either 10 nmol/L R1881 or ethanol. At the indicated time, cell lysate was collected in 0.5 mL of lysis buffer. After a low-speed spin, 0.4 mL of supernatant was incubated with 20 µL of {alpha}-hemagglutinin antibody–conjugated Sepharose (Convance, Richmond, CA) overnight at 4°C. The pellet was washed twice with lysis buffer and dissolved in the sample buffer, then subjected to a 10% SDS-PAGE and probed with primary antibody. Data from Western blot analysis was visualized with an enhanced chemiluminescence detection kit (Amersham, Piscataway, NJ).

COS cells were cultured in 60 mm dish using T medium containing 5% charcoal-striped FBS (Highclone, Logan, UT). Forty-eight hours after transfection, cells were treated with R1881 or ethanol for 5 minutes. The cells were collected and subjected to a pull-down assay as described before (4).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Androgen-induced cell proliferation of prostate cancer modulated by Src or DOC-2/DAB2. In prostate cancer, androgen is a potent mitogen for androgen receptor–positive cell such as LNCaP (Fig. 1A). In the presence of c-Src–specific inhibitor PP1, a significant reduction in the cell proliferation of LNCaP was detected (Fig. 1A). On the other hand, PP3, an inactive c-Src inhibitor, did not alter the cell proliferation of LNCaP (Fig. 1A), indicating that the role of c-Src is involved in androgen receptor–mediated cell growth of LNCaP cells.



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  		Figure 1. Inhibitory effect of DOC-2/DAB2 on androgen-induced cell proliferation of prostate cancer. Cells were incubated with various amounts of dihydrotestosterone, and the cell proliferation was determined by MTT assay. A, the growth of LNCaP cells in the presence of either a c-Src inhibitor PP1 (10 µmol/L) or PP3 (10 µmol/L) determined 5 days after dihydrotestosterone treatment. B, the growth of C4-2 cells treated with PP2 (10 µmol/L) or PP3 (10 µmol/L) and transfected with DOC-2/DAB2 expression vector (p59-23) determined 5 days after dihydrotestosterone treatment. All the experiments were done in quadruplicates and repeated at least twice. Columns, mean from four independent experiments; bars, SD.

 
To examine the role of DOC-2/DAB2 in regulating androgen-induced cell proliferation of prostate cancer cells, C4-2 cell (a LNCaP subline) and its DOC-2/DAB2 transfectant (i.e., p59-23; ref. 13) were treated with dihydrotestosterone. As shown in Fig. 1B, dihydrotestosterone increased cell proliferation of C4-2 cells, which could be blocked by a c-Src–specific inhibitor-PP1, indicating the role of c-Src in dihydrotestosterone-mediated cell proliferation. In contrast, p59-23 cells, a stable DOC-2/DAB2–transfected C4-2 subline (13), failed to respond to dihydrotestosterone stimulation, suggesting the inhibitory role of DOC-2/DAB2 in modulating the mitogenic effect of androgen.

The inhibition of androgen-elicited c-Src activation by DOC-2/DAB2. Although androgen receptor is a typical transcription factor, the androgen receptor activity can be regulated by nongenomic pathway via protein-to-protein interaction (5, 19, 20). It is known that androgen receptor can activate c-Src and Erk protein phosphorylation in a ligand-dependent manner by interacting c-Src protein with its proline-rich domain (5). Because DOC-2/DAB2 is able to bind to c-Src (4), there may be a competition between DOC-2/DAB2 and androgen receptor for the c-Src binding. In LNCaP cells with undetectable endogenous DOC-2/DAB2 protein, a rapid activation of c-Src measured by its Tyr416 phosphorylation (pSrcY416) could be detected 2 minutes shortly after androgen treatment (Fig. 2A). The increased expression of DOC-2/DAB2 in these cells resulted in a dramatic suppression of pSrcY416 level under the same condition. Also, the activation of Akt via the phosphorylation at Ser473 (pAktS473) is also found in androgen receptor–mediated signaling (8). As shown in Fig. 2A, the increased DOC-2/DAB2 levels in LNCaP cells could block pAktS473 levels. Consistently, an elevated Erk phosphorylation, a downstream c-Src–elicited signal pathway, peaked 2 minutes after the androgen treatment, which was also inhibited by DOC-2/DAB2 expression in LNCaP cells (Fig. 2B).



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  		Figure 2. Inhibitory effect of DOC-2/DAB2 on androgen-induced activation of c-Src, Akt, and Erk2. After incubating with 10 nmol/L R1881, cell lysate was subjected to Western blot analysis (A) or coimmunoprecipitation (B). A, inactivation of c-Src and Akt phosphorylation in LNCaP cells by DOC-2/DAB2 protein. B, inactivation of Erk2 phosphorylation in LNCaP cells by DOC-2/DAB2 protein. The fold of induction was calculated based on the ratio between the phosphoprotein and the total protein. Each data point was averaged from three independent experiments. Columns, mean; bars, SD.

 
Enhanced androgen-mediated cell growth in normal prostatic epithelial cells transfected DOC-2/DAB2 RNA interference expression vector. We profiled the steady-state levels of both androgen receptor and DOC-2/DAB2 protein expression from four primary cultures (PrEC1, PrEC2, PrEC3, and SWNPC2) and one immortalized cell (PZ-HPV7; ref. 25) derived from normal prostate, two primary cultures derived from primary prostate cancer (SWPC1 and SWPC3), and five cell lines derived from androgen-independent prostate cancer (LNCaP, C4-2, LAPC4, MDAPCa2a, and MDAPCa2b). Noticeably, the expression patterns of both genes exhibited an inverse correlation in all cell lines tested; the normal cells expressed higher DOC-2/DAB2 than androgen receptor and androgen-independent prostate cancer had the opposite pattern (Fig. 3A).



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  		Figure 3. Enhanced androgen-mediated cell proliferation and increased Src activation in PZ-HPV7 cells transfected with DOC2/DAB2 RNA interference expression vector. Cells were transfected with RNA interference expression vector specific to human DOC-2/DAB2 (2112) or its control vector (U6) and incubated with various amounts of dihydrotestosterone. A, the steady-state levels of DOC-2/DAB2 and androgen receptor proteins in each cell were determined by Western analysis. B, the time course of the dihydrotestosterone-induced growth of PZ-HPV7 cells with reduced endogenous DOC-2/DAB2 protein expression was determined by MTT assay. C, the inactivation of c-Src in PZ-HPV7 cells transfected with DOC-2/DAB2 RNA interference. The intensity of each band was determined by densitometry. The fold of induction was calculated based on the ratio between the phosphoprotein and the total protein. Each data point was averaged from three independent experiments. Columns, mean; bars, SD.

 
Because androgen receptor and DOC-2/DAB2 were detected in PZ-HPV7 (Fig. 3A), the growth of these cells could also be induced by dihydrotestosterone in a dose-dependent manner (Fig. 3B). To determine whether decreased endogenous DOC-2/DAB2 can further enhance the dihydrotestosterone-induced cell growth of this cell, the RNA interference expression vector, DOC-2/DAB2 (2112), or its control (U6) was transfected into PZ-HPV7 cells. In the presence of 2112, decreased endogenous DOC-2/DAB2 protein levels were detected in cells 4 days after transfection (Fig. 3C). Under the same condition, dihydrotestosterone-mediated growth of PZ-HPV7 cells was significantly enhanced compared with that of cells transfected with control vector (Fig. 3B). Concurrently, in the presence of DOC-2/DAB2 RNA interference, the pSrcY416 levels were elevated in PZ-HPV-7 treated with androgen (Fig. 3C). These data support the notion that DOC-2/DAB2 is a key modulator in androgen receptor–elicited signal transduction mediated by Src activation. These data provide further evidence that the steady-state levels of endogenous DOC-2/DAB2 in normal prostatic epithelium is critical for modulating androgen-mediated cell proliferation in these cells.

The competition between androgen receptor and DOC-2/DAB2 for the binding to c-Src protein. To delineate the mechanism of action of DOC-2/DAB2, we examined whether DOC-2/DAB2 could compete with the binding between androgen receptor and c-Src protein. As shown in Fig. 4A, an increased DOC-2/DAB2 protein expression could disrupt the binding between androgen receptor and c-Src protein in cells with or without dihydrotestosterone stimulation. In contrast, an increased androgen receptor protein expression could also block the binding between DOC-2/DAB2 and c-Src protein (Fig. 4B). These data indicate that the presence of DOC-2/DAB2 protein can modulate the interaction between androgen receptor and c-Src proteins and there is a competition between DOC-2/DAB2 and androgen receptor for the interaction with c-Src protein.



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  		Figure 4. Competition between DOC-2/DAB2 and androgen receptor for binding to c-Src proteins. COS (A, B) or LNCaP (C) cells were transfected with increased amounts of either androgen receptor (A) or DOC-2/DAB2 (B, C) expression vector. In every experiment, the same total amount of DNA (2 µg/well) in each transfection was applied by supplementing with a control vector (pCI-neo). After incubating with R1881 (10 nmol/L) for 5 minutes, cell lysates were subjected to a pull-down assay (A, B) done by incubating an equal amount of cell lysate with GST-SH3 domain of c-Src or an immunoprecipitation (C) by androgen receptor antibody then immunoblotted with DOC-2/DAB2 antibody. The lysate was then probed with antibodies against androgen receptor or DOC-2/DAB2. The experiments have been repeated thrice; these represent typical data.

 
Furthermore, using coimmunoprecipitation assay, we observed that androgen was able to promote the interaction between androgen receptor and pSrcY416 in LNCaP cells (Fig. 4C). On the other hand, an elevated DOC-2/DAB2 expression in LNCaP cells could disrupt such interaction (Fig. 4C). Thus, DOC-2/DAB2 is able to modulate the complex of androgen receptor and c-Src protein in prostate cancer cells.

The effect of DOC-2/DAB2 on the transcription activity of androgen receptor. Androgen was first known as a typical transcription factor, binding directly to the specific cis-element (i.e., androgen responsive element) associated with the androgen-responsive gene(s). The effect of DOC-2/DAB2 on the transactivation activity of androgen receptor was largely unknown. We used reporter gene constructs containing the promoter region from two well-characterized androgen-responsive genes, prostate-specific antigen (PSA; ref. 21), and Probasin (22). The PSA promoter activity was studied in LNCaP cells after transfecting with the DOC-2/DAB2 expression vector. The PSA promoter was stimulated upon treatment with androgen, which could be inhibited by an antiandrogen flutamide (Fig. 5A). However, an increased amount of DOC-2/DAB2 expression vector in LNCaP cells failed to suppress the androgen-induced PSA promoter (Fig. 5A), indicating that DOC-2/DAB2 has no effect on the classic androgen action as a transcription factor. In addition, the Probasin promoter activity, which can be induced in LNCaP cells by androgen, also showed no change even when an increased amount of DOC-2/DAB2 vector was transfected into cells (Fig. 5B). We further compared the expression levels of 96 known androgen-regulated genes in C4-2 cells with or without DOC-2/DAB2 using cDNA array and real-time reverse transcription-PCR (RT-PCR). No obvious genes were found in the stable DOC-2/DAB2–transfected cells compared with control cell treated with androgen (see Supplementary Data). These data suggest that DOC-2/DAB2 may not influence the transactivation function of androgen receptor.



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  		Figure 5. Effect of DOC-2/DAB2 on androgen receptor–mediated gene transcription. The PSA (A) or Probasin (B) promoter-driven luciferase reporter gene construct was cotransfected with both increasing amounts of DOC-2/DAB2 expression plasmid and a constant amount of ß-Gal expression plasmid into LNCaP cells. Two days after transfection, cells were treated with 10 nmol/L R1881 for 16 hours. Luciferase activities were determined after normalizing with ß-gal activity. Columns, mean from three independent experiments; bars, SD.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, a dramatic difference in DOC-2/DAB2 expression is observed among prostatic epithelial cells derived from normal tissue, primary tumor, and androgen-independent prostate cancer cells (Fig. 3A). In normal prostatic epithelia and primary tumor, it seems that the higher levels of DOC-2/DAB2 are critical for preventing overgrowth of normal cell stimulated by androgen (Fig. 3B). Any changes in the expression pattern between DOC-2/DAB2 and androgen receptor, such as decreased DOC-2/DAB2 levels, could augment the androgen-induced cell proliferation observed in normal prostatic epithelia (Fig. 3B). Once the alteration becomes permanent, as seen in androgen-independent prostate cancer cells, the reduced DOC-2/DAB2 and increased androgen receptor could give a growth advantage to these cells. At present, the underlying mechanism(s) leading to such alteration is still unclear. Nevertheless, data from our previous study (13) show that DOC-2/DAB2 gene expression can be suppressed by androgen in rat ventral prostate. In addition, our preliminary data suggest that decreased DOC-2/DAB2 expression can be controlled by epigenetic mechanisms such as histone acetylation. Considering androgen receptor as a transcription factor, it is known that several androgen receptor coregulators can modify androgen receptor–elicited gene expression (27–29). Further investigation for the role of androgen receptor on DOC-2/DAB2 gene regulation is warranted.

DOC-2/DAB2 is known as a potent growth inhibitor in prostate cancer cells (13). To understand the potential impact of DOC-2/DAB2 on androgen receptor–mediated function in prostate cancer, we unveiled that DOC-2/DAB2 could suppress the androgen-induced cell proliferation of prostate cancer via the c-Src–mediated pathway (Fig. 2). In prostate cancer, c-Src signal transduction can increase either cell growth or migration capacity of these cells (5, 6, 30). The constitutive activation of c-Src has also been reported in high-grade prostate cancer (31). DOC-2/DAB2 is often absent in metastatic prostate cancer cell lines and increased DOC-2/DAB2 expression is able to suppress the androgen-induced c-Src activation (Fig. 3) and the downstream kinases, including Erk and AKT activation. This effect can be observed in prostate cancer cells only minutes after androgen stimulation, which is characterized as the nongenomic pathway (5, 19, 20). Also, DOC-2/DAB2 does not influence the transcription activity of androgen receptor (Fig. 5). We believe that DOC-2/DAB2 is a key modulator for androgen receptor–mediated nongenomic pathway (Fig. 6) critical for cell proliferation of prostatic epithelia and prostate cancer. We have shown that DOC-2/DAB2 can bind to c-Src via the proline-rich domain of DOC-2/DAB2 and the SH3 domain of c-Src (4). It seems that DOC-2/DAB2 and androgen receptor are competitors for their binding to c-Src (Fig. 4). These data are consistent with our notion that the higher levels of DOC-2/DAB2 are critical to maintain normal physiologic activity of androgen receptor in normal prostatic epithelial cells. In contrast, the reduced endogenous DOC-2/DAB2 in prostatic epithelia (Fig. 3B) could signify the abnormal growth of preneoplastic cells. Thus, we believe that the loss of DOC-2/DAB2 is a key event contributing to the outgrowth of androgen-independent prostate cancer cells associated with elevated androgen receptor levels.



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  		Figure 6. The role of DOC-2/DAB2 in modulating androgen receptor activity. After the binding of androgen to androgen receptor, androgen receptor can activate the c-Src illustrated by the presence of nonphosphorylated Y416 via the interaction between proline-rich domain and SH3 domain, which elicited downstream Erk phosphorylation leading to cell growth. In addition, androgen receptor can initiate gene transcription via binding to the specific cis-acting element of many genes. The presence of DOC-2/DAB2 can disrupt androgen receptor/c-Src complex that leads to the suppression of c-Src phosphorylation and its downstream signal transduction. However, DOC-2/DAB2 does not impact on androgen receptor–mediated gene transcription. Rectangle, inactive form; circle, active form; Y, nonphosphorylated tyrosine; *Y, phosphorylated tyrosine.

 
As a transcription factor, androgen receptor can bind to a specific cis-element on the promoter region of PSA and Probasin gene (21, 22). Interestingly, the increased expression of DOC-2/DAB2 in LNCaP cells did not have any effect on PSA or Probasin promoters stimulated by androgen (Fig. 5). Similar results were also found in C4-2 and DOC-2/DAB2–transfected (p59-23) cells, as detected by cDNA microarray or quantitative RT-PCR (data not shown). Unlike any known antiandrogens, DOC-2/DAB2 seems to have a differential effect on androgen receptor activity (i.e., it blocks the nongenomic pathway of androgen receptor, but not the genomic pathway; Fig. 6). Androgen is required for growth, cell survival, and differentiation of prostatic epithelial cells; however, the underlying signal pathways for these processes are largely undefined. Based on these results, DOC-2/DAB2 could be a potential tool to analyze the functional role of the androgen receptor–mediated genomic pathway during prostate development.

Data from a genome-wide survey of RAS-transformed cells indicate that DOC-2/DAB2 is one of the most significant suppressed genes in these cells (32). In prostate cancer, studies have shown that most metastatic tumors expressed Ras protein, whereas only a fifth of the primary tumors tested did (33–35). These results suggest a link between the down-regulation of DOC-2/DAB2 and Ras protein status in androgen-independent prostate cancer cells. Using NIH3T3 cells, Hocevar et al. (36) have shown that DOC-2/DAB2 also functions as a negative regulator for Wnt signaling by stabilizing ß-catenin degradation. We have also documented that DOC-2/DAB2 can modulate protein kinase C– or peptide growth factor receptor–mediated signal pathways elicited by many exogenous stimuli such as 12-O-tetradecanoylphorbol-13-acetate and EGF in prostate cancer cells (4, 18, 37). This study further unveils the importance of DOC-2/DAB2 as part of a regulatory complex in maintaining the homeostasis of signal transduction of prostatic epithelial cells and suggests that the ratio between DOC-2/DAB2 and androgen receptor mRNA levels could be a potent prognostic indicator during the onset of androgen-independent prostate cancer.


   Acknowledgments
 
Grant support: United States Army grant DAMD17-03-2-0033 and DK63661.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Drs. Claus Roehrborn and Kenneth S. Koeneman for reading the manuscript and helpful discussion, and Ryan Roark for editing the manuscript.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Received 4/28/05. Revised 7/13/05. Accepted 8/16/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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