Transcription of Nrdp1 by the androgen receptor is regulated by nuclear filamin A in prostate cancer - PubMed (original) (raw)

doi: 10.1530/ERC-15-0021. Epub 2015 Mar 10.

Liqun Chen 2, Salma Siddiqui 2, Frank U Melgoza 2, Blythe Durbin-Johnson 2, Christiana Drake 2, Maitreyee K Jathal 1, Swagata Bose 1, Thomas M Steele 2, Benjamin A Mooso 2, Leandro S D'Abronzo 1, William H Fry 2, Kermit L Carraway 3rd 2, Maria Mudryj 1, Paramita M Ghosh 3

Affiliations

Transcription of Nrdp1 by the androgen receptor is regulated by nuclear filamin A in prostate cancer

Rosalinda M Savoy et al. Endocr Relat Cancer. 2015 Jun.

Abstract

Prostate cancer (PCa) progression is regulated by the androgen receptor (AR); however, patients undergoing androgen-deprivation therapy (ADT) for disseminated PCa eventually develop castration-resistant PCa (CRPC). Results of previous studies indicated that AR, a transcription factor, occupies distinct genomic loci in CRPC compared with hormone-naïve PCa; however, the cause of this distinction was unknown. The E3 ubiquitin ligase Nrdp1 is a model AR target modulated by androgens in hormone-naïve PCa but not in CRPC. Using Nrdp1, we investigated how AR switches transcription programs during CRPC progression. The proximal Nrdp1 promoter contains an androgen response element (ARE); we demonstrated AR binding to this ARE in androgen-sensitive PCa. Analysis of hormone-naive human prostatectomy specimens revealed correlation between Nrdp1 and AR expression, supporting AR regulation of NRDP1 levels in androgen-sensitive tissue. However, despite sustained AR levels, AR binding to the Nrdp1 promoter and Nrdp1 expression were suppressed in CRPC. Elucidation of the suppression mechanism demonstrated correlation of NRDP1 levels with nuclear localization of the scaffolding protein filamin A (FLNA) which, as we previously showed, is itself repressed following ADT in many CRPC tumors. Restoration of nuclear FLNA in CRPC stimulated AR binding to Nrdp1 ARE, increased its transcription, and augmented NRDP1 protein expression and responsiveness to ADT, indicating that nuclear FLNA controls AR-mediated androgen-sensitive Nrdp1 transcription. Expression of other AR-regulated genes lost in CRPC was also re-established by nuclear FLNA. Thus, our results indicate that nuclear FLNA promotes androgen-dependent AR-regulated transcription in PCa, while loss of nuclear FLNA in CRPC alters the AR-regulated transcription program.

Keywords: ABP280/filamin A; AR/androgen receptor; FLRF/RNF41/Nrdp1; HER3/ErbB3; castration-resistant prostate cancer.

© 2015 Society for Endocrinology.

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Conflict of interest statement

CONFLICT OF INTEREST

The authors declare no conflict of interest. The work reported here does not represent the views or opinions of the Department of Veteran Affairs or the United States Government.

Figures

Figure 1

Figure 1. Nrdp1 is a transcriptional target of the AR in androgen-dependent LNCaP cells

(A) LNCaP cells were cultured in complete media containing FBS or CSS with and without DHT (1 nM) for 72 hours. Cell lysates were blotted for Nrdp1 and two isoforms detected – 36 kDa and 28 kDa. Exposure to CSS caused a decrease in both 36 kDa and 28 kDa Nrdp1 but this effect was restored by DHT, showing that Nrdp1 is androgen regulated. (B) We identified an ARE in the proximal promoter of Nrdp1 gene. Comparison of PSA AREs and Nrdp1 ARE show that the PSA AREs and Nrdp1 ARE3 both contain a 15bp-palindromic ARE. (C) ChIP assay of AR binding in LNCaP cells to Nrdp1 ARE3. LNCaP cells were cultured in media containing CSS for 48 hours and then switched to complete media containing FBS or CSS with or without DHT. Chromatin samples were immunoprecipitated with an anti-AR antibody and analyzed by qPCR with primers flanking the Nrdp1 ARE3 region against a negative control (ARarfneg2). In LNCaP cells grown in normal FBS media the AR binds to the Nrdp1 ARE3 sequence, this binding no longer occurs in CSS media, but is restored in CSS media with the addition of DHT. In contrast the negative control is unaffected by these manipulations. (D) LNCaP cells were transfected with plasmids expressing luciferase under control of wild-type Nrdp1 ARE3 promoter, mutant Nrdp1 ARE3 promoter (as shown), or with the parental vector (pGL4), and assayed for luciferase activity in the presence of DMSO vehicle, or 1nM DHT, with or without 10µM bicalutamide (Casodex). The luciferase activity of Nrdp1 ARE3 in LNCaP cells was responsive to androgens but was abolished by the mutations.

Figure 2

Figure 2. Nrdp1 is transcribed by both wild-type and mutated AR in a normal prostate-derived cell line

(A) Parental pRNS-1-1 cells derived from a normal prostate were stably transfected with an empty vector, wild-type AR (WT-AR), or mutant AR(T877A). qPCR to determine AR expression in parental pRNS1-1 cells, or those stably expressing AR expression goes up 120 fold in PRNS1-1 WT-AR cells (p ≤ 0.0001) and 53 fold in PRNS1-1 AR T877A (p ≤ 0.0001) compared to PRNS1-1 Parental cells. AR transcript levels were normalized to the corresponding values for β-Actin. (B) qPCR for Nrdp1 expression in parental pRNS1-1 cells, or those stably expressing WT-AR, or AR(T877A). Nrdp1 expression goes up 2.3 fold in pRNS1-1 WT-AR cells (p=0.0004) and 1.6 fold in pRNS1-1 AR T877A (p=0.0062) compared to parental pRNS1-1 cells. Nrdp1 transcript levels were normalized to the corresponding values for β-actin. (C) pRNS1-1 AR T877A cells were transfected with plasmids expressing luciferase under control of the Nrdp1 ARE3 promoter or with the parental vector (pGL4), and assayed for luciferase activity in the presence of DMSO (vehicle), 1 nM DHT, or 10µM bicalutamide (Casodex), and shows responsiveness to androgens. (D) Immunofluorescence of parental pRNS1-1 cells, or those stably expressing WT-AR, or AR(T877A). Cells were stained for Nrdp1 (green) or DAPI (blue) to demonstrate the location of the nucleus. (top) Note that while parental pRNS1-1 express very little Nrdp1, (middle) the expression of wt-AR or (bottom) AR(T877A) increased Nrdp1 in the cytoplasm. Negative control(s) denote staining with secondary antibody alone. Bar=20µm.

Figure 3

Figure 3. Nrdp1 is highly expressed in hormone naïve localized tumors from patients with PCa and correlate with intratumoral AR

(A) Formalin fixed paraffin-embedded human localized prostate cancer specimens obtained by prostatectomy were arranged in a tissue microarray and stained with anti-Nrdp1 antibody. Nrdp1 expression (brown staining) was observed in the nucleus, cytoplasm or both and was scored on a 0 to 3 scale in both benign and cancerous prostate tissues. Shown are typical staining from a benign section (score 1) (left) and a section showing Gleason grade 3 tumor (score 3) (right) bar=20µm. (B) Boxplot depicting the distribution of Nrdp1 in the nucleus or cytoplasm of tumor compared to non-tumor tissue (n=78) demonstrate that the expression of nuclear Nrdp1 remains the same in both cancer and non-tumor tissues, whereas cytoplasmic expression of Nrdp1 increases in tumor compared to non-tumor tissue. (C) Nuclear Nrdp1 levels differed significantly by clinical stage (p < 0.001), with post-hoc testing showing significantly higher expression in Stage 2 (T2) and Stage 3 (T3) patients than in Stage 1 (T1) patients (p = 0.001). (D) (left) Boxplots showing the correlation between Nrdp1 and AR in tumor tissue (p<0.001). (right) IHC of AR and Nrdp1 in two patients with high and low AR vs Nrdp1. Note that the patient who expressed little AR also expressed little Nrdp1, whereas strong AR staining correlated with strong Nrdp1 expression. Bar=50µm.

Figure 3

Figure 3. Nrdp1 is highly expressed in hormone naïve localized tumors from patients with PCa and correlate with intratumoral AR

(A) Formalin fixed paraffin-embedded human localized prostate cancer specimens obtained by prostatectomy were arranged in a tissue microarray and stained with anti-Nrdp1 antibody. Nrdp1 expression (brown staining) was observed in the nucleus, cytoplasm or both and was scored on a 0 to 3 scale in both benign and cancerous prostate tissues. Shown are typical staining from a benign section (score 1) (left) and a section showing Gleason grade 3 tumor (score 3) (right) bar=20µm. (B) Boxplot depicting the distribution of Nrdp1 in the nucleus or cytoplasm of tumor compared to non-tumor tissue (n=78) demonstrate that the expression of nuclear Nrdp1 remains the same in both cancer and non-tumor tissues, whereas cytoplasmic expression of Nrdp1 increases in tumor compared to non-tumor tissue. (C) Nuclear Nrdp1 levels differed significantly by clinical stage (p < 0.001), with post-hoc testing showing significantly higher expression in Stage 2 (T2) and Stage 3 (T3) patients than in Stage 1 (T1) patients (p = 0.001). (D) (left) Boxplots showing the correlation between Nrdp1 and AR in tumor tissue (p<0.001). (right) IHC of AR and Nrdp1 in two patients with high and low AR vs Nrdp1. Note that the patient who expressed little AR also expressed little Nrdp1, whereas strong AR staining correlated with strong Nrdp1 expression. Bar=50µm.

Figure 4

Figure 4. Loss of Nrdp1 expression and AR regulation of Nrdp1 transcription in CRPC compared to hormone naïve tumors

(A) qPCR comparing Nrdp1 expression in LNCaP cells vs those in LNCaP R273H (p=0.0036), C4-2 (p=0.0007), and C4-2B (p=0.0002). Note the decrease in Nrdp1 levels in the latter three cell lines, which are all CRPC. Nrdp1 transcript levels were normalized to the corresponding values for β-Actin. (B) Comparison of AR binding to the Nrdp1 ARE3 in LNCaP vs C4-2 cells. Note the sharp decrease in AR binding in C4-2 compared to LNCaP (p<0.0001). Chromatin samples were immunoprecipitated with an anti-AR antibody and analyzed by qPCR with primers flanking the Nrdp1 ARE3 region with Znf333 as a negative control. **(C) (Top)** Nude mice were subcutaneously implanted with either CWR22 **(right)** or CWR22Rv1 **(left)** tumor and the tumors were allowed to grow for up to 29 days; the mice were euthanized and the tumors excised when tumors>150 cm3 or at the end of that period. Tumor size was measured as described and plotted over time. The Rv1 tumors were more aggressive compared to CWR22 (p=0.003). (D) Formalin fixed paraffin-embedded tumor specimens were stained with anti-Nrdp1 antibody. Note that the cells in CWR22 stained strongly for Nrdp1 (brown), while those in CWR22Rv1 did not (bar=30µm). (Bottom) Boxplot of Nrdp1 in CWR22 (n=6) vs CWR22Rv1 (n=6) in the nucleus (N) and cytoplasm (C). Primary CWR22 tumors expressed higher levels of nuclear Nrdp1 compared to recurrent CWR22Rv1 tumors (p=0.0157). (E) Whole cell lysates of xenografted tumors were run on a Western blotted and stained with AR, PSA and ErbB3, while tubulin levels were used as loading control. Results show that despite the sharp change in Nrdp1 between the two tumor types, there was no significant difference in AR levels (except that CWR22Rv1 tumors also expressed the alternately spliced forms). However, AR transcriptional activity in the CWR22Rv1 was significantly suppressed as shown by a decrease in PSA levels. In support of the decrease in Nrdp1 in CWR22Rv1 compared to CWR22, the former expressed higher levels of the Nrdp1 degradation target ErbB3.

Figure 4

Figure 4. Loss of Nrdp1 expression and AR regulation of Nrdp1 transcription in CRPC compared to hormone naïve tumors

(A) qPCR comparing Nrdp1 expression in LNCaP cells vs those in LNCaP R273H (p=0.0036), C4-2 (p=0.0007), and C4-2B (p=0.0002). Note the decrease in Nrdp1 levels in the latter three cell lines, which are all CRPC. Nrdp1 transcript levels were normalized to the corresponding values for β-Actin. (B) Comparison of AR binding to the Nrdp1 ARE3 in LNCaP vs C4-2 cells. Note the sharp decrease in AR binding in C4-2 compared to LNCaP (p<0.0001). Chromatin samples were immunoprecipitated with an anti-AR antibody and analyzed by qPCR with primers flanking the Nrdp1 ARE3 region with Znf333 as a negative control. **(C) (Top)** Nude mice were subcutaneously implanted with either CWR22 **(right)** or CWR22Rv1 **(left)** tumor and the tumors were allowed to grow for up to 29 days; the mice were euthanized and the tumors excised when tumors>150 cm3 or at the end of that period. Tumor size was measured as described and plotted over time. The Rv1 tumors were more aggressive compared to CWR22 (p=0.003). (D) Formalin fixed paraffin-embedded tumor specimens were stained with anti-Nrdp1 antibody. Note that the cells in CWR22 stained strongly for Nrdp1 (brown), while those in CWR22Rv1 did not (bar=30µm). (Bottom) Boxplot of Nrdp1 in CWR22 (n=6) vs CWR22Rv1 (n=6) in the nucleus (N) and cytoplasm (C). Primary CWR22 tumors expressed higher levels of nuclear Nrdp1 compared to recurrent CWR22Rv1 tumors (p=0.0157). (E) Whole cell lysates of xenografted tumors were run on a Western blotted and stained with AR, PSA and ErbB3, while tubulin levels were used as loading control. Results show that despite the sharp change in Nrdp1 between the two tumor types, there was no significant difference in AR levels (except that CWR22Rv1 tumors also expressed the alternately spliced forms). However, AR transcriptional activity in the CWR22Rv1 was significantly suppressed as shown by a decrease in PSA levels. In support of the decrease in Nrdp1 in CWR22Rv1 compared to CWR22, the former expressed higher levels of the Nrdp1 degradation target ErbB3.

Figure 5

Figure 5. Correlation between Nrdp1 levels and expression of a 90kDa FlnA isoform

(A) (upper) Comparison of Nrdp1 protein levels in LNCaP and C4-2 cell lines cultured in FBS. Cell lysates were immunoblotted with anti-Nrdp1, and anti-tubulin antibodies. Note that C4-2 cells expressed lower levels of this protein compared to LNCaP. (lower) Correspondingly, the levels of 90kDa FlnA was also assessed in the presence or absence of 1nM DHT. LNCaP cells cultured in FBS showed no difference in FlnA levels with DHT, however, in C4-2 cells these levels were much lower and change with DHT was immediately obvious. (B) Protein expression of FlnA in CWR-R1 and CWR22Rv1 cells cultured in FBS. Cell lysates were immunoblotted with anti-FlnA, anti-AR, and anti-tubulin antibodies. Whole cell lysates of CWR-R1 and CWR22Rv1 cells demonstrate the decreased expression of the 90 kDa fragment of FlnA in CWR22Rv1 cells. (C) qPCR for AR and FlnA expression in CWR-R1 and CWR22-Rv1. Higher expression of FlnA was observed in CWR-R1 cells (p=0.0002), although AR expression in the two cell lines was comparable (p>0.05). AR and FlnA transcript levels were normalized to the corresponding values for β-Actin. (D) ChIP assay of AR binding to ARE3 in CWR-R1 and CWR22Rv1 cells. AR binds to Nrdp1 ARE3 in CWR22R1 cells but not CWR22Rv1 cells. Chromatin samples were immunoprecipitated with an anti-AR antibody and analyzed by qPCR with primers flanking the Nrdp1 ARE3 region (p<0.0001), and Znf333 (p>0.05) as a negative control. (E) Androgen sensitivity in CWR-R1 vs. CWR22Rv1 cells. AR transcriptional activity was tested in untransfected cells, or cells transfected cells with luciferase driven by the PSA ARE. The decrease in luciferase activity in CWR-R1 cells but not in CWR22-Rv1 cells in the presence of 10µM bicalutamide indicates that CWR-R1 cells are more androgen sensitive compared to CWR22Rv1 cells, though both are considered castration resistant (bicalutamide decreased AR transcriptional activity on the PSA promoter in CWR-R1 cells by ~38%, p<0.0001).

Figure 6

Figure 6. The 90kDa FlnA isoform localized to the nucleus and promoted apoptosis and growth arrest in a ligand dependent manner

(A) Immunofluorescence of C4-2B cells transfected with empty vector (EV), FlnA 1-15, or FlnA 16-24. Cells were stained for C-terminal FlnA or DAPI to identify the location of the nucleus. (top panel) Control cells (transfected with empty vector) only express cytoplasmic FlnA, as demonstrated by unstained nuclear regions in FlnA stained cells, (2nd panel) and transfection of FlnA 1-15 did not affect the localization, (3rd panel) while those transfected with FlnA 16-24 express both cytoplasmic and nuclear FlnA. (4th panel) Transfection of full-length FlnA did not restore nuclear localization completely. (5th panel) Negative controls were treated the same but did not use the anti-FlnA antibody (bar=30µm). (B) Subcellular fractionation of C4-2 cells transfected with empty vector, FlnA 1-15, or FlnA 16-24. Fractionated cell lysates were immunoblotted with anti-Nrdp1, anti-AR, anti-FlnA (C-terminal), anti-β-Actin (to demonstrate specificity of cytoplasmic fraction), and anti-Lamin A/C (to demonstrate specificity of nuclear fraction) and demonstrates that transfection of FlnA 16-24 caused nuclear expression of FlnA and restores Nrdp1 protein in C4-2 cells, although AR levels were not altered. (C) Protein expression of Nrdp1 in CWR-R1 cells is regulated by the 90 kDa FlnA. Whole cell lysates of CWR-R1 cells that were transfected with empty vector, full length FlnA, FlnA 1-15, or FlnA 16-24 were immunoblotted with anti-FlnA, anti-Nrdp1, and anti-tubulin antibodies. Nrdp1 protein levels increased with the increased levels of the 90 kDa FlnA fragment. (D) FlnA restores Nrdp1 expression in CRPC cells. qPCR for Nrdp1 expression in LNCaP, C4-2, and stably transfected C4-2-FlnA(16-24) showed that Nrdp1 expression was reduced in C4-2 compared to LNCaP cells (p=0.0141), but expression of FlnA16-24 in C4-2 cells restored Nrdp1 expression to a level similar to LNCaP cells (p=0.0002 compared to C4-2). Nrdp1 transcript levels were normalized to the corresponding values for β-Actin. (E) Comparison of Nrdp1 response to changes in AR in CWR-R1 and CWR22-Rv1 cells cultured in FBS, CSS or CSS treated with increasing doses of DHT as indicated. Lysates were immunoblotted with anti-Nrdp1, anti-ErbB3, and anti-tubulin antibodies. While the levels of Nrdp1 in CWR22Rv1 cells were unaltered despite culture in CSS, in CWR-R1 these levels altered slightly. (F) (left) Reporter gene activity of AR on a luciferase-tagged PSA promoter section demonstrates that in control LNCaP cells, 10 µM bicalutamide is able to suppress AR activity whereas in cells where FlnA is downregulated by siRNA, bicalutamide failed to affect AR activity. (right) Western blotting demonstrating the efficacy of FlnA siRNA used.

Figure 6

Figure 6. The 90kDa FlnA isoform localized to the nucleus and promoted apoptosis and growth arrest in a ligand dependent manner

(A) Immunofluorescence of C4-2B cells transfected with empty vector (EV), FlnA 1-15, or FlnA 16-24. Cells were stained for C-terminal FlnA or DAPI to identify the location of the nucleus. (top panel) Control cells (transfected with empty vector) only express cytoplasmic FlnA, as demonstrated by unstained nuclear regions in FlnA stained cells, (2nd panel) and transfection of FlnA 1-15 did not affect the localization, (3rd panel) while those transfected with FlnA 16-24 express both cytoplasmic and nuclear FlnA. (4th panel) Transfection of full-length FlnA did not restore nuclear localization completely. (5th panel) Negative controls were treated the same but did not use the anti-FlnA antibody (bar=30µm). (B) Subcellular fractionation of C4-2 cells transfected with empty vector, FlnA 1-15, or FlnA 16-24. Fractionated cell lysates were immunoblotted with anti-Nrdp1, anti-AR, anti-FlnA (C-terminal), anti-β-Actin (to demonstrate specificity of cytoplasmic fraction), and anti-Lamin A/C (to demonstrate specificity of nuclear fraction) and demonstrates that transfection of FlnA 16-24 caused nuclear expression of FlnA and restores Nrdp1 protein in C4-2 cells, although AR levels were not altered. (C) Protein expression of Nrdp1 in CWR-R1 cells is regulated by the 90 kDa FlnA. Whole cell lysates of CWR-R1 cells that were transfected with empty vector, full length FlnA, FlnA 1-15, or FlnA 16-24 were immunoblotted with anti-FlnA, anti-Nrdp1, and anti-tubulin antibodies. Nrdp1 protein levels increased with the increased levels of the 90 kDa FlnA fragment. (D) FlnA restores Nrdp1 expression in CRPC cells. qPCR for Nrdp1 expression in LNCaP, C4-2, and stably transfected C4-2-FlnA(16-24) showed that Nrdp1 expression was reduced in C4-2 compared to LNCaP cells (p=0.0141), but expression of FlnA16-24 in C4-2 cells restored Nrdp1 expression to a level similar to LNCaP cells (p=0.0002 compared to C4-2). Nrdp1 transcript levels were normalized to the corresponding values for β-Actin. (E) Comparison of Nrdp1 response to changes in AR in CWR-R1 and CWR22-Rv1 cells cultured in FBS, CSS or CSS treated with increasing doses of DHT as indicated. Lysates were immunoblotted with anti-Nrdp1, anti-ErbB3, and anti-tubulin antibodies. While the levels of Nrdp1 in CWR22Rv1 cells were unaltered despite culture in CSS, in CWR-R1 these levels altered slightly. (F) (left) Reporter gene activity of AR on a luciferase-tagged PSA promoter section demonstrates that in control LNCaP cells, 10 µM bicalutamide is able to suppress AR activity whereas in cells where FlnA is downregulated by siRNA, bicalutamide failed to affect AR activity. (right) Western blotting demonstrating the efficacy of FlnA siRNA used.

Figure 7

Figure 7. Expression of the 90kDa FlnA isoform restored AR regulation of Nrdp1 transcription

(A) AR binds to Nrdp1 ARE3 in the presence of FlnA 16-24 (90 kDa). Chromatin samples were immunoprecipitated with an anti-AR antibody and analyzed by qPCR with primers flanking the Nrdp1 ARE3 region with Znf333 as a negative control. Note that AR binding increased 2-fold upon FlnA16-24 transfection (p<0.0001). **(B)** AR transcriptional activity of Nrdp1 ARE3 is androgen regulated in the presence of FlnA 16-24. C4-2 cells transfected with empty vector or FlnA 16-24 were cultured in FBS medium and transfected with control vector, normal Nrdp1 ARE3, or mutant Nrdp1 ARE3. AR transcriptional activity was measured by luciferase assay. Cells were also treated with DMSO, 1nM DHT, or 10µM bicalutamide (Casodex). Luciferase was increased in the presence of FlnA 16-24, and regulated in an androgen dependent manner with normal Nrdp1 ARE3. **(C)** Flow cytometric analysis in PI-stained, ethanol fixed C4-2 cells to determine the effect of transfection with FlnA 16-24 on cell cycle. Cells were grown in FBS or CSS and transfected with either empty vector or FlnA 16-24. Tranfection with FlnA 16-24 has the same effect as growing the cells in CSS, however the combination of the two completely S phase. **(D)** FlnA acts as a regulator of mRNA expression levels in multiple genes. qPCR for GDF15, IL32, TMPRSS2, BHLHE40, and HMOX1 expression in LNCaP, C4-2, and C4-2 FlnA 16-24. The expression of 4 of 5 genes were significantly decreased in C4-2 compared to LNCaP cells [GDF15: p<0.0001 (*); IL-32: p=0.0029 (▪); BHLHE40: p=0.0012 (▲); HMOX1: p=0.0129 (⌂)] while that for TMPRSS2 was not significant (p>0.05; ●). The expression of FlnA 16-24 in C4-2 cells restored expression of these genes to a level similar or higher than LNCaP cells [GDF15: p<0.0001 (*); IL-32: p=0.0014 (◊); BHLHE40: p=0.0003 (►); HMOX1: p=0.0052 (♦); TMPRSS2: p=0.0312 (○)]. All transcript levels were normalized to the corresponding values for β-Actin.

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