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The FKBP52 Cochaperone Acts in Synergy with β-Catenin to Potentiate Androgen Receptor Signaling.

Storer Samaniego C, Suh JH, Chattopadhyay A, Olivares K, Guy N, Sivils JC, Dey P, Yumoto F, Fletterick RJ, Strom AM, Gustafsson JÅ, Webb P, Cox MB - PLoS ONE (2015)

Bottom Line: MJC13 effectively blocks β-catenin interaction with the AR LBD and the synergistic up-regulation of AR by FKBP52 and β-catenin.Our data suggest that co-regulation of AR by FKBP52 and β-catenin does not require FKBP52 PPIase catalytic activity, nor FKBP52 binding to Hsp90.However, the FKBP52 proline-rich loop that overhangs the PPIase pocket is critical for synergy.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences and Border Biomedical Research Center, The University of Texas at El Paso, El Paso, Texas, United States of America; Department of Chemistry and Biochemistry, Kettering University, Flint, Michigan, United States of America.

ABSTRACT
FKBP52 and β-catenin have emerged in recent years as attractive targets for prostate cancer treatment. β-catenin interacts directly with the androgen receptor (AR) and has been characterized as a co-activator of AR-mediated transcription. FKBP52 is a positive regulator of AR in cellular and whole animal models and is required for the development of androgen-dependent tissues. We previously characterized an AR inhibitor termed MJC13 that putatively targets the AR BF3 surface to specifically inhibit FKBP52-regulated AR signaling. Predictive modeling suggests that β-catenin interacts with the AR hormone binding domain on a surface that overlaps with BF3. Here we demonstrate that FKBP52 and β-catenin interact directly in vitro and act in concert to promote a synergistic up-regulation of both hormone-independent and -dependent AR signaling. Our data demonstrate that FKBP52 promotes β-catenin interaction with AR and is required for β-catenin co-activation of AR activity in prostate cancer cells. MJC13 effectively blocks β-catenin interaction with the AR LBD and the synergistic up-regulation of AR by FKBP52 and β-catenin. Our data suggest that co-regulation of AR by FKBP52 and β-catenin does not require FKBP52 PPIase catalytic activity, nor FKBP52 binding to Hsp90. However, the FKBP52 proline-rich loop that overhangs the PPIase pocket is critical for synergy.

No MeSH data available.


Related in: MedlinePlus

FKBP52 is Required for β-Catenin Potentiation of AR in 22Rv1 Prostate Cancer Cells.(A) AR-mediated probasin-luciferase activity was assessed at a range of hormone concentrations in 22Rv1 prostate cancer cells stably transfected with a 19 base pair shRNA directed against FKBP52 (52KD) or wild type 22Rv1 cells. Significant differences at each hormone concentration are indicated (***p < 0.001). The upper panels show Western blots for AR (both the full length and truncated ARs are shown), FKBP52, and GAPDH as a loading control from 52KD and wild type 22Rv1 cell lysates. (B) A luciferase reporter assay using the AR-inducible probasin-luciferase reporter plasmid in wild type and 52KD 22Rv1 cells with and without overexpression of the indicated proteins in the presence or absence of 175 pM dihydrotestosterone (DHT). Reporter expression in the presence of β-catenin (S33A) and DHT is significantly different from all other conditions (p < 0.001). Wild type cells with empty vector alone in the presence of DHT also significantly differed from all other conditions with p values ranging from < 0.05 to < 0.001. No conditions in the presence of FKBP52 knockdown significantly differed from each other in pairwise comparisons (p>0.05).
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pone.0134015.g004: FKBP52 is Required for β-Catenin Potentiation of AR in 22Rv1 Prostate Cancer Cells.(A) AR-mediated probasin-luciferase activity was assessed at a range of hormone concentrations in 22Rv1 prostate cancer cells stably transfected with a 19 base pair shRNA directed against FKBP52 (52KD) or wild type 22Rv1 cells. Significant differences at each hormone concentration are indicated (***p < 0.001). The upper panels show Western blots for AR (both the full length and truncated ARs are shown), FKBP52, and GAPDH as a loading control from 52KD and wild type 22Rv1 cell lysates. (B) A luciferase reporter assay using the AR-inducible probasin-luciferase reporter plasmid in wild type and 52KD 22Rv1 cells with and without overexpression of the indicated proteins in the presence or absence of 175 pM dihydrotestosterone (DHT). Reporter expression in the presence of β-catenin (S33A) and DHT is significantly different from all other conditions (p < 0.001). Wild type cells with empty vector alone in the presence of DHT also significantly differed from all other conditions with p values ranging from < 0.05 to < 0.001. No conditions in the presence of FKBP52 knockdown significantly differed from each other in pairwise comparisons (p>0.05).

Mentions: The data indicate that FKBP52 and β-catenin co-regulate AR activity in HeLa and 52KO MEF cells. Demonstrating that this co-regulatory mechanism is relevant in a prostate cancer setting required a prostate cancer cell line with stable knockdown of FKBP52 protein in which to assess β-catenin effects on AR activity in the presence or absence of significant levels of FKBP52. Stable knockdown of FKBP52 protein levels was attempted in LNCaP and PC3 prostate cancer cell lines without success. Based on available evidence, the knockdown of FKBP52 is expected to significantly reduce AR signaling leading to a loss of proliferation in AR-dependent prostate cancer cells, which could prevent the selection of clonal populations. While this is true for LNCaP cells, PC3 cells are known to lack AR responsiveness and the reasons for lack of colony formation upon FKBP52 knockdown are unknown. However, we were able to achieve stable, shRNA-mediated FKBP52 knockdown in 22Rv1 prostate cancer cells (Fig 4A, top panel), which express both full length AR and a constitutively active, truncated AR protein that supports AR-dependent growth in the absence of full length AR activity. The fact that we were able to achieve knockdown in this cell line suggests that FKBP52 does not regulate activity from the truncated AR protein, which is expected given that FKBP52 is known to act through the receptor LBD [7]. FKBP52 knockdown had no effect on both full length and truncated AR protein levels (Fig 4A, top panel), but did significantly reduce hormone-dependent expression of an AR-mediated luciferase reporter gene (Fig 4A, bottom panel). In DHT-treated cells, overexpression of β-catenin (S33A) in wild type 22Rv1 cells, which endogenously express FKBP52, potentiated AR-mediated expression of a luciferase reporter up to 3-fold as compared with the vector control. shRNA-mediated knockdown of FKBP52 led to overall decreased signaling by AR, both in the absence or presence of overexpressed β-catenin. While the overexpression of β-catenin trended towards increased AR activity in the presence of FKBP52 knockdown, this activity was not statistically different from the vector control (Fig 4B). It is interesting to note that the data in Fig 4B were not normalized to hormone-independent basal AR activity. While many, including us, have reported the constitutive AR activity from the truncated AR protein in this cell line, our reporter assays with the probasin promoter consistently lack this basal activity. While we don't have an answer as to why the truncated AR protein does not regulate this reporter construct, this might suggest that the truncated protein displays promoter specificity.


The FKBP52 Cochaperone Acts in Synergy with β-Catenin to Potentiate Androgen Receptor Signaling.

Storer Samaniego C, Suh JH, Chattopadhyay A, Olivares K, Guy N, Sivils JC, Dey P, Yumoto F, Fletterick RJ, Strom AM, Gustafsson JÅ, Webb P, Cox MB - PLoS ONE (2015)

FKBP52 is Required for β-Catenin Potentiation of AR in 22Rv1 Prostate Cancer Cells.(A) AR-mediated probasin-luciferase activity was assessed at a range of hormone concentrations in 22Rv1 prostate cancer cells stably transfected with a 19 base pair shRNA directed against FKBP52 (52KD) or wild type 22Rv1 cells. Significant differences at each hormone concentration are indicated (***p < 0.001). The upper panels show Western blots for AR (both the full length and truncated ARs are shown), FKBP52, and GAPDH as a loading control from 52KD and wild type 22Rv1 cell lysates. (B) A luciferase reporter assay using the AR-inducible probasin-luciferase reporter plasmid in wild type and 52KD 22Rv1 cells with and without overexpression of the indicated proteins in the presence or absence of 175 pM dihydrotestosterone (DHT). Reporter expression in the presence of β-catenin (S33A) and DHT is significantly different from all other conditions (p < 0.001). Wild type cells with empty vector alone in the presence of DHT also significantly differed from all other conditions with p values ranging from < 0.05 to < 0.001. No conditions in the presence of FKBP52 knockdown significantly differed from each other in pairwise comparisons (p>0.05).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4514735&req=5

pone.0134015.g004: FKBP52 is Required for β-Catenin Potentiation of AR in 22Rv1 Prostate Cancer Cells.(A) AR-mediated probasin-luciferase activity was assessed at a range of hormone concentrations in 22Rv1 prostate cancer cells stably transfected with a 19 base pair shRNA directed against FKBP52 (52KD) or wild type 22Rv1 cells. Significant differences at each hormone concentration are indicated (***p < 0.001). The upper panels show Western blots for AR (both the full length and truncated ARs are shown), FKBP52, and GAPDH as a loading control from 52KD and wild type 22Rv1 cell lysates. (B) A luciferase reporter assay using the AR-inducible probasin-luciferase reporter plasmid in wild type and 52KD 22Rv1 cells with and without overexpression of the indicated proteins in the presence or absence of 175 pM dihydrotestosterone (DHT). Reporter expression in the presence of β-catenin (S33A) and DHT is significantly different from all other conditions (p < 0.001). Wild type cells with empty vector alone in the presence of DHT also significantly differed from all other conditions with p values ranging from < 0.05 to < 0.001. No conditions in the presence of FKBP52 knockdown significantly differed from each other in pairwise comparisons (p>0.05).
Mentions: The data indicate that FKBP52 and β-catenin co-regulate AR activity in HeLa and 52KO MEF cells. Demonstrating that this co-regulatory mechanism is relevant in a prostate cancer setting required a prostate cancer cell line with stable knockdown of FKBP52 protein in which to assess β-catenin effects on AR activity in the presence or absence of significant levels of FKBP52. Stable knockdown of FKBP52 protein levels was attempted in LNCaP and PC3 prostate cancer cell lines without success. Based on available evidence, the knockdown of FKBP52 is expected to significantly reduce AR signaling leading to a loss of proliferation in AR-dependent prostate cancer cells, which could prevent the selection of clonal populations. While this is true for LNCaP cells, PC3 cells are known to lack AR responsiveness and the reasons for lack of colony formation upon FKBP52 knockdown are unknown. However, we were able to achieve stable, shRNA-mediated FKBP52 knockdown in 22Rv1 prostate cancer cells (Fig 4A, top panel), which express both full length AR and a constitutively active, truncated AR protein that supports AR-dependent growth in the absence of full length AR activity. The fact that we were able to achieve knockdown in this cell line suggests that FKBP52 does not regulate activity from the truncated AR protein, which is expected given that FKBP52 is known to act through the receptor LBD [7]. FKBP52 knockdown had no effect on both full length and truncated AR protein levels (Fig 4A, top panel), but did significantly reduce hormone-dependent expression of an AR-mediated luciferase reporter gene (Fig 4A, bottom panel). In DHT-treated cells, overexpression of β-catenin (S33A) in wild type 22Rv1 cells, which endogenously express FKBP52, potentiated AR-mediated expression of a luciferase reporter up to 3-fold as compared with the vector control. shRNA-mediated knockdown of FKBP52 led to overall decreased signaling by AR, both in the absence or presence of overexpressed β-catenin. While the overexpression of β-catenin trended towards increased AR activity in the presence of FKBP52 knockdown, this activity was not statistically different from the vector control (Fig 4B). It is interesting to note that the data in Fig 4B were not normalized to hormone-independent basal AR activity. While many, including us, have reported the constitutive AR activity from the truncated AR protein in this cell line, our reporter assays with the probasin promoter consistently lack this basal activity. While we don't have an answer as to why the truncated AR protein does not regulate this reporter construct, this might suggest that the truncated protein displays promoter specificity.

Bottom Line: MJC13 effectively blocks β-catenin interaction with the AR LBD and the synergistic up-regulation of AR by FKBP52 and β-catenin.Our data suggest that co-regulation of AR by FKBP52 and β-catenin does not require FKBP52 PPIase catalytic activity, nor FKBP52 binding to Hsp90.However, the FKBP52 proline-rich loop that overhangs the PPIase pocket is critical for synergy.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences and Border Biomedical Research Center, The University of Texas at El Paso, El Paso, Texas, United States of America; Department of Chemistry and Biochemistry, Kettering University, Flint, Michigan, United States of America.

ABSTRACT
FKBP52 and β-catenin have emerged in recent years as attractive targets for prostate cancer treatment. β-catenin interacts directly with the androgen receptor (AR) and has been characterized as a co-activator of AR-mediated transcription. FKBP52 is a positive regulator of AR in cellular and whole animal models and is required for the development of androgen-dependent tissues. We previously characterized an AR inhibitor termed MJC13 that putatively targets the AR BF3 surface to specifically inhibit FKBP52-regulated AR signaling. Predictive modeling suggests that β-catenin interacts with the AR hormone binding domain on a surface that overlaps with BF3. Here we demonstrate that FKBP52 and β-catenin interact directly in vitro and act in concert to promote a synergistic up-regulation of both hormone-independent and -dependent AR signaling. Our data demonstrate that FKBP52 promotes β-catenin interaction with AR and is required for β-catenin co-activation of AR activity in prostate cancer cells. MJC13 effectively blocks β-catenin interaction with the AR LBD and the synergistic up-regulation of AR by FKBP52 and β-catenin. Our data suggest that co-regulation of AR by FKBP52 and β-catenin does not require FKBP52 PPIase catalytic activity, nor FKBP52 binding to Hsp90. However, the FKBP52 proline-rich loop that overhangs the PPIase pocket is critical for synergy.

No MeSH data available.


Related in: MedlinePlus