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Complex impacts of PI3K/AKT inhibitors to androgen receptor gene expression in prostate cancer cells.

Liu L, Dong X - PLoS ONE (2014)

Bottom Line: Co-targeting AR and PI3K/AKT signaling has been proposed to be a more effective therapeutic means for CRPC patients.PI3K/AKT inhibitors had various impacts on AR protein expressions primarily through alterations of AR gene transcription initiation and RNA splicing.However, these effects remained unchanged in the presence RNA silencing of the AKT genes.

View Article: PubMed Central - PubMed

Affiliation: The Vancouver Prostate Centre, Department of Urologic Sciences, University of British Columbia, Vancouver, Canada.

ABSTRACT

Background: Androgen deprivation therapy (ADT) is the first-line treatment to metastatic prostate cancer (PCa). However, sustained expression and function of the androgen receptor (AR) gene contribute to the progression of castration resistant prostate cancers (CRPC). Additionally, tumors can adapt the PI3K/AKT survival pathway to escape ADT. Co-targeting AR and PI3K/AKT signaling has been proposed to be a more effective therapeutic means for CRPC patients. Many clinical trials are ongoing to test whether PI3K/AKT inhibitors are beneficial to PCa patients. However whether these inhibitors have any impacts on the expressions of full length AR (AR-FL) and its splice variant (AR-V7) remains unclear.

Methods: Four human prostate cancer cell lines (LNCaP, LNCaP95, VCaP and 22Rv1) with different genetic backgrounds were treated with five PI3K/AKT inhibitors (LY294002, Wortmannin, BKM120, AKTi and AZD5363) and or AKT siRNA. AR and AR-V7 protein and mRNA levels were measured by immunoblotting and real-time PCR assays. AR gene transcription initiation, alternative RNA splicing and AR mRNA degradation rates were also determined.

Results: PI3K/AKT inhibitors had various impacts on AR protein expressions primarily through alterations of AR gene transcription initiation and RNA splicing. However, these effects remained unchanged in the presence RNA silencing of the AKT genes.

Conclusion: PI3K/AKT inhibitors have off-target effects on AR gene expression in prostate cancer cells, which shall be considered when applying these inhibitors to PCa patients, particularly patients under ADT treatment.

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Related in: MedlinePlus

Impacts of AKT knockdown to AR gene expression.LNCaP, LNCaP95, VCaP and 22Rv1 cells were transfected with control or pooled siRNA for AKT 1–3 isoforms for 36 hours. Cells were then further treated with 50 uM LY294002 (A) or 5 uM AZD5363 (B) for another 18 hours. Protein lysates were immunoblotted with AR (N-20), AR-V7, Pan-AKT, p-AKT(ser473) and β-Actin antibodies. Results were repeated in more than three independent experiments. Densitometry analysis of protein bands were measured by the Image J software and plotted as mean+SEM. (C) AR-FL and (D) AR-V7 mRNA expression levels relative to 18rS were determined by real-time PCR. Results were from three independent RNA extractions with triplicate real-time PCR assays on each RNA sample and plotted as mean+SEM. Student's t-tests were performed comparing between vehicle and LY294002/AZD5363 treatments with * as P<0.05; ** as P<0.01 and *** as P<0.001.
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pone-0108780-g004: Impacts of AKT knockdown to AR gene expression.LNCaP, LNCaP95, VCaP and 22Rv1 cells were transfected with control or pooled siRNA for AKT 1–3 isoforms for 36 hours. Cells were then further treated with 50 uM LY294002 (A) or 5 uM AZD5363 (B) for another 18 hours. Protein lysates were immunoblotted with AR (N-20), AR-V7, Pan-AKT, p-AKT(ser473) and β-Actin antibodies. Results were repeated in more than three independent experiments. Densitometry analysis of protein bands were measured by the Image J software and plotted as mean+SEM. (C) AR-FL and (D) AR-V7 mRNA expression levels relative to 18rS were determined by real-time PCR. Results were from three independent RNA extractions with triplicate real-time PCR assays on each RNA sample and plotted as mean+SEM. Student's t-tests were performed comparing between vehicle and LY294002/AZD5363 treatments with * as P<0.05; ** as P<0.01 and *** as P<0.001.

Mentions: To further investigate whether the impacts of LY294002 and AZD5363 on AR protein and mRNA levels were mediated through AKT, we performed AKT knockdown assays using pooled siRNA against all three AKT1–3 isoforms. Efficiency of AKT siRNA was shown by Western blotting (Fig. 4A–B). We observed that regardless the presence of AKT knockdown, LY294002 can still dramatically decrease AR-FL and AR-V7 protein (Fig. 4A) and mRNA (Fig. 4C) levels in LNCaP, LNCaP95 and VCaP cells. LY294002 upregulated AR-FL expression, but decreased AR-V7 expression in 22Rv1 cells. In addition, we observed that AZD5363 significantly increased AR-FL expressions in LNCaP and LNCaP95 cells and decreased AR-FL and AR-V7 in VCaP and 22Rv1 cells, which effects remained unchanged even in the presence of efficient AKT knockdown (Fig. 4B and 4D). Together, these results indicated that the impacts of LY294002 and AZD5363 to AR expressions were independent to AKT and its downstream effectors.


Complex impacts of PI3K/AKT inhibitors to androgen receptor gene expression in prostate cancer cells.

Liu L, Dong X - PLoS ONE (2014)

Impacts of AKT knockdown to AR gene expression.LNCaP, LNCaP95, VCaP and 22Rv1 cells were transfected with control or pooled siRNA for AKT 1–3 isoforms for 36 hours. Cells were then further treated with 50 uM LY294002 (A) or 5 uM AZD5363 (B) for another 18 hours. Protein lysates were immunoblotted with AR (N-20), AR-V7, Pan-AKT, p-AKT(ser473) and β-Actin antibodies. Results were repeated in more than three independent experiments. Densitometry analysis of protein bands were measured by the Image J software and plotted as mean+SEM. (C) AR-FL and (D) AR-V7 mRNA expression levels relative to 18rS were determined by real-time PCR. Results were from three independent RNA extractions with triplicate real-time PCR assays on each RNA sample and plotted as mean+SEM. Student's t-tests were performed comparing between vehicle and LY294002/AZD5363 treatments with * as P<0.05; ** as P<0.01 and *** as P<0.001.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4215833&req=5

pone-0108780-g004: Impacts of AKT knockdown to AR gene expression.LNCaP, LNCaP95, VCaP and 22Rv1 cells were transfected with control or pooled siRNA for AKT 1–3 isoforms for 36 hours. Cells were then further treated with 50 uM LY294002 (A) or 5 uM AZD5363 (B) for another 18 hours. Protein lysates were immunoblotted with AR (N-20), AR-V7, Pan-AKT, p-AKT(ser473) and β-Actin antibodies. Results were repeated in more than three independent experiments. Densitometry analysis of protein bands were measured by the Image J software and plotted as mean+SEM. (C) AR-FL and (D) AR-V7 mRNA expression levels relative to 18rS were determined by real-time PCR. Results were from three independent RNA extractions with triplicate real-time PCR assays on each RNA sample and plotted as mean+SEM. Student's t-tests were performed comparing between vehicle and LY294002/AZD5363 treatments with * as P<0.05; ** as P<0.01 and *** as P<0.001.
Mentions: To further investigate whether the impacts of LY294002 and AZD5363 on AR protein and mRNA levels were mediated through AKT, we performed AKT knockdown assays using pooled siRNA against all three AKT1–3 isoforms. Efficiency of AKT siRNA was shown by Western blotting (Fig. 4A–B). We observed that regardless the presence of AKT knockdown, LY294002 can still dramatically decrease AR-FL and AR-V7 protein (Fig. 4A) and mRNA (Fig. 4C) levels in LNCaP, LNCaP95 and VCaP cells. LY294002 upregulated AR-FL expression, but decreased AR-V7 expression in 22Rv1 cells. In addition, we observed that AZD5363 significantly increased AR-FL expressions in LNCaP and LNCaP95 cells and decreased AR-FL and AR-V7 in VCaP and 22Rv1 cells, which effects remained unchanged even in the presence of efficient AKT knockdown (Fig. 4B and 4D). Together, these results indicated that the impacts of LY294002 and AZD5363 to AR expressions were independent to AKT and its downstream effectors.

Bottom Line: Co-targeting AR and PI3K/AKT signaling has been proposed to be a more effective therapeutic means for CRPC patients.PI3K/AKT inhibitors had various impacts on AR protein expressions primarily through alterations of AR gene transcription initiation and RNA splicing.However, these effects remained unchanged in the presence RNA silencing of the AKT genes.

View Article: PubMed Central - PubMed

Affiliation: The Vancouver Prostate Centre, Department of Urologic Sciences, University of British Columbia, Vancouver, Canada.

ABSTRACT

Background: Androgen deprivation therapy (ADT) is the first-line treatment to metastatic prostate cancer (PCa). However, sustained expression and function of the androgen receptor (AR) gene contribute to the progression of castration resistant prostate cancers (CRPC). Additionally, tumors can adapt the PI3K/AKT survival pathway to escape ADT. Co-targeting AR and PI3K/AKT signaling has been proposed to be a more effective therapeutic means for CRPC patients. Many clinical trials are ongoing to test whether PI3K/AKT inhibitors are beneficial to PCa patients. However whether these inhibitors have any impacts on the expressions of full length AR (AR-FL) and its splice variant (AR-V7) remains unclear.

Methods: Four human prostate cancer cell lines (LNCaP, LNCaP95, VCaP and 22Rv1) with different genetic backgrounds were treated with five PI3K/AKT inhibitors (LY294002, Wortmannin, BKM120, AKTi and AZD5363) and or AKT siRNA. AR and AR-V7 protein and mRNA levels were measured by immunoblotting and real-time PCR assays. AR gene transcription initiation, alternative RNA splicing and AR mRNA degradation rates were also determined.

Results: PI3K/AKT inhibitors had various impacts on AR protein expressions primarily through alterations of AR gene transcription initiation and RNA splicing. However, these effects remained unchanged in the presence RNA silencing of the AKT genes.

Conclusion: PI3K/AKT inhibitors have off-target effects on AR gene expression in prostate cancer cells, which shall be considered when applying these inhibitors to PCa patients, particularly patients under ADT treatment.

Show MeSH
Related in: MedlinePlus