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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

LY294002 and AZD5363 affect AR gene transcription initiation, RNA splicing and AR mRNA stability.(A) LNCaP and LNCaP95 cells were treated with vehicle, 50 uM LY294002 or 5 uM AZD5363 for 18 hours (left). LNCaP and LNCaP95 cells were also transfected with control or AKT1–3 siRNAs for 48 hours (right). Nuclear run-on assays were performed as described in Material and Method section. (B) LNCaP and LNCaP95 cells were transfected with control or pooled siRNA for AKT 1–3 isoforms for 36 hours. Cell were then further treated with 50 uM LY294002 or 5 uM AZD5363 for another 18 hours. RNA splicing assays for AR-FL and AR-V7 were measured as described in Material and Method section. (C) LNCaP and LNCaP95 cells were pretreated with 2 uM actinomycin D for 2 hours. Cells were then treated with vehicle, 50 uM LY294002 or 5 uM AZD5363 for 0, 2, 4, 6, 8, and 16 hours. AR-FL and AR-V7 mRNA expression levels relative to 18rS were determined by real-time PCR. (D) LNCaP and LNCaP95 cells were transfected with control or pooled siRNA for AKT 1–3 isoforms for 36 hours. Cell were pre-treated with 2 uM Actinomycin D for 2 hours and then treated with vehicle, 50 uM LY294002 or 5 uM AZD5363 for 6 hours. AR-FL and 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. One-way ANOVA followed by student t-test was performed with * as P<0.05, ** as P<0.01 and *** as P<0.001.
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pone-0108780-g005: LY294002 and AZD5363 affect AR gene transcription initiation, RNA splicing and AR mRNA stability.(A) LNCaP and LNCaP95 cells were treated with vehicle, 50 uM LY294002 or 5 uM AZD5363 for 18 hours (left). LNCaP and LNCaP95 cells were also transfected with control or AKT1–3 siRNAs for 48 hours (right). Nuclear run-on assays were performed as described in Material and Method section. (B) LNCaP and LNCaP95 cells were transfected with control or pooled siRNA for AKT 1–3 isoforms for 36 hours. Cell were then further treated with 50 uM LY294002 or 5 uM AZD5363 for another 18 hours. RNA splicing assays for AR-FL and AR-V7 were measured as described in Material and Method section. (C) LNCaP and LNCaP95 cells were pretreated with 2 uM actinomycin D for 2 hours. Cells were then treated with vehicle, 50 uM LY294002 or 5 uM AZD5363 for 0, 2, 4, 6, 8, and 16 hours. AR-FL and AR-V7 mRNA expression levels relative to 18rS were determined by real-time PCR. (D) LNCaP and LNCaP95 cells were transfected with control or pooled siRNA for AKT 1–3 isoforms for 36 hours. Cell were pre-treated with 2 uM Actinomycin D for 2 hours and then treated with vehicle, 50 uM LY294002 or 5 uM AZD5363 for 6 hours. AR-FL and 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. One-way ANOVA followed by student t-test was performed with * as P<0.05, ** as P<0.01 and *** as P<0.001.

Mentions: The impacts of LY294002 and AZD5363 on AR mRNA levels could be at multiple possible levels including AR gene transcription initiation, pre-mRNA splicing and AR mRNA degradation. Using nuclear run-on assay, we measured both AR and 18rS gene transcription initiation rates in presence of vehicle or PI3K/AKT inhibitors (Fig. 5A). The 18rS gene was used as an internal control as its mRNA levels were not affected by PI3K/AKT inhibitors. We showed that LY294002 inhibited, while AZD5363 enhanced AR gene transcription initiation rates relative to 18rS in both LNCaP and LNCaP95 cells. Furthermore, knocking down AKT did not alter AR gene transcription rates.


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

Liu L, Dong X - PLoS ONE (2014)

LY294002 and AZD5363 affect AR gene transcription initiation, RNA splicing and AR mRNA stability.(A) LNCaP and LNCaP95 cells were treated with vehicle, 50 uM LY294002 or 5 uM AZD5363 for 18 hours (left). LNCaP and LNCaP95 cells were also transfected with control or AKT1–3 siRNAs for 48 hours (right). Nuclear run-on assays were performed as described in Material and Method section. (B) LNCaP and LNCaP95 cells were transfected with control or pooled siRNA for AKT 1–3 isoforms for 36 hours. Cell were then further treated with 50 uM LY294002 or 5 uM AZD5363 for another 18 hours. RNA splicing assays for AR-FL and AR-V7 were measured as described in Material and Method section. (C) LNCaP and LNCaP95 cells were pretreated with 2 uM actinomycin D for 2 hours. Cells were then treated with vehicle, 50 uM LY294002 or 5 uM AZD5363 for 0, 2, 4, 6, 8, and 16 hours. AR-FL and AR-V7 mRNA expression levels relative to 18rS were determined by real-time PCR. (D) LNCaP and LNCaP95 cells were transfected with control or pooled siRNA for AKT 1–3 isoforms for 36 hours. Cell were pre-treated with 2 uM Actinomycin D for 2 hours and then treated with vehicle, 50 uM LY294002 or 5 uM AZD5363 for 6 hours. AR-FL and 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. One-way ANOVA followed by student t-test was performed with * as P<0.05, ** as P<0.01 and *** as P<0.001.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4215833&req=5

pone-0108780-g005: LY294002 and AZD5363 affect AR gene transcription initiation, RNA splicing and AR mRNA stability.(A) LNCaP and LNCaP95 cells were treated with vehicle, 50 uM LY294002 or 5 uM AZD5363 for 18 hours (left). LNCaP and LNCaP95 cells were also transfected with control or AKT1–3 siRNAs for 48 hours (right). Nuclear run-on assays were performed as described in Material and Method section. (B) LNCaP and LNCaP95 cells were transfected with control or pooled siRNA for AKT 1–3 isoforms for 36 hours. Cell were then further treated with 50 uM LY294002 or 5 uM AZD5363 for another 18 hours. RNA splicing assays for AR-FL and AR-V7 were measured as described in Material and Method section. (C) LNCaP and LNCaP95 cells were pretreated with 2 uM actinomycin D for 2 hours. Cells were then treated with vehicle, 50 uM LY294002 or 5 uM AZD5363 for 0, 2, 4, 6, 8, and 16 hours. AR-FL and AR-V7 mRNA expression levels relative to 18rS were determined by real-time PCR. (D) LNCaP and LNCaP95 cells were transfected with control or pooled siRNA for AKT 1–3 isoforms for 36 hours. Cell were pre-treated with 2 uM Actinomycin D for 2 hours and then treated with vehicle, 50 uM LY294002 or 5 uM AZD5363 for 6 hours. AR-FL and 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. One-way ANOVA followed by student t-test was performed with * as P<0.05, ** as P<0.01 and *** as P<0.001.
Mentions: The impacts of LY294002 and AZD5363 on AR mRNA levels could be at multiple possible levels including AR gene transcription initiation, pre-mRNA splicing and AR mRNA degradation. Using nuclear run-on assay, we measured both AR and 18rS gene transcription initiation rates in presence of vehicle or PI3K/AKT inhibitors (Fig. 5A). The 18rS gene was used as an internal control as its mRNA levels were not affected by PI3K/AKT inhibitors. We showed that LY294002 inhibited, while AZD5363 enhanced AR gene transcription initiation rates relative to 18rS in both LNCaP and LNCaP95 cells. Furthermore, knocking down AKT did not alter AR gene transcription rates.

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