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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 and AR-V7 transcriptional activity in PCa cells.(A) LNCaP and (B) VCaP cells were transfected with control or AKT1–3 siRNAs for 48 hours and treated with vehicle or 1 nM R1881. Cells were also co-treated with vehicle, 50 uM LY294002 or 5 uM AZD5363 for 18 hours. Real-time PCR assays measured PSA and OPRK1 mRNA levels. (C) LNCaP95 and (D) VCaP cells were transfected with control or AKT1–3 siRNAs for 48 hours and treated with vehicle, 50 uM LY294002 or 5 uM AZD5363 in the presence of 5 uM MDV3100 for 18 hours. Real-time PCR assays measured UGT2b17 mRNA levels. Results were from three independent RNA extractions with triplicate real-time PCR assays on each RNA sample. 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-g006: LY294002 and AZD5363 affect AR and AR-V7 transcriptional activity in PCa cells.(A) LNCaP and (B) VCaP cells were transfected with control or AKT1–3 siRNAs for 48 hours and treated with vehicle or 1 nM R1881. Cells were also co-treated with vehicle, 50 uM LY294002 or 5 uM AZD5363 for 18 hours. Real-time PCR assays measured PSA and OPRK1 mRNA levels. (C) LNCaP95 and (D) VCaP cells were transfected with control or AKT1–3 siRNAs for 48 hours and treated with vehicle, 50 uM LY294002 or 5 uM AZD5363 in the presence of 5 uM MDV3100 for 18 hours. Real-time PCR assays measured UGT2b17 mRNA levels. Results were from three independent RNA extractions with triplicate real-time PCR assays on each RNA sample. 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 alterations of AR-FL and AR-V7 protein levels by LY294002 and AZD5363 would impact AR transcriptional activities, we measured the expressions of two AR-FL regulated genes (PSA and OPRK1) and one AR-V7 regulated gene (UGT2b17). In both LNCaP and VCaP cells, AR-FL agonist R1881 stimulated PSA expression, which actions were suppressed by LY294002 (Fig. 6A–B). By contrast, AZD5363 strengthened the R1881 effect in upregulating PSA expression in LNCaP cells, but reduced the R1881 action in VCaP cells. These changes were consistent to the impacts of LY294002 and AZD5363 on AR-FL protein levels in these cells. The expression of OPRK1 gene was suppressed by ligand activated AR-FL [39]. Treatment of LY294002 decreased AR-FL mediated inhibition to OPRK1 expression in both LNCaP cells (69% suppression reduced to 35%) and VCaP cells (94% suppression reduced to 42%). AZD5363 strengthened AR-FL in suppressing OPRK1 expression in LNCaP cells (69% suppression increased to 79%), but reduced AR-FL mediated inhibition of OPRK1 expression in VCaP cells (94% suppression reduced to 86%). However, both impacts of LY294002 and AZD5363 on PSA and OPRK1 expressions were not significantly altered by AKT knockdown. To study the AR-V7 transcriptional activity, we measured the expression of UGT2b17 in VCaP and LNCaP95 cells that were treated with MDV3100 to block AR-FL activity. Both VCaP and LNCaP95 cells express both AR-FL and AR-V7. We have previously demonstrated that in the presence of AR-FL functional blockade, the upregulation of expression of UGT2b17 was determined by AR-V7 [27]. LY294002 inhibited UGT2b17 expression in both LNCaP95 and VCaP cells (Fig. 6C–D). AZD5363 stimulated UGT2b17 expression in LNCaP cells, but reduced its expression in VCaP cells. These changes in UGT2b17 gene expression were consistent to AR-V7 protein levels under LY294002 and AZD5363 treatments. However, these impacts were not altered by AKT knockdown.


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 and AR-V7 transcriptional activity in PCa cells.(A) LNCaP and (B) VCaP cells were transfected with control or AKT1–3 siRNAs for 48 hours and treated with vehicle or 1 nM R1881. Cells were also co-treated with vehicle, 50 uM LY294002 or 5 uM AZD5363 for 18 hours. Real-time PCR assays measured PSA and OPRK1 mRNA levels. (C) LNCaP95 and (D) VCaP cells were transfected with control or AKT1–3 siRNAs for 48 hours and treated with vehicle, 50 uM LY294002 or 5 uM AZD5363 in the presence of 5 uM MDV3100 for 18 hours. Real-time PCR assays measured UGT2b17 mRNA levels. Results were from three independent RNA extractions with triplicate real-time PCR assays on each RNA sample. 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

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getmorefigures.php?uid=PMC4215833&req=5

pone-0108780-g006: LY294002 and AZD5363 affect AR and AR-V7 transcriptional activity in PCa cells.(A) LNCaP and (B) VCaP cells were transfected with control or AKT1–3 siRNAs for 48 hours and treated with vehicle or 1 nM R1881. Cells were also co-treated with vehicle, 50 uM LY294002 or 5 uM AZD5363 for 18 hours. Real-time PCR assays measured PSA and OPRK1 mRNA levels. (C) LNCaP95 and (D) VCaP cells were transfected with control or AKT1–3 siRNAs for 48 hours and treated with vehicle, 50 uM LY294002 or 5 uM AZD5363 in the presence of 5 uM MDV3100 for 18 hours. Real-time PCR assays measured UGT2b17 mRNA levels. Results were from three independent RNA extractions with triplicate real-time PCR assays on each RNA sample. 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 alterations of AR-FL and AR-V7 protein levels by LY294002 and AZD5363 would impact AR transcriptional activities, we measured the expressions of two AR-FL regulated genes (PSA and OPRK1) and one AR-V7 regulated gene (UGT2b17). In both LNCaP and VCaP cells, AR-FL agonist R1881 stimulated PSA expression, which actions were suppressed by LY294002 (Fig. 6A–B). By contrast, AZD5363 strengthened the R1881 effect in upregulating PSA expression in LNCaP cells, but reduced the R1881 action in VCaP cells. These changes were consistent to the impacts of LY294002 and AZD5363 on AR-FL protein levels in these cells. The expression of OPRK1 gene was suppressed by ligand activated AR-FL [39]. Treatment of LY294002 decreased AR-FL mediated inhibition to OPRK1 expression in both LNCaP cells (69% suppression reduced to 35%) and VCaP cells (94% suppression reduced to 42%). AZD5363 strengthened AR-FL in suppressing OPRK1 expression in LNCaP cells (69% suppression increased to 79%), but reduced AR-FL mediated inhibition of OPRK1 expression in VCaP cells (94% suppression reduced to 86%). However, both impacts of LY294002 and AZD5363 on PSA and OPRK1 expressions were not significantly altered by AKT knockdown. To study the AR-V7 transcriptional activity, we measured the expression of UGT2b17 in VCaP and LNCaP95 cells that were treated with MDV3100 to block AR-FL activity. Both VCaP and LNCaP95 cells express both AR-FL and AR-V7. We have previously demonstrated that in the presence of AR-FL functional blockade, the upregulation of expression of UGT2b17 was determined by AR-V7 [27]. LY294002 inhibited UGT2b17 expression in both LNCaP95 and VCaP cells (Fig. 6C–D). AZD5363 stimulated UGT2b17 expression in LNCaP cells, but reduced its expression in VCaP cells. These changes in UGT2b17 gene expression were consistent to AR-V7 protein levels under LY294002 and AZD5363 treatments. However, these impacts were not altered by AKT knockdown.

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