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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 PI3K/AKT inhibitors to AR protein levels in VCaP and 22Rv1.(A) VCaP and (B) 22Rv1 cells were treated with increasing doses of PI3K/AKT inhibitors LY294002 (0, 25, 50 uM), Wortmannin (0, 0.5 and 1 uM), BKM120 (0, 0.5 and 1 uM), AKTi (0, 5 and 10 uM) or AZD5363 (0, 2.5 and 5 uM) for 18 hours. Protein lysates were immunoblotted with AR (N-20), AR-V7, Pan-AKT, phosphor-AKT (ser473) and β-Actin antibodies. (C) Results were repeated at least three independent experiments. Densitometry analysis of protein bands were measured by the Image J software and plotted as mean+SEM. 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-g002: Impacts of PI3K/AKT inhibitors to AR protein levels in VCaP and 22Rv1.(A) VCaP and (B) 22Rv1 cells were treated with increasing doses of PI3K/AKT inhibitors LY294002 (0, 25, 50 uM), Wortmannin (0, 0.5 and 1 uM), BKM120 (0, 0.5 and 1 uM), AKTi (0, 5 and 10 uM) or AZD5363 (0, 2.5 and 5 uM) for 18 hours. Protein lysates were immunoblotted with AR (N-20), AR-V7, Pan-AKT, phosphor-AKT (ser473) and β-Actin antibodies. (C) Results were repeated at least three independent experiments. Densitometry analysis of protein bands were measured by the Image J software and plotted as mean+SEM. 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: LY294002 is a strong competitive inhibitor to PI3K and prevents PI3K from phosphorylating and activating downstream effectors of AKT [33]. Wortmannin is a covalent inhibitor of PI3K that irreversibly suppresses AKT phosphorylation [34]. LY294002 repressed AR-FL protein levels in LNCaP cells (Fig. 1A), inhibited both AR-FL and AR-V7 protein levels in LNCaP95 cells (Fig. 1B). Both LNCaP and LNCaP95 cells are PTEN deficient cells, in which AKT is constitutively active in its phosphorylation form (p-AKT). Both LY294002 and Wortmannin strongly inhibited p-AKT levels in both cell lines. Densitometry analyses on protein expressions of AR-FL, AR-V7 and p-AKT were measured (Fig. 1C). Interestingly, both LY294002 and Wortmannin suppressed AR-FL and AR-V7 protein expressions in VCaP cells (Fig. 2A). However, LY294002 increased AR-FL, but decreased AR-V7 protein levels in 22Rv1 cells (Fig. 2B). Wortmannin inhibited both AR-FL and AR-V7 protein expressions in VCaP and 22Rv1 cells. Densitometry analyses on protein expressions of AR-FL and AR-V7 were analyzed (Fig. 2C). Since VCaP and 22Rv1 cells are PTEN sufficient cells, where p-AKT is at very low/undetectable levels unless its upstream tyrosine kinase receptors are activated, the effects of LY294002 to AR protein expression in VCaP and 22Rv1 cells were not likely related to AKT activation. BKM120 is a pan-PI3K inhibitor, but not to mTOR [35]. BKM120 efficiently suppressed p-AKT levels in both LNCaP and LNCaP95 cells. However, BKM120 had no effects to AR-FL and AR-V7 protein levels in all four PCa cell lines (Fig. 1–2). AKTi is a competitive phosphatidylinositol ether analog that docks into the pleckstrin homolgy (PH) domain of AKT, prevents AKT translocation to PI3K at the plasma membrane thus inhibits AKT phosphorylation and activation [36]. AKTi did not show any suppressive effects to AR-FL or AR-V7 protein levels (Fig. 1–2). AZD5363 is an ATP-competitive inhibitor of AKT [37], [38]. It increased both AR-FL and AR-V7 protein levels and induced p-AKT levels in LNCaP and LNCaP95 cells (Fig. 1). However, it decreased AR-FL and AR-V7 protein levels in PTEN sufficient VCaP and 22Rv1 cells (Fig. 2).


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

Liu L, Dong X - PLoS ONE (2014)

Impacts of PI3K/AKT inhibitors to AR protein levels in VCaP and 22Rv1.(A) VCaP and (B) 22Rv1 cells were treated with increasing doses of PI3K/AKT inhibitors LY294002 (0, 25, 50 uM), Wortmannin (0, 0.5 and 1 uM), BKM120 (0, 0.5 and 1 uM), AKTi (0, 5 and 10 uM) or AZD5363 (0, 2.5 and 5 uM) for 18 hours. Protein lysates were immunoblotted with AR (N-20), AR-V7, Pan-AKT, phosphor-AKT (ser473) and β-Actin antibodies. (C) Results were repeated at least three independent experiments. Densitometry analysis of protein bands were measured by the Image J software and plotted as mean+SEM. One-way ANOVA followed by student t-test was performed with * as P<0.05, ** as P<0.01 and *** as P<0.001.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0108780-g002: Impacts of PI3K/AKT inhibitors to AR protein levels in VCaP and 22Rv1.(A) VCaP and (B) 22Rv1 cells were treated with increasing doses of PI3K/AKT inhibitors LY294002 (0, 25, 50 uM), Wortmannin (0, 0.5 and 1 uM), BKM120 (0, 0.5 and 1 uM), AKTi (0, 5 and 10 uM) or AZD5363 (0, 2.5 and 5 uM) for 18 hours. Protein lysates were immunoblotted with AR (N-20), AR-V7, Pan-AKT, phosphor-AKT (ser473) and β-Actin antibodies. (C) Results were repeated at least three independent experiments. Densitometry analysis of protein bands were measured by the Image J software and plotted as mean+SEM. 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: LY294002 is a strong competitive inhibitor to PI3K and prevents PI3K from phosphorylating and activating downstream effectors of AKT [33]. Wortmannin is a covalent inhibitor of PI3K that irreversibly suppresses AKT phosphorylation [34]. LY294002 repressed AR-FL protein levels in LNCaP cells (Fig. 1A), inhibited both AR-FL and AR-V7 protein levels in LNCaP95 cells (Fig. 1B). Both LNCaP and LNCaP95 cells are PTEN deficient cells, in which AKT is constitutively active in its phosphorylation form (p-AKT). Both LY294002 and Wortmannin strongly inhibited p-AKT levels in both cell lines. Densitometry analyses on protein expressions of AR-FL, AR-V7 and p-AKT were measured (Fig. 1C). Interestingly, both LY294002 and Wortmannin suppressed AR-FL and AR-V7 protein expressions in VCaP cells (Fig. 2A). However, LY294002 increased AR-FL, but decreased AR-V7 protein levels in 22Rv1 cells (Fig. 2B). Wortmannin inhibited both AR-FL and AR-V7 protein expressions in VCaP and 22Rv1 cells. Densitometry analyses on protein expressions of AR-FL and AR-V7 were analyzed (Fig. 2C). Since VCaP and 22Rv1 cells are PTEN sufficient cells, where p-AKT is at very low/undetectable levels unless its upstream tyrosine kinase receptors are activated, the effects of LY294002 to AR protein expression in VCaP and 22Rv1 cells were not likely related to AKT activation. BKM120 is a pan-PI3K inhibitor, but not to mTOR [35]. BKM120 efficiently suppressed p-AKT levels in both LNCaP and LNCaP95 cells. However, BKM120 had no effects to AR-FL and AR-V7 protein levels in all four PCa cell lines (Fig. 1–2). AKTi is a competitive phosphatidylinositol ether analog that docks into the pleckstrin homolgy (PH) domain of AKT, prevents AKT translocation to PI3K at the plasma membrane thus inhibits AKT phosphorylation and activation [36]. AKTi did not show any suppressive effects to AR-FL or AR-V7 protein levels (Fig. 1–2). AZD5363 is an ATP-competitive inhibitor of AKT [37], [38]. It increased both AR-FL and AR-V7 protein levels and induced p-AKT levels in LNCaP and LNCaP95 cells (Fig. 1). However, it decreased AR-FL and AR-V7 protein levels in PTEN sufficient VCaP and 22Rv1 cells (Fig. 2).

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