Impacts of PI3K/AKT inhibitors to AR protein levels in LNCaP and LNCaP95 cells.

<p>(<b>A</b>) LNCaP and (<b>B</b>) LNCaP95 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. (<b>C</b>) 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.</p

Complex Impacts of PI3K/AKT Inhibitors to Androgen Receptor Gene Expression in Prostate Cancer Cells

<div><p>Background</p><p>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.</p><p>Methods</p><p>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.</p><p>Results</p><p>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.</p><p>Conclusion</p><p>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.</p></div

LY294002 and AZD5363 affect AR gene transcription initiation, RNA splicing and AR mRNA stability.

<p>(<b>A</b>) 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108780#s2" target="_blank">Material and Method</a> section. (<b>B</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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108780#s2" target="_blank">Material and Method</a> section. (<b>C</b>) 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. (<b>D</b>) 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.</p

Impacts of PI3K/AKT inhibitors to AR mRNA levels in PCa cells.

<p>LNCaP, LNCaP95, VCaP and 22Rv1 cells were treated with increasing doses of PI3K/AKT inhibitors LY294002 (0, 25, 50 uM), Wortmanin (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. AR-FL (<b>A</b>) and AR-V7 (<b>B</b>) 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. Data was 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.</p

Impacts of PI3K/AKT inhibitors to AR protein levels in VCaP and 22Rv1.

<p>(<b>A</b>) VCaP and (<b>B</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. (<b>C</b>) 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.</p

Prostate Stromal Cells Express the Progesterone Receptor to Control Cancer Cell Mobility

<div><p>Background</p><p>Reciprocal interactions between epithelium and stroma play vital roles for prostate cancer development and progression. Enhanced secretions of cytokines and growth factors by cancer associated fibroblasts in prostate tumors create a favorable microenvironment for cancer cells to grow and metastasize. Our previous work showed that the progesterone receptor (PR) was expressed specifically in prostate stromal fibroblasts and smooth muscle cells. However, the expression levels of PR and its impact to tumor microenvironment in prostate tumors are poorly understood.</p><p>Methods</p><p>Immunohistochemistry assays are applied to human prostate tissue biopsies. Cell migration, invasion and proliferation assays are performed using human prostate cells. Real-time PCR and ELISA are applied to measure gene expression at molecular levels.</p><p>Results</p><p>Immunohistochemistry assays showed that PR protein levels were decreased in cancer associated stroma when compared with paired normal prostate stroma. Using <i>in vitro</i> prostate stromal cell models, we showed that conditioned media collected from PR positive stromal cells inhibited prostate cancer cell migration and invasion, but had minor suppressive impacts on cancer cell proliferation. PR suppressed the secretion of stromal derived factor-1 (SDF-1) and interlukin-6 (IL-6) by stromal cells independent to PR ligands. Blocking PR expression by siRNA or supplementation of exogenous SDF-1 or IL-6 to conditioned media from PR positive stromal cells counteracted the inhibitory effects of PR to cancer cell migration and invasion.</p><p>Conclusions</p><p>Decreased expression of the PR in cancer associated stroma may contribute to the elevated SDF-1 and IL-6 levels in prostate tumors and enhance prostate tumor progression.</p></div

PR negatively regulates prostate cancer migration through a paracrine pathway.

<p>Conditioned media (CM) were collected from parental hCAFs, WPMY-1 or their derived cell lines expressing mock, PRA or PRB in the presence of vehicle or 10 nM P4. PC-3 cells were seeded in 6 well plates and incubated with CM from hCAFs (<b>A</b>) or from WPMY-1 (<b>B</b>) cells for 24 hours in wound healing assays. Representative images after 24 hour CM treatment were captured by an inverted microscope. WPMY-1 and its derived cell lines expressing mock, PRA or PRB were treated with 0, 10 nM and 100 nM of P4 for 24 hours (<b>C and E</b>) or with vehicle, 10 nM of P4 and/or 10 uM of RU486 for 24 hours (<b>D and F</b>). CM were then collected and incubated with PC-3 cells (<b>C–D</b>) and C4-2B (<b>E–F</b>) in cell migration assays as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092714#s2" target="_blank">material and methods</a> section. One-way ANOVA and paired student's t-test calculate the statistical significance set at P<0.05 as * and P<0.001 as ***.</p

Pathological Parameters of Study Patients and PSA Levels.

<p>Pathological Parameters of Study Patients and PSA Levels.</p

PR represses transcription of SDF-1 and IL-6 genes.

<p>WPMY-1 cells expressing mock, PRA or PRB were transiently transfected with control siRNA or siRNA against PR. SDF-1 (<b>A</b>) and IL-6 (<b>B</b>) mRNA levels relative to GAPDH were measured by real-time PCR. hCAFs expressing mock, PRA or PRB isoform were treated with either control or 20 ug/ml of cycloheximide for 16 hours. Real-time PCR assays measured mRNA levels of SDF-1 (<b>C</b>) and IL-6 (<b>D</b>) relative to GAPDH. (<b>E</b>) WPMY-1 cells and their derived cells expressing mock, PRA or PRB were treated with vehicle or 10 nM of P4 for 24 hours. Chromatin immunoprecipitation assays were performed using acetyl-Histone 3 antibody. Eluted DNA fragments were subjected to measure the enrichment of acetyl-Histone 3 levels in SDF-1 and GAPDH promoter regions. One-way ANOVA and student's t-test calculated the significance set with P<0.01 as * and P<0.001 as ***.</p

PR inhibitory effects to cancer cell mobility are mediated by SDF-1 and IL-6.

<p>WPMY-1 cells expressing mock, PRA or PRB were transiently transfected with control siRNA or siRNA against PR for 24 hours. Cells were washed twice with PBS buffer and replenished with serum free DMEM medium for 48 hours. CM were collected and incubated with PC-3 (<b>A and C</b>) or C4-2B (<b>B and D</b>) cells for cell migration assays (<b>A and B</b>) and Matrigel invasion assays (<b>C and D</b>) as described in Material and Method section. (<b>E</b>) CM were collected from hCAFs in the presence of vehicle or 10 nM of P4 and then mixed with vehicle, 10 ng/ml of SDF-1 or 10 ng/ml IL-6 and incubated with PC-3 cells in Matrigel invasion assays. One-way ANOVA and paired student's t-test calculate the level of significance set at P<0.05 as * and P<0.001 as ***.</p