Suppression Versus Induction of Androgen Receptor Functions by the Phosphatidylinositol 3-Kinase/Akt Pathway in Prostate Cancer LNCaP Cells with Different Passage Numbers

Abstract

The phosphatidylinositol 3-kinase (PI3K)/Akt pathway controls several important biological functions, such as cell growth regulation, apoptosis, and migration. However, the way in which PI3K/Akt controls androgen receptor (AR)-mediated prostate cancer cell growth remains unclear and controversial. Here Prostate cancer is the second leading cause of cancer-related death among men in the United States. The normal prostate and prostate cancers at early stages require androgen for growth and survival. In addition to androgen signaling, which plays an essential role in survival of prostate cancer, the phosphatidylinositol 3-kinase (PI3K) 1 /Akt pathway represents another important survival signal for prostate cancer cells. It appears that these two pathways can compensate for each other in growth regulation of prostate cancer LNCaP cells, because androgen treatment can rescue cells from apoptosis induced by application of PI3K inhibitors (1). Furthermore, activation of the PI3K/Akt pathway protects cells from apoptosis induced by serum starvation and androgen deprivation (2).Recent rapid progress of the PI3K/Akt signal pathway studies, as well as its influence on the androgen receptor (AR)-mediated prostate cancer growth, has resulted in many exciting yet controversial results. Here we address these controversial results by summarizing Akt-AR-related results and provide new data, as well as possible explanations for the distinct roles of the PI3K/Akt pathway in AR-mediated prostate cancer growth. Particular emphases will be: 1) Akt suppresses versus induces AR activity, 2) Akt phosphorylation sites on AR protein, and 3) promotion of AR degradation by the PI3K/Akt pathway. EXPERIMENTAL PROCEDURESReagents-pCDNA3 cAkt (3) and mutant AR S210A/S790A were described previously (4). pCDNA3-PTEN was a gift from Dr. Charles L. Sawyers, and pGEX-KG-PTEN was from Dr. Frank B. Furnari. Insulinlike growth factor-1 (IGF-1) and LY294002 was from Calbiochem. 5␣-Dihydrotestosterone (DHT), doxycycline (Dox), and cycloheximide were from Sigma. The anti-AR polyclonal antibody, NH27, was produced as described previously (3). The mouse monoclonal PTEN and prostatespecific antigen (PSA) antibodies and the goat polyclonal ␤-actin antibody were from Santa Cruz Biotechnology. The mouse monoclonal Akt and phospho-Akt (Ser 473 ) antibodies were purchased from Cell Signaling.Cell Culture and Transfections-DU145, PC-3, and COS-1 cell lines were maintained in Dulbecco's minimum essential medium containing penicillin (25 units/ml), streptomycin (25 g/ml), and 10% fetal calf serum (FCS). LNCaP cells were maintained in RPMI 1640 with 10% FCS. Transfections were performed using SuperFect TM according to standard procedures (Qiagen).Luciferase Reporter Assays-Luciferase reporter assay was as described previously with some modifications (5). The cells were transfected with plasmids in 10% charcoal-stripped serum (CSS) medium for 16 h and then treated with ethanol or 10 nM DHT for 16 h. The cells were lysed, and luciferase activity was detected by the dua...

Phosphorylation of the androgen receptor is associated with reduced survival in hormone-refractory prostate cancer patients

“…Cell line work demonstrates that Akt activity increases during androgen ablation to stimulate cell growth and survival when androgen reliance is weaker, and therefore promote development of HRPC (Gao et al, 2003;Ghosh et al, 2003;Lin et al, 2003). Work using human prostate tissue confirms that pAkt 473 is expressed in PIN and invasive prostate cancer, and staining intensity positively correlated with PSA levels and Gleason grades (Ghosh et al, 2003;Altomare and Testa, 2005;Majumder and Sellers, 2005).…”

“…However, during hormone ablation or antihormone treatment, LNCaP cells undergo growth arrest and apoptosis, and Akt activity is upregulated (more than 20-fold higher), resulting in stimulation of cell growth, compensating for the effects of androgen withdrawal (Gao et al, 2003). Data from these experiments suggest that Akt signals for cell growth and survival at low levels of androgen, and therefore may promote development of HRPC (Ghosh et al, 2003;Lin et al, 2003). This is supported by a report that demonstrates that upregulation of the PI3K cascade allows cells to grow in the presence of antiandrogens and contributes to failure of endocrine therapy (Murillo et al, 2001).…”

“…In vitro studies demonstrate that Akt/protein kinase B phosphorylates AR at serine residues (Ser 210 and Ser 790 ) resulting in modulation of AR transcriptional activity (Lin et al, 2001(Lin et al, , 2003 and suggesting that AR phosphorylation might promote development of HRPC. It has also been reported that activation of phosphatidylinositol 3-OH kinase (PI3K)/Akt pathway can induce expression of AR at the protein and mRNA level, again suggesting that this pathway may be involved with hormone-refractory disease .…”

“…It has also been reported that activation of phosphatidylinositol 3-OH kinase (PI3K)/Akt pathway can induce expression of AR at the protein and mRNA level, again suggesting that this pathway may be involved with hormone-refractory disease . Akt is a downstream member of the PI3K cascade, which plays an important role in cell growth, death, adhesion and migration and is frequently activated in cancer cells (Lin et al, 2003). Akt is activated when phosphorylated at threonine 308 and subsequently serine 473.…”

Androgen Action During Prostate Carcinogenesis

“…Indeed, androgen-bound AR physically binds to the p85α regulatory subunit of PI3K and thereby activates PI3K/AKT (4,5). This is truly a two-way crosstalk, since AR expression and activity are also regulated by PI3K/AKT (67)(68)(69). Additional details about the crosstalk between androgen/AR and PI3K/AKT pathways have previously been discussed in several other reviews (61,70).…”