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Keywords:

  • Akt/protein kinase B;
  • PI3K;
  • prostate cancer;
  • PTEN;
  • cell invasion;
  • cell survival

Abstract

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Activated phosphoinositide 3-kinase (PI3K) and its downstream target Akt/PKB are important signaling molecules and key survival factors involved in the control of cell proliferation, apoptosis and oncogenesis. We investigated the role of the PI3K-Akt signaling pathway in the invasion of prostate cancer cell lines and activation of this pathway in primary human prostate tumors. Treatment of human prostate cancer cells viz. LNCaP, PC-3 and DU145 with PI3K pharmacological inhibitor, LY294002, potentially suppressed the invasive properties in each of these cell lines. Restoration of the PTEN gene to highly invasive prostate cancer PC-3 cells or expression of a dominant negative version of the PI3K target, Akt also significantly inhibited invasion and downregulated protein expression of urokinase-type plasminogen activator (uPA) and matrix metalloproteinase (MMP)-9, markers for cell invasion, indicating a central role of the PI3K-Akt pathway in this process. Immunoblot analysis of PI3K and total/activated levels of Akt showed increased protein levels of catalytic (p110α/β) and regulatory (p85) subunits of PI3K and constitutive Akt activation in high-grade tumors compared to low-grade tumor and benign tissue. Immunohistochemical analyses further confirmed a progressive increase in p-Akt (p-Ser473) levels but not of total-Akt (Akt1/2) in cancer tissues compared to benign specimens. A successive increase in p-Akt expression was further noted in specimens serially obtained from individuals with time-course disease progression. Taken together, these results suggest that aberrant activation of PI3K-Akt pathway may contribute to increased cell invasiveness and facilitate prostate cancer progression. © 2007 Wiley-Liss, Inc.

During prostate cancer progression, tumor cells undergo a variety of molecular alterations that lead to the acquisition of uncontrolled growth properties.1 One such set of molecular alterations may be mediated by the PI3K-Akt signaling pathway.2, 3 Phosphoinositide 3-kinase (PI3K) is a heterodimeric protein composed of a catalytic subunit (p110α/β/γ/δ) and a regulatory subunit (p85α/β) that participate in multiple cellular processes, including cell growth, transformation, migration and differentiation.4 The PI3K pathway has been shown to be an essential survival mechanism in a number of cell types and in some forms of human cancer.2 This pathway is upregulated by several different mechanisms. Amplification of the gene coding for the catalytic subunit of PI3K has been observed in cervical and ovarian cancers.5, 6 Following activation of cells by growth factors or cytokines, PI3K is recruited to the plasma membrane, where it catalyzes the conversion of membrane phosphoinositide 4,5-biphosphate (PIP2) in the D3 position to generate phosphoinositide 3,4,5-triphosphate (PIP3). The accumulation of PIP3 creates a docking site for Akt at the plasma membrane, which binds to PIP3 via the pleckstrin homology domain. PIP3 binding induces a conformational change in Akt, exposing the critical Thr308/309 residue in the activation loop tophosphorylation by phosphotidylinositol-dependent kinase 1 (PDK-1). For full length activation, Akt is subsequently phosphorylated at Ser473/474 by an as yet unidentified kinase termed PDK-2.7 Activation of PI3K leads to the generation of PIP3 which can be counterbalanced by the action of PTEN/MMAC1/TEP1, a lipid phosphatase and tumor suppressor that dephosphorylates PIP3 back to PIP2, controlling the activation of Akt.2, 7 PTEN is frequently lost in glioblastoma, breast cancer, endometrial cancer, melanoma and prostate cancer.8, 9, 10, 11

Akt/protein kinase B/RAC-PK is an essential serine/threonine kinase and a core component of the PI3K signaling pathway whose activation has been implicated in the genesis or progression of various human malignancies.2, 7 The genes of PI3K targets, AKT1 and AKT2, are amplified and over-expressed in breast, gastric and ovarian cancers.12, 13 Akt3 activity is often increased in prostate and breast cancer.14 In experimental systems, constitutively active PI3K or Akt is oncogenic in cell culture systems and animal tumor models.15, 16 Several studies have shown that Akt/PKB activates the transcription of a wide variety of genes, especially those involved in immune activation, cell proliferation, apoptosis and cell survival.2, 7, 17 Activated Akt protects cells from apoptotic death by phosphorylating substrates such as BAD, procaspase-9, NF-κB and fork-head transcription family members.17 Akt activation affects cell cycle progression, through regulation of cyclin D stability and inhibition of p27/Kip1and p21/WAF1 protein levels.18 Akt has been shown to prolong cell survival by delaying p53-dependent apoptosis through MDM2 phosphorylation.19 Akt has also been shown to inhibit the Raf-MEK-ERK pathway through phosphorylation of Raf-1 and to overcome constitutively activated MAPK-induced cell cycle arrest.20 It has been proposed that Akt regulates permeability transition pore openings within the mitochondrial membrane by increasing the coupling of glucose metabolism to oxidative phosphorylation.21 Based on its role as a key regulator of cell survival, Akt has emerged as an important factor in tumorigenesis.22

Several studies have shown that various growth factors, cytokines and oncogenes, exert their effects via the PI3K signaling pathway, which, in turn, leads to Akt activation.23, 24 Increased Akt activity facilitates prostate cancer progression through downregulation of the cyclin-dependent kinase inhibitor, p27/Kip1.25 Akt has been shown to suppress androgen-induced apoptosis by phosphorylation and inhibition of androgen receptor.26 Conditional activation of Akt has been shown to promote androgen- independent progression and is essential for neuroendocrine differentiation of prostate cancer.27, 28 Development of hormone-insensitivity in patients who have been on long-term androgen ablation therapy for prostate cancer is associated with reinforcement of the PI3K-Akt pathway.29 Akt/PKB activation has been shown to correlate with increased angiogenesis and metastasis through hypoxia-inducible factor-1α.30 Increase of p-Akt expression, particularly at serine 473, has been shown to correlate with higher Gleason score and is an excellent predictor of poor clinical outcome in prostate cancer patients.31, 32 Although high levels of activated Akt expression have been demonstrated in prostate cancer,31, 32, 33 it is unclear if PI3K-Akt activation plays an essential role during prostate cancer progression. We investigated the role of the PI3K-Akt signaling pathway in this process in prostate cancer cell lines and in primary human prostate cancers.

Material and methods

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Tissue samples

Discarded benign and malignant prostate tissue from patients without any previous form of adjuvant therapy and who underwent surgery was obtained from the Tissue Procurement Facility of University Hospitals Case Medical Center and the Midwestern Division of the Cooperative Human Tissue Network. The Gleason grade and score of adenocarcinoma specimens were assigned by a surgical pathologist experienced in genitourinary pathology. Immediately after procurement, samples were snap frozen in liquid nitrogen and stored at −80°C till further use. In addition, 4-μm paraffin-embedded tissue sections were obtained from 15 benign prostate specimens and from 46 prostate cancer specimens along with 6 cases of retrospective needle biopsy specimens. These studies were approved by the Institutional Review Board at Case Western Reserve University.

Cell lines and reagents

Androgen-responsive human prostate cancer (LNCaP and 22Rv1) and androgen-refractory (DU145 and PC-3) cells were obtained from ATCC (Manassas, VA). Normal human prostate epithelial cells (NHPE) and culture medium were obtained from Clonetics® (Walkersville, MD). RPMI 1640 medium and all other cell culture materials were obtained from Life Technologies (Gaithersburg, MD). Dominant negative Akt in pUSEamp (K179M mutant) (DN-Akt) and the empty vector pUSEamp (+) were purchased from Upstate Cell Signaling (Lake Placid, NY). Lipofectamine™ 2000 and Geneticin (G-418 sulfate) were purchased from Invitrogen (Carlsbad, CA). The PI3K inhibitor, LY294002 was purchased from Sigma Chemical (St Louis, MO). Anti-Akt1/2, anti-PI3K (p110α/β), anti-MMP-9 and anti-uPA antibodies were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA). Antibodies for anti-p-Akt (Ser473), anti-p-Akt (Thr308), anti-PI3K (p85), anti-PTEN and anti-p-GSK-3α/β were purchased from Cell Signaling Technology® (Danvers, MA).

Cell culture treatments

NHPE cells were cultured in PrEBM media and supplements specially designed to support growth of human primary derived cells. Human prostate cancer cells were cultured under recommended condition in RPMI 1640 culture medium with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin. The cells were maintained at 37°C and 5% CO2 in a humid environment. At 60% confluence, cultures were switched to serum-free medium for 16 hr, and then treated with specified doses of PI3K inhibitor, LY294002 (5–20 μM), in complete cell culture medium for various time intervals. After preferred treatments, medium was aspirated, cells were harvested by the addition of Trypsin-EDTA and cell extracts were prepared as previously described.34

For the transfection experiments, PC-3 cells were transfected using Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA) according to the vendor's protocol. Cells in 100-mm dishes were co-transfected with 5 μg of pEGFP-C1-CMV (Clontech, Mountain View, CA) for green fluorescent protein (GFP) expression together with 1 μg of pCMV-XL5 vector or pCMV-XL5 vector encoding human cDNA clone PTEN (NM_000314) (OriGene, Rockville, MD) with pEGFP-C1-CMV G-418 resistance vector. Cells were subcultured at a 1:4 dilution 24 hr after transfection and maintained for 5 days in 500 μg/mL G-418-containing medium to eliminate untransfected cells. Transfection efficiency was determined by observing transfected cells under a UV microscope, 48 hr post-transfection and counting bright cells. For invasion assays, cells were serum starved in RPMI, 0.1% BSA during the last 24 hr of G-418 selection to exclude any effects of growth suppression on cell invasion.

Cell proliferation assay

The effect of LY294002 and DN-Akt on the viability of cells was determined by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyl tetrazoliumbromide) assay and the absorbance was recorded on a microplate reader at the wavelength of 540 nm. The effect of LY294002 and DN-Akt on cell growth inhibition was assessed as percent cell viability where vehicle-treated cells were taken as 100% viable.

Phospho-Akt (Ser473) ELISA assay

Following treatment with PI3K pharmacological inhibitor, the endogenous levels of phosphorylated Akt protein were determined by the PathScan™ Phospho-Akt (Ser473) sandwich ELISA kit (no. 7160) (Cell Signaling Technology) according to vendor's protocol. The PathScan Phospho-Akt ELISA kit detects endogenous levels of Akt only when it is phosphorylated at Serine 473. The magnitude of optical density of this assay is proportional to the quantity of phosphorylated Akt1 protein.

Invasion assay

Invasion was assayed using Biocoat Matrigel Invasion Chambers (24-well size; Becton-Dickinson, Franklin Lakes, NJ) according to manufacturer's protocol. All experiments were performed in triplicate. Cells were serum-starved in RPMI 1640, 0.1% BSA and seeded into the upper well of the invasion chamber in RPMI 1640, 0.1% BSA. RPMI 1640, 10% FBS was added to the bottom well of the chamber to serve as a chemoattractant. For pharmacological inhibition assays, 10,000 cells/well were seeded in the presence (both top and bottom chamber) or absence of inhibitors (treated with DMSO only), and invasion assessed after 24 hr by staining membranes with Diff-Quick cell staining kit (Fisher Scientific, Pittsburgh, PA). At least 5 independent fields were counted for each chamber. For transfection experiments, 10,000 G-418-selected cells were seeded into each chamber. Invasion was assessed after 24 hr by staining membranes with Diff-Quick; again, at least 5 independent fields were counted for each chamber. Invasion of GFP transfected cells was normalized to 1 and results for others were reported relative to GFP cells (relative invasion).

Immunoblot analysis

Frozen tissues (benign or cancer) and cultured cells were processed for tissue/cell extract and the protein content was determined using the DC Bio-Rad protein assay kit, as previously described.34 For immunoblot analysis, 40-μg protein was resolved using 4–20% polyacrylamide gels (Novex, Carlsbad, CA) and transferred to a nitrocellulose membrane. The blot was blocked in blocking buffer (5% nonfat dry milk/1% Tween 20; in 20 mM TBS, pH 7.6) for 2 hr at room temperature, incubated with appropriate primary antibody in blocking buffer for 2 hr at room temperature or overnight at 4°C, followed by incubation with the appropriate IgG secondary antibody conjugated to horseradish peroxidase (Amersham-Pharmacia, Piscataway, NJ) and detected by ECL-chemiluminescence and autoradiography using XAR-5 film (Eastman Kodak, Rochester, NY).

Immunohistochemistry

Immunohistochemical staining for p-Akt was performed using the SignalStain™ phospho-Akt (Ser473) IHC detection kit (Cell Signaling Technology). Briefly, 4-μm-thick paraffin-embedded sections from benign and cancer tissues were deparaffinized, rehydrated, immersed in target retrieval solution, and blocked for endogenous peroxidase activity. The sections were permeabilized in TNB-BB (100 mM Tris, pH 7.5, 150 mM NaCl, 0.5% blocking agent, 0.3% Triton-X and 0.2% saponin) and incubated with primary antibodies of prediluted p-Akt overnight at 4°C. Control sections were incubated with antisera in the presence of 10-fold excess of these antibodies or with isotype-matched IgG normal goat serum. After washing 3 times in TBS, sections were incubated for 2 hr at room temperature with biotinylated secondary antibody. Immunoreactive complexes were detected using diaminobenzidine substrate-chromagen. Slides were then counterstained in Mayer's hematoxylin, mounted in crystal mount media and dried overnight on a level surface. For fluorescence assay of p-Akt (Ser473), sections were subsequently incubated with 4.0 μg/mL Texas Red fragment of donkey anti-mouse IgG (Santa Cruz) and images were captured using a fluorescent microscope (Olympus, BX51).

Immunostaining evaluation

The immunostained sections were examined independently by 3 of the authors (SS, SG and GTM) using light microscopy. Computer-assisted morphometric methods were applied for analysis and nuclear staining of p-Akt in the high-grade cancer cells were used as positive control. Sections were examined with an inverted Olympus BX51 microscope and images were acquired with Olympus MicroSuite™ Five Software (Soft Imaging System, Lakewood, CO). The intensity of staining was graded semiquantitatively and each specimen was assigned a score on a scale from 0 to 3 designated as 0 (negative), 1+ when 10–20% of cells stain (weak), 2+ when 20–50% of cell stain (moderate) and 3+ when >50% of cell stain (strong). The immunoreactive score was designated by the percentage of positive cells and the staining intensity, as previously described.32 The percent of staining for p-Akt was scored by counting the positive-stained cells and total number of cells quantified in random microscopic fields (400× magnification) with the assistance of the software program.

Statistical analysis

Tumor grade (Gleason score) and p-Akt expression determined at different times were summarized by mean, median, range and SEM. The difference in tumor grade, p-Akt expression at various time intervals was examined by paired t-test. All tests were two-sided and p-values less than 0.05 were considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Constitutive PI3K expression and Akt phosphorylation is higher in some prostate cancer cells

To determine whether the PI3K-Akt signaling pathway is activated in human prostate cancer, we evaluated a panel of 5 human prostate-derived cell lines that included normal prostate epithelial (NHPE) cells, androgen-responsive prostate cancer (LNCaP and 22Rv1) and androgen-refractory prostate cancer (DU145 and PC-3) cells. The cells were deprived of serum and/or supplements overnight, and subsequently transferred to complete cell culture medium for 3 hr; following this, expression of PI3K and p-Akt was evaluated in total cell lysate. As shown in Figure 1a, protein levels of catalytic (p110α/β) and regulatory (p85) subunits of PI3K were modestly elevated in androgen responsive-LNCaP and refractory-PC-3 cells compared to NHPE and other human prostate cancer cells. Both LNCaP and PC-3 cells were highly phosphorylated at Ser473, compared to other cell lines examined. In vitro kinase assay using glycogen synthase kinase-3α/β as a substrate was performed to verify that phosphorylated Akt/PKB is enzymatically active in these cells. The highest level of Akt/PKB enzymatic activity was observed in PC-3 and LNCaP cells, which have shown higher levels of p-Akt previously (Figs. 1a and 1b). These results suggest that phosphorylation of Akt at Ser473 correlates precisely with Akt kinase activity.

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Figure 1. Protein expression of (a) catalytic (p110α/β) and regulatory (p85) subunits of PI3K, total Akt, p-Akt and PTEN in normal human prostate epithelial (NHPE), and carcinoma cells (LNCaP, 22Rv1, DU145 and PC-3). Cells were grown in serum/supplement-depleted medium for 16 hr, and switched to complete culture medium for 3 hr. Total cell extracts were prepared and electrophoresed by SDS-PAGE, followed by immunoblotting with anti-p110α/β, anti-p85, anti-Akt, anti-p-Akt (Ser473) and anti-PTEN in the total cell lysate. In vitro kinase assay of Akt immunoprecipitates from normal and carcinoma cells were determined using Akt kinase assay with glycogen synthase kinase (GSK)-3α/β as a substrate. To ensure equal protein loading the blots were stripped and reprobed with anti-α-tubulin antibody. (b) Densitometic analysis of each protein expressed percent relative to normal prostate epithelial cells. The details are described in Material and methods section.

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For further studies we selected 3 primary cancer cell lines, LNCaP, PC-3 and DU145, which are widely used and representative of advanced prostate cancer and are highly tumorigenic.35 LNCaP cells harbor PTEN mutations, and PC-3 cells harbor PTEN deletion.36 However, despite upregulated Akt status, the degree of dependence on the PI3K-Akt pathway for invasiveness varies between LNCaP and PC-3 cells (Table I). Therefore, to determine whether activation of PI3K-Akt may lead to prostate cancer progression, we used another androgen-refractory human prostate cancer cell line, DU145, which expresses modest protein expression of PI3K and p-Akt levels, without mutation of PTEN. As control, we used normal human prostate epithelial (NHPE) cells in these experiments.

Table I. Correlation of PTEN, PI3-Kinase and p-Akt Levels with Invasiveness in Various Human Prostate Epithelial Cells
Cell linePTENPI3Kp-AktInvasion
  1. NHPE, normal prostate epithelial cells; LNCaP and 22Rv1, androgen-responsive cancer cells; DU145 and PC-3, androgen-refractory cancer cells.

NHPENormal−−−−−−
LNCaPMutated+++++++
22Rv1Normal−−−−++
DU145Normal−−−−+++
PC-3Deleted++++++++

Pharmacological inhibition of the PI3K-Akt pathway reduces invasiveness of human prostate cancer cells

To determine whether activated Akt/PKB promotes invasiveness of prostate cancer cells, we used a pharmacological approach to alter PI3K-Akt activity and assessed whether this inhibition leads to diminished cell invasion ability. We used the Matrigel invasion-chamber assay, which measures 2 important factors that contribute to tumor aggressiveness, motility and invasiveness. Cells must first degrade a reconstituted basement membrane that occludes the pores of a filter and then move through these cleared pores to adhere to the other side. As expected, NHPE cells were completely noninvasive using this assay, while the LNCaP, PC-3 and DU145 cells were invasive. A highly specific pharmacological inhibitor of PI3K catalytic activity, LY294002, was used as an initial approach to assess the significance of the PI3K pathway in invasion. To initiate these experiments, for each cell line an optimal concentration had to be established with LY294002 that maximally suppressed its target pathway, but maintained cellular viability over the course of a 24-hr invasion assay. Thus, we determined a dose–response curve with PI3K inhibitor and examined pathway inhibition and effects on cellular viability. In the experiments shown in Figures 2a and 2b, lysates were prepared from all 3 cell lines after 16-hr treatment with LY294002 (5–20 μM) and were blotted with anti-Akt1/2, anti-p-Akt and anti-PI3K antibodies. While LY294002 inhibited Akt phosphorylation as expected, it had no effect on the total Akt1/2 levels in all 3 cancer cell lines. Treatment of all cancer cell lines at these doses of LY294002 up to 16 hr did not exhibit any significant change in cell viability; time chosen for a cut-off point (data not shown).

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Figure 2. Effect of LY294002 treatment on the protein expression of (a) regulatory (p85) subunit of PI3K, total Akt, p-Akt (Ser473) in human prostate cancer cells (LNCaP, PC-3 and DU145). Cells were grown in complete culture medium along with increasing doses of LY294002 for 16 hr. Total cell extracts were prepared and electrophoresed by SDS-PAGE, followed by immunoblotting with anti-p85, anti-Akt and anti-p-Akt (Ser473) in the total cell lysate. To ensure equal protein loading, the membrane was stripped and reprobed with anti-α-tubulin antibody. (b) Akt phosphorylation at Ser473 by ELISA in NHPE and human prostate cancer LNCaP, PC-3 and DU145 cells after treatment with increasing doses of LY294002. (c) For invasion assay, cells were either treated with DMSO (control) or cultured for 16 hr in the presence of increasing concentrations of LY294002. The details are described in Material and methods section. Bars ± SE, *p < 0.05, **p < 0.001, compared to control.

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In the final experiments, the concentration of PI3K inhibitor was chosen for each cell line that inhibited the target pathway by >90% while maintaining >90% cellular viability. Figure 2c shows the effect of LY294002 on cell invasion. While the total Akt level remains unchanged, inhibition of Akt phosphorylation showed positive correlation with decreased invasiveness in all 3 cancer cell lines. Compared to untreated controls, decrease in invasiveness and p-Akt levels were in the order PC-3 > LNCaP > DU145, respectively (Figs. 2b and 2c).

Biochemical manipulation of the PI3K-Akt pathway reducesinvasiveness in human prostate cancer PC-3 cells

Previous results demonstrated strong evidence for an essential role of the PI3K pathway in prostate cancer cell invasion. We sought to confirm these results independently by inhibiting the PI3K pathway with the expression of 2 negative regulators; the antagonist PTEN or a dominant negative version of Akt and its correlation to invasion markers.

PC-3 cells were co-transfected with pEGFP-C1-CMV (for GFP expression) vector encoding G-418 resistance with pCMV-XL5 human cDNA clone PTEN or DN-Akt pUSEamp(+). Cells were placed in G-418 containing media for 5 days to select cells expressing both plasmids, and then the same numbers of cells were seeded into invasion chambers in triplicate. G-418 selection eliminates untransfected cells, resulting in a pure population of expressing cells. As the expression of PTEN or DN-Akt in PC-3 cells may inhibit their growth, cells were serum-starved during the last 24 hr of G-418 selection to suppress growth, excluding contributions of growth suppression in the invasion assay as previously demonstrated.37 After selection, transfected cells were also seeded onto coverslips and stained with DAPI, which revealed no evidence of condensed or fragmented nuclei (morphological changes indicative of apoptosis) in the GFP, GFP-PTEN or GFP/DN-Akt transfectants (data not shown). In addition, in parallel experiments, it was determined that transfection of PTEN or DN-Akt had no significant effect on cell number or viability of selected cells during the 24-hr period of the invasion assay (data not shown). In a separate study, adenoviral-mediated overexpression of MMAC/PTEN in PC-3 cells reduced cell numbers by 46% at 6 days after infection.36 Increasing levels of MMAC/PTEN were associated with more growth inhibition.36, 38 While there was no growth inhibition by PTEN during the 24-hr period used it is likely that the expression levels of PTEN achieved did not approach those obtained with adenoviral-mediated overexpression.

As shown in Figure 3a, the effect of expression of PTEN and DN-Akt on Akt phosphorylation in PC-3 cells was visualized by Western blotting. The expression of PTEN coincides with abolished Akt phosphorylation similar to that observed with 20 μM LY294002 treatment for 24 hr, while expression of DN-Akt reduced Akt phosphorylation by 85%. The extent of inhibition of invasion by PTEN, DN-Akt and LY294002 treatment was quantified, with GFP invading cells normalized to 1 (Fig. 3b). The expression of PTEN inhibited the invasive properties of PC-3 cells by 95%, expression of DN-Akt inhibited invasion by 92% and LY294002 inhibited invasion by 80%. This experiment was repeated 3 times and the results shown in Figure 3b are representative. Interestingly, inhibition of p-Akt expression correlated with decreased protein expression of urokinase-type plasminogen activator (uPA) and matrix metalloproteinase (MMP)-9, markers for cell invasion. These results confirm the observations from our pharmacological inhibition studies, together with biochemical manipulation indicating that the PI3K-Akt pathway is essential in the invasive behavior of prostate cancer cells.

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Figure 3. Inhibition of PC-3 cell invasion by PTEN overexpression, PI3K inhibitor (LY240092) treatment and Akt dominant-negative approach. (a) Cells were transfected with either pEGFP-C1-CMV (for GFP expression) vector encoding G-418 resistance with pCMV-XL5 human cDNA clone PTEN or DN-Akt pUSEamp (+) or treated with 20 μM LY294002 for 24 hr. Total cell extract were prepared and electrophoresed by SDS-PAGE, followed by immunoblotting with anti-PTEN, anti-p-Akt (Ser473), anti-uPA and anti-MMP-9 antibodies. For protein loading the blots were stripped and reprobed with anti-α-tubulin antibody. (b) PTEN- and DN-Akt transfected and LY294002 treated cells were seeded into invasion chambers in triplicate and invasion was measured. The relative number ± SE of invading cells is plotted for each condition. Bars ± SE, **p < 0.001.

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Constitutive PI3K expression and Akt phosphorylation are elevated in human prostate cancer specimens

Next we sought to ascertain whether the PI3K-Akt pathway, Akt phosphorylation in particular, was overactive in human prostate adenocarcinoma. We performed immunoblot analysis for PI3K (catalytic subunit p110α/β and regulatory subunit p85), total- and activated-Akt levels in benign prostate tissue and prostate cancer specimens. As shown in Figure 4a, immunoblot analysis for catalytic and regulatory subunits of PI3K exhibited a significant increase in high-grade cancer tissue (Gleason score 5+4; and 5+5) compared with low-grade cancer (Gleason score 3+3) and benign tissue. A similar pattern of protein expression for activated Akt (p-Akt; Ser473) was observed as previously shown for PI3K with significantly higher levels in high-grade cancer tissue compared to low-grade cancer and benign tissue. However, no significant variation in the protein expression of total Akt (Akt1/2) was observed in these tissues (Fig. 4a). Similar results were observed for p-Akt (Thr308) in these tissues (data not shown). We also compared the protein levels of PTEN in these tissues. For our studies, we used monoclonal antibody produced by immunizing mice with a synthetic peptide (KLH coupled) derived from the carboxyl terminus sequence of human PTEN recognizing a 54-kDa protein. The endogenous PTEN protein was detected in all tissue specimens examined. Compared to benign tissue, PTEN protein expression was higher in low-grade cancer with a loss of expression observed in high-grade cancer tissues (Fig. 4a). Our results are in agreement with previous studies reporting loss of PTEN protein expression in advanced grade cancer, which subsequently leads to prostate cancer progression.39, 40 The densitometric analysis for these proteins are shown in Figure 4b.

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Figure 4. Protein expression of (a) catalytic (p110α/β) and regulatory (p85) subunits of PI3K, total Akt, p-Akt (Ser473) and PTEN in benign prostate tissue and prostate cancer specimens. Total cell extracts from tissues were prepared and electrophoresed by SDS-PAGE, followed by immunoblotting with anti-p110α/β, anti-p85, anti-Akt, anti-p-Akt (Ser473) and anti-PTEN antibodies. Cancer tissue is subdivided into low-grade cancer (Gleason score <7) and high-grade cancer (Gleason score 7–10). (b) Densitometic analysis of each protein normalized to epithelial content by probing the blots with anti-cytokeratin (CK) 18 antibodies. Bars ± SE, *p < 0.05, **p < 0.001, compared to benign tissue. (c) Immunostaining for activated Akt (p-Akt, Ser473) and its evaluation in representative samples of benign prostate tissue and prostate cancer specimens of various Gleason grades. Magnification ×200. Evaluation of total sections based on staining intensity scored as weak, moderate or strong. A detailed staining pattern of benign and prostate cancer specimens is shown below the figures. The details are described in Material and methods section. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

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Next we examined the protein expression of p-Akt (Ser473) by immunohistochemical analysis in benign prostate tissue and prostate cancer specimens. Cancers were analyzed according to Gleason grade and tissue specimens were assigned to 4 subgroups consisting of benign tissue, low-grade cancer (Gleason score 2–4), medium-grade cancer (Gleason score 5–7) and high-grade cancer (Gleason score 8–10). The degree of immunoreactivity of these tissues was assessed by evaluation of the amount and intensity of tissue immunostaining in the sections. As shown in Figure 4c, benign tissues did not exhibit any significant p-Akt expression. A progressive increase in the staining intensity of p-Akt (Ser473) with increasing tumor grade was observed in the prostate cancer specimens. Of 15 benign specimens that were analyzed, 2 exhibited weak expression and 13 were negative for p-Akt. In 6 low-grade tumors, moderate p-Akt expression was observed in 1, 1 exhibited weak staining and 4 showed no p-Akt staining. In 18 medium-grade tumors, strong p-Akt expression was observed in 6, 4 exhibited moderate staining, 3 exhibited weak staining and 5 showed no p-Akt staining. In 22 high-grade tumors, 10 exhibited strong staining, 6 exhibited moderate staining, 3 exhibited weak staining and 3 were negative for p-Akt staining (Fig. 4c).

Additionally, in 6 cases we had an opportunity to evaluate sequential samples of prostate tissue obtained at different periods of the patient's life; for example, 2 initial prostate biopsies followed within 5 years by a transuretheral resection of the prostate. These specimens were studied to address whether levels of p-Akt increases with increased malignancy. An increase in the levels of p-Akt (Ser473) was observed in all these specimens, which correlated with increasing Gleason score (Figs. 5a5c). Since activation of Akt is essential for efficient prostate cancer cell invasion, and tumor invasion correlates strongly with poor prognosis in patients, it seems probable that activation of the PI3K-Akt pathway is critical in the progression of some prostate tumors.

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Figure 5. Time-course evaluation of p-Akt (Ser473) expression and its correlation with tumor progression. (a) Immunoflourescence staining for p-Akt (Ser473) in needle biopsy specimens and resected tissue from same patient (no. 1). The H&E staining is shown above each panel. Magnification ×100. (b) Evaluation of total sections based on staining intensity and its correlation with tumor grade. (c) Statistical analysis of longitudinal data of tumor grade and p-Akt expression in consecutive specimens along with the p-values. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

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Discussion

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Prostate cancer cells utilize multiple molecular pathways to proliferate and invade tissue during the course of tumor progression.1 Among several independent cell survival signaling pathways, upregulation of PI3K-Akt signaling through mutations in the PTEN gene and constitutive activation of growth factor receptors are particularly important.3, 7 Lipid products of PI3K provide an anchor for assembling signaling proteins at specific locations in the membrane in response to cell stimulation. These signaling proteins coordinate complex events that lead to changes in cell metabolism, cell growth, cell motility, invasiveness and survival.7 The PI3K-Akt signaling pathway is believed to play an important role in the genesis of some human cancers.2 However, the role of the PI3K-Akt signaling pathway in the progression of prostate cancer has not been yet established. Because Akt provides strong cell survival signals in response to external stimuli and is activated in numerous human malignancies,5, 6, 12, 13, 14 we hypothesized that PI3K-Akt signaling plays a role in prostate carcinogenesis. In this study, we have shown not only that PI3K and Akt are constitutively activated in human prostate adenocarcinoma, but also that they enhance invasiveness resulting in tissue invasion. In addition, we have shown that increasing Akt activation correlates with increasing grade of prostate cancer, suggesting that Akt immunostaining may be useful as a prognostic indicator of tumor aggressiveness.

Accumulating evidence indicates a potential role for the PI3K pathway in prostate cancer progression. Mutation and/or loss of function in the negative regulator PTEN has been observed in advanced stage human prostate cancer9, 39, 40 and in xenograft models.36, 41 Loss of PTEN function through PTEN mutations in murine models has been shown to be associated with neoplasia in multiple organ sites, including endometrium, liver, gastrointestinal tract, thyroid, thymus and prostate.42 Loss of PTEN function in prostate cancer cells has been shown to be associated with increased proliferation, angiogenesis and tumorigenesis.43, 44 In nearly 50% of prostate cancers, the PI3K-Akt survival pathway has been shown to be constitutively upregulated because of loss of function and/or mutations of tumor suppressor PTEN, which functions as a negative regulator of PI3K through its lipid phosphatase activity.39, 40 Previous studies have demonstrated that loss of PTEN function in some prostate cancer cells leads to higher Akt expression,41 a feature that may promote prostate cancer progression. We have additionally observed that PTEN protein expression diminishes with increasing grade of cancer, with maximum loss of expression observed in high-grade cancer specimens of Gleason score 8–10. These observations suggest that aberrant activation of the PI3K-Akt pathway by any number of mechanisms may contribute to increased tumor invasiveness and cancer progression.

We evaluated the role of constitutive Akt activation in cell invasion by studying a PI3K pharmacological inhibitor, LY294002, as well as a dominant negative mutant construct of Akt (DN-Akt). Highly invasive LNCaP and PC-3 prostate cancer cells harbor mutations or deletions of PTEN, and each has a high level of PI3K activity, judging from the LY294002-mediated inhibition of Akt phosphorylation that we observed. LY294002 suppressed the invasive properties of each of these cell lines, and reconstitution of intact PTEN into PC-3 cells also efficiently suppressed invasion. This indicates that the 3-phosphorylated lipid products of PI3K are necessary for cellular invasion. In support of our findings, a previous study has demonstrated that treatment of prostate cancer cells with PI3K inhibitor, LY294002, resulted in cell cycle-mediated arrest and induction of apoptosis.45 In addition, androgen receptor-mediated Akt activation has also been shown to enhance cell growth and survival of prostate cancer cells, through the nuclear β-catenin signaling pathway.46 More recent studies have demonstrated that conditional Akt activation promotes androgen-independent progression and is essential for neuroendocrine differentiation of prostate cancer.27, 28 While the PI3K-Akt pathway is critical for prostate cancer invasion, several other pathways are known to be involved in tumor cell invasion. These include focal adhesion kinase, C-Jun-N terminal kinase, phospholipase C-δ and Ras/ERK1/2, which are involved in invasion of many cancer types including prostate cancer and their activation is sufficient to induce invasive behavior.47, 48

Activation of the PI3K/Akt pathway confers chemotherapeutic resistance in numerous tumor types including cancers of lung, cervix, ovary, pancreas, bladder and breast.2–5,, 49 Adenoviral-mediated expression of PTEN inhibits proliferation and metastasis in human prostate cancer PC-3 cells.37 Overexpression of PTEN and diminution of Akt phosphorylation restores doxorubicin sensitivity to the doxorubicin-resistant prostate cancer PC-3 cells.50 All of the aforementioned studies suggest that PI3K and Akt may be promising molecular targets in the management of prostate cancer. As the pathway appears to be involved in several cellular processes it will be important to identify biochemical and gene targets of PI3K-Akt activation that specifically lead to increased tumor aggressiveness and chemotherapeutic resistance.

In summary, our studies suggest that constitutive PI3K-Akt activation actively contributes to the progress of prostate cancer from organ-confined disease to highly invasive and potentially metastatic disease. Our results also point to the possibility that p-Akt might be used as a marker for those low-grade tumors that are at risk of progression to high-grade invasive tumors. We found that phosphorylation and activation of Akt increases tumor invasiveness, which increased in parallel with increasing grade of cancer, with maximum activation observed in high-grade cancer specimens (Gleason score 8–10). Thus, PI3K-Akt activation may be an important prognostic indicator of tumor aggressiveness. Furthermore, PI3K-Akt and its associated regulatory signaling pathways are potential targets for therapeutic intervention and molecular-based approaches for management of prostate cancer in humans.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

This work was conducted in The James and Eilleen Dicke Research Laboratory, Department of Urology, Case Western Reserve University, Cleveland, OH.

References

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
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