- Top of page
- Material and methods
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.
- Top of page
- Material and methods
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.