Anti-androgen 2-hydroxyflutamide modulates cadherin, catenin and androgen receptor phosphorylation in androgen-sensitive LNCaP and androgen-independent PC3 prostate cancer cell lines acting via PI3K/Akt and MAPK/ERK1/2 pathways
Introduction
Cadherins and catenins are major contributors to cell-cell adhesion, playing crucial roles in morphogenetic processes and maintaining integrity and homeostasis in adult prostate. Alterations in these proteins are involved in a number of important phenomena related to prostate cancer progression, including cellular de-differentiation, invasiveness of tumor cells and metastasis (Mol et al., 2007). Epithelial cadherin (E-cadherin) is the classical cadherin present on normal prostatic epithelium as well as in tumor cells. Its cytoplasmic domain binds p120 catenin and β-catenin proteins, which play dual role regulating cell adhesion and transcription (reviewed in McEwen et al., 2012). It is well established that disturbance of the E-cadherin/catenin complex during tumor progression is correlated with the onset of an invasive phenotype (Bracke et al., 1996). Multiple mechanisms of E-cadherin/catenin complex inactivation in tumor cells have been described, including genetic mutation and gene inactivation through methylation (Noë et al., 2001). In the invasive prostate cancer N-cadherin expression is enhanced concurrent with E-cadherin downregulation (Kolijn et al., 2015). Over the past decades, evidence continues to mount showing that increase of N-cadherin induce an invasive morphology of tumor cells, stimulate migration and metastasis (Hazan et al., 2000). N-cadherin is highly expressed in PC3, but not in LNCaP and Du145 human prostate cancer cell lines (Nalla et al., 2011).
Function of cadherin/catenin complex is however dependent not only on the expression level of particular proteins, but is regulated by posttranscriptional mechanisms. It was demonstrated that phosphorylation of cadherins modulate β-catenin binding, which is relevant to both adhesive and nuclear signaling functions of β-catenin. Also, β-catenin phosphorylation can affect cadherin binding and adherens junctions stability. Phosphorylation of cadherins and catenins may be mediated by multiple kinases and different kinase-dependent pathways (Daugherty and Gottardi, 2007).
In cancer research a special reference is given to the mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2 (MAPK/ERK1/2) and phosphoinositide-3 kinase/protein kinase B (PI3K/Akt) pathways, since they are critical for cancer cell survival, proliferation and migration (Grant, 2008). The signaling via MAPK/ERK1/2 pathway is usually initiated by small G proteins (e.g. Ras). Signal is further transmitted by Ser/Thr kinase Raf (A-Raf, B-Raf or C-Raf-1), which activates the dual-specificity kinases MEK1 and MEK2. MEK1/2 kinases sequentially phosphorylate Tyr and Thr in Ser/Thr kinase ERK1/2, which in turn regulates a wide variety of proteins (Burotto et al., 2014). Activation of PI3K/Akt pathway is induced by activated PI3K that produces phosphatidylinositol-3,4,5-trisphosphate from phosphatidylinositol (3,4)-bisphosphate. Phosphatidylinositol-3,4,5-trisphosphate binds the N-terminal pleckstrin homology (PH) domain of the Ser/Thr kinase Akt facilitating Akt recruitment to the plasma membrane. At the plasma membrane Akt is activated through dual - Thr 308 and Ser 473 - phosphorylation by 3-phosphoinositide-dependent protein kinase 1 (PDK1) and the mammalian target of rapamycin (mTOR) (Robbins and Hague, 2016). Dysregulation of these pathways is implicated in many advanced prostate cancers (Liao et al., 2003, Edlind and Hsieh, 2014, Zarif and Miranti, 2016). Moreover, cellular effects induced by the activation of particular pathways may differ dependent on the prostate cell characteristics. For example, in androgen-independent PC3 cells the silencing of Akt gene caused inhibition of Akt and β-catenin expression, while in androgen-sensitive LNCaP cell line β-catenin expression remained unchanged (Dulinska-Litewka et al., 2013).
Numerous experimental studies and clinical data clearly indicate that in early prostate cancer androgen signaling is a critical survival pathway for cancer cells. However over time an androgen independent state of prostate cancer develops. In consequence, resistance to androgen deprivation therapy is observed (Watson et al., 2015). An important mechanism of the drug resistance is AR mutation and generation of constitutively active AR splice variants that are no longer dependent on androgen (Korpal et al., 2013). Furthermore, hormone depletion upregulates Akt and ERK1/2 activity in cancer cells to mediate AR downregulation (Hong et al., 2011). Activity of the AR may be also regulated at cytoplasmic level by AR phosphorylation which affects recruitment of co-regulators to the receptor (for review see Koryakina et al., 2014). It was recently reported that anti-androgens may inhibit AR nuclear translocation, resulting in cytoplasmic accumulation of AR and elevated Src signaling (Efstathiou et al., 2015, Zarif et al., 2015). These current findings necessitate the further studies on anti-androgen effects independent of targeting the nuclear functions of AR in prostate cancer cells.
In the study reported herein we focused on anti-androgen mediated cytoplasmic signaling (referred to as non-genomic signaling) with special emphasis on the proteins involved in cell-cell adhesion. Such signaling does not directly or initially influence gene expression, but rather drives rapid effects (in the seconds to minutes range), e.g. changes in proteins' activity (Lösel and Wehling, 2003). The study was designed to 1) assess rapid effects of anti-androgen 2-hydroxyflutamide (HF) on cadherin/catenin complex and AR in androgen-sensitive LNCaP and androgen-independent PC3 prostate cancer cell lines, and 2) to reveal signaling pathways that mediate these effects.
Section snippets
Prostate cell lines and culture conditions
LNCaP (androgen-sensitive) and PC-3 (androgen-independent) cell lines were obtained from American Type Culture Collection (Manassas, Virginia, USA). The cells were cultured in RPMI-1640 medium supplemented with 10% fetal calf serum (FCS) (Gibco, Grand Island, NY, USA), 1% l-glutamine (Sigma–Aldrich, St. Louis, MO, USA) and gentamycin (50 mg/l, KRKA, Warszawa, Poland). Semi-confluent cell cultures, initially seeded in 12-well culture dishes at a density of 1 × 105 per well for lysate preparation,
Effect of hydroxyflutamide on cadherins and β-catenin phosphorylation in LNCaP and PC3 cells
To determine whether HF affects E-cadherin, N-cadherin and β-catenin expression and phosphorylation, cells were treated with 10− 4 M HF for 0, 5, 15, 30, 60 min and the levels of total and phosphorylated cadherins and β-catenin were analyzed by western blotting and immunocytochemistry. The effect of androgen on phosphorylation of these proteins was also evaluated (Fig. 1, Fig. 2).
Irrespective of the incubation period (5–60 min) the levels of total E-cadherin and β-catenin in LNCaP cell line were
Discussion
We and others previously demonstrated that the expression of adherens junction proteins in normal prostate and prostate cancer is modified following anti-androgen treatment (Patriarca et al., 2003, Gorowska et al., 2014). The studies presented here provide new insights into the cytoplasmic effects of anti-androgens on cadherin and catenin phosphorylation in prostate cancer cells. We found that in LNCaP cells incubated with HF rapid increase of E-cadherin phosphorylation at Ser 838/840 residues
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Acknowledgements
This work was supported by a grant no. 9000871 from the Society for Biology of Reproduction, Poland, and by K/DSC/002975 and K/ZDS/006308 funds.
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