ORIGINAL RESEARCH ARTICLE
Journal of
b-Arrestin2 Contributes to Cell Viability and Proliferation via the Down-Regulation of FOXO1 in Castration-Resistant Prostate Cancer
Cellular Physiology
XIAOLU DUAN,1 ZHENZHEN KONG,1 YANG LIU,1 ZHIWEN ZENG,2 SHUJUE LI,1 WENQI WU,1 WEIDONG JI,1 BICHENG YANG,1 ZHIJIAN ZHAO,1 AND GUOHUA ZENG1* 1
Department of Urology, Minimally Invasive Surgery Center, the First Affiliated Hospital of Guangzhou Medical University, Guangdong Key Laboratory of Urology, Guangzhou, Guangdong, China
2
Key Laboratory of Neurogenetics and Channelopathies of Guangdong Province and the Ministry of Education of China, Collaborative Innovation Center for Neurogenetics and Channelopathies, Institute of Neuroscience and the Second Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong, China
b-Arrestin2 has been identified to act as a corepressor of androgen receptor (AR) signaling by binding to AR and serving as a scaffold to affect the activity and expression of AR in androgen-dependent prostate cancer cells; however, little is known regarding its role in castration-resistant prostate cancer (CRPC) progression. Here, our data demonstrated that b-arrestin2 contributes to the cell viability and proliferation of CRPC via the downregulation of FOXO1 activity and expression. Mechanistically, in addition to its requirement for FOXO1 phosphorylation induced by IGF-1, b-arrestin2 could inhibit FOXO1 activity in an Akt-independent manner and delay FOXO1 dephosphorylation through the inhibition of PP2A phosphatase activity and the attenuation of the interaction between FOXO1 and PP2A. Furthermore, b-arrestin2 could downregulate FOXO1 expression via ubiquitylation and proteasomal degradation. Together, our results identified a novel role for b-arrestin2 in the modulation of the CRPC progress through FOXO1. Thus, the characterization of b-arrestin2 may represent an alternative therapeutic target for CRPC treatment. J. Cell. Physiol. 230: 2371–2381, 2015. © 2015 Wiley Periodicals, Inc.
Prostate cancer remains a major cause of cancer-related deaths in men worldwide. Androgen–ablation therapies, including surgical and medical castration, are currently a standard treatment for advanced androgen-dependent prostate cancer (ADPC). Although these therapies initially lead to disease regression, advanced prostate cancer ultimately progresses to an androgen-independent prostate cancer (AIPC) stage, which is refractory to current therapies (also referred to as “castration-resistant prostate cancer, CRPC”). The clarification of the exact molecular mechanism of CRPC and the development of effective therapeutics for CRPC patients remain the major challenges for this disease to date (Feldman and Feldman, 2001; Pienta and Bradley, 2006; Jemal et al., 2010). b-Arrestins, including b-arrestin1 and b-arrestin2, are well-known negative regulators of G-protein-coupled receptor (GPCR) signaling. In recent years, accumulating evidence indicates that b-arrestins can also function as scaffold proteins and interact with a variety of different signaling molecules. Through the formation of scaffolding complexes with signaling molecules, such as Src, Akt, ERK1/2, MDM2, and IkB, b-arrestins act as signal transducers to guide signals from distinct pathways, which play important roles in cell proliferation, apoptosis, and cancer progression (Kovacs et al., 2009; DeFea, 2011). Previous studies have determined that the function of b-arrestins in cancer is largely through the modulation of multiple signaling pathways, including the insulin-like growth factor-1 (IGF-1), transforming growth factor-b (TGF-b), p53, NF-kB, and Notch pathways, which are responsible for tumor viability and metastasis (Hu et al., 2013). Recently, © 2 0 1 5 W I L E Y P E R I O D I C A L S , I N C .
Lakshmikanthan et al. (2009) demonstrated that b-arrestin2 could form a complex with the androgen receptor (AR), and act as an AR corepressor in ADPC LNCaP cells. Accordingly, human prostate tissues evidenced a negative relationship between b-arrestin2 expression and AR activity; however, the role of b-arrestin2 in CRPC remains unknown.
Contract grant sponsor: National Natural Science Foundation of China; Contract grant numbers: 81402430, 81370804, 81170652. Contract grant sponsor: Colleges and universities in Guangzhou Yangcheng scholars research project; Contract grant number: 12A017S. Contract grant sponsor: Science and technology project in Guangzhou; Contract grant number: 201300000096. *Correspondence to: Guohua Zeng, Department of Urology, Minimally Invasive Surgery Center, the First Affiliated Hospital of Guangzhou Medical University, Guangdong Key Laboratory of Urology, Kangda Road 1#, Haizhu District, Guangzhou, Guangdong, China, 510230. Email: [email protected] Manuscript Received: 25 August 2014 Manuscript Accepted: 17 February 2015 Accepted manuscript online in Wiley Online Library (wileyonlinelibrary.com): 9 March 2015. DOI: 10.1002/jcp.24963
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FOXO1, also known as FKHR, is one of the Forkhead box-O transcription factors (FOXOs), which play an important role in tumor suppression via the regulation of multiple genes involved in cell proliferation, differentiation and apoptosis. The function of FOXO1 is regulated by posttranslational modifications, such as phosphorylation, acetylation, ubiquitination and protein-protein interactions. As FOXO1 plays a pivotal role in cell fate decisions, accumulated evidence indicates that FOXO1 functions as a tumor suppressor in a variety of cancers, including prostate cancer (Fu and Tindall, 2008; Weidinger et al., 2008). In prostate cancer, a decreased level of FOXO1 and/or an increased level of its phosphorylated form p-FOXO1 are the indicators of the more aggressive biological features of prostate cancer (Li et al., 2007). In addition, androgen negatively regulates FOXO1 via a proteolytic mechanism, and activated AR inhibits FOXO1-induced cell cycle arrest and apoptosis (Huang et al., 2004); FOXO1 is an important target for both Akt-dependent and -independent survival signals (Li et al., 2003). Thus, the regulation of FOXO1 function is critical for tumorigenesis and the progression of prostate cancer. Here, our data demonstrated that b-arrestin2 contributes to cell viability and proliferation via the downregulation of FOXO1 activity and expression in CRPC cell lines. Mechanistically, b-arrestin2 could inhibit FOXO1 activity in an Akt-independent manner and attenuate FOXO1 dephosphorylation via the inhibition of PP2A phosphatase activity and the attenuation of the interaction between FOXO1 and PP2A. Furthermore, b-arrestin2 could downregulate FOXO1 expression via ubiquitylation and proteasomal degradation. Overall, our findings represent a novel role of b-arrestin2 in the regulation of the CRPC progress via FOXO1. Materials and Methods Cell culture and drugs All prostate cancer cell lines, including RWPE-1, LNCaP, 22Rv1, PC3, and DU145, were purchased from ATCC (Manassas, VA) and cultured as recommended by ATCC in a humidified incubator that contained 5% CO2 at 37°C. HEK293 cells were maintained in DMEM supplemented with 10% (V/V) fetal bovine serum (FBS, Gibico, Life Technologies, Staley Road Grand Island, NY), 1% penicillin/streptomycin and 10 mM Hepes buffer. PC3 monoclonal cells that expressed pEGFP-N1-b-arrestin2 (PC3-b-arr2) or empty vector pEGFP-N1 (PC3-N1) were generated and cultured in the presence of G418 (0.5 mg/ml). IGF-1 and bortezomib were purchased from R&D Systems (Minneapolis, MN). Akt inhibitor VIII was purchased from Sigma–Aldrich (St. Louis, MO). Cell transfection and plasmids Small interfering RNA (siRNA) was synthesized as previously described (Fosbrink et al., 2006; Gan et al., 2009; Rajagopal et al., 2010) by RiBoBio Co. Ltd (Shanghai, China). The sequences were as follows: 50 -GGACCGCAAAGUGUUUGUG-30 (siRNA-b-arrestin2-1), 50 -CCAACCUCAUUGAAUUCGA-30 (siRNA-b-arrestin2-2), 50 -CCAGAUGCCUAUACAAACA-30 (siRNA-FOXO1-1), and 50 -GAGCGUGCCCUACUUCAAG-30 (siRNA-FOXO1-2). The sequence of unrelated siRNA was 50 -UUCUCCGAACGUGUCACGU-30 (siRNA-NC). The full lengths of b-arrestin2, FOXO1, and PP2A were cloned into modified pEGFP-N1 vector, pDsRed2-N1 vector, and pcDNA3.1 vector in-frame with HA, respectively. The truncation mutants of b-arrestin2 were generated as previously described and cloned into modified pcDNA3.1 vector in-frame with HA (Wang et al., 2006). The authenticity of the DNA sequences was confirmed by sequencing. For transient transfections, cells were seeded in 60 mm dishes and transfected at 70% confluence. The transfections were conducted with siRNAs or plasmids using JOURNAL OF CELLULAR PHYSIOLOGY
Lipofectamine 2000 according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA). Cell viability and proliferation assay Cell viability and proliferation were assessed using the Cell Titer 1 96 AQueous One Solution Cell Proliferation Assay kit (MTS, Promega, Madison, WI). For the cell viability assay, 48 h after transfection, equal amounts of cells were seeded in a 96-well plate and cultured in medium, supplemented without serum for 48 h prior to the MTS assay, whereas cells were cultured in the medium supplemented with 10% FBS for 48 h prior to the MTS assay for the cell proliferation assay. For the MTS assay, at the end of the experiment, 10 ml of MTS (5 mg/ml in PBS) were added, and the cells were incubated for 2 h in the humidified incubator that contained 5% CO2 at 37°C. The relative cell viability and proliferation were obtained by scanning with an ELISA reader with a 490 nm filter. For the cell growth array, the cell number was counted in triplicates of samples at five time points after transfection. The cells were trypzinized and resuspended in a 1:1 mixture of PBS and 0.5% trypan blue, and the number of viable cells was counted using a hemocytometer. Cell apoptosis assay Cell apoptosis was detected via flow cytometry using an Annexin V-FITC Apoptosis detection Kit (eBioscience, San Diego, CA). Forty-eight hours after transfection, equal amounts of PC3 or DU145 cells were seeded in the 60 mm cell culture dish and cultured in the medium supplemented without serum for 48 h. The cells were then trypzinized, washed twice with ice-cold PBS, resuspended in Binding Buffer and incubated with Annexin V-FITC for 10 min at room temperature. The cells were washed with ice-cold PBS, resuspended in Binding Buffer and incubated with propidium iodide (20 mg/ml) for 5 min. Apoptosis was subsequently analyzed via flow cytometry (BD FACSCalibur) using BD CellQuestTM Pro software. Western blot analysis Western blot analysis was conducted as previously described (Wu et al., 2013). In brief, appropriately treated cells were lysed in RIPA buffer, and equal amounts of protein were separated on a 10% SDS polyacrylamide gel, transferred to a PVDF membrane (Millipore, Billerica, MA) and immunoblotted with antibodies. The primary antibodies used included antibodies against b-arrestin2, phospho-Akt (Ser473), Akt1, FOXO1 (Cell Signaling Technology, Danvers, MA), phospho-FOXO1 (Ser256, SAB), HA-Tag, GFP-Tag (Abmart, Shanghai, China), PP2A (Epitomics, Cambridge, UK), Ubiquitin, and GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA). The secondary antibodies were anti-mouse and anti-rabbit IgG conjugated with HRP (Santa Cruz Biotechnology). For the detection of FOXO1 ubiquitlylation, the cell extracts were incubated with FOXO1 antibody at 4°C overnight; the samples were subsequently immobilized on protein A/G-Sepharose beads (Santa Cruz Biotechnology) for 4 h, washed three times with RIPA lysis buffer and analyzed using Western blot analysis after elution from beads. The band intensities were quantified with respect to GAPDH using ImageJ software and presented as bar graphs after testing statistical validity. Luciferase Reporter Assay The luciferase reporter 3 IRS-luc, which contains three copies of an FKHR response element from the promoter of the insulin-like growth factor-binding protein-1gene, was constructed as previously described (Tang et al., 1999). After transfections, cell lysates were prepared, and luciferase activity was determined using the Dual–Luciferase Assay (Promega) according to the
b-ARRESTIN2 DOWN-REGULATES FOXO1 IN CRPC CELLS
manufacturer’s instructions. The relative luciferase activity was measured as the ratio of firefly luciferase activity to renilla luciferase activity. PP2A activity assay Cells were lysed in RIPA buffer, and the protein concentrations were determined using the DCTM protein assay regents (Bio-Rad, Hercules, CA). Then, PP2A was immunoprecipitated with PP2A antibody (Epitomics) and protein A/G agarose (Santa Cruz Biotechnology) overnight at 4°C. The beads were subsequently washed three times with the previous lysis buffer, and the phosphatase activity of immunoprecipitated PP2A was assayed with a Serine/ Threonine Phosphatase Assay System (Promega) using RRA (pT) VA as the substrate peptide following the manufacturer’s instructions. The absorbance was measured at 600 nm using an ELISA reader. Assays were performed in quadruplicate in three separate experiments, and the PP2A activity was calculated using the fold change in each experiment. Immunoprecipitation To cross-link interacting proteins prior to IP, cells were treated with 2 mM dithiobis (Pierce, Thermo Scientific, Wilmington, DE) dissolved in dry DMSO. The cells were then mechanistically broken (passage through a 29-gauge needle) in ice-cold Nonidet P-40 buffer (142.5 mM KCl, 5 mM MgCl2, 10 mM HEPES, and 0.1% NP-40) with protease inhibitors and incubated with FOXO1 antibody (Cell Signaling Technology) or control IgG antibody (Santa Cruz Biotechnology) at 4°C overnight. The lysate antibody mixture was centrifuged at 4000 rpm/min for 5 min and washed three times with lysis buffer. The precipitated proteins were eluted with SDS sample buffer for Western blot analyses. Translocation studies Forty-eight hours after the HEK293 cells were transfected with the indicated plasmids, the culture medium was replaced with DMEM supplemented with 10% FBS (or serum-free) for 2 h and counter stained with DAPI for 10 min in the dark. The cytoplasmic/ nuclear location of b-arrestin2 and FOXO1 were subsequently captured using an inverted fluorescence microscope (Olympus IX73). Statistical analysis The data are reported as the means SD of at least three independent experiments. The mean differences were compared using ANOVA and the Student t test. A P value of less than 0.05 was considered to be statistically significant. Results b-Arrestin2 contributes to the cell viability and proliferation in CRPC cell lines
To investigate the role of b-arrestin2 in the regulation of the viability and proliferation of CRPC cells, we first established the b-arrestin2 expression in distinct types of prostate cancer cell lines, such as benign prostate RWPE-1 cells, ADPC LNCaP cells, and CRPC cells (PC3 and DU145). Compared with the benign prostate RWPE-1 cells, the LNCaP cells expressed less b-arrestin2 protein, whereas the CRPC cells expressed more b-arrestin2 protein (Fig. 1A), which suggest that b-arrestin2 may exert a specific regulatory function in CRPC cells that is distinct from ADPC cells. Thus, we detected the effects of siRNA mediated b-arrestin2 inhibition on the cell viability and proliferation in CRPC cells. As shown in Figure 1B, both b-arrestin2 siRNA sequences dramatically decreased the expression of endogenous b-arrestin2. Compared with the JOURNAL OF CELLULAR PHYSIOLOGY
control group, both siRNA sequences that targeted b-arrestin2, but not unrelated siRNA, significantly decreased the cell viability, promoted serum-deprivation -induced cell apoptosis, and inhibited the cell proliferation and growth of PC3 or DU145 cells (Fig. 1C–G), which suggest that b-arrestin2 is required for cell viability and proliferation in CRPC cell lines. FOXO1 is required for b-arrestin2 siRNA-mediated inhibition of cell viability and proliferation in CRPC cells
As a key downstream target of PTEN, FOXO1 plays a crucial role in tumor suppression through the induction of cell growth arrest and apoptosis (Nakamura et al., 2000; Modur et al., 2002). Furthermore, as an important target of Akt, its phosphorylation by Akt can regulate cell apoptosis and proliferation via the operation of its target genes in the nucleus (Song et al., 2005; Huang and Tindall, 2007). To assess the role of FOXO1 in b-arrestin2 siRNA-mediated cell viability and proliferation, b-arrestin2 siRNAs and FOXO1 siRNAs were cotransfected into CRPC cells (Fig. 2A and B); the cell viability and proliferation were subsequently detected. As shown in Figure 2C–G, compared with the control group, the knockdown of the expression of FOXO1 markedly increased the cell viability, promoted the cell proliferation and growth, and inhibited the cell apoptosis induced by serum-deprivation. In addition, compared with the knockdown b-arrestin2 expression alone, the coknockdown of the expression of FOXO1 and b-arrestin2 not only significantly attenuated b-arrestin2 siRNA-mediated inhibition of cell viability, proliferation and growth, but also inhibited b-arrestin2 siRNA-induced cell apoptosis. These findings suggest that FOXO1 is, at least in part, required for b-arrestin2 siRNA-mediated cell viability and proliferation inhibition in CRPC cells. b-Arrestin2-mediated FOXO1 phosphorylation in response to IGF-1 stimulation
As FOXO1 is functionally inhibited by phosphorylation in response to IGF-1 stimulation through PI3K/Akt kinase (Nakae et al., 2000), we investigated the role of b-arrestin2 in IGF-1-induced Akt/FOXO1 signal transduction. Compared with the control group, the knockdown of the endogenous expression of b-arrestin2 significantly decreased IGF-1-induced phosphorylation of Akt and FOXO1, whereas the forced overexpression of b-arrestin2 accelerated the phosphorylation of Akt and FOXO1. However, both transient knockdown and overexpression of b-arrestin2 had no obvious effects on the expression of Akt and FOXO1 (Fig. 3A and B). In addition, we examined the effects of b-arrestin2 truncation mutants, including the b-arrestin2 fragment that comprises amino acids 1–185 and 186–409, on the IGF-1-induced phosphorylation of Akt and FOXO1. The results demonstrated that the overexpression of b-arrestin2 (1–185) or b-arrestin2 (186–409) dramatically reduced the IGF-1-stimulated phosphorylation of Akt and FOXO1 in the PC3 cells (Fig. 3C). As IGF-1 inactivates FoxO1 through the PI3K/Akt pathway (Ni et al., 2007), we investigated whether b-arrestin2 mediated phosphorylation of FOXO1 induced by IGF-1 is Akt dependent. Interestingly, although pretreatment with an Akt inhibitor dramatically attenuated the IGF-induced FOXO1 phosphorylation in both GFP-N1- and GFP-b-arrestin2-transfected PC3 cells (Fig. 3D), the ectopic expression of b-arrestin2 resulted in a significant decrease in the transcriptional activity of endogenous FOXO1 and attenuated the increased transcriptional activity induced by Akt inhibitor pretreatment, which indicates b-arrestin2 could mediate FOXO1 activity in an Akt-independent manner. In
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Fig. 1. b-Arrestin2 contributes to the cell viability and proliferation in CRPC cells. (A) b-Arrestin2 expression in distinct prostate cancer cell lines. The expressions of indicated proteins were detected using Western blot. (B) Ninety-six hours after transfection with the indicated siRNA, the protein levels were determined using Western blot with the indicated antibodies. (C) Forty-eight hours after transfection, equal amounts of cells were seeded in a 96-well plate and cultured in medium supplemented with/without serum for 48 h. The cell viability was subsequently detected using MTS array. (D) The cell apoptosis was detected as described in “Materials and Methods”. (E) The cell proliferation was detected using MTS array and expressed as a percent of the control group. (F) and (G) Cell growth was measured at five time points after transfection using Trypan blue staining and a hemocytometer. & denotes P 0.01, *, # denotes P 0.05 vs. C, and ** denotes P 0.01 vs. C. (C: control, NC: negative control).
JOURNAL OF CELLULAR PHYSIOLOGY
b-ARRESTIN2 DOWN-REGULATES FOXO1 IN CRPC CELLS
Fig. 2. FOXO1 is required for b-arrestin2 siRNA-mediated cell viability and proliferation inhibition in CRPC cells. (A) and (B) Ninety-six hours after transfection with the indicated siRNAs, the expressions of the indicated proteins were detected using Western blot. (C–G) The cell viability, apoptosis, proliferation and growth were determined as described in Fig. 1. & denotes P 0.01, $ denotes P 0.05 vs. C, # denotes P 0.05, ** denotes P 0.01, * denotes P 0.05 vs. siRNA-arr2 þ siRNA-O1.
addition, the ectopic expression of b-arrestin2 also significantly decreased the reporter gene activity activated by exogenous FOXO1 with or without Akt inhibitor pretreatment, which suggests that b-arrestin2 inhibits the transcriptional activities of both endogenous and transfected FOXO1 (Fig. 3E).
phosphatase activity of PP2A (Fig. 4C). In addition, the forced expression of b-arrestin2 attenuated the interaction between PP2A and FOXO1. Together, these findings indicated that the effect of b-arrestin2 on FOXO1 dephosphorylation is at least, in part, achieved through PP2A.
b-Arrestin2-mediated FOXO1 dephosphorylation
b-Arrestin2 attenuates serum-deprivation-induced nucleic accumulation of FOXO1
As signal transduction is tightly regulated by phosphorylation– dephosphorylation cycles, the phosphorylation and dephosphorylation of FOXO1 in response to IGF-I stimulation were investigated. Similar to a previous report (Schachter et al., 2012), IGF-1 stimulation induced the phosphorylation of Akt and FOXO1 in a time-dependent manner (Fig. 4A). In contrast, compared with GFP-N1 transfected PC3 cells, the dephosphorylation of FOXO1 in GFP-b-arrestin2 transfected cells was delayed (Fig. 4B). As PP2A is a pivotal serine/ threonine phosphatase in the regulation of FOXO1 dephosphorylation (Yan et al., 2008), we tested the effect of b-arrestin2 on PP2A activity. The results indicated that the forced expression of b-arrestin2 significantly attenuated the JOURNAL OF CELLULAR PHYSIOLOGY
As a transcription factor, FOXO1 regulates a large spectrum of tumor suppression genes in the nucleus, and its sub-cellular location in the nucleus and cytoplasm is critical for its function. Thus, the effect of b-arrestin2 on the serum-deprivation-induced nucleic accumulation of FOXO1 was investigated. In the culture medium supplemented with 10% FBS, both b-arrestin2 and FOXO1 were mainly located in the cytoplasm. In contrast, two hours after serum deprivation, approximately all FOXO1 translocated into the nucleus, whereas in the cells that cooverexpressed b-arrestin2, the nucleic accumulation of FOXO1 induced by serum-deprivation was dramatically attenuated (Fig. 5).
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Fig. 3. b-Arrestin2-mediated FOXO1 phosphorylation in response to IGF-1 stimulation. (A–C) Forty-eight hours after transfection with the indicated siRNAs or plasmids, PC3 cells were incubated in serum-free medium for 1 h and followed by IGF-1 (100 ng/ml) stimulation for 10 min. The expressions of the indicated proteins were subsequently detected using Western blot. (D) Forty-eight hours after transfection with the indicated plasmids, PC3 cells were incubated in serum-free medium for 1 h followed by Akt inhibitor (10 mM) treatment for 1 h prior to IGF-1 stimulation (100 ng/ml, 10 min). The expressions of the indicated proteins were subsequently detected using Western blot. (E) PC3 cells were transiently cotransfected with the 3 IRS luciferase reporter plasmid along with the indicated plasmids. At 12 h after transfection, the cells were treated with an Akt inhibitor (10 mM) or DMSO for an additional 24 h. Luciferase measurement and data analysis were performed as described in “Materials and Methods”. * denotes P 0.05 and ** denotes P 0.01.
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b-ARRESTIN2 DOWN-REGULATES FOXO1 IN CRPC CELLS
Fig. 4. b-Arrestin2-mediated dephosphorylation of FOXO1. (A) and (B) PC3 cells (or PC3 monoclonal cells that stably expressed GFP-N1 or GFP-b-arrestin2) were incubated in serum-free medium for 1 h followed by IGF-1 stimulation for the indicated times. The expressions of the indicated proteins were subsequently detected using Western blot. (C) PC3 cells were transfected with the indicated plasmids, and the relative phosphatase activity was detected as described in “Materials and Methods” at 48 h after transfection. (D) Decreased association between PP2A and FOXO1 in the PC3 cells that overexpressed GFP-b-arrestin2. Forty-eight hours after transfection with the indicated plasmids, the cell lysates and immunoprecipitated proteins were analyzed using Western blotting with the indicated antibodies. ** denotes P 0.01.
b-Arrestin2-mediated FOXO1 expression and proteasomal degradation
To assess the effect of b-arrestin2 on the protein expression of FOXO1, PC3 monoclonal cells with stable overexpression of pEGFP-N1-b-arrestin2 (PC3-b-arr2) or empty vector pEGFP-N1 (PC3-N1) were generated through G418 selection. As shown in Figure 6A, the FOXO1 expression was significantly decreased in the PC3-b-arr2 cells compared with the PC3-N1 cells. Ubiquitylation is a mechanism to target proteins for proteasomal degradation (Huang and Tindall, 2011). Thus, bortezomib, a proteasome inhibitor (Lakshmikanthan et al., 2009), was used to investigate the potential mechanisms involved in the b-arrestin2-mediated FOXO1 degradation. Bortezomib treatment increased the FOXO1 accumulation in the control PC3-N1 cells, and the decrease in FOXO1 expression (caused by the forced JOURNAL OF CELLULAR PHYSIOLOGY
b-arrestin2 overexpression) was reversed in the PC3-b-arr2 cells (Fig. 6B). Furthermore, bortezomib treatment represented a markedly enhanced ubiquitylation signal in the PC3-b-arr2 cells compared with the PC3-N1 cells, which implies b-arrestin2 causes the FOXO1 degradation in the proteasome via ubiquitylation (Fig. 6C). Discussion
In general, prostate cancer ultimately progresses to become androgen independent after androgen ablation therapies, which thereby renders anti-androgen therapy ineffective. As a lethal form of prostate cancer progresses and metastasizes, CRPC remains untreatable to date (Feldman and Feldman, 2001; Pienta and Bradley, 2006; Jemal et al., 2010; Jacome-Pita et al., 2014). Understanding the mechanism that leads to the development of CRPC will pave the way to effective therapies
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Fig. 5. b-Arrestin2 attenuates serum-deprivation-induced nucleic accumulation of FOXO1. Forty-eight hours after transfection with the indicated plasmids, HEK293 cells were incubated in medium supplemented with free or 10% FBS for 2 h and then counter stained with DAPI to visualize the nuclei. Representative images were captured using an inverted fluorescence microscope.
for CRPC. In the present study, our data indicated that b-arrestin2 is required for cell viability and proliferation in CRPC cells, which is associated with its inhibitory effects on the activity and expression of FOXO1, and thereby represents a novel potential mechanism of CRPC progress mediated by b-arrestin2. b-Arrestins were initially known as negative regulators of GPCR-mediated signaling. However, recent studies have demonstrated that b-arrestins can also function as scaffold proteins and interact with a variety of different signaling molecules in the cytoplasm; thus, they play important roles in the regulation of receptor trafficking and signaling (Ma and Pei, 2007; DeFea, 2011). Accumulating evidence has established that b-arrestins are widely involved in various cancer developmental signal transductions that are responsible for tumor viability and metastasis, which suggests an impressive role of b-arrestins in tumor progression (Hu et al., 2013). More JOURNAL OF CELLULAR PHYSIOLOGY
recently, b-arrestin2 was confirmed to form a complex with AR and act as an AR corepressor in ADPC LNCaP cells, whereas its role in CRPC cells remains uninvestigated (Lakshmikanthan et al., 2009). To investigate the role of b-arrestin2 in CRPC cells, we first investigated the basal expression of b-arrestin2 in ADPC and CRPC cell lines. The results demonstrated that compared with benign prostate RWPE-1 cells, the expression of b-arrestin2 was significantly decreased in ADPC LNCaP cells but dramatically increased in CRPC 22Rv1, PC3 and DU145 cells. These findings suggest b-arrestin2 may exert a different regulatory function in CRPC cells compared with ADPC cells. This result is consistent with a previous report that had not compared the b-arrestin2 expression in RWPE-1 and CRPC cells (Lakshmikanthan et al., 2009). Although the role of b-arrestin2 in cell survival and proliferation has been well established in lung and breast cancers, little is known regarding
b-ARRESTIN2 DOWN-REGULATES FOXO1 IN CRPC CELLS
Fig. 6. b-Arrestin2-mediated FOXO1 expression and proteasomal degradation. (A) FOXO1 expression in PC3-N1 and PC3-b-arr2 cells. PC3 monoclonal cells were cultured in growth medium for 24 h, and the expressions of the indicated proteins were detected using Western blot. (B) PC3 monoclonal cells were cultured in a growth medium for 24 h and treated with or without bortezomib (100 nM) for 6 h. The expressions of the indicated proteins were subsequently detected using Western blot. (C) PC3 monoclonal cells were pretreated with bortezomib (100 nM) for 6 h; the cell lysates were then subjected to immunoprecipitation with anti-FOXO1 antibody, and ubiquitination was detected using Western blot with anti-ubiquitin antibody. The cell lysates were probed with anti-GAPDH antibody to demonstrate the total protein expression. *, # denotes P 0.05 and ** denotes P 0.01.
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its role in prostate cancer. In the present study, our results demonstrated that similar to its role in breast cancer, b-arrestin2 is required for cell viability and proliferation in CRPC cells (Fig. 1B and C), whereas it is different from that in lung cancer, which suggests the function of b-arrestin2 is distinct in different cancers (Raghuwanshi et al., 2008; Zhao et al., 2009). FOXO1 is an important nuclear target for both Akt-dependent and -independent survival signals in prostate cancer progression (Modur et al., 2002; Li et al., 2003). In the present study, the co-knockdown of FOXO1 expression dramatically attenuated the b-arrestin2 siRNA-mediated inhibition of cell viability, proliferation and growth, and significantly inhibited b-arrestin2 siRNA-induced cell apoptosis, which suggests b-arrestin2-mediated regulation of cell viability and proliferation in CRPC cells is at least, in part, mediated by FOXO1. Post-translational inactivation and nuclear exclusion of FOXO1 have been observed in carcinomas of the breast, stomach, thyroid, lung, and prostate (Fu and Tindall, 2008). As a key downstream target of Akt, FOXO1 is functionally inhibited by phosphorylation in response to IGF-1 stimulation through PI3K/Akt kinase (Nakae et al., 2000). Phosphorylated Akt phosphorylates FOXO1 and results in the inactivation of FOXO1 and its translocation from the nucleus to the cytoplasm, which leads to the transcription suppression of proapoptotic genes (Fu and Tindall, 2008). In various cancers, including prostate cancer, IGF-1 activates the PI3K/Akt pathway to promote cell proliferation and survival via binding to its receptor IGF-1R (Pollak, 2008). IGF-1 mediated AR reactivation in low or absent androgen environments is one of the suggested multiple mechanisms by which ADPC cells progress to the androgen-independent stage (Grossmann et al., 2001; Cronauer et al., 2003; Fan et al., 2007). Furthermore, accumulating evidence indicates that IGF-1-induced activation of the PI3K/Akt pathway, which is one of the most important intracellular signaling pathways that favor cell proliferation and survival, greatly contributes to prostate cancer progression (Pollak, 2008). PI3K/Akt is a controversial pathway regulated by b-arrestins, and there is evidence for both the inhibition and stimulation of this pathway mediated by b-arrestins (Povsic et al., 2003; Beaulieu et al., 2005; Wang and DeFea, 2006; Girnita et al., 2007; Wang et al., 2007; Lodeiro et al., 2009; Luan et al., 2009; DeFea, 2011). In contrast, the exact role of b-arrestin2 in the regulation of IGF-1-induced phosphorylation of Akt and FOXO1 in CRPC cells remains unclear. In the present study, our data identified a pivotal role of b-arrestin2 in the regulation of the phosphorylation of Akt and FOXO1 induced by IGF-1 in CRPC cells, which was consistent with the results reported by Luan et al. (2009). However, interestingly, our results demonstrated that b-arrestin2 could regulate FOXO1 activity in both Akt-dependent and -independent manners, which indicates that some other unclear mechanisms were involved in the b-arrestin2-mediated regulation of FOXO1 transcriptional activity in CRPC cells. Reversible phosphorylation catalyzed by kinases and phosphatases is a pivotal regulatory mechanism of FOXO transcriptional activity. In the present study, our results demonstrated that the overexpression of b-arrestin2 significantly delayed FOXO1 dephosphorylation after IGF-1 stimulation via the downregulation of PP2A activity and the attenuation of the interaction between PP2A and FOXO1, which demonstrates a novel mechanism of FOXO1 activity mediated by b-arrestin2. The nucleic/cytoplasmic shuttling that regulates the intracellular FOXO1 distribution is a central regulation mechanism for FOXO1 transcriptional activity (Brunet et al., 1999; Gan et al., 2005). A recent study indicated that JOURNAL OF CELLULAR PHYSIOLOGY
b-arrestin1 is required for clozapine and lithium to suppress the nuclear localization of FOXO (Weeks et al., 2011). Similar to their results, our data demonstrated that the intracellular distribution of b-arrestin2 and FOXO1 represent a high consistency, and b-arrestin2 significantly attenuated the nuclear accumulation of FOXO1 induced by serum-deprivation, which suggests a critical role of b-arrestin2 in the regulation of FOXO1 activity. In general, FOXO1 is dysregulated in breast cancer, prostate cancer, glioblastoma, rhabdomyosarcoma, and leukemia (Fu and Tindall, 2008). The ubiquitination and proteasome degradation of FOXO1 play an important role in tumorigenesis. The ubiquitination of FOXO1 mediated by E3 ligases, including Skp2 and Mdm2, leads to its ubiquitination– proteasome degradation, which thereby favors cell proliferation and survival (Girnita et al., 2005; Huang and Tindall, 2011). As one of the well-characterized binding partners of Mdm2, b-arrestin2 acts as a scaffold for Mdm2, which leads to AR ubiquitylation and degradation in ADPC LNCaP cells (Girnita et al., 2005; Lakshmikanthan et al., 2009; Kommaddi and Shenoy, 2013). Similar to its effect on AR, our results demonstrated that b-arrestin2 induces a decrease in FOXO1 protein level through proteasome degradation. In conclusion, our data identified a novel role of b-arrestin2 in the regulation of FOXO1 activity and expression, as well as the IGF-induced transactivation of Akt/FOXO1 pathway, which may account for its requirement for cell viability and proliferation in CRPC cells. These findings represent a novel potential mechanism of CRPC progress mediated by b-arrestin2. Acknowledgements
This work was financed by grants from the National Natural Science Foundation of China (No. 81402430, NO. 81370804 and NO. 81170652), the Colleges and universities in Guangzhou Yangcheng scholars research project (No. 12A017S), and the Science and technology project in Guangzhou (the people’s livelihood special major science and Technology, No.201300000096). Literature Cited Beaulieu JM, Sotnikova TD, Marion S, Lefkowitz RJ, Gainetdinov RR, Caron MG. 2005. An Akt/beta-arrestin 2/PP2A signaling complex mediates dopaminergic neurotransmission and behavior. Cell 122:261–273. Brunet A, Bonni A, Zigmond MJ, Lin MZ, Juo P, Hu LS, Anderson MJ, Arden KC, Blenis J, Greenberg ME. 1999. Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell 96:857–868. Cronauer MV, Schulz WA, Burchardt T, Anastasiadis AG, de la Taille A, Ackermann R, Burchardt M. 2003. The androgen receptor in hormone-refractory prostate cancer: Relevance of different mechanisms of androgen receptor signaling (Review). Int J Oncol 23:1095–1102. DeFea KA. 2011. Beta-arrestins as regulators of signal termination and transduction: How do they determine what to scaffold?. Cell Signal 23:621–629. Fan W, Yanase T, Morinaga H, Okabe T, Nomura M, Daitoku H, Fukamizu A, Kato S, Takayanagi R, Nawata H. 2007. Insulin-like growth factor 1/insulin signaling activates androgen signaling through direct interactions of Foxo1 with androgen receptor. J Biol Chem 282:7329–7338. Feldman BJ, Feldman D. 2001. The development of androgen-independent prostate cancer. Nat Rev Cancer 1:34–45. Fosbrink M, Niculescu F, Rus V, Shin ML, Rus H. 2006. C5b-9-induced endothelial cell proliferation and migration are dependent on Akt inactivation of forkhead transcription factor FOXO1. J Biol Chem 281:19009–19018. Fu Z, Tindall DJ. 2008. FOXOs, cancer and regulation of apoptosis. Oncogene 27:2312– 2319. Gan L, Chen S, Wang Y, Watahiki A, Bohrer L, Sun Z, Wang Y, Huang H. 2009. Inhibition of the androgen receptor as a novel mechanism of taxol chemotherapy in prostate cancer. Cancer Res 69:8386–8394. Gan L, Zheng W, Chabot JG, Unterman TG, Quirion R. 2005. Nuclear/cytoplasmic shuttling of the transcription factor FoxO1 is regulated by neurotrophic factors. J Neurochem 93:1209–1219. Girnita L, Shenoy SK, Sehat B, Vasilcanu R, Girnita A, Lefkowitz RJ, Larsson O. 2005. {beta}-Arrestin is crucial for ubiquitination and down-regulation of the insulin-like growth factor-1 receptor by acting as adaptor for the MDM2 E3 ligase. J Biol Chem 280:24412– 24419. Girnita L, Shenoy SK, Sehat B, Vasilcanu R, Vasilcanu D, Girnita A, Lefkowitz RJ, Larsson O. 2007. Beta-arrestin and Mdm2 mediate IGF-1 receptor-stimulated ERK activation and cell cycle progression. J Biol Chem 282:11329–11338.
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Ni YG, Wang N, Cao DJ, Sachan N, Morris DJ, Gerard RD, Kuro-O M, Rothermel BA, Hill JA. 2007. FoxO transcription factors activate Akt and attenuate insulin signaling in heart by inhibiting protein phosphatases. Proc Natl Acad Sci USA 104:20517–20522. Pienta KJ, Bradley D. 2006. Mechanisms underlying the development of androgen-independent prostate cancer. Clin Cancer Res 12:1665–1671. Pollak M. 2008. Insulin, insulin-like growth factors and neoplasia. Best Pract Res Clin Endocrinol Metab 22:625–638. Povsic TJ, Kohout TA, Lefkowitz RJ. 2003. Beta-arrestin1 mediates insulin-like growth factor 1 (IGF-1) activation of phosphatidylinositol 3-kinase (PI3K) and anti-apoptosis. J Biol Chem 278:51334–51339. Raghuwanshi SK, Nasser MW, Chen X, Strieter RM, Richardson RM. 2008. Depletion of beta-arrestin-2 promotes tumor growth and angiogenesis in a murine model of lung cancer. J Immunol 180:5699–5706. Rajagopal S, Kim J, Ahn S, Craig S, Lam CM, Gerard NP, Gerard C, Lefkowitz RJ. 2010. Beta-arrestin- but not G protein-mediated signaling by the “decoy” receptor CX CR7. Proc Natl Acad Sci USA 107:628–632. Schachter TN, Shen T, Liu Y, Schneider MF. 2012. Kinetics of nuclear-cytoplasmic translocation of Foxo1 and Foxo3A in adult skeletal muscle fibers. Am J Physiol Cell Physiol 303:C977–C990. Song G, Ouyang G, Bao S. 2005. The activation of Akt/PKB signaling pathway and cell survival. J Cell Mol Med 9:59–71. Tang ED, Nunez G, Barr FG, Guan KL. 1999. Negative regulation of the forkhead transcription factor FKHR by Akt. J Biol Chem 274:16741–16746. Wang P, DeFea KA. 2006. Protease-activated receptor-2 simultaneously directs beta-arrestin-1-dependent inhibition and Galphaq-dependent activation of phosphatidylinositol 3-kinase. Biochemistry 45:9374–9385. Wang P, Kumar P, Wang C, Defea KA. 2007. Differential regulation of class IA phosphoinositide 3-kinase catalytic subunits p110 alpha and beta by protease-activated receptor 2 and beta-arrestins. Biochem J 408:221–230. Wang Y, Tang Y, Teng L, Wu Y, Zhao X, Pei G. 2006. Association of beta-arrestin and TRAF6 negatively regulates Toll-like receptor-interleukin 1 receptor signaling. Nat Immunol 7:139–147. Weeks KR, Dwyer DS, Aamodt EJ. 2011. Clozapine and lithium require Caenorhabditis elegans beta-arrestin and serum- and glucocorticoid-inducible kinase to affect Daf-16 (FOXO) localization. J Neurosci Res 89:1658–1665. Weidinger C, Krause K, Klagge A, Karger S, Fuhrer D. 2008. Forkhead box-O transcription factor: Critical conductors of cancer’s fate. Endocr Relat Cancer 15:917–929. Wu W, Kong Z, Duan X, Zhu H, Li S, Zeng S, Liang Y, Iliakis G, Gui Z, Yang D. 2013. Inhibition of PARP1 by small interfering RNA enhances docetaxel activity against human prostate cancer PC3 cells. Biochem Biophys Res Commun 442:127–132. Yan L, Lavin VA, Moser LR, Cui Q, Kanies C, Yang E. 2008. PP2A regulates the pro-apoptotic activity of FOXO1. J Biol Chem 283:7411–7420. Zhao M, Zhou G, Zhang Y, Chen T, Sun X, Stuart C, Hanley G, Li J, Zhang J, Yin D. 2009. Beta-arrestin2 inhibits opioid-induced breast cancer cell death through Akt and caspase-8 pathways. Neoplasma 56:108–113.
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b-Arrestin2 Contributes to Cell Viability and Proliferation via the Down-Regulation of FOXO1 in Castration-Resistant Prostate Cancer
Cellular Physiology
XIAOLU DUAN,1 ZHENZHEN KONG,1 YANG LIU,1 ZHIWEN ZENG,2 SHUJUE LI,1 WENQI WU,1 WEIDONG JI,1 BICHENG YANG,1 ZHIJIAN ZHAO,1 AND GUOHUA ZENG1* 1
Department of Urology, Minimally Invasive Surgery Center, the First Affiliated Hospital of Guangzhou Medical University, Guangdong Key Laboratory of Urology, Guangzhou, Guangdong, China
2
Key Laboratory of Neurogenetics and Channelopathies of Guangdong Province and the Ministry of Education of China, Collaborative Innovation Center for Neurogenetics and Channelopathies, Institute of Neuroscience and the Second Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong, China
b-Arrestin2 has been identified to act as a corepressor of androgen receptor (AR) signaling by binding to AR and serving as a scaffold to affect the activity and expression of AR in androgen-dependent prostate cancer cells; however, little is known regarding its role in castration-resistant prostate cancer (CRPC) progression. Here, our data demonstrated that b-arrestin2 contributes to the cell viability and proliferation of CRPC via the downregulation of FOXO1 activity and expression. Mechanistically, in addition to its requirement for FOXO1 phosphorylation induced by IGF-1, b-arrestin2 could inhibit FOXO1 activity in an Akt-independent manner and delay FOXO1 dephosphorylation through the inhibition of PP2A phosphatase activity and the attenuation of the interaction between FOXO1 and PP2A. Furthermore, b-arrestin2 could downregulate FOXO1 expression via ubiquitylation and proteasomal degradation. Together, our results identified a novel role for b-arrestin2 in the modulation of the CRPC progress through FOXO1. Thus, the characterization of b-arrestin2 may represent an alternative therapeutic target for CRPC treatment. J. Cell. Physiol. 230: 2371–2381, 2015. © 2015 Wiley Periodicals, Inc.
Prostate cancer remains a major cause of cancer-related deaths in men worldwide. Androgen–ablation therapies, including surgical and medical castration, are currently a standard treatment for advanced androgen-dependent prostate cancer (ADPC). Although these therapies initially lead to disease regression, advanced prostate cancer ultimately progresses to an androgen-independent prostate cancer (AIPC) stage, which is refractory to current therapies (also referred to as “castration-resistant prostate cancer, CRPC”). The clarification of the exact molecular mechanism of CRPC and the development of effective therapeutics for CRPC patients remain the major challenges for this disease to date (Feldman and Feldman, 2001; Pienta and Bradley, 2006; Jemal et al., 2010). b-Arrestins, including b-arrestin1 and b-arrestin2, are well-known negative regulators of G-protein-coupled receptor (GPCR) signaling. In recent years, accumulating evidence indicates that b-arrestins can also function as scaffold proteins and interact with a variety of different signaling molecules. Through the formation of scaffolding complexes with signaling molecules, such as Src, Akt, ERK1/2, MDM2, and IkB, b-arrestins act as signal transducers to guide signals from distinct pathways, which play important roles in cell proliferation, apoptosis, and cancer progression (Kovacs et al., 2009; DeFea, 2011). Previous studies have determined that the function of b-arrestins in cancer is largely through the modulation of multiple signaling pathways, including the insulin-like growth factor-1 (IGF-1), transforming growth factor-b (TGF-b), p53, NF-kB, and Notch pathways, which are responsible for tumor viability and metastasis (Hu et al., 2013). Recently, © 2 0 1 5 W I L E Y P E R I O D I C A L S , I N C .
Lakshmikanthan et al. (2009) demonstrated that b-arrestin2 could form a complex with the androgen receptor (AR), and act as an AR corepressor in ADPC LNCaP cells. Accordingly, human prostate tissues evidenced a negative relationship between b-arrestin2 expression and AR activity; however, the role of b-arrestin2 in CRPC remains unknown.
Contract grant sponsor: National Natural Science Foundation of China; Contract grant numbers: 81402430, 81370804, 81170652. Contract grant sponsor: Colleges and universities in Guangzhou Yangcheng scholars research project; Contract grant number: 12A017S. Contract grant sponsor: Science and technology project in Guangzhou; Contract grant number: 201300000096. *Correspondence to: Guohua Zeng, Department of Urology, Minimally Invasive Surgery Center, the First Affiliated Hospital of Guangzhou Medical University, Guangdong Key Laboratory of Urology, Kangda Road 1#, Haizhu District, Guangzhou, Guangdong, China, 510230. Email: [email protected] Manuscript Received: 25 August 2014 Manuscript Accepted: 17 February 2015 Accepted manuscript online in Wiley Online Library (wileyonlinelibrary.com): 9 March 2015. DOI: 10.1002/jcp.24963
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FOXO1, also known as FKHR, is one of the Forkhead box-O transcription factors (FOXOs), which play an important role in tumor suppression via the regulation of multiple genes involved in cell proliferation, differentiation and apoptosis. The function of FOXO1 is regulated by posttranslational modifications, such as phosphorylation, acetylation, ubiquitination and protein-protein interactions. As FOXO1 plays a pivotal role in cell fate decisions, accumulated evidence indicates that FOXO1 functions as a tumor suppressor in a variety of cancers, including prostate cancer (Fu and Tindall, 2008; Weidinger et al., 2008). In prostate cancer, a decreased level of FOXO1 and/or an increased level of its phosphorylated form p-FOXO1 are the indicators of the more aggressive biological features of prostate cancer (Li et al., 2007). In addition, androgen negatively regulates FOXO1 via a proteolytic mechanism, and activated AR inhibits FOXO1-induced cell cycle arrest and apoptosis (Huang et al., 2004); FOXO1 is an important target for both Akt-dependent and -independent survival signals (Li et al., 2003). Thus, the regulation of FOXO1 function is critical for tumorigenesis and the progression of prostate cancer. Here, our data demonstrated that b-arrestin2 contributes to cell viability and proliferation via the downregulation of FOXO1 activity and expression in CRPC cell lines. Mechanistically, b-arrestin2 could inhibit FOXO1 activity in an Akt-independent manner and attenuate FOXO1 dephosphorylation via the inhibition of PP2A phosphatase activity and the attenuation of the interaction between FOXO1 and PP2A. Furthermore, b-arrestin2 could downregulate FOXO1 expression via ubiquitylation and proteasomal degradation. Overall, our findings represent a novel role of b-arrestin2 in the regulation of the CRPC progress via FOXO1. Materials and Methods Cell culture and drugs All prostate cancer cell lines, including RWPE-1, LNCaP, 22Rv1, PC3, and DU145, were purchased from ATCC (Manassas, VA) and cultured as recommended by ATCC in a humidified incubator that contained 5% CO2 at 37°C. HEK293 cells were maintained in DMEM supplemented with 10% (V/V) fetal bovine serum (FBS, Gibico, Life Technologies, Staley Road Grand Island, NY), 1% penicillin/streptomycin and 10 mM Hepes buffer. PC3 monoclonal cells that expressed pEGFP-N1-b-arrestin2 (PC3-b-arr2) or empty vector pEGFP-N1 (PC3-N1) were generated and cultured in the presence of G418 (0.5 mg/ml). IGF-1 and bortezomib were purchased from R&D Systems (Minneapolis, MN). Akt inhibitor VIII was purchased from Sigma–Aldrich (St. Louis, MO). Cell transfection and plasmids Small interfering RNA (siRNA) was synthesized as previously described (Fosbrink et al., 2006; Gan et al., 2009; Rajagopal et al., 2010) by RiBoBio Co. Ltd (Shanghai, China). The sequences were as follows: 50 -GGACCGCAAAGUGUUUGUG-30 (siRNA-b-arrestin2-1), 50 -CCAACCUCAUUGAAUUCGA-30 (siRNA-b-arrestin2-2), 50 -CCAGAUGCCUAUACAAACA-30 (siRNA-FOXO1-1), and 50 -GAGCGUGCCCUACUUCAAG-30 (siRNA-FOXO1-2). The sequence of unrelated siRNA was 50 -UUCUCCGAACGUGUCACGU-30 (siRNA-NC). The full lengths of b-arrestin2, FOXO1, and PP2A were cloned into modified pEGFP-N1 vector, pDsRed2-N1 vector, and pcDNA3.1 vector in-frame with HA, respectively. The truncation mutants of b-arrestin2 were generated as previously described and cloned into modified pcDNA3.1 vector in-frame with HA (Wang et al., 2006). The authenticity of the DNA sequences was confirmed by sequencing. For transient transfections, cells were seeded in 60 mm dishes and transfected at 70% confluence. The transfections were conducted with siRNAs or plasmids using JOURNAL OF CELLULAR PHYSIOLOGY
Lipofectamine 2000 according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA). Cell viability and proliferation assay Cell viability and proliferation were assessed using the Cell Titer 1 96 AQueous One Solution Cell Proliferation Assay kit (MTS, Promega, Madison, WI). For the cell viability assay, 48 h after transfection, equal amounts of cells were seeded in a 96-well plate and cultured in medium, supplemented without serum for 48 h prior to the MTS assay, whereas cells were cultured in the medium supplemented with 10% FBS for 48 h prior to the MTS assay for the cell proliferation assay. For the MTS assay, at the end of the experiment, 10 ml of MTS (5 mg/ml in PBS) were added, and the cells were incubated for 2 h in the humidified incubator that contained 5% CO2 at 37°C. The relative cell viability and proliferation were obtained by scanning with an ELISA reader with a 490 nm filter. For the cell growth array, the cell number was counted in triplicates of samples at five time points after transfection. The cells were trypzinized and resuspended in a 1:1 mixture of PBS and 0.5% trypan blue, and the number of viable cells was counted using a hemocytometer. Cell apoptosis assay Cell apoptosis was detected via flow cytometry using an Annexin V-FITC Apoptosis detection Kit (eBioscience, San Diego, CA). Forty-eight hours after transfection, equal amounts of PC3 or DU145 cells were seeded in the 60 mm cell culture dish and cultured in the medium supplemented without serum for 48 h. The cells were then trypzinized, washed twice with ice-cold PBS, resuspended in Binding Buffer and incubated with Annexin V-FITC for 10 min at room temperature. The cells were washed with ice-cold PBS, resuspended in Binding Buffer and incubated with propidium iodide (20 mg/ml) for 5 min. Apoptosis was subsequently analyzed via flow cytometry (BD FACSCalibur) using BD CellQuestTM Pro software. Western blot analysis Western blot analysis was conducted as previously described (Wu et al., 2013). In brief, appropriately treated cells were lysed in RIPA buffer, and equal amounts of protein were separated on a 10% SDS polyacrylamide gel, transferred to a PVDF membrane (Millipore, Billerica, MA) and immunoblotted with antibodies. The primary antibodies used included antibodies against b-arrestin2, phospho-Akt (Ser473), Akt1, FOXO1 (Cell Signaling Technology, Danvers, MA), phospho-FOXO1 (Ser256, SAB), HA-Tag, GFP-Tag (Abmart, Shanghai, China), PP2A (Epitomics, Cambridge, UK), Ubiquitin, and GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA). The secondary antibodies were anti-mouse and anti-rabbit IgG conjugated with HRP (Santa Cruz Biotechnology). For the detection of FOXO1 ubiquitlylation, the cell extracts were incubated with FOXO1 antibody at 4°C overnight; the samples were subsequently immobilized on protein A/G-Sepharose beads (Santa Cruz Biotechnology) for 4 h, washed three times with RIPA lysis buffer and analyzed using Western blot analysis after elution from beads. The band intensities were quantified with respect to GAPDH using ImageJ software and presented as bar graphs after testing statistical validity. Luciferase Reporter Assay The luciferase reporter 3 IRS-luc, which contains three copies of an FKHR response element from the promoter of the insulin-like growth factor-binding protein-1gene, was constructed as previously described (Tang et al., 1999). After transfections, cell lysates were prepared, and luciferase activity was determined using the Dual–Luciferase Assay (Promega) according to the
b-ARRESTIN2 DOWN-REGULATES FOXO1 IN CRPC CELLS
manufacturer’s instructions. The relative luciferase activity was measured as the ratio of firefly luciferase activity to renilla luciferase activity. PP2A activity assay Cells were lysed in RIPA buffer, and the protein concentrations were determined using the DCTM protein assay regents (Bio-Rad, Hercules, CA). Then, PP2A was immunoprecipitated with PP2A antibody (Epitomics) and protein A/G agarose (Santa Cruz Biotechnology) overnight at 4°C. The beads were subsequently washed three times with the previous lysis buffer, and the phosphatase activity of immunoprecipitated PP2A was assayed with a Serine/ Threonine Phosphatase Assay System (Promega) using RRA (pT) VA as the substrate peptide following the manufacturer’s instructions. The absorbance was measured at 600 nm using an ELISA reader. Assays were performed in quadruplicate in three separate experiments, and the PP2A activity was calculated using the fold change in each experiment. Immunoprecipitation To cross-link interacting proteins prior to IP, cells were treated with 2 mM dithiobis (Pierce, Thermo Scientific, Wilmington, DE) dissolved in dry DMSO. The cells were then mechanistically broken (passage through a 29-gauge needle) in ice-cold Nonidet P-40 buffer (142.5 mM KCl, 5 mM MgCl2, 10 mM HEPES, and 0.1% NP-40) with protease inhibitors and incubated with FOXO1 antibody (Cell Signaling Technology) or control IgG antibody (Santa Cruz Biotechnology) at 4°C overnight. The lysate antibody mixture was centrifuged at 4000 rpm/min for 5 min and washed three times with lysis buffer. The precipitated proteins were eluted with SDS sample buffer for Western blot analyses. Translocation studies Forty-eight hours after the HEK293 cells were transfected with the indicated plasmids, the culture medium was replaced with DMEM supplemented with 10% FBS (or serum-free) for 2 h and counter stained with DAPI for 10 min in the dark. The cytoplasmic/ nuclear location of b-arrestin2 and FOXO1 were subsequently captured using an inverted fluorescence microscope (Olympus IX73). Statistical analysis The data are reported as the means SD of at least three independent experiments. The mean differences were compared using ANOVA and the Student t test. A P value of less than 0.05 was considered to be statistically significant. Results b-Arrestin2 contributes to the cell viability and proliferation in CRPC cell lines
To investigate the role of b-arrestin2 in the regulation of the viability and proliferation of CRPC cells, we first established the b-arrestin2 expression in distinct types of prostate cancer cell lines, such as benign prostate RWPE-1 cells, ADPC LNCaP cells, and CRPC cells (PC3 and DU145). Compared with the benign prostate RWPE-1 cells, the LNCaP cells expressed less b-arrestin2 protein, whereas the CRPC cells expressed more b-arrestin2 protein (Fig. 1A), which suggest that b-arrestin2 may exert a specific regulatory function in CRPC cells that is distinct from ADPC cells. Thus, we detected the effects of siRNA mediated b-arrestin2 inhibition on the cell viability and proliferation in CRPC cells. As shown in Figure 1B, both b-arrestin2 siRNA sequences dramatically decreased the expression of endogenous b-arrestin2. Compared with the JOURNAL OF CELLULAR PHYSIOLOGY
control group, both siRNA sequences that targeted b-arrestin2, but not unrelated siRNA, significantly decreased the cell viability, promoted serum-deprivation -induced cell apoptosis, and inhibited the cell proliferation and growth of PC3 or DU145 cells (Fig. 1C–G), which suggest that b-arrestin2 is required for cell viability and proliferation in CRPC cell lines. FOXO1 is required for b-arrestin2 siRNA-mediated inhibition of cell viability and proliferation in CRPC cells
As a key downstream target of PTEN, FOXO1 plays a crucial role in tumor suppression through the induction of cell growth arrest and apoptosis (Nakamura et al., 2000; Modur et al., 2002). Furthermore, as an important target of Akt, its phosphorylation by Akt can regulate cell apoptosis and proliferation via the operation of its target genes in the nucleus (Song et al., 2005; Huang and Tindall, 2007). To assess the role of FOXO1 in b-arrestin2 siRNA-mediated cell viability and proliferation, b-arrestin2 siRNAs and FOXO1 siRNAs were cotransfected into CRPC cells (Fig. 2A and B); the cell viability and proliferation were subsequently detected. As shown in Figure 2C–G, compared with the control group, the knockdown of the expression of FOXO1 markedly increased the cell viability, promoted the cell proliferation and growth, and inhibited the cell apoptosis induced by serum-deprivation. In addition, compared with the knockdown b-arrestin2 expression alone, the coknockdown of the expression of FOXO1 and b-arrestin2 not only significantly attenuated b-arrestin2 siRNA-mediated inhibition of cell viability, proliferation and growth, but also inhibited b-arrestin2 siRNA-induced cell apoptosis. These findings suggest that FOXO1 is, at least in part, required for b-arrestin2 siRNA-mediated cell viability and proliferation inhibition in CRPC cells. b-Arrestin2-mediated FOXO1 phosphorylation in response to IGF-1 stimulation
As FOXO1 is functionally inhibited by phosphorylation in response to IGF-1 stimulation through PI3K/Akt kinase (Nakae et al., 2000), we investigated the role of b-arrestin2 in IGF-1-induced Akt/FOXO1 signal transduction. Compared with the control group, the knockdown of the endogenous expression of b-arrestin2 significantly decreased IGF-1-induced phosphorylation of Akt and FOXO1, whereas the forced overexpression of b-arrestin2 accelerated the phosphorylation of Akt and FOXO1. However, both transient knockdown and overexpression of b-arrestin2 had no obvious effects on the expression of Akt and FOXO1 (Fig. 3A and B). In addition, we examined the effects of b-arrestin2 truncation mutants, including the b-arrestin2 fragment that comprises amino acids 1–185 and 186–409, on the IGF-1-induced phosphorylation of Akt and FOXO1. The results demonstrated that the overexpression of b-arrestin2 (1–185) or b-arrestin2 (186–409) dramatically reduced the IGF-1-stimulated phosphorylation of Akt and FOXO1 in the PC3 cells (Fig. 3C). As IGF-1 inactivates FoxO1 through the PI3K/Akt pathway (Ni et al., 2007), we investigated whether b-arrestin2 mediated phosphorylation of FOXO1 induced by IGF-1 is Akt dependent. Interestingly, although pretreatment with an Akt inhibitor dramatically attenuated the IGF-induced FOXO1 phosphorylation in both GFP-N1- and GFP-b-arrestin2-transfected PC3 cells (Fig. 3D), the ectopic expression of b-arrestin2 resulted in a significant decrease in the transcriptional activity of endogenous FOXO1 and attenuated the increased transcriptional activity induced by Akt inhibitor pretreatment, which indicates b-arrestin2 could mediate FOXO1 activity in an Akt-independent manner. In
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Fig. 1. b-Arrestin2 contributes to the cell viability and proliferation in CRPC cells. (A) b-Arrestin2 expression in distinct prostate cancer cell lines. The expressions of indicated proteins were detected using Western blot. (B) Ninety-six hours after transfection with the indicated siRNA, the protein levels were determined using Western blot with the indicated antibodies. (C) Forty-eight hours after transfection, equal amounts of cells were seeded in a 96-well plate and cultured in medium supplemented with/without serum for 48 h. The cell viability was subsequently detected using MTS array. (D) The cell apoptosis was detected as described in “Materials and Methods”. (E) The cell proliferation was detected using MTS array and expressed as a percent of the control group. (F) and (G) Cell growth was measured at five time points after transfection using Trypan blue staining and a hemocytometer. & denotes P 0.01, *, # denotes P 0.05 vs. C, and ** denotes P 0.01 vs. C. (C: control, NC: negative control).
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b-ARRESTIN2 DOWN-REGULATES FOXO1 IN CRPC CELLS
Fig. 2. FOXO1 is required for b-arrestin2 siRNA-mediated cell viability and proliferation inhibition in CRPC cells. (A) and (B) Ninety-six hours after transfection with the indicated siRNAs, the expressions of the indicated proteins were detected using Western blot. (C–G) The cell viability, apoptosis, proliferation and growth were determined as described in Fig. 1. & denotes P 0.01, $ denotes P 0.05 vs. C, # denotes P 0.05, ** denotes P 0.01, * denotes P 0.05 vs. siRNA-arr2 þ siRNA-O1.
addition, the ectopic expression of b-arrestin2 also significantly decreased the reporter gene activity activated by exogenous FOXO1 with or without Akt inhibitor pretreatment, which suggests that b-arrestin2 inhibits the transcriptional activities of both endogenous and transfected FOXO1 (Fig. 3E).
phosphatase activity of PP2A (Fig. 4C). In addition, the forced expression of b-arrestin2 attenuated the interaction between PP2A and FOXO1. Together, these findings indicated that the effect of b-arrestin2 on FOXO1 dephosphorylation is at least, in part, achieved through PP2A.
b-Arrestin2-mediated FOXO1 dephosphorylation
b-Arrestin2 attenuates serum-deprivation-induced nucleic accumulation of FOXO1
As signal transduction is tightly regulated by phosphorylation– dephosphorylation cycles, the phosphorylation and dephosphorylation of FOXO1 in response to IGF-I stimulation were investigated. Similar to a previous report (Schachter et al., 2012), IGF-1 stimulation induced the phosphorylation of Akt and FOXO1 in a time-dependent manner (Fig. 4A). In contrast, compared with GFP-N1 transfected PC3 cells, the dephosphorylation of FOXO1 in GFP-b-arrestin2 transfected cells was delayed (Fig. 4B). As PP2A is a pivotal serine/ threonine phosphatase in the regulation of FOXO1 dephosphorylation (Yan et al., 2008), we tested the effect of b-arrestin2 on PP2A activity. The results indicated that the forced expression of b-arrestin2 significantly attenuated the JOURNAL OF CELLULAR PHYSIOLOGY
As a transcription factor, FOXO1 regulates a large spectrum of tumor suppression genes in the nucleus, and its sub-cellular location in the nucleus and cytoplasm is critical for its function. Thus, the effect of b-arrestin2 on the serum-deprivation-induced nucleic accumulation of FOXO1 was investigated. In the culture medium supplemented with 10% FBS, both b-arrestin2 and FOXO1 were mainly located in the cytoplasm. In contrast, two hours after serum deprivation, approximately all FOXO1 translocated into the nucleus, whereas in the cells that cooverexpressed b-arrestin2, the nucleic accumulation of FOXO1 induced by serum-deprivation was dramatically attenuated (Fig. 5).
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Fig. 3. b-Arrestin2-mediated FOXO1 phosphorylation in response to IGF-1 stimulation. (A–C) Forty-eight hours after transfection with the indicated siRNAs or plasmids, PC3 cells were incubated in serum-free medium for 1 h and followed by IGF-1 (100 ng/ml) stimulation for 10 min. The expressions of the indicated proteins were subsequently detected using Western blot. (D) Forty-eight hours after transfection with the indicated plasmids, PC3 cells were incubated in serum-free medium for 1 h followed by Akt inhibitor (10 mM) treatment for 1 h prior to IGF-1 stimulation (100 ng/ml, 10 min). The expressions of the indicated proteins were subsequently detected using Western blot. (E) PC3 cells were transiently cotransfected with the 3 IRS luciferase reporter plasmid along with the indicated plasmids. At 12 h after transfection, the cells were treated with an Akt inhibitor (10 mM) or DMSO for an additional 24 h. Luciferase measurement and data analysis were performed as described in “Materials and Methods”. * denotes P 0.05 and ** denotes P 0.01.
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Fig. 4. b-Arrestin2-mediated dephosphorylation of FOXO1. (A) and (B) PC3 cells (or PC3 monoclonal cells that stably expressed GFP-N1 or GFP-b-arrestin2) were incubated in serum-free medium for 1 h followed by IGF-1 stimulation for the indicated times. The expressions of the indicated proteins were subsequently detected using Western blot. (C) PC3 cells were transfected with the indicated plasmids, and the relative phosphatase activity was detected as described in “Materials and Methods” at 48 h after transfection. (D) Decreased association between PP2A and FOXO1 in the PC3 cells that overexpressed GFP-b-arrestin2. Forty-eight hours after transfection with the indicated plasmids, the cell lysates and immunoprecipitated proteins were analyzed using Western blotting with the indicated antibodies. ** denotes P 0.01.
b-Arrestin2-mediated FOXO1 expression and proteasomal degradation
To assess the effect of b-arrestin2 on the protein expression of FOXO1, PC3 monoclonal cells with stable overexpression of pEGFP-N1-b-arrestin2 (PC3-b-arr2) or empty vector pEGFP-N1 (PC3-N1) were generated through G418 selection. As shown in Figure 6A, the FOXO1 expression was significantly decreased in the PC3-b-arr2 cells compared with the PC3-N1 cells. Ubiquitylation is a mechanism to target proteins for proteasomal degradation (Huang and Tindall, 2011). Thus, bortezomib, a proteasome inhibitor (Lakshmikanthan et al., 2009), was used to investigate the potential mechanisms involved in the b-arrestin2-mediated FOXO1 degradation. Bortezomib treatment increased the FOXO1 accumulation in the control PC3-N1 cells, and the decrease in FOXO1 expression (caused by the forced JOURNAL OF CELLULAR PHYSIOLOGY
b-arrestin2 overexpression) was reversed in the PC3-b-arr2 cells (Fig. 6B). Furthermore, bortezomib treatment represented a markedly enhanced ubiquitylation signal in the PC3-b-arr2 cells compared with the PC3-N1 cells, which implies b-arrestin2 causes the FOXO1 degradation in the proteasome via ubiquitylation (Fig. 6C). Discussion
In general, prostate cancer ultimately progresses to become androgen independent after androgen ablation therapies, which thereby renders anti-androgen therapy ineffective. As a lethal form of prostate cancer progresses and metastasizes, CRPC remains untreatable to date (Feldman and Feldman, 2001; Pienta and Bradley, 2006; Jemal et al., 2010; Jacome-Pita et al., 2014). Understanding the mechanism that leads to the development of CRPC will pave the way to effective therapies
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Fig. 5. b-Arrestin2 attenuates serum-deprivation-induced nucleic accumulation of FOXO1. Forty-eight hours after transfection with the indicated plasmids, HEK293 cells were incubated in medium supplemented with free or 10% FBS for 2 h and then counter stained with DAPI to visualize the nuclei. Representative images were captured using an inverted fluorescence microscope.
for CRPC. In the present study, our data indicated that b-arrestin2 is required for cell viability and proliferation in CRPC cells, which is associated with its inhibitory effects on the activity and expression of FOXO1, and thereby represents a novel potential mechanism of CRPC progress mediated by b-arrestin2. b-Arrestins were initially known as negative regulators of GPCR-mediated signaling. However, recent studies have demonstrated that b-arrestins can also function as scaffold proteins and interact with a variety of different signaling molecules in the cytoplasm; thus, they play important roles in the regulation of receptor trafficking and signaling (Ma and Pei, 2007; DeFea, 2011). Accumulating evidence has established that b-arrestins are widely involved in various cancer developmental signal transductions that are responsible for tumor viability and metastasis, which suggests an impressive role of b-arrestins in tumor progression (Hu et al., 2013). More JOURNAL OF CELLULAR PHYSIOLOGY
recently, b-arrestin2 was confirmed to form a complex with AR and act as an AR corepressor in ADPC LNCaP cells, whereas its role in CRPC cells remains uninvestigated (Lakshmikanthan et al., 2009). To investigate the role of b-arrestin2 in CRPC cells, we first investigated the basal expression of b-arrestin2 in ADPC and CRPC cell lines. The results demonstrated that compared with benign prostate RWPE-1 cells, the expression of b-arrestin2 was significantly decreased in ADPC LNCaP cells but dramatically increased in CRPC 22Rv1, PC3 and DU145 cells. These findings suggest b-arrestin2 may exert a different regulatory function in CRPC cells compared with ADPC cells. This result is consistent with a previous report that had not compared the b-arrestin2 expression in RWPE-1 and CRPC cells (Lakshmikanthan et al., 2009). Although the role of b-arrestin2 in cell survival and proliferation has been well established in lung and breast cancers, little is known regarding
b-ARRESTIN2 DOWN-REGULATES FOXO1 IN CRPC CELLS
Fig. 6. b-Arrestin2-mediated FOXO1 expression and proteasomal degradation. (A) FOXO1 expression in PC3-N1 and PC3-b-arr2 cells. PC3 monoclonal cells were cultured in growth medium for 24 h, and the expressions of the indicated proteins were detected using Western blot. (B) PC3 monoclonal cells were cultured in a growth medium for 24 h and treated with or without bortezomib (100 nM) for 6 h. The expressions of the indicated proteins were subsequently detected using Western blot. (C) PC3 monoclonal cells were pretreated with bortezomib (100 nM) for 6 h; the cell lysates were then subjected to immunoprecipitation with anti-FOXO1 antibody, and ubiquitination was detected using Western blot with anti-ubiquitin antibody. The cell lysates were probed with anti-GAPDH antibody to demonstrate the total protein expression. *, # denotes P 0.05 and ** denotes P 0.01.
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its role in prostate cancer. In the present study, our results demonstrated that similar to its role in breast cancer, b-arrestin2 is required for cell viability and proliferation in CRPC cells (Fig. 1B and C), whereas it is different from that in lung cancer, which suggests the function of b-arrestin2 is distinct in different cancers (Raghuwanshi et al., 2008; Zhao et al., 2009). FOXO1 is an important nuclear target for both Akt-dependent and -independent survival signals in prostate cancer progression (Modur et al., 2002; Li et al., 2003). In the present study, the co-knockdown of FOXO1 expression dramatically attenuated the b-arrestin2 siRNA-mediated inhibition of cell viability, proliferation and growth, and significantly inhibited b-arrestin2 siRNA-induced cell apoptosis, which suggests b-arrestin2-mediated regulation of cell viability and proliferation in CRPC cells is at least, in part, mediated by FOXO1. Post-translational inactivation and nuclear exclusion of FOXO1 have been observed in carcinomas of the breast, stomach, thyroid, lung, and prostate (Fu and Tindall, 2008). As a key downstream target of Akt, FOXO1 is functionally inhibited by phosphorylation in response to IGF-1 stimulation through PI3K/Akt kinase (Nakae et al., 2000). Phosphorylated Akt phosphorylates FOXO1 and results in the inactivation of FOXO1 and its translocation from the nucleus to the cytoplasm, which leads to the transcription suppression of proapoptotic genes (Fu and Tindall, 2008). In various cancers, including prostate cancer, IGF-1 activates the PI3K/Akt pathway to promote cell proliferation and survival via binding to its receptor IGF-1R (Pollak, 2008). IGF-1 mediated AR reactivation in low or absent androgen environments is one of the suggested multiple mechanisms by which ADPC cells progress to the androgen-independent stage (Grossmann et al., 2001; Cronauer et al., 2003; Fan et al., 2007). Furthermore, accumulating evidence indicates that IGF-1-induced activation of the PI3K/Akt pathway, which is one of the most important intracellular signaling pathways that favor cell proliferation and survival, greatly contributes to prostate cancer progression (Pollak, 2008). PI3K/Akt is a controversial pathway regulated by b-arrestins, and there is evidence for both the inhibition and stimulation of this pathway mediated by b-arrestins (Povsic et al., 2003; Beaulieu et al., 2005; Wang and DeFea, 2006; Girnita et al., 2007; Wang et al., 2007; Lodeiro et al., 2009; Luan et al., 2009; DeFea, 2011). In contrast, the exact role of b-arrestin2 in the regulation of IGF-1-induced phosphorylation of Akt and FOXO1 in CRPC cells remains unclear. In the present study, our data identified a pivotal role of b-arrestin2 in the regulation of the phosphorylation of Akt and FOXO1 induced by IGF-1 in CRPC cells, which was consistent with the results reported by Luan et al. (2009). However, interestingly, our results demonstrated that b-arrestin2 could regulate FOXO1 activity in both Akt-dependent and -independent manners, which indicates that some other unclear mechanisms were involved in the b-arrestin2-mediated regulation of FOXO1 transcriptional activity in CRPC cells. Reversible phosphorylation catalyzed by kinases and phosphatases is a pivotal regulatory mechanism of FOXO transcriptional activity. In the present study, our results demonstrated that the overexpression of b-arrestin2 significantly delayed FOXO1 dephosphorylation after IGF-1 stimulation via the downregulation of PP2A activity and the attenuation of the interaction between PP2A and FOXO1, which demonstrates a novel mechanism of FOXO1 activity mediated by b-arrestin2. The nucleic/cytoplasmic shuttling that regulates the intracellular FOXO1 distribution is a central regulation mechanism for FOXO1 transcriptional activity (Brunet et al., 1999; Gan et al., 2005). A recent study indicated that JOURNAL OF CELLULAR PHYSIOLOGY
b-arrestin1 is required for clozapine and lithium to suppress the nuclear localization of FOXO (Weeks et al., 2011). Similar to their results, our data demonstrated that the intracellular distribution of b-arrestin2 and FOXO1 represent a high consistency, and b-arrestin2 significantly attenuated the nuclear accumulation of FOXO1 induced by serum-deprivation, which suggests a critical role of b-arrestin2 in the regulation of FOXO1 activity. In general, FOXO1 is dysregulated in breast cancer, prostate cancer, glioblastoma, rhabdomyosarcoma, and leukemia (Fu and Tindall, 2008). The ubiquitination and proteasome degradation of FOXO1 play an important role in tumorigenesis. The ubiquitination of FOXO1 mediated by E3 ligases, including Skp2 and Mdm2, leads to its ubiquitination– proteasome degradation, which thereby favors cell proliferation and survival (Girnita et al., 2005; Huang and Tindall, 2011). As one of the well-characterized binding partners of Mdm2, b-arrestin2 acts as a scaffold for Mdm2, which leads to AR ubiquitylation and degradation in ADPC LNCaP cells (Girnita et al., 2005; Lakshmikanthan et al., 2009; Kommaddi and Shenoy, 2013). Similar to its effect on AR, our results demonstrated that b-arrestin2 induces a decrease in FOXO1 protein level through proteasome degradation. In conclusion, our data identified a novel role of b-arrestin2 in the regulation of FOXO1 activity and expression, as well as the IGF-induced transactivation of Akt/FOXO1 pathway, which may account for its requirement for cell viability and proliferation in CRPC cells. These findings represent a novel potential mechanism of CRPC progress mediated by b-arrestin2. Acknowledgements
This work was financed by grants from the National Natural Science Foundation of China (No. 81402430, NO. 81370804 and NO. 81170652), the Colleges and universities in Guangzhou Yangcheng scholars research project (No. 12A017S), and the Science and technology project in Guangzhou (the people’s livelihood special major science and Technology, No.201300000096). Literature Cited Beaulieu JM, Sotnikova TD, Marion S, Lefkowitz RJ, Gainetdinov RR, Caron MG. 2005. An Akt/beta-arrestin 2/PP2A signaling complex mediates dopaminergic neurotransmission and behavior. Cell 122:261–273. Brunet A, Bonni A, Zigmond MJ, Lin MZ, Juo P, Hu LS, Anderson MJ, Arden KC, Blenis J, Greenberg ME. 1999. Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell 96:857–868. Cronauer MV, Schulz WA, Burchardt T, Anastasiadis AG, de la Taille A, Ackermann R, Burchardt M. 2003. The androgen receptor in hormone-refractory prostate cancer: Relevance of different mechanisms of androgen receptor signaling (Review). Int J Oncol 23:1095–1102. DeFea KA. 2011. Beta-arrestins as regulators of signal termination and transduction: How do they determine what to scaffold?. Cell Signal 23:621–629. Fan W, Yanase T, Morinaga H, Okabe T, Nomura M, Daitoku H, Fukamizu A, Kato S, Takayanagi R, Nawata H. 2007. Insulin-like growth factor 1/insulin signaling activates androgen signaling through direct interactions of Foxo1 with androgen receptor. J Biol Chem 282:7329–7338. Feldman BJ, Feldman D. 2001. The development of androgen-independent prostate cancer. Nat Rev Cancer 1:34–45. Fosbrink M, Niculescu F, Rus V, Shin ML, Rus H. 2006. C5b-9-induced endothelial cell proliferation and migration are dependent on Akt inactivation of forkhead transcription factor FOXO1. J Biol Chem 281:19009–19018. Fu Z, Tindall DJ. 2008. FOXOs, cancer and regulation of apoptosis. Oncogene 27:2312– 2319. Gan L, Chen S, Wang Y, Watahiki A, Bohrer L, Sun Z, Wang Y, Huang H. 2009. Inhibition of the androgen receptor as a novel mechanism of taxol chemotherapy in prostate cancer. Cancer Res 69:8386–8394. Gan L, Zheng W, Chabot JG, Unterman TG, Quirion R. 2005. Nuclear/cytoplasmic shuttling of the transcription factor FoxO1 is regulated by neurotrophic factors. J Neurochem 93:1209–1219. Girnita L, Shenoy SK, Sehat B, Vasilcanu R, Girnita A, Lefkowitz RJ, Larsson O. 2005. {beta}-Arrestin is crucial for ubiquitination and down-regulation of the insulin-like growth factor-1 receptor by acting as adaptor for the MDM2 E3 ligase. J Biol Chem 280:24412– 24419. Girnita L, Shenoy SK, Sehat B, Vasilcanu R, Vasilcanu D, Girnita A, Lefkowitz RJ, Larsson O. 2007. Beta-arrestin and Mdm2 mediate IGF-1 receptor-stimulated ERK activation and cell cycle progression. J Biol Chem 282:11329–11338.
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