Med Oncol (2015) 32:49 DOI 10.1007/s12032-015-0486-1
ORIGINAL PAPER
GPCR48/LGR4 promotes tumorigenesis of prostate cancer via PI3K/Akt signaling pathway Fang Liang • Junmin Yue • Junyong Wang Lijuan Zhang • Rui Fan • Hao Zhang • Qingsong Zhang
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Received: 22 December 2014 / Accepted: 22 January 2015 Ó Springer Science+Business Media New York 2015
Abstract G-protein-coupled receptor (GPCR) 48, also known as leucine-rich repeat-containing G-protein-coupled receptor (LGR) 4, is an orphan receptor belonging to the GPCR superfamily, which plays an important role in the development of various organs and multiple cancers. However, the function of GPCR48/LGR4 in prostate cancer has not been fully investigated. Herein, GPCR48/LGR4 was overexpressed and silenced in prostate cancer cells via plasmid and shRNA transfection, respectively. The expression of GPCR48/LGR4 in mRNA and protein levels was analyzed using RT-qPCR and Western blotting, respectively. Subsequently, we demonstrated the effects of GPCR48/LGR4 on the migration, invasion, proliferation and apoptosis of prostate cancer cells, including Du145 and PC-3 cells. Next, we investigated the relationship between GPCR48/LGR4 and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/Akt signaling pathway. The results showed that the overexpression of GPCR48/LGR4 was associated with the up-regulation of Akt, a key effector of PI3K/Akt signaling pathway, which meantime up-regulated the expression of mammalian target of rapamycin
Fang Liang and Junmin Yue have contributed equally to the work. F. Liang L. Zhang Department of Oncology, Zhengzhou Central Hospital Affiliated to Zhengzhou University, Zhengzhou, China J. Yue J. Wang R. Fan H. Zhang Department of Urinary Surgery, Zhengzhou Central Hospital Affiliated to Zhengzhou University, Zhengzhou, China Q. Zhang (&) Department of Breast Surgery, Zhengzhou Central Hospital Affiliated to Zhengzhou University, 195 Tongbai Avenue, Zhengzhou 450007, China e-mail: [email protected]
(mTOR) and glycogen synthase kinase 3b (GSK-3b), while down-regulated forkhead box, class O (FOXO), all of whom are the downstream targets of PI3K/Akt signaling pathway. Hence, the results suggested that GPCR48/LGR4 may regulate prostate cancer cells and tumor growth via the PI3K/Akt signaling pathway and could provide a better therapeutic target for the diagnosis and treatment of prostate cancer. Keywords Tumor
GPCR48/LGR4 Prostate cancer PI3K/Akt
Introduction Prostate cancer is the main cause of cancer-related deaths, which has the second largest population of male patients in the USA [1]. Traditional treating methods include androgen deprivation therapy (ADT), radical prostatectomy, hormonal therapy and radio-chemotherapy [2]. ADT contributes greatly during the early stages of prostate cancer. Hormonal treatment is relatively simple and less painful; however, it may easily lead to drug resistance, and related drugs are ineffective at this stage. To acquire a high cure rate, localized prostate tumor is usually subjected to radical prostatectomy or radio-chemotherapy [3]. What is worse, patients usually suffer from side effects of the radiochemotherapy, such as hair loss or immune system degradation. G-protein-coupled receptor (GPCR) is a big superfamily, which contributes greatly to the development of some organs [4] and even to the formation of some tumors, such as lung cancer and prostate cancer [5–7]. GPCR superfamily also involves a subfamily of leucine-rich repeatcontaining G-protein-coupled receptors (LGR), which
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consist of a transmembrane domain of seven a-helixes and an extracellular area with a long N-terminal fragment containing leucine-rich repeat motifs which function in receptor-ligand interactions [8–10]. As reported, LGR has three kinds of subtypes, including three hormones (luteotropic hormone, follicle-stimulating hormone and thyroid stimulating hormone), LGR 4–6 and LGR 7–8 [11]. Recently, both preclinical and clinical studies have proved that GPCR was overexpressed in prostate cancer tissues [12, 13]; however, the mechanism was still not clear. Current methods such as prostatectomy and radio-chemotherapy are so radical and drastic that patients may suffer from secondary diseases. Besides, we need earlier diagnosis and treatment which benefit a lot to the patients because of the limited focus and functions of cancer cells are easier to deal with. What is more important is that we want to diagnose and treat prostate cancer specifically. These all underline the need for new methods with specific target molecules for prostate cancer [14, 15]. GPCR is widespread in human bodies, and its function has been proved in some cancers, such as gastric cancer and colorectal cancer [16]. Convincing evidence confirms that abnormal activation or expression of GPCR is intimately related to increased cancer cell proliferation, tumor growth and metastasis [12, 13]. These characteristics would highlight GPCR as a novel biomarker and therapeutic target of prostate cancer. Therefore, the identification of a specific GPCR for prostate cancer and a possibly related signaling pathway are two crucial prerequisites for a more detailed way to recover from prostate cancer. GPCR48, also known as LGR4, is an orphan receptor which exists broadly in multiple tissues and organs from embryonic to adult periods [16, 17]. In the PI3K/Akt signaling pathway, what is more, Akt is the pivotal link between the upstream regulation and downstream expression in the development of various organs [18]. Our study aimed to investigate the function of GPCR48/LGR4 in prostate cancer cell lines and its correlation with prostate tumors growth and the PI3K/Akt signaling pathway. We hypothesize that GPCR48/LGR4 is a potential new target for the diagnosis and treatment of prostate cancer.
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Cell culture Du145 (American Type Culture Collection, Manassas, VA, USA) and PC-3 cells (The Cell Bank of Chinese Academy of Sciences, Shanghai) were cultured in RPMI-1640 medium (Hyclone, Logan, UT, USA) supplemented with 10 % (v/v) fetal bovine serum (FBS, Hyclone) and 1 % (v/v) penicillin/streptomycin (Hyclone). Cells were grown in a humidified incubator at 37 °C with 5 % CO2. Plasmid constructions and cell transfection A GPCR48 plasmid encoding full-length human GPCR48 and an empty vector was purchased from Fitgene (Guangzhou, China). For GPCR48 overexpression, the GPCR48 plasmid and the empty vector were separately transfected into cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions, and the stable transfectants were selected in the presence of 600 lg/ml G418 (Amresco, Solon, OH, USA). Transfection with lentivirus-mediated shRNA GPCR48/LGR4 knockdown was performed via transfection with a lentivirus that expresses human GPCR48/ LGR4-specific short hairpin RNA (shRNA). Briefly, the lentivirus vector plasmid encoding human GPCR48/LGR4specific shRNA (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was transfected with capsule and packaging plasmids using Superfect (Qiagen, Dusseldorf, Germany) into HEK293T cells. Supernatant was collected 48 h later and used as infection solution without enrichment. The scramble shRNA obtained from Addgene (Cambridge, MA, USA) was also employed. The non-infected cells were removed by medium containing puromycin (5 lg/ml; Sigma-Aldrich, St. Louis, MO, USA). The GPCR48/LGR4 knockdown was confirmed by RT-qPCR and Western blotting. Transwell assay
Materials and methods Animals Six-week-old male athymic nude mice were purchased from the Experimental Animal Center of Zhengzhou University (Zhengzhou, China) and fed in pathogen-free conditions with free access to boiled water and food. All animal experiments were done in accordance with protocols preapproved by the Institutional Animal Care and Treatment Committee of Zhengzhou Central Hospital.
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Cell migration ability was measured in 24-well transwell chambers (Corning, Tewksbury, MA, USA). The bottom chamber was prepared with 10 % FBS as a chemoattractant. For migration assays, cells in serum-free medium (5 9 105 cells per well) were added to the upper chamber and incubated for 12 h. Non-migrating cells on the upper side of the membrane were removed, and the cells that migrated to the lower chamber were fixed with 4 % paraformaldehyde and stained with hematoxylin–eosin (HE). For invasion assays, Matrigel (BD Biosciences, San Jose, CA, USA) was added to the transwell membrane chambers
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and incubated at 37 °C for 4 h. Cells (1 9 105 cells per well) were seeded and incubated before analysis. The number of migrating or invading cells was then determined in five randomly selected fields under a microscope. Each experiment was performed at least three times. Both migration assay and invasion assay were tested at three time points (24, 36 and 48 h after overexpression or knockdown), respectively. MTT assay An MTT assay was used to detect cell proliferation. Cells were seeded into 96-well plates at a rate of 1,000 cells per well. On the day of harvest (24, 36 and 48 h after overexpression or knockdown), 100 ll of spent culture medium was replaced with an equal volume of fresh medium (10 % FBS). MTT (Sigma-Aldrich) was added to the cells at a final concentration of 0.5 mg/ml, and the plates were incubated for 4 h at 37 °C. Then, 100 ll of DMSO (SigmaAldrich) was added to each well and plates were shaken at room temperature for 10 min. The absorbance was measured at 490 nm. Apoptosis analysis Hoechst staining and TUNEL assay were used to assess the cell apoptosis. Cells were cultured in 96-well plates with a density of 3,000 cells per well after overexpression and silence of GPCR48/LGR4. Cellular nuclear morphology was detected using Hoechst Staining Kit (Beyotime Biotech, Haimen, China). DNA fragmentation morphology was examined by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) staining using TUNEL Apoptosis Detection Kit (Merck Millipore, Billerica, MA, USA). Both staining-positive cells were visualized and counted in randomly selected fields for further analysis. Tumor growth For xenograft tumor transplantation, cells (2 9 106) in 100 ll PBS and Matrigel [1:1 (v/v)] were subcutaneously injected into the flanks of athymic male mice. External caliper measurement of long (L) and short (S) tumor lengths was done to calculate tumor volume using the equation: p Tumor volume ¼ L S2 6 Tumor measurements were taken throughout the duration of the experiment at least three times every 5 days. Tissue mass was measured immediately after killing and surgical excision of tumor. Tissues were divided in half, flash-
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frozen in liquid nitrogen and stored at -80 °C. Experiments were repeated twice. Data represent values from 5 to 10 animals. RT-qPCR RNA was isolated using TRIzol reagent (Invitrogen), and complementary DNA (cDNA) was created from 1 lg RNA using RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA). Product amplification was done using a SYBR Green PCR kit (TAKARA BIOTECHNOLOGY, Dalian, Liaoning, China) with CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). For RT-qPCR, 2 ll of cDNA was amplified in a 20-ll standard PCR system, followed by initial denaturation at 94 °C for 3 min; 45 cycles of 94 °C for 45 s, annealing temperature (different, respectively) for 45 s, 72 °C for 45 s; and a final extension for 10 min at 72 °C, followed by termination at 4 °C. Primer sequences were: GPCR48/LGR4 (sense) 50 - CCA ATC ACC ACT CTT ACA CAA TGG CTA AC -30 and (antisense) 50 - ATT CCC GTA GGA GAT AGC GTC CTA G -30 ; Akt (sense) 50 - ATG GTA CCA TGA GCG ACG TGG C -30 and (antisense) 50 - ATT CTA GAT CAG GCC GTG CCG C -30 ; mTOR (sense) 50 - TGC TAC GGA CGC ACT GAG AT -30 and (antisense) 50 - CGT GAA GAG GGA AGG TGG AAC A-30 ; GSK-3b (sense) 50 - ATG GAA CAC CAG CTC CTG TG -30 and (antisense) 50 - TGT CGG TGT AGA TGC ACA G -30 ; FOXO (sense) 50 - TCA TTG AAC AGC CAC CAC -30 and (antisense) 50 - CCC AGA CTC AGG AGG AAG -30 ; and b-actin (sense) 50 - CAG CGA CAC CCA CTC CTC -30 and (antisense) 50 - TGA GGT CCA CCA CCC TGT -30 . All experiments were run three times using at least triplicate samples. Relative gene level calculation was based on the difference in the Ct of the reference gene (b-actin). Relative gene expression is shown as the fold over the reference gene, determined by the DDCt method. Western blotting Western blotting analysis was used to determine relative protein expression. Cells that grew to 70–80 % confluence (about 48 h after overexpression or knockdown) were washed once with ice-cold PBS, lysed in RIPA buffer (Sigma-Aldrich), supplemented with protease inhibitors PMSF (Sigma-Aldrich) and harvested for protein fractionation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Blots were incubated in blocking buffer composed of 4 % bovine serum albumin (BSA, Sigma-Aldrich) tris-buffered saline/Tween-20 (Sigma-Aldrich) for 1 h, followed by 2-h incubation with primary antibody, all 1:1,000 (v/v) in blocking buffer.
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Fig. 1 Overexpression and silencing of GPCR48/LGR4 in Du145 and PC-3 cells, respectively. a RT-qPCR analysis showed that GPCR48/ LGR4 was overexpressed in mRNA level. b RT-qPCR analysis showed that GPCR48/ LGR4 expression was silenced in mRNA level. Western blotting analysis (c) and quantitative analysis of western results (e) also showed that GPCR48/LGR4 was overexpressed in protein level. Western blotting analysis (d) and quantitative analysis of western results (f) also showed that GPCR48/LGR4 expression was silenced in protein level. Each column represented mean ± SD of at least three independent experiments; *P \ 0.05 and **P \ 0.01 versus untreated Du145 or PC-3 cells; #P \ 0.05 and ##P \ 0.01 versus cells transfected with vector or scramble shRNA
Immunoblots were washed in tris-buffered saline/Tween three times for 5 min each, incubated with the appropriate horseradish peroxidase-conjugated anti-rabbit or antimouse secondary antibody for 1 h and washed. The protein signal was visualized using the Amersham ECLTM Plus Western Blotting Detection kit (GE Healthcare, Piscataway, NJ, USA).
determine significant differences between two-sample data. All values are considered significant if P \ 0.05 (*), extremely significant if P \ 0.01 (**).
Results
Statistical analysis
GPCR48/LGR4 is overexpressed or silenced via plasmid or lentivirus transfection, respectively
All the artworks were created using GraphPad Prism 5 software. All statistical analyses were carried out using the SPSS 19.0 statistical software package. All numerical data are shown as the mean ± standard deviation (SD) of at least three independent experiments or at least five biological samples (tumor). The Student’s t test was used to
Firstly, we wanted to verify the overexpression and knockdown of GPCR48/LGR4 gene in prostate cancer cells. We observed that GPCR48/LGR4 expression was significantly up-regulated after the transfection of gene encoding GPCR48/LGR4 compared with untreated cells and vector cells (transfected with empty vector) in both
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Fig. 2 Effects of GPCR48/ LGR4 on migration, invasion, proliferation and apoptosis. a Cell migration ability was promoted by GPCR48/LGR4 overexpression while inhibited by GPCR48/LGR4 knockdown. b Cell invasion ability was promoted by GPCR48/LGR4 overexpression while inhibited by GPCR48/LGR4 knockdown. Results were presented as the ratios of cells relative to those of Control group. c, d MTT assays showed that proliferation ability was significantly increased after GPCR48/LGR4 overexpression but decreased after GPCR48/LGR4 knockdown. e Hoechst staining results showed that GPCR48/ LGR4 inhibited apoptosis of both Du145 and PC-3 cells. f TUNEL assay also proved that GPCR48/LGR4 inhibited apoptosis. Each column represented mean ± SD of at least three independent experiments; *P \ 0.05 versus untreated Du145 or PC-3 cells
mRNA (Fig. 1a) and protein level (Fig. 1c, e). Also, the expression of GPCR48/LGR4 was inhibited by genes silence (Fig. 1b, d, f). These results demonstrated that we overexpressed and silenced GPCR48/LGR4 gene in both Du145 and PC-3 cells, respectively. The migration, invasion, proliferation and apoptosis of Du145 and PC-3 cells are regulated by GPCR48/ LGR4 Next, to investigate the effects of GPCR48/LGR4 on migration and invasion, Du145 and PC-3 cells were subjected to in vitro transwell assay. The number of GPCR48-
overexpressing cells migrating into the lower chamber was at most 5.3-fold (Du145) and 5.5-fold (PC-3) greater than untreated cells (Control group) in a transwell chamber migration assay (Fig. 2a). The results of a Matrigel invasion assay revealed at most 25.1-fold (Du145) and 26.7-fold (PC3) up-regulation in the invasive capacity of GPCR48-overexpressing cells compared with Control group (Fig. 2b). Similarly, both the migration and invasion ability were attenuated after GPCR48/LGR4 knockdown (Fig. 2a, b). We also assessed the effect of GPCR48 on proliferation and found that Du145/GPCR48(LGR4) plasmid cells increased 3.2-fold (Fig. 2c) and PC-3/GPCR48(LGR4) plasmid cells 2.0-fold (Fig. 2d) more than the Control group. The
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Fig. 3 Effects of GPCR48/LGR4 on tumor growth. a For Du145 cells, curves for average tumor volume showed that GPCR48/LGR4 overexpression significantly increased the tumor volume, while GPCR48/LGR4 silence inhibited tumor growth. b For PC-3 cells, curves for average tumor volume showed that GPCR48/LGR4
Fig. 4 RT-qPCR analysis of the effects of GPCR48/LGR4 on PI3K-/Akt-related genes expression in mRNA level. a Akt expression was significantly promoted via GPCR48/LGR4 overexpression, but its expression was significantly down-regulated by GPCR48/LGR4 knockout. b mTOR. c GSK-3b. d FOXO showed an opposite result from Akt, mTOR and GSK-3b. Each column represented mean ± SD of at least three independent experiments; *P \ 0.05 and **P \ 0.01 versus untreated Du145 or PC-3 cells
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overexpression significantly increased the tumor volume, while GPCR48/LGR4 silence inhibited tumor growth. Each column represented mean ± SD of 5–10 independent experiments; *P \ 0.05 versus mice injected with untreated Du145 and PC-3 cells
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Fig. 5 Western blotting analysis of the effects of GPCR48/LGR4 on PI3K-/Aktrelated genes expression in protein level. Western blotting results of Akt, mTOR, GSK-3b and FOXO. Akt, mTOR and GSK-3b expression was enhanced after GPCR48/LGR4 overexpression but attenuated after GPCR48/LGR4 silencing. On the contrary, FOXO expression was attenuated after GPCR48/LGR4 overexpression but enhanced after GPCR48/ LGR4 silencing in Du145 cells (a) and PC-3 cells (b). c– f Quantitative analysis of the western results. Each column represented mean ± SD of at least three independent experiments; **P \ 0.01 versus untreated Du145 or PC-3 cells
proliferation ability was also inhibited by GPCR48/LGR4 silence (Fig. 2c, d). Lastly, we identified the effects of GPCR48/LGR4 on apoptosis. Hoechst staining results showed that GPCR48/LGR4 overexpression slowed down apoptosis and GPCR48/LGR4 overexpression promoted (Fig. 2e). TUNEL results showed a decrease and increase in positive cells after overexpression and silence of GPCR48/ LGR4, respectively, which kept same pace with abovementioned results (Fig. 2f). Collectively, these results
demonstrated that GPCR48 could not only promote migration, invasion and proliferation of Du145 and PC-3 cells but also inhibit apoptosis. Overexpression of GPCR48/LGR4 promotes prostate tumor growth To test whether GPCR48 overexpression and knockdown impacted the growth of prostate tumor in vivo, Du145 and
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Fig. 6 Akt overexpression after transfection with Akt plasmid in Du145 and PC-3 cells. a Akt was overexpressed in Du145 cells in mRNA level. b Akt was overexpressed in PC-3 cells. Western blotting results showed Akt overexpression in both Du145 (c) and PC-3 cells (d). Quantitative analysis of the above western results showed that Akt expression was significantly up-regulated in protein level after plasmid transfection in Du145 (e) and PC-3 cells (f), respectively. Each column represented mean ± SD of at least three independent experiments; *P \ 0.05 versus untreated Du145 or PC-3 cells; #P \ 0.05 versus vector cells
PC-3 cells were subcutaneously injected into immunodeficient mice. The results showed that GPCR48/LGR4 overexpression increased the tumor volume to 79 % (Du145, Fig. 3a) and 77 % (PC-3, Fig. 3b) compared with Control group, respectively, in contrast to the negative effects of shRNA groups. GPCR48/LGR4 regulates the expression of PI3K/Akt signaling genes Akt served as a pivotal link of Wnt/b-catenin signaling pathway and its downstream genes. Firstly, we measured Akt expression both under the conditions of GPCR48/ LGR4 overexpression and knockdown. RT-qPCR results demonstrated that Akt expression was 4.2 (Du145) and 4.1 (PC-3) times greater in the GPCR48-overexpressing cells than Control group, respectively (Fig. 4a). Subsequently, we measured the expression of mTOR, GSK-3b and FOXO, demonstrating that mTOR and GSK-3b were up-regulated, while FOXO was down-regulated after
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GPCR48/LGR4 overexpression (Fig. 4b–d). Lentivirusmediated shRNA transfection led to an obvious crosscurrent compared with GPCR48/LGR4 overexpression. Western blotting analysis also proved that Akt was increased 5.3-fold and 4.4-fold after GPCR48/LGR4 overexpression in Du145 and PC-3 cells, respectively (Fig. 5a–c). The mTOR, GSK-3b expression in protein level were enhanced, and FOXO expression was attenuated along with GPCR48/LGR4 plasmid transfection (Fig. 5a, b, d–f). GPCR48/LGR4 knockdown almost abolished the expression of Akt, mTOR and GSK-3b while boosted FOXO expression in protein level (Fig. 5a, b, d–f). These results demonstrated a similar tendency between Akt, mTOR and GSK-3b expression and an opposite trend of FOXO expression after GPCR48/LGR4 overexpression or silence. Akt could be overexpressed in Du145 and PC-3 cells From above results, we know that the variation of GPCR48/LGR4 expression could alter the Akt expression
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Fig. 7 Up-regulation of GPCR48/LGR4 expression caused by Akt overexpression. RT-qPCR results showed that GPCR48/LGR4 mRNA expression was significantly promoted by Akt overexpression in Du145 cells (a) and PC-3 cells (b). Western blotting results showed that GPCR48/LGR4 expression was enhanced in protein level in Du145 cells (c) and PC-3 cells (d), respectively. Quantitative analysis of the above western results showed that GPCR48/ LGR4 expression was significantly up-regulated in protein level in Du145 (e) and PC-3 cells (f), respectively. Each column represented mean ± SD of at least three independent experiments; *P \ 0.05 versus untreated Du145 or PC-3 cells; #P \ 0.05 versus vector cells
both in mRNA and in protein level. To investigate whether Akt could in turn influence the expression of GPCR48/ LGR4, here, we wanted to overexpress Akt in Du145 and PC-3 cells. RT-qPCR results showed a 5.6-fold and 4.9fold increase compared with untreated Du145 and vector cells, respectively (Fig. 6a, b). In PC-3 cells, we detected a 3.1-fold and 3.3-fold increase. Western blotting results also proved the overexpression of Akt in protein level (Fig. 6c–f). Overexpression of Akt could enhance GPCR48/LGR4 expression Next, we would identify the alteration of GPCR48/LGR4 expression after Akt overexpression. We detected a 3.6fold up-regulation of GPCR48/LGR4 compared with untreated Du145 cells and a 2.5-fold rise in PC-3 cells (Fig. 7a, b). Furthermore, Western blotting showed an
overexpressed GPCR48/LGR4 after Akt overexpression both in Du145 and PC-3 cells (Fig. 7c–f). These results verified the interactions between Akt and GPCR48/LGR4 expression.
Discussion In this study, we investigated the function and effects of GPCR48/LGR4 in two prostate cancer cell lines. Firstly, GPCR48/LGR4 was overexpressed and silenced via plasmid transfection and lentivirus-mediated shRNA, respectively, in Du145 and PC-3 cells; in vitro, its high expression promoted the migration, invasion and proliferation of the two cell lines while inhibited apoptosis; besides, GPCR48/LGR4 promoted tumor growth in vivo, suggesting that GPCR48/LGR4 contributed to prostate cancer development. Next, we also found that GPCR48/
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LGR4 was positively related with Akt, mTOR and GSK3b, while negatively controlled FOXO. Interestingly, the overexpression of Akt could in turn promote GPCR48/ LGR4 expression. Therefore, the inhibition of GPCR48/ LGR4 via PI3K/Akt signaling could act as a therapeutic method for prostate cancer patients. GPCR is the biggest group of molecules attached to cell surface, which mainly functions in signal transmission and also works in tumor occurrence and development [13]. We know that excessive activation of GPCR promotes prostate cancer progression by causing androgen independence [12, 19–22]. Hence, it is pivotal to inhibit the excessively activated GPCR for prostate cancer patients [23, 24]. And, a group of GPCR named GPCR 49 was reported to be overexpressed in liver cancer and ovarian cancer, and its association cancer cells metastasis was confirmed [25, 26]. GPCR48 is another group, also known as LGR4, which has previously been reported to be involved in many different physiological and pathophysiological processes [27]. It is reported that GPCR48 overexpression could promote the proliferation of multiple cancer cells, including MCF-7, HepG2 and NCI-N87 cells [5]. Recent studies have also confirmed the function of GPCR48 in carcinogenesis [27, 28], for example, GPCR48 expression was shown to be upregulated in lung cancer at both transcription and translation level [17, 27]. However, the relationship between GPCR48/LGR4 expression and prostate cancer development has not been fully investigated. We determined to investigate their cross talk. The PI3K/Akt signaling pathway is a central regulator in cell proliferation and tumorigenesis [29]. Therefore, one of major strategies for cancer chemotherapy is inhibition of this pathway [30]. In this study, we found that overexpression of GPCR48/LGR4 could activate PI3K/Akt pathway, leading to up-regulation of Akt, mTOR, GSK-3b and down-regulation of FOXO. Akt is also known as PKB or Rac, which plays a significant role in cell survival and apoptosis [31]. mTOR serves as a regulator of protein synthesis and is usually activated in human cancer cells [32]. GSK-3b is a key enzyme of glycogen metabolism, which also functions in signaling protein and transcription factor to regulate cell behavior [33]. The co-function of the three genes contributed to promoting cell migration, invasion and proliferation. As we know, FOXO gene could foster cell apoptosis; hence, it is inhibited by GPCR48/ LGR4 overexpression and promoted by GPCR48/LGR4 silencing. We also confirmed that the silencing of GPCR48/LGR4 could inhibit Akt, mTOR and GSK-3b expression, suggesting that GPCR48/LGR4 is worthy of being studied as a therapeutic target. Lastly, we conducted an experiment to overexpress Akt in Du145 and PC-3 cells. It was confirmed that the overexpression of Akt could also up-regulate GPCR48/LGR4
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expression, proving that Akt and GPCR48/LGR4 had an interaction. Akt could serve as a linker between GPCR48/ LGR4 and PI3K/Akt signaling pathway, which deserved further investigation. Acknowledgments Conflict of interest of interest.
None. The authors declare that they have no conflict
References 1. Tenesa A, Dunlop MG. New insights into the aetiology of colorectal cancer from genome-wide association studies. Nat Rev Genet. 2009;10:353–8. 2. Narita S, Tsuchiya N, Habuchi T. Treatment for high-risk localized prostate cancer. Nihon Rinsho. 2014;72:2212–6. 3. Ghosh D, Yu H, Tan XF, Lim TK, Zubaidah RM, Tan HT, et al. Identification of key players for colorectal cancer metastasis by iTRAQ quantitative proteomics profiling of isogenic SW480 and SW620 cell lines. J Proteome Res. 2011;10:4373–87. 4. O’Neill PR, Giri L, Karunarathne WK, Patel AK, Venkatesh KV, Gautam N. The structure of dynamic GPCR signaling networks. Wiley Interdiscip Rev Syst Biol Med. 2014;6:115–23. 5. Zhu YB, Xu L, Chen M, Ma HN, Lou F. GPR48 promotes multiple cancer cell proliferation via activation of Wnt signaling. Asian Pac J Cancer Prev. 2013;14:4775–8. 6. Luo W, Rodriguez M, Valdez JM, Zhu X, Tan K, Li D, et al. Lgr4 is a key regulator of prostate development and prostate stem cell differentiation. Stem Cells. 2013;31:2492–505. 7. Luo J, Zhou W, Zhou X, Li D, Weng J, Yi Z, et al. Regulation of bone formation and remodeling by G-protein-coupled receptor 48. Development. 2009;136:2747–56. 8. Kudo M, Chen T, Nakabayashi K, Hsu SY, Hsueh AJ. The nematode leucine-rich repeat-containing, G protein-coupled receptor (LGR) protein homologous to vertebrate gonadotropin and thyrotropin receptors is constitutively active in mammalian cells. Mol Endocrinol. 2000;14:272–84. 9. Mendive F, Laurent P, Van Schoore G, Skarnes W, Pochet R, Vassart G. Defective postnatal development of the male reproductive tract in LGR4 knockout mice. Dev Biol. 2006;290:421–34. 10. Yamashita R, Takegawa Y, Sakumoto M, Nakahara M, Kawazu H, Hoshii T, et al. Defective development of the gall bladder and cystic duct in Lgr4-hypomorphic mice. Dev Dyn. 2009;238:993–1000. 11. Weng J, Luo J, Cheng X, Jin C, Zhou X, Qu J, et al. Deletion of G protein-coupled receptor 48 leads to ocular anterior segment dysgenesis (ASD) through down-regulation of Pitx2. Proc Natl Acad Sci USA. 2008;105:6081–6. 12. Daaka Y. G proteins in cancer: the prostate cancer paradigm. Sci STKE. 2004;2004:re2. 13. Dorsam RT, Gutkind JS. G-protein-coupled receptors and cancer. Nat Rev Cancer. 2007;7:79–94. 14. Rosenberg J, Small EJ. Prostate cancer update. Curr Opin Oncol. 2003;15:217–21. 15. Harada M, Noguchi M, Itoh K. Target molecules in specific immunotherapy against prostate cancer. Int J Clin Oncol. 2003;8:193–9. 16. Wu J, Xie N, Xie K, Zeng J, Cheng L, Lei Y, et al. GPR48, a poor prognostic factor, promotes tumor metastasis and activates bcatenin/TCF signaling in colorectal cancer. Carcinogenesis. 2013;34:2861–9. 17. Hsu SY, Liang SG, Hsueh AJ. Characterization of two LGR genes homologous to gonadotropin and thyrotropin receptors
Med Oncol (2015) 32:49
18.
19.
20.
21.
22.
23. 24.
25.
with extracellular leucine-rich repeats and a G protein-coupled, seven-transmembrane region. Mol Endocrinol. 1998;12:1830–45. Fresno Vara JA, Casado E, de Castro J, Cejas P, Belda-Iniesta C, Gonzalez-Baron M. PI3K/Akt signalling pathway and cancer. Cancer Treat Rev. 2004;30:193–204. Kasbohm EA, Guo R, Yowell CW, Bagchi G, Kelly P, Arora P, et al. Androgen receptor activation by G(s) signaling in prostate cancer cells. J Biol Chem. 2005;280:11583–9. Dai J, Shen R, Sumitomo M, Stahl R, Navarro D, Gershengorn MC, et al. Synergistic activation of the androgen receptor by bombesin and low-dose androgen. Clin Cancer Res. 2002;8: 2399–405. Lee LF, Guan J, Qiu Y, Kung HJ. Neuropeptide-induced androgen independence in prostate cancer cells: roles of nonreceptor tyrosine kinases Etk/Bmx, Src, and focal adhesion kinase. Mol Cell Biol. 2001;21:8385–97. Xie Y, Wolff DW, Lin MF, Tu Y. Vasoactive intestinal peptide transactivates the androgen receptor through a protein kinase A-dependent extracellular signal-regulated kinase pathway in prostate cancer LNCaP cells. Mol Pharmacol. 2007;72:73–85. Nelson JB. Endothelin inhibition: novel therapy for prostate cancer. J Urol. 2003;170:S65–7 discussion S7–8. Growcott JW. Preclinical anticancer activity of the specific endothelin A receptor antagonist ZD4054. Anticancer Drugs. 2009;20:83–8. Takahashi H, Ishii H, Nishida N, Takemasa I, Mizushima T, Ikeda M, et al. Significance of Lgr5(?ve) cancer stem cells in the colon and rectum. Ann Surg Oncol. 2011;18:1166–74.
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26. Fan XS, Wu HY, Yu HP, Zhou Q, Zhang YF, Huang Q. Expression of Lgr5 in human colorectal carcinogenesis and its potential correlation with b-catenin. Int J Colorectal Dis. 2010;25:583–90. 27. Steffen JS, Simon E, Warneke V, Balschun K, Ebert M, Rocken C. LGR4 and LGR6 are differentially expressed and of putative tumor biological significance in gastric carcinoma. Virchows Arch. 2012;461:355–65. 28. Gao Y, Kitagawa K, Hiramatsu Y, Kikuchi H, Isobe T, Shimada M, et al. Up-regulation of GPR48 induced by down-regulation of p27Kip1 enhances carcinoma cell invasiveness and metastasis. Cancer Res. 2006;66:11623–31. 29. Polivka J Jr, Janku F. Molecular targets for cancer therapy in the PI3K/AKT/mTOR pathway. Pharmacol Ther. 2014;142:164–75. 30. Sabbah DA, Brattain MG, Zhong H. Dual inhibitors of PI3K/ mTOR or mTOR-selective inhibitors: which way shall we go? Curr Med Chem. 2011;18:5528–44. 31. Jiang K, Zhong B, Ritchey C, Gilvary DL, Hong-Geller E, Wei S, et al. Regulation of Akt-dependent cell survival by Syk and Rac. Blood. 2003;101:236–44. 32. Laplante M, Sabatini DM. mTOR signaling in growth control and disease. Cell. 2012;149:274–93. 33. Gupta C, Kaur J, Tikoo K. Regulation of MDA-MB-231 cell proliferation by GSK-3b involves epigenetic modifications under high glucose conditions. Exp Cell Res. 2014;324:75–83.
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ORIGINAL PAPER
GPCR48/LGR4 promotes tumorigenesis of prostate cancer via PI3K/Akt signaling pathway Fang Liang • Junmin Yue • Junyong Wang Lijuan Zhang • Rui Fan • Hao Zhang • Qingsong Zhang
•
Received: 22 December 2014 / Accepted: 22 January 2015 Ó Springer Science+Business Media New York 2015
Abstract G-protein-coupled receptor (GPCR) 48, also known as leucine-rich repeat-containing G-protein-coupled receptor (LGR) 4, is an orphan receptor belonging to the GPCR superfamily, which plays an important role in the development of various organs and multiple cancers. However, the function of GPCR48/LGR4 in prostate cancer has not been fully investigated. Herein, GPCR48/LGR4 was overexpressed and silenced in prostate cancer cells via plasmid and shRNA transfection, respectively. The expression of GPCR48/LGR4 in mRNA and protein levels was analyzed using RT-qPCR and Western blotting, respectively. Subsequently, we demonstrated the effects of GPCR48/LGR4 on the migration, invasion, proliferation and apoptosis of prostate cancer cells, including Du145 and PC-3 cells. Next, we investigated the relationship between GPCR48/LGR4 and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/Akt signaling pathway. The results showed that the overexpression of GPCR48/LGR4 was associated with the up-regulation of Akt, a key effector of PI3K/Akt signaling pathway, which meantime up-regulated the expression of mammalian target of rapamycin
Fang Liang and Junmin Yue have contributed equally to the work. F. Liang L. Zhang Department of Oncology, Zhengzhou Central Hospital Affiliated to Zhengzhou University, Zhengzhou, China J. Yue J. Wang R. Fan H. Zhang Department of Urinary Surgery, Zhengzhou Central Hospital Affiliated to Zhengzhou University, Zhengzhou, China Q. Zhang (&) Department of Breast Surgery, Zhengzhou Central Hospital Affiliated to Zhengzhou University, 195 Tongbai Avenue, Zhengzhou 450007, China e-mail: [email protected]
(mTOR) and glycogen synthase kinase 3b (GSK-3b), while down-regulated forkhead box, class O (FOXO), all of whom are the downstream targets of PI3K/Akt signaling pathway. Hence, the results suggested that GPCR48/LGR4 may regulate prostate cancer cells and tumor growth via the PI3K/Akt signaling pathway and could provide a better therapeutic target for the diagnosis and treatment of prostate cancer. Keywords Tumor
GPCR48/LGR4 Prostate cancer PI3K/Akt
Introduction Prostate cancer is the main cause of cancer-related deaths, which has the second largest population of male patients in the USA [1]. Traditional treating methods include androgen deprivation therapy (ADT), radical prostatectomy, hormonal therapy and radio-chemotherapy [2]. ADT contributes greatly during the early stages of prostate cancer. Hormonal treatment is relatively simple and less painful; however, it may easily lead to drug resistance, and related drugs are ineffective at this stage. To acquire a high cure rate, localized prostate tumor is usually subjected to radical prostatectomy or radio-chemotherapy [3]. What is worse, patients usually suffer from side effects of the radiochemotherapy, such as hair loss or immune system degradation. G-protein-coupled receptor (GPCR) is a big superfamily, which contributes greatly to the development of some organs [4] and even to the formation of some tumors, such as lung cancer and prostate cancer [5–7]. GPCR superfamily also involves a subfamily of leucine-rich repeatcontaining G-protein-coupled receptors (LGR), which
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consist of a transmembrane domain of seven a-helixes and an extracellular area with a long N-terminal fragment containing leucine-rich repeat motifs which function in receptor-ligand interactions [8–10]. As reported, LGR has three kinds of subtypes, including three hormones (luteotropic hormone, follicle-stimulating hormone and thyroid stimulating hormone), LGR 4–6 and LGR 7–8 [11]. Recently, both preclinical and clinical studies have proved that GPCR was overexpressed in prostate cancer tissues [12, 13]; however, the mechanism was still not clear. Current methods such as prostatectomy and radio-chemotherapy are so radical and drastic that patients may suffer from secondary diseases. Besides, we need earlier diagnosis and treatment which benefit a lot to the patients because of the limited focus and functions of cancer cells are easier to deal with. What is more important is that we want to diagnose and treat prostate cancer specifically. These all underline the need for new methods with specific target molecules for prostate cancer [14, 15]. GPCR is widespread in human bodies, and its function has been proved in some cancers, such as gastric cancer and colorectal cancer [16]. Convincing evidence confirms that abnormal activation or expression of GPCR is intimately related to increased cancer cell proliferation, tumor growth and metastasis [12, 13]. These characteristics would highlight GPCR as a novel biomarker and therapeutic target of prostate cancer. Therefore, the identification of a specific GPCR for prostate cancer and a possibly related signaling pathway are two crucial prerequisites for a more detailed way to recover from prostate cancer. GPCR48, also known as LGR4, is an orphan receptor which exists broadly in multiple tissues and organs from embryonic to adult periods [16, 17]. In the PI3K/Akt signaling pathway, what is more, Akt is the pivotal link between the upstream regulation and downstream expression in the development of various organs [18]. Our study aimed to investigate the function of GPCR48/LGR4 in prostate cancer cell lines and its correlation with prostate tumors growth and the PI3K/Akt signaling pathway. We hypothesize that GPCR48/LGR4 is a potential new target for the diagnosis and treatment of prostate cancer.
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Cell culture Du145 (American Type Culture Collection, Manassas, VA, USA) and PC-3 cells (The Cell Bank of Chinese Academy of Sciences, Shanghai) were cultured in RPMI-1640 medium (Hyclone, Logan, UT, USA) supplemented with 10 % (v/v) fetal bovine serum (FBS, Hyclone) and 1 % (v/v) penicillin/streptomycin (Hyclone). Cells were grown in a humidified incubator at 37 °C with 5 % CO2. Plasmid constructions and cell transfection A GPCR48 plasmid encoding full-length human GPCR48 and an empty vector was purchased from Fitgene (Guangzhou, China). For GPCR48 overexpression, the GPCR48 plasmid and the empty vector were separately transfected into cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions, and the stable transfectants were selected in the presence of 600 lg/ml G418 (Amresco, Solon, OH, USA). Transfection with lentivirus-mediated shRNA GPCR48/LGR4 knockdown was performed via transfection with a lentivirus that expresses human GPCR48/ LGR4-specific short hairpin RNA (shRNA). Briefly, the lentivirus vector plasmid encoding human GPCR48/LGR4specific shRNA (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was transfected with capsule and packaging plasmids using Superfect (Qiagen, Dusseldorf, Germany) into HEK293T cells. Supernatant was collected 48 h later and used as infection solution without enrichment. The scramble shRNA obtained from Addgene (Cambridge, MA, USA) was also employed. The non-infected cells were removed by medium containing puromycin (5 lg/ml; Sigma-Aldrich, St. Louis, MO, USA). The GPCR48/LGR4 knockdown was confirmed by RT-qPCR and Western blotting. Transwell assay
Materials and methods Animals Six-week-old male athymic nude mice were purchased from the Experimental Animal Center of Zhengzhou University (Zhengzhou, China) and fed in pathogen-free conditions with free access to boiled water and food. All animal experiments were done in accordance with protocols preapproved by the Institutional Animal Care and Treatment Committee of Zhengzhou Central Hospital.
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Cell migration ability was measured in 24-well transwell chambers (Corning, Tewksbury, MA, USA). The bottom chamber was prepared with 10 % FBS as a chemoattractant. For migration assays, cells in serum-free medium (5 9 105 cells per well) were added to the upper chamber and incubated for 12 h. Non-migrating cells on the upper side of the membrane were removed, and the cells that migrated to the lower chamber were fixed with 4 % paraformaldehyde and stained with hematoxylin–eosin (HE). For invasion assays, Matrigel (BD Biosciences, San Jose, CA, USA) was added to the transwell membrane chambers
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and incubated at 37 °C for 4 h. Cells (1 9 105 cells per well) were seeded and incubated before analysis. The number of migrating or invading cells was then determined in five randomly selected fields under a microscope. Each experiment was performed at least three times. Both migration assay and invasion assay were tested at three time points (24, 36 and 48 h after overexpression or knockdown), respectively. MTT assay An MTT assay was used to detect cell proliferation. Cells were seeded into 96-well plates at a rate of 1,000 cells per well. On the day of harvest (24, 36 and 48 h after overexpression or knockdown), 100 ll of spent culture medium was replaced with an equal volume of fresh medium (10 % FBS). MTT (Sigma-Aldrich) was added to the cells at a final concentration of 0.5 mg/ml, and the plates were incubated for 4 h at 37 °C. Then, 100 ll of DMSO (SigmaAldrich) was added to each well and plates were shaken at room temperature for 10 min. The absorbance was measured at 490 nm. Apoptosis analysis Hoechst staining and TUNEL assay were used to assess the cell apoptosis. Cells were cultured in 96-well plates with a density of 3,000 cells per well after overexpression and silence of GPCR48/LGR4. Cellular nuclear morphology was detected using Hoechst Staining Kit (Beyotime Biotech, Haimen, China). DNA fragmentation morphology was examined by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) staining using TUNEL Apoptosis Detection Kit (Merck Millipore, Billerica, MA, USA). Both staining-positive cells were visualized and counted in randomly selected fields for further analysis. Tumor growth For xenograft tumor transplantation, cells (2 9 106) in 100 ll PBS and Matrigel [1:1 (v/v)] were subcutaneously injected into the flanks of athymic male mice. External caliper measurement of long (L) and short (S) tumor lengths was done to calculate tumor volume using the equation: p Tumor volume ¼ L S2 6 Tumor measurements were taken throughout the duration of the experiment at least three times every 5 days. Tissue mass was measured immediately after killing and surgical excision of tumor. Tissues were divided in half, flash-
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frozen in liquid nitrogen and stored at -80 °C. Experiments were repeated twice. Data represent values from 5 to 10 animals. RT-qPCR RNA was isolated using TRIzol reagent (Invitrogen), and complementary DNA (cDNA) was created from 1 lg RNA using RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA). Product amplification was done using a SYBR Green PCR kit (TAKARA BIOTECHNOLOGY, Dalian, Liaoning, China) with CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). For RT-qPCR, 2 ll of cDNA was amplified in a 20-ll standard PCR system, followed by initial denaturation at 94 °C for 3 min; 45 cycles of 94 °C for 45 s, annealing temperature (different, respectively) for 45 s, 72 °C for 45 s; and a final extension for 10 min at 72 °C, followed by termination at 4 °C. Primer sequences were: GPCR48/LGR4 (sense) 50 - CCA ATC ACC ACT CTT ACA CAA TGG CTA AC -30 and (antisense) 50 - ATT CCC GTA GGA GAT AGC GTC CTA G -30 ; Akt (sense) 50 - ATG GTA CCA TGA GCG ACG TGG C -30 and (antisense) 50 - ATT CTA GAT CAG GCC GTG CCG C -30 ; mTOR (sense) 50 - TGC TAC GGA CGC ACT GAG AT -30 and (antisense) 50 - CGT GAA GAG GGA AGG TGG AAC A-30 ; GSK-3b (sense) 50 - ATG GAA CAC CAG CTC CTG TG -30 and (antisense) 50 - TGT CGG TGT AGA TGC ACA G -30 ; FOXO (sense) 50 - TCA TTG AAC AGC CAC CAC -30 and (antisense) 50 - CCC AGA CTC AGG AGG AAG -30 ; and b-actin (sense) 50 - CAG CGA CAC CCA CTC CTC -30 and (antisense) 50 - TGA GGT CCA CCA CCC TGT -30 . All experiments were run three times using at least triplicate samples. Relative gene level calculation was based on the difference in the Ct of the reference gene (b-actin). Relative gene expression is shown as the fold over the reference gene, determined by the DDCt method. Western blotting Western blotting analysis was used to determine relative protein expression. Cells that grew to 70–80 % confluence (about 48 h after overexpression or knockdown) were washed once with ice-cold PBS, lysed in RIPA buffer (Sigma-Aldrich), supplemented with protease inhibitors PMSF (Sigma-Aldrich) and harvested for protein fractionation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Blots were incubated in blocking buffer composed of 4 % bovine serum albumin (BSA, Sigma-Aldrich) tris-buffered saline/Tween-20 (Sigma-Aldrich) for 1 h, followed by 2-h incubation with primary antibody, all 1:1,000 (v/v) in blocking buffer.
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Fig. 1 Overexpression and silencing of GPCR48/LGR4 in Du145 and PC-3 cells, respectively. a RT-qPCR analysis showed that GPCR48/ LGR4 was overexpressed in mRNA level. b RT-qPCR analysis showed that GPCR48/ LGR4 expression was silenced in mRNA level. Western blotting analysis (c) and quantitative analysis of western results (e) also showed that GPCR48/LGR4 was overexpressed in protein level. Western blotting analysis (d) and quantitative analysis of western results (f) also showed that GPCR48/LGR4 expression was silenced in protein level. Each column represented mean ± SD of at least three independent experiments; *P \ 0.05 and **P \ 0.01 versus untreated Du145 or PC-3 cells; #P \ 0.05 and ##P \ 0.01 versus cells transfected with vector or scramble shRNA
Immunoblots were washed in tris-buffered saline/Tween three times for 5 min each, incubated with the appropriate horseradish peroxidase-conjugated anti-rabbit or antimouse secondary antibody for 1 h and washed. The protein signal was visualized using the Amersham ECLTM Plus Western Blotting Detection kit (GE Healthcare, Piscataway, NJ, USA).
determine significant differences between two-sample data. All values are considered significant if P \ 0.05 (*), extremely significant if P \ 0.01 (**).
Results
Statistical analysis
GPCR48/LGR4 is overexpressed or silenced via plasmid or lentivirus transfection, respectively
All the artworks were created using GraphPad Prism 5 software. All statistical analyses were carried out using the SPSS 19.0 statistical software package. All numerical data are shown as the mean ± standard deviation (SD) of at least three independent experiments or at least five biological samples (tumor). The Student’s t test was used to
Firstly, we wanted to verify the overexpression and knockdown of GPCR48/LGR4 gene in prostate cancer cells. We observed that GPCR48/LGR4 expression was significantly up-regulated after the transfection of gene encoding GPCR48/LGR4 compared with untreated cells and vector cells (transfected with empty vector) in both
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Fig. 2 Effects of GPCR48/ LGR4 on migration, invasion, proliferation and apoptosis. a Cell migration ability was promoted by GPCR48/LGR4 overexpression while inhibited by GPCR48/LGR4 knockdown. b Cell invasion ability was promoted by GPCR48/LGR4 overexpression while inhibited by GPCR48/LGR4 knockdown. Results were presented as the ratios of cells relative to those of Control group. c, d MTT assays showed that proliferation ability was significantly increased after GPCR48/LGR4 overexpression but decreased after GPCR48/LGR4 knockdown. e Hoechst staining results showed that GPCR48/ LGR4 inhibited apoptosis of both Du145 and PC-3 cells. f TUNEL assay also proved that GPCR48/LGR4 inhibited apoptosis. Each column represented mean ± SD of at least three independent experiments; *P \ 0.05 versus untreated Du145 or PC-3 cells
mRNA (Fig. 1a) and protein level (Fig. 1c, e). Also, the expression of GPCR48/LGR4 was inhibited by genes silence (Fig. 1b, d, f). These results demonstrated that we overexpressed and silenced GPCR48/LGR4 gene in both Du145 and PC-3 cells, respectively. The migration, invasion, proliferation and apoptosis of Du145 and PC-3 cells are regulated by GPCR48/ LGR4 Next, to investigate the effects of GPCR48/LGR4 on migration and invasion, Du145 and PC-3 cells were subjected to in vitro transwell assay. The number of GPCR48-
overexpressing cells migrating into the lower chamber was at most 5.3-fold (Du145) and 5.5-fold (PC-3) greater than untreated cells (Control group) in a transwell chamber migration assay (Fig. 2a). The results of a Matrigel invasion assay revealed at most 25.1-fold (Du145) and 26.7-fold (PC3) up-regulation in the invasive capacity of GPCR48-overexpressing cells compared with Control group (Fig. 2b). Similarly, both the migration and invasion ability were attenuated after GPCR48/LGR4 knockdown (Fig. 2a, b). We also assessed the effect of GPCR48 on proliferation and found that Du145/GPCR48(LGR4) plasmid cells increased 3.2-fold (Fig. 2c) and PC-3/GPCR48(LGR4) plasmid cells 2.0-fold (Fig. 2d) more than the Control group. The
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Fig. 3 Effects of GPCR48/LGR4 on tumor growth. a For Du145 cells, curves for average tumor volume showed that GPCR48/LGR4 overexpression significantly increased the tumor volume, while GPCR48/LGR4 silence inhibited tumor growth. b For PC-3 cells, curves for average tumor volume showed that GPCR48/LGR4
Fig. 4 RT-qPCR analysis of the effects of GPCR48/LGR4 on PI3K-/Akt-related genes expression in mRNA level. a Akt expression was significantly promoted via GPCR48/LGR4 overexpression, but its expression was significantly down-regulated by GPCR48/LGR4 knockout. b mTOR. c GSK-3b. d FOXO showed an opposite result from Akt, mTOR and GSK-3b. Each column represented mean ± SD of at least three independent experiments; *P \ 0.05 and **P \ 0.01 versus untreated Du145 or PC-3 cells
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overexpression significantly increased the tumor volume, while GPCR48/LGR4 silence inhibited tumor growth. Each column represented mean ± SD of 5–10 independent experiments; *P \ 0.05 versus mice injected with untreated Du145 and PC-3 cells
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Fig. 5 Western blotting analysis of the effects of GPCR48/LGR4 on PI3K-/Aktrelated genes expression in protein level. Western blotting results of Akt, mTOR, GSK-3b and FOXO. Akt, mTOR and GSK-3b expression was enhanced after GPCR48/LGR4 overexpression but attenuated after GPCR48/LGR4 silencing. On the contrary, FOXO expression was attenuated after GPCR48/LGR4 overexpression but enhanced after GPCR48/ LGR4 silencing in Du145 cells (a) and PC-3 cells (b). c– f Quantitative analysis of the western results. Each column represented mean ± SD of at least three independent experiments; **P \ 0.01 versus untreated Du145 or PC-3 cells
proliferation ability was also inhibited by GPCR48/LGR4 silence (Fig. 2c, d). Lastly, we identified the effects of GPCR48/LGR4 on apoptosis. Hoechst staining results showed that GPCR48/LGR4 overexpression slowed down apoptosis and GPCR48/LGR4 overexpression promoted (Fig. 2e). TUNEL results showed a decrease and increase in positive cells after overexpression and silence of GPCR48/ LGR4, respectively, which kept same pace with abovementioned results (Fig. 2f). Collectively, these results
demonstrated that GPCR48 could not only promote migration, invasion and proliferation of Du145 and PC-3 cells but also inhibit apoptosis. Overexpression of GPCR48/LGR4 promotes prostate tumor growth To test whether GPCR48 overexpression and knockdown impacted the growth of prostate tumor in vivo, Du145 and
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Fig. 6 Akt overexpression after transfection with Akt plasmid in Du145 and PC-3 cells. a Akt was overexpressed in Du145 cells in mRNA level. b Akt was overexpressed in PC-3 cells. Western blotting results showed Akt overexpression in both Du145 (c) and PC-3 cells (d). Quantitative analysis of the above western results showed that Akt expression was significantly up-regulated in protein level after plasmid transfection in Du145 (e) and PC-3 cells (f), respectively. Each column represented mean ± SD of at least three independent experiments; *P \ 0.05 versus untreated Du145 or PC-3 cells; #P \ 0.05 versus vector cells
PC-3 cells were subcutaneously injected into immunodeficient mice. The results showed that GPCR48/LGR4 overexpression increased the tumor volume to 79 % (Du145, Fig. 3a) and 77 % (PC-3, Fig. 3b) compared with Control group, respectively, in contrast to the negative effects of shRNA groups. GPCR48/LGR4 regulates the expression of PI3K/Akt signaling genes Akt served as a pivotal link of Wnt/b-catenin signaling pathway and its downstream genes. Firstly, we measured Akt expression both under the conditions of GPCR48/ LGR4 overexpression and knockdown. RT-qPCR results demonstrated that Akt expression was 4.2 (Du145) and 4.1 (PC-3) times greater in the GPCR48-overexpressing cells than Control group, respectively (Fig. 4a). Subsequently, we measured the expression of mTOR, GSK-3b and FOXO, demonstrating that mTOR and GSK-3b were up-regulated, while FOXO was down-regulated after
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GPCR48/LGR4 overexpression (Fig. 4b–d). Lentivirusmediated shRNA transfection led to an obvious crosscurrent compared with GPCR48/LGR4 overexpression. Western blotting analysis also proved that Akt was increased 5.3-fold and 4.4-fold after GPCR48/LGR4 overexpression in Du145 and PC-3 cells, respectively (Fig. 5a–c). The mTOR, GSK-3b expression in protein level were enhanced, and FOXO expression was attenuated along with GPCR48/LGR4 plasmid transfection (Fig. 5a, b, d–f). GPCR48/LGR4 knockdown almost abolished the expression of Akt, mTOR and GSK-3b while boosted FOXO expression in protein level (Fig. 5a, b, d–f). These results demonstrated a similar tendency between Akt, mTOR and GSK-3b expression and an opposite trend of FOXO expression after GPCR48/LGR4 overexpression or silence. Akt could be overexpressed in Du145 and PC-3 cells From above results, we know that the variation of GPCR48/LGR4 expression could alter the Akt expression
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Fig. 7 Up-regulation of GPCR48/LGR4 expression caused by Akt overexpression. RT-qPCR results showed that GPCR48/LGR4 mRNA expression was significantly promoted by Akt overexpression in Du145 cells (a) and PC-3 cells (b). Western blotting results showed that GPCR48/LGR4 expression was enhanced in protein level in Du145 cells (c) and PC-3 cells (d), respectively. Quantitative analysis of the above western results showed that GPCR48/ LGR4 expression was significantly up-regulated in protein level in Du145 (e) and PC-3 cells (f), respectively. Each column represented mean ± SD of at least three independent experiments; *P \ 0.05 versus untreated Du145 or PC-3 cells; #P \ 0.05 versus vector cells
both in mRNA and in protein level. To investigate whether Akt could in turn influence the expression of GPCR48/ LGR4, here, we wanted to overexpress Akt in Du145 and PC-3 cells. RT-qPCR results showed a 5.6-fold and 4.9fold increase compared with untreated Du145 and vector cells, respectively (Fig. 6a, b). In PC-3 cells, we detected a 3.1-fold and 3.3-fold increase. Western blotting results also proved the overexpression of Akt in protein level (Fig. 6c–f). Overexpression of Akt could enhance GPCR48/LGR4 expression Next, we would identify the alteration of GPCR48/LGR4 expression after Akt overexpression. We detected a 3.6fold up-regulation of GPCR48/LGR4 compared with untreated Du145 cells and a 2.5-fold rise in PC-3 cells (Fig. 7a, b). Furthermore, Western blotting showed an
overexpressed GPCR48/LGR4 after Akt overexpression both in Du145 and PC-3 cells (Fig. 7c–f). These results verified the interactions between Akt and GPCR48/LGR4 expression.
Discussion In this study, we investigated the function and effects of GPCR48/LGR4 in two prostate cancer cell lines. Firstly, GPCR48/LGR4 was overexpressed and silenced via plasmid transfection and lentivirus-mediated shRNA, respectively, in Du145 and PC-3 cells; in vitro, its high expression promoted the migration, invasion and proliferation of the two cell lines while inhibited apoptosis; besides, GPCR48/LGR4 promoted tumor growth in vivo, suggesting that GPCR48/LGR4 contributed to prostate cancer development. Next, we also found that GPCR48/
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LGR4 was positively related with Akt, mTOR and GSK3b, while negatively controlled FOXO. Interestingly, the overexpression of Akt could in turn promote GPCR48/ LGR4 expression. Therefore, the inhibition of GPCR48/ LGR4 via PI3K/Akt signaling could act as a therapeutic method for prostate cancer patients. GPCR is the biggest group of molecules attached to cell surface, which mainly functions in signal transmission and also works in tumor occurrence and development [13]. We know that excessive activation of GPCR promotes prostate cancer progression by causing androgen independence [12, 19–22]. Hence, it is pivotal to inhibit the excessively activated GPCR for prostate cancer patients [23, 24]. And, a group of GPCR named GPCR 49 was reported to be overexpressed in liver cancer and ovarian cancer, and its association cancer cells metastasis was confirmed [25, 26]. GPCR48 is another group, also known as LGR4, which has previously been reported to be involved in many different physiological and pathophysiological processes [27]. It is reported that GPCR48 overexpression could promote the proliferation of multiple cancer cells, including MCF-7, HepG2 and NCI-N87 cells [5]. Recent studies have also confirmed the function of GPCR48 in carcinogenesis [27, 28], for example, GPCR48 expression was shown to be upregulated in lung cancer at both transcription and translation level [17, 27]. However, the relationship between GPCR48/LGR4 expression and prostate cancer development has not been fully investigated. We determined to investigate their cross talk. The PI3K/Akt signaling pathway is a central regulator in cell proliferation and tumorigenesis [29]. Therefore, one of major strategies for cancer chemotherapy is inhibition of this pathway [30]. In this study, we found that overexpression of GPCR48/LGR4 could activate PI3K/Akt pathway, leading to up-regulation of Akt, mTOR, GSK-3b and down-regulation of FOXO. Akt is also known as PKB or Rac, which plays a significant role in cell survival and apoptosis [31]. mTOR serves as a regulator of protein synthesis and is usually activated in human cancer cells [32]. GSK-3b is a key enzyme of glycogen metabolism, which also functions in signaling protein and transcription factor to regulate cell behavior [33]. The co-function of the three genes contributed to promoting cell migration, invasion and proliferation. As we know, FOXO gene could foster cell apoptosis; hence, it is inhibited by GPCR48/ LGR4 overexpression and promoted by GPCR48/LGR4 silencing. We also confirmed that the silencing of GPCR48/LGR4 could inhibit Akt, mTOR and GSK-3b expression, suggesting that GPCR48/LGR4 is worthy of being studied as a therapeutic target. Lastly, we conducted an experiment to overexpress Akt in Du145 and PC-3 cells. It was confirmed that the overexpression of Akt could also up-regulate GPCR48/LGR4
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expression, proving that Akt and GPCR48/LGR4 had an interaction. Akt could serve as a linker between GPCR48/ LGR4 and PI3K/Akt signaling pathway, which deserved further investigation. Acknowledgments Conflict of interest of interest.
None. The authors declare that they have no conflict
References 1. Tenesa A, Dunlop MG. New insights into the aetiology of colorectal cancer from genome-wide association studies. Nat Rev Genet. 2009;10:353–8. 2. Narita S, Tsuchiya N, Habuchi T. Treatment for high-risk localized prostate cancer. Nihon Rinsho. 2014;72:2212–6. 3. Ghosh D, Yu H, Tan XF, Lim TK, Zubaidah RM, Tan HT, et al. Identification of key players for colorectal cancer metastasis by iTRAQ quantitative proteomics profiling of isogenic SW480 and SW620 cell lines. J Proteome Res. 2011;10:4373–87. 4. O’Neill PR, Giri L, Karunarathne WK, Patel AK, Venkatesh KV, Gautam N. The structure of dynamic GPCR signaling networks. Wiley Interdiscip Rev Syst Biol Med. 2014;6:115–23. 5. Zhu YB, Xu L, Chen M, Ma HN, Lou F. GPR48 promotes multiple cancer cell proliferation via activation of Wnt signaling. Asian Pac J Cancer Prev. 2013;14:4775–8. 6. Luo W, Rodriguez M, Valdez JM, Zhu X, Tan K, Li D, et al. Lgr4 is a key regulator of prostate development and prostate stem cell differentiation. Stem Cells. 2013;31:2492–505. 7. Luo J, Zhou W, Zhou X, Li D, Weng J, Yi Z, et al. Regulation of bone formation and remodeling by G-protein-coupled receptor 48. Development. 2009;136:2747–56. 8. Kudo M, Chen T, Nakabayashi K, Hsu SY, Hsueh AJ. The nematode leucine-rich repeat-containing, G protein-coupled receptor (LGR) protein homologous to vertebrate gonadotropin and thyrotropin receptors is constitutively active in mammalian cells. Mol Endocrinol. 2000;14:272–84. 9. Mendive F, Laurent P, Van Schoore G, Skarnes W, Pochet R, Vassart G. Defective postnatal development of the male reproductive tract in LGR4 knockout mice. Dev Biol. 2006;290:421–34. 10. Yamashita R, Takegawa Y, Sakumoto M, Nakahara M, Kawazu H, Hoshii T, et al. Defective development of the gall bladder and cystic duct in Lgr4-hypomorphic mice. Dev Dyn. 2009;238:993–1000. 11. Weng J, Luo J, Cheng X, Jin C, Zhou X, Qu J, et al. Deletion of G protein-coupled receptor 48 leads to ocular anterior segment dysgenesis (ASD) through down-regulation of Pitx2. Proc Natl Acad Sci USA. 2008;105:6081–6. 12. Daaka Y. G proteins in cancer: the prostate cancer paradigm. Sci STKE. 2004;2004:re2. 13. Dorsam RT, Gutkind JS. G-protein-coupled receptors and cancer. Nat Rev Cancer. 2007;7:79–94. 14. Rosenberg J, Small EJ. Prostate cancer update. Curr Opin Oncol. 2003;15:217–21. 15. Harada M, Noguchi M, Itoh K. Target molecules in specific immunotherapy against prostate cancer. Int J Clin Oncol. 2003;8:193–9. 16. Wu J, Xie N, Xie K, Zeng J, Cheng L, Lei Y, et al. GPR48, a poor prognostic factor, promotes tumor metastasis and activates bcatenin/TCF signaling in colorectal cancer. Carcinogenesis. 2013;34:2861–9. 17. Hsu SY, Liang SG, Hsueh AJ. Characterization of two LGR genes homologous to gonadotropin and thyrotropin receptors
Med Oncol (2015) 32:49
18.
19.
20.
21.
22.
23. 24.
25.
with extracellular leucine-rich repeats and a G protein-coupled, seven-transmembrane region. Mol Endocrinol. 1998;12:1830–45. Fresno Vara JA, Casado E, de Castro J, Cejas P, Belda-Iniesta C, Gonzalez-Baron M. PI3K/Akt signalling pathway and cancer. Cancer Treat Rev. 2004;30:193–204. Kasbohm EA, Guo R, Yowell CW, Bagchi G, Kelly P, Arora P, et al. Androgen receptor activation by G(s) signaling in prostate cancer cells. J Biol Chem. 2005;280:11583–9. Dai J, Shen R, Sumitomo M, Stahl R, Navarro D, Gershengorn MC, et al. Synergistic activation of the androgen receptor by bombesin and low-dose androgen. Clin Cancer Res. 2002;8: 2399–405. Lee LF, Guan J, Qiu Y, Kung HJ. Neuropeptide-induced androgen independence in prostate cancer cells: roles of nonreceptor tyrosine kinases Etk/Bmx, Src, and focal adhesion kinase. Mol Cell Biol. 2001;21:8385–97. Xie Y, Wolff DW, Lin MF, Tu Y. Vasoactive intestinal peptide transactivates the androgen receptor through a protein kinase A-dependent extracellular signal-regulated kinase pathway in prostate cancer LNCaP cells. Mol Pharmacol. 2007;72:73–85. Nelson JB. Endothelin inhibition: novel therapy for prostate cancer. J Urol. 2003;170:S65–7 discussion S7–8. Growcott JW. Preclinical anticancer activity of the specific endothelin A receptor antagonist ZD4054. Anticancer Drugs. 2009;20:83–8. Takahashi H, Ishii H, Nishida N, Takemasa I, Mizushima T, Ikeda M, et al. Significance of Lgr5(?ve) cancer stem cells in the colon and rectum. Ann Surg Oncol. 2011;18:1166–74.
Page 11 of 11
49
26. Fan XS, Wu HY, Yu HP, Zhou Q, Zhang YF, Huang Q. Expression of Lgr5 in human colorectal carcinogenesis and its potential correlation with b-catenin. Int J Colorectal Dis. 2010;25:583–90. 27. Steffen JS, Simon E, Warneke V, Balschun K, Ebert M, Rocken C. LGR4 and LGR6 are differentially expressed and of putative tumor biological significance in gastric carcinoma. Virchows Arch. 2012;461:355–65. 28. Gao Y, Kitagawa K, Hiramatsu Y, Kikuchi H, Isobe T, Shimada M, et al. Up-regulation of GPR48 induced by down-regulation of p27Kip1 enhances carcinoma cell invasiveness and metastasis. Cancer Res. 2006;66:11623–31. 29. Polivka J Jr, Janku F. Molecular targets for cancer therapy in the PI3K/AKT/mTOR pathway. Pharmacol Ther. 2014;142:164–75. 30. Sabbah DA, Brattain MG, Zhong H. Dual inhibitors of PI3K/ mTOR or mTOR-selective inhibitors: which way shall we go? Curr Med Chem. 2011;18:5528–44. 31. Jiang K, Zhong B, Ritchey C, Gilvary DL, Hong-Geller E, Wei S, et al. Regulation of Akt-dependent cell survival by Syk and Rac. Blood. 2003;101:236–44. 32. Laplante M, Sabatini DM. mTOR signaling in growth control and disease. Cell. 2012;149:274–93. 33. Gupta C, Kaur J, Tikoo K. Regulation of MDA-MB-231 cell proliferation by GSK-3b involves epigenetic modifications under high glucose conditions. Exp Cell Res. 2014;324:75–83.
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